Nonnucleoside inhibitors of reverse transcriptase, composite binding pocket and methods for use thereof

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The inventors acknowledge and appreciate the assistance of Dr. Elise Sudbeck.
BACKGROUND OF THE INVENTION
Design of potent inhibitors of human immunodeficiency virus (HIV-1) reverse transcriptase (RT), an enzyme responsible for the reverse transcription of the retroviral RNA to proviral DNA, has been a focal point in translational AIDS research efforts (Greene, W. C., New England Journal of Medicine, 1991, 324, 308-317; Mitsuya, H. et al., Science, 1990, 249, 1533-1544; De Clercq, E., J. Acquired Immune Defic. Syndr. Res. Human. Retrovirus, 1992, 8, 119-134). Promising inhibitors include nonnucleoside inhibitors (NNI), which bind to a specific allosteric site of HIV-1 RT near the polymerase site and interfere with reverse transcription by altering either the conformation or mobility of RT, thereby leading to noncompetitive inhibition of the enzyme (Kohlstaedt, L. A. et al., Science, 1992, 256, 1783-1790).
NNI of HIV-1 RT include the following:
(a) 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT; Tanaka, H. et al., J. Med. Chem., 1991, 34, 349-357; Pontikis, R. et al., J. Med. Chem., 1997, 40, 1845-1854; Danel, K., et al., J. Med. Chem., 1996, 39, 2427-2431; Baba, M., et al., Antiviral Res, 1992, 17, 245-264);
(b) tetrahydroimidazobenzodiazepinethiones (TIBO; Pauwels, R. et al., Nature, 1990, 343, 470-474);
(c) bis(heteroaryl)piperazines (BHAP; Romero, D. L. et al., J. Med. Chem., 1993, 36, 1505-1508);
(d) dihydroalkoxybenzyloxopyrimidine (DABO; Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med. Chem., 1997, 40, 1447-1454);
(e) 2'-5'-bis-O-(tertbutyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) pyrimidines (TSAO; Balzarini, J. et al., Proc. Natl. Acad. Sci USA, 1992, 89, 4392-4396); and
(f) phenethylthiazolylthiourea (PETT) derivatives (Bell, F. W. et al., J. Med. Chem., 1995, 38, 4929-4936; Cantrell, A. S. et al., J. Med. Chem., 1996, 39, 4261-4274).
Current protein structure-based drug design efforts rely heavily on crystal structure information of the target binding site. A number of crystal structures of RT complexed with NNIs (including .alpha.-APA, TIBO, Nevirapine, BHAP and HEPT derivatives) have been reported, and such structural information provides the basis for further derivatization of NNI aimed at maximizing binding affinity to RT. However, the number of available crystal structures of RT NNI complexes is limited, and no structural information has been reported for RT-PETT complexes or RT-DABO complexes. Given the lack of structural information, researchers must rely on other design procedures for preparing active PETT and DABO derivatives. One of the first reported strategies for systematic synthesis of PETT derivatives was the analysis of structure-activity relationships independent of the structural properties of RT and led to the development of some PETT derivatives with significant anti-HIV activity (Bell, F. W. et al., J. Med. Chem., 1995, 38, 4929-4936; Cantrell, A. S. et al., J. Med. Chem., 1996, 39, 4261-4274). The inclusion of structural information in the drug design process should lead to more efficient identification of promising RT inhibitors.
Although the crystal structure of an RT-NNI complex can be used to provide useful information for the design of a different type of NNI, its application is limited. For example, an analysis of the RT-APA (.alpha.-anilinophenylacetamide) complex structure would not predict that the chemically dissimilar inhibitor TNK (6-benzyl-1-benzyloxymethyl uracil) could bind in the same region. The RT-APA structure reveals that there would not be enough room in the APA binding site for the 1-benzyloxymethyl group of TNK (Hopkins, A. L. et al., J. Med. Chem., 1996, 39, 1589-1600). Nevertheless TNK is known to bind in this region as evidenced by the crystal structure of RT-TNK which shows that RT residues can adjust to accommodate the 1-benzyloxymethyl group. Conversely, an analysis of the RT-TNK complex would not predict favorable binding of APA in the TNK binding site. The structure does not show how residue E138 can move to accommodate the 2-acetyl group of the A-APA inhibitor.
Thus, any NNI binding pocket model based on an individual RT-NNI crystal structure would have limited potential for predicting the binding of new, chemically distinct inhibitors. To overcome this problem, the invention disclosed herein uses the NNI binding site coordinates of multiple, varied RT-NNI structures to generate a composite molecular surface. A specific embodiment of the invention is a composite molecular surface or binding pocket generated from nine distinct RT-NNI complexes, and reveals a larger than presumed NNI binding pocket not shown or predicted by any of the individual structures alone (FIG. 2A). This novel composite binding pocket, together with a computer docking procedure and a structure-based semi-empirical score function, provides a guide to predict the energetically favorable position of novel PETT, DABO, and HEPT derivatives, as well as other novel compounds, in the NNI binding site of RT.
The invention further provides a number of computational tools which set forth a cogent explanation for the previously unexplained and not understood relative activity differences among NNIs, including PETT, DABO, and HEPT derivatives, and reveals several potential ligand derivatization sites for generating new active derivatives. Disclosed herein is the structure-based design of novel HEPT derivatives and the design and testing of non-cytotoxic PETT and DABO derivatives which abrogate HIV replication in human peripheral blood mononuclear cells at nanomolar concentrations with an unprecedented selectivity index of >10.sup.5.
One procedure useful in structure-based rational drug design is docking (reviewed in Blaney, J. M. and Dixon, J. S., Perspectives in Drug Discovery and Design, 1993, 1, 301). Docking provides a means for using computational tools and available structural data on macromolecules to obtain new information about binding sites and molecular interactions. Docking is the placement of a putative ligand in an appropriate configuration for interacting with a receptor. Docking can be accomplished by geometric matching of a ligand and its receptor, or by minimizing the energy of interaction. Geometric matching is faster and can be based on descriptors or on fragments.
Structure-based drug design efforts often encounter difficulties in obtaining the crystal structure of the target and predicting the binding modes for new compounds. The difficulties in translating the structural information gained from X-ray crystallography into a useful guide for drug synthesis calls for continued effort in the development of computational tools. While qualitative assessments of RT-inhibitor complexes provide helpful information, systematic quantitative prediction of inhibitory activity of new compounds based on structural information remains a challenge.
There is a need for more complete information on the structure and flexibility of the NNI binding pocket and for an improved model of the binding pocket to serve as a basis for rational drug design. In addition, there is a need for more effective inhibitors of reverse transcriptase, particularly HIV-1 reverse transcriptase.
The invention disclosed herein addresses these needs by providing a model for the three-dimensional structure of the RT-NNI binding pocket based on the available backbone structure of RT-DNA complex and full structure of RT complexed with several NNI compounds. Structural information from multiple RT-NNI complexes was combined to provide a suitable working model. In one embodiment, the NNI binding site coordinates of nine RT-NNI structures is used to generate a composite molecular surface revealing a larger than presumed NNI binding pocket. This pocket, together with docking and a structure-based semi-empirical score function, can be used as a guide for the synthesis and analyses of structure-activity relationships for new NNI of RT, including new derivatives of HEPT, DABO, and PETT, as well as novel compounds having little or no relationship to known NNIs. The practical utility of this novel composite model is illustrated and validated by the observed superior potency of new PETT and S-DABO derivatives as anti-HIV agents, described herein.
SUMMARY OF THE INVENTION
The invention provides novel compounds which inhibit reverse transcriptase (RT) and which inhibit replication of a retrovirus, such as human immunodeficiency virus-1 (HIV-1). In one embodiment, the novel compounds of the invention are analogs or derivatives of phenethylthiazolylthiourea (PETT), dihydroalkoxybenzyloxopyrimidine (DABO) or 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). Alternatively, the novel compounds of the invention bind the NNI binding pocket, but are not related to any known NNI. Specific compounds of the invention are described more fully in the Detailed Description and in the Examples below.
The invention additionally provides compositions and methods for inhibiting reverse transcriptase activity of a retrovirus, such as HIV-1, by contacting the RT binding site of the retrovirus with a compound of the invention. The methods of the invention are useful for inhibiting replication of a retrovirus, such as HIV-1 and include treating a retroviral infection in a subject, such as an HIV-1 infection, by administering a compound or composition of the invention, for example, in a pharmaceutical composition.
The invention further provides a composite ligand binding pocket constructed by superimposing multiple structures of ligand-binding site complexes. Preferably, the composite binding pocket is constructed by superimposing the structures of at least one each of the following NNI complexed with RT: a compound, analog or derivative of HEPT or MKC; TNK, APA, Nevipapine, and TIBO. In one embodiment, the composite ligand binding pocket is an HIV-1 reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket constructed by superimposing nine structures of NNI-RT complexes, preferably having the coordinates set forth in Table 9.
Using the novel composite binding pocket of the invention, compounds that bind to the NNI binding site of reverse transcriptase can be identified and/or screened. For example, a useful inhibitor is identified by analyzing the fit of a candidate compound to the composite binding pocket is analyzed. In one embodiment, the comparing comprises analyzing the molecular surface of the composite binding pocket. The extent of contact between the molecular surface of the compound and the molecular surface of the binding pocket can be visualized, and any gap space between the compound and the composite binding pocket can be determined and quantified. The candidate inhibitory compound can be docked in the composite binding pocket, and its binding characteristics analyzed. For example, an estimate of the inhibition constant for the docked compound can be calculated. The value of the inhibition constant is inversely related to the affinity of the candidate compound for the binding pocket.
Using information provided by the composite binding pocket of the invention, novel inhibitors of reverse transcriptase can be designed and screened. Using molecular modeling techniques, a compound can be docked into an RT-NNI binding pocket, and the complex analyzed for its binding characteristics. Gap space or regions that do not demonstrate optimum close contacts between the compound and the binding pocket are identified, permitting the compound to be modified to better occupy the site. In such a method, novel inhibitors of reverse transcriptase are designed and screened.
Also provided by the invention are inhibitors of reverse transcriptase identified or designed by analyzing the compound's structural fit to the binding pocket. Potent inhibitors designed and confirmed using the composite binding pocket of the invention include analogs and derivatives of known NNI, such as phenethylthiazolylthiourea (PETT) analogs, dihydroalkoxybenzyloxopyrimidine (DABO) analogs, and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs.
The compounds of the invention may be combined with carriers and/or agents to enhance delivery to sites of viral infection, such as targeting antibodies, cytokines, or ligands. The compounds may include chemical modifications to enhance entry into cells, or may be encapsulated in various known delivery systems.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a model of the HIV-1 reverse transcriptase (RT) active site, derived primarily from two crystal structures: HIV-1 RT (PDB access code hni) and HIV-1 RT with DNA fragment (PDB access code hmi). The binding site for non-nucleoside inhibitors is labeled NNI. The site for nucleoside inhibitors is labeled dNTP which includes the 3' terminus of DNA. Features describing the geometry of the binding region include the thumb, palm, fingers, and hinge region of RT.
FIG. 1B shows models of compound I-3 (color coded by atom type) and compound I-4 (in blue) in NNI binding site of HIV reverse transcriptase, positioned by docking procedure. Wing 1 and Wing 2 represent two different regions of the NNI binding site.
FIG. 2A shows a composite binding pocket of NNI active site of HIV-1 RT. Grid lines represent the collective van der Waals surface of nine different inhibitor crystal structures superimposed in the active site and highlight the available space for binding (inhibitor structures include HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and two 9-Cl TIBO compounds, with PDB access codes rti, rt1, rt2, hni, vrt, rth, hnv, rev and tvr, respectively). The surface is color-coded for hydrogen bonding (red), hydrophobic (gray) and hydrophilic (blue) groups of the superimposed inhibitors. The hydrogen atoms were not included.
FIG. 2B shows a composite binding pocket (purple) superimposed on the active site residues of RT taken from the crystal structure coordinates of RT complexed with 8-Cl-TIBO (pdb access code: hnv). In the composite binding pocket, there are a number of regions which are larger than those defined by residues in individual crystal structures. Residues shown here which extend past the purple surface and toward the center of the binding site represent regions which are considered flexible and could be displaced by an appropriate inhibitor.
FIG. 3A shows a model of compound trouvirdine docked in the NNI binding site and color-coded by atom type. Spheres represent the sites of the molecular surface which are in contact with protein residues and are unavailable for future modification.
FIG. 3B shows a model of PETT compound I-3 docked in the NNI binding site and color-coded by atom type. Spheres represent the sites of the molecular surface which are in contact with protein residues and are unavailable for future modification.
FIG. 4A shows a stereo model of compound I-2 and grid shown in red which represents gaps between the compound and protein residues (each red line=1 .ANG. distance). Dashed lines show the nearest distance between an atom in the compound and the gap net which does not intersect the spheres shown in FIG. 3A.
FIG. 4B shows a stereo model of PETT compound I-3 and grid shown in red which represents gaps between the compound and protein residues (each red line=1 .ANG. distance). Dashed lines show the nearest distance between an atom in the compound and the gap net which does not intersect the spheres shown in FIG. 3B.
FIG. 5A shows a stereoview of compound trovirdine in the composite binding pocket which was constructed from combined coordinates of RT complexed with nine different NNI compounds.
FIG. 5B shows a stereoview of PETT compounds I-3 (in magenta) and I-4 (multicolor) in the composite binding pocket which was constructed from combined coordinates of RT complexed with nine different NNI compounds.
FIG. 6 shows a model of PETT compound I-4 docked in the NNI binding site and color-coded by atom type, as described above for FIG. 3A. The surface of the composite binding pocket is color-coded for hydrogen bonding (red), hydrophobic (gray) and hydrophilic (blue) groups of the superimposed inhibitors.
FIG. 7A is a view of the composite binding pocket of the NNI active site of HIV-1 RT. The DABO compound 3c is superimposed in the NNI composite binding site of the crystal structure of the RT/MKC-442 complex (hydrogen atoms not shown). MKC-442 (from crystal structure) is shown in pink, and compound 3c (from docking calculations) in multicolor. Compound 3c was docked into the active site of the RT/MKC complex (PDB access code: rt1) and then superimposed into the NNI composite binding pocket based on the matrix used in the pocket construction. The S2 substituent of the DABO compound 3c occupies the same region of the binding pocket as the N1 substituent of the HEPT derivative MKC-442.
FIG. 7B is a view of the composite binding pocket of the NNI active site of HIV-1 RT. An X-ray crystal structure of DABO compound 3b is superimposed on the docked model of DABO compound 3d in the NNI composite binding pocket of RT, demonstrating their remarkably similar conformations.
FIG. 8 is an ORTEP drawing of the room temperature X-ray crystal structure of DABO compound 3b (30% ellipsoids).

DETAILED DESCRIPTION OF THE INVENTION
Definitions
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
As used herein, a "retrovirus" includes any virus that expresses reverse transcriptase. Examples of a retrovirus include, but are not limited to, HIV-1, HIV-2, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV, and MoMuLV.
As used herein, "reverse transcriptase (RT)" refers to an enzyme having an NNI binding site similar to that of HIV-1 RT and to which ligands which bind the composite binding pocket of the invention bind.
As used herein, "reverse transcriptase (RT) activity" means the ability to effect reverse transcription of retroviral RNA to proviral DNA. One means by which RT activity can be determined is by measuring viral replication. One measure of HIV-1 viral replication is the p24 core antigen enzyme immunoassay, for example, using the assay commercially available from Coulter Corporation/Immunotech, Inc. (Westbrooke, Mich.). Another means by which RT activity is analyzed is by assay of recombinant HIV-1 reverse transcriptase (rRT) activity, for example, using the Quan-T-RT assay system commercially available from Amersham (Arlington Heights, Ill.) and described in Bosworth, et al., Nature 1989, 341:167-168.
As used herein, a compound that "inhibits replication of human immunodeficiency virus (HIV)" means a compound that, when contacted with HIV-1, for example, via HIV-infected cells, effects a reduction in the amount of HIV-1 as compared with untreated control. Inhibition of replication of HIV-1 can be measured by various means known in the art, for example, the p24 assay disclosed herein.
As used herein, a "nonnucleoside inhibitor (NNI)" of HIV reverse-transcriptase (HIV-RT) means a compound which binds to an allosteric site of HIV-RT, leading to noncompetitive inhibition of HIV-RT activity. Examples of nonnucleoside inhibitors of HIV-RT include, but are not limited to, tetrahydroimidazobenzodiazepinthiones (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT), bis(heteroaryl)piperazines (BHAP), 2'-5'-bis-O-(tertbutyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) pyrimidines (TSAO), dihydroalkoxybenzyloxopyrimidine (DABO) and phenethylthiazolylthiourea (PETT) analogs. In one embodiment of the invention, the nonnucleoside inhibitor of HIV-RT is a PETT analog. In another embodiment of the invention, the nonnucleoside inhibitor of HIV-RT is a DABO analog. In another embodiment of the invention, the nonnucleoside inhibitor of HIV-RT is a HEPT analog.
As used herein, a "composite HIV reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket" or "composite binding pocket" means a model of the three-dimensional structure of a ligand binding site, such as the nonnucleoside inhibitor binding site of HIV-RT constructed from a composite of multiple ligand-binding site complexes. The composite binding pocket represents a composite molecular surface which reveals regions of flexibility within the binding site. Flexible residues within the NNI binding site include Tyr180, Tyr181, Tyr318, Tyr319, Phe227, Leu234, Trp229, Pro95, and Glu138 (the latter from the p51 subunit of RT). Examples of such a model include, but are not limited to, a composite molecular surface developed with the aid of computer software and based on a composite of coordinates of multiple RT-NNI complexes, as disclosed herein. In one embodiment, the binding pocket has the coordinates set forth in Table 9.
As used herein, a "compound that fits the nonnucleoside inhibitor (NNI) pocket of reverse transcriptase (RT)" means a compound that substantially enters and binds the NNI binding site on RT. In one embodiment, a compound that fits the NNI pocket of RT inhibits RT activity. Generally, compounds which better fit the NNI pocket of RT contact a greater portion of the available molecular surface of the pocket and are more potent inhibitors of RT activity. In one embodiment, the compound that fits the NNI pocket of RT is a PETT analog. In another embodiment, the compound that fits the NNI pocket of RT is a DABO analog. In another embodiment, the compound that fits the NNI pocket of RT is a HEPT analog.
As used herein, "docking" a compound in a binding pocket means positioning a model of a compound in a model of the binding pocket. In one embodiment, the model of the binding pocket can be a composite binding pocket constructed in accordance with the invention. The model of the binding pocket can be, for example, based on coordinates obtained from the crystal structure of RT complexed with a NNI. In one embodiment, the docking is performed with the use of computer software, such as the Affinity program within InsightII (Molecular Simulations Inc., 1996, San Diego, Calif.). Docking permits the identification of positions of the compound within the binding pocket that are favored, for example, due to minimization of energy.
As used herein, "minimization of energy" means achieving an atomic geometry of a molecule or molecular complex via systematic alteration such that any further minor perturbation of the atomic geometry would cause the total energy of the system as measured by a molecular mechanics force-field to increase. Minimization and molecular mechanics force-fields are well understood in computational chemistry (Burkert, U. and Allinger, N. L., Molecular Mechanics, ACS Monograph, 1982, 177, 59-78, American Chemical Society, Washington, D.C.).
As used herein, "comparing" includes visualizing or calculating available space encompassed by the molecular surface of the composite binding pocket of the invention, taking into account the flexibility of residues, such as Tyr180, Tyr181, Tyr318, Tyr319, Phe227, Leu234, Trp229, Pro95, and Glu138 of RT (the latter from the p51 subunit of RT). "Comparing" also includes calculating minimal energy conformations.
As used herein, "gap space" means unoccupied space between the van der Waals surface of a compound positioned within the binding pocket and the surface of the binding pocket defined by residues in the binding site. This gap space between atoms represents volume that could be occupied by new functional groups on a modified version of the compound positioned within the binding pocket.
In the present invention, the terms "analog" or "derivative" are used interchangeably to mean a chemical substance that is related structurally and functionally to another substance. An analog or derivative contains a modified structure from the other substance, and maintains the function of the other substance, in this instance, maintaining the ability to interact with an NNI-RT binding site. The analog or derivative need not, but can be synthesized from the other substance. For example, a HEPT analog means a compound structurally related to HEPT, but not necessarily made from HEPT.
As used herein, "alkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
As used herein, "alkene" includes both branched and straight-chain unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
As used herein, "halogen" includes fluoro, chloro, bromo and iodo.
As used herein "non-hydrogen atom group" includes, but is not limited to, alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, thiol, thiolalkyl, amino, substituted amino, phosphino, substituted phosphino, or nitro. In addition, cycloalkyl, aryl, and aralkyl groups may be included if the non-hydrogen atom group contains a sufficient number of non-hydrogen atoms. Often, a number or range of numbers is specified to indicate the number of non-hydrogen (e.g., C, O, N, S, or P) atoms in the functional group.
As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or (c) salts formed with an organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (d) combinations of (a) and (b) or (c), e.g., a zinc tannate salt; and the like. The preferred acid addition salts are the trifluoroacetate salt and the acetate salt.
As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with a compound of the invention, allows the compound to retain biological activity, such as the ability to inhibit RT activity, and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton Pa. 18042, USA).
Composite Binding Pocket of the Invention
As shown in FIG. 1, the NNI binding site of HIV-RT rests between the palm and thumb regions of the RT molecular structure, adjacent to the hinge region. The NNI binding site includes two distinct regions, indicated in FIG. 1B as Wing 1 and Wing 2, forming a butterfly-shaped binding pocket.
In the method of the invention, a composite ligand binding pocket is constructed by superimposing multiple structures of ligand-binding site complexes, preferably using 5 or more distinct structures. In one embodiment, the composite ligand binding pocket is an HIV-1 reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket constructed by superimposing multiple structures of NNI-RT complexes. The composite binding pocket is preferably an HIV-1 RT-NNI binding pocket.
A preferred binding pocket of the invention can be made by superimposition of coordinates, obtainable from the Protein Data Bank (PDB) via access codes disclosed herein, corresponding to the three-dimensional structure of an RT-NNI complex. The superimposition of coordinates is preferably based on alignment of the coordinates corresponding to the palm region of the binding pocket due to the greater rigidity of this region.
The superimposing of coordinates can also be accomplished by first using models of the protein backbone and DNA phosphate backbone of the RT-DNA complex structure (with PDB access code hmi) onto a model of an RT mutant complexed with an NNI, such as APA ((2-acetyl-5-methylanilino)(2,6-dibromophyl)acetamide) having PDB access code hni. Next, models of one or more additional RT-NNI complexes are superimposed onto the models superimposed above. In one embodiment, the superimposition is based on alignment of the region of RT from residue 100 to 230, preferably by a least squares procedure. In another embodiment, the superimposition is based on alignment of the region of RT from residues 97 to 213. Preferably, the superimposition is based on alignment of the palm region and part of the NNI binding site. Most preferably, the superimposition is based on alignment of the region corresponding to residues 100 to 230 of RT, or on alignment of 117 C alpha atoms of residues 97 to 213, and preferably using a least squares procedure.
A molecular surface of a binding pocket can then be generated that encompasses all superimposed NNI models. One such composite binding pocket constructed from nine individual NNI-RT complex structures, is shown in FIG. 2A. Grid lines in the figure represent the collective van der Waals surface, and highlight space available for binding.
The molecular surface of the complex can be generated, for example, by reading the overlaid coordinates of the complexed inhibitors into a computer program such as GRASP (A. Nicholls, GRASP, Graphical Representation and Analysis of Surface Properties, 1992, N.Y.). Examples of NNI compounds which can be used in the construction of a binding pocket include, but are not limited to, HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and 9-Cl TIBO (PDB access codes, rti, rt1, rt2, hni, vrt, rth, hnv and rev or tvr, respectively).
Using the composite NNI binding pocket, binding of compounds can be modeled to identify available space within the binding pocket. New and more potent NNI inhibitors of RT can be developed by designing compounds to better fit the binding pocket.
In one embodiment, the composite binding pocket is constructed by superimposing structures of NNI-RT complexes comprising RT complexed with: an HEPT or MKC analog; a TNK analog; an APA analog; a Nevirapine analog; and a TIBO analog. In another embodiment, the composite NNI binding pocket is based on the structure of RT complexed with 9 NNI and on the RT-DNA complex. Examples of NNI compounds which can be used in the construction of a binding pocket include, but are not limited to, HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and 9-Cl TIBO structures (PDB access codes, rti, rt1, rt2, hni, vrt, rth, hnv, and tvr and/or rev, respectively). In one embodiment, the resulting composite binding pocket has the coordinates set forth in Table 9.
Construction and Use of the Binding Pocket
A compound that binds the NNI binding site of reverse transcriptase is identified by comparing a test compound to the composite binding pocket of the invention and determining if the compound fits the binding pocket. As shown in FIGS. 7A and 7B, the test compound may be compared to another inhibitory compound, by superimposing the structures in the binding pocket. The test compound is also compared to the binding pocket by calculating the molecular surface of the compound complexed with the composite binding pocket. The extent of contact between the molecular surface of the compound and the molecular surface of the binding pocket can be visualized, and the gap space between the compound and the binding pocket can be calculated. In FIGS. 4A and 4B, gaps between the molecular surface of the binding pocket and the NNI are presented in red, with each red line being 1 angstrom in distance.
To design a novel inhibitor of reverse transcriptase, a compound is docked in the composite binding pocket of the invention. Gap space is identified between the compound and the binding pocket, for example, using an algorithm based on a series of cubic grids surrounding the docked compound, with a user-defined grid spacing. The compound is then modified to more completely occupy the gap space.
Computerized docking procedures can be used to dock the test compound in the binding pocket and analyze the fit. One docking program, DOCK (Kuntz, I. D., et al., J. Mol. Biol., 1982, 161, 269-288; available from University of California, San Francisco), is based on a description of the negative image of a space-filling representation of the receptor that should be filled by the ligand. DOCK includes a force-field for energy evaluation, limited conformational flexibility and consideration of hydrophobicity in the energy evaluation. CAVEAT (Bartlett, P. A. et al., Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc., 1989, 78, 182-196; available from University of California, Berkeley) suggests ligands to a particular receptor based on desired bond vectors. HOOK (Molecular Simulations, Burlington, Mass.) proposes docking sites by using multiple copies of functional groups in simultaneous searches. MACCS-3D (Martin, Y. C., J. Med. Chem., 1992, 35, 2145-2154) is a 3D database system available from MDL Information Systems, San Leandro, Calif. Modeling or docking may be followed by energy minimization with standard molecular mechanics forcefields or dynamics with programs such as CHARMM (Brooks, B. R. et al., J. Comp. Chem., 1983, 4, 187-217) or AMBER (Weiner, S. J. et al., J. Am. Chem. Soc., 1984, 106, 765-784).
LUDI (Bohm, H. J., J. Comp. Aid. Molec. Design, 1992, 6, 61-78; available from Biosym Technologies, San Diego, Calif.) is a program based on fragments rather than on descriptors. LUDI proposes somewhat larger fragments to match with the interaction sites of a macromolecule and scores its hits based on geometric criteria taken from the Cambridge Structural Database (CSD), the Protein Data Bank (PDB) and on criteria based on binding data. Other software which can be used to propose modifications for constructing novel inhibitors include LEGEND (Nishibata, Y. and Itai, A., Tetrahedron, 1991, 47, 8985; available from Molecular Simulations, Burlington, Mass.) and LeapFrog (Tripos Associates, St. Louis, Miss.).
The AUTODOCK program (Goodsell, D. S. and Olson, A. J., Proteins: Struct. Funct. Genet., 1990, 8, 195-202; available from Scripps Research Institute, La Jolla, Calif.) helps in docking ligands to their receptive proteins in a flexible manner using a Monte Carlo simulated annealing approach. The procedure enables a search without bias introduced by the researcher. This bias can influence orientation and conformation of a ligand in the active site. The starting conformation in a rigid docking is normally biased towards an energy minimum conformation of the ligand. However, the binding conformation of the ligand may be of relatively high conformational energy, but offset by the binding energy.
In a preferred embodiment of the invention, docking is performed by using the Affinity program within InsightII (Molecular Simulations Inc., 1996, San Diego, Calif.). As modeling calculations progress during the docking procedure, residues within a defined radius of 5 .ANG. from the NNI molecule are allowed to move in accordance with energy minimization, permitting the identification of promising positions for modification. Initial coordinates of newly designed compounds can be generated using the Sketcher module within InsightII.
In one embodiment, the method further comprises calculating the inhibition constant of the docked compound. Inhibition constants (K.sub.i values) of compounds in the final docking positions can be evaluated using a score function in the program, LUDI (Bohm, H. J., J. Comput. Aided Mol. Des., 1994, 8, 243-256; Bohm, H. J., J. Comput. Aided Mol. Des., 1992, 6, 593-606). Predictions of K.sub.i values can be improved by modifications of the LUDI calculation., for example, those described in Example 1. First, the molecular surface area can be directly calculated from the coordinates of the compounds in docked conformation using the MS program described in Connolly, M. L., 1983 Science 221:709-713. Second, because InsightII does not account for structural rigidity imposed by internal hydrogen bonds, the number of rotatable bonds can be re-evaluated. For example, this re-evaluation can be performed by counting the number of rotatable bonds according to the principle introduced by Bohm (supra) and taking out the number of bonds which are not rotatable due to the conformational restraint imposed by the internal hydrogen bond between the thiourea NH and pyridyl N in PETT derivatives. Third, the calculation can be modified by the assumption that the conserved hydrogen bond with RT does not deviate significantly from the ideal geometry. This assumption is supported by the fact that, in known crystal structures of RT complexes, all hydrogen bonds between NNIs and RT are near the ideal geometry. These constraints provide for more predictive K.sub.i values for modeled compounds.
In a preferred embodiment, the compound has a predicted inhibition constant (K.sub.i) of less than about 1 .mu.M, and the compound in the binding has an estimated molecular surface area greater than 276 .ANG..sup.2.
Candidate inhibitors of RT identified or designed by the methods of the invention can be evaluated for their inhibitory activity using conventional techniques which typically involve determining the location and binding proximity of a given moiety, the occupied space of a bound inhibitor, the deformation energy of binding of a given compound and electrostatic interaction energies. Examples of conventional techniques useful in the above evaluations include, but are not limited to, quantum mechanics, molecular dynamics, Monte Carlo sampling, systematic searches and distance geometry methods (Marshall, G. R., Ann. Ref. Pharmacol. Toxicol., 1987, 27, 193). Examples of computer programs for such uses include, but are not limited to, Gaussian 92, revision E2 (Gaussian, Inc. Pittsburgh, Pennsylvania), AMBER version 4.0 (University of California, San Francisco), QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass.), and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif.). These programs may be implemented, for example, using a Silicon Graphics Indigo2 workstation or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known and of evident applicability to those skilled in the art.
Inhibitors identified or designed by the methods of the invention can be tested for their anti-HIV or anti-RT activity using one of the standard in vitro assays known in the art, such as the p24 enzyme immunoassay disclosed herein.
The invention further provides novel compounds identified by the above methods, which can be used as inhibitors of RT. Novel inhibitors so identified include analogs or derivatives of known NNI compounds such as HEPT, DABO, and PETT, as well as novel compounds designed to fit the composite binding pocket which are unrelated to any known NNI compound.
Compounds of the Invention
Compounds of the invention are useful as nonnucleoside inhibitors of RT. These include, for example, analogs and derivatives of PETT, DABO, and HEPT compounds, as well as novel compounds unrelated to known NNI but designed to fit the composite binding pocket.
PETT compounds:
Novel compounds of the invention include derivatives and analogs of PETT, having the general formula (I): ##STR1##
Z can be phenyl, piperizine, piperidine, or morpholine. Z is preferably substituted with one or more substituents, including alkyl, alkene, halogen, methoxy, alcohol, amino, thio, thioxy, or phosphino. In one embodiment, the compounds of the invention are PETT derivatives or analogs having the following formula (II): ##STR2##
The R's can be the same or different, and represent points of optional substitution. R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 can be hydrogen, or can be substituted, with a non-hydrogen atom group such as halo (Br, Cl, F, I), alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH.sub.2 group, where R is alkyl. Preferably, one or more is alkyl, halo, or alkoxy. Preferred halogens are F, Br, or Cl. One or more of R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, and R.sub.7 can be a C.sub.1 -C.sub.3 alkoxy, e.g., methoxy.
R.sub.8 can also be aryl, aralkyl, ROH, or RNH.sub.2 group, where R is alkyl. Preferably, at least one of R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is not hydrogen. R.sub.4 is a preferably hydrophobic group such as H, an alkyl or alkene, and can be Me, Et, or i-Pr. R.sub.6 and/or R.sub.7 are preferably a 3 or 4 (non-hydrogen)-atom group.
R.sub.6 and R.sub.7 can be a group having 1 to 4 non-hydrogen atoms, whereas R.sub.2, R.sub.3, and R.sub.5 preferably each are a group having 1 to 3 non-hydrogen atoms. Available gap space in the binding pocket near R.sub.8, is approximately 8 angstroms by 5 angstroms, by 3.3 angstroms. Thus, a molecule having a volume of up to about 8.times.6.times.4 angstroms can be used to fill this space, e.g., accommodating a group of about 7 non-hydrogen atoms, or up to about the size of a phenyl ring. R.sub.8 can be halo, alkyl, phenyl, --CH.sub.2 Ph, or alkoxy. R.sub.8 can be X--R, where X is a bridging atom, including, but not limited to, C, S, O, N and P.
In a preferred embodiment, R.sub.8 is bromine, and at least one of R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 is fluoro, chloro, or methoxy.
A compound of the invention preferably conforms to the composite NNI binding pocket of the invention. Most preferably, the compound complexed with an NNI-RT binding pocket, has a predicted K.sub.i of less than about 1 .mu.M.
Preferred modifications of PETT compounds include ortho-halogen, meta-O-Me, and hydrophobic groups at the para position of the ring. Most preferably, the modifications do not disrupt the intramolecular hydrogen bond. Specific compounds include those having the following formulae (III-VIII) shown below. ##STR3##
In another embodiment, the PETT derivative comprises the formula (IX): ##STR4##
The R's can be the same or different, and represent points of optional substitution. R.sub.5, R.sub.6, and R.sub.7 can be hydrogen, or can be substituted, with a non-hydrogen atom group such as halo (Br, Cl, F, I), alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH.sub.2 group, where R is alkyl. Preferably, one or more is alkyl, halo, or alkoxy. Preferred halogens are F, Br, or Cl. One or more of R, R.sub.6, and R.sub.7 can be a C.sub.1 -C.sub.3 alkoxy, e.g., methoxy.
R.sub.6 and/or R.sub.7 are preferably a 3 or 4 (non-hydrogen)-atom group. R.sub.6 and R.sub.7 can be a group having 1 to 4 non-hydrogen atoms, whereas R.sub.5 preferably is a group having 1 to 3 non-hydrogen atoms. R.sub.8 can be a group of about 7 non-hydrogen atoms, or up to about the size of a phenyl ring. R.sub.8 can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, aryl, aralkyl, --CH.sub.2 Ph, alkoxy, ROH or RNH.sub.2, where R is alkyl. R.sub.8 can be X--R, where X is a bridging atom, including, but not limited to, C, S, O, N and P.
X can be CR'R", NR'", or O, where R', R", and R'" can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, or phosphino group. In one embodiment, R.sub.5, R.sub.6, R', R", and R'" are each hydrogen. In an alternative embodiment, X is CR'R" and at least one of R'and R" are fluoro, chloro, bromo, hydroxy, methoxy, or C1-3 alkyl. In a preferred embodiment, R.sub.8 is bromine, and at least one of R.sub.5, R.sub.6, and R.sub.7 is fluoro, chloro, or methoxy.
Preferred compounds include a larger functional group near the ethyl linker, for example R.sub.7 acetamide or methoxy. Also preferred is a bulkier heterocyclic ring such as a bulky piperidinyl ring or an ortho/meta substituted pyridyl ring.
Specific PETT derivatives of the invention include:
N-[2-(1-piperidinoethyl)]-N'-[2-(5-bromopyridyl)]thiourea,
N-[2-(2,5-dimethoxyphenethyl)]-N'-[2-(5-bromopyridyl)]thiourea,
N-[2-(o-Chlorophenethyl)]-N'-[2-(5-bromopyridyl)]thiourea
N-[2-(o-Fluorophenethyl)]-N'-[2-(5-bromopyridyl)]thiourea, and
N-[2-(m-Fluorophenethyl)]-N'-[2-(5-bromopyridyl)]thiourea.
Other specific compounds of the invention are described in the Examples below.
DABO Compounds:
In another embodiment of the invention, the compounds are derivatives of DABO, and have the following general formula (X): ##STR5##
R.sub.1 and R.sub.2 can be alike or different, and can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. Preferably, one or more of R.sub.1 and R.sub.2 is a C.sub.1-3 alkyl, such as methyl (Me), ethyl (Et), or isopropyl (i-Pr). Preferably, R.sub.1 is alkyl, alkenyl, ROH, or RNH.sub.2. R.sub.2 is preferably halo, alkyl, or C1-3 alkoxy.
Y can be S or O, and is preferably S. R.sub.3 can be alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl, and is preferably C1-3 alkyl.
Specific DABO compounds of the invention include:
5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one.
Other specific compounds of the invention are described in the Examples below.
HEPT Compounds:
In another embodiment, the compounds of the invention are HEPT derivatives having the formula (XI): ##STR6## X and Y can be independently S or O. Preferably, at least one of X and Y is S. More preferably, X is S, and in specific embodiments, both X and Y are S.
R.sub.1 and R.sub.2 can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. R.sub.3 can be H, alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl. Preferably, R.sub.1 is alkyl, alkenyl, ROH, or RNH.sub.2, and can be, for example, methyl, ethyl, or isopropyl. R.sub.2 is preferably halo, alkyl, or C.sub.1-3 alkoxy, and is preferably in the ortho or meta position. R.sub.2 can be Br, F, Cl, or O--Me.
Specific HEPT compounds of the invention include:
6-benzyl-5-isopropyl-1[(methylthio)methyl]-2-thiouracil.
Other specific compounds of the invention are described in the Examples below.
The compounds of the invention have the ability to inhibit replication of a retrovirus, such as human immunodeficiency virus (HIV), preferably with an IC.sub.50 of less than 50 .mu.M, for example, as determined by p24 enzyme immunoassay described in the Examples below. More preferably, the compound of the invention inhibits replication of HIV in the p24 assay with an IC.sub.50 of 1 to 5 .mu.M, or less. Most preferably, the compound inhibits replication of HIV in the p24 assay with an IC.sub.50 of less than 5 nM. In some embodiments, the compound inhibits replication of HIV in the p24 assay with an IC.sub.50 of less than 1 nM.
The invention provides a composition comprising a compound or inhibitor of the invention, and optionally, an acceptable carrier. The composition can be a pharmaceutical composition. Compositions of the invention are useful for prevention and treatment of retroviral infection, such as HIV infection.
Methods of Using Compounds of the Invention
The compounds of the invention are useful in methods for inhibiting reverse transcriptase activity of a retrovirus. Retroviral reverse transcriptase is inhibited by contacting RT in vitro or in vivo, with an effective inhibitory amount of a compound of the invention. The compounds of the invention also inhibit replication of retrovirus, particularly of HIV, such as HIV-1. Viral replication is inhibited, for example, by contacting the virus with an effective inhibitory amount of a compound of the invention.
Due to the ability to inhibit replication of retrovirus and to inhibit retroviral RT activity, the invention provides a method for treating or preventing retroviral infection, such as HIV infection, and a method for treating AIDS or AIDS-related complex (ARC). The method comprises administering to a subject an effective inhibitory amount of a compound of the invention or a pharmaceutically acceptable salt of the compound. The compound or inhibitor of the invention is preferably administered in combination with a pharmaceutically acceptable carrier, and may be combined with specific delivery agents, including targeting antibodies and/or cytokines. The compound or inhibitor of the invention may be administered in combination with other antiviral agents, immunomodulators, antibiotics or vaccines.
The compounds of the invention can be administered orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrastemal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. Pharmaceutical compositions of the invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.
For oral administration as a suspension, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation. The compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents. As immediate release tablets, the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
For administration by inhalation or aerosol, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation. The compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.
For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
For rectal administration as suppositories, the compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquify or dissolve in the rectal cavity to release the drug.
Dosage levels of approximately 0.02 to approximately 10.0 grams of a compound of the invention per day are useful in the treatment or prevention of retroviral infection, such as HIV infection, AIDS or ARC, with oral doses 2 to 5 times higher. For example, HIV infection can be treated by administration of from about 0.1 to about 100 milligrams of compound per kilogram of body weight from one to four times per day. In one embodiment, dosages of about 100 to about 400 milligrams of compound are administered orally every six hours to a subject. The specific dosage level and frequency for any particular subject will be varied and will depend upon a variety of factors, including the activity of the specific compound the metabolic stability and length of action of that compound, the age, body weight, general health, sex, and diet of the subject, mode of administration, rate of excretion, drug combination, and severity of the particular condition.
The compound of the invention can be administered in combination with other agents useful in the treatment of HIV infection, AIDS or ARC. For example, the compound of the invention can be administered in combination with effective amounts of an antiviral, immunomodulator, anti-infective, or vaccine. The compound of the invention can be administered prior to, during, or after a period of actual or potential exposure to retrovirus, such as HIV.
Strategies for Design and Synthesis of Inhibitors
It has been proposed that NNI interfere with reverse transcription by altering either the conformation or mobility of RT rather than directly preventing the template-primer binding (Tantillo, C. et al., J Mol Biol, 1994, 243, 369-387). Specifically, binding of NNI to the NNI binding site (approximately 10 .ANG. away from the polymerase catalytic site) inhibits RT by interfering with the mobility of the "thumb" and/or position of the "primer grip" (residues 229-231), which interact with the DNA primer strand (FIG. 1A).
Computer programs can be used to identify unoccupied (aqueous) space between the van der Waals surface of a compound and the surface defined by residues in the binding site. These gaps in atom-atom contact represent volume that could be occupied by new functional groups on a modified version of the lead compound. More efficient use of the unoccupied space in the binding site could lead to a stronger binding compound if the overall energy of such a change is favorable. A region of the binding pocket which has unoccupied volume large enough to accommodate the volume of a group equal to or larger than a covalently bonded carbon atom can be identified as a promising position for functional group substitution. Functional group substitution at this region can constitute substituting something other than a carbon atom, such as oxygen. If the volume is large enough to accommodate a group larger than a carbon atom, a different functional group which would have a high likelihood of interacting with protein residues in this region may be chosen. Features which contribute to interaction with protein residues and identification of promising substitutions include hydrophobicity, size, rigidity and polarity. The combination of docking, K.sub.i estimation, and visual representation of sterically allowed room for improvement permits prediction of potent derivatives.
Design of HEPT Derivatives ##STR7##
New HEPT derivative designs included compounds with added groups at the N-1 (Y-R.sub.3) and C-5 (R.sub.1) positions and those having oxygen (X or Y) atoms replaced by sulfur. Substitution of oxygen by sulfur can aid binding by decreasing the desolvation energy involved in binding. The modifications were made such that the HEPT derivative would fit favorably into the butterfly-shaped RT-NNI binding site, (See FIG. 2A) with the benzyl ring residing in one wing and thyrnine ring in the other. For all designed compounds, the benzyl ring is near Trp229 and the N-1 group is near Pro236, a typical position observed in crystal structures. The modeling calculations, along with the application of the constructed binding pocket, provided a guideline for the synthesis of lead compounds designed to have potent anti-HIV activity. The choice of compounds was also based on synthetic feasibility.
The region of the NNI site of HIV-1 RT located near the thymine ring nitrogen N-1 of the HEPT analogs contains a Pro236 loop region which is large enough to accommodate N-1 substituents. When an inhibitor binds to the NNI site of HIV-1 RT, the presence of a hydrophobic N-1 substituent could influence the Pro loop of this flexible region and provide additional hydrophobic contact leading to stronger binding. Docking results indicated that substitution at N-1 also helps the molecule position itself to achieve the best fit within the pocket.
The LUDI analysis showed a substantial increase in contact (lipo score) between the compound and the pocket and the calculation suggested an increase in hydrophobic contact and stronger binding when the substituent on the N-1 tail (R.sub.3) is larger in size than a methyl moiety.
The Tyr183 residue of the HIV-1 RT is located in the catalytic region which has a conserved YMDD motif characteristic of reverse transcriptases. Therefore, the displacement of this tyrosine residue can interfere with catalysis and render the HIV-1 RT protein inactive. It has been suggested that bulky substituents at the 5th position of the thymine ring (RI) could indirectly accomplish this goal by displacing Tyr181 which is near Tyr183. The composite binding pocket shows sufficient room for at least a 3-carbon group in this region. The addition of a methyl, ethyl or isopropyl group on the 5th position of the thymine ring would lead to a higher affinity for the relatively hydrophobic environment.
LUDI analysis showed that the hydrophobic contact increases as hydrophobic groups at the 5th position (R.sub.1) get bulkier. As it binds to the site, the ethyl or isopropyl group causes the nearby Tyr181 residue to rotate away from the inhibitor. This change in conformation in turn affects the positions of the neighboring Tyr183 and Tyr188 which can lead to the inactivation of HIV-1 RT.
DABO Derivatives
Detailed analysis of HEPT binding revealed that the NI substituents of HEPT derivatives occupy the same region of the binding site as the thio (S2) substituents of DABO compounds (See FIG. 7A). Therefore, new DABO derivatives were designed and their binding into the NNI site of RT modeled using the crystal structure coordinates of the RT/MKC complex (pdb access code: rt1) and a molecular docking procedure. The final coordinates of the docked molecules were then superimposed into the composite binding pocket to evaluate the fit within the RT NNI pocket. Notably, multiple sterically allowed unoccupied spatial gaps in the binding site were identified from the docking studies which could be filled by strategically designed functional groups (See FIG. 7B). ##STR8##
The docked DABO molecule showed significant space surrounding the 6-benzyl ring and the 5th position of the thymine ring, which led to our design and synthesis of new DABO derivatives. Specific DABO compounds are discussed more fully in the Examples, below.
PETT Derivatives ##STR9##
Each PETT derivative described in the Examples below, can be viewed as two chemical groups linked together by a thiourea group. Upon binding RT, the PETT derivative fits into the butterfly-shaped binding site. (See FIG. 6). One half of the molecule is composed of a pyridyl thiourea group (compounds I-1 to 4, II-1 to 9, and III-1 to 3) or a 2-aminothiazole group (PETT) which forms an intramolecular hydrogen-bonded 6-membered heterocyclic ring (shown below). The other half of the molecule is a piperidinyl ring (II-9), a pyridyl ring (trovirdine), or a phenyl ring separated from the thiocarbonyl group by an ethyl linker.
The positions of the compounds having stronger binding and higher scores (evaluated by LUDI function) all fall into the butterfly-shaped binding region with one part residing in Wing 1 and the other in Wing 2, as illustrated in FIG. 1 B. For these compounds the ring closest to the thiocarbonyl group is near the Lys(K) 101 loop and the other pyridyl ring is near Trp(W)229 derivatives.
Analysis of trovirdine, revealed multiple sites which can be used for the incorporation of larger functional groups. In the composite binding pocket, the docked trovirdine molecule showed a lot of usable space surrounding the pyridyl ring, (R.sub.2 -R.sub.6), the ethyl linker (R.sub.7) and near the 5-bromo position (R.sub.8). (See FIG. 5A) ##STR10##
Efficient use of this space by strategically designed functional groups would lead to high affinity binding and ultimately result in better inhibitors. Our modeling studies suggest that designs using the space available in these regions, including (1) substitutions at R.sub.2 -R.sub.6 ; (2) substituting heterocyclic rings for the pyridyl ring of trovirdine; (3) substitutions at R.sub.7 ; (4) substitutions at R.sub.8 ; and (5) maintaining the intramolecular hydrogen bond. As shown in the Examples below, modifications in these areas lead to potent RT inhibitors. ##STR11## Advantages of the Invention
The invention provides a model for the three-dimensional structure of the RT-DNA complex based on the available backbone structure of RT-DNA complex and full structure of RT complexed with several NNI compounds. This is the first model to combine structural information from several complexes into a single composite and provides a suitable working model for the development of novel inhibitory compounds. The use of multiple NNI binding site coordinates from RT-NNI structures, as disclosed herein, permits the generation of a composite molecular surface. Analysis of the composite NNI binding pocket of the invention reveals that the binding pocket is surprisingly and counter-intuitively larger (instead of smaller) and more flexible (instead of more rigid) than expected. This composite NNI binding pocket serves as a guide for the synthesis and analyses of structure-activity relationships for the identification and design of new and more potent NNI of RT. The composite binding pocket additionally provides a model for the design of derivatives of NNIs for which crystal structure information is not available (e.g., PETT, DABO).
The compounds of the invention are useful for inhibition of RT activity and for inhibition of retroviral replication. The compounds disclosed herein provide more potent NNI of RT than known HEPT, DABO and PETT derivatives. With all strategies combined, a number of sites are identified for developing more potent derivatives of PETT, such as the incorporation of a larger functional group near the ethyl linker of PETT. Hitherto unknown piperidinyl substituted and piperozinyl substituted, as well as morpholinyl substituted PETT derivatives are disclosed which show potent anti-HIV activity at nanomolar concentrations.
In addition, the compounds of the invention provide a higher selectivity index (S.I.>10.sup.5) than currently available anti-HIV compounds. This high S.I. permits more effective antiviral activity with a minimum of adverse cytotoxic effects.
EXAMPLES
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.
Example 1
Modeling Procedure
Construction of the Composite NNI binding pocket
A novel model of the NNI binding pocket of RT was constructed by superimposing nine individual RT-NNI crystal structures and then generating a van der Waals surface which encompassed all of the overlaid ligands. This "composite binding pocket" surprisingly reveals a different and unexpectedly larger NNI binding site than shown in or predictable from any of the individual structures and serves as a probe to more accurately define the potentially usable space in the binding site (FIG. 2A).
Modeling studies were based on the construction of a binding pocket which encompassed the superimposed crystal structure coordinates of all known RT-NNI complexes, including nine different structures of RT complexed with HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 9-Cl TIBO (Ren, J. et al., Structure, 1995, 3, 915-926); 9-Cl TIBO (Das, K. et al., J. Mo. Biol., 1996, 264, 1085-1100) and 8-Cl-TIBO (PDB access codes rti, rt1, rt2, hni, vrt, rth, rev, tvr, and hnv, respectively).
The "thumb" region of RT complexes are relatively variable compared with the "palm" region. Therefore, a total of 117 C-alpha atoms of the residues from 97 to 213 which cover part of the NNI binding site and the "palm" region were used for a least-squares superimposing procedure within the program O (Jones, T. A. et al., Acta Crystallogr. A., 1991, 47, 110-119). Each coordinate set was superimposed onto the same initial coordinate set (RT/9-Cl TIBO). the distance between the pair was minimized by rotating and translating one coordinate set onto the other, minimizing distances between x, y, and z coordinates, according to the method of the program "O". The root mean square (RMS) values of the coordinates of the atoms being superimposed are shown to be 1.00, 0.98, 0.99, 0.62, 0.80, 0.87, 0.94 and 0.65 .ANG. for HEPT, MKC, TNK, APA, Cyclopropanyl Nevirapine, N-ethyl Nevirapine derivative and two 9-Cl TIBO compounds, respectively. Next, the coordinates of the corresponding inhibitor molecules were then transformed according to the same matrices derived from the superimposition. Lastly, the overlaid coordinates of all inhibitors were read into the program GRASP (Nicholls, A., GRASP 1992, New York), from which an overall molecular surface was generated providing a binding pocket encompassing all inhibitors.
As shown in FIG. 2A, the surface of the binding pocket was color coded to reflect characteristics of the overlaid inhibitors, such as hydrogen bonding, hydrophilic, and hydrophobic regions. The amide nitrogens on the uracil ring of HEPT and TIBO derivatives are colorcoded red for hydrogen bonding atoms. Oxygen or sulfur atoms of carbonyl, thiocarbonyl, and ester groups, nitrogen atoms of amine groups, and halogen atoms are color-coded blue for polar (hydrophilic) groups. Carbon atoms are considered hydrophobic and are colored grey. This pocket, referred to as the composite binding pocket, was used as a basis for the analysis of inhibitor binding.
To generate the coordinates of the composite binding pocket using the InsightII program, each data point of the net defining the surface of the pocket was represented as a water molecule and was saved in Brookhaven Protein Databank (pdb) format. To provide a visual frame of reference, the coordinates have been superimposed on the pdb coordinates of an existing crystal structure having pdb access code hnv (HIV-1 RT/8-Cl TIBO complex). The coordinates of a composite binding pocket for HIV-1 RT generated by superimposing nine different NNI-RT complexes, are set forth in Table 9.
Docking and K.sub.i prediction
A computer simulation of the binding of PETT, DABO, and HEPT compounds into the NNI binding site of RT was accomplished using a molecular docking procedure. Docking of the compounds into the NNI binding site required the use of X-ray coordinates of an RT-NNI complex (RT/9-Cl-TIBO complex was used for modeling PETT, and the RT/MKC-442 complex was used for modeling DABO and HEPT). Upon binding to RT, the compound can fit into a butterfly-shaped NNI binding site (described by Ding et. al), Ding, J. et al., Nat. Struct. Biol., 1995, 2, 407-415 (FIGS. 1B and 2A). Once the final docked position of the molecule in the NNI site was determined, the molecule was assigned a score (LUDD, from which an estimation of the inhibition constant (K.sub.i value) was determined.
After docking and K.sub.i estimation was completed for the inhibitors, evaluation of the docked compounds in the active site of RT involved placing each compound into the composite binding pocket using the same orientation matrix utilized in construction of the pocket. The potentially flexible regions in the binding site were then readily identified as were atom sites for future derivatization of the compounds. Fixed docking in the Affinity program within InsightII (InsightII, Molecular Simulations Inc., 1996, San Diego, Calif.), was used for docking small molecules to the NNI binding site which was taken from a crystal structure (PDB code rev, RT/9-Cl-TIBO complex). The program has the ability to define a radius of residues within a 5 .ANG. distance from the NNI molecule. As the modeling calculations progressed, the residues within the radius were allowed to move in accordance with the energy minimization. Ten final docking positions were initially chosen for each inhibitor modeling calculation but failed to reveal more than two promising positions. Later, only two calculated positions were set for the search target.
Calculations were carried out on a Silicon Graphics INIDIGO.sup.2 using the CVFF force field in the Discover program and a Monte Carlo search strategy in Affinity (Luty, B. A. et al., J. Comp. Chem., 1995, 16, 454-464). No solvation procedures were used. Since the total number of movable atoms exceeds 200, Conjugated Gradient minimization was used instead of the Newton minimization method. The initial coordinates of the compounds were generated using the Sketcher module within InsightII. Each final docking position was then evaluated by a score function in LUDI. The top scoring model was then compared with the composite binding pocket and the known crystal structure of similar compounds and used for further analyses. The inhibitory constants (K.sub.i values) of the positioned NNI compounds were evaluated using the LUDI score function (Bohm, H. J., J. Comput. Aided Mol. Des., 1994, 8, 243-256; Bohm, H. J., J. Comput. Aided. Mol. Des., 1992, 6, 593-606).
Several modifications were imposed during the calculation of inhibitory constants (K.sub.i values) of the positioned compounds using the LUDI score function (Bohm, H. J. 1994 supra; Bohm, H. J. 1992 supra). First, the molecular surface areas (MS) were directly calculated from the coordinates of the compounds in docked conformations using the MS program. Second, the number of rotatable bonds (NR), which was assessed inaccurately by INSIGHTII (rigidity imposed by hydrogen bonding was not accounted for in the program), was re-evaluated. Third, it was assumed that the conserved hydrogen bond with RT was assumed to not deviate significantly from the ideal geometry. This assumption was supported by the fact that in the known crystal structures of RT complexes, all hydrogen bonds between NNIs and RT are near the ideal geometry. Last, for the trovirdine compounds, an additional penalty was imposed for a charged group or halogen atoms when positioned near the ring plane of a protein residue such as tryptophan 229 because the interaction was not adequately accounted for in the LUDI score. The working modification of the LUDI scoring function for the PETT compounds included subtracting a score of P from the total LUDI score when the ring plane of the Trp229 was within 5 .ANG. from a para substituent (R):
LUDI Score=MS*BS*2.93+85 (H-bond)-NR*24.2-100-P;
where
P=200, when R=a hydrophilic group, e.g. --OH or --NO2;
P=100, when R=a para-halogen atom, e.g. --F, --Cl or --Br;
P=50, when R=a para-methoxy, e.g. --OMe;
P=0, when R=a hydrophobic group, e.g. H, CH3;
Consequently, the K.sub.i values for the modeled compounds were more predictable than they would be without such modification (Bohm, H. J. 1994 supra; Bohm, H. J. 1992 supra).
Contact Surface and Gap Analysis
Independent of the composite binding pocket and as a follow-up to the docking procedure, computer programs were used to analyze the surface complementarity between the compounds and the binding site residues. This analysis provided another useful way to examine binding interactions, based solely upon the structure that was used for docking (RT/9-Cl TIBO for PETT and RT/MKC-442 for DABO and HEPT) (Das, K. et al., J. Mol. Biol., 1996, 264, 1085-1100).
A number of computer programs were written to analyze the surface of the compounds in the NNI binding site of RT and to better visualize any spatial gaps between the compounds and nearby residues of the RT protein. The algorithm used in these programs was based on a series of cubic grids surrounding the compound, with a user-defined grid spacing. All cubes were coded based on the distance and the nature of the interaction with the protein residues and/or compound atoms. The cubes that overlap both protein and compound within the contact radius are displayed as spheres and were selected to represent the buried surface (user-defined contact radius was the van der Waals radius plus an uncertainty factor, dependent on the reliability of source coordinates). All other cubes that did not interact with protein residues and were within a certain distance from the compound were selected to represent the gap space (space unoccupied by compound or protein) and are displayed as rods.
A graphic interface was then used to examine whether the "gap" spheres could be connected with the compounds without intersecting the "contact" spheres. If the criterion was met, the points that stemmed from the surface of the compound were defined as an expandable region (eligible for synthetic modification). The spheres generated by the programs (shown in FIG. 3) represent the sites buried by protein residues, indicating regions of the compound which are probably not available for derivatization.
FIG. 4 shows the binding pocket embellished with a grid of red rods which represent unoccupied space between the compound and active site residues, providing a complementary view to that shown by the spheres. The grid illustrates the candidate sites for derivatization of the compound and, when used as a distance scale (the length of one rod represents 1 .ANG.), also indicates the volume available for new functional groups.
One example of a useful program is the "SeeGap" program, whose code is listed below in Example 11, together with instructions for its use.
Composite NNI Binding Pocket of RT Reveals Protein Flexibility And Future Inhibitor Modification Sites
The integrated structural information surprisingly revealed a much larger binding site than any shown in individual structures and served as a probe to define the potentially usable space in the binding site (FIG. 1). The three-dimensional binding site can be used as a reference point for the analysis of compounds which have been positioned by a docking procedure.
Upon inspection of the pocket it was apparent that although there are no large-scale conformational changes within the NNI binding site, a number of RT protein residues in contact with the inhibitors are relatively flexible and vary from structure to structure. These residues include Tyr180, Tyr181, Tyr318, Try319, Phe227, Leu234, Trp229, Pro95, and Glu138 (the latter from p51 subunit of RT).
As shown in FIG. 2B, the surface of the composite binding pocket which is overlaid with the RT-TIBO binding site is a short distance (<1.5 .ANG.) away from or even extends past RT residues 234-236, Y188, F227, and the backbone of K101. This indicates that these residues are flexible and can be displaced by the right substituent on an inhibitor.
The composite binding pocket of the invention, unlike a single crystal structure, is able to integrate the nature and extent of the flexibility of the active site residues in the NNI binding site of RT. This uniquely permits prediction of potential modification sites on PETT, DABO, and HEPT derivatives after positioning the compounds in the NNI active site of RT. The method for designing new NNI compounds was particularly useful given the fact that no known crystal structures exist for RT-PETT and RT-DABO complexes, a fact which in this case would prevent the successful application of traditional structure-based drug design methods. Importantly, the model was validated by experimentally demonstrating the superior potency of newly designed agents, predicted to have strong RT inhibitory activity, based upon the low K.sub.i values estimated.
Example 2
Predicted Efficacy of HEPT Derivatives
Compounds listed in Table 1 have been modeled into the NNI binding site of RT (RT/MKC 422 complex) using the docking procedure. The modeled positions were compared with the composite binding pocket of the invention, having the coordinates set forth in Table 9. Modeling was followed by analysis with the LUDI score function.
All of the positions of the compounds with top scores fall into the butterfly-shaped binding site, with the benzyl ring residing in wing 1 and the thymine ring in the wing 2 (FIG. 2). For all compounds tested, the benzyl ring is near Trp229 and the N-1 group is near Pro236, a typical position observed in crystal structures (FIG. 1B). The trend of calculated values listed in Table 1 shows that the K.sub.i value decreases as a result of three factors: para substituents (R2) removed from the benzyl ring, larger alkyl groups added to the thymine ring (R.sub.1), and sulfur atoms substituted for oxygen (at X and/or Y). The modeling calculations, along with the application of the composite NNI binding pocket, provided a guideline for the synthesis of lead compounds designed to have potent anti-HIV activity. The choice of compounds was also based on synthetic feasibility.
TABLE 1__________________________________________________________________________Results of modeling calculations for HEPT derivatives ##STR12## Accessible Molecular Buried LUDI LUDI Surface surface Surface Score Score.sup.d KI.sup.dX Y R.sub.1 R.sub.2 R.sub.3 NR.sup.a (.ANG..sup.2) (.ANG..sup.2) (%) (Lipo) (Sum) (.mu.M)__________________________________________________________________________O O Et F Et 6 549 296 n.d. n.d. n.d. n.d.O O Et Br Et 6 576 311 n.d. n.d. n.d. n.d.S O Me OMe Et 6 558 303 n.d. n.d n.d. n.d.O O Me H Et 5 505 269 85 599 463 23O O Et H Et 6 528 284 87 661 501 9.8O O i-Pr H Et 6 541 294 88 688 528 5.2S O Me H Et 5 512 275 87 703 567 2.1S O Et H Et 6 536 290 90 732 572 1.9S O i-Pr H Et 6 550 300 89 741 580 1.5S S Me H Et 5 521 283 86 706 570 2.0S S Et H Et 6 545 297 90 756 595 1.1S S i-Pr H Et 6 557 308 90 777 617 0.68S S Me H Me 4 491 266 84 661 549 3.2S S Et H Me 5 514 280 88 703 567 2.1S S i-Pr H Me 5 527 290 90 738 602 0.95__________________________________________________________________________ Me = methyl, Et = ethyl, iPr = isopropyl n.d. (not determined) means high K.sub.i values resulting from energetically unfavorable rotation of Trp229 which sterically hinders binding in cases of the para substitution, as revealed by modeling. .sup.a NR = number of rotatable bonds in the compound. Used in the LUDI calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom. .sup.b Molecular surface area calculated using the program GRASP, and defined as the boundary of the volume within any probe sphere (meant to represent a water molecule) of given radius sharing no # volume with the hard sphere atoms which make up the molecule. The values are slightly smaller than the ones approximated by LUDI program. The accessible surfac can be defined as the locus of the centers of all possible such probes in contact with the hard sphere atoms. Alternatively it can be defined as th hard sphere surface if each atomic radius is increased by the probe radiu (1.4 .ANG. radius). .sup.c Buried surface represents the percentage of molecular surface in contact with the protein calculated by LUDI based on the docked positions Based on published crystal structures of RT complexes, the calculation shows that these values could be as low as 77% (in RT/HEPT complex) and can be as high as 90% (in RT/APA complex) but most of them including RT/MKC average around 84%. Therefore, the calculated values may be in the worst case slightly overestimated. .sup.d Ideal hydrogen bond distances and angles between the compounds and the protein are assumed in all cases for K.sub.i and Score (sum) calculation. In published crystal structures of RT complexes, hydrogen bond geometry's are indeed close to ideal; the amide carbonyl of residue A101 on a loop demonstrates a substantial flexibility which can accommodate the best geometry for hydrogen bonding.
Synthesis of HEPT Derivatives
The compounds listed in Table 1 above can be synthesized by reaction of substituted aryl acetonitriles and appropriately functionalized 2-bromo ethyl esters, for example in the presence of zinc in refluxing tetrahydrofuran. Products of the reaction are purified by gel chromatography. Generated 3-oxo esters are next converted into 5-alkyl-6-(arylmethyl)-2-thiouracils with chloroacetic acid, e.g., overnight to yield 5-alkyl-6-(arylmethyl)uracils. The final step in the synthesis is reaction of the uracil with hexamethyldisilazane (HMDS) in the presence of ammonium sulfate. Subsequent treatment with acetals and trimethyl silyl triflate in acetonitrile leads to the formation of N-'substituted uracil and thiouracil derivatives.
These and other known methods can be used to synthesize the compounds of the invention.
Example 3
DABO Derivatives
Chemical Synthesis
All chemicals were used as received from Aldrich Chemical Company (Milwaukee, Wisconsin). All reactions were carried out under nitrogen. Column chromatography was performed using EM Science silica gel 60 and one of the following solvents: ethyl acetate, methanol, chloroform, hexane, or methylene chloride. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian (Palo Alto, Calif.) 300 MHz instrument (Mercury 2000 model) and chemical shifts (.delta.) are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard at 0 ppm. .sup.13 C NMR spectra were recorded at 75 MHz in CDCl.sub.3 on the same instrument using a proton decoupling technique. The chemical shifts reported for .sup.13 C NMR are referenced to the chloroform triplet at 77 ppm. Melting points were measured using a Mel-Temp 3.0 (Laboratory Devices Inc., Holliston, Mass.) melting apparatus and are uncorrected. UV spectra were recorded from a Beckmann (Fullerton, Calif.) model DU 7400 UV/Vis spectrometer using a cell path length of 1 cm and methanol solvent. Fourier Transform Infrared spectra were recorded using an FT-Nicolet (Madison, Wis.) model Protege 460 instrument. Mass spectrum analysis was performed using a Hewlett-Packard (Palo Alto, Calif.) Matrix Assisted Laser Description time-of-flight (MALDI-TOF) spectrometer (model G2025A) in the molecular ion detection mode (matrix used was cyanohydroxycinnamic acid). Some samples were analyzed using a Finnigan (Madison, Wis.) MAT 95 instrument. Elemental analysis was performed by Atlantic Microlabs (Norcross, Ga.).
General Procedure for the synthesis of DABO compounds 3a-d:
The 5-alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one derivatives 3a-d were prepared as shown in Scheme 1.
______________________________________Scheme 3 ##STR13## ##STR14## ##STR15## ##STR16##1-3 R.sub.1 R.sub.2______________________________________a H Meb H Etc H i-Prd Me i-Pr______________________________________ Reagents and conditions: a R.sub.2 CHBrCOOEt/Zn/THF, b HCl(aq), c (H.sub.2 N).sub.2 CS/Na/EtOH, d DMF, K.sub.2 CO.sub.3, Chloromethyl methyl sulfide, 15 h.
Ethyl-2-alkyl-4-(phenyl)-3-oxobutyrates 1a-d were obtained from commercially available phenyl acetonitrile. The .beta.-ketoesters were condensed with thiourea in the presence of sodium ethoxide to furnish the corresponding thiouracils 2a-d. Compounds (1a-d and 2a-d) were produced by a methods previously described (Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med. Chem., 1997, 40, 1447-1454; Danel, K. et al., J. Med. Chem., 1998, 41, 191-198).
Subsequent reaction of thiouracil with methylchloromethyl sulfide in N,N-dimethylformamide (DMF) in the presence of potassium carbonate afforded compounds 3a-d in moderate yields A mixture of thiouracil compound 2 (1 mmol), methylchloromethyl sulfide (1 mmol), and potassium carbonate (1 mmol) in anhydrous DMF (5 ml) was stirred overnight at room temperature. After treatment with water (50 ml), the solution was extracted with ethyl acetate (3.times.50 ml). The combined extracts were washed with saturated NaCl (2.times.50 ml), dried (MgSO.sub.4), filtered and concentrated in vacuo to give the crude products 3a-d which were purified by column chromatography (hexane:ethyl acetate eluent).
X-ray Crystallography
Yellow rectangular plates of compound 3b were grown from tetrahydrofuran by slow evaporation at room temperature. X-ray diffraction data for a 0.5.times.0.2.times.0.08 mm plate crystal of compound 3b was collected at room temperature using a SMART CCD X-ray detector (Bruker Analytical X-ray Systems, Madison, Wis.). Structure solution and refinement was performed using the SHELXTL suite of programs (Bruker Analytical X-ray Systems, Madison, Wis.). All nonhydrogen atoms were refined using anisotropic displacement parameters. Hydrogen atoms were placed at ideal positions and refined as riding atoms with relative isotropic displacement parameters.
The refined small molecule X-ray crystal structure of compound 3b is shown as an Oak Ridge Thermal Ellipsoid Program (ORTEP) drawing in FIG. 8. Table 2 lists the crystal data and structure refinement statistics for compound 3b. Data was collected at room temperature (.lambda.=0.71073 .ANG.), refined using full-matrix least-squares refinement on F.sup.2, and corrected for absorption using semi-empirical psi-scans.
TABLE 2______________________________________Unit Cell a = 4.7893(4).ANG. b = 10.8709(10).ANG. c = 30.040(3).ANG. .alpha. = 90.degree. .beta. = 92.474(2).degree. .gamma. = 90.degree.Space Group P2.sub.1 /nUnit Cell Volume 1562.5(2).ANG..sup.3Z 4.theta. range for data collection 1.36 to 28.27.degree.Limiting indices -6.sup.2 h.sup.2 6 -8.sup.2 k.sup.2 14 -39.sup.2 l.sup.2 37Reflections collected 8744Independent reflections 3507 (R.sub.int = 0.0486)Data/restraints/parameters 3507/0/183Goodness-of-fit on F.sup.2 1.095Final R indices [I > 2.sigma.(I)] R1 = 0.0666, wR2 = 0.1384R indices (all data) R1 = 0.1114, wR2 = 0.1569Absorption coefficient 0.338 mm.sup.-1Max. and min. transmission 0.8356 and 0.6542Extinction coefficient 0.0004(11)Largest difference peaks 0.279 and -0.211 e.ANG..sup.-3______________________________________ R.sub.int = .SIGMA..vertline.F.sub.o.sup.2 - <F.sub.o.sup.2 >.vertline./.SIGMA..vertline.F.sub.o.sup.2,R1 = .SIGMA..vertline..vertline.F.sub.o .vertline. - .vertline.F.sub.c .vertline..vertline./.SIGMA..vertline.F.sub.0 .vertline.- wR2 = {.SIGMA.[w(F.sub.o.sup.2 - F.sub.c.sup.2).sup.2 ]/.SIGMA.[w(F.sub.o.sup.2).sup.2]}.sup.1/2 GooF = S = {.SIGMA.[w(F.sub.o.sup.2 - F.sub.c.sup.2).sup.2]/(n - p)}.sup.1/2, where n = reflections, p = parameters
Physical Data Of Synthesized Compounds:
5-methyl-2-[(methylthiomethyl)thiol-6-benzyl-pyrimidin-4-1H-one (3a)
Yield 62%; mp 148-149.degree. C.; .sup.1 H NMR(CDCl.sub.3):.delta. 2.10 (s, 3H), 2.14 (s, 3H), 3.91 (s, 2H), 4.29 (s, 2H), 7.29-7.26 (m, 5H), 12.20 (s, 1H); .sup.13 C NMR(CDCl.sub.3):.delta. 10.7 (CH.sub.3), 15.5 (SCH.sub.3), 36.6 (CH.sub.2 Ph), 41.0 (SCH.sub.2), 116.7 (C-5), 137.6-126.4 (Ph), 155.2 (C-6), 162.0 (C-4), 165.1 (C-2); CI-MS: 293.1 (M+1).
5-ethyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (3b)
Yield 65%; mp 124-126.degree. C.; .sup.1 H NMR(CDCl.sub.3):.delta. 1.08 (t, 3H), 2.12 (s, 3H), 2.58 (q, 2H), 3.91 (s, 2H), 4.26 (s, 2H), 7.28-7.26 (m, 5H), 12.30 (s, 1H); .sup.13 C NMR(CDCl.sub.3):.delta. 13.1 (CH.sub.3), 15.4 (SCH.sub.3), 18.7 (CH.sub.2), 36.4 (CH.sub.2 Ph), 40.3 (SCH.sub.2), 122.4 (C-5), 138.0-126.3 (Ph), 155.4 (C-6), 161.5 (C-4), 165.2 (C-2); Cl-MS: 307.1 (M+1).
5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (3c)
Yield 57%; mp 116-117 C; .sup.1 H NMR(CDCl.sub.3):.delta. 1.22 (d, 6H), 2.07 (s, 3H), 3.03 (q, 1H), 3.88 (s, 2H), 4.21 (s, 2H), 7.24-7.13 (m, 5H), 12.43 (s, 1H); .sup.13 C NMR(CDCl.sub.3):.delta. 15.4 (SCH.sub.3), 19.6 (CH.sub.3), 28.0 (CH), 36.3 (CH.sub.2 Ph), 40.9 (SCH.sub.2), 125.3 (C-5), 138.3-126.3 (Ph), 155.5 (C-6), 161.1 (C-4), 164.5 (C-2); CI-MS 321.1 (M+1).
5-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl)-pyrimidin-4-1H-one (3d)
Yield 67%; mp 116-120.degree. C.; .sup.1 H NMR(CDCl.sub.3):.delta. 1.28 (d, 6H), 2.15 (s, 3H), 2.27 (s, 6H), 3.10 (q, 1H), 3.88 (s, 2H), 4.31 (s, 2H), 6.84 (s, 3H), 12.42 (s, 1H); .sup.13 C NMR(CDCl.sub.3):.delta. 15.3 (SCH.sub.3), 19.6 (CH.sub.3), 21.2 (CH.sub.3), 28.0 (CH), 36.3 (CH.sub.2 Ph), 40.8 (SCH.sub.2), 125.2 (C-5), 138.0-126.5 (Ph), 155.4 (C-6), 161.3 (C-4), 164.7 (C-2); CI-MS: 349.2 (M+1).
Modeling and Design of DABO Compounds:
The calculated molecular coordinates of DABO compounds which were energy-minimized and docked into the NNI binding site adopted a conformation remarkably similar to that of the crystal structure of compound 3b. FIG. 7B shows the modeled coordinates superimposed on the crystal structure coordinates of 3b and illustrates their conformational similarity, suggesting that the final docked positions of the DABO compounds in the NNI pocket were energetically favorable and quite suitable for these studies. Multiple sterically allowed unoccupied spatial gaps in the binding site were identified from the docking studies which could be filled by strategically designed functional groups (FIG. 7B).
The docked DABO molecule (compound 3a) unexpectedly showed significant space surrounding the benzyl ring and the 5th position of the thymine ring, which led to design of compounds 3b, 3c and 3d. The inhibition constants of the docked molecules were calculated based on a LUDI score function and are listed in Table 3. The calculated K.sub.i values suggested that compounds 3c and 3d would be particularly active inhibitors of RT. ##STR17##
Compound 3d, which differs from compound 3c by the addition of two methyl groups to the benzyl ring, provides more hydrophobic contact with the NNI binding pocket and was predicted to be more potent than compound 3c, based on the modeling studies. Calculations indicate that compounds 3a-3d have progressively larger molecular surface areas but still maintain approximately the same percentage of the molecular surface area in contact with the protein residues. Consequently, the calculated contact surface area between the protein and the compound increases in the following order: compound 3a, 3b, 3c, and 3d. This increased surface area in turn dictates a decrease in calculated K.sub.i values, with 3a having the worst value and 3d the best.
The Tyr183 residue of the HIV RT is located in the catalytic region which has a conserved YMDD motif characteristic of reverse transcriptases. Therefore, the displacement of this tyrosine residue can interfere with catalysis and render the HIV-1 RT protein inactive.
Bulky substituents at the 5th position of the thymine ring could indirectly accomplish such inactivation by displacing Tyr181 which is near Tyr183 (Ding, J. et al., Nat. Struct. Biol., 1995, 2, 407-415). The composite binding pocket shows sufficient room for at least a 3-carbon group at the 5th position. The addition of a methyl, ethyl or isopropyl group at the 5th position of the thymine ring is expected to lead to higher affinity for the relatively hydrophobic environment at this location of the binding pocket. The favorable hydrophobic contact increases as the hydrophobic group at the 5th position gets bulkier. As the DABO derivative binds to the site, the ethyl or isopropyl group can also cause the nearby Tyr181 residue to rotate away from the inhibitor.
Modeling studies showed that this change in conformation in turn affects the positions of neighboring Tyr183 and Tyr188 which may contribute to the inactivation of HIV-1 RT. The benzyl ring of compounds 3a-3d is located near a region surrounded by the hydrophobic ring planes of residues Trp229, Pro95, Y188 and Y181. The analysis of compounds 3a-3c in the composite binding pocket suggests that the benzyl ring would be located on the boundary of the pocket, near residue Y 188. A para substituent of the ring is situated perpendicular to the ring plane of nearby Trp229, within van der Waals contact, and leaves a lot of space unfilled between the compound and Pro95. With a slight conformational rotation of the benzyl ring, compound 3d, with the addition of two methyl groups, was found to better fill the composite binding pocket (FIG. 7B). Such observations indicate that further modifications to the benzyl ring could lead to even more potent inhibitors.
TABLE 3______________________________________Dabo Compounds ##STR18## Ludi.sup.aCompound M.S..sup.b B.S..sup.c Lipo K.sub.iNumber R.sub.1 R.sub.2 (.ANG..sup.2) (%) Score (.mu.M)______________________________________3a H Me 275 88 709 3.33b H Et 283 88 730 2.03c H i-Pr 301 89 785 0.563d Me i-Pr 329 89 875 0.05______________________________________ .sup.a Ludi K.sub.i values were calculated based on the empirical score function in Ludi program (Bohm, H. J., J. Comput. Aided. Mol. Des., 1994, 8, 243-256; 1996). Ideal hydrogen bond distances and angles between compounds and protein are assumed in all cases for Ludi K.sub.i and Ludi Score calculation. In published crystal structures of RT complexes, hydrogen bond geometries are indeed close to ideal; the amide carbonyl of residue A101 on a loop demonstrates substantial flexibility which can accommodate the best geometry for hydrogen bonding. The number of rotatable bonds (= 2) is used in the Ludi calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom. .sup.b MS, molecular surface area calculated using Connolly's MS program (Connolly, M. L., Science, 1983, 221, 709-713). Defined as boundary of volume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with hard sphere atoms which make up the molecule. Values are slightly smaller than those approximated by Ludi program. .sup.c BS, buried surface: percentage of molecular surface in contact wit protein calculated by Ludi relative to docked positions. Based on published crystal structures of RT complexes, the calculation shows that these values could be as low as 77% (in RTHEPT complex) and can be as hig as 90% (in RTAPA complex) but most of them including RTMKC average around 84%.
Predictable Activities
The trend of the calculated K.sub.i values based on the modeling and on the use of the composite binding pocket, with surprising accuracy, predicted the trend of the experimentally determined IC.sub.50 values from HIV replication assays. Compounds 3a-3d were tested for RT inhibitory activity in cell-free assays using purified recombinant HIV RT (listed as IC.sub.50 [rRT] in Table 4), as well as by in vitro assays of anti-HIV activity in HTLVIIIB-infected peripheral blood mononuclear cells (IC.sub.50 [p24] in Table 4) (Zarling, J. M. et al., Nature, 1990, 347, 92-95; Erice, A. et al., Antimicrob. Ag. Chemother., 1993, 37, 835; Uckun, F. M. et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383).
Larger compounds which better fill the composite binding pocket and have lower calculated K.sub.i values showed better IC.sub.50 [rRT] values. This is reflected by the enhancement of the inhibitory activity with the addition of progressively larger groups such as methyl (3a), ethyl (3b), and isopropyl (3c) at the C-5 position of the thymine ring (see Table 4). The same trend was also observed for IC.sub.50 [p24] values.
The lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)pyrimidin-4-(1H)-one (compound 3c), elicited potent anti-HIV activity with an IC.sub.50 value less than 1 nM for inhibition of HIV replication (measured by p24 production in HIV-infected human peripheral blood mononuclear cells) and showed no detectable cytotoxicity (inhibition of cellular proliferation was >100 .mu.M as measured by MTA) (Table 4). In contrast to all previously published data for DABO and S-DABO derivatives which were less active than AZT and MKC-442 (Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med. Chem., 1997, 40, 1447-1454; Danel, K. et al., J. Med. Chem., 1998, 41, 191-198) and showed selectivity indices of <1,000, the novel compound 3c was more than 4-fold more active than AZT and MKC-442, and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations with an unprecedented selectivity index (=IC.sub.50 [MTA]/IC.sub.50 [p24] ) of >100,000.
The X-ray crystal structure of 3b was determined to compare its conformation to that of the compound after docking into the NNI binding site. The refined small molecule X-ray crystal structure of compound 3b is represented as an ORTEP drawing in FIG. 8. The calculated molecular coordinates of DABO compounds which were energy-minimized and docked into the NNI binding site adopted a conformation remarkably similar to that of the crystal structure of compound 3b. FIG. 7B shows the modeled coordinates superimposed on the crystal structure coordinates of 3b and illustrates their conformational similarity, suggesting that the final docked positions of the DABO compounds in the NNI pocket were energetically favorable.
TABLE 4______________________________________Inhibitory Activity of DABO Compounds: ##STR19## IC.sub.50 IC.sub.50 CC.sub.50Compound [rRT] [p24] [MTA]Number R.sub.1 R.sub.2 (.mu.M) (.mu.M) (.mu.M) S.I..sup.d______________________________________3a H Me 18.8 4.5 >100 >223b H Et 9.7 0.8 >100 >1253c H i-Pr 6.1 <0.001 >100 >100,0003d Me i-Pr 4.8 n.d n.d. n.d.AZT >100 0.04 50 1250MKC-442 0.004 >100 >25,000______________________________________ .sup.d Selectivity Index is equal to the ratio of fifty percent cytotoxic concentration to IC.sub.50. n.d. = not determined
Example 4
Synthesis of PETT Derivatives
Chemical Synthesis
All chemicals were used as received from Aldrich Chemical Company (Milwaukee, Wis.). All reactions were carried out under nitrogen. Column chromatography was performed using EM Science silica gel 60 and one of the following solvents: ethyl acetate, methanol, chloroform, hexane, or methylene chloride. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian (Palo Alto, Calif.) 300 MHz instrument (Mercury 2000 model) and chemical shifts (.delta.) are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard at 0 ppm. .sup.13 C NMR spectra were recorded at 75 MHz in CDCl.sub.3 on the same instrument using a proton decoupling technique. The chemical shifts reported for .sup.13 C NMR are referenced to the chloroform triplet at 77 ppm. Melting points were measured using a Mel-Temp 3.0 (Laboratory Devices Inc., Holliston, Mass.) melting apparatus and are uncorrected. UV spectra were recorded from a Beckmann (Fullerton, Calif.) model DU 7400 UV/Vis spectrometer using a cell path length of 1 cm and methanol solvent. Fourier Transform Infrared spectra were recorded using an FT-Nicolet (Madison, Wis.) model Protege 460 instrument. Mass spectrum analysis was performed using a Hewlett-Packard (Palo Alto, Calif.) Matrix Assisted Laser Desorption time-of-flight (MALDI-TOF) spectrometer (model G2025A) in the molecular ion detection mode (matrix used was cyanohydroxycinnamic acid). Some samples were analyzed using a Finnigan (Madison, Wis.) MAT 95 instrument. Elemental analysis was performed by Atlantic Microlabs (Norcross, Ga.).
General Procedure for Synthesis of PETT Derivatives
Compounds I-1, I-3, and I-4 were synthesized as described in Scheme 3. Trovirdine (I-2) was synthesized according to the literature procedure (Bell, F. W., et al., J. Med. Chem., 1995, 38,4929-4936). ##STR20## Physical Data Of Synthesized Compounds: N-[2-(2-pyridylethyl)]-N'-[2-(pyridyl)]-thiourea (I-1)
white solid (Ig, 49%); mp 98-100.degree. C.; UV(MeOH).lambda.max: 293, 265, 247 and 209 nm; IR(KBr Disc).nu. 3415, 3222, 3050, 2360, 1600, 1533, 1479, 1436, 1315, 1240, 1151 and 775 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.90 (s, 1H), 8.8 (s, 1H), 8.60-8.58 (d, 1H), 8.03-8.01 (d, 1H), 7.65-7.56 (m, 2H), 7.27-7.14 (m, 2H), 6.93-6.89 (d, 1H), 6.80-6.77 (d, 1H) 4.23-4.15 (q, 2H) and 3.41-3.20 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 179.2, 158.9, 153.0, 149.2, 145.5, 138.5, 136.4, 123.5, 121.4, 117.7, 111.8, 44.9, and 36.9; MALDI-TOF mass found, 257.1(M-1), calculated, 258.3; Anal. (C.sub.13 H.sub.14 N.sub.4 S) C, H, N, S.
N-[2-(1-piperidinoethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (I-3)
white solid (2 g, 74%); mp 150-152.degree. C.; UV (MeOH).lambda.max: 306, 275 and 205 nm; IR(KBr Disc).nu. 3155, 3077, 2935, 2850, 2360, 1591, 1525, 1465, 1319, 1226, 1095, 827 and 756 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.53 (br s, 1H), 9.72 (br s, 1H), 8.22 (d, 1H), 7.72-7.68 (dd, 1H), 6.95-6.92 (d, 1H), 3.84-3.78 (q, 2H), 2.61-2.57 (t, 2H), 2.45 (br s, 4H), 1.64-1.48 (m, 6H); .sup.13 C NMR(CDCl.sub.3).delta. 178.1, 151.8, 146.3, 140.8, 113.5, 112.6, 56.1, 54.0, 43.0, 26.3, and 24.3, MALDI-TOF mass found, 343.5, calculated, 343.3; Anal. (C.sub.13 H.sub.19 BrN.sub.4 S) C, H, N, S, Br.
N-[2-(2,5-dimethoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (I-4)
white solid (2 g, 67%); mp 133-138.degree. C.; UV (MeOH).lambda.max: 202, 205, 231, 276 and 300 nm; IR(KBr Disc).nu. 3209, 3152, 3078, 3028, 2951, 2831, 1595, 1533, 1468, 1306, 1227, 1095, 1059, 1022, 862, 825, 796, 707 cm.sup.-1 ; .sup.1 H NMR(CDCl.sub.3).delta. 11.24 (br s, 1H), 9.30 (br s, 1H), 8.10-8.09 (d, 1H), 7.65 (dd, 1H), 6.82-6.76 (m, 4H), 4.03-3.97 (q, 2H), 3.77 (s, 3H), 3.76 (s, 3H), 3.00-2.96 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 178.7,153.1,151.8, 151.7,146.5,140.9, 128.1,117.7,113.3, 112.6, 111.2, 110.9, 55.7, 55.5, 45.6, and 29.9; MALDI-TOF mass found, 394.0 (M-1), 396.0 (M+1), calculated, 395.0; Anal. (C.sub.16 H.sub.18 BrN.sub.3 O.sub.2 S) C, H, N, S, Br.
Chemical Synthesis II
Compounds II-1-9 were synthesized according to Scheme 4. In brief, 2-amino-5-bromopyridine was condensed with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl derivative (A). Further reaction with appropriately substituted phenylethyl amine gave the target PETT derivatives in good yields.
General procedure for synthesis
Thiocarbonyldiimidazole (8.90 g, 50 mmol) and 2-amino-5-bromo pyridine (8.92 g, 50 mmol) were added to 50 mL of dry acetonitrile at room temperature. The reaction mixture was stirred for 12 h and the precipitate filtered, washed with cold acetonitrile (2.times.25 mL), and dried under vacuum to afford (11.40 g, 80% ) of compound A. To a suspension of compound A (0.55 eqv) in dimethyl formamide (15 mL) an appropriate amine (0.50 eqv) was added. The reaction mixture was heated to 100.degree. C. and stirred for 15 hours. The reaction mixture was poured into ice-cold water and the suspension was stirred for 30 minutes. The product was filtered, washed with water, dried, and further purified by column chromatography to furnish the target compounds I-9 in good yields. ##STR21## Physical data of synthesized compounds: N-12-(2-methoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-1)
yield: 65%; mp 143-145.degree. C.; UV (MeOH).lambda.max: 202, 205, 275 and 306 nm; IR(KBr).nu. 3211, 3153, 3036, 2956, 2835, 1593, 1533, 1462, 1242, 1186, 1036, 1007, 862, 812, 756, 708 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.22 (br s, 1H), 9.37 (br s, 1H), 8.02-8.01 (d, 1H), 7.69-7.65 (dd, 1H), 7.28-7.18 (m, 2H), 6.94-6.80 (m, 3H), 4.04-3.98 (q, 2H), 3.81 (s, 3H), 3.04-2.99 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 178.7, 157.6, 151.7, 146.3, 141.0, 130.7, 127.9, 126.8, 120.3, 113.5, 112.5, 110.3, 55.2, 45.6, 29.8; Maldi Tof found: 366.0 (M+1), calculated: 365.0; Anal. (C.sub.15 H.sub.16 BrN.sub.3 OS) C,H,N,S.
N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-2)
yield: 71%; mp 156-157.degree. C.; UV (MeOH).lambda.max: 209, 256, 274 and 305 nm; IR(KBr).nu. 3446, 3234, 3163, 3055, 2935, 1672, 1595, 1560, 1531, 1466, 1390, 1362, 1311, 1265, 1227, 1169, 1136, 1089, 1003, 864, 825, 756 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.36 (br s, 1H), 9.47 (br s, 1H), 8.05-8.04 (d, 1H), 7.72-7.68(dd, 1H), 7.30-7.03 (m, 4H), 6.87-6.84 (d, 1H), 4.06-3.99 (q, 2H), 3.10-3.05 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 179.1, 163.1, 151.7, 146.2, 141.1, 131.2, 131.1, 128.5, 128.4, 124.1, 115.5, 115.2, 113.6, 112.2, 45.8 and 28.2; .sup.19 F NMR(CDCl.sub.3) 6-42.58 & -42.55 (d); Maldi Tof found: 355.0 (M+1), calculated: 354.0; Anal. (C.sub.14 H.sub.13 BrFN.sub.3 S) C, H, N, S.
N-[2-(2-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-3)
yield: 72%; mp 137-139.degree. C.; UV (MeOH).lambda.max: 208, 213, 256, 275 and 305 nm; IR(KBr).nu. 3433, 3221, 3157, 3089, 3037, 2922, 2866, 1668, 1597, 1535, 1466, 1338, 1263, 1209, 1188, 1130, 1095, 1053, 1001, 864, 823, 750 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.41 (br s, 1H), 9.54 (br s, 1H), 8.17-8.16 (d, 1H), 7.83-7.79 (dd, 1H), 7.50-7.30 (m, 4H), 6.97-6.94 (d, 1H), 4.19-4.13 (q, 2H), 3.30-3.26 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 179.2, 151.7, 146.3, 141.2, 136.3, 134.2, 131.1, 129.6, 128.1, 126.8, 113.6, 112.7, 45.2, and 32.5; Maldi Tof found: 371.8 (M+1), calculated: 371.0; Anal. (C.sub.14 H.sub.13 BrClN.sub.3 S) C, H, N, S, Br.
N-[2-(3-methoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-4)
yield: 68%; mp 155-156.degree. C.; UV (MeOH).lambda.max: 208, 274 and 306 nm; IR(KBr).nu. 3454, 3236, 3147,3030,2951,2869, 2827, 1591, 1545, 1525, 1466, 1304, 1265, 1229, 1188, 1151, 1095, 1051, 1024, 980, 860, 825, 789, 698 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.30 (br s, 1H), 9.25 (br s, 1H), 8.05-8.04 (d, 1H), 7.71-7.67 (dd, 1H), 7.29-7.24 (t, 1H), 6.89-6.78 (m, 4H), 4.05-3.99 (q, 2H), 3.81 (s, 3H), 3.00-2.96 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 178.9, 159.7, 151.6, 146.4, 141.1, 140.3, 129.6, 121.2, 115.0, 113.4, 112.7, 111.6, 55.1, 47.1 and 34.8; Maldi Tof found: 367.0 (M+2), calculated: 365.0; Anal. (C.sub.15 H.sub.16 BrN.sub.3 OS) C, H, N, S.
N-[2-(3-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-5)
yield: 73%; mp 171-172.degree. C.; UV (MeOH).lambda.max: 202, 208, 258, 275 and 306 nm; IR(KBr).nu. 3213, 3155, 3084, 3028, 2866, 1595, 1533, 1477, 1336, 1308, 1229, 1211, 1173, 1136, 1092, 1026, 935, 870, 827, 791, 740 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) 811.33 (br s, 1H), 9.46 (br s, 1H), 8.05-8.04 (d, 1H), 7.73-7.69 (dd, 1H), 7.31-7.26 (m, 1H), 7.08-6.97 (m, 3H), 6.87-6.83 (d, 1H), 4.06-3.99 (q, 2H), 3.05-3.00 (t, 2H); .sup.13 C NMR (CDCl.sub.3).delta. 179.1, 163.1, 151.7, 146.2, 141.2, 130.1,129.9,124.5, 115.9, 115.6, 113.7, 113.5, 113.4, 112.8, 46.7 and 34.6; .sup.19 F NMR(CDCl.sub.3).delta. -37.30&-37.33 (d); Maldi Tof found: 354.0 (M.sup.+), calculated: 354.0; Anal. (C.sub.14 H.sub.13 BrFN.sub.3 S) C, H, N, S.
N-[2-(3-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-6)
yield: 72%; mp 163-165.degree. C.; UV (MeOH).lambda.max: 202, 213, 258, 276 and 305 nm; IR(KBr).nu. 3242, 3161, 3043, 2929, 1593, 1579, 1547, 1527, 1466, 1313, 1227, 1167, 1095, 997, 889, 827, 812, 785, 700 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.33 (br s, 1H), 9.37 (br s, 1H), 8.09-8.08 (d, 1H), 7.73-7.69 (dd, 1H), 7.28-7.15 (m, 4H), 6.85-6.82 (d, 1H), 4.04-3.98 (q, 2H), 3.02-2.97 (t, 2H), .sup.13 C NMR (CDCl.sub.3).delta. 179.1,151.6, 146.3, 141.2, 140.7, 134.2,129.8,129.0, 127.0, 126.8, 113.4,112.8, 46.7 and 34.5; Maldi Tof found: 371.8 (M+1), calculated: 371.0; Anal. (C.sub.14 H.sub.13 BrClN.sub.3 S) C, H, N, S.
N-[2-(4-methoxyphenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-7)
yield: 85%; mp 178-179.degree. C.; UV (MeOH).lambda.max: 205, 226, 275 and 305 nm; IR(KBr).nu. 3221, 3159, 3042, 2931, 2827, 1587, 1510, 1464, 1311, 1225, 1165, 1088, 1034, 820, 773, 708 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.30 (br s, 1H), 9.87 (br s, 1H), 8.00-7.99 (d, 1H), 7.67-7.63 (dd, 1H), 7.21-7.18 (d, 2H), 6.95-6.85 (m, 3H), 4.00-3.93 (q, 2H), 3.81 (s, 3H), 2.96-2.92 (t, 2H); .sup.13 C NMR (CDCl.sub.3).delta. 179.1, 158.0,151.9, 145.8,140.7,130.6, 129.6,113.8,113.7, 112.1, 55.1,46.9 and 33.8; Maldi Tof found: 366.0 (M+1), calculated: 365.0; Anal. (C.sub.15 H.sub.16 BrN.sub.3 OS) C, H, N, S.
N-[2-(4-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-8)
yield: 69%; mp 177-178.degree. C.; UV (MeOH).lambda.max: 208, 211, 274 and 306 mn; IR(KBr).nu. 3456, 3213, 3155, 3086, 3028, 2868, 1595, 1560, 1533, 1477, 1336, 1308, 1238, 1211, 1173, 1136, 1092, 1026, 933, 869, 827, 791, 741, 694 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.29 (br s, 1H), 9.27 (br s, 1H), 8.04-8.03 (d, 1H), 7.73-7.69 (dd, 1H), 7.27-7.22 (m, 2H), 7.04-6.99 (m, 2H), 6.83-6.79 (d, 1H), 4.03-3.96 (q, 2H), 3.02-2.97 (t, 2H); .sup.13 C NMR(CDCl.sub.3).delta. 179.1, 163.2, 151.6, 146.3, 141.2, 134.3, 130.3, 130.2, 115.4, 115.2, 113.5, 112, 47.0, and 34.1; .sup.19 F NMR (CDCl.sub.3) 6-40.55 (m); Maldi Tof found: 354.8 (M+1), calculated: 354.0; Anal. (C.sub.14 H.sub.13 BrFN.sub.3 S) C, H, N, S.
N-[2-(4-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (II-9)
yield: 71%; mp 180-183.degree. C.; UV (MeOH).lambda.max: 206, 209, 219, 256, 275 and 305 nm; IR(KBr)v 3221, 3153, 3086, 3022, 2931, 1674, 1593, 1562, 1533, 1473, 1406, 1340, 1304, 1265, 1227, 1169, 1138, 1092, 1016, 820, 752, 714 cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3).delta. 11.40 (br s, 1H), 9.34 (br s, 1H), 8.15-8.14 (d, 1H), 7.84-7.80 (dd, 1H), 7.46-7.30 (m, 4H), 6.92-6.89 (d, 1H), 4.10-4.07 (q, 2H), 3.13-3.08 (t, 2H); .sup.13 C NMR (CDCl.sub.3).delta. 179.2, 151.6, 146.3, 141.3, 137.1, 130.2, 128.6, 113.5, 112.8, 46.8 and 34.2; Maldi Tof found: 372.0 (M+1), calculated: 371.0; Anal. (C.sub.14 H.sub.13 BrClN.sub.3 S) C, H, N, S.
Chemical Synthesis m
Compounds III-1-3 were prepared as illustrated in Scheme 5. The synthesis involved condensing 2-amino-5-bromopyridine with 1,-thiocarbonyl diimidazole to furnish the required thiocarbonyl derivative. Further reaction of this thiocarbonyl derivative with an appropirate amine gave 1-3 in good yields. ##STR22## Physical data of synthesized compounds: N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (III-1)
Yield: 74%; mp 150-152.degree.; .sup.1 H NMR (CDCl.sub.3).delta. 11.53 (br s, 1H), 9.72 (br s, 1H), 8.22 (d, 1H), 7.72-7.68 (dd, 1H), 6.95-6.92 (d, 1H), 3.84-3.78 (q, 2H), 2.61-2.57 (t, 2H), 2.45 (br s, 4H), 1.64-1.48 (m, 6H); .sup.13 C NMR(CDCl.sub.3).delta. 178.1, 151.8, 146.3, 140.8, 113.5, 112.6, 56.1, 54.0, 43.0, 26.3, and 24.3.
N-[2-(1-piperizinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (III-2)
Yield: 75%; mp 178-180.degree. C.; .sup.1 H NMR (CDCl.sub.3).delta. 11.50 (br s, 1H), 9.77 (br s, 1H), 8.19-8.18 (d, 1H), 7.75-7.71 (dd, 1H), 6.97-6.95 (d, 1H), 3.87-3.86 (m, 2H), 3.63-3.60 (t, 2H), 3.45-3.42 (m, 3H), 2.74-2.69 (t, 2H), 2.59-2.52 (m, 4H); .sup.13 C NMR(CDCl.sub.3).delta. 178.7, 151.8, 146.1, 141.0, 113.7, 112.7, 55.2, 52.0, 51.9 and 45.8.
N-[2-(1-morpholinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (III-3)
Yield: 65%; 124-126.degree. C.; .sup.1 H NMR (CDCl.sub.3).delta. 11.51 (br s, 1H), 9.23 (br s, 1H), 8.25-8.24 (d, 1H), 7.75-7.71 (dd, 1H), 6.85-6.82 (d, 1H), 3.87-3.74 (m, 6H), 2.68-2.54 (m, 6H); .sup.13 C NMR(CDCl.sub.3).delta. 178.5, 151.7, 146.4, 141.0, 113.5, 112.7, 67.2, 55.4, 53.1, 42.5.
______________________________________Compound R Compound R______________________________________I-1 pyridyl II-1 piperidinylI-3 piperidinyl III-2 piperozinylI-4 2,5-dimethoxy phenyl III-3 morpholinylII-1 o-methoxy phenyl II-6 m-chlorophenylII-2 o-fluorophenyl II-7 p-methoxy phenylII-3 o-chlorophenyl II-8 p-flurophenylII-4 m-methoxy phenyl II-9 p-chlorophenylII-5 m-fluorophenyl______________________________________
Example 5
Structure-based Design and Docking of Novel PETT Derivatives into Composite NNI Binding Pocket I
A novel model of the NNI binding pocket of RT was constructed by carefully superimposing the coordinates of 9 individual RT-NNI crystal structures and then generating a van der Waals surface which encompassed all of the overlaid ligands. The integrated structural information of this "composite binding pocket" revealed an unexpectedly different and much larger NNI binding site than shown in or predictable from any of the individual structures and served as a probe to more accurately define the potentially usable space in the binding site (FIG. 2a). A number of protein residues in contact with the inhibitors are relatively flexible and vary from structure to structure. These residues include Tyr180, Tyr181, Tyr318, Try319, Phe227, Leu234, Trp229, Pro95, and Glu138 (from p51 subunit of RT). As shown in FIG. 2b, the surface of the composite binding pocket is a short distance away from (<1.5 .ANG.) or even extends past RT residues 234-236, Y188, F227, and the backbone of K101. This indicates that these residues are flexible and can be displaced by the right inhibitor. The composite binding pocket, unlike an individual crystal structure, is able to summarize the nature and extent of the flexibility of the active site residues. This allowed prediction of potential modification sites on the PETT derivatives I after positioning the compounds in the RT active site (see Methods).
A computer simulation of the binding of PETT compounds into the NNI binding site of RT was accomplished using a molecular docking procedure. Docking of PETT and trovirdine into the NNI binding site required the use of X-ray coordinates of an RT/NNI complex (in this case the RT/9-Cl-TIBO complex).
Upon binding to RT, the compound can fit into a butterfly-shaped NNI binding site (described by Ding, J., et al., Nat. Struct. Biol., 1995, 2, 407-415) (FIGS. 1B and 2). PETT and its derivatives such as compounds I-1-4 could be viewed as two chemical groups linked together by a thiourea group (Table 5). One half of the molecule is composed of a 2-aminothiazole group (PETT) or a pyridylthiourea group (compounds I-1-4) which forms an intramolecular hydrogen-bonded heterocyclic ring. The other half of the molecule is a phenyl or heterocyclic ring separated from the thiocarbonyl group by an ethyl linker.
Once the final docked position of the molecule in the NNI site was determined, the molecule was assigned a score, from which an estimation of the inhibition constant (K.sub.i value) was determined (Table 5). When trovirdine was docked into the NNI binding site of RT it had a higher binding score than PETT and fit into the butterfly-shaped binding region with one part residing in Wing 1 and the other in Wing 2 (FIG. 1B). The ring closest to the thiocarbonyl group resided near the Lys(K)101 loop and the other pyridyl ring was near Trp(W)229.
After docking and K.sub.i estimation was completed for the PETT inhibitors, evaluation of the docked compounds in the active site of RT involved placing each compound into the composite binding pocket using the same orientation matrix utilized in its construction. The potentially flexible regions in the binding site were then readily identified as were atom sites for future derivatization of the compounds. The area within Wing 2 and the residues near the thiourea group seemed to be the most forgiving regions in the binding site of RT. This observation was also supported by the analysis of gaps in atom-to-atom contact between the protein and the inhibitor.
TABLE 5__________________________________________________________________________Interaction scores, calculated K.sub.i values, and measured IC.sub.50data for PETT derivatives I. ##STR23## Ludi.sup.c IC.sub.50 M.S..sup.a B.S..sup.b Lipo Ludi.sup.c K.sub.i p24R.sub.1 R.sub.2 (.ANG..sup.2) (%) Score Score (.mu.M) (.mu.M) S.I..sup.d__________________________________________________________________________PETT phenyl 2-thiazolyl 254 84 625 562 2.4 n.d. n.d.I-1 2-pyridyl 2-pyridyl 260 84 640 577 l.7 0.230 >435I-2 2-pyridyl 2-(5- 276 84 679 616 0.7 0.007Trovirdine bromo) pyridylI-3 1-piperidinyl 2-(5- 278 84 684 621 0.6 <0.001 >10.sup.4 bromo) pyridylI-4 2,5- 2-(5- 317 84 779 716 0.2 <0.001 >10.sup.5 dimethoxy- bromo) phenyl pyridylAZT 0.008 6250__________________________________________________________________________ .sup.a MS, molecular surface area calculated using Connolly's MS program. (Connolly, M. L., Science, 1983, 221, 709-713) Defined as boundary of volume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with hard sphere atoms which make up the molecule. Values are slightly smaller than those approximated by Ludi program. .sup.b BS, buried surface: percentage of molecular surface in contact wit protein calculated by Ludi based on docked positions. Based on published crystal structures of RT complexes, our calculation shows that these values could be as low as 77% (in RTHEPT complex) and can be as high as 90% (in RTAPA complex) but most of them including RTMKC average around 84%. .sup.c Ludi Ki values were calculated based on the empirical score function in Ludi program. (Bohm, H. J., J. Comput. Aided. Mol. Des., 1994 8, 243-256; 1996,) Ideal hydrogen bond distances and angles between compounds and protein are assumed in all cases for Ludi K.sub.i and Ludi Score calculation. In published crystal structures of RT complexes, hydrogen bond geometries are indeed close to ideal; the amide carbonyl of residue A101 on a loop demonstrates substantial #flexibility which can accommodate the best geometry for hydrogen bonding. The number of rotatable bonds (= 2) is used in the Ludi calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom. .sup.d Selectivity Index is equal to the ratio of fifty percent cytotoxic concentration to IC.sub.50. n.d., not determined.
Analysis of the molecular surface of the compounds in the NNI binding site of RT included visualization of spatial gaps between the compounds and nearby residues of the RT protein, as described above for Example 1. The spheres generated are shown in FIG. 3, and indicate regions of the compound which are probably not available for derivatization. FIG. 4 shows the binding pocket embellished with a grid of red rods which represent unoccupied space between the compound and active site residues, providing a complementary view to that shown by the spheres in FIG. 3. The grid illustrates the candidate sites for derivatization of the compound and, when used as a distance scale (the length of one rod represents 1 .ANG.), also indicates the volume available for new functional groups. After the docked PETT compounds were subjected to the grid (gap) analysis, a number of gaps in the binding site were identified (FIGS. 3-4), some of which could be filled by strategically designed functional groups on new PETT derivatives. It was postulated that a more efficient use of such sterically allowed unoccupied spatial gaps in the binding site could be achieved by replacing the 2-pyridyl ring of trovirdine with a 1-piperidinyl (compound I-3) or 2,5-dimethoxyphenyl moiety (compound I-4) and yield potentially more active PETT compounds with larger molecular surface areas, higher Ludi scores, and lower Ki values (Table 5).
Compounds I-1, I-3 and I-4 were subjected to the same docking procedure and K.sub.i calculation used to analyze the parent compounds PETT and trovirdine (compound I-2). The molecular surface area of the compounds calculated after docking increased in the following order: PETT, compound I-1, I-2 (trovirdine), I-3, and I-4. At docked positions, the atom surface area in contact with the protein residues constituted an average of 84% of the entire molecular surface (FIG. 3). We used this average value in the calculation of the inhibitory constant (K.sub.i) based on the Ludi score function. Calculated Ki values for I-3 and I-4 predicted that these compounds would have potency superior to that of trovirdine. The calculated K.sub.i values of our compound I-3 (0.6 .mu.M), and compound I-4 (0.2 .mu.M) were better than those of known compounds PETT (2.4 .mu.M), compound I-1 (1.7 .mu.M) and trovirdine (0.7 .mu.M).
Example 6
In Vitro Assays of Anti-HIV Activity Using PETT Derivatives I
The HIV-1 strain HTLVIIIB (kindly provided by Dr. Neal T. Wetherall, VIROMED Laboratories, Inc.), was propagated in CCRF-CEM cells, and used in in vitro assays of the anti-HIV-1 activity of the synthesized novel derivatives. Cell-free supernatants of HTLVIIIB-infected CCRF-CEM cells were harvested, dispensed into 1 ml aliquots, and frozen at -70.degree. C. Periodic titration of stock virus was performed by examining its cytopathic effects in MT-2 cells following the procedures described in (Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835).
Normal human peripheral blood mononuclear cells (PBMNC) from HIV-negative donors were cultured 72 hours in RPMI 1640 supplemented with 20%(v/v) heat-inactivated fetal bovine serum (FBS), 3% interleukin-2, 2 mM L-glutamine, 25 mM HEPES, 2 g/L NaHCO.sub.3, 50 .mu.g/ml gentamicin, and 4 .mu.g/ml phytohemagglutinin prior to exposure to HIV-l. The incubated cells were then exposed to HIV-1 at a multiplicity of infection (MOI) of 0.1 during a one-hour adsorption period at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. Subsequently, infected cells were cultured in 96-well microtiter plates (100 .mu.l/well; 2.times.10.sup.6 cells/ml) in the presence of test compounds, including AZT as a control. Aliquots of culture supernatants were removed from the wells on the 7th day after infection for p24 antigen assays. The methods used in the P24 assay were as previously described in Uckun, et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383; Zarling, et al., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835.
The applied p24 enzyme immunoassay (EIA) was the unmodified kinetic assay commercially available from Coulter Corporation/Immunotech, Inc. (Westbrooke, Me.). In the assay, a murine monoclonal antibody against HIV core protein is coated onto microwell strips. Antigen (HIV core protein) present in the test culture supernatant samples binds the antibody and the bound antibody-antigen complex is quantitated. Percent viral inhibition was calculated by comparing the p24 values from the test substance-treated infected cells with p24 values from untreated infected cells (i.e., virus controls).
In addition, the activity of the test compounds to inhibit recombinant HIV-1 reverse transcriptase (rRT) activity was determined using the Quan-T-RT assay system (Amersham, Arlington Heights, Ill.), which utilizes the scintillation proximity assay principle. The assay method is described in Bosworth, N., et al., Nature, 1989, 341, 167-168. Data for both bioassays is reported as IC.sub.50 values.
In parallel with the bioactivity assays, the effects of the test compounds on cell viability was also examined, using the Microculture Tetrazolium Assay (MTA) described in Darling, et al., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835. In brief, non-infected PBMNC were treated with test compounds or controls for 7 days under identical experimental conditions and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), was added to quantitative cellular proliferation.
An energy-minimized model of compound I-4 in the RT binding site had the largest molecular surface area in contact with the protein and thus achieved the highest lipophilicity score. The docking studies indicated that the 2-methoxy group of compound I-4 is situated beneath the ethyl linker and fits favorably into a cavity of the binding pocket, providing contact with protein residues that cannot be achieved by trovirdine. Likewise, the 5-methoxy group of compound I-4 provides close contact with residues Pro95 and Trp229. The trend of the calculated K.sub.i values accurately predicted the trend of the experimentally determined IC.sub.50 values from HIV replication assays, as shown in Table 5, thereby providing conclusive evidence of the practical utility of the composite model.
The lead compound, I-4 with the lowest calculated K.sub.i values of the series, was 8-times more potent than trovirdine against purified recombinant HIV-RT using the cell-free Quan-T-RT system (IC50[rRT] was 0.1 .mu.M for I-4 versus 0.8 .mu.M for trovirdine). Compound I-4 also elicited potent anti-HIV activity with IC.sub.50 values of less than 0.001 .mu.M in 3 of 3 independent experiments which was consistently lower than the IC.sub.50 values for trovirdine (0.007 .mu.M) and AZT (0.008 .mu.M). None of the PETT derivatives were cytotoxic at concentrations as high as 100 .mu.M. Therefore, the calculated selectivity index (IC.sub.50 [MTA]/IC.sub.50 [p24]) of compounds I-3 and I-4 were greater than 10.sup.5.
All active PETT compounds listed in Table 5 are able to form an intramolecular hydrogen bond between the nitrogen atom of pyridine or thiazole and an amide hydrogen of the thiourea group, as shown in Wing 1 of FIG. 1B. The intramolecular hydrogen bond was also observed in our small molecule crystal structure of compound I-3 (data not shown). The energy gained by the formation of such a hydrogen bond has been estimated to be about 5 kcal/mol (Bell, et al., J. Med Chem., 1995, 38, 4929-4936). Our docking results showed that the internal hydrogen bond keeps the pyridyl thiourea (or thiazolylthiourea) in a more rigid conformation and allows the molecule to adopt the appropriate geometry to occupy Wing 1 of the binding site, and at the same time maintain a hydrogen bond with a backbone carbonyl of residue Lys101 (FIG. 1B).
Compounds I-3 and I-4 differ from trovirdine at the proposed Wing 2 binding region of the molecule. Compound I-3 has a heterocyclic ring which replaces the pyridyl ring and compound 4 has two methoxy groups added at meta and ortho positions of the phenyl ring. The molecular surface areas of compounds I-3 and I-4 are larger than that of trovirdine, as calculated from the coordinates of the predicted active conformation obtained from docking. This larger surface area results in a better lipophilic score and lower calculated K.sub.i value (Table 5). Both pyridylethyl and piperidinylethyl groups occupy the same region of Wing 2 near Tyr229 (FIG. 2 and 5). Our composite binding pocket shows a space large enough to accommodate a group larger than the pyridyl ring of trovirdine. Docking results and analyses of gaps indicate that the pyridyl ring of trovirdine has multiple sites which can be used for incorporation of larger groups. As shown in FIG. 5, there is sufficient space surrounding the pyridylethyl ring for the addition of a two- to four-atom substituent at any of the ring positions. Both sides of the pyridylethyl ring plane of trovirdine are relatively exposed in the pocket (FIG. 3A) and can accommodate additional substituents (FIG. 4A). This prediction was confirmed by the potency of compound I-4 (which contains ortho, meta-dimethoxy substituents), in inhibitng HIV replication.
The piperidinyl group of I-3 is puckered and therefore occupies a larger overall volume than the planar pyridyl ring of trovirdine and is in close contact with residues Leu234 and Leu100, the latter of which can mutate to isoleucine, frequently found in a drug-resistant RT mutant strain. In contrast to previously reported extensive attempts at expanding within the pyridyl ring plane (Bell, et al., J. Med. Chem., 1995, 38, 4929-4936; Cantrell, A. S., et al., J. Med Chem., 1996, 39, 4261-4274; Ahgren, C., et al., Antimicrob. Agents Chemotherapy, 1995, 39, 1329-1335), the success of our efforts at modification perpendicular to the ring plane introduces new possibilities to develop more potent inhibitors which combine both modifications. The piperidinyl ring is conformationally more flexible than an aromatic ring has the advantage of fitting an uncompromising binding pocket more effectively, despite the expense paid for loss of entropy upon binding. The analysis shown in FIGS. 3, 4, and 5 provides new insights for modifications which are different from those of trovirdine derivatives. Various combinations of double substitutions at axial or equatorial positions of the piperidinyl ring generate derivatives with a broader range of curvatures than trovirdine derivatives and better fit Wing 2 which itself contains some curvature.
In summary, a composite binding pocket was constructed which integrated all available crystal structure information about the NNI binding site of RT. This novel computer-generated model was an unexpectedly effective tool that helped to much better comprehend the flexible nature of the binding pocket and to identify specific areas for structural improvements of the inhibitors. Nine lead NNI compounds from published crystal structures were analyzed. With all strategies combined, a number of previously unknown candidate sites for developing more potent derivatives of PETT were identified, such as substituting a bulkier piperidinyl group or an ortho/meta substituted phenyl group in place of an unsubstituted ring which resulted in enhanced inhibitory activity. The presented experimental results demonstrate that two novel PETT derivatives which resulted from our structure-based design efforts using the composite binding pocket are remarkably potent and noncytotoxic anti-HIV agents with unprecedented selectivity indices of>10.sup.5. The superior activity of these designed PETT compounds would not have been predictable from existing information about trovirdine alone, or from any single crystal structure of RT complexed with an NNI.
Example 7
Structure-based Design and Docking of PETT Derivatives into Composite NNI Binding Pocket II
The PETT derivatives II, synthesized as described above for Example 4, were analyzed for fit into the NNI binding pocket. Target compounds were also analyzed for anti-viral activity in p24 enzyme immunoassays and also for the ability to inhibit HIV reverse transcriptase activity, using rRT. Methods for these biological assays are described above for Example 6. ##STR24##
Potential Modification Sites for PETT Derivatives
A computer simulation of the binding of the target PETT derivatives into the NNI binding site of RT was accomplished using a molecular docking procedure. Docking of the compounds into the NNI binding site required the use of X-ray coordinates of an RT/NNI complex (in this case the RT/9-Cl-TIBO complex).
Trovirdine derivatives could be viewed as two chemical groups linked together by a thiourea group (Table 6). One half of the molecule is composed of a pyridylthiourea group (compounds II-1-9) which forms an intramolecular hydrogen-bonded heterocyclic ring (shown in trovirdine structure). The other half of the molecule is a pyridyl ring separated from the thiocarbonyl group by an ethyl linker.
When trovirdine was docked into the NNI binding site of RT, it fit into the butterfly-shaped binding region (described by Ding, et al., Nat. Struct. Biol., 1995, 2, 407-415) with one part of the molecule residing in Wing 1 and the other in Wing 2. The ring closest to the thiocarbonyl group resided near the Lys(K)101 loop and the other pyridyl ring was near Trp(W)229.
Compounds II-1-9 were positioned according to this binding mode into the RT/9-Cl-TIBO active site by a docking procedure described above for Example 1. The results are shown in FIG. 6. One of the NH groups of the thiourea part of these compounds consistently formed a hydrogen bond with the backbone of K101.
Once the final, energetically favored docked position of the molecule in the NNI site was determined, a LUDI score was assigned, from which an estimation of the inhibition constant (K.sub.i value) was determined (Table 6). The calculated K.sub.i values, ranging from 0.4 .mu.M to 0.8 .mu.M suggested that compounds II-2-7 would be active inhibitors of RT. The modeling data, shown below in Table 6, predicted that compounds II-2 to II-7 would be as potent as or more potent than trovirdine for inhibiting RT. The data for the bioassay of RT inhibition follows this prediction.
TABLE 6______________________________________Interaction scores, K.sub.i values, and measured IC.sub.50 datafor a series of PETT derivatives. ##STR25## K.sub.i IC.sub.50 IC.sub.50Com- MS.sup.a BS.sup.b LIPO (calc) rRT* p24pound X (.ANG..sup.2) (%) Score (.mu.M).sup.c (.mu.M) (.mu.M) SI.sup.d______________________________________II-1 o- 282 82% 678 1.2 1.0 0.01 >1 .times. OMe 10.sup.4II-2 o-F 281 82% 674 0.8 0.6 <0.001 >1 .times. 10.sup.5II-3 o-Cl 285 83% 694 0.5 0.7 <0.001 >1 .times. 10.sup.5II-4 m- 296 84% 729 0.4 0.4 0.003 >3 .times. OMe 10.sup.4II-5 m-F 282 83% 687 0.6 0.7 <0.001 >1 .times. 10.sup.5II-6 m-Cl 283 81% 672 0.8 3.1 N.D. N.D.II-7 p- 302 83% 734 0.6 0.9 0.015 >6 .times. OMe 10.sup.3II-8 p-F 284 81% 674 7.8 6.4 N.D. N.D.II-9 p-Cl 293 81% 696 4.7 2.5 N.D. N.D.trovirdine N.A. 276 84% 679 0.7 0.8 0.007 >1 .times. 10.sup.4AZT N.A. N.A. N.A. N.A. N.A. >100 0.004 7 .times. 10.sup.3______________________________________ *rRT, recombinant HIV reverse transcriptase assay .sup.a MS, molecular surface area calculated using Connolly's MS program. (Connolly, Science, 1983, 221, 709-713) Defined as boundary of volume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with hard sphere atoms which make up the molecule. Values are slightly smaller than those approximated by Ludi program. .sup.b BS, buried surface: percentage of molecular surface in contact wit protein calculated by Ludi based on docked positions. Based on published crystal structures of RT complexes, our calculation shows that these values could be as low as 77% (in RTHEPT complex) and can be as high as 90% (in RTAPA complex) but most of them average around 84%. .sup.c Ludi K.sub.i values were calculated based on modified empirical score function in the Ludi program as described for Example 1. (Bohm, J. Comput. Aided. Mol. Des., 1994, 8, 243-256; 1996,) Ideal hydrogen bond distances and angles between compounds and protein are assumed in all cases for Ludi Score and K.sub.i calculation. In published crystal structures of RT complexes, hydrogen bond geometries # are indeed close to ideal; the amide carbonyl of residue A101 on a loop demonstrates substantial flexibility which can accommodate the best geometry for hydrogen bonding. The number of rotatable bonds (2, or 2 + n for n methox groups) is used in the Ludi calculation to reflect loss of binding energy due to freezing of internal degrees of freedom. .sup.d SI (selectivity index) = IC.sub.50 [MTA]/IC.sub.50 [p24]). IC.sub.50 [MTA] values were >100 .mu.M for compounds II1-9, as well as trovirdine. IC.sub.50 [MTA] for AZT was 50 .mu.M. N.D., not determined, for compounds with IC.sub.50 [rRT] greater than 1.0 .mu.M. N.A., not applicable.
Example 8
In Vitro Assays of PETT Derivatives II
Methoxy Substitutions
The estimated K.sub.i values accurately predicted the trend of the measured IC.sub.50 [rRT] values for the inhibition of recombinant HIV RT. Compound II-4 had the lowest K.sub.i value. The docking results showed that the meta-methoxy group of II-4 is situated near Pro95 and Trp229 in the binding site, providing contact with these protein residues which cannot be achieved by trovirdine (FIG. 5). Based on the IC.sub.50 [rRT] values of all methoxy compounds, the meta-methoxy substituted compound II-4, which had a K.sub.i value of 0.4 .mu.M, showed greater inhibitory activity against recombinant HIV RT and it was approximately 2-fold more potent than trovirdine (IC.sub.50 [rRT] was 0.4 .mu.M for compound II-4 versus 0.8 .mu.M for trovirdine). Compound II-4 abrogated HIV replication in human peripheral blood mononuclear cells at nanomolar concentrations with an IC.sub.50 value of 3 nM and a selectivity index (SI) of >3.times.10.sup.4 (Table 6).
Fluorine Substitutions
Among the fluorine (F) substituted compounds II-2, II-5, and II-8, both meta and ortho fluoro compounds were at least 7-fold more active than trovirdine (IC.sub.50 [p24]<1 nM) (Table 6). Based on the IC.sub.50 [rRT] values, compounds with F substitutions at the meta and ortho positions had nearly the same inhibitory activity against recombinant HIV RT but the para-F substituted compound was 10-fold less active. The color-coded composite binding pocket (FIG. 5) also shows that Wing 2 is mostly hydrophobic except for the region near the ortho positions on both sides of the phenyl ring where polar groups such as halogen atoms would be compatible. Trovirdine, however, lacks such ring substitutents which could provide favorable interactions with these regions of the binding site based on our modeling. Substitutions at the meta position could be on the polar region or the hydrophobic region depending on the chemical group and its consequent conformational change (FIG. 5). The m-F substituent of compound II-5 is probably exposed to the polar (blue) region and therefore is as active as the o-F group which would also be exposed to the polar region according to our modeling. The trend in IC.sub.50 [rRT] values observed for F-substituted compounds may reflect such a preference. The p-F atom, which is small in size but electronegative, may not be compatible with the location of the ring plane of nearby hydrophobic Trp229 and could contribute to the lower activity. We postulate that this same incompatibility should be observed for any other highly hydrophilic group at the para position, and that an additional binding penalty be imposed to better quantitate such features when undertaking modeling studies.
Chlorine Substitutions
Chlorine (Cl) substituted compounds II-3, II-6, and II-9 show a trend of observed biological activities which differs from that of both the fluorine and methoxy compounds. Like the p-F substituted compound which was less active than other F-substituted compounds, the p-Cl compound was less active than the o-Cl compound based on the IC.sub.50 [rRT] values. Unlike the m-F substituted compound which was as active as the o-F substituted compound, the m-Cl compound was not as active as the o-Cl substituted compound. According to our modeling, o-Cl is the most likely substituent to be situated near a limited polar region at Wing 2, an interaction which would be favorable. The o-Cl compound, like the o-F compound discussed above, was in fact more active than trovirdine, as was predicted by the modeling procedure and by the use of the composite binding pocket.
Hydrophobic Group Preferred At The Para Position
When IC.sub.50 [rRT]values of all compounds with para substitutions are compared (II-7-9), a distinct trend is evident: the p-methoxy (OMe) compound (7) is favored over the p-halogen group compounds (II-8 and II-9) (Table 6). Only the p-OMe substituted PETT derivative, compound II-7, is comparable to trovirdine in its inhibitory activity against recombinant HIV RT. Compound II-7 inhibited HIV replication in peripheral blood mononuclear cells with an IC.sub.50 value of 15 nM (Table 6). This p-OMe preference is consistent with the understanding of the color-coded composite binding pocket at Wing 2, where the binding pocket residues near the para position are relatively hydrophobic. One can reasonably assume, based on chemical intuition and the available inhibition data which is consistent with the modeling, that para substituted hydrophobic groups positioned near a hydrophobic region of the pocket are most preferred, followed by halogens, and finally hydrophilic groups.
Conclusions
In summary, the data revealed the following structure-activity relationships affecting the potency of PETT derivatives with substitutions on various positions of the phenyl ring:
1) methoxy substitution is more favorable at the meta position than at the ortho or para positions;
2) fluorine substitution is favorable at ortho and meta positions but not at the para position;
3) chlorine substitution is favorable only at the ortho position;
4) a hydrophobic group is more desirable than a polar group or hydrophilic group at the para position. These results were generally consistent with predictions made during modeling.
The use of the composite NNI binding pocket allowed the identification and structure-based design of at least 3 promising PETT derivatives with ortho-F (II-2), ortho-Cl (II-3), and meta-F (II-5) substituents on the phenyl ring. These novel PETT derivatives were more active than trovirdine (as predicted) or AZT and showed potent anti-HIV activity with IC.sub.50 [p24] values <1 nM and selectivity indices (SI) of >100,000 (Table 6).
Example 9
Design of Heterocyclic PETT Derivatives III
In the course of the search for potent NNIs, a computer model has been developed in which a composite binding pocket was constructed from nine individual crystal structures of RT-NNI complexes. Modeling studies lead to the identification of a number of NNIs with IC.sub.50 values beyond 1 nM for the inhibition of HIV replication (measured by p24 production in HIV-infected human peripheral blood mononuclear cells) and showed no detectable cytotoxicity against human T-lymphocytes (inhibition of cellular proliferation was >100 .mu.M as measured by MTA).
The detailed analysis of trovirdine, a potent PETT derivative, revealed multiple sites which can be used for the incorporation of larger functional groups. In the composite binding pocket, the docked trovirdine molecule showed a lot of usable space surrounding the pyridyl ring, the ethyl linker and near the 5-bromo position (shown in structure of PETT derivative). It was proposed that efficient use of this space by strategically designed functional groups would lead to high affinity binding and ultimately result in better inhibitors.
______________________________________ ##STR26##III 1 2 3______________________________________X = CH.sub.2 NH O______________________________________
The effect of systematic substitutions of the phenyl ring of trovirdine with various heterocyclic rings was studied. This provides an alternative strategy to fit the compound into the relatively flexible and spacious Wing 2 region (as illustrated by the composite binding pocket). In the subsequent modeling studies these heterocyclic rings, which have a larger volume than the pyridyl ring of trovirdine, were shown to better fill the Wing 2 region of the composite binding pocket.
The piperidinyl, piperzinyl and morpholinyl rings of compounds III-1-3 are puckered and therefore occupy a larger overall volume than the planar pyridyl ring of trovirdine and are in close contact with residues Leu234 and Leu100, the latter of which can mutate to isoleucine, frequently found in a drug-resistant RT mutant strain. The encouraging results from efforts to make modifications perpendicular to the ring plane introduces new possibilities to develop more potent inhibitors of RT.
The heterocyclic rings which are conformationally more flexible than an aromatic ring may have the advantage of fitting an uncompromising binding pocket more effectively, despite the expense paid for loss of entropy upon binding. Various combinations of double substitutions at axial or equatorial positions of these heterocyclic rings would generate derivatives with a broader range of curvatures than trovirdine derivatives and would serve to better fit Wing 2 which itself contains some curvature.
Example 10
In Vitro Assays of Anti-HIV-1 Activity Using PETT Derivatives III
Compounds III-1 to III-3 were tested for anti-HIV activity in HTLVIIIB-infected peripheral blood mononuclear cells. Anti-HIV activity was tested using the p24 immunoassay described above for Example 6. Cytotoxicity was also analyzed using a Microculture tetrazolium Assay (MTA), as described above for Example 6.
The data below in Table 7 show the inhibitory effects of PETT derivatives (compounds III-1-3) on p24 production in HIV-infected peripheral blood mononuclear cells and on viability of peripheral blood mononuclear cells. IC.sub.50 values represent the concentration required to inhibit by 50% the activity of HIV replication as measured by assays of p24 production (IC.sub.50 [p24]) or the concentration required to decrease cellular proliferation by 50% as measured by MTA (IC.sub.50 [MTA]) (Uckun, et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383; Zarling, et al., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag Chemother., 1993, 37, 835).
All three compounds III-1-3 were more potent than trovirdine for inhibitition of HIV. Our lead heterocyclic PETT derivatives, N-[2-(1-piperidinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (compound III-1) and N-[2-(1-morpholinylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (compound 3) elicited potent anti-HIV activity with IC.sub.50 values less than 1 nM for the inhibition of HIV replication (measured by p24 production in HIV-infected human peripheral blood mononuclear cells) and showed no detectable cytotoxicity (inhibition of cellular proliferation was >100 .mu.M as measured by MTA) (Table 7).
TABLE 7______________________________________ ##STR27## IC.sub.50 [p24] IC.sub.50 [MTA]Compound R (.mu.M) (.mu.M) SI______________________________________III-1 piperdinyl <0.001 >100 >1 .times. 10.sup.5III-2 piperazinyl 0.002 >100 >5 .times. 10.sup.4III-3 morpholinyl <0.001 >100 >1 .times. 10.sup.5Trovirdine pyridyl 0.007 >100 >1 .times. 10.sup.4AZT -- 0.004 50 7 .times. 10.sup.3______________________________________
Example 11
"SeeGap" Program for Analysis of Gap Space
To analyze the gap space between the binding pocket and complexed NNI, the "SeeGap" program was developed. The following instructions are for use of the program, whose code is listed below in Table 8:
Preparation:
1. Extract the source codes at the lines indicated. The first program is a C-shell command file and should be named as "SeeGap"; the second program should be named as "pdbmax.f"; the third "gridbox.f" and fourth "chgcolor.f".
2. Compile the source codes: for the first, chmod+x SeeGap; the second, third, and fourth by "f77-o file file.f".
3. You should now have the executive versions of the programs named as "SeeGap", "pdbmax", "gridbox" and "chgcolor". The preparation is ready.
Use the program:
1. Open "insightII" window, and read in the coordinates of the protein and the coordinates of the ligand. Next, assign the potential to both coordinates by builder module within "insightiI" (see insight II manual).
2. Position the ligand in the binding site by a docking procedure, if the position of the ligand is unknown.
3. Using subset/interface command, determine the coordinates of the protein that immediately surround the ligand by a defined distance, e.g., 7 angstroms. Write out the coordinates and name it as "bind.pdb"; write out the coordinates of the ligand and name it as "ligand.pdb".
4. Adjust the input parameters in the command file "SeeGap" as appropriate.
5. Run the program by typing "SeeGap ligand.pdb bind.pdb>out&".
6. The results should be in three files: contact.pdb, which represents the grid points on the surface of the ligand and in contact with the protein residues; gap.pdb, which represents the grid points available for modification; and lig.pdb, which represents the grid points covering the ligand.
7. Use a molecular graphics software to display these coordinates.
TABLE 8A______________________________________C-shell command file "SeeGap"______________________________________#######C-shell command file "SeeGap",## cut below#!/bin/csh# chen mao, Nov. 8, 1997grep "ATOM " $1 >fort.1grep "ATOM " $2 >fort.2# modify expansion value (5.0 A) for the ligand/usr2/mao/local/bin/pdbmax <<eof5.0eof.andgate. modify the grid (1.0 A), too-small-grids may waste time/usr2/mao/local/bin/gridbox <<eof1.0eof# modify the distance cutoff considered to be close/usr2/mao/local/bin/chgcolor <<eof2.0eofgrep " H " fort.30>contact.pdbgrep "END " fort.30>>contact.pdbgrep " N " fort.20>lig.pdbgrep "END " fort.30>>lig.pdbgrep " OH2" fort.30>gap.pdbgrep "END " fort.30>>gap.pdb/bin/rm fort.1 fort.2 fort.30 fort.20______________________________________
TABLE 8B______________________________________Program "pdbmax.f" to Determine Boundaries______________________________________## PROGRAM "pdbmax.f" TO DETERMINED THE BOUNDARY OF###THE COORDINATES, cut below #xmin = 9999.0xmax = -9999.0ymin = 9999.0ymax = -9999.0zmin = 9999.0zmax = -9999.0open(unit = 99, file = "boundary.out", status = "unknown")read (*,*)add20 read(1,'(30x,3f8.3) `,end = 999)x,y,zif (x.lt.xmin) xmin = xif (y.lt.ymin) ymin = yif (z.lt.zmin) zmin = zif (x.gt.xmax) xmax = xif (y.gt.ymax) ymax = yif (z.gt.zmax) zmax = zgo to 201000 format(a4,i7,2x,a1,a2,1x,a3,2x,i4,4x,3f8.3,2f6.2)999 continuewrite(*,'("the extreme of the coordinates are")')write(*,'(6(3x,f6.1))')xmin,xmax,ymin,ymax,zmin,zmaxxmin = xmin - addymin = ymin - addzmin = zmin - addxmax = xmax + addymax = ymax + addzmax = zmax + addwrite(99,'(6(3x,f6.1))')xmin,xmax,ymin,ymax,zmin,zmaxstopend______________________________________
TABLE 8C______________________________________Program "gridbox.f" to Generate Grids______________________________________####### PROGRAM "gridbox.f" TO GENERATE GRIDS FOR THE#BINDING SITE, cut belowCHARACTER*1 ATOM1character*2 ATOM2CHARACTER*4 CHNcharacter*4 RESinteger xs, ys, zsparameter q = .perp..0, w = 0.0write(*,`("step size in A")`)open(unit = 99, file = "boundary.out", status = "old",readonly)read(*,*)stepCHN = `ATOM`RES = `TIP3`ATOM1 = `O`ATOM2 = `H2`ICNTS = 0C read the boundary of the box to generate gridwrite(*,`("six min max values ")`)read (99,*)xmin, xmax, ymin, ymax, zmin, zmaxs = (xmax - xmin)/stepxs = ss = (ymax - ymin)/stepys = ss = (zmax - zmin)/stepzs = sif (xs.lt.0.0.or.ys.lt.0.0.or.zs.lt.0.0) thenwrite(*,`("nonsense input")`)go to 999end ifwrite (*,*)xs,ys,zsinum = xs*ys*zswrite(*,*)inumif (inum.gt.25000) thenwrite(*,`("too many grids")`)go to 999end ifdo 100 n = 1, zsdo 100 m = 1, ysdo 100 l = 1, xsx1 = xmin + float(l) *stepy1 = ymin + float(m) *stepz1 = zmin + float(n) *stepicnts = icnts + 1100 write (10,1000) CHN,ICNTS,ATOM1,ATOM2,RES,1 icnts, x1,y1,z1,Q,W1000 format(A4,I7,2X,A1,A2,1X,A4,I6,3X,3F8.3,2F6.2)C write (10,`("END ")`)999 stopend______________________________________
TABLE 8D______________________________________Program "chgcolor.f" to Determine Contact Area and GAP______________________________________##### PROGRAM "chgcolor.f" TO DETERMINE THE CONTACT## AREA AND GAP, cut below #character*1 atom1, zatom1character*2 atom2, zatom2CHARACTER*4 chn, zchncharacter*4 res, zresinteger iatom, izatom, ires, izresreal u, v, w, q, zq, windex, zwC set for delta distance value, please reviseC parameter da=1.5 write(*,`("distance cutoff")`)C da1 is for hydrogen, da2 for other kindsread (*,*)daC read (*,*)da1, da2100 read (10,1000, end=199) chn,iatom,atom1,atom2,res,1 ires, u,v,w,q,windexrewind 1130 read(1, 1000, end=198) zchn,izatom,zatom1,zatom2,zres,1 izres,x,y,z,zq,zwC if (zatom1.eq."H") thenC da=da1C go to 133C end ifC da=da2133 delx=abs(u-x) dely=abs(v-y) delz=abs(w-z) if(delx.lt.da.and.dely.lt.da.and.delz.lt.da ) then dist=sqrt(delx*delx+dely*dely+delz*delz) if(dist.lt.da) then windex=windex+1.0 atom1="N" atom2=" " go to 198 end if end if go to 130198 write (20,1000)chn,iatom,atom1,atom2,res,1 ires, u,v,w,q,windexgo to 100199 continue rewind 20200 read (20,1000, end=299) chn,iatom,atom1,atom2,res,1 ires, u,v,w,q,windexrewind 2230 read(2, 1000, end=298)zchn,izatom,zatom1,zatom2,zres,1 izres,x,y,z,zq,zwC if (zatom1.eq."H") thenC da=da1C go to 233C end ifC da=da2233 delx=abs(u-x) dely=abs(v-y) delz=abs(w-z) if(delx.lt.da.and.dely.lt.da.and.delz.lt.da ) then dist=sqrt(delx*delx+dely*dely+delz*delz) if(dist.lt.da) then windex=windex+1.0 atom1="C" atom2=" " go to 298 end if end if go to 230298 continueif (windex.eq.2.0) thenatom1="H"atom2=" "end ifwrite (30,1000)chn,iatom,atom1,atom2,res,1 ires, u,v,w,q,windexgo to 200299 continuewrite(30,`("END ")`)stop1000 format(A4,I7,2X,A1,A2,1X,A4,1x,I5,3X,3F8.3,2F6.2)end################______________________________________
TABLE 9______________________________________Coordinates of Composite Binding PocketThese coordinates can be entered into a molecular graphics program togenerate a molecular surface representation of the composite bindingpocket, which then can be used to design and evaluate inhibitors of______________________________________RT.ATOM 1 O H2O 1 144.048 -24.778 68.464 inf infATOM 2 O H2O 2 144.416 -24.592 68.433 inf infATOM 3 O H2O 3 144.416 -24.225 68.423 inf infATOM 4 O H2O 4 143.694 -25.486 68.876 inf infATOM 5 O H2O 5 144.048 -25.306 68.683 inf infATOM 6 O H2O 6 144.749 -25.257 68.756 infATOM 7 O H2O 7 143.349 -24.944 68.703 inf infATOM 8 O H2O 8 144.790 -24.969 68.630 inf infATOM 9 O H2O 9 143.080 -24.603 68.775 inf infATOM 10 O H2O 10 145.130 -24.581 68.682 inf infATOM 11 O H2O 11 143.639 -24.225 68.487 inf infATOM 12 O H2O 12 145.513 -24.404 68.846 inf infATOM 13 O H2O 13 143.655 -23.832 68.549 inf infATOM 14 O H2O 14 145.157 -23.856 68.637 inf infATOM 15 O H2O 15 143.471 -23.455 68.774 inf infATOM 16 O H2O 16 144.786 -23.480 68.619 inf infATOM 17 O H2O 17 143.670 -23.285 68.803 inf infATOM 18 O H2O 18 144.785 -23.149 68.737 inf infATOM 19 O H2O 19 144.417 -22.949 68.853 inf infATOM 20 O H2O 20 143.693 -25.667 69.048 inf infATOM 21 O H2O 21 144.417 -25.702 69.012 inf infATOM 22 O H2O 22 143.280 -25.554 69.161 inf infATOM 23 O H2O 23 145.154 -25.515 69.200 inf infATOM 24 O H2O 24 142.936 -24.965 69.009 inf infATOM 25 O H2O 25 142.683 -24.618 69.149 inf infATOM 26 O H2O 26 142.673 -24.225 69.139 inf infATOM 27 O H2O 27 146.037 -24.225 69.239 inf infATOM 28 O H2O 28 146.042 -23.856 69.233 inf infATOM 29 O H2O 29 145.586 -23.456 68.921 inf infATOM 30 O H2O 30 143.152 -23.144 69.225 inf infATOM 31 O H2O 31 145.515 -23.125 69.025 inf infATOM 32 O H2O 32 143.661 -22.890 69.155 inf infATOM 33 O H2O 33 144.786 -22.742 69.007 inf infATOM 34 O H2O 34 144.063 -22.602 69.236 inf infATOM 35 O H2O 35 144.048 -26.097 69.620 inf infATOM 36 O H2O 36 144.417 -25.997 69.413 inf infATOM 37 O H2O 37 143.287 -25.730 69.365 inf infATOM 38 O H2O 38 145.148 -25.868 69.584 inf infATOM 39 O H2O 39 142.892 -25.364 69.350 inf infATOM 40 O H2O 40 142.606 -25.130 69.584 inf infATOM 41 O H2O 41 145.857 -25.125 69.596 inf infATOM 42 O H2O 42 145.964 -24.629 69.323 inf infATOM 43 O H2O 43 146.208 -24.258 69.503 inf infATOM 44 O H2O 44 142.554 -23.662 69.558 inf infATOM 45 O H2O 45 142.828 -23.175 69.610 inf infATOM 46 O H2O 46 143.260 -22.858 69.517 inf infATOM 47 O H2O 47 145.718 -22.739 69.559 inf infATOM 48 O H2O 48 143.886 -22.425 69.590 inf infATOM 49 O H2O 49 144.975 -22.345 69.548 inf infATOM 50 O H2O 50 144.786 -22.277 69.595 inf infATOM 51 O H2O 51 144.048 -26.251 69.938 inf infATOM 52 O H2O 52 144.994 -26.125 69.920 inf infATOM 53 O H2O 53 145.525 -25.701 69.751 inf infATOM 54 O H2O 54 142.858 -25.603 69.941 inf infATOM 55 O H2O 55 142.410 -24.956 69.939 inf infATOM 56 O H2O 56 146.247 -24.586 69.759 inf infATOM 57 O H2O 57 146.322 -24.242 69.726 inf infATOM 58 O H2O 58 146.447 -23.856 69.936 inf infATOM 59 O H2O 59 146.368 -23.509 69.971 inf infATOM 60 O H2O 60 146.277 -23.296 69.932 inf infATOM 61 O H2O 61 145.876 -22.762 69.762 inf infATOM 62 O H2Q 62 143.833 -22.310 69.916 inf infATOM 63 O H2O 63 145.829 -22.628 69.962 inf infATOM 64 O H2O 64 145.143 -22.230 69.948 inf infATOM 65 O H2O 65 144.048 -26.591 70.339 inf infATOM 66 O H2O 66 144.605 -26.461 70.287 inf infATOM 67 O H2O 67 144.849 -26.350 70.242 inf infATOM 68 O H2O 68 143.010 -25.838 70.326 inf infATOM 69 O H2O 69 145.844 -25.653 70.169 inf infATOM 70 O H2O 70 142.505 -25.253 70.305 inf infATOM 71 O H2O 71 146.408 -25.313 70.366 inf infATOM 72 O H2O 72 142.287 -24.619 70.305 inf infATOM 73 O H2O 73 142.270 -24.225 70.305 inf infATOM 74 O H2O 74 146.581 -23.856 70.155 inf infATOM 75 O H2O 75 146.640 -23.667 70.298 inf infATOM 76 O H2O 76 146.387 -23.165 70.341 inf infATOM 77 O H2O 77 146.235 -22.946 70.319 inf infATOM 78 O H2O 78 145.533 -22.364 70.118 inf infATOM 79 O H2O 79 144.038 -22.156 70.305 inf infATOM 80 O H2O 80 145.471 -22.274 70.333 inf infATOM 81 O H2O 81 144.048 -27.016 70.623 inf infATOM 82 O H2O 82 144.634 -26.841 70.626 inf infATOM 83 O H2O 83 144.819 -26.507 70.435 inf infATOM 84 O H2O 84 145.332 -26.427 70.685 inf infATOM 85 O H2O 85 145.880 -26.228 70.717 inf infATOM 86 O H2O 86 142.907 -25.909 70.653 inf infATOM 87 O H2O 87 146.588 -25.657 70.623 inf infATOM 88 O H2O 88 147.374 -25.700 70.660 inf infATOM 89 O H2O 89 148.108 -25.686 70.594 inf infATOM 90 O H2O 90 142.531 -25.283 70.673 inf infATOM 91 O H2O 91 147.001 -25.530 70.644 inf infATOM 92 O H2O 92 148.427 -25.333 70.643 inf infATOM 93 O H2O 93 146.982 -24.943 70.558 inf infATOM 94 O H2O 94 148.109 -25.140 70.625 inf infATOM 95 O H2O 95 147.195 -24.587 70.651 inf infATOM 96 O H2O 96 147.177 -24.225 70.696 inf infATOM 97 O H2O 97 142.471 -23.515 70.647 inf infATOM 98 O H2O 98 142.595 -23.318 70.666 inf infATOM 99 O H2O 99 142.934 -22.926 70.677 inf infATOM 100 O H2O 100 146.583 -22.969 70.735 inf infATOM 101 O H2O 101 146.022 -22.436 70.730 inf infATOM 102 O H2O 102 144.417 -22.087 70.674 inf infATOM 103 O H2O 103 145.844 -22.277 70.742 inf infATOM 104 O H2O 104 144.233 -27.553 71.039 inf infATOM 105 O H2O 105 143.655 -27.432 70.974 inf infATOM 106 O H2O 106 144.442 -27.438 70.968 inf infATOM 107 O H2O 107 142.971 -26.975 71.068 inf infATOM 108 O H2O 108 144.850 -26.872 70.763 inf infATOM 109 O H2O 109 142.790 -26.440 71.066 inf infATOM 110 O H2O 110 145.888 -26.614 71.059 inf infATOM 111 O H2O 111 147.185 -26.441 71.041 inf infATOM 112 O H2O 112 148.109 -26.648 71.020 inf infATOM 113 O H2O 113 148.669 -26.449 71.032 inf infATOM 114 O H2O 114 146.285 -26.324 70.974 inf infATOM 115 O H2O 115 147.001 -26.084 70.828 inf infATOM 116 O H2O 116 148.503 -26.108 70.772 inf infATOM 117 O H2O 117 142.649 -25.772 70.972 inf infATOM 118 O H2O 118 142.535 -25.326 71.039 inf infATOM 119 O H2O 119 142.463 -24.937 71.041 inf infATOM 120 O H2O 120 148.837 -24.973 70.888 inf infATOM 121 O H2O 121 147.762 -24.573 70.772 inf infATOM 122 O H2O 122 149.033 -24.594 71.039 inf infATOM 123 O H2O 123 148.108 -24.225 70.852 inf infATOM 124 O H2O 124 142.459 -23.880 71.019 inf infATOM 125 O H2O 125 148.477 -23.866 70.928 inf infATOM 126 O H2O 126 142.550 -23.661 71.054 inf infATOM 127 O H2O 127 147.710 -23.518 70.952 inf infATOM 128 O H2O 128 148.845 -23.672 71.048 inf infATOM 129 O H2O 129 147.390 -23.272 70.974 inf infATOM 130 O H2O 130 143.004 -22.996 71.018 inf infATOM 131 O H2O 131 147.021 -22.918 71.009 inf infATOM 132 O H2O 132 143.843 -22.331 71.057 inf infATOM 133 O H2O 133 144.057 -22.209 71.039 inf infATOM 134 O H2O 134 145.155 -22.003 70.856 inf infATOM 135 O H2O 135 146.253 -22.218 71.067 inf infATOM 136 O H2O 136 145.894 -21.890 71.108 inf infATOM 137 O H2O 137 143.673 -27.752 71.404 inf infATOM 138 O H2O 138 144.425 -27.759 71.401 inf infATOM 139 O H2O 139 142.960 -27.339 71.427 inf infATOM 140 O H2O 140 145.148 -27.353 71.418 inf infATOM 141 O H2O 141 145.550 -27.062 71.366 inf infATOM 142 O H2O 142 146.233 -26.749 71.320 inf infATOM 143 O H2O 143 147.403 -26.893 71.480 inf infATOM 144 O H2O 144 147.735 -26.822 71.219 inf infATOM 145 O H2O 145 148.468 -26.781 71.247 inf infATOM 146 O H2O 146 142.643 -26.440 71.276 inf infATOM 147 O H2O 147 147.003 -26.730 71.337 inf infATOM 148 O H2O 148 142.446 -26.051 71.452 inf infATOM 149 O H2O 149 149.369 -26.060 71.434 inf infATOM 150 O H2O 150 149.447 -25.719 71.367 inf infATOM 151 O H2O 151 142.424 -24.951 71.422 inf infATOM 152 O H2O 152 149.685 -24.933 71.469 inf infATOM 153 O H2O 153 149.734 -24.594 71.448 inf infATOM 154 O H2O 154 149.268 -24.225 71.124 inf infATOM 155 O H2O 155 142.731 -23.841 71.448 inf infATOM 156 O H2O 156 142.812 -23.520 71.368 inf infATOM 157 O H2O 157 149.748 -23.478 71.434 inf infATOM 158 O H2O 158 147.423 -23.050 71.114 inf infATOM 159 O H2O 159 148.867 -23.117 71.149 inf infATOM 160 O H2O 160 143.329 -22.764 71.216 inf infATOM 161 O H2O 161 147.365 -22.759 71.245 inf infATOM 162 O H2O 162 148.847 -22.748 71.181 inf infATOM 163 O H2O 163 143.692 -22.585 71.396 inf infATOM 164 O H2O 164 147.183 -22.382 71.418 inf infATOM 165 O H2O 165 148.288 -22.374 71.403 inf infATOM 166 O H2O 166 149.548 -22.416 71.339 inf infATOM 167 O H2O 167 144.299 -22.076 71.413 inf infATOM 168 O H2O 168 146.991 -22.215 71.443 inf infATOM 169 O H2O 169 149.576 -22.203 71.431 inf infATOM 170 O H2O 170 145.001 -21.694 71.453 inf infATOM 171 O H2O 171 146.443 -21.649 71.443 inf infATOM 172 O H2O 172 145.894 -21.452 71.403 inf infATOM 173 O H2O 173 143.692 -27.877 71.623 inf infATOM 174 O H2O 174 144.406 -27.883 71.619 inf infATOM 175 O H2O 175 144.818 -27.779 71.750 inf infATOM 176 O H2O 176 142.717 -27.204 71.769 inf infATOM 177 O H2O 177 145.720 -27.202 71.768 inf infATOM 178 O H2O 178 145.935 -27.086 71.728 inf infATOM 179 O H2O 179 147.001 -26.972 71.791 inf infATOM 180 O H2O 180 148.495 -27.054 71.756 inf infATOM 181 O H2O 181 142.384 -26.441 71.781 inf infATOM 182 O H2O 182 142.297 -26.096 71.743 inf infATOM 183 O H2O 183 149.650 -25.913 71.745 inf infATOM 184 O H2O 184 149.804 -25.332 71.759 inf infATOM 185 O H2O 185 142.522 -24.758 71.783 inf infATOM 186 O H2O 186 150.044 -24.594 71.845 inf infATOM 187 O H2O 187 142.920 -23.846 71.616 inf infATOM 188 O H2O 188 150.094 -23.840 71.811 inf infATOM 189 O H2O 189 150.140 -23.487 71.781 inf infATOM 190 O H2O 190 149.996 -23.128 71.524 inf infATOM 191 O H2O 191 143.870 -22.754 71.775 inf infATOM 192 O H2O 192 144.036 -22.552 71.787 inf infATOM 193 O H2O 193 148.080 -22.338 71.501 inf infATOM 194 O H2O 194 147.010 -21.997 71.566 inf infATOM 195 O H2O 195 148.458 -21.971 71.538 inf infATOM 196 O H2O 196 149.817 -21.962 71.710 inf infATOM 197 O H2O 197 144.643 -21.670 71.794 inf infATOM 198 O H2O 198 147.377 -21.815 71.758 inf infATOM 199 O H2O 199 148.660 -21.627 71.771 inf infATOM 200 O H2O 200 149.604 -21.778 71.734 inf infATOM 201 O H2O 201 145.510 -21.250 71.547 inf infATOM 202 O H2O 202 146.868 -21.251 71.710 inf infATOM 203 O H2O 203 145.161 -21.090 71.791 inf infATOM 204 O H2O 204 146.261 -20.905 71.603 inf infATOM 205 O H2O 205 145.710 -20.536 71.791 inf infATOM 206 O H2O 206 146.621 -20.740 71.815 inf infATOM 207 O H2O 207 143.707 -28.248 72.013 inf infATOM 208 O H2O 208 144.405 -28.256 71.996 inf infATOM 209 O H2O 209 143.294 -27.935 71.947 inf infATOM 210 O H2O 210 142.946 -27.729 72.153 inf infATOM 211 O H2O 211 145.390 -27.597 72.111 inf infATOM 212 O H2O 212 145.884 -27.333 72.171 inf infATOM 213 O H2O 213 147.742 -27.170 71.967 inf infATOM 214 O H2O 214 142.440 -26.773 72.151 inf infATOM 215 O H2O 215 147.002 -27.056 72.135 inf infATOM 216 O H2O 216 149.074 -26.861 72.124 inf infATOM 217 O H2O 217 149.521 -26.560 72.216 inf infATOM 218 O H2O 218 142.208 -26.069 71.967 inf infATOM 219 O H2O 219 142.199 -25.701 71.966 inf infATOM 220 O H2O 220 142.187 -25.515 72.147 inf infATOM 221 O H2O 221 142.397 -24.970 72.151 inf infATOM 222 O H2O 222 142.720 -24.572 72.153 inf infATOM 223 O H2O 223 143.061 -24.180 72.171 inf infATOM 224 O H2O 224 143.358 -23.534 71.918 inf infATOM 225 O H2O 225 150.315 -23.667 72.155 inf infATOM 226 O H2O 226 143.910 -23.165 72.103 inf infATOM 227 O H2O 227 144.088 -22.957 72.119 inf infATOM 228 O H2O 228 144.267 -22.388 72.138 inf infATOM 229 O H2O 229 144.380 -22.178 72.162 inf infATOM 230 O H2O 230 150.348 -22.182 72.139 inf infATOM 231 O H2O 231 148.108 -21.617 71.951 inf infATOM 232 O H2O 232 150.013 -21.767 72.104 inf infATOM 233 O H2O 233 147.340 -21.307 72.000 inf infATOM 234 O H2O 234 148.473 -21.440 72.140 inf infATOM 235 O H2O 235 144.704 -20.904 72.218 inf infATOM 236 O H2O 236 147.177 -20.908 72.158 inf infATOM 237 O H2O 237 145.147 -20.533 71.955 inf infATOM 238 O H2O 238 146.825 -20.525 72.144 inf infATOM 239 O H2O 239 144.833 -20.164 72.106 inf infATOM 240 O H2O 240 146.241 -20.189 72.032 inf infATOM 241 O H2O 241 144.952 -19.783 72.107 inf infATOM 242 O H2O 242 146.216 -19.842 72.107 inf infATOM 243 O H2O 243 145.525 -19.468 72.091 inf infATOM 244 O H2O 244 145.524 -19.285 72.215 inf infATOM 245 O H2O 245 144.048 -28.821 72.532 inf infATOM 246 O H2O 246 144.620 -28.691 72.489 inf infATOM 247 O H2O 247 144.840 -28.339 72.255 inf infATOM 248 O H2O 248 145.273 -28.245 72.573 inf infATOM 249 O H2O 249 145.206 -27.957 72.285 inf infATOM 250 O H2O 250 145.561 -27.779 72.473 inf infATOM 251 O H2O 251 142.595 -27.218 72.480 inf infATOM 252 O H2O 252 146.633 -27.181 72.334 inf infATOM 253 O H2O 253 147.370 -27.155 72.339 inf infATOM 254 O H2O 254 142.416 -26.796 72.520 inf infATOM 255 O H2O 255 149.241 -26.847 72.309 inf infATOM 256 O H2O 256 149.756 -26.795 72.547 inf infATOM 257 O H2O 257 150.146 -26.445 72.502 inf infATOM 258 O H2O 258 150.259 -26.038 72.429 inf infATOM 259 O H2O 259 150.293 -25.686 72.382 inf infATOM 260 O H2O 260 150.311 -25.332 72.353 inf infATOM 261 O H2O 261 150.496 -24.963 72.533 inf infATOM 262 O H2O 262 150.406 -24.631 72.557 inf infATOM 263 O H2O 263 150.332 -24.408 72.517 inf infATOM 264 O H2O 264 150.307 -23.852 72.338 inf infATOM 265 O H2O 265 143.671 -23.664 72.523 inf infATOM 266 O H2O 266 144.054 -23.308 72.517 inf infATOM 267 O H2O 267 150.636 -22.748 72.488 inf infATOM 268 O H2O 268 150.564 -22.365 72.506 inf infATOM 269 O H2O 269 144.546 -21.640 72.520 inf infATOM 270 O H2O 270 144.506 -21.295 72.521 inf infATOM 271 O H2O 271 148.847 -21.270 72.335 inf infATOM 272 O H2O 272 149.923 -21.503 72.536 inf infATOM 273 O H2O 273 147.750 -21.063 72.513 inf infATOM 274 O H2O 274 149.215 -21.213 72.561 inf infATOM 275 O H2O 275 144.701 -20.533 72.216 inf infATOM 276 O H2O 276 144.291 -20.164 72.559 inf infATOM 277 O H2O 277 147.001 -20.349 72.520 inf infATOM 278 O H2O 278 146.596 -19.809 72.371 inf infATOM 279 O H2O 279 144.782 -19.424 72.329 inf infATOM 280 O H2O 280 146.486 -19.395 72.481 inf infATOM 281 O H2O 281 145.159 -19.062 72.347 inf infATOM 282 O H2O 282 146.294 -19.210 72.473 inf infATOM 283 O H2O 283 145.525 -18.765 72.451 inf infATOM 284 O H2O 284 145.524 -18.548 72.587 inf infATOM 285 O H2O 285 143.655 -28.924 72.853 inf infATOM 286 O H2O 286 144.789 -28.850 72.884 inf infATOM 287 O H2O 287 142.895 -28.315 72.675 inf infATOM 288 O H2O 288 145.572 -27.954 72.657 inf infATOM 289 O H2O 289 142.485 -27.547 72.922 inf infATOM 290 O H2O 290 146.244 -27.683 72.938 inf infATOM 291 O H2O 291 146.672 -27.456 72.863 inf infATOM 292 O H2O 292 147.551 -27.151 72.889 inf infATOM 293 O H2O 293 148.476 -27.172 72.705 inf infATOM 294 O H2O 294 149.218 -27.105 72.738 inf infATOM 295 O H2O 295 148.109 -27.148 72.891 inf infATOM 296 O H2O 296 149.954 -27.067 72.816 inf infATOM 297 O H2O 297 142.307 -26.459 72.889 inf infATOM 298 O H2O 298 150.882 -26.444 72.884 inf infATOM 299 O H2O 299 151.038 -26.047 72.773 inf infATOM 300 O H2O 300 142.238 -25.701 72.926 inf infATOM 301 O H2O 301 142.319 -25.313 72.918 inf infATOM 302 O H2O 302 142.449 -25.005 72.868 inf infATOM 303 O H2O 303 142.596 -24.804 72.879 inf infATOM 304 O H2O 304 142.983 -24.473 72.868 inf infATOM 305 O H2O 305 150.375 -24.223 72.681 inf infATOM 306 O H2O 306 143.829 -23.829 72.923 inf infATOM 307 O H2O 307 144.187 -23.457 72.919 inf infATOM 308 O H2O 308 144.433 -22.753 72.702 inf infATOM 309 O H2O 309 144.507 -22.378 72.891 inf infATOM 310 O H2O 310 144.506 -21.640 72.865 inf infATOM 311 O H2O 311 149.407 -21.230 72.889 inf infATOM 312 O H2O 312 144.269 -20.878 72.901 inf infATOM 313 O H2O 313 148.093 -21.141 72.906 inf infATOM 314 O H2O 314 144.164 -20.553 72.860 inf infATOM 315 O H2O 315 147.019 -20.158 72.693 inf infATOM 316 O H2O 316 147.029 -19.967 72.873 inf infATOM 317 O H2O 317 146.823 -19.421 72.885 inf infATOM 318 O H2O 318 146.568 -19.105 72.785 inf infATOM 319 O H2O 319 144.769 -18.654 72.654 inf infATOM 320 O H2O 320 146.585 -18.900 72.927 inf infATOM 321 O H2O 321 144.967 -18.297 72.871 inf infATOM 322 O H2O 322 146.251 -18.515 72.901 inf infATOM 323 O H2O 323 143.679 -29.093 73.301 inf infATOM 324 O H2O 324 144.048 -29.055 73.069 inf infATOM 325 O H2O 325 144.770 -28.976 73.106 inf infATOM 326 O H2O 326 142.958 -28.633 73.087 inf infATOM 327 O H2O 327 145.380 -28.694 73.227 inf infATOM 328 O H2O 328 145.577 -28.329 73.048 inf infATOM 329 O H2O 329 142.521 -27.931 73.052 inf infATOM 330 O H2O 330 142.378 -27.547 73.255 inf infATOM 331 O H2O 331 146.704 -27.506 73.258 inf infATOM 332 O H2O 332 148.291 -27.190 73.258 inf infATOM 333 O H2O 333 149.222 -27.345 73.264 inf infATOM 334 O H2O 334 149.954 -27.252 72.999 inf infATOM 335 O H2O 335 142.337 -26.809 73.259 inf infATOM 336 O H2O 336 150.742 -26.848 73.025 inf infATOM 337 O H2O 337 151.074 -26.450 73.061 inf infATOM 338 O H2O 338 151.404 -26.061 73.092 inf infATOM 339 O H2O 339 151.452 -25.701 73.059 inf infATOM 340 O H2O 340 151.507 -25.368 73.295 inf infATOM 341 O H2O 341 151.051 -24.974 73.084 inf infATOM 342 O H2O 342 142.913 -24.761 73.277 inf infATOM 343 O H2O 343 151.019 -24.821 73.275 inf infATOM 344 O H2O 344 143.838 -24.201 73.278 inf infATOM 345 O H2O 345 144.025 -24.018 73.276 inf infATOM 346 O H2O 346 150.577 -23.486 73.276 inf infATOM 347 O H2O 347 150.615 -23.144 73.285 inf infATOM 348 O H2O 348 150.557 -22.367 73.271 inf infATOM 349 O H2O 349 150.114 -21.665 73.249 inf infATOM 350 O H2O 350 144.393 -21.278 73.063 inf infATOM 351 O H2O 351 144.186 -20.933 73.243 inf infATOM 352 O H2O 352 148.455 -21.162 73.234 inf infATOM 353 O H2O 353 143.997 -20.489 73.302 inf infATOM 354 O H2O 354 147.700 -20.766 73.287 inf infATOM 355 O H2O 355 147.358 -20.355 73.264 inf infATOM 356 O H2O 356 147.111 -19.822 73.284 inf infATOM 357 O H2O 357 147.031 -19.598 73.250 inf infATOM 358 O H2O 358 144.017 -18.857 73.243 inf infATOM 359 O H2O 359 144.347 -18.433 73.203 inf infATOM 360 O H2O 360 146.418 -18.342 73.276 inf infATOM 361 O H2O 361 145.524 -18.014 73.198 inf infATOM 362 O H2O 362 143.104 -29.065 73.593 inf infATOM 363 O H2O 363 144.417 -29.211 73.626 inf infATOM 364 O H2O 364 145.333 -29.010 73.634 inf infATOM 365 O H2O 365 142.896 -28.894 73.572 inf infATOM 366 O H2O 366 142.329 -28.332 73.570 inf infATOM 367 O H2O 367 142.209 -28.100 73.633 inf infATOM 368 O H2O 368 142.135 -27.587 73.705 inf infATOM 369 O H2O 369 146.556 -27.657 73.627 inf infATOM 370 O H2O 370 149.585 -27.526 73.452 inf infATOM 371 O H2O 371 142.225 -27.366 73.640 inf infATOM 372 O H2O 372 147.329 -27.239 73.631 inf infATOM 373 O H2O 373 148.110 -27.171 73.444 inf infATOM 374 O H2O 374 150.381 -27.477 73.599 inf infATOM 375 O H2O 375 142.298 -26.809 73.606 inf infATOM 376 O H2O 376 151.190 -26.771 73.646 inf infATOM 377 O H2O 377 151.474 -26.274 73.627 inf infATOM 378 O H2O 378 142.560 -25.327 73.448 inf infATOM 379 O H2O 379 142.899 -24.929 73.464 inf infATOM 380 O H2O 380 151.445 -25.138 73.627 inf infATOM 381 O H2O 381 143.651 -24.539 73.491 inf infATOM 382 O H2O 382 144.023 -24.206 73.468 inf infATOM 383 O H2O 383 150.752 -24.370 73.668 inf infATOM 384 O H2O 384 144.417 -23.671 73.628 inf infATOM 385 O H2O 385 150.501 -23.117 73.625 inf infATOM 386 O H2O 386 150.448 -22.399 73.607 inf infATOM 387 O H2O 387 150.328 -22.007 73.445 inf infATOM 388 O H2O 388 149.971 -21.620 73.455 inf infATOM 389 O H2O 389 148.882 -21.317 73.662 inf infATOM 390 O H2O 390 149.581 -21.467 73.620 inf infATOM 391 O H2O 391 148.436 -21.223 73.625 inf infATOM 392 O H2O 392 147.726 -20.728 73.627 inf infATOM 393 O H2O 393 143.766 -19.820 73.603 inf infATOM 394 O H2O 394 147.031 -19.417 73.430 inf infATOM 395 O H2O 395 147.037 -19.236 73.617 inf infATOM 396 O H2O 396 144.068 -18.517 73.634 inf infATOM 397 O H2O 397 146.682 -18.461 73.619 inf infATOM 398 O H2O 398 144.965 -17.912 73.617 inf infATOM 399 O H2O 399 146.060 -17.991 73.640 inf infATOM 400 O H2O 400 146.632 -33.227 74.059 inf infATOM 401 O H2O 401 145.905 -32.888 74.020 inf infATOM 402 O H2O 402 146.279 -32.707 73.869 inf infATOM 403 O H2O 403 147.184 -32.714 73.999 inf infATOM 404 O H2O 404 146.632 -32.346 73.829 inf infATOM 405 O H2O 405 146.053 -31.960 73.939 inf infATOM 406 O H2O 406 147.180 -31.981 74.010 inf infATOM 407 O H2O 407 143.310 -29.504 74.022 inf infATOM 408 O H2O 408 143.679 -29.392 73.812 inf infATOM 409 O H2O 409 144.417 -29.333 73.852 inf infATOM 410 O H2O 410 145.100 -29.337 73.867 inf infATOM 411 O H2O 411 142.614 -28.981 73.855 inf infATOM 412 O H2O 412 145.487 -29.185 74.026 inf infATOM 413 O H2O 413 142.255 -28.613 73.844 inf infATOM 414 O H2O 414 145.933 -28.503 73.976 inf infATOM 415 O H2O 415 146.257 -28.094 74.002 inf infATOM 416 O H2O 416 146.799 -27.521 74.005 inf infATOM 417 O H2O 417 149.597 -27.646 73.997 inf infATOM 418 O H2O 418 150.489 -27.502 73.997 inf infATOM 419 O H2O 419 147.350 -27.300 74.017 inf infATOM 420 O H2O 420 147.920 -27.149 73.996 inf infATOM 421 O H2O 421 150.704 -27.380 73.997 inf infATOM 422 O H2O 422 148.107 -27.138 73.998 inf infATOM 423 O H2O 423 151.377 -26.473 73.997 inf infATOM 424 O H2O 424 142.586 -25.709 73.804 inf infATOM 425 O H2O 425 142.889 -25.291 73.864 inf infATOM 426 O H2O 426 143.287 -24.929 73.835 inf infATOM 427 O H2O 427 151.393 -25.172 73.984 inf infATOM 428 O H2O 428 151.028 -24.829 73.979 inf infATOM 429 O H2O 429 150.643 -24.442 73.980 inf infATOM 430 O H2O 430 150.377 -23.898 73.952 inf infATOM 431 O H2O 431 150.360 -23.487 73.828 inf infATOM 432 O H2O 432 144.583 -22.748 73.997 inf infATOM 433 O H2O 433 150.148 -22.375 74.006 inf infATOM 434 O H2O 434 150.009 -22.167 74.042 inf infATOM 435 O H2O 435 149.638 -21.773 74.039 inf infATOM 436 O H2O 436 148.639 -21.306 73.985 inf infATOM 437 O H2O 437 147.933 -20.892 73 993 inf infATOM 438 O H2O 438 144.104 -20.681 73.977 inf infATOM 439 O H2O 439 147.510 -20.183 73:997 inf infATOM 440 O H2O 440 143.701 -19.426 73.997 inf infATOM 441 O H2O 441 147.048 -19.102 73.997 inf infATOM 442 O H2O 442 144.040 -18.498 73.997 inf infATOM 443 O H2O 443 144.417 -18.134 73.997 inf infATOM 444 O H2O 444 146.313 -18.058 73.973 inf infATOM 445 O H2O 445 145.525 -17.795 74.005 inf infATOM 446 O H2O 446 145.950 -33.378 74.276 inf infATOM 447 O H2O 447 146.632 -33.439 74.203 inf infATOM 448 O H2O 448 145.564 -33.239 74.415 inf infATOM 449 O H2O 449 146.228 -33.152 74.044 inf infATOM 450 O H2O 450 147.550 -33.080 74.370 inf infATOM 451 O H2O 451 147.629 -32.740 74.292 inf infATOM 452 O H2O 452 147.657 -32.346 74.263 inf infATOM 453 O H2O 453 145.534 -31.982 74.195 inf infATOM 454 O H2O 454 147.742 -32.161 74.363 inf infATOM 455 O H2O 455 146.233 -31.549 74.064 inf infATOM 456 O H2O 456 147.537 -31.618 74.380 inf infATOM 457 O H2O 457 146.263 -31.252 74.213 inf infATOM 458 O H2O 458 147.360 -31.433 74.376 inf infATOM 459 O H2O 459 146.079 -30.868 74.361 inf infATOM 460 O H2O 460 145.894 -30.688 74.389 inf infATOM 461 O H2O 461 144.803 -29.908 74.419 inf infATOM 462 O H2O 462 145.517 -29.942 74.392 inf infATOM 463 O H2O 463 142.742 -29.414 74.359 inf infATOM 464 O H2O 464 144.038 -29.641 74.329 inf infATOM 465 O H2O 465 142.522 -29.258 74.346 inf infATOM 466 O H2O 466 142.029 -28.647 74.369 inf infATOM 467 O H2O 467 141.888 -28.318 74.333 inf infATOM 468 O H2O 468 141.828 -28.102 74.364 inf infATOM 469 O H2O 469 141.799 -27.597 74.416 inf infATOM 470 O H2O 470 146.939 -27.655 74.412 inf infATOM 471 O H2O 471 149.393 -27.598 74.366 inf infATOM 472 O H2O 472 141.868 -27.378 74.376 inf infATOM 473 O H2O 473 148.109 -27.149 74.185 inf infATOM 474 O H2O 474 149.148 -27.501 74.404 inf infATOM 475 O H2O 475 142.122 -26.878 74.401 inf infATOM 476 O H2O 476 142.357 -26.423 74.362 inf infATOM 477 O H2O 477 151.408 -26.071 74.406 inf infATOM 478 O H2O 478 1S1.467 -25.701 74.197 inf infATOM 479 O H2O 479 151.398 -25.349 74.165 inf infATOM 480 O H2O 480 151.142 -25.091 74.387 inf infATOM 481 O H2O 481 143.987 -24.717 74.415 inf infATOM 482 O H2O 482 150.699 -24.589 74.182 inf infATOM 483 O H2O 483 150.364 -24.199 74.209 inf infATOM 484 O H2O 484 150.263 -23.873 74.140 inf infATOM 485 O H2O 485 150.049 -23.117 74.307 inf infATOM 486 O H2O 486 149.850 -22.354 74.410 inf infATOM 487 O H2O 487 149.741 -22.038 74.354 inf infATOM 488 O H2O 488 144.429 -21.268 74.180 inf infATOM 489 O H2O 489 148.486 -21.257 74.184 inf infATOM 490 O H2O 490 144.383 -21.102 74.366 inf infATOM 491 O H2O 491 144.122 -20.597 74.382 inf infATOM 492 O H2O 492 143.992 -20.386 74.384 inf infATOM 493 O H2O 493 147.400 -19.967 74.366 inf infATOM 494 O H2O 494 147.102 -19.084 74.392 inf infATOM 495 O H2O 495 144.063 -18.513 74.361 inf infATOM 496 O H2O 496 144.606 -17.960 74.369 inf infATOM 497 O H2O 497 144.816 -17.851 74.396 inf infATOM 498 O H2O 498 146.240 -17.802 74.395 inf infATOM 499 O H2O 499 146.631 -33.922 74.791 inf infATOM 500 O H2O 500 147.185 -33.821 74.737 inf infATOM 501 O H2O 501 145.852 -33.700 74.693 inf infATOM 502 O H2O 502 145.259 -33.138 74.681 inf infATOM 503 O H2O 503 145.124 -32.910 74.714 inf infATOM 504 O H2O 504 147.981 -32.716 74.697 inf infATOM 505 O H2O 505 144.951 -31.984 74.721 inf infATOM 506 O H2O 506 144.918 -31.629 74.662 inf infATOM 507 O H2O 507 144.780 -31.424 74.728 inf infATOM 508 O H2O 508 147.361 -31.248 74.560 inf infATOM 509 O H2O 509 144.868 -30.868 74.673 inf infATOM 510 O H2O 510 146.643 -30.853 74.512 inf infATOM 511 O H2O 511 144.618 -30.492 74.760 inf infATOM 512 O H2O 512 145.919 -30.476 74.452 inf infATOM 513 O H2O 513 146.961 -30.715 74.785 inf infATOM 514 O H2O 514 144.787 -30.130 74.553 inf infATOM 515 O H2O 515 146.222 -30.173 74.672 inf infATOM 516 O H2O 516 143.514 -29.717 74.735 inf infATOM 517 O H2O 517 145.875 -29.762 74.579 inf infATOM 518 O H2O 518 142.953 -29.549 74.735 inf infATOM 519 O H2O 519 142.311 -29.099 74.735 inf infATOM 520 O H2O 520 142.149 -28.876 74.735 inf infATOM 521 O H2O 521 141.846 -28.328 74.735 inf infATOM 522 O H2O 522 146.679 -27.955 74.528 inf infATOM 523 O H2O 523 147.020 -27.761 74.716 inf infATOM 524 O H2O 524 149.954 -27.692 74.735 inf infATOM 525 O H2O 525 141.934 -27.136 74.735 inf infATOM 526 O H2O 526 148.475 -27.324 74.735 inf infATOM 527 O H2O 527 150.829 -27.130 74.716 inf infATOM 528 O H2O 528 142.224 -26.634 74.735 inf infATOM 529 O H2O 529 151.295 -26.071 74.756 inf infATOM 530 O H2O 530 143.022 -25.598 74.735 inf infATOM 531 O H2O 531 151.176 -25.356 74.711 inf infATOM 532 O H2O 532 150.884 -24.956 74.740 inf infATOM 533 O H2O 533 150.481 -24.647 74.708 inf infATOM 534 O H2O 534 144.506 -24.172 74.736 inf infATOM 535 O H2O 535 149.803 -23.846 74.749 inf infATOM 536 O H2O 536 149.764 -23.486 74.733 inf infATOM 537 O H2O 537 149.926 -23.117 74.538 inf infATOM 538 O H2O 538 144.566 -22.379 74.735 inf infATOM 539 O H2O 539 144.466 -21.675 74.701 inf infATOM 540 O H2O 540 144.375 -21.264 74.735 inf infATOM 541 O H2O 541 148.644 -21.314 74.746 inf infATOM 542 O H2O 542 148.162 -21.002 74.708 inf infATOM 543 O H2O 543 147.801 -20.671 74.714 inf infATOM 544 O H2O 544 143.884 -19.795 74.726 inf infATOM 545 O H2O 545 147.264 -19.407 74.735 inf infATOM 546 O H2O 546 147.020 -18.682 74.542 inf infATOM 547 O H2O 547 146.925 -18.257 74.689 inf infATOM 548 O H2O 548 146.626 -17.957 74.555 inf infATOM 549 O H2O 549 144.985 -17.608 74.749 inf infATOM 550 O H2O 550 146.094 -17.544 74.714 inf infATOM 551 O H2O 551 145.870 -17.469 74.759 inf infATOM 552 O H2O 552 147.370 -34.334 75.146 inf infATOM 553 O H2O 553 145.716 -33.812 75.107 inf infATOM 554 O H2O 554 147.399 -33.881 74.831 inf infATOM 555 O H2O 555 148.040 -33.789 75.022 inf infATOM 556 O H2O 556 147.806 -33.504 74.803 inf infATOM 557 O H2O 557 145.017 -33.068 75.119 inf infATOM 558 O H2O 558 144.915 -32.729 75.090 inf infATOM 559 O H2O 559 148.122 -32.346 74.911 inf infATOM 560 O H2O 560 144.785 -31.977 74.919 inf infATOM 561 O H2O 561 144.709 -31.633 74.842 inf infATOM 562 O H2O 562 147.630 -31.163 75.079 inf infATOM 563 O H2O 563 144.478 -30.848 74.960 inf infATOM 564 O H2O 564 144.415 -30.501 74.916 inf infATOM 565 O H2O 565 144.093 -30.287 75.119 inf infATOM 566 O H2O 566 146.818 -30.130 75.103 inf infATOM 567 O H2O 567 146.272 -29.759 74.910 inf infATOM 568 O H2O 568 142.960 -29.518 75.103 inf infATOM 569 O H2O 569 142.310 -29.100 75.105 inf infATOM 570 O H2O 570 142.004 -28.660 75.104 inf infATOM 571 O H2O 571 146.664 -28.310 74.901 inf infATOM 572 O H2O 572 141.822 -28.102 75.104 inf infATOM 573 O H2O 573 141.816 -27.549 75.102 inf infATOM 574 O H2O 574 149.400 -27.548 75.104 inf infATOM 575 O H2O 575 150.158 -27.593 75.117 inf infATOM 576 O H2O 576 148.118 -27.407 75.105 inf infATOM 577 O H2O 577 150.390 -27.463 75.138 inf infATOM 578 O H2O 578 1S0.738 -27.013 75.131 inf infATOM 579 O H2O 579 142.365 -26.431 75.110 inf infATOM 580 O H2O 580 142.493 -26.199 75.126 inf infATOM 581 O H2O 581 142.936 -25.693 74.921 inf infATOM 582 O H2O 582 151.072 -25.884 75.113 inf infATOM 583 O H2O 583 143.613 -25.401 75.153 inf infATOM 584 O H2O 584 144.049 -24.964 74.919 inf infATOM 585 O H2O 585 150.713 -25.122 75.129 inf infATOM 586 O H2O 586 150.310 -24.801 75.086 inf infATOM 587 O H2O 587 149.795 -24.215 75.122 inf infATOM 588 O H2O 588 149.704 -23.873 75.086 inf infATOM 589 O H2O 589 149.724 -23.104 75.098 inf infATOM 590 O H2O 590 149.695 -22.390 75.104 inf infATOM 591 O H2O 591 144.395 -21.639 75.104 inf infATOM 592 O H2O 592 144.381 -21.272 75.104 inf infATOM 593 O H2O 593 144.325 -20.875 75.104 inf infATOM 594 O H2O 594 148.815 -21.186 75.170 inf infATOM 595 O H2O 595 148.056 -20.769 75.125 inf infATOM 596 O H2O 596 147.678 -20.390 75.125 inf infATOM 597 O H2O 597 147.404 -19.965 75.104 inf infATOM 598 O H2O 598 144.005 -19.056 75.085 inf infATOM 599 O H2O 599 144.069 -18.647 75.104 inf infATOM 600 O H2O 600 147.102 -18.347 75.105 inf infATOM 601 O H2O 601 146.996 -18.137 75.104 inf infATOM 602 O H2O 602 146.281 -17.548 74.905 inf infATOM 603 O H2O 603 145.524 -17.371 75.098 inf infATOM 604 O H2O 604 146.818 -34.556 75.477 inf infATOM 605 O H2O 605 147.558 -34.572 75.466 inf infATOM 606 O H2O 606 146.609 -34.445 75.427 inf infATOM 607 O H2O 607 148.090 -34.346 75.497 inf infATOM 608 O H2O 608 145.900 -33.998 75.476 inf infATOM 609 O H2O 609 145.269 -33.524 75.473 inf infATOM 610 O H2O 610 144.975 -33.082 75.473 inf infATOM 611 O H2O 611 144.848 -32.746 75.442 inf infATOM 612 O H2O 612 144.643 -32.317 75.487 inf infATOM 613 O H2O 613 144.495 -32.038 75.427 inf infATOM 614 O H2O 614 144.393 -31.614 75.278 inf infATOM 615 O H2O 615 144.358 -31.238 75.263 inf infATOM 616 O H2O 616 147.519 -30.893 75.485 inf infATOM 617 O H2O 617 147.394 -30.674 75.466 inf infATOM 618 O H2O 618 147.112 -30.179 75.497 inf infATOM 619 O H2O 619 146.848 -29.751 75.452 inf infATOM 620 O H2O 620 143.269 -29.654 75.474 inf infATOM 621 O H2O 621 142.126 -28.951 75.473 inf infATOM 622 O H2O 622 146.785 -29.022 75.504 inf infATOM 623 O H2O 623 141.848 -28.328 75.473 inf infATOM 624 O H2O 624 141.817 -27.917 75.474 inf infATOM 625 O H2O 625 141.838 -27.732 75.473 inf infATOM 626 O H2O 626 147.681 -27.640 75.504 inf infATOM 627 O H2O 627 142.037 -27.191 75.473 inf infATOM 628 O H2O 628 149.223 -27.409 75.500 inf infATOM 629 O H2O 629 150.467 -27.153 75.440 inf infATOM 630 O H2O 630 150.709 -26.814 75.300 inf infATOM 631 O H2O 631 150.755 -26.460 75.350 inf infATOM 632 O H2O 632 150.774 -26.070 75.404 inf infATOM 633 O H2O 633 150.529 -25.687 75.523 inf infATOM 634 O H2O 634 143.895 -25.381 75.457 inf infATOM 635 O H2O 635 144.281 -24.995 75.457 inf infATOM 636 O H2O 636 150.304 -25.186 75.416 inf infATOM 637 O H2O 637 144.572 -24.581 75.482 inf infATOM 638 O H2O 638 149.655 -24.267 75.432 inf infATOM 639 O H2O 639 149.624 -23.854 75.306 inf infATOM 640 O H2O 640 144.748 -23.121 75.471 inf infATOM 641 O H2O 641 144.627 -22.368 75.472 inf infATOM 642 O H2O 642 144.408 -21.643 75.290 inf infATOM 643 O H2O 643 144.410 -21.271 75.448 inf infATOM 644 O H2O 644 144.365 -20.899 75.428 inf infATOM 645 O H2O 645 144.304 -20.510 75.449 inf infATOM 646 O H2O 646 147.562 -20.160 75.471 inf infATOM 647 O H2O 647 147.421 -19.839 75.516 inf infATOM 648 O H2O 648 147.357 -19.612 75.477 inf infATOM 649 O H2O 649 144.035 -18.870 75.473 inf infATOM 650 O H2O 650 147.106 -18.345 75.473 inf infATOM 651 O H2O 651 144.492 -17.840 75.473 inf infATOM 652 O H2O 652 146.662 -17.735 75.473 inf infATOM 653 O H2O 653 146.234 -17.479 75.473 inf infATOM 654 O H2O 654 146.992 -34.777 75.829 inf infATOM 655 O H2O 655 147.949 -34.619 75.826 inf infATOM 656 O H2O 656 148.430 -34.154 75.686 inf infATOM 657 O H2O 657 145.844 -34.055 75.825 inf infATOM 658 O H2O 658 148.787 -33.485 75.779 inf infATOM 659 O H2O 659 144.868 -32.773 75.857 inf infATOM 660 O H2O 660 148.497 -32.331 75.646 inf infATOM 661 O H2O 661 144.392 -31.991 75.654 inf infATOM 662 O H2O 662 147.996 -31.560 75.843 inf infATOM 663 O H2O 663 144.148 -30.891 75.842 inf infATOM 664 O H2O 664 143.945 -30.444 75.842 inf infATOM 665 O H2O 665 143.776 -30.184 75.817 inf infATOM 666 O H2O 666 143.630 -29.990 75.826 inf infATOM 667 O H2O 667 142.958 -29.536 75.842 inf infATOM 668 O H2O 668 142.129 -28.950 75.843 inf infATOM 669 O H2O 669 141.970 -28.675 75.856 inf infATOM 670 O H2O 670 147.153 -28.263 75.853 inf infATOM 671 O H2O 671 147.325 -28.056 75.861 inf infATOM 672 O H2O 672 147.712 -27.691 75.856 inf infATOM 673 O H2O 673 148.476 -27.460 75.818 inf infATOM 674 O H2O 674 149.961 -27.191 75.678 inf infATOM 675 O H2O 675 142.372 -26.794 75.848 inf infATOM 676 O H2O 676 142.584 -26.450 75.650 inf infATOM 677 O H2O 677 1S0.311 -26.440 75.639 inf infATOM 678 O H2O 678 150.090 -26.071 75.769 inf infATOM 679 O H2O 679 143.513 -25.730 75.833 inf infATOM 680 O H2O 680 150.312 -25.707 75.618 inf infATOM 681 O H2O 681 144.146 -25.246 75.842 inf infATOM 682 O H2O 682 144.326 -25.024 75.842 inf infATOM 683 O H2O 683 144.491 -24.830 75.841 inf infATOM 684 O H2O 684 144.722 -24.243 75.841 inf infATOM 685 O H2O 685 149.443 -23.868 75.861 inf infATOM 686 O H2O 686 144.697 -23.119 75.869 inf infATOM 687 O H2O 687 149.811 -22.759 75.842 inf infATOM 688 O H2O 688 149.741 -22.018 75.846 inf infATOM 689 O H2O 689 149.513 -21.561 75.842 inf infATOM 690 O H2O 690 149.330 -21.342 75.842 inf infATOM 691 O H2O 691 148.497 -20.868 75.653 inf infATOM 692 O H2O 692 144.367 -20.488 75.838 inf infATOM 693 O H2O 693 148.425 -20.796 75.842 inf infATOM 694 O H2O 694 144.320 -19.765 75.813 inf infATOM 695 O H2O 695 147.417 -19.590 75.829 inf infATOM 696 O H2O 696 144.156 -18.666 75.875 inf infATOM 697 O H2O 697 144.426 -17.957 75.656 inf infATOM 698 O H2O 698 146.936 -18.177 75.821 inf infATOM 699 O H2O 699 146.605 -17.792 75.832 inf infATOM 700 O H2O 700 146.213 -17.543 75.793 inf infATOM 701 O H2O 701 146.623 -34.767 76.211 inf infATOM 702 O H2O 702 148.122 -34.581 76.020 inf infATOM 703 O H2O 703 145.815 -34.121 76.211 inf infATOM 704 O H2O 704 145.659 -33.855 76.211 inf infATOM 705 O H2O 705 148.870 -33.460 76.023 inf infATOM 706 O H2O 706 145.207 -33.235 76.194 inf infATOM 707 O H2O 707 148.825 -32.721 76.030 inf infATOM 708 O H2O 708 148.691 -32.327 76.211 inf infATOM 709 O H2O 709 148.344 -31.915 76.201 inf infATOM 710 O H2O 710 148.157 -31.744 76.212 inf infATOM 711 O H2O 711 147.595 -30.852 76.200 inf infATOM 712 O H2O 712 147.486 -30.524 76.233 inf infATOM 713 O H2O 713 147.243 -30.093 76.189 inf infATOM 714 O H2O 714 143.305 -29.767 76.026 inf infATOM 715 O H2O 715 146.980 -29.393 76.031 inf infATOM 716 O H2O 716 142.388 -29.022 76.210 inf infATOM 717 O H2O 717 142.182 -28.852 76.218 inf infATOM 718 O H2O 718 147.259 -28.334 76.162 inf infATOM 719 O H2O 719 147.597 -27.958 76.177 inf infATOM 720 O H2O 720 148.097 -27.696 76.236 inf infATOM 721 O H2O 721 148.836 -27.339 76.212 inf infATOM 722 O H2O 722 142.252 -27.043 76.192 inf infATOM 723 O H2O 723 149.900 -26.768 75.960 inf infATOM 724 O H2O 724 143.063 -26.364 76.249 inf infATOM 725 O H2O 725 143.278 -26.032 76.046 inf infATOM 726 O H2O 726 149.874 -26.071 75.948 inf infATOM 727 O H2O 727 149.486 -25.674 76.261 inf infATOM 728 O H2O 728 144.192 -25.304 76.225 inf infATOM 729 O H2O 729 149.320 -24.989 76.152 inf infATOM 730 O H2O 730 144.672 -24.235 76.231 inf infATOM 731 O H2O 731 144.647 -23.480 76.225 inf infATOM 732 O H2O 732 149.710 -23.100 76.203 inf infATOM 733 O H2O 733 149.803 -22.379 76.220 inf infATOM 734 O H2O 734 149.725 -22.023 76.205 inf infATOM 735 O H2O 735 144.340 -21.302 76.241 inf infATOM 736 O H2O 736 148.651 -20.928 76.211 inf infATOM 737 O H2O 737 148.114 -20.525 76.023 inf infATOM 738 O H2O 738 144.448 -20.352 76.211 inf infATOM 739 O H2O 739 148.028 -20.401 76.212 inf infATOM 740 O H2O 740 147.745 -19.977 76.210 inf infATOM 741 O H2O 741 147.478 -19.503 76.211 inf infATOM 742 O H2O 742 144.293 -18.708 76.171 inf infATOM 743 O H2O 743 144.374 -18.290 76.056 inf infATOM 744 O H2O 744 146.945 -18.347 75.999 inf infATOM 745 O H2O 745 146.641 -17.938 76.034 inf infATOM 746 O H2O 746 145.525 -17.677 76.274 inf infATOM 747 O H2O 747 146.255 -17.777 76.201 inf infATOM 748 O H2O 748 146.623 -34.767 76.581 inf infATOM 749 O H2O 749 148.054 -34.664 76.581 inf infATOM 750 O H2O 750 148.453 -34.352 76.580 inf infATOM 751 O H2O 751 145.414 -33.406 76.555 inf infATOM 752 O H2O 752 145.148 -33.088 76.397 inf infATOM 753 O H2O 753 145.079 -32.953 76.581 inf infATOM 754 O H2O 754 148.690 -32.327 76.581 inf infATOM 755 O H2O 755 148.383 -31.887 76.549 inf infATOM 756 O H2O 756 148.034 -31.532 76.548 inf infATOM 757 O H2O 757 144.168 -30.897 76.599 inf infATOM 758 O H2O 758 144.049 -30.684 76.580 inf infATOM 759 O H2O 759 143.736 -30.259 76.581 inf infATOM 760 O H2O 760 143.396 -29.833 76.580 inf infATOM 761 O H2O 761 143.023 -29.443 76.611 inf infATOM 762 O H2O 762 142.727 -29.055 76.593 inf infATOM 763 O H2O 763 142.348 -28.686 76.620 inf infATOM 764 O H2O 764 142.222 -28.465 76.553 inf infATOM 765 O H2O 765 142.089 -27.916 76.533 inf infATOM 766 O H2O 766 142.120 -27.524 76.455 inf infATOM 767 O H2O 767 148.489 -27.567 76.393 inf infATOM 768 O H2O 768 142.397 -27.188 76.570 inf infATOM 769 O H2O 769 142.597 -26.841 76.377 inf infATOM 770 O H2O 770 149.369 -26.777 76.568 inf infATOM 771 O H2O 771 149.440 -26.451 76.598 inf infATOM 772 O H2O 772 149.554 -26.086 76.381 inf infATOM 773 O H2O 773 144.090 -25.559 76.564 inf infATOM 774 O H2O 774 144.383 -25.134 76.590 inf infATOM 775 O H2O 775 149.180 -24.973 76.379 inf infATOM 776 O H2O 776 144.553 -24.225 76.581 inf infATOM 777 O H2O 777 149.187 -24.036 76.572 inf infATOM 778 O H2O 778 144.436 -23.296 76.593 inf infATOM 779 O H2O 779 149.632 -23.071 76.534 inf infATOM 780 O H2O 780 144.180 -22.411 76.518 inf infATOM 781 O H2O 781 143.935 -22.010 76.652 inf infATOM 782 O H2O 782 144.132 -21.640 76.451 inf infATOM 783 O H2O 783 144.211 -21.266 76.568 inf infATOM 784 O H2O 784 144.246 -20.902 76.588 inf infATOM 785 O H2O 785 148.035 -20.455 76.585 inf infATOM 786 O H2O 786 144.447 -20.164 76.400 inf infATOM 787 O H2O 787 144.455 -19.789 76.575 inf infATOM 788 O H2O 788 144.498 -19.380 76.639 inf infATOM 789 O H2O 789 147.127 -19.114 76.557 inf infATOM 790 O H2O 790 146.773 -18.731 76.537 inf infATOM 791 O H2O 791 144.956 -18.299 76.615 inf infATOM 792 O H2O 792 144.842 -18.005 76.340 inf infATOM 793 O H2O 793 145.525 -17.862 76.483 inf infATOM 794 O H2O 794 146.263 -17.949 76.396 inf infATOM 795 O H2O 795 146.992 -34.775 76.962 inf infATOM 796 O H2O 796 147.948 -34.617 76.966 inf infATOM 797 O H2O 797 148.172 -34.471 76.982 inf infATOM 798 O H2O 798 145.862 -34.028 76.960 inf infATOM 799 O H2O 799 148.818 -33.634 76.949 inf infATOM 800 O H2O 800 144.899 -32.766 76.950 inf infATOM 801 O H2O 801 144.756 -32.543 76.950 inf infATOM 802 O H2O 802 148.483 -31.972 76.764 inf infATOM 803 O H2O 803 148.146 -31.577 76.745 inf infATOM 804 O H2O 804 148.013 -31.204 76.896 inf infATOM 805 O H2O 805 143.937 -30.451 76.950 inf infATOM 806 O H2O 806 143.595 -30.064 76.950 inf infATOM 807 O H2O 807 143.414 -29.843 76.949 inf infATOM 808 O H2O 808 143.066 -29.422 76.951 inf infATOM 809 O H2O 809 142.766 -29.017 76.946 inf infATOM 810 O H2O 810 142.511 -28.654 76.813 inf infATOM 811 O H2O 811 142.289 -28.246 76.666 inf infATOM 812 O H2O 812 147.796 -28.495 76.933 inf infATOM 813 O H2O 813 148.135 -27.948 76.749 inf infATOM 814 O H2O 814 142.551 -27.735 76.967 inf infATOM 815 O H2O 815 142.588 -27.190 76.744 inf infATOM 816 O H2O 816 142.974 -26.850 76.740 inf infATOM 817 O H2O 817 149.258 -26.769 76.927 inf infATOM 818 O H2O 818 149.350 -26.425 76.942 inf infATOM 819 O H2O 819 143.912 -25.774 76.950 inf infATOM 820 O H2O 820 149.159 -25.268 76.950 inf infATOM 821 O H2O 821 144.495 -24.937 76.949 inf infATOM 822 O H2O 822 144.511 -24.224 76.927 inf infATOM 823 O H2O 823 149.125 -23.880 76.977 inf infATOM 824 O H2O 824 149.208 -23.668 76.947 inf infATOM 825 O H2O 825 149.409 -23.120 76.953 inf infATOM 826 O H2O 826 149.465 -22.749 76.993 inf infATOM 827 O H2O 827 149.468 -22.379 76.995 inf infATOM 828 O H2O 828 143.746 -22.010 76.832 inf infATOM 829 O H2O 829 143.784 -21.615 76.871 inf infATOM 830 O H2O 830 149.018 -21.285 76.949 inf infATOM 831 O H2O 831 148.659 -20.910 76.952 inf infATOM 832 O H2O 832 148.105 -20.536 76.766 inf infATOM 833 O H2O 833 147.919 -20.166 76.951 inf infATOM 834 O H2O 834 147.639 -19.735 76.949 inf infATOM 835 O H2O 835 147.450 -19.497 76.982 inf infATOM 836 O H2O 836 144.898 -19.008 76.998 inf infATOM 837 O H2O 837 144.832 -18.706 76.719 inf infATOM 838 O H2O 838 146.438 -18.704 76.938 inf infATOM 839 O H2O 839 145.525 -18.298 76.793 inf infATOM 840 O H2O 840 146.228 -18.561 76.903 inf infATOM 841 O H2O 841 146.812 -34.588 77.327 inf infATOM 842 O H2O 842 146.108 -34.161 77.307 inf infATOM 843 O H2O 843 148.454 -34.172 77.131 inf infATOM 844 O H2O 844 148.536 -34.049 77.317 inf infATOM 845 O H2O 845 148.792 -33.615 77.318 inf infATOM 846 O H2O 846 148.941 -33.083 77.348 inf infATOM 847 O H2O 847 144.635 -32.323 77.319 inf infATOM 848 O H2O 848 144.518 -32.012 77.296 inf infATOM 849 O H2O 849 144.401 -31.803 77.314 inf infATOM 850 O H2O 850 148.131 -31.232 77.124 inf infATOM 851 O H2O 851 148.136 -30.877 77.372 inf infATOM 852 O H2O 852 148.155 -30.500 77.365 inf infATOM 853 O H2O 853 148.121 -30.314 77.312 inf infATOM 854 O H2O 854 147.792 -29.736 77.090 inf infATOM 855 O H2O 855 147.791 -29.427 77.362 inf infATOM 856 O H2O 856 147.740 -29.208 77.319 inf infATOM 857 O H2O 857 147.829 -28.619 77.350 inf infATOM 858 O H2O 858 142.653 -27.916 77.079 inf infATOM 859 O H2O 859 142.874 -27.514 77.192 inf infATOM 860 O H2O 860 148.676 -27.561 77.319 inf infATOM 861 O H2O 861 149.025 -27.174 77.319 inf infATOM 862 O H2O 862 143.372 -26.652 77.299 inf infATOM 863 O H2O 863 143.592 -26.217 77.322 inf infATOM 864 O H2O 864 143.956 -25.794 77.319 inf infATOM 865 O H2O 865 149.164 -25.266 77.318 inf infATOM 866 O H2O 866 149.053 -24.958 77.319 inf infATOM 867 O H2O 867 149.028 -24.225 77.319 inf infATOM 868 O H2O 868 149.073 -23.866 77.320 inf infATOM 869 O H2O 869 144.044 -23.306 77.317 inf infATOM 870 O H2O 870 149.268 -23.115 77.271 inf infATOM 871 O H2O 871 149.314 -22.747 77.287 inf infATOM 872 O H2O 872 143.435 -22.010 77.293 inf infATOM 873 O H2O 873 149.246 -21.694 77.314 inf infATOM 874 O H2O 874 149.163 -21.479 77.317 inf infATOM 875 O H2O 875 148.883 -21.062 77.308 inf infATOM 876 O H2O 876 148.311 -20.516 77.305 inf infATOM 877 O H2O 877 147.989 -20.101 77.300 inf infATOM 878 O H2O 878 147.718 -19.813 77.138 inf infATOM 879 O H2O 879 147.243 -19.369 77.318 inf infATOM 880 O H2O 880 144.989 -19.094 77.300 inf infATOM 881 O H2O 881 145.521 -18.664 77.155 inf infATOM 882 O H2O 882 146.278 -18.820 77.341 inf infATOM 883 O H2O 883 147.002 -34.566 77.680 inf infATOM 884 O H2O 884 146.146 -34.124 77.688 inf infATOM 885 O H2O 885 148.103 -34.368 77.688 inf infATOM 886 O H2O 886 148.519 -34.048 77.688 inf infATOM 887 O H2O 887 148.905 -33.411 77.688 inf infATOM 888 O H2O 888 144.969 -32.717 77.688 inf infATOM 889 O H2O 889 144.771 -32.543 77.686 inf infATOM 890 O H2O 890 144.422 -31.974 77.506 inf infATOM 891 O H2O 891 148.460 -31.619 77.509 inf infATOM 892 O H2O 892 148.409 -31.451 77.688 inf infATOM 893 O H2O 893 148.290 -30.869 77.690 inf infATOM 894 O H2O 894 143.726 -30.284 77.688 inf infATOM 895 O H2O 895 143.405 -29.851 77.688 inf infATOM 896 O H2O 896 143.230 -29.658 77.715 inf infATOM 897 O H2O 897 142.944 -29.207 77.687 inf infATOM 898 O H2O 898 147.962 -28.665 77.672 inf infATOM 899 O H2O 899 148.180 -28.214 77.729 inf infATOM 900 O H2O 900 143.141 -27.558 77.683 inf infATOM 901 O H2O 901 143.263 -27.343 77.701 inf infATOM 902 O H2O 902 143.331 -26.822 77.5OO inf infATOM 903 O H2O 903 143.505 -26.444 77.688 inf infATOM 904 O H2O 904 143.757 -25.995 77.688 inf infATOM 905 O H2O 905 149.278 -25.703 77.648 inf infATOM 906 O H2O 906 144.447 -25.160 77.688 inf infATOM 907 O H2O 907 149.065 -24.595 77.679 inf infATOM 908 O H2O 908 144.422 -24.038 77.688 inf infATOM 909 O H2O 909 143.781 -23.200 77.660 inf infATOM 910 O H2O 910 143.469 -22.759 77.681 inf infATOM 911 O H2O 911 143.357 -22.413 77.654 inf infATOM 912 O H2O 912 143.290 -22.010 77.742 inf infATOM 913 O H2O 913 143.320 -21.827 77.691 inf infATOM 914 O H2O 914 143.394 -21.246 77.663 inf infATOM 915 O H2O 915 148.996 -20.025 77.714 inf infATOM 916 O H2O 916 148.691 -20.510 77.659 inf infATOM 917 O H2O 917 148.121 -20.150 77.497 inf infATOM 918 O H2O 918 144.358 -19.894 77.688 inf infATOM 919 O H2O 919 144.793 -19.431 77.502 inf infATOM 920 O H2O 920 147.430 -19.491 77.688 inf infATOM 921 O H2O 921 145.329 -19.031 77.695 inf infATOM 922 O H2O 922 145.894 -18.883 77.685 inf infATOM 923 O H2O 923 147.370 -34.566 77.873 inf infATOM 924 O H2O 924 146.969 -34.505 78.089 inf infATOM 925 O H2O 925 148.303 -34.206 78.062 inf infATOM 926 O H2O 926 148.500 -34.029 78.066 inf infATOM 927 O H2O 927 148.797 -33.617 78.043 inf infATOM 928 O H2O 928 148.960 -33.084 78.047 inf infATOM 929 O H2O 929 144.545 -32.402 78.057 inf infATOM 930 O H2O 930 144.375 -32.191 78.057 inf infATOM 931 O H2O 931 148.552 -31.689 78.057 inf infATOM 932 O H2O 932 144.010 -30.818 78.056 inf infATOM 933 O H2O 933 148.304 -30.500 78.057 inf infATOM 934 O H2O 934 143.461 -29.784 78.068 inf infATOM 935 O H2O 935 148.000 -29.374 78.057 inf infATOM 936 O H2O 936 147.960 -29.024 78.057 inf infATOM 937 O H2O 937 142.914 -28.286 78.004 inf infATOM 938 O H2O 938 142.972 -28.105 78.048 inf infATOM 939 O H2O 939 143.219 -27.069 78.026 inf infATOM 940 O H2O 940 143.423 -27.162 78.074 inf infATOM 941 O H2O 941 148.874 -27.010 78.079 inf infATOM 942 O H2O 942 149.092 -26.460 78.097 inf infATOM 943 O H2O 943 149.229 -26.070 77.878 inf infATOM 944 O H2O 944 149.223 -25.701 77.874 inf infATOM 945 O H2O 945 149.226 -25.330 77.871 inf infATOM 946 O H2O 946 149.183 -24.972 77.888 inf infATOM 947 O H2O 947 149.286 -24.589 78.141 inf infATOM 948 O H2O 948 144.333 -23.791 78.057 inf infATOM 949 O H2O 949 149.262 -23.856 78.104 inf infATOM 950 O H2O 950 143.757 -23.223 78.057 inf infATOM 951 O H2O 951 149.141 -23.090 78.057 inf infATOM 952 O H2O 952 143.314 -22.422 78.057 inf infATOM 953 O H2O 953 149.200 -22.010 77.874 inf infATOM 954 O H2O 954 143.308 -21.640 78.058 inf infATOM 955 O H2O 955 143.323 -21.229 78.057 inf infATOM 956 O H2O 956 149.141 -20.856 77.994 inf infATOM 957 O H2O 957 148.869 -20.515 77.859 inf infATOM 958 O H2O 958 143.903 -20.204 78.057 inf infATOM 959 O H2O 959 144.102 -20.061 78.057 inf infATOM 960 O H2O 960 148.140 -19.906 78.036 inf infATOM 961 O H2O 961 147.217 -19.379 78.073 inf infATOM 962 O H2O 962 145.351 -19.082 78.050 inf infATOM 963 O H2O 963 145.894 -18.942 78.047 inf infATOM 964 O H2O 964 146.655 -34.309 78.403 inf infATOM 965 O H2O 965 148.036 -34.269 78.391 inf infATOM 966 O H2O 966 148.429 -33.959 78.407 inf infATOM 967 O H2O 967 148.825 -33.447 78.232 inf infATOM 968 O H2O 968 148.820 -33.265 78.411 inf infATOM 969 O H2O 969 148.872 -32.715 78.373 inf infATOM 970 O H2O 970 148.828 -32.533 78.416 inf infATOM 971 O H2O 971 148.613 -32.015 78.397 inf infATOM 972 O H2O 972 148.486 -31.605 78.247 inf infATOM 973 O H2O 973 144.070 -31.051 87.426 inf infATOM 974 O H2O 974 143.705 -30.118 78.229 inf infATOM 975 O H2O 975 143.713 -29.935 78.403 inf infATOM 976 O H2O 976 147.955 -29.384 78.440 inf infATOM 977 O H2O 977 147.914 -29.024 78.422 inf infATOM 978 O H2O 978 143.007 -28.305 78.213 inf infATOM 979 O H2O 979 148.068 -28.084 78.427 inf infATOM 980 O H2O 980 143.495 -27.547 78.425 inf infATOM 981 O H2O 981 148.482 -27.365 78.428 inf infATOM 982 O H2O 982 148.704 -26.823 78.455 inf infATOM 983 O H2O 983 148.825 -26.617 78.412 inf infATOM 984 O H2O 984 144.087 -25.935 78.397 inf infATOM 985 O H2O 985 144.723 -25.275 78.337 inf infATOM 986 O H2O 986 149.296 -25.296 78.429 inf infATOM 987 O H2O 987 144.803 -24.593 78.216 inf infATOM 988 O H2O 988 144.966 -24.227 78.431 inf infATOM 989 O H2O 989 144.615 -23.837 78.404 inf infATOM 990 O H2O 990 144.418 -23.670 78.426 inf infATOM 991 O H2O 991 143.799 -23.182 78.448 inf infATOM 992 O H2O 992 143.625 -22.969 78.444 inf infATOM 993 O H2O 993 143.299 -22.010 78.243 inf infATOM 994 O H2O 994 143.304 -21.641 78.243 inf infATOM 995 O H2O 995 149.378 -21.275 78.433 inf infATOM 996 O H2O 996 143.577 -20.589 78.398 inf infATOM 997 O H2O 997 143.908 -20.209 78.408 inf infATOM 998 O H2O 998 144.390 -19.918 78.444 inf infATOM 999 O H2O 999 148.275 -19.838 78.439 inf infATOM 1000 O H2O 1000 147.386 -19.574 78.437 inf infATOM 1001 O H2O 1001 145.525 -19.060 78.240 inf infATOM 1002 O H2O 1002 146.298 -19.104 78.495 inf infATOM 1003 O H2O 1003 146.946 -19.324 78.399 inf infATOM 1004 O H2O 1004 147.370 -34.263 78.720 inf infATOM 1005 O H2O 1005 148.079 -34.133 78.582 inf infATOM 1006 O H2O 1006 146.972 -34.095 78.883 inf infATOM 1007 O H2O 1007 148.274 -33.804 78.776 inf infATOM 1008 O H2O 1008 148.437 -33.614 78.763 inf infATOM 1009 O H2O 1009 145.901 -33.261 78.790 inf infATOM 1010 O H2O 1010 145.204 -32.755 78.747 inf infATOM 1011 O H2O 1011 144.613 -32.334 78.791 inf infATOM 1012 O H2O 1012 144.452 -32.138 78.784 inf infATOM 1013 O H2O 1013 144.224 -31.610 78.799 inf infATOM 1014 O H2O 1014 144.082 -30.869 78.588 inf infATOM 1015 O H2O 1015 144.159 -30.522 78.827 inf infATOM 1016 O H2O 1016 148.062 -30.097 78.864 inf infATOM 1017 O H2O 1017 143.889 -29.753 78.778 inf infATOM 1018 O H2O 1018 143.845 -29.401 78.815 inf infATOM 1019 O H2O 1019 143.691 -29.202 78.777 inf infATOM 1020 O H2O 1020 147.810 -28.622 78.757 inf infATOM 1021 O H2O 1021 143.377 -27.943 78.544 inf infATOM 1022 O H2O 1022 148.024 -28.046 78.767 inf infATOM 1023 O H2O 1023 148.327 -27.562 78.805 inf infATOM 1024 O H2O 1024 148.431 -27.213 78.830 inf infATOM 1025 O H2O 1025 148.565 -26.776 78.761 inf infATOM 1026 O H2O 1026 148.805 -26.419 78.590 inf infATOM 1027 O H2O 1027 148.782 -26.223 78.763 inf infATOM 1028 O H2O 1028 144.785 -25.884 78.797 inf infATOM 1029 O H2O 1029 145.144 -25.326 78.628 inf infATOM 1030 O H2O 1030 149.355 -25.316 78.810 inf infATOM 1031 O H2O 1031 145.194 -24.592 78.573 inf infATOM 1032 O H2O 1032 145.314 -24.234 78.813 inf infATOM 1033 O H2O 1033 145.184 -24.023 78.773 inf infATOM 1034 O H2O 1034 144.804 -23.642 78.773 inf infATOM 1035 O H2O 1035 144.387 -23.391 78.855 inf infATOM 1036 O H2O 1036 143.727 -22.909 78.771 inf infATOM 1037 O H2O 1037 149.224 -22.381 78.610 inf infATOM 1038 O H2O 1038 149.312 -21.997 78.795 inf infATOM 1039 O H2O 1039 149.393 -21.272 78.795 inf infATOM 1040 O H2O 1040 143.642 -20.694 78.808 inf infATOM 1041 O H2O 1041 144.048 -20.165 78.611 inf infATOM 1042 O H2O 1042 148.936 -20.259 78.795 inf infATOM 1043 O H2O 1043 144.950 -19.764 78.816 inf infATOM 1044 O H2O 1044 145.150 -19.604 78.802 inf infATOM 1045 O H2O 1045 146.986 -19.456 78.596 inf infATOM 1046 O H2O 1046 145.549 -19.317 78.745 inf infATOM 1047 O H2O 1047 146.600 -19.338 78.732 inf infATOM 1048 O H2O 1048 147.370 -33.905 79.095 inf infATOM 1049 O H2O 1049 146.466 -33.434 79.126 inf infATOM 1050 O H2O 1050 149.747 -33.654 79.189 inf infATOM 1051 O H2O 1051 145.890 -33.089 78.985 inf infATOM 1052 O H2O 1052 148.270 -33.084 79.130 inf infATOM 1053 O H2O 1053 145.678 -32.761 79.195 inf infATOM 1054 O H2O 1054 144.772 -32.364 78.991 inf infATOM 1055 O H2O 1055 148.224 -32.373 79.096 inf infATOM 1056 O H2O 1056 144.758 -32.190 79.187 inf infATOM 1057 O H2O 1057 144.352 -31.567 79.085 inf infATOM 1058 O H2O 1058 144.326 -31.238 79.102 inf infATOM 1059 O H2O 1059 147.917 -30.869 79.160 inf infATOM 1060 O H2O 1060 144.523 -30.499 79.242 inf infATOM 1061 O H2O 1061 144.285 -30.110 79.112 inf infATOM 1062 O H2O 1062 144.244 -29.762 79.153 inf infATOM 1063 O H2O 1063 144.049 -29.393 78.978 inf infATOM 1064 O H2O 1064 143.743 -28.992 78.885 inf infATOM 1065 O H2O 1065 147.666 -29.023 78.907 inf infATOM 1066 O H2O 1066 144.421 -28.841 79.149 inf infATOM 1067 O H2O 1067 143.719 -28.306 78.920 inf infATOM 1068 O H2O 1068 147.613 -28.333 79.223 inf infATOM 1069 O H2O 1069 144.614 -27.916 79.137 inf infATOM 1070 O H2O 1070 144.082 -27.547 78.929 inf infATOM 1071 O H2O 1071 148.239 -27.529 79.146 inf infATOM 1072 O H2O 1072 148.329 -27.188 79.180 inf infATOM 1073 O H2O 1073 148.470 -26.807 78.977 inf infATOM 1074 O H2O 1074 144.784 -26.628 79.192 inf infATOM 1075 O H2O 1075 144.471 -26.142 78.891 inf infATOM 1076 O H2O 1076 145.349 -26.076 79.143 inf infATOM 1077 O H2O 1077 145.462 -25.625 79.042 inf infATOM 1078 O H2O 1078 149.042 -25.714 79.167 inf infATOM 1079 O H2O 1079 149.378 -25.317 79.165 inf infATOM 1080 O H2O 1080 145.551 -24.583 78.954 inf infATOM 1081 O H2O 1081 149.589 -24.593 79.164 inf infATOM 1082 O H2O 1082 149.592 -24.408 79.165 inf infATOM 1083 O H2O 1083 149.471 -23.838 79.165 inf infATOM 1084 O H2O 1084 149.364 -23.497 79.159 inf infATOM 1085 O H2O 1085 144.762 -23.337 79.188 inf infATOM 1086 O H2O 1086 144.067 -22.919 79.141 inf infATOM 1087 O H2O 1087 143.827 -22.939 79.201 inf infATOM 1088 O H2O 1088 143.786 -22.037 79.242 inf infATOM 1089 O H2O 1089 149.331 -21.640 79.142 inf infATOM 1090 O H2O 1090 143.684 -21.089 79.161 inf infATOM 1091 O H2O 1091 149.056 -20.517 79.173 inf infATOM 1092 O H2O 1092 144.397 -20.299 79.214 inf infATOM 1093 O H2O 1093 144.814 -20.072 79.106 inf infATOM 1094 O H2O 1094 145.474 -19.728 79.097 inf infATOM 1095 O H2O 1095 147.024 -19.709 79.016 inf infATOM 1096 O H2O 1096 147.923 -19.802 79.162 inf infATOM 1097 O H2O 1097 145.546 -19.480 78.926 inf infATOM 1098 O H2O 1098 146.263 -19.436 78.970 inf infATOM 1099 O H2O 1099 146.670 -33.415 79.234 inf infATOM 1100 O H2O 1100 146.290 -33.048 79.304 inf infATOM 1101 O H2O 1101 147.746 -33.091 79.376 inf infATOM 1102 O H2O 1102 146.287 -32.839 79.474 inf infATOM 1103 O H2O 1103 147.405 -32.750 79.489 inf infATOM 1104 O H2O 1104 145.497 -32.403 79.433 inf infATOM 1105 O H2O 1105 147.562 -32.349 79.544 inf infATOM 1106 O H2O 1106 144.977 -31.971 79.521 inf infATOM 1107 O H2O 1107 147.830 -31.977 79.440 inf infATOM 1108 O H2O 1108 147.618 -31.608 79.623 inf infATOM 1109 O H2O 1109 147.574 -31.229 79.563 inf infATOM 1110 O H2O 1110 147.546 -30.870 79.520 inf infATOM 1111 O H2O 1111 144.944 -30.499 79.596 inf infATOM 1112 O H2O 1112 144.703 -30.131 79.432 inf infATOM 1113 O H2O 1113 144.467 -29.762 79.274 inf infATOM 1114 O H2O 1114 147.355 -29.954 79.512 inf infATOM 1115 O H2O 1115 144.932 -29.374 79.592 inf infATOM 1116 O H2O 1116 147.343 -29.420 79.270 inf infATOM 1117 O H2O 1117 146.780 -29.024 79.478 inf infATOM 1118 O H2O 1118 145.106 -28.633 79.423 inf infATOM 1119 O H2O 1119 146.632 -28.840 79.528 inf infATOM 1120 O H2O 1120 144.827 -28.284 79.236 inf infATOM 1121 O H2O 1121 146.984 -28.453 79.499 inf infATOM 1122 O H2O 1122 145.147 -27.904 79.390 inf infATOM 1123 O H2O 1123 147.366 -28.093 79.525 inf infATOM 1124 O H2O 1124 145.158 -27.547 79.336 inf infATOM 1125 O H2O 1125 148.115 -27.551 79.353 inf infATOM 1126 O H2O 1126 145.449 -27.145 79.458 inf infATOM 1127 O H2O 1127 144.802 -26.808 79.181 inf infATOM 1128 O H2O 1128 148.288 -26.786 79.490 inf infATOM 1129 O H2O 1129 148.431 -26.417 79.326 inf infATOM 1130 O H2O 1130 148.451 -26.238 79.525 inf infATOM 1131 O H2O 1131 146.015 -25.703 79.596 inf infATOM 1132 O H2O 1132 145.967 -25.331 79.614 inf infATOM 1133 O H2O 1133 145.954 -24.963 79.565 inf infATOM 1134 O H2O 1134 149.578 -24.594 79.348 inf infATOM 1135 O H2O 1135 149.516 -24.196 79.563 inf infATOM 1136 O H2O 1136 145.102 -23.522 79.385 inf infATOM 1137 O H2O 1137 144.750 -23.173 79.386 inf infATOM 1138 O H2O 1138 149.221 -23.301 79.535 inf infATOM 1139 O H2O 1139 144.604 -22.746 79.531 inf infATOM 1140 O H2O 1140 144.246 -22.370 79.501 inf infATOM 1141 O H2O 1141 143.933 -22.038 79.431 inf infATOM 1142 O H2O 1142 143.732 -21.641 79.296 inf infATOM 1143 O H2O 1143 149.254 -21.641 79.365 inf infATOM 1144 O H2O 1144 149.257 -21.272 79.367 inf infATOM 1145 O H2O 1145 149.066 -20.891 79.557 inf infATOM 1146 O H2O 1146 144.408 -20.708 79.553 inf infATOM 1147 O H2O 1147 145.524 -20.457 79.457 inf infATOM 1148 O H2O 1148 144.786 -20.414 79.469 inf infATOM 1149 O H2O 1149 145.893 -20.163 79.352 inf infATOM 1150 O H2O 1150 146.630 -20.338 79.547 inf infATOM 1151 O H2O 1151 148.803 -20.392 79.491 inf infATOM 1152 O H2O 1152 147.022 -20.033 79.480 inf infATOM 1153 O H2O 1153 148.104 -19.992 79.525 inf infATOM 1154 O H2O 1154 147.001 -32.658 79.633 inf infATOM 1155 O H2O 1155 147.015 -32.336 79.766 inf infATOM 1156 O H2O 1156 145.879 -31.999 79.770 inf infATOM 1157 O H2O 1157 146.817 -31.979 79.910 inf infATOM 1158 O H2O 1158 145.145 -31.615 79.743 inf infATOM 1159 O H2O 1159 146.446 -31.608 79.913 inf infATOM 1160 O H2O 1160 144.843 -31.238 79.633 inf infATOM 1161 O H2O 1161 145.894 -31.422 79.898 inf infATOM 1162 O H2O 1162 146.632 -31.424 79.899 inf infATOM 1163 O H2O 1163 145.144 -30.861 79.746 inf infATOM 1164 O H2O 1164 146.263 -30.842 79.826 inf infATOM 1165 O H2O 1165 145.162 -30.500 79.703 inf infATOM 1166 O H2O 1166 146.632 -30.500 79.769 inf infATOM 1167 O H2O 1167 145.502 -30.131 79.810 inf infATOM 1168 O H2O 1168 146.994 -30.135 79.693 inf infATOM 1169 O H2O 1169 145.894 -29.762 79.860 inf infATOM 1170 O H2O 1170 145.159 -29.395 79.710 inf infATOM 1171 O H2O 1171 146.584 -29.417 79.648 inf infATOM 1172 O H2O 1172 146.232 -29.048 79.617 inf infATOM 1173 O H2O 1173 146.223 -28.646 79.584 inf infATOM 1174 O H2O 1174 146.633 -28.245 79.609 inf infATOM 1175 O H2O 1175 146.631 -27.946 79.762 inf infATOM 1176 O H2O 1176 146.231 -27.613 79.851 inf infATOM 1177 O H2O 1177 147.356 -27.702 79.859 inf infATOM 1178 O H2O 1178 146.074 -27.180 79.909 inf infATOM 1179 O H2O 1179 148.034 -27.148 79.643 inf infATOM 1180 O H2O 1180 147.887 -26.790 79.848 inf infATOM 1181 O H2O 1181 147.927 -26.441 79.908 inf infATOM 1182 O H2O 1182 146.261 -26.254 79.908 inf infATOM 1183 O H2O 1183 148.287 -26.063 79.894 inf infATOM 1184 O H2O 1184 148.880 -25.742 79.739 inf infATOM 1185 O H2O 1185 148.914 -25.584 79.957 inf infATOM 1186 O H2O 1186 149.250 -25.162 79.923 inf infATOM 1187 O H2O 1187 145.774 -24.206 79.894 inf infATOM 1188 O H2O 1188 145.644 -23.893 79.912 inf infATOM 1189 O H2O 1189 149.162 -23.453 79.936 inf infATOM 1190 O H2O 1190 145.150 -23.307 79.904 inf infATOM 1191 O H2O 1191 149.120 -22.748 79.903 inf infATOM 1192 O H2O 1192 149.115 -22.379 79.903 inf infATOM 1193 O H2O 1193 149.050 -22.004 79.911 inf infATOM 1194 O H2O 1194 144.786 -21.825 79.903 inf infATOM 1195 O H2O 1195 144.404 -21.263 79.748 inf infATOM 1196 O H2O 1196 144.435 -20.921 79.663 inf infATOM 1197 O H2O 1197 145.508 -21.056 79.948 inf infATOM 1198 O H2O 1198 148.851 -21.086 79.908 inf infATOM 1199 O H2O 1199 145.921 -20.597 79.655 inf infATOM 1200 O H2O 1200 146.624 -20.663 79.934 inf infATOM 1201 O H2O 1201 148.805 -20.575 79.677 inf infATOM 1202 O H2O 1202 147.364 -20.127 79.749 inf infATOM 1203 O H2O 1203 148.117 -20.139 79.735 inf infATOM 1204 O H2O 1204 146.632 -31.608 79.925 inf infATOM 1205 O H2O 1205 147.330 -27.507 79.967 inf infATOM 1206 O H2O 1206 147.360 -27.173 80.053 inf infATOM 1207 O H2O 1207 147.366 -26.809 80.072 inf infATOM 1208 O H2O 1208 147.001 -26.440 80.108 inf infATOM 1209 O H2O 1209 147.001 -26.089 80.155 inf infATOM 1210 O H2O 1210 146.620 -25.707 80.106 inf infATOM 1211 O H2O 1211 147.736 -25.880 80.263 inf infATOM 1212 O H2O 1212 146.311 -25.313 80.039 inf infATOM 1213 O H2O 1213 148.793 -25.463 80.218 inf infATOM 1214 O H2O 1214 149.225 -24.966 80.093 inf infATOM 1215 O H2O 1215 149.102 -24.594 80.343 inf infATOM 1216 O H2O 1216 149.096 -24.225 80.337 inf infATOM 1217 O H2O 1217 149.196 -23.862 80.074 inf infATOM 1218 O H2O 1218 145.135 -23.127 80.098 inf infATOM 1219 O H2O 1219 145.162 -22.931 80.268 inf infATOM 1220 O H2O 1220 144.849 -22.043 79.987 inf infATOM 1221 O H2O 1221 148.947 -22.038 80.215 inf infATOM 1222 O H2O 1222 148.825 -21.839 80.254 inf infATOM 1223 O H2O 1223 145.522 -21.455 80.275 inf infATOM 1224 O H2O 1224 148.543 -21.223 80.202 inf infATOM 1225 O H2O 1225 146.236 -21.008 80.324 inf infATOM 1226 O H2O 1226 148.492 -20.896 80.108 inf infATOM 1227 O H2O 1227 147.370 -20.747 80.228 inf infATOM 1228 O H2O 1228 148.090 -20.746 80.207 inf infATOM 1229 O H2O 1229 147.369 -25.639 80.362 inf infATOM 1230 O H2O 1230 146.992 -25.350 80.483 inf infATOM 1231 O H2O 1231 148.489 -25.355 80.491 inf infATOM 1232 O H2O 1232 146.999 -25.152 80.645 inf infATOM 1233 O H2O 1233 148.467 -25.137 80.619 inf infATOM 1234 O H2O 1234 146.318 -24.710 80.600 inf infATOM 1235 O H2O 1235 145.894 -24.225 80.456 inf infATOM 1236 O H2O 1236 145.821 -23.819 80.604 inf infATOM 1237 O H2O 1237 145.558 -23.461 80.437 inf infATOM 1238 O H2O 1238 145.673 -23.136 80.695 inf infATOM 1239 O H2O 1239 145.213 -22.748 80.418 inf infATOM 1240 O H2O 1240 148.880 -22.748 80.480 inf infATOM 1241 O H2O 1241 148.877 -22.379 80.477 inf infATOM 1242 O H2O 1242 148.632 -22.022 80.611 inf infATOM 1243 O H2O 1243 148.288 -21.647 80.633 inf infATOM 1244 O H2O 1244 145.907 -21.466 80.624 inf infATOM 1245 O H2O 1245 147.398 -21.200 80.559 inf infATOM 1246 O H2O 1246 148.109 -21.271 80.458 inf infATOM 1247 O H2O 1247 147.001 -21.124 80.604 inf infATOM 1248 O H2O 1248 148.062 -20.949 80.316 inf infATOM 1249 O H2O 1249 148.108 -24.957 80.817 inf infATOM 1250 O H2O 1250 146.487 -24.554 80.971 inf infATOM 1251 O H2O 1251 148.087 -24.736 80.946 inf infATOM 1252 O H2O 1252 146.304 -24.378 80.959 inf infATOM 1253 O H2O 1253 146.090 -23.851 80.999 inf infATOM 1254 O H2O 1254 146.076 -23.487 81.102 inf infATOM 1255 O H2O 1255 146.081 -23.117 81.006 inf infATOM 1256 O H2O 1256 146.081 -22.748 81.003 inf infATOM 1257 O H2O 1257 145.568 -22.379 80.760 inf infATOM 1258 O H2O 1258 148.489 -22.379 80.843 inf infATOM 1259 O H2O 1259 148.106 -22.196 81.002 inf infATOM 1260 O H2O 1260 146.242 -21.599 80.888 inf infATOM 1261 O H2O 1261 147.014 -21.806 81.056 inf infATOM 1262 O H2O 1262 147.739 -21.845 80.946 inf infATOM 1263 O H2O 1263 146.632 -21.300 80.783 inf infATOM 1264 O H2O 1264 147.370 -24.619 81.232 inf infATOM 1265 O H2O 1265 146.595 -24.262 81.250 inf infATOM 1266 O H2O 1266 147.727 -24.385 81.343 inf infATOM 1267 O H2O 1267 146.275 -23.851 81.183 inf infATOM 1268 O H2O 1268 148.255 -23.836 81.322 inf infATOM 1269 O H2O 1269 148.264 -23.487 81.336 inf infATOM 1270 O H2O 1270 146.617 -23.298 81.405 inf infATOM 1271 O H2O 1271 148.173 -23.069 81.308 inf infATOM 1272 O H2O 1272 147.002 -22.681 81.331 inf infATOM 1273 O H2O 1273 147.751 -22.915 81.420 inf infATOM 1274 O H2O 1274 146.632 -22.380 81.191 inf infATOM 1275 O H2O 1275 147.728 -22.392 81.160 inf infATOM 1276 O H2O 1276 147.001 -22.202 81.123 inf infATOM 1277 O H2O 1277 147.370 -24.195 81.476 inf infATOM 1278 O H2O 1278 147.370 -23.858 81.573 inf infATOM 1279 O H2O 1279 147.002 -23.487 81.559 inf infATOM 1280 O H2O 1280 147.009 -23.134 81.506 inf infTER______________________________________

Nonnucleoside inhibitors of reverse transcriptase, composite binding pocket and methods for use thereof

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Parent Case Info

US Referenced Citations (1)

Foreign Referenced Citations (1)

Non-Patent Literature Citations (43)

Divisions (1)