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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
  • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
  • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
  • C07D307/18—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D307/20—Oxygen atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

• This application is generally in the field of drugs to treat drug resistant pathogens, and in particular relates to protease inhibitors that do not elicit drug-resistant mutations in the pathogens they inhibit, such as the human immunodeficiency virus (HIV).
• HIV human immunodeficiency virus
• Drug resistance generally is a problem with the treatment of most pathogens, including bacteria and viruses.
• a variety of methods have been used, the most common being determining which drugs the pathogen is sensitive to, then treating the patient with a drug that the pathogen is sensitive to.
• Another approach is the use of a "cocktail", a mixture of two or three different drugs, preferably operating by different mechanisms of action, to block the life cycle of the pathogen before it can develop drug resistance.
• a virus such as HIV
• this latter approach has been widely adopted, primarily through the use of one or two nucleoside drugs that inhibit replication by interacalation into the viral nucleic acid, in combination with a protease inhibitor that prevents replication.
• HIV protease gene codes for a protease which, upon expression as part of the gag-pol protein, processes gag and gag-pol polyproteins into individual structural proteins and enzymes for the assembly of HIV virions (Debouck et al. (1987), J. Med. Chem. Res. 21:1-17). Mutation of the active- site residues of HlVPr renders the mutant virus non-infectious (Kohl et al. (1988), Proc. Natl. Acad. Sci. USA 85:4686-4690; Peng et al. (1989), J.
• HlVPr inhibitors have been synthesized and tested (Wlodawer and Erickson (1993), Ann. Rev. Biochem. 62:843-855), among which four have been marketed: saquinavir (Ro 31-8959, Craig et al. (1991), Antiviral Res. 16:295-305), indinavir (L-735,524, Dorsey et al. (1994), J. Med. Chem. 37:3443-3451), ritonivir (ABT-538, Kempf et al. (1995), Proc. Natl. Acad. Sci.
• HINPr aspartic protease
• proteins include the identification of the HTVPr genome, expression and purification of recombinant enzyme, total chemical synthesis (Schneider and Kent (1988), Cell 54:363-368), crystal structure of HIV-1 protease (Wlodawer et al. (1989), Science 245:616-621; ⁇ avia et al. (1989), Nature 337:615-620; Lapatto et al. (1989), Nature 342:299-302) and HIV-2 protease (Mulichak and Watenpaugh (1993), J. Biol. Chem. 268:13103-
• the active HlVPr is a homodimer of 99-residue monomers.
• the active-site cleft is located between two monomers with two Asp 25 residues forming the catalytic apparatus.
• the active-site cleft is covered by two flaps and can accommodate eight substrate residues.
• the specificity of the enzyme is somewhat broad (Poorman et al. (1991), J. Biol. Chem. 266:14554-14561) which is consistent with the sequence differences of the eight natural processing sites.
• An unique specificity of HlVPr is the ability to cleave an X-Pro bond, which appears to be related to the mobility of the active site of the enzyme (Hong et al. (1998), Protein Sci. 7:300-305).
• the specificity of the subsite pockets is also influenced by the side chains bound in adjacent pockets (Ridky et al. (1996), J. Biol. Chem. 271 :4709-4717).
• Each of the four commercial HlVPr inhibitor contains an isostere - CH(OH)-CH 2 - which mimics the transition state in the catalytic mechanism of aspartic proteases (Marciniszyn et al., 1976), J. Biol. Chem. 251:7088- 7094) thereby rendering the tight binding properties of the inhibitor.
• the position of the isostere which is equivalent to that of the scissiled bond in the substrate, defines the subsite binding for the inhibitor residues.
• An example can be seen in a non-commercial inhibitor U-85548 in Figure la.
• the commercial inhibitor drugs which require good pharmacokinetic properties and high potency, are typically shorter and have less well defined residue boundaries ( Figures lb-d).
• HlVPr inhibitors The interaction of subsite residues in HlVPr with the inhibitors are generally known from the crystal structures of the HIVPr-inhibitor complexes (Wlodawer and Erickson, 1993). The ability of HlVPr inhibitors to suppress HIV replication has been demonstrated in tissue culture and in clinical trials (Wei et al., 1995; Ho et al., 1995). The use of HlVPr inhibitors along with other drugs in combination therapy has offered the best results so far in suppressing HIV propagation in vivo (Mellors, 1996). The first transition-state analogue of aspartic proteases discovered was pepstatin by Marciniszyn et al. (1976).
• isosteres were later designed and shown to be effective in aspartic protease inhibitors. These include, in addition to hydroxyethylene, dihydroxyethylene [-CH(OH)-CH(OH)-], hydroxyethylamine [-CH(OH)- CH 2 . NH-], phosphinate [-PO(OH)-CH 2 -] and reduced amide [-CH 2 -NH-] (Reviewed by Vacca, 1994).
• a single transition-state isostere is used in an inhibitor since it mimics a substrate peptide with a single hydrolysis site.
• U-85548 is an HIV protease inhibitor, but it is not marketed as anti-HIV drug. We use U-85548 here to illustrate the principle of HIV Protease inhibitor design.
• the other 3 (lb, lc, Id) are drugs.
• Protease inhibitors especially viral protease inhibitors such as HlVPr inhibitors, which are effective against drug resistance resulting from the mutations in the protease gene have been developed. These compounds contain two or more isosteres - CH(OH)-CH 2 - which each mimic the transition state in the catalytic mechanism of the protease. Design and testing of the inhibitors containing two or more isosters is demonstrated using an HlVPr inhibitor. Unlike known commercial HlVPr inhibitors, these inhibitors do not contain only one isoster having a single orientation which binds to the HlVPr active site at only one mode. These HlVPr inhibitors bind to HlVPr in two or more modes.
• Figures la-Id are formulas of U-85548 (Figure la) and known HlVPr inhibitors which have been clinically marketed; Saquinaur or R031 -8959 ( Figure lb); Indinavir or L-735,525 ( Figure lc); and Ritonavor ABT-538 ( Figure Id).
• Figure 2 is a schematic of UIC 98-056.
• Figure 3 is a schematic of the synthesis of UIC-98-056.
• Figure 4 is a graph of the relative Kj values of three commercial
• the Kj values of the wild-type HlVPr are shown in Table II.
• the relative Kj values (in parenthesis of Table II and in Fig. 4) of the ten resistant mutants were calculated from the ratio of Kj of the mutant HIVPr/Kj of the wild-type HlVPr.
• the key to the numbering of the mutants is shown in the inset of Fig. 4. Detailed Description of the Invention
• HlVPr inhibitors The design and testing of these protease inhibitors is exemplified using HlVPr inhibitors. It is understood, however, that this concept is generally applicable to protease inhibitors, especially aspartic acid protease inhibitors. Design of HlVPr Inhibitors Effective against Drug Resistant Mutants
• Table I HIV-1 protease resistant mutants compiled from the result of clinical trials against three commercial HlVPr inhibitor drugs indinavir, ritonavir and saquinavir.
• the resistant mutants of HlVPr inhibitors have three dimensional structural changes from that of the wild-type enzyme.
• the x-ray crystal structures of several resistant mutants of HlVPr have been studied (Chen et al, 1995, J. Biol.Chem. 270:21433-21436; Baldwin et al., 1995, Nature
• HlVPr Although not all the structural factors involved in resistance is understood at the present, it is known that one of the most frequent structural changes as a consequences of resistant mutation of HlVPr is the change of the subsite side chain pockets of the enzyme which causes the inhibitor to bind less effectively. Some of the resistant mutation sites are not located in the subsite pockets. However, due to the flexibility of HlVPr conformation (Ridky et al., 1996), J. Biol. Chem. 271:4709-4717), the change of subsite pocket conformation can be induced from a distance.
• an isostere is placed in the polypeptide backbone (or equivalent) of the inhibitor to mark the position of the scissile peptide bond and to mimic the transition state.
• the position of the isostere defines the assignments of the side chains (A to F) to different subsites of the enzyme. (There are eight subsites).
• the substrate subsites on the amino-terminal side of the scissile bond are Pi, P 2 , P 3 and P 4 in that order, and the substrate subsites on the carboxyl-terminal side of the scissile bond are Pi', P ', P 3 ' and P 4 '.
• the 8 corresponding subsite binding pockets in the enzyme are named Si (for binding Pi), S 2 (for binding P 2 ) . . . . and so on.
• Inhibitor 1 is named Si (for binding Pi), S 2 (for binding P 2 ) . . . . and so on.
• the side chains of the inhibitor are designed to fill the subsite pockets of HlVPr, thus creating a tight binding.
• each residue (A to F) has two subsite- assignments:
• residue A can be in either P 2 or P and residue E can bind in either P ' or Pi' and so on.
• Efree + Ifree Eli (1)
• Efree ⁇ Ifree EI 2 (2) where Ef ree and If ree are unbound enzyme and inhibitor respectively. Eli and EI 2 are inhibitor bound to enzyme by a first isotere and by the second isostere, respectively.
• the overall inhibition constant, K tract of a two-isostere inhibitor for the enzyme is K ⁇ CK x K ⁇ / CKu + K ⁇ ) (3)
• K, ⁇ and K 1>2 both to be 1 x 10 "9 M, based on equation (3), the overall inhibition constant, K tribe is 0.5 x 10 '9 M, lower than either K,, ⁇ or K,, 2 .
• HlVPr inhibitors Although described herein with specific reference to design of HlVPr inhibitors, it is readily apparent that this concept is generally applicable to the development of effective therapeutics which are targeted against other proteases, especially those aspartic proteases of viral origin.
• human cathespin D is involved in breast cancer metastasis (Rochefort (1990) Semin. Cancer Biol. 1: 153-160) and in the development of Alzheimer disease in the brain (Siman et al. (1993) J. Biol. Chem. 268: 16602-16609).
• the design of transition-state inhibitors for cathespin D to control these diseases has been attempted (Majer et al. (1997) Protein Sci. 6: 1458-1466).
• Human renin an aspartic protease
• proteases in pathogens For example, malaria causing protozoa Plasmodium contains two aspartic proteases, plasmepsin I and II, which are also targets for transition-state inhibitor drugs (Carroll et al. (1998) Bioorg. Med. Chem. 8: 2315-2320; Carroll et al. (1998) Bioorg. Med. Lett. 8: 3203-3206).
• Retroviruses which cause in addition to immunodeficiency and leukemia in human and animals and different tumors, contain aspartic proteases with processing functions similar to that of HIV protease (Weiss et al. (1984) RNA Tumor Viruses, Molecular biology of Tumor Viruses, Second Edition, Vol. l,Cold Spring Harbor, NY). These proteases are all drug design targets for the control of diseases.
• the human genome also contains an endogenous virus which expresses active aspartic protease, which has been studied for inhibition by transition-state, isostere-containing inhibitors (Towler et al. (1998) Biochemistry >1 : 17137-17144).
• Drugs targeted to these protease can benefit from the design utilizing two or more isosteres in a single inhibitor molecule in order to enhance the potency and withstand development of resistance.
• isosteres which mimic the transistion state of aspartic protease catalysis are shown by Vacca, "Design of Tight-Binding Human Immunodeficiency Virus Type 1 Protease Inhibitors", Methods in Enzymology, 241, 313-333 (1994).
• the protease inhibitors described herein are administered to a patient in need of treatment, or prophylactically, using methods and formulations similar to those for other HlVPr inhibitors.
• the protease inhibitor is preferably administered orally.
• the protease inhibitor is most preferably administered as part of a "cocktail" including other anti-HIV compounds such as the nucleosides like AZT.
• the most recent guideline for such therapy by the International AIDS Society is described in Carpenter, Fischel, Hammer et al. (1998) J. Am. Med. Assoc. 280: 78-86.
• the regimens and the choice of drug combinations are dependent on the resistance genotype and phenotype of the HIV strains.
• the therapeutic strategy is summarized by Larder, Richman and Vella (1998) HIV Resistance and Implications for Therapy, MediCom.
• Example 1 Design and Synthesis of Two-isostere HlVPr Inhibitor UIC-98-056. Based on the principle described above and other considerations,
• HlVPr inhibitor UIC-98-056 was designed and synthesized. The structure of this inhibitor is shown in Figure 2.
• HTV Protease Inhibitor UTC-98-056 The synthesis of HIV protease inhibitor UIC-98-056 with hydroxyethylene and hydroxyethylamine isosteres is outlined in Figure 3.
• the known lactone 1 was converted to acid 2 by lithium hydroxide mediated hydrolysis followed by protection of the alcohol functionality as tert- butyldimethylsilyl ether (Ghosh et al., 1998, Synthesis, 937 (Review); Ghosh et al., 1991, J. Org. Chem. 56:6500; Evens et al., 1985).
• the previously described (Ghosh et al., 1992, J. Chem. Soc, Chem.
• azido epoxide 3 was reacted with isobutylamine in 2-propanol at 80°C for 4 h and the resulting azidoalcohol was treated with m-tetrahydropyranyloxybenzenesulfonyl chloride 4 (Metanilic acid was diazotized at 0°C and the resulting salt was boiled with water to obtain 3-hydroxybenzene sulfonic acid which was then treated with thionyl chloride/catalytic DMF/reflux to obtain sulfonyl chloride.
• the hydroxy group of the resulting 3-hydroxybenzene sulfonyl chloride was protected as THP ether by treating with DHP/catalytic PPTS in rnethylene chloride to get 4 as an oil.) in the presence of aqueous NaHCO to provide the sulfonamide derivative 5.
• the azide functionality of 5 was hydrogenated over 10% Pd-C in methanol to afford the corresponding amine which was coupled with the acid 2 in the presence of l-[3-dimethylaminopropyl]-3- ethylcarodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) to afford the amide 6.
• Example 2 Demonstration that Inhibitor UIC-98-056 Can Withstand Resistance.
• the inhibition constant, K, of UIC-98-056 was determined for the wild type HIV-1 protease and 10 mutants resistant to HlVPr inhibitors using the methods described by Ermolieff et al. (1997), Biochemistry 36:12364- 12370.
• the wild-type HIV-1 Pr was produced as recombinant enzyme in E. coli as described by Ido et al. ( 1991) J. Biol. Chem. 266:24359-24366.
• mutant enzymes were made by site-directed mutagenesis of the HTVPr gene either as described by Ermolieff et al., 1997, Biochemistry 36:12364-12370 or by a similar procedure. These mutants were identified in clinical trials and in vitro studies to resist saquinvair (mutants G48V and L90M, Jacobsen et al., 1996, J. Infect.
• indinavir mutants V82A, M46I and L10I, Condra et al., 1995; Lander, Richman and Vella (1998) HIV Resistance and Implications for Therapy MediCom.
• ritonavir mutants L90M, V82A, K20R and M46I, Molla et al, 1996, Nat. Med. 2:760-765; Carder et al. (1998)).
• K, values against the wild- type HlVPr and ten mutants were also determined for saquinavir, indinavir and ritonavir.
• Table II shows these K, values and the ratios (in parenthesis) between the inhibition constant of the mutants, K, )Tnut , to the inhibition constant of the wild-type HINPr taken as 1.0.
• the latter results are also plotted in Figure 3. It can be seen clearly that for three commercial drugs, the K, ⁇ mut values of the resistant mutants are consistently higher than K, of the wild-type HlVPr (Table II and Figure 3), indicating the resistance and cross-resistance properties of the mutants. In contrast, the same comparison for inhibitor UIC-98-056, the K, ⁇ mut values are nearly the same as K, value of the wild- type HINPr for all mutants except mutant I84V, which increased 9-fold.

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