(12) United States Patent
`Erickson et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,470,506 B1
`Dec. 30, 2008
`
`US007470506B1
`
`(54) FITNESS ASSAY AND ASSOCIATED
`METHODS
`
`(75)
`
`Inventors: John W. Erickson, Frederick, MD (US);
`Sergei V. Gulnik, Frederick, MD (US);
`Hlroaki Mitsuya,
`Chase,
`Arun K. Ghosh, River Forest, IL
`
`(73) Assignees: The United States of America as
`represented by the Department of
`Health and Human Services,
`Washington, DC (US); Board of
`Trustees of the University of Illinois,
`Urbana, IL (US)
`
`( * ) Notice:
`
`Subject. to any disclaimer, the term of this
`patent 1S extended or adjusted under 35
`U.S.C. l54(b) by 0 days.
`
`EP
`EP
`EP
`
`Ep
`GB
`
`W0
`W0
`
`W0
`W0
`
`W0
`W0
`W0
`W0
`
`0 528 661 A2
`0 534 511 A1
`0 539 192 B1
`
`0 550 924 A1
`2276621
`
`2/1993
`3/1993
`4/1993
`
`7/1993
`10/1994
`
`WO 90/09191 A1
`W0 94/04492
`
`W0 94/05639
`W0 9404492
`
`W0 9405639
`W0 94/14793
`W0 95/06030
`W0 9506030
`
`8/1990
`3/1994
`
`3/1994
`3/1994
`
`3/1994
`7/1994
`3/1995
`3/1995
`
`*
`*
`
`*
`*
`
`(Continued)
`OTHER PUBLICATIONS
`
`(21) Appl‘ NO‘:
`(22) PCT Filed,
`
`09/720376
`Jun_ 23, 1999
`
`PCT/US99/14119
`
`(86) PCT NO‘.
`§ 371 (CX1),
`(2), (4) Date, Mar_ 7’ 2001
`NO‘,
`PCT Pub. Date: Dec. 29, 1999
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`Related U.S. Application Data
`(60) Provisional application No. 60/090,393, filed on Jun.
`23, 1998.
`
`(51)
`Int. C1.
`2006.01
`C12
`1/70
`....................... 435/5
`...........
`(52) U.S.QCl.
`..................
`h
`435/5.
`S
`58
`F_ 1d fcl
`_fi
`_
`(
`)
`le
`0
`ass] canon 515:/1:572 228 2’
`1.
`.
`fil f
`1
`’
`’ h hf
`’
`‘
`ee app lcauon
`e or Comp ete Seam lstory‘
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`(74) A3307’719% A897”; 07’ F17’m‘LeYd1gs V011 & Mayer: Ltd-
`
`(57)
`
`ABSTRACT
`
`The Pres?“ invemmn Pr°Vi.deS an @559 f°r Fleteirmining the
`biochemical fitness of a biochemical species in a mutant
`replicating biological entity relative to its predecessor. The
`present invention further provides a continuous fluorogenic
`assay formeasuring the anti-HIVprotease activity ofprotease
`inhibitor. The present invention also provides a method of
`administering a therapeutic compound that reduces the
`chances of the emergence of drug resistance in therapy. The
`present invention also provides a compound of formula (I) or
`a pharmaceutically acceptable salt, a prodrug, a composition,
`or an ester thereof, Where1nA1s 21 group of formulas (A), (B),
`(C) or (D); R1, R2, R3, R5 or R6 is H, or an optionally substi-
`tuted and/or heteroatom-bearing alkyl, alkenyl, alkynyl, or
`cyclic group; Y and/or Z are CH2, 0, S, SO, S02, amino,
`amides, carbamates, ureas, or
`thiocarbonyl derivatives
`~
`-
`~
`_
`thereof’OPUQMHYS“bStmlted.W“hana1ky1’a1l‘eny1’°ra1kY
`nyl group, 11 1S from 1 to 5, X IS a bond, an optionally substi-
`d
`h 1
`h 1
`.
`.
`.
`tute met y ene.or et y ene, an4amino, O or S, Q 1S C(O),
`C(S), 02302, H115 fr0m0t0 6; R ‘Is OH, :0 (keto), NH2, or
`alkylannno, Includmg esters, amldes, and salts thereof; and
`W is C(O), C(S), S(O), or S02. Optionally, R5 and R5,
`together with the N—W bond of formula (I), comprises a
`macrocycljc ring.
`
`9 Claims, 5 Drawing Sheets
`
`Janssen Ex. 2019
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR2015-01030
`
`(Page 1 of 31)
`
`

`
`US 7,470,506 B1
`Page 2
`
`FOREIGN PATENT DOCUMENTS
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`W0
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`W0
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`WO 96/28463
`W0 9633187
`WO 97/19055
`WO 99/65870
`WO 99/67254
`WO 99/67417
`WO 99/67417 A2
`W0 9967254
`WO 00/48466 A2
`
`9/1996
`* 10/1996
`5/1997
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`12/1999
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`1993).
`Ghosh et al., .I. Medicinal Chemistry, 36(2), 292-294 (Jan. 1993).
`Ghosh et al.,
`.I. Medicinal Chemistry, 37(16), 2506-2508 (Aug.
`1994).
`Ghosh et al., .I. Medicinal Chemistry, 37, 1177-1188 (Apr. 1994).
`Gulnik et al., Biochemistry, 34(29), 9282-9287 (Jul. 1995).
`Ho et al., .I. Wrology, 68(3), 2016-2020 (Mar. 1994).
`Huff, .I. Med. Chem., 34(8), 2305-2314 (Aug. 1991).
`Kageyama et al., Antimicrob Agents Chemother, 36, 926-933 (May
`1992).
`Kaplan et al., PNAS USA, 91, 5597-5601 (1994).
`Kim et al., .I. Medicinal Chemistry, 38(17). 1181-1182 (1995).
`Klabe et al., Biochemistry, 37(24), 8735-8742 (May 1998).
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`Ghosh et al., Tetrahedron Letters, 39, 4651-4654 (1998).
`Ghosh et al., 39d‘ Interscience Conference on Antimicrobial Agents
`and Chemotherapy, San Francisco, Calif., Session 89F, paper 928,
`(Sep. 26-29, 1999).
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`Holloway et al., .I. Med Chem., 38, 305-317 (1995).
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`L.S. Appl. No. 11/030,632, Utility Patent Application Transmittal
`with Fee Transmittal, filed Jan. 6, 2005.
`L.S. Appl. No. 11/030,632, Application Data Sheet, filed Jan. 6,
`2005.
`L .S. Appl. No. 11/030,632, Certificate ofExpress Mailing, filed Jan.
`6, 2005.
`L .S. Appl. No. 11/030,632. Preliminary Amendment signed Jan. 5,
`2005, filed Jan. 6, 2005.
`L .S. Appl. No. 11/030,632, Specification, Claims, andAbstract, filed
`Jan. 6, 2005.
`L .S. Appl. No. 11/030,632, Drawings, filed Jan. 6,2005.
`L.S. Appl. No. 11/030,632, Combined Declaration and Power of
`Attorney signed by John W. Erickson, Sergei V. Gulnik, and Hiroaki
`Mitsuya, filed Jan. 6, 2005.
`L .S. Appl. No. 11/030,632, Statement Under 37 C.F.R. 1.48(a)(2),
`filed Jan. 6, 2005.
`L.S. Appl. No. 11/030,632, Combined Declaration and Power of
`Attorney signed by Applicant Arun K. Ghosh, filed Jan. 6, 2005.
`I
`.S. Appl. No. 1 1/030,632, Request for Correction oflnventorship of
`Patent Application Under 37 C.F.R. 1.48(a), filed Jan. 6, 2005.
`L .S. Appl. No. 11/030,632. Written Consent ofAssignee (the Gov-
`ernment of the United States .
`.
`. ) Under 37 CFR. 1.48(a)(5), filed
`Jan. 6, 2005.
`L .S. Appl. No. 11/030,632. Written Consent ofAssignee (Board of
`Trustees of the University of Illinois) Under 37 C.F.R. 1.48(a)(5),
`filed Jan. 6, 2005.
`L .S. Appl. No. 11/030,632, Assignment from Aurn K. Ghosh to the
`Board of Trustees of the University of Illinois, filed Jan. 6, 2005.
`
`* cited by examiner
`
`Janssen Ex. 2019
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR2015-01030
`
`(Page 2 of 31)
`
`

`
`U.S. Patent
`
`Dec. 30,2008
`
`Sheet 1 0f5
`
`US 7,470,506 B1
`
`I
`N
`3\‘/A
`
`'
`211'’
`
`+
`
`Zk/NH -3'-P59§———-'>
`
`ao°c. 121:
`
`3
`
`12
`
`HQ ?/ 0Me
`NH°\/.\/N\
`
`I
`
`_
`
`0
`
`O
`
`K
`Ph
`
`15
`
`
`
`'
`
`Pyridine
`
`V
`
`,y
`Ho
`E /
`
`0142
`
`Q \
`O
`
`14
`
`N:
`
`'
`/
`
`Janssen Ex. 2019
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR2015-01030
`
`(Page 3 of 31)
`
`

`
`U.S. Patent
`
`Dec. 30, 2008
`
`Sheet 2 of 5
`
`US 7,470,506 B1
`
`<10
`
`21
`
`N-Iodosuccinimide
`
`Propargyl alcohol
`CH2Cl2, 0° to 23°C
`
`’ CL0
`
`0,, CH2Cl2-Me0H
`Me2S,
`-73° to 23°C
`
`NaBI-I4, EtOH
`-15°C, 1 h
`
`24
`
`Immobilized Lipase 30
`Aczo, DME, 23°C, 3 h
`
`
`
`Janssen Ex. 2019
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR2015-01030
`
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`
`

`
`U.S. Patent
`
`Dec. 30,2008
`
`Sheet 3 0f5
`
`Us 7,470,506 B1
`
`°\
`
`N
`
`. - °
`
`H
`
`x
`
`\\n//
`°
`
`II
`
`0
`
`H
`
`31
`
`Amine 15
`
`c1-Izcl,
`
`0
`
`H
`
`H
`
`"
`
`.
`‘
`E
`OJ
`
`25
`
`Disuccinimidyl
`carbonate
`Et3N, cH,cN
`Tetrahedron Letters
`1995, 36, s05
`
`5
`
`0
`
`H
`
`H
`
`I
`
`'0
`
`O
`
`‘-
`:
`5‘\J/’
`
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`Ph/
`
`I‘-IO
`.
`H
`N\\»//*\\v/’N\\ S
`
`O’ ‘*0
`
`32
`
`0
`
`Fig. 3A
`
`
`
`Et3N,
`
`c1-1,cN
`
`Disuccinimidyl
`carbonate
`
`37
`
`Tetrahedron Letters
`1995, 36, 505
`
`33
`
`Amine 15
`cH,c1,
`
`II-IO
`N
`‘
`E
`\/\_/ \s
`:
`00 \\o
`Ph’/)
`
`34
`
`0
`
`0Me
`
`0Me
`
`Janssen Ex. 2019
`
`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR201501030
`
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`
`

`
`U.S. Patent
`
`Dec. 30,2008
`
`Sheet 4 0f5
`
`US 7,470,506 B1
`
`N3
`
`0
`
`+
`
`(cH2)m
`|
`
`R3
`
`(1)
`
`1?
`NH
`
`=
`
`(ii)
`
`-————-————n.
`
`on
`
`5
`
`1‘
`N\H
`
`N3
`
`(cingm
`
`R’
`
`. ..
`(111)
`
`I-'\ /R6
`
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`
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`|
`11/”
`
`(CH2)m
`
`5
`
`1‘
`N\ /3
`W
`
`6
`
`.
`(V1)
`
`1
`
`03;‘
`Reduction,
`Reductive Amlnation
`‘with R2cH=o
`
`N3
`
`T5
`m\ /R,5
`W
`
`R4
`
`(C )H
`l
`2 m
`
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`
`x
`
`1.:
`
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`
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`
`z
`
`\(cH2 n
`(vii)
`
`1
`
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`
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`
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`J1
`\Q/
`
`R4
`
`Ts
`R5
`N
`\W/
`
`Z
`
`NCHZ n
`
`(CHg).,,
`I
`R3
`
`(32)
`
`Fig. 4
`
`Janssen Ex. 2019
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`Lupin Ltd. v. Janssen Sciences Ireland UC
`|PR2015-01030
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`
`

`
`U.S. Patent
`
`Dec. 30,2008
`
`Sheet 5 0f5
`
`Us 7,470,506 B1
`
`OH
`
`Janssen Ex. 2019
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`|PR2015-01030
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`
`

`
`1
`
`2
`
`US 7,470,506 B1
`
`FITNESS ASSAY AND ASSOCIATED
`METHODS
`
`TECHNICAL FIELD OF THE INVENTION
`
`The present invention relates to a biochemical fitness assay
`and related methods.
`
`BACKGROUND OF THE INVENTION
`
`The development of drug resistance is one of the most
`perplexing challenges in the field of medicine. One of the
`most common causes of drug failure in the treatment of dis-
`eases involving replicating biological entities, for example,
`cancer and infectious diseases, is the emergence of drug resis-
`tance. One of the most dramatic and tragic examples of drug
`resistance can be found in connection with the antiviral
`
`therapy of acquired immune deficiency syndrome (AIDS).
`AIDS is a fatal disease, reported cases of which have
`increased dramatically within the past several years. Esti-
`mates of reported cases in the very near future also continue
`to rise dramatically.
`The AIDS virus was first identified in 1983. It has been
`
`known by several names and acronyms. It is the third known
`T-lymphocyte virus (HTLV-III), and it has the capacity to
`replicate within cells of the immune system, causing pro-
`found cell destruction. The AIDS virus is a retrovirus, a virus
`that uses reverse transcriptase during replication. This par-
`ticular retrovirus is also known as lymphadenopathy-associ-
`ated virus (LAV), AIDS-related virus (ARV) and, most
`recently, as human immunodeficiency virus (HIV). Two dis-
`tinct families of HIV have been described to date, namely
`HIV-1 and HIV-2. The acronym HIV will be used herein to
`refer to HIV viruses generically.
`Specifically, HIV is known to exert a profound cytopathic
`effect on the CD4+ helper/inducer T-cells, thereby severely
`compromising the immune system. HIV infection also results
`in neurological deterioration and, ultimately, in the death of
`the infected individual.
`
`The field of viral chemotherapeutics has developed in
`response to the need for agents effective against retroviruses,
`in particular HIV. For example anti-retroviral agents, such as
`3'-azido-2',3'-dideoxythymidine
`(AZT), 2'3'-dideoxycyti-
`dine (ddC), and 2'3‘-dideoxyinosine (ddI) are known to
`inhibit reverse transcriptase. There also exist antiviral agents
`that inhibit transactivator protein. Nucleoside analogs, such
`as AZT, are currently available for antiviral therapy. Although
`very useful, the utility of AZT and related compounds is
`limited by toxicity and insufficient therapeutic indices for
`fully adequate therapy.
`Retroviral protease inhibitors also have been identified as a
`class of anti-retroviral agents. Retroviral protease processes
`polyprotein precursors into viral structural proteins and rep-
`licative enzymes. This processing is essential for the assem-
`bly and maturation of fully infectious virions. Accordingly,
`the design of protease inhibitors remains an important thera-
`peutic goal in the treatment ofAIDS.
`The use of HIV protease inhibitors, in combination with
`agents that have different antiretroviral mechanisms (e.g.,
`AZT, ddI and ddT), also has been described. For example,
`synergism against HIV-1 has been observed between certain
`C2 symmetric HIV inhibitors and AZT (Kageyarna et al.,
`Antimicrob. Agents Chemother, 36, 926-933 (1992)).
`Numerous classes of potent peptidic inhibitors of protease
`have been designed using the natural cleavage site of the
`precursor polyproteins as a starting point. These inhibitors
`typically are peptide substrate analogs in which the scissile
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`P1-P1‘ amide bond has been replaced by a non-hydrolyzable
`isostere with tetrahedral geometry (Moore et al, Perspect.
`Drug Dis. Design, 1, 85 (1993); Tomasselli et al., Int. J.
`Chem. Biotechnology, 6 (1991); Huff, .1. Med. Chem., 34,
`2305 (1991); Norbeck et al., Ann. Reports Med. Chem., 26,
`141 (1991); and Meek, J. Enzyme Inhibition, 6, 65 (1992)).
`Although these inhibitors are effective in preventing the ret-
`roviral protease from functioning, the inhibitors suffer fro1n
`some distinct disadvantages. Generally, peptidomimetics
`often make poor drugs, due to their potential adverse phar-
`macological properties, i.e., poor oral absorption, poor sta-
`bility and rapid metabolism (Plattner et al, Drug Discovery
`Technologies, Clark et al., eds., Ellish Horwood, Chichester,
`England (1990)).
`The design of the HIV-1 protease inhibitors based on the
`transition state mimetic concept has led to the generation of a
`variety of peptide analogs highly active against viral replica-
`tion in vitro (Erickson et al, Science, 249, 527-533 (1990);
`Kramer et al., Science, 231, 1580-1584 (1986); McQuade et
`al., Science, 247, 454-456 (1990); Meek et al., Nature (Lon-
`don), 343, 90-92 (1990); and Roberts et al., Science, 248,
`358-361 (1990)). These active agents contain a non-hydro-
`lyzable, dipeptidic isostere, such as hydroxyethylene (Mc-
`Quade et al., supra; Meek et al., Nature (London), 343, 90-92
`(1990); and Vacca et al., J. Med. Chem., 34, 1225-1228
`(1991)) or hydroxyethylamine (Ghosh et al., Bioorg. Med.
`Chem. Lett., 8, 687-690 (1998); Ghosh et al., J. Med. Chem.,
`36, 292-295 (1993)); Rich et al., J. Med. Chem., 33, 1285-
`1288 (1990); and Roberts et al., Science, 248, 358-361
`(1 990)) as an active moiety that mimics the putative transition
`state of the aspartic protease-catalyzed reaction.
`Two-fold (C2) symmetric inhibitors of HIV protease rep-
`resent another class of potent HIV protease inhibitors, which
`were created by Erickson et al., on the basis of the three-
`dimensional symmetry of the enzyme active site (Erickson et
`al. (1990), supra). Typically, however, the usefulness of cur-
`rently available HIV protease inhibitors in the treatment of
`AIDS has been limited by relatively short plasma half-life,
`poor oral bioavailability, and the technical difficulty of scale-
`up synthesis (Meek et al. (1992), supra).
`In a continuing effort to address the problem of short
`plasma half-life and poor bioavailability, new HIV protease
`inhibitors have been identified. For example, HIV protease
`inhibitors incorporating the 2,5 -diamino -3 ,4-di sub stituted-1 ,
`6-diphenylhexane isostere are described in Ghosh et al.,
`Bioorg. Med. Chem. Lett., 8, 687-690 (1998) and U.S. Pat.
`Nos. 5,728,718 (Randad et al.). HIV protease inhibitors,
`which incorporate the hydroxyethylamine isostere, are
`described in U.S. Pat. Nos. 5,502,060 (Thompson et al.),
`5,703,076 (Talley et al.), and 5,475,027 (Talley et al.).
`Recent studies, however, have revealed the emergence of
`mutant strains of HIV, in which the protease is resistant to the
`C2 symmetric inhibitors (Otto et al., PNAS USA, 90, 7543
`(1993); Ho et al., J.
`I/irology, 68, 2016-2020 (1994); and
`Kaplan et al., PNAS USA, 91, 5597-5601 (1994)). In one
`study, the most abundant mutation found in response to a C2
`symmetry based inhibitor was Arg to Gln at position 8 (R8Q),
`which strongly affects the S3/S3. subsite of the protease bind-
`ing domain. In this study, the shortening ofthe P3/P3. residues
`resulted in inhibitors that were equipotent towards both wild-
`type and R8Q mutant proteases (Majer et al., 13th American
`Peptide Symposium, Edmonton, Canada (1993)). Inhibitors
`have been truncated to P2/P2‘ without significant loss of
`activity (Lyle et al., J. Med. Chem., 34, 1230 (1991); andBone
`et al., J Am. Chem. Soc., 113, 9382 (1991)). These results
`suggest that inhibitors can be truncated and yet maintain the
`crucial interactions necessary for strong binding. The benefits
`
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`
`US 7,470,506 B1
`
`4
`
`3
`present invention permits the administration ofan inhibitor or
`of such an approach include the elimination of two or more
`peptide bonds, the reduction of molecular weight, and the
`combination of inhibitors to treat a disease in a way that
`dimini shment of the potential for recognition by degradative
`decreases the likelihood that drug resistance will develop.
`enzymes.
`The present invention further provides a continuous fluo-
`More recently, new mutant strains of HIV have emerged
`rogenic assay for measuring the anti-HIV protease activity of
`that are resistant to multiple, structurally diverse, experimen-
`a protease inhibitor. The continuous fluorogenic assay of the
`tal and chemotherapeutic retroviral protease inhibitors. Such
`present invention utilizes a substrate of the formula Ala-Arg-
`multidrug-resistant HIV strains are typically found in
`Val-Tyr-Phe(NOa)-Glu-Ala-Nle-NH2. The continuous fluo-
`infected patients, who had undergone treatment with a com-
`10 rogenic assay of the present invention is highly sensitive and
`bination of HIV protease inhibitors or a series of different
`particularlyuseful forthe predictionoftheantiviral inhibitory
`HIV protease inhibitors. The number of reported cases of
`activity of a ccnincnnd against nintant HIV.
`patients infected with multidrug-resistant HIV.1S rising dra-
`The present invention further provides a method of admin
`matically. Tragically for these patients, the available options
`,
`,
`h
`,
`d h
`,nh,b,
`b,
`h
`,
`1
`for AIDS chemotherapy and/or HIV management is severely
`lstenng a t erapeuuc Cempount
`t ‘at 1
`.1 “Sta loctemlca
`limited or is, otherwise, completely nonexistent.
`target of a disease-causing replicating. biological entity. The
`Dntg tesistanee is nnfertnnatety tne ntest eemmen reason
`thetapeutle e0h1P011hds Wheh adhllhlstered 1h aeeefdahee
`for drug failures generally. One of the most dramatic
`Wlth the I11eth0d Of the Present 1hVeht10h, 1h1h1h11ZeS the
`examples of drug failure due to resistance is in HIV therapy.
`chances that the disease-causing entity will develop drug
`Once HIV resistance is obtained to first-line therapy, the
`chances of future success are greatly diminished because of 20 resistance.As such, the method ofadministering atherapeutic
`the development ofmultidrug cross resistance. Other diseases
`ccnipcnnd in accordance with tnc picscnt invcnticn inipicvcs
`involving infectious agents (e.g., viruses, bacteria, protozoa,
`tne enanees ef1eng_tenn sneeess in therapy.
`,
`and prions) or other disease-causing cells (e.g., tumor cells)
`Th
`h d f dm,
`,
`,
`h
`present similar challenges in that drug resistance is a primary
`e Present met .0
`0. a
`tlmstermg a t erapeuuc Com"
`eanse of dntg fantne.
`25 pound involves the identification of at least one mutant rep-
`In view of the foregoing problems, there exists a need to
`heatlhg hletegleal eht1tY (the mutant) eapahle Of eV01V1hg
`determine whether a mutant will be capable of replicating in
`from the disease-causing replicating biological entity (the
`the presence of a drug. There also exists a need for a method
`predecessor). Biochemical fitness is determined by compar-
`of predicting Whether drug resistance is likely to emerge in a
`ing the biochemical vitality of the mutant’s biochemical tar-
`disease involving areplicatingtbiological entity. There is also 30 get with tnc biccncniicai Vitality cf tnc nicdcccsscns bio-
`a need for a memod of devlsmg a 1oug'term mempeuue
`chemical target. Biochemical fitness is determined in the
`regimen that minimizes the ukelmood, thatresistance ‘mu
`presence ofa drug (e.g, aninhibitor).The biochemical vitality
`occur in a disease involving a replicating biological entity.
`,
`.
`.
`.
`.
`.
`.
`.
`of the mutant s biochemical target is compared to biochemi-
`Moreover, there is a need for a method of preventing or
`1
`,
`1,
`f h
`d
`, b, h
`,
`1
`,
`h
`inhibiting the development of drug resistance in such dis- 35 Ca Vlta my 0 t e pre ecessor 5
`1°C emlca target In t e
`eases.
`presence of the drug. When there are two or more drugs
`The picscnt invention provides Such methods. Tncsc and
`available for treatment, biochemical fitness can be deter-
`other advantages of the present invention, as well as addj-
`mined for each drug in accordance with the present invention.
`tional inventive features, will be apparent from the descrip-
`A therapeutic compound is then administered from among
`ti0I1 Of the iI1VeI1ti0I1 pr0Vided herein.
`40 one of the compounds that produces a lower value for bio-
`chemical fitness with respect to one or more mutants. Admin-
`istration of a therapeutic compound producing a lower fitness
`value for a particular mutant indicates that the predecessor is
`.
`.
`.
`less likely to develop resistance in the presence of that com-
`d
`poun ‘
`The present invention also provides amethod ofpreventing
`the development ofdrug resistance of HIV in an HIV-infected
`mammal by the administration ofa drug resistance-inhibiting
`.
`effeeuve amoum of a eompouud of me formula:
`
`15
`
`BRIEF SUMMARY OF THE INVENTION
`,
`,
`,
`,
`,
`,
`The present invention is predicated on the surprising and
`.
`.
`.
`“ .
`.
`,,
`unexpected discovery that biochemical
`vitality,
`as 45
`described below, can be used to determine the biological
`fitness of a mutant replicating biological entity relative to its
`Ptedeeeeser uhdet the eeteetteh Pressure Of ah thhthtteh The
`Preseut mvemlon prOV1.deS an essay for de.term1m.ng the bloe
`chemical fitness of a biochemical target (i.e., a biomolecule 50
`having a biochemical function), of a mutant replicating bio-
`logical entity relative to its predecessor’s biochemical target,
`in the presence of a compound that acts upon the biochemical
`target. The assay method of the present invention includes
`obtaining the predeces sor, determining the biochemical vital- 55
`ity of the biochemical target of both the predecessor and the
`mutant in the presence of a compound that acts upon the
`biochemical target of the predecessor, and comparing the
`vitality of the mutant’s biochemical target relative to the
`vitality of the predecessor’s biochemical target. Where the 60
`.
`.
`.
`biochemical vitality of the mutant is greater than the bio-
`chemical fitness ofthe predecessor, the mutant is predicted to
`be more biologically fit in the presence ofthe compound. The
`assay method can thus be used to predict the emergence of
`drug resistance for a particular replicating biological entity 65
`(e.g., a disease-causing cell) in the presence a drug (e.g., an
`inhibitor). Utilization of the assay in accordance with the
`
`R2
`|
`/X\ /N
`A
`Q
`
`R4
`
`R5
`
`(n
`‘
`
`/R5,
`
`N\
`
`W
`
`2 m
`(CH )
`|
`R3
`
`or a pharmaceutically acceptable salt, a prodrug, or an ester
`thereof, or a pharmaceutical composition thereof, wherein:
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`

`
`5
`A is a group of the formula:
`
`R1
`
`
`
`R1 is H or an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a
`cycloalkylalkyl, an aryl, an aralkyl, a heterocycloalkyl, a het-
`erocycloalkylalkyl, a heteroaryl, or a heteroaralkyl radical,
`which unsubstituted or substituted;
`Y and Z are the same or different and are each selected from
`
`the group consisting of CH2‘ O, S, SO, SO2, NR8, R8C(O)N,
`R8C(S)N, R8OC(O)N, R8OC(S)N, R8SC(O)N, R8R9NC(O)
`N, and R8R9NC(S)N, wherein R8 and R9 are each H, an alkyl,
`an alkenyl, or an alkynyl;
`n is an integer from 1 to 5;
`X is a covalent bond, CHRIO, CHRIOCH2, CHZCHRIO, O,
`NR1°, or S, wherein R10 is H, an alkyl, an alkenyl, or an
`alkynyl;
`Q is C(O), C(S), or SO2;
`R2 is H, an alkyl, an alkenyl, or an alkynyl;
`m is an integer from 0 to 6;
`R3 is a cycloalkyl, a heterocycloalkyl, an aryl, or a het-
`eroaryl which is unsubstituted or substituted;
`R4 is OH, :O (keto), NH2, or a derivative thereof;
`R5 is H, a C1-C6 alkyl radical, a C2-C6 alkenyl radical, or
`(CH2)qR14, wherein q is an integer form 0 to 5, and R14 is a
`cycloalkyl, a heterocycloalkyl, an aryl, or a heteroaryl which
`is unsubstituted or substituted;
`W is C(O), C(S), S(O), or SO2; and
`R6 is a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl
`which is unsubstituted or substituted.
`
`Optionally, R5 and R6, together with the N—W bond of for-
`mula (I), comprise a macrocyclic ring which can contain at
`least one additional heteroatom in the ring skeleton.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 illustrates the synthesis of a particular sulfonamide
`isostere core of a compound of the present invention.
`FIG. 2 illustrates the synthesis of a bis-tetrahydrofuran
`ligand and the optical resolution thereof.
`FIG. 3A illustrates the synthesis of a compound of the
`present invention via coupling of a bis-tetrahydrofuran ligand
`to a sulfonamide isostere of the present invention.
`FIG. 3B illustrates the synthesis of a compound of the
`present invention via coupling of a bis-tetrahydrofuran ligand
`to a sulfonamide isostere of the present invention.
`FIG. 4 illustrates generally the present method of synthe-
`sizing a compound of the present invention.
`FIGS. 5A-5D illustrate the structures of particular com-
`pounds that were tested against various drug resistant HIV
`mutants.
`
`US 7,470,506 B1
`
`6
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`The present invention is predicated on the surprising and
`unexpected discovery to that the “vitality” of a biochemical
`target of a mutant replicating biological entity relative to that
`of its predecessor’ s biochemical target can be used to predict
`the biological fitness of the mutant under the selection pres-
`sure ofan inhibitor ofthe biochemical target. The “vitality” of
`a biochemical target of a mutant replicating biological entity
`relative to the “vitality” of its predecessor’s biochemical tar-
`get is defined herein as the “biochemical fitness.”
`“Vitality” as utilized herein describes the ability of a par-
`ticular biomolecular “target” (i.e., a biochemical species
`intended to be inhibited by a particular inhibitor) to perfonn
`its biochemical function in the presence of the inhibitor. Bio-
`chemical vitality is a function of at least two variables: the
`ability of a particular inhibitor to inhibit a biochemical target
`ofthe replicating biological entity in question, and the ability
`of the cell’s biochemical target to inherently perform its bio-
`chemical function (irrespective of an inhibitor). Biochemical
`vitality also can include other factors that effect the ability of
`a biochemical target to perform its biochemical function in
`the presence of the inhibitor.
`for
`in question can include,
`The biochemical
`target
`example, a biochemical species with one or more known or
`unknownbiological functions. The biochemical target canbe,
`for example, a biochemical species having one or more spe-
`cific biochemical function, or it can be a biochemical species
`that effects or influences a biochemical function directly or
`indirectly. Suitable biochemical targets include, for example,
`enzymes, proteins, oligomers, receptors, and the like. Suit-
`able enzymes include, for example, reverse transcriptases,
`proteases (e.g., retroviral proteases, plasmepsins, and the
`like), methylases, oxidases, esterases, acyl transferases, and
`the like. Suitable enzymes also include, for example, viral and
`non-viral helicases, topoisomerases, DNA gyrases, DNA and
`RNA polymerases, parasite-encoded proteases, and the like.
`Suitable proteins include, for example, proteins that incor-
`porate a conformational change as a maj or functional require-
`ment, and the like. Examples of such proteins include HIV
`gp4l a11d other fusogenic viral proteins and peptides, topoi-
`somerases, and all DNA enzymes, and the like.
`Suitable oligomers include, for example, oligomers that
`require oligomerization in order to perform their biochemical
`function. Examples of such oligomers include HIV protease,
`retroviral fusion proteins, peptides, HIV gp 41, viral and
`non-viral membrane fusion proteins, tumor suppressor pro-
`teins (e.g., p53, and the like) prions, ribosomes, and the like.
`The ability of a particular inhibitor to inhibit a biochemical
`target of a particular replicating biological entity canbe deter-
`mined by any suitable method and/or can be obtained fro1n
`any suitable source. The ability of a particular inhibitor to
`inhibit a biochemical function of a replicating biological
`entity can be determined, for example, on the basis of a
`measurable property, or a measurable relationship of proper-
`ties, that correlate with the ability ofthe inhibitor to inhibit the
`target. Suitable methods for determining the ability of the
`inhibitor to inhibit the target include, for example, assays, and
`the like. In some instances, the ability of the inhibitor to
`inhibit the target can be obtained from one or more suitable
`sources, for example, assay data from a database, a textbook,
`or the literature.
`
`When the biochemical target is a protein, the ability of an
`inhibitor to inhibit
`the protein can be determined,
`for
`example, by obtaining the equilibrium dissociation constant
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`
`US 7,470,506 B1
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`7
`(Kd) of drug binding to the target where drug binding inter-
`feres with the function of the protein.
`When the biochemical target is an enzyme, the ability of an
`inhibitor to inhibit
`the enzyme can be determined,
`for
`example, by obtaining the inhibition constant (Klllh), or the
`like. The inhibition constant can be in terms ofdrug inhibition
`constant for the effect of the drug on substrate catalysis (e.g.,
`Kl) or dissociation constant for drug binding (e.g., Kd) where
`drug binding correlates with inhibition of enzyme function.
`When the biochemical target is an oligomer, the ability of
`an inhibitor to inhibit the oligomer can be determined, for
`example, by obtaining the equilibrium dissociation constant
`(Kd) for drug binding where drug binding interferes with
`oligomerization of the target.
`Where the biochemical target is a protein that requires a
`conformational change for its function, the ability of an
`inhibito