`
`Lupin Ex. 1084 (Page 1 of 14)
`Lupin Ex. 1084 (Page 1 of 14)
`
`

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`Lupin Ex. 1084 (Page 2 of 14)
`Lupin Ex. 1084 (Page 2 of 14)
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`_i MEDICINAI, -_
`CHEMISTRY
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`Registered in US. Patent and Trademark Office
`© Copyright 1.993 by the American Chemical Society
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`'
`
`volume 36, Number 16
`August 6, 1993
`-
`
`JMCMAR 36(16) 2243—2430 (1e92,)
`IISSN 0022-2623
`
`
`
`COMMUNICATIONS TO THE EDITOR
`
`2420 Application of a Conformationally Restricted Phe—Leu Dipepfide Mmetic to the Design of a Combined Inhibitor of
`Angiotensin I-Converting Enzyme and Neutral Endopeptidase 24.11
`'
`Gary A. Flynn.‘ Douglas W. Beight, Shujaath Mehdi, Jack R. Koehl, Eugene L.‘Giroux, John F. French,
`Paul W. Hake, and Richszd C. Dage
`
`ARTICLES
`
`-_
`
`-
`
`'
`
`'-
`
`2243 Anticonvulsant Activities of Some Arylsemjoerhazonee Displaying Potent Oral Activity in the Maximal Electroshock
`l
`Screen in Rats Accompanied by High Protection Indioes
`J. It. Dimmek,‘ K. E. Sidhu, R. S. Thayer, P. Mack, M. J. Duffy, R. 3. Reid. J. W. Quail, U. Pugashenthi,
`A. Ong, J. A. Bikker, and D. F. Weaver
`
`2253 Nonpeptide 'Angiotensin 1] Receptor Antagonists. 2. Design, Synthesis, and Structure—Activity Relationships of
`2-Alkyl-4—(LH—pyrrol-l-yl)-lH-imidazo'1e Derivatives: Profile of 2-Propyl—1-[[2’-(1H-tetrazol—5—yl)—[1,1’-biphenyl]-4-yl]-
`methyl]-4—{2é(trifluoroeoetyl)—lH-pyrrol-l-yl]-1H—imida.zole—5—carhoxylic Acid (CI-996}.
`Ila Bil-"oer,at John C. Badman-John Quin III, Amy M. Bunker, R. Thomas Winters, Jeremy J. Edmunds,
`Catherine R. Kostlan, Cleo Connolly, Stephen J. Kesten, James M. Hamby, John G. Topliss,
`_Joa_.n_A. Kaiser, and Robert L. Panel:
`2266 Studies-on 'Neurokinin Antes nistii. 3. 'Desig'n and Sh‘ucture—Acfivity-Relationships of'lilewiiBranched Tripeptides
`' N“-{Substituted L-aspartyl, L-omithyl, or L-lysyll-N—methyl-N-(phenylmethylHrphenylslsninamidee as Substance P
`Antagonists
`-
`-
`Daijiro Hagiware, Hirosbi Miyake, Eenji Moreno, Eireshi Morimoto, Masako Murai, Tekashi Fujii,
`Iseo Nakanishi. and Meseaki Matsuo“
`
`2279.: 'Benzoquinazoline Inhibitors of Thymidylste Synthase: Enzyme Inhibitory Activity and Cytotoxicity of Some 3-Amino-
`I
`and 3-Methylbenzomquinazolin-H2Hl-ones __
`William Pendergast,‘ Jay V. Johnson, Scott H. Dickerson, Inderjit K. Dev, David S. Duch, Robert Fer-one,
`
`William B. HeJ1,'Joan Humphmys, Joseph M. Kelly, and David C. Wilson
`
`2292 Synthesis and Structure—Activity Studies of a Series of SpirooxszolidineflA-diones: 4-013 Analogues of the'Muscai-inic
`Agonist 2—Ethyl—8-n1ethyl-gfiédiazaspirol4.5]decane-1,3-dione
`-
`'
`-
`Shin-ichi Tsukemoto,‘ Maseto Iohihara, Fumikezu Wanibuohi, Shinii Usude, Kazuyuki Eidaka,
`Mesetoini Horatio, and Toshinari Tamara
`
`2300 Potent HIV Protease Inhibitors: The Development of Ten-ahydrofursnylglycines as Novel Pringands and Pyrezine
`Amides as Pal-Ligands
`Arun K. Ghosh,* Wayne J. Thompson, M. Katharine Holloway, Seen P. McKee, Tien T. Duong,
`Bee Yoon Lee, Peter M. Manson, Anthony M. Smith, Jenny M. Wei. Paul L. Berke, Joan A. anay,
`Emilio A. Emini, William A. Schleil’, Joel H. Huff, and Paul S. Anderson
`_
`'
`
`_
`2311 Synthesis and Evaluation of Conformationally Restricted
`N—[2-(3,4—Dichloropbenyllethyl]-N-methyl—2-(1-pynolidinybethylamines at o Receptors. 2. Piperezines, Bicyclic Amines,
`Bridged Bicyclio Amines, and Miscellaneous Compounds
`.f
`-
`Brian B. lie Costa,* Xiao-shu He, Joannes T. M. Lindere, Celia Dominguez, Zi Qiang Gu,
`Wanda. Williams, and Wayne D. Bowen
`-
`
`2321 A Novel Constrained Reduced-Amide Inhibitor of HIV-1 Protease Derived from the Sequential Incorporation of 7-Turn
`Mimetics into a Model Substrate
`'
`Kenneth A. Newlender,‘ James F. Callahan, Michael L. Moore, Thaddeus A. Tomaszek, Jr, and
`William F. Huffman
`
`Lupin Ex. 1084 (Page 3 of 14)
`Lupin Ex. 1084 (Page 3 of 14)
`
`

`

` 7KLV PDWHULDO PD\ EH SURWHFWHG E\ &RS\ULJKW ODZ 7LWOH  86 &RGH

`
`Lupin Ex. 1084 (Page 4 of 14)
`
`

`

`Potent HIV Protease Inhibitors
`
`Scheme [1. Synthesis of Amino Acidll
`
`E? ab 53
`
`Etozc
`
`coast
`
`c,d
`
`
`
` =an
`
`«Key. {a} M301, EtaN,Cl-l2012 —10 °c; (bi Nan, casement
`DMF, (c) 1 N NaOH then H304”; (d) CugO (cat), CthN, 80 “C; {e}
`meow, EtsN, THF, 473 =0 then' N—lithio-(Sl-(Ffli-beuzylmzo-
`lidinone; (f1 KNCIMSh, THF, -78 °C, 30 min, then triayl aside, “78
`'C, 2 min, then AcOH,.3{l °C.
`1. h; (g) LiOH, TIE—H30; (h) 5%
`Pde, EtOH--H20.
`I
`yisohutyryl chloridein chloroform at 23 °C followed by
`eaposlire of the resulting chloroacetate derivative to an
`excess of sodium methoxidem tetrahydrofiiran at 23 °C
`for 3 h7
`The synthetic route leading to the (2S,3’R)—tetrahy-
`drofuranylglycine equivalent1s described1n Scheme 11
`Readily prepareda enantiomerically pure (S)-(+)_-3-hy—
`droxytetrahydrofuran (8) was mesylated with mesyl chlo-
`ride and triethylamine in_methylene_._chloride.at 0 PC for
`20 min. --Displacement9 of the resulting mesylate with the
`medium salt of diethyl malonste in DMF at 100 c'C
`furnished the malonate derivative 9. Ester hydrolysis
`followed by copper ion promoted dccarboxylationm fur-
`- -nished the--{,R-)-tetrahydrofuranylacetic acid(10)in very-
`good yield. The highly diastereoselective azidation pro»
`toool developed by Evans11 was then employed to introduce
`the a-mine functionality. Thus, deprotonation of the
`(SJ-(~3-4-benzyloxazolidinone with n—BuLi followed by
`acylation with the mixed anhydrido resulting from the
`reaction of acid 10 with pivaloyl chloride in the presence
`. of-triethylamine provided the carboximide 11 after silica
`\gel chromatography. Treatment of this carboximide with
`potassium hexamethyldisilazide in tetrahydrofdran at —78
`c'C for 30 min provided the potassium enolate which was
`reacted with trisyl aside at -78 “C for 2 min and then
`quenched with glacial acetic acid and warmed to 30 ”C.
`The o—azido carboximide thus obtained was purified by
`silica gel chrOmatographyto furnish the azide 12 as a single
`diastereomer by HPLC and 1H-NhrIR MOO-MHZ) analysis.
`Removal of the chiral auxiliary was effected by exposure
`to lithium hydroxide in aqueous tetrahydrofuran to provide
`the desired acid 13. The resulting azido acid was hydro-
`genated over 5% palladium on charcoal in a mixture of
`r’r
`
`Journolnf Medicinal Chemistry. 1993, Vol. 36, No. 16'
`
`2301
`
`Table I. Structure and Inhibitory Potanciee of Various
`Constrained Pig-Ligands
`
`
`
`' 10w (11M)
`cons,
`J;
`
`0.23170J
`(P3)
`
`CIGps (nM)
`
`221%?
`(11:10)
`
`
`
`
`0.0541002?
`0:4)
`
`as:
`(n=8)
`
`5.4
`
`2.6
`
`100
`
`._
`
`0:?
`
`'
`
`100
`
`0.6
`
`50
`
`3.3
`
`>200"
`
`05
`
`-—
`
`39
`
`—
`
`35,
`
`,. in .
`
`67.9
`
`ethanol and water (2:1) to furnish the amino acid 13a. The
`corresponding (2S,3’S}-tetrahydrofuranylglycine equiV-
`alent Was obtained utilizing LR)-(~)-3-hydroxytetrahydro—
`furan as the starting material Similarly, diaatereomeric
`azido acid 28 and other cyclic amino acids utilized in Table
`I were prepared following a similar course of reaction as
`described in Scheme 11. Various pyrazine derivatives were
`synthesized as shown in Scheme III. The known“
`dichloropyrazine derivative 14 was heated with dimeth—
`ylamine or l-methyl piperazine in 2-propanol at 85 °C for
`12 h to furnish the pyraaine derivative 15a or 15b.
`Hydrolysis ofthe correSponding methyl ester with aqueous
`NaOH in ethanol and subsequent acidification provided
`the acid derivative. For the preparation of pyrazine
`derivative 17, pyrazine 15b was first converted to bromide
`
`Lupin Ex. 1084 (Page 5 of 14)
`Lupin Ex. 1084 (Page 5 of 14)
`
`

`

`2302
`
`Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16
`
`Scheme III. Synthesis of Pyrazine Derivatives“
`NH2
`-
`
`.
`
`Scheme Vl
`
`c1
`
`"ArCOZMB
`I
`/J\l/N
`CI
`14
`
`a
`
`lsaxzrme,
`
`"leW2M9
`('9‘
`
`is.
`
`\_.f
`
`15bx=n We
`
`b
`
`1
`
`Gheeh ct cl.
`
`
`
`17
`
`16
`
`“ Key: (e) dimethylamine or l-methyl piperazine. iPrOI-I, 85 ‘C;
`(b) NeNOa, Bra, I'EBr, AcOH; (e) H2, 5% Pd—C, THF: (d) 1 N NaOI-l,
`EtOI-I then H30't.
`-
`
`Scheme IV‘
`
`
`
`“ Key: (a) iPrOH, 80 °C, 12 h: (1')} Hz, 10% Pd—C, AcOH,MeOH-
`THF, 12 h; (c) aside acid 13, EDC, 11031;, Eth, DMF; (d) H2, 5%
`Pd-C, AcOH, MeOl-I—Tlfi‘, 12 h.
`
`16 by reaction with bromine and sodium nitrite in amixture
`of glacial acetic acid and 48% hydrobromic acid. Bromo
`ester 16 was then debrominated upon hydrogenation over
`10% palladium on charcdal, and the resulting ester was
`saponjfied to furnish acid 17.
`Various hydrosyethylamine isosteres containing tet-
`rahydrofuranylglycine at the P2 position were prepared
`following the general synthetic route outlined in Scheme
`IV. Azido epoxide 7 and dccahydroisoquinolinefl-l‘ de-
`rivative 18 were heated at reflux in 2—propanol for 12 h to
`furnish the azido alcohol 19 in 83% yield after silica gel
`chromatography. Catalytic hydrogenation ofaside 19 with
`10% palladium on charcoalin a mixture oftetrahydrofuran
`and methanol (4:1) in the presence of acetic acid afforded
`the amine 20 in excellent yield. Using a standard peptide
`coupling procedure,15 amine 20 was reacted with the azido
`acid 13 in the presence of N-ethy1—N’-(3—.(dimethylamino)—
`propyl)carbodiimide hydrochloride, triethylamine, and
`1-hydroxybenzotrianole hydrate in DMF to furnish the
`aside 21 which was hydrogenated over 5% palladium on
`charcoal to provide amine 22. The amine 22 was then
`converted to various protease inhibitors by standard
`coupling reaction with the corresponding acid [Scheme
`
`2
`_
`C‘_
`_ view.
`= Key".
`to) quineldic acid, PthOCI. can, THF, —10 °c, 1 s, then
`23 “C, 3 h; (b) pyrazine 14, iPrOH, 34 °C, 12 h; (c) acid”, thPOCl,
`EtsN, THE-10 °C, 2 h, then 2.3 °C. 3 h.
`
`V)._ For example, reaction of qnin'aldie acid with diphe
`nylphoephinic chloride and triethylamine in tetrahydroe
`furan at -10 90-for 1 h followed by addition of- amine 22
`afforded the inhibitor 23 in excellent yield.“5 Similarly,
`reaction of acid 17 with amine 22 resulted in the inhibitor
`2. The N—pyrazinyl compound 27 has been prepared by
`reaction of pyrazine derivative Id and the amine 22 in
`refluxing 2-propanol far 12 )1. ' Various inhibitors with
`tetrahydrothiophene derivatives or sulfolanes at the P2
`pesition were synthesized according t6 Scheme VI. .Azido
`acids 28 were prepared as a mixture of diastereomers
`following Evans‘ asymmetric azidation protocol described
`in Scheme IL The coupling of acid ZBwith amine 20 under
`standard conditions-afforded the coupling products 29
`and 30. The isomers were easily separated by oolnmn__
`chromatography over silica gel, and the stereochemistry
`at the 3-position of the tetrahydrothiophene ring was
`assigned by comparison of ‘H NMR (300 MHz) spedtra
`of the aside 21 and the diastereomer of 21 that contains
`the 2(S)-azido-3(S}-tetrahydrofurany1moiety; The aside
`derivatives 29 and 30 were then converted to inhibitors 31
`and 32 following a similar course of reaction as described
`for inhibitor 23. Further- aSsignments of tetrahydro—
`thiophene ring stereochemistry of compound 31 and 32
`were made based on comparison of 1H NMR of the
`correspondingtetrahydrofursnylderived inhibitors 23 and
`24. Selective oxidation11f of the ring sulfur of inhibitors
`31 and 32 with a catalytic amount of osmium te'traoxide
`and an excess ofN-methy1morpholine N~oxide in amixture
`(3:1) of acetone and water furnished the sulfolsne deriv-
`atives 33 and 34 in good yield. Various'ihhjbitors inTables
`IandIIhavebeen synthesized byfollowingasimilsr course
`as shown in Schemes V and VI.
`'
`
`Results and Discussion
`
`The X-ray crystal structure of the enzyme-inhibitor
`complex of n539,5o2 bound to HIV-1 protease (2.25 A
`
`Lupin Ex. 1084 (Page 6 of 14)
`Lupin Ex. 1084 (Page 6 of 14)
`
`

`

`Potent HIV Protease Inhibitors
`
`Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16 2303
`
`Scheme V}.
`
`Table II. Structure and Inhibitory Potencies of Various
`Constrained Pa-Ligands
`
` u
`
`”9
`H—NW
`
`NH
`
`0
`Ph/
`
`
`103; (nM)
`01095 (11M)
`Compd
`R
`
`' NY“
`
`2?.
`
`use
`
`0371:0003
`(11:2)
`
`12
`
`0.16
`
`3
`
`"‘Alfltjé,
`
`0.39
`
`0.12
`
`12
`
`33.
`
`a
`
`a in,
`0
`
`2.33
`
`39.
`
`|
`
`i i'
`
`-
`
`0.24
`
`25
`
`
`
`I x
`/N
`
`31 x =‘s
`dfiss x = so;
`
`
`
`
`
`32 X = S
`d]:- 34 x = soz
`a Key:
`(:3) amine 20, EDC, H0Bt,Et-.3N,DMF: {b}H2,10% Pd~C,
`MeOH—EtOAc; (c) quineidic acid, P11213001, EtaN, THE, —10 °C, 1
`h, then 23 “C, 3 h; (d)_0s04, 4-methylmorpholine N-oxide, acetone-
`water.
`
`40. Ni in,
`CI
`0
`
`o
`
`0
`
`41.
`
`42.
`
`N :
`H a
`
`i4
`
`“ 1‘4 o
`
`0.06
`
`12
`
`0.15
`
`I
`
`25
`
`0.11
`
`25
`
`
`
`resolution)1a was utilized to. construct the modeled struc-
`ture of compound 1 (Ro 31-8959). The structure was then
`energy minimized in the active site using the Merck
`molecular force field, OPTIMOL,” which is a variant of
`the MM2 program.” An examination of the modeled
`structure of inhibitor 1 revealed that the carbonyl oxygen
`of the asparagine is within hydrogen bonding distance to
`.,_.___
`theaap 29 and asp 30 NH present in the 3; binding domain
`of the HIV-1 protease. 0n the basis ofthis possible insight,
`-
`-
`.
`.
`.
`_
`we hypothesize theta conformationally constrained cyclic _
`etheLoxygeleighLdQnate its. loan pair;_s__-for_ hydrogen
`- magma model myths inhibitor 2-3 wascreatedand based
`..
`bonding to the appropriate residues in the'Sz iregidniofthe 3
`onthesuperimpositionQnthe'inlodel structure of 1 (Figure
`enzyme active site. Such a desigii may effectively rep1a0e _'-_'
`'12}; it appeared that the-tatrahydrofuran oxygen of 23 is
`the Pg-asparagine, providing an inhibitor with improved .
`'.
`"in close-proximityto the carbonyl oxygen of the asparagine
`in uitro potencies and pharhaa'cokinetic prowl-ties. Indeed, "
`Iiloietyfi".3 Thus, the poéition-of the oxygen in 3’(R)-Thfg
`as can be seen from Table 11,-;the- replacement of the '
`-_ __1 seems to set-upforhydrogen-bonding interaction with the
`asparagine of compound :21 with (_2}S,3’R}"—tettah,yti‘ro-n'
`---:'asp 29_an'd asp -'30_ of the HW¥1 protease. The antiviral
`furanylglycine [compound 23} resulted in an over 43013
`potehcies of the inhibitors 23;] and 24 are consistent with
`increaseinitsinhibitorypotency?»Interestingly,'(23,3’8)-'
`their enzyme inhibitory potenCies. As is evident in Table
`teh'ahydrofuranylglycine.(3’_(S)-'Thfg) (Eontain'ing inhibitor
`I, 3’[RI-'l‘hfg-derived'inhibitor23has prevented the spread
`(compound 24) showed a 16-fold less in potency compared
`of HIV-1 !in MT4 human T-lymphoid cells infected with
`to 1. Furthermore, examination ofthe cyciopentyl deri-
`Hib'isolate15 at an average concentration of 8 nM (01095),
`vative 25 established that the ring oxygen is essential for
`. a 3rfold'phtency enhancement over inhibitor 1. In contrast,
`potency enhancement. Also; as expected, the inhibitory
`31$}ng derived inhibitor-'24 has exhibited an antiviral
`potency ofthe corresponding acyclic analog 26 isbver _1_'?-
`' potency of 109 nM. The synthesis of compounds 31-35
`fold less potent than the compound 23. The fact that the _
`was undertaken to evaluate the influence of other het-
`3’(,R)-Thfg-derived inhibitor 23 is more potent than the
`eroato'ms and het‘erocycles on the enzyme inhibitory and
`3’(S)-Thfg—derived inhibitor 24 suggested that 3’(R)-Th.fg
`antiviral potencies. Teflahydrothiofuranylglycine—derived
`is successfufly mimicking the enzyme-bound conformation
`inhibitor 31 with a S’R configuration is significantly less
`of the asparagine side chain of compound 1. Accordingly,
`
`'
`
`I
`
`-
`"Z ‘
`
`\
`
`\.\
`
`:
`
`I;
`.rz
`
`Lupin Ex. 1084 (Page 7 of 14)
`Lupin Ex. 1084 (Page 7 of 14)
`
`

`

`2304 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 16
`
`Check cool
`
`
`
`1, R0 31— 8959
`:04nM
`IC50[[HIV- 1]
`—-0.5nM
`[HIV— 2]
`
` l'
`
`N
`
`2 ICSD [HIV-'1] = 0.07 nM
`[HIV-2]
`= 0
`
`figure 1.
`
`potent (1050 0.6 nM) than the corresponding tetrahydro-
`finan derivative 23. Since the sulfur atom in the tet-
`rahydrothiophene ring is a poor hydrogen bond acceptor?i
`aweak hydrogen-bondhlg interaction may ac count for this
`loss of potency. The stereochemical preference for 31??-
`configuration by this binding region, however, remained
`consistent with our earlier observation with the 3’-Thfg—
`derived inhibitors. Next, in order to examine the effect
`of solfone oxygens in this binding pocket, the correspond-
`ing sulfolane derivatives were prepared. The sulfolane
`derivative with a B’R-configuration is equipotent to the
`corresponding sulfide (compound 33, IC50 0.5 nM). 0n
`the other hand,
`the sulfone derivative with a 3’8-
`configuration turned out to be Ill-fold less potent than
`the sulfide 32. Furthermore, the replacement of the
`3—tetrahydrofuran ring in 23 with 4-tetrahydropyran
`(compound 35} was examined. This substitution has
`resulted in a dramatic loss in enzyme inhibitory potency
`(compound 35, 1050 68 nM). The reason for this loss of
`potency is probably due to the increase in ring size as well
`
`as the position of the Oxygen, which impedes the access
`to the appropriate residues for specific interaction in the
`SQ subsite of HIV-1 protease. Thus, from these studies,
`it is quite apparent that 3’(R)-Th:fg is a highly effective
`asparagine surrogate for the 82 region of the HIV-1
`substrate binding site.
`In an attempt to further improve the in vitro potencies
`and possibly the pharmacokinetic properties of 3’(R) -Thfg-
`derived inhibitor 23, we have incorporated various pyrazine
`derivatives and heterocycles at the P3 position. As shown -
`in Table II, substitution of the 2—quinolinoy] moiety in 26
`with the quinoxalinoyl group resulted in (compound 36)
`a slight improvement in potency. However, incorporation
`of the 2-quinoxalinoyl moiety in 23 afforded the inhibitor
`37 with an IC59 value of 0.12 nM, a 2-fold loss in potency
`compared to 23. The removal of the second aromatic ring
`of the 2-quinoxalinoyl moiety in 37 provided unsubstituted
`pyrazine—derived inhibitor 38 (ICw—238—nM-Ir—w-i-th- a more
`than 20-fold loss in potency. Next, we have examined a
`number of pyrazine derivatives with various functionalities
`in order to determine whether or not substitution onnthe—Fr'
`pyrazine ring would have any significant effect On potency.
`Indeed, compound 39 With 2-amino-5-(dimethylamino)—
`6-chloropyrazinoyl moiety at the P3 position resulted in
`a greater than I‘ll-fold potency enhancement (ICao 0.24
`nM) compared to unsubstituted pyrazine derived com-
`pound 38. The removal of the 3-amino functionality from
`the pyrazine ring of compound 39 has significant potency
`enhancing effect. As shown, compound 40 has displayed
`an enzyme inhibitory potency of 0.06 11M, a 4-fold
`improvement over 39. Further attachment of a basic amine
`functionality (compound 2] appears to have no effect on
`lCm value (0.07 nM) compared to compound 40. The
`marked enhancement of enzyme inhibitory potency of
`compounds 40 and 2 was translated into their antiviral
`potencies. Both compounds consistently exhibited an
`antiviral potency of 12 nM in ce]l culture assay.
`With the goal of minimizing peptide-like character while
`retaining comparable enzyme inhibitory and antiViral
`activity, we then explored the possibility of removal of the
`Pg—Pg amide carbonyl by attaching the 3’(R)-Th.fg—derived
`amine directly to the pyrazine ring. This turned out to
`be a rewarding endeavor. Compound 27 thus obtained
`
`
`
`
`
`Figure 2. Stereoview of the optimized bound conformations of compound 1 (Re 31--8959] {pink} and the inhibitor 23 (green) superimposed
`in the L-689, 502 inhibited HIV— 1 protease active site. “5
`
`Lupin Ex. 1084 (Page 8 of 14)
`Lupin Ex. 1084 (Page 8 of 14)
`
`

`

`Potent H] V Protease Inhibitors
`
`Journal of Medicinal Chemistry, 1993, Vol. 36', No. 16'
`
`2305
`
`Table III. Inhibitory Potsncies (HIV-2) of Selected Compounds
`1050,
`ICED;
`
`nM (HIV-2F”
`compd
`nM [HIV-2135
`0.5
`25
`3.2
`0.18
`27
`24.9
`0.24
`
`compd
`l.
`2
`23
`
`_
`
`showed a similar level of enzyme inhibitory activity (1050
`0.16 11M} as the inhibitor 1. More importantly, there is
`a dramatic improvement in antiviral potency (C1095 3
`nM)25 of compound 27 compared to l. Presumably, various
`functional groups present in the pyrazine ring of compound
`27 are consequential to binding; however, the actual role
`of the pyrazine substituents has not been specificaliy
`examined. A thorough understanding ofthe contributions
`of the various functional groups in this binding region
`should await a detailed structure—acthdty relationship
`investigation. Intriguing changes in activitywere observed
`upon introduction of 2-chromone carboxamide as the Pa
`ligand. Replacement of the 2-quinolinoyl group with a
`2-chromone derivative (compound 41) resulted in 4-fold
`enhancement of both enzyme inhibitory and antiviral
`potencies compared to compound 26. Thus, simple
`replacement of Pg-asparagine and P3-2-quinolinoy] units
`with valine and 2-chromonoyl moieties afforded an in-
`hibitor (compound 41) with comparable in vitro potencies
`to compound 1. Interestingly, substitution of valine in 41
`with 3’(R)—Thfg'has no effect on potency.
`Selected inhibitors from this present series have been
`evaluated for their ability to inhibit HIV-2 protease.” As
`shown in Table 111,-inhibitor 23, derived from 3’(R)«beg
`([050 0.24 MM), is 2-fold more potent than compound 1
`(ICm 0.5 nM). Incontrast, replacement of asparagine in
`l with valine (compound 26} resulted in over (Hold loss
`of potency. Furthermore, the N~pyrazinyl compound 27
`showed an inhibitory potency of 24.9 nM. Compound 2
`with 3"(R)-Thfg at the P2 and substituted pyrazinoyl
`moiety at P3 is the most potent inhibitor (HIV-2) in this
`series. This compound has exhibited an 1050 value of 0.18
`nM'(H IV-2), nearly a 3-fold improvement over compound
`1 (Ro 31-8959).
`'
`
`Conclusion
`' “Tieplacement ofthe Pg-aspsragine and P3-2-quinollnoyl
`ligand ofpresent clinical candidate 1 with 3’.(Rf-Thfg and
`substituted pyrasine derivatives has led-to a novel series
`' of pot-Zeit—inhihitor's—of HIV-1 and HIV-2 proteases.
`Interestingly, the inhibitor containing tetrahydrofura—
`nylglycine with the 2S,§R-c'onfiguration has exhibited
`nearly 100-fold potency enhancement over the 28,38-
`derived inhibitor. This marked difference in activity and
`strong stereochemical preference has suggested some
`specific hydrogen-bonding interaction with the residues
`in the 82-binding domain of HIV-1 protease. Also, it has
`been demonstrated-that substituted pyrazinoyl derivatives
`are effective as Pg-ligands, leading to potent protease
`inhibitors. Of particular interest, compound 2, with 3’-
`(R)~Thfg at P2 and pyrazine derivative at P3, is one of the
`most potent inhibitors of HIV-1 and HIV-2 proteases.
`Since these are the genetically most divergent strains of
`HIV known to exist to date, the protease inhibitors which
`are potent against HIV-1 and HIV-2 strains may have a
`beneficial effect in terms of reducing susceptibility to
`clinical resistance. Another intriguing result in this series
`is the identification of compound 27 in which the Par-P3
`
`amide carbonyl is removed. This has resulted in sub-
`stantial improvement in antiviral potency while retaining
`the enzyme inhibitory potency at a level similar to that
`of compound 1. Further design of compounds from the
`study of ligand binding site interaction may lead to
`structurally novel inhibitors with less peptidelike char-
`acter.
`Investigations along this line are in progress.
`
`Experimental Section
`
`Mmeltingpoints were recordedonaThomas-Hoovercapillary
`melting point apparatus and are uncorrected. Proton magnetic
`resonancespectrawere recordedonaVarianXLSOOspectrometer
`using tetramethylsilane as internal standard. Significant 1H
`NMR data for representative compounds are tabulated in the
`following order: multiplicity (s, singlet; d, doublet; t, triplet; q,
`quartet; m, multiplet), number of protons, coupling coastanus)
`in Hz. FAB mass spectra were recorded on a VG Model 7070
`mass spectrometer, and relevant data are tabulated as m/z.
`Elemental analyses were performed by the analytical department,
`Merck Research Laboratories, West Point, PA, and were within
`i0.4% of the theoretical values. Anhydrous solvents were
`obtained as follows: methylene chloride, distillation from P4010;
`tetrahydrofuran, distillation from sodiumfbenzophenone: dime-
`tbylfcrmamide and pyridine, distillation from CaH2. All other
`solvents were HPLC grade. The abbreviations DME, DMZE‘, THI‘,
`HOBT and EDC refer to 1,2-dimethoxyethane. N,N-dimethyl-
`formamide, tetrshydrofuran, l-hydroxybenzotriazole hydrate,
`and 1—etbyl—3-(3—(dimethylaminolpropy1)carbodiimide hydro-
`chloride. Column chromatographywas performed with E. Merck
`240—400 mesh silica gel under low pressure of 5—10 psi. Thin-
`layer chromatography (TLC) was carried out with E. Merck silica
`gel 60 F—25-i plates.
`trans-«l—Phenyh2-buten-l-ol (4}. A mixture of CuCN (2.43
`g, 27 mmol) was added to a solution of butadiene monooxide (19
`'g, 270 mmol) in 500 :mL of anhydrous tetrahydrofuran, and the
`mixture was cooled to —78 °C. Phenylmagnesium bromide
`solution in ether (32 mmol) was added dropwise to this mixture.
`The reaction mixture was warmed to 0 °C and was stirred until
`the reacfion mixture became homogeneous. The reacfionmixture
`was cooled to -78 “C, and 0.29 mol of phenyhnagnesium bromide
`solution in ether was added dropwise over 30 min. The reaction
`mixture was allowed to warm to room temperature with stirring
`and then quenched by slow addition of saturated NmCl (50 mL)
`followed by NED}! (30 mL), saturated Nl-hCl (200 mL), and
`H20 (100 mL). The aqueous layer was extracted with two 200-
`ml. portions of ethyl acetate. The combined organic layers were
`dried and concentrated. The residue was distilled under vacuum
`(0.1 Torr) at. 100 °C to give trons-bphenyl-Z-buten—l-ol [4, 38.9
`g).
`
`2(3)3(m-Epoxy-A-phenylbuten-l-ol (5). A mixture of
`powdered 4-K molecular sieves (3 g), titanium tetraisopropoxide
`(1.5 mL). and diethyl n—tartrate {1.1 mL'} in anhydrous methylene
`chloride (350 mL) was cooled to —22 “C, and tert-butyl hydro-
`peroxide solution in isooctane (210 mmol) was added slowly with
`stirring. After 30 min at—22 ”C a solution cfd (15.3 g, 100 mmol)
`in anhydrous methylene chloride (50 mL} was added dropwise
`for 20 min at —22 ”C. The reaction mixture was aged at -22 °C
`in a freezer for 20 h. Water (40 mL) was added to the reaction
`mixture, and after 30 min at 0 °C, 30% NaOI-I in brine (8 mL)
`was added. The resulting mixture was stirred for 1 h at room
`temperature. The organic phase was separated, and the aqueous
`layer was extracted with two 30-mL portions of methylene
`chloride. Combined organic layers were dried over Na2804,
`diluted withtoluene (300 mL), and