MEDICINAL
`CHEMISTRY
`
`
`
`"'9
`
`b8905266783 a
`
`JOURNAL OF
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`B‘Il‘JSEEE?
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`Lupin Ex. 1085 (Page 1 of 15)
`Lupin Ex. 1085 (Page 1 of 15)
`
`

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`Lupin Ex. 1085 (Page 2 of 15)
`Lupin Ex. 1085 (Page 2 of 15)
`
`

`

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

`Lupin Ex. 1085 (Page 3 of 15)
`
`

`

`Noripepiidai Ligands for HIV Protease Inhibitors
`
`Journal of Medicinal Chemisto», I996, Vol. 39, No. 17 3279
`
`Scheme 1. Enantioselective Synthesis of the Bicyclic
`Ligands“
`
`OH
`
`Scheme 2. Synthesis and Optical Resolution of the
`Bicyclic Ligands"
`
`Ewac\)\CDzEl
`
`
`
`Ia} LDA, CH-CHCHeBr; (bl LAH. Eth: [cl acetone.
`" Key:
`stOH; (d) Swern oxidation: is! CSA, MeOH; (l) ozonolysis then
`NaBHi; (g) CSA, Cflzcig; (h) Q-BBN, THF, aqueous NaOH. H202.
`
`hydroboration with 9-BBN followed by reaction of the
`resulting alcohol with USA in methylene chloride.
`Similarly, enantiomeric bicyclic ligands 10 and I] were
`synthesized, starting from optically pure 3(S)-diethyl
`malate (9) following the sequence of reactions described
`above.
`racemic synthesis of these ligands
`Alternatively,
`followed by their enzymatic resolution provided an easy
`access to these ligands in optically active form.12 As
`shown in Scheme 2. reaction of commercial 2,3-dihy-
`drofuran (12) with N—iodosuccinimide and propargyl
`alcohol in methylene chloride at 0—23 “C for 3 h resulted
`in the iodo ether 13 in excellent yields (91-95%).
`Radical cyclization of the iodo ether 13 with tributyltin
`hydride13 in refluxing toluene in the presence of a
`catalytic amount of AIBN afforded the bicyclic acetal
`14 in good yield (70-80%) after silica gel chromatog-
`raphy. This radical cyclization was more conveniently
`effected with sodium borohydride reduction in the
`presence of a catalytic amount (10 mol %) of cobaloxime
`(Scheme 2)” in 95% ethanol at 65 °C for 3 h affording
`the bicyclic acetal 14 in comparable yield (70—75%).
`Ozonolytic cleavage followed by the reduction of the
`resulting ketone with sodium borohydride in ethanol at
`- 15 CC furnished the racemic endo alcohol 15 (74—78%)
`after chromatography.18 The optical resolution of the
`racemic alcohol 15 was carried out efficiently by expo-
`sure to Amano lipaseH-mediated acylation as well as
`the hydrolysis of the corresponding acetate. Thus,
`acylation of 15 with immobilized” lipase PS—30 (25%
`by weight with respect to lipase P830) in the presence
`of acetic anhydride in dimethoxyethane at 23 “C for 3 h
`afforded the unacylated alcohol 7 (42% yield) and the
`acylated alcohol 16 (45% yield) which were separated
`by silica gel chromatography. The optical purity of the
`
`0 selfI"!
`
`12
`
`13
`
`b
`
`H
`
`2
`
`,.on
`
`03
`6
`.J 150
`
`4———
`c,d
`
`'4
`
`°‘/
`
`o
`
`,0”
`u
`
`HQ:
`
`:
`OJ
`7
`
`'1' on
`.a
`
`o
`
`+ Hg fi—
`
`lo a =A
`--n R:H
`
`c
`
`°Cl
`.
`
`.-DF|
`
`3
`~. —+- n+-
`
`n
`: on
`«"
`
`"can
`3dr"
`+ H ‘
`x
`
`1|]
`
`'9 R=AC
`_
`(1)17 R—H
`f: 8 R:H
`l3 R=Ac
`la] N—iodosuccinimide, propargyl alcohol. CH2C12, 0—23
`‘1 Key:
`r’C: (bl cobaloxime (can, NaBHi. EtOH; lo] 03, Cflgciz—MEOH.
`Megs. -'?8-23 “C; (d) NaBI—L, EtOH, —15 °C; (e) immobilized
`lipase 30. ACQO, DME, 23 “C; (F: aqueous LiOH, THF_H20; lg}
`immobilized lipase 30. pH 7 buffer. 23 °C.
`
`alcohol 7 (95% cc. [(1123:] -11.9", MeOH) was determined
`by formation of Mosher ester and 19F-NMR analysis.19
`The acylated alcohol 16 was hydrolyzed by treatment
`with aqueous lithium hydroxide to provide optically
`active 11 (87% ee, [M230 +1137", MeOH}. Similarly,
`racemic 17 was synthesized utilizing dihydropyran as
`the starting material. The resolution of 17 was effected
`by formation of the corresponding acetate 18 followed
`by the enzymatic hydrolysis with immobilized lipase PS
`30 in phosphate buffer (pH = 7.0) at 23 “C for 24 h.
`The hydrolyzed alcohol 10 (yield 34%. 90% es) and the
`acetate 19 (40%) were separated by silica gel chroma-
`tography. Ester hydrolysis of 19 furnished the alcohol
`3 in optically active form (94% as). The represented
`absolute configurations of the resolved alcohols were
`assigned based on comparison of their optical rotation
`with the ligands synthesized utilizing 3(S)— and 3(Rl-
`diethyl malates as described above.
`Enantiomerically pure fused tetrahydrofuran 23
`was synthesized from commercial cis-(—)—3.3a,6,6a-
`tetrahydro-ZH—cyclopenta[b]furan-2-one (20} according
`to Scheme 3. As shown, reduction of 20 with LAH in
`tetrahydrofuran at 23 c‘C afforded the diol 21 (isolated
`yield 96%). Treatment of the diol 21 with iodine and
`potassium iodide in methylene chloride at 23 ”C fur-
`nished the iodo ether 22.20 Radical dehalogenation of
`the iodine with tributyltin hydride in refluxing dioxanc
`in the presence of a catalytic amount of AIBN provided
`the bicyclic ligand 23 with defined absolute configura-
`tion.
`
`Synthesis of symmetric bicyclic ligand 26 is outlined
`in Scheme 4. Reaction of 2.3-dihydrofuran (12) with
`mCPBA in l-butanol at —15—0 c'C for 2 h afforded the
`alcohol 24 after distillation (65%). Oxidation of 24 with
`
`Lupin Ex. 1085 (Page 4 of 15)
`Lupin Ex. 1085 (Page 4 of 15)
`
`

`

`Scheme 5. Synthesis of Racemic Bicyclic LigandsCl
`
`Ghosh et at
`
`0;.—
`in x=cu,
`so xncn,
`29 x.o
`
`0:?”
`lb
`
`"
`
`31 X20
`
`2
`
`'
`“'“x
`.14 xzcu;
`35 x=o
`
`2
`
`i
`“‘x
`
`J: x=cnz
`33 X=O
`
`" Key: fa) N—bromosuccinimide. pl'opargyl alcohol. Cchlg. 0—2"
`“C; (bl anSnH, AIBN. Phil. reflux; (CI 0.1. CH2C12-ME'0H. M228.
`-7S—23 °C; (d: NaBHl. EtO‘H, —15 CC.
`
`Scheme 6. Synthesis of the Bicyclic Ligand“
`
`Hl
`
`DH]
`,H
`
`.
`
`0
`
`ago
`
`3
`as ape-ooh .RFH
`
`+y. Hug
`31 RI=H.uF—g-opn
`lb
`
`
`
`
`39
`
`37 A5
`
`DFtl
`
`“ Key: Isl PhOCtSlCl. EtsN, CH2C12; (b) separated by silica gel
`chromatography: th nBuaan, AIBN, PhMe. reflux.
`
`Scheme 7
`
`a)
`
`H
`
`5
`
`H902Pr.
`
`SJ
`
`the major isomer 37 was exposed to the radical deoxy-
`genationgil conditions to provide the ligand 39.
`Synthesis of various inhibitors with bicyclic ethers as
`the P2 ligands and decallydroisoquinolinecarboxamide
`as the P1’ ligand was carried out according to Scheme
`7. The previously described“ hydroxyethylamine iso»
`stere 42 was transformed into the various target inhibi-
`tors listed in Tables 1 and 2 by an alkoxycarbonylation
`of the respective alcoholde‘1 For example, reaction of bis-
`Thf ligand 7 with dipyridyl carbonate (40) and triethyl-
`amine in methylene chloride afforded the active car-
`bonate 41 after chromatography. Reaction of the mixed
`carbonate 41 with amine 42 in methylene chloride
`
`J
`
`Lupin Ex. 1085 (Page 5 of 15)
`Lupin Ex. 1085 (Page 5 of 15)
`
`3280 Jam-no! of Medicinal Chemistry, £996, Vol. 35', No. 17
`
`Scheme 3. Enantioselective Synthesis of the Bicyclic
`Ligands"
`
`H
`
`0
`
`.--°
`
`q");
`
`a
`
`"9:" ic— MOP?"
`
`d.../
`
`(SJ
`
`is] LiAlHl. THF. 23 “C: lb} KI. 12. NaHCOa. CH2C12.23
`” Key:
`“C: Ifc) nBusfi‘mH, AIBN, dioxanc. reflux.
`
`Scheme 4. Synthesis of the Symmetric Bicyclic Ligand“
`_.OH
`
`(1 —-“
`0
`
`'3
`
`: OMH
`CI
`24 .
`
`it). c
`
`H
`"V
`
`O Ill. Gm
`25
`
`0
`
`on
`
`d, e
`«1—
`
`0
`fC‘Z? R=—E-o—N:]
`
`215 R2}!
`
`o
`
`0
`
`o
`
`“ Key: Ea} mCPBA. o-hutanol, 0 “C: I bi Pyr-SOB‘ DMSO, EtaN:
`[cl allylmagnesium bromide. Eth. 0‘23 ‘C; id) 03. CH-zClg—
`MeOl—l. —78—0 “C. then NaBH... EtOH, 0 °C:l'e'IstOH.CH2C12,
`23 ”C; {fl COClg. pyridine, PhCHa. then N~hydroxysuccinimide,
`CH3CN, EtaN‘ 23 “C.
`
`pyridine-803 complex in methylene chloride gave the
`corresponding ketone which was reacted with allyimag—
`nesiurn bromide in diethyl ether at 0—23 “C for 4 h to
`furnish the alcohol 25.21 Ozonolysis of the terminal
`olefin followed by reduction of the ozonide with sodium
`borohydride in ethanol at 0 “'0 provided the correspond-
`ing alcohol which was treated with p-TsOH in methyl»
`ene chloride to afford the symmetric ligand 26. Reaction
`of 26 with phosgene and pyridine in toluene followed
`by reaction with N—hydroxysuccinimide in acetonitrile
`furnished the mixed active carbonate 27 after silica gel
`chromatography.22
`Racemic hicyclic ligands 34 and 35 were prepared
`(Scheme 5) utilizing a similar synthetic route as de-
`scribed for racemic 15. Reaction of cyclopentene with
`N-bromosuccinimide and propargyl alcohol provided
`good yield of the corresponding bromo ether 30 (yield
`72%). However,
`the reaction with 2,5-dihydrofuran
`proceeded with modest yield (35%} of the corresponding
`bromo ether 31. Mbutyltin hydride-mediated radical
`cyclization of 30 and 31 provided the bicyclic olefins 32
`and 33 which were converted to racemic bicyclic ligands
`34 and 35 for structure—activity studies.
`Bicyclic ligand 39 with oxygens in a vicinal relation-
`ship was synthesized in enantiomerically pure form
`starting from commercially available 1.4:3,6-dianhydro-
`IJ-sorbitol
`(36) (Scheme 6). Treatment of 36 with
`commercial chlorothionoformate and triethylamine in
`methylene chloride at 23 “C for 4 h afforded a mixture
`(2:1) of thionocarbonates 3'7 and 38. The isomers were
`separated on silica gel by column chromatography, and
`
`

`

`Norms-pride! Ligands for HIV Protease Inhibitors
`
`Joor'noi of Medicinal Chemistry. 1.9.96, Vol. 39, No. 1‘? 3281
`
`afforded only the inhibitor 49 (white solid, mp 98—101
`”C) by 1H—NMR and HPLC analysis (isoiated yield 76%).
`For the preparation of inhibitor 55, the mixed active
`carbonate 27 was reacted with amine 42 in methylene
`chloride in the presence of 3 equiv of triethylamine at
`23 °C for 12 h to provide 55 after silica gel chromatog-
`raphy. Similarly, various nonpeptidal
`ligands were
`converted to other inhibitors in Tables 1 and 2.
`
`Table 1. Structure and Inhibitory Potencies of Various
`Heteroc'yclic Derivatives
`
`
`
`Results and Discussion
`
`ICfiU (11M)
`
`CIC9S {TIM}
`
`43.
`
`44
`
`.
`
`45.
`
`46.
`
`47.
`
`48.
`
`As described previously? the preliminary X—ray crys-
`tal structure of the enzyme—inhibitor complex of 43 and
`Hl'V-l protease25 has suggested a number of structural
`changes on the tetrahydrofuran ring that could lead to
`improved binding. The urethane of 3(Sl-hydroxysul-
`folane (46) has exhibited nearly a 2-fold potency en-
`hancement compared to 43, presumably due to the close
`proximity of the sulfolane oxygen cis to the 3-hydroxyl
`group to Asp 29 and Asp 30 NH. It is evident in the
`crystal structure that the oxygen atom of the tetrahy-
`drofuran ring in 43 is oriented toward the Asp 29 and
`Asp 30 NH. Recently, researchers from Vertex Labo-
`ratories have reported a similar observation in their
`X-ray crystal structure of protein—ligand complex con-
`taining an inhibitor with 3(S)-tetrahydrofuranyloxy
`group as the P2 ligand?6 The protein—ligand structure
`of inhibitor 43 further provided rationale for incorpora-
`tion of the cis-2-alkyl substitutent of the sulfolane ring
`of inhibitor 46. Optimization of these findings resulted
`in inhibitors with reduced molecular weight and com-
`parable in vitro potency to 1 (Ro 31-8959). A detailed
`account of these investigations has been reported re-
`cently.8 Subsequently, during the introduction of a cis—
`2—methoxymethyl substitutent of the sulfolane ring of
`inhibitor 46, we have observed a very intriguing struc-
`ture—activity relationship. As eVident
`in Table 1,
`replacement of the ring oxygen in 43 with sulfur
`(inhibitor 45) resulted in no change in inhibitory
`potency. Oxidation of the ring sulfur to sulfone has
`provided nearly a 2-fold improvement over 45. Thus
`far, our observation is that the ring sulfones, in general.
`are 2—5-fold more potent than their corresponding ring
`sulfides.” Consistent with our earlier observation,
`incorporation of a cis-Z-methoxymethyl substitutent in
`cyclic sulfide 45 afforded inhibitor 47 with a 6-fold
`potency enhancement. However, in contrast, oxidation
`of ring sulfur to the corresponding sulfone resulted in
`inhibitor 48 with reduction in potency {105.1 23.8 nM).
`To gain insight into the molecular binding properties,
`an energy-minimized active model of inhibitors 46 and
`4'7 was created”5 utilizing the X-ray crystal structure
`of the enzyme—inhibitor complex of L-689,502 bound to
`HIV-1 protease (2.25 A resolution).3u On the basis of
`superimposition of these modeled structures (Figure 1},
`it appeared that in addition to filling the hydrophobic
`pocket in the 82 region, the methoxyl oxygen of inhibitor
`47 is in close proximity to hydrogen bonding with the
`Asp 29 and Asp 30 NH. Thus, the methoxyl oxygen is
`effectively competing for the same binding site as the
`sulfolane oxygen cis to the 3-hydroxyl group of inhibitor
`46. Therefore, oxidation of the ring sulfide in inhibitor
`47 did not provide any additional potency enhancement
`as was observed earlier. These findings subsequently
`provided the basis for a conformationally constrained
`and structurally new class of ligand design.
`
`As can be seen from the superimposed stereoviews of
`46 and 47 (Figure 1), the conformation of methoxy-
`methyl side chain of 47 can be further restricted to form
`another ring cycle to the existing five-membered ring.
`On the basis of this possible molecular insight, we
`speculated that a bicyclic ligand with oxygeus positioned
`appropriately in the ring would interact effectively with
`the Asp 29 and Asp 30 residues of the enzyme active
`site. Also, superimposition of the X-ray crystal struc-
`ture of the protein—ligand complex31 of Ro 31-8959 and
`the modeled structure of 47 {Figure 2) revealed the
`potential benefit of such ligand design. It appears that
`a fused bicyclic tetrahydroi‘uran not only could replace
`the quinaldic amide and asparagine amide of R0 31-
`8959 inhibitor, but it may also provide additional
`binding energy to offset the loss of Pg-hydrophobic
`binding corresponding to the quinoline ring.
`Indeed,
`incorporation of bis-tetrahydrofuran as the P2 ligand
`with 3(R),3a(S),6a(R)-configurations (inhibitor 49}. as
`speculated from the ligand-binding site interactions of
`inhibitors 47 and 1, has shown impressive in vitro
`potencies.g As presented in Table 2, inhibitor 49 has
`shown an enzyme inhibitory potency (105.0) of 1.8 :t 0.2
`nM (n = 6).
`In comparison, the inhibitor with 3(Sl,3a-
`(R),6a{S)—bis-Thf as the P2 ligand (inhibitor 50, 1050 5.4
`Him is less potent than 49. The difference in enzyme
`inhibitory potencies is also reflected in their antiviral
`activities.
`Inhibitor 49 has prevented the spread of
`HIV-1 in MT4 human T-lymphoid cells infected with
`UN) isolate” at an average concentration [a = 4} of 46
`d: 4 nM (01095}. Inhibitor 50. in contrast, has shown
`an antiviral potency of 200 nM.
`In head to head
`
`Lupin Ex. 1085 (Page 6 of 15)
`Lupin Ex. 1085 (Page 6 of 15)
`
`(fr—«Ck.
`
`CI
`
`(70“
`D
`
`v.0
`
`(:T ‘
`
`S
`
`00
`
`n=3}
`
`>300
`
`132230
`
`[(1:72)
`
`694
`
`132
`
`_..a.,_
`
`76:12
`(F14)
`
`364:44
`(11:1?)
`
`093“
`
`H
`
`,0
`
`19.7
`
`23.8
`
`(:1 “
`
`S
`
`_.O
`
`(j ‘“
`0:5“ “-.__/0m!
`0
`
`ch/OMQ
`
`HH
`
`

`

`3282 Joormii offl-lcdfcinui ('Tiiemishir. 39.96, “ii. 3.9, No. 17
`
`Ghosh of r- -'_
`
`Figure l. Stcreovicw ol‘thc optimized bound ctmi'rn'lnations ol'inhihitors 46ll1'tzlgcnlal and 47 rgreeni superimposed in the HIV-T
`protease active site.“
`
`
`
`
`Figure 2. Stcz‘eoview ol‘the X-ray sll‘tlcture nf'inhibitor l Imagenta! bound to HlV-l protease and tiplimized bound conliirtn‘rition
`of inhibitor 47 [ere-cm.
`
`comparison. inhibitor 49 was shown to be equipotcnt
`to 1 tRo 3i-8959: Cnga 23 r‘l' T thm The enhanced
`inhibitory potency as well as the stel'eochen‘iical prefer-
`ence ['or 3tHi.3atSLGalRi-coniigurations in bis—Thi'ligand
`tinhibitor 49]
`indicated a specilic hydrogen—bonding
`interaction with the residues of the 8_ region of the
`enzyme active site.
`Incorporation of 3-hexahydro|‘nro-
`pyran as the P3 ligand has shown reversal of stereo-
`ehemical trends.
`Inhibitor 52 with IlelJiatRLTaESL
`configurations has shown an [Can value ol'1.2 nM. a
`greater than 3-fold potency increase over inhibitor 51
`iIC.‘-:,n 4.2 nMi. Antiviral potency of
`this inhibitor
`leonipound 50: Cng; 50 an is also comparable to that
`of Ru 31~8959 or inhibitor 49. Evaluation oi'compounds
`53 and 54 established that both oxygens are involved
`in binding.
`Incorporation ol'symmetric bis—Thl' ligand
`{inhibitor 551 resulted in at least. a 225—fold loss i [Ci-,1:
`41K) nMJ in inhibitory potency. Similarly. change of the
`ring oxygen positions [inhibitors 56 and 57] also re-
`sulted in significant loss ol‘ potency. Various substitu—
`tion at
`the hicyclic ring {inhibitors 58-601 did not
`improve enzyme inhibitory or antiviral potencies. Thus.
`the above structure-activity studies provided ample
`evidence that in the bis—Thf ligand ol‘inhibitor 49. the
`ring stereocheinistry, ring size. and position of ring
`oxygens all are critical to effective binding in the Sg
`region of the substrate-binding site.
`To gain further insight into the molecular binding
`properties,
`the three—dimensional structure of both
`inhibitors 1 rRo 31-8959] and 49 was determined by
`
`X‘ray dil'l'raetion at a resolution of 2.2 and 2.10 A.
`respectivc—rlyf"I A superimposed stereoview of these
`inhibitors tFigure 3: was then created by superimposi-
`tion of the X—ray crystal structure of 1 (green) on the
`X—i'ay crystal structure ol'49 [ magenta! bound to HIV-l
`protease in the same frame. of reference. The (Rt.
`hydroxyi group of inhibitor 49 is positioned sym—
`metrically between the catalytic aspartates of the HIV—1
`protease. The Pi henzyl side chain and the P1’ decahy-
`droisoquinoline moietyr of 49 are located at the S; and
`81' regions. respectively. in the enzyme active site. The
`N‘terr-butyl group is positioned in the 83’ subsite. A
`comparison of' binding properties of the bis-Thf' ligand
`of 49 and the asparagine ol' 1
`is very intriguing. As
`shown. both the his-Thf oxygen—l and asparagine car—
`bonyl ol' 1 are within hydrogen—bonding distance :32
`and 3.5 A. respectively, between the heavy atoms] to
`the Asp 130 NH of the HIV-1 protease. The bis—'l‘hf
`oxygen-t5 and the PH quinoline amide carbonyl of 1 are
`also appropriately positioned for hydrogen—bonding
`interaction with the Asp 253 NH [bonding distance 3.0
`and 3.3 A. respectively. between the heavy atoms!
`present in the fig-binding domain or the enzyme. Like
`most reported protein—ligand complex structures. the
`P3 bis—'l‘hf urethane carbonyl and the icrbbutyl amide
`carbonyl of 49 hydrogen bond to the structural water
`molecule that interacts with the flap llc 50 NH resi-
`(luesfi‘r' Thus. incorporation of a stcreocheniicaily dc-
`i'tned bis-Thl‘ or fused Thl'—’l‘hp ligand into the Ho 31-
`8959—based hydroxyethylainine
`isostere provided
`
`Lupin Ex. 1085 (Page 7 of 15)
`Lupin Ex. 1085 (Page 7 of 15)
`
`

`

`Nonpeptidal Ligands for HIV Protease Inhibitors
`
`Journal of Medicinal Chemistry. 1.996, Vol. 39. No. 1'? 3233
`
`Table 2. Structure and Inhibitory Potencies of Inhibitors
`Containing Novel Bicyclic P2 Ligands
`
`
`
`
`Comp.
`ICSO {11M}
`CICQS {11M}
`
`l .8i0.2
`(“=7)
`
`46:4
`(n=4)
`
`6.4
`
`200
`
`4.2
`
`100
`
`l .2
`
`50
`
`l 90
`
`17
`
`-
`
`‘“
`
`4 10
`
`>200
`
`1700
`
`---
`
`
`
`essentially replaces two amide bonds and a 10::-
`aromatic system of the Ro 31-8959 inhibitor which is
`currently in advanced clinical trials.“
`In addition to reduction of molecular weight and
`replacement of amide bonds, inhibitors incorporating
`the fused cyclic others as P2 ligands have shown
`significant improvement in aqueous solubility compared
`to the Ro 31-8959 class of inhibitors. As can be seen in
`Table 3, inhibitor 49 has shown an aqueous solubility
`of 0.235 mgme in phosphate buffer (pH = 7.4), a greater
`than 23-fold improvement over 1 [Ro 31-8959; aqueous
`solubility less than 0.01 mg/mL). The 3(Sl-tetrahydro-
`furanyl carbamate in inhibitor 43 also exhibited solubil-
`ity enhancement (aqueous solublity 0.15 mgme). The
`inhibitor containing bis-Thf ligand (inhibitor 49} also
`shows a decreased log P value compared to inhibitor 1.
`The log P values of inhibitors 1 and 49 were measured
`to be 5.7 and 3.5. respectively. Pharmacokinetic studies
`in dogs with inhibitor 52 are encouraging. Inhibitor 52
`was administered in dogs at an oral dose of 10 cog/kg
`dissolved in a 10% citric acid solution. The average {two
`dogs} Cm“ was 4380 nM after 20 min. After 3 h, the
`plasma level was above 200 nM. more than 4-fold over
`its CIC95 value. Oral bioavailability was estimated by
`comparing the area under the curve following oral
`administration with that of iv administration at a dose
`of 2 mgfkg in DMSO. The oral bioavailability was
`determined to be 17% in dogs under the formulation
`protocol described above.”
`Conclusion
`
`In summary, on the basis of X-ray crystal structures
`of various protein-ligand complexes, we have designed
`and synthesized a novel class of nonpeptidal high-
`affinity P2 ligands for the HIV-1 protease substrate-
`binding site. The designed ligands are conformationally
`constrained with a fused bicyclic ether-like feature. The
`inhibitor incorporating a 3(R).3a(S).6a(Rl-bis-Thf as the
`P2 ligand (inhibitor 49} is the most potent compound in
`the series. This inhibitor has exhibited in vitro antiviral
`
`activities comparable to inhibitors in the Ro 31-8959
`(inhibitor ll-based hydroxyethylamine series with both
`P: and P3 ligands. Based on the inhibitor-bound X-ray
`crystal structures of 1 and 49 with the HIV-1 protease,
`it appears that the bis-Thf ring oxygens essentially
`compete for the same binding site as the P2 asparagine
`carboxamide and the Pa quinaldic amide carbonyls of
`inhibitor 1 {.Ro 31-8959). Through various structure—
`activity studies, We have also shown that the stereo-
`chemistry, position of oxygens, substitution in the ring,
`and ring size all are critical
`to optimum binding.
`Incorporation of bis-Thf ligand {inhibitor 49) not only
`provided a protease inhibitor with reduced molecular
`weight but also led to improved aqueous solubility and
`decreased log P value (Figure 4). Basically, fusion of a
`tetrahydrofuran ring to inhibitor 44 resulted in inhibitor
`49 [Figure 4) with an impressive SOD-fold enhancement
`in its inhibitory potency and more than 30-fold enhance-
`ment of its antiviral potency. The molecular weight of
`the bis-Thf is essentially one-half the combined molec-
`ular weight of the P2 asparag’lne and P3 qujholine
`ligands of inhibitor 1. Further molecular design is
`currently underway in our laboratories.
`
`.
`
`49
`
`50.
`
`51.
`
`52.
`
`53.
`
`54.
`
`55,
`
`56.
`
`57.
`
`53.
`
`59.
`
`60.
`
`>3000
`
`l 8. 1
`
`(X=H. Y=MEJ
`
`4. l
`(X=Me. Y=Hl
`
`27. l
`
`>200
`
`>200
`
`200
`
`200
`
`inhibitors (compounds 49 and 52) with comparable in
`vitro antiviral potencies to inhibitors with both P2 and
`P3 ligands. As shown in Figure 3, the bis-Thf ligand
`
`Experimental Section
`All melting points were recorded on a Thomas-Hoover
`capillary melting point apparatus and are uncorrected. Proton
`
`Lupin Ex. 1085 (Page 8 of 15)
`Lupin Ex. 1085 (Page 8 of 15)
`
`

`

`3284
`
`Jnm‘rm.’ :if'.-'l.-fe(llriritrl ("Itl’nltstr'x-u 3.95M. Vm’. 35'. NH. l7
`
`films-h l"! ril
`
`
`
`Figure 3. SLL'I'LEfJViL'W ol' the X‘l'Ily strut-lure of the inhiliimrs 1 IgrcenI and 49 [meet-mm bound to HIV-l protease.
`
`Table 3. Aqueuus Suluhilities ul' Selet'tetl Iiil'iIlnluI's
`iuhiluiturs suluhility I rue-"ml“
`
`]
`ILHI
`4:3
`".15
`.15.;
`ll.
`”if:
`5!}
`[Hill]
`52
`0.1!]?
`
`
`r
`
`H
`.../\_._
`
`"x
`
`’
`rH
`
`Ho
`H
`o
`/N ,/ x/N-x
`/'~::H/“~N'
`ll.
`N
`H H o
`g H i.-
`r. T.
`0' N,
`.
`H
`f
`“xxx”
`
`n
`
`Ph/
`
`1 R.—L‘H3L"UNH‘.
`2 R = t- r; -] |l -'
`I
`I
`I
`
`ll
`
`
`
`44
`
`”'“x
`_
`H. I
`_..J "Y/H
`.N.\_
`.. H
`
`H
`
`l,-
`
`HO
`
`/\
`
`H l
`./*-..
`
`-'
`T H
`
`H
`
`Figure 4.
`
`magnetic resonance spectra were recorded on a Van-mu XI.—
`thJlJ speetrmimter using tetrametliylsiltme as the internal
`standard. Significant. 1l'l-NMR tlfiiu lill' representative mm—
`puulirla are. lahlllmud in the liillnwing nrtier: niuliiplieiiy is.
`singlet:(Li-lmihlt-rt:l.tripletq.quartet-.111. Inultipleti. number
`Hf pruitms. L'llllpllt‘if: cunstanllsl in hertz. FAB mass spectra
`
`were I'eL'urrletl on :I \"(i Mutlel TUTU mass speetrumeter. antl
`I‘t-levum data are tabulated as ”Ir": Elemental analyses were
`perl'nrmerl by the Analytic-til Department. Merck Research
`Luburtiluries. West Point. FA. and were \l'ILlllll
`:;I.I.4'« ul'the
`theoretical values. Alihytlr'ritls solvents were obtained as
`liilluws: melhvlerle t'hlnride. distillation li‘nm |’;< 1;“; Ll-’[.l'i-Il1}-'-
`(lrnliirzm. distillation l‘rnm sndium-"henznphennne: dimethyl-
`lisrmamitle and pyridine. (lislillatiun l'rnm (_‘tiHi. All ether
`snlvt-uts were i-ii’U1 grade.
`(Iltllllll'li'l ehI‘umalugruphy was
`pi-rl'urmL-(l with E. Merck 2rlt| mu mush silica gel under .1 low
`pressure at?)
`In psi,
`'l‘hin-layer Climmat .igraphy r'l‘l.t"-I was
`
`carried mat with IC. Mort-k silica gel h'II F )fi-l plates.
`J—l)i—
`r
`t2R.3Sl-Ethyl 2-Hydr0xy~3-alIylsuecinate M).
`['l-l1}'ll'2”JiHJ-ii-HllXl-Z'l‘lydt'nxyfitlt't'lHal-l”i733. gI was prepared
`Liteilt'tllng tn the pruretlure of D. SL‘t'lJilCl}. J. Aehi. and I}.
`Wusmulh. (Jr-gum}- .“x'_\-i.=filw.~;e.-< 1985. fill. 109 - 12!}. Compound
`4: El-l-NMR I(".[}{"-l;:i r! 5.3 I m.
`1 Hi. :‘i. 1:") I m. 2 H I. 4.3 Im.
`] H ”I.
`-'1.I--1.2I.m.:1ilr.f$.tl|'m. l lll.2.-1-2.7Im.2|ll.1.25I'l.3}'l.
`J — Millzl.1.2tl.3ll.J=ti.EJllzI_
`(ZRBRI-l.2-0-Isopropylidene-S-allylbutane-1.4-diol (5].
`A soluiiuu of $5.4 it; will); Iumul i Ial'fZHJSSi—ethyl 2—h_\-‘(lrr1x_\'—I$-
`:Illylslieeinule |2| in 1:3 mL ni'rlielhyl ether was added t‘lrnp“ Ise
`in a suspensiuu (ii 3.1 g Halli mimill ul' LEAH-l.
`in ether ITU
`IiiI.i at
`I) "l‘. The resulting mixture was stirred at
`I'uum
`ll’l'lipl‘l‘t-Illll't'
`t'ur 12 h. After this perinrl. the mixture was
`heated In reflux for 1 h and then muted to LI "(.‘. The reamirm
`was quencher! hy- set‘luentinl rlrnpwise :‘itltlitiisn ul' water IIll
`mla. 2W5 uqueuus NaOI-l
`ILL]
`tub. and [lien water I8 i'nLi.
`The mixture w“:- .-.:tirred for
`l h. and ‘l‘lll-1 I50 min and
`:mliydmus Nag-50.; were added. The mixture was filtered
`Lhrutlgli CL‘llLL‘. and the tiller [Julie was Llu'uughl).r washed will)
`Till"
`I‘lvnpurutinn or the sulvenls gave 63 g at the rm’re-
`sprawling“r trial. as a eulm'le s all whieh was utilized rlii'(--Cl.l_\-'
`without t'urt her puritieatirm.
`The I't-:s‘11ltingztrio| was dissulved in acetone E500 lllLl. aml
`ISII mg at}:vmluenesulfnnir arirl mmmhyclmte was added. The
`resulting.r mixlure was stirred at 2-4 "t‘. for 3 h. After this
`period. the mixture was L‘lll'll't'l'li rate-(l umler l‘(-'(ll.lL'(-.‘(l pressure.
`and Ihe resulting t‘uéfiitltlt’ was taken up in ethyl Ellfflliile and
`“'L'ttil'lL'El with saturated aqueous N:iH(‘.U;-,. The layers were
`
`
`separated, and the urgunie layel was rli‘it—ttl
`river N‘ 50.1.
`
`Filtration anti evaporation Hi the sulvenl prm’irled u I‘ .sidue
`which was chrrmmtngrupherl over silica gel [5(1‘3 ethyl acetate"
`hexane: to furnish 4.] 1.1159“; “\‘it‘ldl of the. title. emnpeuntl as
`t1 L'nlurless uil:
`il-LNMH IC-DUlnI r‘I 5.?5 lm. l H1. 5.] int. 2 ill.
`
`Lupin Ex. 1085 (Page 9 of 15)
`Lupin Ex. 1085 (Page 9 of 15)
`
`

`

`
`.Wru' -.
`
`Nonpeptidoi Ligands for HIV Protease In