EXHIBIT B1
`EXHIBIT B1
`
`ALVOGEN, Exh. 1055, p. 0084
`
`ALVOGEN, Exh. 1055, p. 0084
`
`

`

`Rate
`
`Editor-in-Chief:
`DALE L. BOGER
`WeegeeMat
`veeyiTe)
`CA 92037
`
`USA
`
`A TETRAHEDRON PUBLICATION
`ASULIZ ctaTPN laTaeeytt
`COMMUNICATIONS ON ALL ASPECTS OF
`ESCAom eaCatAMantate Le
`JOVIenaaTaianTTT RELATED DISCIPLINES
`
`PERGAMON
`
`
`;
`
`
`
`ALVOGEN, Exh. 1055, p. 0085
`
`

`

`BIOORGANIC & MEDICINAL CHEMIST!
`
`
`EXECUTIVE BOARD OF EDITORS FOR TETRAHEDRON PUBLICATIONS
`
`Chairman: Professor H. H. Wasserman
`Department of Chemistry, Yale University, PO Box 208107, New Haven, CT 06520-8107, USA
`ProfessorSir J. E. Baldwin, FRS, Dyson Perrins Laboratory,
`Professor G. Qurisson, Centre National de la Recherche
`Oxford, OX] 30Y, UK
`Scientifique, Centre de Neurochimie, 67084 Strasbourg, Cedex,
`France
`Professor D. L. Boger, Department of Chemistry, The Scripps
`(Associate Editor, Professor G. Solladié)
`ResearchInstitute, 10550 North Torrey Pines Road, La Jolla, CA
`:
`92037, USA
`Professor G, H. Posner, Departmentof Chemistry, Johns Hopkins
`.
`.
`;
`.
`University, Baltimore, MD 21218, USA
`Professor D. Curran, Department of Chemistry, University of
`Pittsburgh, Pittsburgh, PA 15260, USA
`Professor M. Shibasaki, Faculty of Pharmaceutical Sciences,
`University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo | 13, Japa:
`Professor S. G. Davies, Dyson Perrins Laboratory, Oxford,
`°
`;
`-
`?
`,
`pan
`OX] 30Y, UR
`Professor T. Shioiri, Faculty of Pharmaceutical Sciences, Nagoya
`.
`City University, Tanabe-dori, Mizuho-ku, Nagoya 467, Japan
`Professor B. Ganem, Departmentof Chemistry & Chemical
`.
`“
`'
`Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-
`Professor W. Steglich, Institut fiir Organische Chemie der
`1301, USA
`Universitit Miinchen, Butenandtstr. 5-13, D-81377 Miinchen,
`z
`—_
`;
`Germany
`Professor L. Ghosez, Laboratoire de Chimie Organique de
`.
`Synthése, Université Catholique de Louvain, B-1348 Louvain-la-
`Professor R. J. K. Taylor, Departmentof Chemistry, University
`Neuve, Belgium
`of York, Heslington, York, YOLO SDD, UK
`(Associate Editors, Dr P. A. O’Brien and Dr J. M. Vernon)
`Professor N. K. Kochetkov, N. D. ZelinskyInstitute of Organic
`‘
`7
`"
`ae
`Chemistry, Academyof Sciences, Moscow B-334, Russia
`Professor E. J. Thomas, Departmentof Chemistry, University of
`.
`.
`.
`.
`Manchester, Brunswick Street, Manchester, M13 9PL, UK
`Professor Lin Guo-Qiang, Shanghai Institute of Organic
`(Associate Editor, Dr T. J. Donohoe)
`Chemistry, Chinese Academyof Sciences, 354 Fenglin Road,
`:
`Shanghai 200032, China
`Professor C.-H. Wong, Department of Chemistry, The Scripps
`.
`ResearchInstitute, 10550 North Torrey Pines Road, La Jolla, CA
`Professor S. F. Martin, Departmentof Chemistry and
`92037, USA
`Biochemistry, University of Texas, Austin, TX 78712, USA
`Professor Y, Yamamoto, Department of Chemistry, Graduate
`Professor W. B. Motherwell, Department of Chemistry,
`SchoolofScience, Tohoku University, Sendai 980-8578, Japan
`(Associate Editor, Professor M. Hirama)
`University College, 20 Gordon Street, London, WCIH OAS, UK
`
`BOARD OF CONSULTING EDITORS
`C, D, Poulter, Salt Lake City, UT
`A. K, Ganguly, Bloomfield, NJ
`P. S. Anderson, Wilmington, DE
`R, A. Lerner, La Jolla, CA
`J. Rebek, Jr, La Jolla, CA
`D. Gani, St Andrews
`J, K. Barton, Pasadena, CA
`S. J. Lippard, Cambridge, MA
`B. Samuelsson, Stockholm
`5B.Giese, Basel
`.
`K, W. Bair, Summit, NF
`H. Liu, Minneapolis, MN
`R. L. Schowen, Lawrence, KS
`e - es Panicle,A p-ae ooo —
`D. Mansuy,Paris
`.
`S. L, Sehreiber, Cambridge, MA
`A.
`U, Dresiow,
`New
`York,
`,
`4.
`Groves, Princeton,
`A. McKillop, Northumberland
`d: Ay Bristol, Ann Arbor, Ml
`G. L. Granewald, Lawrence, KS
`B. W, Metcalf, King ofPrussia, PAP. G, Schultz, Berkeley, CA
`T. C. Bruice, Santa Barbara, CA
`P, Herrling, Basel
`R. Metternich, Basel
`A. I. Seott, College Station. TX
`A. R. Chamberlin, Irvine, CA
`D. Hilvert, Zurich
`L. A. Mitscher, Lawrence, KS
`C. J. Sih, Madison, WI
`4. P, Collman, Stanford, CA
`R. H. Holm, Cambridge, MA
`ay
`<n
`I
`‘eee
`a
`P. N, Confalone, Wilmington, DE
`D, C. Horwell, Cambridge
`a
`-
`*
`B Lo
`“Tat
`|
`W. H. Moos, San Diego, CA
`J. Stubbe, Cambridge, MA
`?
`E. J, Corey, Cambridge, MA
`J. R. Huff, West Point, PA
`K. Mori, Tokyo
`N. i, Terrett, Sandwich
`5. J. Danishefsky, New York, NY
`B. Imperiali, Pasadena, CA
`K. Nakanishi, New York, NY
`M.D. Tsai, Columbus, OH
`P, B. Dervan, Pasadena, CA
`K. Janda, La Jolla, CA
`K. C. Nicolaou, La Jolla, CA
`G, L. Verdine, Cambridge, MA
`A, Eschenmoser, Ziirich
`W. L. Jorgensen, New Haven, CT
`T. Ogawa, Saitama
`Hi. Waldmann, Karlsruhe
`JoM. Fang, Taipei
`A. R. Katritzky, Gainesville, FL
`M. Ohno, Tokyo
`€. T. Walsh, Boston, MA
`A. R. Fersht, Cambridge
`D. D. Keith, Cambridge, MA
`HW. L. Pearce, Indianapolis, IN
`G. Whitesides, Cambridge, MA
`D. M, Floyd, Princeton, NI
`P, Krogsgaard-Larsen, Copenhagen
`P, Potier, Gif-sur-Yvette
`R. V. Wolfenden, Chapel Hill, NC
`
`
`PUBLISHED TWICE MONTHLY
`Orders, claims, and product enquiries: please contact the Customer Support Departmentat the Regional Sales Office nearest you:
`New York: Elsevier Science, PO Box945, New York, NY 10159-0945, USA;phone: (+1) (212) 633 3730 [toll free number for North American
`customers: 1-888-4ES-INFO (437-4636)]; fax: (+1) (212) 633 3680; e-mail: usinfo-f@elsevier.com
`Amsterdam: Elsevier Science, PO Box 21 1, 1000 AE Amsterdam, The Netherlands; phone: (+31) 20 4853757:fax: (431) 20 4853432: e-mail:
`nlinfo-f@elseviernl
`Tokyo: Elsevier Science, 9-15 Higashi-Azabu I-chome, Minato-ku, Tokyo 106-0044, Japan; phone: (+81) (3) 5561 5033; fax: (+81) (3) 5561
`5047; e-mail: info@elsevier.co.jp
`Singapore: Elsevier Science, No.
`| Temasek Avenue, #17-01 Millenia Tower, Singapore 039192; phone: (+65) 434 3727: fax: (+65) 337 2230;
`e-mail: asiainfo@elsevier.com.sg
`Rio de Janeiro: Elsevier Science, Rua Sete de Setembro 111/16 Andar, 20050-002 Centro, Rio de Janeiro - RJ, Brazil: phone: (+55) (21) 509
`5340; fax: (455) (21) 507 1991: e-mail: elsevier@campus.com.br [Note (Latin America): for orders, claims and help desk information, please
`contact the Regional Sales Office in New York aslisted above]
`
`Copyright © 1999 Elsevier Science Lid
`
`ALVOGEN,Exh. 1055, p. 0086
`
`ALVOGEN, Exh. 1055, p. 0086
`
`

`

`BIOORGANIC & MEDICINAL
`CHEMISTRY LETTERS
`
`
`A Tetrahedron Publication for the Rapid Dissemination of Preliminary Communications on
`all Aspects of Bioorganic Chemistry, Medicinal Chemistry, Bioinorganic Chemistry
`and Related Disciplines
`
`Chairmanofthe Executive Board ofEditorsfor Tetrahedron Publications
`Professor H. H. Wasserman
`
`
`
`PERGAMON
`
`Printedin GreatBritain by Nuffield Press Ltd.
`MN
`
`
`
`
`
`
`
`
`
`
`
`
`0960-894Xx(1999)9:11; 1-R
`
`geet:
`Pen 4 yy
`x a,
`
`a ‘ ' he A Hay Mo
`
`|
`
`JUNG 6 1999
`
`|
`
`\
`‘LIBRAaL “
`
`/
`
`972
`
`soso
`
`ALVOGEN, Exh. 1055, p. 0087
`
`

`

`date ofdispatch.
`
`publisher,
`
`noted above.
`
`(Tel: (01865) 843000: Fax: (01865) 843010),
`
`
`
`PublishingandAdvertising Office. Elsevier Science Lid. The Boulevard. Langford Lane, Kidlington, Oxford. © 31GB
`Publication information, Bioorganic& Medicinal ChemistryLetters (ISSN 0960-894). For 1999, volume 9 1S scheduled
`forpublication. Subscription pricesareavailable upon request fromthePublisheror from the Regional Sales Officenearest
`you or fromthisjournal’s website (hitp:/Avww.elsevier.niMocate/is), Purtherinformationis available on thisjournal and
`otherElsevierScience products through Elsevier’s website (http://www.elsevier.nb), Subscriptions areaccepted on aprepaid
`basis onlyand areentered on acalendaryearbasis. Issues are sent by standard mail (surface within Europe,airdelivery
`outside Europe), Priorityrates are available upon request. Claims formissing issues should be made within six months ofthe
`PeriodicalsPostageispaidatRahway, NJ, Bioorganic & Medicinal ChemistryLetters (ISSN0960-894)is published twice
`monthlyJanuaryto Decemberin one volume byElsevier Science Ltd., The Boulevard, Langford Lane, Kidlington, Oxford
`OX5 IGB, UK. The US subscription price is $1770.00 per year,
`POSTMASTER:Sendaddress correctionsto: Bioorganic & Medicinal ChemistryLetters, ElsevierScience, Customer
`Support Department, PO Box 945, New York, NY 10159-09445.
`Distributedin the USA by Mercury Airfreight International, 365 Blair Road, Avenel, NJ 07001.
`Microform andBackIssues. Back issues ofall previouslypublished volumes, in both hard copyand on microform,are
`available direct from Elsevier Science offices.
`© 1999 Elsevier Science Ltd. All rights reserved.
`Thisjournal and the individual contributions contained in itareprotected undercopyright byElsevierScienceLtd, and the
`following terms and conditions apply to their use:
`Photocopying. Single photocopies ofsingle articles maybe made forpersonal use as allowed bynational copyright laws,
`Permission ofthepublisherand paymentofa fee is requiredforall otherphotocopying, including multiple orsystematic
`copying, copying for advertising or promotional purposes, resale, and all forms ofdocument delivery. Special rates are
`available foreducational institutions that wish to make photocopiesfornon-profit educational classroom use.
`Permissions maybesought directly fromElsevierScience Rights & Permissions Department, PO Box 800, Oxford OX5
`IDX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333. e-mail: permissions@elsevier.co.uk. You mayalso contact
`Rights & Permissions directlythrough Elsevier’s homepage (http:/Avww.elsevier.n]), selectingfirst ‘CustomerSupport’,
`then ‘General Information’, then “Permissions Query Form’,
`In the USA,users mayclearpermissions and makepayments through the Copyright Clearance Center, Inc., 222 Rosewood
`Drive, Danvers, MA01923, USA; phone: (978) 7508400,fax: (978) 7504744, and in the UK through the Copyright
`LicensingAgencyRapidClearanceService (CLARCS), 90Tottenham Court Road, London WIPOLP, UK;phone: (+44)
`171 631 5555:fax: (+44) 171 631 5500. Othercountries mayhave a local reprographicrights agencyforpayments,
`Derivative Works. Subscribers mayre roducetables ofcontents or prepare lists ofarticles including abstracts forinternal
`¥
`rep
`Pp.
`g
`circulation within theirinstitutions. Permission ofthepublisheris required forresale ordistributionoutside the institution,
`Permission ofthe publisheris required for all otherderivative works, including compilations and translations.
`ElectronicStorageor Usage. Permission ofthepublisheris requiredto store or use electronicallyany material contained in
`this journal, including anyarticle or part of an article,
`Exceptas outlinedabove, no partofthis publication maybereproduced,stored in aretrieval systemortransmitted in any
`formorbyany means, electronic, mechanical, photocopying, recording orotherwise, without prior written permission ofthe
`Address permissions requests to: ElsevierScience Rjghts & Permissions Department, at the mail, fax and e-mail addresses
`Notice. No responsibilityis assumed by the Publisherfor anyinjury and/ordamageto persons orpropertyas a matterof
`productsliability, negligenceorotherwise, or fromanyuse oroperation ofany methods, products,instructions or ideas
`contained in the material herein. Becauseofrapid advancesin the medical sciences, in particular, independentverification of
`diagnoses and drug dosages should be made.
`Althoughall advertisingmaterial is expected toconform to ethical (medical) standards, inclusioninthis publication does not
`constituteaguarantee orendorsementofthe quality orvalueofsuch product orofthe claims madeofit byits manufacturer,
`®™Thepaperusedin this publicationmeets therequirements ofANSI/NISO239.48-1992 (PermanenceofPaper),
`
`
`
`ALVOGEN, Exh. 1055, p. 0088
`
`ALVOGEN, Exh. 1055, p. 0088
`
`

`

`Bioorganic & Medicinal Chemistry Letters Vol. 9, No. 11
`
`¥
`
`Vii
`
`1477
`
`1484
`
`Contents
`
`Contributors to this issue
`
`Graphical abstracts
`Uridine phosphorylaseinhibitors: chemical modification of benzyloxybenzyl-
`barbituric acid andits effects on UrdPase inhibition
`
`Nonpeptidic HIV protease inhibitors: S-alkyl-5,6-dihydropyran-2-ones
`possessing achiral 3-(4-amino/carboxamide-2-butyl,5-methylphenyl thio)
`moiety: antiviral activities and pharmacokinetic properties
`
`1487
`
`1493
`
`1499
`
`1505
`
`1517
`
`1521
`
`1527
`
`1537
`
`15441
`
`A new monoclonal anti-idiotypic catalytic antibody with a CPA-like activity
`Synthesis and evaluation of glucocerebrosidase inhibitory activity of anhydro
`deoxyinositols from (+)-epi- and (~)-vibo-quercitols
`
`Synthesis and biological evaluation of a-mannosidase inhibitory activity of
`three deoxy derivatives of mannostatin A
`Synthesis of phosphonate 3-phthalidyl esters as prodrugs for potential
`intracellular delivery of phosphonates
`
`Design of the first highly potent and selective aminopeptidase N (EC
`3.4.14.2) inhibitor
`Liquid phase parallel synthesis of guanidines
`isolation and synthesis of a novel
`immunosuppressive 17a-substituted
`dammarane fromthe flour of the Palmyrah palm (Borassus Habellifer
`The sulfonimidamide as a novel transition state analog for aspartic acid and
`metallo-proteases
`Isolation and characterization of an active-site peptide from a sterol methyl
`transferase with a mechanism-based inhibitor
`Liquid phase synthesis of arylamines and its application to the benz-
`imidazolone via nucleaphilic aryl substitution
`Synthesis and evaluation of hapalosin and analogs as MDR-reversing agents
`
`ogo
`BOCp
`
`J. Guerin, D, Mazeas,
`S. Musale, F.N. M, Naguib,
`N. Al Safarjalani, M. H. el Kouni
`nd R.P. Panzica
`V.N. Vara Prasad, F. E, Boyer,
`M. Domagala, E. L. Ellsworth,
`- Gajda, S. E. Hagen, L. J. Markoski,
`. D, Tait, E. A, Lunney,
`P. J. Tummino, D. Ferguson,
`T. Holler, D. Hupe, C. Nouhan,
`S.J. Gracheck, S. VanderRoest,
`. Saunders, K. lyer, M. Sing and
`. Brodfuehrer
`
`dd M
`
`. Du, Z, Guo and S. Jin
`5, Ogawa,S. Uetsuki, Y. Tezuka,
`T. Morikawa, A. Takahashi and
`K. Sato
`
`5S. Ogawa and T. Morikawa
`
`TO
`. Dang, B. S. Brown,
`D. van Poelje, T. J. Colby and
`M.D. Erion
`
`H. Chen, B. P. Roques and
`M.-C. Fournié-Zaluski
`
`KG, Ho and C.-M. Sun
`L. Révész, P. Hiestand,
`i. La Vecchia, A. Naef, H.-U, Naegeli,
`L. Oberer and H.-J. Hoth
`83. &, Cathers and J. V. Schloss
`
`J. A. Marshall and W. D. Nes
`
`P.-C, Pan and C.-M. Sun
`
`i &, O'Connell, K. A. Salvato,
`ON
`Meng, B. A, Littlefield and
`E. Schwartz
`E,
`
`8.E.Maryanoff, S. 0, Nortey,
`1547
`Potential anxiolytic agents. Part 3: Novel A-ring modified pyridof1,2-
`J.J. MeNally, Po. Sanfilippo,
`a]benzimidazoles
`2. F. MeComsey, B, Dubinsky,
`: Shank and A. B. Reitz
`ALP
`HH.
`Sato, O. Kitagawa,Y. Aida,
`J. Chikazawa,T. Kurimoto, M. Takei,
`Y. Fukuta and K. Yoshida
`Y. Hsiao, K. M. Wells, C. Yang,
`M.S. Jensen, J. YL. Chung,
`N. Yasuda and D. L. Hughes
`v. Wouters and P, Herdewijn
`
`1553
`
`1559
`
`1563
`
`E. Davioud-Charvet, A. Berecibar,
`S. Girault, V. Landry, H. Drobecq and
`C. Sergheraert
`J. Wichmann, G. Adam,
`S. Kolezewski, V. Mutel and
`T. Woltering
`
`1567
`
`1573
`
`Dual-acting agents with &,-adrenoceptor antagonistic and Steroid 5a-
`reductase inhibitory activities. Synthesis and evaluation of arylpiperazine
`derivatives
`Preparationof crystalline p-nitrobenzyl 2-formyl carbapenems by oxidative
`cleavage
`
`5-Substituted pyrimidine 1,5-anhydrohexitols: conformational analysis and
`interaction with Viral thymidine kinase
`Synthesis of polyamine derivatives
`for
`the preparation of
`affinity
`chromatography columns for the search of new Trypanosoma cruzi targets
`Structure—activity relationships of substituted SH-thiazolo[3,2-a]pyrimidines
`as group 2 metabotropic glutamate receptor antagonists
`
`ALVOGEN, Exh. 1055, p. 0089
`
`ALVOGEN, Exh. 1055, p. 0089
`
`

`

`&. Charles and Y, Letourneux
`
`P. Ciuffreda, S. Casati and
`E. Santaniella
`». Caldirola, R. Chowdhury,
`L. Unelius, N, Mohell, U. Hacksell
`and A. M. Johansson
`M. H. Chen, 0. P, Goel, J.-W. Hyun,
`J. Magano and J.B. Rubin
`,
`C, Mao, E. A, Sudbeck,
`TK. Venkatachalam and FM. Uckun
`P, Beuchet, M. Dherbomez,L. Elkiel,
`E. Baston and R. Ww. Hartmann
`D. B. Jordan, T. A. Lessen,
`& Wawrzak, J. d, Bisaha,
`T. C. Gehret, S. 1, Hansen,
`R. S. Schwartz and G, S. Basarab
`G. S. Basarab, D. B. Jordan,
`T. C. Gehret, R. S. Schwariz and
`A. P. Tamiz, E.R. Whittemore,
`R. M. Woodward, R. B. Upasani and
`J. F.W. Keana
`G. W. Muller, R. Chen,S.-Y, Huang,
`L. G. Corral, L. M. Wong,
`RT. Patterson, Y. Chen, G. Kaplan
`and D. |. Stirling
`
`2. Wawrzak
`
`inhibitor
`
` Lipase-catalyzed protection ofthe hydroxy groupsofthe nucieosides inosine
`1577
`and 2'-deoxyinosine: a new chemoenzymatic synthesis of the antiviral drug
`«',3'-dideoxyinosine
`1583 Novel derivatives of3-(dipropylaminojchroman. Interactions with S-HT. and
`D., receptors
`1587 An
`efficient Siereoselective synthesis of (3S5,95)]-3-[[[9-(benzoyl-
`aminojoctahydro-6,10-dioxo-6H-pyridazino-(1,2-a)(12)-diazepin-1-yl]-
`carbonyilamino}-4-oxobutanoic acid, an interleukin converting enzyme (ICE)
`1593
`Rationaldesign of©(2,6-dimethoxyphenylethyl)}-N'-[2-(5-bromopyridyl)}-
`thiourea (HI-236) as a potent non-nucleoside inhibitor of drug-resistant
`human immunodeficiencyvirus
`1599
`Synthesis of 25-aminosterols, new antifungal agents
`1601
`A-Substituted 4-(5-indoly!)benzoie acids. Synthesis and evaluation of steroid
`Sca-reductase type | andIl inhibitory activity
`1607 Design of scylalone dehydratase inhibitors as tice blast fungicides: (N-
`phenoxypropyl)-carboxamides
`1613 Design of scyialone dehydratase inhibitors as fice blast
`fungicides:
`derivatives of norephedrine
`1619
`Structure—activity relationship fora series of2-substituted 1,2,3,4-tetrahydro-
`9H-pyrido[3,4-blindoles: potent subtype-selective inhibitors of N-methyl-p-
`aspartate (NMDA)receptors
`1625
` Amino-substitutedthalidomide analogs: potentinhibitors ofTNF-c production
`
`|
`
`Instructions to contributors
`
`RETGT|
`ae
`
`
`
` http://www.elsevier.niocate/tis
`
`
`http://www.elsevier.com/locate/tis
`
`Indexed/Abstracted in: Chemical Abstracts,Current Contents
`Science Citation Index, SciSearch, Research Alert, EMBASE/Excerpta
`Medica, Elsevier BIOBASE/Current Awareness in Biological Sciences,
`index Medicus, MEDLINE
`
`
`
`Pergamon
`ISSN 0960-894X
`
`
`
`ALVOGEN, Exh. 1055, p. 0090
`
`ALVOGEN, Exh. 1055, p. 0090
`
`

`

`
`
`Pergamon
`
`Bioorganic & Medicinal Chemistry Letters 9 (1999) 1625-1630
`
`AMINO-SUBSTITUTED THALIDOMIDE ANALOGS: POTENT INHIBITORS OF
`TNF-o. PRODUCTION
`George W. Muller,” Roger Chen," Shaei-Yun Huang," Laura G. Corral,“Lu Min Wong,* Rebecca T. Patterson,"
`Yuxi Chen,” Gilla Kaplan,” and David I. Stirling."
`“Celgene Corporation, 7 Powder Horn Drive, Warren, NJ 07059, U.S.A.
`Rockefeller University, 1230 YorkAvenue, New York, N¥1002], USA,
`
`Received 31 March 1999: accepted 30 April 1999
`
`Abstract: Thalidomide, (1), is a knowninhibitor of TNF-c release in LPS stimulated human PBMC. Herein
`we describe the TNF-c inhibitoryactivity of amino substituted analogs of thalidomide (1) andiis isoindolin-1-
`one analog, EM-12 (2). The 4-amino substituted analogs were found to be potent inhibitors ofTNF-a release in
`LPS stimulated human PBMC. © 1999 Elsevier Science Ltd. Alll rights reserved,
`Introduetion: Thalidomide (2-(2,6-dioxo-3-piperidy)isoindoline-1,3-dione), (1) was developed as a sedative
`without the side effects of barbiturates in the 1950’s by Chemie Grunenthal.! Thalidomide quickly became a
`popular sedative in Europe and Australia and was subsequently used for the treatment of morning sickness in
`pregnant women. However, thalidomide was removed from the marketplace whenits use was linked to birth
`defects. Thalidomide’s teratogenic properties made the drug infamous and catalyzed the development of the
`current drug approval regulations. A serendipitous discovery in 1965 by Sheskin while treating erythema
`nodosum leprosum (ENL), an acute inflammatory condition associated with lepromatous leprosy led to the
`discovery that thalidomide possesses immunomodulatory properties.” Since this initial discovery, thalidomide
`has been foundto afford clinical benefit in a variety ofautoimmune and inflammatory disease states?
`
`QA \ /
`
`Oo
`A, H
`4
`-
`if
`Fre oe YON
`oF NX =o
`i
`
`EM-12 (2)
`Thalidomide(1)
`In 1991 it was reported that thalidomide was a selective inhibitor of tumornecrosis factor-c. (TNF-a)
`over production in stimulated human monocytes.’ TNF-c: is a key cytokine in the inflammatory cascade and
`elevated TNF-a levels are associated with inflammatory diseases.> Recent successful clinical
`trials
`in
`rheumatoid arthritis and inflammatory bowel disease with TNF-c antibodies and soluble TNF-c. receptors have
`validated the inhibition of TNF-a. as a clinical treatment.°
`The clinical activity of thalidomide and the importance of TNF-ct inhibitionled us to initiate a progran
`to improve the TNF-a inhibitory activity of thalidomide by structural modification. We have previously
`
`9960-894X/99/S - see front matter © 1999 Elsevier Science Ltd, All rights reserved.
`PH: $0960-894X(99)00250-4
`
`ALVOGEN, Exh. 1055, p. 0091
`
`ALVOGEN, Exh. 1055, p. 0091
`
`

`

`1626
`
`GW. Muller et al. / Bioorg. Med. Cheni. Lett. 9 (2999) 1625-1630
`
`reported a series ofthalidomide analogs derived from §-amino-B-arylpropanoic acid derivatives that are potent
`inhibitors of TNF-c.’ Further studies revealed these compounds to be potent inhibitors of phosphodiesterase
`type 4 (PDE4),* The PDE4 inhibitory potency for most of these compounds has correlated with their TNF-c
`inhibitoryactivity. PDE4 is the major PDE isoenzyme present in monocytes and macrophages, key producers of
`TNF-a. PDE enzymes control the levels of cyclic adenosine monophosphate (cAMP) by hydrolysis of cAMP to
`3°-AMP. Inhibition of PDE4 in stimulated monocytes has been demonstrated to elevate levels of cAMP and
`inhibit of TNF-c production,’
`In further studies to improve the TNF-o inhibitory activity of thalidomide, we prepared a series of
`amino-phthaloyl substituted analogs of thalidomide (1) and its isoindoline-1-one analog, EM-12 (2). Some
`amino substituted thalidomide analogs have previously been reported but were not assayed for their TNF-c
`inhibitory activity." EM-12 (2) has been reported to be a more potent teratogen than thalidomidein rabbis, rats,
`and monkeys.'’ When 2 was evaluated for TNF-o: inhibitory activity in LPS stimulated human PBMC it was
`found to have similar activity to thalidomide. The isoindolinone replacementof the phthaloy! ring increases the
`stability of the molecule and maylead to increased bioavailability. Herein, we report the structure-activity
`relationships of amino substitution of the phthaloy! ring of thalidomide and isoindolinone ring of EM-12 onthe
`TNF-c inhibitoryactivity in LPS stimulated human PBMC.
`
`Scheme 1
`
`a,b
`Cbz-L-glutamine —-—»
`
`Oy
`yoN
`g} HN ~< =0
`
`3
`
`oO
`
`P oH
`c, d ps ON
`ONS
`3 Boo
`KO a,
`ms
`4a X = 4-NO, 5a X = 4-NH,
`4b X = §-NO, Sb X = §-NH,
`Reagents: (a) CDI, THF, reflux; (b) H,, 10% Pd/C, EtOAc/4N HCI;
`{c) 3, ACOH,reflux; (d) 10% Pd/C, acetone,
`
`=O
`
`Chemistry. The amino substituted analogs of thalidomide were prepared asillustrated in Scheme 1.'* The
`aminothalidomide analogs were prepared via the condensation of 3-aminopiperidine-2,6-dione hydrochloride,
`(3). Compound3 wasprepared in two steps from commercially available Cbz-L-glutamine. Treatment of Cbz-
`L-glutamine with carbonyl diimidazole (CDI) in refluxing THF afforded Cbz-aminoglutarimide. The Cbz
`protecting group was readily removed by hydrogenolysis under 50-60 psi of hydrogeninthe presence of 10%
`Pd/C in a mixture of ethyl acetate and 4 N HCl. The hydrochloride (3) was used directly in the anhydride
`
`ALVOGEN, Exh. 1055, p. 0092
`
`$y
`
`&
`
`*
`
`i
`
`ALVOGEN, Exh. 1055, p. 0092
`
`

`

`with ethyl chloroformate.'* Nef’s reagent is a reagent commonly usedin the preparationofchiral N-phthaloy!
`
`i
`
`| |i:E | ii | ;
`
`protected amino acids. Treatment of 10 with the single isomers of t-butyl glutamine afforded the phthaloy!
`glutamine derivatives, (S)- and (R)- 11. The t-butyl group was removed using standard acidic conditions to
`afford (S)- and (R)- 12. To avoid racemization, the ring closure was accomplished using the methodreported by
`Casini and Ferappi'* for the synthesis of the single isomers ofthalidomide to afford (R)- and (S)- of 4a." The
`nitro groups were reduced as described earlier in acetone to afford the single isomers of Sa.
`Scheme 3
`
`Oo 0
`oO
`i _H \.Oo
`if
`Se
`
`_
`— /
`et
`ap
`foo} ON
`| N-CO,Et
`sl = NAY —
`\
`i
`No, ©
`No, ©
`
`R
`
`\
`i7-NH,
`QO
`
`- C H
`aS“ OTN
`Nek
`|
`7
`od LN
`ey —
`x
`oO
`
`‘se
`
`40
`
`“S)- and (R)- 4a X = NO,
`(S)- and (R)- 47 R = t-Butyl
`(S)- and (R)- 8a X = NH,
`(S)- and (R)- 12 R=H
`Reagents: (a) Et,N, (R) or (S) t-butyl glutamine HCI; (b) HCI, CH,Cl,; (c) SOCI,, pyr/Et,N; (d) Hz, 10% Pd/C, acetone.
`
`ALVOGEN, Exh. 1055, p. 0093
`
`GW. Mulleret al. / Bioorg. Med, Chem. Lett. 9 (1999) 1625-1630
`
`1627
`
`condensation reaction without purification. Treatment of 3 with 3- or 4-nitrophthalic anhydrides in reiluxing
`acetic acid affordedthe 4- and 5-nitro substituted thalidomide analogs 4a and 4h, respectively, in goodyields.
`The nitro groups of 4a and 4b were reduced by hydrogenation in a Parr shaker under 50-60 psi of hydrogen in
`the present of 10% Pd/C to afford the desired 4- and 5-amino substituted thalidomide analog 5a and Sb,
`respectively. The amino substituted isoindolinone analogs were prepared as illustrated Scheme 2.'* Treatment
`of3 with the appropriately substituted nitro substituted methy] 2-(bromomethy!)benzoates, 6a-d yielded the four
`isomeric nitro EM-12 analogs 7a-d. The nitro groups were hydrogenated to the desired amino compound as
`described above to afford 8a-d. The four isomeric nitro substituted methyl 2-(bromomethyl)benzoates (6a-d)
`were prepared by benzylic bromination of the corresponding commercially available nitro substituted methyl 2-
`methylbenzoates.
`
`Scheme 2
`
`i
`
`cSom
`‘
`f> | as
`aa
`ON
`|
`Br
`2
`
`7 PQ yw
`RH PN
`bc i oN S=0
`Bie NLS
`Xx 4
`
` t
`
`ye
`
`7a X = 4-NO, 8a X= 4-NH,
`6a 3-NO,
`7b X = 5-NO, 8b X= 5-NH,
`Sb 4-NO,
`7c X = 6-NO, 8c X= 6-NH,
`6c 5-NO,
`7d X = 7-NO, 8d X= 7-NH,
`6d 6-NO,
`Reagents:(a) light, NBS, CCl,, reflux; (b) 3, EL,N, DMF, 80 °C; (d) H,, 10% Pd/C, MeOH
`The R and 8 isomers of 5a were prepared starting from the S- and R-isomers of glutamine /-buty! ester
`(Scheme 3). The nitro substituted Nef’s reagent analog, 10 was prepared bytreatment of 3-nitrophthalimide
`
`ALVOGEN, Exh. 1055, p. 0093
`
`

`

`1628
`
`CG. W.dulleret al. /Bioore. Med. Chem. Lett. 9 (1999) 1625—1630
`
`The 4-amino-c.-methy! analog (14) ofthalidomide was prepared from a-methylglutamic acid (Scheme
`4). Bystandard chemistry a-methylglutamic acid was converted to Cbz-o-methylglutamie acid anhydride (15),
`Treatment ofthe anhydride with ammonia afforded a mixture ofa- and y-amides, 16, This mixture was cyclized
`with CDI to the Cbz-protected aminoglutarimide 17. The Cbz-group was removed by hydrogenation under
`acidic conditions to afford aminoglutarimide hydrochloride 18, Condensation with 3-nitrophthalic anhydride
`followed byreductionof the nitro group afforded 14,
`
`Sy
`a
`a,b,c
`/|
`/ B
`ZHNA po XA ;f=O
`
`Scheme 4
`
`de
`tee
`
`O #
`/—N
`(
`JO
`
`
`
`48
`
`17 X= Z-NH
`19X=NO,
`18 X= ClH,N
`14 = NH,
`Reagents: (a) NH,, CH,Cl,; (b) CDI, THF: (c) H,, 10% Pd/C, ELOH/4N HCE
`{d) 3-NO,-phthalic anydride, AcOH, reflux: (e) H,, Pd/C, acetone.
`Biological Assays. TNF-« inhibitory activity was measured in lipopolysacharide (LPS) stimulated PBMCas
`previouslyreported.’ The human whole blood TNF-a inhibition assay was run in a similarfashion to the PBMC
`assay except heparinized fresh human whole blood was plated directly into microtiter plates. The assay was then
`continued as previously reported for the PBMC assay. The assay for PDE4 enzyme inhibition was ran as
`previously described,*
`Results and Discussion. Thalidomide has been reported to be a selective inhibitor ofTNF-c. in LPS stimulated
`human monocytes.” Thalidomide has a TNF-c [Cs9 of ~200 uMin LPSstirnulated PBMC.’ Previous research
`with thalidomide analogs suggested that phthaloyl substitution could lead to increasesinactivity. Although the
`amino substitution had been previously described, these analogs had not beentested for their ability to inhibit
`TNF-c production (Table 1). The 5-amino analog, Sb, was found to have a TNF-c ICso of ~100 uM. No
`imhibitory activity was observed at the lower concentrations tested (less than or equal to 10 uM). The 4-amino
`analog, Sa, wassignificantly more potent with an ICs9 of 13 nM. Thus,this compound was ~15,000 times more
`potent than thalidomide as a TNF-c inhibitor in vitro. The novel isomeric amino-substituted EM-12 analogs
`were then prepared and tested. Unlike thalidomide where there are only two regio isomers,
`there are four
`possible regio isomers, Sad, Onlythe 4-amino analog 8a potently inhibited TNF-c. production (ICsp less than
`100 4M). Compound 8a was found to have an ICsq of 100 nM (Table 1). This substitution correlates with the
`amino substitution on 5a and demonstrated that the amino group needed to be opposite to the carbonyl ofthe
`isoindolinone for optimal activity. The S- and R-isomers of Sa were prepared and evaluated. The S-isomerof5a
`was found to be the more active isomer with a TNF-c ICso of 3.9 nM. The R-isomer was ~20-fold less active
`with a TNF ICso of 94 nM, Although (2)-Sa’s optical purity was greater than 95% ee, some activity was
`probablydue to residual (S)-isomerin the sample.
`
`& F
`
`s)
`
`ALVOGEN, Exh. 1055, p. 0094
`
`ALVOGEN, Exh. 1055, p. 0094
`
`

`

`G. W. Mulleret al /Bioorg. Med, Chem. Leit. 9 (1999) J625—1630
`
`1629
`
`The o-methyl analog of thalidomide, 13, has also been reported to demonstrate similar TNF-c inhibitory
`activity to thalidomide.'® This compounddoes not contain the racemizable chiral center found in thalidomide.
`The 4-amino analog 14 was a potent inhibitor ofTNF-a with an ICso of 44 nM. Work is iN progress to prepare
`the single isomers of 14 and will be reported onin the future,
`Compounds Sa, 8a, and 14 were evaluated for PDE4 inhibitoryactivity using PDE4 enzymeisolated
`from U937 cells.® All three compounds were inactive (<50% inhibition) at 100 1M, the highest concentration
`assayed. These results strongly suggested that these compounds do not act by PDE4 inhibition. The three active
`analogs, 5a, 8a, and 14 were evaluated for their ability to inhibit TNF-c: levels LPS stimulated human whole
`blood to mimic their activity in vivo. The compounds had only modest declines in activity in this assay. (Table
`
`1):
`
`Table 1 TNF-o Inhibition in LPS Stimulated Human PBMCand Whole Blood
`
`
`
`~CompdTNF-aInhibit.TNF-a
`At 100pM
`ICso
`TNE-o [Cp
`
`5a
`95%
`13 nM
`25 nM
`Sb
`55% -
`~100.000 nM
`ND
`8a
`74%
`100 nM
`480 nM
`8b
`15%
`ND
`ND
`8e
`12%
`ND
`ND
`8d
`18%
`ND
`ND
`14
`98%
`44 nM
`216 nM
`(S)-5a
`99%
`3.9 nM
`14 nM
`(2)-5a
`85%
`93 nM
`73 nM
`
`In summary, we have discovered three high potency inhibitors of TNF-c by 4-amino substitution of
`thalidomide, EM-12, and a-methylthalidomide. The (S)-4-amino substituted analog of 5a was found to be
`~50,000 times more potent than thalidomideat inhibiting TNF-o levels in LPS stimulated human PBMC, None
`the three compounds showed significant activity as a PDE4 inhibitor, A recent publication reported 14 to
`enhance TNF-o productionin 12-O-tetradecanoyl-phorbol 13-acetate stimulated humanleukemia HL-60 cells.!”
`These discordantresults are possibly related to our use of primary humancells stiraulated with LPSin contrast
`to the other investigators use of the HL-60 cell line stimulated with TPA, Further, we have demonstrated that
`these compoundsretain high activity in the milieu of whole human blood. We are presently investigating the
`structure-activity relationships ofother substituted phthaloyl and isoindolinone analogsof thalidomide and EM-
`i2 and will be publishing on the biological profiles of Sa, 8a, and 14,8
`
`ALVOGEN, Exh. 1055, p. 0095
`
`ALVOGEN, Exh. 1055, p. 0095
`
`

`

`1630
`
`G.W. Mulleret al, / Bioorg. Med. Chem. Lett. 9 (1999) 16251630
`
`References and Notes
`
`L. Stirling, D. I. Pharmaceutical News 1996, 3,17.
`2. Sheskin, J. Clinical Pharmacol. Ther. 1965, 6, 303,
`3. fa) Gutierrez-Rodriguez, O. Arth. And Rheum. 1984, 27, 1118. (b) Peterson, D. L.; Georghiou, P. R.:
`Allworth, A. M.: Kemp, R. J. Infection. Clin. Infect. Dis. 1995, 20, 250. (c) Schuler, U.; Ehninger, G. Drug
`Safety, 1995, 12, 364, (d) Vogelsang, G. B.; Hess, A. D.; Gordon, G.; Brundrette, R.: Santos. G. Ww.
`Transplantation Proc. 1987, AIX, 2658. (e) Klausner, J.; Makonkawkeyoon,S.; Akarasewi, P.; Nakata, K.-
`Kasinrerk, W.; Corral, L.; Dewar, R.; Lane, C.; Freedman, V.; Kaplan, G. J Acq. Immun. Def, Syad. 1996,
`/1, 247,(f) Stirling, D.; Sherman, M.; Strauss, S. J. Amer. Pharm. Assoc. 1997, NS37(3).
`4. Sampaio, E. P.; Sarno, E. N.; Galilly, R.; Cohn, Z. A.; Kaplan, G. J. Exp. Med. 1991, 773, 699.
`a). Eigler, A.; Sinha, B.; Hartmann, G.; Endres, S. Immunology Today 1997, 18, 487,
`J.
`J; Tytgat. G: Woody,
`6.
`(a) van Dullemen, H; van Deventer, S; Hommes, D; Bijl, L;
`Jansen,
`Gastroenterology 1995, 109, 129. (b) Elliott, M.; Feldmann, ML: Maini, R. Jat J Immunopharmac. 1998.
`
`i7, 141,
`
`~I
`
`. Muller, G. W.; Corral, L. G; Shire, M. G.; Wang, H.; Moreira, A.; Kaplan, G.; Stirling, D. LJ Afed Chem,
`
`1996, 39, 3238.
`
`oS
`
`. Muller, G.W.; Shire, M. G.; Wong, L. M.; Corral, L. G.; Patterson, R. T.; Chen, Y.; Stirling, D. I, Bioorg.
`Med. Chem. Lett. 1998, 8, 2669.
`9. Torphy, T. J. Am. J. Resp. Crit. Ca