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`BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
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`Oxford, OX1 3QY, UK
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`
`Bioorganic & Medicinal Chemistry Letters Vol. 8, No. 3
`
`Contents
`
`Contributors to this issue
`
`Graphical abstracts
`
`Stereochemistry of the reduction of 24-ethyldesmosterol to sitosterol in tissue
`cultures of Oryza sativa
`
`Selective inhibition of the chymotrypsin-like activity of the 20S proteasome
`by 5-methoxy-1-indanone dipeptide benzamides
`
`Immunomodulatory activity ofthunberginol A and related compounds isolated
`from Hydrangeae Dulcis Folium on splenocyte proliferation activated by
`mitogens
`
`Methyloxime-substituted aminopyrrolidine: a new surrogate for 7-basic group
`of quinolone
`
`Synthesis and esr study of new dihydroxamic acid siderophores S as
`scavengers of hydroxyl radicals
`
`An alternative synthesis of 4,4-dimethyl-5oi-cholesta-8,14,24-trien-SB-ol, an
`intermediate in sterol biosynthesis and a reported activator of meiosis and of
`nuclear orphan receptor LXRoi
`
`Homologated aza analogs of arabinose as antimycobacterial agents
`
`trans-4-Methyl-3-imidazoyl pyrrolidine as a potent, highly selective histamine
`H3 receptor agonist in vivo
`
`Synthesis and structure—aclivity relationships of pyridine-modified analogs
`of 3-[2-((S)-pyrrolidiny|)melhoxy]pyridine, A-84543, a potent nicotinic
`acetylcholine receptor agonist
`
`A practical synthesis of 3-[(1Ff)-1-t-butyldimethylsilyloxyethyl]-4-[(2Ff)-4—ha|o-
`3-oxo-2-buty|]azetidinone, a versatile intermediate for carbapenem antibiotics
`
`Synthesis of constrained or-amino acid derivatives via ring-closing olefin
`metathesis
`
`Design and synthesis of a biotinylated dopamine transporter ligand for the
`purification and labeling of dopaminergic neurons
`
`Synthesis of L|AZAL'", a retinoic acid metabolism blocking agent (RAMBA)
`with potential clinical applications in oncology and dermatology
`
`A paclitaxel analogue with a 2(3—>20)abeotaxane skeleton: synthesis and
`biological evaluation
`Syntheses of new modified Phe—Pro peptides. Use of proline replacements
`in potential HIV inhibitors
`
`Synthesis and structure—activity relationships of CP-122,721, a second-
`generation NK-1 receptor antagonist
`
`Rapid hydrolysis of amides under physiological conditions: Influence of the
`microenvironment on the stability of the amide bond
`
`inhibitors of acyl-CoA:cho|estero| O-acyltransferase (ACAT) as hypo-
`cholesterolemic agents: Synthesis and structure—activity relationships of
`novel
`series
`of
`sulfonamides.
`acylphosphonamides
`and
`acyl-
`phosphoramidales
`
`vii
`
`205
`
`209
`
`215
`
`221
`
`227
`
`233
`
`237
`
`243
`
`249
`
`255
`
`257
`
`261
`
`267
`
`273
`
`277
`
`281
`
`285
`
`289
`
`y_ Fujimoto, N. Sato, T. Okuzumi,
`J_ Yamada and M. Morisaki
`R, T. Lum, M. G. Nelson, A. Joly,
`A. G. Horsma, G. Lee, S. M. Meyer,
`M, M. Wick and S. R. Schow
`H, Matsuda, H. Shimoda,
`J_ Yamahara and M. Yoshikawa
`
`. Hong, Y. K. Kim, Y. H. Lee and
`
`YH
`
`c,
`
`M. V. D. Nguyen, L. Nicolas,
`.Gaudemer and M. E. Brik
`R
`_ uan, W. K. Wilson and
`G. J. Schroepfer Jr.
`
`A 3
`
`AJ
`
`.AW
`
`.
`
`J. A. Maddry, N. Bansal,
`L. E. Bermudez, R. N. Camber,
`| M. Orme, W. J. Suling, L. N. Wilson
`and R. C. Reynolds
`N
`-Y. Shih, R. Aslanian,
`T. Lupo Jr., 5. Orlando,
`J. Piwinski, M. J. Green,
`K. Ganguly, R. West, S. Tozzi,
`Kreutner and J. A. Hey
`-H. Lin, D. E. Gunn, Y. Li, Y. He,
`Bai, K. B. Ryther, T. Kuntzweiler,
`L. Donnelly-Roberts,
`J. Anderson, J. E. Campbell,
`P. Sullivan, 8. P. Arneric and
`.W. Holladay
`
`NHDD M C S
`
`J.
`
`Yang and N. Yasuda
`
`. Kotha and N. Sreenivasachary
`
`K. Zimmermann and B. Hengerer
`
`E. Freyne, A. Raeymaekers,
`M. Venet, G. Sanz, W. Wouters,
`R. De Coster and J. Van Wauwe
`
`J. Soto, J. L. Mascarefias and
`L. Castedo
`
`M. Bouygues, M. Medou,
`G. Quéléver, J. C. Chermann,
`M. Camplo and J. L. Kraus
`
`T. J. Flosen, K. J. Coffman,
`S. McLean, R. T. Crawford,
`D. K. Bryce, Y. Gohda, M. Tsuchiya,
`A. Nagahisa, M. Nakane and
`5- A. Lowe III
`
`K--H. Gliisenkamp, C. Mengede,
`. Drosdziok, E. Jéihde and
`35
`- F. Raiewsky
`-
`. Lee, W. H. Roark, J. A. Picard,
`21-:
`.Sliskovic, B. D. Roth,
`. Stanfield, K. L. Hamelehle,
`‘I'll’-
`. Bousley and B. R. Krause
`
`2302
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`
`l I
`
`\
`
`lT
`
`l l
`
`l l
`
`il
`
`li
`
`ii
`
`R. E. Mewshaw, M. Husbands,
`E. S. Gildersleeve, M. B. Webb,
`X. Shi, H. Mazandarani, M. I. Cockett,
`R. Ochalski, J. A. Brennan,
`M. Abou-Gharbla, K. Marquis,
`G. B. McGaughey, J. Coupet and
`T. H. Andree
`
`J. M. Fevig, J. Buriak Jr., J. Cacciola,
`R. S. Alexander, C. A. Kettner,
`R. M. Knabb, J. R. Pruitt, P. C. Weber
`and R. R. Wexler
`
`W. J. Pitts, J. W. Jetter, D. J. Pinto,
`M. J. Onlvat, D. G. Batt, S. R. Sherk,
`J. J. Petraitis, I. C. Jacobson,
`R. A. Copeland, R. L. Dowling,
`B. D. Jaffee, T. L. Gardner,
`E. A. Jones and R. L. Magolda
`W. D. Vaccaro and H. Ft. Davis Jr.
`
`W. D. Vaccaro, R. Sher and
`H. R. Davis Jr.
`
`295
`
`301
`
`307
`
`313
`
`319
`
`New generation dopaminergic agents. Part 2: Discovery of 3-QH_
`phenoxyethylamine and 3-OH-N’-phenylpiperazine dopaminergic templates
`
`B,B.Dia|ky|_
`inhibitors:
`thrombin
`boropeptide
`of
`design
`Rational
`phenethylglycine P2 analogs of DuP 714 with greater selectivity over
`complement factor I and an improved safety profile
`
`Structure—~activity relationships (SAR) of some tetracyclic heterocycles
`related to the immunosuppressive agent brequinar sodium
`
`Sugar-substituted 2-azetidinone cholesterol absorption inhibitors: enhanced
`potency by modification of the sugar
`
`Carboxy-substituted 2-azetidinones as cholesterol absorption inhibitors
`
`SVSTEM
`
`IEIRAHEDIION
`INFORMMIUN
`
`http://www.e|sevier.nl/locate/tis
`http://www. elsevier.com/locate/tis
`
`Indexed/Abstracted in: Chemical Abstracts,Current Conlerfa’
`Science Citation Index, Scisearch, Research Alert, Excerpla Medgs)
`Database EMBASE, CABS (Current Awareness in Biological Sclenc
`
`
`Pergamon
`
`X
`ISSN 0960-53:8)
`BMCLE8 8 (3) 2o5—322 (1
`
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`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`
`Pergamon
`
`Bioorganic & Medicinal Chemistry Letters 8 (1998) 301-306
`
`BIOORGANIC &
`MEDlC!N AL CI-[EMISTRY
`LETTERS
`
`[5,B-DIALKYL-
`RATIONAL DESIGN OF BOROPEPTIDE THROMBIN INHIBITORS:
`PHENETHYLGLYCINE P2 ANALOGS OF DUP 714 WITH GREATER SELECTIVITY
`OVER COMPLEMENT FACTOR I AND AN IMPROVED SAFETY PROFILE]
`
`John M. Fevig,* Joseph Buriak, Jr., Joseph Cacciola, Richard S. Alexander, Charles A. Kettner,
`Robert M. Knabb, James R. Pruitt, Patricia C. Weber} and Ruth R. Wexler
`
`The DuPont Merck Pharmaceutical Company, P.0. Box 80500, Wilmington, DE 19880-0500
`
`Received 9 October 1997; accepted 29 December 1997
`
`Abstract: The potent boropeptide thrombin inhibitor DuP 714 caused side effects in laboratory animals that
`appear to be related to its ability to inhibit complement factor 1,
`thereby activating the complement cascade.
`Using X-ray crystal structure information, we have designed compounds that have greater selectivity for
`thrombin over factor I and that have reduced tendency to produce these side effects.
`© 1998 The DuPont Merck Pharmaceutical Company. Published by Elsevier Science Ltd.
`
`The serine protease thrombin is a critical enzyme in the blood coagulation cascade and, consequently,
`inhibitors of thrombin have been pursued as potential antithrombotic agents.2 Ac-D-Phe-Pro-boroArg-OH (DuP
`714) (Figure 2) is a potent (Ki = 0.04 nM), orally active thrombin inhibitor that is effective against both arterial
`and venous thrombosis in animal models.3 However, DuP 714 also causes hypotension and elevated levels of
`serum transaminases following bolus iv dosing. Further studies indicated that DuP 714 also caused transient
`
`thrombocytopenia and leukopenia, and that it caused localized inflammation in response to local injections.
`.
`.
`.
`.
`.
`.
`4
`.
`.
`.
`.
`.
`.
`Extensive in vitro and rat in viva studies aimed at determining the mechanism of these toxic side effects
`
`initially focused on mast cell degranulation and/or complement activation as potential inflammatory mediators.
`The histamine release characteristic of known mast cell degranulators such as compound 48/805 was not
`
`observed upon administration of DuP 714, suggesting that DuP 714 is not acting as a mast cell degranulator.
`However, depletion of complement with cobra venom factor (CVF)6 prior to administration of DuP 714 blocked
`the hypotension, serum transaminase elevations and thrombocytopenia normally observed.4 Additionally, DuP
`714 was found to activate complement in vitro at 1-10 uM, indicated by increased serum levels of SC5b-9,
`
`anaphylatoxin C3a and factor Bb, a marker of activation of the alternate complement pathway. Examination of
`
`the complement cascade (Figure 1) reveals that, of its many serine proteases, factor I alone has an attenuating
`
`role, which involves the inactivation of C3b. Subsequently, DuP 714 was found to be a potent inhibitor of
`complement factor I (IC5o = 10 nM).7 Therefore, it was concluded that inhibition of complement factor I by
`
`DuP 714 allows for rapid amplification of the alternate pathway of the complement cascade, which ultimately
`leads to the production of C3a, C5a and SC5b-9.4 The transient high levels of anaphylatoxins C3a and C5a
`presumably either directly or indirectly cause the observed side effects. As a means of reducing these
`
`undesirable side effects, we sought to design inhibitors with greater selectivity for thrombin over factor I. This
`
`manuscript will describe our rational design efforts in this area that have indeed led to compounds that are more
`selective f(;l' thrombin over factor I and that have less propensity to cause hypotension and serum t:ransaminase
`elevations.
`
`
`
`0960—894X/98/$19.00 © 1998 The DuPont Merck Pharmaceutical Company. Published by Elsevier Science Ltd.
`PII: S0960-894X(98)OOOl 3-4
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`
`95;:
`
`Csb
`
`ca
`C7
`C3
`
`C9
`
`Membrane
`
`attack complex
`(SC5b-9)
`
`
`
`302
`
`J. M. Fevig er al. /Bioorg. Med. Chem. Len‘. 8 (1998) 301-306
`
`Om. design efforts were based
`upon a comparison of the amino acid
`.
`9
`sequences of thrombin and factor 1,
`
`enzymes
`both
`that
`revealed
`which
`contained sirrularly shaped and charged
`residues in most areas of the ligand
`.
`.
`.
`binding region.
`However,
`the two
`.
`.
`.
`
`the side
`enzymes differ at residue 99,
`chain of which projects toward the aryl
`binding pocket“)
`Thrombin has
`a
`
`leucine at this position while factor I has a
`
`Figure 1. The Complement Cascade
`
`Classical
`
`P‘-"hwy
`
`C4+C2
`cm”
`
`
`“Ma
`
`C3
`
`csbisb
`
`D
`
`Anernm /
`‘V C3b
`
`P”""“‘y C3bB
`
`C4b2a3b
`
`
`
`Cm)
`
`C5
`
`:
`
`C3bBb3b
`
`
`
`lC3b
`
`The longer
`tyrosine residue.
`longer
`Tyr99 residue should make the
`aryl
`
`binding pocket of factorl smaller than that of thrombin, thereby allowing the opportunity for achieving greater
`
`selectivity by more completely filling the larger thrombin pocket. Factor Xa, another integral serine protease in
`
`the coagulation cascade, also contains the Tyr99 for Leu99 substitution while being similar to both thrombin and
`factor I in the other ligand binding pockets. Due to the apparent similarity between factor Xa and factor I, we
`elected to use the X-ray coordinates of factor Xa“ as a model
`in our design of more selective thrombin
`inhibitors.
`The boropeptide [N-hydrocinnamoyl-N-phenethyl]Gly—boroLys—OH (1)12 (Figure 2) was ideally suited
`as a starting point for these rational design efforts. The X—ray crystal structure of the thrombin:l complex12’13
`reveals that the N-phenethyl residue of 1 occupies a position adjacent to residue Leu99, with the phenyl ring
`being engaged in a favorable aromatic edge-to—face interaction14 with the indole side chain of Trp215 in the aryl
`binding pocket. We expected that the N-phenethyl residue would therefore serve as a handle on which to
`
`append additional functionality to interact with this region of the binding pocket. An overlay of the coordinates
`of the thrombinzl complex with those of factor Xa (Figure 3a) reveals a distance of 4.8 A between the [3 carbon
`of the N-phenethyl residue of 1 and a Leu99 termintd methyl group. The Tyr99 side chain of factor Xa projects
`
`toward the N-phenethyl residue so that the distance between the [3 carbon and the Tyr99 phenolic oxygen in this
`
`model is 2.5 A. Working on the assumption that the overall conformation of factor I will be similar to that of
`
`factor Xa, we expected that disubstitution of the N-phenethyl [3 carbon of 1 would result in compounds which
`would suffer from steiic interactions with Tyr99 upon binding to factor I. Tlirombin, with the smaller Leu99
`
`Figure 2
`
`H‘HC|
`PH
`PH
`H2” H/\/\'/B\oH HC"H2N\/\/\|/B\°H
`NH
`NH
`OT
`Q9
`
`N
`
`"““°
`
`0
`
`Z/\/\|/BKOH
`0
`NH
`’‘ ‘(
`
`N
`
`x
`
`R, R = H, H; Me, Me:
`-cH2<cH2).cH2-; (n = 0-3)
`x: H, Me
`2 = -CHZNH2, -NHCH(=NH).
`
`—NHC(=NH)NH2
`
`N
`
`DuP 714. Ki = 0.04 nM
`
`1, Ki = 0.42 nM
`
`2
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`
`J. M. Fevig er al. /Bioorg. Med. Chem. Lett. 8 (1998) 301és’06
`
`303
`
`residue, was expected to be more tolerant of substitution at this position. As shown in Figure 2, we chose
`
`initially to make compounds 2 in which the [3 carbon was disubstituted with hydrophobic groups to avoid both
`the introduction of a new chiral center and any potential hydrogen bonding interactions with Tyr99.
`
`The B,B—disubstituted phenethylglycines required for preparation of compounds 2 were prepared as
`
`illustrated for N-hydrocinnamoyl-N-[(2,2-dimethyl-2-phenyl)ethyl]glycine 6 shown in Scheme 1. The aldehyde
`3a could be directly dialkylated with 2.2 equiv of methyl iodide and 2.2 equiv of KOt-Bu to give 415 in about
`
`65% yield. Alternatively, dialkylation of ester 3b followed by LAH reduction and PCC oxidation gave
`
`aldehyde 4 in comparable overall yield. The required cycloalkyl aldehydes related to 4 were prepared from
`commercially available 1-pheny1-1-cycloalkanecarboxylic acids by an analogous two step adjustment of
`oxidation state.
`Reductive amination of 4 with glycine ethyl ester hydrochloride salt and sodium
`
`cyanoborohydride smoothly afforded the amine salt 5. Acylation with hydrocinnamoyl chloride followed by
`saponification of the ester gave the desired glycine derivative 6. The conversion of 6 and related derivatives to
`the final boropeptide inhibitors followed established procedures and was straightforward.3a'i2’16
`
`Scheme 1.
`
`a(for3a), or
`a,b,c(for3b)
`X —-—:—>
`60-70%
`
`33 (X=l-I)
`3b(X=OMe)
`
`d
`CHO :>
`M9 M9
`86%
`4
`
`°
`
`°E‘
`I _
`N“ “°' e,f
`-—>
`85%
`
`Me Me
`
`5
`
`°
`
`°”
`T
`
`Me Me
`6
`
`(a) Mel (2.2 equiv), KO:-Bu (2.2 equiv), THF; (b) LiAlH4. Et2O, 0°C; (c) PCC, CH2Cl2; (d) GlyOEt 'HCl,
`Reagents:
`NaCNBH3, MeOH; (e) PhCH2CH2COCl, NMM. THF;
`(1) KOH, MeOH/H20, reflux.
`
`In Table 1 is shown binding and selectivity data for analogs prepared according to our design. Liver
`
`enzyme elevation (ALT) data is included as a measure of toxicity. In general, B43-disubstitution of the phenethyl
`
`residue led to compounds which had greater selectivity for thrombin over factor I, as indicated by increases in
`the calculated selectivity ratio (factor I IC5g/thrombin Ki). The same basic trend is seen in the selectivity for
`
`thrombin over factor Xa, which lends support to our use of factor Xa as a model of factor I. Some exceptions
`
`to this trend are observed in the borolysine series 1, 7-9. The B,[i-dimethyl analog 7 is equipotent to 1 toward
`thrombin and factor I, so there is no increase in the selectivity ratio. However, 7 is more selective versus factor
`
`Xa than is 1. Also, the cyclopropyl analog 8 is more selective for thrombin over both factor I and factor Xa
`
`than is the bulkier cyclopentyl analog 9, although the loss of factorl and factor Xa affinity for 9 relative to 1 is
`
`still consistent with our model and with our design. The effect of [LB-disubstitution is best observed within the
`(formamidino)boroomithine12 series 10-13.
`The B,[S-dimethyl and cyclopropyl analogs 11 and 12,
`
`respectively, are nearly equipotent to the unsubstituted analog 10 toward thrombin but have lower affinities for
`factor I and factor Xa. The bulkier cyclopentyl analog 13 begins to lose affinity for thrombin while dramatically
`
`losing affinity for factor I. Thus, the selectivity ratios for 13 increase > fivefold against factor I and about
`
`eightfold against factor Xa relative to the unsubstituted analog 10. The cyclopropyl analogs are consistently
`more selective for thrombin over factorl than are the corresponding B,B-dimethyl analogs.
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`304
`
`J. M. Fevig et al. /Bioarg. Med. Chem. Lett. 8 (1998) 301-306
`
`The 3,5-dimethylphenethyl series 16-21 also follows the same selectivity trend. The B,B-dimethyl
`
`analogs 17, 19, and 21 are nearly equipotent toward thrombin relative to their unsubstituted counterparts 15,
`18, and 20, respectively, but are considerably less potent toward factor I and factor Xa, with increases in the
`
`selectivity ratio versus factor I ranging from fourfold to > fifteenfold and increases in the selectivity ratio versus
`factor Xa ranging from eightfold to tenfold. Indeed, the effect of [LB-dimethyl substitution in this series is even
`greater than in the phenyl series, where B,B—dimethyl substitution resulted in two- to threefold increases in
`selectivity ratios. This effect might result from one of the meta methyl groups being positioned over Trp2l5,
`which may sterically force the phenethyl tether closer to Ty199 and enhance the effect of [3,B-disubstitution.”
`The measured levels of alanine transaminase (ALT) reported in Table 1 are also worthy of note.
`
`In
`
`accord with the relationship between factor I inhibition and toxicity, only the most potent factor 1 inhibitors,
`
`namely DuP 714 and 14, show significant elevations in ALT levels. The borolysines 1 and 7-9 and the
`(forrnamidino)boroomithines 1(H3 are free from ALT elevations, which, we believe, reflects the fact that they
`are weak factor I inhibitors relative to DuP 714 and 14. Interestingly, even the unsubstituted phenethyl analogs
`
`1 and 10 are relatively weak factor 1 inhibitors, possibly because even the unsubstituted phenethyl residue
`
`provides some degree of steric interaction with the Tyr99 of factorl (Figure 3a).
`
`Table 1. Binding data, selectivity data and toxicity data for
`
`13,0-disubstituted phenethylglycine derivatives.
`
`pH
`Z/\/Y3\°
`
`CTN“
`
`H
`
`Selectivity
`Ratios
`
`Toxicity Data
`
`207
`
`-
`H, H
`Me, Me
`-(CH2)2.
`.(CH,)4.
`H, H
`Me, Me
`
`,
`
`,
`
`-N1—1c(=NH)NH2
`—CH2NH2
`_CH2NH2
`-C1-{ZN}-12
`-CH2NH2
`-NHCH(=NH)
`.N1-[C1-{(=NH)
`-NHCH(=NH)
`-NHCH(=NH)
`-NHC(=NH)NH2
`-NHC(=NH)N1-12
`_cH2NH2
`-CH2NH2
`-NHc]-{(=NH)
`-NHCH(=NH)
`-NHC(:NH)NH2
`-NHC(=NH)NH2
`
`.
`
`.
`
`.
`
`.
`
`10
`1500
`1300
`4300
`19,000
`2730
`6900
`7700
`>50,000
`130
`540
`170
`2450
`400
`2390
`<40
`320
`
`130
`320
`438
`1390
`22
`80
`300
`630
`99
`46
`54
`810
`30
`392
`
`62
`
`225
`310
`889
`1019
`579
`25
`67
`361
`210
`1650
`767
`193
`1976
`81
`740
`
`250
`3570
`3611
`10,000
`7916
`3067
`5750
`9277
`> 1 6,667
`2166
`9000
`607
`5975
`1080
`4509
`<69
`1066
`
`287(2,imo1/kg)
`60
`53
`60
`66
`55
`70
`
`a) Values for inhibitory constant (K_) were determined as described in ref 32:. The majority of compounds are slow-binding
`inhibitors. Reported values are the averages of at least duplicate measurements after steady state velocities were reached.
`b) Values for IC50 were determined as described in ref 7 and are averages of at least duplicate measurements.
`c) Values for inhibitory constant (K-,) were determined as described in ref 3a and are averages of at least duplicate measuremenlS«
`(1) ALT = alanine transaminase. Control level is 57. nt = not tested.
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`J. M. Fevig er al. /Bioorg. Med. Chem. Len‘. 8 (1998) 301-306
`
`305
`
`Figure 3a.
`
`Figure 3b.
`
`
`
`Figure 3a. Factor Xa coordinates (red) superimposed onto coordinates of 1 (green) bound to thrombin (yellow). The distance from
`the phcncthyl B-carbon of 1 to the methyl group of Leu99 of thrombin is 4.8 angstroms (pink dashed line) and to the phenolic
`oxygen of Tyr99 of factor Xa is 2.5 angstroms (yellow dashed line). Figure 3b. Factor Xa coordinates (red) superimposed onto
`coordinates of 1 (green) and 7 (blue) bound to thrombin (yellow). The distance from the methyl group of 7 to the phenolic oxygen
`of Tyr99 of factor Xa in this model is 1.4 angstroms.
`
`The additional effect of factor I selectivity on hypotension is illustrated in Table 2 for a series of
`
`boroarginine inhibitors. The very potent factor I inhibitor DuP 714 causes dramatic ALT elevations along with
`
`hypotension after an iv bolus dose of 2 umol/kg in rats. The boroarginine 14, which is thirteenfold less potent
`
`toward factor 1, causes dramatic ALT elevations along with hypotension only after a tenfold higher dose. The
`
`boroarginine 15, with the same thrombin affinity as 14 but with fourfold lower factor I affinity, causes only
`
`slight ALT elevations and no hypotension at the 20 umol/kg dose. Thus, these results effectively demonstrate
`
`that, among structurally—related compounds having comparable thrombin affinity, the toxic side effects of the
`
`boropeptides can be attenuated in vivo by increasing the selectivity over complement factor I.
`Table 2.
`
`Compound
`
`DuP 714
`
`14
`
`15
`
`0.04
`
`0.06
`
`0.06
`
`10
`
`130
`
`540
`
`No (20 umol/kg)
`
`Thrombin
`K; (nM)
`
`Factor I
`IC50 (nM)
`
`ALT Levels
`rat i.v. bolus (dose)
`
`Hypotension Data*
`rat i.v. bolus (dose)
`
`287 (2 umol/kg)
`
`Yes (2 umol/kg)
`
`21 l (20 umol/kg)
`
`Yes (20 umol/kg)
`
`89 (20 umol/kg)
`
`* Yes defined as a > 40 mmHg drop in blood pressure.
`
`To determine whether the disubstituted analogs listed in Table 1 are binding in the manner in which they
`
`were designed, we solved the X-ray crystal structure of 7 bound to thrombin. Although this compound has
`
`about the same factor I potency as the corresponding unsubstituted analog 1, it is threefold more selective versus
`
`factor Xa than is 1. Figure 3b shows an overlap of the coordinates of the thrombin:7 complex” with those of
`
`both the thrombin:l complex" and factor Xa. While the overall binding conformations of 7 and 1 are similar,
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`306
`
`J. M. Fevig et al. /Bioorg. Med. Chem. Lett. 8 (1998) 301-306
`
`the phenethyl residue of 7 has moved slightly, presumably to accommodate the additional methyl groups. Also,
`the orientation of the phenyl residue with respect to Trp215 has changed, but the edge-to-face interaction is still
`maintained at approximately the same inter-ring angle. The measured distance of 1.4 A between the phenethyl
`methyl group of 7 and the oxygen of Tyr99 of factor Xa in this model lends support to our hypothesis and to
`our results regarding the increased selectivity of substituted analogs of 1.
`Presumably, binding of the
`dialkylated phenethyl analogs to factor I or factor Xa in a conformation similar to 1 would be disrupted by steric
`interactions between the added alkyl groups and Tyr99. To accommodate these added groups the phenethyl
`residue might bind in a different manner, which might be expected to disrupt the favorable edge-to-face
`interaction with Trp215.
`In summary, we have used X-ray crystal structure information to design substituted analogs of the
`boropeptide 1 which have greater selectivity for thrombin over complement factor I. The resulting inhibitors
`have less tendency to cause the side effects which we believe are mediated by inhibition of complement factor I.
`This work is an example of how rational drug design can be used to target a specific binding interaction, based
`on a single amino acid substitution, which can dramatically alter the selectivity and biological activity profile of a
`promising series of enzyme inhibitors.
`Acknowledgements: We wish to thank Joseph Luettgen for perfonning in vivo studies, Lawrence Mersinger and
`Susan Spitz for obtaining compound binding data, Frank Barbera for factor I IC50 determinations and Angela
`Smallwood for assistance in obtaining the X-ray crystal structure of the thrombin:7 complex.
`
`References and Notes
`i Current address: Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033.
`1. Presented in part at the 212th ACS National Meeting, Orlando, FL, August 25-29, 1996, MEDI 127.
`2. Das,J.; Kimball, S. D. Bioorg. Med. Chem. 1995, 3, 999.
`(b) Knabb, R. M.; Kettner, C.
`3.
`(a) Kettner, C.; Mersinger, L.; Knabb, R. J. Biol. Chem. 1990. 265, 18289.
`A.; Timmeimans, P. B. M. W. M.; Reilly, '1‘. M. Thromb. Haemostas. 1992, 67, 56.
`4. For preliminary accounts of this work, see: (a) Kettner, C.; Knabb, Rx, Fevig, J.;
`I-Iugli, T.; Lee, S.; Mantri, P.;
`Pangbum, M.; Reilly, T.; Stouten, P.; Thoolen, M.; Weber, P.; Wexler, R. Book of Abstracts 212th ACS National
`Meeting, Orlando, FL, August 25-29, 1996, MEDI 112;
`(b) Knabb, R. M.; Luettgen, J. M.; Leamy, A. W.;
`Baibera, F. A.; Kettner, C. A.; Pangbum, M. K.; Thoolen, M. J. Circulation 1996,94, Suppl. 1, 696. Full details
`will be reported elsewhere.
`5. Thou. I. L.; Uvnas, B. Acta Physiol. Scand. 1967, 7], 303.
`6. Cochrane. C. G.; Mueller-Eberhard, H. J.; Aikin, B. S. J. Immunol. 1970, I05, 55.
`7. Pangbum, M. K.; Muller-Eberhard, H. J. Biochemistry 1983,22, 178.
`8. Verbeuren and co-workers have also reported on the relationship between factor I selectivity and toxicity for a
`series of boronic acid thrombin inhibitors. See: Rupin, A.; Mennecier, P.; Lila. C.; de Nanteuil, G.; Verbeuren,
`T. J. Thromb. Haemostas. 1997,78, 1221.
`9. Perkins, S. J.; Smith, K. F. Biochem J. 1993.295, 109.
`10. Bode, W.; Turk. D.; Karshikov, A. Protein Sci. 1992, 426.
`11. Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky. A.; Park, C. H.; Bode, W.; Huber, R.; Blankenship, D. T.:
`Cardin, A. D.; Kisiel. W. J. Mol. Biol. 1993, 232, 947.
`12. Galemmo, R. A., Jr.; Fevig,J. M.; Carini, D. J.; Cacciola,J.; Wells,B. L.; Hillyer,G.L.; Buriak,J.,Jr.; Rossi.
`K. A.; Stouten, P. F. W.; Alexander, R. S.; Hilmer, R.; Bostrom, L.; Abelman, M. M.; Lee, S.-L.; Weber, P. C.:
`Kettner, C. A.; Knabb, R. M.; Wexler, R. R. Bioorg. Med. Chem. Lett. 1996, 6, 2913.
`13. A complex of 1 and thrombin was crystallized in space group C2 (21 = 70.7, b = 72.3, c = 72.2 A, alpha =
`90.0°, beta = 100.44", gamma = 90.0°). Data were collected to 1.8 A resolution and the structure refined to an
`R,_,,,,, of 21%. A complex of 7 and thrombin was crystallized in space grou/E C2 (a = 71.2, b = 72.6, c = 72.2 A.
`alpha = 90.0”, beta = 100.5”, gamma = 90.0°). Data were collected to 2.25
`resolution and the structure refined to
`an R,_,,,,, of 20%.
`14. Burley, S. K.; Petsko, G. A. Science 1985, 229, 23.
`15. Satisfactory spectral data were obtained for all new compounds.
`16. Wityak,J.; Earl. R. A.; Abelman, M. M.; Bcthel, Y. B.; Fisher,B.N.; Kauffman, G. S.; Kettner, C. A.; Ma,
`P.; McMi1lan,J. L.; Meisinger, L. J.; Pesti,J.; Pierce, M. E.; Rankin, F. W.; Chorvat, R. J.; Confalone, P. N. J.
`Org. Chem. 1995, 60, 3717.
`17. For an example of this type of binding in a closely related compound. see: Strickland, C. L.; Fcvig, J. M.;
`Galemmo, R. A., Jr.; Wells, B. L.; Kettner, C. A.; Weber, P. C. Acta Crystallographica Section D, in press.
`
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