.5?‘
`
`Vol. 4, No. 20
`
`20 October 1994
`
`N0 5 9
`
`ISSN 0960-394X
`
`A Tetrahedron Publication
`
`for Rapid Dissemination of
`"Preliminary Communications on all aspects of
`Bioorganic Chemistry, Medicinal Chemistry,
`Rioinorganic Chemistry and related disciplines
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`Editor-in-Chief
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`DALE L BOGER
`
`The Scripps Research Institute
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`I ajolla
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`USA
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`Chairman of the Executive
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`Board of Editors for
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`SIR DEREK BARTON
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`Texas A 8 M University
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`BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
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`Copyright © 1994 Elsevier Science Ltd
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`

`
`
`
`Bioorganic & Medicinal Chemistry Letters Vol. 4, No. 20, 1994
`
`J. Bermudez, L. Gaster, J. Gregory,
`g. J(erman, G. F. Joiner, F. D. King and
`.
`Rahman
`
`R. Shimazawa, R. Shirai, Y. Hashimoto
`and S. lwasaki
`
`P. Demonchaux, P. Lenoir, G. Augert
`and P. Dupassieux
`
`N.-H. Lin, Y. He, D. J. Anderson,
`J. T. Wasicak, R. Kasson, D. Sweeny
`and J. P. Sullivan
`
`T.-S. Wu, S.-C. Huang, P.-L. Wu and
`K.—H. Lee
`G. Romeo, F. Russo, S. Guccione,
`R. Chabin, D. Kuo and W. B. Knight
`
`2365
`
`2367
`
`2373
`
`2377
`
`2383
`
`2389
`
`2395
`
`2399
`
`Contents
`
`Contributors to this Issue
`
`Graphical Abstracts
`
`Synthesis and 5-HT3 receptor antagonist potency of novel (endo) 3,9-
`diazabicyclo[3.3.1]nonan-7-amino derivatives
`
`DNA-Binding ability of non-diynene class of dynemicins and aza-anthra-
`quinones
`
`Design of pyrrolo-1,4-benzoxazine derivatives as inhibitors of 5-lip-
`oxygenase and PAF antagonists with antihistaminic properties
`
`Synthesis and structure-—activity relationships of pyrrolidine-modified
`analogs of the potent cholinergic channel activator, ABT 418
`'
`
`»
`
`Structure and synthesis of clausenaquinone-A. A novel carbazolequinone
`alkaloid and bioactive principle from Clausena excavata
`Synthesis of new thiazinoindole derivatives and their evaluation as
`inhibitors of human leukocyte elastase and other related serine proteases
`
`J. Lee, N. E. Lewin, P. M. Blumberg and 2405
`V. E. Marquez
`
`E. K. Lehnert, K. E. Miller,
`J. S. Madalengoitia, T. J. Guzi and
`T. L. Macdonald
`
`2411
`
`Contorrnationally constrained analogues of diacy|g|ycerol—-IX. The effect
`-C) binding affinity of
`of side-chain orientation on the protein kinase C (
`-
`5-lactones
`
`DNA Topoisomerase ll inhibition by substituted 1,2,3,4-tetrahydro-ii-carbo
`line derivatives
`2
`
`S. J. Steiner, J. T. Bien and B. D. Smith 2417
`J. A. Hartley, M. D. Wyatt,
`2421
`B. J. Garbiras, C. Richter and M. Lee
`P. Pradhan, D. L. Luthria and A. Banerji 2425
`~
`
`Diphenylborinic acid is a strong inhibitor of serine proteases
`Probing the importance of the second chloroethyl arm of a benzoic ac%i;:
`mustard derivative of an imidazole-containing analogue of distamycin
`Pimolin, a new class of natural product from Pimpinella monoica: a novel
`dimeric turochromone
`
`B. Kiinig and M. Gréitzel
`
`G. Adlam, I. S. Blagbrough, S. Taylor,
`H. C. Latham, l. S. Haworth and
`A. Rodger
`
`2429
`
`2435
`
`An immunosensor for the detection of human B-lymphocytes
`
`Multiple binding modes with DNA of anthracene-9~carbonyl-N‘-spermins
`probed by LD, CD, normal absorption, and molecular modelling compared
`with those of spermidine and spennine
`
`S. K. Thompson, A. M. Epgley,
`J. S. Frazee, M. G. Darcy,
`. T. Lum,
`T.
`A. Tomaszek, Jr, L. A. lvanoff,
`J.
`F. Morris, E. J. Sternberg,
`D.
`M. Lambert, A. V. Fernandez,
`S.
`W Petteway, Jr, T. D. Meek,
`. Metcalf and J. G. Gleason
`B.
`
`2441
`
`Synthesis and antiviral activity of a novel class of HIV-1 protease inhibitors
`containing a heterocyclic P1'—P2' amide bond isostere
`
`[Continued on inside back cover‘
`
`
`II
`
`
`
`
`
`
`II llll
`
`
`Ill
`
`
`
`
`
`
`llllllllllllllllllll
`
`V
`0960-894X(1994)4:20;1-I
`Indexed/Abstracted in: Chemical Abstracts, Current Contehts,
`Science Citation Index, Scisearch, Research Alert, Excerpta Medica Database EMBASE
`
`.
`
`BMCLE8 4 (20) 2365-2490 (1994l
`
`Pergamon
`
`
`CFAD v. Anacor |PR2015—O1776 ANACOR EX. 2117 — 3/8
`W
`Printed in Great Britain by Nuffield Press Ltd.
`972
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`

`
`Bioorganic 8: Medicinal Chemistry Letters Vol. 4, No. 20, 1994
`
`Contents
`
`[Continued from outside back cover]
`
`Penazetidine A, an alkaloid inhibitor of protein kinase C
`
`Selective inhibitors of protein kinase C in a model of Graft-vs-Host disease
`
`Synthesis and neuraminidase inhibition studies of 4-azido, amino and
`acetamido substituted sialosides
`
`Synthesis of cis-8-phenylmethyl-D-proline using a nitrogen-centered radi-
`cal derived from a chiral oxazindine
`
`Highly water soluble taxol derivatives: 2‘-polyethyleneglycol esters as
`potential prodrugs
`
`Stereospecific bio-reversibility of dinucleoside S-alkyl phosphorothiolates
`to dinucleoside phosphorothioates
`
`Ketones related to the benzoate 5-HT4 receptor antagonist RS-23597 are
`high affinity partial agonists
`
`Synthesis and preliminary phannacological evaluation of 2-benzyloxy
`substituted aryl ketones as 5—HT4 receptor antagonists
`
`Structure—activity studies of N-cyano-3-pyridinecarboxamidines and their
`amide and thioamide congeners
`Additions and Corrections
`
`Instructions to Contributors
`
`2447
`
`2451
`
`2457
`
`2461
`
`2465
`
`2471
`
`2477
`
`2481
`
`2485
`
`2489
`
`K. A. Alvi, M. Jaspars, P. Crews,
`B. Strulovici and E. Oto
`
`J. P. Demers, W. E. Hageman,
`S. G. Johnson, D. H. Klaubert,
`R. A. Look and J. B. Moore
`3. Sabesan
`
`J. Aubé, B. Giilgeze and X. Peng
`
`R. B. Greenwald, A. Pendri. D. Bolikal
`and C. W. Gilbert
`
`R. P. lyer, D. Yu and S. Agrawal
`
`. Clark, A. Jahangir,
`Langston, K. K. Weinhardt,
`Miller, E. Leung and R. M. Eglen
`
`DA
`
`.
`.B
`
`.D
`Clark, A. Jahangir,
`A
`. Langston, K. K. Welnhardt,
`B. Miller, E. Leung, D. W. Bonhaus,
`H. F. Wong and Ft. M. Eglen
`
`Nd
`
`akajima, T. Kashiwabara, T. lzawa
`T.
`an S.Nakaiima
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`

`
`This material may be protected by Copyright law (we 17 u.s. Code)
`
`Pergamon
`
`Hioorganic & Medicinal Chemistry Letrenr, Vol. 4, No. 20, pp. 2417-2420, 1994
`Copyright © 1994 Elsevier Science Ltd
`Printed in Great Britain. All rights reserved
`0960-894X/94 $7.0U+U.OO
`
`0960-894X(94)0O3 50-5
`
`DIPHENYLBORINIC ACID IS A STRONG INHIBITOR OF SERINE
`PROTEASES¢
`
`Steven J. Steiner} Jeffrey T. Bien, Bradley D. Smith‘
`
`Department of Chemistiy and Biochemistry, University ofNotre Dame, Notre Dame, IN 46556, USA
`
`Abstract. Diphenylborinic acid, a commercially available and reasonably air stable compound, was found to be a
`strong competitive inhibitor of three serine proteases. Compared to phenylboronic acid, it was a thirty-fold better
`inhibitor of oi-chymotrypsin, a fifteen-fold better inhibitor of subtilisin BPN', and a sixty-fold better inhibitor of
`bovine trypsin. The pKa and inhibitory ability of methylphenylborinic acid was also determined.
`
`Boronic acids have been studied as competitive inhibitors of serine proteases for more than twenty-five
`
`years.‘ Nonetheless, interest in these compounds remains high due to their potential clinical uses,2 and their
`
`ability to act as structural probes of enzyme binding sites.3 Despite numerous X—ray and NMR studies, some of
`the details concerning the structures of the enzyme/inhibitor complexes remain controversial, particularly when the
`
`inhibitors are simple, "non substrate-like" boronic acids.4 In some cases there is clear evidence for a covalent
`
`tetrahedral adduct with the active-site serine hydroxyl.4»5 In other cases there is no doubt that the boron is
`coordinated to the active-site histidine.5
`
`Our interest in this area stems from our recent efforts to develop molecular transport devices using boron
`
`acids.7 While conducting experiments with diphenylborinic acid, 1, we became curious about its ability to inhibit
`
`serine proteases. Inhibition with asymmetric borinic acids has been reported before,8 the most recent study by the
`
`Jones research group.9 In general, borinic acids are better inhibitors than boronic acids. The major detraction
`with borinic acids is their susceptibility to air oxidation. Diarylborinic acids, however, are reasonably air stable
`
`compounds. For example, a solution of 1 in phosphate buffer, at pH 7.4, was found to be > 90 % pure after
`
`standing on the bench top for 24 hours. Compound 1 has a pKa of 6.2.10 At neutral pH it readily combines with
`vicinal diols to form anionic, tetrahedral "ate" complexes.7 The expected inhibitory ability of 1 was hard to
`predict, a priori, since it was difficult to estimate the relative importance of various opposing factors such as
`
`increased acidity, enzyme binding site specificity, inhibitor hydrophobicity, loss of a potential active-site
`
`hydrogen, etc. We felt that if 1 were a good protease binder then it may have utility in clarifying some of the
`
`structural and mechanistic ambiguities concerning this class of transition-state-analogue inhibitors.
`
`OH
`
`HO
`
`H
`
`3
`
`©/ \©
`
`1
`
`©
`
`2
`
`B\
`
`©’ CH3
`
`3
`
`CH2CH2CH2CH3
`
`B\
`
`4
`
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`2418
`
`S. J. STEINER et al.
`
`Inhibition studies were carried out at 22 °C in sodium phosphate solution buffered at pH 7.4. Due to their
`instability, borinic acids are usually synthesized and stored as their aminoethanol esters. Previous kinetic studies
`with the esters of alkylborinic acids used the esters directly, since they were found to be rapidly hydrolyzed to the
`acids.3~9 The aminoethanol ester of 1, however, is reasonably stable at neutral pH.“ To avoid any ambiguity
`due to slow ester hydrolysis, the aminoethanol group was removed beforehand by acid extraction,“ and the free
`acid used in the experiments. Enzyme activity was monitored spectrophotometrically with the following standard
`substrates, N-succinyl-Ala-Ala-Pro~Phe-4-nitroanilide, 0.015 - 0.075 mM, (ot-chymotrypsin, 56 nM); 4-
`nitrophenylbutyrate, 0.015 - 0.12 mM (subtilisin BPN', 250 nM); and N-benzoyl-DL-arginine-4-nitroanilide,
`0.30 - 0.60 mM, (bovine trypsin, 1.23 LLM). Inhibition constants were determined from Lineweaver-Burk plots
`which were consistent with competitive inhibition.
`
`
`
`T sin
`170
`10200
`—
`
`
`
`
`
`Ch mo sin
`20
`640 (200)d
`30
`
`
`
`Table 1. Inhibition Constants (dissociation) and Ka's.3
`Ki/ttM i 10% (lit.)
`Subtilisin
`
`30
`470 (230)d
`—
`
`Inhibitor
`1
`
`K;,i0.l (lit.)
`5_1 (5_2)b
`8.8 (8.85)°
`8.1
`
`
`
`
`
`
`(1)6
`(8)6
`-
`
`3A]l experimental measurements are the average of at least two independent determinations.
`‘Reference 10. °Reference 7. ‘Reference 12. ‘’Reference 9.
`
`In the event, 1 was found to be a strong competitive inhibitor of the three serine proteases examined.
`Compared to phenylboronic acid, 2, it was a thirty-fold better inhibitor of chymotrypsin, a fifteen-fold better
`inhibitor of subtilisin, and a sixty—fold better inhibitor of trypsin (Table 1). As a way of calibrating our results
`with those of Jones, we also examined methylphenylborinic acid, 3. This compound was synthesized according
`to the method of Brown. 13 It was found to be moderately air sensitive which made inhibition studies problematic.
`Our pragmatic procedure involved synthesizing 3 as its aminoethanol ester, and deprotecting a fresh sample just
`before use.” Evaluation of its inhibition of chymotrypsin produced a Ki of 30 1.1M which correlates reasonably
`well with the Ki of 8 j.tM reported for the structurally related butylphenylborinic acid 4 (Table 1). The sub-
`millimolar inhibition of trypsin by 1 is a notable result as the enzyme has a strong preference for an arginyl
`residue at its S1 specificty site, and therefore is poorly inhibited by arylboronic acids. There is much interest in
`trypsin-like enzymes as they play important roles in regulatory systems such as blood coagulation and
`fibrinolysis.2 Compound 1 should be a useful, and readily available, transition-state probe for NMR and X-ray
`studies of these enzymes.5 With regard to NMR studies, a salient point is that outside the extreme-narrowing
`range, “B NMR signals have the unusual feature of becoming narrower as the molecular correlation time and
`magnetic field strength increase. 14
`
`The increased inhibition exhibited by borinic acids has been attributed, in part, to the increased
`electrophilicity of the boron center,9 although to our knowledge this has never been strictly proved. To confirm
`this reasoning we determined the pK3'S for 1, 2. and 3, to be 6.1, 8.8 and 8.1, respectively (Table 1).” Thus,
`the pKa's reflected, qualitatively, the order of Ki's. There are, of course, other factors that influence enzyme-
`inhibitor binding such as solvation changes, electrostatics, hydrogen bonding, and steric effects. For the
`subtilisin-boronic acid system, in particular, these factors have been examined in detail and will not be discussed
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`
`Diphenylborinic acid
`
`2419
`
`here.3 The lower pKa of 3 compared to 2 is somewhat counter-intuitive if stability of the conjugate base is the
`
`only criterion considered; a methyl group is not expected to stabilize an adjacent negative charge better than a
`
`hydroxyl group. A reasonable explanation is provided by examining both sides of the acid-base equilibrium for 3
`
`and 2. It appears that replacing the methyl group in 3 with a hydroxyl results in stabilization of the acid more than
`the conjugate base.
`
` ,OH
`F’h—B\X +
`2H2O
`,
`acid
`
`_?:'OH
`F'h—B\
`base
`
`X
`
`+
`
`+
`
`H30
`
`1 x = Ph, pKa = 6.2
`2 x = OH, pKa = 8.8
`3 X=CH3,pKa=8.1
`
`The subtilisin inhibition ability of 1 was examined as a function of pH. Previous studies on subtilisin
`
`inhibition by boronic acids produced bell-shaped curves with an invariant pK1 of about 7, and a pK2 that
`
`depended on the boronic acid (usually greater than 8).”-16 Since pK1 corresponded to the pKa of the active-site
`imidazole and pK2 matched the boronic acid pKa, the profile was rationalized, mechanistically, in terms of a
`neutral boronic acid binding with an alkaline enzyme active-site. The plot of 1/K1 versus pH for subtilisin
`
`inhibition by 1 is shown in Figure 1. Although the curve is transposed to lower pH values, with Kimpt) at pH
`
`6.8, the profile is still consistent with the above binding mechanism.”-13 In this case, the pKa of inhibitor 1 is
`lower than the pKa of the enzyme active-site, z'.e., pK2 < pK1.19 The Alberty-Massey equation was used to
`calculate pK values of 6.2 and 7.4.18
`
`1/Ki
`
`X105 M-1
`
`0.05
`
`0.04
`
`0.03
`
`0.02
`
`0.01
`
`0.00
`
`5
`
`6
`
`7
`
`pH
`
`8
`
`9
`
`Figure 1. pH profile for subtilisin inhibition by 1.
`
`In conclusion, diphenylborinic acid, 1, proved to be a strong competitive inhibitor of the three serine
`
`proteases examined. The commercial availability and increased stability of this compound makes it an attractive
`probe for X-ray and NMR studies of enzyme/inhibitor complexes.
`
`Acknowledgment. This work was supported by a grant from the National Science Foundation (CHE 93-
`11584) and an award from the Research Corporation (Cottrell Scholarship to B.D.S).
`J.T.B. thanks the
`
`University of Notre Dame for an Upjohn Fellowship.
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`
`2420
`
`S. J. STEINER et al.
`
`References and Notes.
`
`¢Molecular recognition with boron acids, part 8. For part 7, see: Paugam, M. -F.; Valencia, L. S.; Smith, B. D.
`J. Am. Chem. Soc., submitted.
`
`:f:Current address: Indiana University Medical School, Indianapolis, IN.
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`Simpelkamp, J.; Jones, J. B. Bioorg. Med. Chem. Lett., 1992, 2, 1391-1394. A typographical error in
`Table 1 of this paper leads to the conclusion that borinic acids are weaker inhibitors of chymotrypsin than
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`D-#>—|5"!‘
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`The pKa's were determined potentiometrically. Albert, A.; Serjent, E. P. The Determination ofIonization
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`17. A referee noted that the profile deviates from the classical bell-shape at low pH, suggestive of a more
`
`complicated equilibrium at this pK, such as a two-proton ionization, or weak binding by the
`enzyme/inhibitor system in its incorrectly protonated form.
`
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`19.
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`p. 110 in Reference 18.
`
`(Received in USA 27 July 1994; accepted 13 September 1994)
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2117 - 8/8