United States Patent
`Bachovchin et al.
`
`[19]
`
`[11] Patent Number:
`
`4,935,493
`
`[45] Date of Patent:
`
`Jun. 19, 1990
`
`[57]
`
`ABSTRACT
`
`A compound having the structure
`
`HI
`
`X-Il~I-(|2=-T
`R*—<':—YR2
`
`where T is of the fomrula
`
`D2
`I
`._B_D1'
`
`[54] PROTEASE INHIBITORS
`
`[75]
`
`Inventors: William W. Bachovchin, Melrose;
`Andrew G. Plaut, Lexington, both of
`Mass.; Charles A. Kettner,
`Wilmington, Del.
`
`[73] Assignees: E. I. Du Pont de Nemours and
`Company, Wilmington, Del.; New
`England Medical Center Hospitals,
`Inc., Boston; Tufts University,
`Medford, both of Mass.
`
`[21] Appl. No.: 105,768
`
`[22] Filed:
`
`Oct. 6, 1987
`
`[51]
`
`Int. Cl.5 ....................... A61K 37/02; C07K 5/08;
`C07K 5/10
`[52] U.S. C1. ................................... .. 530/331; 530/330
`[58] Field of Search ............... 514/2, 18, 19; 530/330,
`530/331
`
`where each D1 and D2, independently, is a hydroxyl
`group of a group which is capable of being hydrolysed
`to a hydroxyl group in aqueous solution at physiolog-
`ical pH; a group of the formula
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`-'C— CF2"‘G,
`ll0
`
`where G is either H,F or an alkyl group containing 1 to
`about 20 carbon atoms and optional heteroatoms which
`can be N, S, or O; or a phosphonate group of the for-
`mula
`
`0l
`
`l—P—J
`IO—J
`
`where J is O-alkyl, N-alkyl, or alkyl, each comprising
`about 1-20 carbon atoms and, optionally, heteroatoms
`which can be N, S, or O; T being able to form a
`complex with the catalytic site of an enzyme, X IS a
`group having at least one amino acid,
`
`I
`I
`I
`Y is -c—R4 or R4-c—c—R5 or
`113
`[(3 1116
`
`(Abstract continued on next page.)
`
`4,318,904 3/1982 Shaw et al.
`4,499,082 2/1985 Shenvi et al.
`4,582,821
`4/1986 Kettner et al.
`4,636,492
`1/1987 Kettner et al.
`4,644,055
`2/1987 Kettner et al.
`4,652,552 3/1987 Kettner et al.
`
`....................... .. 424/177
`
`
`
`..
`........................ 514/18
`
`OTHER PUBLICATIONS
`
`R. Baugh et al., Proteinases and Tumor Invasion
`157-179, 165 (ed. Strauli et al., 1980).
`Cordes et a1., Transition States of Biochemical Pro-
`cesses 429-465 (ed. Gandour et al., 1978).
`Matteson et al., 1984, Organometallics 3:l284.
`Thompson, 1973, Biochemistry 12:47.
`Thompson, Methods in Enzymology 46:220-225.
`Yoshimoto et al., 1985, J. Biochem 98:975.
`Report of the National Heart Lung and Blood Institute
`Workshop on Elastase Inhibitors for Treatment of Em-
`physema held in Rockville, Md. (Jun. 10-11, 1985).
`Primary Examiner—Lester L. Lee
`Attorney, Agent, or Firm—Fish & Richardson
`
`Page 1 of 11
`
`Astraleneca Exhibit 2076
`
`Mylan V. Astraleneca
`IPR2015-013-10
`
`AstraZeneca Exhibit 2076
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 11
`
`

`
`Page 2
`
`4,935,493
`
`_cominued
`
`R5 R7
`R4_(|:_(':_(':_
`I
`I
`I
`R3 R5 R8
`
`not lower than Ki of the compound to less than 10*7M)
`with site-specific recognition of the compound by the
`enzyme, and allows a complex to be formed with the
`enzyme.
`
`and each R1, R2, R3, R4, R5, R5, R7, and R5 is separately
`21 group which does not interfere significantly (i.e., does
`
`12 Claims, 2 Drawing Sheets
`
`Page 2 of 11
`
`Page 2 of 11
`
`

`
`US. Patent
`
`Jun. 19, 1990
`
`Sheet 1 012
`
`4,935,493; ,§
`
`CH2
`/ \CH“3/OH
`\OH
`cH\2\
`I
`
`.
`
`cH2/
`
`‘
`CH2\
`
`CH
`
`0
`2’\CH 5': c
`"' — F3
`|
`N
`CH2/ \X
`
`N
`\
`
`X
`
`PF0'Y‘ B°"°“at9
`
`Prolyl Trifluoro alkyl ketone
`
`CH2\/ CH-—P(O)-J
`CH2
`]
`'0.)
`\ N
`CH/ \x
`
`Prolyl phosphonate
`
`F|G.|
`
`Page30f1_1V
`
`Page 3 of 11
`
`

`
`US. Patent
`
`Jun. 19,1990
`
`Sheet 2 of2
`
`A
`
`4,935,493;
`
`0
`.
`[(CH2)3 Si]2 N" Li "'
`(1) Br-- ((31-12):,-CH - s\O .
`
`/
`
`4-bromo-1-chlorobutyl boronate pinacolA
`
`O
`
`(H) Br—(CH2)3 - C'IH- B<O
`./N\ .
`SI
`SI
`/|\ /|\
`
`Distillation
`
`4-bromo-1 [ (bistrimethylsilyl) amino] butyl bornonate pinacol
`
`\|_/
`SI‘
`N
`
`(111)
`
`_..._..____,,
`
`O
`H
`0
`HC|:Dioxane
`~B’
`N
`3/ :1:
`\0 0/ ‘o
`
`I
`
`’
`
`‘ Hcl
`
`.
`1-trimethylsilyl-boroProline pinacol
`
`I
`N
`_boroProline-pinaco|-HC|
`
`BOG-AID'—'N
`
`EH
`
`isobutylchloroformate
`
`1 I
`
`1
`
`/0-<
`
`F|G.2
`
`Boc-Ala-Pro-boroPro-pinacol
`
`Page 4 of 11_
`
`Page 4 of 11
`
`

`
`1
`
`‘ 4,935,493
`
`2
`
`PROTEASE INHIBITORS
`BACKGROUND OF THE INVENTION
`
`This invention relates to protease inhibitors, and par-
`ticularly to transition state analogs.
`Transition state analogs, compounds which are
`thought to resemble the substrates of enzymes, are
`thought to bind more tightly to the enzymes than the
`substrates themselves. Transition state analogs form
`complexes with enzymes at their catalytic sites.
`Baugh et al. (Proteinases and Tumor Invasion ed.
`Strauli et al., Raven Press, N.Y., 1980, p. 165) state that
`transition state analogs containing boronic acid moieties
`or aldehydes form tetrahedral adducts with serine pro-
`teases and are thus good inhibitors of these enzymes.
`Further, they state that some peptide aldehydes have
`been synthetically prepared, that most are of microbial
`origin, and‘ that “it would appear that changing the
`R-group [of synthetic peptides] to" satisfy the specific
`requirements of a given protease should result in both
`potent and specific inhibitors.” They also state that
`transition state analogs containing cyclic ester moieties
`have been used to inhibit chymotrypsin and that “varia-
`tions thereof may become useful as inhibitors of cathep-
`sin G.”
`»
`
`Yoshimoto et al. (J. Biochem .98: 975, 1985) describe
`prolyl endopeptidase inhibitors containing a protinal
`moiety. These inhibitors appear to act non-competi-
`tively.
`Shenvi et al., U.S. Pat. No. 4,499,082, describe pep-
`tides having an a-amino boronic acid residue. These
`peptides are reversible inhibitors of elastase. They have
`the structureal formula
`
`5
`
`10'
`
`15
`
`20
`
`25
`
`30
`
`35
`
`0Y1
`
`R‘-I(A3).(A2),.(A1)1—NI-Icn-B
`R2
`
`\
`
`OY2
`
`where R2 is an alkyl group of one to six carbons which
`may have an aromatic substituent or an in-chain biva-
`lent group.
`
`45
`
`SUMMARY OF THE INVENTION
`
`In a first aspect, the invention features compounds
`having the structure
`
`II-I
`X—N—C-T
`I
`I
`121-?-YR2
`
`(1)
`
`and salts thereof, where T is a boronate group of the
`formula
`
`50
`
`55
`
`_]‘3....Dl’
`D2
`
`is a hydroxyl
`where each D1 and D2, independently,
`group or a group which is capable of being hydrolyzed
`to a hydroxyl group in aqueous solution at physiolog-
`ical pH; or T is a group of the formula
`
`as
`
`O
`II
`-C-CF2-G»
`where G is either H,F or an alkyl group containing 1 to
`about 20 carbon atoms and optional heteroatoms which
`can be N, S, or O; or T is a phosphonate group of the
`formula
`
`(2)
`
`(3)
`
`is O-alkyl, N-alkyl, or
`where each I, independently,
`alkyl (each containing about 1-20 carbon atoms) and,
`optionally, heteroatoms which can be N, S, or 0; where
`T is'a group able to form a complex with the catalytic
`site of an enzyme; X includes one or more amino acids,"
`
`I
`I
`I
`Y is —c—R4 or R4-C--C--R5 or
`I
`\
`\
`R3
`(4)
`
`R6 R8
`I
`I
`I
`R4—c—c—c
`}{3 R5/ R7/
`(5)
`
`and each R1, R2, R3, R4, R5, R5, R7, and R3, indepen-
`dently, is a group which does not interfere significantly
`(i.e., does not raise the Ki of the compound to greater _
`than 10-5M) with site-specific recognition of the com-
`pound by the enzyme, while permitting a complex to be
`formed between the compound and the enzyme.
`In preferred embodiments, T is a boronate group,
`each D1 and D2, independently, is OH or F or D1 and
`D2 together form a ring containing 1 to about 20 carbon
`atoms, and optionally heteratoms which can be N, S, or
`0; each R1'3 is H; X mimics the substrate recognized by
`the enzyme, for example X is pro-, thr-pro-, ala-pro-,
`ala-ala-pro, ser-thr-pro-, pro-ser-, pro-thr- or ser-pro-
`(pro=proline, thr=threonjne, and ser=serine); X con-
`tains both an amino acid and a blocking group, such as
`an acetyl group;
`the enzymes inhibited by the com-
`pounds of the invention are post prolyl cleaving en-
`zymes, most preferably serine proteases, even more
`preferably IgAl proteases; and the analog has a binding
`constant of at least lO—7M, most preferably l0‘1°M.
`In a second aspect,
`the invention features a com-
`pound, which is useful as an intermediate in the synthe-
`sis of compounds of Formula (1), having the formula
`
`(7)
`
`V—l|\I—C|2I-I-Z
`Rl—ClI-YR2
`
`where V is (CH3)3 Si—- or H—,
`Y is
`
`\‘.
`
`Page 5 of 11
`
`Page 5 of 11
`
`

`
`3
`
`4,935,493
`
`4
`
`T3 1115 $7
`If ifs
`I
`-C"'R"'. -‘C-0-. or -C-0-C-.
`:13
`11¢: 11¢
`11¢:
`[Ls
`lles
`(8)
`(9)
`(10)
`
`Drawing
`FIG. 1 shows the general formula of three preferred
`compounds.
`FIG. 2 is a diagrammatic representation of the syn-
`thesis of a boroProline compound.
`
`5
`
`each R1, R2, R3, R4, R5, R6, R7, and R3 is independently
`H, or c,-c1o alkyl or my], and
`
`.
`Z15 -113-'D1’
`D2
`
`(11)
`
`where D1 and D1 are as defined above.
`The invention also features a method for producing
`the above compounds. The method includes the steps of
`reacting a compound of the formula
`
`Q—(|3-Y‘/cH—'1l3—wDl
`R2 Cl
`D2
`
`(12)
`
`where Q is C1 or Br, and R2, Y, Z, and D1 and D2 are as
`defined above, with a compound of the formula
`
`STRUCTURE
`The compounds of the invention have the general
`10 structure recited in the Summary of the Invention
`above. Examples of preferred structures are those re-
`ferred to as preferred embodiments above.
`The structure of the compounds is such that at least a
`portion of the amino acid sequence near the cleavage
`15 site of a post-prolyl cleaving enzyme substrate is dupli-
`cated, or nearly duplicated. This duplication is in part
`responsible for the ability of the compounds to inhibit
`the enzyirilile, by a mechanismhthought to ;nvo‘livehcom-
`petitive i
`ibition between t e compoun an t e ac-
`20 tual enzyme substrate.
`The choice of amino acid sequence affects the inhibi-
`tory activity of the compound, and its specificity. As a
`first step in determining a suitable sequence, the amino
`acid sequence of the substrate is determined near its
`25 cleavage site. In some cases, such as for serine proteases,
`a suitable inhibitor sequence is the amino acid sequence
`[(C1‘13)35il2N‘1-1+
`(13)
`N-terminal to (i.e., to the left of) the proline cleavage
`oint, and includes that
`roline. Peptide fragments can
`in an inert S01Ve11t 315 3 temPe1'at11fe between —73° C- 30 lie synthesized and thenptested to determine their effi-
`and 25° C-, and heating the reaction mixture $0 313 1eaSt
`cacy as inhibitors, using standard techniques. Specific-
`80° C.
`ity is determined in a similar fashion, by testing the
`In preferred embodiments the reacting step is per-
`inhibitory effect of a particular compound on a variety
`formed in the absence of an inert solvent.
`of enzyme activities. The compounds preferably inhibit
`The compounds of the invention are peptide deriva- 35 the activity of enzymes detrimental to an animal or
`tives (or intermediates in their formation) which are
`human patient. and D1'efefab1Y do I10t inhibit necessary
`potent
`inhibitors of proteolyic enzymes (especially
`eI1ZYmeS-
`_
`those produced by pathogens) which are posppl-01y]
`The compounds of the invention can be designed so
`eleaving enzymes, e_g_, the pmteaeee able to act on
`that they are resistant to attack by agents which might
`IgA1 proteins_ The compounds generally have a pro. 40 otherwise cause their catabolic degradation by cleavage
`line, proline analog, a 2_azetidineearboXy1ie acid’ or
`of one or more peptide bonds in the peptides. For exam-
`pipecolic acid linked to a group, for example a boronate
`P15’ an N'1°m‘ma_1 blockmg gmup’ S“°_h 3:5 acetyl’ can
`group’ which mimics the transition state of an enzyme
`increase the half-life of the compounds in vivo, and thus
`substrate, and to a peptide moiety which preferably
`Improves mhlbmom
`.
`mimics the site of the substrate acted upon by the en- 45
`.The structure of .such blqckmg groups can Vary
`.
`.
`.
`.
`widely. In one blocking reaction, a hydrogen atom of
`zyme of interest. It is proposed that the peptide moiety
`.
`t
`.
`1
`.
`is re laced metal]
`h
`is recognized as a substrate by the enzyme to be inhib-
`e ammo erlnma am.m° group
`P.
`’ g
`y
`.
`.
`.
`.
`.
`.
`in a dehydration reaction. Thus, blocking groups such
`ited, and it then enters an active site, catalytic site, or
`as
`transition state binding site of the enzyme, and the tran-
`sition state-inimicing group of the compound of the 50
`invention binds strongly at this site. This binding advan-
`tageously prevents the enzyme from acting upon its
`natural substrate. The high affinity of these compounds
`make them effective at concentrations as low as 10-7M, 55 are readily added to a peptide chain. Others include
`or even 10-1°M.
`
`W
`fl’
`H-C. and —"CH3—'CH2'-C-are
`
`Thus, the present invention also provides composi-
`tions including one or more compounds of formula 1
`above, and methods of using such compounds or com-
`positions in the inhibition of post prolyl cleaving en- 50
`zymes.
`
`0
`O
`H
`H
`-C-01-13. '''C
`
`O
`H
`.and —C-(CH2)2—CH3-
`
`Other features and advantages Of the invention Will
`be apparent from the following description of preferred
`embodiments thereof. and from the Claims.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`The Figures will first briefly be described.
`
`Such N-te1-Ininal blocking groups may be employed not
`only to protect amino.termina1 groups, but also may
`protect-side chain amino group of amino acids which
`65 make up X, such as where X includes Lys or Arg. Simi-
`larly, amino acid residues having acidic or hydroxy side
`chains can be protected in the form of t-butyl, benzyl, or
`other suitable esters or ethers. Short length of the com-
`
`Page 6 of 11
`
`Page 6 of 11
`
`

`
`4,935,493 A
`
`6
`amino acids, especially ones having non-bulky side
`groups, such as Ala, Gly, or Ser.
`Other examples of enzymes which can be inhibited
`according to the invention include other post prolyl
`cleaving enzymes, such as IgA enzymes, enkephalin
`degrading enzymes, vasopressin degrading enzymes,
`and oxytosin degrading enzymes. Further, the serine
`protease, dipeptidyl peptidase Type IV, on T-lym-
`phocytes (Andrews et al., Clin. Lab. Haemato. 7:
`359-368, 1985), which plays a role in regulation of the
`immune response, can be inhibited by suitable such
`compounds. These inhibitors may be useful in treatment
`of Aids. Walters et al., Molecular and Cellular Biochem-
`istry 30: 111-127, 1980 describe other such enzymes, and
`are hereby incorporated by reference.
`
`Synthesis
`
`Synthesis of boroProline
`
`Referring to FIG. 2, the starting compound I is pre-
`pared essentially by the procedure of Matteson et al., 3
`Organometallics 1284, 1984, except that a pinacol ester is
`substituted for the pinanediol ester. Similar compounds
`such as boropipecolic acid and 2-azetodine boronic acid
`can be prepared by making the appropriate selection of
`starting material to yield the pentyl and propyl analogs
`of compound I. Further, Cl can be substituted for Br in
`the formula, and other diol protecting groups can be
`substituted for pinacol
`in the formula, e.g., 2,3-
`butanediol and alpha-pinanediol.
`Compound II is prepared by reacting compound I
`with [(CH3)O3Si]2N~Li+. In this reaction hexamethyl-
`disilazane is dissolved in tetrahydrofuran and an equiva-
`lent of n-butyllithium added at —78° C. After warming
`to room temperature (20° C.) and cooling to -—78° C. an
`equivalent of compound I is added in tetrahydrofuran.
`The mixture is allowed to slowly come to room temper-
`ature and to stir overnight. The alpha-bis[trimethyl-
`silane]-protected amine is isolated by evaporating sol-
`vent and adding hexane under anhydrous conditions.
`Insoluble residue is removed by filtration under a nitro-
`gen blanket, yielding a hexane solution of compound 2.
`Compound III, the N-trimethysilyl protected form of
`boroProline is obtained by the thermal cyclization of
`compound II during the distillation process in which
`compound II is heated to 100°-150° C. and distillate is
`collected which boils 66°—62° C. at 0.06-0.10 mm pres-
`sure.
`
`Compound IV, boroProline-pinacol hydrogen chlo-
`ride, is. obtained by treatment of compound III with
`HCl:dioxane. Excess HCl and by-products are removed
`by trituration with ether. The final product is obtained
`in a high degree of purity by recrystallization from
`ethyl acetate. H-boroProline as the hydrochloride salt is
`preferred, but other salts forms such as the hydrobro-
`mide and trifluoroacetate can readily be obtained by
`substitution of the appropriate acid for HCl.
`The boroProline esters can also be obtained by treat-
`ment of the reaction mixture obtained in the preparation
`of compound II with anhydrous acid to yield l-amino-
`4-bromobutyl boronate pinacol as a salt. Cyclization
`occurs after neutralizing the salt with base and heating
`the reaction.
`
`Synthesis of boroProline Peptides
`
`General methods of coupling of N-protected peptides
`and amino acids with suitable side-chain protecting
`groups to H-boroProline-pinacol are applicable. When
`
`5
`pounds of the invention (2-7, preferably 3 or 4 amino
`acids) is advantageous because it provides stability and
`increased half life.
`
`5
`
`The compounds also include a group (T) which
`causes the compound to complex with an enzyme, not
`only in a competitive fashion, but in a chemically reac-
`tive manner to form a strong bond between the com-
`pound and the enzyme. This group thus acts to bind the
`compound to the enzyme, and increases the inhibitory
`binding constant (Ki) of the compound. Examples of 10
`such groups include boronates, fluoroalkyl ketones and
`substituted phosphonates (of the formulae given in the
`Summary above, examples of which are shown in FIG.
`1). These groups are covalently bonded to the prolyl
`residue of the compound, as in the above formula.
`The proline or proline analog, represented by
`
`15
`
`\
`\
`R1-C—Y
`\
`R2,
`
`(14)
`
`20
`
`25
`
`30
`
`35
`
`above, is chosen so that it mimics the structure of pro-
`line recognized by the active site of the enzyme to be
`inhibited. It can be modified by providing R14 groups
`which do not interfere significantly with this recogni-
`tion and thus do not significantly affect the Ki of a
`compound. Thus, one or more hydroxyl groups can be
`substituted to form hydroxy-proline, and methyl or
`sugar moieties may be linked to these groups. One
`skilled in the art will recognize that these groups are not
`critical in this invention and that a large choice of sub-
`stituents are acceptable for R.
`Examples of compounds having utility as serine pro-
`tease inhibitors include compounds having a peptide
`chain similar to the subtrate of IgA1 and IgA2 pro-
`teases. Plant 1983 Ann Rev. Microbiol 37, 603-622).
`These enzymes hydrolyze specific peptide bonds of
`IGA, the immunoglobulin that provides antibody de- 40
`fense of mucosal surfaces, resulting in a nonfunctional
`immunoglobulin and impairment of the host defense
`system. This is expected to be a strong contributing
`factor to pathogenesis of organisms such as Streptococ-
`cus sanquis, S. pneumoniae, Neisseria ganorrhoae, N.
`meningitidis, and Haemophilus influenzae.
`lgA1 proteases recognize the cleavage site Ser-Thr-
`Pro-Pro-X (where X is any amino acid), hydrolyzing
`between Pro and X (i.e., they are post prolyl cleaving
`enzymes). Accordingly, Ser-Thr-Pro-Pro-T is a suitable
`compound of the invention for inhibiting IGA1 pro-
`teases. The Set or Thr in this compound can be readily
`substituted with any of the 20 naturally occurring amino
`acids, most preferably those having non-bulky side 55
`groups, such as Ala and Gly. It is also possible to substi-
`tute non-naturally occurring amino acids, such as 2-
`azetidinecarboxylic acid or pipecolic acid (which have
`six-membered, and four-membered ring structures re-
`spectively) for either of the Pro residues. Those skilled
`in the art will recognize that there are other such
`changes which can be made without significantly affect-
`ing the inhibitory character of these compounds.
`In the case of IgA2, the cleavage site is Pro-Thr-Pro-
`X, with hydrolysis occurring between Pro and X. Thus,
`a preferred compound of the invention for inhibiting
`IGA2 proteases has the formula Pro-Thr-Pro-T. Thr
`can be substituted by any of the naturally occurring
`
`45
`
`50
`
`60
`
`65
`
`Page 7 of 11
`
`Page 7 of 11
`
`

`
`4,935,493
`
`8
`
`EXAMPLE 1
`
`10
`
`15
`
`7
`needed, side-chain protecting and N-terminal protect-
`ing groups can be removed by treatment with a.nhy-
`drous.HCl, HBr, trifluoroacetic acid, or by catalytic
`hydrogenation. These procedures are known to those
`skilled in the art of peptide synthesis. One exception is
`that in the preparation of a compound with the Pro-
`Thr-boroPro sequence. Removal of acid labile protect-
`ing groups from threonine hydroxyl group results in a
`complex mixture of products. Thus,
`the use of hy-
`drogenolytic protecting groups for threonyl residue is
`preferred.
`The mixed anhydride procedure of Anderson et al., J.
`Am. Chem. Soc.. 89:5012 (1984) is preferred for peptide
`coupling. The mixed anhydride of an N-protected
`amino acid or a peptide varying in length from a dipep-
`tide to tetrapeptide is prepared by dissolving the peptide
`in tetrahydrofuran and adding one equivalent of N-
`methylmorpholine. The solution is cooled to —20° C.
`and an equivalent of isobutyl chloroformate is added. 20
`After 5 minutes, this mixture and one equivalent of
`triethylamine (or other sterically hindered base) are
`added to a solution of H-boroPro-pinacol dissolved in
`either cold chloroform or tetrahydrofuran.
`'
`The reaction mixture is routinely stirred for one hour
`at -20“ C. and 1-2 hours at room temperature (20° C.).
`Solvent is removed by evaporation, and the residue is
`dissolved in ethyl acetate. The organic solution is
`washed with 0.20N hydrochloric acid, 5% aqueous
`sodium bicarbonate, and saturated aqueous sodium
`chloride. The organic phase is dried over anhydrous
`sodium sulfate, filtered, and evaporated. Products are
`purified by either silica gel chromatography or gel per-
`meation chromatography using Sephadex TM LH-20
`and methanol as a solvent.
`.
`
`Preparation of boroProline-pinacol
`(H-boroPro-pinacol)
`The intermediate, 4-Bromo-l-chlorobutyl boronate
`pinacol, was prepared by the method in Matteson et al.,
`Organometallics, (3): 1284-1288 (1984), except that con-
`ditions were modified for large scale preparations and
`the pinacol was substitued for the pinanedoil protecting
`group.
`3-bromopropyl boronate pinacol was prepared by
`hydrogenboronation of allyl bromide (173 ml, 2.00
`moles) with catechol borane (240 ml, 2.00 moles). Cate-
`chol borane was added to allyl bromide and the reaction
`heated for 4 hours at 100“ C. under a nitrogen atmo-
`sphere. The product, 3-bromopropyl boronate catechol
`(bp 95°—102° C., 0.25 mm), was isolated in a yield of
`49% by distillation. The catechol ester (124 g, 0.52
`moles) was transesterified with pinacol (61.5 g, 0.52
`moles) by mixing the component in 50 ml of THF and
`allowing them to stir for 0.5 hours at 0° C. and 0.5 hours
`at room temperature. Solvent was removed by evapora-
`tion and 250 ml of hexane added. Catechol was re-
`moved as a crystalline solid. Quantitative removal was
`achieved by successive dilution to 500 ml and to 1000
`ml with hexane and removing crystals at each dilution.
`Hexane was evaporated and the product distilled to
`yield 177 g (bp 60°—64° C., 0.35 mm).
`4-Bromo-l-chlorobutyl boronate pinacol was pre-
`pared by homologation of the corresponding propyl
`boronate. Methylene chloride (50.54 ml, 0.713 moles)
`- was dissolved in 500 ml of THF, l.54N n-butyllithium
`in hexane (480 ml, 0.780 moles) was slowly added at
`- 100° C. 3-Bromopropyl boronate pinacol
`(178 g,
`0.713 moles) was dissolved in 500 ml of THG, cooled to
`the freezing point of the solution, and added to the
`reaction mixture. Zinc chloride (54.4 g, 0.392 moles)
`was dissolved in 250 ml of THG, cooled to 0° C., and
`added to the reaction mixture in several portions. The
`reaction was allowed to slowly warm to room tempera-
`ture and to stir overnight. Solvent was evaporated and
`the residue dissolved in hexane (1 liter) and washed
`with water (1 liter). Insoluble material was discarded.
`After drying over anhydrous magnesium sulfate and
`filtering, solvent was evaporated. The product was
`distilled to yield 147 g (bp l10°—112° C. 0.200 mm).
`N-Trimethylsilyl-boroProline pinacol was prepared
`first by dissolving hexamethyldisilizane (20.0 g, 80.0
`mmoles) in 30 ml of THF, cooling the solution to —78°
`C. and adding 1.62N n-butyllithium in hexane (49.4 ml,
`80.0 mmoles). The solution was allowed to slowly
`warm to room temperature. It was recooled to — 78° C.
`and 4-bromo-l-chlorobutyl boronate pinacol (23.9 g,
`80.0 mmoles) added in 20 ml of THF. The mixture was
`allowed to slowly warm to room temperature and to stir
`overnight. Solvent was removed by evaporation and
`dry hexane (400 ml) added to yield a precipitant which
`was removed by filtration under an nitrogen atmo-
`sphere. The filtrate was evaporated and the residue
`distilled, yielding 19.4 g of the desired product (bp
`60°—62° C., 0.l—0.06 mm).
`H-boroProline-pinacol.HCl was prepared by cooling
`N-trimethylsilyl-boroProline-pinacol
`(16.0
`g,
`61.7
`mmoles) to -78° C. and adding 4N HCL:dioxane 46
`ml, 185 mmoles). The mixture was stirred 30 minutes at
`—78° C. and 1 hour at room temperature. Solvent was
`evaporated and the residue triturated with ether to yield
`
`25
`
`30
`
`35
`
`Previous studies have shown that the pinacol protect-
`ing group can be removed in situ by preincubation in
`phosphate buffer prior to running biological experi-
`ments; Kettner et al., J. Biol. Chem. 259: 15106-15114
`(1984). Several other methods are also applicable for
`removing pinacol groups from peptides including boro-
`Proline and characterizing the final product. First, the
`peptide can be treated with diethanolamine to yield the
`corresponding diethanolamine boronic acid ester,
`which can be readily hydrolyzed by treatment with
`aqueous acid or a sulfonic acid substituted polystyrene
`resin as described in Kettner et al., Id. Both pinacol and
`pinanediol protecting groups can be removed by treat-
`ing with BCI3 in methylene chloride as described by
`Kinder et al., J. Med. Chem, 28: 1917. Finally, the free
`boronic acid can be converted to the difluoroboron
`
`40
`
`45
`
`50
`
`55
`
`derivative (—-BF2) by treatment with aqueous HF as
`described by Kinder et al., Id.
`Similarly, different ester groups can be introduced by
`reacting the free boronic acid with various di-hydroxy
`compounds (for example, those containing heteroatoms
`such as S or N) in an inert solvent.
`The following abbreviations are used in the examples
`below. THF - tetrahydrofuran; H-Pro-OBzl - the benzyl
`ester of proline; I-I—Thr (OBz1)-OH - the benzyl ether
`derivative of threonine; Boc - the tertiary butyloxycar-
`bonyl group; FABMS - fast atom bombardment mass 55
`spectometry.
`All natural amino acids are in the L-configuration.
`H-boroProline is in the D,L-configuration.
`
`60
`
`Page 8 of 11
`
`Page 8 of 11
`
`

`
`4,935,493
`
`9
`a solid. The crude product was dissolved in chloroform
`and insoluble material removed by filtration. The solu-
`tion was evaporated and the product crystallized from
`ethyl acetate to yield ll.l g of the desired product (mp
`l56.5°—l57° C.).
`
`10
`applied to a 2.5 X 100 colunm of LH-20 in methanol and
`fractions (approximately 7 ml) collected. Fraction 22-28
`contained 0.16 g of the desired product.
`EXAMPLE 5
`
`EXAMPLE 2
`
`Preparation of MeOSuc-Ala-Ala-Pro-boroPro-pinacol.
`
`Preparation of Boc-Ala-Pro-boroPro-pinacol
`Boc-Ala-Pro-boroPro-pinacol was prepared by cou-
`pling Boc-Ala-Pro-OH to H-boroPro-pinacol. First, the
`dipeptide, Boc-Ala-Pro-OBzl, was prepared by the
`mixed anhydride procedure. Boc-Ala-OH (10 g, 52.8
`mmoles) was reacted with N-methylmorpholine (5.8 ml,
`52.8 mmoles) and isobutyl chloroformate (6.8 ml, 52.8
`mmole) for 5 minutes in 50 ml of THF at -20". The
`reaction mixture and additional N-methylmorpholine
`(5.8 ml) were added to H-Pro-OBzl.HCL (12.8 g, 52.8
`mmoles) dissolved in 50 ml of cold chloroform. After
`the mixture was stirred for 1 hour at -20“ C. and 2
`hours at room temperature, it was filtered and the fil-
`trate evaporated. The residue was dissolved in ethyl
`acetate and washed sequentially with 0.2N hydrochlo-
`ric acid, 5% aqueous NaCO3, and saturated aqueous
`NaCl. The organic phase was dried over anhydrous
`Na2SO4, filtered, and evaporated to yield Boc-Ala-Pro-
`OBzl as an oil (14.1 g). The benzyl ester protecting
`group was removed by dissolving Boc-Ala-Pro-OBzl
`(35 g, 92.8 mmoles) in 200 ml of methanol and hydroge-
`nating for 1 hour in the presence of 0.5 g of 10% Pd/C.
`The catalyst was removed by filtration and the solvent
`evaporated. The residue was crystallized from ethyl
`acetate to yield 16.1 g of Boc-Ala-Pro-OH (mp
`153.5°—l54.5° C.).
`Boc-Ala-Pro-OH (1.26 g, 4.28 mmoles) was coupled
`to H-boroPro-pinacol by the general procedure de-
`scribed for the preparation of Boc-Ala-Pro-OBzl. Boc-
`Ala-Pro-OH (1.26 g, 4.28 mmoles) was dissolved in 10
`ml of THF and cooled to —-20° C.; N-methylmorpho-
`line (0.47 ml, 4.28 mmoles) and isobutyl chloroformate
`(0.557 ml, 4.28 mmoles) were added. After stirring for 5
`minutes, 10 ml of cold THF and triethylamine (0.597
`ml, 4.28 mmoles) were added and the mixture added to
`a cold solution of H-boroPro-pinacol.HCl (1.0 g, 4.28
`mmoles) in 5 ml chloroform. After dissolving the reac-
`tion product in ethyl acetate and washing with aqeous
`HCl, NaHCO3, and saturated aqueous NaCl, 0.39 g of
`material were obtained. It was further purified by chro-
`matography on a 2.5X5O cm column of Sephadex
`LH-20 in methanol to yield 0.25 g.
`EXAMPLE 3
`
`Preparation of H-Ala-Pro-boroPro-pinacol.HCL
`
`Boc-Ala-Pro-boroPro-pinacol (0 58 g, 1.25 mmoles)
`was allowed to react with 2.5 mol of 4N HCl:dioxane
`for 30 minutes at room temperature. Ether (50 ml) was
`added to yield 0.22 g of amorphous white solid.
`EXAMPLE 4
`
`Preparation of Ac-Ala-Pro-boroPro-pinacol
`
`0.55
`g,
`H-Ala-Pro-boroPro-pinacol.HCL (0.22
`mmoles) was dissolved in 3 ml of THF and cooled to 0°
`C. Acetic anhydride (0.078 ml, 0.825 mmoles) and tri-
`ethylamine (0.115 ml, 0.825 mmoles) were added and
`the reaction was allowed to come to room temperature.
`After approximately 25 minutes, additional triethylam-
`ine (0.25 ml, 0.179 mmoles) was added. After a total
`reaction time of 45 minutes, the reaction solution was
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`MeOSuc-Ala-Ala-Pro-OH was prepared by the pro-
`cedure described in Kettner et al., J. Biol. Chem., 259:
`15106-15114 (1984). MeOSuc-Ala-Ala-Pro-OH (1.59 g.
`4.28 mmoles) was coupled to, H-boroPro-pinacol.HCl
`(1.00 g, 4.28 mmoles) by the mixed anhydride procedure
`described for the preparation of Boc-Ala-Pro-boroPro-
`pinacol except that, after filtration and evaporation of
`the reaction solvent, it was applied to a 2 cm column
`containing 10 g of silica gel equilibrated with chloro-
`form. The column was eluted with chloroform and
`fractions containing the desired product were evapo-
`rated and triturated with hexane to yield 0.58 g of a
`white solid.
`
`EXAMPLE 6
`
`Preparation of Boc-Pro-Thr(OBzl)-boroPro-pinacol.
`
`Boc-Pro-Thr(OBzl)-OH was prepared by coupling 1
`Boc-Pro-OSu (the N-hydroxysuccinamfcle ester of Boc-
`Pro-OH)
`to H-Thr
`(OBzl)
`- OH. H-Thr
`(OBzl)-
`OH.HCL was dissolved in 25 ml of water, NaHCO3
`(6.21 g, 73.9 mmoles) and a solution of Boc-Pro-Osu
`(5.07 g, 16.25 mmoles) in 25 ml of dioxane were added.
`After stirring 3 hours, the reaction mixture was acidi-
`tied with hydrochloric acid and the product extracted
`into ethyl acetate. It was washed with 0.2N HCl pre-
`pared in saturated aqueous NaCl, saturated NaCl and
`dried over anhydrous Na2SO4. Solvent was evaporated
`to yield 6.24 g (mp l20°—l2.5°).
`Boc-Pro—Thr(OBzl)-boroPro-pinacol was prepared
`by coupling Boc-Pro-Thr(OBzl)-OH (2.70 g, 6.42
`mmoles)
`to H-boroPro-pinacol.HCL(1.50 g,
`6.42
`mmoles) using the procedure described for Boc-Ala-
`Pro-boroPro-pinacol. The product (2.4 g) was purified
`by chromatography on a 2.5 X 50 cm column of LH-20
`in methanol and was obtained as 0.84 g of oil.
`EXAMPLE 7
`
`Preparation of Boc-Pro-Thr-boroPro-Pinacol
`
`Boc-Pro-Thr-boroPro-pinacol was prepared by hy-
`drogenation of Boc-Pro-Thr(OBzl)-boroPro-pinacol
`(from Example 6, 0.585 g, 0.79 mmoles). The protected
`peptide was dissolved in 100 ml of methanol and was
`hydrogenated for 2 hours on a Parr apparatus in the
`presence of 0.5 g of 10% Pd/C. The reaction was fil-
`tered and solvent evaporated. The product was allowed
`to stir with hexane, and hexane insoluble material re-
`moved by filtration. The filtrate was evaporated to
`yield the desired product (0.24 g) as a white foam.
`USE
`
`The compounds of formula 1 above can be adminis-
`tered in an effective amount either alone or in combina-
`tion with a pharmaceutically acceptable carrier or dilu-
`ent.
`
`65
`
`The compounds of formula 1 or compositions thereof
`can be used to treat mammals, e.g., humans, suffering
`from or subject to infections by pathogenic bacteria
`which produce deleterious post prolyl cleaving . en-
`zymes, e.g., the IgAl protease of Haemophilus influ-
`enza. The compounds or compositions can be adminis-
`
`-9
`
`Page 9 of 11
`
`Page 9 of 11
`
`

`
`12
`4. The compound of claim 1 where X is pro-, thr-pro-,
`ala-pro-, ala-ala-pro-, ser-thr-pro-, pro-ser-, pro-thr- or
`ser-pro-.
`5. The compound of claim 1, said enzyme being a
`postprolyl cleaving enzyme.
`6. The compound of claim 5, said enzyme being a
`serine protease.
`7. The compound of claim 5, said enzyme b