(19) United States
`(12) Reissued Patent
`Baker et al.
`
`USO0RE3907lE
`
`(10) Patent Number:
`
`US RE39,071 E
`(45) Date of Reissued Patent:
`Apr. 18, 2006
`
`(54) ANHYDRO-AND ISOMER-A-21978C CYCLIC
`PEPTIDES
`
`(75)
`
`Inventors: Patrick J. Baker, Greenwood, IN (US);
`Manuel Debono, Indianapolis, IN (US);
`Khadiga Z. Farid, Lebanon, IN (US);
`R. Michael Molloy, Avon, IN (US)
`
`(73) Assignee: Eli Lilly and Company, Indianapolis,
`IN (US)
`
`(21) Appl. No.: 09/547,357
`
`(22)
`
`Filed:
`
`Apr. 11, 2000
`
`Related U.S. Patent Documents
`
`Reissue of:
`
`(64) Patent No.:
`Issued:
`Appl. No.:
`Filed:
`
`5,912,226
`Jun. 15, 1999
`07/809,039
`Dec. 16, 1991
`
`U.S. Applications:
`(63) Continuation of application No. 07/670,375, filed on Mar.
`14, 1991, now abandoned, which is a continuation of appli-
`cation No. 07/060,148, filed on Jun. 10, 1987, now aban-
`doned.
`
`(51)
`
`Int. Cl.
`A61K 38/00
`A61K 31/00
`A61K 38/12
`C07K 16/00
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl.
`
`............................. .. 514/9; 514/2; 530/317;
`530/320
`
`(58) Field of Classification Search ................... .. 514/9,
`514/2; 530/317, 320
`See application file for complete search history.
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,399,067 A *
`8/1983 Debono .................... .. 530/323
`
`4,537,717 A *
`8/1985 Abbott et al.
`. . . . . .
`. . . . .. 530/317
`RE32,311 E
`* 12/1986 Debono .................... .. 530/317
`
`OTHER PUBLICATIONS
`
`Goodman et al, American Chemical Society, vol. 12, No. 1
`(Jan. 1979), pp 1—7.*
`Organic Chemistry, 3'“ Edition, Morrision & Boyd, p 225.*
`T. Geiger et al. Dearnidation, Isomerization and Racemiza-
`tion at Apaginyl and Aspartyl residues in Peptides, J. Biol.
`Chem. 262 (2). 785-794 (1987).*
`M. Bodansky et al., “Side Reaction in Peptide Synthesis,”
`Synthesis 1981 (may), 333—338, 351—356.*
`E. A. Hagan et al., “Synthesis of Ac—Asp4}ly—Ser and
`Ac—Asp—Pro—Leu—Gly—NH2”
`Int.
`J. Peptide Protein
`Research. 23, 642-649. (1978).*
`M. Bodanszky et al., “Side Reactions in Peptide Synthesis,”
`Int. J. Peptide Protein Res. 12, 69—74 (1978).*
`B.A. Johnson et al., Enzymatic Protein Caboxyl Methylation
`at Physiological pH: cyclic imide Formation Explains Rapid
`Methyl Turnover, Biochemistry 24, 2581—2586 (1985).*
`
`* cited by examiner
`
`Primary Examiner—Padmashri Ponnaluri
`(74) Attorney, Agent, or Firm—Kenyon & Kenyon
`
`(57)
`
`ABSTRACT
`
`Two new groups ofA-21978C cyclic peptides, anhydro- and
`isomer-A21978C peptide derivatives, have antibacterial
`activity and are useful as intermediates. The two groups are
`prepared via transpeptidation of the parent cyclic peptides.
`Pharmaceutical formulations containing the new peptides as
`active ingredients and methods of treating infections caused
`by susceptible Gram-positive bacteria with the formulations
`are also provided.
`
`The invention also provides an antibacterial composition
`containing the new drug substance LY 146032 in substan-
`tially pure form.
`
`3,953,415 A *
`
`4/1976 Kisfaludy et al.
`
`........ .. 530/306
`
`31 Claims, No Drawings
`
`lofl5
`
`FRESENIUS-KABI, Exh. 1034
`
`
`1 of 15
`
`FRESENIUS-KABI, Exh. 1034
`
`

`
`US RE39,071 E
`
`1
`ANHYDRO-AND ISOMER-A-21978C CYCLIC
`PEPTIDES
`
`Matter enclosed in heavy brackets [ ] appears in the
`original patent but forms no part of this reissue specifi-
`cation; matter printed in italics indicates the additions
`made by reissue.
`This application is a continuation of application Ser. No.
`07/670,375, filed on Mar. 14, 1991, abandoned which is a
`continuation of application Ser. No. 07/060,148, filed on
`Jun. 10, 1987, abandoned.
`
`10
`
`SUMMARY OF THE INVENTION
`
`Gly: glycine
`Kyn: kynurenine
`3-MG: L-threo-3-methylglutamic acid
`Orn: ornithine
`Ser: serine
`Thr: threonine
`
`Trp: tryptophan
`t-BOC: tert-butoxycarbonyl
`Cbz: benzyloxycarbonyl
`DMF: dimethylforrnamide
`THF: tetrahydrofuran
`HPLC: high performance liquid chromatography
`
`NMR: 1H nuclear magnetic resonance
`TLC: thin-layer chromatography
`UV: ultraviolet
`
`Despite the availability of antibacterial agents today, the
`need for improved antibiotics continues. Antibiotics diifer in
`their effectiveness against specific pathogenic organisms. In
`addition, organism strains resistant to known antibiotics
`continue to develop. Furthermore, individual patients fre-
`quently suifer serious reactions to specific antibiotics, due to
`hypersensitivity and/or to toxic effects. There is, therefore, a
`continuing need for new and improved antibiotics.
`This invention relates to new antibiotics and an improved
`form of the known antibiotic LY146032, which inhibit the
`growth of Gram-positive bacteria. In particular, the inven-
`tion relates to two new groups of A-21978C cyclic peptide
`derivatives. The first group of derivatives, the ar1hydro-A-
`21978C peptide derivatives, are compounds which have
`formula 1:
`
`This invention relates to two new groups of derivatives of 1
`A-21978C cyclic peptides, designated “anhydro-A-21978C
`peptide derivatives” (formula 1 compounds) and “isomer-
`A-21978C peptide derivatives” formula 2 compounds). Like
`the previously known A-21978C cyclic peptide derivatives
`(the parent cyclic peptides), the two new groups of deriva-
`tives and their salts are useful semi-synthetic antibacterial
`agents or are intermediates to such agents.
`This invention also provides processes for preparing the
`anhydro- and isomer-derivatives by trans-peptidation of the
`parent peptides.
`In another aspect, this invention provides an improved
`antibacterial composition comprising the new drug sub-
`stance LY146032, or a pharmaceutically-acceptable salt
`thereof, in substantially pure form.
`This invention further provides 1) methods of treating
`infections caused by susceptible Gram-positive bacteria
`which comprises administering a formula 1 or 2 compound
`to the animal to be treated, and 2) pharmaceutical formula-
`tions comprising a formula 1 or 2 compound or LY146032
`in a pharmaceutically purified form as the active ingredient.
`
`5
`
`20
`
`25
`
`30
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`60
`
`In this specification the following abbreviations, most of
`which are commonly known in the art, are used:
`Ala: alanine
`
`65
`
`Asn: asparagine
`Asp: aspartic acid
`
`independently, hydrogen,
`in which R, R1 and R2 are,
`C4—C14-alkyl, optionally substituted C2—C19-alkanoyl,
`C§—C19-alkenoyl or an amino-protecting groripg R3, R4 and
`R are hydrogen or (i) R3 and R1 and/or (ii) R and R and/or
`(iii) R5 and R2, taken together, may represent a C4—C1§
`alkylidene group; provided that 1) at least one of R, R1 or R
`must be other than hydrogen or an amino-protecting group,
`2) at
`least one of R1 or R2 must be hydrogen or an
`
`2of15
`
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`
`

`
`3
`
`4
`
`US RE39,071 E
`
`amino-protecting group, and 3) the R, R1 and R2 groups
`must together contain at least four carbon atoms; and their
`salts.
`
`The second group of A-21978C cyclic peptide
`derivatives, the isomer-A-21978C peptide derivatives, are 5
`compounds which have formula 2:
`
`The term “amino-protecting group” refers to a recgonized
`amino-protecting group which is compatible with the other
`functional groups in the A-21978C molecule. Preferably,
`amino-protecting groups are those which can be readily
`removed from the subsequently acylated compound.
`Examples of suitable protecting groups can be found in
`
`O
`
`N
`H
`
`oH
`
`o
`
`HN
`
`o
`
`OH
`
`N
`H
`
`o
`
`H
`N
`
`o
`
`N
`H
`
`o
`
`o
`
`o
`
`E
`
`H
`
`o
`
`N_R5
`|,
`R
`
`N
`H
`
`0
`
`“Protective Groups in Organic Synthesis” by Theodora W.
`Greene, John Wiley and Sons, New York, 1981, chapter 7.
`Especially preferably amino-protecting groups are the tert-
`butoxycarbonyl and benzyloxycarbonyl groups.
`
`the invention contemplates the
`In subgeneric aspects,
`following preferred embodiments of the compound of for-
`mulas 1 and 2
`
`(a) The compounds wherein R is alkanoyl of the formula
`
`0
`
`CH3<CH2>n—c—,
`
`wherein n is an integer from 3 to 17;
`
`(b) The compounds wherein R is alkanoyl of the formula
`
`0
`
`CH3(CH2)n— C :,
`
`wherein n is 5 to 14;
`
`(c) The compounds wherein R is alkanoyl of the formula
`
`CH3
`
`0
`
`CH3(CH2)nCH(CH2)m— Cm ,
`
`wherein n and m are each, independently, an integer
`from 0 to 14, provided that n+m must be no less than
`1 and no greater than 15; and further provided that,
`when n is 0, m cannot be 8 and, when n is 1, m carmot
`be 6 or 8;
`
`N
`H
`
`NH
`
`0 3
`
`0
`
`35
`
`40
`
`R
`
`_
`
`o
`
`N
`
`O
`
`N
`
`NH;
`
`0H
`
`HN
`
`E
`
`o
`
`o
`
`R1—N
`R3
`
`o
`
`o
`
`Ho
`
`in which R, R1, R2, R3, R4 a11d R3 are as defined supra with
`the same provisos; and their salts.
`The term “C4£14-alkylidenyl” refers to a group of the
`formula
`
`wherein R33 and R43 are hydrogen or an alkyl group of from
`3 to 13 carbon atoms, provided that one of R3 and R4 must
`be other than hydrogenaand further provided that the sum of
`the carbon atoms in R3 and R4 must be no greater than 13.
`Those compounds wherein one of R and R4, R1 and R3 or
`R2 and R3 is C4£14-alkylidenyl are known as Schifi’ s
`bases.
`
`The term “C4—C14-alkyl” refers to a univalent saturated,
`straight- or branched-chain alkyl group containing from 4 to
`14 carbon atoms. Those compounds wherein one of R, R1 or 50
`R2 are C4—C14-alkyl, referred to herein as “reduced Schifi’ s
`bases”, are prepared by reduction of the corresponding
`compounds where R and R4, R1 and R3 or R2 and R3
`represent a C4—C14-alkylidenyl group.
`The terms “optionally substituted C2£19-alkanoyl” and 55
`“C5—C19-alkenoyl” refer to acyl groups derived from car-
`boxylic acids containing from 2 to 19 and 5 to 19 carbon
`atoms, respectively. When the group is alkanoyl, the alkyl
`portion is a univalent saturated, straight-chain or branched-
`chain hydrocarbon radical which can optionally bear one 60
`hydroxyl, carboxyl, or C1—C3-alkoxy group or from one to
`three halo substituents selected from chlorine, bromine, and
`fluorine. When R is alkenoyl,
`the alkenyl portion is a
`univalent, unsaturated, straight-chain or branched-chain
`hydrocarbon radical containing not more than three double 65
`bonds. The double bond portion(s) of the unsaturated hydro-
`carbon chain may be either in the cis or trans configuration.
`
`3of15
`
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`
`

`
`US RE39,071 E
`
`6
`The compounds of the formulas 1 and 2 are capable of
`forming salts. These salts are also part of this invention.
`Such salts are useful, for example,
`for separating and
`purifying the compounds.
`For example, the compounds of formulas 1 and 2 have
`several free carboxyl groups which can form salts. Partial,
`mixed and complete salts of these carboxyl groups are,
`therefore, contemplated as part of this invention. In prepar-
`ing these salts, pH levels greater than 10 should be avoided
`due to the instability of the compounds at such levels.
`Representative and suitable alkali-metal and alkaline-
`earth metal salts of the compounds of formulas 1 and 2
`include the sodium, potassium, lithium, cesium, rubidium,
`barium, calcium and magnesium salts.
`The alkali-metal and alkaline-earth-metal cationic salts of
`
`the compounds for formula 1 and 2 are prepared according
`to procedures commonly used for the preparation of cationic
`salts. For example, the free acid form of a formula 1 or 2
`compound is dissolved in a suitable solvent such as warm
`methanol or ethanol. A solution containing a stoichiometric
`quantity of the desired inorganic base in aqueous methanol
`is added to this solution. The salt thus formed can be isolated
`
`by routine methods, such as filtration or evaporation of the
`solvent. A convenient method of preparing salts is by the use
`of ion-exchange resins.
`Suitable amine salts of the formula 1 and 2 compounds
`include the ammonium and the primary, secondary, and
`tertiary C1—C4-alkylammonium and hydroxy-C2—C4-
`alkylarnmonium salts. Illustrative amine salts include those
`formed by reaction of a formula 1 or 2 compound with
`ammonium hydroxide, methylamine, sec-butylamine,
`isopropylamine, diethylamine, di-isopropylamine,
`cyclohexylamine, ethanolamine, triethylamine, 3-amino-1-
`propanol and the like.
`The salts formed with organic amines can also be pre-
`pared by well known procedures. For example, the gaseous
`or liquid amine can be added to a solution of a formula 1 or
`2 compound in a suitable solvent such as ethanol. The
`solvent and excess amine can be removed by evaporation.
`Because the compounds of this invention also have free
`amino groups, they can, therefore, form acid addition salts.
`Such salts are also part of this invention. Representative and
`suitable acid-addition salts of the compounds of formula 1 or
`2 include those salts formed by standard reaction with both
`organic and inorganic acids such as,
`for example,
`hydrochloric, sulfuric, phosphoric, acetic, succinic, citric,
`lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic,
`D-glutamic, d-camphoric, glutaric, glycolic, phthalic,
`tartaric,
`lauric, stearic, salicylic, methanesulfonic,
`benzenesulfonic, sorbic, picric, benzoic, cinnamic and like
`acids.
`
`Pharmaceutically acceptable alkali-metal, alkaline-earth-
`metal, amine and acid-addition salts are a particularly useful
`group of compounds of this invention.
`The formula 1 and 2 compounds are prepared from
`previously known A-21978C cyclic peptides, which in mm
`are prepared from the A-21978C antibiotics. The A-21978C
`antibiotics, a group of closely related, acidic peptide
`antibiotics, are described by Robert L. Hamill and Marvin
`M. Hoehn in U.S. Pat. No. 4,208,403, issued Jun. 17, 1980.
`As described in U.S. Pat. No. 4,208,403,
`the A-21978
`antibiotic complex contains a major component, factor C,
`which is itself a complex of closely related factors. A-21978
`factor C, which is called the A-21978C complex, contains
`individual factors C0, C1, C2, C3, C4 and C5. Factors C1, C2
`and C3 are major factors; and factors C0, C4 and C5 are
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`5
`(d) The compounds wherein R is cis or trans alkenyl of the
`formula
`
`0
`
`cH3(cH2),,cH=cH(cH2),,,—c—
`
`wherein n and m are each, independently, an integer
`from 0 to 14, provided that ncxm must be no less than
`1 and no greater than 15;
`(e) The compounds where R is cis or trans alkenyl of the
`formula
`
`0 l
`
`l
`cH2:cH(cH2),,c—
`
`wherein n is an integer of from 4 to 15;
`(f) The compounds where R is alkyl of the formula
`CH3(CH2)n— and n is an integer from 5 to 12; and
`(g) The compounds wherein R is:
`
`11
`CH3(CH2)5—C:
`O
`
`ll
`1
`CH3(CH2,)4jCj
`O
`
`ll
`1
`CH3(CH2,)3jCj
`O
`
`ll
`1
`CH3(CH2)8—C:
`O
`
`ll
`1
`CH3(CH2)9—C:
`O
`
`CH3(CH2)10‘C:
`
`ll
`CH3=CH—(CH3)9—C—
`O
`
`CH3(CH2)11:C:
`O
`
`CH3(CH2)12—C:
`
`O H
`
`cH3—(cH2)3—cH=cH—(cH2)7—c—
`o
`
`CH3(CH2)13—C:
`
`O
`
`CH3CH2CH(CH2) * (CH2)4 *Cj
`CH3(CH2) 1 1:
`
`CH3(CH2)s:
`
`CH3(CH2)9:
`
`(h) The compounds wherein R and R4 together are:
`
`CH3(CH2)10OCH:
`
`CH3(CH2)5CH:
`
`CH3(CH2)8CH:
`
`4of15
`
`
`4 of 15
`
`

`
`US RE39,07l E
`
`7
`minor factors. The A-2l978C factors have the structure
`shown in formula 3:
`
`L—AspN/ Gly
`D—Ala
`¢
`Ldfsp
`D—ier
`L—Orn
`31/{G
`\
`/L—Kyn
`Gly
`O
`
`L—Thr/
`
`L—?sp
`L—?sn
`L—Trp
`NIRN
`
`wherein RN represents a specific fatty acid moiety. The
`specific RN groups of the factors are as follows:
`
`A—21978C Factor
`
`RN Moiety
`
`C 1
`C2
`C3
`Co
`C4
`C5
`
`8-methyldecanoyl
`l0-methylundecanoyl
`l0-methyldodecanoyl
`CLO-alkamoyl*
`C12-alkamoyl**
`C1;-alkamoyl**
`
`*A-2l978Co was later found to be a mixture of two compounds in
`approximately 2:1 ratio, the RN of the major component being a brusched
`C10-alkamoyl, and the RN of the minor component being a n-decanoyl
`**Identity not yet determined
`
`The parent A-2l978C cyclic peptides are prepared from
`the A-2l978C antibiotics as described by Abbott, Manuel
`Debono and David S. Fukuda in U.S. Pat. No. 4,537,717.
`The preparation involves removing the fatty acid side chain
`(RN) from the naturally occurring antibiotics with an enzyme
`produced by Actinoplanes utahensis NRRL 12052 to give
`the common A-2l978C cyclic peptide (the A-2l978C
`nucleus). The nucleus, or an appropriately substituted
`derivatives of the nucleus, is then reacylated with the desired
`acyl group to give the parent group of cyclic peptides.
`An improved method for preparing the parent group of
`cyclic peptides is described by Floyd M. Huber, Richard L.
`Pieper and Anthony J. Tietz in the copending U.S. patent
`application Ser. No. 773,762, filed Sep. 9, 1985, entitled
`IMPROVED PROCESS FOR A-2l978C DERIVATIVES.
`
`In the parent group described by Abbott et al., one
`particular compound has been found to have especially
`outstanding activity, i.e. the compound wherein the reacy-
`lated side chain is n-decanoyl. This compound has been
`given the designation “LYl46032”.
`The two groups of cyclic peptides of this invention were
`discovered during work with LYl46032. During that work
`we found that the LYl46032 material contained two impu-
`rities. The impurities were more pronounced when
`LYl46032 was in solution in the pH range of 4 to 6. Our
`work led to the isolation of these materials and to the further
`
`discovery that they were closely related to LYl46032. Like
`
`8
`the new compounds also have antibacterial
`LYl46032,
`activity. Identification of the two materials and subsequent
`studies showed that they were formed by a transpeptidation
`reaction. This reaction involves 3 compounds: 1) the starting
`ot-aspartyl peptide (LYl46032), 2) a stable intermediate and
`3) the [3-aspartyl isomer of LYl46032.
`The stable intermediate was found to be the compound of
`formula 1 wherein R is n-decanoyl and R1, R2, R3, R4 and
`R5 are hydrogen. In discussions herein, this compound is
`designated “anhydro-LYl46032”.
`The third compound was found to be the [3-aspartyl
`isomer of LYl46032, i.e. the formula 2 compound wherein
`R is n-decanoyl and R1, R2, R3, R4, and R5 are hydrogen. In
`discussions herein this compound is designated “isomer-
`LYl46032”.
`
`Thus, the formula 1 and 2 compounds are formed by
`aspartyl transpeptidation of the parent cyclic peptides, which
`include LYl46032. The transpeptidation involves two
`distinct, reversible steps: (1) formation of the compounds of
`formula 1 (the anhydro intermediates) from either the parent
`ot-aspartyl peptide or from the formula 2 peptides (the
`[3-aspartyl peptides) and (2) hydrolysis of the intermediate
`formula 1 compounds to either the parent ot-aspartyl pep-
`tides or to the [3-aspartyl peptides of formula 2.
`The mechanism of transpeptidation involves formation of
`a succinimide intermediate, probably through intramolecu-
`lar dehydration of the free carboxyl group of aspartic acid
`and the amino group of the neighboring glycine. This step is
`followed by nucleophilic hydroxide attack of either the (X- or
`[3-carbonyl of the succinimido intermediate which results in
`formation of the corresponding [3- or ot-aspartyl peptide.
`Formation of the [3-aspartyl peptide predominates by a factor
`of 2—3, presumably because of the greater electrophilicity of
`the ot-carbonyl of the succinimide intermediate. The
`transpeptidation reactions are shown in Scheme 1.
`
`Scheme l
`Transpeptidation of A-2l97C Cycle Peptides
`
`Parent
`
`— - - — ->
`
`Formula I
`
`- - — — ->
`
`Formula 2
`
`0L-aspartyl 4- - - - -
`peptide
`
`peptide intermediate 4- - - - -
`(anhydro)
`
`[5-aspartyl peptide
`(isomer)
`
`In the preparation of formula 1 and 2 compounds, a pH
`range of 4—6 is optimum for the transpeptidation reactions.
`At pH levels below 4 and above 6, other degradation
`processes predominate.
`In another aspect, this invention provides an improved
`antibacterial composition comprising the new drug sub-
`stance LYl46032 in substantially pure form. The term “new
`drug substance LYl46032” refers to LYl46032 in bulk
`pharmaceutical form prior to its formulation as a pharma-
`ceutical. The term “in substantially pure form” refers to
`LYl46032 which contains less than 2.5 percent of a com-
`bined total of anhydro-LYl46032 and isomer-LYl46032.
`Previously, the new drug substance LYl46032 contained a
`combined total amount of ar1hydro- and isomer-LYl46032
`in amounts of at least 6 percent.
`The new derivatives of this invention inhibit the growth of
`a broad spectrum of pathogenic bacteria, especially Gram-
`positive bacteria. Table I summarizes the minimal inhibitory
`concentrations (MIC’s) at which the two illustrative
`compounds, anhydro-LYl46032 and isomer-LYl46032,
`inhibit certain organisms, as determined by standard agar-
`dilution assays.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`5of15
`
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`
`

`
`US RE39,071 E
`
`9
`
`TABLE I
`
`Antibacterial Activig of A-21978C Derivatives“
`
`Compound
`
`Isomer-
`LY146032
`
`Anhydro-
`LY146032
`
`8
`8
`8
`8
`8
`2
`8
`128
`32
`
`4
`4
`8
`4
`4
`1
`4
`64
`16
`
`Organism
`
`Slaphylacaccus aureus XL1
`Slaphylacaccus aureus V411’
`Slaphylacaccus aureus X400“
`Slaphylacaccus aureus S13E
`Slaphylacaccus epidermidis 222
`Szrepzacaccus pyagenes C203
`Szrepzacaccus pneumoniae Park 1
`Szrepzacaccus faecalis X66
`Szrepzacaccus faecalis 2041
`Haemaphilus influenzae CL‘!
`Haemaphilus influenzae 76“
`
`“MIC’s in ugmL
`bPenicillin-resistant strain
`°Methicillin-resistant strain
`dAmpicillin-sensitive strain
`“Ampicillin-resistant strain
`f>128
`
`The new derivatives of this invention have also shown in
`vivo antimicrobial activity against experimental bacterial
`infections. When two doses of test compound were admin-
`istered to mice in experimental
`infections,
`the activity
`observed was measured as an EDSO value [effective dose in
`mg/kg to protect 50% of the test animals: see Warren Wick,
`et al.,
`J. Bacteriol. 81, 233-235 (1961)]. EDSO values
`observed for the two illustrative derivatives are given in
`Table II.
`
`TABLE II
`
`Subcutaneous ED50 Values for A-21978C
`Derivatives against Experimental
`Infections Induced by Organ-positive Bacteria
`
`Infecting Organism
`
`Compound
`
`Anhyd.ro—
`LY146032
`Isomer-
`LY146032
`
`Slaphylocaccus
`aueus
`
`Slreploco00143
`pyagenes
`
`Slreplococcus
`pneumaniae
`
`9.05
`10.68
`
`1.15
`<1.25
`
`3.36
`3.40
`
`Pharmaceutical formulations of the compounds of formu-
`las 1, and 2 or their salts and of pharmaceutically purified
`LY146032 or its salts are also part of this invention. The
`compounds, preferably as a pharmaceutically acceptable
`salts, can be formulated for oral or parenteral administration
`for the therapeutic or prophylactic treatment of bacterial
`infections. For example, a compound of this invention can
`be admixed with conventional pharmaceutical carriers and
`excipients and used in the form of tablets, capsules, elixirs,
`suspensions, syrups, wafers and the like. The compositions
`comprising a compound of this invention will contain from
`about 0.1 to about 90% by weight of the active compound,
`and more generally from about 10 to about 30%.
`The compositions may contain common carriers and
`excipients, such as corn starch or gelatin, lactose, sucrose,
`microcrystalline cellulose, kaolin, mannitol, dicalcium
`phosphate, sodium chloride and alginic acid.
`Disintegrators commonly used in the formulations of this
`invention include croscarmellose sodium, microcrystalline
`cellulose, corn starch, sodium starch glycolate and alginic
`acid.
`
`10
`Tablet binders that can be included are acacia,
`methylcellulose, sodium carboxymethylcellulose, polyvi-
`nylpyrrolidone (Povidone), hydroxypropyl methylcellulose,
`sucrose, starch and ethylcellulose.
`Lubricants that can be used include magnesium stearate or
`other metallic stearates, stearic acid, silicone fluid,
`talc,
`waxes, oils and colloidal silica.
`Flavoring agents such as peppermint, oil of wintergreen,
`cherry flavoring or the like can also be used.
`It may be desirable to add a coloring agent to make the
`dosage form more esthetic in appearance or to help identify
`the product.
`For intravenous (IV) use, a water soluble form of the
`compound can be dissolved in one of the commonly used
`intravenous fluids and administered by infusion. Fluids such
`as, for example, physiological saline can be used.
`For intramuscular preparations, a sterile formulation of a
`suitable soluble salt form of the compound, for example the
`hydrochloride salt, can be dissolved and administered in a
`pharmaceutical diluent such as Water-for-Injection, physi-
`ological saline or 5% glucose. A suitable insoluble form of
`the compound may be prepared and administered as a
`suspension in an aqueous base or a pharmaceutically accept-
`able oil base, e.g. an ester of a long chain fatty acid such as
`ethyl oleate.
`For oral use, solid formulations such as tablets and
`capsules are particularly useful. Sustained release or enteri-
`cally coated preparations may also be devised. For pediatric
`and geriatric applications, suspensions, syrups and chewable
`tablets are especially suitable.
`Alternatively, the unit dosage form of the antibiotic can be
`a solution of the compound or preferably a salt thereof in a
`suitable diluent in sterile, hermetically sealed ampoules. The
`concentration of the antibiotic in the unit dosage may vary,
`e.g. from about 1 percent to about 50 percent, depending on
`the compound used and its solubility and the dose desired by
`the physician.
`In a further aspect, this invention provides a method for
`treating infectious diseases, especially those caused by
`Gram-positive bacteria, in animals. The term “treating” is
`used to denote both the prevention of infectious diseases and
`the control of such diseases after the host animal has become
`
`infected. The method comprises administering to the animal
`an effective dose of a compound of this invention. An
`effective dose is generally between about 0.1 and about 100
`mg/kg of the compound or its pharmaceutically acceptable
`salt. A preferred dose is from about 1 to about 30 mg/kg of
`compound. A typical daily dose for an adult human is from
`about 100 mg to about 1.0 g.
`In practicing this method, the antibiotic compound can be
`administered as a single daily dose or in multiple doses per
`day. The treatment regime may require administration over
`extended periods of time, e.g., for several days or for from
`two to four weeks. The amount per administered dose or the
`total amount administered will depend on such factors as the
`nature and severity of the infection, the age and general
`health of the patient, the tolerance of the patient to the
`antibiotic and the microorganism or microorganisms
`involved in the infection.
`
`A convenient method of practicing the treatment method
`is to administer the antibiotic orally, using tablets, capsules,
`suspensions, syrups and the like. The antibiotic may also be
`administered by other methods, e.g. as a suppository or
`parenterally via IV infusion.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6of15
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`6 of 15
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`

`
`11
`
`12
`
`US RE39,071 E
`
`The methods of making and using the compounds of the
`present invention are illustrated in the following nonlimiting
`examples:
`
`EXAMPLE 1
`
`Isolation of t-BOC-Anhydro-LY146032
`The t-BOC derivative of LY146032 which contained
`
`t-BOC-anhydro-LY146032 (1.494 g) was chromatographed
`over an RP C-18 silica-gel prep-pak column (Prep 500 Unit),
`using a gradient containing H20/MeOH/CH3CN with 0.1%
`pyridinium acetate as follows: 8 L of 2/1/1932 L of 1/1/1,
`collecting 250-mL fractions. Fractions were combined based
`on TLC and UV analysis Fractions containing t-BOC-
`anhydro-LY146032 (Nos. 26—31) were combined, concen-
`trated in vacuo to a volume of 100 mL and lyophilized to
`yield 320 mg of the product as a creme powder.
`UV (EtOH): 358 nm (€3,570), 289(e4,910), 257(e8,120),
`222(e45,550);
`FAB-MS: 1724 (Na+P);
`Integer mol. wt.=1701;
`Calc’d mol. wt. for C77H1O7N17O27=1702.76;
`Amino acid analysis: Asp—1.84(4), Thr—0.457 (1),
`SerA).390(1), GlyA).969(2), Ala—0.508(1), 3-MeGlu—
`0.509(1), KynA).421(1), TrpA).395(1), OrnA).557(1),
`nmoles/mg.
`Weakly bioactive (G+).
`
`EXAMPLE 2
`
`Preparation of Anhydro-LY146032
`
`Part of the t-BOC-anhydro-LY146032 (200 mg, 0.12
`mole) prepared in Example 1 was dissolved in trifluoro-
`acetic acid (10 mL) containing anisole (1 mL) at 5° C. After
`5-minutes,
`the mixture was allowed to warm to room
`temperature (~15 min). The tan solution was concentrated in
`vacuo to a syrup, which was triturated with diethyl ether
`(3><50 mL). The brown solids which formed were separated
`by filtration and redissolved in water (10 mL). The pH was
`adjusted to 6.2 with neat pyridine. Filtration and lyophiliza-
`tion gave 190 mg of cream-colored product.
`UV (EtOH): 365 nm (€4,000), 289(e5,000), 255(e8,750),
`222(e47,000).
`FAB-MS: 1602 (P+1);
`This material (175 mg) was further purified by chroma-
`tography over an RP C-18 silica gel prep-pak column (Prep
`500) as described in Example 1. Fractions were combined on
`the basis of TLC and HPLC assays. Fractions containing
`anhydro-LY146032 (Nos. 22—25) were combined, concen-
`trated and lyophilized to give 53 mg of anhydro-LY146032
`as a light creme powder.
`FAB-MS: integer mol wt. of 1602 (P+1)
`
`EXAMPLE 3
`
`Recovery of Isomer-LY146032
`
`Intermediate quality LY146032 (500 g) was dissolved in
`developing solvent (5 L) and filtered through a Super-Cel
`Hyflo pad to remove insolubles.
`The developing solvent was made up as follows:
`65% by volume of 0.5 M sodium acetate in water, then pH
`adjusted to 4.7 with sodium hydroxide.
`25% by volume of acetonitrile
`10% by volume of methanol
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`This solution was applied to a Pharmacia column con-
`taining Diaion HP-20ss resin (18 L, Mitsubishi Chemical)
`which had previously been equilibrated with the developing
`solvent. The column was eluted at a flow-rate of 0.5 colunm
`
`volumes (CV)/hour. The developing solvent was chilled to
`7° C. prior to applying it to the colunm. After initially
`collecting and discarding a large fraction (90 L, 5 CV), 4-L
`fractions were collected and assayed by analytical HPLC.
`On this basis, fractions containing isomer-LY146032 (Nos.
`9, 10, and 11) were pooled and chilled until they were
`desalted, using Diaion HP-20 resin as follows:
`The pooled fractions were diluted (1:1) with an equal
`volume of chilled deionized water. This solution was applied
`to a column of Diaion HP-20 resin (1 L) in the chillroom.
`The column was eluted at a flow-rate of 6 CV/l1r. Efi‘luent
`was collected and discarded. The colunm was then washed
`with 3 CV of chilled, deionized water, which was discarded.
`Isomer-LY146032 was then eluted from the column with 5
`
`CV of a solution containing 60% acetonitrile and 40%
`chilled, deionized water. The active eluate was concentrated
`and freeze-dried to give 4.4 g of an enriched, desalted
`preparation of isomer-LY146032.
`This preparation was further purified using a reverse-
`phase C18 column,
`followed by a Diaion HP-20 resin
`colunm in reverse mode operation. The material was dis-
`solved in a developing solvent (200 mL) which consisted of
`27% acetonitrile, 28% methanol with 1% acetic acid
`adjusted to pH 4.8 with sodium hydroxide. This solution was
`applied to a Chromatospac 100 column containing reverse-
`phase C18 silica gel (4 L). The column was eluted at a
`flow-rate of 1 CV/l1r, collecting 500-mL fractions and assay-
`ing by analytical HPLC. Fractions containing isomer-
`LY146032 (Nos. 14, 15, 16 and 17) were combined.
`This pooled fraction was further purified by diluting it
`with an equal volume of chilled, deionized water and
`adsorbing it onto Diaion HP-20 resin (100 mL) in a batch
`mode after adjusting the pH to 3.5 with sulfuric acid. After
`the mixture was stirred for at least one hour in the chillroom,
`the effluent was removed by filtration and discarded. The
`charged resin was washed with 2 volumes of chilled, deion-
`ized water, filtering and discarding the wash water. The
`charged resin (100 mL) was slowly added to 10 volumes of
`acetonitrile while stirring;
`then the hydrated resin was
`packed into a column. The resin column was then washed
`with 2.5 CV of a developing solvent consisting of 95%
`acetonitrile and 5% chilled, deionized water. The colunm
`was eluted first with 2.5 colunm volumes of 90% acetonitrile
`
`and 10% chilled, deionized water, collecting five 50-mL
`fractions, and then with 2.5 CV of 85% acetonitrile and 15%
`chilled, deionized water, collecting 50-mL fractions. The
`fractions were assayed by analytical HPLC. The fractions
`containing isomer-LY146032 were combined, concentrated
`under vacuum and freeze-dried to give 470 mg of purified
`isomer-LY146032.
`
`EXAMPLE 4
`
`Preparing New Drug Substance LY146032 in Pure
`Form
`
`New drug substance LY146032 in pure form is prepared
`by purifying LY146032 using procedures like those in
`Examples 1—3 so that the substance contains no more than
`2.5% by weight of a combined total of ar1hydro-LY146032
`and isomer-LY146032.
`
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`7 of 15
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`

`
`US RE39,07l E
`
`13
`EXAMPLE 5
`
`HPLC System
`
`The following analytical high performance liquid chro-
`matography (HPLC) system is useful for following the
`processes of Examples l—3 and for preparing new drug
`substance LYl46032 in substantially purified form:
`
`Analytical HPLC System
`Column: Zorbax C8 150A (DuPont)
`Solvent: 32% CH3CN/68% H20 containing 0.5%
`(NH4)H2PO4
`Flow Rate: 1.5 mL/min
`
`Detection; UV at 214 nm
`
`In this system, the LYl46032 materials have the following
`approximate retention times:
`
`14
`-continued
`
`0
`
`g
`
`0H
`
`O
`
`O
`
`O
`
`0
`
`O
`
`g
`
`O
`
`N
`
`R2
`
`0
`
`0
`
`H NJ\/N
`
`H
`
`0
`
`0
`
`HO
`
`10
`
`15
`
`0
`Rl‘1|‘l
`R3
`
`in which R is C5—C14-alkanoyl; R1, R2, R3, R4 and R5 are
`hydrogen; or a pharmaceutically acceptable salt thereof; and
`wherein the alanine is D-alanine and the serine is D-serine.
`2. [A] An isolated compound of the formula 2
`
`FORMULA 2
`
`o
`