WO2002051855A2 - Motilide compounds - Google Patents
Motilide compounds Download PDFInfo
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- WO2002051855A2 WO2002051855A2 PCT/US2001/043963 US0143963W WO02051855A2 WO 2002051855 A2 WO2002051855 A2 WO 2002051855A2 US 0143963 W US0143963 W US 0143963W WO 02051855 A2 WO02051855 A2 WO 02051855A2
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- substituted
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- alkyl
- alkenyl
- alkynyl
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- 0 C[C@](C[C@@]([C@@]1O)N(C)C)O[C@]1O[C@@](CC(*)[C@@](C)C([C@](C)[C@](C(C)(C)C(*)OC([C@]1C)=O)O)=O)[C@@](C)[C@@]1O[C@](C[C@@]1(C)C=O)O[C@@](C)[C@@]1O Chemical compound C[C@](C[C@@]([C@@]1O)N(C)C)O[C@]1O[C@@](CC(*)[C@@](C)C([C@](C)[C@](C(C)(C)C(*)OC([C@]1C)=O)O)=O)[C@@](C)[C@@]1O[C@](C[C@@]1(C)C=O)O[C@@](C)[C@@]1O 0.000 description 9
- OKKBGEUPRZRWPX-UHFFFAOYSA-N CC(C1O)OC(CO)CC1(C)OC Chemical compound CC(C1O)OC(CO)CC1(C)OC OKKBGEUPRZRWPX-UHFFFAOYSA-N 0.000 description 1
- BUNGIJYWWKIIQK-UHFFFAOYSA-N CC(C1OC1(C1)OC)OC1O Chemical compound CC(C1OC1(C1)OC)OC1O BUNGIJYWWKIIQK-UHFFFAOYSA-N 0.000 description 1
- LJXKJTXWXRBULE-UHFFFAOYSA-N CC(CC(C1OC(C)=O)N(C)C)OC1[O]=C Chemical compound CC(CC(C1OC(C)=O)N(C)C)OC1[O]=C LJXKJTXWXRBULE-UHFFFAOYSA-N 0.000 description 1
- NLXHHRRSKQTWLI-YUPNIOSZSA-N CCCC[C@H]([C@](C)(C[C@@H](C)[C@H](C1)[C@H](C)CC1(C)CCC[C@H]1C)O)NC1=O Chemical compound CCCC[C@H]([C@](C)(C[C@@H](C)[C@H](C1)[C@H](C)CC1(C)CCC[C@H]1C)O)NC1=O NLXHHRRSKQTWLI-YUPNIOSZSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/06—Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/10—Laxatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention provides novel prokinetic agents with superior pharmacological and pharmacokinetic properties for the treatment of gastrointestinal motility disorders.
- the invention relates to the fields of chemistry, medicinal chemistry, medicine, molecular biology, and pharmacology.
- Gastrointestinal motility regulates the orderly movement of ingested material through the gut to insure adequate absorption of nutrients, electrolytes and fluids. Appropriate transit through the esophagus, stomach, small intestine and colon depends on regional control of intraluminal pressure and several sphincters that regulate forward movement and prevent back-flow of GI contents. The normal GI motility pattern may be impaired by a variety of circumstances including disease and surgery.
- Gastroparesis is the delayed emptying of stomach contents. Symptoms of gastroparesis include stomach upset, heartburn, nausea, and vomiting. Acute gastroparesis may be caused by, for example, drugs (e.g., opiates), viral enteritis, and hyperglycemia, and is usually managed by treating the underlying disease rather than the motility disorder. The most common causes of chronic gastroparesis are associated with long standing diabetes or idiopathic pseudo-obstruction, often with so- called "non-ulcer" or "functional" dyspepsia.
- GERD refers to the varied clinical manifestations of reflux of stomach and duodenal contents into the esophagus. The most common symptoms are heartburn and dysphasia; blood loss may also occur from esophageal erosion. GERD may be associated with low tone and inappropriate relaxation of the lower esophageal sphincter and occurs with gastroparesis in about 40% of cases. In most cases, GERD appears to be treatable with agents that reduce the release of acidic irritant by the stomach (e.g., Prilosec) or agents that increase the tone of the lower esophageal sphincter (e.g., cisapride).
- agents that reduce the release of acidic irritant by the stomach e.g., Prilosec
- agents that increase the tone of the lower esophageal sphincter e.g., cisapride.
- disorders whose symptoms include impaired gastrointestinal motility are anorexia, gall bladder stasis, postoperative paralytic ileus, scleroderma, intestinal pseudoobstruction, gastritis, emesis, and chronic constipation (colonic inertia).
- MMC Migrating Motor Complexes
- erythromycin A 1 undergoes an acid catalyzed rearrangement in the stomach to form the enol ether 2 which is then further degraded into the spiroketal 3.
- Both erythromycin A and the enol ether are motilin agonists but the spiroketal is not. Because the enol ether is approximately ten fold more potent as a motilin agonist than erythromycin A and does not also posses antimicrobial activity, the potential clinical uses of enol ether derivatives as prokinetic agents are being investigated.
- Enol ether erythromycin derivatives under clinical investigation include EM-523 (4); EM- 574 (5); LY267,108 (6); GM-611 (7); and ABT-229 (8) whose structures are shown below. See U.S. Patent Nos. 5,578,579; 5,658,888; 5,922,849; 6,077,943; and 6,084,079 which are all incorporated herein by reference.
- lactam enol ethers and lactam epoxide derivatives.
- lactam compounds include A-81648 (9) and A- 173508 (10) whose structures are shown below. See also U.S. Patent Nos. 5,712,253; 5,523,401; 5,523,418; 5,538,961; 5,554,605 which are incorporated herein by reference.
- these and other previously disclosed macrolides are synthetically accessible compounds that are derived from erythromycin A or B. Because nature has not optimized the erythromycin structure for its prokinetic activity, it is likely that the potency of motilide agonists could be greatly enhanced. Compounds resulting from such efforts could be of significant benefit in the treatment of wide variety of diseases and conditions. The present invention provides such compounds.
- the present invention provides novel macrolide compounds (or intermediates thereto) with superior pharmacological and pharmacokinetic properties for the treatment of gastrointestinal disorders where enhanced GI motiliy is indicated or desired.
- the compounds of the present invention typically are derived from "unnatural" erythromycins and generally differ from naturally occurring erythromycins A, B, C, and D by having a non-ethyl group (a group that is not -CH CH 3 ) or a substituted ethyl at C-13 and/or by having a hydrogen instead of a methyl group at C-6 (C-6 desmethyl compounds).
- inventive compounds contain one or more chiral centers. Unless indicated otherwise, all of the stereoisomers of a depicted structure are included within the scope of the invention, as pure compounds as well as mixtures of stereoisomers. Similarly, all geometric isomers are also included within the scope of the invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
- the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts.”
- Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts.
- Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
- suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
- alkali metal salts e.g., sodium or potassium salts
- alkaline earth metal salts e.g., calcium or magnesium salts
- suitable organic ligands e.g., quaternary ammonium salts.
- representative pharmaceutically acceptable salts include the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N- methylglucamine ammonium salt, oleate
- the present invention includes within its scope prodrugs of the compounds of this invention.
- prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound.
- the term “administering” shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
- Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.
- alkyl or “unsubstituted alkyl” refers to a straight, branched or cyclic hydrocarbon.
- alkenyl or “unsubstituted alkenyl” refers to a straight, branched, or cyclic chain hydrocarbon with at least one carbon-carbon double bond.
- Alkynyl or
- unsubstituted alkynyl refers to a straight, branched, or cyclic hydrocarbon with at least one carbon-carbon triple bound.
- Substituted alkyl, substituted alkenyl, or substituted alkynyl refer to the respective alkyl, alkenyl or alkynyl group substituted by one or more substituents.
- aryl or "unsubstituted aryl” refers to an aromatic ring having 6 to 12 carbon atoms and includes heteroaryls (aryls that have one or more heteroatoms such as N, S and O).
- aryl include but are not limited to biphenyl, furyl, imidazolyl, indolyl, isoquinolyl, naphthyl, oxazolyl, phenyl, pyridyl, pyrryl, quinolyl, quinoxalyl, tetrazoyl, thiazoyl, thienyl and the like.
- Substituted aryl refers to an aryl group substituted by, for example, one to four substituents such as substituted and unsubstituted alkyl, alkenyl, alkynyl, and aryl; halo; trifluoromethoxy; trifluoromethyl; hydroxy; alkoxy; cycloalkyloxy; heterocyclooxy; alkanoyl; alkanoyloxy; amino; alkylamino; aralkylamino; cycloalkylamino; heterocycloamino; dialkylamino; alkanoylamino; thio; alkylthio; cycloalkylthio; heterocyclothio; ureido; nitro; cyano; carboxy; carboxyalkyl; carbamyl; alkoxycarbonyl; alkylthiono; arylthiono; alkylsulfonyl; sulfonamido; aryloxy; and
- alkylaryl or "arylalkyl” (or “unsubstituted alkylaryl or “unsubstituted arylalkyl) refer to an aryl group bonded directly through an alkyl group, such as benzyl.
- alkenylaryl and arylalkenyl refer to an aryl group bonded directly through an alkenyl group
- alkynylaryl and arylalkynyl refer to an aryl group bonded directly through an alkynyl group.
- Substituted counterparts of these moieties are the respective moiety that is substituted by one or more substituents.
- halogen refers to fluorine, chlorine, bromine and iodine.
- erythromycin refers to a compound of the formula
- R°, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 8 are as described herein and derivatives and analogs thereof.
- hydroxyl groups in the compounds of the present invention may optionally be protected with a hydroxyl protecting group.
- hydroxy protecting group refers to groups known in the art for such purpose. Commonly used hydroxy protecting groups are disclosed, for example, in T. H. Greene and P.G. M. Wuts, Protective Groups in Organic Synthesis, 2 nd edition, John Wiley & Sons, New York (1991), which is incorporated herein by reference.
- hydroxyl protecting groups include but not limited to tetrahydropyranyl; benzyl; methylthiomethyl; ethythiomethyl; pivaloyl; phenylsulfonyl; triphenylmethyl; trisubstituted silyl such as trimethyl silyl, triethylsilyl, tributylsilyl, tri- isopropylsilyl, t-butyldimethylsilyl, tri-t-butylsilyl, methyldiphenylsilyl, ethyldiphenylsilyl, t-butyldiphenylsilyl and the like; acyl and aroyl such as acetyl, pivaloylbenzoyl, 4- methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryl and the like. Hydroxyl protected versions of the inventive compounds are also encompassed within the scope of the present invention.
- the inventive compounds may include other substitutions where applicable.
- the erythromycin backbone or backbone substituents may be additionally substituted (e.g., by replacing one of the hydrogens or by derivatizing a non-hydrogen group) with one or more substituents such as C ⁇ -C 5 alkyl, C ⁇ -C 5 alkoxy, phenyl, or a functional group.
- suitable functional groups include but are not limited to alcohol, sulfonic acid, phosphine, phosphonate, phosphonic acid, thiol, ketone, aldehyde, ester, ether, amine, quaternary ammonium, imine, amide, imide, imido, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, carbamate, acetal, ketal, boronate, cyanohydrin, hydrazone, oxime, hydrazide, enamine, sulfone, sulfide, sulfenyl, and halogen.
- subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
- terapéuticaally effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
- composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
- R is hydrogen, substituted Ci-Cio alkyl, unsubstituted Ci-Cio alkyl, substituted C 2 - Cio alkenyl, unsubstituted C 2 -C ⁇ o alkenyl, substituted C2-C10 alkynyl, unsubstituted C 2 -C ⁇ o alkynyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl;
- R° is hydroxyl or methoxy
- R 1 is selected from the group consisting of hydrogen, hydroxyl, halide, NH 2 , OR 9 , o o o o
- R 2 and R 3 are each independently selected from the group consisting of hydrogen, substituted Ci-Cio alkyl, unsubstituted C1-C 10 alkyl, substituted C 2 - 0 alkenyl, unsubstituted C2- 0 alkenyl, substituted C2-C1 0 alkynyl, unsubstituted C2-C10 alkynyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, and unsubstituted alkynylaryl, or R and R together form a cycloalkyl or an aryl moiety;
- R 4 is hydrogen or methyl;
- R 5 is hydroxyl or oxo;
- R 6 is hydrogen, hydroxyl or OR 12 where R 12 is substituted C 1 -C 10 alkyl, unsubstituted C1-C 10 alkyl, substituted C 2 -C ⁇ o alkenyl, unsubstituted C 2 -C1 0 alkenyl, substituted C2-C 10 alkynyl, or unsubstituted C2-C 10 alkynyl;
- R 7 is methyl, unsubstituted C 3 -C ⁇ o alkyl, substituted C 1 -C 10 alkyl, substituted C 2 -C 10 alkenyl, unsubstituted C 2 -C10 alkenyl, substituted C 2 -C 10 alkynyl, unsubstituted C 2 -C ⁇ o alkynyl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl;
- R 8 is unsubstituted C ⁇ -C ⁇ 0 alkyl, substituted Ci-Cio alkyl, substituted C 2 -C ⁇ 0 alkenyl, unsubstituted C 2 -C ⁇ o alkenyl, substituted C 2 -C 10 alkynyl, unsubstituted C 2 -C ⁇ 0 al
- compounds are provided of structure I wherein the C-8 carbon is in the R configuration.
- compounds are provided of structure I wherein the C-9 carbon is in the R configuration.
- compounds are provided of structure I wherein the C-8 and C-9 carbons are both in the R configuration.
- R is hydrogen, substituted C ⁇ -C 5 alkyl, unsubstituted C ⁇ -C 5 alkyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, or unsubstituted alkylaryl;
- R° is hydroxyl or methoxy;
- R is hydrogen or hydroxyl;
- R and R are each independently substituted C ⁇ -C 5 alkyl, unsubstituted C 1 -C 5 alkyl, substituted phenyl, unsubstituted phenyl, substituted benzyl or unsubstituted benzyl;
- R 4 is methyl;
- R 5 is hydroxyl or oxo;
- R 6 is hydrogen, hydroxyl or OR 12 wherein R 12 is substituted C ⁇ -C 5 alkyl or unsubstituted C ⁇ -C 5 alkyl;
- R 7 is substituted methyl, unsubstituted methyl, substituted C 3 -
- R is hydrogen, C ⁇ -C 5 alkyl, aryl, or alkylaryl; R° is hydroxyl or methoxy; R 1 is hydrogen or hydroxyl; R 2 and R 3 are each independently C ⁇ -C 5 alkyl, phenyl or benzyl; R 4 is methyl; R 5 is hydroxyl or oxo; R 6 is hydrogen, hydroxyl or methoxy; R 7 and R 8 are amidoalkylaryl.
- R is hydrogen, methyl, ethyl, propyl, isopropyl, phenyl or benzyl;
- R° is hydroxyl or methoxy;
- R 1 is hydrogen or hydroxyl;
- R 2 is methyl;
- R 3 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl or tertbutyl;
- R 4 is methyl;
- R 5 is hydroxyl;
- R 6 is hydroxyl or methoxy;
- R 7 is methyl, vinyl, propyl, isobutyl, pentyl, prop-2-enyl, propargyl, but-3- enyl, 2-azidoethyl, 2-fluoroethyl, 2-chloroethyl, cyclohexyl, phenyl, or benzyl;
- R 8 is methyl, ethyl vinyl, propyl, iso
- R is hydrogen; R° is methoxy; R 1 is hydrogen or hydroxyl; R 2 is methyl; R 3 is methyl, ethyl, or isopropyl; R 4 is methyl; R 5 is hydroxyl; R 6 is hydroxyl; R 7 is propyl, but- 3-enyl, 2-azidoethyl, phenyl, or benzyl; R 8 is ethyl, propyl, but-3-enyl, 2-azidoethyl, phenyl, or benzyl; and, x is a single or a double bond.
- R 1 is hydrogen or hydroxyl
- R 3 is methyl, ethyl, or isopropyl
- R 7 is propyl or fluoroethyl
- R 8 is ethyl, fluoroethyl, or propyl.
- Y is hydrogen, substituted C1-C10 alkyl, unsubstituted C1-C1 0 alkyl, substituted C 2 - Cio alkenyl, unsubstituted C2-C10 alkenyl, substituted C2-C10 alkynyl, unsubstituted C2-C 10 alkynyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, unsubstituted alkynylaryl, unsubstituted cladinose, or substituted cladinose;
- R 3 is hydrogen, substituted C1-C10 alkyl, unsubstituted Ci-Cio alkyl, substituted C 2 - Cio alkenyl, unsubstituted C2-C10 alkenyl, substituted C 2 -C 10 alkynyl, unsubstituted C2-C1 0 alkynyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl;
- R 5 is hydroxyl or oxo;
- R 6 is hydrogen, hydroxyl or OR 12 where R 12 is substituted Ci-Cio alkyl, unsubstituted Ci-Cio alkyl, substituted C 2 -C ⁇ o alkenyl, unsubstituted C2-C1 0 alkenyl, substituted C 2 -C 10 alkynyl, or unsubstituted C2-C10 alkynyl; R 7 is methyl, unsubstituted C 3 -C ⁇ o alkyl, substituted C1-C1 0 alkyl, substituted C 2 -C 10 alkenyl, unsubstituted C 2 -C 10 alkenyl, substituted C2-C 10 alkynyl, unsubstituted C 2 -C 10 alkynyl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted al
- R 13 is hydrogen, unsubstituted -C1 0 alkyl, substituted C 1 -C 10 alkyl, substituted C 2 - Cio alkenyl, unsubstituted C 2 -C1 0 alkenyl, substituted C 2 -C 10 alkynyl, unsubstituted C 2 -C 10 alkynyl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl; and,
- R 17 is hydrogen or methyl.
- R 3 is substituted C 3 -C ⁇ o alkyl, unsubstituted C 3 -C ⁇ o alkyl, substituted C 4 -C ⁇ o alkenyl, unsubstituted C 4 -C ⁇ o alkenyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, or unsubstituted alkenylaryl;
- R 5 and R 6 are both hydroxyl;
- R 7 is propyl or fluoroethyl
- R 17 is hydrogen or methyl
- Y is cladinose, 4-acyl-cladinose, 4-sulfonyl-cladinose, or 4-carbamoyl-cladinose.
- R 3 is hydrogen, substituted -Cio alkyl, unsubstituted Ci-Cio alkyl, substituted C2- C 10 alkenyl, unsubstituted C2-C 10 alkenyl, substituted C 2 -C1 0 alkynyl, unsubstituted C2-C 10 alkynyl, substituted aryl, unsubstituted aryl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl;
- R 7 is methyl, unsubstituted C 3 -C ⁇ o alkyl, substituted C1-C10 alkyl, substituted C2-C1 0 alkenyl, unsubstituted C2-C1 0 alkenyl, substituted C 2 -C 10 alkynyl, unsubstituted C2-C1 0 alkynyl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl;
- R 13 is hydrogen, unsubstituted C 1 -C 10 alkyl, substituted -C 10 alkyl, substituted C 2 - C 10 alkenyl, unsubstituted C 2 -C 10 alkenyl, substituted C 2 -C 10 alkynyl, unsubstituted C 2 -C1 0 alkynyl, substituted alkylaryl, unsubstituted alkylaryl, substituted alkenylaryl, unsubstituted alkenylaryl, substituted alkynylaryl, or unsubstituted alkynylaryl; and
- R is hydrogen, acyl, sulfonyl or carbamoyl.
- R 7 is propyl or 2-fluoroethyl
- R 13 is
- the compounds of the present invention can be prepared in accordance with the methods of the present invention by a combination of recombinant DNA technology and organic chemistry.
- Recombinant techniques are used to provide, in many instances, "unnatural" erythromycins or erythromycin derivatives that differ in one or more positions from the naturally occurring erythromycins A, B, C, or D.
- a useful starting point is the complete 6-dEB synthase gene cluster that has been cloned in vectors and thus is amenable to genetic mampulations in E. coli and expression of the polyketide in Streptomyces. See U.S. Patent Nos. 5,672,491 ; 5,830,750; 5,843,718; 5,712,146; and 5,962,290 which are all incorporated herein by reference.
- the aglycone is formed, it is next hydroxylated and/or glycoslyated and/or methylated at the appropriate positions by a converter strain that possesses the desired functionalities.
- a particularly useful converter strain is an Saccharopolyspora erythraea eryA mutant that is unable to produce 6-dEB but can still carry out the desired conversions (Weber et al., J. Bacteriol. 164(1): 425-433 (1985).
- This mutant strain is able to take exogenously supplied 6-dEB and process it to erythromycin A by converting it into erythronolide B, 3- ⁇ - mycarosylerythronolide B, erythromycin D, erythromycin C, and finally to erythromycin A.
- erythromycin A An alternative route to erythromycin A is through erythromycin B where exogenously supplied 6-dEB is converted into erythronolide B, 3- ⁇ -mycarosylerythronolide B, erythromycin D, erythromycin B, and finally to erythromycin A.
- Other mutant strain such as eryB, eryC, eryG, and/or eryK mutants, or mutant strains having mutations in multiple genes can be used to make compounds having any combinations of hydroxylations at C-6 and C-12, glycosylations at C-3 and C-5, and methylation at C-3"-OH. Any of these products may be used as starting materials for the practice of the present invention.
- the 6- deoxyerythronolide B synthase (“DEBS”) from S. erythraea can be used in a recombinant expression system described in U.S. Patent No. 5,672,491 to produce the aglycone in Streptomyces coelicolor.
- the oleandolide or megalomicin polyketide synthase (“PKS”) genes may be used in this expression system. See U.S. Provisional Patent Application Serial No. 60/158,305 filed October 8, 1999 and utility application Serial No.
- 6-dEB and 6-dEB derivatives such as 13 -substituted 6-dEB are converted into the desired erythromycin starting material by an appropriate converter strain.
- any one of the post PKS products may be used as starting materials such as 13-substituted counterparts (where the ethyl group which normally exists at C-13 is replaced with another substituent) to: erythronolide B, 3- ⁇ -mycarosylerythronolide B, erythromycin D, erythromycin B, erythromycin C, and erythromycin A.
- 13-substituted erythromycin A can be made by fermentation with an eryA mutant that is incapable of producing 6-dEB but can still carry out the desired conversions.
- 13-substituted erythromycin B can be made by fermentation with an eryA mutant that is incapable of producing 6-dEB and in which the ery K (12-hydroxylase) gene has been deleted or otherwise rendered inactive.
- erythromycin B derivatives can be made in a KSl ⁇ ery ⁇ . mutant strain of S. erythaea.
- the general method for using chemobiosynthesis for making modified 6-dEB is illustrated by Example 1 with specific reference to 13-propyl-6-dEB (13-propyl-13- desethyl-6-dEB).
- Example 2 The general method for converting modified 6-dEB compounds to the desired hydroxylated and glycosylated form by using an eryA converter strain is illustrated by Example 2 with specific reference to converting 13-propyl 6-dEB to 13-propyl erythromycin A (13-propyl-13-desethyl-erythromycin A).
- 6-Desmethyl erythromycins a starting material for making the furanyl erythromycins (compounds of formula II or IN) of the present invention, are made by replacing the acyl transferase ("AT") domain of module 4 (encoding a 6-methyl group) of a 6-dEB or 8,8a- deoxyoleandolide synthase with a malonyl specific AT domain (encoding a 6-hydrogen) to provide the 6-desmethyl analog of the erythromycin aglycone.
- AT acyl transferase
- modules 4 encoding a 6-methyl group
- a malonyl specific AT domain encoding a 6-hydrogen
- the AT4 domain of 6-dEB or 8,8a-deoxyoleandolide polyketide synthase is mutated to correspond to AT domains more characteristic of AT domains having malonyl specificity. More particularly, three mutations are made.
- CGC GTC GAC GTG CTC open reading frame encoding AT4
- GAC GAC CTC TAC GCC where bold indicates the altered nucleotide, to change the encoded amino acids from RVDNLQ to DDLYA.
- nucleotides 6316-6318 (CAG) is modified to the sequence CTC to change the encoded amino acid from Q to L.
- nucleotides 6613-6621 (TAC GCC TCC) is modified to the sequence CAC GCC TTC to change the encoded amino acids from YAS to HAF.
- the resulting aglycone is bioconverted to 6-desmethyl erythromycin as described above although some modification for C-6 hydroxylation may be required.
- 6-hydroxy-erythromycin (where the methyl at C-6 has been replaced with a hydroxyl group)
- 6-oxo erythromycin where the methyl at C-6 has been replaced with an oxo group
- 6-methoxy erythromycin where the methyl at C-6 has been replaced with a methoxy group
- 6-desmethyl, 7-hydroxy-erythromycin 6-OH, 6-OMe erythromcyins are made by replacing AT4 of 6-dEB or 8,8a- deoxyoleandolide synthase with an AT domain encoding hydroxymalonate or methoxymalonate. See PCT Publication WO 00/20601 which is incorporated herein by reference.
- the 6-OH and 6-OMe aglycone is bioconverted to 6-desmethyl-6-hydroxy erythromycin and 6-desmethyl-6-methoxy erythromycin respectively by fermentation with an appropriate eryA mutant that is incapable of producing 6-dEB and in which the eryF (C- 6 hydroxylase) function has been deleted or otherwise inactivated. Fermentation of 6-OH or 6-OMe aglycone with an eryA mutant that possesses eryF (or equivalent) function leads to the 6-desmethyl-6-oxo erythromycin.
- 6-desmethyl, 7-hydroxy erythromycins are made by replacing AT4 of a 6-dEB or 8,8a-deoxyoleandolide polyketide synthase with a malonyl specific AT as described above as well as deleting or otherwise inactivating the dehydratase activity of module 3 ("DH3").
- DH3 dehydratase activity of module 3
- the resulting 6-desmethyl, 7-hydroxy aglycone is converted into the corresponding erythromycin derivative by fermentation with an appropriate eryA mutant that is incapable of producing 6-dEB as described above.
- intermediate compounds that are subsequently converted to the corresponding lactams.
- These intermediate compounds include the 6,9-enol-ether, 6,9-epoxide, and furanyl erythromycins.
- the 6,9-enol ether erythromycins are also referred to as 8,9-anhydro erythromycin 6,9-enol ethers, enol ethers or dihydrofurans.
- the 6,9-epoxides are also referred to as epoxides or tetrahydrofurans.
- Scheme 1 A illustrates one embodiment for making the enol ether and epoxide compounds from erythromycin A derivatives (where R is as previously described).
- Enol ether compounds 12 are formed by treating with mild acid the desired erythromycin starting material such as 11.
- the corresponding epoxide 13 is formed by reducing the carbon-carbon double bond between C-8 and C-9 of the enol ether 12.
- Scheme IB illustrates another embodiment for making epoxide 13.
- the free hydroxyls of erythromycin 11 are protected and the C-9 oxo is reduced with sodium borohydride to a 9-dihydro erythromycin intermediate 14 (where C-9 is -CHOH-).
- suitable protecting groups include acetyl for the C-2', a carbonate ester such as Troc, Cbz or Boc for C-4" hydroxyls and a cyclic carbonate for the C-l 1 and C-12 hydroxyls.
- the hydroxyl group at C-9 of compound 14 is subsequently activated and displaced to form epoxide 13.
- the epoxide is formed by treatment with triflic anhydride and pyridine.
- Furanyl erythromycins may be prepared using several different strategies.
- furanyl erythromycins are prepared synthetically by demethylating the naturally occurring methyl group at C-6.
- a suitably protected erythromycin is converted to the 6-O-xanthate via reaction with carbon disulfide and methyl iodide, and the xanthate is pyrolyzed to yield 6,6a-anhydroerythromycin.
- Ozonolysis yields the 6-oxo- erythromycin, which can be converted to the 6,9-epoxide by dehydration from treatment with mild acid or acetic anhydride.
- the 6-oxo-erythromycin may be prepared recombinantly as described previously.
- Scheme 2 illustrates another embodiment using 6- desmethyl erythromycins. SCHEME 2
- 6-Desmethyl erythromycin 15 (where R' and R 6 are hydrogen or hydroxyl and R 8 is as previously described) is treated with mild acid such as dichloroacetic acid to form enol ether 16. Compound 16 is then treated with a mild oxidizing agent such as bromine in base to yield furanyl erythromycin 17.
- 6-desmethyl-7-hydroxy-8,9- anhydro erythromycin 6,9-hemiacetal is (specific embodiment of compound 16 where R' is hydroxyl) is mesylated and subjected to base-catalyzed elimination to yield furanyl erythromycin 17.
- erythromycin lactams are made from 6,9-enol ethers (17 where x is a double bond) and 6,9- epoxides (17 where x is a single bond) as shown by Scheme 3 A.
- Compound 17 is treated with potassium carbonate in methanol to form the 12 membered derivative 18 which is converted into 12, 13 epoxide 19 by treatment with Martin sulfiirane.
- Compound 19 is reacted with NH2R to form erythromycin lactam 20 (where R is as described previously).
- 6,9 enol ether lactam compounds are 20 where x is a double bond and 6,9-ether lactams are 20 where x is a single bond.
- Furanyl erythromycin 21 (where R 8 is as described previously) is treated with potassium carbonate to form the 12 membered derivative 22 which is converted into 12, 13 epoxide 23 by treatment with Martin sulfurane.
- Compound 23 is reacted with NH 2 R to form furanyl erythromycin lactam 20 (where R is as described previously).
- Lactams 20 and 24 may be made by making the desired modifications either before or after lactam formation. In most cases, the timing of the modifications is based on synthetic convenience.
- methods for making 3'-N-desmethyl-3'N-alkyl lactam compounds are provided.
- One or both of the 3'-N-methyl groups are demethylated and the demethylated 3'- nitrogen is subsequently reacted with a substituted or unsubstituted alkyl or aryl group.
- the 3'-N demethylation and subsequent alkylation (or arylation) may be performed using erythromycins, enol ethers, epoxide and furanyl erythromycins as well as their lactam counterparts.
- Scheme 4 illustrates one embodiment where the demethylation and alkylation reactions are illustrated with respect to 6,9-enol ether 12.
- Enol ether 12 formed from erythromycin 11 as described previously by Scheme 1A, is demethylated at the 3'-N by treatment with light, iodine and sodium acetate. Additional reagents and longer reaction times will remove both methyl groups if desired.
- the demethylated enol ether 25 is then alkylated or arylated with the appropriate alkyl halide or aryl halide to yield compound 26.
- Enol ether 26 may be optionally reduced to form its 6,9 epoxide counterpart using the procedures described by Scheme 1 A. Compound 26 or its epoxide counterparts are used as starting materials for the protocols described in Scheme 3 to make the corresponding 3'-N-desmethyl-3'-N-alkyl lactams.
- 4"-desoxy lactams are provided.
- 4"-desoxy erythromycin is made as described by Scheme 5.
- Erythromycin 11 is acetylated at the 2' hydroxyl to yield compound 26.
- the 2'-O-acetyl erythromycin 26 is then treated with thiocarbonyldiimidazole and 4-dimethylaminopyridine in dichloromethane.
- the resulting product is isolated and treated with tributyltin hydride to yield compound 27.
- the corresponding lactam can be made by using compound 27 as starting materials in the protocols described by Schemes 1 and 3.
- an alternate route for making erythromycin lactam is provided.
- erythromycin 11 is converted into 2'-O-acetyl-9-dihydro- erythromycin A 11 , 12, cyclic carbamate 28 which is transformed into the corresponding 6,9-epoxide.
- the 6,9-epoxide is converted into lactam 29 as previously described in Scheme 3.
- Compound 29 may then be subsequently modified using standard procedures that are known in the art. See e.g. Advanced Organic Chemistry 3rd Ed. by Jerry March (1985) which is incorporated herein by reference.
- the 3'-N-methyl group may be demethylated and subsequently modified by reductive amination to yield compound 30.
- a keto group may be formed at C-11 by protecting the 2', 4" and 12 hydroxyls and oxidizing the C-11 hydroxyl to a ketone.
- erythromycin 11 is converted into the 9-oximinoether 32 using an O-alkyl or O-aryl hydroxylamine.
- oximinoethers 32 can be prepared by alkylation of erythromycin 9-oxime.
- One of the 3' dimethyl groups of the desosamine sugar is then demethylated with iodine to yield compound 33.
- the 2' hydroxyl of the desosamine is protected with a protecting group such as acetyl and the 4" hydroxyl of the cladinose is functionalized using for example, an acid chloride in the presence of a base such as DMAP.
- a protecting group such as acetyl
- the 4" hydroxyl of the cladinose is functionalized using for example, an acid chloride in the presence of a base such as DMAP.
- compound 36 is used in this illustration to make the corresponding 4" modified compound.
- Deprotection with methanol yields the desired product which in this case is compound 37.
- the 10, 11-diol of a compound such as compound 36 in this example, is protected through carbonate exchange with ethylene carbonate and the cladinose moiety is removed by mild acid hydrolysis (0.5 N HCl). If further chemistry is desired at the resulting C-3 hydroxyl, the 2* hydroxyl is transiently protected with a protecting group such as an acetyl group to yield compound 38.
- a protecting group such as an acetyl group
- a suitably protected compound such as 38 can be functionalized through carbamoylation to yield compound 39 or through formation of a mixed acetal to yield compound 40.
- methods of using the compounds of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention.
- disorders that may be treated with the inventive compounds include but are not limited to gastroparesis, gastroesophageal reflux disease, anorexia, gall bladder stasis, postoperative paralytic ileus, scleroderma, intestinal pseudoobstruction, gastritis, emesis, and chronic constipation (colonic inertia).
- the therapeutically effective amount can be expressed as a total daily dose of the compound or compounds of this invention and may be administered to a subject in a single or in divided doses.
- the total daily dose can be in amounts, for example, of from about 0.01 to about 25 mg/kg body weight, or more usually, from about 0.1 to about 15 mg/kg body weight.
- Single dose compositions may contain such amounts or submultiples thereof as to make up the daily dose.
- treatment regimens according to the present invention comprise administration to a subject in need of such treatment of from about 10 mg to about 1000 mg of the compound(s) of the present invention per day in single or multiple doses.
- the inventive compound will be part of a pharmaceutical composition or preparation which may be in any suitable form such as solid, semisolid, or liquid form.
- the pharmaceutical preparation will contain one or more of the compounds of the invention as an active ingredient and a pharmaceutically acceptable carrier.
- the active ingredient is in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application.
- the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use.
- Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference.
- the carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form.
- auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
- the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U. S . Patent No. 4,916, 138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference.
- Medium 1 comprises 45 g/L cornstarch; 10 g/L corn steep liquor; 10 g/L dried, inactivated brewers yeast; and 1 g/L CaCO 3 .
- This solution is sterilized by autoclaving for 90 minutes at 121°C. After sterizilization, 1 mL/L of sterile filtered 50 mg/ml thiostrepton in 100% DMSO and 1 mL/L autoclaved 100% antifoam B silicon emulsion (J.T. Baker) are added prior to use.
- the flask is placed in an incubator/shaker maintained at 30 ⁇ 1°C and 175 ⁇ 25 RPM for 48 ⁇ 10 hours.
- the 50 mL culture is then added to a 2.8 L baffled flask containing 500 mL of Medium 1.
- This flask is incubated in an incubator/shaker at 30 ⁇ 1°C and 175 ⁇ 25 RPM for 48 ⁇ 10 hours.
- the 500 mL culture is than used to inoculate a 10 L fermenter containing 5 L of Medium 1.
- the fermenter is controlled at 30°C, pH 6.5 by addition of 2.5 N H SO 4 and 2.5 N NaOH, agitation rate 600 RPM, and air flow rate 1-2 LPM.
- Foam is controlled by the addition of a 50% solution of Antifoam B as needed.
- the fermenter culture is allowed to grow under these conditions for 24 ⁇ 5 hours.
- a 150 L fermenter is prepared by sterilizing 100 L of Medium 1 at 121°C for 45 minutes. After the growth period, the contents from the 10 L fermenter are aseptically added to a 150 L fermenter.
- the fermenter is controlled at 30°C, pH 6.5 by addition of 2.5 N H 2 SO 4 and 2.5 N NaOH, dissolved oxygen > 80% air saturation by agitation rate (500-700 RPM), air flow rate (10-50 LPM), and/or back pressure control (0.1-0.4 bar).
- Foam is controlled by the addition of a 50% solution of Antifoam B as needed.
- (2S, 3R)-2-methyl-3-hydroxypentanoyl-N- acetylcysteamine is added to a final concentration of 2 g/L.
- Propyl diketide is prepared by solubolizing in dimethyl sulfoxide at a ratio of 2:3 (diketide to DMSO) and then filter sterilized (0.2 ⁇ m, nylon filter).
- Production of 13-propyl-6- deoxyerythonolide B (13-propyl-6dEB) ceases on day 8 and the fermenter is harvested.
- the fermentation broth is centrifuged at 20,500 g in an Alpha Laval AS-26 centrifuge. The product is predominantly in the centrate; the centrifuged cell mass is discarded.
- HP20 resin Mitsubishi resin (Mitsubishi). Column size is selected based on centrate volume and titer, so that the loading capacity of 15 g 13-propyl-6-dEB per liter HP20 resin is not exceeded.
- the centrifuged broth is passed through the resin bed at a linear flow rate of 300+20 cm/h. The pressure on the column should not exceed 15 psi.
- the resin is then washed with 2 column volumes (CN) of water and then 2 CV of 30% methanol, each at a rate of 300 ⁇ 20 cm/h. 13-propyl-6dEB is eluted using 7-10 CN 100% methanol at a rate of 300+20 cm/h.
- fractions of X A CV are collected.
- the fractions are then analyzed, and those containing product are combined to yield a product pool containing >95% of the original 13-propyl-6-dEB in the centrifuged broth.
- the product pool is reduced to solids using rotary evaporation.
- Product purity at this stage is 5-35%.
- Methanol-insoluble material is removed from the product pool by suspending the solids in 3 L 100% methanol per 100 L original broth volume, mixing for 20 minutes, and filtering.
- the final purification step is chromatography using HP20SS resin (Mitsubishi). Column size is selected based on amount of product, so that the loading capacity of 15 g 13-propyl- 6-dEB per liter HP20SS resin is not exceeded.
- the filtered methanol solution is diluted by adding an equal volume of water.
- the 50% methanol solution is passed through the resin bed at a linear flow rate of 300 ⁇ 20 cm/h.
- the column is then washed with 2 CV of 50% methanol at a rate of 300 ⁇ 20 cm/h.
- Product is eluted using 12 CV 70% methanol at a rate of 300 ⁇ 20 cm/h. During elution, fractions of 1 CV are collected.
- a 1 mL vial from working cell bank K39-14V (an eryA mutant of S. erythraea that is incapable of producing 6-dEB) is thawed and the contents of the vial are added to 50 mL of Medium 2 in a 250 mL baffled flask.
- Medium 2 comprises 16 g/L cornstarch; 10 g/L corn dextrin; 15 g/L soy meal flour; 4 g/L CaCO 3 ; 5 g/L corn steep liquor; 6 g/L soybean oil; 2.5 g/L NaCl; and 1 g/L
- This solution is sterilized by autoclaving for 60 minutes at 121 °C and 1 mL/L autoclaved 100% antifoam B silicon emulsion (J.T. Baker) is added prior to use.
- the flask is placed in an incubator/shaker maintained at 34 ⁇ 1°C and 175 ⁇ 25 RPM for 48 ⁇ 10 hours.
- the 50 mL culture is then added to a 2.8 L baffled flask containing 500 mL of Medium 2.
- the flask is incubated in an incubator/shaker at 34+1 °C and 175 ⁇ 25 RPM for 48 ⁇ 10 hours.
- the 500 mL culture is than used to inoculate a 10 L fermenter containing 5 L of Medium 2.
- the fermenter is controlled at 34°C, pH 7.0 by addition of 2.5 N H 2 SO 4 and 2.5 N NaOH, agitation rate 600 RPM, and air flow rate 1-2 LPM.
- Foam is controlled by the addition of a 50% solution of Antifoam B as needed.
- the fermenter culture is allowed to grow under these conditions for 24+5 hours.
- a 150 L fermenter is prepared by sterilizing 100 L of Medium 3 at 121°C for 45 minutes.
- Medium 3 comprises 17.5 g/L cornstarch; 16 g/L corn dextrin; 16.5 g/L soy meal flour; 4 g/L CaCO 3 ; 6 g L corn steep liquor; 3 g/L soybean oil; 3.5 g/L NaCl; and 1 g/L
- the contents from the 10 L fermenter are aseptically transferred to the 150 L fermenter.
- the fermenter is controlled at 34°C, pH 7.0 by addition of 2.5 N H 2 SO 4 and 2.5 N NaOH, dissolved oxygen > 80% air saturation by agitation rate (500-700 RPM), air flow rate (15-50 LPM), and/or back pressure control (0.1-0.4 bar).
- Foam is controlled by the addition of a 50% solution of Antifoam B.
- a 58-60 mL/hour 15% dextrin (w/v) feed is initiated.
- the dextrin solution is continuously mixed during the feed period.
- 25 grams of 13-propyl-6dEB are added to the fermenter.
- the 13-propyl-6dEB is prepared by solubolizing 25 grams of 13-propyl-6dEB in 400-600 mL of 100% ethanol and filtering (0.2 ⁇ m, nylon filter). Conversion of 13-propyl-6dEB to 13-propyl-erythromycin A ceases after 60 ⁇ 10 hours and the fermenter is harvested.
- the fermentation broth is centrifuged at 20,500 g in an Alpha Laval AS-26 centrifuge. The product is predominantly in the centrate; the centrifuged cell mass is discarded.
- HP20 resin Mitsubishi resin (Mitsubishi). Column size is selected based on centrate volume and titer, so that the loading capacity of 15 g 13-propyl-erythromycin A per liter HP20 resin is not exceeded.
- the centrifuged broth is adjusted to pH 9, then passed through the resin bed at a linear flow rate of 275 ⁇ 25 cm/h. The pressure on the column should not exceed 15 psi.
- the resin is then washed with 1 column volume (CV) of water at a rate of 275+25 cm/h. 13-propyl-6dEB is eluted using 5 CV 100% methanol at a rate of 275+25 cm/h. During elution, fractions of 1 CV are collected. The fractions are then analyzed, and those containing product are combined to yield a product pool. The product pool is reduced to solids using rotary evaporation.
- Methanol-insoluble material is removed from the product pool by suspending the solids in 1L 100% methanol per 100 L original broth volume, adjusting to pH 9, and filtering.
- the product pool (filtrate) is reduced to solids using rotary evaporation.
- 13-propyl-erythromycin A is extracted from the product pool (solids) by adding 2 L 4:1 hexane: acetone per 100 L original broth volume, mixing for 20 minutes, and filtering. The remaining solids are extracted the same way two more times and filtrates are combined. The product pool is reduced to solids using rotary evaporation.
- the final purification step is chromatography using HP20SS resin (Mitsubishi). Column size is selected based on amount of product, so that the loading capacity of 15 g 13-propyl erythromycin A per liter HP20SS resin is not exceeded.
- the solids from the previous steps are dissolved in 1 L methanol per 100 L original broth volume, and an equal volume of water is added.
- the 50% methanol solution is passed through the resin bed at a linear flow rate of 275+25 cm/h.
- the column is then washed with 1 CV of 50% methanol, then 3 CV 60% methanol, each at a rate of 275 ⁇ 25 cm/h.
- Product is eluted using 3 CV 70% methanol, then 10 CV 75% methanol, each at a rate of 275 ⁇ 25 cm/h. During elution, fractions of V 2 CV are collected. The fractions are then analyzed, and those containing 13-propyl- erythromycin A are combined. The product pool is reduced to solids using rotary evaporation.
- An exemplary NMR data for one of the compounds of the present invention, 8,9- anhydroerythromycin A 6,9-hemiacetal is as follows. ,3 C-NMR (CDC1 3 ): ⁇ 178.2, 151.7, 102.9, 101.4, 94.6, 85.5, 80.1, 78.2, 78.1, 76.3, 75.3, 73.0, 70.8, 70.1, 68.8, 65.8, 65.6, 49.5, 44.7, 43.2, 42.6, 40.3, 34.6, 30.5, 28.7, 26.2, 21.5, 21.3, 21.0, 18.2, 16.1, 15.0, 13.4, 11.9, 11.4, 10.8, 8.6.
- the 3'-N-desmethyl-8,9-anhydroerythromycin 6,9-hemiacetals are prepared by substituting the 8,9-anhydroerythromycin 6,9-hemiacetals for the erythromycin in the above procedure.
- Alkylating agents useful in this procedure include ethyl iodide, benzyl bromide, 2- iodopropane, 4-bromo-l-butene, allyl bromide, propargyl bromide, or sec-butyl iodide, or the corresponding trifluoromethanesulfonates, which give rise to the 3'-N-ethyl, isopropyl, butenyl, allyl, propargyl, or -fee-butyl derivatives, respectively.
- 3'-N-desmethyl-3'-N-alkyl-8,9-anhydroerythromycin 6,9-hemiacetal is prepared by substituting 3'-N-desmethyl-8,9-anhydroerythromycin 6,9-hemiacetal (Example 7) for the 3'-N-desmethyl-erythromycin in the above procedure.
- Step 1 A mixture of 2'-O-acetyl-erythromycin (3.5 mmol; Example 8), thiocarbonyldiimidazole (1 g), and 4-dimethylaminopyridine (0.67 g) in 100 mL of CH2CI2 is stirred overnight at ambient temperature. The mixture is treated with 150 mL of sat. NaHCO , and the organic phase is then washed with water, dried over MgSO , filtered, and evaporated. The product 4"-O-thiocarbonylimidazolide is crystallized. Step 2. The product from Step 1 is dissolved in 60 mL of toluene and heated to 98 °C.
- Tributyltin hydride (1.9 mL) is added followed by 2,2'-azobisisobutyronitrile (60 mg) and heating is continued for 35 minutes. The mixture is concentrated under reduced pressure. The oily residue is dissolved in 340 mL of acetonitrile, washed with 5 portions of hexanes, and concentrated to yield the crude product. Purification by silica gel chromatography yields the pure product.
- NMR data follows for one of the compounds of the present invention, 2'-O-acetyl-4"-deoxyerythromycin A: 13 C-NMR: ⁇ 222.0, 175.6, 170.0, 100.4, 96.8, 83.2, 79.0, 74.8, 74.6, 71.7, 70.5, 68.9, 67.9, 63.2, 61.4, 49.2, 45.3, 45.1, 44.7, 40.7, 38.9, 37.9, 34.1, 30.7, 26.8, 25.5, 25.2, 22.2, 21.9, 21.5, 21.2, 18.2, 16.3, 15.9, 12.0, 10.6, 9.1.
- NMR data follows for one of the compounds of the invention, (PS)-2'-O-acetyl-9-dihydroerythromycin A 11,12-cyclic carbonate: 13 C-NMR: ⁇ 175.8, 169.9, 153.8, 100.1, 96.7, 85.3, 82.3, 81.1, 80.0, 77.7, 76.5, 74.6, 71.7, 70.6, 68.6, 62.9, 61.8, 49.1, 45.3, 44.7, 42.3, 40.7, 34.5, 34.2, 33.6, 30.9, 25.5, 25.1, 22.9, 21.5, 21.4, 21.0, 20.1, 14.5, 14.4, 14.3, 10.7, 9.2.
- Samarium iodide is prepared by stirring a solution of samarium (3.43 mmol) and iodine (3.09 mmol) in 40 mL of tetrahydrofuran at reflux for 2.5 hours. Upon cooling to ambient temperature, 10 mg of N1I 2 is added and the mix is cooled to -78 °C. A solution of the 4"- O-(2,2,2-trichloroethoxycarbonyl)-protected erythromycin derivative (0.386 mmol) in 10 mL of tetrahydrofuran is added, and the mix is stirred for 1 hour at -78 °C. The reaction is quenched by addition of sat. NaHCO 3 , warmed to ambient temperature, and extracted with ether. The extract is dried over MgSO 4 , filtered, and evaporated. The product is purified by silica gel chromatography.
- the mixture was diluted with CH 2 CI 2 and washed sequentially with sat. NaHCO 3 and water, then dried over MgSO 4 , filtered, and evaporated.
- the crude product was partially purified by flash chromatography on silica gel (gradient of 2:1 to 1:1 hexanes/acetone + 1% Et N) to provide 4.5 g of light yellow solid.
- Trichloroethyl chloroformate (2.5 g) is added dropwise to a mixture of 2'-O-acetyl-15- methylerythromycin A (7.9 g) and 4-(dimethylamino)pyridine (1.5 g) in CH 2 C1 (100 mL) at -78 °C, and the mixture is stirred for 24 hours. After warming to ambient temperature, the mixture is diluted with CH 2 C1 2 and washed sequentially with phsphate buffer (5% KH 2 PO 4 + 1% K 2 HPO 4 ) and brine, then dried over MgSO 4 , filtered, and evaporated. The crude product is purified by flash chromatography on silica. EXAMPLE 35
- Example 43 The oximes of Example 43 were further modifed at the desosamine nitrogen using a similar methods except that 3-pyridinecarboxyaldehyde was replaced with the appropriate aldehyde to yield compounds where R 2 is methyl and R 3 is
- the compounds of the present invention are tested using a motilin receptor competitive binding assay.
- An illustrative protocol for such assay is described by Bormans et al, Regul. Peptides, 15: 143 (1986) which is incorporated herein by reference.
- membranes prepared from rabbit antrum or duodenum are incubated with 25-50 pM 125 I-labelled motilin and varying concentration of a test ligand. Protein-bound radioactivity is estimated from parallel reactions to which 100 nm unlabelled motilin is added. Efficacy of a test compound is expressed as IC 5 o, the concentration of the test compound to reduce the specific binding capacity to 50%.
- the compounds of the present invention are tested in a contractile activity assay described by Depoortere et al, Peptides, 11:515-519 (1990) which is incorporated herein by reference. Briefly, integral segments of rabbit small intestine (1.5-2 cm) are vertically suspended in tissue baths (10 ml), continuously gassed with 95% oxygen, 5% carbon dioxide, and kept at 37° C. The tissue baths contain Hepes buffer (pH 7.4) comprising 137 mM NaCl; 5.9 mM KCl; 1.2 mM CaCl 2 ; 1.2 mM MgCl 2 ; 11.6 mM Hepes; and 11.5 mM glucose. Contractions are recorded isotonically.
- Cumulative concentrations response curves are established by adding logarithmically increasing doses of the test compounds in 100 ⁇ l quantities to the bath. From each curve, the negative logarithm of the concentration necessary to induce 50% of the maximal contraction (pED 5 o) is determined by fitting a sigmoid curve to the data.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002552948A JP2004522726A (en) | 2000-12-01 | 2001-11-21 | Motilide compounds |
AU2002225724A AU2002225724A1 (en) | 2000-12-01 | 2001-11-21 | Motilide compounds |
EP01995209A EP1337540A2 (en) | 2000-12-01 | 2001-11-21 | Motilide compounds |
CA002429709A CA2429709A1 (en) | 2000-12-01 | 2001-11-21 | Motilide compounds |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US25064000P | 2000-12-01 | 2000-12-01 | |
US60/250,640 | 2000-12-01 | ||
US26936201P | 2001-02-15 | 2001-02-15 | |
US60/269,362 | 2001-02-15 |
Publications (2)
Publication Number | Publication Date |
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WO2002051855A2 true WO2002051855A2 (en) | 2002-07-04 |
WO2002051855A3 WO2002051855A3 (en) | 2002-10-31 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US2001/043963 WO2002051855A2 (en) | 2000-12-01 | 2001-11-21 | Motilide compounds |
Country Status (5)
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EP (1) | EP1337540A2 (en) |
JP (1) | JP2004522726A (en) |
AU (1) | AU2002225724A1 (en) |
CA (1) | CA2429709A1 (en) |
WO (1) | WO2002051855A2 (en) |
Cited By (11)
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JP2005200318A (en) * | 2004-01-14 | 2005-07-28 | Shionogi & Co Ltd | Erythromycin derivative |
WO2005085266A2 (en) * | 2004-02-27 | 2005-09-15 | Rib-X Pharmaceuticals, Inc. | Macrocyclic compounds and methods of making and using the same |
US7091196B2 (en) | 2002-09-26 | 2006-08-15 | Rib-X Pharmaceuticals, Inc. | Bifunctional heterocyclic compounds and methods of making and using same |
JP2007521320A (en) * | 2003-08-26 | 2007-08-02 | ファイザー インコーポレイテッド | N-desmethyl-N-substituted-11-deoxyerythromycin compounds |
EP1950219A1 (en) * | 2005-10-14 | 2008-07-30 | The Kitasato Institute | Novel dihydropseudoerythromycin derivatives |
WO2008143729A2 (en) * | 2007-02-28 | 2008-11-27 | Rib-X Pharmaceuticals, Inc. | Macrolide compounds and methods of making and using the same |
JP2009506075A (en) * | 2005-08-24 | 2009-02-12 | リブ−エックス ファーマシューティカルズ,インコーポレイテッド | Triazole compounds and methods for making and using the same |
JP4796300B2 (en) * | 2002-08-29 | 2011-10-19 | ファイザー インコーポレイテッド | Motilide compounds |
EP2431380A2 (en) | 2006-09-11 | 2012-03-21 | Tranzyme Pharma, Inc. | Macrocyclic antagonist of the motilin receptor for treatment of gastrointestinal dysmotility disorders |
US8470985B2 (en) | 2005-08-24 | 2013-06-25 | Rib-X Pharmaceuticals, Inc. | Triazole compounds and methods of making and using the same |
CN108101948A (en) * | 2004-02-27 | 2018-06-01 | 瑞伯-X医药品有限公司 | Macrocyclic compound and its making and use method |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992018134A1 (en) * | 1991-04-09 | 1992-10-29 | Abbott Laboratories | Macrocyclic lactam prokinetic agents |
WO2000062783A2 (en) * | 1999-04-16 | 2000-10-26 | Ortho-Mcneil Pharmaceutical, Inc. | Ketolide antibacterials |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08231580A (en) * | 1994-12-27 | 1996-09-10 | Chugai Pharmaceut Co Ltd | Erythromycin derivative |
-
2001
- 2001-11-21 CA CA002429709A patent/CA2429709A1/en not_active Abandoned
- 2001-11-21 JP JP2002552948A patent/JP2004522726A/en not_active Withdrawn
- 2001-11-21 EP EP01995209A patent/EP1337540A2/en not_active Withdrawn
- 2001-11-21 AU AU2002225724A patent/AU2002225724A1/en not_active Abandoned
- 2001-11-21 WO PCT/US2001/043963 patent/WO2002051855A2/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992018134A1 (en) * | 1991-04-09 | 1992-10-29 | Abbott Laboratories | Macrocyclic lactam prokinetic agents |
WO2000062783A2 (en) * | 1999-04-16 | 2000-10-26 | Ortho-Mcneil Pharmaceutical, Inc. | Ketolide antibacterials |
Non-Patent Citations (2)
Title |
---|
FAGHIH R ET AL: "Preparation of 9-deoxo-4-deoxy-6,9-epoxyerythromycin lactams motilactides: potent and orally active prokinetic agents" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 8, no. 7, 7 April 1998 (1998-04-07), pages 805-810, XP004136969 ISSN: 0960-894X * |
PATENT ABSTRACTS OF JAPAN vol. 1997, no. 01, 31 January 1997 (1997-01-31) & JP 08 231580 A (CHUGAI PHARMACEUT CO LTD), 10 September 1996 (1996-09-10) * |
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Also Published As
Publication number | Publication date |
---|---|
WO2002051855A3 (en) | 2002-10-31 |
AU2002225724A1 (en) | 2002-07-08 |
EP1337540A2 (en) | 2003-08-27 |
JP2004522726A (en) | 2004-07-29 |
CA2429709A1 (en) | 2002-07-04 |
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