EP0397779A1 - Peptides inhibiteur de renine avec groupes terminaux polaires - Google Patents

Peptides inhibiteur de renine avec groupes terminaux polaires

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Publication number
EP0397779A1
EP0397779A1 EP89902438A EP89902438A EP0397779A1 EP 0397779 A1 EP0397779 A1 EP 0397779A1 EP 89902438 A EP89902438 A EP 89902438A EP 89902438 A EP89902438 A EP 89902438A EP 0397779 A1 EP0397779 A1 EP 0397779A1
Authority
EP
European Patent Office
Prior art keywords
phe
pro
amino
lva
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP89902438A
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German (de)
English (en)
Inventor
Gordon L. Bundy
Suvit Thaisrivongs
Norman A. Nelson
Jackson B. Hester, Jr.
Jed F. Fisher
Michael F. Lipton
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmacia and Upjohn Co
Original Assignee
Upjohn Co
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Publication date
Application filed by Upjohn Co filed Critical Upjohn Co
Publication of EP0397779A1 publication Critical patent/EP0397779A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
    • C07K9/003—Peptides being substituted by heterocyclic radicals, e.g. bleomycin, phleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0227—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the (partial) peptide sequence -Phe-His-NH-(X)2-C(=0)-, e.g. Renin-inhibitors with n = 2 - 6; for n > 6 see C07K5/06 - C07K5/10
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention provides novel compounds . More particularly, the present invention provides novel renin-inhibiting peptide analogs. Most particularly, the present invention provides renininhibitory compounds having a variety of polar end groups at the N- terminus, specifically polyhydroxy-substituted-alkyl moieties.
  • the renin inhibitors provided herein are useful for the diagnosis and control of renin-dependent hypertension, congestive heart failure, renin dependent hyperaldosteronism, and other renin dependent cardiovascular disorders.
  • Renin is an endopeptidase which specifically cleaves a particular peptide bond of its substrate (angiotensinogen), of which the N-terminal sequence in equine substrate is for example: Renin ⁇ Asp - Arg - Val - Tyr - Ile - His - Pro - Phe - His - Leu- Leu - Val - Tyr - Ser - IA
  • Renin cleaves angiotensinogen to produce angiotensin I, which is converted to the potent pressor angiotensin II.
  • a number of angiotensin I converting enzyme inhibitors are known to be useful in the treatment of hypertension.
  • Inhibitors of renin may also be useful in the treatment of hypertension.
  • Terminal disulfide cycles have also been disclosed in renin inhibiting peptides; see, e.g., U.S. patents 4,477,440 and 4,477,441.
  • Aromatic and aliphatic amino acid residues at the 10,11 position of the renin substrate are disclosed in U.S. patents 4,478,827 and 4,455,303.
  • Renin inhibitors containing a C - terminal amide cycle are disclosed in U. S . patent 4 , 485 , 099 and European published applications 156,320 and 156,318.
  • Certain tetrapeptides are disclosed in European publications 111,266 and 77,027. Further, European published application No.
  • Chem., 50, 4615 (1985) discloses the synthesis of Hydroxyethylene Dipeptide Isosteres. See also, published European patent application 163,237, which discloses certain renin inhibiting peptides. Additionally, published European Applications 45,161 and 53,017 disclose amide derivatives useful as inhibitors of angiotensin converting enzymes.
  • Certain peptides containing dihalostatine have been disclosed in PCT application, Serial No. 000,713, filed 7 April 1986.
  • Certain peptides containing C- terminus truncated epoxy or azido or cyano groups or containing a position 10-11 diol and a position 11-12 retro bond have been disclosed in U.S. patent application, Serial No. 945, 340, filed 22 December 1986.
  • E.P. 189,203 discloses new N-dihydroxyalkyl peptide derivatives which are useful as inhibitors of renin for treating hypertension.
  • E.P. 184,855 discloses new hydroxy substituted-statine peptide derivatives which are useful as inhibitors of renin for treating hypertension.
  • U.S. Patent 4,613,676 discloses certain substituted 5-amino-4hydroxy valeryl derivatives, having 2 amino acid residues between the hydrogen or acyl N-terminus and the transition state moiety.
  • a broad class of acyl substituents, including hydroxy-substituted alkyl and etherified sugar moieties, are disclosed..
  • U.S. Patent 4,729,985 discloses renin inhibitors having a protecting group with a molecular weight less than 500, including, e.g., a (tris-hydroxy)-(t-butyluriedo).
  • U.S. Patent 4,668,770 generically discloses renin inhibitory tripeptides having a poly-hydroxy group at the C-terminus.
  • the present invention particularly provides:
  • the present invention also provides renin inhibitors having the substituents noted above and at least three amino acid residues between the N-terminus and the transition state insert.
  • the compounds of the present invention have a heterocyclic or heterocyclic N-oxide or another polyhydroxy-substituted alkyl moiety at the C terminus.
  • a number of specific examples of such polyhydroxy substituted alkyl moieties are exemplified in the EXAMPLES, CHARTS, and CLAIMS below.
  • the present invention provides the renin inhibitory peptides having a polyhydroxy substituted alkyl substituent and having amino acid residues and transition state inserts as depicted by the following formulas.
  • the variables A, B, C, D, G, H, and I are meant to depict amino acid residues and E-F depicts a stabilized transition state insert.
  • examples of compounds of the present invention are those having amino acid residues of the formula:
  • renin-inhibitory peptides described in European Application 0173481, published 5 March 1986, at pages 2 to 12 (expressly incorporated herein) are particularly suitable for this invention. These compounds are shown in relation to the human renin substrate as follows:
  • renin inhibitory peptide is meant a compound capable of inhibiting the renin enzyme in mammalian metabolism and having three or more amino acid residues linked by peptidic or pseudo-peptidic bonds.
  • a non-cleavable transition state insert is meant a transition state insert which is not cleavable by a hydrolytic enzyme in mammalian metabolism.
  • transition state inserts corresponding to the 10,11-position of the renin substrate, are known in the art. The following references disclose a variety of such inserts and are presented as merely illustrative of, and not as limiting, the transition state inserts encompassed by the present invention:
  • Polyhydroxy-substituted-alkyl moiety is meant a cyclic or acyclic, branched or unbranched alkyl derivative having from three to nine carbon atoms and from two to eight hydroxyl substituents which are attached to the N-terminus of the renin inhibiting peptide by means of a bond or by amide, urea or carbamate linkages.
  • polyhydroxy-substituted-alkyl moieties include sugars and other linear or branched polyhydroxy-substituted-alkyl derivatives which are coupled to the N-terminal nitrogen of the renin inhibitor peptide by means of an amino, a carboxylic acid or an aldehyde or ketone substituent on the polyhydroxy-substituted-alkyl moiety.
  • the phrase "bonded directly through carbon or nitrogen” is meant to exclude the use. of ether linkages between the polyhydroxy substituent and the peptide.
  • suitable amino-substituted polyhydroxy-substituted-alkyl derivatives include the aminodesoxyaldoses and the aminodesoxyketoses and their cyclic and reduced derivatives, 2,3-dihydroxy-2-(hydroxymethyl)propylamine, 2-hydroxy-1,1-bis(hydroxymethyl)ethylamine, 3-hydroxy-2,2-bis(hydroxymethyl)propylamine, 3-amino-1,2-propanediol, 2-amino-1,3-propanediol, 4-hydroxy-3,3- bis(hydroxymethyl)butylamine, and 2,3,4-trihydroxybutylamine.
  • suitable carboxylic acid substituted polyhydroxy-substituted-alkyl derivatives include the onic and uronic acid sugars such as gluconic and glucuronic acids, glyceric acid, 3-hydroxy-2,2-bis(hydroxymethyl)propionic acid, 2-hydroxy-2,2-bis(hydroxymethyl)- acetic acid, 4-hydroxy-3,3-bis(hydroxymethyl)butyric acid, 2,3,4-trihydroxybutyric acid.
  • suitable aldehyde or ketone substituted sugars include the aldoses and ketoses which can be condensed directly with the N-terminal amine of renin inhibitory peptides by means of a reductive alkylation with, for example, sodium cyanoborohydride.
  • reducing sugars can be condensed with the N-terminal amine by means of the acid catalyzed Amadori rearrangement. Examples of this and other reactions suitable 'for incorporating sugars onto peptides having a free amine are set forth in WO published PCT application 87/02756.
  • derivatives of amino acids is meant the well known amino acid derivatives commonly employed in renin inhibitors as set forth in the references described above.
  • the renin inhibitory peptides of the present invention can occur in several isomeric forms, depending on the configuration around the asymmetric carbon atoms. All such isomeric forms are included within the scope of the present invention.
  • the stereochemistry of the amino acids corresponds to that of the naturally-occurring amino acids. Where the stereochemistry of the amino acid is not indicated, it is preferably in the L-configuration.
  • Renin inhibitory peptides commonly have protecting. groups at the N-terminus and the C- terminus. Many of these protecting groups are known in the polypeptide art. Examples of such protecting groups are given below.
  • the renin inhibitory peptides of the present invention have a variety of novel polar end groups at the N-terminus. These polar end groups will increase the water solubility of the peptides, improve the oral absorption characteristics of the peptides and/or slow the metabolic degradation of the peptides, all while retaining the therapeutically useful renin-inhibiting activity of the peptides.
  • Examples of pharmaceutically acceptable acid addition salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2naphthalenesulfonate, nicocinate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyan
  • the carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix (C i -C j ) indicates a moiety of the integer "i" to the integer "j" carbon atoms, inclusive.
  • (C 1 -C 4 )alkyl refers to alkyl of one to 4 carbon atoms, inclusive, or methyl, ethyl, propyl, butyl, and isomeric forms thereof .
  • C 4 -C 7 cyclic amino indicates a monocyclic group containing one nitrogen and 4 to 7 carbon atoms.
  • Examples of (C 3 -C 10 )cycloalkyl which include alkyl-substituted cycloalkyl containing a total of up to 10 total carbon atoms, are cyclopropyl, 2-methylcyclopropyl, 2,2-dimethylcyclopropyl, 2,3-diethylcyclopropyl, 2-butylcyclopropyl, cyclobutyl, 2 -methyl-cyclobutyl, 3-propylcyclobutyl, cyclopentyl, 2,2-dimethylcyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and isomeric forms thereof.
  • aryl examples include phenyl, naphthyl, (o-, m-, p-)tolyl, (o- , m- , p-)ethylphenyl, 2-ethyl-tolyl, 4-ethyl-o-tolyl, 5-ethyl-mtolyl, (o- , m-, or p- )propylphenyl, 2-propyl- (o-, m-, or p-)tolyl, 4isopropyl-2 ,6-xylyl, 3-propyl-4-ethylphenyl, (2,3,4- 2,3,6-, or 2,4, 5-) -trimethylphenyl, (o-, m- , or p-) fluorophenyl, (o-, m-, or p-trifluoromethyl)phenyl, 4-fluoro-2,5-xylyl, (2,4-, 2,5-, 2,6-, 3,
  • Examples of -Het include: 2-, 3-, or 4-pyridyl, imidazolyl, indolyl, N in -formyl-indolyl, N in -C 1 -C 5 alkyl-C(O)-indolyl, [1,2,4]-triazolyl, 2-, 4-, or 5-pyrimidinyl, 2- or 3-thienyl, piperidinyl, pyrryl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pyrazinyl, piperazinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl
  • a heterocycle as defined herein for -Het would not be bonded through oxygen or sulfur or through nitrogen which is within a ring and part of a double bond.
  • Halo is halogen (fluoro, chloro, bromo, or iodo) or trifluoromethyl.
  • pharmaceutically acceptable cations include: pharmacologically acceptable metal cations, ammonium, amine cations, or quaternary ammonium cations.
  • pharmacologically acceptable metal cations are those derived from the alkali metals, e.g., lithium, sodium, and potassium, and from the alkaline earth metals, e.g., magnesium and calcium, although cationic forms of other metals, e.g., aluminum, zinc, and iron are also within the scope of this invention.
  • Pharmacologically acceptable amine cations are those derived from primary, secondary, or tertiary amines.
  • novel peptides herein contain both natural and synthetic amino acid residues. These residues are depicted using standard amino acid abbreviations (see, e.g., Eur. J. Biochem., 138, 9 (1984)) unless otherwise indicated.
  • the compounds of the invention are effective in the treatment of humans .
  • the renin inhibitors of this invention are useful for treating any medical condition for which it is beneficial to reduce the levels of active circulating renin.
  • examples of such conditions include renin-associated hypertension and hyperaldosteronism, hypertension, hypertension under treatment with another antihypertensive and/or a diuretic agent, congestive heart failure, angina, and post-myocardial infarction.
  • the renin-angiotension system may play a role in maintenance of intracellular homeostasis: see Clinical and Experimental Hypertension, 86, 1739-1742 (1984) at page 1740 under Discussion.
  • the compounds will also be useful as molecular probes for the diagnosis and study of the physiology of blood pressure regulation or other physiological functions.
  • the renin inhibitors of this invention may be useful in the treatment of cerebrovascular disorders and disorders of intracellular homeotasis.
  • the possible role of the renin-angiotensin system in the maintenance of intracellular homeostasis is disclosed in Clinical and Experimental Hypertension, 86:1739-1742 (1984).
  • the renin inhibitors of this invention potentiate the antithrombotic activity of a thromboxane antagonist (U.S. patent 4,558,037).
  • the antihypertensive effect of the renin inhibitors of this invention are potentiated by combination with a thromboxane synthetase inhibitor.
  • the compounds of the present invention are preferably orally administered to humans to effect renin inhibition for the purpose of favorably affecting blood pressure.
  • the compounds are administered from 0.1 mg to 1000 mg per kg per dose, administered from 1 to 4 times daily.
  • the compounds of the present invention are preferably orally administered either as the present compound or in the form of pharmacologically acceptable acid addition salts.
  • Preferred pharmacologically acceptable salts for oral administration include the citrate and aspartate salts, although any pharmacologically acceptable salt is useful in this invention, including those listed above. These salts may be in hydrated or solvated form.
  • parenteral by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxoc pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example as a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectibles.
  • the compounds of the present invention may be in the form of pharmaceutically acceptable salts both those which can be produced from the free bases by methods well known in the art and those with which acids have pharmacologically acceptable conjugate bases.
  • the renin-inhibiting compounds of this invention may be administered in combination, with other agents used in antihypertensive therapy such as diuretics, ⁇ and/or ⁇ -adrenergic blocking agents, CNS-acting agents, adrenergic neuron blocking agents, vasodilators, angiotensin I converting enzyme inhibitors, and the like as described, for example, in published European patent application 156 318.
  • agents used in antihypertensive therapy such as diuretics, ⁇ and/or ⁇ -adrenergic blocking agents, CNS-acting agents, adrenergic neuron blocking agents, vasodilators, angiotensin I converting enzyme inhibitors, and the like as described, for example, in published European patent application 156 318.
  • the compounds of this invention can be given in combination with such compounds or salts or other derivative forms thereof as:
  • Diuretics acetazolamide; amiloride; bendroflumethiazide; benzthiazide; bumetanide; chlorothiazide; chlorthalidone; cyclothiazide; ethacrynic acid; furosemide; hydrochlorothiazide; hydroflumethiazide; indacrinone (racemic mixture, or as either the (+) or (-) enantiomer alone, or a manipulated ratio, e.g., 9:1 of said enantiomers, respectively); metolazone; methyclothiazide; muzolimine; polythiazide; quinethazone; sodium ethacrynate; sodium nitroprusside; spironolactone; ticrynaten; trimaterene; trichlormethiazide; ⁇ -Adrenergic Blocking Agents: dibenamine; phentolamine; phenoxybenzamine; prazosin; to
  • antihypertensive agents include: aminophylline; cryp tenamine acetates and tannates; deserpidine; meremethoxylline procaine; pargyline; tri-methaphan camsylate; and the like, as well as admixtures and combinations thereof.
  • the individual daily dosages for these combinations can range from about one-fifth of the minimally recommended clinical dosages to the maximum recommended levels for the entities when they are given singly.
  • Coadministration is most readily accomplished by combining the active ingredients into a suitable unit dosage form containing the proper dosages of each. Other methods of coadministration are, of course, possible.
  • novel peptides of the present invention possess an excellent degree of activity in treating renin-associated hypertension and hyperaldosteronism.
  • Renin inhibitors have also been disclosed to control the rise in intraocular pressure associated with the use of steroidal antiinflammatory drugs as described in International Application PCT/US86/02291 (International Publication Number WO 87/02581 dated 7 May 1987).
  • Chart A In Chart A is described the conversion of N-deprotected Boc-ProPhe-N(Me)His-LVA-Ile-AMP, i.e., Pro-Phe-N(Me)His-LVA-Ile-AMP into its tris(hydroxymethyl)methylamino (THAM) urea derivative.
  • THAM tris(hydroxymethyl)methylamino
  • the preparation of Boc-Pro-Phe-N(Me)His-LVA-Ile-AMP is described in U.S. patent application, Serial No. 147,073, filed 20 January 1988, and in published European patent application 0173481, published 5 March 1986, which are hereby incorporated by reference.
  • reaction of the A-1 compound with the A-2 compound yields the A-3 compound.
  • the triacetate is coupled with the protected LVA intermediate to yield the C-3 compound.
  • Subsequent block coupling with the Boc-Pro- Phe-His(Ts)-OH fragment yields the C-4 compound.
  • the tosyl and silyl protecting groups are simultaneously removed from the final product using tetrabutylammonium fluoride in tetrahydrofuran.
  • N(Me)His THAM amides D-5 (R is acetyl or hydrogen) are prepared in classical stepwise fashion from intermediate C-3 (R is H or acetyl) (Chart C). Removal of N-protecting groups (Boc or Ts) are accomplished using standard procedures. Likewise, the peptide bond formations are done using diethylphosphoryl cyanide, dicyclohexylcarbodiimide or other usual coupling reagents.
  • Chart F describes the preparation of renin inhibitory peptides of the present invention having (R 120 ) (R 124 )N(CH 2 ) n C(O)- at the N- terminal of the peptide.
  • the amines of formula F-1 are either known compounds or are easily prepared by known methods. Conversion of the amines of formula F-1 to the unsymmetrical ureas of formula F-3 can be done by a number of standard methods, a convenient one of which is described in Tetrahedron Letters 1935 (1977).
  • the formula F-1 amine is treated with phenyl chloroformate to obtain the compound of formula F-2.
  • the formula F-2 compound is joined to a peptide such as H-A 6 -B 7 -C 8 -D 9 -E 10 -F 11 -G 12 -H 13 -I 14 -Z to obtain the unsymmetrical ureas of formula F-3.
  • "-NHA 6 -" represents the amino nitrogen from the amino acid A 6 .
  • R 120 and R 124 are protected during the urea formation steps as non-polar derivatives, for example, as ethers, orthoesters or acylates.
  • the unsymmetrical urea products of the formula F-3 are stable to both acidic and basic blocking group removal conditions.
  • CHART G Chart G describes the preparation of renin inhibitory peptides of the present invention having -N(R 120 ) (R 124 ) at the C-terminal of the peptide.
  • the amines of formula G-1 are either known compounds or are easily prepared by known methods.
  • the amides of formula G-2 are prepared from the amines of formula G-1 by standard peptide-forming reactions.
  • "-I 14 C(O)-” represents the carbonyl group from the amino acid I 14 .
  • R 120 and R 124 are best protected as non-polar derivatives during the coupling reactions, for example, esters for the carboxys; ethers, orthoesters or acylates for the hydroxys.
  • this substituent may be prepared from aminocitric acid, a known compound. See A. Dornow, K. Rombusch, Chem. Ber. 88:1334 (1965).
  • this substituent may be prepared from 3-amino-1,2-propanediol (Aldrich Chem. Co.). In peptides wherein R 120 is the structure in (e), this substituent may be prepared by standard techniques from the substituent in (d).
  • CHART H Renin inhibitory peptides of the present invention include compounds with a polar N-terminal group and utilizing the small C-terminal truncated diol as the transition-state insert.
  • the polar groups are attached to the N-terminal proline of the smallest C-terminal truncated renin inhibitory peptide template.
  • CHART I Chart I shows the preparation of the renin inhibitor 1-4.
  • the known phenyl urethane I-1 is reacted with the amine 1-2 to give the urea 1-3.
  • the protecting group is removed with acid to give the desired compound 1-4.
  • the corresponding renin inhibitor having(OCH 3 )Tyr and -His in place of -Phe and -N(Me)His is prepared in an analogous manner.
  • CHART K Chart K shows the preparation of the renin inhibitor K-4.
  • the acid K-1 prepared as the compound J-4 in Chart J, is coupled to the amine K-2 to give the adduct K-3.
  • the protecting group is removed with ammonia in methanol to give the desired compound K-4.
  • the corresponding renin inhibitor having -(OCH 3 )Tyr and -His in place of -Phe and -N(Me)His is prepared in an analogous manner.
  • the starting material is the N(CH 3 )His protected compound L-1, prepared as described in U.S. patent application, Serial No. 147,073, filed 20 January 1988, and in European application 0173481, published 5 March 1986.
  • the Boc protective group is removed in the usual way (trifluoroacetic acid) to give L-2 Polar end groups, derived from shikimic acid and 2,2-bis(hydroxymethyl)propionic acid, are then attached to the N-terminus and, finally, the tosyl protective group on the N(CH 3 )His(Ts) unit is removed.
  • the two final products prepared, L-4a and L-4b are obtained.
  • Chart N illustrates the preparation of the renin inhibitor N-4, which is further described in Example 23 below.
  • Chart O illustrates the preparation of the renin inhibitor O-4, which is further described in Example 24 below.
  • CHART P Renin inhibitors of generic formula P-4 can be prepared as described in Chart P.
  • the renin inhibitor residue preferably contains Pro at the P 4 site, an aromatic amino acid residue at the P 3 site (e.g., Phe, Tyr (O-Me ether, etc.), His or N(Me)His at the P 2 site, a transitionstate unit occupying the P 1 -P 1 sites such as LVA, CVA or other transition-state units as described above and may contain other binding units at the P 2 ' and P 3 ' sites as described above.
  • An example of this synthetic procedure is the preparation of renin inhibitor N-4 as described in Example 23 below and Chart N above.
  • the stereochemistry of the carbinol centers in the sugar-like residues attached to the amino termini can be R or S.
  • the starting materials P-1 are commercially available (e.g., glucamine and HOCH 2 -CHOH-CH 2 -NH 2 ), or are described in the literature. See F. Kagan, M.A. Rebenstorf and R.V. Heinzelman, J. Am. Chem. Soc., 79:3541 (1957); G.W.E. Plant and R.A. Harvey, Methods Enzymol., 18(Issue Pt B):515 (1971); M. Israel and N. Bengal, J. Heterocycl. Chem., 10:209 (1973); S. Hyundai, S. Suzuki and K. Kakehi, J. Chromatography, 396:93 (1987); H. Pischel, A. Holy, J.
  • Renin inhibitors of generic formula Q-2 can be prepared as described in Chart Q.
  • the stereochemistry of the carbinol centers in the sugar-like residues attached to the amino termini can be R or S.
  • the definition of renin inhibitor residue is as defined above.
  • the preferred aldoses are D-arabinose, L-arabinose, D-xylose, D-ribose, D-glucose, D-mannose and D-galactose.
  • the reductive amination is carried out as described earlier for the preparation of renin inhibitors of formula Q-2 wherein n is 3 or 4 and the renin inhibitor residue is -Pro-Phe-N(Me)His-LVA-Ile-AMP in Examples 25 and 26 below. (See also, the references described above for reductive amination of aldoses to produce aldamines).
  • Renin inhibitors of generic formula Q-3 can be prepared in several ways. Where Renin Inhibitor Residue is as defined above and the preferred aldonic acid residues are derived from D-arabinose, L-arabinose, D-xylose, D-ribose, D-glucose, D-mannose and D-galactose, the stereochemistry of the chiral carbinol centers in the aldonic acid unit can be R or S.
  • the common aldonic acids or their lactones are known compounds and some are commercially available (e.g., D-glucono-5-lactone, D-gluconic acid, potassium salt, etc.).
  • Aldonic acids are readily prepared from the corresponding aldoses by hypoiodite oxidation, bromine oxidation or electrolytic oxidation (see Methods in Carbohydrate Chemistry, Volume II, 11-15 (1963) edited by R.L. Whistler, M. L. Wolfrom and S.N. BeMiller).
  • the corresponding ⁇ and/or ⁇ lactones can be' readily prepared (see reference above, pp 16-18).
  • An ammonolysis reaction of an aldonic acid lactone ( ⁇ or ⁇ lactone) and a renin inhibitor having an amino group on the amino terminus leads directly to the desired product Q-3.
  • Another way to obtain products of type Q-3 is to acylate the renin inhibitor having a free amino group on the amino terminus with a fully acetylated aldonyl chloride and then remove the acetyl protective groups by ammonolysis with methanolic ammonia (see conversion of P-3 to P-4).
  • the fully acetylated aldonyl chloride is obtained by treating the fully acetylated aldonic acid with phosphorous pentachloride in ether or other suitable solvent (references below).
  • the fully acetylated aldonic acids are obtained by acetylation of the aldonic acid salts with acetic anhydride in the presence of hydrogen chloride or by stepwise acetylation of aldonic acid lactones with acetic anhydride and fused zinc chloride.
  • Procedures for preparing fully acetylated aldonyl chlorides are, for example, Methods in Carbohydrate Chemistry, Volume II, 21-26 (1963), edited by R.L. Whistler, M.L. Wolfrom and J.N. BeMiller; CE. Braun and CD. Cook, Org. Synth., Coll., Vol 5:887 (1973).
  • the reaction of the acetylated aldonyl chlorides with the basic renin inhibitor can be carried out in, for example, pyridine, and the various products are purified as described earlier for related structures (i.e., extractions, washings, chromatography, etc.).
  • the acylation of a basic renin inhibitor will occur selectively on the terminal amino group. If the transition-state unit contains an amino group (e.g., in the form of a reduced dipeptide unit) it is not necessary to selectively block the amino group of the transitionstate unit. Any acylation that occurs on the imidazole ring of a histidine unit can be reversed by selective hydrolysis of the imidazoyl amide so formed.
  • CHARTS R AND S Charts R and S exemplify the preparation of renin inhibitory peptides obtained by the amide linkage to the N-terminal phenylalanine or N-terminal proline of an alkanedioic acid having attached to its remaining free carboxylic acid moiety an N-linked saccharide, especially monosaccharides.
  • alkanedioic acid include succinic acid, L-aspartic acid, D-aspartic acid, N-protected L- and D-aspartic acid and derivatives thereof.
  • monosaccharides include mannose and 2-amino-2-deoxyglucose and derivatives thereof, including N-acyl derivatives.
  • mannose-asparagine fragment is prepared and coupled as outlined in Chart S.
  • CHART T Chart T describes the preparation of transition-state insert Cha ⁇ [CH(OH)CH 2 ]Val(CVA).
  • U.S. Patent Application Serial No. 213,787 filed 30 June 1988, which is hereby incorporated by reference, provides novel processes and intermediates for the synthesis of the hydroxyethylene isostere dipeptide leu-val and similar transition state moieties. Processes and intermediates analogous to those described therein be used to prepare CVA.
  • Table A describes additional renin inhibitor compounds that may be prepared having polar end groups at the N-terminus and CVA as the transition state insert.
  • the compound of formula XX wherein X is an electron-withdrawing group and anion stabilizing group, for example, -NO 2 and -Br; and wherein R 11 , R 12, and R 13 are each independently trimethylsilyl or t-butyldimethylsilyl; is useful as an intermediate in making renin inhibitor peptides, especially useful as an acylating agent.
  • R 13 are each trimethylsilyl or wherein one of R 11, R 12 and R 13 is t-butyldimethylsilyl and the remaining two are trimethylsilyl.
  • the compound of formula XX wherein R 10 is H and R 11, R 12 and R 13 are each trimethylsilyl is prepared as described in Preparation 49, and is used in the preparation of the renin inhibitor peptide of formula B-4
  • the renin inhibiting polypeptides may be prepared by either polymer assisted or solution phase peptide synthetic procedures analogous to those described hereinafter or to those methods known in the art.
  • the carboxylic moiety of N ⁇ -t-butyloxycarbonyl (Boc)-substituted amino acid derivatives having suitable side chain protecting groups may be condensed with the amino functionality of a suitably protected amino acid, peptide or polymer-bound peptide using a conventional coupling protocol such as dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT) or diethylphosphoryl cyanide (DEPC) and triethylamine (Et 3 N) in methylene chloride or dimethylformamide.
  • DCC dicyclohexylcarbodiimide
  • HOBT 1-hydroxybenzotriazole
  • DEPC diethylphosphoryl cyanide
  • Et 3 N triethylamine
  • peptides may also be prepared by the standard solid phase techniques of Merrifield. Appropriate protecting groups, reagents, and solvents for both the solution and solid phase methods can be found in "The Peptides: Analysis, Synthesis, and Biology,” Vols. 1-5, eds. E. Gross and T. Meienhofer, Academic Press, NY, 1979-1983.
  • the compounds of the present invention may be in either free form or in protected form at one or more of the remaining (not previously protected) peptide, carboxyl, amino, hydroxy, or other reactive groups.
  • the protecting groups may be any of those known in the polypeptide art. Examples of nitrogen and oxygen protection groups are set forth in T.W. Greene, Protecting Groups in Organic Synthesis, Wiley, New York, (1981); J.F.W. McOmie, ed. Protective Groups in Organic Chemistry, Plenum Press (1973); and J. Fuhrhop and G. Benzlin, Organic Synthesis, Verlag Chemie (1983).
  • nitrogen protective groups include t-butoxycarbonyl (Boc), benzyloxycarbonyl, acetyl, allyl, phthalyl, benzyl, benzoyl, trityl and the like.
  • the preferred compounds of this invention are: N-[[[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]carbonyl]-Pro-Phe-N(Me)His-LVA-He-AMP, pyridine N-oxide;
  • L-Histidinamide 1-[[2-deoxy-D-glucopyranos-2-yl)amino]carbonyl]-L-prolyl-L-phenylalanyl-N-[1-(cyclohexylmethyl)-2-hydroxy-5-methyl-4-[[(2-methylbutyl)amino]carbonyl]hexyl]-N.alpha.-methyl-, [1S[1R*,2R*,4R*(R*)]]-.
  • the most preferred compounds of this invention are: N-[[[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]carbonyl]-Pro-Phe-N(Me)His-LVA-Ile-AMP, pyridine N-oxide;
  • L-Histidinamide 1-[[2-deoxy-D-glucopyranos-2-yl)amino]carbonyl]-L-prolyl-L-tyrosyl-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5-methylhexyl]-, [1S-(1R*,2S*,3R*)];
  • L-Histidinamide 1-[[2-deoxy-D-glucopyranos-2-yl)amino]carbonyl]-L-prolyl-L-phenylalanyl-N-[1-(cyclohexylmethyl)-2-hydroxy-5-methyl-4-[[(2-methylbutyl)amino]carbonyl]hexyl]-N.alpha,-methyl-, [1S[1R*,2R*,4R*(R*)]]-;
  • AMP is 2-(aminomethyl)pyridinyl; AMP-NO is (2-pyridylmethyl)amino, pyridine N-oxide; Asn is asparagine; BOC is t-butoxycarbonyl; BOM is benzyloxymethyl; Bz is benzyl; C is centigrade;
  • Celite is a filter aid
  • CVA is Cha ⁇ [CH(OH)CH 2 ]Val
  • DCC is dicyclohexylcarbodiimide
  • DEPC is diethyl cyanophosphonate or diethylphosphoryl cyanide
  • DGLU is (2-deoxy-D-glucopyranos-2-yl)amino
  • DGLUA is (2-deoxy-D-glucopyranos-2-yl)amino tetraacetate ester
  • DHPA is ( ⁇ )-(2,3-dihydroxypropyl)amino; DMF is dimethylformamide;
  • EtOAc is ethyl acetate
  • FTrp is N in -formyl-indolyl; g is grams;
  • GEA is 2-(guanidylethyl)amino
  • GMPMA is (3-(guanidylmethyl)phenyl)methylamino
  • HPLC high performance liquid chromatography
  • IR is infra red spectra
  • L-Selectride is lithium tri-sec-butylborohydride
  • LVA is Leu ⁇ [CH(OH)CH 2 ]Val
  • Lys is lysine; M or mol is mole;
  • MBA is 2-methylbutylamino (racemic or optically active);
  • MBAS is 2S-methylbutylamino
  • Me is methyl; min is minute; ml is milliliter;
  • MS is mass spectroscopy
  • NMHis is N ⁇ -methyl-L-histidine
  • NMR nuclear magnetic resonance
  • NOA1 is (1-na ⁇ hthyloxy)acetyl
  • p-TSA salt is para-toluene sulfonic acid salt
  • Ph is phenyl
  • Phe is phenylalanine
  • Pro is proline
  • POA is phenoxyacetyl
  • RIP means a compound having the formula H-Pro-His-Phe-His-Phe- Phe-Val-Tyr-Lys-OH.2(CH 3 C(O)OH).
  • XH 2 O which is a known renin-inhibiting peptide.
  • Skellysolve B is as defined in the Merck Index, 10th edition; t-BDMS or TBDMS is t-butyldimethylsilyl; TFA is trifluoroacetic acid;
  • THAM is [2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino
  • THAMOA is (1-methyl-2,6,7-trioxabicyclo[2.2.2]oct-4-yl)amino
  • THF is tetrahydrofuran
  • TLC is thin layer chromatography; Tos or TS is p-toluenesulfonyl; Tr is trityl (triphenylmethyl); Tyr is tyrosine; 2HPA is ( ⁇ )-(2-hydroxypropyl)amino;
  • 2HPA(S) is (S)-(2-hydroxypropyl)amino; UV is ultraviolet; and Z is benzyloxycarbonyl.
  • the wedge-shape line indicates a bond which extends above the plane of the paper relative to the plane of the compound thereon.
  • the dotted line indicates a bond which extends below the plane of the paper relative to the plane of the compound thereon.
  • a 5% solution of the Boc protected amine in an equal volume of methylene chloride and trifluoroacetic acid is allowed to stir at room temperature for 1 h and then concentrated in vacuo, A solution of the residue in methylene chloride is washed once with aqueous sodium bicarbonate. The aqueous wash is backwashed twice with methylene chloride. The combined organic fractions are dried over magnesium sulfate and concentrated in vacuo. The residue is then used as is in the next step without further purification.
  • DEPC diethylphosphoryl cyanide
  • Infrared ( ⁇ max , Nujol, cm -1 ): 3465, 3373, 1446, 1408, 1392, 1354, 1297, 1124, 1053, 1012, 994, 983, 893, 864, 860, 816, 760.
  • TLC Silica Gel GF: Rf 0.27 (1% triethylamine/ethyl acetate); Rf 0.36 [5% (4M ammonia-methanol)/95% chloroform.
  • the crude product a white solid, is recrystallized from approximately 25 ml of 30% ethyl acetate/hexane and dried at 25oC for 3 h, thereby affording pure title product, 2.62 g, m.p. 186-188°C
  • the mother liquors from this recrystallization are chromatographed on a 200 g column of silica gel. The column is packed and eluted in 18 ml fractions with 35% ethyl acetate/hexane (the material is introduced onto the top of the column with chloroform). Fractions 26-40 yield an additional 665 mg of clean title product (total yield is 3.285 g).
  • the crude product is chromatographed on a column containing 400 g of 40-60 ⁇ l silica gel.
  • the column is packed and eluted in 25 ml fractions with 4.5% [4M ammonia-methanol)/95.5% chloroform.
  • Fractions 132-160, homogeneous by TLC, are combined and afford 2.40 g of pure urea derivative A-6 (Refer to Chart A).
  • the purified product is to be deprotected in a subsequent step and is therefore allowed to remain a colorless foam (after drying 1 h at 30oC).
  • the chromatographically pure product is precipitated by dripping a concentrated methylene chloride solution into an excess of hexane with stirring. Filtration and drying afford an amorphous, free-flowing white solid. Physical characteristics are as follows:
  • a 0.1 M methanolic hydrochloric acid solution is prepared by adding 0.78 ml of acetyl chloride, with stirring, to 100 ml of dry methanol at 0°C. This solution is used to dissolve 2.40 g of the title product of Example 1, and the resulting mixture is stirred for 2 h at 25°C TLC analysis of the reaction mixture (spotted directly without dilution or workup) shows clean and complete conversion to the title product. The mixture is treated with 12 ml of 1 M aqueous potassium hydroxide (final pH 13.2) and then stirred 30 min longer. The desired title product is then isolated by extraction with chloroform (after first removing the methanol in vacuo).
  • the crude title product is chromatographed on a column containing 180 g of silica gel.
  • the column is packed and eluted in 18 ml fractions with 12% 4 M ammonia-methanol/88% chloroform.
  • Fractions 38-60 are combined and evaporated to dryness.
  • the residue is dissolved in 30 ml of warm methylene chloride, and the solution is dripped into 100 ml of rapidly stirred hexane. Filtration and drying (0.1 mm, 25oC, 2 h) affords 2.036 g of pure title product as a white, easily-handled, amorphous solid.
  • the extracts are washed with additional aqueous sodium bicarbonate and brine, dried over anhydrous sodium sulfate and evaporated.
  • the crude product (4.5 g) is dissolved in 60 ml of methanol and treated with 2.2 g of 1-hydroxybenzotriazole hydrate, and the resulting clear solution is stirred for 12 h at room temperature under argon. The mixture is then evaporated to dryness and the residue is chromatographed immediately on a column containing 450 g of silica gel, packed and eluted once with 1000 ml 6% (4 M ammoniamethanol)/94% chloroform, then 25 ml fractions.
  • Fractions 88-120 are homogeneous by TLC and are combined. Following evaporation of the solvent, the residue is dissolved in 8 ml of methylene chloride, and that solution is added dropwise to 50 ml of rapidly stirred hexane. After filtration and drying (0.1 mm, 25oC, 2 h), the resulting white solid, clean title product weighs 2.25 g.
  • the crude product (4.2 g) is chromatographed on a column containing 400 g of silica gel, packed and eluted once with 750 ml of 12% (4 M ammoniamethanol)/88% chloroform, then 25 ml fractions.
  • TLC Silica Gel GF
  • R f 0.25 [16% (7M ammonia-methanol)/84% chloroform]
  • title compound of Example 4 has R f 0.19 on the same plate.
  • IC 50 1.1 ⁇ 10 -9 M.
  • THAM amide (the title product of Preparation 4) in 5 ml of 1:1 trifluoroacetic acid and methylene chloride is allowed to stand for 1 h at 25oC Following evaporation to dryness on the rotary evaporator, the material is divided in half and each dissolved in 2 ml of dry methylene chloride.
  • the other half of the de-Boc'd product (in 2 ml of methylene chloride) is treated with 45 mg of the silylated LVA intermediate, 22 mg of dicyclohexylcarbodiimide and 14.2 mg of 1-hydroxybenzotriazole hydrate.
  • the resulting solution is stirred for 18 h at 25oC and then filtered to remove the dicyclohexylurea.
  • the filtrate is diluted with more methylene chloride and washed with aqueous sodium bicarbonate.
  • the extracts are dried (sodium sulfate) and concentrated to an oil.
  • the extracts are dried (sodium sulfate) and evaporated, and the crude product is chromatographed on a 21 g column of silica gel. The column is packed and eluted once with 25 ml of 5% methanol/chloroform, then 1-2 ml fractions.
  • the partially purified product is subjected to preparative TLC on a single 500 ⁇ l 8 ⁇ 8 inch plate.
  • the material is spotted onto the plate in chloroform, and the plate is developed with 10% (4 M ammonia-methanol)/90% chloroform and visualized under UV light.
  • the product is recovered by stirring the scraped-off silica band with 20 ml of 20% (4 M ammonia-methanol)/80% chloroform. Filtration and evaporation of the solvent yield a white foam weighing 7 mg.
  • the product is dissolved in 100 ⁇ l of methylene chloride and precipitated by addition of 2 ml of hexane. Filtration and drying afford 6 mg of an amorphous white powder, the desired THAM amide title product, completely clean by TLC.
  • a solution of the Section B product in 60 ml of methylene chloride is cooled to 0°C and treated with 3.45 g of Boc-Leu ⁇ -[CH(OTBS)CH 2 ]Val-OH, followed by 1.13 ml of triethylamine and 1.24 ml of diethylphosphoryl cyanide, the latter reagent being added dropwise over about 5 min.
  • the clear reaction mixture is stirred at 0°C for 1 h and 25oC for 18 h. It is then poured into aqueous sodium bicarbonate, and the product is isolated by extraction with methylene chloride.
  • the extracts are dried (sodium sulfate) and evaporated, and the crude product (8.1 g) is chromatographed on 700 g of silica gel. The column is packed and eluted once with 800 ml of 45% ethyl acetate/hexane, then 25 ml fractions.
  • the purified product from the preceding paragraph is dissolved in 1.5 ml of tetrahydrofuran and treated with 0.8 ml of 1 M tetrabutylammonium fluoride in tetrahydrofuran (Aldrich). After 6 h, another 0.8 ml of tetrabutylammonium fluoride is added, and stirring is continued for an additional 6 h. The mixture is then poured into brine containing a little sodium bicarbonate and extracted three times with chloroform (80 ml). The extracts are washed with brine, dried over sodium sulfate and concentrated. The crude product (780 mg) is chromatographed on 70 g of silica gel.
  • the crude product (8.1 g) is chromatographed on a column containing 700 g of silica gel.
  • the column is packed with 65% ethyl acetate/hexane and eluted with 4000 ml of the same solvent, (one 450 ml portion, then 50 ml fractions), followed by 3000 ml of pure ethyl acetate.
  • the silyl derivative (the title product of Preparation 7 (R is acetyl)) is de-Boc'd (and simultaneously desilylated) using 25 ml of trifluoroacetic acid and 20 ml of methylene chloride at 25oC for 4.5 h.
  • the alcohol, the title product of Preparation 7 (R is H) (0.95 g, also isolated in the preceding experiment) is treated with 5 ml of trifluoroacetic acid and 5 ml of methylene chloride at 25°C for 4.5 h.
  • the crude amine is dissolved in 60 ml of methylene chloride and treated with 1.67 ml of triethylamine, 3.18 g of Boc-Phe, and 1.82 ml of diethylphosphoryl cyanide added dropwise at 0oC
  • the mixture is stirred for 1 h at 0oC, then over a weekend at 25oC
  • Standard methylene chloride/sodium bicarbonate workup affords a crude product (10.1 g), which is chromatographed on a 700 g column of silica gel. The column is packed and eluted once with 800 ml of 90/10 ethyl acetate/hexane, then 50 ml fractions.
  • the crude amine is dissolved in 30 ml of methylene chloride and treated with 572 mg of Boc-Pro, 0.37 ml of triethylamine and 0.4 ml of diethylphosphoryl cyanide. After 1 h at 25oC, TLC indicates that about 20% of the unreacted amine remains, and an additional 172 mg of Boc-Pro, 0.11 ml of triethylamine and 0.12 ml of diethylphosphoryl cyanide are added. After 16 h at 25°C, the reaction mixture is poured into aqueous sodium bicarbonate and extracted with methylene chloride.
  • the crude product is chromatographed on 180 g of silica gel. The column is packed and eluted in 15 ml fractions with 12% (4 M ammonia-methanol)/88% chloroform. Fractions 33-60 are combined and evaporated. The residue is dissolved in 5 ml of methylene chloride, and the solution is added dropwise to 70 ml of stirred hexane. Filtration and drying (0.1 mm, 25° C, 14 h) affords 1.27 g of pure THAM amide, the title product, as an easily-handled amorphous white solid.
  • reaction mixture is diluted with 75 ml of water and transferred to a 400 ml beaker.
  • the use of an air stirrer then solves both the stirring and foam control problems.
  • the suspension is stirred at 25oC for 10 min after the last of the phenyl chloroformate has been added.
  • the solids are isolated by filtration through a medium porosity sintered glass funnel and washed two times with acetone (10 ml) and methanol (10 ml).
  • the crude phenyl carbamate after drying for 16 h at 25oC and 0.1 mm, weighs 6.0 g, and is used immediately in the next step without further purification or characterization.
  • a solution of 5.32 g of the crude phenyl carbamate in 30 ml of pyridine and 10 ml of acetic anhydride is stirred at 25°C for 3 h.
  • the mixture is then cooled in an ice bath) treated with 5 ml of methanol, and stirred for 1 h at 0°C and 1 h at 25oC
  • the mixture is poured into ice and water containing 52 g of sodium bisulfate and extracted with 1:1 ethyl acetate/hexane.
  • the extracts are washed with water, aqueous sodium bicarbonate and brine, dried (sodium sulfate), and evaporated.
  • the crude title product (10.4 g) is chromatographed on a 700 g column of silica gel, packed and eluted once with 2000 ml of 50% ethyl acetate/hexane, then 230 ml fractions. Fractions 4-12 are combined, and upon evaporation of the solvent, the product is readily crystallized. The product is triturated with 25 ml of ether, then diluted with 15 ml of pentane and filtered.
  • the tetraacetate (210 mg) is dissolved in 5 ml of methanol and treated with 1 ml of 4 M ammonia in methanol, and the resulting yellow solution is stirred under nitrogen for 18 h at 25oC
  • the reaction mixture is then evaporated to dryness and the crude product (160 mg; >95% pure by TLC) is chromatographed on 21 g of silica gel.
  • the column is packed with 15% (4 M ammonia-methanol)/85% chloroform and eluted with 100 ml of 20%, followed by 200 ml of 25% (4 M ammonia-methanol)/75% chloroform (1 ⁇ 25 ml, then 2.5 ml fractions).
  • IC 50 4.9 ⁇ 10 -10 M.
  • Example 10 (Glucosaminocarbonyl)-Pro-Phe-N(Me)His-LVA-Ile- AMP, N-oxide.
  • A. BOC Removal A solution of 3.2 of Boc-Pro-Phe-N(Me)His-LVA-Ile-AMP, prepared as described in U.S. patent application, Serial No. 147,073 filed 20 January 1988, and in published European patent application 0173481, in 10 ml of methylene chloride is treated with 5.5 ml of trifluoroacetic acid, and the resulting, clear, light yellow solution is stirred at 25oC for 1 h.
  • reaction mixture is then diluted with 125 ml of methylene chloride and added dropwise over 30 min to a vigorously stirred mixture of 500 ml of water, 8 g of solid sodium bicarbonate and 200 ml of methylene chloride.
  • the aqueous layer is separated and extracted with eight 100 ml portions of methylene chloride.
  • the combined organic layers are dried over anhydrous sodium sulfate and evaporated to dryness.
  • the resulting colorless, amorphous solid (2.9 g) is 95-97% pure by TLC and is used immediately in part B below.
  • TLC Silica Gel GF R f 0.31 (10% 4M ammonia-methanol/90% chloroform; starting material exhibited Rf 0.54 on the same plate).
  • B. Activated glucosamine coupling A stirred solution of the part A product, 3.21 g of N-phenoxycarbonyl-D-glucosamine, tetra-0-acetate and 0.7 ml of triethylamine in 40 ml of dioxane is heated at 50°C for 64 h under nitrogen. The dioxane is then removed at reduced pressure (vacuum pump), and the residue is partitioned between aqueous sodium bicarbonate and chloroform. The aqueous layer is extracted with two additional 100 ml portions of chloroform. Removal of the chloroform at reduced pressure affords the coupled product, containing no unreacted starting peptide and contaminated only by the excess reagent.
  • TLC Silica Gel GF R f 0.54 (10% 4M ammonia-methanol/90% chloroform; starting material exhibited R f 0.31 and the tetra-acetate reagent, R f 0.85 on the same plate).
  • C Pyridine N-oxide formation A solution of the part B product in 75 ml of chloroform is treated with 853 mg of 80-85% pure m-chloroperbenzoic acid (approximately 15% excess) added in one portion. After 1 h at 25°C, the reaction mixture is treated with excess 10% aqueous sodium sulfite, transferred to a separatory funnel, and extracted with several 100 ml portions of chloroform.
  • Acetate removal A solution of 2.2 g of the part C product in 50 ml of methanol is treated with 10 ml of 4M ammonia-methanol, and the resulting solution is stirred for 18 h at 25V. (The solution darkens considerably during this time). Removal of the methanol in vacuo affords a dark residue (2.5 g) which is chromatographed on 200 g of silica gel. The column is packed and eluted (20 ml fractions) with 25% 4M ammonia-methanol/75% chloroform.
  • fractions 58-105 are combined and evaporated to dryness.
  • the residue is dissolved in 6 ml of 10% methanol/chloroform and reprecipitated by the rapid addition of 150 ml of pentane.
  • the methanol/chloroform solution should be added to the pentane. This avoids the formation of large aggregates and yields a more uniform, finely-divided product).
  • the solids are filtered and dried in a vacuum desiccator (25°, 6 h, 0.05 mm), thereby affording 1.15 g of the title product. (A 50 mg portion of the product that was subjected to more vigorous drying - - 48 h, 70°, 0.05 mm - - developed four less polar impurities totaling 1-5%). Physical characteristics are as follows:
  • Example 11 L-Histidinamide, 1-[[(2-deoxy-L-glucitol-2- yl)amino]carbonyl]-L-prolyl-L-phenylalanyl-N-[2- hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2- methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]- butyl]amino]carbonyl]hexyl]-N.alpha,-methyl-, N- oxide.
  • a solution of 110 mg of the title product of Example 10 in 8 ml of absolute ethanol is treated with 110 mg (large excess) of sodium borohydride, added in one portion.
  • the mixture is stirred in a nitrogen atmosphere for 12 h at 25°C.
  • the mixture is then cooled to 0oC and acidified carefully with IM methanolic hydrochloric acid..
  • the mixture is concentrated to dryness on the rotary evaporator, and the residue is taken up in methanol.
  • Ammonia-methanol (approximately 8M) is added until the mixture is basic.
  • the mixture is filtered, and the solids (which by TLC were shown to contain essentially none of the desired product) are discarded. Evaporation of the filtrate affords 215 mg of an amorphous solid.
  • the recovered material from the silica chromatography is purified on a 10 g column of 50-100 ⁇ C 18 reverse phase silica gel (Waters). The column is eluted with 65/35 methanol/water (75 drop fractions). Fractions 28-56 are combined and lyophilized (after first removing the methanol on the rotary evaporator), thereby affording 37 mg of the title product as an amorphous solid. Physical characteristics are as follows:
  • Example 12 L-Histidinamide, 1-[[[2-hydroxy-1,1-bis(hydroxy- methyl)ethyl]amino]carbonyl]-L-prolyl-L-phenylalanyl-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5- methylhexyl]-N.alpha.-methyl-, [1S-(1R*,2S*,3R*)- ]- or THAM-Pro-Phe-N-MeHis-CVG (Formula I-4) Refer to Chart I.
  • Example 13 L-Histadinamide, 1-[[(2-deoxy-D-glucopyranos-2- yl)amino]carbonyl]-L-prolyl-L-phenylalanyl-N-[1- (cyclohexylmethyl)-2,3-dihydroxy-5-methylhexyl]- N.alpha.-methyl-, [1S-(1R*,2S*,3R(*)]]- or Glu- Pro-Phe-N-MeHis-CVG (Formula K-4) Refer to Chart K. A solution of 85 mg of compound K-3 of Preparation 17 in 2 ml of methanol (saturated with ammonia) is allowed to stand overnight.
  • Example 14 L-Histidinamide, 1-[[[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino]carbonyl]-L-prolyl-O-methyl-L- tyrosyl-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5- methylhexyl]-, [1S-(1R*,2S*,3R*)]- or THAM-Pro- Tyr(OCH 3 )-His-CVG.
  • This compound is prepared using procedures analogous to those for the title product of Example 12.
  • Example 16 L-Histidinamide, N-[[[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]carbonyl]-L-phenylalanyl-N-[2- hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2- methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-N.alpha.-methyl-, [1S- [1R*,2R*,4R*(1R*,2R*)]]-.
  • Example 18 L-Histidinamide, 1-[[(2-deoxy-D-glucopyranos-2- yl)amino]carbonyl]-L-prolyl-L-phenylalanyl-N-[4- [[[5-amino-1-[[(4-piperidinylmethyl)amino]carbonyl]pentyl]amino]carbonyl]-2-hydroxy-5-methyl- 1-(2-methylpropyl)hexyl]-N.alpha.-methyl-, [1S- [1R*,2R*,4R*(R*)]]-, triacetate (salt) or Glu- Pro-Phe-N-MeHis-LVA-Lys-Ampip.
  • TLC Rf 0.12 in 6% methanol/methylene chloride with acetic acid present (UV) .
  • serinol hydrochloride Aldrich
  • This material is suspended in 16 ml of pyridine and 8.0 mL of acetic anhydride and the resulting mixture is stirred at room temperature for 4.5 h. The reaction is quenched with methanol and concentrated under reduced pressure. The residue is then diluted with water, acidified to pH between 3 and 4 (concentrated hydrochloric acid) and extracted two times with ethyl acetate. The organic extracts are washed with 1N hydrochloric acid, saturated aqueous sodium bicarbonate and brine, dried (magnesium sulfate), filtered and concentrated to give 3.52 g of the crude product as a yellow oil.
  • Mass spectrum found m/e 947.5743; other ions at m/e 513, 365, 215, 187, 124, 109, 86, 70.
  • TLC R f 0.14 and 0.43 in 10 and 15% (4M ammonia/methanol)/chloroform, respectively (phosphomolybdic acid).
  • Example 21 The title product of Example 21 (0.100 g) and 0.27 g of metachloroperoxybenzoic acid (MCPBA; Aldrich) all in 5 ml of chloroform is stirred at room temperature under a nitrogen atmosphere for 2.5 h.
  • MCPBA metachloroperoxybenzoic acid
  • TLC R f 0.31 in 15% (4M ammonia/methanol)/chloroform (phosphomolybdic acid).
  • This solid is suspended in 12 ml of acetic anhydride and 24 ml of pyridine and the resulting mixture is stirred at room temperature under a nitrogen atmosphere for 20 h.
  • the reaction is quenched with methanol and concentrated under reduced pressure.
  • the concentrate is diluted with water, acidified with concentrated hydrochloric acid and extracted two times with ethyl acetate.
  • the organic extracts are washed with IN hydrochloric acid, saturated aqueous sodium bicarbonate and brine, dried (magnesium sulfate), filtered and concentrated under reduced pressure to give 7.73 g the crude title product as a yellow oil.
  • Example 23 N-[Glucaminocarbonyl]-Pro-Phe-N[CH 3 ]-His-LVA-Ile- AMP (Formula N-4) Refer to Chart N.
  • TLC R f 0.45 in 70:25:5, chloroform/methanol/ammonium hydroxide (sulfuric acid).
  • N-[phenoxycarbonyl]-S-methyl-1-thiolincosaminide This material is suspended in 1.5 ml of acetic anhydride and 2.6 ml of pyridine and the resulting mixture is stirred at room temperature under a nitrogen atmosphere for 19 h. The reaction is quenched with methanol and concentrated under reduced pressure. The concentrate is diluted with water, acidified with concentrated hydrochloric acid and extracted two times with ethyl acetate. The organic extracts are washed with
  • Example 24 N-[S-Methyl -1- thiolincosaminide]carbonyl-Pro- Phe-N[CH 3 ]-His-LVA-Ile-AMP (Formula 0-4) Refer to
  • TLC R f 0.37% (70:25:5 chloroform:methanol:cone ammonium hydroxide) (sulfuric acid).
  • a mixture of the benzyl ester (54.7 g) from Part A, methanol (800 ml) and 10% palladitun-on-carbon catalyst (6.08 g) is hydrogenated at an initial pressure of 50 psi for 2 h.
  • the mixture is filtered and the filtrate concentrated.
  • a solution of the residue in diethyl ether (500 ml) containing 10 drops of methanol is boiled in the steam bath while adding hexane to keep the volume constant.
  • the product crystallizes; it is collected by filtration and dried in vacuo to give 34.9 g of the product.
  • the mixture AB is rechromatographed over a 400 ml silica gel column (elution with 7% ethyl acetate/hexane) to yield an additional 0.3698 g of isomer A, 0.6414 g of a mixture and 0.4980 g of isomer B.
  • the mixture is extracted three times with ethyl acetate and the combined extracts are washed in turn with cold 0.5 N hydrochloric acid, and then water, aqueous sodium bicarbonate and brine at ambient temperature, dried over magnesium sulfate and concentrated in vacuo.
  • the residue is chromatographed over a 150 ml silica gel column (elution with 2% methanol/methylene chloride containing 0.2% ammonium hydroxide) to yield 0.2617 g of the acylated product.
  • the amine is then coupled (coupling procedure B) described before the Experimental Section with N ⁇ -t-butoxycarbonyl-L-prolyl-L-phenylalanime and chromatographed over a 150 ml silica gel column (elution first with 2.5% methanol/methylene chloride containing 0.25% ammonium hydroxide followed by 3.5% methanol/methylene chloride containing 0.35% ammonium hydroxide) to yield first 0.0758 g of the desired coupled product (BocProPhe(imTos)NMHisCVA(OBz)GluAmp followed by 0.017 g of the starting peptide having the Boc and TBDMS groups removed.
  • the aqueous wash is backwashed twice with methylene chloride.
  • the combined organic layers are dried over magnesium sulfate and concentrated in vacuo.
  • the residue is chromatographed over a 100 ml. silica gel column (elution with 3.75% methanol/methylene chloride containing 0.38% ammonium hydroxide) to yield a 0.0495 g of the urea.
  • BocProPhe(imTos)NMHisCVAAmp To a nitrogen covered, ice bath cooled solution of 2.495 g of the Boc amine of Part B in 12.1 ml of methylene chloride is added dropwise 12.1 ml of trifluoroacetic acid over 11 min. The ice bath is removed and after 4.75 h, the solution is added dropwise over 5 min to a vigorously stirred mixture of 14.5 g of sodium bicarbonate in 100 ml of water and 60 ml of methylene chloride. After mixing well, the aqueous layer is separated and washed twice with methylene chloride- The combined organic fractions are dried over magnesium sulfate and concentrated in vacuo to yield 2.11 g of the amine free base.
  • a portion (0.50 g) of the crude amine is coupled (coupling procedure B described before the Experimental Section) with N ⁇ -t-butoxycarbonyl-L-prolyl-L-phenylalanine and chromatographed over a 200 ml silica gel column (elution first with 1.5% methanol/methylene chloride containing 0.15% ammonium hydroxide and then 2.5% methanol/methylene chloride containing 0.25% ammonium hydroxide) to yield in order compound A (0.2618 g), compound B (0.0513 g), and finally the desired coupled product C (0.2445 g).
  • the Boc amine product C of Part C (0.1212 g) is dissolved in methylene chloride (1.0 ml), covered with nitrogen and cooled in an ice bath. Trifluoroacetic acid (1.0 ml) is added dropwise from a pipette over 1 min. Stirring the solution in the cold is continued, and, after 1 h in the cold, a small aliquot is removed and diluted with about 5 drops of methylene chloride. It is then washed with 2 drops of aqueous sodium bicarbonate. The organic fraction is analyzed by TLC (elution with 5% methanol/methylene chloride containing 0.5% ammonium hydroxide). When deprotection is completed, there is a single product spot.
  • reaction mixture is analyzed by TLC (as stated above) and deprotection is completed. After 1 h, 25 min, reaction mixture is concentrated on a rotary evaporator. Residue is dissolved in methylene chloride and washed once with aqueous sodium bicarbonate and backwashed twice with methylene chloride. Pooled organic fractions are dried over magnesium sulfate and concentrated on rotary evaporator to form a hard foamy residue which amounts to 0.3304 g.
  • the product (0.436 g) from the above reactions is combined and dissolved in dioxane (2.3 ml).
  • the mixture is covered with nitrogen and treated with triethylamine (0.053 g) followed by the title product from Preparation 3 (0.25 g).
  • the solution is heated in a oil bath at 50°. After heating for 20 h, 15 min, the oil bath is removed and the reaction mixture is allowed to cool. It is analyzed by TLC (elution with 5% methanol/methylene chloride containing 0.5%. ammonium hydroxide); the reaction is complete.
  • the reaction mixture is concentrated on a rotary evaporator. The residue is dissolved in methylene chloride and washed once with aqueous sodium bicarbonate.
  • the aqueous wash is backwashed twice with methylene chloride.
  • the pooled organic fractions are dried over magnesium sulfate and concentrated on rotary evaporator to a gummy residue which amounts to 0.7861 g.
  • This material is adsorbed onto silica gel and placed on a 150 ml silica gel column and settled in 3% methanol/methylene chloride containing 0.3% ammonium hydroxide.
  • the column is eluted with the same solvent to fraction 181 and then with 3.5% methanol/methylene chloride containing 0.35% ammonium hydroxide; 5.4 ml fractions are collected and fractions 216-302 are combined.
  • the product amounts to 0.2978 g.
  • the peptide of Part D (0.1738 g) is dissolved in chloroform (4.5 ml), covered with nitrogen and m-chloroperoxybenzoic acid (0.044 g) is added. The clear solution is stirred at room temperature for 1.5 h. The mixture is analyzed by TLC (elution with 5% methanol/methylene chloride containing 0.5% ammonium hydroxide. The reaction mixture is poured by pipette into aqueous sodium bicarbonate. After mixing well, the aqueous layer is separated and extracted twice with chloroform. The combined organic fractions are washed once with aqueous sodium bicarbonate and once with dilute brine. The aqueous fractions are backwashed with chloroform.
  • Product D (0.040 g) of Part E which by TLC is a mixture of the titled product (B, C) and the partially hydrolyzed ortho ester product E is dissolved in 1.2 ml of methanol, covered with nitrogen, and 1.46 N hydrochloric acid in diethyl ether (0.08 ml) is added.
  • the BocProPhe(imTos)NMHisCVAAmp prepared as in Part C of Example 33 (0.4959 g) is dissolved in methylene chloride (2.1 ml), covered with nitrogen, and cooled in an ice bath. Trifluoroacetic acid (2.1 ml) is added dropwise from an addition funnel over 2.5 min. Stirring is continued in the cold for 1 h, 15 min. The reaction mixture is concentrated on a rotary evaporator and the residue is dissolved in methylene chloride and washed once with aqueous sodium bicarbonate. The aqueous wash is backwashed twice with methylene chloride.
  • the pooled organic fractions are dried over magnesium sulfate and concentrated on a rotary evaporator to a rigid foamy residue which amounts to 0.4298 g.
  • This material is dissolved in dimethylformamide (9.2 ml), covered with nitrogen and treated with triethylamine (0.065 g) followed by the title product from Preparation 10 (0.46 g).
  • the residue is heated in an oil bath at 50°. After 5.75 h, it is analyzed by TLC (elution with 5% methanol/methylene chloride containing 0.5% ammonium hydroxide). The reaction is complete and the reaction mixture is concentrated on a rotary evaporator at vacuum pump pressure to yield a gummy residue that amounts to 1.0628 g.
  • reaction mixture is concentrated on a rotary evaporator to an amorphous solid residue that amounts to 0.2454 g.
  • This material is adsorbed onto silica gel and placed on a 150 ml silica gel column, settled in 20% methanol/methylene chloride containing 0.5% ammonium hydroxide.
  • the mixture is eluted with the same solvent and 5.6 ml fractions are collected. The following fractions are combined:
  • the DGLUAC(O)ProPhe(im-Tos)NMHisCVAAmp, prepared in Part A of Example 34 (0.2553 g) is dissolved in chloroform (4.3 ml), covered with nitrogen, and m-chloroperbenzoic acid (0.049 g) added. The solution is stirred at room temperature for 1 h, 5 min. The mixture is analyzed by TLC (elution with 5% methanol/methylene chloride containing 0.5% ammonium hydroxide). The reaction is complete and is treated with 5.0 ml of a 10% (10 g/100 ml) aqueous solution of sodium sulfite. It is mixed well and the aqueous fraction is separated and washed three times with chloroform.
  • the pooled organic fractions are dried over magnesium sulfate and concentrated on a rotary evaporator to an amorphous solid that amounts to 0.2661 g.
  • This material is adsorbed onto silica gel and placed on a 150 ml silica gel column, settled in 5% methanol/methylene chloride containing 0.5% ammonium hydroxide. the mixture is eluted with the same solvent and 5.4 ml fractions are collected. Fractions 94-180 are combined to give 0.2309 g. of the product.
  • the protected peptide of Part A (0.2309 g) is dissolved in methanol (5.7 ml), covered with nitrogen, and 5.45 M ammonia in methanol (1.2 ml) is added. After stirring at room temperature for 17 h, 15 min, the reaction takes on a light brown color. It is analyzed by TLC (elution with 20% methanol/methylene chloride containing 0.5% ammonium hydroxide) showing the reaction is complete. One major polar product plus several less polar materials remain, with no starting peptide. The reaction mixture is concentrated on a rotary evaporator to about 1/3 volume. A small amount of silica gel is added and the mixture is concentrated further to a free flowing solid residue.
  • Sample B is solubilized in a small amount of methanol, mixed with 10 ml of water and lyophilized to give 51.5 mg. of the titled product.
  • a suspension of azide R-2 of Preparation 28 (0.64 g) and 5% palladium/calcium carbonate (0.27 g) in absolute ethanol (50 ml) is hydrogenated at atmospheric pressure for 2.25 hours, filtered through Celite, and the filtercake is washed with ethanol. The combined filtrate is concentrated in vacuo to give 0.59 g of crude title product as a greenish-brown solid.
  • TLC Silica Gel GF: R f is 0.42 in 20% acetone/methylene chloride.
  • Preparation 31 (Formula R-5) Refer to Chart R. A suspension of R-4 of Preparation 30 (0.292 g) and 10% palladium/charcoal (0.065) in methanol (25 ml) is hydrogenated at 50 p.s.i. for 4 h, filtered through Celite and the filtercake is washed with methanol (25 ml). The combined filtrate is concentrated in vacuo to give 0.239 g of crude title product. Crude title product is taken into further reaction.
  • TLC Silica Gel GF: R f is 0.44 in 10% methanol/methylene chloride.
  • TLC Silica Gel GF
  • R f is 0.14 in 20% of 5 M ammonia/methanol in methylene chloride .
  • TLC Silica Gel GF: Rf is 0.49 in 10% methanol/methylene chloride.
  • Example 37 ⁇ -N-AcGlc- ⁇ -N-Boc-Asn-Phe-N-MeHis-Leu[CH(OH)CH 2 ]- Val-Ile-AMP (Formula R-11) Refer to Chart R. A solution of R-10 of Preparation 35 (0.219 g) in methanol (7.5 ml) is treated at ambient temperature under argon with water (1.5 ml) followed by 1N potassium hydroxide (1.5 ml).
  • the solution is stirred at room temperature for 2 h, concentrated in vacuo and diluted with ice-cold brine (60 ml) and methylene chloride (60 ml). The resulting suspension is centrifuged to give an aqueous layer, insoluble white residue and methylene chloride layer. The white solid is dissolved in methanol and concentrated in vacuo to give a white solid (0.164 g) which is homogeneous by TLC.
  • TLC Silica Gel GF
  • R f is 0.55 in 1:1 ethyl acetate-hexane.
  • IC 50 3.6 ⁇ 10 -8 M.
  • a suspension of azide S-2 of Preparation 36 (0.55 g) and 5% palladium/calcium carbonate (0.22 g) in absolute ethanol (17 ml) is hydrogenated at atmospheric pressure for 2.5 h at room temperature.
  • the suspension is filtered through Celite, and the filtercake is washed with absolute ethanol.
  • the combined filtrate is concentrated in vacuo to give 0.427 g of off-white solid.
  • the crude product is taken into further reaction. Physical characteristics are as follows:-.
  • the crude product is taken into further reaction without purification.
  • TLC Silica Gel GF: Rf is 0.49 in 10% of 4M ammonia/methanol in methylene chloride.
  • Example 38 ⁇ -Man- ⁇ -N-Boc-Asn-Pro-Phe-N-MeHis-Leu[CH(OH)CH 2 ]- Val-He-AMP (Formula S-7) Refer to Chart S.
  • the suspension is extracted three times with 80 ml of chloroform each time, then three times with 100 ml of ethyl acetate each time, and the organics are combined, dried over sodium sulfate, filtered and concentrated.
  • Example 39 ⁇ -N-AcGlc- ⁇ -N-Boc-Asn-Pro-Phe-N(Me)His-LVA-Ile-AMP-NO
  • TLC Silica Gel GF: R f is 0.13 in 25% of 5 M ammonia/methanol in methylene chloride. R f is 0.30 in 30% of 5 M ammonia/methanol in methylene chloride.
  • Example 40 ⁇ -N-AcGlc-a-Asn-Pro-Phe-N(Me)His-LVA-Ile-AMP Physical characteristics are as follows:
  • TLC Silica Gel GF
  • R f is 0.11 in 30% of 5 M ammonia/methanol in methylene chloride
  • R f is 0.13 in 30% of 9:1 methanol-ammonium hydroxide in methylene chloride.
  • Example 42 ⁇ -N-AcGlc- ⁇ -N-Boc-Asn-Pro-Phe-N(Me)His-LVA-Ile-THAM
  • TLC Silica Gel GF
  • R f is 0.28 in 30% of 4 M ammonia/methanol in methylene chloride.
  • Example 43 ⁇ -N-AcGlc-C(O)-(CH 2 ) 2 -C(O)-Pro-Phe-N(Me)His-LVA-Ile- AMP Physical characteristics are as follows: FAB (High Resolution) Mass Spec: Found: 1132.639. Other m/z: 586, 478, 365, 327, 299, 222, 204, 152, 124, 109, 86, 70.
  • TLC Silica Gel GF
  • R f is 0.26 in 30% of 4.5 M ammonia/methanol in methylene chloride.
  • TLC Silica Gel GF: R f is 0.09 in 20% of 4 M ammonia/methanol in methylene chloride. R f is 0.26 in 30% of 4 M ammonia/methanol in methylene chloride.
  • Example 45 ⁇ -N-AcGlc- ⁇ -N-Boc-Asn-Pro-Phe-His-Cha ⁇ (CH 2 NH)Phe-NH 2 Physical characteristics are as follows: FAB (High Resolution) Mass Spec: Found: 1102.597. Other m/z: 242, 204, 120, 110, 81, 70, 57. TLC (Silica Gel GF) : R f is 0.11 in 20% of 4.5 M ammonia/methanol in methylene chloride.
  • the reaction mixture is stirred at room temperature overnight then divided into two portions and concentrated on the rotary evaporator using dry ice-acetone traps and Hi vacuum to reduce the volume to about 300 ml.
  • the two portions are freeze dried and triturated four times with 200 ml of boiling ethyl acetate to yield 35.32 g and 34.14 g of the crude product.
  • the structure is confirmed by NMR.
  • the mixture is then warmed to 0°C with tap water and then held at that temperature with an ice bath. There is then added 130 ml of 30% hydrogen peroxide over 10 min. The temperature is maintained at 40-45°C during the addition. The ice bath is allowed to melt and the reaction warmed to room temperature and stirred for 16 h. With mechanical stirring the reaction is cooled to 10°C with an ice bath and there is added over 10 min 260 ml of 3 N hydrochloric acid. The cold bath is removed and the mixture is stirred at room temperature for 2.5 h. Brine and diethyl ether are then added to effect a two phase mixture. The aqueous layer is separated and extracted twice with diethyl ether.
  • the residue is diluted with 350 ml of diethyl ether, washed in turn with dilute brine, aqueous sodium bicarbonate and again with dilute brine, dried over magnesium sulfate and concentrated in vacuo.
  • the residue is chromatographed over a 2000 ml silica gel column (elution with 2% ethyl acetate:methylene chloride to fraction 320 and then 4% ethyl acetate:methylene chloride). Thirty-five ml fractions are collected to fraction 290 and then 275 ml fractions are collected. Fractions 295-337 are combined to yield 13.203 g of the tetrahydropyranyl ether. Mass spectrum supports the proposed structure.
  • the analytical sample has m.p., 106-108oC and [ ⁇ ] D -22oC (ethanol).
  • the IR and mass spectra support the proposed structure. The assigned stereochemistry is established by an NOE-Difference experiment. Physical characteristics are as follows: Anal. Found: C, 70.52; H, 10.25. The less polar material from the second column, fractions 112- 146, is recrystallized from diethyl ether-pentane to give 0.20 g, m.p. 120-121 and 0.089 g, m.p. 118-120oC
  • the analytical sample has m.p. 120-121°C and [ ⁇ ] D -10°C (ethanol).
  • the IR, NMR and mass spectra are identical to those of the (2S.4S.5S) enantiomer (Formula T-10, refer to Chart T)
  • a precipitate is collected on a filter and washed with 5% ethyl acetate:hexane.
  • the combined filtrates are concentrated in vacuo.
  • the residue is treated with 250 ml of 5% ethyl acetate:hexane, slurried and then filtered.
  • the solid filter cake is washed with 5% ethyl acetate:hexane and the combined filtrates are concentrated in vacuo.
  • the residue is chromatographed over a 600 ml silica gel column (elution with 45% methylene chloride:hexane) and 22 ml fractions are collected.
  • a mixture of 2.822 g of the azide of Part A and 1.0 g of 5% Pd/C in 100 ml of absolute ethanol is hydrogenated at atmospheric pressure. At intervals of 15 min, 1.5 h, 2.75 h and 3.5 h the system is evacuated and rechanged with hydrogen. After 4.25 h the catalyst is removed by filtration and the filtrate concentrated in vacuo. A solution of the residue in ethyl acetate is washed once with aqueous sodium bicarbonate, once with brine, dried over magnesium sulfate and concentrated in vacuo. The residue is concentrated two additional times from toluene to yield 2.8 g of crude amine. The structure is supported by NMR and mass spectra.
  • the reaction mixture is treated with 16.9 ml of pyridine, cooled to 3-7°C in an ice bath, and finally treated dropwise over 15 min with a solution of 25.0 ml of phenyl chloroformate in a small amount of tetrahydrofuran.
  • the mixture is stirred for 30 min at 5°, then 5-6 h at room temperature.
  • the solids pyridine hydrochloride
  • the filtrate is concentrated in vacuo.
  • the material is maintained on a Kugelrohr apparatus (0.2 mm) until excess MSTFA has been removed.
  • the remaining viscous residue weighs 89.4 g and, due to its hydrolytic instability, is used in subsequent coupling reactions without further purification. Physical characteristics are as follows: NMR ( ⁇ , CDCl 3 ): 7.3-6.9, 3.66, 0.00.
  • Example 46 N-[[[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino]carbonyl]-Pro-Phe-N(Me)His-LVA-Ile-AMP, pyridine N-oxide (Alternative Process for Making the Compound of Formula B-4)
  • a stirred solution of 465 mg of Pro-Phe-N(Me)His-LVA-He-AMP, prepared as described in Example 4, Part A, and 0.56 ml of triethylamine in 4 ml of anhydrous dimethylformamide is treated dropwise with 1.28 g of the tris (trimethylsilyloxymethyl) urethane of Preparation 49, and the resulting mixture is stirred in a nitrogen atmosphere at 35°C for 64 h.
  • the dimethylformamide is then removed in vacuo, and the residue is dissolved in 20 ml of methanol containing a trace of ammonia (pH 9-10).
  • the solution is stirred and treated with 2 ml of 1 M hydrochloric acid/methanol.
  • the resulting solution has a pH under 1.
  • 5 ml of 1 M aqueous potassium hydroxide is added, and the methanol is removed under pressure.
  • the residue is taken up in brine and chloroform and then extracted with three 100 ml portions of chloroform. The extracts are dried over anhydrous sodium sulfate and evaporated, thereby affording a crude product weighing 902 mg.
  • TLC analysis (16% 7 M ammonia-methanol/84% trichloromethane) showed no starting peptide remaining; the major product is THAM urea, the title product of Example 2, contaminated with 1-5% of what appears to be the less polar oxazoline impurity based on TLC mobility.
  • the crude product is dissolved in 10 ml of chloroform and treated with 133 mg of m-chloroperbenzoic acid added in one portion. After 60 min in at 25°, TLC analysis (16% 7 M ammonia-methanol/84% trichloromethane) shows about 90% title product and 10% of something which appears to be starting material. An additional 20 mg of m-chloro-perbenzoic acid is added., but after 30 min, TLC indicates that the reaction has progressed no further. The reaction mixture is evaporated to dryness in vacuo, and the residue is taken up in 12% 7 M ammonia-methanol/88% trichloromethane (the anticipated chromatography solvent). Part of the product dissolves readily, leaving behind a white, easily-filtered solid.
  • the solids are filtered off using a medium-porosity sintered glass funnel and washed with three 2 ml portions of the 12% mixed solvent. Concentration of the combined filtrate yields 900 mg of the crude product. (TLC analysis of the solids shows that they are excess m-chloro-perbenzoic acid and the corresponding reduced acid, and contains none of the desired product.)
  • HPLC Major component 99.36% [30 cm Waters C 18 column 16; 50% 0.05 M ammonium acetate/25% methanol/25% acetonitrile; 1 ml/min; t r 9.2 min].
  • Methanesulfonate A 0.02 M solution of methanesulfonic acid in water is prepared (192.2 mg in 100 ml of water). A suspension of 100 mg of the title product of Example 46 in 5 ml of water and 5 ml of the stock methane-sulfonic acid solution is stirred until it becomes a clear solution. Lyophilization followed by drying of the solids at 65-70oC for 18 h, 0.05 mm, yields 113 mg of the methanesulfonate salt (no electrostatic problems). The salt softens at 135° and becomes clear, but not mobile, at 160-170o.
  • This material shrinks during drying from a very light, fluffy, electrostatically-charged solid into a much denser, low-volume, non-charged, easily-handled amorphous solid, which softens at 125° and becomes clear (not mobile) at 135-145°.
  • Hydrochloride % CHN (corrected for water): C, 58.31; H, 7.45; N, 13.60.
  • Methanesulfonate % CHN (corrected for water): C, 56.09; H, 7.37; N, 12.85.

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Abstract

La présente invention décrit de nouveaux peptides inhibiteurs de rénine comportant un insert à l'état de transition non clivable, qui correspond à la position 10, 11 du substrat de rénine (angiotensinogène), et une fraction alkyle à substitution polyhydroxy liée directement par carbone ou azote à la terminaison N. De tels inhibiteurs sont utiles dans le diagnostic et le traitement de l'hypertension dépendant de la rénine et d'autres maladies associées.
EP89902438A 1988-02-01 1989-01-27 Peptides inhibiteur de renine avec groupes terminaux polaires Pending EP0397779A1 (fr)

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US5583198A (en) * 1989-12-22 1996-12-10 Commonwealth Scientific And Industrial Research Organization Amino acids, peptides or derivatives thereof coupled to fats
DE4037437A1 (de) * 1990-11-24 1992-05-27 Hoechst Ag Aminodiol-derivate
US5691368A (en) * 1995-01-11 1997-11-25 Hoechst Marion Roussel, Inc. Substituted oxazolidine calpain and/or cathepsin B inhibitors
US6004928A (en) * 1997-05-13 1999-12-21 Biomeasure, Incorporated Method of treating hyperlipidemia
US7026289B2 (en) 1997-05-13 2006-04-11 Societe De Conseils De Recherches Et D'applications Scientifiques, Sas Method and compositions for treating hyperlipidemia and other conditions
DK0981363T3 (da) 1997-05-13 2003-12-01 Sod Conseils Rech Applic Somatostatinagonister til reduktion af legemsvægt
US5968903A (en) * 1998-05-07 1999-10-19 Biomeasure, Incorporated Inhibition of H. pylori proliferation
US6455554B1 (en) 1999-06-07 2002-09-24 Targacept, Inc. Oxopyridinyl pharmaceutical compositions and methods for use
DK1401863T3 (da) 2001-06-08 2009-05-25 Ipsen Pharma Kimære somatostatin-dopamin-analoge
US20080207527A1 (en) * 2004-09-17 2008-08-28 Comentis, Inc. Bicyclic Compounds Which Inhibit Beta-Secretase Activity and Methods of Use Thereof
KR20080015079A (ko) 2005-04-08 2008-02-18 코멘티스, 인코포레이티드 베타 세크레타제 활성을 억제하는 화합물 및 이것의 사용방법
CN107446053A (zh) 2008-06-12 2017-12-08 益普生生物创新有限公司 癌症的抑制
KR101642363B1 (ko) 2008-06-12 2016-07-25 입센 바이오이노베이션 리미티드 신경내분비계 질환의 억제
GB0820970D0 (en) 2008-11-17 2008-12-24 Syntaxin Ltd Suppression of cancer
AU2014280847B2 (en) 2013-06-13 2019-07-04 Antisense Therapeutics Ltd Combination therapy
CN108558685B (zh) * 2017-06-08 2020-11-20 西安力邦制药有限公司 2,6-二取代苯酚葡甲胺类衍生物及应用

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EP0163237A3 (fr) * 1984-05-29 1988-04-27 Merck & Co. Inc. Inhibiteurs di- et tri-peptidiques de la rénine
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NL8800100A (nl) * 1987-01-21 1988-08-16 Sandoz Ag Nieuwe peptidederivaten en werkwijzen voor het bereiden en toepassen van deze derivaten.

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Also Published As

Publication number Publication date
JPH03502328A (ja) 1991-05-30
DK183090D0 (da) 1990-07-31
KR900700502A (ko) 1990-08-13
ZA89723B (en) 1989-10-25
IL89110A0 (en) 1989-08-15
DK183090A (da) 1990-09-14
AU3063089A (en) 1989-08-25
EP0329295A1 (fr) 1989-08-23
AU632468B2 (en) 1993-01-07
WO1989007109A1 (fr) 1989-08-10
NZ227786A (en) 1992-02-25

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