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WO2000061631A1 - Modified pentapeptide antagonists of the atrial natriuretic peptide clearance receptor - Google Patents

Modified pentapeptide antagonists of the atrial natriuretic peptide clearance receptor Download PDF

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Publication number
WO2000061631A1
WO2000061631A1 PCT/GB2000/001319 GB0001319W WO0061631A1 WO 2000061631 A1 WO2000061631 A1 WO 2000061631A1 GB 0001319 W GB0001319 W GB 0001319W WO 0061631 A1 WO0061631 A1 WO 0061631A1
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Prior art keywords
phenyl
resin
secbu
para
dmf
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PCT/GB2000/001319
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French (fr)
Inventor
Chris Allan Veale
Philip Duke Edwards
Robert Toms Jacobs
Timothy Wayne Davenport
Paul James Warwick
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AstraZeneca AB
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AstraZeneca AB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • ANP is a member of a family of natriuretic peptide hormones, which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP).
  • the natriuretic peptides have a number of actions on the cardiovascular system, including; natriuresis, diuresis, and relaxation of vascular smooth muscle.
  • ANP is a 28-amino acid cyclic peptide which is produced in atrial myocytes in response to increases in heart rate and atrial stretch.
  • ANP receptors There are two biologically- and functionally-distinct classes of ANP receptors. The first one is linked to guanylate cyclase and is thought to mediate the physiological effects of ANP via increases in intracellular cGMP levels. These guanylate cyclase receptors are further divided into the ANP-A and ANP-B receptors according to their relative affinity for different natriuretic peptides. The second class of ANP receptors do not mediate the cardiovasculature effects of the hormone and are thought to mainly serve a clearing function of ANP from the extracellular circulation. This receptor is known as the atrial natriuretic peptide clearance receptor (ANPCR).
  • ANPCR atrial natriuretic peptide clearance receptor
  • Natriuretic peptides have a very short half life in vivo, and there are thought to be two major modes of their clearance from systemic circulation. One is via proteolytic inactivation by the enzyme neutral endopeptidase (NEP). The other is via binding to the ANP clearance receptor which is expressed on the vascular endothelium. Binding to the clearance receptor is followed by internalization and degradation of the peptide.
  • NEP neutral endopeptidase
  • the lung is thought to play a major role in ANP clearance, and studies have found over 50% of ANP is cleared in a single pass through the lungs. Approaches based on the inhibition of NEP are further complicated by the number of physiologically important peptide hormones which are substrates for this enzyme.
  • the ANPCR is thought to be primarily responsible for removal of ANP in the pulmonary vasculature, and the ANPCR is the dominate ANP receptor in lung tissue. Additionally, blockade of the clearance receptor in the lung was thought to provide a pulmonary selective approach to reduction of pulmonary blood pressure due to the presence of both the ANP-A,B and ANPCR receptors in the lung and by the proximity of these receptors to the site of ANP synthesis. For these reasons blockade of the ANPCR was chosen as the best approach to increase endogenous levels of ANP.
  • An ANPCR antagonist could have therapeutic usefulness in treating pulmonary hypertension secondary to COPD.
  • all natriuretic peptides i.e., ANP, BNP and CNP
  • an ANPCR antagonist may also be useful for protection of the transplanted heart given that plasma levels of BNP are elevated in this situation.
  • An ANPCR antagonist may have the greatest therapeutic utility in the treatment of congestive heart failure (CHF), by virtue of raising plasma concentrations of ANP and BNP.
  • CHF congestive heart failure
  • the present invention is directed to synthetic analogs of atrial peptides and more particularly o synthetic linear peptide analogs which find use as diuretics, natriuretics and/or vasodilators, or as intermediates for or modulators of such useful compounds, together with methods for their production and use.
  • lactams of the generic structure shown below were found to be blockers of the ANP clearance receptor (ANPCR).
  • lactam molecules which contained a D- amino acid or a sarcosine residue in position R7 has good metabolic stability. This is in contrast to the natural hormone ANP which has poor metabolic stability.
  • Figure 1 is a chart showing the dose-response effect of IV or vehicle administered orally 165 min before evaluating the change in right intraventricular peak systolic pressure
  • RVSP mean systemic arterial pressure
  • MAP mean systemic arterial pressure
  • Figure 2 is a chart showing the effect of vehicle or I (30 mg/kg, p.o.; top panel) and IV
  • the compounds of the instant invention are linear peptide compounds having the structure:
  • R 2 should be hydrogen or a C,-C 4 alkyl group, but is preferably hydrogen or methyl.
  • R 6 is a C 3 -C 5 branched or unbranched alkyl group, preferably isobutyl or sec-butyl;
  • R 8 is L-isoleucine-NH 2 , D-isoleucine-NH 2 , -CH 2 -cyclopentyl, -CH 2 -2-tetrahydrofuranyl, tert- butylglycine-NH 2 , n-butyl, NH-cyclopentyl, NHCH 2 -2-furanyl, -NHCH 2 -pyrininyl, -NHCH 2 - cyclohexyl, -NH-2-indolizidinyl, D-leucinol, -NH-isobutyl, L-allo-isoleucine-NH 2 , 1- hydroxycycloleucinol, 2-(aminomethyl)-l-ethyl-pyrrolidine, or (S)-NH-2-methylbutyl, but if R 7 is -NH-2-indolizidine, then R 8 is absent; and
  • Representative compounds according to the present invention include those of the structure:
  • Compounds of the present invention are shown to have natriuretic, diuretic and hypotensive activity in the intact mammal, and may possess vasorelaxant activity or inhibit the release of aldosterone and renin.
  • these compounds, and compositions containing them may be used as therapeutic agents in the treatment of various edematous states such as, for example, congestive heart failure, nephritic syndrome and hepatic cirrhosis, pulmonary disease, in addition to hypertension and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
  • the present invention also provides compositions comprising an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, serve to provide the above-recited therapeutic benefits.
  • Such compositions can also be provided together with physiologically-tolerable liquid, gel or solid diluents, adjuvants and excipients.
  • the compounds of the present invention may also be combined with other compounds known to be adjuvants for, or otherwise used as, therapeutic agents for the above or related indications.
  • compositions may be administered to humans in a manner similar to other therapeutic agents and, additionally, to other mammals for veterinary use, such as with domestic animals.
  • dosage required for therapeutic efficacy will range from about 0.01 to 1000 mg/kg, more usually 0.1 to 100 mg/kg of the host body weight.
  • dosages within these ranges can be administered by constant infusion over an extended period of time until the desired therapeutic benefits have been obtained.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like.
  • compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intravenously.
  • Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and, in some cases, oral formulations.
  • suppositories traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10% preferably l%-2%.
  • Oral formulations include such normally-employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders, and contain 10%-95% of active ingredient, preferably 25%-70%.
  • the peptide compounds may be formulated into compositions as neutral or salt forms.
  • Pharmaceutically-acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • compounds of the present invention may also be employed as intermediates in the synthesis of such useful compounds.
  • compounds of the present invention whose activity levels are reduced or eliminated entirely can serve to modulate the activity of other diuretic, natriuretic or vasorelaxant compounds, including compounds outside the scope of the present invention, by, for example, binding to alternate receptors, stimulating receptor turnover, or providing alternate substrates for degradative enzyme or receptor activity and thus inhibiting these enzymes or receptors.
  • such compounds may be delivered as admixtures with other active compounds or may be delivered separately, for example, in their own carriers.
  • Compounds of the present invention may also be used for preparing antisera for use in immunoassays employing labeled reagents, usually antibodies.
  • the polypeptides can be conjugated to an antigenicity-conferring carrier, if necessary, by means of dialdehydes, carbodiimide or using commercially-available linkers.
  • These compounds and immunologic reagents may be labeled with a variety of labels such as chromophores; fluorophores such as, e.g., fluorescein or rhodamine; radioisotopes such as l25 1, 5 S, ,4 C, or 3 H; or magnetized particles, by means well known in the art.
  • labeled compounds and reagents can find use as, e.g., diagnostic reagents.
  • Samples derived from biological specimens may be assayed for the presence or amount of substances having a common antigenic determinant with compounds of the present invention.
  • monoclonal antibodies may be prepared by methods known in the art, which antibodies can find therapeutic use, e.g., to neutralize overproduction of immunologically -related compounds in vivo.
  • Synthesis Compounds within the scope of the present invention may be synthesized chemically by means well known in the art. One example of such a scheme may be generally depicted as:
  • the starting material is attached to a resin and the compound is constructed by the successive addition of various building blocks.
  • the resin may be attached to a starting material that will end up in a more central location of the desired compound; and through the use of commonly-known protecting groups, the compound may be extended in multiple directions. Examples
  • the resin was swelled in DMF and 250 mL of a 1 M (66.1 g diluted to 500 mL with DMF) solution of benzenethiol sodium salt in DMF was added and shaken for 1 hr. The resin was drained, washed 8X DMF and the remaining 250 mL of the 1 M solution was added and shaken for an additional hour. The resin was filtered and washed 3X DMF, 3X MeOH, 3X DMF, 3X MeOH, 8X CH 2 C1 2 , Npit test positive. Fmoc protection. To the CH 2 C1 2 swelled resin was added a solution of 12 mL DIPEA in 150 mL dry CH 2 C1 2 .
  • Fmoc-Cl (23.73 g) was dissolved in 150 mL dry CH 2 C1 2 followed by 12 mL DIPEA. This was added to the resin and reaction shaken for three hours. The resin was filtered and washed 8X CH 2 C1 2 , Npit Test, negative.
  • Boc-D-methionine (24.2 g, 97.2 mmol), L- isoleucine methyl ester hydrochloride (17.7 g, 97.2 mmol), hydroxybenztriazole hydrate (16.3 g, 117 mmol), 1 -(3 -dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.5 g, 117 mmol) and diisoproplylethylamine (35ml, 200 mmol) were dissolved in DMF (300 mL). The reaction was stirred under nitrogen for 16 hours, then it was diluted with water (1000 mL) and extracted with ethyl acetate (2 x 250 mL).
  • the second crop was determined by HPLC to be of sufficient quality to combine with the first.
  • the combined product was dried in vacuo for 30 minutes at 50 °C to yield 25.3 g white solid (89%).
  • Boc-D-freidingerlactam-L-isoleucene methyl ester t-Boc-D-methionine-L-isoleucine methyl ester (25.3 g, 67.2 mmol) was dissolved in dry methylene chloride (250 mL) under nitrogen and chilled in an ice bath.
  • Trimethyloxonium tetrafluoroborate (9.94 g, 67.2 mmol) was added in one portion. The ice bath was removed and the reaction was allowed to react for 3 hours.
  • the solids were collected by vacuum filtration and washed with hexanes (100 mL). A second crop of solids was obtained from the mother liquor and washed with hexanes (50 mL). The second crop was determined by HPLC to be of sufficient quality to combine with the first. The combined product was dried in vacuo at 50 °C for 30 minutes to yield 17.0 g white solid (77%).
  • Boc-D-zadingerlactam-L-isoluecene Boc-D-kaolin-L-isoleucene methyl ester (15.6 g, 47.5 mmol) was dissolved in THF (60 mL) and methanol (60 mL). Lithium hydroxide (4.2 g, 100 mmol) dissolved in water (60 mL) was added. After one hour, TLC analysis showed no remaining starting material. All the solvent was removed by rotary evaporation. The resulting white solid was dissolved in water (300 mL), washed with methylene chloride (50 mL), and acidified with IM HCl (105 mL). A white precipitate formed.
  • Boc-D- fingerlactam-L-isoluecene (8.86 g, 28.2 mmol) was suspended in methylene chloride and trifluoroacetic acid (30 mL) was added. After 1.5 hours the volatiles were removed by rotary evaporation. Methylene chloride (2 x 50 mL) was added and evaporated to rid remaining TFA. The residue was cooled in an ice bath and dioxane (42ml) and 10% aqueous sodium carbonate (71ml) were added. FMOC chloride (8.8 g, 33.9 mmol) was added to the resulting solution in four portions.
  • the combined organic extracts were dried with Na 2 SO 4 , filtered through celite and concentrated.
  • the material was loaded on a silica gel flash column (5 x 20 cm) and the product was eluted off using a gradient of 10 to 30% EtOAc in hexane.
  • the FMOC-aminopyridone (15.3 g) was dissolved in 3:1 CH 2 C1 2 in TFA (100 mL). After 14 h the solvents were removed under reduced pressure. The residue was concentrated from Et,O (3 x 20 mL) to give the product (N-fluorenylmethyloxycarbonyl pyridone- isoleucine) (13.6 g) as a foam.
  • the residue was purified by silica gel flash chromatography (6 x 23 cm) using a gradient from 20 to 67% EtOAc in hexane to afford the mononitroimidazole product (8.17 g; R f of 0.33 in 30% EtOAc in hexane).
  • the obtained material and 10% Pd-C (1.4 g) were reacted in EtOH (135 mL) under 47 psi H 2 for 2 h.
  • the reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure.
  • the amino-imidazole product (7.05 g) was dissolved in 10% Na 2 CO 3 (aq) (70 mL) and dioxane (42 mL) and the solution was cooled in an ice-water bath.
  • the FMOC-amino compound (5.07 g) was dissolved in 3:1 CH 2 C1 2 :TFA. After 16 h the solvents were removed under reduced pressure. The viscous oil was concentrated from Et 2 O (30 mL x 3) until a foam resulted.
  • t-Butyl ester vii (17.2 g, 60.35 mmol) was dissolved in DMSO (120 mL) and NaNO 2 (16.66 g, 4 eq.) was added in one portion followed by addition of CH 3 CO 2 H (34.55 mL, 10.0 eq.). A condenser was connected to the flask and the stirred mixture was heated to 35 °C overnight. The reaction was cooled to room temperature and quenched with water (200 mL) and allowed to stir for 15 min. The mixture was washed with Et 2 O ( 3 x 150 mL). The Et 2 O layers were combined, washed with brine, then with NaHCO 3 (sat.) (2 x 100 mL).
  • the benzoic acid derivative viii (7.57 g, 32.07 mmol) was dissolved in DMF (100 mL) and K 2 CO 3 (4.4 g, 32.07 mmol) was added as a solid and the mixture was stirred under N 2 for 15 min followed by addition of allyl bromide (2.9 mL 1.05 eq). After 2 hours, the reaction was added to ethyl acetate and washed with water (150 mL) and brine (5 x 200 mL). The organic layer was dried (MgSO 4 ), and concentrated. Silica gel chromatography (5% ethyl acetate in hexane) of the crude oil provided h as a colorless and clear oil (7.5 g, 85%).
  • N-(2-naphthoyl)-3-aminophenylacetic acid and N-(l-naphthoyl)-3- aminophenylacetic acid are examples of N-(2-naphthoyl)-3-aminophenylacetic acid and N-(l-naphthoyl)-3- aminophenylacetic acid.
  • N-(2-Naphthoyl)-3-aminophenylacetic acid A portion of the methyl 3- aminophenylacetate (1.65 g, 10 mmol) was dissolved in methylene chloride (50 mL). DIEA (3.5 mL, 20 mmol) then 2-naphthoyl chloride (2.0 g, 10.5 mmol) dissolved in methylene chloride (10 mL) were added to the resulting solution. After 16 hours the reaction was diluted with ethyl acetate (100 mL) and washed with water (50 mL), saturated ammonium chloride (50 mL), and brine (50 mL).
  • N-(2-NaphthoyI)-2-aminophenylacetic acid N-(2-NaphthoyI)-2-aminophenylacetic acid.
  • Methyl 2-aminophenylacetate 1.6 g, 10 mmol was dissolved in methylene chloride (50 mL) and DIEA (3.5 mL, 20 mmol), then 2- naphthoyl chloride (2.0 g, 10.5 mmol) dissolved in methylene chloride (10 mL) were added. After 16 hours the reaction was diluted with ethyl acetate (100 mL) and washed with saturated ammonium chloride (50 mL) and brine (50 mL). The organic layer was dried over magnesium sulfate, and the solvent was removed by rotary evaporation.
  • the solvent was removed by rotary evaporation.
  • the solid was dissolved in water (1000 mL) containing a small amount of sodium carbonate. This was washed with ethyl acetate.
  • the aqueous phase was acidified with 1 M HCl and extracted with ethyl acetate (4 x 250 mL). The organics were washed with brine and dried over magnesium sulfate.
  • the solvent was removed by rotary evaporation.
  • the resulting solid was recrystallized from refluxing ethyl acetate (1000 mL) to afford N-(l-naphthoyl)-4-aminophenylacetic acid (1.16 g, 67%) as a white solid.
  • the aqueous filtrate was extracted with ethyl acetate (2 x 250 mL).
  • the solid filter cake was dissolved in ethyl acetate (1.5 L) and 0.12 N hydrochloric acid (250 mL) and the phases separated.
  • the ethyl acetate solutions were combined and concentrated in vacuo to afford an off white solid.
  • This product was suspended in ethyl acetate (100 mL), isolated by filtration and dried to afford the title compound as a white solid (18.9 g, 50.6 mmol, 76%).
  • Rink amide resin (1.5 g) was suspended in DMF (20 mL) and was gently agitated for 30 minutes. The solvent was drained from the resin, 20% (v/v) piperidine in DMF (20 mL) was added, and the suspension was gently agitated for 10 minutes. The piperidine solution was drained from the resin and the resin was washed with DMF (2 x 20 mL). The piperidine treatment was repeated.
  • the resin was suspended in DMF (5 mL) and N-fluorenylmethyloxycarbonyl-L-isoleucine (0.795 g), HATU (0.813 g) and IM N,N-diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 3.5 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL).
  • the resin was suspended in DMF (5 mL) and N- fluorenylmethyloxycarbonyl-L-Asp(O-t-Bu)-OH (0.88 g), HATU (0.813 g) and 1M N.N- diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 1.75 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with 20% (v/v) piperidine (20 mL) for 10 min, then was washed with DMF (2 x 20 mL). This treatment was repeated, and the resin was washed with additional DMF (2 x 20 mL).
  • the resin was washed with methanol (2 x 20 mL) and diethyl ether (2 x 20 mL) and was then dried in vacuo.
  • the product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2% (v/v) thioanisole (85 mL) for 4 h.
  • the resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL).
  • the filtrate was concentrated to afford a red oil which was triturated with diethyl ether (4 x 100 mL) to afford the crude product as a yellow solid (0.700 g).
  • the product was purified by preparative HPLC on a 45 mm i.d.
  • the peptide was assembled by a method analogous to that described in Method 1 up to the coupling of the Freidinger lactam component starting with 0.5 g of RINK amide resin. The N-terminal substituent was then appended:
  • the product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2% (v/v) thioanisole (30 mL) for 3 h.
  • the resin was removed by filtration and washed with trifluoroacetic acid (10 mL).
  • the filtrate was concentrated to afford a red oil which was triturated with diethyl ether (3 x 100 mL) to afford the crude product as a yellow solid (0.184 g).
  • the product was purified on a C, 8 SepPak. Fractions containing the desired product were pooled and lyophilized to afford the title compound as a pale yellow solid. Yield: 0.086 g.
  • Ethyl 4-(2-quinoxaloyl)amidophenylacetate A solution of ethyl 4- aminophenylacetate (0.50 g) in dichloromethane (10 mL) was treated with 2-quinoxaloyl chloride (0.51 g) and N,N-diisopropylethylamine (0.38 g). The reaction mixture was stirred at room temperature for 2.5 h, then was diluted with ethyl acetate (50 mL) and washed sequentially with 0.1 N hydrochloric acid (2 x 50 mL), water (50 mL) and brine (50 mL). The aqueous washes were extracted with ethyl acetate (50 mL).
  • Procedure B (compounds where R 8 terminates with aromatics, cvcloalkyls, and heterocvcles) 4-(2-Naphthaloylamido)phenylacetyl-3-(R)-amido-(2-oxopyrrolidine)-l- ⁇ -(l-L-(S)- methylpropyl)acetyl-L-aspartyl-N-(2-indanoyl)carboxamide
  • Polystyrene-PEG-PAC resin 50 g, 0.16 meq./gram was suspended in DMF (300 mL) and was gently agitated for 30 minutes. The solvent was drained from the resin, and the resin was washed with additional DMF (2 x 200 mL). Following the last DMF wash, N-fluorenylmethyloxycarbonyl-L-Asp- ⁇ -(allyl)-OH (15.8 g) in DMF (-30 mL), 1,3- diisopropylcarbodiimide (7.52 mL) and 0.08M of 4-dimethylaminopyridine in DMF (10 mL) were added to the resin.
  • the reaction solution was drained from the resin and the resin was washed with DMF (4 x 4 mL).
  • FT-IR analysis showed no change in intensity of the 1760 cm " ' absorption indicating that double coupling is unnecessary.
  • the resin was treated with 20% (v/v) piperidine (250 mL) for 10 min, then was washed with DMF (2 x 300 mL). This treatment was repeated, and the resin was washed with additional DMF (4 x 250 mL). Coupling of Freidinger lactam.
  • the reaction solution was drained from the resin and the resin was washed with: 5% acetic acid / 2.5% N- methylmorpholine in methylene chloride (5 x 350 mL); methylene chloride (3 x 300 mL); 0.5% sodium dietyldithiocarbamate in DMF (4 x 300 mL); DMF (5 x 300 mL); methylene chloride (4 x 300 mL); 10% acetic acid in methylene chloride (4 x 300 mL); and ether (6 x 300 mL).
  • the resin was dried under high vacuum for 18h. Yield : 45 g.
  • the mixture was gently agitated for 18 hr.
  • the reaction solution was drained from the resin and the resin was washed with DMF (5 x 20 mL), methylene chloride ( 5 x 20 mL), and diethyl ether (5 x 20 mL) and was then dried in vacuo.
  • the product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% (v/v) water (35 mL) for 1.5 h.
  • the resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL).
  • the peptide was assembled by the method described in Procedure B, method 1. The only difference is that the amine components were synthesized on the Bohdan RAM synthesizer using low temperature conditions.
  • the resin was treated with the following solution containing: sarcosinyl-N-[(+/-)- tetrahydrofurfuryl]-carboxamide.HCl (0.125 g), HATU (0.036 g), N,N-diisopropylethylamine (0.054ml) and 1-methylimidazole (0.012 mL) in DMF (4 mL). The mixture was gently agitated for 18 hr. The reaction solution was drained from the resin and the resin was washed with DMF (5 x 20 mL), methylene chloride (5 x 20 mL), and diethyl ether (5 x 20 mL) and was then dried in vacuo.
  • DMF 5 x 20 mL
  • methylene chloride 5 x 20 mL
  • diethyl ether 5 x 20 mL
  • the product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% (v/v) water (35 mL) for 1.5 h.
  • the resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL).
  • the filtrate was concentrated to afford a clear oil which was triturated with diethyl ether (3 x 50 mL) to afford the crude product as a white solid (0.096 g).
  • the reaction was kept at -20 °C for two hours with mixing every 30 minutes by way of nitrogen bubbling.
  • a solution containing tetrahydrofurfurylamine (0.248 mL), 1- methylimidazole (0.264 mL) in THF (4.0 mL) was added using a slow syringe speed.
  • the reaction was kept at -20 °C for four hours with mixing every 30 minutes by way of nitrogen bubbling.
  • the cooling unit was then turned off and the reaction was allowed to warm to room temperature overnight with mixing every 30 minutes by way of nitrogen bubbling.
  • the solvent was removed under vacuum.
  • the peptide was assembled using the Milligen 9050 continuous flow automated peptide synthesizer by the fmoc/t-butyl strategy on Pepsyn KA(100) resin.
  • N-fluorenylmethyloxycarbonyl-L-Asp(OBu)-OH Coupling of N-fluorenylmethyloxycarbonyl-L-Asp(OBu)-OH.
  • a solution of N- fluorenylmethyloxycarbonyl-L-Asp(OBu)-OH (0.720 g), HATU (0.660 g), and N,N- diisopropylethylamine (0.610 mL) in DMF (5.30 mL) was added to the resin using a three- hour coupling cycle.
  • the resin was then treated with 20% (v/v) piperidine / DMF using a 10 min cycle.
  • the product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% (v/v) water (35 mL) for 1.5 h.
  • the resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL).
  • the filtrate was concentrated to afford a clear oil which was triturated with diethyl ether (3 x 50 mL) to afford the crude product as a white solid (0.410 g).
  • the product was purified by preparative HPLC on a 25 mm i.d. x 20 cm Waters 300 A column using an acetonitrile/water (+0.1% (v/v) trifluoroacetic acid) gradient at a flow rate of 12 mL/min.
  • the mixture was stirred with an overhead stirrer for two hours, then transferred to a fritted glass funnel and was washed with a one to one mixture of methanol and DMF (3 x 300 mL), DMF (3 x 300 mL), methylene chloride (5 x 300 mL) and methanol (5 x 300 mL).
  • the resin was dried in vacuo at 40 °C for 16 hours. MAS-NMR showed disappearance of the aldehyde proton.
  • the resin was treated with a second batch of the reaction cocktail (4.2 g N-fluorenylmethyloxycarbonyl- D-Arg(Pbf)-OH, 2.3 g HATU, and 2.7 mL DIEA in 50 mL DMF) for an additional hour.
  • the liquid was drained and the resin was washed with DMF (10 x 50 mL).
  • NPIT test showed the reaction was complete.
  • the resin was treated with 20% piperidine in DMF (2 x 50 mL) then washed with DMF (10 x 50 mL).
  • the liquid was drained and the resin was washed with DMF (10 x 20 mL), methylene chloride (5 x 20 mL) and ether (5 x 20 mL) then dried in vacuo at 40 °C for 30 minutes.
  • the resin was treated with a solution of TFA (50 mL), water (1 mL), thioanisole (1 mL), and TIS (0.5 mL) for one hour.
  • the liquid was filtered from the resin and reduced to about 5 mL by rotary evaporation. Ether (200 mL) was added to precipitate the crude product.

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Abstract

A compound having general formula (A) and methods of using such compounds for the treatment of diseases and pharmaceutical composition comprising such compounds.

Description

MODIFIED PENTAPEPTIDE ANTAGONISTS OF THE ATRIAL NATRIURETIC
PEPTIDE CLEARANCE RECEPTOR
Background ANP is a member of a family of natriuretic peptide hormones, which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The natriuretic peptides have a number of actions on the cardiovascular system, including; natriuresis, diuresis, and relaxation of vascular smooth muscle. ANP is a 28-amino acid cyclic peptide which is produced in atrial myocytes in response to increases in heart rate and atrial stretch.
There are two biologically- and functionally-distinct classes of ANP receptors. The first one is linked to guanylate cyclase and is thought to mediate the physiological effects of ANP via increases in intracellular cGMP levels. These guanylate cyclase receptors are further divided into the ANP-A and ANP-B receptors according to their relative affinity for different natriuretic peptides. The second class of ANP receptors do not mediate the cardiovasculature effects of the hormone and are thought to mainly serve a clearing function of ANP from the extracellular circulation. This receptor is known as the atrial natriuretic peptide clearance receptor (ANPCR).
Natriuretic peptides have a very short half life in vivo, and there are thought to be two major modes of their clearance from systemic circulation. One is via proteolytic inactivation by the enzyme neutral endopeptidase (NEP). The other is via binding to the ANP clearance receptor which is expressed on the vascular endothelium. Binding to the clearance receptor is followed by internalization and degradation of the peptide. The lung is thought to play a major role in ANP clearance, and studies have found over 50% of ANP is cleared in a single pass through the lungs. Approaches based on the inhibition of NEP are further complicated by the number of physiologically important peptide hormones which are substrates for this enzyme. Of the two modes of clearance, the ANPCR is thought to be primarily responsible for removal of ANP in the pulmonary vasculature, and the ANPCR is the dominate ANP receptor in lung tissue. Additionally, blockade of the clearance receptor in the lung was thought to provide a pulmonary selective approach to reduction of pulmonary blood pressure due to the presence of both the ANP-A,B and ANPCR receptors in the lung and by the proximity of these receptors to the site of ANP synthesis. For these reasons blockade of the ANPCR was chosen as the best approach to increase endogenous levels of ANP. It is estimated that a large segment of patients with chronic obstructive pulmonary disease ("COPD") will develop pulmonary hypertension (> 6 million in the US alone), and that the number of patients will increase as diagnostic methods improve. An ANPCR antagonist could have therapeutic usefulness in treating pulmonary hypertension secondary to COPD. In addition, since all natriuretic peptides (i.e., ANP, BNP and CNP) inhibit vascular smooth muscle cell proliferation, an ANPCR antagonist may also be useful for protection of the transplanted heart given that plasma levels of BNP are elevated in this situation. An ANPCR antagonist may have the greatest therapeutic utility in the treatment of congestive heart failure (CHF), by virtue of raising plasma concentrations of ANP and BNP. Infusion of exogenous BNP decreased plasma renin activity, increased plasma cGMP and increased urinary sodium output with concomitant decreases in pulmonary capillary wedge pressure in the dog model of acute heart failure. It has been hypothesized that ANP and BNP's role in the circulation may be to produce venodilation and increase capillary permeability to reduce cardiac preload and prevent pulmonary congestion. Increased plasma ANP and BNP would be expected by blockade of the ANPCR receptor. Summary of the Invention
The present invention is directed to synthetic analogs of atrial peptides and more particularly o synthetic linear peptide analogs which find use as diuretics, natriuretics and/or vasodilators, or as intermediates for or modulators of such useful compounds, together with methods for their production and use.
A series of lactams of the generic structure shown below were found to be blockers of the ANP clearance receptor (ANPCR).
Figure imgf000004_0001
Such a replacement group contains a chiral center at the lactam α-carbon, giving the diastereomeric pair I and II. It has now been found that the R-isomer of the lactam is preferred, as shown in the structures below.
Figure imgf000005_0001
Figure imgf000005_0002
In the course of this work is was found that lactam molecules which contained a D- amino acid or a sarcosine residue in position R7 has good metabolic stability. This is in contrast to the natural hormone ANP which has poor metabolic stability.
Figure imgf000005_0003
— C-Term inal Region -*-;
Im prove m etabolic stability
Work in the n-Cap region found that decreases in molecular weight caused large decreases in potency. For example, truncation to the phenylacetic carboxamide (III) resulted in complete loss of binding affinity, while more modest truncation of the naphthyl group to a series of substituted benzamides generally produced compounds that bind in the 100 nM range.
Figure imgf000006_0001
A number of promising compounds in terms of biological activity resulted from work in the n-Cap region. A series of heterocyclic replacements for the naphthyl group, were most promising. A compound which combined a 2-quinoxalazine as a replacement for the naphthyl ring, coupled with a D-Arg-14 residue (IV) shows promising oral activity.
Figure imgf000006_0002
Brief Description of the Drawings Some data related to the invention will now be described with reference to the accompanying drawings, in which:
Figure 1 is a chart showing the dose-response effect of IV or vehicle administered orally 165 min before evaluating the change in right intraventricular peak systolic pressure
(RVSP; upper panel) and mean systemic arterial pressure (MAP; lower panel); and Figure 2 is a chart showing the effect of vehicle or I (30 mg/kg, p.o.; top panel) and IV
(100 mg/kg, p.o.; bottom panel) on immunoreactive plasma content of ANP in rats exposed to acute hypoxia.
Detailed Description of the Invention
The compounds of the instant invention are linear peptide compounds having the structure:
Figure imgf000007_0001
In this structure: R1 is a hydrocarbon chain containing from one to four carbon atoms and zero-to-two heteroatoms, but is preferably -CH2CH2-, -CH2CH2CH2-, =CH-CH=CH- or -N=CH-;
R2 should be hydrogen or a C,-C4 alkyl group, but is preferably hydrogen or methyl. R3 is a zero-to-four atom chain or aromatic ring containing from zero-to-eight carbon atoms and zero- to-three heteroatoms; R3 is preferably -CH2CH2CH2-, -(E)-CH=CHC(=O)NH-, -CH2CH2C(=O)NH-, para- disubstituted phenyl, ortho-disubstituted phenyl, meta-disubstituted phenyl or a single bond, wherein, in the disubstituted phenyl groups, one substituent is R4 and the other is the methylene group alpha to the amide carbonyl, as shown in the generic structure above; R4 is -NHC(=O , -C(=O)NH- or -S(=O)2NH-; R5 is a substituted or unsubstituted alkylaryl, aryl or heteroaryl compound, preferably 1- naphthyl, 2-naphthyl, -CH2CH2NHCH2CH=CH-phenyl, -CH2CH2-phenyl, -CH=CH-phenyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 6-quinolyl, 3-isoquinolyl, 2-quinoxaline, 5-chloro-2- indolyl, 2-indolyl, 4-chlorophenyl, 4-methylphenyl, 3-methoxyphenyl, 4-cyanophenyl, 3,4- difluorophenyl, 3-chloro-4-fluorophenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 4- chlorophenyl, 3,5-dimethoxyphenyl, 4-tert-butylphenyl, phenyl,4-trifluoromethylphenyl, - CH2CH2CH2 -phenyl, 6-quinolyl-C(=O)-, 2-quinoxaline-C(=O)-, 5-chloro-2-benimidazolyl, fluorenylmethoxycarbonyl, 4-chlorobenzyl, 4-methylbenzyl, 3-quinoxalinyl, 3,4- difluorophenyl, or 4-fluorophenyl;
R6 is a C3-C5 branched or unbranched alkyl group, preferably isobutyl or sec-butyl; R7 is a natural or unnatural amino acid, preferably N-methylglycine, -NHCH2CH2NHC(=O)-, L-arginine, D-arginine, L-ornithine, D-ornithine, histidine, citrulline, proline, hydroxyproline, 3-pyridinylalanine, L-N-methylalanine, D-N-methylalanine, aminobutyric acid, or thiazolidine;
R8 is L-isoleucine-NH2, D-isoleucine-NH2, -CH2-cyclopentyl, -CH2-2-tetrahydrofuranyl, tert- butylglycine-NH2, n-butyl, NH-cyclopentyl, NHCH2-2-furanyl, -NHCH2-pyrininyl, -NHCH2- cyclohexyl, -NH-2-indolizidinyl, D-leucinol, -NH-isobutyl, L-allo-isoleucine-NH2, 1- hydroxycycloleucinol, 2-(aminomethyl)-l-ethyl-pyrrolidine, or (S)-NH-2-methylbutyl, but if R7 is -NH-2-indolizidine, then R8 is absent; and
R9 is a one carbon spacer that is preferably =CH- or -C(=O)-; such that when R9 is =CH-, then =~^ is a double bond, and when R9 is -C(=O)- then =~^ is a single bond, and when R1 is - N=CH- and R9 is =CH-, then the central ring is a disubstituted imidazole.
Representative compounds according to the present invention include those of the structure:
Figure imgf000008_0001
# R' R2 R3 R4 R5
1 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
2 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl J (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
4 (R)-CH2CH2- H a single bond -C(=0)NH- (E)-PhHC=CHCH2NHCH2CH2-
5 (R)-CH,CH2- Me para-phenyl -C(=0)NH- 2-naphthyl
6 (R)-CH2CH2- Me 1,3-propyl -NHC(=0)- PhCH,CH2CH2-
7 (R)-CH2CH2- Me -CH2CH2C(=0)NH- -S(=0)2NH- 2-naphthyl
8 (R)-CH2CH2- Me -CH2CH2C(=0)NH- -S(=0)2NH- (E)-PhHC=CH-
9 (R)-CH2CH2- Me -CH2CH2C(=0)NH- -C(=0)NH- 6-quinolinyl-C(=0)-
10 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 6-quinolinyl-C(=0)-
1 1 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 2-quinoxalinyl-C(=0)-
12 =CH-CH=CH- H para-phenyl -C(=0)NH- 2-naphthyl
13 =CH-CH=CH- H para-phenyl -C(=0)NH- 2-naphthyl
14 =CH-CH=CH- H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
15 =CH-CH=CH- H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
16 (R)-CH2CH2- Me -CH2CH2C(=0)NH- -C(=0)NH- (E)-PhHC=CH-
17 =CH-CH=CH- H para-phenyl -C(=0)NH- 2-naphthyl
18 =CH-CH=CH- H para-phenyl -C(=0)NH- 2-naphthyl
19 (R)-CH2CH2- Me -CH2CH2C(=0)NH- -C(=0)NH- 2-indole
20 imidazole* H para-phenyl -C(=0)NH- 2-naphthyl
21 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 2-naphthyl
22 (R)-CH2CH2- Me -CH2CH2C(=0)NH- -C(=0)NH- (E)-PhHC=CH-
23 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 2-naphthyl
24 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 2-naphthyl
25 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 6-quinolinyl
26 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 3-quinolinyl
27 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 2-quinolinyl
28 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 2-indole
29 (R)-CH2CH2- Me para-phenyl -C(=0)NH- (E)-PhHC=CH-
30 (R)-CH2CH2- Me para-phenyl -C(=0)NH- 4-methylphenyl
31 (R)-CH2CH2- H para-phenyl -NHC(=0)- 4-chlorobenzyl
32 (R)-CH2CH2- H para-phenyl -NHC(=0)- 4-methylbenzyl R1 R2 R3 R4 R5
( )-CH2CH2~ H para-phenyl -C(=0)NH- 2-naphthyl
(Rj-CH^CH^" H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R^CH^CH^- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(Rj-Crl^Crl^- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-quinolinyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 6-quinolinyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinolinyl
(R)-CH2 i2" H para-phenyl -C(=0)NH- 2-naphthyl
(R)-CH2CH2" H para-phenyl -C(=0)NH- 3-quinoxalinyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-quinolinyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinolinyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 6-quinolinyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 3-isoquinolinyl
(R)-CH2CH2~ H para-phenyl -C(=0)NH- 2-quinolinyl
H para-phenyl -C(=0)NH- 2-indolyl
(R)-CH2CH2" H para-phenyl -C(=0)NH- 2-indolyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-chlorophenyl
(Rj-Cr^Cr.^- H para-phenyl -C(=0)NH- 4-trifluoromethylphenyl
(R)-CH2CH2~ H para-phenyl -C(=0)NH- 4-fluorophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-methylphenyl
(R)-CH2CH2~ H para-phenyl -C(=0)NH- 3-methoxyphenyl
(Rj-Cr^CH^- H para-phenyl -C(=0)NH- 4-cyanophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 3 ,4-difluorophenyl
(R)- . 2V-Η2" H para-phenyl -C(=0)NH- 3-chloro-4-fluoropheny]
(R)-CH2CH2" H para-phenyl -C(=0)NH- 3 ,5-dimethoxyphenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2-indolyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 3-methoxyphenyl
(R)-CH2CH2" H para-phenyl -C(=0)NH- 2,4-dichlorophenyl
H para-phenyl -C(=0)NH- 4-chlorophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-chlorophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-trifluoromethylphenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-chlorophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-trifluoromethylphenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 2,4-dichlorophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 4-chlorophenyl
(R)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinolinyl
(R)-CH2CJH2" H para-phenyl -C(=0)NH- 3-quinolinyl
(R)-CH2CH2~ H para-phenyl -C(=0)NH- 2-quinolinyl # R1 R2 R3 R4 R5
89 ^)-CH2CH2- H para-phenyl -C(=0)NH- 3-isoquinolinyl
90 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-quinolinyl
91 )-CH2C.H2" H para-phenyl -C(=0)NH- 2-indolyl
92 ιR)-Cr 2CH2~ H para-phenyl -C(=0)NH- 3-isoquinolinyl
93 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-indolyl
94 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
95 ^R)-CH2CH2" H para-phenyl -C(=0)NH- 2-naphthyl
96 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
97 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
98 VR)-CH2CJH2" H para-phenyl -C(=0)NH- 2-naphthyl
99 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
100 1R)-CJH2CH2~ H para-phenyl -C(=0)NH- 2-naphthyl
101 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
102 ^R^CH^CH^" H para-phenyl -C(=0)NH- 2-naphthyl
103 kR)-CH2CH2" H para-phenyl -C(=0)NH- 2-naphthyl
104 kR)-CH2Cri2" H para-phenyl -C(=0)NH- 2-naphthyl
105 (S)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
106 R)-CH2Cr 2" H para-phenyl -C(=0)NH- 1 -naphthyl
107 (R)-CH2CH2- H ortho-phenyl -C(=0)NH- 2-naphthyl
108 H meta-phenyl -C(=0)NH- 1 -naphthyl
109 (R)-CH2CH2- H meta-phenyl -C(=0)NH- 2-naphthyl
1 10 kR)-CH2CH2- H ortho-phenyl -C(=0)NH- 1 -naphthyl
1 1 1 (R)-CH2CH2- H -CH2CH2C(=0)NH- -NHC(=0)- 2-phenylethyl
1 12 ιR)-CH2CH2" H -(E)-HC=CHC(=0)NH- -NHC(=0)- 2-phenylethyl
1 13 R)-CH2CH2~ H -CH2CH2C(=0)NH- -NHC(=0)- (E)-Ph-HC=CH-
1 14 S)-CH2CH2~ H para-phenyl -C(=0)NH- 1 -naphthyl
1 1 5 (S)-CH2CH2- H ortho-phenyl -C(=0)NH- 2-naphthyl
1 16 (S)-CH2CH2- H meta-phenyl -C(=0)NH- 1 -naphthyl
1 17 - )-CH2Crl2- H meta-phenyl -C(=0)NH- 2-naphthyl
1 1 8 Sj-Cr^CH^- H -CH2CH2C(=0)NH- -C(=0)NH- (E)-Ph-HC=CH-
1 19 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
120 R)-CH2C-ri2- H para-phenyl -C(=0)NH- 2-naphthyl
121 y R)-Cri2CH2" H para-phenyl -C(=0)NH- 2-naphthyl
122 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
123 R)-CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
124 Rj-CH^Cr^- H -CH2CH2C(=0)NH- -C(=0)NH- (E)-Ph-HC=CH-
125 R)-CH2CH2~ H para-phenyl -C(=0)NH- FMOC
126 R^Cr^Cr^- H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
127 kR)-CH2Cr 2" H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
128 kR)-CH2CH2- H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
129 R)-CH2CH2~ H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
130 R)-CH2CH2~ H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
13 1 Rj-Cr^C-H^" H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
132 (R)-CH2CH2- H para-phenyl -C(=0)NH- 6-methyl-3-pyridinyl
133 -R)-CH'jCH2~ H para-phenyl -C(=0)NH- 6-methyl-3-pyridinyl
134 -R)-CH',CH2" H para-phenyl -C(=0)NH- 6-quinolinyl
135 (R)-CH2CH2- H para-phenyl -C(=0)NH- 6-quinolinyl
136 H para-phenyl -C(=0)NH- 3-quinoxalinyl
137 iR)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinoxalinyl
138 (R)-CH2CH2- H para-phenyl -C(=0)NH- 5 -ch loro-2-benzimidazoly 1
139 (R)-CH2CH2- H para-phenyl -C(=0)NH- 5-chloro-2-benzimidazolyl
140 (R)-CH2CH2- H para-phenyl -C(=0)NH- 5-chloro-2-benzimidazolyl
141 (R)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinolinyl
142 (R)-CH2CH2- H para-phenyl -C(=0)NH- 3-isoquinolinyl
143 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-quinolinyl
144 R)-CH-)CH2- H para-phenyl -C(=0)NH- 2-indolyl # R1 R2 R3 R4 R5
145 ( j-Cr^ H^- H para-phenyl -C(=0)NH- 3-quinolinyl
146 (R)-CH2CH2~ H para-phenyl -C(=0)NH- 3-isoquinolinyl
147 (R - H^ -Η^ H para-phenyl -C(=0)NH- 2-quinolinyl
148 (R)-CH2Cri2~ H para-phenyl -C(=0)NH- 2-naphthyl
149 (R)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinolinyl
150 (R)-Cri2CH2- H para-phenyl -C(=0)NH- 3-isoquinolinyl
151 (R)-CH2CH2- H para-phenyl -C(=0)NH- 2-quinolinyl
152 (R)-CH2CH2- H para-phenyl -C(=0)NH- 6-quinolinyl
153 (R)-CH2CH2- H para-phenyl -C(=0)NH- 3-quinoxalinyl
154 (R)-Cri2CH2~ H para-phenyl -C(=0)NH- 2-indolyl
155 (R)-CH2CH2- H para-phenyl -C(=0)NH- 5-chloro-2-indolyl
156 (R)-CH2CH2CH2- H para-phenyl -C(=0)NH- 2-naphthyl
* when R' is imidazole, the central ring contains no carbonyl group.
# R6 R7 R8
1 (L)(S)secBu N-MeGly Ile-NH2
2 (L)(S)secBu -NHCH2CH2NHC(=0)- CH2-cyclopentyl
3 (L)(S)secBu -NHCH2CH2-NHC(=0)- n-Bu
4 (L)(S)secBu N-MeGly Ile-NH2
5 (L)(S)secBu (D)Arg lle-NH2
6 (L)(S)secBu (D)Arg Ile-NH2
7 (L)(S)secBu (D)Arg Ile-NH2
8 (L)(S)secBu (D)Arg Ile-NH2
9 (L)(S)secBu (D)Arg Ile-NH2
10 (LXS)secBu (D)Arg Ile-NH2
1 1 (L)(S)secBu (D)Arg Ile-NH2
12 (DL)(S)secBu (L)Arg Ile-NH2
13 (DL)(S)secBu (D)ornithine Ile-NH2
14 (DL)(S)secBu (D)Arg Ile-NH2
15 (DL)(S)secBu N-MeGly Ile-NH2
16 (L)(S)secBu (D)Arg Ile-NH2
17 (DL)(S)secBu (D)Arg Ile-NH2
18 (DL)(S)secBu N-MeGly Ile-NH2
19 (L)(S)secBu (D)Arg Ile-NH2
20 (LXS)secBu (L)Arg Ile-NH2
21 (L)(S)secBu (L)Arg Ile-NH2
22 (L)(S)secBu ( )Arg Ile-NH2
23 (L)(S)secBu N-MeGly Ile-NH2
24 (L)(S)secBu Gly Ile-NH2
25 (L)(S)secBu N-MeGly Ile-NH2
26 (L)(S)secBu N-MeGly Ile-NH2
27 (L)(S)secBu N-MeGly Ile-NH2
28 (L)(S)secBu N-MeGly Ile-NH2
29 (L)(S)secBu N-MeGly Ile-NH2
30 (LXS)secBu N-MeGly Ile-NH2
31 (L)(S)secBu N-MeGly NH-i-Bu
32 (L)(S)secBu N-MeGly NH-i-Bu
33 (D)(S)secBu (D)Arg Ile-NH2
34 (D)isoBu (L)ornithine NH-i-Bu
35 (D)isoBu (D)Arg Ile-NH2
36 (D)isoBu N-MeGly Ile-NH2
37 (D)isoBu His Ile-NH,
38 (D)isoBu citrulline NH-i-Bu
39 (D)isoBu (D)Arg NH-i-Bu
40 (L)(S)secBu Pro NH-i-Bu R6 R7 R8
(L)(S)secBu Hyp NH-i-Bu
(L)(S)secBu 3-pyridinyl-Ala NH-i-Bu
(L)(S)secBu N-MeAla NH-i-Bu
(LXS)secBu (D)N-MeAla NH-i-Bu
(LXS)secBu aminobutyric acid NH-i-Bu
(L)(S)secBu thiazolidine NH-i-Bu
(L)(S)secBu Pro Ile-NH2
(L)(S)secBu Arg (L)-allo-Ile-NH2
(L)(S)secBu (L)ornithine (L)-allo-Ile-NH2
(L)(S)secBu (D)ornithine Ile-NH2
(L)(S)secBu (D)Arg Ile-NH2
(L)(S)secBu (L)ornithine (D) Ile-NH2
(LXS)secBu Arg (D) lle-NH2
(L)(S)secBu (D)Arg NH-i-Bu
(LXS)secBu (D)Arg Ile-NH2
(LXS)secBu (D)Arg Ile-NH2
(L)(S)secBu (D)Arg Ile-NH2
(L)(S)secBu (D)ornithine NH-i-Bu
(L)(S)secBu (D)Arg Ile-NH2
(L)(S)secBu (D)Arg Ile-NH2
(L/)(S)secBu (D)Arg NH-i-Bu
(L)(S)secBu (D)Arg NH-i-Bu
(L)(S)secBu (D)Arg NH-i-Bu
(L)(S)secBu (D)Arg NH-i-Bu
(L)(S)secBu (D)Arg NH-i-Bu
(LXS)secBu N-MeGly NH-i-Bu
(L)(S)secBu N-MeGly lle-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(LXS)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(LXS)secBu (D)Arg Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly lle-NH2
(L)(S)secBu (D)ornithine NH-i-Bu
(L)(S)secBu (D)Arg NH-i-Bu
(LXS)secBu (D)Arg NH-i-Bu
(L)(S)secBu Pro NH-i-Bu
(L)(S)secBu Pro NH-i-Bu
(L)(S)secBu Pro NH-i-Bu
(L)(S)secBu N-MeGly NH-i-Bu
(LXS)secBu N-MeGly lle-NH2
(L)(S)secBu (D)ornithine Ile-NH2
(L)(S)secBu (D)ornithine Ile-NH2
(L)(S)secBu (D)ornithine Ile-NH2
(L)(S)secBu N-MeGly Ile-NH2
(LXS)secBu (D)ornithine lle-NH2
(L)(S)secBu N-MeGly Ile-NH2
(L)(S)secBu N-MeGly lle-NH2
(L)(S)secBu Arg NH-i-Bu
(L)(S)secBu NH-2-indolizidine [n/a]
(LXS)secBu N-MeGly -CH,-2-THF # R6 R7 R8
97 (LXS)secBu N-MeGly (D)-t-BuGly-NH2
98 (L)(S)secBu N-MeGly (DL)-t-BuGly-NH2
99 (L)(S)secBu N-MeGly -NH-cycloleucinol
100 (L)(S)secBu N-MeGly -NH-2-(NHCH2)- 1 -Et-pyrrolidine
101 (L)(S)secBu N-MeGly -NHCH2-2-furan
102 (L/)(S)secBu N-MeGly (D)-leucinol
103 (L)(S)secBu N-MeGly -NHCH2-2-pyridinyl
104 (L)(S)secBu Arg Ile-NH2
105 (L/)(S)secBu Arg Ile-NH2
106 (L)(S)secBu Arg Ile-NH2
107 (L)(S)secBu Arg Ile-NH2
108 (L)(S)secBu Arg Ile-NH2
109 (L)(S)secBu Arg Ile-NH2
1 10 (L)(S)secBu Arg Ile-NH2
1 1 1 (L)(S)secBu Arg Ile-NH2
1 12 (L)(S)secBu Arg Ile-NH2
1 13 (L)(S)secBu Arg Ile-NH2
1 14 (L)(S)secBu Arg Ile-NH2
1 15 (L)(S)secBu Arg Ile-NH2
1 16 (L)(S)secBu Arg Ile-NH2
1 17 (L)(S)secBu Arg Ile-NH2
1 18 (L)(S)secBu Arg Ile-NH2
1 19 (L)(S)secBu Arg (S)-NH-2-methylbutyl
120 (L)(S)secBu Gly (S)-NH-2-methylbutyl
121 (L)(S)secBu Arg -NH-CH2-cyclohexyl
122 (L)(S)secBu Gly -NH-CH2-cyclohexyl
123 (L)(S)secBu (L)Arg Ile-NH2
124 (L)(S)secBu (L)Arg lle-NH2
125 (L)(S)secBu (D)Arg Ile-NH2
126 (L)(S)secBu (D)Arg Ile-NH2
127 (LXS)secBu N-MeGly Ile-NH2
128 (LXS)secBu (D)ornithine Ile-NH2
129 (L)(S)secBu (D)ornithine NH-i-Bu
130 (L)(S)secBu (D)Arg NH-i-Bu
131 (L)(S)secBu N-MeGly NH-i-Bu
132 (LXS)secBu (D)Arg lle-NH2
133 (L)(S)secBu (D)ornithine Ile-NH2
134 (L)(S)secBu N-MeGly Ile-NH2
135 (L)(S)secBu (D)ornithine lle-NH2
136 (L)(S)secBu N-MeGly lle-NH2
137 (L)(S)secBu (D)ornithine Ile-NH2
138 (L)(S)secBu (D)Arg Ile-NH2
139 (L)(S)secBu N-MeGly Ile-NH2
140 (L)(S)secBu (D)ornithine Ile-NH2
141 (L)(S)secBu (D)ornithine -NH-i-Bu
142 (L)(S)secBu (D)ornithine -NH-i-Bu
143 (L)(S)secBu (D)ornithine -NH-i-Bu
144 (L)(S)secBu (D)ornithine -NH-i-Bu
145 (L)(S)secBu N-MeGly -NH-i-Bu
146 (L)(S)secBu N-MeGly -NH-i-Bu
147 (L)(S)secBu N-MeGly -NH-i-Bu
148 (L)(S)secBu (D)Arg -NHCH2-cyclohexyl
149 (L)(S)secBu (D)Arg -NHCH2-cyclohexyl
150 (L)(S)secBu (D)Arg -NHCH2-cyclohexyl
151 (L)(S)secBu (D)Arg -NHCH2-cyclohexyl
152 (LXS)secBu (D)Arg -NHCH2-cyclohexyl # R6 R7 R8
153 (L)(S)secBu (D)Arg -NHCH2-cyclohexyl
154 (LXS)secBu (D)Arg -NHCH2-cyclohexyl
155 (LXS)secBu (D)Arg -NHCH2-cyclohexyl
156 (L)(S)secBu Arg -NH-isobutyl
The above representative compounds were synthesized according to the Assembly Procedures below, using the resins shown and purification methods shown. i(nM) Assembly Resin Purification MS MS HPLC RT HPLC
Procedure Method Method (min) Method
1 2.17 A Ramage ppt 800.3 ES+ 17.25 a
2 490 B PAC-PEG HPLC 769.3 ES+ 13.09 a
3 986 B PAC-PEG HPLC 743.3 ES+ 12.51 a
4 155 A Ramage ppt 757.4 ES+ 7.15 b
5 0.69 A Ramage SPE 899.5 ES+ 15.66 a
6 14 A Ramage ppt 857.5 ES+ 11.14 c
7 328 A Ramage SPE 930.3 ES+ 10.83 c
8 262 A Ramage SPE 906.4 ES+ 10.42 c
9 451 A Ramage ppt 893.4 ES+ 7.55 c
10 38.6 A Ramage ppt 900.5 ES+ 9.06 c
1 1 19 A Ramage ppt 901.5 ES+ 1 1.49 c
12 2 A Ramage SPE 12.582 ES+ 895.6 c
13 87 A Ramage SPE 853.6 ES+ 12.4 c
14 936 A Ramage SPE 918.4 ES+ 12.81 c
15 410 A Ramage SPE 833.4 ES+ 13.48 c
16 35 A Ramage SPE 870.6 ES+ 9.75 c
17 104 A Ramage SPE 895.5 ES+ 12.6 c
18 165 A Ramage SPE 810.3 ES+ 13.24 c
19 89 A Ramage SPE 883.6 ES+ 10.00 c
20 161 A Ramage SPE 868.6 ES+ 1 1.28 c
21 0.16 A Ramage ppt 890 ES+ 1 1.4 d
22 1.8 A Ramage SPE 870 ES+ 7.5 d
23 4.6 A Ramage SPE 814 ES+ 12.3 d
24 2.4 A Ramage SPE 800 ES+ 17.6 d
25 26.5 A Ramage ppt 815 ES+ 7.46 d
26 6.9 A Ramage ppt 815 ES+ 8.4 d
27 22 A Ramage ppt 815 ES+ 12.6 d
28 23 A Ramage ppt 803 ES+ 11.6 d
29 14 A Ramage ppt 790 ES+ 11.6 d
30 27 A Ramage ppt 832 ES+ 12.7 d
31 233 C Merrifield HPLC 741 ES 27.10 e
32 683 C Merrifield HPLC 722 ES 26.32 e
33 9.9 A Rink SPE 886 FAB+ 6.1 f
34 232 C PS-Aldehyde SPE 786 FAB+ 7.2 f
35 299 A Rink SPE 885 FAB+ 6.5 f
36 321 A Rink SPE 800 FAB+ 7.7 f
37 79 A Rink SPE 866 FAB+ 6.3 f
38 641 C PS-Aldehyde SPE 827 FAB+ 7.5 f
39 83 C PS-Aldehyde SPE 828 FAB+ 7.6 f
40 6.0 C PS-Aldehyde SPE 769 FAB+ 7.6 g
41 8.4 C PS-Aldehyde SPE 785 FAB+ 6.3 g
42 704 C PS-Aldehyde SPE 820 FAB+ 6.9 g
43 69 C PS-Aldehyde SPE 757 APCI+ 8.7 j
44 18 C PS-Aldehyde SPE 757 APCI+ 8.7 j
45 13 C PS-Aldehyde SPE 757 APC1+ 8.6 j
46 12 C PS-Aldehyde SPE 787 APC1+ 8.8 j Ki(nM) Assembly Resin Purification MS MS HPLC RT HPLC
Procedure Method Method (min) Method 7 1 A Rink SPE 827 APCI+ 7.6 j 8 3.5 A Rink SPE 885.4 MALDI 6.14 f 9 4.9 A Rink SPE 843.4 MALDI 5.94 f 0 1 A Rink SPE 843.4 MALDI 6.67 f 1 1.65 A Rink SPE 885.4 MALDI 5.09 f 2 0.68 A Rink SPE 843.4 MALDI 5.77 f 3 0.41 A Rink HPLC 886.4 MALDI 6.06 f 4 51 C PS-Aldehyde SPE 827.9 ES+ 7.92 f 5 4 A Rink SPE 887 ES+ 2.70 f 6 9 A Rink SPE 887 ES+ 2.18 f 7 1 A Rink SPE 887 ES+ 5.78 f 8 23 C PS-Aldehyde SPE 786 ES+ 7.61 f 9 10 A Rink SPE 887.5 ES+ 5.00 f 0 12 A Rink SPE 886.6 ES+ 6.83 f 1 183 C PS-Aldehyde SPE 829 ES+ 3.08 g 2 908 C PS-Aldehyde SPE 829 ES+ 2.47 g 3 550 C PS-Aldehyde SPE 829 ES+ 6.21 g 4 621 C PS-Aldehyde SPE 829 ES+ 6.82 g 5 4 C PS-Aldehyde HPLC 817 ES+ 6.03 g 6 435 C PS-Aldehyde SPE 732.3 ES+ 7.77 g 7 29 A Rink ppt 784.3 API+ 6.12 g 8 31 A Rink ppt 818.3 AP1+ 7.01 g 9 465 A Rink ppt 768.4 API+ 5.06 g 0 131 A Rink ppt 764.4 API+ 5.49 g 1 390 A Rink ppt 780.4 API+ 4.94 g 2 212 A Rink ppt 775.4 API+ 4.52 g 3 793 A Rink ppt 786.3 API+ 5.73 g 4 868 A Rink ppt 802.3 AP1+ 6.27 g 5 478 A Rink ppt 810.4 AP1+ 5.25 g 6 4.1 A Rink SPE 874.5 API+ 5.22 g 7 8 A Rink ppt 780.4 APR 5.28 g 8 6.7 A Rink ppt 818.2 APR 7.39 g 9 297 C PS-Aldehyde HPLC 770.3 ES+ 5.99 o σ 0 649 C PS-Aldehyde HPLC 812.3 APR 6.25 to σ 1 740 C PS-Aldehyde HPLC 846.5 APR 7.03 g 2 180 C PS-Aldehyde ppt 753.3 AP1+ 5.29 h 3 255 C PS-Aldehyde ppt 787.3 APR 5.85 h 4 732 C PS-Aldehyde ppt 787.2 APR 6.36 h 5 898 C PS-Aldehyde SPE 727.3 APR 5.28 h 6 16.0 A Rink SPE 801.3 ES+ 6.09 7 2.1 A Rink ppt 844.5 API+ 5.79 8 5.4 A Rink ppt 844.6 APR 7.60 9 3.0 A Rink ppt 844.5 APR 7.27 0 8.9 A Rink HPLC 801.3 APR 8.04 1 3.1 A Rink ppt 832.5 APR 7.12 2 7.0 A Rink HPLC 801.3 APR 7.66 3 12.7 A Rink HPLC 789.4 APR 7.55 4 0.65 B2 PAC-PEG HPLC Mna+=850 APR 25.44 m 5 947 B2 PAC-PEG HPLC MH+=734 APR 23.89 6 19 B2 PAC-PEG HPLC MNa+=793 APR 25.52 m 7 674 B3 Pepsyn A (lOO) HPLC MH+=801 LC-ES 21.72 8 7.8 B3 Pepsyn KAM (175) HPLC MH+=800 LC-ES 21.93 9 12 B2 PAC-PEG HPLC MH+=785 LC-ES 21.99
100 605 B2 PAC-PEG HPLC MH+=798.9 LC-ES 22.14
101 779 B2 PAC-PEG HPLC MH+=767.5 LC-ES 22.87 i(nM) Assembly Resin Purification MS MS HPLC RT HPLC Procedure Method Method (min) Method
102 24 B2 PAC-PEG HPLC MH+=787.5 LC-ES 21.92 1
103 779 B2 PAC-PEG HPLC MH+=778.5 LC-ES 21.76 1
104 0.01 A Rink ppt 885.3 FAB+ 8.75 n
105 0.41 A Rink ppt 885.7 FAB+ 8.37 n
106 0.70 A Rink ppt 885.5 FAB+ 11.77 n
107 213 A Rink ppt 885.5 FAB+ 12.40 n
108 8.4 A Rink ppt 885.6 FAB+ 1 1.85 n
109 0.15 A Rink ppt 885.6 FAB+ 12.62 n
1 10 248 A Rink ppt 885.5 FAB+ 12.40 n
1 1 1 2.4 A Rink ppt 885.6 FAB+ 1 1.85 n
1 12 1.2 A Rink ppt 885.6 FAB+ 12.62 n
1 13 0.36 A Rink ppt 856.6 FAB+ 7.31 n
1 14 73 A Rink ppt 885.6 FAB+ 1 1.31 n
1 15 45 A Rink ppt 885.6 FAB+ 12.82 n
1 16 178 A Rink ppt 885.6 FAB+ 11.45 n
1 17 174 A Rink ppt 885.6 FAB+ 12.17 n
1 18 326 A Rink ppt 856.5 FAB+ 7.91 n
1 19 0.1 1 C PS-Aldehyde ppt 842.6 FAB+ 14.14 n
120 46 C PS-Aldehyde ppt 743.3 FAB+ 15.22 n
121 0.5 C PS-Aldehyde ppt 868.6 FAB+ 15.22 n
122 310 C PS- Aldehyde ppt 769.4 FAB+ 16.44 n
123 0.36 A Rink ppt 885.6 FAB+ 12.60 n
124 39 A Rink ppt 856.7 FAB+ 7.68 n
125 185 A Rink SPE 953.4 ES+ 7.55 o
126 1.44 A Rink HPLC 908.4 ES+ 6.30 0
127 6.62 A Rink SPE 823.3 ES+ 6.57 0
128 1.99 A Rink HPLC 866.3 ES+ 6 0
129 101 C PS-Aldehyde SPE 809.3 ES+ 5.65 0
130 85.2 C PS-Aldehyde SPE 851.3 ES+ 6.73 0
131 87.4 C PS-Aldehyde SPE 766.3 ES+ 7.05 0
132 70 A Rink HPLC 850.5 ES+ 2.72 0
133 459 A Rink HPLC 808.4 ES+ 3.07 0
134 37 A Rink SPE 801.4 ES+ 3.41 0
135 8 A Rink HPLC 844.5 ES+ 2.76 0
136 63 A Rink SPE 802.3 ES+ 5.41 0
137 1 1 A Rink HPLC 844.5 ES+ 4.99 0
138 14 A Rink SPE 909.5 ES+ 6 0
139 65 A Rink SPE 824.4 ES+ 6 0
140 14 A Rink SPE 867.5 ES+ 6 0
141 416 C PS-Aldehyde SPE 784.3 ES+ 4.06 0
142 460 C PS-Aldehyde SPE 787.3 ES+ 5.88 0
143 822 C PS-Aldehyde SPE 787.3 ES+ 6.34 0
144 838 C PS-Aldehyde SPE 775.3 ES+ 5.75 0
145 177 C PS-Aldehyde SPE 744.4 ES+ 4.41 0
146 317 C PS- Aldehyde SPE 744.3 ES+ 6.36 0
147 648 C PS-Aldehyde SPE 744.4 ES+ 6.82 0
148 52 C PS-Aldehyde SPE 868.6 ES+ 7.24 0
149 296 C PS-Aldehyde SPE 869.6 ES+ 5.19 0
150 280 C PS-Aldehyde SPE 869.6 ES+ 7.02 0
151 730 C PS-Aldehyde SPE 869.6 ES+ 7.38 0
152 663 C PS-Aldehyde SPE 869.6 ES+ 4.07 0
153 915 C PS-Aldehyde SPE 870.6 ES+ 6.58 0
154 857 C PS-Aldehyde HPLC 857.6 ES+ 6.75 0
155 429 C PS-Aldehyde HPLC 891.6 ES+ 7 0
156 16 C PS-Aldehyde HPLC 842.5 ES+ 6.50 0 In Vivo Profiles
Oral dose-response activity of several compounds, including I and IV, has been demonstrated in an Acute Hypoxia Model (AHM, Figure 1). A detailed description of this model can be found in WILLIAM L. RUMSEY ET AL., OXYGEN TRANSPORT TO TISSUE XIX (Harrison and Delpy eds., Plenum Press 1997), herein incorporated by reference. Studies with I show that orally-dosed ANPCR blockers are capable of diminishing the rise in hypoxia- induced pulmonary pressure at doses as low as 10 mg/kg. This compound caused marked effects on the pulmonary side of the circulation; little if any systemic effect on mean arterial pressure (MAP) was observed.
Similar results were observed upon oral administration of peptide IV. Peptide IV decreased the rise in hypoxia-induced pulmonary pressure at 30 mg/kg without significant systemic effects. Radioimmunoassay
Studies were performed (Figure 2) to determine whether oral administration of I would increase plasma levels of ANP concomitant with changes in right ventricular systolic pressure in the acute hypoxia model in vivo. Compared with vehicle controls, rats exposed to I showed a 1.7-fold increase in immunoreactive plasma concentrations of ANP (mean±SE: 19.91±1.24 vs. 33.85±4.54 pg/ml, respectively; Figure 2, top panel). These values were statistically different at the P<0.05 level. Similar results were observed with IV (Figure 2, bottom panel). Administration and Use
Compounds of the present invention are shown to have natriuretic, diuretic and hypotensive activity in the intact mammal, and may possess vasorelaxant activity or inhibit the release of aldosterone and renin. Thus, these compounds, and compositions containing them, may be used as therapeutic agents in the treatment of various edematous states such as, for example, congestive heart failure, nephritic syndrome and hepatic cirrhosis, pulmonary disease, in addition to hypertension and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
The present invention also provides compositions comprising an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, serve to provide the above-recited therapeutic benefits. Such compositions can also be provided together with physiologically-tolerable liquid, gel or solid diluents, adjuvants and excipients. The compounds of the present invention may also be combined with other compounds known to be adjuvants for, or otherwise used as, therapeutic agents for the above or related indications.
These compounds and compositions may be administered to humans in a manner similar to other therapeutic agents and, additionally, to other mammals for veterinary use, such as with domestic animals. In general, the dosage required for therapeutic efficacy will range from about 0.01 to 1000 mg/kg, more usually 0.1 to 100 mg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time until the desired therapeutic benefits have been obtained.
Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like. The compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intravenously. Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and, in some cases, oral formulations. For suppositories, traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10% preferably l%-2%. Oral formulations include such normally-employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders, and contain 10%-95% of active ingredient, preferably 25%-70%.
The peptide compounds may be formulated into compositions as neutral or salt forms. Pharmaceutically-acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
In addition to the compounds of the present invention which display natriuretic, diuretic or vasorelaxant activity, compounds of the present invention may also be employed as intermediates in the synthesis of such useful compounds. Alternatively, by appropriate selection, compounds of the present invention whose activity levels are reduced or eliminated entirely can serve to modulate the activity of other diuretic, natriuretic or vasorelaxant compounds, including compounds outside the scope of the present invention, by, for example, binding to alternate receptors, stimulating receptor turnover, or providing alternate substrates for degradative enzyme or receptor activity and thus inhibiting these enzymes or receptors. When employed in this manner, such compounds may be delivered as admixtures with other active compounds or may be delivered separately, for example, in their own carriers.
Compounds of the present invention may also be used for preparing antisera for use in immunoassays employing labeled reagents, usually antibodies. Conveniently, the polypeptides can be conjugated to an antigenicity-conferring carrier, if necessary, by means of dialdehydes, carbodiimide or using commercially-available linkers. These compounds and immunologic reagents may be labeled with a variety of labels such as chromophores; fluorophores such as, e.g., fluorescein or rhodamine; radioisotopes such as l251, 5S, ,4C, or 3H; or magnetized particles, by means well known in the art.
These labeled compounds and reagents, or labeled reagents capable of recognizing and specifically binding to them, can find use as, e.g., diagnostic reagents. Samples derived from biological specimens may be assayed for the presence or amount of substances having a common antigenic determinant with compounds of the present invention. In addition, monoclonal antibodies may be prepared by methods known in the art, which antibodies can find therapeutic use, e.g., to neutralize overproduction of immunologically -related compounds in vivo. Synthesis Compounds within the scope of the present invention may be synthesized chemically by means well known in the art. One example of such a scheme may be generally depicted as:
R^
Rc- - resin R£ -resin
Figure imgf000020_0001
Figure imgf000020_0002
where the starting material is attached to a resin and the compound is constructed by the successive addition of various building blocks. Alternatively, the resin may be attached to a starting material that will end up in a more central location of the desired compound; and through the use of commonly-known protecting groups, the compound may be extended in multiple directions. Examples
Purification methods:
SPE = solid phase extraction; ppt = precipitation from ether; HPLC = preparative HPLC.
HPLC methods:
(a) 20% to 80% ACN in 25 min. Monitored at 254 nm; (b) 20% to 80% ACN in 25 min. Monitored at 210 nm;
(c) 10% to 60% ACN in 10 min. Monitored at 210 nm and 254 nm;
(d) 20-80% ACN/H2O (both w/ 0.1% TFA) over 20 min hold 80% ACN for 5 min.;
(e) 10-50% ACN/H2O/0.1%TFA over 30 min on a Dynamax C18, 60 A, 4.6 mm x 300 mm column at 210 nm and 254nm;
(f) 4.6 mm x 25 cm Vydac C!8 Peptide/Protein Column (5 mm) 30-70% CH3CN/H2O (+0.1% CF3CO2H) over 20 min. 1.5 mL/min, T=35 °C; 1 = 220 nm;
(g) 4.6 mm x 25 cm Vydac C18 Peptide/Protein Column (5 mm) 30-60% CH3CN/H2O (+0.1% CF3CO2H) over 10 min. 1.5 mL/min, T=35 °C; 1 = 220 nm; (h) 4.6 mm x 5 cm Varian Microsorb Column (3 mm) 30-60% CH3CN/H2O (+0.1 %
CF3CO2H) over 8 min. 1.0 mL/min, T=35 °C; 1 = 220 nm;
(i) 4.6 mm x 5 cm Varian Microsorb Column (3 mm) 5-80% CH3CN/H2O (+0.1% CF3CO2H) over 12 min. 1.0 mL/min, T=35 °C; 1 = 220 nm;
0) 4.6 mm x 5 cm Varian Microsorb Column (3 mm) 10-90% CH3CN/H2O (+0.1% CF3CO2H) over 10 min. 1.0 mL/min, T=35 °C; 1 = 220 nm;
(k) Preparative Method: Using a Waters LC 4000 HPLC system with Waters 991 PDA detector. Column: Dynamax 25 mm id. x 20cm 300 A column No. C18-83-223-C with Guard column using a water + 0.1% [v/v] trifluoroacetic acid /acetonitrile + 0.1% [v/v]trifluoroacetic acid gradient at a flow rate of 12 mL/min. 1=220 nm; (1) Using a Waters LC 4000 HPLC system with Waters 991 PDA detector. Column:
YMC 4.6 mm x 250mm ODS-A S-5 C18 120 A column spherical particle-5m using a 20 to 70% water + 0.1% [v/v] trifluoroacetic acid /acetonitrile + 0.1% [v/v]trifluoroacetic acid gradient over 20 min. at a flow rate of 1.4ml/min. 1=220 nm;
(m) Using a Waters LC 600E HPLC system with Waters tunable-absorbance UV detector. Column: Vydac 218TP54 4.6 mm x 250mm C18 with guard column 300 A column 5 μm particle size using a 10 to 50% water + 0.1% [v/v] trifluoroacetic acid /ACN + 0.1% [v/v]trifluoroacetic acid gradient over 30 min. at a flow rate of 1.5ml/min. 1=220 nm;
(n) Dynamax C18 column, 25 cm x 4.6 mm, 60A, 8 μm, 1.5 mL/min, 20%-60% ACN/ H2O (0.1%TFA) over 20 min, 215 nm and 254 nm; (o) Dynamax C,8 column, 5 cm x 4.6 mm, 100 A, 3 μm, 1 mL/min, 20%-60%
ACN/H2O (0.1%TFA) over 7.5 min, 215 nm and 254 nm. Central Ring Intermediate Examples: Synthesis of N-fluorenylmethyloxycarbonyl-N-Me-D-freidingerlactam-L- isoleucine.
Loading of N-fluorenylmethyloxycarbonylfreidingerlactam-L-isoleuicine to 2- chlorotritylchloride resin, 1% DVB. The 2-chlorotritylchloride resin (25g) was swelled in CH2C12 (300 mL) and drained. DIPEA (12.5 mL) was dissolved in 175 mL dry CH2C12 and added to the swelled resin. The N-fluorenylmethyloxycarbonylfreidingerlactam-L-isoleucine (11.82g) was dissolved in 175 mL of dry CH2C12, followed by 12.5 mL DIPEA with vigorous stirring. This was added to the resin and reaction was shaken on a mechanical shaker for three hours. The resin was filtered, washed 500 mL 17:2:1 CH2Cl2:MeOH:DIPEA, and CH2C12 (8X). The recovered filtrates were combined washed 2X IN HC1, stripped to an off white solid, weight recovered starting material 1.98g (17%).
2-Nitrobenzenesulfonamide protection. The resin was swelled in DMF and 20% piperidine/DMF was added (200 mL) and N2 was bubbled through for 20 minutes. The resin was filtered and the deprotection repeated. Resin was washed 8X DMF, Kaiser test, strong positive. The resin was washed 8X dry THF. A solution of 24 mL DIPEA in 500 mL dry THF was added to the resin, followed by portion- wise addition of 20.32 g 2- nitrobenzenesulfonyl chloride dissolved/diluted to 92 mL with CH2C12. The resin was shaken for 4 hrs when the cocktail was filtered and resin washed 8X THF, Kaiser test negative. Mitsunobu. To the THF-swelled resin was added 30.05 g triphenylphosphine dissolved/diluted to 57 mL in dry THF, followed by a solution of 9.3 mL dry MeOH in 375 mL dry THF. Diethylazodicarboxylate (DEAD) (18.0 mL) was dissolved/diluted to 114 mL with dry THF and added to the resin. After shaking the reaction for 1.5 hrs the cocktail was filtered and the resin washed 8X THF, 8X CH2C12, 8X Et2O, dried over N2 and stored under refrigeration over night. Sulfonamide cleavage. The resin was swelled in DMF and 250 mL of a 1 M (66.1 g diluted to 500 mL with DMF) solution of benzenethiol sodium salt in DMF was added and shaken for 1 hr. The resin was drained, washed 8X DMF and the remaining 250 mL of the 1 M solution was added and shaken for an additional hour. The resin was filtered and washed 3X DMF, 3X MeOH, 3X DMF, 3X MeOH, 8X CH2C12, Npit test positive. Fmoc protection. To the CH2C12 swelled resin was added a solution of 12 mL DIPEA in 150 mL dry CH2C12. Fmoc-Cl (23.73 g) was dissolved in 150 mL dry CH2C12 followed by 12 mL DIPEA. This was added to the resin and reaction shaken for three hours. The resin was filtered and washed 8X CH2C12, Npit Test, negative.
Cleavage of N-fluorenylmethyloxycarbonyl-N-Me-D-freidingerlactam-L- isoleucine from the resin. A 1% TFA/CH2C12 solution (250 mL) was added to the resin and shaken for 20 minutes and drained into a round-bottom flask, this was repeated again with 250 mL fresh 1% TFA/ CH2C12 for 25 minutes and collected. The organics were striped, leaving a light brown solid which was placed under vacuum overnight. Weight of material 10.80 g (quantitative yields, based on loaded starting material) APCI-MS: M+l , 451 (20%); M-l 8, 433 (50%); M-46, 405 (50%); M-222, 229 (100%) Η NMR (d6-DMSO/TFA shake, 300 MHz) δ 0.839 (m, 3H, CH3) δ 0.941 (m, 3H, CH3) δ 1.033-1.127 (m, IH, CH2) δ 1.374 (m, IH, CH) δ 1.985 (m, 2H, CH2) δ 2.219 (m, IH, CH2) δ 2.702 (s, 3H, CH3) δ 3.345-3.430 (m, 2H, CH2) δ 4.263-4.424 (m, 4H, 2CH, CH2) δ 4.680-4.794 (m, IH, CH) δ 7.354 (t, 2H, CH, J=7.2) δ 7.434 (t, 2H, CH, J= 7.4) δ 7.668 (d, 2H, CH, J=7.2) δ 7.893 (d, 2H, CH, J= 7.5) Synthesis of N-Fluorenylmethyloxycarbonyl-D-freidingerlactam-L-isoleucine t-Boc-D-methionine-L-isoleucine methyl ester. Boc-D-methionine (24.2 g, 97.2 mmol), L- isoleucine methyl ester hydrochloride (17.7 g, 97.2 mmol), hydroxybenztriazole hydrate (16.3 g, 117 mmol), 1 -(3 -dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22.5 g, 117 mmol) and diisoproplylethylamine (35ml, 200 mmol) were dissolved in DMF (300 mL). The reaction was stirred under nitrogen for 16 hours, then it was diluted with water (1000 mL) and extracted with ethyl acetate (2 x 250 mL). The combined organics were washed with IM HCl (100 mL), water (2 x 100 mL), saturated aqueous sodium bicarbonate (100 mL) and brine (100 mL). The organics were dried with magnesium sulfate and concentrated by rotary evaporation. The resulting oil was diluted with ether (25 mL) and seeded with crystals. The product was allowed to crystallize for 30 minutes then collected by vacuum filtration, then washed with a 1 to 5 ether/hexanes mixture (100 mL). A second crop of crystalline product was recovered from the mother liquors. It was washed with a 1 to 10 ether/hexanes mixture. The second crop was determined by HPLC to be of sufficient quality to combine with the first. The combined product was dried in vacuo for 30 minutes at 50 °C to yield 25.3 g white solid (89%). Boc-D-freidingerlactam-L-isoleucene methyl ester. t-Boc-D-methionine-L-isoleucine methyl ester (25.3 g, 67.2 mmol) was dissolved in dry methylene chloride (250 mL) under nitrogen and chilled in an ice bath. Trimethyloxonium tetrafluoroborate (9.94 g, 67.2 mmol) was added in one portion. The ice bath was removed and the reaction was allowed to react for 3 hours. HPLC analysis of an aliquot showed there was no remaining starting material. Dry, powdered potassium carbonate (27.9 g, 202 mmol) was added and the reaction was stirred vigorously with an overhead stirrer and refluxed for 16 hours. HPLC analysis of an aliquot showed there was no remaining intermediate. The reaction was diluted with methylene chloride (400 mL) and washed with water (5 x 500 mL) then brine (200 mL). The organics were dried with magnesium sulfate and rotary evaporated to a white solid. The residue was dissolved in refluxing methylene chloride (25 mL), and precipitated by addition of hexanes (300 mL). The solids were collected by vacuum filtration and washed with hexanes (100 mL). A second crop of solids was obtained from the mother liquor and washed with hexanes (50 mL). The second crop was determined by HPLC to be of sufficient quality to combine with the first. The combined product was dried in vacuo at 50 °C for 30 minutes to yield 17.0 g white solid (77%).
Boc-D-freidingerlactam-L-isoluecene. Boc-D-freidingerlactam-L-isoleucene methyl ester (15.6 g, 47.5 mmol) was dissolved in THF (60 mL) and methanol (60 mL). Lithium hydroxide (4.2 g, 100 mmol) dissolved in water (60 mL) was added. After one hour, TLC analysis showed no remaining starting material. All the solvent was removed by rotary evaporation. The resulting white solid was dissolved in water (300 mL), washed with methylene chloride (50 mL), and acidified with IM HCl (105 mL). A white precipitate formed. It was extracted from the aqueous phase with ethyl acetate (700 mL), washed with brine (50 mL), dried with magnesium sulfate, and concentrated to a white solid (11.4 g) by rotary evaporation. HLPC analysis showed 17% epimerization. The single diastereomer was obtained by refluxing the solid in ethyl acetate (125 mL), allowing it to stand at room temperature for one hour, collecting the solid by vacuum filtration and drying in vacuo at 50 °C for 30 minutes. The resulting white solid (8.9 g, 60%) was determined to be the pure single diastereomer by HPLC and the correct product by Η NMR.
N-Fluorenylmethyloxycarbonyl-D-freidingerlactam-L-isoleucine. Boc-D- freidingerlactam-L-isoluecene (8.86 g, 28.2 mmol) was suspended in methylene chloride and trifluoroacetic acid (30 mL) was added. After 1.5 hours the volatiles were removed by rotary evaporation. Methylene chloride (2 x 50 mL) was added and evaporated to rid remaining TFA. The residue was cooled in an ice bath and dioxane (42ml) and 10% aqueous sodium carbonate (71ml) were added. FMOC chloride (8.8 g, 33.9 mmol) was added to the resulting solution in four portions. After 18 hours the reaction was diluted with water until a clear solution was obtained (400 mL total volume). This was washed with ether (50 mL), and the aqueous phase was acidified with IM HCl to pH = 3. The aqueous layer was extracted with methylene chloride (4 x 100 mL). The combined methylene chloride extracts were washed with brine (50 mL) and dried with magnesium sulfate. The solvent was removed by rotary evaporation to give a white foam. This was dissolved in n-butyl acetate (300 mL), and crystallized by addition of hexanes (200 mL). The white solid was collected by vacuum filtration and dried in vacuo 50 °C for 30 minutes (9.36 g, 76%).
Synthesis of N-fluorenylmethyloxycarbonylpyridone-D,L-isoleucine. A solution of isoleucine t-butyl ester hydrochloride (16.7 g) in EtOH (48 mL) was added to a solution of diethyl 3-ethoxyallylidenemalonate (18.0 g) in EtOH (100 mL) followed by diisopropylethylamine (19.4 mL). After 2 h, NaOEt in EtOH (55.5 mL of a 21 wt.% solution) was added. After 2.5 h, H2O (70 mL) was added to the reaction. The reaction solution was acidified to pH = 5 by the addition of IN HCl 15 h later. The ethanol was removed from the reaction under reduced pressure and the residue was partitioned between IN HCl (300 mL) and CHC13 (300 mL). The organic layer was dried with Na2SO4, filtered through celite and concentrated to a viscous oil. The material was purified by silica gel flash chromatography (6 cm x 25 cm) using a gradient from 20 to 100% EtOAc in hexane. The product (a 3- carboxypyridone)(13.9 g) was obtained as an oil (Rf = 0.4 in 30% EtOAc in hexane). Diphenylphosphorylazide (11.2 mL) and triethylamine (7.5 mL) were added to a solution of the substituted 3-carboxypyridone (13.8 g) in dioxane (100 mL). After heating to 100 °C for 1 h, benzyl alcohol (5.2 mL) was added and the reaction mixture was continually heated at 100 °C for 16h. The reaction was cooled to room temperature, the solvent was removed under reduced pressure and the obtained residue was partitioned between EtOAc (300 mL) and 1 :1 IN HCl and sat. NaCl solution (200 mL). The organic layer was washed with 1 : 1 IN HCl and sat. NaCl solution (200 mL). The organic layer was dried with Na2SO4, filtered through celite and concentrated. The material was purified by silica gel flash chromatography (5 cm x 30 cm) using a gradient from 10 to 50% EtOAc in hexane. The benzyloxycarbonyl N-protected product obtained (15.2 g; Rf = 0.7 in 30% EtOAc in hexane) was dissolved in EtOH (150 mL) and 10% Pd-C (3 g) was added. The reaction mixture was kept under H2 (45 psi) for 5 h. The reaction solution was filtered through celite and the filtrate was concentrated giving the corresponding 3-aminopyridone (9.9 g). 9-Fluorenylmethyl chloroformate(l 1.0 g) was added in small batches over the course of 1 h to a cooled solution (4 °C) of the 3-aminopyridone (9.9 g) in 10% aqueous Na2CO3 (89 mL) and dioxane (53 mL). The reaction was allowed to warm to room temperature over 1 h. The solvents were removed under reduced pressure and the residue was partitioned between EtOAc (300 mL) and IN HCl (200 mL). The organic layer was washed with IN HCl (100 mL). The combined aqueous washings were extracted with EtOAc (40 mL). The combined organic extracts were dried with Na2SO4, filtered through celite and concentrated. The material was loaded on a silica gel flash column (5 x 20 cm) and the product was eluted off using a gradient of 10 to 30% EtOAc in hexane. The FMOC- protected product (15.4 g; Rf = 0.75 in 30% EtOAc in hexane) was obtained as an off-white foam. The FMOC-aminopyridone (15.3 g) was dissolved in 3:1 CH2C12 in TFA (100 mL). After 14 h the solvents were removed under reduced pressure. The residue was concentrated from Et,O (3 x 20 mL) to give the product (N-fluorenylmethyloxycarbonyl pyridone- isoleucine) (13.6 g) as a foam.
Synthesis of N-fluorenylmethyloxycarbonylimidazole-L-isoleucine. A solution of L-isoleucine-O-t-butyl ester hydrochloride (7.10 g) and NaHCΟ3 (2.80 g) in MeOH (150 mL) and H2O (80 mL) were added to 1 ,4-dinitroimidazole (5.0 g) at 0 °C. After 4.5 h the MeOH was removed under reduced pressure and the remaining solution was partitioned between EtOAc (300 mL) and IN HCl (200 mL). The organic layer was washed with IN HCl (200 mL x 2), dried with Na2SO4, filtered through celite and concentrated. The residue was purified by silica gel flash chromatography (6 x 23 cm) using a gradient from 20 to 67% EtOAc in hexane to afford the mononitroimidazole product (8.17 g; Rf of 0.33 in 30% EtOAc in hexane). The obtained material and 10% Pd-C (1.4 g) were reacted in EtOH (135 mL) under 47 psi H2 for 2 h. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure. The amino-imidazole product (7.05 g) was dissolved in 10% Na2CO3 (aq) (70 mL) and dioxane (42 mL) and the solution was cooled in an ice-water bath. Then, 9- fluorenylmethyl chloroformate (7.93 g) was added in small portions over the course of 35 min. The ice bath was removed and the reaction was continued for another 3 h. The reaction mixture was partitioned between 250 mL EtOAc and 350 mL IN HCl. The organic layer was washed with IN HCl (200 mL x 2), dried with Na2SO4, filtered through celite and concentrated. The residue was applied to a silica gel flash column (6 x 23 cm). The product (5.07 g; Rf = 0.5 in 50% EtOAc/hexane) was eluted off the column using a gradient of 20 to 67% EtOAc in hexane. The FMOC-amino compound (5.07 g) was dissolved in 3:1 CH2C12:TFA. After 16 h the solvents were removed under reduced pressure. The viscous oil was concentrated from Et2O (30 mL x 3) until a foam resulted.
Synthesis of N-benzyloxycarbonyl-D-freidingerlactam-L-isoleucine
Figure imgf000027_0001
Figure imgf000027_0002
Aldehyde i (2.18 g, 5.16 mmol) and L-isoleucine methyl ester hydrochloride (1.09 g, ό.Ommol, 1.2 eq) were dissolved in 30 mL methanol. Sodium cyanoborohydride (0.95 g, 15 mmol) was added and the mixture was stirred for 2 hours, then diluted with 150 mL ethyl acetate and washed with aqueous 10% sodium carbonate, water and brine. The organics were dried with anhydrous magnesium sulfate and the volatiles were removed by rotary evaporation. Column chromatography on silica gel using 25% ethyl acetate in hexanes afforded 1.45 g (51%) of intermediate ii as a colorless oil. A portion (0.75 g, 1.36 mmol) of intermediate ii was dissolved in 10 mL methylene chloride and 10 mL trifluoroacetic acid was added. After 2 hours the volatiles were removed by rotary evaporation, and the residue was dissolved in 25 mL dry THF to which was added DIEA (1.1 mL, 4.5 eq), HOBT (0.42 g, 2.2 eq), and EDC (0.58 g, 2.2 eq). The reaction was stirred under nitrogen atmosphere for 16 hours, then diluted with ethyl acetate and washed with water and brine. Column chromatography on silica gel with a gradient of ethyl acetate in methylene chloride afforded 0.35 g (69%) of intermediate iii. All of intermediate iii was dissolved in 30 mL ethanol, and hydrogenated for 3 hours on a Parr shaker with 50 psi hydrogen and Pearlman's catalyst. R5-R4-R3-CH:CO:H Intermediate Examples
Figure imgf000028_0001
VI
4-(2-Naphthaloylamido)phenylacetic acid p-Aminophenylacetic acid (5.0 g, 33.07 mmol) was dissolved in MeOH (50 mL) and placed in an ice water bath followed by drop-wise addition of H2SO4 (4 mL). After stirring for 10 min, the dark colored reaction mixture was heated to 60 °C for one hour. The reaction was allowed to cool to room temperature and while stirring the mixture was slowly quenched by addition of NaHCO3 (sat. 100 mL). The reaction mixture was washed with ether (3 x 200 mL). The combined organics were washed with water and brine, then dried (MgSO4), and evaporated under vacuum to provide 4.26 g (76%) of the dark crude methyl ester iv that was used directly in the next step. The crude methyl ester (4.26 g, 25.81 mmol) was dissolved in CH2C12 (50 mL) and Et3N (3.96 mL) was added. 2-Naphthoyl chloride (4.92 g, 25.81 mmol) dissolved in CH2C12 (5 mL) was added drop-wise and reaction allowed to stir for 2 hours under N2. The reaction was quenched with NaHCO3 (100 mL) and washed with CH2C12 (3 x 150 mL). The organics were combined and washed with brine, dried (MgSO4), and evaporated under vacuum. The material was precipitated with Et2O, filtered and washed with Et2O several times and dried in an vacuum oven at 50 °C to provide 6.86 g (83%) of amide v as an off-white solid that was used directly in the next step. The crude off-white solid (6.3 g, 19.7 mmol) was dissolved in THF (253 mL) and MeOH (63 mL). Next, LiOH.H20 (1.66 g) dissolved in water (63 mL) was added drop-wise. The reaction mixture was stirred for 30 min and reaction adjusted to pH 2 with IN HCl. Reaction mixture was washed with EtOAc (3 x 300 mL). The combined organics were washed with brine, dried (MgSO4), and evaporated under vacuum. The crude material was precipitated with Et2O, filtered and washed with Et2O several times and dried in an vacuum oven at 50 °C to provide 5.75 g (95.7%) of a pale white solid vi that was used for peptide N-capping. Η NMR (DMSO-d6) δ 10.42 (s, IH), 8.58 (s, IH), 8.11-8.00 (m, 4H), 7.75 (d, J = 8.4 Hz, 2H), 7.67-7.60 (m, 2H), 7.25 (d, J = 8.4 Hz, 2H), 3.55 (s, 2H).
4-(Allylacetate)phenylacetic acid
Figure imgf000029_0001
vii viii
Figure imgf000029_0002
x ix
4-(Bromomethyl)phenylacetic acid (20.0 g, 87.3 mmol) was stirred in anhydrous CH2C12 (200 mL) under N2 and cooled in an ice-water bath. DMF (cat., 0.4 mL, 0.25 eq) was added followed by drop-wise addition of oxalyl chloride (9.1 mL) to the stirring cooled solution and the 4-(bromomethyl)phenylacetic acid began to slowly dissolve. After 2 hours, the solvent and excess oxalyl chloride was removed under vacuum at 50 °C and the crude acid chloride was chased with anhydrous toluene (2x 10 mL). In a separate 3 -neck flask with a condenser charged with t-BuOH (500 mL, Aldrich distilled), the t-BuOH was purged with a stream of N2 while stirring under N2 for 15 min. The t-BuOH was heated to 55 °C and DIPEA (22.8 mL) was added followed by addition of the crude acid chloride dissolved in CH2C12 (100 mL). After 30 min, the excess t-BuOH was evaporated under vacuum at 60 °C and the salts were precipitated with Et2O, filtered, washed with Et2O, and discarded. The Et2O mother liquors were combined and concentrated. Silica gel flash chromatography of the crude product (5% ethyl acetate-hexane) provided 17.2 g ( 69%) of the t-butyl ester vii as a clear and colorless oil. Η NMR (CDC13) δ 7.3 (d, J= 8.3 Hz, 2H), 7.2 (d, J= 8.3 Hz, 2H), 4.5 (s, 2H), 3.5 (s, 2H), 1.3 (s, 9H). Mass spectroscopy did not provide any interpretable peaks or fragments. t-Butyl ester vii (17.2 g, 60.35 mmol) was dissolved in DMSO (120 mL) and NaNO2 (16.66 g, 4 eq.) was added in one portion followed by addition of CH3CO2H (34.55 mL, 10.0 eq.). A condenser was connected to the flask and the stirred mixture was heated to 35 °C overnight. The reaction was cooled to room temperature and quenched with water (200 mL) and allowed to stir for 15 min. The mixture was washed with Et2O ( 3 x 150 mL). The Et2O layers were combined, washed with brine, then with NaHCO3 (sat.) (2 x 100 mL). The NaHCO3 layers were combined and acidified to pH = 4 with slow addition of concentrated HCl. The acidic aqueous layer was washed with CH2C12 (3 x 150 mL), the organics were combined and washed with brine, dried (MgSO4), and concentrated to provide viii (8.2 g, 60%) as a white solid. 'H NMR (DMSO-d6) δ 7.9 (d, J= 8.4 Hz, 2H), 7.3 (d, J= 8.4 Hz, 2H), 3.6 (s, 2H), 1.4 (s, 9H). Mass spectroscopy did not provide any interpretable peaks or fragmentation's. The benzoic acid derivative viii (7.57 g, 32.07 mmol) was dissolved in DMF (100 mL) and K2CO3 (4.4 g, 32.07 mmol) was added as a solid and the mixture was stirred under N2 for 15 min followed by addition of allyl bromide (2.9 mL 1.05 eq). After 2 hours, the reaction was added to ethyl acetate and washed with water (150 mL) and brine (5 x 200 mL). The organic layer was dried (MgSO4), and concentrated. Silica gel chromatography (5% ethyl acetate in hexane) of the crude oil provided h as a colorless and clear oil (7.5 g, 85%). Η NMR (DMSO-d6) δ 7.9 (d, J= 8.4 Hz, 2H), 7.4 (d, J= 8.1 Hz, 2H), 6.0 (m, IH), 5.4(dd, J= 3 Hz, J= 17.2 Hz, IH), 5.2 (dd, J= 3 Hz, J= 10.5 Hz, IH) 4.8 (d, J= 5.4 Hz, 2H), 3.7 (s, 2H), 1.4 (s, 9H). Mass spectroscopy provided fragmentation of 221 indicating lose of the t-butyl group (-57). Compound ix (7.5 g, 27.17 mmol) was dissolved in CH2C12 (200 mL) and TFA (63 mL) dissolved in CH2C12 (100 mL) was added drop-wise to the stirring solution. After 4 hours, the solvent was removed under vacuum. Silica gel flash chromatography of the crude product ( 5% MeOH/ CH2C12 ) provided x as a white solid (5.9 g, 95%). 'H NMR (DMSO-d6) δ 7.9 (d, J= 8.4 Hz, 2H), 7.4 (d, J= 8.1 Hz, 2H), 6.0 (m, IH), 5.4(dd, J= 3 Hz, J= 17 Hz, IH), 5.2 (dd, J= 3.0, Hz, J= 10.5 Hz, IH) 4.8 (d, J= 5.1 Hz, 2H), 3.7 (s, 2H). MS 220.3 (M+), 163.2 (-57)
N-(2-naphthoyl)-3-aminophenylacetic acid and N-(l-naphthoyl)-3- aminophenylacetic acid.
Methyl 3-aminophenylacetate. 3-Aminophenylacetic acid (3.8 g, 25 mmol) was dissolved in methanol (50 mL) and sulfuric acid (2 mL, 36 mmol). After the reaction was stirred for 8 hours the solvents were removed by rotary evaporation. The residue was partitioned between ethyl acetate (100 mL) and 10% sodium carbonate (50 mL). The organic phase was washed with brine (30 mL) and dried over magnesium sulfate. The solvent was removed by rotary evaporation to afford methyl 3-aminophenylacetate (4.0 g, 96%) as a yellow oil.
N-(2-Naphthoyl)-3-aminophenylacetic acid. A portion of the methyl 3- aminophenylacetate (1.65 g, 10 mmol) was dissolved in methylene chloride (50 mL). DIEA (3.5 mL, 20 mmol) then 2-naphthoyl chloride (2.0 g, 10.5 mmol) dissolved in methylene chloride (10 mL) were added to the resulting solution. After 16 hours the reaction was diluted with ethyl acetate (100 mL) and washed with water (50 mL), saturated ammonium chloride (50 mL), and brine (50 mL). The organics were dried over magnesium sulfate and the solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate. Methyl N-(2-naphthoyl)-3-aminophenylacetate (2.1 g, 66%) was obtained as an off-white solid. This was dissolved in methanol (20 mL) and THF (20 mL). A solution of lithium hydroxide (0.84 g, 20 mmol) dissolved in water (15 mL) was added and the reaction was allowed to stir for 4 hours. The solvent was removed by rotary evaporation. The resulting solid was dissolved in water and washed with ethyl acetate (30 mL). The aqueous phase was acidified to pH = 3 with IM HCl and extracted with ethyl acetate. The organics were washed with brine and dried over magnesium sulfate. The solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate to afford pure N-(2- naphthoyl)-3-aminophenylacetic acid (1.4 g, 70%) as a white solid. N-(l-Naphthoyl)-3-aminophenylacetic acid. A second portion of methyl 3- aminophenylacetate (1.65 g, 10 mmol) was dissolved in methylene chloride (50 mL). DIEA (3.5 mL, 20 mmol) then 1-naphthoyl chloride (2.0 g, 10.5 mmol) dissolved in methylene chloride (10 mL) was added to the resulting solution. After 16 hours the solvent was removed by rotary evaporation. The resulting solid was dissolved in ethyl acetate (100 mL) and methylene chloride (25 mL) and washed with water (50 mL), saturated ammonium chloride (50 mL) and brine (50 mL). The organic layer was dried over magnesium sulfate and the solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate. Methyl N-(l-naphthoyl)-3 -aminophenylacetate (2.2 g, 69%) was obtained as a white solid. This was dissolved in methanol (20 mL) and THF (20 mL). A solution of lithium hydroxide (0.76 g, 18 mmol) was added to the solution and the reaction was allowed to stir for 5 hours. The solvent was removed by rotary evaporation. The resulting solid was dissolved in water (100 mL) and washed with ethyl acetate (30 mL). The aqueous phase was acidified to pH = 3 with IM HCl and extracted with ethyl acetate. The organics were washed with brine and dried over magnesium sulfate. The solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate to afford pure N-(l-naphthoyl)-3-aminophenylacetic acid (1.4 g, 68%) as a white solid. N-(2-Naphthoyl)-2-aminophenylacetic acid and N-(l-naphthoyl)-2- aminophenylactic acid.
Methyl 2-aminophenylacetate. 2-Nitrophenylacetic acid (5.4 g, 30 mmol) was dissolved in methanol (50 mL). Sulfuric acid (1.7 mL, 30 mmol) was added and the reaction was stirred for 16 hours. The solvent was removed by rotary evaporation. The resulting oil was dissolved in ethyl acetate (100 mL) and washed with 10% sodium carbonate (50 mL), brine (50 mL). The organic layer was dried over magnesium sulfate and the solvent was removed to give the methyl ester as a yellow oil (5.2 g, 89%). This was dissolved in methanol (100 mL) and Pearlman's catalyst (50 mg) was added. This was hydrogenated under 50 psi hydrogen for 3 hours. The catalyst was removed by vacuum filtration through a pad of celite. The solvent was removed by rotary evaporation to give methyl 2-aminophenylacetate (4.4 g, 99%) as a colorless oil.
N-(2-NaphthoyI)-2-aminophenylacetic acid. Methyl 2-aminophenylacetate (1.6 g, 10 mmol) was dissolved in methylene chloride (50 mL) and DIEA (3.5 mL, 20 mmol), then 2- naphthoyl chloride (2.0 g, 10.5 mmol) dissolved in methylene chloride (10 mL) were added. After 16 hours the reaction was diluted with ethyl acetate (100 mL) and washed with saturated ammonium chloride (50 mL) and brine (50 mL). The organic layer was dried over magnesium sulfate, and the solvent was removed by rotary evaporation. The crude product was chromatographed on silica gel with methylene chloride to give pure methyl N-(2-naphthoyl)- 2-aminophenylacetate (2.3 g, 72%) as a yellow oil that crystallized on standing. This was dissolved in methanol (20 mL) and THF (20 mL) and a solution of lithium hydroxide (0.92 g, 22 mmol) in water (20 mL) was added and stirred for 4 hours. The solvent was removed by rotary evaporation. The resulting solid was dissolved in water (100 mL) and washed with ethyl acetate (3 x 20 mL). The aqueous phase was acidified to pH = 3 with 1 M HCl, and extracted with ethyl acetate. The organics were washed with brine and dried over magnesium sulfate. The solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate to afford pure N-(2-naphthoyl)-2-aminophenylacetic acid (1.15 g, 52%) as a white solid. N-(l-Naphthoyl)-2-aminophenylactic acid. A second portion of methyl 2- aminophenylacetate (1.65 g, 10 mmol) was dissolve in methylene chloride (50 mL). DIEA (3.5ml, 20 mmol) then 1-naphthoyl chloride (2.0 g, 10.5 mmol) in methylene chloride (10 mL) was added to the solution. After 16 hours the solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate (100 mL), washed with water (50 mL), saturated ammonium chloride (50 mL), and brine (50 mL). The organic layer was dried over magnesium chloride. The product was recrystallized from refluxing ethyl acetate to give clean methyl N-(l-naphthoyl)-2 -aminophenylacetate (2.0 g, 63%) as a pink solid. This was dissolved in methanol (10 mL) and THF (10 mL) and a solution of lithium hydroxide (0.76 g, 18 mmol) in water (15 mL) was added and stirred for 4 hours. The solvent was removed by rotary evaporation. The resulting solid was dissolved in water (100 mL) and washed with ethyl acetate (30 mL). The aqueous phase was acidified to pH = 3 with IM HCl and extracted with ethyl acetate. The organics were washed with brine and dried over magnesium sulfate. The solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate to afford pure N-(l-naphthoyl)-2-aminophenylacetic acid (1.36 g, 63%) as a white solid.
N-(l-Naphthoyl)-4-aminophenylacetic acid. Methyl 4-aminophenylacetate (1.6 g, 9.6 mmol) was dissolved in methylene chloride (100 mL). DIEA (3.5 mL, 20 mmol) then 1- naphthpyl chloride (1.9 g, 10 mmol) dissolved in methylene chloride (10 mL) were added. After 4 hours the solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate (100 mL) and saturated ammonium chloride (50 mL). The organic phase was washed with 1 M HCl (20 mL), water (2 x 50 mL), and brine (50 mL) then dried over magnesium sulfate. The solvent was removed by rotary evaporation. The resulting brown solid was recrystallized from refluxing ethyl acetate (the mother liquors were diluted with ether) to afford methyl N-(l-naphthoyl)-4-aminophenylacetate (1.9 g, 63%) as a white solid. This was dissolved in methanol (20 mL) and THF (20 mL) and a solution of lithium hydroxide (0.84 g, 20 mmol) dissolved in water (15 mL) was added and stirred for 16 hours. The solvent was removed by rotary evaporation. The solid was dissolved in water (1000 mL) containing a small amount of sodium carbonate. This was washed with ethyl acetate. The aqueous phase was acidified with 1 M HCl and extracted with ethyl acetate (4 x 250 mL). The organics were washed with brine and dried over magnesium sulfate. The solvent was removed by rotary evaporation. The resulting solid was recrystallized from refluxing ethyl acetate (1000 mL) to afford N-(l-naphthoyl)-4-aminophenylacetic acid (1.16 g, 67%) as a white solid.
4-(9-Fluorenylmethoxycarbonylamino)phenylacetic acid. A mechanically-stirred suspension of 4-aminophenylacetic acid (10.0 g, 66.2 mmol) in dioxane (100 mL) and IM aqueous sodium carbonate (165 mL) was cooled to 0 °C and 9-fluorenylmethyl chloroformate (20.54 g, 79.4 mmol) was added. The reaction mixture was allowed to warm to room temperature and was stirred for 16 h. The resulting suspension was acidified to pH = 2 by addition of 12 N hydrochloric acid and the solid product isolated by filtration. The aqueous filtrate was extracted with ethyl acetate (2 x 250 mL). The solid filter cake was dissolved in ethyl acetate (1.5 L) and 0.12 N hydrochloric acid (250 mL) and the phases separated. The ethyl acetate solutions were combined and concentrated in vacuo to afford an off white solid. This product was suspended in ethyl acetate (100 mL), isolated by filtration and dried to afford the title compound as a white solid (18.9 g, 50.6 mmol, 76%). 'H NMR (CDC13): δ 12.28 (s, IH, CO2H); 9.69 (s, IH, NH); 7.91 (d, J = 7.2 Hz, 2H); 7.75 (d, J= 7.2 Hz, 2H); 7.51-7.22 (m, 6H); 7.15 (d, J = 8.1 Hz); 4.48 (d, J= 6.6 Hz, 2H); 4.31 (t, J = 6.6 Hz, IH); 3.49 (s, 2H).
N-(E)-Cinnamyl-β-alanine. β-Alanine methyl ester hydrochloride (2.79g) was stirred in a solution containing methanol (80 mL) and 0.5N sodium methoxide in methanol (40 mL). The volume was reduced and the reaction mixture was filtered to remove salts. To the filtrate trans-cinnamaldehyde (2.5ml) was added. Upon stirring for 18 h the reaction was cooled to 0 °C and sodium borohydride (1.89 g) was added portion-wise over 2.5 hours. The methanol was removed under reduced pressure and the residue was dissolved in methylene chloride. The organic solution was washed with aqueous sodium bicarbonate and brine then it was concentrated and dried completely on vacuum pump. The crude product was dissolved in THF (50 mL) and a solution of di-t-butyl dicarbonate (17.46 g) and triethylamine (13.9 mL) in THF (60 mL) was added drop- wise. Upon stirring for 18 h the reaction was concentrated and the residue dissolved in ethyl acetate washed with water and brine; concentrated and purified by column chromatography to afford a yellow oil (2.55 g). Hydrolysis of the methyl ester was completed by stirring with sodium hydroxide (IN, 16 mL), water (1.1 mL) and methanol(50 mL) for 5 h. The solution was concentrated under reduced pressure and the remaining aqueous layer was acidified. This was extracted with methylene chloride (3 x 50 mL), concentrated and dried on vacuum pump to afford product, N-cinnamyl-β-alanine (2g). Assemblv Procedure A (compounds where R8 terminates with -C(=O)NH2) 4-(2-Quinoxaloylamido)phenylacetyI-3-(R)-amido-(2-oxopyrrolidine)-l-α-(l-L-(S)- methylpropyl)acetyl-L-aspartyl-D-argininyl-L-isoleucinylcarboxamide (Procedure A, Method 1):
Coupling of He. Rink amide resin (1.5 g) was suspended in DMF (20 mL) and was gently agitated for 30 minutes. The solvent was drained from the resin, 20% (v/v) piperidine in DMF (20 mL) was added, and the suspension was gently agitated for 10 minutes. The piperidine solution was drained from the resin and the resin was washed with DMF (2 x 20 mL). The piperidine treatment was repeated. Following the last DMF wash, the resin was suspended in DMF (5 mL) and N-fluorenylmethyloxycarbonyl-L-isoleucine (0.795 g), HATU (0.813 g) and IM N,N-diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 3.5 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). A Kaiser test indicated the presence of free amine, therefore the resin was resuspended in DMF (5 mL) and N-fluorenylmethyloxycarbonyl-L-isoleucine (0.795 g), HATU (0.813 g) and IM N,N-diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 2.25 h, the reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with 20% (v/v) piperidine (20 mL) for 10 min, then was washed with DMF (2 x 20 mL). This treatment was repeated, and the resin was washed with additional DMF (2 x 20 mL).
Coupling of D-Arg. The resin was suspended in DMF (5 mL) and N- fluorenylmethyloxycarbonyl-D-Arg(Pbf)-OH (1.39 g), HATU (0.813 g) and IM N,N- diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 2 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with 20% (v/v) piperidine (20 mL) for 10 min, then was washed with DMF (2 x 20 mL). This treatment was repeated, and the resin was washed with additional DMF (2 x 20 mL).
Coupling of Asp. The resin was suspended in DMF (5 mL) and N- fluorenylmethyloxycarbonyl-L-Asp(O-t-Bu)-OH (0.88 g), HATU (0.813 g) and 1M N.N- diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 1.75 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with 20% (v/v) piperidine (20 mL) for 10 min, then was washed with DMF (2 x 20 mL). This treatment was repeated, and the resin was washed with additional DMF (2 x 20 mL).
Coupling of Freidinger lactam. The resin was suspended in DMF (5 mL) and N- fluorenylmethyloxycarbonyl-L-Ile(lactam)-OH (0.932 g), HATU (0.813 g) and 1M N.N- diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 1 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with 20% (v/v) piperidine (20 mL) for 10 min, then was washed with DMF (2 x 20 mL). This treatment was repeated, and the resin was washed with additional DMF (2 x 20 mL). Coupling of 4-aminophenylacetic acid. The resin was suspended in DMF (5 mL) and N-fluorenylmethyloxycarbonyl-4-aminophenylacetic acid (0.798 g), HATU (0.813 g) and IM N,N-diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 1 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with 20% (v/v) piperidine (20 mL) for 10 min, then was washed with DMF (2 x 20 mL). This treatment was repeated, and the resin was washed with additional DMF (2 x 20 mL).
Coupling of 2-quinoxaloyl chloride. The resin was suspended in DMF (5 mL) and 2-quinoxaloyl chloride (0.412 g) and IM N,N-diisopropylethylamine in DMF (4.3 mL) were added. The mixture was gently agitated for 5.5 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). A second coupling of 2-quinoxaloyl chloride (0.412 g) and IM N,N-diisopropylethyl amine in DMF (4.3 mL) was conducted for 12 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL).
The resin was washed with methanol (2 x 20 mL) and diethyl ether (2 x 20 mL) and was then dried in vacuo. The product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2% (v/v) thioanisole (85 mL) for 4 h. The resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL). The filtrate was concentrated to afford a red oil which was triturated with diethyl ether (4 x 100 mL) to afford the crude product as a yellow solid (0.700 g). The product was purified by preparative HPLC on a 45 mm i.d. x 30 cm Dynamax Clg 300 A column using an acetonitrile/water (+0.1% (v/v) trifluoroacetic acid) gradient at a flow rate of 60 mL/min. Fractions containing the desired product were pooled and lyophilized to afford the pure title compound as a pale yellow solid. Yield: 0.47 g. HPLC (Method f): 6.97 min. MS (ES+): m/z 887.6 Da (M+H)+ 'H NMR (d6-DMSO + TFA-d): δ 9.59 (s, IH), 8.32 (m, IH), 8.25 (m, IH), 8.04 (m, 2H), 7.90 (d, J= 8.7 Hz, 2H), 7.35 (d, J= 8.7 Hz, 2H), 4.52 (m, 2H), 4.39 (m, IH), 4.30 (d, J= 11.1 Hz, IH), 4.18 (m, IH), 3.76 (m, IH), 3.51 (s, 2H), 3.30 (m, 2H), 3.11 (m, 2H), 2.70 (m, 2H), 2.48 (m, 2H), 1.77 (m, 4H), 1.43 (m, 6H), 1.05 (m, 2H), 0.85 (m, 12H).
4-(2-Quinoxaloylamido)phenylacetyl-3(R)-amido-(2-oxopyrrolidine)-l-α-(l-L-(S)- methylpropyI)acetyl-L-aspartyl-D-argininyl-L-isoleucinyl carboxamide (Procedure A, Method 2):
The peptide was assembled by a method analogous to that described in Method 1 up to the coupling of the Freidinger lactam component starting with 0.5 g of RINK amide resin. The N-terminal substituent was then appended:
Coupling of 4-(2-quinoxaloyl)-amidophenylacetic acid. The resin was suspended in DMF (5 mL) and 4-(2-quinoxaloyl)-amidophenylacetic acid (0.23 g), HATU (0.285 g) and IM N,N-diisopropylethylamine in DMF (1.5 mL) were added. The mixture was gently agitated for 1 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was washed with methanol (2 x 20 mL) and diethyl ether (2 x 20 mL) and was then dried in vacuo. The product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2% (v/v) thioanisole (30 mL) for 3 h. The resin was removed by filtration and washed with trifluoroacetic acid (10 mL). The filtrate was concentrated to afford a red oil which was triturated with diethyl ether (3 x 100 mL) to afford the crude product as a yellow solid (0.184 g). The product was purified on a C,8 SepPak. Fractions containing the desired product were pooled and lyophilized to afford the title compound as a pale yellow solid. Yield: 0.086 g.
Ethyl 4-(2-quinoxaloyl)amidophenylacetate. A solution of ethyl 4- aminophenylacetate (0.50 g) in dichloromethane (10 mL) was treated with 2-quinoxaloyl chloride (0.51 g) and N,N-diisopropylethylamine (0.38 g). The reaction mixture was stirred at room temperature for 2.5 h, then was diluted with ethyl acetate (50 mL) and washed sequentially with 0.1 N hydrochloric acid (2 x 50 mL), water (50 mL) and brine (50 mL). The aqueous washes were extracted with ethyl acetate (50 mL). The organic extracts were combined, dried over MgSO4, filtered and evaporated to yield the title compound as a yellow solid. Yield 0.81 g. 'H NMR (CDC13) δ 9.87 (s, IH), 9.79 (s, IH), 8.23 (m, 2H), 7.91 (m, 2H), 7.81 (d, J= 8.1 Hz, 2H), 7.36 (d, J= 8.1 Hz, 2H), 4.17 (q, J= 6.9 Hz, 2H), 3.64 (s, 2H), 1.27 (t, J= 6.9 Hz, 3H). MS (APCI) m/z 358 (M+Na)\ 336 (M+H)+.
4-(2-quinoxaloyl)amidophenylacetic acid. A solution of ethyl 4-(2- quinoxaloyl)amido phenylacetate (0.8 g) and lithium hydroxide monohydrate (0.2 g) in tetrahydrofuran (20 mL), methanol (15 mL) and water (15 mL) was stirred at room temperature for 16 h. The mixture was then diluted with water (20 mL) and concentrated in vacuo to a final volume of ca. 40 mL. The solution was acidified to pH = 2 by addition of IN hydrochloric acid to afford the product as a yellow precipitate, which was isolated by filtration, washed with water (2 x 10 mL) and dried. Yield: 0.68 g. Η NMR (d6-DMSO): δ 12.3 (brs, IH), 10.8 (s, IH), 9.56 (s, IH), 8.31 (m, IH), 8.24 (m, IH), 8.03 (m, 2H), 7.88 (d, J= 8.4 Hz, 2H), 7.26 (d, J= 8.4 Hz, 2H), 3.58 (s, 2H).
Procedure B (compounds where R8 terminates with aromatics, cvcloalkyls, and heterocvcles) 4-(2-Naphthaloylamido)phenylacetyl-3-(R)-amido-(2-oxopyrrolidine)-l-α-(l-L-(S)- methylpropyl)acetyl-L-aspartyl-N-(2-indanoyl)carboxamide (Procedure B, Method 1)
Coupling of Asp. Polystyrene-PEG-PAC resin (50 g, 0.16 meq./gram) was suspended in DMF (300 mL) and was gently agitated for 30 minutes. The solvent was drained from the resin, and the resin was washed with additional DMF (2 x 200 mL). Following the last DMF wash, N-fluorenylmethyloxycarbonyl-L-Asp-α-(allyl)-OH (15.8 g) in DMF (-30 mL), 1,3- diisopropylcarbodiimide (7.52 mL) and 0.08M of 4-dimethylaminopyridine in DMF (10 mL) were added to the resin. The mixture was gently agitated for 4.5 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 200 mL). FT-IR analysis showed a strong absorption at 1760 cm"'. To determine if the resin had been fully derivatized, 0.1 g of derivatized resin was treated with N-fluorenylmethyloxycarbonyl-L-Asp-α-(allyl)-OH (0.0316 g.), 1,3 -diisopropylcarbodiimide (15 mL) and 0.08M of 4-dimethylaminopyridine in DMF (0.10 mL). The mixture was gently agitated for 3.5 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 4 mL). FT-IR analysis showed no change in intensity of the 1760 cm"' absorption indicating that double coupling is unnecessary. The resin was treated with 20% (v/v) piperidine (250 mL) for 10 min, then was washed with DMF (2 x 300 mL). This treatment was repeated, and the resin was washed with additional DMF (4 x 250 mL). Coupling of Freidinger lactam. A solution of N-fluorenylmethyloxycarbonyl-L- Ile(lactam)-OH (5.24 g), HATU (4.41 g), N,N-diisopropylethylamine (3.3 mL) in DMF (150 mL) was added to the resin. The mixture was gently agitated for 4 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 200 mL). A Kaiser test indicated the presence of free amine, therefore the resin was treated with N- fluorenylmethyloxycarbonyl-L-Ile(lactam)-OH (1.45 g), HATU (1.22 g) and N,N- diisopropylethylamine (0.91 mL) in DMF (45 mL). The mixture was gently agitated overnight. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 200 mL). A Kaiser test indicated no free amine. The resin was treated with 20% (v/v) piperidine (250 mL) for 10 min, then was washed with DMF (2 x 300 mL). This treatment was repeated, and the resin was washed with additional DMF (4 x 250 mL). Coupling of 4-(2-naphthaloylamido) phenylacetic Acid. A solution of 4-(2- naphthaloylamido)phenylacetic acid (7.32 g), HATU (8.97 g) and N,N-diisopropylethylamine (6.56 mL) in DMF (150 mL) was added to the resin. The mixture was gently agitated for 4 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 200 mL). A Kaiser test indicated no free amine. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 250 mL).
Removal of the allyl protecting group from Asp-α-allyl ester. The resin was washed with: DMF (2 x 300 mL); methylene chloride (5 x 300 mL); and 5% acetic acid / 2.5% N- methylmorpholine in methylene chloride (2 x 350 mL). The washed resin was suspended in 5% acetic acid / 2.5% N-methylmorpholine in methylene chloride(800 mL). Tetrakis (triphenylphosphine)palladium(O) (14 g) was added and mixed gently for 48 hr. The reaction solution was drained from the resin and the resin was washed with: 5% acetic acid / 2.5% N- methylmorpholine in methylene chloride (5 x 350 mL); methylene chloride (3 x 300 mL); 0.5% sodium dietyldithiocarbamate in DMF (4 x 300 mL); DMF (5 x 300 mL); methylene chloride (4 x 300 mL); 10% acetic acid in methylene chloride (4 x 300 mL); and ether (6 x 300 mL). The resin was dried under high vacuum for 18h. Yield : 45 g.
Coupling of 2-aminoindan. A solution of 2-aminoindan hydrochloride (0.271 g), HATU (0.597 g), and N,N-diisopropylethylamine (0.56 mL) in DMF (4 mL) was added to the resin (2.0 g). The mixture was gently agitated for 4 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with a solution of 2-aminoindan hydrochloride (0.271 g), HATU (0.597 g). and N,N- diisopropylethylamine (0.56 mL) in DMF (4 mL). The mixture was gently agitated for 18 hr. The reaction solution was drained from the resin and the resin was washed with DMF (5 x 20 mL), methylene chloride ( 5 x 20 mL), and diethyl ether (5 x 20 mL) and was then dried in vacuo. The product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% (v/v) water (35 mL) for 1.5 h. The resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL). The filtrate was concentrated to afford a tan oil which was triturated with diethyl ether (3 x 50 mL) to afford the crude product as a white solid (0.200 g). The product was purified by preparative HPLC on a 25 mm i.d. x 20 cm Waters 300 A column using an acetonitrile/water (+0.1% (v/v) trifluoroacetic acid) gradient at a flow rate of 12 mL/min. Fractions containing the desired product were pooled and lyophilized to afford the pure title compound as a white solid. Yield : 0.083g. MS (API+): 734.
4-(2-Naphthaloylamido)phenyIacetyl-3-(R)-amido-(2-oxopyrrolidine)-l-α-(l-L- (S)-methylpropyl)acetyl-L-aspartyl-sarcosinyl-N-[(+/-)-tetrahydrofurfuryI]carboxamide (Procedure B, Method 2):
Chiral
Figure imgf000040_0001
The peptide was assembled by the method described in Procedure B, method 1. The only difference is that the amine components were synthesized on the Bohdan RAM synthesizer using low temperature conditions.
Coupling of sarcosinyl-N-[(+/-)-tetrahydrofurfuryl]carboxamide.HCl. The following solution containing: sarcosinyl-N-[(+/-)-tetrahydrofurfuryl]carboxamide.HCl (0.125 g), HATU (0.036 g), N,N-diisopropylethylamine (0.054ml) and 1-methylimidazole (0.012ml) in DMF (4 mL) was added to the resin (0.6 g). The mixture was gently agitated for 4 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). The resin was treated with the following solution containing: sarcosinyl-N-[(+/-)- tetrahydrofurfuryl]-carboxamide.HCl (0.125 g), HATU (0.036 g), N,N-diisopropylethylamine (0.054ml) and 1-methylimidazole (0.012 mL) in DMF (4 mL). The mixture was gently agitated for 18 hr. The reaction solution was drained from the resin and the resin was washed with DMF (5 x 20 mL), methylene chloride (5 x 20 mL), and diethyl ether (5 x 20 mL) and was then dried in vacuo. The product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% (v/v) water (35 mL) for 1.5 h. The resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL). The filtrate was concentrated to afford a clear oil which was triturated with diethyl ether (3 x 50 mL) to afford the crude product as a white solid (0.096 g).
The product was purified by preparative HPLC on a 25 mm i.d. x 20 cm Waters 300 A column using an acetonitrile/water (+0.1% (v/v) trifluoroacetic acid) gradient at a flow rate of 12 mL/min. Fractions containing the desired product were pooled and lyophilized to afford the pure title compound as a white solid. Yield : 0.060g. HPLC 25.52 min. MS (API-Na+): 793.
Preparation of sarcosinyl-N-[(+/-)-tetrahydrofurfuryl]carboxamide.HCl t-Boc-sarcosinyl-N-[(+/-)-tetrahydrofurfuryl] carboxamide. In the Bohdan RAM synthesizer using the cooling block reactor, a solution containing t-boc-sarcosine (0.38 g) and N-methylmorpholine (0.242 mL) in THF (6.0 mL) was cooled to -20 °C. To this cooled solution, isobutyl chloroformate (0.262 mL) in THF (3.0 mL) was added using a slow syringe speed. The reaction was kept at -20 °C for two hours with mixing every 30 minutes by way of nitrogen bubbling. A solution containing tetrahydrofurfurylamine (0.248 mL), 1- methylimidazole (0.264 mL) in THF (4.0 mL) was added using a slow syringe speed. The reaction was kept at -20 °C for four hours with mixing every 30 minutes by way of nitrogen bubbling. The cooling unit was then turned off and the reaction was allowed to warm to room temperature overnight with mixing every 30 minutes by way of nitrogen bubbling. The solvent was removed under vacuum. The residue was dissolved in ethyl acetate (75 mL) and washed with water (2 x 10 mL), acetic acid ( 10% aqueous, 3 x 15 mL), and NaOH (IM, 2 x 10 mL). The organic extract was dried over MgSO4 , filtered and evaporated. HPLC 9.5min. MS (API+): 273.
Sarcosinyl-N-[(+/-)-tetrahydrofurfuryl]carboxamide.HCl. t-Boc-sarcosinyl-N-[(+/- )-tetrahydrofurfuryl]carboxamide was dissolved in 6 N HCl (10 mL) and for four hours. The reaction was diluted with water (30 mL), shell-frozen and lyophilized to afford the pure title compound as a white solid. Yield : 0.410g. HPLC 3.5min. MS (API+): 173. 4-(2-Naphthaloylamido)phenylacetyl-3-(R)-amido-(2-oxopyrrolidine)-l-α-(l-L-(S)- methylpropyl)acetyl-L-aspartyl-sarcosinyl-D-tertiary-leucine (Procedure B, Method 3):
Figure imgf000042_0001
The peptide was assembled using the Milligen 9050 continuous flow automated peptide synthesizer by the fmoc/t-butyl strategy on Pepsyn KA(100) resin.
Coupling of D-tertiary leucine. Pepsyn KA(IOO) resin (5.0 g, 0.10 meq./gram) was suspended in DMF (30 mL) and was gently agitated for 30 minutes. The solvent was drained from the resin, and the resin was washed with additional DMF (2 x 20 mL). Following the last DMF wash, N-fluorenylmethyloxycarbonyl-D-tertiary leucine (0.884 g) in DMF (~4 mL), 1,3- diisopropylcarbodiimide (0.47 mL) and 0.030 g of 4-dimethylaminopyridine in DMF (2 mL) were added. The mixture was gently agitated for 4.5 h. The reaction solution was drained from the resin and the resin was washed with DMF (4 x 20 mL). This derivatized resin was then packed into a 17 mm x 150 mm Omni high pressure borosilicate glass column with
PTFE adjustable end piece and attached to the Milligen 9050 automated peptide synthesizer to complete the synthesis.
Coupling of N-fluorenylmethyloxycarbonyl-Sar-OH. On the Milligen 9050, the fmoc protecting group was removed by use of 20% (v/v) piperidine / DMF using a 10 min cycle. The efficiency of the deprotection and coupling cycles were monitored by recording both pre- and post-column UV absorption (-300-350 nm). The following solution containing: N-fmoc-Sar-OH (0.545 g), HATU (0.660 g), N,N-diisopropylethylamine (0.610 mL) in DMF (5.30 mL) was added to the resin using a two- hour coupling cycle. The resin was then treated with 20% (v/v) piperidine / DMF using a 10 min cycle. The UV monitoring indicated satisfactory coupling and deprotection cycles for these steps.
Coupling of N-fluorenylmethyloxycarbonyl-L-Asp(OBu)-OH. A solution of N- fluorenylmethyloxycarbonyl-L-Asp(OBu)-OH (0.720 g), HATU (0.660 g), and N,N- diisopropylethylamine (0.610 mL) in DMF (5.30 mL) was added to the resin using a three- hour coupling cycle. The resin was then treated with 20% (v/v) piperidine / DMF using a 10 min cycle. The UV monitoring indicated satisfactory coupling and deprotection cycles for these steps.
Coupling of Freidinger lactam. A solution of N-fluorenylmethyloxycarbonyl-L- Ile(lactam)-OH (0.764 g), HATU (0.660 g), and N,N-diisopropylethylamine (0.610 mL) in DMF (5.30 mL) was added to the resin using a three-hour coupling cycle. The resin was then treated with 20% (v/v) piperidine / DMF using a 10 min cycle. The UV monitoring indicated satisfactory coupling and deprotection cycles for these steps.
Coupling of 4-(2-naphthaloylamido)phenylacetic acid. A solution of 4-(2- naphthaloylamido)phenylacetic acid (0.534 g), HATU (0.660 g), and N,N- diisopropylethylamine (0.610 mL) in DMF (5.30 mL) was added to the resin using a three- hour coupling cycle. The UV monitoring indicated satisfactory coupling cycle for this step. The resin was washed with DMF (5 x 20 mL), methylene chloride (5 x 20 mL), and diethyl ether (5 x 20 mL) and was then dried in vacuo. The product peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% (v/v) water (35 mL) for 1.5 h. The resin was removed by filtration and washed with trifluoroacetic acid (2 x 10 mL). The filtrate was concentrated to afford a clear oil which was triturated with diethyl ether (3 x 50 mL) to afford the crude product as a white solid (0.410 g). The product was purified by preparative HPLC on a 25 mm i.d. x 20 cm Waters 300 A column using an acetonitrile/water (+0.1% (v/v) trifluoroacetic acid) gradient at a flow rate of 12 mL/min. Fractions containing the desired product were pooled and lyophilized to afford the pure title compound as a white solid. Yield : 0.271g. HPLC 21.72 min. MS (LC-ES+): 801. Procedure C (compounds where R8 terminates with -NH-alkvD 4-(2-quinoIinylamido)phenylacetyl-3-(R)-amido-(2-oxopyrrolidine)-l-α-(l-L-(S)- methylpropyl)acetyl-L-aspartyl-D-argininyl-isobutyl
Coupling of isobutylamine. Polystyrene resin with the Ellman's aldehyde linker (50 mmol) was swelled in DMF (1000 mL) and acetic acid (10 mL) for 10 minutes. Isobutylamine (32 mL, 325 mmol) and sodium triacetoxyborohydride (69.5 g, 328 mmol) were added. The mixture was stirred with an overhead stirrer for two hours, then transferred to a fritted glass funnel and was washed with a one to one mixture of methanol and DMF (3 x 300 mL), DMF (3 x 300 mL), methylene chloride (5 x 300 mL) and methanol (5 x 300 mL). The resin was dried in vacuo at 40 °C for 16 hours. MAS-NMR showed disappearance of the aldehyde proton.
Coupling of D- Arg. A portion of this material (8 g, 6.5 mmol) was swelled in DMF for 15 minutes, then added a solution of N-fluorenylmethyloxycarbonyl-D-Arg(Pbf)-OH (5 g, 7.5 mmol), HATU (2.8 g, 7.4 mmol), and DIEA (2.7 mL, 15 mmol) in DMF (50 mL). The reaction was mixed with a gentle nitrogen gas flow for two hours. The liquid was drained and the resin was washed with DMF (10 x 50 mL). NPIT test showed unreacted amine. The resin was treated with a second batch of the reaction cocktail (4.2 g N-fluorenylmethyloxycarbonyl- D-Arg(Pbf)-OH, 2.3 g HATU, and 2.7 mL DIEA in 50 mL DMF) for an additional hour. The liquid was drained and the resin was washed with DMF (10 x 50 mL). NPIT test showed the reaction was complete. The resin was treated with 20% piperidine in DMF (2 x 50 mL) then washed with DMF (10 x 50 mL).
Coupling of Asp. A solution of N-fluorenylmethyloxycarbonyl-L-Asp(O-t-Bu)-OH (6.2 g, 15 mmol), HATU (5.0 g, 13 mmol) and DIEA (5 mL, 30 mmol) in DMF (50 mL) was added to the resin and mixed with a gentle nitrogen gas flow for 16 hours. The liquid was drained and the resin was washed with DMF (10 x 50 mL). Kaiser test showed the reaction was complete. The resin was treated with 20% piperidine in DMF (2 x 50 mL) then washed with DMF (10 x 50 mL).
Coupling of Freidinger lactam. A solution of N-fluorenylmethyloxycarbonyl-D- freidingerlactam-L-isoleucine (6.6 g, 15 mmol), HATU (5.0 g, 13 mmol) and DIEA (5.0 mL, 30 mmol) in DMF (50 mL) was added to the resin and mixed with a gentle nitrogen flow for 3 hours. The liquid was drained and the resin was washed with DMF (10 x 50 mL). Kaiser test showed the reaction was complete. The resin was treated with 20% piperidine in DMF (2 x 50 mL) then washed with DMF (10 x 50 mL). Coupling of 4-aminophenylacetic acid. A solution of N- fluorenylmethyloxycarbonyl-4-aminophenylacetic acid (4.1 g, 11 mmol), HATU (3.8 g, 10 mmol) and DIEA (2.6 mL, 20 mmol) in DMF (50 mL) was added to the resin and mixed with a gentle flow of nitrogen gas for 16 hours. The liquid was drained and the resin was washed with DMF (10 x 50 mL). Kaiser test showed the reaction was complete. The resin was washed with methylene chloride (5 x 50 mL) and ether (5 x 50 mL) then dried in vacuo at 40 °C for 5 hours. A portion of this resin (2.4 g, 1 mmol) was swelled in DMF (20 mL) for 30 minutes, treated with 20% piperidine in DMF (2 x 20 mL) then washed with DMF (10 x 20 mL). Coupling of quinaldic acid. A solution of quinaldic acid (0.52 g, 3 mmol), HATU (0.95 g, 2.5 mmol) and DIEA (1 mL, 6 mmol) in DMF (20 mL) was added to the resin and mixed with a gentle flow of nitrogen gas for 16 hours. The liquid was drained and the resin was washed with DMF (10 x 20 mL), methylene chloride (5 x 20 mL) and ether (5 x 20 mL) then dried in vacuo at 40 °C for 30 minutes. The resin was treated with a solution of TFA (50 mL), water (1 mL), thioanisole (1 mL), and TIS (0.5 mL) for one hour. The liquid was filtered from the resin and reduced to about 5 mL by rotary evaporation. Ether (200 mL) was added to precipitate the crude product. Purified by preparative HPLC on C,8 Dynamax column (21.4 mm x 25 cm, 60A) using a gradient of 20% to 40% acetonitrile in water with 0.5% TFA. Fractions containing pure product were combined and lyophilized to give 0.40 g (42%) of the final product as a yellow solid. HPLC: 93% purity 6.51 min C18 Dynamax (5 cm x 4.6 mm, 3 μm particle, 100 A pore) 20 - 60% acetonitrile/ water (each containing 0.5% TFA) over 7.5 min. at 1 mL/min. MS electrospray M+ 829.4 parent, 415.4 base. HNMR 250 MHz (DMSO- d6, TFA-d,) 0.86 m 12H, 0.94 m 2H, 1.48 m 4H, 1.78 m 3H, 1.94 m IH, 2.32 m IH, 2.60 m 2H, 2.91 d J= 5.5hz 2H, 3.11 t j=5.3hz 2H, 3.35 m 2H, 3.52 s 2H, 4.21 m IH, 4.26 d
J=23.3hz IH, 4.48 m 2H, 7.36 d J=7.0 2H, 7.78 t J=6.0 IH, 7.92 m 2H, 8.14 d J=6.5 IH, 8.30 d J=7.0 2H 8.66 d J=7.0 1H

Claims

Claims:
1. A linear peptide of the formula:
Figure imgf000046_0001
wherein:
R' is -CH2CH2-, -CH2CH2CH2-, =CH-CH=CH- or -N=CH-;
R2 is H or CH3;
R3 is -CH2CH2CH2-, -(E)-CH=CHC(=O)NH-, -CH2CH2C(=O)NH-, para-disubstituted phenyl, ortho-disubstituted phenyl, meta-disubstituted phenyl or a single bond;
R4 is -NHC(=O , -C(=O)NH- or -S(=O)2NH-;
R5 is 1 -naphthyl, 2-naphthyl, -CH2CH2NHCH2CH=CH-phenyl, -CH2CH2-phenyl, - CH=CH-phenyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 6-quinolyl, 3-isoquinolyl, 2-quinoxaline, 5-chloro-2-indolyl, 2-indolyl, 4-chlorophenyl, 4-methylphenyl, 3-methoxyphenyl, 4- cyanophenyl, 3,4-difluorophenyl, 3-chloro-4-fluorophenyl, 2,4-dichlorophenyl, 3,4- dichlorophenyl, 4-chlorophenyl, 3,5-dimethoxyphenyl, 4-tert-butylphenyl, phenyl, 4- trifluoromethylphenyl, -CH2CH2CH2-phenyl, 6-quinolyl-C(=O)-, 2-quinoxaline-C(=O)-, 5- chloro-2-benimidazolyl, fluorenylmethoxycarbonyl, 4-chlorobenzyl, 4-methylbenzyl, 3- quinoxalinyl, 3,4-difluorophenyl, or 4-fluorophenyl; R6 is isobutyl or sec-butyl;
R7 is N-methylglycine, -NHCH2CH2NHC(=O)-, L-arginine, D-arginine, L-ornithine, D- ornithine, histidine, citrulline, proline, hydroxyproline, 3-pyridinylalanine, L-N- methylalanine, D-N-methylalanine, aminobutyric acid, N-2-indolizidinyl or thiazolidine;
R8 is L-isoleucine-NH2, D-isoleucine-NH2, -CH2-cyclopentyl, -CH2-2- tetrahydrofuranyl, tert-butylglycine-NH2, n-butyl, isobutyl, -NH-cyclopentyl, -NHCH2-2- furanyl, -NHCH2-pyrininyl, -NHCH2-cyclohexyl, D-leucinol, -NH-isobutyl, L-allo-isoleucine- NH2, 1 -hydroxycycloleucinol, 2-(aminomethyl)-l-ethyl-pyrrolidine, or (S)-NH-2-methylbutyl, or R8 is absent when R7 is N-2-indolizidinyl;
R9 is =CH- or -C(=O)-; and =~^ represents a double bond when R9 is =CH- and a single bond when R9 is - C(=O)-.
2. The compound of Claim 1, wherein R1 is -N=CH-; and R9 is =CH-.
3. The compound of Claim 1, wherein:
R' is -CH2CH2-, -CH2CH2CH2- or =CH-CH=CH-; and R9 is -C(=O)-.
4. The compound of Claim 3, wherein R1 is -CH2CH2-;
R3 is para-disubstituted phenyl; R4 is -C(=O)NH-;
R5 is 1 -naphthyl, 2-naphthyl, -CH2CH2NHCH2CH=CH-phenyl, -CH2CH2-phenyl, -CH=CH-phenyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 6-quinolyl, 3-isoquinolyl, 2-quinoxaline, 5-chloro-2-indolyl, 2-indolyl, 4-chlorophenyl, 4-methylphenyl, 3-methoxyphenyl, 4-cyanophenyl, 3,4-difluorophenyl, 3-chloro-4-fluorophenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 4-chlorophenyl, 3,5-dimethoxyphenyl, 4-tert-butylphenyl, phenyl or 4-trifluorophenyl; and R6 is sec-butyl.
5. The compound as recited in Claim 4, wherein
R2 is H;
R5 is 2-naphthyl;
R7 is L-arginine or D-arginine; and R8 is isobutyl.
6. The compound of Claim 4, wherein
R2 is H;
R5 is 2-quinoxaline; R7 is L-arginine or D-arginine; and R8 is L-isoleucine-NH2 or D-isoleucine-NH2.
7. A pharmaceutical composition having natriuretic, diuretic or vasodilator activity in mammals, comprising a pharmaceutically effective amount of a linear peptide of Claim 1.
8. A method for treating one or more conditions selected from the group consisting of pulmonary hypertension, congestive heart failure, nephritic syndrome, hepatic cirrhosis, pulmonary disease, pulmonary hypertension and renal failure, comprising the step of administering a pharmaceutically-effective amount of a compound according to Claim 1.
9. A method for treating one or more conditions selected from the group consisting of pulmonary hypertension, congestive heart failure, nephritic syndrome, hepatic cirrhosis, pulmonary disease, pulmonary hypertension and renal failure, comprising the step of administering a pharmaceutically-effective amount of a compound according to Claim 4.
PCT/GB2000/001319 1999-04-12 2000-04-07 Modified pentapeptide antagonists of the atrial natriuretic peptide clearance receptor Ceased WO2000061631A1 (en)

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Cited By (21)

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Publication number Priority date Publication date Assignee Title
EP1189881A4 (en) * 1999-05-07 2002-11-27 Texas Biotechnology Corp Propanoic acid derivatives that inhibit the binding of integrins to their receptors
US6723711B2 (en) 1999-05-07 2004-04-20 Texas Biotechnology Corporation Propanoic acid derivatives that inhibit the binding of integrins to their receptors
US6972296B2 (en) 1999-05-07 2005-12-06 Encysive Pharmaceuticals Inc. Carboxylic acid derivatives that inhibit the binding of integrins to their receptors
US7812038B2 (en) 1999-05-07 2010-10-12 Encysive Pharmaceuticals, Inc. Carboxylic acid derivatives that inhibit the binding of integrins to their receptors
WO2001068047A3 (en) * 2000-03-17 2002-03-07 Merck Patent Gmbh Preparation containing quinoxaline derivatives
US7276481B2 (en) 2001-03-20 2007-10-02 Prochon Biotech Ltd. Method and composition for treatment of skeletal dysplasias
US8211853B2 (en) 2002-07-29 2012-07-03 Hmgene Inc. Method of promoting apoptosis of differentiated adipocytes and increasing endogenous expression of SFRP-5 peptide by administration of SFRP-5 peptide
US7879544B2 (en) 2002-07-29 2011-02-01 Hmgene Inc. Methods of identifying adipocyte specific genes, the genes identified, and their uses
US8377884B2 (en) 2007-11-21 2013-02-19 Biomarin Pharmaceutical Inc. Variants of C-type natriuretic peptides
USRE46707E1 (en) 2009-05-20 2018-02-13 Biomarin Pharmaceutical Inc. Variants of C-type natriuretic peptide
WO2010135541A2 (en) 2009-05-20 2010-11-25 Biomarin Pharmaceutical Inc. Variants of c-type natriuretic peptide
US8198242B2 (en) 2009-05-20 2012-06-12 Biomarin Pharmaceutical Inc. Variants of C-type natriuretic peptide
US8598121B2 (en) 2009-05-20 2013-12-03 Biomarin Pharmaceutical Inc. Variants of C-type natriuretic peptide
EP3175863A1 (en) 2009-05-20 2017-06-07 BioMarin Pharmaceutical Inc. Variants of c-type natriuretic peptide
USRE48267E1 (en) 2009-05-20 2020-10-20 Biomarin Pharmaceutical Inc. Variants of C-type natriuretic peptide
EP4029512A1 (en) 2009-05-20 2022-07-20 BioMarin Pharmaceutical Inc. Variants of c-type natriuretic peptide
EP4523749A2 (en) 2009-05-20 2025-03-19 BioMarin Pharmaceutical Inc. Variants of c-type natriuretic peptide
EP4591934A2 (en) 2009-05-20 2025-07-30 BioMarin Pharmaceutical Inc. Variants of c-type natriuretic peptide
US8633220B2 (en) 2010-07-02 2014-01-21 Allergan, Inc. Therapeutic agents for ocular hypertension
US9714238B2 (en) 2010-07-02 2017-07-25 Allergan, Inc. Therapeutic agents for ocular hypertension
WO2012003145A3 (en) * 2010-07-02 2012-05-03 Allergan, Inc. Therapeutic agents for ocular hypertension

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