OXYLIPINS, PROCESSES FOR MAKING THE SAME, AND METHODS FOR USING THE SAME
20240002325 ยท 2024-01-04
Inventors
- Mithun SINHA (Indianapolis, IN, US)
- Robert MINTO (Indianapolis, IN, US)
- Imran Khan MOHAMMED (Indianapolis, IN, US)
Cpc classification
C07B2200/05
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to oxylipins, processes for making the same, and methods of using. The process may form deuterated oxylipins that can be used as mass standards for mass spectroscopy.
Claims
1. A process for forming a compound of the formula I ##STR00038## or a salt thereof, wherein R.sup.1 is C.sub.1-C.sub.10 alkyl or C.sub.1-C.sub.10 alkyl having one or more of the hydrogen atoms in C.sub.1-C.sub.10 alkyl being independently, substituted by deuterium; R.sup.2 is H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl, optionally wherein one or more hydrogen atoms in C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl is independently substituted by deuterium; R.sup.c is H or D; and n is an integer from 1-7; the process comprising the following procedures: I) (a) contacting a compound of formula II ##STR00039## wherein R.sup.1 is as defined above, and each of R.sup.a and R.sup.b is independently OC.sub.1-C.sub.6 alkyl, Oaryl, Oheteroaryl; with a compound of formula III ##STR00040## wherein R.sup.2 and n are as defined above, in the presence of a base to form a compound of formula IV ##STR00041## wherein R.sup.1 and R.sup.2 are as defined above; and (b) contacting a compound of formula IV with a reducing agent to form the compound of formula I ##STR00042## wherein R.sup.1 and R.sup.2 are as defined above; and optionally hydrolyzing the compound of formula I wherein R.sup.2 is not H to form a compound of formula I wherein R.sup.2 is H; or II) (c) contacting a compound of formula V
PG-OC.sub.1-C.sub.9 alkyl-LG V wherein PG is a hydroxy protecting group and LG is a leaving group, and each hydrogen atom in C.sub.1-C.sub.9 alkyl is optionally substituted by deuterium, with a compound of formula R.sup.3-MX, wherein R.sup.3 is C.sub.1-C.sub.9 alkyl, wherein each hydrogen atom in C.sub.1-C.sup.9 alkyl is optionally substituted by deuterium, M is a metal, and X is a halogen, to form a compound of formula VI
PG-OCH.sub.2R.sup.1 VI wherein R.sup.1 is as defined above and each hydrogen atom in CH.sub.2R.sup.1 is optionally substituted by deuterium; or (d) deprotecting a compound of formula VI into a compound of formula VII
HOCH.sup.2R.sup.1 VII wherein each hydrogen atom in CH.sup.2R.sup.1 is optionally substituted by deuterium; (e) oxidizing a compound of formula VII to form a compound of formula VIII
R.sup.DO(O)CR.sup.1 VIII wherein R.sup.D is H or C.sub.1-C.sub.6 alkyl; and (f) contacting a compound of formula VIII with (R.sub.a)(R.sub.b)(CH.sub.3)P(O) in the presence of a base to form a compound of formula II; and (g) oxidatively cleaving a C.sub.3-C.sub.10 cycloalkene with a reducing agent to produce a linearized dicarbonyl and optionally esterifying the product to form a compound of formula III which can be used to generate the compound of /Formula I using steps (a) and (b).
2. The process of claim 1 comprising step (a) and step (b).
3. The process of claim 2 further comprising steps (c)-(g).
4. The process of claim 1, comprising steps (c)-(g).
5. The process of claim 1 wherein R.sup.c is H; and R.sup.1 is C.sub.2-C.sub.10 alkyl, or C.sub.2-C.sub.10 alkyl where one or more hydrogen atoms in C.sub.2-C.sub.10 alkyl is independently, substituted by deuterium.
6. The process of claim 5, wherein R.sup.1 is C.sub.6-C.sub.10 alkyl, or C.sub.6-C.sub.10 alkyl where one or more hydrogen atoms in C.sub.6-C.sub.10 alkyl is independently, substituted by deuterium
7. The process of claim 6, wherein R.sup.1 comprises at least three deuteriums.
8. The process of claim 1, wherein the compound of formula I is ##STR00043## or a salt thereof.
9. A compound of formula I ##STR00044## wherein R.sup.1 is C.sub.1-C.sub.10 alkyl and at least one hydrogen atom in C.sub.1-C.sub.10 alkyl is substituted by deuterium; R.sup.2 is H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl, wherein each hydrogen atom in C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, or 3-7 membered heteroaryl is optionally substituted by deuterium; R.sup.c is H or D; and n is an integer from 1-7; or a salt thereof.
10. The compound or salt of claim 9, wherein R.sup.2 is H.
11. The compound or salt of claim 10, wherein R.sup.C is H.
12. The compound or salt of claim 11, wherein R.sup.1 includes at least three deuteriums.
13. The compound or salt of claim 12, wherein the compound is ##STR00045##
14. A method for detecting a biofilm in a patient, the method comprising collecting a sample from a patient; adding to the sample a compound or salt thereof of claim 9, to form a spiked sample; and analyzing the spiked sample with mass spectrometry.
15. The method of claim 14, wherein said compound has the general formula of ##STR00046## or a salt thereof.
16. The method of claim 14, wherein the sample is from a breast implant.
17. A method for detecting a biofilm in a patient, the method comprising collecting a sample from a patient; adding to the sample a compound or salt thereof made according to the process of claim 1, to form a spiked sample; and analyzing the spiked sample with mass spectrometry.
18. The method of claim 17, wherein said compound has the general formula of ##STR00047## or a salt thereof.
19. The method of claim 18, wherein the sample is from a breast implant.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0087]
[0088]
DETAILED DESCRIPTION
[0089] Definitions
[0090] In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
[0091] Whenever a yield is given as a percentage, such yield refers to a mass of the entity for which the yield is given with respect to the maximum amount of the same entity that could be obtained under the particular stoichiometric conditions. Concentrations that are given as percentages refer to mass ratios, unless indicated differently.
[0092] Except as otherwise noted, the methods and techniques of the present embodiments are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001.
[0093] Chemical nomenclature for compounds described herein has generally been derived using the commercially available ACD/Name 2014 (ACD/Labs) or ChemBioDraw Ultra 19.0 (Perkin Elmer).
[0094] As used herein, the terms including, containing, and comprising are used in their open, non-limiting sense.
[0095] The term about as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term about is also intended to encompass the embodiment of the stated absolute value or range of values.
[0096] As used herein, the term purified and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term purified does not require absolute purity; rather, it is intended as a relative definition.
[0097] The term isolated requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
[0098] As used herein, the term pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
[0099] As used herein, the term treating includes alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
[0100] As used herein an effective amount or a therapeutically effective amount of a drug refers to a nontoxic but enough of the drug to provide the desired effect. The amount that is effective will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact effective amount. However, an appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
[0101] As used herein the term patient without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans receiving a therapeutic treatment with or without physician oversight.
[0102] The term inhibit defines a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
[0103] As used herein, the term alkyl refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. For example, C.sub.1-C.sub.6 alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, iso-butyl, sec-butyl, tert-butyl, and the like. As used herein, the term alkylene refers to a straight or branched, saturated, aliphatic diradical having the number of carbon atoms indicated. For example, C.sub.1-C.sub.6 alkyl includes, but is not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like. It will be appreciated that alkyl and alkylene groups can be optionally substituted with one or more substituents by replacement of one or more hydrogen atoms on the alkyl and alkylene group.
[0104] As used herein, the term heteroaryl refers to a monocyclic or fused ring group of 5 to 12 ring atoms containing one, two, three or four ring heteroatoms selected from nitrogen, oxygen and sulfur, the remaining ring atoms being carbon atoms, and also having a completely conjugated pi-electron system. It will be understood that in certain embodiments, heteroaryl may be advantageously of limited size such as 3- to 7-membered heteroaryl, 5- to 7-membered heteroaryl, and the like. Heteroaryl may be unsubstituted, or substituted as described for alkyl or as described in the various embodiments provided herein. Illustrative heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, tetrazolyl, triazinyl, pyrazinyl, tetrazinyl, quinazolinyl, quinoxalinyl, thienyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl and carbazoloyl, and the like. Illustrative examples of heteroaryl groups shown in graphical representations, include the following entities, in the form of properly bonded moieties:
##STR00014##
[0105] As used herein, halogen or halo refers to fluorine, chlorine, bromine, or iodine.
[0106] As used herein, bond refers to a covalent bond.
[0107] The term substituted means that the specified group or moiety bears one or more substituents. The term unsubstituted means that the specified group bears no substituents. Where the term substituted is used to describe a structural system, the substitution is meant to occur at any valency-allowed position on the system. In some embodiments, substituted means that the specified group or moiety bears one, two, or three substituents. In other embodiments, substituted means that the specified group or moiety bears one or two substituents. In still other embodiments, substituted means the specified group or moiety bears one substituent.
[0108] As used herein, optional or optionally means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, wherein each hydrogen atom in C.sub.1-C.sub.6 alkyl is independently optionally substituted by CN means that a cyano may be but need not be present on the C.sub.1-C.sub.6 alkyl by replacing a hydrogen atom for each cyano group, and the description includes situations where the C.sub.1-C.sub.6 alkyl is substituted with a cyano group and situations where the C.sub.1-C.sub.6 alkyl is not substituted with the cyano group.
[0109] As used herein, independently means that the subsequently described event or circumstance is to be read on its own relative to other similar events or circumstances. For example, in a circumstance where several equivalent hydrogen groups are optionally substituted by another group described in the circumstance, the use of independently optionally means that each instance of a hydrogen atom on the group may be substituted by another group, where the groups replacing each of the hydrogen atoms may be the same or different. Or for example, where multiple groups exist all of which can be selected from a set of possibilities, the use of independently means that each of the groups can be selected from the set of possibilities separate from any other group, and the groups selected in the circumstance may be the same or different.
[0110] As used herein, the term pharmaceutically acceptable salt refers to those salts with counter ions which may be used in pharmaceuticals. See, generally, S. M. Berge, et al., Pharmaceutical Salts, J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without undue toxicity, irritation, or allergic response. A compound described herein may possess a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Such salts include: [0111] (1) acid addition salts, which can be obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D)- or (L)-malic acid, maleic acid, methane sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like; or [0112] (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, and the like.
[0113] Acceptable salts are well known to those skilled in the art, and any such acceptable salt may be contemplated in connection with the embodiments described herein. Examples of acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, -hydroxybutyrates, glycolates, tartrates, and mandelates. Lists of other suitable acceptable salts are found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985.
[0114] Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Certain specific isotopic embodiments are also described herein. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as .sup.2H, .sup.3H, .sup.11C, .sup.13C, .sup.14C , .sup.15N , .sup.18O, .sup.17O, .sup.31P, .sup.32P, .sup.35S, .sup.18F, .sup.36Cl, and .sup.125I, respectively. Such isotopically labelled compounds are useful in metabolic studies (preferably with .sup.14C), reaction kinetic studies (with, for example .sup.2H or .sup.3H), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)] including drug or substrate tissue distribution assays, or in radioactive treatment of patients. Further, substitution with heavier isotopes such as deuterium (i.e., .sup.2H) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and also for example for use as a standard in mass spectrometry. Isotopically labeled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
[0115] A biofilm-derived oxylipin may be in increased abundance in BII subjects. The oxylipin may lead to activation of CD4.sup.+ Th1 cells in an in vitro and in vivo mouse model indicating its role in the establishment of an autoimmune response often observed to be associated with BII. Compounds described herein may be useful in identifying the presence of oxylipins in biofilm samples.
[0116] Representative Embodiments
[0117] The compounds described herein are directed to a compound of the formula I
##STR00015## [0118] or a salt thereof.
[0119] In some embodiments, R.sup.1 is C.sub.1-C.sub.10 alkyl. In some embodiments, each hydrogen atom in C.sub.1-C.sub.10 alkyl is optionally substituted by deuterium. In one embodiment one or more of the hydrogen atoms in the C.sub.1-C.sub.10 alkyl R.sup.1 substituent is substituted by deuterium. In some embodiments, R.sup.1 includes at least 2, at least 3, at least 4, at least 5, or at least 9 deuteriums In some embodiments, R.sup.1 comprises up to about 17, up to about 14, up to about 11, or up to about 9 deuteriums. In some embodiments, R.sup.1 is octyl. In some embodiments, R.sup.1 is octyl and includes 8 deuteriums.
[0120] In some embodiments, R.sup.2 is H, deuterium, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl. In one embodiment one or more of the hydrogen atoms in the C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl R.sup.2 substituent is substituted by deuterium. Illustratively, each hydrogen atom in C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl is optionally substituted by deuterium. In some embodiments, R.sup.2 is H.
[0121] In some embodiments, R.sup.C is H or D. In some embodiments, R.sup.C is H. In some embodiments, R.sup.C is D.
[0122] In some embodiments, n is an integer selected from the range of 1-7. In some embodiments, n is 1, 2, 3, 4, 5, 6, or 7. In some embodiments, n is 5.
TABLE-US-00001 TABLE 1 Compound Number Structure Chemical Name 1 (E)-10-hydroxyoctadec-8-enoic acid I-d.sub.5 (E)-10-hydroxyoctadec-8-enoic- 17,17,18,18,18-d.sub.5 acid
[0123] The compounds and process of the present disclosure are described in detail below. A step of the process of the present disclosure can be described according to Scheme 1.
##STR00016## [0124] wherein n is an integer from 1 to 7, preferably 2-7, even more preferably 4 to 7. R.sup.2 is H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl, wherein each hydrogen atom in C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, heterocycloalkyl, cycloalkyl, or heteroaryl is optionally substituted by deuterium. In some embodiments, R.sup.2OH is preferably methanol. In some embodiments, the compound of formula III can be transesterfied to form other compounds of formula III. The integer n is equal to the integer m-1. The reaction in Scheme 1 can be performed in the presence of a base such as an inorganic base. In some embodiments, the inorganic base is sodium carbonate. Illustratively, the reaction can be worked up with an acetate salt and an anhydride, preferably sodium acetate and acetic anhydride.
[0125] In some embodiments, the process of the present disclosure includes a step according to Scheme 2.
##STR00017## [0126] wherein n is an integer from 1-9, PG is a protecting group for a hydroxyl, LG is a leaving group, R.sup.1 is C.sub.1-C.sub.10 alkyl and each hydrogen atom in C.sub.1-C.sub.10 alkyl is optionally substituted by deuterium, R.sup.D is H or C.sub.1-C.sub.6 alkyl such as methyl, and R.sup.3 is C.sub.1-C.sub.9 alkyl, wherein each hydrogen atom in C.sub.1-C.sup.9 alkyl is optionally substituted by deuterium.
[0127] Step (a) may be performed by treating the diol with a base in the presence of the protecting group precursor to protect one of the hydroxyl groups. Illustrative protecting group precursors include aryl halides, such as benzyl bromide. Step a may be performed in a solvent such as THF. The base may be sodium hydride. Illustratively, hydroxy protecting group means a protecting group that is protecting a hydroxy functional group.
[0128] Step (b) may be performed by treating the protected alcohol with a base in the presence of a leaving group precursor. Illustrative protecting group precursors include arylsulfonyl halides such as tosyl chloride. Step b may be performed in a solvent such as dichloromethane. The base may be an organic base, preferably triethylamine. Step (b) may also include a catalyst such as dimethylaminopyridine (DMAP).
[0129] In Step (c), the product of step (b) is contacted by R.sup.3MX. The metal, M, may be magnesium. The X may be a halide, preferably bromide. Step (c) may be performed in a solvent, preferably THF. Step (c) may be performed in the presence of a catalyst. Illustrative catalyst include copper chloride (CuCl.sub.2). Step (c) may also be performed in the presence of a complexing ligand, such as 1-phenylpropyne.
[0130] Step (d) removes the protecting group form PG-OCH.sub.2R.sup.1. In an illustrative embodiment, if the protecting group is benzyl, the benzyl is removed with boron trichloride. Alternative embodiments using different protecting groups are envisioned and the removing the protecting group is known in the art.
[0131] Step (e) oxidizes the product of step (d) to a compound of formula VIII. Step (e) may be performed using trichloroisocyanuric acid in the presence of methanol to yield a compound of formula VIII wherein R.sup.D is methyl. Alternative embodiments may be performed in the presence of other alcohols. Step (e) may be performed in a solvent such as dichloromethane.
[0132] In some embodiments, the process of the present disclosure includes a step according to Scheme 3.
##STR00018## [0133] wherein R.sup.D is H or C.sub.1-C.sub.6 alkyl such as methyl, and each of R.sup.a and R.sup.b is independently OC.sub.1-C.sub.6 alkyl, Oaryl, Oheteroaryl; and R.sup.1 is C.sub.1-C.sub.10 alkyl, wherein each hydrogen atom in C.sub.1-C.sub.10 alkyl is optionally substituted by deuterium, optionally wherein the R.sup.1 alkyl chain substituent comprises at least 3 deuterium atoms substituting for hydrogen. In some embodiments, the phosphonate is contacted with a base prior to contacting the compound of formula VIII. Illustrative bases include alkyllithiums, such as butyllithium. In some embodiments, the reaction in scheme 3 is performed at about 78 C. In some embodiments, the compound of formula II can be purified by distillation.
[0134] In some embodiments, the process of the present disclosure includes a step according to Scheme 4.
##STR00019## [0135] wherein R.sup.1, R.sub.a, R.sub.b, R.sup.2, and n are defined above. In some embodiments, the compound of formula II is contacted with a base prior to contacting the compound of formula III. Illustrative bases include sodium hydride. In some embodiments, the reaction in Scheme 4 is performed at a reduced temperature. In some embodiments, the reaction temperature increases as the reaction occurs.
[0136] In some embodiments, the process of the present disclosure includes a step according to Scheme 5.
##STR00020## [0137] wherein R.sup.1, R.sup.2 and n are defined above. In some embodiments, Step a includes contacting the compound of formula IV with a reducing agent, preferably sodium borohydride. In some embodiments, the reducing agent delivers a deuteride instead of a hydride. In some embodiments, Step a is performed in a solvent such as a blend of an alcohol and water. In some embodiments, the reducing agent may be chiral or complexed with a chiral ligand; in some embodiments, this ligand may be the Corey-Bakshi-Shibata oxazaborolidine.
[0138] Step b is optional. If performed, Step b hydrolyzes the ester to convert R.sup.2 (wherein R.sup.2 is described above but is not H) to a compound of formula I wherein R.sup.2 is H. In some embodiments, Step b is performed in the presence of a base in an water-organic solvent mixture. In some embodiments, the base is sodium methoxide.
[0139] In illustrative embodiments, the compound of formula I can be made by a process as described below.
[0140] In some embodiments, the process includes step (a) of contacting a compound of formula II
##STR00021## [0141] wherein R.sup.1 is C.sub.1-C.sub.10 alkyl and each hydrogen atom in C.sub.1-C.sub.10 alkyl is optionally substituted by deuterium, and each of R.sub.a and R.sub.b is independently OC.sub.1-C.sub.6 alkyl, Oaryl, Oheteroaryl; with a compound of formula III
##STR00022## [0142] wherein R.sup.2 and n are as defined above, in the presence of a base to form a compound of formula IV
##STR00023## [0143] wherein R.sup.1 and R.sup.2 are as defined above.
[0144] In some embodiments, step (a) is performed in a solvent such as diethyl ether. In some embodiments, the compound of formula III is added to the compound of formula II. In some embodiments, the compound of formula II is treated with base, such as sodium hydride, prior to adding the compound of formula III. In some embodiments, the base is stoichiometric.
[0145] In some embodiments, the process includes step (b) contacting a compound of formula IV with a reducing agent to form the compound of formula I
##STR00024## [0146] wherein R.sup.1 and R.sup.2 are as defined above, except for when R.sup.2 is H. In some embodiments, the reducing agent is a borohydride, such as sodium borohydride or sodium cyanoborohydride. In some embodiments, the reducing agent provides a deuterium. In some embodiments, step (b) further includes hydrolyzing the compound of formula I, wherein R.sup.2 is not H, to form a compound of formula I wherein R.sup.2 is H.
[0147] In some embodiments, the process includes step (c), contacting a compound of formula V
PG-OC.sub.1-C.sub.9 alkyl-LG V [0148] wherein PG is a hydroxy protecting group and LG is a leaving group. Illustrative protecting groups include benzyl and others known in the art. Illustrative leaving groups include O-tosylates and those known in the art. Each hydrogen atom in C.sub.1-C.sub.9 alkyl is optionally substituted by deuterium. The compound of formula V is contacted with a compound of formula R.sup.3-MX, wherein R.sup.3 is C.sub.1-C.sub.9 alkyl, wherein each hydrogen atom in C.sub.1-C.sup.9 alkyl is optionally substituted by deuterium, M is a metal such as magnesium, and X is a halogen such as bromo, to form a compound of formula VI
PG-OCH.sub.2R.sup.1 VI
[0149] Illustratively, each hydrogen atom in CH.sub.2R.sup.1 is optionally substituted by deuterium.
[0150] In some embodiments, the process includes step (d), deprotecting a compound of formula VI into a compound of formula VII
HOCH.sub.2R.sup.1 VII [0151] wherein each hydrogen atom in CH.sub.2R.sup.1 is optionally substituted by deuterium. The deprotection step may depend on the nature of the protecting group. For example, if PG is a benzyl group, the deprotection may be performed by BCl.sub.3 or any other suitable agent.
[0152] In some embodiments, the process includes step (e) oxidizing the compound of formula VII to form a compound of formula VIII
R.sup.DO(O)CR.sup.1 VIII [0153] wherein Ris H or C.sub.1-C.sub.6 alkyl such as methyl. In some embodiments, the process includes trichloroisocyanuric acid. In some embodiments, step (e) is performed in an inert atmosphere. In some embodiments, step (e) is performed in a solvent that may include more than one solvent. In illustrative embodiments, the solvent includes dichloromethane and methanol. In some embodiments, the ratio of the compound of formula VII to methanol is about 1:10.
[0154] In some embodiments, the process includes step (f), contacting a compound of formula VII with (C.sub.1-C.sub.6 alkoxy).sub.2(CH.sub.3)P(O) in the presence of a base to form a compound of formula II. In some embodiments, the base is stoichiometric. In some embodiments, the base is n-butyllithium. In some embodiments, the (C.sub.1-C.sub.6 alkoxy).sub.2 (CH.sub.3)P(O) is added to a flask containing the base prior to contacting the compound of formula VII.
[0155] In some embodiments, the process includes step (g), oxidatively cleaving a C.sub.3-C10 cycloalkene and optionally esterifying the product to form a compound of formula III. In some embodiments, the oxidative cleavage is performed with ozone.
[0156] Those skilled in the art will recognize that the species listed or illustrated herein are not exhaustive, and that additional species within the scope of these defined terms may be selected.
[0157] Chemical Synthesis
[0158] Exemplary chemical entities useful in methods of the description will now be described by reference to illustrative synthetic schemes for their general preparation below and the specific examples that follow. Artisans will recognize that, to obtain the various compounds herein, starting materials may be suitably selected so that the ultimately desired substituents will be carried through the reaction scheme with or without protection as appropriate to yield the desired product. Alternatively, it may be necessary or desirable to employ, in the place of the ultimately desired substituent, a suitable group that may be carried through the reaction scheme and replaced as appropriate with the desired substituent. Furthermore, one of skill in the art will recognize that the transformations shown in the schemes below may be performed in any order that is compatible with the functionality of the particular pendant groups.
[0159] Glassware was oven-dried at 120 C. or flame-dried under a stream of nitrogen. Tetrahydrofuran was dried either with a commercial molecular sieves-based solvent purification system or by distillation from sodium/benzophenone. Anhydrous 1,2-dimethoxyethane was used as received. Reagents were acquired from Aldrich/Sigma Chemicals unless specified. Sodium methoxide was acquired from TCI America. NaH and phenylhydrazine were received from Merck AG and Oakwood Chemicals, respectively. Magnesium and sodium borohydride were purchased from Fisher Scientific. Butyllithium was titrated by the double Gilman titration method. The concentration of Grignard reagents was determined using salicylaldehyde phenylhydrazone. Ozone was generated from dry oxygen with an HTU-500AC unit (Azco Industries). GC-MS samples of alcohols were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide containing 2% chlorotrimethylsilane at 80 C. for 1 h. All chromatography was carried out using flash grade silica gel 60-200 mesh (60 pore size) from SiliCycle (Quebec City, Canada) driven by air pressure.
[0160] All proton NMR spectra were collected in CDCl.sub.3 using Bruker Avance II 500-MHz or Avance III 400-MHz NMR spectrometers. Broadband-decoupled and DEPT .sup.13C spectra were acquired in the same instruments at 125 and 100 MHz, respectively. All data was solvent referenced to 7.26 (.sup.1H) and 77.0 ppm (.sup.13C). In the case of 10-HOME acid and Me ester, it was essential to deacidify CDCl.sub.3 used for NMR samples by passage of the solvent through a plug of basic alumina. GC-MS data was collected with an Agilent 7890/5975C system using a VF-23 ms column (30 m, 250 m film, 0.25 mm diameter) with an oven program starting at 60 C., ramp 7 C./min to 150 C., hold at 150 C. for 7 min, ramp 10 C./min to 220 C., hold 1 min, ramp 50 C./min to 250 C., and hold 2 min; He flow 1.9 mL min High-resolution mass spectra (HRMS) were collected in dual electrospray ionization (ESI) mode using an Agilent 6520 accurate mass instrument [fragmentor voltage 125V, nebulizer gas temperature 325 C., solvent: 90:10 acetonitrile:water with 0.1% formic acid].
[0161] Abbreviations The examples described herein use materials, including but not limited to, those described by the following abbreviations known to those skilled in the art:
TABLE-US-00002 eq. or equiv. equivalent DME 1,2-dimethoxyethane TLC thin-layer chromatography min minutes h hour EtOAc ethyl acetate N Normal THF tetrahydrofuran MHz megahertz d doublet q quintet s singlet br broad NMR nuclear magnetic resonance t triplet m multiplet
##STR00025##
[0162] Methyl 8-oxooctanoate (2): Prepared by the Schreiber variation of ozonolysis for cycloalkenes, where the ozonolysis was performed under basic conditions (NaHCO.sub.3) in a mixture of methanol and methylene chloride, followed by treating the reaction mixture with triethylamine and acetic anhydride to produce the ester-aldehyde functionalities. The crude aldehyde was purified by distillation (53-64 C./45 mTorr) resulting in a 74% yield of 2. .sup.1H NMR 9.67 (t, J=0.9 Hz, 1H), 3.57 (s, 3H), 2.34 (m, 2H), 2.21 (m, 2H), 1.54 (m, 4H), 1.24 (m, 4H).
##STR00026##
[0163] Methyl nonanoate (non-deuterated version of 5) was prepared in 97% yield by the esterification of nonanoic acid catalyzed by 0.005 equivalents of p-toluenesulfonic acid in the presence of methanol (10 equiv) and 2,2-dimethoxypropane (1 equiv).
##STR00027##
[0164] 1-(7-Benzyloxyheptyl) tosylate (3) was prepared both by the benzylation of 1,7-heptanediol using NaH, where the intermediate benzyloxy alcohol was purified prior to tosylation, or by a KOH-based method without the intermediate purification.
[0165] Intermediate for stepwise procedure: 7-Benzyloxy-1-heptanol .sup.1H NMR 7.27-7.34 (m, 5H), 4.50 (s, 2H), 3.63 (t, J=6.6, 2H), 3.47 (t, J=7.1, 2H), 1.54-1.63 (m, 5H including br OH), 1.34-1.39 (m, 6H). (300 MHz, CDCl.sub.3) 7.38-7.23 (m, 5H, ArH), 4.49 (s, 2H), 3.58 (t, J=6.5 Hz, 2H), 3.46 (t, J=6.5 Hz, 2H), 1.94 (s, 1H, OH), 1.67-1.46 (m, 5H), 1.44-1.27 (m, 6H); (75 MHz, CDCl.sub.3) 138.5, 128.2, 127.5, 127.4, 72.7, 70.3, 62.7, 32.5, 29.6, 29.1, 26.0, 25.6.
[0166] 1-Benzyloxy-7-tosylheptane (3): .sup.1H NMR 7.78 (d, J=7.9, 2H), 7.25-7.33 (m, 7H), 4.48 (s, 2H), 4.01 (t, J=6.4, 2H), 3.43 (t, J=6.5, 2H), 2.44 (s, 3H), 1.54-1.63 (m, 4H), 1.24-1.32 (m, 6H). HRMS (ESI) [M+H].sup.+ Calc. 377.1781, Exptl. 377.1790. .sup.1H NMR (CDCl.sub.3, 300 MHz) 7.76-7.81 (m, 2H), 7.25-7.36 (m, 7H), 4.49 (s, 2H), 4.01 (t, J=6.4, 2H), 3.44 (t, J=6.7, 2H), 2.44 (s, 3H), 1.51-1.66 (m, 4H), 1.20-1.36 (m, 6H).
##STR00028##
[0167] 1-Benzyloxynonane-8,8,9,9,9-d.sub.5 (4-d.sub.5): In a flame-dried 50-mL Schlenk flask capped with a septum under nitrogen was placed a football stir bar, Mg turnings (360.7 mg, 15.0 mmol, 3.4 equiv), and THF (10 mL). Following the addition of 50 L of 1,2-dibromoethane as an initiator, C.sub.2H.sub.5Br (750 L, 10.0 mmol, 2.3 equiv; passed through a plug of basic alumina to remove traces of DBr) was added via a gastight syringe in portions over 26 min. The septum was replaced with a reflux condenser and the reaction was refluxed for 1 h. The concentration of the Grignard reagent measured by the phenylhydrazone method was 0.88 M. Tosylate 3 (1.6947 g, 4.38 mmol, 1.0 equiv) was placed in a dry 50-mL single-necked, round-bottomed flask containing a football-shaped magnetic stirbar. CuCl.sub.2 (41.4 mg, 0.31 mmol, 0.7 equiv) was added, followed by 8 mL of THF, and 1-phenylpropyne (115 L, 0.92 mmol, 0.2 equiv). The Grignard reagent was added to the tosylate-containing flask dropwise over approximately 10 minutes, resulting in a range of color changes commencing from colorless then orange brown, green, and colorless, before becoming indigo blue then finally a deep amethyst purple. The reaction was stirred for 18 h at room temperature, during which it gains a brownish tint. The reaction mixture was quenched with 1N HCl (5 mL) and saturated NH.sub.4Cl (10 mL). The layers were separated and the aqueous layer was extracted twice with diethyl ether (10 mL each). The combined organics were washed with brine (10 mL), dried over MgSO.sub.4, vacuum filtered through Celite and the solvent was evaporated with a rotary evaporator leading to a yellow oil (1.4018 g). The ether was purified by column chromatography (1:40 EtOAc/hexanes, 100 mL SiO.sub.2; product R.sub.f 0.32) resulting in an isolated yield of 0.8113 g of 4-d.sub.5 (76%). .sup.1H NMR 7.29-7.44 (m, 5H), 4.54 (t, J=6.7, 2H), 1.66 (m, 2H), 1.23-1.45 (m, 10H). .sup.13C NMR 138.71, 128.25, 127.5, 127.4, 72.79, 70.48, 31.56, 29.75, 29.54, 29.46, 29.21, 26.17, 21.6 (quint, .sup.1J.sub.CD=19.2), 12.9 (m, .sup.1J.sub.CD=18.6).
[0168] .sup.1H NMR for protio coupling 7.27-7.35 (s, 5H), 4.50 (s, 2H), 3.47 (t, J=6.7, 2H), 1.26-1.95 (m, 14H), 0.88 (t, J=6.9, 3H). IR (neat) 1103 (CO); .sup.1H NMR 7.27 (s, 5 H), 4.46 (s, 2 H), 3.43 (t, 2 H), 0.65-1.95 (m, 17 H).
##STR00029##
[0169] Methyl nonanoate-d.sub.5 (5-d.sub.5):
[0170] Deprotection: In a 200-mL flask under nitrogen gas that contained benzyl ether 4-d.sub.5 (0.760 g, 3.18 mmol, 1.0 equiv) was added a stir bar and methylene chloride (63 mL). The flask was cooled to 78 C. in a dry ice-acetone bath. BCl.sub.3 (6.35 mL, 1M in CH.sub.2Cl.sub.2, 2.0 equiv) was added by syringe over 10 min, the solution changing to a light yellow-orange color. The cooling bath was swapped for an ice-water bath, and the reaction stirred for 2 h. Subsequently, the reaction mixture was re-cooled to 78 C., quenched with 2 mL of MeOH, and then allowed to warm to 0 C. in an ice-water bath. The mixture was washed with 10 mL of water and the aqueous layer was then extracted with CH.sub.2Cl.sub.2 (10 mL). The combined organic layers were dried over MgSO.sub.4, vacuum filtered through Celite, and concentrated with a rotary evaporator. The initial yellow oil, which spontaneously turns green, was rapidly loaded onto a flash column (1:3 EtOAc/hexanes, 40 mL SiO.sub.2, 1.5-cm column diameter; product R.sub.f 0.49) then re-chromatographed with 1:5 EtOAc/hexanes producing 1-nonanol-d.sub.5 (389.9 mg, 82% yield) as a colorless oil. .sup.13C NMR 63.11, 32.82, 31.57, 29.57, 29.42, 29.21, 25.73, deuterated carbons not observed. Literature data for 1-nonanol: .sup.1H NMR (CDCl.sub.3, 300 MHz) 3.59 (m, 2H), 2.40 (s, 1H), 1.52-1.58 (m, 2H), 1.18-1.30 (m, 12H), 0.89 (m, 3H); .sup.13C NMR (CDCl.sub.3, 75 MHz) 62.89, 32.70, 31.97, 29.70, 29.54, 29.39, 25.84, 22.73, 14.13.
[0171] In a 25-mL flask containing the deuterated nonanol (384.8 mg, 2.59 mmol, 1.0 equiv) and a stir bar was added CH.sub.2Cl.sub.2, MeOH (1.1 mL, 10 equiv). Solid trichloroisocyanuric acid (716.5 mg, 3.02 mmol, 1.17 equiv) was added and the reaction was allowed to stir for 17 h under a static N.sub.2 atmosphere. The reaction was diluted with CH.sub.2Cl.sub.2 (15 mL) and vacuum filtered through Celite. The clear reaction mixture was washed twice with saturated sodium sulfite (5 mL), after which it was oxidant negative with a KI-starch test, 1N NaOH (5 mL), and saturated brine (5 mL). The methylene chloride solution was dried over MgSO.sub.4, vacuum filtered and carefully concentrated with a room temperature bath using a rotary evaporator to a colorless oil (421.0 mg, 91% yield). .sup.1H NMR (CDCl.sub.3, 300 MHz) 3.66 (s, 3H), 2.30 (t, J=7.6, 2H), 1.58-1.62 (m, 2H), 1.23-1.32 (m, 8H). .sup.13C NMR (CDCl.sub.3, 75 MHz) 174.3, 51.4, 34.1, 31.5, 29.21, 29.15, 29.06, 25.0, deuterated carbon signals not detected.
##STR00030##
[0172] Dimethyl 2-oxodecylphosphonate (6):
[0173] In a flame-dried 1-L, 3-necked round-bottomed flask equipped with a graduated dropping funnel, septum, magnetic stir bar, and thermocouple, was placed 325 mL of tetrahydrofuran (THF) and dimethyl methylphosphonate (20.03 g, 162 mmol, 2.01 eq). The flask was cooled in a dry ice/acetone bath to an internal temperature of 70 C. n-Butyllithium (107 mL, 1.503 M in hexanes, 161 mmol, 2.0 equiv) was added dropwise via the dropping funnel over 30 min, maintaining a temperature below 60 C. and resulting in a yellow solution. After an additional 15 min of stirring, methyl nonanoate (13.866 g, 80.6 mmol, 1.0 equiv) was weighed into a transfer flask and diluted with 50 mL of THF. Over 11 minutes, the ester was added by cannula to the lithiated phosphonate solution and the reaction was stirred an additional 25 minutes. The cooling bath was replaced with a cool water bath, and the reaction mixture was allowed to warm to 0.5 C. The reaction was quenched with 100 mL of 1N HCl, the organic layer collected, and the aqueous layer was extracted with three 25-mL portions of CHCl.sub.3. The combined organic layers were dried over MgSO.sub.4, vacuum filtered, and concentrated with a rotary evaporator leading to 24.743 g of a crude yellow oil [containing approximately 59% w/w product]. The ketophosphonate was purified by Kuglerohr distillation at 91 C./100 mTorr, whereby side-products and excess reagents condensed in the collection bulb and the product 6 remained in the distillation pot (15.54 g, 78% yield). .sup.1H NMR 3.66 (d, J=11.2, 6H), 2.97 (d, J=22.7, 2H), 2.48 (t, J=7.3, 2H), 1.45 (m, 2H), 1.05-1.2 (m, 10H), 0.75 (t, J=6.7, 3H). .sup.13C NMR 201.7 (d, .sup.2J.sub.CP=6.2), 52.7 (d, .sup.2J.sub.CP=6.5), 43.9, 40.9 (.sup.1J.sub.CP=128), 31.5, 29.0, 28.8, 28.6, 23.1, 22.3, 13.8.
##STR00031##
[0174] Dimethyl 2-oxodecylphosphonate-d.sub.5 (6-d.sub.5): An oven-dried 25-mL one-neck, round-bottomed flask containing a stirbar was cooled to 78 C. with a dry ice/acetone bath. THF (8 mL) was added by syringe followed by the slow addition of n-butyllithium (5.5 mL, 1.503 M in hexanes, 8.27 mmol, 3.5 equiv). Neat dimethyl methylphosphonate (895 L, 8.26 mmol, 3.5 equiv) was added over 10 minutes by syringe resulting in a creamy suspension. Methyl nonanoate-d.sub.5 (418 mg, 2.36 mmol, 1.0 equiv) dissolved in 1 mL of THF was added dropwise over 30 minutes, followed by an additional 0.3 mL of THF used to ensure complete transfer of the ester from the weighing flask. Dry ice was removed from the bath and the temperature was allowed to slowly rise to 18 C. (approx. 40 min). The reaction was quenched with 1 mL of glacial acetic acid followed by saturated NH.sub.4Cl (10 mL). The organic layer was collected and the aqueous layer was extracted thrice with ethyl acetate (10 mL each). The combined organic layers were washed with brine (10 mL) and dried overnight with MgSO.sub.4 Filtration and concentration with the aid of a rotary evaporator led to 1.6757 g of an oil. The crude product was purified by column chromatography (pure EtOAc, 100 mL SiO.sub.2, 2.5-cm column diameter) providing a colorless oil 6-d.sub.5 (571.1 mg, 90% yield). .sup.1H NMR 3.73 (d, J=11.2, 6H), 3.03 (d, J=22.7, 2H), 2.55 (t, J=7.4, 2H), 1.52 (m, 2H), 1.05-1.2 (m, 8H). .sup.13C NMR 201.9 (d, J=6.0), 52.9 (d, J=6.4), 44.1, 41.1 (.sup.1J.sub.CP=126), 31.4, 29.2, 28.9, 28.8, 23.3, 21.4 (m), deuterated Me group not observed. HRMS (ESI) [M+H].sup.+ m/z Calc. 270.1877, Exptl. 270.1887.
##STR00032##
[0175] Methyl 10-oxooctadec-8-enoate (7):
[0176] In an oven-dried 25-mL 3-necked round-bottomed flask, equipped with a small stir bar and three septa was placed NaH (62.1 mg, 60% w/w in paraffin oil, 1.55 mmol, 1.1 equiv). The paraffin oil was removed by thrice washing with as-received hexanes (1-mL aliquots) then 5 mL of 1,2-dimethoxyethane (DME) was added, and the flask cooled to 0 C. in an ice-water bath. In a separate flask, a solution of phosphonate 6 (439.4 mg, 1.66 mmol, 1.2 equiv) was dissolved in 2.5 mL of DME. The phosphonate solution was added rapidly dropwise to the stirred NaH suspension by syringe, leading to a yellow solution, then the cooling bath was removed. After stirring for 1 h, neat methyl 8-oxooctanoate (240.0 mg, 1.395 mmol, 1.0 equiv) was added by syringe leading to a dense cream-colored suspension. The reaction mixture was stirred for 16 h, after which thin-layer chromatography (TLC, 1:5 v/v EtOAc/hexanes) showed that the aldehyde (R.sub.f 0.29) was consumed and product had formed (R.sub.f 0.53). The reaction was diluted with 10 mL of ether, and the organic phase was washed sequentially with 1N NaOH (10 mL) and brine (10 mL), then dried over MgSO.sub.4, and concentrated by means of a rotary evaporator leading to the crude product (488.5 mg). The product was purified by flash column chromatography (1:7 EtOAc/hexanes, 50 mL SiO.sub.2, 1.5-cm column diameter) leading to 278.2 mg of a yellowish oil (7, 57% yield). .sup.1H NMR 6.78 (dt, J=13.8, 6.9, 1H), 6.05 (dm, J=15.9, 1H), 3.63 (s, 3H), 2.48 (t, J=7.3, 2H), 2.27 (t, J=7.4, 2H), 2.17 (q, J=6.5, 2H), 1.57 (m, 4H), 1.44 (m, 2H), 1.12-1.31 (m, 16H), 0.84 (t, 3H). .sup.13C NMR 200.8, 174.0, 146.8, 130.3, 51.3, 40.0, 33.9, 32.2, 31.7, 29.31, 29.25, 29.1, 28.8, 28.7, 27.8, 24.7, 24.3, 22.6, 14.0. GC-EIMS (22.6 min retention time, VF-23 ms column) 310, 279, 251, 212, 167 (base), 137, 119, 95, 74, 55. The (Z) isomer appeared in the lead column fraction at 20.4 min with the same fragmentation pattern. HRMS (ESI) [M+H].sup.+ m/z calc. 211.2581, exptl. 211.2573.
##STR00033##
[0177] Methyl 10-oxooctadec-8-enoated.sub.5 (7-d.sub.5)
[0178] For the d.sub.5-isotopologue, a flask was loaded with 84.6 mg of NaH (2.12 mmol, 60% w/w in oil, 1.0 equiv), and the paraffin oil removed by washing with dry hexanes before the base was suspended in 8 mL of DME. The d.sub.5-phosphonate (6-d.sub.5, 571.1 mg, 2.12 mmol, 1.0 equiv) was dissolved in 4-mL of DME, and added to the NaH suspension that had been cooled to 0 C. At this concentration, a creamy solid presumed to be the ylide precipitated, additional solvent was added (3 mL), and the mixture was stirred for 1 h. Neat aldehyde (428.5 mg, 1.3 equiv) was added by syringe in two portions to the flask, where mixing of the dense mixture was initially aided by manual swirling. The reaction was allowed to stir for an additional 24 h at room temperature and then diluted with 15 mL of diethyl ether. The organic phase was washed with 1N NaOH (15 mL) and then brine (15 mL) before drying with MgSO.sub.4 and vacuum filtering the solution through a pad of Celite. Evaporation of the solvent with a rotary evaporator led to a yellow oil (904.2 mg) that was purified by flash column chromatography (1:8 EtOAc/hexanes, 80 mL SiO.sub.2; product R.sub.f 0.33) leading to 7-d.sub.5 as a pale yellow oil (384.7 mg, 63.5% yield). .sup.1H NMR 6.80 (dt, J=6.9, 1H), 6.07 (dt, J=15.8, 1.4, 1H), 3.63 (s, 3H), 2.51 (t, J=7.3, 2H), 2.29 (t, J=7.5, 2H), 2.20 (q, J=6.5, 2H), 1.60 (m, 4H), 1.47 (m, 2H), 1.22-1.34 (m, 14H). .sup.13C NMR 200.0, 173.4, 146.3, 130.0, 50.9, 39.6, 33.5, 31.9, 31.1, 29.0, 28.9, 28.7, 28.44, 28.38, 27.5, 24.4, 23.9, 21.2 (quint, .sup.1J.sub.CH=18.9), 12.5 (m, .sup.1J.sub.CH=18.8).
##STR00034##
[0179] Methyl 10-hydroxyoctadec-8-enoate (10-HOME Me ester, 8):
[0180] The ketone (222 mg, 0.71 mmol, 1.0 equiv) was dissolved in 2.5 mL of methanol in a 25-mL round-bottomed flask. A slurry of NaBH.sub.4 (54 mg, 1.42 mmol, 2.0 equiv) in 2.5-mL Me0H was transferred into the reaction flask and the mixture was magnetically stirred for 1 h at room temperature. After 1 h, additional sodium borohydride (5 mg, 0.2 equiv) was added and the mixture stirred for an additional 15 min. The solvent was removed by rotary evaporation. The residue was dissolved in 10 mL EtOAc and washed with an equal volume of brine. The organic layer was dried over MgSO.sub.4, yielding 212.5 mg of crude product. Impurities were removed by flash column chromatography (1:7 EtOAc/hexanes, 50 mL SiO.sub.2, 1.5-cm column) yielding 138 mg (62% yield). .sup.1H NMR 5.61 (dt, J=15.3, 6.8, 1H), 5.43 (ddt, J=15.4, 7.1, 1.3, 1H), 4.02 (q, J=6.7, 1H), 3.66 (s, 3H), 2.30 (t, J=7.5, 2H), 2.01 (q, J=6.5, 2 H), 1.23-1.64 (m, 25H), 0.87 (t, J=6.8, 3H). .sup.13C NMR 174.3, 133.2, 131.9, 73.2, 51.4, 37.4, 34.0, 32.0, 31.9, 29.6, 29.2, 28.9, 28.7, 25.5, 24.8, 22.6, 14.1. EIMS of TMS ether m/z 384, 337, 271 (base), 241, 149, 129, 107, 73.
##STR00035##
[0181] Methyl 10-hydroxyoctadec-8-enoate d.sub.5 (10-HOME Me ester, 8-d.sub.5):
[0182] For the d.sub.5 -isotopologue 8-d.sub.5, a flask was loaded with the ketoester (382 mg, 1.20 mmol, 1.0 equiv), methanol (10 mL), and a magnetic stir bar. NaBH.sub.4 (46 mg, 1.21 mmol per addition) was added to the reaction three times, each spaced by 20-30 minutes at ambient temperature (20 C.), leading to the consumption of the ketone. The crude product (377.0 mg) was purified as for the non-deuterated compound. that was purified by flash column chromatography (1:8 EtOAc/hexanes, 80 mL SiO.sub.2; product R.sub.f 0.33) leading to a pale yellow oil (384.7 mg, 63.5% yield). .sup.1H NMR 5.61 (dt, J=15.7, 6.8, 1H), 5.43 (dd, J=15.7, 7.1, 1H), 4.02 (q, J=6.7, 1H), 3.66 (s, 3H), 2.30 (t, J=7.5, 2H), 2.01 (q, J=6.8, 2H), 1.63 (m, 4H), 1.2-1.65 (m, 19H). .sup.13C NMR 174.2, 133.2, 131.8, 73.2, 51.4, 37.4, 34.0, 32.0, 31.6, 29.6, 29.2, 28.9, 28.7, 25.5, 24.9, deuterated carbons not observed.
##STR00036##
[0183] (E)-10-Hydroxyoctadec-8-enoic acid (10-HOME, 1-d.sub.5): A 2-M solution of sodium methoxide was prepared in 15% aqueous methanol (800 L of 5 M NaOMe in MeOH, 300-L water, 900-L MeOH). One milliliter of the methoxide solution was added to the deuterated 10-HOME Me ester (31.6 mg, 0.10 mmol, 1 equiv) in a glass centrifuge tube. The mixture was vortex mixed and heated for 2 h at 60 C. After cooling to room temperature, the reaction was quenched with 1 mL of 0.5 M acetic acid and 3 mL of 1 M HCl. The reaction mixture was extracted 3 with a 1:1 mixture of Et0Ac:hexanes (2 mL) and concentrated using a rotary evaporator and high vacuum pump leading to the white crystalline acid 1-d.sub.5 (29.0 mg, 96% yield). An analogous procedure was used to hydrolyze 20.0 mg of 2 leading to white crystalline 1 (17.1 mg, 89%).
[0184] 10-HOME-d.sub.5 (1-d.sub.5). .sup.1H NMR 6.1 (br COOH, 1H), 5.59 (dt, J=15.3, 6.7, 1H), 5.43 (dd, J=7.2, 15.3, 1H), 4.03 (q, J=6.7, 1H), 2.33 (t, J=7.5, 2H), 2.03 (q, J=6.7, 2 H), 1.62 (virt quint, J=7.1-7.4, 2H), 1.53 (m, 1H), 1.46 (m, 1H), 1.25-1.43 (m, 19H). .sup.13C NMR 179.4, 133.0, 132.0, 73.3, 37.2, 34.0, 32.0, 31.6, 29.54, 29.52, 29.2, 28.8, 28.75, 28.6, 25.4, 24.6. HRMS (ESI) [MH].sup. m/z Calc. 302.2749, Exptl. 302.2734; fragment C.sub.11H.sub.13O.sub.3 m/z Calc. 193.0870, Exptl. 193.0897.
##STR00037##
[0185] 10-HOME (1) was prepared in a similar manner as above. .sup.1H NMR 5.60 (dtd, J=0.8, 15.3, 6.7, 1H), 5.44 (tdd, J=1.3, 7.1, 15.3, 1H), 4.03 (q, J=7.8, 1H), 2.34 (t, J=7.5, 2H), 2.02 (virt q, J=6.7, 2 H), 1.63 (virt quint, J=7.1-7.4, 2H), 1.47 (m, 1H), 1.38 (m, 1H), 1.26-1.43 (m, 20H), 0.87 (t, J=7.0, 3H). .sup.13C NMR 178.9, 133.1, 132.0, 73.3, 37.3, 33.8, 32.0, 31.9, 29.5, 29.2, 28.81, 28.76, 28.6, 25.5, 24.6, 22.6, 14.1. HRMS (ESI) [MH].sup. m/z Calc. 297.2435, Exptl. 297.2445; fragment C.sub.11H.sub.13O.sub.3 m/z Calc. 193.0870, Exptl. 193.0915.
[0186] Assays
[0187] Human subjects. Subjects participating in the study were patients diagnosed with BII. Demographic characteristics of patients presented in (Table 2). All human studies were approved by The Indiana University School of Medicine Institutional Review Board. Declaration of Helsinki protocols was followed, and patients gave their written informed consent.
[0188] Bacterial strains. Staphylococcus epidermidis (Winslow and Winslow) Evans (ATCC 35984) were grown on tryptic soy agar plate at 37 C.
[0189] Scanning Electron Microscope Imaging. The samples were collected in glutaraldehyde fixation buffer, dehydrated with graded ethanol, and treated with hexamethyldisilazane (HMDS, Ted Pella Inc.) and left overnight for drying. Before scanning, samples were mounted and coated with gold. Imaging of the samples will be done by using a FEI NOVA nanoSEM scanning electron microscope (FEI, Hillsboro, OR) equipped with a field-emission gun electron source.
[0190] Lipid Extraction For LCMS.
[0191] LC-MS/MS targeted analysis from capsule and breast adipose tissue was performed. Samples were weighed and transferred to a 2-mL vials with 1.4-mm ceramic beads and 1 mL of water with 0.1% formic acid was added. The standard solution was prepared by aliquoting 1 g of each stock solution into a new tube drying the original solvent and solubilizing in 1 mL of 100% ethanol to obtain a final concentration of 1 ng/ml each. Samples were homogenized using Precellys24 tissue homogenizer (Bertin Technologies, Rockville, MD, USA). The total volume of the homogenate was extracted with ethyl acetate in a 1:1 volume ratio. Samples were vortexed for 1 minute and centrifuged at 14,000 rpm for 10 minutes. The organic phase was collected and transferred to a new vial to be evaporated and stored at 80 C. until analysis. The dried lipid extracts were reconstituted with 50 L of methanol/water at 1:1 volume ratio and submitted for targeted quantification by liquid chromatography tandem MS (LC/MS/MS). The LC column used was an Acquity UPLC BEH C18 1.7 m 2.1100 (Waters, Milford, MA). The binary pump flow rate was set at 0.3 mL/min in an Agilent UPLC (G7120A) using water and 0.1% formic acid as mobile phase A and acetonitrile and 0.1% formic acid as mobile phase B. The LC column was pre-equilibrated with 80% A for 1 min. The binary pump was set in a linear gradient to 100% B in 8 min and held for 2.50 min. It was then returned to 80% A and re-equilibrated for 4 min. Ten L of the reconstituted sample was delivered to the column through a multisampler (G7167B) into a QQQ6470A triple quadrupole mass spectrometer (Agilent Technologies, San Jose, CA) equipped with ESI Jet Stream ion source. In the mass spectrometer the capillary voltage was 3500 V on the negative ion mode, the gas temperature was 325 C., gas flow was set at 8 l/min, the sheath gas heater at 250 C. and the sheath gas flow at 7. The fragmentation voltage was 100 and the cell accelerator voltage was 4 V. The MRMs (parent-fragment) for the acquisition was performed Data processing was carried out by using Mass Hunter (B.06.00).
[0192] Bacterial biofilm was observed in implant-associated capsules through scanning electron microscopy (
[0193] Increased Abundance of Biofilm Derived 10-HOME in BII Subjects
[0194] The oxylipin 10-hydroxy-(8E)-octadecenoic acid (10-HOME) is formed by the oxidation of oleic acid (