WO2018132876A1 - Novel glycine transport inhibitors for the treatment of pain - Google Patents

Novel glycine transport inhibitors for the treatment of pain Download PDF

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WO2018132876A1
WO2018132876A1 PCT/AU2018/050035 AU2018050035W WO2018132876A1 WO 2018132876 A1 WO2018132876 A1 WO 2018132876A1 AU 2018050035 W AU2018050035 W AU 2018050035W WO 2018132876 A1 WO2018132876 A1 WO 2018132876A1
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nmr
mhz
compound according
dmso
amino
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Robert Vandenberg
Renae Monique RYAN
Tristan RAWLING
Wendy IMLACH
Macdonald Christie
Jane CARLAND
Shannon MOSTYN
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The University Of Sydney
University Of Technology Sydney
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Priority claimed from AU2017900163A external-priority patent/AU2017900163A0/en
Application filed by The University Of Sydney, University Of Technology Sydney filed Critical The University Of Sydney
Publication of WO2018132876A1 publication Critical patent/WO2018132876A1/en

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    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/49—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
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    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
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    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine

Definitions

  • the present invention relates to novel glycine transport inhibitor compounds and their use for treating pain.
  • Background of the invention
  • Chronic pain is a significant global health, economic and social problem, with the annual economic burden in the USA recently estimated at $600 billion.
  • the disease burden of pain has been documented in a rigorous epidemiological study, which revealed that -20% of adults have poor self-rated health as a consequence of chronic pain.
  • the economic burden of unrelieved persistent pain in Australia is large and continuing to escalate, with a total cost of $34 billion in 2007.
  • the major analgesics used clinically were limited to non-steroidal, local anaesthetic, opioid and anticonvulsant drugs that provide adequate pain relief in only a small proportion of chronic pain patients.
  • newer classes of analgesics also have limited efficacy, with the most effective, gabapentin, at best providing only 50% reduction in pain scores in chronic pain, with the number needed to treat for a clinical response being > 4.
  • GlyT2 glycine transporter 2
  • Neurotransmitter transporters are responsible for regulating the synaptic concentration of monoamines (5-hydroxytryptamine, dopamine, noradrenaline) and amino acid ( ⁇ -aminobutyric acid, glycine) neurotransmitters. These membrane-bound proteins use electrochemical gradients to drive the transport of neurotransmitters across neuronal and glial membranes, serving to terminate neurotransmission and replenish intracellular levels of neurotransmitter for future release.
  • neurotransmitter transporters to selectively enhance or diminish neuronal signalling makes them attractive drug targets. Indeed, a wide range of therapeutically useful drugs, including antidepressants and anxiolytics, target these proteins. Diminished glycinergic transmission is an important CNS mechanism underlying neuropathic pain including mechanical hyperalgesia and allodynia (a painful response to normally innocuous tactile stimuli). Drugs that can enhance dysfunctional glycerinergic transmission in neuropathic pain, and in particular glycine transporter inhibitors, are potential chronic pain therapeutics.
  • GlyT1 and GlyT2 which serve to regulate glycine concentrations.
  • GlyT1 is expressed throughout the CNS predominantly by astrocytes surrounding both inhibitory and excitatory synapses to influence Glycine and NMDA receptors (NMDAR). Enhancement of NMDAR activity by GlyT1 inhibition is an established therapeutic target in diseases with NMDAR hypofunction, such as schizophrenia, but is undesirable in chronic pain states where suppression of NMDAR function is desirable.
  • GlyT2 is considered a promising therapeutic target for chronic pain.
  • GlyT2 shows much more restricted expression patterns than GlyT1 and is predominantly expressed by glycinergic terminals in the spinal cord and brain stem.
  • GlyT2 plays two important roles at glycinergic inhibitory synapses: regulation of extracellular glycine concentrations to control inhibitory glycinergic tone, and to recycle released glycine to provide sufficient intracellular glycine for repackaging into synaptic vesicles for subsequent release.
  • GlyT2 Glycine receptors
  • ALX1393 shows only a 10-fold selectivity for GlyT2 over GlyT1 , and the dose required for analgesia may also result in partial inhibition of GlyT1 and increases in glycine at excitatory synapses leading to an initial increase in NMDAR-related pain before analgesia is achieved.
  • ORG25543 causes respiratory depression and loss of motor control at high doses which has been attributed to irreversible inhibition of GlyT2 with subsequent reductions in glycine loading of synaptic vesicles. Partial knock down of GlyT2 using siRNAs indicates that partial inhibitors of GlyT2 have the potential to be therapeutic in the treatment of neuropathic pain.
  • GlyT2 Knockdown of GlyT2 to approximately 30% of wild type levels (which may mimic partial inhibition of transport) provides analgesia in rat models of allodynia associated with neuropathic pain.
  • the equilibrium glycine concentration gradient achieved by GlyT2 is determined by the ion gradients across the cell membrane, whereas the rate at which equilibrium is achieved will be determined by the number of glycine transporters.
  • partial inhibition will not alter the equilibrium glycine concentration gradient, but will prolong the time required to reach equilibrium, allowing greater glycine receptor activity. It appears that partial inhibition of glycine transport in the siRNA knock-down experiments allows sufficient glycine uptake for loading of presynaptic glycinergic vesicles to maintain, or even prolong, glycinergic neurotransmission.
  • the present invention provides a compound of formula (I):
  • X is an amino acid or derivative thereof
  • L is an amide or retro amide
  • Y is C 10 - C 24 alkyl or alkenyl
  • X, L and Y may be independently substituted or unsubstituted
  • the present invention provides a compound of formula (I) as defined above wherein the compound is not one or more of the compounds selected from the group consisting of:
  • a pharmaceutical composition comprising a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof, together with one or more pharmaceutically acceptable carriers or adjuvants.
  • a method of treating pain by administering an effective amount of a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof, to a subject in need thereof.
  • a compound according to the invention for use in treatment of pain in a subject in need thereof.
  • a method of treating pain by administering an effective amount of at least one compound selected from the group consisting of:
  • the pain may be chronic or acute.
  • the pain is chronic pain, more preferably neuropathic pain.
  • Fig. 1 shows plasma concentration vs time profiles for Compounds 28, 29, 8, 1 and 36, following incubations at 37°C in human ( ⁇ ) and rat ( ⁇ ) plasma.
  • Fig. 2 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of Compound 28.
  • Fig. 3 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of Compound 39.
  • Fig. 4 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of Oleoyl-L-carnitine.
  • Fig. 5 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of N-arachidonyl glycine.
  • Fig. 6 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs Oleoyl-L-glycine.
  • Fig. 7 shows plasma and brain concentrations of Compound 29 in male Sprague Dawley rats following IP administration at 27.5 mg/kg.
  • Fig. 8 shows the agonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on S1 PR1 .
  • Fig. 9 shows the antagonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65,
  • FIG. 10 shows the agonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB2.
  • Fig. 11 shows the antagonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB2.
  • Fig. 12 shows the agonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB1 .
  • Fig. 13 shows the antagonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB1 .
  • Fig. 14 shows the analgesic effect of intrathecal administration of Compound 29.
  • Fig. 15 shows the analgesic effect of intraperitoneal administration of Compound
  • the present invention provides a compound of formula (I):
  • X is an amino acid or derivative thereof
  • L is an amide or retro amide
  • Y is Cio - C 24 alkyl or alkenyl
  • X, L and Y may be independently substituted or unsubstituted
  • the present invention provides a compound of formula (I) as defined above wherein the compound is not one or more of the compounds selected from the group consisting of:
  • X is a naturally occurring or non-naturally occurring amino acid.
  • X is a naturally occurring hydrophobic and/or aromatic amino acid, more preferably L-tryptophan.
  • X is a non- naturally occurring amino acid, more preferably a D-amino acid.
  • X is selected from one or more of the group consisting of glycine, L-carnitine, L-serine, D-serine, L-lysine, D-lysine, L-lysine derivative wherein the carbon chain of the lysine side group is of C1 -C3 in length, L-arginine, L-methionine, L-leucine, D-leucine, L-alanine, D-alanine, B-alanine, L-valine,- D-valine, D-phenylalanine, L- phenylalanine derivative wherein the carbon chain of the phenylalanine side group is of two carbons in length, L-tryptophan, D-tryptophan, L-tyrosinol- L-dopamine, L-aspartate, D-aspartate, L-glutamate, L-histidine, D-histidine, L-lysine OME, L-norleu
  • X is unsubstituted.
  • X is substituted with 1 , 2, or 3 C 1 - C 3 alkyl groups at the a carbon of the amino acid or derivative thereof.
  • the C 1 - C 3 alkyl group is methyl.
  • X is lysine substituted with 1 , 2, or 3 methyl groups at the a carbon of the lysine. Substituting X as described herein, advantageously increases the metabolic stability and efficacy of the compounds of the invention by minimising in vivo degradation of the linking group, L.
  • L is an amide
  • L is unsubstituted.
  • L is substituted with a C 1 - C 3 alkyl group at the nitrogen of the amide or retro amide.
  • the C 1 -C 3 alkyl group is methyl.
  • Y includes 1 - 4 cis double bonds, more preferably Y is monounsaturated and includes 1 cis double bond.
  • the single cis double bond may be at any position of the C 10 - C 24 alkenyl chain, preferably 5 - 15 carbons from L, more preferably 8 - 10 carbons from L.
  • Y includes 1 -4 trans double bonds. More preferably, Y is monounsaturated and includes 1 trans double bond.
  • the single trans double bond may '1Ut>
  • the do - C 24 alkenyl chain preferably 5 - 15 carbons from L, more preferably 8 - 10 carbons from L, more preferably 9 carbons from L.
  • Y is a monounsaturated Ci 8 , Ci 6 or Ci 4 chain, preferably a monounsaturated Ci 8 chain.
  • the monounsaturated Ci 8 carbon chain includes a cis-double bond in the ⁇ 3, 5, 6, 7, 8, 9, 10, 1 1 , or 12 position, more preferably in the ⁇ 9 position.
  • the monounsaturated Ci 6 carbon chain includes a cis-double bond in the ⁇ 5, 6, 7, 9, or 1 1 position.
  • Y is a saturated Ci 6 or Cu carbon chain.
  • the compound is selected from one or more of compounds 2 - 7, 9 - 12, 1 7 - 21 , 23, 26 - 44, 47 - 54 of Table 1 .
  • the compound is selected from one or more of compounds 2 - 7, 1 0 - 1 2, 17, 18, 20, 21 , 23, 28 - 34, 37, 42, 43, 48 and 52 of Table 1 .
  • the invention also relates to methods of treating pain by administering an effective amount of a compound according to the invention, a salt or pharmaceutically acceptable derivative thereof, to a subject in need thereof.
  • a compound according to the invention a salt or a pharmaceutically acceptable derivative thereof for use in treatment of pain in a subject in need thereof.
  • the pain may be acute or chronic, preferably chronic.
  • the pain is neuropathic pain.
  • amino acid as used herein is intended to encompass compounds having an amino group and a carboxyl group in which the amino group and the carboxyl group are separated by at least one carbon atom.
  • the amino acid may be a L- or D- isomer and may have a naturally occurring side chain or a non-naturally occurring side chain.
  • An amino acid having a non-naturally occurring side chain refers to an amino acid having a side chain that does not occur in the naturally occurring L-a-amino acids.
  • non-natural amino acids and derivatives include, but are not limited to amino acids substituted with 1 , 2 or 3 C1-C3 alkyl groups at the a carbon of the amino acid, L-carnitine, gamma aminobutyric acid (GABA), dopamine, L-lysine OMe and/or D- isomers of amino acids.
  • amino acids substituted with 1 , 2 or 3 C1-C3 alkyl groups at the a carbon of the amino acid L-carnitine, gamma aminobutyric acid (GABA), dopamine, L-lysine OMe and/or D- isomers of amino acids.
  • amino acid derivative as used herein is intended to encompass derivatives of naturally occurring and non-naturally occurring amino acids, preferably ester derivatives of naturally occurring and non-naturally occurring amino acids. It also encompasses amino acids that have had their side chain modified, for example, the hydrocarbon chain shortened or extended. Some non-limiting examples of these are shown below for lysine and phenylalanine:
  • retro amide refers to a reverse amide bond wherein the nitrogen of the amide bond is located on the proximal side of the carbonyl group, ie (NHCO).
  • alkyl refers to a saturated, straight-chain or branched hydrocarbon group. Specific examples of alkyl groups are methyl, ethyl, propyl, / ' so-propyl, n-butyl, / ' so-butyl, sec-butyl, te/t-butyl, n-pentyl, / ' so-pentyl, n-hexyl and 2,2-dimethylbutyl.
  • alkenyl refers to an at least partially unsaturated, straight-chain or branched hydrocarbon group that contains at least two carbon atoms (i.e. C2 alkenyl).
  • alkenyl groups are ethenyl (vinyl), propenyl (allyl), / ' so-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl, propargyl, / ' so-prenyl and hex-2-enyl group.
  • alkenyl groups have one or two double bond(s).
  • This expression also refers to a group that is substituted by one, two, three or more alkyl, alkenyl or heteroalkyl (e.g. -OCH 3 , -OCH 2 CH 3 , -CH 2 NHCH 3 and -CH 2 NH 2 ) groups. These groups may themselves be substituted.
  • an alkyl group substituent may be substituted by one or more halogen atoms (i.e. may be a haloalkyl group, such as trifluoromethyl, dichloroethyl, dichloromethyl and iodoethyl).
  • halogen atoms i.e. may be a haloalkyl group, such as trifluoromethyl, dichloroethyl, dichloromethyl and iodoethyl.
  • a wording defining the limits of a range of length such as, for example, "from 1 to 5" means any integer from 1 to 5, i.e. 1 , 2, 3, 4 and 5.
  • any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.
  • pharmaceutically acceptable derivative may include any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound which upon administration to a subject, is capable of providing (directly or indirectly) a compound of the present invention or a pharmaceutically active metabolite or residue thereof.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic (such as acetic, HOOC-(CH 2 ) n -COOH where n is any integer from 0 to 6, i.e.
  • acids such as hydrochloric, phosphoric, hydrobromic, malic, glyco
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent (such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile), or in a mixture of the two.
  • an organic solvent such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile
  • a “prodrug” is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound provided herein.
  • a prodrug may be an acylated derivative of a compound as provided herein.
  • Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein.
  • Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.
  • composition as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutically acceptable it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • This invention thus further provides a pharmaceutical formulation or composition
  • a pharmaceutical formulation or composition comprising a compound of the invention or a pharmaceutically acceptable salt or derivative thereof together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients.
  • the carriers(s) must be "acceptable” in the sense of being compatible with other ingredients of the formulation and not deleterious to the recipient thereof.
  • compositions or compositions include those for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • the compounds of the invention may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids as solutions, suspensions, emulsions, elixirs or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
  • the subjects treated in the above method are mammals, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species, and preferably a human being, male or female.
  • the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired treatment of the pain, or pain prevention. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods.
  • a therapeutic, or treatment, effective amount is an amount of the compound which, when administered according to a desired dosing regimen, is sufficient to at least partially attain the desired therapeutic effect, or delay the onset of, or inhibit the progression of or halt or partially or fully reverse the onset or progression of pain.
  • a prevention effective amount is an amount of compound which when administered according to the desired dosing regimen is sufficient to at least partially prevent or delay the onset of pain.
  • administering should be understood to mean providing a compound of the invention to the subject in need of treatment.
  • treating encompasses curing and ameliorating pain.
  • the pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds which are usually applied in the treatment of pain. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage.
  • the dosage is preferably in the range of 1 pg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage.
  • the dosage is in the range of 1 mg to 500 mg per kg of body weight per dosage.
  • the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage.
  • the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage.
  • the dosage is in the range of 1 g to 1 mg per kg of body weight per dosage.
  • Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the type and/or severity of pain as well as the general age, health and weight of the subject.
  • the active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical formulation.
  • the present invention therefore relates to the use of a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable derivative thereof, for treating pain.
  • the present invention also provides a pharmaceutical composition for use in treating pain, in any of the embodiments described in the specification.
  • the present invention also relates to the use of a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for treating pain.
  • the present invention also relates to a compound of formula (I), or a pharmaceutically acceptable derivative thereof, when used in a method of treating pain.
  • the present invention also relates to a composition having an active ingredient for use in treating pain, wherein the active ingredient is a compound of formula (I), or a pharmaceutically acceptable derivative thereof.
  • the present invention also relates to the use of a pharmaceutical composition containing a compound of the formula (I), or a pharmaceutically acceptable derivative thereof, in treating pain, such as described above.
  • the compound of formula (I) is essentially the only active ingredient of the composition.
  • the pain is neuropathic pain.
  • the aqueous phase was further extracted with diethyl ether (2x80ml), and the combined extracts were washed with water (300ml), and brine (100ml), and dried over Na 2 SO 4 anhydrous, concentrated under vacuo, and the residue was purified by silica gel dry column vacuum chromatography ( DCVC) by stepwise gradient elution with dichloromethane/hexane (50:50 to 100:0).
  • DCVC silica gel dry column vacuum chromatography
  • Glycine transport by GlyT2 and GlyT1 is coupled to 3 Na+/1 CI- and 2Na+/1 Cl- ions respectively, creating an electrogenic process and allowing the two electrode voltage clamp technique to be used to measure glycine transport.
  • Defoliculated stage V- VI oocytes were injected with 4.6 ng of cRNA encoding the transporter (Drummond Nanoinject, Drummond Scientific Co., Broomall, PA, USA).
  • glycine transport currents were measured at -60 mV using Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) with a Powerlab 2/20 chart recorder (ADInstruments, Sydney, Australia) using chart software (ADInstruments).
  • Glycine was applied, followed by co-administration of glycine (EC50) in the presence of inhibitor, until the inhibitory response was observed to plateau.
  • EC50 glycine
  • Each compound was tested to a maximal concentration of 3 ⁇ because these compounds form micelles at higher concentrations.
  • the compounds were applied at concentrations below their CMC in ND96 which was determined using a CMC assay. Reversibility of each compound was tested by applying the EC50 dose of glycine every 5 minutes following inhibition for a 30 minute time course or until recovery was reached. Recovery is defined as glycine currents within +/- 5 % of the pre-inhibitory glycine response. Concentration response curves for the active compounds were then performed.
  • NADPH NADPH is the cofactor required for CYP450-mediated metabolism
  • Control samples were included (and quenched at 2, 30 and 60 minutes) to monitor for potential degradation in the absence of cofactor.
  • Metabolite screening was performed using accurate mass measurements only. Compounds were incubated at a low substrate concentration and as such, structure confirmation and elucidation using MS/MS scans was not conducted. Calculations
  • Test compound concentration versus time data were fitted to an exponential decay function to determine the first-order rate constant for substrate depletion.
  • Table 3 Metabolic stability parameters for nine compounds based NADPH-dependent degradation profiles in human and rat liver microsomes.
  • Plasma samples were quantified relative to calibration standards prepared using blank plasma of the same species. Calibration standards were spiked with test compound over a range of 0.5 to 10,000 ng/mL. Internal standard (leucine enkephalin) was added to calibration standards and incubation samples, and then immediately quenched using two volumes of acetonitrile to precipitate plasma proteins. Samples were vortex mixed and centrifuged (10,000 rpm for 3 minutes) in a microcentrifuge and the supernatant analysed by LC-MS using conditions tabulated below.
  • the mean and standard deviation of measured plasma concentrations were calculated for each time point and expressed as a percentage remaining, relative to the initial time point (2 min). Where measurable degradation was detected, and assuming first order degradation kinetics, the data were fit using a mono-exponential decay function to obtain the apparent first-order degradation rate constant (k, min-1 ) and degradation half-life.
  • NMDG- based recovery ACSF NMDG-based recovery ACSF
  • rACSF NMDG-based recovery ACSF
  • 95% O 2 and 5% C0 2 composed of (mM): 93 NMDG, 2.5 KCI, 1 .2 NaH 2 P0 4 , 30 NaHC0 3 , 20 HEPES, 25 Glucose, 5 Na ascorbate, 2 thiourea, 3 Na pyruvate, 10 MgSO 4 and 0.5 CaCI 2 , and adjusted to pH 7.4 with HCI.
  • slices were transferred to normal oxygenated ACSF where they were allowed to recover for 1 hour at 34° C and then maintained at room temperature prior to transfer to the recording chamber.
  • Normal ACSF had the following composition (imM): 125 NaCI, 2.5 KCI, 1 .25 NaH 2 PO 4 , 1 .2 MgCI 2 , 2.5 CaCI 2 , 25 glucose, and 1 1 NaHCO 3 and was equilibrated with 95% O 2 and 5% CO 2 .
  • Electrophysioloqy Slices were transferred to a recording chamber and superfused continuously at 2 ml/min with normal ACSF that had been equilibrated with 95% O 2 and 5% CO 2 and maintained at 34°C with an inline heater and monitored by a thermister in the slice chamber.
  • Dodt-contrast optics was used to identify lamina II neurons in the translucent substantia gelatinosa layer of the superficial dorsal horn.
  • a Cs + -based internal solution which should minimise postsynaptic effects, was used to record electrically evoked inhibitory post-synaptic currents (elPSCs) and tonic current, and contained (imM): 140 CsCI, 10 EGTA, 5 HEPES, 2 CaCI 2 , 2 MgATP, 0.3 NaGTP, 5 QX-314.CI, 2 Lucifer Yellow CH dipotassium salt and 0.1 % biocytin (osmolarity 285-295 mosmol ⁇ 1 ). Patch electrodes had resistances between 3 and 5 ⁇ . Synaptic currents were measured in whole-cell voltage-clamp (-70 mV, not corrected for a liquid junction potential of 4 mV) from lamina II cells.
  • Bipolar tungsten electrodes placed in the inner laminae were used to elicit elPSCs using a stimulus strength sufficient to evoke reliable elPSCs.
  • Neurons ventral to lamina II, in regions that are known to contain glycinergic neurons were electrically stimulated.
  • All elPSCs were recorded in CNQX (10 ⁇ ), AP5 (100 ⁇ ) and picrotoxin (80 ⁇ ).
  • strychnine 0.5 ⁇ was added to the superfusion solution to confirm that recorded currents were glycine- mediated IPSCs.
  • Drugs were superfused onto slices at a rate of 2ml/min in normal oxygenated ACSF at 34°C.
  • the formulation was prepared by dissolving solid compound in DMSO prior to addition of 1 % Solutol in 50mM PBS pH 7.4 solution, which after sonication and heating yielded a very fine off-white suspension with an apparent pH of 7.05.
  • This suspension of oleoyl-D-lysine was administered in a dose volume of 5 imL/kg via intraperitoneal injection (via 27G 1 /2" needle) resulting in a nominal oleoyl-D-lysine dose of 27.5 mg/kg.
  • Plasma concentration versus time data were analysed using non-compartmental methods (PKSolver Version 2.0). Standard calculations for each pharmacokinetic parameter are listed below.
  • the concentration of oleoyl-D-lysine in brain parenchyma was calculated on the basis of the measured concentration in brain homogenate, after correcting for the contribution of compound contained within the vascular space of brain samples as follows:
  • Plasma standards were freshly prepared, with each set of standards comprising at least six different analyte concentrations.
  • Solution standards were diluted from a stock solution (1 mg/mL in DMSO) with 50% acetonitrile in water.
  • Plasma standards were prepared by spiking blank plasma (50 ⁇ _) with solution standards (10 ⁇ _) and the internal standard, diazepam (10 ⁇ _, 5 Mg/mL). Plasma samples were similarly prepared, except that blank acetonitrile (10 ⁇ _) was added instead of solution standards.
  • Protein precipitation was carried out by the addition of acetonitrile (120 ⁇ _), vortexing (20 s) and centrifugation (10,000 rpm) in a microcentrifuge for 3 minutes. The supernatant was subsequently separated and 3 ⁇ _ injected directly onto the column for LC-MS analysis using conditions presented in the method summary section. All concentrations are expressed as the non-salt equivalent.
  • Pre-weighed rat brains were homogenised in 3-volume/weight of stabilisation mixture (composed of 0.1 M EDTA and 4 g/L KF in water) using a gentleMACSTM dissociator. Extraction of the test compound from the resulting tissue homogenate was conducted using protein precipitation with methanol. Tissue homogenate standards were freshly prepared, with each set of standards comprising at least six different analyte concentrations. Tissue standards were prepared by spiking blank tissue homogenate (200 ⁇ _) with solution standards (10 ⁇ _) and the internal standard, diazepam (10 ⁇ _, 5 pg/mL). Tissue samples were similarly prepared, except that blank methanol (10 ⁇ _) was added instead of solution standards.
  • Protein precipitation was carried out by the addition of methanol (600 ⁇ _), vortexing (20 s) and centrifugation (10,000 rpm) in a microcentrifuge for 3 minutes. The supernatant was subsequently separated and 3 ⁇ _ injected directly onto the column for LC-MS analysis using conditions presented in the method summary section. Results
  • Plasma and brain concentration versus time profiles are presented in Figure 6, whilst calculated plasma and brain exposure parameters, values for individual rats, together with the corresponding brain-to-plasma (B:P) ratios are summarised in Tables 5 and 6.
  • B:P ratios increased over the 24 h exposure period, and maximum values (ranging from 0.6-1 .6) were observed at 24 h post-dose. This may be indicative of a slow rate of compound equilibration between plasma and brain.
  • B:P partitioning ratio values (based on AUC 0 -24h values in plasma and brain) may provide a better indication of the distribution of oleoyl-D-lysine into the brain, although it may still be an underestimation as it is lower than the B:P at 24 h.
  • Table 5 Exposure parameters for oleoyl-D-lysine in male Sprague Dawley rats following IP administration at 27.5 mg/kg
  • S1 PR1 Assays The purpose of these assays was to determine whether the compounds of the present invention have any off-target effects.
  • the S1 PR1 , CB1 and CB2 receptors were chosen as they have been known to be agonised or antagonised by lipid-based molecules. S1 PR1 Assay
  • sphingolipid G-protein-coupled receptor, 1 NCBI protein database NP_001391 .2
  • This cell line has been tested negative for Mycoplasma sp.
  • This cell line has been tested positive for Endothelial Differentiation, Sphingolipid G-Protein-coupled Receptor, 1 specific response.
  • the receptor specific activity is stable for 10 weeks continuous passage.
  • Growth medium 90% DMEM, 10% FBS, 250 ⁇ g/ml G418 and 1 ⁇ g/ml puromycin
  • Freezing medium 10% DMSO, 90% growth medium Testing compounds on ACTOne-SI PR1 cells
  • ACTOne-S1 PR1 cells (CB-80300-250) were maintained in cell culture medium consisting of 90% Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250 ⁇ g/ml G418 and 1 ⁇ g/ml puromycin. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in the growth medium. 20 ⁇ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • the agonist control picked here was S1 P (125 uM stock in 4 mg/ml Fatty acid free BSA, Avanti Polar Lipids 860492P).
  • the agonist stimulation solutions were prepared as below:
  • testing compounds were first diluted in DMSO to 1 imM each. It was further diluted (1 :200) in agonist dilution buffer to 5 ⁇ each.
  • Isoproterenol is used to stimulate the adenylyl cyclase through the activation of Gs-coupled endogenous ⁇ -adrenoceptor.
  • Ro20-1724 is a PDE4 specific inhibitor.
  • the cell plates were placed on a Molecular Devices SpectraMax Gemini EM and the baselines (F0) were read before the addition of any compound. 10 ⁇ of above agonist stimulation solutions (5X) was added into each well. The plates were recorded again on Gemini EM 50 min (Ft) after the compound addition. The ratio of Ft/FO (Fold) was calculated for each well; data was analyzed and graphed using GraphPad Prism.
  • the assays were again performed on 384-well plates. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in growth medium. 20 ⁇ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
  • testing compounds (1 mM in DMSO) were diluted (1 :200) in 1 X DPBS to 5 ⁇ each. 10 ⁇ of such solution was added into each well of 384-well plates and incubated at room temperature for 20 min. 10 ⁇ of W146 (Final) was used as a positive control.
  • the cell plates were read on Gemini EM (FO). Afterwards, 12.5 ⁇ of 5X agonist stimulation solution (125 ⁇ Ro 20-1724, 1 .5 ⁇ isoproterenol and 500 nM S1 P prepared in DPBS with 0.1 mg/ml BSA) was added into each well. The cell plates were read again after 50 min (Ft).
  • 5X agonist stimulation solution 125 ⁇ Ro 20-1724, 1 .5 ⁇ isoproterenol and 500 nM S1 P prepared in DPBS with 0.1 mg/ml BSA
  • the compounds have no antagonist activity on S1 PR1 .
  • CB2 CELL LINE DESIGNATION Cannabinoid receptor 2 cell line
  • RECEPTOR INTRODUCED Human Cannabinoid receptor 2. (NCBI protein database NP_001832 with SNP at amino acid position 63.)
  • This cell line has been tested negative for Mycoplasma sp.
  • This cell line has been tested positive for CB2 specific response.
  • the receptor specific activity is stable for 10 weeks continuous passage. Cell culture condition
  • Growth medium for Cannabinoid receptor 2 cell line 90% DMEM with Glutamine, 10% FBS, 250 ⁇ g/ml G418 and 1 ⁇ g/ml puromycin 2. Freezing medium: 10% DMSO, 90% growth medium
  • ACTOne-CB2 cells (CB-80300-225) were maintained in cell culture medium consisting of 90% Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250 ⁇ g/ml G418 and 1 ⁇ g/ml puromycin. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in the growth medium. 20 ⁇ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • the agonist control picked here was CP-55940 (10mM stock in DMSO, Sigma C1 1 12). Dilute 10mM CP-55940 stock in DMSO containing 30 ⁇ Isoproterenol and 2.5 imM Ro 20-1724. These concentrations are 100X the expected final testing concentrations.
  • Isoproterenol is used to stimulate the adenylyl cyclase through the activation of Gs-coupled endogenous ⁇ adrenoceptor.
  • Ro20-1724 is a PDE4 specific inhibitor. Table 8. An example of CP-55940 concentrations in a compound dilution plate
  • the compound concentration is 5X testing concentration.
  • the testing compounds were first diluted in DMSO to 2 mM each. They were further diluted to 100 ⁇ in DMSO containing 30 ⁇ Isoproterenol and 2.5 mM Ro 20- 1724. Further dilute the solutions 1 :20 with 1 X DPBS in compound plates. At this step, the compound concentration is 5X testing concentration.
  • the cell plates were placed on a Molecular Devices SpectraMax Gemini EM and the baselines (F0) were read before the addition of any compound. 10 ⁇ of above agonist stimulation solutions (5X) was added into each well. The plates were recorded again on Gemini EM 50 min (Ft) after the compound addition. The ratio of Ft/FO (Fold) was calculated for each well; data was analyzed and graphed using GraphPad Prism.
  • the assays were again performed on 384-well plates. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in growth medium. 20 ⁇ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
  • testing compounds (2 mM in DMSO) were diluted (1 :400) in 1 X DPBS to 5 ⁇ each. 10 ⁇ of such solution was added into each well of 384-well plates and incubated at room temperature for 20 min. 10 ⁇ of AM251 (Final) was used as a positive control.
  • the cell plates were read on on Gemini EM (F0). Afterwards, 12.5 ⁇ of 5X agonist stimulation solution (125 ⁇ Ro 20-1724, 1 .5 ⁇ isoproterenol and 200 nM CP- 5590 in DPBS) (diluted from 100X solution prepared in DMSO) was added into each well. The cell plates were read again after 50 min (Ft).
  • 5X agonist stimulation solution 125 ⁇ Ro 20-1724, 1 .5 ⁇ isoproterenol and 200 nM CP- 5590 in DPBS
  • This cell line has been tested negative for Mycoplasma sp.
  • This cell line has been tested positive for CB1 specific response.
  • the receptor specific activity is stable for 10 weeks continuous passage.
  • Growth medium for Cannabinoid receptor 1 cell line 90% DMEM with Glutamine, 10% FBS, 250 ⁇ g/ml G418 and 1 ⁇ g/ml puromycin
  • Freezing medium 10% DMSO, 90% growth medium Testing compounds on ACTOne-CB1 cells
  • ACTOne-CB1 cells (CB-80300-205) were maintained in cell culture medium consisting of 90% Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250 ⁇ g/ml G418 and 1 ⁇ g/ml puromycin. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in the growth medium. 20 ⁇ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • G418 fetal bovine serum
  • puromycin 1 ⁇ g/ml puromycin
  • the cell plates were taken out from the incubator and an equal volume (20 ⁇ ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 ⁇ .
  • the plates will be referred to as the cell plates.
  • the compound concentration is 5X testing concentration.
  • the testing compounds were first diluted in DMSO to 2 mM each. I. They are further diluted to 100 ⁇ in DMSO containing 30 ⁇ Isoproterenol and 2.5 mM Ro 20- 1724. Further dilute the solutions 1 :20 with 1 X DPBS in a compound plate. At this step, the compound concentration is 5X testing concentration
  • the cell plates were placed on a Molecular Devices SpectraMax Gemini EM and the baselines (F0) were read before the addition of any compound. 10 ⁇ of above agonist stimulation solutions (5X) was added into each well. The plates were recorded again on Gemini EM 60 min (Ft) after the compound addition. The ratio of Ft/FO (Fold) was calculated for each well; data was analyzed and graphed using GraphPad Prism. Results
  • testing compounds (1 imM in DMSO) were diluted (1 :200) in 1 X DPBS to 5 ⁇ each. 10 ⁇ of such solution was added into each well of 384-well plates and incubated at room temperature for 20 min. 10 ⁇ of AM251 (Final) was used as a positive control.
  • the cell plates were read on on Gemini EM (F0). Afterwards, 12.5 ⁇ of 5X agonist stimulation solution (125 ⁇ Ro 20-1724, 1 .5 ⁇ isoproterenol and 2 ⁇ CP- 5590 in DPBS) (diluted from 100X solution prepared in DMSO) was added into each well. The cell plates were read again after 60 min (Ft). Results
  • Compound 29 was tested in rats suffering from neuropathic pain induced by partial ligation of the sciatic nerve. Two routes of compound administration were used - intrathecal and intraperitoneal.
  • Lu Y et al. A feed-forward spinal cord glycinergic neural circuit gates mechanical allodynia. J. Clin Invest. 2013;123:4050-62. 7. Dohi T et al. Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol & Therap. 2009;123:54-79.

Abstract

The present invention relates to novel glycine transport inhibitor compounds and their use for treating pain.

Description

Novel glycine transport inhibitors for the treatment of pain
Field of the invention
The present invention relates to novel glycine transport inhibitor compounds and their use for treating pain. Background of the invention
Chronic pain is a significant global health, economic and social problem, with the annual economic burden in the USA recently estimated at $600 billion. In Australia, the disease burden of pain has been documented in a rigorous epidemiological study, which revealed that -20% of adults have poor self-rated health as a consequence of chronic pain. The economic burden of unrelieved persistent pain in Australia is large and continuing to escalate, with a total cost of $34 billion in 2007. Until recently, the major analgesics used clinically were limited to non-steroidal, local anaesthetic, opioid and anticonvulsant drugs that provide adequate pain relief in only a small proportion of chronic pain patients. Unfortunately, newer classes of analgesics also have limited efficacy, with the most effective, gabapentin, at best providing only 50% reduction in pain scores in chronic pain, with the number needed to treat for a clinical response being > 4.
Recent insights into the physiological adaptations underlying chronic pain have provided an impetus to discover new classes of pain-relieving drugs, particularly for neuropathic pain. One such target is the glycine transporter 2 (GlyT2).
Neurotransmitter transporters are responsible for regulating the synaptic concentration of monoamines (5-hydroxytryptamine, dopamine, noradrenaline) and amino acid (γ-aminobutyric acid, glycine) neurotransmitters. These membrane-bound proteins use electrochemical gradients to drive the transport of neurotransmitters across neuronal and glial membranes, serving to terminate neurotransmission and replenish intracellular levels of neurotransmitter for future release.
The capacity of neurotransmitter transporters to selectively enhance or diminish neuronal signalling makes them attractive drug targets. Indeed, a wide range of therapeutically useful drugs, including antidepressants and anxiolytics, target these proteins. Diminished glycinergic transmission is an important CNS mechanism underlying neuropathic pain including mechanical hyperalgesia and allodynia (a painful response to normally innocuous tactile stimuli). Drugs that can enhance dysfunctional glycerinergic transmission in neuropathic pain, and in particular glycine transporter inhibitors, are potential chronic pain therapeutics.
There are two closely related glycine transporters, GlyT1 and GlyT2, which serve to regulate glycine concentrations. GlyT1 is expressed throughout the CNS predominantly by astrocytes surrounding both inhibitory and excitatory synapses to influence Glycine and NMDA receptors (NMDAR). Enhancement of NMDAR activity by GlyT1 inhibition is an established therapeutic target in diseases with NMDAR hypofunction, such as schizophrenia, but is undesirable in chronic pain states where suppression of NMDAR function is desirable. By contrast, GlyT2 is considered a promising therapeutic target for chronic pain. GlyT2 shows much more restricted expression patterns than GlyT1 and is predominantly expressed by glycinergic terminals in the spinal cord and brain stem. GlyT2 plays two important roles at glycinergic inhibitory synapses: regulation of extracellular glycine concentrations to control inhibitory glycinergic tone, and to recycle released glycine to provide sufficient intracellular glycine for repackaging into synaptic vesicles for subsequent release.
The sensation of pain can be suppressed by inhibiting GlyT2, which increases glycine concentrations in the synaptic cleft and enhances neurotransmission through activation of Glycine receptors (GlyRa3) in the spinal cord. The two GlyT2 inhibitors that have been most extensively studied are ALX1393 and ORG25543. Both compounds show efficacy as analgesics in the treatment of various forms of acute and chronic pain, but there are limitations in their use. ALX1393 shows only a 10-fold selectivity for GlyT2 over GlyT1 , and the dose required for analgesia may also result in partial inhibition of GlyT1 and increases in glycine at excitatory synapses leading to an initial increase in NMDAR-related pain before analgesia is achieved. ORG25543 causes respiratory depression and loss of motor control at high doses which has been attributed to irreversible inhibition of GlyT2 with subsequent reductions in glycine loading of synaptic vesicles. Partial knock down of GlyT2 using siRNAs indicates that partial inhibitors of GlyT2 have the potential to be therapeutic in the treatment of neuropathic pain. Knockdown of GlyT2 to approximately 30% of wild type levels (which may mimic partial inhibition of transport) provides analgesia in rat models of allodynia associated with neuropathic pain. The equilibrium glycine concentration gradient achieved by GlyT2 is determined by the ion gradients across the cell membrane, whereas the rate at which equilibrium is achieved will be determined by the number of glycine transporters. Thus, partial inhibition will not alter the equilibrium glycine concentration gradient, but will prolong the time required to reach equilibrium, allowing greater glycine receptor activity. It appears that partial inhibition of glycine transport in the siRNA knock-down experiments allows sufficient glycine uptake for loading of presynaptic glycinergic vesicles to maintain, or even prolong, glycinergic neurotransmission.
There is a need for new compounds that mimic the actions of the partial knockdown of GlyT2 for use as analgesic drugs for the treatment of various forms of pain.
Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.
Summary of the invention
In one aspect, the present invention provides a compound of formula (I):
X-L-Y
(I)
or pharmaceutically acceptable derivatives thereof, wherein:
X is an amino acid or derivative thereof;
L is an amide or retro amide;
Y is C10 - C24 alkyl or alkenyl;
X, L and Y may be independently substituted or unsubstituted; and
wherein the compound is not
Figure imgf000005_0001
Figure imgf000006_0001
In another aspect, the present invention provides a compound of formula (I) as defined above wherein the compound is not one or more of the compounds selected from the group consisting of:
Figure imgf000006_0002
Figure imgf000007_0001
Figure imgf000008_0001
In another aspect, there is provided a pharmaceutical composition comprising a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof, together with one or more pharmaceutically acceptable carriers or adjuvants. In another aspect, there is provided a method of treating pain by administering an effective amount of a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof, to a subject in need thereof.
In another aspect, there is provided a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof, for use in treatment of pain in a subject in need thereof.
In another aspect, there is provided use of a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for treatment of pain in a subject in need thereof.
In another aspect, there is provided a method of treating pain by administering an effective amount of at least one compound selected from the group consisting of:
Figure imgf000008_0002
5
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
a salt, or a pharmaceutically acceptable derivative thereof, to a subject in need thereof.
In another aspect there is provided a compound selected from the group consisting of:
Figure imgf000011_0002
Figure imgf000012_0001
11
Figure imgf000013_0001
Figure imgf000014_0001
a salt, or a pharmaceutically acceptable derivative thereof, for use in treatment of pain in a subject in need thereof.
In another aspect there is provided a compound selected from the group consisting of:
Figure imgf000014_0002
Figure imgf000015_0001
Figure imgf000016_0001
 a salt, or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for treatment of pain in a subject in need thereof.
The pain may be chronic or acute. Preferably the pain is chronic pain, more preferably neuropathic pain. Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.
Brief description of the drawings
Fig. 1 shows plasma concentration vs time profiles for Compounds 28, 29, 8, 1 and 36, following incubations at 37°C in human (·) and rat (□) plasma.
Fig. 2 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of Compound 28.
Fig. 3 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of Compound 39. Fig. 4 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of Oleoyl-L-carnitine.
Fig. 5 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs of N-arachidonyl glycine.
Fig. 6 shows elPSC amplitude (A), tonic current (B) and decay time constant (C) graphs Oleoyl-L-glycine.
Fig. 7 shows plasma and brain concentrations of Compound 29 in male Sprague Dawley rats following IP administration at 27.5 mg/kg.
Fig. 8 shows the agonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on S1 PR1 . Fig. 9 shows the antagonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65,
27 and 39 on S1 PR1 . Fig. 10 shows the agonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB2.
Fig. 11 shows the antagonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB2.
Fig. 12 shows the agonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB1 .
Fig. 13 shows the antagonist activity of Compounds 1 , 2, 16, 25, 28, 29, 36, 65, 27 and 39 on CB1 .
Fig. 14 shows the analgesic effect of intrathecal administration of Compound 29. Fig. 15 shows the analgesic effect of intraperitoneal administration of Compound
29.
Detailed description of the embodiments
In one aspect, the present invention provides a compound of formula (I):
X-L-Y
(I)
or pharmaceutically acceptable derivatives thereof, wherein:
X is an amino acid or derivative thereof;
L is an amide or retro amide;
Y is Cio - C24 alkyl or alkenyl;
X, L and Y may be independently substituted or unsubstituted; and
wherein the compound is not
Figure imgf000018_0001
Figure imgf000019_0001
In another aspect, the present invention provides a compound of formula (I) as defined above wherein the compound is not one or more of the compounds selected from the group consisting of:
Figure imgf000020_0001
Figure imgf000021_0001
In one embodiment, X is a naturally occurring or non-naturally occurring amino acid. In a preferred embodiment, X is a naturally occurring hydrophobic and/or aromatic amino acid, more preferably L-tryptophan. In another preferred embodiment, X is a non- naturally occurring amino acid, more preferably a D-amino acid. In a particularly preferred embodiment X is selected from one or more of the group consisting of glycine, L-carnitine, L-serine, D-serine, L-lysine, D-lysine, L-lysine derivative wherein the carbon chain of the lysine side group is of C1 -C3 in length, L-arginine, L-methionine, L-leucine, D-leucine, L-alanine, D-alanine, B-alanine, L-valine,- D-valine, D-phenylalanine, L- phenylalanine derivative wherein the carbon chain of the phenylalanine side group is of two carbons in length, L-tryptophan, D-tryptophan, L-tyrosinol- L-dopamine, L-aspartate, D-aspartate, L-glutamate, L-histidine, D-histidine, L-lysine OME, L-norleucine. In a particularly preferred embodiment, X is selected from the group consisting of glycine, D- lysine, L-tryptophan L-lysine methylester, and L-leucine.
In one embodiment, X is unsubstituted. In another embodiment, X is substituted with 1 , 2, or 3 C1 - C3 alkyl groups at the a carbon of the amino acid or derivative thereof. Preferably, the C1 - C3 alkyl group is methyl. In a particularly preferred embodiment, X is lysine substituted with 1 , 2, or 3 methyl groups at the a carbon of the lysine. Substituting X as described herein, advantageously increases the metabolic stability and efficacy of the compounds of the invention by minimising in vivo degradation of the linking group, L.
In a preferred embodiment, L is an amide.
In one embodiment, L is unsubstituted. In another embodiment, L is substituted with a C1 - C3 alkyl group at the nitrogen of the amide or retro amide. Preferably, the C1-C3 alkyl group is methyl. Substituting L as described herein and/or reversing the amide, advantageously increases the metabolic stability and efficacy of the compounds of the invention by minimising in vivo degradation of the linking group, L.
In a preferred embodiment, Y includes 1 - 4 cis double bonds, more preferably Y is monounsaturated and includes 1 cis double bond. The single cis double bond may be at any position of the C10 - C24 alkenyl chain, preferably 5 - 15 carbons from L, more preferably 8 - 10 carbons from L.
In one embodiment, Y includes 1 -4 trans double bonds. More preferably, Y is monounsaturated and includes 1 trans double bond. The single trans double bond may '1Ut>
be at any position of the do - C24 alkenyl chain, preferably 5 - 15 carbons from L, more preferably 8 - 10 carbons from L, more preferably 9 carbons from L.
In a preferred embodiment, Y is a monounsaturated Ci8, Ci6 or Ci4 chain, preferably a monounsaturated Ci8 chain. Preferably the monounsaturated Ci8 carbon chain includes a cis-double bond in the ω 3, 5, 6, 7, 8, 9, 10, 1 1 , or 12 position, more preferably in the ω 9 position. Preferably, the monounsaturated Ci6 carbon chain includes a cis-double bond in the ω 5, 6, 7, 9, or 1 1 position.
In another embodiment, Y is a saturated Ci6 or Cu carbon chain.
Preferably, the compound is selected from one or more of compounds 2 - 7, 9 - 12, 1 7 - 21 , 23, 26 - 44, 47 - 54 of Table 1 . In another preferred embodiment the compound is selected from one or more of compounds 2 - 7, 1 0 - 1 2, 17, 18, 20, 21 , 23, 28 - 34, 37, 42, 43, 48 and 52 of Table 1 .
Table 1. Exemplary compounds of the present invention
Figure imgf000023_0001
'1Ut>
Figure imgf000024_0001
'1Ut>
Figure imgf000025_0001
'1Ut>
Figure imgf000026_0001
'1Ut>
Figure imgf000027_0001
'1Ut>
Figure imgf000028_0001
'1Ut>
Figure imgf000029_0001
'1Ut>
Figure imgf000030_0001
'1Ut>
Figure imgf000031_0001
The invention also relates to methods of treating pain by administering an effective amount of a compound according to the invention, a salt or pharmaceutically acceptable derivative thereof, to a subject in need thereof.
In another aspect, there is provided a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof for use in treatment of pain in a subject in need thereof.
In another aspect, there is provided use of a compound according to the invention, a salt or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for treatment of pain in a subject in need thereof.
In another embodiment the invention relates to methods of treating pain by administering an effective amount of at least one compound selected from the group consisting of:
Figure imgf000032_0001
Figure imgf000033_0001
32
Figure imgf000034_0001
33
Figure imgf000035_0001
a salt, or pharmaceutically acceptable derivative thereof, to a subject in need thereof.
In another aspect there is provided a compound selected from the group consisting of:
Figure imgf000035_0002
Figure imgf000036_0001
Figure imgf000037_0001
 a salt, or a pharmaceutically acceptable derivative thereof, for use in treatment of pain in a subject in need thereof.
In another aspect there is provided a compound selected from the group consisting of:
Figure imgf000038_0001
Figure imgf000039_0001

Figure imgf000040_0001
a salt, or a pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for treatment of pain in a subject in need thereof.
The pain may be acute or chronic, preferably chronic. In a particularly preferred embodiment, the pain is neuropathic pain. Definitions
The term "amino acid" as used herein is intended to encompass compounds having an amino group and a carboxyl group in which the amino group and the carboxyl group are separated by at least one carbon atom. The amino acid may be a L- or D- isomer and may have a naturally occurring side chain or a non-naturally occurring side chain. An amino acid having a non-naturally occurring side chain refers to an amino acid having a side chain that does not occur in the naturally occurring L-a-amino acids. Examples of non-natural amino acids and derivatives include, but are not limited to amino acids substituted with 1 , 2 or 3 C1-C3 alkyl groups at the a carbon of the amino acid, L-carnitine, gamma aminobutyric acid (GABA), dopamine, L-lysine OMe and/or D- isomers of amino acids.
The term "amino acid derivative" as used herein is intended to encompass derivatives of naturally occurring and non-naturally occurring amino acids, preferably ester derivatives of naturally occurring and non-naturally occurring amino acids. It also encompasses amino acids that have had their side chain modified, for example, the hydrocarbon chain shortened or extended. Some non-limiting examples of these are shown below for lysine and phenylalanine:
Figure imgf000041_0001
The term "retro amide" as used herein refers to a reverse amide bond wherein the nitrogen of the amide bond is located on the proximal side of the carbonyl group, ie (NHCO).
The term "alkyl" refers to a saturated, straight-chain or branched hydrocarbon group. Specific examples of alkyl groups are methyl, ethyl, propyl, /'so-propyl, n-butyl, /'so-butyl, sec-butyl, te/t-butyl, n-pentyl, /'so-pentyl, n-hexyl and 2,2-dimethylbutyl.
The term "alkenyl" refers to an at least partially unsaturated, straight-chain or branched hydrocarbon group that contains at least two carbon atoms (i.e. C2 alkenyl). Specific examples of alkenyl groups are ethenyl (vinyl), propenyl (allyl), /'so-propenyl, butenyl, ethinyl, propinyl, butinyl, acetylenyl, propargyl, /'so-prenyl and hex-2-enyl group. Preferably, alkenyl groups have one or two double bond(s).
The term "substituted" refers to a group in which one, two, three or more hydrogen atoms have been replaced independently of each other by halogen (for example, fluorine, chlorine, bromine or iodine atoms) and/or by, for example, OH, =O, SH, SO3H, NH2, N-alkyl, NH-alkyl, N3 or NO2 groups. This expression also refers to a group that is substituted by one, two, three or more alkyl, alkenyl or heteroalkyl (e.g. -OCH3, -OCH2CH3, -CH2NHCH3 and -CH2NH2) groups. These groups may themselves be substituted. For example, an alkyl group substituent may be substituted by one or more halogen atoms (i.e. may be a haloalkyl group, such as trifluoromethyl, dichloroethyl, dichloromethyl and iodoethyl).
As used herein a wording defining the limits of a range of length such as, for example, "from 1 to 5" means any integer from 1 to 5, i.e. 1 , 2, 3, 4 and 5. In other words, any range defined by two integers explicitly mentioned is meant to comprise and disclose any integer defining said limits and any integer comprised in said range.
Compounds according to the formula provided herein, which have one or more stereogenic centres, have an enantiomeric excess of at least 50%. For example, such compounds may have an enantiomeric excess of at least 60%, 70%, 80%, 85%, 90%, 95%, or 98%. Some embodiments of the compounds have an enantiomeric excess of at least 99%. It will be apparent that single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors, biosynthesis or by resolution of the racemates, for example, enzymatic resolution or resolution by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.
Certain compounds are described herein using a general formula that includes variables such as X, L and Y. Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. Therefore, for example, if a group is shown to be substituted with 0, 1 or 2 FT, the group may be unsubstituted or substituted with up to two R* groups and R* at each occurrence is selected independently from the definition of R*. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds, i.e., compounds that can be isolated, characterized and tested for biological activity. The term "pharmaceutically acceptable derivative" may include any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound which upon administration to a subject, is capable of providing (directly or indirectly) a compound of the present invention or a pharmaceutically active metabolite or residue thereof. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic (such as acetic, HOOC-(CH2)n-COOH where n is any integer from 0 to 6, i.e. 0, 1 , 2, 3, 4, 5 or 6), and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. A person skilled in the art will recognize further pharmaceutically acceptable salts for the compounds provided herein. In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent (such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile), or in a mixture of the two.
A "prodrug" is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound provided herein. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.
The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
This invention thus further provides a pharmaceutical formulation or composition comprising a compound of the invention or a pharmaceutically acceptable salt or derivative thereof together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients. The carriers(s) must be "acceptable" in the sense of being compatible with other ingredients of the formulation and not deleterious to the recipient thereof.
Pharmaceutical formulations or compositions include those for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The compounds of the invention, together with a conventional adjuvant, carrier or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids as solutions, suspensions, emulsions, elixirs or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
The subjects treated in the above method are mammals, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species, and preferably a human being, male or female.
As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired treatment of the pain, or pain prevention. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. A therapeutic, or treatment, effective amount is an amount of the compound which, when administered according to a desired dosing regimen, is sufficient to at least partially attain the desired therapeutic effect, or delay the onset of, or inhibit the progression of or halt or partially or fully reverse the onset or progression of pain. A prevention effective amount is an amount of compound which when administered according to the desired dosing regimen is sufficient to at least partially prevent or delay the onset of pain.
The terms "administration of" and/or "administering a" compound should be understood to mean providing a compound of the invention to the subject in need of treatment.
The terms "treating", "treatment" and "therapy" are used herein to refer to curative therapy. Therefore, in the context of the present disclosure, the term "treating" encompasses curing and ameliorating pain. The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds which are usually applied in the treatment of pain. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage. The dosage is preferably in the range of 1 pg to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another preferred embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 g to 1 mg per kg of body weight per dosage.
Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the type and/or severity of pain as well as the general age, health and weight of the subject.
The active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical formulation.
The present invention therefore relates to the use of a therapeutically effective amount of a compound of formula (I), or a pharmaceutically-acceptable derivative thereof, for treating pain. The present invention also provides a pharmaceutical composition for use in treating pain, in any of the embodiments described in the specification. The present invention also relates to the use of a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for treating pain.
The present invention also relates to a compound of formula (I), or a pharmaceutically acceptable derivative thereof, when used in a method of treating pain. The present invention also relates to a composition having an active ingredient for use in treating pain, wherein the active ingredient is a compound of formula (I), or a pharmaceutically acceptable derivative thereof. The present invention also relates to the use of a pharmaceutical composition containing a compound of the formula (I), or a pharmaceutically acceptable derivative thereof, in treating pain, such as described above. In one embodiment, the compound of formula (I) is essentially the only active ingredient of the composition. In one embodiment, the pain is neuropathic pain. In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples.
Compounds of formula (I) may be prepared using the methods depicted or described herein or known in the art for the preparation of compounds of analogous structure. It will be understood that minor modifications to methods described herein or known in the art may be required to synthesise particular compounds of formula (I).
Figure imgf000048_0001
General procedure for the synthesis of ethyl esters
To a solution of the carboxylic acid (5.51 mmol) in ethanol (50 mL) was added acetyl chloride (16.5 mmol). The solution was stirred at room temperature for 4 h, then concentrated under reduced pressure. The residue was dissolved in di ethyl ether (70 mL), and washed with sat. NaHCO3 (3 x 70 mL), water (70 mL) and brine (70 mL). The organic phase was dried with NaSO4 and concentrated under reduced pressure, affording the ethyl ester as a white solid.
Ethyl 8-Bromododecanoate
1H NMR (500 MHz, CDCI3), δ 4.08 (q, J=7.0Hz, 2H), 3.40 (t, J=7.5Hz 2H), 2.28 (t, J=7.5Hz 2H), 1 .84 (p, J=7.5Hz, 2H), 1 .60-1 .57 (m, 2H), 1 .41 -1 .38 (m, 2H), 1 .34-1 .30 (m, 4H), 1 .24 (t, J=7.5Hz, 3H).
Ethyl 11-Hydroxyundecanoate
1H NMR (500 MHz CDCI3), δ 4.10 (q, J=7.5Hz, 2H), 3.38 (t, J=7.5, 2H), 2.24 (t, J=7.5Hz 2H), 1 .60-1 .96 (m, 4H), 1 .34-1 .30 (m, 13H). Ethyl 12-Bromododecanoate
1H NMR (500 MHz, CDCI3), δ 4.08 (q, J=7.0Hz, 2H), 3.37 (t, J=7.0Hz, 2H), 2.24 (t, J=7.5Hz, 2H), 1 .80 (p, J=7.5Hz, 2H), 1 .60-1 .56 (m, 2H), 1 .40-1 .37 (m, 2H), 1 .24-1 .22 (m, 12H), 1 .20 (t, J=7.5Hz, 3H).
Ethyl 12-Hydroxydodecanoate 1H NMR (500 MHz, CDCI3), δ 4.10 (q, J=7.0Hz, 2H), 3.36 (t, J=6.5Hz 2H), 2.25
(t, J=7.5Hz 2H), 1 .60-1 .52 (m, 4H), 1 .24-1 .22 (m, 15H). General procedure for the synthesis of cyano ethyl ester
To a solution of the bromo alkyl ethyl ester (8.88 mmol) and sodium cyanide (26.66mmol) in DMSO (20ml) was stirred under nitrogen atmosphere at 60°C for 18 hours. Water (70ml) was added to the reaction mixture, and the product extracted with ethyl acetate (3x 50ml). The combined extracts were washed with brine (50ml), dried over sodium sulfate, and the solvent was removed in vacuo.
Ethyl 4-cyanobutanoate
1H NMR (500 MHz, CDCI3): δ 4.06 (q, J=7.0Hz, 2H), 2.39 (t, J=7.5Hz, 2H), 2.37 (t, J=7.5Hz, 2H) 1 .91 -1 .88 (m, 2H), 1 .20 (t, J=7.0Hz, 3H) Ethyl 6-cyanohexanoate
1H NMR (500 MHz CDCI3): δ 4.10(q, J=7.0Hz, 2H), 2.30 (t, J=7.0Hz, 2H), 2.27 (t, J=7.0Hz, 2H), 1 .63-1 .58(m, 4H), 1 .44-1 .42 (m, 2H), 1 .20 (t, J=7.0Hz, 3H)
Ethyl 7-cyanoheptanoate
1H NMR (500 MHz, CDCI3): δ 4.10 (q, J=7.0Hz, 2H), 2.31 (t, J=7.0Hz, 2H), 2.27 (t, J=7.0Hz, 2H), 1 .65-1 .60(m, 4H), 1 .45-1 .44 (m, 2H), 1 .35-1 .33 (m, 2H), 1 .22 (t, J=7.5Hz, 3H)
Ethyl 8-cyanooctanoate
1H NMR (500 MHz, CDCI3): δ 4.12 (q, J=7.0Hz, 2H), 2.32 (t, J=7.0Hz, 2H), 2.27 (t, J=7.0Hz, 2H), 1 .66-1 .60 (m, 4H), 1 .44-1 .41 (m, 2H), 1 .34-1 .31 (m, 4H), 1 .24 (t, J=7.5Hz, 3H)
Ethyl 12-cyanododecanoate
1H NMR (500 MHz, CDCI3): δ 4.12 (q, J=7.0Hz, 2H), 2.30 (t, J=7.0Hz, 2H), 2.24 (t, J=7.0Hz, 2H), 1 .65-1 .56(m, 4H), 1 .43-1 .38 (m, 2H), 1 .30-1 .26 (m, 12H), 1 .24 (t, J=7.5Hz, 3H) General method for the synthesis of aldehydes via cyano intermediates
To a solution of the cyano ethyl esters (4.8mmol) in pyridine (26ml) were added water (13ml), acetic acid (13ml), sodium hyposphite (39.06mmol) and Raney nickel. The suspension was then stirred at 40°C for 2hr. The catalyst was removed by filtration, and washed with ethanol (5ml). Water (300ml) and diethyl ether (80ml) were added to the filtrate, and the organic layer was separated. The aqueous phase was further extracted with diethyl ether (2x80ml), and the combined extracts were washed with water (300ml), and brine (100ml), and dried over Na2SO4 anhydrous, concentrated under vacuo, and the residue was purified by silica gel dry column vacuum chromatography ( DCVC) by stepwise gradient elution with dichloromethane/hexane (50:50 to 100:0).
Ethyl-5-oxopentanoate
1H NMR (500 MHz, CDCI3), δ 9.76 (t, J=1 .5Hz, 1 H), 4.1 1 (q, J=7.5Hz, 2H), 2.51 (td, J=7.5, 1 .5Hz, 2H), 2.34 (t, J=7.5Hz 2H), 1 .94-1 .91 (m, 2H), 1 .23 (t, J=7.5Hz, 3H).
Ethyl 7-oxoheptanoate 1H NMR (500 MHz, CDCI3), δ 9.74 (t, J=1 .5Hz, 1 H), 4.10 (q, J=7.5Hz, 2H), 2.41
(td, J=7.5, 1 .5Hz, 2H), 2.28 (t, J=7.5Hz 2H), 1 .65-1 .62 (m, 4H), 1 .34-1 .32 (m, 2H), 1 .23 (t, J=7.5Hz, 3H).
Ethyl 8-oxooctanoate
1H NMR (500 MHz, CDCI3), δ 9.76 (t, J=1 .5Hz, 1 H), 4.12 (q, J=7.5Hz, 2H), 2.42 (td, J=7.5, 1 .5Hz, 2H), 2.27 (t, J=.5Hz 2H), 1 .66-1 .60 (m, 4H), 1 .35-1 .32 (m, 4H), 1 .24 (t, J=.5Hz, 3H).
Ethyl 9-oxononanoate
1H NMR (500 MHz, CDCI3), δ 9.71 (t, J=1 .5Hz, 1 H), 4.07 (q, J=7.5Hz, 2H), 2.38 (td, J=7.5, 1 .5Hz, 2H), 2.24 (t, J=7.5Hz 2H), 1 .60-1 .56 (m, 4H), 1 .30-1 .24 (m, 6H), 1 .21 (t, J=7.5Hz, 3H) Ethyl 13-oxotridecanoate
1H NMR (500 MHz, CDCI3), δ 9.71 (t, J=1 .5Hz, 1 H), 4.10 (q, J=7.0Hz, 2H), 2.38 (td, J=7.5, 1 .5Hz, 2H), 2.26 (t, J=7.5Hz 2H), 1 .61 -1 .56 (m, 4H), 1 .27-1 .24 (m, 14H), 1 .21 (t, J=7.5Hz, 3H) General procedure for synthesis of aldehydes via alcohol intermediates
To a suspension of PCC (6.68 mmol) and Celite (1 .440 g) in anhydrous DCM (20 mL) under nitrogen, was slowly added the alcohol (3.93 mmol) in anhydrous DCM (6 mL). The mixture was stirred for 2 hr under nitrogen, after which diethyl ether (50 mL) was slowly added. The resulting mixture was stirred for 10 min, and then filtered over Celite. The Celite was washed with ether (2 x 20 mL), and the filtrate concentrated in vacuo. The residue was purified on silica gel by stepwise gradient elution with dichloromethane/hexane (40:60 to 100:0).
Ethyl 11-oxoundecanoate
1H NMR (500 MHz, CDCI3), δ 9.71 (t, J=2.0Hz, 1 H), 4.10 (t, J=7.5Hz, 2H), 2.36 (td, J=7.5, 1 .5Hz, 2H), 2.28 (t, J=7.5Hz 2H), 1 .66-1 .58 (m, 4H), 1 .34-1 .29 (m, 10H), 1 .25 (t, J=7.5Hz, 3H)
Ethyl 12-oxododecanoate
1H NMR (500 MHz, CDCI3), δ 9.66 (t, J=1 .5Hz, 1 H), 4.04 (t, J=7.5Hz, 2H), 2.32 (td, J=7.5, 1 .5Hz, 2H), 2.19 (t, J=7.5Hz 2H), 1 .54-1 .50 (m, 4H), 1 .20-1 .70 (m, 12H), 1 .15 (t, J=7.5Hz, 3H)
General procedure for the synthesis of triphenylphosphouium bromides
A solution of triphenylphosphine (12.0 mmol) and the bromo-alkane (12 mmol) in deoxygenated anhydrous toluene (30ml) was refluxed for 3 days under a nitrogen atmosphere. The solvent was removed under educed pressure, and the resulting oil was triturated with n-hexane until a solid product was produced. The product was then isolated as a white solid by filtration. Triphenyl(octyl)phosphounium bromide
1H NMR (500 MHz, CDCI3): δ 7.85-7.80 (m, 6H), 7.79-7.76 (m, 3H), 7.70-7.67 (m, 6H), 3.77-3.75 (m 2H), 1 .61 -1 .59 (m, 4H), 1 .23-1 .17 (m, 8H), 0.82 (t, J=7Hz, 3H)
Triphenyl(nonyl)phosphounium bromide 1H NMR (500 MHz CDCI3): δ 7.86-7.80 (m, 6H), 7.78-7.76 (m, 3H), 7.70-7.66 (m,
6H), 3.79-3.78 (m 2H), 1 .60-1 .59 (m, 4H), 1 .24-1 .17 (m, 10H), 0.83 (t, J=7Hz, 3H)
Triphenyl(decyl)phosphounium bromide
1H NMR (500 MHz, CDCI3): δ 7.82-7.78 (m, 6H), 7.77-7.75 (m, 3H), 7.70-7.66 (m, 6H), 3.72-3.66 (m 2H), 1 .58-1 .57 (m, 4H), 1 .21 -1 .15 (m, 12H), 0.82 (t, J=7Hz, 3H) Triphenyl(undecyl)phosphounium bromide
1H NMR (500 MHz, CDCI3): δ 7.86-7.78 (m, 6H), 7.77-7.71 (m, 3H), 7.70-7.66 (m, 6H), 3.82-3.78 (m 2H), 1 .61 -1 .60 (m, 4H), 1 .27-1 .17 (m, 14H), 0.85 (t, J=7Hz, 3H)
Triphenyl(tridecyl)phosphounium bromide
1H NMR (500 MHz, CDCI3): δ 7.84 (m, 6H), 7.86-7.80 (m, 6H), 7.78-7.71 (m, 3H), 7.70-7.67 (m, 6H) 3.79-7.78 (m 2H), 1 .61 -1 .60 (m, 4H), 1 .28-1 .17 (m, 18H), 0.85 (t, J=7Hz, 3H)
General procedure for the synthesis of MUFA ethyl esters
To a suspension of n-alkyl triphenylphosphonium bromide (7.07 mmol) in anhydrous THF (10 mL) at 0°C under a nitrogen atmosphere, was added NaN(TMS)2 (1 .0 M in THF, 6.43 mmol). The resulting orange mixture was stirred for 40 min at room temperature. The mixture was then cooled to -78°C, and the aldehyde (3.21 mmol) in THF (5 mL) was added dropwise by syringe. Stirring at -78°C was continued for 30 min after which the reaction mixture was allowed to warm to room temperature. After further stirring for 2 hr, the reaction was quenched with saturated aqueous NH4CI (20 mL), and extracted with DCM (3 x 40 mL). The combined extracts were dried over Na2SO4, and the DCM removed in vacuo. The residue was purified on silica gel by stepwise gradient elution with dichloromethane/hexane (20:80 to 50:50). ethyl (132)-octadec-13-enoate
1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 4.1 1 (q, J=7.0Hz, 2H), 2.28 (t, J=7.5Hz, 2H), 2.02-2.01 (m, 4H), 1 .60 (p, J=7.0Hz, 2H), 1 .34-1 .23 (m, 23H), 0.88 (t, J=7.0Hz, 3H) ethyl (122)-octadec-12-enoate
1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 4.1 1 (q, J=7.0Hz, 2H), 2.26 (t, J=7.0Hz, 2H), 2.03-2.02 (m, 4H), 1 .60 (p, J=7.0Hz, 2H), 1 .35-1 .27 (m, 23H), 0.88 (t, J=7.0Hz, 3H) methyl (112)-octadec-11-enoate
1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 2.30 (t, J=7.0Hz, 2H), 2.01 -2.00 (m, 4H), 1 .60 (p, J=7.0Hz, 2H), 1 .31 -1 .28 (m, 23H), 0.88 (t, J=7.0Hz, 3H) ethyl (82)-octadec-8-enoate 1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 4.1 1 (q, J=7.0Hz, 2H), 2.24 (t,
J=7.5Hz, 2H), 2.01 -2.00 (m, 4H), 1 .62 (p, J=7.5Hz, 2H), 1 .32-1 .26 (m, 23H), 0.88 (t, J=7.0Hz, 3H) ethyl (72)-octadec-7-enoate
1H NMR (500 MHz, CDCI3): δ 5.32-5.30 (m, 2H), 4.1 1 (q, J=7.0Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 2.04-2.01 (m, 4H), 1 .61 (p, J=7.0Hz, 2H), 1 .36-1 .26 (m, 23H), 0.88 (t, J=7.0Hz, 3H) ethyl (112)-hexadec-11-enoate
1H NMR (500 MHz, CDCI3): δ 5.36-5.34 (m, 2H), 4.1 1 (q, J=7.0Hz, 2H), 2.34 (t, J=7.5Hz, 2H), 2.03- 2.01 (m, 4H), 1 .62 (p, J=7.0Hz, 2H), 1 .34-1 .23 (m, 19H), 0.88 (t, J=7.0Hz, 3H) methyl (10Z)-hexadec-10-enoate
1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 3.66 (s, 3H), 2.30 (t, J=7.0Hz, 2H), 2.03-2.00 (m, 4H), 1 .60 (p, J=7.0Hz, 2H), 1 .34-1 .24 (m, 16H), 0.88 (t, J=7.0Hz, 3H) ethyl (72)-hexadec-7-enoate 1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 4.12 (q, J=7.0Hz, 2H), 2.30 (t,
J=7.5Hz, 2H), 2.04-1 .98 (m, 4H), 1 .62 (p, J=7.5Hz, 2H), 1 .35-1 .23 (m, 19H), 0.88 (t, J=7.0Hz, 3H) ethyl (52)-hexadec-5-enoate
1H NMR (500 MHz, CDCI3): δ 5.40-5.30 (m, 2H), 4.1 1 (q, J=7.0Hz, 2H), 2.30 (t, J=7.5Hz, 2H), 2.09-2.00 (m, 4H), 1 .66 (p, J=7.5Hz, 2H), 1 .34-1 .22 (m, 19H), 0.88 (t, J=7.0Hz, 3H) methyl (4Z)-hexadec-4-enoate
1H NMR (500 MHz, CDCI3): δ 5.36-5.30 (m, 2H), 3.64 (s, 3H), 2.34-2.31 (m, 4H), 2.02 (q, J=7.0Hz, 2H), 1 .34-1 .22 (m, 18H), 0.88 (t, J=7.0Hz, 3H) General procedure for the synthesis of MUFAs
To a solution of the fatty acid ethyl ester (1 .66 mmol) in ethanol (25 mL) was added 1 .5M NaOH (12 mL). The mixture was heated to 40°C for 5 min to obtain a clear solution. Stirring was continued at room temperature for 2 hr. The volume of the reaction mixture was reduced to 20 mL by rotary evaporation, and the solution was adjusted to pH 3 with 1 .0M HCI. The product was extracted with (3x25ml) dichloromethane, then dried over Na2SO4, filtered, and solvent was removed in vacuo.
(13Z)-octadec-13-enoic acid
1H NMR (500 MHz, CDCI3): δ 5.36-5.34 (m, 2H), 2.35 (t, J=7.5Hz, 2H), 1 .99-2.06 (m, 4H), 1 .64 (p, J=7.0Hz, 2H), 1 .34-1 .27 (m, 20H), 0.89 (t, J=7.0Hz, 3H) (12Z)-octadec-12-enoic acid
1H NMR (500 MHz, CDCI3): δ 5.36-5.34 (m, 2H), 2.35 (t, J=7.5Hz, 2H), 1 .99-2.04 (m, 4H), 1 .64 (p, J=7.0Hz, 2H), 1 .34-1 .27 (m, 20H), 0.89 (t, J=7.0Hz, 3H)
(11 Z)-octadec-11 -enoic acid 1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 2.30 (t, J=7.5Hz, 2H), 1 .99- 2.04
(m, 4H), 1 .60 (p, J=7.0Hz, 2H), 1 .31 -1 .28 (m, 20H), 0.88 (t, J=7.0Hz, 3H)
(8Z)-octadec-8-enoic acid
1H NMR (500 MHz, CDCI3): δ 5.35-5.34 (m, 2H), 2.35 (t, J=7.5Hz, 2H), 1 .98-2.04 (m, 4H), 1 .62 (p, J=7.0Hz, 2H), 1 .32-1 .26 (m, 20H), 0.89 (t, J=7.0Hz, 3H) (7Z)-octadec-7-enoic acid
1H NMR (500 MHz, CDCI3): δ 5.36-5.34 (m, 2H), 2.35 (t, J=7.5Hz, 2H), 1 .98-2.04 (m, 4H), 1 .64 (p, J=7.0Hz, 2H), 1 .34-1 .27 (m, 20H), 0.89 (t, J=7.0Hz, 3H)
(11 Z)-hexadec-11 -enoic acid
1H NMR (500 MHz, CDCI3): δ 5.36-5.34 (m, 2H), 2.35 (t, J=7.5Hz, 2H), 2.03- 2.01 (m, 4H), 1 .64 (p, J=7.0Hz, 2H), 1 .34-1 .27(m, 16H), 0.89 (t, J=7.0Hz, 3H)
(10Z)-hexadec-10-enoic acid
1H NMR (500 MHz, CDCI3): δ 5.36-5.34 (m, 2H), 2.35 (t, J=7.5Hz, 2H), 2.03-2.01 (m, 4H), 1 .63 (p, J=7.0Hz, 2H), 1 .35-1 .25 (m, 16H), 0.89 (t, J=7.0Hz, 3H)
(7Z)-hexadec-7-enoic acid 1H NMR (500 MHz, CDCI3): δ 5.35-5.33 (m, 2H), 2.30 (t, J=7.5Hz, 2H), 2.03-2.01
(m, 4H), 1 .60 (p, J=7.0Hz, 2H), 1 .30-1 .23 (m, 16H), 0.88 (t, J=7.0Hz, 3H) (5Z)-hexadec-5-enoic acid
1H NMR (500 MHz, CDCI3): δ 5.34-5.30 (m, 2H), 2.36(t, J=7.5Hz, 2H), 2.01 -1 .98 (m, 4H), 1 .70 (p, J=7.5Hz, 2H), 1 .33-1 .26 (m, 16H), 0.88 (t, J=7.0Hz, 3H)
(4Z)-hexadec-4-enoic acid 1H NMR (500 MHz, CDCI3): δ 5.40-5.34 (m, 2H), 2.35-2.40 (m, 4H), 2.05 (q,
J=7.0Hz, 2H), 1 .26-1 .07 (m, 18H), 0.88 (t, J=7.0Hz, 3H)
General procedure for synthesis of 2a - 7a, 9a - 12a, 17a - 22a, 24a
To a solution of fatty acid (2.0 mmol) in anhydrous DMF (10 imL) was added hydroxybenzotriazole hydrate (2.40 mmol), and EDCI (2.80 mmol). The mixture was stirred at room temperature for 1 h, then glycine (1 .0 mmol) and triethylamine (0.607 g, 6.0 mmol) were added. The reaction mixture was stirred for 18 h, then diluted with water (50 mL). The crude product was extrated with ethyl acetate (3 x 25 imL) and purified on silica gel by stepwise gradient elution with chloroform/isopropanol (100:0 to 90:10), yielding the amides as white solids. ethyl 2-[[(Z)-octadec-15-enoyl]amino]acetate (2a)
1H NMR (500 MHz, CDCI3) δ 5.92 (s, 1 H), 5.40-5.30 (m, 2H), 4.22 (q, J=7.0Hz, 2H), 4.04 (d, J=5.5Hz, 2H), 2.24 (t, J=7.5Hz, 2H), 2.08-1 .95 (m, 4H), 1 .65 (p, J=7.0Hz, 2H), 1 .35-1 .20 (m, 23H), 0.96 (t, J=7.5Hz, 3H) ethyl 2-[[(Z)-octadec-13-enoyl]amino]acetate (3a) 1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.06
(q, J=7.0Hz, 2H), 3.76 (d, J=6.0Hz, 2H), 2.10 (t, J=7.5Hz, 2H),1 .99-1 .95 (m, 4H), 1 .46 (p, J=7.0Hz, 2H), 1 .30-1 .23(m, 20H), 1 .17(t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-octadec-12-enoyl]amino]acetate (4a)
1H NMR (500 MHz, DMSO-d6) δ 8.19 (t, J=6.0Hz, 1 H), 5.35-5.33 (m, 2H), 4.21 (q, J=7.0Hz, 2H), 3.76 (d, J=5.5Hz, 2H, 2H), 2.20 (t, J=7.5Hz, 2H), 2.04-1 .99 (m, 4H), 1 .46 (p, J=7.0Hz, 2H), 1 .30-1 .23 (m, 20H), 1 .17 (t, J=7.0Hz, 3H), 0.88 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-octadec-11-enoyl]amino]acetate (5a)
1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 3.76 (d, J=6.0Hz, 2H, 2H), 2.10(t, J=7.5Hz, 2H), 2.00-1 .95(m, 4H),
1 .46 (p, J=7.0Hz, 2H), 1 .30-1 .23(m, 20H), 1 .17(t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(E)-octadec-11-enoyl]amino]acetate (6a)
1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1 H), 5.35-5.30 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 3.76 (d, J=6.0Hz, 2H, 2H), 2.09 (t, J=7.5Hz, 2H), 1 .98-1 .88(m, 4H),
1 .47 (p, J=7.0Hz, 2H), 1 .32-1 .20 (m, 20H), 1 .17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-octadec-10-enoyl]amino]acetate (7a) 1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.05
(q, J=7.0Hz, 2H), 3.76 (d, J=6.0Hz, 2H, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1 .95 (m, 4H), 1 .47 (p, J=7.5Hz, 2H), 1 .30-1 .23 (m, 20H), 1 .17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(E)-octadec-9-enoyl]amino]acetate (9a)
1H NMR (500 MHz, DMSO-d6) δ 8.18(t, J=6.0Hz, 1 H), 5.36-5.34 (m, 2H), 4.06(q, J=7.0Hz, 2H), 3.76(d, J=6.0Hz, 2H, 2H), 2.09(t, J=7.5Hz, 2H), 1 .94- 1 .91 (m, 4H), 1 .47 (p, J=7.0Hz, 2H), 1 .30-1 .23(m, 20H), 1 .17(t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-octadec-8-enoyl]amino]acetate (10a)
1H NMR (500 MHz, DMSO-d6) δ 8.19 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.07 (q, J=7.0Hz, 2H), 3.77 (d, J=6.0Hz, 2H, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1 .95 (m, 4H), 1 .47 (p, J=7.5Hz, 2H), 1 .28-1 .20 (m, 20H), 1 .17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-octadec-7-enoyl]amino]acetate (11a)
1H NMR (500 MHz, DMSO-d6) δ 8.20 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.06 (q, J=7.5Hz, 2H), 3.76 (d, J=6.0Hz, 2H, 2H), 2.10 (t, J=7.0Hz, 2H), 1 .99-1 .95 (m, 4H), 1 .48 (p, J=7.0Hz, 2H), 1 .31 -1 .23 (m, 20H), 1 .17 (t, J=7.0Hz, 3H), 0.88 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-octadec-6-enoyl]amino]acetate (12a)
1H NMR (500 MHz, DMSO-d6) δ 8.19 (t, J=6.0Hz, 1H), 5.35-5.33 (m, 2H), 4.06 (q, J=7.5Hz, 2H), 3.76 (d, J=6.0Hz, 2H, 2H), 2.10 (t, J=7.0Hz, 2H), 1.99-1.95 (m, 4H), 1.48 (p, J=7.0Hz, 2H), 1.30-1.22 (m, 20H), 1.17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-hexadec-11-enoyl]amino]acetate (17a)
1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1H), 5.33-5.31 (m, 2H), 4.07 (q, J-7.0HZ, 2H), 3.76 (d, J-6.0Hz, 2H), 2.09 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.46 (p, J=7.0Hz, 2H), 1.28-1.23 (m, 16H), 1.16 (t, J=7.0Hz, 3H), 0.88 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-hexadec-10-enoyl]amino]acetate (18a) 1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1H), 5.35-5.33 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 3.76 (d, J=5.5Hz, 2H), 2.09 (t, J=7.5Hz, 2H), 1.99-1.95 (m, 4H), 1.46 (p, J=7.0Hz, 2H), 1.30-1.23 (m, 16H), 1.17(t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-hexadec-9-enoyl]amino]acetate (19a)
1H NMR (500 MHz, DMSO-d6) δ 8.15 (t, J=6.0Hz, 1H), 5.29-5.27 (m, 2H), 4.05 (q, J=7.0Hz, 2H), 3.74 (d, J=5.5Hz, 2H), 2.07 (t, J=7.5Hz, 2H), 1.96-1.92 (m, 4H), 1.45 (p, J=7.0Hz, 2H), 1.27-1.21 (m, 16H), 1.15 (t, J=7.0Hz, 3H), 0.82 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-hexadec-7-enoyl]amino]acetate (20a)
1H NMR (500 MHz, DMSO-d6) δ 8.18 (t, J=6.0Hz, 1H), 5.32-5.30 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 3.77 (d, J=6.0Hz, 2H), 2.09 (t, J=7.5Hz, 2H), 1.97-1.95 (m, 4H), 1.48 (p, J=7.5Hz, 2H), 1.30-1.23 (m, 16H), 1.17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-hexadec-5-enoyl]amino]acetate (21a)
1H NMR (500 MHz, DMSO-d6) δ 8.20 (t, J=5.5Hz, 1H), 5.34-5.30 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 3.77 (d, J=6.0Hz, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1.95 (m, 4H), 1.52 (p, J=7.0Hz, 2H), 1.28-1.23 (m, 16H), 1.17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-hexadec-4-enoyl]amino]acetate (22a)
1H NMR (500 MHz, DMSO-d6) δ 8.23 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.06 (q, J=7.0Hz, 2H), 3.77 (d, J=6.0Hz, 2H), 2.20 (t, J=7.0Hz, 2H), 2.15-2.12 (m, 2H), 1 .99- 1 .97 (m, 2H), 1 .29-1 .23(m, 18H), 1 .17 (t, J=7.0Hz, 3H), 0.84 (t, J=7.0Hz, 3H) ethyl 2-[[(Z)-tetradec-9-enoyl]amino]acetate (24a)
1H NMR (500 MHz, DMSO-d6): δ 8.19 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.07 (q, J=7.0Hz, 2H), 3.77 (d, J=6.0Hz, 2H), 2.10 (t, J=7.5Hz, 2H), 2.05-1 .90 (m, 4H), 1 .47 (p, J=7.5Hz,2H), 1 .30-1 .20 (m, 12H), 1 .17 (t, J=7.0Hz, 2H), 0.85 (t, J=7.0Hz, 3H)
General procedure for synthesis of 2 - 7, 9 - 12, 16 - 22, 24 To a solution of the ethyl ester (0.51 mmol) in ethanol (30 mL), was added 1 M
NaOH (10 mL). The solution was stirred at 40°C for 3 h. The ethanol was removed under reduced pressure, and the aqueous residue was adjusted to pH 2 with 0.5M HCI. The resulting suspension was filtered and the solid product washed with water (10 mL) and ethanol (5 mL). 2-[[(Z)-octadec-15-enoyl]amino]acetic acid (2)
1H NMR (500 MHz, CDCI3) δ 6.02 (s, 1 H), 5.40-5.30 (m, 2H), 4.09 (d, J=5.5Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 2.10-1 .95 (m, 4H), 1 .65 (p, J=7.0Hz, 2H), 1 .35-1 .20 (m, 20H), 0.96 (t, J=7.5Hz, 3H)
2-[[(Z)-octadec-13-enoyl]amino]acetic acid (3) 1H NMR (500 MHz, DMSO-d6): δ 8.06 (t, J=5.5Hz, 1 H), 5.32-5.30 (m, 2H), 3.70
(d, J=6.0HZ, 2H), 2.09 (t, J=7.5Hz, 2H), 1 .98-1 .95 (m, 4H), 1 .47 (p, J=7.0Hz, 2H), 1 .31 - 1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-octadec-12-enoyl]amino]acetic acid (4)
1H NMR (500 MHz, DMSO-d6): δ 7.90 (t, J=5.5Hz, 1 H), 5.32-5.30 (m, 2H), 3.61 (d, J=5.5HZ, 2H), 2.08(t, J=7.5Hz, 2H), 1 .99-1 .95(m, 4H), 1 .46 (p, J=7.0Hz, 2H), 1 .30-1 .22 (m, 20H), 0.84 (t, J=7.0Hz, 3H) 2-[[(Z)-octadec-11-enoyl]amino]acetic acid (5)
1H NMR (500 MHz, DMSO-d6): δ 8.00 (t, J=5.5Hz, 1 H), 5.32-5.30 (m, 2H), 3.65 (d, J=6.0Hz, 2H), 2.07 (t, J=7.5Hz, 2H), 1 .99-1 .95(m, 4H), 1 .46 (p, J=6.5Hz, 2H), 1 .30- 1 .22(m, 20H), 0.84 (t, J=7.0Hz, 3H) 2-[[(E)-octadec-11-enoyl]amino]acetic acid (6)
1H NMR (500 MHz, DMSO-d6): δ 7.94 (t, J=5.5Hz, 1 H), 5.36-5.34 (m, 2H), 3.65 (d, J=6.0Hz, 2H), 2.08 (t, J=7.0Hz, 2H), 1 .93-1 .92 (m, 4H), 1 .46 (p, J=6.5Hz, 2H), 1 .28- 1 .22(m, 20H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-octadec-10-enoyl]amino]acetic acid (7) 1H NMR (500 MHz, DMSO-d6): δ 8.04 (t, J=5.5Hz, 1 H), 5.32-5.30 (m, 2H), 3.70
(d, J=6.0HZ, 2H), 2.08 (t, J=7.5Hz, 2H), 1 .99-1 .95 (m, 4H), 1 .47 (p, J=7.0Hz, 2H), 1 .30- 1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H)
2-[[(E)-octadec-9-enoyl]amino]acetic acid (9)
1H NMR (500 MHz, DMSO-d6): δ 8.04(t, J=5.5Hz, 1 H), 5.36-5.40 (m, 2H), 3.70 (d, J=6.0Hz, 2H), 2.10 (t, J=7.0Hz, 2H), 1 .94-1 .91 (m, 4H), 1 .48 (p, J=7.0Hz, 2H), 1 .30- 1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-octadec-8-enoyl]amino]acetic acid (10)
1H NMR (500 MHz, DMSO-d6): δ 8.02 (t, J=5.0Hz, 1 H), 5.32-5.30 (m, 2H), 3.70 (d, J=6.0HZ, 2H), 2.09(t, J=7.5Hz, 2H), 1 .98-1 .95 (m, 4H), 1 .46 (p, J=7.0Hz, 2H), 1 .31 - 1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-octadec-7-enoyl]amino]acetic acid (11)
1H NMR (500 MHz, DMSO-d6): δ 8.10 (t, J=5.5Hz, 1 H), 5.32-5.30 (m, 2H), 3.70 (d, J=6.0HZ, 2H), 2.10 (t, J=7.0Hz, 2H), 1 .97 (m, 4H), 1 .48 (p, J=7.0Hz, 2H), 1 .31 -1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H) 2-[[(Z)-octadec-6-enoyl]amino]acetic acid (12)
1H NMR (500 MHz, DMSO-d6): δ 8.04 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 3.70 (d, J=6.0Hz, 2H), 2.10 (t, J=7.0Hz, 2H), 1.98-1.95 (m, 4H), 1.48 (p, J=7.5Hz, 2H), 1.30- 1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H) 2-[[(Z)-hexadec-11 -enoyl]amino]acetic acid (17)
1H NMR (500 MHz, DMSO-d6): δ 8.07 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 3.70 (d, J=5.5Hz, 2H), 2.09 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.46 (p, J=7.0Hz, 2H), 1.31- 1.23(m, 16H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-hexadec-10-enoyl]amino]acetic acid (18) 1H NMR (500 MHz, DMSO-d6): δ 8.02 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H),
3.68(d, J=6.0Hz, 2H), 2.08 (t, J=7.5Hz, 2H), 1.99-1.95 (m, 4H), 1.46 (p, J=7.0Hz, 2H), 1.30-1.22 (m, 16H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-hexadec-9-enoyl]amino]acetic acid (19)
1H NMR (500 MHz, DMSO-d6): δ 8.05 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 3.68 (d, J=6.0HZ, 2H), 2.09 (t, J=7.5Hz, 2H), 1.98-1.96(m, 4H), 1.46 (p, J=6.5Hz, 2H), 1.28- 1.23 (m, 16H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-hexadec-7-enoyl]amino]acetic acid (20)
1H NMR (500 MHz, DMSO-d6): δ 8.02 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 3.67 (d, J=5.5Hz, 2H), 2.09 (t, J=7.5Hz, 2H), 1.97-1.96 (m, 4H), 1.47 (p, J=7.0Hz, 2H), 1.30- 1.23 (m, 16H), 0.84 (t, J=7.0Hz, 3H)
2-[[(Z)-hexadec-5-enoyl]amino]acetic acid (21)
1H NMR (500 MHz, DMSO-d6): δ 8.00 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 3.60 (d, J=5.5Hz, 2H), 2.10(t, J=7.5Hz, 2H), 1.97-1.95 (m, 4H), 1.51 (p, J=7.5Hz, 2H), 1.28- 1.23(m, 16H), 0.84 (t, J=7.0Hz, 3H) 2-[[(Z)-hexadec-4-enoyl]amino]acetic acid (22)
1H NMR (500 MHz, DMSO-d6): δ 7.70 (t, J=5.5Hz, 1 H), 5.32-5.30 (m, 2H), 3.51 (d, J=5.0Hz, 2H), 2.20 (m, 2H), 2.10 (t, J=6.5Hz,2H), 1 .98-1 .95 (m, 2H), 1 .28-1 .23 (m, 18H), 0.84 (t, J=7.0Hz, 3H) 2-[[(Z)-tetradec-9-enoyl]amino]acetic acid (24)
1H NMR (500 MHz, DMSO-d6): δ 8.09 (t, J=6.0Hz, 1 H), 5.32-5.30 (m, 2H), 3.69 (d, J=6.0Hz, 2H), 2.09 (t, J=7.5Hz, 2H), 2.05-1 .90 (m, 4H), 1 .45 (p, J=7.5Hz,2H), 1 .30- 1 .18 (m, 12H), 0.84 (t, J=7.0Hz, 3H)
Figure imgf000064_0001
General procedure for synthesis of 28a - 33a, 35a, 61a, 65a - 66a
To a solution of fatty acid (2.0 mmol) in anhydrous DMF (10 mL) was added hydroxybenzotriazole hydrate (2.40 mmol), and EDCI (2.80 mmol). The mixture was stirred at room temperature for 1 h, then the BOC protected amino acid (1 .0 mmol) and triethylamine (6.0 mmol) were added. The reaction mixture was stirred for 18 h, then diluted with water (50 mL). The crude product was extrated with ethyl acetate (3 x 25 mL) and purified on silica gel by stepwise gradient elution with chloroform/isopropanol (100:0 to 90:10), yielding the desired product in yeilds ranging from 40 - 90 %. methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-9-enoyl]amino] hexanoate (28a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.04 (d, J=7.0Hz, 1H), 5.35-5.33 (m, 2H), 4.62-4.58 (m, 1H), 3.74 (s, 3H), 3.10 (m, 2H), 2.22 (t, J=7.5Hz, 2H), 2.01-1.99 (m, 4H), 1.86-1.80 (m, 1H), 1.72-1.60 (m, 4H), 1.52-1.49 (m, 2H), 1.44-1.42 (m, 9H), 1.37-1.26 (m, 22H), 0.88 (t, J=7.0Hz, 3H). methyl (2R)-6-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-9-enoyl]amino] hexanoate (29a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.05-6.03 (d, J=7.0Hz, 1H), 5.35- 5.33 (m, 2H), 4.60-4.57 (m, 1H), 3.74 (s, 3H), 3.11-3.08 (m, 2H), 2.21 (t, J=7.5Hz, 2H), 2.02-1.98 (m, 4H), 1.86-1.80 (m, 1H), 1.70-1.60 (m, 4H), 1.52-1.45 (m, 2H), 1.44-1.42 (m, 9H), 1.34-1.25 (m, 22H), 0.88 (t, J=7.0Hz, 3H). methyl (2S)-5-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-9-enoyl]amino] pentanoate (30a) White powder.1H NMR (500 MHz, CDCI3): δ 8.13(d, J=8.0Hz, 1H), 6.76-6.75 (m,
1H), 5.32-5.30 (m, 2H), 4.18-4.17 (m. 1H), 3.60 (s, 3H), 2.88 (q, J=6.5Hz, 2H), 2.10- 2.08 (m, 2H), 1.98-1.96 (m, 4H), 1.65-1.62(m, 1H), 1.52-1.44 (m, 3H), 1.41-1.38 (m, 2H), 1.35-1.33 (m, 9H), 1.28-1.22 (m, 20H), 0.84 (t, J=7.0Hz, 3H). methyl-(2S)-4-(tert-butoxycarbonylamino)-2-[[(Z octadec-9-enoyl]amino] butanoate (31a)
White powder. 1H NMR (500 MHz, CDCI3): δ 8.13 (d, J=7.5Hz, 1H), 6.79-6.77 (m,1H), 5.32-5.30 (m, 2H), 4.24-4.20 (m.1H), 3.60 (s, 3H), 2.69-2.90 (m, 2H), 2.09 (t, J=7.0Hz, 2H), 1.97 (q, J=7.0Hz, 4H), 1.85-1.78 (m, 1H), 1.69-1.64 (m, 1H), 1.46 (p, J=7.0Hz, 2H), 1.37-1.35(πι, 9H), 1.30-1.20 (m, 20H), 0.84 (t, J=7.0Hz, 3H). methyl (2S)-3-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-9-enoyl]amino] propanoate (32a)
White powder.1H NMR (500 MHz, CDCI3): δ: 8.02 (d, J=7.5Hz, 1H), 6.85-6.83 (m, 1H), 5.32-5.30 (m, 2H), 4.27-4.25 (q, J=7.0Hz, 1H), 3.58 (s, 3H), 3.24-3.22 (m, 2H), 2.09 (t, J=7.5Hz, 2H), 1.97 (q, J=7.0Hz, 4H), 1.46 (p, J=7.0Hz, 2H), 1.37-1.35(m, 9H), 1.30-1.20 (m, 20H), 0.84 (t, J=7.0Hz, 3H). methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[(ir)-octadec-9-enoyl]amino] hexanoate (33a) White powder.1H NMR (500 MHz, CDCI3): δ 6.04 (br, 1H), 5.35-5.33 (m, 2H),
4.62-4.58 (m, 2H), 3.75 (s, 3H), 3.10 (m, 2H), 2.22 (t, J=7.5Hz, 2H), 2.00-1.90 (m, 4H), 1.86-1.80 (m, 1H), 1.70-1.60 (m, 4H), 1.501.4 (m, 11 H), 1.37-1.26 (m, 22H), 0.86 (t, J=7.0Hz, 3H). methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-13-enoyl]amino] hexanoate (35a)
White powder.1H NMR (500 MHz, DMSO d6): δ 8.10 (d, J=7.5Hz, 1H), 6.74 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 4.19-4.14 (m, 1H), 3.60 (s, 3H), 2.88-2.84 (m, 2H), 2.08 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.66-1.60 (m, 1H), 1.58-1.51 (m, 1H), 1.47- 1.44 (m, 2H), 1.35 (m, 9H), 1.30-1.22 (m, 24H), 0.85 (t J=7.0Hz, 3H). methyl (2R)-6-(tert-butoxycarbonylamino)-2-[[(2)-hexadec-13- enoyl]amino]hexanoate (61a)
White powder.1H NMR (500 MHz, CDCI3): 6.09-6.07 (m, 1H), 5.34-5.30 (m, 2H), 4.60-4.58 (m, 2H), 3.72 (s, 3H), 3.10-3.07 (m, 2H), 2.20 (t, J=7.5Hz, 2H), 2.05-1.98 (m, 4H), 1.83-1.79 (m, 1H), 1.68-1.64 (m, 1H), 1.63-1.60 (m, 2H), 1.41 (m, 9H), 1.37-1.24 (m, 20H), 0.93 (t, J=7.0Hz, 3H). methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[(2)-hexadec-13-enoyl]amino ]hexanoate (65a)
White powder.1H NMR (500 MHz, CDCI3): δ 8.09 (d, J=7.5Hz, 1H), 6.75 (m, 1H), 5.33-5.26 (m, 2H), 4.17-4.16 (m, 1H) 3.59 (s, 3H), 2.88-2.84 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1.96 (m, 4H), 1.70-1.65 (m, 1H), 1.56-1.52 (m, 1H), 1.47-1.44 (m, 2H), 1.35(m, 9H), 1.30-1.23 (m, 20H), 0.90 (t, J=7.0Hz, 3H). methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[(2)-hexadec-9-enoyl]amino] hexanoate. (66a)
White powder.1H NMR (500 MHz, CDCI3): δ 7.94 (d, J=7.5Hz, 1H), 673 (m, 1H)), 5.34-5.26 (m, 2H), 4.12-4.10 (m, 1H), 2.88-2.84 (m, 2H), 2.11-2.06 (m, 2H), 1.99-1.95 (m, 4H), 1.65-1.62 (m, 1 H), 1.54-1.50 (m, 1 H), 1.48-1.44 (m, 2H), 1.35(m, 9H) 1.27-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).
General procedure for synthesis of 28b - 33b, 35b, 61b, 65b - 66b
To a solution of 28a - 33a, 35a, 61a, 65a - 66a (0.50 mmol) in ethanol (30 mL) was added 1M NaOH (10 mL). The solution was stirred at 40°C for 3 h. The ethanol was removed under reduced pressure, and the aqueous residue was adjusted to pH 2 with 0.5M HCI. The resulting suspension was filtered and the solid product washed with water (10 mL) and ethanol (5 mL), yielding 28b - 33b, 35b, 61b, 65b - 66b in yields ranging from 60 - 95%.
(2S)-6-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-9-enoyl]amino]hexanoic acid (28b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.95 (d, J=7.5Hz, 1H), 6.74 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 4.12-4.11 (m, 1H), 2.88-2.84 (m, 2H), 2.10-2.08 (m, 2H), 1.98-1.96 (m, 6H), 1.65-1.62 (m, 1H), 1.54-1.51 (m, 1H), 1.49-1.44 (m, 2H), 1.35- 1.33 (m, 9H), 1.28-1.22 (m, 22H), 0.84 (t, J=7.0Hz, 3H). (2R)-6-(tert-butoxycarbonylamino)-2-[[(Z>octadec-9-enoyl]amino]hexanoic acid (29b)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 7.95(d, J=8.0Hz, 1H), 6.73 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 4.13-4.10 (m. 1H), 2.87-2.84 (m, 2H), 2.10-2.06 (m, 2H), 1.98-1.96 (m, 5H), 1.68-1.60 (m, 1H), 1.54-1.51 (m, 1H), 1.49-1.44 (m, 2H), 1.35- 1.33 (m, 9H), 1.28-1.22 (m, 23H), 0.84 (t, J=7.0Hz, 3H). (2S)-5-(tert-butoxycarbonylamino)-2-[[(2)-octadec-9-enoyl]amino]pentanoic acid (30b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.78 (d, J=8.0Hz, 1H), 678-6.77 (m,1H), 5.32-5.30 (m, 2H), 4.14-4.11 (m.1H), 2.88 (q, J=6.5Hz, 2H), 2.10-2.08 (m, 2H), 1.98-1.96 (m, 5H), 1.65-1.62 (m, 1H), 1.51-1.38 (m, 4H), 1.35-1.33 (m, 9H), 1.28-1.22 (m, 20H), 0.84 (t, J=7.0Hz, 3H).
(2S)-4-(tert-butoxycarbonylamino)-2-[[(2)-octadec-9-enoyl]amino]butanoic acid (31b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.00 (d, J=8.0Hz, 1H), 6.76-6.74 (m, 1H), 5.32-5.30 (m, 2H), 4.17-4.14 (m.1H), 2.99-2.88 (m, 2H), 2.09 (t, J=7.0Hz, 2H), 1.98-1.96 (m, 4H), 1.84-1.79 (m, 1H), 1.66-1.60 (m, 1H), 1.48-1.45 (m, 2H), 1.38-1.34 (m, 9H), 1.28-1.22 (m, 20H), 0.84 (t, J=7.0Hz, 3H).
(2S)-3-(tert-butoxycarbonylamino)-2-[[(2)-octadec-9-enoyl]amino]propanoic acid (32b) White powder.1H NMR (500 MHz, DMSO-d6): δ 7.86 (d, J=8.0Hz, 1 H), 6.75-6.73
(m,1H), 5.32-5.30 (m, 2H), 4.24-4.20 (m, 1H), 3.24-3.22 (m, 2H), 2.09 (t, J=7.0Hz, 2H), 1.97 (q, J=7.0Hz, 4H), 1.48-1.45 (m, 2H), 1.38-1.34 (m, 9H), 1.28-1.22 (m, 20H), 0.84 (t, J=7.0Hz, 3H).
(2S)-6-(tert-butoxycarbonylamino)-2-[[(E>octadec-9-enoyl]amino]hexanoic acid (33b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.06 (d, J=8.0Hz, 1H), 7.96 (br, 3H), 5.35 (m, 2H), 4.12 (m, 1H), 2.72 (br, 2H), δ 2.098 (t, J=7.5Hz, 2H), 1.98- 1.80 (m, 4H), 1.48-1.45 (m, 2H), 1.38-1.34 (m, 9H), 1.28-1.22 (m, 20H), 0.82 (t, J=7.0Hz, 3H).
(2S)-6-(tert-butoxycarbonylamino)-2-[[(Z)-octadec-13-enoyl]amino]hexanoic acid (35b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.95 (d, J=7.5Hz, 1H), 6.74 (t, J=5.5Hz, 1H), 5.32-5.30 (m, 2H), 4.11-4.10 (m, 1H), 2.88-1.84 (m, 2H), 2.10-2.08 (m, 2H), 1.98-1.96 (m, 4H), 1.68-1.62(m, 1H), 1.54-1.51 (m, 1H), 1.49-1.44 (m, 2H), 1.35- 1.33 (m, 9H), 1.28-1.22 (m, 24H), 0.84 (t, J=7.0Hz, 3H).
(2R)-6-(tert-butoxycarbonylamino)-2-[[(Z)-hexadec-13-enoyl]amino]hexanoic acid (61b) White powder.1H NMR (500 MHz, DMSO-d6): δ 7.94 (d, J=7.5Hz, 1H), 674 (m,
1 H)), 5.34-5.26 (m, 2H), 4.11 -4.10 (m, 1 H), 2.88-2.84 (m, 2H), 2.11 -2.06 (m, 2H), 1.99- 1.95 (m, 4H), 1.65-1.62 (m, 1H), 1.54-1.50 (m, 1H), 1.48-1.44 (m, 2H), 1.35(m, 9H) 1.27-1.23 (m, 20H), 0.90 (t, J=7.0Hz, 3H).
(2S)-6-(tert-butoxycarbonylamino)-2-[[(2)-hexadec-13-enoyl]amino]hexanoic acid (65b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.05 (d, J=7.5Hz, 1H), 6.75 (m, 1H) 5.33-5.26 (m, 2H), 4.16-4.12 (m, 1H), 2.88-2.84 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1.96 (m, 4H), 1.70-1.65 (m, 1H), 1.60-1.46 (m, 5H), 1.35(m, 9H), 1.37-1.23 (m, 18H), 0.90 (t, J=7.0Hz, 3H). (2S)-6-(tert-butoxycarbonylamino)-2-[[(2)-hexadec-9-enoyl]amino]hexanoic acid (66b)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.94 (d, J=7.5Hz, 1H), 673 (m, 1 H)), 5.34-5.26 (m, 2H), 4.12-4.10 (m, 1 H), 2.88-2.84 (m, 2H), 2.11 -2.06 (m, 2H), 1.99- 1.95 (m, 4H), 1.65-1.62 (m, 1H), 1.54-1.50 (m, 1H), 1.48-1.44 (m, 2H), 1.35(m, 9H) 1.27-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).
General procedure for synthesis of 28 - 33, 35, 61, 65 - 66
28b - 33b, 35b, 61b, 65b - 66b were dissolved in dichloromethane (2 mL) and 2M HCI in diethyl ether (2 mL) was added. The solution was stirred at room temperature for 4 hours and then concentrated in vacuo. The resulting solid was washed with diethyl ether, yielding 28 - 33, 35, 61 , 65 - 66. (2S)-6-amino-2-[[(Z)-octadec-9-enoyl]amino]hexanoic acid.HCI (28)
Light yellow powder. 1H NMR (500 MHz, DMSO d6): δ 8.05 (d, J=7.5Hz, 1H), 7.90 (s, 3H), 5.32-5.30 (m, 2H), 4.14-4.12 (m, 1H), 2.74-2.71 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 1.98-1.9 (m, 4H), 1.67-1.65 (m, 1 H), 1.57-1.44 (m, 5H), 1.31 -1.23 (m, 22H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.69, 172.31, 129.60(2C), 51.49, 38.38, 34.99, 31.24, 30.33, 29.10, 29.06, 28.79, 28.66(2C), 28.64, 28.59, 28.55, 26.59, 26.54, 26.44, 25.21, 22.40, 22.06, 13.92. HRMS (ESI) m/z [M+H]+ calcd for C24H46N2O3, 411.3581; found, 411.3589.
(2R)-6-amino-2-[[(Z)-octadec-9-enoyl]amino]hexanoic acid.HCI (29) Light yellow powder. 1H NMR (500 MHz, DMSO d6): δ 8.05 (d, J=7.5Hz, 1H),
7.90 (s, 3H), 5.32-5.30 (m, 2H), 4.14-4.12 (m, 1H), 2.72 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.66 (m, 1H), 1.57-1.44 (m, 5H), 1.31-1.23 (m, 22H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.72, 172.36, 129.63(2C), 51.53, 38.42, 35.03, 31.27, 30.33, 29.14, 29.10, 28.83, 28.70(2C), 28.68, 28.63, 28.59, 26.62, 26.58, 26.48, 25.25, 22.44, 22.09, 13.96. HRMS (ESI) m/z [M+H]+ calcd for C24H46N2O3, 411.3581; found, 411.3579.
(2S)-5-amino-2-[[(Z)-octadec-9-enoyl]amino]pentanoic acid.HCI (30)
Light yellow powder. 1H NMR (500 MHz, DMSO d6): δ 8.10 (d, J=8.0Hz, 1H), 7.94 (s, 3H), 5.32-5.30 (m, 2H), 4.17-4.16 (m, 1H), 2.75-2.73 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 1.97 (q, J=6.0Hz, 4H), 1.76-1.74 (m, 1H), 1.60-1.56 (m, 3H), 1.47-1.45 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.44, 171.37, 129.64(2C), 51.23, 38.34, 35.06, 31.28, 29.15, 29.09(2C), 28.82, 28.72(2C), 28.68, 28.58, 28.00, 26.63, 26.57, 25.25, 22.80, 22.09, 13.96. HRMS (ESI) m/z [M+H]+ calcd for C23H44N2O3, 397.3425; found 397.3428. (2S)-4-amino-2-[[(Z)-octadec-9-enoyl]amino]butanoic acid.HCI (31)
Light yellow powder. 1H NMR (500 MHz, DMSO d6): δ 8.22 (d, J=7.5Hz, 1H), 7.97 (s, 3H), 5.32-5.30 (m, 2H), 4.27-4.26 (m, 1H), 2.79-2.77 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 5H), 1.88-1.86 (m, 1H), 1.48-1.46 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100 MHz, DMSO-d6): δ 172.77, 172.54, 129.63(2C), 49.68, 36.15, 35.18, 31.26, 29.14, 29.08, 28.97(2C), 28.82, 28.71, 28.67, 28.62, 28.58, 26.62, 26.56, 25.19, 22.08, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C22H42N2O3, 383.3268; found, 383.3273.
(2S)-3-amino-2-[[(Z)-octadec-9-enoyl]amino]propanoic acid.HCI : (32) Light yellow powder.1H NMR (500 MHz, DMSO-d6): δ 8.31 (d, J=8.0Hz, 1 H), 8.0
(s, 3H), 5.32-5.30 (m, 2H), 4.47-4.44 (m, 1H), 3.22-2.19 (m, 1H), 2.99-2.97 (m, 1H), 2.15-2.13 (m, 2H), 1.97(q, J=6.0Hz, 4H), 1.49-1.47 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.92, 170.81, 129.63(2C), 49.82, 35.18, 31.26, 29.14, 29.08(2C), 28.82(2C), 28.74, 28.67, 28.62, 28.58, 26.62, 26.56, 24.88, 22.08, 13.95. HRMS (ESI) m/z [M+H]+ calcd for C2iH4oN2O3, 369.3112; found, 369.3109.
(2S)-6-amino-2-[[(E)-octadec-9-enoyl]amino]hexanoic acid.HCI (33)
1H NMR (500MHz, DMSO-d6): δ 8.06 (d, J=7.5Hz,1H), 7.96 (br, 3H), 5.35-5.30 (m, 2H), 4.16 - 4.12 (m, 1H), 2.72 (br, 2H), 2.09 (t, 2H, J=7.5Hz), 1.92 (m, 4Hz), 1.66 (m, 1H), 1.56- 1.46 (m, 5H), 1.33 - 1.21 (m, 22H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100MHz, CDCI3): δ 174.16, 172.80, 130.50 (2C), 51.99, 38.84, 35.47, 32.40, 31.72, 30.81, 29.12, 29.03, 28.77, 28.65(2C), 28.63, 28.60, 28.56, 26.92, 25.69, 22.89, 22.54, 14.40. HRMS (ESI) mlz [M + H]+ calcd for C24H46N2O3, 411.3581 ; found, 411.3597.
(2S)-6-amino-2-[[(Z)-octadec-13-enoyl]amino]hexanoic acid.HCI (35) Light yellow powder. 1H NMR (500 MHz, DMSO-d6): δ 8.04 (d, J=7.5Hz, 1H),
7.89 (s, 3H), 5.32-5.30 (m, 2H), 4.15-4.11 (m.1H), 2.72 (m, 2H), 2.09 (t, J=7.0Hz, 2H), 1.97 (m, 4H), 1.67-1.65 (m, 1H), 1.58-1.44 (m, 5H), 1.31-1.23 (m, 22H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100 MHz, DMSO-d6): δ 173.71, 172.35, 129.62, 129.58, 51.49, 38.43, 35.03, 31.36, 30.37, 29.09, 29.01, 28.97, 28.86(2C), 28.82, 28.64, 28.58, 28.56, 26.47, 26.31 , 25.25, 22.44, 21.71 , 13.81. HRMS (ESI) m/z [M+H]+ calcd for C24H46N2O3, 411.3581 ;found,411.3589. (2R)-6-amino-2-[[(Z)-hexadec-13-enoyl]amino]hexanoic acid.HCI (61 )
Light yellow powder. 1 H NMR (500 MHz, DMSO-d6): δ 8.03 (d, J=8.0Hz, 1 H), 7.82 (s, 3H), 5.33-5.27 (m, 2H), 4.15-4.12 (m, 1 H), 2.75-2.71 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1 .96 (m, 4H), 1 .68-1 .65 (m, 1 H), 1 .58-1 .45 (m, 5H), 1 .37-1 .23 (m, 18H), 0.90 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.73, 172.35, 131 .27, 129.01 , 51 .49, 38.48, 35.04, 30.36, 29.1 1 , 29.01 (2C), 28.96(2C), 28.88, 28.81 , 28.62, 28.59, 26.47, 25.24, 22.42, 20.00, 14.24 HRMS (ESI) m/z [M+H]+ calcd for C22H42N2O3, 383.3268; found, 383. 3263.
(2S)-6-amino-2-[[(Z)-hexadec-13-enoyl]amino]hexanoic acid.HCI (65) Light yellow powder. 1 H NMR (500 MHz, DMSO-d6): δ 8.05 (d, J=7.5Hz, 1 H),
7.90 (s, 3H), 5.34-5.26 (m, 2H), 4.16-4.12 (m, 1 H), 2.75-2.71 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 2.00-1 .96 (m, 4H), 1 .70-1 .65 (m, 1 H), 1 .60-1 .46 (m, 5H), 1 .37-1 .23 (m, 18H), 0.90 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.73, 172.35, 131 .27, 129.01 , 51 .49, 36.66, 35.03, 30.36, 29.1 1 , 29.01 (2C), 28.96(2C), 28.88, 28.81 , 28.62, 28.59, 26.47, 25.24, 22.42, 20.00, 14.24. HRMS (ESI) m/z [M+H]+ calcd for C22H42N2O3, 383.3268; found, 383. 3268.
(2S)-6-amino-2-[[(Z)-hexadec-9-enoyl]amino]hexanoic acid.HCI (66)
Light yellow powder. 1 H NMR (500 MHz, DMSO-d6): δ 8.04 (d, J=7.5Hz, 1 H), 7.90 (s, 3H), 5.32-5.30 (m, 2H), 4.13-4.12 (m, 1 H), 2.74-2.71 (m, 2H), 2.09 (t, J=8.0Hz, 2H), 1 .99-1 .95 (m, 4H), 1 .68-1 .65 (m, 1 H), 1 .56-1 .45 (m, 5H), 1 .32-1 .23 (m, 18H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.73, 172.35, 129.65(2C), 51 .51 , 38.42, 35.06, 31 .12, 30.37, 29.12(2C), 29.08, 28.67, 28.61 , 28.56, 28.26, 26.60, 26.47, 25.24, 22.43, 22.07, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C22H42N2O3, 383.3268; found, 383. 3266. Synthesis of 36
28a was dissolved in dichloromethane (2 imL) and 2M HCI in diethyl ether (2 imL) was added. The solution was stirred at room temperature for 4 hours and then concentrated in vacuo. The resulting solid was washed with diethyl ether, yielding 36. methyl (2S)-6-amino-2-[[(Z)-octadec-9-enoyl]amino]hexanoate.HCI (36)
Wax, 1H NMR (500 MHz, DMSO d6): δ 8.20-8.18 (d, J=7.0Hz, 1 H), 7.86 (s, 3H), 5.32-5.30 (m, 2H), 4.19-4.17 (m, 1 H), 3.60 (s, 3H), 2.75-2.71 (m, 2H), 2.10 (t, J=7.5Hz, 2H), 1 .99-1 .95 (m, 4H), 1 .68-1 .50 (m, 4H), 1 .48-1 .45 (m, 2H), 1 . 28-1 .23 (m, 22H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz DMSO-d6): δ 172.71 , 172.47, 129.64, 129.62, 51 .73, 51 .63, 38.39, 34.66, 32.26, 30.21 , 29.1 1 , 29.08(2C), 28.81 (2C), 28.66(2C), 28.56, 26.60, 26.56, 26.43, 25.18, 22.31 , 22.08, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C25H48N2O3, 425.3738; found,425.3740.
Figure imgf000073_0001
General procedure for synthesis of 26a, 27a, 34a, 37a-50a, 53a-60a, 62a-64a
To a solution of oleic acid (2.0 mmol) in anhydrous DMF (10 mL) was added hydroxybenzotriazole hydrate (2.40 mmol), and EDCI (2.80 mmol). The mixture was stirred at room temperature for 1 h, then the substituted amine (1 .0 mmol) and triethylamine (6.0 mmol) were added. The reaction mixture was stirred for 18 h, then diluted with water (50 mL). The crude product was extrated with ethyl acetate (3 x 25 mL) and purified on silica gel by stepwise gradient elution with chloroform/isopropanol (100:0 to 90:10), yielding the desired products in yields from 40 - 85%. methyl (2S)-3-hydroxy-2-[[(Z>octadec-9-enoyl]amino]propanoate (26a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.40 (d, J=7.5Hz, 1H), 5.35-5.32 (m, 2H), 4.70-4.67 (m, 1H), 3.99-3.96 (dd, J=4.0, 11.0Hz, 1H), 3.93-3.91 (dd, J=3.5, 11.0Hz, 1H), 3.79 (s, 3H), 2.26 (t, J=7.5Hz, 2H), 2.04-1.99 (m, 4H), 1.65 (p, J=7.0Hz, 2H), 1.34-1.24 (m, 20H), 0.88 (t, J=7.0Hz, 3H). methyl (2R)-3-hydroxy-2-[[(Z)-octadec-9-enoyl]amino]propanoate (27a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.36 (d, J=7.5Hz, 1H), 5.35-5.32 (m, 2H), 4.70-4.67 (m, 1H), 3.98-3.93 (m, 2H), 3.79 (s, 3H), 2.46 (t, J=6.0Hz, 3H).2.26 (t, J=7.5Hz, 2H), 2.03-1.99 (m, 4H), 1.65 (p, J=7.0Hz, 2H), 1.31-1.27 (m, 20H), 0.88 (t, J=7.0Hz, 3H). methyl (2S)-4-methyl-2-[[(2)-octadec-9-enoyl]amino]pentanoate (34a)
White powder.1H NMR (500 MHz, CDCI3): δ 5.80 (d, J=8.5Hz, 1H), 5.34-5.32 (m, 2H), 4.66-4.64 (m, 1H), 3.73 (s, 3H), 2.20 (t, J=7.5Hz, 2H), 2.00 (q, J=6.5Hz, 4H), 1.66-1.62 (m, 5H), 1.53-1.50 (m, 1H), 1.30-1.26 (m, 20H), 0.95-0.93 (m, 6H), 0.87 (t, J=7.0Hz, 3H). methyl (2S)-5-guanidino-2-[[(Z)-octadec-9-enoyl]amino]pentanoate (37a)
White powder.1H NMR (500 MHz, CDCI3): δ 7.64(m, 1H), 7.38 (d, J=7.5Hz, 1H), 6.92 (m, 3H), 5.35-5.31 (m, 2H), ), 4.57 (m, 1H), 4.45-4.40 (m, 1H), 3.74 (s, 3H), 3.32- 3.20 (m, 2H), 2.28 (t J=7.5Hz„ 2H),1.99 (q, J=6.0Hz, 4H), 1.90-1.76 (m, 2H), 1.68-1.58 (m, 4H), 1.31 -1.23 (m, 20H), 0.88 (t, J=7.0Hz, 3H). methyl (2S)-4-methylsulfanyl-2-[[(Z)-octadec-9-enoyl] amino] butanoate
(38a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.14 (d, J=7.5Hz, 1H), 5.34-5.32 (m, 2H), 4.74-4.70 (m, 1H), 3.75 (s, 3H), 2.52-2.48 (m, 2H), 2.22 (t, J=7.5Hz, 2H).2.19- 2.14 (m, 1H), 2.08 (s, 3H), 1.99-1.94 (m, 5H), 1.63 (p, J= 7.0 Hz, 2H), 1.32-1.24 (m, 20H), 0.87 (t, J=7.0Hz, 3H). methyl (2S)-4-methyl-2-[[(2)-octadec-9-enoyl]amino]pentanoate (39a)
White powder.1H NMR (500 MHz, CDCI3): δ 5.80 (d, J=8.5Hz, 1H), 5.34-5.32 (m, 2H), 4.66-4.64 (m, 1H), 3.73 (s, 3H), 2.20 (t, J=7.5Hz, 2H), 2.00 (q, J=6.5Hz, 4H), 1.66-1.62 (m, 5H), 1.53-1.50 (m, 1H), 1.30-1.26 (m, 20H), 0.95-0.93 (m, 6H), 0.87 (t, J=7.0Hz, 3H). methyl (2R)-4-methyl-2-[[(Z)-octadec-9-enoyl]amino]pentanoate (40a)
White powder.1H NMR (500 MHz, DMSO-d6): δ 5.80 (d, J=8.5Hz, 1H), 5.34-5.32 (m, 2H), 4.66-4.64 (m, 1H), 3.73 (s, 3H), 2.20 (t, J=7.5Hz, 2H), 2.00 (q, J=6.5Hz, 4H), 1.66-1.62 (m, 5H), 1.53-1.50 (m, 1H), 1.30-1.26(m, 20H), 0.95-0.93(m, 6H), 0.87 (t, J=7.0Hz, 3H). methyl (2S)-2-[[(Z)-octadec-9-enoyl]amino]hexanoate (41a)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.12 (d, J=7.5Hz, 1H), 5.31-5.30 (m, 2H), 4.20-4.17 (m, 1H), 3.59 (s, 3H), 2.12-2.06 (m, 2H), 1.97 (q, J=6.5Hz, 4H), 1.68- 1.62 (m, 1H), 1.60-1.54 (m, 1H), 1.46 (p, J=6.5Hz, 2H), 1.29-1.22 (m, 24H), 0.84 (t, J=7.0Hz, 6H). methyl (2S)-[[(Z)-octadec-9-enoyl]amino]propanoate (42a)
White powder.1H NMR (500 MHz DMSO-d6): δ 8.17 (d, J=6.5Hz, 1H), 5.32-5.30 (m, 2H), 4.24-4.21 (m, 1H), 3.59 (s, 3H), 2.07 (t, J=7.5Hz, 2H), 1.97 (q, J=7.0Hz, 4H), 1.45 (p, J=7.0Hz, 2H), 1.30-1.23 (m, 23H), 0.84 (t, J=7.0Hz, 3H). methyl (2R)-2-[[(2)-octadec-9-enoyl]amino]propanoate (43a)
White powder.1H NMR (500 MHz DMSO-d6): δ 8.17 (d, J=7.0Hz, 1H), 5.32-5.30 (m, 2H), 4.24-4.21 (m, 1H) 3.59 (s, 3H), 2.07 (t, J=7.5Hz, 2H), 1.97 (q, J=7.0Hz, 4H), 1.45 (p, J=7.5Hz, 2H), 1.30- 1.23 (m, 23H), 0.84 (t, J=7.0Hz, 3H). ethyl 3-[[(Z)-octadec-9-enoyl]amino]propanoate (44a) White powder.1H NMR (500 MHz, CDCI3): δ 6.04 (m, 1H), 5.35-5.32 (m, 2H),
4.17-4.13 (q, J=7.0Hz, 2H) 3.51 (q, J=7.0Hz, 2H), 2.52 (t, J=6.0Hz, 2H), 2.13 (t, J=7.5.0Hz, 2H), 1 .97 (q, J=6.0Hz, 4H), 1 .61 -1 .58 (m, 2H), 1 .31 -1 .23 (m, 23H), 0.88 (t, J=7.0Hz, 3H). methyl (2S)-3-methyl-2-[[(2)-octadec-9-enoyl]amino]butanoate (45a)
White powder. 1H NMR (500 MHz DMSO-d6): δ 8.03 (d, J=8.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.17-4.14 (m, 1 H) 3.61 (s, 3H), 2.18-2.08 (m 2H), 2.02-1 .95 (m, 5H), 1 .48-1 .44 (m, 2H) 1 .28-1 .23 (m, 20H), 0.87 (dt, J=7.0, 9.50 Hz, 9H). methyl (2R)-3-methyl-2-[[(Z)-octadec-9-enoyl]amino]butanoate (46a)
White powder. 1H NMR (500 MHz DMSO-d6): δ 8.03 (d, J=8.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.16-4.13 (m, 1 H) 3.60 (s, 3H), 2.17-2.07 (m 2H), 2.01 - 1 .95 (m, 5H), 1 .47- 1 .44 (m, 2H) 1 .28- 1 .23 (m, 20H), 0.87 (dt, J=7.0, 9.50 Hz, 9H). methyl (2R)-2-[[(Z)-octadec-9-enoyl]amino]-3-phenyl-propanoate (47a)
White powder. 1H NMR (500 MHz, CDCI3): δ 7.32-7.26 (m, 3H), 7.12-7.10 (d, J=7.0Hz, 2H), 5.89-5.87 (d, J=7.5Hz, 1 H), 5.37-5.35 (m, 2H), 4.94-4.91 (m, 1 H), 3.75 (s, 3H), 3.20-3.16 (dd, J=6.0, 14.0Hz, 1 H) 3.13-3.09 (dd, J=5.5, 14.0Hz, 1 H), 2.20 (t, J=7.5Hz, 2H), 2.04 (q, J=6.5Hz, 4.0H), 1 .60 (p, J=7.0Hz, 2H), 1 .30-1 .17 (m, 20H), 0.89 (t, J=7.0Hz, 3H). ethyl (2S)-2-[[(Z)-octadec-9-enoyl]amino]-4-phenyl-butanoate (48a)
White powder. 1 H NMR (500 MHz, DMSO-d6): δ 8.25-8.20 (m, 1 H), 7.30-7.24 (m, 2H), 7.20-7.14 (m, 3H), 5.35-5.25 (m, 2H), 4.18-4.12 (m, 1 H), 4.10-4.00 (m, 2H), 2.66- 2.52 (m, 2H), 2.16-2.08 (m, 2H), 2.00-1 .80 (m, 6H), 1 .49 (d, J=7.0Hz, 2H), 1 .30-1 .20 (m, 20H), 1 .15 (t, J=7.0Hz, 2H), 0.83 (d, J=7.0Hz, 3H). ethyl (2R)-2-[[(Z)-octadec-9-enoyl]amino]-4-phenyl-butanoate (49a)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 8.22 (d, J=7.5Hz, 1 H), 7.28-7.24 (m, 2H), 7.18-7.15 (m, 3H), 5.31 -5.28 (m, 2H), 4.18-4.12 (m, 1 H), 4.10-3.98 (m, 2H), 2.70-2.50 (m, 2H), 2.12 (dp, J=7.0, 4.0Hz, 2H), 2.00-1 .80 (m, 6H), 1 .49 (d, J=7.0Hz, 2H), 1 .32-1 .18 (m, 20H), 1 .15 (t, J=7.0Hz, 2H), 0.83 (d, J=7.0Hz, 3H). methyl (2S)-3-(1H-indol-3-yl)-2-[[(2)-octadec-9-enoyl]amino]propanoate
(50a)
White powder.1H NMR (500 MHz, CDCI3): δ 8.14 (s, 1H), 7.53 (d, J=8.0Hz, 1H), 7.35 (d, J=8.0Hz, 1H), 7.20 (t, J=7.5Hz, 1H), 7.12 (t, J=7.5Hz, 1H), 6.98 (d, J=7.5Hz, 1H), 5.95 (d, J=7.5Hz, 1H), 5.35-5.33 (m, 2H), 4.14-4.10 (m, 1H), 3.70 (s, 3H), 3.33- 3.31 (t, J=5.0Hz, 2H), 2.15-2.12 (t, J=7.5Hz, 2H), 2.02-1.98 (m, 4H), 1.59-1.56 (m, 2H), 1.32-1.25 (m, 20H), 0.88 (t, J=7.0Hz, 3H) dimethyl (2S)-2-[[(Z)-octadec-9-enoyl]amino]butanedioate (53a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.44 (d, J=8.5Hz, 1H), 5.35-5.33 (m, 2H), 4.88-4.86 (m, 1H), 3.79 (s, 3H), 3.72 (s, 3H), 3.09-3.04 (dd, J=4.5, 17.0Hz, 1H), 2.91-2.86 (dd, J=4.5, 17.0Hz, 1H), 2.25 (t, J=7.0Hz, 2H), 2.04 (q, J=7.0Hz, 4H), 1.68 (p, J=7.0Hz, 2H), 1.36-1.30 (m, 20H), 0.90 (t, J=7.0Hz, 3H). dimethyl (2R)-2-[[(Z)-octadec-9-enoyl]amino]butanedioate (54a)
White powder, 1H NMR (500 MHz, CDCI3): δ 6.44 (d, J=8.5Hz, 1H), 5.35-5.33 (m, 2H), 4.88-4.86 (m, 1H), 3.76 (s, 3H), 3.69 (s, 3H), 3.06-3.02 (dd, J=4.5, 17.0Hz, 1H), 2.87-2.83 (dd, J=4.5, 17.0Hz, 1H), 2.22 (t, J=7.5Hz, 2H), 2.0 (q, J=6.5Hz, 4H), 1.63 (p, J=7.0Hz, 2H), 1.34-1.26 (m, 20H), 0.88 (t, J=7.0Hz, 3H). methyl (2S)-4-amino-2-[[(Z)-octadec-9-enoyl]amino]-4-oxo-butanoate (55a)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.10 (d, J=8.0Hz, 1H), 7.35 (s, 1 H), 6.88 (s, 1 H), 5.34-5.28 (m, 2H), 4.55-4.50 (m, 1 H), 3.57 (s, 3H), 2.55-2.40 (m, 2H), 2.06 (t, J=7.5Hz, 2H), 2.00-1.94 (m, 4H), 1.45 (p, J=7.0Hz, 2H), 1.32-1.18 (m, 20H), 0.84 (d, J=7.0Hz, 3H). dimethyl (2S)-2-[[(Z)-octadec-9-enoyl]amino]pentanedioate (56a)
White powder.1H NMR (500 MHz, CDCI3): δ 6.12 (d, J=7.5Hz, 1H), 5.35-5.33 (m, 2H), 4.66-4.61 (m, 1H), 3.75 (s, 3H), 3.68 (s, 3H), 2.45-2.33 (m, 2H), 2.23-2.19 (m, 2H), 2.03-1.98 (m, 5H), 1.63 (p, J=7.0Hz, 2H), 1.30-1.26 (m, 20H), 0.88 (t, J=7.0Hz, 3H). dimethyl (2R)-2-[[(Z)-octadec-9-enoyl]amino]pentanedioate (57a)
White powder. 1H NMR (500 MHz, CDCI3): δ 6.20 (d, J=7.5Hz, 1 H), 5.33-5.31 (m, 2H), 4.63-4.59 (m, 1 H), 3.73 (s, 3H), 3.66 (s, 3H), 2.43-2.31 (m, 2H), 2.22-2.17 (m, 2H), 2.01 -1 .95 (m, 5H), 1 .61 (p, J=7.0Hz, 2H), 1 .30-1 .25 (m, 20H), 0.86 (t, J=7.0Hz, 3H). methyl (2S)-3-(1 H-imidazol-5-yl)-2-[[(Z)-octadec-9-enoyl]amino]propanoate (58a)
White powder. 1H NMR (500 MHz, CDCI3), δ 7.56(s, 1 H), 7.10 (s, 1 H), 6.80 (s, 1 H), 5.35-5.33 (m, 2H), 4.83-4.80 (m, 1 H), 3.60 (s, 3H), 3.08 (m, 2H), 2.23 (t, J=7.5Hz, 2H), 2.01 -1 .98 (m, 2H), 1 .64-1 .60 (m, 4H), 1 .30-1 .26 (m, 20H), 0.88 (t, J=7.0Hz, 3H) methyl (2S)-2-[[(Z)-octadec-9-enoyl]amino]-3-phenyl-propanoate (59a)
White powder. 1H NMR (500 MHz, CDCI3): δ 7.32-7.26 (m, 3H), 7.12-7.10 (d, J=7.0Hz, 2H), 5.89-5.87 (d, J=7.5Hz, 1 H), 5.37-5.35 (m, 2H), 4.94-4.91 (m, 1 H), 3.75 (s, 3H), 3.20-3.16 (dd, J=6.0, 14.0Hz, 1 H) 3.13-3.09 (dd, J=5.5, 14.0Hz, 1 H), 2.20 (t, J=7.5Hz, 2H), 2.04 (q, J=6.5Hz, 4.0H), 1 .60 (p, J=7.0Hz, 2H), 1 .30-1 .17 (m, 20H), 0.89 (t, J=7.0Hz, 3H). methyl (2R)-4-methylsulfanyl-2-[[(Z)-octadec-9-enoyl] amino] butanoate
(60a) White powder. 1 H NMR (500 MHz, CDCI3): δ 6.12 (d, J=7.5Hz, 1 H), 5.34-5.32
(m, 2H), 4.74-4.70 (m, 1 H), 3.75 (s, 3H), 2.52-2.48 (m, 2H), 2.22 (t, J=7.5Hz, 2H). 2.19- 2.14 (m, 1 H), 2.08 (s, 3H), 1 .99-1 .94 (m, 5H), 1 .63 (p, J= 7.0 Hz, 2H), 1 .32-1 .24 (m, 20H), 0.87 (t, J=7.0Hz, 3H). methyl (2R)-5-guanidino-2-[[(Z)-octadec-9-enoyl]amino]pentanoate (62a) White powder. 1H NMR (500 MHz, CDCI3): δ 7.83 (m, 1 H), 7.20 (d, J=7.5Hz, 1 H),
7.05-7.03 (m, 3H), 5.34-5.32(m, 2H), 4.45-4.40 (m, 1 H), 3.74 (s, 3H), 3.36-3.20 (m, 2H), 2.28 (t J=7.5Hz, 2H), 1 .99 (q, J=6.0Hz, 4H), 1 .90-1 .76 (m, 2H), 1 .68-1 .58 (m, 4H), 1 .31 -1 .23 (m, 20H), 0.88 (t, J=7.0Hz, 3H). methyl (2R)-3-(1H-indol-3-yl)-2-[[(2)-octadec-9-enoyl]amino]propanoate
(63a)
White powder.1H NMR (500 MHz, DMSO-d6): δ 10.84 (s, 1H), 8.18 (d, J=8.0Hz, 1H), 7.47 (d, J=8.0Hz, 1H), 7.31 (t, J=7.5Hz, 1H), 7.11 (t, J=7.5Hz, 1H), 7.03 (d, J=7.5Hz, 1H), 6.96 (d, J=7.5Hz, 1H), 5.35-5.33 (m, 2H), 4.49-4.47 (m, 1H), 3.70 (s, 3H), 3.33-3.31 (t, J=5.0Hz, 2H), 2.15-2.12 (t, J=7.5Hz, 2H), 2.02-1.98 (m, 4H), 1.59-1.56 (m, 2H), 1.32-1.25 (m, 20H), 0.88 (t, J=7.0Hz, 3H). methyl (2R)-3-(1H-imidazol-5-yl)-2-[[(Z)-octadec-9-enoyl]amino]propanoate
(64a) White powder. 1H NMR (500 MHz, CDCI3): δ 8.75 (s, 1H), 7.42 (s, 1H), 7.16 (s,
1H), 5.34-5.33 (m, 2H), 4.83-4.80 (m, 1H), 3.76 (s, 3H), 3.40-3.38 (m, 1H), 3.25-3.21 (m, 1H), 2.30 (t, J=7.5Hz, 2H), 2.01-1.98 (m, 4H), 1.64-1.60 (m, 2H), 1.30-1.26 (m, 20H), 0.88 (t, J=7.0Hz, 3H).
General procedure for synthesis of 26, 27, 34, 37-50, 53-60, 62-64 To a solution of the ester (0.51 mmol) in ethanol (30 mL), was added 1M NaOH
(10 mL). The solution was stirred at 40°C for 3 h. The ethanol was removed under reduced pressure, and the aqueous residue was adjusted to pH 2 with 0.5M HCI. The resulting suspension was filtered and the solid product washed with water (10 mL) and ethanol (5 mL). (2S)-3-hydroxy-2-[[(Z)-octadec-9-enoyl]amino]propanoic acid (26)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.87 (d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.23-4.21 (m, 1H), 3.66-3.63 (dd, J=5.5,11.0Hz), 1H), 3.59-3.55 (dd, J=4.5, 11.0Hz, 1H), 2.10 (t, J=7.5Hz, 2H), 1.98-1.97 (m, 4H), 1.46 (p, J=7.0Hz, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.37, 171.38, 129.63, 129.59, 40.82, 35.09, 31.36, 29.08, 29.00(2C), 28.94, 28.85, 28.81, 28.62, 28.57, 26.55, 26.30, 25.19, 21.70, 13.81. HRMS (ESI) m/z [M+H]+ calcd for C2iH39NO4, 370.2952; found, 370.2953. (2R)-3-hydroxy-2-[[(2)-octadec-9-enoyl]amino]propanoic acid (27)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.88 (d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.24-4.22 (m, 1H), 3.66-3.63 (dd, J=5.5, 11.0Hz, 1H), 3.59-3.55 (dd, J=4.5, 11.0Hz, 1 H), 2.11 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.46 (p, J=7.0Hz, 2H), 1.31 -1.23 (m, 20H ), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.27, 172.16, 129.65, 129.63, 61.46, 54.52, 35.03, 31.27, 29.13, 29.10, 28.83, 28.72, 28.69, 28.64, 28.59, 28.57, 26.62, 26.57, 25.21, 22.09, 13.95. HRMS (ESI) m/z [M+H]+ calcd for C2iH39NO4, 370.2952; found, 370.2955.
(2S)-4-methyl-2-[[(Z)-octadec-9-enoyl]amino]pentanoic acid (34) White powder.1H NMR (500 MHz, DMSO-d6): δ 7.98 (d, J=8.0Hz, 1 H), 5.32-5.30
(m, 2H), 4.20-4.18 (m, 1H), 2.09-2.06 (m, 2H), 1.97 (q, J=6.5Hz, 4H), 1.62-1.58 (m, 1H), 1.50-1.43 (m, 4H), 1.30-1.17 (m, 20H), 0.83 (t, J=7.0Hz, 9H).13C NMR (100 MHz, DMSO-de): δ 174.32, 172.19, 129.62(2C), 50.00, 35.02, 31.26, 29.08, 28.81, 28.67, 28.62, 28.59(3C), 28.57, 28.49, 26.62, 26.57, 25.25, 24.32, 22.89, 22.08, 21.17, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C24H45NO3, 396.3472; found, 396.3472
(2S)-5-guanidino-2-[[(2)-octadec-9-enoyl]amino]pentanoic acid.HCI (37)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 7.49 (d, J=7.5Hz, 1H), 5.32- 5.30 (m, 2H), 3.92-3.90 (m, 1H), 3.01 (t, J=6.5Hz, 2H), 2.11-2.09 (m, 2H), 1.98-1.95 (m, 4H), 1.61-1.38 (m, 6H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100 MHz, DMSO-d6): δ 175.98, 170.99, 157.32, 129.64, 129.60, 53.50, 35.03, 31.28, 29.77, 29.15, 29.10(2C), 28.83, 28.77, 28.74, 28.68, 28.61, 28.58, 26.63, 26.57, 25.42, 25.16, 22.09, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C24H46N4O3, 439.3643; found,439.3644.
(2S)-4-methylsulfanyl-2-[[(Z)-octadec-9-enoyl]amino] butanoic acid (38)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.04 (d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.29-4.26 (m, 1H), 2.47-2.40 (m, 2H), 2.11-2.07 (m, 2H), 2.02 (s, 3H), 1.98- 1.97 (m, 4H), 1.93-1.90 (m, 1H), 1.83-1.80 (m, 1H), 1.46 (p, J=7.0Hz, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.48, 172.34, 129.60(2C), 50.75, 35.04, 31.28, 30.70, 29.74, 29.12, 29.10(2C), 28.84, 28.68, 28.67, 28.59, 28.56, 26.62, 26.57, 25.22, 22.09, 14.57, 13.92. HRMS (ESI) m/z [M+H]+ calcd for C23H43NO3, 414.3036; found, 414.3038.
(2R)-4-methyl-2-[[(2)-octadec-9-enoyl]amino]pentanoic acid (39)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.98 (d, J=8.0Hz, 1H), 5.32-5.30 (m, 2H), 4.20-4.18 (m, 1 H), 2.09-2.06 (m, 2H), 1.97 (q, J=6.5Hz, 4H), 1.62-1.58 (m, 1 H), 1.50-1.43 (m, 4H), 1.30-1.17 (m, 20H), 0.83 (t, J=7.0Hz, 9H). 13C NMR (100 MHz, DMSO-de): δ 174.32, 172.19, 129.62(2C), 50.00, 35.02, 31.26, 29.08, 28.81, 28.67, 28.62, 28.59(3C), 28.57, 28.49, 26.62, 26.57, 25.25, 24.32, 22.89, 22.08, 21.17, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C24H45NO3, 396.3472; found, 396.3472 (2S)-4-methyl-2-[[(Z)-octadec-9-enoyl]amino]pentanoic acid (40)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.98 (d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.21-4.17 (m, 1H), 2.10-2.06 (m, 2H), 1.97 (q, J=6.5 Hz, 4H), 1.63-1.58 (m, 1H), 1.52-1.42 (m, 4H), 1.27-1.20 (m, 20H), 0.84 (t, J=7.0Hz, 9H).13C NMR (100 MHz, DMSO-d6): δ 174.31, 172.18, 129.61 (2C), 49.99, 35.01, 31.27, 29.09, 28.82, 28.67, 28.63, 28.60(3C), 28.58, 28.50, 26.62, 26.56, 25.25, 24.31, 22.87, 22.08, 21.15, 13.93. HRMS (ESI) m/z [M+H]+ calcd for C24H45NO3, 396.3472; found, 396.3470.
(2S)-2-[[(2)-octadec-9-enoyl]amino]hexanoic acid (41)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.97(d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.14-4.01 (m, 1H), 2.11-2.09(m, 2H), 1.98-1.95 (m, 4H), 1.61-1.53 (m, 2H), 1.47-1.43 (m, 2H), 1.31-1.23 (m, 24H), 0.84 (t, J=7.0Hz, 6H).13C NMR (100 MHz, DMSO-d6): δ 173.92, 172.21, 129.62, 129.61, 51.55, 34.99, 31.27, 30.72, 29.10, 29.09, 28.82, 28.67, 28.66, 28.59, 28.58, 28.53, 27.59, 26.61, 26.57, 25.27, 22.08, 21.69, 13.93, 13.78. HRMS (ESI) m/z [M+H]+ calcd for C24H46NO3, 396.3472; found, 396.3470.
(2S)-2-[[(2)-octadec-9-enoyl]amino]propanoic acid (42) White powder.1H NMR (500 MHz DMSO-d6): δ 8.00 (d, J=8.0Hz, 1 H), 5.32-5.30
(m, 2H), 4.16-4.13 (m, 1H) 2.06 (t, J=8.0Hz, 2H), 1.97 (m, 4H), 1.45 (p, J=7.0Hz, 2H), 1.28-1.23 (m, 23H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 174.31, 171.94, 129.63, 129.62, 47.30, 34.98, 31.28, 29.12, 29.10, 28.84(2C), 28.69, 28.61, 28.59, 28.57, 26.61 , 26.57, 25.18, 22.09, 17.21 , 13.93. HRMS (ESI) m/z [M+H]+ calcd for C21 H39NO3, 354.3003; found, 354.3009.
(2R)-2-[[(2)-octadec-9-enyl] amino]propanoic acid (43)
White powder. 1H NMR (500 MHz DMSO-d6): δ 8.02 (d, J=7.5Hz, 1 H), 5.32-5.30 (m, 2H), 4.18-4.15 (m, 1 H) 2.06 (t, J=7.5Hz, 2H), 1 .99-1 .95 (m, 4H), 1 .45 (p, J=7.5Hz, 2H), 1 .28-1 .23 (m, 23H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 174.28, 171 .93, 129.61 , 129.60, 47.30, 34.98, 31 .28, 29.13, 29.1 1 , 28.84(2C), 28.69, 28.61 , 28.59, 28.57, 26.61 , 26.56, 25.18, 22.09, 17.21 , 13.93. HRMS (ESI) m/z [M+H]+ calcd for C21 H39NO3, 354.3003; found, 354.3003. 3-[[(2)-octadec-9-enoyl]amino]propanoic acid (44)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 7.81 (t, J=5.0Hz, 1 H), 5.32-5.30 (m, 2H), 3.19 (q, J=7.0Hz, 4H), 2.32 (t, J=7.0Hz, 2H), 1 .97 (q, J=6.0Hz, 4H), 1 .49-1 .46 (m, 2H), 1 .31 -1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.10, 172.32, 129.89(2C), 35.40, 34.88, 34.18, 31 .39, 29.23, 29.21 , 28.94, 28.80, 28.77, 28.74, 28.69, 28.64, 26.71 , 26.68, 25.36, 22.20, 14.06. HRMS (ESI) m/z [M+H]+ calcd for C20H37NO3, 340.2846; found, 340.2850.HRMS (ESI) m/z [M+H]+ calcd for C21 H39NO3, 354.3003; found, 354.3003.
(2S)-3-methyl-2-[[(Z)-octadec-9-enoyl]amino]butanoic acid (45)
White powder. 1H NMR (500 MHz DMSO-d6): δ 7.85(d, J=8.5Hz, 1 H), 5.32-5.30 (m, 2H), 4.50-4.47 (m, 1 H) 2.18-2.09 (m 2H), 2.04-1 .95 (m, 5H), 1 .48-1 .44 (m, 2H), 1 .28-1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 9H). 13C NMR (100 MHz, DMSO-d6): δ 172.25, 172.36, 129.59(2C), 57.07, 34.98, 31 .28, 29.78, 30.66, 29.83, 29.10, 28.82(2C), 28.69(2C), 28.65, 28.59, 26.61 , 26.56, 25.37, 22.08, 19.17, 18.02, 13.92. HRMS (ESI) m/z [M+H]+ calcd for C23H43NO3, 382.3316; found, 382.3319. (2R)-3-methyl-2-[[(Z)-octadec-9-enoyl]amino]butanoic acid (46)
White powder. 1H NMR (500 MHz DMSO-d6): δ 7.85 (d, J=9.0Hz, 1 H), 5.32-5.30 (m, 2H), 4.13-4.10 (m, 1 H) 2.16-2.10 (m 2H), 2.00-1 .95 (m, 5H), 1 .48-1 .44 (m, 2H) 1 .28- 1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 9H). 13C NMR (100 MHz, DMSO-d6): δ 172.96, 172.14, 129.38(2C), 56.80, 34.72, 31 .28, 30.43, 29.53, 28.84, 28.83(2C), 28.56, 28.42, 28.38, 28.32, 28.31 , 26.34, 26.30, 25.1 1 , 21 .83, 18.93, 17.79, 13.92. HRMS (ESI) m/z [M+H]+ calcd for C23H43NO3, 382.3316; found, 382.3318.
(2R)-2-[[(2)-octadec-9-enoyl]amino]-3-phenyl-propanoic acid (47) White powder. 1H NMR (500 MHz, DMSO-d6): δ 7.77 (d, J=8.0Hz, 1 H), 7.20-7.12
(m,5H), 5.32-5.30 (m, 2H), 4.27-4.25 (m, 1 H), 3.07-3.06 (dd, J=5.5, 14.0Hz, 1 H), 2.85- 2.80 (dd, J=6.0, 14.0Hz, 1 H), 1 .98-1 .95 (m, 6H), 1 .36 (p, J=7.0Hz, 2H), 1 .30-1 .17 (m, 18H), 1 .1 1 -2.09 (m, 2H)), 0.84 (t, J=7.0Hz, 3H). ). 13C NMR (100 MHz, DMSO-d6): δ 173.62, 171 .46, 138.69, 129.65, 129.60, 129.27, 127.76, 125.83, 54.34, 37.24, 35.41 , 31 .29, 29.16, 29.12, 28.85(2C), 28.75, 28.70(2C), 28.61 , 28.58, 28.55, 26.65, 26.59, 25.30, 22.10, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C27H43NO3, 430.3315; found, 430.3319.
(2S)-2-[[(Z)-octadec-9-enoyl]amino]-4-phenyl-butanoic acid (48)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 8.10 (d, J=8.0Hz, 1 H), 7.28-7.24 (m, 2H), 7.18-7.14 (m, 3H), 5.33-5.26 (m, 2H), 4.16-4.10 (m, 1 H), 2.66-2.52 (m, 2H), 2.20-2.08 (m, 2H), 1 .98-1 .80 (m, 6H), 1 .49 (d, J=7.0Hz, 2H), 1 .32-1 .18 (m, 20H), 0.84 (d, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.74, 172.32, 170.28, 141 .04, 129.59, 129.58, 129.28, 125.87, 59.73, 51 .20, 35.06, 32.86, 32.50, 31 .27, 29.12, 29.09, 28.83, 28.68, 28.61 (2C), 28.58, 26.62, 26.56, 25.28, 22.09, 20.74, 14.07, 13.92. HRMS (ESI) m/z [M+H]+ calcd for C28H45NO3, 443.3472; found, 443.3469.
(2R)-2-[[(Z)-octadec-9-enoyl]amino]-4-phenyl-butanoic acid (49)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 8.09 (d, J=8.0Hz, 1 H), 7.28-7.24 (m, 2H), 7.18-7.14 (m, 3H), 5.34-5.28 (m, 2H), 4.14-4.08 (m, 1 H), 2.66-2.52 (m, 2H), 2.18-2.08 (m, 2H), 2.00-1 .80 (m, 6H), 1 .49 (d, J=7.0Hz, 2H), 1 .32-1 .18 (m, 20H), 0.84 (d, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.74, 172.31 , 141 .09, 129.62, 129.61 , 128.30, 125.89, 51 .15, 35.02, 32.88, 31 .49, 31 .26, 29.10, 29.07, 28.81 , 28.66(4C), 28.59(3C), 28.57, 26.60, 26.55, 25.27, 22.08, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C28H45NO3, 443.3472; found, 443.3475. (2S)-3-(1 H-indol-3-yl)-2-[[(2)-octadec-9-enoyl]amino]propanoic acid.HCI (50)
Off-white solid.1H NMR (500 MHz, DMSO-d6): δ 10.80 (s, 1H), 7.9 (d, J=8.0Hz, 1H), 7.52-7.50 (d, J=7.50Hz, 1H), 7.31-7.30 (d, J=8.0Hz, 1H), 7.11-7.10 (d, J=2.0Hz, 1H), 7.03 (t, J=7.0Hz, 1H), 6.95 (t, J=7.0Hz, 1H), 5.32-5.30 (m, 2H), 4.45-4.44 (m, 1H), 3.16-3.12 (dd, J=5.5, 14.0Hz, 1H), 2.99-2.95 (m, 1H), 2.06-2.04 (m, 2H), 1.98-1.95 (m, 4H), 1.39-1.37 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100 MHz, DMSO-de): δ 173.58, 172.10, 136.06, 129.66, 129.60, 127.23, 123.45, 120.82, 118.25, 118.17, 110.10, 110.06, 52.86, 35.08, 31.26, 29.12, 29.09, 28.82, 28.67, 28.58, 28.54, 28.52, 27.13(2C), 26.62, 26.57, 25.27, 22.08, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C29H44N2O3, 469.3425; found, 469.3429.
(2S)-2-[[(Z)-octadec-9-enoyl]amino]butanedioic acid (53)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.95(d, J=7.5Hz, 1H), 7.31 (s, 1H), 6.86 (s, 1H), 5.32-5.30 (m, 2H), 4.47-4.45 (m, 1H), 2.54-2.50 (m, 1H), 2.43-2.38 (dd, J=7.5, 15.5Hz, 1 H), 2.05 (t, J=7.0Hz, 2H), 1.97 (q, J=6.0Hz, 4H), 1.46-1.43 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.56, 172.03, 171.67, 129.65, 129.62, 48.48, 35.02, 31.26, 29.12, 29.09, 28.82(2C), 28.68, 28.67, 28.57, 28.55, 28.53, 26.61, 26.56, 25.19, 22.08, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C22H39NO5, 398.2901, found, 398.2909.
(2R)-2-[[(2)-octadec-9-enoyl]amino]butanedioic acid (54) White powder.1H NMR (500 MHz, DMSO-d6): δ 8.07 (d, J=8.0Hz, 1 H), 5.32-5.30
(m, 2H), 4.50-4.47 (m, 1H), 2.67-2.62 (dd, J=5.5, 16.0Hz, 1H), 2.54-2.50 (m, 1H), 2.05 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.45-1.43 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.64, 172.22, 171.77, 129.72, 129.69, 48.58, 36.15, 35.11, 31.36, 29.22, 29.18, 28.92, 28.78, 28.77, 28.67, 28.65, 28.62, 26.70, 26.65, 25.28, 22.17, 14.01. HRMS (ESI) m/z [M+H]+ calcd for C22H39NO5, 398.2901, found, 398.2901.
(2S)-4-amino-2-[[(Z)-octadec-9-enoyl]amino]-4-oxo-butanoic acid (55)
White powder.1H NMR (500 MHz, DMSO-d6): δ 7.95 (d, J=8.0Hz, 1H), 7.31 (s, 1H), 6.86 (s, 1H), 5.34-5.28 (m, 2H), 4.48-4.43 (m, 1H), 2.54-2.38 (m, 2H), 2.06 (t, J=8.0Hz, 2H), 2.00-1.94 (m, 4H), 1.44 (p, J=7.0Hz, 2H), 1.32-1.18 (m, 20H), 0.84 (d, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.97, 171.95, 171.21, 129.63, 129.60, 48.67, 36.78, 35.06, 31.26, 29.13(2C), 29.08, 28.81, 28.70, 28.66(2C), 28.57, 26.61, 26.56, 25.21, 22.07, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C22H40N2O4, 397.3061; found, 397.3065.
(2S)-2-[[(Z)-octadec-9-enoyl] amino] pentanedioic acid (56)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.0 (d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.21-4.16 (m, 1H), 2.27-2.23 (m, 2H), 2.09-2.07 (m, 2H), 1.98-1.95 (m, 5H), 1.78-1.72 (m, 1H), 1.46-1.43 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.75, 173.50, 172.40, 129.64, 129.60, 51.06, 35.09, 31.37, 30.13, 29.23, 29.20(2C), 28.94, 28.79, 28.69(3C), 26.70, 26.65, 26.41, 25.30, 22.18, 13.95. HRMS (ESI) m/z [M+H]+ calcd for C23H4iNO5, 412.3058; found, 412.3061.
(2R)-2-[[(2)-octadec-9-enoyl]amino]pentanedioic acid (57)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.0 (d, J=8.0Hz, 1H), 5.32-5.30 (m, 2H), 4.20-4.15 (m, 1H), 2.26-2.23 (m, 2H), 2.09-2.07 (m, 2H), 1.98-1.95 (m, 5H), 1.78-1.72 (m, 1H), 1.47-1.45 (m, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.71, 173.46, 172.33, 129.63(2C), 50.99, 35.02, 31.27, 30.11, 29.12 (2C), 29.09, 28.82, 28.67(3C), 28.58, 26.61, 26.56, 26.34, 25.22, 22.08, 13.94.95. HRMS (ESI) m/z [M+H]+ calcd for C23H4iNO5, 412.3058; found, 412.3060. (2S)-3-(1H-imidazol-5-yl)-2-[[(Z)-octadec-9-enoyl] amino] propanoic acid.HCI
(58)
White powder. 1H NMR (500 MHz, DMSO-d6), δ 8.80(s, 1H), 8.28(d, J=8.0Hz, 1H), 7.27(s, 1H), 5.32-5.30 (m, 2H), 4.50-4.47 (m, 1H), 3.08(dd, J=5.0, 15.0Hz, 1H), 2.97-2.95 (m, 2H), 2.05 (t, J=7.5Hz, 2H), 1.98-1.95 (m, 4H), 1.42-1.40 (m, 2H), 1.31- 1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100 MHz, DMSO-d6): δ 172.36, 172.32, 133.73, 129.67, 129.63, 116.74, 51.26, 36.72, 31.28, 29.16, 29.02, 28.84(2C), 28.68(2C), 28.59, 28.56, 28.51, 26.63, 26.58, 26.45, 25.14, 22.10, 13.97. HRMS (ESI) m/z [M+H]+ calcd for C24H41N3O3, 420.3221 ; found, 420.3224. (2S)-2-[[(2)-octadec-9-enoyl]amino]-3-phenyl-propanoic acid (59)
White powder, 1H NMR (500 MHz, DMSO-d6): δ 7.77 (d, J=8.0Hz, 1H), 7.20-7.12 (m,5H), 5.32-5.30 (m, 2H), 4.27-4.25 (m, 1H), 3.07-3.06 (dd, J=5.5, 14.0Hz, 1H), 2.85- 2.80 (dd, J=6.0, 14.0Hz, 1H), 1.98-1.95 (m, 6H), 1.36 (p, J=7.0Hz, 2H), 1.30-1.17 (m, 18H), 1.11-2.09 (m, 2H)), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.62, 171.46, 138.69, 129.65, 129.60, 129.27, 127.76, 125.83, 54.34, 37.24, 35.41,
31.29, 29.16, 29.12, 28.85(2C), 28.75, 28.70(2C), 28.61, 28.58, 28.55, 26.65, 26.59,
25.30, 22.10, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C27H43NO3, 430.3315; found, 430.3317. (2R)-4-methylsulfanyl-2-[[(Z)-octadec-9-enoyl]amino] butanoic acid (60)
White powder.1H NMR (500 MHz, DMSO-d6): δ 8.04 (d, J=7.5Hz, 1H), 5.32-5.30 (m, 2H), 4.29-4.26 (m, 1H), 2.47-2.42 (m, 2H), 2.11-2.07 (m, 2H), 2.02 (s, 3H), 1.98- 1.97 (m, 4H), 1.93-1.90 (m, 1H), 1.83-1.80 (m, 1H), 1.46 (p, J=7.0Hz, 2H), 1.31-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 173.48, 172.34, 129.60(2C), 50.75, 35.04, 31.28, 30.70, 29.74, 29.12, 29.10(2C), 28.84, 28.68, 28.67, 28.59, 28.56, 26.62, 26.57, 25.22, 22.09, 14.57, 13.92. HRMS (ESI) m/z [M+H]+ calcd for C23H43NO3, 414.3036; found, 414.3039.
(2R)-5-guanidino-2-[[(2)-octadec-9-enoyl]amino]pentanoic acid.HCI (62)
White powder. 1H NMR (500 MHz, DMSO-d6): δ 8.05 (d, J=7.5Hz, 1H), 7.71 (t, J=5.5Hz, 1H), 7.42-7.13 (m, 3H), 5.32-5.30 (m, 2H), 4.18-4.13 (m, 1H), 3.01 (t, J=6.5Hz, 2H), 2.09-2.04 (m, 2H), 1.98-1.95 (m, 4H), 1.72-1.69 (m, 2H), 1.58-1.55 (m, 2H), 1.45-1.36 (m, 2H), 1.30-1.23 (m, 20H), 0.84 (t, J=7.0Hz, 3H).13C NMR (100 MHz, DMSO-de): δ 175.98, 170.99, 157.32, 129.64, 129.60, 53.50, 35.03, 31.28, 29.77, 29.15, 29.10(2C), 28.83, 28.77, 28.74, 28.68, 28.61, 28.58, 26.63, 26.57, 25.42, 25.16, 22.09, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C24H46N4O3, 439.3643; found,439.3644.
(2R)-3-(1 H-indol-3-yl)-2-[[(Z)-octadec-9-enoyl]amino]propanoic acid.HCI (63)
White solid.1H NMR (500 MHz, DMSO-d6): δ 10.80 (s, 1H), 7.99 (d, J=8.0Hz, 1H), 7.52-7.50 (d, J=7.50Hz, 1H), 7.31-7.30 (d, J=8.0Hz, 1H), 7.11-7.10 (d, J=2.0Hz, 1H), 7.03 (t, J=7.0Hz, 1H), 6.95 (t, J=7.0Hz, 1H), 5.32-5.30 (m, 2H), 4.45-4.44 (m, 1H), 3.16-3.12 (dd, J=5.5, 14.0Hz, 1 H), 2.99-2.95 (m, 1 H), 2.06-2.04 (m, 2H), 1 .98-1 .95 (q, J=6.0Hz, 4H), 1 .39-1 .37 (m, 2H), 1 .31 -1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-de): δ 173.58, 172.10, 136.06, 129.66, 129.60, 127.23, 123.45, 120.82, 1 18.25, 1 18.17, 1 10.10, 1 10.06, 52.86, 35.08, 31 .26, 29.12, 29.09, 28.82, 28.67, 28.58, 28.54, 28.52, 27.13(2C), 26.62, 26.57, 25.27, 22.08, 13.94. HRMS (ESI) m/z [M+H]+ calcd for C29H44N2O3, 469.3425; found, 469.3420.
(2R)-3-(1 H-imidazol-5-yl)-2-[[(Z)-octadec-9-enoyl] amino] propanoic acid.HCI
(64)
White powder. 1H NMR (500 MHz, DMSO-d6), δ 8.86 (s, 1 H), 8.22 (d, J=8.0Hz, 1 H), 7.30 (s, 1 H), 5.32-5.30 (m, 2H), 4.50-4.47 (m, 1 H), 3.12-3.08 (m, 2H), 2.98-2.92 (m, 1 H), 2.05 (t, J=7.5Hz, 2H), 1 .98-1 .95 (m, 4H), 1 .42-1 .40 (m, 2H), 1 .31 -1 .23 (m, 20H), 0.84 (t, J=7.0Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 172.36, 172.32, 133.73, 129.67, 129.63, 1 16.74, 51 .26, 36.72, 31 .28, 29.16, 29.02, 28.84, 28.68(2C), 28.59, 28.56, 28.51 , 28.47, 26.63, 26.58, 26.45, 25.14, 22.10, 13.97 HRMS (ESI) m/z [M+H]+ calcd for C24H41 N3O3, 420.3221 ; found, 420.3220.
Inhibitory activity
Methods for assessing the activity of compounds of the invention is described in Carland et al (2013), which is incorporated herein by reference.
Briefly, inhibition of glycine transporters, GlyT1 and GlyT2, by compounds of the invention was examined by injecting RNA encoding these proteins into Xenopus laevis oocytes and measuring the concentration dependent glycine currents in the presence and absence of the compounds.
Glycine transport by GlyT2 and GlyT1 is coupled to 3 Na+/1 CI- and 2Na+/1 Cl- ions respectively, creating an electrogenic process and allowing the two electrode voltage clamp technique to be used to measure glycine transport. Defoliculated stage V- VI oocytes were injected with 4.6 ng of cRNA encoding the transporter (Drummond Nanoinject, Drummond Scientific Co., Broomall, PA, USA). When transporter expression levels are sufficient 2-5 days following injection, glycine transport currents were measured at -60 mV using Geneclamp 500 amplifier (Axon Instruments, Foster City, CA, USA) with a Powerlab 2/20 chart recorder (ADInstruments, Sydney, Australia) using chart software (ADInstruments).
Glycine was applied, followed by co-administration of glycine (EC50) in the presence of inhibitor, until the inhibitory response was observed to plateau. Each compound was tested to a maximal concentration of 3 μΜ because these compounds form micelles at higher concentrations. The compounds were applied at concentrations below their CMC in ND96 which was determined using a CMC assay. Reversibility of each compound was tested by applying the EC50 dose of glycine every 5 minutes following inhibition for a 30 minute time course or until recovery was reached. Recovery is defined as glycine currents within +/- 5 % of the pre-inhibitory glycine response. Concentration response curves for the active compounds were then performed.
The activity assay results are provided in Table 2, below.
Table 2. Inhibitory activity of exemplary compounds of the present invention
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
The physical properties of the compounds of the invention have distinct advantages over traditional compounds, because they do not allow complete and prolonged inhibition, which are responsible for the toxic side effects of existing drugs. Metabolic stability in rat and human liver microsomes
The metabolic stability of exemplary compounds of the invention was tested using human and rat liver microsomes in vitro.
Incubation methods The metabolic stability assay was performed by incubating each test compound
(1 μΜ) with human (Xenotech, Lot #1410230) and rat (Xenotech, Lot #1410271 ) liver microsomes at 37°C and 0.4 img/mL protein concentration. The metabolic reaction was initiated by the addition of a NADPH-regenerating system (i.e. NADPH is the cofactor required for CYP450-mediated metabolism) and quenched at various time points over the 60 minute incubation period by the addition of acetonitrile containing diazepam as internal standard. Control samples (containing no NADPH) were included (and quenched at 2, 30 and 60 minutes) to monitor for potential degradation in the absence of cofactor.
Sample preparation
Following protein precipitation with acetonitrile, samples were centrifuged for 4 minutes at 4,500 rpm. The supernatant was removed and analysed by LC/MS (see below).
Analytical conditions
Figure imgf000095_0001
Metabolite identification:
Metabolite screening was performed using accurate mass measurements only. Compounds were incubated at a low substrate concentration and as such, structure confirmation and elucidation using MS/MS scans was not conducted. Calculations
Test compound concentration versus time data were fitted to an exponential decay function to determine the first-order rate constant for substrate depletion.
The in vitro half-life of each of the test compounds in the presence and absence of NADPH, along with metabolic stability calculations (in vitro intrinsic clearance, predicted blood clearance and predicted hepatic extraction ratio) are presented in Table 3.
Table 3: Metabolic stability parameters for nine compounds based NADPH-dependent degradation profiles in human and rat liver microsomes.
Figure imgf000097_0001
Note: degradation half-life in non-NADPH control samples is an estimate based on up to three time points 1 Apparent non-NADPH mediated degradation was observed in metabolism control samples and the apparent degradation half-life in control incubations is shown in parentheses. Predicted clearance parameters are therefore not determined (ND) as the assumptions for in vivo clearance predictions are no longer valid (Obach, 1999). 2 No measurable concentration of the parent compound was detected at the initial time point (i.e. 2 minutes), hence, the clearance parameters could not be calculated (c.n.c).
3 This compound showed minimal microsomal degradation (<15%) over the course of the incubation.
There was no measurable degradation of compounds 29, 36 and 50 in microsomal control incubations, suggesting that there was no major non-cofactor dependent microsomal metabolism contributing to the overall rates of metabolism. These compounds would be expected to show low hepatic clearance in vivo.
Metabolic stability in rat and human plasma
The metabolic stability of exemplary compounds of the invention was tested using human and rat plasma in vitro.
Incubation
Human plasma (pooled; n=3 donors) was separated from whole blood procured from the Australian Red Cross Blood Service. Rat plasma (pooled; multiple rats) from male Sprague Dawley rats was procured from Animal Resources Centre (ARC; Perth). Plasma was stored frozen at -80°C, on the day of the experiment, plasma was thawed in a water bath maintained at 37°C.
Aliquots of plasma were spiked with DMSO/acetonitrile/water solutions of test compound to a nominal compound concentration of 1000 ng/mL. The maximum final DMSO and acetonitrile concentrations were 0.2% (v/v) and 0.4% (v/v) respectively. Immediately after compound spiking, plasma was vortex mixed and aliquots of spiked plasma (50 μΙ_) were transferred into fresh micro centrifuge tubes and were maintained at 37°C. At various time points over the 240 min incubation period, triplicate plasma samples were taken and immediately snap-frozen in dry ice. All samples were stored frozen at -80°C until analysis by LC-MS. Bioanalysis
Plasma samples were quantified relative to calibration standards prepared using blank plasma of the same species. Calibration standards were spiked with test compound over a range of 0.5 to 10,000 ng/mL. Internal standard (leucine enkephalin) was added to calibration standards and incubation samples, and then immediately quenched using two volumes of acetonitrile to precipitate plasma proteins. Samples were vortex mixed and centrifuged (10,000 rpm for 3 minutes) in a microcentrifuge and the supernatant analysed by LC-MS using conditions tabulated below.
Figure imgf000099_0001
Data Analysis
The mean and standard deviation of measured plasma concentrations were calculated for each time point and expressed as a percentage remaining, relative to the initial time point (2 min). Where measurable degradation was detected, and assuming first order degradation kinetics, the data were fit using a mono-exponential decay function to obtain the apparent first-order degradation rate constant (k, min-1 ) and degradation half-life.
Results
The results of the metabolic study are shown in Table 4, below, and in Figure 1 . In Figure 1 , data is expressed as mean ± SD (n = 3) and where measurable degradation was observed, the data were fitted using a mono-exponential decay function. Table 4. Metabolic stability of five compounds in human and rat plasma in vitro
Figure imgf000100_0001
Where compounds exhibited loss that was less than 15% over the course of the 240 min incubation, the degradation half-life was estimated to be >1022 min.
Spinal cord electrophysiology
Preparation of spinal cord slices
Adult male Sprague-Dawley rats (6-8 weeks at the time of slice preparation) were anaesthetized with isoflurane, decapitated and the lumbar region of the spinal cord was removed. Parasagittal slices (340 pm thick) of spinal cord were cut on a vibratome (Leica VT 1200s) in oxygenated ice-cold sucrose-based artificial CSF (sACSF) that contained (mM): 100 sucrose, 63 NaCI, 2.5 KCI, 1 .2 NaH2PO4, 1 .2 MgCI2, 25 glucose, and 25 NaHCO3. Slices were transferred to a submerged chamber containing NMDG- based recovery ACSF (rACSF) for 15 minutes at 34°C, equilibrated with 95% O2 and 5% C02 and composed of (mM): 93 NMDG, 2.5 KCI, 1 .2 NaH2P04, 30 NaHC03, 20 HEPES, 25 Glucose, 5 Na ascorbate, 2 thiourea, 3 Na pyruvate, 10 MgSO4 and 0.5 CaCI2, and adjusted to pH 7.4 with HCI. Following the recovery incubation, slices were transferred to normal oxygenated ACSF where they were allowed to recover for 1 hour at 34° C and then maintained at room temperature prior to transfer to the recording chamber. Normal ACSF had the following composition (imM): 125 NaCI, 2.5 KCI, 1 .25 NaH2PO4, 1 .2 MgCI2, 2.5 CaCI2, 25 glucose, and 1 1 NaHCO3 and was equilibrated with 95% O2 and 5% CO2.
Electrophysioloqy Slices were transferred to a recording chamber and superfused continuously at 2 ml/min with normal ACSF that had been equilibrated with 95% O2 and 5% CO2 and maintained at 34°C with an inline heater and monitored by a thermister in the slice chamber. Dodt-contrast optics was used to identify lamina II neurons in the translucent substantia gelatinosa layer of the superficial dorsal horn. A Cs+-based internal solution, which should minimise postsynaptic effects, was used to record electrically evoked inhibitory post-synaptic currents (elPSCs) and tonic current, and contained (imM): 140 CsCI, 10 EGTA, 5 HEPES, 2 CaCI2, 2 MgATP, 0.3 NaGTP, 5 QX-314.CI, 2 Lucifer Yellow CH dipotassium salt and 0.1 % biocytin (osmolarity 285-295 mosmol Γ1). Patch electrodes had resistances between 3 and 5 ΜΩ. Synaptic currents were measured in whole-cell voltage-clamp (-70 mV, not corrected for a liquid junction potential of 4 mV) from lamina II cells. Bipolar tungsten electrodes placed in the inner laminae (lamina III region) were used to elicit elPSCs using a stimulus strength sufficient to evoke reliable elPSCs. Neurons ventral to lamina II, in regions that are known to contain glycinergic neurons were electrically stimulated. All elPSCs were recorded in CNQX (10 μΜ), AP5 (100 μΜ) and picrotoxin (80 μΜ). At the conclusion of each experiment strychnine (0.5 μΜ) was added to the superfusion solution to confirm that recorded currents were glycine- mediated IPSCs. Drugs were superfused onto slices at a rate of 2ml/min in normal oxygenated ACSF at 34°C.
Results The results of the spinal cord electrophysiology are presented in Figures 2 - 6.
The results show that Compounds 28 and 39 are effective at increasing tonic current, elPSC amplitude and decay time constants. Plasma and brain exposure of oleyl-D-lysine following IP administration
To assess the plasma and brain exposure of oleyl-D-lysine, Sprague Dawley rates were administered (via IP) a 27.5 mg/kg dose of the compound, according to the following procedure. Summary of procedure
Figure imgf000102_0001
Formulation
Small scale formulation trials (~200 μΙ_ volume) indicated oleoyl-D-lysine was a solution when formulated using a vehicle of 10% (v/v) DMSO, with 1 % Solutol in 50 imM PBS 7.4. On the day of dosing the formulation was scaled up to a volume of 24.9 imL, which yielded a fine uniform suspension.
The formulation was prepared by dissolving solid compound in DMSO prior to addition of 1 % Solutol in 50mM PBS pH 7.4 solution, which after sonication and heating yielded a very fine off-white suspension with an apparent pH of 7.05. This suspension of oleoyl-D-lysine was administered in a dose volume of 5 imL/kg via intraperitoneal injection (via 27G 1 /2" needle) resulting in a nominal oleoyl-D-lysine dose of 27.5 mg/kg.
Rat Exposure
All animal studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee.
The systemic exposure of oleoyl-D-lysine was studied in non-fasted male Sprague Dawley rats weighing 250 - 306 g. Rats had access to food and water ad libitum throughout the pre- and postdose sampling period. A single blood sample (1 imL) was collected via cardiac puncture from each rat
(anaesthetised using gaseous Isoflurane) at designated times up to 24 h (3 rats per time point). At the time of blood sampling, the whole brain was also rapidly removed from the carcass. No urine samples were collected as rats were housed in bedded cages.
Blood was collected into polypropylene Eppendorf tubes containing heparin as anticoagulant and stabilisation cocktail containing Complete® (a protease inhibitor cocktail) and potassium fluoride) to minimise the potential for ex vivo compound degradation in blood/plasma samples. Once collected, blood samples were centrifuged immediately, supernatant plasma was removed, and stored at - 80°C until analysis by LC-MS. The whole brains were blotted to remove excess blood, placed into pre- weighed polypropylene vials and weighed. The brains were snap frozen in dry ice and subsequently stored frozen (-80°C) until analysis. A summary of the bioanalytical method and assay validation details is included in Appendix 2.
Standard Calculations
Plasma concentration versus time data were analysed using non-compartmental methods (PKSolver Version 2.0). Standard calculations for each pharmacokinetic parameter are listed below.
Figure imgf000104_0001
Calculation of Brain Exposure Parameters
The concentration of oleoyl-D-lysine in brain parenchyma was calculated on the basis of the measured concentration in brain homogenate, after correcting for the contribution of compound contained within the vascular space of brain samples as follows:
103
Substitute Sheet
(Rule26)RO/AU
Figure imgf000105_0001
Sample processing and analysis The extraction of the test compound from plasma samples was conducted using protein precipitation with acetonitrile. Both solution and plasma standards were freshly prepared, with each set of standards comprising at least six different analyte concentrations. Solution standards were diluted from a stock solution (1 mg/mL in DMSO) with 50% acetonitrile in water. Plasma standards were prepared by spiking blank plasma (50 μΙ_) with solution standards (10 μΙ_) and the internal standard, diazepam (10 μΙ_, 5 Mg/mL). Plasma samples were similarly prepared, except that blank acetonitrile (10 μΙ_) was added instead of solution standards. Protein precipitation was carried out by the addition of acetonitrile (120 μΙ_), vortexing (20 s) and centrifugation (10,000 rpm) in a microcentrifuge for 3 minutes. The supernatant was subsequently separated and 3 μΙ_ injected directly onto the column for LC-MS analysis using conditions presented in the method summary section. All concentrations are expressed as the non-salt equivalent.
Pre-weighed rat brains were homogenised in 3-volume/weight of stabilisation mixture (composed of 0.1 M EDTA and 4 g/L KF in water) using a gentleMACS™ dissociator. Extraction of the test compound from the resulting tissue homogenate was conducted using protein precipitation with methanol. Tissue homogenate standards were freshly prepared, with each set of standards comprising at least six different analyte concentrations. Tissue standards were prepared by spiking blank tissue homogenate (200 μΙ_) with solution standards (10 μΙ_) and the internal standard, diazepam (10 μΙ_, 5 pg/mL). Tissue samples were similarly prepared, except that blank methanol (10 μΙ_) was added instead of solution standards. Protein precipitation was carried out by the addition of methanol (600 μΙ_), vortexing (20 s) and centrifugation (10,000 rpm) in a microcentrifuge for 3 minutes. The supernatant was subsequently separated and 3 μΙ_ injected directly onto the column for LC-MS analysis using conditions presented in the method summary section. Results
Following IP administration of oleoyl-D-lysine, all rats exhibited mild piloerection and reduced activity, however all rats appeared to recover approximately 2 hours post- dose.
Plasma and brain concentration versus time profiles are presented in Figure 6, whilst calculated plasma and brain exposure parameters, values for individual rats, together with the corresponding brain-to-plasma (B:P) ratios are summarised in Tables 5 and 6.
In plasma and brain collected from a single rat that was not dosed with oleoyl-D- lysine, there was no detectable level of oleoyl-D-lysine. This supports the specificity of the LC-MS method and infers that the measured concentrations observed in rats that were dosed with the compound are due to the oleoyl-D-lysine itself, and not an endogenous interference.
Following IP administration, oleoyl-D-lysine was rapidly absorbed with maximum plasma concentrations observed at the first sampling time. The apparent terminal half- life of the plasma profile (based on the last 3 time points) was ~ 10 h.
In contrast to the plasma concentration-time profile, brain concentrations remained relatively constant for the duration of the 24 h sampling period. B:P ratios increased over the 24 h exposure period, and maximum values (ranging from 0.6-1 .6) were observed at 24 h post-dose. This may be indicative of a slow rate of compound equilibration between plasma and brain. As such, the time-averaged brain-to-plasma (B:P) partitioning ratio values (based on AUC0-24h values in plasma and brain) may provide a better indication of the distribution of oleoyl-D-lysine into the brain, although it may still be an underestimation as it is lower than the B:P at 24 h. Table 5: Exposure parameters for oleoyl-D-lysine in male Sprague Dawley rats following IP administration at 27.5 mg/kg
Figure imgf000107_0001
c.n.c. - Could not calculate as the flat profile precluded an estimation of half-life and extrapolation to infinity
Table 6: Individual and mean ± SD (n=3) plasma and brain concentrations and brain to plasma (B:P) ratios of oleoyl-D-lysine in male Sprague Dawley rats following IP administration at 27.5 mg/kg
Figure imgf000108_0001
ND - Not detected Cell-based agonist and antagonist assays on ACTOne-CB1 , CB2 and
S1 PR1 cells
The purpose of these assays was to determine whether the compounds of the present invention have any off-target effects. The S1 PR1 , CB1 and CB2 receptors were chosen as they have been known to be agonised or antagonised by lipid-based molecules. S1 PR1 Assay
ACTOne-S1 PR1 cells used for the assay
CELL LINE DESIGNATION Endothelial Differentiation, Sphingolipid
G-Protein-coupled Receptor, 1 cell line (CB-80300-250)
ORIGIN (PARENTAL CELL) HEK 293-CNG cell (CB-80200-200)
GENE INTRODUCED Genbank Locus ID 1901
RECEPTOR INTRODUCED Human endothelial differentiation,
sphingolipid G-protein-coupled receptor, 1 (NCBI protein database NP_001391 .2)
Usage cAMP assay for Gi-coupled human Endothelial Differentiation, Sphingolipid G- Protein-coupled Receptor, 1 (S1 PR1 )
Quality control
1 . This cell line has been tested negative for Mycoplasma sp.
2. This cell line has been tested positive for Endothelial Differentiation, Sphingolipid G-Protein-coupled Receptor, 1 specific response.
3. Surviving rate: More than 2.5 million/vial on the second day after thawing.
4. The receptor specific activity is stable for 10 weeks continuous passage. Cell culture condition
1 . Growth medium: 90% DMEM, 10% FBS, 250 μg/ml G418 and 1 μg/ml puromycin
2. Freezing medium: 10% DMSO, 90% growth medium Testing compounds on ACTOne-SI PR1 cells
ACTOne-S1 PR1 cells (CB-80300-250) were maintained in cell culture medium consisting of 90% Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250 μg/ml G418 and 1 μg/ml puromycin. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in the growth medium. 20 μΙ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
On the second day, the cell plates were taken out from the incubator and an equal volume (20 μΙ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 μΙ. The plates will be referred to as the cell plates. i) Agonist Assay
The agonist control picked here was S1 P (125 uM stock in 4 mg/ml Fatty acid free BSA, Avanti Polar Lipids 860492P). The agonist stimulation solutions were prepared as below:
1 . Dilute Ro 20-1724 (Sigma B8279) stock to 125 μΜ and isoproterenol (Sigma I6504) stock to 1 .5 μΜ in 1X DPBS with 0.1 mg/ml BSA (Sigma A8806). It was referred as agonist dilution buffer. 2. Dilute 125 μΜ S1 P stock in agonist dilution buffer as shown in Table 1 . These concentrations are 5X of the expected final testing concentrations.
3. The testing compounds were first diluted in DMSO to 1 imM each. It was further diluted (1 :200) in agonist dilution buffer to 5 μΜ each.
Isoproterenol is used to stimulate the adenylyl cyclase through the activation of Gs-coupled endogenous β-adrenoceptor. Ro20-1724 is a PDE4 specific inhibitor.
The cell plates were placed on a Molecular Devices SpectraMax Gemini EM and the baselines (F0) were read before the addition of any compound. 10 μΙ of above agonist stimulation solutions (5X) was added into each well. The plates were recorded again on Gemini EM 50 min (Ft) after the compound addition. The ratio of Ft/FO (Fold) was calculated for each well; data was analyzed and graphed using GraphPad Prism.
The final concentrations of S1 P used are listed in Table 7.
Table 7. An example of the final testing concentrations of S1 P
Figure imgf000111_0001
The testing compounds were assayed the same way. Results
As can be seen from Figure 8, the compounds tested have no agonist activity on S1 PR1 . ii) Antagonist Assay
Based on the above data, 100 nM S1 P (final) was used in the antagonist assay.
The assays were again performed on 384-well plates. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in growth medium. 20 μΙ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
On the second day, the cell plates were taken out from the incubator and an equal volume (20 μΙ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 μΙ.
The testing compounds (1 mM in DMSO) were diluted (1 :200) in 1 X DPBS to 5 μΜ each. 10 μΙ of such solution was added into each well of 384-well plates and incubated at room temperature for 20 min. 10 μΜ of W146 (Final) was used as a positive control.
The cell plates were read on Gemini EM (FO). Afterwards, 12.5 μΙ of 5X agonist stimulation solution (125 μΜ Ro 20-1724, 1 .5 μΜ isoproterenol and 500 nM S1 P prepared in DPBS with 0.1 mg/ml BSA) was added into each well. The cell plates were read again after 50 min (Ft).
Results
As can be seen from Figure 9, the compounds have no antagonist activity on S1 PR1 .
CB2 Assay
ACTOne-CB2 cells used for the assay
CELL LINE DESIGNATION Cannabinoid receptor 2 cell line (CB2)
(CB-80300-225)
ORIGIN (PARENTAL CELL) HEK 293-CNG cell (CB-80200-200)
GENE INTRODUCED Genbank LocusID 1269
RECEPTOR INTRODUCED: Human Cannabinoid receptor 2. (NCBI protein database NP_001832 with SNP at amino acid position 63.)
Usage cAMP assay for Gi-coupled human cannabinoid receptor 2 (CB2).
Quality control
1 . This cell line has been tested negative for Mycoplasma sp.
2. This cell line has been tested positive for CB2 specific response.
3. Surviving rate: More than 2.5 million/vial on the second day after thawing.
Ill 4. The receptor specific activity is stable for 10 weeks continuous passage. Cell culture condition
1 . Growth medium for Cannabinoid receptor 2 cell line: 90% DMEM with Glutamine, 10% FBS, 250 μg/ml G418 and 1 μg/ml puromycin 2. Freezing medium: 10% DMSO, 90% growth medium
Testing compounds on ACTOne-CB2 cells
ACTOne-CB2 cells (CB-80300-225) were maintained in cell culture medium consisting of 90% Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250 μg/ml G418 and 1 μg/ml puromycin. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in the growth medium. 20 μΙ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
On the second day, the cell plates were taken out from the incubator and an equal volume (20 μΙ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 μΙ. The plates will be referred to as the cell plates. i) Agonist Assay
The agonist control picked here was CP-55940 (10mM stock in DMSO, Sigma C1 1 12). Dilute 10mM CP-55940 stock in DMSO containing 30 μΜ Isoproterenol and 2.5 imM Ro 20-1724. These concentrations are 100X the expected final testing concentrations.
Isoproterenol is used to stimulate the adenylyl cyclase through the activation of Gs-coupled endogenous β adrenoceptor. Ro20-1724 is a PDE4 specific inhibitor. Table 8. An example of CP-55940 concentrations in a compound dilution plate
Figure imgf000114_0001
CP-55940 (μΜ)
Further dilute the solutions 1 :20 with 1 X DPBS in compound plates. At this step, the compound concentration is 5X testing concentration.
Table 9. An example of the final testing concentrations of CP-55940 in a cell assay plate
Figure imgf000114_0002
CP-55940 (nM)
The testing compounds were first diluted in DMSO to 2 mM each. They were further diluted to 100 μΜ in DMSO containing 30 μΜ Isoproterenol and 2.5 mM Ro 20- 1724. Further dilute the solutions 1 :20 with 1 X DPBS in compound plates. At this step, the compound concentration is 5X testing concentration.
The cell plates were placed on a Molecular Devices SpectraMax Gemini EM and the baselines (F0) were read before the addition of any compound. 10 μΙ of above agonist stimulation solutions (5X) was added into each well. The plates were recorded again on Gemini EM 50 min (Ft) after the compound addition. The ratio of Ft/FO (Fold) was calculated for each well; data was analyzed and graphed using GraphPad Prism.
Results
As can be seen from Figure 10 the compounds have no agonist activity on CB2. ii) Antagonist Assay
Based on the above data, 200 nM CP-55940 (final) was used in the antagonist assay.
The assays were again performed on 384-well plates. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in growth medium. 20 μΙ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight.
On the second, the cell plates were taken out from the incubator and an equal volume (20 μΙ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 μΙ.
The testing compounds (2 mM in DMSO) were diluted (1 :400) in 1 X DPBS to 5 μΜ each. 10 μΙ of such solution was added into each well of 384-well plates and incubated at room temperature for 20 min. 10 μΜ of AM251 (Final) was used as a positive control.
The cell plates were read on on Gemini EM (F0). Afterwards, 12.5 μΙ of 5X agonist stimulation solution (125 μΜ Ro 20-1724, 1 .5 μΜ isoproterenol and 200 nM CP- 5590 in DPBS) (diluted from 100X solution prepared in DMSO) was added into each well. The cell plates were read again after 50 min (Ft).
Results
As can be seen from Figure 1 1 , the compounds have no antagonist activity on
CB2. CB1 Assay
ACTOne-CB1 cells used for the assay
CELL LINE DESIGNATION Cannabinoid receptor 1 cell line
(CB-80300-205)
ORIGIN (PARENTAL CELL) HEK 293-CNG cell (CB-80200-200)
GENE INTRODUCED Genbank LocusID 1268
RECEPTOR INTRODUCED Human cannabinoid receptor 1 (NCBI protein database P21554)
Usage cAMP assay for Gi-coupled human cannabinoid receptor 1 (CB1 ). Quality control
1 . This cell line has been tested negative for Mycoplasma sp.
2. This cell line has been tested positive for CB1 specific response.
3. Surviving rate: More than 2.5 million/vial on the second day after thawing
4. The receptor specific activity is stable for 10 weeks continuous passage. Cell culture condition
1 . Growth medium for Cannabinoid receptor 1 cell line: 90% DMEM with Glutamine, 10% FBS, 250 μg/ml G418 and 1 μg/ml puromycin
2. Freezing medium: 10% DMSO, 90% growth medium Testing compounds on ACTOne-CB1 cells
ACTOne-CB1 cells (CB-80300-205) were maintained in cell culture medium consisting of 90% Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), 250 μg/ml G418 and 1 μg/ml puromycin. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in the growth medium. 20 μΙ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight. On the second day, the cell plates were taken out from the incubator and an equal volume (20 μΙ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 μΙ. The plates will be referred to as the cell plates.
Table 10. An example of CP-55940 concentrations in a compound dilution plate
Figure imgf000117_0001
Further dilute the solutions 1 :20 with 1 X DPBS in compound plates. At this step, the compound concentration is 5X testing concentration.
Table 11. An example of the final testing concentrations of CP-55940 in a cell assay plate
Figure imgf000117_0002
The testing compounds were first diluted in DMSO to 2 mM each. I. They are further diluted to 100 μΜ in DMSO containing 30 μΜ Isoproterenol and 2.5 mM Ro 20- 1724. Further dilute the solutions 1 :20 with 1 X DPBS in a compound plate. At this step, the compound concentration is 5X testing concentration
The cell plates were placed on a Molecular Devices SpectraMax Gemini EM and the baselines (F0) were read before the addition of any compound. 10 μΙ of above agonist stimulation solutions (5X) was added into each well. The plates were recorded again on Gemini EM 60 min (Ft) after the compound addition. The ratio of Ft/FO (Fold) was calculated for each well; data was analyzed and graphed using GraphPad Prism. Results
As can be seen from Figure 12 the compounds have no agonist activity on CB1 . ii) Antagonist Assay
Based on the above data, 400 nM CP-55940 (final) was used in the antagonist assay. The assays were again performed on 384-well plates. The day before the assay, the cells were trypsinized and diluted to the final concentration of 600K cells/ml in growth medium. 20 μΙ of such cell suspension was added into each well of 384-well plates. Each well contained 12K cells. The plates were then transferred to a cell culture incubator and the cells were growing overnight. On the second day, the cell plates were taken out from the incubator and an equal volume (20 μΙ) of 1X ACTOne membrane potential dye was added into each well and the plates were kept at room temperature in the dark for 2 hrs. The total volume after this step was 40 μΙ.
The testing compounds (1 imM in DMSO) were diluted (1 :200) in 1 X DPBS to 5 μΜ each. 10 μΙ of such solution was added into each well of 384-well plates and incubated at room temperature for 20 min. 10 μΜ of AM251 (Final) was used as a positive control.
The cell plates were read on on Gemini EM (F0). Afterwards, 12.5 μΙ of 5X agonist stimulation solution (125 μΜ Ro 20-1724, 1 .5 μΜ isoproterenol and 2 μΜ CP- 5590 in DPBS) (diluted from 100X solution prepared in DMSO) was added into each well. The cell plates were read again after 60 min (Ft). Results
As can be seen from Figure 13, the compounds have no antagonist activity on
CB1 . in vivo analgesic effects of Compound 29
Compound 29 was tested in rats suffering from neuropathic pain induced by partial ligation of the sciatic nerve. Two routes of compound administration were used - intrathecal and intraperitoneal.
Assay 1 - intrathecal injections
• Species: Rats; Sprague Dawley
• Injury: Partial nerve ligation (PNL)
• Test: von Frey test for allodynia
• Route of administration: Intrathecal
• Vehicle: 25% DMSO, 15% ethanol, 60% saline
• Tested: 2 weeks post injury, 4 days post catheter placement
• Comparison: GlyT2 inhibitor ALX1393 (40 μg bolus injection) The results are shown in Figure 14.
Assay 2 - intraperitoneal injection
• Species: Rats; Sprague Dawley
• Injury: Partial nerve ligation (PNL)
• Test: von Frey test for allodynia • Route of administration: Intaperitoneal
• Vehicle: 10% DMSO, 1 % Soludol in saline
• Tested: 2.5 weeks post injury
• Compound 29: 30 mg/kg (N = 3), and 3 mg/kg (N = 3) · Comparison: GlyT2 inhibitor ORG25543 3 mg/kg N = 2
The results are shown in Figure 15.
As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
References
1 . Gaskin DJ, Richard P. The economic costs of pain in the United States. J Pain :2012;13:715-24.
2. The high price of pain: the economic impact of persistent pain in Australia. Access Economics Reports. 2007.
3. Wiffen PJ et al Gabapentin for acute and chronic pain. The Cochrane database of systematic reviews. 2005:CD005452.
4. von Hehn CA et al. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron. 2012;73:638-52. 5 Vandenberg, R. J., Ryan, R. M., Carland, J. E., Imlach, W. L. & Christie,
M. J. Glycine transport inhibitors for the treatment of pain. Trends in pharmacological sciences 35, 423-430 (2014).
6. Lu Y et al. A feed-forward spinal cord glycinergic neural circuit gates mechanical allodynia. J. Clin Invest. 2013;123:4050-62. 7. Dohi T et al. Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol & Therap. 2009;123:54-79.
8. Roux MJ, Supplisson S. Neuronal and Glial Glycine Transporters have Different toichiometries. Neuron. 2000;25:373-83.
9. Wiles, A. L, Pearlman, R. J., Rosvall, M., Aubrey, K. R. & Vandenberg, R. J. N-Arachidonyl-glycine inhibits the glycine transporter, GLYT2a. Journal of neurochemistry 99, 781 -786 (2006).
10. Vuong, L. A., Mitchell, V. A. & Vaughan, C. W. Actions of N-arachidonyl- glycine in a rat neuropathic pain model. Neuropharmacology 54, 189-193 (2008).
1 1 . Carland, J. E., Mansfield, R. E., Ryan, R. M. & Vandenberg, R. J. Oleoyl- L-carnitine inhibits glycine transport by GlyT2. British journal of pharmacology 168, 891 -
902 (2013).

Claims

CLAIMS 1. A compound of formula (I):
Figure imgf000122_0002
or pharmaceutically acceptable derivatives thereof, wherein:
X is an amino acid or derivative thereof;
L is an amide or retro amide;
Y is Cio - C24 alkyl or alkenyl;
X, L and Y may be independently substituted or unsubstituted; and wherein the compound is not
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
(41 ).
3. A compound according to claim 1 or 2, wherein X is selected from the group consisting of: non-naturally occurring amino acids, naturally occurring aromatic amino acids and naturally occurring hydrophobic amino acids. 4. A compound according to any one of claims 1 -3, wherein X is substituted with 1 , 2, or 3 Ci - C3 alkyl groups at the a carbon of the amino acid or derivative thereof.
5. A compound according to claim 4, wherein the Ci - C3 alkyl is methyl.
6. A compound according to any one of the preceding claims, wherein X is selected from the group consisting of: glycine, L-carnitine, L-serine, D-serine, L-lysine, D-lysine, L-lysine derivative wherein the carbon chain of the lysine side group is of C1 -C3 in length, L-arginine, L-methionine, L-leucine, D-leucine, L-alanine, D-alanine, B-alanine, L-valine,- D-valine, D-phenylalanine, L-phenylalanine derivative wherein the carbon chain of the phenylalanine side group is of two carbons in length, L-tryptophan, L- tyrosinol- L-dopamine, L-aspartate, D-aspartate, L-glutamate, L-histidine, L-lysine methylester, L-norleucine. 7. A compound according to claim 6, wherein X is selected from the group consisting of: glycine, D-lysine, L-tryptophan L-lysine methylester, and L-leucine.
8. A compound according to any one of the preceding claims, wherein L is substituted with a C1 - C3 alkyl group at the nitrogen of the amide or retro amide.
9. A compound according to claim 8, wherein the Ci - C3 alkyl is methyl. 1 0. A compound according to any one of the preceding claims, wherein L is an amide.
1 1 . A compound according to any one of the preceding claims, wherein Y includes 1 - 4 cis double bonds.
1 2. A compound according to any one of the preceding claims, wherein Y is monounsatu rated and includes 1 cis double bond.
1 3. A compound according to claim 1 2, wherein the cis double bond is 5 - 1 5 carbons from L.
14. A compound according to claim 1 2 or 1 3, wherein the cis double bond is 8 - 10 carbons from L. 1 5. A compound according to any one of claims 12 to 14, wherein Y is a
monounsatu rated Ci8 carbon chain.
16. A compound according to any one of claims 12 to 14, wherein Y is a monounsatu rated C16 carbon chain.
17. A compound according to claim 16, wherein the monounsaturated Ci6 carbon chain includes a cis-double bond in the ω 5, 6, 7, 9, or 1 1 position. 18. A compound according to any one of claims 1 to 10, wherein Y is a saturated C14 carbon chain.
19. A compound according to claim 1 selected from the group consisting of:
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
130
Figure imgf000132_0001
131
Figure imgf000133_0001
Figure imgf000133_0002
132
Figure imgf000134_0001
ı33
Figure imgf000135_0001
134
Figure imgf000136_0001
A compound according to claim 1 selected from the group consisting of
Figure imgf000136_0002
Figure imgf000137_0001
22. A method of treating pain by administering an effective amount of a compound according to any one of the preceding claims, a salt or pharmaceutically acceptable derivative thereof, to a subject in need thereof.
23. A method of treating pain by administering an effective amount of at least one compound selected from the group consisting of:
Figure imgf000137_0002
Figure imgf000138_0001

Figure imgf000139_0001
138
Figure imgf000140_0001
a salt, or pharmaceutically acceptable derivative thereof, to a subject in need thereof.
24. Use of an effective amount of a compound according to any one of claims 1 to 21 , a salt or pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for treatment of pain in a subject in need thereof.
Use of an effective amount of at least one compound selected from the group isting of:
Figure imgf000140_0002
Figure imgf000141_0001
6
a salt, or pharmaceutically acceptable derivative thereof, in the manufacture of a medicament for treatment of pain in a subject in need thereof.
26. A compound according to any one of claims 1 to 21 , a salt or pharmaceutically acceptable derivative thereof, for use in treating pain in a subject in need thereof. 27. A compound selected from the group consisting of:
Figure imgf000143_0001
Figure imgf000144_0001
143
Figure imgf000145_0001
Figure imgf000145_0002
a salt, or pharmaceutically acceptable derivative thereof, for use in treating pain in a subject in need thereof.
28. A method according to claim 22 or 23, a use according to claim 24 or 25, or a compound according to claim 26 or 27, wherein the pain is chronic pain.
29. A method according to claim 22 or 23, a use according to claim 24 or 25, or a compound according to claim 26 or 27, wherein the pain is neuropathic pain.
30. A pharmaceutical composition comprising a compound according to any one of claims 1 to 21 , together with one or more pharmaceutically acceptable carriers.
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