WO2019135386A1

WO2019135386A1 - Deuterated phospholipid and method for producing same

Info

Publication number: WO2019135386A1
Application number: PCT/JP2018/048187
Authority: WO
Inventors: 直道馬場; 三澤　嘉久; 忠広対馬
Original assignee: 備前化成株式会社
Priority date: 2018-01-04
Filing date: 2018-12-27
Publication date: 2019-07-11
Also published as: JP7279941B2; JPWO2019135386A1

Abstract

The present invention provides a novel deuterated phospholipid and a method for producing same. The synthetic deuterated phospholipid according to the present invention can be used to address problems in the field. Antiphospholipid antibody syndrome has a clinical problem regarding the difficulty in establishing a detection standard thereof, due to the diversity in antiphospholipid antibodies. The present invention also provides a diagnostic agent which can facilitate such detection. The present invention is also applicable to measurement of degradation of food by tracking changes in the phospholipid of the present invention added to food.

Description

Deuterated phospholipid and method for producing the same

The present invention relates to deuterated phospholipids, a process for their preparation and their use.

A large number of molecular species with different structures exist in the phospholipid group indispensable as cell membrane components or biological signal transduction substances. The basic structure of glycerophospholipids is that the same or different fatty acids such as fatty acids such as palmitic acid, stearic acid, icosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid are bound to two hydroxyl groups of the glycerol skeleton, and the third hydroxyl group is bound Polar groups such as phosphocholine, phosphoserine, phosphoethanolamine, etc. are bound. Therefore, a huge number of kinds of phospholipid molecular species exist in living tissues, depending on the number of combinations thereof. In addition, their molecular species exhibit different biological functions. The association between individual phospholipids and pathological conditions has also been studied for a long time, and in recent years analysis has been performed using a lipidomics method combining liquid chromatography and mass spectrometry techniques.

However, there are cases where functional studies of phospholipids are difficult even using such analytical techniques. For example, it is extremely difficult to trace at the molecular level the process of metabolism and chemical conversion of a specific phospholipid molecular species. As a means to solve this problem, phosphatidylcholine in which one of three methyl groups bonded to the nitrogen atom of the polar group of choline is substituted with deuterium, which is a stable isotope, was synthesized by Baba et al. Patent documents H. Tominaga, T. Ishihara, A. K. M. Azad Shah, R. Shimizu, A. N. Onyango, H. Ito, T. Suzuki, Y. Kondo, H. Koaze, K. Takahashi, Naomichi Baba, American Journal of Analytical Chemistry, 2013, 4, 16, 26).

This phospholipid was found to be detected with extremely high selectivity by detection in the product ion mode of electrospray ionization mass spectrometry (ESI-MS). Even in the coexistence of all kinds of phospholipids present in living tissues, deuterated phospholipids and metabolites in the acyl moiety thereof can be detected very specifically and sensitively. This method revealed the metabolic process in the blood of phospholipid peroxide synthesized from linoleic acid. (Non-patent document 2)
The fatty acids bound in phospholipids have different functions. For example, arachidonic acid is essential for the growth and reproduction of infants, EPA is known to contribute to blood homeostasis controlling heart disease and the like, and DHA is known to be deeply involved in brain function homeostasis. These omega-3 fatty acids are usually bound to triglycerides and phospholipids in the living body, and are cut out by metabolic enzymes as necessary to play their role. For example, when inflammation occurs, phospholipase A2 is expressed at that site, and EPA, DPA and DHA bound to the surrounding cell membrane are released from phospholipids, and these are metabolized by lipoxygenase (LOX) and cyclooxygenase (COX), and resolvin A metabolite called SPM (specialized proresolving mediator) represented by protectin and mercerin is produced and involved in suppression of inflammation. In addition, important phospholipids to which such metabolites are bound have also been discovered. However, omega-3 fatty acids bound in phospholipids may produce phospholipid-type metabolites by the direct action of the above-mentioned enzymes, and the details are not known. It is known that soybean lipoxygenase acts on omega-3 fatty acids bound in phospholipids to introduce a hydroperoxy group. (Non-patent document 1)
In addition, neutrophils activated by inflammation, natural killer cells, various active oxygen produced by macrophages, such as hydrogen peroxide and ozone, attack bacteria at the site of inflammation, but they are extremely highly reactive. These reactive oxygen species nonenzymatically oxidize omega 3 fatty acids bound in surrounding omega 3 fatty acids and phospholipids to produce other phospholipids that have undergone structural change and exhibit some biological function There is a possibility, but such a mechanism is hardly clear. The deuterated phospholipids provided by the present invention can be an important tool in elucidating these unknown mechanisms.

Omega-3 fatty acids taken in by the diet are present in the blood as triglycerides and phospholipids, and are transferred to each tissue as needed. For example, it has recently been revealed that DHA is lysophospholipid-bound in the liver and translocates into the brain through the brain barrier with blood flow. It is also known that DPA that has been transferred into the brain is once converted to C24 (5n-3) and then turned to 6n-3 by the desaturase, which again becomes DHA with a decrease of 2 carbons. Although the mechanism is unclear. Arachidonic acid bound in phospholipids undergoes various complex oxidation reactions in the metabolic process, and as a result, phospholipids in which various fatty acids that have undergone oxidative degradation are bound have been identified. And, interestingly, these molecular species are reported to be inflammatory or show anti-inflammatory activity. (Non-patent document 3)
However, in this study, a mixture of various phospholipid oxidized metabolites generated from phospholipids bound with arachidonic acid is used. Therefore, it is not clear which molecular species show an inflammatory action or an anti-inflammatory action. In addition, since this study is limited to arachidonic acid, it is unclear what function the respective oxidation products of EPA, DPA, and DHA bound phospholipids exhibit.

As described above, the functions of phospholipids have not yet been fully elucidated, and new tools for their analysis are desired. The synthetic deuterated phospholipids provided by the present invention can be applied to the solution of problems in such fields. Furthermore, antiphospholipid antibody syndrome has clinical problems that are said to be difficult to standardize its detection due to the diversity of antiphospholipid antibodies, and the present invention can facilitate such detection. It also provides diagnostic agents.

Accordingly, the present invention provides the following.
(Item 1)
The following formula I

(In the formula,
R ¹ and R ² are each independently —H or —C (= O) R,
R is a linear or branched saturated or unsaturated hydrocarbon group,
Wherein the hydrocarbon group contains 8 to 36 carbon atoms,
The hydrocarbon group contains 0-8 double bonds,
At least one of carbons on the hydrocarbon group may be substituted with a substituent selected from the group consisting of = O, -OH and -OOH,
At least one of two adjacent sets of carbons on the hydrocarbon group is substituted with O to be an epoxy group

May form a
Each R ^A is independently selected from hydrogen, deuterium or tritium)
Or a salt thereof.
(Item 2)
The compound or a salt thereof according to item 1, wherein all R ^A on the same carbon atom is the same hydrogen atom selected from hydrogen, deuterium or tritium.
(Item 3)
A method for producing a compound according to item 1 or a salt thereof, comprising
Step 1) Trideuterio methyl group introduction reagent

Preparing the
Process 2) The following structure

Synthesizing a choline containing a deuterated methyl group selected from
Step 3) protecting 1-hydroxy group and 3-hydroxy group of glycerol,
Step 4) protecting the 2-hydroxy group of glycerol,
Step 5) Deprotecting 1-hydroxy group and 3-hydroxy group of glycerol,
Step 6) Step of introducing fatty acid of R ¹ -OH structure into 1-hydroxy group of glycerol, step 7) Introduction of choline containing phosphate group and deuterated methyl group of step 2 into 3-hydroxy group of glycerol The process to
Step 8) Deprotecting 2-hydroxy group of glycerol,
Step 9) Deuteration including at least one of the steps of introducing a fatty acid of the structure of R ² —OH into 2-hydroxy group of glycerol, optionally further introduced in the following step 10) step 7 A production method comprising the steps of: converting choline containing a methyl group into aminoethanol; and introducing a deuterated methyl group into aminoethanol.
(Item 4)
A composition comprising the compound according to item 1 or 2 or a salt thereof.
(Item 5)
5. A composition according to item 4 for the measurement of phospholipid metabolism disorders.
(Item 6)
5. A composition according to item 4 for the diagnosis of a disease or condition.
(Item 7)
7. The composition according to item 6, wherein the disease or condition is a disease causing an abnormality in lipid metabolism.
(Item 8)
7. The composition according to item 6, wherein the disease or condition is selected from the group consisting of obesity, hyperlipidemia, non-insulin dependent diabetes and osteoarthritis of the knee.
(Item 9)
7. The composition according to item 6, wherein the disease or condition is selected from the group consisting of cancer, inflammation, arteriosclerosis, Alzheimer's disease, sepsis and antiphospholipid antibody syndrome.
(Item 10)
A method for producing palmitoyl glycerol palmitoylated with unsaturated fatty acid, which is selected from the group consisting of (i) Formula (A), Formula (B), and Formula (C)

(Wherein, R ₁ to R ₁₁ are independently H or an alkyl group having 1 to 12 carbon atoms), and
(Ii) unsaturated fatty acids,
Performing a lipase-enzyme reaction using a substrate as a substrate.
(Wherein R ₁ to R _{11 each} independently represent H or an alkyl group having 1 to 12 carbon atoms, H or an alkyl group having 1 to 11 carbon atoms, H or an alkyl group having 1 to 10 carbon atoms, H or 1 carbon atom) -9 alkyl group, H or alkyl group having 1 to 8 carbon atoms, H or alkyl group having 1 to 7 carbon atoms, H or alkyl group having 1 to 6 carbon atoms, H or alkyl group having 1 to 5 carbon atoms, H or an alkyl group having 1 to 4 carbon atoms, or H or an alkyl group having 1 to 3 carbon atoms)
(Item 11)
11. The method according to item 10, wherein said unsaturated fatty acid contains 8 to 36 carbon atoms and contains 1 to 8 double bonds.
(Item 12)
11. The method according to item 10, wherein at least one of carbons on the unsaturated fatty acid may be substituted with a substituent selected from the group consisting of = O, -OH and -OOH, At least one of two adjacent sets of carbons on a hydrocarbon group is substituted with O to be an epoxy group

May form a way.
(Item 13)
11. The method according to item 10, wherein the lipase enzyme reaction is performed at a temperature of −20 ° C. to 50 ° C. for 0.5 hour to 24 hours.
(Item 14)
The method according to item 10, further using an acylating agent.
(Item 15)
The method according to item 10, further using a solvent.

In the present invention, it is contemplated that the one or more features described above may be provided in further combination in addition to the explicitly stated combination. Additional embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, as appropriate.

The present invention can contribute to the functional study of phospholipids. In addition, the present invention can provide new diagnostic agents for diseases associated with phospholipids.

Hereinafter, the present invention will be described. Throughout the specification, it is to be understood that the singular form also includes the concepts of the plural, unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meanings commonly used in the art unless otherwise stated. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Also, in the present specification, "wt%" is used interchangeably with "mass percent concentration". "%" Means "wt%" or "w / w%" or "weight percent concentration" unless otherwise stated.

In the present specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or". In the present specification, when “within the range” of “two values” is specified, the range also includes the two values themselves.

References cited herein, such as scientific literature, patents, patent applications, etc., are incorporated herein by reference in their entirety to the same extent as specifically described.

(Definition)
Hereinafter, definitions of terms and / or basic technical contents specifically used in the present specification will be appropriately described.

The term "unsaturated fatty acid" as used herein refers to a fatty acid with one or more unsaturated carbon bonds. An unsaturated carbon bond is an unsaturated bond between carbons in a carbon molecular chain, that is, a carbon double bond or a triple bond. Naturally occurring unsaturated fatty acids have one or more double bonds, and by replacing saturated fatty acids in fatty acids, they change the properties of fats such as melting point and fluidity.

As used herein, the term "ω3 fatty acid" refers to an unsaturated fatty acid in which a double bond is present from the third carbon at the methyl terminal (ω3 position), and typically, α-linolenic acid, stearidone Acids, eicosatetraenoic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPAn-3), docosahexaenoic acid (DHA), tetracosapentaenoic acid, and tetracosahexaenoic acid. It is not limited. As used herein, the term "modified omega-3 fatty acid" refers to a substance modified with omega-3 fatty acid and includes an oxide of omega-3 fatty acid (eg, oxidized metabolite), typically 13- Hydroxy-α-linolenic acid, 13-hydroxystearidonic acid, 15-hydroxyeicosatetraenoic acid, 15-hydroxyEPA, 17-hydroxyDPAn-3, 17-hydroxy DHA, 19-hydroxytetracosapentaenoic acid, and And 19-hydroxytetracosahexaenoic acid, but is not limited thereto.

As used herein, the term "docosahexaenoic acid" is used interchangeably with "DHA" and includes both free fatty acid forms and forms esterified to ethanol, glycerin, phospholipids.

As used herein, the term "eicosapentaenoic acid" is used interchangeably with "EPA" and includes both the free fatty acid form and the form esterified to ethanol, glycerin, phospholipids Do.

As used herein, the term "docosapentaenoic acid" is used interchangeably with "DPA" and includes both the free fatty acid form and the esterified form of ethanol, glycerin. In addition, “DPA” not specified as either “n-3” or “n-6” is “n-3 DPA” (ie, all-cis-7, 10, 13, 16, 19-docosa) Mean pentaenoic acid). "N-3 DPA" is "ω-3
Used interchangeably with DPA.

The term "n-6 DPA" as used herein is used interchangeably with "all-cis-4,7,10,13,16-docosapentaenoic acid" or "ω6 DPA" and is free It includes both fatty acid forms and forms esterified to ethanol and glycerin.

As used herein, the term "stearidonic acid" is used interchangeably with "SDA" and includes both the free fatty acid form and the esterified form of ethanol, glycerin.

As used herein, the term "alpha-linolenic acid" is used interchangeably with "ALA" and encompasses both the free fatty acid form and the esterified form of ethanol, glycerin.

As used herein, the term "eicosatetraenoic acid" is used interchangeably with "ETA" and encompasses both the free fatty acid form and the esterified form of ethanol, glycerin.

The term "hydrogen" as used herein refers to ¹ H of the isotopes of hydrogen atoms, also denoted as H. The hydrogen atoms contained in naturally occurring compounds include isotopes such as deuterium and tritium in a certain proportion, but when using hydrogen or H in the present specification, all strictly contained in the compounds does not mean that the hydrogen atoms are ^{1 H,} it does not exclude that the isotopes of hydrogen, such as deuterium and tritium are included in the normal degree included.

The term "deuterium" as used herein refers to the ² H of the isotopes of hydrogen atoms, also denoted D or d.

The term "tritium" as used herein refers to the ³ H of the isotopes of hydrogen atoms, also denoted as T or t.

The term "hydrogen species" as used herein, is used to described without distinguishing isotopes of hydrogen atom ^{(1 H,} 2 ^H and ^{3 H),} hydrogen atom ^{1 H,} ² H And indicate that it is any of ³ H.

The term "phospholipid" as used herein refers to an amphiphilic substance having a hydrophobic group composed of a long chain alkyl group in the molecule and a hydrophilic group containing a phosphate group. Examples of phospholipids include phosphatidyl choline (= lecithin), phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, sphingophospholipid, diphosphatidyl phospholipid, palmitoyl oleoyl phosphatidyl choline, lysophosphatidyl choline, lysophosphatidyl ethanolamine , Dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol (DPPG), dioleoylphosphatidyl choline (DOPC), distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl glyc Seroru (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoyl distearoylphosphatidylcholine (PSPC), palmitoylstearoyl distearoyl phosphatidylglycerol (PSPG), dilinoleoyl phosphatidylcholine, and the like without limitation.

The terms "phosphatidyl choline" or "lecithin", as used herein, is a compound used interchangeably and also denoted as PC and of glycerol (propane-1,2,3-triol, also referred to as glycerol) An acyl group is attached to the sn-1 position and the sn-2 position, and choline is attached to the phosphate linked to the sn-3 position.

As used herein, the term "lysophosphatidylcholine" is a compound also referred to as lyso-PC, wherein an acyl group is attached to either the sn-1 position or the sn-2 position of glycerol, It refers to one in which choline is linked to the phosphate linked to the 3-position.

The term "sn-" as used herein is an abbreviation for Stereospecifically Numbered and is used when the carbon atom of a glycerol derivative is designated by stereospecific numbering. When the glycerol derivative is a racemate, rac- is attached, and when the stereochemistry is unknown, X- is attached. There are two types of phospholipids, sn-1-phospholipid and sn-2-phospholipid, depending on the difference in the bonding position of the acyl group. For the actual designation, the position of the acyl group is used as it is, as in 1-acyl-phosphatidylcholine, 2-acyl-phosphatidylcholine. In the present specification, when there is no information on sn, it is the case where it is described without distinction or used as a generic term (upper-level concept).

As used herein, the term "acyl (group)" is used in the usual sense in the art to refer to a group formed by removing a hydroxyl group from an organic acid (carboxylic acid; fatty acid). Broadly, formyl group HCO-, acetyl group CH ₃ CO-, malonyl group -COCH ₂ CO-, benzoyl group C ₆ H ₅ CO-, cinnamoyl group C ₆ H ₅ CH = CHCO- and the like, ketone derivatives And so on. The acyl group contained in phosphatidylinositol phosphate and lysophosphatidylinositol phosphates is also referred to as a fatty acid group because it forms a fatty acid in a preferred embodiment. As used herein, a fatty acid can be represented by its carbon number and the number of double bonds, for example, arachidonic acid can be represented as (20: 4). When specifying the position of the double bond further, specify all positions for the indication of cis or trans, or the indication of E or Z, or indicate in a system such as omega (ω) 3 system or omega 6 system can do.

In the present specification, generally, the following fatty acids and acyl groups based thereon are used, but it is understood that fatty acids having any chain length and any double bond can be used without limitation thereto. For example, in terms of carbon number, one or more, typically from 1 to 30, usually from 4 to 30, may be mentioned, and one, two, three, four, five, six, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 18, 19, 20, 21, 22, 23. , 24, 26, 27, 28, 29, 30, etc., but is not limited thereto. Further, the number of double bonds may be 0, 1, 2, 3, 4, 5, 6, 7, etc. According to the number of carbon atoms, any permissible number can be adopted. The position of the double bond is typically omega-3, omega-6, omega-9, etc. In addition to this, omega-1, omega-4, omega-5, omega-7, etc. are also confirmed. Any of these available can be used. The fatty acid may contain triple bonds, and the number thereof is 0, 1, 2, 3, 4, 5, 6, 6, 7 or any other acceptable number of carbon atoms. A number can be adopted.

As used herein, the term "diagnosis" identifies various parameters associated with a disease, disorder, condition (eg, a disease caused by a lipid mediator, disorder, condition etc.) in a subject, etc. To determine the current or future state of a disease, disorder, or condition. By using the method, device and system of the present invention, the condition in the body can be examined, and such information can be used to formulate a treatment, treatment or prophylaxis to be administered for a disease, disorder, condition or subject in a subject. Or various parameters, such as a method, can be selected. In the present specification, in a narrow sense, "diagnosis" refers to diagnosing the current state, but broadly includes "early diagnosis", "predictive diagnosis", "pre-diagnosis" and the like. The diagnostic method of the present invention is industrially useful in principle because it can be used from the body and can be performed with the hands of medical workers such as doctors removed. In the present specification, in order to clarify that it can be carried out with the hands of medical workers such as doctors, "predictive diagnosis, prior diagnosis or diagnosis" may be referred to as "support". The techniques of the present invention are applicable to such diagnostic techniques. The techniques of the present invention can be used to identify the presence of deuterated phospholipids and be applied to such a variety of diagnostics.

The term "prognosis" as used herein is meant to predict the possibility of death or progression due to a disorder such as cancer, a disease due to lipid mediators, a disorder or the like. Prognostic factors are variables relating to the natural course of the disease, which affect the recurrence rate etc. of the patient once having developed the disease. Clinical indicators associated with aggravated prognosis include, for example, any cellular indicator used in the present invention. Prognostic factors are often used to classify patients into subgroups with different disease states. The ability to identify the presence of deuterated phospholipids using the techniques of the present invention may be useful as a technique for providing a prognostic factor since it can be linked to a specific disease state.

As used herein, the term "diagnostic agent (agent)" broadly refers to any agent capable of diagnosing a condition of interest (eg, cancer, a disease or disorder associated with a lipid mediator, etc.).

The term "cancer" as used herein refers to any cancer that can be treated or prevented with a vaccine or drug, such as hepatocellular carcinoma, esophageal squamous cell carcinoma, breast cancer, pancreatic cancer, Squamous cell or adenocarcinoma of the head and neck, colorectal cancer, renal cancer, brain cancer (tumor), prostate cancer, small cell and non small cell lung cancer, bladder cancer, bone or joint cancer, Uterine cancer, cervical cancer, multiple myeloma, hematopoietic malignancy, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma, squamous cell cancer, leukemia, lung cancer, ovarian cancer, gastric cancer, Kaposi Sarcoma, laryngeal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, pituitary cancer, adrenal cancer, cholangiocellular carcinoma, endometriosis, esophageal cancer, liver cancer, NSCLC, osteosarcoma Includes pancreatic cancer, SCLC, soft tissue tumor, AML, and CML That, but is not limited to these.

As used herein, the term "lipid metabolism disorder" is a disorder caused by an abnormality in lipid or fatty acid metabolism. Lipid metabolism disorders include, but are not limited to, for example, obesity, hyperlipidemia, non-insulin dependent diabetes.

In the present specification, “inflammation” refers to any detectable inflammation, and includes activation of blood cells associated with inflammation such as macrophages (chemotaxis of inflammatory cells, active oxygen production, phagocytosis, enzyme secretion reaction, etc.) May accompany. Exemplary inflammations include, for example, arthritis, tendinitis, bursitis, psoriasis, cystic fibrosis, Sjögren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), spondylitis, multiple Myositis, dermatomyositis, pemphigus, pemphigus, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD such as Crohn's disease, ulcerative colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, Silicosis, bronchiectasis, talcum lung, pneumoconiosis, sarcoidosis, delayed hypersensitivity reaction (eg, poison ivy dermatitis), airway inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reaction, asthma, hay fever, allergy , Acute anaphylaxis, reperfusion injury, rheumatoid arthritis, glomerulonephritis, pyelonephritis, cellulitis, cystitis, cholecystitis, allograft rejection, host versus graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis ,bronchitis Cervicalitis, cholangitis, chorioamnionitis, conjunctivitis, lacrimal glanditis, dermatomyositis, endocarditis, endometritis, enterocolitis, enterocolitis, epicondylitis, epididymitis, fasciitis, conjunctivitis , Gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, umbilitis, ovarian disease, orchitis, otitis media, pancreatitis, parotiditis, pericarditis , Pharyngitis, pleuritis, phlebitis, pneumonia, proctitis, prostatitis, rhinitis, odontitis, sinusitis, stomatitis, synovitis, testicular inflammation, tonsillitis, urethritis, cystitis, uveitis, vagina Diseases or disorders associated with acute or chronic inflammation, including, but not limited to, inflammation, vasculitis, vulvovaginitis, vulvovaginitis, vasculitis, osteomyelitis, optic neuritis, myelitis, and fasciitis .

As used herein, the term "marker" refers to or is at risk for a condition (eg, functional, transformed, diseased, impaired, or proliferative capacity, level of differentiation, etc). A substance that serves as an indicator to track whether there is a sex. In the present invention, detection, diagnosis, preliminary detection, prediction or prior diagnosis for a certain condition (eg, disease such as disease state, health condition, cell or tissue differentiation disorder, etc.) is not limited to the detection method provided by the present invention. , A marker-specific agent, agent, factor or means, or a composition, kit, system or the like containing them.

As used herein, the term "cancer marker" is also referred to as a tumor marker, and is a substance found in biological substances or organisms effective for cancer diagnosis and follow-up after treatment, detection of recurrence and metastasis. Say. Typically, by measuring substances in the blood, cancer diagnosis and the like can be performed. In this case, the substance in the blood corresponds to a cancer marker.

(Deuterated phospholipid)
In one aspect, the present invention provides compounds of formula I

It may be formed,
Each R ^A is independently selected from hydrogen, deuterium or tritium)
Or a salt thereof.

In addition, fatty acids containing the above R include, for example, the following moieties, but are not limited thereto:
1) Saturated fatty acid: a saturated fatty acid having a carbon chain of C8 to C36.
2) Unsaturated fatty acid: fatty acid containing carbon at C8 to C36 and containing 1 to 8 double bonds.
3) Hydroxyl-binding type fatty acid: fatty acid having hydroxyl group bonded. The number of hydroxyl groups to be added is 1 to 3 per fatty acid.
4) Epoxy type fatty acid: fatty acid obtained by epoxidizing the double bond of unsaturated fatty acid of 2). Epoxy group is 1-2 per fatty acid.
5) Oxo type fatty acid: those having a carbonyl group or an aldehyde group in the fatty acid in addition to the carbonyl group of the carboxyl group of the fatty acid are also called oxo type fatty acids. The carbonyl group is 1 to 3 per fatty acid.
6) Hydroperoxy-type fatty acid ..... a fatty acid having a hydroperoxy group bonded. The hydroperoxy group is 1 to 3 per fatty acid.
7) Fatty acids in which the functional groups shown in the above 2) to 6) are complexly bonded to fatty acids 8) In the case of fatty acids in which a carbonyl group or an epoxy structure is bonded together with a hydroxyl group or a hydroperoxy group, only hydroxyl groups and hydroperoxy groups are protected Just do it.

In addition, DCC and DMAP are used in chloroform as fatty acid binding reagents to the hydroxyl group of sn-2, but generally, many other reagents such as 1-hydroxybenzotriazole and diphenyl phosphate azide can be used. .

In one embodiment, R is a linear saturated or unsaturated hydrocarbon group. In one embodiment, R is a linear saturated hydrocarbon group. In one embodiment, R is a linear unsaturated hydrocarbon group. In one embodiment, the hydrocarbon group contains 0, 1, 2, 3, 4, 5, 6, 7 or 8 double bonds. In one embodiment, the hydrocarbon group comprises a double bond in at least one of the positions selected from omega 1, omega 3, omega 4, omega 5, omega 6, omega 7 or omega 9. In one embodiment, R ¹ is a lauryl group, myristoyl group, palmitoyl group, stearyl group, 9,12-octadecanedienoyl group, 9,12,15-octadecanetrienoyl group, 5,8,11,14- Eicosatetraenoyl group, 4, 7, 10, 13, 16-docosapentaenoyl group, 7, 10, 13, 16, 19-docosapentaenoyl group, 4, 7, 10, 13, 16, 19- It is selected from the group consisting of docosahexaenoyl groups. In one embodiment, R ¹ is an acyl group comprising a linear or branched saturated hydrocarbon group containing 8 to 36 carbon atoms selected, for example a lauryl group, a myristoyl group, a palmitoyl group, Stearyl group and the like. In one embodiment, R ² represents a lauryl group, myristoyl group, palmitoyl group, stearyl group, 9,12-octadecanedienoyl group, 9,12,15-octadecanetrienoyl group, 5,8,11,14- Eicosatetraenoyl group, 4, 7, 10, 13, 16-docosapentaenoyl group, 7, 10, 13, 16, 19-docosapentaenoyl group, 4, 7, 10, 13, 16, 19- It is selected from the group consisting of a docosahexaenoyl group and

In one embodiment, all R ^A on the same carbon atom are the same hydrogen species selected from hydrogen, deuterium or tritium. In one embodiment, only R ^A present in one of three methyl groups attached to the N of the R ^A is deuterium or tritium, all R ^A present on the other methyl group It is hydrogen. In one embodiment, only R ^A present in one of three methyl groups attached to the N of the R ^A is hydrogen, all R ^A present on the other methyl group are deuterium or tritium It is hydrogen. In one embodiment, all of R ^A present on three methyl groups attached to the N of the R ^A is deuterium or tritium. In one embodiment, two R ^A on carbon directly attached to the phosphate group are deuterium or tritium. In one embodiment, two R ^A on carbon directly attached to the phosphate group are deuterium or tritium. In one embodiment, two R ^A on carbon R ^A two bound of the carbon directly bonded to N is deuterium or tritium.

In one embodiment, the compound of the present invention is a compound in which hydrogen in a naturally occurring compound is replaced with deuterium or tritium. In one embodiment, the compound of the present invention is a compound in which hydrogen in a compound known to be present in food is replaced by deuterium or tritium. In one embodiment, the compound of the present invention is a compound in which hydrogen in a compound present in a mammal is replaced with deuterium or tritium. In one embodiment, the compound of the present invention is a compound in which hydrogen in a compound known to exist in human is replaced by deuterium or tritium.

(Production of deuterated phospholipids)
In one aspect, the invention provides a method of producing a compound of the invention.

Each step in the method for producing the compound of the present invention is described below. In the following production examples, although deuterium is used for illustration, the same reaction can be performed using tritium instead of deuterium.

Synthetic scheme 1

(Step 1: Preparation of trideuteriomethyl group introducing reagent)

The reaction of p-toluenesulfonyl chloride with tetradeuteriomethanol gives trideuteriomethyl p-toluenesulfonate. This reaction can be allowed to proceed, for example, by slowly adding 35% aqueous sodium hydroxide solution to the mixture at -5 to 0 ° C. In addition, the phospholipid which couple | bonds with the dejuteio methyl group or the mono-deuterio methyl group is also available. Instead of p-toluenesulfonyl chloride, another reagent such as 2,4,6-triisopropylbenzene sulfonyl chloride, or various aryl and alkyl sulfonic acid halogenation can also be suitably used.

(Step 2: Synthesis of Choline Containing Deuterated Methyl Group)

The deuterated choline is obtained by reacting N, N-dimethylethanolamine with the trideuteriomethyl p-toluenesulfonate obtained above. Here, instead of N, N-dimethylaminoethanol, its deuterium substitution can be used. As the deuterium-substituted choline obtained in this manner, for example,

There is a structure of, but not limited to. By using these deuterium-substituted cholines in place of compound (5), compounds containing these deuterium-substituted derivatives as final products can be obtained.

It is also possible to obtain 9-deuterated phospholipid (18) by using choline in which three methyl groups are deuterated to phosphatidic acid and using 2,4,6-triisopropylbenzenesulfonyl chloride as a condensing agent.

(Step 3: protection of 1- and 3-hydroxy groups of glycerol)

Glycerol and benzaldehyde are reacted under acidic conditions to protect glycerol 1- and 3-hydroxy groups. Instead of benzaldehyde, a compound that provides another suitable protecting group that protects the 1- and 3-hydroxy groups of glycerol may be used if necessary.

(Step 4: protection of 2-hydroxy group of glycerol)

Introduce a protecting group to the 2-hydroxy group of glycerol. For example, by adding benzyl chloride under basic conditions, a benzyl group can be introduced as -OR '. -OR 'is any suitable substituent and is preferably selected so as not to be removed under the conditions for removing the protecting groups of 1- and 3-hydroxy groups of glycerol. In one embodiment, -OR 'may be selected a substituent that readily causes elimination. In one embodiment, -OR 'can be benzyl, methoxy, ethoxy, isopropyloxy and the like.

(Step 5: Deprotection of 1- and 3-hydroxy groups of glycerol)

The protecting groups of the 1- and 3-hydroxy groups of glycerol are removed. For example, when protected with benzaldehyde, it can be deprotected by hydrolysis under acidic conditions. As the reaction conditions at this time, it is preferable to use conditions in which -OR 'is not removed.

(Step 6: Introduction of fatty acid to 1-hydroxy group of glycerol)

A fatty acid is introduced into the 1-hydroxy group of glycerol. R ¹ is —C (= O) R and R is a linear or branched saturated or unsaturated hydrocarbon group, wherein the hydrocarbon group contains 8 to 36 carbon atoms, The hydrocarbon group contains 0 to 8 double bonds, and at least one of carbons on the hydrocarbon group is substituted with a substituent selected from the group consisting of = O, —OH and —OOH It may be It may be introduced enzymatically by a lipase, or may be introduced by a known chemical esterification method. When this reaction is carried out by a lipase, for example, R ′ is selected to have a substituent that becomes a substrate and recognized by the lipase such as benzyl group, methoxy group, ethoxy group, isopropyloxy group, etc. Temperature, solvent, concentration etc) are properly controlled. When the reaction is carried out by a lipase, a product with high optical purity can be obtained. When the reaction is carried out by a chemical esterification method, the product can be obtained as a racemate.

Any lipase using a lipid such as triglyceride as a substrate can be used as the lipase of the present invention. The lipase used in the present invention is preferably a lipase (lipolytic enzyme) that selectively hydrolyzes the 1- and / or 3-position of triglyceride. The lipase may be a natural enzyme or a recombinant enzyme. Furthermore, the "lipase" used in the present invention may be in the form of a solution or in an immobilized form. Examples of microorganism-derived lipases include, for example, Lipase OF (trade name, manufactured by Meito Sangyo Co., Ltd.), Alcaligenes sp. -Derived lipase QLM, lipase QLC, lipase PL (all are trade names, manufactured by Meisho Sangyo Co., Ltd.), Lipase PS derived from Burkholderia cepacia (trade name, manufactured by Amano Enzyme Inc.), lipase AK derived from Pseudomonas fluorescens Examples include trade names, manufactured by Amano Enzyme Co., Ltd., and the like, but are not limited thereto. Preferably, the lipase used in the present invention is a microorganism-derived lipase that selectively hydrolyzes positions 1 and 3 of triglyceride. In the present invention, immobilized lipase, for example, immobilized lipase derived from Thermomyces lanuginosa, Lipozyme TLIM (trade name, manufactured by Novozyme Co., Ltd.) can be used, but it is not limited thereto. A particularly preferred lipase according to the invention is the lipase AmanoPS. The lipase enzyme protein preferably has general stability such as physical and biochemical, and more preferably chemically stable to an organic solvent. Those skilled in the art can obtain desired stereoselectivity by appropriately selecting and adding a solvent, an acylating agent, a temperature, a concentration and the like. The preferred solvent for the lipase reaction is dichloromethane. The preferred temperature during the lipase reaction is -20 ° C to 50 ° C, more preferably -10 ° C to 30 ° C, still more preferably -5 ° C to 20 ° C, most preferably -2 ° C to 10 ° C, eg 10 ° C It is. The preferred reaction time of the lipase reaction is 0.5 hours to 24 hours, more preferably 1 hour to 10 hours, still more preferably 2 hours to 7 hours, most preferably 3 hours to 6 hours, eg 6 hours . As the acylating agent, not only palmitic acid vinyl ester but also palmitic acid isopropenyl ester, palmitic acid trifluoroethyl ester, palmitic acid anhydride, 2-O-benzyl glyceride or vinyl ester of other saturated fatty acid is used It is possible. The enantiomeric excess achieved by the present invention is 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, 99% or more, or 99.5 or more. % Or more.

In general, the type of lipase, the type of substrate, the type of acylating agent, and the type of solvent indicate whether the product has high or low stereoselectivity in a lipase-catalyzed acylation reaction, that is, high optical purity. It is influenced by many factors such as enzyme concentration, concentration of acylating agent, concentration of substrate, temperature and the like. Also, some theoretical and empirical conditions for obtaining high stereoselectivity are known. On the other hand, the lower the temperature, for example, the higher the stereoselectivity, but the lower the reaction temperature, the lower the reaction yield. Based on such current situation, in this application, palmitoyl group is stereoselectively bound to the hydroxyl group of enantiotopic substrate called 2-O-Benzylgcerol to obtain 2-O-benzyl-1-palmitoylglycerol (9) with high optical purity. We examined the conditions for this. As a result, a solution of 2-O-benzylglycerol (8) (0.494 g, 2.5 mmol) and vinyl palmitate (1.41 g, 5.0 mmol) in dichloromethane (5.2 mL) was cooled to 10 ° C. and stirred. Add Lipase PS-IM-Amano (100 mg) (Amano Enzyme Co., Ltd.) and stir for 6 hours to obtain 2-O-benzyl-1-palmitoylglycerol (9) with optical purity (98% or more). The first thing to be done was clarified. Incidentally, 2-O-benzyl-1-palmitoylglycerol (9) with high optical purity thus obtained is a very important compound as a common intermediate for the synthesis of various optically active glycerophospholipids. Furthermore, if such conditions are determined, it is extremely easy for those skilled in the art to expand the reaction scale. In contrast, in the case of chiral columns, it is possible to obtain 2-O-benzyl-1-palmitoylglycerol (9) with high optical purity, but the amount of substrate throughput by the expensive optically active column is very small and the throughput is increased As a result, increasing the column size not only makes the cost very high but also requires unnecessary solvent treatment to drain the column.

The enzyme reaction of lipase may be carried out in an organic solvent (chloroform, n-hexane, isopropyl ether, etc.). When the reaction is carried out in dichloromethane, optically active phospholipids (products with high optical purity) can be obtained relatively easily.

Appropriate modifications can be made to this reaction, for example, by referring to the following documents as appropriate. M. Murata et al. , Efficient lipase-catalysed synthesis of chiral glycerol derivatives. Chem. Pharm. Bull. , 1989, 37, 2670-2672, O.I. Ghisalva et al. , Enzymatic preparation of acylglycols of high optical purity. Recl. Trav. Chim. Pay-Bas. 1991, 110, 263-64, Murata, M. et al., Design of enzyme-catalyzed asymmetric reactions in organic solvents and their application to optically active pharmaceutical synthesis. Organic Synthetic Chemistry, 1991, 49, 1127-1141, and S.A. Hazarika, P .; Goswami, and N. N. Dutta, Lipase catalysed transformation of 2-o-benzylglycerol with vinyl acetate: solvent effect. Chemical Engineering Journal, 2003, 94, 1-10.

(Protecting group)
In the present invention, when a protecting group is used and lipase or palmitoylation of glycerol is carried out by a method other than that to synthesize palmitoyl glycerol which is a high optical purity intermediate, the protecting group used is 2-O-benzyl. It is not limited to glycerol.

The first requirement of the protecting groups used is that the substituent attached to position 2 of the glycerol backbone needs to be removable by hydrogenolysis. In the present application, as the simplest example, a benzyl group (in the structure A of the following chemical formula, R ₁ -R ₇ = H). However, the method of the present invention can be practiced using a compound of the structural formula B or C of the following chemical formula. The hydrolytic deprotection does not cleave the palmitoyl group esterified by the lipase.

When using a compound having any of structural formulas A, B or C, preferably R ₁ = R ₂ . In the case of R ₁ ≠ R ₂ , the carbon to which they are attached, C *, is an asymmetric center, and the racemic form of the structural formula D is obtained. When a racemic form is obtained, it is also possible to optically resolve these racemates using the OH group remaining in the glycerol skeleton. However, since the asymmetric center to which R ₁ and R ₂ are bonded and the remaining hydroxyl group are too far apart, high stereoselectivity can not be expected in optical resolution of the OH group by lipase. Therefore, in the present invention, preferably, R ₁ = R ₂ .

Furthermore, when using a compound of any of structural formulas A, B or C, any one of R ₁ to R ₁₁ is independently substituted with methyl group, ethyl group, propyl group, and / or isopropyl group, etc. Can be a substrate for lipases sufficiently and can give high stereoselectivity.

The method for producing palmitoyl glycerol using a lipase according to the present invention includes, for example, a step of subjecting a compound having any of the structural formulas A, B or C described above and a fatty acid as a substrate to a lipase enzymatic reaction. In compounds having any of the above structural formulas A, B or C, R ₁ to R ₁₁ are each independently H, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 5 carbon atoms, 1 to 5 carbon atoms It is an alkyl group of 4 or an alkyl group having 1 to 3 carbon atoms. And / or in a compound having any of the structural formulas A, B or C, preferably, R ₁ = R ₂ .

(Step 7: Introduction of phosphate group and choline to 3-hydroxy group of glycerol)

A phosphoric acid group and choline are introduced into the 3-hydroxy group of glycerol by reacting with phosphoryl chloride and the p-toluenesulfonate of deuterated choline synthesized in step 2 (compound 5). The reaction is carried out, for example, by slowly adding dry triethylamine and the product of step 6 to a solution of phosphoryl chloride while stirring under a nitrogen atmosphere at 0-5 ° C., followed by adding dry pyridine and choline deuterated. The reaction can be carried out by adding p-toluenesulfonate and reacting at room temperature.

(Step 8: Deprotection of 2-hydroxy group of glycerol)

Remove the protecting group introduced into 2-hydroxy group of glycerol. In this reaction, for example, when a benzyl group is introduced as a protecting group R ′, the product of step 7 (compound 10) is dissolved in a mixed solvent of methanol and water, and treated with palladium hydroxide on carbon and hydrogen gas Can proceed with

(Step 9: Introduction of fatty acid to 2-hydroxy group of glycerol)

A second fatty acid is introduced into the 2-hydroxy group of glycerol by reacting deuterated lysophosphatidylcholine with a fatty acid. In the reaction, for example, the deuterated lysophosphatidylcholine prepared in step 8 (compound 11) and the fatty acid R ² -COOH to be introduced are dissolved in dry chloroform containing no ethanol, to which dicyclohexylcarbodiimide (DCC) and dimethylamino are added. It can be advanced by adding pyridine (DMAP) and a very small amount of butylated hydroxytoluene and stirring under a nitrogen atmosphere. Although DCC and DMAP are used in the above examples, the reaction is not limited to these reagents, and one of ordinary skill in the art would appreciate other known reagents such as 1-hydroxybenzotriazole and diphenyl phosphate azide. It can be selected appropriately.

In one embodiment, R ² can be a C ₈ -C ₃₆ saturated or unsaturated hydrocarbon group, wherein the hydrocarbon group contains 0-8 double bonds and is a hydroxyl group (—OH ), 0 to 2 epoxy groups, 0 to 2 carbonyl groups, and 0 to 3 hydroperoxy groups (—OOH). In one embodiment, R ² can be a C ₈ -C ₃₆ saturated hydrocarbon group. In one embodiment, R ² can be a C ₈ -C ₃₆ unsaturated hydrocarbon group containing 1 to 8 double bonds. In one embodiment, R ² may be a C ₈ to C ₃₆ saturated hydrocarbon group containing 1 to 3 hydroxyl groups (—OH). When R ² is a hydrocarbon group containing a hydroxyl group, a fatty acid whose hydroxyl group is protected as an ester or a silyl ether is reacted with deuterated lysophosphatidylcholine, followed by deprotection of the hydroxyl group to obtain a hydroxyl group. R ² can be introduced. In one embodiment, R ² may be a C ₈ to C ₃₆ saturated hydrocarbon group containing 1 to 2 epoxy groups. In one embodiment, R ² may be a C ₈ -C ₃₆ saturated hydrocarbon group containing 1 to 2 carbonyl groups. In one embodiment, R ² may be a C ₈ to C ₃₆ saturated hydrocarbon group containing 1 to 3 hydroperoxy groups (—OOH). When R ² is a hydrocarbon group containing a hydroperoxy group, a fatty acid having this hydroperoxy group protected as a peracetal is reacted with deuterated lysophosphatidylcholine, followed by deprotection of the hydroperoxy group. , R ² containing a hydroperoxy group can be introduced. When R ² contains two or more of double bond, hydroxyl group (-OH), epoxy group, carbonyl group, and hydroperoxy group (-OOH), only hydroxyl group and / or hydroperoxy group is protected; The desired R ² can be introduced without protecting the functional group.

Furthermore, choline can be optionally converted to deuterium-substituted choline by the following steps 10 and 11. For these steps, for example, H.U. Gally, W. Niederberger, and J.J. Seeling, Conformation and motion of the choline head group in bilayers of dipalmitoyl-3-sn-phosphatidylcholine. Biochemistry, * 1975 *, 14, 3674-3652, G.I. S. Harbison and R. G. Grifin, Improved method for the synthesis of phosphatidylcholines. Journal of Lipid Research, * 1984 *, 25, 1141-1144. , And K. M. Patel, J.A. D. Morrisett, and J.J. T. Sparrow, the conversion of phosphatidyl erythritol into phosphatidyl choline labeled in the choline group using methyl iodide, 18-crown-6 and potassium carbonate. Lipids, * 1979 *, 14, 596- 597), S.I. Sunder et al. Infrared and Raman spectra of specifically desorbed 1,2-dipalmitoyl-sn-glycero-3-phosphocholines, Chemistry and Physics of Lipids, 1981, 28, 137-148. See etc.

In addition, (14) is a non-deuterated phospholipid from (12), which is reacted with 2-aminoethanol to obtain (13), which is then reacted with deuterated methyl iodide for synthesis. Yes (deuterated later).

Also, 2-deuterium substituted 2-aminoethanol is reacted with (12) to give (13). This can be reacted with deuterated methyl iodide to obtain an 11-deuterated phospholipid (14).

(Step 10: conversion of choline to aminoethanol)

In step 10, choline is replaced with 2-aminoethanol by reacting the product of step 9 (compound 12) with 2-aminoethanol in the presence of phospholipase D. As 2-aminoethanol, its deuterium substitution may be used if necessary, and as such deuterium substitution, for example, 2,2-dijuterio-2-aminoethanol, 1,1-dijuterio -2-aminoethanol and 1,1,2,2-tetradeuterio-2-aminoethanol etc. can be used, and when these deuterium substitutes are used more weight as final product A compound containing hydrogen is obtained.

(Step 11: Introduction of deuterated methyl group to aminoethanol)

Step 11 introduces a deuterated methyl group into the product of step 10 (compound 13). This reaction may proceed, for example, by mixing the product of step 10 with methyl deuteroiodide (CD ₂ I) under basic conditions such as potassium hydroxide.

In one aspect, the following synthetic methods can be used other than the above synthetic route.
5) Alternative;
In one embodiment, e.g. Xia and YZ. Hui, Synthesis of a small library of mixed-acid phospholipids from D-Mannitol as a homochiral starting material. Chem. Pharm. Bull. , 1999, 47, 1659-1663. It is possible to use a method of converting to optically active phosphatidylcholine (PC) without changing the configuration of optically active mannitol with reference to the etc., in which deuterated N, N-dimethylethanolamine (compound 4) is introduced. Compound 12 can be obtained.

In one embodiment, for example, R. Rosseto et al. , A new approach to the synthesis of lysophospholipids: preparation of lysophosphate acid and lysophosphate phosphate from p-nitrophenyl glycerate. With reference to Tetrahedron Letters, 2004, 45, 7371-7373 etc., a method for converting p-nitrophenyl glycerate to phosphatidyl choline can be used, and it is possible to use p-nitrophenyl glycerate having an optically active glyceride skeleton. Compound 12 can be obtained by introducing the above-mentioned deuterated choline.

In one embodiment, e.g. Lindberg et al. , Efficient synthesis of phospholipids from glycidyl phosphates. J. Org. Chem. A commercially available method for converting optically active glycidol to phosphatidyl choline can be used, as described in J. Chem., 2002, 67, 194-199 etc., and the above-mentioned deuterated choline is introduced into glycidol having an optically active glyceride skeleton. Compound 12 can be obtained.

In one embodiment, for example, P.I. D'Arrigo and S. Servi, Synthesis of Lysophospholipids. Journal of Organic Chemistry, * 2002 *, 67, 194-199. The compound 12 can also be synthesized by reacting choline tosylate (compound 5) with optically active glycidol to synthesize a lysophospholipid and binding an unsaturated fatty acid thereto (step 9).

(Use of deuterated phospholipids)
Many papers have been published on the importance of phospholipids containing fatty acids such as EPA, DPA and DHA in biological functions. However, in living tissues such as blood, various phospholipid molecular species having similar chemical structures are mixed, their distribution differs among the tissues, and the chemical structures change constantly in the metabolic process. Therefore, it is extremely difficult to track the structural change of a specific phospholipid molecule species over time. In recent years, lipidomics analysis by mass spectrometer has been conducted, but such analysis has not been sufficiently conducted. On the other hand, using the deuterated phospholipids of the present invention, structural changes associated with specific metabolism of phospholipids may be traceable.

In one aspect, the invention provides a composition comprising a phospholipid of the invention. Deuterated phospholipids exhibit similar physicochemical properties as the corresponding non-deuterated phospholipids and are considered to exhibit similar absorption, distribution, metabolism or excretion in vivo. However, because deuterated phospholipids differ in mass from the corresponding non-deuterated phospholipids, deuterated phospholipids or their metabolites can be easily distinguished by mass spectrometric analysis Can. Therefore, it can be usefully used to trace the structural change of phospholipids, particularly, associated with metabolism.

In one embodiment, the composition of the invention is used to detect or measure absorption, metabolism, distribution or excretion of phospholipids.

In one embodiment, a composition of the invention is administered to a subject. In one embodiment, the subject may be a mammal, such as, but not limited to, humans, mice, rats, dogs, monkeys, rabbits and the like. Any suitable mode of administration may be used, for example oral administration, injection and the like.

In one embodiment, a composition of the invention is contacted with a sample in vitro. In one embodiment, the sample is a biological sample, such as, but not limited to, tissue, blood, urine, saliva, tears, and the like.

In one embodiment, the composition of the present invention may be used for food quality assessment. Since phospholipids containing fatty acids such as EPA, DHA and DPA are abundant in marine products and their processed foods, and these phospholipids can be enzymatically and chemically changed due to the deterioration of the food, the composition of the present invention It can be used to analyze this change. For example, it may be possible to measure the deterioration of the food by adding the composition of the present invention to the food beforehand and tracking the change thereafter.

(Measurement of phospholipid metabolism disorder)
In one embodiment, the composition of the present invention may be used for the measurement of phospholipid metabolism disorders. For example, by adding it to tissues, blood, serum or plasma collected from patients and healthy people and detecting the chemical changes that occur there (eg, measuring the type and amount of isotope labeled compounds produced) Thus, it is possible to determine whether or not the phospholipid metabolism of the patient is abnormal as compared to a healthy person.

(Use for the diagnosis of a disease or condition)
In one embodiment, the composition of the present invention may be used for the diagnosis of a disease. The disease may be any disease associated with phospholipids, including, for example, diseases causing abnormalities in lipid metabolism such as obesity, hyperlipidemia, non-insulin dependent diabetes mellitus, and osteoarthritis of the knee May be, but not limited to, cancer, inflammation, arteriosclerosis, Alzheimer's disease, sepsis, antiphospholipid antibody syndrome (age-related macular degeneration, systemic lupus erythematosus, etc.), etc. For example, adding the composition of the present invention to a tissue, blood, serum or plasma collected from a living body and detecting the chemical change that occurs there (for example, measuring the type and amount of isotope labeled compounds produced To make it possible to diagnose the pathological condition and the degree of progression.

(1) Diagnosis of Alzheimer's Disease Although pathological methods are taken exclusively for the diagnosis of Alzheimer's disease, many problems still remain in diagnostic methods using biomarkers. A paper published by Gonzalez-Dominguez et al. In 2016 in this respect (Current Alzheimer's)
r Research, 2016, 13, 641- 65) states that chemical changes in phospholipids in serum are closely related to the pathogenesis of Alzheimer's disease and mild dementia, and the following important contents Is pointed out.
"The use of blood in Alzheimer's research has never been looked at so far because the relationship between blood-based measurements and processes in the brain was thought to be difficult. A close link between metabolic abnormalities and brain samples has recently been demonstrated using a transformed Alzheimer's disease model, and there is growing evidence that Alzheimer's disease is a systemic disease. The use of peripheral blood has been shown to be important in the study of the pathological mechanisms involved in Alzheimer's disease, which is related to the metabolic injury of phospholipids and sphingolipids and the acyls that bind them Chain length and degree of unsaturation play a crucial role in Alzheimer's disease, which was once caused by phospholipases in the brain. Excess degradation of the plasma membrane, which leads to the destruction of the cell membrane, and the disappearance of unsaturated fatty acids such as DHA and arachidonic acid, which are present in high concentrations in neural tissue, by oxidative stress causes damage to the cell membrane. This is related to the pathology of Alzheimer's disease, and this rationality is due to the fact that saturated fatty acid bound phosphatidyl choline and phosphatidyl ethanolamine increase and simultaneously unsaturated fatty acid bound phosphatidyl choline such as linoleic acid, arachidonic acid and DHA decrease significantly. Confirmed. ”From such background, as described below, the phospholipids of the present invention can provide excellent diagnostic agents.

There have been several reports on the relationship between the type and amount of phospholipids in plasma and Alzheimer's disease. For example, phosphatidylcholine (PC16: 0/20: 5, PC16: 0/22: 6) which binds palmitic acid to sn-1 position from plasma of Alzheimer's disease patients with dementia and binds EPA or DHA to sn-2 position. In 2013, results were reported that three molecular species of phosphatidylcholine (PC18: 0/22: 6) that bind stearic acid at the sn-1 position and sn-1 position and DHA at the sn-2 position are reduced . Based on these results, the authors say that specific phosphatidylcholine is involved in the pathology of Alzheimer's disease. (Luke Whiley et al, Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease, Neurobiology of Aging, 35, 2014, 1-8)
In addition, in many disease states including Alzheimer's disease, it is also well known that phospholipase A2 activity in plasma using phospholipid as a substrate is changed. (J. Sundaram, E. S Chan, C. P Poore, T. K Pareek, W. F Cheong G Shui, N. Tang C. M Low, M. R Wenk, and S Kesavapany. (2012) Cdk5 / p25 -Induced cytosolic PLA 2-mediated lysophosphoridylcholine production regulation neuroinflammation and triggers neurodegeneration. The Journal of Neuroscience, 32, 2012, 1020-1034)
Therefore, phospholipase A2 is deeply involved in the type and quantitative change of phospholipids corresponding to the pathology of Alzheimer's disease. When this enzyme acts on phospholipids, lysophospholipids and fatty acids are produced. It is also consistent with the fact that DHA levels are reduced in the blood and brain of patients with Alzheimer's disease (Javier Orazaran, et al., A blood-based, 7-metabolite signature for the early diagnosis of Alzheimer's disease , Journal of Alzheimer's disease, 45, 2015, 1157-1173).

Based on these facts, diagnosis of Alzheimer's disease is reported to be possible. However, the types of biological phospholipids, fatty acids and lysophospholipids are extremely diverse, and it is not easy to conduct their analysis simultaneously with high reliability using the lipidomics method.

In the ESI MASS analysis of the deuterated phospholipid synthesized in the present invention, the product ion is input as m / z 187 (inputting m / z 187 does not detect any phospholipid other than the deuterated phospholipid). Thus, deuterated phospholipids and those that have undergone structural change can all be detected simultaneously with extremely high selectivity.

Thus, for example, ESI of a sample collected after adding deuterated phospholipids (PC16: 0/20: 5, PC16: 0/22: 6, PC18: 0/22: 6) to blood and incubating for a certain period of time From the MASS spectrum, it is possible to diagnose Alzheimer's disease by simultaneously analyzing the variations of residual deuterated phospholipids and lysophosphatidylcholine generated therefrom.

For example, it has been reported that the ratio of lysophosphatidylcholine to phosphatidylcholine in cerebrospinal fluid but not in blood is related to Alzheimer's disease. (C. J. J. Mulder et al., Decreased lysophosphoryl choline / phosphoryl choline ratio in cerebrospinal fluid in alzheimer's disease, journal of Neural Transmission 110, 2003, 949-55)
The three kinds of molecular species reported in the above-mentioned Luke Whiley et al are none other than those in which deuterium of deuterated phosphatidyl choline synthesized in the present invention is replaced with the original hydrogen atom.

In addition, unsaturated fatty acids and unsaturated fatty acids that bind to phospholipids are oxidatively stressed in the living body to form peroxides thereof, and it has long been known that these are related to various pathological conditions including inflammation. It is done. A number of reports have also been made in the tissues of patients with Alzheimer's disease (Elic Tonnies et al., Oxidative stress, synaptic dysfunction, and Alzheimer's disease, Journal of Alzheimer's Disease, 57, 2017, 1105-1121).

While EPA, DPA or DHA bound to deuterated phospholipids synthesized in the present invention play an important role in the living body, they are extremely easily oxidized due to oxidative stress, inflammation and the like. Moreover, the composition of the oxidation product is not simple and contains various molecular species. By inputting the product ion as m / z 187 and measuring ESI MASS, almost all oxidation product species of deuterated phospholipid can be analyzed at the same time (Hiroko Tominaga et al., Molecular proves in tandem electrospray ionization mass spectrometry: application to tracing chemical changes of specific phospholipid molecular species, American Journal of Analytical Chemistry, 4, 2013, 16 to 26)
This analysis can be applied to diagnosis if the correlation between the pathological condition such as Alzheimer's disease and the phospholipid oxide pattern in ESI MASS can be confirmed. However, either of the above methods alone is difficult to selectively diagnose a specific disease.

In such a case, in ESI MASS measurement using m / z 187, quantitative change of deuterated phospholipid itself, formation of lysophosphatidylcholine, and simultaneous analysis of the pattern of peroxide molecular species and pathological condition including Alzheimer's disease and Can be applied to diagnosis with higher accuracy.

Based on the above-mentioned prior art, healthy individuals express phospholipase A2 enzyme at a higher expression level or have increased enzyme activity than Alzheimer's disease patients. Furthermore, phosphatidylcholine D3 which is an omega-3 fatty acid and which is deeply involved in anti-inflammatory properties, phosphatidylcholine D3 which binds EPA, DHA or DPA and phosphatidylcholine D3 which an inflammatory arachidonic acid which is an omega 6 fatty acid binds each lysophosphatidylcholine D3 by the action of phospholipase A2. Generate And, the ratio of PCD3 / Lyso-PCD3 in each enzyme reaction is expected to be different depending on the presence / absence and type of pathological condition. In addition, it is long known that phospholipase A2 activated with inflammation underlying the pathological condition liberates arachidonic acid bound to membrane phospholipids, and this produces various inflammatory substances through the arachidonic acid cascade. Therefore, the isotopically-labeled compound of the present invention is considered to be available as an indicator of the inflammatory condition attributable to Alzheimer's disease in the brain, and hence for the diagnosis of Alzheimer's disease and the diagnosis of the disease condition of Alzheimer's disease It is predicted to be possible. Based on the above understanding, diagnosis of Alzheimer's disease is possible by utilizing the present invention. For example, the isotopically labeled phospholipid of the present invention is mixed with a body fluid to be diagnosed (eg, a body fluid such as blood, saliva, urine or cerebrospinal fluid) to determine the type and amount of labeled phospholipid in the reaction mixture It is possible to make a diagnosis of Alzheimer's disease. For example, phosphatidylcholine D3 that binds EPA, DHA, DPA or AA (arachidonic acid) as an isotope-labeled phospholipid is administered to a body fluid, incubated for a fixed time, and then a lipid sample containing phospholipid D3 is extracted. It is possible to diagnose Alzheimer's disease by analyzing by and measuring the PCD3 / Lyso-PCD3 ratio.

(2) Osteoarthritis of the knee The research has been reported that the degree of progression of knee osteoarthritis is related to the lysophosphatidylcholine / phosphatidylcholine ratio in plasma and can be used for diagnosis. (Weidong Zhang et al., Lysophosphotidylcholines to phosphatidylcholines ratio predicts advanced knee osteoarthritis, Rheumatology, 55, 2016, 1566-1574) The deuterated phospholipids synthesized in this patent may also be applied to the efficiency and accuracy improvement of this method it is conceivable that.

In addition to plasma, the method is also applicable to saliva and the like. (Arkasubhra Ghosh and Krishnatej Nishtala, Biofluid lipidome: a source for potential diagnostic biomarkers, Critical and Translational Medicine, 6, 2017, 1 to 10) Therefore, as with Alzheimer's disease diagnosis for osteoarthritis of the knee, also EPA, DHA, DPA or AA The diagnosis is made possible by the ratio of the amount of phosphatidyl choline D3 binding to each lysophosphatidyl choline D3 produced therefrom. The reason is described below.

Based on the above-mentioned prior art, a healthy individual has a higher expression level of phospholipase A2 enzyme or a higher enzyme activity than osteoarthritic knee patients. Furthermore, phosphatidylcholine D3 which is an omega-3 fatty acid and which is deeply involved in anti-inflammatory properties, phosphatidylcholine D3 which binds EPA, DHA or DPA and phosphatidylcholine D3 which an inflammatory arachidonic acid which is an omega 6 fatty acid binds each lysophosphatidylcholine D3 by the action of phospholipase A2. Generate And, the ratio of PCD3 / Lyso-PCD3 in each enzyme reaction is expected to be different depending on the presence or type of the pathological condition. In addition, it is long known that phospholipase A2 activated with inflammation underlying the pathological condition liberates arachidonic acid bound to membrane phospholipids, and this produces various inflammatory substances through the arachidonic acid cascade. Therefore, the isotopically-labeled compound of the present invention is considered to be usable as an indicator of the inflammatory condition of various tissues, and therefore, a diagnosis of osteoarthritis of the knee and a diagnosis of a disease condition of the knee osteoarthritis It is expected to be available to Based on the above understanding, it is possible to diagnose osteoarthritis of the knee by utilizing the present invention. For example, the isotopically labeled phospholipid of the present invention is mixed with a body fluid to be diagnosed (eg, a body fluid such as blood, saliva, urine or cerebrospinal fluid) to determine the type and amount of labeled phospholipid in the reaction mixture It is possible to make a diagnosis of osteoarthritis of the knee. For example, phosphatidylcholine D3 that binds EPA, DHA, DPA or AA (arachidonic acid) as an isotope-labeled phospholipid is administered to a body fluid, incubated for a fixed time, and then a lipid sample containing phospholipid D3 is extracted. It is possible to diagnose osteoarthritis of the knee by analyzing by and measuring PCD3 / Lyso-PCD3 ratio.

The same can be applied to diagnosis by each PCD3 / Lyso-PCD3 ratio in other cases as well. For example, by combining the values of each collected tissue and each body fluid and the values of other biomarkers, it is possible to diagnose various pathological conditions.

(Features of the present invention)
A representative phosphatidylcholine, which accounts for the majority of biological phospholipids, is optically active because the carbon at position 2 of the glycerol backbone is an asymmetric center. Because this asymmetric center has a very high enantiomeric excess, it is not easy to chemically construct a glycerol backbone with such asymmetric centers. Under such circumstances, there has been taken a method of partially decomposing optically active glycerophospholipids present in the natural world while maintaining an asymmetric center, and reconstituting them into a target phospholipid. However, in this method, it is almost impossible to introduce deuterium into the choline portion of the fragment structure containing the asymmetric center. There are other methods using optically active natural products such as mannitol, but all of them are complicated in operation. In this application, in order to solve this problem, optically active glycerol intermediate is synthesized by attaching palmitoyl group to glycerol skeleton stereoselectively using enzyme-derived enzyme lipase, and deuterium synthesized in advance is synthesized to this. The method of binding the choline and, after deprotection, finally combining EPA, DPA or DHA was carried out.

In addition, according to the past report example, since introduction | transduction of the palmitoyl group by lipase does not necessarily give high stereoselectivity, in this application, H. Palmitate acid vinyl ester is used as an acylating agent with reference to Hazarika et al. (Chemical Engineering Journal, * 2003 *, 94, 1-10), and lipase PS (Amano) IM is used as a catalyst in dichloromethane at 10 ° C. It was possible to synthesize an intermediate with a very high enantiomeric excess by carrying out the reaction in As the acylating agent, not only palmitic acid vinyl ester but also palmitic acid isopropenyl ester, palmitic acid trifluoroethyl ester or palmitic acid anhydride, or vinyl esters of other saturated fatty acids can be used. The enantiomeric excess achieved by the present invention is 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, 99% or more, or 99.5 or more. % Or more.

Among the deuterated phospholipids targeted by the present invention, particularly those that bind DPA, one of the problems was that the availability of high purity DPA necessary for the synthesis was not easy. However, in the present invention, this is made possible by the development of a novel method for chemically synthesizing high purity DPA from EPA by a short route.

(Effect of the present invention)
In living tissues, there are many phospholipid molecular species that bind various fatty acids including EPA, DPA and / or DHA, and play a role in each. In addition, although the roles and metabolism of these molecular species are closely related to homeostasis and pathophysiology of the living body, in addition to the diversity of phospholipid molecular species, the association with these pathopathies is extremely complicated. Therefore, specific phospholipids that bind EPA, DPA or DHA, which have recently been attracting attention as nutrients extremely important to health, are temporarily contained in natural and synthetic standard substances (known substances) having the same chemical structure as them. Even then, it is not easy to selectively detect them from complex biological phospholipids. At present, the most powerful method to solve this problem is metabolomic analysis, but in this method, phospholipid components in a biological sample have to be separated by high performance liquid chromatography prior to mass analysis. On the other hand, deuterated synthetic phospholipids binding with EPA, DPA or DHA synthesized in the present application, when added to a living tissue, do not require chemical changes in the chemical changes therein, which require separation by liquid chromatography, It can be tracked very specifically by precursor ion detection in mass spectrometry. From this point of view, the novel deuterium-substituted phospholipid binding EPA, DPA or DHA synthesized in the present application exhibits extremely excellent effects as compared with known substances, and in the future, not only basic research on phospholipid metabolism but also diagnosis Application to medicine is expected.

The present invention has been described above with the preferred embodiments shown for ease of understanding. EXAMPLES The present invention will be described below based on examples, but the above description and the following examples are provided for the purpose of illustration only and are not provided for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

Examples are described below.

Example 1
Synthesis of 2-O-docosapentaenoyl-1-O-palmitoyl-glycerophosphocholine D3 (13) Synthetic scheme

The above synthetic scheme shows a synthetic pathway of deuterated phosphatidyl choline to which DPA is bound, but it can be similarly synthesized in the case of EPA and DHA. Previously reported methods for synthesizing phospholipid peroxides (Naomichi Baba, Kenji Yoneda, Shoichi
Tahara, Junkichi Iwasa, Takao Kaneko, Mitsuyoshi Matsuo, J.A. Chem. Soc. Chem. Commun. Appropriate modifications can be made with reference to, for example, J. Chem.

Step 1: Synthesis of sulfonic acid (2) :
35% aqueous caustic soda solution (5.3 g) in a mixture of commercially available p-toluenesulfonyl chloride (8.03 g, 42.0 mmol) and tetradeuteriomethanol (8.88 g, 24.6 mmol) at -5 to 0 ° C It dripped slowly. After completion of the reaction, the reaction solution was transferred to a separatory funnel using diethyl ether (100 mL). This was washed twice with saturated aqueous sodium bicarbonate solution and then dried over anhydrous sodium sulfate. After distilling off the ether under reduced pressure, the residue was distilled under reduced pressure to obtain 7.00 g of a product (yield 88.1%).

Step 2: Synthesis of N-trideuteriomethyl-N, N-dimethylaminoethanol p-toluenesulfonate (3) :
Trideuteriomethyl p-toluenesulfonate (7.0 g, 0.037 mol) and N, N-dimethylaminoethanol (3.30 g, 0.037 mol) are added to a mortar, and stirring with a spatula immediately causes an exothermic reaction to solidify. did. After 20 minutes, the solid was crushed, suspended in excess dry acetone and filtered quickly. The crystals on the filter were washed twice with dry acetone, quickly placed in a desiccator and dried under vacuum with phosphorus pentoxide to give 6.90 g of product (yield 67.0%).

Step 3: Protection of # 1.3 of Glycerol :
Glycerol (370.8 g, 4.03 mol) and benzaldehyde (316.6 g, 2.99 mol) were reacted under acidic conditions to give 173.1 g (33.2%) of a compound of (6).

Step 4: Protection of the Second Place of Glycerol :
The compound of (6) (48.4 g, 0.27 mol) and benzyl chloride (427.3 g, 3.37 mol) are reacted with powdered KOH (96.5 g, 1.6 mol) to give a compound of (7) 54.5 g (71.0%) were obtained.

Step 5: Deprotection of 1, 3 position of glycerol :
The compound of (7) (12.6 g, 0.047 mol) was hydrolyzed in an acidic solution to give 2.83 g (33.3%) of the compound of (8).

Step 6: Synthesis of 2-O-benzyl-1-O-palmitoyl glycerol (9) :
The dichloromethane (5.2 mL) of 2-O-benzyl glycerol (8) (0.494 g, 2.5 mmol) and vinyl palmitate (1.41 g, 5.0 mmol) was cooled to 10 ° C. under stirring, Lipase PS -IM-Amano (100 mg) (Amano Enzyme Co., Ltd., Aichi Prefecture) was added, stirred and reacted for 6 hours. The enzyme was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified through a silica gel column to give 0.90 g of a product (yield 82.6%).

Step 7: Synthesis of 2-O-benzyl-1-O-palmitoylphosphatidylcholine D3 (10):
In a reaction flask equipped with a side tube, phosphoryl chloride (0.19 mL, 2.0 mmol) and a stirrer were placed, and the dropping funnel and thermometer were attached and cooled to 0-5 ° C. in a nitrogen atmosphere. Dry chloroform (0.38 mL, 2.5 mmol) and 2-O-benzyl-1-O-palmitoyl glycerol (9) (0.74 g, 1.7 mmol) in dry chloroform so that the temperature does not rise above this temperature while stirring The (19 mL) solution was slowly added dropwise. After dropping, the mixture was stirred at this temperature for 1 hour, and further stirred at room temperature for 1 hour. To this solution was added dry pyridine (0.64 mL, 8.0 mmol) and N-trideuteriomethyl-N, N-dimethylaminoethanol p-toluenesulfonic acid (3) (0.66 g, 2.4 mmol) at room temperature Stir for 48 hours. Water (8.6 mL) was added, and after stirring for 30 minutes, the mixture was transferred to a separatory funnel, and 1N hydrochloric acid and methanol were added and shaken, and then the desired substance was extracted three times with chloroform. The solution was washed with 1N hydrochloric acid and then dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was purified by silica gel column to obtain 0.57 g of product (yield 55.8%).

Step 8: Synthesis of Lyso-PCD 3 (11) :
2-O-benzyl-1-O-palmitoylphosphatidylcholine D3 (10) (2.62 g, 44.5 mmol) is dissolved in a mixed solvent of methanol (30 mL) and water (3 mL), and carbon-supported palladium hydroxide (1. 50 g) was added and stirred for 3 days with a balloon containing hydrogen gas. After completion of the reaction, the solution was filtered using filter paper and the filtrate was concentrated. As a result of performing azeotropic distillation with isopropanol in order to remove the remaining water, 1.89 g of powdery lyso-PCD3 (11) was obtained (yield: 85.2%).

Step 9: Synthesis of 2-O-docosapentaenoyl-1-O-palmitoyl-glycerophosphocholine D3 (13) :
Lyso-PCD 3 (11) (0.30 g, 0.60 mmol) and docosahexaenoic acid (0.5 g, 1.50 mmol) were dissolved in dry chloroform (5.0 mL) containing no ethanol, and dicyclohexyl carbodiimide (0. 2) was dissolved therein. 44 g (2.0 mmol), dimethylaminopyridine (26 mg, 0.2 mmol) and a very small amount of butylated hydroxytoluene as an antioxidant were added and stirred for 2 days under a nitrogen atmosphere. After completion of the reaction, it was concentrated, and the residue was purified by silica gel column to obtain 0.36 g of product (yield: 74.0%).

¹ H NMR (600 MHz; CD ₃ OD): δ 0.88 (3 H, t, j = 7.8, terminal CH _{3 of} palmitoyl group), 0.97 (3 H, t, j = 7.8, docosa) Terminal CH _{3 of} pentaenoyl group, 1.28 (26 H, m, CH ₂ x 13), 1. 38 ( ₂ H, m, CCC-C-CH ₂ -C), 1.61 (2 H, m , CH ₃ -CH ₂ -C = C), 2.08 (2H, m, COCH ₂ CH ₂ ), 2.3 (4H, m, OCOCH _{2 of} each fatty acid chain), 2.85 (8H, m , C = C-CH ₂ -C = C x 4), 3.22 (6 H, s, N [CH ₃ ] ₂ ), 3. 64 ( ₂ H, m, CH ₂ N), 4.00-4.45 ( m, 5H, protons on the glycerol _{backbone), 4.28 (2H, br,} P-O-CH 2), 5.24-5.40 (m 10H, olefinic proton)
ESI MS observed _{_{m / z = 811.6006, [C}} 47 H 83 D 3 NO 8 P + H] + Calculated = 811.6039.

(Example 2)
Synthesis of 2-O-eicosapentaenoyl-1-O-palmitoyl-glycerophosphocholine D3 EPA-PCd3 was synthesized in the same manner as in Example 1. 0.40 g (84.7%)
¹ H NMR (600 MHz; CD ₃ OD): δ 0.80 (3 H, t, j = 7.8, terminal CH _{3 of} palmitoyl group), 0.86 (3 H, t, j = 7.8, eicosa Pentaenoiru terminal _CH 3 _{groups), 1.19 (26H, m,} CH 2 x13), 1.59 (2H, m, CH 3 -CH 2 -C = C), 2.00 (2H, m, _{_{COCH 2 CH 2), 2.25 (}} 4H, m, OCOCH 2) of each fatty _{chain, 2.75 (8H, m, C} = C-CH 2 -C = Cx4), 3.12 (6H, s , N [CH ₃ ] ₂ ), 3.54 (2H, m, CH ₂ N), 3.86-4. 35 (m, 5H, proton on glycerol skeleton), 4.32 (2H, br, P _{-O-CH 2), 5.18-5.33 (} m, 10H, olefinic protons).
ESI MS observation m / z = 783.57349, calculated value for [C ₄₄ H ₇₅ D ₃ NO ₈ P + H] ⁺ = 783.57382
(Example 3)
Synthesis of 2-O-docosahexaenoyl-1-O-palmitoyl-glycerophosphocholine D3 DHA-PCd3 was synthesized in the same manner as in Example 1. 0.20 g (28.4%)
¹ H NMR (600 MHz; CD ₃ OD): δ 0.91 (3 H, t, j = 7.8, terminal CH _{3 of} palmitoyl group), 0.97 (3 H, t, j = 7.8, docosahexa) Terminal CH _{3 of the} enoyl group, 1.28 (26 H, m, CH ₂ x 13), 1.59 (2 H, m, CH ₃ -CH ₂ -C = C), 2.19 (2 H, m, COCH) ₂ CH ₂ ), 2.30-2.42 (4 H, m, OCOCH _{2 of} each fatty acid chain), 2.8 1-2. 90 (10 H, m, C = C-CH ₂ -C = C x 5), _{_{3.30 (6H, s, N [}} CH 3] 2), 3.64 (2H, m, CH 2 N), 4.00-4.43 (m, 5H, protons on the glycerol backbone), 4. _{27 (2H, br, P-} O-CH 2), 5.23-5.40 (m, 10H, olefin-flop Tons).
ESI MS observed _{_{m / z = 809.5837, [C}} 47 H 81 D 3 NO 8 P + H] + Calculated = 809.5883
(Note)
As mentioned above, although the present invention is illustrated using a preferred embodiment of the present invention, it is understood that the present invention should be interpreted the scope only by a claim. Patents, patent applications and documents cited in the present specification should be incorporated by reference to the present specification as the content itself is specifically described in the present specification. Be understood.

The present invention can be widely used in the pharmaceutical and food fields such as samples for research on diseases involving phospholipids, diagnostic agents, and reagents for evaluating the quality and deterioration of foods containing phospholipids.

Claims

The following formula I

(In the formula,
R 1 and R 2 are each independently —H or —C (= O) R,
R is a linear or branched saturated or unsaturated hydrocarbon group,
Wherein the hydrocarbon group contains 8 to 36 carbon atoms,
The hydrocarbon group contains 0-8 double bonds,
At least one of carbons on the hydrocarbon group may be substituted with a substituent selected from the group consisting of = O, -OH and -OOH,
At least one of two adjacent sets of carbons on the hydrocarbon group is substituted with O to be an epoxy group

May form a
Each R A is independently selected from hydrogen, deuterium or tritium)
Or a salt thereof.
The compound or a salt thereof according to claim 1, wherein all R A on the same carbon atom are the same hydrogen atom selected from hydrogen, deuterium or tritium.
A method for producing a compound according to claim 1 or a salt thereof,
Step 1) Trideuterio methyl group introduction reagent

Preparing the
Process 2) The following structure

Synthesizing a choline containing a deuterated methyl group selected from
Step 3) protecting 1-hydroxy group and 3-hydroxy group of glycerol,
Step 4) protecting the 2-hydroxy group of glycerol,
Step 5) Deprotecting 1-hydroxy group and 3-hydroxy group of glycerol,
Step 6) Step of introducing fatty acid of R 1 -OH structure into 1-hydroxy group of glycerol, step 7) Introduction of choline containing phosphate group and deuterated methyl group of step 2 into 3-hydroxy group of glycerol The process to
Step 8) Deprotecting 2-hydroxy group of glycerol,
Step 9) At least one of the steps of the step of introducing the fatty acid of the structure of R 2 -OH into 2-hydroxy group of glycerol, and optionally the weight introduced further in the following step 10) step 7 A production method comprising the steps of: converting choline containing a methylated methyl group into aminoethanol; and introducing a deuterated methyl group into aminoethanol.
A composition comprising the compound according to claim 1 or a salt thereof.
A composition according to claim 4 for the measurement of phospholipid metabolism disorders.
A composition according to claim 4 for the diagnosis of a disease or condition.
The composition according to claim 6, wherein the disease or condition is a disease causing an abnormality in lipid metabolism.
The composition according to claim 6, wherein the disease or condition is selected from the group consisting of obesity, hyperlipidemia, non-insulin dependent diabetes and osteoarthritis of the knee.
7. The composition of claim 6, wherein the disease or condition is selected from the group consisting of cancer, inflammation, arteriosclerosis, Alzheimer's disease, sepsis, and antiphospholipid antibody syndrome.
A method for producing palmitoyl glycerol palmitoylated with unsaturated fatty acid, which is selected from the group consisting of (i) Formula (A), Formula (B), and Formula (C)

(Wherein, R 1 to R 11 are each independently H or an alkyl group having 1 to 6 carbon atoms);
(Ii) unsaturated fatty acids,
Performing a lipase-enzyme reaction using a substrate as a substrate.
The method according to claim 10, wherein the unsaturated fatty acid contains 8 to 36 carbon atoms and contains 1 to 8 double bonds.
The method according to claim 10, wherein at least one of carbons on the unsaturated fatty acid may be substituted with a substituent selected from the group consisting of = O, -OH and -OOH.
At least one of two adjacent sets of carbons on the hydrocarbon group is substituted with O to be an epoxy group

May form a way.
The method according to claim 10, wherein the lipase enzyme reaction is performed at a temperature of -20 ° C to 50 ° C for 0.5 hour to 24 hours.
The method according to claim 10, further comprising using an acylating agent.
The method according to claim 10, further using a solvent.