JP7279941B2 - Deuterated phospholipid and method for producing same - Google Patents

Description

The present invention relates to deuterated phospholipids, methods for their production and uses thereof.

Phospholipids, which are indispensable as cell membrane constituents or biological signal transmitters, include many molecular species with different structures. The basic structure of glycerophospholipids is that the same or different fatty acids such as palmitic acid, stearic acid, icosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid are bound to two hydroxyl groups of the glycerol skeleton, and the third hydroxyl group is Polar groups such as phosphocholine, phosphoserine, and phosphoethanolamine are bound. Therefore, a huge number of types of phospholipid molecular species exist in biological tissues due to the number of combinations thereof. In addition, each of these molecular species exhibits different biological functions. The relationship 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 technique that combines liquid chromatography and mass spectrometry techniques.

However, functional studies of phospholipids can be difficult even with such analytical techniques. For example, it is extremely difficult to follow the process of metabolism and chemical conversion of a particular phospholipid molecular species at the molecular level. As a means of solving this problem, Baba et al. synthesized phosphatidylcholine in which one of the three methyl groups attached to the nitrogen atom of the polar group of choline was substituted with deuterium, a stable isotope (Tominaga 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). Deuterium-substituted phospholipids and metabolites in their acyl moieties can be detected very specifically and sensitively even when all kinds of phospholipids present in living tissue coexist. By this method, the metabolic process in the blood of phospholipid peroxide synthesized from linoleic acid was clarified. (Non-Patent Document 2)
Fatty acids bound in phospholipids have different functions. For example, arachidonic acid is essential for the growth and reproductive function of infants, EPA is known to contribute to blood homeostasis that controls heart disease and the like, and DHA is deeply involved in brain function homeostasis. These omega-3 fatty acids are usually bound to triglycerides and phospholipids in vivo, and are cut out by metabolic enzymes as necessary to play their roles. For example, when inflammation occurs, phospholipase A2 is expressed at the 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) to resolvin. , metabolites called SPMs (specialized pro-resolving mediators) typified by protectin and merceline are produced and participate in the suppression of inflammation. Important phospholipids with such metabolites attached have also been discovered. However, omega-3 fatty acids bound in phospholipids may produce phospholipid-type metabolites through the direct action of the above enzymes, and the details are unknown. It is known that soybean lipoxygenase acts on omega-3 fatty acids bound in phospholipids to introduce hydroperoxy groups. (Non-Patent Document 1)
In addition, various reactive oxygen species such as hydrogen peroxide and ozone produced by neutrophils, natural killer cells, and macrophages, which are activated by inflammation, attack bacteria at the site of inflammation, but are chemically extremely reactive. These reactive oxygen species non-enzymatically oxidize omega-3 fatty acids present in the vicinity and omega-3 fatty acids bound in phospholipids, producing different phospholipids that have undergone structural changes, exhibiting some biological functions. Although possible, such a mechanism is largely unknown. The deuterated phospholipids provided by the present invention can be important tools in elucidating these unknown mechanisms.

Omega-3 fatty acids taken in through meals are present in the blood as triglycerides and phospholipids, and are transferred to each tissue as needed. For example, it has recently been clarified that DHA becomes a lysophospholipid-bonded form in the liver and moves into the brain through the brain barrier along with blood flow. It is also known that DPA that has entered the brain is once converted to C24 (5n-3), then converted to 6n-3 by desaturase, which is accompanied by a reduction of 2 carbons again to become DHA. However, the mechanism is not clear. Arachidonic acid bound in phospholipids undergoes various complex oxidation reactions in the metabolic process, and as a result, phospholipids bound with various fatty acids that have undergone oxidative decomposition have been identified. Interestingly, these molecular species have been reported to be inflammatory or exhibit anti-inflammatory effects. (Non-Patent Document 3)
However, this study used a mixture of various phospholipid oxidation metabolites generated from phospholipids bound to arachidonic acid. Therefore, it is not clear which molecular species exhibits inflammatory or anti-inflammatory activity. In addition, since this research is limited to arachidonic acid, it is not clear what kind of action each oxidation product of phospholipid bound with EPA, DPA, and DHA exhibits.

S. C. Dyall, Long-chain omega-3 fat acids and the brain: a review of the independent and shared effects of EPA, DPA and DHA, Frontiers in Aging Neuroscience, 2015,7,1-16 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 V. N. Bochkov, Inflammatory profile of oxidized phospholipids, Thromb Haemost 2007;97:348-354

As described above, the functions of phospholipids have not yet been fully clarified, and new tools for their analysis are desired. The synthetic deuterated phospholipids provided by the present invention can be applied to solve problems in such fields. In addition, antiphospholipid antibody syndrome has a clinical problem that standardization of its detection is said to be difficult due to the diversity of antiphospholipid antibodies, and the present invention can facilitate such detection. We also offer diagnostics.

Accordingly, the present invention provides:
(Item 1)
Formula I below

(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 said hydrocarbon group contains 8 to 36 carbon atoms,
the hydrocarbon group contains 0 to 8 double bonds;
at least one of the carbons on the hydrocarbon group is optionally substituted with a substituent selected from the group consisting of =O, -OH and -OOH;
At least one of two adjacent carbon pairs on the hydrocarbon group is substituted with O to form an epoxy group.

may form
each ^RA is independently selected from hydrogen, deuterium or tritium)
A compound having the structure of or a salt thereof.
(Item 2)
A compound according to item 1 or a salt thereof, wherein all ^RA on the same carbon atom are the same hydrogen atom selected from hydrogen, deuterium or tritium.
(Item 3)
A method for producing the compound or a salt thereof according to item 1,
Step 1) Trideuteriomethyl group-introducing reagent

a step of preparing
Step 2) Structure below

synthesizing a choline containing a deuterated methyl group selected from
Step 3) protecting the 1-hydroxy and 3-hydroxy groups of glycerol;
Step 4) protecting the 2-hydroxy group of glycerol,
Step 5) deprotecting the 1-hydroxy and 3-hydroxy groups of glycerol;
Step 6) Step of introducing a fatty acid having a structure of R ¹ -OH to the 1-hydroxy group of glycerol, Step 7) Introducing a choline containing a phosphate group and a deuterated methyl group of step 2 to the 3-hydroxy group of glycerol. the process of
Step 8) deprotecting the 2-hydroxy group of glycerol;
step 9) introducing a fatty acid of the structure R ² —OH to the 2-hydroxy group of glycerol, optionally further comprising step 10) deuteration introduced in step 7 A production method comprising the steps of converting choline containing a methyl group into aminoethanol, and step 11) introducing a deuterated methyl group into aminoethanol.
(Item 4)
A composition comprising the compound of item 1 or 2 or a salt thereof.
(Item 5)
A composition according to item 4 for the determination of phospholipid metabolism disorders.
(Item 6)
5. A composition according to item 4 for diagnosis of a disease or condition.
(Item 7)
The composition according to item 6, wherein the disease or condition is a disease that causes abnormalities in lipid metabolism.
(Item 8)
7. The composition of item 6, wherein said disease or condition is selected from the group consisting of obesity, hyperlipidemia, non-insulin dependent diabetes mellitus, and knee osteoarthritis.
(Item 9)
7. The composition of item 6, wherein said 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 palmitoylglycerol palmitoylated with an unsaturated fatty acid, comprising (i) a compound selected from the group consisting of 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,
A method comprising the step of performing a lipase enzymatic reaction using as a substrate.
(R ₁ to R ₁₁ are independently 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 ~9 alkyl group, H or an alkyl group having 1 to 8 carbon atoms, H or an alkyl group having 1 to 7 carbon atoms, H or an alkyl group having 1 to 6 carbon atoms, H or an 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 of item 10, wherein the unsaturated fatty acid contains 8-36 carbon atoms and contains 1-8 double bonds.
(Item 12)
11. The method of item 10, wherein at least one of the carbons on said unsaturated fatty acid is optionally substituted with a substituent selected from the group consisting of =O, -OH and -OOH, At least one of two adjacent carbon pairs on the hydrocarbon group is substituted with O to form an epoxy group

A method that may form a
(Item 13)
11. The method of item 10, wherein the lipase enzymatic reaction is carried out at a temperature of -20°C to 50°C for 0.5 hours to 24 hours.
(Item 14)
11. The method of item 10, further using an acylating agent.
(Item 15)
11. The method of item 10, further using a solvent.

It is contemplated in the present invention that one or more of the features described above may be provided in further combinations in addition to the explicit combinations. Further embodiments and advantages of the present invention will be appreciated by those skilled in the art, if necessary, upon reading and understanding the following detailed description.

The present invention can contribute to functional studies of phospholipids. In addition, the present invention can provide new diagnostic agents for phospholipid-related diseases.

The present invention will be described below. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Also, it should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Thus, unless defined otherwise, 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, "wt%" is used interchangeably with "mass percent concentration" herein. "%" means "wt %" or "w/w %" or "mass percent concentration" unless otherwise specified.

In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "or". When we say "within a range" of "two values" herein, the range includes the two values themselves.

All references, such as scientific articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entireties to the same extent as if each were specifically set forth.

(definition)
Definitions of terms and/or basic technical content particularly used in the present specification will be described below as appropriate.

As used herein, the term "unsaturated fatty acid" refers to a fatty acid with one or more unsaturated carbon bonds. An unsaturated carbon bond is an unsaturated bond between carbon atoms in a carbon molecular chain, ie, a carbon double bond or a triple bond. Unsaturated fatty acids found in nature 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.

The term "ω3 fatty acid" as used herein refers to an unsaturated fatty acid having a double bond from the third carbon (ω3 position) from the methyl end, typically α-linolenic acid, stearidone acids, eicosatetraenoic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPAn-3), docosahexaenoic acid (DHA), tetracosapentaenoic acid, and tetracosahexaenoic acid. Not limited. As used herein, the term "modified ω3 fatty acid" is a substance that modifies ω3 fatty acid, and includes oxides of ω3 fatty acid (e.g., oxidized metabolites), typically 13- Hydroxy-α-linolenic acid, 13-hydroxystearidonic acid, 15-hydroxyeicosatetraenoic acid, 15-hydroxyEPA, 17-hydroxyDPAn-3, 17-hydroxyDHA, 19-hydroxytetracosapentaenoic acid, and , 19-hydroxytetracosahexaenoic acid.

The term "docosahexaenoic acid" as used herein is used interchangeably with "DHA" and includes both the free fatty acid form and the form ester-bonded to ethanol, glycerin, and phospholipids.

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

The term "docosapentaenoic acid" used herein is used interchangeably with "DPA" and includes both the free fatty acid form and the form ester-bonded to ethanol and glycerin. Also, "DPA" not specified as either "n-3" or "n-6" is referred to as "n-3 DPA" (i.e., all-cis-7,10,13,16,19-docosa pentaenoic acid). "n-3 DPA" is "ω-3
DPA" is used interchangeably.

As used herein, the term "n-6 DPA" is used interchangeably with "all-cis-4,7,10,13,16-docosapentaenoic acid" or "ω6 DPA" and the free Both the fatty acid form and the form ester-bonded to ethanol and glycerin are included.

The term "stearidonic acid" used herein is used interchangeably with "SDA" and includes both the free fatty acid form and the form ester-linked to ethanol and glycerin.

The term "α-linolenic acid" used herein is used interchangeably with "ALA" and includes both the free fatty acid form and the form ester-bonded to ethanol and glycerol.

As used herein, the term "eicosatetraenoic acid" is used interchangeably with "ETA" and includes both the free fatty acid form and the form ester-bonded to ethanol and glycerin.

As used herein, the term “hydrogen” refers to the ¹ H isotope of the hydrogen atom, also denoted H. Although the hydrogen atoms contained in naturally occurring compounds contain a certain proportion of isotopes such as deuterium and tritium, when hydrogen or H is used herein, strictly speaking all contained in the compound. is ¹ H, but does not exclude the inclusion of isotopes of hydrogen, such as deuterium and tritium, to the extent they are normally included.

As used herein, the term “deuterium” refers to the ² H isotope of the hydrogen atom, also denoted D or d.

As used herein, the term “tritium” refers to the ³ H isotope of the hydrogen atom, also denoted T or t.

As used herein, the term “hydrogen species” is used when describing without distinction between isotopes of a hydrogen atom ( ¹ H, ² H and ³ H), and hydrogen atoms are ¹ H, ² H and ³ H.

The term "phospholipid" used herein refers to an amphiphilic substance having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group containing a phosphate group in the molecule. Examples of phospholipids include phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, sphingophospholipids, diphosphatidyl phospholipids, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine , dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), dilinoleoylphosphatidylcholine, and the like.

As used herein, the terms “phosphatidylcholine” or “lecithin” are used interchangeably and are compounds also designated as PC and glycerin (propane-1,2,3-triol, also known as glycerol). Acyl groups are bound to the sn-1 and sn-2 positions, and choline is bound to the phosphoric acid bound to the sn-3 position.

As used herein, the term "lysophosphatidylcholine" is a compound also denoted as lyso-PC, in which an acyl group is attached to either the sn-1 or sn-2 position of glycerin, and the sn- Choline is bound to the phosphate bound to the 3-position.

As used herein, the term "sn-" is an abbreviation for Stereospecifically Numbered and is used when the carbon atoms of glycerin derivatives are represented by stereospecific numbering. When the glycerin derivative is racemic, it is marked with rac-, and when the stereochemistry is unknown, it is marked with X-. Phospholipids are classified into two types, sn-1-phospholipids and sn-2-phospholipids, depending on the bonding positions of their acyl groups. For actual designations, acyl group positions are used as is, 1-acyl-phosphatidylcholine, 2-acyl-phosphatidylcholine, and so on. In the present specification, when there is no information about sn, it is a case of being described without particular distinction or being used as a generic term (higher concept).

As used herein, the term "acyl (group)" is used in the ordinary sense in the art and refers to a group formed by removing a hydroxyl group from an organic acid (carboxylic acid; fatty acid). In a broad sense, it includes formyl group HCO-, acetyl group _CH3CO- , malonyl group _-COCH2CO- , benzoyl group _C6H5CO- , cinnamoyl group _C6H5CH =CHCO- and the like, and ketone _{derivatives .} And so on. Acyl groups contained in phosphatidylinositol phosphates and lysophosphatidylinositol phosphates, in a preferred embodiment, form fatty acids and are therefore also referred to as fatty acid groups. Fatty acids can be represented herein by their carbon number and number of double bonds, for example, arachidonic acid can be represented as (20:4). If the position of the double bond is further specified, specify all positions for cis or trans designation, or E or Z designation, or display in the omega (ω) 3 series, omega 6 series, etc. can do.

Although the following fatty acids and acyl groups based thereon are generally used herein, it is understood that fatty acids having any chain length and any double bond can be used without being limited thereto. For example, the number of carbon atoms is 1 or more, typically 1 to 30, and usually in the range of 4 to 30. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, etc., but are not limited thereto. Also, the number of double bonds may be 0, 1, 2, 3, 4, 5, 6, 7, or any allowable number depending on the number of carbon atoms. The position of the double bond is typically omega-3, omega-6, omega-9, etc. In addition, omega-1, omega-4, omega-5, and omega-7 have also been confirmed. , any of which are available. Fatty acids may contain triple bonds, the number of which is 0, 1, 2, 3, 4, 5, 6, 7, etc. number can be employed.

As used herein, the term "diagnosis" refers to identifying various parameters associated with a disease, disorder, condition, etc. (e.g., a disease, disorder, condition, etc., attributed to a lipid mediator) in a subject, and identifying such parameters. Determining the present or future status of any disease, disorder or condition. By using the methods, devices, and systems of the present invention, conditions within the body can be investigated, and such information can be used to determine the disease, disorder, condition, treatment or prophylactic formulation to be administered in a subject. Alternatively, various parameters such as method can be selected. In the present specification, "diagnosis" in a narrow sense means diagnosing the current situation, but in a broader sense it includes "early diagnosis", "predictive diagnosis", "pre-diagnosis" and the like. INDUSTRIAL APPLICABILITY The diagnostic method of the present invention is industrially useful because, in principle, it is possible to use what comes out of the body, and it can be performed without the hands of medical professionals such as doctors. In this specification, in order to clarify that it can be performed without the hands of medical personnel such as doctors, "predictive diagnosis, preliminary diagnosis or diagnosis" is sometimes referred to as "assisting". The technology of the present invention can be applied to such diagnostic technology. The techniques of the present invention can be used to identify the presence of deuterated phospholipids and have various such diagnostic applications.

As used herein, the term "prognosis" means predicting the likelihood of death or progression due to a disorder such as cancer, a disease caused by lipid mediators, a disorder, and the like. Prognostic factors are variables related to the natural course of a disease, and they affect the recurrence rate of patients who have once developed the disease. Clinical indicators associated with poor prognosis include, for example, any cellular indicator used in the present invention. Prognostic factors are often used to divide patients into subgroups with different pathologies. The ability to identify the presence of deuterated phospholipids using the techniques of the present invention can be useful as a technique for providing prognostic factors because it can be associated with certain disease states.

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

The term "cancer" as used herein refers to any cancer that can be treated or prevented with a vaccine or pharmaceutical, such as hepatocellular carcinoma, esophageal squamous cell carcinoma, breast cancer, pancreatic cancer, squamous cell or adenocarcinoma of the head and neck, colorectal cancer, kidney cancer, brain cancer (tumor), prostate cancer, small and non-small cell lung cancer, bladder cancer, bone or joint cancer, Uterine cancer, cervical cancer, multiple myeloma, hematopoietic malignancies, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma, squamous cell carcinoma, leukemia, lung cancer, ovarian cancer, gastric cancer, Kaposi sarcoma, laryngeal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, pituitary cancer, adrenal cancer, cholangiocarcinoma, endometriosis, esophageal cancer, liver cancer, NSCLC, osteosarcoma , pancreatic cancer, SCLC, soft tissue tumors, AML, and CML.

As used herein, the term “lipid metabolism disease” refers to a disease whose at least one cause is abnormal lipid or fatty acid metabolism. Lipid metabolism disorders include, but are not limited to, obesity, hyperlipidemia, and non-insulin dependent diabetes mellitus.

As used herein, "inflammation" refers to any detectable inflammation, and activation of blood cells related to inflammation such as macrophages (chemotaxis of inflammatory cells, active oxygen production, phagocytosis, enzyme secretory reaction, etc.) can accompany Exemplary inflammations include, for example, arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, Sjögren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), spondylitis, multiple myositis, dermatomyositis, pemphigus, pemphigoid, Hashimoto's thyroiditis, cholangitis, inflammatory bowel disease (IBD, e.g. Crohn's disease, ulcerative colitis), colitis, inflammatory skin disease, pneumonia, asbestosis, Silicosis, bronchiectasis, talc, pneumoconiosis, sarcoidosis, delayed hypersensitivity reactions (e.g. poison ivy dermatitis), airway inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hay fever, allergies , acute anaphylaxis, reperfusion injury, rheumatoid arthritis, glomerulonephritis, pyelonephritis, cellulitis, cystitis, cholecystitis, allograft rejection, host versus graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis , bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryodenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis , fibromyalgia, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, ophthalmitis, oophoritis, orchitis, osteitis, otitis media, pancreatitis, parotitis , pericarditis, pharyngitis, pleuritis, phlebitis, pneumonia, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, orchitis, tonsillitis, urethritis, cystitis, grapes diseases or disorders involving acute or chronic inflammation such as meningitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, vasculitis, osteomyelitis, optic neuritis, myelitis, and fasciitis, but It is not limited to these.

As used herein, the term "marker" refers to a state (e.g., functional, transformed state, disease state, disordered state, or proliferative potential, level of differentiation state, presence or absence, etc.) of or at risk of A substance that serves as an index for tracking whether or not there is In the present invention, the detection, diagnosis, preliminary detection, prediction or pre-diagnosis of a condition (e.g., disease state, health condition, disease such as cell or tissue differentiation disorder) may be performed in addition to the detection methods provided by the present invention. , a marker-specific drug, agent, factor or means, or a composition, kit or system containing them, or the like.

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

(deuterated phospholipids)
In one aspect, the present invention provides a compound of formula I below

(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 said hydrocarbon group contains 8 to 36 carbon atoms,
the hydrocarbon group contains 0 to 8 double bonds;
at least one of the carbons on the hydrocarbon group is optionally substituted with a substituent selected from the group consisting of =O, -OH and -OOH;
At least one of two adjacent carbon pairs on the hydrocarbon group is substituted with O to form an epoxy group.

may form
each ^RA is independently selected from hydrogen, deuterium or tritium)
A compound having the structure or a salt thereof is provided.

In addition, examples of fatty acids containing R include, but are not limited to, the following moieties:
1) Saturated fatty acids: Saturated fatty acids with C8 to C36 carbon chains.
2) Unsaturated fatty acid: Fatty acid containing C8 to C36 carbon atoms and 1 to 8 double bonds.
3) Hydroxyl-bonded fatty acid: Fatty acid to which a hydroxyl group is bonded. 1 to 3 hydroxyl groups are added per fatty acid.
4) Epoxy-type fatty acid: A fatty acid obtained by epoxidizing the double bond of the unsaturated fatty acid in 2). 1 to 2 epoxy groups per fatty acid.
5) Oxo-type fatty acid: A fatty acid having a carbonyl group or an aldehyde group in addition to the carbonyl group of the carboxyl group of the fatty acid is also called an oxo-type fatty acid. 1 to 3 carbonyl groups per fatty acid.
6) Hydroperoxy-type fatty acid: Fatty acid with a hydroperoxy group attached. 1 to 3 hydroperoxy groups per fatty acid.
7) Fatty acids in which the functional groups shown in 2) to 6) above are combined with fatty acids 8) In the case of fatty acids in which a carbonyl group or an epoxy structure is combined with a hydroxyl group or a hydroperoxy group, only the hydroxyl group and the hydroperoxy group should be 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 in general, 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 includes 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 lauryl, myristoyl, palmitoyl, stearyl, 9,12-octadecanedienoyl, 9,12,15-octadecanetrienoyl, 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- is selected from the group consisting of docosahexaenoyl groups; In one embodiment, R ¹ is a selected acyl group comprising a linear or branched saturated hydrocarbon group containing 8 to 36 carbon atoms, such as lauryl, myristoyl, palmitoyl, and a stearyl group. In one embodiment, R ² is lauryl, myristoyl, palmitoyl, stearyl, 9,12-octadecanedienoyl, 9,12,15-octadecanetrienoyl, 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- is selected from the group consisting of a docosahexaenoyl group and

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

In one embodiment, the compounds of the present invention are compounds in which hydrogen in naturally occurring compounds is replaced with deuterium or tritium. In one embodiment, the compounds of the present invention are compounds in which hydrogen in compounds known to be present in foods is replaced with deuterium or tritium. In one embodiment, the compounds of the present invention are compounds in which hydrogen in compounds present in mammals is replaced with deuterium or tritium. In one embodiment, the compounds of the invention are compounds in which hydrogen in compounds known to exist in humans is replaced with deuterium or tritium.

(Production of deuterated phospholipids)
In one aspect, the invention provides methods for making the compounds of the invention.

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

Synthetic scheme 1

(Step 1: Preparation of trideuteriomethyl group-introducing reagent)

Reaction of p-toluenesulfonyl chloride with tetradeuteriomethanol gives trideuteriomethyl p-toluenesulfonate. This reaction can be carried out, for example, by slowly adding dropwise a 35% aqueous solution of caustic soda to the mixture at -5 to 0°C. Phospholipids that bind di-deuteriomethyl groups or mono-deuteriomethyl groups are also available. Instead of p-toluenesulfonyl chloride, other reagents such as 2,4,6-triisopropylbenzenesulfonyl chloride, or various aryl and alkylsulfonic acid halogenations can be used as appropriate.

(Step 2: Synthesis of choline containing deuterated methyl group)

Deuterium-introduced choline can be obtained by reacting N,N-dimethylethanolamine with the trideuteriomethyl p-toluenesulfonate obtained above. Here, instead of N,N-dimethylaminoethanol, its deuterium-substituted product can also be used. Deuterium-substituted cholines thus obtained include, for example,

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

It is also possible to obtain 9-deuterated phospholipid (18) by using choline in which 3 methyl groups are deuterated in phosphatidic acid and 2,4,6-triisopropylbenzosulphonyl 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 the 1- and 3-hydroxy groups of glycerol. Instead of benzaldehyde, any other suitable protecting group-providing compound may be used to protect the 1- and 3-hydroxy groups of glycerol, if desired.

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

A protective group is introduced to the 2-hydroxy group of glycerol. For example, a benzyl group can be introduced as —OR′ by adding benzyl chloride under basic conditions. —OR′ is any suitable substituent and is preferably selected so that it is not removed under the conditions that remove the protecting groups of the 1- and 3-hydroxy groups of glycerol. In one embodiment, —OR′ can be selected as a substituent that facilitates an elimination reaction. 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 for 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, 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 and at least one of the carbons on the hydrocarbon group is substituted with a substituent selected from the group consisting of =O, -OH and -OOH. may be It may be enzymatically introduced by lipase, or may be introduced by a known chemical esterification method. When this reaction is performed with a lipase, R' is selected from a substituent recognized by the lipase such as a benzyl group, a methoxy group, an ethoxy group, and an isopropyloxy group to be a substrate, and the type of lipase and reaction conditions ( temperature, solvent, concentration, etc.) are appropriately controlled. When performing this reaction with a lipase, an optically pure product can be obtained. When carrying out this reaction by chemical esterification methods, the product can be obtained as a racemate.

Any lipase that uses 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-positions of triglycerides. Lipases may be natural enzymes or recombinant enzymes. Also, the "lipase" used in the present invention may be in the form of a solution or an immobilized form. Examples of lipases derived from microorganisms include Candida cylindoracea-derived lipase OF (trade name, manufactured by Meito Sangyo Co., Ltd.), Alcaligenes sp. Lipase QLM, Lipase QLC, Lipase PL derived from (all trade names, manufactured by Meito Sangyo Co., Ltd.), Lipase PS derived from Burkholderia cepacia (trade name, manufactured by Amano Enzyme Co., Ltd.), Lipase AK derived from Pseudomonas fluorescens ( product name, manufactured by Amano Enzyme Co., Ltd.), but not limited thereto. Preferably, the lipase used in the present invention is a microbial lipase that selectively hydrolyzes the 1- and 3-positions of triglycerides. In the present invention, immobilized lipase, for example, Thermomyces lanuginosa-derived immobilized lipase, Lipozyme TLIM (trade name, manufactured by Novozyme) can be used, but is not limited thereto. A particularly preferred lipase in the present invention is lipase AmanoPS. The lipase enzyme protein is desired to have general stability such as physical and biochemical stability, and more preferably to be chemically stable in an organic solvent. Those skilled in the art can obtain the desired stereoselectivity by appropriately selecting and adding the solvent, acylating agent, temperature, concentration, and the like. A preferred solvent for the lipase reaction is dichloromethane. The preferred temperature for the lipase reaction is -20°C to 50°C, more preferably -10°C to 30°C, even more preferably -5°C to 20°C, most preferably -2°C to 10°C, for example, 10°C. is. A preferred reaction time for the lipase reaction is 0.5 hours to 24 hours, more preferably 1 hour to 10 hours, even more preferably 2 hours to 7 hours, most preferably 3 hours to 6 hours, such as 6 hours. . As the acylating agent, not only palmitic acid vinyl ester but also palmitic acid isopropenyl ester, palmitic acid trifluoroethyl ester, palmitic anhydride, 2-O-benzyl glyceride, or other vinyl esters of saturated fatty acids are 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.

High or low stereoselectivity in a lipase-catalyzed acylation reaction, i.e., whether it gives a product of high enantiopure, is generally determined by the type of lipase, the type of substrate, the type of acylating agent, and the type of solvent. , enzyme concentration, acylating agent concentration, substrate concentration, and temperature. In addition, some theoretical and empirical conditions for obtaining high stereoselectivity are known. On the other hand, for example, the lower the temperature, the higher the stereoselectivity, but when the reaction temperature is low, there is a problem that the reaction yield decreases. In view of this situation, in the present application, a palmitoyl group is stereoselectively bound to the hydroxyl group of an enantiotropic substrate called 2-O-Benzylglycerol to obtain 2-O-benzoyl-1-palmitoylglycerol (9) with high optical purity. We investigated the conditions for 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. Below, Lipase PS-IM-Amano (100 mg) (Amano Enzyme Co., Ltd.) was added and stirred for 6 hours to obtain 2-O-benzoyl-1-palmitoylglycerol (9) with optical purity (98% or more). I made it clear for the first time that I could. The high optical purity 2-O-benzoyl-1-palmitoylglycerol (9) thus obtained is a very important compound as a common intermediate for synthesizing various optically active glycerophospholipids. Furthermore, once such conditions are established, it is very easy for those skilled in the art to extend the scale of the reaction. In contrast, in the case of a chiral column, it is possible to obtain 2-O-benzoyl-1-palmitoylglycerol (9) with high optical purity, but the amount of substrate processed by an expensive optically active column is very small, so the amount of processing is increased. Therefore, if the column size is increased, not only is the cost extremely high, but also unnecessary solvent treatment to be discharged from the column is required.

The enzymatic reaction of lipase may be carried out in an organic solvent (chloroform, n-hexane, isopropyl ether, etc.). If 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 by appropriately referring to, for example, the following literature. M. Murata et al. , Efficient lipase-catalyzed synthesis of chiral glycerol derivatives. Chem. Pharm. Bull. , 1989, 37, 2670-2672; Ghisalva et al. , Enzymatic preparation of acylglycerols of high optical purity. Recl. Trav. Chim. Pay-Bas. 1991, 110, 263-64, Masakazu Murata et al., Design of enzyme-catalyzed asymmetric reaction in organic solvent and its application to synthesis of optically active pharmaceuticals. Synthetic Organic Chemistry, 1991, 49, 1127-1141; Hazarika, P.; Goswami, and N.L. N. Dutta, Lipase catalyzed transesterification 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 to palmitoylate glycerol with lipase or another method to synthesize palmitoylglycerol, which is an intermediate of high optical purity, the protecting group used is 2-O-benzyl It is not limited to glycerol.

A first requirement in the protecting groups used is that the substituent attached to the 2-position of the glycerol backbone must be removable by hydrogenolysis. The simplest example in this application is a Benzyl group (R ₁ -R ₇ =H in structure A of the chemical formula below). is used, it is also possible to practice the method of the present invention with compounds of structural formula B or C of the formula below. Note that this hydrogenolytic deprotection does not cleave the palmitoyl group ester-bonded with lipase.

When using compounds having either structural formula A, B or C, preferably R ₁ =R ₂ . When R ₁ ≠R ₂ , the carbon to which they are attached, C★, becomes the center of chirality and gives the racemic form of structure D. When a racemic form is obtained, it is also possible to optically resolve these racemates by utilizing the OH groups remaining in the glycerol skeleton. However, since the asymmetric center to which R ₁ and R ₂ are bonded is too far from the remaining hydroxyl group, high stereoselectivity cannot be expected in optical resolution of OH groups by lipase. Therefore, in the present invention preferably R ₁ =R ₂ .

Further, when using compounds of any of Structural Formulas A, B or C, any of R ₁ -R ₁₁ are independently substituted with methyl, ethyl, propyl, and/or isopropyl groups, etc. It is a sufficient substrate for lipase and can give high stereoselectivity.

A method of producing palmitoylglycerol using lipase according to the present invention includes, for example, a step of carrying out a lipase enzymatic reaction using a compound having any of the above structural formulas A, B or C and a fatty acid as substrates. In compounds having any of the above structural formulas A, B or C, R ₁ to R ₁₁ are independently H, alkyl groups having 1 to 6 carbon atoms, alkyl groups having 1 to 5 carbon atoms, alkyl groups having 1 to 5 carbon atoms, 4 alkyl group or an alkyl group having 1 to 3 carbon atoms. and/or in compounds having any of structural formulas A, B or C above, preferably R ₁ =R ₂ .

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

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

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

The protective group introduced to the 2-hydroxy group of glycerol is removed. For this reaction, for example, when a benzyl group is introduced as the 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. For example, the deuterated lysophosphatidylcholine prepared in step 8 (compound 11) and the fatty acid R ² —COOH to be introduced are dissolved in ethanol-free dry chloroform, and dicyclohexylcarbodiimide (DCC) and dimethylamino It can be proceeded 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 those skilled in the art will appreciate other known reagents such as 1-hydroxybenzotriazole and diphenyl phosphate azide. It can be selected as appropriate.

In one embodiment, R ² can be a C ₈ to C ₃₆ saturated or unsaturated hydrocarbon group, wherein the hydrocarbon group contains 0 to 8 double bonds and 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 1-8 double bonds. In one embodiment, R ² can be a C ₈ -C ₃₆ saturated hydrocarbon group containing 1-3 hydroxyl groups (—OH). When R ² is a hydrocarbon group containing a hydroxyl group, a fatty acid in which the hydroxyl group is protected as an ester or a silyl ether is reacted with deuterated lysophosphatidylcholine, and then the hydroxyl group is deprotected to obtain a hydroxyl group-containing ^R2 can be introduced. In one embodiment, R ² can be a C ₈ -C ₃₆ saturated hydrocarbon group containing 1-2 epoxy groups. In one embodiment, R ² can be a C ₈ -C ₃₆ saturated hydrocarbon group containing 1-2 carbonyl groups. In one embodiment, R ² can be a C ₈ -C ₃₆ saturated hydrocarbon group containing 1-3 hydroperoxy groups (--OOH). When R ² is a hydrocarbon group containing a hydroperoxy group, a fatty acid in which the hydroperoxy group is protected as a peracetal is reacted with deuterated lysophosphatidylcholine, followed by deprotection of the hydroperoxy group. , can introduce R ² containing a hydroperoxy group. When R ² contains more than one of double bonds, hydroxyl groups (-OH), epoxy groups, carbonyl groups, and hydroperoxy groups (-OOH), only the hydroxyl and/or hydroperoxy groups are protected; The desired ^R2 can be introduced without protecting the functional group.

Additionally, choline can optionally be converted to deuterated choline by steps 10 and 11 below. These steps are described, for example, in HU. Gally, W.; Niederberger, andJ. Seeling, Conformation and motion of the choline head group in bilayers of dipalmitoyl-3-sn-phosphatidylcholine. Biochemistry, *1975*, 14, 3674-3652; S. Harbison andR. G. Griffin, Improved method for the synthesis of phosphatidylcholines. Journal of Lipid Research, *1984*, 25, 1141-1144. , and K. M. Patel, J.; D. Morrisette, andJ. T. Sparrow, The conversion of phosphatidylethanolamine into phosphatidylcholine labeled in the choline group using methyl iodide, 18-crown-6 and potassium carbonate. Lipids, *1979*, 14, 596-597), S. Sunder et al. , Infrared and Raman spectrum of specifically deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholines, Chemistry and Physics of Lipids, 1981, 28, 137-14 8. See Etc.

Also, since (14) is a non-deuterated phospholipid (12), it can be synthesized by reacting with 2-aminoethanol to obtain (13), which is then reacted with deuterated methyl iodide. possible (later deuteration).

(13) is obtained by reacting 2-aminoethanol in which two deuterium atoms are substituted with (12). This can be reacted with deuterated methyl iodide to give the 11-deuterated phospholipid (14).

(Step 10: Conversion of Choline to Aminoethanol)

Step 10 replaces choline with 2-aminoethanol by reacting the product of step 9 (compound 12) with 2-aminoethanol in the presence of phospholipase D. 2-Aminoethanol may optionally be used in its deuterium-substituted form. -2-aminoethanol and 1,1,2,2-tetradeuterio-2-aminoethanol, etc., can be used, and when these deuterium-substituted forms are used, the final product will be more heavy. A compound containing hydrogen is obtained.

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

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

In one aspect, the following synthetic methods can be used in addition to the synthetic routes described above.
5) Alternatively;
In one embodiment, for example, J.P. 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. etc., a method of converting optically active mannitol into phosphatidylcholine (PC) without changing the configuration can be used, and in this method 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 lysophosphatidic acid and lysophosphatidylcholine from p-nitrophenyl glyc Erate. With reference to Tetrahedron Letters, 2004, 45, 7371-7373, etc., a method of converting p-nitrophenylglycerate to phosphatidylcholine can be used, and p-nitrophenylglycerate having an optically active glyceride skeleton can be used. Compound 12 can be obtained by introducing a deuterated choline as described above.

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

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

(Use of deuterated phospholipids)
Many papers have been published on the biofunction importance of phospholipids, including fatty acids such as EPA, DPA and DHA. However, in living tissues such as blood, various phospholipid molecular species with similar chemical structures are mixed, and their distribution differs even between tissues, and their chemical structures change from moment to moment during metabolic processes. Therefore, it is extremely difficult to track structural changes of a specific phospholipid molecular species over time. In recent years, lipidomics analysis using a mass spectrometer has been performed, but the above analysis has not been sufficiently performed. 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 compositions comprising the phospholipids of the invention. Deuterated phospholipids are believed to exhibit similar physicochemical properties and similar absorption, distribution, metabolism, or excretion in vivo as their non-deuterated counterparts. However, deuterated phospholipids or their metabolites are readily distinguishable by mass spectrometric analysis because deuterated phospholipids differ in mass from their nondeuterated counterparts. can be done. Therefore, it can be particularly useful for tracking structural changes in phospholipids accompanying metabolism.

In one embodiment, the compositions of the invention are used to detect or measure phospholipid absorption, metabolism, distribution, or excretion.

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

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

(Measurement of abnormal phospholipid metabolism)
In one embodiment, the compositions of the invention can be used for the measurement of phospholipid metabolism disorders. For example, by adding to tissues, blood, serum or plasma collected from patients and healthy subjects and detecting chemical changes that occur there (for example, measuring the type and amount of isotopically labeled compounds produced) , making it possible to determine whether a patient's phospholipid metabolism is abnormal compared to healthy subjects.

(Use for Diagnosis of Diseases or Conditions)
In one embodiment, the compositions of the invention can be used for diagnosis of disease. The disease can be any disease associated with phospholipids, such as diseases that cause abnormalities in lipid metabolism (e.g., obesity, hyperlipidemia, non-insulin dependent diabetes mellitus, and knee osteoarthritis). but not limited to), cancer, inflammation, arteriosclerosis, Alzheimer's disease, sepsis, antiphospholipid antibody syndrome (age-related macular degeneration, systemic lupus erythematosus, etc.), etc., but not limited thereto. For example, adding the composition of the present invention to tissue, blood, serum, or plasma collected from a living body, and detecting chemical changes that occur there (for example, measuring the type and amount of isotopically labeled compounds produced By doing so, it may be possible to diagnose the pathology and the degree of progression.

(1) Diagnosis of Alzheimer's disease Diagnosis of Alzheimer's disease is mostly based on pathological techniques, but many problems still remain in diagnostic methods using biomarkers. Regarding this point, Gonzalez-Dominguez et al. published a paper in 2016 (Current Alzheime
r Research, 2016, 13, 641-653) states that chemical changes in serum phospholipids are closely related to the pathology of Alzheimer's disease and mild dementia, and the following important content: has been pointed out.
"The use of blood in Alzheimer's disease research has so far been neglected because the relationship between blood-based measurements and processes in the brain was thought to be difficult. A close relationship between metabolic disorders and brain samples was recently demonstrated using a transformed model of Alzheimer's disease, and there is increasing evidence that Alzheimer's disease is a systemic disease. The use of peripheral blood has proven important in the study of the pathological mechanisms associated with Alzheimer's disease, which is associated with impaired metabolism of phospholipids and sphingolipids and the acyl binding to them. Chain length and degree of unsaturation play a decisive role: Alzheimer's disease was once associated with excessive degradation of brain phospholipids by phospholipases, leading to disruption of cell membranes, and neuronal tissue. Loss of unsaturated fatty acids such as DHA and arachidonic acid present in high concentrations due to oxidative stress causes cell membrane damage, which is implicated in the pathology of Alzheimer's disease.This rationale suggests that saturated fatty acid-bound phosphatidylcholine and phosphatidylethanolamine phosphatidylcholines bound to unsaturated fatty acids such as linoleic acid, arachidonic acid and DHA are significantly decreased.” Against this background, as described below, the phospholipids of the present invention It can provide an excellent diagnostic agent.

There have been several reports on the relationship between the types and amounts of phospholipids in plasma and Alzheimer's disease. For example, phosphatidylcholine (PC16:0/20:5, PC16:0/22:6, PC16:0/20:5, PC16:0/22:6 ) and phosphatidylcholine (PC18:0/22:6) with stearic acid at sn-1 and DHA at sn-2 (PC18:0/22:6) decreased in 2013. . These results implicate specific phosphatidylcholines in the pathology of Alzheimer's disease, the authors say. (Luke Whiley et al, Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease, Neurobiology of Aging, 35, 2014, 1-8)
It is also well known that plasma phospholipase A2 activity, which uses phospholipids as a substrate, is altered in many pathological conditions including Alzheimer's disease. (J. R 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 PLA2-Mediated lysophosphatidylcholine production regulates neuroinflammation and triggers neurodegeneration.The Journal of Neuroscience, 3 2, 2012, 1020-1034)
Therefore, phospholipase A2 is deeply involved in the types and quantitative changes in phospholipids that correspond 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 Alzheimer's disease patients (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).

It is reported that diagnosis of Alzheimer's disease is possible based on these facts. However, the types of biological phospholipids, fatty acids, and lysophospholipids are extremely diverse, and it is not easy to analyze them simultaneously with high reliability even using lipidomics techniques.

When the deuterated phospholipids synthesized in the present invention are analyzed by ESI MASS, the product ion is input as m/z 187 (By inputting m/z 187, no phospholipids other than deuterated phospholipids are detected. ), deuterated phospholipids and those that have undergone structural changes can all be detected simultaneously with extremely high selectivity.

Thus, for example, deuterated phospholipids (PC16:0/20:5, PC16:0/22:6, PC18:0/22:6) were added to blood and the ESI of samples collected after incubation for a period of time. From the MASS spectrum, it is possible to diagnose Alzheimer's disease by simultaneously analyzing the residual deuterated phospholipids and the fluctuations of the lysophosphatidylcholine produced from them.

For example, it has been reported that the ratio of lysophosphatidylcholine to phosphatidylcholine in cerebrospinal fluid, but not blood, is associated with Alzheimer's disease. (CJJ Mulder et al., Decreased lysophosphatidylcholine/phosphotidylcholine ratio in cerebrospinal fluid in alzheimer's disease, journal of Neural Trans mission 110, 2003, 949-55)
All of the three types of molecular species reported by Luke Whiley et al. above are nothing but those in which the deuterium of the deuterated phosphatidylcholine synthesized in the present invention is replaced with the original hydrogen atom.

In addition, it has long been known that unsaturated fatty acids and unsaturated fatty acids that bind to phospholipids undergo oxidative stress in living organisms and generate their peroxides, which are associated with various pathologies including inflammation. It is It has also been reported in many tissues of Alzheimer's disease patients (Elic Tonnies et al., Oxidative stress, synaptic dysfunction, and Alzheimer's disease, Journal of Alzheimer's Disease, 57, 2017, 1105 -1121).

EPA, DPA, or DHA that binds to deuterated phospholipids synthesized in the present invention plays an important role in the body, but is extremely susceptible to oxidation 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 molecular species of deuterated phospholipids can be analyzed simultaneously (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 C Chemistry, 4, 2013, 16-26)
If this analysis can confirm the correlation between pathological conditions such as Alzheimer's disease and the phospholipid oxide pattern in ESI MASS, it can be applied to diagnosis. However, it is difficult to selectively diagnose a specific disease using any of the above methods alone.

In such a case, in ESI MASS measurement using m / z 187, simultaneous analysis of quantitative changes in deuterated phospholipids themselves, formation of lysophosphatidylcholine, and patterns of peroxide molecular species and pathology including Alzheimer's disease can be applied to diagnosis with higher accuracy.

Based on the above-described conventional technology, healthy subjects have a higher expression level of phospholipase A2 enzyme, or an increased enzyme activity, than Alzheimer's disease patients. Furthermore, phosphatidylcholine D3, which binds EPA, DHA, or DPA, which is an omega-3 fatty acid and is deeply involved in anti-inflammatory properties, and phosphatidylcholine D3, which binds inflammatory arachidonic acid, which is an omega-6 fatty acid, lysophosphatidylcholine D3 through the action of phospholipase A2. Generate. The ratio of PCD3/Lyso-PCD3 in each enzyme reaction is expected to vary depending on the presence/absence and type of pathology. It has long been known that phospholipase A2, which is activated by inflammation underlying the pathology, releases arachidonic acid that binds to membrane phospholipids, and that this releases various inflammatory substances through the arachidonic acid cascade. Therefore, it is believed that the isotopically-labeled compounds of the present invention can be used as indicators of inflammatory conditions caused by Alzheimer's disease in the brain, and are therefore useful in diagnosing Alzheimer's disease and disease states of Alzheimer's disease. expected to be possible. Based on the above understanding, it is possible to diagnose Alzheimer's disease by using the present invention. For example, the isotopically labeled phospholipid of the present invention is mixed with a body fluid to be diagnosed (e.g., body fluid such as blood, saliva, urine, or cerebrospinal fluid), and the type and amount of labeled phospholipid in the reaction mixture is determined. Thus, it is possible to diagnose Alzheimer's disease. For example, as an isotope-labeled phospholipid, phosphatidylcholine D3 that binds EPA, DHA, DPA or AA (arachidonic acid) is administered to a body fluid, incubated for a certain period of time, a lipid sample containing phospholipid D3 is extracted, and ESI MASS is performed. By analyzing by and measuring the PCD3/Lyso-PCD3 ratio, it is possible to make a diagnosis of Alzheimer's disease.

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

In addition, this method can be applied to saliva as well as blood plasma. (Arkasubhra Ghosh and Krishnatej Nishtala, Biofluid lipidome: a source for potential diagnostic biomarkers, Critical and Translational Medicine, 6, 2017, 1- 10) Therefore, EPA, DHA, DPA or AA is used for osteoarthritis of the knee as well as Alzheimer's disease diagnosis. and lysophosphatidylcholine D3 produced from them. The reason is described below.

Based on the above-described conventional technology, healthy subjects have a higher expression level of phospholipase A2 enzyme or an increased enzyme activity than knee osteoarthritis patients. Furthermore, phosphatidylcholine D3, which binds EPA, DHA, or DPA, which is an omega-3 fatty acid and is deeply involved in anti-inflammatory properties, and phosphatidylcholine D3, which binds inflammatory arachidonic acid, which is an omega-6 fatty acid, lysophosphatidylcholine D3 through the action of phospholipase A2. Generate. The ratio of PCD3/Lyso-PCD3 in each enzymatic reaction is expected to vary depending on the presence/absence and type of pathology. It has long been known that phospholipase A2, which is activated by inflammation underlying the pathology, releases arachidonic acid that binds to membrane phospholipids, and that this releases various inflammatory substances through the arachidonic acid cascade. Therefore, it is believed that the isotopically labeled compounds of the present invention can be used as indicators of inflammatory conditions in various tissues, and are therefore useful for diagnosing knee osteoarthritis and disease states of knee osteoarthritis. is expected to be available for Based on the above understanding, it is possible to diagnose knee osteoarthritis by using the present invention. For example, the isotopically labeled phospholipid of the present invention is mixed with a body fluid to be diagnosed (e.g., body fluid such as blood, saliva, urine, or cerebrospinal fluid), and the type and amount of labeled phospholipid in the reaction mixture is determined. Therefore, it is possible to diagnose knee osteoarthritis. For example, as an isotope-labeled phospholipid, phosphatidylcholine D3 that binds EPA, DHA, DPA or AA (arachidonic acid) is administered to a body fluid, incubated for a certain period of time, a lipid sample containing phospholipid D3 is extracted, and ESI MASS is performed. By analyzing by and measuring the PCD3/Lyso-PCD3 ratio, it is possible to make a diagnosis of knee osteoarthritis.

In addition, other cases can be similarly applied to diagnosis based on each PCD3/Lyso-PCD3 ratio. For example, there is a possibility of diagnosing various pathological conditions by combining the numerical values of each tissue and each body fluid collected and the values of other biomarkers.

(Characteristics of the present invention)
A representative phosphatidylcholine, which occupies most of biological phospholipids, has an asymmetric center at the 2-position carbon of the glycerol skeleton, and is therefore optically active. Since this chiral center has a very high enantiomeric excess, it is not easy to chemically construct a glycerol skeleton with such a chiral center. Under these circumstances, a method has been adopted in which naturally occurring optically active glycerophospholipids are partially decomposed while leaving the chiral center, and reconstructed into the target phospholipid. However, by this method, it is almost impossible to introduce deuterium into the choline moiety of the fragment structure containing the chiral center. Although there are other methods using optically active natural products such as mannitol, the operations are complicated in each case. In the present application, in order to solve this problem, an optically active glycerol intermediate is synthesized by stereoselectively binding a palmitoyl group to a glycerol skeleton using a microorganism-derived enzyme lipase, and pre-synthesized deuterium is added to this intermediate. A method was carried out in which choline was coupled, and after deprotection, finally EPA, DPA or DHA was coupled.

From past reports, the introduction of palmitoyl groups by lipase does not necessarily give high stereoselectivity. Hazarika et al. (Chemical Engineering Journal, *2003*, 94, 1-10), vinyl palmitate was used as an acylating agent, lipase PS (Amano) IM was used as a catalyst, and 10°C in dichloromethane was used. We were able to synthesize an intermediate with a very high enantiomeric excess by reacting with . As the acylating agent, not only vinyl palmitate but also isopropenyl palmitate, trifluoroethyl palmitate, palmitic anhydride, or other vinyl esters of 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.

Among the deuterated phospholipids of interest in the present invention, those that bind DPA in particular have a problem in that high-purity DPA required for synthesis is not readily available. However, in the present invention, it has become possible by developing a novel method for chemically synthesizing high-purity DPA from EPA in a short route.

(Effect of the present invention)
Numerous phospholipid molecular species that bind various fatty acids, including EPA, DPA and/or DHA, exist in biological tissues and play their respective roles. In addition, the role and metabolism of these molecular species are deeply related to the homeostasis and pathology of living organisms, but in addition to the diversity of phospholipid molecular species, their relationships with these pathologies are extremely complicated. Therefore, if there are specific phospholipids that bind EPA, DPA, or DHA, which have been attracting attention in recent years as extremely important nutrients for health, there are natural/synthetic standard substances (publicly known substances) that have the same chemical structure as those. Even so, it is not easy to selectively detect them among complex biological phospholipids. Metabolome analysis is currently the most influential method for this problem, but this method requires separation of the phospholipid components in the biological sample by high-performance liquid chromatography prior to mass spectrometry. On the other hand, the deuterium-substituted synthetic phospholipid that binds EPA, DPA, or DHA synthesized in the present application does not require a separation operation by liquid chromatography to cause a chemical change therein when added to a biological tissue. It can be traced very specifically by precursor ion detection in mass spectrometry. From this point of view, the novel deuterium-substituted phospholipid that binds EPA, DPA, or DHA synthesized in the present application exhibits extremely superior effects compared to known substances, and will be used not only for basic research on phospholipid metabolism but also for diagnostics in the future. Application to medicine is expected.

The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described with reference to examples, which are provided for illustrative purposes only and not 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

Although the above synthesis scheme shows a synthetic route of deuterated phosphatidylcholine bound with DPA, EPA and DHA can be similarly synthesized. A previously reported synthesis method for phospholipid peroxides (Naomichi Baba, Kenji Yoneda, Shoichi
Tahara, Junkichi Iwasa, Takao Kaneko, Mitsui Matsuo, J.P. Chem. Soc. , Chem. Commun. , 1990, 1281-1282), etc., with appropriate modifications.

Step 1: Synthesis of sulfonic acid (2) :
To a mixture of commercially available p-toluenesulfonyl chloride (8.03 g, 42.0 mmol) and tetradeuteriomethanol (8.88 g, 24.6 mmol) was added 35% aqueous sodium hydroxide solution (5.3 g) at -5 to 0°C. dripped slowly. After completion of the reaction, the reaction solution was transferred to a separating funnel using diethyl ether (100 mL). This was washed twice with saturated aqueous sodium bicarbonate 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 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) were added to a mortar and stirred with a spatula. bottom. After 20 minutes the solid was pulverized, suspended in excess dry acetone and quickly vacuum filtered. The crystals on the filter paper were washed twice with dry acetone, quickly placed in a desiccator and dried under reduced pressure with phosphorus pentoxide to give 6.90 g of product (yield 67.0%).

Step 3: Protection of 1.3 positions 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 compound (6).

Step 4: Protection of Glycerol Position 2 :
Compound (6) (48.4 g, 0.27 mol) and benzyl chloride (427.3 g, 3.37 mol) were reacted with powdered KOH (96.5 g, 1.6 mol) to give compound (7). 54.5 g (71.0%) was obtained.

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

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

Step 7: Synthesis of 2-O-benzyl-1-O-palmitoylphosphatidylcholine D3 (10):
A reaction flask with side tube was charged with phosphoryl chloride (0.19 mL, 2.0 mmol) and a stir bar, equipped with a dropping funnel and a thermometer, cooled to 0-5° C. under a nitrogen atmosphere. Dry triethylamine (0.38 mL, 2.5 mmol) and 2-O-benzyl-1-O-palmitoylglycerol (9) (0.74 g, 1.7 mmol) in dry chloroform while stirring so as not to exceed this temperature. (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. Dry pyridine (0.64 mL, 8.0 mmol) and N-trideuteriomethyl-N,N-dimethylaminoethanol p-toluenesulfonic acid (3) (0.66 g, 2.4 mmol) were added to this solution and the mixture was stirred at room temperature. Stirred for 48 hours. After adding water (8.6 mL) and stirring for 30 minutes, the mixture was transferred to a separating funnel, appropriate amounts of 1N-hydrochloric acid and methanol were added, and after shaking, the desired product was extracted with chloroform three times. This solution was washed with 1N-hydrochloric acid and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was purified with a silica gel column to obtain 0.57 g of product (yield 55.8%).

Step 8: Synthesis of Lyso-PCD3 (11) :
2-O-benzyl-1-O-palmitoylphosphatidylcholine D3 (10) (2.62 g, 44.5 mmol) was dissolved in a mixed solvent of methanol (30 mL) and water (3 mL), and palladium hydroxide on carbon (1. 50 g) was added and stirred for 3 days with a balloon containing hydrogen gas attached. After completion of the reaction, the mixture was filtered using filter paper, and the filtrate was concentrated. Azeotropic vacuum distillation with isopropanol was performed to remove residual water, resulting in 1.89 g of powdery lyso-PCD3 (11) (85.2% yield).

Step 9: Synthesis of 2-O-docosapentaenoyl-1-O-palmitoyl-glycerophosphocholine D3 (13) :
Lyso-PCD3(11) (0.30 g, 0.60 mmol) and docosahexaenoic acid (0.5 g, 1.50 mmol) were dissolved in ethanol-free dry chloroform (5.0 mL), to which was added dicyclohexylcarbodiimide (0.5 g, 1.50 mmol). 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 nitrogen atmosphere. After completion of the reaction, it was concentrated and the residue was purified with a silica gel column to obtain 0.36 g of product (yield 74.0%).

¹ H NMR (600 MHz; CD ₃ OD): δ 0.88 (3H, t, j=7.8, terminal CH ₃ of palmitoyl group), 0.97 (3H, t, j=7.8, docosasa Pentaenoyl terminal CH ₃ ), 1.28 (26H, m, CH ₂ x 13), 1.38 (2H, m, C=C—C—CH ₂ —C), 1.61 (2H, 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×4), 3.22 (6H, s, N[CH ₃ ] ₂ ), 3.64 (2H, m, CH ₂ N), 4.00-4.45 ( m, 5H, proton on glycerol skeleton), 4.28 (2H, br, PO-CH ₂ ), 5.24-5.40 (m, 10H, proton of olefin)
ESI MS observed m _/ z ₌ 811.6006, calculated _for [ _C47H83D3NO8P +H] ⁺ = 811.6039.

(Example 2)
Synthesis of 2-O-eicosapentaenoyl-1-O-palmitoyl-glycerophosphocholine D3 Analogously to Example 1, EPA-PCd3 was synthesized. 0.40g (84.7%)
¹ H NMR (600 MHz; CD ₃ OD): δ 0.80 (3H, t, j=7.8, terminal CH ₃ of palmitoyl group), 0.86 (3H, t, j=7.8, eicosa Pentaenoyl group terminal CH ₃ ), 1.19 (26H, m, CH ₂ x 13), 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 acid 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, protons on the glycerol backbone), 4.32 (2H, br, P —O—CH ₂ ), 5.18-5.33 (m, 10H, proton of olefin).
_ESI MS observed m/z ₌ 783.57349, calculated for [ _C44H75D3NO8P +H] ⁺ = 783.57382 _.
(Example 3)
Synthesis of 2-O-docosahexaenoyl-1-O-palmitoyl-glycerophosphocholine D3 Similar to Example 1, DHA-PCd3 was synthesized. 0.20g (28.4%)
¹ H NMR (600 MHz; CD ₃ OD): δ 0.91 (3H, t, j=7.8, terminal CH ₃ of palmitoyl group), 0.97 (3H, t, j=7.8, docosahexa enoyl group terminal CH ₃ ), 1.28 (26H, m, CH ₂ x 13), 1.59 (2H, m, CH ₃ —CH ₂ —C=C), 2.19 (2H, m, COCH ₂ CH ₂ ), 2.30-2.42 (4H, m, OCOCH ₂ of each fatty acid chain), 2.81-2.90 (10H, m, C=C-CH ₂ -C=Cx5), 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, proton of olefin).
ESI MS observed m/z ₌ 809.5837, calculated _for [ _C47H81D3NO8P +H] ⁺ = 809.5883 _.
(Note)
While the invention has been illustrated using the preferred embodiments thereof, it is understood that the invention is to be construed in scope only by the appended claims. It is understood that the patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety to the same extent as if the content itself were specifically set forth herein. understood.

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

Claims (2)

A process for the preparation of a compound having the structure of Formula I or a salt thereof comprising:

(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 said hydrocarbon group contains 8 to 36 carbon atoms,
the hydrocarbon group contains 0 to 8 double bonds;
at least one of the carbons on the hydrocarbon group is optionally substituted with a substituent selected from the group consisting of =O, -OH and -OOH;
At least one of two adjacent carbon pairs on the hydrocarbon group is substituted with O to form an epoxy group.

may form
each RA ^is independently selected from hydrogen, deuterium or tritium)
Step 1) Trideuteriomethyl group-introducing reagent

a step of preparing
Step 2) Structure below

synthesizing a choline containing a deuterated methyl group selected from
Step 3) protecting the 1-hydroxy and 3-hydroxy groups of glycerol;
Step 4) protecting the 2-hydroxy group of glycerol,
Step 5) deprotecting the 1-hydroxy and 3-hydroxy groups of glycerol;
Step 6) Step of introducing a fatty acid having a structure of R ¹ -OH to the 1-hydroxy group of glycerol, Step 7) Introducing a choline containing a phosphate group and a deuterated methyl group of step 2 to the 3-hydroxy group of glycerol. the process of
Step 8) deprotecting the 2-hydroxy group of glycerol;
Step 9) introducing a fatty acid of the structure R ² —OH to the 2-hydroxy group of glycerol, optionally further comprising:
A production method comprising the steps of step 10) converting choline containing a deuterated methyl group introduced in step 7 into aminoethanol, and step 11) introducing a deuterated methyl group into aminoethanol. all R on the same carbon atom ^A. are the same hydrogen atoms selected from hydrogen, deuterium or tritium.