WO2020137880A1 - Oxytocin derivative and use thereof - Google Patents

Abstract

The present invention is a compound represented by Formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof (in Formula (1), X is a sulfur atom or a methylene group and R3 is a group represented by Formula (2) or Formula (3)).

Description

Oxytocin derivative and use thereof

The present invention relates to an oxytocin derivative and its use. More specifically, the present invention relates to a novel oxytocin derivative, oxytocin receptor agonist, and oxytocin receptor agonist pharmaceutical composition. The present application claims priority based on Japanese Patent Application No. 2018-246141 filed in Japan on December 27, 2018, and the content thereof is incorporated herein.

Oxytocin (CAS number: 50-56-6) is a compound represented by the following formula (A), which is a peptide hormone secreted from the posterior pituitary.

Oxytocin is present in peripheral organs such as the uterus and mammary gland and in the central nervous system, and is known to induce uterine contraction and lactation. Oxytocin is also known to enhance social communication such as social behavior, cognition and memory in mammals including humans.

By the way, autism spectrum disorder (ASD) is a congenital disease characterized by social communication, social interaction, repetitive behavior, limited interest, etc. Although a treatment method for ASD is being investigated, the current situation is that there is no effective therapeutic drug.

Oxytocin may be associated with abnormal social communication behavior in ASD patients. For example, ASD patients have been reported to have low levels of oxytocin in blood or salivary glands. In addition, it has been reported that high frequency gene mutations are detected in the coding region and non-coding region of the oxytocin receptor in ASD patients. It is known that a gene mutation of CD38, which is a transmembrane protein related to oxytocin signal transduction, causes autism-like symptoms in mice. Further, it is known that the single nucleotide polymorphism of CD38 is associated with ASD. These events suggest the importance of oxytocin signaling in social behavior.

Therefore, oxytocin may be a valuable lead compound for the development of new drugs that restore normal social behavior in patients with neurodevelopmental disorders or psychiatric disorders.

Carbetocin (CAS number: 37025-55-1) is a derivative of oxytocin and is used as a uterine contractor together with oxytocin. However, it is known that oxytocin and carbetocin lack selectivity for vasopressin receptors, particularly V ₂ receptors. Therefore, side effects such as antidiuresis and hyponatremia may be observed during administration of oxytocin and carbetocin.

Various oxytocin derivatives have been synthesized in order to improve the pharmacological properties of oxytocin (see, for example, Patent Document 1).

Japanese Patent No. 5539310

Against this background, the present invention aims to provide a novel oxytocin derivative.

The present invention includes the following aspects.
[1] A compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof (wherein, in the following formula (1), X is a sulfur atom or a methylene group, and R ³ is It is a group represented by the following formula (2) or the following formula (3).

[2] An oxytocin receptor agonist comprising the compound according to [1], a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient.
[3] The oxytocin receptor agonist according to [2], which is a therapeutic agent for neurodevelopmental disorders or mental disorders or a uterine contractor.
[4] An oxytocin receptor agonist pharmaceutical composition comprising the oxytocin receptor agonist according to [2] or [3] and a pharmacologically acceptable carrier.

According to the present invention, a novel oxytocin derivative can be provided.

9 is a graph showing the results of a radiation competition assay in Experimental Example 2. (A)-(d) is a graph showing the measurement results of intracellular calcium ion concentration in Experimental Example 3. 9 is a graph showing the results of the tail suspension test in Experimental Example 5. (A) And (b) is a graph which shows the result of having quantified the consumption of water and a sucrose solution in Experimental example 6. (A) to (c) are graphs showing the results of measuring the plasma concentration of each compound in Experimental Example 7. 9 is a graph showing the measurement results of the plasma concentration of each compound in Experimental Example 8. (A)-(c) is a graph showing the exercise competition curve measured in Experimental Example 9. 11 is a graph showing the results of measuring the uterine contractility inducing effect of each compound in Experimental Example 10.

[Oxytocin derivative]
In one embodiment, the present invention provides a compound represented by the following formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof. In the following formula (1), X is a sulfur atom or a methylene group, and R ³ is a group represented by the following formula (2) or the following formula (3).

In the above formula (1), X is preferably a sulfur atom. As will be described later in Examples, the inventors of the present invention have shown that in the above formula (1), X is a sulfur atom and R ³ is a group represented by the above formula (2) (hereinafter, referred to as “compound 2”). Has a super agonist activity of oxytocin receptor. Here, having a super agonist activity means having an agonist activity having a higher oxytocin receptor activation ability than oxytocin, which is a natural ligand for the oxytocin receptor. The chemical formula of compound 2 is shown in the following formula (4).

Further, in the formula (1), the inventors of the present invention have a compound in which X is a sulfur atom and R ³ is a group represented by the formula (3) (hereinafter, may be referred to as “compound 5”). Was an agonist showing a long-term and lasting activation effect on the oxytocin receptor. Here, the term “long term” means that the duration of activation of the oxytocin receptor is longer than that of oxytocin, which is a natural ligand for the oxytocin receptor, and for example, even after 24 hours after acting on the oxytocin receptor. It may be that the activation effect of the oxytocin receptor is maintained. The chemical formula of compound 5 is shown in the following formula (5).

In the present specification, examples of the pharmaceutically acceptable salt include salts commonly used pharmaceutically, and examples thereof include metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like. More specifically, for example, hydrochloride, sulfate, hydrobromide, nitrate, inorganic acid salt such as phosphate, acetate, mesylate, succinate, maleate, fumarate, Organic acid salts such as citrate and tartrate, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, metal salts such as aluminum salt and zinc salt, ammonium salt, tetra salt Examples thereof include ammonium salts such as methylammonium salt, organic amine addition salts such as morpholine and piperidine, amino acid addition salts such as glycine, phenylalanine, lysine, aspartic acid and glutamic acid.

Further, the solvate of the compound represented by formula (1) and the solvate of the salt of the compound represented by formula (1) are not particularly limited as long as they are pharmaceutically acceptable solvates. Examples include hydrates and organic solvates.

[Oxytocin receptor agonist]
In one embodiment, the present invention provides an oxytocin receptor agonist comprising a compound represented by the above formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient.

As will be described later in Examples, the inventors have clarified that the compound represented by the above formula (1) has an agonistic activity on the oxytocin receptor. Among them, the compound 2 represented by the above formula (4) exhibits superagonist activity on the oxytocin receptor. In addition, the compound 5 represented by the above formula (5) is an agonist that exhibits a long-lasting activating effect on the oxytocin receptor.

The salts and solvates of the oxytocin receptor agonist of the present embodiment are the same as those described above. The oxytocin receptor agonist may be a therapeutic agent for neurodevelopmental disorders or mental disorders, or a uterine contractile agent.

Neurodevelopmental disorders or mental disorders include autism spectrum disorder (ASD; Asperger's syndrome, high functioning autism, autism including Kanner's syndrome), schizophrenia, attention deficit hyperactivity disorder (ADHD), Parkinson's disease Mental disorders associated with

The uterine contractile agent can be used as a hemostatic agent (uterine contractile hemostatic agent) or the like that induces labor, promotes labor, and prevents blood loss from the uterus after labor.

[Pharmaceutical composition for activating oxytocin receptor]
In one embodiment, the above-mentioned oxytocin receptor agonist may be formulated as a oxytocin receptor agonist-containing pharmaceutical composition containing a pharmacologically acceptable carrier.

The above-mentioned pharmaceutical composition is orally administered in the form of tablets, capsules, elixirs, microcapsules and the like, or parenterally in the form of injections, suppositories, external preparations for the skin, etc. You can Specific examples of the external preparation for skin include dosage forms such as ointments and patches.

As the pharmaceutically acceptable carrier, those usually used for preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injection such as water, ethanol and glycerin; Examples thereof include adhesives such as rubber-based adhesives and silicone-based adhesives.

The pharmaceutical composition may contain additives. As additives, lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavoring agents such as peppermint and red oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid Examples thereof include buffers such as salts and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives.

The pharmaceutical composition is prepared by admixing the above-mentioned oxytocin receptor agonist and the above-mentioned pharmaceutically acceptable carrier and additive in an appropriate combination in a unit dose form required for generally accepted pharmaceutical practice. Can be converted.

The dose of the pharmaceutical composition varies depending on the symptoms, weight, age, sex, etc. of the patient and cannot be determined unconditionally, but in the case of oral administration, for example, 0.1 to 100 μg/kg body weight per unit dosage form is used. The active ingredient (oxytocin receptor agonist) may be administered. In the case of an injection, for example, 0.01 to 50 μg/kg body weight of the active ingredient may be administered per dosage unit form.

Further, the daily dose of the pharmaceutical composition varies depending on the symptoms, weight, age, sex, etc. of the patient and cannot be determined unconditionally, but for example, an adult dose of 0.1-100 μg/kg body weight is effective. The components may be administered once a day or in 2 to 4 divided doses.

[Other Embodiments]
In one embodiment, the present invention provides a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof or a solvate thereof for use as a medicine (in the above formula (1), X is a sulfur atom or a methylene group, and R ³ is a group represented by the above formula (2) or the above formula (3).

In one embodiment, the present invention provides a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof or a solvate thereof for treating a neurodevelopmental disorder or a psychiatric disorder (the above formula ( 1), X is a sulfur atom or a methylene group, and R ³ is a group represented by the above formula (2) or the above formula (3).

In one embodiment, the present invention provides a compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof or a solvate thereof for the treatment of blood loss from the uterus (the above formula (1) In the above, X is a sulfur atom or a methylene group, and R ³ is a group represented by the above formula (2) or the above formula (3).

In one embodiment, the present invention provides a compound represented by the above formula (1) for producing a therapeutic agent for neurodevelopmental disorder or psychiatric disorder or a uterine contractor, a pharmaceutically acceptable salt thereof, or a salt thereof. The use of a solvate (in the above formula (1), X is a sulfur atom or a methylene group, and R ³ is a group represented by the above formula (2) or the above formula (3)) is provided.

In one embodiment, the present invention provides a compound represented by the above formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof (wherein, X is a sulfur atom or a methylene group). And R ³ is a group represented by the above formula (2) or the above formula (3).) A neurodevelopmental disorder or a mental illness, which comprises administering to a patient in need of treatment an effective amount of To provide a treatment method.

In one embodiment, the present invention provides a compound represented by the above formula (1), a pharmaceutically acceptable salt thereof, or a solvate thereof (wherein, X is a sulfur atom or a methylene group). And R ³ is a group represented by the above formula (2) or the above formula (3).) Treatment of blood loss from the uterus, which comprises administering to a patient in need thereof an effective amount. Provide a way.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Synthesis of oxytocin derivative)
In the compounds represented by the following formula (F), compounds in which R ¹ , R ² , R ³ , R ⁴ and X are compounds 1 to 6 in the following Table 1 were respectively synthesized. Compounds 2, 3, 5, 6 are new compounds.

Compounds 2, 3, 5 and 6 were synthesized as summarized in Scheme 1 below. In addition, known compounds, Compounds 1 and 4, were also synthesized for comparison in Experimental Examples described later.

First, a linear peptide was synthesized on a resin by a general solid-phase peptide synthesis method using a 9-fluorenylmethyloxycarbonyl group (Fmoc group) as a protecting group. For the synthesis of compound 2 and compound 5, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt) were used as protected amino acids. -OH, Fmoc-Ile-OH and Fmoc-Tyr(tBu)-OH were used. Furthermore, the synthesis of Compound 3 and Compound 6, a protected amino acid, Fmoc-Gly-OH, Fmoc -Leu-OH, Fmoc-Cys ((CH 2) 2 CH (NH-pNZ) CO 2 Allyl) -OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH and Fmoc-Tyr(tBu)-OH were used.

For the condensation reaction of amino acids, Fmoc-amino acid (3 equivalents), DIC (diisopropylcarbodiimide, 3 equivalents), HOAt (1-hydroxy-7-azabenzotriazole, 3 equivalents), and DMF (N,N-dimethylformamide) as a solvent are used. ) Was used. However, for the N-alkylglycine residue, N-acylation was performed in NMP (N-methylpyrrolidone) using bromoacetic acid (4 equivalents), DIC (4 equivalents), HOAt (4 equivalents), and then Coupling was done by treatment with p-fluorobenzylamine (10 eq) or 3-t-butoxypropylamine (10 eq) in NMP. A piperidine/DMF (1:4) solution was used for the removal of the Fmoc group. A mixed solution of TFA (trifluoroacetic acid)/TIS (triisopropylsilane)/water (99:2:2) was used to cut out the protected peptide from the resin.

In the synthesis of Compounds 2 and 5, after peptide synthesis on the resin, the terminal Fmoc group was removed, the chain precursors of Compounds 2 and 5 were cleaved from the resin, and a water/acetonitrile (1:4) mixture was prepared. After dissolution, I ₂ -methanol solution (0.1 M) was added to carry out a cyclization reaction, and after purification by reverse phase HPLC (water-acetonitrile solvent containing 0.1% TFA), desired compounds 2 and 5 were obtained. Obtained as the trifluoroacetate salt. The chemical formula of compound 2 is shown in the above formula (4). The chemical formula of compound 5 is shown in the above formula (5).

Compound 2:
HPLC: Rt=11.1 min, solvent A/B=75/25; Purity: 97.8%; LRMS(ESI) m/z 1075.45 [(M+H) ⁺ ], 1097.43 [(M+Na) ^+. ]; HRMS(ESI) calcd for C ₄₇ H ₆₇ FN ₁₂ O ₁₂ S ₂ Na: 1097.4319 [(M+Na) ⁺ ], found: 1097.4321.

Compound 5:
HPLC: Rt=5.61 min, solvent A/B=80/20; Purity: 98.8%; LRMS(ESI) m/z 1025.45[(M+H) ⁺ ], 1047.44[(M+Na) ^+. ]; HRMS(ESI) calcd for C ₄₃ H ₆₉ N ₁₂ O ₁₃ S ₂ Na: 1025.4543 [(M+Na) ⁺ ], found: 1025.4540.

In the synthesis of compounds 3 and 6, after peptide synthesis on the resin, chloroform/N-methylmorpholine/acetic acid (37:1:2) containing Pd(PPh ₃ ) ₄ (palladium tetrakistriphenylphosphine, 3 equivalents) was used. Treatment with the mixture removed the allyl group, followed by removal of the terminal Fmoc group with piperazine. Further, PyBop (hexafluorophosphate (benzotriazol-1-yloxy)tripyrrolidinophosphonium, 5 equivalents), HOAt (5 equivalents), and diisopropylethylamine (10 equivalents) were treated in DMF to carry out the cyclization reaction. .. Then, after cleaving the peptide from the resin, the obtained cyclic peptide was subjected to catalytic hydrogenation reaction in a mixed solvent of ethanol/methanol with 10% Pd-C (0.1 equivalent) and hydrogen at 1 atm, and the reverse After purification by phase HPLC (water-acetonitrile solvent containing 0.1% TFA), the deprotected desired compounds 3 and 6 were obtained as the trifluoroacetate salt. The chemical formula of compound 3 is shown in the following formula (6). Further, the chemical formula of compound 6 is shown in the following formula (7).

Compound 3:
HPLC: Rt=5.4 min, solvent A/B=70/30; Purity: 97.4%; LRMS(ESI) m/z 1057.50 [(M+H) ⁺ ], 1079.48 [(M+Na) ^+. ]; HRMS(ESI) calcd for C ₄₈ H ₆₉ FN ₁₂ O ₁₂ SNa: 1079.4755 [(M+Na) ⁺ ], found: 1079.4773.

Compound 6:
HPLC: Rt=4.9 min, solvent A/B=80/20; Purity: 96.1%; LRMS(ESI) m/z 1007.50 [(M+H) ⁺ ], 1029.48 [(M+Na) ^+. ]; HRMS(ESI) calcd for C ₄₄ H ₇₀ N ₁₂ O ₁₃ SNa: 1029.4798 [(M+Na) ⁺ ], found: 1029.4812.

[Experimental Example 2]
(Receptor binding assay)
First, the binding of oxytocin to the human oxytocin receptor (hOTR) was examined. Specifically, first, hOTR was forcibly expressed in human embryonic kidney HEK-293 cells. Subsequently, [ ³ H]oxytocin binding to hOTR in the crude membrane fraction of HEK-293 cells expressing hOTR was examined. As a result, it was revealed that [ ³ H]oxytocin specifically and reversibly binds to hOTR with a Kd value of 0.3±0.06 nM. Non-specific binding was determined in the presence of 1 μM unlabeled oxytocin.

Subsequently, the binding affinity of oxytocin, vasopressin, carbetosine, and compounds 1 to 6 to hOTR was measured by a radiation competition assay. Specifically, the displacement of [ ³ H]oxytocin bound to hOTR in the presence of 1 pM to 1 μM of each compound was measured.

FIG. 1 is a graph showing the results of a radiation competition assay. In FIG. 1, “OT” represents oxytocin, “AVP” represents vasopressin, and “1” to “6” represent compounds 1 to 6, respectively. The horizontal axis of FIG. 1 represents the concentration of each compound, and the vertical axis represents the ratio (%) of the remaining [ ³ H]oxytocin. Table 2 also shows the results of the radiation binding assay.

As a result, it was revealed that vasopressin and compounds 1 to 6 all showed strong binding affinity to hOTR (Ki≦3.0 nM). In particular, compound 2 showed a high binding affinity for hOTR (Ki=0.51 nM), which was comparable to the binding affinity of oxytocin for hOTR (Ki=0.58 nM). Compounds other than compound 2 showed slightly lower binding affinity compared to the binding affinity of oxytocin for hOTR.

Compounds 2 and 3 showed higher binding affinity compared to compounds 5 and 6. The measured binding affinity of carbetocin for hOTR (Ki=1.81 nM) was in agreement with the reported values.

[Experimental Example 3]
(Measurement of intracellular calcium ion concentration)
The hOTR agonist activity of oxytocin, vasopressin, carbetocin, and compounds 1 to 6 was measured. Specifically, HEK-293 cells forcibly expressing hOTR for mobilization of Ca ²⁺ from intracellular inositol-1,4,5-trisphosphate-sensitive Ca ²⁺ storage organelles following receptor activation, To measure free Ca ²⁺ ion concentration in HEK-293 cells forcibly expressing human vasopressin 1a receptor (hV _1a R) and HEK-293 cells forcibly expressing human vasopressin 1b receptor (hV _1b R). It was measured by. Free Ca ²⁺ ion concentration was measured using a fluorescent probe, fura-2/AM, and a fluorescent microscope. The measurement results of the free Ca ²⁺ ion concentration are shown in FIGS. 2(a) to (d). Table 3 shows the EC ₅₀ , selectivity, and E _max of each compound calculated based on the measurement results of the free Ca ²⁺ ion concentration.

FIG. 2( a) is a graph showing the measurement results of the agonist activity of Compound 2 against hOTR, hV _1a R and hV _1b R. FIG. 2( b) is a graph showing the measurement results of the agonist activity of Compound 5 against hOTR, hV _1a R and hV _1b R.

In FIG. 2 (a) and (b), "OTR" indicates HOTR, _{"V 1A"} denotes the _{hV 1a} R, _{"V 1B"} indicates the _{hV 1b} R, the horizontal axis represents the concentration of each compound and the vertical axis shows the proportion (%) of the free ^{Ca 2+} ion concentration in the case of the free ^{Ca 2+} ion concentration in the case of addition of oxytocin as 100%.

FIG. 2(c) is a graph showing the measurement results of hOTR activation by oxytocin, vasopressin, compound 2 and compound 5. In FIG. 2(c), “OT” indicates oxytocin, “AVP” indicates vasopressin, “2” indicates compound 2, “5” indicates compound 5, and the horizontal axis indicates the concentration of each compound. and the vertical axis shows the proportion (%) of the free ^{Ca 2+} ion concentration in the case of the free ^{Ca 2+} ion concentration in the case of addition of oxytocin as 100%.

FIG. 2( d) is a graph showing the measurement results of hV _1a R activation by oxytocin, vasopressin, compound 2 and compound 5. In FIG. 2(d), “OT” represents oxytocin, “AVP” represents vasopressin, “2” represents compound 2, “5” represents compound 5, and the horizontal axis represents the concentration of each compound. and the vertical axis shows the proportion (%) of the free ^{Ca 2+} ion concentration in the case of the free ^{Ca 2+} ion concentration in the case of addition of vasopressin as 100%.

As a result, it was revealed that oxytocin, carbetocin and vasopressin all activate hOTR, hV _1a R and hV _1b R. The agonistic activity of these compounds on hOTR, ranked based on EC ₅₀ , is oxytocin (0.010 nM)=carbetosine (0.010 nM)>compound 1 (0.025 nM)≈compound 2 (0.028 nM)≈compound 5 (0.031 nM)>Compound 3 (0.089 nM)≈vasopressin (0.10 nM)≈Compound 6 (0.15 nM)>Compound 4 (0.27 nM).

The hOTR selectivity for hV _1a R and hV _1b R was slightly different. Oxytocin, compounds 2 and 3, showed an approximately 20-75 fold increase in selectivity for hOTR over hV _1a R. Carbetocin showed an approximately 200-fold increased selectivity for hOTR over hV _1a R. Compounds 1, 4, 5 and 6 showed >1000-fold increased selectivity for hOTR over hV _1a R. The data for compounds 1 and 4 (Table 2) were in good agreement with the reported values. The selectivity of hOTR for hV _1a R of compound 4 was high, but the selectivity of hOTR for hV _1b R was not high.

It was revealed that compounds 1, 3, 4, and 6 maintain partial agonist activity of hOTR. Compound 1 had an E _max of 61%, compound 3 had an E _max of 82%, compound 4 had an E _max of 57%, and compound 6 had an E _max of 50%.

It was revealed that compound 5 maintains almost complete agonist activity of hOTR. The E _{max of} compound 5 was 88%.

Surprisingly, it was revealed that compound 2 has a hOTR superagonist activity. _{E max} of the compounds 2 in the case of the _{E max} of oxytocin as 100% was 131%.

[Experimental Example 4]
(Production of CD38KOCC mouse)
All animal experiments were conducted in accordance with the basic guidelines for the proper implementation of animal experiments and related activities at academic research institutions under the jurisdiction of MEXT, and were approved by the Kanazawa University Animal Experiment Committee (ethical certification Code AP-173824).

First, we created a mouse that knocked out CD38 by editing the genome (hereinafter sometimes referred to as "CD38KOCC mouse"). The sgRNA was designed to target exon 2 of the Cd38 locus. hCas9 mRNA and sgRNA were synthesized using an in vitro RNA transcription kit (trade name "mMESSAGE mMACHINE T7 Transcription Kit", Thermo Fisher Scientific Co.) and an electroporator (model "NEPA21", NEPA GENE Co.) was used. Introduced into mouse eggs.

DNA was isolated from the F ₀ generation mouse tail, PCR was used to amplify the genomic region spanning the sgRNA binding site, and individuals having a mutation in the Cd38 gene were screened. Then, 30 or more pups were screened using a commercially available kit (trade name "Guide-it Mutation detection kit", Takara Bio Inc.). As a result, pups having a 2-base pair insertion in the Cd38 gene were identified. This insertion forms a stop codon within the open reading frame of the Cd38 gene, resulting in a loss-of-function mutation of the Cd38 gene.

The pups of CD38KOCC mice were weaned at 21 to 28 days of age, and were kept in the same sex group of 5 mice under the standard conditions of 24° C., 12 hours of light/dark cycle, and lighting at 8:00 am, and used in the experiment described later.

[Experimental Example 5]
(Tail suspension test)
The tail suspension test (TST) is a test for measuring the struggling time of a mouse suspended upside down, and is generally one of the tests for evaluating depression-like behavior. The suspended mouse moves about to escape, but the time it does not move (immobility time) gradually increases. It is known that the immobility time in the tail suspension test is reduced in mice treated with antidepressants. A longer immobility time is interpreted as an increase in depression-like behavior, and a decrease in immobility time is interpreted as a decrease in depression-like behavior. Also, the short duration of inactivity can be interpreted as reflecting attention-deficit/hyperactivity disorder-like behavior, and the duration of inactivity can be used to test the effect on attention-deficit/hyperactivity disorder. it can.

In this experimental example, the tail of the mouse was taped to a suspension bar in a suspension box made of Plastec having a height of 55 cm, a width of 60 cm and a depth of 11.5 cm. The test time was 6 minutes, and the total immobility time in the last 4 minutes was measured.

As a result of the tail suspension test, it was revealed that the CD38KOCC mouse exhibited shorter attentionless time than the wild-type ICR mouse, and exhibited attention-deficit/hyperactivity disorder-like behavior.

Oxytocin, Compounds 2 and 5 were each dissolved in phosphate buffered saline (PBS) at a concentration of 100 ng/mL, and a single dose of 0.3 mL/mouse was intraperitoneally administered to CD38KOCC male mice. A group to which only PBS was administered was also prepared as a control. Subsequently, a microsuspension test was performed 30 minutes and 24 hours after the administration.

FIG. 3 is a graph showing the results of the tail suspension test. In FIG. 3, “OT” represents oxytocin, “2” represents compound 2, and “5” represents compound 5. The vertical axis represents the length of idle time (seconds). Also, "*" indicates that there is a significant difference at p<0.05.

As a result, in the CD38KOCC mice administered with PBS, the immobility time 30 minutes after administration was 74.2±2.9 seconds (n=5). In contrast, in CD38KOCC mice administered with oxytocin, compound 2 and compound 5, the immobility time 30 minutes after administration was 171±13.46 seconds (n=5, p=0.0012), 149.5, respectively. ±13.8 seconds (n=8, p=0.029) and 151.83±21.3 seconds (n=6), showing a marked increase, and recovery to the wild type was observed.

24 hours after administration, the immobility time of PBS-administered CD38KOCC mice was 56.5±18.8 seconds (n=5, p=0.135). On the other hand, the immobility time of CD38KOCC mice administered with Compound 5 was 157.3±21.4 seconds (n=5, p=0.039), and only the group administered with Compound 5 was administered with PBS. It showed a marked increase in absent time compared to the group and showed recovery to wild type.

2-way ANOVA by statistical analysis results, the time significance _{(F 1,31 = 7.103, P =} 0.0121) and significance of the treatment _{(F 3,31 = 10.55, p =} 0.0001) Was recognized. On the other hand, at 24 hours after administration, no statistically significant difference was observed in CD38KOCC mice administered with oxytocin or compound 2 as compared with PBS.

[Experimental example 6]
(Sucrose preference test)
The sucrose preference test is considered to reflect the depression state of human depression or Parkinson's disease and the apathy (unpleasant sensation) that is observed due to a decrease in the willingness to socialize (QOL), and indifference to others. There is. In mice that are overstressed, there is a behavioral change in which they no longer take the sucrose water that they originally prefer. It is also known that this reaction is improved by treatment with antidepressant drugs.

In this experimental example, first, wild type mice and CD38KOCC male mice were placed in a state in which they could freely select between water and a 1% sucrose solution. As a result, it was revealed that the consumption of the sucrose solution was significantly lower in the CD38KOCC male mouse than in the wild-type mouse.

Subsequently, oxytocin, Compounds 2 and 5 were each dissolved in phosphate buffered saline (PBS) at a concentration of 100 ng/mL, and each was intraperitoneally administered once to each CD38KOCC male mouse at a dose of 0.3 mL/mouse. .. A group to which only PBS was administered was also prepared as a control.

Subsequently, the mice in each group were placed in a state where they could freely choose between water and 1% sucrose solution, and the consumption of water and sucrose solution was quantified 1 hour and 24 hours after administration. did.

FIGS. 4A and 4B are graphs showing the results of quantifying the consumption amounts of water and sucrose solution. FIG. 4(a) is a graph showing the results 1 hour after the administration of each compound, and FIG. 4(b) is a graph showing the results 24 hours after the administration of each compound. In FIGS. 4A and 4B, “OT” represents oxytocin, “2” represents compound 2, “5” represents compound 5, and the vertical axis represents the consumption of water or sucrose solution. Indicates a percentage. Also, "*" indicates that there is a significant difference at p<0.05.

As a result, CD38KOCC mice administered with PBS did not show sucrose preference (0.54±0.04, p=0.21, n=8). In contrast, oxytocin-administered CD38KOCC mice showed a significant recovery of sucrose preference 1 hour after administration (0.21±0.02 to 0.80±0.02, p=0.0001, n=5). In addition, the CD38KOCC mice administered with Compound 2 showed a significant recovery of sucrose preference 1 hour after the administration (0.20±0.02 to 0.79±0.02, p=0.0001, n). = 5). In addition, CD38KOCC mice administered with compound 5 showed a significant recovery of sucrose preference 1 hour after administration (0.29±0.05 to 0.72±0.05, p=0.001,n). = 5).

Also, 24 hours after the administration of each compound, only CD38KOCC mice to which the compound 5 was administered showed a significant recovery of sucrose preference (0.41±0.03 to 0.59±0.03, p= 0.004, n=5). On the other hand, it was revealed that the administration of oxytocin and compound 2 did not show the effect of restoring the sucrose preference 24 hours after the administration. From this result, it became clear that the compound 5 has a lasting effect.

[Experimental Example 7]
(Pharmacokinetic study)
To understand the pharmacological potency and duration of oxytocin, compound 2, compound 5, the pharmacokinetics of these compounds were tested. Specifically, first, oxytocin, compound 2 and compound 5 were tail vein-administered at 3 μg/mouse to male ICR mice each weighing 30 g. Then, each mouse was killed immediately after administration, 5 minutes, 15 minutes, and 30 minutes later, and blood was collected. Cerebrospinal fluid was collected 30 minutes and 12 hours after administration. The concentration of each compound in plasma and cerebrospinal fluid was measured by the LC-MS/MS method.

FIGS. 5(a) to 5(c) are graphs showing the measurement results of plasma concentrations of oxytocin, compound 2 and compound 5 measured by the LC-MS/MS method. 5A shows the measurement result of oxytocin, FIG. 5B shows the measurement result of compound 2, and FIG. 5C shows the measurement result of compound 5. In measuring the compounds 2 and 5, the concentration of endogenous oxytocin was also measured at the same time.

In FIGS. 5A to 5C, “OT” indicates oxytocin, “2” indicates compound 2, and “5” indicates compound 5. The horizontal axis shows the time (minutes) after administration, and the vertical axis shows the plasma concentration (ng/mL) of each compound.

Also, Table 4 below shows the measurement results of pharmacokinetic parameters. In Table 4, "Fast period" indicates that the calculation was performed based on the measured value 15 minutes after the administration of the compound, and "Slow period" was calculated based on the measured value 15 to 60 minutes after the administration of the compound. "CSF" indicates cerebrospinal fluid.

As a result, the oxytocin concentration in plasma immediately after administration was 334±155 ng/mL (n=5). The concentration of Compound 2 in plasma immediately after administration was 118±60 ng/mL (n=3). The concentration of compound 5 in plasma immediately after administration was 108±35 ng/mL (n=3). These three compounds cleared rapidly from plasma with a half-life of approximately 10 minutes. The fast phase t _1/2 of oxytocin, Compound 2 and Compound 5 were 2.91 min, 2.84 min and 3.1 min, respectively.

Also, it was revealed that Compound 2 was removed from blood more quickly than Oxytocin and Compound 5. The concentration of Compound 2 in the cerebrospinal fluid 30 minutes after administration was 100 pg/mL. Surprisingly, the concentration of Compound 2 in cerebrospinal fluid 12 hours after administration was higher, reaching 380 pg/mL. The concentration of Compound 5 in the cerebrospinal fluid was below the detection limit and therefore could not be measured.

[Experimental Example 8]
(Study of plasma stability)
The plasma stability of oxytocin, Compound 2 and Compound 5 was examined. Specifically, oxytocin, Compound 2 or Compound 5 was added to 100 μL of pooled mouse plasma at a final concentration of 50 ng/mL, and incubated at 37° C. for 1, 5, 15, 30, 60 and 120 minutes. Then, 400 μL of acetonitrile/methanol=1:1 (v/v) containing an internal standard was added, and the concentration of each compound was measured by the LC-MS/MS method.

FIG. 6 is a graph showing the measurement results of the plasma concentration of each compound. In FIG. 6, “OT” represents oxytocin, “2” represents compound 2, and “5” represents compound 5. The horizontal axis represents time (minutes) and the vertical axis represents the ratio (%) to the initial concentration.

As a result, the concentration of all compounds was within the range of 85 to 115% with respect to the initial concentration, and it was considered that they were stable even after incubation in plasma at 37°C for 2 hours. This result indicates that the disappearance of oxytocin, Compound 2 and Compound 5 in blood is not due to decomposition by enzymatic digestion, but is due to clearance or adsorption to various tissues and organs.

[Experimental Example 9]
(Kinetic analysis)
The Motulsky and Mahan equations were used to calculate the kinetic parameters for oxytocin, compound 2 and compound 5.

First, the kinetic parameters of [ ³ H]oxytocin were determined. Specifically, three different [ ³ H]oxytocin concentration ranges were used to generate the relevant kinetic curves. The results are shown in Table 5 below.

Subsequently, a kinetic competition curve was measured for each of oxytocin, compound 2, and compound 5 at three concentrations (near Ki, 5-fold Ki, and 10-fold Ki). Specifically, 500 pM [ ³ H]oxytocin and various concentrations of competing compounds were added to the crude membrane fraction of HEK-293 cells forcibly expressing hOTR, and after incubation for various times, [ ³ H] The amount of oxytocin bound was measured. Non-specific binding was determined in the presence of 1 μM unlabeled oxytocin.

7(a) to 7(c) are graphs showing measured exercise competition curves. FIG. 7( a) shows the results for oxytocin, FIG. 7( b) shows the results for compound 2, and FIG. 7( c) shows the results for compound 5. Table 5 below shows the measurement results of reaction kinetic parameters of each compound.

As a result, Compound 2 showed a t _1/2 of about 1/2 that of [ ³ H]oxytocin. Moreover, the compound 2 showed t _1/2 of about 1/6 of the compound 5. Compound 5 also showed faster association and dissociation rates compared to [ ³ H]oxytocin.

From the results of this experimental example, it is not possible to explain the reason why administration of Compound 5 to CD38KOCC mice showed a long-term effect in Experimental Examples 5 and 6 described above. From this point as well, it is an unexpected effect that administration of Compound 5 to CD38KOCC mice shows a long-term effect.

[Experimental Example 10]
(Study of uterine contraction inducing ability)
The effect of oxytocin, vasopressin, carbetocin, and compounds 1 to 6 on inducing uterine contraction in mice was evaluated ex vivo by isometric force measurement. First, after removing the endometrium of the mouse, the uterine horn was longitudinally cut to form a 1 mm wide myometrium. Subsequently, the myometrium was attached between two tungsten wires in a culture tank filled with PBS maintained at 37° C. in a 95% O ₂ -5% CO ₂ environment. One wire was stationary and the other wire was connected to a transducer (AD instrument).

Next, the myometrium was stimulated with 60 mM potassium ion every 15 minutes until a constant response was obtained. Oxytocin, vasopressin, carbetocin, and compounds 1 to 6 were added to the culture layer to induce uterine muscular contraction, and the force area (area under the curve) was calculated with LabChat software (AD instrument). The measured responses were normalized with the data of constant spontaneous contractions measured before applying each compound. Table 6 shows the evaluation results of the uterine contractility inducing effect of each compound. Further, FIG. 8 is a graph showing the measurement results of the uterine muscle contraction inducing effect of oxytocin, carbetocin, compound 2 and compound 5. In FIG. 8, the horizontal axis represents the concentration of each compound, and the vertical axis represents the ratio (%) with the spontaneous contraction value as 100%.

As a result, the EC _{50 of} Compound 2 was 55.5 pM, which was the same among natural oxytocin (238.8 pM), carbetosine (2.7 nM), Compound 1 (182.3 pM) and Compound 5 (3.1 nM). It became clear that it was the highest. Further, the EC _{50 of} Compound 3 was >10 nM and the EC _{50 of} Compound 6 was 8.1 nM.

This result further supports the above-mentioned result that compound 2 has an affinity close to that of natural oxytocin and exhibits superagonist activity against hOTR.

According to the present invention, a novel oxytocin derivative can be provided.

Claims (4)

1. A compound represented by the following formula (1), a pharmacologically acceptable salt thereof, or a solvate thereof (in the following formula (1), X is a sulfur atom or a methylene group, and R ³ is the following formula ( 2) or a group represented by the following formula (3).
   

   
2. An oxytocin receptor agonist comprising the compound according to claim 1, a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient.
3. The oxytocin receptor agonist according to claim 2, which is a therapeutic agent for neurodevelopmental disorders or mental disorders or a uterine contractile agent.
4. An oxytocin receptor agonist pharmaceutical composition comprising the oxytocin receptor agonist according to claim 2 or 3 and a pharmacologically acceptable carrier.

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