JPH0827086A - N-acyl-n-substituted cinnamoyl ethylene diamine derivative - Google Patents

Abstract

PURPOSE:To obtain the subject new compound having a human neutrophile O<->2 production-inhibiting activity, an intracellular phospholipase-inhibiting activ ity, and a nerve growth factor production-stimulating activity, and useful as an antiinflammatory agent and as a treating agent for dementia, spinal cord damages, diabetic neuropathy, etc. CONSTITUTION:This compound is expressed by formula I (R<1> is the acyl residue of a >=16C higher unsaturated fatty acid; R<2>, R<3> are H, lower; (n) is 1,2), for example, N-[3-(3,4-dihydroxyphenylpropenoyl)]-N'-(4,7,10,13,16,19-docosahexaenoyl) ethylene diamine. The compound of formula I is obtained by subjecting a compound of formula II and a compound of formula III to a condensation reaction.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a human neutrophil O ₂ ^- production inhibitory activity, intracellular phospholipase A ₂ inhibitory activity, and /
Alternatively, it relates to an N-acyl-N-substituted cinnamoylethylenediamine derivative having a nerve growth factor (NGF) production promoting activity.

[0002]

2. Description of the Related Art The inflammatory reaction is a kind of biological defense effect that is activated when a harmful stimulus enters the body, but as a result, it causes less swelling, pain, organ dysfunction, etc. and is less likely to die. Absent. Specifically, from acute inflammation associated with type 1 allergy to external effects to chronic inflammation caused by nephritis and rheumatic diseases, the causes, onset processes, and symptoms are extremely wide-ranging and complicated. A drug called an anti-inflammatory agent is used as the symptomatic therapeutic agent, and is broadly classified into a steroidal anti-inflammatory agent and a non-steroidal anti-inflammatory agent, and various glucocorticoids, indomethacin and the like are representative. However, steroidal anti-inflammatory drugs have a protein synthesis inhibitory action including various protein mediators, and although they have a wide range of pharmacological effects and a large curative effect, they are known to cause serious side effects. It was recently revealed that the separation of the two is almost impossible. On the other hand, the effects of non-steroidal anti-inflammatory drugs are limited because the cyclooxygenase inhibition (prostaglandin production inhibition) action is the main pharmacological action. Therefore and development of anti-inflammatory agent is required based on a new pharmacological action, O ₂ ^- production inhibitory activity, the compounds having intracellular phospholipase A ₂ inhibitory activity is considered a substance that meets the purpose.

[0003] That is, various active oxygen species are the most central mediators involved in tissue damage during inflammation, whose production is O ₂ by leukocytes (neutrophils, macrophages) ^- due to the production. This active oxygen causes duodenal ulcer, gastric ulcer,
It is known to be involved in various diseases such as arteriosclerosis, ischemic diseases of the brain and heart, cancer, aging, cataract, autoimmune disease, inflammation, arthritis, edema, etc. Is being investigated [Pharmacia, Vol.29,
No. 9, 1014, 1029 (1993)].

Intracellular phospholipase A ₂ is an enzyme whose existence has been clarified only recently, and its activation commonly initiates the enzymatic production of prostaglandins, leukotrienes and PAF, which are the major mediators of inflammation. It is known that [protein, nucleic acid, enzyme, Vo
l.36, No.3, 325 (1991)]. PAF is a potent inflammatory mediator along with prostaglandins and leukotrienes, but its synthetic inhibitor has not yet been known. Therefore, intracellular phospholipase A ₂ is considered to be useful as a preventive or therapeutic agent for inflammatory disorders and allergic diseases.

On the other hand, NGF not only differentiates nerve cells in vitro to promote neurite outgrowth and maintains the survival of nerve cells, but in animal experiments, when NGF is administered into the brain, memory-learning effect in aged rats. Are known to be improved. It is also known to prevent the death of nerve cells by cerebral ischemia [J. Neurosci., 6, 2155 (19
86), Brain Res., 293, 305 (1985), Science 235, 214
(1986), Proc. Natl. Acad. Sci. USA, 83, 9231 (1986),
Annals of Neurology, 120, 275 (1986), Chemistry and Biology, Vol.29, No.10, 640 (1991)].

In Alzheimer-type senile dementia, it has been confirmed in many cases that most of the cholinergic neurons of the Meinert nucleus, which is a nerve cell that controls memory and thought, are dead and lost. It has been revealed that NGF is essential for survival and differentiation of nerve cells [Geriatric Neurology, 3,751 (1986)]. Lance and Olson et al.
GF (6.6 mg / 3 months) was intraventricularly administered to confirm improvement of dementia (Symposium on Alzheimer's disease treatment in 1991).

Further, NGF acts as a trophic factor for not only central nerves but also peripheral sensory and sympathetic nervous systems and is an essential factor for nerve regeneration. That is, spinal cord injury, peripheral nerve injury,
It is considered that it can be used for treatment of diabetic neuropathy and amyotrophic lateral sclerosis.

However, in order to actually use NGF for the treatment of humans, there is no established means for mass production of NGF, and since NGF does not pass through the blood-brain barrier, it cannot be administered from the periphery, which is inconvenient. is there.

[0009]

Therefore, an object of the present invention is to provide an anti-inflammatory substance and / or NGF production promoting substance having a novel structure.

[0010]

The present inventors have SUMMARY OF THE INVENTION As a result of various search, certain N- acyl -N- substituted cinnamoyl ethylenediamine derivatives, human neutrophil O ₂ ^- production inhibitory activity,
The present invention was completed by finding that it has an intracellular phospholipase A ₂ inhibitory activity and / or an NGF production promoting activity.

That is, the present invention has the following general formula

[0012]

Embedded image

An N-acyl-N-substituted cinnamoylethylenediamine derivative represented by the formula (wherein R ¹ has 16 carbon atoms)
The above represents an acyl residue of a higher unsaturated fatty acid, R ² and R ³ independently represent a hydrogen atom or a lower alkyl group, and n is 1 or 2.).

As the acyl residue of the higher unsaturated fatty acid having 16 or more carbon atoms in the above general formula, hexadecanoyl group,
Examples thereof include an oleoyl group, a linoleoyl group, a linolenoyl group, an eicosatrienoyl group, an arachidonoyl group, an eicosapentaenoyl group, a docosatetraenoyl group, and a docosahexaenoyl group. Examples of the lower alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, pentyl group and hexyl group.

The N-acyl-N-substituted cinnamoylethylenediamine derivative of the present invention can be produced, for example, according to the following scheme.

[0016]

Embedded image

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples.

[0018]

【Example】

Reference example 1. N-4,7,10,13,16,19-docosahexaenoylethylenediamine (DHA-ED
Synthesis of A) 3.28 g (10 m) of docosahexaenoic acid (95% or more)
mol) and N, N-carbonyldiimidazole 1.78.
g (11 mmol) was added to anhydrous tetrahydrofuran (hereinafter,
It was dissolved in 10 mL of THF (abbreviated as THF) and reacted at room temperature under a nitrogen stream for about 1 hour. Then, 1.5 g (25 mmo) of ethylenediamine (hereinafter abbreviated as EDA) was added to the reaction solution.
1) and 2.55 g (25 mmol) of triethylamine dissolved in 10 mL of anhydrous THF were added under ice cooling, and 2
Allowed to react for hours. After the completion of the reaction, 0.1m hydrochloric acid 60m
L and chloroform-methanol (2: 1, 300 mL) were added for liquid separation, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrated solution was dissolved in a small amount of chloroform and placed on a silica gel (160 g) column previously activated with chloroform, and 500 mL of chloroform, 100 mL of chloroform-methanol (98: 2), chloroform-methanol-water (85:15: 1) 1000 mL, chloroform-methanol-concentrated aqueous ammonia (85: 15: 1)
The eluate was flowed in this order to elute. Using the thin layer chromatography (TLC) analysis as an index, the obtained eluate fraction was collected as a target fraction and concentrated to obtain 2.86 g (yield: 77.3%) of the target product as a pale yellow oily substance. It was This substance is
TLC analysis (silica gel plate, developing solvent: chloroform-methanol-water (75: 25: 2)) showed a single spot by detection with iodine, 0.2% ninhydrin reagent and 50% sulfuric acid-methanol reagent. It was EI-MS (m / z): 370 (M ⁺ )

Reference Example 2. N-5, 8, 11, 14, 1
7-Eicopentaenoylethylenediamine (EPA-
Synthesis of EDA) Eicosapentaenoic acid (95% or more) 3.03 g (10
mmol) and N, N-carbonyldiimidazole 1.7.
8 g (11 mmol) was dissolved in 10 mL of anhydrous THF,
The reaction was carried out at room temperature under a nitrogen stream for about 1 hour. Next, 1.5 g (25 mmol) of EDA and 2.55 g (25 mmol) of triethylamine were added to this reaction solution in anhydrous THF (10 m).
The solution dissolved in L was added under ice cooling and reacted for 2 hours.
After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrate was placed on a silica gel (160 g) column, chloroform-methanol-water (85: 15: 1) 1000 mL, chloroform-methanol-concentrated ammonia water (85: 15: 1).
The eluate was flowed in this order to elute. Using the thin layer chromatography (TLC) analysis as an index, the obtained elution fractions were collected as target fractions and concentrated to obtain 2.72 g (yield 79%) of the target product. This substance was subjected to TLC analysis (silica gel plate, developing solvent: chloroform-methanol-water (75: 25: 2)), and iodine, 0.2%
Detection with ninhydrin reagent and 50% sulfuric acid-methanol reagent showed a single spot. EI-MS (m / z): 344 (M ⁺ )

Reference Example 3. Synthesis of N-9,12-octadecadienoylethylenediamine (LA-EDA) 2.8 g (10 mmol) of linoleic acid (95% or more) and 1.78 g (11 m of N, N-carbonyldiimidazole)
mol) in 10 mL of anhydrous THF, and under a nitrogen stream,
The reaction was carried out at room temperature for about 1 hour. Then add ED to this reaction solution.
A 1.5 g (25 mmol) and triethylamine 2.5
A solution prepared by dissolving 5 g (25 mmol) in 10 mL of anhydrous THF was added under ice cooling and reacted for 2 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The concentrate obtained was treated with silica gel (16
0 g) and loaded on a column, and then eluted with chloroform-methanol-water (85: 15: 1) 1000 mL and chloroform-methanol-concentrated ammonia water (85: 15: 1) in this order. Using the thin layer chromatography (TLC) analysis as an index, the obtained eluate fractions were collected as desired fractions and concentrated to give 2.43 g of the desired product (yield 75.5).
%) Was obtained. This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-water (75:
25: 2)) was performed and a single spot was detected by detection with iodine, 0.2% ninhydrin reagent and 50% sulfuric acid-methanol reagent. EI-MS (m / z): 322 (M ⁺ )

Example 1. N- [3- (3,4-dihydride
Roxyphenylpropenoyl)]-N '-(4,7,1
0,13,16,19-docosahexaenoyl) ethyl
Synthesis of diamine (DHA-EDA-CA) 3,4-dihydroxycinnamic acid (caffeic acid) 7
09 mg (3.94 mmol) and N-hydroxy alcohol
Cubic imide (hereinafter abbreviated as HOSu) 543 mg (4.
73 mmol) N, N-dimethylformamide 3 mL
And 1 mL of dichloromethane after dissolving in 5 mL of dichloromethane.
(3-Dimethylaminopropyl) -carbodiimide hydrochloric acid
Salt (hereinafter abbreviated as WSC) 903 mg (4.73 mmo)
1) was added and reacted for 2 hours. Then, in this reaction solution
1.457 g of DHA-EDA obtained in Reference Example 1 (3.
94 mmol) and triethylamine 482 mg (4.7
3 mmol) dissolved in 5 mL of dichloromethane
In addition to ice cooling, the mixture was reacted at room temperature for 6 hours under a nitrogen stream.
After the completion, add 60 mL of 0.1N hydrochloric acid and chloroform to the reaction solution.
-Add 300 mL of methanol (2: 1) to separate the layers,
The layers were separated and concentrated under reduced pressure. Small amount of concentrate obtained
Dissolve in chloroform and activate with chloroform beforehand.
Place on a column of activated silica gel (100 g) and apply chloro
Form 500 mL, chloroform-methanol (98:
2) 500 mL, chloroform-methanol (95:
5) The eluate was flown in the order of 500 mL to elute. Got
Thin layer chromatography (TLC) fraction from the eluted fractions
The target fraction was collected using the analysis as an index and concentrated. this
Repeat the purification by column chromatography to
The desired product (472 mg, yield 23.6%) was obtained. In addition, the real thing
Quality is TLC analysis (silica gel plate, developing solvent: black
Loloform-methanol-water (85: 15: 1))
After a while, iodine, UV and 50% sulfuric acid-methanol
Reagent detection showed a single spot.¹ H-NMR (400Hz, C_FiveD_FiveN): δ 0.91 (3H, t, -CH₃), 2.03 (2H_,m,
-CH ₂CH₃), 2.16 (2H, t, -CH₂CH ₂CONH-), 2.27 (2H, m, -CH = CHC
H₂CH = CHCH ₂CH₂-), 2.79 (10H, m, -CH = CHCH ₂CH = CH-), 3.20 ~
3.50 (4H, m, -NHCH ₂CH ₂NH-), 5.33 (12H, m, -CH= CHCH₂CH= CH
-), 6.35 (1H, d, -CH = CHC₆H₃(OH)₂), 6.90 ~ 7.10 (3H, t, -C ₆ H
₃(OH)₂), 7.42 (1H, d, -CH= CHC₆H₃(OH)₂), 8.09 (1H, t, -CONH
CH₂CH₂-), 8.35 (1H, t, -NHCOCH = CH-).

Example 2. N- [3- (3,4-dihydroxyphenylpropenoyl)]-N '-(5,8,1
Synthesis of 1,14,17-eicopentaenoyl) ethylenediamine (EPA-EDA-CA) 3,4-dihydroxycinnamic acid (caffeic acid) 7
83 mg (4.35 mmol) and HOSu600 mg
After dissolving (5.22 mmol) in 3 mL of N, N-dimethylformamide and 5 mL of dichloromethane, WSC997 was used.
mg (5.22 mmol) was added and reacted for 2 hours.
Then, the EPA-ED obtained in Reference Example 2 was added to this reaction solution.
A 1.497 g (4.35 mmol) and triethylamine 532 mg (5.22 mmol) were added to dichloromethane 5
The solution dissolved in mL was added under ice cooling, and the mixture was reacted at room temperature for 6 hours under a nitrogen stream. After the completion, add 0.1N hydrochloric acid 6 to the reaction solution.
0 mL, chloroform-methanol (2: 1) 300 m
L was added thereto for liquid separation, the lower layer was separated and concentrated under reduced pressure.
The obtained concentrate was dissolved in a small amount of chloroform, placed on a silica gel (100 g) column, and 482 mg (yield 20.9%) of the desired product was obtained from the fraction eluted with chloroform-methanol (95: 5). In addition, when this substance was subjected to TLC analysis (silica gel plate, developing solvent: chloroform-methanol-water (85: 15: 1)), a single spot was detected by detection with iodine, ultraviolet rays and 50% sulfuric acid-methanol reagent. Indicated. ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.92 (3H, t, -CH ₃ ), 1.56 (2H, m,
-CH ₂ CH = CHC H ₂ CH _2- ), 2.05 (4H, m, -C H ₂ CH ₃ , -CH ₂ C H ₂ CH ₂ CONH
-), 2.11 (2H, m, -CH ₂ CONH-), 2.80 (8H, m, -CH = CHC H ₂ CH = CH
-), 3.30 ~ 3.50 (4H, m, -NHC H ₂ C H ₂ NH-), 5.33 (10H, m, -C H = C H
CH ₂ C H = C H- ), 6.35 (1H, d, -CH = C H C ₆ H ₃ (OH) ₂ ), 6.90 ~ 7.10 (3
H, t, -C ₆ H ₃ (OH) ₂ ), 7.42 (1H, d, -C H = CHC ₆ H ₃ (OH) ₂ ), 8.09 (1
H, t, -CON H CH ₂ CH _2- ), 8.35 (1H, t, -N H COCH = CH-).

Example 3. N- [3- (3,4-dihydroxyphenylpropenoyl)]-N '-(9,12-
Octadecadienoyl) ethylenediamine (LA-ED
A-CA) Synthesis 3,4-dihydroxycinnamate (caffeic acid) 3
96 mg (2.2 mmol) and HOSu 304 mg
(2.6 mmol) was added to N, N-dimethylformamide 3
mLSC and dichloromethane 5mL, WSC504m
g (2.6 mmol) was added and reacted for 2 hours. Then, LA-EDA71 obtained in Reference Example 3 was added to this reaction solution.
8 g (2.2 mmol) and 269 mg of triethylamine
A solution of (2.64 mmol) dissolved in 5 mL of anhydrous THF was added under ice cooling, and the mixture was reacted under a nitrogen stream at room temperature for 6 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The concentrate obtained was dissolved in a small amount of chloroform, and the silica gel (100
g) Place on a column, chloroform-methanol (95:
5) The desired product (380 mg, yield 39%) was obtained from the eluted fraction. This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-water (85:
15: 1)), iodine, UV and 50%
Detection with sulfuric acid-methanol reagent showed a single spot. ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.85 (3H, t, -CH ₃ ), 1.23 to 1.26
(16H, m, -CH _2- ), 1.49 (2H, t, -C H ₂ CONH-), 2.00 (4H, m, -CH = C
HCH ₂ CH = CHC H _2- ), 2.73 (2H, m, -CH = CHC H ₂ CH = CH-), 3.30 ~ 3.
50 (4H, m, -NHC H ₂ C H ₂ NH-), 5.31 (4H, m, -C H = C H- ), 6.35 (1H,
d, -CH = C H C ₆ H ₃ (OH) ₂ ), 6.90 to 7.10 (3H, t, -C ₆ H ₃ (OH) ₂ ), 7.4
2 (1H, d, -C H = CHC ₆ H ₃ (OH) ₂ ), 8.09 (1H, t, -CON H CH ₂ CH _2- ), 8.
35 (1H, t, -N H COCH = CH-).

Example 4. N- [3- (4-hydroxy-3-methoxyphenylpropenoyl)]-N'-
(4,7,10,13,16,19-docosahexaenoyl) ethylenediamine (DHA-EDA-HMCA)
Synthesis of 4-hydroxy-3-methoxycinnamic acid (ferulic acid) 524 mg (2.7 mmol) and HOSu372
After dissolving mg (3.24 mmol) in 3 mL of N, N-dimethylformamide and 5 mL of dichloromethane, WSC6
19 mg (3.24 mmol) was added and reacted for 2 hours. Then, the DHA-obtained in Reference Example 1 was added to this reaction solution.
EDA 986 mg (2.7 mmol) and triethylamine 330 mg (3.24 mmol) were added to dichloromethane 5
The solution dissolved in mL was added under ice-cooling, and the mixture was reacted under a nitrogen stream at room temperature for 4 hours. After the completion, add 0.1N hydrochloric acid 6 to the reaction solution.
0 mL, chloroform-methanol (2: 1) 300 m
L was added thereto for liquid separation, the lower layer was separated and concentrated under reduced pressure.
The obtained concentrate was placed on a silica gel (100 g) column, and 775 mg (yield 53.2%) of the desired product was obtained from the fraction eluted with chloroform-methanol (98: 2). In addition,
This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-water (85:15:
1)) was carried out, iodine, UV and 50% sulfuric acid
Detection with methanol reagent showed a single spot. ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.91 (3H, t, -CH ₃ ), 2.03 (2H _, m,
-C H ₂ CH ₃ ), 2.10 (2H, t, -CH ₂ C H ₂ CONH-), 2.25 (2H, m, -CH = CHC
H ₂ CH = CHC H ₂ CH _2- ), 2.79 (10H, m, -CH = CHC H ₂ CH = CH-), 3.10 ~
3.40 (4H, m, -NHC H ₂ C H ₂ NH-), 3.79 (3H, s, -C ₆ H ₃ (OC H ₃ ) OH),
5.33 (12H, m, -C H = C H CH ₂ C H = C H- ), 6.25 (1H, d, -CH = C H C ₆ H ₃ (O
CH ₃ ) OH), 6.79 ~ 7.00 (3H, t, -C ₆ H ₃ (OCH ₃ ) OH), 7.30 (1H, d,-
C H = CHC ₆ H ₃ (OCH ₃ ) OH), 7.80 (1H, t, -N H COCH = CH-), 7.95 (1H,
t, -CON H CH ₂ CH _2- ). Note that EPA-EDA-HMCA, LA-EDA-HM
CA and LA-DET-HMCA also have EPA-EDA, LA-EDA and LA-, respectively, instead of DHA-EDA.
A similar synthesis was performed using DET.

Embodiment 5 FIG. N- [3,4-dimethoxyphenylpropenoyl)]-N '-(4,7,10,1
Synthesis of 3,16,19-docosahexaenoyl) ethylenediamine (DHA-EDA-DMCA) 3,4-dimethoxycinnamic acid 479 mg (2.3 m
mmol) and HOSu317 mg (2.76 mmol)
Was dissolved in 3 mL of N, N-dimethylformamide and 5 mL of dichloromethane, and then 527 mg of WSC (2.76 mmo)
1) was added and reacted for 2 hours. Then, 828 mg (2.2 of DHA-EDA obtained in Reference Example 1 was added to this reaction solution.
4 mmol) and 274 mg of triethylamine (2.7 m
(mol) in 5 mL of dichloromethane was added under ice cooling, and the mixture was reacted under a nitrogen stream at room temperature for 3 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrate was placed on a silica gel (60 g) column, and 923 mg (yield: 7) of the target product was obtained from the fraction eluted with chloroform-methanol (98: 2).
3.6%). In addition, when this substance was subjected to TLC analysis (silica gel plate, developing solvent: chloroform-methanol-water (85: 15: 1)), a single spot was detected by detection with iodine, ultraviolet rays and 50% sulfuric acid-methanol reagent. Indicated. ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.91 (3H, t, -CH ₃ ), 2.03 (2H _, m,
-C H ₂ CH ₃ ), 2.16 (2H, t, -CH ₂ C H ₂ CONH-), 2.27 (2H, m, -CH = CHC
H ₂ CH = CHC H ₂ CH _2- ), 2.79 (10H, m, -CH = CHC H ₂ CH = CH-), 3.20 ~
3.50 (4H, m, -NHC H ₂ C H ₂ NH-), 3.79 (6H, s, -C ₆ H ₃ (OC H ₃ ) ₂ ), 5.
33 (12H, m, -C H = C H CH ₂ C H = C H- ), 6.51 (1H, d, -CH = C H C ₆ H ₃ (OCH
₃ ) ₂ ), 6.95-7.15 (3H, t, -C ₆ H ₃ (OCH ₃ ) ₂ ), 7.41 (1H, d, -C H = C
HC ₆ H ₃ (OCH ₃ ) ₂ ), 8.16 (1H, t, -CON H CH ₂ CH _2- ), 8.39 (1H, t, -N
H COCH = CH-). In addition, EPA-EDA-DMCA and LA-EDA-D
MCA is also EPA-ED instead of DHA-EDA.
Similar synthesis was performed using A and LA-EDA.

Reference Example 4. Synthesis of N-9,12-octadecadienoyldiethylenetriamine (LA-DET) Linoleic acid (95% or more) 2.8 g (10 mmol) and N, N-carbonyldiimidazole 1.78 g (11 m
mol) in 10 mL of anhydrous THF, and under a nitrogen stream,
The reaction was carried out at room temperature for about 1 hour. Next, 2.06 g of diethylenetriamine (hereinafter abbreviated as DET) was added to this reaction liquid.
A solution prepared by dissolving (20 mmol) and 2.06 g of triethylamine in 10 mL of anhydrous THF was added under ice cooling and the reaction was carried out for 4 hours. After the completion, add 60 mL of 0.1N hydrochloric acid to the reaction solution,
Chloroform-methanol (2: 1) (300 mL) was added for liquid separation, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrated liquid was placed on a silica gel (160 g) column, and chloroform, chloroform-methanol (95: 5), chloroform-methanol-water (80: 20: 1) mL, chloroform-methanol-concentrated aqueous ammonia (80: 2).
The eluate was flown in the order of 0: 1) to elute. The target fraction was collected from the obtained eluate fractions using thin layer chromatography (TLC) analysis as an index and concentrated to obtain 1.78 g (yield 48%) of the target product as a pale yellow oily substance. This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-concentrated aqueous ammonia (80:20:
When 1)) was performed, detection with iodine, 0.2% ninhydrin reagent and 50% sulfuric acid-methanol reagent showed a single spot. EI-MS (m / z): 365 (M ⁺ )

Reference Example 5. N-5, 8, 11, 14, 1
7-Eicopentaenoyldiethylenetriamine (EP
Synthesis of A-DET 3.03 g (10%) of eicosapentaenoic acid (95% or more)
mmol) and N, N-carbonyldiimidazole 1.7.
8 g (11 mmol) was dissolved in 10 mL of anhydrous THF,
The reaction was carried out at room temperature under a nitrogen stream for about 1 hour. Next, a solution prepared by dissolving 2.06 g (20 mmol) of DET and 2.06 g of triethylamine in 10 mL of anhydrous THF was added to this reaction solution under ice cooling, and the reaction was carried out for 4 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The concentrated liquid thus obtained was treated with silica gel (16
0 g) on a column, and the eluate was run in the order of chloroform, chloroform-methanol (95: 5), chloroform-methanol-water (80: 20: 1), chloroform-methanol-concentrated ammonia water (80: 20: 1). Was eluted. Using the TLC analysis as an index, the obtained eluate fractions were collected and concentrated to obtain 1.85 g (yield 47.8%) of the desired product as a pale yellow oil. This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-concentrated aqueous ammonia (80:20:
When 1)) was performed, detection with iodine, 0.2% ninhydrin reagent and 50% sulfuric acid-methanol reagent showed a single spot. EI-MS (m / z): 387 (M ⁺ )

Reference Example 6. N-4, 7, 10, 13, 1
Synthesis of 6,19-docosahexaenoyldiethylenetriamine (DHA-DET) docosahexaenoic acid (95% or more) 3.28 g (10 m
mol) and N, N-carbonyldiimidazole 1.78.
g (11 mmol) was dissolved in 10 mL of anhydrous THF, and the mixture was reacted under a nitrogen stream at room temperature for about 1 hour. Next, a solution prepared by dissolving 2.06 g (20 mmol) of DET and 2.06 g of triethylamine in 10 mL of anhydrous THF was added to this reaction solution under ice cooling, and the reaction was carried out for 4 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The concentrated liquid thus obtained was treated with silica gel (16
0 g) on a column, and the eluate was run in the order of chloroform, chloroform-methanol (95: 5), chloroform-methanol-water (80: 20: 1), chloroform-methanol-concentrated ammonia water (80: 20: 1). Was eluted. Using the TLC analysis as an index, the obtained eluate fractions were collected as target fractions and concentrated to obtain 2.06 g (yield 50.0%) of the target product as a pale yellow oil. This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-concentrated aqueous ammonia (80:20:
When 1)) was performed, detection with iodine, 0.2% ninhydrin reagent and 50% sulfuric acid-methanol reagent showed a single spot. EI-MS (m / z): 413 (M ⁺ )

Example 6. N- [3- (3,4-dimeth)
Xyphenylpropenoyl)]-N '-(9,12-o
Cutadecadienoyl) diethylenetriamine (LA-D
Synthesis of ET-DMCA) 424 mg (2.04) 3,4-dimethoxycinnamic acid
mmmol) and N, N-carbonyldiimidazole 39
Dissolve 6 mg (2.4 mmol) in 10 mL of anhydrous THF
Then, the mixture was reacted at room temperature for 2 hours under a nitrogen stream. Then this
746 g of LA-DET obtained above (2.
04 mmol) and triethylamine 208 mg (2.6
4 mmol) in 10 mL of anhydrous THF was added.
Well, it was reacted for about 4 hours. After the completion, add 0.1N salt to the reaction solution.
Acid 60 mL, chloroform-methanol (2: 1) 30
0 mL was added for liquid separation, the lower layer was separated and concentrated under reduced pressure.
Was. The concentrate obtained was applied to a silica gel (60 g) column.
Chloroform-methanol-acetic acid (85:15:
It was eluted in 1). The obtained elution fraction is used as an index for TLC analysis.
The target fraction was collected as, and concentrated to give a pale white powder.
Thus, 674 mg of the desired product was obtained (yield: 45.6%). Genuine
Quality is TLC analysis (silica gel plate, developing solvent: black
Loloform-methanol-water (85: 15: 1))
After a while, iodine, UV and 50% sulfuric acid-methanol
Reagent detection showed a single spot.¹ H-NMR (400Hz, C_FiveD_FiveN): δ 0.85 (3H, t, -CH₃), 1.23 to 1.26
(16H, m, -CH₂-), 1.49 (2H, t, -CH ₂CONH-), 2.00 (4H, m, -CH = C
HCH₂CH = CHCH ₂-), 2.73 (2H, m, -CH = CHCH ₂CH = CH-), 2.94-3.
00 (4H, t, -CH ₂NHCH ₂-), 3.31-3.43 (4H, m, -NHCH ₂CH₂NHCH₂
CH ₂NH-), 3.78 (6H, s, -C₆H₃(OCH ₃)₂), 5.31 (4H, m, -CH= CH
-), 6.51 (1H, d, -CH = CHC₆H₃(OCH₃)₂), 6.95-7.15 (3H, t, -C
₆ H ₃(OCH₃)₂), 7.40 (1H, d, -CH= CHC₆H₃(OCH₃)₂), 8.06 (1H,
t, -CONHCH₂CH₂-), 8.33 (1H, t, -NHCOCH = CH-).

Example 7. N- [3- (3,4-dimethoxyphenylpropenoyl)]-N '-(5,8,1
Synthesis of 1,14,17-eicopentaenoyl) diethylenetriamine (EPA-DET-DMCA) 362 mg (1.74) of 3,4-dimethoxycinnamic acid
mmmol) and N, N-carbonyldiimidazole 33
8 mg (2.08 mmol) was dissolved in 10 mL of anhydrous THF and reacted under a nitrogen stream at room temperature for 2 hours. Then
To this reaction solution, 674 g of EPA-DET obtained above
A solution of (1.74 mmol) and 177 mg of triethylamine dissolved in 10 mL of anhydrous THF was added, and the mixture was reacted for about 4 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrate was placed on a silica gel (60 g) column and eluted with chloroform-methanol-acetic acid (85: 15: 1).
The obtained elution fraction was collected as a target fraction using TLC analysis as an index, and concentrated to obtain the target product 440 as a pale white powder.
mg (yield 43.8%) was obtained. When this substance was subjected to TLC analysis (silica gel plate, developing solvent: chloroform-methanol-water (85: 15: 1)), it showed a single spot by detection with iodine, ultraviolet rays and 50% sulfuric acid-methanol reagent. . ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.92 (3H, t, -CH ₃ ), 1.56 (2H, m,
-CH ₂ CH = CHC H ₂ CH _2- ), 2.05 (4H, m, -C H ₂ CH ₃ , -CH ₂ C H ₂ CH ₂ CONH
-), 2.11 (2H, t, -CH ₂ CONH-), 2.80 (8H, m, -CH = CHC H ₂ CH = CH
-), 2.97 ~ 3.02 (4H, m, -C H ₂ NHC H _2- ), 3.34 ~ 3.36 (4H, m, -NH
_{C H 2 CH 2 NHCH 2 C} H 2 NH -), 3.79 (6H, s, -C 6 H 3 (OC H 3) 2), 5.33 (10
H, m, -C H = C H CH ₂ C H = C H- ), 6.51 (1H, d, -CH = C H C ₆ H ₃ (OCH ₃ ) ₂ ),
6.95 ~ 7.15 (3H, t, -C ₆ H ₃ (OCH ₃ ) ₂ ), 7.40 (1H, d, -C H = CHC ₆ H ₃
(OCH ₃ ) ₂ ), 8.09 (1H, t, -CON H CH ₂ CH _2- ), 8.35 (1H, t, -N H COCH
= CH-).

Example 8. N- [3- (3,4-dimethoxyphenylpropenoyl)]-N '-(4,7,1
Synthesis of 0,13,16,19-docosahexaenoyl) diethylenetriamine (DHA-DET-DMCA) 3,4-dimethoxycinnamic acid 406 mg (1.94)
mmmol) and N, N-carbonyldiimidazole 37
9 mg (2.34 mmol) was dissolved in 10 mL of anhydrous THF, and the mixture was reacted under a nitrogen stream at room temperature for 2 hours. Then
805 g of DHA-DET obtained above in this reaction solution
A solution of (1.95 mmol) and 199 mg of triethylamine dissolved in 10 mL of anhydrous THF was added, and the mixture was reacted for about 4 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrate was placed on a silica gel (60 g) column and eluted with chloroform-methanol-acetic acid (85: 15: 1).
Using the TLC analysis as an index, the obtained elution fractions were collected as target fractions and concentrated to give the target product 498 as a pale yellow powder.
mg (yield 42.5%) was obtained. When this substance was subjected to TLC analysis (silica gel plate, developing solvent: chloroform-methanol-water (85: 15: 1)), it showed a single spot by detection with iodine, ultraviolet rays and 50% sulfuric acid-methanol reagent. . ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.91 (3H, t, -CH ₃ ), 2.03 (2H _, m,
-C H ₂ CH ₃ ), 2.16 (2H, t, -CH ₂ C H ₂ CONH-), 2.27 (2H, m, -CH = CHC
H ₂ CH = CHC H ₂ CH _2- ), 2.79 (10H, m, -CH = CHC H ₂ CH = CH-), 3.00 ~
3.05 (4H, t, -C H ₂ NHC H _2- ), 3.11-3.22 (4H, m, -NHC H ₂ CH ₂ NHC
H ₂ C H ₂ NH-), 3.79 (6H, s, -C ₆ H ₃ (OC H ₃ ) ₂ ), 5.33 (12H, m, -C H = C
H CH ₂ C H = C H- ), 6.51 (1H, d, -CH = C H C ₆ H ₃ (OCH ₃ ) ₂ ), 6.95 ~ 7.1
5 (3H, t, -C ₆ H ₃ (OCH ₃ ) ₂ ), 7.41 (1H, d, -C H = CHC ₆ H ₃ (OCH ₃ ) ₂ ),
8.16 (1H, t, -CON H CH ₂ CH _2- ), 8.39 (1H, t, -N H COCH = CH-).

Test Example 1. O ₂ in human neutrophils ^- production inhibitory activity measuring human acute myelogenous leukemia cells HL-60, containing 10% fetal calf serum and 1.4% dimethylsulfoxide RPM
The cells were suspended in I-1640 medium at 3 × 10 ⁵ cells / mL and cultured in a CO ₂ incubator (37 ° C., CO ₂ 5.0%, humidity 100%) for 4 days to give neutrophils. Differentiated into. Differentiated human neutrophils were added to MEM medium without addition of phenol red, the cell concentration was adjusted to 2 × 10 ⁶ cells / mL, and 25 μL thereof was dispensed into a 96-well plate. The compound of the present invention was made into a methanol solution having a predetermined concentration of 200 times, and 2 μL of the same was added to 200 μL of the same medium, and 25 μL was added to the cell solution.
The mixture was further incubated in a CO ₂ incubator for 15 minutes. At this time, 25 μL of the same medium was added to the sample-free group and the 100% inhibition control group. After culturing for 15 minutes, a 100% inhibition control group was treated with 4 mg / mL physiological saline solution of cytochrome C and TPA20.
Add 0 μg / mL ethanol solution to 1 μL / mL and SOD1
50 μL of a solution to which 40 μL / mL of 5000 U / mL (= 4.2 mg / mL) physiological saline solution was added, and 5 to the other in which only TPA was added to the cytochrome C solution at the same concentration
0 μL was added, and the cells were incubated in a CO ₂ incubator for 1 hour to develop color. 550nm on plate reader (control, 570n
The absorbance of m) was measured to quantify the reduced cytochrome C produced by O ₂ ⁻ , and the 50% production inhibitory concentration (IC ₅₀ ) was determined from the O ₂ ⁻ production inhibition rate relative to the control group. The results are shown in Table 1.

[0033]

[Table 1]

Test Example 2. Phospholipase A ₂ Inhibitory Activity Measurement As phospholipase A ₂ (PLA ₂ ), 85 kDa cytosolic PLA 2 (cPLA 2) purified from rabbit platelets based on a previous report (FEBS Lett., 282, 326-330, 1991) was used, and its inhibitory activity against this enzyme was used. It measured as follows.
The compound of the present invention was dissolved in methanol and used as a test solution. 25 μL of 1 M Tris-hydrochloric acid (pH 9.0), 5
The test solution and the cPLA2 solution were added to a mixed buffer solution of 20 μL of 0 mM calcium chloride solution to make 200 μL, and the mixture was reacted at 37 ° C. for 20 minutes. Then, the substrate 1-palmitoyl-2- [ ¹⁴ C] arachidonoyl-glycerophosphoethanolamine (0.5 nmol / 50000 dpm /
200 μL) was added, and the mixture was further reacted at 37 ° C. for 20 minutes. 1.25 mL of Dole's reagent (isopropanol: heptane: 1N sulfuric acid = 10: 40: 1) was added to stop the reaction, and the [ ¹⁴ C] arachidonic acid fraction was collected by the method of Dole and its radioactivity was measured by a liquid scintillation counter. The enzyme activity was measured by measuring with. Table 2 shows the results
Shown in

[0035]

[Table 2]

Test Example 3. NGF production promoting activity on L-M cells 199 containing 0.5% peptone at a concentration of 2 × 10 ⁴ / ml
0.2 ml each of the LM cells suspended in the medium was inoculated into each well of the 96-well multiplate and cultured for 2-3 days. Then, these cells were exchanged with a test medium (199 medium containing 0.5% bovine serum albumin) each containing the compound of the present invention at various concentrations, and further cultured for 24 hours. After completion of the culture, the amount of NGF produced by the LM cells and released in the culture supernatant was measured by the enzyme immunoassay method shown below.

[Method for measuring NGF] 96 made of polystyrene
Hole multi plate (Sumitomo Bakelite MS-349
6F), an anti-mouse beta NGF polyclonal antibody (produced by the inventors according to a conventional method using beta NGF prepared from mouse submandibular gland as an antigen, [S. Furukawa, I. Kamo,
Y.Furukawa, S.Akazawa, E.Satoyoshi, K.Itoh and K.Haya
shi., J. Neurochem., 40, 734-744 (1983)]) (pH 8.3) was dispensed into each well in an amount of 50 microliters and left at 37 ° C for 4 hours. After removing the antibody not adsorbed on the microplate, each well was washed three times with a washing solution. A standard beta NGF (manufactured by Toyobo) solution or 40 microliters of the culture supernatant of LM cells obtained by the above experiment was dispensed into each well and allowed to stand at 4 ° C. for 18 hours, and then the standard solution or sample The solution was removed. Furthermore, each hole was washed 3 times.

Beta-galactosidase labeled anti-beta NG
F monoclonal antibody (Boehringer Mannheim)
A solution (40 mU / ml, pH 7.6) was dispensed into each well in an amount of 50 microliters and left at 37 ° C. for 4 hours.
The enzyme-labeled antibody was removed, and each hole was washed 3 times in the same manner as above. 4-Methylumbelliferyl-beta-D
-A galactoside (manufactured by Sigma) solution (20 microgram / ml, pH 7.6) was dispensed into each well in an amount of 100 microliter, and after reacting at room temperature for 1.5 hours, 0.2
N-glycine-sodium hydroxide buffer (pH 10.3)
The enzyme reaction was stopped by aliquoting 100 microliters into each well, and the fluorescence intensity of the produced 4-methylumbelliferone was measured with a plate reader.
The amount was calculated.

[0039]

[Table 3]

[0040]

N- acyl -N- substituted cinnamoyl ethylenediamine derivatives of the present invention exhibits, O ₂ of human neutrophils ^- having production inhibitory activity, intracellular phospholipase A ₂ inhibitory activity,
Use as a medicine such as an anti-inflammatory agent, NGF
Since it has a production promoting action, it can be used as a therapeutic agent for dementia, spinal cord injury, peripheral nerve injury, diabetic neuropathy, amyotrophic lateral sclerosis and the like.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] May 30, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

Example 5. N- [3- (3,4-dimeth
Xyphenylpropenoyl)]-N '-(4,7,1
0,13,16,19-docosahexaenoyl) ethyl
Synthesis of diamine (DHA-EDA-DMCA) 3,4-dimethoxycinnamic acid 479 mg (2.3 m
mmol) and HOSu317 mg (2.76 mmol)
With N, N-dimethylformamide (3 mL) and dichlorometa
After dissolving in 5 mL of WSC, 527 mg of WSC (2.76 mmo
1) was added and reacted for 2 hours. Then, in this reaction solution
828 mg (2.2 of DHA-EDA obtained in Reference Example 1)
4 mmol) and 274 mg of triethylamine (2.7 m
(mol) dissolved in 5 mL of dichloromethane
In addition to the above, the mixture was reacted at room temperature for 3 hours under a nitrogen stream. End
After that, 60 mL of 0.1N hydrochloric acid and chloroform-medium were added to the reaction solution.
Add 300 mL of tanol (2: 1) to separate the layers,
It was separated and concentrated under reduced pressure. The concentrate obtained is treated with silica gel.
(60 g) column, chloroform-methanol
From the (98: 2) eluted fraction, 923 mg of the desired product (yield 7
3.6%). This substance was analyzed by TLC
Kagel plate, developing solvent: chloroform-methanol
-Water (85: 15: 1)), iodine, UV
Line and 50% sulfuric acid-methanol reagent for single scan
Showed the pot.¹ H-NMR (400Hz, C_FiveD_FiveN): δ 0.91 (3H, t, -CH₃), 2.03 (2H_,m,
-CH ₂CH₃), 2.16 (2H, t, -CH₂CH ₂CONH-), 2.27 (2H, m, -CH = CHC
H₂CH = CHCH ₂CH ₂-), 2.79 (10H, m, -CH = CHCH ₂CH = CH-), 3.20 ~
3.50 (4H, m, -NHCH ₂CH ₂NH-), 3.79 (6H, s, -C₆H₃(OCH ₃)₂),Five.
33 (12H, m, -CH= CHCH₂CH= CH-), 6.51 (1H, d, -CH = CHC₆H₃(OCH
₃)₂), 6.95-7.15 (3H, t, -C ₆ H ₃(OCH₃)₂), 7.41 (1H, d, -CH= C
HC₆H₃(OCH₃)₂), 8.16 (1H, t, -CONHCH₂CH₂-), 8.39 (1H, t, -N
HCOCH = CH-). In addition, EPA-EDA-DMCA and LA-EDA-D
MCA is also EPA-ED instead of DHA-EDA.
Similar synthesis was performed using A and LA-EDA.

[Procedure amendment]

[Submission date] July 7, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

Example 3. N- [3- (3,4-dihydroxyphenylpropenoyl)]-N '-(9,12-
Octadecadienoyl) ethylenediamine (LA-ED
A-CA) Synthesis 3,4-dihydroxycinnamate (caffeic acid) 3
96 mg (2.2 mmol) and HOSu 304 mg
(2.6 mmol) was added to N, N-dimethylformamide 3
mLSC and dichloromethane 5mL, WSC504m
g (2.6 mmol) was added and reacted for 2 hours. Then, LA-EDA71 obtained in Reference Example 3 was added to this reaction solution.
8 m g (2.2mmol) and triethylamine 269m
A solution of g (2.64 mmol) dissolved in 5 mL of anhydrous THF was added under ice cooling, and the mixture was reacted at room temperature under a nitrogen stream for 6 hours. After the completion, 60 mL of 0.1N hydrochloric acid and 300 mL of chloroform-methanol (2: 1) were added to the reaction solution to separate the layers, and the lower layer was separated and concentrated under reduced pressure. The concentrate obtained was dissolved in a small amount of chloroform, and the silica gel (100
g) Place on a column, chloroform-methanol (95:
5) The desired product (380 mg, yield 39%) was obtained from the eluted fraction. This substance was analyzed by TLC (silica gel plate, developing solvent: chloroform-methanol-water (85:
15: 1)), iodine, UV and 50%
Detection with sulfuric acid-methanol reagent showed a single spot. ¹ H-NMR (400Hz, C ₅ D ₅ N): δ 0.85 (3H, t, -CH ₃ ), 1.23 to 1.26
(16H, m, -CH _2- ), 1.49 (2H, t, -C H ₂ CONH-), 2.00 (4H, m, -CH = C
HCH ₂ CH = CHC H _2- ), 2.73 (2H, m, -CH = CHC H ₂ CH = CH-), 3.30 ~ 3.
50 (4H, m, -NHC H ₂ C H ₂ NH-), 5.31 (4H, m, -C H = C H- ), 6.35 (1H,
d, -CH = C H C ₆ H ₃ (OH) ₂ ), 6.90 to 7.10 (3H, t, -C ₆ H ₃ (OH) ₂ ), 7.4
2 (1H, d, -C H = CHC ₆ H ₃ (OH) ₂ ), 8.09 (1H, t, -CON H CH ₂ CH _2- ), 8.
35 (1H, t, -N H COCH = CH-).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

Reference Example 4. Synthesis of N-9,12-octadecadienoyldiethylenetriamine (LA-DET) Linoleic acid (95% or more) 2.8 g (10 mmol) and N, N-carbonyldiimidazole 1.78 g (11 m
mol) in 10 mL of anhydrous THF, and under a nitrogen stream,
The reaction was carried out at room temperature for about 1 hour. Next, 2.06 g of diethylenetriamine (hereinafter abbreviated as DET) was added to this reaction liquid.
A solution prepared by dissolving (20 mmol) and 2.06 g of triethylamine in 10 mL of anhydrous THF was added under ice cooling and the reaction was carried out for 4 hours. After the completion, add 60 mL of 0.1N hydrochloric acid to the reaction solution,
Chloroform-methanol (2: 1) (300 mL) was added for liquid separation, and the lower layer was separated and concentrated under reduced pressure. The obtained concentrated liquid was placed on a silica gel (160 g) column, and chloroform, chloroform-methanol (95: 5), chloroform-methanol-water (80: 20: 1 ), chloroform-methanol-concentrated aqueous ammonia (80:20 :).
The eluate was passed in the order of 1) to elute. The target fraction was collected from the obtained eluate fractions using thin layer chromatography (TLC) analysis as an index and concentrated to obtain 1.78 g (yield 48%) of the target product as a pale yellow oily substance. This substance is
TLC analysis (silica gel plate, developing solvent: chloroform-methanol-concentrated aqueous ammonia (80:20:
When 1)) was performed, detection with iodine, 0.2% ninhydrin reagent and 50% sulfuric acid-methanol reagent showed a single spot. EI-MS (m / z): 365 (M ⁺ )

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

Example 6. N- [3- (3,4-dimeth)
Xyphenylpropenoyl)]-N '-(9,12-o
Cutadecadienoyl) diethylenetriamine (LA-D
Synthesis of ET-DMCA) 424 mg (2.04) 3,4-dimethoxycinnamic acid
mmmol) and N, N-carbonyldiimidazole 39
Dissolve 6 mg (2.4 mmol) in 10 mL of anhydrous THF
Then, the mixture was reacted at room temperature for 2 hours under a nitrogen stream. Then this
LA-DET746 obtained above in the reaction liquid ofmg
(2.04 mmol) and 208 mg of triethylamine
(2.64 mmol) was dissolved in 10 mL of anhydrous THF.
The solution was added and reacted for about 4 hours. When the reaction is complete,
60 mL of 0.1N hydrochloric acid, chloroform-methanol
(2: 1) Add 300 mL for liquid separation, and collect the lower layer
It was concentrated under reduced pressure. The concentrate obtained was treated with silica gel (60
g) Place on a column, chloroform-methanol-acetic acid
Elution at (85: 15: 1). The elution fraction obtained is T
Target fractions were collected using LC analysis as an index and concentrated.
To give 674 mg of the desired product as a pale white powder (yield: 45.6).
%). This substance was analyzed by TLC analysis (silica gel
Developing solvent: chloroform-methanol-water (85:
15: 1)), iodine, UV and 50%
Detection with sulfuric acid-methanol reagent shows a single spot
Was.¹ H-NMR (400Hz, C_FiveD_FiveN): δ 0.85 (3H, t, -CH₃), 1.23 to 1.26
(16H, m, -CH₂-), 1.49 (2H, t, -CH ₂CONH-), 2.00 (4H, m, -CH = C
HCH₂CH = CHCH ₂-), 2.73 (2H, m, -CH = CHCH ₂CH = CH-), 2.94-3.
00 (4H, t, -CH ₂NHCH ₂-), 3.31-3.43 (4H, m, -NHCH ₂CH₂NHCH₂
CH ₂NH-), 3.78 (6H, s, -C₆H₃(OCH ₃)₂), 5.31 (4H, m, -CH= CH
-), 6.51 (1H, d, -CH = CHC₆H₃(OCH₃)₂), 6.95-7.15 (3H, t, -C
₆ H ₃(OCH₃)₂), 7.40 (1H, d, -CH= CHC₆H₃(OCH₃)₂), 8.06 (1H,
t, -CONHCH₂CH₂-), 8.33 (1H, t, -NHCOCH = CH-).

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0030

[Correction method] Change

[Correction content]

Example 7. N- [3- (3,4-dimeth)
Xyphenylpropenoyl)]-N '-(5,8,1
1,14,17-Eicopentaenoyl) diethyleneto
Synthesis of Liamine (EPA-DET-DMCA) 362 mg (1.74) 3,4-dimethoxycinnamic acid
mmmol) and N, N-carbonyldiimidazole 33
Dissolve 8 mg (2.08 mmol) in 10 mL of anhydrous THF
The solution was thawed and reacted at room temperature for 2 hours under a nitrogen stream. Then
EPA-DET674 obtained above was added to this reaction solution.mg
(1.74 mmol) and 177 mg of triethylamine
Add a solution dissolved in 10 mL of anhydrous THF and let stand for about 4 hours.
I responded. After the completion, add 60 mL of 0.1N hydrochloric acid to the reaction solution,
Add 300 mL of loroform-methanol (2: 1)
The layers were separated, the lower layer was separated, and concentrated under reduced pressure. Obtained rich
Place the residue on a silica gel (60 g) column and apply chloroform.
Elution with methanol-methanol-acetic acid (85: 15: 1).
Using the TLC analysis as an index, the target fraction
Collected and concentrated to give the desired product 440 as a pale white powder.
mg (yield 43.8%) was obtained. This substance is analyzed by TLC
(Silica gel plate, developing solvent: chloroform-meta
When water (85: 15: 1) was added,
Detection with elemental, UV and 50% sulfuric acid-methanol reagents
Indicates a single spot.¹ H-NMR (400Hz, C_FiveD_FiveN): δ 0.92 (3H, t, -CH₃), 1.56 (2H, m,
-CH₂CH = CHCH ₂CH₂-), 2.05 (4H, m, -CH ₂CH₃, -CH₂CH ₂CH₂CONH
-), 2.11 (2H, t, -CH₂CONH-), 2.80 (8H, m, -CH = CHCH ₂CH = CH
-), 2.97 ~ 3.02 (4H, m, -CH ₂NHCH ₂-), 3.34 ~ 3.36 (4H, m, -NH
CH ₂CH₂NHCH₂CH ₂NH-), 3.79 (6H, s, -C₆H₃(OCH ₃)₂), 5.33 (10
H, m, -CH= CHCH₂CH= CH-), 6.51 (1H, d, -CH = CHC₆H ₃(OCH₃)₂),
6.95 ~ 7.15 (3H, t, -C ₆ H ₃(OCH₃)₂), 7.40 (1H, d, -CH= CHC₆H₃
(OCH₃)₂), 8.09 (1H, t, -CONHCH₂CH₂-), 8.35 (1H, t, -NHCOCH
= CH-).

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction content]

Example 8. N- [3- (3,4-dimeth)
Xyphenylpropenoyl)]-N '-(4,7,1
0,13,16,19-docosahexaenoyl) diech
Synthesis of Rentriamine (DHA-DET-DMCA) 3,4-Dimethoxycinnamic acid 406 mg (1.94)
mmmol) and N, N-carbonyldiimidazole 37
Dissolve 9 mg (2.34 mmol) in 10 mL of anhydrous THF
The solution was thawed and reacted at room temperature for 2 hours under a nitrogen stream. Then
DHA-DET805 obtained above was added to this reaction solution.mg
(1.95 mmol) and 199 mg of triethylamine
Add a solution dissolved in 10 mL of anhydrous THF and let stand for about 4 hours.
I responded. After the completion, add 60 mL of 0.1N hydrochloric acid to the reaction solution,
Add 300 mL of loroform-methanol (2: 1)
The layers were separated, the lower layer was separated, and concentrated under reduced pressure. Obtained rich
Place the residue on a silica gel (60 g) column and apply chloroform.
Elution with methanol-methanol-acetic acid (85: 15: 1).
Using the TLC analysis as an index, the target fraction
Collected and concentrated to give the desired product 498 as a pale yellow powder.
mg (yield 42.5%) was obtained. This substance is analyzed by TLC
(Silica gel plate, developing solvent: chloroform-meta
When water (85: 15: 1) was added,
Detection with elemental, UV and 50% sulfuric acid-methanol reagents
Indicates a single spot.¹ H-NMR (400Hz, C_FiveD_FiveN): δ 0.91 (3H, t, -CH₃), 2.03 (2H_,m,
-CH ₂CH₃), 2.16 (2H, t, -CH ₂CH ₂CONH-), 2.27 (2H, m, -CH = CHC
H₂CH = CHCH ₂CH₂-), 2.79 (10H, m, -CH = CHCH ₂CH = CH-), 3.00〜
3.05 (4H, t, -CH ₂NHCH ₂-), 3.11-3.22 (4H, m, -NHCH ₂CH₂NHC
H₂CH ₂NH-), 3.79 (6H, s, -C₆H₃(OCH ₃)₂), 5.33 (12H, m, -CH= C
HCH₂CH= CH-), 6.51 (1H, d, -CH = CHC₆H₃(OCH₃) ₂), 6.95 to 7.1
5 (3H, t, -C ₆ H ₃(OCH₃)₂), 7.41 (1H, d, -CH= CHC₆H₃(OCH₃)₂),
8.16 (1H, t, -CONHCH₂CH₂-), 8.39 (1H, t, -NHCOCH = CH-).

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Tomoko Tsuji 6-12-2 Nomidai, Kanazawa-ku, Yokohama, Kanagawa Prefecture (72) Inventor Koji Yamaguchi 1-9-2, Minamidai, Sagamihara, Kanagawa Prefecture (72) Inventor, Yasuji Soda 4-5-11 Motoyama Kitamachi, Higashinada-ku, Kobe City, Hyogo Prefecture

Claims (1)

[Claims] 1. The following general formula: N-acyl-N-substituted cinnamoylethylenediamine derivative represented by the formula (wherein R ¹ represents an acyl residue of a higher unsaturated fatty acid having 16 or more carbon atoms, and R ² and R ³ are independently a hydrogen atom or a lower atom). Represents an alkyl group, n is 1 or 2).