WO1995000518A1

WO1995000518A1 - 14-substituted morphinan derivative and use thereof

Info

Publication number: WO1995000518A1
Application number: PCT/KR1994/000075
Authority: WO
Inventors: Gi Beom Kwon; Keun Jae Kim
Original assignee: Dong Kook Pharmaceutical Co., Ltd.
Priority date: 1993-06-19
Filing date: 1994-06-18
Publication date: 1995-01-05
Also published as: KR950000705A

Abstract

A novel morphinan derivative represented by general formula (I), which is useful as an analgesic free from the problem of habituation and toxicity and is highly physiologically active. In said formula (I) R1 represents C¿1?-C4 alkyl, allyl, cyclopropylmethyl, or benzyl substituted by alkyl, nitro, etc.; R?2¿ represents fluoroalkyl such as trifluoromethyl, pentafluoroethyl, etc.; R3 represents hydrogen, methyl or ethyl; and X and Y represent each hydrogen or hydroxy, or X and Y are combined with each other to represent oxo.

Description

Details

14-Substituted morphinan analogs and their uses

The present invention is a general formula (I) useful as a painkiller

[In the formula, R ¹ represents alkyl, aryl, cyclopropylmethyl, alkyl having 1 to 4 carbon atoms or benzyl substituted by nitro, etc., and R ² represents a fluorine-substituted alkynole group such as trifluoromethyl or pentafluoroethyl. R3 represents a hydrogen atom, a methyl group or an ethyl group, X and Y each represent a hydrogen atom or a hydroxyl group, and X and Y together represent an oxo group.] Jour its induction congregation -從來, various compounds as鎭痛劑force morphine or the like is used ^f, analgesic Effect large Qiao鎭痛劑, e.g.痲藥such as morphine addictive and toxic Strong and limited in its use Despite the requirement for an ideal painkiller to be non-hazardous and non-toxic in the human body, Noble Since the pain was not developed, the development of anesthetic analgesics is only disclosed in a few patents [D. Ayer, USP, 3, 137, 701 (1964), GA Boswell , ibid, 4, 241, 065, (1980)], but few compounds that can be applied as pharmaceuticals have been developed. Considering that attempts have been made to introduce fluorine groups into the skeleton of various compounds, the present inventor is similar to morphinan A fluorine-substituted alkyl group such as trifluoromethyl is introduced at the 14th position of the body, and a compound that is a hydrogen atom and a lower alkyl group is synthesized at the 5th position. As a result, the present invention was completed by finding a substance having high physiological activity and no habit and toxicity. The purpose of the present invention is to provide a compound of the general formula (1) which is effective as the aforementioned painkiller It is to provide a morphinan derivative.

Another object of the present invention is to provide a use as an analgesic containing an effective amount of the morphinan derivative of the general formula (I).

Hereinafter, the present invention will be described in detail.

The compounds of the present invention can be produced by the following methods. However, the present invention is not limited to these methods.

[First step]

After dissolving 6,7,8,14-tetradehydro-4,5-epoxy-3,6-dimethoxy-17-methylmorphinan of the general formula (II) in acetonitrile, disopropylazodicarboxylate To give 6,7,8,14-tetradehydro-4,5-epoxy-3,6-dimethoxy-17-morphinan of the general formula (ΠΙ), and then react it with N, N- In a solvent such as dimethylporumamide, dimethylsulfoxide, acetone, and acetonitrile, sodium hydroxide, hydroxylic acid, and sodium carbonate are added, and an acyl halide is added to the compound of the general formula (IV). of

6,7,8,14-tetradehydro-4,5-epoxy-3,6-dimethoxy-17-substituted-morphinan was obtained.

—The morphinan derivative of the general formula (IV) is reacted with an aldehyde having a fluorine group of the general formula (V) to obtain a heterodiyl-alda compound of the general formula (VI). Possible solvents include, for example, benzene, toluene, xylene, methylene chloride, or ether. The reaction temperature is preferably 0 ° C to 60 ° C, and the reaction time is preferably 10 to 24 hours.

The heterodilus-alda compound (VI) obtained above was reacted under an acid catalyst to obtain a compound having a fluorine group introduced at the 14-position of the general formula (VH). Chloroic acid, sulfuric acid, Lewis acid, etc. are suitable, and tetrahydrofuran and ether are used as solvents. The reaction temperature is in the range of 0 ° C to 30 ° C, and the reaction time is preferably 6 to 12 hours. The compound (VII) itself obtained in this way also exhibits an excellent analgesic effect.

(VI)

[Step 2

The β-fluorinated ketone compound obtained above was reduced in an alcohol solvent in the presence of sodium borohydride and a reducing agent such as cesium chloride to obtain optical isomers of the general formulas (™) and (IX). The reaction temperature and time are preferably from room temperature to 50 ° C and 5 to 20 minutes.

Hereinafter, the present invention will be described in detail using examples and reference examples.

Reference example 1

6,7,8,14-tetradehydro 4,5-epoxy-1,3,6-dimethoxy 17-synthesis of monorefinan

6,7,8,14-tetradehydro 4,5-epoxy-1,3,6-dimethoxy-17-methinolemoslefinin 45 g (0.144 mol) is dissolved in acetonitrile 240 ml, and disopropyl azodicarboxy is dissolved. Silrat 29. lg (0.144 mol) was dissolved in 60 ml of acetonitrile, stirred under reflux, added with 22 g of pyridin hydrochloride, refluxed for another 30 minutes, and returned to room temperature when a white solid began to form. , Filtered, and the filtered white solid was washed several times with 100 ml of methyl alcohol, followed by vacuum evaporation. The residue was dried with hexane to obtain 6,7,8,14-tetrahydro-4,5-epoxy-3,6-dimethoxymorphinanine hydrochloride (36 g, yield 75%).

This was stirred for 5 minutes with a 10% aqueous solution of potassium carbonate and methyl chloride chloride, and the organic layer was separated and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to remove the solvent. 10.7 g of tetrahydro-4,5-epoxy-3,6-dimethoxy-17-morphinan was obtained.

mp; 149'C ~ 154 ° C,

m / e 298 [M +]

Reference Example 2 Synthesis of N- (p_nitrobenzyl) -6,7,8,14-tetradehydro-4,5-epoxy-3,6-dimethoxy-17-morphinan

In a reaction vessel, dissolve 0.216 g (l mmol) of α-bromo-p-nitrotoluene in 10 ml of N, N-dimethylporumamide, add 0.168 g of sodium hydrogencarbonate (1 in.), And stir for 30 minutes. Then, add 0.335 g (l rnnol) of 6,7,8,14-tetradehydro 4,5-epoxy-1,3,6-dimethoxy-17-moclefinan synthesized in Reference Example 1 and stir at room temperature overnight. Then, this was poured into cold water, extracted three times with 20 ml of methyllen chloride, washed several times with water and brine, and dried over anhydrous magnesium sulfate. The residue was purified by column chromatography-[elution solvent: n-hexane: ethyl acrylate = 2: 1] to give 0.233 g (yield: 53.9%) of the title compound as crystal II.

mp; 145 ° C,

m / e 433 [M +]

_{1H NMR (CDC1 3, TMS)} δ: 8.23 (d, J = 8.32Hz, 2H), 7.62 (d, J = 8.62Hz, 2H), 6.71 (d, J = 8.22Hz, 1H), 6.66 (d, J = 8.22Hz, 1H), 5.53 (d, J = 6.43Hz, 1H), 5.35 (s, 1H), 5.09 (d, J = 6.43Hz, 1H), 3.89 (m, 5H), 3.65 (s, 3H), 3.61 (d, J = 7.06Hz, 1H), 3.36 (d, J = 17.13Hz, 1H), 3.05 (dt, J = 13.08Hz, J = 3.50Hz, 1H), 2.84 (dd, J = 17.96Hz, J = 7.15Hz, IH), 2.67 (dd, J = 13.15Hz, J = 4.59Hz, IH), 2.29 (dt, J = 12.70Hz, J = 4.98Hz, IH), 1.74 (bd, J = 10.84Hz , IH)

Reference example 3

Synthesis of N- (cyclopropylmethyl) -6, fu, 8,14-tetradehydro-4,5-epoxy-3,6-dimethoxy-17-morphinan

Cyclopropylmethyl chloride was reacted in the same manner as in Reference Example 2 to obtain the title compound. Mp;

m / e 352 [M +]

! H賺_{(CDC1 3, TMS) δ:} 6.71 (d, J = 8.22Hz, IH), 6.66 (d, J = 8.22Hz, IH), 5.53 (d, J = 6.43Hz, IH), 5.09 (d , J = 6.43Hz, IH), 3.89 (s, 3H), 3.65 (s, 3H), 3.61 (d, J = 7.06Hz, IH), 3.5-3.4 (m, 2H), 3.36 (d, J = 17.13Hz, IH),

3.05 (dt, J = 13.08Hz, J = 3.50Hz, IH), 2.84 (dd, J = 17.96Hz, J = 7.15Hz, 1H),

2.67 (dd, J = 13.15Hz, J = 4.59Hz, IH), 2.29 (dt, 12.70Hz, J = 4.98Hz, IH),

1.74 (bd, J = 10.84Hz, IH), 0.97-0.85 (m, IH), 0.58-0.50 (m, 2H), 0.14-0.ll (m, IH) Reference example 4

'Synthesis of N-methyl-3,6-dimethoxy-6,14-etheno-8ot-trifluoromethyl-7-oxamorphinane

6,7,8,14-Τtradehydro-4,5-epoxy-3,6-dimethoxy-17-methylmorphinan Excess amount synthesized by piercing or the like in a solution of lg (3.2 fractions ol) in 7 ml of penzen Trifluoroacetoaldehyde was injected at -78 ° C. This was sealed, reacted at 50 ° C for 24 hours, the solvent was distilled off, and the remaining S was subjected to column chromatography-[elution solvent: acetic acid Ethyl: n-hexane = 1: 2] to give the title compound (970 mg, yield 91%).

mp; 114t, m / e 409 [M +]

1H NMR (CDC13, TMS) δ: 6.67 (d, J = 8.2Hz, 1H), 6.58 (d, J = 8.2Hz, 1H), 6.15 (d, J = 8.7Hz, 1H), 5.06 (d, J = 8.8Hz, 1H), 5.23 (q, J = 7.7Hz, 1H), 4.57 (d, J = l.1Hz, 1H), 3.81 (s, 3H), 3.60 (d, J = 6.7Hz, 1H) , 3.54 (s, 3H), 3.28 (d, 18.7Hz, 1H), 2.62-2.44 (m, 2H), 2.36 (m, 3H), 2.26-2.15 (m, 1H), 1.95-1.49 (m, 1H )

Reference example 5

Synthesis of N-methyl-5-methyl-3,6-dimethoxy-6,14-etheno-8α-trifluo mouth-7-oxamorphinan

6,7,8,14-Tetradehydro-4,5-epoxy-3,6-dimethoxy-17-methylmorphinan 10 g (32.1 mmol) is dissolved in 800 ml of tetrahydrofuran, and the mixture is dissolved at -78 ° C under nitrogen gas. 30 ml of butyllithium (1.74M in hexane) was added at 15 minutes. After 15 minutes, 4.6 ml of dimethyl sulfate (48.6, fraction 1) was added, and the mixture was stirred overnight at room temperature. 10 m of water was added, and the mixture was concentrated. The residue obtained was dissolved in 250 ml of porcine, extracted with water and brine, dried over sodium sulfate, concentrated, and recrystallized from ethanol to give the title compound.

-§-¾ 157-158'C,

m / e 325 [M: 25], 269 [100], 310 [15], 183 [11]

_{l NMR (CDC1 3, TMS)} δ: 1.71 (m, 1Η, H = 15mq), 1.74 (s, 3Η, 5 = e), 2.12 (m, 1H, H = 15ax), 2.45 (s, 3H, N = Me), 2.62 (dd, 1H, H = 10x, J (10x, 9), 7.0Hz, J (10a, 10β)-18.0Hz), 2.7 (m, 2H, H = 16eq, H = 16ax) , 3.28 (d, 1H, H = 10β, J (10β, lOx)-18.0Hz),

3.55 (s, 3H, 6-OMe), 3.63 (d, 1H, H-9, J (9, 10a) 6.8Hz), 3.83 (s, 3H, 3-0Me), 4.92 (d, 1H, H- 8, J (8, 7) 6.8Hz), 5.46 (d, 1H, H-7, J (7, 8), 6.8Hz), 6.58 (d, 1H, Hl, J (1, 2) 8.0Hz) , 6.64 (d, 1H, H-2, J (2, 1) 8.0Hz)

Reference example 6 Synthesis of N-methyl_3,6-dimethoxy-6,14-etheno-8ot-pentafluoromethyl-7-oxamorphinan

As in Example 5, pentafluoropropyl aldehyde was generated from 0-ethylpentafluoropropyl aldehyde by miriacetal to form 6,7,8,14-tetradehyde as in Example 5. The title compound was obtained by reacting with -4,5-epoxy-3,6-dimethoxy-17-methylmorphinan.

m / e 459 [M +]

Thigh _{(CDC1 3, TMS) δ:} 6.67 (d, J = 8.17Hz, 1H), 6.59 (d, J = 8.16Hz, 1H), 6.16 (d, J = 8.88Hz, 1H), 5.68 (dd, J = 8.78Hz, J = 4.7Hz, 1H), 5.35 (dd, J = 23.11Hz, J = 5.03Hz, 1H), 4.55 (d, J = 1.06Hz, 1H), 3.81 (s, 3H), 3.66 (d, J = 6.64Hz, 1H), 3.51 (s, 3H), 3.29 (d,

J = 18.69Hz, 1H), 2.58-2.47 (m, 3H), 2.35 (s, 3H), 2.28-2.18 (m, 1H), 1.92 (dd, 13.7Hz, J = 2.41Hz, 1H)

Reference example 7

Synthesis of N-methyl-3,6-dimethoxy-6,14-etheno-8o-heptafluoropropyl-7-oxamorphinan

6,7,8,14-Tetradehydro-4,5-epoxy-3,6-dimethoxy-17-methylmorphinan (200 mg, 0.64 mol) and heptafluobutylbutyraldehyde (200 mg, 1.04 mnol) were added to 3 ml of penzen. The fluoro π-butyl aldehyde was reduced to 40 ° C or less for 24 hours at 50 ° C in a sealed tube, and the residue was subjected to column chromatography [elution solvent: ethyl acetate: n-hexane = 2: 1]. The title compound was obtained as an oil (160 mg, cattle: 85%).

m / e 509 [M +]

_{! HNR (CDC1 3, TMS)} δ: 6.67 (d, J = 8.2Hz, 1H), 6.59 (d, J = 8.2Hz, 1H), 6.15 (d, J = 8.9Hz, 1H), 5.69 (dd, J = 8.9Hz, J = 4.2Hz, 1H), 5.43 (dd, J = 21.9Hz, J = 5.7Hz, 1H), 4.55 (d, J = l.1Hz, 1H), 3.80 (s, 3H), 3.60 (d, J = 5.5Hz, 1H), 3.51 (s.3H), 3.28 (d, J = 18.7Hz , 1H), 2.58-2.47 (1 ^ 2H), 2.35 (s, 3H), 2.32-2.1 (m, 1H), 1.95 (dd, J = 13.8Hz, J = 2.6Hz, 1H)

Reference Example 8

Synthesis of N-cyclopropylmethyl-3,6-dimethoxy-6,14-etheno-8 (χ-trifluoromethyl-7-oxamorphinan

Ν- (cyclopropylmethyl) -6,7,8,14-tetradehydro-4,5-epoxy-3,6-dimethoxy-17-morphinan synthesized in Reference Example 3 The title compound was obtained by the reaction described above.

mp; 128'C,

m / e 412 [M +]

_{MR (CDC1 3, TMS) δ} : 6.67 (d, J = 10.58Hz, 1H), 6.58 (d, J = 10.49Hz, 1H), 6.15 (d, J = 8.81Hz, 1H), 5.26 (d, J = 6.58Hz, 1H), 5.35 (q, J = 6.58Hz, 1H), 5.20 (q, J = 3.66Hz, 1H), 4.91 (d, J = 1.09Hz, 1H), 4.00 (d, J = 8.36 Hz, 1H), 3.81 (s, 3H), 3.55 (s, 3H), 3.41-3.36 (m, 1H), 3.19 (d, J = 18.5 Hz, 1H), 2.3 (b, 2H), 2.82- 2.75 (m, 1H), 2.55-2.42 (m, 1H), 2.27-2.18 (m, 1H), 1.96-1.91 (m, 1H), 1.32- 1.23 (m, 1H), 0.97-0.85 (tn , 1H), 0.48-0.50 (m, 1H), 0.14-0.ll (m, 1H)

Example 1

Synthesis of 3-methoxy-4,5-epoxy-6-carbonyl-7,8-dehydro-14-(-trifluoromethyl-Γ-hydroxy) morphinan (Compound No. 1)

N-methyl-3,6-dimethoxy-6,14-ethoxy-8α-trifluoromethyl-7-oxamorphinan synthesized in Reference Example 5 was dissolved in 400 mg (0.8imiol) of tetrahydrochloride in 0.5 ml of concentrated hydrochloric acid. The solution was added to 25 ml of Drofuran and stirred at room temperature for 2 hours. Extracted with methylene chloride, washed with water, dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography-[elution solvent: ethyl acetate: n-hexane = (2: 1)]. This gave 520 tng of the title compound (yield; 88%).

mp; 162 ° C (^-Sfl),

m / e 393 [Μ +]

_{! HR (CDC1 3, TMS)} δ: 6.67 (d, J = 8.20Hz, IH), 6.66 (d, J = 6.2Hz, IH), 6.54 (dq, J = 10.2Hz, J = 3Hz, IH), 6.16 (d, J = 13.70Hz, IH), 6.12 (d, J = 13.7Hz, IH), 5.71 (dq, J = 9.5Hz, J = 6.7Hz, IH), 4.84 (s, 1H), 3.79 ( s, 3H), 3.50 (d, J = 5.4Hz, IH), 3.24 (d, J = 18.7Hz, J = 5.5Hz, IH), 2.94 (d, J = 9.4Hz, IH), 2.64-2.53 ( m.3H), 2.48 (dd, J = 18.7Hz, J = 5.5Hz, IH), 2.45 (s, 3H), 2.51-2.41 (m, IH), 1.66-1.61 (m, IH)

Example 2

Of 3-Methoxy-4,5-epoxy-6-hydroxy-7,8-dehydro-14- (1'-trifluoromethyl-1'-hydroxy) morphinan (Compound Nos. 2 and 3) Synthesis

The compound obtained in Example 1 was reduced to give 3-methoxy-6CX-hydroxy-7,8-dehydro-14-R- (1′-trifluoromethyl-1, -hydroxy) morphinan (compound No. 2) and 3-methoxy-6β-hydroxy-7,8-dehydro-14- (1′-trifluoromethyl-1′-hydroxy) morphinan (Compound No. 3) The physical properties of Compound No. 2 are as follows. M / e 397 [M +] (100)

IR (neat) cm-l: 3506, 3402, 2951, 1508, 1446, 1280, 1157, 1118, 1084, 1049, 794

^{_{] HNR (CDC1 3, TMS)}} δ: 6.70-6.56 (m, 2H), 6.25-6.22 (m, IH), 4.86- 4.92 (m, 2H), 4.32 (t, J = 5.21Hz, IH), 3.84 (s, 3H), 3.29 (d, J = .9Hz, 1H), 3.13 (d, J = 18.Hz, 1H), 3.0 (bs _f 1H), 2.36 (a, 3H), 2.1-2.21 (m, 3H), 1.64-1.56 ( m, 2H)

Example 3

3-Methoxy-4,5-epoxy-5-methyl-6-carbonyl-7,8-dehydro-14-(-trifluoromethyl-Γ-hydroxy) morphinan (Compound Nos. 4 and 5) Synthesis

Using Ν-methyl-5-methyl-3,6-dimethoxy-6,14-etheno-80C-trifluoromethyl-7-oxamorphinan obtained in Reference Example 5 in the same manner as in Example 1. Reaction with 3-methoxy-5-methinole-60-canolepodinole-7,8-dehydro-14- (1'-trifluoromethyl-1,1-hydroxy) morphinan (Compound No. 4) and 3-Methoxy-5-methyl-6β-capilloninole-7,8-dehydro-14- (1'-trifluoromethyl-1, -hydroxy) morphinan (Compound No. 5) Obtained.

Example 4

Synthesis of 3-methoxy-4,5-epoxy-5-methyl-6-hydroxy-7,8-dehydro-14- (Γ-trifluoromethyl- -hydroxy) morphinan

The title compound was obtained by reducing the compound obtained in Example 3.

Example 5

Synthesis of 3-Methoxy-4, 5-epoxy-6-carbonyl-7, 8-dehydro-14- (1, -pentafluoroethyl-Γ-hydroxy) morphinan (Compound No. 1)

Using Ν-methyl-3,6-dimethoxy-6,14-etheno-8α-pentafluoromethyl-7-oxamorphinan obtained in Reference Example 6 above, in the same manner as in Example 1. Reaction gave the title compound.mp; 147-150 ° C,

m / e 44 [M +]

_{1H NMR (CDC1 3, TMS)} δ: 6.69 (d, J = 8.22Hz, 1H), 6.63 (d, J = 8.39Hz, 1H), 6.62- 6.56 (ra, 1H), 6.23 (d, J = 10.10Hz, 1H), 6.56 (m, 1H), 6.23 (d, J = 10.10 Hz, 1H), 5.94-5.86 (m, 1H), 4.90 ( s, 1H), 3.83 (s, 3H), 3.54 (d, J = 5.46Hz, 1H), 3.21 (d, J = 18.41Hz, 1H), 3.00 (bs, 1H), 2.65-2.60 (m, 1H ), 2.47-2.30 (m, 3H), 2.37 (s, 3H), 1.75-1.64 (m, 3H) The following are test examples for the compounds of the present invention. In the pain effect test, 8 to 11 mice per group were administered at the dosages shown in Table 1 below, and the pain threshold was measured 30 minutes and 90 minutes after administration. The results are shown in Table 1.

Pain threshold (mmHg)

Compound dose test

Number (m ^ kg., S.c.)

30 minutes 90 minutes

2 0.2 9 222.41 + 17.94 ** 224.41 + 13.38 ** 3 0.2 10 183.52 + 10.82 + * 208.58 + 16.38 ** 4 0.2 9 213.72 + 12.73 + * 207.67 + 18.90 **

0.2 8 214.57 + 19.88 * + 199.02 ± 16.11 + * intact 11 174.61 + 6.67 184.94 + 5.83 Control 8 114.59 + 7.07 * 116.63 ± 10.59 * Morphine 2.0 9 228.26 + 13.96 ** 216.45 ± 13.38 **

* Significant value for the control group 0.01> Ρ, ** 0.001> Ρ). From the above test results, it is found that the activity of the compound of the present invention is about 10 times as pharmacological as compared to morphine. The compounds of the present invention were subjected to 1) an acute toxicity test upon intraperitoneal administration, 2) a reversion test using microorganisms and 3) a chromosome test using cultured mammalian cells. (Acute toxicity test when given intraperitoneally) 1. Test materials and test method 1) Test substance

The test substance was prepared by storing Compound No. 2 in a cold storage for dilution, and diluting it to an appropriate concentration in sterile physiological saline at an appropriate concentration.

2) Test animals

SPF (Specific Pathogen Free) 6 ~ 7 weeks old ICR mouse (sa) was used in Korea Research Institute of Science and Technology, Dove Control Center-Animal Center, 27 ± 1 in animal laboratory. C, Humidity 57 ± 5, Fluorescent light Dark 12hr cycle, MS 300-600 lx, reared in a polycass bonnet (OW X 240L X 120H mm) box, 6 animals were bred. The experimental animal feed was supplied to the experimental animals and water was supplied ad libitum. After the animals were allowed to sacrifice, only animals that were normal and visually observed after one week of acclimatization and breeding were subjected to the experiment.

3) Group separation and identification

After the acclimatization period, the weights of all animals are measured, animals within the specified range are randomly selected and distributed, and each test group is identified using the tag marking method by box.

4) Yubei test and main test

Using the acute toxicity test method of Dietrich Lorke (1), three animals were placed in each group, and 10 ml / kg of the test substance suspended in 0.9% sterile saline was intraperitoneally injected. The administered dose-finding test was carried out, the concentration between LD ₀ and LD ₁₀₀ was set to 5 and the group was set to 3 animals. After intraperitoneal injection, the animals were observed for 10 days, followed by Litchfield and Wilcoxon method (2). Accordingly

Use PHARM / PCS software (3) to determine LDsn

2. Results and discussion

1) Yube test

According to the Lorke's method, the initial doses were 10, 100, and 1000 mg / kg, and each group was tested with three animals.The results are summarized in Table 2. Table 2. Preliminary test

Capacity Observation period (days) Number of dead animals Lethal rate (mg / kg) / Number of animals used (%)

2 3 4 5 6 8 9 10

10 0 0 0 0 0 0 0 0 0 0 0/3 0

100 0 0 0 0 0 0 0 0 0 0 0/3 0

1000 0 0 0 0 0 0 0 0 0 0 0/3 0

As shown in Table 2, there was no death at 10 or 100 mg / kg or even at the highest concentration of 1000 mg / kg. Result, liver

(Liver), spleen (Spleen), kidney (Kideny), and other major organs, of course, were almost anatomically toxic and could not be observed. Based on the preliminary test results, a secondary test was performed by the capacity calculation method according to the Lorke method. The results are summarized in Table 2. The capacities were 5000 mg / kg at maximum, 2900 and 1600 mg / kg. Each group received three animals, and the capacity was set as shown in Table 3.

Capacity Observation period (say) Number of dead animals Mortality (mg / kg) / Number of animals used (%)

1 2 3 4 5 6 7 8 9 10

1600 0 0 0 0 0 0 0 0 0 0 0/3

2900 0 0 0 0 0 0 0 0 0 0 0/3

5000 0 0 0 0 0 0 0 0 0 0 0/3

As a result of the acute toxicity test, a slight splenic hypertrophy was observed in each administration group, and the animals were dissected after 10 days and observed macroscopically. Almost no toxicologically significant changes could be observed in the organs.

2) Main experiment In this experiment, as described above, a 0.5¾CMC solution was used, and a sample was carefully prepared using mortar and pestle to make the sample substance fine particles so that intraperitoneal injection was perfect. The results are shown in Table 4. Table 4. This experiment

Capacity Observation period (days) Number of lethal animals Lethal rate (mg / k) / Number of animals used ()

2 3 4 5 6 8 9 10

10 0 0/5

100 0 0/5

1000 0 0/5

1600 0 0/5

2900 0 0 I 5

5000 0/5

The results of this experiment were intraperitoneally injected at a maximum dose of 5000 mg / kg, fully referring to the results of the above experiment, and as a result, one animal in the 5000 mg / kg administration group died and was dissected. As a result of the observation, no abnormalities were seen, and the time to death was too short to recognize the absorption of the sample substance and the toxicity due to the absorption of the sample substance. In all other animals and all groups, no deaths were observed until the observation period, and no difference in general behavior could be observed. To ascertain the spleen hypertrophy observed in the experiment, a control group to which only 0.5% CMC solution was applied was separately subjected to this experiment. After some observations, no toxicological findings could be observed.體重 and spleen were harvested phase 對的 spleen weight (relative spleen weight, mg / g of body weight) of animals of Te to Baie unto a control group and a 1: 匕铰 result shown in Table 5. Table 5. Effect of Compound No. 2 on relative spleen weight of male ICR SPF mice Dose group Relative spleen weight

(mg / g of body weight, mean + S.E.

0 5.59 Sat 0.72

100 4.26 k 0.29

1000 5.95 Sat 0.89

1600 5.84 Sat 0.72

2900 5.26 Sat 0.31

5000 4.71 + 0.44 The relative spleen weight relative to body weight was determined to confirm the spleen hypertrophy observed in the results of the above experiment. As a result, no statistically significant change (P <0.05) was observed in all groups given the control compared to the control group. However, no significant toxicological changes or macroscopic anatomical findings were observed.

Compound No. intraperitoneal injection in mice號2 (i. Ρ ·) in accordance LD ₅₀ (50% lethal dose) is Shiryo about 5000mg / kg (= 5g / kg ) or more, the special matter in macroscopic autopsy report No toxicity change could be observed.

Also, suspend the compound No. 5 in a 0.5% CMC solution, and prepare the sample with great care so that the sample material becomes fine particles with mortar and pestle, and complete intraperitoneal injection. The results are shown in Table 5-1. Table 5-1

Dose Application time Application date Final test mg / kg / lOm / 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Death 5000 0 2 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5/5 3600 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2/5 2900 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/5 1600 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5 1000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5

Five everyone died within 4 hours at 5000 mg / kg, the highest dose, and 2 animals force ^f deaths at 3600 mg / kg, and .160 Omg / kg died one animal force at 290 Omg / kg ToAtae group 100 Omg / kg and the control group did not show any deaths.The above results were obtained using the PHARM / PCS software by the Litchfield Wilcoxon method: when determining the LD50; LD, ₀ value was 3574.5 mg / kg ( (The area between 95¾ and 賴) was 2801.4 mg / kg and 4560.9 mg / kg).

In general behavior, died individuals showed decreased momentum and dyspnea, died, and no other symptoms were observed. In terms of anatomical pathology, splenic hypertrophy (Spenomegaly) was observed in the highest dose group. After the observation period, autopsy was performed and gross observations were performed. As a result, no change in the toxicity of not only the main organs such as the liver and the kidney but also the anomalies could not be observed. (Return mutation test using microorganisms)

1. Materials and methods

1) Test strain

The Salmonella typhimurium TA98 and TA100 strains used in the test were obtained from Professor BN Ames of Berkley, University of California, USA Dimethyl sulfoxide (DMS0) which had been cryopreserved in a doping control center at a temperature of -70 ° C under a strong refrigeration condition of 10% of the culture broth was used. The experimental characteristics were as shown in Table 6. Before the test, the characteristics of each test strain against histidine requirement, crystal violet sensitivity, UV sensitivity, and self-returning property were confirmed. Ampicillin resistance was confirmed for TA98 and TA100, especially those that possess R factor, and used in this test. Table 6. Genetic characteristics of each test strain

Test strains heritage characteristics reference

Salmone l l a typhi murium

TA 98 hi sD3052 rfa AuvrB pKMlOl Maron & Ames (1983)

TA 100 hi sG46 rfa AuvrB pKMlOl Maron & Ames (1983)

2. Medium

1) Juice nutrient solution Juice nutrient solution 16 g NaCl 10 g distilled water lOOOOra / 121, 15 lbs, steam pressure sterilization for 20 minutes before use.

2) Pokeru Bona medium E (50x)

MgS04 7H20 10g Cuenoic acid monohydrate 100g K2HP04 500g NaHNH4P04 4H20 175g Distilled water 670mi 121 'C, 15 lbs, sterilized by steam pressure for 20 minutes, used. 40% glucose 50m /

Pokel Bona medium E (50x) 20 (

Agar 15g

D. W. 930mi

The 40% glucose was separately manufactured and sterilized. The flask containing Bacto agar was sterilized with steam at 121, 151bs for 20 minutes under steam pressure, and 40% darcose and Pokel Bona medium E (50x) were prepared. After mixing well, dispense into a gamma-ray sterilized ぺ dish (13 x xπι ^ Corning, Cat No. 430167), solidify, and place the plate down. After leaving for ~ 3 days, they were used for testing.

4) Top agar

It was made into 0.6% Bacto agar (Bacto agar) and 0.5% NaCl, sterilized at 121 ° C, 151bs, steam pressure for 20 minutes, and kept cold. After dissolution, a 0.5 mM histidine HC1 / 0.5m D-pyotin solution of lOnrf was added and used.

3) Preparation of S-9 mixture

The S-9 fraction was produced using rat (Rat) according to the method of Maron & Ames (19), and was -70. C, stored and used under strong cold conditions. S-9 The composition of the mixture is as follows.

S-9 fraction Ι. Οταί

MgC12-KCl salt solution 1.0m /

1M Darcos-6-post 0.25nrf

0.1M NADP 2.0mt

0.2 M sodium phosphate buffer, pH 7.4 25. Oral

Aseptic distilled water 19. 19.5ταΐ

4) Preparation and concentration of test substance The test substance is a white powder of Compound No. 2. As a result of a preliminary toxicological test using Salmonella typhimurium TA100 before this test, it was found that even at the highest concentration of 5000 / ig / plate test, Since no toxicity was observed, 5000 / «/ plate was used as the highest concentration in this test, and five concentrations were set with azeotrope 3. The solvent used for preparing the test material was DMS0.

5) Drugs

A broth nutrient solution, Bacto agar, was obtained from Difco Lab. U.S.A. Other reagents were obtained and used even from Sigma Chem. Co. U.S.

6) Test method

The test strain was inoculated into the broth for nutrient solution and shake-cultured at 37 ° C for about 12 to 14 hours, and the test tube (glass, 13x100 nm) was used. ,

Corning), add the DMS0 solution of the 0.1 test substance in the culture solution 0. Ιτηί, 0.2Μ sodium phosphate (ρΗ7.4) 0.5 m S-9mixture 0.5ταί in the metabolic activation method, mix, and mix in a 37 ° C water bath. After preincubation for 30 minutes in a plate, 2nrf of top agar was added, mixed, placed on a minimum Dalkose agar plate, and allowed to solidify for a few minutes. After cultivation, the number of revertant colonies was counted. Using three plates at each concentration, the results were shown by the standard deviation, and the number of revertant colonies was more than twice as many as that of the solvent. Suddenly, it is judged to be mutagenic.

2. Result

Compound No. 2, which is the test substance in this test method, was used for the test strains Salmonella typhimurium TA 98 and TA 100 when the S-9 mixture was not used and when the metabolic activation method using the S-9 mixture was used. As shown in Table 7, it did not show more than twice the number of reverted colonies as compared to the solvent control.

Table 7. Salmonella typhimuriumf! His + return sudden change / plate

1ϋ¾ 仅 9¾ ri mi lure

UStl V? Ω Ω + 7 lO 0 + 11 s

, Uchi iiU'mWC ώ 4 + 4 Q 113 3 + 11 Q uu Π 04 _L-1

J JU iQ 0-J ς. N υ i.0 J 9fi + 7 1 JL 11? 7 + 1

UO iqg n + 21 R 107 3 5 7

7-NF m 191 5 + 37 5

G 5 1987 5 + 23 3

DMSO +? 9 0 + 5 3 108 3 + 23 5

# 12 5000 + 16 5 + 0 118.0 + 9.9

1667 + 30.3 ± 2.9 116.3 ± 14.7

556 + 21.7 ± 3.1 117.3 ± 21.0

185 + 27.5 ± 0.7 126.7 ± 3.5

62 + 29.3 ± 4.9 118.0 ± 6.1

2-AF 10 + 1005.0 ± 9.9 1460.5 ± 15.3

2-NF; 2-Nitrate fluorene,

MNNG; N-methyl-N, -dito-N-ditroneguanine,

2-AF; 2-Amisofluorene

Test Example 4 (Chromosome test)

1. Test materials and methods

1) Test substance and solvent

Compound No. 2, which is the test substance, has a molecular weight of 397. It was used after being diluted to an appropriate concentration by DMS0. The volume of the test substance in the medium was 0.5% or less.

2) Cells used and culture method

Using Chinese hamster fibroblast (CHL) cell line, this cell line was We obtained a part from Dr. Ishidate and used the frozen storage preservative at the Korean Consumer Science and Technology Institute's Dove Control Center. The culture medium used was 5¾Feta] Bovine Serum (FBS, Gibco, Cat No. 200) -6140) and 1 Essential Antibiotic-Antimycotic solution (100x, Gibco, Cat No. 600-5240) [Eagle's Minimum Essential edium (EMEM, Gibco, Cat No. 410-1100)] The cells were cultured in an incubator (Vision Science Cat No. VI9011-C) supplied with 5% C02 under saturated humidity.

3) Metabolic activator

As a metabolic activation system to test whether the test substance was activated by metabolism and showed genetic toxicities, an S-9 mixture having the following composition was prepared and used.

Aseptic distilled water 1.42m /

0.02M sodium phosphate buffer (pH 7) 1.8n ^

0.1M NADP 0. \ mi

1M Darcos-6-post 0.018m /

MgC12-KC] Salt solution 0. lUmt

S-9 percent 1.48m /

5.006nrf in total

4) Preliminary test for setting the concentration of the test substance

Preliminary test was used to determine the concentration of 50% cell culture inhibition (IC ₅₀ ) of the cultured cells of the cells. For cell transfer, 2.5 x 105cel ls / m / 24 l plate (Costar) After inoculation, the cells were cultured for 24 hours in a C02 incubator at 37 ° C. After the culture, the growth was confirmed using an inverted microscope (inverted microscope), and the cells were pre-treated in a 37 ° C water bath. Washed with 2 W of warm phosphate buffer. After washing, 4 pellets per concentration were assigned, and the test material was diluted with the culture solution and treated while decreasing the concentration to 5 levels. The sixth element is a group of no illusions .. 24 Hours 37 ° C, C0 ₂ and cultured in an incubator with AraiKiyoshi twice with phosphate Salt buffer medium was discarded after culture - after was, methanol -. Were fixed Le 2m fixed after air氣中completely It was dried, stained with 5% Kiemsa [Giemsa (Gurr R66 BDH)] staining solution for 15 minutes, washed with distilled water, and dried. Observed on an impeded microscopic microscope, 50% The reproductive inhibition concentration was estimated.

5) Chromosome test

The maximum concentration was the 50% cytotoxic concentration determined in the preliminary test, and three concentrations were set as azeotropic concentrations to determine the test concentration.The absence of metabolic activity (without S-9 mixture or- S-9) and in the presence (with S-9 mixture or + S-9), the solvent control group (0.5¾ (v / v) DMS0) and the positive control group (-S-9 Benzopyrene was tested for tomycin C and + S-9.

① Testing in the absence of metabolic activity

Cultured cells were seeded such that the lx l0 ⁵ cel ls / 5ml in cell culture di Mesh of 60ran. 37'C, and cultured for 3 days in C0 ₂ incubator.試驗substance and positive to each cell culture Day Tsushu after culture The medium was replaced with a culture medium containing a control substance, etc., and cultured for 22 hours. After 22 hours, colcemid [colcemid (Gibco. Cat No. 120-0574)] was treated to 各 μΙ in each cell culture dish. After that, the total culture time was set to 24 hours.

After culturing the medium containing the S-9 mixture (proportion in the medium: 20%), the test substance, the positive control substance, etc. for 6 hours, the medium is replaced with a normal medium and cultured for 16 hours. The subsequent process was performed in the same manner as the test in the absence of metabolic activity.

6) Preparation of chromosome specimen

The cells were collected in a centrifuge tube with 15 ml of 0.05% tribsine-EDTA, and then spun down at 1,500 rpm for 5 minutes. Discard the supernatant and suspend well at 37 with stock solution (0.075M KC1) 5m /. After turbidity, it was left on a water bath at 37 ° C for 15-20 minutes.After dropping 10 drops of fixative (methanol: ft acid = 3: 1) stored at -20 ° C, Centrifuge at 1,500rpm for 5 minutes, discard the upper layer solution, add 5m / of fixative, mix and mix. After centrifugation at 1,500 rpm for 5 minutes, fixation solution treatment was performed again, and kept cool for 1 day. Use a bathtel pipe to discard the appropriate amount and discard the suspension. Submerge the washed slide in distilled water, store in a cold storage, and place the cell suspension on the slide. After dropping a drop, the slide was tilted, air-dried, dyed with 5% Giemsa dyeing solution for 5 minutes, washed with distilled water and dried.

7) Observation of the chromosome

Using a light microscope (X 1,000), 100 well-expanded steam fission cells were observed for each test group. Cells that did not satisfy the karyotype of the cell line used for the test were removed.

8) Judgment of chromosome heterogeneity test results

The total frequency of structural anomalies in the Jiao group is determined as follows according to the toxicity rating criteria of Ishidate.

Negative (-): less than 5%

Bisexual (person): 5% or more and less than 10%

Positive (+): less than 10%

2. Results and discussion

1) Toyo Toxicity Test

First, 10-fold dilution was performed with 10-2M as the highest concentration, and the test was performed at 10-5M. As a result, it showed more than 70-80% cytotoxicity up to the 10--13M area. 10_ ³ by setting the concentration gradient M and 10_ ⁴ Myuganma曰was試驗. as a result, the concentration showing an IC ₅₀ was 49. 63 / «/ m / a. azeotropically 2 this concentration as the highest concentration of 3 Wood tests were performed at different concentrations.

2) Chromosome test

In this experiment, mitomycin C, a positive control group in the absence of metabolic activity, and the presence of metabolic activity The chromosome catabolism-inducing ability of benzo (the) pyrene was 28% and 16%, respectively, and it was confirmed that the cells used were normal chromosome chromosomes by inducing sufficient chromosome heterogeneity as a positive control substance. As a negative control group, 0.5% DMSO was set to 0 to 1 それぞれ or less, respectively, without affecting the cells. Compound No. 2, which is a test substance, showed 5% as shown in Table 1 as a result of the chromosome heterogeneity test. The frequency of induction of unsatisfactory chromosome heterogenesis was observed, and the result was negative. Compound 2 was negative for chromosome heterogeneity in the absence of metabolic activity and in the presence of metabolic activity. It was about 10 times as effective as the conventional morphine, had extremely low toxicity, did not show any chromosomal abnormalities, and did not undergo any sudden changes in a sudden return test using microorganisms.

Claims

Scope of Claim

1. The general formula (I

[In the formula, R ¹ represents alkyl having 1 to 4 carbon atoms, arinole, cyclopropylmethyl, alkyl or benzyl substituted by nitro, etc., and R ² represents a fluorine-substituted alkynole group such as trifluoromethyl, pentafluoroethyl and the like. R ³ represents a hydrogen atom, a methyl group or an ethyl group, X and Y each represent a hydrogen atom or a hydroxyl group, and X and Y together represent an oxo group.] A 14-substituted morphinan analog of

2. An analgesic containing as an active ingredient a 14-substituted morphinan of the above general formula (I) or an analog thereof.