JP3535207B2 - Anticancer agent and GRP receptor antagonist - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel use of a compound represented by the following formula (I).

[0002]

[Chemical 3]

In the formula, R represents hydrogen or 3-methyl-2-butenyl. The compound according to the present invention has a gastrin-releasing peptide (GRP) receptor antagonistic activity which is extremely stronger than conventional non-peptide GRP receptor antagonists, and is therefore useful as an anti-cancer agent and various other pharmaceutical agents. .

[0004]

GRP is found as a peptide having a strong gastrin secretion stimulating action in mammals and is classified as a peptide belonging to the bombesin family (Nippon Rinji, Vol. 48, No. 5, p. 105, 199 or less).
0 years).

The GRP receptor is present in the brain, pituitary gland and pancreatic acinar cells, and promotes secretion of gastrointestinal hormones, promotion of gastric acid / pancreatic exocrine secretion, gallbladder contraction, blood pressure increase, antidiuretic action, luteinizing hormone secretion stimulation, It is involved in various physiological activities such as suppression of thyroid-stimulating hormone secretion. The presence of the receptor has also been confirmed in cancer cells of small cell lung cancer (SCLC), which accounts for about 25% of human lung cancer, and GRP is an autocrine of cancer cells such as small cell lung cancer and prostate cancer. It has been suggested to act as a growth factor for growth (Nature, vol. 361, 823-82).
6 pages, 1985). The GRP receptor antagonist is expected to be used as a medicine by suppressing the growth of these cancer cells (Cancer Research (Ca
ncerRes. ), Vol. 51, 5980-5986
P., 1991).

There are almost no reports of non-peptidic compounds having GRP receptor antagonistic activity, and only a small amount of CP-700
30 and CP-75998 were known (Bioorganic & Med Chemistry Letters (Bioorganic & Med).
icinal Chemistry Letter
s), Vol. 12, No. 4, pp. 333-338, 1992.
Year).

[0007]

The first object of the present invention was to find a non-peptide compound having a strong GRP receptor antagonistic activity. In addition, as a second purpose, based on this action, an anti-cancer agent and a variety of other medicinal products having a mechanism of action different from conventional ones were created.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a substance showing a GRP receptor antagonistic activity is contained in an underground extract of a cultivated mulberry,
Upon isolation, this was found to be a compound of formula (I). In the formula, R is hydrogen when it is Kuwanon G, and R is 3-methyl-2-
Butenyl is a known substance for Kuwanon H. Then GR about these things
When the activity of the P receptor as an antagonist was investigated, it was found that it has an excellent antagonist activity far superior to those of CP-70030 and CP-75998, and the present invention was completed.

Moraceous plant mulberry (Morus bomby
cis) has been cultivated for a long time as a bait for silkworms, but its root bark is marketed as a crude drug mulberry bark and has been used for the purpose of anti-inflammatory, diuretic, expectorant, etc. It is known that the extract of mulberry root bark has a hypotensive action, and its active ingredients are quanones containing guanone G and quanon H, and marberofurans. Other phenolic components, triterpenes, alkaloids, etc. have been isolated, and in particular, the phenolic components are known to have a cAMP phosphodiesterase inhibitory action.

[0010] Therefore, quanon G and quanon H are known substances, but it has never been known that they have GRP receptor antagonistic activity. According to the studies by the present inventors, the conventionally known non-peptide GRP receptor antagonists CP-70030 and CP-7599
8 has been reported to have an IC ₅₀ of 1.5 to 3 μM, whereas quanon G and quanone H are reported to be 3-10.
It had twice as much activity.

The above-mentioned Quanon G and Quanon H are GRP
Is effective for small cell lung cancer, prostate cancer, etc., which are thought to act as growth factors for autocrine proliferation. Also,
It can also act as a regulator of various physiological activities caused by binding of GRP to a receptor. Therefore,
The drug containing quanon G or quanon H as an active ingredient is, for example, a gastric acid secretion inhibitor, a bile secretion inhibitor, an intestinal agent, a blood pressure lowering agent, a diuretic, a luteinizing hormone secretion inhibitor, a thyroid stimulating hormone secretagogue, etc. It can also be used for applications.

[0012] Quanone G or Quanone H is administered to animals including humans as it is or together with a pharmaceutically acceptable non-toxic and inert carrier. Quanon G and Quanon H
Each can be administered alone or as a mixture.

As the carrier, one or more solid, semi-solid or liquid diluents, fillers, and other auxiliary agents for formulation are used, for example, 0.1% to 99.5%, preferably 0.5.
Used at a rate of ~ 90%. The anticancer agent or GR of the present invention
The P receptor antagonist can be safely administered orally or parenterally. As a parenteral administration form, for example, local administration such as tissue administration, subcutaneous administration, intramuscular administration,
Intramuscular / intravenous administration, transrectal administration and the like can be mentioned. Formulation forms suitable for these administration methods may be prepared using well-known and conventional technical means.

The dose as an anti-cancer agent or GRP receptor antagonist is preferably set in consideration of the patient's age, body weight, administration route, type and degree of disease, etc.
In the case of administration to humans, it is general to administer 10 to 2000 mg / day, preferably 50 to 1000 mg / day, orally as an active ingredient amount to an adult.
Also, parenterally, depending on the route of administration,
Usually 1 to 200 mg / day, preferably 5 to 100 mg
It may be administered in the range of / day. In some cases, lower doses may be sufficient, and conversely, higher doses may be required. It can also be divided into 2 to 4 times a day for administration.

Oral administration can be carried out in solid or liquid dosage units such as powders, powders, granules, tablets, capsules, syrups, elixirs or suspensions and other dosage forms.

Powders are prepared by milling the active substance into suitable granules. The powders are made up of a suitable finely divided active substance and then a finely divided pharmaceutical carrier, such as starch,
It is manufactured by mixing with edible carbohydrates such as mannitol and other excipients. If desired, a flavoring agent, a preservative, a dispersant, a coloring agent, a fragrance and the like may be mixed.

The capsule is manufactured by first filling the powdered powder, powder or granules as described above into the capsule shell such as a gelatin capsule. Further, a lubricant or a fluidizing agent such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol may be optionally mixed before filling. Disintegrators and solubilizers such as carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, croscarmellose sodium, sodium carboxystarch, calcium carbonate, sodium carbonate, etc. The effectiveness of the drug can be improved.

Further, the fine powder of this product may be suspended and dispersed in vegetable oil, polyethylene glycol, glycerin and a surfactant, and this may be wrapped with a gelatin sheet to give a soft capsule.

The granules are prepared by mixing the powdered active substance with the above-mentioned excipients and disintegrants, and if necessary, a binder (for example, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, Gelatin, polyvinylpyrrolidone, polyvinyl alcohol, etc.) and wetting agent (eg, syrup, starch paste, gum arabic, cellulose solution or polymer solution, etc.) and kneading, and then forcibly passing through a sieve to prepare You can Instead of granulating the powder in this way, first put it on a tablet machine and then
The resulting imperfect form of the slag can also be crushed into granules. A dissolution retarder (eg, paraffin, wax, hydrogenated castor oil, etc.), resorbent (eg, quaternary salt, etc.) or adsorbent (eg, bentonite, kaolin, dicalcium phosphate, etc.) is mixed in advance. You may stay.

A tablet can be prepared by adding stearic acid, a stearate salt, talc, mineral oil or the like as a lubricant to the granules thus produced and tableting. The uncoated tablets thus produced may be further subjected to film coating or sugar coating.

The active substance may be directly tableted after mixing with a fluid inert carrier without undergoing the steps of granulation and slag formation as described above. A transparent or translucent protective coating consisting of a shellac sealing coating, a coating of sugar or polymer material, and a polish coating consisting of wax can also be used.

Other oral dosage forms such as syrups, elixirs and suspensions may also be in dosage unit form so that a given quantity contains a certain amount of active substance. Syrups are prepared by dissolving the active substance in a suitable flavor aqueous solution, and elixirs are prepared by using a non-toxic alcoholic carrier. Suspensions are formulated by dispersing the active substance in a nontoxic carrier. Suspending agents and emulsifying agents (eg, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters), preservatives, flavoring agents (eg, peppermint oil, saccharin), etc. can also be optionally added. .

If desired, dosage unit formulations for oral administration can be microencapsulated. This formulation can also be provided with coatings or by embedding the active substance in polymers, wax or the like to prolong the duration of action and to provide sustained release.

Subcutaneous, intramuscular or intra-arterial / intravenous administration can be carried out in the form of a liquid dosage unit, for example, an injection in the form of a solution or suspension. These are prepared by dissolving or suspending a fixed amount of the active substance in a non-toxic liquid carrier compatible with the purpose of injection, such as an aqueous or oily solvent, and then sterilizing the solution or suspension. It
Alternatively, a fixed amount of the powdered or lyophilized active substance may be placed in a vial, and then the vial and its contents may be sterilized and sealed. In this case, a preliminary vial or carrier may be prepared for dissolution or mixing immediately before administration.
A non-toxic salt or salt solution may be added to make the injection solution isotonic, and a stabilizer, a preservative, a suspending agent, an emulsifier and the like may be used in combination.

For rectal administration, a hydrophobic or hydrophilic suppository base such as polyethylene glycol, cocoa butter, higher esters (eg myristyl palmitate) and mixtures thereof with the active substance is incorporated. Can be prepared by.

[0026]

EXAMPLES The present invention will be described in more detail below with reference to Examples, Test Examples and Formulation Examples, but the scope of the present invention is not limited thereto.

Example 1 Isolation of Quanone G and Quanone H 5 kg of underground mulberry cultivated seeds were mixed with 15 L of methyl alcohol.
The extract was dried under reduced pressure to dryness twice for 4 days. 60 g of 150 g of the obtained extract was partitioned with ethyl acetate and water, the ethyl acetate layer was dried under reduced pressure, and further partitioned with n-hexane and 90% methyl alcohol. Half of the residue (25.81 g) obtained by drying the 90% methyl alcohol layer under reduced pressure was chromatolex ODS (500 g, manufactured by Fuji Davisson), and the eluate was separated by high performance liquid chromatography (develocyl ODS). H
G-5, 4.6 × 150 mm, eluted with a 7: 3 mixed solution of methyl alcohol-water). After elution with a 1: 1 mixed solution of methyl alcohol-water (1000 ml) and a 2: 1 mixed solution (900 ml), a 2: 1 mixed solution (25
(0 ml) and 3: 1 mixed solution (200 ml) were sequentially eluted, and a fraction showing a peak with a retention time of 16.2 min was obtained. After this fraction was collected and further eluted with a 3: 1 mixed solution of methyl alcohol-water (450 ml), a fraction showing a peak with a retention time of 23.7 minutes was obtained. The remaining half amount of the sample was treated in the same manner, and the retention time was 16.2.
2.33 g of fractions showing a peak of minutes, retention time 2
A total of 2.68 g of fractions showing a peak at 3.7 minutes were obtained.

Of the fractions showing a peak with a retention time of 16.2 minutes, 1.44 g was further subjected to centrifugal liquid-liquid partition chromatography (n-hexane-methylene chloride-methyl alcohol-).
The mixture was separated by a 10: 40: 34: 16 mixed solution of water, a descending method) to obtain 539 mg of a fraction. 105 mg of which
By high performance liquid chromatography (Develocyl ODSH
G-5, 20 × 250 mm, eluted with a 4: 1 mixed solution of methyl alcohol-water and recycled 3 times) to obtain 80 mg of Compound 1.

Further, 2.28 g of the fraction showing a peak with a retention time of 23.7 minutes was subjected to high performance liquid chromatography (develocyl ODS HG-5, 20 × 250 mm, 4: 1 mixed solution of methyl alcohol-water). Elution) and then centrifugal liquid-liquid partition chromatography (n-hexane-methylene chloride-methyl alcohol-water 10:40:34:
Separation by a mixed solution of 16 and ascending method) to obtain 913 mg of a fraction. 103 mg of that was analyzed by high performance liquid chromatography (Developerosil ODS HG-5, 20 × 250).
mm, eluted with a 4: 1 mixed solution of methyl alcohol-water,
The product was purified by recycling 4 times) to obtain 59 mg of compound 2.

Compound 1 obtained by purifying a fraction having a peak with a retention time of 16.2 minutes had an mp of 199-204.
℃, IR absorption peak (Nujol) ν _max (cm ^-1 )
= 3292, 2914, 2848, 1649, 162
0, 1456, [α] _D = −491.2 ° (MeOH,
c = 0.582), which was in agreement with the data of the known Quannon G preparation. Moreover, the data of HPLC and TLC were also in agreement with those of the known standard product of Kwanon G, and Compound 1 was identified as Kwanon G. Compound 1 400MHz ¹ H-
In the NMR spectrum, the signal becomes broader, and some conformers (conf) increase with increasing measurement temperature.
The existence of an (ormer) was suggested. ¹ H-NMR δ (D
MSO-d _6): 1.43,1.51,1.60,1.
82, 3.06, 3.60, 4.21, 4.41, 5.
08, 5.11, 5.88, 5.91, 5.93, 5.
96, 6.10, 6.38, 6.49, 6.52, 7.
04, 7.25.

Compound 2 obtained by purifying the fraction having a peak with a retention time of 23.7 minutes had mp = 177-182.
℃, IR absorption peak (Nujol) ν _max (cm ^-1 )
= 3298, 2914, 2848, 1648, 162
6, 1458, [α] _D = -474.6 ° (MeOH,
c = 0.539), which was in agreement with the data of the standard product of Quanon H. Moreover, the data of HPLC and TLC were also in agreement with those of the standard product of Quanon H, and Compound 2 was identified as Quanon H. In the 400 MHz ¹ H-NMR spectrum of Compound 2, the signal became broad and some conformers were observed when the measurement temperature was increased.
The presence of ¹ H-NMR δ (DMSO-
d _6): 1.42,1.50,1.55,1.60,
1.62, 1.80, 3.01, 3.09, 3.60,
4.21, 4.41, 4.99, 5.07, 5.91,
5.94, 5.96, 6.10, 6.37, 6.49,
7.04, 7.12.

Formulation Example 1 Quanone G 100 mg Lactose 109 mg Corn starch 55 mg Low-substituted hydroxypropylcellulose 30 mg Hydroxypropylmethylcellulose 4.5 mg Magnesium stearate 1.5 mg Total 300 mg The above ingredients except hydroxypropylmethylcellulose and magnesium stearate are uniformly mixed After mixing
Granules for tableting were produced by a wet granulation method using a 5% (w / w) aqueous solution of hydroxypropylmethyl cellulose as a binder. This was mixed with magnesium stearate and then molded into a tablet with a diameter of 9 mm and a tablet weight of 300 mg using a tableting machine.

Formulation Example 2 Quanone G 100 mg Lactose 442.5 mg Mannitol 442.5 mg Hydroxypropylcellulose 15 mg Total 1000 mg The above ingredients except hydroxypropylmethylcellulose were uniformly mixed. Wet granulation was performed using a 5% (w / w) aqueous solution of hydroxypropyl cellulose as a binder to obtain a fine granule.

Formulation Example 3 Quanone H 100 mg Lactose 109 mg Corn starch 55 mg Low-substituted hydroxypropylcellulose 30 mg Hydroxypropylmethylcellulose 4.5 mg Magnesium stearate 1.5 mg Total 300 mg The above ingredients except hydroxypropylmethylcellulose and magnesium stearate were uniformly mixed After mixing
Granules for tableting were produced by a wet granulation method using a 5% (w / w) aqueous solution of hydroxypropylmethyl cellulose as a binder. This was mixed with magnesium stearate and then molded into a tablet with a diameter of 9 mm and a tablet weight of 300 mg using a tableting machine.

Formulation Example 4 Quanone H 100 mg Lactose 442.5 mg Mannitol 442.5 mg Hydroxypropylcellulose 15 mg Total 1000 mg The above ingredients except for hydroxypropylmethylcellulose were uniformly mixed. Wet granulation was performed using a 5% (w / w) aqueous solution of hydroxypropyl cellulose as a binder to obtain a fine granule.

Test Example 1 Inhibition of binding of ¹²⁵ I-labeled GRP to a receptor
Action Mouse fibroblasts cultured in a 24-well culture plate were incubated with 100 pM ¹²⁵ I-labeled GRP in the presence and absence of various concentrations of guanone G or guanone H.
Incubated at 0 ° C for 1 hour. After the reaction was completed, the cells were thoroughly washed, the collected cells were placed on a gamma counter,
Radioactivity based on ¹²⁵ I-labelled GRP bound to cells was measured. The specific binding was determined by subtracting the non-specific binding obtained under the condition containing 10 ^-6 M of non-radioactive GRP. 5 specific binding of ¹²⁵ I-labeled GRP to cells
Concentration of guanone G and quinone H that inhibit 0% (I
C ₅₀ ) is shown in Table 1.

[0037]

[Table 1]

[0038]

EFFECT OF THE INVENTION Quanone G and Quanone H according to the present invention
Is a few non-peptide GRP receptor antagonists, and has an extremely strong action as compared with conventional non-peptide antagonists. Therefore, drugs containing these as active ingredients are used as anti-cancer agents and GRP receptor antagonists. It is useful.

─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kazuya Tokura 29 Shimotake Nishi-Momonohonmachi, Kita-ku, Kyoto (56) References JP-A-3-68515 (JP, A) JP-A-3-68516 (JP, A) JP-A-3-68517 (JP, A) JP-A-3-68518 (JP, A) JP-A-5-345767 (JP, A) Valentine, James J. et al, CP-70, 030 and CP-75, 998: the first non-peptide of tagonists of bombs in and gastrin releas linguistics, Biological, Biological, & 1992. 2, no. 4, pp. 333-338 (58) Fields surveyed (Int.Cl. ⁷ , DB name) A61K 31/351 A61K 35/78 A61P 35/00 A61P 43/00 111 C07D 311/30 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. An anticancer agent comprising a compound represented by formula (I) as an active ingredient. [Chemical 1] In the formula, R represents hydrogen or 3-methyl-2-butenyl. 2. The anticancer agent according to claim 1, wherein the anticancer agent is an antipulmonary cancer agent. 3. The anticancer agent according to claim 1, wherein the anticancer agent is an antiprostate cancer agent. 4. A GRP receptor antagonist comprising a compound represented by formula (I) as an active ingredient. [Chemical 2] In the formula, R represents hydrogen or 3-methyl-2-butenyl.