WO2006043338A1

WO2006043338A1 - Novel polyethylene glycol derivative or pharmaceutically acceptable salt thereof

Info

Publication number: WO2006043338A1
Application number: PCT/JP2004/016220
Authority: WO
Inventors: Yoshinobu Tsukada
Original assignee: Actice Co., Ltd.
Priority date: 2004-10-18
Filing date: 2004-11-01
Publication date: 2006-04-27
Also published as: WO2006043353A1

Abstract

A novel compound having apoptosis-inducing activity. It is represented by the following general formula (1) and is isolated from a culture of a filamentous fungus. (In the formula, R represents -C(CH3)2CH2-C(CH3)3 and n is an integer of 5 to 13.)

Description

Specification

Novel polyethylene glycol derivatives or pharmaceutically acceptable salts thereof

[0001] The present invention relates to a polyethylene glycol derivative having a substituted phenyl group.

Background art

[0002] Various compounds are known as compounds that induce cell death or compounds that enhance the action of compounds that induce cell death, and are useful as active ingredients in pharmaceuticals for the prevention or treatment of various diseases. The use of is being actively studied.

[0003] JP-A-2004-51513 describes an anti-tumor agent comprising a mouth-opening anthocyanidin component derived from apple, which is a substance that induces apoptosis, and JP-A-2002-28690 A prophylactic / therapeutic agent for malignant tumors using TNF_a and IL-4 as site force-in as active ingredients and utilizing their apoptosis-inducing action is described. Furthermore, Japanese Patent Application Laid-Open No. 2000-72749 describes that an apoptosis inducer containing a quinoline derivative as an active ingredient can be used as an anticancer agent. Japanese Patent Application Laid-Open No. 2003-238567 describes that a benzocraton'enimide compound derived from a bacterium belonging to the genus Pseudomonas has cytotoxic activity and is useful as an active ingredient of an antitumor agent.

Disclosure of the invention

[0004] Substances and compounds that induce apoptosis in cancer cells and have a low probability of causing apoptosis in normal cells can be expected to be used as components of anticancer agents. An object of the present invention is to provide a novel compound that can also be used as an active ingredient of an anticancer agent.

[0005] The present invention provides the following general formula (1): [0006] [Chemical 1]

R

(1)

(In the above formula, R represents _C (CH) CH -C (CH), and n represents an integer of 5-13).

3 2 2 3 3

And a pharmaceutically acceptable salt thereof.

[0007] According to the present invention, it is possible to provide a polyethylene glycol derivative and a pharmaceutically acceptable salt thereof that can be suitably used as an active ingredient of a cell death inducer or an anticancer agent. Brief Description of Drawings

[0008] FIG. 1 is a spectrum chart showing the results of NMR.

FIG. 2 is a spectrum chart showing the results of LS / MS measurement.

BEST MODE FOR CARRYING OUT THE INVENTION

[0009] The polyethylene glycol derivative according to the present invention has a structure having an alkyl-substituted phenyl group at the end of the polyethylene glycol chain as shown in the general formula (1), and is a cell death inducer or anticancer agent. Useful as an active ingredient.

[0010] Among the compounds of the general formula (1), a compound in which n is 9 and a compound in which n is 10 are particularly preferable. A compound in which n is 9 or 10 can be said to be a polyethylene glycol adduct of 2,4-di-tert-octylphenol or polyethylene glycol mono-, p-diisooctylphenol ether.

[0011] The compound of the general formula (1) is a fungus (Asupergirus

ilimigatus) SRT strain (FERM BP-5030) (As of March 3, 1995, at the time of the Ministry of International Trade and Industry, Institute of Biotechnology, Institute of Biotechnology, Tsukuba Ito, Ibaraki, Japan 1-3 Extracted, separated and purified from the deposit under the Budapest Treaty) It is a thing. This compound of the general formula (1) is amphiphilic and has the property of inducing apoptosis in cancer cells and hardly causing apoptosis in normal cells. In addition, it has DNA synthesis inhibitory or inhibitory action and is useful as an active ingredient in anticancer agents

[0012] The pharmacologically acceptable salt of the compound of the above general formula (1) can be obtained by a conventional method, for example, a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid or the like. Alternatively, it can be used as a salt with an organic acid such as formic acid, acetic acid, fumaric acid, citrate, maleic acid, oxalic acid, malic acid, tartaric acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid.

[0013] The cell death inducer or anticancer agent in the present invention is at least i selected from the compound of the above general formula (1) and a pharmacologically acceptable salt thereof as a reagent or a carrier or diluent for preparation. It can be prepared by mixing with a reagent or preparation. Examples of the dosage form include solid preparations such as tablets, pills, powders, granules, capsules and suppositories, liquid preparations such as injections, suspensions, syrups and emulsions, and semi-preparations such as patches. A solid agent etc. can be mentioned, and it can formulate by selecting suitably the carrier according to these dosage forms, a diluent, an excipient | filler, and various additives.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0015] (Example 1) Preparation of an extract from a filamentous fungus

Asupergirus

iumigatus) SRT strain (FERM BP-5030) spores at an inoculum of ³ × 10 ⁵ cells / ml in 100 ml of Potato Dextrose Broth (Difco) 35. C. Shaking culture was performed for 5 days as a preculture under the conditions of 120 rpm. Next, the total volume of the obtained culture broth lOOmL was added to Potato

The mixture was mixed with 2 L of Dextrose Broth (Difco) and shake cultured for 5 days as the main culture at 35 ° C. and 120 μm. The cells were collected from the obtained culture broth, and this was subjected to freezing treatment at 120 ° C for at least 20 hours and thawing treatment by standing at room temperature for 3 hours three times in this order. The last thawed cells in this repeated freeze-thaw treatment were loosened in PBS (-) and further stirred in a mixer. After further adding 10 g of Triton_X to this bacterial cell sample, the whole amount was added to PBS (-), stirred to 2 L, and allowed to stand at 4 ° C. This bacterial cell sample Suction filtration was repeated using filters with different sizes (6 xm, 0.7 xm, 0.45 μm) in order to remove the cells. The obtained filtrate was mixed with ammonium sulfate to reach 90% saturation, and left overnight at 4 ° C. The solid content of the upper layer generated in the filtrate after standing was recovered by centrifugation (ll, 000 X g, 10 minutes), dissolved in 300 mL of distilled water, and then dialyzed against distilled water. From the extract obtained by this dialysis treatment, Bio-Beads

Triton_X was removed using SM-2 adsorbent. The extract from which Triton_X was removed was dialyzed again against distilled water and freeze-dried to obtain sample A.

(Example 2) HPLC fractionation

The sample A obtained in Example 1 was subjected to HPLC under the following conditions.

(l) Preparation of samples for HPLC

(1 1) Preparation of analytical solution

To 10 mg of sample A, 20 ml of acetone in a container was added, sealed, and shaken for about 2 hours. The obtained acetone extract was transferred to a centrifuge tube, centrifuged for about 5 minutes, and the supernatant was filtered with a 0.2 × m pore size filter. The filtrate was concentrated to dryness under a nitrogen stream. DMSO (250 mL) was added to the dried solid to dissolve it, and the insoluble matter in the obtained solution was removed with a 0.2 μm pore size filter to obtain an analytical solution.

(1 2) HPLC conditions

Equipment: LC 1 OAvp (Shimadzu Corporation)

HPLC column: Cosmosil

5C18 4.6 X 150mm

Column temperature: 40 ° C

Mobile phase: A / B (100Z0) (5 minutes) one (20 minutes) _ΑΖΒ (75/25) (0 minutes) one (20 minutes) —A / Β (5/95) (20 minutes)

Α: Water

B: Acetonitrinore

Flow rate: 0.7 mL / min

Sample injection volume: 50

UV detector: 216nm (1 1 3) Results

A fraction showing a plurality of peaks including a fraction having a peak at a retention time of about 59 minutes (fraction 1) was obtained. The highest peak, one in fraction 1, was used for the following operations.

(2) LC / MS analysis

The sample A was subjected to LSZMS measurement under the following conditions.

Apparatus: LCT mass spectrometer (micromass)

HPLC: HP1100

HPLC column: Cosmosil

5C18 4.6 X 150mm, 5 μm, Nakarai Tex

Column temperature: 40 ° C

Mobile phase: A / B (100/0) (5 minutes) one (20 minutes) A / B (75/25) (0 minutes) one (20 minutes) A / B (5/95) (20 minutes)

A: water

B: acetonitrile

Flow rate: 0.7 ml / min

Sample injection volume: 10 μΐ

Ionization method: ESI

Measurement ion: Positive ion

Spray voltage: 3000V

Cone voltage: 30V

Desolvation gas: Nitrogen (7001 / hour)

Ion source temperature: 150 ° C

Desolvation temperature: 250 ° C

Strike range: m / zl 50—1000 (2sec / range)

UV detector: 190-400

When the ESI mass spectrum was launched for the peak eluting at about 59 minutes, the results shown in Fig. 2 were obtained. The following results were obtained for (M + Na) ⁺ .

Observation ion (m / z) 517, 561, 605, 649, 693, 737, 781 [(M + Na)]

In the mass spectrum, ions were observed every 44 Da, and it was confirmed to have a PEG (polyethylene glycol) structure. PGE is a 44Da _ (CH CH ○) _ repeating unit

twenty two

It has a structure with many bonds.

[0017] (Example 3) MTT assembly for LC preparative fraction

MTT assembly was performed on fraction 1 obtained in Example 2.

[0018] First, fraction 1 was freeze-dried to obtain sample 1 for assembly. Atsy sample (20mg)

Add to Dulbecco's MEM medium in medium (4 mL) containing 10% by weight urine fetal serum (FBS), 2.5% by weight urinary serum (HS), penicillin and streptomycin, and stir with vortex mixer for 1 minute After mixing, the mixture was allowed to stand overnight at 4 ° C., further stirred for 1 minute, and sterilized by filtration through a 0.22 / im pore size filter. The filtrate was diluted in a medium having the same composition as above to prepare samples of 100 μg / mL, 200 μg / mL, and 40 μg / mL, respectively. Each sample was subjected to the following MTT assembly.

[0019] MIAPaCa-2 (Dainippon Pharmaceutical Co., Ltd.), a human knee cancer-derived cell, was seeded at ⁵ IX 10 per well on a 96-well plate in an incubator at 37 ° C, 5 volumes ⁰ / oC Dulbecco's MEM

2

The medium was cultured for 2 hours in a medium containing 10% by weight urine fetal serum (FBS), 2.5% by weight urinary serum (HS), penicillin and streptomycin. Replace the medium with the above-mentioned Sampu Nolet and incubate for 72 hours in an incubator at 37 ° C and 5% volume by volume, and then add 5mgZmlMTT solution.

2

Was added at a rate of 10 zL / well. Incubate the well further at 37 ° C and 5% by volume.

2

Incubate for 4 hours in beta and add 150 zL of isopropanol / HC1. After 1 hour, pipetting was performed sufficiently to dissolve formazan and the absorbance at 560 nm was measured. The results obtained are shown in Table 1.

[0020] [Table 1] Table 1 (average of 0. D. 5 60 nm, n = 2)

(Reference: In case of medium only: 1.6 0 8) From the results in Table 1, a significant growth inhibitory effect was observed for sample 1 and 5 mgZmL.

Example 4 Molecular Structure Analysis of Sample A

16 mg of Sample A was sealed with 35 ml of acetone and shaken for about 2 hours. The extract was transferred to a centrifuge tube and centrifuged for about 5 minutes. The supernatant was filtered using a filter with a pore size of 0.45 zm. The filtrate was concentrated to dryness on a rotary evaporator. The dried product was filtered by adding DMS2 50 μΐ to the dried product, and the filtrate was used as a preparative solution. Further, the same operation was performed using 200 mg of the sample bowl to obtain a solution for preparative separation.

Next, the preparative solution and the preparative solution were each subjected to HPLC treatment under the same conditions as in Example 2, and a retention time peak of about 59 minutes (the fraction of Example 2). (Corresponding to minute 1). Each of the obtained fractions was concentrated with a rotary evaporator, transferred to a vial, and concentrated / dried under a nitrogen stream. The purity of the dried product obtained from the preparative liquid was measured by a conventional chromatographic method, and the purity was 95% or more.

[0023] Next, the structure of the dried solid from the preparative solution was analyzed by NMR.

NMR conditions

Equipment: Varian UNITY

INOVA 500

Observation frequency: ¹ H: 499. 8 MHz, ¹³ C: 125. 7 MHz

Solvent: CDC1

Three

Concentration: Total amount ZO. 65mL

Standard: TMS

Temperature: 25 ° C

Measurement item

¹ HNMR measurement, ¹³ CNMR measurement, DEPT (Distortionless Enhancement by Polarization Transfer) measurement (measured with CH, CH positive, CH negative signal), HSQC

3 2

(Heteronuclear Single Quantum Coherecel) measurement

DQF-COSY (Double Quantum Filtered Correlation)

Spectroscopy Y) measurement (H— ¹ H-shift correlation two-dimensional NMR method), HMBC (Heteronuclear Multiple Bond Correlation) measurement (one method of long range ¹ H_ ¹³ C shift correlation 2D NMR method), NOESY (Nuclear Overhauser Effect and exchange)

Spectroscopy Y) measurement (N〇E correlated two-dimensional NMR method)

The obtained results (NMR attribution table) are shown below.

[Table 2] Table 2 (NMR attribution table)

* 2: s: singlet, d: dopublet, t: triplet, m: mul t lpiet

* 3: (Hz)

* 4: Overlapping signal From the results of Table 2 above, it was confirmed that the compound obtained from fraction 1 had the structure of the general formula (1). Furthermore, the ions mZ z = 561, 605, 649, 693, 737, and 781 observed by LSZMS (ESI) described in Example 2 are Na of n = 5, 6, 7, 8, 9, and 10, respectively. Since it corresponds to an ion, n (integer) is considered to be a force (M + Na) + to match a 5–10 Na addition ion. That is, it was confirmed that n = 5-10 was included.

[0025] On the other hand, the 3.89 ppm (2H) and 4.09 ppm (2H) signals in the HNMR results are attributed to the terminal portion of (OCH CH), so n = 9 is the main component from the integral ratio (I). There is

2 2 n

Was confirmed. {I / (I +1) = 8, n = 8 + l}

(3.5— 3.8ppm) (3.89ppm) (4.09ppm)

The compound of the general formula (1) where n = 9 can be separated from other compounds by a conventional method.

(Example 5) Fractionation of fraction 2 and its analysis

(1) Preparation of sample for analysis

To 50 mg of the sample A obtained in Example 1, 100 ml of acetone was added, and the mixture was extracted by stirring at room temperature for 2 hours. After extraction, the mixture was centrifuged, and the supernatant was collected, concentrated to about 20 ml under reduced pressure, filtered through Millex-L H (pore size: 0.45 / im), and the filtrate was dried under reduced pressure. Residue 11.4mg DMSO

1. Soluble in 25 mL, filtered through Millex-LH (pore size: 0.45 μm), and the filtrate was fractionated by HPL under the following conditions.

HPLC conditions

Column: TOSOH TSKgel

ODS 80Ts

Flow rate: 0.7 mL / min

Column temperature: 40 ° C

Mobile phase:

A: 0. 1% TFA

B: 0. 1% TFA, 80% CH CN

Three

Gradient

0 min (B: 0%), 5 min (B: 0%), 25 min (B: 25%), 40 min (B: 100%), 70 min (B: 100%) Detection wavelength: 215nm In the above HPLC, a peak (fraction 2) eluting at about 60 minutes was collected, evaporated to dryness, and used for structural analysis. The structural analysis was the same as in Example 4. Further, LC / MS measurement was performed in the same manner as in Example 2. The results of C one ¹ H NMR in the results obtained in FIG. 1 shows the results of LCZMS in FIG.

[0027] From the NMR analysis results, Fraction 2 fractionated under the above conditions is equivalent to Fraction 1. Further, from LSZMS (ESI) measurement, molecular ions of M + Na and M + H A peak was seen at n = 6 1 13. From the results, it was confirmed that the compound of general formula (1) was mixed with n = 6-13. Furthermore, LS / MS (ESI) measurement force M + H = 759, M + Na = 781, and there are molecular peaks at 44 mass intervals of (1 CH CH 0 1).

twenty two

From this, n = 10 was obtained. The compound of the general formula (1) in which n = 10 can be separated from other compounds by a conventional method.

Industrial applicability

[0028] The novel polyethylene glycol derivative and pharmaceutically acceptable salt thereof according to the present invention can be suitably used as an active ingredient of a cell death inducer or an anticancer agent.

Claims

A polyethylene glycol derivative represented by the following general formula (1) or a pharmaceutically acceptable salt. [Chemical 1] R

(1)

(In the formula, R represents one C (CH) CH -C (CH), and n represents an integer of 5-13).

3 2 2 3 3

n is 9,

The polyethylene glycol derivative or pharmaceutically acceptable salt according to claim 1. n is 10,

The polyethylene glycol derivative or pharmaceutically acceptable salt according to claim 1.