JP2009242266A - Fuzanins a, b, c and d - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new chemical substances as resources. <P>SOLUTION: Disclosed are compounds represented by the chemical formula. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to fuzanins A, B, C and D which are novel compounds, and further relates to a drug using them.

  In our current life, chemicals contained in the body of natural animals and plants, microorganisms, etc. (hereinafter referred to as “natural products”) are effective in the use of crude drugs and pharmaceuticals that have a beneficial effect on the human body. Used as an ingredient. Moreover, such a thing has various roles also as a research material for developing a more useful pharmaceutical, and is very important.

  As described above, as a report on the search for natural products that have a beneficial effect on the human body, for example, Non-Patent Document 1 listed below extracts a bisindole compound, a naphthoquinone compound, a glyceride compound, and the like from a deformed bacterium.

Masami Ishibashi, “Recycling unused fungi: Search for natural products from deformed bacteria”, Journal of Synthetic Organic Chemistry, 2003, Vol. 61, No. 2, pp. 152-163

  However, on the other hand, despite the fact that many people have been searching for natural products, less than 10% of all the species on Earth have been studied and investigated as search materials. It is said.

  An object of this invention is to provide a novel chemical substance as a resource.

Fuzanin A according to one means of the present invention is represented by the following formula.

Further, fuzanin B according to another means of the present invention is represented by the following formula.

Further, fuzanin C according to another means of the present invention is represented by the following formula.

Further, fuzanin D according to another means of the present invention is represented by the following formula.

Moreover, the chemical | medical agent which concerns on another means of this invention contains at least any one of the fuzanin A shown by a following formula, and its salt as an active ingredient.

Moreover, the chemical | medical agent which concerns on another means of this invention contains at least any one of the fuzanin B shown by a following formula, and its salt as an active ingredient.

In addition, a drug according to another means of the present invention contains at least one of fuzanin C represented by the following formula and a salt thereof as an active ingredient.

Moreover, the chemical | medical agent which concerns on another means of this invention contains at least any one of the fuzanin D shown by a following formula, and its salt as an active ingredient.

  In addition, although said medicine is not necessarily limited, it is anticipated that it will be useful as a cancer therapeutic agent.

  According to the present invention, a novel chemical substance can be provided as a resource. In particular, the chemical substance according to the present invention is expected to be used, for example, as a therapeutic agent for cancer because it exhibits a cell growth inhibitory effect on cancer cells.

  Hereinafter, embodiments of the present invention will be described. However, the present invention can be implemented in many different forms, and should not be construed narrowly only in the description of the following embodiments.

Fuzanins A, B, C, and D according to an embodiment of the present invention are respectively shown below.

  As will be apparent from the examples described later, fuzanins A, B, C and D according to this embodiment are actinomycetes Kitasatospora, preferably actinomycetes Kitasatospora sp. Although it can extract from IFM10917 stock | strain, it is not limited to this, It is also possible to synthesize | combine.

  Fuzanins A, B, C, and D according to this embodiment are expected to be used as drugs, for example, cancer therapeutics, because they exert a cell growth inhibitory action on cancer cells. In addition, when using fuzanin A, B, C, and D as a therapeutic agent for cancer, it is preferable to contain at least one of fuzanin A, B, C, and D and a salt thereof as an active ingredient.

  In addition, the therapeutic agent for cancer according to the present embodiment includes at least one of the fuzanins A, B, C, and D, and salts thereof, as well as normal pharmaceutically acceptable carriers, binders, and stabilizers. , Excipients, diluents (eg distilled water), pH buffering agents (eg phosphate buffered saline), disintegrating agents, solubilizers, solubilizers, isotonic agents, etc. be able to.

  Moreover, the therapeutic agent for this cancer can be administered orally or parenterally in an appropriate dose suitable for the sex, weight and symptoms of the patient. For oral administration, commonly used administration forms such as powders, granules, tablets, capsules, solutions, suspensions, oils, emulsifiers and the like can be employed. In addition, as parenteral administration, a commonly used administration form, for example, a method of administering the above-mentioned solution, suspension or the like directly to the affected area, a form of administration by injection or the like can be adopted.

  In this example, the actinomycete Kitasatospora sp. The result of having extracted and examined fuzanin A, B, C, and D from IFM 10917 strain is shown. FIG. 1 outlines a scheme for the isolation of fuzanins A, B, C and D.

  First, the actinomycetes Kitasatospora sp. Isolate IFM 10917 strain, waxman agar medium (glucose 2%, peptone 0.5%, meat extract 0.5%, yeast extract 0.3%, sodium chloride 0.5%, calcium carbonate 0.3%, Agar 1.5%) and cultured at 28 ° C. for 1 week. Then, after confirming the formation of colonies and spores, these were scraped, and the Waxmann liquid medium in the Sakaguchi flask (glucose 2%, peptone 0.5%, meat extract 0.5%, yeast extract 0.3%, sodium chloride 0 0.5%, calcium carbonate 0.3%), followed by shaking culture at 28 ° C. for 3 days. Then, in order to culture the strain in large quantities, newly prepared Waxman liquid medium (glucose 2%, peptone 0.5%, meat extract 0.5%, yeast extract 0.3%, sodium chloride 0.5%, The pre-cultured culture solution was inoculated on calcium carbonate (0.3%), and shake culture was performed at 28 ° C. for 5 days using a turnip flask. After culturing, 3 L of the obtained culture broth was centrifuged and divided into cells and supernatant. And the microbial cell extracted with methanol, and obtained the microbial cell methanol extract. The supernatant was partitioned with ethyl acetate and butanol to obtain each soluble part.

Next, the obtained ethyl acetate soluble part was attached to a column (φ20 × 290 mm) using Sephadex LH-20 as a carrier, eluted with methanol, and each fraction from 1A to 1F was obtained in the order of elution. . In addition, 1C (463.9 mg) eluted above was applied to a column (φ30 × 160 mm) using Silicagel 60N as a carrier, eluted with a hexane-ethyl acetate mixed solution and methanol, and each fraction from 2A to 2I was eluted. Got the minute. Further, the eluted 2D (74.2 mg) was subjected to HPLC fractionation using the HPLC conditions (1) shown in Table 1 below, and fuzanin A (14.5 mg), B (23.2 mg), C (1 0.5 mg) and D (1.8 mg) were isolated respectively.

(Fuzanin A)
Fuzanin A was isolated as an oil. High-resolution FABMS was performed on fuzanin A, and a peak of m / z 304.0976 estimated to be [M + K] ⁺ was observed. As a result, the molecular formula was determined as C ₁₄ H ₁₉ NO ₄ .

Further, infrared absorption measurement (hereinafter referred to as “IR measurement”) was performed on fuzanin A to obtain an infrared absorption spectrum (hereinafter referred to as “IR spectrum”). As a result, absorptions derived from a hydroxy group and a carbonyl group were observed at 3369 and 1733 cm ⁻¹ , respectively.

  Further, ultraviolet absorption measurement (hereinafter referred to as “UV measurement”) was performed on fuzanin A to obtain an ultraviolet absorption spectrum (hereinafter referred to as “UV spectrum”). This ultraviolet absorption measurement was performed using methanol as a solvent with a concentration of 0.1 mol / l and a cell length of 1 cm. As a result, it had an absorption peak at 232 nm and could be estimated to be an aromatic compound or a conjugated double bond compound. FIG. 2 shows the UV spectrum of fuzanin A.

In addition, the optical rotation angle [α] _D was measured for fuzanin A. As a result, the optical rotation angle was +30.0 degrees, and it was confirmed to have optical activity. Table 2 below shows the results of optical rotation angle, high resolution FABMS, UV measurement, and IR measurement.

Further, ¹ H NMR and ¹³ C NMR were also measured for fuzanin A. The results are shown in Table 3, FIG. 3 and FIG. ^{In 1} H NMR, signals of two methyl groups and four transolefin hydrogens having a binding constant of about 15 Hz can be observed. In ¹³ C NMR, 14 methyl groups containing two methyl groups and six olefinic carbons can be observed. The signal could be observed. The carbonyl carbon signal was observed at δc155.9 ppm, which is higher than usual, and the results of HRFBMS indicate that this compound has a carbamate structure in which a nitrogen atom and an oxygen atom are adjacent to the carbonyl carbon in the molecule. Estimated.

In addition, HMQC, HMBC, and ¹ H- ¹ H COSY were also determined for fuzanin A. This spectrum is shown in FIGS. As a result, the present compound was found to have a linear structure having a conjugated diene and a condensed ring structure having a 5-membered ring containing a carbamate group and a 3-methyltetrahydropyridine ring. Regarding the stereochemistry of this compound, NOE correlations were observed between the 2α-position hydrogen and the 3-position hydrogen, the 6-position hydrogen and the 2β-position hydrogen, and the 6-position hydrogen and the 7-position hydrogen. From these, the relative configuration of the fused ring portion was determined. Regarding the absolute configuration, the bis (S) -MTPA ester and bis (R) -MTPA ester of this compound were prepared, and the results of the examination by the improved Mosher method and the NOE correlation data were combined, and the 3rd, 6th, The absolute configuration at position 1, 7 and 12 was determined as 3R, 6S, 7R and 12R.

  From the above results, the structure of fuzanin A was determined as shown by the above formula.

(Fuzanin B)
Fuzanin B was isolated as an oil. High-resolution FABMS was performed on fuzanin B, and a peak at m / z 266.1403 estimated to be [M + H] ⁺ was observed. As a result, the molecular formula was determined as C ₁₄ H ₁₉ NO ₄ .

Further, IR measurement was performed on fuzanin B to obtain an IR spectrum. This result was very similar to the above fuzanin A, and absorptions derived from a hydroxy group and a carbonyl group were observed at 3369 and 1733 cm ⁻¹ , respectively.

  Further, UV measurement was performed on fuzanin B to obtain a UV spectrum. This UV measurement was performed using methanol as a solvent with a concentration of 0.2 mol / l and a cell length of 1 cm. This result was similar to Fuzanin A above. That is, it has an absorption peak at 232 nm, and it was estimated that it was an aromatic compound or a conjugated double bond compound. FIG. 8 shows the UV spectrum of fuzanin A.

In addition, the optical rotation angle [α] _D was measured for fuzanin A. As a result, the optical rotation angle was −19.0 degrees, and it was confirmed to have optical activity. Table 4 below shows the results of optical rotation angle, high resolution FABMS, UV measurement, and IR measurement.

Further, ¹ H NMR and ¹³ C NMR were also measured for fuzanin B. The results are shown in Table 5, FIG. 9 and FIG. The ¹ H NMR spectrum was similar to that of fuzanin A, but it was confirmed that the chemical shift values of the 6th, 7th and 8th hydrogens differed by 0.3 to 0.5 ppm compared to that of fuzanin A. It was done.

Further, HMQC, HMBC, and ¹ H- ¹ H COSY were also obtained for fuzanin B. This spectrum is shown in FIGS. As a result, it was found that fuzanin B has the same planar structure as fuzanin A. In addition, in order to clarify the three-dimensional structure of the present compound, a NOE experiment was first conducted. As a result, NOE correlation was observed between hydrogen at the 2α position and hydrogen at the 3rd position, hydrogen at the 6th position and hydrogen at the 2β position, and hydrogen at the 6th position and hydrogen at the 8th position. In comparison, it was found that the 7-position early withdrawal relative configuration was different. As for the absolute configuration, the bis (S) -MTPA ester and bis (R) -MTPA ester of this compound were prepared, and the 3rd position of this compound was determined by combining the examination by the improved Mosher method and NOE correlation data. The absolute configurations at the 6th, 7th and 12th positions were determined as 3R, 6S, 7S and 12S.

  From the above results, the structure of fuzanin B was determined as shown by the above formula.

(Fuzanin C)
Fuzanin C was isolated as an oil. HREIMS was performed on fuzanin C, and a peak at m / z 219.1293 estimated to be [M ⁺ ] was observed. As a result, the molecular formula was determined as C ₁₃ H ₁₇ NO ₂ .

Further, IR measurement was performed on fuzanin C to obtain an IR spectrum. As a result, absorption derived from a hydroxy group was observed at 3350 cm ⁻¹ .

Further, UV measurement was performed on fuzanin C to obtain a UV spectrum. This ultraviolet absorption measurement was performed using CHCl ₃ as a solvent with a concentration of 0.05 mol / l and a cell length of 1 cm. As a result, it had absorption peaks at 314.0 nm and 269 nm, respectively, and could be estimated to be an aromatic compound or a conjugated double bond compound. FIG. 14 shows the UV spectrum of fuzanin C.

In addition, the optical rotation angle [α] _D was measured for fuzanin C. As a result, the optical rotation angle was +34.5 degrees, and it was confirmed to have optical activity. Table 6 below shows the results of optical rotation angle, high resolution FABMS, UV measurement, and IR measurement.

Further, ¹ H NMR and ¹³ C NMR were also measured for fuzanin C. The results are shown in Table 7, FIG. 15 and FIG. ^{In the 1} H NMR spectrum, the signals of four olefinic hydrogens in a trans configuration (δH 6.00, 6.57, 6.82, 7.40-7.45 overwrapped) with two methyl groups and a binding constant around 15 Hz are shown. Observed. In the ¹³ C NMR spectrum, signals of 4 olefinic carbons and 5 aromatic carbons were observed at two methyl groups of 120 to 155 ppm. In the ¹ H NMR spectrum of this compound, the chemical shift value of hydrogen at position 2 was observed in a low magnetic field of δH8.41, so it was estimated that the heterocycle was pyridine and the nitrogen atom was adjacent to position 2. It was.

Further, HMQC, HMBC, and ¹ H- ¹ H COSY were also obtained for fuzanin C. The spectrum of HMBC is shown in FIG. Detailed analysis of the HMBC spectrum revealed that this compound had a structure in which a trans-conjugated conjugated diene and a side chain having an adjacent diol structure were bonded to the 2-position of the 3-methylpyridine ring. The stereochemistry at the 11th and 12th positions was determined by comparing the bond multiplier constant of the compound having a diol structure adjacent to the double bond in the trans configuration and the bond constant between the 11th and 12th hydrogens. That is, in a compound having a diol structure adjacent to a double bond in a trans configuration, when the hydroxy group of 1,2-diol is in an anti configuration, a bond multiplier constant between hydrogens bonded to the same carbon as each hydroxy group Is 3 Hz and syn is known to be 6.2 Hz in one arrangement. Since the binding constant between the 11th and 12th hydrogens of this compound was 6.5 Hz, it was suggested that the 11th and 12th hydroxyl groups were in the syn configuration, and the relative configuration at the 11th and 12th positions was changed. each ^11R *, was determined to be 12R ^*.

  From the above results, the structure of fuzanin C was determined as shown by the above formula.

(Fuzanin D)
Fuzanin D was isolated as an oil. HREIMS was performed on fuzanin C, and a peak at m / z 219.124 estimated to be [M ⁺ ] was observed. As a result, the molecular formula was determined as C ₁₃ H ₁₇ NO ₂ .

  Further, IR measurement and UV measurement were performed on fuzanin C, and IR spectrum and UV spectrum were obtained. This result was almost the same as that of fuzanin A. FIG. 18 shows the UV spectrum of fuzanin D.

Further, the optical rotation angle [α] _D was measured for fuzanin D. As a result, the optical rotation angle was -32.9 degrees, and it was confirmed to have optical activity. Table 8 below shows the results of optical rotation angle, HREIBMS, UV measurement, and IR measurement.

Further, ¹ H NMR and ¹³ C NMR were also measured for fuzanin D. The results are shown in Table 9 and FIG. ¹ H NMR spectrum was very similar to fuzanin C, but chemical shift values at 11th position (δ _H 4.22, J = 6.7 Hz) and 12th position (δ _H 3.92, J = 6.5 Hz). Shifted to the low magnetic field side compared with fuzanin C, and their coupling constants were also different.

Further, HMQC, HMBC, and ¹ H- ¹ H COSY were also determined for fuzanin D. This spectrum is shown in FIGS. This compound was determined to have the same planar structure as fuzanin AC by detailed analysis of the HMBC spectrum. Regarding the stereochemistry of this compound, as with fuzanin C, the compound having a diol structure adjacent to the double bond in the trans configuration and the bond constant between the 11th and 12th hydrogens were 3.8 Hz. It is suggested that the hydroxy groups at the 11th and 12th positions have an anti configuration, and the relative configurations at the 11th and 12th positions were determined as 11S ^* and 12R ^* , respectively.

  From the above results, the structure of fuzanin CD was determined as shown by the above formula.

(Cell growth inhibitory activity)
Next, cell growth inhibitory activity against human colon cancer cell lines (HCT116, DLD1) and human gastric cancer cell line AGS was examined for the isolated fuzanin D. The results are shown in Table 10 below. As a result, fuzanin D showed cell growth inhibitory activity against DLD1, and its IC ₅₀ value was 41.2 μM. On the other hand, it was confirmed that fuzanin D has selectivity without having cell growth inhibitory activity in 293T, which is a normal cell line.

  The present invention comprises a novel compound as an active ingredient and has industrial applicability as a drug.

It is a figure which shows the outline of the isolation scheme of fuzanin A, B, C, and D in an Example. It is a UV spectrum of fuzanin A in an example. It is a ¹ H NMR spectrum of fuzanin A in an example. It is a ¹³ C NMR spectrum of fuzanin A in an example. It is a HMQC spectrum regarding the fuzanin A in an Example. It is a HMBC spectrum regarding the fuzanin A in an Example. It is a ¹ H- ¹ H COZY spectra for Fuzanin A in Example. It is a UV spectrum of fuzanin B in an example. ^{1 is} a ¹ H NMR spectrum of fuzanin B in an example. It is a ¹³ C NMR spectrum of fuzanin B in an example. It is a HMQC spectrum regarding the fuzanin B in an Example. It is a HMBC spectrum regarding the fuzanin B in an Example. It is a ¹ H- ¹ H COZY spectra for Fuzanin B in Example. It is a UV spectrum of the fuzanin C in an Example. ^{1 is} a ¹ H NMR spectrum of fuzanin C in an example. It is a ¹³ C NMR spectrum of fuzanin C in an example. It is a HMBC spectrum regarding the fuzanin C in an Example. It is a UV spectrum of fuzanin D in an Example. It is a ¹ H NMR spectrum of fuzanin D in an example. It is a HMQC spectrum regarding the fuzanin D in an Example. It is a HMBC spectrum regarding the fuzanin D in an Example. It is a ¹ H- ¹ H COZY spectra for Fuzanin D in Example.

Claims (9)

Fuzanin A represented by the following formula.
Fuzanin B represented by the following formula.
Fuzanin C represented by the following formula.
Fuzanin D represented by the following formula.
A drug containing at least one of fuzanin A represented by the following formula and a salt thereof as an active ingredient.
A drug containing at least one of fuzanin B represented by the following formula and a salt thereof as an active ingredient.
The chemical | medical agent which contains at least any one of the fuzanin C shown by a following formula, and its salt as an active ingredient.
A drug containing at least one of fuzanin D represented by the following formula and a salt thereof as an active ingredient.
  The drug according to claim 4, 5, 6, or 7, which is a therapeutic drug for cancer.