A phenylacetic ester compound, and a preparation method and application thereof

Abstract

This invention belongs to the field of microbiology and novel pharmaceutical and veterinary drug technology, specifically relating to a phenylacetic acid ester compound derived from *Variococcus marineans*, and further disclosing its preparation method and application. This invention utilizes preserved *Variococcus marineans* (… Verrucosispora gifhornensis. Two phenylacetic acid ester compounds with antitumor potential were isolated and screened from FIM06-0036, and a method for extracting and isolating these compounds from the fermentation broth of this *Verticillium* strain FIM06-0036 was provided. The phenylacetic acid ester compounds described in this invention provide potential compounds for the research and development of new antitumor drugs and are of significant value for the development and utilization of China's marine drug resources.

Description

Technical Field

[0001] This invention belongs to the field of microbial and novel pharmaceutical and veterinary drug technology, specifically relating to a phenylacetic acid ester compound derived from marine verrucous spores, and further disclosing its preparation method and application. Background Technology

[0002] Actinomycetes are a major source of microbial drugs, primarily Streptomyces and some rarer genera. However, since the 1970s, the probability of discovering new compounds from Streptomyces has decreased, while the rate of repetition of new compounds has increased. In recent years, rare actinomycetes, such as Verrucospora and Nocardia, which have fewer known species, have received increasing attention. In particular, the secondary metabolites of rare actinomycetes contain a large number of novel active compounds, increasing the probability of obtaining target active compounds.

[0003] Since 2003, when it first came from Verrucosispora maris Since the discovery of abyssomicins (abysomycin) family compounds in AB-18-032, research on secondary metabolites of *Verticillium* has developed rapidly. In 2005, proximicin AC was reported as a novel aminofuran antibiotic and antitumor compound, attracting widespread attention. Subsequently, a series of structurally novel compounds were discovered, including surugamides in 2009, gifhornenolones in 2013, a kidneyycin analog in 2019, and polyketanesomycin in 2021, further enriching the structural diversity of *Verticillium* secondary metabolites.

[0004] In summary, screening novel or novel-mechanism antitumor drugs from *Verticillium* fungi is a practically feasible approach. A search of the SCIFINDER database revealed that phenylacetate-containing small molecule compounds are relatively rare in the natural product category, with no relevant literature reports found. This field anticipates discovering new types of phenylacetate-containing active substances from *Verticillium* fungi, which would contribute to further expanding the current antitumor drug pool and safeguarding human health. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a phenylacetic acid ester compound derived from marine verrucous fungi, said phenylacetic acid ester compound having antitumor potential; The second technical problem to be solved by the present invention is to provide a method for preparing and applying the above-mentioned phenylacetic acid ester compounds derived from marine verrucous fungi.

[0006] To solve the above-mentioned technical problems, the phenylacetic acid ester compounds of the present invention include compounds having the following formula (P): ; The configuration of position C-9 is either R-type or S-type.

[0007] Specifically, the phenylacetic acid ester compounds of the present invention include compound C-1 having the structure shown in formula (I) and compound C-2 having the structure shown in formula (II): .

[0008] The present invention also discloses a method for preparing the phenylacetic acid ester compound as described above, comprising the step of inoculating marine wart spore strain FIM06-0036 into a suitable fermentation medium for fermentation culture; The marine wart spore strain FIM06-0036 is classified as follows: Verrucosispora gifhornensis It has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 16548.

[0009] Specifically, the preparation method of the phenylacetic acid ester compound includes the following steps: (1) Seed culture: The preserved marine wart spore strain FIM06-0036 was inoculated into seed culture medium for seed culture, and the seed culture was collected for later use; (2) Fermentation broth culture: The seed liquid is transferred to the fermentation medium for fermentation culture to obtain the fermentation product containing the desired phenylacetic acid ester compounds.

[0010] Specifically, in the preparation method of the phenylacetic acid ester compounds, in step (1), the seed culture medium comprises: 1.2-1.8 wt% soluble starch, 0.4-0.6 wt% yeast powder, 0.4-0.6 wt% peptone, 0.4-0.6 wt% glucose, 0.04-0.06 wt% K2HPO4, 0.04-0.06 wt% MgSO4·7H2O, 0.04-0.06 wt% (NH4)2SO4, 1.5-1.8 wt% sea salt, 0.08-0.12 g / L CaCO3, prepared with pure water, and adjusted to pH 7.0-8.0; Preferably, the temperature for the seed culture step is 25-35℃, and the culture time is 1-3 days.

[0011] Preferably, the seed culture medium comprises: 1.5 wt% soluble starch, 0.5 wt% yeast extract, 0.5 wt% peptone, 0.5 wt% glucose, 0.05 wt% K₂HPO₄, 0.05 wt% MgSO₄·7H₂O, 0.04-0.06 wt% (NH₄)₂SO₄, 1.65 wt% sea salt, 0.1 g / L CaCO₃, prepared with pure water, and pH 7.5 before sterilization.

[0012] Specifically, in the preparation method of the phenylacetic acid ester compound, in step (2), the components of the fermentation culture medium include: peptone 0.1-0.3wt%, molasses 0.4-0.6wt%, sucrose 1.5-2.5wt%, MgSO4·7H2O 0.01-0.03wt%, FeSO4·7H2O 0.005-0.02wt%, KI 0.04-0.07wt%, sea salt 1.5-1.8wt%, CaCO3 0.4-0.6wt%, prepared with pure water, and adjusted to pH 7.0-8.0; Preferably, the fermentation culture step is carried out at a temperature of 25-35℃ for 4-6 days.

[0013] Preferably, the fermentation medium comprises: 0.2 wt% peptone, 0.5 wt% molasses, 2 wt% sucrose, 0.02 wt% MgSO4·7H2O, 0.01 wt% FeSO4·7H2O, 0.05 wt% KI, 1.5-1.8 wt% sea salt, 0.5 wt% CaCO3, prepared with pure water, and pH 7.5 before sterilization.

[0014] Specifically, the method for preparing the phenylacetic acid ester compound further includes an extraction step of the phenylacetic acid ester compound, specifically comprising: (3) Extraction: The collected fermentation products are separated by solid-liquid separation to obtain fermentation broth and mycelium respectively; the fermentation broth is added to macroporous resin for adsorption, and desorbed by ethanol solution to collect crude extract A; the mycelium is extracted by alcohol solvent, and the extract is collected and concentrated to obtain crude extract B; Preferably, the macroporous resin includes at least one of D101, AB-8, HZ816, Amberlite XAD-4, XAD-8, XAD-16 or HP20 resin; Preferably, the mass ratio of the macroporous resin to the fermentation broth is 1:10 to 1:30.

[0015] Specifically, the method for preparing the phenylacetic acid ester compound further includes a purification step of the phenylacetic acid ester compound, specifically including: (4) Purification: Crude extract A and crude extract B are mixed and added to a normal phase silica gel column for chromatography. Gradient elution is performed with a 100%-0% v / v dichloromethane-methanol solution (elution is performed with a gradient concentration of 1% v / v, and the elution volume is based on the complete elution of the separated substances). The separation and purification process is detected by high performance liquid chromatography. The eluent is collected in segments, and the 30%-60% v / v dichloromethane-methanol solution segment is collected to obtain the eluent containing C-1 and C-2 compounds. Preferably, the mass ratio of the normal-phase silica gel column to the total amount of crude extract A and crude extract B is 1:15-25, and more preferably 1:20.

[0016] Specifically, the method for preparing the phenylacetic acid ester compound further includes a purification step of the phenylacetic acid ester compound, specifically including: (5) Purification: The obtained eluent was prepared by preparative C18 reversed-phase high-performance liquid chromatography and isocratic elution with 65% methanol-water by volume. Substances with different retention times were collected to obtain pure products containing phenylacetic acid ester compounds C-1 (tR 12.5 min) and C-2 (tR 14.3 min).

[0017] Specifically, in the preparation method of the phenylacetic acid ester compound, step (4) and / or (5) further includes a step of HPLC monitoring using an Ailgent SB C18 (φ4.6*250mm) analytical column; Preferably, the mobile phase system is controlled as 10%-100% v / v methanol-water, eluted with a gradient concentration of 1% v / v, the analysis time is 60 min, the flow rate is 0.5-1.2 ml / min, and the detection wavelength is 254 nm.

[0018] This invention also discloses the use of the phenylacetic acid ester compounds in the preparation of antitumor drugs, wherein the tumor includes human esophageal squamous cell carcinoma, particularly human esophageal squamous cell carcinoma cells KYSE30; wherein, The compound C-1 has inhibitory activity against human esophageal squamous cell carcinoma KYSE30.

[0019] This invention utilizes preserved marine wart spores ( Verrucosispora gifhornensis. Two phenylacetic acid ester compounds with antitumor potential were isolated and screened from FIM06-0036, and a method for extracting and isolating these compounds from the fermentation broth of this *Verticillium* strain FIM06-0036 was provided. The phenylacetic acid ester compounds described in this invention provide potential compounds for the research and development of new antitumor drugs and are of significant value for the development and utilization of China's marine drug resources.

[0020] This invention also provides a marine warts strain ( Verrucosispora gifhornensis.This invention discloses a method for culturing FIM06-0036 and obtaining the fermentation broth, and a method for extracting and isolating a series of phenylacetic acid ester-containing compounds C-1 and C-2 from the fermentation broth. Compound C-1 exhibits antitumor activity. Activity screening of compounds C-1 and C-2 showed that compound C-1, containing phenylacetic acid esters, possesses certain anti-human esophageal squamous cell carcinoma cells KYSE30. This invention provides potential compounds for the research and development of new antitumor drugs and is of significant value for the development and utilization of China's marine drug resources.

[0021] The C-1 and C-2 substances in this invention contain phenylacetic acid ester compounds. C-1 is a novel structural substance that exhibits activity against human esophageal squamous cell carcinoma cells KYSE30 and can be further studied as a potential anti-tumor drug. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is the hydrogen nuclear magnetic resonance image of compound C-1 of the phenylacetic acid ester in this invention. 1 H spectrum; Figure 2 This is the carbon NMR spectrum of the phenylacetic acid ester compound C-1 in this invention. 13 C spectrum; Figure 3 It is the phenylacetic acid ester compound C-1 in this invention. 1 H- 1 HCOSY diagram; Figure 4 This is the HSQC correlation spectrum of phenylacetic acid ester compound C-1 in this invention; Figure 5 This is the HMBC correlation spectrum of phenylacetic acid ester compound C-1 in this invention; Figure 6 This is the hydrogen nuclear magnetic resonance image of phenylacetic acid ester compound C-2 in this invention. 1 H spectrum; Figure 7 This is the carbon NMR spectrum of the phenylacetic acid ester compound C-2 in this invention. 13 C spectrum. Detailed Implementation

[0023] In the following embodiments of the present invention, a phenylacetic acid ester compound derived from marine verrucous spores is provided, including compound C-1 having the structure shown in formula (I) and compound C-2 having the structure shown in formula (II): .

[0024] The phenylacetic acid ester compounds C-1 and C-2 provided by this invention have been verified to have biological activity. Compound C-1 has anti-human esophageal squamous cell carcinoma cell line KYSE30. This invention provides a promising compound for the research and development of new anti-tumor active drugs and is of great value for the development and utilization of China's marine drug resources.

[0025] In the following embodiments of the present invention, a method for preparing the phenylacetic acid ester compound as described above is also disclosed, comprising the step of inoculating marine wart spore strain FIM06-0036 into a suitable fermentation medium for fermentation culture; The marine wart spore strain FIM06-0036 is classified as *Warlothorax*. Verrucosispora gifhornensis It was deposited on September 28, 2018 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 16548.

[0026] Specifically, the preparation method of the phenylacetic acid ester compound includes the following steps: (1) Seed culture: The preserved marine wart spore strain FIM06-0036 was inoculated into seed culture medium for seed culture, and the seed culture was collected for later use; (2) Fermentation broth culture: The seed liquid is transferred to a fermentation medium for fermentation culture to obtain a fermentation product containing the desired phenylacetic acid ester compounds; (3) Extraction: The collected fermentation products are separated by solid-liquid separation to obtain fermentation broth and mycelium respectively; the fermentation broth is added to macroporous resin for adsorption, and desorbed by ethanol solution to collect crude extract A; the mycelium is extracted by alcohol solvent, and the extract is collected and concentrated to obtain crude extract B; (4) Purification: Mix crude extract A and crude extract B, mix them with normal phase silica gel, and perform normal phase silica gel column chromatography at a mass ratio of 1:15-25. Use a gradient elution with a 100%-0% volume ratio of dichloromethane-methanol solution. The separation and purification process is detected by high performance liquid chromatography. Collect the eluent in segments, and collect the eluent containing C-1 and C-2 compounds in 30%-60% segments. (5) Purification: The obtained eluent was prepared by preparative C18 reversed-phase high-performance liquid chromatography and isocratic elution with 65% methanol-water by volume. Substances with different retention times were collected to obtain pure products containing phenylacetic acid ester compounds C-1 (tR 12.5 min) and C-2 (tR 14.3 min).

[0027] In some specific embodiments, the present invention, as shown in the following embodiments, also provides a method using marine verrucous spores (… Verrucosispora gifhornensis.The preparation method of the phenylacetic acid ester compound produced by FIM06-0036 specifically includes the following steps: (1) Seed culture: Marine verrucosa spp. Verrucosispora sp FIM06-0036 was inoculated onto ISP2 lipid slant culture and then inoculated into liquid seed medium at 25-35℃ for 1-3 days. The seed culture medium is composed of (wt%): 1.2-1.8wt% soluble starch, 0.4-0.6wt% yeast extract, 0.4-0.6wt% peptone, 0.4-0.6wt% glucose, 0.04-0.06wt% K₂HPO₄, 0.04-0.06wt% MgSO₄·7H₂O, 0.04-0.06wt% (NH₄)₂SO₄, 1.5-1.8wt% sea salt, and 0.08-0.12g / L CaCO₃, prepared with pure water and adjusted to pH 7.0-8.0. (2) Fermentation broth culture: Transfer the collected seed liquid to the fermentation medium, culture at 25-35℃ with shaking for 4-6 days, and then use it. The fermentation medium in this embodiment consists of the following components (wt%): peptone 0.1-0.3wt%, molasses 0.4-0.6wt%, sucrose 1.5-2.5wt%, MgSO4·7H2O 0.01-0.03wt%, FeSO4·7H2O 0.005-0.02wt%, KI 0.04-0.07wt%, sea salt 1.5-1.8wt%, CaCO3 0.4-0.6wt%, prepared with pure water, and adjusted to pH 7.0-8.0. (3) Extraction: The collected fermentation broth and mycelium are separated into solid and liquid components. The fermentation broth is adsorbed with macroporous resin (including D101, AB-8, HZ816, Amberlite XAD-4, XAD-8, XAD-16, HP20, etc., but not limited to the above resin types). After adsorption, the crude extract A is obtained by desorption and concentration with ethanol. The mycelium is extracted with ethanol or methanol, and the soaking solution is concentrated under reduced pressure to obtain crude extract B. (4) Purification: After mixing the crude extract with normal-phase silica gel, normal-phase silica gel column chromatography was performed at a mass ratio of 1:20. Gradient elution was performed with dichloromethane-methanol solution at a volume ratio of 100%-0% and a gradient concentration of 1% v / v. The elution volume was based on the complete separation of the substances. The separation and purification process was detected by high performance liquid chromatography. Specifically, an Ailgent SB C18 (φ4.6×250mm) analytical column was used for HPLC monitoring. The mobile phase system was 10-100% methanol-water, and elution was performed with a gradient concentration of 1% v / v. The analysis time was 60 min, the flow rate was 0.5-1.2 ml / min, and the detection wavelength was 254 nm. The eluent containing C-1 and C-2 series substances was collected. (5) Purification: The obtained eluent was prepared by preparative C18 reversed-phase high-performance liquid chromatography and isocratic elution with 65% methanol-water by volume. Substances with different retention times were collected to obtain pure products containing phenylacetic acid ester compounds C-1 (tR 12.5 min) and C-2 (tR 14.3 min).

[0028] In the following embodiments of the present invention, the strain FIM06-0036 was identified by 16S rRNA gene analysis as belonging to the genus Verrucocephala within the actinomycetes, and was classified as Verrucocephala. Verrucosispora gifhornensis It was deposited on September 28, 2018 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.16548, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] This embodiment uses a preserved strain of marine verrucous (Varicospora spp.) Verrucosispora sp. A novel phenylacetic acid ester compound, C-1, and C-2, was extracted and isolated from the fermentation broth of FIM06-0036. Example 1

[0030] Preserved wart fungus Verrucosispora sp FIM06-0036 ISP2 agar slant culture was inoculated into liquid seed culture medium and cultured at 32℃ for 2 days to obtain seed solution.

[0031] The liquid seed culture medium used in this embodiment has the following composition (wt%): soluble starch 1.5wt%, yeast extract 0.5wt%, peptone 0.5wt%, glucose 0.5wt%, K₂HPO₄ 0.05wt%, MgSO₄·7H₂O 0.05wt%, (NH₃)₂ 4） 0.04-0.06 wt% SO42-, 1.65 wt% sea salt, 0.1 g / L CaCO3, prepared with pure water, pH 7.5 before sterilization, sterilized at 121℃ for 30 min, ready for use.

[0032] The obtained seed liquid was mixed with the prepared fermentation medium at a volume ratio of 1:10, and fermented by shaking culture at 28℃ for 5 days to obtain the fermentation product.

[0033] The fermentation medium used in this embodiment has the following composition (wt%): peptone 0.2wt%, molasses 0.5wt%, sucrose 2wt%, MgSO4·7H2O 0.02wt%, FeSO4·7H2O 0.01wt%, KI 0.05wt%, sea salt 1.5-1.8wt%, CaCO3 0.5wt%, prepared with pure water, pH 7.5 before sterilization, sterilized at 121℃ for 30 min, and ready for use. Example 2

[0034] The fermentation products obtained in Example 1 were collected, and the fermentation broth and mycelium were collected separately after filtration.

[0035] At a mass ratio of 1:20, macroporous resin HP20 was mixed with fermentation broth, and the mixture was then loaded onto a resin column for adsorption. After adsorption, the mixture was washed with distilled water and then desorbed with 100% ethanol. The crude extract A was obtained after recovering the ethanol solvent. The mycelium was extracted with ethanol three times, and the soaking solution was concentrated under reduced pressure to obtain crude extract B.

[0036] Crude extracts A and B were combined and mixed with normal-phase silica gel. The mixture was then added to a normal-phase silica gel column at a mass ratio of 1:20 for normal-phase silica gel column chromatography. Gradient elution was performed using a 100%–0% (v / v) dichloromethane-methanol solution at a 1% v / v gradient concentration. The elution volume was determined based on complete elution of the substances. The separation and purification process was monitored using high-performance liquid chromatography (HPLC), specifically an Ailgent SB C18 (φ4.6×250 mm) column. The solvent system was 10%–100% v / v methanol-water, eluted at a 1% v / v gradient concentration, with an analysis time of 60 min, a flow rate of 1.0 ml / min, and a detection wavelength of 254 nm. The 30%–60% v / v methanol-water eluent was collected in fractions to obtain eluents containing C-1 and C-2 series substances.

[0037] The resulting eluent was prepared by preparative C18 reversed-phase high-performance liquid chromatography, and isocratic elution was performed with 65% methanol-water (v / v). Substances with different retention times were collected to obtain a series of substances containing phenylacetic acid esters, namely C-1 (tR 12.5 min) and C-2 (tR 14.3 min). Pure products of phenylacetic acid ester compounds C-1 and C-2 with antitumor potential activity were obtained. Example 3

[0038] This embodiment focuses on the structural analysis of the isolated compounds C-1 and C-2.

[0039] The structures of phenylacetic acid ester compounds C-1 and C-2 were identified by MS and NMR techniques. C-1 and C-2 have the following characteristics.

[0040] Compound C-1 is a pale yellow oil with the molecular formula C. 12 H 16 O3, High-resolution mass spectrometry: Measured values m / z 207.0633 [M+Na]+, theoretical value m / z 207.0628 [M+Na]+, soluble in organic solvents such as methanol, acetonitrile, ethyl acetate and dimethyl sulfoxide.

[0041] Compound C-2 is a pale yellow oil with the molecular formula C. 12 H 16 O3, high-resolution mass spectrometry: measured value m / z 221.0786 [M+Na]+, theoretical value m / z 221.0784 [M+Na]+, soluble in organic solvents such as methanol, acetonitrile, ethyl acetate and dimethyl sulfoxide.

[0042] Compound C-1 1 H nuclear magnetic resonance spectroscopy: 1 H NMR (600 MHz, DMSO- D 6) δ 7.28 (t, J = 7.4Hz, 2H), 7.22 (dd, J = 12.4, 7.2 Hz, 3H), 4.72 (d, J = 5.1 Hz, 1H), 4.57 (qd, J =6.4, 5.0 Hz, 1H), 3.60 (s, 2H), 3.55 (h, J = 6.4 Hz, 1H), 1.07 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H).

[0043] Compound C-1 13 C nuclear magnetic resonance spectroscopy: 13 C NMR (150 MHz, DMSO- D 6) δ 171.21, 135.07, 129.81, 128.85, 127.28, 74.97, 68.32, 41.18, 19.46, 15.87.

[0044] Compound C-2 1 H nuclear magnetic resonance spectroscopy: 1 H NMR (600 MHz, DMSO- D 6) δ 7.30 – 7.25 (m,2H), 7.24 – 7.20 (m, 3H), 4.69 – 4.60 (m, 1H), 3.61 (s, 2H), 3.58 (dd, J = 6.4, 5.2 Hz, 1H), 1.05 (d, J = 6.5 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H).

[0045] Compound C-2 13 C nuclear magnetic resonance spectroscopy: 13 C NMR (150 MHz, DMSO- D 6) δ 171.28, 135.12, 129.80, 128.82, 127.25, 74.47, 67.78, 41.13, 18.85, 15.63.

[0046] In addition, the present invention also determined multiple NMR spectra of the C-1 and C-2 of this compound, as shown in the appendix. Figures 1-7 The relevant test data are shown in Tables 1-2 below.

[0047] Table 1. Compounds C-1 1 H and 13 C(DMSO- d 6) Ownership

[0048] Table 2. C-2 compounds 1 H and 13 C(DMSO- d 6) Ownership

[0049] This determined the assignment of all carbon and hydrogen atoms at C-1 and C-2, as well as the chemical structure of the compound, identifying it as a novel phenylacetic acid ester compound with the following structural formula: ; .

[0050] Example 4 This embodiment is based on the bioactivity test of the isolated phenylacetic acid ester compound C-1, and the results are shown in Table 3 below.

[0051] Table 3. Cancer cell inhibition rate at 20 µM concentration of the compound

[0052] It is evident that compound C-1 has a certain inhibitory effect on human esophageal squamous cell carcinoma KYSE30, and it has potential applications in resisting multiple cancer cell types.

[0053] In summary, this embodiment screened the anticancer activity of phenylacetic acid ester compound C-1, and the results showed that compound C-1 has a certain inhibitory effect on human esophageal squamous cell carcinoma cells KYSE30. Therefore, phenylacetic acid ester compound C-1 is expected to be a potential antitumor active substance and is a compound with potential antitumor activity.

[0054] It should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described in this application.

Claims

1. A phenylacetic acid ester compound, characterized in that, Including compounds having the following formula (P): ； The configuration of position C-9 is either R-type or S-type.

2. The phenylacetic acid ester compound according to claim 1, characterized in that, The compounds include compound C-1 having the structure shown in formula (I) and compound C-2 having the structure shown in formula (II); 。 3. A method for preparing a phenylacetic acid ester compound as described in claim 1 or 2, characterized in that, This includes the step of inoculating marine wart spore strain FIM06-0036 into a suitable fermentation medium for fermentation culture; The marine wart spore strain FIM06-0036 is classified as follows: Verrucosispora gifhornensis It was deposited on September 28, 2018 at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo.16548.

4. The method for preparing the phenylacetic acid ester compound according to claim 3, characterized in that, Includes the following steps: (1) Seed culture: The preserved marine wart spore strain FIM06-0036 was inoculated into seed culture medium for seed culture, and the seed culture was collected for later use; (2) Fermentation broth culture: The seed liquid is transferred to the fermentation medium for fermentation culture to obtain the fermentation product containing the desired phenylacetic acid ester compounds.

5. The method for preparing the phenylacetic acid ester compound according to claim 4, characterized in that, In step (1), the seed culture medium comprises: 1.2-1.8 wt% soluble starch, 0.4-0.6 wt% yeast extract, 0.4-0.6 wt% peptone, 0.4-0.6 wt% glucose, 0.04-0.06 wt% K2HPO4, 0.04-0.06 wt% MgSO4·7H2O, 0.04-0.06 wt% (NH4)2SO4, 1.5-1.8 wt% sea salt, 0.08-0.12 g / L CaCO3, and pH 7.0-8.

0. Preferably, the temperature for the seed culture step is 25-35℃, and the culture time is 1-3 days.

6. The method for preparing the phenylacetic acid ester compound according to claim 4 or 5, characterized in that, In step (2), the fermentation medium comprises: 0.1-0.3 wt% peptone, 0.4-0.6 wt% molasses, 1.5-2.5 wt% sucrose, 0.01-0.03 wt% MgSO4·7H2O, 0.005-0.02 wt% FeSO4·7H2O, 0.04-0.07 wt% KI, 1.5-1.8 wt% sea salt, 0.4-0.6 wt% CaCO3, and pH 7.0-8.

0. Preferably, the fermentation culture step is carried out at a temperature of 25-35℃ for 4-6 days.

7. The method for preparing the phenylacetic acid ester compound according to any one of claims 3-6, characterized in that, The method further includes a step of extracting the phenylacetic acid ester compound, specifically including: (3) Extraction: The collected fermentation products are separated by solid-liquid separation to obtain fermentation broth and mycelium respectively; the fermentation broth is added to macroporous resin for adsorption, and desorbed by ethanol solution to collect crude extract A; the mycelium is extracted by alcohol solvent, and the extract is collected and concentrated to obtain crude extract B; Preferably, the macroporous resin includes at least one of D101, AB-8, HZ816, Amberlite XAD-4, XAD-8, XAD-16 or HP20 resin; Preferably, the mass ratio of the macroporous resin to the fermentation broth is 1:10 to 1:

30.

8. The method for preparing the phenylacetic acid ester compound according to claim 7, characterized in that, The method further includes a purification step of the phenylacetic acid ester compound, specifically including: (4) Purification: Crude extract A and crude extract B are mixed and added to a normal phase silica gel column for chromatography. Gradient elution is performed with a 100%-0% (v / v) dichloromethane-methanol solution. During the separation and purification process, high performance liquid chromatography is used for detection. The eluent containing C-1 and C-2 compounds in the 30-60% v / v range is collected. Preferably, the mass ratio of the normal-phase silica gel column to the total amount of crude extract A and crude extract B is 1:15-25, and more preferably 1:

20.

9. The method for preparing the phenylacetic acid ester compound according to claim 8, characterized in that, The method further includes a purification step of the phenylacetic acid ester compound, specifically including: (5) Purification: The obtained eluent was prepared by preparative C18 reversed-phase high-performance liquid chromatography and isocratic elution with 65% methanol-water solution by volume. Substances with different retention times were collected to obtain pure products containing phenylacetic acid ester compounds C-1 and C-2.

10. The use of the phenylacetic acid ester compound according to claim 1 or 2 in the preparation of an antitumor drug, characterized in that, The tumors include human esophageal squamous cell carcinoma; Preferably, compound C-1 has inhibitory activity against human esophageal squamous cell carcinoma KYSE30.

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