RESUMEN
Ten new (1-10) and nine known (11-19) austocystins, along with four known anthraquinones (20-23), were isolated from the culture of Aspergillus ustus NRRL 5856 by bioactivity-guided fractionation. The structures of the new compounds were elucidated by spectroscopic data analysis, X-ray crystallographic study, the modified Mosher's method, [Rh2(OCOCF3)4]-induced ECD spectral analysis, and comparison of the experimental ECD spectra with those of the similar analogues. Compounds 1-8 represent the first examples of austocystins with a C-4' oxygenated substitution. The absolute configuration of 1â³-hydroxy austocystin D (11) was determined by single-crystal X-ray diffraction and consideration of its biosynthetic origin. Compounds 5, 9, and 11 exhibited significant inhibitory effects against the proliferation of ConA-induced T cells with IC50 values of 1.1, 1.0, and 0.93 µM, respectively. Furthermore, these compounds suppressed the expression of IL-6 in a dose-dependent manner. Compounds 10-12 and 14 showed pronounced cytotoxicities against MCF-7 with IC50 values of 3.9, 1.3, 0.46, and 2.3 µM, respectively.
Asunto(s)
Aspergillus , Inmunosupresores , Aspergillus/química , Humanos , Inmunosupresores/farmacología , Inmunosupresores/química , Inmunosupresores/aislamiento & purificación , Estructura Molecular , Cristalografía por Rayos X , Interleucina-6/metabolismo , Antraquinonas/farmacología , Antraquinonas/química , Animales , Ensayos de Selección de Medicamentos Antitumorales , Linfocitos T/efectos de los fármacos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Proliferación Celular/efectos de los fármacosRESUMEN
Twelve new austalide meroterpenoids (1-12) were isolated from the endophytic fungus Diaporthe sp. XC1211. Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1, 3, 4, and 6 were established by single-crystal X-ray diffraction, whereas those for the others were established by experimental electronic circular dichroism (ECD) data analysis. Compounds 1-12 represent a rare class of austalides with a 24α-CH3. Compounds 2 and 5 demonstrated potent proliferation inhibitory effects against LPS-induced B cells with IC50 values of 6.7 (SI = 3.6) and 3.8 (SI > 13) µM, respectively. Compounds 2 and 5 decreased the secretion of IL-6 in LPS-induced B cells in a dose-dependent manner.
Asunto(s)
Hongos , Lipopolisacáridos , Estructura Molecular , Lipopolisacáridos/farmacología , Cristalografía por Rayos X , Dicroismo CircularRESUMEN
Peniandranoids A-E (1-5), five new meroterpenoids, together with three known analogues (6-8), were isolated from the fermentation of a soil-derived fungus, Penicillium sp.sb62. Their structures including absolute configurations were determined by extensive spectroscopic analysis, and the absolute configurations of compounds 1 and 5 were further elucidated by single-crystal X-ray diffraction. Peniandranoids A-E belong to a rare class of andrastin-type meroterpenoids incorporating an extra polyketide unit (a C10 polyketide unit for 1 and 2, a C9 polyketide unit for 3 and 4, and a furancarboxylic acid unit for 5). Compounds 1 and 6 exhibited favorable inhibitory activities against influenza virus A (H1N1) with EC50 values of 19 and 14 µg/mL, respectively. Compounds 3-8 exhibited potent immunosuppressive activities against concanavalin A-induced T cell proliferation with EC50 values ranging from 4.3 to 27 µM and lipopolysaccharide-induced B cell proliferation with EC50 values ranging from 7.5 to 23 µM, respectively.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Penicillium , Policétidos , Estructura Molecular , Penicillium/química , Antivirales/farmacología , Antivirales/químicaRESUMEN
Twelve new hypothemycin-type resorcylic acid lactones, three 10-membered (1-3) and nine 14-membered (4-12), together with seven known analogues (13-19), were obtained from the solid rice-based culture of Podospora sp. G214. Their structures were elucidated utilizing spectroscopic analysis, and the absolute configurations were determined by modified Mosher's method, Mo2(OAc)4-induced electronic circular dichroism experiments, and single-crystal X-ray diffraction. Compounds 1, 5, 10, and 12-19 exhibited potent immunosuppressive activities against concanavalin A-induced T cell proliferation with IC50 values ranging from 6.0 to 25.1 µM and lipopolysaccharide-induced B cell proliferation with IC50 values ranging from 6.2 to 29.1 µM. Further studies revealed that 1 induced apoptosis in activated T cells through the JNK-mediated mitochondrial pathway.
Asunto(s)
Linfocitos B/efectos de los fármacos , Inmunosupresores/farmacología , Lactonas/farmacología , Podospora/química , Linfocitos T/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , China , Inmunosupresores/aislamiento & purificación , Lactonas/aislamiento & purificación , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Raíces de Plantas/microbiología , Sanguisorba/microbiología , Bazo/citología , Zearalenona/análogos & derivados , Zearalenona/aislamiento & purificación , Zearalenona/farmacologíaRESUMEN
Twelve new [11]-chaetoglobosins, chaetopseudeurins A-L (1-12), were identified from the fermentation of the endophytic fungus Pseudeurotium bakeri P1-1-1. Their structures with absolute configurations were elucidated by detailed interpretation of NMR spectroscopy, mass spectrometry, and single-crystal X-ray diffraction. Compounds 2 and 5-7 exhibited significant cytotoxicities against seven human cancer cell lines, with IC50 values ranging from 4.7 ± 1.0 to 12.2 ± 0.7 µM, and induced G2/M cell cycle arrest and apoptosis in MCF-7 and A427 cells dose dependently. Western blot analysis showed that compound 2 induced apoptosis of MCF-7 cells via the Bcl-2/caspase-3/PARP pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Ascomicetos/química , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Alcaloides Indólicos/farmacología , Línea Celular Tumoral , China , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Alcaloides Indólicos/aislamiento & purificación , Células MCF-7 , Estructura MolecularRESUMEN
Ten novel (1, 2, 3a, 3b, 4a, 4b, 5a, 5b, 6a, and 6b) furancarboxylic acids including four pairs of epimers (3a, 3b; 4a, 4b; 5a, 5b; 6a, 6b), together with seven known analogues (7a, 7b, 8a, 8b, 9a, 9b, and 10), were isolated from the fermentation of the soil-derived fungus Penicillium sp. sb62. Their structures were established on the basis of spectroscopic data analysis, and the absolute configurations were determined by time-dependent density functional theory electronic circular dichroism calculations, comparison of the specific optical rotation values, and modified Mosher's method. Compounds 1-4 represent the first class of natural furancarboxylic acids featuring a thiophene moiety. Compounds 1-7 showed antimicrobial inhibitory activities against Escherichia coli, Staphylococcus aureus, and Candida albicans with MIC values ranging from 0.9 to 7.0 µg/mL, from 1.7 to 3.5 µg/mL, and from 3.3 to 7.0 µg/mL, respectively.
Asunto(s)
Antiinfecciosos/aislamiento & purificación , Ácidos Carboxílicos/aislamiento & purificación , Penicillium/química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Candida albicans/efectos de los fármacos , Ácidos Carboxílicos/química , Escherichia coli/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Staphylococcus aureus/efectos de los fármacosRESUMEN
Twelve new resorcylic acid lactones (RALs) including three new 16-membered RALs (1a, 1b and 2), eight new 14-membered RALs (3-10), and one new 12-membered RAL (11), along with five known 14-membered RALs (12-16), were identified from the fermentation of the soil-derived fungus Ilyonectria sp. sb65. Their structures were established by detailed analyses of 1D and 2D NMR, HRESIMS, and X-ray diffraction crystallography. All new compounds were evaluated for their cytotoxic effects against three human cancer cell lines, along with their potential as TRAIL sensitizers in TRAIL-resistant A549 human lung adenocarcinoma cells and their in vitro immunosuppressive effects against ConA-induced T-cell and LPS-induced B-cell proliferation.
Asunto(s)
Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Hypocreales/química , Lactonas/química , Lactonas/farmacología , Resorcinoles/química , Animales , Línea Celular Tumoral , Cristalografía por Rayos X , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Fermentación , Células HeLa , Humanos , Inmunosupresores/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Resorcinoles/farmacologíaRESUMEN
Parasubindoles A-G (1-7), seven eremophilanyl indoles with an unprecedented 12 H-cyclopentane[ b]naphthalenespiro-1,3'-indole skeleton, were isolated from the whole plant of Parasenecio albus. Their structures with absolute configurations were elucidated by spectroscopic methods, single-crystal X-ray diffraction, and ECD analyses. Plausible biosynthetic pathways of 1-7 were postulated.
Asunto(s)
Asteraceae/química , Indoles/química , Asteraceae/metabolismo , Indoles/metabolismo , Modelos Moleculares , Conformación Molecular , EstereoisomerismoRESUMEN
Ten new alkaloids (1-10), including two pairs of enantiomeric mixtures (5a,b and 6a,b), and 15 known analogues (11-25) were obtained from the bark of Pausinystalia yohimbe. The structures of 1-25 were established by spectroscopic methods, and the absolute configurations of compounds 1-10 were resolved by X-ray diffraction and ECD data analyses. The in vitro immunosuppressive activities of selected isolates were tested. Compounds 11 and 16 exhibited moderate inhibition with IC50 values of 16.8 and 27.6 µM against ConA-induced T lymphocyte proliferation and 13.5 and 40.5 µM against LPS-induced B lymphocyte proliferation, respectively.
Asunto(s)
Alcaloides/química , Alcaloides/farmacología , Inmunosupresores/química , Inmunosupresores/farmacología , Pausinystalia/química , Corteza de la Planta/química , Animales , Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dicroismo Circular , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Difracción de Rayos XRESUMEN
Nine new lignans (1-9) and ten known analogues (10-19) were isolated from the fruits of Schisandra bicolor var. tuberculata. The structures of compounds 1-9 were established on the basis of spectroscopic data analysis. The absolute configuration of compound 1 was determined by single-crystal X-ray diffraction analysis with Cu Kα irradiation techniques, and the absolute configurations of compounds 2-9 were deduced by comparing their experimental ECD spectra and optical rotations with those of compound 1 or similar compounds. All isolates were evaluated for their neuroprotective activities against CoCl2, H2O2, and Aß25-35-induced SH-SY5Y cell injury, and were found to exhibit different degrees of neuroprotective effects. At a low concentration of 3.2 nM, compounds 3, 8, 9, and 14-19 in CoCl2-induced, compounds 7, 8, 13, 17, and 18 in H2O2-induced, and compounds 2, 6, 7, 9, 10, and 12-19 in Aß25-35-induced SH-SY5Y cell injury models, showed statistically significant neuroprotective activities, when compared with each negative control group.
Asunto(s)
Frutas/química , Lignanos/aislamiento & purificación , Lignanos/farmacología , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/farmacología , Schisandra/química , Péptidos beta-Amiloides/efectos de los fármacos , Peróxido de Hidrógeno/análisis , Lignanos/química , Conformación Molecular , Estructura Molecular , Fármacos Neuroprotectores/química , Fragmentos de Péptidos/efectos de los fármacos , Difracción de Rayos XRESUMEN
Twenty-two new highly oxygenated bisabolane-type sesquiterpenoids, pararubin A-V (1-22), were isolated from the whole plant of Parasenecio rubescens. The structure determination of 1-22, including the assignment of their relative configurations, was accomplished by extensive 1D and 2D NMR spectroscopy. The absolute configuration of pararubin A (1) was established by single-crystal X-ray crystallographic analysis. The isolated compounds were evaluated for their cytotoxic effects against three cancer cell lines (B16 mouse melanoma cells, HepG2 human hepatocellular carcinoma cells, and MCF7 human breast adenocarcinoma cells) and for their antimicrobial effects against Staphylococcus aureus, Escherichia coli, and Monilia albicans.
Asunto(s)
Asteraceae/química , Medicamentos Herbarios Chinos/aislamiento & purificación , Sesquiterpenos/aislamiento & purificación , Animales , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/farmacología , Escherichia coli/efectos de los fármacos , Células Hep G2 , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Sesquiterpenos/química , Sesquiterpenos/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
Two rare 7,8-seco-lignans (1, 2), three new lignan glycosides (3, 4a, 4b), and 10 known lignans (5-14) were isolated from the fruit of Schisandra glaucescens Diels. The absolute configurations of 1 and 2 were determined by comparing their experimental and calculated electronic circular dichroism spectra. The molecular structures of the new compounds (3, 4a, and 4b), including their absolute configurations, were determined using various spectroscopic methods and hydrolysis reactions. The antioxidant activities of the isolated compounds were tested using 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays. Compounds 4, 7, 8, 10, 11, and 12 exhibited antioxidant activities of varying potential in both assays. Of these compounds, 7 showed the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, with IC50 values of 15.7 (150 µM DPPH) and 34.6 µM (300 µM DPPH), respectively, and 4, 12, and 7 displayed higher total antioxidant activities than Trolox in the ferric reducing antioxidant power assay. The neuroprotective effects of these compounds against Aß25-35-induced cell death in SH-SY5Y cells were also investigated. Compounds 1, 2, 6, 7, 8, 11, and 12 exhibited statistically significant neuroprotective effects against Aß25-35-induced SH-SY5Y cell death compared with the group treated only with Aß25-35.
Asunto(s)
Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Medicamentos Herbarios Chinos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Glicósidos/aislamiento & purificación , Glicósidos/farmacología , Lignanos/aislamiento & purificación , Lignanos/farmacología , Fármacos Neuroprotectores/aislamiento & purificación , Fármacos Neuroprotectores/farmacología , Schisandra/química , Péptidos beta-Amiloides/farmacología , Antioxidantes/química , Compuestos de Bifenilo/farmacología , Cromanos , Medicamentos Herbarios Chinos/química , Frutas/química , Glicósidos/química , Concentración 50 Inhibidora , Lignanos/química , Estructura Molecular , Fármacos Neuroprotectores/química , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Fragmentos de Péptidos/farmacología , Picratos/farmacología , Extractos Vegetales/químicaRESUMEN
Two new alkaloids, lycosprenine (1) and 2α-methoxy-6-O-methyllycorenine (2), along with 22 known alkaloids (3-23b), were isolated from the bulb of Lycoris sprengeri. Their structures were elucidated on the basis of spectral analysis and by comparison of the spectroscopic data with those of known compounds. Selected compounds (1-3 and 6-9) were tested for their neuroprotective activities against H2O2-, CoCl2- and Aß25-35-induced SH-SY5Y cell injury, most of which exhibited neuroprotective effects of different degrees.
Asunto(s)
Alcaloides/aislamiento & purificación , Lycoris/química , Fármacos Neuroprotectores/aislamiento & purificación , Alcaloides/química , Alcaloides/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Estructura Molecular , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Raíces de Plantas/químicaRESUMEN
In order to discover anticancer agents from natural sources, an ethanol-soluble extract of the root bark of Juglans cathayensis was investigated and showed cytotoxic effects against various human cancer cell lines. A subsequent phytochemical study on the EtOAc-soluble fraction determined 2-methoxyjuglone (1) as one of the main active constituents. Compound 1 was shown to be cytotoxic against HepG2 cells. Morphological features of apoptosis were observed in 1-treated HepG2 cells, including cell shrinkage, membrane blebbing, nuclear condensation, and apoptotic body formation. Cell cycle analysis with propidium iodide staining showed that 1 induced cell cycle arrest at the S phase in HepG2 cells. Flow cytometric analysis with annexin V and propidium iodide staining demonstrated that 1 induced HepG2 cell apoptotic events in a dose-dependent manner (0-8 µg/mL). Western blot analysis of apoptosis-related proteins revealed that 1 induces HepG2 cell apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway (intrinsic pathway). An in vivo experiment using tumor-bearing mice showed that treatment with 1 at 0.5 and 1.0 mg/kg per day decreased the tumor mass by 56% and 67%, respectively.
Asunto(s)
Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Juglans/química , Naftoquinonas/aislamiento & purificación , Naftoquinonas/farmacología , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células Hep G2 , Humanos , Ratones , Naftoquinonas/química , Fase S/efectos de los fármacosRESUMEN
Three new ursane-type triterpenoids, 2α,3ß-dihydroxy-11α,12α-epoxy-urs-28,13ß-olide (1), 2α,3ß,24-trihydroxy-11α,12α-epoxy-urs-28,13ß-olide (2), and 2α,3α,24-trihydroxy-11,20(30)-dien-urs-28,13ß-olide (6), together with six known ursane-type triterpenoids (3-5, 7-9), were isolated from the EtOAc extract of the aerial parts of Isodon excisoides. Their structures were elucidated on the basis of 1D NMR and 2D NMR analyses as well as HRMS experiments.
Asunto(s)
Medicamentos Herbarios Chinos/aislamiento & purificación , Isodon/química , Triterpenos/aislamiento & purificación , Medicamentos Herbarios Chinos/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Triterpenos/químicaRESUMEN
Four undescribed neolignan glycosides, bletineosides A-D (1-4) were isolated from the pseudobulbs of Bletilla striata. Their structures with absolute configurations were elucidated on the basis of spectroscopic analyses, along with acidic hydrolysis reactions and ECD experiments. All isolates were evaluated for their neuroprotective activities against glutamate-induced PC12 cell injury. Compound 3 and 4 showed significantly neuroprotective effects at the concentration of 10 µM when compared with the model group. Compounds 1-4 represented the first examples of neolignan glycosides from the genus Bletilla. This study disclosed the potency of Bletilla striata as a new source of anti-neurodegenerative agents.
Asunto(s)
Lignanos , Orchidaceae , Estructura Molecular , Glutamatos , Glicósidos/farmacología , Lignanos/farmacologíaRESUMEN
Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His(8)-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K(M) values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 µM, respectively. Average turnover number (k(cat)) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s(-1). FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.
Asunto(s)
Biología Computacional , Dimetilaliltranstransferasa/genética , Dimetilaliltranstransferasa/metabolismo , Indoles/metabolismo , Neosartorya/enzimología , Neosartorya/genética , Cromosomas Fúngicos/genética , Técnicas de Cultivo , Dimetilaliltranstransferasa/biosíntesis , Dimetilaliltranstransferasa/química , Indenos/metabolismo , Cinética , Familia de Multigenes/genética , Neosartorya/metabolismo , Prenilación , Análisis de Secuencia , Especificidad por SustratoRESUMEN
Two new tetrahydrofuran lignans, schiglaucin A and B (1-2), together with eight known analogues (3-10), were isolated from the stems of Schisandra glaucescens Diels. Their structures were elucidated on the basis of spectroscopic techniques (HRESIMS, UV, IR, NMR, and CD experiments). All of the compounds were tested for their neuroprotective activities against H2O2- and CoCl2-induced cell injuries in SH-SY5Y cells, respectively. Compounds 1-10 showed significant neuroprotective effects against H2O2-induced SH-SY5Y cell death, while compounds 1-5 and 8-10 exhibited significant neuroprotective effects against CoCl2-induced SH-SY5Y cell injury.
Asunto(s)
Lignanos/farmacología , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Schisandra/química , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cobalto , Humanos , Peróxido de Hidrógeno , Estructura Molecular , Neuroblastoma/tratamiento farmacológico , Tallos de la Planta/química , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Phytochemical investigations of the root bark of Juglans cathayensis DODE. led to the isolation of three new naphthalenyl glycosides, Jugnaphthalenoside A-C (1-3). Their structures were elucidated on the basis of extensive analysis of spectroscopic data. The cytotoxicities of the three new compounds were also evaluated.
Asunto(s)
Antineoplásicos/química , Glicósidos/química , Juglans/química , Corteza de la Planta/química , Raíces de Plantas/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
2-Methoxyjuglone, a member of the 1,4-naphthoquinone family, was first obtained through semi-synthesis based on 2-hydroxyjuglone as the precursor in 1966. It has been isolated and identified from different plant species of the Juglandaceae, Sterculiaceae, and Proteaceae families. 2-Methoxyjuglone has been demonstrated to possess a wide range of biological activities, including antitumor, antifungal, and antibacterial activities; in addition, it has been shown to poison fish and inhibit seed germination. These properties make 2-methoxyjuglone a promising bioactive compound for pharmaceutical and agricultural purposes. This review article provides an overview of the current research progress on 2-methoxyjuglone for the first time, with a primary focus on the plant sources, extraction, identification, synthesis, and biological activities associated with this compound for further development.