C.G. Ferraz, et al.
Fitoterapia138(2019)104346
A sequence of reactions as delineated in (Fig. 6) produce produces
The mixture was then diluted with 12.50 mL of distilled water and
cooled in an ice bath. Then, ice cold NaNO2 solution (43%) was added
to the mixture. After vacuum filtration, nine grams of nitro-
somethylurea were obtained. The samples were methylated in an
four- and five-membered rings as shown in intermediate IV. Further
prenylations and intramolecular cyclizations lead to the formation of
burlemarxione A, which is a tautomer of burlemarxione B (Fig. 6).
Fig. 6. Putative biosynthesis pathway of burlemarxione A.
Glioblastoma is an extremely resistant brain tumor due to the in-
herent hurdle for the delivery of chemotherapy agents across the blood
brain barrier into the brain. For these reasons, temozolomide is one of
the few chemotherapy agents applied to treat glioblastoma brain tu-
mors. Nevertheless, patients survival expectations for this type of tumor
is approximately 11 months if they undergo surgery, radiotherapy and
chemotherapy with temozolomide. Cytotoxicity of 1a and sampsonine
N was assessed against GL-15 multiforme glioblastoma-derived human
cell line. Compound 1a showed EC50 of 31.96 μM, whereas EC50 for
sampsonine N was 26.00 μM. Temozolomide as positive control had an
EC50 of 716 μM.
ethereal diazomethane solution recently prepared by a reaction invol-
ving the slow addition of nitrosomethylurea to an aqueous KOH solu-
tion (50%) in sulfur ether in an ice bath The sample to be methylated
was solubilized in CHCl3 and the ethereal ice-cold diazomethane solu-
tion was added gradually until no further gas evolution was observed.
The solvent was evaporated at room temperature and the product was
examined by 1H NMR to ensure completion of the methylation reaction.
3.3. Plant material
C. burle-marxii trunk was collected at Mucugê city (Bahia state,
Brazil). The specimens were identified by Maria Lenise da Silva Guedes,
PhD from the Biology Institute of the Federal University of Bahia,
3. Experimental section
Brazil.
A voucher specimen (ALCB-61584) was deposited in the
3.1. General experimental procedures
Herbarium Alexandre Leal Costa at Federal University of Bahia.
Optical rotations were recorded on a PerkinElmer model 343 po-
larimeter. IR spectra were recorded on Perkin Elmer (FT-IR Spectrum
1000) spectrophotometer. NMR spectra were measured on Varian
Gemini 2000 (INOVA-500) and Bruker spectrometers. The resonances
of residual CHCl3 (7.26 and 77.0 ppm) and benzene (7.20 and
128.0 ppm) were used as internal references for 1H and 13C NMR
spectra acquisition. HRESIMS were recorded on a SHIMADZU LCMS-IT-
TOF.
3.4. Extraction and isolation
Dried powdered trunk (10.0 kg) was extracted with n-hexane three
times by maceration. The extract (121.17 g) was fractionated with
hexane and hexane/EtOAc mixtures in a silica gel column. Fractions 3
and 4 were combined and methylated with diazomethane. The me-
thylation was necessary to improve the chromatographic separations of
mixture whose 1H NMR spectrum showed signals between δ 15 and δ 16
indicating the probable presence of tautomers in keto-enol equilibrium.
The methylated fraction was fractioned with hexane/EtOAc mixtures in
a silica gel CC. From the fraction 5 of this column, after successive
chromatography on silica gel column, and preparative TLC (silica gel;
hexane-EtOAc 9:1) compound 1a (260 mg) was isolated. Fraction 7 was
also submitted to successive chromatography on silica gel column and
one of the fractions was purified with HPLC on silica gel (hexane/AcOEt
85:15) giving the compounds 2a (43.0 mg) and 3a (34 mg). Sampsonine
N (19.0 mg) and obdeltifolione C (7.0 mg) were isolated from fractions
3.2. Methylation reaction
Nitrosomethylurea was initially prepared by slowly stirring an
aqueous solution of NaOH (38.5%), Br2 (22.0 g) and acetamide
(14.75 g). This mixture was heated to 100 °C and then cooled in an ice
bath for two hours. Acetyl methyl urea crystals (12.25 g) were obtained
after vacuum filtration. The acetyl methyl urea crystals were dissolved
in 12.50 mL of concentrated HCl and heated to 100 °C for about 12 min.
6