Design and functionalization of bioactive benzoxazines. An unexpected: Ortho -substitution effect

Article abstract of DOI:10.1039/c8nj06440g

A family of o, p- and N-functionalized dihydro-2H-benzo-1,3-oxazines was synthesized and characterized. In vitro antimicrobial activity of the new benzoxazines was assessed against pathogenic fungi and Gram-negative and Gram-positive bacteria. The screene

Full text of DOI:10.1039/c8nj06440g

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Szafert and J. Ejfler, New J. Chem., 2019, DOI: 10.1039/C8NJ06440G.
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Design and functionalization of bioactive benzoxazines. An
unexpected ortho-substitution effect
Agata Arendt-Pindel,a Aleksandra Marszałek-Harych,a Elżbieta Gębarowska,b Tomasz Gębarowski,c
Dawid Jędrzkiewicz,a Elżbieta Pląskowska,b Dariusz Zalewski,d Nurbey Gulia,a Sławomir Szafert,a
Jolanta Ejfler*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A family of o, p- and N-functionalized dihydro-2H-benzo-1,3-oxazines was synthesized and characterized. In vitro
antimicrobial activity of the new benzoxazines was assessed against pathogenic fungi and Gram-negative and Gram-
positive bacteria. The screened compounds showed significant in vitro antimicrobial effect, but introduction of the bulky
substituent in the ortho position of the aryl ring caused a loss of their antibacterial efficacy. Cytotoxic assay results
revealed that a molecule containing long alkyl chain substituents offered a remarkable viability of mouse fibroblast cells.
Additionally, the liquid nature of alkyl-chain-modified monomers and the high processing windows indicate their high
application potential.
benzoxazines is still of great importance. Here, we report on
the synthesis and characterization of new benzoxazines as
potential antibacterial agents and valuable monomers for
Introduction
A search for new antibacterial compounds is a challenging task
since bacteria continuously develop resistance to such
compounds. Infections due to such bacterial strains are
infrequent but potentially dangerous or fatal, especially for
immunosuppressed patients. Similarly, fungal infections are a
commonly observed complication in the course of many
diseases. Therefore, it is still essential to modify the structural
motif of biologically active compounds or design novel motifs
showing a similar or better activity. Compounds based on the
1,3-benzoxazine architecture show different antimicrobial
properties, e.g. bactericidal or fungicidal, and exhibit a wide
spectrum of pharmacological features, such as antitumor, anti-
tuberculosis, antimalarial, or anthelmintic activity.1 Therefore,
these types of compounds have been an unceasingly
important subject of interdisciplinary investigations. In
addition, N-substituted 3,4-dihydro-2H-1,3-benzoxazines are
monomers to form polybenzoxazines, which have gained
immense attention because of their excellent mechanical and
thermal properties.2 Hence, the search for new functional
polybenzoxazine formation.
Experimental section
General
Solvents for reactions were purified by standard methods: THF
and triethylamine were distilled from Na/benzophenone.
Other solvents (also for workup) were used as received.
Column chromatography was performed on silica gel 60 Å 230-
400 mesh (Macherey Nagel) and Macherey Nagel TLC plates
(silica gel 60 Å, UV 254). All chemicals were obtained from
commercial sources and used without further purification: 4-
tert-buthylphenol (99%), 2,4-di-tert-buthylphenol (99%),
dodecylamine (98%), p-cresol (98%), 4-(2,4,4-trimethylpentan-
2-yl)phenol
(98%),
p-bromophenethylamine
(98%),
formaldehyde (37% solution in H2O) were purchased from
Aldrich. [Pd(PPh3)2]Cl2 (Aldrich, 98%), CuI (Aldrich, 99.9%),
ethynyl(trimethyl)silane (Fluorochem, 97%), K2CO3 (anhydrous,
1
Aldrich, 99%). The H, 13C NMR spectra were obtained using
Bruker Avance 500 MHz spectrometer. The chemical shifts are
given in ppm relative to the residual signals of the solvent
(CDCl3, 1H: 7.26 ppm, 13C: 77.2 ppm). HRMS spectra were
recorded using Bruker MicOTOF-Q spectrometers with ESI ion
source and time-of-flight mass analyzer. IR spectra were
a. Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie, 50-383 Wrocław,
Poland
b. Department of Plant Protection, University of Environmental and Life Science, 24A
pl. Grunwaldzki, 53-363 Wrocław, Poland
c. Department of Basic Medical Science, Wroclaw Medical University, Borowska
211, 50-556 Wrocław, Poland
d. Department of Genetics, Plant Breeding and Seed Production, University of
Environmental and Life Science, pl. Grunwaldzki 24A, 53-363 Wrocław, Poland
*Corresponding author: jolanta.ejfler@chem.uni.wroc.pl
Electronic Supplementary Information (ESI) available: spectroscopic data (1H,
13C{1H} NMR, spectra, X-ray experimental data and refinement, antimicrobial data.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/x0xx00000x
recorded using
a
Bruker 66/s FTIR spectrometer.
Microanalyses were conducted with an Elementar CHNS Vario
EL III analyzer. Melting points were characterized by
differential scanning calorimetry conducted using a Mettler-
Toledo DSC 3 from 30 to 250 C at a heating rate of 10 C/min
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under nitrogen. Thermogravimetric analysis (TGA) was Cells
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Mouse embryo fibroblasts (BALB/3TD3OI:c1l0o.n10e39/AC38N1)J06w44e0rGe
purchased from Sigma-Aldrich.
determined with a SETARAM Setsys TG-DTA 16/18 at a heating
rate of 10 C/min under N2.
X-ray data collection and reduction
Determination of minimum inhibitory concentration (MIC),
minimum bactericidal (MCB) and fungicidal (MFC) concentration
X-ray diffraction data for a suitable monocrystal of the
measured samples were collected using a KUMA KM4 CCD and
Xcalibur CCD Onyx or Ruby (see ESI, Table S1) with  scan
technique. The data collection and processing utilized CrysAlis
suit of programs.3 The space groups were determined based
on systematic absences and intensity statistics. Lorentz
polarization corrections were applied. The structures were
solved by direct methods and refined by full-matrix least-
squares on F2. All calculations were performed using the
SHELXTL-2013 suite of programs.4 All non-hydrogen atoms
were refined with anisotropic displacement parameters.
Hydrogen atom positions were calculated with geometry and
fixed. Thermal ellipsoid plots were prepared with 30%
probability displacements for non-hydrogen atoms using
Mercury 3.1 program.5 All data have been deposited with the
Cambridge Crystallographic Data Centre CCDC-1547956 for 1, -
1547957 for 2, -1547955 for 3, -1835726 for 4, -1860484 for 8,
-1835932 for 12, -1860482 for 13, -1860480 for 15, -1835916
for 16, -1860481 for 20, and -1860483 for 21. Copies of the
data can be obtained free of charge by application to CCDC, 12
Union Road, Cambridge CB21EZ, UK or e-mail:
deposit@ccdc.cam.ac.uk.
The minimum inhibitory concentrations (MIC) were
determined by a serial dilution method in 48-well plates
(Tissue Culture Plates VWR European).6a The bacterial strains
were grown on Mueller-Hinton Agar (MHA, Sigma-Aldrich) at
35±2 C for 24h but plant pathogens of Pectobacterium spp.
were incubated at 28 C. The fungal species were grown on
Sabouraud Dextrose agar (SDA, Oxoid, Thermo Scientific) at
28±2 C for 24 h (yeast) or 48 h (filamentous fungi). Optical
density of bacterial and yeast suspension was standardized to
0.5 McFarland (spectrophotometer VIS-723G, Rayleigh,
Beijing). Suspensions turbidity of the molds were confirmed by
viable counting in a Thoma chamber. The final inoculum size
was 5105 cfucm-3 for the antibacterial assay and 1104
cfucm-3 for the antifungal assay. Compounds were dissolved
in dimethyl sulfoxide (DMSO) and tested at a concentration
range from 400 to 6,25 µgcm-3. Mueller-Hinton Broth (for
bacteria) or Sabouraud Dextrose Broth (for fungi) were used
for serial dilution. Tetracycline (Sigma-Aldrich) and Aphotericin
B (Gibco, Life Technologies) were used as negative controls (at
the dose of 30 µgcm-3) and solvent (DMSO) as positive
control. The microbial growth was visualized by adding 50 µl of
rezasurine aqueous solution on well (pink colour of medium
indicates growth of microorganisms and blue means inhibition
of growth).6b Minimum inhibitory concentration (MIC) was
defined as the lowest concentration of the tested chemical
compounds that inhibited visible growth, while minimum
bactericidal/fungicidal concentration (MBC/MFB) was defined
as the lowest compounds concentration that killed 99% of
microorganisms cells.6c To determine MBC/MFC, cultures were
taken from each well without visible growth and inoculated in
MHA for bacteria or in SDA for yeasts and molds (time and
temperature of incubation as described above). The
experiments were done in 3 series at 3 repetition.
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Biological assay, Antimicrobial screening , Test microorganisms
The compounds 1-8 were tested against
a panel of
microorganisms including Gram-positive cocci: Staphylococcus
aureus PCM 2054, S. pseudintermedius KP-Spi1, Streptococcus
agalactiae KP-Sag1, S. canis KP-Sac1; Gram-positive endospore
forming rods: Bacillus megaterium PCM 1400, B. subtilis PCM
1949, B. cereus PCM 1948, B. mycoides PCM 2024; Gram-
negative rods: Escherichia coli PCM 2057, Pseudomonas
aeruginosa PCM 2058, Salmonella galinarum KP-Sg1, Proteus
vulgaris PCM 542; plant pataogenes: Pectobacterium
carotovora sbp. carotovora IOR (strain 1822 and 1815), P.
atrosepticum IOR (strain 1826 and 1825); yeasts: Candida
krusei KW-F117, C. albicans KP-Ca1, C. glabrata KP-Cg1;
filamentous fungi: Fusarium culmorum KW-F, F. moniliforme
KW-Fm1, Aspergillus niger KW-An1, A. flavus KW-Af1,
Trichoderma harzianum sbp. aggressivum KW-Tha1. The
strains came from the following collections: KW - own
collection (Agricultural Microbiology Lab, Department of Plant
Protection, University of Environmental and Life Sciences,
Wrocław, Poland), KP - Microbiology Lab, Department of
Pathology (University of Environmental and Life Sciences,
Wrocław, Poland), PCM - Polish Collection of Microorganisms
(Institute of Immunology and Experimental Therapy, Polish
The data were subjected to analysis of variance using the
Tukey test (p ˃ 0.05) using STATISTICA 12.0 software for
Windows. All data were presented as the mean values ±
standard deviation (SD).
Preparation of resazurin solution
300 mg resazurin sodium salt (Sigma-Aldrich) was dissolved in
40 ml sterile water. A vortex mixer was used to ensure that it
was a well-dissolved and homogenous solution.6b
Agar disk diffusion assay
Academy of Sciences, Wrocław, Poland), IOR
- Culture The antimicrobial activity of 1-8 was evaluated by the disc
Collection of Plant Pathogens at Institute of Plant Protection diffusion technique by determination of growth inhibition
(Poznań, Poland). Cultures of bacteria were maintained on TSA zones.7 Briefly, a volume of 100 μL of suspension of the test
(Tryptic Soy Agar, Fluka, Sigma-Aldrich), yeast and filamentous microorganisms containing 1.5108 µgcm-3 of bacteria or
fungi on PDA (Potato Dextrose Agar, Emapol).
1.5106 µgcm-3 of yeast was spread on Mueller Hinton Agar
2 | J. Name., 2012, 00, 1-3
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or Sabouraud’s dextrose agar medium, respectively. The ex 488 / em 525) and dead in red (Texas Red, ex531 / em593)
turbidity of the tested strains was adjusted according to Mac- (manual of kit).
Farland (spectrophotometer VIS-723G, Rayleigh, Beijing) scale
0.5 which was used as standard inoculum. Paper discs Synthetic procedures
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DOI: 10.1039/C8NJ06440G
(Whatman no. 1, England, 6 mm diameter) were placed on the
3-(4-bromophenethyl)-6-(tert-butyl)-3,4-dihydro-2H-benzo[e]
agar surface and impregnated with 20 μL of stock solutions of
[1,3]oxazine, [1]. To a round-bottom flask equipped with a stir
50 and 100 µgcm-3. The solvent of dimethylsulfoxide (10%
bar and a condenser the solution of 4-tert-buthylphenol (1.362
g, 9.07 mmol) in 20 mL of methanol, p-
bromophenethyloamine (1.41 mL, 9.07 mmol) and
DMSO) was used as a negative control. In addition, filter discs
impregnated with 30 µg/mL tetracycline (Sigma-Aldrich) or
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Aphotericin B (Gibco, Life Technologies) were used as positive
formaldehyde 37% solution in water (24.2 mmol, 1.80 mL)
reference control for bacteria and fungi, respectively. The
were added. The resulting mixture was heated at reflux for 72
h. Next the solvent was removed by rotary evaporation, oily
residue was digested in 10 mL of methanol and put in freezer
(-30 C). After 2 days white solid precipitated which was
plates, after remaining at 4 C for 2 h, were incubated at 35±2
C (20 h) for bacterial strains and at 28±2 C (48 h) for yeasts.
All tests were performed in triplicate and susceptibility was
assessed by measuring the zone of inhibition.
filtered off and washed with 15 mL of cold methanol. The solid
was recrystallized from methanol to give colorless crystals.
Cytostatic activity
Yield: 1.629 g (4.35 mmol), 48%; mp 73 C. HRMS(ESI): calcd
1
The cytotoxicity evaluation of the lead compounds against for C20H25BrNO: 374.1114 [M + H]+, found 374.1113. H NMR
mammalian cells on fibroblast 3T3 mouse BALB was (500 MHz, CDCl3) δ: 7.41 – 7.38 (m, 2H, ArCH), 7.15 (dd, JHH
determined by SRB method.8 Cells were grown in the culture 8.6, 2.5 Hz, 1H, ArCH), 7.11 – 7.08 (m, 2H, ArCH), 6.94 (d, JHH
=
=
media recommended by cell line supplier. The cells 3T3BALB 2.4 Hz, 1H, ArCH), 6.72 (d, JHH = 8.6 Hz, 1H, ArCH), 4.84 (s, 2H,
were grown at 37 °C in DMEM Medium supplemented with O-CH2-N), 4.01 (s, 2H, Ar-CH2-N-), 3.03-3.01 (m, 2H, CH2-CH2-N
10% (v/v) foetal bovine serum (FBS) and antimycotic and ), 2.85 – 2.82 (m, 2H, CH2-CH2-N), 1.28 (s, 9H, C(CH3)3). 13C
antibacterial solutions (Lonza) in humidified atmosphere NMR (126 MHz, CDCl3) δ: 152.0 (s), 143.5 (s), 139.0 (s), 131.6
having 5% CO2. Before the test, adherent cells were detached (s), 130.6 (s), 124.8 (s), 124.2 (s), 120.1 (s), 119.4 (s), 116.0 (s),
with the trypsin EDTA solution, washed twice in phosphate- 82.4 (s), 52.9 (s), 50.9 (s), 34.4 (s), 34.2 (s), 31.7 (s). IR (cm-1,
buffered saline (PBS), spun down, counted, stained with a 0.4 nujol mull) 1229 (C-O-C), 938 (out-of-plane bending of C–H in
% solution of trypan blue, and inspected under a microscope benzoxazine).
for cell viability. Then, cells were plated on 96-well plastic 3-(4-bromophenethyl)-6-(2,4,4-trimethylpentan-2-yl)-3,4-
culture plates (2103 cells/well in 200 µl DMEM medium) and dihydro-2H-benzo[e][1,3]oxazine, [2]. To a round-bottom flask
incubated at 37 C in a CO2-incubator for 24 h, afterwards, the equipped with a stir bar and a condenser the solution of 4-
tested compound were added in final concentration 5-100 (2,4,4-trimethylpentan-2-yl)phenol (1.359 g, 6.58 mmol) in 40
µgcm-3 and the cultures were incubated for 48 h in CO2- mL of methanol, p-bromophenethyloamine (1.02 mL, 6.58
incubator at 37 C. Then, cells were harvested and intended mmol) and and formaldehyde 37% solution in water (17.6
for cell proliferation test.
Cell density/cell proliferation was estimated with the at reflux for 48 h. Next the solvent was removed by rotary
sulforhodamine (SRB)-colorimetric assay. Briefly, cell evaporation, oily residue was digested in 10 mL of methanol
mmol, 1.31 mL) were added. The resulting mixture was heated
B
cultures were fixed with cold TCA (final concentration 10% and put in freezer (-30 C). After 24 h white solid precipitated
(w/v)) in cultures of adherent cells for 1 h at 4 C, then washed which was filtered off and washed with 15 mL of cold
four times with tap water and air-dried at room temperature methanol. The solid was recrystallized from methanol to give
(20-25 C). A mildly acidic SRB solution (0.4% dye solution in colorless crystals. Yield: 1.947 g (4.52 mmol), 69%; mp 71 C.
1% acetic acid) was added to each well for 30 min at 25 C and HRMS(ESI) calcd for C24H3rBrNO: 430.1740 [M + H]+, found
1
then, unbound stain was removed by rinsing with an aqueous 430.1738. H NMR (500 MHz, CDCl3) δ: 7.41 – 7.38 (m, 2H,
solution of 1% (v/v) acetic acid. Culture plates were then ArH), 7.11 (dd, JHH = 8.6, 2.4 Hz, 1H, ArH), 7.09 – 7.06 (m, 2H,
allowed to dry at room temperature. The SRB bound to the ArH), 6.91 (d, JHH = 2.3 Hz, 1H, ArH), 6.69 (d, JHH = 8.6 Hz, 1H,
intracellular proteins was dissolved in 10 mM Trizma-base ArH), 4.85 (s, 2H, O-CH2-N ), 4.00 (s, 2H, Ar-CH2-N), 3.02 – 2.97
solution (pH 10.5) for 10 min on a gyratory shaker and (m, 2H, CH2-CH2-N), 2.84 – 2.81 (m, 2H, CH2-CH2-N), 1.67 (s,
absorbance of the SRB solution was estimated at 540 nm in a 2H, CH2 ), 1.33 (s, 6H, CH3), 0.73 (s, 9H, CH3). 13C NMR (126
Victor 2 microplate reader (Perkin-Elmer, MA, USA).
MHz, CDCl3) δ: 151.8 (s), 142.4 (s), 139.1 (s), 131.6 (s), 130.6
Microscopic evaluation of cell culture was performed in the (s), 125.7 (s), 125.1 (s), 120.1 (s), 119.0 (s), 115.7 (s), 82.5 (s),
detection of cytopathic and cytotoxic effects. Changes in the 57.2 (s), 53.0 (s), 50.9 (s), 38.1 (s), 34.4 (s), 32.5 (s), 31.9 (s),
cells (vacuolization, change of shape) testify to the toxicity of 31.7 (s). IR (cm-1, nujol mull) 1234 (C-O-C), 937 (out-of-plane
test compounds for cell culture. To assess cell viability of bending of C–H in benzoxazine).
mouse fibroblast, was performed staining using the LIVE / 3-(4-bromophenethyl)-6-methyl-3,4-dihydro-2H-benzo[e]
DEAD Cell Imaging Kit and evaluated in the Evos microscope [1,3]oxazine, [3]. To a round-bottom flask equipped with a stir
(FL, ThermoFisher Scientific). The dyed cells live in green (FITC, bar and a condenser the solution of p-cresol (1.369 g, 12.66
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mmol) in 40 mL of methanol, p-bromophenethyloamine (1.96 Next the solvent was removed by rotary evaporation, oily
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mL, 12.66 mmol) and formaldehyde 37% solution in water residue was digested in 10 mL of methaDnOolI:a10n.d10p39u/tCi8nNJfr0e6e4z4e0Gr.
(26.7 mmol, 2.0 mL) were added. The resulting mixture was After 2 days white solid precipitated which was filtered off and
heated at reflux for 72 h. Next the solvent was removed by washed with 15 mL of cold methanol. The solid was
rotary evaporation, oily residue was digested in 10 mL of recrystallized from methanol to give white precipitate (at -20
methanol and put in freezer. After 2 days white solid C) which was filtered off at low temperature (<0 C). Product
precipitated which was filtered off and washed with 15 mL of melted at room temperature. Yield: 3.14 g (7.55 mmol), 78%,
cold methanol. The solid was recrystallized from methanol to colorless oil. HRMS(ESI) calcd for C21H35NO: 416.32 [M + H]+,
give colorless crystals. Yield: 3.085 g (9.29 mmol), 73%; mp 94 found 416.38. 1H NMR (500 MHz, CDCl3) δ: 7.10 (dd, J = 8.6, 2.4
C. HRMS(ESI) calcd for C17H19BrNO: 332.0644 [M + H]+, found Hz, 1H, ArCH ), 6.91 (d, J = 2.4 Hz, 1H, ArCH), 6.68 (d, J = 8.6 Hz,
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332.0649. H NMR (500 MHz, CDCl3) δ: 7.39 (d, JHH = 8.3 Hz, 1H, ArCH), 4.84 (s, 2H, NCH2O), 3.97 (s, 2H, NCH2Ar), 2.81 –
2H, ArH), 7.08 (d, JHH = 8.2 Hz, 2H, ArH), 6.92 (d, JHH = 7.7 Hz, 2.64 (m, 2H, NCH2), 1.67 (s, 2H, CH2), 1.56 (dd, J = 14.4, 7.2 Hz,
1H, ArH), 6.76 (s, 1H, ArH), 6.68 (d, JHH = 8.3 Hz, 1H, ArH), 4.84 2H, N-CH2-CH2), 1.32 (s, 6H, CH3), 1.31 – 1.21 (m, 18H, CH2-
(s, 2H, O-CH2-N ), 3.98 (s, 2H, Ar-CH2-N ), 3.00 (t, JHH = 7.5 Hz, (CH2)9-CH3), 0.88 (t, J = 7.0 Hz, 3H, CH2 -CH3), 0.72 (s, 9H,
2H, CH2-CH2-N ), 2.82 (t, JHH = 7.5 Hz, 2H, O-CH2-N-), 2.25 (s, C(CH3)3 ). 13C NMR (126 MHz, CDCl3) δ: 151.9 (s, 1C, ArC-O),
3H, CH3).13C NMR (151 MHz, CDCl3) δ: 152.1 (s), 139.0 (s), 142.2 (s, 1C, ArC), 125.5 (s, 1C, ArCH), 125.1 (s, 1C, ArCH),
131.6 (s), 130.6 (s), 130.0 (s), 128.5 (s), 127.9 (s), 120.1 (s), 119.3 (s, 1C, ArC-CH2), 115.5 (s, 1C, ArCH), 82.5 (s, 1C, OCH2N),
119.9 (s), 116.3 (s), 82.5 (s), 52.9 (s), 50.6 (s), 34.4 (s), 20.7 (s). 57.2 (s, 1C, CH2), 51.6 (s, 1C, NCH2), 50.8 (s, 1C, NCH2Ar), 38.1
IR (cm-1, nujol mull) 1226 (C-O-C), 936 (out-of-plane bending of (s, 1C, C(CH3)2), 32.5 (s, 2C, (CH3)2), 32.0 (s, 1C, C(CH3)3), 31.7
C–H in benzoxazine).
(s, 8C, (CH2)8 ), 29.8 (s, 3C, C(CH3)3), 29.5 (s, 1C, N-CH2-CH2),
3-(4-bromophenethyl)-6,8-di-tert-butyl-3,4-dihydro-2H-benzo 22.8 (s, 1C, CH2-CH3), 14.3 (s, 1C, CH3). IR (cm-1, nujol mull)
[e][1,3]oxazine, [4]. The compound obtained according to the 1232 (C-O-C), 937 (out-of-plane bending of C–H in
literature procedure.9
benzoxazine).
6-(tert-butyl)-3-dodecyl-3,4-dihydro-2H-benzo[e][1,3]oxazine, 3-dodecyl-6-methyl-3,4-dihydro-2H-benzo[e][1,3]oxazine, [7].
[5]. To a round-bottom flask equipped with a stir bar and a To a round-bottom flask equipped with a stir bar and a
condenser the solution of 4-tert-butylphenol (2 g, 13.31 mmol) condenser the solution of 4-methylphenol (2.0 g, 18.5 mmol)
in 10 mL of methanol, dodecan-1-amine (2.47 g,13.31 mmol) in 10 mL of methanol, dodecan-1-amine (3.42 g, 18.5 mmol)
and formaldehyde 37% solution in water (29.8 mmol, 2.22 mL) and formaldehyde 37% solution in water (40.68 mmol, 3.08
were added. The resulting mixture was heated at reflux for 24 mL) were added. The resulting mixture was heated at reflux for
h. Next the solvent was removed by rotary evaporation, oily 24 h. Next the solvent was removed by rotary evaporation, oily
residue was digested in 10 mL of methanol and put in freezer. residue was digested in 10 mL of methanol and put in freezer.
After 2 days white solid precipitated which was filtered off and After 2 days white solid precipitated which was filtered off and
washed with 15 mL of cold methanol. The solid was washed with 15 mL of cold methanol. The solid was
recrystallized from methanol to give white precipitate (at - recrystallized from methanol to give white precipitate (at -20
20 C) which was filtered off at low temperature (<0 C). C) which was filtered off at low temperature (<0 C). Product
Product melted at room temperature. Yield: 3.30 g (9.18 melted at room temperature yield: 4.20 g (13.23 mmol), 71%,
mmol), 68%, colorless oil. HRMS(ESI) calcd for C24H41NO: colorless oil. HRMS(ESI) calcd for C21H35NO: 318.3 [M + H]+,
1
360.3261 [M + H]+, found 360.3186. H NMR (500 MHz, CDCl3) found 318.27. 1H NMR (500 MHz, CDCl3) δ 6.92 (dd, J = 8.2, 1.5
δ: 7.14 (d, J = 2.5 Hz, 1H, ArCH), 6.96 (t, J = 3.8 Hz, 1H, ArCH), Hz, 1H, ArCH), 6.77 (d, J = 0.7 Hz, 1H, ArCH), 6.68 (d, J = 8.3 Hz,
6.71 (d, J = 5.0 Hz, 1H, ArCH), 4.86 (d, J = 25.7 Hz, 2H, NCH2O), 1H, ArCH), 4.84 (s, 2H, , NCH2O), 3.96 (s, 2H, NCH2Ar), 2.79 –
3.97 (d, J = 20.6 Hz, 2H, NCH2Ar), 2.78 – 2.70 (m, 2H, N-CH2), 2.66 (m, 2H, NCH2), 2.26 (s, 3H, CH3), 1.59 – 1.44 (m, 2H, N-
1.61 - 1.53 (m, 2H, N-CH2-CH2), 1.29 (m, 18H, CH2-(CH2)9-CH3), CH2-CH2), 1.44 – 1.15 (m, 27H, CH2-(CH2)9-CH3, C(CH3)3), 0.89 (t,
1.27 (m, 9H, C(CH3)), 0.89 (t, J = 7.0 Hz, 3H, CH2-CH3). 13C NMR J = 7.0 Hz, 3H, CH3). 13C NMR (126 MHz, CDCl3) δ 152.12 (s, 1C,
(126 MHz, CDCl3) δ: 152.0 (s, 1C, ArC-O), 143.2 (s, 1C, ArC), ArC-O), 129.74 (s, 1C, ArC), 128.30 (s, 1C, ArCH), 127.97 (s, 1C,
124.6 (s, 1C, ArCH), 124.1 (s, 1C, ArCH), 119.5 (s, 1C, ArC-CH2), ArCH), 120.08 (s, 1C, ArC-CH2), 116.20 (s, 1C, ArCH), 82.52 (s,
115.8 (s, 1C, ArCH), 82.3 (s, 1C, OCH2N), 51.5 (s, 1C, NCH2), 50.6 1C, OCH2N), 51.55 (s, 1C, NCH2), 50.43 (s, 1C, NCH2Ar), 32.06 (s,
(s, 1C, NCH2Ar), 34.1 (s, 1C, C(CH3)3), 32.0 (s, 1C, C(CH3)3 ), 29.7 1C, N-CH2-CH2), 29.75 (s, 8C, C(CH2)8), 20.70 (s, 1C, CH3), 14.25
(s, 8C, C(CH2)8), 28.1 (s, 1C, N-CH2-CH2), 22.7 (s, 1C, CH2-CH3), (s, 1C, CH3). IR (cm-1, nujol mull) 1228 (C-O-C), 939 (out-of-
14.2 (s, 1C, CH3). IR (cm-1, nujol mull) 1232 (C-O-C), 939 (out-of- plane bending of C–H in benzoxazine).
plane bending of C–H in benzoxazine).
6,8-di-tert-butyl-3-dodecyl-3,4-dihydro-2H-benzo[e][1,3]
3-dodecyl-6-(2,4,4-trimethylpentan-2-yl)-3,4-dihydro-2H-
oxazine, [8]. To a round-bottom flask equipped with a stir bar
benzo[e][1,3]oxazine, [6]. To a round-bottom flask equipped and a condenser the solution of 2,4-di-tert-butylphenol (2.0 g,
with a stir bar and a condenser the solution of 4-(2,4,4- 9.7 mmol) in 10 mL of methanol, dodecan-1-amine (1.8 g, 9.7
trimethylpentan-2-yl)phenol (2.0 g, 9.69 mmol) in 10 mL of mmol) and formaldehyde 37% solution in water (21.5 mmol,
methanol, dodecan-1-amine (1.79 g, 9.69 mmol) and 1.6 mL) were added. The resulting mixture was heated at
formaldehyde 37% solution in water (21.3 mmol, 1.6 mL) were reflux for 24 h. Next the solvent was removed by rotary
added. The resulting mixture was heated at reflux for 24 h. evaporation, oily residue was digested in 10 mL of methanol
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and put in freezer. After 2 days white solid precipitated which 2.97 (m, 2H, NCH2CH2), 2.89 – 2.83 (m, 2H, NCH2CH2), 1.67 (s,
was filtered off and washed with 15 mL of cold methanol. The 2H, CH2), 1.32 (s, 6H, CH3), 0.72 (s, 9H, C(CH3)3), 0.24 (s, 9H,
View Article Online
DOI: 10.1039/C8NJ06440G
solid was recrystallized from methanol to give white SiMe3). 13C NMR (126 MHz, CDCl3) δ: 151.8 (CPhO), 142.3
precipitate (at -15 C). Yield: 3.0 g (7.22 mmol), 74%; mp 41.7 (CPhC(CH3)2), 140.7 (CPhCH2CH2), 132.1 (CH of C6H4), 128.7 (CH
C. HRMS(ESI) calcd for C28H49NO: 416.3887 [M + H]+, found of C6H4), 125.6 (CH of C6H3), 125.0 (CH of C6H3), 121.0
1
416.4042. H NMR (500 MHz, CDCl3) δ: 7.16 (d, J = 2.5 Hz, 1H, (CPhCH2N), 119.1 (CPhC≡C), 115.7 (CH of C6H3), 105.3 (SiC≡C),
ArCH), 7.81 (d, J = 2.3 Hz, 1H, ArCH), 4.85 (s, 2H, NCH2O), 4.00 93.7 (C6H4C≡C), 82.5 (OCH2N), 57.2 (CH2), 53.0 (NCH2CH2), 50.9
(s, 2H, NCH2Ar), 2.79 - 2.68 (m, 2H, NCH2), 1.68 – 1.54 (m, 2H, (CPhCH2N), 38.1 (CPhC(CH3)2), 35.1 (NCH2CH2), 32.5 (C(CH3)3),
N-CH2-CH2), 1.46 - 1.38 (m, 9H, C(CH3)3 ), 1.36 - 1.22 (m, 27H, 31.9 (C(CH3)3), 31.7 (C(CH3)2), 0.2 (SiMe3). IR (cm-1, nujol mull)
CH2-(CH2)9-CH3, C(CH3)3), 0.90 (t, J = 6.9 Hz, 3H, CH3). 13C NMR 2120 (C≡C), 1249 (C–O–C), 1157 (C–N–C), 937 (out-of-plane
(126 MHz, CDCl3) δ: 150.7 (s, 1C, ArC-O), 141.9 (s, 1C, ArC), bending of C–H in benzoxazine).
136.5 (s, 1C, ArC-C), 121.9 (s, 1C, ArCH), 121.8 (s, 1C, ArCH), 6-methyl-3-(4-((trimethylsilyl)ethynyl)phenethyl)-3,4-dihydro-
119.2 (s, 1C, ArC-CH2), 81.6 (s, 1C, OCH2N), 51.6 (s, 1C, NCH2), 2H-benzo[e][1,3]oxazine [11]. Compound was synthesized
51.2 (s, 1C NCH2Ar), 34.9 (s, 1C, C(CH3)3), 34.3 (s, 1C, C(CH3)3), according to the procedure for 9. Used: 2 (0.797 g, 2.40 mmol),
32.0 (s, 3C, C(CH3)3), 31.7 (s, 3C, C(CH3), 8C, (CH2)8), 29.6 (s, 1C, NEt3 (20 ml), [Pd(PPh3)2]Cl2 (0.084 g, 0.12 mmol),
N-CH2-CH2), 22.7 (s, 1C, CH2-CH3), 14.1 (s, 1C, CH3). IR (cm-1, ethynyl(trimethyl)silane (980 µl, 7.20 mmol). Reaction time: 48
nujol mull) 1224 (C-O-C), 948 (out-of-plane bending of C–H in h, chromatography condition: silica gel, Et2O/hexane, v/v, 1/2.
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benzoxazine).
Yield 69% (0.578 g, 1.65 mmol) of 11 as a yellow powder.
HRMS(ESI) calcd for C22H28NOSi: 350.1935 [M + H]+, found
6-(tert-butyl)-3-(4-((trimethylsilyl)ethynyl)phenethyl)-3,4-
dihydro-2H-benzo[e][1,3]oxazine, [9]. The solution of 1 (0.392 350.1930. 1H NMR (500 MHz, CDCl3) δ: 7.43–7.40 (m, 2H,
g, 1.05 mmol) and [Pd(PPh3)2]Cl2 (0.037 g, 0.05 mmol) in dry C6H4), 7.17 (d, JHH = 8.3 Hz, 2H, C6H4), 6.98 – 6.94 (m, 1H, C6H3),
triethylamine (15 ml) was degassed by freeze-pump-thaw (3 6.79 (d, JHH = 1.1 Hz, 1H, C6H3), 6.74 – 6.71 (m, 1H, C6H3), 4.86
times) technique. Then ethynyl(trimethyl)silane (426 μl, 3.14 (s, 2H, OCH2N), 4.00 (s, 2H, NCH2Ph), 3.06 – 3.01 (m, 2H,
mmol) was added in one portion and the mixture was warmed NCH2CH2), 2.91 – 2.87 (m, 2H, NCH2CH2), 2.29 (s, 3H, CH3), 0.30
up (55 C) and mixed in the oil bath. After 24 h next portion of (s, 9H, SiMe3). 13C NMR (126 MHz, CDCl3) δ: 152.1 (CPhO), 140.7
[Pd(PPh3)2]Cl2 (0.037 g, 0.05 mmol), ethynyl(trimethyl)silane (CPhCH2CH2), 132.1 (CH of C6H4), 129.9 (CCH3), 128.7 (CH of
(426 μl, 3.14 mmol) were added and the mixture was placed C6H4), 128.4 (CH of C6H3), 127.9 (CH of C6H3), 121.0 (CPhCH2N),
back in the oil bath (55 C) for next 48 h. During that time the 119.9 (CPhC≡C), 116.3 (CH of C6H3), 105.3 (SiC≡C), 93.8
reaction was monitored by 1H NMR. The solvent was (C6H4C≡C), 82.5 (OCH2N), 52.9 (NCH2CH2), 50.6 (CPhCH2N), 35.0
evaporated and the product isolated by column (NCH2CH2), 20.7 (CH3), 0.2 (SiMe3). IR (cm-1, nujol mull) 2116
chromatography (silica gel, Et2O/hexane, v/v, 1/2). Yield 72% (C≡C), 1249 (C–O–C), 1157 (C–N–C), 937 (out-of-plane bending
(0.293 g, 0.75 mmol) of 9 as a yellow oil. HRMS(ESI) calcd for of C–H in benzoxazine).
1
C25H33NOSi: 392.2404 [M + H]+, found 392.2419. H NMR (500 6,8-di-tert-butyl-3-(4-((trimethylsilyl)ethynyl)phenethyl)-3,4-
MHz, CDCl3) δ: 7.39 – 7.36 (m, 2H, C6H4), 7.15 (dd, J = 8.4, 1.9 dihydro-2H-benzo[e][1,3]oxazine, [12]. The compound
Hz, 3H: 2H of C6H4, 1H of C6H3), 6.94 (d, J = 2.4 Hz, 1H, C6H3), obtained according to the literature procedure.3
6.72 (t, J = 7.2 Hz, 1H, C6H3), 4.84 (s, 2H, OCH2N), 4.02 (d, J = 6-(tert-butyl)-3-(4-ethynylphenethyl)-3,4-dihydro-2H-
10.3 Hz, 2H, NCH2Ph), 3.05 – 3.00 (m, 2H, NCH2CH2), 2.91 – benzo[e][1,3]oxazine, [13]. To the solution of 9 (0.033 g, 0.083
2.84 (m, 2H, NCH2CH2), 1.28 (s, 9H, C(CH3)3), 0.24 (s, 9H, mmol) in MeOH/i-PrOH (v/v, 1/3, 30 mL) anhydrous K2CO3
SiMe3). 13C NMR (126 MHz, CDCl3) δ: 152.0 (CPhO), 143.5 (0.035 g, 0.25 mmol) was added and the suspension was mixed
(CPhC(CH3)3), 140.7 (CPhCH2CH2), 132.1 (CH of C6H4), 128.8 (CH for 24 h. The solvent was evaporated under reduced pressure,
of C6H4), 124.8 (CH of C6H3), 124.2 (CH of C6H3), 121.0 residue was suspended in water (50 mL) and product was
(CPhCH2N), 119.5 (CPhC≡C), 116.0 (CH of C6H3), 105.3 (SiC≡C), extracted with diethyl ether (3 x 20 mL). The organic phase
93.8 (C6H4C≡C), 82.4 (OCH2N), 52.9 (NCH2CH2), 50.9 (CPhCH2N), was dried (anhydrous MgSO4), filtered and evaporated. The
35.1 (NCH2CH2), 34.2 (C(CH3)3), 31.6 (C(CH3)3), 0.1 (SiMe3).
6-(2,4,4-trimethylpentan-2-yl)-3-(4-
((trimethylsilyl)ethynyl)phenethyl)-3,4-dihydro-2H-
product did not require additional purification. Yield 98%
(0.026 g, 0.082 mmol) of 13 as a yellowish powder; mp 97 C.
HRMS(ESI) calcd for C22H26NO: 320.2009 [M + H]+, found
1
benzo[e][1,3]oxazine, [10]. Compound was synthesized 320.2004. H NMR (500 MHz, CDCl3) δ: 7.42 – 7.39 (m, 2H,
according to the procedure for 9. Used: 2 (0.128 g, 0.30 mmol), C6H4), 7.17 (d, JHH = 8.3 Hz, 2H, C6H4), 7.14 (dd, JHH = 8.6, 2.4 Hz,
NEt3 (7 mL), [Pd(PPh3)2]Cl2 (0.010 g, 0.02 mmol), 1H, C6H3), 6.94 (d, JHH = 2.4 Hz, 1H, C6H3), 6.72 (d, JHH = 8.6 Hz,
ethynyl(trimethyl)silane (121 µl, 0.89 mmol) were used. 1H, C6H3), 4.85 (d, JHH = 2.7 Hz, 2H, OCH2N), 4.02 (s, 2H,
Reaction time: 48 h, chromatography condition: silica gel, NCH2Ph), 3.05 – 3.01 (m, 3H, NCH2CH2, HC≡C), 2.90 – 2.86 (m,
Et2O/hexane, v/v, 1/2. Yield 57% (0.077 g, 0.17 mmol) of 10 as 2H, NCH2CH2), 1.28 (s, 9H, C(CH3)3). 13C NMR (126 MHz, CDCl3)
a yellow powder; mp 94 C. HRMS(ESI) calcd for C29H42NOSi: δ: 151.9 (CPhO), 143.4 (CPhC(CH3)3), 140.1 (CPhCH2CH2), 132.2
1
448.3030 [M + H]+, found 448.3042. H NMR (500 MHz, CDCl3) (CH of C6H4), 128.8 (CH of C6H4), 124.7 (CH of C6H3), 124.1 (CH
δ: 7.39 – 7.35 (m, 2H, C6H4), 7.14 – 7.11 (m, 3H: 2H of C6H4, 1H of C6H3), 119.9 (CPhCH2N), 119.4 (CPhC≡C), 116.0 (CH of C6H3),
of C6H3), 6.90 (d, J = 2.3 Hz, 1H, C6H3), 6.69 (dd, J = 8.6, 2.7 Hz, 83.8 (C6H4C≡C), 82.3 (OCH2N), 77.0 (C≡CH), 52.8 (NCH2CH2),
1H, C6H3), 4.84 (s, 2H, OCH2N), 3.99 (s, 2H, NCH2Ph), 3.02 – 50.8 (CPhCH2N), 34. 9 (NCH2CH2), 34.1 (C(CH3)3), 31.6 (C(CH3)3).
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IR (cm-1, nujol mull) 3265 (C≡CH), 1980 (C≡C), 1231 (C–O–C), separated fractions by column chromatographVyiew(sAirltiicclae Ognleinle,
1157 (C–N–C), 933 (out-of-plane bending of C–H in Et2O/heksan, v/v, 1/1). Yield 25% (0.067 g, 0.162 mmol) of 17
DOI: 10.1039/C8NJ06440G
benzoxazine).
as an orange grease and 8% (0.033 g, 0.05 mmol) of 20 as a
3-(4-ethynylphenethyl)-6-(2,4,4-trimethylpentan-2-yl)-3,4-
yellow solid.
dihydro-2H-benzo[e][1,3]oxazine, [14]. Compound was 17: HRMS(ESI) calcd for C27H34NOSi: 416.2404 [M + H]+, found
1
synthesized according to the procedure for 13. Used: 10 (0.297 416.2408. H NMR (500 MHz, CDCl3) δ: 7.41 – 7.38 (m, 2H,
g, 0.67 mmol), MeOH/i-PrOH (v/v, 1/3, 30 mL), K2CO3 (0.277 g, C6H4), 7.18 – 7.13 (m, 3H: 2H of C6H4, 1H z C6H3), 6.94 (d, JHH
=
2.00 mmol). Reaction time 24 h. Yield 75% (0.187 g, 0.50 2.4 Hz, 1H, C6H3), 6.74 – 6.70 (m, 1H, C6H3), 4.84(s, 2H, OCH2N),
mmol) of 14 as an orange solid. HRMS(ESI) calcd for C26H35NO: 4.01 (s, 2H, NCH2Ph), 3.02 (dd, JHH = 8.5, 6.5 Hz, 2H, NCH2CH2),
10
11
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14
15
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17
18
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376.2635 [M + H]+, found 376.2634. H NMR (500 MHz, CDCl3) 2.90 – 2.86 (m, 2H, NCH2CH2), 1.28 (s, 9H, C(CH3)3), 0.23 (s, 9H,
δ: 7.34 – 7.31 (m, 2H, C6H4), 7.08 (d, JHH = 8.3 Hz, 2H, C6H4), SiMe3). 13C NMR (126 MHz, CDCl3) δ: 152.0 (CPhO), 143.5
7.04 (dd, JHH = 8.6, 2.4 Hz, 1H, C6H3), 6.83 (d, JHH = 2.4 Hz, 1H, (CPhC(CH3)3), 141.8 (CPhCH2CH2), 132.8 (CH of C6H4), 129.0 (CH
C6H3), 6.61 (d, JHH = 8.6 Hz, 1H, C6H3), 4.78 (s, 2H, OCH2N), 3.93 of C6H4), 124.8 (CH of C6H3), 124.2 (CH of C6H3), 119.4
(s, 2H, NCH2Ph), 2.96 – 2.91 (m, 3H, NCH2CH2, HC≡C), 2.82 – (CPhCH2N), 119.2 (CPhC≡C), 116.0 (CH of C6H3), 90.5 (SiC≡C),
2.78 (m, 2H, NCH2CH2), 1.59 (s, 2H, CH2), 1.25 (s, 6H, CH3), 0.65 88.1 (C≡C), 82.4 (OCH2N), 77.0 (C6H4C≡C), 74.0 (C≡C), 52.8
(s, 9H, C(CH3)3). 13C NMR (126 MHz, CDCl3) δ: 151.8 (CPhO), (NCH2CH2), 50.9 (CPhCH2N), 35.0 (NCH2CH2), 34.2 (C(CH3)3), 31.6
142.4 (CPhC(CH3)2), 141.1 (CPhCH2CH2), 132.3 (CH of C6H4), (C(CH3)3), -0.2 (SiMe3).
128.9 (CH of C6H4), 125.7 (CH of C6H3), 125.1 (CH of C6H3), 20: mp 151 C. HRMS(ESI) calcd for C44H49N2O2: 637.3789 [M +
1
120.0 (CPhCH2N), 119.0 (CPhC≡C), 115.7 (CH of C6H3), 83.8 H]+, found 637.3790. H NMR (500 MHz, CDCl3) δ: 7.45 – 7.41
(C6H4C≡C), 82.5 (OCH2N), 57.2 (CH2), 52.9 (NCH2CH2), 50.9 (m, 2H, C6H4), 7.19 (d, JHH = 8.3 Hz, 2H, C6H4), 7.14 (dd, JHH
=
(CPhCH2N), 38.1 (CPhC(CH3)2), 35.0 (NCH2CH2), 32.5 (C(CH3)3), 8.6, 2.5 Hz, 1H, C6H3), 6.94 (d, JHH = 2.4 Hz, 1H, C6H3), 6.72 (d,
31.9 (C(CH3)3), 31.7 (C(CH3)2), 1C missing. IR (cm-1, nujol mull) JHH = 8.6 Hz, 1H, C6H3), 4.85 (s, 2H, OCH2N), 4.02 (s, 2H,
3266 (C≡CH), 1980 (C≡C), 1240 (C–O–C), 1157 (C–N–C), 937 NCH2Ph), 3.03 (dd, JHH = 8.5, 6.6 Hz, 2H, NCH2CH2), 2.89 (t, JHH
(out-of-plane bending of C–H in benzoxazine).
3-(4-ethynylphenethyl)-6-methyl-3,4-dihydro-2H-benzo[e]
=
7.6 Hz, 2H, NCH2CH2), 1.28 (s, 9H, C(CH3)3). 13C NMR (126 MHz,
CDCl3) δ: 152.0 (CPhO), 143.5 (CPhC(CH3)3), 141.6 (CPhCH2CH2),
[1,3]oxazine, [15]. Compound was synthesized according to 132.6 (CH of C6H4), 129.0 (CH of C6H4), 124.9 (CH of C6H3),
the procedure for 13. Used: 11 (0.578 g, 1.65 mmol), MeOH/i- 124.2 (CH of C6H3), 119.7 (CPhCH2N), 119.4 (CPhC≡C), 116.0 (CH
PrOH (v/v, 1/3, 30 mL), K2CO3 (0.686 g, 4.96 mmol). Reaction of C6H3), 82.5 (OCH2N), 81.6 (C6H4C≡C), 73.8 (C≡C), 52.8
time 24 h. Yield 37% (0.171 g, 0.62 mmol) of 15 as an orange (NCH2CH2), 50.9 (CPhCH2N), 35.1 (NCH2CH2), 34.2 (C(CH3)3), 31.6
solid; mp 85 °C. HRMS(ESI) calcd for C19H20NO: 278.1539 [M + (C(CH3)3). IR (cm-1, nujol mull) 2116 (C≡C), 1908 (C≡C), 1231 (C–
1
H]+, found 278.1539. H NMR (500 MHz, CDCl3) δ: 7.35 – 7.31 O–C), 1156 (C–N–C), 937 (out-of-plane bending of C–H in
(m, 2H, C6H4), 7.09 (d, JHH = 8.3 Hz, 2H, C6H4), 6.84 (dt, JHH = 5.6, benzoxazine).
3.2 Hz, 1H, C6H3), 6.68 (d, JHH = 1.0 Hz, 1H, C6H3), 6.60 (dd, JHH
=
6-(2,4,4-trimethylpentan-2-yl)-3-(4-((trimethylsilyl)buta-1,3-
8.2, 3.8 Hz, 1H, C6H3), 4.77 (s, 2H, OCH2N), 3.91 (s, 2H, diyn-1-yl)phenethyl)-3,4-dihydro-2H-benzo[e][1,3]oxazine,
NCH2Ph), 2.96 – 2.91 (m, 3H, NCH2CH2, HC≡C), 2.82 – 2.77 (m, [18]. Compound was synthesized according to the procedure
2H, NCH2CH2), 2.17 (s, 3H, CH3). 13C NMR (126 MHz, CDCl3) δ: for 17. Used: 14 (0.182 g, 0.49 mmol), THF (15 mL), CuI (0.005
152.1 (CPhO), 141.0 (CPhCH2CH2), 132.3 (CH of C6H4), 129.9 g, 0.02 mmol), [Pd(PPh3)2]Cl2 (0.017 g, 0.02 mmol),
(CCH3), 128.9 (CH of C6H4), 128.4 (CH of C6H3), 127.9 (CH of bromoethynyl(trimethyl)silane (0.129 g, 0.73 mmol),
C6H3), 120.0 (CPhCH2N), 119.9 (CPhC≡C), 116.3 (CH of C6H3), 83.8 diisopropylamine (172 µL, 1.21 mmol). Reaction time: 4 h,
(C6H4C≡C), 82.5 (OCH2N), 52.9 (NCH2CH2), 50.6 (CPhCH2N), 35.0 chromatography conditions: (silica gel, Et2O/heksan, v/v, 1/1).
(NCH2CH2), 20.7 (CH3), 1C missing. IR (cm-1, nujol mull) 3265 Yield 19% (0.044 g, 0.09 mmol) of 18 as a brown grease.
(C≡CH), 1227 (C–O–C), 1160 (C–N–C), 936 (out-of-plane HRMS(ESI) calcd for C31H42NOSi: 472.3030 [M + H]+, found
1
bending of C–H in benzoxazine).
6,8-di-tert-butyl-3-(4-((trimethylsilyl)ethynyl)phenethyl)-3,4-
472.3028. H NMR (500 MHz, CDCl3) δ: 7.41 – 7.38 (m, 2H,
C6H4), 7.16 (t, JHH = 7.0 Hz, 2H, C6H4), 7.11 (dd, JHH = 8.6, 2.4 Hz,
dihydro-2H-benzo[e][1,3]oxazine, [16]. The compound 1H, C6H3), 6.90 (d, JHH = 2.3 Hz, 1H, C6H3), 6.68 (d, JHH = 8.6 Hz,
obtained according to the literature procedure.9
6-(tert-butyl)-3-(4-((trimethylsilyl)buta-1,3-diyn-1-
1H, C6H3), 4.84 (s, 2H, OCH2N), 3.99 (s, 2H, NCH2Ph), 3.00 (dd,
JHH = 8.6, 6.4 Hz, 2H, NCH2CH2), 2.89 – 2.84 (m, 2H, NCH2CH2),
yl)phenethyl)-3,4-dihydro-2H-benzo[e][1,3]oxazine, [17] and 1.67 (s, 2H, CH2), 1.32 (s, 6H, 2 × CH3), 0.72 (s, 9H, C(CH3)3),
1,4-bis(4-(2-(6-(tert-butyl)-2H-benzo[e][1,3]oxazin-3(4H)-
0.23 (s, 9H, SiMe3). 13C NMR (126 MHz, CDCl3) δ: 151.8 (CPhO),
yl)ethyl)phenyl)buta-1,3-diyne, [20]. To the solution of 13 142.4 (CPhC(CH3)3), 141.9 (CPhCH2CH2), 132.9 (CH of C6H4),
(0.206 g, 0.65 mmol), CuI (0.0061 g, 0.032 mmol), 129.0 (CH of C6H4), 125.7 (CH of C6H3), 125.1 (CH of C6H3),
[Pd(PPh3)2]Cl2
(0.023
g,
0.03
mmol)
and 119.2 (CPhCH2N), 119.0 (CPhC≡C), 115.7 (CH of C6H3), 90.5
bromoethynyl(trimethyl)silane (0.171 g, 0.97 mmol)10 in dry (SiC≡C), 89.1 (C≡C), 82.5 (OCH2N), 77.0 (C6H4C≡C), 74.0 (C≡C),
oxygen-free THF under nitrogen atmosphere diisopropylamine 57.2 (CH2), 52.8 (NCH2CH2), 50.9 (CPhCH2N), 38.1 (CPhC(CH3)2),
(230 µl, 1.61 mmol) was added dropwise. The mixture was 35.1 (NCH2CH2), 32.5 (C(CH3)3), 31.9 (C(CH3)3), 31.7 (C(CH3)2), -
stirred in room temperature for 5 h. The solvent was 0.2 (SiMe3).
evaporated and the product 17 and 20 were isolated as
6 | J. Name., 2012, 00, 1-3
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Page 7 of 14
New Journal of Chemistry
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Journal Name
6-methyl-3-(4-((trimethylsilyl)buta-1,3-diyn-1-yl)phenethyl)-
ARTICLE
1
2
3
4
5
6
7
8
R
OH
O
N
View Article Online
HCHO 2.0-2.7 eq. DOI: 10.1039/C8NJ06440G
R1
R1
3,4-dihydro-2H-benzo[e][1,3]oxazine, [19] and 1,4-bis(4-(2-(6-
methyl-2H-benzo[e][1,3]oxazin-3(4H)-yl)ethyl)phenyl)buta-
1,3-diyne [21]. Compounds were obtained according to the
procedure for 17 and 20. Used: 15 (0.171 g, 0.62 mmol), THF
(15 mL), CuI (0.006 g, 0.032 mmol), [Pd(PPh3)2]Cl2 (0.022 g,
0.03 mmol), bromoethynyl(trimethyl)silane (0.135 g, 0.92
mmol), diisopropylamine (217 µL, 1.54 mmol). Reaction time:
24 h, chromatography condition: (silica gel, Et2O/heksan, v/v,
1/1). Yield 48% (0.111 g, 0.30 mmol) of 19 as a yellowish oil
and 3% (0.008 g, 0.014 mmol) of 21 as an orange grease.
+
R NH2
MeOH, reflux
24 - 72 h
R2
R2
O
N
O
N
O
N
O
N
t-Bu
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
t-Bu
O
Br
t-Oct
Br
Br
t-Bu
Br
1
2
3
4
, 64%
, 48%
C12H25
, 69%
, 73%
C12H25
C12H25
C12H25
N
O
N
O
N
O
N
t-Bu
19: HRMS(ESI) calcd for C24H28NOSi: 374.1935 [M + H]+, found
1
374.1940. H NMR (500 MHz, CDCl3) δ: 7.40 (dq, JHH = 6.2, 2.1
t-Bu
t-Oct
t-Bu
Hz, 2H, C6H4), 7.16 (d, JHH = 8.3 Hz, 2H, C6H4), 6.92 (dd, JHH
=
5
6
7
8
, 74%
, 68%
, 78%
, 71%
8.3, 1.5 Hz, 1H, C6H3), 6.75 (d, JHH = 1.3 Hz, 1H, C6H3), 6.68 (dd,
JHH = 8.3, 1.8 Hz, 1H, C6H3), 4.84 (s, 2H, OCH2N), 3.97 (s, 2H,
NCH2Ph), 3.03 – 2.98 (m, 2H, NCH2CH2), 2.89 – 2.84 (m, 2H,
NCH2CH2), 2.25 (s, 3H, CH3), 0.23 (s, 9H, SiMe3). 13C NMR (126
MHz, CDCl3) δ: 152.0 (CPhO), 141.8 (CPhCH2CH2), 132.9 (CH of
C6H4), 130.0 (CCH3), 129.0 (CH of C6H4), 128.5 (CH of C6H3),
127.9 (CH of C6H3), 119.8 (CPhCH2N), 119.2 (CPhC≡C), 116.3 (CH
of C6H3), 90.5 (SiC≡C), 88.1 (C≡C), 82.5 (OCH2N), 77.0
(C6H4C≡C), 74.0 (C≡C), 52.8 (NCH2CH2), 50.6 (CPhCH2N), 35.0
((NCH2CH2), 20.7 (CH3), -0.2 (SiMe3).
Scheme 1. Synthesis of benzoxazines 1–8 (for the synthesis of 4, see ref 3).
In the next step, benzoxazines 1–4 were functionalized
through the Sonogashira cross-coupling using TMSC≡CH and
[Pd(PPh3)2]Cl2 or [Pd(PPh3)2]Cl2/CuII catalytic system in
triethylamine (Scheme 2). The procedure typically used for
such reactions gave a trace of products.11 In all cases, the
addition of two portions of ethynyl(trimethyl)silane was used
to obtain products with the reported yields. Benzoxazines 9–
11 were synthesized with an additional portion of the catalyst
introduced after 24 h. Standard work-up procedures followed
by column chromatography gave trimethylsilylacetylene
benzoxazine derivatives 9–12 with good yields (57–72%).
Deprotection with K2CO3 in an i-PrOH/MeOH mixture led to
terminal acetylene-functionalized benzoxazines 13–16. The
elongation of acetylene to butadiynes was implemented
through the Cadiot-Chodkiewicz protocol. The desired
benzoxazines 17–19 were isolated by column chromatography
as the main products. During the procedure, homocoupling
product formation was observed as well. Small amounts of the
benzoxazine-end-capped butadiynes 20 and 21 were obtained
as a minor fraction (Scheme 2).
21: HRMS(ESI) calcd for C38H37N2O2: 553.2850 [M + H]+, found
1
553.2847. H NMR (500 MHz, CDCl3) δ: 7.45 – 7.41 (m, 2H,
C6H4), 7.17 (d, JHH = 8.2 Hz, 2H, C6H4), 6.92 (dd, JHH = 8.3, 1.6 Hz,
1H, C6H3), 6.76 (s, 1H, C6H3), 6.67 (d, JHH = 8.3 Hz, 1H, C6H3),
4.84 (s, 2H, OCH2N), 3.98 (s, 2H, NCH2Ph), 3.01 (dd, JHH = 8.5,
6.5 Hz, 2H, NCH2CH2), 2.89 – 2.85 (m, 2H, NCH2CH2), 2.25 (s,
3H, CH3). 13C NMR (126 MHz, CDCl3) δ: 152.0 (CPhO), 141.6
(CPhCH2CH2), 132.6 (CH of C6H4), 130.0 (CCH3), 129.0 (CH of
C6H4), 128.5 (CH of C6H3), 128.0 (CH of C6H3), 119.9 (CPhCH2N),
119.7 (CPhC≡C), 116.3 (CH of C6H3), 82.5 (OCH2N), 81.6
(C6H4C≡C), 73.8 (C≡C), 52.8 (NCH2CH2), 50.6 (CPhCH2N), 35.0
(NCH2CH2), 20.7 (CH3).
Results and discussion
The new benzoxazines 1–8 (Scheme 1) with simple
functionalization both as arms linked to a nitrogen atom and
substituents in the ortho/para position of the aryl core have
been obtained through the standard one-pot Mannich
coupling
while
using
p-bromophenylethylamine
or
dodecylamine, formaldehyde (37% solution in H2O), and the
corresponding phenols: p-cresol, 4-tert-buthylphenol, and 4-
(2,4,4-trimethylpentan-2-yl)phenol. Recrystallization of the
crude products 1–8 gave the corresponding benzoxazines with
moderate to good yields (48–78%). Benzoxazines 6 and 7 are
liquid at room temperature, nevertheless, low-temperature
recrystallization from methanol proceeds smoothly.
Compound 4 has previously been prepared through a similar
procedure.9
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Page 8 of 14
ARTICLE
Journal Name
1
2
3
4
5
TMSC2H 3.0 eq.
View Article Online
DOI: 10.1039/C8NJ06440G
O
N
O
N
[Pd(PPh3)2]Cl2
or [Pd(PPh3)2]Cl2/CuII
R1
R1
K2CO3
1-4
NEt3, 55 o
48-72 h
C
MeOH, i-PrOH
RT, 24 h
R2
R2
6
7
8
Si
; R1 H; R2 t-Bu, 72%
9
13
14
15
16
; R1 H; R2 t-Bu 98%
; R1 H; R2 t-Oct, 97%
; R1 H; R2 Me, 37%
; R1 t-Bu; R2 t-Bu 95%
10
11
12
; R1 H; R2 t-Oct, 57%
; R1 H; R2 Me, 69%
; R1 t-Bu; R2 t-Bu 68%
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
R1
O
N
TMSC2Br 2.0-3.0 eq.
CuI, [Pd(PPh3)2]Cl2,
R1
13-15
+
R2
NH(i-Pr)2, THF, RT
4-24 h
R2
R2
O
Fig. 1. Molecular structures of benzoxazines 1-4 with atom labeling. The thermal
ellipsoids are drawn at the 50% probability level. H atoms are excluded for
clarity.
Si
R1
N
17
18
19
; R1 H; R2 t-Bu, 25%
; R1 H; R2 t-Oct, 19%
; R1 H; R2 Me, 48%
20; R1 H; R2 t-Bu, 8%
21
; R1 H; R2 Me, 3%
Scheme 2. Synthesis of acetylene- and butadiyne-functionalized benzoxazines 9–
21.
All of the obtained benzoxazines were characterized by NMR,
HRMS, spectroscopic, and thermo-gravimetric methods. The
diagnostic 1H NMR resonances due to Ar-CH2-N- and O-CH2-N-,
attributed to the benzoxazine motif, were observed at 3.97–
4.05 and 4.84–4.88 ppm in CDCl3. Chemical shifts associated
with aromatic protons, trimethylsilyl, and alkyl substituents
were typical for all the compounds. The signals representing
the -CH2-CH2-N linker appeared at nearly the same positions as
two triplets for 1–4 and 9–21. The dodecyl fragment located
on the nitrogen atom for 5–8 represented a typical set of
methylene groups appearing as multiplets and triplets for the
methyl group located at the end of the alkyl chain. The
13CNMR spectra were used to detect the acetylene group.
Chemical shifts of alkyne carbons appeared in the 72.0–105.3
ppm range. For some compounds, one of the acetylene signals
overlapped with the solvent peak (CDCl3, 77.2 ppm). The IR
spectra were similar and exhibited bands at 936–938 and
1226–1234 cm-1, characteristic benzoxazine fingerprints.
Typical alkyne C≡C bands (1980–2120 cm-1) also occured in the
IR spectra of acetylene benzoxazine derivatives. The molecular
structure in the solid state was confirmed for functionalized
benzoxazines by bromobenzyl (Fig. 1, benzoxazine 1-4), alkyl
chain (Fig. 2, benzoxazine 8), acetylene (Fig. 3, benzoxazine 13,
15 - 16), and butadiyne (Fig. 4) through X-ray analysis. X-ray
experiment details and the selected bond lengths and angles
are summarized in the supporting information file, Tables S1
and S2.
Fig. 2. Molecular structure of benzoxazine 8 with atom labeling. The thermal
ellipsoids are drawn at the 50% probability level. H atoms are excluded for
clarity.
Fig. 3. Molecular structures of benzoxazines 13, 15, 16 (two molecules in
asymmetric unit) with atom labeling. The thermal ellipsoids are drawn at the
50% probability level. H atoms are excluded for clarity.
8 | J. Name., 2012, 00, 1-3
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Page 9 of 14
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Journal Name
ARTICLE
1
2
3
4
View Article Online
DOI: 10.1039/C8NJ06440G
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 4. Molecular structures of benzoxazines 20 (up), 21 (bottom) with atom
labeling. The thermal ellipsoids are drawn at the 50% probability level. H atoms
are excluded for clarity.
The bond lengths and angles are comparable with those of
similar benzoxazines previously described in the literature.12
However, there are not so many X-ray data of 1,3-
benzoxazines in the crystallographic database. Such
information is valuable with regard to theoretical studies that
usually use X-ray data as the starting point for geometry
optimization. CSD accommodates only 36 structures with the
1,3-benzoxazine structural motif and only one that contains an
acetylene fragment. The expected irregular chair conformation
was observed for all the compounds, which is overall
considered a driving force for the polymerization process.
The study of the thermal properties of benzoxazines 1–8
delivered information on their thermal stability, which is
crucial for polybenzoxazine formation. Overall, the thermal
polymerization of benzoxazine was carried out in the
temperature range of 200–250 C, depending on the structural
motif of the monomers. The results of the TGA analysis are
summarized in Fig. 5.
Fig. 5. TGA thermograms of uncured benzoxazine monomers.
The TGA profiles for 1–8 compounds showed T5% in the 227–
253 C range and T10% weight loss for temperatures between
247 and 270 C. Unfortunately, benzoxazines 1–3 do not
polymerize before thermal decomposition, in analogy to
compound 4.9 Despite the assumed design goal of
functionalized benzoxazines they undergo destruction into
difficult to identify products which disqualifies them in these
applications. Therefore, their acetylene derivatives, which are
usually less stable, were not tested in this regard. In parallel,
we paid more attention to the thermal properties and the
polymerization process of the long-alkyl-chain benzoxazines 5–
8. Figure 6 presents the DSC thermogram for benzoxazines 5–8
with the same alkyl chain arm and different modifications in
the aromatic ring. The sharp exotherms with the maximum
temperatures at 258, 257, 256, and 271 C correspond to the
ring opening of the oxazine ring. The first exotherm at lower
temperatures with the maximums at 139, 169, 173, and 179 C
is probably attributed to a cross-linking process similar to that
described earlier in the literature, while multiple exothermic
behavior patterns have been reported for alkyl-substituted
benzoxazine motifs.13
heat of
heat of
first exotherm
polymerization
second exotherm
polymerization
onset
max.
(°C)
onset
(°C)
max.
(°C)
monomer
(°C)
ΔH (J/g)
64.39
55.14
60.23
55.31
ΔH (J/g)
46.39
74.30
85.92
32.83
5
6
7
8
147.96
147.77
157.08
113.01
173.83
169.49
180.00
146.82
231.82
230.92
230.54
246.91
257.82
258.65
257.33
271.13
Fig. 6. DSC profile of benzoxazines 5–8.
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DOI: 10.1039/C8NJ06440G
Journal Name
6
7
8
9
ARTICLE
10
11
12
13
14
15
16
17
18
19
20
21
22
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45
46
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48
49
50
51
52
53
54
55
56
57
58
59
60
The benzoxazines were converted to polybenzoxazines by Benzoxazines have a suitable framework for biologically active
monomers melted and heated under vacuum at 180 C for 2 h, species design. The functionalization of benzoxazines by
next at 240 C, and at 250 C for 2 h in an air-circulating oven. acetylenic groups resulted in poorer solubility in ordinary
The obtained polybenzoxazines were brown and transparent. solvents, which led to difficulties during the study of their
The thermal stability of polybenzoxazines 5–8 are presented biological activity. Therefore their precursors (1-4) have been
by the TGA profiles shown in Fig. 7. Benzoxazines 1–4 undergo tested. A modification of the aryl core to include a steric
degradation during the polymerization process, and it was obstruction located in the para position and the removal of
impossible to obtain identifiable products. Polybenzoxazines the acetylenic fragment improved both the solubility and
are high-performance thermoset materials and therefore stability of the new benzoxazines. The preliminary
should have excellent thermal stability with high char yield. antimicrobial tests for compounds 1–8 were performed by disc
The polymers presented here showed a 10% weight loss diffusion assay used for the estimation of their potential
temperature between 306 and 325 C; the char yield was biological activity (see ESI, Table S6).7 On the basis of diffusion
determined at 800 C in the range of 15–22%.
tests, the potential of antimicrobial properties of tested
compounds could not be unambiguously determined, due to
their poor diffusion (3, 4), and additionally in the case of
compounds 6, 7 very poor solubility and precipitation in the
biological medium have been noticed. These preliminary
screening tests enabled the selection of the most active
compounds. Already at this stage, benzoxazines with a
substituent located in the ortho position relative to the oxygen
of the heterocyclic ring showed no activity against the tested
microorganisms. Despite this fact, tests were carried out using
the serial dilution method for the remaining benzoxazines. In
each case, no significant activities were obtained for the
highest concentrations as indicated under Table 1 as >400. For
the compounds that obtained the highest activity in screening
tests, detailed research was carried out, the minimal inhibitory
concentration (MIC) was determined using the serial dilution
method.8 Tetracycline and amphotericin B were used as
reference antimicrobial/antifungal agents at the therapeutic
dose of 30 µgcm-3. The investigated bacterial species were:
Gram-positive cocci (S. aureus, S. pseudintermedius, S.
agalactiae, S. canis), Gram-positive endospore-forming rods
(Bacillus spp.), and Gram-negative rods (E. coli, S. galinarum, P.
vulgaris, Pectobacterium spp.). The antifungal evaluation was
determined against yeast (C. krusei, C albicans, C. glabrata)
and filamentous fungi (Fusarium spp., Aspergillus spp., T.
harzianum). The microbial activity data (MIC or MBC/MFC) are
presented in Table 1.
sample
T5%[ºC]
317
325
307
306
T10%[ºC]
338
349
332
326
Yc[%]
18
15
22
19
5
6
7
8
Fig. 7. TGA of polybenzoxazines obtained from monomers 5–8.
The glass transition temperatures are extremely low in
comparison to classical benzoxazine derivatives. The liquid
nature of alkyl-chain-modified monomers and the high
processing windows indicate their high processable potential.
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DOI: 10.1039/C8NJ06440G
Journal Name
5
6
7
8
9
ARTICLE
10
11
12
13
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20
21
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Table 1. Antimicrobial activity of tested benzoxazines.
MIC µgcm-3
MBC/MFC µgcm-3
Microorganisms
1
2
5
1
2
5
Gram-positive cocci
S. agalactiae KP-Sag1
S. canis KP-Sac1
15.6±3a
15.6±3a
16.3±3a
16.3±3a
29.2±3b
50.0±0d
47.5±6cd
56.3±6d
50.0±0d
25.0±0a
25.0±0a
31.9±4a
28.8±3a
91.7±6c
70.8±10b
75.0±10bc
91.7±6c
75.0±0bc
87.5±6bc
87.5±6bc
81.3±6bc
33.3±3b
37.5±5b
45.8±3cb
S. pseudintermedius KP-Spi1
S. aureus PCM 2054
Gram-positive endospore forming rods
B. cereus PCM 1948
B. subtilis PCM 1949
25.0±5a
25.0±5a
25.0±5a
31.3±6ab
45.8±3cd
40.0±6bc
50.0±0cd
58.3±6d
81.3±9f
69.0±6def
100±0g
31.3±3a
35.0±5a
43.8±6b
56.3±6ab
62.5±9c
80.0±4d
92.0±6de
92.0±6de
100±0e
81.3±6d
100±0e
100±0e
B. mycoides PCM 2024
B. megatherium PCM 1400
100±0g
Gram- negative rods
E. coli PCM 2057
S. galinarum KP-Sg1
P. vulgaris PCM 542
P. aeruginosa PCM 2058
200±0c
400
400
400
33.3±6a
55.0±2b
60.0±0b
400
400
400
400
400
400±0c
400
400
400
50.0±0a
105±10b
120.0±0b
400
400
400
400
400
Gram-negative rods (plant pathogen)
P. sbp. carotovora IOR 1815
P. sbp. carotovora IOR 1822
P. atrosepticum IOR 1825
P. atrosepticum IOR 1826
46.9±3a
46.9±3a
50.0±0a
56.3±6a
48.3±1a
48.3±1a
48.3±1a
48.3±1a
187.5±10b
187.5±10b
400
62.5±6a
62.5±6a
75.0±9ab
75.0±9ab
96.7±2c
88.3±5bc
96.7±2c
96.7±2c
400
400
400
400
400
Fungal strains
C. albicans KP-Ca1
C. krusei KW-F117
38.1±5a
29.1±3a
50.0±0b
200±0c
200±0c
400±0e
400±0e
400±0e
200±0c
200±0c
200±0c
400
350±27d
350±27d
400e
47.5±2a
50.0±0a
50.0±0a
400±0c
400±0c
400
400±0c
400±0c
400±0c
400
350±27b
350±27b
400
C. globrata KP-Cg1
F. culmorum KP-F1
T. sbp. aggressivum KP-Tha1
F. moniliforme KP-Fm1
A. niger KP-An1
400
400
400
400
400
400
400
400
400
400
400
400
400
400
400
A. flavus KP-Af1
400
400
400
400
400
>400 – Inactivity at a dose 12.5 to 400 µgcm-3, data for 3, 4, 6-8 described are >400, and therefore they have been omitted for the clarity; ± – SD (standard deviation); Values followed by the same letter, are
not significantly different (p > 0.05 ). Tukey's multiple-range test was done alone for MIC and MBC values and alone for cocci species, for Bacillus spp., for plant pathogens, for gram-negative bacteria and for
fungi.
Compounds 1 and 5 did not exhibit any antibacterial activity to MCB = 105 µgcm-3), and P. vulgaris (MIC = 60 µgcm-1 and
Gram-negative bacteria (P. aeruginosa, P. vulgaris, S. MBC = 120 µgcm-1). Additionally, 1 and 2 were effective
galinarum, E. coli) at the tested concentrations, but compound against Gram-negative plant pathogenic Pectobacterium
2 exhibited strong activity against E. coli (MIC = 33 µgcm-3 strains (the range of MIC and MCB was 46.9–56.3 µgcm-3 and
and MCB = 50 µgcm-3), S. galinarum (MIC = 55 µgcm-3 and 62.5–96.7 µgcm-3, respectively). The most sensitive to 1, 2,
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and 5 were Gram-positive cocci and endospore-forming rods. was observed for 8 and 5, and the difference between them is
Among all the tested compounds, 1 showed the strongest also only in the aryl core, free or sizable substituents in the
View Article Online
DOI: 10.1039/C8NJ06440G
antibacterial activity against Streptococcus and Staphylococcus ortho position. These results could be a guide for further
species. The applied MIC and MBC doses (15.6–25.0 µgcm-3) biological studies because most of the previously tested
were up to 2- or 3-fold lower compared to 2 and 5. The lowest benzoxazine moieties were based on the aryl ring with free
degree of inhibition against endospore-forming rods was substituents or with methyl substituents bonded in this
observed for 1 (MIC ranged from 25.0 to 31.3 µgcm-3), and position.
5
6
7
8
9
the bactericidal effect ranged from 31.3 to 56.3 µgcm-3.
Finally, benzoxazines 1, 2, and 5 were studied for their
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The antifungal activity of the tested compounds was cytotoxic effect against BALB/3T3 mouse fibroblasts by SRB
determined against selected yeast and filamentous fungi. assay.9 The morphological anomalies of cells caused by the
Although, none of the benzoxazines showed higher efficacy studied benzoxazines were evidenced under
a contrast
than the referenced amphotericin B against the tested fungi, 1 microscope. The influence of the tested benzoxazines on the in
was found to be significantly potent against Candida species vitro growth of mouse fibroblasts is shown in Fig. 8.
with low values of MIC and MFB (29.1–50 µgcm-3). The
exchange of bromophenyl substituents in 1 for the alkyl chain
as in 5 led to the reduction of antifungal activity, and 5 showed
only anticandidal activity (MIC and MFB = 350 µgcm-3).
Overall, the tested compounds 1, 2, and 5 showed the
broadest range of biological activity exhibiting the highest
activity against Gram-positive, lower but interesting activity
against Gram-negative Pectobacterium spp., and additionally
antifungal effect against C. krusei, C. albicans, F. culmorum,
and T. aggressivum. Benzoxazine
1 indicated effective
inhibitory effect against Gram-positive bacteria strains and is
equipotent to the reference standard drug (tetracycline) used
against the tested bacterium. The structure-activity
relationships showed that the antimicrobial activity profile of
1,3-benzoxazines depends on the substituents present in the
aryl core. Benzoxazines, specifically those with the 1,3-motif,
show a wide range of biological activity, which causes
continuous interest in modifying and functionalizing the
structure of these compounds in the context of potential
bioapplications. Examples of such studies, concerning the
influence of the benzoxazine aromatic ring substituents on
biological activity are rare. Typically, the studies are focused
on the modification of the substituents on the heterocyclic
ring. Recently, the new class of anti-malarials based on
benzoxazine with phytophenols core derived from natural
product has been developed,1a where the main focus of the
studies was to highlight the effect of substituents located in
the benzene ring that may play an important role in the
biological activity. In this context, the antitubercular agents
based on 1,3-benzoxazine motifs have been evaluated.
Substitution pattern of aryl ring bonded to nitrogen atoms
clearly shows the differential sensitivity of Mycobacterial
strains.1b However, the modification of aryl core of
benzoxazine has not been discussed in this aspect. Whereas,
the appropriate compounds have been synthesized, what
more a similar relationships can be observed as presented in
this work, because the analog with free ortho position was the
Fig. 8. Impact of 1, 2, and 5 on the in vitro growth of 3T3 BALB fibroblasts. A –
cell growth stimulation, B – cell growth inhibition, C – cytotoxic effect, control Tz
(start test) – time added compounds to the cell culture and the tested dose,
Control (100%) – cell growth.
Benzoxazines 1 and 2 inhibit the growth of fibroblasts by about
50% at a dose of 5 µgcm-3, but compound 5 inhibited cell
growth (50%) at a five-fold higher dose (~25 µgcm-3).
Compounds 1 and 2 inhibit cell growth, at a similar level, in the
ranges of 5–25 µgcm-3 and 5–35 µgcm-3, respectively. Above
these concentrations, benzoxazines 1 and 2 exhibit cytotoxic
effects on cells. In contrast, 5 decreases cell growth at a higher
dose, ranging from 10 to 65 µgcm-3. In addition, compound 5,
at a dose of 5 µgcm-3, causes the growth of cell proliferation,
which suggests a positive effect on cells (vitality and growth
increases). The photographs (ESI Photos 1–12 and Fig. S11–18)
showed the effect of benzoxazines 1 and 5 on the vacuolation
of fibroblast cells and cytopathic effects. Observed changes in
the cells included vacuolation and the change of shape.
Microscopic observations confirm the results of the SRB test.
Conclusions
most active compared to substituted one. The bulky The family of new N-substituted benzoxazines were
obstruction in the ortho position in relation to the oxygen synthesized and fully characterized. Their molecular structure
atom of the oxazine ring resulted in the complete loss of was determined using spectroscopic methods and X-ray
benzoxazine antimicrobial efficacy. Benzoxazine 1 with the crystallography. The focus of our study was the experimental
free ortho position displayed significant activity and, in verification of the relation of these simple molecular motifs to
contrast, the analog 4, containing tert-butyl substituents, is potential applications as antimicrobial species. One of them, 1,
inactive against all the screened microbes. Similar behavior showed significant activity with MIC values in a range
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E. Matuschek, D. F. J. Brown and G. Kahlmeter, Clin.
equipotent and in some examples better than tetracycline
against the tested bacterium. These results may be useful for
the application of 1 as a new antibacterial and biocidal agent.
The ortho position of the aryl core is crucial for the biological
activity of 1,3-benzoxazines; sizable substituents caused the
deactivation of these antimicrobial agents. Analysis of the
literature data leads to the same conclusions. The cytotoxic
assay results revealed that benzoxazines with long alkyl chains
offered remarkable cell viability. The new benzoxazine
monomers (5-8) have been polymerized. The glass transition
temperatures are extremely low in comparison to classical
benzoxazine derivatives. The liquid nature of alkyl-chain-
modified monomers and the high processing windows indicate
their high processable potential.
View Article Online
Microbiol. Infect., 2014, 20, 255–266. DOI: 10.1039/C8NJ06440G
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The author gratefully acknowledge the National Science Centre
in Poland (Grant 2017/25/B/ST5/00597).
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