Fluorescent Probes from Aromatic Polycyclic Nitrile Oxides: Isoxazoles versus Dihydro-1λ3,3,2λ4-Oxazaborinines

Article abstract of DOI:10.1002/open.201900137

Anthracenenitrile oxide undergoes 1,3-dipolar cycloaddition reaction with propargyl bromide affording the expected isoxazole as single regioisomer, suitably synthetically elaborated and functionalized with a protected triple bond. The introduction of a br

Full text of DOI:10.1002/open.201900137

DOI: 10.1002/open.201900137
Full Papers
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Fluorescent Probes from Aromatic Polycyclic Nitrile Oxides:
Isoxazoles versus Dihydro-1λ3,3,2λ4-Oxazaborinines
Mattia Moiola,[a] Antonio Bova,[a] Stefano Crespi,[a] Misal G. Memeo,[a] Mariella Mella,[a]
Herman S. Overkleeft,[b] Bogdan I. Florea,[b] and Paolo Quadrelli*[a]
Dedicated to the memory of Prof. Pierluigi Caramella
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Anthracenenitrile oxide undergoes 1,3-dipolar cycloaddition
reaction with propargyl bromide affording the expected
isoxazole as single regioisomer, suitably synthetically elaborated
and functionalized with a protected triple bond. The introduc-
tion of a bromine atom at the position C10 of the anthracene
moiety allows for inserting a variety of aromatic and hetero-
cyclic substituents through Suzuki coupling. A two-way syn-
thetic route can lead to simple isoxazole derivatives or, after
NÀ O bond reductive cleavage and BF3 complexation, enamino
ketone boron complexes. The photophysical properties of both
the substituted isoxazoles and the corresponding boron
complexes were investigated to show the potentialities for the
employment as fluorescent tags in imaging techniques. The
quite good quantum yield values confirm the suitability of
these compounds in the cellular environment. Scope and
limitations of the methodology are discussed.
1. Introduction
The chemistry of isoxazoles dates from 1888,[1] when Claisen
proposed the correct structure for a compound isolated some
years before from the reaction of hydroxylamine with benzoyla-
cetone. In 1891, Claisen published a paper in which the
fundamental outline of the isoxazole chemistry was reported;
after that fundamental work, a few other authors explored the
isoxazole chemistry but the reemergence of interest in these
heterocycles[2] must be ascribed to Quilico and co-workers as a
consequence of their research on the reaction of nitric acid with
C�C triple bond-bearing compounds initiated in the thirties of
last century.[3] 1,3-Dipolar cycloadditions of nitrile oxides 1 to
alkynes of type 2 can be undoubtedly considered a pillar of the
synthesis of isoxazoles 3 (Scheme 1). The uses of isoxazoles
themselves and their elaborated compounds in organic syn-
thesis have been already accounted in several books and
papers, being a mature subject of organic chemistry.[2,4] Among
these methods, it is worth mentioning the readily cleavage at
the NÀ O bond by several reducing agents,[2] and metal carbonyl
[Mo(CO)6] in particular, to afford the β-enamino ketones of type
Scheme 1. From nitrile oxides to 3,5-disubstituted isoxazoles, NÀ O bond
reductive cleavage and boron complexation: the synthetic route to
fluorescent probes. Ar=Anthryl.
4.[5] The importance of these structures lies into the presence of
two functional groups, the amino and the carbonyl groups that,
among others, can serve as ligands in coordinative compounds.
A typical case is that of boron complexation leading to the 2,2-
difluoro-dihydro-1λ3,3,2λ4-oxazaborinine of type 5. The versatil-
ity of this strategy was recently applied to synthesize structures
designed for imaging. Albeit the number of works devoted to
this topic is increasing, the applications of the probes
constructed via this pathway remain quite low so far.[6]
In a recent conceptual work we successfully employed the
1,3-dipolar cycloaddition of stable aromatic nitrile oxides to
afford novel fluorescent compounds. The reported strategy is
the cornerstone to furnish boron-substituted complexes suit-
able for biochemical applications; the nitrile oxide-based
protocol is cleaner and selective with dipolarophiles and can be
a useful alternative to the use of azides. Indeed, once properly
derivatized can found proper use in the activity-based protein
[a] Dr. M. Moiola, Dr. A. Bova, Dr. S. Crespi, Dr. M. G. Memeo, Prof. M. Mella,
Prof. P. Quadrelli
Department of Chemistry
University of Pavia
Viale Taramelli 12, 27100 – Pavia (Italy)
E-mail: paolo.quadrelli@unipv.it
[b] Prof. H. S. Overkleeft, Dr. B. I. Florea
Institute of Chemistry
University of Leiden
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900137
©2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial NoDerivs License, which permits use and dis-
tribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
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profiling (ABPP).[7] ABPP has emerged as one of the most
powerful tools to gain insights into complex biological
systems,[8] and the aim resides in the visualization of the active
forms of the enzymes using chemical probes directed to the
active site of a target protein, resulting in the selective labeling
of the sole catalytically active form of the enzyme.[9]
ties and possible differences in terms of selectivity. To detect
the fluorescence of the probes adducts to epoxomicin while
they bind the proteasome, several different light sources were
tried to excite the fluorophores, but unluckily they did not
match the excitation wavelength of our probes and no
fluorescence were registered. Some possible explanations were
considered. We cannot state if these probes maintained their
fluorescence during the interaction with the proteasome; it was
also possible that the fluorophores maintained their optical
properties but the excitation was not efficient. Alternatively, we
cannot exclude a potential quenching or degradation (insta-
bility of the boron complex) during the interaction with the
proteasome.[10]
For these reasons, we started revising and enlarging the
library of compounds prepared on the base of isoxazole
chemistry in order to synchronize better synthesis and
application, fluorescent activity and stability of products.
Suitably designed isoxazoles can show intense fluorescent
properties, too, even stronger than the corresponding boron
complexes. That is why in this study, we present the synthetic
approach to fluorescent brand-new compounds, comparing the
photochemical behavior of the uncomplexed and complexed
probe couples with the aim to enhance the fluorescence
quantum yields and stability of compounds in order to become
competitive and possibly more convenient than commercial
ABPP traditional probes.
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5
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From a structural point of view, chemical probes consist of
three different parts: a tag for recognition, a linker of variable
length and a warhead (ligation handle) containing the func-
tional groups to link the probe with the target substrate with
highly specific interactions that make the probe selective for a
well-defined biological structure (see Scheme 2). In our previous
work we detailed the synthetic strategy and the fluorescence
study of compound 5 that structurally follows the aforemen-
tioned paradigms. The compound is synthesized starting from a
1,3-dipolar cycloaddition reaction between a 4-(prop-2-yn-1-
yloxy)benzene of type 2 (R=À C�CÀ TMS), affording the 5-
substituted isoxazole 3 in very good yields as a single
regioisomer. The outcome of the transformation nicely follows
the predictions based on the frontier orbital theory.[7] Reductive
cleavage of the NÀ O bond and complexation with BF3 furnishes
the corresponding fluorescent boron complex 5 (Scheme 1).
The molecule bears an anthryl substituent (Ar=9-anthryl) on
the tag and a triple C�C bond on the warhead end terminus
(R=À C�CH) of the probe, suitable for click-chemistry applica-
tions and late-stage functionalization. Furthermore, we also
presented a strategy to prepare chemical probes that maintain
the same tag structure, while bearing warheads prone to an
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orthogonal functionalization compared to 5. Indeed, in com- 2. Results and Discussion
pound 5 the triple bond requires the use of a substrate bearing
a dipole (typically an azido-derivative) to connect the two sides
of the chemical reporter (Scheme 2). The newly designed
compound 6 bears an oxime moiety on the warhead that is
suitable to be oxidized to nitrile oxide. Hence, the probe is
another 1,3-dipole that can be attached to a double (or triple)
bond located on a target substrate. Being a nitrile oxide, probe
6 can enter the ligation process without the help of metal ions
that normally are needed in azide-based protocols, affording a
metal-free methodology to be easily applied in a cellular
environment.
The two chemical probes 5 and 6 have been compared
from the photophysical point of view and tested by coupling
them with differently functionalized epoxomicin derivatives.
Ultimately, competitive ABPP assays will be performed with the
aim to verify the maintenance of proteasome inhibitor proper-
Anthracenenitrile oxide 1 was prepared in very good yields
(81%) from the commercially available corresponding oxime
°
under standard NCS treatment in chloroform solution at 0 C for
3 h, by adapting literature reported procedures.[11,12] The solid
stable aromatic nitrile oxide 1 can be stored for months at low
temperature and was used, without any further purification or
particular adaptations of experimental procedures, in the 1,3-
dipolar cycloaddition with propargyl bromide (2) (Scheme 3).[7]
The 3-(anthracen-9-yl)-5-(bromomethyl)isoxazole (3) was ob-
tained as single regioisomer in 75% yield as a yellow solid (m.p.
°
81–85 C from ethanol) and was submitted to bromination
°
reaction under mild conditions (Br2, DCM at 63 C for 2 h) to
insert a bromine atom in the position 10 of the anthracene
moiety, to afford the 3-(10-bromoanthracen-9-yl)-5-(bromo-
methyl)isoxazole (7) in 80% yield as a straw coloured high
[13]
°
melting solid (m.p. 129–132 C from ethanol).
The commercially available 4-iodophenol (8) was derivatized
with ethynyltrimethylsilane (9) under typical Pd(0)-catalyzed
conditions to afford the 4-((trimethylsilyl)ethynyl)phenol (10) in
80% yield. The ethynyl derivative 10 was coupled with the
isoxazole derivative 7 by treatment with mild basic conditions,
°
to afford compound 11 (80% yield, m.p. 126–130 C from
ethanol) that represent the starting point for a series of
derivatizations at the carbon C10 of the anthracene moiety.
From the structural point of view all the compounds were
1
fully characterized. In particular compound 3 shows in the H
Scheme 2. Probe structures: dipolarophile and dipole precursor ligation
handles structures.
NMR spectrum (CDCl3) the diagnostic signal of the H4 isoxazole
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Scheme 4. Probe synthesis (I): from isoxazole derivative 11 to compounds
14a–h.
Table 1. Yields, physical chemical data of compounds 13a–h and 14a–h.
Compd. Yield
(%)
Mp ( C)
(from Cy/
AcOEt)
1H NMR
(δ, CDCl3)
°
13
a
b
c
d
e
f
80
72
63
66
30
74
211–215
178–182
124–128
Sticky oil
Sticky oil
Semi solid/oil
–
–
1.49 (s, 3H, CH3)
3.99 (s, 3H, OCH3)
3.59 (s, 6H, OCH3)
1.50 (t, 3H, CH3); 4.51 (q, 2H,
Scheme 3. Synthetic pathway to the 3-(10-bromoanthracen-9-yl)-5-((4-((tri-
methylsilyl)ethynyl) phenoxy)methyl)isoxazole (11).
OCH2)
g
h
–
44
–
–
–
211–214
proton at δ 6.59 as a singlet, also found in the bromo-derivative
14
1
7 at δ 6.57. In the H NMR spectrum (CDCl3) of compound 11
a
b
c
d
e
f
70
81
59
66
4
206–208
176–178
198–200
181–183
175–180
65–68
–
–
the signal of the TMS protecting group can be found at δ 0.27
along with the H4 isoxazole proton singlet at δ 6.61.
1.91 (s, 3H, CH3)
3.99 (s, 3H, OCH3)
3.59 (s, 6H, OCH3)
1.51 (t, 3H, CH3); 4.51 (q, 2H,
The isoxazole derivative 11 was then coupled with a series
of boronic acids 12a–j according to a typical Pd(0)-catalyzed
Suzuki procedure[14] to afford the 10-substituted anthracen-9-yl-
isoxazoles 13a–h in good yields whose triple bond was
deported under standard TBAF conditions[7] leading to the
desired fluorescent isoxazole derivatives 14a–h in very good
yields (Scheme 4).
Table 1 collects the chemical yields and the relevant
physical-chemical data of both compounds 13 and 14. As a
general comment, the yields are pretty good in all the cases,
except for 13e and 14e that were found quite unstable, and
the synthetic procedures were standardized for all the sub-
stituents introduced and were found robust and reliable.
Just in one case the intermediate 13g could not be isolated
because TMS deprotection occurred under the experimental
conditions affording the final compound 14g. Most of the
products are solids with a few exceptions.
32
OCH2)
g
h
84
81
104–105
185–190
–
–
A second synthetic route was followed to prepare the boron
complexes relative to the isoxazoles compounds 14a–h and the
experimental steps are shown in Scheme 5. The NÀ O bond
cleavage[15] in the isoxazole moieties was performed using Mo
°
(CO)6 in a mixed solvent of CH3CNÀ H2O (8:2, 70 C, 4 h) to give
the (Z)-4-amino-4-(10-aryl-substituted-anthracen-9-yl)-1-(4-((tri-
methylsilyl)ethynyl)phenoxy) but-3-en-2-ones 15a–h as a yel-
low/yellowish solids in 50–80% yields. As previously
1
reported,[7,10] the signals shown in the H NMR spectrum denote
the existence of an intramolecular hydrogen bond between the
amino group and the carbonyl functionality obtained from the
NÀ O bond cleavage. As a consequence, one of the NH2 protons
is found in the range δ 5.63–5.90 ppm while the other one is
strongly deshielded in the range δ 10.44–10.53 ppm. These
latters are the direct evidence of the six-membered cyclic array
kept together by the hydrogen bonding. Likewise, the newly
The structures were confirmed on the basis of the
corresponding analytical and spectroscopic data. In particular,
1
in the H NMR spectra (CDCl3) the most relevant changes in
compounds 13, compared with 11, is the thicken of the
aromatic region between δ 7.0–8.5 ppm and the presence of
new signals in the cases of products 13c–f due to the phenyl
ring substitution.
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(CDCl3) the presence of the �CH proton signals at δ 3.03 ppm
for all the compounds clearly indicated the successful depro-
tection. With reference to the synthetic route shown in
Scheme 5, we precise that the entire described methodology is
valid for all the compounds with the single exception of 17g
that was obtained directly from 14g, since the TMS protection
could not be saved during the synthesis. The experimental
section reports the characterization of the enamino ketone
15 g’ having the structure shown in Scheme 5 without the TMS
group.
We performed some UV-Vis and fluorescence studies on
both the isoxazole derivatives 14a–h and the corresponding
boron complexes 17a–h, comparing the results with those
previously obtained for compounds 5 and 6 (see Scheme 2).[7,10]
These studies will be propaedeutic to evaluate the potential use
of the new compounds as chemical probes in ABPP inves-
tigations.
The oxime derivative 6 showed an absorption band at
384 nm (ɛ=5.00·103 molÀ 1) with a shoulder at 238 nm and
vibrational peaks. Analogously, the BF2 complex 5 shows an
absorption band at 387 nm (ɛ=8.20·103 molÀ 1) along with
several vibrational peaks.[7,10] Table 2 resumes the main photo-
physical characteristics of compounds 14a–h and 17a–h. The
absorption and emission spectra of compounds 14c and 17c in
DCM are depicted in Figure 1, here reported as paradigmatic
examples. All the remaining spectra in all the solvents are
collected in the Supporting Information. Both the isoxazoles
and the BF2 complexes possess an absorption around 400 nm,
characterized by a vibrational structure, attributable to the
anthryl moiety. Interestingly, the change in functional groups
on the phenyl ring does not drastically shift the absorption
peak in the near UV.
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Scheme 5. Probe synthesis (II): from isoxazole derivatives 13a–h to com-
pounds 17a–h.
formed carbonyl groups can be found in the 13C NMR spectra
around δ 194 ppm.
The enaminoketones 15a–h were used as ligand for boron
complexation,[16] achieved by addition of 10 equivalents of
BF3 ·Et2O in an anhydrous DCM solution, in the presence of
excess Et3N. Protected boron complexes 16a–h were not
isolated since the reaction conditions promoted partial depro-
tection from TMS group in all the cases. As a consequence, the
synthetic protocol was properly adapted in order to skip
immediately on the final products 17a–h by treating the
resulting reaction mixtures under standard TBAF conditions
leading to the desired boron complexes 17a–h. The yields are
in the range 30–60%. With the exception of compound 17e
that could not be synthetized because of the very low yields of
13e available, the structures of all the other boron complexes
were confirmed by full characterization by analytical and
spectroscopic methods and, in particular, in the 1H NMR spectra
The fluorescence emission spectra of 14a–h and 17a–h are
not influenced in their relative shapes and λem (emission
wavelength) by the change of solvents. The fluorescence of
Table 2. Quantum Yields (ΦF) values for compounds 14a–h and 17a–h in the listed solvents.a
DCM
λads
MeOH
λads
DMSO/H2O
χ
χ
χ
ɛ
λem
Stkb
ɛ
ΦF
λem
Stkb
ɛ
ΦF
λads
λem
Stkb
ΦF
14
a
b
c
d
e
f
394
393
394
395
394
394
395
395
418
436
434
441
432
438
430
429
24
43
40
46
38
44
35
34
8.37.103
8.71.103
6.76.103
1.17.104
6.99.103
7.02.103
7.44.103
9.70.103
0.00
0.90
0.99
0.14
0.94
0.73
0.13
0.81
391
390
391
392
391
391
392
392
419
427
425
443
425
434
423
424
28
37
34
51
34
43
31
32
8.09.103
7.65.103
6.48.103
9.20.103
6.41.103
6.16.103
6.76.103
7.84.103
0.00
0.92
>0.99
0.11
0.96
0.97
0.10
0.74
391
394
396
397
396
398
393
396
421
433
432
452
437
442
435
434
30
39
36
55
41
44
42
38
6.89.103
6.07.103
6.62.103
7.45.103
2.18.103
7.48.103
8.37.103
9.40.103
0.00
0.17
0.91
0.05
0.65
0.71
0.22
0.89
g
h
17
a
b
c
d
e
f
396
395
395
397
–
396
396
396
437
491
496
511
–
492
483
488
41
96
101
114
–
96
87
92
3.48.105
7.88.103
6.91.103
8.38.103
–
0.02
0.10
0.07
0.08
–
0.09
0.01
0.12
392
391
392
393
–
392
392
392
430
495
496
493
–
490
460
486
38
9.97.103
7.89.103
6.56.103
9.10.103
–
0.00
0.02
0.02
0.01
–
0.04
0.01
0.03
396
396
396
397
–
396
396
402
434
491
497
463
–
482
455
460
38
95
101
66
–
86
59
58
8.91.103
8.01.103
6.68.103
7.17.103
–
0.00
0.03
0.02
0.05
–
0.04
0.01
0.08
104
104
100
–
98
68
1.19.104
7.82.103
3.90.103
1.27.104
7.77.103
7.25.103
7.51.103
7.30.103
1.27.103
g
h
94
a. Concentration range 3·10À 7–5·10À 5 M in all the solvents. b. Stk, Stokes shifts. – Dielectric constants: DCM, 8.9; MeOH, 33; DMSO/H2O, 55. – Viscosities (cP at
20 C): DCM, 0.44; MeOH, 0.55; DMSO/H2O, 3.30. c. Molar absorptivity, ɛ (L.molÀ 1.cmÀ 1).
18
°
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omatics 14h) give better results if compared with electron rich
rings (14d and 14g). In compounds 5 and 6,[7,10] the change in
viscosity and polarity of the solvent mixture – hence moving
from DCM, to MeOH and DMSOÀ H2O 1:1 – is accompanied
with a decrease in the ΦF. Such an effect is not pronounced in
the series of the isoxazoles. Indeed compounds 14c, e and 14f
are promising for further testing their applicability as molecular
probes.
1
2
3
4
5
6
7
8
All the BF2 derivatives are, on the other hand, weakly
fluorescent suggesting a potential limitation in biological
environment. All the nitro derivatives are outliers in both the
two series of compounds analyzed. The compounds are non-
fluorescent, except for 17a in DCM (ΦF =0.02) and possess the
lower Stokes shift. The facile Intersystem Crossing to the triplet
state due to the presence of the NO2 chromophore could
explain the absence of the fluorescence in the nitro substituted
molecules.[19]
The difference between the two series of compounds
analyzed is mirrored by the nature of the excited state
populated after irradiation, calculated at the TD-CAM-B3LYP/
def2-TZVP//B3LYP/6-31G(d) level of theory.[20] In the case of
compounds 14, the excitation is confined to the anthryl moiety
(exemplified in Figure 2 by compound 14b). On the other hand,
compounds 17 show also a partial charge transfer to the
boracycle (17b in Figure 2).
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10
11
12
13
14
15
16
17
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Figure 1. UV-vis (black) and fluorescence (blue) spectra of compounds 14c
(A) and 17c (B) in DCM (2·10À 5 M solutions).
3. Conclusions
In conclusion, this work has contributed to show the possible
and reliable application of the nitrile oxide chemistry in the
biomedical field, especially as starting material for the synthesis
of some fluorescent probes. The chemistry of isoxazoles in the
field of imaging probes seems to be promising and a valuable
alternative to azides, avoiding the use of copper (metals are
detrimental in a biological environment) and the chemistry is
cleaner and safer; the construction of the boron complexes is
simple, reliable and robust.[13] The methodology can be applied
to a variety of structures having the aldehydes as starting
compounds. In this light, we have demonstrated the possibility
to expand the application of 1,3-dipolar cycloaddition reactions
in order to obtain a group of easy-derivatizable fluorescent
probes. Starting from a known stable nitrile oxide and adapting
a simple chemical approach, we were able to obtain two
families of fluorescent compounds of type 14 and 17. One of
the key points of the present project was the enhancement of
the fluorescence quantum yields; this goal was achieved by
preparing derivatized isoxazole compounds.
The optical properties of these compounds suggest the
potential for the employment as fluorescent tags in imaging
techniques. In spite of the drops of quantum yields values for
compound 6 with respect to 5, the quite good quantum yields
values maintained by some of these compounds in the DMSO/
H2O mixture confirm the suitability of these compounds in the
cellular environment. The ABPP assays performed did not give
us an ultimate answer about the behavior of compounds 5 and
6 in SDS-page analyses. The coupling tests performed showed a
14a–h is characterized by a λem shifted around ca. 40 nm
compared to the absorption wavelength (λabs). Compounds
17a–h are characterized by higher Stokes shifts that reach the
100 nm. These findings suggest that the dipole moments of the
molecule in the ground and excited states are almost the
same.[17] These results show the same trend observed for
compound 5 and 6.[2] The quantum yields of fluorescence
emission (ΦF) are considerably higher in the isoxazole deriva-
tives. Compound 14c possess almost quantitative emission in
the solvents examined, with MeOH the one in which the better
performances are obtained (ΦF >0.99). The nature of the
substituents has a role in the observed ΦF in compounds 14a–
h. Both the electronic and steric nature of the moieties modify
the emission properties of the final compound. Indeed, the
most fluorescent compounds possess ortho substituents in the
aryl moiety attached to the anthryl one (14c and 14e). It is
possible that the increased steric hindrance diminishes the
electronic interactions between the anthryl moiety and its
substituents, or shields the chromophore from the interaction
with solvent molecules that could deactivate the excited state.
In this case, the electronic nature of the substituent in ortho
is not relevant for the ΦF. We can postulate that the ortho
substituents block the anthryl and aryl rings in perpendicular
fashion, more than the para ones. In the latter case the
electronic effects are more pronounced. Hence, the electron
attracting groups (14b and 14f and electron poor heteroar-
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Figure 2. Electronic orbitals involved in the lower allowed electronic transition from (A) HOMO (top) to LUMO (bottom) of 14b; (B) HOMO (top) to LUMO
(bottom) of 17b. The TD-CAM-B3LYP/def2-TZVP//B3LYP/6-31G(d) level of theory was applied.
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procedure.[12] Solvents and other reagents were purchased and
used without any further purification.
good reactivity for the probes 5 and 6 even with short peptide
chains such as the Epoxomicin. Improvements on the
fluorescence properties of enamino ketone-based boron com-
plexes were needed but the results demonstrated the better
performances of the isoxazoles. These results reasonably
indicate the isoxazole as candidate for imaging studies. The
tuning of the optical properties of the probes is a crucial point
for the application in imaging techniques: at the moment the
shift towards higher wavelengths remains a target to be
achieved with the proposed chemical approach. Future devel-
opments of the synthetic strategy are currently under inves-
tigation; specifically we have designed the synthetic approach
to fully conjugated isoxazole structures of type 18 and the
corresponding boron complexes of type 19 with the specific
target to have further insights on the relative bathochromic
shifts in the presence of the reported substituents on the
aromatic moieties (Figure 3).
1
2
3
4
5
6
7
8
9
Synthesis of the 3-(anthracen-9-yl)-5-(bromomethyl)isoxazole 3. In a
250 mL flask 4.45 g (20.3 mmol) of anthracenenitrile oxide (1),
dissolved in 50 mL anhydrous DCM, were added dropwise to 80 mL
anhydrous DCM solution of 2.65 g (2 mL, 22.3 mmol) of propargyl
bromide (2). The reaction was kept in the dark and under stirring at
room temperature for 48 hours. Then the mixture was diluted with
DCM (30 mL) and the solution was washed with Brine (3×50 mL)
and dried over anhydrous Na2SO4.
The solvent was evaporated and the crude residue was purified by
column chromatography, giving a yellow solid of 3 in 75% yield
(5.20 g).
10
11
12
13
14
15
16
17
18
19
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Mp: 81–85 C from cyclohexane/ethyl acetate. FT-IR: νC=N 1598 cmÀ 1
;
°
νC=C 1626 cmÀ 1
.
1H-NMR (δ, CDCl3): 4.68 (s, 2H, CH2); 6.59 (s, 1H,
C=CÀ H); 7.51 (m, 4H, anthr.); 7.87 (m, 2H, anthr.); 8.08 (m, 2H,
anthr.); 8.60 (s, 1H, anthr.). 13C-NMR (δ, CDCl3): 18.6; 107.2; 122.4;
125.0; 125.3; 125.4; 126.6; 128.5; 129.1; 130.5; 131.0; 161.2; 167.8.
Elemental Analysis: Calculated for C18H12BrNO (MW=338.20): C,
63.93; H, 3.58; N, 4.14. Found: C, 63.94; H, 3.58; N, 4.13.
Synthesis of the 3-(10-bromoanthracen-9-yl)-5-(bromomethyl)isoxa-
zole 7. In a 500 mL round-bottom flask 2.60 g (0.833 mL, 16.3 mmol)
of Br2, diluted in 40 mL anhydrous DCM, were added dropwise to
250 mL anhydrous DCM solution of 5.00 g (14.8 mmol) of cyclo-
adduct 3. The reaction was kept in the dark heating at reflux and
under stirring for 2 hours. Then the mixture was diluted with DCM
(20 mL) and the solution was washed with 5% Na2S2O4 solution (3×
35 mL) and water (3×50 mL) and finally dried over anhydrous
Na2SO4.
Figure 3. Future perspectives in the synthesis of fluorescent compounds.
The solvent was evaporated and the crude residue was purified by
column chromatography, giving a yellow solid of 7 in 90% yield
(5.56 g).
Having some of the synthetized isoxazoles unexpected
intense fluorescent properties, even stronger than the corre-
sponding boron complexes, the planned SDS-page analyses on
coupled epoxomicin compounds will direct future develop-
ments in the application of nitrile oxide chemistry to the
synthesis of fluorescent tags.
°
Mp: 128–132 C from cyclohexane/ethyl acetate. FT-IR: νC=N
1598 cmÀ 1; νC=C 1636 cmÀ 1. 1H-NMR (δ, CDCl3): 4.68 (s, 2H, CH2); 6.57
(s, 1H, C=CÀ H); 7.51 (m, 2H, anthr.); 7.63 (m, 2H, anthr.); 7.48 (d, 2H,
J=9 Hz, anthr.); 8.63 (d, 2H, J=9 Hz, anthr.). 13C-NMR (δ, CDCl3):
18.1; 106.8; 122.2; 122.8; 125.4; 126.4; 127.7; 129.7; 130.6; 160.6;
167.7. Elemental Analysis: Calculated for C18H11Br2NO (MW=417.10):
C, 51.83; H, 2.66; N, 3.36. Found: C, 51.84; H, 2.67; N, 3.35.
Synthesis of the 3-(10-bromoanthracen-9-yl)-5-((4-((trimethylsilyl)
ethynyl)phenoxy)methyl)isoxazole 11. In a 250 mL round-bottom
flask 7.70 g (55.6 mmol) of K2CO3 were added to 120 mL acetone
solution of 2.90 g (6.95 mmol) of compound 7 and the mixture
Experimental Section
General. Melting points (mp) are uncorrected. Elemental analyses
were performed on a FlashSmart™ elemental analyzer. IR spectra
were registered using a FT-IR Perkin-Elmer RX-1 spectrometer [Nujol
°
heated at 56 C. An acetone solution (100 mL) of 1.71 g (9.04 mmol)
of 4-((trimethylsilyl)ethynyl)phenol (10) was then added dropwise
and the mixture was allowed to react at reflux and under stirring
overnight. The mixture was then filtered and the solvent evapo-
rated at reduced pressure. The brown residue was taken up with
DCM (100 mL) and the organic phase washed with brine (3×40 mL)
and finally dried over anhydrous Na2SO4.
1
mulls or dissolving the analyzed products in DCM (film)]. H-NMR
and 13C-NMR were registered on Bruker AVANCE 300 spectrometers
in deuterated solvent solutions as specified. The chemical shifts are
expressed in δ (ppm), using tetramethylsilane as internal reference.
Coupling constants (J) are expressed in Hertz (Hz).
Evaporation of the solvent left the crude compound 11 that was
purified by column chromatography, giving a yellow fluorescent
solid in 82% yield (3.00 g).
Chromatographic columns were performed using Kieselgel 60, 70–
230 (Merck), with a BIOTAGE MPLC or BIOTAGE Isolera One with KP-
SIL columns; the elution solvents were cyclohexane/ethyl acetate
from 9:1 to pure ethyl acetate.
°
Mp: 126–130 C from cyclohexane/ethyl acetate. FT-IR: νC=N
1598 cmÀ 1; νC=C 1676 cmÀ 1. 1H-NMR (δ, CDCl3): 0.27 (s, 9H, CH3); 5.40
(s, 2H, CH2); 6.61 (s, 1H, C=CÀ H); 7.00 (AA’BB’, 2H, arom.); 7.51 (m,
4H, anthr.); 7.66 (m, 2H, anthr.); 7.81 (AA’BB’, 2H, arom.); 8.64 (m, 2H,
anthr.). 13C-NMR (δ, CDCl3): À 0.1; 61.5; 77.1; 107.0; 114.7; 125.9;
126.7; 127.1; 128.1; 130.1; 131.1; 133.6; 157.7; 160.7; 168.1.
Elemental Analysis: Calculated for C29H24BrNO2Si (MW=526.51): C,
66.16; H, 4.59; N, 2.66. Found: C, 66.15; H, 4.58; N, 2.65.
UV-Vis spectra were registered with
a Jasco V-550 UV/VIS
Spectrophotometer; the fluorescence spectra were registered with
a Perkin-Elmer LS 55 Luminescence Spectrometer.
Materials. Anthracenenitrile oxide
1 was prepared from the
corresponding commercially available oxime according to known
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General procedure for the synthesis of the 3-(10-aryl-subnstituted-
anthracen-9-yl)-5-((4-((trimethylsilyl)ethynyl)phenoxy)methyl)isoxazole
13a–h. In a 100 mL round-bottom flask 0.50 g (0.95 mmol) of
compound 11 were dissolved in 15 mL toluene. An ethanol solution
(10 mL) of 1.90 mmol of the boronic acids 12a–h was mixed with
the previously prepared solution of 11. A water solution (5 mL) of
700 mg (6.60 mmol) of Na2CO3 was then added and the mixtures
were allowed to react at room temperature under stirring and
nitrogen atmosphere for 30 minutes. Pd(PPh3)4 (17 mg, 0.014 mmol)
were then added to the reaction mixtures and the resulting
2H, arom.); 8.32 (m, 2H, anthr.). 13C-NMR (δ, CDCl3): À 0.0; 14.4; 61.2;
61.7; 107.1; 114.9; 125.6; 125.7; 126.4; 126.8; 129.5; 129.7; 130.3;
131.3; 133.7; 157.9; 161.2; 166.5; 168.1. Elemental Analysis: Calcu-
lated for C38H33NO4Si (MW=595.77): C, 76.61; H, 5.58; N, 2.35.
Found: C, 76.60; H, 5.55; N, 2.34.
1
2
3
4
5
6
7
8
9
13g: this compound could not be isolated because, under the
reaction conditions, deprotection occurred to afford 14g.
°
13h: Yield: 44%. Mp: 211–214 C from ethyl acetate. FT-IR: νC=N
1610 cmÀ 1
.
1H-NMR (δ, CDCl3): 0.27 (s, 9H, CH3); 5.45 (s, 2H, CH2);
°
solution was heated at 80 C for 3 h. The mixtures were then cooled
6.73 (s, 1H, C=CÀ H); 7.02 (d, 2H, arom.); 7.51 (m, 7H, anthr.); 7.93 (d,
2H, arom.); 8.22 (d, 1H, arom.); 8.62 (s, 1H, arom.); 9.54 (s, 1H, arom.).
13C-NMR (δ, CDCl3): À 0.0; 61.5; 107.1; 114.7; 125.3; 125.8; 125.9;
126.4; 126.5; 127.8; 128.0; 128.2; 130.0; 130.2; 130.7; 133.0; 152.2;
157.7; 161.0; 168.0. Elemental Analysis: Calculated for C44H34N2O2Si
(MW=650.85): C, 81.20; H, 5.27; N, 4.30. Found: C, 81.20; H, 5.26; N,
4.31.
down, 50 mL water were added and the organic phases separated.
The water phases were furtherly extracted with DCM (3×30 mL)
and finally dried over anhydrous Na2SO4.
10
11
12
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15
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18
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Evaporation of the solvent left the crude compounds 13a–h that
were purified by column chromatography, giving
fluorescent solids in very good yield.
a yellow
General procedure for the synthesis of the 3-(10-aryl-substituted-
°
13a: Yield: 80%. Mp: 211–215 C from ethyl acetate. FT-IR: νC=N
1560 cmÀ 1; νC=C 1603 cmÀ 1. 1H-NMR (δ, CDCl3): 0.27 (s, 9H, CH3); 5.43
(s, 2H, CH2); 6.68 (s, 1H, C=CÀ H); 7.02 (AA’BB’, 2H, arom.); 7.50 (m,
4H, anthr.); 7.68 (m, 2H, anthr.); 7.89 (AA’BB’, 2H, arom.); 8.51 (m, 2H,
anthr.). 13C-NMR (δ, CDCl3): À 0.1; 61.5; 77.1; 106.9; 114.7; 123.7;
125.8; 126.0; 126.1; 126.4; 129.2; 130.1; 132.1; 133.6; 145.8; 147.6;
157.7; 160.9; 168.1. Elemental Analysis: Calculated for C35H28N2O4Si
(MW=568.70): C, 73.92; H, 4.96; N, 4.93. Found: C, 73.92; H, 4.97; N,
4.94.
anthracen-9-yl)-5-((4-ethynylphenoxy)methyl)isoxazole 14a–h. In
a
100 mL flask a DCM solution of compounds 13a–h (0.50 g) was
treated with 1 mL of TBAF solution (1 M in THF) for 30 minutes. The
organic phases were then washed with water (3×40 mL) and dried
over anhydrous Na2SO4.
Evaporation of the solvent left the crude compounds 14a–h that
were purified by column chromatography, giving
fluorescent solids in very good yield.
a yellow
°
13b: Yield: 72%. Mp: 178–182 C from ethyl acetate. FT-IR: νC=N
°
14a: Yield: 70% Mp: 206–208 C from ethyl acetate. FT-IR: νC=N 1604;
1604 cmÀ 1
.
1H-NMR (δ, CDCl3): 0.28 (s, 9H, CH3); 5.42 (s, 2H, CH2);
νC�C 2306; ν�CÀ H 3296 cmÀ 1
.
1H-NMR (δ, CDCl3): 5.44 (s, 2H, CH2);
6.67 (s, 1H, C=CÀ H); 7.03 (AA’BB’, 2H, arom.); 7.44 (m, 4H, anthr.);
7.62 (m, 4H, arom. and anthr.); 7.90 (m, 2H, anthr.). 13C-NMR (δ,
CDCl3): À 0.1; 61.5; 77.1; 107.0; 114.7; 125.3; 125.4; 125.6; 125.7;
126.3; 126.5; 129.5; 130.1; 131.4; 133.6; 142.5; 157.7; 161.0; 168.0.
Elemental Analysis: Calculated for C36H28F3NO2Si (MW=591.61): C,
73.08; H, 4.77; N, 2.37. Found: C, 73.06; H, 4.78; N, 2.36.
6.68 (s, 1H, C=CÀ H); 7.05 (AA’BB’, 2H, arom.); 7.53 (m, 4H, anthr.);
7.68 (m, 2H, anthr.); 7.89 (AA’BB’, 2H, arom.); 8.51 (m, 2H, anthr.).
13C-NMR (δ, CDCl3): 61.5; 107.0; 114.8; 123.7; 125.8; 126.0; 126.1;
126.4; 129.2; 130.1; 132.1; 133.7; 145.8; 147.6; 157.9; 160.9; 168.0.
Elemental Analysis: Calculated for C32H20N2O4 (MW=496.52): C,
77.41; H, 4.06; N, 5.64. Found: C, 77.40; H, 4.07; N, 5.65.
°
13c: Yield: 63%. Mp: 124–128 C from ethyl acetate. FT-IR: νC=N
°
14b: Yield: 81%. Mp: 176–178 C from ethyl acetate. FT-IR: νC=N
1604 cmÀ 1
.
1H-NMR (δ, CDCl3): 0.34 (s, 9H, CH3); 1.49 (s, 3H, CH3);
1604; νC�C 2306; ν�CÀ H 3296 cmÀ 1
.
1H-NMR (δ, CDCl3): 3.06 (s, 1H,
5.38 (s, 2H, CH2); 6.69 (s, 1H, C=CÀ H); 7.02 (d, 2H, J=9 Hz, arom.);
7.44 (m, 8H, anthr.); 7.93 (d, 2H, J=9 Hz, arom.). 13C-NMR (δ, CDCl3):
À 0.0; 26.9; 61.4; 107.2; 114.8; 125.5; 125.7; 125.9; 126.3; 126.8; 128.1;
129.5; 130.1; 130.4; 130.9; 133.6; 137.6; 157.8; 161.2; 167.9.
Elemental Analysis: Calculated for C36H31NO2Si (MW=537.73): C,
80.41; H, 5.81; N, 2.60. Found: C, 80.40; H, 5.82; N, 2.59.
�CH); 5.43 (s, 2H, CH2); 6.68 (s, 1H, C=CÀ H); 7.03 (AA’BB’, 2H, arom.);
7.44 (m, 4H, anthr.); 7.62 (m, 4H, arom. and anthr.); 7.90 (m, 2H,
anthr.). 13C-NMR (δ, CDCl3): 61.5; 107.0; 114.8; 125.3; 125.4; 125.6;
125.7; 126.3; 126.5; 129.5; 130.1; 131.4; 133.7; 137.7; 157.9; 161.0;
167.9. Elemental Analysis: Calculated for C33H20F3NO2 (MW=519.52):
C, 76.29; H, 3.88; N, 2.70. Found: C, 76.28; H, 3.88; N, 2.72.
13d: Yield: 66%. Sticky oil. FT-IR: νC=N 1602 cmÀ 1. 1H-NMR (δ, CDCl3):
0.28 (s, 9H, CH3); 3.99 (s, 3H, OCH3); 5.41 (s, 2H, CH2); 6.66 (s, 1H,
C=CÀ H); 7.02 (AA’BB’, 2H, arom.); 7.17 (d, 2H, anthr.); 7.41 (m, 4H,
anthr.); 7.77 (AA’BB’, 2H, arom.); 7.85 (m, 2H, anthr.). 13C-NMR (δ,
CDCl3): À 0.0; 55.4; 61.6; 107.2; 113.9; 114.8; 125.2; 125.6; 126.3;
127.4; 130.2; 130.4; 132.2; 133.7; 157.9; 159.3; 161.4; 167.9.
Elemental Analysis: Calculated for C36H31NO3Si (MW=553.73): C,
78.09; H, 5.64; N, 2.53. Found: C, 78.09; H, 5.66; N, 2.53.
°
14c: Yield: 59%. Mp: 198–200 C from ethyl acetate. FT-IR: νC=N
1606; νC�C 2306; ν�CÀ H 3297 cmÀ 1
.
1H-NMR (δ, CDCl3): 1.91 (s, 3H,
CH3); 3.07 (s, 1H, �CH); 5.43 (s, 2H, CH2); 6.70 (s, 1H, C=CÀ H); 7.05 (d,
2H, J=9 Hz, arom.); 7.48 (m, 8H, anthr.); 7.87 (d, 2H, J=9 Hz, arom.).
13C-NMR (δ, CDCl3): 19.3; 61.1; 106.7; 114.4; 125.0; 125.2; 125.4;
125.8; 126.4; 127.6; 129.0; 129.6; 129.9; 130.5; 133.3; 157.6; 160.9;
167.3. Elemental Analysis: Calculated for C33H23NO2 (MW=465.55):
C, 85.14; H, 4.98; N, 3.01. Found: C, 85.14; H, 4.99; N, 3.00.
13e: Yield: 30%. Sticky oil. FT-IR: νC=N 1603 cmÀ 1. 1H-NMR (δ, CDCl3):
0.28 (s, 9H, CH3); 3.59 (s, 6H, OCH3); 5.39 (s, 2H, CH2); 6.68 (s, 1H,
C=CÀ H); 6.84 (AA’BB’, 2H, arom.); 7.01 (d, 2H, anthr.); 7.41 (m, 4H,
anthr.); 7.66 (AA’BB’, 2H, arom.); 7.85 (m, 2H, anthr.). 13C-NMR (δ,
CDCl3): À 0.0; 55.8; 61.5; 104.4; 114.7; 124.9; 125.6; 126.0; 126.8;
129.8; 130.1; 130.5; 133.6; 157.8; 158.8; 161.5; 167.5. Elemental
Analysis: Calculated for C37H33NO4Si (MW=583.76): C, 76.13; H, 5.70;
N, 2.40. Found: C, 76.15; H, 5.71; N, 2.41.
°
14d: Yield: 66%. Mp: 181–183 C from ethyl acetate. FT-IR: νC=N
1601; νC�C 2306; ν�CÀ H 3232 cmÀ 1
.
1H-NMR (δ, CDCl3): 3.06 (s, 1H,
�CH); 3.99 (s, 3H, OCH3); 5.42 (s, 2H, CH2); 6.66 (s, 1H, C=CÀ H); 7.04
(AA’BB’, 2H, arom.); 7.17 (d, 2H, anthr.); 7.44 (m, 4H, anthr.); 7.78
(AA’BB’, 2H, arom.); 7.86 (m, 2H, anthr.). 13C-NMR (δ, CDCl3): 55.3;
61.5; 107.1; 113.8; 114.9; 125.1; 125.4; 126.1; 127.2; 130.1; 130.3;
132.1; 133.7; 158.0; 159.1; 161.3; 167.7. Elemental Analysis: Calcu-
lated for C33H23NO3 (MW=481.55): C, 82.31; H, 4.81; N, 2.91. Found:
C, 82.30; H, 4.82; N, 2.92.
13f: Yield: 74%. Sticky oil (semi-solid compound). FT-IR: νC=N 1609,
1
νC=O 1716 cmÀ 1. H-NMR (δ, CDCl3): 0.27 (s, 9H, CH3); 1.50 (t, 3H, J=
°
14e: Yield: 4%. Mp: 175–180 C from ethyl acetate. FT-IR: νC=N 1605;
1
νC�C 2107; ν�CÀ H 3289 cmÀ 1. H-NMR (δ, CDCl3): 3.06 (s, 1H, �CH);
7 Hz, CH3); 4.51 (q, 2H, J=7 Hz, OCH2); 5.42 (s, 2H, CH2); 6.66 (s, 1H,
C=CÀ H); 7.02 (AA’BB’, 2H, arom.); 7.51 (m, 6H, anthr.); 7.86 (AA’BB’,
3.59 (s, 6H, OCH3); 5.39 (s, 2H, CH2); 6.68 (s, 1H, C=CÀ H); 6.83
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(AA’BB’, 2H, arom.); 7.03 (d, 2H, anthr.); 7.44 (m, 4H, anthr.); 7.67
(AA’BB’, 2H, arom.); 7.85 (m, 2H, anthr.). 13C-NMR (δ, CDCl3): 55.4;
61.1; 103.8; 114.4; 124.5; 125.2; 125.6; 126.4; 129.4; 129.7; 130.1;
133.3; 157.6; 158.4; 161.1; 167.1. Elemental Analysis: Calculated for
C34H25NO4 (MW=511.58): C, 79.83; H, 5.93; N, 2.74. Found: C, 79.84;
H, 5.92; N, 2.73.
anthr.); 8.13 (d, 2H, arom.); 10.49 (bs, 1H, NH). 13C-NMR (δ, CDCl3):
À 0.1; 26.8; 71.8; 92.6; 95.8; 104.9; 114.5; 115.8; 125.4; 125.8; 126.4;
126.5; 128.0; 129.3; 131.3; 131.4; 133.4; 142.2; 158.3; 161.8; 194.5.
Elemental Analysis: Calculated for C36H30NO2Si (MW=593.72): C,
72.83; H, 5.09; N, 2.36. Found: C, 72.82; H, 5.07; N, 2.35.
1
2
3
4
5
6
7
8
9
°
15c: Yield: 50%. Mp: 212–214 C from ethyl acetate. FT-IR: νC=O
1
14f: Yield: 32%. Mp: 65–68 C from ethyl acetate. FT-IR: νC=N 1606;
1587; νC�C 2155; νNH2 3472 and 3944 cmÀ 1. H-NMR (δ, CDCl3): 0.27
°
1
νC=O 1715; νC�C 2107; ν�CÀ H 3292 cmÀ 1. H-NMR (δ, CDCl3): 1.51 (t,
(s, 9H, CH3); 1.89 (s, 3H, CH3); 4.68 (s, 2H, CH2); 5.69 (bs, 1H, NH); 5.85
(s, 1H, C=CÀ H); 6.86 (d, 2H, anthr.); 7.42 (m, 8H, arom. and anthr.);
8.13 (d, 2H, anthr.); 10.53 (bs, 1H, NH). 13C-NMR (δ, CDCl3): À 0.1;
19.7; 71.8; 95.8; 105.0; 114.5; 125.4; 125.5; 126.4; 126.7; 128.0; 129.3;
130.5; 130.8; 130.9; 133.4; 137.6; 158.3; 162.3; 194.2. Elemental
Analysis: Calculated for C36H33NO2Si (MW=539.75): C, 80.11; H, 6.16;
N, 2.60. Found: C, 80.12; H, 6.16; N, 2.61.
3H, J=7 Hz, CH3); 3.06 (s, 1H, �CH); 4.51 (q, 2H, J=7 Hz, OCH2); 5.42
(s, 2H, CH2); 6.68 (s, 1H, C=CÀ H); 7.04 (AA’BB’, 2H, arom.); 7.51 (m,
6H, anthr.); 7.88 (AA’BB’, 2H, arom.); 8.32 (m, 2H, anthr.). 13C-NMR (δ,
CDCl3): 14.3; 61.1; 61.5; 107.1; 114.8; 125.6; 126.3; 126.7; 128.0;
129.4; 129.6; 130.6; 131.1; 133.7; 158.0; 161.1; 166.4; 168.3.
Elemental Analysis: Calculated for C34H25NO4 (MW=523.18): C,
80.29; H, 4.81; N, 2.68. Found: C, 80.30; H, 4.86; N, 2.62.
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
°
15d: Yield: 70%. Mp: 126–129 C from ethyl acetate. FT-IR: νC=O
1
14g: Yield: 84%. Mp: 104–105 C from ethyl acetate. FT-IR: νC=N
1604; νC�C 2155; νNH2 3469 and 3943 cmÀ 1. H-NMR (δ, CDCl3): 0.26
°
1606; νC�C 2306; ν�CÀ H 3297 cmÀ 1
.
1H-NMR (δ, CDCl3): 3.06 (s, 1H,
(s, 9H, CH3); 3.98 (s, 3H, OCH3); 4.69 (s, 2H, CH2); 5.70 (bs, 1H, NH);
5.83 (s, 1H, C=CÀ H); 6.86 (AA’BB’, 2H, arom.); 7.16 (d, 2H, anthr.);
7.41 (m, 6H, anthr); 7.73 (AA’BB’, 2H, arom.); 8.13 (d, 2H, anthr.);
10.49 (bs, 1H, NH). 13C-NMR (δ, CDCl3): À 0.1; 26.8; 55.3; 71.8; 95.8;
105.0; 113.9; 114.7; 125.2; 125.3; 126.3; 126.8; 129.9; 130.2; 130.5;
130.8; 130.9; 133.4; 137.6; 158.3; 162.5; 194.2. Elemental Analysis:
Calculated for C36H33NO3Si (MW=555.75): C, 77.80; H, 5.99; N, 2.52.
Found: C, 77.81; H, 5.98; N, 2.53.
�CH); 5.41 (s, 2H, CH2); 6.65 (s, 1H, C=CÀ H); 6.77 (m, 1H, fur.); 7.05
(m, 2H, anthr.); 7.49 (m, 5H, anthr.); 7.85 (m, 2H, fur.); 7.97 (m, 1H,
anthr.). 13C-NMR (δ, CDCl3): 61.5; 107.0; 114.8; 125.6; 125.9; 126.0;
126.3; 126.5; 126.7; 127.2; 127.9; 128.2; 130.2; 131.1; 143.1; 158.0;
161.0; 167.9. Elemental Analysis: Calculated for C36H23NO3 (MW=
517.58): C, 83.54; H, 4.48; N, 2.71. Found: C, 83.53; H, 4.45; N, 2.75.
°
14h: Yield: 81%. Mp: 185–190 C from ethyl acetate. FT-IR: νC=N
1605; νC�C 2305; ν�CÀ H 3295 cmÀ 1
.
1H-NMR (δ, CDCl3): 3.07 (s, 1H,
15e: This compound was not prepared because of the extremely
low amounts of the precursor.
�CH); 5.46 (s, 2H, CH2); 6.74 (s, 1H, C=CÀ H); 7.06 (AA’BB’, 2H, arom.);
7.16 (d, 1H, J=9 Hz, isoq.); 7.32–7.56 (m, 9H, anthr.); 7.70 (t, 1H, J=
7 Hz, isoq.); 7.93 (AA’BB, 2H, arom.); 8.21 (d, 1H, J=8 Hz, isoq.); 8.63
(s, 1H, anthr.); 9.54 (s, 1H, isoq.). 13C-NMR (δ, CDCl3): 61.1; 75.9; 82.7;
106.7; 114.4; 115.2; 123.6; 124.9; 125.4; 125.5; 126.0; 126.2; 127.4;
127.6; 127.8; 129.8; 130.3; 130.8; 132.7; 133.3; 135.8; 143.7; 151.9;
157.6; 160.7; 167.6. Elemental Analysis: Calculated for C35H22N2O2
(MW=502.57): C, 83.65; H, 4.41; N, 5.57. Found: C, 83.64; H, 4.41; N,
5.56.
°
15f: Yield: 72%. Mp: 114–117 C from ethyl acetate. FT-IR: νC=O
1
1715; νC=O 1602; νC�C 2155; νNH2 3392 and 3944 cmÀ 1. H-NMR (δ,
CDCl3): 0.27 (s, 9H, CH3); 1.48 (t, 3H, J=7 Hz, CH3); 4.46 (q, 2H, J=
7 Hz, OCH2); 4.62 (s, 2H, CH2); 5.81 (s, 1H, C=CÀ H); 5.90 (bs, 1H, NH);
6.81 (AA’BB’, 2H, arom.); 7.35 (d, 2H, anthr.); 7.48 (m, 2H, anthr); 7.59
(d, 2H, anthr); 8.13 (AA’BB’, 2H, arom.); 8.26 (m, 2H, anthr.); 10.46
(bs, 1H, NH). 13C-NMR (δ, CDCl3): À 0.1; 14.3; 26.8; 61.1; 71.6; 95.7;
105.0; 114.5; 115.7; 125.4; 125.7; 126.4; 126.5; 127.9; 129.2; 129.6;
129.9; 131.0; 131.1; 133.4; 137.8; 143.2; 158.2; 162.2; 166.3; 194.3.
Elemental Analysis: Calculated for C38H35NO4Si (MW=597.79): C,
76.35; H, 5.90; N, 2.34. Found: C, 76.36; H, 5.90; N, 2.34.
General procedure for the synthesis of the 4-amino-4-(10-aryl-
substitutedanthracen-9-yl)-1-(4-((trimethylsilyl)ethynyl)phenoxy)but-3-
en-2-one 15a–h. In a 100 mL round-bottom flask 0.47 mmol of
compounds 13a–h were dissolved in 50 mL MeCN/H2O 8:2
solution. The solutions were kept under stirring under nitrogen
atmosphere for 15 minutes. After this period of time 0.47 mmol of
Mo(CO)6 were added and the mixtures were allowed to react
°
15g’: Yield: 58%. Mp: 166–168 C from ethyl acetate. FT-IR: νC=O
1
1735; νC=O 1654; νC�C 2305; νNH2 3296 and 3470 cmÀ 1. H-NMR (δ,
CDCl3): 3.03 (s, 1H, �CH); 4.68 (s, 2H, CH2); 5.81 (s, 1H, C=CÀ H); 5.90
(bs, 1H, NH); 6.81 (AA’BB’, 2H, arom.); 7.35 (d, 2H, anthr.); 7.48 (m,
2H, anthr); 7.59 (d, 2H, anthr); 8.13 (AA’BB’, 2H, arom.); 8.26 (m, 2H,
anthr.); 10.44 (bs, 1H, NH). 13C-NMR (δ, CDCl3): 14.1; 60.3; 71.7; 75.8;
110.9; 112.5; 114.6; 125.3; 125.5; 125.6; 126.2; 126.4; 126.9; 127.3;
128.0; 128.3; 129.0; 130.1; 133.5; 143.1; 149.7; 158.4; 166.3; 194.3.
Elemental Analysis: Calculated for C38H35NO4Si (MW=597.79): C,
76.35; H, 5.90; N, 2.34. Found: C, 76.36; H, 5.90; N, 2.34.
°
heating at 70 C for 4 h. The mixtures were then cooled down,
80 mL of a mixture of DCM/CHCl3 1:1 were added and the organic
phases separated. The organic phases were washed with 3×40 mL
brine. The collected organic phases were then dried over anhydrous
Na2SO4.
Evaporation of the solvent left the crude compounds 15a–h that
were purified by column chromatography, giving
fluorescent solids in very good yield.
a yellow
°
15h: Yield: 56%. Mp: >240 C from ethyl acetate. FT-IR: νC=N 1603;
νC�C 2157 cmÀ 1
.
1H-NMR (δ, CDCl3): 0.27 (s, 9H, CH3); 4.70 (s, 2H,
°
15a: Yield: 79%. Mp: 169–173 C from ethyl acetate. FT-IR: νC=O
CH2); 5.70 (bs, 1H, NH); 5.89 (s, 1H, C=CÀ H); 6.88 (m, 2H, isoq.); 7.41
(m, 11H, isoq. And anthr.); 8.17 (m, 3H, anthr); 8.47 (s, 1H, isoq.);
9.54 (s, 1H, anthr.); 10.55 (bs, 1H, NH). 13C-NMR (δ, CDCl3): À 0.1; 71.7;
77.1; 92.6; 95.8; 104.9; 114.5; 115.8; 125.1; 125.4; 125.6; 126.2; 126.3;
126.4; 126.6; 128.0; 128.1; 128.2; 130.5; 131.6; 132.0; 132.1; 133.4;
142.7; 151.7; 158.3; 161.7. Elemental Analysis: Calculated for
C38H32N2O2Si (MW=576.77): C, 79.13; H, 5.59; N, 4.88. Found: C,
79.13; H, 5.60; N, 4.47.
1
1590; νC�C 2305; νNH2 3296 and 3476 cmÀ 1. H-NMR (δ, CDCl3): 0.25
(s, 9H, CH3); 4.69 (s, 2H, CH2); 5.64 (bs, 1H, NH); 5.82 (s, 1H, C=CÀ H);
6.85 (AA’BB’, 2H, arom.); 7.53 (m, 6H, anthr.); 8.15 (d, 2H, anthr.);
8.49 (AA’BB’, 2H, arom.); 10.49 (bs, 1H, NH). 13C-NMR (δ, CDCl3):
À 0.1; 29.6; 71.8; 77.3; 95.8; 114.5; 123.7; 125.5; 126.0; 126.2; 126.6;
127.5; 129.1; 132.0; 132.1; 133.4; 136.1; 145.6; 158.2; 161.5; 194.6.
Elemental Analysis: Calculated for C35H30N2O4Si (MW=570.72): C,
73.66; H, 5.30; N, 4.91. Found: C, 73.65; H, 5.31; N, 4.91.
General procedure for the synthesis of the 4-(10-aryl-substituted-
anthracen-9-yl)-6-((4-ethynylphenoxy)methyl)-2,2-difluoro-2,3-dihydro-
1λ3,3,2λ4-oxazaborinine 17a–h. In a 100 mL flask 0.30 mmol of
compounds 15a–h were dissolved in 30 mL anhydrous DCM and
7.9 mmol distilled Et3N were added. The solutions were kept under
°
15b: Yield: 71%. Mp: 116–119 C from ethyl acetate. FT-IR: νC=O
1
1586; νC�C 2154; νNH2 3472 and 3943 cmÀ 1. H-NMR (δ, CDCl3): 0.26
(s, 9H, CH3); 4.68 (s, 2H, CH2); 5.63 (bs, 1H, NH); 5.83 (s, 1H, C=CÀ H);
6.84 (d, 2H, anthr.); 7.52 (m, 7H, arom. and anthr.); 7.89 (d, 2H,
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stirring under nitrogen atmosphere for 10 minutes. After this period
of time excess BF3 ·Et2O (1 mL) was added and the mixtures were
allowed to react at room temperature for 2 h. The mixtures were
then diluted with 40 mL DCM and the organic phases washed with
3×40 mL brine. The collected organic phases were then dried over
anhydrous Na2SO4.
(s, 1H, �CÀ H); 4.96 (s, 2H, CH2); 6.27 (s, 1H, C=CÀ H); 6.77 (m, 1H,
fur.); 6.86 (m, 2H, anthr.); 7.42–7.85 (m, 7H, fur., arom. and NH); 7.98
(d, 2H, anthr.). 13C-NMR (δ, CDCl3): 67.4; 77.1; 83.0; 97.8; 111.0; 112.9;
114.5; 123.9; 126.5; 126.9; 127.4; 130.7; 133.7; 149.1; 157.4; 173.9;
177.5. Elemental Analysis: Calculated for C30H20BF2NO3 (MW=
491.30): C, 73.34; H, 4.10; N, 2.85. Found: C, 73.36; H, 4.11; N, 2.85.
1
2
3
4
5
6
7
8
9
°
In a 100 mL flask a DCM solution of compounds 16a–h was treated
with 1 mL of TBAF solution (1 M in THF) for 30 minutes. The organic
phases were then washed with water (3×40 mL) and dried over
anhydrous Na2SO4.
17h: Yield: 12%. Mp: >240 C from ethyl acetate. FT-IR: νC=C 1637;
1
νC�C 2157 cmÀ 1, νC�H 3296; νNH 3360 cmÀ 1. H-NMR (δ, DMSO): 5.10
(s, 2H, CH2); 6.20 (s, 1H, NH); 6.27 (s, 1H, C=CÀ H); 7.07 (m, 2H, isoq.);
7.41 (m, 12H, isoq. And anthr.); 7.83 (m, 2H, anthr); 8.38 (s, 1H,
isoq.); 8.59 (s, 1H, anthr.); 11.30 (bs, 1H, NH). 13C-NMR (δ, DMSO):
67.1; 79.6; 83.2; 97.7; 115.2; 124.4; 124.5; 126.3; 126.4; 126.8; 127.0;
127.5; 128.3; 128.4; 129.7; 131.5; 132.9; 133.6; 135.1; 135.2; 144.0;
144.3; 153.2; 157.7; 172.2; 175.5. Elemental Analysis: Calculated for
C35H23BF2N2O2 (MW=552.39): C, 76.10; H, 4.20; N, 5.07. Found: C,
76.11; H, 4.21; N, 5.06.
Evaporation of the solvent left the crude compounds 17a–h that
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
were purified by column chromatography, giving
a yellow
fluorescent solids in very good yield. Compound 17e was not
synthetized because of the extremely low yields of compound 13e
available for all the synthetic steps.
°
17a: Yield: 41%. Mp: 190–198 C from ethyl acetate. FT-IR: νC=C
UV-Vis and Fluorescence Spectroscopic Analysis. In order to assess
the optical properties of the synthetized fluorescent probes 14a–h
and 17a–h and in order to evaluate their potential use in ABPP, we
have performed some UV-Vis and fluorescence spectroscopic
analysis. These analyses were performed in 3 different solvents with
different polarity (DCM, MeOH), considering also the possible
applications in the cellular environment (DMSO/H2O 1:1).
1
1606 cmÀ 1; νC�C 2306; νC�H 3340; νNH 3361 cmÀ 1. H-NMR (δ, CDCl3):
3.04 (s, 1H, �CÀ H); 4.98 (s, 2H, CH2); 6.30 (s, 1H, C=CÀ H); 6.87
(AA’BB’, 2H, arom.); 7.47 (m, 3H, anthr.); 7.61 (m, 5H, anthr.); 7.74
(bs, 1H, NH); 7.89 (d, 2H, anthr.); 8.53 (AA’BB’, 2H, arom.). 13C-NMR
(δ, CDCl3): 26.8; 97.8; 114.5; 123.8; 124.2; 126.5; 126.6; 127.3; 127.8;
129.0; 131.9; 133.7; 144.9; 157.4; 177.7. Elemental Analysis: Calcu-
lated for C32H21BF2N2O4 (MW=546.34): C, 70.35; H, 3.87; N, 5.13.
Found: C, 70.35; H, 3.88; N, 5.14.
UV-Vis spectra of the compounds 14a–h and 17a–h were
registered in some different solvents (DCM, MeOH and DMSO/H2O
1:1) with a Jasco V-550 UV/VIS Spectrophotometer. The maxima of
absorption were acquired and all the obtained spectra are reported
in graphics in the SI.
°
17b: Yield: 52%. Mp: 240–244 C from ethyl acetate. FT-IR: νC=C
1618 cmÀ 1; νC�C 2305; νC�H 3341; νNH 3361 cmÀ 1. H-NMR (δ, CDCl3):
1
3.03 (s, 1H, �CÀ H); 4.97 (s, 2H, CH2); 6.30 (s, 1H, C=CÀ H); 6.87 (d, 2H,
arom.); 7.46 (m, 3H, anthr.); 7.59 (m, 5H, anthr.); 7.79 (bs, 1H, NH);
7.90 (m, 2H, arom. and anthr.). 13C-NMR (δ, CDCl3): 76.3; 97.9; 114.5;
124.1; 125.5; 125.6; 126.2; 126.9; 127.4; 127.6; 129.2; 131.2; 133.7;
157.5; 177.5. Elemental Analysis: Calculated for C33H21BF5NO2 (MW=
569.34): C, 69.62; H, 3.72; N, 2.46. Found: C, 69.61; H, 3.71; N, 2.45.
Fluorescence spectra of compounds 14a–h and 17a–h were
registered in different solvents (DCM, MeOH and DMSO/H2O 1:1)
with a Perkin-Elmer LS 55 Luminescence Spectrometer. The maxima
of excitation and emission were acquired and the fluorescence
spectra are reported in the graphics in the SI.
°
17c: Yield: 58%. Mp: 139–144 C from ethyl acetate. FT-IR: νC=C
1619 cmÀ 1; νC�C 2305; νC�H 3297; νNH 3360 cmÀ 1. H-NMR (δ, CDCl3):
1
1.89 (s, 3H, CH3); 3.03 (s, 1H, �CÀ H); 4.97 (s, 2H, CH2); 6.30 (s, 1H,
C=CÀ H); 6.89 (d, 2H, arom.); 7.49 (m, 8H, arom. and anthr.); 7.77 (b,
1H, NH); 7.88 (m, 2H, anthr.). 13C-NMR (δ, CDCl3): 26.8; 77.1; 98.0;
114.5; 124.0; 125.9; 127.2; 127.6; 128.3; 129.2; 130.1; 130.6; 133.7;
157.5; 177.1. Elemental Analysis: Calculated for C33H24BF2NO2 (MW=
515.37): C, 76.91; H, 4.69; N, 2.72. Found: C, 76.92; H, 4.70; N, 2.73.
Acknowledgements
Financial support by the University of Pavia and COST Action
(CM1004) “Synthetic Probes for Chemical Proteomics and Elucida-
tion of Biosynthetic Pathways” are gratefully acknowledged. We
also thank “VIPCAT – Value Added Innovative Protocols for
Catalytic Transformations” project (CUP: E46D17000110009) for
valuable financial support.
°
17d: Yield: 39%. Mp: 210–215 C from ethyl acetate. FT-IR: νC=C
1607 cmÀ 1; νC�C 2305; νC�H 3298; νNH 3361 cmÀ 1. H-NMR (δ, CDCl3):
1
3.03 (s, 1H, �CÀ H); 3.98 (s, 3H, OCH3); 4.96 (s, 2H, CH2); 6.30 (s, 1H,
C=CÀ H); 6.86 (AA’BB’, 2H, arom.); 7.15 (d, 2H, anthr.); 7.42 (m, 2H+
1H, anthr. and NH); 7.54 (m, 2H, anthr.); 7.77 (AA’BB’, 2H, arom.);
7.85 (d, 2H, anthr.). 13C-NMR (δ, CDCl3): 55.3; 67.4; 76.3; 83.0; 98.0;
113.9; 114.5; 123.8; 125.6; 127.4; 127.5; 127.6; 129.6; 129.8; 131.8;
133.7; 157.5; 159.3; 174.2; 177.1. Elemental Analysis: Calculated for
C33H24BF2NO3 (MW=531.37): C, 74.59; H, 4.55; N, 2.64. Found: C,
74.60; H, 4.52; N, 2.64.
Conflict of Interest
The authors declare no conflict of interest.
°
17f: Yield: 28%. Mp: >225 C (dec.) from ethyl acetate. FT-IR: νC=C
1624; νC=O 1717 cmÀ 1; νC�C 2305; νC�H 3299; νNH 3360 cmÀ 1. H-NMR
1
Keywords: isoxazoles · enamino-ketones · fluorescent probes ·
boron complexes · photophysical studies
(δ, CDCl3): 1.48 (t, 3H, J=7 Hz, CH3); 3.03 (s, 1H, �CÀ H); 3.98 (s, 3H,
OCH3); 4.49 (q, 2H, OCH2); 4.97 (s, 2H, CH2); 6.30 (s, 1H, C=CÀ H); 6.86
(AA’BB’, 2H, arom.); 7.53 (m, 8H+1H, anthr. and NH); 7.87 (AA’BB’,
2H, arom.); 8.29 (d, 2H, anthr.). 13C-NMR (δ, CDCl3): 14.3; 61.2; 76.4;
97.9; 114.5; 124.0; 126.1; 127.4; 127.5; 127.6; 129.6; 129.8; 131.8;
133.7; 157.5; 166.2; 177.3. Elemental Analysis: Calculated for
C35H26BF2NO4 (MW=573.40): C, 73.31; H, 4.57; N, 2.44. Found: C,
73.32; H, 4.58; N, 2.44.
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°
17g: Yield: 40%. Mp: 175–179 C from ethyl acetate. FT-IR: νC=C
1618; νC�C 2305; νC�H 3296; νNH 3360 cmÀ 1. H-NMR (δ, CDCl3): 3.03
1
[5] M. Nitta, T. Kobayashi, J. Chem. Soc., Chem. Commun. 1982, 877–878.
ChemistryOpen 2019, 8, 770–780
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Full Papers
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Manuscript received: April 16, 2019
Revised manuscript received: May 17, 2019
ChemistryOpen 2019, 8, 770–780
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© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA