Synthesis of 3,5-Disubstituted Isoxazoles through a 1,3-Dipolar Cycloaddition Reaction between Alkynes and Nitrile Oxides Generated from O-Silylated Hydroxamic Acids

Article abstract of DOI:10.1002/ejoc.201901045

In this paper, we report the regioselective synthesis of 3,5-disubstituted isoxazoles by 1,3-dipolar cycloaddition between alkynyl dipolarophiles and nitrile oxide dipoles generated in-situ from O-silylated hydroxamic acids in the presence of trifluoromethanesulfonic anhydride and NEt₃. Thanks to the mild, metal-free and oxidant-free conditions that this strategy offers, the reaction was successfully applied to a wide variety of alkynyl dipolarophiles, demonstrating the tolerance of this approach to diverse functional groups. In particular, we have shown that the method was compatible with biological molecules such as peptides and peptide nucleic acids (PNA). This protocol constitutes another example of metal-free 1,3-dipolar cycloaddition leading to the regioselective formation of isoxazoles.

Full text of DOI:10.1002/ejoc.201901045

DOI: 10.1002/ejoc.201901045
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Click Reactions
Synthesis of 3,5-Disubstituted Isoxazoles through a 1,3-Dipolar
Cycloaddition Reaction between Alkynes and Nitrile Oxides
Generated from O-Silylated Hydroxamic Acids
Laure-Elie Carloni,[a] Stefan Mohnani,[a] and Davide Bonifazi*[b]
Abstract: In this paper, we report the regioselective synthesis of alkynyl dipolarophiles, demonstrating the tolerance of this
of 3,5-disubstituted isoxazoles by 1,3-dipolar cycloaddition be- approach to diverse functional groups. In particular, we have
tween alkynyl dipolarophiles and nitrile oxide dipoles gener- shown that the method was compatible with biological mol-
ated in-situ from O-silylated hydroxamic acids in the presence ecules such as peptides and peptide nucleic acids (PNA). This
of trifluoromethanesulfonic anhydride and NEt3. Thanks to the protocol constitutes another example of metal-free 1,3-dipolar
mild, metal-free and oxidant-free conditions that this strategy cycloaddition leading to the regioselective formation of isox-
offers, the reaction was successfully applied to a wide variety azoles.
Introduction
approach, via a preferred in-situ preparation of the di-
poles.[1b,2,12a,13] Indeed, as Quilico demonstrated in 1970, nitrile
oxides in the presence of none or poor trapping agents, readily
dimerize to furoxans.[14] They can also dimerize to 1,2,4-oxadi-
azole 4-oxide in the presence of NEt3.[13a,13b,15]
Identified in countless biologically active derivatives, such as
chlorophyll, amino acids, nucleobases and vitamins, five- and
six-membered heterocycles are of greatest importance to life,
drug discovery and medicinal chemistry.[1] Among them, isox-
azoles form a major class of five-membered heterocycles with
two heteroatoms (Figure 1).[2] Isoxazoles are key pharmaco-
phores occurring in many natural products, e.g. ibotenic acid 1
and muscimol 2;[3] in a variety of bioactive compounds, such
as anti-inflammatories 3 and 4,[4] monoamine oxidase inhibitor
5,[5] penicillin antibiotic 6,[6] and herbicidal isoxaflutole 7.[7]
Isoxazoles have other biological properties as antitubulin, anti-
nociceptive and anticancer.[8] They also find applications in ma-
terial science, e.g. molecular switches and polymer syntheses.[9]
Furthermore, these heterocycles constitute an important or-
ganic synthetic tool. Indeed, although isoxazoles are hydrolyti-
cally stable, they can be cleaved under reducing or basic condi-
tions, leading to different important latent functionalities,
namely 1,3-dicarbonyl compounds 8, enaminones 9, and ꢀ-
amino carbonyls 10 (Figure 1).[2,10] As a result, many research
groups have used isoxazole derivatives as masked functions in
synthetic strategies towards heterocyclic compounds and natu-
ral products.[11] Isoxazoles, and their partially saturated ana-
logues, can be prepared using diverse synthetic strategies.[12]
Among them, the 1,3-dipolar cycloaddition reaction between
nitrile oxides and corresponding alkenyl and alkynyl dipolaro-
philes is probably the most convenient, attractive and direct
Several methods towards the in-situ generation of nitrile ox-
ides have thus been developed, two of which are by far the
most popular and well-established protocols. The first ap-
proach, namely the Mukaiyama method (Figure 2, Method a),
involves the dehydration of nitroalkanes by aryl isocyanates in
the presence of NEt3.[13c] The use of POCl3, MeSO2Cl and BzCl
as dehydrating agents are improved variants of the original
Mukaiyama procedure.[13a,13b,13d,16] The action of DMAP and
Boc2O[17] or DMTMM[18] on nitroalkanes was also successfully
used to give nitrile oxide dipoles. The second method relies on
the base-mediated dehydrochlorination of hydroximoyl chlor-
ides to yield the desired nitrile oxides (Figure 2, Method b).[13l]
This procedure is often carried out as a halogenation/dehydro-
halogenation reaction on the parent aldoximes, especially for
unstable hydroximoyl chlorides.[13a,13b] NCS, tBuOCl or NaOCl in
the presence of NEt3, or chloramine-T without additional bases,
are frequently employed in these procedures.[13e,13f] On top of
these approaches, the direct oxidation of aldoximes through
the use of MnO2[19] or organic hypervalent iodine reagents[20] is
also a promising route to the in-situ generation of nitrile oxides
(Figure 2, Method c). Another strategy was described by Carreira
and co-workers in 2000.[21] It involves the treatment of O-silyl-
ated hydroxamic acids with Tf2O in the presence of NEt3 (Figure
2, Method d). In 2019, Dai et al. reported a novel direct Csp3-H
bond functionalization of 2-methyl ketones and 2-methylquin-
oline with tBuONO, generating nitrile oxide in-situ via a radical
mechanism (Figure 2, Method e).[22]
[a] Department of Chemistry and Namur Research College (NARC),
University of Namur,
Rue de Bruxelles 61, Namur, 5000, Belgium
[b] Cardiff University, School of Chemistry, Park Place, Main Building, CF10
3AT, Cardiff, Wales, UK.
Although the cycloaddition between nitrile oxides and alk-
enyl derivatives is well-developed, the reaction with alkynyl di-
polarophiles is usually affected by side reactions, often leading
E-mail: bonifazid@cardiff.ac.uk
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201901045.
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Figure 1. Examples of natural products (1–2) and bioactive compounds (3–7) bearing an isoxazole function. Isoxazoles can also be used as masked functional
groups for various synthetic strategies e.g. towards 8–10.
and ethyl 2-nitroacetate with terminal alkynes leading to isox-
azoles under green conditions.[28] Recently, the Csp3-H metal-
free radical functionalization/cycloaddition cascade from ket-
ones, alkynes and tBuONO proposed by Dai et al. allowed the
synthesis of 3-acyl and 3-quinoline isoxazoles in moderate to
good yield.[22]
While studying metal-free methodologies for the bioconju-
gation of structures of biological interest with functional or-
ganic dyes,[29] our group became interested in developing an
alternative bioconjugation approach that would use the 1,3-
dipolar cycloaddition reaction between a nitrile oxide and an
Figure 2. Overview of the synthetic approaches for preparing nitrile oxides
in-situ: a) dehydration of nitroalkanes; b) dehydrochlorination of hydroximoyl
alkynyl derivative to produce isoxazole linkers. During these en-
deavors, we focused our attention on the Carreira's protocol,[21]
chlorides; c) oxidation of aldoximes; d) oxidation of O-silylated hydroxamic
acids; and e) Csp3-H bond functionalization of 2-methylketones with tBuONO.
that produces isoxazolines via cycloaddition between an alkenyl
derivative and a nitrile oxide, the latter being produced in-situ
from O-silylated hydroxamic acids upon treatment with Tf2O
and NEt3 (Figure 2, Method d). In addition to the mild, metal-
free and oxidant-free conditions that this strategy offers, we
conjectured that the straightforward preparation of the parent
O-silylated hydroxamic acids 11R1 could also be easily adapted
on a large variety of substrates. Indeed, O-silylated hydroxamic
acids can be readily obtained from the corresponding hydrox-
amic acids 12R1 by silylation with tert-butyl(chloro)diphenyl-
silane in the presence of NaH (Scheme 1a). They can also be
prepared from the corresponding carboxylic acids 13R1 by reac-
tion with O-silylated hydroxylamine 14[30] following activation
of the carboxylic acid moiety with HATU in the presence of
DIEA (Scheme 1b). To our surprise, this method has not been
used to prepare isoxazoles. It is with this aim that herein we
report the successful adaptation of the Carreira's protocol to
the regioselective preparation of 3,5-disubstituted isoxazoles
(Scheme 1c) through the 1,3-dipolar cycloaddition reaction be-
tween in-situ generated nitrile oxides (15R1) and alkynyl di-
to by-products and low yields. This is due to the relatively inert
character of the triple bond. Following the Cu(I)-catalyzed az-
ide-alkyne cycloaddition protocol,[23] it was also demonstrated
that the addition of Cu(I)-[24] or Ru(II)-based[25] catalysts to a
mixture of alkynes and nitrile oxides, the latter species being
generated in-situ respectively from the parent aldoximes and
hydroximoyl chloride, led to the formation of 3,5-di or 3,4-di
and 3,4,5-trisubstituted isoxazoles in high yields. Among the
metal-free protocols, the group of Heaney conjugated aryl moi-
eties with DNA on solid phase through a 1,3-dipolar cycloaddi-
tion between nitrile oxide and alkynyl moieties.[26] In their ap-
proach, nitrile oxide was generated in-situ by dehydrogenation
of aryl aldoxime. In 2011, the group of Van Delft described an
efficient phenyliodine bis(trifluoroacetate) (PIFA)-mediated syn-
thesis of isoxazoles.[20b] In a parallel avenue, Kankala et al. re-
ported in 2011 the nucleophilic organocarbene-catalyzed 1,3-
dipolar cycloaddition of nitrile oxides with alkynes.[27] In 2014,
the group of Pal reported the use of polyethylene glycol to
facilitate the 1,3-dipolar cycloaddition of benzoylnitromethane
polarophiles 16R2
.
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from nitrile oxide 15a and are independent on the dipolaro-
phile. With this information in hand, we attempted the optimi-
zation of the reaction, with the idea of favoring the intermolec-
ular cycloaddition reaction over the other side-reactions. Sev-
eral conditions were explored, mainly focusing on the nitrile
oxide generation.[33] Parameters such as the excess of the di-
polarophile, time, temperature, the order of addition of the rea-
gents, as well as the concentration of the reaction mixture were
studied. Although none of these tests gave improved yields, we
could conclude that: (i) the side-products mainly derived from
the reaction of nitrile oxide 15a with itself or with 20a (Scheme
3); (ii) these side-reactions do not take place below 0 °C; and
(iii) they are faster than the 1,3-dipolar cycloaddition with 1-
octyne at r.t. In light of these observations, we decided to moni-
tor the generation of nitrile oxide at different temperatures, and
noticed that it is formed within three minutes at –40 °C. This
observation allowed us to improve the protocol. Namely, the
reaction mixture was allowed to stir only for 3 min at –40 °C
after the addition of Tf2O to a solution of 11a, before it was
cannulated over 1 h onto neat 1-octyne 16a at r.t. The solution
was then stirred overnight at r.t. (Scheme 1c). Following this
procedure, desired isoxazole 17a was formed in 75 % yield, with
an overall increase of 26 %. To the best of our knowledge, this
is one of the most efficient synthetic strategies to form 5-hexyl-
3-phenylisoxazole 17a.[34] Similar yields were obtained either
using the base-mediated conversion of propargylic N-hydroxyl-
amines, through a detosylative 5-endo-dig-cyclisation,[34a] or ex-
ploiting hypervalent iodine to generate in-situ the nitrile oxide
species.[34b] Having in our hands the optimal conditions for our
transformation, we extended the protocol to a variety of O-
silylated hydroxamic acids 11R1 and of alkynyl substrates 16R2
(Scheme 4).
Scheme 1. Synthetic protocol proposed in this work: formation of O-silylated
hydroxamic acids 11R1 a) from hydroxamic acid derivatives 12R1, and b) from
parent acids 13R1. Yields were 72 % and 44 % for R1 = Ph, respectively.
c) Optimized 1,3-dipolar cycloaddition developed in this work for preparing
3,5-disubstituted isoxazoles 17R1,R2
.
Results and Discussion
We commenced our studies with the investigation of the basic
reactivity of alkynes respect to alkenes in the 1,3-dipolar cyclo-
addition reaction with O-silylated hydroxamic acid 11a (R1
=
Ph). This was achieved using aliphatic compounds 1-octyne 16a
and 1-octene 18, following Carreira's procedure (Scheme 2).[21]
Hence, after dropwise addition of 1
M Tf2O in CH2Cl2 to a solu-
tion of 11a and NEt3 in CH2Cl2 at –40 °C, the reaction mixture
was allowed to stir for 1 h at 0 °C, after which an 8-fold excess of
dipolarophile[31] 16a or 18 was added, and the solution stirred
overnight at r.t. Desired isoxazole 17a and isoxazoline 19 were
successfully synthesized in 49 % and 79 % isolated yield, respec-
tively. The 30 % difference in yield between the two reactions
confirmed the expected lower reactivity of alkynes.[32]
Scheme 3. Reaction side-products 20 are formed during the formation of
nitrile oxide. By-products 21 derive from the dimerization of nitrile oxides.
Reported structures 21 are proposals which were assigned with the support
of literature.[13a,15]
Scheme 2. Evaluation of the reactivity of 1-octyne 16a respect to 1-octene
18 in the 1,3-dipolar cycloaddition reaction with nitrile oxide 15a, generated
in-situ from O-silylated hydroxamic acid 11a.
Next, we attempted to improve the cycloaddition yield with
We started by studying the effect of electron-withdrawing
1-octyne 16a.[33] First, all side-products were isolated and com- groups (EWG) and electron-donating groups (EDG) on the alk-
pared to those obtained in a second trial in which no dipolaro- ynyl reagent.[32] Reactions were carried out between O-silylated
phile had been added to the reaction mixture. We observed hydroxamic acid 11a and aromatic alkynes: ethynylbenzene
that these side-products (Scheme 3, 20/21), of which structures 16b as a reference, electron-poor 1-ethynyl-4-(trifluoromethyl)
21 were proposed with the support of literature,[13a,15] were benzene 16c and electron-rich 4-ethynyl-N,N-dimethylaniline
identical in the absence and in the presence of 1-octyne. This 16d. When adding the alkyne in one batch onto benzonitrile
observation suggested that the side-reactions mainly derive oxide (according to Scheme 2),[21] both activated dipolarophiles
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Scheme 4. Scope of this work: the optimized protocol for the 1,3-dipolar cycloaddition reaction between alkynyl dipolarophile 16R2 and nitrile oxide 15R1
generated in-situ from O-silylated hydroxamic acid 11R1 was applied to a variety of substrates leading to diverse isoxazoles 17R1,R2. Isolated yields are
reported. [a] 65 % with original Carreira's protocol. [b] Measured %area of desired cycloadduct in the RP-HPLC chromatogram of the crude reaction mixture.
[c] Calculated from 49 % conversion, as determined from the RP-HPLC chromatogram of the crude reaction mixture.
16c and 16d gave the corresponding desired isoxazoles in isoxazoles 17b and 17c in 78 % of yield. On the other hand, 4-
higher yield than reference ethynylbenzene 16b (ca. 65 % iso- ethynyl-N,N-dimethylaniline 16d seemed to have decomposed
lated yield respect to 54 %).
to some extent under these conditions, thus giving a yield of
57 %. Although the isolated yield for 3,5-diphenyl isoxazole 17b
is slightly inferior to the protocols using PIFA (90 %)[20b] or a
The cycloaddition reaction yields were increased with ethyn-
ylbenzene 16b and 1-ethynyl-4-(trifluoromethyl) benzene 16c
by cannulating the nitrile oxide into a solution of the dipolaro- mixture of KCl and oxone in H2O (87 %),[35] our method gave
phile (Scheme 1c). Indeed, both dipolarophiles afforded desired similar results as those obtained with Cu(I)-catalyzed synthesis
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Scheme 5. Synthesis of ethynylrhodamine 16f and further 1,3-dipolar cycloaddition reaction with nitrile oxide 15a. Upon formation of desired isoxazole 17f,
the ring-opening of the spirolactam form was observed in the presence of acid.
of isoxazoles (72 %),[24] as well as to other metal-based oxid- phenylboronic acid 24 with MeOH in SOCl2.[39] Reaction with
ative[19,20c,20d,34b] and dehydrating strategies.[17]
1,3,5-tribromobenzene 25 in the presence of a catalytic amount
of [Pd(PPh3)4] and Na2CO3 under microwave conditions,[40] gave
desired tricarboxylic acid derivative 26 after saponification with
aqueous NaOH of resulting tris-ester 27.[41] Activation of tris-
carboxylic acid 26 with HATU in the presence of DIEA, followed
by reaction with O-silylated hydroxylamine 14, afforded desired
nitrile oxide precursor 11e. Finally, subsequent cycloaddition
reaction with ethynyltrimethylsilane 16e provided desired tris-
isoxazole 17l in 39 % yield, i.e. 73 % yield per functionality.
Lastly, we have studied the cycloaddition reaction between
O-silylated hydroxamic acid 11a and 1,2-diphenylethyne 16g
and with 1,4-diethynylbenzene 16h. 3,4,5-triphenylisoxazole
17m was obtained in less than 15 % yield. This is in line with
the limited reactivity of internal alkynes with nitrile oxides, as
it is well established that isoxazoles would exclusively be ob-
tained from electron-deficient internal acetylenic dipolaro-
philes.[2b,42] On the other hand, only one of the two alkynyl
moiety of 1,4-diethynylbenzene 16h was successfully converted
to isoxazole, leading to derivative 17n in 18 % yield. We ex-
plained this result by considering the constraint imposed by
1,4-diethynylbenzene 16h to work with an excess of nitrile ox-
ide, favoring decomposition of the latter into by-products over
its reaction with alkynyl dipolarophile 16h (Scheme 3).
Using the optimized procedure, we then evaluated the com-
patibility of the reaction conditions with other functional
groups. The protocol afforded 3-phenyl-5-(trimethylsilyl)isoxaz-
ole 17e in 69 % when starting from O-silylated hydroxamic acid
precursor 11a and ethynyltrimethylsilane 16e. The reaction was
also successfully applied to ethynylrhodamine 16f (Scheme
5).[36] The 1,3-dipolar cycloaddition reaction with nitrile oxide
15a led to the formation of desired isoxazole 17f in 66 %. Upon
formation of desired cycloadduct 17f, the ring-opening of the
spirolactam moiety was observed, as described in Scheme 5.[37]
Considering its application as molecular sensor, the result ob-
tained with rhodamine B 22 was particularly interesting as this
strategy offers a new route of functionalization of the fluoro-
phore.[38] Indeed, modification of the N-terminus of the spiro-
amide moiety of 16f with a conveniently functionalized dipole
precursor, could allow its conjugation to various receptors for
the targets of interests.
The 1,3-dipolar cycloaddition reaction was tested using eth-
ynyltrimethylsilane 16e with polycyclic aromatic dipoles 11b
and 11c, namely with an anthracenyl and a naphthyl core, re-
spectively. The corresponding O-silylated hydroxamic acids
were synthesized according to Scheme 1b. The desired isox-
azoles 17g and 17h were respectively generated in 71 % and
67 % isolated yields. The 1,3-dipolar cycloaddition reaction was
also performed between naphthyl precursor 11c and ethynyl-
benzene 16b as well as 4-ethynyl-N,N-dimethylaniline 16d. Cor-
responding isoxazoles 17i and 17j were formed in 72 % and
61 % yield, respectively.
The 1,3-dipolar cycloaddition reaction was finally used to
functionalize peptide and peptide nucleic acid (PNA) species.
Both were synthesized on a solid phase semi-automatic peptide
synthesizer (Focus XC), using a classical 9-fluorenylmethyl carb-
amate (Fmoc) solid-phase procedure (Scheme SI1) on a Rink
Amide MBHA resin, with the required amino acids (aa) or PNA
The reaction was also extended to di- and tri-topic sub- monomers.[43] A peptide sequence (KFRVGVADVC) displaying
strates, starting with the simple di-substitution pattern consist- potential function in antitumor therapies[44] was selected, and
ing of a 1,4-benzene core. The corresponding O-silylated synthesized (Section 6 in the SI). Functionalization at the N-
hydroxamic acid 11d was synthesized according to Scheme 1b terminal position with an acetylenic moiety was subsequently
from terephthalic acid in 48 % isolated yield, corresponding to achieved to allow the 1,3-dipolar cycloaddition with nitrile ox-
69 % per functionality. The 1,3-dipolar cycloaddition reaction ides generated from O-silylated hydroxamic acid 11a (Scheme
with ethynyltrimethylsilane 16e provided desired 17k in 50 % 7). The ethynyl moiety was introduced in the sequence by cou-
yield, corresponding to 71 % yield per hydroxamic acid group. pling 5-hexynoic acid with the relevant peptide precursor 28d
Next, we focused on a tris-1,3,5-benzene substituted core pat- under the standard solid-phase synthesis conditions (Scheme
tern. O-silylated hydroxamic acid derivative 11e was synthe- 7a). The 1,3-dipolar cycloaddition reaction with nitrile oxide 15a
sized in four steps (Scheme 6). First, 4-methoxycarbonylphenyl- was then performed on solid phase (Scheme 7b). To this end, a
boronic acid 23 was prepared by esterification of 4-carboxy- 10-fold excess of nitrile oxide, generated in-situ from O-silylated
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Scheme 6. Synthesis of tris-O-silylated hydroxamic acid precursor 11e and subsequent 1,3-dipolar cycloaddition reaction with ethynyltrimethylsilane 16e.
Scheme 7. a) Synthesis of resin-bound ethynyl-functionalized peptide 16i; b) 1,3-dipolar cycloaddition reaction on solid phase between 16i and nitrile oxide
15a. The yield was calculated based on RP-HPLC chromatogram (%area) of the crude reaction mixture and on the measured 49 % conversion. PS = polystyrene;
R1 = amino acid side chain. Sequence: KFRVGVADVC.
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ABCR, and were used as received from the commercial suppliers.
Resins for solid-phase synthesis were purchased from Peptides Inter-
national and aapptec. Solvents were purchased from Sigma Aldrich
and Acros Organics. Deuterated solvents were purchased from
Eurisotop. General solvents were distilled in vacuo. Anhydrous
CH2Cl2 was distilled from phosphorus pentoxide. Anhydrous DMF
was purchased from Acros Organics. Low-temperature baths were
prepared using different solvent mixtures depending on the desired
temperature: –40 °C with CH3CN/liquid N2, –10 °C with ice/brine,
and 0 °C with ice/H2O. Anhydrous conditions were achieved by dry-
hydroxamic acid 11a with Tf2O in the presence of NEt3 at
–40 °C, was cannulated at the same temperature onto a suspen-
sion of resin-bound ethynyl-functionalized peptide 16i in DMF.
The resulting mixture was heated at 50 °C and shaken for 16 h.
This was followed by cleavage from the resin, complete depro-
tection of the side chains using an acidic cleavage mixture TFA/
H2O/EDT/TIPS, 94:2.5:2.5:1, precipitation with Et2O, and analysis
of the resulting white solids by reverse phase HPLC (RP-HPLC).
The RP-HPLC analysis of crude 17o displayed two major prod-
ucts, namely isoxazole 17o, and cleaved starting material ing Schlenk lines, 2-neck flasks or 3-neck flasks by flaming with a
heat gun under vacuum and then purging with argon. The inert
atmosphere was maintained using argon-filled balloons equipped
with a syringe and needle that was used to penetrate the silicon
stoppers used to close the flasks' necks. The addition of liquid rea-
gents was done by means of dried plastic syringes or by cannula-
tion, using standard inert atmosphere techniques. Microwave reac-
tions were performed on a Biotage AB Initiator microwave instru-
ment producing controlled irradiation at 2.45 GHz. Solid-phase
peptide syntheses were performed on a semi-automatic FOCUS XC
peptide synthesizer, coming with a computer/control system from
aapptec. Reactions were monitored by thin-layer chromatography
(TLC) using pre-coated aluminum sheets with 0.20 mm Machevery-
Nagel Alugram SIL G/UV234 with fluorescent indicator UV254 or
UV366. Components were visualized by illumination with short-
wavelength UV light. All products were purified by flash column
chromatography on Grace silica gel 60 (particle size 40–63 μm).
Peptide and PNA oligomers were analyzed and purified by high-
performance liquid chromatography (HPLC) on a Varian 940-LC liq-
uid chromatograph system with a Varian Pursuit C18, 5 μm,
250 × 4.6 mm analytical column and a Varian Pursuit C18, 5 μm,
250 × 21.2 mm preparative column. 0.1 % TFA in H2O and 0.1 %
TFA in CH3CN were used as eluents in all cases. Lyophilisation was
performed on a Christ Freeze Dryer ALPHA 2–4 LDplus, connected to
a Vacuubrand Chemistry-HYBRID-pump, with an ice condenser tem-
perature of approx. –85 °C and a vacuum of approx. 2.10–3 mbar.
1H and 13C NMR spectra were obtained on a 400 MHz NMR (Jeol
JNM EX-400). Chemical shifts were reported in ppm according to
tetramethylsilane using the solvent residual signal as an internal
ethynyl-peptide 16i. Incomplete conversion was observed likely
due to the favored side-reactions of nitrile oxide over the reac-
tion with the alkynyl moieties when the former is in excess. Yet,
the 1,3-dipolar cycloaddition reaction was shown to be compat-
ible with peptidic structures and solid-phase synthesis. Based
on the analytical RP-HPLC chromatogram of the crude reaction
mixture, 49 % conversion of N-terminal ethynyl-peptide 16i was
obtained, of which 41 % corresponded to desired cyclo-added
peptide 17o, and 8 % to unidentified side-products, thereby
resulting in 84 % yield of peptide-isoxazole conjugate.
Next, the reaction was attempted on self-complementary
PNA (sequence: (AATT)3-Lys), which was synthesized from the
relevant Fmoc/benzyloxycarbonyl (Cbz)-protected PNA mono-
mers. Solid phase synthesis, terminal functionalization with an
acetylenic moiety and reaction with nitrile oxide 15a following
the same strategies as that described above for ethynyl-peptide
16i, gave PNA-isoxazole conjugate 17p formed in 43 % (yield
calculated on the %area from the RP-HPLC chromatogram of
the crude reaction mixture). No trace of ethynyl-functionalized
PNA 16j was detected.[33]
Conclusions
In conclusion, we have successfully demonstrated the catalyst-
and oxidant-free regioselective synthesis of 3,5-disubstituted
isoxazoles by 1,3-dipolar cycloaddition reactions between alk-
ynyl dipolarophiles and nitrile oxides, with the latter reagent
being produced in-situ under mild conditions. The method is
experimentally straightforward and convenient as it makes use
of stable crystalline O-silylated hydroxamic acids dipole precur-
sors readily synthesized from the corresponding hydroxamic or
carboxylic acids. Through the application of the protocol to a
variety of dipoles and dipolarophiles, we have observed that
the mildness of this approach provides a tolerance to diverse
functional groups as different 3,5-disubstituted isoxazoles were
successfully synthesized in moderate to good yields. In particu-
lar, we have shown that the method was compatible with bio-
logical molecules such as peptides and PNA,[45] thus opening
the way to the biorthogonal applications.[46] An isoxazole deriv-
ative of rhodamine B was also successfully formed, indicating
that the strategy could provide a promising new functionaliza-
tion route towards labelling and sensing applications.
reference (CDCl3: δH = 7.26 ppm, δC = 77.16 ppm; CD2Cl2: δH
5.32 ppm, δC = 53.84 ppm; [D6]DMSO: δH = 2.50 ppm, δC
=
=
39.52 ppm). Coupling constants (J) were given in Hz and were aver-
aged. Resonance multiplicity was described as s (singlet), d (dou-
blet), t (triplet), q (quartet), quin (quintet), m (multiplet), br (broad
signal), dd (doublet of doublets). Carbon spectra were acquired with
a complete decoupling for the proton.
General Experimental Procedure for the Preparation of O-(tert-
Butyldiphenylsilyl)hydroxamic acids (11) – Hydroxamic acid
route (Method A): A solution of benzhydroxamic acid (1 equiv.) in
THF was treated at 0 °C with NaH (60 % in mineral oil, 2 equiv.) in
two portions at 20 min intervals, under an inert atmosphere of
argon. After 30 min, when all H2 gas evolution had stopped, the
solution was cooled to 0 °C, and tert-butylchlorodiphenylsilane
(1.1 equiv.) was added dropwise over 30 min. The reaction mixture
was then warmed up to r.t. and was stirred for additional 2 h. It
was subsequently diluted with H2O and extracted with EtOAc. The
organic extracts were washed with H2O and dried with anhydrous
Na2SO4, filtered and concentrated to dryness in vacuo. The residue
was purified by silica gel column chromatography to afford desired
product 11. With this procedure, we prepared derivative 11a.
Experimental Section
General Experimental Procedure for the Preparation of O-(tert-
Butyldiphenylsilyl)hydroxamic acids (11) – Carboxylic acid route
General Information: Chemicals were purchased from Sigma Al-
drich, Acros Organics, Fluorochem, TCI, aapptec, carbosynth, and (Method B): To a suspension of benzoic acid (1 equiv.) in anhydrous
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DMF were added DIEA (2 equiv.) and HATU (1.1 equiv.), in this order, 1516.3, 1651.8, 2859.4, 2955.5, 3190.5; MS (ESI-HRMS):
under an inert atmosphere of argon. The resulting reaction mixture
was stirred for 30 min at r.t., after which O-silylated hydroxylamine
14 (1.5 equiv.) was added at 0 °C and the resulting solution stirred
overnight (approx. 16 h) at r.t. It was then concentrated to dryness
in vacuo to yield a solid residue, which was dissolved in CH2Cl2 and
washed with H2O. The aqueous phase was extracted with CH2Cl2
and the combined organic phases washed with H2O and brine,
dried with anhydrous Na2SO4, filtered and concentrated to dryness
in vacuo. The residue was purified by silica gel column chromatog-
raphy to afford desired product 11. With this procedure, we pre-
pared derivatives 11a–e.
Found 673.2905 [M + H]+, C40H45N2O4Si2 requires = 673.2912.
N4,N4′′-Bis((tert-butyldiphenylsilyl)oxy)-5′-(4-(((tert-butyldi-
phenylsilyl)oxy)carbamoyl)phenyl)-[1,1′:3′,1′′-terphenyl]-4,4′′-
dicarboxamide (11e) synthesis according to Method B: Yield
26 % (104 mg; 64 % per functionality) of desired product 11e as a
1
white solid. Rf = 0.39 (CH2Cl2). m.p. 104–107 °C; H NMR (400 MHz,
[D6]DMSO, *denotes rotamer peaks): δ = 11.48 & 11.04* (s, 3H, NH),
7.94 (s, 3H, CH), 7.91 (d, J = 8.2 Hz, 6H, CH), 7.81–7.74* (m, 13.5H,
CH), 7.64* (d, J = 8.2 Hz, 4.5H, CH), 7.49–7.40 (m, 18H, CH), 1.13 (s,
27H, CH); 13C NMR (100 MHz, [D6]DMSO, *denotes rotamer peaks):
δ = 166.0, 142.6, 140.7, 135.6, 135.2, 134.8*, 134.5*, 133.5*, 132.1,
131.8*, 130.1, 129.7*, 129.2*, 127.7, 127.6, 127.5*, 127.3*, 127.1,
N-((tert-Butyldiphenylsilyl)oxy)benzamide (11a): Synthesis ac-
cording to Method A: Yield 72 % (492 mg) of desired product 11a
as a white solid. Synthesis according to Method B: Yield 44 % (2.69 g,
white solid). Rf = 0.42 (Cyclohexane(Cy)/EtOAc, 95:5). m.p. 134–
137 °C; 1H NMR (400 MHz, [D6]DMSO, *denotes rotamer peaks): δ =
11.40 & 10.92* (s, 1H, NH), 7.76 (m, 4H, CH), 7.53–7.37 (m, 11H, CH),
1.11 (s, 9H, CH); 13C NMR (100 MHz, [D6]DMSO): δ = 166.5, 135.5,
132.7, 132.1, 131.4, 130.1, 128.4, 127.6, 127.2, 26.8, 19.2. All other
spectroscopic and analytical properties were identical to those re-
ported in the literature.[21]
126.2*, 125.1, 27.2*, 26.8, 26.5*, 19.2; IR (cm–1): ν = 486.8, 502.4,
˜
612.4, 621.6, 649.4, 697.9, 738.7, 760.0, 797.9, 821.9, 876.7, 909.9,
952.9, 998.3, 1013.8, 1035.5, 1106.78, 1113.9, 1158.7, 1187.5, 1233.5,
1291.5, 1314.0, 1362.5, 1392.1, 1409.1, 1427.4, 1441.4, 1371.6,
1487.7, 1526.8, 1588.4, 1609.8, 1681.5, 1899.7, 2857.0, 2893.2,
2930.3, 3048.8, 3071.8, 3192.4, 3408.3; MS (ESI-HRMS):
Found 1198.5030 [M + H]+, C75H76N3O6Si3 requires = 1198.5036,
found 1199.0054 [2M + 2H]2+, C150H152N6O12Si6 requires =
1199.0053.
O-(tert-Butyldiphenylsilyl)hydroxylamine (14): To a stirred sus-
pension of hydroxylamine hydrochloride (500 mg, 7.19 mmol) in
anhydrous CH2Cl2 (20 mL) was added NEt3 (1.60 g, 2.21 mL,
15.83 mmol) under an inert atmosphere of argon. The mixture was
allowed to stir for 1 h at r.t. Neat tert-butylchlorodiphenylsilane
(2.18 g, 2.06 mL, 7.91 mmol) was added, and the reaction mixture
allowed to stir overnight (approx. 16 h) at r.t. The mixture was then
concentrated to dryness in vacuo and THF (10 mL) added to the
crude. Triethylamine hydrochloride was removed as a white solid
by filtration. The flask and precipitate were washed (3 × 5 mL) with
THF, and the resulting solution concentrated to dryness in vacuo.
Ice-cold pentane (15 mL) was added to the residue, the mixture
briefly sonicated and the compound allowed to crystallise in the
fridge for 2 h. The solid was collected by filtration and washed with
ice-cold pentane, yielding 14 (1.77 g, 91 %) as a white crystalline
N-((tert-Butyldiphenylsilyl)oxy)anthracenyl-9-carboxamide
(11b), synthesis according to Method B: Yield 41 % (351 mg) of
desired product 11b as a yellow solid. Rf = 0.62 (CH2Cl2/MeOH,
95:5). m.p. 91–94 °C; 1H NMR (400 MHz, [D6]DMSO): δ = 11.67 (s,
1H, NH), 8.60 (s, 1H, CH), 8.04 (m, 2H, CH), 7.88 (m, 4H, CH), 7.57 (m,
2H, CH), 7.51–7.44 (m, 6H, CH), 7.30–7.23 (m, 4H, CH) 1.17 (s, 9H,
CH), the proton assignment has been done by 2D analysis; 13C NMR
(100 MHz, [D6]DMSO): δ = 165.7, 135.9, 131.4, 130.4, 130.3, 129.7,
128.2, 127.8, 127.7, 126.4, 125.5, 125.1, 26.9, 19.2 (one of the quater-
nary aromatic carbons overlaps with another signal); IR (cm–1): ν =
˜
507.3, 698.7, 709.2, 727.8, 737.9, 807.9, 888.5, 1060.0, 1116.1, 1427.5,
1471.0, 1498.4, 1648.9, 2856.9, 2929.8, 2955.5, 3052.6, 3200.4; MS
(APCI-HR-MS): Found 476.2043 [M + H]+, C31H29NO2Si requires =
476.2046.
1
N-((tert-Butyldiphenylsilyl)oxy)-1-naphthamide (11c), synthesis
according to Method B: Yield 48 % (237 mg) of desired product
11c as a white solid. Rf = 0.45 (Cy/EtOAc, 93:7). m.p. 164–167 °C; 1H
NMR (400 MHz, [D6]DMSO): δ = 11.48 (s, 1H, NH), 7.95 (m, 1H, CH),
7.89 (m, 1H, CH), 7.81 (m, 4H, CH), 7.51–7.42 (m, 9H, CH), 7.36–7.34
(m, 1H, CH), 7.27 (d, J = 6.9 Hz, 1H, CH), 1.14 (s, 9H, CH), the proton
assignment has been done by 2D analysis; 13C NMR (100 MHz,
[D6]DMSO): δ = 166.5, 135.8, 132.9, 131.9, 131.7, 130.2, 130.1, 129.9,
128.0, 127.6, 126.6, 126.2, 125.5, 125.1, 124.7, 26.9, 19.2; IR (cm–1):
solid. m.p. 68–70 °C; H NMR (400 MHz, CD2Cl2): δ = 7.75–7.71 (m,
4H, CH), 7.44–7.39 (m, 6H, CH), 1.08 (s, 9H, CH), NH protons were
not observed; 13C NMR (100 MHz, CD2Cl2): δ = 135.8, 134.1, 130.0,
128.0, 27.4, 19.4. All other spectroscopic and analytical properties
were identical to those reported in the literature.[30]
3′,6′-bis(Diethylamino)-2-(prop-2-yn-1-yl)spiro[isoindoline-1,9′-
xanthen]-3-one (16f): To a solution of rhodamine B 22 (1 g,
2.09 mmol) in anhydrous 1,2-dichloroethane (82 mL) was added
dropwise POCl3 (2.08 g, 1.26 mL, 13.58 mmol) with vigorous stirring,
under an inert atmosphere of argon. The mixture was stirred under
reflux at 80 °C for 6 h, after which it was cooled to r.t. and concen-
trated to dryness in vacuo. The resulting residue was dissolved in
THF (30 mL) and treated with NEt3 (1.21 g, 1.66 mL, 12.0 mmol),
followed by propargylamine (121 mg, 0.14 mL, 2.2 mmol). The solu-
tion was stirred overnight (approx. 16 h) at r.t., after which it was
concentrated to dryness in vacuo and subjected to silica gel column
chromatography (eluent: CH2Cl2) yielding 68 % (685 mg) of desired
ν = 503.2, 526.6, 567.3, 593.9, 615.1, 623.3, 689.0, 701.7, 711.6, 734.2,
˜
754.1, 782.6, 805.3, 822.8, 899.5, 1054.6, 1116.5, 1427.2, 1512.4,
1651.9; MS (ESI-HRMS): Found 426.1882 [M + H]+, C27H28NO2Si re-
quires = 426.1884, found 448.1703 [M + Na]+, C27H27NNaO2Si re-
quires = 448.1703.
N1,N4-bis((tert-Butyldiphenylsilyl)oxy)terephthalamide (11d),
synthesis according to Method B: Yield 48 % (581 mg; 69 % per
functionality) of desired product 11d as a white solid. Rf = 0.51
(Cy/EtOAc, 90:10). m.p. decomposition at 121–123 °C; 1H NMR product 16f as a pale pink solid. Rf = 0.82 (CH2Cl2). m.p. 191–193 °C;
(400 MHz, [D6]DMSO, *denotes rotamer peaks): δ = 11.47 & 11.13*
(s, 2H, NH), 7.73–7.72 (m, 8H, CH), 7.49–7.38 (m, 16H, CH), 1.09 (s,
18H, CH); 13C NMR (100 MHz, [D6]DMSO): δ = 135.4, 134.6, 132.3,
1H NMR (400 MHz, CDCl3): δ = 7.93–7.90 (m, 1H, CH), 7.44–7.39 (m,
2H, CH), 7.10–7.08 (m, 1H, CH), 6.46 (d, J = 8.9 Hz, 2H, CH), 6.39 (d,
J = 2.5 Hz, 2H, CH), 6.26 (dd, J1 = 2.5 Hz, J2 = 8.9 Hz, 2H, CH), 3.94
130.0, 127.6, 126.8, 26.9, 19.2, quaternary carbonyl carbon signal (d, J = 2.5 Hz, 2H, CH), 3.33 (q, J = 7.1 Hz, CH), 1.75 (t, J = 2.5 Hz,
was not observed; IR (cm–1): ν = 507.2, 615.9, 622.7, 697.1, 710.9,
757.7, 764.8, 822.6, 852.8, 894.8, 1013.1, 1031.2, 1116.0, 1167.2,
1194.4, 1261.1, 1275.7, 1312.3, 1362.1, 1427.7, 1471.1, 1486.0,
1H, CH), 1.15 (t, J = 7.1 Hz, 12H, CH); 13C NMR (100 MHz, CDCl3):
δ = 167.4, 153.8, 153.5, 148.8, 132.6, 130.4, 129.1, 128.0, 123.8, 122.9,
107.9, 105.1, 97.8, 78.3, 70.1, 64.8, 44.4, 28.5, 12.6. All other spectro-
˜
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scopic and analytical properties were identical to those reported in oil. Synthesis according to Method B: Yield 75 % (35 mg, viscous light-
the literature.[36]
yellow oil). Rf = 0.58 (CH2Cl2). 1H NMR (400 MHz, CD2Cl2): δ = 7.79–
7.76 (m, 2H, CH), 7.44–7.42 (m, 3H, CH), 6.31 (s, 1H, CH), 2.77 (m,
2H, CH), 1.74–1.68 (m, 2H, CH), 1.40–1.29 (m, 6H, CH), 0.89 (t, J =
7.1 Hz, 3H, CH), the proton assignment has been done by 2D analy-
sis; 13C NMR (100 MHz, CD2Cl2): δ = 174.9, 162.6, 130.1, 129.9, 129.2,
127.1, 99.1, 31.9, 29.2, 27.9, 27.2, 22.9, 14.2; MS (ESI-HRMS):
Found 230.1541 [M + H]+, C15H20NO requires = 230.1539. All other
spectroscopic and analytical properties were identical to those re-
ported in the literature.[34a]
VGVA Acetylenic Peptide (16i): Acetylenic-peptide 16i was syn-
thesised on 200 mg scale, following the strategy described on
Scheme SI1, using the appropriate amino acids. Following synthesis
of the desired sequence, 5-hexynoic acid (68.9 mg, 68 μL,
0.615 mmol) was used in place of an amino acid, and a double
coupling was achieved in order to ensure a quantitative reaction.
An aliquot of the resulting crude was isolated, cleaved from the
resin and analyzed by RP-HPLC. The cleavage cocktail mixture used
was TFA/H2O/EDT/TIPS, 94:2.5:2.5:1. The cleavage took place for 4 h,
under an inert atmosphere of argon. Peptide 16i was isolated as a
white solid, soluble in a mixture of H2O/CH3CN, 1:1 + 0.1 % TFA.
HPLC retention time: 29.4 min. MS (ESI-HRMS): Found 1186.6520 [M
+ H]+, 593.8314 [M+2H]2+, which resolved to 1185.6456 0.0053
[M], C54H87N15O13S requires = 1185.6328. Additional synthetic and
analytical details are described in the supporting information.
3,5-Diphenylisoxazole (17b): Synthesis according to Method A:
Yield 54 % (16 mg) of desired product 17b as a white solid. Synthe-
sis according to Method B: Yield 78 % (23 mg, white solid). Rf = 0.58
(CH2Cl2). m.p. 137–139 °C; 1H NMR (400 MHz, CD2Cl2): δ = 7.85–7.83
(m, 4H, CH), 7.50–7.47 (m, 6H, CH), 6.88 (s, 1H, CH); 13C NMR
(100 MHz, CD2Cl2): δ = 170.7, 163.3, 130.6, 130.4, 129.6, 129.4, 129.3,
127.8, 127.1, 126.1, 97.9. All other spectroscopic and analytical prop-
erties were identical to those reported in the literature.[20c]
(AATT)3 Lys Acetylenic PNA (16j): Acetylenic PNA dodecamer 16j
was synthesised on 250 mg scale, following the strategy described
on Scheme SI1, using the appropriate amino acid (lysine) and PNA
monomers. Following synthesis of the desired sequence, 5-hexynoic
acid (68.9 mg, 68 μL, 0.615 mmol) was used in place of a PNA
monomer, and a double coupling was achieved in order to ensure a
quantitative reaction. An aliquot of the resulting crude was isolated,
cleaved from the resin. The Cbz-protecting groups were depro-
tected. Resulting deprotected PNA 16j was isolated as a white solid,
soluble in H2O. HPLC retention time: 11.4 min. MS (MALDI-MS):
Found 3487.5 [M + H]+, C144H184N69O38 requires = 3487.46, found
3509.5 [M + Na]+, C144H183N69NaO38 requires = 3509.44, found
3525.4 [M + K]+, C144H183N69KO38 requires = 3525.42. Additional
synthetic and analytical details are described in the supporting in-
formation.
3-Phenyl-5-(4-(trifluoromethyl)phenyl)isoxazole (17c): Synthesis
according to Method A: Yield 64 % (37 mg) of desired product 17c
as a white crystalline solid. Synthesis according to Method B: Yield
78 % (30 mg, white crystalline solid). Rf = 0.63 (CH2Cl2). m.p. 172–
1
175 °C; H NMR (400 MHz, CD2Cl2): δ = 7.97 (d, J = 8.7 Hz, 2H, CH),
7.88–7.85 (m, 2H, CH), 7.76 (d, J = 8.7 Hz, 2H, CH), 7.50–7.48 (m, 3H,
CH), 6.99 (s, 1H, CH); 13C NMR (100 MHz, CD2Cl2): δ = 169.2, 163.5,
131.1, 130.6, 129.4, 129.2, 127.2, 126.6, 126.5, 126.4, 126.3, 99.5. All
other spectroscopic and analytical properties were identical to
those reported in the literature.[12c]
N,N-Dimethyl-4-(3-phenylisoxazol-5-yl)aniline (17d): Synthesis
according to Method A: Yield 65 % (34 mg) of desired product 17d
as a as a pale yellow solid. Synthesis according to Method B: Yield
57 % (20 mg, pale yellow solid). Rf = 0.52 (CH2Cl2). m.p. 155–158 °C;
1H NMR (400 MHz, CD2Cl2): δ = 7.86–7.83 (m, 2H, CH), 7.68 (d, J =
8.9 Hz, 2H, CH), 7.47–7.45 (m, 3H, CH), 6.75 (d, J = 8.9 Hz, 2H, CH),
6.64 (s, 1H, CH), 3.01 (s, 6H, CH); 13C NMR (100 MHz, CD2Cl2): δ =
171.6, 163.1, 152.0, 130.1, 130.0, 129.2, 127.3, 127.1, 115.4, 112.1,
General Experimental Procedure for the Preparation of 3,5-di-
substituted Isoxazoles (17) – Method A:[21] To a solution of O-
silylated hydroxamic acid 11 (1 equiv.) in anhydrous CH2Cl2 was
added NEt3 (3 equiv.) under an inert atmosphere of argon. This was
followed by the dropwise addition of a 1 M solution of Tf2O in
CH2Cl2 (1.1 equiv.) at –40 °C. Once the addition was completed, the
reaction mixture was allowed to stir for 1 h at 0 °C, after which
alkyne 16 (8 equiv.) was added, and the resulting mixture stirred
overnight (approx. 16 h) at r.t. Next, the solution was washed with
H2O. The organic layer was dried with anhydrous Na2SO4, filtered
and concentrated to dryness in vacuo.[3] The residue was purified
by silica gel column chromatography to afford desired isoxazole 17.
With this procedure, we prepared cycloadducts 17a-d.
94.9, 40.3; IR (cm–1): ν = 689.2, 771.9, 814.4, 926.1, 949.1, 1169.9,
˜
1198.7, 1230.9, 1326.0, 1368.4, 1397.0, 1413.4, 1446.2, 1466.0,
1507.6, 1524.4, 1578.2, 1614.1, 2922.8; MS (ESI-HRMS):
Found 265.1334 [M + H]+, C17H17N2O requires = 265.1335.
3-Phenyl-5-(trimethylsilyl)isoxazole (17e), synthesis according
to Method B: Yield 69 % (20 mg) of desired product 17e as a pale
1
yellow oil. Rf = 0.57 (CH2Cl2). H NMR (400 MHz, CD2Cl2): δ = 7.82–
7.79 (m, 2H, CH), 7.47–7.40 (m, 3H, CH), 6.78 (s, 1H, CH), 0.37 (s, 9H,
CH); 13C NMR (100 MHz, CD2Cl2): δ = 179.3, 161.1, 130.1, 129.7,
General Experimental Procedure for the Preparation of 3,5-di-
substituted Isoxazoles (17) – Method B: To a solution of O-silyl-
ated hydroxamic acid 11 (1 equiv.) in anhydrous CH2Cl2 was added
NEt3 (3 equiv.) under an inert atmosphere of argon. This was fol-
lowed by the dropwise addition of a 1 M solution of Tf2O in CH2Cl2
(1.1 equiv.) at –40 °C. Once the addition was completed, the reac-
tion mixture was allowed to stir for 3 min at –40 °C, after which
it was cannulated batchwise (every 15 min over 45 min) at that
temperature, onto neat alkyne 16 kept at r.t. (8 equiv.). The resulting
mixture was stirred overnight (approx. 16 h) at r.t. Next, the solution
was washed with H2O. The organic layer was dried with anhydrous
Na2SO4, filtered and concentrated to dryness in vacuo. The residue
was purified by silica gel column chromatography to afford desired
isoxazole 17. With this procedure, we prepared cycloadducts
17a–n.
129.3, 127.3, 111.1, –1.83; IR (cm–1): ν = 505.8, 631.1, 684.6, 691.2,
˜
760.2, 839.1, 897.5, 949.4, 971.7, 1026.5, 1056.2, 1077.9, 1097.9,
1252.2, 1385.1, 1424.1, 1458.9, 1505.2, 1548.8, 2959.9; MS (ESI-
HRMS): Found 218.0995 [M + H]+, C12H16NOSi requires = 218.0996.
3′,6′-Bis(diethylamino)-2-((3-phenylisoxazol-5-yl)methyl)spiro-
[isoindoline-1,9′-xanthen]-3-one (17f), synthesis according to
Method B: Yield 66 % (53 mg) of desired product 17f as a pale
1
pink solid. Rf = 0.17 (CH2Cl2/MeOH, 9:1). m.p. 215–217 °C; H NMR
(400 MHz, CD2Cl2): δ = 7.92–7.89 (m, 1H, CH), 7.55–7.53 (m, 2H, CH),
7.49–7.47 (m, 2H, CH), 7.36–7.35 (m, 3H, CH), 7.07–7.06 (m, 1H, CH),
6.31–6.29 (m, 4H, CH), 6.14 (m, 2H, CH), 5.80 (s, 1H, CH), 4.41 (s, 2H,
CH), 3.20 (q, J = 7.1 Hz, 8H, CH), 1.03 (t, J = 7.1 Hz, 12H, CH), the
proton assignment has been done by 2D analysis; 13C NMR could
not be well acquired due to the closed-opened equilibrium of rhod-
5-Hexyl-3-phenylisoxazole (17a): Synthesis according to Method A: amine's core, which hampered the 13C NMR recording of a pure
Yield 49 % (23 mg) of desired product 17a as a viscous light-yellow
isomer;[37] IR (cm–1): ν = 699.6, 760, 787.3, 818.1, 920.3, 950, 1018.7,
˜
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1089.3, 1118.5, 1152.2, 1219.4, 1265.3, 1305.1, 1329.3, 1357.3, 1376, 1H NMR (400 MHz, CDCl3): δ = 7.90 (s, 4H, CH), 6.82 (s, 2H, CH), 0.37
1424.5, 1443.2, 1467.6, 1514.8, 1547.9, 1614.6, 1634, 1698.3, 2928.5, (s, 18H, CH); 13C NMR (100 MHz, CDCl3): δ = 179.6, 160.5, 130.9,
2970; MS (ESI-HRMS): Found 599.3008 [M + H]+, C38H39N4O3 re- 127.8, 111.1, –1.85; IR (cm–1): ν = 514.2, 529.8, 608.0, 634.6, 691.7,
˜
quires = 599.3017, found 300.1552 [M + 2H]2+, C38H40N4O3 re- 706.1, 762.8, 798.1, 822.8, 841.5, 897.5, 947.7, 971.1, 1021.5, 1063.7,
quires = 300.1545; UV/Vis (CH2Cl2, r.t.): λmax [nm] (ε [M–1 cm–1]): 240 1101.8, 1217.6, 1251.9, 1276.0, 1352.4, 1373.6, 1410.6, 1447.2,
(2.1×107), 275 (9×106), 316 (3.7×106).
1523.0, 1559.4, 2854.1, 2924.8, 2960.4, 3122.6; MS (ESI-HRMS):
Found 357.1451 [M + H]+, C18H25N2O2Si2 requires = 357.1449.
3-(Anthracen-9-yl)-5-(trimethylsilyl)isoxazole (17g), synthesis
according to Method B: Yield 71 % (30 mg) of desired product
17g as a viscous yellow oil. Rf = 0.51 (CH2Cl2). 1H NMR (400 MHz,
CDCl3): δ = 8.57 (s, 1H, CH), 8.05 (m, 2H, CH), 7.80 (m, 2H, CH),
7.51–7.43 (m, 4H, CH), 6.69 (s, 1H, CH), 0.48 (s, 9H, CH), the proton
assignment has been done by 2D analysis; 13C NMR (100 MHz,
CDCl3): δ = 178.6, 159.1, 131.3, 130.8, 128.7, 128.6, 126.4, 125.9,
3,3′-(5′-(4-(5-(Trimethylsilyl)isoxazol-3-yl)phenyl)-[1,1′:3′,1′′-ter-
phenyl]-4,4′′-diyl)bis(5-(trimethylsilyl)isoxazole) (17l), Synthesis
according to Method B: Yield 39 % (38 mg; 73 % per functionality)
of desired product 17l as a white solid. Rf = 0.38 (CH2Cl2). m.p. 42–
44 °C; 1H NMR (400 MHz, CDCl3): δ = 7.95 (d, J = 8.6 Hz, 6H, CH),
7.93 (s, 3H, CH), 7.85 (d, J = 8.6 Hz, 6H, CH), 6.84 (s, 3H, CH), 0.39 (s,
27H, CH); 13C NMR (100 MHz, CDCl3): δ = 179.5, 160.7, 142.4, 142.1,
125.5, 123.7, 116.2, –1.5; IR (cm–1): ν = 622.5, 734.8, 759.4, 844.8,
˜
129.1, 128.2, 127.9, 125.7, 111.1, –1.79; IR (cm–1): ν = 505.6, 535.0,
1073.0, 1252.8, 1307.2, 1381.3, 1617.4, 1637.8, 2850.3, 2922.9,
2959.2, 3050.2, 3411.5, 3476.2, 3554.9; MS (APCI-HR-MS): Found
318.1313 [M + H]+, C20H20NOSi requires = 318.1314.
˜
575.2, 609.3, 630.5, 672.2, 702.0, 736.9, 758.5, 804.9, 836.5, 898.8,
949.7, 971.6, 1018.3, 1050.3, 1098.9, 1209.0, 1251.8, 1303.2, 1377.2,
1403.2, 1417.9, 1449.6, 1516.8, 1545.7, 1571.5, 1596.0, 1611.4,
1918.0, 2899.7, 2958.7, 3050.3; MS (ESI-HRMS): Found 724.2832 [M +
3-(Naphthalen-1-yl)-5-(trimethylsilyl)isoxazole (17h), synthesis
according to Method B: Yield 67 % (24 mg) of desired product
17h as a viscous colorless oil. Rf = 0.49 (CH2Cl2). 1H NMR (400 MHz,
CD2Cl2): δ = 8.37–8.35 (m, 1H, CH), 7.96–7.91 (m, 2H, CH), 7.70–7.67
(m, 1H, CH), 7.56–7.52 (m, 3H, CH), 6.77 (s, 1H, CH), 0.42 (s, 9H, CH),
the proton assignment has been done by 2D analysis; 13C NMR
(100 MHz, CD2Cl2): δ = 178.4, 161.0, 134.2, 131.5, 130.2, 128.8, 128.1,
H]+, C42H46N3O3Si3 requires = 724.2841, found 362.6461 [M + 2H]2+
,
C42H47N3O3Si3 requires = 362.6457.
3,4,5-Triphenylisoxazole (17m), synthesis according to Method
B: Desired derivative 17m was isolated by silica gel column chroma-
tography (eluent: CH2Cl2), along with traces of an unidentified by-
product. Further purification was attempted by means of prepara-
tive TLC plates (3 ×; eluent: CH2Cl2). However, 17m could not be
isolated pure. 6 mg of that mixture was obtained as a white solid,
thus affording less than 15 % yield in 17m. Rf = 0.69 (CH2Cl2). 1H
NMR (400 MHz, CD2Cl2): δ = 8.15 (m, CH), 7.52–7.44 (m, CH); MS (ESI-
HRMS): Found 298.1225 [M + H]+, C21H16NO requires = 298.1226.
127.6, 127.3, 126.6, 126.1, 125.6, 114.5, –1.76; IR (cm–1): ν = 535.0,
˜
561.6, 630.9, 658.8, 703.3, 759.8, 773.9, 798.9, 840.2, 887.1, 934.6,
971.8, 1026.7, 1076.0, 1130.3, 1180.4, 1214.7, 1252.3, 1325.2, 1358.4,
1371.1, 1395.4, 1512.9, 1579.5, 1597.5, 1730.4, 2900.2, 2959.1,
3050.1; MS (ESI-HRMS): Found 268.1153 [M + H]+, C16H18NOSi re-
quires = 268.1152.
5-(4-Ethynylphenyl)-3-phenylisoxazole (17n), synthesis accord-
3-(Naphthalen-1-yl)-5-phenylisoxazole (17i), synthesis accord-
ing to Method B: Yield 18 % (7 mg) of product 17n as a white
ing to Method B: Yield 72 % (26 mg) of desired product 17i as a
1
solid. Rf = 0.58 (CH2Cl2). m.p. 96–98 °C; H NMR (400 MHz, CD2Cl2):
1
white solid. Rf = 0.53 (CH2Cl2). m.p. 108–110 °C; H NMR (400 MHz,
δ = 7.86–7.84 (m, 2H, CH), 7.80 (d, J = 8.6 Hz, 2H, CH), 7.60 (d, J =
8.6 Hz, 2H, CH), 7.49–7.47 (m, 3H, CH), 6.90 (s, 1H, CH), 3.26 (s, 1H,
CH); 13C NMR (100 MHz, CD2Cl2): δ = 169.8, 163.4, 133.1, 130.5,
129.3, 129.1, 127.9, 127.1, 126.0, 124.3, 98.7, 83.2, 79.5; IR (cm–1):
CD2Cl2): δ = 8.45–8.42 (m, 1H, CH), 7.98 (m, 1H, CH), 7.93 (m, 1H,
CH), 7.88 (m, 2H, CH), 7.75 (m, 1H, CH), 7.59–7.46 (m, 6H, CH), 6.88
(s, 1H, CH), the proton assignment has been done by 2D analysis;
13C NMR (100 MHz, CD2Cl2): δ = 170.1, 163.5, 134.3, 131.4, 130.7,
130.6, 129.5, 128.9, 128.1, 127.8, 127.4, 127.3, 126.7, 126.2, 126.0,
ν = 511.4, 543.2, 615.2, 627.1, 663.7, 692.7, 764.0, 819.0, 845.3, 916.6,
˜
949.9, 1044.9, 1090.1, 1112.1, 1399.2, 1413.7, 1462.4, 1493.7, 2920.0,
3298.2; MS (ESI-HRMS): Found 246.0914 [M + H]+, C17H12NO re-
quires = 246.0913.
125.6, 101.5; IR (cm–1): ν = 535.5, 565.7, 653.8, 668.1, 687.9, 763.1,
˜
773.7, 797.9, 864.6, 915.7, 936.0, 948.1, 970.7, 1026.4, 1098.1, 1130.6,
1143.5, 1162.7, 1180.6, 1214.2, 1261.7, 1292.2, 1335.2, 1364.7,
1378.6, 1408.4, 1446.6, 1475.4, 1497.3, 1514.5, 1571.5, 1590.6,
1612.4, 1730.7, 1818.8, 1951.1, 2850.8, 2921.6, 3051.9, 3126.1; MS
(ESI-HRMS): Found 272.1068 [M + H]+, C19H14NO requires =
272.1070.
VGVA Peptide-isoxazole conjugate (17o): To a solution of O-silyl-
ated hydroxamic acid of phenyl 11a (180 mg, 0.48 mmol) in anhy-
drous CH2Cl2 (7.2 mL) was added NEt3 (146 mg, 202 μL, 1.44 mmol)
under an inert atmosphere of argon. This was followed by the drop-
wise addition of a 1 M solution of Tf2O in CH2Cl2 (528 μL,
0.528 mmol) at –40 °C. Once the addition was completed, the reac-
tion mixture was allowed to stir for 3 min at –40 °C, after which it
was added to a suspension of resin-bound ethynyl-functionalized
peptide 16i (100 mg, 0.048 mmol) in DMF (2 mL). The resulting
mixture was shaken overnight (approx. 16 h) at 50 °C. The solution
was then filtered and washed with DMF (5 × 5 mL) and CH2Cl2
(7 × 5 mL). The resulting crude was isolated, cleaved from the resin,
analyzed and purified by RP-HPLC. The cleavage cocktail mixture
used was TFA/H2O/EDT/TIPS, 94:2.5:2.5:1. The cleavage took place
for 4 h, under an inert atmosphere of argon. Peptide 17o was iso-
lated as a white solid, soluble in a mixture of H2O/CH3CN, 1:1 +
0.1 % TFA. HPLC retention time: 26.7 min. MS (MALDI-HRMS): Found
1305.6781 [M + H]+, C61H93N16O14S requires = 1305.6778, found
1327.7 [M + Na]+, C61H92N16NaO14S requires = 1327.66, found
1343.6 [M + K]+, C61H92N16KO14S requires = 1343.63. Based on the
analytical RP-HPLC chromatogram, it was calculated that 49 % con-
N,N-Dimethyl-4-(3-(naphthalen-1-yl)isoxazol-5-yl)aniline (17j),
synthesis according to Method B: Yield 61 % (25.5 mg) of desired
product 17j as a pale yellow solid. Rf = 0.44 (CH2Cl2). m.p. 122–
125 °C; 1H NMR (400 MHz, CD2Cl2): δ = 8.47–8.45 (m, 1H, CH), 7.97–
7.92 (m, 2H, CH), 7.75–7.71 (m, 3H, CH), 7.58–7.53 (m, 3H, CH), 6.77
(m, 2H, CH), 6.65 (s, 1H, CH), 3.02 (s, 6H, CH), the proton assignment
has been done by 2D analysis; 13C NMR (100 MHz, CD2Cl2): δ =
170.9, 163.3; 152.0, 134.2, 131.5, 130.3, 128.8, 127.9, 127.8, 127.4,
127.2, 126.6, 126.2, 125.6, 115.4, 112.2, 98.5, 40.3; IR (cm–1): ν =
˜
659.7, 776.3, 785.4, 803.3, 819.5, 935.7, 949.9, 1063.1, 1123.3, 1169.0,
1195.8, 1226.9, 1263.7, 1362.5, 1408.4, 1441.8, 1480.7, 1518.3,
1609.6, 2894.5; MS (ESI-HRMS): Found 315.1491 [M + H]+, C21H19N2O
requires = 315.1492.
1,4-Bis(5-(Trimethylsilyl)isoxazol-3-yl)benzene (17k), synthesis
according to Method B: Yield 50 % (10.6 mg; 71 % per function)
of desired product 17k as a viscous colorless oil. Rf = 0.54 (CH2Cl2).
Eur. J. Org. Chem. 0000, 0–0
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10
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
version was obtained, of which 41 % corresponded to desired cyclo-
added peptide 17o, and 8 % to unidentified subproducts. 51 % of
starting acetylenic-peptide 16i were recovered thereby resulting in
84 % yield of peptide–isoxazole conjugate. Percentages are given
as area%. Additional synthetic and analytical details are described
in the supporting information.
NMR (400 MHz, CDCl3): δ = 8.29 (d, J = 8.0 Hz, 2H, CH), 8.17 (d, J =
8.0 Hz, 2H, CH), 3.98 (s, 3H, CH), BOH proton signals were not ob-
served; 13C NMR (100 MHz, CDCl3): δ = 167.1, 135.7, 134.0, 133.6,
129.1, 52.5. All other spectroscopic and analytical properties were
identical to those reported in the literature.[39,47]
5′-(4-Carboxyphenyl)-[1,1′:3′,1′′-terphenyl]-4,4′′-dicarboxylic
acid (26): Tri-ester 27 (370 mg, 0.77 mmol) was dissolved in a mix-
(AATT)3 Lys PNA-isoxazole conjugate (17p): To a solution of
O-silylated hydroxamic acid of phenyl 11a (19 mg, 0.040 mmol) in
anhydrous CH2Cl2 (50 μL) was added NEt3 (12.2 mg, 16.6 μL,
ture of THF/MeOH, 1:1 (14 mL), and 7 mL of 4.8
M aq. solution of
NaOH (1.344 g, 33.6 mmol) added at 0 °C. The reaction mixture was
0.12 mmol) under an inert atmosphere of argon. This was followed stirred overnight (approx. 16 h) at r.t., after which the solvents were
by the dropwise addition of a 1 M solution of Tf2O in CH2Cl2 (44 μL,
0.044 mmol) at –40 °C. Once the addition was completed, the reac-
tion mixture was allowed to stir for 3 min at –40 °C, after which it
was added to a suspension of resin-bound Cbz-protected ethynyl-
functionalized PNA 16j (10 mg, 0.0041 mmol) in DMF (200 μL). The
resulting mixture was shaken overnight (approx. 16 h) at 50 °C. The
solution was then filtered and washed with DMF (5 × 5 mL) and
CH2Cl2 (7 × 5 mL). The resulting crude was isolated, cleaved from
the resin and the Cbz groups deprotected. Resulting deprotected
PNA 17p was analyzed and purified. PNA 17p was isolated as a
white solid, soluble in a mixture of H2O/CH3CN, 1:1 + 0.1 % TFA.
evaporated in vacuo. The residue was solubilised in H2O (minimum
amount, i.e. 7 mL) and the resulting solution acidified to pH ca. 2
with 1 M aq. solution of HCl. The resulting white precipitate was
filtered, collected, resuspended in H2O (5 mL), sonicated and filtered
throughce more. Through this procedure, NaCl salts were washed
out. The white solid was collected and dried under high vacuum
(233 mg, 70 % yield in 26). m.p. 316–319 °C; 1H NMR (400 MHz,
[D6]DMSO): δ = 8.09 (s, 3H, CH), 8.06 (s, 12H, CH), COOH proton
signals were not observed; 13C NMR (100 MHz, [D6]DMSO): δ =
167.1, 143.8, 140.8, 130.0, 129.9, 127.4, 125.6. All other spectroscopic
and analytical properties were identical to those reported in the
HPLC retention time: 18.1 min. MS (MALDI-HRMS): Found 3606.6648 literature.[41,48]
[M + H]+, C151H189N70O39 requires = 3606.4952, found 3628.6714 [M
Dimethyl 5′-(4-(methoxycarbonyl)phenyl)-[1,1′:3′,1′′-terphenyl]-
+ Na]+, C151H188N70NaO39 requires = 3628.4772, found 3644.6257
[M + K]+, C151H188N70KO39 requires = 3644.4511. 17p was formed in
43 % (%area of desired cycloadduct in the RP-HPLC chromatogram).
Additional synthetic and analytical details are described in the sup-
porting information.
4,4′′-dicarboxylate (27): Na2CO3 (1.01 g, 9.53 mmol), 4-methoxy-
carbonylphenylboronic acid 23 (1.28 g, 7.15 mmol), 1,3,5-tribromo-
benzene 25 (500 mg, 1.59 mmol) and [Pd(PPh3)4] (184 mg,
0.159 mmol) were weighed into a 10–20 mL microwave flask, under
an inert atmosphere of argon. A degassed mixture of toluene/EtOH,
1:1 (17 mL) was then added to the solids (Note: toluene and EtOH
are degassed in two different two-neck flasks; simple argon bub-
5-Hexyl-3-phenyl-4,5-dihydroisoxazole (19): To a solution of O-
silylated hydroxamic acid of phenyl 11a (75 mg, 0.2 mmol) in anhy-
drous CH2Cl2 (3 mL) was added NEt3 (60.7 mg, 83.6 μL, 0.6 mmol) bling (with two argon balloons and two thick needle outlets), for
under an inert atmosphere of argon. This was followed by the drop-
wise addition of a 1 M solution of Tf2O in CH2Cl2 (220 μL,
0.22 mmol) at –40 °C. Once the addition was completed, the reac-
tion mixture was allowed to stir for 1 h at 0 °C, after which 1-octene
45 min is enough to degas the solvents for this reaction; drops of
water are added in EtOH). The resulting mixture was stirred for
5 min before being heated at 110 °C for 4 h under microwave condi-
tions (the pressure rose up to 8 bars). The solution was then diluted
18 (179.6 mg, 252 μL, 1.6 mmol) was added, and the resulting mix- with CH2Cl2 (250 mL) and extracted with H2O (3 × 100 mL) and
ture stirred overnight (approx. 16 h) at r.t. Next, the solution was
washed with H2O (2 × 1 mL). The organic layer was dried with anhy-
drous Na2SO4, filtered and concentrated to dryness in vacuo. The
residue was purified by silica gel column chromatography (eluent:
CH2Cl2), yielding 19 (36.6 mg, 79 %) as a white solid. Rf = 0.56
(CH2Cl2). m.p. 49–52 °C; 1H NMR (400 MHz, CD2Cl2, *denotes rota-
mer peaks): δ = 7.65–7.62 (m, 2H, CH), 7.39–7.37 (m, 3H, CH), 4.67–
4.65 (m, 1H, CH), 3.40–3.33 & 2.97–2.90* (m, 2H, CH), 1.78–1.70 &
1.62–1.55* (m, 2H, CH), 1.47–1.24 (m, 8H, CH), 0.89–0.85 (t, J =
7.0 Hz, 3H, CH), the proton assignment has been done by 2D analy-
sis; 13C NMR (100 MHz, CD2Cl2): δ = 156.7, 130.5, 130.1, 129.0, 126.8,
81.9, 40.2, 35.7, 32.1, 29.5, 25.9, 22.9, 14.2. All other spectroscopic
and analytical properties were identical to those reported in the
literature.[20c]
brine (1 × 100 mL). The organic phase was dried with anhydrous
MgSO4, filtered and concentrated to dryness in vacuo. The residue
was purified by silica gel column chromatography (eluent: Cy to Cy/
EtOAc, 4:6) yielding 48 % of desired product 27 (370 mg) as a white
1
solid. Rf = 0.45 (Cy/EtOAc, 4:6). H NMR (400 MHz, CDCl3): δ = 8.16
(d, J = 8.2 Hz, 6H, CH), 7.86 (s, 3H, CH), 7.77 (d, J = 8.5 Hz, 6H, CH),
3.96 (s, 9H, CH).[40,49] The next step, i.e. saponification, was per-
formed on the whole batch, without further characterizations of
triester derivative 27.
Acknowledgments
D. B. gratefully acknowledges the EU through the ERC (project
COLORLANDS) and EC Marie Curie RTN PRAIRIES (MRTN-CT-
2006-035810) funding schemes and Cardiff University for gener-
ous financial support. L.-E. C. the FRS-FNRS for her FRIA PhD
fellowship.
(4-(Methoxycarbonyl)phenyl)boronic acid (23): To a solution of
4-carboxyphenyl boronic acid 24 (1 g, 6.03 mmol) in anhydrous
MeOH (17.4 g, 22 mL, 543 mmol) was slowly added SOCl2 (4.5 g,
2.7 mL, 37.39 mmol) under an inert atmosphere of argon. The re-
sulting mixture was stirred for 2 h 30 at 50 °C, after which it was
concentrated to dryness in vacuo. The compound was also dried
under high vacuum to remove any residual trace SOCl2. The residue
was dissolved in EtOAc (50 mL) and washed with brine (20 mL).
The aqueous phase was extracted with EtOAc (2 × 30 mL) and the
combined organic phases dried with anhydrous MgSO4, filtered and
concentrated to dryness in vacuo to yield 23 as a white solid (1 g,
92 %). No further purification was required. m.p. 232–233 °C; 1H
Keywords: Cycloaddition reactions · Isoxazole · Click
reactions · Nitrile Oxides · Biorthogonal reactions
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Click Reactions
L.-E. Carloni, S. Mohnani,
D. Bonifazi* ....................................... 1–14
Synthesis of 3,5-Disubstituted Isox-
azoles through a 1,3-Dipolar Cyclo-
addition Reaction between Alkynes
and Nitrile Oxides Generated from
O-Silylated Hydroxamic Acids
The regioselective synthesis of 3,5-di- large variety of substrates due to the
substituted isoxazoles through a 1,3- mild, metal-free and oxidant-free reac-
dipolar cycloaddition reaction be- tion conditions. The method can be
tween alkynyl dipolarophiles and in used to introduce fluorescent as well
situ formed nitrile oxide dipoles. This as peptidic and PNA substituents.
approach provides a tolerance to a
DOI: 10.1002/ejoc.201901045
Eur. J. Org. Chem. 0000, 0–0
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14
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim