Synthesis of functionalized 2-isoxazolines as three-dimensional fragments for fragment-based drug discovery

Article abstract of DOI:10.1016/j.tetlet.2015.05.035

Abstract The design of new sp³ and spiro-enriched fragments has been achieved from 1,3-dipolar cycloaddition between alkenes and chloro-oximes. The selection of reagents was performed to afford a panel of 2-isoxazoline-containing fragments that show desirable three dimensional (3D) characteristics to allow the probing of biologically-relevant chemical space. Principal moments of inertia (PMI) were calculated to evaluate the 3D diversity. The resulting 3D fragments with suitable physicochemical properties, especially a good solubility, will be used to improve the hit rate of our fragment-based screening.

Full text of DOI:10.1016/j.tetlet.2015.05.035

Accepted Manuscript
Synthesis of functionalized 2-isoxazolines as three-dimensional fragments for
fragment-based drug discovery
Ngoc Chau Tran, Heleen Dhondt, Marion Flipo, Benoit Deprez, Nicolas Willand
PII:
S0040-4039(15)00844-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.05.035
TETL 46307
Reference:
Tetrahedron Letters
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Received Date:
Revised Date:
Accepted Date:
13 March 2015
4 May 2015
11 May 2015
Please cite this article as: Tran, N.C., Dhondt, H., Flipo, M., Deprez, B., Willand, N., Synthesis of functionalized
2-isoxazolines as three-dimensional fragments for fragment-based drug discovery, Tetrahedron Letters (2015), doi:
http://dx.doi.org/10.1016/j.tetlet.2015.05.035
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Ngoc Chau Tran, Heleen Dhondt, Marion Flipo, Benoit Deprez and Nicolas Willand

Tetrahedron Letters
1
Tetrahedron Letters
journal homepage: www.elsevier.com
Synthesis of functionalized 2-isoxazolines as three-dimensional fragments for
fragment-based drug discovery
Ngoc Chau Trana,b, Heleen Dhondta, Marion Flipoa, Benoit Depreza and Nicolas Willanda,∗
a Drugs and Molecules for Living Systems, INSERM UMR1177, Pasteur Institute Lille - University Lille 2, 59000 Lille, France
b Department of Medicinal Chemistry, School of Pharmacy, University of Medicine and Pharmacy, 41 Dinh Tien Hoang, Dist.1, Ho Chi Minh City, Viet Nam
ARTICLE INFO
ABSTRACT
Article history:
Received
The design of new sp3 and spiro-enriched fragments has been achieved from 1,3-dipolar
cycloaddition between alkenes and chloro-oximes. The selection of reagents was performed to
afford a panel of 2-isoxazoline-containing fragments that show desirable three dimensional (3D)
characteristics to allow the probing of biologically-relevant chemical space. Principal moments of
inertia (PMI) were calculated to evaluate the 3D diversity. The resulting 3D fragments with
suitable physicochemical properties, especially a good solubility, will be used to improve the hit
rate of our fragment-based screening.
Received in revised form
Accepted
Available online
Keywords:
1,3-Dipolar cycloaddition
2-isoxazoline
© 2015 Elsevier Ltd. All rights reserved.
Fragment library
3D-fragments
sp3-rich fragments
The approval of anti-melanoma drug vemurafenib in 2011 and
the development of several drug candidates that originated from
fragment-based lead discovery are testaments of the efficacy of
the use of fragments in the drug-discovery process. Fragment-
based approach relies on the screening of small chemical entities
that can easily probe specific biological targets. Identified hits are
then rapidly optimized using rational design to drug-like
compounds showing a better pharmacological profile.1
medicinal chemistry (Figure 1).4 The most notable compounds
are acivicin and (+)-calafianin. Acivicin5 is a fermentation
product of Streptomyces sviceus which has shown promising
anti-neoplastic activity and is under clinical trials. (+)-calafianin6,
a bromotyrosine-derived spiro-isoxazoline has been isolated from
marine sponge Aplysina gerardogreeni. Recently, Kaur et al.
have pointed out many 2-isoxazoline-containing natural products
which exhibit potential anticancer activity.7 Beside its natural
occurence, 2-isoxazoline is also an important skeleton found in
many synthetic bioactive compounds. Many isoxazoline
derivatives have been reported in the literature to exhibit a broad
range of biological activities, e.g. antimicrobial8, anticancer9,
antiplatelet10, anti-inflammatory11 activities, and regular effects
on central nervous system12 or sympathetic nervous system.13
The design and availability of fragment libraries is therefore
considered as a pivotal element in the success of fragment-based
screenings. However, current fragment libraries have been
limited to planar sp2-rich scaffolds which may not always allow
generating hits for several challenging targets.2 Less planar
fragments are also better starting point to explore the spatial
features of target proteins during hit-to-lead process. Therefore,
strategies to increase the proportion of sp3-rich fragments are
now considered key to improve hit-rate in fragment-based
screening.3
Prompted by these observations, we focused our work on the
design of new three-dimensional fragments based on 2-
isoxazoline motif and we report herein their synthesis. The key
element of our approach was the optimization of the one-pot 1,3-
dipolar cycloaddition starting from aldoximes and chloro-oximes
to yield a diverse set of 3D-fragments.
Many papers have accounted in the last decade for the
synthesis and occurrence of 2-isoxazoline in Nature and
———
∗ Corresponding author. Tel.: +33-320-964-991; fax: +33-320-964-709 ; E-mail address: nicolas.willand@univ-lille2.fr (N. Willand)

2
Tetrahedron Letters
Several synthetic methods used for the formation of 2-
isoxazoline have been described in the literature.4,14 Among
them, 1,3-dipolar cycloaddition has proved to be an excellent and
general synthetic route.15 The retrosynthetic analysis of 2-
isoxazoline is depicted in Scheme 1. Our objective was to
generate 2-isoxazoline via 1,3-dipolar cycloaddition between
nitrile oxide (dipole) and 1,1-disubstitued alkene (dipolarophile).
Many studies on the regioselectivity of isoxazoline synthesis by
intermolecular 1,3-dipolar cycloaddition emphasized that 1,1-
disubstituted alkenes react with nitrile oxides to predominantly
afford 5-subtituted isoxazolines.16 Nitrile oxides are reactive
intermediates that are usually generated in situ from stable
precursors. They can be generated from primary nitroalkane via
dehydration or by a direct oxidation of aldoxime or halogenation
of aldoxime to N-hydroxyiminoyl halides followed by dehydro-
halogenation.4 The latter is the most usual method in nitrile oxide
generation.17
Table 1. 3D-fragments synthesized from benzaldoxime
Name
Structure
Yielda
(%)
MW
(Da)
Fsp3
0.46
Sol.
(mM)d
4a
44b
216.3c
>2
4b
4c
93
249.3
0.38
0.40
1.0
35
88
60
291.3
277.3
249.2
1.0
1.5
>2
Benzaldoxime 2a and N-Boc-4-methylenepiperidine 3 served
as model substrates in the initial experiments (Scheme 2). 2-
Isoxazoline was firstly prepared using an aqueous solution
sodium hypochlorite (1.6N).19 Unfortunately, the reaction mixture
contained by-products and the targeted isoxazoline 4a-Boc was
obtained in poor yield (32%). To improve yield, we considered
other described conditions.20 Benzaldoxime was firstly
chlorinated by N-chlorosuccinimide (NCS) in N,N-
dimethylformamide (DMF). The N-hydroxyiminoyl chloride 2a’
was isolated with quantitative yield. This intermediate was then
converted to nitrile oxide followed by a 1,3-dipolar cycloaddition
with N-Boc-4-methylenepiperidine 3 in tetrahydrofurane (THF)
to yield 47% of the desired isoxazoline 4a-Boc.
4d
4e
0.36
0.25
4f
4g
94
56
16
44
217.2
205.2
212.3
212.3
0.33
0.27
0.38
0.38
-
However, the main inconvenience of this synthetic pathway is
that this process comprises two steps where the intermediate and
the final product must be separately purified. This drawback
becomes more challenging when a large number of isoxazoline
fragments are synthesized. Furthermore, several studies in the
literature have reported that some hydroxyiminoyl chloride
(chloro-oxime) are unstable and must be used shortly after
preparation.21
1.8
0.4
0.3
4h
4h’
Finally, an improvement of this synthetic method was
achieved. The reaction was conducted using a one-pot procedure
with a mixture of dichloromethane (DCM) and DMF (10:1)
thanks to the adaptation of a published protocol.22 Benzaldoxime
was converted after 1h into hydroxy-iminoyl chloride in situ
using NCS. Addition of Boc-4-methylidenepiperidine and
triethylamine allowed the formation of the targeted spiro-
isoxazoline 4a-Boc in an overall yield of 60%. Interestingly, the
tert-butoxycarbonyl (Boc) protecting group was not cleaved
under these mild conditions. 4a-Boc was then deprotected using
acidic conditions to yield 4a.
a Isolated yield after purification
bYield obtained after 3 steps including Boc deprotection.
cMolecular weight calculated as free base
dMeasured in PBS at pH 7.4
As expected, 1,3-dipolar cycloaddition is controlled by the
reactivity of alkenes. When alkenes are substituted with electron
withdrawing groups (4b, 4d, 4e, 4f and 4g), the reaction was
completed after 16 hours and isoxazolines were obtained in
moderate to good yield (56 to 94%). Interestingly, the presence
of carboxylic function (compounds 4e or 4g) did not affect the
reactivity. On the contrary, a low reactivity was observed with
alkene 4c. In the case of 3-methylenecyclobutane carbonitrile
used as starting alkene, reaction yielded two diastereomers 4h
and 4h’. After separation by flash-chromatography, the allocation
of their structures was achieved using 1D-NOESY.
With the optimized reagents and synthetic conditions in hands
(method 3, Scheme 2), the scope of the reaction was explored
with benzaldoxime and a large set of gem-disubstituted alkenes
in order to have at least one tetra substituted carbon atom, or one
spiranic carbon in the structure (Table 1). Itaconic acid dimethyl
ester was successfully synthesized according to a published
protocol.23 Non-commercially available alkenes were obtained
from the corresponding ketones using Wittig conditions.18
Original 2-isoxazoline containing fragments 4a-4h’ were
obtained in good to high yields.
Fsp3 value is defined as the ratio of sp3-hybridized carbon
atoms on the total carbon count. This value was calculated for
fragments 4a-4h’.24 The result showed that Fsp3 range from 0.25
to 0.46 (Table 1). In an effort to generate more sp3-rich
fragments, benzaldoxime was then replaced by commercially
available
acetaldoxime
or
synthesized
cyclopropylcarbaldoxime.25 This led to the synthesis of nine
spiroisoxazoline-containing fragments (Table 2) with higher
Fsp3, ranging from 0.42 to 0.92. Fragments 5e and 5g were
obtained in good yields thanks to the high reactivity of the

Tetrahedron Letters
starting alkene, where electron density is lowered by the lactone.
3
Table 3. Synthesis of isoxazolines 7a-7c starting from lactone 4f
The seven remaining fragments were synthesized with low to
moderate yields. Boc derivatives (5a-Boc to 5d-Boc) were then
deprotected using acidic conditions to afford the corresponding
hydrochloride salts.26
N
O
N
O
O
H
MW
R
H2N
+
O
80oC, 15min
NH
R
O
3 eq.
O
4f
7a-c
Table 2. 3D-fragments synthesized from acetaldoxime or
cyclopropylcarbaldoxime
Name
Structure
Yielda
(%)
MW
Fsp3
Sol.
(mM)
(Da)
272.3
276.3
278.3
1) NCS, rt, 1h
2) alkene
3) TEA, rt, 16h
b
OH
H
O
R'
N
R'
N
N
N
N
O
+
OH
OH
OH
R
R''
R''
R
DCM-DMF (10:1)
7a
7b
7c
82
0.33
0.47
0.43
>2
>2
1.9
N
O
O
H
2b: R = Me
2c: R = cyclopropyl
5a - 5d (Boc)
5e - 5i
Yielda
(%)
MW
(Da)
Fsp3
Sol.
(mM)d
78
Name
Structure
N
H
O
O
O
O
65
N
N
NH.HCl
NH.HCl
NH.HCl
5a
5b
5c
5d
30 b
31b
50b
47b
154.2 c
168.2 c
140.2 c
180.3 c
0.88
0.89
0.86
0.90
>2
>2
>2
>2
N
H
OH
O
aIsolated yield after purification
bMeasured in PBS at pH 7.4
O
N
Finally, we analyzed the overall shape coverage of our entire
set of fragments by monitoring normalized PMI ratios
(npr1/npr2), according to Sauer’s method.28 The PMI plot is
shown in Figure 2. The data obtained for our 3D-fragments were
compared to a set of 471 members of our in house library of
commercially available fragments, whose molecular weight was
equivalent to the synthesized compounds.
O
N
NH.HCl
O
N
O
5e
5f
72
10
155.1
193.3
0.71
0.92
-
O
The graph shows that our set of substituted isoxazoline
fragments (in pink) cover a large space of shapes (Figure 2) with
a greater proportion of rod-like or sphere-like shapes than
commercially available fragments (in cyan). The calculation of
O
N
1.9
O
N
O
5g
5h
87
57
181.2
225.2
0.78
0.42
-
the mean of NPR1+NPR2 with both sets: 1.19
0.08 for 2-
O
isoxazoline containing fragments and 1.09 0.07 for the 471
commercially available fragments confirms this tendency.
Interestingly, two fragments 5h and 5i which were designed from
aromatic alkenes (with medium Fsp3) showed also sphere-like
and rod-like properties. These results are conformed to a recent
study in which the authors showed that a positive-correlation
between Fsp3 and 3-dimentionality of molecule are not always
observed.29
O
0.8
N
F
F
N
O
5i
32
215.3
0.50
1.6
aIsolated yield after purification
bYield obtained after 3 steps including Boc deprotection.
cMolecular weight calculated as free base
dMeasured in PBS at pH 7.4
Finally, a critical property of fragments for screening is their
solubility in aqueous media. To validate the impact of the three-
dimensionality on solubility, we measured this parameter in an
aqueous phosphate buffered saline solution (PBS) at pH =7.4 for
a set of 18 fragments. Interestingly, 15 fragments (83%) show
solubility higher than 1 mM, including aliphatic or aromatic
fragments 5f and 5i.In summary, we optimized a reliable protocol
based on 1,3-dipolar cycloaddition for the synthesis of a diverse
set of fragments containing 2-isoxazoline motif. Among the 21
compounds described, 16 (4b, 4c, 4h, 4h’, 5a-i, 7a-c) are
original. Lactone containing fragment 4f was shown to be further
functionalized with amines under microwave irradiation. Using
chemoinformatic analysis, we quantified the shapes of the novel
synthesized fragments. Finally, the majority of these fragments
were proved to be soluble in PBS at a concentration higher than 1
mM. The resulting compounds will be screened for their affinity
to therapeutically relevant proteins using Thermal Shift Assay
and the results will be reported in due course.
In some of the 1,3-dipolar cycloadditions, we observed the
formation of a by-product. This side-product was supposed to
arise from the dimerization of the corresponding nitrile oxide in
the absence of a reactive dipolarophile as trapping agent to yield
1,2,5-oxadiazole-2-oxide derivative.27 To confirm this
hypothesis, we conducted reactions with benzaldoxime and
acetaldoxime in the absence of alkene. We only observed the
formation of furoxan 6a which is consistent with the literature
(Scheme 3).17
In an effort to exemplify how diverse structures can be rapidly
obtained from 4f, we performed the opening of the lactone with
amines to yield isoxazolines 7a-7c (Table 3). This reaction was
carried out using 3 equivalents of the corresponding amine. The
mixtures were heated at 80°C under microwave irradiations to
afford the targeted compounds in good yields. This method could
also be applied to intermediates 5e and 5g to extend the scope of
the reaction and increase diversity.
Acknowledgments
We are grateful to the institutions that support our laboratory:
Inserm, Université Lille Nord de France, Institut Pasteur de Lille,
EU, Région Nord-Pas de Calais, and PRIM, Pôle de Recherche

4
Tetrahedron Letters
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Interdisciplinaire du Médicament. N.C. Tran acknowledges
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a
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Tetrahedron Letters
5
Captions
Figure 1. Structure of natural products containing
2-isoxazoline motif.
Figure 2. Molecular shape analysis of set
isoxazoline containing 3D-fragments
Scheme 1. Retrosynthetic analysis of 2-isoxazoline
Scheme 2. Synthetic conditions used for the
optimization of the synthesis of compound 4a-Boc
Scheme 3. Formation of the by-product furoxan 6a.

6
Tetrahedron Letters
1
N
O
H
O
O
2
5
N
3
Cl
OH
4
NH2
acivicin
2-isoxazoline
O
O
O
N
O
O
Br
N
H
H
N
Br
O
N
O
O
(+)-calafianin

Tetrahedron Letters
7

8
Tetrahedron Letters

Tetrahedron Letters
9

10
Tetrahedron Letters
O
N
OH
H
OH
Cl
O
O
N
N
N
TEA
NCS
N
R
R
R
R
R
in situ
O
O
O
N
O
N
N
N
H3C
CH3
furoxan 6a
6b (not formed)