A base promoted multigram synthesis of aminoisoxazoles: Valuable building blocks for drug discovery and peptidomimetics

Article abstract of DOI:10.1039/c6ra02365g

A practical multigram metal free synthesis of isoxazole-containing building blocks from commercially available amino acids was elaborated. The key reaction was a regioselective [3 + 2]-cycloaddition of in situ generated nitrile oxides with alkynes/enamines. The obtained building blocks were used in the preparation of bioactive compounds and peptidomimetics.

Full text of DOI:10.1039/c6ra02365g

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Kandaurova, K. Hrebeniuk, O. Manoilenko, I. Kulik, R. Iminov, V. Kubyshkin, A. Tverdokhlebov, P. K.
Mykhailiuk and O. Ablialimov, RSC Adv., 2016, DOI: 10.1039/C6RA02365G.
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A base promoted multigram synthesis of aminoisoxazoles:
valuable building blocks for drug discovery and peptidomimetics.
Bohdan A. Chalyk,a Inna Y. Kandaurova,b Kateryna V. Hrebeniuk,b Olga V. Manoilenko,a
Irene B. Kulik,d Rustam T. Iminov,a Vladimir Kubyshkin,e Anton V. Tverdokhlebov,a* Osman
K. Ablialimov,a* and Pavel K. Mykhailiuka,c,f
*
aEnamine Ltd., Chervonotkatska 78, 01103 Kyiv, Ukraine, www.enamine.net
A.tverdohlebov@mail.enamine.net (AT),
O.ablialimov@mail.enamine.net (OA),
Pavel.Mykhailiuk@gmail.com, Pavel.Mykhailiuk@mail.enamine.net (PM)
bDepartment of Chemistry, Kyiv Polytechnic Institute, Prosp. Peremohy 37, Kyiv 03056, Ukraine.
cDepartment of Chemistry, Taras Shevchenko National University of Kyiv, Volodymyrska Street 64, Kyiv
01601, Ukraine
dInstitute of Bioorganic Chemistry & Petrochemistry, NAS of Ukraine, Murmanska 1, Kyiv 02660, Ukraine
eInstitute of Chemistry, Technical University of Berlin, MüllerꢀBreslauꢀStr. 10, Berlin 10623, Germany
eUkrOrgSyntez Ltd. (UORSY), Chervonotkatska 78, 01103 Kyiv, Ukraine, www.uorsy.com
[3+2]
cycloaddition
R
Bioactive agents
Peptidomimetics
CO2H
N
N
5ꢀ6 steps
O
H
N
Boc
Diverse amino acids
ca 100 building blocks
Abstract
A practical multigram metal free synthesis of isoxazoleꢀcontaining building blocks
from commercially available amino acids was elaborated. The key reaction was a
regioselective [3+2]ꢀcycloaddition of in situꢀgenerated nitrile oxides with alkynes/enamines.
The obtained building blocks were used in the preparation of bioactive compounds and
peptidomimetics.
Keywords
Isoxazole, [3 + 2]ꢀcycloaddition, nitrile oxides, building blocks, drug discovery.
1

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Introduction
Isoxazole core is a structural element in many commercial pharmaceuticals (Figure
1). The range of biological activities of the isoxazoleꢀcontaining drugs is broad: analgesic,1
antiꢀinflammatory,1a,c,2 anthelmintic,3 antiꢀdepressant,4 antiꢀbacterial,5 anticancer,6
insecticidal,7 nootropic,8 anxiolytic,8a neuroprotective,9 and HIVꢀinhibitory agents.10 In this
context, the development of practical methods towards isoxazoleꢀbased building blocks
from common cheap starting materials is truly desirable.
CF3
SO2NH2
O
S
O
O
O
NH
CF3
N
N
N
O
O
O
Vald
ec
oxib
Leflunomide
Isoxaflutole
antiꢀreumatic
antiꢀ
inflam
m
atory
herbicide
O
S
HO
NH
H2N
O
NH2
N
N
O
O
Sulfamethoxazole
Muscimol
antibiotic
sedative
Figure 1. Commertialized biologically active derivatives of isoxazole.
Nitrile oxides are useful intermediates towards the synthesis of a wide plethora of
isoxazoleꢀbased architectures.11aꢀh However; most of these methods either utilize toxic
metals or hypervalent iodine compounds11h which in turn, are not environmentally friendly.
At the same time, there are just a few known reports dealing with metalꢀfree conditions and
most common are based on the use of triethylamine12aꢀb and sodium
perchlorate/triethylamine.12c Therefore, the development of metalꢀfree protocols towards
the synthesis of compounds bearing isoxazole core are of particular interest.12d
The use of amino acids13aꢀh in construction of isoxazoleꢀcontaining compounds has
been known before. However, these reports, dealt with the specific amino acids, and the
studies have not been performed systematically on large variety of substrates. Taking into
account the need of exploration of metalꢀfree protocols as well as search for easily
available substrates, herein we would like to elaborate on a general practical multigram
2

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metalꢀfree synthesis of isoxazoleꢀcore building blocks starting from commonly available
and diverse αꢀ, βꢀ and γꢀamino acids.
Results and discussion
Synthesis
1. Synthesis of chloroximes from amino acids. The initial synthesis step was
reduction of the amino acid carboxylꢀgroups in a number of NꢀBoc protected amino acids.
Resulting NꢀBoc amino alcohols were subsequently oxidized into the corresponding
aldehydes (Table 1). These aldehydes formed oximes upon treatment with hydroxylamine
under mildly basic conditions (sodium hydrogencarbonate). The oximes were then treated
with Nꢀchlorosuccinimide under acidic conditions to afford the target NꢀBoc protected
chloroximes 1-7 in good yields. The products were white powders that could be stored at
room temperature for at least several months as exemplified in Table 1.
Table 1. Chloroximes synthesized starting from NꢀBoc amino acids.
Bocꢀamino acid
Chloroxime
BocHN
Yielda
49%
Storageb
>2 years
Cl
BocꢀGly
1
2
BocHN
BocHN
BocHN
CO2H
CO2H
CO2H
N
OH
Cl
Bocꢀ(S)ꢀAla
Bocꢀ(R)ꢀAla
Bocꢀ(S)ꢀPro
68%
67%
70%
≤0.5 yearsc
≤0.5 yearsc
>2 years
BocHN
BocHN
N
N
OH
Cl
3
4
5
OH
Cl
N
N
CO2H
Boc
N
N
Boc
OH
Cl
N
Bocꢀ(R)ꢀPro
71%
>2 years
N
CO2H
Boc
Boc
OH
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Boc
N
Boc
Boc
N
Cl
BocꢀAze
BocꢀPip
6
65%
73%
>2 years
CO2H
N
OH
Boc
N
N
Cl
7
>2 years
CO2H
N
OH
a Overall yield over four steps. b The products were stored in closed flasks at the room temperature. c The products were stored at 0 ºC
under argon. After six months ca. 10% decomposition was found according to 1H NMR.
2. Synthesis of NꢀBoc aminoisoxazoles via [3+2]ꢀcycloaddition.
It is worth mentioning that our methodology for the synthesis of variety isoxazoles is
known and here we would just mention some selected examples where the similar
conditions like base or reaction temperatures were applied. Johnson et al reported on the
regioselective synthesis of 3,4ꢀdisubstituted isoxazoles between enolsilanes and nitrile
oxides.14 In the same vein, the Zhu group and later Wang and coꢀworkers reported on the
synthesis of 3,4ꢀdisubstituted isoxazoles in high yields.15a,b The key point of the reported
protocols was the reaction of in situ generated enamines from corresponding ketones or
ᵦꢀketo esters and nitrile oxides. Caddick and coꢀworkers reported and interesting strategy
towards the synthesis of 3,5ꢀdisubstituted isoxazoles bearing sulfonamide group at the
5ꢀposition.16 Later the Shibata group developed the strategy for the synthesis of isoxazole
triflones.17 Hamme et al reported on the synthesis of 3,5ꢀdisubstituted isoxazoles with
electron withdrawing group at the 5ꢀposition by the reaction of 1,1ꢀdisubstituted
bromoalkenes with nitrile oxides.18a,b Last but not least Schmidt and coꢀworkers reported
on the regioselective synthesis of 4ꢀtrifluorosubstituted isoxazoles in good yields.19 All the
aforementioned methods avoided the usage of metalꢀmediated reactions which prompted
us to follow the similar conditions in order to synthesize a large panel of aminoisoxazoles
in large scale.
In our case, synthesis of isoxazoles from the corresponding in situꢀgenerated nitrile oxides
and alkynes/enamines was analogous to the mentioned above methods. Namely,
treatment of chloroximes 1ꢀ7 with a mild base (sodium hydrogen carbonate or
triethylamine) at either the room temperature or at 0 ºC generated corresponding nitrile
oxides (Table 2). Subsequent addition of enamines 8ꢀ10 (1.2 eq) to these in situ generated
nitlile oxdes lead to [3+2]ꢀcycloaddition intermediates that readily eliminated
dimethylamine to give the 3,4ꢀdisubstituted isoxazoles in a regioselective fashion as
previously reported with push–pull enamines (Table 2, A).20 In contrast, terminal alkynes
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11, 12 afforded a mixture of 3,4ꢀ/3,5ꢀisomers ~30/70 when reaction was performed at the
room temperature.21 Nevertheless, the same reaction at 0 ºC exhibited an improved
regioselectivity of ~10/90, and the major 3,5ꢀdisubstituted isomers were easily isolated by
column chromatography.22
While the above described [3+2] cycloaddition reactions were performed in ethyl
acetate the synthesis also worked well in tetrahydrofuran, dichloromethane,
dimethoxymethane or chloroform, though with compromised regioselectivity. At the same
time synthesis of isoxazoles in water as environmentally benign solvent is of high
interest.23 It is also worth mentioning, that these reactions were performed under obligatory
high dilution – substrate/solvent = 1/10 (wt:v), – otherwise formation of side furoxans was
observed.24
As the result we obtained a variety of NꢀBoc aminoisoxasoles initially starting from
amino acids, and the synthetic approach was scalable such that up to 50 g of a product
(Bocꢀ6ꢀH) was obtained in a single synthesis run.
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Table 2. The synthesis of NꢀBoc aminoisoxazoles via [3+2]ꢀcycloaddition.
A
(CO Me)
2
X
8
9
10
X
N
(CN)
(COMe)
X
N
Boc
Me2N
via
Boc
N
EtOAc
rt
NMe2
N
O
O
intermediate
Cl
NaHCO3
[3+2]ꢀcycloaddition
N
N
N+
EtOAc
rt
Boc
Oꢀ
Boc N
B
OH
11
12
(CO Me)
2
X
N
Boc
N
N
in situꢀgenerated
(TMS)
Boc
Boc
N
N+
EtOAc
0 o
Oꢀ
N
O
X
C
O
side product
up to 50 g
in one synthesis run
A
Boc
N
Boc
N
CO2Me
CO2Me
BocHN
CO2Me
N
Boc
CO2Me
N
Boc
CO2Me
CO2Me
CO2Me
BocHN
BocHN
N
N
N
N
N
N
N
O
O
O
O
O
O
O
Bocꢀ1A-CO2Me
Bocꢀ2A-CO2Me
Bocꢀ3A-CO2Me
Bocꢀ4A-CO2Me
Bocꢀ5A-CO2Me
Bocꢀ6A-CO2Me
Bocꢀ7A-CO2Me
(95%)
(97%)
(98%)
(92%)
(90%)
(91%)
(91%)
Boc
Boc
N
N
CN
CN
CN
N
Boc
CN
N
Boc
CN
CN
CN
BocHN
BocHN
BocHN
N
N
O
N
O
N
N
O
N
O
N
O
O
O
Bocꢀ1A-CN
Bocꢀ2A-CN
Bocꢀ3A-CN
Bocꢀ4A-CN
Bocꢀ5A-CN
Bocꢀ6A-CN
Bocꢀ7A-CN
(89%)
(96%)
(97%)
(95%)
(93%)
(94%)
(92%)
Boc
Boc
XꢀRay
COMe
N
N
COMe
COMe
COMe
N
Boc
N
COMe
COMe
BocHN
BocHN
BocHN
COMe
Boc
N
N
N
O
Bocꢀ3A-COMe
N
N
O
Bocꢀ5A-COMe
N
O
Bocꢀ6A-COMe
N
O
Bocꢀ7A-COMe
O
O
O
Bocꢀ1A-COMe
Bocꢀ2A-COMe
Bocꢀ4A-COMe
(88%)
(89%)
(91%)
(87%)
(89%)
(95%)
(94%)
Boc
B
Boc
N
N
N
Boc
N
Boc
BocHN
BocHN
BocHN
N
N
N
N
N
N
N
CO2Me
CO2Me
CO2Me
O
CO2Me
CO2Me
O
CO2Me
O
CO2Me
O
O
O
O
Bocꢀ1B-CO2Me
Bocꢀ2B-CO2Me
Bocꢀ3B-CO2Me
Bocꢀ4B-CO2Me
Bocꢀ5B-CO2Me
Bocꢀ6B-CO2Me
Bocꢀ7B-CO2Me
(89%)
(87%)
(88%)
(91%)
(92%)
(95%)
(88%)
Boc
Boc
N
a
N
N
Boc
N
Boc
BocHN
BocHN
BocHN
N
N
N
N
N
N
N
TMS
TMS
TMS
O
TMS
TMS
O
TMS
O
TMS
O
O
O
O
Bocꢀ1B-TMS
Bocꢀ2B-TMS
Bocꢀ3B-TMS
Bocꢀ4B-TMS
Bocꢀ5B-TMS
Bocꢀ6B-TMS
Bocꢀ7B-TMS
(87%)
(75%)
(77%)
(85%)
(84%)
(76%)
(63%)
a Et3N was used instead of NaHCO3 to generate the nitrile oxide, 5 eq. of 12 were used.
3. Synthesis of isoxazoleꢀcontaining amines and amino acids.
We also made several forays toward the cleavage of TMSꢀgroup utilizing K2CO3 or
trifluoroacetic acid (TFA) as TMSꢀscavengers,25 but unfortunately in our case the described
procedure was not that effective, furnishing the desired products in moderate yields.
Moreover, attempts to cleave TMSꢀgroup with 4M HCl in dioxane were also not so effective
in terms of low yield. Though, these forays were not successful, it allowed us to improve
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our tactics. As a result, we found that cleavage of the TMSꢀgroup from the isoxazole core
was easily achieved by treatment with catalytic amounts of KHF2 in methanol – water
mixture. Resulting 3ꢀsubstituted isoxazoles were obtained in excellent yields (Table 3).
Table 3. Cleavage of TMSꢀgroup from the isoxazole ring.
Boc
Boc
N
N
N
Boc
N
Boc
BocHN
BocHN
BocHN
N
N
N
N
N
N
N
O
O
O
O
O
O
O
Bocꢀ1-H
(94%)
Bocꢀ2-H
(92%)
Bocꢀ3-H
(93%)
Bocꢀ4-H
(95%)
Bocꢀ5-H
(93%)
Bocꢀ6-H
(89%)
Bocꢀ7-H
(91%)
As next, cleavage of NꢀBoc group from all compounds was performed under acidic
conditions to afford the target building blocks – aminoisoxazoles (Table 4).
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Table 4. Cleavage of the NꢀBoc protection group from the amino moiety.
HN
HN
CO2Me
CO2Me
CO2Me
HN
CO2Me
HN
CO2Me
CO2Me
H2N
H2N
CO2Me
H2N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
1A-CO2Me • HCl
2A-CO2Me • HCl
3A-CO2Me • HCl
4A-CO2Me • HCl
5A-CO2Me • HCl
6A-CO2Me • HCl
7A-CO2Me • HCl
(98%)
(99%)
(97%)
(96%)
(94%)
(93%)
(95%)
HN
HN
CN
CN
CN
HN
CN
HN
CN
CN
CN
H2N
H2N
H2N
N
O
1A-CN • HCl
N
N
N
N
N
N
O
O
O
O
O
O
2A-CN • HCl
3A-CN • HCl
4A-CN • HCl
5A-CN • HCl
6A-CN • HCl
7A-CN • HCl
(99%)
(97%)
(98%)
(96%)
(95%)
(94%)
(92%)
HN
HN
COMe
COMe
COMe
HN
COMe
HN
COMe
COMe
H2N
H2N
H2N
COMe
N
O
N
N
N
N
N
N
O
O
O
O
O
O
1A-COMe • HCl
2A-COMe • HCl
3A-COMe • HCl
4A-COMe • HCl
5A-COMe • HCl
6A-COMe • HCl
7A-COMe • HCl
(95%)
(96%)
(94%)
(97%)
(98%)
(93%)
(90%)
HN
HN
HN
HN
H2N
H2N
H2N
N
N
N
N
N
N
N
CO2Me
O
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
O
O
O
O
O
O
1B-CO2Me • HCl
2B-CO2Me • HCl
3B-CO2Me • HCl
4B-CO2Me • HCl
5B-CO2Me • HCl
6B-CO2Me • HCl
7B-CO2Me • HCl
(98%)
(99%)
(97%)
(96%)
(95%)
(94%)
(93%)
HN
HN
HN
HN
H2N
H2N
H2N
N
O
1B-H
N
N
N
N
N
N
O
O
O
O
O
O
2B-H • HCl
(96%)
3B-H • HCl
4B-H • HCl
5B-H • HCl
(94%)
6B-H • HCl
(98%)
7B-H
(84%)
(86%)
(97%)
(95%)
Carboxymethylꢀsubstituted isoxazoles were saponificated with sodium hydroxide in
methanol to afford corresponding free amino acids in good yields (Table 5).
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Table 5. Synthesis of isoxazoleꢀcontaining amino acids.
NaOH
MeOH
N
Boc
N
Boc
CO2Me
CO2H
N
N
O
O
Boc
Boc
XꢀRay
N
N
CO2H
CO2H
CO2H
N
Boc
CO2H
N
Boc
CO2H
CO2H
CO2H
BocHN
BocHN
BocHN
N
N
N
N
N
N
N
O
O
O
O
O
O
O
Bocꢀ1A-CO2H
Bocꢀ2A-CO2H
Bocꢀ3A-CO2H
Bocꢀ4A-CO2H
Bocꢀ5A-CO2H
Bocꢀ6A-CO2H
Bocꢀ7A-CO2H
(95%)
(97%)
(98%)
(92%)
(90%)
(91%)
(91%)
Boc
Boc
N
N
N
Boc
N
Boc
BocHN
BocHN
BocHN
N
N
N
N
N
N
N
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
O
O
O
O
O
O
O
Bocꢀ1B-CO2H
Bocꢀ2B-CO2H
Bocꢀ3B-CO2H
Bocꢀ4B-CO2H
Bocꢀ5B-CO2H
Bocꢀ6B-CO2H
Bocꢀ7B-CO2H
(84%)
(90%)
(89%)
(87%)
(83%)
(85%)
(81%)
The structures of two final compounds were confirmed by Xꢀray analysis (Figure 2).
Interestingly, the structure of Bocꢀ4AꢀCOMe indicates a repulsion occurring between the
carbamate carbonyl oxygen and the isoxazole nitrogen, similar to what has been recently
reported for analogous structures with 1,3ꢀoxa/thiazine in place of the isoxasole.26
Bocꢀ4AꢀCOMe
Bocꢀ6AꢀCO2H
Figure 2. Xꢀray crystal structures of Bocꢀ4AꢀCOMe and Bocꢀ6AꢀCO2H
.
Practical applications.
After having established a practical general synthetic protocol towards isoxazoleꢀ
containing building blocks, we also aimed to demonstrate applicability of the synthesized
structures in other chemical areas.
1. Synthesis of a drug candidate. ABT-418 is a known nootropic agent.27a Recently,
chemists at Abbott Laboratories synthesized its bioisostere 16 in four steps and 7.3% total
yield starting from 4 and 2ꢀbromopropene.27b Herein, we have developed an alternative
approach to afford compound 16 (Scheme 1). First, [3+2]ꢀcycloaddition between
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allylbromide and nitrile oxide generated from chloroxime 4 gave isoxazoline 13 in 95 %
yield. Next, elimination of hydrogen bromide under basic conditions gave isoxazole 14.
Cleavage of the NꢀBoc group produced free amine 15, which was the subjected to
reductive amination with aqueous formaldehyde to afford the target product 16 in gram
quantities. The overall yield of the synthesis was 55% over 5 steps.
Scheme 1. Synthesis of isoxazole 16 – bioisostere of the known nootropic agent ABT- 418.
2. Synthesis of peptidomimetics. We also envisioned that chiral aminoisoxazoles
could be used to prepare conformationally restricted peptidomimetics.28 In this case the
heterocyclic fragment will act as a rigid peptide bond substitute29 (Scheme 2).
a)
O
O
O
O
H
N
N
N
N
N
H
H
H
H
O
N O
αꢀPeptide
conformationally flexible
αꢀPeptidomimetic
conformationally restricted
b)
O
O
O
O
enzymatic
H
N
N
N
N
N
N
H
H
H
H
X
O
HO
X: O,S
postꢀtranslationally modified
genetically encoded
Scheme 2. (a) αꢀpeptide, and its conformationallyꢀrestricted analogue with isoxazole backbone; (b)
construction of a postꢀtranslationally modified peptide with oxa/thiasole backbone.
In addition, isomeric oxazoles and thiazoles are common in the backbones30 of
various ribosomally synthesized and postꢀtranslationally modified peptides.31 Construction
of an azole ring is generally afforded in nature by cyclization of serine or threonine side
chain on the downstream amide bond.32,33,34 Total synthesis of respective peptidomimetics
has been developed in order to provide naturally occurring antimicrobial agents or toxins
as well as their chemically modified analogues.35,36 Though, oxa/thiazole to isoxazole
mutations have not been explored. Therefore, next we attempted the synthesis of
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isoxazoleꢀcontaining peptidomimetics by solid phase peptide synthesis (SPPS) in order to
highlight this opportunity.
Initially we synthesized the Fmocꢀprotected amino acid FmocꢀAlaIsoꢀOH (17)
(Scheme 3). Peptide chains were grown on Rinkꢀamide resin, and TBTU/HOBt/DIPEA
system was used for coupling. Only in the case of AcꢀLeuꢀAlaIsoꢀValꢀNH2 (18) we
observed minor degree of epimerization (≤ 15 %), whereas in AcꢀLeuꢀValꢀAlaIsoꢀNH2 (19)
no racemization was observed.37 In addition, no degradation was found after cleavage
from the resin upon treatment with TFA/TIS. Generally the amino acid FmocꢀAlaIsoꢀOH
turned out to be compatible with the standard SPPS, allowing synthesis and isolation of
both target peptides by conventional methods.38
O
O
H
N
NH2
N
H
N
H
N O
O
O
O
18
(AcꢀLeuꢀAlaIsoꢀValꢀNH2)
1) HCl
SPPS
CO2H
CO2H
BocHN
FmocHN
2) FmocCl
Na2CO3
87%
N O
Bocꢀ2BꢀCO2H
N O
O
O
H
N
17
N
H
N
NH2
(FmocꢀAlaIsoꢀOH)
H
N O
O
19
(AcꢀLeuꢀValꢀAlaIsoꢀNH2)
Scheme 3. Synthesis of isoxazoleꢀcontaining peptidomimetics 18 and 19.
Conclusions
We have developed a general metalꢀfree practical protocol towards isoxazoleꢀ
containing building blocks starting from commonly available αꢀ, βꢀ and γꢀamino acids. The
target products were obtained in up to 50 g scale. The high potential of the developed
method was demonstrated by synthesis of compound 16 – bioisostere of the known
nootropic agent ABT–418, ꢀ and preparation of isoxazoleꢀcontaining peptidomimetics 18,
19 by solid phase peptide synthesis.
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Experimental Section.
General: All reagents were available from Enamine Ltd. Solvents were purified according
to standard procedures. When organic solutions were concentrated under reduced
pressure, 35–40 °C bath temperature was used. Column chromatography was performed
1
with silica gel 60 (230–400 mesh) as the stationary phase. H, 13C NMR spectra were
recorded at the NMR spectrometers operating at 400 and 500 1H frequency (101 and 126
MHz for 13C experiments). NMR chemical shifts are reported in ppm, in the δ scale and are
1
referenced using residual NMR solvent peaks at 7.26 and 77.16 ppm for H and 13C in
1
1
CDCl3, 2.50 and 39.52 ppm for H and 13C in DMSOꢀd6, for 4.79 and H in D2O. For
peptides NMR analysis was performed in 5 mM peptide solutions in CD3OD at 700 MHz
1
(1H frequency) spectrometer. H NMR spectra were recorded 25 min after the peptides
were dissolved (fresh) and therefore contained few of the nonꢀexchanged amide
resonances. The 1H NMR spectra were in addition recorded after overnight measurements
when the NHꢀtoꢀND exchange was already completed. 1H HOHAHA (dipsi2 of 60 ms) and
1
ROESY (spinꢀlock 300 ms) spectra were recorded to complete the assignment of the H
1
resonances. 13C {1H} dept45 and H {13C} HSQC experiments were performed in order to
assign accompanied 13C resonances. The following abbreviations are used in reported
NMR data: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), brs (broad
singlet). Coupling constants (J) are in Hz. Spectra are reported as follows: chemical shift
(δ, ppm), multiplicity, integration, coupling constants (Hz). Measured melting points are
uncorrected. Boiling points were measured at 0.8 mm Hg unless otherwise specified
herein. LCꢀMS data were acquired on Agilent 1200 HPLC system equipped with
DAD/ELSD/LCMSꢀ6120 diodeꢀmatrix and massꢀselective detector, column: Poroshell 120
SBꢀC18, 4.6 mm × 30 mm. Eluent, A, acetonitrile − water with 0.1 % of FA (99 : 1); B,
water with 0.1% of FA. Optical rotations were measured on polarimeter in methanol using
1ꢀdm cell; optical rotation values are given in 10–1 deg cm2 g–1; concentrations (c) are given
in mmol/L, wavelength 589 nm at 20 °C. The enantiomeric excess and retention time (tR )
was determined for major signal by HPLCs: Daicel CHIRALPACK IA, 5 ꢁm, 4.6×250 mm,
Daicel CHIRALPACK IB, 5 ꢁm, 4.6×250 mm, Daicel CHIRALPACK OJꢀH, 5 ꢁm, 4.6×250
mm, Daicel CHIRALPACK ASꢀH, 5 ꢁm, 4.6×250 mm chiral columns, injection volume 0.1
ꢁL, eluent (hexanes : 2ꢀpropanol). Solid compounds were recrystallized from acetonitrile
unless other is specified.
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Synthesis of 3,4-disubstituted isoxazoles, representative procedure
methyl
3-(((tert-butoxycarbonyl)amino)methyl)isoxazole-4-carboxylate
(Boc-1A-
CO2Me)
A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding
chloroxime 1 (2 g, 9.6 mmol) and EtOAc (20 mL). To the resulting vigorously stirred
homogeneous solution (E)ꢀmethyl 3ꢀ(dimethylamino) acrylate 8 (1.48 g, 11.5 mmol) was
added followed by addition of NaHCO3 (1.6 g, 19.2 mmol) at the ambient temperature. The
resulting mixture was stirred overnight. The progress of the reaction was monitored by
NMR spectroscopy. After the reaction was complete, the organic phase was washed with
10% NaHSO4(aq) (3×5 mL), 10% K2CO3(aq) (2×5 mL) and brine (1×5 mL). Organic phase
was dried over anhydrous Na2SO4. The resulting mixture was filtered through a silicagel
pad and concentrated to yield Boc-1A-CO2Me (95%, 2.3 g) as a beige powder, mp 65 °C.
1H NMR (500 MHz, DMSOꢀd6): δ 9.59 (s, 1H, isoxaz), 7.24 (brs, 1H, NH), 4.41 (d, 2H,
CH2), 3.80 (s, 3H, OCH3), 1.39 (s, 9H, CH3ꢀBoc). 13C NMR (125 MHz, DMSOꢀd6): δ 164.9,
161.1, 160.0, 155.4, 111.9, 78.2, 51.9, 35.9, 28.1. MS (APCI) m/z [MꢀBoc+1H]+ calculated
for C10H13N2O5: 255.1; found: 255.4. Anal. calcd for C11H16N2O5: C, 51.56; H, 6.29; N,
10.93. Found: C, 51.35; H, 6.07; N, 10.52.
Synthesis of 3,5-disubstituted isoxazoles, representative procedure
methyl
3-(((tert-butoxycarbonyl)amino)methyl)isoxazole-5-carboxylate
(Boc-1B-
CO2Me)
A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding
chloroxime 1 (2 g, 9.6 mmol) and EtOAc (20 mL). Then, the resulting vigorously stirred
homogeneous solution was cooled to ꢀ5 – 0 °C and methyl propiolate (0.9 mL, 10.1 mmol)
was added followed by addition of NaHCO3 (1.6 g, 19.2 mmol). (In case of reaction 7 (2 g,
7.6 mmol) with ethynyltrimethylsilane 12 (5.4 mL, 38 mmol) dropwise addition of Et3N
(1.6 mL, 11.5 mmol) as a solution in EtOAc (100 mL) was required). The resulting mixture
was stirred overnight. The progress of the reaction was monitored by NMR spectroscopy.
Workꢀup was done by simple filtration through silica pad, then the resulting solution was
concentrated and dried in vacuo to yield the corresponding product Boc-1B-CO2Me (89%,
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2.2 g) as a white powder, mp 84ꢀ86 °C. For reaction of compound 7 with 12 when the
reaction was complete, the organic phase was washed with 10% K2CO3(aq) (2×20 mL) and
1
brine (1×20 mL). Organic phase was dried over anhydrous Na2SO4. H NMR (500 MHz,
DMSOꢀd6): δ 7.49 (s, 1H, NH), 7.05 (s, 1H, isoxaz), 4.24ꢀ4.23 (d, 2H, J = 5.5, CH2), 3.89
(s, 3H, OCH3), 1.39 (br, 9H, CH3ꢀBoc). 13C NMR (125 MHz, DMSOꢀd6): δ 163.7, 159.5,
156.7, 155.7, 109.0, 78.5, 52.9, 35.6, 28.1. MS (APCI) m/z [MꢀBoc+1H]+ calculated for
C10H13N2O5: 255.1; found: 255.4. Anal. calcd for C11H16N2O5: C, 51.56; H, 6.29; N, 10.93.
Found: C, 51.27; H, 6.54; N, 10.63.
Representative procedure for cleavage of tert-butyloxycarbonyl group
1-(3-(aminomethyl)isoxazol-4-yl)ethan-1-one hydrochloride (1A-COMe•HCl)
A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding
Bocꢀprotected isoxazole Boc-1A-COMe (2.5 g, 9.8 mmol) and EtOAc (25 mL). The flask
was submerged into an ice cooling bath; and to the vigorously stirred homogeneous
solution equimolar ratio of MeOH (1.05 eq) was added followed by dropwise addition of
AcCl (1.05 eq). As the result white precipitate was formed. After the reaction was complete
(NMR control) resulting solid was filtered and washed with cold EtOAc and recrystallized
from MeCN (if it was necessary) affording 1A-COMe•HCl as a white powder. In case of
EWG = CO2Meꢀgroup AcCl (1.05 eq) in MeOH was used.
1
1A-COMe•HCl: 95%, white powder, mp 94ꢀ96 °C. H NMR (500 MHz, D2Oꢀd2): δ 9.53
(s, 1H, isoxaz), 4.54 (s, 2H, CH2), 2.60 (s, 3H, CH3ꢀAc). 13C NMR (125 MHz, DMSOꢀd6):
δ 192.0, 166.1, 155.9, 119.8, 34.4, 29.0. MS (APCI) m/z [M+1H]+ calculated for C6H9N2O2:
141.1; found: 141.2. Anal. calcd for C6H9ClN2O2: C, 40.81; H, 5.14; N, 15.86. Found: C,
40.25; H, 5.33; N, 15.96.
Representative procedure for the hydrolysis of isoxazole-based esters
3-(((tert-butoxycarbonyl)amino)methyl)isoxazole-4-carboxylic acid (Boc-1A-CO2H)
A 250 ml round bottomed flask was charged with a magnetic stirrer, corresponding
isoxazole (Boc-1A-CO2Me, 2 g, 7.8 mmol) and MeOH (18 mL). Next, the flask was
submerged into an ice cooling bath and to the resulting vigorously stirred homogeneous
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solution 2 equiv. of 10% NaOH(aq) was added dropwise. After the NMR control indicated
the reaction completion (10 – 24 h), the solution was concentrated in vacuo. HCl(aq.), or
NaHSO4(aq.), was added dropwise until pH = 3 was reached and solid NaCl was added until
the resulting solution become saturated. The product was extracted by EtOAc (3×20 mL).
Combined organic fractions were dried over anhydrous MgSO4, concentrated and dried in
1
vacuo yielding Boc-1A-CO2H as a white powder, (95%, 1.8 g), mp 129ꢀ131 °C. H NMR
(500 MHz, DMSOꢀd6): δ 13.25 (br, 1H, CꢀOOH), 9.45 (s, 1H, isoxaz), 7.17 (brs, 1H, NHꢀ
major rotamer), 6.80 (brs, NHꢀminor of rotamer), 4.40 (d, 2H, J = 4.5, CH2), 1.38 (s, 9H,
CH3ꢀBoc). 13C NMR (125 MHz, DMSOꢀd6): δ 164.6, 162.4, 160.3, 155.5, 113.2, 78.2, 36.1,
28.2. MS (APCI) m/z [Mꢀ1H]+ calculated for C10H13N2O5: 241.1; found: 241.2. Anal. calcd
for C10H14N2O5: C, 49.58; H, 5.83; N, 11.56. Found: C, 49.38; H, 5.66; N, 11.43.
Representative procedure for the for the synthesis of 3-substituted isoxazoles
tert-butyl (isoxazol-3-ylmethyl)carbamate (Boc-1-H)
A round bottomed flask (100 mL) was charged with a magnetic stirrer, substrate Boc-1-H-
TMS (5.5 g, 20.3 mmol) and MeOH (25 mL). KHF2 5 mol% (80 mg, 1 mmol) with H2O(cat)
(100 µL) were added and the resulting homogeneous solution was vigorously stirred for 24
h at the ambient temperature while the progress was monitored by NMR spectroscopy.
After the reaction was completed the solvent was evaporated and the remaining residue
was dissolved in EtOAc (50 mL). Organic phase was filtered through anhydrous Na2SO4
1
pad and concentrated to yield Boc-1-H as a white powder (94%, 3.8 g,). H NMR (500
MHz, DMSOꢀd6): δ 8.79 (m, 1H, CHꢀisoxaz), 7.43ꢀ7.35 (brs, 1H, NH), 6.42 (s, 1H, isoxaz),
4.20ꢀ4.19 (m, 2H, CH2), 1.39 (s, 9H, CH3ꢀBoc). 13C NMR (125 MHz, DMSOꢀd6): δ 161.5,
159.8, 155.7, 103.8, 78.3, 35.5, 28.2. Anal. calcd for C9H14N2O3: C, 54.53; H, 7.12; N,
14.13. Found: C, 54.36; H, 7.18; N, 14.01.
Synthesis of the ABT- 418 bioisostere 16
tert-butyl (2S)-2-(5-(bromomethyl)-4,5-dihydroisoxazol-3-yl)pyrrolidine-1-carboxylate
(13)
A 500 mL round bottomed flask was charged with a magnetic stirrer, corresponding
chloroxime 4 (10 g, 40.2 mmol) and EtOAc (100 mL). To the resulting vigorously stirred
homogeneous solution allyl bromide (5.6 g, 46.23 mmol) was added followed by addition of
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NaHCO3 (6.7 g, 80.4 mmol). The resulting mixture was stirred overnight. The progress of
the reaction was monitored by NMR spectroscopy. After the reaction was complete,
organic phase was dried over anhydrous Na2SO4 and filtered through the silica gel pad.
The resulting solution was concentrated to yield 13 (95 %, 12.95 g) as a white powder, mp
60 °C.
13ꢀSꢀisomer: 95%, white powder, [α] = ꢀ80.00 (с = 35.42). 99.6% ee, tR = 12.2 min;
13ꢀRꢀisomer: 97%, white powder, [α] = +81.84 (с = 35.42), 99.8% ee, tR = 10.5 min.
1H NMR (500 MHz, CDCl3):
δ
4.85ꢀ4.78 (m, 1H, isoxazolineꢀCH), 4.63 (brs,
1H, ꢀpyrrolidineꢀCH), 3.48ꢀ3.32 (m, 4H, CH2ꢀpyrrolidine + CH2ꢀBr), 2.94ꢀ2.90 (m, 2H,
CH2ꢀisoxazoline), 2.17ꢀ1.91 (m, 4H, CH2ꢀpyrrolidine), 1.46 (s, 9H, CH3ꢀBoc). 13C NMR (125
MHz, DMSOꢀd6, mixture of both rotamers): δ 160.3, 159.7, 153.5, 153.1, 78.8, 78.3, 54.0,
46.3, 46.0, 38.7, 35.7, 35.2, 34.8, 30.4, 30.2, 29.6, 29.3, 28.0, 23.9, 23.0. MS (APCI) m/z
[MꢀBoc]+ calculated for C8H13BrN2O: 235.0; found: 235.1. Anal. calcd for C13H21BrN2O3: C,
46.86; H, 6.35; N, 8.41. Found: C, 46.77; H, 6.44; N, 8.57.
tert-butyl (S)-2-(5-methylisoxazol-3-yl)pyrrolidine-1-carboxylate (14)
A 250 mL round bottomed flask was charged with a magnetic stirrer, corresponding
isoxazoline 13 (12.5 g, 37.5 mmol) and DMF (50 mL). K2CO3 (10.4 g, 75 mmol) was added
and the resulting mixture was stirred overnight at 100 °C. The progress of the reaction was
monitored by NMR spectroscopy. After the reaction was complete, the reaction mixture
was concentrated the residue was poured into iceꢀbath and aqueous phase was extracted
with EtOAc. The combined orhanic phases were washed with brine (4×20 mL). Organic
phase was dried over anhydrous Na2SO4. The resulting mixture was filtered through the
silicagel pad and the solution was concentrated. The residue is colorless oil which was
solidified upon standing 77%.
14ꢀSꢀisomer: 77%, [α] = ꢀ78.8 (с = 3.96), 99.9% ee, tR = 9.5 min;
14ꢀRꢀisomer: 75%, [α] = +79.63 (с = 3.96), 99.9% ee, tR = 7.6 min.
1H NMR (500 MHz, DMSOꢀd6): δ 6.10 (brs, 1H, isoxaz), 4.83ꢀ4.77 (m, 1H, CHꢀpyrrolidine),
3.43ꢀ3.38 (m, 2H, CH2ꢀpyrrolidine), 2.36 (s, 3H, CH3), 2.21 (br, 1H, CHꢀpyrrolidine), 1.85
(br, 3H, CHꢀpyrrolidine), 1.39, (s, 9H, CH3ꢀBoc, major rotamer), 1.24 (s, 9H, CH3ꢀBoc,
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minor rotamer). 13C NMR (125 MHz, DMSOꢀd6): δ 169.02, 166.4, 166.0, 153.5, 153.2,
100.4, 99.8, 78.6, 78.5, 53.3, 53.1, 46.3, 46.0, 32.6, 31.3, 28.1, 27.9, 23.6, 22.9, 11.7.
MS (APCI) m/z [M+3H]+ calculated for C13H23N2O3: 255.3; found: 255.2. Anal. calcd for
C13H20N2O3: C, 61.88; H, 7.99; N, 11.10. Found: C, 61.95; H, 7.83; N, 11.14.
(S)-5-methyl-3-(pyrrolidin-2-yl)isoxazole hydrochloride (15*HCl)
The same experimental procedure as previously described for 3,4ꢀ and 3,5ꢀanalogues for
the cleavage of Bocꢀgroup was used.
15ꢀSꢀisomer, white powder, 92%, [α] = ꢀ21.63 (с = 53.00);
15ꢀRꢀisomer, white powder, 93%, [α] = +22.95 (с = 53.00).
1H NMR (500 MHz, DMSOꢀd6): δ 10.63 (br, 1H, NH•HCl), 9.57 (br, 1H, NH•HCl), 6.55
(s, 1H, isoxaz), 4.66 (brs, 1H, CH), 3.27ꢀ3.22 (m, 2H, CH2ꢀpyrrolidine), 2.43 (s, 3H, CH3),
2.35ꢀ2.33 (m, 1H, CHꢀpyrrolidine), 2.02 (br, 3H, CH2ꢀ pyrrolidine). 13C NMR (125 MHz,
DMSOꢀd6): δ 170.6, 160.1, 101.4, 54.0, 44.4, 29.4, 22.8, 11.9. MS (APCI) m/z [MꢀCl+H]+
calculated for C8H19N2O: 153.1; found: 153.2. Anal. calcd for C8H13ClN2O: C, 50.93; H,
6.95; N, 14.85. Found: C, 50.87; H, 7.08; N, 14.89.
(S)-5-methyl-3-(1-methylpyrrolidin-2-yl)isoxazole (16)
A 500 mL round bottomed flask was charged with magnetic stirrer and corresponding
isoxazole 15 (2.1 g, 10.60 mmol) which was suspended in dichloroethane (250 mL). MeOH
(50 mL) was added followed by addition of 30% CH2O(aq) (10 mL) and NaBH(OAC)3 (7.0 g,
33.03 mmol). Then, the resulting mixture was stirred overnight at the ambient temperature.
Next, the reaction mixture was concentrated, EtOAc (250 mL) was added and K2CO3 was
added until the pH = 10 was reached (Caution: carbon dioxide evolution!). The reaction
mixture was then stirred over Na2SO4 for ca 10 min filtered and concentrated. The product
was purified by distillation yielding 16 (1.52 g, bp. 75ꢀ80 °C at 5.5 mmHg) as a colorless
liquid (with pyrrolidine odour) which is extremely well soluble in water.
16ꢀSꢀisomer, 82%, [α] = ꢀ8.00 (с = 60.16);
16ꢀRꢀisomer, 83%, [α] = +7.86 (с = 60.16).
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1H NMR (500 MHz, DMSOꢀd6): δ 6.14 (brs, 1H, isoxaz), 3.24ꢀ3.19 (t, 1H, J = 10.0,
CHꢀpyrrolidine), 3.07ꢀ3.03 (m, 1H, CHꢀpyrrolidine), 2.36 (s, 3H, CH3ꢀNH), 2.26ꢀ2.19 (m,
1H, CHꢀpyrrolidine), 2.11 (s, 4H, CH3 + CH), 1.84ꢀ1.71 (m, 3H, CHꢀpyrrolidine). 13C NMR
(125 MHz, DMSOꢀd6): δ 169.1, 166.1, 99.7, 61.3, 56.0, 31.7, 22.4, 11.8. MS (APCI) m/z
[M+H]+ calculated for C9H15N2O: 167.1; found: 167.2. Anal. calcd for C9H14N2O: C, 65.03;
H, 8.49; N, 16.85. Found: C, 65.14; H, 8.33; N, 16.94.
ASSOCIATED CONTENT Supporting Information Available: Experimental procedure and
characterization of all new compounds (PDF). This material is available free of charge via
the Internet at
Notes and references
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37. Curiously, epimerization of the AlaIso residue in AcꢀLeuꢀAlaIsoꢀValꢀNH2 (but not in AcꢀLeuꢀ
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An easy access to a large panel of aminoisoxazoles starting from commonly available amino acids.