A novel synthetic approach to the racemic Neuraminidase inhibitor Peramivir

Article abstract of DOI:10.1016/j.tet.2016.10.039

An advanced intermediate in the synthesis of the racemic Neuraminidase inhibitor Peramivir was synthesised in a new and versatile manner starting from a stereoselective 1,3-dipolar cycloaddition reaction between the nitrile oxide deriving from 2-ethylbutanal and the commercially available and inexpensive bicyclo[2.2.1]hepta-2,5-diene. The reaction mainly afforded the exo-isoxazolino-norbornene derivative from which the oxidative cleavage of the carbon[sbnd]carbon double bond followed by subsequent dehydration led to the corresponding anhydride intermediate. Amines and alcohols were used as nucleophiles for opening the anhydride, with amines providing the better results. Both the monoester–monoacid and the monoester–monoamide were transformed into the monoester–monoamino intermediate from which the synthesis continued using previously published methods. In the best protocol, the total yield of this key intermediate was increased up to 17% from bicyclo[2.2.1]hepta-2,5-diene.

Full text of DOI:10.1016/j.tet.2016.10.039

Accepted Manuscript
A novel synthetic approach to the racemic Neuraminidase inhibitor Peramivir
Emanuela Erba, Concetta La Rosa
PII:
S0040-4020(16)31073-0
10.1016/j.tet.2016.10.039
TET 28180
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 26 May 2016
Revised Date: 3 October 2016
Accepted Date: 14 October 2016
Please cite this article as: Erba E, La Rosa C, A novel synthetic approach to the racemic Neuraminidase
inhibitor Peramivir, Tetrahedron (2016), doi: 10.1016/j.tet.2016.10.039.
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A novel synthetic approach to the racemic
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Neuraminidase inhibitor Peramivir
Emanuela Erba and Concetta La Rosa*
Università degli Studi di Milano - DISFARM - Sezione di Chimica Generale e Organica “A. Marchesini”,
V. Venezian 21, I-20133 Milano, Italy

ACCEPTED MANUSCRIPT
A novel synthetic approach to the racemic Neuraminidase inhibitor
Peramivir
*
Emanuela Erba and Concetta La Rosa
Università degli Studi di Milano
DISFARM - Sezione di Chimica Generale e Organica “A. Marchesini”, V. Venezian 21, I-20133 Milano, Italy
Abstract – An advanced intermediate in the synthesis of the racemic Neuraminidase inhibitor Peramivir
was synthesised in a new and versatile manner starting from a stereoselective 1,3-dipolar cycloaddition
reaction between the nitrile oxide deriving from 2-ethylbutanal and the commercially available and
inexpensive bicyclo[2.2.1]hepta-2,5-diene. The reaction mainly afforded the exo-isoxazolino-norbornene
derivative from which the oxidative cleavage of the carbon-carbon double bond followed by subsequent
dehydration led to the corresponding anhydride intermediate. Amines and alcohols were used as
nucleophiles for opening the anhydride, with amines providing the better results. Both the monoester-
monoacid and the monoester-monoamide were transformed into the monoester-monoamino intermediate
from which the synthesis continued using previously published methods. In the best protocol, the total
yield of this key intermediate was increased up to 17% from bicyclo[2.2.1]hepta-2,5-diene.
Keywords: Peramivir; Neuraminidase inhibitor; Bicyclo[2.2.1]hepta-2,5-diene; 1,3-Dipolar cycloaddition.
1. Introduction
1
Influenza viruses infect an enormous number of people each year, and the resulting epidemics lead to
2
substantial costs for the community and a high mortality rate. The infection is particularly dangerous for
3
children, the elderly, and people with immunosuppression.
*
Corresponding author’s e-mail address: concetta.larosa@unimi.it; Tel.: +39 02 50314473; Fax: +39 02 50314476.
1

ACCEPTED MANUSCRIPT
Vaccination remains the primary preventive measure although it provides limited control, partially
because influenza viruses mutate very rapidly and there is a continuous emergence of new strains to
4
which even previously vaccinated subjects remain sensitive.
The target of the compounds that have most recently been used in influenza prophylaxis is influenza
5
Neuraminidase, a viral surface enzyme belonging to the family of Glycoside Hydrolases. Neuraminidase
catalyses the cleavage of the glycosidic bond between a terminal sialic acid and Hemagglutinin, thus
allowing the virion to escape from infected host cells and spread the infection in the respiratory tract.
The first Neuraminidase inhibitors, which prevent the virus from reproducing and therefore slow the
6
7
spread of the infection, were Zanamivir 1 and Oseltamivir 2 (Fig. 1).
Figure 1
The clinical use of Zanamivir is hampered by the fact that its limited oral bioavailability means that it
must be administered by oral inhalation, whereas the second-generation Neuraminidase inhibitor
Oseltamivir has good oral activity as a pro-drug. Both products have side effects: headache and diarrhoea
8
in the case of Zanamivir, and nausea and vomiting in the case of Oseltamivir. The new Peramivir
Neuraminidase inhibitor 3 has recently proved to be very potent against a variety of strains of influenza,
9
and more effective than Zanamivir or Oseltamivir, but its poor bioavailability has aroused considerable
1
0
interest in designing and synthesising analogous structures. Finding new, versatile and efficient means
of synthesis could promote the structure-activity relationship (SAR) studies necessary for the
development of new inhibitors.
1
1
A number of studies of Peramivir synthesis have been published. The starting material in the first
11a,b
11d
studies,
and in a recent improvement,
was (-)-(1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one 4
1
2
(
Vince’s lactam), which is commercially available as an expensive pure enantiomer. Using a different
2

ACCEPTED MANUSCRIPT
1
1c
approach, Miller started with the racemic 2-oxa-3-aza-bicyclo[2.2.1]hept-5-ene-3-carboxylic acid tert-
butyl ester 5 obtained from the hetero-Diels-Alder cycloaddition between N-Boc-protected
hydroxylamine and cyclopentadiene. Both syntheses have the bicyclic isoxazoline 6a as a common key
intermediate (Scheme 1).
Scheme 1
Given our interest in the stereoselective synthesis of highly functionalised heterocycles using 1,3-dipolar
1
3
cycloaddition reactions, we here describe a new approach to the synthesis of Peramivir in which the
isoxazoline ring of intermediate 6a was generated by a 1,3-dipolar cycloaddition between a suitable nitrile
oxide and the commercially available and inexpensive bicyclo[2.2.1]hepta-2,5-diene (2,5-norbornadiene-
NBD) 7, as outlined in Scheme 2.
Scheme 2
The cycloaddition reaction does not pose any problems of regioselectivity, only stereoselectivity. Only
the exo-adduct has the cyclopentane ring with the correct steric relationships of the four substituents of
Peramivir. Furthermore, published data concerning 1,3-dipolar cycloaddition reactions between 2,5-
norbornadiene and nitrile oxides have always shown stereo-selectivity in favour of the exo-isomer, thus
14
predicting a similar result in our case. The next norbornene opening takes advantage of the presence of
the carbon-carbon double bond: the two carboxylic groups can be introduced by means of oxidative
cleavage, and appropriately transformed into the acid and substituted amino groups on intermediate 6a of
3

ACCEPTED MANUSCRIPT
Peramivir. This approach should provide greater versatility in synthesising structural analogues of
Peramivir, particularly in relation to the substitution (R≠CH(CH CH ) ).
2
3 2
2
. Results and discussion
The synthetic scheme was developed for the racemic Peramivir. Oxime 8, the precursor of the nitrile
oxide, was prepared from the corresponding commercially available 2-ethylbutanal and hydroxylamine at
1
5
reflux in MeOH (Scheme 3), and then transformed into the corresponding 1,3-dipole by means of
treatment with NaOCl and TEA in a biphasic system, in the presence of an excess of 2,5-norbornadiene 7.
The reaction gave a mixture of the exo- and endo-adducts 9 and 10 in a 95:5 ratio regardless of the
observed yield, which depends on the ratio between 2,5-norbornadiene 7 and oxime 8 is used: total yield
was 42% with a ratio of 2:1, but up to 75% with a ratio of 4:1 (the excess 2,5-norbornadiene can be
recovered at the end of the reaction by means of distillation). A mixture of the two regioisomers 11 and
1
2 was also obtained from exo-adduct 9 as a result of a double 1,3-dipolar cycloaddition. Exo-adduct 9
was easily separated from the other products by means of chromatography on silica gel (Scheme 3).
Scheme 3
NMR spectroscopy was used to assign exo and endo to mono-adducts 9 and 10. In exo-adduct 9, the
coupling constant of the bridgehead protons H-3a and H-7a with the adjacent endo-protons H-4 and H-7
was 0 Hz (JH3a,H4 = JH7a,7 = 0 Hz) whereas, in endo-adduct 10, the corresponding coupling constant of
protons H-3a and H-7a with the adjacent exo-protons H-4 and H-7 was 4.0 Hz (JH3a,H4 = JH7a,7 = 4.0 Hz).
14a,e
These values are in line with published findings concerning analogous compounds,
and supported by
4

ACCEPTED MANUSCRIPT
1
4d
calculations based on the Karplus equation and molecular geometries. Adducts 11 and 12 were also
isolated, and their exact regiochemistry attributed by comparison of the relative NMR spectroscopic data
14a,d,e
with those of analogous compounds.
The synthetic approach considered the oxidative cleavage of the carbon-carbon double bond with the
insertion of two carboxylic groups, so the exo-adduct 9 was transformed into the dicarboxylic acid 13 by
treating it with KMnO under controlled conditions (Scheme 4).
4
Scheme 4
A key point of the synthesis was expected to be the regioselective differentiation of the two carboxylic
groups. We decided to synthesise the intermediate cyclic anhydride 14, which could be opened by a
suitable nucleophile to obtain a dicarboxylic acid mono-acyl azide or mono-amide from which to obtain
an amino group using Curtius or Hoffmann rearrangements. We verified both possibilities. Anhydride 14
was prepared in quantitative yield by heating the diacid 13 with acetic anhydride, then turned into a 50:50
mixture of the two mono-ester, mono-acid regioisomers 15 and 16 by heating with MeOH (Scheme 4).
This mixture was inseparable, therefore it was used as such in the next step. Numerous ways of carrying
1
6
out Curtius rearrangement were tested, but only activation of the carboxylic group with ClCO Et
2
1
6e
followed by treatment with sodium azide and heating in toluene/tBuOH led to a mixture of the
11b
unreacted compounds 15 and 16 and the regioisomeric products 6a and 6b, and then with a total yield
of only 13% (Scheme 5).
Scheme 5
5

ACCEPTED MANUSCRIPT
Considering these results unsatisfactory, we turned our attention to Hoffmann rearrangement for which it
was necessary to prepare the mono-ester, mono-amide derivative. A THF solution of anhydride 14 was
first treated with gaseous ammonia in methanol and then with TMSiCHN to yield a mixture of the two
2
regioisomeric compounds 17a and 18a in a ratio of 60:40, as estimated by integrating the peaks of proton
1
H-6a in the H NMR spectra of the crude reaction mixture (Scheme 6).
Scheme 6
C O O H
C O N H R
C O O H
C O O C H 3
C O N H R
T M S iC H N
C H O H
2
O
O
O
O
O
O
R N H
5° C
2
3
N
N
+
N
N
+
N
2
2 5 ° C
O
O
(
C O N H R
C O N H R
C O O C H 3
±
) 1 4
(
±
)
1 7 a ,b
(
±
)
1 8 a ,b
a ) R = H b ) R = tB u
R = H
9 9 % , 6 0 : 4 0
R = tB u 8 0 % , 5 0 : 5 0
The exact regiochemistry of the two products was established by comparing them with the amides
obtained from deprotecting the corresponding N-tBu-amides, as described below. The observed
regioselectivity of the opening of the anhydride was unsatisfactory, and it was not possible to separate the
two mono-amides by means of flash chromatography, not even as mono-acid intermediates. With the aim
of improving regioselectivity, anhydride 14 was treated with the more sterically hindered amine tBu-NH2.
Although regioselectivity remained poor, it was possible to separate the mono-amide/mono-ester
regioisomers 17b and 18b by means of flash chromatography and confirm their structures on the basis of
analytical and spectroscopic data (HSQC, HMBC and COSY experiments). Structures 17b and 18b were
respectively assigned to the regioisomers having the H-6a proton signal at 5.31 and 5.01 δ. Finally the t-
butyl group was removed by treating compound 17b with trifluoroacetic acid, thus obtaining amide 17a
(Scheme 7).
Scheme 7
6

ACCEPTED MANUSCRIPT
Unsuccessful attempts were made to develop Hoffmann rearrangements of 17a using aqueous NaOH/Br2
1
7a
or CH ONa/Br in methanol. The protocol that provided the best results was heating amide 17a with
3
2
1
7b,c
lead tetracetate in a mixture of t-butanol and DMF.
Under these conditions, the reaction directly
afforded the amino N-Boc protected derivative 6a (Scheme 7). The synthesis of Peramivir was completed
11b,d
from 6a using previously reported procedures and with similar results.
By means of this sequence, the isoxazoline intermediate 6a was obtained from 2,5-norbornadiene in six
steps, with a total yield of about 11.5%, which is comparable with that obtained by Miller (10.8% from
1
1c
hydroxylamine hydrochloride in seven steps), but lower than that reported by Chand (52% from
1
1b
Vince’s lactam in two steps). This was mainly due to the lack of regioselectivity observed when
opening the anhydride 14 with tBu-NH2.
With the aim of testing regioselectivity when opening the anhydride with other nucleophiles, 14 was
treated with the sterically hindered alcohol benzhydrol. The reaction required a long time at high
temperatures, but provided a mixture of the two ester regioisomers 19 and 20 in a 70:30 ratio (Scheme 8).
Scheme 8
Compound 19 was easily separated from the other product by means of chromatography on silica gel, but
the yield was only 23%.
Given the difficulty in obtaining the regioselective opening of anhydride 14 with alcohols or sterically
hindered amines, we reconsidered using ammonia. As intermediate 6a can be separated
11b
chromatographically from its regioisomer 6b,
the anhydride was opened with ammonia, and the
subsequent reactions were carried out on the mixture of the two amides 17a and 18a. In this way, the
desired bicyclic isoxazoline 6a was isolated, from 2,5-norbornadiene 7 in five steps, with an overall yield
of 17% (Scheme 9).
Scheme 9
7

ACCEPTED MANUSCRIPT
C O O H
O
O
N a C lO
T E A
N
A c 2
O
O
O
K M n O
4
H O
N
C
N
N
+
H
9
9 %
9 5 %
O
O
C O O H
1 3
7
1 %
7
8
(
±
)
9
(
±
)
(
±
)
1 4
C O O C H 3
C O N H 2
C O O C H 3
O
O
O
N
N
P b (O A c)
4
N
1
)
N H
3
+
P e ra m ivir
2
2 2
)T M S C H N
C O N H 2
C O O C H 3
tB u O H /D M F
N H B o c
co lu m n
2
6 %
(
±
)
1 7 a
±
( ) 1 8 a
(
± ) 6 a
3
. Conclusions
We describe new strategy for obtaining racemic Peramivir, a potent Neuraminidase inhibitor approved by
FDA as an anti-influenza drug. In order to synthesise the tricyclic system from which to obtain the
cyclopentane nucleus, we used a 1,3-dipolar cycloaddition reaction of the appropriate nitrile oxide on 2,5-
norbornadiene. In this way, intermediate 6a was synthesised using inexpensive, commercially available
reagents, and simple reactions. This approach represents an improvement of the previously reported
processes, and makes it possible to obtain potential Neuraminidase inhibiting analogues of Peramivir by
varying the alkyl group of the nitrile oxide used.
4. Experimental Section
4
.1. General methods
1
13
Melting points were determined on a Büchi B-540 apparatus . H and C NMR spectra were recorded
using a Varian-Gemini 200 MHz spectrometer. Chemical shifts (δ) are given in ppm in relation to TMS;
the solvent was CDCl unless otherwise specified. All of the coupling constants (J) are in Hertz.
3
Elemental analyses were performed by the Microanalytical Laboratory of the Department. The MS
spectra were recorded with a Thermo-Finnigan LCQ advantage AP electrospray/ion trap equipped
instrument using a syringe pump device to directly inject sample solutions. Bicyclo[2.2.1]hepta-2,5-diene
1
5
and 2-ethylbutanal were obtained from commercial sources. Oxime 8 was prepared following the
reported method.
8

ACCEPTED MANUSCRIPT
4
.2. 1,3-Dipolar cycloaddition of 2,5-norbornadiene 7 with 2-ethyl-butanenitrile oxide. A 4.5%
aqueous solution of NaClO (229.4 mL) was added dropwise to a solution of bicyclo[2.2.1]hepta-2,5-diene
7
(25.61 g, 278.4 mmol, 4 equiv.), oxime 8 (8.0 g, 69.6 mmol, 1 equiv.) and TEA (0.64 mL, 4.64 mmol,
0
.06 equiv.) in CH Cl (100 mL). The mixture was stirred for 20 h at 45°C, and then cooled to room
2
2
temperature, the phases were separated and the aqueous phase was extracted using CH Cl (3x30 mL).
2
2
The combined extracts were dried over anhydrous Na SO and concentrated at reduced pressure.
2
4
Compounds 9, 10, 11 and 12 were separated by means of column chromatography on silica gel (from
hexane/ethyl acetate=95/5 to hexane/ethyl acetate=75/25).
1
4
.2.1 (±)-exo-Cycloadduct (9): Colorless oil (R 0.55, hexane/ethyl acetate=90/10, I2), (10.1 g, 71%). H
f
NMR:
δ 0.90 (t, J=7.3, 6H, 2CH ); 1.43-1.79 (m, 6H, 2H-8 + 2CH ); 2.16-2.28 (m, 1H, CH); 2.98 (broad
3 2
s, 1H, H-4); 3.17 (broad s, 1H, H-7); 3.26 (d, J=8.1, 1H, H-3a); 4.75 (d, J=8.1, 1H, H-7a); 6.00-6.05,
1
3
6
.20-6.24 (2m, 2H, H-5 + H-6). C NMR:
δ 11.6, 12.3 (2CH ); 24.7, 26.1 (2CH ); 41.7 (CH); 43.4 (C-
3 2
8
); 44.9, 49.7 (C-4, C-7); 59.3 (C-3a); 87.9 (C-7a); 135.9, 140.1 (C-5, C-6); 160.2 (C-3). IR (nujol): 705
(
δC-H, CH=CH), 1604 (vC=N, C=N), 1706 (vC=C, C=C). Anal. Calcd for C H NO: C, 76.06; H, 9.33; N,
13 19
+
+
+
6
.82. Found: C, 76.01; H 9.29; N, 6.78. MS-ESI (m/z): 206 [M+H] , 228 [M+Na] .
1
4
.2.2 (±)-endo-Cycloadduct (10): Colorless oil (R 0.40, hexane/ethyl acetate=90/10, I2), (0.57 g, 4%). H
f
NMR:
δ
0.88, 0.89 (2t, J=7.4, 6H, 2CH ); 1.44-1.69 (m, 6H, 2H-8 + 2CH ); 2.07-2.17 (m, 1H, CH); 3.10
3
2
(broad s, 1H, H-4); 3.29 (broad s, 1H, H-7); 3.64 (dd, J=9.5, 4.0, 1H, H-3a); 5.20 (dd, J=9.5, 4.0, 1H, H-
1
3
7
a); 6.02-6.13, 6.15-6.20 (2m, 2H, H-5 + H-6). C NMR:
δ
11.4, 12.3 (2CH ); 23.8, 25.2 (2CH ); 41.3
3
2
(
CH); 43.4 (C-8); 46.6, 47.7 (C-4, C-7); 58.5 (C-3a); 85.9 (C-7a); 134.3, 135.3 (C-5, C-6); 160.9 (C-3).
IR (nujol): 726 ( C-H, CH=CH), 1602 (vC=N, C=N), 1727 (vC=C, C=C). Anal. Calcd for C H NO: C,
6.06; H, 9.33; N, 6.82. Found: C, 76.02; H 9.28; N, 6.77. MS-ESI (m/z): 206 [M+H] , 228 [M+Na] .
δ
13
19
+
+
+
7
9

ACCEPTED MANUSCRIPT
4
6
2
.2.3 (±)-Cycloadduct (11): Colorless amorphous glass (R 0.20, hexane/ethyl acetate=90/10, I2), (0.7 g,
f
1
.4%). H NMR:
δ 0.86 (t, J=7.3, 12H, 4CH ); 1.46-1.60 (m, 10H, 2H-9 + 4CH ); 2.22-229 (m, 2H,
3 2
1
3
CH); 2.69 (broad s, 2H, H-4, H-8); 3.00 (d, J=8.4, 2H, H-3a, H-7a); 4.47 (d, J=8.1, 2H, H-4a, H-8a). C
NMR:
δ
11.5, 12.2 (4CH ); 24.7, 26.1 (4CH ); 27.0 (C-9); 41.5 (2CH); 45.5 (C-4, C-8); 54.4 (C-3a, C-
3
2
7
a); 85.1 (C-4a, C-8a); 160.0 (C-3, C-7). IR (nujol): 1614 (vC=N, C=N). Anal. Calcd for C H N O : C,
19
30
2
2
+
+
+
7
1.66; H, 9.50; N, 8.80. Found: C, 71.62; H 9.45; N, 8.71. MS-ESI (m/z): 319 [M+H] , 341 [M+Na] .
4
5
2
2
5
.2.4 (±)-Cycloadduct (12): Colorless amorphous glass (R 0.10, hexane/ethyl acetate=90/10, I2), (0.62 g,
f
1
.6%). H NMR:
δ 0.91 (t, J=7.3, 12H, 4CH ); 1.53-1.69 (m, 10H, 2H-9 + 4CH ); 2.22-2.29 (m, 2H,
3 2
CH); 2.46 (broad s, 1H, H-4); 2.94 (broad s, 1H, H-8); 3.13 (d, J=8.1, 2H, H-3a + H-4a); 4.46 (d, J=8.1,
1
3
H, H-7a + H-8a). C NMR:
δ 11.4, 12.3 (4CH ); 24.6, 26.0 (4CH ); 27.2 (C-9); 41.2 (2CH); 41.3 (C-4);
3 2
0.3 (C-8); 58.4 (C-3a, C-4a); 82.1 (C-7a, C-8a); 159.9 (C-3, C-5). IR (nujol): 1609 (vC=N, C=N). Anal.
+
Calcd for C H N O : C, 71.66; H, 9.50; N, 8.80. Found: C, 71.59; H 9.37; N, 8.68. MS-ESI (m/z): 319
1
9
30
2
2
+
+
[
M+H] , 341 [M+Na] .
4
.3. (±)-3-(1-Ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-4,6-dicarboxylic acid (13).
A solution of (±) exo-cycloadduct 9 (4.3 g, 21.0 mmol) in acetone (38 mL) was added dropwise to a
solution of KMnO (11.0 g, 69.6 mmol) in H O (38 mL) and cooled to 0°C, while maintaining the
4
2
temperature between 0 and 5° C. The mixture was stirred for 3 h at room temperature, cooled to 0°C and
treated with Na S O (13.22 g, 69.6 mmol), stirred for 0.5 h at room temperature, and then acidified to pH
2
2
5
2
with concentrated HCl and extracted using ethyl acetate. The organic phase was separated and dried
over anhydrous Na SO , and the solvent was evaporated in vacuo. Compound (±)-13 was obtained as a
2
4
colourless solid, (5.6 g, 99%); (R 0.25, toluene/dioxane/AcOH=4.5/1/0.1, bromocresol green); m.p. 133-
f
1
3
2
5
5 °C (diisopropyl ether). H NMR (CD OD):
δ
0.89, 0.93 (2t, J=6.9, 6H, 2CH ); 1.53-1.70 (m, 4H,
3
3
CH ); 2.17-2.42 (m, 3H, CH + 2H-5); 2.89-3.01 (m, 2H, H-4 + H-6); 4.09 (dd, J=9.9, 5.5, 1H, H-3a);
2
1
3
.21 (dd, J=9.9, 4.8, 1H, H-6a). C NMR (CD OD):
δ 10.2, 11.4 (2CH ); 24.0, 25.4 (2CH ); 32.2 (C-5);
3 2
3
1
0

ACCEPTED MANUSCRIPT
4
0.9 (CH); 48.0, 52.3 (C-4, C-6); 57.2 (C-3a); 87.4 (C-6a); 162.8 (C-3); 173.8, 174.9 (2C=O). The
assignments of the signals was made based on the HSQC NMR spectra. IR (nujol): 2970 (vO-H, O-H),
1
5
726 (vC=O, C=O), 1612 (vC=N, C=N). Anal. Calcd for C H NO : C, 57.98; H, 7.11; N, 5.20. Found: C,
13 19 5
+
+
+
7.92; H 7.05; N, 5.11. MS-ESI (m/z): 270 [M+H] , 292 [M+Na] .
2
,6
4
.4. (±)-5-(1-Ethyl-propyl)-3,9-dioxa-4-aza-tricyclo[5.3.1.0 ]undec-4-ene-8,10-dione (14). A solution of
diacid (±)-13 (1.0 g, 3.72 mmol) in acetic anhydride (8 mL) was stirred for 18 h at 90°C. The acetic
anhydride was evaporated in vacuo to afford the compound (±)-14 as a light brown solid that was used
1
without further purification, (0.92 g, 99%); m.p. 124-26 °C. H NMR:
δ 0.92 (t, J=7.3, 6H, 2CH ); 1.59-
3
1
.68 (m, 4H, 2CH ); 1.92-2.09 (m, 1H, CH); 2.28-2.36 (m, 2H, 2H-11); 3.31 (d, J=3.2, 1H, H-7); 3.58 (d,
2
J=3.2, 1H, H-1); 3.88 (d, J=8.2, 1H, H-6); 5.13 (d, J=8.2, 1H, H-2).
4
.5. (±)-3-(1-Ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-4,6-dicarboxylic acid 6-
methyl ester (15) and (±)-3-(1-Ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-4,6-
dicarboxylic acid 4-methyl ester (16). A solution of anhydride (±)-14 (0.92 g, 3.67 mmol) in anhydrous
CH OH (5 mL) was stirred for 18 h at 65°C. The solvent was evaporated in vacuo to afford an
3
inseparable 50:50 mixture of the two mono-ester, mono-acid regioisomers (±)-15 and (±)-16 as shown by
1
1
the H NMR spectrum, (1.03 g, 99%); (R 0.5, CHCl /CH OH/AcOH=97/2/1, bromocresol green). H
f
3
3
NMR:
δ 0.89, 0.90 (2t, J=6.9, 6H, 2CH ); 1.54-1.67 (m, 4H, 2CH ); 2.27-2.37 (m, 3H, CH + 2H-5); 2.91-
3 2
2
.95, 3.08-3.14 (m, 2H, H-4 + H-6); 3.70, 3.72 (2s, 3H, OCH ); 4.08 (dd, J=9.2, 4.0, 1H, H-3a); 5.29 (dt,
3
1
3
J=9.5, 3.3, 1H, H-6a); 8.40 (broad s, 1H, COOH). C NMR:
δ 11.1, 12.3 (2CH ); 24.2, 25.7 (2CH );
3 2
3
2.0 (C-5); 40.9 (CH); 47.9, 52.3 (C-4, C-6); 52.0, 52.7 (OCH ); 56.9, 57.1 (C-3a); 87.1, 87.3 (C-6a);
3
1
61.9, 162.0 (C-3); 173.1, 173.5 (C=O); 177.5, 177.8 (C=O). IR (nujol): 2964 (vO-H, O-H), 1737 (vC=O
,
+
+
C=O), 1612 (vC=N, C=N). MS-ESI (m/z): 284 [M+H] .
1
1

ACCEPTED MANUSCRIPT
4
.6. (±)-4-Carbamoyl-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-6-carboxylic
acid
methyl
ester
(17a) and
(±)-6-Carbamoyl-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-
cyclopenta[d]isoxazole-4-carboxylic acid methyl ester (18a). NH (7N in CH OH) (3.0 mL, 21.0 mmol)
3
3
was added to a solution of anhydride (±)-14 (0.92 g, 3.67 mmol) in anhydrous THF (10 mL). The mixture
was stirred for 18 h at room temperature, and then the solvent was evaporated in vacuo. The residue was
treated with H O, acidified to pH 2 with concentrated HCl, and extracted using ethyl acetate. The organic
2
phase was dried over anhydrous Na SO , and the solvent was evaporated off. The residue was then
2
4
dissolved in anhydrous THF (10 mL) and treated with TMSiCHN 2N in hexane (5.5 mL, 11.0 mmol).
2
The mixture was stirred for 18 h at room temperature, after which CH OH (3.0 mL) was added and the
3
solvent was evaporated in vacuo to afford an inseparable 60:40 mixture of the two mono-ester, mono-
1
amide regioisomers (±)-17a and (±)-18a, as shown by the H NMR spectrum, (1.02 g, 99%); (R 0.35,
f
1
hexane/ethyl acetate =25/75, I ). H NMR:
δ 0.89, 0.91 (2t, J=7.6, 6H, 2CH ); 1.52-1.82 (m, 4H, 2CH );
3 2
2
2
5
.2-2.34 (m, 3H, CH + 2H-5); 2.74- 3.12 (m, 2H, H-4 + H-6); 3.73, 3.74 (2s, 3H, OCH ); 4.12 (dd, J=9.9,
3
.9, 1H, H-3a); 5.07 (dd, J=10.3, 5.5, 1H (40%), H-6a); 5.31 (dd, J=10.3, 4.4, 1H (60%), H-6a); 5.61-5.86
(
broad m, 2H, NH ). The products were fully characterised after being obtained pure (see below).
2
4
.7.
carboxylic acid methyl ester (17b) and (±)-6-tert-Butylcarbamoyl-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-
aH-cyclopenta[d]isoxazole-4-carboxylic acid methyl ester (18b). tertBuNH (1.6 mL, 14.9 mmol) was
(±)-4-tert-Butylcarbamoyl-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-6-
3
2
added to a solution of anhydride (±)-14 (1.87 g, 7.43 mmol) in anhydrous THF (15 mL). The mixture was
stirred for 18 h at room temperature, after which the solvent was evaporated in vacuo. The residue was
treated with H O, acidified to pH 2 with concentrated HCl, and extracted using ethyl acetate. The organic
2
phase was dried over anhydrous Na SO , and the solvent was evaporated off. The residue was then
2
4
dissolved in anhydrous THF (10 mL) and treated with TMSiCHN 2N in hexane (11.15 mL, 22.3 mmol).
2
The mixture was stirred for 18 h at room temperature, after which CH OH (6.0 mL) was added and the
3
solvent was evaporated in vacuo to give a 50:50 mixture of the two mono-ester, mono-amide
1
2

ACCEPTED MANUSCRIPT
regioisomers (±)-17b and (±)-18b, as shown by the NMR spectrum. The products were separated by
means of flash chromatography (silica gel, hexane/ethyl acetate =65/35), and their exact regiochemistry
was assigned by HSQC, HMBC and COSY experiments.
1
(
±)-17b (1.0 g, 40%); colourless solid, m.p. 108-111 °C; (R 0.35, hexane/ethyl acetate =65/35, I ). H
f
2
NMR:
δ 0.90, 0.92 (2t, J=7.3, 6H, 2CH ); 1.36 (s, 9H, C(CH ) ); 1.50-1.65 (m, 4H, 2CH ); 2.18-2.29 (m,
3 3 3 2
3
6
2
H, C-H + 2H-5); 2.53-2.60 (m, 1H, H-4); 2.96-3.06 (m, 1H, H-6); 3.73 (s, 3H, OCH ); 4.09 (dd, J=10.2,
3
1
3
.4, 1H, H-3a); 5.31 (dd, J=10.2, 4.6, 1H, H-6a); 5.53 (broad s, 1H, NH). C NMR:
δ 11.4, 12.5 (2CH3);
4.3, 25.7 (2CH ); 29.1 (C(CH ) ); 34.9 (C-5); 41.3 (CH); 51.1 (C-4); 51.9 (C(CH ) ); 52.6 (OCH ); 53.1
2
3 3
3 3
3
(C-6); 57.1 (C-3a); 87.8 (C-6a); 162.7 (C-3); 171.2 (N-C=O); 173.0 (O-C=O). Anal. Calcd for
+
+
C H N O : C, 63.88; H, 8.93; N, 8.28. Found: C, 63.81; H 8.85; N, 8.19. MS-ESI (m/z): 339 [M+H] ,
18
30
2
4
+
3
61 [M+Na] .
1
(
±)-18b (1.0 g, 40%); colourless solid, m.p. 85-88 °C; (R 0.5, hexane/ethyl acetate =65/35, I ). H NMR:
f
2
δ
2
4
1
5
0.86, 0.89 (2t, J=7.3, 6H, 2CH ); 1.32 (s, 9H, C(CH ) ); 1.47-1.68 (m, 4H, 2CH ); 2.20, 2.21 (2t, J=7.8,
3 3 3 2
H, 2H-5); 2.28-2.35 (m, 1H, C-H); 2.55-2.63 (m, 1H, H-6); 2.78-2.89 (m, 1H, H-4); 3.73 (s, 3H, OCH3);
1
3
.10 (dd, J=10.1, 5.9, 1H, H-3a); 5.01 (dd, J=10.1, 5.5, 1H, H-6a); 5.60 (broad s, 1H, NH). C NMR:
δ
1.2, 12.5 (2CH ); 24.3, 25.7 (2CH ); 29.1 (C(CH ) ); 32.4 (C-5); 40.9 (CH); 47.7 (C-4); 51.8 (C(CH ) );
3
2
3 3
3 3
2.7 (OCH ); 54.4 (C-6); 57.4 (C-3a); 87.9 (C-6a); 162.8 (C-3); 171.0 (N-C=O); 173.5 (O-C=O). Anal.
3
+
Calcd for C H N O : C, 63.88; H, 8.93; N, 8.28. Found: C, 63.79; H 8.84; N, 8.18. MS-ESI (m/z): 339
1
8
30
2
4
+
+
[
M+H] , 361 [M+Na] .
4
.8. (±)-4-Carbamoyl-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-6-carboxylic
acid methyl ester (17a.) A solution of (±)-17b (0.2 g, 0.6 mmol) in anhydrous TFA (3 mL) was heated to
0°C for 24 h. The mixture was cooled on ice, and a saturated solution of Na CO was added until pH=7.
8
2
3
The solution was extracted using ethyl acetate, after which the extracts were dried over anhydrous
Na SO and the solvent was evaporated off to give compound (±)-17a as an amorphous glass (0.125 g,
2
4
1
7
5%). H NMR:
δ
0.89, 0.90 (2t, J=7.3, 6H, 2CH ); 1.48-1.62 (m, 4H, 2CH ); 2.20-2.31 (m, 3H, CH +
3
2
1
3

ACCEPTED MANUSCRIPT
2
H-5); 2.74-2.84 (m, 1H, H-4); 3.01-3.11 (m, 1H, H-6); 3.73 (s, 3H, OCH ); 4.12 (dd, J=9.9, 5.5, 1H, H-
3
1
3
3
a); 5.31 (dd, J=10.3, 4.4, 1H, H-6a); 5.61 (broad s, 2H, NH ). C NMR:
δ 11.1, 12.3 (2CH ); 24.1, 25.6
3
2
(
2CH ); 34.1 (C-5); 41.0 (CH); 49.2 (C-4); 52.5 (OCH ); 52.9 (C-6); 56.9 (C-3a); 87.5 (C-6a); 162.3 (C-
2
3
3
5
); 173.0 (N-C=O); 174.7 (O-C=O). Anal. Calcd for C H N O : C, 59.56; H, 7.85; N, 9.92. Found: C,
14 22 2 4
+
+
9.48; H 7.76; N, 9.84. MS-ESI (m/z): 305 [M+Na] .
4
.9. (±)-6-Carbamoyl-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-4-carboxylic
acid methyl ester (18a). Compound (±)-18a was obtained as described for compound (±)-17a.
1
Amorphous glass (80%). H NMR:
δ 0.88, 0.91 (2t, J=7.3, 6H, 2CH ); 1.52-1.66 (m, 4H, 2CH ); 2.24 (t,
3 2
J=8.4, 2H, 2H-5); 2.29-2.35 (m, 1H, CH); 2.74-2.95 (m, 2H, H-4 + H-6); 3.74 (s, 3H, OCH ); 4.06-4.16
3
13
(
m, 1H, H-3a); 5.07 (dd, J=10.3, 5.5, 1H, H-6a); 5.85 (broad s, 2H, NH ). C NMR:
δ
11.0, 12.3 (2CH3);
2
2
1
4.1, 25.5 (2CH ); 32.3 (C-5); 40.8 (CH); 47.6 (C-4); 52.7 (OCH ); 53.0 (C-6); 57.2 (C-3a); 87.7 (C-6a);
2
3
62.6 (C-3); 173.4 (N-C=O); 174.3 (O-C=O). Anal. Calcd for C H N O : C, 59.56; H, 7.85; N, 9.92.
1
4
22
2
4
+
+
Found: C, 59.46; H 7.79; N, 9.88. MS-ESI (m/z): 305 [M+Na] .
4
.10.
(±)-4-tert-Butoxycarbonylamino-3-(1-ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-
cyclopenta[d]isoxazole-6-carboxylic acid methyl ester (6a). A solution of (±)-17a (0.244 g, 0.77 mmol)
in a mixture of tBuOH (4 mL) and DMF (2 mL) was heated to 40°C. After the addition of Pb(OAc) (1.7
4
g, 2.31 mmol), the mixture was heated to 80°C for 8 h. The solvent was evaporated, and the residue was
treated with water and extracted using ethyl acetate (3x15 mL). The combined organic layers were dried
over anhydrous Na SO , filtered and concentrated by means of rotary evaporation. The resulting residue
2
4
was purified by means of column chromatography (silica gel, hexane/ethyl acetate =85/15) to give
1
compound (±)-6a as a white solid (0.169 g, 55%), m.p. 108-109 °C. H NMR:
δ 0.88, 0.91 (2t, J=7.3, 6H,
2
3
CH ); 1.43 (s, 9H, tBu); 1.57-1.73 (m, 4H, 2CH ); 2.02-2.11 (m, 2H, 2H-5); 2.43-2.53 (m, 1H, CH);
3 2
.18 (broad d, 1H, J=6.6, H-6); 3.57 (d, 1H, J=9.2, H-3a); 3.75 (s, 3H, OCH ); 4.21 (m, 1H, H-4); 5.19 (d,
3
1
4

ACCEPTED MANUSCRIPT
J=9.2, 1H, H-6a); 5.58 (broad d, J=7.7, 1H, NH). Anal. Calcd for C H N O : C, 61.00; H, 8.53; N, 7.90.
1
8
30
2
5
+
+
Found: C, 60.89; H 8.47; N, 7.83. MS-ESI (m/z): 377 [M+Na] .
4
.11. (±)-3-(1-Ethyl-propyl)-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-4,6-dicarboxylic acid 6-
benzhydryl ester (19). Benzhydrol (0.68 g, 3.67 mmol) and DMAP (0.05 g, 0.37 mmol) were added to a
solution of anhydride (±)-14 (0.92 g, 3.67 mmol) in anhydrous toluene (10 mL), and the mixture was
stirred at reflux temperature for 10 h. After the solvent had been distilled, the residue was treated with
brine and extracted using ethyl acetate (3x10 mL). The combined organic layers were dried over
anhydrous Na SO , filtered, and then concentrated by means of rotary evaporation. Distillation of the
2
4
solvent afforded a 70:30 mixture of the two mono-ester, mono-acid regioisomers (±)-19 and (±)-20, as
1
shown by the H NMR spectrum. Compound (±)-19 was purified by means of column chromatography
(silica gel, hexane/ethyl acetate =65/35). Its exact regiochemistry was assigned by HMBC and COSY
experiments. Colourless solid (0.37 g, 23%), m.p. 130-133 °C; (R 0.55, hexane/ethyl acetate =50/50,
f
1
bromocresol green). H NMR:
δ 0.90, 0.92 (2t, J=7.3, 6H, 2CH ); 1.51-1.72 (m, 4H, 2CH ); 2.28-2.40
3 2
(m, 3H, C-H + 2H-5); 2.89-2.99 (m, 1H, H-4); 3.21-3.26 (m, 1H, H-6); 4.08 (dd, J=9.7, 5.0, 1H, H-3a);
1
3
5
.30 (dd, J=9.7, 3.9, 1H, H-6a); 6.90 (s, 1H, CHPh ); 7.36 (s, 10H, 2Ph); 8.7 (broad s, 1H, OH). C
2
NMR:
δ 11.3, 12.5 (2CH ); 24.4, 25.9 (2CH ); 32.2 (C-5); 41.1 (CH); 48.0 (C-4); 52.5 (C-6); 57.1 (C-
3 2
3
a); 78.1 (CPh ); 87.5 (C-6a); 127.4-140.1 (Ph); 162.1 (C-3); 171.8 (O-C=O); 178.2 (HO-C=O). Anal.
2
+
Calcd for C H NO : C, 71.70; H, 6.71; N, 3.22. Found: C, 71.58; H 6.66; N, 3.15. MS-ESI (m/z): 458
2
6
29
5
+
[
M+Na] .
Acknowledgements
The authors thank Dr. Federica Gurini for her helpful technical assistance, and MIUR – PRIN 2010
project No. 20109Z2XRJ) for its financial support.
(
References
1
5

ACCEPTED MANUSCRIPT
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1
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1
6. For examples relating to the Curtius rearrangement of cyclopentanecarboxylic acids see: (a)
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2
010, 21, 519. (e) Boeckman Jr., R. K.; Miller, Y.; Savage, D.; Summerton, J. E. Tetrahedron Lett.
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2
1
7. (a) Radlick, P.; Brown, L. R. Synthesis 1974, 290. (b) Ben Cheikh, A.; Craine, L. E.; Recher, S. G.;
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Caption Figure 1
Figure 1. Structure of three Neuraminidase inhibitors.
1
8