A novel simplified synthesis of acivicin

Article abstract of DOI:10.1016/j.tetasy.2009.02.010

This report describes an efficient synthesis of the natural isomer of acivicin, which is the only one provided with a noteworthy biological activity. The present procedure allowed the synthesis of (+)-1 in just five steps with a 34% overall yield. Due to

Full text of DOI:10.1016/j.tetasy.2009.02.010

Tetrahedron: Asymmetry 20 (2009) 508–511
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Tetrahedron: Asymmetry
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A novel simplified synthesis of acivicin
*
Andrea Pinto, Paola Conti , Lucia Tamborini, Carlo De Micheli
Dipartimento di Scienze Farmaceutiche‘Pietro Pratesi’, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
This report describes an efficient synthesis of the natural isomer of acivicin, which is the only one pro-
vided with a noteworthy biological activity. The present procedure allowed the synthesis of (+)-1 in just
five steps with a 34% overall yield. Due to the easy separation of the two diastereomers and to the avail-
ability of the starting material at low cost, the present procedure can be scaled-up to gram quantities.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 27 January 2009
Accepted 10 February 2009
Available online 9 March 2009
1. Introduction
namidine ribonucleotide synthetase,11
-GT),12 asparagine and glutamate synthase (GltS).13
Since rapidly proliferating tumor cells have elevated activities
c-glutamyltranspeptidase
(c
Acivicin
[(a
S,5S)-a-amino-3-chloro-4,5-dihydroisoxazol-5-yl
acetic acid] (+)-1, a heterocyclic analogue of
L
-glutamine 2 (Fig. 1),
of the glutamine amidotransferases, due to their involvement in
the de novo biosynthesis of purine and pyrimidine nucleotides,
the antitumor activity of acivicin is believed to be a consequence
of the inhibition of the above-mentioned enzymes.
All the glutamine amidotransferases possess, in the pocket of
the catalytic site, an essential cysteine residue that attacks the
was originally isolated from the fermentation broths of Streptomyces
sviceus.1,2 It is provided with potent anticancer activity, and a num-
ber of experimental evidences suggested its potential use in the
treatment of tumors such as leukemia, melanoma, and a variety of
carcinomas.3,4 Unfortunately, during clinical trials the administra-
tion of acivicin was associated with the appearance of many severe
gastrointestinal and neurological side effects.5–8 The neurotoxic side
effects of acivicin were dose related and spanned from lethargy,
headaches and confusion to hallucinations, amnesia, and transient
unconsciousness. Since these neurological side effects did not corre-
late with peak levels of acivicin, it was suggested that the drug is
metabolized into a neurotransmitter antagonist.3
amide bond of glutamine to form a covalent c-glutamyl thioester
intermediate.14,15 Acivicin contains a 4,5-dihydroisoxazole ring
bearing a chloro substituent at position 3 which could be displaced
by a nucleophilic attack of the thiolate anion of such a cysteine res-
idue. The result is the formation of a covalent thioether bond with
the dihydroisoxazole ring of acivicin and the irreversible inactiva-
tion of the enzyme. This mechanism of inhibition has been eluci-
dated through X-ray diffraction analysis of the complex of
acivicin with the carbamoyl phosphate synthetase from Escherichia
coli.16 A recent report on the crystal structure of the complex
Cl
H2N
between
c-glutamyltranspeptidase and acivicin shows that the
CO2H
NH2
CO2H
NH2
N
O
adduct was not derived simply by the nucleophilic displacement
of the 3-chloro substituent of acivicin, but involved the opening
of its 4,5-dihydroisoxazole nucleus followed by a closure with a
shift of the double bond to positions 4 and 5.17
O
2
(+)-1
In the past, we had prepared a series of conformationally
constrained analogues of acivicin in order to study its structure–
activity relationship.18 We evaluated the anticancer activity of
new compounds in vitro against a panel of human tumor cell lines;
we also measured their ability to inhibit glutamate synthase (GltS)
from Azospirillum brasilense.18
With the aim of extending our investigation to different targets,
for example, the capacity to inhibit trypanosome CTP-synthetase, a
target for the treatment of African sleeping sickness,19 we needed
substantial amounts of acivicin to be used as the reference com-
pound. Although acivicin is available through fermentation,
large-scale production has been hampered to some extent by the
occurrence of contaminants from which it is not easily separable.
Figure 1. Model compounds.
Despite its failure as a drug candidate, acivicin has undergone
extensive biochemical and pharmacological investigations to find
out its mode of action as an antitumor agent.3 It has been shown
that acivicin behaves as a glutamine antimetabolite9 that irrevers-
ibly inactivates several L-glutamine amidotransferases, including
cytosine triphosphate synthetase (CTP-synthetase),10 formylglyci-
* Corresponding author. Tel.: +39 02 50319329; fax: +39 02 50319326.
E-mail address: paola.conti@unimi.it (P. Conti).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.02.010

A. Pinto et al. / Tetrahedron: Asymmetry 20 (2009) 508–511
509
For such a reason, there has been interest in the total synthesis of
acivicin with results ranging from stereorandom to stereoselective
formation of the (
S,5S) natural diastereomer.20–25
Br
Cl
a
a
N
N
OH
O
O
O
Herein we report the total synthesis of (+)-1 through the use of the
bromonitrileN-oxidecycloaddition todipolarophile(S)-3. We accom-
plished the exchange of bromide with chloride at the 3 position of the
86 %
N
NH2
Boc
4
6
(+)-
Br
(+)-
D
2-isoxazoline nucleus just by treating the appropriate intermediate
with a THF solution of hydrochloric acid. The exchange is quantitative
and takes place at room temperature in a few minutes.
Br
b
N
N
2. Results and discussion
OH
NHBoc
OH
O
O
NH2
As already described in a previous paper,26 the 1,3-dipolar
cycloaddition of bromonitrile oxide to (S)-3-(tert-butoxycarbonyl)-
2,2-dimethyl-4-vinyloxazolidine27 [(S)-3; Scheme 1] yielded the
two pairs of diastereomeric cycloadducts erythro-(+)-4/threo-(ꢀ)-5
in a 65:35 ratio and in a 88% overall yield. The two isomers were eas-
ily separated by silica gel column chromatography.
9
( )-
( )-8
Cl
d-f
c
(+)-6
(+)-1
N
85%
OH
NHBoc
82%
O
(+)-7
Br
Scheme 2. Reagents and conditions: (a) 10 M HCl/THF; (b) CF3COOH/CH2Cl2; (c)
Boc2O, TEA/CH2Cl2; (d) PDC/DMF; (e) 4 N HCl/dioxane; (f) Amberlite IR-120 plus,
0.5 N NH4OH (aq).
O
N
N
O
O
O
N
N
Boc
Boc
a
3
(S)-
noteworthy biological activity. The present procedure allowed
the synthesis of (+)-1 in just five steps with a 34% overall yield.
The limiting step is the 1,3-dipolar cycloaddition since the desired
isomer (+)-4 is obtained in high yield, but as a 65:35 mixture with
its diastereomer (–)-5. Nevertheless, due to the easy separation of
the two diastereomers and to the availability of the starting
material at low cost, the present procedure can be scaled-up to
gram quantities, and represents a significant improvement over
the previously reported synthetic strategies.
(+)-4
88%
+
Br
Br
Br
N
OH
O
N
Boc
(−)-5
Scheme 1. Reagents: (a) NaHCO3/AcOEt.
4. Experimental
The erythro cycloadduct (+)-4 wasdissolved ina 10 MTHF solution
ofHClandwasleftstirringfor1 hatroomtemperature.Aftertheusual
work-up, amino alcohol (+)-6 was recovered in 86% yield (Scheme 2).
In order to exclude the presence of trace amounts of the correspond-
ing 3-bromo-amino alcohol, we carried out an accurate analytical
investigation based on a HRGC–MS analysis. For such a purpose, we
prepared a pure sample of compound ( )-9 by treating intermediate
( )-828 with a dichloromethane solution of trifluoroacetic acid. As de-
tailed in Section 4, derivatives (+)-6 and ( )-9 possess different reten-
tion times, and consequently, we excluded the presence of the bromo
derivative ( )-9 in the reaction mixture of (+)-6. Furthermore, we
found that the exchange is rapid and complete in 5 min.
4.1. Material and methods
All reagents were purchased from Sigma. Dibromoformaldoxime
was prepared according to a literature procedure.23 Dipolarophile
(S)-3-(tert-butoxycarbonyl)-2,2-dimethyl-4-vinyloxazolidine27 (S)-
3 was prepared from (R)-Garner’s aldehyde following the procedure
reported in the literature.
1H NMR and 13C NMR spectra were recorded with a Varian
Mercury 300 (300 MHz) spectrometer. Chemical shifts (d) are
expressed in ppm, and coupling constants (J) are expressed in Hertz.
Rotary power determinations were carried out with a Jasco J-810
spectropolarimeter coupled with a Haake N3-B thermostat. TLC
analyseswereperformedon commercialSilicaGel 60F254 aluminum
sheets; spots were further evidenced by spraying with a dilute alka-
line potassium permanganate solution or with ninhydrin. Melting
points were determined on a model B 540 Büchi apparatus, and
are uncorrected. The HRGC-FID analyses were performed using an
Agilent Technologies (Palo Alto, CA) model HP 6890 series equipped
with a split–splitless injector, electronic pressure control, HP 6890
autosampler, and flame ionization detector (FID); the column used
Finally, the primary amino group of (+)-6 was temporarily pro-
tected as a tert-butyl carbamate to give intermediate (+)-7, which
was fully characterized. The primary alcohol was then oxidized
to the corresponding carboxylic acid by treatment with pyridinium
dichromate followed by removal of the amino protective group by
treatment with a 4 N dioxane solution of HCl. After purification by
cation exchange chromatography, acivicin (+)-1 was obtained in its
zwitterionic form. Crystallization from aqueous methanol afforded
white crystals of (+)-1, whose chiroptical properties matched those
of the material obtained from different synthetic procedures, for
was a J&W Scientific DB-5 (15 m ꢂ 0.32 mm i.d., 0.25
lm); nitrogen
example, ½a 2D0
ꢁ
¼ þ157:6 (c 0.13, H2O) versus ½a D20
¼ þ139 (c 0.14,
ꢁ
was used as the carrier at a flow rate of 1.3 mL/min; the injector and
detector temperatures were 280 and 300 °C, respectively; the col-
umn temperature was programmed from an initial 100 °C to
300 °C at 10 °C/min; the total analysis time was 23 min. Direct infu-
sion mass analyses were performed using a Varian 320-MS Triple
Quadrupole. Microanalyses (C, H, N) of new compounds agreed with
the theoretical value within 0.4%.
H2O)25 or ½a 2D0
ꢁ
¼ þ148 (c 0.845, H2O).21
3. Conclusion
In summary, this report describes an efficient synthesis of the
natural isomer of acivicin, which is the only one provided with a

510
A. Pinto et al. / Tetrahedron: Asymmetry 20 (2009) 508–511
4.1.1. Synthesis of (2R,5S)-2-amino-2-(3-chloro-4,5-dihydro-
isoxazol-5-yl)-ethanol (+)-6
4.1.4. Synthesis of (aS,5S)-a-amino-3-chloro-4,5-dihydro
isoxazol-5-yl acetic acid (+)-1
4-(3-Bromo-4,5-dihydro-isoxazol-5-yl)-2,2-dimethyl-oxazoli-
dine-3-carboxylic acid tert-butyl ester (+)-4 (7.00 g, 20.0 mmol)
was dissolved in a 10 N THF solution of HCl (50 mL). The reaction
mixture was stirred for 1 h. The reaction was monitored by
HRGC-FID analysis to exclude the presence of the corresponding
bromo-amino alcohol 9. The solvent was evaporated, water
(50 mL) was added, and the aqueous phase was extracted with
AcOEt (2 ꢂ 100 mL). The aqueous layer was made basic with
0.5 N NaOH and was newly extracted with AcOEt (4 ꢂ 100 mL).
The organic extracts were pooled and dried over anhydrous
sodium sulfate. The solvent was removed under vacuum, and the
crude material was purified by column chromatography (eluent:
dichloromethane/methanol 8:2), obtaining 2.81 g of compound
(+)-6 (17.1 mmol, 86% yield).
(a) To a solution of compound (+)-7 (3.84 g, 14.5 mmol) in DMF
(80 mL) pyridinium dichromate (85 g, 225 mmol) was added, and
the mixture was stirred at room temperature for 6 h. The progress
of the reaction was monitored by TLC (CH2Cl2/MeOH 95:5 + 1%
acetic acid). Water was added (150 mL), and the mixture was
extracted with AcOEt (3 ꢂ 150 mL). The organic layer was
extracted with 1 M NaOH (3 ꢂ 50 mL), and the aqueous phase
was made acidic with 2 M HCl and was extracted with AcOEt
(3 ꢂ 50 mL). The organic extracts were washed with brine, dried
over anhydrous Na2SO4, and the solvent was evaporated to give
3.62 g of the crude carboxylic acid.
(b) The crude material obtained from the previous step (3.62 g,
13.0 mmol) was treated with a 4 M HCl/dioxane solution. The reac-
tion mixture was stirred at room temperature until disappearance
of the starting material (1 h). The volatiles were removed under
vacuum, and the residue was dissolved in water and submitted
to cation exchange chromatography using Amberlite IR-120 plus.
The acidic solution was slowly eluted onto the resin, and then
the column was washed with water until the pH was neutral.
The compound was eluted off the resin with 0.5 N aqueous ammo-
nia, and the product-containing fractions (detected with ninhydrin
stain on a TLC plate) were combined and the solvent was freeze-
dried to give 2.13 g (11.9 mmol, 82% yield after two steps) of (+)-
1 as a white solid.
(+)-6: pale yellow oil; Rf (dichloromethane/methanol 8:2) 0.5;
½
a 2D0
ꢁ
¼ þ95:7 (c 1.00, CHCl3); 1H NMR (CDCl3): 2.07 (br s, 3H);
3.13 (dd, J = 10.7, 17.6, 1H); 3.15 (ddd, 4.4, 5.5, 5.8, 1H); 3.25 (dd,
J = 9.0, 17.6, 1H); 3.54 (dd, J = 5.8, 11.0, 1H); 3.66 (dd, J = 4.4,
11.0, 1H); 4.73 (ddd, J = 5.5, 9.0, 10.7, 1H); 13C NMR (CDCl3):
39.9; 54.4; 63.4; 84.1; 150.0; HRGC retention time: 3.7 min;
[M+H]+ = 165.1. Anal. Calcd for C5H9ClN2O2 (164.59): C, 36.49; H,
5.51; N, 17.02. Found: C, 36.70; H, 5.69; N, 16.69.
*
*
4.1.2. Synthesis of (2R ,5S )-2-amino-2-(3-bromo-4,5-dihydro-
isoxazol-5-yl)-ethanol ( )-9
Recrystallization from aqueous methanol gave colorless prisms
Intermediate ( )-8 (100 mg, 0.32 mmol) was treated with a 30%
of (+)-1, mp dec >180 °C, Rf: 0.55 (MeOH–H2O–pyridine: 20:5:1);
dichloromethane solution of trifluoroacetic acid (250
l
L) at 0 °C.
½
½
a 2D0
a 2D0
ꢁ
¼ þ157:6 (c 0.13, H2O) {lit. ½a D20
ꢁ
¼ þ139 (c 0.14, H2O)25 or
The reaction mixture was stirred at room temperature until disap-
pearance of the starting material (4 h). The volatiles were removed
under vacuum. Water (5 mL) was added, and the aqueous phase
was extracted with AcOEt (2 ꢂ 5 mL). The aqueous layer was made
basic with 0.5 N NaOH and was newly extracted with AcOEt
(4 ꢂ 10 mL). The organic extracts were pooled and dried over
anhydrous sodium sulfate. The solvent was removed under vac-
uum and purified by column chromatography (eluent: dichloro-
methane/methanol 8:2), obtaining 58 mg of compound ( )-9
(0.28 mmol, 85% yield).
( )-9: pale yellow oil; Rf (dichloromethane/methanol 8:2) 0.5;
1H NMR (CDCl3): 1.65 (br s, 3H); 3.12–3.24 (m, 2H); 3.30 (dd,
8.8, 17.3, 1H); 3.56 (dd, J = 5.8, 11.0, 1H); 3.68 (dd, J = 4.4, 11.0,
1H); 4.67 (ddd, J = 5.2, 8.8, 10.4, 1H); 13C NMR (CDCl3): 42.9;
54.3; 63.8; 83.6; 138.3; HRGC retention time: 4.6 min;
[M+H]+ = 208.9. Anal. Calcd for C5H9BrN2O2 (209.04): C, 28.73; H,
4.34; N, 13.40. Found: C, 29.01; H, 5.81; N, 16.40.
ꢁ
¼ þ148 (c 0.845, H2O)21}; 1H NMR (D2O): 3.34 (dd, J = 8.3,
18.0, 1H); 3.43 (dd, J = 11.0, 18.0, 1H); 3.94 (d, J = 3.3, 1H), 5.18
(ddd, J = 3.3, 8.3, 11.0, 1H); 13C NMR (D2O): 39.8; 56.1; 80.5; 152.4;
170.1; [MꢀH]ꢀ = 176.5. Anal. Calcd for C5H7ClN2O3 (178.57): C,
33.63; H, 3.95; N, 15.69. Found: C, 33.42; H, 4.00; N, 15.59.
Acknowledgments
We are grateful to MIUR (PRIN 2007, Rome) and Università degli
Studi di Milano (PUR) for their financial support. The technical
assistance of Mr. S. Arnoldi and Mr. G. L. Visconti is gratefully
acknowledged.
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yl)-2-hydroxy-ethyl]-carbamic acid tert-butyl ester (+)-7
To a stirred solution of compound (+)-6 (2.81 g, 17.1 mmol) in
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