Synthesis and biological evaluation of new amino acids structurally related to the antitumor agent acivicin

Article abstract of DOI:10.1016/S0014-827X(03)00107-1

A set of racemic conformationally constrained analogues of the antitumor antibiotic acivicin (+)-1 has been prepared through a strategy based on 1,3-dipolar cycloaddition of bromonitrile oxide to suitable dipolarophiles. The bromo analogue (2) of acivicin was also synthesized and tested as a reference compound, together with its stereoisomer 3. The antitumor properties of novel amino acids 4-7 were evaluated in vitro against human tumor cell lines. Their efficacy to inhibit glutamate synthase (GltS) from Azospirillum brasilense was also assayed. None of the studied compounds, but 2, showed significant activity.

Full text of DOI:10.1016/S0014-827X(03)00107-1

Il Farmaco 58 (2003) 683Á690
/
www.elsevier.com/locate/farmac
Synthesis and biological evaluation of new amino acids structurally
related to the antitumor agent acivicin
a,
Paola Conti *, Gabriella Roda , Helena Stabile , Maria Antonietta Vanoni ,
a
b
c
b
Bruno Curti , Marco De Amici *
a,
a
Istituto di Chimica Farmaceutica, Universit a` di Milano, viale Abruzzi 42, Milano 20131, Italy
b
Dipartimento di Fisiologia e Biochimica Generali, Universit a` di Milano, Via Celoria 26, Milano 20131, Italy
Dipartimento di Scienze Chimiche, Fisiche e Matematiche, Universit a` dell’Insubria, Via Valleggio 11, Como 22100, Italy
Received 10 October 2002; accepted 31 December 2002
c
Abstract
A set of racemic conformationally constrained analogues of the antitumor antibiotic acivicin (ꢀ
strategy based on 1,3-dipolar cycloaddition of bromonitrile oxide to suitable dipolarophiles. The bromo analogue (2) of acivicin was
/)-1 has been prepared through a
also synthesized and tested as a reference compound, together with its stereoisomer 3. The antitumor properties of novel amino acids
4
brasilense was also assayed. None of the studied compounds, but 2, showed significant activity.
Á
/
7 were evaluated in vitro against human tumor cell lines. Their efficacy to inhibit glutamate synthase (GltS) from Azospirillum
´
2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
#
Keywords: Amino acids; Antitumor agent; Acivicin
1
. Introduction
number of severe side effects associated with the use of
acivicin emerged during clinical trials, e.g. myelotoxicity
and damages of the central nervous system [3]. There-
fore, acivicin represents an excellent lead for the design
of novel structurally related derivatives with an im-
proved selectivity and a reduced toxicity. We started an
Acivicin [(aS,5S)-a-amino-3-chloro-4,5-dihydro-iso-
xazol-5-yl acetic acid, AT-125] (ꢀ)-1 (Fig. 1) is an
/
antitumor agent known to inhibit cell growth [1].
Acivicin was first isolated as a fermentation product of
Streptomyces sviceus [1], and it was shown to behave as
a glutamine antimetabolite [2], able to inhibit a number
investigation on the structureÁactivity relationship of
/
acivicin through the design and synthesis of a set of
conformationally constrained analogues of acivicin.
The novel derivatives are characterized by the pre-
of
sine triphosphate synthetase (CTP-synthetase), xantho-
sine monophosphate aminase (XMP-aminase),
L-glutamine-dependent amidotransferases, e.g. cyto-
2
sence in their structure of the 3-bromo-D -isoxazoline
moiety, at variance with acivicin, which contains the 3-
carbamoyl-phosphate synthetase II [3], anthranilate
synthase, asparagine synthetase and glutamate synthase
2
chloro-D -isoxazoline portion. Previous investigations
[
8] demonstrated that such a structural modification
(
GltS) [4]. Moreover, acivicin behaves as a potent g-
does not significantly affect the pharmacological profile
of the compounds. As a matter of fact, when assayed in
vivo for antitumor activity in the L1210 murine
glutamyl transpeptidase (g-GT) inhibitor [5].
A number of experimental evidences suggested that
acivicin could be potentially useful in the treatment of
certain tumors, i.e. myeloid leukemia [6,7]. However, a
leukemia test at a dose of 8 mg/kg (9)-1 produced a
/
T/C (%) equal to 185 (T being the median survival time
of treated animals and C being the median survival time
of control animals) versus a value of 169 displayed by its
*
Corresponding authors.
E-mail address: paola.conti@unimi.it (P. Conti).
bromo analogue (9)-2 [8]. Accordingly, due to its easier
/
´
0
014-827X/03/$ - see front matter # 2003 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00107-1

684
P. Conti et al. / Il Farmaco 58 (2003) 683Á690
/
Fig. 1. Chemical structure of model and target compounds.
and safer synthetic access [8Á
and used as the reference compound.
This paper deals with the synthesis of bicyclic
derivatives (9)-4Á(9)-7 (Fig. 1), which represent dif-
ferent locked conformations of (9)-2. The antitumor
activity of (9)-2, its stereoisomer (9)-3 [8Á10] and
novel derivatives (9)-4Á(9)-7 was evaluated at the
/
10], (9
/
)-2 was prepared
/
/
/
/
/
/
/
/
/
/
National Cancer Institute (Bethesda, MD) in a panel of
in vitro disease-oriented primary antitumor tests.
Furthermore, the ability of the newly synthesized
compounds to inhibit Azospirillum brasilense GltS, a
well-characterized enzyme belonging to L-glutamine-
dependent amidotransferase class [11,12] was deter-
mined.
Scheme 2.
fractional crystallization. Intermediates (9
/
)-8 and (9)-9
/
were heated at reflux with 4 N HCl followed by ion
exchange chromatography to yield final amino acids
2
. Chemistry
(
(
9
/
)-4 and (9
Following a similar approach, amino acids (9
)-7 (Fig. 1) were prepared through 1,3-dipolar
/
)-5 in a pure form.
2
/)-6 and
The synthesis of 3-bromo-D -isoxazolinylprolines
)-4 and (9)-5 was accomplished according to the
9
/
(
9
/
/
cycloaddition of bromonitrile oxide to cyclopentene
derivatives (9)-14 or (9)-15 (Scheme 2). Dipolarophiles
)-14 and (9)-15 were synthesized according to the
reaction sequence illustrated in Scheme 1. As previously
reported [13], 1,3-dipolar cycloaddition of bromonitrile
oxide to racemic N-BOC-3,4-didehydroproline methyl
ester yielded three out of four possible stereoisomers in
similar amounts. The separation of the cycloadducts was
rather laborious, since a column chromatography of the
reaction mixture gave two fractions containing pure
/
/
(
9
/
/
reaction sequence reported in Scheme 2. Based on a
procedure previously reported in the literature [14],
commercially available 2-oxo-cyclopentane-carboxylic
acid ethyl ester (9)-11 was transformed into intermedi-
/
(
9
/
)-8 and a mixture of (9
/
)-9 and (9
/
)-10. Fortunately,
ate diester 12. The keto derivative 12 was reduced to the
related secondary alcohol with sodium borohydride and
then transformed into the corresponding methanesulfo-
nate, which was subsequently reacted with 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) at reflux in toluene to
yield alkene 13. This intermediate was treated with 1
equiv. of sodium hydroxide to afford the corresponding
monoacid, which was then submitted to the Curtius
rearrangement. The emerging primary amino group was
reacted with di-tert-butyl dicarbonate, under a standard
cycloadduct (9)-9 could be separated from (9
/
/)-10 by
procedure, to give N-BOC derivative (9
transformation of alkene (9)-14 into (9)-15 needed
harsher conditions, since it was necessary to reflux a
THF solution of (9)-14 and excess di-tert-butyl dicar-
/)-14. The
/
/
/
bonate in the presence of a catalytic amount of 4-
Scheme 1.
(dimethylamino)pyridine (DMAP).

P. Conti et al. / Il Farmaco 58 (2003) 683Á
/690
685
For analytical purposes, amino esters (9
8d were separately reconverted under standard condi-
tions (see Section 4) into the corresponding mono-BOC
derivatives (9)-16aÁ(9)-16d and di-BOC derivatives
)-17aÁ(9)-17d. The relative percentages of stereo-
isomers (9)-16aÁ(9)-16d and (9)-17aÁ(9)-17d, re-
/
)-18aÁ
/
(9
/
)-
1
/
/
/
(
9
/
/
/
/
/
/
/
/
/
Fig. 2. Representation of the transition state leading to (9)-16b.
/
ported in Scheme 2, were obtained by HPLC analysis
of the two crude reaction mixtures.
The structure to the cycloadducts was assigned on the
It is worth pointing out that the pericyclic reaction
produces all four possible stereoisomers 16a/16b/16c/16d
in a relative ratio of 7:50:15:28. By far, the major
1
basis of the H NMR spectrum of the corresponding
amines (9
/
)-18aÁ
/
(9)-18d. The multiplicity of H-5, the
/
stereoisomer of the reaction mixture is (9
already observed in similar instances [15Á
transition state leading to (9)-16b is stabilized by a
/)-16b. As
most deshielded proton, is diagnostic for the assignment
of the regiochemistry since it resonates as a doublet in
/17], the
/
cycloadducts (9
/
)-18a and (9
/
)-18b, and as a multiplet in
)-18d. The structure to the
hydrogen bond between the NHBOC group of the
dipolarophile and the negatively charged oxygen of the
nitrile oxide, as depicted in Fig. 2.
cycloadducts (9
/
)-18c and (9
/
couples of stereoisomers 18a/18b and 18c/18d was
assigned by taking into account the upfield shift of
proton H-4 and H-5, respectively, observed in com-
In order to reverse the stereopreference observed with
dipolarophile (9
,3-dipolar cycloaddition of bromonitrile oxide to
alkene (9)-15. In this case, we caused both an increase
in the bulkiness of the carbamate group and the removal
of the hydrogen bond interaction. The pericyclic reac-
tion gave a mixture of four cycloadducts 17a/17b/17c/
/
)-14, we carried out the corresponding
pounds (9
due to the influence of the spatially close amino group.
As a matter of fact, H-4 of (9)-18c resonates at 3.42
ppm versus 4.03 ppm observed in derivative (9)-18d.
The same considerations hold true for H-5 in derivatives
)-18a and (9)-18b (4.73 ppm versus 5.08 ppm).
/
)-18a and (9
/
)-18c. Such a shielding effect is
1
/
/
/
(
9
/
/
1
7d in a relative ratio of 46:26:17:11 (Scheme 2). As
expected, (9)-17b (26%) is not any longer the major
stereoisomer of the reaction mixture, but is overcome by
)-17a (46%).
In both cases, the separation of four cycloadducts
could only be performed after their conversion into the
corresponding primary amines (9)-18aÁ(9)-18d. As
shown in Scheme 3, the treatment of the crude reaction
mixtures (9)-16aÁ(9)-16d and (9)-17aÁ(9)-17d with
a dichloromethane solution of trifluoroacetic acid
afforded a mixture of amino esters (9)-18aÁ(9)-18d
in quantitative yield, which were separated by column
chromatography. Intermediates (9)-18a and (9)-18b
were converted into final zwitterionic amino acids (9)-6
and (9)-7 by hydrolysis with 1 N NaOH, followed by
/
3. Results and discussion
(
9
/
Amino acids (9
/
)-2, (9
/
)-3, (9
/
)-4, (9
/
)-5, (9)-6 and
/
(
Developmental Therapeutics Program, which consists in
9
/
)-7 were submitted to the National Cancer Institute
/
/
/
an in vitro disease-oriented primary antitumor screening
18,19]. This investigation utilizes 60 different human
/
/
/
/
/
/
[
tumor cell lines, representative of leukemia, melanoma
and cancers of the lung, colon, brain, ovary, breast,
prostate and kidney. In agreement with previously
/
/
/
/
/
reported data, our reference compound (9)-2 showed
/
/
cell growth inhibition. According to the protocol, the
activity is expressed using three parameters: GI , TGI
/
50
ion exchange chromatography.
and LC , which indicate the log of the molar
10
5
0
concentration that produces, respectively, 50% cell
growth inhibition, total cell growth inhibition and 50%
cell death. The average values displayed by compound
(
9
/
)-2, referred to all cell lines, are GI ꢁ
/
ꢂ
/
4.60,
4.00. These values are
comparable with those reported for acivicin (GI ꢁ
5
0
TGIꢁ
/
ꢂ
/
4.01 and LC ꢁ
/
ꢂ
/
5
0
/
ꢂ
/
5
0
4
.74, TGIꢁ
nately, none of the novel compounds (9
showed comparable levels of cell growth inhibition,
since they exhibited values of GI , TGI and LC ]
/
ꢂ
/
4.00 and LC ꢁ
/
ꢂ
/
4.00) [20]. Unfortu-
5
0
/
)-3Á(9)-7
/
/
/
ꢂ
/
5
0
50
4.00.
In parallel, the set of new compounds has been tested
in a relatively simple assay, based on the inhibition and
inactivation of A. brasilense GltS. It has been reported
that acivicin behaves as an inhibitor of several amido-
transferases, by competing with glutamine for the same
Scheme 3.

686
P. Conti et al. / Il Farmaco 58 (2003) 683Á690
/
binding site [4]. Incubation of these enzymes with
acivicin leads to a time-dependent loss of activity, due
to the formation of a covalent addition product with the
catalytically essential cysteine residue located in the
glutamine amidotransferase (GAT) site of these enzymes
4. Experimental
4.1. Material and methods
Dibromoformaldoxime [8], compounds (9
/
)-2, (9)-3
/
[
12]. Acivicin was also shown to act as an irreversible
inhibitor of the Escherichia coli GltS (Kinactꢁ0.57 mM;
0.19 min ) [4], and to compete with -gluta-
mine (K ꢁ0.56 mM) for the same binding site [4]. Based
[8Á10], (9)-8Á(9)-10 [13] and 12 [14] were prepared
according to literature procedures. H NMR spectra
were recorded with a 300 MHz Varian spectrometer in
/
/
/
/
1
/
ꢂ1
kinact
ꢁ
/
L
CDCl or D O at 20 8C; the signal assignments are the
/
3
2
i
results of a combination of 1D and 2D COSY. Chemical
shifts (d) are expressed in ppm and coupling constants
(J) in hertz. HPLC analyses were performed on a
LiChrospher Si 60 Merck column, using a Jasco PU-
on the sequence homology among GltSs [11] as well as
the structural similarity between GAT domain of GltSs
and that of other Type-II amidotransferases [11,12,21],
we tested the activity of acivicin bioisostere (9
inhibitor/inactivator of the well-characterized A. brasi-
lense GltS. Compound (9)-2 behaved as an inhibitor of
the enzyme, competitive with -glutamine, with a K ꢁ
.9490.44 mM. This value is similar to that obtained
for acivicin with E. coli GltS [4], if we take into account
that compound (9)-2 was tested as a racemate, whereas
the value reported for acivicin refers to the eutomer (ꢀ)-
only. Furthermore, compound (9)-2 was able to
/)-2 as an
9
9
80 pump equipped with a UVÁ
/
Vis detector Jasco UV-
75. TLC was performed on commercial silica gel 60
/
F254 aluminum sheets; spots were further evidenced by
spraying with dilute alkaline potassium permanganate
solution or with ninhydrin. Melting points were deter-
mined with a capillary method on a B u¨ chi B 540
apparatus and are uncorrected. Liquid compounds
were characterized by the oven temperature for Kugel-
rohr distillations. Microanalyses of new compounds
L
/
i
3
/
/
/
1
/
inactivate A. brasilense GltS with an observed rate of
ꢂ1
agreed with theoretical values of 90.3%.
/
approximately 0.02 min when the enzyme (3 mM) was
incubated with compound (9
-glutamine analogue
binds to GltS with high affinity (K :
/
)-2 (1 mM) at 25 8C. The
-methionine sulfone, which
2.5 mM) [22], fully
protects the enzyme, demonstrating that compound
)-2 inactivates the enzyme through its interaction
with the glutamine binding site.
None of the newly synthesized compounds [(9
)-7] exhibited any inhibitory effect of GltS catalytic
4
.1.1. Synthesis of amino acids (9
/
)-4 and (9
/
)-5
L
L
Compound (9)-8 [13] (300 mg, 0.86 mmol) was
/
/
i
refluxed with 4 N HCl (9 ml) for 3 h. After evaporation
of the solvent, 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 10% aqueous pyridine, and the
product-containing fractions (detected with ninhydrin
stain on a TLC plate) were combined and concentrated
(
9
/
/
)-3Á
/
(
9
/
activity nor inactivated the enzyme upon incubation for
several hours at 25 8C. These results match those
obtained in the in vitro antitumor tests. As a conse-
quence, the evaluation of inhibition or inactivation of A.
brasilense GltS could represent a convenient model for a
pre-screening of new molecules designed as acivicin
analogues. Furthermore, since the three-dimensional
structures of several GATs have been solved [12],
including those of A. brasilense [21] and Synechocystis
GltSs [23], the rational design of novel acivicin analo-
gues is now feasible.
under vacuum to give (9
/
)-4 (129 mg, 0.55 mmol) in 64%
yield.
(
9
/
)-(3aS,6R,6aS)-3-Bromo-4,5,6,6a-tetrahydro-
3
aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid ((9)-4);
/
m.p. (dec.)ꢀ
/
175 8C; colorless prisms from H O/EtOH;
2
1
H NMR (D O): 3.57 (dd, 1, Jꢁ
/8.1, 12.9 Hz), 3.83 (d,
2
1
, Jꢁ
/
12.9 Hz), 4.46 (dd, 1, Jꢁ
/
8.1, 9.3 Hz), 4.48 (d, 1,
Jꢁ
/
5.3 Hz), 5.69 (dd, 1, Jꢁ5.3, 9.3). Anal.
/
The results of the present investigation evidence that
the conformation of the side chain of acivicin is a critical
requirement for its binding to the target enzymes.
Indeed, the modification of the torsional angles between
the a-amino acid group and the isoxazoline ring brings
about the loss of any biological activity. Thus, in the
design of new antitumor agents acting as acivicin
analogues, the only ‘‘allowed’’ structural modifications
appear to be those that do not alter the conformational
requirements of the parent compound. On the basis of
these results, we have now designed a set of novel amino
acids, in which the nature of the heterocyclic ring of
(
C H BrN O ) C, H, N.
6
7
2
3
Compound (9)-9 [13] (400 mg, 1.15 mmol) was
/
refluxed with 4 N HCl (12 ml) for 3 h. After evaporation
of the solvent, the residue was dissolved in water. Ion
exchange chromatography (Amberlite IR-120 plus ca-
tion exchange: eluted with 10% pyridine in water)
afforded (9
(9)-(3aS,6S,6aS)-3-Bromo-4,5,6,6a-tetrahydro-
3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid ((9
/
)-5 (190 mg, 0.81 mmol) in 70% yield.
/
/
)-5);
m.p. (dec.)ꢀ
/
172 8C; colorless prisms from H O/MeOH;
2
1
H NMR (D O): 3.68 (dd, 1, Jꢁ
/
7.9, 12.9 Hz), 3.84 (d,
7.9, 9.3 Hz), 4.51 (s, 1),
2
1, Jꢁ
5.62 (d, 1, Jꢁ9.3 Hz). Anal. (C H BrN O ) C, H, N.
6 7 2 3
/
12.9 Hz), 4.48 (dd, 1, Jꢁ
/
model compound (9)-2 has been modified.
/
/

P. Conti et al. / Il Farmaco 58 (2003) 683Á
/690
687
4.1.2. Synthesis of diethylcyclopent-2-ene-1,1-
dicarboxylate (13)
To a stirred solution of 12 [14] (30.7 g, 134.7 mmol) in
EtOH (300 ml), cooled at 0 8C, sodium borohydride (2.5
g, 67.3 mmol) was added portionwise. After stirring for
oformate (3.44 ml, 36.1 mmol). The mixture was stirred
at r.t. for 30 min, then cooled at 0 8C. A solution of
sodium azide (2.63 g, 40.4 mmol) in water (5 ml) was
slowly added and the mixture was stirred for 1 h at 0 8C.
The progress of the reaction was monitored by TLC
3
0 min at 0 8C, the reaction was quenched by a dropwise
(eluant: petroleum etherÁ
evaporation of the acetone at reduced pressure, the
aqueous phase was extracted with Et O (2ꢃ10 ml) and
the pooled organic extracts were dried and concentrated.
The residue, containing acyl-azide, was dissolved in
benzene (100 ml) and refluxed for 3 h to give the
corresponding isocyanate. The progress of the reaction
/
ethyl acetate, 4:1). After
addition of 1 N HCl. After evaporation at reduced
pressure of the volatiles, the residue was dissolved in
water and extracted with dichloromethane. The organic
extracts were dried over anhydrous sodium sulfate and
concentrated. The crude material was submitted to
column chromatography on silica gel (eluant: petroleum
/
2
etherÁ/ethyl acetate, 4:1) to give the corresponding
was monitored by TLC (eluant: petroleum etherÁethyl
/
alcohol derivative as a yellow oil (14.54 g; yield: 47%).
The above prepared alcohol (14.54 g, 63.21 mmol)
was reacted with 1.5 equiv. of triethylamine (TEA) (13.1
ml, 94.8 mmol) and 1.5 equiv. of methanesulfonylchlor-
ide (7.4 ml, 94.8 mmol), in dichloromethane, at 0 8C.
After stirring for further 30 min at room temperature
acetate, 9:1). When the conversion was complete, the
solvent was evaporated under vacuum and the residue
was dissolved in THF (100 ml) and reacted with 2 N
HCl (100 ml) overnight. THF was evaporated at
reduced pressure, the aqueous phase was extracted
with dichloromethane (2ꢃ
/
30 ml), then made alkaline
(r.t.), 1 N HCl (6 ml) was added to the reaction mixture.
The organic layer was separated and washed with an
with solid K CO . The product was extracted with ethyl
2
3
acetate (5ꢃ
/
30 ml). The combined organic extracts were
aqueous solution of NaHCO . After the usual work up,
3
dried over anhydrous Na SO and concentrated under
4
2
the crude material was purified by column chromato-
reduced pressure to give 2.05 g (50% yield) of the desired
amino ester as a yellow oil.
graphy on silica gel (eluant: petroleum etherÁethyl
/
acetate, 9:1) to give 11.1 g of mesylate in 57% yield.
A solution of the mesylate obtained in the previous
step (11.1 g, 36.04 mmol) and DBU (27 ml, 180.2 mmol)
in toluene (90 ml) was heated at reflux for 48 h. After
cooling at r.t. the reaction mixture was washed with 2 N
A dichloromethane solution (30 ml) of the amino
ester (2.05 g, 13.2 mmol) and TEA (2.76 ml, 19.8 mmol)
was stirred and cooled at 0 8C. To the mixture, a
solution of di-tert-butyl dicarbonate (4.3 g, 19.8
mmol) in CH Cl (10 ml) was added dropwise and
2
2
HCl (2ꢃ
/
30 ml) and water (1ꢃ
/
30 ml). The organic
stirred at r.t. overnight. After treating with 1 N HCl, the
organic layer was dried and concentrated under vacuum.
The residue was purified by a silica gel column
phase was dried over anhydrous Na SO and concen-
2
4
trated under reduced pressure. The crude material was
purified by column chromatography on silica gel
chromatography (eluant: petroleum etherÁ
9:1) to give alkene (9)-14 as a yellow oil (2.9 g, 86%
yield).
(9)-Ethyl-N-tert-butoxycarbonyl-1-amino-cyclo-
pent-2-ene-1-carboxylate ((9
1.26 (t, 3, Jꢁ7.2 Hz), 1.43 (s, 9), 2.05 (m, 1), 2.55 (m, 2),
2.70 (m, 1), 4.19 (q, 2, Jꢁ7.2 Hz), 5.18 (bs, 1), 5.67 (m,
), 6.07 (m, 1). Anal. (C H NO ) C, H, N.
/ethyl acetate,
(
6
eluant: petroleum etherÁ
.88 g (90% yield) of 13 as a colorless liquid.
Diethylcyclopent-2-ene-1,1-dicarboxylate (13); color-
/
ethyl acetate, 95:5) to give
/
/
1
1
less oil, b.p.: 70 8C/0.35 mbar; H NMR (CDCl ): 1.24
/
)-14); H NMR (CDCl ):
3
3
(
t, 6, Jꢁ
/
6.8 Hz), 2.46 (m, 4), 4.18 (q, 4, Jꢁ
/6.8 Hz), 5.82
/
(
m, 1), 5.98 (m, 1). Anal. (C H O ) C, H, N.
/
1
1
16
4
1
1
3
21
4
4
cyclopent-2-ene-1-carboxylate (14)
.1.3. (9)-Ethyl-N-tert-butoxycarbonyl-1-amino-
/
4.1.4. (9
cyclopent-2-ene-1-carboxylate (15)
To a solution of alkene (9)-14 (1.6 g, 6.27 mmol) in
THF (25 ml) under stirring was added DMAP (76 mg,
0.627 mmol) followed by the dropwise addition of a
THF solution (5 ml) of di-tert-butyl dicarbonate (2.05 g,
9.4 mmol). After refluxing the mixture for 3 days,
/
)-Ethyl-N-di-tert-butoxycarbonyl-1-amino-
Alkene 13 (6.88 g, 32.5 mmol) was dissolved in EtOH
30 ml) and treated with 1 N NaOH (32.5 ml, 32.5
(
mmol) at r.t. for 15 h. After evaporation of the ethanol
at reduced pressure, the aqueous layer was extracted
/
with dichloromethane (3ꢃ
3. The aqueous layer was then acidified with 2 N HCl
and extracted with dichloromethane (3ꢃ15 ml). The
/10 ml) to remove unreacted
1
/
further BOC O was added (2.74 g, 12.54 mmol) and
2
organic phase was dried and evaporated to give 4.93 g of
the corresponding monoacid of 13 which was not
characterized but directly used in the next step.
the reaction mixture was refluxed for additional 3 days.
The progress of the reaction was monitored by TLC
(eluant: petroleum etherÁethyl acetate, 9:1). After
/
The above-prepared monoacid derivative (4.93 g, 26.8
mmol) was dissolved in acetone (45 ml) and cooled at
evaporation of the solvent, the crude material was
purified by column chromatography on silica gel
0 8C. Triethylamine (4.48 ml, 32.2 mmol) was added at
once followed by the dropwise addition of ethylchlor-
(eluant: petroleum etherÁ
/
ethyl acetate, 95:5) to give
alkene (9)-15 as a yellow oil (1.96 g, 88% yield).
/

688
P. Conti et al. / Il Farmaco 58 (2003) 683Á690
/
(
9
/
)-Ethyl-N-di-tert-butoxycarbonyl-1-amino-cyclo-
1
)-15); H NMR (CDCl ):
acetate, 1:4); (9
clohexaneÁethyl acetate, 1:4).
In analogy, the mixture of (9
3.69 mmol) was treated under the same conditions to
give (9)-18d (112 mg); (9)-18b (220 mg); (9)-18a (385
mg) and (9)-18c (90 mg).
Amino ester (9
Jꢁ7.4 Hz), 1.58 (bs, 2), 1.69 (m, 1), 2.00Á
3.89 (dd, 1, Jꢁ8.2, 8.6 Hz), 4.23 (q, 2, Jꢁ7.4 Hz), 4.73
(d, 1, Jꢁ8.6 Hz).
Amino ester (9
Jꢁ7.3 Hz), 1.58 (m, 1), 1.85 (bs, 2), 1.99 (m, 2), 2.11 (m,
1), 3.84 (m, 1), 4.18 (q, 2, Jꢁ7.3 Hz), 5.08 (d, 1, Jꢁ9.5
Hz).
Amino ester (9
Jꢁ7.4 Hz), 1.62 (bs, 2), 1.69 (m, 1), 2.15Á
3.42 (d, 1, Jꢁ8.2 Hz), 4.20 (q, 2, Jꢁ7.4 Hz), 5.31 (m,
1).
Amino ester (9
Jꢁ7.4 Hz), 1.77 (bs, 2), 1.86 (m, 1), 2.20 (m, 3), 4.03 (d,
1, Jꢁ9.6 Hz), 4.23 (q, 2, Jꢁ7.4 Hz), 5.28 (m, 1).
/
)-18c (160 mg), R ꢁ
/
0.41 (cy-
F
pent-2-ene-1-carboxylate ((9
.24 (t, 3, Jꢁ
), 3.01 (m, 1), 4.16 (q, 2, Jꢁ
m, 1). Anal. (C H NO ) C, H, N.
/
/
3
1
2
(
/
6.9 Hz), 1.47 (s, 18), 2.05 (m, 1), 2.53 (m,
6.9 Hz), 5.72 (m, 1), 6.01
/
)-17aÁ
/
(9
/
)-17d (1.76 g,
/
/
/
/
1
8
29
6
/
1
4
.1.5. 1,3-Dipolar cycloaddition of bromonitrile oxide to
)-14
To a solution of (9
acetate (15 ml) was added dibromoformaldoxime (1.9 g,
.4 mmol) and solid NaHCO (3.5 g). The mixture was
/
)-18a; H NMR (CDCl ): 1.30 (t, 3,
3
(
9
/
/
/2.38 (m, 3),
/)-14 (1.2 g, 4.7 mmol) in ethyl
/
/
/
1
9
/
)-18b; H NMR (CDCl ): 1.26 (t, 3,
3
3
vigorously stirred for 3 days. The progress of the
reaction was monitored by TLC (eluant: petroleum
etherÁethyl acetate, 4:1). Water (10 ml) was added to
the reaction mixture and the organic layer was separated
/
/
/
/
1
/
)-18c; H NMR (CDCl ): 1.29 (t, 3,
3
and dried over anhydrous Na SO . The crude material,
2
/
/2.52 (m, 3),
4
obtained after evaporation of the solvent, was purified
/
/
by column chromatography on silica gel (eluant: petro-
ethyl acetate, 85:15) to give 1.52 g (86%
)-
1
leum etherÁ
/
/
)-18d; H NMR (CDCl ): 1.31 (t, 3,
3
yield) of an unsplittable mixture of cycloadducts (9
/
/
1
6aÁ
/
(9
/)16d.
/
/
4
(
.1.6. 1,3-Dipolar cycloaddition of bromonitrile oxide to
)-15
To a solution of (9
4.1.8. Conversion of (9
corresponding derivatives (9
17aÁ(9)-17d
20 mg of each amino ester (9
treated with 1.5 equiv. of TEA and 1.5 equiv. of di-tert-
butyl dicarbonate in dichloromethane to give the
/
)-18aÁ
/
(9
/
)-18d into the
9
/
/
)-16aÁ
/
(9)-16d and (9)-
/
/
/
)-15 (1.9 g, 5.35 mmol) in ethyl
/
/
acetate (20 ml) was added dibromoformaldoxime (2.17
g, 10.7 mmol) and solid NaHCO (4 g). The mixture was
vigorously stirred for 3 days. The progress of the
reaction was monitored by TLC (eluant: petroleum
/
)-18aÁ
/
(9)-18d was
/
3
corresponding N-BOC derivatives (9
/
)-16aÁ
/
(9)-16d,
/
etherÁ
/
ethyl acetate, 9:1). After a further addition of 1
which were not isolated but directly used as standards
for HPLC analysis (column: LiChrospher Si 60 Merck;
equiv. of dibromoformaldoxime (1.08 g, 5.35 mmol), the
reaction was stirred for additional 3 days, until the
disappearance of alkene. The crude material, obtained
after the previously reported work up, was purified by
column chromatography on silica gel (eluant: petroleum
eluant: petroleum etherÁ
ml/min; lꢁ254 nm). Retention times: 16b, 11.65 min;
16d, 13.23 min; 16c, 16.05 min; 16a, 18.15 min.
In analogy, 20 mg of each amino ester (9)-18aÁ(9)-
/ethyl acetate, 85:15; flow: 0.5
/
/
/
/
etherÁ
/
ethyl acetate, 95:5) to give 1.76 g (69% yield) of an
18d was dissolved in THF and refluxed in the presence
of 1.5 equiv. of di-tert-butyl dicarbonate and a catalytic
amount of DMAP to give the corresponding derivatives
unseparable mixture of the cycloadducts (9
/
)-17aÁ(9)-
/
/
1
7d.
(
9
/
)-17aÁ
HPLC analysis (column: LiChrospher Si 60 Merck;
eluant: petroleum etherÁethyl acetate, 85:15; flow: 0.2
ml/min; lꢁ254 nm). Retention times: 17c, 20.35 min;
17d, 22.45 min; 17b, 24.36 min; 17a, 27.90 min.
/
(9
/
)-17d, which were used as standards for
4
(
.1.7. Synthesis of intermediates (9
)-18c and (9)-18d
The mixture of (9)-16aÁ
/
)-18a, (9)-18b,
/
9
/
/
/
/
/
(9
/
)-16d (1.52 g, 4.03 mmol)
/
was treated with a 30% dichloromethane solution of
trifluoroacetic acid (10.3 ml) at 0 8C. The reaction
mixture was stirred at r.t. until disappearance of the
starting material (3 h). The volatiles were removed
under vacuum and the residue was treated with a 10%
K CO solution (25 ml) and extracted with ethyl acetate
4.1.9. Synthesis of amino acids (9
A solution of (9)-18a (380 mg, 1.37 mmol) in ethanol
(3 ml) was treated with 1 N NaOH (1.5 ml). The solution
was stirred at r.t. until TLC (eluant: petroleum etherÁ
/
)-6 and (9
/
)-7
/
/
2
3
(
anhydrous Na SO and concentrated under vacuum.
4ꢃ
/
10 ml). The pooled organic extracts were dried over
ethyl acetate, 1:4) showed the disappearance of the
starting material (4 h). The solution was then made
acidic (pH 2) with 2 N HCl and submitted to cation
exchange chromatography, using Amberlite IR-120
plus. The acidic solution was slowly eluted onto the
resin, then the column was washed with water until the
pH was neutral. The compound was eluted off the resin
with 10% aqueous pyridine, and the product-containing
2
4
The residue was chromatographed on silica gel (eluant:
petroleum etherÁethyl acetate, 1:1) to give four fractions
which were eluted in the following order: (9)-18d (298
mg), R ꢁ0.80 (cyclohexaneÁethyl acetate, 1:4); (9)-
8b (530 mg), R ꢁ0.65 (cyclohexaneÁethyl acetate,
)-18a (75 mg), R ꢁ0.52 (cyclohexaneÁethyl
/
/
/
/
/
F
1
/
/
F
1
:4); (9
/
/
/
F

P. Conti et al. / Il Farmaco 58 (2003) 683Á
/690
689
fractions (detected with ninhydrin stain on a TLC plate)
were combined and concentrated under vacuum to give
Acknowledgements
1
57 mg (46%) of (9
waterÁethanol as white prisms.
)-(3aS,6S,6aS)-6-Amino-3-bromo-4,5,6,6a-tetra-
hydro-3aH-cyclopenta[d]isoxazole-6-carboxylic acid
(9)-6): R ꢁ0.48 (n-butanolÁwaterÁacetic acid,
0:25:15); m.p. (dec.)ꢀ180 8C; H NMR (D O): 2.01
m, 1), 2.15Á2.42 (m, 3), 4.20 (t, 1, Jꢁ
, Jꢁ8.5 Hz). Anal. (C H BrN O ) C, H, N.
The above-reported protocol applied to (9
mg, 1.8 mmol) gave 237 mg (yield: 53%) of (9
was crystallized from waterÁethanol as white prisms.
)-(3aS,6R,6aS)-6-Amino-3-bromo-4,5,6,6a-tetra-
hydro-3aH-cyclopenta[d]isoxazole-6-carboxylic acid
(9)-7): R ꢁ0.58 (n-butanolÁwaterÁacetic acid,
0:25:15); m.p. (dec.)ꢀ174 8C; H NMR (D O): 1.84
m, 1), 2.04Á2.40 (m, 3), 4.22 (t, 1, Jꢁ
8.8 Hz). Anal. (C H BrN O ) C, H, N.
/
)-6, which was crystallized from
The financial support from National Council of
Research (CNR, Rome) and from University of Milan
(FIRST) is gratefully acknowledged.
/
(
9
/
(
6
(
1
/
/
/
/
F
1
/
2
References
/
/8.5 Hz), 5.14 (d,
/
7
9
2
3
[1] L.J. Hanka, D.G. Martin, G.L. Neil, A new antitumor anti-
metabolite, (aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazolea-
cetic acid (NSC-163501): antimicrobial reversal studies and
preliminary evaluation against L1210 mouse leukemia in vivo,
/
)-18b (500
)-7, which
/
/
Cancer Chemother. Rep. 57 (1973) 141Á
2] H.N. Jayaram, D.A. Cooney, J.A. Ryan, G. Neil, R.L. Dion,
V.H. Bono,
-[aS,5S]-a-amino-3-chloro-4,5-dihydro-5-isoxazo-
leacetic acid (NSC-163501): a new amino acid antibiotic with
the properties of an antagonist of -glutamine, Cancer Che-
mother. Rep. 59 (1975) 481Á491.
3] R.H. Earhart, G.L. Neil, Acivicin in 1985, Adv. Enzyme Regul.
4 (1985) 179Á205.
4] J.Y. Tso, S.G. Bower, H. Zalkin, Mechanism of inactivation of
glutamine amidotransferases by the antitumor drug
-(aS,5S)-a-
amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), J.
Biol. Chem. 255 (1980) 6734Á6738.
/
148.
(9
/
[
L
(
6
(
1
/
/
/
/
F
1
L
/
2
/
/
/8.8 Hz), 5.25 (d,
[
[
, Jꢁ
/
7
9
2
3
2
/
L
4
.2. Biological testing
/
[
5] L. Allen, R. Meck, A. Yunis, The inhibition of g-glutamyl
transpeptidase from human pancreatic carcinoma cells by
4
.2.1. Anticancer screening
Evaluation of anticancer activity of compounds (9
(9)-7 was performed at the National Cancer Institute
(
aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic
(AT-125; NSC-163501), Res. Commun. Chem. Pathol. Pharma-
col. 27 (1980) 175Á182.
acid
/)-
/
2
Á
/
/
[
6] K.E. Nichols, S.R. Chitneni, J.O. Moore, J.B. Weinberg, Mono-
cytoid differentiation of freshly isolated human myeloid leukemia
cells and HL-60 cells induced by the glutamine antagonist
(
NCI) of Bethesda, MD, following the well-known in
vitro disease-oriented antitumor screening program
18,19], which is based upon the use of multiple panels
[
acivicin, Blood 74 (1989) 1728Á1737.
/
of 60 human tumor cell lines against which the
compounds under study were tested at 10-fold dilutions
[7] B. Bauvois, A. Laouar, D. Rouillard, J. Wietzerbin, Inhibition of
g-glutamyl transpeptidase activity at the surface of human
myeloid cells is correlated with macrophage maturation and
transforming growth factor beta production, Cell Growth Differ.
ꢂ4
of five different concentrations, ranging from 10
ꢂ8
to
M. The percentage growth was evaluated spectro-
1
0
6
(1995) 1163Á
8] D.M. Vyas, Y. Chiang, T.W. Doyle, A short, efficient synthesis of
(9)-acivicin and (9)-bromo-acivicin, Tetrahedron Lett. 25 (1984)
87Á490.
/
1170.
photometrically versus controls not treated with test
agents. A 48-h continuous drug exposure protocol was
followed and a sulfurhodamine B (SRB) protein assay
was used to estimate cell viability or growth.
[
[
/
/
4
/
9] A.A. Hagedorn, B.J. Miller, J.O. Nagy, Direct synthesis of the
antitumor agent erythro-a-amino-3-bromo-4,5-dihydroisoxazole-
5
10] E.C. Boyd, R.M. Paton, Stereoselective cycloaddition of nitrile
-acetic acid, Tetrahedron Lett. 21 (1980) 229Á230.
/
[
4
.2.2. Biochemical methods
Recombinant A. brasilense GltS was produced, pur-
oxides to 4-vinyl-oxazolines and oxazolidines, Tetrahedron Lett.
3
4 (1993) 3169Á
11] M.A. Vanoni, B. Curti, Glutamate synthase: a complex iron-
sulfur flavoprotein, Cell. Mol. Life Sci. 55 (1999) 617Á638.
12] H. Zalkin, J. Smith, Enzymes utilizing glutamine as an amide
donor, Adv. Enzymol. Relat. Areas 72 (1998) 87Á144.
/
3172.
[
[
ified and assayed as described [22]. Steady-state kinetic
measurements were carried out at 25 8C in 50 mM
HepesÁ
acivicin analogues was determined by assaying enzyme
activity in the presence of varying concentrations of
/
/KOH buffer, pH 7.5. The inhibitory effect of
/
[13] P. Conti, C. Dallanoce, M. De Amici, C. De Micheli, R. Fruttero,
Synthesis of new bicyclic analogues of glutamic acid, Tetrahedron
L
-
5
5 (1999) 5623Á5634.
/
glutamine, 2-oxoglutarate or NADPH and increasing
concentrations of the compound under analysis. To test
GltS inactivation, the enzyme (3 mM) was incubated in
[
14] J.P. Ferris, B.G. Wright, C.C. Crawford, The acylation of b-keto
esters. Control of the position of acylation by variation of the
acylating agent and solvent, J. Org. Chem. 30 (1965) 2367Á2371.
/
2
5 mM HepesÁ
glycerol, in the presence of the compound under analysis
1 mM) for up to 8 h. At different times after addition of
/
KOH buffer, pH 7.5, 1 mM EDTA, 10%
[15] D.P. Curran, S.-M. Choi, S.A. Gothe, F.-T. Lin, Directed nitrile
oxide cycloaddition reactions. The use of hydrogen bonding to
direct regio- and stereochemistry in nitrile oxide cycloadditions
(
with cyclopentenylamides, J. Org. Chem. 55 (1990) 3710Á3712.
/
the potential inactivator, enzyme activity was measured
upon dilution in reaction mixtures containing saturating
concentrations of the enzyme substrates [22].
[
16] D.P. Curran, S.A. Gothe, S.-M. Choi, Hydrogen bond directed
nitrile oxide cycloaddition reactions of allylic 28-amides, Hetero-
cycles 35 (1993) 1371Á1395.
/

690
P. Conti et al. / Il Farmaco 58 (2003) 683Á690
/
[
17] K.-H. Park, M.J. Kurth, Diastereoselective synthesis of hydan-
toin- and isoxazoline-substituted dispirocyclobutanoids, J. Org.
[20] This information is available at the web site: http://dtp.nci.nih.-
gov.
Chem. 65 (2000) 3520Á
/
3524.
[21] C. Binda, R. Bossi, S. Wakatsuki, S. Artz, A. Coda, B. Curti,
M.A. Vanoni, A. Mattevi, Cross-talk and ammonia channeling
between active centers in the unexpected domain arrangement of
[
18] A. Monks, D.A. Scudiero, P. Skehan, R. Shoemaker, K.D. Paull,
D.T. Vistica, C. Hose, J. Langley, P. Cronise, M. Vaigro-Wolf,
M. Gray-Goodrich, H. Campbell, M.R. Mayo, Feasibility of a
high-flux anticancer drug screen using a diverse panel of cultured
glutamate synthase, Structure 8 (2000) 1299Á1308.
/
[22] H. Stabile, B. Curti, M.A. Vanoni, Functional properties of
human tumor cell lines, J. Natl. Cancer Inst. 83 (1991) 757Á
/766.
recombinant Azospirillum brasilense glutamate synthase, a com-
[
19] M.R. Boyd, K.D. Paull, Some practical considerations and
applications of the National Cancer Institute in vitro anticancer
plex iron-sulfur flavoprotein, Eur. J. Biochem. 267 (2000) 2720Á
2730.
/
drug discovery screen, Drug Dev. Res. 34 (1995) 91Á
/109.
[23] A. Mattevi, M.A. Vanoni, B. Curti, unpublished results.