Chemical Basis for the Biological Activity of Imexon and Related Cyanoaziridines

Article abstract of DOI:10.1021/jm030225v

Chemical aspects of mode of action of imexon and related cyanoaziridines were studied. These compounds do not alkylate DNA nor react with the ε-amino groups of L-lysine, despite the presence of an aziridine ring. They do react readily with biologically im

Full text of DOI:10.1021/jm030225v

2
18
J . Med. Chem. 2004, 47, 218-223
Ch em ica l Ba sis for th e Biologica l Activity of Im exon a n d Rela ted
Cya n oa zir id in es
Bhashyam S. Iyengar, Robert T. Dorr, and William A. Remers*
Arizona Cancer Center and Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona 85721
Received May 13, 2003
Chemical aspects of mode of action of imexon and related cyanoaziridines were studied. These
compounds do not alkylate DNA nor react with the ꢀ-amino groups of L-lysine, despite the
presence of an aziridine ring. They do react readily with biologically important sulfhydryl
compounds to give products derived from either aziridine ring opening, interaction with the
cyano group of cyanoaziridines, or opening of the iminopyrrolidone ring of imexon. The products
from reactions of imexon and related cyanoaziridines with thiols are not as potent as their
parent compounds against tumor cells. These results are consistent with biological studies
that show that the mechanism of cytotoxicity involves thiol depletion followed by oxidative
stress leading to apoptosis.
In tr od u ction
In 1975 Bicker reported a new type of carcinostatic
1
aziridine, 2-cyanoaziridine-1-carboxamide (1). Subse-
quent publications from his laboratory described the
cytotoxicity of analogues in which the amide nitrogen
was substitiuted with a variety of functional groups
2
,3
including methyl, phenyl, and 4-sulfamylphenyl. Some
of these compounds showed immunomodulatory proper-
ties, which prompted the suggestion that they act
1
indirectly by an effect on immunological mechanisms.
More recently, a series of N-substituted 2-cyanoaziri-
dine-1-carboxamides was shown to be cytotoxic in a
large panel of cultured tumor cells including many
4
species resistant to standard chemotherapeutic agents.
thiazolidine, but the more reasonable thiazoline struc-
ture 5 would give the molecular ion of 307 found in its
mass spectrum. It is noteworthy that the latter product
would be formed by reaction of the sulfur atom with the
cyano group in preference to the aziridine ring.
They gave a correlation between lipophilicity and po-
tency.
When 1 was treated with KOH in methanol, it
underwent cyclization to 4-imino-1,3-diazabicyclo[3.1.0]-
5
hexan-2-one (2), which was named imexon. Imexon is
Biological studies by Dorr and colleagues support the
active against various transplanted syngeneic tumors
1
1
idea that imexon does not alkylate DNA. Instead, it
is cytotoxic by a unique mechanistic pathway: induction
of reactive oxygen species (ROS). They demonstrated
that depletion of cellular thiols was dose- and time-
6
in rodents and transplanted human tumors in SCID
7
mice. Objective responses were found in a phase I
clinical trial.8 In fresh human tumors in clonogenic
assay, imexon was selectively cytotoxic to multiple
1
1
dependent. Oxidative stress was detected by confocal
microscopy, which showed increased levels of 8-hydroxy-
guanosine in the cytoplasm, but not in the nucleus of
imexon-treated 8226 myeloma cells. Interestingly, fur-
ther studies have shown damage to mitochondrial but
not nuclear DNA. These imexon-treated cells also
showed the classic morphological features of apoptosis.
Further depletion of glutathione by buthionine sulfox-
imide increased imexon cytotoxicity, and sulfhydryl
replenishment with N-acetylcysteine blocked cytotox-
icity. The cytotoxicity of imexon was shown to be time
and concentration dependent with maximal effects
9
myeloma. The activity in immunodeficient SCID mice,
and in cell cultures lacking immune cell components,
suggests that immunomodulation is not the mechanism
of cytotoxic action.
One surprising property of 2-cyanoaziridine-1-car-
boxamide (1) was its failure to react with 4-(4-nitroben-
zyl)pyridine in the standard assay for evaluating po-
1
tential alkylating agents. In contrast, cyanoaziridine
(
3) was reported to react readily with the stronger
1
nucleophile cysteine. Furthermore, ciamexon (4), a
cyanoaziridine derivative which received brief clinical
study in the late1980s, was found to deplete glutathione
in liver microsomes, and to react readily with cysteine
in vivo.10 The product of this reaction was said to be a
1
1
noted after 48 h exposure at 37 °C. Another study
showed that 8226 myeloma cells which are sensitive to
imexon undergo a significant loss of mitochondrial
1
2
membrane potential (MMP). This loss was shown to
follow the generation of ROS. The release of cytochrome
C from mitochondria was also documented to follow loss
*
To whom correspondence should be addressed. Arizona Cancer
Center, 1515 N. Campbell Ave., Tucson, AZ 85724. Phone 520-626-
532. E-mail remers@pharmacy.arizona.edu.
4
1
0.1021/jm030225v CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

Imexon and Related Cyanoaziridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 219
Ta ble 1. Relative Alkylating Abilities of Imexon and
Cyanoaziridines
mixture. No attempt was made to isolate and identify
the products. According to this procedure, imexon, 6,
and 7 remained unchanged, whereas melphalan reacted
almost completely within 30 min. Another study was
made using L-lysine as a model for the reactive groups
in proteins. No reaction between imexon and L-lysine
was observed under a variety of conditions including
variation of pH from 7.0 to 9.4, and temperatures in the
range from room temperature (21 °C) to 42 °C.
a
compd
relative absorbance at 560 nm
blank
0.03
0.05
0.04
0.04
0.36
0.47
2
3
6
7
9
a
This assay was conducted according to a standard procedure
Preussmann, R.; Schneider, H.; Epple, F. Investigation on the
Detection of Alkylating Agents. Artzneimittelforsch. 1969, 19,
Imexon, 3, and 8 each reacted readily with cysteine
at room temperature. The products were isolated by
preparative TLC (Experimental Section), and their
(
1
059-1073). Visible light absorbance was measured on a Perkin-
Elmer Lambda 3A spectrophotometer.
of MMP. Overall, these studies suggest a sequential
pathway for imexon-induced cytotoxicity, consisting of
binding to sulfhydryls, accumulation of ROS, loss of
MMP, release of mitochondrial cytochrome C, and
induction of apoptosis.12
Ch em istr y
To gain a clearer picture of the alkylating ability of
cyanoaziridines and imexon, we have undertaken a
study on the comparative reactivity of certain of these
compounds toward nucleophiles including 4-(4-nitroben-
zyl)pyridine, 2′-deoxyguanosine, L-lysine, and cysteine.
Furthermore, we have determined the structures of
products from the reactions of these compounds with
cysteine and other thiols of biological importance in
vitro. Thus, a set of compounds including imexon (2),
structures were determined by proton NMR and by
mass spectrometry. The structures of the products
varied with the natures of the compounds. Thus, imexon
reacted with cysteine at room temperature to furnish
the thiazoline conjugate 11 in which the five-membered
ring has opened to provide a carboxamido group. The
2
-cyanoaziridine (3), 2-cyanoaziridine-1-(N-phenylcar-
boxamide) (6), aziridine-1-(N-phenylcarboxamide) (7),
and melphalan (9) were each treated with 4-(4-nitroben-
2
,13
zyl)pyridine at 100 °C under standard conditions.
In
this assay, reactive compounds alkylate the pyridine
nitrogen, and then treatment with piperidine removes
a benzylic hydrogen to give a conjugated system with a
purple color. The extent of reaction is determined by
measuring light absorption at 560 nm. Compound 7,
which lacks a cyano group, was used to determine the
importance of this substituent on alkylation. The bi-
functional alkylating agent melphalan (9) was used as
the positive control. The results of this assay are listed
in Table 1. They show that imexon, 3, and 6 were not
alkylating agents; however, 7 was almost as reactive
as melphalan. This observation suggests that the pres-
ence of a cyano group on the aziridine ring is sufficient
to drastically reduce its alkylating ability. (Acylation of
the aziridine nitrogen promotes reaction with nucleo-
philes because it participates in delocalization of the
negative charge formed on the nitrogen in the transition
state for nucleophilic attack on a carbon in the aziridine
ring.)14 These results contradict the previous report by
Bicker and Fuhse that compound 6 was a more active
alkylating agent than 7.2
mechanism of this process is unknown, but it can be
visualized as an attack of the cysteine sulfur atom on
the cyclic amidine functionality of imexon, with ring
opening facilitated by relief of ring strain. Cyclization
of the hypothetical intermediate 10 would then give the
thiazoline ring of 11 with loss of ammonia. An alterna-
tive mechanism might be opening of the five-membered
ring of 2 to give 1, followed by reaction with cysteine to
give 11. We have observed that 1 is slowly formed when
2 is kept in aqueous solution, although it takes days to
form appreciable quantities of 1.
When imexon and cysteine were reacted at 50 °C,
thiazoline 11 was still obtained, but it was accompanied
by 12, the product of aziridine ring opening. Compound
A qualitative comparison was made on the reactivity
of some of the above compounds with 2′-deoxyguanosine
in water at 100 °C. This assay involved visual observa-
tion of the disappearance of starting material and the
appearance of a new spot or spots on TLC of the
1
12 was identified by its H NMR spectrum. When the
NMR solution was concentrated after two weeks and
used to determine the mass spectrum, it was found that

2
20 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Iyengar et al.
the imine group had hydrolyzed to give imidazolidinedi-
one derivative 13. The composition of 13 was deter-
mined by HRMS.
and 2.83 ppm were absent, and new peaks at 2.95-3.2
and 3.85-3.95 ppm, corresponding to the open-chain
structure 18, were present. The HRMS analysis indi-
cated that a NH had been replaced by O, suggesting
that the imine group had been hydrolyzed to a carbonyl
group, as occurred in compound 13. Aziridine ring
opening when imexon reacts with glutathione is reason-
able because the γ-glutamyl substituent on the nitrogen
of the cysteine residue of glutathione prevents imida-
zoline formation.
2
-Cyanoaziridine (3) reacted with cysteine by way of
its cyano group to give the thiazoline 14. Bicker sug-
gested previously that 3 reacts at its cyano group, but
1
proposed no structure for the product. In contrast, we
have found that 2-cyanoaziridine-1-(N-phenylcarboxa-
mide) (6) reacted with cysteine through its aziridine ring
to furnish compound 15. This difference is attributed
The development of imexon resistance in 8226 my-
eloma cells is correlated in part with an increase in the
1
6
enzyme thioredoxin. This result suggested that it
might be valuable to investigate the reaction of imexon
and related cyanoaziridines with thiols more like cys-
teine incorporated into enzymes than to free cysteine.
N-Acetylcysteine was chosen for this purpose because
its amino group is substituted and thus not available
for thiazoline formation. Glutathione is another example
of this type of compound.
Imexon reacted with N-acetylcycteine at 37 °C in
water to afford a product that appeared to be a mixture
of iminoimidazolidolinone 19 and the corresponding
imidazolidinedione 20 according to mass spectrometry,
to the fact that 6 had to be heated to 100 °C because of
its very low water solubility. Even then it reacted as a
suspension. Apparently the high temperature promotes
aziridine ring opening. This reaction occurs to some
extent when imexon is heated to 50 °C, as described
1
above. Although the H NMR spectrum of 15 is consis-
tent with the depicted structure, it does not rule out
one in which the aziridine ring has been opened by
nucleophilic attack of sulfur on the carbon bearing the
cyano group. There is, however, literature precedent for
opening of the cyanoaziridine rings by nucleophilic
attack on the less hindered methylene group of the
aziridine. The corresponding compound 7, without a
cyano group, also has very low water solubility and had
to be reacted at 100 °C. It can only react by opening of
its aziridine ring. Although the crude product of this
reaction was so insoluble that it could not be character-
ized completely, its mass spectrum showed a molecular
ion and daughter peaks which were consistent with the
expected product 16. The 2-cyanoaziridine-1-(N-meth-
ylcarboxamide) (8) has good water solubility and it
reacted readily with cysteine at room temperature to
provide 17.
+
which showed appropriate MH ions at 284 and 285 m/z,
1
5
respectively. The proton NMR spectrum was consistent
with these structures. When this solution was kept for
days, the peak at 284 disappeared, suggesting com-
7
plete hydrolysis to dione 20. A new peak appeared at
m/z 347, but the mixture was not investigated further.
In contrast to imexon, 2-cyanoaziridine-1-(N-methyl-
carboxamide) (8) failed to react with N-acetylcysteine
in water at 37 °C. Decomposition resulted at 55 °C.
The foregoing results suggest that imexon is more
reactive than related cyanoaziridines toward thiols. A
direct competition experiment was conducted to gain
more information on this possibility. In this experiment,
a solution containing equivalent amounts (molar basis)
of imexon, 2-cyanoaziridine-1-carboxamide (1), and cys-
teine in D2O was examined by proton NMR spectrom-
etry. The amounts of unreacted imexon and 1 were
estimated by comparing the integrals of the doublets
for their CH protons at 3.8 and 3.38 ppm, respectively.
Solutions containing only imexon, 1, or cysteine in D2O
were run as standards. This experiment showed that
imexon was consumed approximately two times faster
than 1.
Previous biological experiments indicated that imexon
reacts readily with glutathione to deplete this important
thiol.11 We found that equimolar amounts of imexon and
glutathione react completely within 1 h at room tem-
perature. A completely pure sample of the product 18
To gain insight into the relative reactivity between
imexon and standard thiol trapping agents, a competi-
tion between imexon and N-methylmaleimide was con-
ducted. N-Methylmaleimide was chosen because the two
protons on a double bond are clearly separated from all
imexon protons, and the methyl group can serve as a
standard for the relative amounts of other protons.
was not obtained; however, the 1H NMR spectrum
indicated that the aziridine ring of imexon had been
opened with incorporation of glutathione. In particular,
the peaks for protons on the aziridine ring at 2.0, 2.14,

Imexon and Related Cyanoaziridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 221
Ta ble 2. Cytotoxicity of Cyanoaziridines and Related
Compounds
is interesting in view of the finding described above that
7
appears to alkylate DNA, whereas 6 does not. 2-Cy-
IC50 (µM)a
anoaziridine (3) and 2-cyanoaziridine-1-carboxamide (1)
were not active against either L1210 leukemia or 8226
myeloma cells in culture; however, the N-methyl deriva-
tive 8 and the N-phenyl derivative 6 of 1 were cytotoxic
in these cell lines. This difference may be caused by the
greater lipophilicity of 6 and 8. A correlation between
lipophilicity and cytotoxicity was previously found in a
compd
L1210/S
L1210/MDR
myeloma 8226/S
6
7
1
1
3
4
8
7
5
0
1.2, 38.4
2.1, 27.1
>500
>500
>500
>500
25.1
>500
73.9
73.7
11, 41.2
5.8, 28.4
>500
>500
>500
>500
36.7
>500
>500
116.4
5.3
5.5
>500
>500
>500
>500
15.5
1
1
4
larger set of 2-cyanoaziridine-1-carboxamides. Table 2
shows that none of the products containing both an
aziridine ring and a thiazoline ring (11, 14, and 17) was
cytotoxic. Somewhat surprisingly, compounds 15 and
20, in which the aziridine ring has been opened by
reaction with cysteine, retain some cytotoxicity. They
are less potent than cyanoaziridines 6 and 8, but clearly
more potent than compounds 11, 14, and 17, which
retain the aziridine ring. It is possible that they act with
cells by a mechanism different from that of imexon and
cyanoaziridines.
1
1
2
>500
44.9
56.3
a
Cytotoxicity against the L1210 leukemia cells, both sensitive
and multidrug resistant (MDR), and the human myeloma 8226
cells was determined by the MTT assay (Alley, M. C.; Scudiero,
D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J .; Fine, D. L.;
Abbott, B. J .; Mayo, J . G.; Schoemaker, R. H.; Boyd, M. R.
Feasibility of Drug Screening with Panels of Human Tumor Cell
Lines Using a Microculture Tetrazolium Assay. Cancer Res. 1998,
4
8, 589-601.
Furthermore, it is widely used in biological studies
involving thiols. In this experiment, 1 equiv of cysteine
Con clu sion s
The results of chemical studies on the mode of action
of imexon and related cyanoaziridines with nucleophiles
were consistent with the published results of biological
and biochemical studies. These compounds did not
appear to be DNA alkylators, according to both the 4-(4-
nitrobenzyl)pyridine color test and direct treatment with
in water was treated with 1 equiv of imexon and 1 equiv
of N-methylmaleimide. The 1H NMR spectrum was
determined after 6 h. It showed almost complete loss of
the two protons on the double bond of N-methylmale-
imide and the generation of three protons on aliphatic
carbons formed by saturation of the double bond. The
protons attributable to imexon were unchanged in
location and intensity. Standards consisting of imexon,
N-methylmaleimide, and cysteine singly in D2O were
used in this comparison.
2
′-deoxyguanosine. They also did not react with the
ꢀ
-amino group of L-lysine. In contrast, they reacted
readily with cysteine. Cysteine reacts preferentially
with the cyano group of cyanoaziridines or the amidine
system of imexon at room or physiological temperatures.
At higher temperatures, some aziridine ring opening
occurs. Glutathione, which is incapable of forming a
thiazoline, reacts by opening the aziridine rings of
imexon or cyanoaziridines. Imexon reacts more rapidly
than the cyanoaziridines. Imexon also reacts with
N-acetylcysteine, a model compound for cysteine resi-
dues in proteins, but cyanoaziridine 8 fails to react with
N-acetylcysteine even at 42 °C. The aziridine ring of
cyanoaziridines enhances reactivity of the cyano group
toward thiols, as indicated by the unreactivity of aceto-
nitrile (21). Imexon reacts readily with thiols, but more
slowly than N-methylmaleimide.
The products of reaction of imexon or related cy-
anoaziridine-1-carboxamides lose cytotoxic potency; how-
ever, this decrease is greater for compounds that retain
their aziridine rings and form thiazolines than for those
that lose their aziridine rings and retain their cyano
groups.
It appears that the aziridine ring is required for
significant potency in imexon and its analogues, but it
might not be the actual target for nucleophilic attack
by biological thiols in some analogues containing cyano
groups.
The current results show that the aziridine ring
activates the cyano group toward reaction with cysteine.
Condensation of cysteine with the simpler cyano com-
pound, acetonitrile (21), to afford thiazoline 22 is
described in the literature;17 however, it requires the
presence of a base (sodium ethoxide) and reflux tem-
perature, and the product is the sodium salt of the
carboxylic acid group. We confirmed this reaction, but
found that there was no reaction when the base was
omitted. This finding suggests that small amounts of
the thiolate anion, formed in equilibrium, are required
for addition to the cyano group of 21. Enhanced reactiv-
ity of 2-cyanoaziridine over acetonitrile toward cysteine
presumably results from the electron-withdrawing prop-
1
erty of the R-nitrogen atom. The H NMR spectrum of
the acidic form of 22 was useful for assigning the
protons in the spectra of the thiazolines described above.
Biology
Exp er im en ta l Section
The potencies of imexon and related cyanoaziridines
Melting points were recorded on a Mel-Temp apparatus and
in cultures of various tumor cells have been reported
1
previously.4 In Table 2 the relative potencies of the
are uncorrected. H NMR spectra were recorded on a Varian
Unity-300 spectrometer using the indicated solvents and
internal standards. NMR shift values are expressed in ppm.
Com p a r ison of th e Alk yla tin g Abilities of Im exon ,
Cya n oa zir id in es, a n d Melp h a la n . A 1 mL quantity of 0.005
mM solutions of each compound, including imexon (2), cy-
noncyano compound aziridine-1-(N-phenylcarboxamide)
(7) and its 2-cyano analogue 6 are compared. Both
compounds were active in the micromolar range against
sensitive L1210 murine leukemia cells. This observation

2
22 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Iyengar et al.
anoaziridine (3), 2-cyanoaziridine-1-(N-phenylcarboxamide)
6), aziridine-1-(N-phenylcarboxamide) (7), and melphalan (9)
concentration to ensure that all of the deuterium was ex-
changed for hydrogen. When the resulting solid was examined
by mass spectrometry using the conditions described above,
there was no peak at m/z 233, but there was one at m/z 234.
(
in 2-methoxyethanol was treated with 1 mL of a 5% solution
of 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol, and each
resulting solution was heated at 100 °C for 1 h. A control
solution involving 5% 4-(4-nitrobenzyl)pyridine in 2-methoxy-
ethanol at the same concentration was used. The solutions
were then cooled and treated with 0.5 mL of piperidine and
HRMS on this peak showed a MW of 234.0544, which is
+
appropriate for the chemical formula (MH ) C
7
H
12
N
3
O
4
(calcd
234.0548). This change is probably produced by hydrolysis of
the CdNH group to a CdO group to afford imidazolidinedione
13.
2
.5 mL of 2-methoxyethanol. The solutions from 2, 3, and 6
and the control remained colorless, whereas those from 7 and
turned purple. The absorbance of each solution was mea-
When imexon was reacted with dl-cysteine at 37 °C and the
same concentrations as used in method A, the products were
the same as those found in method A except for a trace amount
of product possibly produced by hydrolysis of the imine group
to a carbonyl group.
Rea ction of 2-Cya n oa zir id in e w ith DL-Cystein e. A
mixture of 2-cyanoaziridine (3, 136 mg, 2 mmol) and DL-
cysteine in 2.5 mL of deionized water was warmed to ap-
proximately 40 °C to produce a solution and then immediately
cooled to room temperature. After 2 h, TLC analysis (3:7
9
sured at 560 nM. The results are given in Table 1.
Com p a r a tive Rea ctivities of Im exon , Cya n oa zir id in es,
a n d Melp h a la n w ith 2-Deoxygu a n osin e. A mixture of each
of the following compounds: imexon, 6, 7, and melphalan, with
1
equiv of 2′-deoxyguanosine in water was heated at reflux
temperature. The reaction was monitored by thin-layer chro-
matography on silica gel with methanol/chloroform (2:8 v/v)
as solvent. Melphalan reacted within 30 min, with two new
spots formed and only a trace of starting material remaining.
Only starting materials were observed with imexon and 6 even
after 48 h. Compound 7 decomposed partially, but there was
no change in the 2′-deoxyguanosine present.
Rea ction of Im exon w ith Lysin e. Solutions containing
imexon (10 mg, 0.09 mmol) and L-lysine (15 mg, 0.09 mmol)
in deionized water were treated under the following sets of
conditions: (A) pH 9.4 and room temperature for 48 h; (B) pH
3
MeOH-CHCL v/v on silica gel) showed at least four new
spots, but no starting material remained. This mixture was
concentrated under reduced pressure and separated by pre-
parative TLC on a silica gel plate (20 cm × 20 cm × 0.2 cm)
using the solvent system described above. The major band
(slow moving) was extracted with methanol, and the extract
was filtered and evaporated under reduced pressure to afford
11 mg of 14 as pale yellow solid which decomposed above 220
1
7
3
.0 (tris buffer) and room temperature for 48 h; (C) pH 7.0 at
°C; H NMR (D
2
O, DSS) δ 1.93 (split doublet, 1), 2.21 (d,1),
7-42 °C for two h. These solutions were monitored by TLC
3.07 (m,1) 3.35-3.64 (m, 2), 4.95 (bs s, partially under DOH);
MS (LR, ES) m/z 173 (MH and base peak), 128, 101, 69, 59;
HRMS (FAB ) calcd for C
+
on silica gel plates with MeOH-CHCl
3
(2:8 v/v) as solvent.
+
+
No new spots were detected in any of the chromatograms.
Rea ction s of Im exon w ith Cystein e. A. At Room Tem -
p er a tu r e. A solution of imexon (2, 10 mg, 0.09 mmol) and DL-
cysteine (12 mg, 0.1 mmol) in deionized water was kept at room
6
H
8
N
2
O
2
S (MH ) 174.0418; found
174.0420.
R ea ct ion of 2-Cya n o-1-(N-p h en ylca r b oxa m id e) w it h
DL-Cystein e. A mixture of 6 (57 mg, 0.3 mmol) and DL-cysteine
(40 mg, 0.33 mmol) in 1 mL of deionized water was heated to
about 100 °C for 15 min and then stirred at room temperature
3
temperature for 2 h. TLC analysis (2:8 v/v MeOH-CHCl on
silica gel) then indicated complete conversion of starting
material. The solution was concentrated under reduced pres-
sure, and the white solid residue was was stirred 30 min with
for 3 h. TLC analysis (2:8 MeOH-CHCl on silica gel) then
3
showed no remaining starting material. The mixture was
concentrated under reduced pressure, and the residue was
dissolved in 1 mL of MeOH and purified by preparative TLC
on a silica gel plate (20 × 20 × 0.2 cm) with 3:7 MeOH-CHCl
(v/v) as solvent. The slow moving band was extracted with
MeOH, and this extract was filtered and concentrated. This
ethanol and then filtered. The residue 11 was dried under high
1
vacuum. It did not melt below 250 °C; H NMR (D
2
O + DSS)
δ 2.86 (d, 2), 2.91 (d,1), 3.09 (br s, 1), 4.8 (under DOH, 1); MS
elctrospray) indicated MH ) 216; HRMS (FAB ) calcd for
S 216.04426, found MH 216.04420.
B. At Eleva ted Tem p er a tu r es. A mixture of 30 mg (0.27
mmol) of imexon (2), 36 mg (0.297 mmol) of DL-cysteine, and
.0 mL of deionized water was warmed at about 50 °C to
3
+
+
(
C
+
7
H
10
N
3
O
3
procedure afforded 10 mg of 15 as off-white solid which
1
decomposed above 200 °C; H NMR (DMSO-d
6
, TMS) δ 3-4
+
1
(m, 6), 7-7.5 (br s, 5); MS (LR, ES) m/z 309 (MH ); HRMS
(FAB ) calcd For C13H N O S 309.1021, found 309.1029.
16 4 3
+
maintain a clear solution and then allowed to cool to room
temperature during 1 h. TLC (silica gel with methanol-
chloroform 1:4 v/v) then showed no starting material. The
mixture was concentrated by rotary evaporation, and the
residual solid was divided into two approximately equal
portions. An HPLC separation of this mixture using an
analytical C18 column and solvent consisting of 10% acetoni-
trile and 90% water containing 0.1% trifluoroacetic acid under
isocratic conditions gave two main peaks. The solid residue
obtained from concentration of the larger and faster-moving
Rea ction of 2-Cya n oa zir id in e-1-(N-m eth ylca r boxa m -
id e) w ith DL-Cystein e. A solution of 25 mg of 8 and 25 mg of
DL-cysteine in 1 mL of deionized water was stirred at room
temperature for 2 h and then evaporated under reduced
pressure. The white solid residue was stirred with 2 mL of
ethanol, the mixture was filtered, and the solids were dried
1
under high vacuum. Product 17 did not melt below 200 °C; H
NMR (D O, DSS) δ 2.7 (s, 3), 2.9-3.0 (br s, 2), 3.4-3.7 (m, 3),
2
+
+
5.0 (t, 1); MS (ESI) m/z 230 (MH , base peak) and 173 (MH
- CH NCO); HRMS (FAB ) calcd for C H N O S (MH )
230.0599; found 230.0590.
1
+
+
peak (12) decomposed without melting when heated; H NMR
3
8
11
3
3
(
3
7
D
2
O + DSS) δ 3.06 (d, 1, J ) 9 Hz), 3.13 (d, 1, J ) 4.2 Hz),
.20 (d, 1, J ) 7.2 Hz), 3.25 (d, 1, J ) 4.5 Hz), 4.22 (dd, 1, J )
.2 Hz, 4.5 Hz), 4.57 (dd, 1, J ) 9 hz, 4.2 Hz).
Rea ction of Im exon w ith Glu ta th ion e. A mixture of
imexon (13 mg, 0.12 mol), glutathione (36 mg, 0.12 mmol), and
1 mL of deionized water was stirred for 1 h at room temper-
ature. The resulting solution then showed complete consump-
tion of the imexon according to TLC on silica gel with 2:8
The material from the smaller, slower-moving peak was
identical in Rf value on HPLC with the sample of 11 obtained
in method A.
The second portion of the crude product was subjected to
HPLC-mass spectrometry using an identical instrument,
column, and conditions as above for the HPLC separation. The
ionization mode was electrospray. The spectrum obtained from
the larger, faster-moving peak showed a molecular ion (MH )
at m/z 233 and fragment ions at 217, 173, 161, 145, 122, 112,
3
MeOH-CHCl (v/v) as solvent. It was concentrated under
reduced pressure, and the white solid residue was stirred 30
min with ethanol (2 mL) and filtered. The resulting solid 18
was dried under high vacuum. It did not melt below 250 °C;
+
1
H NMR (D
2
O, DSS) δ 2.1 (d, 2, CH
SH), 2.95-3.2 (m, 2, CH
3.75 (t, 1, CH(NH )CO H), 3.80 (s, 2, CH
CH derived from imexon), 4.55 (t, 1, CH(NH
2
CO), 2.5 (m, 2, CH
derived from imexon),
), 3.85-3.95 (m, 1,
)CO H); HRMS
S (MH ) 420.11885; found 420.1195.
2 2
CH ),
2.9 (m, 2, CH
2
2
1
2
2
00, 88, and 69, all appropriate for 12. HRMS (FAB) on the
2
2
2
33 peak gave a MW of 233.0684. Calcd for C
7
H
13
N
4
O
3
:
2
2
+
+
33.0708.
(FAB ) calcd for C14
H
21
N
5
O
8
1
After two weeks, the H NMR sample of 12 was concentrated
Syn th esis of Azir id in e-1-(N-p h en ylca r boxa m id e), 7.
to dryness and treated repeatedly with H
2
O followed by
Ethyleneimine was prepared from 2-aminoethyl hydrogen

Imexon and Related Cyanoaziridines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 223
sulfate (11.3 g) and 25 mL of 40% aqueous sodium hydroxide
as described in the literature.18
Ack n ow led gm en t. This research was funded by
Grant 2 PO1 CA17094-25 awarded by the National
Cancer Institute, DHHS. We thank Dr. Thomas Mc-
Clure of the University of Arizona College of Pharmacy
Analytical Center for low-resolution mass spectra and
Dr. Arpad Somogyi of the University of Arizona Depart-
ment of Chemistry for high-resolution mass spectra.
A 250 mg (6 mmol) portion of the ethyleneimine was treated
with 0.65 mL (6 mmol) of phenyl isocyanate in 2 mL of toluene
at 0-5 °C for 12 h. Removal of the toluene under reduced
pressure afforded the product 7 in 55% yield. It had mp 78-
1
8
7
0 °C; H NMR (CDCl
3
, TMS) δ 2.2 (s, 4, aziridine), 7.1 (t,1),
.3 (t,2), 7.5 (br s, 2), 7.9 (br s, 1, NH).
Rea ction of Azir id in e 1-(N-p h en ylca r boxa m id e) w ith
DL-Cystein e. A mixture of aziridine-1-(N-phenylcarboxamide)
16 mg, 0.1 mmol) and DL-cysteine (12 mg, 0.1 mmol) in 1 mL
(
Refer en ces
of deionized water was heated to 95-100 °C for 6 h in a tightly
capped glass vial. The resulting mixture was cooled to room
temperature and filtered. The solids were washed with 10 mL
of ethyl acetate to remove unreacted starting material. They
were then dried under vacuum. This crude product was not
soluble in water or organic solvents. The only data obtained
was a mass spectrum determined in the ABCI mode; m/z 283.9
(1) Bicker, U. F. Immunopharmacological Properties of 2-Cyanoazir-
idine Derivatives. In Immune Modulation Agents and Their
Mechanisms; Fenical, R. G., Chirigos, M. A., Eds.; Marcel
Dekker: New York and Basle, 1984; pp 447-473.
(
2) Bicker, U.; Fichse, P. Carcinostatic Action of 2-Cyanoaziridines
Against Sarcoma in Rats. Exp. Pathol. 1975, 10, 279-284.
3) Schaumann, W.; Determan, H.; Bicker, U.; Rothe, W.; Letten-
bauer, G.; Kampe, W. Use of 2- Cyanoaziridine and its deriva-
tives for Immunostimulation. Ger. Offen. 2,736,296, Feb 22,
(
+
+
+
(
M ), 196.9 (M - H
2
CdC(NH
NCO), 162.9 (M - cysteine), 136.9 (C
suggests the possible presence of compound 16.
2
)CO
2
H), 164.9 (M - C
6 5
H -
+
6
H
5
NHCONH ). It
2
1
979.
Rea ction of Im exon w ith N-Acetylcystein e. A solution
of imexon (2,110 mg, 1 mmol) and N-acetylcysteine (165 mg,
(4) Iyengar, B. S.; Dorr, R. T.; Alberts, D. S.; Hersh, E. M.; Salmon,
S. E.; Remers, W. A. Novel Antitumor 2-Cyanoaziridine-1-
carboxamides. J . Med. Chem. 1999, 42, 510-514.
5) Bicker, U.; Kampe, W.; Steingross, W. U.S. Patent 4,083,987,
Apr 11, 1978.
1
mmol) in 2 mL of Tris buffer (pH 7.0) was warmed at 37 °C
(
for 24 h. TLC on silica gel with MeOH-CHCl (2:8 v/v) then
3
indicated that all starting material was consumed. The solu-
tion was concentrated under reduced pressure, and the
residual solid was extracted with 5 mL of MeOH. This extract
was filtered and concentrated under reduced pressure, afford-
(6) Bicker, U.; Hebold, G. Cancerostatic Action of the Immune-
Stimulating Compound 4-imino- 1,3-diazabicyclo[3.1.0]hexan-
2
-one, BM 06 002 (Proposed INN Name Imexon) on various
Transplantation Tumors. IRCS Med. Sci.: Cancer; Hematol.
Immunol. Allergy, Pharmacol. 1977, 5, 428.
ing 67 mg (24.3% yield) of a mixture of 19 and 20 as white
1
crystals, which had mp 102-105 °C; H NMR (D
2
O, DSS) δ
(
7) Hersh, E. M.; Dorr, R. T.; Curtis, R. A.; Prince, J . E. Antitumor
Effects of Imexon Combined with Myelosuppressive and Non
Myelosuppressive Anticancer Agents. Proc. Am. Assoc. Cancer
Res. 1995, 36, 294.
2
.06 (d, 3, N-CH
3
), 2.9-3.2 (m, 4, CH
2
), 4.365 (1, CH), 4.51
+
+
(
1, CH); MS (APCI) m/z 275 (MH ) and 233 (MH - CH
2
CO)
+
+
for 19 (C
2
2
9
H
N
13
N
3
O
5
S), 276 (MH ) and 234 (MH - CH
2
CO) for
+
0 (C
9
H
13
4
O
5
S); HRMS (FAB) 275.0818 (calcd for 19 MH ,
(8) Micksche, M.; Kokoschka, E. M.; Sagaster, P.; Bicker, U. Phase
I Study for a new Immunostimulant Drug, BM 06 002, in Man.
In Immune Modulation and Control of Neoplasia by Adjuvant
Therapy; Chirigos, M. A., Ed.; Raven Press: New York, 1978;
pp 403-413.
+
75.08137), 276.06630 (calcd for 20 MH , 276.06538).
The sample from NMR was concentrated under reduced
pressure, and the residue was dissolved in water, kept at room
temperature for one week, and evaporated under reduced
pressure. The mass spectrum (APCI) then showed only the
peak at m/z 276 and no peak at m/z 275, indicating completion
of the hydrolysis of 19 to 20.
(
9) Hersh, E. M.; Gschwind, C. R.; Taylor, C. W.; Dorr, R. T.; Taetle,
R.; Salmon, S. E. Antiproliferative and Antitumor Activity of
the 2-Cyanoaziridine Compound Imexon on Tumor Cell Lines
and Fresh Human Tumor Cells In Vitro. J . Natl. Cancer Inst.
1955, 84, 1238-1244.
Rela tive Rea ctivities of Im exon a n d 2-Cya n oa zir id in e-
1
-ca r boxa m id e w ith Cystein e. A solution of 11 mg (0.1
(10) Tingle, M. D.; Lavens, S. E.; Park, B. K. Depletion of Hepatic
mmol) of imexon (2), 11 mg (0.1 mmol) of 2-cyanoaziridine-1-
carboxamide (1), and 12 mg (0.1 mmol) of DL-cysteine in 0.6
Glutathione by Ciamexon in the Rat. Hum. Exp. Toxicol. 1991,
1
0, 489-490.
(
11) Dvorakova, K.; Payne, C.; Tome, M.; Briehl, M.; McClure, T.;
Dorr, R. T. Induction of Oxidative Stress and Apoptosis in
Myeloma Cells by Imexon. Biochem. Pharmacol. 2000, 60, 749-
mL of D
2
O, containing DSS as internal standard, was analyzed
by H NMR spectrometry after 6 h. The integrals of the dd at
.8 ppm for the CH of imexon and the dd at 3.38 ppm for the
1
3
7
58.
CH in 1 were compared with the corresponding peaks in
separate solutions of each of these compounds in the same
solvent. These spectra showed that the ratio of imexon to 1
consumed was 4:1.
Rela tive Rea ctivities of Im exon a n d N-Meth ylm a le-
im id e. A solution of imexon (11 mg, 0.1 mmol), N-methyl-
maleimide (11 mg, 0.1 mmol), and cysteine (12 mg, 0.1 mmol)
(
(
(
12) Dvorakova, K.; Payne, C.; Tome, M.; Briehl, M.; Dorr, R. T.
Induction of Mitochondrial Changes in Myeloma Cells by Im-
exon. Blood 2001, 97, 1-8.
13) Preussman, R.; Schneider, H.; Epple, F. Investigation on the
Detection of Alkylating Agents. Artzneimittelforsch. 1969, 19,
1059-1073.
14) Dermer, O. C.; Ham, G. E. Ethyleneimine and other Aziridines.
Chemistry and Applications; New York, Academic Press: 1969.
(15) Wagner, E.; Xiang, Y. B.; Baumann, K.; Gueck, J .; Eschenmoser,
A. Chemistry of R-Aminonitrile. Aziridine-2-carbonitrile, A
Source of Racemic O-3-Phosphoserinenitrile and Glycolaldehyde
Phosphate. Helv. Chim. Acta 1990, 73, 1391-1409.
2
in 0.6 mL of D O containing DSS as an internal standard was
1
examined by H NMR spectrometry after 6 h at room temper-
ature. The resulting spectrum was compared with reference
spectra of imexon and of N-methyleimide without cysteine in
the same solvent. These spectra showed that the imexon in
the mixture was unchanged, whereas the N-methyleimide was
nearly completely reacted, as indicated by the almost total loss
of the vinyl proton singlet at 6.83 ppm.
(
16) Dvorakova, K.; Waltmire, C. N.; Payne, C. M.; Vasquez, M. A.;
Tome, M. E.; Briehl, M. M.; Dorr, R. T. Characterization of an
RPMI 8226 Myeloma Cell Line Resistant to the Anticancer Drug
Imexon., Proc. Am. Assoc. Cancer. Res. 2001, 42, 644.
Tr ea tm en t of Aceton itr ile w ith Cystein e. A mixture of
acetonitrile (21, 0.5 mL) and DL-cysteine (40.9 mg) in 2 mL of
deionized water was stirred at room temperature and moni-
tored by TLC on a silica gel plate with 10% methanol in
chloroform as solvent. There was no reaction indicated at 6 h.
Increasing the time another 6 h at 40 °C did not produce any
reaction. In contrast, when the reaction was run in the
presence of sodium ethoxide as described in the literature,17
the sodium salt of 2-methylthiazoline-4-carboxylic acid (22)
was formed.
(17) Ohta, G.; Nagase, O.; Hosokawa, Y.; Tagawa, H.; Shimizu, M.
Pantothenic Acid and Its Related Compounds. III. Chemical
Studies. 2. Synthesis of Di-D-panthenoyl-L-cystine, Chem. Pharm.
Bull. (Tokyo) 1967, 15, 644-647.
18) Allen, C. F. H.; Spangler, F. W.; Webster, E. R. Ethyleneimine.
Organic Syntheses; Wiley: New York, 1963; Collect. Vol. IV, p
p 433.
(
(19) Najer, H.; Guidicelli, J .; Menin, J .; Morel, C. Isomerization of
N,N-Ethylene-N-arylureas. Bull. Soc. Chim. Fr. 1963, 323-328.
J M030225V