Synthesis of novel [1-aziridinyl-(hydroxyimino)methyl]arenes and their cytotoxic activity

Article abstract of DOI:10.1007/s10593-009-0245-3

The synthesis has been developed for novel potential anticancer agents whose structures contain an aziridine amidoxime group. The cytotoxic activity of all of the target compounds on different cell lines are given together with the in vitro IC₅₀ and LD₅₀ values found. The most promising anticancer agents were found to be the methyl 4-[1-aziridinyl(hydroxyimino) methyl]benzoate, 1-aziridinyl(2-naphthyl)-methanone oxime, and 1-aziridinyl(2-quinolyl)methanone oxime.

Full text of DOI:10.1007/s10593-009-0245-3

Chemistry of Heterocyclic Compounds, Vol. 45, No. 2, 2009
SYNTHESIS OF NOVEL [1-AZIRIDINYL-
(HYDROXYIMINO)METHYL]ARENES
AND THEIR CYTOTOXIC ACTIVITY
A. Grigorjeva1*, A. Jirgensons1, I. Domracheva1, E. Yashchenko1,
I. Shestakova1, V. Andrianov1, and I. Kalvinsh1
The synthesis has been developed for novel potential anticancer agents whose structures contain an
aziridine amidoxime group. The cytotoxic activity of all of the target compounds on different cell lines
are given together with the in vitro IC50 and LD50 values found. The most promising anticancer agents
were found to be the methyl 4-[1-aziridinyl(hydroxyimino)methyl]benzoate, 1-aziridinyl(2-naphthyl)-
methanone oxime, and 1-aziridinyl(2-quinolyl)methanone oxime.
Keywords: aziridine, amidoximes, hydroxyimidoyl chlorides, oximes, cytotoxic activity.
The development of efficient anticancer preparations is a complex problem in contemporary
pharmaceutical chemistry [1]. Problems in cancer medication include especially the emergence of resistance of
infected cells against new preparations and also the need to discover substances suppressing the reproduction of
cancer infected cells without affecting metabolic processes in the host organism cells [2, 3].
Prviously have developed the synthesis of a novel class of cytotoxic agents which contain two
functional groups of an amidoxime [4, 5]. There was aziridine-containing amidoxime derivatives due mostly is
to their high cytotoxicity and anticancer activity. Selected examples of the most active of these compounds were
compounds 1a,b. However, a limitation of these compounds is the restricted possibility of modification of the
OH
N
OH
N
N
N
N
N
OH
OH
N
N
N
1b
1a
_______
* To whom correspondence should be addressed, e-mail: anna@osi.lv.
1Latvian Institute of Organic Synthesis, Riga LV-1006, Latvia, e-mail: aigars@osi.lv.
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 203-213, February, 2009. Original
article submitted July 4, 2008.
0009-3122/09/4502-0161©2009 Springer Science+Business Media, Inc.
161

basic structure which hinders the improvement of pharmacokinetic properties of novel compounds. In this
connection a search for novel cytotoxic compounds has been carried out. We chose structural analogs of
compounds 1a,b which contain one aziridine amidoxime functional group having the structural formulae 2-5 as
synthetic targets (Table 1).
O
MeO
N
O
N
N
N
X
OH
OH
OH
R1
OMe
N
N
N
4, 5
2
3
4 X = CH; 5 X = N, R1 = H, CONH2, CO2Me
Synthesis of compound 2 was carried out in several steps using dimethyl pyridine-2,6-dicarboxylate 6 as
starting material.
NaBH4
PCC
OMe
MeO
MeO
N
N
MeOH
58%
CH2Cl2
64%
O
O
O
OH
6
7
NH2OH · HCl
NCS
MeO
MeO
Py, MeOH
66%
DMF
40%
N
N
O
O
O
O
NOH
9
8
,
HN
Et3N
NOH
MeO
NOH
MeO
N
N
MeCN
50%
O
N
Cl
10
2
PCC = pyridinium chlorochromate, NCS = N-chlorosuccinimide
Sodium borohydride (ether solution, 3 equivalents) was used to achieve the selective reduction of one of
the ester groups in the pyridine-2,6-dicarboxylate 6 and a large quantity of methanol was optimum conditions
for preparing compound 7 [6]. Aldehyde 8 was prepared from compound 7 using pyridinium chlorochromate as
oxidant.
The corresponding oxime 9 was obtained in moderate yield using pyridine as the base. The
hydroxyimidoyl chloride 10 was prepared by treating methyl 6-(hydroxyiminomethyl)pyridine-2-carboxylate (9)
with N-chlorosuccinimide in DMF. The reaction was carried out at temperature sligthly increased to 40ºC.
Reaction of hydroxyimidoyl chloride 10 with aziridine gave the final product methyl 6-[1-
aziridinyl(hydroxyimino)methyl]pyridine-2-carboxylate (2). Formation of side products was also observed due
to instability of the aziridine ring.
Hydrolysis of the methyl ester group in compound 2 was unsuccessful, a full degradation of the
aziridine ring occurring under these reaction conditions.
162

TABLE 1. Characteristics of Compounds 2-5
Found, %
——————
Calculated, %
Com-
pound
Empirical
formula
mp, °C
Yield, %
С
Н
N
2
50.68
50.21
59.85
59.99
72.39
72.04
66.34
66.47
62.53
61.99
58.64
58.86
5.48
5.48
5.49
5.49
5.69
5.81
5.11
5.30
4.98
4.83
4.66
4.94
17.08
17.56
12.39
12.72
12.83
12.92
19.19
19.38
15.01
15.49
20.51
21.12
92-96
147-151
141-142
110-112
59-62
50
44
14
17
36
25
C10H11N3O3 ⋅H2O
C10H12N2O3
3
4
C13H12N2O ⋅ 0.25H2O
C12H11N3O ⋅ 0.2H2O
C14H13N3O3
5а
5b
5c
C13H12N4O2 ⋅ 0.2H2O
157-159
Commercially available 4-hydroxymethylbenzoic acid (11) was used as the starting material for the
synthesis of 3. The carboxyl group was first esterified [7] and the product 12 obtained was oxidized by using the
Dess-Martin reagent. Further synthesis of compound 3 followed the method reported above for compound 2.
OMe
O
O
Me
DMP
O
O
HCl
HO
MeOH
71%
CH2Cl2
18%
O
OH
OMe
OH
13
12
11
OMe
OMe
HN
NCS
O
NH2OH · HCl
O
O
Et3N, MeCN
44%
NOH
DMF
68%
Py/MeOH
61%
NOH
N
NOH
Cl
14
15
3
DMP− Dess-Martin reagent
Hydrolysis of the ester group in compound 3 (on attempt to obtain sodium salt) was unsuccessful as in
the preceding experiment with ester 2.
For the preparation of compound 4 the naphthalenecarbaldehyde 16 was treated with hydroxylamine in
the presence of sodium bicarbonate in THF solution. Conversion to the hydroxyimidoyl chloride 18 and
subsequent reaction with aziridine gave the target compound 4.
NH2OH · HCl
NCS
NaHCO3
O
N
THF
88%
DMF
98%
OH
H
17
16
,
Et3N
HN
NOH
N
MeCN
14 %
OH
Cl
N
4
18
163

The target compounds 5a-c were synthesized differently to the method reported above. Reaction of the
quinolinecarbonitrile 19 with hydroxylamine in the presence of sodium bicarbonate in acetonitrile solution at
room temperature gave the N-hydroxyquinoline-2-carboxamidine (20) in 77% yield [8].
NH2OH · HCl
NaHCO3
NaNO2
HON
MeOH, H2O
77%
N
conc. HCl
84%
N
N
NH2
20
19
R
,
HN
Et3N
HON
HON
MeCN
N
N
Cl
N
21
R
5a–c
5 a R = H, b R = C(O)NH2, c R = CO2Me
A subsequent diazotization reaction was carried out under quite rigid conditions treating the amidoxime
20 with sodium nitrite in conc. HCl. The hydroxyimidoyl chloride 21 obtained as a result was separated in a
pure state. The product was washed with a large quantity of cold water to remove excess hydrochloric acid.
Compound 21 served as the starting material for preparation of the target compounds 5a-c by using aziridine,
the amide or methyl ester of aziridine carboxylic acid as reaction components.
The cytotoxic effect (IC50) of the target compounds 2-4, 5a-c was tested on monolayer cell lines
obtained from the ATCC collection (Tables 2 and 3).
The following cell lines were used: 3T3 – mouse embryonic fibroblasts, HT-1080 – human connective
tissue fibrosarcoma, MG-22A – mouse hepatoma, MDA-MB-453s (MDA) – human breast adenocarcinoma,
estrogen negative, CCRF S-180II (CCL-8) – mouse sarcoma, monolayer, MCF-7 – human adenocarcinoma,
estrogen positive, Caco-2 – human intestinal adenocarcinoma, H9C2 – rat cardiomyocytes, HepG2 – human
hepatocyte carcinoma, Capan-2 – human pancreatic adenocarcinoma, PANC-1 – human pancreatic carcinoma, and
HPAF-II – human pancreatic carcinoma. The MTT and CV tests were used to investigate the IC50 cytotoxic effect.
The results of the CV and MTT tests depended on the level of degradation of the membrane structure
and/or the degree of suppression of the oxidation-reduction system in the cell. To avoid in vivo experiments for
determination of the LD50 in vitro test on the cell line 3T3 was used for calculation the LD50 value. Staining was
carried out with Neutral Red as reported in the method "Interagency Coordinating Committee on the Validation
of Alternative Methods and National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods".
Compound 4 and 5a were comparable in their efficiency with 1a and 1b (for compound 4 the effect is on the
Capan-2 line weaker and this may be connected with features of the tumor metabolism). Compound 3 was less
effective but also less toxic, its IC50 is comparable with known preparation. Compound 2 had little effect and
compounds 5b and 5c were found to be inactive. Compounds 3, 4, 5a could be considered as promising.
EXPERIMENTAL
1H NMR spectra were recorded on a Varian Mercury VX-200 instrument (200 MHz) using DMSO-d6 as
solvent (compounds 2-4, 5a-c, 7-10, 14, 15, 17, 18, 20, 21) or CDCl3 (compounds 12 ad 13) with TMS as
164

165

internal standard. The course of the reaction and the purity of the product were monitored using TLC on
Kieselgel 60 F254 plates (Merck) in the system ethyl acetate–hexane in different ratios and vizualized using a UV
lamp with 254 nm filter. Column chromatography used Kieselgel silica gel (35-70 and 60-200 µm). Melting
points were determined on an Optimelt apparatus and are not corrected.
Methyl 6-Hydroxymethylpyridine-2-carboxylate (7). Dimethyl pyridine-2,6-dicarboxylate (6) (1.95 g,
10 mmol) was dissolved in methanol (100 ml), sodium borohydride (1.13 g, 30 mmol) was added, and the reaction
mixture was stirred for 1.5 h at room temperature to reach full conversion of the starting material (TLC
monitoring). At the end of the reaction the filtrate was evaporated. The obtained residue was treated with cold water
(100 ml) and extracted with chloroform (3×50 ml). The extract was washed with water to give 0.97 g (57%) as
colorless crystalline product. 1H NMR spectrum, δ, ppm (J, Hz): 3.86 (3H, s, OCH3); 4.61 (2H, d, J = 6, CH2OH);
5.56 (1H, t, J = 6, CH2OH); 7.71 (1H, d, J = 7.4, 5-CH pyridine); 7.93-8.04 (2H, m, 3,4-CH pyridine).
Methyl 6-Formylpyridine-2-carboxylate (8). Compound 7 (1 g, 6 mmol) was dissolved in
dichloromethane (70 ml), pyridinium chlorochromate (1.94 g, 8.98 mmol) was added, and the mixture stirred for
4 h at room temperature (monitored by TLC). The mixture was filtered through Celite and the filtrate evaporated
and repeatedly filtered through silica gel. After evaporation, the residue was purified by column chromatography
1
eluting with petroleum ether–ethyl acetate (1:1). Yield 0.63 g (64%). H NMR spectrum, δ, ppm (J, Hz): 3.94
(3H, s, OCH3); 8.16 (1H, dd, J = 1.4 and J = 6.2, 3-CH pyridine); 8.25 (1H, t, J = 8, 4-CH pyridine); 8.33 (1H,
dd, J = 1.4 and J = 6.0, 5-CH pyridine); 10.02 (1H, s, CHO).
Methyl 6-(Hydroxyiminomethyl)pyridine-2-carboxylate (9). Compound 8 (250 mg, 1.5 mmol) and
hydroxylamine hydrochloride (118 mg, 1.7 mmol) were mixed dry, a mixture of pyridine and methanol (1:1)
was added, and the reaction mixture was refluxed to completion of the reaction (TLC monitoring), then was
evaporated and residue was suspending in dichloromethane and washed with aqueous sodium bicarbonate
solution (10 ml saturated sodium bicarbonate + 40 ml water). The organic layer was separated, dried over
sodium sulphate, and evaporated. Product yield 0.236 g (66%). 1H NMR spectrum, δ, ppm: 3.88 (3H, s, OCH3);
8.02 (3H, s, pyridine); 8.13 (1H, s, CHNOH); 11.87 (1H, s, CHNOH).
Preparation of Compounds 10, 15, 18 (General Method). N-Chlorosuccinimide (1.20 mmol) was
added to a solution of the starting oxime (1 mmol) in DMF (5 ml). The reaction mixture was stirred for 2 h at
room temperature and then held for 1 h at 40ºC (for the synthesis of compound 18 the temperature was raised to
80ºC). The mixture was cooled and water with ice (50 ml) was added. After cooling, the precipitate was filtered
and thoroughly washed with cold water.
1
Compound 10. Yield 55%. H NMR spectrum, δ, ppm: 3.91 (3H, s, OCH3); 8.08-8.12 (3H, m,
pyridine).
1
Compound 15. Yield 68%. H NMR spectrum, δ, ppm: 3.87 (3H, s, OCH3); 7.99 (4H, dd, J = 8.8 and
J = 1.3, C6H5).
Compound 18. Yield 98%. 1H NMR spectrum, δ, ppm: 7.60 (2H, m, C10H7); 8.06 (5H, m, C10H7); 8.35
(1H, s, 1-CH C10H7); 12.53 (1H, s, NOH).
Methyl 4-Hydroxymethylbenzoate (12). 4-Hydroxymethylbenzoic acid (11) (1.09 g, 6.57 mmol) was
dissolved in methanol (30 ml), acidified with HCl (3 ml), and refluxed for 4 h. After cooling, the reaction mixture
was neutralized with triethylamine (TEA) to pH 7 and evaporated. The residue obtained was dissolved in
dichloromethane (20 ml) and washed with a 5% aqueous solution of sodium bicarbonate (2×20 ml). The organic
layer was separated, dried over sodium sulphate, and evaporated. Yield 782 mg (72%). 1H NMR spectrum, δ, ppm
(J, Hz): 3.11 (1H, br. s, OH); 3.85 (3H, s, OCH3); 4.67 (2H, s, CH2); 7.37 (2H, d, J = 8, 2,6-CH Ph); 7.95 (2H, d,
J = 8, 3,5-CH Ph).
Methyl Formylbenzoate (13). The Dess-Martin reagent (0.54 ml, 1.7 mmol) was added to a solution of
compound 12 (0.264 g, 1.6 mmol) in dichloromethane (10 ml), the reaction mixture was stirred at room
temperature for 12 h, extracted with ether. The organic layer was washed with a saturated solution of sodium
bicarbonate and separated. The aqueous layer was repeatedly extracted with ether. The extracts were
166

combined, washed with saturated sodium chloride solution, and dried over sodium sulphate. Evaporation gave
the product which was purified by column chromatography using ethyl acetate–petroleum ether (1:4) as eluent.
1
Yield 49 mg (18%). H NMR spectrum, δ, ppm (J, Hz): 3.96 (3H, s, OCH3); 7.96 (2H, d, J = 8, 2,6-CH Ph);
8.18 (2H, d, J = 8, 3,5-CH Ph); 10.09 (1H, s, COH).
Methyl 4-(Hydroxyiminomethyl)benzoate (14) was prepared by the method reported above for
1
compound 9. Yield 61%. H NMR spectrum, δ, ppm (J, Hz): 3.85 (3H, s, OCH3); 7.73 (2H, d, J = 8.8, 2,6-CH
Ph); 7.97 (2H, d, J = 8.2, 3,5-CH Ph); 8.22 (1H, s, CHNOH); 11.87 (1H, s, NOH).
2-Naphthalenecarbaldehyde Oxime (17). Aqueous sodium bicarbonate solution (3 ml) was added to a
solution of compound 16 (312 mg, 2 mmol) in THF (7 ml). After 10 min hydroxylamine hydrochloride (278 mg,
4 mmol) was added. The reaction mixture was held for 12 h at room temperature, extraction was carried out with
ethyl acetate, extract dried over sodium sulphate and evaporated. Column chromatography of the mixture
obtained gave compound 17 (eluent petroleum ether–ethyl acetate, 1:2). Yield 88%. 1H NMR spectrum, δ, ppm
(J, Hz): 7.57 (2H, m, C10H7); 7.82 (1H, d, J = 8.8, H-4 C10H7); 7.91 (3H, m, C10H7); 8.00 (1H, s, H-1 C10H7);
8.29 (1H, s, CHNOH); 11.34 (1H, s, NOH).
N-Hydroxyquinoline-2-carboxamidine (20). Hydroxylamine hydrochloride (139 mg, 2 mmol) was
dissolved in methanol (15 ml), cooled in an ice bath, and an aqueous solution of sodium bicarbonate (168 mg,
2 mmol/5 ml of water) was added with stirring at room temperature for 30 min. Freshly prepared hydroxylamine
solution was added to a solution of the 3-quinolinecarbonitrile (19) (308 mg, 2 mmol) in methanol (15 ml). The
reaction mixture was stirred at room temperature for 1.5 h and then for 1.5 h at 40-50ºC. The precipitate formed
1
was filtered off and washed with a large amount of water. Yield 77%. H NMR spectrum, δ, ppm (J, Hz): 5.99
(2H, br. s, NH2); 7.62 (1H, dt, J = 1.6 and J = 7.2, 6-CH quinoline); 7.79 (1H, dt, J = 1.4 and 7.2, 7-CH
quinoline); 8.01 (3H, dd, J = 4.2 and J = 7.8, quinoline); 8.33 (1H, d, J = 8.6, 8-CH quinoline); 10.21 (1H, s,
NOH).
Quinoline-2-carboximidoyl Chloride (21). A solution of compound 20 (280 mg, 1.5 mmol) in conc.
HCl (5 ml) was cooled to 0-5ºC, an aqueous solution of sodium nitrite (332 mg, 4 mmol/1 ml water) was added
and stirred for 1 h at 0-4ºC and then at room temperature for 12 h. The precipitate formed was filtered off and
1
dried in vacuo. Yield of product 0.26 g (84%). H NMR spectrum, δ, ppm (J, Hz): 7.68 (1H, t, J = 7.4, 6-CH
quinoline); 7.83 (1H, t, J = 7.6, 7-CH quinoline); 8.05 (3H, dd, J = 6.4 and J = 9.4, quinoline); 8.44 (1H, d,
J = 8.6, 8-CH quinoline); 10.23 (1H, s, NOH).
Preparation of Target Compounds 2-5 (General Method). A mixture of TEA and aziridine in
acetonitrile was cooled to 0ºC. A suspension of the hydroxyimidoyl chloride in acetonitrile was added (2 ml of
solvent for 0.5 mmol of starting material, the reaction being carried out in an inert atmosphere) and the reaction
mixture was held for 30 min at the low temperature and then at room temperature until the reaction was
complete (TLC monitoring). In some cases a precipitate of triethylamine hydrochloride was formed and this was
filtered off. The filtrate was evaporated and the tarry residue was triturated with ether using a glass rod. The
precipitated material was filtered off and washed on the filter with a small amount of ether.
Methyl 6-[1-Aziridinyl(hydroxyimino)methyl]pyridine-2-carboxylate (2) was prepared from TEA
(0.29 ml, 2.1 mmol), aziridine (0.08 ml, 1.6 mmol), and the starting product 10 (113 mg, 0.53 mmol). Yield of
compound 2 70 mg (50%). 1H NMR spectrum, δ, ppm: 2.24 (4H, s, CH2 aziridine); 3.90 (3H, s, OCH3); 8.0 (3H,
m, CH pyridine); 10.79 (1H, s, NOH).
Methyl 4-[1-Aziridinyl(hydroxyimino)methyl]benzoate (3). TEA (0.28 ml, 2 mmol), aziridine (0.08
ml, 1.6 mmol), and the starting product 15 (106 mg, 0.5 mmol) were stirred at room temperature for 1 h. Yield
1
of compound 3 48 mg (44%). H NMR spectrum, δ, ppm (J, Hz): 2.21 (4H, s, CH2 aziridine); 3.86 (3H, s,
OCH3); 7.80 (2H, d, J = 8.6, CH Ph); 7.97 (2H, d, J = 8.6, CH Ph); 10.71 (1H, s, NOH).
1-Aziridinyl(2-naphthyl)methanone Oxime (4). TEA (0.56 ml, 4 mmol), aziridine (0.15 ml, 3 mmol),
and the starting product 18 (210 mg, 1 mmol) were stirred for 1.5 h at room temperature. Yield of compound 4
30 mg (14%). 1H NMR spectrum, δ, ppm: 2.27 (4H, s, CH2 aziridine); 7.54 (2H, m, C10H7); 7.90 (4H, m, C10H7);
8.21 (1H, s, 1-CH C10H7); 10.54 (1H, s, NOH).
167

1-Aziridinyl(2-quinolyl)methanone Oxime (5a) TEA (0.56 ml, 4 mmol), aziridine (0.15 ml, 3 mmol),
and starting product 21 (207 mg, 1 mmol) were stirred at room temperature for 1 h. Yield of compound 5a 37
mg (17%). 1H NMR spectrum, δ, ppm (J, Hz): 2.31 (4H, s, CH2 aziridine); 7.67 (1H, t, J = 6.4, 6-CH quinoline);
7.78 (1H, t, J = 6.4, 7-CH quinoline); 8.00 (3H, m, quinoline); 8.32 (1H, d, J = 8.8, 8-CH quinoline); 10.89
(1H, s, NOH).
1-[Hydroxyimino(2-quinolyl)methyl]aziridine-2-carboxamide (5b). TEA (0.44 ml, 3.2 mmol),
aziridinecarboxamide (206 mg, 2.4 mmol), and the starting product 21 (150 mg, 0.8 mmol) were stirred at room
temperature for 1.5 h. The product was filtered off, washed with ethyl acetate, and dried in vacuo. Yield of
compound 5b 40 mg (25%). 1H NMR spectrum, δ, ppm (J, Hz): 2.50 (2H, CH2 aziridine, proton signal situated
under the solvent signal); 3.02 (1H, dd, J = 3.6 and J = 1.5, CH aziridine); 7.09 (2H, s, NH2); 7.58 (1H, s, CH
quinoline); 7.64 (1H, t, J = 6.6, CH quinoline); 7.95 (3H, m, CH quinoline); 8.28 (1H, d, J = 8.8, 8-CH
quinoline); 10.94 (1H, s, NOH).
Methyl 1-[Hydroxyimino(2-quinolyl)methyl]aziridine-2-carboxylate (5c) TEA (0.56 ml, 3.2 mmol),
methyl aziridine carboxylate (300 mg, 3 mmol) and the starting product 21 (190 mg, 1.0 mmol) wered stirred for
3 h at room temperature. After evaporation, the oily product was purified by column chromatography eluting
1
with a mixture of ethyl acetate and petroleum ether (1:1). Yield of compound 5c 100 mg (36%). H NMR
spectrum, δ, ppm (J, Hz): 2.67 (1H, d, J = 3.4, CH aziridine); 2.73 (1H, d, J = 6, CH aziridine); 3.23 (1H, proton
signal partly situated under the D2O signal, CH aziridine); 3.61 (3H, s, OCH3); 7.62 (1H, t, J = 6.6, CH
quinoline); 7.78 (1H, dt, J = 1.4 and J = 8, CH quinoline); 7.94 (3H, dd, J = 4.2 and J = 4.2, CH quinoline); 8.32
(1H, d, J = 8.8, 8-CH quinoline); 11.07 (1H, s, NOH).
Biological Screening. The cytotoxic properties of the target compounds in vitro were determined on 96
well panels using the vital dyes MTT and CV in accordance with methods [9, 10] validated [11].
This work was carried out with the support of the European Regional Development Fund
(VPD1/ERAF/CFLA/05/APK/2.5.1/000029/014).
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