Synthesis and biological evaluation of aziridin-1-yl oxime-based vorinostat analogs as anticancer agents

Article abstract of DOI:10.1007/s10593-015-1752-z

The suberoyl anilide hydroxamic acid (vorinostat) analogs with the aziridin-1-yl oxime moiety as a possible metal chelating functionality have been synthesized. Their biological activity and stability under physiological conditions have been evaluated. Although some of the synthesized compounds demonstrated high antiproliferative activity against human HT1080 fibrosarcoma (HT1080, IC₅₀ 0.3-7.7 μM) comparable to vorinostat (HT1080, IC₅₀ 2.4 μM), they showed only weak histone deacetylase inhibition activity in HeLa cell line extracts.

Full text of DOI:10.1007/s10593-015-1752-z

DOI 10.1007/s10593-015-1752-z
Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
Synthesis and biological evaluation of aziridin-1-yl
oxime-based vorinostat analogs as anticancer agents
Anna Nikitjuka1*, Irina Shestakova1, Nadezhda Romanchikova1, Aigars Jirgensons1
1 Latvian Institute of Organic Synthesis,
21 Aizkraukles St., Riga LV-1009, Latvia; е-mail: anna@osi.lv
Submitted May 28, 2015
Accepted June 16, 2015
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2015, 51(7), 647–657
The suberoyl anilide hydroxamic acid (vorinostat) analogs with the aziridin-1-yl oxime moiety as a possible metal chelating functionality
have been synthesized. Their biological activity and stability under physiological conditions have been evaluated. Although some of the
synthesized compounds demonstrated high antiproliferative activity against human HT1080 fibrosarcoma (HT1080, IC50 0.3–7.7 M)
comparable to vorinostat (HT1080, IC50 2.4 M), they showed only weak histone deacetylase inhibition activity in HeLa cell line extracts.
Keywords: aldoxime, aziridin-1-yl oxime, histone deacetylase, hydroximoyl chloride, suberoyl anilide hydroxamic acid, cytotoxic activity.
Vorinostat (suberoyl anilide hydroxamic acid or SAHA) (1)
is a linear hydroxamic acid compound that is a potent
enzyme inhibitor with multiple targets including I and II
class histone deacetylase (HDAC). SAHA consists of a
chelating group (hydroxamic acid moiety) and six-carbon
spacer (connection unit) attached to a hydrophobic group
(phenyl). It has been shown that hydroxamic acid moiety of
SAHA binds to a zinc ion in the HDAC catalytic site
allowing the rest of the molecule to lie along the protein
surface with the phenyl ring oriented out of the catalytic
pocket.1
N-nucleophile (aziridine, aziridine derivative) to the nitrile
oxide generated from the hydroximoyl chloride in situ. An
alternative approach through the appropriate nitrile oxide
starting from the corresponding amidoxime has been
investigated by us previously.3 Unfortunately this approach
failed to provide nitrile oxides bearing an alkyl moiety.
The routes used for synthesis of SAHA analogs 5a–l, 6–8
are depicted in Schemes 1–7. Our initial attempt to
synthesize suberoyl anilide was the condensation of suberic
acid with aniline at high temperatures. However, the
desired compound was formed in low yield together with
by-products. This prompted us to use a different synthetic
route proposed by Mai.4 Thus, suberoyl anhydride (9) was
obtained in good yields by heating suberic acid with acetic
anhydride.4 Anhydride 9 was treated with aniline or aniline
derivatives to afford monoamides 10b–e,g,h which were
converted into the corresponding esters 11b–e,g,h by
reaction with methyl iodide in the presence of potassium
carbonate (Scheme 1, Method I). The second approach to
prepare intermediates 11 involved the conversion of
suberic acid anhydride (9) to its monomethyl ester 12
which was coupled with aniline or aniline derivatives using
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDCI)
(Scheme 1, Method II).
The aziridin-1-yl oxime group shows high structural
similarity with hydroxamic acid. Moreover the aziridin-
1-yl oxime moiety has a potential to form a covalent
interaction, particularly when activated with zinc ions.
Following this idea we aimed to synthesize a small
molecule library of SAHA analogs and test their
anticancer, as well as HDAC inhibitory activity. It has been
already shown that aromatic compounds containing
aziridin-1-yl oxime groups show high cytotoxic activity
against different cancer cell lines.2
The key intermediates in the described strategy are the
aliphatic aldoximes 2a–h, 3, and 4, which were converted
to the aziridin-1-yl oximes in a two-step one-pot reaction:
formation of hydroximoyl chloride and addition of
Esters 11a–h were converted to aldehydes by the
reduction of the ester functionality with lithium aluminum
hydride (LAH) in THF followed by the oxidation with
pyridinium chlorochromate (PCC). The reaction of aldehy-
des with hydroxylamine hydrochloride in the presence of
NaHCO3 or NaOAc afforded aldoximes 2a–h.
0009-3122/15/5107-0647©2015 Springer Science+Business Media New York
647

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
Scheme 1
NH2
H
O
O
O
N
R
6COOH
O
THF, rt, 30 min
Method I
94
10b e g h
R
 , ,
MeOH,
, 3h
MeI, K2CO3,
DMF, rt, 16 h
Method II
NH2
1. LiAlH4, THF, 0°C
2. PCC, CH2Cl2, rt
H
N
H
N
N
R
OH
6COOMe
6
HOOC 6 COOMe
EDCI, HOBt,
TEA, DMF, rt
O
3. NH2OHHCl,
NaOAc, H2O, EtOH,
rt, 16 h
O
H
R
R
12
11a h
2a h


2, (10,) 11
a
b
c
d
e
f
g
h
4-F
I
4-Me 4-MeO 2,6-Me2 4-PhO 4-EtO
4-Et
I
R
H
II
Method
I
I
I
II
I
Scheme 2
Synthesis of oxime 3 was accomplished by the reaction
of cycloheptanone (13) with potassium persulfate in
ethanol in the presence of sulfuric acid, and the resulting
6-hydroxyheptanoic acid ethyl ester was oxidized with
PCC (Scheme 2).5 The aldehyde group of the obtained
intermediate 14 was protected as acetal to give compound
15. Basic hydrolysis of the ester group and the coupling of
the resulting acid with aniline was followed by acetal
group cleavage. The resulting aldehyde 16 was suc-
cessfully converted to aldoxime 3 in the presence of
NaHCO3.
give the anilide alcohol which was subsequently oxidized
with PCC to afford aldehyde 186 which was converted to
the aldoxime 4.
Aziridin-1-yl oximes were prepared by chlorination of
oximes 2a–h, 3, 4. This provided hydroximoyl chlorides7
directly converted in situ to the nitrile oxide by treatment
the reaction mixture with triethylamine in great excess. The
following addition of aziridine allowed to obtain final
compounds 5a–l in low or moderate yields (Schemes 4, 5).
When N-chlorosuccinimide (NCS) in DMF was used for
the chlorination of aldoxime 2a, hydroximoyl chlorides
with chlorinated benzene ring were formed. This resulted,
after the treatment with aziridine, in monochlorinated
product 5a and dichlorinated product 5b (Scheme 4).
The synthetic route to the intermediate 4 starting from
ε-caprolactone (17) is outlined in Scheme 3. The capro-
lactone cycle was opened in the reaction with aniline to
Scheme 3
Scheme 4
648

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
The formation of hydroximoyl chloride without phenyl
ring chlorination was successfully carried out with NCS
and catalytic amount of pyridine in chloroform or dichloro-
methane (Scheme 5).8
Scheme 5
Figure 1. The Z- and E-isomers of compound 5a.
fibroplasts 3T3, human fibrosarcoma HT1080, mouse
hepatoma MG22A) using different colorimetric methods
(NR – neutral red, MTT – 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, and CV – crystal violet)
(Table 1).
Our results demonstrate that there is a difference in
cytotoxic activity of compounds bearing diverse substi-
tuents at the phenyl moiety. Thus, compounds having
doubly substituted phenyl rings 5b,g are inactive, however,
the activities of other SAHA analogs are comparable. A
variety of substitution is tolerated at position 4 of the
phenyl group when n = 6.
A series of homologous compounds was synthesized
with a different length of the spacer unit between phenyl
and aziridin-1-yl oxime groups. Thus, compounds 5k,l,
containing five- and four-carbon spacer, respectively,
dramatically lost cytotoxic activity in comparison with
compound 5c containing a six-carbon spacer.
The influence of the substitution at the aziridine cycle on
the example of compounds 6 and 7 was also examined. The
introduction of azabicyclic function (compound 6) or
carboxamide substituent (compound 7) contributes to the
loss of the cytotoxic activity. The aziridine substitution
with amidoxime functionality resulted in inactive com-
pound 8, but it might also be argued that its weak cytotoxic
activity resulted from the shorter spacer unit.
The obtained final compounds can be allocated in one of
the four toxicity categories based on their acute oral
toxicity properties according to the cut-off criteria
established by the current EU regulations.12 Thus, most of
the newly synthesized SAHA analogs 5a,c–f,h,j can be
classified as slightly toxic compounds (category 3), while
compounds 5b,g,i,k,l, 6–8 are practically non-toxic (cate-
gory 4).
To investigate the structure–activity relationship of
aziridin-1-yl oxime moiety, substituted analogs 6 and 7
were prepared following a similar synthetic protocol to that
shown in Scheme 4. 7-Azabicyclo[4.1.0]heptane required
for the synthesis of compound 6 was obtained according to
the procedure described in literature starting from
cyclohexene oxide (Scheme 6).9
Finally, the synthesis of amidoxime 8 was realized
starting from the commercially available 6-bromohexanoic
acid (19) which was converted to the corresponding
6-cyanohexanoic acid in the reaction with sodium cyanide,
followed by the reaction with aniline in the presence
of EDCI.10 Transformation of the nitrile group in com-
pound 20 to amidoxime group by the reaction with
hydroxylamine in isopropanol gave the desired product 8 in
quantitative yield.
Azirindin-1-yl oxime 5a was used as a model compound
for the investigation of the configuration of oxime. The
(Z)-configuration of the compound was confirmed by 2D
NMR NOESY spectra which showed a NOE between the
hydroxyl proton and the aziridine methylene protons (Fig. 1).
The stability of compound 5a was assessed by HPLC
method. These studies indicated that compound 5a was
stable at least 4 h in 0.05 M phosphate buffer solution (pH
7.4, 25°C), however after 24 h, it content in the analyzed
solution was 86%, and after 120 h, only 32%.
All synthesized aziridin-1-yl oximes 5a–l, 6–8, struc-
tural analogs of SAHA, were tested for their cytotoxic
activity on different cell lines (mouse embryonic
Selected SAHA analogs 5a,c,d,f,h–j were tested for
their ability to act as a histone deacetylase inhibitors in
Hella extract using SAHA as a reference inhibitor (Table 2).
Scheme 6
H
Scheme 7
H
N
1. NaCN, DMF
NH2OH HCl, NaHCO3
2-PrOH,
N
NH2
OH
Br
COOH
O
8
5N
5
5
N
2. PhNH2, EDCI,
HOBt, DMF
O
19
20
649

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
Table 1. Cytotoxic effect of SAHA analogs on different monolayer cell lines
HT1080
IC50  M (CV)
HT1080
IC50,  M (MTT)
MG22A
IC50,  M (CV)
MG22A
IC50,  M (MTT)
Com-
pound
3T3
n
R
LD50, mg/kg (NR)
SAHA
5a
5b
5c
5d
5e
5f
6
6
6
6
6
6
6
6
6
6
6
5
4
6
6
5
–
4-Cl
2,4-Cl2
H
NT*
97
2.4**
0.6
2.4**
0.6
NT
1.2
NT
0.9
NT
1.0
0.9
4.6
2.7
>10
0.5
3.0
0.6
3.6
7.7
>10
NT
>10
358
87
>10
0.6
NT
0.3
>10
1.0
4-F
92
0.9
0.6
0.6
4-Me
4-MeO
2,6-Me2
4-PhO
4-EtO
4-Et
H
121
128
482
153
233
146
220
261
275
2191
873
0.8
1.3
3.6
1.0
1.3
2.7
>10
NT
NT
NT
7.3
>10
1.0
>10
NT
NT
NT
7.3
5g
5h
5i
1.5
0.9
5j
3.6
5k
5l
H
3.8
3.8
7.6
–
>10
>10
>10
>10
NT
>10
>10
>10
>10
6
7
8
–
–
* NT – not tested.
** See ref.11
The results in Table 2 indicate that aziridin-1-yl oxime
analogs were inactive in HDAC inhibition test. The
synthesized SAHA analogs had significantly lower IC50
values than SAHA. The poor HDAC inhibition of aziridin-
1-yl oximes may indicate that HDAC is not the primary
pharmacological target responsible for their biological
activity.
In summary, we have synthesized a series of SAHA
analogs in which hydroxamic acid moiety is replaced by
aziridin-1-yl oxime group. We have shown that some of the
obtained compounds exhibit significant antiproliferative
activity against the growth of monolayer cell lines
including human HT1080 fibrosarcoma. However, all
tested compounds are weak inhibitors of HDAC suggesting
that aziridin-1-yl oxime analogs of SAHA do not enter
covalent interaction with zinc atom in the HDAC catalytic
pocket. The new enzymatic target of the obtained com-
pound library should be established in the future.
Experimental
1H NMR spectra were recorded on Varian 400 Mercury
(400 MHz), Bruker Fourier 300 (300 MHz), and Varian
200 (200 MHz) spectrometers. 13C NMR spectra were
recorded on a Varian 400 Mercury spectrometer (100 MHz).
The 1H chemical shifts are given relative to residual proton
signal of DMSO-d6 signal (2.50 ppm) or CDCl3 (7.26
ppm), the 13C chemical shifts – relative to DMSO-d6 signal
(39.5 ppm). Ultra-performance liquid chromatography
(UPLC) data were obtained on a Waters mass spectrometer
(column Acquity UPLC BEH-C18) using electrospray
ionization (ESI) in positive mode. Elemental analysis was
performed on a Carlo Erba EA1108 elemental analyzer.
Melting points were determined on a Standard Research
Systems Optimelt melting point apparatus and were uncor-
rected. Purification of compounds was performed by flash
silica gel chromatography using Merck Kieselgel (230–400
mesh), eluting with ethyl acetate and light petroleum ether
in different ratios. Thin-layer chromatography was
performed on silica gel and was visualized by staining with
KMnO4 or in UV at 210 or 254 nm. Reagents and starting
materials were obtained from commercial sources and used
as received.
Table 2. HDAC inhibition activity
of selected aziridin-1-yl oximes
IC50,  M,
30 min incubation
IC50,  M,
3 h incubation
Compound
SAHA
5a
5c
5d
5f
5h
5i
0.1
177
61
36
13
32
28
40
0.1
NT
70
37
20
31
30
24
Synthesis of target compounds 5a–c was realized
starting from intermediate 12 (Method II) as follows.
5j
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Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
8-Methoxy-8-oxooctanoic acid (12). The solution of
6.98 (1H, t, J = 7.4, H-4 Ph); 7.19–7.29 (2H, m, H-3,5 Ph);
7.55 (2H, d, J = 7.5, H-2,6 Ph); 9.63 (1H, t, J = 1.6,
CH2CHO); 9.80 (1H, s, NH).
compound 9 (2.0 g, 12.8 mmol) in methanol (6.5 ml) was
stirred at reflux in pressure tube during 3 h. The reaction
mixture was evaporated to dryness and treated with ether.
The white precipitate formed was filtered off. Yield 1.92 g
8-(Hydroxyimino)-N-phenyloctanamide (2a). The
obtained 8-oxo-N-phenyloctanamide (0.31 g, 1.3 mmol)
was dissolved in ethanol (5 ml), and sodium acetate (0.16 g,
2.0 mmol, 1.5 equiv) was added to the solution in one
portion followed by the addition of hydroxylamine
hydrochloride (0.14 g, 2.0 mmol, 1.5 equiv) solution in
water (10 ml). The reaction mixture was stirred for 16 h at
room temperature, and the precipitate that formed was
filtered off and dried in vacuo. Yield 0.29 g (89%), amor-
1
(80%). H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.15–1.28 (4H, m, (CH2)2(CH2)2CO2CH3); 1.37–
1.52 (4H, m, HO2C(CH2CH2CH2)2CO2CH3); 2.14 (2H, t,
J = 7.4, CH2CO2CH3); 2.24 (2H, t, J = 7.4, CH2CO2H);
3.54 (3H, s, CO2CH3, partially overlapped with water
signal); 11.94 (1H, br. s, CO2H).
Methyl 8-oxo-8-(phenylamino)octanoate (11a). Triethyl-
amine (1.7 ml, 12 mmol, 1.5 equiv), EDCI (2.10 g,
10.4 mmol, 1.3 equiv), and HOBT (1.40 g, 10.4 mmol,
1.3 equiv) were added to a solution of the intermediate 12
(1.38 g, 8 mmol) in DMF (8 ml), followed by the addition
of aniline (0.73 g, 8 mmol). The reaction mixture was
stirred overnight at room temperature, treated with water
(20 ml), and extracted with AcOEt. The organic phase was
washed with water, dried over Na2SO4, filtrated, and evapo-
rated. The residue was purified by flash chromatography
(eluent AcOEt–hexane, 1:2). Yield 1.62 g (77%), white
1
phous solid. H NMR spectrum (400 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.24–1.31 (4H, m, (CH2)2(CH2)2CHNOH);
1.34–1.41 (2H, m, NHC(O)CH2CH2); 1.49–1.58 (2H, m,
CH2CH2CHNOH); 2.15–2.22 (2H, m, NHC(O)CH2); 2.25
(2H, t, J = 7.4, CH2CHNOH); 6.60 (1H, t, J = 5.4,
CHNOH); 6.95–7.00 (1H, m, H-4 Ph); 7.20–7.27 (2H, m,
H-3,5 Ph); 7.49–7.55 (2H, m, H-2,6 Ph); 9.80 (1H, s, NH);
10.68 (1H, s, NOH).
(Z)-8-(Aziridin-1-yl)-N-(4-chlorophenyl)-8-(hydroxy-
imino)octanamide (5a). Aldoxime 2a (0.30 g, 1.21 mmol)
was dissolved in DMF (3 ml); NCS (0.34 g, 2.54 mmol,
2.1 equiv) was added in one portion. The reaction mixture
was kept at 70°C for 1.5 h, then cooled to 0°C, and
aziridine (0.78 ml, 15 mmol) was added. The reaction
mixture was allowed to warm up to room temperature,
stirred for an additional 1 h, diluted with AcOEt, and
extracted with water. The organic phase was washed
several times with AcOEt, dried over Na2SO4, filtrated, and
evaporated at water bath temperature 30°C. The residue
was purified by flash chromatography eluting with 5%
Et3N in AcOEt. Yield 165 mg (42%), white solid, mp 127.5–
1
amorphous solid. H NMR spectrum (200 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.19–1.34 (4H, m, (CH2)2(CH2)2CO2CH3);
1.42–1.62 (4H, m, NH(CH2CH2CH2)2CO2CH3); 2.21–2.29
(4H, m, NHCH2(CH2)4CH2CO2CH3); 3.54 (3H, s,
COOCH3); 6.98 (1H, t, J = 7.4, H-4 Ph); 7.24 (2H, t,
J = 7.9, H-3,5 Ph); 7.54 (2H, d, J = 7.4, H-2,6 Ph); 9.80
(1H, s, NH).
8-Hydroxy-N-phenyloctanamide. Lithium alumohydride
(0.12 g, 3.0 mmol, 1.1 equiv) was slowly added to a solution
of intermediate 11a (0.73 g, 2.8 mmol) in dry THF (14 ml)
at 0°C. After the full conversion of the starting material
(TLC control), the reaction mixture was treated with metha-
nol (1 ml), evaporated to dryness, then diluted with ethyl
acetate, and extracted with water. The organic phase was
dried over Na2SO4, filtered, and evaporated. The obtained
crude product was purified by flash chromatography
(eluent AcOEt–hexane, 1:2, gradient to AcOEt). Yield 0.51 g
(79%), white solid. 1H NMR spectrum (200 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.21–1.27 (6H, m, (CH2)3(CH2)2OH); 1.34–
1.40 (2H, m, NHC(O)CH2CH2); 1.49–1.56 (2H, m,
CH2CH2OH); 2.25 (2H, t, J = 7.4, NHC(O)CH2); 3.29–3.34
(2H, m, CH2OH, partially overlapped with water signal);
4.28 (1H, t, J = 5.1, OH); 6.95–7.00 (1H, m, H-4 Ph); 7.22–
7.27 (2H, m, H-3,5 Ph); 7.52–7.57 (2H, m, H-2,6 Ph); 9.79
(1H, s, NH).
1
129°C. H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.19–1.37 (4H, m, (CH2)2(CH2)2C(NC2H4)NOH);
1.34–1.68 (4H, m, (CH2CH2CH2)2C(NC2H4)NOH); 1.99
(4H, s, 2CH2 aziridine); 2.02–2.09 (2H, m, partially over-
lapped with aziridine signal, NHC(O)CH2); 2.27 (2H, t,
J = 7.4, CH2C(NC2H4)NOH); 7.31 (2H, d, J = 8.9, H-3,5 Ar);
7.59 (2H, d, J = 8.9, H-2,6 Ar); 9.49 (1H, s, NOH,); 9.94
(1H, s, NH). 13C NMR spectrum (100 MHz, DMSO-d6),
δ, ppm: 25.4; 26.3; 26.6; 28.8; 28.9; 31.4; 36.8; 121.0;
126.9; 129.0; 138.8; 156.9 (C=NOH); 171.9 (C=O). Found, %:
C 58.40; H 6.70; N 12.60. C16H22ClN3O2. Calculated (with
1.4% H2O): C 58.53; H 6.91; N 12.80.
(Z)-8-(Aziridin-1-yl)-N-(2,4-dichlorophenyl)-8-(hydroxy-
imino)octanamide (5b). Aldoxime 2a (0.50 g, 2 mmol)
was dissolved in DMF (10 ml); NCS (0.81 g, 6 mmol, 3 equiv)
was added, and the reaction mixture was kept at 70°C for 1 h.
An additional amount of NCS (0.27 g, 2 mmol) was added,
and reaction mixture was heated for 3 h (full conversion of
starting material, TLC control), then cooled to 0°C, and
aziridine (0.52 ml, 10 mmol) was added. The reaction
mixture was allowed to warm up to room temperature,
stirred for an additional 1 h, diluted with AcOEt, and
extracted with water. The organic phase was washed
several times with AcOEt, dried over Na2SO4, filtrated, and
evaporated at water bath temperature 30°C. The residue
was treated with ether, and the precipitate that formed was
8-Oxo-N-phenyloctanamide. A solution of the obtained
8-hydroxy-N-phenyloctanamide (0.43 g, 1.8 mmol) in
CH2Cl2 (18 ml) was added to a suspension of PCC (0.79 g,
3.7 mmol, 2 equiv) and Celite (0.79 g) in CH2Cl2 (18 ml).
The reaction was stirred until the full conversion of the
starting material (TLC control) and filtered through a short
silica gel column, eluting with ether. The filtrate was
1
evaporated. Yield 0.32 g (74%), white solid. H NMR
spectrum (200 MHz, DMSO-d6), δ, ppm (J, Hz): 1.22–1.30
(4H, m, (CH2)2(CH2)2CHO); 1.44–1.60 (4H, m,
NHC(O)(CH2CH2CH2)2CHO); 2.25 (2H, t, J = 7.4,
NHC(O)CH2); 2.38 (2H, td, J = 7.4, J = 1.6, CH2CHO);
651

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
filtered and dried. Yield 210 mg (29%), white solid, mp 112–
2 mmol) in DMF (2 ml) in one portion, and the mixture
was stirred for 30 min. Methyl iodide (75 l, 1.2 mmol)
was then added, and the reaction mixture was stirred for 16 h at
room temperature. Then it was diluted with water (20 ml)
and extracted with ethyl acetate (2×10 ml). The organic
layers were combined and washed with water (2×20 ml)
and brine (20 ml), dried over potassium sulfate, filtered,
evaporated, and treated with Et2O. Yield 0.36 g (64%).
1H NMR spectrum (200 MHz, DMSO-d6), δ, ppm (J, Hz):
1.18–1.33 (4H, m, (CH2)2(CH2)2CO2CH3); 1.38–1.66
(4H, m, (CH2CH2CH2)2CO2CH3); 2.19–2.33 (4H, m,
NHC(O)CH2(CH2)4CH2CO2CH3); 3.56 (3H, s, CO2CH3);
7.04–7.16 (2H, m, H-3,5 Ar); 7.58 (2H, dd, J = 9.1, J = 5.0,
H-2,6 Ar); 9.89 (1H, s, NH).
114°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.22–1.37 (4H, m, (CH2)2(CH2)2C(NC2H4)NOH);
1.44–1.63 (4H, m, (CH2CH2CH2)2C(NC2H4)NOH); 2.02
(4H, s, 2CH2 aziridine); 2.08 (2H, t, J = 7.2, NHC(O)CH2);
2.37 (2H, t, J = 7.2, CH2C(NC2H4)NOH); 7.39 (1H, dd,
J = 8.8, J = 2.3, H Ar); 7.63 (1H, d, J = 2.3, H Ar); 7.67–
7.74 (1H, m, H Ar); 9.49 (1H, s, NOH); 9.52 (1H, s, NH).
13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm: 25.4;
26.3; 26.6; 28.7; 28.8; 31.3; 36.1; 127.9; 129.3; 129.7;
134.7; 156.9 (C=NOH); 172.1 (C=O). Found, %: C 51.50;
H 5.58; N 11.14. C16H21Cl2N3O2. Calculated (with 4.8%
HCl), %: C 51.04; H 5.76; N 11.16.
(Z)-8-(Aziridin-1-yl)-8-(hydroxyimino)-N-phenyloctan-
amide (5c). Pyridine (0.1 ml) was added to a solution of
NCS (28 mg, 0.2 mmol) in CH2Cl2 (2 ml). The solution
was stirred for 15 min, then aldoxime 2a (50 mg, 0.2 mmol)
was added, and the stirring was continued at 35°C until the
reaction mixture became clear. After cooling to room tem-
perature, aziridine (32 l, 0.6 mmol, 3 equiv) and triethyl-
amine (58 l, 0.4 mmol, 2 equiv) were added to the reac-
tion mixture, which was then stirred for 2.5 h at room
temperature. The reaction mixture was treated with water
and extracted with AcOEt. The organic phase was dried
over Na2SO4, filtered, and evaporated at water bath tempe-
rature 30°C. The residue was purified by flash chromato-
graphy (eluent 5% Et3N in AcOEt). Yield 26% (15 mg), white
Synthesis of aldoxime 2b was carried out starting from
compound 11b analogously to the synthesis of compound 2a.
N-(4-Fluorophenyl)-8-hydroxyoctanamide was pre-
1
cipitated from Et2O. Yield 68%. H NMR spectrum
(200 MHz, DMSO-d6), δ, ppm (J, Hz): 1.18–1.44 (8H, m,
(CH2)4(CH2)2OH); 1.48–1.63 (2H, m, CH2CH2OH); 2.26
(2H, t, J = 7.3, NHC(O)CH2); 3.32–3.36 (2H, m, CH2OH
partially overlapped with water signal); 4.32 (1H, t, J = 5.2,
OH); 7.03–7.17 (2H, m, H-3,5 Ar); 7.58 (2H, dd, J = 9.2,
J = 5.1, H-2,6 Ar); 9.89 (1H, s, NH).
N-(4-Fluorophenyl)-8-oxooctanamide was purified by
flash chromatography (eluent AcOEt–hexane, 1:1). Yield
72%. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.17–1.36 (4H, m, (CH2)2(CH2)2CHO); 1.42–1.64
(4H, m, (CH2CH2CH2)2CHO); 2.25 (2H, t, J = 7.1,
NHC(O)CH2); 2.39 (2H, t, J = 7.1, CH2CHO, partially
overlapped with DMSO signal); 7.03–7.15 (2H, m,
H-3,5 Ar); 7.57 (2H, dd, J = 8.9, J = 5.2, H-2,6 Ar); 9.64
(1H, s, CHO ); 9.88 (1H, s, NH).
1
powder, mp 115–117°C. H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 1.23–1.32 (4H, m,
(CH2)2(CH2)2C(NC2H4)NOH);
1.40–1.62
(4H,
m,
(CH2CH2CH2)2C(NC2H4)NOH); 1.98 (4H, s, 2CH2 aziridine);
2.04 (2H, t, J = 7.4, NHC(O)CH2); 2.25 (2H, t, J = 7.4,
CH2C(NC2H4)NOH); 6.97 (1H, t, J = 7.4, H-4 Ph); 7.24
(2H, t, J = 7.8, H-3,5 Ph); 7.54 (2H, d, J = 7.8, H-2,6 Ph);
9.46 (1H, s, NOH); 9.80 (1H, s, NH). 13C NMR spectrum
(100 MHz, DMSO-d6), δ, ppm: 25.5; 26.3; 26.6; 28.8; 28.9;
31.3; 36.8; 119.4; 123.3; 129.0; 139.8; 156.9 (C=NOH);
171.6 (C=O). Found, %: C 65.34; H 8.02; N 13.78.
C16H23N3O2. Calculated (with 7% AcOEt), %: C 65.57;
H 8.09; N 13.49.
(4-Fluorophenyl)-8-(hydroxyimino)octanamide (2b) was
1
precipitated from Et2O. Yield 56%. H NMR spectrum
(400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.20–1.45 (6H, m,
(CH2)3(CH2)2CHNOH); 1.48–1.66 (2H, m, CH2CH2CHNOH);
2.12–2.32 (4H, m, NHC(O)CH2(CH2)4CH2CHNOH); 6.61
(1H, t, J = 5.0, CHNOH); 7.10 (2H, m, H-3,5 Ar); 7.57
(2H, dd, J = 9.0, J = 5.0, H-2,6 Ar); 9.89 (1H, s, NH);
10.70 (1H, s, NOH).
Synthesis of the target compound 5d was realized
starting from intermediate 10b (Method I) as follows.
8-[(4-Fluorophenyl)amino]-8-oxooctanoic acid (10b).
4-Fluorophenylaniline (117 l, 1.2 mmol) was added to a
stirred solution of suberic acid anhydride (9) (190 mg,
1.2 mmol) in anhydrous THF (12 ml). After stirring at room
temperature for 30 min, the solid (bisamide) was filtered
off (yield 10–15%), and the filtrate was evaporated. The
residue was treated with diethyl ether, and the precipitate
was filtered and dried in vacuo at room temperature. Yield
190 mg (63%). 1H NMR spectrum (400 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.19–1.26 (4H, m, (CH2)2(CH2)2CO2H);
1.39–1.49 (4H, m, (CH2CH2CH2)2CO2H); 2.15 (4H, t,
J = 7.4, NHC(O)CH2(CH2)4CH2CO2H); 6.46–6.52 (2H, m,
H-3,5 Ar); 6.76–6.82 (2H, m, H-2,6 Ar); 9.90 (1H, s, NH);
11.92 (1H, br. s, COOH).
(Z)-8-(Aziridin-1-yl)-N-(4-fluorophenyl)-8-(hydroxy-
imino)octanamide (5d). Pyridine (0.1 ml) was added to a
solution of NCS (25 mg, 0.19 mmol) in CH2Cl2 (2 ml) and
stirred for 15 min. Aldoxime 2b (50 mg, 0.19 mmol) was
then added, and the reaction mixture was heated at 35°C
until the reaction mixture became clear. After cooling to
room temperature, aziridine (29 l, 0.57 mmol, 3 equiv)
and triethylamine (53 l, 0.38 mmol, 2 equiv) were added,
the stirring was continued for 1.5 h at room temperature.
The reaction mixture was treated with water and extracted
with AcOEt. The organic phase was dried over Na2SO4,
filtered, and evaporated at water bath temperature 30°C.
The residue was treated with acetonitrile. Yield 24 mg
1
(41%), white powder, mp 126–128°C (decomp.). H NMR
spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.23–1.33
(4H, m, (CH2)2(CH2)2C(NC2H4)NOH); 1.41–1.59 (4H, m,
(CH2CH2CH2)2C(NC2H4)NOH); 1.98 (4H, s, 2CH2 aziridine);
2.04 (2H, t, J = 7.4, NHC(O)CH2); 2.24 (2H, t, J = 7.4,
Methyl 8-[(4-fluorophenyl)amino]-8-oxooctanoate (11b).
Anhydrous potassium carbonate (0.552 g, 4 mmol) was
added to a stirred solution of intermediate 10b (0.534 g,
652

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
CH2C(NC2H4)NOH); 7.04–7.11 (2H, m, H-3,5 Ar); 7.49–
Methyl 8-[(4-methoxyphenyl)amino]-8-oxooctanoate
(11d) was precipitated from water. Yield 65%. 1H NMR
spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.22–
1.28 (4H, m, (CH2)2(CH2)2CO2CH3); 1.44–1.59
(4H, m, (CH2CH2CH2)2CO2CH3); 2.19–2.27 (4H, m,
NHC(O)CH2(CH2)4CH2CO2CH3); 3.54 (3H, s, CO2CH3);
3.67 (3H, s, CH3OAr); 6.82 (2H, d, J = 9.0, H-3,5 Ar); 7.44
(2H, d, J = 9.0, H-2,6 Ar); 9.65 (1H, s, NH).
7.63 (2H, m, H-2,6 Ar); 9.46 (1H, s, NOH); 9.88 (1H, s,
NH). 13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm
(J, Hz): 25.4; 26.3; 26.6; 28.8; 28.9; 31.4; 36.8; 115.0 (d,
J = 22.1); 120.6 (d, J = 7.6); 135.6 (d, J = 2.3); 156.5; 156.9
(C=NOH); 171.9 (C=O). Found, %: C 62.49; H 7.13;
N 12.96. C16H22FN3O2. Calculated (with 5.4% AcOEt), %:
C 62.09; H 7.32; N 12.93.
Synthesis of the target compound 5e was realized
starting from intermediate 10c1 (Method I) according to the
procedure described for compound 5c.
8-Hydroxy-N-(4-methoxyphenyl)octanamide was pre-
cipitated from Et2O. Yield 59%. 1H NMR spectrum
(400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.24–1.31 (6H, m,
(CH2)3(CH2)2OH); 1.32–1.41 (2H, m, NHC(O)CH2CH2);
1.45–1.62 (2H, m, CH2CH2OH); 2.19–2.22 (2H, m,
NHC(O)CH2); 3.29–3.50 (2H, m, CH2OH partially
overlapped with water signal); 3.67 (3H, s, CH3OAr); 4.28
(1H, t, J = 5.1, OH); 6.82 (2H, d, J = 9.0, H-3,5 Ar); 7.44
(2H, d, J = 9.0, H-2,6 Ar); 9.65 (1H, s, NH).
N-(4-Methoxyphenyl)-8-oxooctanamide. Yield 77%.
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.20–1.30 (4H, m, (CH2)2(CH2)2CHO); 1.44–1.56 (4H, m,
(CH2CH2CH2)2CHO); 2.18–2.25 (2H, m, NHC(O)CH2); 2.38
(2H, td, J = 7.2, J = 1.6, CH2CHO); 3.67 (3H, s, CH3OAr);
6.82 (2H, d, J = 9.0, H-3,5 Ar); 7.44 (2H, d, J = 9.0, H-2,6 Ar);
9.62 (1H, t, J = 1.6, CHO); 9.65 (1H, s, NH).
Methyl 8-[(4-methylphenyl)amino]-8-oxooctanoate (11c)
was precipitated from Et2O. Yield 60%. 1H NMR spectrum
(400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.16–1.32
(4H, m, (CH2)2(CH2)2CO2CH3); 1.45–1.56 (4H, m,
(CH2CH2CH2)2 CO2CH3); 2.20 (3H, s, CH3Ar); 2.19–2.33
(4H, m, NHC(O)CH2(CH2)4CH2CO2CH3); 3.54 (3H, s,
COOCH3); 7.04 (2H, d, J = 8.4, H-3,5 Ar); 7.42 (2H, d,
J = 8.4,H-2,6 Ar); 9.71 (1H, s, NH).
8-Hydroxy-N-(4-methylphenyl)octanamide was purified
by flash chromatography (eluent AcOEt–hexane, 1:2). Yield
67%. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.20–1.28 (4H, m, (CH2)2(CH2)3OH); 1.32–1.41
(2H, m, NHC(O)CH2CH2); 1.48–1.57 (2H, m, CH2(CH2)2OH);
2.20 (3H, s, CH3Ar); 2.19–2.28 (4H, m, NHC(O)CH2);
3.30–3.36 (2H, m, CH2OH, partially overlapped with water
signal); 4.28 (1H, m, OH); 7.04 (2H, d, J = 8.2, H-3,5 Ar);
7.33–7.49 (2H, m, H-2,6 Ar); 9.70 (1H, s, NH).
N-(4-Methylphenyl)-8-oxooctanamide. Yield 71%.
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.19–1.30 (4H, m, (CH2)2(CH2)2CHO); 1.46–1.56 (4H, m,
(CH2CH2CH2)2CHO); 2.20 (3H, s, CH3Ar); 2.21–2.25 (2H,
m, NHC(O)CH2); 2.38 (2H, td, J = 7.2, J = 1.6, CH2CHO);
7.04 (2H, d, J = 8.4, H-3,5 Ar); 7.42 (2H, d, J = 8.4, H-2,6 Ar);
9.62 (1H, t, J = 1.6, CHO); 9.70 (1H, s, NH).
8-(Hydroxyimino)-N-(4-methoxyphenyl)octanamide (2d).
Yield 72%.1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.22–1.31 (4H, m, (CH2)2(CH2)2CHNOH); 1.34–1.43
(2H, m, NHC(O)CH2CH2); 1.49–1.58 (2H, m, CH2CH2CHNOH);
2.16–2.25 (4H, m, NHC(O)CH2(CH2)4CH2CHNOH); 3.67
(3H, s, CH3OAr); 6.60 (1H, t, J = 5.3, CHNOH); 6.82 (2H, d,
J = 9.0, H-3,5 Ar); 7.44 (2H, d, J = 9.0, H-2,6 Ar); 9.66 (1H,
s, NH); 10.67 (1H, s, NOH).
(Z)-8-(Aziridin-1-yl)-8-(hydroxyimino)-N-(4-methoxy-
phenyl)octanamide (5f). Yield 70%, white powder, mp 107.5–
1
109°C. H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
8-(Hydroxyimino)-N-(4-methylphenyl)octanamide (2c).
Yield 74%. 1H NMR spectrum (400 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.20–1.35 (4H, m, (CH2)2(CH2)2CHNOH);
1.42–1.61 (4H, m, (CH2CH2CH2)2CHNOH); 2.15–2.25
(4H, m, NHC(O)CH2CH2(CH2)2CH2CH2CHNOH); 2.19
(3H, s, CH3Ar); 6.60 (1H, t, J = 5.2, CH2CHNOH); 7.04
(2H, d, J = 8.4, H-3,5 Ph); 7.35 (2H, d, J = 8.4, H-2,6 Ph);
9.70 (1H, s, NH); 10.67 (1H, s, NOH).
(Z)-8-(Aziridin-1-yl)-8-(hydroxyimino)-N-(4-methyl-
phenyl)octanamide (5e). Yield 11%, white powder, mp 111–
113°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.18–1.35 (m, 4H, (CH2)2(CH2)2C(NC2H4)NOH);
1.42–1.58 (4H, m, (CH2CH2CH2)2C(NC2H4)NOH); 1.98
(4H, s, 2CH2 aziridine); 2.01–2.05 (2H, m, NHC(O)CH2);
2.19 (3H, s, CH3Ar); 2.23 (2H, t, J = 7.5, CH2C(NC2H4)NOH);
6.99–7.07 (2H, m, H-3,5 Ar); 7.30–7.47 (2H, m, H-2,6 Ar);
9.47 (1H, s, NOH); 9.71 (1H, s, NH). 13C NMR spectrum
(100 MHz, DMSO-d6), δ, ppm: 20.8 (CH3Ar); 25.5; 26.3;
26.6; 28.8; 28.9; 31.3; 36.8; 119.5 (C-3,5 Ar); 129.4 (C-2,6
Ar); 132.2 (C-4 Ar); 137.2 (C-1 Ar); 156.9 (C=NOH);
171.4 (C=O).
(J, Hz): 1.19–1.33 (4H, m, (CH2)2(CH2)2C(NC2H4)NOH);
1.42–1.58 (4H, m, (CH2CH2CH2)2C(NC2H4)NOH); 1.98
(4H, s, 2CH2 aziridine); 2.03 (2H, t, J = 7.4, NHC(O)CH2);
2.21 (2H, t, J = 7.4, CH2C(NC2H4)NOH); 3.67 (3H, s,
CH3OAr); 6.81 (2H, d, J = 9.0, H-3,5 Ar); 7.44 (2H, d,
J = 9.0, H-2,6 Ar); 9.48 (1H, s, NOH); 9.67 (1H, s, NH).
13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm: 25.6;
26.3 26.6; 28.8; 28.9; 31.3; 36.7; 55.6 (CH3OAr); 114.2
(C-3,5 Ar); 121.1 (C-2,6 Ar); 132.9 (C-1 Ar); 155.4
(C-4 Ar); 156.9 (C=NOH); 171.2 (C=O). Found, %:
C 61.67; H 7.70; N 10.61 C17H25N3O3. Calculated (with
21% AcOEt), %: C 61.90; H 8.16; N 10.31.
Synthesis of the target compound 5g was realized
starting from intermediate 10e1 (Method I) according to the
procedure described for compound 5c.
Methyl 8-[(2,6-dimethylphenyl)amino]-8-oxooctano-
ate (11e) was precipitated from water. Yield 85%. 1H NMR
spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.24–1.39
(4H, m, (CH2)2(CH2)2CO2CH3); 1.45–1.64 (4H, m,
(CH2CH2CH2)2CO2CH3); 2.08 (6H, br. s, 2CH3Ar); 2.23–
2.31 (4H, m, NHC(O)CH2(CH2)4CH2CO2CH3); 3.54 (3H,
s, CO2CH3); 7.01 (3H, s, H-3,4,5 Ar); 9.12 (1H, br. s, NH).
N-(2,6-Dimethylphenyl)-8-hydroxyoctanamide. Yield
54%. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
Synthesis of the target compound 5f was realized
starting from intermediate 10d1 (Method I) according to the
procedure described for compound 5c.
653

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
(J, Hz): 1.20–1.39 (6H, m, (CH2)3(CH2)2OH); 1.46–1.64 (4H,
δ, ppm (J, Hz): 1.24–1.31 (4H, m, (CH2)2(CH2)2CHNOH);
1.35–1.69 (4H, m, (CH2CH2CH2)2CHNOH); 2.16–2.27
(4H, m, NHC(O)CH2(CH2)4CH2CHNOH); 6.60 (1H, t,
J = 5.3, CHNOH); 6.90–6.94 (4H, m, H Ar); 7.02–7.07
(1H, m, H Ar); 7.29–7.35 (2H, m, H Ar); 7.56 (2H, d,
J = 8.9, H Ar); 9.84 (1H, br. s, NH); 10.67 (1H, s, NOH).
(Z)-8-(Aziridin-1-yl)-8-(hydroxyimino)-N-(4-phenoxy-
phenyl)octanamide (5h). Yield 4%, colorless oil. 1H NMR
spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 1.24–1.44
(6H, m, (CH2)2(CH2)2C(NC2H4)NOH); 1.54–1.76 (4H, m,
(CH2CH2CH2)2C(NC2H4)NOH); 2.12–2.21 (2H, m,
NHC(O)CH2); 2.17 (4H, s, 2CH2 aziridine); 2.28–2.34 (2H,
m, CH2C(NC2H4)NOH); 6.88–6.99 (4H, m, H Ar); 7.05
(1H, t, J = 7.4, H Ar); 7.26–7.31 (2H, m, H Ar partially
overlapped with CDCl3); 7.43–7.51 (2H, m, H Ar). The
signals of NH and NOH protons are not observed due to
exchange with traces of water.
m, CH2(CH2)3CH2CH2OH); 2.08 (6H, br. s, 2CH3Ar); 2.22
(2H, t, J = 7.4, NHC(O)CH2); 3.30–3.48 (2H, m, CH2OH
partially overlapped with water signal); 4.29 (1H, t, J = 5.2,
OH); 7.01 (3H, s, H-3,4,5 Ar); 9.12 (1H, br. s, NH).
N-(2,6-Dimethylphenyl)-8-oxooctanamide. Yield 58%.
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.22–1.39 (4H, m, (CH2)2(CH2)2CHO); 1.38–1.70 (4H, m,
(CH2CH2CH2)2CHO); 2.07 (6H, br. s, 2CH3Ar); 2.24–2.29
(2H, m, NHC(O)CH2); 2.39 (2H, td, J = 7.2, J = 1.6,
CH2CHO); 7.01 (3H, s, H-3,4,5 Ar); 9.12 (1H, br. s, NH);
9.64 (1H, t, J = 1.6, CHO).
N-(2,6-Dimethylphenyl)-8-(hydroxyimino)octanamide (2e).
Yield 86%. 1H NMR spectrum (400 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.21–1.49 (6H, m, (CH2)3(CH2)2CHNOH);
1.55–1.64 (2H, m, CH2CH2CHNOH); 2.08 (6H, s, 2CH3Ar);
2.15–2.33 (4H, m, NHC(O)CH2(CH2)4CH2CHNOH); 6.61
(1H, t, J = 5.3, CHNOH); 7.01 (3H, s, H-3,4,5 Ar); 9.13
(1H, br. s, NH); 10.68 (1H, s, NOH).
(Z)-8-(Aziridin-1-yl)-N-(2,6-dimethylphenyl)-8-(hydroxy-
imino)octanamide (5g). Yield 16%, amorphous powder,
mp 125–126.5°C. 1H NMR spectrum (400 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.22–1.40 (4H, m, (CH2)2(CH2)2C(NC2H4)NOH);
1.43–1.63 (4H, m, (CH2CH2CH2)2C(NC2H4)NOH); 1.98
(4H, s, 2CH2 aziridine); 2.08 (6H, s, 2CH3Ar); 2.03–2.10
(2H, m, NHC(O)CH2); 2.27 (2H, t, J = 7.3, CH2C(NC2H4)NOH);
7.01 (3H, s, H-3,4,5 Ar); 9.14 (1H, s, NH); 9.49 (1H, s,
NOH). 13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm:
18.5 (2CH3Ar); 25.8; 26.5; 28.7; 28.9; 31.3; 35.8; 39.2;
126.7 (C-4 Ar); 128.0 (C-3,5 Ar); 135.6 (C-2,6 Ar); 135.7
(C-1 Ar); 156.9 (C=NOH); 171.2 (C=O).
Synthesis of the target compound 5i was realized
starting from the intermediate 10g (Method I) according to
the procedure described for compound 5c.
8-[(4-Ethoxyphenyl)amino]-8-oxooctanoic acid (10g).
Yield 73%.1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.19–1.32 (7H, m, (CH2)2(CH2)2CO2H, OCH2CH3);
1.40–1.60 (4H, m, (CH2CH2CH2)2CO2H); 2.11–2.26 (4H,
m, NHC(O)CH2(CH2)4CH2CO2H); 3.93 (2H, q, J = 6.9,
OCH2CH3); 6.80 (2H, dd, J = 9.0, J = 1.1, H-3,5 Ar); 7.37–
7.48 (2H, m, H-2,6 Ar); 9.64 (1H, br. s, NH); 11.93 (1H, s,
CO2H).
Methyl 8-[(4-ethoxyphenyl)amino]-8-oxooctanoate (11g)
was precipitated from water. Yield 85%. 1H NMR spectrum
(400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.23–1.32 (m, 7H,
m, (CH2)2(CH2)2CO2CH3, OCH2CH3); 1.44–1.59 (4H,
m, (CH2CH2CH2)2CO2CH3); 2.08–2.35 (4H, m,
NHC(O)CH2(CH2)4CH2CO2CH3); 3.54 (3H, s, CO2CH3);
3.93 (2H, q, J = 6.9, OCH2CH3); 6.80 (2H, dd, J = 9.0,
J = 1.2, H-3,5 Ar); 7.22–7.66 (2H, m, H-2,6 Ar); 9.64 (1H,
br. s, NH).
N-(4-Ethoxyphenyl)-8-hydroxyoctanamide was precipita-
ted from Et2O. Yield 47%. 1H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 1.18–1.28 (9H, m, (CH2)3(CH2)2OH,
OCH2CH3); 1.32–1.41 (2H, m, NHC(O)CH2CH2); 1.46–1.64
(2H, m, CH2CH2OH); 2.21 (2H, t, J = 7.4, NHC(O)CH2);
3.29–3.50 (2H, m, CH2OH partially overlapped with water
signal); 3.93 (2H, q, J = 7.0, OCH2CH3); 4.28 (1H, t,
J = 5.1, OH); 6.71–6.85 (2H, m, H-3,5 Ar); 7.36–7.47 (2H,
m, H-2,6 Ar); 9.64 (1H, br. s, NH).
N-(4-Ethoxyphenyl)-8-oxooctanamide. Yield 43%.
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.22–1.30 (7H, m, (CH2)2(CH2)2CHO, OCH2CH3); 1.45–
1.59 (4H, m, (CH2CH2CH2)2CHO); 2.19–2.22 (2H, m,
NHC(O)CH2); 2.38 (2H, td, J = 7.2, J = 1.6, CH2CHO);
3.93 (2H, q, J = 7.0, OCH2CH3); 6.78–6.83 (2H, m, H-3,5 Ar);
7.34–7.48 ( 2H, m, H-2,6 Ar); 9.62 (1H, s, CHO); 9.64
(1H, br. s, NH).
N-(4-Ethoxyphenyl)-8-(hydroxyimino)octanamide (2g).
Yield 90%.1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.23–1.31 (7H, m, (CH2)2(CH2)2CHNOH, OCH2CH3);
1.33–1.42 (2H, m, NHC(O)CH2CH2); 1.47–1.57
(2H, m, CH2CH2CHNOH); 2.15–2.25 (4H, m,
Synthesis of the target compound 5h was realized
starting from intermediate 12 (Method II) according to the
procedure described for compound 5a.
Methyl 8-oxo-8-[(4-phenoxyphenyl)amino]octanoate
1
(11f). Yield 62%. H NMR spectrum (400 MHz, DMSO),
δ, ppm: 1.21–1.28 (4H, m, (CH2)2(CH2)2CO2CH3); 1.44–
1.58 (4H, m, (CH2CH2CH2)2CO2H); 2.23–2.28 (4H, m,
NHC(O)CH2(CH2)4CH2CO2H); 3.54 (3H, s, CO2CH3); 6.86–
6.96 (4H, m, H Ph); 7.00–7.09 (1H, m, H Ph); 7.27–7.36 (2H,
m, H Ar); 7.47–7.61 (2H, m, H Ar); 9.84 (1H. br. s, NH).
8-Hydroxy-N-(4-phenoxyphenyl)octanamide was recrys-
tallized from Et2O. Yield 42%.1H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 1.22–1.28 (6H, br. s,
(CH2)3(CH2)2OH); 1.33–1.58 (4H, m, CH2(CH2)3CH2CH2OH);
2.25 (2H, t, J = 7.4, NHC(O)CH2); 3.34 (2H, q, J = 6.5,
CH2OH); 4.29 (1H, t, J = 5.2, OH); 6.89–6.95 (4H, m,
H Ar); 7.03–7.08 (1H, m, H Ar); 7.29–7.35 (2H, m, H Ar);
7.54–7.58 (2H, m, H Ar); 9.84 (1H, br. s, NH).
8-Oxo-N-(4-phenoxyphenyl)octanamide. Yield 66 %.
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.24–1.31 (4H, m, (CH2)2(CH2)2CHO); 1.46–1.59 (4H, m,
(CH2CH2CH2)2CHO); 2.25 (2H, t, J = 7.4, NHC(O)CH2);
2.34 (2H, td J = 7.2, J = 1.6, CH2CHO); 6.90–6.95 (4H, m,
H Ar); 7.03–7.08 (1H, m, H Ar); 7.29–7.35 (2H, m, H Ar);
7.54–7.58 (2H, m, H Ar); 9.63 (1H, t, J = 1.6, CHO); 9.84
(1H, br. s, NH).
8-(Hydroxyimino)-N-(4-phenoxyphenyl)octanamide (2f).
Yield 92%. 1H NMR spectrum (400 MHz, DMSO-d6),
654

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
NHC(O)CH2(CH2)4CH2CHNOH); 3.93 (2H, q, J = 7.0,
(1H, t, J = 5.4, CHNOH); 7.07 (2H, d, J = 8.4, H-3,5 Ar);
7.44 (2H, d, J = 8.4, H-2,6 Ar); 9.71 (1H, s, NH); 10.67
(1H, s, NOH).
OCH2CH3); 6.60 (1H, t, J = 5.3, CHNOH); 6.80 (2H, d,
J = 8.9, H-3,5 Ar); 7.43 (2H, d, J = 8.4, H-2,6 Ar); 9.64
(1H, s, NH); 10.67 (1H, s, OH).
(Z)-8-(Aziridin-1-yl)-N-(4-ethylphenyl)-8-(hydroxy-
imino)octanamide (5j) was purified by flash chromato-
graphy (eluent 5% Et3N in AcOEt). Yield 42%, yellowish
oil. 1H NMR spectrum (400 MHz, DMSO-d6) δ, ppm
(J, Hz): 1.11 (3H, t, J = 7.6, CH2CH3); 1.18–1.31 (4H, m,
(Z)-8-(Aziridin-1-yl)-N-(3-ethoxyphenyl)-8-(hydroxy-
imino)octanamide (5i) was precipitated from Et2O. Yield
42%, oil with tendency to crystallize, mp 91°C (decomp.).
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.05 (3H, t, J = 7.0, OCH2CH3); 1.21–1.33 (4H, m,
(CH2)2(CH2)2C(NC2H4)NOH);
1.42–1.58
(4H,
m,
(CH2)2(CH2)2C(NC2H4)NOH);
1.35–1.59
(4H,
m,
(CH2CH2CH2)2C(NC2H4)NOH); 1.98 (4H, s, 2CH2 aziridine);
2.00–2.06 (2H, m, NHC(O)CH2); 2.23 (2H, t, J = 7.4,
CH2C(NC2H4)NOH); 2.50 (2H, q, J = 7.6, CH2CH3 partially
overlapped with DMSO signal); 7.07 (2H, d, J = 8.2,
H-3,5 Ar); 7.44 (2H, d, J = 8.2, H-2,6 Ar); 9.46 (1H, s,
NOH); 9.72 (1H, s, NH). 13C NMR spectrum (100 MHz,
DMSO-d6), δ, ppm: 14.8 (CH3CH2); 25.5; 26.3; 26.6; 28.1
(CH3CH2); 28.8; 28.9; 31.3; 36.8; 119.5 (C-3,5 Ar); 129.4
(C-2,6 Ar); 132.2 (C-4 Ar); 137.2 (C-1 Ar); 156.9
(C=NOH); 171.4 (C=O).
(CH2CH2CH2)2C(NC2H4)NOH); 1.98 (4H, s, 2CH2 aziridine);
2.04 (2H, t, J = 7.4, NHC(O)CH2); 2.21 (2H, t, J = 7.4,
CH2C(NC2H4)NOH); 3.93 (2H, q, J = 7.0, OCH2CH3); 6.80
(2H, d, J = 8.9, H-3,5 Ar); 7.43 (2H, d, J = 8.4, H-2,6 Ar);
9.46 (1H, s, NOH); 9.65 (1H, s, NH). 13C NMR spectrum
(100 MHz, DMSO-d6), δ, ppm: 14.8 (CH3CH2O); 25.8;
26.3; 28.7; 28.9; 31.3; 35.8; 39.2; 58.2 (CH3CH2O); 114.2
(C-3,5 Ar); 121.1 (C-2,6 Ar); 132.9 (C-1 Ar); 155.4 (C-4
Ar); 156.9 (C=NOH); 171.0 (C=O).
Synthesis of the target compound 5j was realized
starting from intermediate 10h1 (Method I) according to the
procedure described for compound 5c.
Synthesis of the target compound 5k was realized
starting from the commercially available cycloheptanone (13)
as follows.
Methyl 8-[(4-ethylphenyl)amino]-8-oxooctanoate (11h)
was purified by flash chromatography (eluent AcOEt–
hexane, 1:2). Yield 23%. 1H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 1.11 (3H, t, J = 7.6, CH2CH3);
1.21–1.29 (4H, m, (CH2)2(CH2)2CO2H); 1.45–1.57
(4H, m, (CH2CH2CH2)2CO2CH3); 2.21–2.27 (4H, m,
NHC(O)CH2(CH2)4CH2CO2CH3); 2.50 (2H, q, J = 7.6,
CH2CH3 partially overlapped with DMSO signal); 3.54
(3H, s, COOCH3); 7.07 (2H, d, J = 8.5, H-3,5 Ar); 7.44
(2H, d, J = 8.5, H-2,6 Ar); 9.71 (1H, s, NH).
N-(4-Ethylphenyl)-8-hydroxyoctanamide was precipi-
tated from Et2O. Yield 83%. 1H NMR spectrum (400 MHz,
DMSO-d6), δ, ppm (J, Hz): 1.11 (3H, t, J = 7.6, CH2CH3);
1.20–1.28 (6H, m, (CH2)3(CH2)2OH); 1.33–1.39 (2H, m,
NHC(O)CH2CH2); 1.49–1.57 (2H, m, CH2CH2OH); 2.23
(2H, t, J = 7.4, NHC(O)CH2); 2.50 (2H, q, J = 7.6,
CH2CH3 partially overlapped with DMSO signal); 3.34
(2H, m, CH2OH partially overlapped with water signal);
4.29 (1H, t, J = 5.2, OH); 7.07 (2H, d, J = 8.5, H-3,5 Ar);
7.44 (2H, d, J = 8.5, H-2,6 Ar); 9.71 (1H, s, NH).
Ethyl 6-(1,3-dioxolan-2-yl)hexanoate (15). p-Toluene-
sulfonic acid (245 mg, 1.3 mmol, 0.2 equiv) and ethylene
glycol (4.26 ml, 77.6 mmol) were added to a solution of
ethyl 7-oxoheptanoate (14)5 (1.12 g, 6.5 mmol) in benzene
(70 ml). The reaction mixture was stirred at reflux with a
Dean–Stark trap for 3 h. Then the reaction mixture was
evaporated to dryness. Yield 1.10 g (78%). 1H NMR
spectrum (200 MHz, DMSO-d6), δ, ppm (J, Hz): 1.16 (3H, t,
J = 7.3, OCH2CH3); 1.23–1.60 (8H, m, (CH2)4CH(OC2H4O));
2.26 (2H, t, J = 7.3, C(O)CH2); 3.69–3.89 (4H, m,
CH(OC2H4O)); 4.02 (2H, q, J = 7.3 OCH2CH3); 4.69–4.77
(1H, m, CH(OC2H4O)).
6-(1,3-Dioxolan-2-yl)hexanoic acid. Aqueous NaOH
solution (1 M, 4.61 ml) was added to a solution of inter-
mediate 15 (1.06 g, 4.61 mmol) in THF (30 ml). The
reaction mixture was stirred overnight at room temperature
and evaporated to dryness to give 710 mg (73%) of white
crystalline product which was submitted to the next reac-
tion step without additional purification and characteri-
zation.
N-(4-Ethylphenyl)-8-oxooctanamide was purified by
flash chromatography (eluent AcOEt–hexane, 1:4). Yield
79%. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.11 (3H, t, J = 7.6, CH2CH3); 1.22–1.29 (4H, m,
(CH2)2(CH2)2CHO); 1.42–1.58 (4H, m, (CH2CH2CH2)2CHO);
2.23 (2H, t, J = 7.4, NHC(O)CH2); 2.38 (2H, td, J = 7.4,
J = 1.6, CH2CHO); 2.50 (2H, q, J = 7.6, CH2CH3 partially
overlapped with DMSO signal); 7.07 (2H, d, J = 8.4,
H-3,5 Ar); 7.44 (2H, d, J = 8.4, H-2,6 Ar); 9.62 (1H, s,
CHO); 9.71 (1H, s, NH).
N-(4-Ethylphenyl)-8-(hydroxyimino)octanamide (2h).
Yield 75%.1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.11 (3H, t, J = 7.6, CH2CH3); 1.23–1.31 (4H, m,
(CH2)2(CH2)2CHNOH); 1.32–1.42 (2H, m, NHC(O)CH2CH2);
1.47–1.59 (2H, m, CH2CH2CHNOH); 2.15–2.27 (4H, m,
NHC(O)CH2(CH2)4CH2CHNOH); 2.50 (2H, q, J = 7.6,
CH2CH3 partially overlapped with DMSO signal); 6.60
6-(1,3-Dioxolan-2-yl)-N-phenylhexanamide. The crude
6-(1,3-dioxolan-2-yl)hexanoic acid (325 mg, 1.5 mmol)
was suspended in DMF (16 ml), and isobutyl chloro-
formate (223 l, 1.7 mmol, 1.1 equiv) was added. The reac-
tion mixture was stirred for 1 h at room temperature.
Aniline (156 l, 1.7 mmol, 1.1 equiv) was subsequently
added dropwise, and the reaction mixture was stirred for 2 h at
room temperature. Aqueous potassium bisulfate solution
(1 M, 50 ml) was added to the reaction mixture, and the
product was extracted with AcOEt, the organic phase was
washed several times with water, dried over Na2SO4,
filtrated, evaporated. The residue was purified by flash
chromatography, eluent AcOEt–hexane, 1:1. Yield 385 mg
1
(94%). H NMR spectrum (200 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.20–1.65 (6H, m, (CH2)3CH2CH(OC2H4O));
2.16 (2H, t, J = 7.2, NHC(O)CH2); 2.26 (2H, t, J = 7.2,
CH2CH(OC2H4O)); 3.63–3.91 (4H, m, CH(OC2H4O)); 4.68–4.78
655

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
(1H, m, CH(OC2H4O)); 6.94–7.04 (1H, m, H-4 Ph); 7.25
(2H, t, J = 7.9, H-3,5 Ph); 7.52–7.59 (2H, m, H-2,6 Ph);
9.82 (1H, br. s, NH).
(C=NOH); 171.6 (C=O). Found, %: C 63.49; H 7.29;
N 15.28. C14H19N3O2. Calculated (with 8% AcOEt), %:
C 63.58; H 7.47; N 14.83.
7-Oxo-N-phenylheptanamide (16). 6-(1,3-Dioxolan-2-yl)-
N-phenylhexanamide (385 mg, 1.46 mmol) was dissolved
in a mixture of THF (10 ml) and aqueous HCl (1 M,
10 ml). The reaction mixture was stirred for 3 h at 70°C,
cooled to room temperature, and extracted with Et2O. The
organic phase was separated and dried over Na2SO4,
filtered and evaporated. Yield 185 mg (54%), colorless oil.
1H NMR spectrum (200 MHz, DMSO-d6), δ, ppm (J, Hz):
1.17–1.62 (6H, m, (CH2)3CH2CHO); 2.10–2.35 (4H, m,
NHC(O)CH2(CH2)3CH2CHO); 6.99 (1H, t, J = 7.6,
H-4 Ph); 7.25 (2H, t, J = 7.8, H-3,5 Ph); 7.55 (2H, d,
J = 8.2, H-2,6 Ph); 9.63 (1H, s, CHO ); 9.82 (1H, br. s, NH).
7-(Hydroxyimino)-N-phenylheptanamide (3) was synthe-
sized using the procedure described for the synthesis of
compound 2a, except by using Na2CO3 instead of NaOAc.
Yield 60%. 1H NMR spectrum (200 MHz, DMSO-d6),
δ, ppm (J, Hz): 1.12–1.74 (6H, m, (CH2)3CH2CHNOH);
2.09–2.32 (4H, m, NHC(O)CH2(CH2)3CH2CHNOH); 6.59–
6.62 (1H, m, CHNOH); 6.97–7.01 (1H, m, H-4 Ph); 7.25
(2H, t, J = 7.6, H-3,5 Ph); 7.56 (2H, t, J = 7.6, H-2,6 Ph);
9.82 (1H, s, NH); 10.71 (1H, s, NOH).
(Z)-7-(Aziridin-1-yl)-7-(hydroxyimino)-N-phenylheptan-
amide (5k) was synthesized from compound 3 following
the procedure for the synthesis of compound 5c and puri-
fied by flash chromatography (eluent 5% Et3N in AcOEt).
Yield 6%, white powder, mp 131–132°C. 1H NMR
spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz): 1.23–1.35
(2H, m, CH2(CH2)2C(NC2H4)NOH); 1.42–1.62 (4H, m,
(CH2CH2)2CN(C2H4)NOH); 1.99 (4H, s, 2CH2 aziridine);
2.05 (2H, t, J = 7.4, NHC(O)CH2); 2.25 (2H, t, J = 7.4,
CH2C(NC2H4)NOH); 6.97 (1H, t, J = 7.9, H-4 Ph); 7.24
(2H, t, J = 7.9, H-3,5 Ph); 7.54 (2H, d, J = 7.6, H-2,6 Ph);
9.46 (1H, s, NOH); 9.80 (1H, s, NH).13C NMR spectrum
(100 MHz, DMSO-d6), δ, ppm: 25.5; 26.4; 26.6; 28.9; 31.3;
36.6; 119.3; 123.3; 129.0; 139.8; 156.9 (C=NOH); 171.6
(C=O). Found %: C 65.46; H 7.63; N 15.60. C15H21N3O2.
Calculated, %: C 65.43; H 7.69; N 15.26.
6-(Hydroxyimino)-N-phenylhexanamide (4) was synthe-
sized from compound 186 using the procedure for the
synthesis of compound 2a, except by using Na2CO3 instead
of NaOAc. The product was isolated by filtration, dried,
and used for the next step without additional purification
and characterization.
(Z)-6-(Aziridin-1-yl)-6-(hydroxyimino)-N-phenylhexan-
amide (5l) was synthesized from compound 4 following
the procedure for the synthesis of compound 5c and
purified by flash chromatography (eluent 5% Et3N in
AcOEt). Yield 6%, white powder, mp 128–131°C.
1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm (J, Hz):
1.43–1.65 (4H, m, (CH2)2CH2C(NC2H4)NOH); 1.99 (4H, s,
2CH2 aziridine); 2.08 (2H, t, J = 7.3, NHC(O)CH2); 2.27
(2H, t, J = 7.3, CH2C(NC2H4)NOH); 6.99 (1H, t, J = 7.4,
H-4 Ph); 7.24 (2H, t, J = 7.8, H-3,5 Ph); 7.54 (2H, d,
J = 7.8, H-2,6 Ph); 9.48 (1H, s, NOH); 9.81 (1H, s, NH).
13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm: 25.5;
26.3; 26.6; 31.3; 36.8; 119.4; 123.3; 129.0; 139.8; 156.9
(Z)-8-(7-Azabicyclo[4.1.0]heptan-7-yl)-8-(hydroxyimino)-
N-phenyloctanamide (6) was synthesized following the
procedure for the synthesis of compound 5c. Yield 34%,
oil. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.07–1.18 (4H, m, 3,4-CH2 azabicycle); 1.22–1.35
(4H, m, (CH2)2(CH2)2C(NC6H10)NOH); 1.43–1.59 (4H, m,
(CH2CH2CH2)2C(NC6H10)NOH); 1.63–1.74 (2H, m) and
1.74–1.85 (2H, m, 2,5-CH2 azabicycle); 1.97–2.04 (2H, m,
NHC(O)CH2); 2.25 (2H, t, J = 7.4, CH2C(NC6H10)NOH);
2.31–2.35 (2H, m, 1,6-CH azabicycle); 6.94–7.00 (1H, m,
H-4 Ph); 7.14–7.28 (2H, m, H-3,5 Ph); 7.54 (2H, dd,
J = 10.9, J = 3.4, H-2,6 Ph); 9.35 (1H, s, NOH); 9.81 (1H,
s, NH). 13C NMR spectrum (100 MHz, DMSO-d6), δ, ppm:
20.8; 25.5; 26.3; 26.6; 28.8; 28.9; 31.3; 36.8; 39.2; 119.5
(C-4 Ph); 129.4 (C-3,6 Ph); 132.2 (C-2,6 Ph); 137.2 (C-1 Ph);
156.9 (C=NOH); 171.4 (C=O). Mass spectrum, m/z: 344
[M+H]+.
1-[(1Z)-8-Anilino-N-hydroxy-8-oxooctanimidoyl]aziridine-
2-carboxamide (7) was synthesized following the proce-
dure for the synthesis of compound 5c. Yield 14%, oil.
1H NMR spectrum (400 MHz, CDCl3), δ, ppm (J, Hz): 1.23–
1.41 (4H, m, (CH2)2(CH2)2CN(C2H3CONH2)NOH); 1.44–
1.51 (2H, m, NHC(O)CH2CH2); 1.52–1.61 (2H, m,
CH2CH2CN(C2H3CONH2)NOH);
1.62–1.73 (2H, m,
NHC(O)CH2); 2.22–2.32 (3H, m, CH2CNOH, CH aziridine);
2.37–2.48 (1H, m, CH aziridine); 2.51–2.60 (1H, m, CH
aziridine); 6.99–7.07 (1H, m, H-4 Ph); 7.21–7.35 (5H, m,
H-3,5 Ph, NH, NH2 partially overlapped with solvent signal);
7.41–7.47 (2H, m, H-2,6 Ph); 9.10 (1H, s, NOH). Mass
spectrum, m/z: 333 [M+H]+.
(Z)-7-Amino-7-(hydroxyimino)-N-phenylheptanamide (8).
NaHCO3 (185 mg, 2.2 mmol, 2.2 equiv) was added to a
solution of hydroxylamine hydrochloride (104 mg, 1.5 mmol,
1.5 equiv) in isopropyl alcohol (2.0 ml). The resulting
mixture was stirred for 15 min, and compound 2010 (216 mg,
1 mmol) was added. The stirring was continued at 80°C for
36 h until the consumption of the starting material. After
completion of the reaction, the mixture was cooled to room
temperature, precipitate was filtered off, washed with a
small amount of water, and dried in vacuo to provide
product 8 in quantitative yield. White solid, mp 78–80°C
1
(decomp.). H NMR spectrum (400 MHz, DMSO-d6), δ, ppm
(J, Hz): 1.33–1.39 (2H, m, CH2(CH2)2C(NH2)NOH); 1.53–
1.69 (4H, m, (CH2CH2)2C(NH2)NOH); 2.02 (2H, t, J = 7.6,
NHC(O)CH2); 2.35 (2H, t, J = 7.4, CH2C(NH2)NOH); 5.04
(2H, br. s, C(NH2)NOH); 7.08 (1H, t, J = 7.4, H-4 Ph);
7.34 (2H, t, J = 8.2, H-3,5 Ph); 7.65 (2H, d, J = 8.2, H-2,6 Ph);
8.75 (1H, s, NOH); 9.91 (1H, s, NH). 13C NMR spectrum
(100 MHz, DMSO-d6), δ, ppm: 25.4; 26.6; 28.7; 31.1; 36.8;
119.4 (C-4 Ph); 123.3 (C-3,6); 129.0 (C-2,6 Ph); 139.8
(C-1 Ph); 153.3 (C(NH2)NOH); 171.6(C=O). Mass
spectrum, m/z: 250 [M+H]+.
Determination of IC50 and LD50. Evaluation of anti-
cancer activity was performed by examining in vitro anti-
proliferative effects of the synthesized compounds in
monolayer tumor cell lines HT1080 (human connective
656

Chemistry of Heterocyclic Compounds 2015, 51(7), 647–657
tissue fibrosarcoma) and MG22A (mouse hepatosarcoma).
HDAC assay buffer. An aliquot (50 µl) of each solution of
the test compounds, as well as the positive control without
inhibitor, were added to the wells containing HeLa extract
(0.5 µl, except for the blank experiment) and HDAC
fluorescent Fluor de Lys® Substrate (250 µM), employing
Trichostatin A as a positive control. The HDAC reactions
were allowed to proceed for 30 min at 37°C and then
stopped by the addition of Fluor de Lys® Developer
(50 µl). The well plate was incubated at room temperature
for additional 15 min. The relative fluorescent units (RFU)
were measured in a Tecan Infinite M1000 reader (excita-
tion and emission wavelength 350 and 460 nm, respecti-
vely). The set of the RFU values for four different concen-
trations of each test compound were used to calculate the
relative HDAC inhibitory activity. Calculation of IC50 was
done with the GraphPad Prism 5.03 software (GraphPad
Software, La Jolla, CA). All data are the mean of three
independent experiments.
The borderline concentration, relevant to the highest tole-
rated dose, was determined for all target compounds 5a–l,
6–8 using the 3T3 (mouse Swiss Albino embryo fibro-
blasts) cell line. All cells were obtained from the ATCC
collection. The basal cytotoxicity was used to predict
starting doses for in vivo acute oral LD50 values in rodent.
Results are summarized in Table 1.
Measurement of cell viability. HT1080 and MG22A
cells were seeded in 96-well plates in Dulbecco's modified
Eagle's (DMEM) medium containing 10% fetal bovine
serum, 4 mM L-glutamine, without antibiotics, and culti-
vated for 72 h by exposure to the different concentrations
of compounds. After the incubation with the test
compounds, the culture medium was removed, and fresh
medium with 0.2 mg/ml MTT was added in each well of
the plate. After a further incubation (3 h, 37°C, 5% CO2),
the medium with MTT was removed, and 200 μl DMSO
was added at once to each sample. The samples were tested
at 540 nm on a Tecan multiplate reader Infinite100. The
IC50 was calculated using the program Graph Pad Prism® 3.0.
Basal cytotoxicity test. The neutral red uptake assay
was performed according to the standard protocol by
Stokes et al.13 Balb/c 3T3 cells (9000 cells/well) were
placed into 96-well plates for 24 h in DMEM containing
5% fetal bovine serum and then exposed to the test
compounds over a range of eight concentrations (1000,
316, 100, 31, 10, 3, 1 g/ml) for 24 h. Untreated cells were
used as a control. After 24 h, the medium was removed
from all plates, and the cells were washed with 200 μl of
phosphate buffered saline (PBS) for each well. Afterwards,
250 μl of neutral red solution was added (0.05 mg/ml NR
in DMEM 24 h preincubated at 37°C and then filtered
before use through 0.22 µm syringe filter). The plates were
incubated for 3 h, and the cells were washed three times
with PBS. The dye within viable cells was released by
extraction with a mixture of acetic acid–ethanol–water,
1:50:49. The absorbance of neutral red was measured using
a spectrophotometer multiplate reader (TECAN Infinite
M1000) at 540 nm. The optical density (OD) was calcu-
lated using the formula: OD (treated cells) × 100/OD (control
cells). The IC50 values were calculated using the program
Graph Pad Prism® 3.0. The in vivo starting dose is the
estimated LD50 value calculated by inserting the in vitro
IC50 value into a regression formula: log LD50 (mmol/kg) =
0.439 log IC50 (mmol/l) + 0.621.14
References
1. Oger, F.; Lecorgne, A.; Sala, E.; Nardese, V.; Dernay, F.;
Chevance, S.; Desravines, D. C.; Aleksandrova, N.;
Le Guével, R.; Lorenzi, S.; Beccari, A. R.; Barath, P.; Hart, D. J.;
Bondon, A.; Carretoni, D.; Simonneaux, G.; Salbert, G.
J. Med. Chem. 2010, 53, 1937.
2. Nikitjuka, A.; Jirgensons, A. Chem. Hetereocycl. Compd.
2013, 49, 1544. [Khim. Geterotskl. Soedin. 2013, 1669.]
3. Grigorjeva, A.; Jirgensons, A.; Domracheva, I.; Yashchenko, E.;
Shestakova, I.; Andrianov, V.; Kalvinsh, I. Chem. Hetereocycl.
Compd. 2009, 45, 161. [Khim. Geterotskl. Soedin. 2009, 203.]
4. Mai, A.; Esposito, M.; Sbardella, G.; Massa, S. Org. Prep.
Proced. Int. 2001, 33, 391.
5. Bosone, E.; Farina, P.; Guazzi, G.; Innocenti, S.; Marotta, V.;
Valcavi, U. Synthesis 1983, 942.
6. Choi, S. E.; Weerasinghe, S. V. W.; Pflum, M. K. H. Biorg.
Med. Chem. Lett. 2011, 21, 6139.
7. Liu K.-C.; Shelton B. R.; Howe R. K. J. Org. Chem. 1980, 45,
3916.
8. Gucma, M.; Gołebiewski, W. M. Synthesis 2008, 13, 1997.
9. Fang, Y.-Q.; Jacobsen, E. N. J. Am. Chem. Soc. 2008, 130,
5660.
10. Ghosh, S.; Lobera, M.; Pohlhaus, D. T.; Trump, R. P.; Zhang, C.;
Zuercher, W. WO Patent 2013066836.
11. Dai, Y.; Guo, Y.; Curtin, M. L.; Li, J.; Pease, L. J.; Guo, J.;
Marcotte, P. A.; Glaser, K. B.; Davidsen, S. K.;
Michaelides, M. R. Bioorg. Med. Chem. Lett. 2003, 13, 3817.
12. Regulation (EC) No 1272/2008 on classification, labelling, and
packaging of substances and mixtures, amending and repealing,
Directives 67/548/EEC and 1999/45/EC, and amending
Regulation (EC) No 1907/2006. http://eur-lex.europa.eu/ LexUriServ/
LexUriServ.do?uri=OJ:L:2008:353:0001:1355:EN:PDF
13. Stokes, W. S.; Casati, S.; Strickland, J.; Paris, M. In Current
Protocols in Toxicology; John Wiley & Sons: New York,
2008, Vol. 36, p. 20.4.1.
HDAC inhibition activity evaluation of selected novel
compounds 5a, 6a,b,d,f,g,h were performed using the
HDAC Fluorimetric Assay/Drug Discovery Kit, a Fluor de
Lys® Fluorescent Assay System (Enzo Life Sci.), accor-
ding to the manufacturer's protocol. Aziridin-1-yl oximes
5a,c,d,f,h–j were dissolved in DMSO to obtain 10 mM
solution and diluted to the desired test concentration in
14. Canonica, S.; Ferrari, M.; Sisti, M. Org. Prep. Proced. Int.
1989, 21, 253.
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