Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain

Article abstract of DOI:10.1016/j.bmc.2013.03.081

A novel way of chemical modification of the macrolide antibiotic oligomycin A (1) at the side chain was developed. Mesylation of 1 with methane sulfonyl chloride in the presence of 4-dimethylaminopyridine produced 33-O-mesyl oligomycin in 56% yield. Reactions of this intermediate with sodium azide produced the key derivative 33-azido-33-deoxy-oligomycin A in 60% yield. 1,3-Dipolar cycloaddition reaction with propiolic acid, methyl ester of propiolic acid, and phenyl acetylene resulted in 33-deoxy-33-(1,2,3-triazol-1- yl)oligomycin A derivatives substituted at N4 of the triazole cycle. The mesylated oligomycin A and 33-deoxy-33-azidooligomycin A did not inhibit F ₀F₁ ATFase ATPase; however, 33-azido-33-deoxy-oligomycin A and the derivatives containing 4-phenyltriazole, 4-methoxycarbonyl-triazole and 3-dimethylaminoethyl amide of carboxyltriazole substituents demonstrated a high cytotoxicity against K562 leukemia and HCT116 human colon carcinoma cell lines whereas non-malignant skin fibroblasts were less sensitive to these compounds. Novel series of oligomycin A derivatives allow for the search of intracellular molecules beyond F₀F₁ ATP synthase relevant to the cytotoxic properties of this perspective chemical class.

Full text of DOI:10.1016/j.bmc.2013.03.081

Bioorganic & Medicinal Chemistry 21 (2013) 2918–2924
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxicity of oligomycin A derivatives modified
in the side chain
Lyudmila N. Lysenkova a, Konstantin F. Turchin a, Alexander M. Korolev a, Lyubov G. Dezhenkova a,
Olga B. Bekker b,c, Alexander A. Shtil c,d, Valery N. Danilenko b,c, Maria N. Preobrazhenskaya a,c,
⇑
a Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11 B. Pirogovskaya Street, Moscow 119021, Russian Federation
b Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkin Street, Moscow 119991, Russian Federation
c Autonomous Non-Commercial Research Center of Biotechnology of Antibiotics BIOAN, 3 Gubkin Street, Moscow 119991, Russian Federation
d Blokhin Cancer Center, Russian Academy of Medical Sciences, 24 Kashirskoye Shosse, Moscow 115478, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel way of chemical modification of the macrolide antibiotic oligomycin A (1) at the side chain was
developed. Mesylation of 1 with methane sulfonyl chloride in the presence of 4-dimethylaminopyridine
produced 33-O-mesyl oligomycin in 56% yield. Reactions of this intermediate with sodium azide
produced the key derivative 33-azido-33-deoxy-oligomycin A in 60% yield. 1,3-Dipolar cycloaddition
reaction with propiolic acid, methyl ester of propiolic acid, and phenyl acetylene resulted in 33-deoxy-
33-(1,2,3-triazol-1-yl)oligomycin A derivatives substituted at N4 of the triazole cycle. The mesylated
oligomycin A and 33-deoxy-33-azidooligomycin A did not inhibit F0F1 ATFase ATPase; however,
33-azido-33-deoxy-oligomycin A and the derivatives containing 4-phenyltriazole, 4-methoxycarbonyl-
triazole and 3-dimethylaminoethyl amide of carboxyltriazole substituents demonstrated a high cytotox-
icity against K562 leukemia and HCT116 human colon carcinoma cell lines whereas non-malignant skin
fibroblasts were less sensitive to these compounds. Novel series of oligomycin A derivatives allow for the
search of intracellular molecules beyond F0F1 ATP synthase relevant to the cytotoxic properties of this
perspective chemical class.
Received 23 January 2013
Revised 21 March 2013
Accepted 26 March 2013
Available online 6 April 2013
Keywords:
Oligomycin A
33-O-Mesyl oligomycin A
33-Azido-33-deoxy-oligomycin A
Huisgen 1,3-dipolar cycloaddition reaction
33-Deoxy-33-(1,2,3-triazol-1-
yl)oligomycins
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
the parent antibiotics.5 2,3-Dihydrooligomycin A obtained by re-
gio-specific biotransformation of oligomycin A was twice more ac-
Oligomycins belong to the class of highly functionalized macro-
lide antibiotics that contain the hydroxyl groups, the lactone and
spiro moieties, as well as the double bonds. Design of new com-
pounds using oligomycin A (1) scaffold is of interest as F0F1 ATP
synthase inhibitors are perspective drug candidates for treatment
of bacterial infections and cancer.1,2 Recently, we reported the
modification at the C7 carbonyl group and the C2@C3 double bond
of 13 and bromination of 1 leading to the tetrahydropyrane ring
annelated with macrolactone and containing Br in the position
C16.4 However, these modifications seriously changed the structure
of the macrolactone ring that was paralleled by a decrease of cyto-
toxic potency. Earlier it was shown that sodium borohydride
reduction of the 7- and 11-keto groups of oligomycins to the corre-
sponding hydroxyl groups led to the reduced compounds that
inhibited ADP dependent (state 3) respiration, Pi formation and
proton extrusion, the events linked to ATP hydrolysis.5 However,
these compounds exerted no effect on other respiration-linked
activities in intact rat liver mitochondria and were less potent than
tive than the antibiotic against Saccharomyces cerevisiae.2
Studies of other chemical classes of antitumor antibiotics have
demonstrated that the modification at the side chains may yield
the compounds with retained or even higher cytotoxicity and im-
proved pharmacological profile.6–8 The aim of this work was to
investigate the methods of selective modification of the side chain
in 1 and to test the ability of novel compounds to kill human tumor
cells.
2. Results and discussion
2.1. Chemistry
Our attempts to substitute the side chain hydroxyl of 1 by the
interaction with acetylating agents failed. However, we succeeded
in substituting 33-hydroxyl group by methane sulfonyl group in
the reaction of 1 with methane sulfonyl chloride in pyridine in
the presence of DMAP. The 33-O-mesyl oligomycin A (2) was
isolated by column chromatography in 56% yield (Scheme 1). The
interaction of 2 with NaN3 in dry DMF at 60–65 °C led to 33-
azido-33-deoxy-oligomycin A (3) (yield 60%). In the IR spectrum
⇑
Corresponding author. Tel.: +7 4992453753; fax: +7 4992450295.
E-mail address: mnp@space.ru (M.N. Preobrazhenskaya).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.081

L. N. Lysenkova et al. / Bioorg. Med. Chem. 21 (2013) 2918–2924
2919
H3C
CH CH3 CH3
H3C OH
3
H3C
CH CH3 CH3
H3C OH
3
7
13
9
11
O
OH
7
13
9
11
O
OH
15
5
15
5
OH
OH
O
OH
OH
O
CH3
17
H3C
CH3
3
17
H3C
3
MeSO2Cl, DMAP,
pyridine
O
19
O
CH3
25
19
CH3
25
1
1
23
O
23
27
O
21
27
21
O
O
CH3
OSO2CH3
CH3
OH
CH
O
O
29
CH
33
29
3
31
33
3
31
CH3
2
CH3
1
NaN3, DMF
H3C
CH3 CH3 CH3
H3C
13
OH
H3C
CH CH3 CH3
H3C OH
13
3
7
9
OH
11
7
5
15
OH
5
15
OH
O
OH
O
OH O
19
OH
O
CH3
17
H3C
3
CH3
17
H3C
3
O
R
19
21
O
CH3
25
1
CH3
25
1
23
O
23
27
N
O
N
N
4a-b
Cu, CuI for
27
R
without Cu, CuI for 4c
O
21
O
CH3
CH3
O
N3
CH
O
29
CH
33
3
31
29
33
3
31
CH3
CH3
4a-с
3
R = COOH
H2NCH2CH2NMe2,,
PyBOP
H3C
CH CH3 CH3
H3C OH
29
3
7
33
13
9
11
O
OH
31
OH
15
5
OH
O
CH3
17
H3C
3
O
19
CH3
25
1
23
O
N
N
N
O
CONH(CH2)2NMe2
CH3
O
4d
CH3
CH3
a. R = Ph, b. R =COOCH3, c. R = COOH
Scheme 1. Synthesis of 33-O-mesyl oligomycin A (2), 33-azido-33-deoxyoligomycin A (3) and 33-deoxy-33-(1,2,3-triazol-1-yl)oligomycin A derivatives (4a–d).
of 3 the band at 2130 cmꢀ1 characteristic for azide compounds is
present. The structures of 2 and 3 were confirmed by mass spec-
troscopy and 1H and 13C NMR (Tables 1 and 2). A sizeable down-
33-O-mesylate of oligomycin A (2). Because the stereochemistry
of 1 and its 33-O-mesylate at C33 atom was (R), and the substitu-
tion of O-mesyl group for azide belongs to SN2 type reactions, we
can ascribe (S) configuration to the C33 atom of azidooligomycin 3.
The Cu(I)-catalyzed version of the Huisgen 1,3-dipolar cycload-
dition reaction of organic azides and alkynes is one of the most
field shift of C33H signal (
D
dH ꢁ0.9 ppm) and C33
(D
dC ꢁ14 ppm)
in comparison with the corresponding signals in 1, as well as the
presence of CH3SO2 signal at d 3.00 ppm proved the structure of

Table 1
13C and 1H NMR spectra of compounds 1–3, 4b (dC, dH ppm, JH,H Hz)
Compd no.
Oligomycin A (1) CDCl3
33-O-Mesyl-oligomycin A (2) CDCl3
33-Azido-33-deoxy-oligomycin A (3) CDCl3
33-Deoxy-33-(4-methoxycarbonyl-1,2,3-triazol-1-
yl)oligomycin A (4b) CDCl3
dC
dH
JH,H
dC
dH
JH,H
dC
dH
JH,H
dC
dH
JH,H
1
2
3
4
5
6
7
8
165.02
122.61
148.29
40.06
O–CO
165.02
122.61
148.29
40.06
O–CO
165.01
122.63
148.32
40.00
O-CO
164.76
122.55
148.28
40.01
O-CO
CH 5.80d
CH 5.80dd
CH 6.62dd
CH 2.36tq
CH 3.75dd
CH 2.70dq
15.6, 0.7
15.6, 10.1
10.0, 6.6
10.1, 1.3
1.3, 7.4
CH 5.80dd
CH 6.62dd
CH 2.73tq
CH 3.76dd
CH 2.71qd
15.7, 0.7
15.6, 10.0
ꢁ 10.0, 6.5
10.1, 1.2
7.3, 1.3
CH 5.81dd
CH 6.63dd
CH 2.37tq
CH 3.77dd
CH 2.71qd
15.7, 0.7
15.6, 10.1
ꢁ10.1, 6.6
10.1, 1.2
7.4, 1.2
15.7
CH 6.60dd
CH 2.37tq
CH 3.75dd
CH 2.70dq
CO
CH 3.59dq
CH 3.94dd
CH 2.74dq
CO
15.7, 10.1
10.1, 6.5
10.0, 1.1
1.1, 7.3
72.88
72.88
72.88
72.86
46.38
46.52
46.59
46.53
220.16a CO
220.18a CO
220.24a CO
220.16a
41.81b
72.54
41.88b
72.57
CH 3.59dq
CH 3.94dd
CH 2.74dq
8.6, 6.9
8.6, 3.1
3.0, 7.1
41.80b
72.54
CH 3.58dq
CH 3.94dd
CH 2.75dd
8.4, 6.9
8.4, 3.0
7.0, 3.0
41.81b
72.49
CH 3.58dq
CH 3.94dd
CH 2.75qd
8.5, 6.9
8.5, 3.2
7.0, 3.1
8.4, 6.9
8.5, 3.0
3.0, 7.0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
45.63b
45.65b
45.60b
45.63b
219.73a
82.89
219.93a CO
219.80a CO
219.80a CO
82.91
72.15
33.41
38.33
129.30
132.30
130.19
137.64
45.94
31.38
30.90
68.95
35.74
76.10
37.61
99.11
25.88
26.41
30.38
67.14
42.46
64.57
24.66
17.84
8.21
Cq-O
CH 3.89d
CH 1.88m
CH 2.17bd; 2.98dt
CH 5.42ddd
CH 6.00ddd
CH 5.90dd
CH 5.21dd
CH 1.85m
CH2 1.52m; 1.35m
CH2 1.59ddd; ꢁ1.02m
CH3.78ddd
CH 2.11ddq
CH 4.91dd
CH 1.78dq
O–C–O
CH2 1.88m; 1.22m
CH2 2.07; .38m
CH 1.54m
82.90
72.18
33.39
38.35
Cq-O
CH 3.92d
CH 1.85m
CH 2.18m
CH 5.44ddd
CH 6.01ddd
CH 5.92dd
CH 5.24dd
CH 1.85m
82.90
72.22
33.41
38.35
Cq-O
Cq-O
1.9
1.8
CH 3.94c
72.18
33.41
38.34
CHc
CH 1.91m
CH 1.87m
CH2 2.15m; 1.98dt
CH 5.44ddd
CH 6.00ddd
CH 5.92dd
CH 5.19dd
CH 1.85m
CH2 1.32m; 1.20m
CH2 1.55m; 1.00m
CH 3.47ddd
CH 2.07m
CH 4.75dd
CH 1.75dq
O–C–O
CH2 1.90m; 1.22m
CH2 2.05m; 1.40m
CH 1.68m
CH 3.70ddd
CH2 2.15m, 1.85m
CH 4.81ddq
CH3 1.59d
CH3 1.15d
CH3 1.05d
CH3 1.07d
CH3 1.01d
CH3 1.11s
CH3 0.98d
CH2 1.34m, 1.22m
CH3 0.81t
1.98dt
CH2 2.18m, 1.99m
CH 5.45ddd
CH 6.02ddd
CH 5.93dd
CH 5.23dd
CH 1.87m
CH2 ꢁ1.40m
CH2 1.61m; 1.07m
CH 3.70ddd
CH 2.12m
CH 4.95dd
CH 1.78dq
14.8, 10.5, 4.1 129.31
14.7, 10.4, 1.4 132.34
14.7, 10.6, 3.9 129.40
14.6, 10.3, 1.6 132.27
14.6, 10.4, 3.7 129.54
14.6, 10.4, 1.6 132.22
14.9, 11.0, 4.0
14.8, 10.5, 1.3
14.8, 10.5
14.9, 10.5
14.8, 9.6
130.31
137.55
45.65
31.40
30.38
96.14
35.55
75.82
37.55
99.24
25.77
26.09
29.63
67.87
40.60
78.15
21.97
17.78
8.21
14.7, 10.4
14.7, 9.6
130.35
137.47
45.71
31.53
30.65
69.03
35.70
75.92
37.54
99.28
25.86
26.22
29.12
67.86
39.07
54.54
19.83
17.77
8.18
14.7, 10.4
14.7, 9.7
130.55
137.13
45.54
31.53
30.39
68.25
35.46
75.61
37.41
99.25
25.90
26.03
29.79
67.85
40.37
55.06
21.87
7.78
8.23
13.98
9.29
20.93
14.39
28.19
12.00
5.81
14.9, 9.6
CH 1.40m
CH 1.59m; 1.07m
CH 3.71ddd
CH 2.11ddq
CH 4.93dd
CH 1.78dq
O-C-O
9.8, 2.7, 2.4
5.0, 2.2, 6.9
11.4, 5.0
10.0, 3.7, 2.1
ꢁ5.0, 2.0, 7.0
11.4, 4.9
9.8, 3.2, 2.1
11.4, 5.0
11.4, 6.6
11.5, 5.0
11.5, 6.6
11.4,6.6
11.4, 6.6
O–C–O
CH2 1.88m
CH2 2.08m
CH ꢁ1.60m
CH 3.83ddd
CH2 1.83m, 1.73m
CH 4.87ddq
CH3 1.476d
CH3 1.157d
CH3 1.048d
CH3 1.084d
CH3 1.015d
CH3 1.116s
CH3 0.977d
CH2 ꢁ1.35m, ꢁ1.25m
CH3 0.812t
CH3 0.816d
CH3 0.937d
CH3 0.906d
SCH3 : 39.14
CH2 1.93m; 1.24m
CH2 2.09m; 1.41m
CH ꢁ1.63m
CH 3.81ddd
CH2 1.69m, 1.43m
CH 3.46ddq
CH3 1.299d
CH3 1.165d
CH3 1.056d
CH3 1.092d
CH3 1.022d
CH3 1.121s
CH3 0.984d
CH2 ꢁ1.30m, ꢁ1.20m
CH3 0.824t
CH3 0.823d
CH3 0.957d
CH3 0.884d
CH 3.96dt
10.3, 2.5
8.2, 5.0, 2.5
7.3, 6.2, 2.6
8.7, 4.2, 2.5
CH2 1.55m, 1.25m
CH 4.00ddq
CH3 1.213d
CH3 1.159d
CH3 1.047d
CH3 1.085d
CH3 1.011d
CH3 1.110s
CH3 0.977d
9.2, 3.1, 6.2
ꢁ6.2(x5)
6.2
6.5
7.3
6.9
8.1, 5.3, 6.5
6.4, 6.6, 6.8
6.2
6.6
7.3
6.9
7.0
6.5
6.6
7.3
7.0
7.0
6.7
6.4
7.3
6.9
6.9
13.99
9.16
13.93
9.26
13.93
9.26
7.0
20.92
14.39
28.42
11.98
5.97
20.90
14.37
28.16
11.99
5.87
20.90
14.67
28.36
11.86
5.92
6.6
6.6
6.6
6.7
CH2 ꢁ1.35m, ꢁ1.25m
CH3 0.799t
7.4
6.9
6.6
6.9
7.4
6.9
6.6
7.0
7.3
6.9
6.6
7.0
7.3
6.9
6.6
7.1
CH3 0.822d
CH3 0.950d
CH3 0.884d
CH3 0.78d
CH3 0.92d
CH3 0.90d
Azol
11.67
11.13
11.67
10.98
11.63
10.82
11.72
10.99
2.99s
125.60
139.47
161.18
25.01
CH sp2 8.12s
=Cq-N
COO
(C46H)
(C47
(C48
)
)
CH3 sp3 3.92s
(C49H3)
a
b
c
Reverse assignments of signals is possible.
Reverse assignments of signals is possible.
overlap of 13H and 9H chemical shifts.

L. N. Lysenkova et al. / Bioorg. Med. Chem. 21 (2013) 2918–2924
2921
Table 2
Spectra NMR of triazole derivatives of oligomycin A (4a, c, d) (dC, dH, ppm)
Compound
(solvent)
Oligomycin A residue
Triazole residue
d46-H
111.79 147.08 7.84s(1H) R = C6H5
C1 (C48): dC 130.00
dCH3
dCH2
dCH
dCquat
dC46
dC47
R
4a (CDCl3)
21.72, 20.97,
17.83, 14.40,
13.95, 11.95,
11.68, 11.04, 9.24,
8.26, 5.89
40.51,
38.34,
31.50,
30.50,
148.51, 137.43, 132.30, 130.35, 129.30,
122.51 75.80, 72.88, 72.64, 72.29, 69.37,
67.41, 54.31, 46.42, 45.71, 45.70, 41.85,
40.08, 37.48, 35.58, 33.44, 29.33
220.03,
220.01,
165.01,
99.27,
CoH (C49H): dC 125.66; dH
7.85d(2H)
CmH (C50H): dC 128.72;
28.08,
82.90
26.02, 25.87
dH 7.40t(2H)
CpH (C51H): dC 128.15; dH
7.31t(1H)
4c (DMSO-
d6)
22.19, 21.17,
17.47, 15.02,
14.91, 12.09,
11.73, 10.92, 9.96,
8.13, 6.14
40.14,
38.16,
31.14,
30.18,
150.03, 136.05, 132.04, 131.08, 130.86,
121.63 75.18, 72.72, 72.21, 71.53, 68.58,
67.38, 53.73, 47.62, 45.18, 43.52, 42.04,
41.46, 37.23, 35.37, 33.56, 29.12
221.79,
215.69,
164.65,
98.71,
124.75 145.22 8.48s(1H) R = COOH
48: dC 163.86
C
27.98,
82.71
25.90, 25.63
40.64,
38.35,
31.60,
30.44,
4d (CDCl3)
22.10, 20.97,
17.72, 14.43,
13.93, 12.04,
11.81, 11.09, 9.43,
8.38, 5.82
148.71, 137.03, 132.21, 130.63, 129.66,
122.45 75.84, 72.89, 72.52, 72.26, 69.11,
68.47, 55.46, 46.65, 45.75, 45.49, 41.91,
40.09, 37.50, 35.46, 33.44, 30.17
220.13,
219.79,
164.92,
99.21,
123.34 142.49 8.15s(1H) R = CONHCH2CH2N(CH3)2
CONH (C48): dC 160.87;
dNH 7.99bs(1H)
CH2 (C49): dC 35.08; dH
3.81m(2H)
28.31,
82.94
26.11, 26.03
CH2 (C50): dC 57.05; dH
3.08bs(2H)
(CH3)2 (C51 52
, ) : dC 43.86;
dH 2.70bs(6H)
versatile ligation methods known as the click reaction.9 We used
this methodology for derivatization of azidooligomycin A (3).
Cycloaddition of 3 to phenylacetylene or methyl propiolate was
performed in tert-butanol–water mixture in the presence of Cu
powder and Cu1+ salts to give triazole containing derivatives
(4a,b) (Scheme 1). The copper-catalyzed variant of Huisgen 1,3-
dipolar cycloaddition reaction allows for the synthesis of 1,4-
disubstituted regioisomers.10,11 However an attempt to obtain
carboxytriazole derivative 4c by the reaction of azide 3 with propi-
olic acid in the presence of Cu salts led to a complex mixture of
products. In contrast, in the absence of Cu salts compound 3 and
propiolic acid gave carboxytriazole 4c in 50% yield. Esterification
of 4c produced methyl ester identical to 4b obtained by the inter-
action of 3 with methyl propiolate in the presence of Cu salts (as
demonstrated by HPLC).
Table 3
Growth inhibition of Streptomyces fradiae ATCC-19609 strain by oligomycin A and its
derivatives with the modified side chaina
Compound
Streptomyces fradiae ATCC-19609
Concentration, nmol/disk Halo diameter, mm
1
2
3
4a
4b
4c
4d
0.001
0.1
10.0 0.5a
9.5 0.5
7.5 0.5
7.5 0.5
8.0 0.5
8.0 0.5
8.0 0.5
1.0
0.1
10.0
100.0
1.0
a
Mean S.D. of 3 independent measurements.
Mycobacterium tuberculosis and Escherichia coli the amino acids in
these positions are different and these bacteria are resistant to 1.
Next, we tested the ability of 1 and its novel derivatives to in-
duce death of human K562 leukemia and HCT116 colon carcinoma
cell lines. In these experiments cells were treated with each com-
pound for 72 h followed by determination of cell viability in an
MTT test.17 Importantly, 3 and 4a,b were equally potent as the par-
ent antibiotic 1 whereas 33-O-mesyl derivative 2 and 33-deoxy-
33-(4-carboxytriazol-1-yl)oligomycin 4c were significantly less
cytotoxic (Table 5). Interestingly, 4c that carries the carboxyltriazol
group virtually lost the cytotoxicity whereas the amide containing
compound 4d retained this activity. We tend to attribute these re-
sults to a low ability of negatively charged 4c to enter the cell, the
effect abrogated by the presence of positively charged N atoms in
4d.
Finally, the potency of 1 and its derivatives 3, 4a, 4b and 4d ac-
tive against tumor cell lines was compared with the cytotoxicity of
these compounds for non-malignant cells. Importantly, human
skin fibroblasts were substantially more resistant to 1, 3, 4a, 4b
and 4d than K562 leukemia or HCT116 colon carcinoma cell lines
(Table 5), suggesting that oligomycins can be preferentially potent
for malignant cells.
The acid 4c was transformed into amide of N,N-Dimethylethyl-
endiamine using PyBOP reagent. The compound was purified by
column chromatography on silica gel and then on Sephadex LH-
20 in methanol. A detailed comparison of 13C and 1H NMR spectra
of 1 and methoxycarbonyl-triazole 4b is presented in Table 1. NMR
spectra of compounds 4a,c,d are shown in Table 2.
2.2. Biology
Gram-negative and Gram-positive bacteria are known to be
resistant to 1 except actinobacteria Streptomyces, including S. livi-
dans.12,13 We studied the antibacterial activity of novel compounds
against Streptomyces fradiae ATCC-19609 strain extremely sensi-
tive to 1 (<0.001 nmol/mL or 0.0005 nmol/disk).14 This test system
has been validated by us as an adequate model for the analysis of
sensitivity to 1.14,15 The derivatives studied with this test system
are shown in Table 3. Compound 2 was two orders of magnitude
weaker than the parental antibiotic 1. The potencies of other tested
compounds were even less pronounced.
Recently, we identified the primary sequence of F1F0 ATP syn-
thase c-subunit of S. fradiae ATCC-19609 (http://www.ncbi.nlm.
nih.gov/nuccore/KC169997.1). For S. fradiae and Saccharomyces
cerevisiae the amino acids responsible for binding of 1 to c-subunits
are identical (Table 4).16 It is interesting to note that in
The present study demonstrates that, similarly to the congeners
with changed structure of the macrolactone cycle in 1, modifica-
tions of the side chain significantly dropped the ability to inhibit

2922
L. N. Lysenkova et al. / Bioorg. Med. Chem. 21 (2013) 2918–2924
Table 4
Comparison of oligomycin binding domains in c-subunits of F1F0 ATPsynthases in oligomycin sensitive and -resistant bacteria
Microorganism
Oligomycin binding domain in F1F0 ATPsynthase c-subunit
First a.a.
Amino acid sequence, a.a.
Last a.a.
S. fradiaea
54
54
52
51
54
54
ILGFAFCEAL ALIGLc
ILGFAFCEAL ALIGL
ILGFALSEAT GLFCL
ILGFALSEAMGLFCL
FITVGLVEAA YFINL
FIVMGLVDAI PMIAV
68
68
66
65
68
68
S. lividansa
S. cerevisiaea
H. sapiensa
M. tuberculosisb
E. colib
a
b
C
Oligomycin sensitive organisms.
Oligomycin resistant organisms.
Amino acids involved in binding to oligomycin are rendered bold.
Table 5
against S. fradiae cells. The azido congener and 33-(1,2,3-triazol-
1-yl)oligomycin A derivatives containing phenyl or methoxycar-
bonyl moieties were similarly cytotoxic as 1 for human K562
leukemia and HCT116 colon carcinoma cell lines. The derivative
33-(4-carboxy-1,2,3-triazol-1-yl)oligomycin A was virtually non-
toxic whereas its N-(2-dimethylaminoethyl)amide analogue
retained the activity, suggesting a role for electric charges in the
side chain substituents in the cytotoxic potency of novel deriva-
tives of 1. Importantly, the cytotoxicity for non-malignant skin
fibroblasts was substantially less pronounced. We believe that
these SAR considerations provide valuable information for design
of drugs based on 1.
Cytotoxicity of 1 and its derivatives for tumor and non-malignant cell linesa
Compound
Cell line
HCT116
hFB-hTERT6
R562
1
2
3
4a
4b
4c
4d
0.16 0.02
5.80 2.10
0.14 0.02
0.15 0.04
0.15 0.03
>12.50
1.00 0.20
6.40 0.90
0.20 0.03
ND
ND
ND
7.50 0.90
ND
1.90 0.27
3.00 0.45
2.00 0.20
>50
0.22 0.03
0.70 0.14
2.00 0.27
ND, not determined.
a
IC50, lM (MTT test after 72 h of cell exposure; mean SD of 3 independent
measurements).
4. Experimental
4.1. Chemistry
growth of bacterial test culture [see also Ref. 4,18]. One may
hypothesize that, by altering the structure of 1, the affinity to
F0F1 ATP synthase would be lowered regardless of whether the
macrolactone ring or the side chain was changed. In contrast, the
integrity of the macrolactone cycle appears to be critical for killing
tumor cells. This differential potency strongly suggests that F0F1
ATPsynthase may not be a key target for antitumor characteristics
of 1 and its derivatives. Or else the modifications of 1 may switch
the preference of a particular derivative from one specific target to
another.
The analysis of a crystal structure of yeast ATPsynthase c ring
complexed with 116 shows that the side chain hydroxyl group of
1 is hydrogen bonded to the side chain of Glu59 (yeast numbering
system) via a water molecule. The hydroxyl group at C33 is buried
between two subunits. Sequence alignment between yeast and S.
fradiae ATPsynthase shows that residues involved in binding of 1
to yeast c ring are highly conserved; S. fradiae Glu61 can interact
with the side chain of 1 like yeast Glu59. If 1 binds to ATP synthase
of Streptomyces similarly to the yeast ATPsynthase, a bulky substi-
tuent at C33 would disturb the interaction.
4.1.1. General procedures
Oligomycin A (1) (purity 95%) was produced at Autonomous
Non-Commercial Research Center of Biotechnology of Antibiotics
BIOAN, Moscow using Streptomyces avermitilis NIC B62. Fermen-
tation was performed for 8 days at 28 °C in liquid medium. Iso-
lation and purification were performed by extraction with
acetone–hexane mixture followed by crystallization. All other re-
agents and solvents were purchased from Aldrich, Fluka and
Merck. The solutions were dried over sodium sulfate and evapo-
rated under reduced pressure on a Buchi rotary evaporator at
<35 °C.The progress reaction products, column eluates and all fi-
nal samples were analyzed by TLC on Merck G60F254 precoated
plates. Reaction products were purified by column chromatogra-
phy on Merck silica gel 60 (0.04–0.063 mm). The NMR spectra
were recorded on Unity+400 (Varian, Palo Alto, CA) spectrometer
(operating frequencies for 1H: 400.0 MHz, for 13C:100.6 MHz),
and on Avance 600 (Bruker, Karlsruhe, Germany) spectrometer
(1H: 600.0 MHz, 13C: 150.9 MHz). The NMR elucidation was
made using homo 1H,1H (COSY) and hetero 1H, 13C (HETCOR,
HSQC) 2D-correlations as well as 1H and 13C 1D spectra of 1
and its derivatives. ESI-mass spectra were recorded on micrO-
TOF-Q II instrument (Bruker). HPLC analyses were performed
on a Shimadzu HPLC instrument of the LC 10 series and Dia-
spher-110-C18 column (BioChemMac, Russia; ID-4 ꢂ 150 mm,
Our series of derivatives with individual variants of the complex
structure of 1 allow for the search of intracellular molecules be-
yond F0F1 ATPsynthase relevant to the cytotoxic properties of this
clinically perspective chemical class.
3. Conclusion
particle size 5 lm), injection volume 10 ll, wavelength 230 nm.
The concentration of samples was 0.02–0.05 mg/mL. The systems
contained (A) H2O or (B) acetonitrile. The proportion of acetoni-
trile varied from 70 to 90% for 10 min and then 90% for 10 min,
flow rate 1 mL/min. Retention time for 1 was 10.85 min. UV
spectra were obtained on a UV/Vis double beam spectrometer
UNICO in methanol. IR spectra were recorded on Thermo Nicolet
iS10 (Thermo Scientific, Waltham, MA). Optical rotation was
measured on AA55 Polarimeter (Optical Activity Ltd, Huntingdon,
UK).
A method of chemical modification of the macrolide antibiotic
oligomycin A (1) at the side chain was developed. Selective mesy-
lation of 1 at the position 33 led to 33-O-mesyl oligomycin that
was transformed into 33-azido-33-deoxyoligomycin A. The latter
was used in Huisgen 1,3-dipolar cycloaddition reaction with phen-
ylacetylene, propiolic acid and its methyl ester to produce 33-
deoxy-33-(1,2,3-triazol-1-yl)oligomycin A derivatives. All newly
synthesized derivatives were significantly less potent than 1

L. N. Lysenkova et al. / Bioorg. Med. Chem. 21 (2013) 2918–2924
2923
4.1.2. 33-O-Mesyl oligomycin A (2)
4b as colorless amorphous powder in 52% yield. MS (ESI): calcd
for
49H77N3O12Na (M+Na+): 922.5405. Found: 922.5406. UV
(MeOH) knm ): 223 (86364), 232 sh. (73636), 239 sh. (43536).
To a solution (0.2 g, 0.2 mmol) of 1 in 6 mL of dry pyridine,
DMAP (0.1 g, 0.001 mmol) and mesyl chloride (0.06 ml, 0.8 mmol)
in chloroform (1 mL) were added. The reaction mixture was stirred
for 1.5 h at room temperature (rt). Then mesyl chloride (0.04 mL,
0.5 mmol) in chloroform (1 mL) and mesyl chloride (0.02 mL,
0.25 mmol) in chloroform (1 mL) were added 1 h apart. The reac-
tion was controlled by TLC (hexane/acetone 1:1). The reaction mix-
ture was poured on ice, acidified by 1 N HCl to pH 3, and the
product was extracted with ethyl acetate (2 ꢂ 50 mL). The extract
was washed by water to neutral pH, dried and concentrated in va-
cuo. After purification by column chromatography on silica gel
(hexane/acetone 4:1) 0.11 g (56%) of compound 2 was obtained
as colorless amorphous powder. Rf 0.47 (hexane/acetone 1:1), Rt
12.4 min (gradient elution in water/acetonitrile). MS (ESI) calcd
for C46H76 NaO13S (M+Na+): 891.4904, found: 891.4939. Calcd for
C
(e
IR (film): 3486, 2972, 2934, 1704, 1456, 1385, 1278, 1226, 1099,
1046, 987(cmꢀ1). ½a D24
ꢀ66° (c 0.18, CH3OH). Rt 14.274 (A: 0.01 M
ꢃ
H3PO4 pH 2.6; B: MeCN), A 30%, B 70%.
4.1.6. 33-Deoxy-33-(4-carboxytriazol-1-yl)–oligomycin A (4c)
To a solution of 3 (0.05 g, 0.06 mmol) in 5 mL of tert-butanol pro-
piolic acid (0.2 mL, 0.2 mmol) was added, and the reaction mixture
was stirred at 45 °C for 24 h. The portions of propiolic acid
(0.05 mL, 0.05 mmol) were added every 6 h. The reaction was con-
trolled by TLC (CHCl3 /MeOH/acetic acid 10:2:0.1). The reaction mix-
ture was diluted with water, tert-butanol was evaporated and the
reaction product was extracted with ethyl acetate (15 mL ꢂ 2). The
extract was washed with 1% NaHCO3 and evaporated. The product
was purified by column chromatography (CHCl3 /MeOH/acetic acid
20:2:0.1), washed with water, dried (Na2SO4), concentrated in vacuo
and gave after addition of hexane 0.027 g (50%) of 4c as colorless
amorphous powder. MS (ESI): calcd for C48H75N3O12Na (M+Na+):
C46H76 KO13S (M+K+): 907.4643. Found: 907.4638. UV knm
(e)
(MeOH): 220 (31193), 225 (34312), 232 (30183), 242 (16972). IR
(film) 3478, 2978, 2930, 1700, 1642, 1458, 1285, 1231, 1175,
1138, 1098, 985, 926 (cmꢀ1). ½a D20
ꢀ43° (c 0.14, MeOH).
ꢃ
908.5249. Found: 908.5281. UV (MeOH) knm
(e): 213–223
4.1.3. 33-Azido-33-deoxyoligomycin A (3)
(58,571), 232 sh. (51,428), 241 sh. (31,428) (nm). IR (film): 3462,
To a solution (0.5 g, 0.6 mmol) of 33-O-mesyl oligomycin 2 in
dry DMF (10 mL) dry sodium azide was added (0.25 g, 4 mmol),
and the reaction mixture was stirred at 60–65°C for 2 h. The reac-
tion was controlled by TLC (hexane/acetone 1:1; chloroform/meth-
anol 10:0.5). Then sodium azide (0.125 g, 2 mmol) was added. The
reaction mixture was extracted with toluene/ethyl acetate (1:1),
washed with water and dried (Na2SO4). The compound 3 was iso-
lated by column chromatography on silica gel (chloroform/metha-
nol 30:0.5). The azide 3 was obtained in 60% yield as colorless
amorphous powder, Rf 0.68 (chloroform/methanol 10:05); Rt
23.0 min. MS (ESI) Calcd for C45H73N3O10Na (M+Na+): 838.5194.
2972, 2936, 2810, 1704, 1644, 1548, 1456, 1385, 1280, 1226, 1193,
1135, 1033. 1048, 986, 922, 880 (cmꢀ1). ½a D24
ꢀ91° (c 0.176, CH3OH).
ꢃ
Rt 10.30 (A: 0.01 M H3PO4 pH 2.6; B:MeCN), A 30 %, B 70%.
4.1.7. N-(2-Dimethylaminoethyl)amide of 33-deoxy-33-(4-
carboxytriazol-1-yl)–oligomycin A (4d)
To a solution of 4c (0.05 g, 0.056 mmol) in 5 mL DMF PyBOP
(0.05 g,
0.1 mmol)
and
2-dimethylaminoethylenediamine
(0.012 mL, 0.1 mmol) were added and stirred for 24 h. The reaction
was controlled by TLC (CHCl3/MeOH/acetic acid 10:3:0.2). Then the
reaction mixture was diluted with water, NaCl was added followed
by extraction with ethyl acetate (15 mL ꢂ 2). The extract was
washed with 1% NaHCO3 and evaporated. The product was purified
by column chromatography (CHCl3/MeOH/acetic acid 10:2:0.1),
the fraction containing pure 4d was washed with 1% NaHCO3,
evaporated and purified on Sephadex LH-20 in methanol to yield
0.008 g (15%) of amide 4d as colorless amorphous powder. MS
(ESI): calcd for C52H86N5O11 (M+H+): 956.6324. Found: 956.6323.
Found: 838.5178. UV: knm (e) (MeOH): 220 (31230), 225 (33450),
232 (33020), 242 (17500) (nm); IR (film) 3478, 2978, 2930, (azide)
2100, 1700, 1642, 1458, 1285, 1231, 1175, 1138, 1098, 985, 926
(cmꢀ1). ½a 2D0
ꢀ24° (c 0.1, MeOH).
ꢃ
4.1.4. 33-Deoxy-33-(4-phenyltriazol-1-yl)oligomycin A (4a)
To a solution of 3 (0.05 g, 0.06 mmol) in 5 mL of mixture (tert-
butanol/water, 1:1) phenylacetylene (0.06 g, 0.6 mmol), sodium
ascorbate as a powder (0.018 g, 0.06 mmol), Cu powder (0.06 g,
0.95 mmol) and CuI (0.05 g, 0.26 mmol) were added and stirred
at 60–65 °C for 18 h under argon atmosphere. The reaction was
controlled by TLC (hexane/acetone 1:1) and diluted with water.
The product was extracted with ethyl acetate (15 mL ꢂ 2), the ex-
tract was filtered, dried (Na2SO4) and evaporated. The compound
was purified by column chromatography (hexane/aceton 4:1) to
yield 0.022 g of 4a as colorless amorphous powder in 39% yield.
Rt 16.053 (A: H2O; B: MeCN), A 20%, B 80%. MS (ESI): calcd for
UV (MeOH) knm (e): 215–225 (53246), 232 sh. (46752), 241 sh.
(28570) (nm). IR (film): 3360, 2967, 2928, 1705, 1659, 1574,
1508, 1458, 1385, 1277, 1226, 1191, 1097, 1049, 987, 923
(cmꢀ1). ½a 2D4
ꢀ18° (c 0.22, CH3OH). Rt 9, 28 (A: 0.2% COONH4 pH
ꢃ
4.5; B: MeCN), A 30%, B 70%.
4.2. Biology
The antibacterial activity of 1 and its novel derivatives was
determined as the diameter of growth inhibition zone of Strepto-
myces fradiae cells around paper disks impregnated with tested
compounds. The spore suspension was mixed with agarized MG
medium (0.7% agar), pH 7.5 and plated on Petri dishes with agar-
ized MG medium (2% agar) (107 spores per dish). After solidifica-
tion of agar the dishes were overlaid with paper disks containing
tested compounds. The lawn was grown for 24 h at 28 °C, then
the halo diameter around the disks was measured.14 Tumor cell
lines, culture conditions and cytotoxicity tests were carried out
as reported.17,18 The hFB-hTERT6 non-malignant human skin fibro-
blast cell line was established in Center for Medical Genetics, Rus-
sian Academy of Medical Sciences, Moscow.
C
53H79N3O10Na (M+Na+): 940.5663. Found: 940.5712. UV: knm
(e)
(MeOH): 205 (60,000), 225 sh. (54,000), 232 (56,000), 241 sh.
(45000) (nm). IR: 3466, 2971, 2931, 2870, 1699, 1643, 1456,
1384, 1278, 1226, 1191, 1097, 1049, 966 (cmꢀ1). ½a D24
ꢀ65° (c
ꢃ
0.184, CH3OH).
4.1.5. 33-Deoxy-33-(4-methoxycarbonyl-triazol-1-
yl)oligomycin A (4b)
To a solution of 3 (0.06 g, 0.075 mmol) in 5 mL of tert-butanol/
water (1:1) methyl propiolate (0.03 ml, 0.37 mmol) and powder of
Cu (0.06 g, 0.95 mmol) were added and stirred at rt for 6 h. The
reaction products were controlled by TLC (toluene/ethyl acetate
2:1). The reaction mixture was diluted with water, the product
was extracted by ethyl acetate (15 mL ꢂ 2), filtered, dried (Na2SO4)
and evaporated. The product was purified by column chromatogra-
phy (toluene/ethyl acetate 25:1) to yield 0.034 g, (0.037 mmol) of
Acknowledgments
This study was supported by the program ‘Research and
development of priorities of scientific and technological complex

2924
L. N. Lysenkova et al. / Bioorg. Med. Chem. 21 (2013) 2918–2924
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8. Tevyashova, A. N.; Shtil, A. A.; Olsufyeva, E. N.; Dezhenkova, L. G.; Isakova, E. B.;
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Supplementary data
Supplementary data (NMR and IR spectra of the compounds)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.03.081. These data include
MOL files and InChiKeys of the most important compounds de-
scribed in this article.
14. Alekseeva, M. G.; Elizarov, S. M.; Bekker, O. B.; Lubimova, I. K.; Danilenko, V. N.
Biochemistry (Moscow) Suppl. A Membr. Cell Biol. 2009, 3, 16.
15. Lysenkova, L.N.; Korolev, A.M.; Shtil, A.A.; Bekker, O.B.; Elizarov, S.M.;
Preobrazhenskaya, M.N.; Danilenko, V.N. Patent No 2454420 (Russian
Federation).
16. Symersky, J.; Osowski, D.; Walters, D. E.; Mueller, D. M. PNAS 2012, 109, 13961.
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