Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти
Eight new phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles were designed and tested for their ability to inhibit cancer cell growth. These compounds were evaluated against complete human tumor cell lines NCI-60. Only three compounds showed antitumor activity in the single dose assay, which were tak...
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V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine
2023
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Ukrainica Bioorganica Acta| _version_ | 1871193589248163840 |
|---|---|
| author | Bahrieieva, Oksana S. Golovchenko, Oleksandr V. Golovchenko, Oksana I. Zhirnov, Victor V. Brovarets, Volodymyr S. |
| author_facet | Bahrieieva, Oksana S. Golovchenko, Oleksandr V. Golovchenko, Oksana I. Zhirnov, Victor V. Brovarets, Volodymyr S. |
| author_institution_txt_mv | [
{
"author": "Oksana S. Bahrieieva",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, Kyiv, Ukraine"
},
{
"author": "Oleksandr V. Golovchenko",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, Kyiv, Ukraine; O.O. Bogomolets National Medical University, Kyiv, Ukraine"
},
{
"author": "Oksana I. Golovchenko",
"institution": "O.O. Bogomolets National Medical University, Kyiv, Ukraine"
},
{
"author": "Victor V. Zhirnov",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, Kyiv, Ukraine"
},
{
"author": "Volodymyr S. Brovarets",
"institution": "V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, Kyiv, Ukraine"
}
] |
| author_sort | Bahrieieva, Oksana S. |
| baseUrl_str | https://bioorganica.com.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-19T14:56:54Z |
| description | Eight new phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles were designed and tested for their ability to inhibit cancer cell growth. These compounds were evaluated against complete human tumor cell lines NCI-60. Only three compounds showed antitumor activity in the single dose assay, which were taken in the five dose assay. Compounds 7 and 8 showed the same average antiproliferative activity and cytotoxicity against sensitive cell lines of the general panel. However, compound 8 showed cytotoxicity to more lines than 7. By all parameters, these compounds were more active than compounds 5. Compounds 7 and 8 also showed high and similar antiproliferative activity in the concentration range GI50: 1-6 and TGI: 6-14 μM against all subpanels. Their cytotoxicity was in the concentration range of 25-54 μM. Compound 5 showed the same activity, with the exception of the leukemia, non-small cell lung cancer and ovarian cancer subpanels against which their activity was lower. When analyzing the structure-activity, it turned out that among the phosphorylated oxazole derivatives, only compounds containing the triphenylphosphonium cation (TPP+) in the 4th position of the oxazole ring exhibit antitumor activity. Moreover, the replacement of the phenyl radical in the 2nd position of the oxazole scaffold with a methyl radical led to the disappearance of the activity. The COMPARE algorithm reveals a high correlation of the antiproliferative activity of the tested compounds with the antitumor agents phyllantoside and chromomycin A3 in the GI50 vector and moderate with phyllantoside in the TGI vector. The target of all standard drugs that correlate with the cytotoxicity of the studied compounds, with the exception of didemnin, is DNA. Unlike standard compounds, synthesized active compounds carry a delocalized TPP+, which delivers them predominantly to mitochondria due to a much more hyperpolarized potential of the mitochondrial membrane in cancer cells than in normal ones. Therefore, their anticancer activity is most likely due to a disturbation of the structural and functional state of the latter due to interference with their intrinsic protein-synthesizing apparatus of mitochondria. The data obtained allow us to consider 5-(hydroxyalkylamino)-1,3-oxazoles loaded with TPP+ as leading compounds for further in-depth study and synthesis of new TPP+-containing 1,3-oxazole derivatives with antitumor activity |
| doi_str_mv | 10.15407/bioorganica2023.01.032 |
| first_indexed | 2025-07-17T12:19:51Z |
| format | Article |
| fulltext |
ISSN 1814-9758. Ukr. Bioorg. Acta, 2023, Vol. 18, N 1
UDC 547.787+547.789:57.083.3
DOI: https://doi.org/10.15407/bioorganica2023.01.032
32
Ukrainica Bioorganica Acta
www.bi oorgan ica .org .ua
RESEARCH ARTICLE
New phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles as potential
anticancer agents
Oksana S. Bahrieieva1, Oleksandr V. Golovchenko1,2, Oksana I. Golovchenko2,
Victor V. Zhirnov1, Volodymyr S. Brovarets1*
1 V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the NAS of Ukraine, Kyiv, Ukraine
2 Bogomolets National Medical Universiry, Kyiv, Ukraine
Abstract: Eight new phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles were designed and tested for their ability to inhibit cancer cell
growth. These compounds were evaluated against complete human tumor cell lines NCI-60. Only three compounds showed antitumor
activity in the single dose assay, which were taken in the five dose assay. Compounds 7 and 8 showed the same average antiproliferative
activity and cytotoxicity against sensitive cell lines of the general panel. However, compound 8 showed cytotoxicity to more lines than 7.
By all parameters, these compounds were more active than compounds 5. Compounds 7 and 8 also showed high and similar
antiproliferative activity in the concentration range GI50: 1-6 and TGI: 6-14 μM against all subpanels. Their cytotoxicity was in the
concentration range of 25-54 μM. Compound 5 showed the same activity, with the exception of the leukemia, non-small cell lung cancer
and ovarian cancer subpanels against which their activity was lower. When analyzing the structure-activity, it turned out that among the
phosphorylated oxazole derivatives, only compounds containing the triphenylphosphonium cation (TPP+) in the 4th position of the oxazole
ring exhibit antitumor activity. Moreover, the replacement of the phenyl radical in the 2nd position of the oxazole scaffold with a methyl
radical led to the disappearance of the activity. The COMPARE algorithm reveals a high correlation of the antiproliferative activity of the
tested compounds with the antitumor agents phyllantoside and chromomycin A3 in the GI50 vector and moderate with phyllantoside in the
TGI vector. The target of all standard drugs that correlate with the cytotoxicity of the studied compounds, with the exception of didemnin,
is DNA. Unlike standard compounds, synthesized active compounds carry a delocalized TPP+, which delivers them predominantly to
mitochondria due to a much more hyperpolarized potential of the mitochondrial membrane in cancer cells than in normal ones. Therefore,
their anticancer activity is most likely due to a disturbation of the structural and functional state of the latter due to interference with their
intrinsic protein-synthesizing apparatus of mitochondria. The data obtained allow us to consider 5-(hydroxyalkylamino)-1,3-oxazoles
loaded with TPP+ as leading compounds for further in-depth study and synthesis of new TPP+-containing 1,3-oxazole derivatives with
antitumor activity.
Keywords: phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles; synthesis; anticancer activity; COMPARE correlations.
Introduction
Today, the solution to the problem of treating cancer,
which is one of the leading causes of morbidity and
mortality worldwide, is considered one of the main tasks of
Received:
Revised:
Accepted:
Published online:
17.04.2023
21.04.2023
19.05.2023
30.06.2023
Corresponding author. Tel.: +380-44-573-2596;
e-mail: brovarets@bpci.kiev.ua (V. S. Brovarets)
ORCID: 0000-0001-6668-3412
medicine. Among the various types of treatment,
chemotherapy continues to play a leading role in the
treatment of cancer. However, due to the serious side effects
inherent in tumor chemotherapy, as well as the development
of drug resistance to them, significant efforts are being
made to find and develop new drugs with increased
antitumor efficacy and reduced side effects. A significant
place among them belongs to 1,3-oxazole derivatives,
which have a wide spectrum of antitumor activity due to
their inherent multiplicity of mechanisms of action [1, 2].
The biological activity of phosphorylated oxazoles has
been little studied, with the exception of compounds
containing phosphonium ion, used for drug delivery to cell
mitochondria. It has been shown that the antifungal activity
of diethyl ethers of 1,3-oxazole-4-phosphonic acids appears
© O.S. Bahrieieva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
mailto:brovarets@bpci.kiev.ua
https://orcid.org/0000-0001-6668-3412
O.S. Bahrieieva, O.V. Golovchenko et al.
33
to be mediated by inhibition of aldolase II in Candida
albicans [3], and antihypertensive activity of 4-phospho-
nyloxazoles in rats due to inhibition of phosphodiesterase
III [4]. The antitumor activity of 4-triphenylphosphonium
salts of 1,3-oxazol have been assessed against NCI-60
cancer cell lines, which showed high anticancer activity
(GI50: 0.3-1.1, TGI: 1.2-2.5 and LC50: 5-6 μM) [5].
In this work, a series of novel phosphorylated 5-(hydro-
xylalkylamino)-1,3-oxazoles was constructed and tested for
their ability to inhibit the growth of the full NCI-60 human
tumor cell lines.
Results and Discussion
Chemistry
The synthesis of compounds 1-8 was carried out by
known methods [6-9]. Diethyl 5-(hydroxyalkyl)amino-2-R-
1,3-oxazol-4-phosphonates 1-4 were obtained by the
interaction of available diethyl esters of 1-aroylamino-2,2,2-
trichloroethylphosphonic acids Ia,b with an excess of the
corresponding aminoalcohols in methanol at room
temperature (Scheme 1).
R N
H
CCl3
P(O)(OEt)2O
N
O
P(O)(OEt)2
NR1R2R
+ HNR1R2
(excess) 18-25 oC
MeOH
I R R R1 R2
a Ph 1 4-O2NC6H4 Me (CH2)2OH
b 4-O2NC6H4 2 Ph H (CH2)3OH
3 Ph H CH2CH(OH)CH2OH
4 Ph Me CH2(CH(OH))4CH2OH
Scheme 1. Synthesis of diethyl 5-(hydroxyalkyl)amino-2-aryl-1,3-
oxazol-4-phosphonates 1-4.
Phosphonium salts 5-8 were synthesized in a similar
way. For this, 1-acylamino-2,2-dichloroethenylphospho-
nium chlorides IIa,b were used as starting reagents. The
interaction of compounds IIa, b with an excess of
aminoalkanol takes place in a methanol environment at 20-
25 ºС and leads to the formation of 2-R-5-(hydroxy-
alkylamino)-1,3-oxazol-4-yl(triphenyl)phosphonium chlo-
rides. For the convenience of isolation and obtaining
analytically pure samples, these substances were
transformed into the corresponding perchlorates 5-8 by
treating the reaction mixture with a saturated solution of
sodium perchlorate (Scheme 2).
The structure of all obtained compounds was proven by
means of elemental analysis, 1H-, 13C-, 31P-NMR
spectroscopy and mass spectrometry. Thus, the values of
the proton signals in the 1H-NMR spectra of compounds 1-8
fully correspond to the presented structures. Particular
attention is drawn to the data of 13C-NMR spectra, in
particular, the signals of the carbon nuclei of the oxazole
cycle and ethoxyl groups of products 1-4, as well as phenyl
rings in compounds 5-8, which appear in the form of
doublets due to their interaction with the nucleus of the
Phosphorus atom. Thus, in phosphonates 1-4, the signals of
carbon nuclei in position 5 of the oxazole cycle appear in
the region of 163.5-161.26 ppm with a spin-spin interaction
constant (J) of 38.9-37.9 Hz, for C(2) in the range of 150.7-
147.8 ppm with the J value of 22.4-21.9 Hz, and for С(4) in
the region of 101.5-96.0 ppm and with J = 254.8-254.3 Hz.
The signals of the methylene group of the dietho-
xyphosphoryl fragment are in the range of 62.0-61.6 ppm
with spin-spin interaction constant 5.5-5.0 Hz, and the
methyl group at 16.2 ppm with J 6.5-6.0 Hz.
Replacement of the 1,3-oxazole ring of the diethoxy-
phosphoryl group in position 4 with a more electron-
accepting triphenylphosphonium does not significantly
affect the position of the signals of the Carbon nuclei in
positions 2 and 5, however, a shift of the signals of the
Carbon C(4) nuclei to a stronger field region of the
spectrum is observed. Thus, the signals of Carbon C(5)
nuclei appear in the range of 165.31-164.0 ppm (29.8-27.6
Hz), C(2) at 153.3-152.8 ppm (21.3-20.5 Hz), and C(4) at
85.84-81.62 ppm with spin-spin interaction constant 154.8-
153.3 Hz. The signals of the phenyl fragment of the
triphenylphosphonium residue of oxazoles 5-8 in the
13C NMR spectra are in the region: С(4) 135.45-135.31 ppm
(3.0-2.9 Hz), С(3) 134.76-134.51 ppm (11.0-10.5 Hz), С(2)
R N
H
CCl2
+PPh3ClO
+ R1HN
OH
n
-
2. NaClO4 (sat.)
1. MeOH, 18-25 oC
N
O
+PPh3ClO4
N
R1
R
-
n
OH
5-8
(excess)
II R R R1 n
a Me 5 Ph H 1
b Ph 6 Me H 1
7 Ph Me 1
8 Ph H 2
Scheme 2. Synthesis of 5-(hydroxyalkyl)amino-2-R-1,3-oxazol-4-(triphenyl)phosphonium perchlorates 5-8.
ISSN 1814-9758. Ukr. Bioorg. Acta, 2023, Vol. 18, N 1
34
130.63-130.58 ppm (13.2-12.5 Hz) and C(1) 120.54-119.40
ppm (93.9-93.2 Hz). Signals of phosphorus nuclei in the
31P-NMR spectra of compounds 1-8 are fixed in the range
of 14.3-10.5 ppm.
In the IR spectra of compounds 1-8, intense absorption
bands of C=N groups of the oxazole cycle were found in the
region of 1628-1605 cm-1 and 1601-1584 cm-1. In
phosphonates 1-4, the absorption bands of the P=O group
are fixed at 1245-1200 cm-1, and those of the P-O-C bonds
at 1049-1016 cm-1 and 976-961 cm-1. The absorption bands
of perchlorate anion in the range of 1110-1106 cm-1 are
observed in phosphonium salts 5-8.
Biology
One dose assay
According to the mean growth inhibition percent of the
total panel, indicated in brackets, the tested compounds
according to the increase in activity are arranged in a row:
1 = 2 = 3 = 4 = 6 (≤6) < 5 (92) < 7 = 8 (98). Hence, only
compounds 5, 7 and 8 showed antitumor activity, complete
data for which are presented in Table S1. The data of
statistical analysis of the antitumor activity of compounds 5,
7 and 8 by subpanels are presented in Table 1.
Table 1. Average growth inhibition of the NCI-60 cancer
cell line subpanels by compounds 5, 7 and 8 in one dose
assay.
Subpanel Compound
5 7 8
Leukemia 99±6 98±7 100±7
NSCLC 84±8 97±5 92±6
Colon cancer 96±16 102±14 103±13
CNS cancer 90±3 99±7 95±2
Melanoma 112±16 124±12 126±13
Ovarian cancer 83±15 83±15 84±14
Renal cancer 48±12 69±12 66±10
Breast cancer 112±8 116±9 102±11
The compounds added at a concentration (1∙10-5 M) and
the culture incubated for 48 h. The number reported for the
one-dose assay is growth inhibition (%) relative to the no-
drug control, and relative to the time zero number of cells.
NSCL - Non-Small Cell Lung cancer. Data represent as
mean ± SE, %.
Compounds 5, 7 and 8 showed equally effective growth
inhibition with the highest and lowest activity against the
melanoma and renal cancer subpanels, respectively.
According to the results of statistical processing of the
obtained data, these compounds were taken for a five dose
assay.
Five dose assay
Total panel
The calculated parameters of the antitumor activity of the
tested compounds against the cell lines of the total panel are
presented in the Table S2, and the results of the statistical
analysis in the Table 2.
Table 2. Average values of parameters of anticancer
activity and the number of cell lines sensitive to the action
of the tested compounds against cell lines of the total panel
in a five-dose analysis.
Parameter Compound
5 7 8
GI50 5.37±0.73 (59) 2.85±0.38 (59) 3.26±0.38 (59)
TGI 21.43±2.37
(56)
13.02±1.24
(59)
11.80±0.84 (59)
LC50 51.23±3.55
(37)
40.73±2.50
(45)
40.70±2.32 (52)
Parameter values are expressed in µM (M±m). Numbers
in parentheses indicate the number of sensitive lines, i.e.,
for which the parameter values are less than 100 µM.
Compounds 7 and 8 showed the same average
antiproliferative activity and cytotoxicity against sensitive
cell lines of the total panel. However, compound 8 showed
cytotoxicity to more lines than 7. In all respects, these
compounds were statistically significantly more active than
compounds 5.
A similar pattern was noted for the dose distribution of
the activity of the tested compounds (Figures S1-S3). At
concentrations up to 10 μM, compounds 7 and 8 showed
50% inhibition in 95 and 92% of cell lines, respectively,
versus 83% for compound 5. Cytostatic activity of
compounds 7 and 8 was observed against 92 and 96% of
cells at concentrations up to 30 μM, then as this parameter
for compound 5 was 76%. Compound 8 at concentrations
up to 100 μM was cytotoxic to 86% of cells, while
compounds 7 and 5 were cytotoxic against 72 and 60%,
respectively.
Subpanels
The data of statistical analysis of the antitumor activity
of compounds 5, 7 and 8 by subpanels are presented in
Table 3. Parameter values are expressed in µM (M±m). The
numbers in parentheses indicate the number of sensitive
lines, i.e., for which the parameter values are ≤ 100 μM.
Compounds 7 and 8 demonstrated high and identical
antiproliferative activity against all subpanels in all
calculated parameters characterizing their antitumor activity
for exception of NCI/ADR-RES ovarian cancer cell line
(Table S2). Thus, the value of 50% inhibition of cell growth
for these compounds was in the concentration range of 1-6
μM, and for cytostatic activity was in the range of 6-14 μM.
O.S. Bahrieieva, O.V. Golovchenko et al.
35
Table 3. Average antitumor activity parameters of compounds 5, 7 and 8 against the NCI-60 cancer subpanels in five dose
assay.
Subpanel 5 7 8
GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50
Leukemia 2.9±0.7
(6)
19.3±4.1
(6)
70.1±8.3
(2)
1.5±0.4
(6)
9.5±1.9
(6)
61.3±11.0
(3)
1.9±0.5
(6)
11.1±1.8
(6)
53.7±10.1
(5)
NSLC 7.1±2.0
(9)
30.7±6.5
(8)
44.6±4.1
(3)
2.7±0.4
(9)
13.0±3.0
(9)
41.9±4.0
(7)
3.5±0.8
(9)
13.0±1.8
(9)
42.4±2.4
(9)
Colon cancer 7.3±3.8
(7)
19.8±5.1
(6)
59.1±12.3
(4)
3.9±1.6
(7)
16.2±3.6
(7)
40.3±10.3
(5)
4.0±1.6
(7)
13.8±2.9
(7)
44.8±4.8
(5)
CNS cancer 4.6±1.3
(6)
15.8±2.4
(6)
60.7±7.0
(6)
2.4±0.3
(6)
10.6±1.3
(6)
37.9±3.6
(6)
2.6±0.4
(6)
10.2±1.3
(6)
36.3±4.0
(6)
Melanoma 2.7±0.3
(9)
9.7±1.8
(9)
37.4±7.7
(9)
1.8±0.2
(9)
8.6±1.1
(9)
30.6±4.0
(9)
2.1±0.2
(9)
8.3±1.6
(9)
25.0±4.1
(8)
Ovararian
cancer
3.5±0.4
(6)
24.2±8.0
(6)
60.9±9.9
(4)
2.2±0.4
(6)
13.3±3.1
(6)
48.7±5.8
(4)
2.4±0.3
(6)
10.9±1.6
(6)
36.8±2.3
(5)
Renal cancer 11.3±2.5
(8)
39.8±9.8
(8)
49.8±8.9
(4)
6.4±2.0
(8)
24.5±5.7
(8)
46.2±7.7
(5)
6.7±1.7
(8)
19.0±3.2
(8)
51.9±7.1
(8)
Breast cancer 2.2±0.3
(6)
8.6±1.8
(5)
45.7±15.6
(4)
1.3±0.3
(6)
6.6±1.4
(6)
37.0±12.3
(5)
2.1±0.3
(6)
6.1±1.0
(6)
34.4±11.4
(5)
Their cytotoxicity was in the concentration range of 25-
54 μM. Compound 5 showed the same activity with the
exception of leukemia, NSCL and ovarian cancer, against
which their potency was lower.
When analyzing the structure-activity, it can be seen that
among the phosphorylated oxazole derivatives, only
compounds containing a triphenylphosphonium cation
(TPP+) in the 4th position of the oxazole ring exhibit
antitumor activity. Moreover, the replacement of the phenyl
radical in the 2nd position by the methyl one (compound 6)
led to the elimination of the activity. A significant effect on
the activity of TPP+ oxazole derivatives by the presence of
an aromatic substituent in the 2nd position was also shown
by previously obtained data [5]. In this work, it was shown
that derivatives in which aromatic radicals (phenyl,
aminophenyl, tolyl or methylphenylsulfonyl) in this
position of oxazole were replaced by methyl radical were
either inactive or weakly active against NCI-60 cell lines.
The activity of compound 5 against the total panel was
inferior in all parameters to compounds 7 and 8, the only
difference between which was the substitution in the 5th
position of 2- (hydroxyethyl)(methyl)amine (7) or 3-hydro-
xypropylamine (8) on 2-hydroxyethylamine. Apparently,
this substitution made some contribution to the elimination
of the activity of compound 6. The essential significance of
the functionalization of the 5th position is confirmed by the
data given in the above cited work. Thus, tested analogues
of compounds 7 and 8 containing in this position aromatic
radicals (methylphenylsulfanyl, methylphenylamine or
phenylamine) instead hydroxyalkylamines showed higher
antitumor potency.
Compare correlation
GI50, TGI and LC50 vectors were analyzed by
COMPARE, generating correlation coefficients for the
compounds with known mechanisms of anticancer activity
presented in public databases to suggest their likely
molecular mechanisms or specific targets. The quantitative
evaluation of the obtained results was carried out according
to the Chaddock scale [10].
Reported mechanisms of the standard drugs correlating
with GI50 and TGI vectors of tested compounds
The COMPARE algorithm reveals a high correlation of
antiproliferative potency of tested compounds with
anticancer agents phyllanthoside and chromomycin A3 by
the GI50 vector, and moderate with phyllanthoside by the
TGI vector. Chromomycin A3 is a GC-specific DNA ligand
which inhibits transcription selectively inhibiting the DNA-
directed RNA polymerase reaction. Does not intercalate
[11, 12]. In addition, it is a selective Sp1 inhibitor that
specifically suppresses the expression of the anti-apoptotic
gene associated with the Sp1 protein and induces caspase-
dependent apoptosis [13]. Phyllanthoside suppresses protein
synthesis in cells by inhibiting the elongation mechanism
during translation [14, 15] that correlate with its effects on
cellular protein synthesis [16].
The presence of a strong correlation of the test
compounds with phyllantoside and chromomycin A3
indicates the possible involvement of molecular
mechanisms inherent in these drugs in the antiproliferative
effect of the test compounds.
ISSN 1814-9758. Ukr. Bioorg. Acta, 2023, Vol. 18, N 1
36
Table 4. Standard agent COMPARE correlations for compounds 5, 7 and 8 tested by five dose assay
Compd. GI50 TGI LC50
5
Phyllanthoside (0.75)
Chromomycin A3 (0.72)
Phyllanthoside (0.68) Trimelamol (0.67), Actinomycin D (0.65)
7
Chromomycin A3 (0.81)
Phyllanthoside (0.78)
Phyllanthoside (0.67)
Didemnin B (0.69)
Morpholino doxorubicin (0.67)
Chromomycin A3 (0.65)
8
Chromomycin A3 (0.78)
Phyllanthoside (0.74)
Phyllanthoside (0.67)
MX2 HCl (0.58), daunorubicin,
doxorubicin, cyanomorpholino
doxorubicin, actinomycin D, and
tetraplatin (0.56)
Reported mechanisms of the standard drugs correlating
with LC50 vector of tested compounds
The target of all standard drugs that correlate with the
cytotoxicity of the studied compounds, with the exception
of didemnine is DNA. These drugs act primarily as DNA
intercalating and alkylating agents, interfering with DNA
metabolism and RNA production. These process itself
results in a distortion of the DNA conformation that causes
the inhibition of topoisomerase II. Cytotoxicity is primarily
due to inhibition of topoisomerase II after the enzyme
induces a break in DNA, preventing religation of the break
and leading to cell death [17-22].
Didemnin B like Phyllanthoside inhibits the elongation
of mRNA translation on the ribosome during protein
synthesis, and induces cell apoptosis. Although in some
cancer cell types, apoptosis is only induced by
concentrations at least one order of magnitude higher than
needed to inhibit translation [23, 24]. The moderate
correlation of the cytotoxicity vector of the tested
compounds with that of the above agents, on the one hand,
does not provide sufficient grounds for classifying their
mechanisms as those of the tested compounds, and on the
other hand, the absence of drugs with other mechanisms of
action in this list increases the probability of their inclusion.
It should be noted that these compounds contain bulky
TPP+, which prevents DNA intercalation, which, however,
does not exclude the possibility of their binding to certain
regions of nucleic acids, like didemnin, phyllantoside,
trimelamol, and chromomycin A3, preventing the processes
of transcription and translation in the cell. In addition, a
moderate correlation may be due to the fact that the
subcellular target of TPP+-loaded compounds is
mitochondria, since the mitochondrial membrane potential
in cancer cells (-220 mV, negative inside) is more
hyperpolarized than in normal cells (-140 mV), which
creates a 100-1000-fold concentration gradient of such
compounds in favor of mitochondria [25]. That is, they
predominantly interact with their intrinsic mitochondrial
protein-synthesizing apparatus, and not with the general
cellular one, which cannot but affect the results of a
COMPARE analysis. Therefore, the functionalization of
known antitumor agents with TPP+ is often used for their
selective accumulation in the mitochondria of cancer cells
[26]. It helps to increase selectivity, anticancer activity,
overcome drug resistance and reduce side effects [27, 28].
In addition, TPP+ is not an inert transporter, and can
promote proton leakage and uncoupling of oxidative
phosphorylation, which reduces the efficiency of ATP
generation [29]. Therefore, unlike standard compounds,
oxazole TPP+ derivatives delivered to mitochondria are
likely to selectively disrupt mitochondrial protein synthesis,
which results in excessive production of reactive oxygen
species, inhibition of cell proliferation, release of
cytochrome c, and apoptosis through the mitochondrial
pathway [30, 31].
From this standpoint, the tested 5-(hydroxyalkylamino)-
1,3-oxazoles loaded with TPP+ should be considered as
promising compounds with high potential antitumor activity
and can be taken for further functionalization in order to
increase their antitumor activity and reduce toxicity to
normal cells.
Conclusions
Eight new phosphorylated 5-(hydroxyalkylamino)-1,3-
oxazoles were tested for their ability to inhibit cancer cell
growth. These compounds were evaluated against complete
human tumor cell lines NCI-60. Structura-active analysis
showed that among the phosphorylated oxazole derivatives,
only compounds containing the triphenylphosphonium
cation in the 4th position of the oxazole ring exhibit
antitumor activity against all the studied cancer cell lines.
Moreover, among them, only compounds containing a
phenyl radical in the 2nd position of the oxazole skeleton
turned out to be active. Compounds 7 and 8 showed strong
and similar antiproliferative activity against all subpanels.
Compound 5 showed the same activity, except for the
leukemia, NSCL and ovarian cancer subpanels where their
efficacy was lower. A COMPARATIVE analysis showed
the highest GI50 correlation of compound 5 with
phyllantoside, and compounds 7 and 8 with chromomycin
A3. The cytostatic activity of all active compounds
moderately correlates with drugs whose cytotoxicity is
mediated by interaction with nucleic acids, which leads to
disruption of the protein-synthesizing function of the cell.
Unlike standard compounds, test compounds containing
TPP+ accumulate predominantly in mitochondria.
Therefore, their anticancer activity is most likely due to a
violation of the structural and functional state of the latter
O.S. Bahrieieva, O.V. Golovchenko et al.
37
due to interference with their intrinsic protein-synthesizing
apparatus of mitochondria. The data obtained allow us to
consider the studied compounds as leading ones for further
in-depth study and synthesis of new TPP+-containing
1,3-oxazole derivatives with antitumor activity.
Notes
Acknowledgments. We would like to thank US Public
Health Service and National Cancer Institute, USA for in
vitro evaluation of anticancer activity (providing the NCl-
60 cell testing) within the framework of Developmental
Therapeutic Program (http://dtp.cancer.gov), ENAMINE
Ltd. for the material and technical support for this work.
Disclaimer. This material should not be interpreted as
representing the viewpoint of the viewpoint of the US
National Institutes of Health, or the National Cancer
Institute.
Experimental section
Chemistry
1H, 13C, 31P NMR spectra were obtained with a Bruker
AVANCE DRX-500 spectrometer (TMS as internal
reference or 85% phosphoric acid as external reference) in
DMSO-d6. IR spectra were recorded with a Vertex 70
spectrometer in KBr pellets or film. LC-MS spectra were
recorded on an LC-MS system-HPLC Agilent 1100 Series
equipped with a diode array detector Agilent LC\MSD SL.
Parameters of LC-MS analysis: Zorbax SB-C18 column
(1.8 lm, 4.6-15 mm, PN 821975-932), solvent water-
acetonitrile mixture (95:5), 0.1% of aqueous trifluoroacetic
acid; eluent flow 3 mL min1; injection volume 1 lL; UV
detection at 215, 254, 265 nm; chemical ionization at
atmospheric pressure (APCI), scan range m/z 80-1000.
Elemental analysis was carried out in the Analytical
Laboratory of the Institute of Bioorganic and
Petrochemistry of the National Academy of Sciences of
Ukraine by manual methods. The carbon and hydrogen
contents were determined using the Pregl gravimetric
method, while nitrogen was determined using the Duma’s
gasometrical micromethod. Chlorine content was
determined by the mercurometric method, phosphorus
content was determined by the colorimetric method. M.p.
were determined with a Fisher-Johns apparatus and are
uncorrected. Reactions and purity of the products were
monitored by thin-layer chromatography on DC-
Fertigfolien ALUGRAM Xtra SIL G/UV254 plates using 9:1
(v/v) chloroform–methanol as eluent. All reagents and
solvents were purchased from Aldrich and used as received.
General procedure for preparing diethyl 5-(hydroxy-
alkyl)amino-2-R-1,3-oxazol-4-phosphonates (1-3).
To a solution of 0.001 mol of compounds Ia,b in 50 mL
of methanol was added 0.045 mol of aminoalkanole. The
mixture was stirred for 12-24 h at a temperature of 18-
25 °C. After the solvent removal in vacuo, the residue
treated with 20 ml distilled water and 20 mL of tert-butyl
methyl ether. Organic phase dried over sodium sulfate. The
solvent was removed in a vacuum, and the reaction products
1–3 were purified by column chromatography
(dichloromethane – methanol, 95:5)
Diethyl 5-((hydroxyethyl)(methyl)amino)-2-(4-nitrophe-
nyl)-1,3-oxazol-4-phosphonate (1) [6].
Yeild: 73%; mp 144-147 ᵒC. IR (KBr): ν = 3423 (OH),
1612, 1591 (C=N), 1508, 1343 (N–O), 1245 (Р=О), 1049,
969 (P-O-C). 1H NMR (500 MHz, DMSO-d6): 8.33
(d, 2Н, 4-O2NC6H4, J 8.8 Hz), 8.03 (d, J 8.3 Hz, 2Н,
4-O2NC6H4,), 4.82 (br s, 1Н, ОН), 4.11-4.01 (m, 4Н,
2ОСН2СН3), 3.73-3.62 (m, 4Н, 2СН2), 3.27 (s, 3Н, CH3),
1.28 (t, 3J 7.0 Hz, 6Н, 2ОСН2СН3). 13C NMR (125.69
MHz, DMSO-d6): 161.8 (d, JPC 37.9 Hz, C5 oxazole), 147.8
(d, JPC 21.9 Hz, C2 oxazole), 147.2, 132.0, 125.6, 124.5
(4-O2NС6Н4), 101.5 (d, JPC 254.3 Hz, C4 oxazole), 62.0
(d, 2JPC 5.5 Hz, РОСН2СН3), 58.7, 54.6, 38.4, 16.2
(d, 3JPC 6.5 Hz, РОСН2СН3). 31P NMR (202.38 MHz,
DMSO-d6): 12.7. LCMS: 400[M + 1]. Anal. Calcd. for
C16H22N3O7P (399.34): С, 48.12; H 5.51; N 10.52; P 7.76.
Found: C, 48.01; H, 5.62; N, 10.50; P, 7.73.
Diethyl 5-((hydroxypropyl)amino)-2-(phenyl)-1,3-oxa-
zol-4-phosphonate (2) [7].
Yeild: 72%; mp 61-63 ᵒC. IR (KBr): ν = 3286 (OH, NH),
1628, 1585 (C=N), 1200 (Р=О), 1020, 975 (P-O-C).
1H NMR (500 MHz, DMSO-d6): 7.83 (d, 3J 7.4 Hz, 2Н,
C6H5), 7.53-7.35 (m, 3Н, C6H5), 6.70-6.63 (m, 1Н, NH),
4.63 (br s, 1Н, ОН), 4.09-3.93 (m, 4Н, 2ОСН2СН3), 3.58-
3.50 (m, 2Н, СН2OH), 3.50-3.41 (m, 2Н, NСН2), 1.80-1.70
(m, 2Н, СН2), 1.31-1.17 (m, 6Н, 2ОСН2СН3). 13C NMR
(125.69 MHz, DMSO-d6): 163.1 (d, 2JPC 38.9 Hz,
C5 oxazole), 150.6 (d, 3JPC 21.9 Hz, C2 oxazole), 129.6, 129.1,
127.5, 124.9 (С6Н5), 98.9 (d, 1JPC 254.8 Hz, C4 oxazole), 61.6
(d, 2JPC 5.0 Hz, РОСН2СН3), 58.4, 40.4, 32.7, 16.2
(d, 3JPC 6.0 Hz, РОСН2СН3). 31P NMR (202.38 MHz,
DMSO-d6): 13.5. LCMS: [M + 1]: 355. Anal. Calcd. for
C16H23N2O5P (354.34): C, 54.23; H, 6.54; N, 7.91; P, 8.74.
Found: C, 54.39; H, 6.49; N, 8.27; P, 8.53.
Diethyl 5-((2,3-dihydroxypropyl)amino)-2-(phenyl)-1,3-
oxazol-4-phosphonate (3) [6].
Yeild: 63%; mp 110-112 ᵒC. IR (KBr): ν = 3334 (OH,
NH), 1626 br (C=N), 1215 (Р=О), 1020, 976 (P-O-C).
1H NMR (500 MHz, DMSO-d6): 7.83 (d, 3J 7.1 Hz, 2Н,
C6H5), 7.54-7.41 (m, 3Н, C6H5), 6.49 (br s, 1Н, NH), 5.02
(br s, 1Н, ОН), 4.72 (br s, 1Н, ОН), 4.10-3.92 (m, 4Н,
2ОСН2СН3), 3.70-3.61 (m, 1Н, СН), 3.57-3.49 (m, 1Н,
СН), 3.44-3.37 (m, 1Н, СН), 3.36-3.24 (m, 2Н, СН), 1.26-
1.21 (m, 6Н, 2ОСН2СН3). 13C NMR (125.69 MHz,
DMSO-d6): 163.5 (d, 2JPC 38.9 Hz C5 oxazole), 150.7
(d, 3JPC 22.4 Hz, C2 oxazole), 129.6, 129.1, 126.6, 124.9
(С6Н5), 96.0 (d, 1JPC 254.8 Hz, C4 oxazole), 70.2, 63.5,
61.6 (d, 2JPC 5.0 Hz, РОСН2СН3), 46.1, 16.2 (d, 3JPC 6.5 Hz,
РОСН2СН3). 31P NMR (202.38 MHz, DMSO-d6):
13.6. LCMS: [M + 1]: 371. Anal. Calcd. for C16H23N2O6P
http://dtp.cancer.gov/
ISSN 1814-9758. Ukr. Bioorg. Acta, 2023, Vol. 18, N 1
38
(370.34): C, 51.89; H, 6.26; N, 7.56; P, 8.36. Found: C,
52.00; H, 6.10; N, 7.40; P, 8.68.
Diethyl 5-{methyl-[(2R,3S,4S,5S)-2,3,4,5,6-pentahydro-
xyhexyl]amino}-2-phenyl-1,3-oxazol-4-phosphonate (4) [8].
To a solution of 0.001 mol of compound IIb in 50 mL of
methanol was added 0.0045 mol of N-methyl-D-glucamine.
The mixture was stirred for 6-12 h at a temperature of 18-
25 °C. The precipitated N-methyl-D-glucamine hydro-
chloride was filtered off. After the solvent removal, 20 mL
of tetrahydrofuran was added to oily residue. The
precipitate was filtered off, the solvent was removed in a
vacuum, and the reaction product 4 were analyzed without
further purification. Yeild: 90%; [α]D +23.30ᵒ; Oil.
IR (film): ν = 3346 (O-H), 1605, 1584 (C=N),, 1205 (Р=О),
1016, 961 (P-O-C). 1H NMR (500 MHz, DMSO-d6): 7.83
(d, 3J 7.1 Hz, 2Н, C6H5), 7.49-7.43 (m, 3Н, C6H5), 4.93 (br
s, 1Н, ОН), 4.54 (br s, 1Н, ОН), 4.45 (br s, 2Н, ОН), 4.35
(br s, 1Н, ОН), 4.10-4.00 (m, 4Н, 2ОСН2СН3), 3.98-3.93
(m, 1Н, СН), 3.69-3.47 (m, 7Н, 2СН2, 3СН), 3.24 (s, 3Н,
NCH3), 1.26 (t, 3J 7.1 Hz, 6Н, 2ОСН2СН3,). 13C NMR
(125.69 MHz, DMSO-d6): 161.26 (d, 2JPC 38.4 Hz,
C5 oxazole), 149.54 (d, 3JPC 22.4 Hz, C2 oxazole), 129.51,
129.07, 126.66, 124.91 (С6Н5), 98.67 (d, 1JPC 254.6 Hz,
C4 oxazole), 71.81, 71.56, 70.89, 70.24, 63.52, 61.89 (d, 2JPC
5.0 Hz, РОСН2СН3), 55.31, 39.08, 16.22 (d, 3JPC
6.5 Hz, РОСН2СН3). 31P NMR (202.38 MHz, DMSO-d6):
14.30. LCMS: 475 [M + 1]. Anal. Calcd. for
C20H31N2O9P (474.44): C, 50.63; H, 6.59; N, 5.90; P, 6.59.
Found: C, 50.91; H, 6.48; N, 5.99; P, 6.60.
General procedure for preparing 2-R-5-[(2-
hydroxyalkyl)amino]-1,3-oxazol-4-yltriphenylphosphonium
perchlorates (5, 6, 8).
To a solution of 0.001 mol of compound II a, b in 50 ml
of methanol was added 0.0035 mol of alkanolamine, the
mixture was stirred for 8 h at 18-25 °C. The solvent was
removed in vacuo to 1/3 volume, 30 ml of methyl tert-butyl
ether was added, stirred and left for 0.5 h, the solvents were
decanted, the oily residue was kept in vacuo until the
solvents were completely removed. 40 ml of water was
added, filtered and 5 ml of saturated aqueous NaClO4 was
added to the aqueous solution. The precipitate was filtered
and the compounds 5-8 were purified by recrystallization
from methanol.
[5-(2-Hydroxyethyl)amino]-2-phenyl-4-oxazolyl]triphe-
nylphosphonium perchlorate (5) [9].
Yeild: 81%; colorless solid; mp 180-182 °С. IR (KBr):
ν = 3437, 3274, 1622, 1584, 1437, 1110, 1063, 725, 690,
621, 566, 518 cm-1. 1H NMR (500 MHz, DMSO-d6):
7.97-7.88 (m, 3Н, С6Н5), 7.85-7.72 (m, 14Н, С6Н5), 7.55-
7.46 (m, 3Н, С6Н5), 7.00 (t, 1Н, J 5.3 Hz, NH), 4.85 (t, 1Н,
J 5.3 Hz, ОН), 3.49-3.45 (m, 2Н, СН2), 3.41-3.35 (m, 2Н,
CH2). 13C NMR (100.61 MHz, DMSO-d6): 164.37 (d, JPC
29.3 Hz, C5 oxazol), 153.30 (d, JPC 20.5 Hz, C2 oxazol), 135.42
(d, JPC 2.9 Hz, C4 C6H5), 134.64 (d, JPC 11.0 Hz, C3 C6H5),
130.91, 130.63 (d, JPC 13.2 Hz, C2 C6H5), 129.65, 126.24,
125.74, 119.43 (d, JPC 93.9 Hz, C1 C6H5), 84.15 (d, JPC
153.3 Hz, C4 oxazol), 60.22 (СН2), 46.52 (CH2). 31P NMR
(202.38 MHz, DMSO-d6): 10.70. LCMS: [M-M(ClO4
-)]+ :
465.2. Anal. Calcd. for C29H26ClN2O6P (564.95): C, 61.65;
H, 4.64; Cl, 6.28; N, 4.96; P, 5.48. Found: C, 61.93; H,
4.71; Cl, 6.37; N, 5.29; P, 5.63.
[5-(2-Hydroxyethyl)amino]-2-methyl-4-oxazolyl]triphe-
nylphosphonium perchlorate (6) [9].
Yeild: 67%; colorless solid; mp 163-165 °С. IR (KBr):
ν = 3507, 3352, 1626, 1601, 1438, 1107, 724, 690, 621,
583, 546, 522 cm-1. 1H NMR (500 MHz, DMSO-d6):
7.91-7.84 (m, 3Н, C6H5), 7.78-7.66 (m, 12Н, C6H5), 6.63 (t,
1Н, J 5.6 Hz, NH), 4.74 (t, 1Н, J 5.3 Hz, ОН), 3.38-3.30
(m, 2Н, СН2), 3.20 (m, 2Н, NCH2), 2.33 (s, 3Н, СН3).
13C NMR (100.61 MHz, DMSO-d6): 164.37 (d, JPC 29.3
Hz, C5 oxazol), 153.29 (d, JPC 21.3 Hz, C2 oxazol), 135.31 (d,
JPC 2.9 Hz, C4 C6H5), 134.51 (d, JPC 11.0 Hz, C3 C6H5),
130.58 (d, JPC 12.5 Hz, C2 C6H5), 119.60 (d, JPC 93.9 Hz,
C1 C6H5), 81.62 (d, JPC 154.8 Hz, C4 oxazol), 60.14 (СН2),
46.36 (CH2), 13.85 (СН3). 31P NMR (202.38 MHz, DMSO-
d6): 10.50. LCMS: [M-M(ClO4
-)]+: 404.2. Anal. Calcd. for
C24H24ClN2O6P (502.88): C, 57.32; H, 4.81; Cl, 7.05; N,
5.57; P, 6.16. Found: C, 57.51; H, 5.02; Cl, 7.13; N, 5.89; P,
6.07.
[5-(2-Hydroxypropyl)amino]-2-phenyl-4-oxazolyl]tri-
phenylphosphonium perchlorate (8) [9].
Yeild: 77%; colorless solid; mp 194-196 °С. IR (KBr):
ν = 3509, 3263, 1623, 1584, 1437, 1371, 1163, 1106, 1050,
762, 722, 687, 563, 515 cm-1. 1H NMR (400 MHz, DMSO-
d6): 7.94-7.87 (m, 3H, C6H5), 7.85-7.74 (m, 12H, C6H5),
7.66-7.58 (m, 2H, C6H5), 7.53-7.47 (m, 3H, C6H5), 7.23 (t, J
4.7 Hz, 1H, NH), 4.55 (br s, 1H, OH), 3.43-3.13 (m, 4H,
2CH2), 1.67-1.56 (m, 2H, CH2). 13C NMR (100.61 MHz,
DMSO-d6): 164.00 (d, JPC 29.3 Hz, C5 oxazol), 152.86 (d,
JPC 20.5 Hz, C2 oxazol), 135.33 (d, JPC 2.9 Hz, C4 C6H5),
134.69 (d, JPC 11.0 Hz, C3 C6H5), 130.82, 130.58 (d, JPC
13.2 Hz, C2 C6H5), 129.64, 126.30, 125.67, 119.40 (d, JPC
93.2 Hz, C1 C6H5), 83.79 (d, JPC 154.1 Hz, C4 oxazol),
58.54, 41.46, 32.57. 31P NMR (202.38 MHz, DMSO-d6):
10.62. LCMS: [M-M(ClO4
-)]+: 479.2. Anal. Calcd. for
C30H28ClN2O6P (578.98): C, 62.23; H, 4.87; Cl, 6.12; N,
4.84; P, 5.35. Found: C, 62.49; H, 4.93; Cl, 6.37; N, 5.11; P,
5.21.
5-[(2-hydroxyethyl)(methyl)amino]-2-phenyl-1,3-oxazol-
4-yltriphenylphosphonium perchlorate (7) [9].
To a solution of 0.001 mol of compound IIb in 50 ml of
methanol at -20 ºC was added 0.26 g (0.0035 mol) of N-
methyl monoethanolamine, the mixture was gently heated
to 0-5 ºC and maintained at this temperature for 8 hours.
The solvent was removed in vacuo to 1/3 volume, 30 ml of
methyl tert-butyl ether was added, stirred and left for 0.5 h,
the solvents were decanted, the oily residue was kept in
vacuo until the solvents were completely removed. 40 ml of
water was added, filtered and 5 ml of saturated aqueous
NaClO4 was added to the aqueous solution. The precipitate
O.S. Bahrieieva, O.V. Golovchenko et al.
39
was filtered and the compound 7 were purified by
recrystallization from methanol. Yeild: 61%; colorless
solid; mp 218-220 °С. IR (KBr): ν = 3494, 1624, 1603,
1585, 1567, 1440, 1396, 1108, 1070, 1021, 992, 755, 723,
692, 620, 560, 518 cm-1. 1H NMR (400 MHz, DMSO-d6):
7.96-7.84 (m, 9H, C6H5), 7.83-7.72 (m, 8H, C6H5), 7.54-
7.46 (m, 3H, C6H5), 4.89 (t, J 4.9 Hz, 1H, OH), 3.51-3.45
(m, 2H, CH2), 3.34 (s, 3H, CH3), 3.27 (t, J 5.4 Hz, 2H,
CH2). 13C NMR (125.69 MHz, DMSO-d6): 165.31 (d, JPC
27.6 Hz, C5 oxazol), 152.80 (d, JPC 21.1 Hz, C2 oxazol), 135.45
(d, JPC 3.0 Hz, C4 C6H5), 134.76 (d, JPC 10.5 Hz, C3 C6H5),
131.00, 130.63 (d, JPC 13.0 Hz, C2 C6H5), 129.62, 126.10,
125.69, 120.54 (d, JPC 93.4 Hz, C1 C6H5), 85.84 (d, JPC
153.6 Hz, C4 oxazol), 58.26, 55.71. 31P NMR (202.38 MHz,
DMSO-d6): 13.97. LCMS: [M-M(ClO4
-)]+: 479.2. Anal.
Calcd. for C30H28ClN2O6P (578.98): C, 62.23; H, 4.87; Cl,
6.12; N, 4.84; P, 5.35. Found: C, 62.41; H, 5.06; Cl, 6.09;
N, 4.99; P, 5.18.
Biology
In vitro anticancer screening of the tested compounds
One-dose assay
Synthesized compounds 1-8 were submitted to National
Cancer Institute (NCI), Bethesda, Maryland, US, under the
Developmental Therapeutic Program DTP. The cell line
panel engaged a total of 60 different human tumor cell lines
derived from nine cancer types, including lung, colon,
melanoma, renal, ovarian, brain, leukemia, breast and
prostate.
Primary in vitro one-dose anticancer screening was
initiated by cell inoculating of each 60 panel lines into a
series of standard 96-well microliter plates at 5000-40000
cells/well in RPMI 1640 medium containing 5% fetal
bovine serum and 2 mM L-glutamine (day 0), and then
preincubated in absence of drug at 37 °C and 5% CO2 for
24 h. Test compounds were then added into the plates at one
concentration of 10 -5 M (day 1), followed by incubation for
a further 48 h at the same conditions. Then the media was
removed, the cells were fixed in situ, washed, and dried
(day 3). The sulforhodamine B assay was used for cell
density determination, based on the measurement of cellular
protein content. After an incubation period, cell monolayers
were fixed with 10% (wt/vol) trichloroacetic acid and
stained for 30 min, after which the excess dye was removed
by washing repeatedly with 1% (vol/vol) acetic acid. The
bound stain was resolubilized in 10 mM Tris base solution
and measured spectrophotometrically on automated
microplate readers for OD determination at 510 nm.
Five-dose assay
Compounds which exhibit significant growth inhibition
in the one dose screen were evaluated against the 60 cell
panel at five concentration levels (0.01, 0.1, 1, 10 and
100 µM). The outcomes were used to create three dose-
response parameters (GI50, TGI and LC50) calculated for
each cell line. The GI50 value (50% growth inhibition) is
measure of the sensitivity of a cell to the effect of the drug,
and corresponds to the concentration of the compound
causing 50% decrease in net cell growth. The TGI (total
growth inhibition) refers to the maximum effect of a drug
and is the concentration of the study drug that causes total
inhibition of cell growth. The LC50 value (cytotoxic
activity) is the concentration of the compound causing net
50% loss of initial cells at the end of the incubation period
of 48 h.
The three dose-response parameters GI50, TGI and LC50
were calculated for each experimental compound. Data
calculations were performed according to the method
described by the NCI/NIH Development Therapeutics
Program https://dtp.cancer.gov/discovery_development/nci-
60/methodology.htm.
Growth inhibition of 50% (GI50) is calculated from: [(T-
T0)/(C-T0)]×100 = 50. The TGI is calculated from: 100×(T-
T0)/(C-T0) = 0. Thus, the TGI signifies a cytostatic effect.
The LC50, which means a cytotoxic effect, is calculated as:
[(T-T0)/T0]×100 = -50, where: T0 is the cell count at day 0;
C is the vehicle control (cell count without drug), and T is
the cell count at the test concentration of drug at the end of
the incubation period.
Data analysis
Statistical Data analysis
Statistical analysis of the results and the histograms was
performed by means of the program of Statistica v 6.0 for
Windows. Statistically significant difference between two
groups were evaluated using the unpaired Student t-test (p <
0.05). The data are given as means ± SEM (standard error
of mean).
COMPARE correlations
The graph of mean values for each of test compounds 5,
7 and 8 was subsequently used to run the COMPARE
algorithm from the Developmental Therapeutics Program
(NCI) and calculate the correlation coefficient
(https://dtp.cancer.gov/databases_tools/compare.htm) with
respect to compounds from the standard agent database with
a known mechanism of action. Pairwise correlation
coefficients of greater than 0.5 were used as the cut-off for
assessing whether two agents were likely to share a similar
mechanism of action. Briefly, the GI50, TGI and LC50
concentration vectors for the test compound were compared
to those from the complete public database of standard
agents.
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O.S. Bahrieieva, O.V. Golovchenko et al.
41
Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні
протипухлинні агенти
О.С. Багрєєва1, О.В. Головченко1,2, О.І. Головченко2, В.В. Жирнов1, В.С. Броварець1
1 Інститут біоорганічної хімії та нафтохімії ім. В.П. Кухаря НАН України, Київ, Україна
2 Національний медичний університет імені О.О. Богомольця, Київ, Україна
Резюме: Вісім нових фосфорильованих 5-(гідроксіалкіламіно)-1,3-оксазолів були розроблені та перевірені на їх здатність пригнічувати ріст
ракових клітин. Ці сполуки оцінювали проти повних клітинних ліній пухлини людини NCI-60. Лише 3 сполуки показали протипухлинну
активність в аналізі одноразової дози, які були взяті в аналізі п’яти доз. Сполуки 7 і 8 показали однакову середню антипроліферативну активність
і цитотоксичність проти чутливих клітинних ліній загальної панелі. Однак сполука 8 виявила цитотоксичність для більшої кількості ліній, ніж 7.
За всіма параметрами ці сполуки були більш активними, ніж сполуки 5. Сполуки 7 і 8 також показали високу та подібну антипроліферативну
активність у діапазоні концентрацій GI50: 1-6 і TGI: 6-14 мкМ проти всіх субпанелей. Їх цитотоксичність була в діапазоні концентрацій 25-54
мкМ. Сполука 5 показала таку ж активність, за винятком субпанелі лейкемії, недрібноклітинного раку легенів і раку яєчників, проти яких їх
активність була нижчою. При аналізі структури-активності виявилося, що серед фосфорильованих похідних оксазолу протипухлинну активність
виявляють лише сполуки, які містять катіон трифенілфосфонію (ТФР+) у 4-му положенні оксазольного циклу. Крім того, заміна фенільного
радикала у 2-му положенні оксазольного скелета на метильний радикал призводила до зникнення активності. Алгоритм COMPARE виявляє
високу кореляцію антипроліферативної активності досліджуваних сполук з протипухлинними агентами філантозидом і хром оміцином А3 у
векторі GI50 і помірну з філантозидом у векторі TGI. Мішенню всіх стандартних препаратів, які корелюють з цитотоксичністю досліджуваних
сполук, за винятком дідемніну, є ДНК. На відміну від стандартних сполук, синтезовані активні сполуки несуть делокалізований TPP+, який
доставляє їх переважно до мітохондрій завдяки значно більшому гіперполяризованому потенціалу мітохондріальної мембрани в рако вих
клітинах, ніж у нормальних. Тому їх протипухлинна активність, швидше за все, зумовлена порушенням структурно-функціонального стану
останніх через втручання в їхній внутрішній білоксинтезуючий апарат мітохондрій. Отримані дані дозволяють розглядати 5 -гідроксіалкіл-аміно-
1,3-оксазоли, наповнені TFP+, як провідні сполуки для подальшого поглибленого вивчення та синтезу нових TPP+-вмісних похідних 1,3-оксазолу
з протипухлинною активністю.
Ключові слова: фосфорильовані 5-гідроксіалкіламіно-1,3-оксазоли; синтез; протипухлинна активність; порівняння кореляцій.
In vitro anticancer screening of the tested compounds
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| id | oai:ojs2.bioorganica.com.ua:article-54 |
| institution | Ukrainica Bioorganica Acta |
| keywords_txt_mv | keywords |
| language | English |
| last_indexed | 2026-07-20T01:01:07Z |
| publishDate | 2023 |
| publisher | V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine |
| record_format | ojs |
| resource_txt_mv | bioorganicacomua/ed/17f8f8cacef95d12b40e94e6d0a79bed.pdf |
| spelling | oai:ojs2.bioorganica.com.ua:article-542026-07-19T14:56:54Z New phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles as potential anticancer agents Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти Bahrieieva, Oksana S. Golovchenko, Oleksandr V. Golovchenko, Oksana I. Zhirnov, Victor V. Brovarets, Volodymyr S. Eight new phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles were designed and tested for their ability to inhibit cancer cell growth. These compounds were evaluated against complete human tumor cell lines NCI-60. Only three compounds showed antitumor activity in the single dose assay, which were taken in the five dose assay. Compounds 7 and 8 showed the same average antiproliferative activity and cytotoxicity against sensitive cell lines of the general panel. However, compound 8 showed cytotoxicity to more lines than 7. By all parameters, these compounds were more active than compounds 5. Compounds 7 and 8 also showed high and similar antiproliferative activity in the concentration range GI50: 1-6 and TGI: 6-14 μM against all subpanels. Their cytotoxicity was in the concentration range of 25-54 μM. Compound 5 showed the same activity, with the exception of the leukemia, non-small cell lung cancer and ovarian cancer subpanels against which their activity was lower. When analyzing the structure-activity, it turned out that among the phosphorylated oxazole derivatives, only compounds containing the triphenylphosphonium cation (TPP+) in the 4th position of the oxazole ring exhibit antitumor activity. Moreover, the replacement of the phenyl radical in the 2nd position of the oxazole scaffold with a methyl radical led to the disappearance of the activity. The COMPARE algorithm reveals a high correlation of the antiproliferative activity of the tested compounds with the antitumor agents phyllantoside and chromomycin A3 in the GI50 vector and moderate with phyllantoside in the TGI vector. The target of all standard drugs that correlate with the cytotoxicity of the studied compounds, with the exception of didemnin, is DNA. Unlike standard compounds, synthesized active compounds carry a delocalized TPP+, which delivers them predominantly to mitochondria due to a much more hyperpolarized potential of the mitochondrial membrane in cancer cells than in normal ones. Therefore, their anticancer activity is most likely due to a disturbation of the structural and functional state of the latter due to interference with their intrinsic protein-synthesizing apparatus of mitochondria. The data obtained allow us to consider 5-(hydroxyalkylamino)-1,3-oxazoles loaded with TPP+ as leading compounds for further in-depth study and synthesis of new TPP+-containing 1,3-oxazole derivatives with antitumor activity Вісім нових фосфорильованих 5-(гідроксіалкіламіно)-1,3-оксазолів були розроблені та перевірені на їх здатність пригнічувати ріст ракових клітин. Ці сполуки оцінювали проти повних клітинних ліній пухлини людини NCI-60. Лише 3 сполуки показали протипухлинну активність в аналізі одноразової дози, які були взяті в аналізі п’яти доз. Сполуки 7 і 8 показали однакову середню антипроліферативну активність і цитотоксичність проти чутливих клітинних ліній загальної панелі. Однак сполука 8 виявила цитотоксичність для більшої кількості ліній, ніж 7. За всіма параметрами ці сполуки були більш активними, ніж сполуки 5. Сполуки 7 і 8 також показали високу та подібну антипроліферативну активність у діапазоні концентрацій GI50: 1-6 і TGI: 6-14 мкМ проти всіх субпанелей. Їх цитотоксичність була в діапазоні концентрацій 25-54 мкМ. Сполука 5 показала таку ж активність, за винятком субпанелі лейкемії, недрібноклітинного раку легенів і раку яєчників, проти яких їх активність була нижчою. При аналізі структури-активності виявилося, що серед фосфорильованих похідних оксазолу протипухлинну активність виявляють лише сполуки, які містять катіон трифенілфосфонію (ТФР+) у 4-му положенні оксазольного циклу. Крім того, заміна фенільного радикала у 2-му положенні оксазольного скелета на метильний радикал призводила до зникнення активності. Алгоритм COMPARE виявляє високу кореляцію антипроліферативної активності досліджуваних сполук з протипухлинними агентами філантозидом і хромоміцином А3 у векторі GI50 і помірну з філантозидом у векторі TGI. Мішенню всіх стандартних препаратів, які корелюють з цитотоксичністю досліджуваних сполук, за винятком дідемніну, є ДНК. На відміну від стандартних сполук, синтезовані активні сполуки несуть делокалізований TPP+, який доставляє їх переважно до мітохондрій завдяки значно більшому гіперполяризованому потенціалу мітохондріальної мембрани в ракових клітинах, ніж у нормальних. Тому їх протипухлинна активність, швидше за все, зумовлена порушенням структурно-функціонального стану останніх через втручання в їхній внутрішній білоксинтезуючий апарат мітохондрій. Отримані дані дозволяють розглядати 5-гідроксіалкіл-аміно-1,3-оксазоли, наповнені TFP+, як провідні сполуки для подальшого поглибленого вивчення та синтезу нових TPP+-вмісних похідних 1,3-оксазолу з протипухлинною активністю. V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry of the National Academy of Sciences of Ukraine 2023-06-30 Article Article application/pdf application/pdf https://bioorganica.com.ua/index.php/journal/article/view/54 10.15407/bioorganica2023.01.032 Ukrainica Bioorganica Acta; Vol. 18 No. 1 (2023): Ukrainica Bioorganica Acta; 32-41 Ukrainica Bioorganica Acta; Том 18 № 1 (2023): Ukrainica Bioorganica Acta; 32-41 1814-9766 1814-9758 10.15407/bioorganica2023.01 en https://bioorganica.com.ua/index.php/journal/article/view/54/66 https://bioorganica.com.ua/index.php/journal/article/view/54/67 Copyright (c) 2023 Oksana S. Bahrieieva, Oleksandr V. Golovchenko, Oksana I. Golovchenko, Victor V. Zhirnov, Volodymyr S. Brovarets https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | Bahrieieva, Oksana S. Golovchenko, Oleksandr V. Golovchenko, Oksana I. Zhirnov, Victor V. Brovarets, Volodymyr S. Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| title | Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| title_alt | New phosphorylated 5-(hydroxyalkylamino)-1,3-oxazoles as potential anticancer agents |
| title_full | Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| title_fullStr | Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| title_full_unstemmed | Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| title_short | Нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| title_sort | нові фосфорильовані 5-(гідроксіалкіламіно)-1,3-оксазоли як потенційні протипухлинні агенти |
| url | https://bioorganica.com.ua/index.php/journal/article/view/54 |
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