Synthesis of flutamide-conjugates

Article abstract of DOI:10.1016/j.bmcl.2020.127507

In this paper, we designed and extended modification basing on the flutamide structure. A series of flutamide-conjugates were obtained with methyl bromoacetate and ethylenediamine. Through the synthesis of two conjugates with 3,5-bis(dodecyloxy)benzoate derivatives, these flutamide conjugates were tested for anticancer activity. Among the compounds tested, the flutamide-conjugates showed good inhibition activity against cancer cell lines U-251, PC-3 and K-562. The conjugates showed a better inhibitory effect than free flutamide and did not show activity against normal COS-7 monkey kidney fibroblast cells. It was also observed that the flutamide conjugates had an inhibitory effect against human colorectal adenocarcinoma HCT-15.

Full text of DOI:10.1016/j.bmcl.2020.127507

Bioorganic & Medicinal Chemistry Letters 30 (2020) 127507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of flutamide-conjugates
a
a
b
Julio César Medina-Rojas , Irving Osiel Castillo-Rodríguez , Elena Martínez-Klimova ,
a
a
a,⁎
Teresa Ramírez-Ápan , Simón Hernández-Ortega , Marcos Martínez-García
a
b
Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Exterior, Coyoacán, C.P. 04510, México, D.F., México
Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Interior, Coyoacán, C.P. 04510, México, D.F., México
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this paper, we designed and extended modification basing on the flutamide structure. A series of flutamide-
conjugates were obtained with methyl bromoacetate and ethylenediamine. Through the synthesis of two con-
jugates with 3,5-bis(dodecyloxy)benzoate derivatives, these flutamide conjugates were tested for anticancer
activity. Among the compounds tested, the flutamide-conjugates showed good inhibition activity against cancer
cell lines U-251, PC-3 and K-562. The conjugates showed a better inhibitory effect than free flutamide and did
not show activity against normal COS-7 monkey kidney fibroblast cells. It was also observed that the flutamide
conjugates had an inhibitory effect against human colorectal adenocarcinoma HCT-15.
Flutamide conjugates
Antiproliferative
Cancer
Therapy
PC-3 inhibitors
Flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]) propa-
nanoscaffolding, to which one can covalently connect many different
1
,2
20
namide is used as an antineoplastic and antiandrogen drug. It is a
powerful nonsteroidal androgen antagonist which has treated prostate
cancer and is believed to block androgen receptor sites, namely for
testosterone and dihydrotestosterone. Hindering the binding of andro-
functional groups, showing specific biochemical functions. In the
present work, we designed and synthesized a new type of dendrimers
with flutamide-derivatives and evaluated its potential for use as a ve-
hicle for drug delivery.
2
gens to the androgen receptors in the prostate gland impedes tumor
The N-alkylation of the amide group by methyl bromoacetate was
3
4,5
growth. In the organism, flutamide is metabolized in plasma,
or
used for the synthesis of the flutamide-derivative methyl N-isobutyryl-
6
21
excreted in the urine. Flutamide shows low solubility in water, poor
membrane permeability and does not participate in acid-base equili-
N-(4-nitro-3-(trifluoromethyl)phenyl)glycinate 2 (Scheme 1).
The
compound 2 was reacted with ethylenediamine at room temperature in
methanol to obtain the flutamide-derivative 3.
7
8
bria. These properties do not permit an optimal drug-receptor binding
1
as solubility and permeability are considered prerequisites for optimal
bioactivity of the drugs. To increase the solubility and membrane per-
In the H NMR spectra of the compound 3, the following signals
were observed: at δ
H
0.94 and δ
H
H
0.97 two singlets due to the CH
3
9
,10
meability of the drugs and decrease the side effects,
flutamide has
groups of the isopropyl group, at δ
group of the isopropyl proton, at δ
CH -CH -N group, at δ 3.86 one doublet due to the CH
with coupling constant J = 5.7 Hz, at δ 6.76–7.12 two singlets as-
signed to the aromatic proton with a coupling constant J = 8.7 Hz, at
7.72 one multiplet due to the aromatic proton, at δ 8.05 one
2.27 one multiplet due to the CH
3.11 a broad signal assigned to the
-C]O group
1
1
been modified in the aromatic ring , in the branched alkyl chain α to
H
1
2
13
the amide carbonyl, and in the amide carbonyl to improve the an-
ticancer activity. Structure-activity relationships showed that the best
activity was observed with the compounds containing electron-with-
2
2
H
2
H
1
4
drawing substituents. Also, several formulation strategies have been
obtained, including the use of macromolecules. In the last few years,
macromolecules as dendrimers have shown good abilities in drug de-
δ
H
H
doublet due to the other proton of the aromatic ring with a coupling
constants J = 9.0 Hz. Finally, one broad signal at δ 8.20 due to the NH
H
1
5,16
17,18
livery,
and in gene delivery.
The dendrimers' amphiphillic
group.
nature i.e., hydrophilic exterior and hydrophobic interior, makes them
analogous to unimolecular micelles. Dendrimers are considered static
unimolecular micelles and their micellar structure remains stable even
On the other hand, the compounds 4–7 were obtained (Scheme 2)
starting from the 3,5-dihidroxibenzoic acid followed by the esterifica-
tion reaction with methanol in presence of sulfuric acid to obtain the
compound 4. After that, a reaction with dodecyl bromide under the
Williamson reaction, yielded the compound 5. The ester 5 was reacted
with ethylenediamine in a mixture of methanol toluene to obtain the
1
9
at higher concentrations of solvents. Micelle-like behavior of den-
drimers resulted in their application to solubilize hydrophobic drugs. A
developed and well-defined dendrimer structure constitutes excellent
⁎
Corresponding author.
E-mail address: margar@unam.mx (M. Martínez-García).
https://doi.org/10.1016/j.bmcl.2020.127507
Received 1 July 2020; Received in revised form 13 August 2020; Accepted 17 August 2020
Available online 28 August 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.

J.C. Medina-Rojas, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127507
Scheme 1. Synthesis of the flutamide-derivative 3.
Fig 2. 1H NMR spectra of the flutamide-derivative conjugates 8 and 9.
Scheme 2. Synthesis of the compounds 4–7.
reaction was carried out in methanol at reflux.
1
The compounds 8 and 9 were characterized by H NMR spectro-
scopy and in the NMR spectra (Fig. 2) the following signals were ob-
served: at δ
H
0.89, 1.27 and at δ 1.43 one triplet and two broad signals
H
H
due to the aliphatic chain, at δ
groups at the isopropyl group. At δ
group at the isopropyl group. At δ
the NH-CH -CH -NH groups. At 3.77 and 3.97 two triplets assigned to
the N-CH -C]O and Ar-CH -O groups with a coupling constant
J = 6.6 Hz and J = 7.2 Hz respectively. At δ 6.67 one triplet signal
due to the NH groups with a coupling constant J = 2.4 Hz. Finally, from
1.13 one signal assigned to the CH
2.37 one multiplet due to the CH
3.44 one broad signal assigned to
3
H
H
2
2
2
2
H
δ 6.91 to 7.76 the signals for the aromatic protons were observed (see
H
Supplementary Material).
Scheme 3. Synthesis of the flutamide-derivative conjugates compounds 8 and
The synthesized compounds 2, 3, 5–9 and the starting drug fluta-
mide were screened for their cytotoxic activity against: U-251 (human
glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human
chronic myelogenous leukemia cells), HCT-15 (human colorectal ade-
nocarcinoma) cancer cell lines and monkey kidney fibroblast cells
9
.
compound 6. Finally, the compound 7 was treated with methyl acrylate
to obtain the diester terminal groups compound 7 (See Scheme 3).
In the case of the compound 5, crystals were grown with the quality
to perform X-ray studies. Fig. 1 shows the structure of compound 5.
2
2
(COS-7) according to the MTT method.
The data is shown in Table 1, revealing that the free flutamide and
the flutamide-derivatives 2 and 3 have very similar antiproliferative
activity against the cancer cell lines tested. Cytotoxic activity tests re-
vealed that free flutamide showed strong activity against the U-251, PC-
3 and K-562 cell lines, as well the flutamide-derivatives 2 and 3. The
flutamide-derivatives 2 and 3 showed higher activity against normal
COS-7 cells in comparison with free flutamide. The compounds 5–7
were not toxic to the listed above cancer cell lines. The flutamide
conjugates 8 and 9 showed better anticancer activity against all cancer
cell lines than the free flutamide and the flutamide-derivatives 2 and 3.
The flutamide conjugate 8 showed high anticancer activity IC50 against
U-251 (human glioblastoma) 14.29 µM and PC-3 (human prostatic
adenocarcinoma) 13.3 µM. The anticancer activity was better when the
poly(amidoamine) chain was extended as observed for the flutamide
conjugate 9 with IC50 value of 8.59 µM for U-251. In the case of the line
PC-3, the IC50 was 7.16 µM. The concentration of the conjugate 9 was
diluted 2 times to have the anticancer activity for one molecule of the
drug to compare to the free drug and to the conjugate 8. The flutamide
conjugates 8 and 9 showed increased anticancer activity compared to
the free flutamide and the flutamide conjugates 2 and 3. It was also
observed that the conjugates 8 and 9 showed good anticancer activity
against HCT-15 (human colorectal adenocarcinoma). Finally, the most
important is that the conjugates 5–9 did not show any activity against
the normal monkey kidney fibroblast cells (COS-7).
1
The compound 7 was characterized by H NMR spectroscopy and in
the spectrum the following signals were observed; one triplet at δ
H
0.85
assigned to the –CH
3
groups with coupling constant J = 6.7, at δ
H
1.24,
1
.41 and 1.74 three signals due to the –CH – groups from the aliphatic
2
chain, at δ
H
2.42 one triplet assigned to the CH
2.59, 2.72 and at δ
-N with coupling constant J = 6.3 and
3.56 one singlet assigned to the O-CH
3.95 one triplet due to the CH -O groups at the aliphatic
chain with a coupling constant J = 6.4 Hz, at 6.51 and at δ 6.97 one
triplet and one doublet due to the aromatic protons with coupling
2
-C]O group with
coupling constant J = 6.3 Hz, at δ
H
H
3.52 three
doublets due to the CH
J = 5.7 Hz respectively, at δ
group, at δ
2
-NH, CH
2
H
3
H
2
H
constant J = 2.1 Hz. Finally, one triplet was observed at δ 7.07 as-
H
signed to the NH groups with a coupling constant J = 4.8 Hz (see
Supplementary Material).
Finally, the flutamide-derivative 3 was coupled to the compounds 5
and 7 to obtain the conjugates of first 8 and second generation 9, the
In this paper, we processed structural design and extended mod-
ification based on the flutamide structure. A series of flutamide-deri-
vatives were obtained with methyl bromoacetate and ethylenediamine
and through the synthesis of two conjugates with 3,5-bis(dodecyloxy)
Fig. 1. X-ray structure of compound 5.
2

J.C. Medina-Rojas, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127507
Table 1
In vitro growth inhibitory effect of compounds 2, 3, 5–9 in human cell lines U-251, PC-3, K-562, HCT-15 and monkey kidney fibroblast COS-7.
Compound
IC50(μM)
Cell line
U-251
PC-3
K-562
HCT-15
COS-7
Flutamide
22.11 ± 5.14
20.11 ± 3.14
24.10 ± 1.34
> 1000
20.3 ± 2.14
19.12 ± 1.04
20.10 ± 1.54
> 1000
29.2 ± 1.16
14.10 ± 5.14
18 ± 1.16
> 1000
> 100
51.51 ± 1.19
12.10 ± 2.25
15.17 ± 3.14
> 1000
2
3
5
6
7
8
9
> 100
> 100
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
14.29 ± 4.01
8.59 ± 3.14
13.3 ± 2.10
7.16 ± 2.11
15.39 ± 1.52
6.79 ± 8.23
19.15 ± 4.09
22.35 ± 2.29
> 1000
> 1000
a
IC50 (μM) values obtained from MTT assays. Cells were incubated with compounds for 48 h. The results represent the mean ± SD of two independent trials,
performed in triplicate.
benzoate derivatives. These flutamide conjugates were tested for an-
ticancer activity. Among the compounds tested, flutamide-derivatives
showed good inhibition activity against U-251, PC-3 and K-562. The
conjugates did not show activity against normal monkey kidney fibro-
blast COS-7 cells. It was also observed that the flutamide conjugates 8
and 9 had an inhibitory effect against human colorectal adenocarci-
noma HCT-15. The presence of a longer poly(amidoamine) chain in the
conjugates increases the anticancer activity in vitro against human
glioblastoma, human prostatic adenocarcinoma and human chronic
myelogenous leukemia cell lines. In future works, we will extend the
effect of the poly(amidoamide) chain to increase the inhibitory prop-
erties.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127507.
References
1. Baker JW, Bachman GL, Schumacher I, Roman GP, Tharp A. J Med Chem.
1
967;10:93–95.
2
3
.
.
Verma A, Singh MK, Kumar B. Int J Pharm Pharm Sci. 2011;3:60–65.
Elkhodairy KA, Hassan MA, Afifi SA. Saudi Pharm J. 2014;22:53–61.
4. Aizawa Y, Ikemoto I, Kishimoto K, et al. Mol Cell Biochem. 2003;252:149–156.
5
6
7
8
9
.
.
.
.
.
Schulz M, Schmoldt A, Donn F, Becker H. Eur J Clin Pharmacol. 1988;34:633–636.
Sarrabay A, Hilmi C, Tinwell H, et al. Appl Pharmacol. 2015;289:515–524.
Brittain HG, Sternal R, Nugara N. San Diego: Academic Press, 2001.
Jeevana JB, Sreelakshmi K. J Young Pharm. 2011;3:4–8.
Declaration of Competing Interest
Gomez JL, Dupont A, Cusan L, et al. Am J Med. 1992;92:465–470.
1
0. Cetin M, Demirci D, Unal A, Altinbas M, Guven M, Uluhizarci K. Hum Exp Toxicol.
1
991;18:137–140.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
1
1
1. Morri JJ, Hughes LR, Glen AT, Taylor PJ. J Med Chem. 1991;34:447–455.
2. Tucker H, Crook JW, Chesterson GJ. J Med Chem. 1988;31:954–959.
13. Battmann T, Bonfils A, Branche C, et al. Steroid Biochem Mol Biol. 1994;48:55–60.
1
1
4. Singh SM, Gauthier S, Labrie F. Curr Med Chem. 2000;7:211–247.
5. Kurtoglu YE, Mishra MK, Kannan S, Kannan RM. Int J Pharm. 2010;384:189–194.
Acknowledgments
16. Khandare J, Kohle P, Pillai O, Kannan S, Lieh-Lai M, Kannan RM. Bioconj Chem.
2
005;16:330–337.
1
1
7. Grillaud M, Russier J, Bianco A. J Am Chem Soc. 2014;136:810–819.
8. Lee S, Son JS, Song JS, Ha HT, Choi SJ. Polymers. 2017;9:224–237.
This research was funded by DGAPA, grant number IN232220 and
EMK was supported by a postdoctoral fellowship from CONACyT. We
would like to thank Huerta S. E., Patiño M. M. R., Peña Gonzalez M. A.,
Rios Ruiz L. F. Orta-Pérez Ma., and Gaviño Ramírez R.L., for technical
assistance.
19. Gupta U, Agashe HB, Asthana A, Jain NK. Biomacromolecules. 2006;7:649–658.
2
2
0. Kojima C, Regino C, Umeda Y, Kobayashi H, Kono K. Int J Pharm. 2010;383:293–296.
1. Pedro-Hernández LD, Martínez-Klimova E, Martínez-Klimov ME, et al. Anti-Cancer
Agents Med Chem. 2018;18:993–1000.
22. Monks A, Scudiero D, Skehan P, et al. J Natl Cancer Inst. 1991;83:757–766.
3