Cytotoxic activity of synthetic chiral amino acid derivatives

Article abstract of DOI:10.21577/0103-5053.20190157

Cancer is a chronic degenerative disease considered one of the most important causes of death worldwide. In this context, a series of dual-protected amino acid derivatives was synthesized and evaluated as potential novel anticancer agents. The 40 derivatives were prepared in up to three reaction steps. The cytotoxic activities were screened in vitro against a panel of tumor and non-tumor cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among the synthesized derivatives, three of them showed promising activity against cancer cells with half-maximal inhibitory concentration (IC₅₀) ranging between 1.7-6.1 μM. The most promising derivative, bearing both a lipophilic N-alkyl diamine moiety and a protected amino acid scaffold showed a selectivity index of 3.4 towards tumor cells. The N-alkyl diamine moiety seems to play a crucial role in the enhancement of the anticancer activity. On the other hand, the incorporation of an amino acid scaffold resulted in increase in the selectivity towards cancer cell lines.

Full text of DOI:10.21577/0103-5053.20190157

http://dx.doi.org/10.21577/0103-5053.20190157
J. Braz. Chem. Soc., Vol. 00, No. 00, 1-8, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
Cytotoxic Activity of Synthetic Chiral Amino Acid Derivatives
a
Pedro P. de Castro,#,a Raoni P. Siqueira,#,b Luiza Conforte,a Chris H. J. Franco,
Gustavo C. Bressan*,b and Giovanni W. Amarante *,a
aDepartamento de Química, Universidade Federal de Juiz de Fora, Campus Martelos,
36036-900 Juiz de Fora-MG, Brazil
bDepartamento de Bioquímica e Biologia Molecular, Universidade Federal de Vicosa,
36570-900 Vicosa-MG, Brazil
Cancer is a chronic degenerative disease considered one of the most important causes of death
worldwide. In this context, a series of dual-protected amino acid derivatives was synthesized and
evaluated as potential novel anticancer agents. The 40 derivatives were prepared in up to three
reaction steps. The cytotoxic activities were screened in vitro against a panel of tumor and non-
tumor cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Among the synthesized derivatives, three of them showed promising activity against cancer cells
with half-maximal inhibitory concentration (IC50) ranging between 1.7-6.1 µM. The most promising
derivative, bearing both a lipophilic N-alkyl diamine moiety and a protected amino acid scaffold
showed a selectivity index of 3.4 towards tumor cells. The N-alkyl diamine moiety seems to play
a crucial role in the enhancement of the anticancer activity. On the other hand, the incorporation
of an amino acid scaffold resulted in increase in the selectivity towards cancer cell lines.
Keywords: amino acid, carbamate, diamine, anticancer activity
Introduction
complex chemical structures, such as paclitaxel and
vinblastine, which demands several synthetic steps for
their preparation, with a direct impact in cancer treatment
cost.6 Thus, the discovery of new candidates based on small
molecules, bearing simple and accessible precursors is
essential for increasing the accessibility of cancer treatment.
Due to the great potential of carbamate in medicinal
chemistry, we envisioned that it is a promising building-
block for the design of new anticancer candidates.7-10 For
example, many important clinically employed anticancer
drugs, with different mechanisms of action, bear this
scaffold, such as the nucleoside analogue capecitabine, the
topoisomerase I inhibitor irinotecan and the taxanes, such
as paclitaxel, docetaxel and carbazitaxel.
Moreover, amino acids also appears as interesting
moieties in organic synthesis and it use has several
advantages, such as the low cost of natural-occurring amino
acids, the easy access, the presence of one or more defined
stereocenters and the possibility of functionalization in
both the amine or carboxyl moiety, greatly enhancing the
molecule complexity in only one or a few reaction steps.11-15
Furthermore, the increased amino acid uptake has been
described for some types of cancer, making it a promising
building block for enhancing the selectivity towards cancer
Nowadays cancer clinically appears as one of the most
important diseases worldwide, with growing incidence and
mortality rates. In 2018, only in the United States, more
than 1.7 million new cancer cases and more than 0.6 million
cancer deaths are expected to occur.1 Clinically, lung, breast,
colorectal and prostate appears among the most diagnosed
cancer types, being responsible for 42.7% of new cases.2
Besides, since chemotherapy drugs presents only moderate
selectivity towards growing abnormal cells, the death of
healthy cells frequently occurs, resulting in toxicity and
several side-effects for the patient during cancer treatment.3,4
In this context, the development of new anticancer
agents, especially those with new mechanisms of action,
fewer side effects and lower toxicity in the clinic appears
as a great challenge in medicinal chemistry.5 In addition,
another great clinical challenge is the advent of new
anticancer drugs capable of selectively acting in different
types of cancer cells, making the treatment more general
and simpler. Finally, most cancer drugs consists of very
*e-mail: gustavo.bressan@ufv.br; giovanni.amarante@ufjf.edu.br
#These authors contributed equally to this work.

2
Cytotoxic Activity of Synthetic Chiral Amino Acid Derivatives
J. Braz. Chem. Soc.
cells.16 Finally, some anticancer drugs, such as Melphalan
and Eflornithine, presents a substituted amino acid scaffold.
Over the last years our research group has prepared
several complex amino acid derivatives, some of them
with promising biological activity.17,18 Thus, we envisioned
that the preparation of new derivatives by the coupling
of a carbamate and an amino acid moiety would result
in new derivatives with potential applications in cancer
treatment. Besides, the modification of the amino acids
employed, associated with carboxyl group functionalization
by different amines, would allow the access to several
biologically promising stable amide derivatives. In
this framework, we describe herein the preparation of
thirty-eight dual-protected amino acid derivatives and
two analogues, as well as their cytotoxic profile against
cancerous and non-cancerous cells.
Then, the solvent was rotary evaporated and a liquid-liquid
extraction employing water/dichloromethane performed.
The organic layer was dried and the solid recrystallized in
hot hexane.After filtration, the desired mono-alkyl diamine
derivatives were isolated as white solids. In an attempt to
compare the amino acid moiety influence in the anticancer
activity, the half-maximal inhibitory concentration (IC50)
of these intermediates were also assayed.
General procedure for the preparation of amino acid
derivatives
The amino acid derivatives were prepared employing
a methodology developed by our research group.19
Initially, the coupling between the tert-butyloxycarbonyl
(Boc) protecting group and amino acids (L-alanine,
D,L-alanine, D-alanine, L-valine, L-leucine, L-isoleucine
or L-phenylalanine, 1.0 mmol) was done by adding to
a solution of 1,4-dioxane/water (40:60 v/v) the amino
acid and 50 mg of Na2CO3. The mixture was stirred until
complete dissolution and then Boc2O (1.5 mmol) was
added. The reaction was kept at magnetic stirring for 18 h
and then purified employing literature protocols.20
For the amide bond formation, the previously prepared
Boc amino acids (1.0 mmol) were transferred to a flame-
dried glassware vial with dichloromethane (5.0 mL) and
then EDC (3-dimethylaminopropyl)-N-ethylcarbodiimide)
hydrochloride (1.1 mmol) was added. The reaction stirred
at 0 °C for 30 min, and then washed twice with distilled
water. The organic layer was immediately transferred to
a vial with the amine nucleophile (2.0 mmol) and the
( )-camphorsulfonic acid organocatalyst (0.1 mmol,
10 mol%). After 24 h the dichloromethane was removed
and the desired compounds purified by recrystallization,
liquid-liquid extraction or column chromatography.
Boc deprotection of two of the previously prepared
products was carried out following literature protocols,21
in which the derivatives (1 mmol) and trifluoroacetic acid
(22 mmol) were added in dichloromethane (5 mL) and kept
under magnetic stirring for 3 h, affording the salts 33 and
34 after solvent removal.
Experimental
General remarks
All chemicals were used as purchased, without
further purification. Solvents were dried following
standard procedures and all reaction mixtures were
carried out in flame-dried glassware. Analytical thin layer
chromatography (TLC) was performed on TLC plates and
visualized employing a ninhydrin (2,2-dihydroxyindane-
1,3-dione) indicator. Yields refers to chromatographically
purified and spectroscopically pure derivatives. The
chemical shifts are reported in ppm relative to the solvent
1
residual peak (chloroform 7.28 ppm). The H nuclear
magnetic resonance (NMR) spectra were recorded at
500 MHz and 13C NMR at 125 MHz. Chemical shifts
are reported in ppm using the following peak pattern
abbreviations: br, broad; s, singlet; d, doublet; t, triplet;
q, quartet; pent, pentet; m, multiplet. Infrared (IR) spectra
were recorded on a PerkinElmer 1720 FTIR spectrometer
in the region of 4000-600 cm-1 as an average of 120 scans.
High resolution mass spectra were recorded in the positive
ion mode using a time-of-flight (TOF) mass spectrometer
equipped with an electron spray ionization (ESI) source.
Melting points and optical rotations were recorded,
respectively, on a melting point apparatus and a polarimeter.
Characterization data
General procedure for the synthesis of N-alkyl diamines
tert-Butyl (S)-(1-(butylamino)-1-oxopropan-2-yl)carbamate
(1)
The two N-alkyl diamine derivatives were synthesized
employing literature protocols.5 Initially, 40 mmol of the
diamine was added in methanol (50 mL) and then alkyl
bromide or chloride (8 mmol) was slowly added to the
reaction mixture. The reaction was kept at reflux for 48 h.
The product was prepared according to the general
procedure and obtained after recrystallization in ethanol/
water as a yellow solid (302 mg, 69%); mp 72.1-72.0 °C;
[α]D20 –10 (c 1.0, CH2Cl2); IR (KBr) ν / cm-1 3316, 3262,
2964, 2925, 2865, 1686, 1653, 1520, 1447, 1242, 1163,

Vol. 00, No. 00, 2019
de Castro et al.
3
1
+
1023; H NMR (500 MHz, CDCl3) d 6.30 (br, 1H), 5.09
(br, 1H), 4.11 (br, 1H), 3.23 (q, 2H, J 6.1 Hz), 1.51-1.41 (m,
11H), 1.37-1.28 (m, 5H), 0.90 (t, 3H, J 7.3 Hz); 13C NMR
(125 MHz, CDCl3) d 172.7, 155.7, 80.2, 50.2, 39.3, 31.7,
28.4, 20.1, 18.5, 13.8; HRMS (ESI-TOF) m/z, calcd. for
C12H24N2NaO3+ [M + Na]+: 267.1685, found: 267.1702.
(ESI-TOF) m/z, calcd. for C15H22N2NaO4 [M + Na]+:
317.1477, found: 317.1502.
tert-Butyl (R)-(1-((4-bromophenyl)amino)-1-oxopropan-2-yl)
carbamate (38)
The product was prepared according to the general
procedure and obtained after recrystallization with ethanol/
water as a white solid (265 mg, 78%); mp 156.6-157.2 °C;
[α]D20 –10° (c 1.0, CH2Cl2); IR (KBr) ν / cm-1 3394, 3261,
tert-Butyl (R)-(1-(diethylamino)-1-oxopropan-2-yl)
carbamate (35)
1
2977, 1678, 1506, 1367, 1248, 1162, 817; H NMR
The product was prepared according to the general
procedure and obtained after liquid-liquid extraction in
dichloromethane/water as a yellow oil (358 mg, 73%);
(500 MHz, CDCl3) d 8.73 (br, 1H), 7.37-7.35 (m, 4H), 5.11
(d, 1H, J 6.4 Hz), 4.33 (br, 1H), 1.45 (s, 9H), 1.42 (d, 3H,
J 7.1 Hz); C NMR (125 MHz, CDCl3) d 171.2, 156.4,
137.1, 132.0, 121.4, 116.9, 81.0, 50.9, 28.4, 17.5; HRMS
(ESI-TOF) m/z, calcd. for C14H19BrN2NaO3+ [M + Na]+:
365.0477, found: 365.0474.
13
20
[α]D –10° (c 1.0, CH2Cl2); IR (KBr) ν / cm-1 3430,
3295, 2973, 2928, 2876, 1704, 1634, 1453, 1362, 1247,
1169, 1054; 1H NMR (500 MHz, CDCl3) d 5.44 (d, 1H,
J 7.5 Hz), 4.48 (pent, 1H, J 7.0 Hz), 3.42-3.38 (m, 1H),
3.28-3.26 (m, 2H), 3.23-3.16 (m, 1H), 1.35 (s, 9H), 1.21
(d, 3H, J 6.8 Hz), 1.14 (t, 3H, J 7.1 Hz), 1.03 (t, 3H,
Cell culture
13
J 7.1 Hz); C NMR (125 MHz, CDCl3) d 172.1, 155.1,
Murine metastatic melanoma (B16F10), murine
fibroblast (NIH3T3), and human hepatocellular carcinoma
(HepG2) cells were kindly provided by DrAnésiaAparecida
dos Santos (Departamento de Biologia Geral, Universidade
Federal de Viçosa, Minas Gerais, Brazil). Human breast
adenocarcinoma (MDA-MB-231) cell was kindly provided
by Dr Sandra Martha Gomes Dias (Centro Nacional de
Pesquisa em Energia e Materiais, Laboratório Nacional
de Biociências, Campinas, São Paulo, Brazil). Human
cervix adenocarcinoma (HeLa) and human embryonic
kidney (HEK293) cells were kindly provided by Dr Jörg
Kobarg (Instituto de Biologia, Universidade Estadual de
Campinas, Campinas, São Paulo, Brazil). Cell lines were
grown in RPMI-1640 (Roswell Park Memorial Institute,
Sigma, Darmstadt, Germany) or DMEM (Dulbecco’s
Modified Eagle Medium, Gibco, Dublin, Ireland) medium
supplemented with 10% (v/v) fetal bovine serum (FBS)
(LGC Biotecnologia, São Paulo, Brazil), 100 g mL-1
streptomycin and 100 units per mL penicillin (Sigma,
Darmstadt, Germany) at pH 7.2 and 37 °C under 5% CO2
atmosphere.
79.3, 46.1, 41.7, 40.3, 28.3, 19.6, 14.5, 12.8; HRMS (ESI-
TOF) m/z, calcd. for C12H24N2NaO3+ [M + Na]+: 267.1668,
found: 267.1685.
tert-Butyl (R)-(1-morpholino-1-oxopropan-2-yl)carbamate
(36)
The product was prepared according to the general
procedure and obtained after liquid-liquid extraction in
dichloromethane/water as a yellow oil (421 mg, 73%);
[α]D20 –14° (c 1.0, CH2Cl2); IR (KBr) ν / cm-1 3427, 3308,
2977, 2924, 2850, 1698, 1639, 1440, 1234, 1162, 1109,
1016; 1H NMR (500 MHz, CDCl3) d 5.50 (d, 1H, J 6.9 Hz),
4.57 (pent, 1H, J 7.2 Hz), 3.69-3.65 (m, 6H), 3.59-3.49
13
(m, 2H), 1.42 (s, 9H), 1.28 (d, 3H, J 6.9 Hz); C NMR
(125 MHz, CDCl3) d 171.5, 155.2, 79.8, 67.1, 66.9, 52.1,
46.0, 42.5, 28.5, 19.4; HRMS (ESI-TOF) m/z, calcd. for
C12H22N2NaO4+ [M + Na]+: 281.1477, found: 281.1490.
tert-Butyl (R)-(1-((2-methoxyphenyl)amino)-1-oxopropan-
2-yl)carbamate (37)
The product was prepared according to the general
procedure and obtained after recrystallization with ethanol/
water as a white solid (236 mg, 74%); mp 129.1-130.0 °C;
[α]D20 –12° (c 1.0, CH2Cl2); IR (KBr) ν / cm-1 3328, 3268,
2977, 2924, 1673, 1658, 1540, 1519, 1460, 1254, 1155,
Cell viability assay
Each cell line were seeded in 96-well plates at the density
of 104 cell per well containing 100 µL of complete medium
and 100 µL of each compound solution at 10 µM or at
different concentrations (0-200 µM) for IC50 determination.
The compounds were diluted in RPMI medium with
10% FBS and 0.4% dimethylsulfoxide (DMSO) (v/v,
Sigma, Darmstadt, Germany). After 48 h of culture,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
1
1022, 744; H NMR (500 MHz, CDCl3) d 8.43 (br, 1H),
8.35 (dd, 1H, J 8.0 Hz, J 1.1 Hz), 7.05 (t, 1H, J 7.3 Hz),
6.95 (t, 1H, J 7.7 Hz), 6.87 (d, 1H, J 7.8 Hz), 5.06 (br, 1H),
4.34 (br, 1H), 3.87 (s, 3H), 1.46-1.44 (m, 12H); 13C NMR
(125 MHz, CDCl3) d 170.8, 155.6, 148.2, 127.5, 124.0,
121.2, 119.9, 110.1, 80.4, 55.8, 51.2, 28.5, 18.5; HRMS

4
Cytotoxic Activity of Synthetic Chiral Amino Acid Derivatives
J. Braz. Chem. Soc.
bromide (MTT) (5 mg mL-1, Sigma, Darmstadt, Germany)
was added to the wells.After 3 h at 37 °C, the MTT solution
was removed and it was added 100 µL per well of DMSO
to solubilize the formazan. Absorbance was measured at
540 nm in a microplate reader (SpectraMax M5, Molecular
Devices). Analyses were performed using Microsoft
Excel (Microsoft Office Software) and GraphPad Prism
(GraphPad Software Inc.).22 Each experimental procedure
was performed in triplicate.
Results and Discussion
By employing the synthetic methodology developed by
our research group,23 32 derivatives from L-amino acids
and 2 from D,L-alanine were prepared in moderate to
good yields. Furthermore, the previously prepared N-alkyl
diamine 39 was successfully employed as nucleophile in
the attainment of the derivative 8. A detailed list of the
synthesized compounds is described in Scheme 1.
*Compounds 23 and 29: racemic mixtures.
Scheme 1. Synthesized L-amino acid derivatives.

Vol. 00, No. 00, 2019
de Castro et al.
5
Scheme 2. (a) Synthesized D-amino acid derivatives; (b) synthesized N-alkyl diamines.
Besides, in an attempt to compare the influence of the
The most promising derivatives in the previous assay
product stereochemistry in the anticancer activity, four
new derivatives were prepared employing the D-alanine
as initial substrate (Scheme 2a). Finally, in addition to
the N-alkyl diamine 39, a more lipophilic derivative was
prepared (40) aiming to evaluate the side-chain size in the
biological activity (Scheme 2b). It is worth mentioning that
in addition to other techniques for the characterization of
the relative and absolute stereochemistry of the products,
the derivative 37 had its X-ray crystallographic structure
determined (Figure 1).
were then selected and had their half-maximal inhibitory
concentration determined for the four cell lines previously
employed and for two other tumor cells: MDA-MB-231, a
breast adenocarcinoma cell, and HepG2, a hepatocellular
carcinoma.
In general, three of the derivatives (8, 39 and 40)
showed promising cytotoxicity, mostly towards cancer
cells. Interestingly, these most active compounds have in
their structure an N-alkyl diamine moiety. Considering all
forty compounds, compound 7 (in which propanediamine
was employed as nucleophile instead of an N-alkyl diamine)
showed no inhibition of tumor cells. On the other hand,
a similar analogue with the incorporation of a lipophilic
sidechain (compound 8) was the only derivative to inhibit
B16F10 tumor cell line in more than 90%. It is important
to mention that metastatic B16F10 is known to be very
resistant to anticancer agents,25 which highlight the result
obtained.
In the tested concentration, most of the compounds
were almost inactive towards the tumor cells evaluated,
with a growth percentage relative to control ranging
between 70-100%. All derivatives prepared though the use
of secondary amines nucleophiles (18-32) presented poor
inhibition over B16F10 cells (above 70% of cell viability
after 48 h of treatment) and most of them stimulated the
growth of HeLa cells. Moreover, the use of Boc deprotected
derivatives (33-34) as well as all four derivatives from
D-alanine (35-38) failed to effectively inhibit the cancer
cells.
Furthermore, by analyzing the data some interesting
observations could be found. For example, compound 14
stimulated the cellular growth of tumor cells, while
showing a small inhibition over non-tumor cells. Besides,
a direct influence of the increase in the activity and the
lipophilicity of the derivatives could not be traced.As when
Figure 1. X-ray crystallographic structure of 37 (anisotropic displacement
ellipsoids are drawn at the 50% probability level).
The cytotoxic effects of the synthesized amino
acid derivatives 1-40 were firstly screened employing
murine metastatic melanoma-B16F10, human cervix
adenocarcinoma-HeLa, murine fibroblast-NIH3T3
(non-tumor cell line), and human embryonic kidney-
HEK293 (non-tumor cell line), using the well-established
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (MTT) assay.24 The results are shown in Table 1.

6
Cytotoxic Activity of Synthetic Chiral Amino Acid Derivatives
J. Braz. Chem. Soc.
Table 1. Effect of synthesized amino acid derivatives on cell viability. Cell viability was determined using the MTT assay. The percentage of inhibition
was calculated considering the cells treated with the vehicle (DMSO), considered as 100% of cell viability
Cell line / % of viable cell
Compounda / 10 µM
B16F10b
74.9 8.7
80.8 4.3
66.2 5.4
63.0 0.9
72.3 11.5
74.8 24.1
98.1 27.5
9.4 0.5
HeLac
NIH3T3d
69.0 9.4
53.7 11.9
66.5 5.0
78.1 9.0
70.2 19.0
75.5 1.3
66.0 16.2
34.5 4.5
94.5 18.3
58.8 6.9
77.5 18.7
68.2 18.8
62.2 28.8
74.0 19.1
41.8 5.0
46.4 3.8
57.5 3.6
53.1 4.9
62.5 3.8
69.2 16.0
79.4 4.1
87.7 13.4
65.7 6.7
74.3 20.6
87.6 11.5
64.1 18.5
69.3 15.8
73.6 4.7
83.3 7.5
75.9 17.5
91.3 11.2
80.4 2.6
98.6 3.8
62.2 18.2
54.3 6.0
86.2 0.4
75.1 11.5
96.1 10.3
36.1 5.2
26.9 4.7
HEK293e
96.8 2.3
95.3 1.3
96.1 4.1
94.3 6.4
92.3 5.7
92.1 5.1
93.9 4.4
17.8 5.3
92.5 5.8
95.6 5.3
95.5 5.6
96.2 2.7
96.9 2.4
92.2 4.9
91.0 2.8
94.9 7.0
95.4 5.2
94.6 4.1
98.4 0.4
94.6 3.5
88.0 2.4
94.9 3.4
94.4 3.6
96.1 3.7
90.9 5.7
96.3 4.0
98.5 0.7
91.4 2.0
93.1 5.8
90.2 3.6
94.8 6.9
93.7 5.7
92.2 5.5
93.9 4.8
94.3 3.8
94.3 4.9
90.0 0.8
95.2 5.1
13.5 0.7
55.7 19.8
1
91.5 1.3
94.7 12.7
39.8 0.6
40.6 1.0
90.3 10.9
108.6 3.1
104.6 1.4
20.1 0.7
93.0 1.8
99.5 5.2
126.4 4.9
90.6 20.4
119.9 2.2
115.5 5.5
89.8 9.4
103.7 7.0
102.5 15.6
98.9 8.7
105.2 4.0
96.8 1.6
109.6 5.2
104.1 9.0
118.5 4.7
126.7 3.0
120.2 3.6
95.3 4.7
99.3 7.7
97.4 2.2
117.5 1.4
124.7 3.1
114.2 8.5
118.7 15.6
101.7 4.1
104.3 1.1
111.8 9.2
84.3 15.7
87.6 3.3
84.7 4.9
25.1 1.1
18.7 1.0
2
3
4
5
6
7
8
9
84.2 12.0
75.2 29.2
77.2 10.1
83.1 10.5
75.6 1.8
106.8 13.6
92.4 25.1
96.2 11.9
70.4 4.6
75.9 19.6
71.7 13.2
84.3 10.4
71.5 13.8
84.2 4.0
82.4 12.5
82.4 9.1
81.9 6.7
76.3 7.8
81.3 4.1
76.2 4.4
92.5 12.9
74.6 11.1
73.3 1.7
74.6 11.0
73.0 9.1
79.2 9.9
80.6 5.8
79.5 3.6
88.0 4.2
88.3 5.1
10.3 0.2
12.0 0.2
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
aB16F10, HeLa, NIH3T3, and HEK293 cells were treated with 10 µM of each compound for 48 h. The values are expressed as the means standard
deviation of triplicated experiments; bB16F10: murine metastatic melanoma; cHeLa: human cervix adenocarcinoma; dNIH3T3: murine fibroblast (non-tumor
cell line); eHEK293: human embryonic kidney (non-tumor cell line).

Vol. 00, No. 00, 2019
de Castro et al.
7
analyzing the series of compounds 1-6, the most active
compounds were the mid-sized chain derivatives 3 and 4,
with considerable decrease in cellular growth inhibition by
altering the carbon chain size.
probably inhibits cell growth by acting in the polyamine
biosynthesis/metabolism pathway, as occur in other
diamine-based in drugs (such as Eflornithine).26 On the
other hand, the enhanced selectivity of compound 8 might
be involved with the increase in amino acid uptake in some
cancer cells, by the overexpression of L-type amino acid
transporter-1 (LAT-1).9
Finally, the selectivity index was calculated by diving
the mean IC50 for normal cells by the mean IC50 for tumor
strains and the results are described in Table 3. Once
more, the compound 8 showed the best overall result,
with a selectivity index of 3.4. Although the selectivity
index and the activity can be further enhanced, we
believe that derivative 8 is a promising building block
for the development of new anticancer candidates. Thus,
the preparation of new amino acid derivatives, bearing
N-alkyl diamines of different sidechain lengths is under
development by our research group and will be reported in
the due course along with more in-depth studies regarding
the mechanism of action in the intracellular context.
In the following experiments, the three most promising
compounds (8, 39 and 40) had their half-maximal inhibitory
concentration determined (Table 2) for six cell lines.
The results indicated that these three derivatives showed
anticancer activity with IC50 ranging from 1.9 to 6.1 µM.
While in N-alkyl diamine 39 the IC50 for one of the tumor
cell lines (MDA-MB-231) was higher than for the tested
normal cells, the same profile was not observed for the
longer chain derivative 40. On the other hand, compound
40 in some cases presented similar half-maximal inhibitory
concentrations for normal and tumor cells, such as for
MDA-MB-231 (IC50 3.1 µM) and NIH3T3 (IC50 4.4 µM),
indicating a poor selectivity.
The most promising result was detected for the
derivative 8, bearing both the N-alkyl diamine aliphatic
sidechain and the amino acid moiety, with IC50 for all
cancer cells near 2.0 µM and for normal cells above
6.0 µM. Most interesting, the comparison of compound 8
(the Boc-protected amino acid bearing a N-alkyl diamine
moiety) and 39 (the N-alkyl diamine) showed considerable
differences in the activity. While the incorporation of the
amino acid scaffold reduced the IC50 for all tumor cells
(especially for MDA-MB-231, with an almost three-fold
decrease in IC50), the inverse behavior was detected in
normal cells, with a small increase in IC50 for HEK293 and
a three-fold increase for NIH3T3. These three compounds
Conclusions
In summary, a series of 40 compounds (including thirty
two L-amino acid, two D,L-amino acid and four D-amino
acid derivatives, as well as, two protected amino acid salts
and two N-alkyl diamines) were synthesized and evaluated
towards their cytotoxic activity over four tumor and two
non-tumor cells. Among the synthesized derivatives, three
bearing an N-alkyl diamine sub-unity exhibited the best
Table 2. Half-maximal inhibitory concentration (IC50) values for the treatments with compounds 8, 39 and 40
Cell line - IC50 / µMa
Compound
B16F10b
1.8 2.1
2.2 1.4
2.4 2.4
MDA-MB-231c
2.3 4.3
HepG2d
1.7 3.0
1.9 2.7
2.3 2.8
HeLae
NIH3T3f
6.6 5.2
2.2 3.8
4.4 5.0
HEK293g
6.0 1.1
5.8 1.2
9.7 1.8
8
1.7 4.3
2.0 7.6
2.1 5.0
39
40
6.1 2.5
3.1 1.9
aIndicated cell lines were treated with increasing concentrations (0-200 µM) of each compound for 48 h. Cell viability was determined using the MTT assay.
b
The values are expressed as the means standard deviation of triplicated experiments; B16F10: murine metastatic melanoma; cMDA-MB-231: breast
adenocarcinoma; dHepG2: hepatocellular carcinoma; eHeLa: cervix adenocarcinoma; fNIH3T3: murine fibroblast (non-tumor cell line); gHEK293: human
embryonic kidney (non-tumor cell line).
Table 3. Selectivity index determination and mean half-maximal inhibitory concentration (IC50) values for the treatments in normal and cancer cells
IC50 / µM
Compound
Non-tumor cell lines
6.3 3.2
Tumor cell lines
1.9 3.4
Selectivity indexa
8
3.4
1.3
2.8
39
40
4.0 2.5
3.1 3.6
7.1 3.4
2.5 3.0
aSelective indexes were calculated by dividing the IC50 mean value for non-tumor cell lines by the IC50 mean values for tumor cells.

8
Cytotoxic Activity of Synthetic Chiral Amino Acid Derivatives
J. Braz. Chem. Soc.
overall inhibitory activity, presenting IC50 values at the
low micromolar range. Notably, compound 8 was the most
potent derivative, presenting IC50 about 2.0 µM on tumor
cells. Furthermore, the inclusion of an amino acid moiety in
the N-alkyl diamine seems to play an important role in the
selectivity towards cancer cells, increasing both the activity
and the selectivity index. To the best of our knowledge,
this consists the first time that the class of dual-protected
amino acid derivatives hereby reported were assayed toward
their anticancer activity. Due to the promising results found
during this study, the preparation of new derivatives based
on the structure of compound 8 is ongoing in our laboratory.
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Crystallographic data (excluding structure factors)
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data can be obtained, free of charge, via www.ccdc.
cam.ac.uk/conts/retrieving.html or from the Cambridge
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Cambridge CB2 1EZ, UK; fax: +44 1223 336033. E-mail:
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1
Supplementary data (copies of H NMR, 13C NMR,
FTIR and XRD) are available free of charge at
http://jbcs.sbq.org.br as PDF file.
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This work was supported in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior-Brazil
(CAPES)-Finance Code 001. The authors also acknowledge
the financial support from FAPEMIG, CNPq, and Rede
Mineira de Química.
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Published online: July 12, 2019
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