β-amino alcohols and their respective 2-phenyl-N-alkyl aziridines as potential DNA minor groove binders

Article abstract of DOI:10.1016/j.ejmech.2018.07.055

It is known that aziridines and nitrogen mustards exert their biological activities, especially in chemotherapy, via DNA alkylation. The studied scaffold, 2-phenyl-1-aziridine, provides a distinct conformation compared to commonly used aziridines, and the

Full text of DOI:10.1016/j.ejmech.2018.07.055

Accepted Manuscript
β-amino alcohols and their respective 2-phenyl-N-alkyl aziridines as potential DNA
minor groove binders
Miguel M. Vaidergorn, Zumira A. Carneiro, Carla D. Lopes, Sérgio de Albuquerque,
Felipe C.C. Reis, Sofia Nikolaou, Juliana F.R. Mello, Giovani L. Genesi, Gustavo H.G.
Trossini, A. Ganesan, Flavio S. Emery
PII:
S0223-5234(18)30620-2
10.1016/j.ejmech.2018.07.055
EJMECH 10590
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 May 2018
Revised Date: 17 July 2018
Accepted Date: 23 July 2018
Please cite this article as: M.M. Vaidergorn, Z.A. Carneiro, C.D. Lopes, Sé. de Albuquerque, F.C.C.
Reis, S. Nikolaou, J.F.R. Mello, G.L. Genesi, G.H.G. Trossini, A. Ganesan, F.S. Emery, β-amino
alcohols and their respective 2-phenyl-N-alkyl aziridines as potential DNA minor groove binders,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.055.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
β-amino alcohols and their respective 2-phenyl-N-alkyl aziridines as potential
DNA minor groove binders
Miguel M. Vaidergorn1*, Zumira A.Carneiro2, , Carla D. Lopes2, Sérgio de Albuquerque2,Felipe
C.C Reis3, , Sofia NIkolaou3, Juliana F.R. Mello4, Giovani L.Genesi4, Gustavo H. G. Trossini4, A.
Ganesan5, Flavio S. Emery1*
1 Departament of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão
Preto – FCFRP-USP. University of São Paulo, Avenida do Café s/n, 14040-903, Ribeirão Preto,
SP, Brazil.
2 Department of Clinical Analyses, Toxicology and Food Science, School of Pharmaceutical
Sciences of Ribeirão Preto – FCFRP-USP. University of São Paulo
3Departament of Chemistry, Faculty of Philosophy, Sciences and letters at Ribeirão Preto –
FFCLRP-USP
4 LITEC, Departament of Pharmacy, School of Pharmaceutical Sciences – University of São
Paulo, Avenida Prof. Lineu Prestes, 580, bloco 13 superior. São Paulo, SP, Brasil, 05508-000
5School of Pharmacy, University of East Anglia, Norwich, UK
* flavioemery@usp.br
KEYWORDS: Aziridines, Triple Negative Breast Cancer, minor groove binding, β-amino
alcohols
ABSTRACT: It is known that aziridines and nitrogen mustards exert their biological activities,
especially in chemotherapy, via DNA alkylation. The studied scaffold, 2-phenyl-1-aziridine,
provides a distinct conformation compared to commonly used aziridines, and therefore, leads to
a change in high-strained ring reactivity towards biological nucleophiles, such as DNA. The
above series of compounds was tested in three breast cell lines: MCF-10, a healthy cell; MCF-7,
a hormone responsive cancer cell; and MDA-MB-231, a triple negative breast cancer cell. Both
aziridines and their precursors, β-amino alcohols, showed activity towards these cells, and
some of the compounds showed higher selectivity index than cisplatin, the drug used as control.
When the type of cell death was investigated, the synthesized compounds demonstrated higher
apoptosis and lower necrosis rates than cisplatin, and when the mechanism of action was
studied, the compounds were shown to interact with DNA via its minor groove instead of
alkylation or intercalation.
1.Introduction
Targeting DNA and drug-DNA interactions has been of great importance since the
second half of the 20th century.1 Small molecules can interfere with the DNA functions
either by modulating transcription or by interfering with cell growth and replication as
inhibitors.2 Although they have been studied for decades, the mechanisms of DNA-drug
interactions are still not fully comprehended.
There are two main modes of drug-DNA binding.3 The covalent type found in alkylating
agents may act via three mechanisms:4 irreversible covalent binding with guanine
(such as cisplatin and aziridines); cross-link formation between atoms of DNA (two
bases are linked by an alkylating agent that has two binding sites, such as nitrogen
mustards); and nucleotide mispairing, resulting in a mutation by covalent interaction,
and consequently leading to apoptosis 5
The non-covalent binding involves
;
intercalation and minor groove binders. The first one is found in planar organic
compounds and act by π-π stacking between DNA base pairs and the aromatic portion
of the molecule. This leads to structural changes in the DNA itself, because
intercalation can unwind the DNA double helix. 4
Minor groove binders are small molecules, usually in a crescent-like shape (Figure 1),
that interact by Van der Waals interactions and hydrogen bonds in the minor groove of
the DNA double strand.1, 3, 6 They typically have several heterocyclic aromatic rings and

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are commonly employed to bind to specific adenine-thymine (AT)-rich sequences.
Netropsin, a polyamide with antibiotic and viral properties, and pyrrole-imidazole
polyamides are examples of DNA-binding agents.
Figure 1. Crescent-like shape molecules used as minor groove binders
Minor groove binders are also used in chemotherapy in several types of cancer, such
as carcinoma and ovary and breast cancer (BRCA).7 Among them, the latter is the
most prevalent type of cancer in women worldwide, representing 12% of all new cases
of cancer and 25–30% of all cancers in women.8, 9 According to the American Cancer
Society, in the USA alone, approximately 266,120 (30% of all) new cases of invasive
breast cancer will be diagnosed in women and more than 41,400 (14%) women are
estimated to die in 2018 from it.10 BRCA is a heterogenous disease in which multiple
subtypes are identified—each one showing distinctive profiles of prognosis,
physiopathology, and treatments.
Several BRCA subtypes present well established therapy guidelines such as the use of
hormonal adjuvant treatment in hormone-sensitive cancers to block estrogen effects for
estrogen- (ER) and progesterone-positive (PR) cancers.10 When the subtype is PR and
ER negative but HER2 positive, monoclonal antibodies and tyrosine kinase inhibitors
12
(such as trastuzumab and lapatinib, respectively) are effective.11,
When
immunohistochemical analysis shows no hormone receptors on breast cancer cell and
the cell is HER2 negative, it is called triple negative breast cancer (TNBC). TNBC
13
accounts for 15–20% of all cases.
Although TNBC is primarily treated with
chemotherapy, the lack of specific molecular targets leads to low efficacy, consequently
leading to a difficult, more aggressive, and less effective treatment. TNBC usually leads
to poor prognosis and a high propensity for metastasis progression and chemotherapy
resistance.13 Since there are no targeted therapies specifically for this type of cancer,
cytotoxic chemotherapy remains the best option to date.14 Alkylating-like agents such
as platinum derivatives associated with doxorubicin and bevacizumab are used for the
treatment of TNBC.15-17
In this work, as part of our continuous efforts to find cytotoxic antitumor candidates, we
herein report the discovery of aziridine and β-amino alcohol minor-groove binders
possessing cytotoxicity against TBNC cells. First, we synthetized both β-amino
alcohols and their correspondent azidirines and evaluated their biological activity
towards healthy and cancer cell lines. Then, since the compounds presented activity
and selectivity, we studied the mechanism of action by flow cytometry and drug-DNA
interactions, which demonstrated a non-conventional mechanism of action. Finally, we
compared these results to molecular docking, which corroborated the experimental
results.
2. Results and discussion
2.1 Chemistry
. The synthesis of 2-phenyl-1-aziridine series 5a-d involved intramolecular cyclization of the
corresponding β-amino alcohol 3, which was prepared by aminolysis of the epoxide starting

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material, via a modified Wenker synthesis18-20 (Scheme 1). This strategy facilitated the
development of a homologue series of N-substituted side chain to study the influence of the
length of alkyl chain between each of the aromatic portions in both amino alcohols and
aziridines towards the presented biological activity.
Initially, amino alcohols 3a-h were obtained by ring-opening of styrene oxides (1a and 1b) using
several amines (2a-g) in a solvent-free methodology. Since aminolysis is not regiospecific, both
the regioisomers were obtained, which were easily separated by crystallization or
chromatography. Then, the desired amino alcohol was allowed to react with chlorosulfonic acid
or sulfuryl chloride to produce the corresponding sulfonyl esters 4a-d. Finally, after treatment of
sulfonyl esters with a base, aziridines were successfully synthesized in varying yields (35–70%
in a 1 mmol scale).
Scheme 1. Synthetic approach for compounds of interest. Conditions: (A) 200°C, 4 h or room temperature (r.t)
H2O, 6 h (when aniline is used); (B) 1, 2 eq. ClSO3H or SO2Cl2, 0°C, Et 2O/DCM; (C) NaOH 6 M/toluene (10:1),
100°C, 18 h
2.2 Biological evaluation
The small library of amino alcohols and respective aziridines were screened against the
following targets: i) healthy breast tissue MCF-10; ii) hormone-responsive breast cancer MCF-;
and iii) TNBC cell line MB-231. Their half maximal inhibitory concentration (IC50), LD50, and
selectivity index (SI) are shown in Table 1. The MTT assays were conducted by following the
Mosmann methodology21. The cytotoxicity of the studied compounds was compared with
cisplatin, which is a drug prescribed for several types of breast cancers and an alkylating-like
agent. Based on the results shown in Table 1, some compounds of the two classes studied
presented higher selectivity towards cancer cell lines than cisplatin, and higher SI. However,
cisplatin showed lower toxicity (higher CC50). It was observed that the longer the alkyl chain
between the aromatic rings (from methyl to butyl—as seen in scheme 1), the higher the activity
in both amino alcohols and aziridines. Furthermore, a difference in selectivity between β-amino
alcohols and their corresponding aziridines was observed. In the MCF7 cell line, β-amino
alcohols presented a higher SI, while aziridines showed higher activity and SI towards the MB-
231 cell line.
MB-231 cell line was of special interest because of the lack of therapeutic drugs with high SI or
efficacy for the long-term treatment of TNBC. The synthetized compounds presented cytotoxic
effects, especially the longest alkyl chain β-amino alcohol 3d and its respective aziridine 5d,
when compared to cisplatin in terms of both IC50 (50.76, 11.09, and 78.14 µM, respectively) and
SI (2.11, 4.08, and 0.92, respectively). Aziridines 5a, 5b, and 5c also showed higher activity
than cisplatin, but with a higher IC50 (40.42, 19.00, and 19.8 µM) and lower SI (1.01, 1.97, and
1.77). In addition, 3f presented a high activity comparable to aziridines, indicating that a minor
change in one of the aromatic moieties can lead to an increase in activity. These same
compounds presented higher activity than cisplatin against the MCF-7 cell line. However, β-
amino alcohols with longer chains presented a higher SI (2.02, 2,88, 4.04, and 2.18 for 3c, 3d,
3f and 3g, respectively, whereas cisplatin showed a value of 1.45), although they did not
necessarily present the best values for IC50.

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Table 1: Anti-tumor activity (IC50), cytotoxicity (CC50), and selectivity indices (SI) of the
compounds.
Compounds IC50MCF-7 (µM)
IC50
CC50 MCF-10(µM) SIMC SIMDA-
MDA-
MB231(µM)
F-7
MB231
3a
3b
209.60 ± 3.10
318.50 ± 0.45
96.40 ± 2.77
37.17 ± 1.34
279.60 ± 1.5
14.48 ± 0.61
53.01 ± 2.66
236.20 ± 5.35
27.83 ± 0.42
169.70 ± 5.99
24.50 ± 1.11
24.72 ± 0.5
225.30 ± 3.85 201.60 ± 4.45 0.96
189.90 ± 5.9 197.50 ± 5.40 0.62
101.80 ± 1.79 195.60 ± 0.75 2.02
50.76 ± 2.47 107.30 ± 4.7 2.88
259.40 ± 4.17 187.70 ± 5.76 0.67
0.89
1.04
1.92
2.11
0.72
2.57
1.56
1.33
1.01
1.97
1.77
4.08
0.91
3c
3d
3e
3f
22.71 ± 0.21
73.89 ± 3.12
58.54 ± 1.33
4.04
3g
115.60 ± 0.95 2.18
3h
236.90 ± 4.92 316.16 ± 4.85 1.33
5a
40.42 ± 1.34
19.00 ± 0.90
19.89 ± 2.23
11.09 ± 0.39
78.14 ± 2,4
40.99 ± 0.25
37.57 ± 1.5
35.43 ± 1.31
43.30 ± 0.03
72.14 ± 4.12
1.47
0.2
5b
5c
1.44
1.83
1.45
5d
Cisplatin
49.26 ± 0.21
2.3 Mechanism of action investigation
Since compounds 3d, 3f, and 5d showed high tumorigenic activity, especially against
the MB-231 cell line, they were selected for further studies to elucidate the mechanism
by which they induced cell death. In a simple approach, the detection of externalization
of phosphatidylserine was performed by staining with Annexin V/propidium iodide (PI)
(Figure 2A) via flow cytometry. Both 3f- and 5d-treated MB-231 cells induced apoptosis
above 75% of MB-231 population (3f = 91.3% and 5d = 77%, Figure 2B). The
percentage of early apoptotic cells (Annexin V simple stain) was higher in 3f-treated
cells (33.6%; Figure 2C). Notably, 5d treatment resulted in late apoptosis/necrosis in
the cells (Annexin V/PI stained) (Figure 2D). This result was in agreement with MTT
results and suggests that 3f and 5d are cytotoxic to cancer cells, and they probably
induced apoptosis by different mechanisms.

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Figure 2. Phenyl aziridines selectively lead to apoptosis of breast tumor cells. Representative dot plot of each
compound (A). Treatment with 3f and 5d showed higher tumorigenicity (B). 3f treatment led to early apoptosis
(C). 5d and 3f together showed the same ability to lead to late apoptosis/necrosis (D). Results are presented as
the mean of triplicate experiments.
Based on the results of flow cytometry assay, we aimed to investigate the mechanism
of action of these compounds and why they direct cell death to a different direction
when compared to cisplatin. Although it is known that nitrogen mustards and aziridines
act as electrophiles for biological nucleophiles such as nitrogenous bases in the DNA,
we were not sure whether the same mechanism would apply for the compounds tested
in this study because of the electronic and conformational differences between the
different types of compounds.
It is well documented in literature that polymethylene analogs of drug candidates can
enhance biological activity.22, 23 This can be caused either by increasing the interaction
with active site or by altering the physicochemical properties related to hydrophobicity,
such as partition coefficient, solubility in water, and critical micelle concentration. This
effect seems to be corroborated by the cancer activity results obtained in the present
study. To investigate this result further, we performed an assay to probe the potential
interaction between DNA and the studied structures (full experimental details of ct-DNA
interaction experiment in the experimental procedure). Based on the results obtained in
the cancer cells, two compounds, 3d and its respective aziridine 5d, were chosen to
study the potential interaction with DNA. These two molecules have the longest alkyl
chain between the aromatic moieties tested and the highest activities when there are
no substituents in the aromatic ring. 3d and 5d were incubated with ct-DNA in buffer
solution at 32°C for 5 min before recording each electronic spectrum. The binding
constant (Kb) for each compound-DNA adduct was obtained using the Benesi-
Hildebrand equation (see supplementary information) to verify the relative strength of
interaction of 3d and 5d with DNA.
Depending on the type of interaction with DNA, ultraviolet (UV)-visible spectra exhibited
distinct profile changes. Compounds that bound through intercalation exhibited
hypochromism on the DNA band centered at 260 nm. To intercalate, the compound
must have a planar and rigid structure, which is usually provided by the presence of
24-26
fused aromatic rings.
In contrast, hyperchromic effect in DNA absorption indicates
the changes in the secondary structure of double helix, which in turn exposes the
nitrogenous bases to the solvent, thereby increasing the absorption of these

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chromophores. This kind of change may arise from covalent, groove, or electrostatic
binding.18 It is rather difficult to distinguish them exclusively based on
spectrophotometric titration. However, previous studies have reported that a
combination of hyper-and hypsochromism indicates a covalent interaction such as
alkylation, while hyperchromism alone is observed when a non-covalent binding takes
place.17-21 As seen in Figure 4, both 3d and 5d caused hyperchromism in absorption
band of DNA at 260 nm following DNA-compound interaction, without any
hypsochromic or bathchromic changes.4, 25, 27
Figure 4. UV-Vis spectra of (A) 3d (10-6 mol L-1) and (B) 5d (1,25x10-8 mol L-1) with increasing concentrations of
ct-DNA (from 4.9x10-5 to 4.9x10-4 mol L-1) in Tris buffer (5 mM tris(hydroxymethyl)aminomethane hydrochloride
(Tris), 50 mM NaCl, pH 7.4). Insert: double reciprocal Benesi-Hildebrand plot.
Owing to this observation, it is suggested that minor groove binding is a more favorable
type of interaction.24 This kind of weak interaction is typical for structures bearing
unfused aromatic rings, such as phenyl groups of 3d and 5d, bound to flexible carbon
chains, which have torsional freedom.20 These features present in the molecules under
investigation in this study, leads to accommodation in the minor groove of DNA, which
might be directed by hydrophobic interaction, hydrogen bonding, and/or electrostatic
interactions. Usually minor groove binding is preferred to major groove binding owing to
a closer in-space interaction of the former and steric hindrance of the latter due to the
presence of the amine group on C2 of guanine.20 Presumably, in our case, the
interaction of both species are hydrophobic in nature because of their relatively long
alkyl chain. In the case of compound 3d, it is reasonable to assume the assistance of
hydrogen bonds, since this compound bears OH and NH groups in its structure. These
assumptions are corroborated by the docking results (vide infra). Finally, if DNA is
assumed to be the target responsible for the observed cytotoxicity, the different kinds of
interactions of 3d and 5d (groove binding) when compared to cisplatin (alkylation) is
probably responsible for triggering different mechanisms of cell death.
The Kb values observed in this study (3d Kb = 1,592 x 105, and 5d Kb = 5,233 x 105)
are consistent with data reported earlier from similar spectrophotometric titration,
suggesting a strong interaction between 3d and 5d with ct-DNA.25 To confirm the
binding mode in DNA, molecular docking was performed for the most active
derivatives. Autodock 4.2 software was used for calculations, and the Lamarckian
Genetic Algorithm was selected as a search method.28 Ligands were constructed using
Spartan 10 WAVEFUNCTION, and Merck Molecular Force Field (MMFF) was applied
for conformer distribution. The charges were calculated using PM6 following the
density functional theory (DFT) method.
2.4 Molecular docking validation
Molecular docking validation was performed in two steps. First step 1 (VS 1) was
performed using re-docking of netropsin in all DNA structures, checking whether the

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selected parameters were able to identify the correct region of interaction. Netropsin
was selected for docking validation as it is a well-known minor groove ligand. In
validation step 2 (VS 2), a reduced grid box was applied to perform a more accurate
assessment of interactions with the DNA region.
The parameters used in docking validation, which considered all DNA, were able to
produce most of the conformational states of netropsin bound in the minor groove, as
expected. The binding region at the minor groove was rich in A-T base pairs and was in
accordance with the known binding mode. After identification of the binding region,
calculations were focused in the minor groove region. The best conformer presented a
predicted binding energy of -7.74 kcal/mol. This binding mode was similar to the
binding mode obtained by Ricci et al. for netropsin docking29.
The best parameter set was applied in the molecular docking of Hoechst-33258 (minor
groove binder), 3d, 3f, 5a, and 5c derivatives.
An additional evaluation of the method was executed using Hoechst-33258, a DNA
30
inhibitor that binds at the minor groove region. Results showed that parameters of
both VS 1 and VS 2 were able to identify the correct region of binding as well as the
correct AT-rich region of binding. Molecular re-docking focusing on minor groove
returned the conformer with a predicted binding energy of -4.80 kcal/mol (SM).
The molecular docking of 3f was performed using both methods, and the results
showed that the minor groove was the preferred region of binding, which was in
accordance with the experimental assays (Figure 4). This amino alcohol derivative (3f)
showed interactions with AT-rich region, and the best conformer exhibited a predicted
binding energy of -8.51 kcal/mol. The proximity of the aromatic ring of the ligand and
DNA backbone suggests interactions between these groups (Figure 5). 3f is also
involved in one hydrogen bond with the backbone at regions DA19 and DT18, besides
a hydrogen bond with thymine of the DT18 base pair (Figure 5b). Although this
interaction profile was not very common for inhibitors that interact with the minor
groove, the role of ligand-DNA backbone interactions could be important for elucidating
the mechanism of action. Interactions between ligands and the sugar atoms of DNA
backbone could promote stabilization of the complex31.
The proximity of the aromatic ring of the ligand and the sugar atoms of DNA backbone
is not exclusive to 3f derivative. Other derivatives also showed this profile, suggesting
interactions between these atoms of the DNA. The best conformer of this derivative
showed a predicted binding energy of -8.42 kcal/mol. The derivatives 5a and 5c
interact mainly through hydrophobic interactions with DNA (Figure 5c-d). Consequently,
their predicted energies of binding were poorer than 3d and 3f derivatives. The
predicted binding energies for 5a and 5c were -3.96 and 5.36 kcal/mol, respectively. 3d
derivative showed two hydrogen bonds with the DNA backbone at DT6 and DA7
regions, besides one hydrogen bond with thymine of the DT6 base pair (Figure 5e).
These results corroborated the experimental tests and provided a new insight of how β-
amino alcohols and aziridines can interact with DNA and the importance of an aromatic
moiety in the scaffold.

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Figure 5. Molecular docking showing minor groove binding in AT-rich regions (A) of derivatives 3f (B), 5a (C),
5c (D) and 3d (E).
3. Conclusion
In conclusion, we synthesized amino alcohols 3a-h and 2-phenyl-1-aziridine series 5a-
d and biologically evaluated these compounds. The results of this study provide new
insights into the mechanism of action of β-amino alcohols and 2-phenyl-N-alkyl
aziridines and their interaction with DNA, especially in two breast cancer cell lines. The
mechanism of action was found to be spatial conformation, leading to a crescent-like
shaped molecule, although they are small molecules. Moreover, the new compounds
were more selective than cisplatin in both the cell lines and may provide evidence for
further studies on new therapies for TNBC.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Acknowledgment
The authors thank ‘Fundação de Amparo à Pesquisa de São Paulo” (processes 2014/03812-9
and 2015/20302-7) for providing financial support during this research.
4. Experimental session
4.1 Chemistry
4.1.1 General information

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1H and 13C NMR NMR spectra were determined on a BRUKER® Fourier300 – Ultra
Shield® spectrometer in CDCl3 or DMSO-d6. The reagents were all analytical grade or
chemically pure acquired from Sigma-Aldrich. Flash chromatography was performed on
silica gel (60Å, 230-400mesh, 40-63µm).
4.1.2 General procedure for synthesis of the β-aminoalcohols
Epoxide (1a-b, 2 mmol, 1 eq) was slowly added during 1 hour to the amine (4mmol,
2eq) in a round-bottom flask in reflux. After the addition was completed, the reaction
mixture was stirred at the amine boiling point for 3h and then cooled to room
temperature. The oily product solidified over time and was recrystallized in
dichlorometane-hexane (2:8). When necessary, an additional purification was carried
out in a short flash chromatography column (Ethyl Acetate-Methanol).
2-(benzilamine)-1-phenylethan-1-ol (3a)
1
White crystals, 55% yield. H RMN (300 MHz, MeOD) δ 7,39 – 7,16 (m, 10H), 4,77
(dd, J = 8,5, 4,5 Hz, 1H), 3,78 (dt, J = 22,2, 7,7 Hz, 1H), 2,86 – 2,65 (m, 2H).13C NMR
(75 MHz, MeOD) δ 144,60; 140,5; 129,5; 129,4; 128,5; 128,3; 127,0; 73,4; 57,4; 54,2.
2-(phenetilamine)-1-phenyletan-1-ol (3b)
1
White crystals, 54% yield. H RMN (300 MHz, CDCl3) δ 7,34 – 7,10 (m, 10H), 4,74
(dd, J = 9.4, 3.4 Hz, 1H), 2,92 – 2,84 (m, 2H), 2,83 – 2,76 (m, 2H), 2,75 – 2,67 (m, 2H).
13C NMR (75 MHz, CDCl3) δ 142,1; 142,0; 139,1; 128,8; 128,6; 128,5; 127,7; 126,5;
125,8; 71,2; 56,6; 50,5; 35,7.
1-phenyl-2-((3-phenylpropyl)amino)ethan-1-ol (3c)
White crystals,42% yield. 1H RMN (300 MHz, CDCl3) δ 7,42 – 7,00 (m, 10H), 4,66 (dd,
J = 8,5; 4,5 Hz, 1H), 2,65 (t, J = 6.,7 Hz, 2H), 2,62 – 2,50 (m, 4H), 1,73 (dt, J = 15,1;
7,5 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 147,1; 145,6; 131,9; 131,9; 131,1; 129,5;
129,4; 75,8; 60,6; 37,0; 34,8
1-phenyl-2-((4-phenylbutyl)amino)ethan-1-ol (3d)
Yellow solid, 45% yield. 1H RMN (300 MHz, MeOD) δ 7,44 – 6,89 (m, 10H), 4,68 (dd, J
= 7,8; 5,3 Hz, 1H), 2,69 (dd, J = 7,9; 4,8 Hz, 2H), 2,57 (ddd, J = 14,7; 10,0; 5,2 Hz, 4H),
13
1,64 – 1,41 (m, 4H). C NMR (75 MHz, CDCl3) δ 142,2; 128,5; 128,5; 128,4; 127,7;
125,9; 77,1; 71,1; 56,7; 49,1; 35,7; 29,0; 28,8.
2-(butylamino)-1-phenylethan-1-ol (3e)
Yellow solid, 42% yield.1H RMN (300 MHz, CDCl3) δ 7,36 – 7,12 (m, 5H), 4,92 (dd, J =
9,8; 2,9 Hz, 1H), 2,88 (ddd, J = 5,96; 11,3; 4,8 Hz, 2H), 2,72 (tdd, J = 11,9; 7,8; 4,3 Hz,
2H), 1,28 (dt, J = 15,0; 7,4 Hz, 4H), 0,76 (t, J = 7,4 Hz, 3H) 13C NMR (75 MHz, CDCl3)
δ 141,7; 128,8; 127,7; 125,8; 70,5; 65,9; 48,8; 31,4; 20,3; 13,8.
1-(4-bromophenyl)-2-((4-phenylbutyl)amino)ethan-1-ol (3f)
1
Yellowish solid, 15% yield. H RMN (300 MHz, CDCl3) δ 7,64 – 6,97 (m, 14H), 5,08
(dd, J = 10,0; 2,5 Hz, 1H), 3,07 – 2,97 (m, 1H), 2,95 – 2,77 (m, 3H), 2,63 (dt, J = 15,0;
5,6 Hz, 3H), 1,84 – 1,60 (m, 5H). 13C NMR (75 MHz, CDCl3) δ 141,6; 140,0; 131,7;
128,4; 128,3; 127,5; 126,0; 121,9; 69,4; 55,7; 48,7; 35,4; 28,6; 27,1.

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2-(benzylamino)-1-(4-bromophenyl)ethan-1-ol (3g)
Yellow solid, 10% yield. 1H RMN (300 MHz, DMSO) δ 7,50 – 7,27 (m, 9H), 4,65 (t, J =
6,1 Hz, 1H), 3,73 (d, J = 2,7 Hz, 2H), 2,61 (d, J = 6,2 Hz, 2H). 13C NMR (75 MHz,
DMSO) δ 143.9, 140.3, 130.7, 128.1, 128.1, 127.9, 126.6, 119.7, 70.7, 56.5, 52.5.
1-phenyl-2-(phenylamino)ethan-1-ol (3h)
Orange oil, 70% yield. 1H RMN (300 MHz, CDCl3) δ 7,49 – 6,50 (m, 10H), 4,53 (dd, J =
6,9; 4,2 Hz, 1H), 3,95 (dd, J = 11,1; 4,2 Hz, 1H), 3,76 (dd, J = 11,1; 7,0 Hz, 1H). 13C
NMR (75 MHz, CDCl3) δ 146.1, 139.4, 129.1, 128.8, 128.6, 127.7, 126.9, 118.7, 114.7,
77.4, 77.0, 76.6, 66.9, 60.8.
4.1.3 – General procedure for the synthesis of aziridines
To a solution of 3 (1mmol) in ethyl ether/dichlorometane (8:2) at 0°C , chlorossulfonic
acid or sulfonyl chloride was slowly added (1.2mmol). After stirring at 0°C for 1h, the
reaction mixture was stirred at room temperature for 2h30. The resulting white solid
was washed and filtered using 0°C ethyl ether (3x10 ml), isopropyl alcohol (3x10ml) and
ethyl ether (3x10ml) and was dried under low pressure, resulting in 4(a-d) in
quantitative yields. To a solution of 4 (a-d - 1mmol) in toluene (5ml), NaOH of 6,2M
(4,96g in 20ml) was added and the reaction was refluxed for 18h. The reaction mixture
was then extracted with ethyl acetate (3x20ml), dried over Na2SO4 and the solvent was
evaporated. The resulting oil was purified over deactivated silica gel (1% Et3N) in
hexane: ethyl acetate. The purified compounds were kept under refrigeration and
covered from light.
1-benzyl-2-phenylaziridine (5a)
Incolor oil, 55% yield. 1H RMN (300 MHz, CDCl3) δ 7,50 – 7,15 (m, 10H), 3,72 (d, J =
13,7 Hz, 1H), 3,63 (d, J = 13,8 Hz, 1H), 2,53 (dd, J = 6,5; 3,4 Hz, 1H), 2,01 (d, J = 3,3
Hz, 1H), 1,88 (d, J = 6,5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 140.1, 139.1, 128.3,
128.3, 127.8, 126.9, 126.9, 126.2, 64.7, 41.5, 37.9.
1-phenethyl-2-phenylaziridine (5b)
1
Incolor oil, 60% yield. H RMN (300 MHz, CDCl3) δ 7,46 – 7,12 (m, 10H), 2,94 (t, J =
7,6 Hz, 2H), 2,68 (m, J = 11,4; 7,4 Hz, 2H), 2,30 (dd, J = 6,5; 3,4 Hz, 1H), 1,92 (d, J =
3,4 Hz, 1H), 1,67 (d, J = 6,6Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 140,2; 140,0; 128,8;
128,3; 128,2; 127,1; 126,1; 126,0; 63,3; 41,4; 37,9; 36,4.
2-phenyl-1-(3-phenylpropyl)aziridine (5c)
Incolor oil, 50% yield. 1H RMN (500 MHz, CDCl3) (300 MHz, CDCl3) δ 7,36 – 7,13 (m,
10H), 2,76 – 2,70 (m, 2H), 2,54 (dt, J = 11,6; 7,2 Hz, 1H), 2,44 – 2,33 (m, 1H), 2,31
(dd, J = 6,5; 3,3 Hz, 1H), 1,98 (dd, J = 15,5; 7,1 Hz, 2H), 1,91 (d, J = 3,4 Hz, 1H), 1,67
13
(d, J = 6,5 Hz, 1H). ). C NMR (75 MHz, CDCl3) δ 142,2; 140,4; 128,5; 128,4; 128,4;
126,9; 126,2; 125,8; 61,1; 41,4; 37,8; 33,7, 31,4.
2-phenyl-1-(4-phenylbutyl)aziridine (5d)
Incolor oil, 65% yield.1H RMN (300 MHz, CDCl3) (300 MHz, CDCl3) δ 7,49 – 6,82 (m,
10H), 2,65 (t, J = 7,3 Hz, 2H), 2,51 (dt, J = 11,7; 6,8 Hz, 1H), 2,44 – 2,35 (m, 1H), 2,31
(dd, J = 6,5; 3,3 Hz, 1H), 1,90 (d, J = 3,2 Hz, 1H), 1,81 – 1,70 (m, 4H), 1,67 (d, J = 6,6

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Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 142,4; 140,4; 128,4; 128,2; 126,8; 126,2; 125,6;
61,6; 41,2; 37,8; 35,8; 29,4; 29.2.
4.2 MTT assay
The cytotoxity was assessed using the classical [3 – (4,5-dimethylthiazol-2-yl) -2,5-
diphenyl tetrazolium bromide] (MTT) colorimetric assay16. To this end, 5.0 x 105 cells
(MCF-10, MCF-7 and MDA-MB231) were incubated for 24 hours in 96-well cell culture.
After this incubation period, the compounds or cisplatine was added (concentrations
500 to 3.9 µM in serial dilution / compounds solubilized in DMSO 0,5%) in a final
volume of 200 ꢀL. Cells were incubated for 48 hours at 37 °C. Aft er incubation with the
compounds, the medium was removed and added 50 ꢀL of MTT (2.0 mg/mL) diluted in
phosphate buffered saline (PBS). The precipitated blue MTT formazan was then
dissolved in 50 µL of DMSO, and the absorbance was measured at 570 nm in a
VARIAN CARY-50 plate reader MPR multiwell. Cell viability was expressed as the
percentage of absorption values in treated cells compared with untreated (control)
cells. IC50 curve fitting was carried out using GraphPad Prism 5 Software (GraphPad
Software Inc., San Diego, CA).
4.3 Flow cytometry assay
To evaluate the apoptosis events, MDA-MB-231 cells were seeded in 12-well plates at
5.0 x 106 cells/well for 24 h. Next, they were treated with 3f and 5d compounds or
cisplatin as positive control for 48 h. After, the cells were trypsinized, washed with ice-
cold PBS and re-suspended in binding buffer, according to the kit instructions. The
cells were then incubated with FITC-conjugated Annexin V (1:100) for another 15 min
as recommended by FITC Annexin V Apoptosis Detection Kit (BD Pharmigen ™).
Propidium iodide (1 µg/mL) was added immediately before BD FAXCANTO™ flow
cytometer analysis. The excitation/emission used for FITC- conjugated Annexin V was
494/518 nm while to propidium iodide, the excitation/emission was 585/617 nm. A total
of 10,000 events were counted per sample and analyzed by the software BD
FACSDIVA (BD Biosciences).
4.4 DNA samples and binding experiments
Calf-thymus deoxyribonucleic acid (ct-DNA) was obtained from Sigma-Aldrich. It was
used as received and stored below 277 K. Ct-DNA was dissolved in a buffer solution (5
mM tris(hydroxymethyl)aminomethane trishydrochloride (Tris), 50 mM NaCl, pH 7.4)
and stirred for about 12 hours. The resulting solution of ct-DNA gave ratios of UV
absorbance at 260 and 280 nm, A260/A280, in between 1.8-1.91, suggesting that the ct-
DNA was sufficiently free of proteins. In this case, the ratio of A260/A280 was 1.86. The
concentration of nucleic acid solutions was determined spectrophotometrically, using
= 6600 M-1 cm-1 at 260 nm and it was expressed in terms of base-pair equivalents per
dm3.
First, using a fixed complex concentration (3d = 2.00 µM; 5d = 1.25 µM) it was added
aliquots of DNA solution ([DNA] = 3.00 mM) to the sample. The experiment was
performed using an UV/Visible Spectrophotometer - UV380G – Gehakain, at 305 K and
the complex-DNA solutions were allowed to incubate for 5 minutes before each spectra

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recording. The intrinsic binding constants (Kb) of the interaction between compounds
and DNA can be calculated using the Benesi-Hildelbrand equation:
ꢀꢁꢂꢃ
ꢈꢉ
1
ꢇ
ꢌ
ꢄꢁ ꢅ ꢁꢂꢆ ꢄꢈꢊꢋꢉ ꢅ ꢈꢉꢆ ꢍꢎꢀꢏꢐꢁꢃ
where, A0 e A are the absorbances of the drug and the durg-DNA adduct, respectively.
ꢈꢉ and ꢈꢊꢋꢉ are the absorption coefficients of the drug and the drug–DNA adduct,
respectively. Kb is the binding constant and it is obtained from intercept-to-slope ratios
of a 1/(A – A0) versus 1/[DNA] plots.
4.5 Docking validation
Step 1 (VS 1): The grid box dimensions were 96x96x96 points, considering all DNA
structure. Molecular docking genetic algorithm parameters changed from default were
population size of 50, number of evaluation of 50000000, for 25 docking runs.
Step 2 (VS 2): In these calculations, the re-docking of neutropsin was performed in a
reduced grid box with dimensions of 60x70x50 points, focusing in the region of binding.
The parameters changed of the default were population size of 50, number of
evaluations of 25000000 and 25 docking runs.
Figure 6. Molecular docking validation using re-docking of neutropsin (green), VS 1 (A) and VS 2 (B). Molecular
docking between Hoechst-33258 (pink) and DNA using parameters of VS 1 (C) and VS 2 (D). A-T rich region in
blue and G-C rich region in purple.
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Highlights:
- A new insight of 2-phenyl-1-alkylaziridines and β-amino alcohols towards cancer cells
- Aziridine 5d was the most potent compound towards TNBC cell line
- β-amino alcohol 3f was the most active compound against MCF 7 cell line
- The compounds induced cell death most probably via an apoptotic pathway
- The compounds acted via a mechanism of action found on crescent-like shape molecules