Synthesis and Biological Evaluation of Amidinourea and Triazine Congeners as Inhibitors of MDA-MB-231 Human Breast Cancer Cell Proliferation

Article abstract of DOI:10.1002/cmdc.201600580

A series of novel amidinourea derivatives was synthesized, and the compounds were evaluated as inhibitors of MDA-MB-231 human breast cancer cell proliferation. In addition, a second series of triazine derivatives designed as rigid congeners of the amidinoureas was synthesized, and the compounds were evaluated for their antiproliferative activity. Among the two series, amidinourea 3 d (N-[N-[8-[[N-(morpholine-4-carbonyl)carbamimidoyl]amino]octyl]carbamimidoyl]morpholine-4-carboxamide) emerged as a potent anticancer hit compound with an IC₅₀value of 0.76 μm, similar to that of tamoxifen.

Full text of DOI:10.1002/cmdc.201600580

Accepted Article
Title: Synthesis and biological evaluation of novel amidinourea and
triazine congeners as inhibitors of MDA-MB-231 human breast
cancer cell proliferation
Authors: Daniele Castagnolo, Rosemary Bass, Sarah Jenkinson,
Jennifer Wright, Tora Smulders-Srinivasan, and Jamie C
Marshall
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To be cited as: ChemMedChem 10.1002/cmdc.201600580
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10.1002/cmdc.201600580
ChemMedChem
COMMUNICATION
Synthesis and biological evaluation of novel amidinourea and
triazine congeners as inhibitors of MDA-MB-231 human breast
cancer cell proliferation
Rosemary Bass,[b] Sarah Jenkinson,[b] Jennifer Wright,[b] Tora Smulders-Srinivasan,[b] Jamie C.
Marshall,[b] Daniele Castagnolo[a],*
[a]
[b]
Dr. D. Castagnolo*
Institute of Pharmaceutical Science,
King’s College London,
150 Stamford Street SE1 9NH
London, United Kingdom
E-mail: daniele.castagnolo@kcl.ac.uk
Dr. R. Bass, Dr. S. Jenkinson, Dr. J. Wright, Mr. J.C. Marshall
Department of Applied Sciences,
Northumbria University,
Ellison Building, Ellison Place, NE1 8ST
Newcastle upon Tyne, United Kingdom
Supporting information for this article is given via a link at the end of the document.
Abstract: A series of novel amidinourea derivatives has been
synthesised and the new compounds have been evaluated as
inhibitors of MDA-MB-231 human breast cancer cell proliferation. In
addition, a second series of triazine derivatives designed as rigid
congeners of the amidinoureas was synthesised as well and the
compounds were evaluated for their antiproliferative activity. Among
the two series, the amidinourea 3d emerged as a potent anticancer
hit compound with IC50 = 0.76 M comparable to tamoxifen.
the biguanides metformin and phenformin recently showed
potential anticancer effects.14 Similarly, Woster and coworkers
reported that biguanides and bis-urea derivatives possess
anticancer activity, including activity against breast cancer, acting
as epigenetic modulators.15 Due to our previous experience in the
synthesis of linear amidinoureas11-12 and their structural
correlation with biguanides, we decided to synthesise a narrow
library of amidinourea derivatives and evaluate them as potential
anticancer agents.
Breast cancers are solid tumors which result from a series
of non-random molecular alterations, transforming normal cells
into cancer cells with invasive and metastatic potential. However,
the steps of tumor progression are not yet well elucidated in
breast cancer.1 Breast cancer represents today the most common
malignant tumor and the second most lethal cancer among
women preceded only by lung cancer.2-3 Women have a 1 in 8
lifetime risk of developing breast cancer and 1 in 35 risk of breast
cancer causing death in the US and Europe. Several studies have
established that estrogens are predominantly involved in the
initiation and proliferation of breast cancer and much efforts are
now being devoted to block estrogen formation and action.4 Most
common breast cancer therapies are based on the use of drugs
that stop estrogen and progesterone from helping breast cancer
cells grow.5 These drugs include the natural drug paclitaxel,6
aromatase inhbitors7 such as the triazole letrezole and the
estrogen receptor modulators tamoxifen and raloxifen.8 (Figure 1).
However, there is constant of need to find novel anticancer
molecules with improved activity, selctivity and reduced side
effects.
Amidinoureas represent an interesting and underexplored
class of compounds.9-10 We recently discovered both macrocyclic
and linear amidinurea derivatives endowed with antifungal11 and
antiviral activity.12 Some amidinurea derivatives also showed
antiproliferative properties13 probably due to their ability to
mimicking the natural nucleobases and thus to interact with DNA.
Amidinoureas can be considered as bio-isosters of biguanides
and bis-ureas, two classes of organic molecules endowed with
inhibitory activity toward a range of cancer cells. As an example,
Figure 1. Common drugs active on breast cancer cells. General
structures of amidinoureas and triazines.
1
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In particular, as an extension of our previous work, we describe
the design and synthesis of two series of mono and bis-
amidinourea derivatives (A and B, Figure 1) and the evaluation of
their anti-proliferative activity against MDA-MB-231 human breast
cancer cells. In addition, a series of triazine analogues of
amidinoureas was designed. In fact, triazines with the general
structure C represent the rigid congeners of A as shown in Figure
1. Chemical rigidification is an established approach in drug
discovery which allows to derivatise and improve the activity of a
drug by reducing the conformations that a molecule can adopt.
Here, we decided to rigidify the amidinourea group into a triazine
bioisosteric moiety. Triazines have been shown to possess
antitumoral properties,16 but their activity on breast cancer cells
has not yet been fully investigated,17 thus the synthesis of a
narrow library of triazine congeners of amidinoureas was planned.
We first focused on the synthesis of amidinoureas with general
structures A and B. Table 1. The thiopseudourea 1 was reacted
with 1,8-diaminooctane affording the biguanide 2. The latter was
then treated with different primary and secondary amines in
refluxing THF affording the desired Boc-protected bis-
amidinoureas which were in turn converted into the desired
products 3a-f upon treatment with freshly prepared HCl/AcOEt.
Similarly, treatment of 1 with benzylamine or p-Cl-aniline led to
guanidines 4a-b, which were reacted with appropriate amines
leading, after Boc group cleavage, to the desired amidinureas 5a-
e.12 The triazine analogues were synthesised as described in
Table 2. Cyanuric chloride 6 was first reacted with different
amines/anilines affording the derivatives 7a-c. These latter were
then reacted with a series of piperazines leading to the final
products 8a-g. A bis-triazine 9 was also synthesised by reacting
6 with p-xylylenediamine. Compound 9 was further functionalised
through reaction with benzylamine leading to derivative 10.
Table 1. Synthesis of amidinourea derivatives 3 and 5
Table 2. Synthesis of triazine derivatives 8, 9 and 10
Cmpd
3a
R
R1
BnNH
-
3b
3c
4-Cl-Ph-NH
-
-
Cmpd
8a
R
R1
4-Cl-Ph
Ph
PhCH2CH2NH
8b
8c
Bn
Ph
Ph
3d
-
PhCH2CH2
3e
3f
-
-
8d
8e
Bn
Bn
Boc
5a
5b
Bn
8f
Bn
Bn
4-Cl-Ph
8g
[a] Reagents and conditions: i. Amine, DCE, -40 oC, 3h. ii. Piperazine-R1, DCE,
80 oC, MW, 20 min. iii. p-xylylenediamine, DCE, -40 oC, 3h. iv. Benzylamine,
DCE, 80 oC, MW, 20 min.
5c
5d
Bn
Bn
All the compounds were then evaluated for their anti-proliferative
effects on MDA-MB-231 human breast cancer cells. The inhibition
of proliferation was monitored after 30 and 60 hours as shown in
Figure 2 and Figure 3 (for compound 3d). A number of
compounds were shown to inhibit cellular proliferation at 50-100
M. The triazines, with the exception of 8a, 8d and 8e, proved to
be inactive, whilst most of the bis-amidinoureas showed a good
activity profile. In particular, 3b produced a cell proliferation
inhibition of 80% when used at 1-10M.
5e
4-Cl-Ph
[a] Reagents and conditions: i. 1,8-diamonoctane, DCM, r.t., 12h. ii. Amine,
THF, reflux, 12h. iii. Amine, DCM, r.t., 12h.
2
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Table 3. Inhibitory efficency of amidinourea and triazine derivatives against the breast cancer cell line MDA-MB-231
Compounds
3a
3b
3d
3e
3f
5e
8a
8e
Tamoxifen
IC50 (M)
MDA-MB-231
67.5
4.9
0.76
1.3
1.5
22.1
12
74.7
0.6618
Among compounds 3, the derivative 3b bearing a p-Cl-phenyl
moiety on the amidinurea group proved to be the most promising
compound in term of inhibition of cell proliferation. The
replacement of the aryl moiety with a benzyl group (3a), or a
heterocycle (morpholine in 3d, pyrrolidine in 3e, pyrroline in 3f)
led to derivatives still able to inhibit the cell proliferation but at
higher dose than 3b. Interestingly, compound 3c bearing a longer
side chain did not show any activity against MDA-MB-231 cells.
The compounds 5a-e also proved to be not active, thus
accounting for the importance of a long aliphatic backbone for the
anticancer activity. Similarly, the triazine analogues of
compounds 5 showed poor inhibition of MDA-MB-231 cells cell
proliferation. However, at higher concentrations (50-100 M) the
derivatives 7c and 8a proved to be able to inhibit the growth of
MDA-MB-231 cells at >80%.
Figure 3. Effect of 3d on MDA-MB-231 cell growth. The effect of
compound 3d on the proliferation of MDA-MB-231 is shown in the
Figure. The cells were incubated for 24 hours prior the addition of
3d (point A) at 0, 1, 10, 50 100 M. The cell growth was evaluated
after 30h (point B) and 60h (point C) in the presence of 3d.
In conclusion, this work showed the potentiality of amidinourea
compounds as potential anticancer agents, leading to the
identification of a new promising hit candidate compound 3d able
to inhibit breast cancer cells proliferation at submicromolar
concentration. The design and synthesis of additional derivatives
are currently under investigation in our lab.
Experimental Section
The procedures for the synthesis of all compounds, the cell
proliferation screening and the IC50 determination are reported in
the Supporting Information.
Figure 2. Anti-proliferative activity of amidinourea and triazine
derivatives on MDA-MB-231 human breast cancer cells
Acknowledgements
The inhibitory efficiency for some of the most active compounds
was then evaluated against the breast cancer cell line MDA-MB-
231. The IC50 values are summarised in Table 3 and were
compared with the data reported for tamoxifen.18 The three
amidinoureas 3d-f confirmed the data previously observed, thus
emerging as potent breast cancer inhibitors.
Northumbria University and Royal Society of Chemistry
(Research Fund 2015) are acknowledged for financial support.
Keywords: amidinourea • triazine • breast cancer • MDA-MB-
231 cells • cell proliferation
In particular compound 3d, bearing a morpholine substituent on
the amidinurea moiety showed IC50 = 0.76 M, close to that of
tamoxifen, thus proving to be a valuable candidate for further
development. Also the derivatives 3e and 3f showed a good
activity with IC50 values of 1.3 M and 1.5 M respectively, as well
as the aryl amidinurea 3b which showed an IC50 = 4.9M. Again,
the triazine derivatives 8a and 8e and the amidinurea 5e showed
poor inhibition with IC50 values >12M.
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Novel amidinourea derivatives have been synthesized and evaluated as inhibitors of breast cancer MDA-MB-231 cellular proliferation.
The amidinourea 3d was found to be active against MDA-MB-231 cells with IC50 = 0.76 M, close to the activity of Tamoxifen.
5
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