Synthesis of minoxidil conjugates and their evaluation as HL-60 differentiation agents

Article abstract of DOI:10.1016/j.bmcl.2016.01.048

Activation of minoxidil (MNX) with N,N′-carbonyldiimidazole and coupling with natural polyamines (PAs) and commercially available aliphatic or aromatic amines provided a series of new conjugates which were evaluated for their ability to induce differentia

Full text of DOI:10.1016/j.bmcl.2016.01.048

Accepted Manuscript
Synthesis of minoxidil conjugates and their evaluation as HL-60 differentiation
agents
Sonia Stoica, George E. Magoulas, Antonia I. Antoniou, Sherif Suleiman,
Analisse Cassar, Lucienne Gatt, Dionissios Papaioannou, Constantinos M.
Athanassopoulos, Pierre Schembri-Wismayer
PII:
S0960-894X(16)30048-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.048
BMCL 23513
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 December 2015
15 January 2016
18 January 2016
Please cite this article as: Stoica, S., Magoulas, G.E., Antoniou, A.I., Suleiman, S., Cassar, A., Gatt, L., Papaioannou,
D., Athanassopoulos, C.M., Schembri-Wismayer, P., Synthesis of minoxidil conjugates and their evaluation as
HL-60 differentiation agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.01.048
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Synthesis of minoxidil conjugates and their evaluation as HL-60 differentiation agents
Sonia Stoicaa, George E. Magoulasb, Antonia I. Antonioub, Sherif Suleiman a Analisse Cassar a,
a
b
Lucienne Gatt , Dionissios Papaioannou , Constantinos M. Athanassopoulosb*, and Pierre
Schembri-Wismayer a*
a Anatomy Department, Faculty of Medicine and Surgery, University of Malta, Malta
b Laboratory of Synthetic Organic Chemistry, Department of Chemistry,
University of Patras, GR-26504 Patras, Greece
*Corresponding Authors: C.M. Athanassopoulos, e-mail: kath@chemistry.upatras.gr and
P. Schembri-Wismayer, e-mail: pierre.schembri-wismayer@um.edu.mt
Abstract
Activation of minoxidil (MNX) with N,N′-carbonyldiimidazole and coupling with natural
polyamines (PAs) and commercially available aliphatic or aromatic amines provided a series of
new conjugates which were evaluated for their ability to induce differentiation to HL-60 acute
myeloid leukemia cancer cells, using a modified NBTZ reduction test. Although neither MNX
nor 4,4'-methylenedianiline (MDA) or 2,7-diaminofluorene (DAF), alone or in combination, had
any effect, the MNX-spermine (SPM) conjugate (11) and the conjugates 7 and 8 of MNX with
MDA and DAF exhibited a differentiation-inducing effect at a concentration of 10 μΜ without
being toxic on proliferating human peripheral blood mononuclear cells.
Keywords: Minoxidil, conjugates, polyamines, differentiation, HL-60
Cancer is a leading cause of death worldwide with projections estimating 13.1 million
deaths in the year 2030.1 Whilst several treatments do exist, many of them are relatively toxic to
normal healthy cells. One goal of current research is to identify novel, safer and more effective
treatments for a wider spectrum of neoplastic conditions in the hope of decreasing morbidity and
mortality. This has resulted in more targeted therapies for cancer, including differentiation
therapy.2 -7 Acute promyelotic leukemia (APL), an uncommon subtype of acute myeloid
3456
leukemia (AML), has been the main success story of differentiation therapy so far.8 This disease
of young adults was almost invariably fatal in the past, due to severe coagulopathy, until the
1

revolutionary introduction of ATRA (all–trans retinoic acid) as a treatment.9 The drug which is a
ligand of the RARα receptor component of the fusion protein, when given in supra-physiological
concentrations, is able to relieve the differentiation block posed by PML+RARα and effectively
induce white cell maturation, managing to achieve a relapse-free survival of about 90%,10 in
combination with low doses of traditional chemotherapy. The serious side-effects of non-targeted
standard chemotherapeutic drugs11,12 are thus avoided.
Apart from suppressing the uncontrolled proliferation of leukemia cells themselves, usual
chemotherapeutic drugs also result in suppression of the normal bone marrow, leading to anemia,
immune-compromise and bleeding disorders, which may result in fatality. AML is the leukemia
with the worst prognosis with average 10 year survival rates of around 10%.2,13,14 Since ATRA
works effectively only on the rare APL genetic subtype of AML, extensive research is being
directed towards causing differentiation in other types of immature leukemic cells.2,7,15,16 Initial
testing makes use of cell lines derived from leukemic patients which are easily available and
easily differentiated with positive controls chemicals (although these are usually too toxic to be
used clinically) and therefore are used as common standards across many research laboratories.
Minoxidil (MNX) is a vasodilator primarily marketed as a drug for the reduction and
slowing of androgenetic alopecia.17 It exhibits its effect by a calcium blocking capacity and may
also stimulate the production of vascular endothelial growth factor (VEGF).18 It is not known to
have any recognized cytotoxicity at micromolar concentrations although some non-toxic
proliferation of fibroblasts and keratinocytes has been reported.19-21 One of the advantages of
20
using MNX as a target for chemical modification is its ease of entrance into cells without the
need of a recognized receptor-dependent pathway.22 On the other hand, natural polyamines (PAs)
such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are known to play an
important role in the processes of differentiation and proliferation in leukemia,23,24 due to their
effects on the ornithine decarboxylase pathway.
In connection with our studies towards the antioxidant activity of MNX-PA conjugates25
and given that certain antioxidants exhibit also antiproliferative activity,26 we tested the effect of
two previously described MNX-PA conjugates, namely compounds 9 and 11,27 on the
differentiation and proliferation of the HL-60 leukemia cell line. The initial evaluation of these
compounds revealed induction of differentiation with compound 11, which prompted us to
2

further extent the study with eight new MNX conjugates (2-8 and 10, Fig. 1). With these
compounds we wanted to establish the role of certain structural modifications on the
differentiation-inducing activity of the resulting analogues. These modifications included: (a) the
presence of an amino group in the chain (conjugates 3 and 9), (b) the length of the aliphatic chain
(conjugates 9 and 10), (c) the nature of the side chain (aliphatic, aromatic or cyano- or amino-
group incorporating aliphatic chain) (conjugates 2 and 4-8), (d) additional substituted (with an
amino or methoxy group) or unsubstituted phenyl rings and the anticipated increase in
lipophilicity (conjugates 4 and 5-8), (e) the site of conjugation (conjugates 4-6 and 7-8), and (f)
the increase of the rigidity of the amine counterpart (conjugates 7 and 8).
N
N
N
N
N
N
N
N
O
O
R
H2N
N
H
H2N
NH2
H2N
N
N
N
H2N
N
O
N
O
N
N
O
N
H
H
H
O
O
4
MNX
1
2: R = CN
3: R = CH2CH3
NH2
R
N
N
N
N
N
N
O
O
O
H2N
N
N
NH2
H2N
N
N
O
H2N
N
H
N
N
H
N
O
N
O
H
H
H
H
R
5: R = H
6: R = OMe
7
8
N
N
N
N
O
O
H
NH2
x TFA
N
NH2
x 3TFA
H2N
N
N
H2N
N
N
N
H
N
O
N
O
n
H
H
H
H
9: n = 1
10: n = 3
11
Figure 1. Structures of compounds related to the present work.
The synthesis of the new compounds is depicted in Scheme 1. MNX was activated with N,N’-
carbonyldiimidazole in DMF to give MNX-derived acylating agent 1, according to published
procedure.27 This in turn was reacted with commercially available amines and N1-tritylhexane-
1,6-diamine to afford conjugates 2-8 and 12 in 40-60% yield. Conjugate 12, upon trifluoroacetic
acid (TFA)-mediated trityl (Trt) group removal, afforded conjugate 10 in 95% yield.
3

N
N
N
N
N
N
N
i
ii
iii
O
10
R
[12]
H2N
H2N
N
N
H2N
NH2
N
O
N
O
N
H
H
O
O
1
2-8, 12
MNX
OMe
CN
2: R =
7: R =
3: R =
4: R =
5: R =
6: R =
OMe
12: R = (CH2)6NH-Trt
10: R = (CH2)6NH2 x TFA
8: R =
NH2
NH2
Scheme 1. Synthesis of MNX conjugates 2-8 and 10. Reagents and conditions: (i) N,N’-
carbonyldiimidazole, DMF, 60 oC, 3h, 60%; (ii) R-NH2, DMF, 60 oC, 2.5-16h, 40-60%; (iii) 30%
TFA in CH2Cl2, 2h, RT, 95%.
The synthesized compounds were tested for their ability to induce differentiation using a
high passage HL-60 sub-line, which is very poorly responsive to ATRA-induced differentiation,
at the concentrations of 0.1, 1 and 10 μΜ for 3 and 5 days of exposure. Conjugates 5, 7, 8 and 11
showed an increased NBT/MTT differentiation index on HL-60 cells at 10 μM, similar to that
shown by a positive control both after 3 and 5 days of exposure (Fig. 2).
4

Figure 2. Graph depicting differentiation of HL-60 cells exposed to MNX and MNX
conjugates 2-11, as well as to positive and negative controls for 3 and 5 days. PMA: phorbol
myristate acetate, DMSO: Dimethyl Sulfoxide. Each value is an average of three replicates for
each of two independent experiments. Each data point is the measure of NBTZ reduction divided
by MTT activity to give a proxy for respiratory burst activity (a differentiation marker) per cell
and equalized to the value of the cells in unexposed media (RPMI). Error bars are calculated
using standard error for each data point.
Compound 10 was only active at day 3, but not at day 5 of differentiation, suggesting it
may induce monocytoid differentiation. ATRA does not have any effect on this late HL-60
passage and neither MNX itself elevates the NBT/MTT differentiation index, suggesting that the
unmodified MNX does not have any differentiation-inducing activity. MTT activity for all
conjugates showed approximately a four-fold drop both at day 3 and 5 of differentiation, as it is
often expected in differentiating cells, due to the reduction of cell proliferation associated with
differentiation (Table 1; IC50 values).
Table 1. IC50 and Clog P values for MNX and MNX-conjugates 2-11 on HL-60 myeloid
leukemia cells.
Clog Pa
Compound
Amine counterpart
IC50 μΜ (±SD)
> 10
0.02
-0.02
1.88
2.06
3.41
3.25
3.04
2.91
-0.01
1.05
-0.26
MNX
3-aminopropionitrile
n-butylamine
> 10
2
3
> 10
benzylamine
> 10
4
benzhydrylamine
4,4’-dimethoxybenzhydrylamine
4,4’-methylenedianiline
2,7-diaminofluorene
PUT
3.05 ± 0.06
> 10
5
6
3.02 ± 0.08
3.04 ± 0.06
> 10
7
8
9
1,6-diaminohexane
3.30 ± 0.10
10
SPM
3.02 ± 0.02
11
aC-QSAR Database, Biobyte Corp., 201 West 4th Str., Suite 204, Claremont CA, California 91711, USA.
5

The potent compounds 5, 7, 8, 10 and 11 were also tested on peripheral blood mononuclear cells
(PBMCs) to see if they would show any cytotoxic activity at the concentrations effective on HL-
60 leukemia cells. In contrast to the clearly toxic effect of 10% DMSO, no cytotoxic effects for
these compounds were detectable on human PBMCs from healthy donors (Fig. 3), at the doses
noted to increase the differentiation index.
Regarding structure-activity relationships based on the biological evaluation results, we note that
conjugates incorporating the amines β-aminonitrile (conjugate 2), n-butylamine (conjugate 3),
benzylamine (conjugate 4) and PUT (conjugate 9) exhibited very low activity. When PUT was
replaced by 1,6-diaminohexane (conjugate 10), or SPM (conjugate 11) the activity of the
conjugate was much improved. In addition, when benzylamine was replaced by
benzhydrylamine (conjugate 5) the conjugate showed comparable activity with the conjugate 11
of SPM. However, when the replacement involved a 4,4’-dimethoxybenzhydrylamine
moiety (conjugate 6) the activity was dramatically decreased, suggesting that methoxy
substituents on the aromatic rings should be avoided. Slightly better results were obtained when
benzhydrylamine was replaced by the aromatic amine MDA (conjugate 7) or its
conformationally restricted analogue, namely the aromatic amine DAF (conjugate 8). Although
three of the most active (IC50 3.02-3.05 μM) conjugates 5, 7 and 8 are associated with high Clog
P values (2.91-3.41), the much less lipophilic (Clog P = 1.18) MNX-SPM conjugate (11)
presents equipotent differentiation activity to MNX-MDA conjugate (7) and the second most
lipophilic conjugate 6 (Clog P = 3.25) has very low activity (IC50 > 10 μM). These results
suggest that lipophilicity seems not to be a prerequisite for the differentiation induction potential.
6

Figure 3. Graph depicting the cell growth curves of peripheral blood mononuclear cells from
normal individual volunteers exposed to MNX-conjugates 5, 7, 8, 10 and 11. Each data point is
an average of the MTT values of three separate replicates. 1.6% DMSO is a normal
differentiation control, whilst 10% DMSO is a toxic control. Error bars are calculated using
standard error for each data point.
In order to establish our initial observation that conjugation is indeed necessary for
improving the biological profile of MNX and also to exclude the involvement of the amine
counterpart, two of the most active compounds, conjugates 7 and 8, were compared against co-
administered unconjugated MNX and the corresponding amine counterparts 4,4’-
methylenedianiline (MDA) and 2,7-diaminofluorene (DAF). Interestingly, such co-treatments as
well as treatments with the amines MDA and DAF alone, did not show any particular
differentiation or cytotoxic activities at 10 μM, in contrast to compounds 7 and 8 (Fig.4).
7

Figure 4. Graph depicting differentiation of HL-60 cells exposed to MNX conjugates 7 and 8 as
well as the parent compound MNX and co-dosage with MNX and the conjugated amines for 3
days. Each value is an average of three replicates. Each data point represents the measure of
NBTZ reduction divided by MTT activity to give a proxy for respiratory burst activity (a
differentiation marker) per cell and equalized to the value of the cells in unexposed media
(RPMI). Error bars are calculated using standard error for each data point. Negative and positive
controls are also included.
The HL-60 cells exposed to compounds 7, 8 and 10 were also assessed for morphological
evidence of differentiation after staining with Leishman’s stain. Differentiation was also
morphologically recognizable in addition to the NBT/MTT ratio. HL-60 cells exposed to the
MNX-conjugates 7, 8 and 10 can be compared to those differentiated with the positive controls
DMSO and PMA as well as with undifferentiated HL-60 cells. Cells treated with the parent
compound MNX are also shown (Fig. 5).
8

a
d
b
e
c
f
g
h
Figure 5. Images taken after cytocentrifugation of cells exposed to different compounds for 3
days onto slides. Each slide is then air dried for fixing and stained with Leishman’s stain. Images
are at 40x magnification. a. RPMI medium alone. b. MNX, 10 μM. c. MDA, 10 μM. d.
compound 7, 10 μM. e. compound 8, 10 μM. f. compound 10, 10 μM. g. PMA, 10 μM. h. 1.6%
DMSO,
It is apparent from this figure that MNX alone, as well as MDA (counterpart amine of 7), have
very little effect on differentiation and the cells retain euchromatic nuclear material and large
nuclei. On the contrary, conjugates 7, 8 and 10 cause a number of changes characteristic of
differentiation, namely the reduction in cell number, the condensation of nuclear chromatin and
the irregularity of nuclear and cytoplasmic shape in some cells. Despite showing very high
NBT/MTT ratios they do not show the complete differentiation induced by DMSO and PMA.
This may be explained by the fact that oxidative burst enzymatic activity capable of reducing
NBT is an earlier marker of differentiation, being already highly expressed in cells just beyond
the promyelocyte stage of maturation and long before the final band cell and granulocyte stage28.
It can be also noted that regarding HL-60, being leukemia cells and not classical myeloid blasts,
9

even agents producing near full differentiation do not always produce classical looking
granulocytes and macrophages.
Regarding the mechanism through which this differentiation induction is being caused is at
present unclear. Natural PAs has been shown to be involved both in differentiation and in
differentiation-blocking, particularly where reduction of SPD and PUT as well as the
accumulation of SPM block HL-60 differentiation by multiple inducers.29 -32 Analogs of SPM
3031
have actually been shown to be effective cytotoxic agents in the K562 Chronic Myeloid
Leukemia cell line.33
One of the mechanisms through which many molecules can induce differentiation in myeloid
leukemia cells is through the induction of DNA breaks.34 This activity has already been
confirmed for the amine counterpart of conjugate 7, that is MDA.35 In fact, MDA has been
shown to be carcinogenic in some animal studies when added at 150 to 300 parts per million in
drinking water for rodents.36 However, whilst increasing the risk of certain tissue cancers, which
could relate to their potential for DNA damage, the same study also showed that MDA produced
reduction in leukemia risk, which agrees with our results. Despite this fact which can be a cause
of concern, numerous other tissues exposed to MDA in other animal studies at lower dosages,
including normal peripheral blood lymphocytes, do not show any marked toxicity or DNA
adduct formation, despite adduct formation in blood proteins.37 This correlates well with the lack
of toxicity seen here on the peripheral blood mononuclear cells. However, changes in PUT and
SPM levels (amine counterparts in MNX conjugates 9 and 11, respectively) are also known to be
involved in the differentiation of other cells types, including keratinocytes,38 suggesting that the
effect can not be purely associated to the DNA damage in the present compounds.
It should be noted that the reduction of SPM in HL-60 cells, induced to differentiate with PMA,
was not affected by inhibitors of the enzymes involved in the normal biosynthetic polyamine
pathway (including ornithine decarboxylase). On the other hand, major changes in activity of
these enzymes did not either occur with PMA differentiation, despite the changes in intracellular
polyamine levels.36 It is thus suspected that other pathways involving the PAs are being affected
in this differentiation and in fact these pathways might well be where the current MNX
conjugates are being involved. In complementary research, increasing SPM levels artificially in
the cells, abrogates myeloid leukemia cell differentiation.39
10

Conjugate 11 might possibly act as a polyamine analog activating spermine oxidase which
catabolizes SPM40. Upregulation of the catabolic oxidase has been seen both with PA analogs
and other compounds41. Thus reduction of SPM levels due to the presence of conjugate 11 might
result in differentiation induction. Since H2O2 is also produced by this reaction, this might play a
role in causing DNA breaks, also resulting in differentiation.
Furthermore, it has been shown that the N1-acetylated 1,6-diaminohexane is deacetylated upon
cell entry.42 1,6-diaminohexane, which is the amine counterpart of conjugate 10, is known to
have a strong differentiation effect on erythroleukemia. Thus, conjugate 10 might act in a similar
manner, resulting in this case in myeloid differentiation.
In conclusion, it is clear that conjugation of MNX to selected amines and PAs can result in
strong differentiation-inducing agents, which may offer methods of treatment for resistant forms
of AML. It appears that the best candidates should contain as amine counterpart an aromatic
amine connected through a methylene group to another aryl moiety or a long-chain PA. Further
work along the line of preparing a more extended library of MNX-amine and -PA conjugates,
taking into consideration our preliminary results, as well as studies towards the elucidation of the
mechanism(s) of differentiation induction caused by these compounds, are now in progress.
Acknowledgements
The authors wish to acknowledge the help of the COST action CM 1106 and especially Prof.
Nadine Martinet from the University of Nice-Sophia Antipolis, France for establishing this
collaboration. Dr. A. Leo and Biobyte Corp. (201 West 4th Str., Suite 204, Claremont CA
California, 91711, USA) are also acknowledged for free access to the C-QSAR program.
Supplementary data
Supplementary data (experimental procedures and characterization data for all
1
compounds, as well as copies of H and 13C NMR spectra) associated with this article can be
found in the online version at http://
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