Synthesis and antikinetoplastid activity of a series of N,N×-substituted diamines

Article abstract of DOI:10.1016/j.bmcl.2011.12.101

A series of 25 N,N×-substituted diamines were prepared by controlled reductive amination of free aliphatic diamines with different substituted benzaldehydes. The library was screened in vitro for antiparasitic activity on the causative agents of human Afr

Full text of DOI:10.1016/j.bmcl.2011.12.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1712–1715
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antikinetoplastid activity of a series of N,N0-substituted diamines
Andrea P. Caminos a, Esteban A. Panozzo-Zenere a, Shane R. Wilkinson b, Babu L. Tekwani c,
Guillermo R. Labadie a,
⇑
a Instituto de Química Rosario (IQUIR-CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK, Rosario, Argentina
b Queen Mary, University of London, Mile End Road, London E1 4NS, UK
c National Center for Natural Products Research & Department of Pharmacology, School of Pharmacy, University of Mississippi, MS 38677, USA
a r t i c l e i n f o
a b s t r a c t
A series of 25 N,N0-substituted diamines were prepared by controlled reductive amination of free ali-
phatic diamines with different substituted benzaldehydes. The library was screened in vitro for antipar-
asitic activity on the causative agents of human African trypanosomiasis, Chagas’ disease and visceral
leishmaniasis. The most potent compounds were derived from a subset of diamines that contained a
4-OBn substitution, having a 50% parasite growth inhibition in the submicromolar (against Trypanosoma
cruzi) or nanomolar (against Trypanosoma brucei and Leishmania donovani) range. We conclude that mem-
bers of this series of N,N0-substituted diamines provide new lead structures that have potential to treat
trypanosomal and leishmanial infections.
Article history:
Received 6 November 2011
Revised 18 December 2011
Accepted 20 December 2011
Available online 28 December 2011
Keywords:
Leishmaniasis
Chagas’ disease
Human African trypanosomiasis
Diamines
Ó 2011 Elsevier Ltd. All rights reserved.
Reductive amination
Neglected diseases are prevalent throughout the tropics and
subtropics, representing a major public health problem in regions
of the world least able to deal with the associated economic bur-
den. In some cases, cost effective therapies are available, for exam-
ple lymphatic filariasis can be treated at a cost as low a $2 per
patient per episode,1 but for most infections this is not the case.
In these situations the current regimes are failing being beset with
a series of problems ranging from toxicity and lack of efficacy
through to drug resistance and cost.2 This is exemplified by kineto-
plastid infections such as Chagas disease, leishmaniasis and human
African trypanosomiasis (HAT)3 where front-line treatments are
based on heavy metal-based compounds or on agents that are
expensive. Against this backdrop there is urgent requirement for
new orally administered agents targeting such infections that are
cheap and present limited/no side effects.
Polyamine derivatives have been extensively studied as antican-
cer agents during the 90s,4 with the use of such scaffolds being eval-
uated to address whether polyamine metabolism was a potential
target for antiparasitic drugs.5 Despite the considerable number of
reports where different sets of substituted polyamine analogs have
been prepared,6 few systematic studies involving the synthesis of
substituted diamines have been performed. In a limited number of
cases the antiparasitic activityof such derivatives has been explored,
like the pioneering work of Bitonti et al. where the bis(benzyl)poly-
amine analog, MDL 27,695 was reported.7 More recently, libraries of
N,N-substituted diamines based on putrescine and 1,3-propanedi-
amine prepared by solid phase parallel synthesis8 and a series of
long chains N-monoalkylated diamines (n = 2–6).9,10 The former
agents are reported to show submicromolar activities against P. fal-
ciparum and L. donovani8 while the latter exhibit leishmanicidal8 and
trypanocidal10 activities. The examples mentioned lack the ade-
quate target validation, but based on their structure it has been
suggested that they interfere with polyamine metabolism.11 There-
fore, some of precedents described before have validated diamines
derivatives as an attractive scaffold for the development of new anti-
parasitic drugs, and were chosen as our starting template to prepare
new sets of analogs.
The backbone of the substituted N,N0-diamine derivatives gener-
ated here is shown in Scheme 1. We prepared a series of N,N0-diamine
derivatives that contained (1) different length carbon chains between
the two amino groups and/or (2) substitutions on the amine group;
the type of side chain incorporated onto the amine was guided by
H2N
NH2
n
H
N
H
N
a
R
R
+
n
CHO
n= 3, 4, 6, 8, 10
R= H; 4-OMe; 4-OBn;
3-OH,4-OMe
R
3-OMe,4-OBn
⇑
Corresponding author.
E-mail address: labadie@iquir-conicet.gov.ar (G.R. Labadie).
Scheme 1. Synthesis of N,N’-derivatives. Reagents: (a) (1) DCM/MeOH (3:1) reflux;
(2) NaBH4 DCM/MeOH (1:1).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.101

A. P. Caminos et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1712–1715
1713
Table 1
previous reports where the most of the biologically activity analogs
Structure and yields of N,N0-substituted diamines prepared
contained, but not exclusively,12 aromatic rings.13 Those rings were
introduced inthe formof benzyl groups for synthetic reasonsand also
because they will give the compounds more flexibility compared to
aniline analogs and will preserve the basicity of the amine, which is
necessaryforimpartingspecificphysiologicalfunctiontopolyamines.
The logical approach to synthesize the analogs requires a reduc-
tive amination between a diamine and an aldehyde. Initially, to
prepare the compounds the well established methodology using
sodium triacetoxyborohydride was employed.14 That methodology
has been successfully used to produce reductive amination of alde-
hydes in situ, without the intermediary step of imine formation.
Putrescine (1,4-diaminobutane) was used to test the reaction using
a 1:2.2 ratio between diamine and benzaldehyde. Under those con-
ditions a mixture of products was obtained which was composed
by N-Bn, N,N0-dibenzyl and the N,N,N0-tribenzyl 1,4-diaminobu-
tane. It was clear that after the N,N0- product was formed, a third
molecule of aldehyde reacted with the product instead of reacting
with the remaining free amine. Based on those results, we had to
move to the stepwise procedure that allows for a more controllable
diimine formation step. Using a procedure reported by Phanstiel
and co-workers15 putrescine was allowed to react with benzalde-
hyde in DCM/MeOH (3:1) overnight, with an aliquot evaporated
and analyzed by NMR revealing a complete diimine formation.
The solvent was evaporated and the intermediate redissolved in
DCM/MeOH (1:1) and reduced with sodium borohydride over-
night. The reaction provided exclusively the expected N,N0-diben-
zyl-1,4-butilendiamine in 73% yield. The reaction conditions did
not require further optimization based on the similar selectivity
and yield obtained when using other diamines and benzaldehydes.
Having found the adequate conditions,16 a collection of 25 prod-
ucts was prepared. The library has two sources of diversity; the
carbon chain length and the substituent on the aromatic ring.
We selected five linear diamines covering different chain lengths
(Scheme 1), with ‘n’ varying from n = 3 to n = 10 and five benzalde-
hydes with different degrees of substitution; being R = H, R = 4-
OMe, R = 4-OBn, R = 3-OH,4-OMe and R = 3-OMe,4-OBn. Isovanillin
(R = 3-OH,4-OMe) was chosen over vanillin (R = 3-OMe,4-OH) be-
cause different reports17 have shown vanillin to be less reactive
on reductive aminations and both share a similar arrangement of
substituents, being, in that sense, equivalent.
Compound
N
R
Yielda (%)
1
2
3
4
5
3
4
6
8
10
H
H
H
H
H
79
73
46
94
89
6
7
8
9
10
3
4
6
8
10
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
78
77
30
59
66
11
12
13
14
15
3
4
6
8
10
4-OBn
4-OBn
4-OBn
4-OBn
4-OBn
67
77
86
99
60
16
17
18
19
20
3
4
6
8
10
3-OH, 4-OMe
3-OH, 4-OMe
3-OH, 4-OMe
3-OH, 4-OMe
3-OH, 4-OMe
96
80
82
78
72
21
22
23
24
25
3
4
6
8
10
3-OMe, 4-OBn
3-OMe, 4-OBn
3-OMe, 4-OBn
3-OMe, 4-OBn
3-OMe, 4-OBn
50
84
81
88
89
a
After purification.
African green monkey kidney epithelial (Vero) cells. For each agent,
dose response curves were generated from which the concentration
of drug that inhibited cell growth by 50% (IC50) determined (Table
2).
Based on this data it is clear that some scaffolds represent new
potent antiparasitic structures, showing activity across all three
species tested with few showing signs of cytotoxicity toward the
mammalian line. In general, each of the five substituted diamines
sub-sets displayed a lower trypanocidal activity against T. cruzi
than towards the other parasites. For T. cruzi, 11 compounds exhib-
ited growth inhibitory properties below 10 lM with 8 having an
effect at sub-micromolar levels. In contrast, most scaffolds affected
T. brucei (21 out of 25) or L. donovani (17 out of 25) growth at
concentrations below the 10 lM cutoff with several having an
The synthesis of 25 compounds through this synthetic approach
provided the expected products in good yields (Table 1). The reac-
tivity of some aldehydes resulted in the reduction of the carbonyl
to the corresponding benzyl alcohol, resulting in difficulty in puri-
fication of these analogs.
The average yield of purified compounds with different carbon
chain lengths, and ordered by the substituent on the benzyl ring,
were: 76% for R = H, 62% for R = 4-MeO, 78% for R = 4-BnO, 82%
for R = 3-OH,4-MeO and 80% for R = 3-OMe,4-BnO; giving a global
average yield of 76% (Table 1).
It is well established that kinetoplastid parasites share many bio-
chemical and several life cycle features. In terms of their polyamine
biosynthetic pathways, these pathogens show both differences and
similarities.11 The variations make it extremely difficult to find com-
pounds that have potential to target all parasite species while the
similarities are sufficient, with some qualitative and quantitative
variations,11 to indicate that compounds designed to block specific
components could produce an inhibitory effect against the different
kinetoplastid parasites. Based on this assumption, we decided to as-
say all compounds against the etiological agents of those parasitic
diseases. Using well characterized growth inhibition assays,18 all
diamine compounds were tested for antiparasitic activity against
Trypanosma cruzi epimastigotes (Chagas’ disease), Trypanosoma bru-
cei bloodstream form (HAT) and Leishmania donovani promastigotes
(visceral leishmaniasis) and for mammalian cytotoxicity using
effect at the nanomolar range. This difference in the compound
susceptibilities may reflect differences in the parasites polyamine
biosynthetic systems: T. cruzi lacks a functional ornithine decar-
boxylase, therefore is auxotrophic for putrescine, while T. brucei
and L. donovani have capacity for de novo polyamine biosynthesis.5
For one diamine sub-set (16–20) addition of a hydroxyl group at
the 3-position on the benzyl side chain generally resulted in sev-
eral derivatives exhibiting no activity against any of the parasites
screened. Additionally those scaffolds that did have an affect
generally showed lower growth inhibitory properties than the
equivalent compounds in other sub-sets: compare the IC50 value
of 19 against T. brucei and L. donovani with the IC50 values obtained
with 4, 9, 14, and 24.
This anomaly may be explained when considering that the phe-
nol group increases the compound’s polarity therefore altering that
scaffolds ability to cross biological membranes. Incorporation of a
4-benzyloxy group onto the benzyl group results in a noticeable
enhancement of the growth inhibitory activity. In fact, compounds
11–15 with only a 4-benzyloxy substituent on the phenyl ring show
an average IC50 of around 1
lM toward T. cruzi and L. donovani and
below 0.40 M toward T. brucei. For this group of compounds, the
l
antiparasitic activity is generally dependent on the carbon
chain length with longer chains resulting in lower trypanocidal/

1714
A. P. Caminos et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1712–1715
Table 2
Growth inhibitory properties of N,N0-substituted diaminesa
Compound
T. cruzi
S.I.b
T. brucei
S.I.b
L. donovani
S.I.b
Vero cells
IC50
(l
M)
IC50
(l
M)
IC50
(
lM)
IC50 (lM)
1
2
3
4
5
>10
>10
>10
>10
8.17
—
—
—
—
>3
6.45
7.08
2.03
1.18
0.61
>3
>3
>8
>128
>22
11.01
18.63
13.83
10.17
1.75
>27
>1
>1
>1
>8
NC
NC
NC
NC
NC
6
7
8
9
>10
>10
>10
>10
2.30
—
—
—
—
>5
2.04
3.20
0.22
5.50
>10
>7
>5
60
>2
—
1.24
8.52
6.73
1.56
1.45
>12
>2
2
>5
>8
NC
NC
8.32
NC
NC
10
11
12
13
14
15
0.78
0.76
1.83
0.99
>10
8
8
>5
>9
—
0.062
0.097
0.14
0.20
0.62
97
60
>67
>45
>14
0.26
0.031
0.24
1.14
4.60
23
6.00
5.83
NC
NC
NC
187
>40
>8
>2
16
17
18
19
20
>10
>10
>10
>10
>10
—
—
—
—
—
>10
>10
>10
5.50
0.96
—
—
—
>2
>11
>100
>100
43.76
44.41
4.50
—
—
>0.3
>0.3
>2
NC
NC
NC
NC
NC
21
22
23
24
25
0.81
0.73
0.74
0.82
0.80
8
0.19
0.17
0.19
0.19
0.11
35
52
>44
24
>67
0.23
0.24
0.21
0.22
1.20
29
37
>40
21
>6
6.65
2.70
NC
2.70
NC
12
>11
6
>10
Amphotericin B
Pentamidine
Nifurtimox
0.32
6.20
2.10
1.70
NC = not cytotoxic at the maximum concentration tested (4.76 lg/mL).
a
Trypanocidal assays were run by quadruplicate, leishmanicidal and cytotoxic assays were run by triplicate.
S.I. = Selectivity index IC50 Vero cells/IC50 parasite.
b
O
leishmanical properties than compared with shorter arrangements
(Fig. 1). For example, for compound 15 (n = 10) IC50 values of >10,
H
N
N
H
0.62 and 4.6
respectively, whereas for compound 12 (n = 4), IC50 values of 0.76,
0.097 and 0.031 M were determined. In addition to the 4-benzyl-
oxy substitution, compounds 21–25 also contain a 3-methoxy
grouping. These are even more active than 11–15 showing an aver-
lM were recorded for T cruzi, T. brucei and L. donovani,
12
O
L. donovani IC50 = 31 nM SI=187
l
T. cruzi
IC50 = 760 nM SI=8
IC50 = 97 nM SI=60
T. brucei
age IC50 of 0.80
lM for T. cruzi, 0.17 lM for T. brucei and around
Figure 2. Compound with the best activity balance over the three parasites.
0.22 M for L. donovani. In contrast to 11–15, this last series seems
l
to have an activity that is independent of the chain length.
Based on growth inhibition and selectivity data, analog 12
(Fig. 2)19 seems to be the best compound against all three parasite
species and represents a lead structure for developing future anti-
parasitic drugs.
It has been reported that structurally similar compounds with
trypanocidal activity inhibits trypanothione reductase (TR).20
Studies to validate TR and other enzymes on the trypanothione
biosynthetic pathway as targets of the N,N0-disubstituted diamines
are currently underway and results will be reported elsewhere.
Figure 1. Antiparasitic activity profile of analogs with R = OBn (11–15).

A. P. Caminos et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1712–1715
1715
10. Giordani, R. B.; Araújo, D. P.; Duarte, M.; Zuanazzi, J. A.; Tasca, T.; De Almeida,
M. V. Biomed. Pharmacother. 2011, 65, 60.
Acknowledgments
11. (a) Heby, O.; Persson, L.; Rentala, M. Amino Acids 2007, 33, 359; (b) Clark, K.;
Niemand, J.; Reeksting, S.; Smit, S.; van Brummelen, A.; Williams, M.; Louw, A.;
Birkholtz, L. Amino Acids 2010, 38, 633.
12. Alkylated polyamines have also been reported as chemotherapeutic agents
including antiparasitic activity. A detailed revision can be found on: Casero, R.
A., Jr.; Woster, P. M. J. Med. Chem. 2001, 44, 1.
13. (a) O’Sullivan, M. C.; Zhoua, Q.; Lia, Z.; Durham, T. B.; Rattendi, D.; Lane, S.;
Bacchi, C. J. Bioorg. Med. Chem. Lett. 1997, 5, 2145; (b) Li, Z.; Fennie, M. W.;
Ganem, B.; Hancock, M. T.; Kobaslija, M.; Rattendi, D.; Bacchi, C. J.; O’Sullivan,
M. C. Bioorg. Med. Chem. Lett. 2001, 11, 251; (c) Oliveira, J.; Ralton, L.; Tavares, J.;
Codeiro-da-silva, A.; Bestwick, C. S.; McPherson, A.; Thoo Lin, P. K. Bioorg. Med.
Chem. 2007, 15, 541.
14. (a) Abdel-Magid, A. F.; Harris, B. D.; Maryanoff, C. A. Synlett 1994, 81; (b) Abdel-
Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J. Org.
Chem. 1996, 61, 3849.
15. (a) Gardner, R. A.; Belting, M.; Svensson, K.; Phanstiel, O. t. J. Med. Chem. 2007,
50, 308; (b) Kaur, N.; Delcros, J.-G.; Imran, J.; Khaled, A.; Chehtane, M.;
Tschammer, N.; Martin, B.; Phanstiel, O. t. J. Med. Chem. 2008, 51, 1393.
16. General procedure for diamine analogues preparation: To a solution of diamine
(1 equiv) in an anhydrous mixture of DCM/methanol (3:1), aldehyde (3 equiv
dissolved in the same mixture of solvents) was added. The mixture was
refluxed overnight, the solvent was evaporated and the crude di-imine was
dissolved in an anhydrous mixture of DCM/methanol (1:1). The solution was
cooled at 0 °C, and sodium borohydride (3 equiv) was added portionwise. The
reaction was allowed to warm to room temperature and stirred for additional
12 h. Excess of sodium borohydride was quenched acidifying with 10% HCl,
with subsequent addition of ammonium hydroxide in order to reach a basic
pH. Phases were separated and the aqueous phase extracted with DCM
(3 ꢀ 20 mL). Combined organic extracts were dried with sodium sulphate and
evaporated. Products were purified by flash chromatograph.
Financial support through CONICET (Consejo Nacionalde Investi-
gaciones Científicas y Técnicas, PIP 2009-0796) and Fundación Bun-
ge y Born (FBB 31/10) is gratefully acknowledged. We thank John
Trott for cytotoxicity assays and Surendra K. Jain for antileishmanial
assays. NCNPR, University of Mississippi is partially supported
through a cooperative scientific agreement No. 58-6408-2-0009
from USDA Agricultural Research Service. This investigation also re-
ceived financial support from the UNICEF/UNDP/WORLD BANK/
WHO Special Programme for Research and Training in Tropical Dis-
eases (TDR) to G.R.L. and Award # W81XWH-09-2-0093 by US Army
Medicalresearchand MaterielCommandtoB.L.T. WealsothankPro-
fessor Paulo Carvalho for critical reading of this manuscript and
insightful comments.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.101.
References and notes
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19. N1,N4-Bis(4-(benzyloxy)benzyl)butane-1,4-diamine (12): 1H NMR (CDCl3):
d = 7.42 (m, 10H, aromatic, 40-benciloxy), 7.22 (d, 4H, J = 8.7 Hz, C20–H), 6.93
(d, 4H, J = 8.7 Hz, C30–H), 5.05 (s, 4H, Ar-CH2-O), 3.71 (s, 4H, Ar-CH2-N), 2.62 (t,
4H, J = 6,6 Hz, N–CH2), 1.53 (m, 4H, C2–H and C4–H. 13C NMR (CDCl3): d = 157.8
(C40–O), 137.1 (C100–CH2–O), 132.9 (C10), 129.3 (CH, C300 and C500), 128.6 (CH,
C20 and C60), 127.9 (CH, C400), 127.5 (CH, C200 and C600), 114,8 (CH, C30 and C50),
70.1 (CH2, O–CH2), 53.4 (CH2, Ar-CH2-N), 49.2 (CH2, N–CH2), 27.9 (CH2, C2 and
C4). IR:
m
= 3294 (R-NH-R1); 3035 (Ar-H); 1167 (C–N–C); 1247 and 1015 (Ar-O-
C); 814 (aromatic) cmꢁ1. ESI-HRMS: calcd for C32H37N2O2 (M+H+) 481.2855;
found 481.2849.
20. (a) O’Sullivan, M. C.; Zhoua, Q.; Lia, Z.; Durham, T. B.; Rattendi, D.; Laneb, S.;
Bacchi, C. J. Bioorg. Med. Chem. Lett. 1997, 5, 2145; (b) Li, Z.; Fennie, M. W.;
Ganem, B.; Hancock, M. T.; Kobaslija, M.; Rattendi, D.; Bacchi, C. J.; O’Sullivan,
M. C. Bioorg. Med. Chem. Lett. 2001, 11, 251.