Glycosylated metal chelators as anti-parasitic agents with tunable selectivity

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Sangenito, A. de Azevedo Guedes, M. H. Branquinha, K. Kavanagh, J. McGinley, A. L. Santos and T.

Velasco-Torrijos, Dalton Trans., 2017, DOI: 10.1039/C6DT04615K.

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ISSN 1477-9226

PAPER

Joseph T. Hupp, Omar K. Farha et al.

Eﬃcient extraction of sulfate from water using

framework

a Zr-metal–organic

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Glycosylated Metal Chelators as Anti-Parasitic Agents with

Tunable Selectivity

Andrew Reddy, ^aLeandro Stefano Sangenito,^bArthur de Azevedo Guedes,^bMarta Helena

Branquinha,^bKevin Kavanagh,^cJohn McGinley,^dAndré Luis Souza dos Santos^*band Trinidad

Velasco-Torrijos ^*a

¹Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

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Trypanosoma cruzi and Leishmania amazonensis are the causative agents of Chagas’ disease and leishmaniasis,

respectively. These conditions affect millions of people worldwide, especially in developing countries. As such, there is an

urgent need for novel, efficient and cost-effective treatments for these diseases, given the growing resistance and side-

effects of current therapies. This work details the synthesis and evaluation of the anti-parasitic activity of novel amino- and

iminopyridyl metal chelators, their glycosylated derivatives and some of their metal complexes. Our results revealed the

potent and metal-dependent activity for the aminopyridyl compounds: Cu(II) complexes were most effective against T.

cruzi trypomastigotes, while Zn(II) complexes presented excellent activity against L. amazonensis promastigotes. In

addition, the compounds showed excellent selectivity indexes and very low relative toxicity as judged by in vitro and in

vivo

studies,

respectively,

using

RAW

macrophages

and

Galleria

mellonella

larvae

model.

million cases of leishmaniasis reported annually and is fatal if

left untreated.¹⁰VL is caused by the infection of the host’s

macrophages with Leishmania amastigotes following infection

with promastigotes which are transmitted by the female sand

fly.¹¹First line treatment typically employs pentavalent

antimonials such as sodium stibogluconate. However, there is

widespread resistance to these compounds which leads to the

use of amphotericin B as the preferred treatment for VL.^12-14

Unfortunately, the use of this drug in endemic regions is

limited by its cost, toxicity and difficulties of administration.¹⁵

These challenges demonstrate the urgent need for novel, safe

and cost-effective therapies for these diseases. In particular,

drugs with activity against multiple kinetoplastic parasites are

especially attractive due to their broad spectrum of activity.¹⁶

Metallodrugs are an emerging therapeutic approach in the

treatment of parasitic diseases.^17,18Accordingly, several metal

complexes have been investigated against CD and

leishmaniasis.^{4, 19-21}The combination of metal ions with drugs

and organic ligands can enhance their activity while at the

same time, reducing the toxicity and undesired side effects

that metal ions on their own could impart.^22-24

Introduction

Neglected tropical diseases (NTD) affect in excess of 1 billion

people in the most impoverished areas of the world, causing

more than 500,000 deaths every year.¹Of the major NTDs, the

kinetoplastid parasite diseases leishmaniasis and Chagas

disease (CD) are considered among the most challenging to

tackle due to their high mortality rates and extremely limited

treatment options.^{2, 3}

CD is a chronic, debilitating parasitic disease caused by

Trypanosoma cruzi and is transmitted by blood-sucking insects

of the subfamily Triatominae.⁴The disease is a significant

healthcare problem and is estimated to infect more than 10

million people worldwide and presents

a substantial

socioeconomic burden in addition to a high morbidity rate.¹

The current treatments for CD are nifurtimox and

benznidazole, both of which present undesirable side effects

and limited efficacy in chronic patients.^5,6Moreover, there is a

growing resistance of T. cruzi towards these treatments which

further necessitates novel therapeutics without cross

resistance with currently used drugs.^7,8

In this study, the synthesis of a series of amino- and

iminopyridyl ligands and some of their metal complexes is

reported. In addition, their anti-parasitic activity against L.

amazonensis promastigotes and T. cruzi trypomastigotes is

evaluated. With a view to further improve their selectivity and

bioavailability, the synthesis and assessment of the anti-

parasitic activity of some per-acetylated glucosyl analogues

has also been performed. Carbohydrate conjugation is an

attractive means of improving the selectivity of bioactive

molecules through a targeting strategy.^25,26However, the

Leishmaniasis is a group of tropical diseases resulting from

infection of parasites of the genus Leishmania and presents a

substantial healthcare problem worldwide.⁹Leishmaniasis

occurs in several forms, the most severe of which is visceral

leishmaniasis (VL) which accounts for nearly 500,000 of the 2

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h; (iii) CuCl₂.2H₂O, ethanol, rt, 16 h; (iv) Zn(ClO₄)₂.6H₂O, ethanol, rt; (v) 2-picolylamine,

ethanol, rt, 4-6 h; (vi) Cu(ClO₄)₂.2H₂O, ethanol, 5 h.

water-soluble character of saccharides can compromise their

ability to cross cellular membranes. Therefore, the use of

acetylated sugars, with increased hydrophobicity, which

undergo hydrolysis after administration is often preferred.²⁷

Thus, the glycosylation of metal chelators seems a logical

approach to enhance their therapeutic potential.²⁸

Results and discussion

Synthesis

The synthesis of the aminopyridyl ligands 2a²⁹and 2b (N₂

series, Scheme 1a) was achieved by the reductive amination of

aldehydes 1a and 1b ³⁰

respectively. The reduction of the

,

corresponding imines was carried out in situ since they were

found to be very sensitive to hydrolysis. The metal complexes

studied were obtained by the direct reaction of ethanolic

solutions of the ligands and metal salts. Complexes with Cu(II)

(1:1 complexes 3a and 3b) were formed from ligands 2a and

2b, while the complex of 2a with Zn(II) was formed in a 2:1

A

B

Fig. 1 X-ray crystal structures of 3a with atomic displacement parameters shown at

50% probability. Hydrogen atoms omitted for clarity.

The chloride ligands are coordinated symmetrically with Cu-Cl

bonds of 2.313 Å. The structure has a τ₅value of 0.07

corresponding to a square pyramidal geometry.³¹There is a

short range interaction of 3 Å between the Cu(II) coordination

centre and a chloride ligand of a neighbouring molecule.

The dimer on the other hand shows a bond length of 2.257 Å

for the axial Cu-Cl bond while the bridging Cu-Cl bonds are

asymmetrically elongated with lengths of 2.302 Å and 2.692 Å.

2a binds more tightly to the metal centre compared to the

mononuclear complex with lengths of 2.010 Å and 2.036 Å for

the Cu-N_pyrand Cu-N_amineinteractions and a slightly smaller

bite angle of 82.25°. This molecule has a τ₅value of 0.15 which

indicates a higher degree of distortion from the ideal square

pyramidal geometry than the mononuclear acetonitrile adduct

described above. As the elemental analysis of the compound

indicates the presence of a water molecule, it is likely that the

material obtained is a 5-coordinate complex analogous to the

acetonitrile adduct with water occupying the axial position in

place of the acetonitrile ligand.

ratio (complex 4).

Crystals suitable for X-ray crystallography of 3a were obtained

from a solution of the complex in acetonitrile. This was found

to contain two distinct 5-coordinate Cu(II) species; an

acetonitrile adduct [Cu(2a)(CH₃CN)Cl₂] (Fig. 1A) encapsulated

inside

a unit cell with a binuclear bridged species

[Cu₂(2a)₂Cl₂(μ₂-Cl)₂] (Fig. 1B) occupying the vertices (see also

ESI-1.1).

In the mononuclear species, the ligand 2a is coordinated in a

bidentate fashion with a bite angle of 82.60°. The Cu-N_pyrand

Cu-N_amineare bonded at distances of 2.025 Å and 2.046 Å

respectively, while acetonitrile adduct shows a Cu-N_MeCNbond

length of 2.302 Å.

We examined the stability of the Cu(II) complex 3a by

recording UV/Vis spectra of a solution of 3a in DMSO/water a

0 h, 24 h and 72 h. No appreciable changes were observed.

(Fig. 2.1-ESI) We also examined the stability of the Zn(II)

complex 4 4 in d₆-

by recording ¹H NMR spectra of a solution of

DMSO/D₂O a 0 h and 72 h (Fig. 2.2-ESI) and again, no

appreciable changes were observed.

Contrary to the N₂compounds, the imine ligands

corresponding to the N₂O series (Scheme 1b) were readily

prepared by the direct reaction of 2-picolylamine with

aldehydes 5a-d without the need for acid catalysis or

desiccating agents. The presence of the ortho hydroxyl group

provides a favourable hydrogen bond interaction with the

imine lone pair of electrons which results in the shifting of the

¹H NMR resonance of the ortho phenolic protons of imines 6b-

d

to 13-14 ppm from 11.4 ppm in the precursor aldehydes.

Scheme 1 Synthesis of a) aminopyridyl ligands (N₂series) and b) iminopyridyl (N₂O

series) ligands and some of their metal complexes. Reagents and conditions: (i) 2-

picolylamine, ethanol, MgSO₄, 80 ˚C, 4 h; (ii) NaBH₄, ethanol/acetic acid, -10 ˚C to rt, 16

This hydrogen bonding is also observed in the crystal

structures of 6a and 6c (ESI-1.2, 1.3) and confers an unusual

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stability on these imines and a remarkable resistance to activities comparable to the control. All of the compounds

hydrolysis, even when stored under ambient conditions. exhibited low toxicity towards RAW cells (Fig. 3.1-ESI) with

Preliminary investigations into the optimum experimental complex 3a presenting the highest toxicity (106.2 μM). In

conditions for the synthesis of metal complexes with ligands of contrast, the least toxic compound was Zn(II) complex which

the N₂O series focused on those for ligand 6a. The Cu(II) and exhibited CC₅₀above the highest concentration used

Zn(II) complexes, and , respectively, presented a 1:1 (400μM); however, given that this compound was found to

stoichiometry. However, it was observed by NMR spectroscopy hydrolyze in DMSO, it is likely that any activity observed is due

that Zn(II) complex was unstable in solution and hydrolyzes to hydrolysis products. These data suggests that both amino-

to the aldehyde 5a and the bis(2-picolylamine)zinc perchlorate and iminopyridyl derivatives are well tolerated by mammalian

complex . The identity of this compound was confirmed by cells.

NMR and X-ray crystallography of the crystals obtained from

the NMR sample of in d₈-THF (Fig. 2, see also ESI-1.4). Anti-T. Cruzi Activity

8

a

7

8

9

8

Interestingly, this adopts an unusual coordination geometry

with a ꢀ_ꢁ^ꢂvalue of 0.70 which suggests a distorted see-saw

structure (ꢀ_ꢁ^ꢂ: 0.64). This is consistent with previously reported

see-saw structures which exhibit ꢀ_ꢁ^ꢂvalues of 0.70 ±0.02.^32,33

This geometry has only been reported once in the literature

for a zinc compound which contained Zn(0)³⁴and, to the best

of our knowledge, it has not been yet reported for a Zn(II)

The anti-parasitic activity of the ligands and their metal

complexes was then evaluated. The library of compounds was

initially screened for inhibitory activity against the viability of

T. cruzi trypomastigotes at a concentration of 50 μM. Seven

compounds (2a-b, 3a-b, 4, 6b, 7) significantly reduced the

viability of T. cruzi. (Fig. 4A). The simple metal salts CuCl₂.2H₂O

and ZnCl₂showed similar activity to the control. All members

of the N₂series showed activity, while from the N₂O series,

complex. Complex

9 can also be synthesized by the direct

reaction of 2-picolyamine and Zn(ClO₄)₂.6H₂O. The NMR data

of the hydrolysis product was identical to that of the complex

only the glycosylated ligand 6b and the Cu(II) complex

7 were

active. All of the compounds were then tested at 10 μM (Fig.

4B) and the LD₅₀values of the most active compounds were

determined (Fig. 4C, Table 1). The Cu(II) complexes 3a-b (LD₅₀

values of 1.7 and 1.8 μM, respectively) showed significantly

better efficacy than the current clinical drug benznidazole

(LD₅₀value 3.8 μM) towards T. cruzi, while the non metallated

9

synthesized purposely. The analogous Cu(II) complex

7

exhibits a much greater stability in solution at room

temperature. However, the corresponding Cu(II) picolylamine

complex [Cu(2-picolylamine)₂(ClO₄)₂]³⁵can be formed by

refluxing the complex

7 for 24 h in ethanol, causing the

hydrolysis of the ligand. The 2-picolylamine ligands in the Cu(II)

picolylamine complex were reported to coordinate in an anti

fashion while crystallographic data showed the ligands in Zn(II)

ligands 2a-b and the Zn(II) complex

4 (LD₅₀values 4.6, 5.3, 5.2

μM, respectively) showed comparable activity.

N₂Series

OAc

O

complex 9 binding in a syn manner.

AcO

HO

O

N

H

N

OAc

N

H

N

2a

2b

H

H₂

O

Cl

N

HO

N

HO

Cu

.2ClO₄

OH

Cl

N

Zn

N

OAc

O

N

H

AcO

H

O

Cl

Cu

3a

Cl

OAc

4

N

H

3b

N2O Series

OAc

O

AcO

O

HO

OH

OAc

N

OH

Fig. 2 Molecular structure of 9 with atomic displacement parameters shown at 50%

probability. Hydrogen atoms omitted for clarity. Only symmetry unique atoms are

labelled. Perchlorate oxygen atoms are modelled in two positions with occupancies of

69/31%.

N

6a

6b

OAc

O

AcO

N

O

HO

N

OAc

N

Evaluation of in vitro cytotoxicity to macrophage cells

O

OH

O

OH

N

6c

6d

A library of eleven compounds (Fig. 3) was initially screened to

test the cytotoxicity of the compounds. They were

administered to murine macrophages and their viability was

estimated using the MTT assay in order to determine the

cytotoxicity concentration (CC₅₀). The simple metal salts

CuCl₂.2H₂O and ZnCl₂did not show significant toxicity, with

OH₂

Cu

HO

O

HO

O

Zn

N

.ClO₄

.ClO₄, 2H₂

O

N

8

7

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Fig. 3 Chemical structure of the amino- and iminopyridyl ligands and metal complexes

evaluated for anti-parasitic activity.

The effect of the active compounds on the morphology of T. Anti-L. amazonensis Activity

cruzi was then evaluated. Compounds from the N₂series (2a-b

,

The ability of the compounds to inhibit the proliferation of L.

amazonensis promastigotes was then evaluated. Firstly, the

compounds were screened at initial concentrations of 50 and

3a-b, 4) were administered to the parasite at their LD₅₀

concentrations and cell morphology was inspected by means

of optical microscopy after 24 h exposure (Fig. 5). Non-treated

trypomastigote cells retained their typical elongated shape, in

which the flagellum is attached along the length of the

parasite cell body, with an undulating membrane provided by

flagellum movement and an unaltered surface. The treatment

of trypomastigotes with the test compounds caused some

significant morphological changes when compared to the

typical appearance of non-treated parasites, including

rounding in shape with reduced cell size, swelling of the cell

body and shortening or loss of flagellum (Fig. 5).

10 μM, respectively (Fig. 6A, 6B). Only compounds 2a-b

and showed inhibitory effects on promastigote proliferation.

The Zn(II) complex from the N₂ligand series had the highest

activity, whilst the hydrolysis products of Zn(II) complex had

, 4, 6b

8

4

8

comparable activity to those of the glycosylated ligands 2b and

6b. As observed in the previously discussed biological assays,

the simple metal salts CuCl₂.2H₂O and ZnCl₂had no anti-

parasitic effect. The five most active compounds were then

tested across a range of concentrations to establish the IC₅₀

values (Fig. 6C, Table 1). All compounds showed dose-

dependent activities: the activity of free ligand 2a (IC₅₀= 25.3

μM) was greatly enhanced by the presence of Zn(II) in complex

4

(IC₅₀= 1.3 μM) and, to a lesser extent, by conjugation to

glucose, as evidenced by activity of 2b (IC₅₀= 7.1 μM). On the

other hand, the compounds from the N₂O series showed

similar activities, with IC₅₀values of 17.1 μM for the hydrolysis

products of Zn(II) complex

8 and 18.4 μM for glycosylated

ligand 6b. These results highlight the potential for

development of the active compounds, especially when

considering the severe toxicity of current treatments for

leishmaniasis.

Fig. 5 Effect of selected compounds on the morphology of T. cruzi trypomastigotes.

Giemsa-stained smears of trypomastigotes were prepared after incubation in the

presence of each compound at LD₅₀concentration for 24 h. Non-treated

trypomastigotes (control) present

a kinetoplast (k), a central nucleus (n) and a

flagellum (f) attached to the parasite cell body. Compound administration induced

numerous morphological changes, such as parasites becoming round in shape with

reduced cell size (arrowhead), swelling of the cell body (black thin arrow), and

shortening or loss of flagellum (white arrow). All images are shown in the same scale

(bar = 10 µm).

Fig.

4 Effects of amino/iminopyridyl derivatives on the viability of T. cruzi

trypomastigotes. (A) Viability rate of trypomastigotes (initial inoculum of 106 cells/mL)

at 37 °C in absence/presence of the compounds (50 µM). The parasites were counted

24 h after addition of the compounds; inset: control with the simple Cu(II) and Zn(II)

salts. (B) Compounds screened at 10 µM as described in A. (C) Cell death profile with

selected compounds at appropriated concentrations (0.1 to 10 µM). The viable

parasites were counted by Trypan blue exclusion and motility in a Neubauer chamber

after 24 h of incubation. Benznidazole was used as a positive control. Results are

expressed as number of viable cells. DMSO (solvent used), was used as a control and

did not affect the parasite viability (data not shown). Data shown are the mean ±

standard deviation of three independent experiments performed in triplicate. The

symbols represent the significant differences in relation to the control (P < 0.01).

As with T. cruzi, the L. amazonensis parasites were inspected

using light microscopy 72 h after the administration of each

compound at its IC₅₀value (Fig.7). Untreated promastigotes

exhibited their typical elongated shape with a long flagellum

emerging at the anterior end of the parasite. The treated

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used as a control and did not affect the parasite viability (data not shown). Data shown

are the mean ± standard deviation of three independent experiments performed in

triplicate. The symbols represent the significant differences in relation to the control (P

< 0.01).

parasites displayed significant changes to their morphology

when compared to these control cells. These changes include

loss of flagellum and a reduced cell size. Further morphological

alterations, such as a swelling and rounding of the cell, were

also observed.

Evaluation of in vivo cytotoxicity to Galleria mellonella larvae

Overall, these results indicate that the compounds from the N₂

The active compounds 2a-b, 3a-b, 4, 6a-b and 8 were also series (aminopyridyl derivatives) are more effective anti-

screened for toxicity against larvae of G. mellonella, a model parasitic agents than their corresponding iminopyridyl

organism used as a surrogate to probe the response of the derivatives (N₂O series). Considering the anti-T. cruzi activities,

human innate immune system.^36-39

Compounds were the selectivity indexes for the Cu(II) complexes 3a-b (62.5 and

administered at concentrations of 1, 10 and 100 μM and the 70.9, respectively, Table 1) are significantly higher than that

viability of the larvae was assessed at 24 and 48 h. All of the for reference drug benznidazole (47.9), currently used in the

larvae treated with the compounds survived and none of those treatment for CD. Other Cu(II) complexes with known drugs,

displayed any melanization of their cuticle, which is indicative like risedronate or levofloxacin^{40, 41}present IC₅₀values as low as

of an immune response (Fig. 3.2-ESI).

1.6 μM, however the corresponding selectivity indexes were as

high

Fig. 7 Effect of selected compounds on the morphology of L. amazonesis. Giemsa-

stained smears of promastigotes were prepared after incubation in the presence of

each compound at LD₅₀concentration for 72 h. Non-treated promastigotes (control)

present a kinetoplast (k), a central nucleus (n) and a flagellum (f) attached to the

parasite cell body. The treatment induced morphological changes, such as parasites

becoming round in shape with/or reduced cell size (arrowhead), swelling of the cell

body (black thin arrow) and loss of flagellum (white arrow). All images are shown in

the same scale (bar = 10 μm).

Fig.

6 Effects of amino/iminopyridyl derivatives on the proliferative rate of L.

amazonensis promastigotes. (A) The proliferation of promastigotes (initial inoculum of

105 cells/mL) was followed at 28 °C in absence/presence of the compounds (50 µM).

The parasites were counted 72 h after addition of the compounds; inset: control with

the simple Cu(II) and Zn(II) salts. (B) Compounds screened at 10 µM as described in (A).

(C) Cell proliferation profile with selected compounds at appropriated concentrations

(0.1 to 50 µM). The viable parasites were counted by Trypan blue exclusion and motility

in a Neubauer chamber after 72 h of incubation. Amphotericin B was used as a positive

control. Results are expressed as number of viable cells. DMSO (solvent used), was

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Table 1 Selectivity indexes (SI) of compounds exhibiting the highest antiparasiticactivit

The influence of the coordinated metal in the anti-parasitic

activities is more evident considering the results from the anti-

leishmanial assays. In this case, none of the Cu(II) complexes

evaluated was able to inhibit the proliferation of L.

amazonensis promastigotes. On the other hand, the Zn(II)

RAW

CC₅₀

T.

L.

RAW/T.

cruzi

RAW/L.

cruzi amazonensis

amazonensis

Compd

LD₅₀

[µM]

IC₅₀

SI

complex

4 showed a remarkably low IC₅₀value of 5.2 ꢀM

2a

2b

3a

3b

4

155.4

234.5

106.2

127.6

118.2

196.1

>400

4.6

5.3

1.7

1.8

5.2

-

25.3

7.1

-

33.8

44.2

62.5

70.9

22.7

-

6.1

33

-

together with a selectivity index of 90.9. Zn(II) species have

been used to treat cutaneous leishmaniasis as both nutritional

supplements and topical drugs.^{45, 46}Zn(II) complexes featuring

the 2-picolyamine binding motif have been recently reported

to inhibit the growth of L. major, with good activity in vivo.⁴⁷

We found that the hydrolysis products from complex 8,

-

1.3

18.4

17.1

-

90.9

10.7

>23.4

-

namely Zn(II) complex

some extent (17.1

9

, also showed anti-parasitic activity to

6b

ꢀ

M). Several modes of action for Zn(II)

8

-

complexes with anti-leishmanial properties have been

discussed in the literature: some compounds have been

reported to inhibit enzymes essential for the parasite’s

carbohydrate metabolism, such as those involved in the

Embden-Meyerhof pathway.⁴⁸Some studies have indicated

that Zn(II) complexes selectively recognize anionic cell surfaces

through a combination of electrostatic attraction and specific

Benznidazole

182.1

3.8

47.9

Amphotericin

B

37.4

-

0.012

-

3.116

as 19.⁴While there are some examples of Cu(II) complexes

capable of inhibiting the growth of T. cruzi, ^{24, 40-43}our results

represent a marked improvement in the development of

selective Cu(II)-based therapies for CD. It seems clear that

Cu(II) coordination enhances the anti-parasitic activity of the

ligands: a reduction in the IC₅₀value of the free ligand 2a from

coordination of the zinc cations with phospholipids in the cell

49, 50

membrane.

Given the changes in parasite morphology

effected by the Zn(II) complexes described herein resulting in

loss of the distinct shape of the parasite, a structural

component of the parasite membrane could be proposed as a

plausible target. However, in depth studies, including TEM

imaging, are necessary in order to ascertain this hypothesis. It

should be also noted that glucosylated ligands 2b and 6b

4.6

decrease); the glucosylated ligand 2b presents an IC₅₀value of

5.3 M while the corresponding Cu(II) complex 3b has 1.8

ꢀM to 1.7 ꢀM for complex 3a was observed (a 2.7-fold

showed significant anti-leishmanial activities (7.1 ꢀM and 18.4

M, respectively). The mechanism of action of these ligands

ꢀ

ꢀM

ꢀ

(a 2.9-fold decrease). This moderate improvement in the

activity suggests that metal complexation does not prompt the

synergistic effects as reported for some anti-trypanosomal

complexes. These effects are characterized by a dramatic

increase in ligand activity upon metal coordination.²³In our

case it seems more likely that Cu(II) provides additional affinity

for the target binding site(s), which would explain the slight

increase in activity compared to the ligands on their own. The

maybe multifunctional, and further investigations are required

in order to elucidate it. Finally, compounds 6c, in which the

phenolic hydroxyl has been alkylated and 6d, its glucosylated

analogue, showed low activity against both of the parasites

studied in this work. This highlights the importance of the

phenolic glycoside structural feature for the biological activity

of the compounds, suggesting that only glycosylated

compounds susceptible to the action of glucosidases would

show anti-parasitic activity.

Zn(II) complex

4, with a 2:1 stoichiometry, possesses

coordination environment and molecular geometry rather

different to the Cu(II) complexes, which may not be so

favourable for binding with the target and could therefore

Conclusions

account for the reduced activity (5.2

trypanosomal Cu(II) complexes reported in the literature bind

to DNA, which could lead to their anti-parasitic activity.^41,42

ꢀM). Some of the anti-

In summary, a collection of amino- (N₂) and iminopyridyl (N₂O)

ligands, their glucosylated analogues and some of their

divalent metal complexes have been synthesized and their

anti-parasitic activity towards T. cruzi and L. amazonensis

evaluated. The compounds from the N₂series were found to

be more active than their corresponding N₂O analogues. The

nature of the metal ion strongly influences the biological

activities of the complexes investigated: Cu(II) complexes 3a

and 3b showed potent activity against T. cruzi while the Zn(II)

A

common structural feature of these is the presence of a

dinitrogen chelating motif, such as 1,10-phenanthroline and

2,2’-bipyridine, which are known cytotoxic agents.^41-44

However, the compounds described herein lack the extended

aromatic planar surfaces required for efficient DNA

intercalation.The high tolerance of the N₂ligand scaffolds, as

evidenced by their high selectivity indexes (Table 1) and the G.

mellonella assays suggest that, from a structural perspective,

the compounds are not likely to effect high affinity DNA

binding. However, further studies would be necessary in order

to determine their mechanism of action.

complex

4 was most efficient at inhibiting the growth of L.

amazonensis. Moreover, the most potent compounds

presented remarkable selectivity indexes; in particular, the

anti-trypanosomal compounds were considerably higher than

6 | J. Name., 2012, 00, 1-3

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ARTICLE

the control drug currently in use for the treatment of CD. ice/acetone/NaCl bath under nitrogen. Acetic acid (0.12 mL,

These preliminary studies suggest that these compounds could 2.2 mmol) was added immediately followed by the dropwise

be extremely attractive candidates for further development as addition of a solution of sodium borohydride (0.33 g, 8.8

novel anti-kinetoplastid therapeutic agents.

mmol) in ethanol (30 mL). The solution was allowed to warm

to rt and was stirred for 16 h before being quenched by the

addition of water (20 mL). The mixture was heated to 80 ˚C for

30 min until the precipitation of boron salts ceased. The

precipitate was removed by filtration and the filtrate was

evaporated under reduced pressure. The resulting residue was

dissolved in water (20 mL) and aq NaHCO₃saturated solution

(30 mL) was added to adjust the pH to approximately 9. It was

then extracted with ethyl acetate (5 x 40 mL). The combined

organic layers were washed with brine (100 mL), dried

(Na₂SO₄) and the solvent was removed in vacuo: brown syrup

Experimental

Chemistry general methods

All chemicals purchased were reagent grade and used without

further purification unless stated otherwise. Dichloromethane

and acetonitrile were freshly distilled over CaH₂prior use.

Ethanol was dried over 3 Å molecular sieves, which were flame

dried, prior to use. Reactions were monitored with thin layer

chromatography (TLC) on Merck Silica Gel F₂₅₄plates.

Detection was effected by UV or charring in a mixture of 5%

sulfuric acid-ethanol. All glassware used for anhydrous

reactions was flame dried under vacuum prior to use. NMR

spectra were obtained on a Bruker Ascend 500 using the

residual solvent peak as internal standard. Chemical shifts are

reported in ppm. Flash chromatography was performed with

Merck Silica Gel 60. Microwave reactions were carried out

using a CEM Discover Microwave Synthesizer. Optical rotations

were obtained from an AA-100 polarimeter. [α]_ꢃvalues are

given in 10⁻¹cm²·g⁻¹. The melting points were obtained using a

Stuart Scientific SMP1 melting point apparatus and are

uncorrected. Purity was confirmed by elemental analysis using

a FLASH EA 1112 Series Elemental Analyzer with Eager 300

operating software. High resolution mass spectrometry

(HRMS) was performed on an Agilent-LC 1200 Series coupled

to a 6210 Agilent Time-Of-Flight (TOF) mass spectrometer

equipped with an electrospray source in both positive and

1

(0.91 g, 76%); H NMR (500 MHz, CDCl₃) δ 8.51 (d, J = 4.5 Hz,

1H, Pyr-H), 7.60 (t, J = 7.6 Hz, 1H, Pyr-H), 7.27 – 7.23 (m, 3H,

Pyr-H, Ar-H), 7.15 – 7.11 (m, 1H, Pyr-H), 6.91 (d, J = 8.4 Hz, 2H,

Ar-H), 5.24 (m, 2H, H-2, H-4), 5.12 (t, J = 9.3 Hz, 1H, H-3), 5.02

(d, J = 7.1 Hz, 1H, H-1), 4.24 (dd, J = 12.4, 5.3 Hz, 1H, H-6), 4.13

(dd, J = 12.4, 2.0 Hz, 1H, H-6’), 3.87 (s, 2H, PyrCH₂), 3.81 (ddd, J

= 7.6, 5.3, 2.0 Hz, 1H, H-5), 3.76 (s, 2H, ArCH₂), 2.94 (bs, 1H, N-

H), 2.03, 2.02, 2.00, 1.99 (each s, 3H, OAc). ¹³C NMR (126 MHz,

CDCl₃) δ 170.7, 170.4, 169.5, 169.4 (each C=O), 159.5 (Pyr-C),

156.2 (Ar-C), 149.4 (Pyr-CH), 136.6 (Pyr-CH), 129.7 (Ar-C),

128.6 (Ar-CH), 122.6 (Pyr-CH), 122.2 (Pyr-CH), 117.2 (Ar-CH),

99.5 (C-1), 72.9 (C-4), 72.2 (C-5), 71.4 (C-3), 68.5 (C-2), 62.1 (C-

6), 54.3 (PyrCH₂), 52.8 (ArCH₂), 20.85, 20.82, 20.77, 20.75

(each OAc). IR (film on NaCl): 3427, 2945, 3922, 1751, 1610,

1592, 1511, 1474, 1435, 1369, 1214, 1069, 1042, 908, 831,

761, 600 cm^-1. HRMS (ESI⁺): m/z calculated for C₂₇H₃₂N₂O₁₀+ H⁺

[M + H⁺]: 545.2135. Found: 545.2139. [α]^ꢄ_ꢃ^ꢄ: -0.07 (c 0.5,

dichloromethane). Elemental analysis calculated (%) for

C₂₇H₃₂N₂O₂: C 59.55, H 5.92, N 5.14. Found: C 59.27, H 6.10, N

5.03.

negative

(ESI+/−)

modes.

Magneꢀc

suscepꢀbility

Johnson

measurements were carried out at rt using

a

Cis-aquadichloro(N-[4-(hydroxyphenyl)methyl]-2-

Matthey Magnetic Susceptibility Balance with [HgCo(SCN)₄]

as reference. Infrared spectra were obtained as a film on NaCl

plates or as KBr disks in the region 4000–400 cm⁻¹on a Perkin

Elmer Spectrum 100 FT-IR spectrophotometer. Purity of tested

compounds was 95% or higher, as determined by elemental

analysis (results within 0.4% of theoretical values). Caution:

Although not encountered in our experiments, perchlorate

salts of metal ions are potentially explosive and should be

manipulated with care.

pyridinemethamino)copper (3a)

Compound 2a (438 mg, 2 mmol) was dissolved in ethanol (10

mL) and was added to a solution of CuCl₂.2H₂O (348 mg, 2

mmol) in ethanol (15 mL). A green precipitate was formed and

the reaction was allowed to stir at rt overnight. The solvent

was reduced by half in vacuo and was then cooled on ice for

30 min and a green solid was collected by vacuum filtration.

This was washed with cold ethanol and dried under vacuum:

Green solid (526 mg, 69%). Mp 178˚C. IR (KBr disk): 3495,

3377, 3198, 2970, 2918, 2873, 1610, 1516, 1484, 1444, 1234,

Synthesis

1217, 1049, 823, 768, 579, 510 cm^-1Magnetic moment: 1.78

.

Compounds 1b,³⁰2a ²⁹5b ⁵¹5c⁵²have been previously

,

B.M. Elemental analysis calculated (%) for C₁₃H₁₆N₂Cl₂O₂Cu: C

42.58, H 4.40, N 7.64. Found: C 42.66, H 4.39, N 7.30. (X-ray

crystallography, see section 4.1-SI).

reported. For optimized synthetic procedures and complete

characterization, see ESI.

Cis-dichloro(N-{[4-(2,3,4,6-tetra-O-acetyl-β-

glucopyranosyloxy)pheny]lmethyl}-2-

pyridinemethamino)copper (3b)

D-

N-{[4-(2,3,4,6-tetra-O-acetyl-β-D-

glucopyranosyloxy)pheny]lmethyl}-2-pyridinemethamine

(2b)

Compound 2b (294 mg, 0.54 mmol) was dissolved in ethanol

(10 mL) at rt and was added to a solution of CuCl₂.2H₂O (110

mg, 0.65 mmol) in ethanol (10 mL). The reaction was allowed

to stir overnight. The resulting green solution was allowed to

stand for 3 days. A green precipitate formed and was collected

1b (1 g, 2.2 mmol) and 2-picolylamine (0.24 mL, 2.2 mmol)

were stirred in ethanol (25 mL) with anhydrous MgSO₄(0.8 g,

6.6 mmol) at 80 ˚C for 4 h under a nitrogen atmosphere. The

mixture was filtered and cooled to -10 ˚C in an

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Journal Name

by vacuum filtration. This was washed with cold ethanol and 1123, 1044, 989 cm^-1. HRMS (ESI+): m/z calculated for

dried under vacuum: Green solid (216 mg, 59%). Mp 146-147 C₂₃H₂₉O₁₃+ H⁺[M + H⁺]: 513.1608. Found: 513.1609. [α]_ꢃ^ꢄꢅ: -

˚C. IR (KBr disk): 3456, 3233, 2998, 2941, 2887, 1753, 1628, 0.11˚ (c 1, methanol).

1610, 1512, 1486, 1447, 1431, 1376, 1231, 1045, 908, 769, 600 General procedure for imine ligand synthesis (N₂O series): 2-

cm^-1. Magnetic Susceptibility: 1.81 B.M. Elemental analysis picolylamine and the corresponding aldehyde (1 equiv) were

calculated (%) for C₂₇H₃₂N₂Cl₂O₁₀Cu: C 47.76, H 4.75, N 4.13. dissolved in ethanol (~0.3 M). The reaction mixture was

Found: C 47.46, H 4.85, N 3.82.

allowed to stir at rt until TLC analysis showed disappearance of

the starting material (~4-6 h). The solvent was reduced by half

in a rotatory evaporator and cooled on ice to enhance

Bis(N-[4-(hydroxyphenyl)methyl]-2-

Pyridinemethamino)zinc perchlorate monohydrate (4)

To

a

solution

of

N-[4-(hydroxyphenyl)methyl]-2- precipitation if required. The precipitate was collected by

pyridinemethamine 2a (397 mg, 1.8 mmol) in ethanol (10 mL) filtration, washed with cold ethanol and dried under vacuum.

was added a solution of Zn(ClO₄)₂.6H₂O (229 mg, 0.6 mmol) in 4-{[(2-methylpyridinyl)-E-imino]methyl}-benzene-1,3-diol

1

ethanol (10 mL) at rt to immediately yield a beige precipitate. (6a). Yellow solid (3.12 g, 95%). Mp: 159-160 ˚C. H NMR (500

This was stirred for 5 h and the precipitate was collected by MHz, CDCl₃) δ 8.60 (d, J = 4.9 Hz, 1H, Pyr-H), 8.25 (s, 1H,

vacuum filtration to yield a hygroscopic brown solid. This was C(=N)H), 7.77 (dd, J = 8.5, 6.9 Hz, 1H, Pyr-H), 7.42 (d, J = 7.8 Hz,

then lyophilized for 48 h to yield the product: Beige solid 1H, Pyr-H), 7.35 – 7.26 (m, 1H, Pyr-H), 6.87 (d, J = 8.4 Hz, 1H,

1

(lyophilized after filtration, 207 mg, 62%). Mp: 161-163˚C. H Ar-H), 6.29 (d, J = 2.2 Hz, 1H, Ar-H), 6.20 (dd, J = 8.4, 2.2 Hz, 1H,

NMR (500 MHz, CDCl₃) δ 8.11 (bs 1H) 8.03 (bs, 1H), 7.49 (bs, Ar-H), 4.83 (s, 2H, CH₂). ¹³C NMR (126 MHz, CDCl₃) δ 166.1 (Ar-

1H), 7.09 (bs, 2H), 6.77 (bs, 2H, Ar-H), 3.91 (s, 2H, Pyr-CH₂), C), 164.2 (Ar-C), 161.1 (C=N), 157.7 (Pyr-C), 148.9 (Pyr-CH),

3.64 (Ar-CH₂). ¹³C NMR (126 MHz, DMSO) δ 157.1 (Pyr-C), 137.8 (Pyr-CH), 133.4 (Ar-CH), 123.2 (Pyr-CH), 123.0 (Pyr-CH),

148.1 (Ar-C), 147.3 (Pyr-CH), 140.4 (Pyr-CH), 130.5 (Pyr-CH), 112.3 (Ar-C), 107.3 (Ar-CH), 103.3 (Ar-CH), 63.9 (CH₂). IR (KBr

130.2 (Ar-C), 124.1 (Ar-CH), 115.7 (Ar-CH), 56.6 (CH₂), 52.0 disk): 3435, 3064, 2763, 2676, 2618, 1621, 1442, 1331, 1271,

(CH₂). IR (KBr disk): 3430, 2935, 2968, 1613, 1574, 1517, 1489, 1120 cm^-1HRMS (ESI+): m/z calculated for C₁₃H₁₂N₂O₂[M⁺]:

.

1446, 1375, 1269, 1222, 1177, 1108, 1051, 928, 833, 764, 623, 228.0899. Found. 228.0905. Elemental analysis calculated (%)

509 cm^-1

Elemental analysis calculated (%) for for C₁₃H₁₂N₂O₂: C 68.41, H 5.30, N 12.27. Found: C 68.63, H

C₂₆H₃₀N₄Cl₂O₁₁Zn: C 43.93, H 4.25, N 7.88. Found: C 44.02, H 5.44, N 12.28. (X-ray crystallography, see section 4.2-SI).

.

4.49, N 8.20.

5-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-2-{[(2-

2-hydroxy-4-[2-(2,3,4,6-tetra-O-acetyl-β-

D

-

methylpyridinyl)-E-imino]methylphenol (6b).

1

Orange solid (1.12 g, 95%). Mp: 77-78˚C. H NMR (500 MHz,

glucopyranosyloxy)ethoxy]benzaldehyde (5d)

To solution of 2-chloroethyl 2,3,4,6-tetra-O-acetyl-

a

β

-D-

CDCl₃) δ 13.72 (s, 1H, OH), 8.58 (d, J = 4.9 Hz, 1H, Pyr-H), 8.45

glucopyranoside⁵³(1 g, 2.4 mmol) and sodium iodide (359 mg, (s, 1H, C(H) =N), 7.69 (td, J = 7.8, 1.9 Hz, 1H, Pyr-H), 7.34 (d, J =

2.4 mmol) in anhydrous acetonitrile (15 mL) was added a 7.8 Hz, 1H, Pyr-H), 7.21 (m, 2H, Pyr-H, Ar-H), 6.56 (d, J = 2.3 Hz,

solution of 2, 4-dihydroxybenzaldehyde (660 mg, 4.8 mmol) 1H, Ar-H), 6.50 (dd, J = 8.5, 2.3 Hz, 1H, Ar-H), 5.36 – 5.20 (m,

and DBU (0.359 mL, 2.4 mmol) in anhydrous acetonitrile (10 2H, H-1, H-2), 5.14 (m, 2H, H-3, H-4), 4.91 (s, 2H, CH_2-Pyr), 4.29

mL). The mixture was sealed in a microwave tube under argon (dd, J = 12.3, 5.7 Hz, 1H, H-6), 4.18 (dd, J = 12.3, 2.3 Hz, 1H, H-

and heated under microwave irradiation to 125 ˚C for 4 h. The 6’), 3.89 (ddd, J = 10.1, 5.7, 2.3 Hz, 1H, H-5), 2.10, 2.07, 2.05,

solvent was then removed in vacuo and the residue was 2.04 (each s, 3H, OAc). ¹³C NMR (126 MHz, CDCl₃) δ 170.8,

dissolved in dichloromethane (20 mL) and washed with water 170.3, 169.5, 169.4 (each C=O), 165.9 (C=N), 163.8 (Pyr-C),

(2 x 20 mL), aq Na₂S₂O₄1 M solution (20 mL), aq NaHCO₃160.2 (Ar-C), 157.9 (Ar-C), 149.6 (Pyr-CH), 137.0 (Pyr-CH),

saturated solution (2 x 20 mL), brine (20 mL) and dried 132.9 (Ar-CH), 122.5 (Pyr-CH), 122.0 (Pyr-CH), 114.6 (Ar-C),

(Na₂SO₄). The solvent was removed in the rotatory evaporator 108.2 (Ar-CH), 104.2 (Ar-CH), 98.3 (C-1), 72.8 (C-2), 72.3 (C-3),

to yield

a brown oil which was purified by column 71.1 (C-5), 68.3 (C-4), 64.6 (CH_2-Pyr), 61.9 (C-6), 20.8, 20.7,

chromatography (3:1 petroleum ether/ethyl acetate, R_f: 0.17): 20.7, 20.7 (each OAc). IR (KBr disk): 3472, 3053, 3017, 2965,

dark yellow oil (862 mg, 66%). ¹H NMR (500 MHz, CDCl₃) δ 2870, 1749, 1635, 1589, 1505, 1368, 1255, 1227, 1176, 1079,

11.39 (s, 1H, OH), 9.67 (s, 1H, HC=O), 7.40 (d, J = 8.7 Hz, 1H, Ar- 1056, 749 cm^-1_.HRMS (ESI+): m/z calculated for C₂₇H₃₀N₂O₁₁

+

H), 6.49 (dd, J = 8.7, 2.3 Hz, 1H, Ar-H), 6.36 (d, J = 2.3 Hz, 1H, H⁺[M + H⁺]: 559.1928. Found: 559.1895. [α]_ꢃ^ꢄꢆ-0.23˚ (c 2,

:

Ar-H), 5.18 (t, J = 9.6 Hz, 1H, H-3), 5.05 (t, J = 9.7 Hz, 1H, H-4), dichloromethane). Elemental analysis calculated (%) for

4.97 (dd, J = 9.6, 8.0 Hz, 1H, H-2), 4.61 (d, J = 8.0 Hz, 1H, H-1), C₂₇H₃₀N₂O₁₁: C 58.06, H 5.41, N 5.02. Found: C 58.24, H 5.42, N

4.22 (dd, J = 12.3, 4.7 Hz, 1H, H-6), 4.16 – 4.05 (m, 4H, H- 5.25.

6’,OCH, CH₂Cl), 3.91 (dt, J = 9.6, 3.5 Hz, 1H, OCH’), 3.69 (ddd, J 5-(2-hydroxyethoxy)-2-{[(2-methylpyridinyl)-E-

= 9.7, 4.7, 2.4 Hz, 1H, H-5), 2.03, 1.98, 1.95, 1.91 (each s, 3H, imino]methyl}phenol (6c). Yellow solid (1.22 g, 86%). Mp:

1

OAc). ¹³C NMR (126 MHz, CDCl₃) δ 194.4 (HC=O), 170.6, 170.2, 124˚C. H NMR (500 MHz, CDCl₃) δ 13.83 (s, 1H, Ar-OH), 8.58

169.4, 169.3 (each C=O), 165.7 (Ar-C), 164.4(Ar-C), 135.4 (Ar- (d, J = 4.9 Hz, 1H, Pyr), 8.41 (s, 1H, C(=N)H), 7.69 (td, J = 7.7, 1.8

CH), 115.4 (Ar-C), 108.4 (Ar-CH), 101.3(Ar-CH), 101.0 (C-1), Hz, 1H, Pyr), 7.35 (d, J = 7.7 Hz, 1H, Ar-H), 7.22 – 7.20 (m, 1H,

72.7 (C-3), 71.9 (C-5), 71.1 (C-4), 68.3 (C-2), 67.7 (C-6), 67.4 Ar-H), 7.18 (d, J = 8.9 Hz, 1H, Ar-H), 6.46 (m, 2H, Ar-H), 6.44 (d,

(CH₂OAr)), 61.8 (OCH₂), 20.7, 20.6 , 20.5, 20.5 (each OAc). IR J = 2.4 Hz, 1H, Ar-H), 4.89 (s, 2H, Pyr-CH₂), 4.18 – 4.06 (t, J = 4.9

(KBr disk): 3471, 2967, 1745, 1633, 1581, 1430, 1379, 1230, Hz, 2H, ArO-CH₂), 4.01 – 3.92 (t, J = 4.9 Hz, 2H, CH₂OH). ¹³C

8 | J. Name., 2012, 00, 1-3

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NMR (126 MHz, CDCl₃) δ 165.8 (C=N), 164.5 (Ar-C), 162.6 (Ar- OH). IR (KBr disk): 3437, 3335, 3278, 3090, 2954, 2011, 1611,

C), 158.1 (Pyr-C), 149.4 (Pyr-CH), 136.9 (Pyr-CH), 132.9 (Ar-CH), 1589, 1489, 1439, 1381, 1293, 1089, 1027, 625 cm^-1_.Mp: 107-

122.4 (Pyr-CH), 121.9 (Pyr-CH), 112.9 (Ar-C), 106.8 (Ar-CH), 109˚C Elemental analysis calculated (%) for C₁₃H₁₅N₂ClO₈Zn: C

101.9 (Ar-CH), 69.3 (ArOCH₂), 64.2 (Pyr-CH₂₎, 61.3 (CH₂OH). IR 38.07, H 3.20, N 8.65. Found: C 37.89, H 3.31, N 8.40. A

(KBr disk): 3406, 3269, 2943, 2927, 2896, 1630, 1596, 1179.5, satisfactory ¹³C NMR spectrum could not to be obtained due to

1138, 1096, 1044, 844, 763, 614 cm^-1HRMS (ESI+): m/z the hydrolysis of the compound over the time scale necessary

.

calculated for C₁₅H₁₆N₂O₃+ H⁺[M + H⁺]: 273.1239. Found: to obtain the data.

273.1233. Elemental analysis calculated (%) for C₁₅H₁₄N₂O₃: C Bis(2-picolylamine)zinc perchlorate (9). Zn(ClO₄)₂.6H₂O (723

66.54, H5.92, N 10.29. Found: C 66.35, H 6.05, N 10.26. (X-ray mg, 1.9 mmol) and 2-picolylamine (0.4 mL, 3.8 mmol) were

crystallography, see section 4.3-SI). stirred at rt in ethanol (20 mL) for 4 h and cooled on ice for 30

5-[2-(2,3,4,6-tetra-O-acetyl- -glucopyranosyloxy)ethoxy)-2- min. This yielded a white precipitate which was collected by

β-D

{[(2-methylpyridinyl)-E-imino]methyl}phenol (6d). Pale yellow vacuum filtration, washed with cold ethanol and dried under

solid (1.13 g, 78%). Mp: 118-119˚C. ¹H NMR (500 MHz, CDCl₃) δ vacuum: white solid (855 mg, 94%). Mp: Compound

13.80 (s, 1H, Ar-OH), 8.58 (d, J = 4.0 Hz, 1H, Pyr-H), 8.41 (s, 1H, decomposes at 245 °C. ¹H NMR (500 MHz, DMSO) δ 8.30 (d, J =

C(H)=N), 7.69 (td, J = 7.7, 1.8 Hz, 1H, Pyr-H), 7.35 (d, J = 7.8 Hz, 2.9 Hz, 2H, Pyr-H), 8.05 (t, J = 7.2 Hz, 2H, Pyr-H), 7.59 (d, J = 7.9

1H, Pyr-H), 7.21 (dd, J = 7.0, 5.4 Hz, 1H, Pyr-H), 7.19 – 7.14 (m, Hz, 2H, Pyr-H), 7.56 – 7.50 (m, 2H, Pyr-H), 4.08 (s, 4H, CH₂),

1H, Ar-H), 6.44 – 6.39 (m, 2H, Ar-H), 5.22 (t, J = 9.6 Hz, 1H, H- 3.91 (s, 4H, NH₂).¹³C NMR (126 MHz, DMSO) δ 157.4 (Pyr-C),

3), 5.10 (t, J = 9.7 Hz, 1H, H-4), 5.02 (dd, J = 9.6, 8.0 Hz, 1H, H- 146.8 (Pyr-CH), 139.7 (Pyr-CH), 123.8 (Pyr-CH), 123.5 (Pyr-CH),

2), 4.89 (s, 2H, CH₂-Pyr), 4.66 (d, J = 8.0 Hz, 1H, H-1), 4.27 (dd, J 42.6 (CH₂). IR (KBr disk): 3439, 3281, 3204, 3133, 2912, 1603,

= 12.3, 4.7 Hz, 1H, H-6), 4.19 – 4.06 (m, 4H, H-6’, OCH₂CH₂OAr, 1590, 1569, 1487, 1439, 1383, 1334, 1294, 1141, 1114, 1090,

OCHCH₂OAr), 4.01 – 3.86 (m, 1H, OCH’CH₂OAr), 3.72 (ddd, J = 1032, 1017, 940, 772, 636, 626 cm^-1. Elemental analysis

10.1, 4.7, 2.4 Hz, 1H, H-5), 2.08, 2.03, 2.00, 1.96 (each s, 3H, calculated (%) for C₁₂H₁₆N₄Cl₂O₈Zn: C 29.99, H 3.36, N 11.6.

OAc). ¹³C NMR (126 MHz, CDCl₃) δ 170.8, 170.4, 169.5, 169.5 Found: C 29.51, H, 3.32, N 11.45.

(each C=O), 165.8 (C=N), 164.5 (Ar-C), 162.5 (Ar-C), 158.2 (Pyr-

C), 149.5 (Pyr-CH), 136.9 (Pyr-CH), 133.1 (Ar-CH), 122.5 (Pyr- Biological assays

CH), 121.9 (Pyr-CH), 112.9 (Ar-C), 106.8 (Ar-CH), 101.8 (Ar-CH),

Cultivation of mammalian cells. The LLC-MK₂epithelial cells

101.2 (C-1), 72.8 (C-3), 72.0 (C-5), 71.2 (C-2), 68.4 (C-4), 68.1

and RAW 264.7 murine macrophages were maintained in

(CH₂OAr), 67.2 (OCH₂), 64.2 (CH₂-Pyr), 61.9 (C-6), 20.8, 20.7,

Dulbecco’s modified Eagle’s medium (DMEM) supplemented

with 10% of fetal bovine serum (FBS) at 37 ^°C in an atmosphere

20.7, 20.7 (each OAc). IR (KBr disk): 3438, 2964, 2935, 2885,

1744, 1630, 1591, 1518, 1433, 1370, 1254, 1224, 1178, 1141,

1093, 1046, 981, 846, 606 cm^-1HRMS (ESI+): m/z calculated

containing 5% CO₂.

.

Murine Macrophage Viability Assay. The effect of each

for C₂₉H₃₄N₂O₁₂+ H⁺[M + H⁺]: 603.2190. Found: 603.2177.

compound on RAW 264.7 murine macrophages viability was

evaluated by MTT assay. Firstly, macrophage cells (10⁵/mL)

[α]^ꢄ_ꢃ^ꢆ-0.16˚ (c 2, dichloromethane). Elemental analysis

calculated (%) for C₂₉H₃₄N₂O₁₂: C 57.80, H 5.69, N 4.65. Found:

:

were allowed to adhere in 96-well tissue culture plates for 24 h

°

at 37 C, in a 5% CO₂atmosphere. Non-adherent cells were

C 58.04, H 5.68, N 4.65.

General procedure for metal complexation (N₂O series): The

removed by washes with PBS and the wells refilled with DMEM

imino ligand was dissolved in ethanol (~2M) and added to a

medium supplemented with 10% FBS. The macrophages were

solution of the corresponding salt (0.5 equiv for

, ~2M) in ethanol. The reaction mixture was allowed to reflux

for 5 h (for complex ) or 3 h (for complex ) under nitrogen.

7, 1 equiv for

then incubated with increasing concentration of the test

compounds (0.39 to 400 µM) and incubated for additional 24 h

°

at 37 C, in a 5% CO₂atmosphere. Subsequently, the culture

8

7

8

The solvent was reduced by half in a rotatory evaporator and

cooled on ice to enhance precipitation. The precipitate was

collected by filtration, washed with cold ethanol and dried

under vacuum.

medium was discharged and the formation of formazan was

measured by adding MTT (5 mg/mL in PBS, 50 µg/well) and

°

incubating the wells for 3 h in the dark at 37 C. The plates

were subsequently centrifuged at 1300 rpm for 8 minutes, the

supernatant was removed, the pellet was dissolved in 200 mL

of DMSO and the absorbance measured in an ELISA reader at

570 nm (Bio-Tek Instruments). The 50% cytotoxicity inhibitory

concentration (CC₅₀) was determined by linear regression

analysis.

Aqua(5-hydroxy-2-{[(2-methylpyridinyl)-E-

imino]methyl}phenoate)copper perchlorate dihydrate (7)

.

Green solid (158 mg, 40%). Mp: 189-191 ˚C. IR (KBr Disk): 3454,

3208, 3121, 2938, 2913, 2021, 1636, 1615, 1541, 1490, 1334,

1231, 1170, 1057, 625 cm^-1_.Magnetic Susceptibility: 1.77 B.M.

Elemental analysis calculated (%) for C₁₃H₂₃N₂ClO₉Cu: C 35.14,

H 3.86, N 6.31. Found: C 34.96, H 3.75, N 6.79.

Parasites and cultivation. Tissue culture trypomastigote forms

of T. cruzi Y strain, which was isolated from a human case,

were obtained from the supernatants of infected LLC-MK₂

(5-hydroxy-2-{[(2-methylpyridinyl)-E-

imino]methyl}phenolate)zinc perchlorate monohydrate (8)

.

epithelial cells after

5 days of incubation in DMEM

Yellow solid (859 mg, 65%, stored under argon). Mp: 107-

109˚C. ¹H NMR (500 MHz, CD₃CN) δ 8.52 (bs, 2H Pyr-H,

HC(=N)), 8.11 (bs, 1H, Pyr-H), 7.61 (bs, 2H, Pyr-H), 7.44 (bs, 1H

Ar-H), 6.60 (bs, 2H, Ar-H), 5.08 (bs, 2H, CH₂), 3.81 (bs, H₂O,

supplemented with 2% heat-inactivated fetal bovine serum

°

(FBS) at 37 C in a humidified 5% CO₂. L. amazonensis (Josefa

strain) promastigotes were maintained by weekly transfers in

J. Name., 2013, 00, 1-3 | 9

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25-cm³culture flasks with Schneider’s insect medium, pH 7.0, statistically by means of Student’s t-test using EPIINFO 6.04

supplemented with 10% FBS at 28 ^°C.

(Database and Statistics Program for Public Health) computer

T. cruzi viability. The effects of the compounds (Fig. 1) on T. software. P values of 0.05 or less were considered statistically

cruzi trypomastigotes were assessed by incubation of the significant.

parasites in DMEM with 2% heat-inactivated FBS. Briefly,

trypomastigotes were counted using a Neubauer chamber and

resuspended in fresh medium to a final concentration of 10⁶

viable cells per mL. The viability was assessed by motility and

lack of Trypan blue staining. Each compound was added to the

culture at final concentrations ranging from 0.1 to 50 µM.

After incubation for 24 h at 37 ^°C, the number of motile

parasites was quantified under light microscopy. The 50%

lethal dose (LD₅₀) was determined after 24 h by linear

regression. The simple salts ZnCl₂and CuCl₂and

dimethylsulfoxide (DMSO) were used as appropriated controls.

L. amazonensis viability. The effects of the compounds (Fig. 1)

on L. amazonensis promastigotes were assessed by incubation

Acknowledgements

We would like to thank Dr Malachy McCann (Maynooth

University) for his advice and guidance and Dr Brendan

Twamley (Trinity College Dublin) for his help with X-ray

crystallography. We would like to acknowledge Maynooth

University for the provision of the John and Pat Hume

Scholarship (A. Reddy).

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Table of contents

Metal complexation imparts selective anti-parasitic activity to aminopyridyl ligands: Zn(II) and Cu(II)

complexes show potent activity and remarkable selectivity indexes.

Article Doi

Glycosylated metal chelators as anti-parasitic agents with tunable selectivity

DOI: 10.1039/c6dt04615k

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