Structural characteristics of chloroquine-bridged ferrocenophane analogues of ferroquine may obviate malaria drug-resistance mechanisms

Article

pubs.acs.org/jmc

Structural Characteristics of Chloroquine-Bridged Ferrocenophane

Analogues of Ferroquine May Obviate Malaria Drug-Resistance

Mechanisms

†

†,‡

†

Paloma F. Salas, Christoph Herrmann, Jacqueline F. Cawthray, Corinna Nimphius,

†

§

§,∥

Alexander Kenkel, Jessie Chen, Carmen de Kock, Peter J. Smith, Brian O. Patrick,

Michael J. Adam, and Chris Orvig*

⊥

,†

^†Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall,

Vancouver, British Columbia V6T 1Z1, Canada

‡

Advanced Applied Physics Solutions, 4004 Wesbrook Mall, Vancouver, British Columbia V6T 2A3, Canada

§

Department of Medicine, University of Cape Town Medical School, Observatory 7925, South Africa

^∥Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa

^⊥TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia V6T 2A3, Canada

*

S Supporting Information

ABSTRACT: Five compounds displaying an unprecedented

binding mode of chloroquine to ferrocene through the bridg-

ing of the cyclopentadienyl rings were studied alongside their

monosubstituted ferrocene analogues and organic fragments.

The antiplasmodial activity was evaluated against strains of the

malaria parasite (Plasmodium falciparum). While the chlor-

oquine-bridged ferrocenyl derivatives were less active than

their ﬁve monosubstituted ferrocenyl analogues, they retained activity in the drug-resistant strains. The biological and physical

properties were correlated to antiplasmodial activity. Intramolecular hydrogen bonding was associated with increased

antiplasmodial action, but it is not the determining factor. Instead, balance between lipophilicity and hydrophilicity had a greater

inﬂuence. It was found that calculated partition coeﬃcient (log P) values of 4.5−5.0 and topological polar surfaces area (tPSA)

2

values of ∼26.0 Å give the best balance. The particular conformation, compact size, and lipophilicity/hydrophilicity balance

observed in the bridged compounds provide them with the structural characteristics needed to escape the mechanisms

responsible for resistance.

9

INTRODUCTION

clinical trials. Several ferrocenyl analogues of chloroquine, in

an exploration of diﬀerent connectivities between the ferrocene

and the chloroquine moieties, have been synthesized and

■

Despite decades of research invested in its prevention and

treatment, malaria remains one of the main causes of mortality

and morbidity in the world. As reported by the World Health

Organization (WHO), 3.3 billion people (almost half of the

world population) were at risk of malaria in 2011. A new study

has revealed how the most recent peak of the disease was in

5

−7,10−13

studied.

A representative group is included in Chart 1,

and a complete set of FQ analogues have been reviewed

1

4−16

1

elsewhere.

The mechanism of action of ferroquine has been studied.

17,18

2

Although it is only partially understood, it is estimated to re-

semble to some extent that of the parent drug chloroquine. Like

chloroquine, ferroquine is thought to interrupt the formation of

hemozoin, the detoxiﬁcation product of the free heme that

results from the degradation of human hemoglobin during the

intraerythrocyte stage of the disease. The buildup of large

concentrations of heme perturbs the barrier properties of

cellular membranes, causing electronic and membrane stress,

disturbing ion homeostasis, and eventually causing the

2

004, taking the lives of 1 817 000 that year only. With no

vaccine available, malaria prophylaxis, diagnosis, and treatment

are the only current approaches to the problem. The ability of

the parasite (Plasmodium falciparum) to promptly develop resis-

tance against antimalarial treatment has hindered the treatment

of this disease. Malaria therapy has relied heavily on the use of

artemisinins since early 2000. Unfortunately, reports of artem-

isinin resistance have surfaced since 2009, perhaps foreshadow-

ing the collapse of the current drug treatment.

Ferroquine (FQ, Chart 1) demonstrated an exceptional ability

to overcome the resistance to the parent drug chloroquine.

3

19

destruction of membranes and parasite death. The increased

therapeutic eﬀectiveness of ferroquine has been attributed to its

4

,5

Ferroquine is highly active against several strains of chloroquine

sensitive and chloroquine resistant Plasmodium parasites in

Received: September 29, 2012

4

−8

vitro and in vivo

and has recently completed phase IIb

©

XXXX American Chemical Society

A

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Chart 1. Ferroquine (FQ) and a Selection of Known Structural Analogues

Chart 2. Chloroquine Derivatives (Series 3), Monosubstituted Chloroquine Ferrocenyl Compounds (Series 4), and 1,1′-

Disubstituted Bridged Chloroquine Ferrocenyl Conjugates (Series 5)

greater accumulation in the parasite food vacuole compared to

that of the parent drug CQ. In addition to thwarting the

In this study, two sets of ferrocenyl chloroquine conjugates

with two distinct substitution patterns (series 4 and 5) were

synthesized using 4-aminoquinolines (series 3) (Chart 2). Both

series of ferrocenyl compounds (4 and 5) are structural analogues

of ferroquine; the monosubstituted ferrocenyl 4-aminoquino-

line derivatives (4a−e) present a ferrocene moiety covalently

attached at the end of the N-alkylamino side chain of the

4-aminoquinoline, while compounds 5a−e incorporate the same

elements of series 4 in a more closed conformation, showcasing

a previously unexplored mode of substitution, with the

chloroquine derivative bridging together both cyclopentadienyl

rings of the ferrocene (Chart 2), forming a ferrocenophane.

The diﬀerence in conformation, ﬂexibility, lipophilicity, and

hydrophilicity of these compounds will illustrate the impact of

these characteristics on the potency and ability of these ferro-

quine analogues to overcome drug resistance.

20

formation of hemozoin, ferroquine creates oxidative stress due

20−22

to the redox activity of the ferrocene/ferrocenium pair.

After reaching its site of action, FQ acts ﬁrst as a strong

inhibitor of β-hematin formation (even more potent than CQ)

and second as redox activator generating reactive oxygen

species (ROS) via a Fenton-like reaction, irreversibly damaging

2

0−22

the parasite.

Ferroquine’s weaker basic properties, higher lipophilicity, and

conformation, inﬂuenced by the presence of an intramolecular

hydrogen bond, allow this compound better permeation

through membranes and therefore a higher accumulation at

the site of action inside the parasite while targeting the

1

7,18,21,23

lipophilic site of hemozoin formation more eﬃciently.

Despite structural similarities to chloroquine and ferroquine,

none of the ferroquine analogues were able to outdo FQ in

performance and, possibly more importantly, studies have not

been able to explain unequivocally the role of the iron and how

it contributes to the overcoming of resistance when ferrocene is

bound to chloroquine. This has made apparent that a very

structurally speciﬁc set of characteristics in ferroquine gives this

compound the speciﬁcity necessary to escape the mechanisms

that encompass chloroquine resistance. The exploration of the

structural features that make ferroquine escape the resistance

mechanisms remains incomplete and is of utmost importance,

since more rational drug designs will be needed urgently if

resistance to current antimalarials continues to grow worldwide.

The antiplasmodial activity of these compounds (series 4 and

series 5) was studied with those of the respective organic

quinoline fragments (3a−e), 4-aminoquinolines with an alkyl

side chain of four or less carbon atoms, both linear and

branched. It has been observed that 4-aminoquinoline

derivatives of chloroquine containing side alkyl chains of

altered lengths can retain the antiplasmodial activity of the

parent drug against certain chloroquine-resistant strains of

24−26

Plasmodium falciparum.

The number of carbon atoms

between the two nitrogens in the diaminealkane side chain is a

major factor for activity against chloroquine-resistant parasite

27,28

strains.

Compounds presenting a shorter rather than longer

B

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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a

Scheme 1. General Route for the Synthesis of Bridged Ferrocenyl 4-Aminoquinoline Derivatives (5a−e)

a

+ −

(

i) n-BuLi, TMEDA, hexanes, 12 h, rt; (ii) CH N(CH ) I , THF, 10 min reﬂux, 24 h rt; (iii) MeI, MeOH, 10 min reﬂux; (iv) NaOH, CH CN,

2

3

2

3

6

h at 110 °C (in microwave reactor).

2

7,29

side chain tend to be more active, especially those with propyl

and butyl

in no product as observed by LR-MS. Compound 5e was

obtained in a 3−5% yield. Unlike the other syntheses, the crude

of 5e contains more side products, thus the need for

preparative TLC puriﬁcation. We hypothesize that formation

of 5 takes place by the stepwise attack of the primary terminal

amine of the chloroquine derivative series 3 on the

trimethylammonium groups of 2. After the ﬁrst attack of 3e

to 2, a strong hydrogen bonding interaction turns the quinoline

away from the other trimethylammonium group (as seen in the

3

0,31

alkyl chains. Several of the monosubstituted

ferrocenyl 4-aminoquinoline derivatives (4a−e) have been

studied in the past and were taken here as a point of reference

7

to develop a robust comparison with the bridged ferrocenyl

4-aminoquinoline derivatives (5a−e).

Structure−activity relationships were established by correla-

tion of the antiplasmodial activity with other biological and

physicochemical properties. These correlations were carried out

in order to elucidate the structural factors that determine the

antimalarial action of ferrocenyl chloroquine derivatives.

36

single crystal structure of the monosubstituted analogue 4e ),

hampering the second attack and displacement of the latter,

resulting in lower yields for its formation. The synthesis of

compound 5b under a diﬀerent methodology has been reported

RESULTS AND DISCUSSION

■

37

Synthesis. The synthesis of each 4-aminoquinoline

chloroquine derivative (3a−e) from the commercially available

,7-dichloroquinoline and the corresponding alkyldiamine was

by others. We oﬀer here a simpler and more general metho-

dology to produce these chloroquine ferrocenyl derivatives, as

well as their full characterization and detailed study of physical

and biological properties.

4

30,32

as previously described

with slight modiﬁcations. These

syntheses were carried out using a microwave reactor, reducing

considerably the reaction time when compared to traditional

methods. The monosubstituted ferrocenyl 4-aminoquinoline

derivatives (4a−e) were prepared following methodology

NMR Characterization. The inﬂuence of the two diﬀerent

bonding modes of chloroquine to the ferrocene moiety (in the

1

13

compound series 4 and series 5) was explored by H and

C

1

NMR spectroscopy. The H NMR chemical shifts suﬀered by

the 4-aminoquinoline hydrogens upon derivatization with

ferrocene are interpreted as an alteration of the structural

environment of the chloroquine-like molecule and thus as a

potential alteration in the interaction (i.e., binding aﬃnity)

between the compound and the drug target hematin. The

7

previously reported with slight modiﬁcations. These com-

pounds were formed via a reductive amination between

commercially available ferrocenecarboxaldehyde and the

corresponding 4-aminoquinoline derivatives 3a−e.

The bridged ferrocenyl 4-aminoquinoline derivatives (5a−e)

were formed by the condensation reaction of 1,1′-bis(N,N′-

trimethylaminomethyl)ferrocene iodide (2) and the corre-

sponding 4-aminoquinoline derivatives 3a−e (Scheme 1). The

nucleophilic displacement of the trimethylammonium group

using the 4-aminoquinoline in the presence of a base is a known

biggest deviation in NMR is observed for the secondary amine

1

hydrogen at the aromatic heterocycle ( H−N ). This could

Ar

potentially be related to the formation or removal of

intramolecular hydrogen bonds between the NH proton in

the 4-position of the quinoline ring and the N atom in the

33

1

reaction for ferrocene substrates with yields of up to 80%

β-position to the cyclopentadienyl ring. Table 1 shows the H

3

4,35

when employing conditions similar to those used here.

The

trimethylammonium methylferrocene iodide is known to be an

excellent electrophile due to the good leaving group property of

the trimethylamine. Methodologies using conventional heat

methods and a microwave reactor were used, with the latter

method being advantageous because it reduces the reaction

time by 8-fold. The average yield obtained was 20% (except for

1

Table 1. Comparison of the H NMR Chemical Shifts of the

1

H−N_A_rSignals in the Quinolines 3a−e vs the Ferrocenes

a

4

a−e and 5a−e

3

a

3b

3c

3d

3e

Δδ (4), ppm

Δδ (5), ppm

0.09

0.38

0.52

0.07

0.09

0.53

0.08

5e). The use of up to 10 equiv of softer bases such as pyridine

−1.68

−0.78

−3.35

a

and sodium carbonate yielded approximately half the amount of

product. When a stronger base such as NaH was employed, no

product was obtained. Instead, an unidentiﬁed ferrocenyl

product (not containing the quinoline fragment) was observed

on the column, possibly a product of decomposition during the

reaction. The use of a protic solvent such as methanol resulted

Δδ is given in ppm, measured in CDCl 400 MHz.

3,

1

NMR chemical shifts of the NH protons ( H−N ) expressed

Ar

as Δδ (ppm) for the monosubstituted (4a−e) and the

disubstituted (5a−e) compounds from the respective quinoline

C

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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3

6

Figure 1. ORTEP diagrams for 5b (top) and 4b (bottom) with 50% thermal ellipsoid level.

1

derivative 3. A positive Δδ indicates the shifting of the H−N

are observed for the H ′ and H ′ hydrogens and the Cp

Ar

α,α

β,β

to higher frequencies (lower ﬁeld) upon coupling to ferrocene,

and a negative Δδ indicates the shifting of the H−N to lower

singlet is absent. The only exception in the group is compound

5d, whose nonsymmetric branched alkyl chain gives four

diﬀerent triplets for the substituted Cp rings. These peaks

appear at higher ﬁeld (4.02−4.20 ppm) in an eﬀect opposite to

that observed for the previous series 4. This might be related to

an electron donating eﬀect of the nitrogen bridging both Cp

1

Ar

frequencies (higher ﬁeld).

1

The H NMR signals of the H−N proton are slightly

shifted to higher frequencies in the respective monosubstituted

ferrocene compounds with the C side chain (4a and 4d);

Ar

2

1

3

however, upon formation of the bridging unit in 5a and 5d, the

shift to higher frequencies is even more pronounced as shown

in Table 1. This shift can be attributed to the deshielding eﬀect

of the formation of an intramolecular hydrogen bond between

the aromatic NH and the alkyl N. The diﬀerence in the

intensity can be explained as a consequence of the alkyl

nitrogen being a secondary amine (in series 4) and a tertiary

amine (in series 5). Compounds with a propyl or butyl chain

rings. Spectroscopic characterization results with C NMR

1

spectroscopy is consistent with that from H NMR.

X-ray Single Crystal Structure Characterization. The

X-ray crystal structures and characteristic bond lengths and

angles for the monosubstituted ferrocenyl compounds 4b and

36

4e were previously reported by our group. In their solid state,

the side chain orientation in 4e brings the NH proton in the

4-position of the quinoline ring and the N atom in the β-position

to the cyclopentadienyl ring to a close proximity so that

intramolecular hydrogen bonding occurs at a distance of

(

b, c, and e) present shifting to higher frequency upon single

attachment of the quinoline derivative to ferrocene (series 4),

again indicating the formation of a hydrogen bond. However,

when connected in the bridging fashion to ferrocene (series 5),

a very pronounced shifting to lower frequencies is observed.

This could indicate that the hydrogen bonding is lost, possibly

because of conformational restrictions in the geometry of 5b,

38

2.11(2) Å, consistent with a hydrogen bonding interaction.

To allow a comparison of the solid state of the two types of

ferrocene compound structures, the single crystal structures of

compounds 4b and 5b are shown in Figure 1. The characteristic

bond lengths and angles in 5b are summarized in Table 2.

Relevant bond lengths and angles in 4b were previously

5

c, and 5e. The fact that the H-bonding in series 5 is

36

maintained and even intensiﬁed when the length of the alkyl

chain is n = 2 (branched or unbranched 5a and 5d) but is lost

when the length of the alkyl chain is n = 3 or higher (5b, 5c, 5e)

has been attributed to the distance between the NH-aromatic

and the alkyl N groups that in 5a and 5d can be brought to

close proximity, whereas in the longer chain analogues, the

distance that separates these two groups does not allow the

H-bonding.

reported by our group and can be found in the Supporting

Information. In 4b, the distance between the corresponding N

and H is 4.86 Å, ruling out the presence of an intramolecular

hydrogen bond in the solid state. When compared to 4b, 5b

presents signiﬁcant diﬀerences in conformation. The quinoline

ring and the ferrocene skeleton are parallel to each other but

contained in two distinct planes, connected by a propyl chain

that displays a nonstaggered arrangement with the quinoline

ring almost perpendicular to the propyl chain. The quinoline

ring is perpendicularly oriented to the plane that contains the

cyclopentadienyl rings of the ferrocene, and no intramolecular

hydrogen bonding can be observed in 5b.

Another interesting characteristic of 5b is that it is a strained

ferrocenophane, speciﬁcally an aza[3]ferrocenophane. As

expected for a ferrocene with an ansa [3] bridge, the normal

geometry of ferrocene has been aﬀected. The parameters used

to describe this type of structure are listed in Table 3. The

degree of ring tilt is expressed as the tilt angle (α), or the

For the ferrocene signals, the typical displacements for both

types of substituted ferrocene are observed. In monosubstituted

products (4a−e) one singlet is observed for the unsubstituted

Cp ring, normally at δ 4.14−4.18 (ppm) and an AA′BB′-type

spectrum with two triplets is observed for the H ′ and H ′

α,α

β,β

hydrogens in the substituted Cp ring. The hydrogens of the

singly substituted cyclopentadienyl are located at lower ﬁeld

(compared to the Cp singlet) because of the electron withdrawing

eﬀect of the chloroquine derivative. As is expected for the

disubstituted products (5a−e), two sets of triplets/multiplets

D

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Table 2. Selected Bond Distances (Å) and Angles (deg) in 5b

dihedral angle between the two planes that contain each of the

Cp rings. The tilt angle (α) of 12.12° is not considered to

induce high strain (tilt angles of up to 32° are considered to

bond distance (Å)

C7−N1

C6−N1

N1−C13

C16−N2

N2−C15

N2−H2_N

1.4808(16)

1.4757(16)

1.4764(14)

1.3514(14)

1.4580(15)

0.8800

39

induce signiﬁcant of strain). This aza[3]ferrocenophane is

structurally similar to a [3]ferrocenophane (a propane chain

bridging the two cyclopentadienyl rings), since the latter has tilt

39

angles up to 12.6°. Ferrocenophanes in general are expected

to show unique chemical properties due to the functionality in

the side arm. Strain energy can be imbedded in this type of

compound (5a−e) and that might be a focus of research, since

this energy and the eﬀects on its conformation could possibly

inﬂuence antiplasmodial activity. Alternatively, the introduction

of conjugated groups could tune the electronic character of the

system. Aza[n]ferrocenophanes are known to undergo

reversible electron transfer between the Fe and N atoms in

angle (deg)

H2 −N2−C16

N

118.6

C15−N2−C16

C7−N1−C13

C13−N1−C6

C6−N1−C7

C8−C7−N1

122.77(10)

109.70(10)

109.03(9)

111.12(10)

113.01(10)

114.36(10)

112.14(10)

112.29(10)

113.98(10)

51.15(17)

−72.96(14)

−69.95(13)

40

the oxidized state.

C5−C6−N1

In Vitro Antiplasmodial Activity Studies. The anti-

plasmodial activities of the compounds 3a−e, 4a−e, and 5a−e

were evaluated in vitro against the chloroquine-sensitive (CQS)

D10 and the chloroquine-resistant (CQR) Dd2 and K1 strains

of P. falciparum. The activities of these compounds were com-

pared to the reference drug chloroquine (CQ). A full dose−

response was performed for all compounds to determine the

concentration inhibiting 50% of parasite growth (IC ). The

C13−C14−C15

N1−C13−C14

N2−C15−C14

N1−C7−C8−C9

C14−C15−N2−C16

N1−C13−C14−C15

5

0

Table 3. Structural Parameters of Strained Ferrocenophane

Compound 5b

results are presented in Table 4. The activities of certain com-

pounds in the second chloroquine-resistant strain (K1) could

not be measured because of insuﬃcient sample to carry the

testing of all compounds in parallel.

All disubstituted bridged ferrocenyl compounds 5a−e

demonstrated activity against all the tested parasite strains.

The most active compound of this group is 5c with IC values

of 91.3 and 152.2 nM in the parasite strains D10 and Dd2,

respectively. On average, compounds 5a−e are more active in

the Dd2 (CQR) strain. When compared against the control,

5a−e are less active than chloroquine against D10 (CQS), as

expected. However, 5a, 5b, and 5c demonstrate more activity

than chloroquine against Dd2 (CQR), as did 5b and 5d against

K1 (CQR), overcoming the parasitic resistance associated with

5

0

parameter

α (deg)

12.12

δ (deg)

171.3(4)

3.2517(2)

0.745

Cp_c_e_n_t_r_o_i_d−Cp_c_e_n_t_r_o_i_d(Å)

N deviation from C5−C6−C7−C8 (Å)

C6 deviation from Cp plane (Å)

C7 deviation from Cp′ plane (Å)

0.138(2)

0.120(2)

Table 4. In Vitro Antiplasmodial Activity and Resistance Indices against P. falciparum CQS D10, CQR Dd2, and CQR K1

a

Strains

b

c

compd

IC , CQS D10 (nM)

IC , CQR Dd2 (nM)

50

RI

IC , CQR K1 (nM)

50

RI

5

0

3

4

5

a

b

c

36.1 ± 13.5 (n = 2)

50.9 ± 29.7 (n = 2)

76.1

261.6 ± 18.0 (n = 2)

398.8 ± 42.4 (n = 2)

1697.8 ± 20.0 (n = 2)

80.6 ± 42.4 (n = 2)

30.3 ± 15.2 (n = 2)

35.7 ± 4.76 (n = 2)

297.4 ± 69.2 (n = 2)

100.5 ± 22.3 (n = 2)

23.1 ± 4.6 (n = 2)

7.3

7.8

ND

22.3

2.7

2.0

0.6

2.2

2.5

0.5

3.0

0.7

1.6

0.6

0.7

1.06

6.2

ND

d

e

a

b

c

29.7 ± 17.0 (n = 2)

15.2 ± 7.6 (n = 2)

60.8

160.1 ± 59.4 (n = 3)

72.0 ± 0.76 (n = 3)

1467.6

5.3

4.8

24.1

0.1

2.8

5.9

0.3

138.3

16.1

40.2

114.1 ± 35.7 (n = 3)

260.5 ± 87.6 (n = 3)

90.9

d

e

a

b

c

43.8 ± 20.7 (n = 2)

368.1

176.0

323.0

91.3

1136.8 ± 21.6 (n = 2)

129.7 ± 23.2 (n = 2)

224.3 ± 6.73 (n = 2)

152.2 ± 6.52 (n = 2)

269.2 ± 4.48 (n = 2)

506.5 ± 21.1 (n = 2)

ND

307.3 ± 170.5 (n = 3)

ND

0.9

0.7

d

e

444.2

669.0

291.6 ± 2.24 (n = 3)

ND

1

4

14

FQ

18.0

29.1 ± 7.75 (n = 19)

19.0

14.0

0.8

CQ

180.3 ± 77.4 (n = 15)

758.0

26.0

a

b

c

n = number of data sets averaged. ND = not determined. Resistance index (RI) = IC (Dd2)/IC (D10). Resistance index (RI) = IC (K1)/

50

IC (D10).

50

E

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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the quinoline fragment. The monosubstituted ferrocenyl com-

pounds 4a−e also demonstrate activity in both CQS and CQR

strains of P. falciparum. The most active compounds against

Dd2 are 4a and 4d, while the most active compounds against

the K1 strain are 4b and 4e. Unlike series 5, these compounds

are generally more active in the CQS D10 strain than in the

CQR Dd2 and K1. The organic components, the chloroquine

derivatives 3a−e, demonstrate a proﬁle similar to that of

chloroquine; they are active against D10 but lose activity

against Dd2 and K1. The exceptions are the most active

compounds 3d and 3e.

The activities of compounds in the series 4 and 5 are

sensitive to the level of drug resistance of the parasite strains.

The diﬀerent responses observed against Dd2 and K1 may

suggest some level of strain speciﬁcity on the activity of these

compounds. Nevertheless, on average (excluding certain

exceptions), activities of 5a−e were superior in the resistant

strains vs the sensitive strain while the opposite was observed

for 4a−e and 3a−e.

or lesser innoculum eﬀect, and the fact that some compounds

were tested as suspensions.

The resistance index (RI) provides a valuable tool for

analysis of drug candidates. Small RI values indicate activity

regardless of the susceptibility of the parasite strain. On the

contrary, large values indicate loss of activity due to resistance

development or likelihood of resistance development. A

promising drug lead will necessarily have a small resistance

index as a sign of not being detected by whatever resistance

mechanism is operating. RI values for each compound ranged

from 0.5 to 24.7 (Table 4). As expected, the quinoline

derivatives 3a−e showed the largest tendency to develop

resistance due to their structural similarity to chloroquine. The

lowest values of RI were found for the disubstituted bridged

ferrocenyl compounds 5a−e (Table 4). Figure 2 illustrates the

The eﬀects of the substitution on the chloroquine derivatives

will be discussed in terms of each set of speciﬁc length of the

methylene spacer. For the ethyl chain quinoline (a), while the

ferrocenyl derivatives are less eﬀective than the organic fraction

3a in the CQS D10 strain, an improvement of 7-fold and 2-fold

in the CQR Dd2 strain is observed for the monosubstituted

ferrocenyl 4a and for the bridged ferrocenyl 5a, respectively.

However, against the CQR K1 strain, 4a loses all activity,

becoming almost 2-fold less active than chloroquine itself. A

similar situation is observed for the propyl (b) and butyl (c)

chain derivatives. The ferrocenyl derivatives 4b and 5b are 1.3-

and 2-fold more active than the organic component 3b in the

chloroquine-resistant Dd2 strain, respectively. Even more

dramatically, 4c and 5c are 17 and 11 times more active than

Figure 2. Resistance index (RI) expressed as the correlation of the in

vitro antiplasmodial activity against P. falciparum CQS D10 and CQR

Dd2 parasite strains.

3c in the CQR Dd2 strain, respectively. A diﬀerent trend is

observed for the branched methylene spacer derivatives d

and e. In the case of the isopropyl derivatives (d), the activity of

the organic component 3d is superior to that of the ferrocenyl

derivatives against all strains except for 4d. In the case of the

resistance index calculated for these compounds for the parasite

strains CQS D10 and CQR Dd2. The diagonal line represents

an equal antiplasmodial potency regardless of the chloroquine

susceptibility of the strain (RI = 1). Ferroquine (FQ), for

example, not only displays low IC₅₀values for both parasite

2,2′-dimethylpropyl derivatives (e), the activity of 3e is far

superior to that of the ferrocenyl derivatives in both D10 and

Dd2 strains but almost equal against K1. 3e is up to 44 times

more active than 5e in D10 and 38 times more active than 4e in

Dd2. It was found then that the length of the methylene spacer

was not as strong an inﬂuence in the activity as is the degree of

branching. These results illustrate a diﬀerent activity trend for

the linear and the branched quinoline derivatives: linear

derivatives can overcome chloroquine resistance by derivatiza-

tion with ferrocene; however, the bulkier branched derivatives

lose all activity when derivatized with ferrocene. Even though

ferroquine (FQ) was not used as control during this

^e^x^p^e^rⁱ^m^eⁿ^t^,^I^C⁵0 values in these parasite strains have been

14

strains but also has an RI value close to 1, which makes this

compound very attractive as an antiplasmodial drug lead.

Compounds 5a−e are not as potent as FQ but remain close to

the ideal line, unlike compound series 3 and 4 that are more

potent but develop resistance later. The fact that some of these

compounds are equally or more potent in drug-resistant

parasite strains than in drug-sensitive strains suggests that,

rather than following the mechanism of accumulation of

chloroquine, these are following a diﬀerent mechanism allowing

them not to be detected by the transmembrane proteins

responsible for lower accumulation of the drug (origin of

resistance). Ferroquine indiscriminative action in parasite

14

reported in literature.^F^e^r^r^o^q^uⁱⁿ^e^h^a^s^I^C⁵0 values of 18, 19,

and 14 nM in the chloroquine sensitive strain D10 and the

1

8,41

strains has been explained in a similar fashion.

14

chloroquine resistant strains Dd2 and K1, respectively.

A

In Vitro Antitumor Activity and Cytotoxicity Assay.

The toxicity of the compounds was assessed in vitro. Two

human cell lines originally from human breast tissue were

employed. The normal breast epithelial cells MCF-10A (CRL-

10317) and the breast cancer cells MDA-MB-435S (HTB-129)

were used as references for a healthy and cancerous cell line,

respectively. Chloroquine and other quinoline-containing drugs

traditionally used as antimalarials have also been studied as

number of the averaged values of the antiplasmodial activity

were obtained with uncertainty reﬂected in the discrepancy

between independent screenings of the compounds. Diverse

factors beyond our control contribute to this inconsistency.

These include the use of human blood for parasite culture,

where the age and source vary in almost every assay performed,

the use of synchronized or nonsynchronized cultures, the

parasitemia and hematocrit levels that can give rise to a greater

42

anticancer agents in human breast tissue. Antitumor activity

F

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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4

3

and cytotoxicity were assayed using the MTT methodology.

availability and incorporation into hemozoin and hence halting

2

8,46

Cisplatin (cis-diamminedichloroplatinum(II)) was used as a

positive control for cell death. Chloroquine and ferroquine

were also used as control. All tested compounds were found to

lower the viability of the tested cell cultures, with increasing

detrimental eﬀect with increasing compound concentration.

IC₅₀values calculated for all tested compounds are shown in

Table 5.

the detoxiﬁcation process of the parasite.

For instance,

monomeric Fe(III)PPIX and chloroquine form a π−π com-

47,48

49

plex

in neutral or weakly acidic media. Thus, the antiplas-

modial activity of 4-aminoquinoline compounds can be

correlated to their ability to coordinate hematin, and this can

be monitored through UV−vis methods. The π → π* elec-

tronic transitions in the porphyrin ring give rise to a Soret band

46

at 402 nm. A strong hypochromism is then observed upon

complex formation between the Fe(III)PPIX and the quinoline

compound. This eﬀect can be quantiﬁed and ﬁtted to a

model of association to determine the strength of complex

Table 5. Toxicity of the Compounds (Expressed as IC )

5

0

against the Human Normal Breast Epithelial Cell Line MCF-

0A and the Human Breast Cancer Cell Line MDA-MB-435S

1

4

6,50

formation.

IC₅₀(μM)

The formation of the complex between Fe(III)PPIX and the

CQ derivative was monitored as a function of the change in

absorbance of the Soret band of hematin at 402 nm upon

coordination to the quinoline moiety. Figure 3 shows a set of

compd

MCF-10A

MDA-MB-435

3

a

b

c

74.7 ± 3.7

103.0 ± 6.0

47.9 ± 1.4

107.0 ± 2.1

105.3 ± 3.1

4.1 ± 0.5

2.8 ± 0.3

3.8 ± 0.6

5.5 ± 0.1

5.4 ± 0.1

2.4 ± 0.2

4.0 ± 1.0

4.2 ± 0.9

1.4 ± 0.1

0.3 ± 0.1

11.1 ± 1.5

26.5 ± 2.0

69.7 ± 4.8

17.5 ± 1.4

21.90 ± 1.3

13.56 ± 2.4

25.67 ± 0.7

22.71 ± 1.2

3.1 ± 0.2

3.1 ± 0.1

3.0 ± 0.2

3.2 ± 0.1

2.6 ± 0.1

1.7 ± 0.1

3.1 ± 0.3

2.8 ± 0.2

1.4 ± 0.1

0.6 ± 0.1

92.3 ± 19.8

12.3 ± 0.8

20.2 ± 1.2

3

4

5

d

e

a

b

c

d

e

a

b

c

d

e

cisplatin

FQ

CQ

In the normal breast epithelial cell line MCF-10A, the

chloroquine derivatives 3a−e are mild to nontoxic. Once

derivatized with ferrocene, the series of compounds 4a−e and

Figure 3. Spectroscopic changes observed when Fe(III)PPIX is

titrated with compound 4a.

5

a−e show a noticeable increase in toxicity. Against the breast

cancer cell line MDA-MB-435S, 3a−e are more active, with

IC values that are roughly a ﬁfth of the values displayed in the

50

normal MCF-10A cell line. The IC₅₀values obtained for the

spectra collected when the hematin solution was titrated with

increasing concentrations of 4a. The spectral changes observed

show a decrease in the Soret band of Fe(III)PPIX at 402 nm

and an increase in the quinoline band at around 338 nm that is

explained by the increasing concentration of the quinoline

derivative. The data obtained were ﬁt to a 1:1 association model

to calculate the association constant as log K. This 1:1 associa-

tion model is the best ﬁt model for the parent drug chloroquine

series of compounds 4a−e and 5a−e against the MDA-MB-

435S cell line are similar to those obtained for the normal

MCF-10A cell line, indicating that any eﬀect is mainly driven by

the toxicity associated with the compounds.

In general, the addition of the metallocene fragment

increases greatly the toxicity of the compounds, and this is

comparable to what has been observed for other ferrocene

3

6,44,45

50

chloroquine compounds.

However, FQ seems to be

under these conditions. Titration data for a selection of com-

somewhat less toxic than the compounds on series 4 and 5. The

mode of substitution, either monosubstituted in one ferrocene

ring or in the bridged form, does not seem to have a direct

inﬂuence on the overall activity of the compounds and neither

does the length of the side chain or its degree of branching.

Although the ferrocenyl compounds were more toxic than

cisplatin and chloroquine, these data only show the in vitro

response to two cell lines. More extensive studies will be

required to determine the toxicity proﬁle of these compounds

and thus their real appeal as pharmacophores.

pounds are shown in the Supporting Information. The associa-

tion constants (as log K) calculated for the compounds are

presented in Table 6.

All compounds interacted with Fe(III)PPIX. The association

constants (log K) obtained for the quinoline fragments (3a−e)

were, as expected, relatively high regardless of the length or

branching of the side alkyl chain. These values are close to the

value obtained for chloroquine that was used as a control in this

study (log K = 5.07 ± 0.05). The association constants for both

4a−e and 5a−e were on average lower than those observed for

the organic fragments. Within the monosubstituted ferrocenyl

compounds, 4a, with an unbranched ethyl side alkyl chain,

exhibits the highest value for this series (log K = 4.71). It was

observed that a longer and more branched side alkyl chain

Hematin Association Assay. Extensive evidence supports

Fe(III)PPIX (hematin) as the molecular drug target of

46

quinoline antimalarial drugs. Most 4-aminoquinolines are

thought to act by complexing Fe(III)PPIX, thus reducing its

G

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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a

b

c

Table 6. Association Constants (log K), ClogP and PSA

of 3a−e, 4a−e, and 5a−e in Their Neutral State

give a ﬁnal concentration of 4.5 M and pH 4.5. After incubation

at 60 °C for 1 h, the process was quenched by the addition of a

5

% (v/v) pyridine solution and buﬀered at pH 7.5 to give the

compd

log K

ClogP

PSA (Å²)

ideal conditions for the formation of a Fe(III)PPIX−pyridine

complex with the hematin remaining in solution. The

absorbance of the complex formed by Fe(III)PPIX and

pyridine is measured at 405 nm. The relationship between

absorbance and concentration is linear; higher concentrations

of hematin in solution indicate the potent eﬀect of the drug to

divert the consumption of hematin from the formation of

β-hematin.

3

4

5

a

b

c

4.67 ± 0.05

4.85 ± 0.02

4.86 ± 0.04

4.62 ± 0.05

4.80 ± 0.03

4.71 ± 0.03

4.42 ± 0.10

4.13 ± 0.05

3.68 ± 0.04

1.27

1.38

1.83

1.59

2.49

4.48

4.84

4.83

4.79

5.64

6.38

6.74

6.71

6.69

7.54

5.1

51.4

51.0

47.5

38.7

39.0

38.9

36.0

33.6

27.4

25.3

26.0

25.1

21.7

26.2

27.2

d

e

a

b

c

A selection of collected spectra is shown in Figure 4. When

no equivalents of the compounds are present, a small amount

d

e

a

b

c

4.18 ± 0.05

4.23 ± 0.15

4.76 ± 0.03

4.47 ± 0.07

4.41 ± 0.13

5.52 ± 0.03

5.07 ± 0.05

d

e

2

1

FQ

CQ

4.63

a

Obtained from spectrophotometric titrations of Fe(III)PPIX with

compounds 3a−e, 4a−e, and 5a−e in 40% DMSO, apparent pH 7.5,

b

c

0

.020 M HEPES buﬀer at 25 °C. ClogP = calculated log P. PSA =

calculated polar surface area.

presented a lower value of log K. The association constant of

compound 4e could not be determined because of insolubility.

Unlike compounds 4a−e, the disubstituted bridged ferrocenyl

compounds 5a−e displayed log K values that increased with

length and branching of the side alkyl chain. The highest

association constant was obtained for compound 5c, the

unbranched butyl side chain (log K = 4.76). The diﬀerent

structural conformations for these two types of ferrocenyl

substitution seem to alter signiﬁcantly the association of the

quinoline fragment with Fe(III)PPIX. In 5b, the ferrocene

fragment is twisted into close proximity to the quinoline

fragment which could sterically hinder the π−π association with

Fe(III)PPIX, lowering the association constant. In 4b, the

ferrocene is directed further away from the quinoline fragment

and hence does not disturb the quinoline fragment association

with hematin, which is reﬂected in a higher value of log K. In

turn, it is expected that the longer chain in the bridged

derivative 5c provides a less sterically hindered interaction

which would explain the elevated log K value for 5c. The

association constant reported for ferroquine, log K = 5.52 ±

2

1

28

0

.03, and the value reported for CQ (log K = 5.52 ± 0.05)

and found in this study (log K = 5.07 ± 0.05) are still

comparable to the values obtained for the association constants

of both series of ferrocenyl derivatives.

β-Hematin Formation Inhibition Assay. The antimalarial

properties of these compounds were also explored as a function

of their capacity to inhibit the formation of β-hematin

Figure 4. Interaction of pyridine and hematin present in solution after

incubation for the formation of β-hematin. The sets of spectra

correspond to increasing amounts of compounds 4b and 5b. The

absorbance of the Soret band of the Fe(III)PPIX−pyridine complex

occurs at 405 nm. The legend indicates the number of equivalents of

test compound added in each experiment. D indicates drug only, a

blank using the maximum concentration of drug in the 5% pyridine

aqueous solution.

(

synthetic hemozoin). This was evaluated quantitatively using

51

the pyridine hemochrome UV−vis assay. In aqueous solu-

tions of hematin, formation of the solid β-hematin is stimulated

by addition of sodium acetate. The degree of inhibition of

β-hematin formation achieved by the tested compound is

measured by calculating the concentration of free hematin

5

1,52

remaining at the end of the assay.

Hematin solution in

of hematin is observed; however, at any other number of

equivalents, no absorbance is detected for this complex

indicating that no free hematin is left in solution. The low

absorbance of the Fe(III)PPIX−pyridine complex at 1 or 2

0

.1 M NaOH was incubated in the presence of the studied

compounds with increasing numbers of equivalents. β-Hematin

formation was initiated by the addition of acetate solution to

H

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

equiv of the compound quickly decays after the number of

equivalents increase. These could lead to false negative results.

If these compounds do not inhibit formation of β-hematin, all

spectra obtained would be expected to be identical to the

spectra obtained at zero equiv of the compound. This pheno-

menon of quenching of the absorbance of the hematin that

should remain in solution, even after a large portion of it has

been used to form β-hematin, is explained by solubility restric-

tions. The weak inhibitory properties are related to the poor

solubility of the compounds in aqueous media. The compounds

in stock solutions, completely dissolved in DMSO, were added

to the aqueous media where they could precipitate from large

amounts of water. It has been noted that poorly water-soluble

of each group, as expected. These ClogP values are similar to

17

those obtained for chloroquine (4.63) and for FQ (5.1). The

high lipophilicity of FQ has been postulated to be key in

the good absorption and transport properties across cell

58

membranes.

Computational methods can be used to predict important

pharmacophoric properties of drug candidates such as the ability

59

of these compounds to permeate biological membranes and

17,21,60,61

have been used in the past to rationalize antimalarial activity.

The molecular electrostatic potential (MEP) surface gives an

indication of the charged surface area, its accessibility by polar

solvents (giving also an idea of the hydrophilicity of the

compounds), and an idea of the importance of the orientation of

the molecule for the activity of the drug candidate. Therefore, MEP

maps were generated to probe key structural features for the

compounds such as steric, electrostatic interactions, hydrogen

donor/acceptor properties, and lipophilicity. Figure 5 shows a

51

compounds trap unreacted hematin in the solid state. This

would explain the complete absence of the Fe(III)PPIX−

pyridine complex absorbance peak. It is known that compounds

with very low water solubility, such as the quinoline drug

halofantrine, indeed inhibit β-hematin formation and appear to

prevent solubilization of hematin in aqueous pyridine medium,

51

giving false negative results. Moreover, the cloudiness arising

from partial precipitation of the compounds and/or from the

transport of miniparticles of β-hematin can interfere with the

measurement of absorbance. The use of other solvents to

facilitate the dissolution of the studied products was not tested,

since the solvent eﬀect on this assay has not been explored and

could considerably aﬀect the interactions that cause the

formation of β-hematin. The addition of other organic solvents

could produce misleading results, since by analogy increasing

the DMSO concentration in the association with hematin assay

weakens the interaction of some aminoquinolines with

5

3

Fe(III)PPIX.

Partition Coeﬃcient and Molecular Shape Analysis.

Accumulation of chloroquine-based compounds in the parasite

food vacuole lacks a special transport mechanism and is

determined by physical characteristics that will allow the

crossing of membranes and the retention upon protonation

49

inside the vacuole. Therefore, physical characteristics such as

lipophilicity and hydrophilicity have been suggested to play a

determining role in the accumulation of drugs inside the acidic

1

9,54,55

food vacuole of the malaria parasite.

For ideal

pharmacokinetic and pharmacodynamic properties in a drug,

the ideal distribution coeﬃcient is a moderate intermediate

between hydrophilic and hydrophobic. The partition coeﬃcient

(

log P) is used frequently to deﬁne the lipophilic character of a

drug. Theoretically calculated values of log P (ClogP) are pre-

sented in Table 6, calculated using the commercially available

56,57

ACDLABS 11.0 program.

In general these compounds are

all lipophilic (log P > 0). Chloroquine derivatives 3a−e experi-

ence, as expected, an increase in lipophilicity as the alkyl side

chain increases in length and in branching. The calculated log P

values show a considerably larger lipophilicity for compounds

4

a−e and 5a−e compared to 3a−e. The disubstituted bridged

Figure 5. Comparison of the molecular electrostatic potential (MEP)

surfaces of a selection of compounds from this study.

5

ferrocenyl compounds are 10 -fold more lipophilic than the

parent chloroquine fragment while the monosubstituted

ferrocenyl compounds are 10 more lipophilic. This approx-

4

imate 10-fold diﬀerence in lipophilicity between series 4a−e

and series 5a−e arises exclusively from the diﬀerence in ferro-

cene substitution and structural orientation. The more closed

conformation of the bridged compounds likely provides a

more hydrophobic surface and therefore a higher value of log P.

Within each of the ferrocenyl series, the lipophilicity of the ﬁrst

four members is relatively similar, but the 2,2′-dimethylpropyl

side chain ferrocenyl derivative (4e and 5e) is the most lipophilic

comparison of the molecular electrostatic potential (MEP)

surfaces generated for a selection of relevant studied com-

pounds at the DFT-B3LYP level of theory for the neutral state.

In a comparison of the MEP maps of the two modes of

ferrocenyl substitution, exempliﬁed by 5b and 4b, the most

noticeable diﬀerence comes from the nitrogen that connects

the alkyl chain and the ferrocene. In 4b, this nitrogen is more

electronically available with the lone pair probably oriented

I

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

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toward the outer part of the molecule and therefore more

accessible to interactions with the surroundings. In the bridged

analogue, the nitrogen lone pair is only partially available.

Because of steric hindrance, this lone pair is buried in the

structure and unavailable for interaction with solvents or other

molecules. From the MEP surface of the corresponding 4-

aminoquinoline fragment 3b, two more electronegative sites

can be observed. As expected, the primary terminal amino

group that is unavailable in 5b and partly available in 4b is

completely available in 3b, displaying an increased electro-

negative surface. The most interesting diﬀerence stems from

the electronegatively charged zone of the nitrogen of the 4-

position in the quinoline ring. Absent in both ferrocenyl

derivatives, the electron density associated with this nitrogen is

thought to be aﬀected by the inductive and electronic changes

generated by the attachment of the ferrocene. When compared

to the MEP surface of ferroquine, the principal diﬀerence noticed

with both types of ferrocene derivatives, series 5 and series 4, is

the availability of the alkyl nitrogen. The conformation and the

electrostatic distribution of charge in ferroquine appear to be

similar to those observed in series 5, with the electron density

of the tertiary nitrogen partially available and hidden in a pocket of

the structure. Ferrocenyl derivatives in general, compared to the

organic 4-aminoquinoline fragment, have a reduced negative

charged surface area due to a hindered alkyl nitrogen and at the

nitrogen at the 4-position in the quinoline ring.

Structure−Activity Relationship. Structural features play

a remarkable role in the interaction of these compounds with

the parasite transmembrane proteins that allow the accumu-

lation of the drug inside the parasite food vacuole, inﬂuence its

ability to accumulate in the vacuole, and therefore overcome

drug resistance. The following discussion encompasses a cor-

relation of each physical property discussed previously with the

antiplasmodial activity observed mainly against the chloroquine-

sensitive strain D10 and the chloroquine-resistant strain Dd2.

Antiplasmodial Activity, Hydrogen Bonding and

Conformation. The presence of intramolecular hydrogen

bonding has been associated with the improved action of

1

7,21

ferroquine.

It is believed that intramolecular hydrogen

bonding will give rise to a closed conformation that can

penetrate more eﬃciently the parasite membranes to reach the

17

drug site of action. Intramolecular hydrogen bonding between

the aromatic NH on the 4-position of the quinoline ring and

the nitrogen atom in the β-position of the cyclopentadienyl ring

of ferrocene was observed in a low dielectric constant solvent

1

(chloroform) by the means of H NMR spectroscopy. All

1

compounds that exhibited shifts in the H−N NMR signals

Ar

toward higher frequencies (indicating the deshielding eﬀect of

the formation of intramolecular hydrogen bonding) also

exhibited reasonable antiplasmodial activity. The exception of

the group is compound 4e, the 2,2′-dimethylpropylalkyl chain

derivative that, despite the most pronounced positive shifting, is

The polar surface area (PSA) along with the partition

coeﬃcients can estimate successfully the capability of

compounds to permeate cell membranes, and this is strictly

related to an increased accumulation in the cellular

inactive, especially in the chloroquine-resistant strain Dd2.

1

Compounds that suﬀered shifts in the H−N NMR signals

Ar

toward lower frequencies (indicating weakening/loss of hydro-

gen bonding, likely due to conformational restrictions) had

mixed results in terms of antiplasmodial activity. Compound 5c,

with a relatively small shifting to lower frequencies, is one of the

most active of the group; however, 5b and 5e, which shifted

−1.68 and −3.35 ppm, respectively, were among the least

active. These observations conﬁrm that the intramolecular

H-bonding observed in solution is associated, with certain

exceptions, with an increase in antiplasmodial activity and that

the opposite is also true, loss of intramolecular H-bonding

interaction is associated with decrease of antiplasmodial action.

This correlation is illustrated in Figure 6. Intramolecular

hydrogen bonding was also observed in the solid state.

Compound 4e, one of the least active compounds, presents

intramolecular H-bonding, while 4b and 5b that surpass its

antiplasmodial activity against both parasite strains lack this

bonding in the solid state. These observations agree with our

previous observations that the branching of the side chain is a

bigger inﬂuence than intramolecular H-bonding when

determining antiplasmodial activity.

6

2,63

organelle.

The topological polar surface area (PSA) of a

molecule is a measure of the surface rich in oxygen or nitrogen

atoms, or hydrogen atoms attached to nitrogen or oxygen

atoms, and serves as an indication of how heavily hydrated it

64

will be in an aqueous environment. This parameter estimates

the H-bonding ability of a molecule and provides good

correlation with experimental transport data, making it an

65

attractive potential predictor of membrane transport. During

the absorption process, a compound must be able to shed its

hydration sphere to enter the lipophilic membrane environ-

ment. If the compound is heavily solvated, this process is likely

to be energetically unfavorable, and so absorption will be

6

4

hindered. Consequently, compounds with lower PSA values,

2

less than 60−140 Å , are good at permeating cell

6

2,64,66

membranes.

PSA values for a selection of compounds

in their neutral state are presented in Table 6. All studied

2

compounds have PSA of less than 60−140 Å cutoﬀ. All the

bridged ferrocenyl compounds seem to have similar relatively

2

low values of PSA ranging from 22 to 27 Å . The

It has been suggested that a N-quinoline to N-methylene

chain distance in the range of 7.52−10.21 Å is required for the

formation of a heme−drug complex and therefore for anti-

monosubstituted ferrocenyl compounds 4a−e display higher

PSA values, likely stemming from the alkyl nitrogen availability

observable in their MEP surfaces (Figure 5). The correspond-

ing quinoline analogues 3a−e, as expected, have even higher

PSA values, almost double those registered for the bridged

ferrocenyl group. This is explained by the additional electronic

surface (Figure 5). Interestingly, the PSA value of ferroquine

26

plasmodial activity. The corresponding distances are 8.912,

6.685, and 7.653 Å in compounds 4b, 4e, and 5b, respectively.

It is worth noting that the only compound in this group that

does not follow this rule is the least active of the three (4e) and

one of the least active compounds of the complete group. The

intramolecular H-bonding and the conformation observed in

the solid state structure of 4e are similar to those of ferroquine

(

FQ) falls in the range of values obtained for the bridged

ferrocenyl compounds 5, conﬁrming the similarity observed in

the map of electrostatic potential of these compounds.

Although the PSA value is not the only parameter to consider,

it indicates that ferroquine and the bridged ferrocenyl

compounds should have similar permeability/absorption

proﬁles.

17

with the quinoline ring found on top of the ferrocene skeleton.

Nonetheless, their antiplasmodial activities are remarkably

diﬀerent. The conformation of 5b (Figure 1) is unusual in

ferrocenyl quinoline antimalarials, with the quinoline ring

parallel to the ferrocene skeleton but in two distinct planes.

J

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

explored by examining the calculated lipophilicity with the PSA

values and the antiplasmodial activity observed. This is likely to

be relevant in determining the accumulation of the ferrocenyl

drug, considering that the drug must cross a number of

membranes to reach the inner part of the parasite food vacuole.

When the partition coeﬃcient (log P) values are correlated to

their antiplasmodial action in the chloroquine-sensitive D10

strain, the increase of lipophilicity is detrimental, since all

ferrocene derivatives are less active than their corresponding

organic components. Much more interesting is the correlation

observed in the chloroquine-resistant parasite strains. In the

organic series 3a−e, an increase in lipophilicity helped to

improve the antiplasmodial action, with the most active com-

pound being 3e. In the case of the monosubstituted ferrocenyl

compounds 4a−e, the increase in lipophilicity due to the

ferrocene produced a 4- to 15-fold improvement in the activity

for compounds 4a−d. For compound 4e, the most lipophilic of

the group (ClogP = 5.64), a signiﬁcant decrease in activity is

observed. Finally, in the case of the bridged ferrocenyl

compounds 5a−e, the closed conformation represents an

even greater increase in lipophilicity compared to series 4. This

is reﬂected in a decrease in the antiplasmodial activity of the

ﬁrst four compounds. Thus, an increase in lipophilicity was

beneﬁcial in improving the activity of the compounds against

drug-resistant strains but only to a certain level. When the

compounds become too lipophilic, antiplasmodial activity

decreases, as can be appreciated in Figure 7. This would

1

Figure 6. H NMR spectroscopic shift of the H−N (as indicator of

Ar

formation/loss of intramolecular hydrogen bond) vs the in vitro

antiplasmodial activity against P. falciparum CQS D10 and CQR Dd2

parasite strains. The most active compounds (low IC ) fall to the

right of the diagram corresponding to the shifting of the H−N

hydrogen toward higher frequencies (forming of hydrogen bond).

50

1

Ar

These conformational characteristics, observed in chloroform

and in the solid state, are likely to remain similar in the

presence of low dielectric media such as the lipid environment

where these compounds accumulate and act.

Monosubstituted derivatization with ferrocene (such in 4a−e)

seems to increase the formation of intramolecular H-bonding.

On the contrary, formation of the bridged ferrocenyl

compounds (such in 5a−e) seems to cause the loss of the

intramolecular H-bond, likely due to conformational restric-

tions exerted by the bridge structure. The presence of an

intramolecular H-bond is generally associated with the increase

of antiplasmodial action, and in turn, the loss of this bonding

seems to decrease the action. However, the exceptions to this

rule point out that other factors might have a bigger inﬂuence

in determining the activity. This would suggest that H-bonding

is relevant but not determinant for antiplasmodial activity.

Antiplasmodial Activity, Hematin Association, and

β-Hematin Formation Inhibition. All compounds exhibited

interaction with Fe(III)PPIX. The trend observed was the

decrease of association constants (expressed as log K) from the

purely organic fragments series 3 to the ferrocenyl derivatives,

both series 4 and 5, regardless of length or branching of the

methylene side chain. In general, large association constants

were observed in compounds that were the most active,

although the opposite was not observed, as is the case of 4d and

Figure 7. Theoretically calculated partition coeﬃcients (ClogP) vs the

in vitro antiplasmodial activity against P. falciparum CQR Dd2 parasite

strain. The most active compounds (low IC ) present partition

50

5a that, with the lowest found values of association constants,

coeﬃcients in the range of ClogP = 4.5−5.0.

log K = 3.68 and 4.18, respectively, were among the most active

compounds tested. The association constants and the

antiplasmodial activity against the chloroquine-resistant Dd2

parasite strain show no clear correlation. This supports the

notion that while association with hematin is important, a more

important process is the delivery of relevant amounts of the

drug to the site of action through the crossing of membranes.

Unfortunately, solubility requirements made it impossible to

prove the β-hematin inhibition property of these compounds.

Antiplasmodial Activity, Lipophilicity, and Hydro-

philicity. The inﬂuence of the ferrocene in the overall balance

between lipophilicity and hydrophilicity of the molecule was

explain the reduced activity observed for the 2,2′-dimethylpro-

pylferrocenyl derivatives. The better activity observed for 5e (vs 4e)

could be explained by its more closed conformation that

possibly aids in its transportation across membranes with its

reduced surface. Indeed, an intermediate lipophilicity that

delivers the best activity seems to be in the range of ClogP =

4.5−5.1, which is coincidentally where the lipophilicity of

ferroquine falls (log P = 5.1).

The hydrophilicity of these compounds was studied by

means of molecular electrostatic potential (MEP) surfaces and

K

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

polar surface values (PSA). The bridged compounds 5a−e

presented MEP surfaces of reduced electronegatively charged

area. The lone pair of the methylene nitrogen is described as

buried in the structure and mostly inaccessible for interaction

with solvents or other molecules and less prone to participate in

solvation. Interestingly, the two compounds that showed partial

availability of the electronic cloud associated with this nitrogen,

ring without disturbing its interaction with hematin. The

hydrophobic ferrocene can then establish favorable van der

Waals interactions with lipid surfaces at the interface with the

membranes to be crossed and aid in the accumulation of the

drug. This particular conformation, its compact size, and

lipophilicity/hydrophilicity balance could provide these com-

pounds with the structural characteristics needed to escape the

resistance mechanism of the transmembrane proteins and

achieve signiﬁcant accumulation inside the parasite food

vacuole. This would explain why their activity is increased in

drug-resistant strains.

5a and 5c, were also the most potent compounds of this series

and those resembling most the MEP surface obtained for

ferroquine. Compounds of the series 4a−e and 3a−e have

increased electrostatic potential surface. While this improved

the antiplasmodial action in most but not in all of the

compounds of series 4, the further increase in electronegatively

charged area in the purely organic derivatives series 3 decreases

the antiplasmodial eﬀectiveness against the drug-resistant strain.

These observations are consistent with the values observed for

the polar surface area (PSA). The bridged compounds 5a−e

CONCLUSION

■

Five disubstituted ferrocene chloroquine derivatives with the

terminal nitrogen of the chloroquine derivative bridging the

two cyclopentadienyl rings of ferrocene (5a−e) were

synthesized and structurally compared to the corresponding

monosubstituted ferrocene chloroquine (4a−e) analogues and

to the corresponding organic fragments (3a−e). All com-

pounds were characterized by a variety of physical methods.

The presence or absence of hydrogen bonding interactions, the

conformation, the degree of rigidity, and the lipophilicity of

these derivatives are correlated to the biological responses of

these compounds against diﬀerent strains of P. falciparum

parasites. All compounds were active against both chloroquine-

sensitive and resistant parasite strains. Bridged disubstituted

ferrocenyl compounds 5a−e were able to overcome resistance

and, in all cases except one (5e), were more active in the

resistant strain than in the sensitive one, giving them appealing

resistance indices of <1. The branching of the methylene spacer

was found to be a strong inﬂuence in activity, with the

derivatives losing eﬃcacy as they grow in branching. The length

of this spacer was not a strong inﬂuence. The presence of an

intramolecular H-bond is relevant but not determinant for

antiplasmodial activity. Association with hematin was observed

for all compounds and did not seem to determine the activity in

vitro. A balance between the lipophilicity and the hydrophilicity

was found to be determinant in the activity displayed by the

compounds. The addition of the ferrocenyl unit contributed

largely to increase the lipophilicity of the compound, and that

has been related in the past to improved transport of the drug

2

have relatively low values of PSA, from 21 to 27 Å . The most

2

active compound of this series, 5c, has a PSA value of 26.0 Å ,

2

very similar to that of ferroquine, 26.2 Å . Compound 5e has, as

expected, the lowest value of PSA, but that is also accompanied

by relatively low activity. Higher PSA values for 3b and 4b are

correlated to lower activity. Therefore, it seems that more than

a trend, a balance, must be reached where the amount of

electronegative surface is slightly more than that observed for

the bridged compounds series 5 but not as much as that

observed for compounds series 4, much like what is observed

for ferroquine. The ideal hydrophilicity that delivers the best

activity seems to be in the approximate range of PSA = 25.9−

2

6.2 Å . Even more illustrative is the correlation between the

resistance index (RI) and PSA values shown in Figure 8. PSA

17

through membranes. It was found that log P values of 4.5−5.0

2

and PSA values of ∼26.0 Å give the best balance, and that is

witnessed in the increase of activity. The values found for both

parameters happen to be consistent with what is observed in

ferroquine.

Thus, compounds 5 would beneﬁt from being slightly more

hydrophilic with more electronegative area available and from

having an intramolecular hydrogen-bonding interaction that

could allow the “ﬂip-ﬂop” mechanism that has been postulated

for aiding in the accumulation of ferroquine when crossing

membranes. With these characteristics in mind and with the

parameters observed to be determinant in antiplasmodial

activity, in silico calculation could aid in the design of more

ferroquine analogues with tuned lipophilicity/hydrophilicity

and intramolecular hydrogen bonding, giving analogues more

potent than ferroquine.

Figure 8. Resistance index (RI, IC (CQR Dd2)/IC (CQS D10)) vs

50

the topological polar surface area (tPSA) values. The compounds that

demonstrated the best capacity to avoid resistance development (RI

2

close to 1) present tPSA values in the range 25.0−28.0 Å . Points

corresponding to 3a (RI = 7.3) and 3b (RI = 7.8) are not visible

because of the break in the x axis.

2

values in the range 25.9−26.2 Å seem to confer lower resis-

tance indices, suggesting that this physical property is

determinant for the overcoming of resistance mechanisms.

It is hypothesized that the closed conformation observed in

compounds 5a−e, which resemble ferroquine, gives the com-

pounds a close to ideal balance where the very lipophilic

ferrocene is sticking to the side but parallel to the quinoline

EXPERIMENTAL SECTION

■

Materials and Instrumentation. All syntheses were performed

using dried solvents under an inert atmosphere (nitrogen or argon)

using Schlenk-type glassware unless otherwise indicated. Solvents were

dried and distilled prior to use. All other chemicals were purchased

L

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

from Sigma Aldrich or Acros Organics and used without further

puriﬁcation. Selected syntheses were carried using a microwave reactor

Initiator 2.5 (Biotage). The products were puriﬁed to >95% and were

conﬁrmed by HPLC (using linear gradient of 100% water to 100%

acetonitrile) and/or elemental analysis. Column chromatography was

performed using UltraPure silica gel (230−400 mesh) (SiliCycle) or

alumina neutral (60−325 mesh) (Fisher Scientiﬁc). An automated

column system CombiFlashRf (Teledyne ISCO, Inc.) was also

employed using RediSepRf gold silica high performance columns.

dichloromethane (30 mL), and diethyl ether (2 × 25 mL) before

being dried under vacuum to yield 1,1′-bis(N,N′-trimethylaminomethyl)-

1

ferrocene iodide 2 as a yellow powder (3.0 g, 6.2 mmol, 83%). H NMR

3

(300 MHz, CD OD): δ (ppm) 4.69 (s, 4H), 4.66 (t, J = 1.83 Hz, 4H),

3

− +

4.56 (t, J = 1.96 Hz, 4H), 3.07 (s, 18H). ESI-MS(+): 457.1 [M − I ] .

Anal. Calcd for C H FeI N : C, 37.01; H, 5.18; N, 4.08. Found: C,

1

8

30

2

37.34; H, 5.13; N, 4.58.

General Procedure for the Synthesis of 4-Aminoquinoline

Derivatives (3a−e). N-(7-Chloroquinolin-4-yl)ethane-1,2-diamine

(3a), N-(7-chloroquinolin-4-yl)propane-1,3-diamine (3b), N-(7-chlor-

oquinolin-4-yl)butane-1,4-diamine (3c), N-(7-chloroquinolin-4-yl)-

propane-1,2-diamine (3d), and N-(7-chloroquinolin-4-yl)-2,2-dime-

thylpropane-1,3-diamine (3e) were prepared by following modiﬁca-

1

13

H and C NMR spectra were recorded at room temperature on

Bruker AV-300, AV-400dir, AV-400inv, and AV-600 instruments. The

NMR spectra are expressed on the δ (ppm) scale and referenced to

residual solvent peaks or internal tetramethylsilane. The NMR

7

,30,32,68

1

tions from literature procedures.

5.04 mmol, 1 equiv) was dissolved in the respective diamine (approximately

0.4 mmol, 10 equiv) in a 20 mL microwave vial. The mixture was

4,7-Dichloroquinoline (1 g,

assignments were supported by additional 2D experiments ( H− H

1

13

1

13

COSY, H− C HSQC, H− C HMBC). High resolution mass spectro-

metry and elemental analyses were performed at the UBC Chemistry

Mass Spectrometry and Microanalysis Services. Low resolution mass

spectra were obtained using a Bruker Esquire ion trap ESI-MS spectro-

meter. High resolution mass spectra were obtained on a Waters/

Micromass LCT spectrometer. Elemental analyses were performed on

a Carlo Erba elemental analyzer EA 1108. Semipreparative high-

performance liquid chromatography (HPLC) was performed on a

system outﬁtted with a Waters W600 solvent gradient controller and a

Waters 2487 dual wavelength absorbance detector set, and a XTerraRP

column (187 μm, 19 mm × 300 mm) was used. Solvent ﬂow rate was

5

heated using a heat gun until complete dissolution. The reaction was

carried out in a microwave reactor for 30 min at 165 °C. After the vial

was cooled to room temperature, aqueous NaOH (1 N, 10 mL) was

added and the mixture was extracted with methylene chloride (3 ×

1

5 mL). The organic fractions were combined and successively washed

with water (3 × 10 mL) and brine (3 × 10 mL). The organic layer was

dried over anhydrous MgSO , and the solvent was removed under

4

reduced pressure. The respective products (3a−e) were obtained by

precipitation after the addition of a 8:2 hexane/methylene chloride

solution.

1

0 mL/min with a 25 min gradient from 100% H O (0.1% triﬂuoro-

2

N-(7-Chloroquinolin-4-yl)ethane-1,2-diamine (3a). This com-

acetic acid, TFA) to 100% CH CN. X-ray data were collected and

3

1

pound was obtained as a yellow solid (0.74 g, 3.33 mmol, 65%). H

processed using a Bruker X8 APEX II diﬀractometer with graphite

monochromated Mo Kα radiation. The structures were solved using

the Bruker SAINT software package (SAINT, version 7.60A, Bruker

AXS Inc., Madison, WI, U.S., 1997−2009). All non-hydrogen atoms

were reﬁned anisotropically. All hydrogen atoms were placed in

calculated positions. All N−H hydrogen atoms were located in

diﬀerence maps and reﬁned isotropically. All other hydrogen atoms

were included in calculated positions but not reﬁned. Further crystal

structure exploration and analysis were conducted using the Mercury

software package (version.2.3, CCDC, Cambridge, U.K.).

3

NMR (300 MHz, CDCl ): δ (ppm) 8.52 (d, J = 5.5 Hz, 1H), 7.95 (d,

3

4

3

4

J = 2.1 Hz, 1H), 7.74 (d, J = 9.1 Hz, 1H), 7.35 (dd, J = 9.1 Hz, J =

3

2.1 Hz, 1H), 6.40 (d, J = 5.5 Hz, 1H), 5.82 (br s, 1H), 3.32 (m, 2H),

3.12 (m, 2H), 1.48 (br s, 2H). ¹³C NMR (75 MHz, CDCl

): δ (ppm)

3

152.2, 150.1, 149.3, 135.0, 128.9, 125.4, 121.4, 117.6, 99.4, 44.9, 40.4.

IR (ATR): ν 3247 (broad), 2894 (w), 2852 (w), 1582 (s), 1541 (s),

1454 (m), 1426 (m), 1340 (m), 1325 (m), 1163 (w), 1141 (w), 1020

−1

(w), 867 (m), 800 (s), 758 (m) [cm ]. Anal. Calcd for C11

H12ClN : C,

3

59.60, H, 5.46, N, 18.95. Found: C, 59.34, H, 5.42, N, 18.87. ESI-MS

+

1

,1′-Bis(N,N′-dimethylaminomethyl)ferrocene (1). This com-

(HR) calculated for C11H13ClN : 222.0798. Found: 222.0793 [M + H] .

3

67

pound was prepared as previously reported with slight modiﬁcations.

n-Butyllithium (1.6 M in hexanes, 32.8 mL, 52.5 mmol, 2.1 equiv) was

added dropwise to a stirred solution of freshly distilled TMEDA

N-(7-Chloroquinolin-4-yl)propane-1,3-diamine (3b). This

compound was obtained as a beige solid (1.08 g, 4.55 mmol, 89%).

1

3

H NMR (300 MHz, CDCl

): δ (ppm) 8.50 (d, J = 5.3 Hz, 1H), 7.93

3

4

3

(

2

7.83 mL, 52.5 mmol, 2.1 equiv) in hexanes (10 mL). Ferrocene (4.65 g,

5.0 mmol, 1.0 equiv) was dissolved in hexanes (180 mL) and added

(d, J = 1.9 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.44 (br s, 1H), 7.31

(dd, J = 9.0 Hz, J = 1.9 Hz, 1H), 6.33 (d, J = 5.3 Hz, 1H), 3.42 (m,

3

4

3

dropwise by syringe over 1 h. The reaction mixture was stirred under

inert atmosphere at room temperature for 12 h. The solution was

further diluted with THF (200 mL), and (N,N′-dimethyl)methylene-

ammonium iodide (9.7 g, 52.5 mmol, 2.1 equiv) was added in one

portion. The reaction mixture was heated under reﬂux for 10 min and

then allowed to cool to room temperature and stirred under inert

atmosphere for 24 h. Water (30 mL) was added to terminate the

reaction. The organic layer was separated, and the aqueous layer was

extracted with portions of diethyl ether (3 × 25 mL). The combined

2H), 2.87 (t, J = 5.7 Hz, 2H), 1.90 (m, J = 5.7 Hz, 2H), 1.65 (br s,

2H). ¹³C NMR (75 MHz, CDCl

): δ (ppm) 152.3, 150.6, 149.4,

3

134.8, 128.7, 125.1, 122.2, 117.7, 98.4, 43.9, 41.7, 30.1. IR (ATR):

ν 3255 (broad), 2934 (w), 2866 (w), 2238 (w), 1580 (s), 1542 (m),

1474 (w), 1434 (m), 1371 (m), 1331 (m), 1284 (m), 1202 (w), 1137

−1

(m), 1078 (w), 899 (s), 851 (s), 800 (s), 818 (m), 763 (s) [cm ].

+

ESI-MS (HR) calculated for C12

[M + H] .

H

15ClN

: 236.0955. Found: 236.0953

3

+

N-(7-Chloroquinolin-4-yl)butane-1,4-diamine (3c). This com-

1

organic fractions were dried over anhydrous MgSO and the solvent

pound was obtained as an oﬀ-white solid (0.62 g, 2.48 mmol, 48%). H

4

3

was removed under reduced pressure to aﬀord an orange-brown oil.

Puriﬁcation of the desired product was done by ﬂash column

chromatography on neutral alumina Brockman activity V. Elution was

achieved with petroleum ether to obtain unreacted ferrocene, with

diethyl ether/petroleum ether (3:7) to obtain the monodimethylami-

nomethyl derivative, and with methanol/diethyl ether (3:97) to obtain

NMR (400 MHz, CDCl

3

): δ (ppm) 8.53 (d, J = 5.5 Hz, 1H), 7.95 (d,

4

3

4

J = 2.1 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.34 (dd, J = 8.8, J = 2.1

Hz, 1H), 6.39 (d, J = 5.5 Hz, 1H), 6.00 (br s, 1H), 3.32 (m, 2H), 2.84

3

(t, J = 6.5 Hz, 2H), 1.88 (quin, J = 6.9 Hz, 2H), 1.67 (quin, J = 6.9

Hz, 2H), 1.44 (br s, 2H). ¹³C NMR (101 MHz, CDCl ): δ (ppm)

3

152.1, 149.2, 144.1, 134.7, 128.8, 125.0, 121.4, 117.3, 98.8, 43.2, 41.5,

30.8, 26.1. IR (ATR): ν 3247 (broad), 2930 (w), 2858 (w), 1576 (s),

1540 (m), 1449 (m), 1431 (m), 1368 (s), 1331 (m), 1284 (w), 1252

(m), 1198 (w), 1130 (m), 972 (w), 898 (s), 849 (s), 817 (m), 794 (s),

1

product 1 as an orange-brown oil (2.64 g, 8.79 mmol, 35%). H NMR

3

(

400 MHz, CDCl ): δ (ppm) 4.10 (t, J = 1.71 Hz, 4H), 4.07 (t, J =

3

1

.71 Hz, 4H), 3.27 (s, 4H), 2.17 (s, 12H).

−

1

+

1

,1′-Bis(N,N′-trimethylaminomethyl)ferrocene iodide (2).

766 (s) [cm ]. ESI-MS (HR) calculated for C H ClN : 250.1111.

13 17 3

67

+

On the basis of a previously published method, iodomethane (1.4 mL,

2.4 mmol, 3 equiv) was added slowly to a solution of 1,1′-bis(N,N-

Found: 250.1114 [M + H] .

2

N-(7-Chloroquinolin-4-yl)propane-1,2-diamine (3d). This

dimethylaminomethyl)ferrocene (1, 2.24 g, 7.47 mmol, 1 equiv) in

methanol (40 mL) under inert atmosphere. The resulting solution was

heated at reﬂux for 10 min before cooling to room temperature.

Diethyl ether (150 mL) was then added and the product precipitated

as a pale yellow solid, which was collected by ﬁltration, washed with

compound was obtained as an oﬀ-white solid (0.68 g, 2.88 mmol,

1

3

56%). H NMR (400 MHz, CDCl ): δ (ppm) 8.53 (d, J = 5.5 Hz,

3

4

3

1H), 7.96 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.38 (dd, J =

8.9 Hz, J = 2.0 Hz, 1H), 6.40 (d, J = 5.5 Hz, 1H), 5.85 (br s, 1H),

3.32 (m, 2H), 3.00 (m, 1H), 1.64 (s, 2H), 1.27 (d, J = 6.1 Hz, 3H).

4

3

M

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

3

−1

C NMR (101 MHz, CDCl ): δ (ppm) 152.4, 150.2, 149.5, 135.1,

(m), 817 (s), 801 (s), 767 (m), 643 (m), 620 (m) [cm ]. Anal. Calcd

for C23 24ClFeN : C, 63.69, H, 5.58, N, 9.69. Found: C, 63.67, H,

3

1

(

29.1, 125.6, 121.5, 117.7, 99.5, 50.3, 46.1, 23.3. IR (ATR): ν 3258

broad), 3010 (w), 2962 (w), 1576 (s), 1538 (m), 1490 (w), 1451

m), 1368 (m), 1334 (m), 1312 (w), 1282 (w), 1254 (w), 1198 (w),

H

3

+

5.59, N, 9.62. ESI-MS (HR) calculated for C23

H

25ClFeN

: 434.1086.

3

+

Found: 434.1089 [M + H] .

1

135 (m), 1085 (w), 1014 (w), 959 (m), 919 (s), 901 (s), 846 (s), 794

N-(7-Chloro-4-quinolyl)-N′-ferrocenyl-butane-1,4-diamine

−1

(

s), 765 (m) [cm ]. Anal. Calcd for C H ClN : C, 61.15, H, 5.99,

(4c). This product was obtained as a light yellow solid (0.323 g,

12 14 3

1

3

N, 17.83. Found: C, 61.22, H, 5.96, N, 17.76. ESI-MS (HR) calculated

for C H ClN : 236.0955. Found: 236.0948 [M + H] .

0.72 mmol, 72%). H NMR (300 MHz, CDCl

3

): δ (ppm) 8.52 (d, J =

+

3

+

4

3

5.5 Hz, 1H, 11-H), 7.95 (d, J = 2.1 Hz, 1H, 13-H), 7.74 (d, J = 8.9

Hz, 1H, 16-H), 7.31 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H, 15-H), 6.37 (d,

J = 5.5 Hz, 1H, 10-H), 6.07 (br, 1H, Ar-NH-), 4.20 (d, J = 1.8 Hz,

12

15

3

4

N-(7-Chloroquinolin-4-yl)-2,2-dimethylpropane-1,3-diamine

3

(

3e). This compound was obtained as a yellowish solid (0.70 g, 2.62

1

mmol, 52%). H NMR (300 MHz, CDCl ): δ (ppm) 8.48 (br s, 1H),

2H, 2,3-H), 4.14 (s, 5H, Cp-H), 3.59 (s, 2H, 4-H), 3.30 (m, 2H, 8-H),

3

4

3

8

9

5

.47 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.72 (d, J =

.0 Hz, 1H), 7.29 (dd, J = 9.0 Hz, J = 2.2 Hz, 1H), 6.27 (d, J =

2

1

(

.76 (m, 2H, 5-H), 2.32 (br s, 1H, CH NHCH ), 1.86 (m, 2H, 7-H),

2 2

3

4

3

.72 (m, 2H, 6-H). ¹³C NMR (75 MHz, CDCl ): δ (ppm) 155.5

3

.4 Hz, 1H), 3.18 (br s, 2H), 2.87 (br s, 2H), 1.68 (br s, 2H), 1.08

C11), 155.1 (C9), 151.9 (C12), 134.8 (C14), 128.5 (C13), 125.1

(C15), 121.6 (C16), 117.3 (C17), 98.8 (C10), 85.3 (C1), 68.6 (C2),

8.5 (Cp), 68.1 (C3), 48.8 (C4), 48.1 (C8), 43.1 (C5), 27.3 (C7),

6.1 (C6). IR (ATR): ν 3227 (w), 2918 (w), 2847 (w), 1581 (s), 1429

(

s, 6H). ¹³C NMR (75 MHz, CDCl ): δ (ppm) 152.3, 151.1, 149.4,

3

1

(

1

(

34.7, 128.6, 124.9, 122.5, 117.9, 98.0, 55.9, 52.9, 34.0, 24.7. IR

ATR): ν 3169 (broad), 2959 (w), 2839 (w), 1579 (s), 1541 (m),

448 (m), 1429 (m), 1364 (m), 1312 (m), 1277 (m), 1246 (w), 1202

m), 1138 (m), 1073 (w), 899 (m), 872 (m), 848 (m), 793 (m), 764

6

2

(

m), 1384 (w), 1358 (m), 1330 (m), 1277 (m), 1232 (m), 1198 (w),

1

139 (m), 1091 (m), 1077 (m), 1019 (w), 1000 (m), 971 (m), 871

−1

(m), 675 (m), 638 (s) [cm ]. Anal. Calcd for C H ClN : C, 63.75,

−1

14

18

3

(s), 850 (m), 801 (s), 765 (m), 642 (m) [cm ]. Anal. Calcd for

C H ClFeN : C, 64.37, H, 5.85, N, 9.38. Found: C, 64.01, H, 6.11,

H, 6.88, N, 15.93. Found: C, 63.16, H, 6.83, N, 15.85. ESI-MS (HR)

calculated for C H ClN : 264.1268. Found: 264.1270 [M + H] .

2

4

26

3

+

14

19

3

N, 8.98. ESI-MS (HR) calculated for C H ClFeN : 448.1243.

Found: 448.1249 [M + H] .

24 27 3

General Procedure for the Synthesis of Monosubstituted

+

Ferrocenyl 4-Aminoquinoline Derivatives (4a−e). These com-

pounds were prepared as previously reported (with the exception of

4

1

N-(7-Chloro-4-quinolyl)-N′-ferrocenyl-propane-1,2-diamine

(

4d). This product was obtained as a yellow solid (0.366 g, 0.84 mmol,

7

d) with modiﬁcations. Ferrocenecarboxaldehyde (0.214 g, 1.0 mmol,

equiv) and the corresponding 4-aminoquinoline derivatives 3a−e

1

3

8

1

5

(

4

(

1

9

4

2

4%). H NMR (300 MHz, CDCl ): δ (ppm) 8.53 (d, J = 5.3 Hz,

3

4

3

H, 10-H), 7.96 (d, J = 2.0 Hz, 1H, 12-H), 7.66 (d, J = 8.9 Hz, 1H,

5-H), 7.36 (dd, J = 8.9 Hz, J = 2.0 Hz, 1H, 14-H), 6.37 (d, J =

.5 Hz, 1H, 9-H), 5.94 (br s, 1H, Ar-NH-), 4.21 (s, 1H, 2-H), 4.18

(

1.10 mmol, 1.1 equiv) were dissolved in dry methanol (20 mL). The

3

4

3

mixture was stirred at room temperature overnight. Sodium boro-

hydride (0.114 g, 3.0 mmol, 3 equiv) was added, and the resulting

mixture was stirred for an additional 2 h. The reaction mixture was

quenched with the addition of 5% NaHCO solution (10 mL), and the

resulting mixture was extracted with diethyl ether (3 × 20 mL). The

combined organic fractions were dried over anhydrous Na SO , and

solvent was removed under reduced pressure. Puriﬁcation of the

desired product was done by ﬂash column chromatography on silica

with a mixture of ethyl acetate/methanol/TEA in increasing polarity,

s, 1H, 3-H), 4.14 (s, 2H, 2′,3′-H), 4.13 (s, 5H, Cp-H), 3.59 (m, 2H,

-H), 3.32 (m, 1H, 5-H), 3.15 (m, 1H, 5′-H), 3.02 (m, 1H, 6-H), 1.58

3

13

br s, 1H, CH NHCH ), 1.27 (d, J = 6.1 Hz, 3H, 7-H). C NMR

2 2

75 MHz, CDCl ): δ (ppm) 150.8 (C10), 150.3 (C8), 141.9 (C11),

3

2

4

35.6 (C13), 126.9 (C12), 125.6 (C14), 122.2 (C15), 117.1 (C16),

8.6 (C9), 89.9 (C1), 68.8 (C2), 68.6 (Cp), 68.4 (C3), 50.5 (C4),

6.7 (C6), 45.4 (C5), 17.5 (C7). IR (ATR): ν 3286 (b), 3087 (w),

918 (w), 2849 (w), 1576 (s), 1528 (m), 1447 (m), 1368 (m), 1330

94:4:2 to 75:15:10 ratios.

(

9

m), 1275 (w), 1238 (m), 1200 (w), 1134 (m), 1104 (m), 1077 (m),

N-(7-Chloro-4-quinolyl)-N′-ferrocenyl-ethane-1,2-diamine

−1

99 (m), 876 (m), 847 (m), 803 (s), 765 (m), 642 (m) [cm ]. Anal.

(

4a). This product was obtained as an orange solid (0.254 g, 0.61

1

3

Calcd for C H ClFeN : C, 63.69, H, 5.58, N, 9.69. Found: C, 63.69,

mmol, 61%). H NMR (300 MHz, CDCl ): δ (ppm) 8.52 (d, J =

23 24 3

3

+

4

3

H, 5.94, N, 9.55. ESI-MS (HR) calculated for C H ClFeN :

23 25 3

434.1086. Found: 434.1090 [M + H] .

5

1

5

2

3

.4 Hz, 1H, 9-H), 7.95 (d, J = 2.1 Hz, 1H, 11-H), 7.70 (d, J = 8.9 Hz,

+

3

4

3

H, 14-H), 7.35 (dd, J = 8.9 Hz, J = 2.1 Hz, 1H, 13-H), 6.38 (d, J =

.4 Hz, 1H, 8-H), 5.91 (br, 1H, Ar-NH-), 4.19 (m, 2H, 2-H), 4.14 (m,

H, 3-H), 4.13 (s, 5H, Cp-H), 3.59 (s, 2H, 4-H), 3.31 (m, 2H, 6-H),

N-(7-Chloro-4-quinolyl)-N′-ferrocenyl-2,2-dimethylpropane-

1

,3-diamine (4e). This product was obtained as a light yellow solid

1

13

(0.313 g, 0.68 mmol, 68%). H NMR (300 MHz, CDCl ): δ (ppm)

3

.05 (m, 2H, 5-H), 1.92 (vb, 1H, CH NHCH ). C NMR (75 MHz,

2

3

4

8

2

.56 (br, 1H, Ar-NH-), 8.47 (d, J = 5.4 Hz, 1H, 12-H), 7.90 (d, J =

.0 Hz, 1H, 14-H), 7.60 (d, J = 8.9 Hz, 1H, 17-H), 7.19 (dd, J = 8.9

Hz, J = 2.0 Hz, 1H, 16-H), 6.25 (d, J = 5.4 Hz, 1H, 11-H), 4.24 (m,

CDCl ): δ (ppm) 152.3 (C9), 150.1 (C7), 149.3 (C10), 135.0 (C12),

3

1

28.8 (C11), 125.4 (C13), 121.6 (C14), 117.6 (C15), 99.4 (C8), 86.5

4

3

(C1), 68.7 (Cp), 68.6 (C2), 68.2 (C3), 48.5 (C4), 47.0 (C6), 42.1

(C5). IR (ATR): ν 3245 (broad), 3083 (w), 2924 (w), 2851 (w), 1577

(s), 1532 (m), 1449 (m), 1367 (m), 1328 (m), 1277 (w), 1231 (w),

2

3

1

H, 2-H), 4.23 (m, 2H, 3-H), 4.15 (s, 5H, Cp-H), 3.58 (br, 2H, 4-H),

.14 (m, 2H, 7-H), 2.77 (br, 2H, 5-H), 1.50 (vb, 1H, CH NHCH ),

2

.09 (s, 6H, 8,9-H). ¹³C NMR (75 MHz, CDCl ): δ (ppm) 152.4

1

6

1

138 (w), 1104 (m), 1079 (w), 1023 (w), 999 (m), 803 (s), 765 (m),

42 (m) [cm ]. Anal. Calcd for C H ClFeN : C, 62.95, H, 5.28, N,

0.01. Found: C, 62.72, H, 5.38, N, 9.82. ESI-MS (HR) calculated for

3

−1

(C12), 151.1 (C10), 149.4 (C13), 134.6 (C15), 128.6 (C14), 125.0

C16), 123.0 (C17), 117.0 (C18), 97.9 (C11), 85.9 (C1), 69.1 (C2),

68.7 (Cp), 68.5 (C3), 61.3 (C7), 56.1 (C4), 50.4 (C5), 33.8 (C6),

5.2 (C8-9). IR (ATR): ν 3081 (w), 2952 (w), 1578 (s), 1465 (m),

1444 (m), 1432 (m), 1380 (w), 1361 (m), 1330 (m), 1310 (m), 1272

(w), 1232 (w), 1202 (w), 1166 (w), 1137 (m), 1086 (m), 1030 (m),

22

3

(

+

C H ClFeN : 420.0930. Found: 420.0931 [M + H] .

22

23

3

2

N-(7-Chloro-4-quinolyl)-N′-ferrocenyl-propane-1,3-diamine

(

4b). This product was obtained as a yellow solid (0.356 g, 0.82 mmol,

2%). H NMR (300 MHz, CDCl ): δ (ppm) 8.50 (d, J = 5.4 Hz,

H, 10-H), 7.96 (br, 1H, Ar-NH-), 7.93 (d, J = 2.0 Hz, 1H,

1

3

8

1

2

4

2

3

4

999 (w), 901 (m), 849 (m), 801 (s), 768 (m), 701 (m), 645 (m)

3

4

−1

2-H), 7.62 (d, J = 8.9 Hz, 1H, 15-H), 7.27 (dd, J = 8.9 Hz, J =

[cm ]. Anal. Calcd for C25

H

28ClFeN

3

: C, 65.02, H, 6.11, N, 9.10.

3

.0 Hz, 1H, 14-H), 6.29 (d, J = 5.4 Hz, 1H, 9-H), 4.24 (m, 2H, 2-H),

.22 (m, 2H, 3-H), 4.17 (s, 5H, Cp-H), 3.62 (s, 2H, 4-H), 3.38 (m,

H, 7-H), 2.98 (m, 2H, 5-H), 1.92 (m, 2H, 6-H), 1.80 (vb, 1H,

Found: C, 64.91, H, 6.38, N, 8.62. ESI-MS (HR) calculated for

+

C H ClFeN : 462.1399. Found: 462.1413 [M + H] .

25 29 3

General Procedure for the Synthesis of Bridged Ferrocenyl

13

CH NHCH ). C NMR (75 MHz, CDCl ): δ (ppm) 152.4 (C10),

4-Aminoquinoline Derivatives (5a−e). Method A. 1,1′-Bis(N,N′-

trimethylaminomethyl)ferrocene iodide 2 (0.10 g, 0.17 mmol, 1 equiv),

the corresponding 4-aminoquinoline derivatives 3a−e (0.34 mmol,

2 equiv), and NaOH (0.04 g, 1 mmol, 6 equiv) were combined with

20 mL of acetonitrile in a 20 mL microwave vial. The reaction was

carried out in a microwave reactor for 6 h at 110 °C. The solvent was

removed under reduced pressure.

2

3

150.8 (C8), 149.4 (C11), 134.7 (C13), 128.7 (C12), 125.1 (C14),

122.7 (C15), 117.8 (C16), 98.3 (C9), 86.0 (C1), 68.9 (C2), 68.7

(

Cp), 68.4 (C3), 49.8 (C4), 49.7 (C7), 44.5 (C5), 27.4 (C6). IR

ATR): ν 3198 (w), 3088 (w), 2945 (w), 2826 (w), 1578 (s), 1539

m), 1490 (w), 1445 (m), 1432 (m), 1364 (m), 1327 (m), 1283 (m),

1240 (m), 1139 (m), 1102 (m), 1056 (w), 1000 (m), 899 (w), 854

N

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Method B. 1,1′-Bis(N,N′-trimethylaminomethyl)ferrocene iodide 2

126.6 (C14), 123.9 (C15), 118.8 (C13), 100.5 (C9), 84.5 (C1), 71.2

(C2), 71.0 (C2′), 70.4 (C3, C3′), 59.7 (C4), 47.5 (C5), 31.2 (C6),

12.5 (C7). IR (ATR): ν 3380 (w), 2918 (m), 2849 (m), 2181 (w),

1664 (m), 1608 (m), 1579 (s), 1524 (m), 1470 (m), 1448 (m), 1379

(m), 1331 (m), 1298 (w), 1260 (w), 1234 (m), 1158 (m), 1088 (m),

1028 (m), 966 (w), 940 (w), 924 (w), 878 (m), 848 (m), 801 (s), 760

(

0.10 g, 0.17 mmol, 1 equiv), the corresponding 4-aminoquinoline

derivatives 3a−e (0.34 mmol, 2 equiv), and NaOH (0.04 g, 1 mmol, 6

equiv) were reﬂuxed in acetonitrile (20 mL) for 48 h. Semipuriﬁcation

of the desired product was done by ﬂash column chromatography on

silica (5a,b,d,e) or on preparative TLC silica gel plate (5c) with an

acetone/dichloromethane/triethylamine (5:4:1) mixture as eluent. The

products were puriﬁed to >95% by HPLC using a linear gradient of

−1

+

(m) [cm ]. ESI-MS (HR) calculated for C H ClFeN : 446.1086.

24

25

3

+

Found: 446.1075 [M + H] .

1

00% water to 100% acetonitrile.

N-{[N′-(7-Chloroquinolin-4-yl)]ethane-1,2-diamine}-1,1′-

N-{[N′-(7-Chloroquinolin-4-yl)]-2,2-dimethylpropane-1,3-di-

amine}-1,1′-(N,N-dimethane-yl)ferrocene (5e). This product was

1

(

N,N-dimethane-yl)ferrocene (5a). This product was obtained as

obtained as a yellow solid (0.004 g, 0.009 mmol, 5%). H NMR (400

1

3

a yellow solid (0.016 g, 0.037 mmol, 22%). H NMR (600 MHz,

MHz, CD

3

OD): δ (ppm) 8.39 (d, J = 6.1 Hz, 1H, 12-H), 8.25 (d, J =

3

4

3

CD OD): δ (ppm) 8.41 (d, J = 5.6 Hz, 1H, 9-H), 8.13 (d, 3J =

8.9 Hz, 1H, 17-H), 7.82 (d, J = 2.4 Hz, 1H, 14-H), 7.50 (dd, J = 8.9,

J = 2.0 Hz, 1H, 16-H), 6.76 (d, J = 6.1 Hz, 1H, 11-H), 4.13 (m, 4H,

2-H), 4.08 (m, 4H, 3-H), 3.46 (m, 2H, 5-H), 3.04 (m, 2H, 7-H), 2.69

3

4

3

4

3

8

.7 Hz, 1H, 14-H), 7.82 (d, J = 2.0 Hz, 1H, 11-H), 7.49 (dd, J = 8.7,

4

3

J = 2.0 Hz, 1H, 13-H), 6.66 (d, J = 5.6 Hz, 1H, 8-H), 4.10 (t, 4H,

3

13

2

6

-H), 4.07 (t, 4H, 3-H), 3.65 (t, J = 6.4 Hz, 2H, 6-H), 3.13 (t, J =

(m,4H, 4-H), 1.16 (s, 6H, 8,9-H). C NMR (101 MHz, CD OD): δ

3

.7 Hz, 2H, 5-H), 3.06 (br s, 4H, 4-H). ¹³C NMR (151 MHz, CD OD):

(ppm) 161.8 (C10), 156.3 (C12), 148.4 (C18), 136.8 (C13), 129.0

(C14), 126.6 (C16), 124.6 (C17), 122.1 (C15), 100.3 (C9), 85.6

(C1), 70.7 (C2), 70.2 (C3), 61.7 (C4), 52.7 (C7), 39.9 (C5), 32.2

(C6), 14.6 (C8,9). IR (ATR): ν 3346 (w), 3180 (w), 2878 (m), 2182

(w), 1670 (m), 1600 (m), 1576 (s), 1520 (m), 1460 (m), 1438 (m),

1374 (m), 1330 (m), 1294 (w), 1260 (w), 1222 (m), 1146 (m), 1088

3

δ (ppm) 158.4 (C7), 149.4 (C9), 148.8 (C15), 133.4 (C10), 124.0

(

C11), 123.0 (C13), 120.6 (C14), 115. 4 (C12), 96.6 (C8), 80.7 (C1),

6

3

7.5 (C3), 66.8 (C2), 52.8 (C5), 49.7 (C4), 38.5 (C6). IR (ATR): ν

228 (w), 3014 (w), 2958 (w), 2918 (m), 2180 (w), 1659 (m), 1613

(

m), 1580 (s), 1453 (m), 1433 (m), 1380 (m), 1302 (m), 1228 (m),

−1

1

8

149 (m), 1112 (m), 1086 (w), 1026 (m), 969 (w), 930 (w), 901 (w),

77 (m), 849 (m), 797 (s), 646 (m) [cm ]. ESI-MS (HR) calculated

(m), 1020 (m), 956 (m), 936 (w), 838 (m), 804 (s), 765 (m) [cm ].

−1

+

ESI-MS (HR) calculated for C26

474.1398 [M + H] .

H

29ClFeN

: 474.1399. Found:

3

+

for C H ClFeN : 432.0930. Found: 432.0927 [M + H] .

23

3

N-{[N′-(7-Chloroquinolin-4-yl)]propane-1,3-diamine}-1,1′-

In Vitro Antiplasmodial Activity Studies. The test samples were

tested in triplicate on one, two, or three occasions against the

chloroquine-sensitive (CQS) D10 strain and the chloroquine-resistant

(CQR) Dd2 and K1 strains of Plasmodium falciparum. Continuous in

vitro cultures of asexual erythrocyte stages of P. falciparum were

maintained using a modiﬁed method of Trager and Jensen. Quan-

titative assessment of antiplasmodial activity in vitro was determined

(

N,N-dimethane-yl)ferrocene (5b). This compound can be further

puriﬁed by recrystallization in an acetone/dichloromethane (1:1)

1

mixture, yielding orange crystals (0.015 g, 0.03 mmol, 20%). H NMR

3

(

600 MHz, CD OD): δ (ppm) 8.37 (d, J = 5.6 Hz, 1H, 10-H), 8.14

3

4

69

(

d, J = 8.7 Hz, 1H, 15-H), 7.78 (d, J = 2.0 Hz, 1H, 12-H), 7.42 (dd,

J = 8.7 Hz, J = 2.0 Hz, 1H, 14-H), 6.63 (d, J = 5.6 Hz, 1H, 9-H),

3

4

3

4

.10 (t, 4H, 2-H), 4.05 (t, 4H, 3-H), 3.54 (t, 2H, 5-H), 2.96 (br s, 4H,

via the parasite lactate dehydrogenase assay using a modiﬁed method

described by Makler. The test samples were prepared as a 2 mg/mL

13

70

4-H), 2.87 (t, 2H, 7-H), 2.09 (m, 2H, 6-H). C NMR (151 MHz,

CD OD): δ (ppm) 153.4 (C8), 152.4 (C10), 149.7 (C16), 136.8

stock solution in 10% DMSO or 20 mg/mL in 100% DMSO and

sonicated to enhance solubility. Samples of 4b, 4d, 4e, and 5b were

used as suspensions in the assay, leading to a tendency to under-

estimate the IC50 values, so the given results are conservative

calculations. Stock solutions were stored at −20 °C. Further dilutions

were prepared on the day of the experiment. Chloroquine (CQ) was

used as the reference drug in all experiments. Test samples were

initially tested at three concentrations (10, 5, and 2.5 μg/mL). CQ was

tested at three concentrations (30, 15, and 7.5 ng/mL). A full dose−

response was performed for all compounds to determine the

concentration inhibiting 50% of parasite growth (IC ). For D10,

test samples were tested at a starting concentration of 100 μg/mL,

which was then serially diluted 2-fold in complete medium to give 10

concentrations, with the lowest concentration being 0.2 μg/mL. The

same dilution technique was used for all samples. CQ was tested at a

starting concentration of 100 ng/mL. Several compounds were retested

at a starting concentration of either 1000 ng/mL or 100 ng/mL. For Dd2

and K1, samples were tested at a starting concentration of 1000 ng/mL,

which was then serially diluted 2-fold in complete medium to give

10 concentrations, with the lowest concentration being 2 ng/mL. The

same dilution technique was used for all samples. CQ was tested at a

starting concentration of 1000 ng/mL. The highest concentration of

solvent to which the parasites were exposed had no measurable eﬀect

on the parasite viability (data not shown). The IC50 values were

obtained using a nonlinear dose−response curve ﬁtting analysis via

Graph Pad Prism, version 4.0, software.

3

(

C11), 127.6 (C12), 126.4 (C14), 124.7 (C15), 119.0 (C13), 100.0

C9), 84.3 (C1), 71.2 (C2), 70.5 (C3), 56.5 (C7), 53.5 (C4), 42.6

C5), 27.7 (C6). IR (ATR): ν 3226 (w), 3092 (w), 2919 (s), 2850

m), 2188 (w), 1611 (m), 1575 (s), 1452 (s), 1367 (m), 1318 (w),

1

282 (w), 1238 (w), 1214 (m), 1168 (m), 1103 (m), 1027 (m), 964

−1

(

w), 930 (w), 902 (w), 880 (m), 849 (m), 801 (s), 768 (m) [cm ].

+

ESI-MS (HR) calculated for C H ClFeN : 446.1086. Found:

2

4

25

3

+

4

46.1093 [M + H] .

N-{[N′-(7-Chloroquinolin-4-yl)]butane-1,4-diamine}-1,1′-

N,N-dimethane-yl)ferrocene (5c). This product was obtained as a

(

5

0

1

yellow solid (0.015 g, 0.033 mmol, 19%). H NMR (400 MHz,

CD OD): δ (ppm) 8.38 (d, J = 6.0 Hz, 1H, 11-H), 8.19 (d, J = 8.8

Hz, 1H, 16-H), 7.80 (d, J = 2.2 Hz, 1H, 13-H), 7.48 (dd, J = 9.1, J =

3

4

3

4

3

2

4

2

.2 Hz, 1H, 15-H), 6.67 (d, J = 6.2 Hz, 1H, 10-H), 4.10 (m, 4H, 2-H),

.07 (m, 4H, 3-H), 3.53 (m, 2H, 8-H), 3.04 (br s, 4H, 4-H), 2.89 (m,

H, 5-H), 1.94 (s, 2H, 7-H), 1.88 (m, 2H, 6-H). C NMR (101 MHz,

13

CD OD): δ (ppm) 152.6 (C9), 149.2 (C11), 146.5 (C17), 136.1

3

(

C14), 125.4 (C15), 124.8 (C13), 123.4 (C16), 117.5 (C12), 98.5

C10), 81.8 (C1), 69.7 (C2), 69.2 (C3), 57.1 (C5), 51.7 (C4), 42.8

C8), 26.0 (C7), 24.3 (C6). IR (ATR): ν 3234 (w), 3190 (w), 2919

m), 2850 (m), 2182 (w), 1664 (m), 1610 (m), 1578 (s), 1450 (m),

1430 (m), 1367 (m), 1334 (m), 1279 (w), 1254 (w), 1212 (w), 1200

(

w), 1100 (m), 1078 (m), 1027 (m), 848 (m), 803 (s), 765 (m)

−1

+

[

cm ]. ESI-MS (HR) calculated for C H ClFeN : 460.1243.

25 27 3

+

Found: 460.1255 [M + H] .

N-{[N′-(7-Chloroquinolin-4-yl)]propane-1,2-diamine}-1,1′-

In Vitro Antitumor Activity and Cytotoxicity Assay. Human

breast cancer cells MDA-MB-435S (HTB-129) were purchased from

ATCC. Cells were grown as monolayers in medium (89% Leibovitz’s

L-15 medium with 2 mM L-glutamine, 0.01 mg/mL bovine insulin, 1%

penicillin/streptomycin, and 10% fetal bovine serum) and maintained

at 37 °C in a humidiﬁed atmosphere. Human normal breast epithelial

cells MCF-10A (CRL-10317) were purchased from ATCC. Cells were

grown as monolayers in medium (93% Dulbecco’s modiﬁed Eagle

medium F12, 5% fetal bovine serum, 1% penicillin/streptomycin, 1%

2 mM glutamine, 0.01 μg/mL human epidermal growth factor, 0.5 μg/mL

(

N,N-dimethane-yl)ferrocene (5d). This product was obtained as a

1

yellow solid (0.018 g, 0.04 mmol, 23%). H NMR (600 MHz, CD OD):

3

δ (ppm) 8.41 (d, J = 5.4 Hz, 1H, 10-H), 8.13 (d, J = 8.9 Hz, 1H,

5-H), 7.84 (d, J = 2.3 Hz, 1H, 12-H), 7.53 (dd, J = 8.9, J = 1.9 Hz,

H, 14-H), 6.61 (d, J = 5.4 Hz, 1H, 9-H), 4.17 (m, 2H, 2-H), 4.07

4

3

4

1

(

6

(

3

m, 2H, 2′-H), 4.02 (m, 2H, 3-H), 3.99 (m, 2H, 3′-H), 3.57 (m, 1H,

-H), 3.46 (m, 2H, 5-H), 3.20 (m, 2H, 4-H), 2.99 (m, 2H, 4′-H), 1.26

2

13

d, J = 6.6 Hz, 3H, 7-H). C NMR (151 MHz, CD OD): δ (ppm)

3

1

57.9 (C8), 152.8 (C10), 150.0 (C16), 136.9 (C11), 128.1 (C12),

O

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

hydrocortisone, 10 μg/mL insulin) and maintained at 37 °C in a

solution of the test compounds were added to give 0−10 equiv relative

to hematin in the ﬁnal solution. After mixing, 11.74 μL of the 12.9 M

acetate solution maintained at 70 °C was added to each Eppendorf

tube. The ﬁnal hematin concentration was 1 mM, and the ﬁnal

solution pH was 4.5. The vortexed solutions were then incubated at

60 °C for 1 h. The solutions were quenched at room temperature with

900 μL of a 5% pyridine solution (200 mM HEPES, pH 8.2), followed

by addition of 1100 μL of 5% pyridine solution (20 mM HEPES, pH

7.5). Each tube was vortexed to ensure the complete dissolution of

hematin and then allowed to settle for 30 min at room temperature.

The supernatant was transferred to a cuvette without disturbing the

precipitate. Absorbances were read at 405 nm at a Cary 100 Bio

(Varian) UV−visible spectrophotometer. Graphs were produced using

the OriginPro 7.5 software.

Partition Coeﬃcient and Molecular Shape Analysis. The-

oretical log P values were calculated using the commercially available

ACD/Labs 11.0 program. Molecular modeling was performed using

the Gaussian 09 and GaussView packages (Gaussian, Inc.). Density

functional theory (DFT), with the B3LYP functional employing the

6-31+G(d,p) basis set, was used to obtain the optimized geometry and

the electron density. Frequency calculations were performed to verify

that the optimized structures were a minima, having no imaginary

frequencies. The electrostatic potential was mapped onto the

calculated electron density surface. Molden package software was

used to obtain the topological polar surface area (tPSA) from the

optimized structures.

humidiﬁed atmosphere containing 5% CO . Cell growth inhibition was

2

4

3

assessed using the colorimetric cell proliferation MTT assay. Cells

were detached from culture ﬂasks with 0.25% trypsin and 0.03%

EDTA (Sigma) and resuspended in fresh culture medium at a density

5

of 3 × 10 cells/mL (MDA-MB435S) or 1 × 10 cells/mL (MCF-10A).

By use of a Falcon 96-well, ﬂat-bottom plate, 100 μL of the cell sus-

pension was added to each of the wells. The cells were incubated for 24 h,

after which time the cells were treated with each compound (in triplicate)

at concentrations ranging from 0.1 to 200 μg/mL. To assist dissolution,

the complexes were ﬁrst dissolved in DMSO to provide stock solutions

and then serially diluted with medium to give the ﬁnal concentrations

(ﬁnal concentration of DMSO was 0.5%). A stock solution of chloroquine

diphosphate salt was prepared in 0.5% DMSO in medium and serially

diluted. Cisplatin was used as positive control, and stock solutions were

prepared in 0.5% DMSO in medium and serially diluted. After incubation

with the compounds for 72 h, 50 μL of a 2.5 mg/mL solution of MTT in

PBS was added to each well and further incubated for 3−4 h. The

supernatant was removed, and the cells were dissolved in 150 μL of

DMSO. Cell survival was determined by means of metabolic reduction of

MTT. Optical density from the MTT assay was measured at 570 nm on a

Beckman Coulter DTX 880 multimode detector. Absorbance values were

corrected to the control and blank. Tests were run in duplicate or

triplicate. The IC₅₀values were obtained from plots of cell survival (%)

against log of drug concentration using a nonlinear dose−response curve

ﬁtting analysis via Graph Pad Prism, version 4.0, software.

Hematin Association Assay. The association constants of the

studied compounds with hematin were measured as described

ASSOCIATED CONTENT

■

50

previously in the literature. Hemin (bovine, ≥90.0%) was obtained

*

S

Supporting Information

from Sigma-Aldrich, and a stock solution was prepared by dissolving

1

Additional H NMR data, H NMR spectra, antiplasmodial

data, survival and association plots, therapeutic indices, and

molecular electronic potential surfaces. This material is

available free of charge via the Internet at http://pubs.acs.org.

6

−8 mg of hemin in 10 mL of AR grade DMSO and stored in the

dark. The working hemin solution (2 μM) was prepared fresh from

0 μL of the hemin stock solution diluted to a ﬁnal volume of 10 mL

2

with the buﬀered solution (40% v/v DMSO, 0.020 M HEPES,

apparent pH 7.5). Hematin−compound interactions were monitored

by titrating the hematin solution with each compound and measuring

the absorbance of the Soret band at 402 nm. In a UV−vis cell, 2 mL of

the 2 μM hematin solution was titrated with a 2 mM solution of the

compound in the same buﬀered solution (40% v/v DMSO, 0.020 M

HEPES, apparent pH 7.5). The temperature was maintained at 25 °C

using a water circulating bath in a thermostated cell holder, and

absorbance readings were recorded for each addition, after 5 min of

stabilization, using a Hewlett-Packard 8543 diode array spectropho-

tometer outﬁtted with a Fisher Scientiﬁc 1016D Isotemp thermostated

water cooling system. Absorbance data were collected using the

Agilent 845x UV−visible spectroscopy system (Agilent Technologies,

Inc., 2003−2008). Diﬀerent concentrations of compound were

analyzed (0, 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,

and 100 equiv of hematin). A reference cell with the buﬀered solution

AUTHOR INFORMATION

Corresponding Author

Phone: 604-822-4449. E-mail: orvig@chem.ubc.ca.

Notes

■

*

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank Dr. Elena Polishchuk from UBC Biological

Services for her advice and support. We are grateful for a

Discovery grant from the Natural Sciences and Engineering

Research Council of Canada (NSERC) and support from

Advanced Applied Physics Solutions (AAPS) C.O. acknowl-

edges the Canada Council for the Arts for a Killam Research

Fellowship (2011−2013).

(

40% v/v DMSO, 0.020 M HEPES, apparent pH 7.5) was also titrated

with the same equivalent of each compound in order to zero the

absorbance of the drug. Titrations were repeated in triplicate for each

compound and blank. Absorbance data were corrected for dilution,

analyzed at 402 nm, and ﬁtted to a 1:1 association model using

nonlinear least-squares ﬁtting analysis via Graph Pad Prism, version

.0, software to obtain the association constant (reported as log K).

Graphs were produced using the OriginPro 7.5 software.

β-Hematin Formation Inhibition Assay. The inhibition of

ABBREVIATIONS USED

■

Cp, cyclopentadienyl; CQ, chloroquine; CQR, chloroquine-

resistant; CQS, chloroquine-sensitive; DMSO, dimethylsulf-

oxide; Fe(III)PPIX, ferriprotoporphyrin IX; FQ, ferroquine;

HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;

IC50, half-maximum inhibitory concentration; ClogP, calculated

partition coeﬃcient; MeI, iodomethane; MeOH, methanol; MEP,

molecular electrostatic potential; MFA, molecular ﬁeld analysis;

MSA, molecular shape analysis; n-BuLi, n-butyllithium; NMR,

nuclear magnetic resonance; tPSA, topological polar surface area;

4

β-hematin formation was assayed as described previously in the litera-

51

ture. A 12.9 M acetate stock solution was prepared using sodium

51

acetate trihydrate and kept at 70 °C throughout the experiment. The

pH of the stock solution was measured as 5.05. A 1.68 mM stock

solution of hematin was prepared by dissolving 5.5 mg of hemin

(

bovine, ≥90.0%, obtained from Sigma-Aldrich) in 5.0 mL of 0.1 M

3

D-QSAR, three dimensional quantitative structure−activity

NaOH. Stock solutions of each compound were prepared in DMSO in

concentrations that ranged from 0.15 to 0.20 M. Hematin stock

solution (20.2 μL, 0.035 μmol) was dispensed to a series of Eppendorf

tubes (11 tubes for each compound tested). The 0.1 M NaOH

solution was neutralized with 2.02 μL of 1.0 M HCl prior to the

addition of the compounds. Predetermined volumes of the stock

relationship; RI, resistance index; rt, room temperature; SAR,

structure−activity relationship; TEA, triethylamine; TFA,

triﬂuoroacetic acid; THF, tetrahydrofuran; TI, therapeutic

index; TLC, thin layer chromatography; TMEDA, N,N,N′,N′-

tetramethylethylenediamine; WHO, World Health Organization

P

dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Eds.; Topics in Organometallic Chemistry, Vol. 32; Springer: Berlin,

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dx.doi.org/10.1021/jm301422h | J. Med. Chem. XXXX, XXX, XXX−XXX

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Structural characteristics of chloroquine-bridged ferrocenophane analogues of ferroquine may obviate malaria drug-resistance mechanisms

DOI: 10.1021/jm301422h

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