A series of structurally simple chloroquine chemosensitizing dibemethin derivatives that inhibit chloroquine transport by PfCRT

European Journal of Medicinal Chemistry 46 (20 1 1 ) 1 729e1 742

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Original article

A series of structurally simple chloroquine chemosensitizing dibemethin

derivatives that inhibit chloroquine transport by PfCRT

Vincent K. Zishiri ^a, Roger Hunter ^a, Peter J. Smith ^b, Dale Taylor ^b, Robert Summers ^c, Kiaran Kirk ^c,

,

*

Rowena E. Martin ^{c d}, Timothy J. Egan ^a

^aDepartment of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa

^bDivision of Pharmacology, Department of Medicine, University of Cape Town, Private Bag, Rondebosch 7701, South Africa

^cResearch School of Biology, The Australian National University, Canberra, ACT 0200, Australia

^dSchool of Botany, University of Melbourne, Parkville, VIC 3010, Australia

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of 12 new dibemethin (N-benzyl-N-methyl-1-phenylmethanamine) derivatives bearing an N-

aminomethyl group attached to the one phenyl ring and an H, Cl, OCH₃or N(CH₃)₂group on the other

have been synthesized. These compounds all showed strong chloroquine chemosensitizing activity,

Received 4 November 2010

Received in revised form

6 February 2011

Accepted 13 February 2011

Available online 22 February 2011

comparable to verapamil, when present at 1 mM in an in vitro culture of the chloroquine-resistant W2

strain of the human malaria parasite, Plasmodium falciparum. Their N-formylated derivatives also

exhibited resistance-reversing activity, but only at substantially higher IC₁₀concentrations. A number of

the dibemethin derivatives were shown to inhibit chloroquine transport via the parasite’s ‘chloroquine

resistance transporter’ (PfCRT) in a Xenopus laevis oocyte expression system. The reduced resistance-

reversing activity of the formylated compounds relative to their free amine counterparts can probably be

ascribed to two factors: decreased accumulation of the formylated dibemethins within the parasite’s

internal digestive vacuole (believed to be the site of action of chloroquine), and a reduced ability to

inhibit PfCRT. The resistance-reversing activity of the compounds described here demonstrates that the

amino group need not be attached to the two aromatic rings via a three or four carbon chain as has been

suggested by previous QSAR studies. These compounds may be useful as potential side chains for

attaching to a 4,7-dichloroquinoline group in order to generate new resistance-reversing chloroquine

analogues with inherent antimalarial activity.

Keywords:

Malaria

Chloroquine resistance

Resistance-reverser

Chemosensitizer

Dibemethin

PfCRT

1. Introduction

The WHO now recommends that uncomplicated falciparum

malaria be treated with artemisinin-combination therapies. These

Malaria is a leading cause of death in the developing world and

is the principal human parasite-borne disease in terms of mortality,

morbidity, and social and economic cost [1,2]. The majority of

deaths from malaria are a consequence of infection by Plasmodium

falciparum in Africa, with children under the age of ﬁve years and

pregnant women being at greatest risk [3]. Historically, the quin-

oline chloroquine (CQ) (1) (Fig. 1) was the most important anti-

malarial; it is cheap and safe, even during pregnancy, and was

highly effective [4]. However, the emergence of CQ-resistant

parasites, starting in Southeast Asia and South America and even-

tually spreading to Africa and other regions, rendered the drug

largely ineffective by the 1990s [5].

pair an artemisinin derivative with a second drug, usually a quino-

line such as amodiaquine, meﬂoquine or a related compound such

as lumefantrine [6,7]. However, artemisinin appears to be losing

potency in Cambodia and it is feared that artemisinin-resistant

parasites may spread throughout malaria endemic regions [8,9].

There is, therefore, an urgent need to develop new antimalarial

strategies and chemotherapies.

One strategy that is currently being pursued involves the

development of new quinolines that evade the mechanism

underlying CQ resistance. Such compounds include ferroquine,

which is currently in clinical development, and isoquine which

progressed as far as phase 1 clinical trials [6,10]. Alternatively, it

may be possible to reverse resistance to CQ using a chemo-

sensitizer, also known as a CQ ‘resistance-reverser’ [7]. This strategy

arose after the discovery in 1987 that the calcium channel blocker

verapamil (2) partially restores the sensitivity of resistant parasites

* Corresponding author. Tel.: þ27 21 650 2528; fax: þ27 21 650 5195.

E-mail address: Timothy.Egan@uct.ac.za (T.J. Egan).

doi:10.1016/j.ejmech.2011.02.026

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V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

to CQ [11]. A range of other compounds have also been shown to

reverse CQ resistance [12e14]. These include antipsychotics such as

imipramine (3) (Fig. 1), and antihistamines (e.g., cyproheptidine

and chlorpheniramine). Small-scale clinical trials have provided

evidence that CQ is more effective against CQ-resistant malaria

when administered with chlorpheniramine [15,16], but the clinical

application of resistance-reversers is precluded by the unaccept-

ably high concentrations required to effect resistance-reversal,

a consequence of the afﬁnity of these compounds for a₁-glyco-

protein [17].

digestive vacuole, away from its site of action, whereas the CQ-

sensitive form (PfCRT^CQS) does not [23]. Furthermore, it was

shown that CQ transport via PfCRT^CQRis inhibited by the resis-

tance-reversers verapamil and primaquine, thus providing

a mechanistic explanation for the ability of these compounds to

reverse CQ resistance [23].

The design of reversed-CQ compounds is assisted by advances in

our understanding of the structureeactivity relationships (SARs) in

CQ and related compounds [24e27], together with work published

by Bhattacharjee et al. and Alibert et al. on the SAR for resistance-

reversing agents [28,29]. According to the proposal of Bhattacharjee

et al., the key requirements for resistance-reversal in imipramine-

like resistance-reversers are two hydrophobic aromatic rings fused

to a 7-membered ring containing a nitrogen atom, which bears

a three-carbon chain terminated by an H-bond acceptor group

(preferably N). Other resistance-reversers were shown to contain

many of the same features, typically the two aromatic rings and the

chain terminated bya basic N atom [28]. Based on this model, Peyton

and Burgess have taken out an extensive patent on quinolines

bearing side chains exhibiting such features [30]. The claim includes

molecules bearing a dibemethin group (5) (Fig. 1).

A comparison of the structures of CQ chemosensitizers reveals

that linkers between the two aromatic rings range from a direct bond

between rings (nifedipine (6) and amlodipine (7)), to a single atom

spacer (dibenzazepines and phenothiazines), two-atom spacer

(ﬂuoxetine (8)) oreven long ﬂexible chainsof sixor seven atoms (e.g.,

verapamil) (Fig. 1) [12]. Indeed, some resistance-reversers such as

primaquine (9) possess only a single aromatic ring system. This

suggests that the linkage between the two aromatic rings is quite

permissive. Based on the structure of the resistance-reverser nomi-

fensine (10) (Fig. 1), in which the amino group is part of a fused six-

membered ring system, we hypothesized that the three carbon chain

bearing the terminal N atom is not an absolute requirement for

resistance reversal. This led us to propose that aminomethyl

Burgess et al. have described a 4-amino-7-chloroquinoline with

a

resistance-reverser structure closely related to imipramine

attached as a side chain linked through the 4-amino group on the

quinoline (4) (Fig. 1) [18]. To date, these “reversed-CQ” compounds

have not been directly demonstrated to exhibit CQ resistance-

reversal activity. However, compound 4 has been shown to be

strongly active against CQ-resistant parasites in vitro [18].

Furthermore, a recent study has shown that an acridone derivative

exhibits additive activity when used in combination with CQ

against a CQ-sensitive strain of P. falciparum (D6), but synergistic

activity against a CQ-resistant strain (Dd2) [19]. This latter study

strongly indicates that this novel approach is viable and, if

successful, could result in new quinoline antimalarials that are

efﬁcacious against CQ-resistant and CQ-sensitive parasites alike.

CQ is believed to act by inhibiting the formation of hemozoin

in the digestive vacuole of the parasite, a site where the drug

accumulates by weak-base trapping [20,21]. CQ resistance in

P. falciparum, on the other hand, arises primarily from mutations

in the ‘chloroquine resistance transporter’ (PfCRT), located in the

membrane of the parasite’s digestive vacuole [22]. Expression of

PfCRT at the plasma membrane of Xenopus laevis oocytes has

enabled the activity of the protein to be studied directly, leading

to the demonstration that the resistance-conferring form of

PfCRT (PfCRT^CQR) has the ability to transport CQ out of the

Fig. 1. Chloroquine (CQ) (1), the CQ resistance reversers verapamil (2) and imipramine (3), “reversed-CQ” compounds containing an imipramine (4) or dibemethin (5) side chain

and the resistance reversers nifedipine (6), amlodipine (7), ﬂuoxetine (8), primaquine (9) and nomifensine (10).

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

1731

dibemethin would exhibit resistance-reversing properties. If so, this

would open the way to connect dibemethin to the quinoline nucleus

in a completely new way which avoids the long connecting chains

present in 4 and 5. Here we report on the synthesis and in vitro CQ

resistance-reversing activity of a series of twenty-four aminomethyl

dibemethin derivatives (Fig. 2, 11(o)e14f(p)). These dibemethins

have been designed with an aminomethyl chain on one phenyl ring

to replace the three carbon aminoalkyl chain of the currently known

resistance-reversers. This primary terminal amine was also for-

mylated to probe the role, if any, of the basicity of this group. Three

different substituents on the second phenyl ring were introduced to

investigate the effect of electronic and hydrophobic properties of

groups attached to the dibemethin nucleus. The groups selected

were Cl (electron withdrawing/hydrophobic), OCH₃(electron

releasing, similar hydrophobicity to H), and N(CH₃)₂(strongly elec-

tron releasing/hydrophobic).

23 or 24) to afford azidomethyl-substituted N-methyldiarylamines

(11(m)ae14(p)a), which could be successfully chromatographed

from the bis(azidomethyl) by-product. While benzylamines (22, 23)

with X ¼ Cl and OMe respectively could be synthesized via substi-

tution of para-substituted benzyl chlorides (18 and 19) with

methylamine according to Scheme 2, reagent (24) needed to be

accessed via reductive amination of para-substituted benzaldehyde

(20) in view of the instability of the corresponding benzyl chloride

with X as the electron-releasing NMe₂. Thereafter, azides (11(m)

ae14(p)a) could be converted into target benzylamines (11(m)e14

(p)) using the Staudinger reaction, which involved reaction with

triphenylphosphine in THF at room temperature for 0.5 h to afford

an iminophosphorane intermediate that was reacted with water in

reﬂuxing THF to liberate the polar amine and triphenylphosphine

oxide. Following removal of solvents, the amine product could be

puriﬁed chromatographically using

a

polar mobile phase

(EtOAc:MeOH:NEt₃¼ 93:5:2). A further series of derivatives (11f

(o)e14f(p), Fig. 2) lacking the basicity of the primary amine was

prepared by formylating compounds (11(o)e14(p)) via reaction

with excess ethyl formate.

2. Chemistry

The o-aminomethyl dibemethin model compound (11(o)) was

synthesized in a four-step sequence from N-benzylmethylamine

(15) as shown in Scheme 1. To this end, alkylation of (15) with

benzyl bromide (16) produced dibemethin (11(o)a), which was

reacted with t-butyllithium in an ortho-assisted regioselective

deprotonation to form the mono-lithio anion, which was for-

mylated with anhydrous dimethylformamide to produce

substituted benzaldehyde (11(o)b). The latter was converted to an

oxime (11(o)c) by reaction with hydroxylammonium chloride,

before being reduced to the desired amine product (11(o)) with

LiAlH₄. The ortho-assisted metallation strategy (DOM) could not be

extended to the meta- and para-aminomethyl dibemethin variants,

so an alternative strategy that could access all three variants was

All products were characterized by ¹H NMR, ¹³C NMR, and

infrared spectroscopy as well as mass spectrometry. Since all of the

compounds were isolated as oils, high resolution mass spectrom-

etry was performed to verify synthesis of the desired products.

3. Biological testing

Initial experiments entailed testing the in vitro antimalarial

activities of 11(o)e14f(p) against the CQ-resistant W2 strain of the

parasite. The IC₅₀values are reported in Table 1. Each compound

was then tested for the ability to decrease the CQ IC₅₀when present

at an extracellular concentration of 1

that all of the dibemethins tested exhibited inherent in vitro anti-

malarial IC₅₀values signiﬁcantly higher than 1 M, with only 11(o),

11(m), 12(o) and 13(m) exhibiting substantial growth inhibition at

M. Indeed, with the exception of these four compounds, the IC₅₀

values are all above 10 M. All of the compounds bearing free

mM (Table 2). The data show

devised involving the substitution chemistry of o-, m-, and p-

dibromoxylenes (17(o), 17(m) and 17(p)), Scheme 2.

a, -

a⁰

m

To this end, each of 17(o), 17(m) or 17(p) were independently

reacted with sodium azide (1 equiv.) in DMF at room temperature to

afford a mixture of the desired azidomethyl/bromomethyl (21(o), 21

(m) or 21(p)) and undesired bis(azidomethyl) aromatic compounds

that were difﬁcult to separate by column chromatography. Thus, the

mixture was carried through to the next step involving benzylic

substitution with para-substituted N-methylbenzylamines (15, 22,

1

m

amino groups showed signiﬁcant resistance-reversing activity

(p < 0.01), whereas the entire formylated series exhibited little or

no activity at 1

mM (only 11f(o) and 14f(m) caused statistically

signiﬁcant changes in the CQ IC₅₀

;

p <

0.05). The former

Fig. 2. The series of twenty-four dibemethin derivatives synthesized in this work. Compounds varied with respect to ortho, meta or para arrangement of the left hand (xylene-

diamine) ring and substituents on the primary/secondary amino group (R) and at the para position on the right hand (benzyl) ring (X). Compounds with the same X group and R ¼ H

are given the same number followed by o, m or p to indicate the substitution pattern of the xylene moiety (11 series, X ¼ H; 12 series, X ¼ Cl; 13 series, X ¼ OCH₃; 14 series, X ¼ N

(CH₃)₂). Compounds with R ¼ formyl are given the corresponding number, but are distinguished by the letter f.

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V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

Scheme 1. Reagents and conditions for synthesis of 11(o): (i) CH₂Cl₂, NEt₃(1.5 equiv), 0 ^ꢀC; (ii) t-BuLi (1.2 equiv), Et₂O, ꢁ78 ^ꢀCe0 ^ꢀC, then DMF (1 equiv); (iii) HONH₃^þCl^ꢁ

(2.75 equiv), NaOH (5.5 equiv), EtOH,

D, 18 h; (iv) LiAlH₄(0.5 mol equiv), Et₂O, D, 18 h.

compounds show chemosensitizing activity similar to verapamil,

with some compounds being substantially better (Table 2). Since

compounds 11(o)e14(p) are dibasic, and their formylated deriva-

tives 11f(o)e14f(p) only monobasic, it is conceivable that the

reduced activity of the latter series arises from decreased

accumulation via ‘weak-base trapping’ (i.e. the trapping of the

membrane-impermeant charged form(s) of the compound) in the

acidic digestive vacuole. The activity of the formylated derivatives

was therefore tested at higher concentrations. For this purpose, the

IC₁₀values (i.e. the concentration at which growth was inhibited by

10%) of the formylated series was determined (Table 3) and the CQ

resistance-reversing activity of each compound was tested at its

IC₁₀concentration. This revealed that the formylated compounds

do indeed exhibit CQ resistance-reversing activity, but at higher

concentrations than the corresponding free amines. With the

exception of 14f(m), which was ineffective at this concentration,

each of the formylated compounds caused a signiﬁcant (p < 0.01)

decrease in the IC₅₀of CQ. However, for most of these compounds

the resistance modiﬁcation index (RMI), even at these high

concentrations, did not attain that measured in the presence of

1

mM of the corresponding free amine.

Since the CQ resistance-reversing activity of the dibemethin

derivatives is likely to result, at least in part, from interactions with

PfCRT^CQR, we tested a subset of these compounds for the ability to

inhibit the ﬂux of CQ through PfCRT^CQR. These experiments made

use of the recently described system for expressing PfCRT in

Xenopus oocytes (unfertilised frog eggs) [23], with which direct

measurements of CQ transport via PfCRT^CQR, and its inhibition by

Scheme 2. Reagents and conditions for synthesis of 11(m)e14(p): (i) DMF, rt, 16 h; (ii) THF, 0 ^ꢀC, then rt, 16 h; (iii) CH₃CN, 0 ^ꢀC, 2 h; (iv) NaCNBH₃(3.5 equiv), rt, 4 h; (v) CH₃CN,

K₂CO₃(1.5 equiv), 0 ^ꢀC; (vi)

, 6 h; (vii) PPh₃(1.2 equiv), THF, rt, 0.5 h; (viii) H₂O, , 6 h. Yields: 22e24 and 11(m)a and 11(p)a (from starting materials) 81, 87, 73, 57 and 59%

respectively; 12(o)ae14(p)a 58, 61, 67, 64, 61, 57, 57, 61 and 63% respectively; 11(m)e14(p) 88, 89, 93, 93, 93, 83, 88, 89, 83, 81 and 89% respectively.

D

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

1733

Table 1

Table 3

The in vitro IC₅₀values of compounds 11(o)e14f(p) against the CQ-resistant W2

strain of P. falciparum.

The IC₁₀values of compounds 11f(o)e14f(p) in the W2 strain of P. falciparum and the

IC₅₀of CQ against this strain at the corresponding IC₁₀concentration of each of the

compounds 11f(o)e14f(p) together with their RMI values under these conditions.

Compound Number

Compound Code

IC₅₀(mM)

Compound

Number

IC₁₀

(

m

M)

IC₅₀of CQ at the IC₁₀

of each compound (nM)

RMI

11(o)

11(m)

11(p)

12(o)

12(m)

12(p)

13(o)

13(m)

13(p)

14(o)

14(m)

14(p)

11f(o)

11f(m)

11f(p)

12f(o)

12f(m)

12f(p)

13f(o)

13f(m)

13f(p)

14f(o)

14f(m)

14f(p)

VZ1A

VZ2A

VZ3A

4.1 ꢂ 0.2

2.8 ꢂ 0.5

29 ꢂ 7

e

136 ꢂ 16^a

60 ꢂ 10

40 ꢂ 7

59 ꢂ 9

46 ꢂ 5

69 ꢂ 8

63 ꢂ 15

43 ꢂ 6

70 ꢂ 8

63 ꢂ 7

66 ꢂ 7

137 ꢂ 7

62 ꢂ 4

1.000

0.44

0.29

0.43

0.34

0.51

0.46

0.32

0.51

0.46

0.49

1.00

0.46

2VZ1A

2VZ2A

2VZ3A

3VZ1A

3VZ2A

3VZ3A

4VZ1A

4VZ2A

4VZ3A

VZ1F

5.7 ꢂ 0.7

30 ꢂ 12

63 ꢂ 26

> 233

11f(o)

11f(m)

11f(p)

12f(o)

12f(m)

12f(p)

13f(o)

13f(m)

13f(p)

14f(o)

14f(m)

14f(p)

1.7 ꢂ 0.4

9.7 ꢂ 0.9

18 ꢂ 4

12 ꢂ 2

4 ꢂ 1

6.8 ꢂ 0.5

91 ꢂ 67

37 ꢂ 9

5.4 ꢂ 0.6

12 ꢂ 3

9 ꢂ 2

30 ꢂ 3

11.7 ꢂ 0.7

11 ꢂ 2

16 ꢂ 2

15 ꢂ 1

b

VZ2F

VZ3F

21 ꢂ 2

ꢁ

56 ꢂ 25

119 ꢂ 40

16 ꢂ 3

15.2 ꢂ 0.6

2VZ1F

2VZ2F

2VZ3F

3VZ1F

3VZ2F

3VZ3F

4VZ1F

4VZ2F

4VZ3F

a

CQ alone.

b

Dose-response curve did not reach 90% cell growth level, compound tested at

M.

29 ꢂ 7

15.2

m

29 ꢂ 4

48 ꢂ 18

37 ꢂ 9

a formylated derivative exhibiting one of the weakest activities (14f

(p)). The concentration dependence of inhibition of CQ transport

was determined for each of these compounds, and the results are

presented in Fig. 3 and Table 4. The four compounds varied

signiﬁcantly in their ability to inhibit PfCRT^CQR, with the magnitude

of the IC₅₀value increasing in the order 11(o) < 12(m) < 12(p) < 14f

(p) (each IC₅₀value was signiﬁcantly different from the other three

values; P < 0.05, unpaired t-test). Compound 11(o) has a lower IC₅₀

than verapamil (Table 4), while the others are less active. It should

be noted that the micromolar concentrations of the compounds

used here to inhibit PfCRT^CQRare physiologically relevant, given

that dibasic compounds such as compounds 11(o)e14(p) could be

expected to accumulate to high micromolar, and even millimolar,

concentrations within the acidic environment of the parasite’s

digestive vacuole via weak-base trapping.

42 ꢂ 7

17 ꢂ 17

33 ꢂ 5

potential resistance-reversers, can be made. The direction of CQ

transport in the PfCRT expression system is from the acidic extra-

cellular medium (pH 6.0) into the oocyte cytosol (pH 7.2) [23],

which corresponds to the efﬂux of CQ from the acidic digestive

vacuole (pH w5) into the parasite cytosol (pH 7.3) [31]. Four

compounds were selected for testing: one exhibiting a strong RMI

(11(o)), two with intermediate RMI values (12(m) and 12(p)), and

Table 2

The IC₅₀of CQ (nM) in the presence of 1

mM concentration of 2, 11(o)e14f(p) and the

corresponding resistance modiﬁcation index (RMI) in the CQ-resistant W2 strain.

4. Discussion

Compound Number

At 1

m

M compound

RMI^a

e

2^c

157 ꢂ 13^b

1.00

0.26^f

0.03

0.08

0.24

0.096

0.31

0.25

0.35

0.25

0.33

0.36

0.24

0.21

0.75

0.84

1.13

1.07

41 ꢂ 5

The dibemethins are a structurally simple class of achiral

compound that can be synthesized in three-steps in good overall

yield from cheap starting materials. None of these compounds, nor

their formylated derivatives (11f(o)e14f(p)), possess strong

inherent in vitro antimalarial activity in the CQ-resistant W2 strain

of P. falciparum. However, all of the free amines, but not the

formylated derivatives (11f(o)e14f(p)) showed CQ resistance-

11(o)

11(m)

11(p)

12(o)

12(m)

12(p)

13(o)

13(m)

13(p)

14(o)

14(m)

14(p)

11f(o)

11f(m)

11f(p)

12f(o)

12f(m)

12f(p)

13f(o)

13f(m)

13f(p)

14f(o)

14f(m)

14f(p)

4^d

13 ꢂ 10

37 ꢂ 6

15 ꢂ 7

49 ꢂ 2

39 ꢂ 9

55 ꢂ 7

40 ꢂ 15

52 ꢂ 12

56 ꢂ 10

38 ꢂ 5

reversing activity when tested against W2 parasites at 1

mM

concentration with RMI values ranging from 0.03 to 0.36 (Table 2),

which are comparable to, and in some cases much better than that

measured for the well known chemosensitizer verapamil (2) (0.26,

Table 2). They also compare favourably with the reported RMI value

of ﬂuoxetine in the same strain at the same concentration (0.18)

[32], while the best ones compare favourably with the reported

value of 0.05 for imipramine in the same strain, but at a slightly

higher concentration of 1.6 mM [28]. Furthermore, most, including

the formylated compounds, exhibited signiﬁcant CQ resistance-

reversing activity at their respective IC₁₀values.

33 ꢂ 4

118 ꢂ 8

132 ꢂ 38

177 ꢂ 45

168 ꢂ 34

e

ꢁ

142 ꢂ 17

191 ꢂ 45

187 ꢂ 106

191 ꢂ 58

157 ꢂ 24

98 ꢂ 36

0.90

1.22

1.19

1.22

1.00

0.62

1.36

The dibasic free-amines 11(o)e14(p) are likely to accumulate in

the digestive vacuole of the parasite through weak-base trapping to

higher concentrations than the monobasic formylated compounds

11f(o)e14f(p) [33], possibly accounting for the reduced CQ resis-

tance-reversing activity of latter. This would be consistent with

a model in which the chemosensitizer binds directly to PfCRT^CQRon

the vacuolar side of the membrane, blocking CQ transport via

214 ꢂ 126

a

IC₅₀of CQ þ compound/IC₅₀of CQ alone.

b

c

d

e

f

In presence of CQ alone.

Verapamil.

Estimated graphically, ﬁt did not converge.

Compound too toxic to parasite to test.

cf. 0.21 in Ref. [32].

PfCRT^CQR[34], and/or ‘out-competing’ CQ for transport by PfCRT^CQR

.

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V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

Fig. 3. Concentration-dependent effects of 11(o), 12(m), 12(p) and 14f(p) on the uptake of [³H]CQ into oocytes expressing Dd2 PfCRT^CQR(closed circles) or D10 PfCRT^CQS(open

circles). IC₅₀values derived from these data are shown in Table 4. In all panels uptake is shown as the mean ꢂ s.e.m. from 4 to 5 separate experiments, within each of which

measurements were made from 10 oocytes per treatment. It should be noted that non-injected oocytes and oocytes expressing PfCRT^CQStake up [³H]CQ to similar (low) levels via

simple diffusion of the neutral species; this represents the ‘background’ level of CQ accumulation in oocytes (refer to publication 23 for full data and a detailed discussion).

There was little difference in the chemosensitizing activity of 11

(o)e14(p), other than compounds 11(o), 11(m) and 12(o), which

showed substantially lower RMI values (Table 2). This may perhaps

be in part because they possesses some direct toxicity to the

parasite, but 13(m) which also possesses signiﬁcant inherent

activity against the parasite at this concentration does not exhibit

such a low RMI value.

In the Xenopus PfCRT expression system all four compounds

tested inhibited CQ transport via the mutant transporter, albeit

with markedly different IC₅₀values. Compound 11(o), which yiel-

ded the lowest RMI value and which therefore possesses the

highest level of resistance-reversing activity, was also the most

effective inhibitor of PfCRT^CQR-mediated CQ uptake. Compounds 12

(m) and 12(p), which yielded substantially higher RMI values than

11(o), were comparatively weaker inhibitors of PfCRT^CQR. The least

potent inhibitor of PfCRT^CQR, compound 14f(p), was the least

effective chemosensitizer. Thus, the data for this limited series of

compounds suggest that the CQ chemosensitizing activities of

these compounds correlate with their respective abilities to inhibit

PfCRT^CQR. Furthermore, the ﬁnding that compound 14f(p) inhibited

PfCRT^CQR-mediated CQ transport with an IC₅₀that was much higher

Table 4

IC₅₀values for the inhibition of PfCRT^CQR-mediated CQ

transport by 11(o), 12(m), 12(p), and 14f(p). PfCRT^CQR-medi-

ated CQ transport was calculated by subtracting the uptake

measured in oocytes expressing PfCRT^CQSfrom that in

oocytes expressing PfCRT^CQR. The data are shown in Fig. 3.

IC₅₀values were derived by least-squares ﬁt of the equation

Y ¼ Y_minþ [(Y_maxꢁ Y_min)/(1 þ ([inhibitor]/IC₅₀)^C)] where Y is

PfCRT^CQR-mediated CQ transport, Y_minand Y_maxare the

minimum and maximum values of Y, and C is a constant. All

values are mean ꢂ s.e.m. from 4 to 5 separate experiments,

within which measurements were made from 10 oocytes per

treatment.

(657 mM) than that seen for the other compounds is consistent with

the N-formylated compounds being relatively poor inhibitors of

PfCRT^CQR-mediated CQ transport. This, together with the lower

accumulation (via weak-base trapping) in the parasite’s acidic

digestive vacuole relative to its dibasic free-amine counterparts,

could account for the observation that the N-formylated

compounds are relatively poor CQ resistance-reversers.

Compound

IC₅₀(mM)

2^a

30 ꢂ 3 (n ¼ 4)^b

24 ꢂ 2 (n ¼ 5)

101 ꢂ 8 (n ¼ 5)

140 ꢂ 10 (n ¼ 4)

657 ꢂ 67 (n ¼ 4)

5. Conclusions

11(o)

12(m)

12(p)

14f(p)

The aminomethyl derivatives of dibemethin represent a new

class of CQ resistance-reversing agent in P. falciparum. Apart from

possessing potent resistance-reversing activities, these compounds

are suitable for attachment to a quinoline; a single step reaction can

a

Verapamil.

From Ref. [23].

b

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

1735

couple any one of compounds 11(o)e14(p) to 4,7-dichloroquino-

line. This would yield a 4-amino-7-chloroquinoline with a poten-

tially resistance-reversing side chain. A signiﬁcant ﬁnding is that

the three carbon aminoalkyl chain positioned between the

aromatic rings is not essential for resistance-reversal activity. The

extent to which the compounds accumulate within the digestive

vacuole through weak-base trapping may contribute signiﬁcantly

to their respective abilities to reverse CQ resistance in vitro. Another

key determinant of chemosensitizing activity may be the strength

chromatographed on silica-gel using mixtures of ethyl acetate:-

hexane (0:100) to (5: 95) as eluent. The product was dried in vacuo

to give a yellow oil, 11(o)b, (2.61 g, 77%): IR (CHCl₃) n_max(cm^ꢁ1

)

3066, 3029, 3010, 2949, 2880, 2843, 2793,1689 (C]O),1600 (Ar_C]C);

¹H NMR (CDCl₃, 300 MHz)

10.42 (1H, s, H-1), 7.92 (1H, dd, J ¼ 1.5,

7.5 Hz, H-7), 7.50e7.23 (8H, m, ArH), 3.84 (2H, s, H-8), 3.53 (2H, s, H-

10), 2.13 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

192.0 (C-1),

d

141.7 (Ar_qu), 138.5 (Ar_qu), 134.9 (Ar_qu), 133.0 (Ar_CeH), 130.4 (C-4),

128.9 (C-7), 128.8 (C-12/16), 128.1 (C-13/15), 127.6 (Ar_CeH), 127.0

(Ar_CeH), 61.8 (C-10), 58.9 (C-8), 41.6 (NeCH₃); HRMS (ES): Found

239.1303 (M^þ): C₁₆H₁₇NO (M^þ) requires 239.1310.

with which these compounds inhibit CQ transport via PfCRT^CQR

.

6. Experimental

6.2.3. o-(N-Benzyl-N-methylamino)methylbenzaldehyde oxime

(11(o)c)

6.1. Chemistry

To a stirred solution of 11(o)b (2.50 g, 10.4 mmol) in absolute

ethanol (50 mL), was added hydroxylammonium chloride (1.97 g,

28.3 mmol) followed by a solution of NaOH (2.26 g, 56.5 mmol) in

deionised water (3 mL). The reaction was allowed to reﬂux over-

night under an atmosphere of nitrogen. The mixture was then

worked up with deionised water and extracted with ethyl acetate

(3 ꢃ 50 mL). The organic extracts were dried over anhydrous

MgSO₄and the solvent evaporated under reduced pressure. The

crude product was chromatographed on silica-gel using a mixture

of ethyl acetate:hexane (10:90) as eluent. The product was dried in

vacuo to give 11(o)c as a pale-yellow oil (2.17 g, 85%): IR (CHCl₃) n_max

(cm^ꢁ1) 3305, 3065, 3028, 3011, 2950 (Ar_CH), 2881, 2841, 2791, 1601

Solvents, acids, and common salts were obtained from Sarchem,

Krugersdorp, South Africa. All other starting materials were

obtained from SigmaeAldrich, Vorna Valley, South Africa. Pre-

coated silica-gel plates as well as silica and alumina for column

chromatography were obtained from Merck, South Africa. ¹H and

¹³C NMR spectra were recorded on a Varian Mercury spectrometer

at 300 MHz, and a Varian Unity spectrometer at 400 MHz. All

spectra were recorded in d3-chloroform or d4-methanol. Infrared

spectra were recorded on a PerkineElmer Paragon 1000 FT-IR

spectrophotometer in the range 3600e800 cm^ꢁ1. Mass spectra

were recorded on a VG Micromass 16F spectrometer operating at

70 eV with an accelerating voltage of 4 kV and a variable temper-

ature source. Accurate mass determinations were performed on

a Kratos Limited MS9/50 spectrometer. All mass spectra were

obtained using electron-impact techniques.

(Ar_C]C); ¹H NMR (CDCl₃, 300 MHz)

d 8.80 (1H, br s, OH), 8.69 (1H, s,

H-1), 7.80 (1H, dd, J ¼ 1.7, 6.5 Hz, H-7), 7.35e7.25 (8H, m, ArH), 3.64

(2H, s, H-8), 3.54 (2H, s, H-10), 2.15 (3H, s, NeCH₃); ¹³C NMR (CDCl₃,

75 MHz)

d 149.2 (C-1), 138.8 (Ar_qu), 137.7 (Ar_qu), 131.4 (Ar_qu), 130.7

(Ar_CeH), 129.4 (Ar_CeH),129.1 (C-12/6),128.3 (C-13/15),127.6 (Ar_CeH),

127.0 (Ar_CeH), 126.4 (C-7), 62.1 (C-10), 60.0 (C-8), 41.8 (NeCH₃);

HRMS (ES): Found 254.1427, (M^þ): C₁₆H₁₈N₂O (M^þ) requires

254.1419.

6.2. Synthetic procedures and characterization

6.2.1. N,N-Dibenzylmethylamine (dibemethin) (11(o)a)

To

a stirred solution of N-benzylmethylamine (10.6 mL,

82.5 mmol) in DCM (50 mL) and triethylamine (17.2 mL,123.8 mmol)

at 0 ^ꢀC under an atmosphere of nitrogen, benzyl bromide (9.8 mL,

82.5 mmol) was added slowly and the reaction allowed to progress

for 2 h after which time a white precipitate had formed. The reaction

mixture was worked up by the addition of a saturated solution of

aqueous Na₂CO₃and the product extracted into ethyl acetate

(3 ꢃ 50 mL), the organic extracts dried over anhydrous MgSO₄and

the solvent removed under reduced pressure to give a crude product

(17.05 g), which was chromatographed on silica-gel (170 g) using

10% ethyl acetate in hexane as eluent. The product was dried invacuo

to give 11(o)a as a pale-yellow oil (11.62 g, 67%): ¹H NMR (300 MHz,

6.2.4. o-[(N-Benzyl-N-methyl)aminomethyl]-benzylamine (11(o))

To a stirred solution of 11(o)c (1.00 g, 3.90 mmol) in anhydrous

diethyl ether (10 mL), was added LiAlH₄(75 mg, 1.96 mmol) slowly

and the reaction allowed to reﬂux overnight under an atmosphere

of nitrogen. After the reaction mixture was allowed to cool to room

temperature, a saturated solution of aqueous Na₂SO₄(10 mL) with

a few drops of triethylamine was added to the reaction mixture.

This mixture was stirred for 30 min, the resultant precipitate

ﬁltered through Celite and the residue washed with a solution of 5%

triethylamine in THF (100 mL). The solvent was removed under

reduced pressure with a small volume of toluene being added to

facilitate the azeotropic removal of any water. The crude product

was puriﬁed by column chromatography using 100% ethyl acetate

followed by EtOAc:MeOH:NEt₃(94:5:1) as eluent to give 11(o) as

a dark-orange oil, (0.83 g, 89%): IR (DMSO) n_max(cm^ꢁ1) 3523e3422,

3287, 2791, 2143, 2050, 1973, 1711, 1663 (Ar_C]C), 1579, 1491; ¹H

CDCl₃)

d

7.57e7.38 (10H, m, ArH), 3.70 (4H, s, CH₂), 2.37 (3H, s, CH₃);

139.2 (Ar_quat), 128.8 (Ar_ortho), 128.1

¹³C NMR (CDCl₃, 75 MHz)

d

(Ar_meta), 126.8 (Ar_para), 61.8 (CH₂), 42.1 (CH₃).

6.2.2. o-(N-Benzyl-N-methylamino)methylbenzaldehyde (11(o)b)

t-Butyllithium (10.0 mL, 1.7 M, 17.0 mmol) was slowly added

drop-wise to a stirred solution of 11(o)a (3.00 g, 14.2 mmol) in

anhydrous diethyl ether (100 mL) at ꢁ78 ^ꢀC under an atmosphere

of nitrogen. The mixture was warmed up to 0 ^ꢀC and allowed to stir

until the colour of the mixture changed from orange to pale-yellow

and a white precipitate of the lithium-salt had formed. Anhydrous

DMF (1.1 mL, 14.3 mmol) was then slowly added drop-wise at 0 ^ꢀC

and the reaction slowly warmed to room temperature while stir-

ring for 3 h after which time the reaction mixture was clear of

precipitate. Diethyl ether was evaporated off on a rotary evaporator,

the resulting solid worked up with deionised water and the product

extracted into ethyl acetate (3 ꢃ 100 mL). The organic extracts were

dried over anhydrous MgSO₄and the solvent removed under

reduced pressure to give a crude product (3.2 g), which was

NMR (CDCl₃, 300 MHz)

NH₂), 3.94 (2H, s, H-1), 3.58 (2H, s, H-8), 3.57 (2H, s, H-10), 2.07 (3H,

s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

137.5 (Ar_qu), 137.1 (Ar_qu),

d 7.32e7.23 (9H, m, ArH), 6.88 (2H, br s,

d

136.8 (Ar_qu), 131.3 (C-7), 130.7 (C-4), 129.5 (C-12/16); 128.5 (C-13/

15); 128.4 (Ar_CeH), 128.0 (Ar_CeH), 127.4 (Ar_CeH), 62.3 (C-10), 60.8 (C-

8), 43.0 (C-1), 40.9 (NeCH₃); HRMS (ES): Found 240.1602 (M^þ):

C₁₆H₂₀N₂(M^þ) requires 240.1626.

6.2.5.

Sodium azide (1.0 equiv.) was added to a stirred solution of the

appropriate

⁰-dibromoxylene (o, m or p) in anhydrous DMF

a,a

⁰-2, 3 and 4 Azido-bromo xylenes (21(o), 21(m), 21(p))

a, a

under N₂, and the reaction allowed to stir at room temperature

overnight. The reaction mixture was diluted with ethyl acetate

(100 mL) and the organic layer washed with saturated brine

1736

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

products were used without further puriﬁcation.

Water (2.39 mL, 132.60 mmol) was added and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to

room temperature and solvent removed by rotary evaporation

with water removed by azeotroping with toluene. The crude

product was puriﬁed directly by column chromatography using

EtOAc (100%) followed by EtOAc:MeOH:NEt₃(94:5:1) as eluent to

give 11(p) as a dark-yellow oil (0.56 g, 89%): IR (DMSO) n_max

3531e3408, 3273, 2131, 2057, 1973, 1659 (Ar_C]C), 1612, 1512; ¹H

6.2.6. N-[{3-(Azidomethyl)phenyl}methyl]-N-benzylmethylamine

(11(m)a)

To a stirred solution of crude 21(m) (0.22 g, 0.99 mmol) and K₂CO₃

(0.27 g, 2.00 mmol) in anhydrous acetonitrile (25 mL) at 0 ^ꢀC, was

added N-benzylmethylamine (0.19 mL, 1.49 mmol) and the reaction

heated and allowed to progress under reﬂux overnight. Acetonitrile

was then removed under reduced pressure and the reaction mixture

worked up with ethyl acetate (100 mL) and saturated aqueous

Na₂CO₃(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The

crude product was puriﬁed by column chromatography using

mixtures of ethyl acetate:hexane (10:90) to (20:80) as eluent to give

11(m)a as a yellow oil (0.15 g, 57%); ¹H NMR (CDCl₃, 300 MHz)

NMR (CD₃OD, 400 MHz)

3.38 (2H, s, H-8), 3.37 (2H, s, H-10), 2.03 (3H, s, NeCH₃); ¹³C NMR

(CD₃OD, 75 MHz) 140.3 (Ar_qu), 139.8 (Ar_qu), 139.2 (Ar_qu), 130.7

d 7.21e7.10 (9H, m, ArH), 3.73 (2H, s, H-1),

d

(C-13/15), 130.3 (C-12/16); 129.3 (C-4/6), 128.8 (C-3/7), 128.3

(C-14), 62.7 (C-8), 62.4 (C-10), 45.9 (C-1), 42.4 (NeCH₃); HRMS

(ES): Found 240.1616 (M^þ): C₁₆H₂₀N₂(M^þ) requires 240.1626.

d

7.40e7.14 (9H, m, ArH), 4.35 (1H, s, H-1), 3.57 (2H, s, H-8), 3.57 (2H, s,

6.2.10. (4-Chlorobenzyl)methylamine (22)

H-10), 2.22 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d

140.0 (Ar_qu),

To a stirred solution of 4-chlorobenzylchloride (18, 3.00 g,

18.63 mmol) in THF (300 mL) under N₂at 0 ^ꢀC, was added

methylamine solution in water (40% v/v) (9.03 mL, 93.48 mmol)

and the reaction allowed to progress at room temperature over-

night. The solvent was removed by rotary evaporation with water

removed by azeotroping with toluene. The crude product was

puriﬁed directly by column chromatography using EtOAc (100%)

followed by EtOAc:MeOH:NEt₃(92:5:3) as eluent to give 22 as

138.9 (Ar_qu), 135.3 (Ar_qu), 128.9 (Ar_CeH), 128.9 (C-12/16), 128.7

(Ar_CeH), 128.7 (Ar_CeH), 128.2 (C-13/15), 127.0 (Ar_CeH), 126.8 (Ar_CeH),

61.8 (C-8), 61.5 (C-10), 54.8 (C-1), 42.1 (NeCH₃); HRMS (ES): Found

266.1495 (M^þ): C₁₆H₁₈N₄(M^þ) requires 266.1531.

6.2.7. m-[(N-Benzyl-N-methyl)aminomethyl]-benzylamine (11(m))

To a stirred solution of 11(m)a (0.44g, 1.65 mmol) in THF

(4.50 mL) under N₂, was added PPh₃(0.44 g, 1.66 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water was added (1.50 mL, 83.33 mmol) and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to

room temperature and solvent removed by rotary evaporation

with water removed by azeotroping with toluene. The crude

product was puriﬁed directly by column chromatography using

EtOAc (100%) followed by EtOAc:MeOH:NEt₃(94:5:1) as eluent to

give 11(m) as a dark-yellow oil (0.35 g, 88%): IR (DMSO) n_max

3567e3374, 3273, 2840, 2784, 2149, 2056, 1968, 1711, 1661 (Ar_C]

a yellow oil (2.49 g, 81%); ¹H NMR (CDCl₃, 300 MHz)

(4H, m, ArH), 3.66 (2H, s, CH₂), 2.38 (3H, s, CH₃), 1.65 (1H, s, NH);

¹³C NMR (CDCl₃, 100 MHz)

138.6 (Ar_qu), 132.6 (Ar_qu), 129.5

d 7.25e7.18

d

(2 ꢃ Ar_CeH), 128.4 (2 ꢃ Ar_CeH), 55.2 (CH₂), 35.8 (CH₃). HRMS (ES):

Found 156.0556 (M þ H)^þ: C₈H₁₁NCl (M þ H)^þrequires 156.0575.

6.2.11. N-[{2-(Azidomethyl)phenyl}methyl]-N-(4-chlorobenzyl)

methylamine (12(o)a)

To a stirred solution of 22 (0.60 g, 3.85 mmol) and K₂CO₃(0.71 g,

5.14 mmol) in anhydrous acetonitrile (150 mL) under N₂at 0 ^ꢀC, was

added crude 21(o) (0.58 g, 2.57 mmol) and the reaction heated and

allowed to progress under reﬂux for 6 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated aqueous Na₂CO₃

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography using mixtures of

ethyl acetate:hexane (5:95) to (20:80) as eluent to give12(o)a as

_C), 1579; ¹H NMR (CD₃OD, 300 MHz)

(8H, m, ArH), 4.10 (2H, s, H-1), 3.67 (2H, s, H-8), 3.67 (2H, s, H-10),

2.23 (3H, s, NeCH₃); ¹³C NMR (CD₃OD, 75 MHz)

139.6 (Ar_qu),

138.3 (Ar_qu), 136.1 (Ar_qu), 131.0 (Ar_CeH), 131.0 (Ar_CeH), 130.7 (C-12/

16), 130.1 (Ar_CeH), 129.5 (C-13/15), 129.1 (Ar_CeH), 128.7 (Ar_CeH),

62.6 (C-8), 62.1 (C-10), 44.5 (C-1), 41.9 (NeCH₃) HRMS (ES): Found

241.1682 (M þ H)^þ: C₁₆H₂₁N₂(M þ H)^þrequires 241.1699.

d 7.60 (1H, s, H-3), 7.44e7.24

d

a yellow oil (0.45 g, 58%); ¹H NMR (CDCl₃, 400 MHz)

(9H, m, ArH), 4.55 (2H, s, H-1), 3.58 (2H, s, H-8), 3.50 (2H, s, H-10),

2.13 (3H, s, H-9); ¹³C NMR (CDCl₃, 75 MHz)

137.4 (Ar_qu), 137.1

d 7.39e7.26

6.2.8. N-[{4-(Azidomethyl)phenyl}methyl]-N-benzylmethylamine

(11(p)a)

d

To a stirred solution of 21(p) (0.50 g, 2.25 mmol) and K₂CO₃

(0.61, 4.50 mmol) in anhydrous acetonitrile (50 mL) at 0 ^ꢀC, was

added N-benzylmethylamine (0.43 mL, 3.40 mmol) and the reac-

tion heated and allowed to progress under reﬂux overnight.

Acetonitrile was then removed under reduced pressure and the

reaction mixture partitioned between ethyl acetate (100 mL) and

saturated Na₂CO₃(3 ꢃ 50 mL). The organic extracts dried (MgSO₄)

and concentrated in vacuo. The crude product was puriﬁed by

column chromatography using mixtures of ethyl acetate:hexane

(10:90) to (20:80) as eluent to give 11(p)a as a yellow oil (0.35 g,

(Ar_qu), 134.7 (Ar_qu), 132.7 (Ar_qu), 130.7 (Ar_CeH), 130.2 (C-12/16),

129.6 (Ar_CeH), 128.3 (C-13/15), 128.1 (Ar_CeH), 127.7 (Ar_CeH), 61.5

(C-10), 59.9 (C-8), 52.0 (C-1), 41.9 (C-9). HRMS (ES): Found 301.1211

(M þ H)^þ: C₁₆H₁₈ClN₄(M þ H)^þrequires 301.1215.

6.2.12. o-[(N-4-Chlorobenzyl-N-methyl)aminomethyl]-

benzylamine (12(o))

To a stirred solution of 12(o)a (0.40g, 1.33 mmol) in THF

(3.60 mL) under N₂, was added PPh₃(0.42 g, 1.60 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water was added (1.20 mL, 66.67 mmol) and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to

room temperature and solvent removed by rotary evaporation

with water removed by azeotroping with toluene. The crude

product was puriﬁed directly by column chromatography using

EtOAc (100%) followed by EtOAc:MeOH:NEt₃(94:5:1) as eluent to

give 12(o) as a yellow oil (0.34 g, 93%); ¹H NMR (CDCl₃, 300 MHz)

59%); ¹H NMR (CDCl₃, 400 MHz)

s, H-1), 3.56 (2H, s, H-8), 3.56 (2H, s, H-10), 2.22 (3H, s, H-9); ¹³C

NMR (CDCl₃, 75 MHz) 139.7 (Ar_qu), 139.2 (Ar_qu), 134.1 (Ar_qu), 129.4

d 7.43e7.26 (9H, m, ArH), 4.35 (1H,

d

(2ꢃ Ar_CeH), 129.0 (2ꢃ Ar_CeH), 128.3 (2ꢃ Ar_CeH), 128.2 (2 ꢃ Ar_CeH),

127.0 (Ar_CeH), 61.9 (C-8), 61.5 (C-10), 54.7 (C-1), 42.1 (C-9); HRMS

(ES): Found 266.1539 (M^þ): C₁₆H₁₈N₄(M^þ) requires 266.1531.

6.2.9. p-[(N-Benzyl-N-methyl)aminomethyl]-benzylamine (11(p))

To a stirred solution of 11(p)a (0.70 g, 2.63 mmol) in THF

(7.20 mL) under N₂, was added PPh₃(0.44 g, 1.66 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

d

7.35e7.16 (9H, m, ArH), 3.87 (2H, s, H-1), 3.55 (2H, s, H-8), 3.52

(2H, s, H-10), 2.07 (3H, s, H-9); ¹³C NMR (CDCl₃, 75 MHz)

d

141.3

(Ar_qu), 136.7 (Ar_qu), 136.4 (Ar_qu), 133.0 (Ar_qu), 131.1 (Ar_CeH), 130.6

(C-12/16), 129.4 (Ar_CeH), 128.5 (C-13/15), 128.2 (Ar_CeH), 127.1

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

1737

(Ar_CeH), 61.7 (C-10), 60.7 (C-8), 44.1 (C-1), 41.4 (C-9); HRMS (ES):

reaction mixture allowed to stir for 30 min at room temperature.

Water (2.40 mL, 133.33 mmol) was added and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to room

temperature and solvent removed by rotary evaporation with

water removed by azeotroping with toluene. The crude product

was puriﬁed directly by column chromatography using EtOAc

(100%) followed by EtOAc:MeOH:NEt₃(94:5:1) as eluent to give

Found 275.1324 (M þ H)^þ: C₁₆H₂₀ClN₂(M þ H)^þrequires 275.1310.

6.2.13. N-[{3-(Azidomethyl)phenyl}methyl]-N-(4-chlorobenzyl)

methylamine (12(m)a)

To a stirred solution of 22 (0.80 g, 5.13 mmol) and K₂CO₃(0.95 g,

6.85 mmol) in anhydrous acetonitrile (200 mL) at 0 ^ꢀC, was added

crude 21(m) (0.77 g, 3.43 mmol), and the reaction heated and

allowed to progress under reﬂux for 6 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated aqueous Na₂CO₃

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography using mixtures of

ethyl acetate:hexane (5:95) to (20:80) as eluent to give12(m)a as

12(p) as a dark-orange oil (0.68 g, 93%): IR (DMSO) n_max(cm^ꢁ1

3551e3388, 3287, 2790, 2138, 2063, 1957, 1708, 1662 (Ar_C]C), 1513,

1490; ¹H NMR (CDCl₃, 300 MHz)

7.32e7.24 (8H, m, ArH), 3.83 (2H,

s, H-1), 3.48 (2H, s, H-8), 3.44 (2H, s, H-10), 3.16 (2H, br s, NH₂), 2.14

(2H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

137.7 (Ar_qu), 132.3

)

d

(Ar_qu), 131.7 (Ar_qu), 129.9 (C-13/15), 128.9 (C-12/16), 128.1 (C-4/6);

127.7 (Ar_qu), 127.0 (C-3/7), 61.3 (C-8), 60.7 (C-10), 45.5 (C-1), 41.9

(NeCH₃); HRMS (ES): Found 275.1315 (M þ H)^þ: C₁₆H₂₀ClN₂

(M þ H)^þrequires 275.1310.

a yellow oil (0.63 g, 61%); ¹H NMR (CDCl₃, 300 MHz)

m, ArH), 7.22 (1H, m, H-3), 4.35 (1H, s, H-1), 3.54 (2H, s, H-8), 3.50

(2H, s, H-10), 2.19 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

139.4

d 7.36e7.31 (7H,

d

(Ar_qu), 137.7 (Ar_qu), 135.3 (Ar_qu), 132.6 (Ar_qu), 130.1 (C-12/16), 128.8

(C-13/15), 128.7 (Ar_CeH), 128.6 (Ar_CeH), 128.3 (Ar_CeH), 126.9 (Ar_CeH),

61.5 (C-8), 61.0 (C-10), 54.7 (C-1), 42.1 (NeCH₃); HRMS (ES): Found

301.1221 (M þ H)^þ: C₁₆H₁₈ClN₄(M þ H)^þrequires 301.1215.

6.2.17. (4-Methoxybenzyl)methylamine (23)

To a stirred solution of 4-methoxybenzylchloride (19, 3.00 g,

19.18 mmol) in THF (350 mL) under N₂at 0 ^ꢀC, was added

methylamine solution in water (40% v/v) (9.30 mL, 95.86 mmol)

and the reaction allowed to progress at room temperature over-

night. The solvent was removed by rotary evaporation with water

removed by azeotroping with toluene. The crude product was

puriﬁed directly by column chromatography using EtOAc (100%)

followed by EtOAc:MeOH:NEt₃(92:5:3) as eluent to give 23 as

6.2.14. m-[(N-4-Chlorobenzyl-N-methyl)aminomethyl]-

benzylamine (12(m))

To a stirred solution of 12(m)a (0.60 g, 2.00 mmol) in THF

(5.40 mL) under N₂, was added PPh₃(0.63 g, 2.40 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water was added (1.80 mL, 100.00 mmol) and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to room

temperature and solvent removed by rotary evaporation with

water removed by azeotroping with toluene. The crude product

was puriﬁed directly by column chromatography using EtOAc

(100%) followed by EtOAc:MeOH:NEt₃(94:5:1) as eluent to give

a yellow oil (2.72 g, 87%); ¹H NMR (CDCl₃, 300 MHz)

d 7.27 (2H, d,

J ¼ 6.6 Hz, H-3/5), 6.87 (2H, d, J ¼ 6.6 Hz, H-2/6), 3.75 (3H, s, OCH₃),

3.64 (2H, s, CH₂), 2.33 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 100 MHz)

d

159.0 (Ar_qu), 132.5 (Ar_qu), 129.6 (C-3/5), 113.8 (C-2/6), 54.9 (OCH₃/

CH₂), 35.1 (NeCH₃). HRMS (ES): Found 152.1070 (M þ H)^þ:

C₉H₁₄NO (M þ H)^þrequires 152.1075.

12(m) as a light-orange oil (0.56 g, 93%): IR (DMSO) n_max(cm^ꢁ1

3547-3399, 3271, 2835, 2783, 2144, 2048, 1707, 1663 (Ar_C]C), 1605,

1491; ¹H NMR (CDCl₃, 300 MHz)

7.31e7.18 (8H, m, ArH), 3.85 (2H,

s, H-1), 3.49 (2H, s, H-8), 3.46 (2H, s, H-10), 3.02 (2H, br s, NH₂), 2.15

(3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

142.0 (Ar_qu), 139.3

)

6.2.18. N-[{2-(Azidomethyl)phenyl}methyl]-N-(4-methoxybenzyl)

methylamine (13(o)a)

d

To a stirred solution of 23 (1.00 g, 6.61 mmol) and K₂CO₃(1.22 g,

8.87 mmol) in anhydrous acetonitrile (300 mL) at 0 ^ꢀC, was added

crude 21(o) (0.99 g, 4.38 mmol) and the reaction heated and

allowed to progress under reﬂux for 6 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated aqueous Na₂CO₃

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography using mixtures of

ethyl acetate:hexane (10:90) to (25:75) as eluent to give 13(o)a as

d

(Ar_qu), 137.7 (Ar_qu), 132.4 (Ar_qu), 130.0 (C-12/16), 128.3 (Ar_qu), 125.8

(Ar_CeH), 128.2 (C-13/15), 127.6 (Ar_CeH), 127.4 (Ar_CeH), 61.6 (C-8), 61.0

(C-10), 45.8 (C-1), 42.0 (NeCH₃). HRMS (ES): Found 275.1316

(M þ H)^þ: C₁₆H₂₀ClN₂(M þ H)^þrequires 275.1310.

6.2.15. N-[{4-(Azidomethyl)phenyl}ethyl]-N-(4-chlorobenzyl)

methylamine (12(p)a)

a yellow oil (0.83 g, 64%); ¹H NMR (CDCl₃, 300 MHz)

d 7.42e7.20

To a stirred solution of 22 (1.00 g, 6.41 mmol) and K₂CO₃(1.19 g,

8.60 mmol) in anhydrous acetonitrile (300.00 mL) at 0 ^ꢀC, was added

crude 21(p) (0.96 g, 4.25 mmol) and the reaction heated and allowed

to progress under reﬂux for 6 h. Acetonitrile was then removed under

reduced pressure and the reaction mixture worked up with ethyl

acetate (100 mL) and saturated Na₂CO₃(3 ꢃ 50 mL), dried (MgSO₄)

and concentrated invacuo. The crude product was puriﬁed bycolumn

chromatography using mixtures of ethyl acetate:hexane (5:95) to

(20:80) as eluent to afford 12(p)a as a light-yellow oil (0.85 g, 67%);

(6H, m, ArH), 6.90 (2H, dd, J ¼ 2.3, 6.8 Hz, H-12/16), 4.56 (2H, s, H-1),

3.83 (3H, s, OMe), 3.57 (2H, s, H-8), 3.51 (2H, s, H-10), 2.15 (3H, s,

NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 159.1 (Ar_qu), 137.8 (Ar_qu), 135.1

(Ar_qu), 131.3 (Ar_qu), 131.1 (Ar_CeH), 130.5 (C-13/15), 129.7 (Ar_CeH),

128.3 (Ar_CeH), 128.3 (Ar_CeH), 114.0 (C-13/15), 62.1 (C-8), 60.0 (C-10),

55.5 (OMe), 52.2 (C-1), 42.3 (NeCH₃); HRMS (ES): Found 297.1716

(M þ H)^þ: C₁₇H₂₁N₄O (M þ H)^þrequires 297.1710.

6.2.19. o-[(N-4-Methoxybenzyl-N-methyl)aminomethyl]-

benzylamine (13(o))

¹H NMR (CDCl₃, 400 MHz)

3.56 (2H, s, H-8), 3.52 (2H, s, H-10), 2.22 (3H, s, NeCH₃); ¹³C NMR

(CDCl₃, 75 MHz) 139.3 (Ar_qu), 137.7 (Ar_qu), 134.0 (Ar_qu), 132.4 (Ar_qu),

d

7.44e7.31 (8H, m, ArH), 4.35 (2H, s, H-1),

To a stirred solution of 13(o)a (0.80 g, 2.70 mmol) in THF

(7.29 mL) under N₂, was added PPh₃(0.85 g, 3.24 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water (2.43 mL, 135.00 mmol) was added and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to room

temperature and solvent removed by rotary evaporation with

water removed by azeotroping with toluene. The crude product

was puriﬁed directly by column chromatography using EtOAc

(100%) followed by EtOAc:MeOH:NEt₃(93:5:2) as eluent to give

d

130.0 (C-12/16); 129.1 (C-13/15), 128.2 (C-4/6), 128.0 (C-3/7); 61.3

(C-8), 61.0 (C-10), 54.4 (C-1), 42.0 (NeCH₃); HRMS (ES): Found

301.1223 (M þ H)^þ: C₁₆H₁₈ClN₄(M þ H)^þrequires 301.1215.

6.2.16. p-[(N-4-Chlorobenzyl-N-methyl)aminomethyl]-

benzylamine (12(p))

To a stirred solution of 12(p)a (0.80g, 2.67 mmol) in THF

(7.20 mL) under N₂, was added PPh₃(0.92 g, 3.20 mmol) and the

13(o) as a light-brown oil (0.61 g, 83%): IR (DMSO) n_max(cm^ꢁ1

)

1738

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

3592e3324, 2593, 2142, 2061, 1981, 1664 (Ar_C]C), 1510; ¹H NMR

(CDCl₃, 300 MHz)

6.5 Hz, H-12/16), 3.85 (2H, s, H-1), 3.77 (3H, s, OMe), 3.53 (2H, s,

ArH), 4.37 (2H, s, H-1), 3.77 (3H, s, OMe), 3.52 (2H, s, H-8), 3.48 (2H,

s, H-10), 2.15 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75.5 MHz)

159.2

(Ar_qu), 140.2 (Ar_qu), 134.6 (Ar_qu), 131.6 (Ar_qu), 130.2 (C-4/6), 128.5

(C-3/7), 129.4 (C-13/15), 113.9 (C-12/16), 61.4 (C-8), 61.4 (C-10), 54.9

(OMe), 54.4 (C-1), 41.8 (NeCH₃); HRMS (ES): Found 297.1706

(M þ H)^þ: C₁₇H₂₁N₄O (M þ H)^þrequires 297.1710.

d

7.32e7.15 (6H, m, ArH), 6.83 (2H, dd, J ¼ 2.0,

d

H-8), 3.49 (2H, s, H-10), 3.40 (2H, br s, NH₂), 2.09 (3H, s, NeCH₃);

¹³C NMR (CDCl₃, 75 MHz)

d 158.7 (Ar_qu), 142.1 (Ar_qu), 136.7 (Ar_qu),

131.0 (Ar_CeH), 130.5 (Ar_qu), 130.4 (C-13/15), 129.1 (Ar_CeH), 128.0

(Ar_CeH), 127.0 (Ar_CeH), 113.6 (C-12/16), 62.0 (C-8), 60.5 (C-10), 55.1

(OMe), 44.3 (C-1), 41.4 (NeCH₃); HRMS (ES): Found 271.1813

(M þ H)^þ: C₁₇H₂₃N₂O (M þ H)^þrequires 271.1805.

6.2.23. p-[(N-4-Methoxybenzyl-N-methyl)aminomethyl]-

benzylamine (13(p))

To a stirred solution of 13(p)a (0.70 g, 2.36 mmol) in THF

(6.39 mL) under N₂, was added PPh₃(0.74 g, 2.84 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water (2.13 mL, 118.13 mmol) was added and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to

room temperature and solvent removed by rotary evaporation

with water removed by azeotroping with toluene. The crude

product was puriﬁed directly by column chromatography using

EtOAc (100%) followed by EtOAc:MeOH:NEt₃(92:5:3) as eluent to

give 13(p) as a light-brown oil (0.57 g, 89%): IR (DMSO) n_max

(cm^ꢁ1) 3374e3272, 2833, 2785, 2145, 2056, 1978, 1903, 1662

6.2.20. N-[{3-(Azidomethyl)phenyl}methyl]-N-(4-methoxybenzyl)

methylamine (13(m)a)

To a stirred solution of 23 (1.00 g, 6.61 mmol) and K₂CO₃(1.22 g,

8.87 mmol) in anhydrous acetonitrile (300 mL) at 0 ^ꢀC, was added

crude 21(m) (0.99 g, 4.38 mmol) and the reaction heated and

allowed to progress under reﬂux for 5 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated aqueous Na₂CO₃

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography using mixtures of

ethyl acetate:hexane (10:90) to (25:75) as eluent to give 13(m)a as

(Ar_C]C), 1610, 1583; ¹H NMR (CDCl₃, 400 MHz)

d 7.32e7.27 (4H,

a yellow oil (0.79 g, 61%); ¹H NMR (CDCl₃, 300 MHz)

d

7.35e7.18

m, ArH), 7.23 (2H, dd, J ¼ 2.0, 6.8 Hz, H-13/18), 6.85 (2H, dd,

J ¼ 2.0, 6.8 Hz, H-12/17), 4.53 (2H, br s, NH₂), 3.83 (2H, s, H-1),

3.77 (3H, s, OMe), 3.46 (2H, s, H-8), 3.43 (2H, s, H-10), 2.13 (3H, s,

(4H, m, ArH), 7.28 (2H, dd, J ¼ 2.1, 6.4 Hz, H-13/15), 6.87 (2H, dd,

J ¼ 2.1, 6.4 Hz, H-12/16), 4.34 (2H, s, H-1), 3.81 (3H, s, OMe), 3.53

(2H, s, H-8), 3.49 (2H, s, H-10), 2.18 (3H, s, NeCH₃); ¹³C NMR (CDCl₃,

NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 158.5 (Ar_qu), 138.6 (Ar_qu),

75 MHz)

d

158.7 (Ar_qu), 140.1 (Ar_qu), 135.3 (Ar_qu), 131.0 (Ar_qu), 130.1

138.4 (Ar_qu), 131.0 (Ar_qu), 129.9 (C-13/15), 129.1 (C-4/6), 127.4 (C-

3/7), 113.5 (C-12/16), 61.1 (C-8), 61.0 (C-10), 55.1 (OMe), 44.9 (C-

(C-12/16), 128.9 (Ar_CeH), 128.7 (Ar_CeH), 128.7 (Ar_CeH), 126.8 (Ar_CeH),

113.6 (C-13/15), 61.3 (C-8), 61.2 (C-10), 55.2 (OMe), 54.8 (C-1), 42.0

(NeCH₃); HRMS (ES): Found 297.1727 (M þ H)^þ: C₁₇H₂₁N₄O

(M þ H)^þrequires 297.1710.

1), 41.8 (NeCH₃); HRMS (ES): Found 271.1810 (M

þ

H)^þ:

C₁₇H₂₃N₂O (M þ H)^þrequires 271.1805.

6.2.24. (4-N,N-Dimethylaminobenzyl)-methylamine (24)

6.2.21. m-[(N-4-Methoxybenzyl-N-methyl)aminomethyl]-

benzylamine (13(m))

To a stirred solution of 4-N, N-dimethylaminobenzaldehyde (20,

3.00 g, 18.3 mmol) in acetonitrile (400 mL) under N₂at 0 ^ꢀC was

added methylamine solution in water (40% v/v) (10.0 mL,

103 mmol) and the reaction allowed to progress for 2 h. Then

NaCNBH₃(3.46 g, 64.1 mmol) was added and the reaction allowed

to progress at room temperature for 4 h. The solvent was removed

on the rotary evaporator with water removed by azeotroping with

toluene. The crude product was puriﬁed directly by column chro-

matography using EtOAc (100%) followed by EtOAc:MeOH:NEt₃

(92:5:3) as eluent to give 24 as a yellow oil (2.20 g, 73%); ¹H NMR

To a stirred solution of 13(m)a (0.75 g, 2.53 mmol) in THF

(6.83 mL) under N₂, was added PPh₃(0.80 g, 3.04 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water was added (2.28 mL, 126.56 mmol) and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to

room temperature and solvent removed by rotary evaporation

with water removed by azeotroping with toluene. The crude

product was puriﬁed directly by column chromatography using

EtOAc (100%) followed by EtOAc:MeOH:NEt₃(93:5:2) as eluent to

give 13(m) as a dark-yellow oil (0.60 g, 88%): IR (DMSO) n_max

(cm^ꢁ1) 3531e3410, 3286, 2137, 2060, 1986, 1661 (Ar_C]C), 1611,

(CDCl₃, 300 MHz)

J ¼ 8.7 Hz, H-2/6), 3.59 (2H, s, H-7), 2.88 (6H, s, NMe₂), 2.34 (3H, s,

H-8); ¹³C NMR (CDCl₃, 100 MHz)

d

7.17 (2H, d, J ¼ 8.7 Hz, H-3/5), 6.70 (2H, d,

d

150.0 (2ꢃ Ar_qu), 129.1 (C-3/5),

1511 (NH); ¹H NMR (CDCl₃, 400 MHz)

d

7.29e6.80 (8H, m, ArH),

112.7 (C-2/6), 55.6 (C-7), 40.3 (C-8), 35.6 (NMe₂); HRMS (ES): Found

4.53 (2H, br s, NH₂), 3.73 (2H, s, H-1), 3.77 (3H, s, OMe), 3.46 (2H,

165.1390 (M þ H)^þ: C₁₀H₁₇N₂(M þ H)^þrequires 165.1386.

s, H-8), 3.43 (2H, s, H-10), 2.13 (3H, s, NeCH₃); ¹³C NMR (CD₃OD,

75 MHz)

d

158.5 (Ar_qu), 142.6 (Ar_qu), 136.2 (Ar_qu), 131.1(Ar_CeH),

6.2.25. N-[{2-(Azidomethyl)phenyl}methyl]-N-(4-

129.9 (Ar_qu), 129.7 (C-12/16), 129.0 (Ar_CeH), 127.6 (Ar_CeH), 127.0

(Ar_CeH), 113.2 (C-13/15), 61.7 (C-8), 60.8 (C-10), 55.3 (OMe), 54.3

(C-1), 41.7 (NeCH₃); HRMS (ES): Found 271.1810 (M þ H)^þ:

C₁₇H₂₃N₂O (M þ H)^þrequires 271.1805.

dimethylaminobenzyl)methylamine (14(o)a)

To a stirred solution of 24 (1.00 g, 6.06 mmol) and K₂CO₃(1.25 g,

9.09 mmol) in anhydrous acetonitrile (150 mL) at 0 ^ꢀC, was added

crude 21(o) (1.14 g, 5.05 mmol) and the reaction heated and

allowed to progress under reﬂux for 6 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated aqueous Na₂CO₃

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography using mixtures of

ethyl acetate:hexane (5:95) to (30:70) as eluent to give 14(o)a as

6.2.22. N-[{4-(Azidomethyl)phenyl}methyl]-N-(4-methoxybenzyl)

methylamine (13(p)a)

To a stirred solution of 23 (1.00 g, 6.61 mmol) and K₂CO₃(1.22 g,

8.87 mmol) in anhydrous acetonitrile (300 mL) at 0 ^ꢀC, was added

crude 21(p) (0.99 g, 4.38 mmol) and the reaction heated and

allowed to progress under reﬂux for 6 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated Na₂CO₃(3 ꢃ 50 mL),

dried (MgSO₄) and concentrated in vacuo. The crude product was

puriﬁed by column chromatography using mixtures of ethyl ace-

tate:hexane (10:90) to (25:75) as eluent to afford 13(p)a as a yellow

a yellow oil (0.89 g, 57%): ¹H NMR (CDCl₃, 400 MHz)

d 7.48e7.42

(4H, m, ArH), 7.40 (2H, d, J ¼ 8.7 Hz, H-12/16), 6.90 (2H, d, J ¼ 8.7 Hz,

H-13/15), 4.69 (2H, s, H-1), 3.69 (2H, s, H-8), 3.64 (2H, s, H-10),3.09

(6H, s, NMe₂), 2.31 (3H, s, H-9); ¹³C NMR (CDCl₃, 75 MHz)

d 149.7

(Ar_qu), 137.4 (Ar_qu), 134.7 (Ar_qu),130.5 (Ar_CeH), 129.8 (C-12/16), 129.7

(Ar_CeH), 127.6 (Ar_CeH), 127.2 (Ar_CeH), 126.4 (Ar_qu), 112.2 (C-13/15),

61.8 (C-10), 59.4 (C-8), 51.6 (C-1), 41.7 (C-9), 40.3 (NMe₂); HRMS

oil (0.74 g, 57%); ¹H NMR (CDCl₃, 400 MHz)

d 7.45e6.91 (8H, m,

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

1739

(ES): Found 310.2032 (M þ H)^þ: C₁₈H₂₄N₅(M þ H)^þrequires

6.2.29. N-[{4-(Azidomethyl)phenyl}methyl]-N-(4-

dimethylaminobenzyl)methylamine (14(p)a)

310.2026.

To a stirred solution of 24 (1.00 g, 6.06 mmol) and K₂CO₃(1.25 g,

9.09 mmol) in anhydrous acetonitrile (150 mL) at 0 ^ꢀC, was added

crude 21(p) (1.14 g, 5.05 mmol) and the reaction heated and

allowed to progress under reﬂux for 6 h. Acetonitrile was then

removed under reduced pressure and the reaction mixture worked

up with ethyl acetate (100 mL) and saturated aqueous Na₂CO₃

(3 ꢃ 50 mL), dried (MgSO₄) and concentrated in vacuo. The crude

product was puriﬁed by column chromatography using mixtures of

ethyl acetate:hexane (5:95) to (30:70) as eluent to give 14(p)a as

6.2.26. o-[(N-4-Dimethylaminobenzyl-N-methyl)aminomethyl]-

benzylamine (14(o))

To a stirred solution of 14(o)a (0.80 g, 2.58 mmol) inTHF (7.20 mL)

under N₂, was added PPh₃(0.81 g, 3.10 mmol) and the reaction

mixture allowed to stir for 30 min at room temperature. Water

(2.40 mL,133 mmol) was added and the reaction heated and allowed

to reﬂux for 6 h. The reaction was then cooled to room temperature

and solvent removed on the rotary evaporator with water removed

byazeotroping with toluene. The crude product was puriﬁed directly

by column chromatography using EtOAc (100%) followed by

EtOAc:MeOH:NEt₃(93:5:2) as eluent to give 14(o) as a light-brown

oil (0.61 g, 83%): IR (DMSO) n_max(cm^ꢁ1) 3550e3401, 3293, 2132,

a yellow oil (0.99 g, 63%): ¹H NMR (CDCl₃, 400 MHz)

d 7.40e7.25

(4H, m, ArH), 7.23 (2H, d, J ¼ 8.6 Hz, H-12/16), 6.72 (2H, d, J ¼ 8.6 Hz,

H-13/17), 4.32 (2H, s, H-1), 3.51 (2H, s, H-8), 3.46 (2H, s, H-10), 2.92

(6H, s, NMe₂), 2.15 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 149.8

2068, 1980, 1663, 1613, 1522; ¹H NMR (CDCl₃, 300 MHz)

d

7.31e7.17

(Ar_qu), 139.9 (Ar_qu), 133.8 (Ar_qu), 129.8 (Ar_CeH), 129.3 (C-12/16),

128.1 (Ar_CeH), 126.9 (Ar_qu), 112.5 (C-13/15), 61.4 (C-10), 61.1 (C-8),

54.6 (C-1), 42.1 (NeCH₃), 40.7 (NMe₂); HRMS (ES): Found 310.2024

(M þ H)^þ: C₁₈H₂₄N₅(M þ H)^þrequires 310.2026.

(6H, m, ArH), 6.71 (2H, d, J ¼ 9.0 Hz, H-13/15), 4.05 (2H, br s, NH₂),

3.85 (2H, s, H-1), 3.53 (2H, s, H-8), 3.48 (2H, s, H-10), 2.92 (6H, s,

NMe₂), 2.07 (3H, s, H-9); ¹³C NMR (CDCl₃, 75.5 MHz)

d 149.9 (Ar_qu),

141.3 (Ar_qu),137.0 (Ar_qu),131.1 (Ar_CeH),130.3 (C-12/16),129.5 (Ar_CeH),

128.0 (Ar_CeH), 127.0 (Ar_CeH), 125.8 (Ar_qu), 112.5 (C-13/15), 62.1 (C-8),

60.5 (C-10), 44.2 (C-1), 41.2 (C-9), 40.6 (NMe₂); HRMS (ES): Found

284.2123 (M þ H)^þ: C₁₈H₂₆N₃(M þ H)^þrequires 284.2121.

6.2.30. p-[(N-4-Dimethylaminobenzyl-N-methyl)aminomethyl]-

benzylamine (14(p))

To a stirred solution of 14(p)a (0.80 g, 2.58 mmol) in THF

(7.20 mL) under N₂, was added PPh₃(0.81 g, 3.10 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water (2.40 mL, 133 mmol) was added and the reaction heated and

allowed to reﬂux for 6 h. The reaction was then cooled to room

temperature and solvent removed on the rotary evaporator with

water removed by azeotroping with toluene. The crude product

was puriﬁed directly by column chromatography using EtOAc

(100%) followed by EtOAc:MeOH:NEt₃(93:5:2) as eluent to give

6.2.27. N-[{3-(Azidomethyl)phenyl}methyl]-N-(4-

dimethylaminobenzyl)methylamine (14(m)a)

To a stirred solution of 24 (1.00 g, 6.06 mmol) and K₂CO₃(1.25 g,

9.09 mmol) in anhydrous acetonitrile (150 mL) at 0 ^ꢀC, was added

crude 21(m) (1.14 g, 5.05 mmol) and the reaction heated and allowed

to progress under reﬂux for 6 h. Acetonitrile was then removed

under reduced pressure and the reaction mixture worked up with

ethyl acetate (100 mL) and saturated aqueous Na₂CO₃(3 ꢃ 50 mL),

dried (MgSO₄) and concentrated in vacuo. The crude product was

puriﬁed by column chromatography using mixtures of ethyl aceta-

te:hexane (5:95) to (30:70) as eluent to give 14(m)a as a yellow oil

14(p) as a light-brown oil (0.65 g, 89%): IR (DMSO) n_max(cm^ꢁ1

3545e3385, 3285, 2582, 2141, 2066, 1985, 1904, 1661, 1521; ¹H

NMR (CDCl₃, 400 MHz) 7.31e21 (4H, m, ArH), 7.19 (2H, d,

)

d

J ¼ 8.8 Hz, H-12/16), 6.69 (2H, d, J ¼ 8.8 Hz, H-13/15), 3.81 (2H, s, H-

(0.95 g, 61%): ¹H NMR (CDCl₃, 300 MHz)

d

7.35e7.26 (4H, m, ArH),

1), 3.46 (2H, s, H-8), 3.43 (2H, s, H-10), 2.91 (6H, s, NMe₂), 2.49 (2H,

7.23 (2H, d, J ¼ 8.9 Hz, H-12/16), 6. 73 (2H, d, J ¼ 8.9 Hz, H-13/15), 4.34

br s, NH₂), 2.14 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 149.8

(2H, s, H-1), 3.52 (2H, s, H-8), 3.47 (2H, s, H-10), 2.94 (6H, s, NMe₂),

(Ar_qu),141.2 (Ar_qu), 138.0 (Ar_qu), 129.8 (C-4/6), 129.2 (C-12/16), 127.0

(C-3/7); 126.8 (Ar_qu), 112.5 (C-13/15), 61.2 or 61.1 (C-8 or 10), 61.2 or

61.1 (C-10 or 8), 46.0 (C-1), 41.9 (NeCH₃), 40.7 (NMe2); HRMS (ES):

Found 284.2115 (M þ H)^þ: C₁₈H₂₆N₃(M þ H)^þrequires 284.2121.

2.19 (3H, s, H-9); ¹³C NMR (CDCl₃, 75 MHz)

d 1.49.9 (Ar_qu), 140.4

(Ar_qu), 135.2 (Ar_qu), 129.9 (C-12/16), 128.9 (Ar_CeH), 128.7 (Ar_CeH),

128.6 (Ar_CeH), 126.8 (Ar_qu), 126.7 (Ar_CeH), 113.6 (C-13/15), 61.4 (C-8),

61.2 (C-10), 54.8 (C-1), 42.0 (C-9), 40.7 (NMe₂); HRMS (ES): Found

310.2032 (M þ H)^þ: C₁₈H₂₄N₅(M þ H)^þrequires 310.2026.

6.2.31. s-trans-N-[{2-(N-Benzyl-N-methylaminomethyl)phenyl}

methyl]formamide (11f(o))

6.2.28. m-[(N-4-Dimethylaminobenzyl-N-methyl)aminomethyl]-

benzylamine (14(m))

A stirred solution of 11(o) (0.35 g, 1.44 mmol) in excess ethyl

formate (5.75 mL, 72.45 mmol) under N₂was reﬂuxed overnight.

The excess ethyl formate was removed under reduced pressure and

the crude product puriﬁed by column chromatography using

mixtures of ethyl acetate:hexane (50:50) to (95:5) as eluent to give

11f(o) as a thick-yellow oil and predominantly (>90%) as an s-trans

rotamer, (0.15 g, 92%): IR (DMSO) n_max(cm^ꢁ1) 3621e3211, 2595,

2148, 2059, 1979, 1907, 1656 (Ar_C]C); ¹H NMR (CDCl₃, 300 MHz)

To a stirred solution of 14(m)a (0.80 g, 2.58 mmol) in THF

(7.20 mL) under N₂, was added PPh₃(0.81 g, 3.10 mmol) and the

reaction mixture allowed to stir for 30 min at room temperature.

Water (2.40 mL, 133 mmol) was added and the reaction heated

and allowed to reﬂux for 6 h. The reaction was then cooled to

room temperature and solvent removed on the rotary evaporator

with water removed by azeotroping with toluene. The crude

product was puriﬁed directly by column chromatography using

EtOAc (100%) followed by EtOAc:MeOH:NEt₃(93:5:2) as eluent to

give 14(m) as a light-brown oil (0.59 g, 81%): IR (DMSO) n_max

(cm^ꢁ1) 3531e3410, 3286, 2137, 2060, 1986, 1661, 1611, 1511; ¹H

d

8.33 (1H, br s, NH), 8.08 (1H, s, H-1), 7.41e7.22 (9H, m, ArH), 4.44

(2H, d, J ¼ 5.7 Hz, H-2), 3.58 (2H, s, H-9), 3.55 (2H, s, H-11), 2.12 (3H,

s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

160.3 (C-1), 137.9 (Ar_qu),

d

137.8 (Ar_qu), 136.7 (Ar_qu), 131.7 (C-8), 130.6 (C-5), 129.4 (C-13/17);

128.6 (Ar_CeH), 128.5 (C-14/16); 127.7 (Ar_CeH), 127.6 (Ar_CeH), 62.2

(C-9), 61.4 (C-11), 42.0 (NeCH₃), 41.3 (C-2); HRMS (ES): Found

268.1588 (M^þ): C₁₇H₂₀N₂O (M^þ). requires 268.1576.

NMR (CDCl₃, 300 MHz)

J ¼ 8.7 Hz, H-13/15), 3.86 (2H, s, H-1), 3.79 (6H, s, NMe₂), 3.49 (2H,

s, H-8), 3.47(2H, s, H-10), 2.50 (2H, br s, NH₂), 2.16 (3H, s, H-9); ¹³

NMR (CDCl₃, 75 MHz) 158.6 (Ar_qu), 142.4 (Ar_qu), 139.6 (Ar_qu),

d 7.29e7.18 (6H, m, ArH), 6.86 (2H, d,

C

d

6.2.32. s-trans-N-[{3-(N-Benzyl-N-methylaminomethyl)phenyl}

methyl]formamide (11f(m))

A stirred solution of 11(m) (0.15 g, 0.63 mmol) in excess ethyl

formate (2.50 mL, 31.50 mmol) under N₂was reﬂuxed overnight.

The excess ethyl formate was removed under reduced pressure and

131.1 (Ar_qu), 130.0 (C-12/16), 128.3 (Ar_CeH), 127.6 (Ar_CeH), 127.5

(Ar_CeH), 125.7 (Ar_CeH), 113.5 (C-13/15), 61.45 (C-10), 61.2 (C-8), 55.1

(NMe₂), 46.1 (C-1), 42.0 (C-9); HRMS (ES): Found 284.2137

(M þ H)^þ: C₁₈H₂₆N₃(M þ H)^þrequires 284.2121.

1740

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

the crude product puriﬁed by column chromatography using

mixtures of ethyl acetate:hexane (50:50) to (95:5) as eluent to give

11f(m) as a thick-yellow oil and predominantly as an s-trans

rotamer, (0.16 g, 95%): IR (DMSO) n_max3591e3275, 2587, 2321, 2137,

(NeCH₃); HRMS (ES): Found 303.1258 (M þ H)^þ: C₁₇H₂₀ClN₂O

(M þ H)^þrequires 303.1259.

6.2.36. s-trans-N-[{4-(N-p-Chlorobenzyl-N-methylaminomethyl)

phenyl}methyl] formamide (12f(p))

2070, 1988, 1904, 1655 (Ar_C]C); ¹H NMR (CDCl₃, 300 MHz)

d 8.25

(1H, s, H-1), 7.38e7.16 (9H, m, ArH), 6.02 (1H, br s, NH), 4.48 (2H, d,

J ¼ 5.9 Hz, H-2), 3.57 (2H, s, H-9), 3.54 (2H, s, H-11), 2.20 (3H, s,

A stirred solution of 12(p) (0.10 g, 0.36 mmol) in excess ethyl

formate (2.88 mL, 36.5 mmol) under N₂was reﬂuxed overnight. The

excess ethyl formate was removed under reduced pressure and the

crude product puriﬁed by column chromatography using mixtures

of ethyl acetate:hexane (50:50) to (100%) as eluent to give 12f(p) as

a thick-yellow oil and predominantly as an s-trans rotamer,

(0.094 g, 86%): IR n_max(cm^ꢁ1) 3630e3180, 2592, 2143, 2053,

NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 161.0 (C-1), 139.3 (Ar_qu), 138.3

(Ar_qu), 137.7 (Ar_qu), 129.1 (C-13/17), 128.7 (Ar_CeH), 128.4 (Ar_CeH),

128.4 (Ar_CeH), 128.3 (C-14/16), 127.2 (Ar_CeH), 126.6 (Ar_CeH), 61.8 (C-

9), 61.5 (C-11), 42.1 (C-2), 42.0 (NeCH₃); HRMS (ES): Found

268.1576 (M^þ): C₁₇H₂₀N₂O (M^þ) requires 268.1576.

w1972, 1909, w1659 (Ar_C]C); ¹H NMR (CDCl₃, 300 MHz)

d 8.21 (1H,

6.2.33. s-trans-N-[{4-(N-Benzyl-N-methylaminomethyl)phenyl}

methyl]formamide (11f(p))

s, H-1), 7.31e7.14 (8H, m, ArH), 6.15 (1H, br s, NH), 4.40 (2H,

d, J ¼ 4.5 Hz, H-2), 3.45 (2H, s, H-9), 3.42 (2H, s, H-11), 2.11 (3H, s,

A stirred solution of 11(p) (0.15 g, 0.63 mmol) in excess ethyl

formate(2.50 mL, 31.50 mmol) under N₂was reﬂuxed overnight. The

excess ethyl formate was removed under reduced pressure and the

crude product puriﬁed by column chromatography using mixtures

of ethyl acetate:hexane (50:50) to (95:5) as eluent to give 11f(p) as

a thick-yellow oil and predominantly as an s-trans rotamer, (0.15 g,

89%): IR (DMSO) n_max3532e3413, 3300, 2582, 2363, 2325, 2262,

NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 161.0 (C-1), 138.6 (Ar_qu), 137.7

(Ar_qu), 136.3 (Ar_qu), 132.5 (Ar_qu), 130.0 (C-14/16), 129.3 (C-13/17),

128.3 (C-5/7), 127.6 (C-4/8), 61.3 (C-9), 61.0 (C-11), 42.1 (NeCH₃),

41.8 (C-2); HRMS (ES): Found 303.1257 (M þ H)^þ: C₁₇H₂₀ClN₂O

(M þ H)^þrequires 303.1259.

6.2.37. s-trans-N-[{2-(N-4-Methoxybenzyl-N-methylaminomethyl)

phenyl}methyl] formamide (13f(o))

2215, 2101,1994,1903,¹H NMR (CDCl₃, 400 MHz)

d 8.15 (1H, m, H-1),

7.34e7.20 (9H, s, ArH), 6.36 (1H, br s, NH), 4.41 (2H, d, J ¼ 5.9 Hz, H-2),

A stirred solution of 13(o) (0.10 g, 0.37 mmol) in excess ethyl

formate (2.92 mL, 37.03 mmol) under N₂was heated to reﬂux

overnight. The excess ethyl formate was removed under reduced

pressure and the crude product puriﬁed bycolumn chromatography

using mixtures of hexane:ethyl acetate:methanol (50:50:0) to

(0:97:3) as eluent to give 13f(o) as a thick-yellow oil and predomi-

3.50 (2H, s, H-9), 3.49 (2H, s, H-11), 2.17 (3H, s, NeCH₃); ¹³C NMR

(CDCl₃, 75 MHz) d 161.2 (C-1), 139.2 (Ar_qu), 138.8 (Ar_qu), 136.4 (Ar_qu),

129.3 (C-14/16), 128.9 (C-13/17), 128.3 (C-5/7), 127.7 (C-4/8), 127.0

(C-15), 61.8 (C-11), 61.5 (C-9), 42.2 (C-2), 41.9 (NeCH₃); HRMS (ES):

Found 268.1574 (M^þ): C₁₇H₂₀N₂O (M^þ) requires 268.1576.

nantly as an s-trans rotamer, (0.087 g, 79%): IR (DMSO) n_max(cm^ꢁ1

3593e3257, 2593, 2146, 2063, 1978, 1905, 1658 (Ar_C]C); ¹H NMR

(CDCl₃, 400 MHz)

)

6.2.34. s-trans-N-[{2-(N-p-Chlorobenzyl-N-methylaminomethyl)

phenyl}methyl]formamide (12f(o))

d

8.50 (1H, br s, NH), 8.15 (1H, br t, J ¼ 1.2, 2.0 Hz, H-

A stirred solution of 12(o) (0.10 g, 0.36 mmol) in excess ethyl

formate (1.44 mL, 18.25 mmol) under N₂was heated to reﬂux

overnight. The excess ethyl formate was removed under reduced

pressure and the crude product puriﬁed by column chromatog-

raphy using mixtures of ethyl acetate:hexane (50:50) to (100%)

as eluent to give 12f(o) as a thick-cream paste and predominantly as

1), 7.39e7.12 (6H, m, ArH), 7.14 (2H, dd, J ¼ 2.2, 6.6 Hz, H-14/16), 6.85

(2H, dd, J ¼ 2.2, 6.6 Hz, H-13/17), 4.43 (2H, d, J ¼ 6.0 Hz, H-2), 3.80

(3H, s, OMe), 3.55 (2H, s, H-9), 3.49 (2H, s, H-11), 2.09 (3H, s, NeCH₃);

¹³C NMR (CDCl₃, 75 MHz)

d 160.6 (C-1), 159.3 (Ar_qu), 138.1 (Ar_qu),

137.1 (Ar_qu),131.9 (Ar_CeH),131.0 (Ar_qu),130.8(C-14/16),130.0 (Ar_CeH),

128.8 (Ar_CeH), 127.9 (Ar_CeH), 114.1 (C-13/17), 61.8 (C-11), 61.4 (C-9),

55.5 (OMe), 41.3 (C-2), 41.0 (NeCH₃). HRMS (ES): Found 299.1754

(M þ H)^þ: C₁₈H₂₃N₂O₂(M þ H)^þrequires 299.1754(M þ H)^þ.

an s-trans rotamer, (0.098 g, 89%): IR (DMSO) n_max(cm^ꢁ1

3627e3228, 2597, 2146, 2060, 1976, 1903, 1661 (Ar_C]C); ¹H NMR

(CDCl₃, 300 MHz)

)

d

8.09 (1H, br t, J ¼ 0.9, 1.8 Hz, H-1), 8.05 (1H, br s,

NH), 7.39e7.14 (9H, m, ArH), 4.65 (2H, d, J ¼ 5.4 Hz, H-2), 3.57 (2H, s,

6.2.38. s-trans-N-[{3-(N-4-Methoxybenzyl-N-methylaminomethyl)

phenyl}methyl] formamide (13f(m))

H-9), 3.51 (2H, s, H-11), 2.10 (3H, s, H-10); ¹³C NMR (CDCl₃, 75 MHz)

d

160.3 (C-1), 137.7 (Ar_qu), 136.5 (Ar_qu), 136.4 (Ar_qu), 133.3 (Ar_qu),

A stirred solution of 13(m) (0.10 g, 0.37 mmol) in excess ethyl

formate (2.92 mL, 37.03 mmol) under N₂was heated and allowed

to reﬂux overnight. The excess ethyl formate was removed under

reduced pressure and the crude product puriﬁed by column

chromatography using mixtures of hexane:ethyl acetate:methanol

(50:50:0) to (0:97:3) as eluent to give 13f(m) as a thick-yellow oil

and predominantly as an s-trans rotamer, (0.084 g, 76%): IR

(DMSO) n_max(cm^ꢁ1) 3599e3278, 2592, 2140, 2066, 1982, 1904,

131.6 (Ar_CeH), 130.6 (C-13/17), 130.4 (Ar_CeH), 128.6 (C-14/16), 128.6

(Ar_CeH), 128.5 (Ar_CeH), 127.7 (Ar_CeH), 61.4 (C-11), 61.1 (C-9), 41.2

(C-10), 40.5 (C-2); HRMS (ES): Found 303.1260 (M

þ

H)^þ:

C₁₇H₂₀ClN₂O (M þ H)^þrequires 303.1259.

6.2.35. s-trans-N-[{3-(N-p-Chlorobenzyl-N-methylaminomethyl)

phenyl}methyl] formamide (12f(m))

A stirred solution of 12(m) (0.10 g, 0.36 mmol) in excess ethyl

formate (2.88 mL, 36.5 mmol) under N₂was heated and allowed to

reﬂux overnight. The excess ethyl formate was removed under

reduced pressure and the crude product puriﬁed by column chro-

matography using mixtures of ethyl acetate:hexane (50:50) to

(100%) as eluent to give 12f(m) as a thick-yellow oil and predomi-

1660 (Ar_C]C); ¹H NMR (CDCl₃, 400 MHz)

d 8.28 (1H, s, H-1),

7.33e7.17 (6H, m, ArH), 6.87 (2H, dd, J ¼ 2.2, 6.6 Hz, H-13/17), 5.85

(1H, br s, NH), 4.50 (2H, d, J ¼ 6.4 Hz, H-2), 3.81 (3H, s, OMe), 2.19

(2H, s, H-9), 2.18 (2H, s, H-11), 2.18 (3H, s, NeCH₃); ¹³C NMR

(CDCl₃, 75 MHz)

d 160.9 (C-1), 158.7 (Ar_qu), 140.1 (Ar_qu), 137.5

(Ar_qu), 131.1 (Ar_qu), 130.1 (C-14/16), 128.7 (Ar_CeH), 128.3 (Ar_CeH),

128.3 (Ar_CeH), 126.4 (Ar_CeH), 113.6 (C-13/17), 61.4 (C-11), 61.3 (C-9),

55.2 (OMe), 42.1 (C-2), 42.1 (NeCH₃); HRMS (ES): Found 299.1746

(M þ H)^þ: C₁₈H₂₃N₂O₂(M þ H)^þrequires 299.1754.

nantly as an s-trans rotamer, (0.093 g, 84%): IR (DMSO) n_max(cm^ꢁ1

3628e3257, 2592, 2147, 2065, 1982, 1906, w1658 (Ar_C]C); ¹H NMR

(CDCl₃, 400 MHz) 8.25 (1H, s, H-1), 7.26e723 (7H, m, ArH), 7.15

)

d

(1H, m, H-4), 6.0 (1H, br s, NH), 4.42 (2H, d, J ¼ Hz, H-2), 3.48 (2H, s,

H-9), 3.43 (2H, s, H-11), 2.13 (3H, s, NeCH₃); ¹³C NMR (CDCl₃,

6.2.39. s-trans-N-[{4-(N-4-Methoxybenzyl-N-methylaminomethyl)

phenyl}methyl] formamide (13f(p))

75 MHz)

d 161.0 (C-1), 140.0 (Ar_qu), 137.7 (Ar_qu), 137.6 (Ar_qu), 132.6

(Ar_qu), 130.1 (C-13/17), 128.7 (Ar_CeH), 128.3 (C-14/16), 128.1 (Ar_CeH),

128.1 (Ar_CeH), 126.4 (Ar_CeH), 61.6 (C-9), 61.1 (C-11), 42.2 (C-2), 42.1

A stirred solution of 13(p) (0.10 g, 0.37 mmol) in excess ethyl

formate (2.92 mL, 37.03 mmol) under N₂was reﬂuxed overnight.

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

1741

The excess ethyl formate was removed under reduced pressure and

the crude product puriﬁed by column chromatography using

mixtures of hexane:ethyl acetate:methanol (50:50:0) to (0:97:3) as

eluent to give 13f(p) as a thick-yellow oil and predominantly as an

s-trans rotamer, (0.087 g, 79%): IR (DMSO) n_max(cm^ꢁ1) 3582e3295,

2595, 2137, 2067, 1983, 1906, 1660 (Ar_C]C); ¹H NMR (CDCl₃,

(CDCl₃, 300 MHz) d 8.16 (1H, s, H-1), 7.32e7.20 (4H, m, ArH), 7.19

(2H, d, J ¼ 8.8 Hz, H-13/17), 6.69 (2H, d, J ¼ 8.8 Hz, H-14/16), 5.41

(1H, br s, NH), 4.41 (2H, d, J ¼ 5.7 Hz, H-2), 3.51 (2H, s, H-9), 3.47

(2H, s, H-11), 2.90 (6H, s, NMe₂), 2.14 (3H, s, NeCH₃); ¹³C NMR

(CDCl₃, 75 MHz) d 161.1 (C-1), 149.9 (Ar_qu), 138.0 (Ar_qu), 136.4 (Ar_qu),

130.0 (C-5/7), 129.5 (C-13/17), 127.6 (C-4), 127.4 (C-8), 125.6 (Ar_qu),

112.4 (C-14/16), 60.9 (C-9 or 11), 60.6 (C-11 or 9), 41.8 (C-2), 41.5

(C-10), 40.6 (NMe₂); HRMS (ES): Found 312.2089 (M þ H)^þ:

C₁₉H₂₆N₃O (M þ H)^þrequires 312.2070.

300 MHz)

d

8.21 (1H, br t, J ¼ 0.9, 1.5 Hz, H-1), 7.35e7.17 (6H, m,

ArH), 6.85 (2H, dd, J ¼ 2.0, 6.8 Hz, H-13/18), 6.06 (1H, br s, NH), 4.44

(2H, d, J ¼ 5.7 Hz, H-2), 3.77 (3H, s, OMe), 3.48 (2H, s, H-9), 3.45 (2H,

s, H-11), 2.15 (3H, s, NeCH₃); ¹³C NMR (CDCl₃, 75 MHz)

d 161.0 (C-1),

158.6 (Ar_qu), 138.8 (Ar_qu), 136.2 (Ar_qu), 131.0 (Ar_qu), 130.0 (C-14/16),

129.3 (C-5/7), 127.7 (C-4/8), 113.6 (C-13/17), 61.1 (C-9), 61.1 (C-11),

55.2 (OMe), 42.0 (C-2/10), 41.9 (C-10/2); HRMS (ES): Found

299.1756 (M þ H)^þ: C₁₈H₂₃N₂O₂(M þ H)^þrequires 299.1754.

6.3. Biological evaluation

6.3.1. Culture of P. falciparum and antiplasmodial activity assays

The ability of the compounds to inhibit parasite malaria prolif-

eration, and to reverse CQ resistance, wase tested in triplicate using

the CQ-resistant W2 strain of P. falciparum. Continuous in vitro

cultures of asexual erythrocyte stages of P. falciparum were main-

tained using a modiﬁcation of the method described by Trager and

Jensen [35]. Quantitative assessment of the ability of the

compounds to inhibit parasite proliferation in vitro was determined

via the parasite lactate dehydrogenase assay using a modiﬁcation of

the method described by Makler [36].

Stock solutions of the compounds were prepared at various

concentrations in 100% methanol. The solutions were stored at

ꢁ20 ^ꢀC, either in undiluted form, or diluted (using sonication) to

2 mg/mL in 10% methanol. Further dilutions of stock solutions in

‘complete’ culture medium were prepared on the day of the exper-

iment. CQ diphosphate was used as a reference drug in all experi-

ments. A full dose-response was performed for all compounds to

determine the concentration inhibiting 50% of parasite growth

6.2.40. s-trans-N-[{2-(N-4-Dimethylaminobenzyl-N-

methylaminomethyl)phenyl}methyl] formamide (14f(o))

A stirred solution of 14(o) (0.10 g, 0.35 mmol) in excess ethyl

formate (3.00 mL, 38.1 mmol) under N₂was heated to reﬂux

overnight. The excess ethyl formate was removed under reduced

pressure and the crude product puriﬁed by column chromatog-

raphy using mixtures of hexane:ethyl acetate:methanol (50:50:0)

to (0:93:7) as eluent to give 14f(o) as a thick-yellow oil and

predominantly as an s-trans rotamer, (0.090 g, 82%): IR (DMSO) n_max

(cm^ꢁ1) 3606e3245, 2587, 2147, 2067, 1981, 1903, 1659; ¹H NMR

(CDCl₃, 400 MHz)

d 8.71 (1H, br s, NH), 8.08 (1H, s, H-1), 7.39e7.18

(4H, m, ArH), 7.09 (2H, d, J ¼ 8.2 Hz, H-14/16), 6.68 (2H, d, J ¼ 8.2 Hz,

H-13/17), 4.42 (2H, d, J ¼ 5.2 Hz, H-2), 3.55 (2H, s, H-9), 3.46 (2H, s,

H-11), 2.93 (6H, s, NMe₂), 2.08 (3H, s, H-10); ¹³C NMR (CDCl₃,

75 MHz)

d 160.6 (C-1), 160.6 (Ar_qu), 138.2 (Ar_qu), 137.0 (Ar_qu), 132.0

(Ar_CeH), 130.9 (Ar_CeH), 130.6 (C-13/17), 129.4 (Ar_qu), 128.7 (Ar_CeH),

127.8 (Ar_CeH), 112.6 (C-14/16), 62.0 (C-11), 61.4 (C-9), 41.2 (C-2), 41.0

(C-10), 40.8 (NMe₂); HRMS (ES): Found 312.2070 (M þ H)^þ:

C₁₉H₂₆N₃O (M þ H)^þrequires 312.2070.

(IC₅₀). Compounds were tested at a starting concentration of 100

mL and then serially diluted 2-fold in complete medium to give 10

different concentrations, with the lowest being 0.2 g/mL. CQ was

tested at a starting concentration of 100 ng/mL. The IC₅₀values were

obtained using a non-linear dose-response curve ﬁtting analysis via

Graph Pad Prism v.4.0 software [37].

mg/

m

6.2.41. s-trans-N-[{3-(N-4-Dimethylaminobenzyl-N-

methylaminomethyl)phenyl}methyl] formamide (14f(m))

CQ resistance-reversal experiments were carried out using the

dose-response method described above, with CQ sensitivity assays

carried out (by growing parasites in the presence of a range of 10

different CQ concentrations) in the presence and absence of a single

concentration of each compound of interest. For each compound

the resistance modiﬁcation index (RMI) was calculated from the

ratio of the CQ IC₅₀measured in the presence of the compound of

interest to the CQ IC₅₀measured in its absence.

A stirred solution of 14(m) (0.10 g, 0.35 mmol) in excess ethyl

formate (3.00 mL, 38.1 mmol) under N₂was heated to reﬂux

overnight. The excess ethyl formate was removed under reduced

pressure and the crude product puriﬁed by column chromatog-

raphy using mixtures of hexane:ethyl acetate:methanol (50:50:0)

to (0:93:7) as eluent to give 14f(m) as a thick-yellow oil and

predominantly as an s-trans rotamer, (0.097 g, 89%): IR (DMSO) n_max

(cm^ꢁ1) 3599e3278, 2592, 2140, 2066, 1982, 1904, 1660; ¹H NMR

(CDCl₃, 400 MHz)

d

8.21 (1H, s, H-1), 7.30e7.27 (3H, m, ArH), 7.21

6.3.2. Radioisotope inﬂux measurements in X. laevis oocytes

expressing PfCRT

(2H, d, J ¼ 8.8 Hz, H-13/17), 7.15 (1H, m, H-4), 6.72 (2H, d, J ¼ 8.8 Hz,

H-14/16), 6.22 (1H, br s, NH), 4.45 (2H, d, J ¼ 6.0 Hz, H-2), 3.49 (2H,

Expression of mutant and wildtype forms of PfCRT (from the

strains Dd2 and D10, respectively) at the plasma membrane of X.

laevis oocytes was performed as described elsewhere [23]. Brieﬂy,

oocytes were injected with cRNA encoding PfCRT (30 ng per oocyte)

s, H-9), 3.46 (2H, s, H-11), 2.94 (6H, s, NMe₂), 2.17 (3H, s, H-10); ¹³

C

NMR (CDCl₃, 75 MHz)

d 161.1 (C-1), 149.9 (Ar_qu), 140.1 (Ar_qu), 137.5

(Ar_qu), 130.0 (C-13/17), 129.9 (Ar_qu), 128.6 (Ar_CeH), 128.4 (Ar_CeH),

128.4 (Ar_CeH), 126.4 (Ar_CeH), 113.6 (C-14/16), 61.4 (C-9), 61.3 (C-11),

42.0 (C-2), 42.0 (C-10), 40.7 (NMe₂); HRMS (ES): Found 312.2063

(M þ H)^þ: C₁₉H₂₆N₃O (M þ H)^þrequires 312.2070.

and the uptake of [³H]CQ (0.15

mM; 15 Ci/mmol) was measured 4e6

days post-injection using a method described previously [38]. The

inﬂux measurements were made over 1e2 h at 27.5 ^ꢀC and in

medium that contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2,

6.2.42. s-trans-N-[{4-(N-4-Dimethylaminobenzyl-N-

1.8 mM CaCl2, 10 mM MES, 10 mM Tris-base (pH 6.0) and 15 mM

methylaminomethyl)phenyl}methyl] formamide (14f(p))

unlabelled CQ. Statistical comparisons were made with the

A stirred solution of 14(p) (0.10 g, 0.35 mmol) in excess ethyl

formate (3.00 mL, 38.1 mmol) under N₂was heated to reﬂux

overnight. The excess ethyl formate was removed under reduced

pressure and the crude product puriﬁed by column chromatog-

raphy using mixtures of hexane:ethyl acetate:methanol (50:50:0)

to (0:93:7) as eluent to give 14f(p) as a thick-yellow oil and

predominantly as an s-trans rotamer, (0.085 g, 78%): IR (DMSO) n_max

(cm^ꢁ1) 3615e3235, 2595, 2142, 2061, 1973, 1901, 1657; ¹H NMR

Student’s t-test for unpaired samples.

Acknowledgements

This work was primarily supported by the South African Malaria

Initiative (SAMI). We also acknowledge the National Research

Foundation (Grant No. 2061833), the Australian National Health

and Medical Research Council (NHMRC) (grant 471472), the

1742

V.K. Zishiri et al. / European Journal of Medicinal Chemistry 46 (2011) 1729e1742

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Article Doi

A series of structurally simple chloroquine chemosensitizing dibemethin derivatives that inhibit chloroquine transport by PfCRT

DOI: 10.1016/j.ejmech.2011.02.026

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