Synthesis and pharmacological testing of polyaminoquinolines as blockers of the apamin-sensitive Ca2+-activated K+ channel (SKCa)

Bioorganic & Medicinal Chemistry 15 (2007) 5457–5479

Synthesis and pharmacological testing of polyaminoquinolines

as blockers of the apamin-sensitive Ca²⁺-activated

K⁺channel (SK_Ca)

David I. Fletcher,^a,ꢀC. Robin Ganellin,^a,*Alessandro Piergentili,^a,ꢁ

Philip M. Dunn^b,§and Donald H. Jenkinson^b

^aDepartment of Chemistry, University College London, 20, Gordon Street, London WC1H 0AJ, UK

^bDepartment of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK

Received 2 August 2006; revised 19 May 2007; accepted 23 May 2007

Available online 26 May 2007

Abstract—The synthesis and pharmacological testing of a series of non-peptidic blockers of the SK_Ca(SK-3) channel is described.

Target compounds were designed to mimic the spatial relationships of selected key residues in the energy-minimised structure of the

octadecapeptide apamin, which are a highly potent blocker of this channel. Structures consist of a central unit, either a fumaric acid

or an aromatic ring, to which are attached two alkylguanidine or two to four alkylaminoquinoline substituents. Potency was tested

by the ability to inhibit the SK_Cachannel-mediated after-hyperpolarization (AHP) in cultured rat sympathetic neurones. It was

found that bis-aminoquinoline derivatives are signiﬁcantly more potent as channel blockers than are the corresponding guanidines.

This adds to the earlier evidence that delocalisation of positive charge through the more extensive aminoquinolinium ring system is

important for eﬀective channel binding. It was also found that an increase in activity can be gained by the addition of a third ami-

noquinoline residue to give non-quaternized amines which have submicromolar potencies (IC₅₀= 0.13–0.36 lM). Extension to four

aminoquinoline residues increased the potency to IC₅₀= 93 nM.

1. Introduction

peutic applications.⁶The latter include eﬀects on periph-

eral tissues (e.g., the insulin releasing cells of the

pancreas⁷) as well as on the CNS where there is evidence

for a role of SK_Cachannels in memory processes, both

general⁸and speciﬁcally hippocampal.⁹

Small conductance Ca²⁺activated K⁺(SK_Ca) channels

are found in a wide variety of cells, both electrically

excitable and inexcitable. These include, for example,

many neurones, intestinal myocytes, endothelial cells

and hepatocytes^1–4. Accordingly, there is much current

interest in the synthesis of blockers of these channels

for use as pharmacological tools⁵and in possible thera-

The work described herein extends an ongoing study of

the pharmacology and medicinal chemistry of SK_Ca

blockers which was begun before the composition and

sequence of these channels had been determined. The

principal measure of blocking potency utilised through-

out has been the ability of the test agents to inhibit the

SK_Ca-mediated after-hyperpolarization (AHP) that fol-

lows the action potential in rat sympathetic ganglion

cells in short term tissue culture. Subsequent to the clon-

ing and sequencing of SK_Cachannels,¹⁰of which three

variants (SK1-3) were identiﬁed, it was established that

this AHP is mediated by the SK3 subtype.¹¹This occurs

in the nerve endings¹²and cell bodies of many CNS neu-

rones as well as in peripheral tissues.⁴

Keywords: Quinoline; Aminoquinoline; Guanidine; Apamin; Potas-

sium ion channel; Calcium activated; SK_Ca; SK3; AHP; After-

hyperpolarization.

*

Corresponding author at present address: University College Lon-

don, Department of Chemistry, Christopher Ingold Laboratories, 20,

Gordon Street, London WC1H 0AJ, UK. Tel.: +44 20 7679 4624;

fax: +44 20 7679 7463; e-mail: c.r.ganellin@ucl.ac.uk

Present address: AstraZeneca, Bakewell Road, Loughborough, Leics

LE11 5RH, UK.

ꢀ

ꢁ

§

Present address: Universita degli Studi di Camerino, Dpto de Scienze

Chimiche, Via S. Agostino,1, 62032 Camerino, Italy.

Present address: Autonomic Neuroscience Institute, The Royal Free

and University College Medical School, Rowland Hill Street, London

NW3 2PF, UK.

The ﬁrst naturally occurring, highly potent, selective

blocker of the SK_Cachannel discovered was apamin^13,14

(Fig. 1). Other peptidic toxins such as scyllatoxin

doi:10.1016/j.bmc.2007.05.054

5458

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

S

Cys-Asn-Cys-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Cys-Ala-Arg-Arg-Cys-Gln-Gln-HisNH₂

S

apamin

+

Et₃N(CH₂)₂O

+

O(CH₂)₂NEt₃

(CH₂)₁₀N⁺

NH₂

H₂N

N⁺

O(CH₂)₂N⁺Et₃

Me

gallamine

dequalinium

HN

NH

N⁺

(CH₂)₅

HN

NH

N⁺

(CH₂)₅

UCL 1684

UCL 1848

MeO

C₂H₅

N

C₂H₅

N

Me

MeO

Cl

N

MeO

N

OMe

1

2

3

Figure 1. Structures of some blockers (apamin, gallamine, dequalinium, UCL 1684, UCL 1848 and 1–3) of SK_Cachannels (SK3).

(leiurotoxin 1)¹⁵and PO₅also have similar aﬃnity.

Several non-peptidic compounds have been shown to

be blockers of SK_Cachannels, such as tubocurarine,^17,18

pancuronium, gallamine (Fig. 1)¹⁹and dequalinium²⁰

(Fig. 1), albeit with aﬃnity several orders of magnitude

lower than the natural toxins. The latter provided a lead

for development of several series of novel selective SK_Ca

blockers.^21–24The most active of these has been the bis-

quinolinium cyclophanes, some of which are equipotent

with apamin,^23,24, for example, UCL 1684 and UCL

1848 (Fig. 1).

as a starting point methylated derivatives of the monot-

ertiary alkaloids bicuculline and laudanosine.²⁶In this

paper, the authors note that one of the compounds (3,

Fig. 1) synthesized is a tertiary amine which may there-

fore be useful as a lead for the development of com-

pounds able to cross the blood–brain barrier. The

potency is, however, rather low, having a K_iof 5.8 lM.

16

We have also previously described^21,22bis non-quatern-

ized amines of similar potencies. These are based on

1,10-diaminodecane with bis substitution by nitrogen

heterocycles such as pyridine, quinoline and acridine

(IC₅₀values = 2.5–6.7 lM). The present paper identiﬁes

tris non-quaternized amines (12–14) which are more

potent by at least an order of magnitude (IC₅₀val-

ues = 0.13–0.36 lM) and where the basic centres are

the quinoline nitrogen atoms.

Bridged bis-aminoquinolines and bridged bis-diamino-

quinazolines (e.g., 1 and 2, Fig. 1) have also been descri-

bed^25a,bin a series of patents from scientists at the Bayer

company. Compounds 1 and 2 are tertiary diamines

where the basic centres are the heterocyclic nitrogen

atoms. These compounds and their analogues have been

claimed to be useful for the treatment of degenerative

diseases of the central nervous system. Another recent

approach to the development of SK_Cablockers takes

Structure–activity studies carried out on apamin,^28,29

16,32

leiurotoxin^30,31and PO₅

the importance for pharmacological activity of two

analogues have indicated

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5459

CONH(CH₂)₃NH

HN

arginines (Arg-13 and Arg-14 in apamin), located on the

same side of the a-helix section of the molecule, whose

cationic guanidine groups are separated by approxi-

NH

(CH ) HNOC

H₂N

NH₂

HN

2

3

mately 11 A.¹⁹However, the high aﬃnity of these toxins

˚

4

for the binding sites does not appear to be explained

exclusively by the presence of the two arginine

groups.^29,33Closely located Gln and His residues may

also contribute. For example, the binding aﬃnity is

reduced to 1.3% of apamin on removal of His-18, and

reduced to 0.01% without both His-18 and Gln-17.

Removal of Gln-16, in addition, resulted in no further

change in aﬃnity.²⁹

(CH₂)₈NH₂

CON

NH

(CH ) HNOC

(CH₂)₃NH

HN

2

3

NH₂

HN

5

The evidence to date indicates that the nature of the

binding is electrostatic, with a region of negative charge

in the channel mouth.^2,31,32Arg-13, Arg-14 and His-18

are all protonated at physiological pH. The Gln-17 res-

idue is located on the helix one turn below Arg-14, and

one may envisage that it could participate in binding

through hydrogen bonding with the electronegative

area. A common feature of the majority of the non-pep-

tidic compounds prepared to date as apamin analogues

is the possession of only two basic groups. In the selec-

tion of the present series of compounds, the nature of

this interaction was explored by incorporating further

positively charged residues to mimic the interaction of

Gln-17 and His-18.

NH

(CH₂)₈NH₂

HN CH₂)₃HNOC

CO

N

(CH₂)₃NH

HN

NH₂

6

CH₂NH₂

CON

NH

H₂N

(CH₂)₃NH

(CH ) HNOC

HN

2

3

NH₂

HN

The spatial conformation of apamin has been well stud-

ied, and the structure optimised by computer molecular

modelling.³⁴One of our research group had built a CPK

model according to this structure and target molecules

were selected by overlaying on this and also by molecu-

lar modelling studies using ChemX.⁴⁵We began with

elaborations of a bis-guanidiniumalkyl diamide of fuma-

ric acid 4 (Fig. 2), which had been originally synthesized

to mimic the Arg-13-Arg-14 groups of apamin and had

been found to exhibit only weak activity (30% inhibition

at 300 M).³³By inspection of the CPK model of apamin it

could be seen that an appropriate spatial relationship of

Gln-17 NH₂to the two arginine residues could be

achieved by incorporation of an octylamino group

(assuming it to be in a fully extended trans conformation)

on one of the amide nitrogens of 4 by rotation of the alkyl

chain, leading to the selection of 5. The cis isomer, based

upon the diamide of maleic acid, was also prepared.

7

HN

NH

HN

NH

H₂N

NH₂

8

Figure 2. Structures of bisguanidines 4–8.

structure–activity studies, suggested²¹that there is a

need for a ring-shaped positive electrostatic ﬁeld around

the charged part of the molecule for blockade of the

SK_Cachannel. It was of interest, therefore, to compare

compounds with guanidine moieties with the corre-

sponding aminoquinolines. Two examples were ex-

plored. In the aminooctyl bis-guanidiniumalkyl

fumaric acid diamide 5 the two guanidine groups were

replaced by aminoquinoline residues to give 9 (Fig. 3)

as a synthetic accessible target; in the bis-aminoquinoli-

no-cis-stilbene 10 (Fig. 3), that we had previously re-

ported on,³⁵the aminoquinoline groups were replaced

by guanidines to give 8 (Fig. 2).

It should be noted that the exact position of the basic

substituents may vary widely in solution due to E/Z con-

formational isomerisation about the amide bond and

ﬂexibility of the alkyl linkages. In order to explore this

further a third target compound, 7, was prepared having

the n-octyl chain of 5 replaced by a biphenyl group, this

˚

unit being much more rigid but of similar length (15 A)

to the alkyl analogue in its fully extended conformation.

In the model compound 10, the aminoquinoline groups

are positively charged through being quaternized

whereas in 9, they are not quaternized. Although 4-ami-

noquinoline is less basic than guanidine, and non-quat-

ernized, quaternization has been shown not to be an

essential feature for channel blockade, as the heterocycle

is suﬃciently basic so that it is likely to be protonated at

physiological pH.²¹

Much of our prior structure–activity studies have in-

volved the synthesis and testing of dequalinium ana-

logues. Dequalinium which contains two quinolinium

rings is approximately 500 times more potent as an

SK_Cachannel blocker than is decamethonium in which

the positive charges are carried by two trimethylammo-

nium groups. This observation, together with our early

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D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

(CH₂)₈NH₂

CO

N

(CH₂)₃NH

NH(CH₂)₃HNOC

N⁺

NH₂

H₂N

N⁺

N

9

10

Figure 3. Structures of the bis-aminoquinoline derivatives 9 and 10 corresponding to the bisguanidines 5 and 8.

As discussed in Section 4, inclusion of the 4-aminoquino-

line group was demonstrated to provide a major

improvement in blocking potency of compounds relative

to those containing guanidine groups; it was of interest

therefore to extend the bis-quinolinium structures by

incorporating a third (or even a fourth) 4-aminoquino-

line group. Since the positive charge in the ring structure

is much more diﬀusely spread over a larger area than in

guanidine, the apamin molecule as modelled with its two

guanidine groups is no longer a reliable guide for show-

ing the precise location to be taken by the aminoquino-

line groups; the structural requirements were therefore

probed more empirically.

tion relative to R₁; each R group has a ﬁve-atom

chain to a quinoline ring. Compound 12 is the lower

homologue where the amino group is present as an ani-

line instead of a benzylamine, thereby reducing consid-

erably the basicity of this nitrogen link. Attempts to

synthesize the lower homologues of 11 and 12, with each

R group having a four-atom chain, were thwarted by the

ready elimination of the leaving group X from the re-

agent X(CH₂)₂NHÆBoc. Synthesis of an isomer of 12,

in which the oxypropylaminoquinoline chains were in

the 2,4-positions, was investigated but was unsuccessful.

In compound 13, a shorter chain to the quinoline group

for R₁was achieved by using dopamine as the starting

material. In this case R₃is in the 4-position. Here again,

R₂and R₃have ﬁve-atom chains to the quinoline rings.

Several attempts to synthesize lower homologues using a

CH₂CH₂link were unsuccessful.

A further series of target compounds was prepared in

which an aromatic central unit was used as a framework

for assembly of three or four aminoquinolinyl groups.

Molecular modelling studies have indicated that a 1,6-

disubstituted indane (Fig. 4b) is a good mimic for two

adjacent residues of an a-helix (Fig. 4a).³⁶A syntheti-

cally more accessible target is a m-substituted aromatic

(Fig. 4c). The ring is able to act as a spacer approxi-

mately equivalent to one turn on the helix, with the re-

sult that it can provide a suitable template for the

three key residues of interest in binding to the SK_Ca

channel. The adjacent Arg-13 and Arg-14 residues in

apamin are represented by m-substituents (R₁and R₂),

and Gln-17 by an appropriate substituent in the 4- or

5-position (R₃).

A homologous analogue (14) of gallamine, having the

quaternary ammonium groups replaced by aminoquino-

linyl, was prepared from pyrogallol. Since gallamine is a

tris-triethylammonium quaternary, the analogue (14)

was also quaternized with ethyl iodide, giving 15 to pro-

vide further data for investigating the need for quatern-

ization (Fig. 6). Isomers of 14, having a 1,2,4

substitution pattern or the fully symmetrical 1,3,5 pat-

tern, were planned but the syntheses posed problems

that could not be overcome.

While the separation between the aminoquinolinyl sub-

stituents representing the two arginines was maintained

As a further measure of a potential statistical contribu-

tion to the binding, two target compounds 16 and 17

having four aminoquinolinyl substituents (Fig. 7) were

prepared, by elaboration of the alkylamino linkage.

The length of the additional linkage chain was selected

to mimic His-18 in apamin based on the modelling

results.

˚

at approximately 11 A, the position of the third substi-

tuent was varied. The following test compounds

(11–13, Fig. 5) were prepared from amongst the various

potential alternatives by examining not only fulﬁlment

of the appropriate structural requirements, but also in

terms of accessibility of starting materials and ease of

synthesis.

2. Biological testing

Compound 11, as a benzylamine, accords with the struc-

ture depicted in Figure 4c with R₃being in the 5-posi-

The potencies of compounds as blockers of the SK3 var-

iant of the SK_Cachannel were determined from their

ability to inhibit the after-hyperpolarization which fol-

lows the action potential in rat sympathetic neurones,

as previously described.^23,38,43The amplitude of the

AHP was measured before, during and after the pres-

ence of the compound. Dequalinium was also applied,

serving as a reference agent in each assay. Because the

eﬀectiveness of dequalinium varied a little between

experiments, equieﬀective molar concentration ratios

(EMR with respect to dequalinium) were determined.

R1

O

R1

R2

H

R2

N

H

O

R2

R3

(a)

(b)

(c)

Figure 4. 1,6-Disubstituted indane (b) is a good mimic for two

adjacent residues of an a-helix (a). This can be represented by a

m-substituted benzene ring (c).

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5461

N

NH

HN(CH )

O(CH₂)₃

₃O

2

NH

HN(CH )

O(CH₂)₃

O

3

2

NH(CH₂)₃NH

NH(CH )

₃NH

2

N

11

12

N

HN(CH₂)₃O

O(CH₂)₃NH

NH

N

13

Figure 5. Trisubstituted benzene derivatives (11–13) having three aminoquinolinyl groups.

Et

N⁺

N

Et

N⁺

N

NH(CH₂)₃O

O(CH₂)₃NH

N⁺

Et

N

14

15

Figure 6. Gallamine analogues (14–15) in which the quaternary ammonium groups of gallamine are replaced by aminoquinolinyl or amino-N-

ethylquinolinium.

The EMR values have been used for the comparisons

between compounds. IC₅₀values, the concentration re-

quired to achieve 50% inhibition of the AHP, were also

calculated.

1). Appropriate choice of protecting groups allowed

the two t-Boc groups to be removed under acidic

conditions for subsequent guanidination (using 3,5-dim-

ethylpyrazole-1-carboxamidine nitrate) before removing

the triﬂuoroacetyl protecting group under basic condi-

tions in the ﬁnal step to leave the primary amine substi-

tuent. A similar synthetic strategy was adopted for 7

using the bis-protected biphenyl analogue 7e (Scheme

3). An approach to 6 via attempted reaction of 5g with

protected aminopropyl maleamide resulted in complete

isomerisation to the trans isomer, and so this target

3. Chemistry

Compound 5 was prepared via reaction between two key

N-protected components, 5g, derived from 1,8-diami-

nooctane, and aminopropyl fumaramide 5c (Scheme

5462

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

N

HN(CH₂)₃O

O(CH₂)₃NH

HN(CH₂)₃O

O(CH₂)₃NH

N

(CH₂)₅NH

HN(CH₂)₃

(CH₂)₅NH

NH(CH₂)₃

N

16

17

Figure 7. Compounds (16–17) having four aminoquinolinyl groups.

compound was prepared by an alternative route via

reaction of 5g with maleic anhydride (Scheme 2). Com-

pound 8 was synthesized via 4,4⁰-bis(aminomethyl)-cis-

stilbene which had been prepared from the known³⁹

dibromide through a Gabriel synthesis, by treatment

with potassium phthalimide followed by hydrazinolysis,

and then by bis-guanidination. In all syntheses, guani-

dination of the deprotected triamines required careful

pH control in order to achieve a balance between re-

moval of the triﬂuoroacetyl group at high pH and pro-

tonation at low pH, which would inhibit reaction.

oxidation. Compound 15 was prepared by alkylation

of 14 in ethyl iodide (Scheme 8), adding the minimum

quantity of DMF throughout the reaction to ensure dis-

solution of the intermediate mono- and di-quaternary

salts.

Preparation of 16 and 17 (Schemes 9, 10, respectively)

followed strategies similar to those for 11 and 12. Reac-

tion of the protected secondary amine with phloroglu-

cinol gave exclusively the monosubstituted product

17a. For 16b an eﬀective procedure was developed for

separating amines by selective extraction, utilising their

diﬀerent base strengths, in which the more basic compo-

nent is extracted into the aqueous layer of a two-phase

aqueous–organic mixture, by addition of a stoichiome-

tric amount of acid.

The general strategy for preparation of the amino-

quinolinyl target compounds involved heating the

amine precursor with 4-chloroquinoline and diisopropyl

ethylamine in n-pentanol. In the synthesis of 9 this was

carried out at early stages of the synthesis, since in sub-

sequent transformations the aminoquinolinyl moiety is

unreactive. After linking of the two key intermediates

9d and 9e, the triﬂuoroacetyl group was removed to give

9 (Scheme 4).

Table 1. Results for inhibition of the after-hyperpolarization of

cultured rat sympathetic neurones

Compound

IC₅₀(lM) SD

EMR^aSD

Apamin

Gallamine

0.0023 0.0007¹²

68 83⁴³

Target compounds (11–14) in which the alkylaminoquino linyl

groups are linked to an aromatic ring as an ether were pre-

pared by Williamson synthesis in which the phenol was

treated with potassium tert-butoxide and a t-Boc-pro-

tected bromopropylamine. Subsequent removal of the

protecting groups liberated the primary amines for

N-alkylation as the ﬁnal step (Schemes 5–8). Removal

of the t-Boc protecting group was usually carried out with

triﬂuoroacetic acid since isolation or further processing of

the product was straightforward, following evaporation

of the solvent under mild conditions, whereas using

water-based acids, the high aqueous solubility of many

of the primary amine compounds made them diﬃcult to

extract without decomposition.

Dequalinium

4

0.6 0.05⁴³

>100^b

1

5

27^c

> > 30

6

7

17

25

33

59 26

6

8

9

10

0.49 0.02

0.19 0.03³⁵

0.36 0.06

0.22 0.03

0.27 0.03

0.13 0.03

0.086 0.009

0.13 0.03

0.093 0.020

0.084 0.020³⁷

0.003 0.001⁴⁴

0.002 0.0005²⁴

0.59 0.03

0.24 0.08

0.48 0.14

0.26 0.07

0.31 0.07

0.23 0.05

0.16 0.02

0.23 0.05

0.17 0.04

0.10 0.005

0.01 0.001

0.003 0.001

11

12

13

14

15

16

17

18^d

For 12 the amine linkage to the aromatic ring

was formed by reaction of 5a with phloroglucinol,

achievable since the phenolic OH has suﬃcient ketonic

character to be susceptible to nucleophilic attack. This

produced a mixture of anilines, which was separated,

and the mono-aniline was converted into 12 using previ-

ous methodology. Attempted conversion of the di-ani-

line 12b was unsuccessful due to rapid aerial

UCL 1684

UCL 1848

^aEquieﬀective molar ratio: the ratio of the concentrations of the test

compound and dequalinium to cause 50% inhibition of the AHP.

^b18 3% inhibition at 100 lM.³³

^cCompound may have had additional actions.

^dRef. 37 wherein dequalinium had EMR 0.78 0.05.

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5463

O

NH

N(CH₂)₈OH

d

5d

O

e

CO₂Et

H₂N(CH₂)₃NH₂

a

HO.OC

H₂N(CH₂)₈OH

H₂N(CH₂)₃NHBoc

5e

b

5a

f

CO₂Et

HNOC

BocHN(CH₂)₃

F₃CCONH(CH₂)₈OH

5b

5f

c

g

CO₂H

F₃CCONH(CH₂)₈NH(CH₂)₃NHBoc

HNOC

BocHN(CH₂)₃

5g

5c

h

(CH₂)₈NHCOCF₃

(CH₂)₃NHBoc

CO

N

BocN(CH₂)₃HNOC

5h

i

(CH₂)₈NHCOCF₃

(CH₂)₃NH₂

CO

N

H₂N(CH₂)₃HNOC

5i

j

(CH₂)₈NH₂

CO

N

NH

(CH ) HNOC

H₂N

(CH₂)₃NH

NH₂

HN

2

3

HN

5

Scheme 1. Reagents: (a) (t-Boc)₂O, MeOH; (b) DCC, HOBT, THF; (c) KOH, H₂O/EtOH; HCl; (d) Br(CH₂)₈OH, K₂CO₃, DMF; (e) NH₂NH₂,

EtOH; (f) F₃CCO₂Me, MeOH; (g) TsCl, NEt₃CHCl₃; 5a, DMF; (h) DCC, HOBT, THF; (i) HCl, MeOH; (j) DMPCN, NaOH, H₂O (pH 7.0–8.5);

NaOH; HNO₃.

4. Results and discussion

tency to submicromolar activity. This is in accord with

the even larger potency diﬀerence found between the

guanidine 8 and the aminoquinoline 10 and supports

the assertion²¹that delocalisation of positive charge is

an important factor in determining potency, by improv-

ing the interaction with the negatively charged region in

the channel.

The results of the biological testing of the target com-

pounds are summarised in Table 1. Although the bis-

guanidines with an additional basic group 5 and 7

were more potent than the simple bisguanidine 4, used

as the starting point, they were much less active than

dequalinium. Direct replacement of the guanidine

groups of 5 with 4-aminoquinolinyl, giving 9, resulted

in a substantial (approximately 50-fold) increase in po-

Compound 9 was only some twofold more potent

than dequalinium (based on comparison of equieﬀective

5464

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

(CH₂)₈NHCOCF₃

(CH₂)₃NHBoc

BocNH(CH₂)₃NH(CH₂)₈NHCOCF₃

HO₂C

CO

N

a

5g

6a

b

(CH₂)₈NHCOCF₃

c

(CH₂)₈NHCOCF₃

(CH₂)₃NH₂

Boc HN(CH₂)₃HNOC

CO

N

H₂N(CH₂)₃HNOC

CO

N

(CH₂)₃NHBoc

6c

6b

d

NH

H₂N

(CH₂)₈NH₂

HN CH₂)₃HNOC

CO

N

H

(CH₂)₃N

NH₂

6

HN

Scheme 2. Reagents: (a) Maleic anhydride, MeOH; (b) NHS, DCC, CH₂Cl₂; 5a; (c) HCl, MeOH; (d) DMPCN, NaOH, H₂O (pH 7.0–8.5); NaOH,

HNO₃.

molar ratios, EMRs) and equipotent with most other

compounds prepared previously³⁵which contain two

aminoquinolinyl groups which are not rigidly held.

Evidently, the aminooctyl group in 9 has not located a

suitable additional binding site. It is noteworthy, how-

ever, that the conformation about the linking trans

double bond in 5 and 7 is important since the alternative

cis form 6 is much less active. This may be due to hydro-

gen bonding between the two cis-amide groups in the

unbound molecule, which would be lost during channel

the addition of yet another aminoquinoline group to

give compounds having four quinoline rings provided

a further twofold increase in potency. This is demon-

strated with compounds 11 and 16, or 13 and 17 where,

in each case, the additional aminoquinoline group is

linked to the rest of the molecule by a penta-methylene

chain.

The similarity in activity between 14 and 15 conﬁrms the

previous conclusion²¹that quaternization is not a prere-

quisite for activity. It is also of interest to compare 15

with gallamine where we again see a demonstration of

the importance of the aminoquinoline group. In this

case, 4-amino-1-ethyl-quinolinium moieties replace each

of the three triethylammonium groups in gallamine to

provide 15 which shows an approximately 800-fold in-

crease in potency. This is consistent with our previously

reported²¹observations on decamethonium.

1

binding. There is evidence in the H NMR spectra of

compounds 5, 6 and 7 for the existence of rotamers

about the amide bonds, which may impact on the ener-

getic favourability of binding.

Though compound 11, having three aminoquinoline

groups, was only a little more potent than 9, all the other

target compounds (12–15) with three aminoquinolinyl

groups were 2–3 times more active than 9. This may

be because of the statistical eﬀect of having three rather

than two potential binding groups present in the mole-

cule, as previously suggested.³⁷In keeping with this,

None of these polyquinolinic compounds (11–17) ap-

pears to be as potent as the tris-(4-aminoquinolinium)

18 (Fig. 8) which had³⁷approximately 8 times the po-

tency of dequalinium. There are two main changes to

the compound structure which might account for the

diﬀerences in potency. First, in 18, each quinoline ring

is on a four-carbon atom chain from the same central

carbon atom (thus there are nine-carbon atoms bridging

the quinoline rings), whereas for 11–17, the quinoline

rings are on ﬁve or six-atom chains (which include het-

eroatoms) that lead from a central benzene ring and

therefore the molecules are less compact (11, for exam-

ple, is bridged by thirteen atom chains). If we consider

all the combinations for the bridging chains between

all the quinoline rings then, for compounds 11–17, they

fall between 11- and 16-atom chains. The chain length

does not seem to be a critical diﬀerence however since,

in a series of dequalinium homologues, there was little

inﬂuence of bridging chain length (CH₂)_nfor n = 5–12

H₂N

NH₂

N⁺

NH₂

Figure 8. The tris-(4-aminoquinolinium) compound 18 described in

Ref. 37.

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5465

Br

CN

CH₂NH₂

a

b

c

CO₂Me

CO₂H

CO₂Me

CH₂OH

d

7a

7b

7c

CH₂NHCOCF₃

e

7d

7e

CH₂OH

CH₂NH(CH₂)₃NHBoc

f

CH₂NHCOCF₃

CON

(CH₂)₃NHBoc

BocNH(CH₂)₃HNOC

7f

g

CH₂NH₂

NH

CON

H₂N

(CH₂)₃NH

HN

NH(CH₂)₃HNOC

NH₂

7

Scheme 3. Reagents: (a) SOCl₂; MeOH; (b) CuCN, DMF; (c) LiBH₄, THF; (d) F₃CCO₂Me, MeOH; (e) TsCl, NEt₃, CHCl₃; 5a, DMF, (f) 5c, DCC,

HOBT, DMF; (g) HCl, MeOH; DMPCN, NaOH, H₂O (pH 7.0–8.5); NaOH; HNO₃.

between the quinoline rings, although potency was re-

duced when the chain was shorter than ﬁve carbon

atoms.⁴⁰Second, in 18, each quinoline ring is attached

to the linking chain by the ring-nitrogen atom at posi-

tion-1, whereas for compounds 11–17 the chain is at-

tached at the 4-amino groups. It has been demonstrated

that attaching the chain at the 4-amino group instead of

at the 1-position to give a non-quaternized dequalinium

isomer does indeed lower the potency by a few fold.²²

can be aromatic groups, alkane chains or a combination

of the two.^23,41,44Counting the least number of carbon

atoms between the points of attachment for the aro-

matic groups and including heteroatoms then: when

the number of bridging atoms varies between 8 and

13, the EMR activities range over one order of magni-

tude, between 0.11 and 1.1. For shorter chains, however,

potency rapidly and strikingly increases as the distances

between the quinolinium rings become optimised so that

UCL 1684 (Fig. 1) has two bridges of 8 and 5 atoms,⁴⁴

respectively, and UCL 1848 (Fig. 1) has correspondingly

7 and 5 atoms.²⁴Molecular modelling studies on these

compounds have been reported^23,27but the compounds

of the present study are too large and ﬂexible to warrant

such modelling.

It is of interest in this connection to observe that the

most active non-peptidic K_Cablockers so far described

are cyclophanes which have two quinolinium groups

joined by two bridges and are attached at both the

4-amino group and the nitrogen 1-position. The bridges

5466

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

Boc

Cl

N

NH(CH₂)₃NH

NH(CH₂)₃NH₂

a

b

N

9b

9a

c

NH(CH₂)₃NH₂

CONH(CH₂)₃NH

Et₂OC

N

9c

N

e

d

NH(CH₂)₃NH(CH₂)₈NHCOCF₃

CONH(CH₂)₃NH

H₂OC

9d

N

f

9e

N

(CH₂)₈NHCOCF₃

CO

N

NH

N

(CH₂)₃

OC

NH(CH₂)₃HN

9f

N

g

(CH₂)₈NH₂

CO

N

NH

(CH₂)₃

OC

NH(CH₂)₃HN

9

N

Scheme 4. Reagents: (a) 5a, NⁱPrEt₂, n-pentanol; (b) TFA; NaOH, H₂O; (c) monoethyl fumarate, DCC, THF; (d) NaOH, MeOH/H₂O; (e)

F₃CCOÆNH(CH₂)₈OTs, NEt₃, DMF; (f) DCC, NⁱPrEt₂, DMF; (g) NaOH, MeOH/H₂O.

5. Conclusion

The variety in central linking units across the series indi-

cates that this unit, whether it is an aromatic ring or a

rigid aliphatic structure, provides an appropriate frame-

work for the active aminoquinolinium substituents. The

degree of similarity within this series and with com-

pounds in the cyclophane series linked by 8–10 atoms

indicates a relatively long range interaction, in which

the compounds are not able to form a tight ﬁt with

the channel. This contrasts with the substantial increase

in activity that was seen in the cyclophane^23,24,27,44series

The synthesis and pharmacological testing of novel

blocking agents of the SK_Cachannel has been described.

The results add to the structure–activity knowledge and

are consistent with previous reported studies, in that

delocalisation of positive charge is an important feature

for potent binding, and the presence of more than two

positively charged substituents causes a small increase

in activity.

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5467

HO

OH

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

b

11b

OH

Br(CH₂)₃NH₂

Br(CH₂)₃NH.Boc

a

11a

c

NH(CH₂)₃NH₂

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

9b

11c

Cl

d

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

NH(CH₂)₃NH

11d

N

e

N

HN(CH₂)₃O

O(CH₂)₃NH

NH(CH₂)₃NH

11

N

Scheme 5. Reagents: (a) (t-Boc)₂O, NaOMe, MeOH; (b) KOBu, 11a, DMF; (c) TsCl, NEt₃, CHCl₃; (d) DMF; (e) TFA; NaOH, MeOH/H₂O; 4ClQ,

NⁱPrEt₂, n-pentanol.

on reducing the number of linking atoms to give much

more rigid structures.

were recorded on either a Bruker AC200 (200 MHz) or

AC400 (400 MHz) spectrometer, and chemical shifts

(ppm) are reported relative to TMS. Signals are desig-

nated as follows: s, singlet; br s, broad singlet; d, dou-

blet; t, triplet; q, quartet; qu, quintet; m, multiplet.

Infrared (IR) spectra were run on a Perkin-Elmer

16PC Fourier Transform spectrophotometer. Mass

spectra (MS) were run on a V.G. Quattro spectrome-

ter. Analytical high performance liquid chromatogra-

phy (HPLC) was carried out on a Varian Star Work

Station, using Varian 9065 Polychrome DAD detector,

6. Experimental

6.1. General

Commercially available compounds were obtained

from either Aldrich Chemical Company or Lancaster

Synthesis. Nuclear magnetic resonance (NMR) spectra

5468

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

HO

OH

HO

OH

HO

NH(CH₂)₃NHBoc

+

a

12a

12b

NH(CH₂)₃NHBoc

OH

NH(CH₂)₃NHBoc

b

N

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

HN(CH₂)₃O

O(CH₂)₃NH

c

NH(CH₂)₃NHBoc

12c

NH(CH₂)₃NH

12

N

Scheme 6. Reagents: (a) (t-Boc)₂O, NaOMe, MeOH; (b) KOBu, 11a, DMF; (c) TFA; NaOH, MeOH/H₂O; 4ClQ, NⁱPrEt₂, n-pentanol.

OH

BocHN(CH₂)₃O

OH

O(CH₂)₃NHBoc

a

b

13b

13a

NHBoc

NH₂HCl

NHBoc

c

N

HN(CH₂)₃O

O(CH₂)₃NH

13

NH

N

Scheme 7. Reagents: (a) 5a, THF; (b) KOBu, 11a, DMF; (c) TFA; NaOH, MeOH/H₂O; 4ClQ, NⁱPrEt₂, n-pentanol.

9010 Pump and Rheodyne 7125 Injector valve with

20 lL loop. Melting points (mp) were obtained on an

Electrothermal melting point apparatus and are uncor-

rected. All ﬁnal products had satisfactory (within

0.4%) C, H and N analyses unless otherwise indicated

(Supplementary Table 2). Elemental analyses were per-

formed by Wyeth Research (UK) analytical laborato-

ries, Taplow, Berkshire, UK. Molecular modelling

was carried out using ChemX.⁴⁵

6.2. Abbreviations

DMF, dimethylformamide; MeOH, methanol; EtOH, eth-

anol; EtOAc, ethyl acetate; t-Boc, tert-butyloxycarbonyl;

NEt₃, triethylamine; NaOMe, sodium methoxide; CMA,

chloroform/methanol/0.91 w/w aq ammonia (100:20:5);

Et₂O, diethyl ether; KOBu, potassium tert-butoxide;

HOBT, 1-hydroxybenzotriazole; DCC, 1,3-dicyclohexyl-

carbodiimide; TsCl, p-toluenesulfonyl chloride; 4-ClQ,

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5469

OH

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

OH

a

b

O(CH₂)₃NHBoc

14a

Et

N⁺

N

Et

N⁺

N

HN(CH₂)₃O

O(CH₂)₃NH

c

O(CH₂)₃NH

15

14

N⁺

Et

N

Scheme 8. Reagents: (a) KOBu, 11a, DMF; (b) TFA; NaOH, MeOH/H₂O; 4ClQ, NⁱPrEt₂, n-pentanol; (c) EtI, DMF.

4-chloroquinoline; NⁱPr₂Et, di-isopropylethylamine; TMS,

trimethylsilane; tlc, thin layer chromatography.

HC@CH), 8.1 (br t, 1H, NHÆCO); IR (Nujol) t_max

3230, 1720, 1660 cm^ꢀ1

.

6.3. N-(3-Guanidinopropyl)-N-(8-aminooctyl)-3-[(N⁰-(N⁰⁰-

3-guanidinopropyl)-carbamoyl)]-trans-propenamide trini-

trate trihydrate (5)

6.3.3. 3-[N-(3-(tert-Butoxycarbonylamino)-propyl)]-car-

bamoyl-trans-propenoic acid (5c). A solution of 5b

(50 g, 0.17 mol) and KOH (11 g, 0.20 mol) in EtOH

(300 mL) and water (25 mL) was stirred for 18 h at

20 ꢁC, then acidiﬁed to pH 2 with concd HCl (25 mL),

diluted with water (500 mL) and extracted with

CHCl₃(4· 250 mL). The extracts were combined and

concentrated to low volume to give an oil, which was di-

luted with EtOAc (500 mL) to precipitate the product as

6.3.1. 3-(tert-Butoxycarbonylamino)-propylamine (5a). A

solution of di tert-butyl dicarbonate (327 g, 1.5 mol)

in MeOH (1.0 L) was added to 1,3-diaminopropane

(450 mL, 7.0 mol) in MeOH (1.5 L) at 10 ꢁC. The

resulting suspension was stirred for 18 h and ﬁltered.

The ﬁltrate was concentrated under vacuum to low

volume, then diluted with water (2 L), ﬁltered to re-

move di-(t-Boc)-1,3-diaminopropane and extracted

with Et₂O (5· 500 mL). The extracts were combined

and concentrated to give the product as a yellow

oil (118 g, 55% yield). ¹H NMR (CDCl₃) d 1.2 (s,

2H, NH₂), 1.3 (s, 9H, C(CH₃)₃), 1.6 (m, 2H, CH₂),

2.7 (t, 2H, CH₂NH₂), 3.15 (q, 2H, CH₂NH), 6.0

(br t, 1H, NHÆCO).

1

a white powder (32 g, 70% yield): mp 143–145 ꢁC; H

NMR (DMSO-d₆) d 1.35 (s, 9H, C(CH₃)₃), 1.55 (m,

2H, CH₂), 2.95 (q, 2H, CH₂N), 3.15 (q, 2H, CH₂N),

6.5 (d, J = 15 Hz, 1H, HC@CH), 6.9 (d, J = 15 Hz,

1H, HC@CH), 8.4 (t, 1H, NHÆCO), 12.8 (br s, 1H,

CO₂H); MS (FB⁺) 273 (M⁺+ 1).

6.3.4. 8-(N-Phthalimido)-octan-1-ol (5d). A mixture of

phthalimide (42.8 g, 0.29 mol), 8-bromooctan-1-ol

(60.5 g, 0.29 mol) and K₂CO₃(140 g, 1.0 mol) was heated

in DMF at 60 ꢁC for 18 h, then cooled to 20 ꢁC, ﬁltered

and concentrated under vacuum. The resulting oil was

diluted with brine (300 mL) and extracted with CH₂Cl₂

(3· 250 mL). The extracts were combined and concen-

trated to low volume to give an oil, which was diluted with

Et₂O, ﬁltered, and the ﬁltrate concentrated to give the

product as a cream powder (33 g, 42% yield): mp

6.3.2. Ethyl 3-[N-(3-(tert-butoxycarbonylamino)-propyl)]-

carbamoyl-trans-propenoate (5b). A mixture of monoeth-

yl fumarate (43.2 g, 0.3 mol), HOBT (40.5 g, 0.3 mol)

and DCC (62.0 g, 0.3 mol) in THF (500 mL) was stirred

at 20 ꢁC for 18 h. A solution of 5a (52.2 g, 0.3 mol) in

THF (100 mL) was added and after stirring for 2 h the

reaction mixture was concentrated to low volume, di-

luted with CHCl₃(500 mL), acidiﬁed to pH 2 with

2 M citric acid and then ﬁltered. The organic phase

was washed with aqueous NaHCO₃(300 mL), then the

solvent removed under vacuum to give the product as

an oﬀ-white powder (62 g, 70% yield): mp 95–97 ꢁC;

¹H NMR (CDCl₃) d 1.35 (s, 12H, C(CH₃)₃+ CH₃),

1.65 (m, 2H, CH₂), 3.0 (q, 2H, CH₂N), 3.3 (q, 2H,

CH₂N), 4.15 (q, 2H, CH₂O), 6.05 (br t, 1H, NHÆCO),

6.5 (d, J = 15Hz, 1H, HC@CH), 6.9 (d, J = 15 Hz, 1H,

1

60–62 ꢁC; H NMR (CDCl₃) d 1.3–1.7 (m, 13H, 6CH₂

+OH), 3.6 (m, 4H, 2CH₂), 7.8 (br s, 4H, 4Ar–H).

6.3.5. 8-Amino-octan-1-ol (5e). A solution of 5d (33 g,

0.12 mol) and hydrazine monohydrate (23.5 g, 0.47 mol)

in EtOH (200 mL) was stirred for 18 h at 20 ꢁC. The reac-

tion mixture was diluted with 4 M HCl (400 mL), ﬁltered

and the ﬁltrate washed with CH₂Cl₂(200 mL), before

being basiﬁed to pH 14 with aq NaOH and extracted with

5470

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

H₂N(CH₂)₅NH₂

a

H₂N(CH₂)₅NHBoc

16a

b

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

(CH₂)₅NHBoc

HN

(CH₂)₃NHBoc

Cl

16b

11c

c

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

N

(CH₂)₅NHBoc

BocNH(CH₂)₃

16c

d

N

HN(CH₂)₃O

O(CH₂)₃NH

N

(CH₂)₅NH

NH(CH₂)₃

N

16

N

Scheme 9. Reagents: (a) (t-Boc)₂O, MeOH; (b) 11a, K₂CO₃, DMF; (c) K₂CO₃, DMF; (d) TFA; 4ClQ, NaOMe, NⁱPrEt₂, n-pentanol.

CH₂Cl₂(5· 150 mL). The extracts were combined and

concentrated to give an oil, which was diluted with diiso-

propyl ether to precipitate the product as a white powder

(15 g, 82% yield): mp 59–61 ꢁC; H NMR (CDCl₃) d

1.3–1.7 (m, 12H, 6CH₂), 1.8 (br s, 3H, NH₂and OH),

2.7 (t, 2H, CH₂N), 3.6 (t, 2H, CH₂O).

6.3.7. N-(Triﬂuoroacetyl)-N⁰-[3-(tert-butoxycarbonylami-

no)-propyl]-1,8-diaminooctane (5g). A solution of 5f

(10.6 g, 44 mmol) and TsCl (13.8 g, 73 mmol) in CHCl₃

(75 mL) was treated with NEt₃(8.6 g, 85 mmol) at

20 ꢁC. After 2 h the solvent was removed by distillation

and the residue diluted with EtOAc (100 mL). The pre-

cipitated NEt₃ÆHCl was ﬁltered oﬀ and the ﬁltrate con-

centrated to give the crude tosylate as an oil, which

was stirred in DMF (50 mL) with 5a (33 g, 170 mmol)

at 20 ꢁC for 18 h. The reaction mixture was then di-

luted with 2 M HCl (300 mL) and extracted with

CH₂Cl₂(2· 100 mL). The extracts were combined,

treated with excess NEt₃(15 g) and washed with water

(2· 100 mL) before concentrating under vacuum. The

resulting oil was absorbed onto silica gel and chro-

matographed with MeOH/0.91 aq NH₃(100:1) to give

1

6.3.6. 8-(N-Triﬂuoroacetamido)-octan-1-ol (5f). A solu-

tion of methyl triﬂuoroacetate (7.1 g, 50 mmol) and 5e

(7.3 g, 50 mmol) in MeOH (25 mL) was stirred for 1 h

at 15 ꢁC, before being concentrated to give an oil, which

was diluted with cyclohexane (100 mL) to precipitate the

product as a cream powder (11.2 g, 93% yield): mp 48–

1

50 ꢁC; H NMR (CDCl₃) d 1.3–1.7 (br s, 12H, 6CH₂),

2.9 (s, 1H, OH), 3.2 (q, 2H, CH₂N), 3.5 (t, 2H,

CH₂O), 7.5 (br s, 1H, NHÆCO).

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5471

HO

OH

BocHN(CH₂)₃O

O(CH₂)₃NHBoc

(CH₂)₅NHBoc

HO

OH

a

b

N

OH

BocNH(CH₂)₃

(CH₂)₅NHBoc

BocNH(CH₂)₃

17a

17b

c

N

HN(CH₂)₃O

O(CH₂)₃NH

N

HN(CH₂)₃

(CH₂)₅NH

17

N

Scheme 10. Reagents: (a) 16b, THF/toluene; (b) KOBu, 11a, DMF; (c) TFA; 4ClQ, NaOMe, NⁱPrEt₂, n-pentanol.

the product as a cream powder, precipitated from

diisopropyl ether (12 g, 68% yield): mp 55–57 ꢁC; IR

20 ꢁC, then diluted with water, cooled to ꢀ5 ꢁC, basiﬁed

to pH 9 with 40% w/w aq NaOH and extracted with

CHCl₃(6· 50 mL). The extracts were combined and

concentrated under vacuum to give 5i (1.6 g) as a yellow

oil. A solution of the oil and 3,5-dimethylpyrazole-1-car-

boxamidine nitrate (2.6 g, 13 mmol) in water (10 mL)

was heated at 60 ꢁC for 24 h, maintaining the pH at

7.0–8.5 by periodic addition of 1 M NaOH solution.

The reaction mixture was then cooled, basiﬁed to pH

14 with 40% w/w NaOH solution (1 mL) and stirred

for 2 h at 20 ꢁC before being washed with CHCl₃(2·

20 mL). 50% w/w aq nitric acid was then added to the

aqueous phase to pH 1 and the solution concentrated

under vacuum to dryness. The residue was stirred in

EtOH, ﬁltered at 70 ꢁC, then cooled to produce an or-

ange oil. Multiple precipitations from EtOH gave the

product as a yellow glass (300 mg, 23% yield); ¹H

NMR (DMSO-d₆) d 1.35 (br s, 8H, 4CH₂), 1.65 (m,

4H, 2CH₂), 1.8 (m, 4H, 2CH₂), 2.75 (m, 2H, CH₂N),

3.2 (m, 6H, 3CH₂N), 3.45 (m, 4H, 2CH₂N), 6.85 (d,

J = 15 Hz, 1H, HC@CH), 7.1 (br s, 8H, 4NH₂^þ), 7.2

(d, J = 15 Hz, 1H, HC@CH), 7.5 (m, 2H, NH), 7.7 (br

s, 3H, NH₃^þ), 8.55 (m, 1H, NHÆCO)—2 rotamers 2:1;

IR (ﬁlm) t_max3350, 3200, 1665, 1635 cm^ꢀ1; HPLC

Lichrosorb RP Select B (MeOH/0.05% aq TFA, 80:20,

1.0 mL/min), 224 nm, product at 3.5 min; MS (LC/MS,

TS⁺) 440 (M⁺+1), fragments at 398, 324, 244. Anal.

(C₂₀H₄₁N₉O₂, 3HNO₃, 3H₂O) C, H, N. Anal. Calcd

for C₂₀H₄₁N₉O₂Æ3HNO₃Æ3H₂O: C, 35.19; H, 7.38; N,

24.62. Found: C, 35.37; H, 7.31; N, 24.48.

(Nujol) t_max3260, 1700 cm^{ꢀ1 1}H NMR (CDCl₃) d

;

1.3 (br s, 8H, 4CH₂), 1.45 (s, 9H, C(CH₃)₃), 1.65 (m,

6H, 3CH₂), 2.55 (t, 2H, CH₂N), 2.75 (t, 2H, CH₂N),

3.2 (q, 2H, CH₂N), 3.4 (q, 2H, CH₂N), 5.3 (br s, 1H,

NHÆCO), 7.25 (br s, 1H, NHÆCO); MS (EI⁺) 397

(M⁺), fragment at 324.

6.3.8.

N-[3-(tert-Butoxycarbonylamino)-propyl]-N-

(8-triﬂuoroacetamidooctyl)-3-[N⁰-3-(tert-butoxycarbon-

ylamino)-propyl]-carbamoyl-trans-propenamide (5h). A

mixture of 5c (8.6 g, 32 mmol), HOBT (4.7 g, 35 mmol)

and DCC (7.2 g, 35 mmol) was stirred in THF

(150 mL) for 18 h at 20 ꢁC to give a thick suspension,

which was treated with 5g (11.0 g, 28 mmol) in THF

(50 mL). After 6 h the dicyclohexylurea was ﬁltered

oﬀ and the ﬁltrate concentrated by distillation. The

residue was diluted with CH₂Cl₂(100 mL) and 1 M

aq citric acid (75 mL). The organic phase was washed

sequentially with aq K₂CO₃(100 mL) and water

(100 mL), before removing the solvent by distillation

under vacuum to give an oily solid. This was absorbed

onto silica gel and chromatographed with EtOAc/

MeOH (20:1) to give 5h, crystallised as white needles

from Et₂O (17.4 g, 95% yield): mp 100–102 ꢁC; ¹H

NMR (CDCl₃) d 1.3 (br s, 8H, 4CH₂), 1.45 (s, 18H,

2C(CH₃)₃), 1.55–1.75 (m, 8H, 4CH₂), 3.05–3.25 (m,

4H, 2CH₂N), 3.3–3.55 (m, 8H, 4CH₂N), 4.85 (br t,

1H, NHÆCO), 5.4 (br t, 1H, NHÆCO), 6.9 (d,

J = 15 Hz, 1H, HC@CH), 7.1 (br s, 2H, 2NHÆCO),

7.3 (d, J = 15 Hz, 1H, HC@CH).

6.4. N-(3-Guanidinopropyl)-N-(8-aminooctyl)-3-[N⁰-(N⁰⁰-

3-guanidino propyl)-carbamoyl]-cis-propenamide trini-

trate trihydrate (6)

6.3.9. N-(3-Guanidinopropyl)-N-(8-aminooctyl)-3-[(N⁰-

(N⁰⁰-3-guanidinopropyl)-carbamoyl)]-trans-propenamide

trinitrate trihydrate (5). A solution of 5h (2.0 g, 3 mmol)

in 15% w/w HCl in MeOH (25 mL) was stirred for 2 h at

6.4.1. N-[3-(tert-Butoxycarbonylamino)-propyl]-N-(8-tri-

ﬂuoroacetamidooctyl)maleamide (6a). A solution of

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D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

maleic anhydride (1.4 g, 1.43 mmol) and 5g (5.3 g,

1.34 mmol) in MeOH (30 mL) was stirred at 20 ꢁC for

2 h, before concentrating then diluting with CH₂Cl₂

(100 mL) and 1 M HCl (50 mL). The organic phase

was concentrated to low volume, and the product pre-

cipitated as a white powder from diisopropylether

C₂₀H₄₁N₉O₂Æ3HNO₃Æ3.5H₂O: C, 34.73; H, 7.43; N,

24.30. Found: C, 34.46; H, 7.13; N, 24.12.

6.5. N-(3-Guanidinopropyl)-N-[4-(4⁰-aminomethylphe-

nyl)]-benzyl-3-N⁰⁰-(3-guanidinopropyl)-carbamoyl-

trans-propenamide trinitrate, (7)

1

(6.5 g, 98% yield): mp 92–94 ꢁC; H NMR (CDCl₃) d

1.35 (m, 8H, 4CH₂), 1.45 (s, 9H, C(CH₃)₃), 1.5 (m,

4H, 2CH₂), 1.75 (qu, 2H, CH₂), 3.1 (m, 2H, CH₂N),

3.25 (q, 4H, 2CH₂N), 3.45 (m, 2H, CH₂N), 5.0 (br t,

1H, NHÆCO), 7.30 (d, J = 15 Hz, 1H, HC@CH), 7.45

(d, J = 15 Hz, 1H, HC@CH), 8.55 (bt t, 1H, NHÆCO);

6.5.1. Methyl 4-(4⁰-bromophenyl)-benzoate (7a). 4-(4⁰-

Bromophenyl)benzoic acid⁴²(257 g) was heated in

SOCl₂(2 L) under reﬂux for 1 h. The solvent was then

replaced by distillation with toluene, before concentrat-

ing the solution under vacuum. The resulting oil was

diluted with MeOH (1.5 L) to give the product as

red–brown crystals (244 g, 90% yield): mp 138–142 ꢁC;

¹H NMR (CDCl₃) d 3.95 (s, 3H, OCH₃), 7.50 (d, 2H,

2Ar–H), 7.55 (d, 2H, 2Ar–H), 7.65 (d, 2H, 2Ar–H),

IR (Nujol) t_max3380, 3250, 1680, 1650 cm^ꢀ1

.

6.4.2. N-(3-(tert-Butoxycarbonylamino)-propyl)-N-(8-triﬂuo-

roacetamidooctyl)-3-[N⁰⁰-3-(tert-butoxycarbonylamino)-pro-

pyl]-carbamoyl-cis-propenamide (6b). A solution of 6a (6.5 g,

13.2 mmol), N-hydroxysuccinimide (1.8 g, 16 mmol) and

DCC (3.0 g, 14.5 mmol) in CH₂Cl₂(60 mL) was stirred for

16 h at 20 ꢁC before adding 5a (2.4 g, 14.5 mmol). After

3 h the reaction mixture was ﬁltered and the ﬁltrate was

washed with water (30 mL), then concentrated under vac-

uum to give an oil. This was absorbed onto silica gel and

chromatographed with EtOAc/MeOH 20:1 to give the prod-

uct as a yellow gum (6.9 g, 80% yield); ¹H NMR (CDCl₃) d

1.3 (m, 8H, 4CH₂), 1.45 (s, 18H, 2C(CH₃)₃), 1.5 (m, 4H,

2CH₂), 1.6 (m, 2H, CH₂), 1.7 (m, 2H, CH₂), 3.1 (m, 4H,

2CH₂N), 3.3 (m, 6H, 3CH₂N), 3.45 (m, 2H, CH₂N), 5.1

(br t, 1H, NHÆCO), 5.4 (br t, 1H, NHÆCO), 6.1 (d,

J = 12.7 Hz, 1H, HC@CH), 6.4 (d, J = 12.7 Hz, 1H,

HC@CH), 7.1 (br t, 1H, NHÆCO), 8.0 (br t, 1H, NHÆCO);

IR (ﬁlm) t_max3350, 1705, 1700 cm^ꢀ1; MS (TS⁺) 652 (M⁺).

8.15 (d, 2H, 2Ar–H); IR (Nujol) t_max1715 cm^ꢀ1

.

6.5.2. Methyl 4-(4⁰-cyanophenyl)-benzoate (7b). Com-

pound 7a (240 g, 0.8 mol) was heated in DMF (1.5 L)

with CuCN (170 g, 1.9 mol) at 130 ꢁC for 16 h. The result-

ing suspension was cooled to 20 ꢁC then added to water

(3 L) and CH₂Cl₂(2 L), and ﬁltered. The organic phase

was washed with water (2 L) then concentrated to give

an oil, which was diluted with diisopropylether (1 L) to

precipitate the product as a yellow powder (79 g, 40%

yield): mp 142–144 ꢁC; ¹H NMR (CDCl₃) d 3.95 (s, 3H,

OCH₃), 7.65 (d, 2H, 2Ar–H), 7.75 (s, 4H, 4Ar–H), 8.15

(d, 2H, 2Ar–H); IR (Nujol) t_max2230, 1710 cm^ꢀ1

.

6.5.3. 4-(4⁰-Aminomethylphenyl)-benzyl alcohol (7c). Com-

pound 7b (78 g) was treated in THF (800 mL) with 2 M

LiBH₄in THF (200 mL). The resulting solution washeated

under reﬂux for 4 h to give a thick suspension, which was

then cooled and added to water (1.5 L) and CHCl₃(1 L).

The mixture was ﬁltered and the layers separated. The

aqueous phase was extracted with CHCl₃/MeOH 5:1 (2·

1 L). The organic layers were combined and solvent re-

moved by distillation to give an oily solid. This was ab-

sorbed onto silica gel, chromatographed with CHCl₃/

MeOH 5:1 and the product precipitated as a yellow powder

from diisopropylether (10 g, 14% yield): mp 164–168 ꢁC;

¹H NMR (CDCl₃) d 3.2 (br s, 2H, NH₂), 3.8 (s, 2H,

CH₂N), 4.55 (s, 2H, CH₂O), 7.40 (d, 2H, 2Ar–H), 7.45

(d, 2H, 2Ar–H), 7.60 (d, 2H, 2Ar–H), 7.65 (d, 2H, 2Ar–H).

6.4.3.

N-(3-Guanidinopropyl)-N-(8-aminooctyl)-3-[N⁰-

(N⁰⁰-3-guanidinopropyl)-carbamoyl]-cis-propenamide trin-

itrate trihydrate (6). Compound 6b (3.2 g, 5 mmol) was

stirred in a solution of HCl in MeOH (22% w/w,

30 mL) at 20 ꢁC for 2 h, before adjusting to pH 6 with

25% w/w NaOMe in MeOH and ﬁltering. The MeOH

was removed from the ﬁltrate by distillation under vac-

uum and the resulting oil 6c diluted with water

(20 mL). 3,5-Dimethylpyrazole-1-carboxamidine nitrate

(3.7 g, 18 mmol) was added and the pH adjusted to 8 with

1 M aq NaOH. The reaction mixture was heated at 60 ꢁC

for 26 h, maintaining the pH between 7.0 and 8.5 by peri-

odic addition of 1 M aq NaOH. After cooling to ꢀ5 ꢁC,

40% w/w aq NaOH (1 mL) was added (to pH 14), and the

solution stirred for 1 h before washing with CHCl₃(2·

30 mL). The aqueous phase was acidiﬁed to pH 1 with

50% w/w nitric acid and concentrated to dryness under

vacuum. The resulting oil was stirred in MeOH

(30 mL), ﬁltered and reconcentrated. The residue was

precipitated twice from EtOH (2· 30 mL) to give the

product 6 as a yellow glass (100 mg, 5%); ¹H NMR

(DMSO-d₆) d 1.2 (br m, 8H, 4CH₂), 1.4 (m, 4H, 2CH₂),

1.8 (m, 4H, 2CH₂), 2.8 (m, 4H, 2CH₂N), 3.3 (m, 6H,

3CH₂N), 3.55 (m, 2H, CH₂N), 6.0 (m, J = 12.7 Hz, 1H,

HC@CH), 6.5 (m, J = 12.7 Hz, 1H, HC@CH), 7.1 (br s,

8H, 4NH₂^þ), 7.5 (m, 2H, 2NH), 7.7 (br s, 3H, NH₃^þ),

8.40 (br s, 1H, NHÆCO)—2 rotamers 1:1; HPLC Lichro-

sorb PR Select B (MeOH/0.5% aq TFA; 80:20, 1.0 mL/

min), 224 nm, product at 2.9 min; MS (TS⁺) 440

(M⁺+1), fragment at 244. Anal. Calcd for

6.5.4. 4-[4⁰-(Triﬂuoroacetylamidomethyl)-phenyl]-benzyl

alcohol (7d). A suspension of 7c (7.8 g, 37 mmol) in

MeOH (100 mL) was treated with methyl triﬂuoroace-

tate (7.0 g, 55 mmol) until a solution was obtained. The

solvent was then removed by distillation under vacuum.

The residue was absorbed onto silica gel and chromato-

graphed with CH₂Cl₂/MeOH 5:1 to give the product as a

white powder, precipitated from diisopropylether (5.9 g,

53% yield): mp 179–181 ꢁC; ¹H NMR (CDCl₃) d 4.45 (d,

2H, CH₂N), 4.55 (d, 2H, CH₂O), 5.25 (t, 1H, OH), 7.40

(d, 2H, 2Ar–H), 7.45 (d, 2H, 2Ar–H), 7.60 (d, 2H, 2Ar–

H), 7.65 (d, 2H, 2Ar–H), 10.05 (br t, 1H, NHÆCO); IR

(Nujol) t_max3290, 1700, 1560 cm^ꢀ1

.

6.5.5. 4-[4⁰-(Triﬂuoroacetamidomethyl)-phenyl]-N-[3-(tert-

butoxycarbonyl)-aminopropyl]benzylamine (7e). DMF

(8 mL) was added to a mixture of 7d (3.7 g, 12 mmol),

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5473

TsCl (5.1 g, 27 mmol) and NEt₃(1.8 g, 18 mmol) in

CHCl₃(30 mL) until a solution was obtained. This

was heated to reﬂux and more NEt₃(0.8 g, 0.7 mol/

mol) added dropwise until the pH increased to 7. The

solution was cooled, washed with 1 M HCl (50 mL),

water (50 mL) and concentrated under vacuum to give

an oil. This was dissolved in DMF and stirred with

the amine 5a (3.4 g, 20 mmol) for 2 h at 20 ꢁC, then di-

luted with water and extracted with CH₂Cl₂(2· 30 mL).

The extracts were combined, washed with water (15 mL)

and concentrated under vacuum to give an oil, which

was absorbed onto silica gel and chromatographed with

CH₂Cl₂/MeOH 3:1 to give the product as a white pow-

der, precipitated from diisopropylether (1.6 g, 69%

yield): mp 195–197 ꢁC; ¹H NMR (CDCl₃) d 1.45 (s,

9H, C(CH₃)₃), 1.7 (m, 2H, CH₂), 2.75 (t, 2H, CH₂N),

3.35 (t, 2H, CH₂N), 3.85 (s, 2H, CH₂N), 4.45 (d, 2H,

CH₂N), 5.35 (br t, 1H, NHÆCO), 6.90 (br t, 1H, NHÆ-

CO), 7.35 (d, 2H, 2Ar–H), 7.40 (d, 2H, 2Ar–H), 7.50

(d, 2H, 2Ar–H), 7.60 (d, 2H, 2Ar–H); MS (FB⁺) 466

(M⁺+1), fragments at 410, 292.

(30 mL), ﬁltered and reconcentrated. The resulting

gum was precipitated twice from EtOH (2· 40 mL) to

give the product as a yellow glass (120 mg, 14% yield);

¹H NMR (DMSO-d₆) d 1.90 (m, 4H, 2CH₂)-satellite

signals due to rotamers, 3.00 (m, 2H, CH₂N), 3.20

(m, 2H, CH₂N), 3.35 (m, 2H, CH₂N), 3.55 (m, 2H,

CH₂N), 4.10 (m, 2H, CH₂N), 4.70 (m, 2H, CH₂N),

7.10 (d, J = 15 Hz, 1H, HC@CH), 7.35 (d, J = 15 Hz,

1H, HC@CH), 7.40 (m, 2H, 2Ar–H), 7.70–7.55 (m,

8H, 4Ar–H), 7.85 (br s, 8H, 4NH₂), 8.35 (br s, 3H,

NH₃^þ), 8.70 (m, 1H, NHÆCO)—2 rotamers 1:1; HPLC

Lichrosorb RP Select B (MeOH/0.05M aq NH₄OAc,

95:5, 1.0 mL/min), 210 nm, product at 4.3 min; MS

(FAB⁺) 509 (M⁺+1), fragments at 467, 425. HPLC

Lichrosorb RP Select B (MeOH/0.05 M aq NH₄OAc,

95/5, 1.0 mL/min), 210 nm, product at 4.3 min. Anal.

Calcd for C₂₆H₃₇N₉O₂Æ3HNO₃Æ0.3EtOHÆ13% w/w

inorganic material: C, 39.20; H, 5.12; N, 20.50. Found:

C, 39.20; H, 5.10; N, 20.30.

6.6. 4,4⁰Bis(guanidinomethyl)-cis-stilbene triﬂuoroacetate

(8)

6.5.6. N-[3-(tert-Butoxycarbonylamino)-propyl]-N⁰-[4-(4⁰-

triﬂuoroacetamidomethyl)-phenyl-benzyl]-3-N⁰-[3-(tert-but-

oxycarbonylamino)-propyl]-carbamoyl-trans-propenamide

(7f). A solution of 5c (0.7 g, 2.57 mmol), HOBT (0.35 g,

2.60 mmol) and DCC (0.55 g, 2.70 mmol) in DMF

(7 mL) was stirred at 20 ꢁC for 2 h. The resulting suspen-

sion was treated with 7e (1.1 g, 2.35 mmol) before being

stirred for a further 2 h. The reaction mixture was ﬁl-

tered, diluted with water (30 mL), extracted with CH₂Cl₂

(60 mL) and the extract was concentrated to low volume.

The resulting oil was absorbed onto silica gel and chro-

matographed with EtOAc to give the product as a white

powder, precipitated from diisopropyl ether (1.3 g, 77%

yield): mp 134–136 ꢁC; ¹H NMR (CDCl₃) d 1.35 (s,

18H, 2C(CH₃)₃)-satellite signals due to rotamers, 1.55

(m, 2H, CH₂), 1.65 (m, 2H, CH₂), 2.95 (m, 4H,

2CH₂N), 3.15 (m, 2H, CH₂N), 3.35 (m, 2H, CH₂N),

4.45 (d, 2H, CH₂N), 4.7 (s, 2H, CH₂N), 6.7 (d,

J = 15 Hz, 1H, HC@CH), 7.15 (d, J = 15 Hz, 1H,

HC@CH), 7.50 (d, 4H, 4Ar–H), 7.60 (d, 4H, 4Ar–H),

10.05 (t, 1H, NHÆCO); IR (Nujol) t_max3320, 1705,

1680 cm^ꢀ1; MS (TS⁺) 720 (M⁺+1), fragments at 292, 427.

4,4⁰-Bis (bromomethyl)-cis-stilbene³⁸(2.65 g, 7.2 mmol)

and potassium phthalimide (3.5 g, 19 mmol) in 10 mL

of anhydrous DMF were heated with stirring for 12 h

at 90 ꢁC, then cooled, treated with 15 mL of water, ﬁl-

tered and washed with water. Crystallisation from

MeOH aﬀorded 3.2 g (89% yield) of the diphthalimide

derivative 8a, mp 180–181 ꢁC.

The latter (8a), 3.2 g in ethanol (130 mL), was heated

under reﬂux with 1 mL of hydrazine hydrate for 5 h.

The mixture was cooled, water (15 mL) was added and

the ethanol was removed by distillation. The residual so-

lid was washed with water and then chromatographed

on silica gel using a mixture of CHCl₃/EtOAc/MeOH

to give the bis-amine product 8b (1.21 g, 79% yield),

mp 123–125 ꢁC decomp.

The bis-amine 8b (0.5 g) was stirred with 3,5-dim-

ethylpyrazole-1-carboxamidine nitrate (1.2 g) in water

(25 mL) at 80 ꢁC. The mixture was cooled to 4 ꢁC and

the product was isolated by preparative chromatogra-

phy using 30% aqueous methanol rising to 50% metha-

nol over 20 min. and then taken into isopropanol,

ﬁltered cold and concentrated in vacuo to give 8 as an

oil (0.05 g, 36% yield).¹H NMR (DMSO-d₆)d 4.33 (br

s, 4H, CH₂N), 6.62 (s, 2H, CH@CH), 7.17 (d, 4H,

C₆H₄), 7.20 (br s, 8H, NH⁺), 7.23 (d, 4H, C₆H₄), 7.99

(br s, 2H, NH). MS(FAB⁺) 323 (M⁺+1), fragments at

306, 263, 149, 102. Anal. Calcd for C₁₈H₂₂N₆Æ2.3CF_3-

CO₂HÆCH₃OH: C, 45.95; H, 4.63; N, 13.63. Found: C,

46.14; H, 4.65; N, 13.52.

6.5.7. N-(3-Guanidinopropyl)-N-[4-(4⁰-aminomethylphe-

nyl)]-benzyl-3-N⁰-(3-guanidino propyl)-carbamoyl-trans-

propenamide trinitrate (7). A solution of 7f (1.2 g,

1.7 mmol) was stirred in HCl in MeOH (17% w/w,

10 mL) at 20 ꢁC for 2 h, before adjusting the pH to 6

with 25% w/w NaOMe in MeOH and ﬁltering. The

MeOH was removed by distillation under vacuum,

and the residue diluted with water (20 mL). 3,5-

Dimethylpyrazole-1-carboxamidine nitrate (1.6 g,

8.0 mmol) was then added and the pH adjusted to 8

with 1 M aq NaOH before heating at 60 ꢁC for 18 h,

maintaining the pH between 7.5 and 8.5 by periodic

addition of 1 M aq NaOH. After cooling to 20 ꢁC,

10 M aq NaOH (0.5 mL) was added to pH 14, and

the solution stirred for 1 h before washing with CHCl₃

(2· 25 mL). The aqueous phase was acidiﬁed to pH 1

with 50% w/w aq HNO₃and concentrated under vac-

uum to give an oil, which was stirred in MeOH

6.7. N-[3-(4-Quinolinyl)-aminopropyl]-N-(8-aminooctyl)-

3-N⁰-[3-(4-quinolinyl)-aminopropyl]-carbamoyl-trans pro-

penamide trihydrate (9)

6.7.1. N-(tert-Butoxycarbonyl)-N⁰-(4-quinolinyl)-1,3-diami-

nopropane (9a). A solution of 5a (32.6 g, 0.187 mmol),

NⁱPr₂Et (25.2 g, 195 mmol) and 4-chloroquinoline

(24.5 g, 150 mmol) in n-pentanol (200 mL) was heated un-

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D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

der reﬂux for 6 h, before concentrated to low volume. The

resulting oil was diluted with CH₂Cl₂(250 mL) and

washed with 2 M NaOH (120 mL). After the second wash

a precipitate was produced, which was ﬁltered oﬀ and

washed with toluene (50 mL). The organic phase from

the ﬁltrate was concentrated and the residue diluted with

toluene (150 mL) to give a further precipitate, which was

also ﬁltered oﬀ and washed with toluene. The two crops

were combined and recrystallised from toluene (300 mL)

to provide the product as a cream powder (38 g, 84%

Ar–H), 8.60 (d, 1H, Ar–H), 8.85 (t, 1H, NHÆCO),

9.50 (t, 1H, CO₂H).

6.7.5. N-(Triﬂuoroacetamido)-N⁰-[3-(4-quinolinylamino)-

propyl]-1,8-diaminooctane (9e). A solution of 9b (5.0 g,

50 mmol), 8-triﬂuoroacetamido-1-octyl tosylate (10.0 g,

26 mmol) and NⁱPr₂Et (3.5 g, 27.5 mmol) in DMF

was stirred for 16 h at 20 ꢁC, before diluting with water

(150 mL) and extracting with CH₂Cl₂(2· 100 mL). The

combined extracts were washed with water (100 mL)

and concentrated to give an orange oil, which was ab-

sorbed onto silica gel and chromatographed with CMA

to give the product as a pale yellow gum (3.5 g, 34%

yield); ¹H NMR (CDCl₃) d 1.25 (br s, 8H, 4CH₂),

1.50 (m, 4H, 2CH₂), 1.95 (qu, 2H, CH₂), 2.70 (m,

2H, CH₂N), 2.95 (m, 2H, CH₂N), 3.35 (q, 2H,

CH₂N), 3.45 (m, 2H, CH₂N), 6.35 (d, 1H, Ar–H),

6.70 (br s, 1H, NH), 7.40 (m, 1H, Ar–H), 7.70 (m,

1H, Ar–H), 7.75 (br s, 1H, NHÆCO), 7.85 (m, 1H,

Ar–H), 7.95 (m, 1H, Ar–H), 8.50 (d, 1H, Ar–H).

1

yield): mp 165–167 ꢁC; H NMR (CDCl₃) d 1.50 (s, 9H,

C(CH₃)₃), 1.85 (qu, 2H, CH₂), 3.30 (q, 2H, CH₂N), 3.45

(q, 2H, CH₂N), 4.85 (br s, 1H, NH), 6.0 (br s, 1H,

NHÆCO), 6.45 (d, 1H, Ar–H), 7.45 (m, 1H, Ar–H), 7.60

(m, 1H, Ar–H), 7.95 (d, 1H, Ar–H), 8.0 (d, 1H, Ar–H),

8.50 (d, 1H, Ar–H).

6.7.2. N-(4-Quinolinyl)-1,3-diaminopropane (9b). A solu-

tion of 9a (30.1 g, 0.10 mol) in triﬂuoroacetic acid

(200 mL) was stirred at 20 ꢁC for 1 h, diluted with tolu-

ene (200 mL) and concentrated to remove the bulk of

the solvent. The resulting oil was diluted with water

(200 mL), basiﬁed to pH 14 with 40% w/w aq NaOH

(50 mL) and extracted with CHCl₃(2· 150 mL). The

combined extracts were concentrated under vacuum to

give the product as a white powder (19 g, 95% yield):

6.7.6. N-[3-(4-Quinolinylamino)-propyl]-N-(8-triﬂuoroace-

tamidooctyl)-N⁰-[3-(4-quinolinylamino)-propyl]-carbamoyl-

trans propenamide (9f). Compound 9d (3.4 g, 10 mmol)

and DCC (2.5 g, 12 mmol) were stirred in DMF

(20 mL) for 3 h to give a thick suspension, which was

treated with 9e (4.3 g, 10 mmol) and NⁱPr₂Et (1.6 g,

12.5 mmol). After 2 h, the mixture was ﬁltered to remove

dicyclohexylurea, diluted with water (100 mL) and ex-

tracted with CH₂Cl₂(2· 100 mL). The combined extracts

were washed with water (50 mL) and concentrated to

give a yellow oil, which was absorbed onto silica gel

and chromatographed with toluene/EtOH/0.91 w/w aq

NH₃(80:20:1) to give the product as oﬀ-white needles,

crystallised from Et₂O (1.5 g, 22% yield): mp 127–

129 ꢁC; ¹H NMR (CDCl₃) d 1.20 (br s, 8H, 4CH₂),

1.95 (m, 4H, 2CH₂), 1.55 (m, 4H, 2CH₂), 3.35 (m, 8H,

4CH₂N), 3.55 (m, 4H, 2CH₂N), 6.10 (t, 1H, NHÆCO),

6.35 (d, 2H, 2Ar–H), 6.55 (t, 2H, 2NH), 7.05 (d,

J = 15 Hz, 1H, HC@CH), 7.35 (m, 2H, 2Ar–H), 7.40

(d, J = 15 Hz, 1H, HC@CH), 7.40 (m, 2H, 2Ar–H),

7.60 (m, 2H, 2Ar–H), 7.85 (m, 1H, NHÆCO), 7.95 (m,

4H, 2Ar–H), 8.50 (m, 2H, 2Ar–H); IR (Nujol) t_max

1

mp 75–77 ꢁC; H NMR (CDCl₃) d 1.90 (qu, 2H, CH₂),

3.05 (t, 2H, CH₂N), 3.45 (t, 2H, CH₂N), 6.35 (d, 1H,

Ar–H), 7.25 (br t, 1H, NH), 7.40 (m, 1H, Ar–H), 7.65

(m, 1H, Ar–H), 7.80 (d, 1H, Ar–H), 7.95 (d, 1H,

Ar–H), 8.55 (d, 1H, Ar–H).

6.7.3. Ethyl 3-[N-(4-quinolinylamino)-propyl]-carbamoyl-

trans-propenoate (9c). Monoethylfumarate (8.6 g,

60 mmol) and DCC (13.6 g, 70 mmol) were stirred in

THF (120 mL) for 2 h at 20 ꢁC to give a thick suspen-

sion, to which a solution of 9b (12.0 g, 60 mmol) in

THF (50 mL) was added. After a further 2 h, the mix-

ture was ﬁltered and the ﬁltrate concentrated under vac-

uum to give the product as a cream powder, precipitated

from diisopropyl ether (11.7 g, 60% yield): mp 194–

1

196 ꢁC; H NMR (CDCl₃) d 1.30 (t, 3H, CH₃), 1.90

(m, 2H, CH₂), 3.40 (m, 2H, CH₂N), 3.55 (m, 2H,

CH₂N), 4.30 (q, 2H, CH₂O), 6.30 (d, 1H, Ar–H), 6.50

(t, 1H, NHÆCO), 6.90 (d, J = 15 Hz, 1H, HC@CH),

7.00 (d, J = 15 Hz, 1H, HC=CH), 7.50 (m, 1H, Ar–H),

7.65 (m, 1H, Ar–H), 7.80 (d, 1H, Ar–H), 8.00 (m, 2H,

Ar–H), 8.50 (d, 1H, Ar–H).

3400, 1710, 1630 cm^ꢀ1

.

6.7.7. N-[3-(4-Quinolinylamino)-propyl]-N-(8-aminooc-

tyl)-3-N⁰-[3-(4-quinolinylamino)-propyl]-carbamoyl-trans

propenamide trihydrate (9). 40% w/w aq NaOH

(0.5 mL) was added to 9f (0.4 g) in a mixture of MeOH

(5 mL) and water (3 mL). After 1 h at 20 ꢁC, the solu-

tion was extracted with CHCl₃(2· 20 mL). The ex-

tracts were combined and concentrated to give a pale

yellow gum, which was redissolved in CHCl₃/MeOH

(20:1, 10 mL), washed with 0.5 M aq NaOH (2·

5 mL) and reconcentrated. The residue was dissolved

in EtOH and diluted with Et₂O to precipitate the prod-

uct as a clear glass (30 mg, 8% yield); ¹H NMR

(CDCl₃) d 1.30 (br s, 8H, 4CH₂), 1.45 (m, 2H, CH₂),

1.65 (m, 2H, CH₂), 1.90 (m, 4H, 2CH₂), 2.65 (m, 2H,

CH₂N), 3.30 (m, 2H, CH₂N), 3.40 (m, 4H, 2CH₂N),

3.55 (m, 4H, 2CH₂N), 6.15 (t, 2H, NH₂), 6.40 (m,

2H, 2Ar–H), 6.55 (t, 1H, NH), 6.90 (t, 1H, NH),

6.7.4. 3-[N-(4-Quinolinylamino)-propyl]-carbamoyl-trans-

propenoic acid hydrochloride (9d). A suspension of 9c

(9.85 g, 30 mmol) in MeOH (30 mL) and water

(120 mL) was stirred at 20 ꢁC while adding 40% w/w

aq NaOH (30 mL, 30 mmol). The mixture was heated

to 50 ꢁC to give a solution, which was cooled to

20 ꢁC, and concd HCl (60 g, 70 mmol) added to precip-

itate the product as white needles (3.0 g, 30% yield):

mp 179–181 ꢁC; ¹H NMR (DMSO-d₆) d 1.90 (qu,

2H, CH₂), 2.70 (m, 2H, CH₂N), 2.95 (m, 2H,

CH₂N), 6.50 (d, J = 15 Hz, 1H, HC CH), 6.80 (d,

1H, Ar–H), 6.95 (d, J = 15 Hz, 1H, HC@CH), 7.70

(m, 1H, Ar–H), 7.95 (m, 2H, 2Ar–H), 8.50 (d, 1H,

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5475

7.05 (d, J = 15 Hz, 1H, HC@CH), 7.40 (m, 2H, 2Ar–

H), 7.45 (d, J = 15 Hz, 1H, HC@CH), 7.60 (m, 2H,

2Ar–H), 7.95 (m, 4H, 4Ar–H), 8.55 (m, 2H, 2Ar–H);

IR (Nujol) t_max3250, 1665 cm^ꢀ1; HPLC (Phenyl

BDS, MeOH/0.05 M aq NH₄OAc 90:10, 215 nm,

1.0 mL/min), product at 3.5 min; MS (TS⁺) 610

(M⁺+1). Anal. Calcd for C₂₆H₄₇N₇O₂Æ3.5H₂OÆ0.3E-

tOH: C, 63.20; H, 8.00; N, 14.01. Found: C, 63.24;

H, 7.42; N, 13.86.

(20 mL) was stirred at 20 ꢁC for 16 h, before diluting with

water (80 mL) and extracting with EtOAc (160 mL). The

extracts were combined, washed with water and concen-

trated under vacuum. The resulting residue was absorbed

onto silica gel and chromatographed with CMA to give

1

the product as a yellow oil (2.6 g, 83% yield); H NMR

(CDCl₃) d 1.35 (s, 18H, 2C(CH₃)₃), 1.80 (qu, 2H, CH₂),

1.85 (qu, 2H, CH₂), 2.80 (t, 2H, CH₂), 3.20 (q, 4H,

2CH₂N), 3.30 (t, 4H, 2CH₂N), 3.70 (s, 2H, CH₂N), 3.80

(t, 4H, 2CH₂O), 4.80 (br s, 2H, NHÆCO), 6.25 (d, 1H,

Ar–H), 6.30 (s, 1H, Ar–H), 6.40 (s, 2H, Ar–H), 7.25 (m,

2H, Ar–H), 7.60 (m, 1H, Ar–H), 7.90 (d, 1H, Ar–H),

8.45 (d, 1H, Ar–H); IR (ﬁlm) t_max3280, 1700 cm^ꢀ1; MS

(ES⁺) 638 (M⁺+1), fragment at 356.

6.8. N-[3-(4-Quinolinylamino)-propyl]-3,5-di-[3-(4-quin-

olinylamino)-propoxy]-benzylamine, (11)

6.8.1. 3-Bromo-N-(tert-butoxycarbonyl)-propylamine (11a).

A solution of 3-bromopropylamineÆHBr (217 g, 1.0 mol)

and di-t-butyl dicarbonate (229 g, 1.05 mol) in MeOH

(400 mL) was treated with 25% w/w NaOMe in MeOH

(450 mL, 2.1 mol) over 1 h at 25 ꢁC. After stirring for

0.5 h the reaction mixture was diluted with water (3 L)

and extracted with CH₂Cl₂(3 L). The extract was washed

with 1 M HCl (1.5 L) then with saturated NaHCO₃

(1.5 L), before being concentrated under vacuum to give

the product as white crystals, precipitated from diiso-

propylether (204 g, 86% yield); ¹H NMR (CDCl₃) d

1.35 (s, 9H, C(CH₃)₃), 1.95 (qu, 2H, CH₂), 3.15 (q, 2H,

CH₂N), 3.30 (t, 2H, CH₂Br), 5.00 (t, 1H, NHÆCO); IR

6.8.5. N-[3-(4-Quinolinylamino)-propyl]-3,5-di-[3-(4-quino-

linylamino)-propoxy]-benzylamine dihydrate (11). A solu-

tion of 11d (1.9 g, 3.0 mmol) in triﬂuoroacetic acid

(20 mL) was stirred at 20 ꢁC for 1 h. The excess acid was

then removed by distillation and the resulting oil dis-

solved in MeOH. 25% w/w NaOMe in MeOH was added

to pH 10 and the solution concentrated to low volume be-

fore diluting with n-pentanol (20 mL). NⁱPr₂Et (2.0 g,

15 mmol) and 4-chloroquinoline (2.0 g, 12 mmol) were

added and the solution heated under reﬂux for 4 h, before

concentrating, diluting with 1 M aq NaOH (50 mL) and

extracting with CH₂Cl₂/MeOH (10:1, 2· 50 mL). The

combined extracts were concentrated and the residue ab-

sorbed onto silica gel and chromatographed with CMA.

The resulting oil was dissolved in CH₂Cl₂, ﬁltered to clar-

ify, then the solvent removed by distillation and the resi-

due triturated with EtOAc to give the product as a pale

yellow foam (0.2 g, 10% yield); ¹H NMR (CDCl₃) d 1.90

(m, 2H, CH₂), 2.05 (m, 2H, CH₂), 2.85 (m, 2H, CH₂),

3.35 (m, 4H, 2CH₂N), 3.45 (m, 4H, 2CH₂N), 3.80 (s,

2H, ArCH₂N), 4.90 (t, 4H, 2CH₂O), 6.40 (m, 3H, 3Ar–

H), 6.50 (s, 2H, 2Ar–H), 7.30 (m, 3H, 3Ar–H), 7.50 (m,

3H, 3Ar–H), 7.75 (m, 3H, 3Ar–H), 7.90 (d, 1H, Ar–H)

7.95 (m, 3H, 3Ar–H), 8.55 (m, 3H, 3Ar–H); IR (Nujol)

(Nujol) t_max3350, 1680 cm^ꢀ1

.

6.8.2. 3,5-Di-[3-(tert-butoxycarbonylamino)-propoxy]-benzyl

alcohol (11b). A solution of KOBu (25 g, 0.22 mol) in

DMF (100 mL) was added to 3,5-dihydroxybenzyl alco-

hol (14 g, 0.1 mol) in DMF (50 mL) at 20–45 ꢁC. The

resulting suspension was treated with 11a (50 g,

0.21 mol) to give a solution, which was diluted with water

(300 mL) and extracted with EtOAc (3· 150 mL). The

combined extracts were washed with 1 M aq NaOH

(100 mL) then water (100 mL) and concentrated under

vacuum to give the product as a cream powder, precipi-

tated from diisopropylether (30.2 g, 71% yield); ¹H

NMR (CDCl₃) d 1.45 (s, 18H, 2C(CH₃)₃), 1.95 (qu, 4H,

2CH₂), 3.30 (q, 4H, 2CH₂N), 4.00 (t, 4H, 2CH₂O), 4.60

(s, 2H, CH₂O), 4.75 (br s, 2H, 2NHÆCO), 6.35 (s, 1H,

Ar–H), 6.50 (s, 2H, 2Ar–H); IR (Nujol) t_max3380,

t

_max3250, 1710 cm^ꢀ1; MS (ES⁺) 692 (M⁺+1). Anal. Calcd

for C₄₃H₄₅N₇O₂Æ2.5H₂O: C, 70.00; H, 6.78; N, 13.29.

Found: C, 70.49; H, 6.77; N, 12.88.

1690 cm^ꢀ1

.

6.9. N-[3-(4-Quinolinylamino)-propyl]-3,5-di-[3-(4-quinoli-

nylamino)-propoxy]-aniline dihydrate (12)

6.8.3. 3,5-Di-[3-(tert-butoxycarbonylamino)-propoxy]-

benzyl chloride (11c). A mixture of 11b (11.3 g, 25 mmol),

NEt₃(3.3 g, 32 mmol) and TsCl (6.0 g, 31 mmol) in

CHCl₃(100 mL) was stirred at 20 ꢁC for 16 h. The solvent

was then replaced with EtOAc by distillation to precipi-

tate NEt₃ÆHCl, which was ﬁltered oﬀ and the ﬁltrate con-

centrated. The residue was absorbed onto silica gel and

chromatographed with EtOAc to give the product as a

yellow oil, which was crystallised from diisopropylether

6.9.1.

N-(3,5-Dihydroxyphenyl)-N⁰-(t-butoxycar-

bonyl)-1,3-diaminopropane (12a) and 3,5-di-[3⁰-(tert-

butoxycarbonylamino)-propyl]-aminophenol (12b). A

solution of phloroglucinol (63 g, 0.50 mol) and 5a

(100 g, 0.58 mol) in THF was heated under reﬂux in

(500 mL) for 0.5 h. Toluene (800 mL) was then added

and the solvent removed under vacuum. The residue

was absorbed onto silica gel and chromatographed

with EtOAc/CH₂Cl₂(1:1) to give an oil, which was

stirred in EtOAc (500 mL) to precipitate 12b as a

dark green powder (28 g, 13% yield). The mother li-

quors were concentrated under vacuum then ab-

sorbed onto silica gel and chromatographed with

Et₂O to give 12a as a brown gum (80 g, 56% yield);

¹H NMR (DMSO-d₆) d 1.40 (s, 9H, C(CH₃)₃), 1.80

(m, 2H, CH₂), 3.00 (m, 2H, CH₂N), 3.10 (m, 2H,

CH₂N), 5.30 (t, 1H, NH), 5.50 (m, 3H, 3Ar–H), 6.80

1

(5.0 g, 42% yield): mp 129–131 ꢁC; H NMR (CDCl₃) d

1.45 (s, 18H, 2C(CH₃)₃), 1.95 (qu, 4H, 2CH₂), 3.30 (q,

4H, 2CH₂N), 4.00 (t, 4H, 2CH₂O), 4.50 (s, 2H, CH₂Cl),

4.75 (br s, 2H, NHÆCO), 6.40 (s, 1H, Ar–H), 6.50 (s,

2H, Ar–H); IR (Nujol) t_max3380, 1690 cm^ꢀ1

.

6.8.4. N-[3-(4-Quinolinylamino)-propyl]-3,5-di-[3-(tert-but-

oxycarbonylamino)-propoxy]-benzylamine (11d). A solution

of 9b (4.0 g, 20 mmol) and 11c (2.35 g , 5 mmol) in DMF

5476

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

(t, 1H, NHÆCO), 8.70 (br s, 2H, 2OH); IR (ﬁlm) t_max

3400, 1690, 1630 cm^ꢀ1

(s, 9H, C(CH₃)₃), 2.45 (t, 2H, ArCH₂) 3.10 (q, 2H,

CH₂N), 6.35 (m, 1H, Ar–H), 6.60 (m, 1H, Ar–H),

6.65 (d, 1H, Ar–H), 6.80 (t, 1H, NHÆCO), 8.80 (br s,

2H, 2OH). MS (EI⁺) 253 (M⁺+1); IR (Nujol) t_max

.

6.9.2. N-[(tert-Butoxycarbonylamino)-propyl]-3,5-di-[(tert-

butoxycarbonylamino)-propoxy]-aniline (12c). A solution

of 12a (5.7 g, 20 mmol) in DMF (50 mL) was treated

sequentially with KOBu (5.3 g, 46 mmol) and 11a (9.5 g,

40 mmol). After 1 h the solution was diluted with water

(200 mL) and extracted with CH₂Cl₂(2· 150 mL). The

combined extracts were washed with 1 M aq NaOH and

concentrated to give a brown oil. This was absorbed onto

silica gel and chromatographed with Et₂O to give the

product as cream crystals (2.3 g, 20% yield): mp 125–

3500, 3380, 1680 cm^ꢀ1

.

6.10.2. N-(tert-Butoxycarbonyl)-3,4-di-[3-(tert-butoxycar-

bonylamino)-propoxy]-phenethyl-amine (13b). A solution

of 13a (12.6 g, 50 mmol) in DMF (200 mL) was treated

with KOBu (13.0 g, 115 mmol) and 11a (25.0 g,

105 mmol) at 20 ꢁC. After 1 h, the reaction mixture was

diluted with water (800 mL) and extracted with CH₂Cl₂

(600 mL). The extract was washed with 1 M aq NaOH,

then concentrated. The residue was absorbed onto silica

gel and chromatographed with Et₂O to give the product

as an oﬀ-white powder, precipitated from diisopropyl-

ether (8.5 g, 33% yield): mp 91–93 ꢁC; ¹H NMR (CDCl₃)

d 1.40 (s, 18H, 2C(CH₃)₃), 2.00 (m, 4H, 2CH₂), 2.70 (t,

2H, CH₂Ar), 3.35 (m, 6H, 3CH₂N), 4.00 (m, 4H,

2CH₂O), 4.60 (br s, 1H, NHÆCO), 5.30 (br s, 2H,

NHÆCO), 6.70 (m, 2H, 2Ar–H), 6.80 (d, 1H, Ar–H); IR

(Nujol) t_max3350, 1680 cm^ꢀ1; MS (TS⁺) 568 (M⁺+1).

1

127 ꢁC; H NMR (CDCl₃) d 1.40 (s, 27H, 3C(CH₃)₃),

1.80 (m, 2H, CH₂), 1.95 (m, 4H, 2CH₂), 3.10 (m, 2H,

CH₂N), 3.20 (m, 2H, CH₂N), 3.30 (q, 4H, 2CH₂N), 3.95

(m, 4H, 2CH₂O), 4.80 (t, 1H, NHÆCO), 4.85 (t, 2H, 2NHÆ-

CO), 5.80 (m, 2H, 2Ar–H), 5.85 (m, 1H, Ar–H); IR (Nujol)

t_max3380, 1700, 1620 cm^ꢀ1; MS (ES⁺) 597 (M⁺+1).

6.9.3. N-[3-(4-Quinolinylamino)-propyl]-3,5-di-[3-(4-quino-

linylamino)-propoxy]-aniline dihydrate (12). A solution of

12c (2.0 g, 3.3 mmol) in triﬂuoroacetic acid (30 mL) was

stirred at ambient temperature for 1 h, then concentrated

under vacuum, diluted with MeOH, and 25% w/w

NaOMe in MeOH added until pH 12. This solution was

concentrated to give a green oil, which was dissolved in

n-pentanol (30 mL) and the solution heated with 4-chlo-

roquinoline (2.2 g, 13 mmol) plus NPr₂Et (2.2 g,

16.5 mmol) for 4 h. The reaction mixture was then con-

centrated, diluted with 1 M aq NaOH (100 mL) and ex-

tracted with CH₂Cl₂/MeOH (10:1, 2· 100 mL). The

combined extracts were washed with 1 M aq NaOH

(50 mL) and concentrated. The residue was absorbed

onto silica gel and chromatographed with CMA to give

the product as a clear glass, precipitated from Et₂O

6.10.3. N-(4-Quinolinylamino)-3,4-di-[3-(4-quinolinylami-

no)-propoxy]-phenethylamine dihydrate (13). A solution

of 13b (5.7 g, 10 mmol) in triﬂuoroacetic acid (30 mL)

was stirred at 20 ꢁC for 1 h, then concentrated under

vacuum to give a yellow oil. This was dissolved in

MeOH (50 mL) and 25% w/w NaOMe in MeOH added

until pH 10. The MeOH was then removed by distilla-

tion and the residue diluted with n-pentanol (50 mL).

4-Chloroquinoline (3.3 g, 40 mmol) and NⁱPr₂Et (3.0g,

40 mmol) were added and the reaction mixture heated

under reﬂux for 7 h, before concentrating under vac-

uum. The residue was absorbed onto silica gel and chro-

matographed with CMA to give a gum, which was

triturated with Et₂O to precipitate the product as a clear

1

(0.5 g, 23% yield): mp 102–107 ꢁC; H NMR (DMSO-

1

d₆) d 1.90 (qu, 2H, CH₂), 2.10 (qu, 4H, 2CH₂), 3.20 (t,

2H, CH₂N), 3.45 (t, 2H, CH₂N), 3.50 (t, 4H, 2CH₂N),

3.95 (t, 4H, 2CH₂O), 5.70 (t, 1H, NH), 5.80 (m, 2H, Ar–

H), 5.85 (m, 1H, Ar–H), 6.45 (m, 3H, 3Ar–H), 7.20 (m,

3H, 3NH), 7.40 (m, 3H, 3Ar–H), 7.60 (m, 3H, 3Ar–H),

7.75 (m, 3H, 3Ar–H), 8.25 (d, 3H, 3Ar–H), 8.40 (m, 3H,

3Ar–H); HPLC (IB-SIL Phenyl-BDS, MeOH/0.05M aq

NH₄OAc, 90:10, 215 nm, 1.0 mL/min), product at

2.6 min, impurity at 1.8 min; MS (EI⁺) 678 (M⁺+1). Anal.

Calcd for C₄₂H₄₃N₇O₂Æ2H₂O: C, 70.58; H, 6.58; N, 13.72.

Found: C, 70.30; H, 6.50; N, 13.38.

glass (30 mg, 5% yield); H NMR (CD₃OD) d 2.05 (m,

2H, CH₂), 2.15 (m, 2H, CH₂), 2.90 (t, 2H, CH₂Ar),

3.45 (m, 2H, CH₂N), 3.55 (m, 4H, 2CH₂N), 4.00 (t,

2H, CH₂O), 4.20 (t, 2H, CH₂O), 6.45 (m, 3H, 3Ar–H),

6.85 (m, 2H, 2Ar–H), 6.95 (d, 1H, Ar–H), 7.35 (m,

3H, 3Ar–H), 7.55 (m, 3H, 3Ar–H), 7.75 (m, 3H, 3Ar–

H), 8.00 (m, 3H, 3Ar–H), 8.20 (d, 2H, 2Ar–H), 8.30

(d, 1H, Ar–H); IR (Nujol) t_max3250, 1700 cm^ꢀ1; HPLC

(Partisil ODS3, MeOH/0.05 M aq NH₄OAc (pH 4)

90:10, 1.0 mL/min, 215 nm), product at 5.6 min, impu-

rity at 4.1 min: MS (ES⁺) 649 (M⁺+1). Anal. Calcd for

C₄₁H₄₀N₆O₂Æ2H₂O: C, 71.82; H, 6.42; N, 12.26. Found:

C, 71.82; H, 6.17; N, 11.88.

6.10. N-(4-Quinolinyl)-3,4-di-[3-(4-quinolinylamino)-

propoxy]-phenethylamine dihydrate (13)

6.11. 1,2,3-Tri-[3-(4-Quinolinylamino)-propoxy]-benzene

hydrate (14)

6.10.1. N-(tert-Butoxycarbonyl)-dopamine (13a).

A

solution of dopamineÆHCl (38.0 g, 0.20 mol) and di-

tert-butyl dicarbonate (48.0 g, 0.22 mol) in MeOH

(200 mL) was treated with 25% w/w NaOMe in MeOH

(50 mL, 0.23 mol) at 25 ꢁC. After 0.5 h, the reaction

mixture was concentrated to low volume, before dilut-

ing with EtOAc (800 mL). The precipitated inorganic

salts were ﬁltered oﬀ and the ﬁltrate concentrated to

give an oil, which was stirred in diisopropylether to

precipitate the product as a grey powder (32.9 g, 65%

6.11.1. 1,2,3-Tri-[3-(tert-butoxycarbonylamino)-propoxy]-

benzene (14a). A solution of pyrogallol (3.2 g, 25 mmol)

in DMF (20 mL) was treated sequentially at 40 ꢁC with

KOBu (9.3 g, 82 mmol) in DMF (50 mL) and 11a

(18.0 g, 75 mmol). The reaction mixture was then cooled

to 20 ꢁC and diluted with water (200 mL) and extracted

with EtOAc (200 mL). The combined extracts were

washed with 1 M aq NaOH (100 mL) then concentrated

under vacuum to give an oil, which was absorbed onto

1

yield): mp 132–134 ꢁC; H NMR (DMSO-d₆) d 1.35

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5477

silica gel and chromatographed with diisopropyl ether/

Et₂O, 1:1 to give the product, crystallised from Et₂O

(3.0 g, 20% yield); H NMR (CDCl₃) d 1.40 (s, 27H,

cipitate a red gum, which was further puriﬁed by

reprecipitation from EtOH to give the product as a yel-

low powder (80 mg, 6% yield); H NMR (DMSO-d₆) d

1

3C(CH₃)₃), 1.90 (qu, 2H, CH₂), 2.00 (qu, 4H, 2CH₂),

3.30 (m, 4H, 2CH₂N), 3.40 (m, 2H, CH₂N), 4.05 (q,

6H, 3CH₂O), 5.15 (br s, 2H, NHÆCO), 5.45 (br s, 1H,

NHÆCO), 6.55 (d, 2H, 2Ar–H), 6.95 (t, 1H, Ar–H); IR

(Nujol) t_max3280, 1700 cm^ꢀ1; MS (ES⁺) 598 (M⁺+H).

1.40 (t, 9H, 3CH₃), 2.10 (qu, 6H, 3CH₂), 3.75 (q, 6H,

3CH₂N), 4.15 (t, 6H, 3CH₂O), 4.60 (q, 6H, 3CH₂N⁺)

6.70 (d, 2H, 2Ar–H), 6.90 (m, 3H, 3Ar–H), 7.05 (t,

1H, Ar–H), 7.70 (m, 3H, 3Ar–H), 8.10 (m, 3H, 3Ar–

H), 8.50 (m, 3H, 3Ar–H), 8.65 (m, 3H, 3r-H), 9.15

(m, 3H, 3Ar–H); MS (ES⁺) 763 (M⁺ꢀ2). Anal. Calcd

for [C₄₈H₅₇N₆O₃]³⁺Æ3I^ꢀ. 4H₂O: C, 47.29; H, 5.37; N,

6.89. Found: C, 47.12; H, 5.02; N, 6.86.

6.11.2. 1,2,3-Tri-[3-(4-quinolinylamino)-propoxy]-benzene

hydrate (14). A solution of 14a (2.4 g, 4 mmol) in triﬂu-

oroacetic acid (20 mL) was stirred at 20 ꢁC for 1 h, be-

fore concentrating under vacuum. The resulting oil was

diluted with MeOH (50 mL) before basifying to pH 10

with 25% w/w NaOMe in MeOH. The solvent was then

replaced with n-pentanol (80 mL) before adding 4-chlo-

roquinoline (2.6 g, 16 mmol) and NⁱPr₂Et (2.6 g,

20 mmol). The solution was then heated under reﬂux

for 6 h, concentrated, diluted with 1 M aq NaOH

(100 mL) and extracted with CH₂Cl₂/MeOH (20:1; 2·

100 mL). The extracts were combined, concentrated un-

der vacuum, and the residue absorbed onto silica gel

and chromatographed with CMA to give an oil, which

was recrystallised from EtOAc to give the product as a

6.13. N-[3-(4-Quinolinylamino)-propyl]-N-[5-(4-quinoli-

nylamino)-pentyl]-3,5-di- [3-(4-quinolinylamino)-prop-

oxy]-benzylamine hydrate (16)

6.13.1. 5-(tert-Butoxycarbonylamino)-pentylamine (16a).

A solution of di tert-butyl dicarbonate (60 g, 275 mmol)

in MeOH (200 mL) was added to 1,5-diaminopentane

(100 g, 1.0 mol) in MeOH (100 mL) at 20 ꢁC. The result-

ing suspension was concentrated to 50% volume by dis-

tillation, then diluted with 1 M aq NaOH (1200 mL),

ﬁltered to remove di-(t-Boc)-1,5-diaminopentane and

extracted with t-butyl methyl ether (5· 500 mL). The ex-

tracts were combined and concentrated under vacuum

1

white powder (200 mg, 8% yield); H NMR (CDCl₃) d

1

2.15 (m, 6H, 3CH₂), 3.45 (m, 6H, 3CH₂N), 4.15 (t,

4H, 2CH₂O), 4.25 (t, 2H, CH₂O), 5.25 (t, 2H, 2NH),

6.20 (t, 1H, NH), 6.30 (m, 3H, 3Ar–H), 6.65 (d, 2H,

2Ar–H), 7.05 (t, 1H, Ar–H), 7.35 (m, 3H, 3Ar–H),

7.55 (m, 3H, 3Ar–H), 7.70 (m, 3H, 3Ar–H), 8.00 (m,

3H, 3Ar–H), 8.50 (m, 3H, 3Ar–H); MS (ES⁺) 679

(M⁺+H). Anal. Calcd for C₄₂H₄₂N₆O₃Æ1.25H₂OÆ0.2E-

tOAc: C, 71.50; H, 6.46; N, 11.69. Found: C, 71.49;

H, 6.24; N, 11.68.

to give the product as a red oil (35 g, 64% yield); H

NMR (CDCl₃) d 1.2 (s, 2H, NH₂), 1.30 (s, 9H,

C(CH₃)₃), 1.60 (qu, 2H, CH₂), 1.95 (qu, 4H, 2CH₂),

2.40 (m, 4H, 2CH₂N), 6.0 (br t, 1H, NHÆCO).

6.13.2. N-[(tert-Butoxycarbonylamino)-propyl]-N⁰-5-(tert-

butoxycarbonylamino)-1,5-diaminopentane (16b). A solu-

tion of 11a (35.6 g, 150 mmol) and 16a (30.3 g,

150 mmol) in DMF (200 mL) was stirred with K₂CO₃

(27 g, 450 mmol) at 20 ꢁC for 16 h, then diluted with

water (600 mL) and extracted with tert-butyl methyl

ether (2· 1 L). The extracts were combined and stirred

with water (600 mL), adding in portions approximately

80% of the theoretical amount of concd HCl needed to

neutralise the amine function in 16b until tlc monitoring

showed that the product had been extracted into the

aqueous phase. This was separated oﬀ, basiﬁed to pH

14 with 40% w/w aq NaOH and extracted with tert-butyl

methyl ether (2· 600 mL). The extracts were combined

and concentrated under vacuum to give the product as

6.12. 1,2,3-Tri-[3-(4-(N-ethyl)-quinoliniumylamino)-prop-

oxy]-benzene triiodide (15)

A solution of 14a (10 g, 16.7 mmol) in triﬂuoroacetic

acid (40 mL) was stirred at 20 ꢁC for 1 h, before concen-

trating under vacuum to low volume, diluting with

MeOH (50 mL) and basifying to pH 10 with 25% w/w

NaOMe in MeOH. The solvent was then replaced with

n-pentanol by distillation before adding 4-chloroquino-

line (11.0 g, 67.5 mmol) and NⁱPr₂Et (10.8 g,

83.5 mmol). The solution was heated under reﬂux for

6 h, before concentrating under vacuum, diluting with

1 M aq NaOH (150 mL) and extracting with CH₂Cl₂/

MeOH (10:1, 2· 100 mL). The combined extracts were

concentrated and the residue absorbed onto silica gel

and chromatographed with CMA. The crude product

14 was diluted with EtI (25 mL) and the suspension

heated to reﬂux. DMF (2 mL) was added to the result-

ing oily suspension to give a clear solution. Reﬂux was

then maintained for a further 10 h, adding more DMF,

in 1 mL portions (total 10 mL), to redissolve the liber-

ated oil, throughout the reﬂux period. The solution

was then cooled before diluting with water (100 mL)

to precipitate a gum. The supernatant liquor was re-

moved, after which more water (100 mL) was added

and the supernatant removed. The remaining residue

was stirred in MeOH (80 mL) at 60 ꢁC, ﬁltered to re-

move insoluble material and the ﬁltrate cooled to pre-

1

an orange powder (20 g, 39% yield); H NMR (CDCl₃)

d 1.4 (s, 18H, 2C(CH₃)₃), 1.60 (qu, 2H, CH₂), 1.65 (m,

2H, CH₂), 1.95 (qu, 4H, 2CH₂), 2.7 (m, 2H, CH₂N),

3.15 (m, 2H, CH₂N), 3.35 (q, 4H, 2CH₂N), 5.00 (br s,

1H, NHÆCO), 5.15 (br s, 1H, NHÆCO).

6.13.3. N-[(tert-Butoxycarbonylamino)-propyl]-N-[5-(tert-

butoxycarbonylamino)-pentyl]-3,5,-di-[(tert-butoxycar-

bonylamino)-propoxy]-benzylamine (16c). A solution of

11c (3.3 g, 7.0 mmol) and 16b (2.7 g, 7.7 mmol) in

DMF (30 mL) was heated with K₂CO₃(1.3 g, 9.5 mmol)

at 80 ꢁC for 3 h. The mixture was then cooled, diluted

with water (100 mL) and extracted with EtOAc (2·

100 mL). The extracts were combined, washed with

water (100 mL) and concentrated under vacuum to give

an oil, which was absorbed onto silica gel and chro-

matographed with EtOAc to give the product as a pale

yellow oil (5.6 g, 100% yield); ¹H NMR (CDCl₃) d 1.4 (s,

5478

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

36H, 4C(CH₃)₃), 1.45 (m, 2H, CH₂), 1.60 (qu, 2H, CH₂),

1.75 (m, 4H, 2CH₂), 1.95 (qu, 4H, 2CH₂), 2.40 (m, 4H,

2CH₂N), 3.15 (m, 2H, CH₂N), 3.35 (q, 4H, 2CH₂N),

3.45 (s, 4H, 2CH₂N), 4.00 (t, 4H, 2CH₂O), 4.75 (t, 4H,

2NHÆCO), 5.00 (br s, 1H, NHÆCO), 5.15 (br s, 1H, NHÆ-

CO), 6.30 (s, 1H, Ar–H), 6.50 (s, 2H, 2Ar–H); MS (ES⁺)

796 (M⁺+1).

20 ꢁC, diluted with water (400 mL) and extracted with

EtOAc (3· 300 mL). The combined extracts were

washed with 1 M aq NaOH (200 mL) and concentrated

under vacuum. The residue was absorbed onto silica gel

and chromatographed with Et₂O to give the product as

1

a brown oil (7.0 g, 45%); H NMR (CDCl₃) d 1.40 (s,

38H, 4C(CH₃)₃+ CH₂), 1.65 (m, 4H, 2CH₂), 1.75 (m,

2H, CH₂), 1.95 (m, 4H, 2CH₂), 3.15 (m, 4H, 2CH₂N),

3.35 (m, 8H, 4CH₂N), 4.00 (m, 4H, 2CH₂O), 4.75 (br

s, 1H, NHÆCO), 4.85 (br s, 2H, NHÆCO), 4.95 (br s,

1H, NHÆCO), 5.85 (s, 2H, 2Ar–H), 6.05 (s, 1H, Ar–H);

6.13.4. N-[3-(4-Quinolinylamino)-propyl]-N-[5-(4-quinoli-

nylamino)-pentyl]-3,5-di-[3-(4-quinolinylamino)-propoxy]-

benzylamine hydrate (16). Compound 16c (4.8 g,

6.0 mmol) was stirred in triﬂuoroacetic acid (40 mL) at

20 ꢁC for 1 h, then concentrated under vacuum to low

volume. The resulting oil was dissolved in MeOH

(50 mL) and 25% w/w NaOMe in MeOH added until

pH 10, before replacing the solvent with n-pentanol

by distillation. 4-Chloroquinoline (4.9 g, 30 mmol) and

NⁱPr₂Et (4.5 g, 35 mmol) were added and the solution

heated under reﬂux for 8 h, before concentrating under

vacuum to low volume, diluting with 1M aq NaOH

(100 mL) and extracting with CH₂Cl₂(2· 100 mL). The

extracts were combined and concentrated to give an

oil, which was absorbed onto silica gel and chromato-

graphed with CMA. The resulting crude product was

reprecipitated twice from EtOAc, ﬁltering the hot super-

natant each time, to give a cream foam (100 mg, 2%

IR (ﬁlm) m_max3380, 1700, 1620 cm^ꢀ1

.

6.14.3. N-[3-(4-Quinolinylamino)-propyl]-N-[5-(4-quinoli-

nylamino)-pentyl]-3,5-di-[3-(4-quinolinylamino)-propoxy]-

aniline trihydrate (17). Compound 17b (5.9 g, 7.5 mmol)

was stirred in triﬂuoroacetic acid (30 mL) at 20 ꢁC for

1 h, then concentrated under vacuum. The resulting oil

was dissolved in MeOH (100 mL), 25% w/w NaOMe

in MeOH added until pH 10, before replacing the sol-

vent with n-pentanol by distillation. 4-Chloroquinoline

(7.3 g, 45 mmol) and NⁱPr₂Et (6.8 g, 53 mmol) were

added and the solution heated under reﬂux for 6 h, be-

fore concentrating to low volume, diluting with water

(150 mL) and extracting with CH₂Cl₂/MeOH (20:1, 2·

100 mL). The extracts were combined, concentrated,

and the residue absorbed onto silica gel and chromato-

graphed with CMA. The resulting crude product was

puriﬁed by precipitation twice from EtOAc, ﬁltering

the hot supernatant each time, to give a fawn foam

1

yield); H NMR (CDCl₃) d 1.45 (m, 2H, CH₂), 1.65

(m, 4H, 2CH₂), 1.85 (m, 2H, CH₂), 2.00 (m, 4H,

2CH₂), 2.60 (m, 4H, 2CH₂N), 3.25 (m, 2H, CH₂N),

3.35 (m, 4H, 2CH₂N), 3.55 (m, 4H, 2CH₂N), 3.80 (m,

4H, 2CH₂O), 5.10 (t, 1H, NH), 5.65 (t, 2H, NH), 6.25

(t, 1H, Ar–H), 6.35 (m, 4H, 4Ar–H), 6.45 (s, 2H, 2Ar–

H), 6.85 (t, 1H, NH), 7.35 (m, 4H, 4Ar–H), 7.55 (m,

4H, 4Ar–H), 7.75 (m, 4H, 4Ar–H), 7.95 (m, 4H, 4Ar–

H), 8.50 (m, 4H, 4Ar–H); TLC CMA/silica gel, r.f 0.3;

MS (ES⁺) 905 (M⁺+1). Anal. Calcd for

C₅₇H₆₁N₉O₂Æ1.75H₂OÆ0.66EtOAc: C, 72.08; H, 7.08;

N, 12.68. Found: C, 71.98; H, 6.91; N, 12.69.

1

(200 mg, 3%); H NMR (CDCl₃) d 1.35 (m, 2H, CH₂),

1.65 (m, 4H, 2CH₂), 2.10 (m, 6H, CH₂), 3.3–3.5 (m,

12H, 6CH₂N), 3.95 (t, 4H, 2CH₂O), 5.90 (s, 3H, 3Ar–

H), 6.40 (m, 4H, 4Ar–H), 7.35 (m, 4H, 4Ar–H), 7.60

(m, 4H, 4Ar–H), 7.75 (m, 4H, 4Ar–H), 7.95 (m, 4H,

4Ar–H), 8.55 (m, 4H, 4Ar–H); MS (EI⁺) 890 (M⁺+1).

Anal. Calcd for C₅₆H₅₉N₉O₂Æ3.5H₂OÆ0.5EtOAc: C,

68.98; H, 7.07; N, 12.63. Found: C, 68.99; H, 6.77; N,

12.50.

6.14. N-[3-(4-Quinolinylamino)-propyl]-N-[5-(4-quinolin-

ylamino)-pentyl]-3,5-di-[3-(4-quinolinylamino)-propoxy]-

aniline trihydrate (17)

6.15. Biological assay

6.14.1. N-[3-(tert-Butoxycarbonylamino)-propyl]-N⁰-[5-

(tert-butoxycarbonylamino)-pentyl]-3,5-dihydroxyaniline

(17a). Phloroglucinol (4.4 g, 35 mmol) and 16b (12.0 g,

35 mmol) were heated in a mixture of THF (30 mL) and

toluene (50 mL) under reﬂux for 2 h. The solution was

then concentrated under vacuum to give an oil, which

was absorbed onto silica gel and chromatographed with

EtOAc, to give the product as an orange gum (10 g,

61% yield); ¹H NMR (DMSO-d₆) d 1.40 (s, 18H,

2C(CH₃)₃), 1.45 (qu, 2H, CH₂), 1.65 (qu, 4H, 2CH₂),

1.75 (m, 2H, CH₂), 2.90 (m, 4H, 2CH₂N), 3.15 (m, 4H,

2CH₂N), 5.55 (s, 1H, Ar–H), 5.65 (s, 2H, Ar–H), 6.68

(t, 1H, NHÆCO), 6.85 (t, 1H, NHÆCO), 8.80 (s, 2H, 2OH).

Intracellular recordings were made, using microelec-

trodes ﬁlled with 1 M KCl, from rat sympathetic neuro-

nes incubated in tissue culture for 6–10 days. Action

potentials were invoked by injection of depolarizing cur-

rent pulses, and test compounds were applied in the

external solution at 2–4 concentrations, on at least three

neurones.

Equieﬀective molar concentration ratios (EMR with

respect to dequalinium) were determined by simulta-

neous non-linear weighted least squares ﬁtting (using

Origin (versions 4–7), OriginLab, Northampton, Mas-

sachusetts, USA) of the data obtained with each

compound, taken together with the values observed

with dequalinium in that set of assays. The Hill

equation was employed to ﬁt the data: a common

Hill coeﬃcient and maximum inhibition were as-

sumed for each assay; IC₅₀values and the concentra-

tion required to achieve 50% inhibition of the AHP

were calculated.

6.14.2. N-[(tert-Butoxycarbonylamino)-propyl]-N-[5-(tert-

butoxycarbonylamino)-pentyl]-3,5-[3-(tert-butoxycarbon-

ylamino)-propoxy]-aniline (17b). Compound 17a (9.4 g,

20 mmol) was sequentially treated in DMF (100 mL)

with KOBu (4.9 g, 44 mmol) at 30 ꢁC and 11a (10.0 g,

42 mmol) at 60 ꢁC. The solution was then cooled to

D. I. Fletcher et al. / Bioorg. Med. Chem. 15 (2007) 5457–5479

5479

20. Castle, N. A.; Haylett, D. G.; Morgan, J. M.; Jenkinson,

D. H. Eur. J. Pharmacol. 1993, 236, 201.

Acknowledgments

21. Galanakis, D.; Davis, C. A.; Del Rey Herrero, B.;

Ganellin, C. R.; Dunn, P. M.; Jenkinson, D. H. J. Med.

Chem. 1995, 38, 595.

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Med. Chem. 2000, 43, 420.

This work was partially supported by the Wellcome

Trust which provided a fellowship for P.M.D. D.F. is

grateful to Wyeth Research, UK, for supporting his re-

search and providing facilities for chemical synthesis,

spectra, and chemical analysis. Thanks are also due to

Dr. K. Heatherington for help with obtaining and inter-

preting the analytical and spectroscopic data. We also

´

thank Vicky Taylor (nee Richardson) for building the

24. Chen, J.Q. Galanakis, D.; Ganellin, C. R.; Dunn, P. M.;

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Article Doi

Synthesis and pharmacological testing of polyaminoquinolines as blockers of the apamin-sensitive Ca2+-activated K+ channel (SKCa)

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