Structure-activity relationships of methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists. 2. Role of polymethylene chain lengths separating amine functions and of substituents on the terminal nitrogen atoms

1860

J . Med. Chem. 2002, 45, 1860-1878

Str u ctu r e-Activity Rela tion sh ip s of Meth octr a m in e-Rela ted P olya m in es a s

Mu scu la r Nicotin ic Recep tor Non com p etitive An ta gon ists. 2.¹Role of

P olym eth ylen e Ch a in Len gth s Sep a r a tin g Am in e F u n ction s a n d of Su bstitu en ts

on th e Ter m in a l Nitr ogen Atom s

Michela Rosini,^†M. Gabriele Bixel,^‡Gabriella Marucci,^§Roberta Budriesi,^†Michael Krauss,^‡

Maria L. Bolognesi,^†Anna Minarini,^†Vincenzo Tumiatti,^†Ferdinand Hucho,^‡and Carlo Melchiorre*^,†

Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy,

Institut fu¨r Chemie-Biochemie, Freie Universita¨t Berlin, Thielallee 63, 14195 Berlin, Germany, and

Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy

Received October 19, 2001

Polymethylene tetraamine methoctramine (1) is a prototypical antimuscarinic ligand endowed

with a significant affinity for muscular nAChRs. Thus, according to the universal template

approach, structural modifications were performed on 1 in order to improve affinity and

selectivity for the muscle-type nAChR. The polyamine derivatives synthesized were tested at

both frog rectus and Torpedo nAChRs and at guinea pig left atria (M₂) and ileum longitudinal

muscle (M₃) mAChRs. All of the compounds, like prototype 1, were noncompetitive antagonists

of nicotinic receptors while being competitive antagonists at M₂and M₃mAChRs. The biological

profile of polyamines 4-7 revealed that increasing the number of amine functions and the

chain length separating these nitrogen atoms led to a significant improvement in potency at

nAChRs. Moreover, the role of the number and type of amine functions in the interaction with

nAChRs was further investigated through the synthesis of compounds 9 and 10. Tetraamines

8 and 11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible

polymethylene chain, displayed a profile similar to that of 1 at nAChRs, whereas a significant

decrease in potency was observed at mAChRs. Tetraamine 12, bearing a 2-methoxyphenethyl

group, was less potent than 1, whereas tetraamine 13, carrying a diphenylethyl moiety, was

more potent than 1, confirming that an increase in size of the hydrophobic group on the terminal

nitrogen atoms increases significantly the binding affinity for nAChRs. Tetraamines 14-17

were significantly more potent than prototype 2 at both frog rectus and Torpedo nAChRs,

confirming that an increase in the distance between the amine functions results in a parallel

increase in the affinity for nAChRs. To gain insight into the mode of interaction of polymethylene

tetraamines with nAChRs, photolabile (19 and 20) and fluorescent (21 and 22) compounds

were synthesized. A most intriguing finding was the observation that 19, which bears two

identical azido groups on the terminal nitrogen atoms, was found to bind the Torpedo nAChR

with a 1:1 stoichiometry, suggesting a U-shaped conformation in the receptor interaction.

Moreover, the high potency shown by fluorescent compounds 21 and 22 appears promising for

a further characterization of the polymethylene tetraamines binding site with the muscle-

type nAChR.

In tr od u ction

The nAChR, a prototypical member of the superfamily

of ligand-gated ion channels, is a heteropentameric

transmembrane protein with the subunit stoichiometry

R₂âγδ.^6-8The five receptor subunits are arranged

around a central pore, which is permeable for cations

upon agonist binding. Each subunit contains four trans-

membrane sequences M1-M4. The M2 sequences of all

subunits contribute structurally to the formation of the

ion pore, thereby facing the lumen of the channel.^9,10

The selectivity filter for cations is formed by several

rings of negatively charged amino acid side chains

protruding into the lumen of the pore.^11-13Multiple

ligand binding domains have been characterized on the

nAChR. Two binding sites for agonists and competitive

antagonists are located in the extracellular region,

mainly on each of the two R-subunits at the R-δ and

R-γ interfaces,^14,15whereas several binding sites for

noncompetitive antagonists, such as triphenylmeth-

Recently, we have modified the structure of methoc-

tramine (1, Figure 1), the prototype polymethylene

tetraamine for antagonism of muscarinic acetylcholine

receptors (mAChRs),²to produce polyamines that have

high affinity and selectivity for the muscle-type nicotinic

acetylcholine receptor (nAChR).^1,3,4The rationale for

these structural modifications was based on the obser-

vation that 1, at micromolar concentrations, antagonizes

nAChRs, hence providing the prospect of applying the

universal template approach⁵to the design of polyamines

as nAChR ligands.¹

* To whom correspondence should be addressed. Phone:

+39-051-2099706. Fax: +39-051-2099734. E-mail: camelch@

kaiser.alma.unibo.it.

^†University of Bologna.

^‡Freie Universita¨t Berlin.

^§University of Camerino.

Published on Web 03/21/2002

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1861

F igu r e 1. Design strategy for the synthesis of 4-8, 11, 14-17, and 19-22 by modifying the polymethylene tetraamine backbone

of methoctramine (1) and by inserting selected aryl substituents of the structural features of philanthotoxin PhTX-343 (2) on the

terminal nitrogen atoms of 1-related compounds.

ylphosphonium bromide (TPMP⁺), have been found

within the ion channel close to the M2 transmembrane

domain.^16,17Luminal noncompetitive antagonists are

assumed to enter the open channel and to bind deep in

the ion channel close to the negatively charged selectiv-

ity filter, thereby inhibiting the ion flow.¹⁸Therefore,

it may well be that the positively charged backbone of

tetraamines is able to interact with the negatively

charged amino acid residues located at the constriction

of the channel and on the extracellular side of this

constriction as well, provided that the distance separat-

ing the amine functions of the ligand fits the distance

between the carboxylate residues of the receptor.

Increasing the number of methylenes separating the

inner nitrogen atoms of 1 resulted in a significant

improvement in potency at the nAChR while decreasing

the affinity for the M₂mAChR. Furthermore, the

tetraamine backbone of 1 was modified by replacing the

terminal 2-methoxybenzyl groups by other pharma-

cophores with the aim of achieving selectivity for the

muscle-type nAChR.¹To this end, we choose the bu-

tyryltyrosyl moiety of philanthotoxin-343 (PhTX-343) (2,

1862 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

Figure 1), a synthetic analogue of a wasp (Philanthus

triangulum) toxin PhTX-433,¹⁹since it is known that

an aromatic moiety at one end and a primary amine

function at the other end of the molecule are important

for philanthotoxin binding at the Torpedo nAChR.^20,21

By combining the structural features of tetraamine 1

and philanthotoxin 2, we designed novel inhibitors of

the muscle-type nAChR. These novel polyamines were

potent noncompetitive antagonists of the nAChR while

being competitive mAChR antagonists. The promising

results obtained following these structural modifications

prompted us to modify further the structure of 1 to

achieve additional information on the structural re-

quirements that allow differentiation between muscle-

type nAChRs and mAChRs.

site. In direct binding studies, a binding stoichiometry

of two molecules of 18 per nAChR monomer was

determined, suggesting that each of the aromatic moi-

eties of the two 18 molecules interact with one of the

two R-subunits.⁴Thus, the symmetrically substituted

photolabile tetraamines 19 and 20 were designed to

verify whether these compounds interact with the

receptor’s R-subunit with only one of the two hydropho-

bic moieties or with both of them. To this end, compound

19 was selected for iodination with ¹²⁵I at both aromatic

rings, generating ¹²⁵I₂-19, since this compound was

slightly more potent in binding assays than its higher

homologue 20. Finally, to gain insight into the mode of

interaction of polymethylene tetraamines with the

nAChR, we have designed the fluorescent analogues 21

and 22.

The finding that the higher homologue 3 of 1 was 12-

fold and 250-fold more potent than prototypes 1 and 2,

respectively, suggests clearly that the length of the

spacer between the inner nitrogen atoms is important

for potency at both nAChRs and mAChRs.¹To improve

hopefully the selectivity, we have designed polyamines

4 and 5, in which the total number of methylenes has

been preserved with respect to 3 while inserting ad-

ditional amine functions, and hexaamines 6 and 7, in

which additional nitrogen atoms have been included at

the extremities of the polyamine backbone of prototypes

1 and 3, respectively. These structural modifications

might give an indication of the number of protonable

nitrogen atoms for optimal interaction with the nAChR

ion channel. To investigate further the role of the

number and type of amine functions for potency at

nAChRs, dioxadiamine 9²²and N,N′,N′′,N′′′-tetrameth-

yltetraamine 10²²have been included in this study.

Tetraamines 8, which carries a (4′′-aminomethyl-

[1,1′;4′,1′′]terphenyl-4-yl)methylamine fragment instead

of a 1,12-dodecanediamine residue, and 11, which bears

a p-xylene moiety instead of a hexamethylene chain

between the inner and outer nitrogen atoms of 3, may

contribute in determining whether flexibility is an

important determinant of potency with respect to nAChR

antagonism. Tetraamines 12³⁵and 13³⁵were also

investigated to verify whether the terminal 2-methoxy-

benzyl groups in symmetrically substituted methoctra-

mine-related tetraamines are relevant for potency at the

nAChR.

All of the compounds synthesized in this study were

tested on muscle-type nAChRs, and most of them were

tested as well on peripheral M₂and M₃mAChRs.

Ch em istr y

The design strategy for our compounds is shown

schematically in Figure 1.

All the compounds were synthesized according to

standard procedures (Schemes 1-10) and were charac-

1

terized by IR, H NMR, mass spectra, and elemental

analysis.

N-[(Benzyloxy)carbonyl]-6-aminocaproic acid was ami-

dated with N-[(benzyloxy)carbonyl]-1,6-hexanediamine¹

to give 23. Removal of the N-(benzyloxy)carbonyl group

was achieved by catalytic hydrogenation over 10%

palladium on charcoal. Thus, hydrogenolysis of 23 gave

24,²³which was reacted with N-[(benzyloxy)carbonyl]-

6-aminocaproic acid to afford 25. Removal of the N-

(benzyloxy)carbonyl group of 25 by hydrolysis with HBr

gave 26, which was treated with 2-methoxybenzalde-

hyde followed by reduction with NaBH₄of the formed

Schiff base to diaminetriamide 27, which in turn was

reduced with borane to 4 (Scheme 1).

N-[(Benzyloxy)carbonyl]-6-aminocaproic acid was ami-

dated with N,N′-dibenzylhexane-1,6-diamine²⁴to give

28. Removal of the N-(benzyloxy)carbonyl group of 28

by hydrolysis with HBr gave diaminediamide 29, which

was reduced with borane to tetraamine 30. The addition

of acrylonitrile to 30 gave a mixture of 31 and 32 that

were separated by chromatography. Dinitrile 32 was

reduced with Raney Ni into the corresponding amine

33. Hydrogenolysis of 33 gave 34, which was treated

with 2-methoxybenzaldehyde followed by reduction with

NaBH₄of the formed Schiff base to hexaamine 5

(Scheme 2).

Selective protection of amine functions of 35²and 36²

was achieved by following an adapted procedure de-

scribed for spermine.²⁵Thus, tetraamines 35 and 36

were treated first with ethyl trifluoroacetate and then

with di-tert-butyl dicarbonate followed by basic hydroly-

sis to give 37 and 39, and 38 and 40, respectively. The

addition of acrylonitrile to 39 and 40 gave 41 and 42,

respectively, which were transformed into the corre-

sponding 43 and 44. These nitriles were reduced with

Raney Ni into 45 and 46, which were treated with

2-methoxybenzaldehyde followed by reduction with

NaBH₄of the formed Schiff base followed by acidic

hydrolysis to give hexaamines 6 and 7 (Scheme 3).

Unsymmetrical hybrid polyamines 14-17, bearing

the structural features of prototypes 1 and 2, were

designed to define whether the chain length between

the nitrogen atoms plays a similar role in asymmetri-

cally and symmetrically substituted polyamines. Inter-

estingly, using fluorescent titration and photo-cross-

linking, we have shown recently that the asymmetrically

substituted photolabile tetraamine 18 binds in the

vestibule, that is, in the wide water-filled entrance of

the pore, located above the narrow part of the channel

and of the selectivity filter and the gating mechanism

of the nAChR.^3,4The terminal aromatic moiety that

carries the photolabile azido group was found to pho-

tolabel the hydrophobic sequence R186-R189 of the

R-subunit. This sequence is located in the upper part of

the ion channel close to the agonist binding site.

Furthermore, the long, positively charged polyamine tail

of 18 overlaps in the narrow part of the nAChR ion

channel with the high-affinity noncompetitive inhibitor

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1863

Sch em e 1^a

a

Cbz ) C₆H₅CH₂OCO-.

Sch em e 2^a

a

Cbz ) C₆H₅CH₂OCO-; Bn ) C₆H₅CH₂-.

Tetraamine 8 was obtained through the condensation

of dialdehyde 47²⁶with diamine 48²⁷followed by reduc-

tion with NaBH₄of the intermediate Schiff base (Scheme

4). It was obtained in poor yields because of its very low

solubility.

addition of acrylonitrile to 38 gave 58, which in turn

was transformed into 59. Reduction of 59 with Raney

Ni afforded 60, which in turn was coupled to 55,²⁸

yielding 61. Removal of the N-(tert-butoxy)carbonyl

groups of 61 by hydrolysis with CF₃COOH gave 62,

which, upon catalytic hydrogenation, afforded 16. The

2-methoxybenzyl group on the terminal nitrogen of 16

was introduced as for 15, giving 17 (Scheme 7).

1,12-Dodecanedioic acid was amidated with amine 49¹

to give 50, which in turn was treated with HBr to afford

diaminediamide 51. Condensation of 51 with 2-meth-

oxybenzaldehyde in the presence of NaBH₄gave 52,

which was reduced with borane to 11 (Scheme 5).

Amine functions of nitrile 31 were protected with di-

tert-butyl dicarbonate to give 53, which was reduced

with Raney Ni to 54. Coupling 54 with the activated

ester 55²⁸afforded 56. Removal of the N-(tert-butoxy)-

carbonyl group of 56 by acidic hydrolysis gave 57, which,

upon catalytic hydrogenation, afforded hybrid poly-

amineamide 14. The introduction of a 2-methoxybenzyl

moiety on the terminal amine function of 14 gave 15

(Scheme 6). The higher homologues 16 and 17 were

synthesized by a different synthetic pathway. The

Symmetrically substituted analogues 19 and 20 of the

photolabile tetraamine 18¹were synthesized as shown

in Scheme 8. Thus, azidosalicilic acid N-hydroxysuccin-

imide ester²⁹was amidated with 45 and 46 to give 63

or 64, which, upon treatment with CF₃COOH to remove

the protecting groups, afforded the photolabile com-

pounds 19 and 20, respectively.

Nitrile 65¹was treated with di-tert-butyl dicarbonate

to give 66, which, upon reduction with Raney Ni,

afforded 67. Amidation of 2,5-dioxotetrahydro-1H-pyr-

rolyl-7-methoxy-2-oxo-2H-3-chromene carboxylate with

1864 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

Sch em e 3^a

a

Boc ) Me₃COCO-.

Sch em e 4

Sch em e 5^a

a

Cbz ) C₆H₅CH₂OCO-.

67 gave 68, which, following acidic hydrolysis with CF₃-

COOH, afforded the fluorescent compound 21 (Scheme

9).

A different route was used to synthesize the iodinated

analogue 22 of 21. Addition of acrylonitrile to 37 gave

69, which was reduced with Raney Ni to 70. Treatment

with ethyl trifluoroacetate afforded the protection of the

primary amine function of 70, giving 71. Subsequent

acidic hydrolysis with CF₃COOH of 71 gave the mono-

protected pentaamine 72, which could be selectively

alkylated on the other free terminal amine function

through condensation with 2-methoxy-5-iodo-benzalde-

hyde³⁰and reduction with NaBH₄to afford 73. Treat-

ment of 73 with di-tert-butyl dicarbonate gave 74, which,

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1865

Sch em e 6^a

a

Boc ) Me₃COCO-; Bn ) C₆H₅CH₂-.

Sch em e 7^a

a

Boc ) Me₃COCO-; Bn ) C₆H₅CH₂-.

upon basic hydrolysis with aqueous ammonia, afforded

75, whose free terminal amine function was allowed to

react with 2,5-dioxotetrahydro-1H-pyrrolyl-7-methoxy-

2-oxo-2H-3-chromene carboxylate, giving 76, which,

following acidic hydrolysis with CF₃COOH to remove

the protecting groups, yielded the unsymmetrically

substituted tetraamine 22 (Scheme 10).

method (Scheme 8).³¹However, it was not ascertained

whether the iodine atom entered at position 3 or 5 of

each terminal phenyl ring.

Biology

The effects of compounds 1-22 on M₂mAChRs were

determined using guinea pig left atria stimulated

electrically at 1 Hz. The guinea pig ileum longitudinal

muscle was used to study their effects on M₃mAChRs.¹

Photolabile compound 19 was radioactively iodinated

with ¹²⁵I to afford ¹²⁵I₂-19, using the chloramine T

1866 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

Sch em e 8^a

a

Boc ) Me₃COCO-.

Sch em e 9^a

a

Boc ) Me₃COCO-.

In both cases the agonist was arecaidine propargyl ester

(APE). The biological data are expressed as the negative

logarithm of the apparent dissociation constants (pK_B)

according to Furchgott.³²

The effects of compounds 1-22 on muscle-type

nAChRs were studied using the frog rectus abdominis

muscle and carbachol-induced contractions as the mea-

sured parameter.³³The results are expressed as pIC₅₀

values, i.e., the negative logarithm of the concentrations

required to inhibit the maximal response to carbachol

by 50%.

displacement assay.³Binding of the radioactively la-

beled polyamine ¹²⁵I₂-19 to the Torpedo nAChR was also

investigated.⁴

Polyamines 1 and 2 and the noncompetitive nAChR

antagonist TPMP⁺were used as standards, and their

pIC₅₀, pK_app, or pK_Bvalues were within the error of

previous determinations.

Resu lts a n d Discu ssion

Summaries of the results are presented in Table 1

and Figures 2 and 3. Over the concentration range

investigated, all of the newly synthesized compounds

were noncompetitive antagonists of the muscle-type

nAChR. The maximum response to carbachol was

reduced, and the magnitude of this reduction was

Apparent binding affinities (pK_app) of compounds 1-3,

5-8, 11, and 14-22 at the noncompetitive binding site

of Torpedo nAChRs were determined by using the

fluorescent noncompetitive inhibitor ethidium in a

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1867

Sch em e 10^a

a

Boc ) Me₃COCO-.

dependent on the concentration of antagonist. In all

cases, washing the muscle in drug-free saline reversed

the antagonism. To determine the binding affinities of

the various polyamine derivatives at nAChRs, we have

used the well-characterized luminal noncompetitive

inhibitor ethidium in a fluorescent displacement assay.³

Ethidium, when bound to the high-affinity site for

noncompetitive antagonists of the nAChR in its desen-

sitized state, shows an intensive emission maximum at

590 nM upon excitation at 480 nm.^3,34Since bound

ethidium is displaceable by well-characterized luminal

noncompetitive antagonists, ethidium can be used as a

reference fluorophor to characterize new ligands of this

binding site. It turned out that the polyamine deriva-

tives investigated in this study compete with bound

ethidium, indicating that these compounds also overlap

with the high-affinity binding site for noncompetitive

antagonists. This observation is in agreement with the

noncompetitive mechanism of action observed at the

frog rectus muscle nAChR. Interestingly, the analysis

of the results reported in Table 1 reveals that the pIC₅₀

values obtained at frog rectus nAChRs are comparable

with pK_appvalues calculated at Torpedo nAChRs, since

the difference was within (0.5 log units with the

exception of 2, 3, 14-16, and 18, whose pIC₅₀and pK_app

values differed to a larger extent. In contrast to the data

for nAChRs, compounds 1-15, 18, 21, and 22 competi-

tively antagonized mAChRs, as revealed by the paral-

lelism of the dose-response data for APE obtained in

the presence and absence of the compounds.

Taking 1-3 as reference compounds, it can be ob-

served how their potency at nAChRs and mAChRs can

be modified by introducing structural modifications.

Replacing the two inner amine functions of 1 with two

oxygen atoms, giving 9,²²resulted in a significant

decrease in the potency for nAChRs and mAChRs,

suggesting that the inner amine functions may play a

similar role at both nAChRs and mAChRs. The methy-

lation of the four secondary amines of 1, affording 10,²⁷

did not modify the potency at nAChRs while decreasing

markedly the potency at mAChRs. Replacing the 2-meth-

oxybenzyl groups of 1 with 2-methoxyphenethyl func-

tions, giving 12,³⁵caused a decrease in the potency for

all receptors tested, whereas the replacement with a

diphenylethyl moiety, affording 13,³⁵increased the

potency at nAChRs. This finding is in agreement with

the observation that an increase in size of the hydro-

1868 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

Ta ble 1. Antagonist Affinities, Expressed as pIC₅₀or pK_BValues, at Nicotinic Acetylcholine Receptors (nAChRs) of the Isolated Frog

Rectus Abdominis Muscle (FRA) and Torpedo and at Muscarinic Acetylcholine Receptors (mAChRs) of Guinea Pig Left Atrium (M₂)

and Longitudinal Ileum (M₃)

nAChR

pK_app(Torpedo)

mAChR

no.^a

R₁

R₂

X

n

pIC₅₀(FRA)

pK_B(M₂)

7.91 ( 0.03^b

pK_B(M₃)

1

2

3

4

5

6

7

8

a

8

5.93 ( 0.03^b

4.62 ( 0.03^b

7.02 ( 0.04^b

6.68 ( 0.06

6.42 ( 0.04

6.44 ( 0.09

6.66 ( 0.02

6.30 ( 0.02

5.14 ( 0.04

5.88 ( 0.08

6.43 ( 0.02

5.36 ( 0.06

6.31 ( 0.11

5.62 ( 0.07

5.48 ( 0.11

6.30 ( 0.02

6.27 ( 0.01

6.39 ( 0.01^b

5.80 ( 0.02

6.17 ( 0.07

6.21 ( 0.04

7.11 ( 0.05

6.05 ( 0.09

6.21 ( 0.10

3.94^c

5.66 ( 0.05

6.14 ( 0.06^b

<5^b

a

d

a

e

d

12

6

8

12

7.35 ( 0.09^b

6.98 ( 0.05^b

6.15 ( 0.01

5.98 ( 0.07^b

5.30 ( 0.11^b

6.40 ( 0.09

6.66 ( 0.11

7.04 ( 0.09

6.35 ( 0.06

<5

<6

6.50 ( 0.27

<5

<5^d

6.79 ( 0.11^e

<5

6.64 ( 0.10^f

5.98 ( 0.15^f

<5

<6

<5

<5^d

<5^e

<5

9

a

H

CH₃

O

10

11

12

13

14

15

16

17

18

19

20

21

22

NCH₃

6.19 ( 0.07

c

f

i

H

a

H

a

H

g

NH

5.32 ( 0.06^f

5.40 ( 0.20^f

5.16 ( 0.08

5.08 ( 0.03

6

12

8

12

8

4.63 ( 0.08

6.12 ( 0.03

5.28 ( 0.06

5.89 ( 0.07

5.46 ( 0.06

5.86 ( 0.06

5.72 ( 0.02

6.41 ( 0.05

6.80 ( 0.07

5.70 ( 0.12

5.87 ( 0.08

i

g

h

7.01 ( 0.09^b

7.35 ( 0.03^b

a

b

7.77 ( 0.11

6.88 ( 0.13

6.19 ( 0.08

5.68 ( 0.23

8

TPMP^{+ g}

a

1, 3, 8, 10-13, 15, 17, tetrahydrochlorides; 2, trihydrochloride; 4, pentahydrochloride; 5-7, hexahydrochlorides; 9, dihydrochloride;

14, 16, 18-22, tetratrifluoroacetates. Data from ref 1. ^cData from ref 3. Data from ref 22. ^eData from ref 27. ^fData from ref 35.

Triphenylmethylphosphonium bromide.

b

d

g

phobic headgroup of 2-related compounds increased

significantly the binding affinity for nAChRs.³

5 retained most of the potency at nAChRs, being only

2-fold and 4-fold less potent than prototype 3, respec-

tively. This finding clearly suggests that additional

protonable nitrogen atoms did not alter significantly the

binding with the receptor and might well interact with

negatively charged or aromatic amino acid residues by

way of formation of cation-anion or cation-π interac-

tions. It derives that an appropriate modification of the

chain length separating the nitrogen atoms might

improve the potency for nAChRs. To this end, we have

studied hexaamines 6 and 7, bearing 8 and 12 methyl-

enes between the inner nitrogen atoms. Again, when

the length of the spacer between the inner nitrogen

atoms was increased, there was an increase in potency

Very recently,¹we have demonstrated that increasing

the number of methylenes separating the inner nitrogen

atoms of 1 resulted in a significant increase of potency

at nAChRs, the most potent homologue being 3. How-

ever, when the fluorescent noncompetitive antagonist

ethidium was used in a displacement assay, 3 was found

to be 23-fold less potent than in functional assays (see

below). Keeping constant the two 2-methoxybenzyl

groups on the terminal amine functions and the total

number of methylenes of 3, we investigated 4 and 5,

bearing one and two additional nitrogen atoms, respec-

tively. It turned out that pentaamine 4 and hexaamine

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1869

and outer nitrogen atoms and of the octamethylene

chain between the inner nitrogen atoms of prototype 1

did not influence activity at the nAChR and the M₃

mAChR, while decreasing markedly the potency at the

M₂mAChR.¹Tetraamine 8 carries a (4′′-aminomethyl-

[1,1′;4′,1′′]terphenyl-4-yl)methylamine function instead

of the 1,12-dodecanediamine fragment of 3, whereas

tetraamine 11 contains a p-xylene moiety instead of a

hexamethylene spacer between the inner and outer

nitrogen atoms of 3. Unlike 3, compound 8 showed the

same potency at both frog rectus and Torpedo nAChRs

and was 5-fold less potent than 3 at the frog rectus

nAChR and 5-fold more potent than 3 at the Torpedo

nAChR. Furthermore, 8, unlike 3, was devoid of activity

at both M₂and M₃mAChRs. This finding may have

relevance because the replacement of the polymethylene

chain between the inner nitrogen atoms of tetraamines

with a rigid spacer is highly detrimental for the interac-

tion with mAChRs while not affecting the affinity for

nAChRs. Moreover, in 8, the inner nitrogen atoms are

unlikely to be less than 15 Å apart because, owing to

the rigidity of the terphenyl moiety, the only possibility

of altering the distance between the two inner amine

functions is restricted to the rotation along the axis of

the two bonds between the inner nitrogen atoms and

the 4′′,4-carbon atoms. It follows that this distance is

important for the interaction with two anionic sites of

the channel located most likely at a distance comparable

with that between the inner nitrogen atoms of 8.

F igu r e 2. (a) Binding of ¹²⁵I₂-19 to the Torpedo nicotinic

receptors. Nicotinic receptor-rich membranes (0.3 mg/mL

protein) were incubated with increasing concentrations of ¹²⁵I₂-

19. The specific binding of ¹²⁵I₂-19 was determined by sub-

tracting the binding in the presence of a 100-fold molar excess

of I₂-19 from the total binding. Each value is the mean ( SD

of three separate experiments. (b) Scatchard plot of ¹²⁵I₂-19

binding to Torpedo nicotinic receptors. The K_appvalue was 1.0

( 0.2 µM. When the R-BTX binding assay was used to

determine the number of receptor monomers per microgram

of protein, the binding stoichiometry of ¹²⁵I₂-19 was calculated

to be 1 mole of ¹²⁵I₂-19 per mole of nicotinic receptor monomer.

Interestingly, compound 11 displayed a biological

profile at nAChRs and mAChRs similar to that observed

for 8. This finding clearly suggests that also an ap-

propriate rigid spacer between the inner and outer

nitrogen atoms may allow significant differentiation

between muscle-type nAChRs and mAChRs.

The results obtained for hybrid tetraamines 14-17

deserve comment. All of the compounds were signifi-

cantly more potent than prototype 2 at both frog rectus

and Torpedo nAChRs, confirming that an increase in

the distance between the amine functions results in a

parallel increase in the affinity for nAChRs. However,

the higher homologues 14 and 16 were (like 2 but to a

greater extent) more potent at frog rectus versus

Torpedo nAChRs. The insertion of a 2-methoxybenzyl

group on the terminal primary amine function of 14 and

16, affording 15 and 17, respectively, caused a slight

decrease in potency for the frog rectus nAChR and a

significant increase in affinity for the Torpedo nAChR.

The increase was more pronounced for 15, which,

contrary to the higher homologue 17, was more potent

(4-fold) at the Torpedo nAChR relative to the frog rectus

nAChR. This finding and the results obtained for 3

clearly suggest that there are likely subtle differences

in the mode of interaction of polyamines with the ion

channel of frog rectus and Torpedo nAChRs. We do not

know at present whether the different potencies dis-

played by 14 and 16 and by 15 and 17 as well as by 3

at the frog rectus relative to the Torpedo nAChR are

the result of structural differences in the ion channel

or simply a different bioavailability of the compounds

at the receptor site.

F igu r e 3. Photoaffinity labeling of nicotinic receptors with

¹²⁵I₂-19. Nicotinic receptor-rich membranes (25 µg) were pho-

tolabeled with 10 µM ¹²⁵I₂-19, and labeled receptor subunits

were separated on a 10% SDS polyacrylamide gel. The gel was

stained with Coomassie (lane 2), and radioactive protein bands

were visualized by autoradiography (lanes 3). Lane 1 shows

the molecular mass markers in kDa: phosphorylase b (97

kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa),

and carbonic anhydrase (30 kDa). Exposure time was 1 day.

at both frog rectus and Torpedo nAChRs. Interestingly,

hexaamine 7 was 4-fold more potent than the lower

homologue 5 and 24-fold more potent than the prototype

3 at the Torpedo nAChR. Furthermore, unlike 3, it was

more potent at the Torpedo nAChR than at the frog

rectus nAChR.

The asymmetrically substituted photolabile tet-

raamine 18¹behaved like 14 and 16, since it was almost

10-fold more potent at frog rectus nAChRs than at

Previously, we showed that the reduction in the

flexibility of the hexamethylene chain between the inner

1870 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

Torpedo nAChRs, confirming that a terminal primary

amine function may interact differently with the nAChR

of different species. Again, the inclusion of an aromatic

group on the terminal amine function of 18, affording

19, caused an increase in potency for the Torpedo

nAChR. The higher homologue 20 retained high affinity

for both frog rectus and Torpedo nAChRs, but potency

was higher for the frog rectus nAChR.

In a reversible binding assay, the radioactively labeled

¹²⁵I₂-19 was shown to bind with an affinity of pK_app

)

6.0 ( 0.1. The non-iodinated 19 binds with pK_app) 5.86

( 0.06. The introduction of two iodine atoms in 19

results in a moderate increase in the binding affinity of

19, an effect that was previously observed when iodi-

nating the asymmetrical 18.⁴The straight line obtained

in the Scatchard plot indicates that ¹²⁵I₂-19 binds to a

single class of binding sites (Figure 2). When ¹²⁵I-labeled

R-BTX in a binding assay was used to determine the

number of receptor monomers per microgram of pro-

tein,³⁶the binding stoichiometry of ¹²⁵I₂-19 was calcu-

lated to be one molecule per receptor monomer. As

shown in Figure 3, ¹²⁵I₂-19 was found to photolabel

exclusively the nAChR R-subunits. No radioactivity was

incorporated into the other receptor subunits at detect-

able levels. Considering the biological profile of ¹²⁵I-18

(see Introduction),⁴our present results clearly demon-

strate that symmetrically substituted tetraamines in-

teract, at least with the Torpedo nAChR, differently

from monosubstituted tetraamines bearing a terminal

primary amine function. It was found that two mol-

ecules of ¹²⁵I-18 label one receptor monomer with a 2:1

stoichiometry whereas ¹²⁵I₂-19 interacted with the

receptor with a 1:1 stoichiometry. It is derived that most

likely the positively charged terminal primary amine

function of 18 and the second N-aryl-substituted moiety

of 19 bind at different sites within the receptor lumen.

We have advanced that the positively charged tet-

raamine tail of 18 interacts deep in the lumen of the

nAChR ion channel with the high-affinity noncompeti-

tive inhibitor site. This binding site is close to the

negatively charged amino acids that are part of the

selectivity filter, suggesting that the positively charged

tetraamine tail interacts with these negative amino acid

residues, which is in agreement with previous models

for polyamines binding,^12,13while the aryl moiety of this

compound binds to a hydrophobic region in the upper

part of the ion channel. As demonstrated in ¹²⁵I-18 in

photoaffinity labeling studies, this site lies within the

hydrophobic sequence HWVY (residues R186-R189)

containing three aromatic residues and is located close

to the agonist binding site.⁴The presence of noncom-

petitive antagonists prevent ¹²⁵I-18 photo-cross-linking;

thus, the binding site must be accessible from the water-

filled lumen of the channel.⁴Since the photolabile azido

group of 18 is linked to the aromatic headgroup, the site

of ¹²⁵I-18 cross-linking identified the region to which the

aryl moiety of the molecule binds. Since two molecules

of ¹²⁵I-18 bind within the nAChR channel, each of the

aromatic moieties of the two ¹²⁵I-18 interacts most likely

with one of the two R-subunits of the nAChR monomer.

It is derived that ¹²⁵I₂-19, which bears two identical

azido groups at both ends of its polyamine tail and

interacts with the receptor molecule with a 1:1 stoichi-

ometry, apparently shows a conformation in which ¹²⁵I₂-

F igu r e 4. Model of ¹²⁵I₂-19 binding in the Torpedo nAChR

ion channel. Each of the two aromatic moieties of ¹²⁵I₂-19 most

likely interacts with one of the two R-subunits of the nAChR

monomer. The long positively charged polyamine chain, which

overlaps with the noncompetitive inhibitor site, reaches further

down into the ion channel and is located close to the negatively

charged selectivity filter. (1) ¹²⁵I₂-19; (2) agonist binding site;

(3) high-affinity noncompetitive inhibitor site.

19 is in contact with both R-subunits of the nAChR

(Figure 4). Consequently, to achieve cross-linking with

both receptor R-subunits, 19 might assume a folded

U-shaped conformation with the long, positively charged

polyamine tail reaching down the ion channel. An

attractive working hypothesis may be that a U-shaped

conformation of 19 recognizes the hydrophobic sequence

HWVY of each receptor R-subunit by way of the two

terminal aryl moieties while interacting with two nega-

tively charged rings of the receptor channel, thanks to

the positive charges of the polymethylene tetraamine

backbone. This model is in line with the observation that

¹²⁵I₂-19 exclusively photolabels the R-subunit (Figure

3). Because of the generally low cross-linking yield of

1-2% per azido group in photoaffinity-labeling experi-

ments, it was not possible to detect R-subunit dimers

cross-linked together by ¹²⁵I₂-19, since the probability

that both azido groups of ¹²⁵I₂-19 react with an R-sub-

unit is only 0.04-0.01%. This view satisfactorily ratio-

nalizes the high affinity of hexaamines 5-7 toward the

nAChR by admitting that in a U-shaped conformation

these compounds may interact in an improved manner

with the negative charges of the selectivity filter of the

ion channel lumen, thanks to the six positively charged

nitrogen atoms that are lined in a manner such that

they are able to face the negative charges of the

receptor.

To gain insight into the interaction mode of polyamines

bearing aryl substituents on the terminal nitrogen

atoms, we have investigated asymmetrically disubsti-

tuted tetraamines 21 and 22. These compounds bear a

fluorescent moiety on one terminus and a substituted

benzyl group on the other terminus. Iodine was intro-

duced into the aryl moiety of 21, affording 22, because

it is known that this element may determine quenching

of the fluorescence when facing the fluorophor group.

Consequently, in a conformation in which the two

aromatic moieties of compound 22 would be in proxim-

ity, i.e., if the two aromatic moieties of 22 would refold

to generate a polyamine ring structure with the two

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1871

carbonyl]-6-aminocaproic acid (2.0 g, 7.5 mmol) and triethyl-

amine (1.05 mL, 7.5 mmol) in dioxane (50 mL), followed after

standing for 30 min by the addition of N-[(benzyloxy)carbonyl]-

1,6-hexanediamine¹(1.88 g, 7.5 mmol) in dioxane (15 mL).

After being stirred at room temperature overnight, the mixture

was evaporated, affording a residue that was suspended in

water (400 mL). The precipitate was filtered and washed with

2 N KHSO₄, aqueous NaHCO₃saturated solution, and water

to give 23: 78% yield; mp 115-118 °C; ¹H NMR (CDCl₃) δ

1.23-1.69 (complex m, 14), 2.17 (t, 2), 3.16-3.22 (m, 6), 4.81

(br s, 2, exchangeable with D₂O), 5.03 (s, 4), 5.58 (br s, 1,

exchangeable with D₂O), 7.30-7.39 (m, 10).

6-Am in oh exa n oic Acid (6-Am in oh exyl)a m id e (24). A

solution of 23 (2.93 g, 5.88 mmol) in MeOH (150 mL) was

hydrogenated over 10% Pd on charcoal (wet, Degussa type

E101 NE/W) (290 mg) for 2 h. Following catalyst removal, the

solvent was evaporated, affording 24 in a quantitative yield:

¹H NMR (CDCl₃) δ 1.29-1.72 (complex m, 14 + 4 exchangeable

with D₂O), 2.18 (t, 2), 2.68 (t, 4), 3.22 (q, 2), 5.70 (br s, 1,

exchangeable with D₂O).

(5-{6-[6-(6-Ben zyloxyca r bon yla m in oh exa n oyla m in o)-

h exa n oyla m in o]h exylca r ba m oyl}p en tyl)ca r ba m ic Acid

Ben zyl Ester (25). It was obtained as a white solid from 24

(1.35 g, 5.88 mmol) and N-[(benzyloxy)carbonyl]-6-aminocap-

roic acid (3.11 g, 11.7 mmol) following the procedure described

for 23: 70% yield; mp 132-134 °C; ¹H NMR (DMSO-d₆) δ

1.12-1.51 (complex m, 26), 2.01 (t, 6), 2.91-3.06 (m, 10), 5.00

(s, 4), 7.21 (br s, 2, exchangeable with D₂O), 7.31-7.35 (m,

10), 7.73 (br s, 3, exchangeable with D₂O).

6-Am in oh exa n oic Acid {6-[6-(6-Am in oh exa n oyla m in o)-

h exa n oyla m in o]h exyl}a m id e (26). A solution of 30% HBr

in acetic acid (20 mL) was added to a solution of 25 (2.98 g,

4.06 mmol) in acetic acid and CF₃COOH (1:1; 60 mL), and the

resulting mixture was stirred for 2 h. Ether (100 mL) was then

added, yielding a solid that was dissolved in water. The

solution was made basic with NaOH pellets and extracted with

CHCl₃(3 × 50 mL). Removal of the dried solvent gave in a

quantitative yield 26: ¹H NMR (CD₃OD) δ 1.27-1.70 (complex

m, 26), 2.12-2.25 (m, 6), 2.70 (t, 4), 3.16 (t, 6).

aromatic moieties of 22 interacting by stacking forces,

the fluorescence intensity of 22 should be lower than

that of 21, which lacks iodine. Preliminary results have

shown that the presence of iodine in 22 did not affect

the intensity of fluorescence relative to 21 (Bixel et al.,

unpublished results). This observation would not sup-

port the hypothesis that symmetrical polyamine binds

in a U-shaped conformation as shown in Figure 4.

However, it still may be possible that the distance

between the fluorophor and the iodine may not be

suitable for quenching in at least two reasonable

aspects. First, a spacer of five atoms and one atom

separates the fluorophor and the iodine-bearing aryl

group of 22, respectively, from the basic nitrogen atoms

to which they are linked. Assuming that these nitrogen

atoms interact with two anionic sites and that each of

them is located in a similar position on both R-subunits,

it is derived that the fluorophor and the aryl moiety can

hardly be facing each other, thus preventing the quench-

ing of the fluorescence. Second, the distance between

the sites on the R-subunits where the fluorophor and

the aryl moieties bind might be large enough to prevent

quenching of the fluorescence. Besides this intriguing

aspect, the results obtained for 21 and 22 appear

promising. Tetraamine 21 displayed a biological profile

similar to that of prototype 1 at all the receptors tested.

Interestingly enough, the introduction of an iodine atom

in 21, affording 22, resulted in a significant increase in

potency at nAChRs while giving a decrease in potency

at mAChRs. As a matter of fact, compound 22 resulted

in the most potent noncompetitive antagonist at the frog

rectus nAChR while being only slightly less potent than

hexaamine 7, which resulted the most potent at the

Torpedo nAChR. It is derived that the insertion of

appropriate substituents on the benzene ring of 1 may

be important for making relevant the structure-activity

relationship studies for improving affinity and selectiv-

ity for muscle-type nAChRs.

6-(2-Meth oxyben zyla m in o)h exa n oic Acid (6-{6-[6-(2-

Meth oxyben zyla m in o)h exa n oyla m in o]h exa n oyla m in o}-

h exyl)a m id e (27). A mixture of 26 (1.89 g, 4.06 mmol) and

2-methoxybenzaldehyde (1.21 g, 8.95 mmol) in refluxing MeOH

(100 mL) was stirred for 2 h. After the mixture was cooled,

NaBH₄(0.34 g, 8.95 mmol) was added and stirring was

continued at room temperature for 5 h. The mixture was then

cautiously made acidic with 3 N HCl, filtered, and evaporated.

The residue was dissolved in water, and the resulting solution

was washed with ether, made basic with 2 N NaOH, and

extracted with CHCl₃. Removal of the dried solvent afforded

Exp er im en ta l Section

Ch em istr y. Melting points were taken in glass capillary

tubes on a Bu¨chi SMP-20 apparatus and are uncorrected. IR,

MALDI-TOF-MS, and NMR spectra were recorded on Perkin-

Elmer 297, Bruker Biflex III, and Varian VXR 300 instru-

ments, respectively. Chemical shifts are reported in parts per

million (ppm) relative to tetramethylsilane (TMS), and spin

multiplicities are given as s (singlet), br s (broad singlet), d

(doublet), t (triplet), q (quartet), or m (multiplet). Although

IR spectra data are not included (because of the lack of unusual

features), they were obtained for all compounds reported and

were consistent with the assigned structures. The elemental

compositions of the compounds agreed to within (0.4% of the

calculated value. When the elemental analysis is not included,

crude compounds were used in the next step without further

purification. Chromatographic separations were performed on

silica gel columns by flash (Kieselgel 40, 0.040-0.063 mm;

Merck), medium pressure (Biotage, KP-SIL 60, 0.032-0.063

nm), or gravity column (Kieselgel 60, 0.063-0.200 mm; Merck)

chromatography. Reactions were followed by thin-layer chro-

matography (TLC) on Merck (0.25 mm) glass-packed precoated

silica gel plates (60 F₂₅₄) that were visualized in an iodine

chamber. The term “dried” refers to the use of anhydrous

sodium sulfate.

1

27 as a foam solid: 78% yield; H NMR (CDCl₃) δ 1.20-1.82

(complex m, 26 + 2 exchangeable with D₂O), 2.18 (t, 6), 2.54

(t, 4), 3.11-3.26 (m, 6), 3.70-3.83 (m, 10), 5.95 (br s, 3,

exchangeable with D₂O), 6.79-6.93 (m, 4), 7.14-7.28 (m, 4).

N-(2-Meth oxyben zyl)-N′-(6-{6-[6-(2-m eth oxyben zyla m i-

n o)h exyla m in o]h exyla m in o}h exyl)h exa n e-1,6-d ia m in e

P en ta h yd r och lor id e (4). A solution of 10 M BH₃‚MeSMe (2.2

mL) in dry diglyme (2 mL) was added dropwise at room

temperature to a solution of 27 (2.2 g, 3.16 mmol) in dry

diglyme (100 mL) with stirring under a stream of dry nitrogen.

When the addition was completed, the reaction mixture was

heated at 120 °C for 10 h. After the mixture was cooled at 0

°C, excess borane was destroyed by cautious dropwise addition

of MeOH (10 mL). The resulting mixture was left to stand for

4 h at room temperature, cooled at 0 °C, treated with HCl gas

for 15 min, and then heated at 120 °C for 4 h. After the mixture

was cooled, ether was added and the resulting mixture was

stirred overnight at room temperature, yielding a solid that

was filtered and dissolved in water. The solution was made

basic with 2 N NaOH and extracted with CHCl₃, affording the

free base that was purified by flash chromatography. Elution

with MeOH/aqueous 28% ammonia (8.5:1.7) afforded crude 4

that was converted into the pentahydrochloride salt: 25%

yield; mp 233-235 °C (from EtOH/MeOH); ¹H NMR (free base)

[6-(6-Ben zyloxyca r bon yla m in oh exa n oyla m in o)h exyl]-

ca r ba m ic Acid Ben zyl E st er (23). Ethyl chlorocarbonate

(0.72 mL, 7.6 mmol) in dry dioxane (5 mL) was added dropwise

to a stirred and cooled (5 °C) solution of N-[(benzyloxy)-

1872 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

(CDCl₃) δ 1.21-1.58 (complex m, 32 + 5 exchangeable with

D₂O), 2.56 (t, 16), 3.76 (s, 4), 3.86 (s, 6), 6.80-6.94 (m, 4), 7.14-

7.28 (m, 4). Anal. (C₄₀H₇₆Cl₅N₅O₂) C, H, N.

N-[3-(2-Met h oxyb en zyla m in o)p r op yl]-N′-(6-{6-[3-(2-

m eth oxyben zyla m in o)p r op yla m in o]h exyla m in o}h exyl)-

h exa n e-1,6-d ia m in e Hexa h yd r och lor id e (5). A mixture of

34 (free base, 0.24 g, 0.55 mmol), molecular sieves (3 Å), and

2-methoxybenzaldehyde (167 mg, 1.23 mmol) in EtOH (20 mL)

was stirred for 30 min at room temperature, then NaBH₄(47

mg, 1.23 mmol) was added, and the stirring was continued

overnight. Following removal of molecular sieves, the solution

was made acidic with 6 N HCl (3 mL). Removal of the solvent

gave a residue that was dissolved in water (20 mL). The

solution was washed with ether (3 × 20 mL) to remove

nonbasic materials, then was made basic with 2 N NaOH, and

finally was extracted with CHCl₃(3 × 20 mL). Removal of

washed (brine) and dried solvents gave 5 that was transformed

into the hexahydrochloride salt: 40% yield; mp 270 °C (dec)

(from MeOH); ¹H NMR (free base) (CDCl₃) δ 1.18-1.44

(complex m, 24), 1.58-1.68 (m, 4 + 6 exchangeable with D₂O),

2.42-2.61 (m, 20), 3.68 (s, 4), 3.73 (s, 6), 6.76-6.88 (m, 4),

7.16-7.20 (m, 4). Anal. (C₄₀H₇₈Cl₆N₆O₂) C, H, N.

{8-[(6-Am in oh exyl)-ter t-bu toxyca r bon yla m in o]octyl}-

(6-ter t-bu toxyca r bon yla m in oh exyl)ca r ba m ic Acid ter t-

Bu tyl Ester (37) a n d (6-Am in oh exyl)-{8-[(6-a m in oh exyl)-

ter t-b u t oxyca r b on yla m in o]oct yl}ca r b a m ic Acid ter t-

Bu tyl Ester (39). Ethyl trifluoroacetate (0.35 mL, 2.9 mmol)

was added to a stirred and cooled (-80 °C) solution of 35²(1.0

g, 2.9 mmol) in MeOH (65 mL). Following stirring at -80 °C

for 1 h and then to 0 °C over 1 h, di-tert-butyl dicarbonate

(2.55 g, 11.7 mmol) was added to the solution. After 1 h of

stirring at room temperature, the pH was increased to 11 with

aqueous 28% ammonia and stirring was vigorously continued

for 3 days. Removal of the solvent gave a residue that was

dissolved in water (30 mL), and the resulting solution was

extracted with CHCl₃(3 × 20 mL). Evaporation of the dried

solvent afforded a mixture of compounds that were separated

by flash chromatography. Elution with CHCl₃/MeOH/aqueous

28% ammonia (9:1:0.13) gave 37 and 39 as yellow oils.

37: 35% yield; R_f) 0.50 [eluent system, CHCl₃/MeOH/

aqueous 28% ammonia (9:1:0.1)]; ¹H NMR (CDCl₃) δ 1.16-

1.55 (complex m, 55 + 2 exchangeable with D₂O), 2.71-2.84

(m, 4), 3.06-3.19 (m, 8), 4.62 (br s, 1, exchangeable with D₂O).

39: 30% yield; R_f) 0.25 [eluent system, CHCl₃/MeOH/

aqueous 28% ammonia (9:1:0.1)]; ¹H NMR (CDCl₃) δ 1.29-

1.51 (complex m, 46 + 4 exchangeable with D₂O), 2.69 (t, 4),

3.11-3.22 (m, 8).

{12-[(6-Am in oh exyl)-ter t-bu toxyca r bon yla m in o]d od e-

cyl}-(6-ter t-bu toxyca r bon yla m in oh exyl)ca r ba m ic Acid

ter t-Bu tyl Ester (38) a n d (6-Am in oh exyl)-{12-[(6-a m in o-

h exyl)-ter t-bu toxycar bon ylam in o]dodecyl}car bam ic Acid

ter t-Bu tyl Ester (40). Ethyl trifluoroacetate (0.15 mL, 1.25

mmol) was added to a stirred and cooled (-80 °C) solution of

36²(0.50 g, 1.25 mmol) in MeOH (30 mL). Stirring was

continued at -80 °C for 1 h and then to 0 °C for an additional

1 h. Di-tert-butyl dicarbonate (1.09 g, 5 mmol) was added to

the mixture that was stirred at room temperature for 1 h.

Removal of the solvent gave a residue that was taken up in

MeOH (40 mL) and water (2 mL). The resulting solution was

treated with K₂CO₃(6 g, 0.05 mol) and heated under reflux

for 2 h. The residue was treated with 40% NaOH and extracted

with CHCl₃(3 × 20 mL). Removal of the dried solvent gave a

mixture of compounds that were purified by flash chromatog-

raphy. Elution with CHCl₃/MeOH/aqueous 28% ammonia (9:

1:0.05) gave 38 and 40 as yellow oils:

[5-(Be n zyl-{6-[b e n zyl-(6-b e n zyloxyca r b on yla m in o-

h exa n oyl)a m in o]h exyl}ca r ba m oyl)p en tyl]ca r ba m ic Acid

Ben zyl Ester (28). It was synthesized from N,N′-dibenzyl-

hexane-1,6-diamine²⁴(5.73 g, 19.3 mmol) and N-[(benzyloxy)-

carbonyl]-6-aminocaproic acid (10.5 g, 39.6 mmol) following the

procedure reported for 23 and purified by flash chromatogra-

phy. Elution with a step gradient system of EtOAc/cyclohexane

1

(6:4 to 7:3) afforded 28 as a foam solid: 70% yield; H NMR

(CDCl₃) δ 1.23-1.75 (complex m, 20), 2.22-2.38 (m, 4), 3.09-

3.35 (m, 8), 4.52 (d, 4), 4.83 (br s, 2, exchangeable with D₂O),

5.09 (s, 4), 7.11-7.39 (m, 20).

6-Am in oh exa n oic Acid {6-[(6-Am in oh exa n oyl)ben zyl-

a m in o]h exyl}ben zyla m id e (29). It was synthesized from 28

(8.83 g, 11.2 mmol) and 30% HBr in acetic acid (65 mL)

following the procedure described for 26: 90% yield; mp 204-

205 °C; ¹H NMR (CDCl₃) δ 1.13-1.69 (complex m, 20 + 4,

exchangeable with D₂O), 2.21-2.35 (m, 4), 3.56-3.68 (m, 4),

3.09 (t, 2), 3.27 (t, 2), 4.48 (d, 4), 7.07-7.28 (m, 10).

N¹-{6-[(6-Am in oh exyl)ben zyla m in o]h exyl}-N¹-ben zyl-

h exa n e-1,6-d ia m in e (30). This compound was obtained by

reduction of 29 (5.3 g, 10.14 mmol) with 10 M BH₃‚MeSMe (5

mL) following the procedure described for 4, and the free base

was purified by medium-pressure chromatography. Elution

with CHCl₃/MeOH/aqueous 28% ammonia (8.5:1.5:0.15) gave

1

30 as a foam solid: 40% yield; H NMR (CDCl₃) δ 1.24-1.44

(complex m, 24 + 4, exchangeable with D₂O), 2.37 (t, 8), 2.65

(t, 4), 3.52 (s, 4), 7.18-7.30 (m, 10).

3-[6-({6-[(6-Am in oh exyl)b en zyla m in o]h exyl}b en zyl-

a m in o)h exyla m in o]p r op ion itr ile (31) a n d 3-{6-[Ben zyl-

(6-{b en zyl-[6-(2-cya n oet h yla m in o)h exyl]a m in o}h exyl)-

a m in o]h exyla m in o}p r op ion itr ile (32). A solution of acry-

lonitrile (0.14 mL, 2.06 mmol) in MeOH (3 mL) was added

dropwise to a cooled (0 °C) and stirred solution of 30 (1.02 g,

2.06 mmol) in MeOH (20 mL). Stirring and cooling were

continued for 3 h, then removal of the solvent gave a mixture

of compounds that were separated by flash chromatography.

Elution with CHCl₃/MeOH/aqueous 28% ammonia (9.5:0.5:

0.05) gave 31 and 32 as yellow oils.

31: 35% yield; R_f) 0.52 [eluent system, CHCl₃/MeOH/

aqueous 28% ammonia (9.5:1.5:0.15)]; ¹H NMR (CDCl₃) δ

1.18-1.58 (complex m, 24 + 3, exchangeable with D₂O), 2.31-

2.39 (m, 8), 2.44 (t, 2), 2.58 (t, 2), 2.63 (t, 2), 2.88 (t, 2), 3.52 (s,

4), 7.21-7.33 (m, 10).

32: 30% yield; R_f) 0.95 [eluent system, CHCl₃/MeOH/

aqueous 28% ammonia (9.5:1.5:0.15)]; ¹H NMR (CDCl₃) δ

1.18-1.62 (complex m, 24 + 2, exchangeable with D₂O), 2.31-

2.38 (m, 8), 2.42-2.61 (m, 8), 2.87 (t, 4), 3.51 (s, 4), 7.19-7.31

(m, 10).

N-(3-Am in op r op yl)-N′-(6-{[6-(3-a m in op r op yla m in o)-

h e x y l]b e n zy la m in o }h e x y l)-N ′-b e n zy lh e x a n e -1,6-d i-

a m in e (33). A suspension of 32 (0.37 g, 0.62 mmol), aqueous

40% NaOH (0.4 mL), and Raney Ni (nickel sponge; suspension

in water) (0.25 g) in EtOH (8 mL) was hydrogenated for 6 h

at room temperature and a pressure of 15 psi. Following

catalyst removal, the solvent was evaporated, yielding a

residue that was dissolved in water (50 mL) and extracted with

CHCl₃(3 × 20 mL). Removal of dried solvents gave a residue

that was purified by flash chromatography. Elution with

CHCl₃/MeOH/aqueous 28% ammonia (5:4.5:1) afforded 33 as

1

40: yield 25%; R_f) 0.45 [eluent system, CHCl₃/MeOH/

aqueous 28% ammonia (8.5:1.5:0.2)]; ¹H NMR (CDCl₃) δ 1.12-

1.55 (complex m, 54), 1.72 (br s, 4, exchangeable with D₂O),

2.63 (t, 4), 3.05-3.18 (m, 8).

an oil: 90% yield; H NMR (CDCl₃) δ 1.24-1.44 (complex m,

24), 1.51-1.68 (m, 4), 1.90 (br s, 6, exchangeable with D₂O),

2.31-2.38 (m, 8), 2.52-2.76 (m, 12), 3.51 (s, 4), 7.19-7.38 (m,

10).

38: yield 45%; R_f) 0.70 [eluent system, CHCl₃/MeOH/

aqueous 28% ammonia (8.5:1.5:0.2)]; ¹H NMR (CDCl₃) δ 1.05-

1.40 (complex m, 63 + 2 exchangeable with D₂O), 2.50-2.63

(m, 2), 2.82-3.10 (m, 10), 4.89 (br s, 1, exchangeable with D₂O).

(8-{ter t-Bu toxyca r bon yl-[6-(2-cya n oeth yla m in o)h exyl]-

am in o}octyl)-[6-(2-cyan oeth ylam in o)h exyl]car bam ic Acid

ter t-Bu tyl Ester (41). A solution of acrylonitrile (0.13 mL,

2.02 mmol) in MeOH (4 mL) was added dropwise to a solution

N -(3-Am in op r op yl)-N ′-{6-[6-(3-a m in op r op yla m in o)-

h exyla m in o]h exyl}h exa n e-1,6-d ia m in e (34). A solution of

33 (0.34 g, 0.55 mmol) and CF₃COOH (0.5 mL) in EtOH (15

mL) was hydrogenated over 10% Pd on charcoal (wet, Degussa

type E101 NE/W) (34 mg) for 2 h. Following catalyst removal

the solvent was evaporated, affording 34 as a white solid:

quantitative yield; mp 143-145 °C; ¹H NMR (D₂O) δ 1.12-

1.55 (complex, 24), 1.81-2.00 (m, 4), 2.78-3.02 (m, 20).

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1873

of 39 (0.55 g, 1.01 mmol) in MeOH (40 mL). After being stirred

at room temperature overnight, the mixture was evaporated,

affording a residue that was purified by flash chromatography.

Elution with petroleum ether/CH₂Cl₂/EtOH/aqueous 28% am-

monia (6:3:1:0.02) gave 41 as an oil: 95% yield; ¹H NMR

(CDCl₃) δ 1.15-1.41 (complex m, 46 + 2 exchangeable with

D₂O), 2.41 (t, 4), 2.52 (t, 4), 2.82 (t, 4), 2.98-3.08 (m, 8).

hexahydrochloride salt: 50% yield; mp 283-285 °C (from

EtOH/Et₂O); ¹H NMR (free base) (CDCl₃) δ 1.18-1.60 (complex

m, 36), 1.65-1.80 (m, 4), 1.97 (br s, 6, exchangeable with D₂O),

2.50-2.78 (complex m, 20), 3.78 (s, 4), 3.82 (s, 6), 6.82-6.98

(m, 4), 7.20-7.35 (m, 4). Anal. (C₄₆H₉₀Cl₆N₆O₂) C, H, N.

N-(2-Meth oxyben zyl)-N′-(4′′-{[6-(2-m eth oxyben zyla m i-

n o)h exyla m in o]m eth yl}-[1,1′;4′,1′′]ter p h en yl-4-ylm eth yl)-

h exa n e-1,6-d ia m in e Tetr a h yd r och lor id e (8). A solution of

47²⁶(0.17 g, 0.59 mmol) and 48²⁷(0.37 g, 1.19 mmol) in EtOH

(20 mL) was stirred at 60 °C for 2 h, then NaBH₄(0.05 g, 1.32

mmol) was added, and the stirring was continued overnight

at room temperature. Removal of the solvent gave a residue

that was purified by flash chromatography. Elution with CH₂-

Cl₂/MeOH/aqueous 28% ammonia (9:1:0.1) gave 8 as free base

that was transformed into the tetrahydrochloride salt: 14%

yield; mp >300 °C (from EtOH/Et₂O); ¹H NMR (free base)

(CDCl₃) δ 1.26-1.73 (complex m, 16 + 4 exchangeable with

D₂O), 2.50-2.60 (m, 8), 3.78 (s, 4), 3.83 (s, 10), 6.83-6.95 (m,

4), 7.20-7.26 (m, 4), 7.39-7.43 (m, 4), 7.59-7.67 (m, 8);

MALDI-MS calcd for C₄₈H₆₃N₄O₂727.49 [M + H⁺], found

727.44. Anal. (C₄₈H₆₆Cl₄N₄O₂) C, H, N.

(12-{ter t-Bu toxycar bon yl-[6-(2-cyan oeth ylam in o)h exyl]-

a m in o}d od ecyl)-[6-(2-cya n oet h yla m in o)h exyl]ca r b a m -

ic Acid ter t-Bu tyl Ester (42). This compound was obtained

from 40 (0.55 g, 0.918 mmol) and acrylonitrile (0.18 mL, 0.669

mmol) as described for 41. Eluting with petroleum ether/CH₂-

Cl₂/EtOH/aqueous 28% ammonia (6:3:1:0.01) gave 42 as an

1

oil: 80% yield; H NMR (CDCl₃) δ 1.15-1.55 (complex m, 54

+ 2 exchangeable with D₂O), 2.45-2.65 (m, 8), 3.05-3.20 (m,

8), 3.91 (t, 4).

(6-{ter t-Bu t oxyca r b on yl-[8-(ter t-b u t oxyca r b on yl-{6-

[ter t-bu toxycar bon yl-(2-cyan oeth yl)am in o]h exyl}am in o)-

octyl]a m in o}h exyl)-(2-cya n oeth yl)ca r ba m ic Acid ter t-

Bu tyl Ester (43). A solution of 41 (0.63 g, 0.97 mmol) and

di-tert-butyl dicarbonate (0.45 g, 2.08 mmol) in dry CHCl₃was

stirred overnight and then was washed with aqueous saturated

NaHCO₃solution (2 × 30 mL) and brine (2 × 30 mL). Removal

of the dried solvent gave 43 in a quantitative yield: ¹H NMR

(CDCl₃) δ 1.09-1.45 (complex m, 64), 2.36-2.53 (m, 4), 2.95-

3.18 (m, 12), 3.37 (t, 4).

[4-({11-[4-(Ben zyloxyca r b on yla m in om et h yl)b en zyl-

ca r ba m oyl]u n d eca n oyla m in o}m eth yl)ben zyl]ca r ba m ic

Acid Ben zyl Ester (50). It was obtained as a white solid from

1,12-dodecanedioic acid (0.35 g, 1.5 mmol) and 49¹(0.83 g, 3.0

mmol) following the procedure described for 23: 80% yield;

1

mp 209 °C; H NMR (DMSO-d₆) δ 1.18-1.31 (m, 12), 1.40-

(6-{ter t-Bu t oxyca r b on yl-[12-(ter t-b u t oxyca r b on yl-{6-

[ter t-bu toxycar bon yl-(2-cyan oeth yl)am in o]h exyl}am in o)-

d od ecyl]a m in o}h exyl)-(2-cya n oeth yl)ca r ba m ic Acid ter t-

Bu tyl Ester (44). It was obtained in quantitative yield from

42 (0.50 g, 0.728 mmol) and di-tert-butyl dicarbonate (0.35 g,

1.6 mmol) following the procedure described for 43: ¹H NMR

(CDCl₃) δ 1.15-1.55 (complex m, 72), 2.45-2.62 (m, 4), 3.03-

3.12 (m, 8), 3.20 (t, 4), 3.40 (t, 4).

1.52 (m, 4), 2.03-2.21 (m, 4), 4.16-4.25 (m, 8), 5.04 (s, 4),

7.12-7.38 (m, 18), 7.81 (t, 2, exchangeable with D₂O), 8.30 (t,

2, exchangeable with D₂O).

Dod eca n ed ioic Acid Bis(4-a m in om eth ylben zyla m id e)

(51). It was obtained in a quantitative yield by hydrolysis with

HBr of 50 (0.9 g, 1.2 mmol) following the procedure described

1

for 26: mp 195-200 °C; H NMR (D₂O) δ 1.15-1.39 (m, 12),

1.51-1.79 (m, 4), 2.25-2.50 (m, 4), 4.12-4.45 (m, 8), 7.31-

7.57 (m, 8).

(3-Am in op r op yl)-(6-{[8-({6-[(3-a m in op r op yl)-ter t-b u -

toxyca r bon yla m in o]h exyl}-ter t-bu toxyca r bon yla m in o)-

octyl]-ter t-bu toxyca r bon yla m in o}h exyl)ca r ba m ic Acid

ter t-Bu tyl Ester (45). This compound was obtained by

reduction of 43 (0.81 g, 0.95 mmol) with Raney Ni (nickel

sponge; suspension in water) (0.30 g) as described for 33.

Removal of the dried solvent gave a residue that was purified

by flash chromatography. Elution with CHCl₃/MeOH/aqueous

28% ammonia (9:1:0.05) gave 45 as an oil: 50% yield; ¹H NMR

(CDCl₃) δ 1.14-1.50 (complex m, 64), 1.55-1.63 (m, 4), 1.95

(br s, 4, exchangeable with D₂O), 2.62 (t, 4), 3.01-3.22 (m, 16).

Dod eca n ed ioic Acid Bis{4-[(2-m eth oxyben zyla m in o)-

m eth yl]ben zyla m id e} (52). It was obtained in low yield

(15%) because of its insolubility from 51 (0.65 g, 1.4 mmol)

and 2-methoxybenzaldehyde (0.39 g, 2.9 mmol) following the

procedure described for 27: mp 120-125 °C; ¹H NMR (CDCl₃)

δ 1.09-1.39 (m, 12), 1.53-1.71 (m, 4), 1.82 (br s, 2, exchange-

able with D₂O), 2.18 (t, 4), 3.74 (s, 4), 3.78 (s, 4), 3.82 (s, 6),

4.39 (d, 4), 5.82 (br t, 2, exchangeable with D₂O), 6.85-6.96

(m, 4), 7.20-7.31 (m, 12).

N,N′-Bis{4-[(2-m et h oxyb en zyla m in o)m et h yl]b en zyl}-

d od eca n e-1,12-d ia m in e Tetr a h yd r och lor id e (11). It was

obtained as a tetrahydrochloride salt by reduction with BH₃‚

MeSMe (0.1 mL) of 52 (0.15 g, 0.21 mmol) following the

procedure described for 4: mp >300 °C (from EtOH/Et₂O); ¹H

NMR (D₂O) δ 1.03-1.22 (m, 16), 1.42-1.61 (m, 4), 2.85-2.93

(m, 4), 3.70 (s, 6), 4.07 (s, 4), 4.11 (s, 4), 4.14 (s, 4), 6.83-6.94

(m, 4), 7.14-7.41 (m, 12). Anal. (C₄₄H₆₆Cl₄N₄O₂) C, H, N.

[6-(Ben zyl-{6-[b en zyl-(6-ter t-b u t oxyca r b on yla m in o-

h exyl)am in o]h exyl}am in o)h exyl]-(2-cyan oeth yl)car bam ic

Acid ter t-Bu tyl Ester (53). It was obtained in a quantitative

yield from 31 (0.39 g, 0.71 mmol) and di-tert-butyl dicarbonate

(0.34 g, 1.47 mmol) following the procedure described for 43:

(3-Am in op r op yl)-(6-{[12-({6-[(3-a m in op r op yl)-ter t-bu -

toxyca r bon yla m in o]h exyl}-ter t-bu toxyca r bon yla m in o)-

dodecyl]-ter t-bu toxycar bon ylam in o}h exyl)car bam ic Acid

ter t-Bu tyl Ester (46). It was obtained by reduction of 44 (0.76

g, 0.38 mmol) with Raney Ni as described for 33: 78% yield;

¹H NMR (CDCl₃) δ 1.18-1.55 (complex m, 72 + 4 exchangeable

with D₂O), 1.58-1.66 (m, 4), 2.65 (t, 4), 3.05-3.20 (m, 16).

N,N′-Bis-{6-[3-(2-m eth oxyben zyla m in o)p r op yla m in o]-

h exyl}octa n e-1,8-d ia m in e Hexa h yd r och lor id e (6). It was

synthesized from 45 (0.19 g, 0.22 mmol) and 2-methoxyben-

zaldehyde (0.066 g, 0.49 mmol) following a slightly modified

procedure described for 5. Following removal of molecular

sieves, the solvent was evaporated to give a residue that was

taken up in 3 N HCl (10 mL). The resulting mixture was

stirred for 3 h at room temperature, was washed with ether

to remove nonbasic materials, then was made basic with 2 N

NaOH, and finally was extracted with CHCl₃(3 × 20 mL).

Removal of the dried solvent gave 6 as free base that was

transformed into the hexahydrochloride salt: 30% yield; mp

295 °C (dec); ¹H NMR (free base) (CDCl₃) δ 1.14-1.40 (m, 18),

1.45-1.65 (m, 10), 1.75-1.85 (m, 4), 2.26 (br s, 6, exchangeable

with D₂O), 2.52-2.80 (m, 20), 3.78 (s, 6), 3.88 (s, 4), 6.85-

6.98 (m, 4), 7.20-7.32 (m, 4). Anal. (C₄₂H₈₂Cl₆N₆O₂) C, H, N.

1

oil; H NMR (CDCl₃) δ 1.18-1.56 (complex m, 42), 2.32-2.38

(m, 8), 2.50-2.61 (m, 2), 3.01-3.12 (m, 2), 3.18-3.23 (m, 2),

3.39-3.46 (m, 2), 3.48-3.55 (m, 4), 4.48 (br s, 1, exchangeable

with D₂O), 7.19-7.38 (m, 10).

(3-Am in op r op yl)-[6-(ben zyl-{6-[ben zyl-(6-ter t-bu toxy-

car bon ylam in oh exyl)am in o]h exyl}am in o)h exyl]car bam ic

Acid ter t-Bu tyl Ester (54). It was obtained by reduction of

53 (0.52 g, 0.72 mmol) with Raney Ni (nickel sponge; suspen-

sion in water) (0.20 g) as described for 33. Removal of dried

solvents gave a residue that was purified by flash chromatog-

raphy. Elution with CHCl₃/MeOH/aqueous 28% ammonia (9:

1

N,N′-Bis-{6-[3-(2-m eth oxyben zyla m in o)p r op yla m in o]-

h exyl}d od eca n e-1,12-d ia m in e Hexa h yd r och lor id e (7). It

was synthesized from 46 (0.30 g, 0.33 mmol) and 2-methoxy-

benzaldehyde (0.098 g, 0.72 mmol) following the procedure

described for 6. The free base was transformed into the

1:0.07) afforded 54 as an oil: 70% yield; H NMR (CDCl₃) δ

1.11-1.48 (complex m, 42), 1.53-1.63 (m, 2), 2.01 (br s, 2,

exchangeable with D₂O), 2.22-2.35 (m, 8), 2.66 (t, 2), 2.98-

3.26 (m, 6), 3.48 (s, 4), 4.54 (br s, 1, exchangeable with D₂O),

7.18-7.31 (m, 10).

1874 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

[6-(Ben zyl-{6-[b en zyl-(6-ter t-b u t oxyca r b on yla m in o-

h exyl)am in o]h exyl}am in o)h exyl]{3-[2-bu tyr ylam in o-3-(4-

p h en oxyp h en yl)p r op ion yla m in o]p r op yl}ca r ba m ic Acid

ter t-Bu tyl Ester (56). A solution of 54 (0.35 g, 0.49 mmol) in

MeOH (10 mL) was added dropwise with stirring at room

temperature to a suspension of 55²⁸(0.25 g, 0.54 mmol) in

MeOH (10 mL). After 1 h, the reaction mixture was evaporated

to give a residue that was purified by flash chromatography.

Elution with a step gradient system of CH₂Cl₂/EtOH (9.5:0.5

1.95 (m, 2), 2.60-2.75 (m, 2), 3.0-3.30 (m, 12), 3.57 (t, 2), 4.71

(br s, 1, exchangeable with D₂O).

[6-(ter t-Bu t oxyca r b on yl-{12-[ter t-b u t oxyca r b on yl-(6-

ter t-bu toxyca r bon yla m in oh exyl)a m in o]d od ecyl}a m in o)-

h exyl]-{3-[2-b u t yr yla m in o-3-(4-p h en oxyp h en yl)p r op io-

n yla m in o]p r op yl}ca r ba m ic Acid ter t-Bu tyl Ester (61). A

solution of 60 (0.41 g, 0.48 mmol) in CHCl₃(10 mL) was added

dropwise to a suspension of 55²⁸(0.22 g, 0.48 mmol) in MeOH

(10 mL). After the mixture was stirred overnight, removal of

the solvent gave a residue that was purified by flash chroma-

tography. Elution with a step gradient system of EtOAc/

1

to 9:1) gave 56 as an oil: 67% yield; H NMR (CDCl₃) δ 0.88

(t, 3), 1.21-1.44 (complex m, 44), 1.58-1.63 (m, 2), 2.18-2.22

(m, 2), 2.35-2.39 (m, 8), 3.02-3.15 (m, 10), 3.53 (s, 4), 4.52

(br s, 1, exchangeable with D₂O), 4.62-4.69 (m, 1), 5.01 (s, 2),

6.16 (br s, 1, exchangeable with D₂O), 6.87 (d, 2), 7.10 (d, 2),

7.26-7.39 (m, 15).

1

petroleum ether (5:5 to 7:3) afforded 61: 50% yield; H NMR

(CDCl₃) δ 0.85 (t, 3), 1.18-1.52 (complex m, 72), 1.59 (q, 2),

1.88-1.92 (m, 2), 2.10-2.20 (m, 2), 2.95-3.20 (m, 18), 4.60-

4.68 (m, 1), 5.0 (s, 2), 6.19 (d, 1, exchangeable with D₂O), 6.86

(d, 2), 7.10 (d, 2), 7.18 (br s, 1, exchangeable with D₂O), 7.28-

7.40 (m, 5).

N-[1-(3-{6-[12-(6-Am in oh e xyla m in o)d od e cyla m in o]-

h exyla m in o}p r op ylca r ba m oyl)-2-(4-p h en oxyp h en yl)eth -

yl]bu tyr a m id e Tetr a tr iflu or oa ceta te (62). A solution of 61

(0.27 g, 0.229 mmol) in CHCl₃(2.5 mL) and CF₃COOH (0.5

mL) was stirred for 2 h. Removal of the solvent gave a residue

that was triturated with ether to afford in a quantitative yield

62 as a tetratrifluoroacetate salt: ¹H NMR (CD₃OD) δ 0.82 (t,

3), 1.20-1.80 (complex m, 40), 2.16 (t, 2), 2.80-3.10 (m, 16),

3.12-3.35 (m, 2), 4.45 (t, 1), 5.0 (s, 2), 6.89 (d, 2), 7.16 (d, 2),

7.22-7.43 (m, 5).

N -[1-{3-[6-({6-[(6-Am in oh e xyl)b e n zyla m in o]h e xyl}-

b en zyla m in o)h exyla m in o]p r op ylca r b a m oyl}-2-(4-p h e-

n oxyph en yl)eth yl]bu tyr am ide Tetr atr iflu or oacetate (57).

A solution of 56 (0.34 g, 0.33 mmol) and CF₃COOH (0.5 mL)

in dry CH₂Cl₂(6 mL) was stirred at room temperature for 2

h. Removal of the solvent gave 57: quantitative yield; ¹H NMR

(CD₃OD) δ 0.84 (t, 3), 1.28-1.80 (complex m, 28), 2.17 (t, 2),

2.85-3.22 (complex m, 18), 4.35 (s, 4), 4.38-4.45 (m, 1), 5.04

(s, 2), 6.92 (d, 2), 7.16 (d, 2), 7.31-7.49 (m, 15).

N -[1-(3-{6-[6-(6-Am i n o h e x y la m i n o )h e x y la m i n o ]-

h exyla m in o}p r op ylca r ba m oyl)-2-(4-h yd r oxyp h en yl)eth -

yl]bu tyr a m id e Tetr a tr iflu or oa ceta te (14). A solution of 57

(0.28 g, 0.32 mmol) in MeOH (15 mL) was hydrogenated over

10% Pd on charcoal (wet, Degussa type E101 NE/W) (28 mg)

for 3 h. Following catalyst removal, the solvent was evapo-

rated, yielding 14 as a foam solid: ¹H NMR (CD₃OD) δ 0.85

(t, 3), 1.43-1.80 (complex m, 28), 2.16 (t, 2), 2.83-3.00 (m,

16), 3.20-3.24 (m, 2), 4.38-4.41 (m, 1), 6.70 (d, 2), 7.04 (d, 2).

Anal. (C₄₂H₆₈F₁₂N₆O₁₁) C, H, N.

N-[1-(3-{6-[12-(6-Am in oh e xyla m in o)d od e cyla m in o]-

h exyla m in o}p r op ylca r ba m oyl)-2-(4-h yd r oxyp h en yl)eth -

yl]bu tyr a m id e Tetr a tr iflu or oa ceta te (16). A solution of 62

(0.26 g, 0.21 mmol) in MeOH (10 mL) was hydrogenated over

10% Pd on charcoal (wet, Degussa type E101 NE/W) (26 mg)

for 2 h. Following catalyst removal, the solvent was evapo-

rated, affording 16 as a tetratrifluoroacetate salt: quantitative

1

N-{2-(4-H yd r oxyp h en yl)-1-[3-(6-{6-[6-(2-m et h oxyb en -

zyla m in o)h exyla m in o]h exyla m in o}h exyla m in o)p r op yl-

ca r ba m oyl]eth yl}bu tyr a m id e Tetr a h yd r och lor id e (15).

It was synthesized from 14 (free base) (0.11 g, 0.18 mmol) and

2-methoxybenzaldehyde (27 mg, 0.20 mmol) following the

procedure described for 5. Removal of the dried solvent gave

a residue that was purified by flash chromatography. Elution

with CHCl₃/MeOH/aqueous 28% ammonia (5:4.5:0.6) afforded

crude 15 that was transformed into the tetrahydrochloride

yield; H NMR (CD₃OD) δ 0.86 (t, 3), 1.22-1.83 (complex m,

40), 2.20 (t, 2), 2.78-3.08 (m, 16), 3.18-3.30 (m, 2), 4.38-4.42

(m, 1), 6.73 (d, 2), 7.07 (d, 2), 8.19 (d, 1, exchangeable with

D₂O). Anal. (C₄₈H₈₀F₁₂N₆O₁₁) C, H, N.

N-{2-(4-Hyd r oxyp h en yl)-1-[3-(6-{12-[6-(2-m eth oxyben -

yla m in o)h exyla m in o]d od ecyla m in o}h exyla m in o)p r o-

pylcar bam oyl]eth yl}bu tyr am ide Tetr ah ydr och lor ide (17).

It was obtained starting from 16 (free base) (0.11 g, 0.16 mmol)

and 2-methoxybenzaldehyde (0.024 g, 0.176 mmol) following

the procedure described for 5. The residue was purified by flash

chromatography. Elution with a step gradient system of

CHCl₃/MeOH/aqueous 28% ammonia (5:4.5:0.4 to 5:4.5:0.5)

afforded 17 as free base that was transformed into the

tetrahydrochloride salt: 60% yield; mp 205-207 °C (from

EtOH/Et₂O); ¹H NMR (CD₃OD) δ 0.84 (t, 3), 1.30-1.81

(complex m, 40), 2.18 (t, 2), 2.80-2.82 (m, 2), 2.90-3.08 (m,

14), 3.15-3.22 (m, 2), 3.92 (s, 3), 4.20 (s, 2), 4.38 (t, 1), 6.70 (d,

2), 7.0-7.13 (m, 4), 7.38-7.50 (m, 2), 8.2 (t, 1, exchangeable

with D₂O). Anal. (C₄₈H₈₈Cl₄N₆O₄) C, H, N.

(6-{[3-(4-Azid o-2-h yd r oxyb en zoyla m in o)p r op yl]-ter t-

b u t oxyca r b on yla m in o}h exyl)-{8-[(6-{[3-(4-a zid o-2-h y-

droxybenzoylamino)propyl]-tert-butoxycarbonylamino}hexyl)-

ter t-bu toxyca r bon yla m in o]octyl}ca r ba m ic Acid ter t-Bu -

tyl Ester (63). A solution of 4-azidosalicylic acid N-hydrox-

ysuccinimide ester²⁹(0.14 g, 0.50 mmol) in dry CHCl₃(5 mL)

was added dropwise to a stirred solution of 45 (0.20 g, 0.23

mmol) in distilled CHCl₃(10 mL). After the mixture was

stirred for 2 h at room temperature, the solvent was evapo-

rated to give a residue that was purified by flash chromatog-

raphy. Elution with petroleum ether/acetone (8.5:2) afforded

63 as an oil: 55% yield; ¹H NMR (CDCl₃) δ 1.22-1.58 (complex

m, 64), 1.72-1.78 (m, 4), 3.10-3.19 (m, 16), 3.36-3.43 (m, 4),

6.50-6.62 (m, 4), 7.62 (d, 2), 8.40 (br s, 2, exchangeable with

D₂O).

1

salt: 65% yield; mp 195-198 °C; H NMR (D₂O) δ 0.83 (t, 3),

1.34-1.82 (complex m, 28), 2.25 (t, 2), 2.83 (t, 2), 2.99-3.10

(m, 14), 3.20-3.28 (m, 2), 3.95 (s, 3), 4.28 (s, 2), 4.46 (t, 1),

6.90 (d, 2), 7.07-7.22 (m, 4), 7.41 (d, 1), 7.55 (t, 1). Anal.

(C₄₂H₇₆Cl₄N₆O₄) C, H, N.

(6-ter t-Bu toxyca r bon yla m in oh exyl)-(12-{ter t-bu toxy-

ca r b on yl-[6-(2-cya n oe t h yla m in o)h e xyl]a m in o}d od e c-

ylca r ba m ic Acid ter t-Bu tyl Ester (58). A solution of acry-

lonitrile (0.042 mL, 0.64 mmol) in CHCl₃(2 mL) was added

dropwise to 38 (0.45 g, 0.643 mmol) in CHCl₃(40 mL). After

the mixture was stirred for 48 h, removal of the solvent gave

a residue that was purified by flash chromatography. Elution

with EtOAc/EtOH (9.5:0.4) gave 58: 60% yield; ¹H NMR

(CDCl₃) δ 1.15-1.55 (complex m, 63), 1.67 (br s, 1, exchange-

able with D₂O), 2.49 (t, 2), 2.60 (t, 2), 2.90 (t, 2), 3.0-3.20 (m,

10), 4.71 (br, s, 1, exchangeable with D₂O).

[6-(ter t-Bu t oxyca r b on yl-{12-[ter t-b u t oxyca r b on yl-(6-

ter t-bu toxyca r bon yla m in oh exyl)a m in o]d od ecyl}a m in o)-

h exyl]-(2-cyan oeth yl)car bam ic Acid ter t-Bu tyl Ester (59).

It was obtained in a quantitative yield from 58 (0.20 g, 0.27

mmol) and di-tert-butyl dicarbonate (65 mg, 0.30 mmol)

following the procedure described for 43: oil; ¹H NMR (CDCl₃)

δ 1.05-1.50 (complex m, 72), 2.45-2.58 (m, 2), 2.90-3.13 (m,

10), 3.17 (t, 2), 3.37 (t, 2), 4.55 (br s, 1, exchangeable with D₂O).

(3-Am in op r op yl)-[6-(ter t-bu toxyca r bon yl-{12-[ter t-bu -

toxyca r bon yl-(6-ter t-bu toxyca r bon yla m in oh exyl)a m in o]-

d od ecyl}a m in o)h exyl]ca r b a m ic Acid ter t-Bu t yl E st er

(60). It was obtained by reduction of 59 (0.55 g, 0.65 mmol)

with Raney Ni (nickel sponge; suspension in water) as de-

scribed for 33: 75% yield; ¹H NMR (CDCl₃) δ 1.12-1.70

(complex m, 72), 1.76 (br s, 2, exchangeable with D₂O), 1.87-

(6-{[3-(4-Azid o-2-h yd r oxyb en zoyla m in o)p r op yl]-ter t-

b u t oxyca r b on yla m in o}h exyl)-{12-[(6-{[3-(4-a zid o-2-h y-

dr oxyben zoylam in o)pr opyl]-ter t-bu toxycar bon ylam in o}-

h exyl)-ter t-bu toxycar bon ylam in o]dodecyl}car bam ic Acid

ter t-Bu tyl Ester (64). It was obtained from 4-azidosalicylic

acid N-hydroxysuccinimide ester²⁹(0.20 g, 0.72 mmol) and 46

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1875

(0.30 g, 0.33 mmol) following the procedure described for 63.

It was purified first by gravity chromatography (eluting

system, petroleum ether/acetone (8:2)) and then by flash

chromatography. Elution with petroleum ether/acetone (9:1)

trile (0.06 mL, 0.93 mmol) following the procedure described

for 41 and purified by flash chromatography. Elution with a

step gradient system of petroleum ether/EtOAc (5:5 to 9:1)

1

gave 69 as an oil: 70% yield; H NMR (CDCl₃) δ 1.11-1.46

1

gave 64: 30% yield; H NMR (CDCl₃) δ 1.10-1.62 (complex

(complex m, 55), 1.81 (br s, 1, exchangeable with D₂O), 2.51

(t, 2), 2.62 (t, 2), 2.91 (t, 2), 3.08-3.14 (m, 10), 4.60 (br s, 1,

exchangeable with D₂O).

m, 72), 1.63-1.82 (m, 4), 3.10-3.22 (m, 12), 3.25-3.50 (m, 8),

6.50-6.63 (m, 4), 7.65 (d, 2), 8.42 (br s, 2, exchangeable with

D₂O).

{6-[(3-Am in op r op yl)-ter t-b u t oxyca r b on yla m in o]h ex-

yl}-{8-[ter t-bu toxyca r bon yl-(6-ter t-bu toxyca r bon yla m i-

n oh exyl)a m in o]oct yl}ca r b a m ic Acid ter t-Bu t yl E st er

(70). This compound was obtained by reduction of 69 (0.44 g,

0.63 mmol) with Raney Ni (nickel sponge; suspension in water)

(0.15 g) as described for 33. Removal of the dried solvent gave

70 as an oil: 90% yield; ¹H NMR (CDCl₃) δ 1.18-1.61 (complex

m, 57 + 3 exchangeable with D₂O), 2.50-2.75 (m, 6), 2.93-

3.18 (m, 10), 4.81 (br s, 1, exchangeable with D₂O).

N¹-[3-({6-[(8-{[6-({3-[(4-Azido-2-h ydr oxyben zoyl)am in o]-

p r op yl}a m in o)h exyl]a m in o}octyl)a m in o]h exyl}a m in o)-

p r op yl]-4-a zid o-2-h yd r oxyben za m id e Tetr a tr iflu or oa ce-

ta te (19). It was obtained as a foam solid from 63 (0.14 g,

0.115 mmol) and CF₃COOH (0.30 mL) following the procedure

reported for 57: quantitative yield; ¹H NMR (CD₃OD) δ 1.38-

1.44 (complex m, 16), 1.62-1.76 (m, 12), 1.95-2.02 (m, 4),

2.90-3.05 (complex m, 16), 3.49 (t, 4), 6.50-6.61 (m, 4), 7.78

(d, 2). Anal. (C₄₈H₇₀F₁₂N₁₂O₁₂) C, H, N.

N¹-[3-({6-[(12-{[6-({3-[(4-Azido-2-h ydr oxyben zoyl)am in o]-

pr opyl}am in o)h exyl]am in o}dodecyl)am in o]h exyl}am in o)-

p r op yl]-4-a zid o-2-h yd r oxyben za m id e Tetr a tr iflu or oa ce-

ta te (20). It was obtained as a foam solid from 64 (0.12 g,

0.092 mmol) and CF₃COOH (0.30 mL) following the procedure

reported for 57: quantitative yield; ¹H NMR (CD₃OD) δ 1.30-

1.82 (complex m, 36), 1.92-2.11 (m, 4), 2.92-3.18 (complex

m, 16), 3.53 (t, 4), 6.58-6.70 (m, 4), 7.82 (d, 2). Anal.

(C₅₂H₇₈F₁₂N₁₂O₁₂) C, H, N.

{8-[ter t-Bu toxyca r bon yl-(6-ter t-bu toxyca r bon yla m in o-

h e xyl)a m in o]oct yl}-(6-{t er t -b u t oxyca r b on yl-[3-(2,2,2-

tr iflu or oacetylam in o)pr opyl]am in o}h exyl)car bam ic Acid

ter t-Bu tyl Ester (71). Ethyl trifluoroacetate (0.07 mL, 0.57

mmol) was added to a stirred and cooled (-80 °C) solution of

70 (0.4 g, 0.57 mmol) in MeOH (30 mL). Stirring was continued

for 1 h at -80 °C and then to 0 °C over 1 h. Removal of the

solvent gave a residue that was purified by flash chromatog-

raphy. Elution with CHCl₃/MeOH (9:1) afforded 71 as an oil:

1

90% yield; H NMR (CDCl₃) δ 1.18-1.51 (complex m, 55 + 1

exchangeable with D₂O), 1.63-1.68 (m, 2), 2.68 (t, 2), 2.88 (t,

2), 3.04-3.18 (m, 10), 3.41-3.48 (m, 2), 4.61 (br s, 1, exchange-

able with D₂O).

[8-(ter t-Bu t oxyca r b on yl-{6-[ter t-b u t oxyca r b on yl-(2-

cya n oet h yl)a m in o]h exyl}a m in o)oct yl]-{6-[t er t-b u t oxy-

ca r bon yl-(2-m eth oxyben zyl)a m in o]h exyl}ca r ba m ic Acid

ter t-Bu tyl Ester (66). It was obtained in quantitative yield

from 65¹(0.31 g, 0.6 mmol) and di-tert-butyl dicarbonate (0.58

g, 2.64 mmol) following the procedure described for 43: ¹H

NMR (CDCl₃) δ 1.08-1.43 (complex m, 64), 2.42-2.53 (m, 2),

3.93-3.18 (m, 12), 3.38 (t, 2), 3.72 (s, 3), 4.38 (d, 2), 6.72-6.83

(m, 2), 7.02-7.18 (m, 2).

[8-({6-[(3-Am in opr opyl)-ter t-bu toxycar bon ylam in o]h ex-

yl}-ter t-bu toxyca r bon yla m in o)octyl]-{6-[ter t-bu toxyca r -

bon yl-(2-m eth oxyben zyl)am in o]h exyl}car bam ic Acid ter t-

Bu tyl Ester (67). This compound was obtained in a quantita-

tive yield as an oil by reduction of 66 (0.49 g, 0.58 mmol) with

Raney Ni (nickel sponge; suspension in water) (0.15 g) as

described for 33: ¹H NMR (CDCl₃) δ 1.12-1.52 (complex m,

64), 1.56-1.62 (m, 2 + 2 exchangeable with D₂O), 2.61 (t, 2),

3.02-3.22 (m, 14), 3.77 (s, 3), 4.38 (d, 2), 6.78 (d, 1), 6.82 (t,

1), 7.03-7.18 (m, 2).

(8-{ter t-Bu t oxyca r b on yl-[6-(ter t-b u t oxyca r b on yl-{3-

[(7-m e t h oxy-2-oxo-2H -ch r om e n e -3-ca r b on yl)a m in o]-

p r op yl}a m in o)h exyl]a m in o}octyl)-{6-[ter t-bu toxyca r bo-

n yl-(2-m eth oxyben zyl)a m in o]h exyl}ca r ba m ic Acid ter t-

Bu tyl Ester (68). A solution of 2,5-dioxotetrahydro-1H-1-

pyrrolyl-7-methoxy-2-oxo-2H-3-chromene carboxylate (53 mg,

0.17 mmol) in dry DMF (4 mL) was added dropwise with

stirring at room temperature to a solution of 67 (0.14 g, 0.15

mmol) in dry DMF (6 mL). After 2 h, the reaction mixture was

evaporated to give a residue that was purified by flash

chromatography. Elution with petroleum ether/acetone/toluene

(7:2:1) afforded 68 as an oil: 82% yield; ¹H NMR (CDCl₃) δ

0.80-1.48 (complex m, 64), 1.81 (t, 2), 3.05-3.28 (m, 14), 3.38-

3.46 (m, 2), 3.79 (s, 3), 3.88 (s, 3), 4.41 (d, 2), 6.80-7.19 (m, 6),

7.55 (d, 1), 8.80 (s, 1).

7-Meth oxy-2-oxo-2H-ch r om en e-3-ca r boxylic Acid [3-(6-

{8-[6-(2-Meth oxyben zyla m in o)h exyla m in o]octyla m in o}-

h exyla m in o)p r op yl]a m id e Tetr a tr iflu or oa ceta te (21). It

was synthesized in a quantitative yield from 68 (0.14 g, 0.12

mmol) and CF₃COOH (0.4 mL) as described for 57: mp 157-

159 °C; ¹H NMR (CD₃OD) δ 1.37-1.41 (complex m, 16), 1.68-

1.70 (complex m, 12), 1.97-2.03 (m, 2), 2.94-3.06 (m, 14), 3.54

(t, 2), 3.90 (s, 3), 3.93 (s, 3), 4.18 (s, 2), 6.97-7.09 (m, 4), 7.37-

7.43 (m, 2), 7.71 (d, 1), 8.77 (s, 1). Anal. (C₅₀H₇₁F₁₂N₅O₁₃) C,

H, N.

N-(3-{6-[8-(6-Am in oh exyla m in o)octyla m in o]h exyla m i-

n o}p r op yl)-2,2,2-tr iflu or oa ceta m id e (72). CF₃COOH (2

mL) was added to a stirred solution of 71 (0.40 g, 0.50 mmol)

in dry CH₂Cl₂(5 mL). After the mixture was stirred for 2 h at

room temperature, the solvent was removed to give a residue

that was taken up in 0.5 N NaOH and immediately extracted

with CHCl₃(1 × 30 mL). Removal of the dried solvent gave

72 as a clear oil: 56% yield; ¹H NMR (CDCl₃) δ 1.18-1.51

(complex m, 28 + 5 exchangeable with D₂O), 1.62-1.69 (m,

2), 2.48-2.56 (m, 10), 2.64 (t, 2), 2.78 (t, 2), 3.43 (t, 2).

2,2,2-Tr iflu or o-N-[3-(6-{8-[6-(5-iod o-2-m et h oxyb en zil-

am in o)h exylam in o]octylam in o}h exylam in o)pr opyl]acet-

a m id e (73). A solution of 72 (0.14 g, 0.28 mmol) and 5-iodo-

2-methoxybenzaldehyde³⁰(74 mg, 0.28 mmol) in toluene (30

mL) was heated under reflux, and the water formed was

continuously removed for 5 h. The cooled mixture was evapo-

rated to give the corresponding Schiff base that was dissolved

in EtOH (25 mL) and treated with NaBH₄(11 mg, 0.29 mmol).

After the mixture was stirred at 0 °C for 4 h, the solvent was

evaporated to give a residue that was treated with petroleum

ether (3 × 20 mL). Removal of the combined organic layers

afforded 73 as an oil: 60% yield; ¹H NMR (CDCl₃) δ 1.18-

1.41 (complex m, 28 + 4 exchangeable with D₂O), 1.61-1.66

(m, 2), 2.43-2.55 (m, 12), 2.78 (t, 2), 3.41 (t, 2), 3.63 (s, 2),

3.74 (s, 3), 6.58 (d, 1), 7.42-7.51 (m, 2).

[8-(ter t-Bu t oxyca r b on yl-{6-[ter t-b u t oxyca r b on yl-(5-

iodo-2-m eth oxyben zyl)am in o]h exyl}am in o)octyl]-(6-{ter t-

bu toxyca r bon yl-[3-(2,2,2-tr iflu or oa cetyla m in o)p r op yl]-

a m in o}h exyl)ca r ba m ic Acid ter t-Bu tyl Ester (74). It was

obtained from 73 (0.13 g, 0.17 mmol) and di-tert-butyl dicar-

bonate (168 mg, 0.17 mmol) following the procedure described

for 43 and purified by flash chromatography. Elution with a

step gradient system of CHCl₃/toluene/EtOH (5:4.5:0.5 to 5:4.5:

1

1.1) gave 74 as an oil: 45% yield; H NMR (CDCl₃) δ 1.18-

1.41 (complex m, 64), 1.61-1.78 (m, 2), 3.05-3.38 (m, 16), 3.80

(s, 3), 4.38 (d, 2), 6.62 (d, 1), 7.38-7.58 (m, 2), 8.19 (br s, 1,

exchangeable with D₂O).

{6-[(3-Am in opr opyl)-ter t-bu toxycar bon ylam in o]h exyl}-

[8-(ter t-bu toxyca r bon yl-{6-[ter t-bu toxyca r bon yl-(5-iod o-

2-m et h oxyb en zyl)a m in o]h exyl}a m in o)oct yl]ca r b a m ic

Acid ter t-Bu tyl Ester (75). Aqueous 28% ammonia was

added to a vigorously stirred solution of 74 (90 mg, 0.07 mmol)

in MeOH (20 mL) until the pH was increased to 11. After the

mixture was stirred at room temperature overnight, the

solvent was evaporated to give a residue that was dissolved

{8-[ter t-Bu t oxyca r b on yl-(6-ter t-b u t oxyca r b on yla m i-

n oh exyl)a m in o]octyl}-{6-[ter t-bu toxyca r bon yl-(2-cya n o-

eth yl)a m in o]h exyl}ca r ba m ic Acid ter t-Bu tyl Ester (69).

It was synthesized from 37 (0.60 g, 0.93 mmol) and acryloni-

1876 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9

Rosini et al.

in water (20 mL) and extracted with CHCl₃(3 × 30 mL).

Removal of the dried solvent gave 75 in quantitative yield; ¹H

NMR (CDCl₃) δ 1.18-1.44 (complex m, 64 + 2 exchangeable

with D₂O), 1.58-1.66 (m, 2), 2.63 (t, 2), 3.03-3.22 (m, 14), 3.78

(s, 3), 4.31 (d, 2), 6.59 (d, 1), 7.38-7.51 (m, 2).

pulses (0.6-0.8 ms, 1 Hz, 1-5 V). Inotropic activity was

recorded isometrically. Tissues were equilibrated for 1 h, and

cumulative concentration-response curves to arecaidine pro-

pargyl ester (APE) (0.01-1 µM) were constructed. Following

incubation with the antagonist for 60 min, a new concentra-

tion-response curve to APE was obtained.

[8-(ter t-Bu t oxyca r b on yl-{6-[ter t-b u t oxyca r b on yl-(5-

iodo-2-m eth oxyben zyl)am in o]h exyl}am in o)octyl]-[6-(ter t-

b u t oxyca r b on yl-{3-[(7-m et h oxy-2-oxo-2H -ch r om en e-3-

car bon yl)am in o]pr opyl}am in o)h exyl]car bam ic Acid ter t-

Bu tyl Ester (76). A solution of 2,5-dioxotetrahydro-1H-1-

pyrrolyl-7-methoxy-2-oxo-2H-3-chromene carboxylate (27 mg,

0.084 mmol) in dry DMF (2 mL) was added dropwise at room

temperature to a solution of 75 (80 mg, 0.077 mmol) in dry

DMF (5 mL). After the mixture was stirred overnight, the

reaction mixture was evaporated to give a residue that was

purified by flash chromatography. Elution with petroleum

ether/acetone/toluene (7:2:1) afforded 76 as an oil: 72% yield;

¹H NMR (CDCl₃) δ 1.18-1.51 (complex m, 64), 1.81 (t, 2), 3.02-

3.23 (m, 14), 3.41-3.48 (m, 2), 3.77 (s, 3), 3.90 (s, 3), 4.38 (d,

2), 6.60 (d, 1), 6.82-6.96 (m, 2), 7.38-7.51 (m, 2), 7.59 (d, 1),

8.81 (s, 1).

Gu in ea P ig Ileu m Lon gitu d in a l Mu scle. The terminal

portion of the ileum was excised after discarding the 8-10 cm

nearest the ileo-caecal junction. The tissue was cleaned, and

segments 2-3 cm long of ileum longitudinal muscle were

separated from the underlying circular muscle and set up

under 1 g of tension at 37 °C in organ baths containing Tyrode

solution of the following composition (mM): NaCl, 118; KCl,

4.75; CaCl₂, 2.54; MgSO₄, 1.2; KH₂PO₄‚2H₂O, 1.19; NaHCO₃,

25; glucose, 11. Tension changes were recorded isotonically.

Tissues were allowed to equilibrate for at least 30 min during

which time the bathing solution was changed every 10 min.

Concentration-response curves to APE (0.01-0.5 µM) were

obtained at 30 min intervals, the first one being discarded and

the second one taken as control. Following incubation with the

antagonist for 60 min, a new concentration-response curve

to the agonist was obtained.

F r og Rectu s Abd om in is Mu scle. The rectus abdominis

muscle of frogs was set up at room temperature in Clark frog

Ringer solution of the following composition (mM): NaCl, 111;

KCl, 1.88; CaCl₂, 1.08; NaH₂PO₄, 0.08; NaHCO₃, 2.38; glucose,

11.1. The tissues were equilibrated under 1 g of tension for

60 min. Two cumulative concentration-response curves to

carbachol (1-100 µM) were constructed at 45 min intervals,

the first one being discarded and the second one taken as

control. Following incubation with the antagonist for 30 min

7-Meth oxy-2-oxo-2H-ch r om en e-3-ca r boxylic Acid [3-(6-

{8-[6-(5-Iod o-2-m eth oxyben zyla m in o)h exyla m in o]octyl-

a m in o}h exyla m in o)p r op yl]a m id e Tetr a tr iflu or oa ceta te

(22). It was obtained in quantitative yield as a foam solid from

76 (80 mg, 0.055 mmol) and CF₃COOH (0.25 mL) following

the procedure reported for 57: ¹H NMR (CD₃OD) δ 1.37-1.43

(complex m, 16), 1.62-1.76 (complex m, 12), 1.97-2.04 (m, 2),

2.94-3.05 (m, 14), 3.55 (t, 2), 3.88 (s, 3), 3.94 (s, 3), 4.18 (s, 2),

6.94-7.03 (m, 3), 7.73-7.79 (m, 3), 8.80 (s, 1). Anal. (C₅₀H₇₀F₁₂

IN₅O₁₃) C, H, N.

-

Syn th esis a n d P u r ifica tion of ¹²⁵I₂-19. Compound 19 was

radioactively iodinated with ¹²⁵I using the chloramine T

method.³¹A total of 60 µL of chloramine T (2.3 mg/mL in 100

mM NaPi, pH 7.4) was added in the dark to a solution

containing 50 µL of 19 (5 mg/mL), 140 µL of K¹²⁵I (30 000 cpm/

nmol), and 380 µL of 100 mM NaPi, pH 7.4. After 5 min of

incubation at room temperature, the iodination reaction was

stopped by applying the solution onto a reverse-phase high-

performance liquid chromatography (RP-HPLC) column (Vy-

dac C₁₈, Waters model 626, Eschborn, Germany). For elution

of the di-¹²⁵I derivative, the following linear gradient (1 mL/

min) was applied: solvent A (aqueous solution containing 0.1%

trifluoroacetic acid (TFA)) and solvent B (acetonitrile contain-

ing 0.085% TFA). The UV absorption of the eluent was

determined at 305 nm, and the radioactivity of each fraction

was detected using a γ-counter. ¹²⁵I₂-19 was characterized by

matrix-assisted laser desorption-ionization mass spectrometry

(MALDI-MS).

Biology. F u n ction a l An ta gon ism . Guinea pigs of either

sex (200-400 g) and frogs (10-20 g) were sacrificed under

ketamine or ether anesthesia, respectively, and the organs

required were set up rapidly under a suitable resting tension

in 15 mL organ baths containing physiological salt solution

kept at the appropriate temperature (see below) and aerated

with 5% CO₂/95% O₂at pH 7.4. Concentration-response

curves were constructed by cumulative addition of the agonist.

The concentration of agonist in the organ bath was increased

approximately 5-fold at each step, with each addition being

made only after the response to the previous addition had

attained a maximal level and remained steady. Contractions

were recorded by means of a force displacement transducer

(FT.03 Grass and 7003 Basile) connected to a four-channel pen

recorder (Battaglia-Rangoni KV 380). In all cases, parallel

experiments in which tissues did not receive any antagonist

were run in order to check for variations in sensitivity.

a

new concentration-response curve to the agonist was

obtained.

¹²⁵I₂-19 Bin d in g Assa ys. nAChR-rich membranes were

prepared from frozen Torpedo californica electric organ as

described earlier.³⁷Increasing concentrations of ¹²⁵I₂-19 (80 000

cpm/nmol) were added to a constant amount of nAChR-rich

membranes (0.3 mg/mL protein, diluted in 50 mM NaCl, 0.1%

TritonX-100, 10 mM Na₃PO₄, pH 7.5, total volume per sample,

200 µL) and were incubated for 30 min at room temperature.

Nonspecific binding was determined from bound ¹²⁵I₂-19 in the

presence of a 100-fold molar excess of I₂-19. Incubations were

terminated by vacuum filtration through Whatman GF/B

filters presoaked in (0.3%) polyethylenimine. The filters were

washed three times with 4 mL aliquots of 50 mM NaCl, 0.1%

TritonX-100, 10 mM Na₃PO₄, pH 7.5. [¹²⁵I]R-BTX binding

assays and the calculation of binding sites have been per-

formed as described previously.³⁶

P h oto-Cr oss-Lin k in g Exp er im en ts. nAChR-rich mem-

branes (25 µg) were diluted in 0.1 M NaPi, pH 7.4, to a final

receptor concentration of 140 nM. The radioactive ¹²⁵I₂-19 (10

µM; 1 Mio cpm/nmol) was mixed with the sample solution,

incubated for 30 min at room temperature, and irradiated with

UV light of 254 nm (distance, 15 cm; quartz lamp; Desaga,

Heidelberg, Germany) at 254 nm for 30 s. Unbound ¹²⁵I₂-19

was separated from ¹²⁵I₂-19 bound to nAChR-rich membranes

by centrifugation (15 000g, 15 min). The pellet was dissolved

and separated by SDS-PAGE using a 10% SDS-PAG.³⁸The

stained gel was dried, and radioactive receptor subunits were

visualized by autoradiography.

F lu or escen ce Titr a tion . All fluorescence spectra were

recorded using an Aminco Bowman spectrometer series 2

(Rochester, NY). For fluorescence titration experiments ali-

quots of competing ligand were added stepwise to a solution

containing nAChR-rich membranes (1 µM receptor concentra-

tion), ethidium (7 µM), and carbachol (1 mM) in 50 mM NaPi,

pH 7.4. Ethidium fluorescence was measured by employing

an excitation wavelength of 480 nm (slit widths, 4 nm/4 nm)

while monitoring the emission from 540 to 740 nm.

Da ta An a lysis. Antagonism of mAChRs, expressed as pK_B

values, was estimated according to the Furchgott equation:

K_B) [antagonist]/(DR - 1), where DR is the ratio between

individual EC₅₀values in the presence and in the absence of

antagonists.³²The potency of the agonist, i.e., the concentration

Gu in ea P ig Left Atr ia . The guinea pig heart was rapidly

removed and washed by perfusion through the aorta with

oxygenated physiological salt solution, and right and left atria

were separated out. The left atria were mounted under 0.2-

0.3 g of tension at 35 °C in Tyrode solution of the following

composition (mM): NaCl, 136.9; KCl, 5.4; MgSO₄‚7H₂O, 1.0;

CaCl₂, 2.52; NaH₂PO₄, 0.4; NaHCO₃, 11.9; glucose, 5.5. Tissues

were stimulated through platinum electrodes by square-wave

Polyamines as Antagonists

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1877

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Ack n ow led gm en t. Grants from the University of

Bologna, the European Community (Grant BMH4-

CT97-2395), MURST, the Deutsche Forschungsgemein-

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J M011067F

Article Doi

Structure-activity relationships of methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists. 2. Role of polymethylene chain lengths separating amine functions and of substituents on the terminal nitrogen atoms

DOI: 10.1021/jm011067f

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