Pentamidine analogs as inhibitors of [3H]MK-801 and [3H]ifenprodil binding to rat brain NMDA receptors

Article abstract of DOI:10.1016/j.bmc.2015.06.012

The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia, but in addition has several other biological targets, including the NMDA receptor (NR). Here we describe the inhibitory potencies of 76 pentamidine analogs at 2 bi

Full text of DOI:10.1016/j.bmc.2015.06.012

Accepted Manuscript
Pentamidine analogs as inhibitors of [3H]MK-801 and [3H]ifenprodil binding to
rat brain NMDA receptors
Michael L. Berger, Dorota Maciejewska, Jean Jacques Vanden Eynde,
Madhusoodanan Mottamal, Jerzy Żabiński, Paweł Kaźmierczak, Mateusz
Rezler, Ivana Jarak, Ivo Piantanida, Grace Karminski-Zamola, Annie Mayence,
Patrick Rebernik, Arvind Kumar, Mohamed A. Ismail, David W. Boykin, Tien
L. Huang
PII:
S0968-0896(15)00506-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.012
BMC 12368
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 October 2014
5 June 2015
5 June 2015
Please cite this article as: Berger, M.L., Maciejewska, D., Vanden Eynde, J.J., Mottamal, M., Żabiński, J.,
Kaźmierczak, P., Rezler, M., Jarak, I., Piantanida, I., Karminski-Zamola, G., Mayence, A., Rebernik, P., Kumar,
A., Ismail, M.A., Boykin, D.W., Huang, T.L., Pentamidine analogs as inhibitors of [3H]MK-801 and [3H]ifenprodil
binding to rat brain NMDA receptors, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bmc.2015.06.012
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Pentamidine analogs as inhibitors of [3H]MK-801
and [3H]ifenprodil binding to rat brain NMDA
receptors
Michael L. Bergera,*, Dorota Maciejewskab, Jean Jacques Vanden Eyndec, Madhusoodanan
Mottamald, Jerzy Żabińskib, Paweł Kaźmierczakb, Mateusz Rezlerb, Ivana Jarake, Ivo
Piantanidae, Grace Karminski-Zamolae, Annie Mayenced, Patrick Rebernika, Arvind Kumarf,
Mohamed A Ismailf, David W. Boykinf, Tien L. Huangd
b
aCenter for Brain Research, Medical University of Vienna, Vienna, Austria; Department of
c
Organic Chemistry, Medical University of Warsaw, Warsaw, Poland; Laboratory of Organic
d
Chemistry, University of Mons-UMONS, Mons, Belgium; College of Pharmacy, Xavier
University of Louisiana, New Orleans, U.S.A.; eDepartment of Organic Chemistry, University
f
of Zagreb, Zagreb, Croatia; Department of Chemistry, Georgia State University, Atlanta,
Georgia, U.S.A.
*Tel. (+431) 40160 34081, Fax (+431) 40160 9 34053,
e-mail michael.berger@meduniwien.ac.at
1

Abstract
The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia,
but in addition has several other biological targets, including the NMDA receptor (NR). Here
we describe the inhibitory potencies of 76 pentamidine analogs at 2 binding sites of the NR,
the channel binding site labeled with [3H]MK-801 and the [3H]ifenprodil binding site. Most
analogs acted weaker at the ifenprodil than at the channel site. The spermine-sensitivity of NR
inhibition by the majority of the compounds was reminiscent of other long-chain dicationic
NR blockers. The potency of the parent compound as NR blocker was increased by modifying
the heteroatoms in the bridge connecting the 2 benzamidine moieties and also by integrating
the bridge into a seven-membered ring. Docking of the 45 most spermine-sensitive
bisbenzamidines to a recently described acidic interface between the N-terminal domains of
GluN1 and GluN2B mediating polyamine stimulation of the NR revealed the domain
contributed by GluN1 as the most relevant target.
2

Chart 1
O
O
H2N
NH2
+
NH2
1
+
NH2
N
N
H2N
+
H2N
NH2
+
NH2
24
1. Introduction
Pentamidine (1), a bisbenzamidine with two aromatic nuclei and an unbranched saturated
five-carbon chain inserted between 2 phenolic ether oxygens (Chart 1), introduced as a
trypanocidal agent,1 gained importance with the HIV pandemic as one of the most effective
agents against opportunistic Pneumocystis pneumonia.2 Despite successes of antiretroviral
therapy, still 20% of HIV-infected patients suffer from neurocognitive disorders, most likely
caused by mediators like quinolinic acid released by invading glia.3,4 By virtue of its
dicationic elongated structure, 1 is predestined to fit into the DNA minor groove,5,6 an
interaction possibly underlying its chemotherapeutic properties. However, 1 has several other
biological targets in addition, including the NMDA receptor (NR),7 the imidazoline I2 binding
site,8 acid-sensing ion channels,9 and the potassium channel KIR2.1,10 as recently reviewed.11
The NR is one of the most common excitatory neurotransmitter receptors,12 activated by
glutamic acid; pathologically increased interstitial quinolinic acid might lead to neurotoxicity
by acting as agonist at this receptor.13 Pentamidine has been shown to block the NR at low
micromolar concentration, but barely reaches the brain after systemic application.14 The
conformationally restricted pentamidine analog N,N’-bis(4-amidinophenyl)homopiperazine
(24; chart 1) inhibits more potently than pentamidine the growth of Pneumocystis carinii in
culture15 and the NR on rat brain membranes.16 Inhibition of the NR by 1 and 24 is sensitive
to polyamines,7,17 suggesting the involvement of the polyamine regulatory site of the NR.18
3

The endogenous polyamines spermidine and spermine increase the opening frequency of the
NR-associated ion channel by allowing the closure of an interface in the extracellular N-
terminal domain between subunits GluN1 and GluN2B; this is brought about by neutralization
of negative surface charges, which in the absence of polyamines repel each other and keep the
interface open.18 Here, we present inhibitory potencies of several pentamidine analogs at the
NR on rat brain membranes, with particular reference to the partial reversal of this inhibition
by the polyamine spermine (polyamine sensitivity). While antimicrobial activities of these
compounds have been evaluated in vitro, most of them have never been tested at the NR. We
test these compounds as inhibitors of specific binding of the channel ligand [3H]MK-801,19 in
absence and in presence of 30 µM spermine, and several of them also as inhibitors of
[3H]ifenprodil binding.20 Also Ifenprodil binds to the GluN1/2B interface, but somewhat
more externally.21 The interaction is mainly mediated by H-bonds and hydrophobic
interactions, not by charge neutralization. MK-801 is an open-channel blocker that, upon
opening of the NR-associated channel, gains access to a site deeply located close to the
narrow restriction of the passage way; specific binding of non-saturating concentrations of
[3H]MK-801 can be taken as indicator for the accessibility and opening frequency of the
channel.19 Finally, we present first attempts to dock most of these compounds to a recently
described binding pocket shared by the NR subunits GluN1 and GluN2B.22
NC
RP
F
RP
F
CN
Scheme 1. Preparation of
the dinitrile precursors of
9 and 11. Atom numbers
in accordance with NMR
data.
Y
HN
RN
NH
RN
K2CO3
115 - 150 °C
2 - 8 h
DMSO, NMP or DMF
7'
CN
2
2'
NC7
RP
RP
1
3
1'
6'
3'
8
8'
Y
6
4
4'
N
N
5
9
9'
5'
RN
RN
9a (RP = OMe, RN = H, Y = CH2)
11a (RP = H, RN = Me, Y = O)
4

2. Syntheses
All structures are shown in Tables 1-4. The syntheses of several pentamidine analogs with
heteroatom variation in the bridge have been described23,24,25,26,27,28 (see Table S1 in the
supporting information for assignment). The syntheses of 9 and 11 and of their precursors 9a
and 11a are described here for the first time (using established procedures). In short, 2 eq of
the appropriate 4-fluorobenzonitrile were subjected to nucleophilic displacement by 1 eq of a
diamino spacer (Scheme 1). The spacers were 1,5-diaminopentane and bis(2-aminoethyl)
ether, respectively, and yielded the terminal nitrogens for the bridge. The resulting
bisbenzonitriles 9a and 11a were converted in a sealed flask with ethanolic HCl to the
iminoesters and thereafter, again in a sealed flask, under alkaline conditions to the respective
bisbenzamidines 9 and 11 (Procedure A).23
Scheme 2. Preparation of the dinitrile precursor of
15. Atom numbers in accordance with NMR data.
NC
O
O
CN
O S O
N
OH
HO
Cl
Cl
K2CO3
NMP
145 °C
6 h
11
4''
3''
5''
6''
10
10'
2''
7'
1''
2
5
2'
5'
NC7
O
O
O
CN
O S O
N
1
3
1'
6'
3'
4'
8
8'
6
4
O
9
9'
15a
5

For the synthesis of 15a, the nitrile precursor of 15, the terminal heteroatoms in the bridge
were (as in pentamidine) oxygens; this time, the leaving halogens were on the spacer N,N-
bis(2-chloroethyl)-p-toluenesulfonamide, and the bridge oxygens were introduced with the
benzonitrile (Scheme 2).
The piperazine and homopiperazine derivatives are known compounds15,29,30; also several of
the open chain analogs have been described31; 40, 43 and 44 are described here for the first
time and were prepared via the bisbenzonitriles in 3 steps. The first one was equivalent to step
one in Scheme 1 (with DMF as the dry solvent). As difunctional spacers we used N,N’-
dimethylpropylenediamine, 1,7-diaminoheptane and 1,8-diaminooctane, respectively. The
obtained bisbenzonitriles were converted into the corresponding bisbenzamidines via the
bisbenzamidoximes (Procedure B).31
Scheme 3. Preparation of the
dinitrile precursor of 71.
S
S
NC
Br
Br
CN
Sn
Sn
S
Pd(PPh3)4
reflux
12 h
1,4-dioxane
S
S
NC
CN
S
71a
Most of the remaining compounds are known32,33,34,35,36,37,38 (see Table S1). The dinitrile
precursor 71a for the terthiophene derivative 71 was prepared by Pd-catalyzed coupling of 2
eq 5-bromo-2-cyanothiophene to 1 eq 2,5-bis(trimethylstannyl) thiophene (Scheme 3) as
6

described for the addition of 2 phenyl residues to a central thiophene.39 The 3,3’-bipyridine
bisbenzonitrile 76a was prepared in 2 steps, starting with Pd-catalyzed coupling of 2,5-
dibromopyridine to 4-cyanophenylboronic acid (Scheme 4) as described for the synthesis of
para-terphenyl.36 The resulting bromo nitrile 76b was dimerized (once more under Pd
catalysis) to the bisbenzonitrile 76a. Both bisbenzonitriles 71a and 76a were finally converted
to the respective bisbenzamidines 71 and 76 by LHMDS in THF (Procedure C).36
Scheme 4. Preparation of the dinitrile
precursor of 76.
OH
OH
HO
HO
CN
B
Br
Br
B
CN
Br
Br
N
N
Pd(PPh3)4, Na2CO3, toluene
N2 12 h 80 °C
CN
Br
Br
CN
N
N
76b
(Bu3Sn)2,
N2
Pd(PPh3)4,
toluene
6 h
120 °C
CN
CN
N
N
76a
3. Results and Discussion
3.1. Variation of bridge heteroatoms
The polyamine-sensitivity of the NR is one of the main physiological parameters evaluated in
this study. We define this sensitivity as the ratio of two IC50s; the IC50 of a polyamine-
sensitive inhibitor of [3H]MK-801 binding will be higher (weaker) in the presence of the
polyamine (here 30 µM spermine) than in its absence. Replacing in the connecting chain of 1
the middle methylene group by oxygen (2; sometimes called ‘γ-oxa-pentamidine’), but not by
7

sulphur (4), converted a moderately spermine-sensitive inhibitor into a highly spermine-
sensitive one (in the absence of added spermine the potencies of 1, 2 and 4 were similar).
Changing the 2 distal oxygens in the highly spermine-sensitive 2 to sulphurs yielded with 5 a
compound more potent and more spermine-sensitive than pentamidine (although not reaching
to the spermine-sensitivity of 2). Introducing nitrogens instead of O or S had even more
pronounced consequences. A nitrogen atom in the center of the pentamidine bridge introduces
a positive charge at physiological pH, while at the distal positions nitrogen has no such
consequences (due to the neighboring aromatic nuclei). Indeed, 12 with central nitrogen acted
much weaker than pentamidine, while at the two distal positions (7) N did not change at all
the properties of pentamidine (inhibition of the NR by this compound has already been
described40). Again, also in this molecule an oxygen in the center (10) conferred very high
sensitivity to spermine (as in 2), but this time also the absolute potency was increased (by a
factor 4.0). Remarkably, the potency of 7 (so to say ‘diaza-pentamidine’) was increased
considerably (by a factor 5.5) by substituting the Ns with methyl groups (giving 8; without
changing sensitivity to spermine). The high potency of 10, with O in the middle and Ns
vicinal to the aromatic rings, was not further increased by N-methylation (11), and the
extreme spermine sensitivity of 10 was reset to ‘normal’ by this modification.
X
X
O
O
Y
O
H2N
RY
NH2
NH2
HN
NH
RP
RP
6
NH2
NH
HN
1-5, 7-15
Table 1. Inhibition of [3H]MK-801 and [3H]ifenprodil binding to rat cortical membranes by
pentamidine analogs with heteroatom variations in the bridge. Number of experiments (n) in
parentheses; if n = 2, no SD is indicated (see supporting information for single values); spm,
spermine; n.d., not determined. The column ‘ratio’ lists the mean ratios IC50 (30 µM spm) /
8

IC50 (no spm). N12N, diaminododecane; N4T8N, 5-(4-aminobutyl)-2-thiopheneoctanamine41;
Bs, benzenesulfonyl.
[3H]MK-801
IC50 SD (n), µM
[3H]ifenprodil
IC50 SD (n)
no spm
X
-
YRY
-
RP
no spm
30 µM spm
196 (2)
ratio
4.60 1.71 (4)
arcaine
-
33.6 (2)
24.4 (2)
960 (2)
49.3 (2)
32.5 (2)
63.8 (2)
43.7 (2)
19.2 (2)
n.d.
7.05 2.38 (11) 245 30 (5) 32.9 6.3 (5)
0.52 0.32 (46) 6.10 1.43 (24) 15.1 7.1 (24)
2.68 0.65 (9) 24.9 5.7 (7) 9.1 2.5 (7)
3.41 0.52 (4) 148 22 (3) 40.6 4.2 (3)
N12N
-
-
-
N4T8N
-
-
-
O
O
O
O
S
O
CH2
O
-
1
2
3
4
5
6
7
-
10.6 3.9 (4)
2.20 0.32 (3)
343 27 (3) 33.1 11.7 (3)
29.1 (2) 12.3 (2)
O
OMe
S
-
-
-
-
-
1.22 0.23 (3) 23.8 2.7 (3) 20.2 6.1 (3)
317 20 (3)
O
O
785 (2)
2.59 (2)
n.d.
3.10 0.11 (3) 42.3 1.9 (3) 13.7 0.6 (3)
0.56 0.08 (4) 5.62 2.88 (4) 9.2 3.5 (4)
NH CH2
NMe CH2
20.6 5.4 (3)
16.6 (2)
4.54 1.43 (3)
11.4 3.7 (3)
21.9 5.2 (3)
n.d.
8
NH CH2 OMe
2.88 (2)
20.7 (2)
7.2 (2)
9
0.85 0.23 (7) 38.5 10.4 (6) 50.6 19.7 (6)
0.76 0.15 (4) 12.5 1.8 (3) 15.2 2.2 (3)
21.2 5.1 (3)
NH
NMe
O
O
O
-
-
-
-
-
10
11
12
NH+Me
N-Bs
NTs
143 (2)
7.65 (2)
0.23 0.06 (4) 2.13 0.17 (3) 10.4 1.56 (3)
0.24 0.08 (5) 1.32 0.23 (4) 6.28 1.18 (4)
3.77 1.37 (4) 28.7 4.4 (3) 8.11 2.91 (3)
O
1.47 0.55 (3)
0.41 0.13 (3)
0.84 0.48 (4)
13
14
15
O
O
NTs OMe
9

Already these first observations demonstrate that absolute potencies and spermine-
sensitivities of pentamidine congeners follow separate SARs. Substitution with methoxy at
the aryl nuclei had minor or unfavorable influence (3 weaker than 2; 9 similar to 7; 15 weaker
than 14), but aromatic substituents at a central N were always favorable, most likely because
they take away the central positive charge. This latter strategy resulted in the most potent
compounds of this series (13, 14), with spermine-sensitivities equivalent to or slightly weaker
than that of pentamidine. Since the loss in potency after central introduction of N was not only
equalized, but even reversed to a considerable gain, the aromatic residue may find a partner
for favorable interaction in this middle part of the pharmacophore, as already suggested by
previous studies.41,42,43 Imidazoline instead of amidine substituents (as in 6) were not tolerated
at this target, as already shown for other pentamidine derivatives.44
3.2. Piperazine and homopiperazine as bridge
N,N’-Bis(4-amidinophenyl)homopiperazine (24)15 may be seen as related to the diaza-analogs
7 and 8 described above, constraining the bridge between the 2 Ns into a strictly defined ring.
All homopiperazine- and piperazine-bridged bisbenzamidines inhibited the NR with similar
spermine-sensitivities (most of them slightly above the sensitivity of 1), but the prototypic 24
clearly stands out from the rest by its absolute potency: none of the 17 analogs in Table 2 (16-
23, 25-33) came close to this value (not even by a factor 10). All modifications, be it by
switching from a seven- to a six-membered ring, be it by adding alkyl substituents to the
terminal amidino groups or by joining these groups to five- or six-membered rings, resulted in
a clear loss of biological activity. Remarkably, the advantage of the prototypic
homopiperazine 24 (IC50 0.25 µM) over the prototypic piperazine 16 (IC50 3.08 µM)
disappeared upon amidine alkylation (IC50s of all compounds around 10 µM). This is in
10

contrast to favorable effects of amidine alkylation in pentamidine analogs with furan- and
pyridine dicarboxamide bridges (see below, Table 3, 63-66).
H
N
+
N
H
H
N
+
N
H
R NH
+
NH2
+
HN R
N
N
N
N
R
R
R
H2N
16-21
22, 23
H
R N
H
N
+
N
H
H
N R
H
N
+
N
H
N
N
N
N
+
+
NH2
R
32, 33
H2N
24-31
R'
R
N
N
N
H2N
+
H2N
N
H2N
+
H2N
NH2
+
H2N
NH2
+
NH2
R
R'
39, 40
34-38
Table 2. Inhibition of [3H]MK-801 and [3H]ifenprodil binding to rat cortical membranes by
pentamidine analogs with piperazine and homopiperazine bridge (including open-chain
isosters). Definitions as in caption to Table 1; c-Pent, cyclopentyl.
[3H]MK-801
IC50 SD (n), µM
[3H]ifenprodil
IC50 SD (n)
no spm
R
H
R’
-
no spm
30 µM spm
70.7 (2)
ratio
3.08 0.59 (4)
21.4 5.2 (4)
19.4 7.4 (4)
15.9 4.9 (4)
10.3 3.6 (5)
26.2 (2)
17.0 (2)
n.d.
16
17
18
19
20
21
22
-
544 34 (3) 32.9 5.8 (3)
i-Pr
-
c-Pr
n-Pr
n-Bu
c-Pent
(CH2)2
466 (2)
371 (2)
21.8 (2)
21.8 (2)
n.d.
-
n.d.
-
139 32 (4) 16.0 3.2 (4)
n.d.
-
9.25 3.85 (5) 159 59 (4) 19.3 6.4 (4)
6.43 1.64 (4) 106 20 (3) 15.1 4.1 (3)
n.d.
-
n.d.
11

-
-
-
-
-
-
-
-
-
-
-
23.8 8.9 (5)
264 26 (3) 13.6 5.2 (3)
(CH2)3
H
n.d.
19.1 (2)
n.d.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.25 0.07 (14) 5.47 1.42 (6) 20.4 7.3 (6)
17.7 4.8 (4)
11.2 2.9 (4)
34.6 4.1 (3)
9.94 3.72 (3)
389 85 (3) 25.3 10.5 (3)
212 32 (4) 19.9 5.7 (4)
i-Pr
c-Pr
t-Bu
n-Bu
c-Pent
2-HO-Et
3-HO-Pr
(CH2)2
(CH2)3
H
n.d.
562 (2)
16.8 (2)
n.d.
103 3 (3)
11.2 3.3 (3)
n.d.
8.13 0.29 (3) 85.7 7.8 (3) 10.6 1.2 (3)
9.44 2.70 (6) 324 34 (5) 35.7 6.9 (5)
n.d.
n.d.
41.2 13.2 (4)
3.03 0.21 (3)
12.8 4.3 (3)
427 (2)
92.2 (2)
9.72 (2)
30.1 (2)
n.d.
n.d.
143 15 (3) 11.7 2.5 (3)
n.d.
2.01 0.76 (6) 50.7 13.8 (5) 30.2 10.4 (5)
H
H
0.76 0.05 (3)
7.14 (2)
13.0 (2)
16.9 (2)
9.16 (2)
77.7 (2)
31.2 (2)
3.18 0.60 (3)
Me
85.3 (2)
30.2 (2)
2.61 0.55 (4) 48.1 6.5 (3) 19.3 7.8 (3)
Et
H
2.71 0.41 (3)
1.14 0.19 (3)
10.0 2.3 (3)
Me
Me
Et
54.5 (2)
13.8 (2)
148 (2)
20.9 (2)
11.8 (2)
17.0 (2)
Et
H
H
1.84 0.65 (4) 24.7 0.8 (3) 14.7 5.3 (3)
Me
Me
Breaking up the successful homopiperazine ring sets back the high potency of 24 to the
‘normal’ level of pentamidine (34 with short –CH2CH2- bridge) or even below (39 with long –
CH2CH2CH2- bridge), while keeping the somewhat increased sensitivity to spermine. This is
surprising, since formally the longer propyl bridge is closer to the pentyl bridge of
pentamidine than the shorter ethyl bridge. Apparently, the 5 carbons chain in pentamidine
forms a rather sharp loop, bringing the 2 benzamidine moieties as close as in 34 and closer
12

than in 39. The potency of 34 was not increased by providing methyl or ethyl substituents to
the ethyl bridge: 35-37 exhibited all pentamidine-like potencies (only the diethyl derivative 38
with IC50 1.14 µM exceeded somewhat this potency). On the other hand, the rather poor
performance of the propyl bridge (in 39 with IC50 10 µM) was raised to the pentamidine level
by methyl substituents (in 40 with IC50 1.84 µM). These results suggest a double role of the
bridge, tuning the extension and orientation of the molecule and providing itself favorable
attachment points. It might be worth to explore larger substituents, given the success of
central aromatic substituents in the first series (13 & 14).
3.3. Bridges of increasing length
The shorter chain homologs of pentamidine (butamidine and propamidine) have been tested
as inhibitors of [3H]MK-801 binding to rat brain membranes,40 returning the IC50s 2.59, 17.7
and 5.54 µM for 5, 4 and 3 methylene groups between the oxygens, respectively. Also in our
diaza-analogs we observed two optima, one with an ethyl and another with a pentyl bridge (34
and 7; Table 3).
H2
X
C
X
X
Ar
X
n
Y
Y
Y
Y
H
H
N
NH2
+
NH2
H2N
N
R
R
+
NH2
+
H2N
+
56-67
7, 34, 39, 41-55
NH2
Table 3. Inhibition of [3H]MK-801 and [3H]ifenprodil binding to rat cortical membranes by
pentamidine analogs with aliphatic linkers of increasing length, and with aromatic linkers.
Definitions as in caption to Table 1; Fur, furandiyl; Pyr, pyridinediyl.
[3H]MK-801
IC50 SD (n), µM
[3H]ifenprodil
IC50 SD (n)
13

X
Y
n
R
-
Ar
-
no spm
30 µM spm
ratio
no spm
0.76 0.05 (3)
77.7 (2)
2.01 0.76 (6) 50.7 13.8 (5) 30.2 10.4 (5)
NH CH2
NH CH2
NH CH2
NH CH2
NH CH2
NH CH2
NH CH2
0
1
2
3
4
5
6
34
39
41
7
10.0 2.3 (3)
4.55 1.74 (3)
-
-
148 (2)
84.2 (2)
17.0 (2)
17.2 (2)
-
-
n.d.
3.10 0.11 (3) 42.3 1.9 (3) 13.7 0.6 (3)
-
-
20.6 5.4 (3)
4.37 0.32 (3)
4.67 0.38 (3)
7.26 (2)
-
-
-
-
-
-
-
-
-
-
-
-
23.9 (2)
25.4 (2)
31.1 (2)
5.26 (2)
5.37 (2)
4.37 (2)
n.d.
n.d.
42
43
44
n.d.
230 97 (4) 675 131 (3) 2.81 0.76 (3)
NH CO 3
NH CO 4
NH CO 5
54.5, 65.6 (2)
n.d.
45
46
47
48
49
165 68 (3)
>1000
437 (2)
>1000
3.46 (2)
n.d.
n.d.
22.9 6.7 (4) 170 15 (3) 7.25 1.95 (3)
26.5 2.6 (3) 111 13 (3) 4.25 0.94 (3)
15.7 2.2 (3) 62.6 4.8 (3) 4.07 0.96 (3)
9.63 1.45 (3) 27.7 6.9 (3) 2.98 0.58 (3)
NH CO 6
NH CO 7
-
-
-
-
n.d.
n.d.
NH CO 8
NH CO 10
CO NH 3
CO NH 4
CO NH 5
CO NH 6
-
-
-
-
-
-
-
-
-
-
-
-
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
50
51
52
53
54
55
56
57
>1000
>1000
688 (2)
266 (2)
>1000
>1000
n.d.
n.d.
>1000
n.d.
1208 (2)
4.59 (2)
2.19 0.85 (5) 18.4 3.2 (4) 10.1 3.2 (4)
1.46 0.60 (4) 5.54 1.23 (3) 4.00 0.93 (3)
0.79 0.32 (4) 6.70 0.89 (4) 9.04 1.93 (4)
5.24 1.12 (4) 25.7 0.9 (3) 5.25 1.29 (3)
CH2
O
-
-
-
-
H 1,2-Ph
H 1,2-Ph
H 1,3-Ph
H 1,4-Ph
7.29 2.85 (4)
1.42, 2.05 (2)
3.97 1.59 (3)
10.8, 16.8 (2)
38.6, 27.0 (2)
O CH2
O CH2
O CH2
58
59
60
10.8 1.7 (3)
O
CO - H 1,4-Ph
46.6 (2)
3.98 (2)
14

42.3 6.8 (4)
53.9 2.4 (3)
152 10 (3)
NH CO - H 1,3-Ph
NH CO - H 1,4-Ph
NH CO - H 2,5-Fur
NH CO - i-Pr 2,5-Fur
NH CO - H 2,6-Pyr
36.7 (2)
52.9 (2)
0.97 (2)
0.96 (2)
n.d.
n.d.
61
62
63
64
65
153 7 (3) 1.01 0.02 (3)
n.d.
10.6 1.3 (3) 174 45 (3) 16.9 6.0 (3)
58.2 (2) 70.9 (2) 1.22 (2)
6.94 1.22 (3) 74.9 13.5 (3) 10.8 1.2 (3)
n.d.
>100
NH CO - i-Pr 2,6-Pyr
50.1, 49.4 (2)
n.d.
66
67
CO NH - H 2,6-Pyr
125 (2)
233 (2)
1.85 (2)
We also tested the influence of chain length with two other types of bridges. In the first one,
the carbons vicinal to the nitrogens were carbonyl groups, changing the chain geometry at this
position from sp3 (tetraedric) to sp2 (planar), with dramatic consequences. Although the
dianilide 45 was equivalent in total bridge length to pentamidine, it turned out 100-fold
weaker, with practically no sensitivity to spermine; the same was true for 46, with butyl
between the two amide groups (-NH-CO-butyl-CO-NH-). The next homolog 47 was still
weaker (IC50 > 1 mM). Surprisingly, activity returned in the higher homologs 48-51, even
with some indication for spermine sensitivity (48). Potencies were very weak, if the amide
groups were inverted (-CO-NH-butyl-NH-CO-, 53), and also here the weakest compounds
were those of middle size. We conclude that the chain linking the two benzamidine moieties
should either be short or should exhibit enough degrees of freedom for folding to adopt the
critical short distance between the end charges. With rigid segments like amide bonds, such a
distance appears difficult to achieve.
3.4. Aryl inserted into the bridge
Finally, we explored the influence of aromatic nuclei inserted into the bridge linking the two
benzamidine moieties. This kind of modification was not only well tolerated, but even
15

increased the potency, if the geometry of pentamidine was maintained as in 58 (IC50 0.79
µM). In this compound, still a continuous chain of 5 carbon atoms separated the 2 oxygens.
Also a chain of 4 carbon atoms as in 57 was compatible with high potency (IC50 1.46 µM),
but only with quite low spermine sensitivity. Interestingly, this sensitivity was restored simply
by switching atom order from -O-C-(1,2-Ph)-C-O- (57) to -C-O-(1,2-Ph)-O-C- (56), with no
loss in potency. Inserting the phenyl nucleus in 1,4 orientation (59) was less favorable.
Still weaker compounds resulted from insertions of an aromatic group between two amide
bonds, nevertheless returning some surprising SARs. While two ester bonds on both sides of a
central phenyl nucleus (with the alcoholic oxygens at the outer positions; 60) were tolerated
almost as well as the ether bonds in 59, amide bonds (with the nitrogens at the outer positions;
61 and 62) led to a strong drop in potency and to the complete loss of spermine sensitivity.
Replacing the phenyl nucleus between these amide bonds by a furan nucleus (63) lowered
these figures even more, but to our surprise potency returned (IC50 10.6 µM) upon alkylation
of the distal amidine groups (64), with parallel restoration of spermine sensitivity. An
analogous phenomenon was observed after insertion of a pyridine nucleus: weak potency
without (65), but appreciable potency with amidine alkylation (66; IC50 6.94 µM), also here
with remarkable restoration of sensitivity to spermine. It should be taken into consideration,
however, that these alkylations achieved medium potencies starting from a rather weak level
(similar potencies had been achieved by alkylations of 16 and 24 - Table 2 - starting from a
much higher level).
16

Table 4. Inhibition of [3H]MK-801 binding to rat cortical
membranes by pentamidine analogs with additional
aromatic rings. Definitions as in caption to Table 1.
IC50 SD (n), µM
no spm 30 µM spm
ratio
4.23 2.47 7.48 2.51 2.37 1.69
(4) (4) (4)
H2N
+
NH2
+
O
68
NH2
H2N
HN
HN
6.97 2.15 24.0 19.2 3.47 2.20
H2N
+
O
NH2
+
(3)
(3)
(3)
69
NH
12.2
(2)
30.7
(2)
2.56
(2)
H2N
+
O
NH2
+
70
NH
NH2
NH2
S
S
5.34
(2)
6.76
(2)
1.50
(2)
+
NH2
+
H2N
S
71
6.68 1.25 17.7 6.3 2.61 0.61
H2N
+
H2N
S
NH2
+
NH2
(4)
(4)
72
(4)
F
9.9 4.4
(3)
34.2
(2)
4.63
(2)
NH2
+
NH2
H2N
+
H2N
73
H
N
NH2
3.21
(2)
13.4
(2)
4.14
(2)
H2N
+
H2N
+
NH2
S
N
74
H2N
NH2
S
5.01 3.28
(3)
11.7
(2)
4.26
(2)
O
+
H2N
+
NH2
O
S
75
H2N
NH2
+
26.8 13.1 30.0 6.7 1.24 0.38
+
H2N
N
N
NH2
76
(3)
(3)
(3)
17

If aryl nuclei were inserted without interspersed flexible linkers, compounds were obtained
with absolute potencies (Table 4) either similar to pentamidine (68, 71, 74, 75) or slightly
weaker (69, 70, 72, 73, 76). In all these compounds, however, sensitivity to spermine was
practically absent. In the bioisosteric sequence 71, 72 and 73, potencies seem to weaken with
increasing distance between the distal charges (IC50s 5.3 < 6.7 < 9.9 µM). The low potency of
the tetracyclic 76 may be due to the still longer distance between them. Although 75 was also
a tetracyclic compound, here the chain of five-membered rings would allow for some
adjustment of length, possibly explaining the higher potency of this compound. Such
flexibility cannot explain the relatively high potency of the benzimidazole-containing 74, one
of the more extended molecules in this series; here, the particular imidazole signature may
have contributed favorably to receptor interaction.
3.5. Inhibition of [3H]ifenprodil binding
Polyamines activate the NR at an interface between the subunits GluN1 and GluN2B.18,21 This
site is formed by acidic amino acid residues contributed by both extra-cellular N-terminal
domains; their neutralization by polyamines is hypothesized to result in the close apposition
of these domains, facilitating the activation of the NR by agonists. A second site located more
externally mediates inhibition of the NR by ifenprodil.21 No high affinity ligands have been
described binding directly to the first site; however, the outer site can be labeled with
[3H]ifenprodil. Compounds can usually be tested directly for their affinity to this site via their
potency to displace this radioligand from neuronal membranes, with the precaution that
indirect influences of polyamines on [3H]Ifenprodil binding have been demonstrated.45
18

A
7
6
5
4
3
13
14
24
8
N4T8N
40
10
38
56
58
11
4
57
34
37
16
36
2
1
9
7
15
35
59
66
3
N12N
arcaine
60
39
45
3
4
5
6
7
-Log IC50 ([3H]Ifenprodil)
B
2.0
1.6
1.2
0.8
0.4
10
2
arcaine
N12N
35
34
3
16
37
24
39
36
11
7
N4T8N
66
40
38
56
4
8
13
1
58
15
9
14
59
57
60
45
3
4
5
6
7
-Log IC50 ([3H]Ifenprodil)
Figure 1. Inhibition by 31 dicationic compounds at the
ifenprodil site of the NR correlates with inhibition at the
channel binding site (A, interrupted line; r2 = 0.22, p <
0.01), but not with the sensitivity of this inhibition to 30
µM spermine (B; r2 = 0.08, n.s.); data from Tables 1-3.
Some of our test compounds were quite potent displacers of [3H]ifenprodil (last column in
Tables 1-3). The most potent was 14, a pentamidine with tosylated N in the middle of the
bridge (IC50 0.41 µM), that was also a potent displacer of [3H]MK-801 (0.24 µM). Quite
potent too were 15 (the methoxy derivative of 14; 0.84 µM), 13 (also a congener of 14; 1.47
µM), 34 (the diaza analog with the shortest bridge; 0.76 µM), and 57 (with phenyl in the
19

bridge; 1.74 µM). Of all 31 compounds tested at both sites, only 3 were more potent at the
‘IF-site’, the other 28 more potent at the ‘MK-site’ (9 of them even more than 10-fold).
Affinities of 31 compounds tested at both sites (including 3 prototypic polyamine inverse
agonists) were significantly correlated to each other (Figure 1A). The correlation line had no
unity slope; high affinity compounds were more similar at both sites, but low affinity
compounds more dissimilar. On the other hand, affinity at the IF-site was no predictor of
spermine-sensitivity at the MK-site (Figure 1B).
3.6. Modeling of potential sites of action
In an attempt to identify the possible molecular target mediating spermine-sensitive inhibition
of the NR by bisbenzamidines, we performed docking calculations for the 45 most spermine-
sensitive bisbenzamidines (see supporting information, Figure S1) at a site proposed to
mediate polyamine-stimulation of the NR.18,21,22 The endogenous polyamines spermidine and
spermine (and also Mg2+) by virtue of their positive charges neutralize surface charges on the
N-terminal domains of subunits GluN1 and GluN2B. In the non-stimulated state (Low Po
mode; Po stands for open probability), these negative surface charges keep the GluN1/2B
domains apart from each other. Polyamines induce the High Po mode by inserting themselves
between the domains, leading them together. Antagonists interfering with this mechanism can
be expected to stabilize the Low Po mode, by binding to only one of the 2 surfaces (without
leading them together). Although stimulation of the NR by polyamines exhibits parallels to
stimulation by increased pH, not only GluN1/2B, but also GluN1/2A and GluN1/2D
combinations are subject to proton inhibition.46 Systematic examination of mutated receptors
did not result in correlations between spermine and proton sensitivities,18 and also in our own
studies,42,47 inhibition by similar dicationic compound families was differently influenced by
spermine and pH. Thus, stimulation by polyamines seems to depend on the pattern of specific
20

acidic amino acid residues on the domain surfaces, rather than on the general neutralization of
a local acidic milieu. Bisbenzamidines were docked separately to the putative polyamine
domains either of GluN1 or of GluN2B, departing from the whole GluN1/N2B complex in the
Low Po mode, i.e. with the domains separated from each other. We obtained highly significant
(r2 = 0.42; p<0.001) correlation for GluN1 (Figure 2A), but not for GluN2B (Figure 2B). No
correlation was obtained with the domains close to each other (not shown). We conclude that
more than 40% of the variability in the potency of these bisbenzamidines is explained by their
interaction with targets on the polyamine domain of GluN1 in the Low Po mode. A still higher
correlation may have been obtained at pure GluN1/2B heterodimeric NRs; the majority of
native NRs as used by us, however, seem to consist of GluN1/2A/2B heterotrimers,48 with
consequences for the GluN1/2B interface.49 The GluN1/2B selective antagonist ifenprodil
blocks with high affinity only 23% of NRs in our native membranes prepared from rat
frontoparietal cortex and hippocampus.50 Nevertheless, we tried to dock the same compounds
also to the ifenprodil site proposed more externally at the GluN1/2B interface.21 The obtained
correlation (r2 = 0.18; Figure S2B), while less pronounced than for GluN1, was significant
(p<0.01). Thus, also this site may have contributed to the variability of inhibitory potency;
however, for none of these compounds we observed partial inhibition of [3H]MK-801 binding
as typical for inhibition of [3H]MK-801 binding by ifenprodil, and in general binding of
[3H]MK-801 was more potently inhibited than binding of [3H]ifenprodil (Figure 1A). To
some extent, significant docking of bisbenzamidines also to the IF-site may be explained by
mutual influences between spermine and ifenprodil at native NRs.44
21

A
60
40
20
58
10
13
56
4
15
5
9
14
48
8
1
7
35
2
37
11
41
31
38
39
12
34
16
3
30
36
24
40
66
64
21
17
28
20
32
29
18 33
26
19
23
22
27
25
0
4
5
6
7
-Log IC50 ([3H]MK-801)
B
60
31
38
13
30
41
9
8
64
48
12
5
56
3
7
35
66
39
20
10
58
4
15
2
29
28
21
26
34
40
14
36
37
25
19
1
18
27
11
40
20
0
24
16
32
22
17
23
33
4
5
6
7
-Log IC50 ([3H]MK-801)
Figure 2. Relationship between goodness of fit to a
polyamine site modeled at the GluN1/2B interface and
potency to inhibit the binding of [3H]MK-801 to native rat
brain membranes. (A) Docking to GluN1, r2 = 0.42. (B)
Docking to GluN2B, r2 = 0.06.
22

Figure 3. Binding mode of an overlay of 1 with the most
potent analogs (5, 8, 10, 11, 13, 14, 38, and 58 - with
omission of 24) to the spermine binding site of GluN1 in
the Low Po mode. Six amino acid residues of GluN1
known to influence spermine stimulation and the
bisbenzamidines are represented as sticks with carbon
atoms in coral and green, respectively. Surface
representation of 1 is colored light blue.
23

Finally, we created an overlay of pentamidine (1) with 8 of the most potent analogs (5, 8, 10,
11, 13, 14, 38, 58), docked to the amino acid residues on GluN1 allegedly mediating
stimulation by polyamines (Figure 3). Bisbenzamidines with flexible linker (as 1 itself) were
able to partially wrap the helix α5 containing amino acid residues E185, E186 and E181, the
amidine groups approaching the negatively charged ω-carboxylic groups. On the other hand,
given the high number of acidic amino acid residues at this interface, several other modes of
interaction may be possible as well, and not just the one suggested in the figure. The
homopiperazine-bridged compound 24 did not superimpose with these compounds (not
shown); although of high potency, it did not yield high docking scores in this model. Also
other bisbenzamidines with inflexible bridge (22, 25) yielded Gold Fitness scores below
expectation (see Figure 2A).
3.7. Competition or no competition?
Already Reynolds & Aizenman,7 the discoverers of pentamidine as NR antagonist,
demonstrated that inhibition of [3H]MK-801 binding by pentamidine was counteracted by the
endogenous polyamine spermidine (the shorter-chain homolog of spermine). Since in their
hands pentamidine did not only shift der spermidine dose/response curve to the right, but also
reduced maximum stimulation, they excluded competitive interaction. Similar data were
produced by us for pentamidine and 2 congeners, compounds 10 and 13 (Fig. 1S,
supplementary information) in ‘competition’ with spermine. Earlier studies by two of us
(MLB, PR)50 had demonstrated that interaction between dicationic long-chain inhibitors as
diaminododecane (N12N) and pentamidine with spermine-stimulation of [3H]MK-801
binding to native rat membranes was not pronounced enough to be explained by direct
competition alone. Also another observation argues against simple direct competition: While
polyamine-stimulation of NRs is limited to NRs with GluN2B subunit,18 the prototypic
24

inverse polyamine agonist arcaine inhibits also NRs without GluN2B.51 It also remains to be
explained why ‘polyamine inverse agonists’ reduce [3H]MK-801 binding in well washed
neuronal membranes (with no effective amounts of the endogenous polyamines spermidine
and spermine left). Do they even then drive open to the Low Po mode the GluN1/N2 interface,
and against which ‘polyamine-like’ agent? Evidently, spermine and spermidine on the one
side and dicationic inhibitors on the other side have additional effects. We interpret our
docking data as indication that a considerable part of benzamidines’ inhibition is mediated by
targets at the GluN1/2B interface also mediating stimulation by spermine. This includes
competition, but leaves ample room for other activities of both compound families.
4. Conclusions
Motivated by the high potency of the homopiperazine-bridged bisbenzamidine 24 as
polyamine-sensitive NR inhibitor, we undertook a detailed SAR study with 76 pentamidine
analogs prepared in 5 different synthetic laboratories. We identified 9 spermine-sensitive
inhibitors with higher potency than 1 (see Figure S4). Four of them (5, 8, 10, 11) were
isosteric to 1, with heteroatom variations in the bridge. Three others had aromatic rings
inserted in (58) or attached to the bridge (13, 14), confirming earlier observations pointing to
a role for such aromatic rings in target interaction.41-43 Among analogs with shorter bridge,
only 38 with 2 ethyl substituents was more potent than 1 (ANOVA, p < 0.05). Ironically, the
lead homopiperazine 24 stayed the only high-potency bisbenzamidine of this structural type;
none of the other homopiperazines came close. In addition, highly significant docking to the
GluN1 part of the polyamine-sensitive GluN1/2B interface did not include 24: the docking
score for 24 was even lower than that obtained for 1. Thus to provide a theoretical explanation
for the particular behavior of 24, we still need more refined docking models based on the
molecular landscape of the real (and still unknown) polyamine-sensitive GluN1/2B interface
25

in heterotrimeric NRs constituting the majority in native rat hippocampal and cortical
neurons.49
5. Experimental Section
All chemicals were purchased from major chemical suppliers as high or highest purity grade
and used without further purification. Melting points were determined with an Electrothermal
9001 Digital Melting Point. 1H NMR spectra in solution were recorded with a Bruker Avance
DMX 400 or with Varian 300 V NMR S, and chemical shifts δ (ppm) in solutions were
referenced to TMS (the solvent is given with the spectral data). Atom numbers are given in
Schemes 1 & 2. Elemental analyses or HRMS are provided for new compounds.
5.1. New syntheses
The bisbenzamidines 9, 11, 15, 40, 43, 44, 71 and 76 were prepared as described below. For
the conversion of the precursor bisbenzonitriles into bisbenzamidine hydrochlorides, 3
different procedures were used:
Procedure A
The appropriate dinitrile (5 mmol) was stirred in a sealed flask with a saturated solution of
HCl in anhydrous ethanol (25-50 ml) for 20-40 h at rt. The solvent was evaporated in vacuo
(or dry diethyl ether was added until a solid was precipitated). The solid residue was filtered,
washed with diethyl ether and dried under vacuum over anhydrous CaCl2 for 2-6 h. The crude
iminoester obtained in high yield was added to a saturated solution of NH3 in anhydrous
ethanol (25-50 ml). The mixture was stirred at rt for 24-48 h in a sealed vessel. The solvent
was evaporated in vacuo, and the solid residue was mixed with 10% aq NaOH (10 ml). The
26

precipitate of free amidine was filtered, washed with water, and dried under reduced pressure
over anhydrous CaCl2. The white microcrystalline solid was suspended in anhydrous
ethanolic HCl (10 ml) and boiled for a few min to obtain the appropriate dihydrochloride salt.
Procedure B
A mixture of hydroxylamine hydrochloride (4.15 g, 60 mmol) and sodium methoxide (3.24 g,
60 mmol) in DMF (15 mL) was heated at 50 °C for 1 h. The precipitate was discarded and the
bisbenzonitrile (5 mmol) was added to the filtrate. The so-obtained mixture was heated at 50
°C for 24 h.
The reaction medium was then poured into water. The obtained
bisbenzamidoxime was collected by filtration and thoroughly washed with water, ethanol, and
finally ether. In the next step, a mixture of the bisbenzamidoxime (3 mmol), ammonium
formate (1.80 g, 30 mmol), and 10% Pd on carbon (0.3 g) in acetic acid (15 mL) was heated
under reflux for 2 h. After cooling the mixture was filtered on a cake of alumina and the
filtrate was concentrated under reduced pressure. Addition of a solution of HCl in isopropanol
(6 M, 15 mL) afforded a precipitate that was washed with water and ethanol to yield the
desired bisbenzamidine (dihydrochloride).
Procedure C
The dinitrile (1 mmol) was allowed to stir overnight in a 1 M solution of LHMDS in THF (20
mL) as described for the conversion of the dinitrile of para-terphenyl to the respective
bisamidine.36
9a 1,5-Bis(4-cyano-2-methoxyphenylamino)pentane
3-Methoxy-4-fluorobenzonitrile (3.02 g, 20 mmol), 1,5-diaminopentane dihydrochloride (1.75
g, 10 mmol) and anhydrous K2CO3 (11.04 g, 80 mmol) were stirred together at 150 °C in
NMP (30 ml) for 3 h (progress of the reaction was followed by TLC). The hot reaction
27

mixture was poured into ice water (400 ml). The obtained solid was filtered, washed with
water and dried. Crystallization from EtOH gave 0.92 g of yellow solid 9a (yield 24%), mp
153-155 oC. 1H NMR (299.87 MHz, CDCl3) δ: 1.53-1.55 (m, 2H, Y=CH2) , 1.72 (quintet, J =
7.2 Hz, 4H, 9- and 9’-CH2), 3.19 (t, J = 6.9 Hz, 4H, 8- and 8’-CH2), 3.85 (s, 6H, 2 x
RP=OCH3), 4.75 (broad s, 2H, 2x RN=NH), 6.50 (d, J = 8.1 Hz, 2H, H-5,-5’), 6.89 (broad s,
2H, H-2,-2’), 7.18 (broad d, J = 8.1 Hz, 2H, H-6,-6’). For C21H24N4O2 (364.45 g/mol) calcd:
C 69.21, H 6.64, N 15.37, found: C 68.92, H 6.91, N 15.03.
9 1,5-Bis(4-amidino-2-methoxyphenylamino)pentane dihydrochloride
This diamidine was obtained by Procedure A from the bisbenzonitrile 9a. The solvent was
evaporated in vacuo. Crude product was dissolved in water and then precipitated by addition
of dry acetone. Precipitated solid was filtered, washed with acetone and dried to give 9 as a
1
yellow solid (yield 37%), mp 283.5-284.5 °C. H NMR (299.87 MHz, DMSO-d6) δ: 1.38-
1.40 (m, 2H, Y=CH2), 1.59 (quintet, J = 7.2 Hz, 4H, 9- and 9’-CH2), 3.18 (m, 4H, 8- and 8’-
CH2), 3.87 (s, 6H, 2 x RP=OCH3), 6.01 (broad s, 2H, 2 x RN=NH), 6.64 (d, J = 8.4 Hz, 2H, H-
5,-5’), 7.33 (d, J = 1.5 Hz, 2H, H-2,-2’), 7.46 (dd, J1 = 1.5 Hz, J2 = 8.4 Hz, 2H, H-6,-6’), 8.56
(broad s, 4H, NH2), 8.90 (broad s, 4H, NH2). For C21H30N6O2 · 2 HCl · 1½ H2O (498.46
g/mol) calcd: C 50.60, H 7.02, N 16.85, found: C 50.43, H 7.05, N 16.48.
11a 1,5-Bis[(4-cyanophenyl)N-methylamino]-3-oxapentane
The bisbenzonitrile 11a was obtained from 2 eq 4-fluorobenzonitrile and 1 eq 2,2’-
methylaminodiethyl ether with K2CO3 in DMF (Scheme 1) as a white solid, mp 88.2-89.4 °C.
1H NMR (299.87 MHz, CDCl3) δ: 2.98 (s, 6H, 2 x RN=CH3), 3.53-3.62 (m, 8 H, 8-, 8’, 9 and
9’-CH2), 6.62-6.65 (m, 4H, H-5,-3,-5’,-3’), 7.42-7.45 (m, 4H, H-6,-2,-6’,-2’). HRMS (ESI-
ToF) M+Na C20H22N4O + Na, calcd: m/z 357.1691, found: m/z 357.1697.
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11 1,5-Bis[(4-amidinophenyl)N-methylamino]-3-oxapentane
This diamidine was obtained by Procedure A from the dinitrile 11a. The white solid was
filtered off and dried at 80° C for 1 h to obtain 0.356 g of 11 (yield 90%) mp 276-279 °C
1
(dec). H NMR (299.87 MHz, DMSO-d6) δ: 2.98 (s, 6H, 2 x RN=CH3), 3.57-3.60 (m, 8 H, H-
8,-9,-8’,-9’) , 6.79-6.82 (m, 4H, H-5,-3,-5’,-3’), 7.70-7.73 (m, 4H, H-6,-2,-6’,-2’), 8.48 (broad
s, 4H, NH2), 8.87 (broad s, 4H, NH2). For C20H28N6O · 2 HCl (441.40 g/mol) calcd: C 54.42,
H 6.85,N 19.04, found: C 54.28, H 6.96, N 18.89.
15a N,N-Bis[2-(4-cyano-2-methoxyphenoxy)ethyl]-4-methylbenzenesulfonamide
3-Methoxy-4-hydroxybenzonitrile (1.49 g, 10 mmol) and N,N-bis(2-chloroethyl)-4-
methylbenzenesulfonamide (1.48 g, 5 mmol) in NMP (20 ml) were stirred at 145 °C together
with anhydrous K2CO3 (2.76 g, 20 mmol) (progress of the reaction was followed by TLC).
After 6 h the reaction mixture was poured into ice water (300 ml). The obtained solid was
filtered, washed with water and dried. The crude product was dissolved in hot EtOH and
filtered hot. The precipitate was purified by column chromatography (Merck Silicagel 60,
230-400 mesh ASTM, eluent CH2Cl2:MeOH – 98:2) to afford 1.31 g of 15a (yield 50%), mp
177-179 °C. 1H NMR (400.13 MHz, CDCl3) δ: 2.34 (s, 3H, 11-CH3), 3.61 (t, J = 6.0 Hz, 4H,
9- and 9’-CH2), 3.69 (s, 6H, 10- and 10’-OCH3), 4.24 (t, J = 6.0 Hz, 4H, 8- and 8’-CH2), 6.82
(d, J = 8.4 Hz, 2H, H-5,-5’), 6.98 (d, J = 2 Hz, 2H, H-2,-2’), 7.16-7.21 (m, 4H, H-6,-6’,-3’’,-
5’’), 7.64-7.66 (m, 2H, H-2’’,-6’’). For C27H27N3O6S (521.60 g/mol) calcd: C 62.17, H 5.22,
N 8.06, S 6.15, found: C 62.03, H 5.47, N 7.86, S 6.32.
15
N,N-Bis[2-(4-amidino-2-methoxyphenoxy)ethyl]-4-methylbenzenesulfonamide
dihydrochloride
This diamidine was obtained by Procedure A from the bisbenzonitrile 15a. The solvent was
1
evaporated in vacuo to give 15 as a yellow solid (yield 86%), mp 233-234 °C. H NMR
29