Allosteric modulators of the tertiary alkanebisamino-type. Variation of the substitution of the middle chain nitrogens

Article abstract of DOI:10.1007/s00706-004-0218-2

Synthesis pathways of high flexibility for variously substituted alkanebisamine-type allosteric modulators of muscarinic receptors capable of passing the blood-brain barrier were developed starting either from N,N′-(hexane-1,6-diyl)bistosylamide or adipic

Full text of DOI:10.1007/s00706-004-0218-2

Monatshefte f€ur Chemie 135, 1539–1556 (2004)
DOI 10.1007/s00706-004-0218-2
Allosteric Modulators of the Tertiary
Alkanebisamino-Type. Variation
of the Substitution of the Middle
Chain Nitrogens
1
2
2
¨
Rainer Pick , Seraina Duda-Johner , Christian Trankle ,
Klaus Mohr2, and Ulrike Holzgrabe1;ꢀ
1
€
Institute of Pharmacy and Food Chemistry, University of Wurzburg,
€
97074 Wurzburg, Germany
2
Institute of Pharmacy, Pharmacology and Toxicology, University of Bonn,
53121 Bonn, Germany
Received April 16, 2004; accepted April 29, 2004
Published online July 23, 2004 # Springer-Verlag 2004
Summary. Synthesis pathways of high flexibility for variously substituted alkanebisamine-type allo-
steric modulators of muscarinic receptors capable of passing the blood-brain barrier were developed
starting either from N,N0-(hexane-1,6-diyl)bistosylamide or adipic acid chloride. Pharmacological
evaluation of some representative compounds revealed the allosteric potency to fall in a submicromolar
range.
Keywords. Alkanebisamine-type compounds; M2-receptors; Structure-activity relationships; Drug
research.
Introduction
Allosteric agents are capable of influencing the binding of agonists and antagonists
to the orthosteric site of a receptor protein. The orthosteric site is the receptor
domain where the endogenous messenger compound binds. Allosteric modulators
are known for a variety of receptors, such as the muscarine, purine, histamine, and
benzodiazepine receptors [1–4]. The treatment of Alzheimer’s disease, pain, or
organophosphate poisoning may take advantage of the use of modulators of the
muscarinic acetylcholine receptors enhancing the equilibrium binding of either
agonists or antagonists.
Muscarinic allosteric modulators described so far [5] are structurally divergent
and many of them are characterized by being positively charged, e.g. alcuronium-,
ꢀ
Corresponding author. E-mail: u.holzgrabe@pharmazie.uni-wuerzburg.de

1540
R. Pick et al.
Fig. 1. Structural formulae of the hexamethonio-type lead compounds W84 and dimethyl-W84
Fig. 2. Structural formulae of the alkanebisamino compounds

Allosteric Modulators
1541
caracurine-, and strychnine-like compounds [6–10], tubocurarine [11], gallamine
[12], alkane-bisammonium-type derivatives such as W84 (see Fig. 1) [13, 14],
Chin3=6 [15], and bispyridinium-type derivatives, e.g. WDUO and IWDUO com-
pounds [16]. The treatment of Alzheimer’s disease and pain requires drugs being
able to pass the blood-brain barrier. Due to their permanent positive charge(s), the
aforementioned compounds are not appropriate for these purposes. Thus, the aim
of this study was to synthesize tertiary alkane-bisphthalimidopropylamine com-
pounds with varying substituents attached to both nitrogens of the middle chain
(see Fig. 2).
Results and Discussion
Synthesis
In a first approach it was tried to synthesize the compounds via an alkylation of
the secondary analogue of W84. In order to obtain the secondary W84, bromo-
propylphthalimide was treated with hexanediamine in polar solvents. Despite
Fig. 3. Synthesis pathway of compounds 1d and 1e

1542
R. Pick et al.
many variations of the reaction solvent and time the phthalic acid 4 could be
isolated only (see Fig. 3). Thus, the Hinsberg method was applied starting with
the synthesis of the N,N0-hexane-1,6-diyl-bistosylamide (2) from hexane-1,6-dia-
mine and tosylchloride in pyridine. The obtained diamide was alkylated by means
of bromopropylphthalimide to give 2 and the tosyl group was eliminated using
48% HBr in glacial acid to obtain W84 3. In order to introduce varying alkyl
groups, the hydrobromide had to be carefully converted into the base. It was im-
possible to alkylate the base with corresponding alkylhalogenides because again
ring-opening resulting in the corresponding phthalic acid occurred in all cases.
However, alkylation via Michael addition using acrylonitrile and ethyl acrylate
gave the cyanoethyl and the ethyl propionate substituted compounds 1d
and 1e.
Since the alkylation of the secondary W84 was not successful another synthesis
strategy had to be applied. Reaction of adipoyl chloride with the corresponding
amine in pyridine resulted in the amides 5h–5o in a short reaction time and in very
high yields. The bisamides 5 could be reduced using diborane-THF to give the
hexanebisamines 6h–6o in rather good yields. The hexanebisamines 6a and 6c
were obtained by amination of dibromohexane with allylamine and aminopropanol
in acetonitrile (see Fig. 4). The compounds 6b, 6f, and 6g having a propine, a
Fig. 4. Synthesis pathway of compounds 1a, 1c, and 1h–1n

Allosteric Modulators
1543
butanone, and a propionamide substituent could not be obtained on either synthesis
pathway.
The tertiary bisamines 1a, 1c, 1i, 1j, 1l, and 1m were directly available by
alkylation with bromopropylphthalimide in presence of an excess of anhydrous
potassium carbonate in acetonitrile. In order to achieve high yields the reaction
time had to be increased up to two weeks. For the synthesis of 1h, 1k, 1m, and 1o a
circuitous route had to be used consisting of alkylation of the bisamine via a
Michael addition with acrylonitrile to give 7h, 7k, 7m, and 7o, reduction of the
nitrile by means of diborane-THF to yield the corresponding 8, and forming the
imide using phthalic acid anhydride. Using methylphthalic anhydride and cyclo-
hexyl substituted 8k compound 9 could be obtained (see Fig. 4). In addition, a
compound was synthesized having the middle chain nitrogen incorporated in a
piperidine ring. Therefore bipiperidine, which is characterized by an inter-nitrogen
distance of six methylene groups, was alkylated by means of bromopropanephthal-
imide to give 10 in an acceptable yield (see Fig. 5).
Physicochemical Properties
Since the compounds have to be capable of passing the blood-brain barrier by
passive diffusion they should be characterized by relatively high lipophilicity.
Therefore, the lipophilicity was evaluated via the capacity factors in HPLC [17].
Using a RP-18 stationary phase and a mixture of methanol=phosphate buffer a
calibration curve was established with references of similar lipophilicity. Thus,
the log P values of 1a, 1c, 1d, 1j, 1k, 1o, and 10 were found to be in the range
of 3 to 4.5 indicating an appropriate lipophilicity for passing the blood-brain bar-
rier (see Table 2). The log P values of the other compounds were higher than 4.5.
Fig. 5. Synthesis pathway of compound 10

1544
R. Pick et al.
With respect to Lipinski’s rule of five [18], log P values higher than 5 will create
problems in terms of solubility.
In order to estimate the pKa values of the new compounds, the pKa values of the
model compounds dimethylaminopropylphthalimide and the secondary W84 were
determined by means of a potentiometric method [25] using the microtitrator PCA
101. The pKa values were found to be in the range of 9 to 10 indicating that 90% of
the compounds will be protonated at pH 7.4 of the physiological media. The pKa
values of the other compounds could not be determined because they are not
soluble over the entire pH range measured. However, the pKa values are very likely
to fall in the same range.
Taken together, it can be stated that the compounds having a log P lower than 4
will be soluble enough for pharmacological evaluation, especially due to the fact
that they are protonated at pH 7.4. On the other hand they will be capable of
passing the blood-brain barrier because a considerable fraction will be neutral
and lipophilic.
Pharmacological Evaluation
The allosteric potency to inhibit the dissociation of the orthosteric agent [3H]N-
methylscopolamine ([3H]NMS) was evaluated as described previously [19, 13]. In
short, homogenates containing muscarinic M2 receptors were prepared from por-
cine heart ventricles, incubated with [3H]NMS in buffer (50 mM Tris-HCl, 3 mM
MgHPO4, pH 7.3, 37ꢁC), and allosteric actions similar to those found under organ-
bath conditions were observed. [3H]NMS-dissociation was measured after addi-
tion of 1 ꢀM atropine to displace the radioligand. Test compounds were added
simultaneously with atropine. The concentration for a half-maximal inhibition of
[3H]NMS-dissociation, EC50,diss, can be taken as a measure for the equilibrium dis-
sociation constant of allosteric modulator binding to [3H]NMS-occupied recep-
tors [20]. Results of the binding experiments of compounds 1k and 9 are displayed
in Table 1 in comparison with W84, and the corresponding tertiary W84. In order
Table 1. Allosteric potencies of representative compounds; pEC50: concentration for a half maximum
inhibition of [3H]NMS dissociation (mean values ꢂ S.E.M from 3 to 7 independent experiments)
Entry
pEC50
W84
6.08 ꢂ 0.07
5.94 ꢂ 0.04
7.08 ꢂ 0.10
6.13 ꢂ 0.18
6.00 ꢂ 0.09
7.35 ꢂ 0.04
4.56 ꢂ 0.13b
7.21 ꢂ 0.12
5.55 ꢂ 0.08
tert. W84
Dimethyl-W84a
1d
1h
1k
2
9
10
a
Data taken from Ref. [26]; b extrapolated value from a curve with the highest data point at 10ꢃ5 M

Allosteric Modulators
1545
to speed up the pharmacological evaluation, compounds 1d, 1h, and 10 as well as
the intermediate tosylamide 2 were checked at two concentrations (10ꢃ6 and
10ꢃ5 M) that allowed to estimate the concentration-effect-relationships.
Structure-Activity Relationships
The lead compounds W84 and dimethyl-W84 are characterized by pEC50 values of
6.08 and 7.08. Omitting one of the N-methyl groups at each of the two nitrogens in
the middle chain of W84 yielding tertiary W84 resulted in almost the same allo-
steric potency. Thus, tertiary amino compounds appear to be as potent as the corre-
sponding quaternary analogues. Even the secondary W84 exhibits an allosteric
potency in the same range of concentration [21]. Thus, a permanent positive charge
is not necessary for a high allosteric potency. However, 2, whose nitrogens are not
basic and, thus, unlikely to be protonated at pH 7.4, is more than tenfold less active
than W84 suggesting that the secondary and tertiary compounds are protonated
upon binding to the allosteric binding site.
Inclusion of the middle chain nitrogen in a piperidine ring (10) hardly influ-
enced the allosteric potency. Similarly, attaching a cyanoethyl (1d) or a phenyl-
propyl group (1h) to the nitrogen did not affect the allosteric potency to inhibit
[3H]NMS-dissociation. In contrast, 1k carrying a cyclohexyl ring was very potent
with pEC50>7. In analogy to the pair W84=Dimethyl-W84 (see Fig. 1), methyl
residues were attached to the lateral phthalimido moieties of 1k yielding 9.
Remarkably, in this pair of compounds (1k=9) lateral ring methylation did not
yield a further gain in activity. Due to low solubility not all of the compounds
could be pharmacologically evaluated properly.
Conclusion
Synthesis pathways for bis(phthalimidopropyl)hexanebisamine compounds of
varying substitution patterns on the middle chain nitrogens were opened. Thus,
allosteric modulators are available, which are probably capable of passing the
blood-brain barrier. A selection of compounds tested gave promising results indi-
cating that changing the quaternary to tertiary nitrogens does not reduce the allo-
steric activity. Furthermore, the substituent attached to the tertiary nitrogen can
enhance the allosteric potency.
Experimental
Melting points were determined with a Gallenkamp MPD350:BM3.5 melting point apparatus (B€uchi,
CH). 1H (299.956 MHz) and 13C (75.43MHz) NMR spectra were recorded on a Varian XL 300 spec-
trometer (Varian Inc. Darmstadt, Germany). CDCl3 was applied as the solvent and the center of the
signals of CDCl3 and DMSO-d6 were used as internal references. IR spectra were obtained using a
Perkin Elmer IR spectrometer 298. The pKa values were recorded on a Sirius PCA 101 microtitrator
(Sirius Analytical Instruments, Forest Row, East Sussex, UK). Elemental analyses were run on a
microanalyzer (Leco 932, St. Josef, MI, USA). The results agreed favorable with the calculated values.
Dry solvents were used throughout. Chemicals were of analytical grade and were purchased from
Aldrich (Steinheim, FRG) or Acros (Schwerte, FRG).

1546
R. Pick et al.
N,N0-(Hexane-1,6-diyl)bistosylamide (2)
In analogy to Ref. [22] 5.8 g of 1,6-hexanediamine (50 mmol) were dissolved in 30 cm3 of pyridine and
19.0g of tosylchloride (100mmol) were added under ice cooling. The solution was refluxed for 1 h,
afterwards poured onto crushed ice, and acidified with conc. HCl. The obtained precipitate was filtered,
washed several times with diluted HCl, and subsequently with H2O. The product was recrystallized
from ethanol to give 19.4g of 2 (91%); 1H NMR (DMSO-d6): ꢁ ¼ 1.07 (m, CH2–CH2), 1.27 (m, CH2–
CH2), 2.36 (s, CH3), 2.68 (q, N–CH2), 7.35 (d, Har), 7.65 (d, Har) ppm.
N,N0-Bis-[(1,3-dioxo-1,3-dihydro-isoindol-2-yl)propyl]hexane-1,6-diammonium
dihydrobromide (3, sec. W84, Ref. [23])
In analogy to Ref. [23] 4.0 g of 2 (5mmol) and 2.8 g of phenol (30 mmol) were dissolved in 50cm3 of
HBr (33% in glacial acid). The solution was stirred at 50ꢁC under N2 for 32h and after 24h a second
portion of 10cm3 of HBr was added. In order to precipitate the product 200 cm3 of diethyl ether
were added under ice cooling. The obtained precipitate was filtered, and washed with H2O, Na2S2O7
solution (3%), H2O, and hot methanol. Yield 2.68g (82%); mp 276ꢁC; 1H NMR (DMSO-d6): ꢁ ¼ 1.30
(m, N–CH2–CH2–CH2), 1.55 (m, N–CH2–CH2), 1.96 (quin, Nphthal–CH2–CH2), 2.86 (t), 2.95
(t, N–CH2), 3.64 (t, Nphthal–CH2), 7.86 (m, Har), 8.47 (s, Nþ–H) ppm; IR (KBr): ꢂꢀ¼ 720, 1040,
1380, 1700, 1770, 2780, 2930, 3460 cmꢃ1
.
3-[[6-[(2-Cyanoethyl)[3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)propyl]amino]hexyl][3-(1,3-
dioxo-1,3-dihydroisoindol-2-yl)propyl]amino]propionitril (1d, C34H40N6O4) and Ethyl
3-[[3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)propyl][6-[[3-(1,3-dioxo-1,3-dihydroisoindol-2-
yl)propyl][2-(ethoxycarbonyl)ethyl]amino]hexyl]amino]propionate (1e, C38H50N4O8)
In order to obtain the base 4 mmol of 3 dihydrobromide were suspended in CHCl3 and stirred with
anhydr. K2CO3 for 3 min under ice cooling. The solution was filtered and the solvent was evaporated in
vacuo. The oil and 10mmol of acrylonitrile or ethyl acryl carboxylate were dissolved in 30cm3 of
ethanol and stirred for 24 h at room temperature. The solvent was evaporated in vacuo and the residue
purified by means of column chromatography (silica gel, CHCl3=MeOH=NH3 ¼ 132=12=1, Rf ¼ 0.9).
The obtained oil was dissolved in ethanol and perchloric acid (70%) was added till precipitation. The
solution was heated for a few minutes and allowed to stand for 3 days at 5ꢁC. The perchlorate was
filtered off.
1d diperchlorate: Yield 64%; mp 191ꢁC (dec.); 1H NMR (CDCl3): ꢁ ¼ 1.27 (br, –CH2–), 1.39 (br,
–CH2–), 2.43 (t, N–CH2–), 2.51 (t, N–CH2–), 1.79 (quin, –CH2–), 2.46 (t, N–CH2–), 2.77 (t, NC–
CH2–), 3.72 (t, Npthth–CH2–), 7.70 (m), 7.80 (m) ppm; 13C NMR (CDCl3): ꢁ ¼ 16.29, 26.48, 27.20,
27.20, 36.21, 49.59, 51.44, 53.50, 123.02, 118.91 (–CꢄN), 133.74, 132.00, 168.13 ppm; IR (KBr):
ꢂꢀ¼ 720, 1040, 1395, 1610, 1705, 1770, 2250, 2860, 2940 cmꢃ1
.
1e diperchlorate: Yield 51%; mp 132ꢁC; 1H NMR (CDCl3): ꢁ ¼ 1.21 (t, O–CH2–CH3), 1.22 (br,
–CH2–), 1.36 (br, –CH2–), 1.78 (quin, –CH2–), 2.39 (t, N–CH2–), 2.46 (t, N–CH2–), 2.36 (t, N–CH2–),
2.74 (t, O¼C–CH2–), 3.68 (t, Npthth–CH2–), 4.07 (q, O–CH2–CH3), 7.69 (m), 7.79 (m) ppm; 13C
NMR (HClO4, DMSO-d6): ꢁ ¼ 13.88 (–CH3), 22.79, 25.35, 28.31, 34.66, 38.67, 47.58, 50.39, 52.22,
60.51, 122.76, 131.37, 134.11, 167.65, 169.62ppm; IR (KBr): ꢂꢀ¼ 720, 1030, 1295, 1395, 1620, 1710,
1770, 2810, 2920 cmꢃ1
.
2,20[(1,6-Hexanediyl)bis(iminio-3,1-propanediyliminocarbonyl)]bis[benzoate]
(4, C28H36N4O6)
Compound 4 is obtained as a by-product in the synthesis of 1d and 1e in varying yields. Mp 169ꢁC
(dec.); 1H NMR (D2O): ꢁ ¼ 1.44 (br, N–CH2–CH2–CH2), 1.73 (m, N–CH2–CH2), 1.98 (quin, O¼C–

Allosteric Modulators
1547
NH–CH2–CH2), 3.06 (t, N–CH2), 3.15 (t, N–CH2), 3.45 (t, O¼C–NH–CH2), 7.54 (m, ph–H) ppm;
13C NMR (D2O): ꢁ ¼ 27.77, 27.97, 28.36, 39.19, 47.69, 50.18, 129.63, 130.56, 131.93, 132.97, 136.70,
140.32, 175.97, 177.89 ppm; IR (KBr): ꢂꢀ¼ 715, 770, 850, 1385, 1580, 2850, 3050, 3405cmꢃ1
.
Synthesis of Substituted Adipoyl Amides 5h–5o
The corresponding primary amine (0.1mol) was dissolved in 30 cm3 of pyridine, 7.3 cm3 of adipoyl
chloride (0.05 mol) were carefully added under ice cooling and the reaction mixture was stirred for 45
to 60 min. Three different work up procedures were applied.
Method A: The reaction mixture was poured into crushed ice and acidified with conc. HCl. The
obtained precipitate was filtered off, washed several times with dil. aqueous HCl and H2O, and
recrystallised from a mixture of EtOH=H2O.
Method B: The reaction mixture was poured into crushed ice, acidified with conc. HCl, and
extracted three times with CHCl3. The combined organic layers were dried (Na2SO4), filtered, and
evaporated in vacuo. The resulting oil was crystallized from EtOH=diethyl ether.
Method C: The reaction mixture was poured into crushed ice, alkalized with dil. NaOH, and
extracted three times with CHCl3. The combined organic layers were dried (Na2SO4), filtered, and
evaporated in vacuo. The white precipitate was suspended in hot diethyl ether and MeOH added until
dissolution. The solution was allowed to stand for several hours at 5ꢁC.
Hexanedioic acid bis[(3-phenylpropyl)amide] (5h, C24H32N2O2)
1
Method A. Yield 84%; mp 139ꢁC; H NMR (DMSO-d6): ꢁ ¼ 1.49 (m, O¼C–CH2–CH2–), 2.07 (t,
O¼C–CH2–CH2), 1.68 (quin, ph–CH2–CH2), 2.55 (t, ph–CH2), 3.45 (q, HN–CH2), 7.18 (m, H–ph),
7.74 (t, N–H) ppm; 13C NMR (DMSO-d6): ꢁ ¼ 24.83, 30.83, 32.25, 35.26, 37.92, 125.58, 127.76,
127.92, 141.42, 171.50 ppm; IR (KBr): ꢂꢀ¼ 690, 1275, 1540, 1630, 2920, 3090, 3300 cmꢃ1
.
Hexanedioic acid bis[(2-phenylethyl)amide] (5i, C22H28N2O2)
1
Method A. Yield 94%; mp 185ꢁC; H NMR (DMSO-d6): ꢁ ¼ 1.45 (m, O¼C–CH2–CH2–), 1.98 (t,
O¼C–CH2–CH2), 2.65 (t, ph–CH2), 3.21 (q, HN–CH2), 7.18 (m, H–ph), 7.76 (t, N–H) ppm; 13C NMR
(DMSO-d6): ꢁ ¼ 25.28, 36.14, 40.78, 75.88, 126.43, 128.57, 128.70, 138.76, 165.64ppm; IR (KBr):
ꢂꢀ¼ 675, 725, 1180, 1245, 1530, 1615, 2920, 3050, 3290 cmꢃ1
.
Hexanedioic acid bisbenzylamide (5j, C20H24N2O2)
Method A. Yield 90%; mp 190ꢁC; 1H NMR (CDCl3): ꢁ ¼ 1.55 (m, O¼C–CH2–CH2–), 2.15 (t, O¼C–
CH2–CH2), 4.26 (t, ph–CH2), 7.25 (m, H–ph), 8.19 (t, N–H) ppm; 13C NMR (CDCl3): ꢁ ¼ 24.91,
35.05, 41.90, 126.25, 126.81, 127.82, 139.35, 171.54 ppm; IR (KBr): ꢂꢀ¼ 670, 705, 1010, 1190, 1260,
1440, 1535, 1620, 2915, 3080, 3280 cmꢃ1
.
Hexanedioic acid biscyclohexylamide (5k, C18H32N2O2)
1
Method A. Yield 89%; mp 244ꢁC; H NMR (CDCl3): ꢁ ¼ 1.13 (m), 1.33 (m), 1.65 (m, O¼C–CH2–
CH2–), 1.90 (m, CH2-ring), 2.16 (t, O¼C–CH2–CH2), 3.75 (m, HN–CH-ring), 5.52 (t, N–H) ppm; 13
C
NMR (CDCl3): ꢁ ¼ 24.97, 25.18, 25.61, 33.27, 36.54, 48.18, 171.61ppm; IR (KBr): ꢂꢀ¼ 720, 1140,
1435, 1540, 1630, 2840, 2930, 3280 cmꢃ1
.
Hexanedioic acid biscyclopentylamide (5l, C16H28N2O2)
Method A. Yield 90%; mp 228ꢁC (dec.); 1H NMR (CDCl3): ꢁ ¼ 1.42 (m), 1.65 (m, O¼C–CH2–CH2–),
1.96 (m), 2.21 (t, O¼C–CH2–CH2), 2.42 (m, CH2-ring), 4.18 (m, HN–CH-ring), 6.02 (t, N–H) ppm;

1548
R. Pick et al.
13C NMR (CDCl3): ꢁ ¼ 23.73, 24.91, 33.03, 35.98, 51.56, 172.57 ppm; IR (KBr): ꢂꢀ¼ 1200, 1540,
1635, 2880, 2950, 3310 cmꢃ1
.
Hexanedioic acid biscyclopropylamide (5m, C12H20N2O2)
1
Method B. Yield 72%; mp 181ꢁC; H NMR (MeOH-d4): ꢁ ¼ 0.46 (m), 0.68 (m, CH2-ring), 1.57 (m,
O¼C–CH2–CH2–), 2.13 (m, O¼C–CH2–CH2), 2.63 (m, HN–CH-ring) ppm; 13C NMR (CDCl3):
ꢁ ¼ 6.49, 23.32, 26.52, 36.52, 23.32, 177.16 ppm; IR (KBr): ꢂꢀ¼ 690, 880, 1100, 1180, 1270, 1345,
1400, 1445, 1530, 1625, 2940, 3040, 3230 cmꢃ1
.
Hexanedioic acid biscyclopropylmethylamide (5n, C14H24N2O2)
1
Method B. Yield 75%; mp 176ꢁC (dec.); H NMR (CDCl3): ꢁ ¼ 0.17 (m), 0.45 (m, CH2-ring), 0.91
(HN–CH-ring) 1.66 (m, O¼C–CH2–CH2–), 2.21 (t, O¼C–CH2–CH2), 6.23 (t, N–H) ppm; 13C NMR
(CDCl3): ꢁ ¼ 3.45, 10.75, 25.15, 36.14, 44.30, 172.62ppm; IR (KBr): ꢂꢀ¼ 1185, 1240, 1355, 1525,
1610, 2920, 3290 cmꢃ1
.
Hexanedioic acid bis[(2-morpholino-4-ylethyl)amide] (5o, C18H34N4O4)
1
Method C. Yield 71%; mp 160ꢁC; H NMR (CDCl3): ꢁ ¼ 1.61 (m), 2.15 (br), 6.18 (br), 2.41 (m,
N(CH2–)3), 3.28 (q, HN–CH2), 3.64 (t, O(CH2–)2) ppm; 13C NMR (CDCl3): ꢁ ¼ 25.04, 35.58, 36.11,
53.25; 57.01, 66.77, 172.41ppm; IR (KBr): ꢂꢀ¼ 870, 1110, 1260, 1545, 1635, 2810, 2940, 3295cmꢃ1
.
Synthesis of Substituted Hexane-1,6-diamines 6h–6o
The corresponding diamide 5h–5o (2mmol) was dissolved in 100 cm3 of THF and B2H6 in THF (1M,
20mmol) added dropwise in 15 min under N2 and ice cooling. The solution was stirred for 30 min at
room temperature and for 1 h at 60ꢁC. After cooling with ice 100 cm3 of 2 M HCl were added, the
resulting solution was refluxed for 30min, after cooling with ice, 25 cm3 of conc. NaOH solution were
added and the aqueous solution was extracted with CHCl3. The combined organic layers were washed
with H2O, dried (Na2SO4), and evaporated in vacuo. The colorless oil was dissolved in diethyl ether, the
solution filtered, and evaporated in vacuo to give the oily 6 which was used without further purification.
N,N0-Bis(3-phenylpropyl)hexane-1,6-diamine (6h, C24H36N2)
1
Yield 86%; H NMR (CDCl3): ꢁ ¼ 1.26 (m, N–CH2–CH2–CH2–), 1.35 (m, N–CH2–CH2–), 1.67
(quin, ph–CH2–CH2), 2.45 (t, N–CH2–CH2–), 2.05 (br, N–H), 2.48 (t, N–CH2), 2.58 (t, ph–CH2),
7.20 (m, H–ph) ppm; 13C NMR (CDCl3): ꢁ ¼ 26.79, 29.59, 31.31, 32.90, 48.73, 49.25, 125.21, 127.85,
127.91, 141.96 ppm; IR (KBr): ꢂꢀ¼ 700, 745, 1120, 1450, 1650, 2935, 3020, 3300 cmꢃ1
.
N,N0-Bis(2-phenylethyl)hexane-1,6-diamine (6i, C22H32N2)
Yield 83%; 1H NMR (CDCl3): ꢁ ¼ 1.32 (m, N–CH2–CH2–CH2–), 1.48 (m, N–CH2–CH2–), 1.89 (br,
N–H), 2.88 (m, N–CH2–CH2–ph), 2.64 (t, N–CH2), 7.31 (m, H–ph) ppm; 13C NMR (CDCl3):
ꢁ ¼ 27.21, 29.94, 36.31, 49.74, 51.14, 126.06, 128.40, 128.64, 140.04ppm; IR (KBr): ꢂꢀ¼ 700, 745,
1120, 1455, 1605, 2860, 2920, 3030, 3250 cmꢃ1
.
N,N0-Dibenzylhexane-1,6-diamine (6j, C20H28N2)
Yield 79%; 1H NMR (CDCl3): ꢁ ¼ 1.33 (m, N–CH2–CH2–CH2–), 1.51 (m, N–CH2–CH2–), 1.51 (br,
N–H), 2.62 (t, N–CH2), 3.79 (s, –CH2–ph), 7.31 (m, H–ph) ppm; 13C NMR (CDCl3): ꢁ ¼ 27.32, 30.10,

Allosteric Modulators
1549
49.41, 54.08, 126.71, 127.96, 128.21, 140.40 ppm; IR (KBr): ꢂꢀ¼ 700, 745, 1170, 1380, 1450, 2920,
3300 cmꢃ1
.
N,N0-Dicyclohexylhexane-1,6-diamine (6k, C18H36N2)
Yield 91%; 1H NMR (CDCl3): ꢁ ¼ 1.13 (m), 1.26 (m, N–CH2–CH2–CH2–), 1.43 (m, N–CH2–CH2–),
1.57 (br, N–H), 1.83 (m, CH2-ring), 2.34 (s, NH–CH2-ring), 2.55 (t, N–CH2) ppm; 13C NMR (CDCl3):
ꢁ ¼ 25.06, 26.10, 27.33, 30.21, 33.40, 46.80, 56.84 ppm; IR (KBr): ꢂꢀ¼ 1170, 1370, 1445, 1640, 2850,
2920, 3300 cmꢃ1
.
N,N0-Dicyclopentylhexane-1,6-diamine (6l, C16H32N2)
Yield 72%; 1H NMR (CDCl3): ꢁ ¼ 1.27 (m, N–CH2–CH2–CH2–), 1.47 (m, N–CH2–CH2–), 1.60 (m,
CH2-ring), 2.14 (br, N–H), 2.99 (m, NH–CH2-ring), 3.53 (t, CH2-ring) ppm; 13C NMR (CDCl3):
ꢁ ¼ 23.94, 27.27, 30.09, 32.97, 48.56, 59.78 ppm; IR (KBr): ꢂꢀ¼ 1070, 1120, 1350, 1450, 1650, 2880,
2920, 3250 cmꢃ1
.
N,N0-Dicyclopropylhexane-1,6-diamine (6m, C12H24N2)
Yield 75%; 1H NMR (CDCl3): ꢁ ¼ 0.31 (m), 0.41 (m, CH2), 1.30 (m, N–CH2–CH2–CH2–), 1.46 (m,
N–CH2–CH2–), 1.76 (br, N–H), 2.65 (m, NH–CH2-ring), 2.08 ppm (t, N–CH-ring); 13C NMR
(CDCl3): ꢁ ¼ 6.28, 27.37, 30.07, 49.61, 30.41 ppm; IR (KBr): ꢂꢀ¼ 890, 1070, 1255, 1370, 1445,
1585, 2920, 3250 cmꢃ1
.
N,N0-Biscyclopropylmethylhexane-1,6-diamine (6n, C14H28N2)
1
Yield 76%; H NMR (CDCl3): ꢁ ¼ 0.42 (m), 0.88 (m, CH2), 1.30 (m, N–CH2–CH2–CH2–), 1.47
(m, N–CH2–CH2–), 2.30 (br, N–H), 2.41 (d, NH–CH2-ring), 2.58 (m, NH–CH2–CH2), 3.56 (t, CH-
ring) ppm; 13C NMR (CDCl3): ꢁ ¼ 3.41, 11.17, 27.33, 30.00, 49.73, 55.00 ppm; IR (KBr): ꢂꢀ¼ 890,
1070, 1300, 1450, 1570, 2920, 3300 cmꢃ1
.
N,N0-Bis(2-morpholino-4-ylethyl)hexane-1,6-diamine (6o, C18H38N4O2)
Yield 66%; 1H NMR (CDCl3): ꢁ ¼ 1.31 (m, N–CH2–CH2–CH2–), 1.47 (m, N–CH2–CH2–), 2.00 (br,
N–H), 2.41 (m, NH–CH2), 2.46 (m, Nmorpholine–CH2), 2.57 (m, Nmorpholine–CH2), 3.67 (t, O–CH2)
ppm; 13C NMR (CDCl3): ꢁ ¼ 27.31, 30.04, 46.10, 50.03, 53.73, 58.27, 66.96 ppm; IR (KBr): ꢂꢀ¼ 1070,
1110, 1450, 1650, 2920, 3300 cmꢃ1
.
Synthesis of the Allylamine and Hydroxypropyl Substituted Hexanediamines
6a and 6c
Allylamine or 3-aminopropanol (20 mmol) was dissolved in 20cm3 of acetonitrile, 1.5 cm3 of 1,6-
dibromopropane (10 mmol) were added and it was stirred for 5 h at room temperature. The precipitate
was filtered off and recrystallized from MeOH=diethyl ether.
N,N0-Diallylhexane-1,6-diamine (6a ꢅ 2HBr, C12H26Br2N2)
Yield 1.5 g (21%); 1H NMR (DMSO-d6): ꢁ ¼ 1.29 (br, N–CH2–CH2–CH2–), 1.60 (br, N–CH2–CH2–),
2.84 (t, N–CH2–CH2–), 3.56 (d, –CH2–CH¼CH2), 5.40 (dd, –CH2–CH¼CH2), 5.89 (m, –CH2–
CH¼CH2), 8.77ppm (br, Nþ–H); 13C NMR (DMSO-d6): ꢁ ¼ 25.24, 25.60, 45.98, 48.75, 122.69
(–CH¼CH2), 129.15 ppm (–CH¼CH2); IR (KBr): ꢂꢀ¼ 940, 1430, 1630, 2800, 2940, 3400cmꢃ1
.

1550
R. Pick et al.
3-[6-(3-Hydroxypropylamino)hexylamino]propan-1-ol (6c ꢅ 2HBr, C12H30 Br2N2O2)
Yield 3.0 g (38%); 1H NMR (DMSO-d6): ꢁ ¼ 1.29 (br, N–CH2–CH2–CH2–), 1.59 (br, N–CH2–CH2–),
2.87 (t, N–CH2–CH2–), 1.74 q, –CH2–), 2.91 (t, N–CH2), 3.44 (t, –CH2–OH), 5.97 (br, Nþ–H) ppm;
IR (KBr): ꢂꢀ¼ 720, 1250, 1430, 2800, 2930, 3400cmꢃ1
.
Cyanoethylation of the Substituted Hexanebisamine 6 to the Bisnitriles
7h, 7k, 7m, and 7o
The corresponding hexanebisamines 6h, 6k, 6m, and 6o were dissolved in 30 cm3 of ethanol and
0.66cm3 of acrylonitrile (10 mmol) dropwise added at 30ꢁC. The reaction solution was allowed to
stand overnight, afterwards the solvent was evaporated in vacuo, and the resulting oil purified by means
of column chromatography (silica gel, mobile phase: CHCl3=MeOH=NH3 ¼ 132=12=1).
3-[[6-[(2-Cyanoethyl)-(3-phenylpropyl)amino]hexyl]-
(3-phenylpropyl)amino]propionitrile (7h, C30H42N4)
Yield 89%; 1H NMR (CDCl3): ꢁ ¼ 1.28 (br, N–CH2–CH2–CH2–), 1.32 (br, N–CH2–CH2–), 1.68 (m,
N–CH2–CH2–), 2.38 (t, –CH2–N–CH2), 2.40 (t, –CH2–N–CH2), 2.48 (t, N–CH2–), 2.51 (t, ph–CH2–),
2.63 (t, NC–CH2), 7.19 (m, ph–H) ppm; 13C NMR (CDCl3): ꢁ ¼ 16.23, 27.24, 27.28, 28.95, 33.35,
49.51, 53.04, 53.57, 119.02, 125.61, 128.15, 128.22, 141.87ppm; IR (KBr): ꢂꢀ¼ 700, 745, 1070, 1360,
1450, 1640, 2250, 2900 cmꢃ1
.
3-[[6-[(2-Cyanoethyl)cyclohexylamino]hexyl]cyclohexylamino]propionitrile
(7k, C24H42N4)
Yield 68%; 1H NMR (CDCl3): ꢁ ¼ 1.00–1.20 (m), 1.50–1.80 (m, –CH2-ring), 1.27 (br, N–CH2–CH2–
CH2–), 1.38 (br, N–CH2–CH2–), 2.36 (t, –CH2–N–CH2), 2.41 (t, –CH2–N–CH2), 2.43 (t, N–CH2-
ring), 2.74 (t, NC–CH2) ppm; 13C NMR (CDCl3): ꢁ ¼ 18.93, 26.11, 26.11, 26.27, 27.20, 29.32, 46.49,
50.75, 60.20, 119.08 ppm; IR (KBr): ꢂꢀ¼ 890, 1100, 1260, 1370, 1450, 2250, 2850, 2920cmꢃ1
.
3-[[6-[(2-Cyanoethyl)cyclopropylamino]hexyl]cyclopropylamino]propionitrile
(7m, C18H30N4)
Yield 58%; 1H NMR (CDCl3): ꢁ ¼ 0.43 (m), 0.48 (m, –CH2-cycl.C3H5), 127 (br, N–CH2–CH2–CH2–),
147 (br, N–CH2–CH2–), 1.75 (m, –CH-ring), 2.51 (t, –CH2–N–CH2), 2.54 (t, –CH2–N–CH2), 2.92
(t, NC–CH2) ppm; 13C NMR (CDCl3): ꢁ ¼ 6.99, 16.00, 27.06, 27.35, 35.92, 50.72, 55.07ppm; IR
(KBr): ꢂꢀ¼ 750, 1020, 1210, 1350, 1460, 2250, 2920 cmꢃ1
.
3-[[6-[(2-Cyanoethyl)-(3-morpholin-4-ylpropxyl)amino]hexyl]-
(3-morpholin-4-ylpropyl)amino]propionitrile (7o, C24H44N6O2)
Yield 68%; 1H NMR (CDCl3): ꢁ ¼ 1.27 (br, N–CH2–CH2–CH2–), 1.40 (m, N–CH2–CH2–), 2.42 (t,
–CH2–N–CH2), 2.42 (m, N–CH2), 2.42 (m, Nmorph–CH2), 2.59 (t, –CH2–N–CH2), 2.79 (t, NC–CH2),
3.67 (t, O–CH2) ppm; 13C NMR (CDCl3): ꢁ ¼ 16.53, 27.18, 27.41, 50.04, 51.04, 54.11, 54.31, 57.16,
66.86, 118.90ppm; IR (KBr): ꢂꢀ¼ 865, 1070, 1215, 1300, 1450, 1650, 2250, 2920 cmꢃ1
.
Reduction of Bisnitriles 7 to Tertiary Spermine Analogues 8
The bisnitrils 7 (2mmol) were dissolved in 100 cm3 of THF and 20 cm3 of B2H6 (1M, THF) was added
within 15 min under N2 and cooling. The reaction mixture was stirred for 30min at room temperature

Allosteric Modulators
1551
and refluxed for 1 h. 100 cm3 of 2 M HCl were added under ice cooling and the resulting solution was
refluxed for 30 min. After cooling to room temperature 50cm3 of 3 M NaOH were added and the
solution was extracted three times with CHCl3. The combined organic layers were washed with H2O,
dried (Na2SO4), and the solvent was evaporated in vacuo. The resulting oil was dissolved in diethyl
ether, filtered, and again evaporated in vacuo to give oily 8, which was used for the next step without
further purification.
N,N0-Bis(3-aminopropyl)-N,N0-bis(3-phenylpropyl)hexane-1,6-diamine (8h, C30H50N4)
Yield 64%; 1H NMR (CDCl3): ꢁ ¼ 1.26 (m), 1.40 (m), 1.59 (quin), 1.75 (quin), 2.41 (m), 2.59 (t), 2.75
(t), 2.90 (br, –NH2), 7.18 (m, ph–H) ppm; 13C NMR (CDCl3): ꢁ ¼ 26.25, 26.45, 27.51, 28.86, 29.16,
32.12, 40.82, 48.38, 50.45, 59.55 ppm; IR (KBr): ꢂꢀ¼ 700, 745, 1155, 1450, 1600, 2860, 2940,
3360 cmꢃ1
.
N,N0-Bis(3-aminopropyl)-N,N0-dicyclohexylhexane-1,6-diamine (8k, C24H50N4)
Yield 84%; 1H NMR (CDCl3): ꢁ ¼ 1.11 (m), 1.21 (m), 1.34 (m), 1.53 (m), 1.70 (br), 2.23 (br, –NH2),
2.38 (m), 2.68 (t, 4H) ppm; 13C NMR (CDCl3): ꢁ ¼ 26.98, 27.50, 28.63, 29.85, 33.75, 40.75, 52.12,
53.53, 53.87, 125.51, 128.09, 128.17, 142.12 ppm; IR (KBr): ꢂꢀ¼ 895, 1170, 1450, 1590, 2860, 2915,
3280 cmꢃ1
.
N,N0-Bis(3-aminopropyl)-N,N0-dicyclopropylhexane-1,6-diamine (8m, C18H38N4)
Yield 62%; 1H NMR (CDCl3): ꢁ ¼ 0.39 (m), 0.45 (m), 1.23 (m), 1.44 (m), 1.65 (m), 2.48 (m), 2.88 (t),
3.17 (br, –NH2), 3.55 (m) ppm; 13C NMR (CDCl3): ꢁ ¼ 6.81, 6.91, 15.91, 26.96, 27.27, 35.84, 50.63,
55.00 ppm; IR (KBr): ꢂꢀ¼ 750, 1020, 1210, 1350, 1460, 2920, 3320 cmꢃ1
.
N,N0-Bis(3-aminopropyl)-N,N0-bis(3-morpholin-4-ylpropyl)hexane-1,6-diamine
(8o, C24H52N4O2)
Yield 57%; 1H NMR (CDCl3): ꢁ ¼ 1.28 (br), 1.42 (br), 1.67 (m), 2.46 (m), 2.84 (m), 3.28 (br, –NH2),
3.71 (m) ppm; 13C NMR (CDCl3): ꢁ ¼ 27.51, 27.53, 40.55, 54.07, 54.10, 54.21, 66.83, 66.87, 66.94,
67.04 ppm; IR (KBr): ꢂꢀ¼ 865, 1070, 1215, 1300, 1450, 1650, 2920, 3310 cmꢃ1
.
Alkylation of Hexanebisamines 6 with Bromopropylphthalimide
to 1a, 1c, 1i, 1j, 1l, and 1n
The corresponding hexanebisamine 6 (3.5mmol) was dissolved in 20 cm3 of acetonitrile and 1.5 g of
K2CO3 were added. 1.88 g of Bromopropylphthalimide (7mmol) were added and the suspension was
stirred for 5 to 14 days at room temperature (TLC control on silica gel, mobile phase:
CHCl3=MeOH=NH3 ¼ 132=12=1, Rf ꢆ 0.9). After the reaction was completed the suspension was
filtered, the solvent evaporated in vacuo, and the resulting oil purified by means of column chromato-
graphy (silica gel, mobile phase: CHCl3=MeOH=NH3 ¼ 132=12=1). The oily bases were converted
into salts using the following procedures.
A (diperchlorate): The corresponding base was dissolved in ethanol and conc. HClO4 was added
dropwise. When the first precipitate appeared, the solution was refluxed for a couple of min and the
resulting clear solution was allowed to stand at 5ꢁC for several days until crystallization.
B (dihydrochloride): The corresponding base was dissolved in acetone and eth. HCl solution was
added dropwise. When the first precipitate appeared, the solution was refluxed for a couple of min and
the resulting clear solution was allowed to stand at 5ꢁC for several days until crystallization.

1552
R. Pick et al.
2-[3-[Allyl[6-[allyl[3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-
propyl]amino]hexyl]amino]propyl]isoindole-1,3-dione (1a, C34H44Cl2N4O4)
Crystallization method A or B. Yield 67%; mp 194ꢁC (base); 1H NMR (DMSO-d6, dihydrochloride):
ꢁ ¼ 1.11 (br, –CH2–), 1.63 (br, –CH2–), 2.04 (m, –CH2–), 2.97 (m, N–CH2–), 3.09 (m, N–CH2–),
3.63 (t, Npthth–CH2–), 3.73 (m, –CH2–CH¼CH2), 5.46 (m, –CH2–CH¼CH2), 5.94 (m, –CH2–
CH¼CH2), 7.85 (m, CH–ph), 10.15 (br, Nþ–H) ppm; 13C NMR (DMSO-d6): ꢁ ¼ 22.84, 25.63,
35.00, 49.65, 51.61, 54.22, 123.23 (–CH¼CH2), 125.02 (CHphth), 127.39 (–CH¼CH2), 131.88
(CHphth), 134.61 (Cphth), 168.17 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 680, 910, 1030, 1110, 1300, 1370,
1680, 1740, 2670, 2870, 3420 cmꢃ1
.
2-[3-[[[6-[[3-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)propyl](3-hydroxypropyl)amino]hexyl](3-
hydroxypropyl)]amino]propyl]isoindole-1,3-dione (1c, C34H48Cl2N4O6)
1
Crystallization method A or B. Yield 69%; mp 210ꢁC (dec. base); H NMR (CDCl3, base): ꢁ ¼ 1.25
(br, –CH2–), 1.44 (br, –CH2–), 1.66 (quin, –CH2–), 1.85 (q, –CH2–), 2.41 (t, N–CH2–), 2.51 (t, N–
CH2–), 2.62 (t, N–CH2), 3.69 (t, Npthth–CH2–), 3.76 (t, HO–CH2), 4.84 (br, –OH), 7.70 (m), 7.80 (m)
ppm; 13C NMR (CDCl3, base): ꢁ ¼ 25.82, 26.46, 27.22, 27.95, 36.17, 51.45, 53.81, 54.12, 63.85,
123.10 (CHphth), 131.98 (Cphth), 133.83 (CHphth), 168.19 (C¼O) ppm; IR (KBr, dihydrochloride):
ꢂꢀ¼ 700, 1000, 1380, 1680, 1760, 2940, 3370 cmꢃ1
.
2-[3-[[6-[[3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)propyl](3-phenylethyl)amino]hexyl](2-
phenylethyl)amino]propyl]isoindole-1,3-dione (1i, C44H50N4O4)
Yield 55% oil (base); 1H NMR (CDCl3, base): ꢁ ¼ 1.26 (br, –CH2–), 1.43 (br, –CH2–), 1.66 (quin,
–CH2–), 1.86 (q, –CH2–), 2.49 (t, N–CH2–), 2.59 (t, N–CH2–), 2.71 (br, N–CH2–CH2–ph), 3.72 (t,
Npthth–CH2–), 7.24 (m, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 26.15, 26.91, 27.42, 33.34, 36.46,
51.50, 53.74, 55.71, 123.06 (CHphth), 125.79 (–CHphenyl), 128.67 (–CHphenyl), 128.21 (–CHphenyl),
132.12 (Cphth), 133.76 (CHphth), 140.52 (C–Cphenyl), 168.27 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 715, 1030,
1395, 1605, 1705, 1770, 2850, 2930, 3010 cmꢃ1
.
2-[3-[Benzyl[6-[benzyl[3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-
propyl]amino]hexyl]amino]propyl]isoindole-1,3-dione (1j, C42H48Cl2N4O4)
Crystallization method B. Yield 34%; mp 246ꢁC (dec. salt); 1H NMR (DMSO-d6 þ D2O, dihydrochlo-
ride): ꢁ ¼ 1.21 (br, –CH2–), 1.59 (br, –CH2–), 1.92 (m, –CH2–), 2.98 (m, N–CH2–), 3.51 (br, Npthth
–
CH2–), 4.35 (br, –CH2–ph), 7.15 (m, CH–ph) ppm; 13C NMR (DMSO-d6 þ D2O, dihydrochloride):
ꢁ ¼ 23.57, 24.24, 26.60, 35.91, 49.98, 54.04, 57.95, 124.84 (CHphth), 130.45, 130.64, 132.45
(–CHphenyl), 131.33 (Cphth), 131.96 (CHphth), 136.40 (C–Cphenyl), 170.56 (C¼O) ppm; IR (KBr):
ꢂꢀ¼ 720, 1030, 1395, 1450, 1610, 1705, 1770, 2930, 3020 cmꢃ1
.
2-[3-[Cyclopentyl[6-[cyclopentyl[3-(1,3-dioxo-1,3-dihydro-isoindol-2-
yl)propyl]amino]hexyl]amino]propyl]isoindole-1,3-dione (1l, C38H50N4O4)
1
Yield 51%; mp 60ꢁC (base); H NMR (CDCl3, base): ꢁ ¼ 1.22 (br, –CH2–), 1.45 (m, CH2cycpent),
1.63 (br, –CH2–), 1.84 (quin, –CH2–), 2.51 (t, N–CH2–), 2.60 (t, N–CH2–), 2.96 (m, N–CHcyclpent),
3.68 (t, Npthth–CH2–), 7.72 (m, CH–ph), 7.81 (m, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 23.93,
25.90, 26.67, 27.52, 29.73, 36.55, 49.07, 51.50, 63.72, 123.12 (CHphth), 132.15 (Cphth), 133.82
(CHphth), 168.30 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 715, 880, 1030, 1185, 1390, 1610, 1705, 1770, 2860,
2930 cmꢃ1
.

Allosteric Modulators
1553
2-[3-[Cyclopropylmethyl[6-[cyclopropylmethyl[3-(1,3-dioxo-1,3-dihydro-isoindol-2-
yl)propyl]amino]hexyl]amino]propyl]isoindole-1,3-dione (1n, C36H48Cl2N4O12)
1
Crystallization method A. Yield 52%; mp 68ꢁC (dec. base); H NMR (CDCl3, base): ꢁ ¼ 0.08 (m,
CH2cycloprop), 0.45 (m, CH2cycloprop), 0.83 (m, –CHcycloprop), 1.26 (br, –CH2–), 1.42 (br, –CH2–), 1.84
(q, –CH2–), 2.34 (t, N–CH2), 2.50 (t, N–CH2–), 2.62 (t, N–CH2–), 3.72 (t, Npthth–CH2–), 7.71 (m,
CH–ph), 7.81 (m, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 3.81, 8.24, 25.77, 26.52, 27.37, 36.44,
51.15, 53.69; 58.45, 123.01 (CHphth), 132.07 (Cphth), 133.71 (CHphth), 168.25 (C¼O) ppm; IR (KBr):
ꢂꢀ¼ 700, 1380, 1705, 1760, 2930 cmꢃ1
.
Condensation of the Amines 7h, 7k, 7m, and 7o with Phthalic Anhydride
to 1h, 1k, 1m, 1o, and 9
The corresponding primary amine 7 (4mmol) was dissolved in 60cm3 of toluene and 8 mmol of
phthalic anhydride or methylphthalic anhydride were added. The solution was refluxed for 2 h using a
water separator. Afterwards the solvent was evaporated in vacuo. The resulting oil was purified by
means of column chromatography on silica gel (mobile phase: CHCl3=MeOH=NH3 ¼ 132=12=1,
Rf ꢆ 0.9).
2-[3-[[6-[[3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)propyl](3-phenylpropyl)amino]hexyl]-
(2-phenylpropyl)amino]propyl]isoindole-1,3-dione (1h, C46H56Cl2N4O12)
Crystallization method A. Yield 67%; mp 82ꢁC (base); 1H NMR (CDCl3, base): ꢁ ¼ 1.24 (br, –CH2–),
1.73 (t, N–CH2–CH2–), 1.81 (quin, –CH2–), 2.39 (t, N–CH2–), 2.44 (t, N–CH2–CH2–), 2.50 (t, N–
CH2–), 2.60 (t, ph–CH2–), 3.71 (t, Npthth–CH2–), 7.70 (m, CH–ph), 7.81 (m, CH–ph) ppm; 13C NMR
(CDCl3, base): ꢁ ¼ 26.19, 26.97, 27.58, 28.76, 33.72, 36.58, 51.52, 53.40, 53.84, 122.99 (CHphth),
125.47 (–CHphenyl), 128.09 (–CHphenyl), 128.21 (–CHphenyl), 132.03 (Cphth), 133.64 (CHphth), 142.26
(C–Cphenyl), 168.13 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 720, 1030, 1395, 1600, 1705, 1765, 2800, 2930,
3010 cmꢃ1
.
2-[3-[Cyclohexyl[6-[cyclohexyl[3-(1,3-dioxo-1,3-dihydro-isoindol-2-
yl)propyl]amino]hexyl]amino]propyl]isoindole-1,3-dione (1k, C40H56Cl2N4O4)
Crystallization method B. Yield 72%; mp 104ꢁC (base); 1H NMR (CDCl3, base): ꢁ ¼ 1.24 (br, –CH2–),
1.24 (m), 1.39 (br, –CH2–), 1.58 (d, –CHCyclohexyl), 1.74 (m, –CH2Cyclohexyl), 1.80 (quin, –CH2–), 2.42
(t, N–CH2–), 2.54 (t, N–CH2–), 2.60 (t, ph–CH2–), 3.68 (t, Npthth–CH2–), 7.69 (m, CH–ph), 7.79
(m, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 26.23, 26.23, 26.39, 27.46, 27.77, 28.92, 36.60, 48.10,
50.48, 60.08, 122.96 (CHphth), 132.09 (Cphth), 133.64 (CHphth), 168.14 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 720,
895, 1030, 1395, 1610, 1705, 1765, 2850, 2920cmꢃ1
.
2-[3-[Cyclopropyl[6-[cyclopropyl[3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-
propyl]amino]hexyl]amino]propyl]isoindole-1,3-dione (1m, C34H42N4O4)
1
Yield 53%; mp 100ꢁC (base); H NMR (CDCl3, base): ꢁ ¼ 0.42 (m, CH2cycloprop), 1.22 (br, –CH2–),
1.45 (br, –CH2–), 1.65 (t, N–CHcycloprop), 1.88 (quin, –CH2–), 2.53 (t, N–CH2–), 2.65 (t, N–CH2–),
3.68 (t, Npthth–CH2–), 7.72 (m, CH–ph), 7.81 (m, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 6.57,
25.83, 26.55, 27.52, 29.82, 36.50, 36.62, 52.78, 55.39, 123.01 (CHphth), 132.10 (Cphth), 133.71
(CHphth), 168.25 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 710, 825, 890, 1010, 1180, 1375, 1705, 1770, 2910,
3005 cmꢃ1
.

1554
R. Pick et al.
2-[3-[[6-[[3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)propyl]-(2-morpholin-4-
ylethyl)amino]hexyl](2-morpholin-4-ylethyl)amino]propyl]isoindole-1,3-dione
(1o, C40H58Cl2N6O14)
Crystallization method A. Yield 56%; mp 93ꢁC (base); 1H NMR (CDCl3, base): ꢁ ¼ 1.23 (br, –CH2–),
1.36 (br, –CH2–), 1.81 (quin, –CH2–), 2.45 (m, 3ꢅN–CH2–), 3.69 (t, Npthth–CH2–), 3.69 (m, O–
CH2–), 7.70 (m, CH–ph), 7.80 (m, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 26.22, 27.02, 27.00,
36.52, 51.00, 52.08, 54.15, 54.50, 56.94, 66.89, 123.02 (CHphth), 132.06 (Cphth), 133.72 (CHphth),
168.13 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 720, 865, 1030, 1110, 1395, 1615, 1705, 1770, 2900, 2930 cmꢃ1
.
2-[3-[Cyclohexyl[6-[cyclohexyl[3-(5-methyl-1,3-dioxo-1,3-dihydroisoindol-2-
yl)propyl]amino]hexyl]amino]propyl]-5-methylisoindole-1,3-dione (9, C42H60Cl2N4O4)
1
Crystallization method B. Yield 68%; mp 127ꢁC (base); H NMR (CDCl3, base): ꢁ ¼ 1.18 (m), 1.24
(br, –CH2–), 1.38 (br, –CH2–), 1.58 (d, CH2cyclhex), 1.72 (m, CH2cyclhex), 1.75 (quin, –CH2–), 2.40 (t,
N–CH2–), 2.52 (t, N–CH2–), 3.66 (t, Npthth–CH2–), 3.69 (m, O–CH2–), 7.46 (d, CH–ph), 7.61 (s,
CH–ph), 7.68 (d, CH–ph) ppm; 13C NMR (CDCl3, base): ꢁ ¼ 22.00 (–CH3), 26.27, 26.45, 27.51,
27.94, 29.00, 29.16, 36.56, 48.10, 50.51, 60.01, 122.86 (CHphth), 123.52 (CHphth), 134.13 (CHphth),
129.52 (Cphth), 132.50 (Cphth), 144.80 (Cphth), 168.23 (C¼O), 168.33 (C¼O) ppm; IR (KBr, salt):
ꢂꢀ¼ 730, 1030, 1385, 1435, 1705, 1760, 2860, 2940, 3420 cmꢃ1
.
Synthesis of 1,10-Bis[1,3-dioxo-1,3-dihydroisoindol-2-ylpropyl]-
[4,40]-bipiperidinyl-1,10-diium dibromide (10, C32H40Br2N4O4)
Bipiperidine dihydrochloride (1.5g, 6.25 mmol) was suspended in 30cm3 of ethanol, potassium tert.
butylate (1.48 g, 12.5mmol) added and stirred for a couple of min. Afterwards the solution was filtered,
N-(3-bromopropyl)phthalimide (3.35 g, 12.5 mmol) added and the solution refluxed for 15h. After
cooling the product crystallizes, the crystals were filtered off, washed with diethyl ether, and recrys-
1
tallized from H2O=ethanol. Yield 1.5 g (34% dihydrobromide); mp 280ꢁC; H NMR (DMSO-d6):
ꢁ ¼ 1.33 (m, CH2–CHbipiperidine), 1.85 (m, CH2bipiperidin), 2.81 (m, Nþ–CH2), 3.09 (m, Nþ–CH2),
3.48 (m, Nþ–CH2), 3.64 (t, Nphthal–CH2), 7.87 (m, CH–ph), 9.10 (br, Nþ–H) ppm; 13C NMR
(DMSO-d6): ꢁ ¼ 22.86, 25.91, 34.72, 51.76, 53.47, 36.90, 122.75, 134.11 (C–Haromat.),
131.45 (Cqaromat.), 167.60 (C¼O) ppm; IR (KBr): ꢂꢀ¼ 715, 960, 1020, 1360, 1395, 1610, 1705,
1770, 2535, 2630, 2910, 3460 cmꢃ1
.
Determination of the Lipophilicity
The test and reference compounds (2-phenylethylamine, 2-phenylethanol, benzene, N,N0-dimethylani-
line, chlorobenzene, toluene, ethylbenzene, cumene, biphenyl, anthracene) were dissolved in methanol
(4mg=cm3). Using a RP column (LiChroCart+ 125-4 HPLC cartridge; LiChrospher+ 100, RP 18, 5 mm,
endcapped, Merck) and a mobile phase of methanol=phosphate buffer pH 7.4 ¼ 70=30 (0.02% N,N-
dimethylamine added) the retention times were determined and converted to k0-values according to Eq.
(1) where TR ¼ retention time of the test compound and T0 ¼ hold-up time.
k0 ¼ ðTr ꢃ T0Þ=T0
ð1Þ
The log k0-values of the reference substances were correlated with the logP values reported in
Ref. [24]. The calibration curve was established (see Fig. 6) and the logP values of the test compounds
calculated. The log P values are summarized in Table 2.
Determination of the pKa Values
The pKa values were determined potentiometrically in water using a Sirius PCA 101 apparatus.
Exactly 0.004 to 0.007 mg of the compounds were dissolved in 8–12cm3 of aqueous methanol

Allosteric Modulators
1555
Fig. 6. Calibration curve for references’ k0 and log P values from Ref. [24]
Table 2. Lipophilicity values log P of representative compounds 1 and W84 derivatives
Entry
log k0
log P
sec. W84(3)
0.26
1.02
2.29
3.94
0.53
1.25
3.21
3.11
3.48
4.22
4.47
6.25
5.55
5.56
tert. W84
W84
ꢃ0.54
ꢃ0.21
0.69
Dimethyl-W84
1a
1c
1d
1j
0.64
0.81
1.15
1k
1l
1.26
2.08
1m
1n
1.76
1.76
(50%) and diluted to 20.0 cm3 with a 0.15 M KCl. The titration was performed starting from pH 11.
Using the Yasuda-Shedlovsky plot [25] the pKa values were calculated and extrapolated to 0% metha-
nol. The pKa of dimethylaminopropylphthalimide was found to amount to 9.4 and that of tert. W84 to
9.0.
Acknowledgement
The skilful technical assistance of Ms. I. Jusen is gratefully acknowledged. Thanks are due to the DFG
(HO 1368=7-1; MO 821=1-2) for financial support.

1556
R. Pick et al.: Allosteric Modulators
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