Synthesis and pharmacological activity of fluorescent histamine H2 receptor antagonists related to potentidine

Article abstract of DOI:10.1016/S0960-894X(03)00235-X

Fluorescently labeled histamine H₂ receptor antagonists were synthesized starting from N-cyano-N′-[3-(3-piperidin-1-ylmethylphenoxy)propyl]guanidines with an additional N″-ω-aminoalkyl substituent (chain lengths 2-8 methylene groups) or from 3-(3-piperidin-1-ylmethylphenoxy)propylamine. The primary amino group was derivatized with various fluorophores (fluorescein, acridine, dansyl, nitrobenzoxadiazole (NBD), indolo[2,3-a]quinolizine). On the isolated spontaneously beating guinea pig right atrium most of the fluorescent probes were only weakly active, however, the NBD-labeled substances proved to be potent histamine H₂ receptor antagonists achieving pA₂ values in the range of 7.5-8.0, comparable to the activity of famotidine.

Full text of DOI:10.1016/S0960-894X(03)00235-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1717–1720
Synthesis and Pharmacological Activity of Fluorescent Histamine
H2 Receptor Antagonists Related to Potentidine1
Liantao Li,a Julia Kracht,b Shiqi Peng,a Gunther Bernhardt,b
Sigurd Elzb and Armin Buschauerb,*
aCollege of Pharmaceutical Sciences, Peking University, 100083, Beijing, China
bInstitute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
Received 24 December 2002; revised 6 February 2003; accepted 3 March 2003
Abstract—Fluorescently labeled histamine H2 receptor antagonists were synthesized starting from N-cyano-N0-[3-(3-piperidin-1-
ylmethylphenoxy)propyl]guanidines with an additional N00-o-aminoalkyl substituent (chain lengths 2–8 methylene groups) or from
3-(3-piperidin-1-ylmethylphenoxy)propylamine. The primary amino group was derivatized with various fluorophores (fluorescein,
acridine, dansyl, nitrobenzoxadiazole (NBD), indolo[2,3-a]quinolizine). On the isolated spontaneously beating guinea pig right
atrium most of the fluorescent probes were only weakly active, however, the NBD-labeled substances proved to be potent histamine
H2 receptor antagonists achieving pA2 values in the range of 7.5–8.0, comparable to the activity of famotidine.
# 2003 Elsevier Science Ltd. All rights reserved.
As part of a program to develop fluorescence-based
methods for the study of ligand receptor interactions at
G-protein coupled receptors (GPCRs) we have recently
demonstrated that the affinity of agonists and antago-
nists can be determined by flow cytometry under equili-
brium conditions by using cyanine5-labeled neuro
peptide Y.2 This approach is very promising in case of
peptides, and it could generally be a very attractive
alternative to radioligand binding if it were also applic-
able to the investigation of small molecules acting at
GPCRs such as biogenic amines and their antagonists.
Therefore, suitable fluorescent probes are needed to
investigate the applicability of such methods. We selec-
ted histamine receptors3,4 as a model to study low
molecular weight ligands which interact with GPCRs.
Very recently, we reported on fluorescent histamine H1
receptor antagonists related to mepyramine.5 In the
present study fluorescently labeled H2 antagonists are
described.1 As spacefilling residues such as radiolabeled
partial structures including spacer groups are tolerated
in the aminopotentidine series,6 the title compounds
were designed by analogy with the approach that has
been successfully applied to the development of the high
affinity radioligand for the H2 receptor, [125I]iodoami-
nopotentidine6,7 (Scheme 1).
Scheme 1.
Chemistry
The fluorescent histamine H2 receptor antagonists 7a,c,
8a,c, 9a, 10a, 11a–g, 12a were synthesized as outlined in
Scheme 2. 3-(3-Piperidin-1-ylmethylphenoxy)propyl-
amine (2) was prepared in three steps from 3-hydro-
xybenzaldehyde (1) as described elsewhere.8 The
synthesis of the o-aminoalkyl-substituted cyanoguani-
dines 5a–g from 2 was accomplished by analogy with
the procedures described in the literature6,8 using
diphenoxymethylenecyanamide (3) and the pertinent
diamines of various chain lengths (4a–g). The primary
amino group was conjugated to fluorescent dyes by
treating 5a–g with the corresponding succinimidyl esters
(6A/B, 6D) or acid chlorides (6C, 6F) or with 4-chloro-
7-nitrobenzo[2,1,3]oxadiazole (6E).
*Corresponding author. Tel.:+49-941-9434827; fax: +49-941-
9434820; e-mail: armin.buschauer@chemie.uni-regensburg.de
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00235-X

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L. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1717–1720
Table 1. Histamine H2 receptor antagonistic activities of fluorescent
ligands and reference compounds on the isolated guinea pig right
atrium
Compd
lmax Ex
(nm)a
lmax Em
(nm)a
Guinea pig atriumb
pA2ÆSEM
Cimetidine
Ranitidine
Roxatidine acetate
Famotidine
6.00Æ0.06c
6.77Æ0.10d
7.41Æ0.06e
7.74f
5a
7a
7c
8a
8c
9a
10a
11b
11e
11g
12a
15
5.96Æ0.11
4.18Æ0.13
5.05Æ0.09
4.35Æ0.37
5.00Æ0.18
6.74Æ0.08
7.59Æ0.09
7.43Æ0.15
7.49Æ0.12
7.96Æ0.05g
5.71Æ0.16
5.74Æ0.02
n.d.
n.d.
497
n.d.
360
482
483
482
483
333
421h
n.d.
n.d.
525
n.d.
433
539
539
539
539
539
513h
aLongest wavelength absorption and fluorescence emission maximum
determined with a Kontron UV–Vis-Spectrometer and a Perkin–Elmer
LS-50B spectrofluorimeter, the compounds were dissolved in a buffer
(NaCl 120 mM, KCl 5 mM, MgCl2 2 mM, CaCl2 1.5 mM, Hepes 25
mM, glucose 10 mM, adjusted to pH 7.4 with NaOH); n.d.: not
determined.
bInhibition of histamine-stimulated positive chronotropic response;
pA2: mean values calculated from the rightward-shift of isometrically
recorded cumulative concentration–response curves for histamine in
the absence and presence of the H2 receptor antagonists at 32.5 ꢀC;
n=2–10; antagonist concentrations from 0.1 to 100 mM (depending on
the activity the test compound). Schild-plots were constructed for
cimetidine, roxatidine acetate and 11g.
Scheme 2.
cn=16, slope: 0.85Æ0.04.
dSlope not significantly different from unity.
The coupling reactions with the succinimidyl esters of
5-/6-carboxyfluorescein (commercially available mix-
ture of the isomers 6A and 6B) and the NBD-sub-
stituted aminohexanoic acid 6D were carried out in
anhydrous dichloromethane/DMSO in the dark.9 Sub-
sequently, the isomers 7a/8a and 7c/8c were separated
by HPLC on a semipreparative scale. Analogously, the
derivatization of the amines with acridine-9-carbonyl
chloride (6C) NBD-Cl (6E), and dansyl chloride (6F)
was accomplished in anhydrous solvents in the pre-
sence of triethylamine or diisopropylethylamine
(DIPEA).
en=15; slope: 0.85Æ0.04; ref 13: pA2=6.56.
fRef 14.
gcf. Fig. 1.
hIn MeOH: lmax Ex: 420 nm, lmax Em: 495 nm.
Pharmacology
The fluorescent compounds were investigated for hista-
mine H2 receptor antagonistic activity on the isolated
spontaneously beating guinea pig right atrium11
according to standard experimental protocols.12 The
results are summarized in Table 1. Representative con-
centration-response curves and a Schild-plot are shown
for compound 11g in Figure 1.
In addition to the preparation of the cyanoguanidines the
amine 2 was converted to the amide 15 with succinic acid
monoester 14 after activation with carbonyldiimidazole
(Scheme 3). Compound 14 was obtained from (S)-tryp-
tophane by a multistep procedure via 13 as described10
followed by conversion of the alcohol to 14 with succinic
anhydride in the presence of 4-dimethylaminopyridine.
Results and Discussion
Amide derivatives of compound 2, such as roxatidine,
as well as related compounds having N,N0-disubstituted
cyanoguanidine or nitroethenediamine partial structures
are known as rather potent histamine H2 antagonists
(for review see ref 15). It is characteristic of these 3-(3-
piperidin-1-ylmethylphenoxy)propylamine derivatives
that relatively bulky residues may be tolerated without
loss of activity. Depending on their structure the sub-
stituents may even confer additional H2 receptor affi-
nity, as demonstrated by [125I]iodoaminopotentidine,
the high affinity radioligand for the histamine H2
receptor.6,7 Therefore, by analogy with the design of the
latter, except compound 18, the fluorescent probes for
the H2 receptor described in this study were structurally
Scheme 3.

L. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1717–1720
1719
NBD dyes. Such fluorescent probes may be useful to
study the GPCRs in tissue preparations and on cells.
Although, due to its spectral properties and low quan-
tum yield in physiological buffers, NBD is not an ideal
fluorophore, we demonstrated by the strategy described
in this paper, that it is possible to obtain fluorescent
histamine H2 receptor ligands which should be useful
pharmacological tools to investigate ligand receptor
interactions, for example in fluorimetric binding assays
and functional studies.
Acknowledgements
Figure 1. Positive chronotropic effect of histamine in isolated, iso-
metrically set up (resting tension 5 mN), spontaneously beating gui-
nea-pig right atria in the absence (*, n=10) and presence of
compound 11g: 0.1 mM (&, n=3, Emax of histamine 78Æ5%), 0.3 mM
(!, n=3, 77Æ2%), and 1 mM (~, n=4, 74Æ1%). (Æ)-Propranolol
(0.3 mM) was present throughout the experiment. Symbols represent
the arithmetic meanÆSEM. Inset: Schild-plot regression yielded a pA2
value of 7.96Æ0.05 (n=10, 95% confidence limits: 7.84–8.09) and a
slope not significantly different from unity (0.93Æ0.13, p>0.5, two-
tailed t-test).
The authors are grateful to the DAAD for a scholarship
to L. Li (DAAD ‘sandwich PhD program’) and to the
Fonds der Chemischen Industrie for grants to G.B.,
S.E. and A.B.
References and Notes
derived from aminoalkyl-substituted N-[3-(3-piperidin-
1-ylmethylphenoxy)propyl]cyanoguanidine.
1. Presented in part at the Annual Meeting of the German
Pharmaceutical Society, Halle, Oct 2001; Abstract: Li, L.;
Kracht, J.; Elz, S.; Bernhardt, G.; Peng, S.; Buschauer, A.
Arch. Pharm. Pharm. Med. Chem. 2001, 334 (Suppl. 2), 41. A
similar approach has been reported in an abstract: Malan,
S. F.; Menge, W. M.; Hoffmann, M.; Timmerman, H.; Leurs,
R. Drugs Fut. 2002, 27 Suppl. A, 334.
2. (a) Mayer, M.; Li, L.; Kracht, J.; Bernhardt, G.;
Buschauer, A. Arch. Pharm. Pharm. Med. Chem. 2000, 333
(Suppl. 2), 51. (b) Mayer, M. PhD Thesis, 2002, University of
Regensburg. (c) Schneider, E.; Mayer, M.; Bernhardt, G.;
Buschauer, A. Arch. Pharm. Pharm. Med. Chem. 2002, 335
(Suppl. 2), 46.
3. Hill, S. J.; Ganellin, C. R.; Timmerman, H.; Schwartz, J.-
C.; Shankley, N. P.; Young, J. M.; Schunack, W.; Levi, R.;
Haas, H. L. Pharmacol. Rev. 1997, 49, 253.
4. Van der Goot, H.; Timmerman, H. Eur. J. Med. Chem.
2000, 35, 5.
5. Li, L.; Kracht, J.; Peng, S.; Bernhardt, G.; Buschauer, A.
Bioorg. Med. Chem. Lett. 2003, 13, 1245.
6. Hirschfeld, J.; Buschauer, A.; Elz, S.; Schunack, W.; Ruat,
M.; Traiffort, E.; Schwartz, J.-C. J. Med. Chem. 1992, 35,
2231.
The results of the investigation for H2 antagonism on
the isolated guinea pig right atrium (Table 1) show that
the nitrobenzoxadiazole group was by far the best one
to obtain compounds with reasonable antagonistic
activity. These NBD derivatives achieve activities in the
range of pA2 7.5–8.0. A spacer with 6–8 methylene
groups proved to be favorable, whereas an additional
chain as in compound 10a (CO(CH2)5) did not increase
the antagonistic activity further. For instance, the pA2
value of 7.96 found for compound 11g on the guinea pig
atrium (Fig. 1) is comparable to that of famotidine,
which is the most potent therapeutically used H2
antagonist. Obviously, the fluorophore contributes to
the increase in the receptor affinity of the ligand by
interaction with an extra binding site, as the primary
amines used as starting material were only weak or
moderately active H2 antagonists (e.g., 5a).
7. Ruat, M.; Traiffort, E.; Bouthenet, M. L.; Schwartz, J. C.;
Hirschfeld, J.; Buschauer, A.; Schunack, W. Proc. Natl. Acad.
Sci. U.S.A. 1990, 87, 1658.
8. Buschauer, A.; Postius, S.; Szelenyi, I.; Schunack, W. Arz-
neimittelforschung 1985, 35, 1025.
All the other fluorophores were inappropriate to confer
high histamine H2 receptor antagonistic activity. This is
in contrast to a series of H1 antagonists related to
mepyramine, where both the NBD as well as the car-
boxyfluorescein moiety were found to have an activity-
enhancing effect. As the NBD group is the smallest
among the fluorescent labeling agents tested in this
study, it may be speculated that the bulk of the fluor-
ophore and not the length of the connecting chains is
the limiting factor in case of the H2 antagonists related
to iodoaminopotentidine.
9. Labeling of 5a–g. (a) Fluorescein-labeled compounds
7a,c,8a,c: To the solution of 5a,c (0.023 mmol) in 1.5 mL of
anhydrous CH2Cl2 9.0 mg (0.019 mmol) of 6A/B, dissolved in
200 mL of anhydrous DMSO, were added and the mixture was
stirred at room temperature in the dark for 24 h. After evapora-
tion of the solvent the isomers were chromatographed analyti-
cally and separated on a semipreparative scale by RP-HPLC
according to a previously described method.5 7a and 8a: Yield:
90%; +FAB-MS (Varian MAT 95, xenon, MeOH-glycerol): m/z
(%)=717 ([M+H]+, 100); HR-+FAB-MS C40H41N6O7, calcd:
717.3037, found: 717.3005; 7c and 8c: Yield: 87%; +FAB-MS:
m/z (%)=745 ([M+H]+, 100), HR-+FAB-MS: C42H45N6O7,
calcd: 745.3350, found: 745.3333.
Conclusions
The results summarized in Table 1 show that potent
fluorescence-labeled H2 antagonists of the N-[3-(3-piper-
(b) N-[2-(Acridine-9-carbonyl)aminoethyl)]-N0-cyano-N00-[3-
(3-piperidin-1-ylmethylphenoxy)propyl]guanidine (9a) was
synthesized from 6C (0.41 mmol) and 5a (130 mg, 0.36 mmol)
in 15 mL of anhydrous CHCl3 in the presence of 200 mL
idin-1-ylmethylphenoxy)propyl]cyanoguanidine
type
can be obtained by derivatization of corresponding pri-
mary amines having appropriate spacer lengths with

1720
L. Li et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1717–1720
DIPEA (N2 atmosphere). The product was isolated by chro-
matography (ChromatotronTM model 8924 (Harrison
Research), rotors with 4 mm layers of silica gel PF254 con-
taining gypsum, EtOAc:MeOH, gradient 1:0 to 1:1). Yield
85%, mp 115 ꢀC (from MeOH). ESI-MS (MeOH + 1%
HOAc): m/z (%)=564 ([M+H]+, 100), 282 ([M+2H]2+, 95);
IR (KBr): n (cmÀ1)=2168 (CꢁN), 1586 (C¼N). Analysis
78%, mp 67 ꢀC. HR-+FAB-MS: C31H41N7SO3, calcd:
592.3070 found: 592.3088.
N-[3-(3-Piperidin-1-ylmethylphenoxy)propyl]succinamic acid
(6S)-3-acetyl-4-oxo-4,6,7,12-tetrahydroindolo[2,3-a]quinolizin-
6-ylmethyl ester (15). Succinic anhydride (80 mg, 0.8 mmol)
was added to the solution of 13 (80 mg, 0.26 mmol) and
DMAP (80 mg, 0.65 mmol) in 5 mL CH2Cl2. After stirring at
room temperature for 2 h the reaction was quenched with
MeOH (0.6 mL), diluted with CH2Cl2, and neutralised with
cold 10% aqueous citric acid. The organic layer was washed
with water, saturated aqueous NaCl, dried over Na2SO4 and
filtered. After evaporation of the solvent 14 was obtained as
yellow powder (yield 85%), mp 94 ꢀC (decomp.). +FAB-MS:
.
(C33H37N7O2 H2O (581.7)) calcd: C 68.14, H 6.76, N 16.86;
found: C 68.05, H 6.78, N 16.40.
(c)
N-Cyano-N0-{2-[5-(7-nitrobenzoxadiazol-4-yl)amino-
pentyl)carbonylaminoethyl]-N00-[3-(3-piperidin-1-ylmethylphe-
noxy)propyl]guanidine (10a): The succinimidyl ester 6D (24
mg, 0.066 mmol), dissolved in 200 mL of DMSO, was added to
the solution of 5a (20 mg, 0.06 mmol) in 2 mL of anhydrous
CH2Cl2. The mixture was stirred at room temperature for 24 h
in the dark and chromatographed (Chromatotron,
CHCl3:MeOH=3:1) to obtain 10a as orange powder (yield
56%). HR-+FAB-MS: C31H43N10O5, calcd: 635.3417, found:
635.3417.
m/z (%)=409 ([M+H]+, 31), 309 (100). IR (KBr):
n
(cmÀ1)=1734 (ester C¼O), 1653 (C¼O). Analysis (C,H,N):
C22H20N2O6 (408.4), calcd C 64.70, H 4.94, N 6.86, found C
25
64.59, H 4.88, N 6.80. ½aꢂD =À2ꢀ (c=0.1 g; DMSO). After
activation of 14 (140 mg, 0.34 mmol) with CDI (56 mg, 0.34
mmol) in 5 mL of anhydrous THF, the solution of 2 (140 mg,
0.556 mmol) in 5 mL of dry THF was added and the mixture
was stirred overnight. After evaporation in vacuo the residual
solid was triturated with water for 4 h and chromatographed
(Chromatotron; CHCl3: MeOH=90:10). Yield 81% 15 as
yellow solid, mp 73–75 ꢀC (MeOH). ESI-MS (MeOH+1%
HOAc): m/z (%)=639 ([M+H]+, 100); 1H NMR (DMSO-
d6): d (ppm)=11.90 (s, 1H), 8.12 (d, J=7.9 Hz, 1H), 7.88 (t,
J=5.5 Hz, 1H), 7.63 (d, J=7.9 Hz, 1H), 7.43 (d, J=8.3 Hz,
1H), 7.26 (2dd, J=6.7/1.2 Hz, 1H), 7.16 (t, J=7.9 Hz, 1H),
7.08 (2dd, J=6.7/0.8 Hz, 1H), 6.84 (d, J=7.9 Hz, 1H), 6.81–
6.78 (m, 2H), 6.75 (dd, J=7.9/2.4 Hz, 1H), 5.68 (q, J=6.3 Hz,
1H), 4.17 (dd, J=11.1/7.13 Hz, 1H), 4.00 (dd, J=11.1/6.34
Hz, 1H), 3.91 (t, J=6.3 Hz, 2H), 3.33 (s, 2H), 3.27–3.12 (m,
4H), 2.57 (s, 3H), 2.40–2.16 (m, 8H), 1.80 (m, J=6.6 Hz, 2H),
(d) N-Cyano-N0-[o-(7-nitrobenzoxadiazol-4-yl)aminoalkyl]-
N00-[3-(3-piperidin-1-ylmethylphenoxy)propyl]guanidines 11a–g:
22 mg (0.11 mmol) of 6E was added to the solution of 0.1 mmol
of 5a–g and 30 mL of Et3N in 5 mL of anhydrous CHCl3. The
mixture was stirred at room temperature for 10 h in the dark.
After evaporation of the solvent the residue was chromato-
graphed (Chromototron, eluent: CHCl3 : MeOH=3:1). 11a:
Yield: 23%; HR-+FAB-MS: C25H32N9O4, calcd: 522.2577,
found: 522.2572. IR (KBr): n(cmÀ1)=2169 (CꢁN), 1297 (NO2).
11b: Yield: 38%; HR-+FAB-MS: C26H34N9O4, calcd: 536.2733,
found: 536.2726. 11c: Yield: 37%; HR-+FAB-MS:
C27H36N9O4, calcd: 550.2890, found: 550.2870. 11d: Yield: 44%;
HR-+FAB-MS: C28H38N9O4, calcd: 564.3046, found: 564.3033.
11e: Yield: 26%; HR-+FAB-MS: C29H40N9O4, calcd: 578.3203,
found: 578.3179. 11f: Yield: 49%; HR-+FAB-MS: C30H42N9O4,
calcd: 592.3359, found: 592.3334. 11g: Yield: 37%; HR-+FAB-
.
1.52–1.28 (m, 6H). Analysis [C37H42N4O6 H2O (656.8)] calcd:
C 67.66, H 6.75, N 8.53, found: C 67.75, H 6.69, N 8.43;
25
MS: C31H44N9O4, calcd: 606.3516, found: 606.3515; H NMR
½aꢂD =À19ꢀ (c=0.1, DMSO).
1
(CDCl3): d (ppm)=8.47 (d, J=8.7 Hz, 1H), 7.21 (t, J=8.0 Hz,
1H), 6.95–6.73 (m, 4H), 7.18 (d, J=8.7 Hz, 1H), 5.71 (t, J=5.1
Hz, 1H), 5.36 (t, J=5.1 Hz, 1H), 4.08 (t, J=5.5 Hz, 2H), 3.50–
3.44 (m, 6H), 3.16–3.08 (q, J=6.7 Hz), 2.39 (s, 4H), 2.12–2.00
(m, J=5.8 Hz, 2H), 1.85–1.25 (m, 18H). IR (KBr):
n(cmÀ1)=2165 (CꢁN), 1585 (C¼N), 1297 (NO2).
10. Zhao, M.; Wang, C.; Guo, M.; Peng, S. Q. J. Prakt.
Chem. 1999, 341, 667.
11. Black, J. W.; Duncan, W. A. M.; Durant, C. J.; Ganellin,
C. R.; Parsons, E. M. Nature 1972, 236, 385.
12. Pertz, H.; Elz, S. J. Pharm. Pharmacol. 1995, 47, 310.
13. Tarutani, M.; Sakuma, H.; Shiratsuchi, K.; Mieda, M.
Arzneimittelforschung 1985, 35, 703.
14. Black, J. W.; Leff, P.; Shankley, N. P. Br. J. Pharmacol.
1985, 86, 581.
15. Van der Goot, H.; Timmerman, H. In Histamine and
Histamine Antagonists; Uvnas, B., Ed.; Handbook of Exp.
Pharmacol. Vol 97; Springer: Berlin, 1991; p 573.
(e) N-Cyano-N0-[2-(5-dimethylaminonaphthalene-1-sulfo-nyl)
aminoethyl] - N00 - [3 - (3 - piperidin - 1 - ylmethylphenoxy)propyl]-
guanidine (12a): The mixture of 5a (55.6 mg, 0.155 mmol), 6F
(50 mg, 0.185 mmol) and Et3N (66 mL) in 2 mL of CH2Cl2 was
stirred at room temperature for 14 h and the product was iso-
lated with a Chromatotron (CHCl3: MeOH=95:5). Yield