Truxillic acid derivatives, neuromuscular blocking agents with very high affinity for the allosteric binding site of muscarinic acetylcholine receptors

Article abstract of DOI:10.1135/cccc19991980

Bis-quaternary salts of 3-piperidinopropyl esters of α-truxillic acid were synthesized in order to study their allosteric action on muscarinic acetylcholine receptor. Using radioligand binding studies, it has been demonstrated that most of prepared compounds bind with high affinity to the allosteric binding site of M₂ muscarinic receptor subtype (K_d values in the range 1-10 nM). Bulky substitution of the quaternary ammonium center led to effective positive modulators of [³H]N-methylscopolamine binding to M₂ receptors. Due to its high allosteric potency, the structure of phenacyl derivative seems to be the most promising candidate for future design of photoaffinity probes or radiolabelled ligands for mapping the allosteric binding site.

Full text of DOI:10.1135/cccc19991980

1980
Urbanský, Proška, Říčný, Drašar:
TRUXILLIC ACID DERIVATIVES, NEUROMUSCULAR BLOCKING
AGENTS WITH VERY HIGH AFFINITY FOR THE ALLOSTERIC
BINDING SITE OF MUSCARINIC ACETYLCHOLINE RECEPTORS
b
c
a2
Marek URBANSKÝa1*, Jan PROŠKA , Jan ŘÍČNÝ and Pavel DRAŠAR
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: 1 maur@uochb.cas.cz,
2 drasar@uochb.cas.cz
b Research Institute for Pharmacy and Biochemistry, Kouřimská 17, 130 60 Prague 3,
Czech Republic; e-mail: rcr.c@telecom.cz
c Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083,
142 20 Prague 4, Czech Republic; e-mail: ricny@sun1.biomed.cas.cz
Received May 27, 1999
Accepted July 30, 1999
Bis-quatern ary salts of 3-piperidin opropyl esters of α-truxillic acid were syn th esized in order
to study th eir allosteric action on m uscarin ic acetylch olin e receptor. Usin g radioligan d bin d-
in g studies, it h as been dem on strated th at m ost of prepared com poun ds bin d with h igh af-
fin ity to th e allosteric bin din g site of M2 m uscarin ic receptor subtype (Kd values in th e ran ge
1–10 n M). Bulky substitution of th e quatern ary am m on ium cen ter led to effective positive
m odulators of [3H]N-m eth ylscopolam in e bin din g to M2 receptors. Due to its h igh allosteric
poten cy, th e structure of ph en acyl derivative seem s to be th e m ost prom isin g can didate for
future design of ph otoaffin ity probes or radiolabelled ligan ds for m appin g th e allosteric
bin din g site.
Key w ord s: Allosteric bin din g; Cooperativity; Muscarin ic receptors; Nicotin ic receptors;
Truxillic acid; Quatern ary am m on ium salts; Cyclobutan es.
Muscarin ic acetylch olin e receptors (m ACh R) belon g to a superfam ily of
G-protein coupled receptors1 (GPCR) wh ose fun ction is to tran sduce ch em i-
cal sign als across cell m em bran es. Bin din g of th e n eurotran sm itter to th e
GPCR leads to th e association with a G-protein . Th e G-protein s eith er stim -
ulate or in h ibit th e production of a secon d m essen ger an d th us th e in itial
sign al is tran sduced to som e effector system 2. In th e m uscarin ic receptors,
th e extracellular ch em ical sign al is m ediated by a m olecule of acetylch o-
lin e. Th ere are five subtypes of m uscarin ic receptors wh ich h ave been iden -
tified in clon in g studies as distin ct m olecular en tities3. All subtypes exh ibit
stron g con servation of sequen ce in th e region s th at are con sidered to bin d
“orth osteric” ligan ds (e.g., acetylch olin e, atropin e-like drugs, R-(–)-3- qui-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Truxillic Acid Derivatives
1981
n uclidin yl ben zilate). As a con sequen ce, attem pts to discover subtype-
specific ligan ds h ave failed. Sufficien tly selective ligan d with affin ity for
on e of th e subtypes at least 100-fold h igh er th an affin ity for th e oth er sub-
types is n ot available.
In addition to th e bin din g site for orth osteric ligan ds, m uscarin ic recep-
tors also con tain an allosteric bin din g site4. It was form erly predicted on
th e basis of kin etic studies th at th is site m ay be located close to an d just
extracellular to th e “classical” bin din g site5. Som e site-directed m utagen esis
studies brough t an experim en tal support of th at prediction 6. However, little
is kn own about th e m olecular n ature of th e site an d th ere is n o un am bigu-
ous eviden ce of th e crucial im portan ce of an y am in o acid residue in th e re-
ceptor for an in teraction with th e allosteric ligan ds. Allosterically actin g
drugs represen t a n ovel approach for affectin g m uscarin ic receptors7. Th e
in teraction s follow th e allosteric tern ary com plex m odel. Recen tly, som e
allosteric ligan ds h ave been con sidered to in crease th e affin ity of acetylch o-
lin e for som e of th e five subtypes of m uscarin ic receptors8,9
.
Search for suitable allosteric m odulators is h igh ly dem an ded to gain in -
sigh t in to th e m ech an ism of ligan d–receptor in teraction , to determ in e th e
location of bin din g sites for differen t types of ligan ds, an d to correlate be-
tween ligan d structure an d th eir affin ity to particular receptor subtypes.
Curren tly, a radioligan d is n ot com m ercially available to label an allosteric
recogn ition site an d th us, direct com petition experim en ts with un labelled
m odulators are n ot possible. Discovery of con ven ien t ligan ds for better un -
derstan din g of th e ph en om en a can th us be regarded as a crucial step to-
wards future developm en t of allosteric drugs10
. Kn own allosteric
m odulators were foun d m ostly am on g n eurom uscular blockers an d
acetylch olin esterase in h ibitors an d reactivators. Th ey exh ibit wide struc-
tural diversity, wh ich raises th e question wh eth er structural elem en ts or
ph ysico-ch em ical properties wh ich govern th e allosteric poten cy can be de-
fin ed11–13. Perh aps th e on ly com m on structural feature is th e presen ce of
quatern ary or proton ated tertiary n itrogen . Gallam in e14, d-tubocurarin e15
,
tacrin e16 an d obidoxim e17 are exam ples of th e substan ces wh ich h ave been
foun d to decrease th e bin din g of radiolabelled m uscarin ic an tagon ists (n eg-
ative cooperativity). Alcuron ium 18, strych n in e an d eburn am on in e19 belon g
to th e less n um erous group of positive allosteric m odulators.
Th e presen t study focused on bis-quatern ary salts of α-truxillic
(2α,4β-diph en ylcyclobutan e-1α,3β-dicarboxylic) acid20,21. Am on g th ese
bis-quatern ary structures, an atruxon ium (1) an d cyclobuton ium (3) were
recogn ized as n on -depolarizin g m yorelaxan ts. Th e fact th at th eir th erapeu-
tic values were reduced by reported sign ifican t side effects on th e cardiac
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1982
Urbanský, Proška, Říčný, Drašar:
m uscle, led us to th e presum ption th at sim ilar derivatives could beh ave as
allosteric m odulators of cardiac M2 receptor subtype. Prelim in ary experi-
m en ts with auth en tic sam ples of com poun ds 1–3 sh owed th at th e
3-piperidinopropyl derivative 1 acts as high-affinity negative allosteric m odu-
lator. We aim ed th erefore to prepare set of yet un described bis-quatern ary
an alogues of 1. Th e com poun ds were exam in ed in radioligan d bin din g
studies with [3H]N-m eth ylscopolam in e at cardiac m uscarin ic M2 receptors.
In a parallel exam in ation , ch an ge in origin al n eurom uscular poten cy in
con n ection with particular structural ch an ges was assessed by in h ibition of
evoked con traction of n ervus ph ren icus-diaph ragm preparation .
Et
Et
COO(CH2)3N Me
Et
COO(CH2)nN
Et
N (CH2)nOOC
Et
Me N (CH2)3OOC
Et
2 I
2 I
1, n = 3 Anatruxonium
2, n = 4 Truxillonium
3, Cyclobutonium
EXPERIMENTAL
Meltin g poin ts were determ in ed on a Kofler block an d are un corrected. In frared spectra were
recorded on a Perkin –Elm er PE 580 spectrom eter (waven um bers in cm –1). 1H NMR spectra
were taken on a Varian UNITY-200 (200 MHz, FT m ode) at 23 °C. Ch em ical sh ifts are given
in ppm (δ-scale), couplin g con stan ts (J) an d width of sym m etrical m ultiplets (W) in Hz. FAB
m ass spectra were recorded on a VG Analytical ZAB-EQ spectrom eter. Thin-layer chrom atography
was perform ed on precoated alum in ium sh eets Alum in ium oxide 60 F254 n eutral, type E
(Merck). For preparative flash colum n ch rom atograph y alum in ium oxide accordin g to
Brockm an n , type II n eutral (Rean al, Hun gary) was used. 4-(2-Hydroxyeth yl)piperazin e-
1-eth an esulfon ic acid (HEPES), gallam in e trieth iodide an d atropin e sulfate were purch ased
from Sigm a–Aldrich Co Ltd., (–)-[N-m eth yl-3H]scopolam in e m eth yl ch loride ([3H]NMS) was
obtain ed from Am ersh am , U.K. Bis(3-piperidin opropyl) α-truxillate (4) was prepared accord-
in g to Aren daruk20
.
(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl)dim eth yl Bis(3-piperidin opropion ate)
Dih ydroch loride (5)
Method A. DCC (4.3 g, 20.8 m m ol) was added to a stirred m ixture of 2α,4β-diph en yl-
cyclobutan e-1α,3β-dim eth an ol22 (2.24 g, 8.3 m m ol), 3-piperidin opropion ic acid (2.8 g, 17.8
m m ol) an d 4-m eth ylben zen esulfon ic acid m on oh ydrate (80 m g, 0.4 m m ol) in dry pyridin e
(40 m l). Th e m ixture was stirred at room tem perature for 8 h an d th en allowed to stan d for
another 16 h. Pyridine was rem oved in vacuo, the residue treated with 5% sulfuric acid (200 m l),
filtered, th e filtrate treated with con cen trated am m on ia (25 m l) an d extracted with eth er.
Th e eth eral extract was wash ed with water an d dried over an h ydrous sodium sulfate. After
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Truxillic Acid Derivatives
1983
evaporation of th e solven t, th e crude product was ch rom atograph ed on a colum n of alu-
m in ium oxide in ch loroform –eth yl acetate–trieth ylam in e (1 : 1 : 0.06) yieldin g free base of 5
as colorless oil (3.54 g). Th is was dissolved in dich lorom eth an e (100 m l) an d saturated with
dry gaseous h ydrogen ch loride. Th e solven t was rem oved an d th e residue crystallized from
m eth an ol–aceton e to give 3.5 g (67%) of th e dih ydroch loride 5, m .p. 205–206 °C. 1H NMR
(D2O): 7.48 m , 10 H (arom . H, W = 48); 4.10 m , 4 H (2 × CH2O, W = 38); 3.78 dd, 2 H (H-2,
H-4, J2,1 = 11, J2,3 = 7); 3.78 m , 2 H (H-1, H-3, W = 24); 3.28 br an d 2.76 br, 8 H (4 × CH2N);
3.04 t, 4 H (2 × CH2O, J = 7); 2.54 t, 4 H (2 × CH2N, J = 7); 1.96–1.30 m , 12 H (6 ×
CH2CH2CH2). IR (KBr): 2 631, 2 612, 2 576, 2 528, 2 479, 2 464, 2 403, 2 279 (NH+); 1 740,
1 732 (C=O); 1 256, 1 221 (C–O); 3 056, 3 032 (C–H, arom .); 1 601, 1 497, 1 453 (arom .
rin g). MS, m/z (%): 583 (6, M – Cl); 547 (100, M – Cl – HCl); 462 (6); 408 (7); 274 (16, (M –
2 Cl)2+). For C34H48Cl2N2O4 (619.7) calculated: 65.90% C, 7.81% H, 11.44% Cl, 4.52% N;
foun d: 65.73% C, 7.69% H, 11.27% Cl, 4.39% N.
Method B. To fin ely powdered 3-piperidin opropion ic acid h ydroch loride (4.33 g, 22.4 m m ol),
on e drop of DMF an d th ion yl ch loride (16 m l, 223 m m ol) were added. After stirrin g for 30
m in at 50 °C, th e m ixture was coevaporated th ree tim es with dry toluen e an d th e residue
suspen ded in dry ch loroform (50 m l). 2α,4β-Diph en ylcyclobutan e-1α,3β-dim eth an ol (3 g,
11.2 m m ol) in ch loroform (50 m l) an d N,N-diisopropyleth ylam in e (4 m l, 22.9 m m ol) were
added at 0 °C. After stirrin g for 1 h at room tem perature th e solven t was evaporated an d th e
residue treated with 5% sulfuric acid. Th e acidic extract was wash ed with eth er, decolorized
by addition of ch arcoal, filtered an d basified (to pH ≈ 9) by addition of con cen trated am m o-
n ia un der coolin g. Resultin g oily base was taken in to eth eral solution an d after dryin g an d
rem oval of th e solven t con verted to h ydroch loride 5 (6.1 g, 88% yield) by th e sam e proce-
dure as described above.
Bis(3-piperidin opropyl) 2α,4β-Diph en ylcyclobutan e-1α,3β-dicarboxylate
Dih ydrobrom ide (6)
Meth an olic solution of h ydrogen brom ide was added to a solution of am in e 4 (1.0 g, 1.8
m m ol) in m eth an ol (10 m l). Evaporation to dryn ess an d recrystallization from m eth an ol
yielded 1.12 g (86%) of dih ydrobrom ide 6, m .p. 218–220 °C. 1H NMR (D2O): 7.45 m , 10 H
(arom . H, W = 38); 4.54 dd, 2 H (H-2, H-4, J2,1 = 11, J2,3 = 7); 4.13 dd, 2 H (H-1, H-3, J1,2
=
11, J1,4 = 7); 3.87 m , 4 H (2 × CH2O, W = 40); 3.36 br an d 2.79 br, 8 H (4 × CH2N); 2.65 t,
4 H (2 × CH2N, J ≈ 8); 2.04–1.45 m , 16 H (8 × CH2CH2CH2). IR (KBr): 2 623, 2 531, 1 425
(NH+); 1 727 (C=O); 1 177, 1 056 (C–O); 3 061, 3 032 (C–H, arom .); 1 604, 1 586, 1 497,
1 455, 1 001 (arom . rin g); 2 944, 1 475, 1 464 (CH2). MS, m/z (%): 627 (11, M – Br); 547
(100, M – Br – HBr); 436 (23); 422 (5); 377 (6); 274 (53, (M – 2 Br)2+). For C34H48Br2N2O4
(708.6) calculated: 57.63% C, 6.83% H, 22.55% Br, 3.95% N; foun d: 57.21% C, 6.77% H,
22.28% Br, 3.87% N.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) Bis(carbon yloxypropan e-3,1-diyl)]-
bis(N-eth ylpiperidin ium ) Dibrom ide (7)
A m ixture of eth yl brom ide (9 m l) an d am in e 4 (1.0 g, 1.8 m m ol) was refluxed for 8 h . Th e
solid residue obtain ed by evaporation of th e excess of th e reagen t was crystallized from a
m ixture of eth an ol an d eth yl acetate to give th e salt 7 (1.09 g, 78%), m .p. 200–204 °C, dec.
1H NMR (D2O): 7.47 s, 10 H (arom . H, W = 31); 4.55 dd, 2 H (H-2, H-4, J2,1 = 11, J2,3 = 7);
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1984
Urbanský, Proška, Říčný, Drašar:
4.12 dd, 2 H (H-1, H-3, J1,2 = 11, J1,4 = 7); 3.85 m , 4 H (2 × CH2O, W = 46); 3.27 q, 8 H (2 ×
CH2CH3, J = 7); 3.20 m , 8 H (4 × CH2N, W = 48); 2.83 m , 4 H (2 × CH2N, W = 60);
1.93–1.58 m , 16 H (8 × CH2CH2CH2); 1.19 t, 6 H (2 × CH2CH3, J = 7). IR (KBr): 1 714 (C=O);
1 198, 1 056 (C–O); 3 057, 3 025, 3 003 (C-H, arom .); 1 603, 1 584, 1 497, 1 453, 1 008 (arom .
rin g); 2 945, 2 864, 1 484, 1 474 (CH2); 2 970, 2 889 (CH3). MS, m/z (%): 685 (66, M – Br);
639 (25); 603 (9, M – Br – HBr); 575 (100, M – Br – C2H5Br); 490 (27); 464 (46); 450 (19);
302, (M – 2 Br)2+). For C38H56Br2N2O4 (764.7) calculated: 59.69% C, 7.38% H, 20.90% Br,
3.66% N; foun d: 59.78% C, 7.21% H, 20.73% Br, 3.49% N.
Gen eral Procedure for Preparation of Quatern ary Salts 8–13
Am in e 4 (1.0 g, 1.8 m m ol) in dry m eth an ol (15 m l) was refluxed with correspon din g alkyl
brom ide (4.0 m m ol) un der an atm osph ere of argon for 5 h . Rem oval of th e volatiles an d
crystallization from appropriate solven ts yielded th e quatern ary am m on ium salts as wh ite
solids.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) bis(carbon yloxypropan e-3,1-diyl)]-bis(N-
allylpiperidin ium ) dibrom ide (8). Yield 1.26 g (87%), m .p. 147–155 °C, dec. (eth an ol–eth yl
acetate). 1H NMR (D2O): 7.45 s, 10 H (arom . H, W = 32); 5.93–5.60 m , 6 H (2 × CH2=CH);
4.53 dd, 2 H (H-2, H-4, J2,1 = 11, J2,3 = 7); 4.10 dd, 2 H (H-1, H-3, J1,2 = 11, J1,4 = 7);
3.92–3.47 m , 4 H (2 × CH2O); 3.82 d, 2 H (2 × CH2=CH–CH2); 3.30–3.10 m , 8 H (4 × CH2N);
2.79 m , 4 H (2 × CH2N, W = 60); 1.92–1.58 m , 16 H (8 × CH2CH2CH2). IR (KBr): 1 724
(C=O); 1 191, 1 057 (C–O); 3 057, 3 028, 2 999 (C–H, arom .); 1 603, 1 584, 1 497, 1 455,
1 006 (arom . rin g); 1 439, 998, 908, (CH2=CH); 2 945, 2 868, 1 479, 1 473 (CH2). MS, m/z
(%): 707 (21, M – Br); 627 (10, M – Br – HBr); 587 (100, M – Br – C3H5Br); 502 (15); 462
(17); 314 (95, (M – 2 Br)2+). For C40H56Br2N2O4 (788.7) calculated: 60.92% C, 7.16% H,
20.26% Br, 3.55% N; foun d: 60.04% C, 7.06% H, 19.87% Br, 3.41% N.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) bis(carbon yloxypropan e-3,1-diyl)]-bis(N-
ben zylpiperidin ium ) dibrom ide (9). Yield 1.30 g (80%), m .p. 146–149 °C (water–aceton e).
1H NMR (DMSO-d6): 7.56 m , 10 H (2 × ben zyl, W = 21); 7.21 m , 10 H (2 × ph en yl, W = 18);
4.35 dd, 2 H (H-2, H-4, J2,1 = 11, J2,3 = 7); 3.92 dd, 2 H (H-1, H-3, J1,2 = 11, J1,4 = 7);
3.58–2.62 m , 16 H (2 × CH2O, 6 × CH2N); 1.98–1.41 m , 16 H (8 × CH2CH2CH2). IR (KBr):
1 726 (C=O); 1 187, 1 180, 1 057 (C–O); 3 057, 3 030 (C–H, arom .); 1 603, 1 583, 1 497,
1 453, 1 002 (arom . rin g); 2 945, 1 473 (CH2). MS, m/z (%): 807 (11, M – Br); 764 (7); 727 (7,
M – Br – HBr); 637 (100, M – Br – C7H7Br); 545 (6); 512 (7); 364, (30, (M – 2 Br)2+). For
C
48H60Br2N2O4 (888.8) calculated: 64.86% C, 6.80% H, 17.98% Br, 3.15% N; foun d:
64.69% C, 6.67% H, 17.66% Br, 2.98% N.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) bis(carbon yloxypropan e-3,1-diyl)]-bis(N-
ph en acylpiperidin ium ) dibrom ide (10). Yield 1.55 g (89%), m .p. 217–222 °C, dec. (eth an ol–
eth yl acetate). 1H NMR (DMSO-d6): 8.08–7.58 m , 10 H (2 × ph en acyl); 7.28 m , 10 H (2 ×
ph en yl, W = 40); 5.29 s, 4 H (2 × COCH2); 4.33 dd, 2 H (H-2, H-4, J2,1 = 10.5, J2,3 = 7.0);
3.87 dd, 2 H (H-1, H-3, J1,2 = 10.5, J1,4 = 7.0); 3.80–3.40 m , 16 H (2 × CH2O, 6 × CH2N);
1.96–1.55 m , 16 H (8 × CH2CH2CH2). IR (KBr): 1 727 (C=O, ester); 1 698 (C=O); 1 189,
1 231, 1 226, 1 054 (C–O); 3 085, 3 061, 3 030 (C–H, arom .); 1 597, 1 582, 1 496, 1 450,
1 003 (arom . rin g); 2 937, 2 871, 1 477, 1 466 (CH2). MS, m/z (%): 863 (27, M – Br); 845 (5);
783 (42, M – Br – HBr); 665 (100, M – Br – C8H7BrO); 581 (18); 554 (37); 540 (7); 695 (6);
392 (48, (M – 2 Br)2+). For C50H60Br2N2O6 (944.8) calculated: 63.56% C, 6.40% H,
16.91% Br, 2.96% N; foun d: 63.69% C, 6.34% H, 16.98% Br, 2.94% N.
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Truxillic Acid Derivatives
1985
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) bis(carbon yloxypropan e-3,1-diyl)]-bis{N-
[2-(2-n aph th yl)-2-oxoeth yl]piperidin ium } dibrom ide (11). Yield 1.75 g (92%), m .p. 211–215 °C,
dec. (m eth an ol–aceton e). 1H NMR (DMSO-d6): 8.85 s, 2 H (2 × H1-n aph th yl); 8.09 m , 8 H
(n aph th yl, W = 53); 7.73 m , 4 H (n aph th yl, W = 28); 7.22 m , 10 H (2 × ph en yl, W = 28);
5.42 s, 4 H (2 × COCH2); 4.32 dd, 2 H (H-2, H-4, J2,1 = 10.5, J2,3 = 7.0); 3.84 dd, 2 H (H-1,
H-3, J1,2 = 10.5, J1,4 = 7.0); 3.80–3.42 m , 16 H (2 × CH2O, 6 × CH2N); 2.06–1.54 m , 16 H (8 ×
CH2CH2CH2). IR (KBr): 1 719 (C=O, ester); 1 686 (C=O); 1 191, 1 185, 1 049 (C–O); 3 052,
3 031, 3 011 (C–H, arom .); 1 623, 1 594, 1 570, 1 508, 1 493, 1 452 (arom . rin g). MS, m/z
(%): 963 (30, M – Br); 883 (25, M – Br – HBr); 715 (100, M – Br – C12H9BrO); 630 (16); 590
(11); 545 (17); 442 (60, (M – 2 Br)2+). For C58H64Br2N2O6 (1 045.0) calculated: 66.67% C,
6.17% H, 15.29% Br, 2.68% N; foun d: 66.10% C, 6.05% H, 15.52% Br, 2.63% N.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) bis(carbon yloxypropan e-3,1-diyl)]-bis[N-
(4-ben zyloxyben zyl)piperidin ium ] dibrom ide (12). Yield 1.77 g (88%), m .p. 208–209 °C, dec.
(eth an ol–eth yl acetate). 1H NMR (DMSO-d6): 7.52–7.11 m , 28 H (arom atic H); 5.20 s, 4 H
(2 × ben zylic CH2); 4.44 m , 4 H (2 × ben zylic CH2); 4.38 dd, 2 H (H-2, H-4, J2,1 = 11, J2,3 = 7);
3.96 dd, 2 H (H-1, H-3, J1,2 = 11, J1,4 = 7); 3.94–3.44 m , 4 H (2 × CH2O); 3.20 m , 8 H (W = 40,
CH2N); 2.69 m , 4 H (W = 36, CH2N); 1.96–1.40 m , 16 H (8 × CH2CH2CH2). IR (KBr): 1 731,
1 717, 1 701 (C=O); 1 254, 1 224, 1 192, 1 183 (C–O); 3 057, 3 030 (C–H, arom .); 1 610, 1 582,
1 514, 1 497, 1 453 (arom. ring). MS, m/z (%): 1 019 (10, M – Br); 975 (23); 939 (4, M – Br – HBr);
743 (100, M – Br – C12H13BrO); 659 (7); 617 (5); 547 (28); 470 (47, (M – 2 Br)2+). For
C
62H72Br2N2O6 (1 101.1) calculated: 67.63% C, 6.59% H, 14.51% Br, 2.54% N; foun d:
67.38% C, 6.52% H, 14.39% Br, 2.52% N.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) bis(carbon yloxypropan e-3,1-diyl)]-bis{N-
[2-(4-azidoph en yl)-2-oxoeth yl]piperidin ium } dibrom ide (13). Yield 1.28 g (68%), decom poses
above 160 °C (eth an ol). 1H NMR (DMSO-d6): 8.18 d, 4 H (2 × ph en acyl, J = 8.5); 7.43 d, 4 H
(2 × ph en acyl, J = 8.8); 7.38 m , 10 H (2 × ph en yl, W = 15); 5.34 s, 4 H (2 × COCH2); 4.42
dd, 2 H (H-2, H-4, J2,1 = 11, J2,3 = 7); 3.95 dd, 2 H (H-1, H-3, J1,2 = 11, J1,4 = 7); 3.89–3.46 m ,
16 H (2 × CH2O, 6 × CH2N); 2.11–1.62 m , 16 H (8 × CH2CH2CH2). IR (KBr): 2 132 (N3);
1 728 (C=O, ester); 1 685 (C=O); 1 283, 1 239, 1 180 (C–O); 3 029 (C–H, arom .); 1 597,
1 574, 1 506, 1 496, 1 455, 1 419 (arom . rin g). MS, m/z (%): 945 (47, M – Br); 917 (9, M –
Br – N2); 901 (20); 865 (62, M – Br – HBr); 839 (13); 809 (6); 706 (100, M – Br – C8H6BrN3O);
680 (35); 622 (18); 595 (44); 545 (96); 433 (58, (M – 2 Br)2+); 403 (40, (M – 2 Br – 2 N2)2+).
For C50H58Br2N8O6 (1 026.9) calculated: 58.48% C, 5.69% H, 15.56% Br, 10.91% N; foun d:
58.03% C, 5.84% H, 15.8% Br, 10.42% N.
N,N′-[(2α,4β-Diph en ylcyclobutan e-1α,3β-diyl) Bis(carbon yloxypropan e-3,1-diyl)]-
bis[N-(2-cycloh eh yleth yl)piperidin ium ] Dibrom ide (14)
Am in e 4 (300 m g, 0.55 m m ol) was stirred with 2-cycloh exyleth yl brom ide (1.0 m l) for 2 h
at 50 °C, th en for addition al 3 h at 100 °C. Cooled reaction m ixture was diluted with tolu-
en e, resultin g precipitate filtered off, wash ed with several portion s of toluen e an d crystal-
lized from aceton e–m eth an ol to give 338 m g (66%) of 14, m .p. 232–233 °C. 1H NMR
(DMSO-d6): 7.39 m , 10 H (2 × ph en yl, W = 40); 4.42 dd, 2 H (H-2, H-4, J2,1 = 10.5, J2,3
=
7.0); 3.97 dd, 2 H (H-1, H-3, J1,2 = 10.5, J1,4 = 7.0); 3.67 m , 4 H (2 × CH2O, W = 84);
3.30–2.92 m , 16 H (8 × CH2N); 1.84–0.81 m , 42 H (20 × CH2, 2 × CH). IR (KBr): 1 725
(C=O); 1 258, 1 231, 1 186, 1 180 (C–O); 1 603, 1 495, 1 448 (arom . rin g). MS, m/z (%): 847
(53, M – Br); 801 (5); 767 (8, M – Br – HBr); 657 (51, M – Br – C8H13Br); 572 (24); 532 (19);
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1986
Urbanský, Proška, Říčný, Drašar:
487 (10); 384 (100, (M – 2 Br)2+). For C50H76Br2N2O4 (929.0) calculated: 64.65% C, 8.25% H,
17.20% Br, 3.02% N; foun d: 64.42% C, 8.08% H, 17.10% Br, 3.25% N.
Reaction of 4 with Ph en eth yl Iodide
Solution of am in e 4 (1.0 g, 1.8 m m ol) an d (2-iodoeth yl)ben zen e (930 m g, 4.0 m m ol) in
ch loroform (5 m l) in a sealed tube was h eated to 40 °C for 5 days. Th e solven t was rem oved
an d th e residue crystallized from a m ixture of m eth an ol an d aceton e (1 : 2) to yield 992 m g
of crystallin e m aterial wh ich was iden tified as dih ydroiodide 15, m .p. 248–250 °C. 1H NMR
(DMSO-d6): 8.92 br, 2 H (2 × NH); 7.49 m , 10 H (arom . H, W = 38); 4.51 dd, 2 H (H-2, H-4,
J2,1 = 11, J2,3 = 7); 4.06 dd, 2 H (H-1, H-3, J1,2 = 11, J1,4 = 7); 3.87 m , 4 H (2 × CH2O, W = 74);
3.34 br an d 2.84 br, 12 H (6 × CH2N); 2.01–1.43 m , 16 H (8 × CH2CH2CH2). IR (KBr): 2 710,
2 683, 2 675, 2 637, 2 578, 2 538 (NH+); 1 729, 1 681 (C=O); 1 263, 1 186, 1 176 (C–O); 3 027
(C–H, arom .); 1 602, 1 496, 1 452 (arom . rin g). MS, m/z (%): 675 (12, M – I); 547 (100, M – I
– HI); 422 (3); 378 (8); 274 (30, (M – 2 I)2+). For C34H48I2N2O4 (802.6) calculated: 50.88% C,
6.03% H, 31.62% I, 3.49% N; foun d: 51.11% C, 6.27% H, 30.37% I, 3.41% N.
Radioligan d Bin din g Assays
Tissue preparation. Experim en ts were perform ed on h om ogen ates of h eart atria taken from
m ale Wistar rats (200–230 g body weigh t). Tissue was h om ogen ized with Polytron h om oge-
n izer, in 10-fold volum e of ice-cold m edium . Th e h om ogen ization an d in cubation m edium
con sisted of 100 m M NaCl an d 20 m M HEPES, pH 7.4. Th e h om ogen ates were diluted with
th e h om ogen ization m edium to 2-fold volum e an d cen trifuged at –4 °C for 15 m in at 700 g,
th e pellets were discarded an d supern atan ts were divided in to th e Eppen dorf tubes an d kept
frozen at –80 °C un til th e day of experim en t.
Incubations. In cubation s were perform ed at 25 °C in a total volum e of 1.6 m l an d lasted
3 h un less in dicated oth erwise. Th e con cen tration of [3H]NMS was 200 pM, close to h alf of
th e apparen t dissociation con stan t KRL for com plex [3H]NMS–receptor (KRL = 436 pM, un der
th e con dition used). Tissue con cen tration was 2 m g (origin al wet weigh t) per tube.
Non -specific radioligan d bin din g was defin ed usin g atropin e (5 µM). In cubation s were term i-
n ated by an addition of 3 m l of ice-cold water an d subsequen t vacuum filtration on
Wh atm an GF/B glass fiber filters (presoaked in water for 3 h ), with two wash es of filters
with 3 m l of water. Radioactivity was determ in ed by scin tillation coun tin g.
Data analysis. Data treatm en t was perform ed as described in refs5,23. Data were an alyzed
with n on lin ear regression an alysis usin g Sigm aPlot 4.0 (SPSS, Erkrath , Germ an y). Un less
stated oth erwise, data are presen ted as m ean ± stan dard error of th e m ean (S.E.M.).
Sym bols R, L an d A den ote th e receptor, th e orth osteric ligan d [3H]NMS an d th e allosteric
ligan d, respectively. KRL, KAR den ote th e apparen t dissociation con stan ts of th e correspon -
den t com plexes. Th e cooperativity coefficien t α expresses th e m utual poten cy of th e
allosteric an d orth osteric ligan ds to in duce by receptor occupation a sh ift of each oth ers
bin din g affin ity24. Values of α < 1 suggest positive cooperativity, values of α > 1 suggest n eg-
ative cooperativity.
Nervus Ph ren icus-Diaph ragm Preparation
Wistar fem ale rats weigh in g 200–300 g were used for experim en ts. Left ph ren ic n erve-
h em idiaph ragm preparation was in cubated in th erm ostated organ bath (37 °C, volum e
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Truxillic Acid Derivatives
1987
10 m l), in Tyrode′s solution of th e followin g com position (in m M) NaCl 137.0; KCl 2.7;
CaCl2 1.8; MgCl2 1.0; NaH2PO4 0.4; NaHCO3 11.9; glucose 11.2; ch olin e 0.001; gassed in ten -
sively with O2–CO2 an d attach ed to a ten som eter un der a ten sion of 2 g in an electrode
h older wh ich allowed in direct stim ulation of th e striated m uscle by a n erve electrode (Grass
stim ulator, pulse duration 0.2 m s, 8 V, 0.2 Hz or 50 Hz). Th e ph ren ic n erve was stim ulated
con tin uously at 0.2 Hz. After equilibration an d con solidation of baselin e (durin g approxi-
m ately 10–30 m in , with th e exch an ge of m edium every 10 m in ) th e experim en tal cycles
started. Th e m edium was exch an ged an d th e preparation stim ulated con tin uously for a
given period (for 15 or 30 m in in th ese experim en ts). At th e en d of th is cycle, a train of
tetan ic stim ulation (50 Hz for 3 s) was applied. Th e m edium was th en exch an ged an d th e
cycle repeated. An tagon ists were applied in cum ulatively in creased con cen tration . Th e re-
spon se of th e ten som eter was electron ically recorded by a com puterised A/D con verter. In
som e experim en ts, recovery of twitch es after wash out of th e drug was recorded. Th e tetan ic
fadin g was used to m easure th e effect of an an tagon ist. Spon tan eous decrease of th e tetan ic
con traction s durin g tim e was recorded in a separate experim en t an d th e respon ses from an -
tagon ist-affected experim en ts were corrected by data calculated from th e lin ear regression .
EC50 value for a given substan ce was calculated from a dose-respon se plot of tetan us fadin g
versus log con cen tration of th e drug fitted by n on -lin ear regression to
a sigm oidal
dose-respon se equation usin g a program Prism (Graph Pad Software, In c.).
RESULTS AND DISCUSSION
We foun d out th at an atruxon ium (1) slows down th e dissociation rate of
tritiated m uscarin ic an tagon ist, [3H]NMS, from M2 receptor subtype. Its af-
fin ity is superior to m an y of yet kn own allosteric m odulators. Sin ce
th ree-m em bered aliph atic spacer of th e m olecule seem s to be optim um for
its in teraction with allosteric site, we decided in itially to ch eck wh eth er
proton ated tertiary am in o group would retain som e of th e activity of th e
bis-quatern ary substan ce. Diam in e 4 was prepared by con den sation of
α-truxillic acid dich loride with 3-piperidin opropan ol accordin g to
Aren daruk20. Dih ydrobrom ide 6 was used for th e bioassay. Preparation of
isom eric diester 5 by couplin g of 2α,4β-diph en ylcyclobutan e-1α,3β-dim eth -
an ol22 with 3-piperidin opropion ic acid is rath er less trivial. Activation of
th e acid is com plicated due to virtual in solubility of its proton ated form in
m ost organ ic solven ts an d due to reverse Mich ael reaction wh ich takes
place un der basic con dition s. We exploited two m eth ods: DCC-m ediated
couplin g in pyridin e an d activation in n eat reaction with excess of th ion yl
ch loride.
Bis-quatern ary am m on ium brom ides 7–14 were prepared by stan dard
alkylation reaction of th e diam in e 4 with correspon din g alkyl brom ides. Re-
active brom ides were used in a sligh t stoich iom etric excess an d th e reac-
tion s were com pleted with in 5 h reflux of m eth an olic solution of both
com pon en ts. Eth yl- an d cycloh exyleth yl brom ides were used in a large ex-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1988
Urbanský, Proška, Říčný, Drašar:
cess as reaction m edia. As sh own below, in troduction of ben zyl- an d
ph en acyl groups led to substan ces with m ost con ven ien t bin din g proper-
ties. An attem pt to prepare
a ph en eth yl an alogue th us followed.
2-Haloeth ylben zen es are reported to un dergo substitution reaction with
1,4-diazabicyclo[2.2.2]octan e at 54 °C with out observable form ation of sty-
ren e25. However, in all an alogous experim en ts with our substrate 4, elim i-
n ation reaction prevailed over substitution , yieldin g on ly am m on ium
h alides 6 an d 15. Th e syn th esized com poun ds were ch aracterized by th eir
1H NMR, IR an d FAB m ass spectra.
H
COO(CH2)3N
CH2OOC(CH2)2N
N(H2C)3OOC
N(H2C)2COOH2C
H
2 Cl
4
5
R
COO(CH2)3N
N(H2C)3OOC
R
2 X
R
X
X
R
H
Br
Br
6
7
CH2O
CH2
Br
Br
12
CH3CH2
CH2=CHCH2
Br
8
N3
COCH2
13
Br
9
CH2
CH2CH2
Br
I
14
15
Br
10
11
COCH2
COCH2
H
Br
All an atruxon ium an alogues exam in ed were foun d to be purely allosteric
m odulators of [3H]NMS bin din g. It h as been dem on strated th at sim ple
m odification , such as quatern ization , of paren t com poun d 6 led to a n um -
ber of com poun ds with differen t effects on [3H]NMS bin din g (Table I). We
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Truxillic Acid Derivatives
1989
foun d out th at ben zyl, ph en acyl an d (2-n aph th yl)acetyl groups led to th e
positive m odulatory effects, alth ough eth yl an d allyl groups afforded m odu-
lators with th e n egative effect. Th e effects of n egative an d positive m odula-
tors are dem on strated in Figs 1 an d 2. Am m on ium salts 5 an d 6 exh ibited
n egative m odulatory action as well. In terestin gly, in com parison with com -
poun d 6 th e isom eric ester fun ction ality of 5 caused a substan tial decrease
in affin ity for receptor, wh ile th e n ature of in teraction did n ot ch an ge.
A 2-cycloh exyleth yl substitution was used in order to test th e workin g
h ypoth esis th at th e presen ce of a ben zen e rin g in th e substituen t is n eces-
sary for positive cooperativity with NMS. Som ewh at surprisin gly, th e ab-
sen ce of arom atic rin g did n ot exclude th e ability of com poun d 14 to
in crease an affin ity of [3H]NMS for receptor. Moreover, th e affin ity of 14 for
un ligan ded receptor was foun d to be th e h igh est am on g th e positive m odu-
lators. Th e presen ce of ben zen e rin g sh ould n ot th erefore be regarded as a
crucial structural feature of positive allosteric m odulators of [3H]NMS bin d-
in g to M2 receptors. It appears th at th e volum e of ch osen N-substituen ts
TABLE I
Quan titative param eters of th e bin din g an d action of allosteric m odulators
pKAR ± S.E.M., M
(n um ber of experim en ts)
Com poun d
α
Gallam in e
6.830 ± 0.031 (4)
8.234 ± 0.193 (9)
7.602 ± 0.180 (5)
6.504 ± 0.152 (3)
7.644 ± 0.168 (4)
7.725 ± 0.435 (3)
7.253 ± 0.085 (3)
6.873 ± 0.141 (4)
6.991 ± 0.104 (3)
7.186 ± 0.199 (4)
7.609 ± 0.162 (3)
21.40
21.25
11.08
23.30
18.57
18.91
0.322
0.158
0.171
0.211
0.515
1
3
5
6
8a
9
10
11
13b
14
a
b
Com poun d stron gly h ygroscopic. Data obtain ed un der con dition s of low ion ic stren gh t
(20 m M HEPES + 10 mM NaCl), apparen t dissociation con stan t for [3H]NMS was KRL
=
145 pM.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1990
Urbanský, Proška, Říčný, Drašar:
m ay play im portan t role in tun in g th e n ature of th e cooperative in teraction
at m uscarin ic receptors.
We exam in ed som e of th ese com poun ds in a fun ction al assay on n ico-
tin ic acetylch olin e receptors. In h ibition of evoked con traction of n ervus
ph ren icus-diaph ragm preparation was used in th e arran gem en t described
by Wessler26. Data in Table II im ply, in con trast to situation on m uscarin ic
receptors, th e sim ple in verse relation between th e volum e of N-substituen ts
an d poten cy of com poun ds (expressed in term s of EC50). An explan ation of-
fers th at direct steric h in dran ce to in teraction of quatern ary n itrogen s with
receptor results in a decrease of th e affin ity of bulky-substituted com -
poun ds for n icotin ic receptors.
120
240
100
200
80
160
60
120
40
80
20
40
0
0
–11
–9
–7
–5
–3
–11
–9
–7
–5
–3
Allosteric modulator
Allosteric modulator
FIG. 2
FIG. 1
Ch an ges in th e specific bin din g of
[3H]N-m eth ylscopolam in e to rat atrial m em -
bran es in duced by 10 (ꢀ). Abscissa: log10 of
th e m olar con cen tration of th e drug. Ordi-
n ate: [3H]N-m eth ylscopolam in e bin din g in
th e presen ce of th e drug, expressed as per
cen t of th e bin din g in th e absen ce of th e
drug. Poin ts are m ean s of four experim en ts
with in cubation perform ed in duplicate.
Full lin e represen ts th e best fit of data to
Eq. 6 in th e study of Eh lert24 with in th e
con cen tration ran ge of 10–11.5 to 10–6.5 M,
wh ere an equilibrium was reach ed5
Allosteric in h ibition of th e specific bin din g
of [3H]N-m eth ylscopolam in e to rat atrial
m em bran es in duced by 1 (ꢀ), 3 (ꢀ) an d th e
prototypic allosteric in h ibitor gallam in e (ꢀ).
Abscissa: log10 of th e m olar con cen tration of
th e drug. Ordin ate: [3H]N-m eth ylscopol-
am in e bin din g in th e presen ce of th e drug,
expressed as per cen t of th e bin din g in th e
absen ce of th e drug. Full lin es represen t th e
best fits of data to Eq. 6 in the study of Ehlert24
.
Poin ts are m ean s ± S.E.M. of n in e (1), four
(3) and four (gallam ine) experim ents with in-
cubations perform ed in duplicate
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Truxillic Acid Derivatives
1991
A relatively direct approach to elucidate th e allosteric bin din g site at th e
m olecular level m igh t be to syn th esize a reactive ligan d th at covalen tly
bin ds to th is site. Th us, am in o acid residues would be iden tified to wh ich
th is ligan d bin ds27. It is well kn own th at aryl azides after irradiation gen er-
ate h igh ly reactive in term ediates. Hen ce, with respect to com m ercial avail-
ability of 2-(4-azidoph en yl)-2-oxoeth yl brom ide, we decided prim arily to
prepare th e reactive an alogue of positive allosteric m odulator 10. Th e n ew
com poun d retain ed th e feature of positive allosteric m odulator of [3H]NMS
bin din g to M2 receptors, alth ough th e low-ion ic-stren gth in cubation m e-
dium h ad to be used to overcom e th e decrease in solubility. Th e pKAR
(7.186) for th is bifun ction al ph otoreactive probe 13 was obtain ed from ex-
perim en ts with out irradiation . Con sequen tly, a direct com parison of th e
bin din g param eters of reactive probe 13 with th ose of tem plate com poun d
10 is n ot available. Neverth eless, 13 appears to be a prom isin g prototype of
ph otoaffin ity probe. Con siderin g m utual positive cooperativity (α = 0.211),
th e affin ity of 13 for th e receptor occupied by [3H]NMS in creases approxi-
m ately five tim es relative to th e un ligan ded receptor.
In con clusion , th e results of th is research dem on strate th at relatively
sm all structural m odification of paren t structure can sign ifican tly affect th e
m ech an ism of action of an allosteric m odulator. We assum e th at th e
ach ieved results, th ough in directly, support th e con cept th at both positive
an d n egative m uscarin ic allosteric m odulators sh are iden tical or overlap-
pin g bin din g sites.
TABLE II
In h ibitory effects of m uscarin ic allosteric m odulators on evoked con cen tration of n ervus
ph ren icus-diaph ragm
Com poun d
pEC50 ± S.E.M.
Relative poten cy
d-Tubocurarin e
6.876 ± 0.036
6.659 ± 0.024
6.088 ± 0.015
5.983 ± 0.008
5.467 ± 0.019
4.149 ± 0.055
1.000
0.607
0.163
0.128
0.039
0.002
1
8
9
10
11
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

1992
Urbanský, Proška, Říčný, Drašar:
The authors are indebted to Dr S. A. Shelkovnikov from Sankt-Petersburg for providing authentic
samples of anatruxonium and cyclobutonium. Their thanks are due to Dr P. Fiedler for taking and in-
terpretation of IR spectra, the staff of the Laboratory of NMR Spectroscopy (Dr M. Buděšínský, Head),
the Laboratory of Mass Spectroscopy (Dr K. Ubik, Head), and the Analytical Laboratory (Dr V.
Pechanec, Head) of the Institute of Organic Chemistry and Biochemistry. The project was supported by
the Grant Agency of the Czech Republic (grant No. 203/97/0302).
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