Scheme:
Preparation
of 3&4
J Pharm Pharmaceut Sci (www.cspscanada.org) 8(3):578-585, 2005
Synthesis of 2-azabicyclo[3.2.2]nonanes from bicyclo[2.2.2]octan-2-ones and their activities against Trypanosoma brucei rhodesiense and Plasmodium falciparum K1
Werner Seebacher1, Robert Weis1, Marcel Kaiser2, Reto Brun2, Robert Saf3
1Institute of Pharmaceutical Sciences,
Pharmaceutical Chemistry, Karl-Franzens-University,
2Swiss Tropical Institute,
3Institute of Chemical Technology of Organic
Materials,
Received April 21, 2005; Revised October 19, 2005; Accepted October 21, 2005, Published October 28, 2005
Corresponding Author:
Werner Seebacher,
ABSTRACT Purpose: New 2-azabicyclo[3.2.2]nonanes were prepared from antiprotozoal bicyclo[2.2.2]octan-2-ones to investigate the influence of the replacement of the rigid bicyclo-octane structure by the more flexible bicyclo-nonane system on the antiplasmodial and antitrypanosomal activity. Methods: The 2-azabicyclo[3.2.2]nonanes were synthesized via a one-step procedure from bicyclo[2.2.2]octan-2-ones and tested for their activities against Trypanosoma b. rhodesiense and Plasmodium falciparum K1 (resistant to chloroquine and pyrimethamine) using in vitro microplate assays. Results: 2-azabicyclo[3.2.2]non-5-ylamines exhibit higher antiprotozoal activities than 4-aminobicyclo[2.2.2]octanes, 4-aminobicycl [2.2.2]octan-2-ones and 4-amino-2-azabicyclo[3.2.2]nonan-3-ones. (7, 8-Diphenyl-2-azabicyclo[3.2.2]non-5-yl)-dimethylamine shows enhanced anti-trypanosomal (IC50 = 0.60 mM) and remarkable antiplasmodial (IC50 = 0.28 mM) activity. However, the in vivo activity of this compound against Plasmodium berghei in mice is moderate. Conclusions: Due to their promising in vitro antiprotozoal activity and their low cytotoxicity, 2-azabicyclo[3.2.2]nonanes should serve as lead compounds for further modifications.
Trypanosoma brucei gambiense and T. b.
rhodesiense are protozoan parasites causing Human African trypanosomiasis.
About 0.5 million people are infected with this disease
in central
Malaria is a
parasitic disease of major global health significance that causes an estimated
2.7 million deaths each year (10). It is caused by
four species and Plasmodium falciparum is the most virulent and
potentially deadly. It is responsible for more than one million deaths in
African children per year (11). Many of the antimalarial drugs are loosing
their effectiveness in the face of multidrug-resistant strains of Plasmodium
(12-14) so that traditional therapeutics such as chloroquine and pyrimethamine
that were once highly effective, are almost useless in many parts of the world
(15, 16). Even for the most recently introduced artemisinine derivatives
loss of sensitivity has been observed (17-21). Hence that,
there is great demand for new antimalarials which are active against resistant
strains of Plasmodium falciparum.
Recently, we reported
the synthesis of 4‑aminobicyclo[2.2.2]octane-2-ones
1 from acyclic starting material (22) via a one-pot procedure, which
could be advantageous for a later industrial synthetic process in large-scale.
Those compounds and their reduction products 2 showed activity against Plasmodium
falciparum K1, which is resistant to chloroquine and
pyrimethamine, and Trypanosoma b. rhodesiense (23,24).
Now we present the synthesis of 2-azabicyclo[3.2.2]nonane
derivatives 3 from 1 in one step and their reduction to bicyclic
amines 4. All new compounds were tested against
P. falciparum K1, T. b. rhodesiense and for their
cytotoxicity against L6 cells.
The tests were performed using in vitro microplate assays. The results are compared to the activities of the previously synthesized compounds.
Melting
points were obtained on a digital melting point
apparatus
Compounds 1 have been
prepared from benzylidene acetone and dialkylammonium thiocyanates in one-pot
reactions (22,23). Their reduction by a Wolff-Kishner
procedure yielded the bicyclic amines 2 (24). The formation of the 2-azabicyclo[3.2.2]nonan-3-ones 3 succeeded by a Beckmann
rearrangement of compounds 1 using hydroxylamine-O-sulfonic acid in
glacial acetic acid. The 2-azabicyclo[3.2.2]nonanes 4
were prepared in good yields by the reduction of 3 with LiAlH4
in ether. The 5-amino-2-azabicyclo[3.2.2]nonane
derivatives 3 and 4 are representatives of a new class of
compounds (Scheme).
Their
structures were established by NMR spectroscopy.
Compound 3a was investigated thoroughly by 2D-NMR experiments. We
observed a cross peak from N-H to 1-H in H,H-COSY
measurements. The assignments were done with the aid
of through-space interactions (NOE) and w-couplings.
The synthesis of compounds 1
and 2 is briefly described above.
Ketones 1 were
suspended in glacial acetic acid and hydroxylamine-O-sulfonic
acid was added and the mixture refluxed over night at 145°C. The brown
solution was poured on ice, alkalized with 2M NaOH and extracted 5 times with
CH2Cl2. The combined organic layers were
washed 3 times with water, dried over Na2SO4 and filtered.
After evaporation of the solvent in vacuo, the residue was
dissolved in the minimum amount of hot ethanol. The products 3
crystallized over night and were further purified.
(7RS,8RS)-()-5-Dimethylamino-7,8-diphenyl-2-azabicyclo[3.2.2]nonan-3-one (3a). Compound 1a (2.94 g, 9.2 mmol) and hydroxylamine-O-sulfonic acid (3.12 g,
27.6 mmol) in glacial acetic acid (35 ml) gave after workup a precipitate which
was purified by means of CC, using CH2Cl2 : MeOH = 8:2 as
eluent giving pure 3a as a colourless resin. Yield: 1.2 g (39%); m.p.
(ethanol): 212°C; IR (KBr, 
): 1646 (s, C=O), 754 (s, =CH, deform.), 702 (s, =CH
deform.)
cm-1; UV (CH2Cl2, l (loge)): 230 (3.121), 290 (2.012) nm; 1H NMR
(CDCl3, d, 400
MHz): 2.02 (ddd, J = 13.4, 11.3, 2.1 Hz, 1H, 6-H), 2.10 (ddd, J =
13.7, 10.6, 1.5 Hz, 1H, 9-H), 2.21 (ddd, J = 13.8, 8.7, 2.1 Hz, 1H,
9-H), 2.32 (s, 6H, N(CH3)2), 2.38 (ddd, J = 13.3,
8.1, 1.5 Hz, 1H, 6-H), 2.71 (dd, J = 17.8, 2.3 Hz, 1H, 4-H), 2.94 (d, J
= 17.7 Hz, 1H, 4-H), 3.23 (d, J = 7.1 Hz, 1H, 1-H), 3.33 (br, t, J
= 9.6 Hz, 1H, 8-H), 3.47 (dd, J = 11.3, 8.1 Hz, 1H, 7-H), 6.15 (d, J
= 6.6 Hz, 1H, N-H), 7.22 - 7.41 (m, 10H, aromatic H) ppm; 13C NMR (CDCl3, d, 100 MHz): 35.32 (C-9), 35.75 (C-6), 37.86 (N(CH3)2),
40.94 (C-4), 41.59 (C-8), 46.38 (C-7), 55.06 (C-5), 57.67 (C-1), 126.72,
127.05, 127.84, 128.63, 128.97, 141.96, 142.99 (aromatic C), 173.96 (C-3) ppm;
MS (base, EI+): m/z (%) =
334 (49.0) [M+], 275 (100.0), 229 (32.3), 184 (42.2), 153 (24.6),
139 (14.8), 104 (12.9), 91 (23.0), 85 (60.2), 70 (25.0); Anal. Calcd for C22H26N2O
(334.46): C 79.01, H 7.84, N 8.38; found: C 78.74, H 8.09, N 8.32; HRMS
(EI+): calcd. (C22H26N2O):
334.20451; found: 334.20446.
(7RS,8RS)-()-5-(1-Morpholinyl)-7,8-diphenyl-2-azabicyclo[3.2.2]nonan-3-one (3b). Compound 1b (3.6 g,
9.9 mmol) and hydroxylamine-O-sulfonic acid (3.5 g, 31 mmol) in glacial acetic
acid (30 ml) gave after workup a precipitate. Yield: 2.3 g
(62%). For analytical and biological test purposes the
crystallizate was purified by means of CC, using CH2Cl2 :
MeOH = 9:1 as eluent giving 3b as a colourless resin.; m.p. (ethanol): 219°C; IR (KBr, ): 1659 (s, C=O), 756 (m, =CH deform.), 704 (m, =CH
deform) cm-1;
UV (CH2Cl2, l (loge)): 231 (2.955) nm; 1H NMR (CDCl3,
d, 400 MHz): 1.98 (ddd, J = 13.6, 11.4, 1.7 Hz, 1H, 6-H), 2.05 (br,
t, J = 11.9 Hz, 1H, 9-H), 2.19 (ddd, J = 13.4, 9.1, 1.7 Hz, 1H,
9-H), 2.32 (ddd, J = 12.9, 8.3, 1.5 Hz, 1H, 6-H), 2.55 - 2.71 (m, 5H,
4-H, N(CH2)2), 2.93 (d, J = 17.6 Hz, 1H, 4-H),
3.27 (d, J = 6.9 Hz, 1H, 1-H), 3.34 (br, t, J = 9.7 Hz, 1H, 8-H),
3.45 (dd, J = 11.4, 8.1 Hz, 1H, 7-H), 3.68 - 3.71 (m, 4H, O(CH2)2),
6.56 (d, J = 6.9 Hz, 1H, N-H), 7.22 - 7.40 (m, 10H, aromatic H) ppm; 13C NMR (CDCl3, d, 100 MHz): 35.27 (C-9), 35.42 (C-6), 41.65 (C-8),
42.32 (C-4), 45.75 (N(CH2)2), 46.26 (C-7), 55.21 (C-5),
57.56 (C-1), 67.47 (O(CH2)2), 126.67, 127.09, 127.80,
128.66, 128.98, 141.83, 142.92 (aromatic C), 173.64 (C-3) ppm; MS (base, EI+):
m/z (%) = 376 (59.9) [M+],
317 (100.0), 271 (27.2), 226 (23.3), 195 (21.0), 127 (24.1), 91 (27.2); Anal. Calcd for C24H28N2O2
(376.49): C 76.56, H 7.50, N 7.44; found: C 76.26, H 7.75, N 7.33; HRMS
(EI+): calcd. (C24H28N2O2):
376.21508; found: 376.21536.
(7RS,8RS)-()-7,8-Diphenyl-5-(1-pyrrolidyl)-2-azabicyclo[3.2.2]nonan-3-one (3c). Compound 1c (3.2 g, 9.3 mmol)
and hydroxylamine-O-sulfonic acid (3.2 g, 28 mmol) in glacial acetic acid (25
ml) gave after workup a precipitate. Yield: 2.1 g (63%).
For analytical and biological test purposes further purification was done by
means of CC, using CH2Cl2 : MeOH
= 9:1 as eluent giving 3c as a colourless resin. IR (KBr, ): 1656 (s, C=O), 756 (s, =CH deform.), 700 (s, =CH,
deform.)
cm-1; UV (CH2Cl2, l (loge)): 230 (3.220) nm; 1H NMR (CDCl3,
d, 400 MHz): 1.78 (br, s, 4H, 2 CH2), 2.10 (br, dd, J =
12.7, 11.7 Hz, 1H, 9-H), 2.16 (br, dd, J = 12.4, 12.1 Hz, 1H, 6-H), 2.26
- 2.35 (m, 2H, 6-H, 9-H), 2.68 - 2.78 (m, 4H, N(CH2)2),
2.81 (d, J = 18.0 Hz, 1H, 4-H), 2.95 (d, J = 17.7 Hz, 1H, 4-H),
3.24 (d, J = 7.1 Hz, 1H, 1-H), 3.34 (br, t, J = 9.6 Hz, 1H, 8-H),
3.48 (dd, J = 11.4, 8.4 Hz, 1H, 7-H), 6.53 (d, J = 7.1 Hz, 1H,
N-H), 7.19 - 7.40 (m, 10H, aromatic H) ppm; 13C
NMR (CDCl3, d,
100 MHz): 23.67 (2CH2), 35.09 (C-9), 37.04 (C-6), 41.48 (C-8), 42.58
(C-4), 45.19 (N(CH2)2), 46.42 (C-7), 54.16 (C-5), 57.81
(C-1), 126.73, 127.01, 127.83, 128.61, 128.95, 142.00, 143.06 (aromatic C),
174.14 (C-3) ppm; MS (base, EI+): m/z (%) = 360 (31.1) [M+], 301 (100.0), 255 (23.3), 199
(21.0), 179 (16.3), 111 (35.0), 91 (19.5); Anal. Calcd for C24H28N2O
(360.49): C 79.96, H 7.83, N 7.77; found: C 79.72, H 8.10, N 7.66; HRMS
(EI+): calcd. (C24H28N2O):
360.22016; found: 360.31889.
(7RS,8RS)-()-7,8-Diphenyl-5-(1-piperidyl)-2-azabicyclo[3.2.2]nonan-3-one (3d). Compound 1d (3.9 g, 10.7 mmol) and hydroxylamine-O-sulfonic acid (3.7 g,
32.7 mmol) in glacial acetic acid (30 ml) gave after workup a precipitate. Yield: 2.18 g (53%). For analytical and biological test
purposes further purification was done by means of CC, using CH2Cl2
: MeOH = 9:1 as eluent giving pure 3d as a colourless resin. m.p. (ethanol):
204°C; IR (KBr, ): 1656 (s, C=O), 754 (m, =CH deform.), 699 (s, =CH
deform.)
cm-1; UV (CH2Cl2, l (loge)): 230 (3.309) nm; 1H NMR (CDCl3,
d, 400 MHz): 1.42 - 1.50 (m, 2H, CH2), 1.59 (br, s, 4H, 2CH2),
2.00 (br, dd, J = 12.3, 12.1 Hz, 1H, 6-H), 2.10 (br, dd, J =
13.1, 10.9 Hz, 1H, 9-H), 2.21 (br, dd, J = 13.1, 8.6 Hz, 1H, 9-H), 2.40
(br, dd, J = 13.1, 8.0 Hz, 1H, 6-H), 2.50 - 2.68 (m, 4H, N(CH2)2),
2.68 (d, J = 18.0 Hz, 1H, 4-H), 2.94 (d, J = 17.5 Hz, 1H, 4-H),
3.24 (d, J = 6.9 Hz, 1H, 1-H), 3.32 (br, t, J = 9.5 Hz, 1H, 8-H),
3.43 (dd, J = 11.5, 8.0 Hz, 1H, 7-H), 6.46 (d, J = 6.7 Hz, 1H,
N-H), 7.19 - 7.38 (m, 10H, aromatic H) ppm; 13C
NMR (CDCl3, d,
100 MHz): 24.92 (CH2), 26.72 (2CH2), 35.53 (C-6), 36.14
(C-9), 41.74 (C-8), 41.92 (C-4), 46.36 (N(CH2)2), 46.41
(C-7), 55.55 (C-5), 57.71 (C-1), 126.75, 127.01, 127.83, 128.62, 128.93,
142.08, 143.10 (aromatic C), 174.15 (C-3) ppm; MS (base, EI+): m/z (%) = 374 (43.6) [M+],
315 (100.0), 283 (18.7), 269 (27.2), 224 (22.6), 193 (19.5), 125 (21.8), 91
(17.9); Anal. Calcd for C25H30N2O
(374.52): C 80.17, H 8.07, N 7.48; found: C 79.95, H 8.14, N 7.42; HRMS
(EI+): calcd. (C25H30N2O):
374.23581; found: 374.23665.
5-Dialkylamino-7,8-diphenyl-2-azabicyclo[3.2.2]nonan-3-ones 3 were suspended in dry
ether. After cooling on an ice bath, LiAlH4 was
added in portions. The reaction mixture was refluxed
at 55°C over night. After cooling to room temperature, the reaction was cooled with an ice bath and quenched carefully with ice water
and 2M NaOH. The mixture was extracted 5 times with ether,
the combined organic layers were washed 3 times with water, dried over sodium
sulfate, filtered and the solvent evaporated. The remaining brownish oil was purified by distillation giving pure 4a-4d as oils. The
dihydrochlorides were prepared by treatment of a solution of 4 in dichloromethane with etheral HCl (2M) and subsequent evaporation of
the solvents in vacuo. The residues crystallized from ethanol/ethyl acetate.
(7RS,8RS)-()-(7,8-Diphenyl-2-azabicyclo[3.2.2]non-5-yl)-dimethylamine (4a). Compound 3a
(1.3 g, 3.9 mmol) in dry ether (50 ml) gave with LiAlH4
(560 mg, 14.8 mmol) after the above described workup 800 mg (64%) of 4a as a colourless oil. m.p. (HCl): 306°C; IR (HCl, KBr, ): 2785,
2609, 2480 (s, N+HR3), 700 (s, =CH deform.) cm-1; UV (base, CH2Cl2, l (loge)): 208 (4.261) nm; 1H
NMR (base, CDCl3, d, 400 MHz): 1.88 - 1.94
(m, 3H, 4-H, 6-H), 2.12 (dd, J = 13.2, 10.8 Hz, 1H, 9-H), 2.25 (ddd, J
= 13.4, 8.9, 2.3 Hz, 1H, 9-H), 2.31 (s, 6H, N(CH3)2),
2.33 (ddd, J = 12.8, 9.1, 2.1 Hz, 1H, 6-H), 3.10 - 3.15 (m, 3H, 1-H,
3-H), 3.29 (ddd, J = 11.0, 8.5, 2.5 Hz, 1H, 8-H), 3.44 (t, J =
9.4 Hz, 1H, 7-H), 7.18 - 7.40 (m, 10H, aromatic H) ppm; 13C NMR (base, CDCl3, d, 100 MHz): 31.70 (C-4), 35.66 (C-9), 35.59
(C-6), 37.95 (N(CH3)2), 39.62 (C-8), 41.86 (C-3), 47.11 (C-7),
57.99 (C-5), 61.54 (C-1), 126.10, 126.24, 127.15, 127.78, 128.44, 128.55,
144.20, 145.51 (aromatic C) ppm; MS (base, EI+): m/z (%) = 320 (93.8) [M+],
275 (37.5), 188 (24.2), 176 (100.0), 145 (48.4), 104 (19.9), 91 (28.1), 85
(92.6), 70 (25.0), 44 (25.8); Anal. Calcd for
C22H30N2Cl2 (393.39): C 67.17, H
7.69, N 7.12, Cl 18.02; found: C 66.91, H 7.91, N 6.94, Cl 18.01; HRMS
(EI+): calcd. (C22H28N2):
320.22525; found: 320.22451.
(7RS,8RS)-()-1-(7,8-Diphenyl-2-azabicyclo[3.2.2]non-5-yl)-morpholine (4b). Compound 3b
(1.42 g, 3.8 mmol) in dry
ether (48 ml) gave with LiAlH4 (540
mg, 14.2 mmol) after the above described workup 818 mg (60%) of 4b. m.p. (HCl): 310°C (decomp.); IR (HCl, KBr, ): 2870,
2663, 2424 (s, N+HR3), 703 (s, =CH deform) cm-1; UV (HCl, CH3OH, l (loge)): 209 (4.213) nm; 1H
NMR (base, CDCl3, d, 400 MHz): 1.87 (dd, J
= 13.0, 10.1 Hz, 2H, 6-H, NH), 1.93 - 1.96 (m, 2H, 4-H), 2.11 (dd, J =
13.3, 10.8 Hz, 1H, 9-H), 2.22 (ddd, J = 13.3, 11.2, 2.3 Hz, 1H, 9-H),
2.29 (ddd, J = 12.8, 9.0, 2.3 Hz, 1H, 6-H), 2.59 - 2.71 (m, 4H, N(CH2)2),
3.09 - 3.17 (m, 3H, 1-H, 3-H), 3.29 (ddd, J = 11.1, 8.5, 2.6 Hz, 1H,
8-H), 3.42 (br, t, J = 9.5 Hz, 1H, 7-H), 3.69 - 3.74 (m, 4H, O(CH2)2),
7.18 - 7.39 (m, 10H, aromatic H) ppm; 13C NMR (base, CDCl3, d, 100 MHz): 32.98 (C-4), 35.62 (C-9), 36.24 (C-6),
39.69 (C-8), 41.83 (C-3), 45.66 (N(CH2)2), 47.09 (C-7),
58.18 (C-5), 61.35 (C-1), 67.63 (O(CH2)2), 126.13,
126.26, 127.06, 127.72, 128.45, 128.55, 144.13, 145.45 (aromatic C) ppm; MS
(base, EI+): m/z (%) = 362
(100.0) [M+], 275 (31.1), 230 (22.6), 218 (91.8), 171 (27.2), 145
(68.5), 127 (35.8), 104 (24.9), 91 (24.1); Anal.
Calcd for C24H30N2O (362.51): C 79.52, H 8.34,
N 7.73; found: C 79.25, H 8.61, N 7.69; HRMS (EI+): calcd. (C24H30N2O): 362.23581;
found: 362.23411.
(7RS,8RS)-()-1-(7,8-Diphenyl-2-azabicyclo[3.2.2]non-5-yl)-pyrrolidine (4c). Compound
3c (1.16 g, 3.2 mmol) in dry ether (47 ml) gave with LiAlH4
(521 mg, 13.7 mmol) after the above described workup 850 mg (76%) of 4c. m.p. (HCl): 306°C; IR
(HCl, KBr, ): 2876,
2677, 2435 (s, N+HR3), 700 (s, =CH deform.) cm-1; UV (HCl, CH3OH, l (loge)): 207 (4.182) nm; 1H
NMR (base, CDCl3, d, 400 MHz): 1.75 - 1.79
(m, 4H, 2CH2), 1.91 - 2.06 (m, 4H, 4-H, 6-H, NH), 2.18 - 2.35 (m,
3H, 6-H, 9-H), 2.69 - 2.77 (m, 4H, N(CH2)2), 3.10 - 3.20
(m, 3H, 1-H, 3-H), 3.32 (ddd, J = 11.1, 8.5, 2.6 Hz, 1H, 8-H), 3.46 (br,
t, J = 9.5 Hz, 1H, 7-H), 7.17 - 7.41 (m, 10H, aromatic H) ppm; 13C NMR (base, CDCl3, d, 100 MHz): 23.61 (2CH2), 33.55
(C-4), 36.08 (C-9), 37.43 (C-6), 39.53 (C-8), 41.94 (C-3), 45.17 (N(CH2)2),
47.13 (C-7), 57.05 (C-5), 61.76 (C-1), 126.06, 126.21, 127.21, 127.80, 128.44,
128.55, 144.29, 145.70 (aromatic C) ppm; MS (base, EI+): m/z (%) = 346 (100.0) [M+],
301 (49.8), 255 (35.0), 241 (24.9), 214 (26.5), 202 (59.1), 145 (23.3), 130
(20.2), 111 (78.6), 91 (27.2); Anal. Calcd for
C24H30N2 (346.51): C 83.19, H 8.73, N 8.08;
found: C 82.93, H 8.52, N 8.04; HRMS (EI+): calcd. (C24H30N2): 346.24090;
found: 346.24023.
(7RS,8RS)-()-1-7,8-Diphenyl-2-azabicyclo[3.2.2]non-5-yl)-piperidine (4d). Compound 3d (1.3 g, 3.5 mmol) in dry ether (45
ml) gave with LiAlH4 (500 mg, 13.2 mmol) after the above described
workup 710 mg (57%) of 4d. m.p. (HCl): 260°C; IR (HCl, KBr, ): 2868,
2661, 2533 (s, N+HR3), 702 (s, =CH deform.) cm-1; UV (HCl, CH3OH, l (loge)): 209 (4.137) nm; 1H
NMR (base, CDCl3, d, 400 MHz): 1.42 - 1.48
(m, 2H, CH2), 1.55 - 1.64 (m, 4H, 2CH2), 1.88 (dd, J
= 12.6, 10.7 Hz, 2H, 6-H, NH), 1.94 - 1.98 (m, 2H, 4-H), 2.13 (dd, J =
13.1, 10.7 Hz, 1H, 9-H), 2.25 (ddd, J = 13.1, 9.2, 2.2 Hz, 1H, 9-H),
2.33 (ddd, J = 12.9, 8.7, 2.2 Hz, 1H, 6-H), 2.52 - 2.68 (m, 4H, N(CH2)2),
3.07 - 3.20 (m, 3H, 1-H, 3-H), 3.29 (ddd, J = 11.0, 8.7, 2.4 Hz, 1H,
8-H), 3.40 (br, t, J = 9.5 Hz, 1H, 7-H), 7.18 - 7.40 (m, 10H, aromatic
H) ppm; 13C NMR (base, CDCl3, d, 100 MHz): 25.06 (CH2), 26.79
(2CH2), 32.94 (C-4), 35.53 (C-9), 36.59 (C-6), 40.09 (C-8), 42.16
(C-3), 46.26 (N(CH2)2), 47.62 (C-7), 58.59 (C-5), 61.42
(C-1), 126.07, 126.18, 127.08, 127.73, 128.44, 128.51, 144.36, 145.68 (aromatic
C) ppm; MS (base, EI+): m/z
(%) = 360 (100.0) [M+], 315 (34.2), 269 (31.1), 255 (16.3), 228
(21.8), 216 (44.4), 171 (17.1), 145 (19.5), 125 (48.2), 104 (20.2), 91 (18.7);
Anal. Calcd for C25H32N2×0.125H2O
(362.80): C 82.77, H 8.96, N 7.72; found: C 82.85, H 9.11, N 7.54; HRMS
(EI+): calcd. (C25H32N2):
360.25655; found: 360.25496.
Antiplasmodial activity was examined using the K1 strain of P. falciparum (resistant to chloroquine
and pyrimethamine). Viability was determined by the incorporation of [3H]-hypoxanthine
into living protozoal cells by a modification of a reported assay (25).
Briefly, infected human red blood cells in RPMI 1640 medium with 5% Albumax
were exposed to serial drug dilutions ranging from 5
to 0.078 mg/ml in microtiter plates. After 48 hours of
incubation at 37°C in a reduced oxygen atmosphere, 0.5 mCi 3H-hypoxanthine
were added to each well. Cultures were
incubated for a further 24 h before they were harvested onto glass-fiber
filters and washed with distilled water. The radioactivity was
counted using a BetaplateTM liquid scintillation counter (Wallac,
Minimum Essential Medium (50
µl) supplemented according to a known procedure (26) with 2-mercaptoethanol and
15% heat-inactivated horse serum was added to each well of a 96-well microtiter
plate. Serial drug dilutions were prepared covering a range from 90 to 0.123 µg/ml. Then 104 bloodstream forms of Trypanosoma b. rhodesiense STIB 900 in
50 µl were added to each well and the plate incubated
at 37°C under a 5% CO2 atmosphere for 72 hours. Alamar Blue (10 µl containing 12.5 mg resazurin dissolved in 1000 ml
distilled water) was then added to each well and incubation continued for a
further 2-4 hours. The Alamar blue dye is an indicator of cellular growth and /
or viability. The blue, non fluorescent, oxidized form
becomes pink and fluorescent upon reduction by living cells. The plate was then
read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices
Cooperation,
Male mice (Fü albino; specific
pathogen free) weighing 20± 2g were infected intravenously with 2x107 P.berghei ANKA strain-infected
erythrocytes from donor mice on day 0 of the experiment. Heparinized blood was taken from donor mice with approximately 30% parasitemia
and was diluted in physiological saline to 108 parasitized
erythrocytes/ml. An aliquot (0.2 ml) of this suspension was
injected intravenously into experimental groups of three mice and a
control group of five mice.
In untreated control mice,
parasitemias rise regularly to 30% to 40% by day +3 after infection
and to 70 to 80% by day +4. The mice die between days +5
and +7 after infection.
On day +3 (48 hours
after treatment) blood smears of all animals were prepared and stained with
Giemsa. Parasitemia was determined microscopically by counting 1000 red blood
cells. For low parasitemias (<1%) 2000 rbc’s have
to be counted. The difference between the mean value for the control group
(taken as 100%) and that for each experimental group was calculated and
expressed as percent reduction (=activity).
The mean survival
day (MSD, in days) was recorded as well as
observations concerning any side effects of the drug.
Compound 4a was prepared at appropriate concentrations, as solutions
containing 3% ethanol and 7% Tween 80. They were administered intraperitoneal
(i.p.) and intravenous (i.v.) in a total volume of 0.01 ml per g of body weight
on day +1 (24 hours after infection) of the experiment.
In vivo studies were carried out by a protocol approved by an animal ethics committee.
The new compounds 3a-3d
and 4a-4d were screened using the assays
described above. Antiprotozoal activities and cytotoxicity of
these new compounds and as well as those of compounds 1 and 2 are
presented in
table 1.
Compound 4a with the highest antiplasmodial
activity was screened in an in vivo assay against Plasmodium berghei. The results are presented in
table 2.
Table 1. In vitro activities of 1-4, expressed as IC50 (mM)a
|
Comp. |
Trypanosoma brucei rhodesiense |
Plasmodium falciparum K1 |
Cytotoxi-city L6 |
|
1a |
9.99 |
>10.57 |
24.57 |
|
1b |
116.3 |
>11.89 |
n.t. |
|
1c |
8.03 |
1.19 |
26.45 |
|
1d |
8.12 |
3.95 |
46.82 |
|
2a |
1.64 |
2.50 |
23.40 |
|
2b |
14.85 |
5.77 |
48.18 |
|
2c |
1.47 |
3.64 |
16.03 |
|
2d |
1.49 |
1.55 |
11.52 |
|
3a |
37.97 |
1.40 |
>269.1 |
|
3b |
138.4 |
>13.28 |
>239.1 |
|
3c |
37.94 |
8.76 |
>249.7 |
|
3d |
36.60 |
13.00 |
233.6 |
|
4a |
0.60 |
0.28 |
108.8 |
|
4b |
9.44 |
6.84 |
>206.7 |
|
4c |
1.16 |
0.56 |
120.4 |
|
4d |
6.57 |
0.64 |
89.74 |
|
mel |
0.0039 |
|
7.78 |
|
sur |
0.0075 |
|
4724.5 |
|
art |
|
0.0064 |
450.5 |
|
chl |
|
0.12b |
188.5 |
|
mef |
|
|
11.37 |
aValues
represent the average of four determinations
(two
determinations of two independent experiments)
bagainst
sensitive P. falciparum strains
n.t.: not tested. art = artemisinin, chl =
chloroquine
mel = melarsoprol, sur =
suramine, mef = mefloquine
Table 2.
In vivo activity of
4a against P. berghei
|
Comp. |
dose in mg/kg |
application |
MSD (days) |
Activity in % |
|
4a |
4×50 |
i.p. |
6.3 |
35.1 |
|
4a |
4×10 |
i.v. |
6.3 |
18.4 |
|
ctrl |
----- |
|
5.9 |
--- |
|
chl |
4×10 |
i.p. |
20 |
99.6 |
chl = chloroquine, ctrl = control
The ring enlargement from
bicyclo-octanones 1 to bicyclo-nonanones 3 leads to a remarkable
decrease of cytotoxicity against L6 cells. In general the antiprotozoal
activity is lost, however, the 5-dimethylamino-2-azabicyclo[3.2.2]nonan-3-one
3a exhibits distinctly higher antiplasmodial activity (IC50 =
1.40 mM) than its bicyclo-octan-2-one analogue 1a (IC50
> 10.57 mM). The positive contribution of the dimethylamino
group to the antiplasmodial activity has also been observed
for compounds 4. Due to the good antiplasmodial activity and the very
low cytotoxicity (IC50 > 269.1 mM) of compound 3a
it is the most promising antiplasmodial agent of the so far prepared
aminobicyclo-alkanones. The low cytotoxicity of compounds 3 results
obviously from the replacement of the keto by an amido group. Maybe, the change
from the bicyclo-octane to the far more flexible bicyclo-nonane skeleton is the
reason for an additional sterical contribution. The bicyclo[2.2.2]octan-4-amines
2 which were prepared from 1 using a Wolff-Kishner procedure, possess
higher antitrypanosomal activity than the ketones 1. Their
antiplasmodial properties and their cytotoxicity are comparable to those of
compounds 1.
In contrast, compounds 4a,c show distinctly higher antiplasmodial (IC50 = 0.28 - 0.56 mM) and antitrypanosomal activities (IC50 = 0.60 - 1.16 mM). (7,8-Diphenyl-2-azabicyclo[3.2.2]non-5-yl)-dimethylamine 4a is the most active compound of both series against Plasmodium falciparum K1 and Trypanosoma b. rhodesiense. The antitrypanosomal acitivity of 4a (IC50 = 0.60 mM) is distinctly higher than that of all other compounds of the bicyclo-octane and the bicyclo-nonane series, but it is far less active than melarsoprol (IC50 = 0.0039 mM) and suramine (IC50 = 0.0075 mM). With reference to the commonly used antimalarial chloroquine (IC50 = 0.12 mM against sensitive strains) 4a possesses a comparable antiplasmodial activity (IC50 = 0.28 mM) and at the same time low cytotoxicity (IC50 = 108.8 mM). However, the in vivo activity of 4a against Plasmodium berghei is moderate (i.p.: 35.1% inhibition; i.v.: 18.4% inhibition) in the case of both applications. This could be caused by fast degradation in the mouse liver or insufficient bioavailability due to their relatively high lipophilicity or binding to other sites. Therefore, further structural modifications are in progress.
2-Azabicyclo[3.2.2]nonanes exhibit promising in vitro antiprotozoal activity and low cytotoxicity. Therefore, they will serve as lead compounds for further modifications. More hydrophilic compounds will be prepared by alkylation of the nitrogen in ring position 2 with substituents bearing polar groups. In order to investigate the influence of its basicity, acylation to amides is considered. In both cases, residues with differing length, polarity and acidity will be inserted.
This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (Austrian Science Fund, grant no. P-15928).
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Published by the Canadian Society for Pharmaceutical Sciences.
Copyright © 1998 by the Canadian Society for Pharmaceutical Sciences.