2750 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Raynes et al.
corresponding to major fragment losses. Melting points were
performed using a Gallenkamp melting point apparatus and
are reported uncorrected.
7-Ch lor o-4-[5′-p r op yl-4′-h yd r oxyl-3′-[(ter t-bu tyla m in o)-
m eth yl]p h en yl]a m in oqu in olin e (9c): off-white solid (54%);
mp 168-169 °C (from aqueous EtOH); H NMR δ 0.9 (t, J )
1
7 Hz, 3H, -CH2CH3 ) 1.1 (s, 9H, tert-butyl), 1.6 (m, 2H, -CH2-
CH2-CH3), 2.6 (q, J ) 7 Hz, - CH2-CH2-CH3), 3.7 (s, 2H,
Ar-CH2-NH-), 6.5 (br s, 1H, -NH-), 6.6 (d, J ) 5 Hz, H-3),
6.8 (s, 1H, Ar), 6.9 (s, 1H, Ar), 7.4 (dd, J ) 9, 2 Hz, 1H, H-6),
7.8 (dd, J ) 9, 2 Hz, 1H, H-5), 7.9 (d, J ) 2 Hz, 1H, H-8), 8.4
(d, J ) 5 Hz, 1H, H-2); MS m/z 398.5 (M + 1). Anal. (C23H28N3-
OCl) C, H, N.
o-Cycloh exylp h en ol (6). The condensation of phenol with
cyclohexyl chloride was performed using a modified procedure
of Abdurasuleva et al.20 A mixture of phenol (0.5 mol),
cyclohexyl chloride (0.1 mol), and ferric chloride (5.5 mmol)
was heated (170 °C) with continuous stirring for 2 h. After
the mixture cooled to room temperature, sodium hydroxide was
added (20%, 20 mL) and the mixture stirred. To facilitate the
precipitation of the sodium salt of the product, diethyl ether
was added (50 mL). The salt was collected by filtration and
dissolved in diluted hydrochloric acid (1 M, 50 mL), and the
solution was extracted with dichloromethane to give a mixture
of p- and o-cyclohexylphenols (7.5 g, 42%). The two isomers
were separated using a silica column and dichloromethane as
the mobile phase (o-cyclohexylphenol, Rf ) 0.48; p-cyclohexyl-
phenol, Rf ) 0.80).
7-Ch lor o-4-[5′-isop r op yl-4′-h yd r oxyl-3′-[(ter t-bu tyla m i-
n o)m eth yl]ph en yl]am in oqu in olin e (9d): yellow solid (52%);
1
mp 188.5-190.0 °C (from aqueous EtOH); H NMR δ 1.1 (m,
15H, tert-butyl, 2 -CH3), 1.6 (m, 2H, -CH2-CH3), 3.7 (s, 2H,
Ar-CH2-NH-), 6.5 (br s, 1H, -NH-), 6.6 (d, J ) 5 Hz, H-3),
6.7 (s, 1H, Ar), 7.0 (s, 1H, Ar), 7.4 (dd, J ) 9, 2 Hz, 1H, H-6),
7.8 (dd, J ) 9, 2 Hz, 1H, H-5), 7.9 (d, J ) 2 Hz, 1H, H-8), 8.4
(d, J ) 5 Hz, 1H, H-2); MS m/z 398.5 (M + 1). Anal. (C23H28N3-
OCl) C, H, N.
3-Alk yl-4-h yd r oxyla ceta n ilid es 7a -7g. All the acetanil-
ide derivatives were prepared from the method of Kesten et
al.16 Yields and melting point data were all comparable to the
literature values.
7-Ch lor o-4-[5′-(sec-bu tyl)-4′-h ydr oxyl-3′-[(ter t-bu tylam i-
n o)m eth yl]ph en yl]am in oqu in olin e (9e): fawny solid (48%);
mp 217 °C (from aqueous EtOH); 1H NMR δ 0.9 (t, J ) 7 Hz,
3H, -CH2CH3), 1.1 (m, 12H, tert-butyl, -CH3), 1.6 (m, 2H,
-CH2-CH3), 3.0 (m, -CH(CH3)-CH2-CH3), 3.7 (s, 2H, Ar-
CH2-NH-), 6.5 (br s, 1H, -NH-), 6.6 (d, J ) 5 Hz, H-3), 6.7
(s, 1H, Ar), 7.0 (s, 1H, Ar), 7.4 (dd, J ) 9, 2 Hz, 1H, H-6), 7.8
(dd, J ) 9, 2 Hz, 1H, H-5), 7.9 (d, J ) 2 Hz, 1H, H-8), 8.4 (d,
J ) 5 Hz, 1H, H-2); MS m/z 412.5 (M + 1). Anal. (C24H30N3-
OCl) C, H, N.
5′-Meth yl-4′-h yd r oxyl-3′-[(ter t-bu tyla m in o)m eth yl]a c-
eta n ilid e (8a ). 3-Methyl-4-hydroxylacetanilide (7a ) (5 mmol)
was subjected to a Mannich reaction with N-tert-butylamine
(10 mmol) and aqueous formaldehyde (10 mmol) in ethanol.
After refluxing for 48 h the solvent was removed under reduced
pressure and the residue dissolved in dichloromethane (10
mL). The organic solution was extracted with dilute hydro-
chloric acid (0.1 M, 2 × 20 mL). This solution was basified
(pH 9-10) and extracted with dichloromethane (3 × 20 mL).
The combined extracts were washed with water (1 × 20 mL)
and dried (MgSO4), and the was solvent evaporated under
reduced pressure to give the product as a crude oil. Recrys-
tallization was achieved using toluene/light petroleum ether
(40-60 °C) (20/80 v/v) to afford a light-brown solid, sufficiently
pure for the next reaction (65%, mp 245-247 °C): 1H NMR δ
1.1 (s, 12H, tert-butyl, -CH3), 1.9 (s, 3H, NH-C(O)-CH3), 2.2
(3H, -CH3), 3.7 (s, 2H, Ar-CH2-NH-C(O)-CH3), 6.8 (s, 1H,
Ar), 7.0 (s, 1H, Ar), 8.1 (s, 1H, NH-C(O)-).
7-Ch lor o-4-[5′-(ter t-bu tyl)-4′-h ydr oxyl-3′-[(ter t-bu tylam i-
n o)m eth yl]p h en yl]a m in oqu in olin e (9f): fawny solid (38%);
1
mp 225-226 °C (from aqueous EtOH); H NMR δ 1.1 (s, 9H,
tert-butyl), 1.3 (s, 9H, tert-butyl), 3.7 (s, 3H, Ar-CH2-NH-),
6.5 (br s, 1H, -NH-), 6.6 (d, J ) 5 Hz, H-3), 6.7 (s, 1H, Ar),
7.0 (s, 1H, Ar), 7.4 (dd, J ) 9, 2 Hz, 1H, H-6), 7.8 (dd, J ) 9,
2 Hz, 1H, H-5), 7.9 (d, J ) 2 Hz, 1H, H-8), 8.4 (d, J ) 5 Hz,
1H, H-2); MS m/z 412.5 (M + 1). Anal. (C24H30N3OCl) C, H,
N.
7-Ch lor o-4-[5′-(cycloh exyl)-4′-h yd r oxyl-3′-[(ter t-bu tyl-
a m in o)m eth yl]p h en yl]a m in oqu in olin e (9g): fawny solid
The synthesis of 8b-8g was completed by a similar proce-
dure as that of 8a , where 7a was replaced by the appropriate
acetanilide 7b - 7g.
1
(39%); mp 189-190 °C (from aqueous EtOH); H NMR δ 1.1
(s, 9H, tert-butyl), 1.3-1.8 (m, 10H, cyclohexyl), 3.0 (m, 1H,
-CH of cyclohexyl), 3.7 (s, 3H, Ar-CH2-NH-), 6.5 (br s, 1H,
-NH-), 6.6 (d, J ) 5 Hz, H-3), 6.7 (s, 1H, Ar), 7.0 (s, 1H, Ar),
7.4 (dd, J ) 9, 2 Hz, 1H, H-6), 7.8 (dd, J ) 9, 2 Hz, 1H, H-5),
7.9 (d, J ) 2 Hz, 1H, H-8), 8.4 (d, J ) 5 Hz, 1H, H-2); MS m/z
438.5 (M + 1). Anal. (C26H32N3OCl) C, H, N.
7-Ch lor o-4-[5′-m eth yl-4′-h ydr oxyl-3′-[(ter t-bu tylam in o)-
m eth yl]p h en yl]a m in oqu in olin e (9a ). A solution of 8a (5.0
g, 23 mmol) in hydrochloric acid (25 mL of 6 N HCl) was heated
under reflux for 1 h. This solution was concentrated by reduced
pressure and then coevaporated with ethanol. The residue was
dissolved in ethanol (30 mL), 4,7-dichloroquinoline (4.5 g, 23
mmol) was added, and the solution heated under reflux for 2
h. The solution was concentrated by reduced pressure to give
a viscous residue which was poured into ice-cold ammonium
hydroxide (5%, 200 mL). The sticky solid which separated was
dissolved in dichloromethane (100 mL) and separated from the
basic solution. The organic solution was washed with water
(100 mL), dried (MgSO4), and evaporated to dryness under
reduced pressure to give the crude product. This solid was
recrystallized twice from aqueous ethanol to give an analytical
product (48%, mp 209-210 °C): 1H NMR δ 1.1 (s, 12H, tert-
butyl, -CH3), 2.2 (3H, -CH3), 3.9 (s, 2H, Ar-CH2-NH-C(O)-
CH3), 6.5 (br s, 1H, -NH-), 6.6 (d, J ) 5 Hz, H-3), 6.7 (s, 1H,
Ar), 7.0 (s, 1H, Ar), 7.4 (dd, J ) 9, 2 Hz, 1H, H-6), 7.8 (dd, J
) 9, 2 Hz, 1H, H-5), 7.9 (d, J ) 2 Hz, 1H, H-8), 8.4 (d, J ) 5
Hz, 1H, H-2); MS m/z 369.5 (M + 1). Anal. (C21H24N3OCl) C,
H, N.
7-Ch lor o-4-[5′-eth yl-4′-h yd r oxyl-3′-[(ter t-bu tyla m in o)-
m eth yl]p h en yl]a m in oqu in olin e (9b): yellow solid (51%);
mp 195-196 °C (from aqueous EtOH); 1H NMR δ 1.1 (s, 12H,
tert-butyl, -CH2CH3), 2.6 (q, J ) 7 Hz, -CH2CH3), 3.7 (s, 2H,
Ar-CH2-NH-), 6.5 (br s, 1H, -NH-), 6.6 (d, J ) 5 Hz, H-3),
6.8 (s, 1H, Ar), 6.9 (s, 1H, Ar), 7.4 (dd, J ) 9, 2 Hz, 1H, H-6),
7.8 (dd, J ) 9, 2 Hz, 1H, H-5), 7.9 (d, J ) 2 Hz, 1H, H-8), 8.4
(d, J ) 5 Hz, 1H, H-2); MS m/z 383.5 (M + 1). Anal. (C22H26N3-
OCl) C, H, N.
An tim a la r ia l Activity. Two strains of P. falciparum from
Thailand were used in this study: (a) the uncloned K1 strain
which is known to be CQ-resistant and (b) the HB3 strain
which is sensitive to all antimalarials. Parasites were main-
tained in continuous culture using the method of Trager and
J enson.31 Cultures were grown in culture flasks containing
human erythrocytes (2-5%) with parasitemia in the range of
1-10% suspended in RPMI 1640 medium, supplemented with
25 mM HEPES and 32 mM NaHCO3, and 10% human serum
(complete medium). Cultures were gassed with a mixture of
3% O2, 4% CO2, and 93% N2.
(a ) In Vitr o Testin g. Antimalarial activity was assessed
using an adaption of the 48-h sensitivity assay of Desjardins
et al.32 using [3H]hypoxanthine incorporation as an assessment
of parasite growth. Stock drug solutions were prepared in
100% dimethyl sulfoxide (DMSO) and diluted to the appropri-
ate concentration using complete medium. Assays were per-
formed in sterile 96-well microtiter plates; each plate contained
200 µL of parasite culture (2% parasitemia, 0.5% hematocrit)
with or without 10-µL drug dilutions. Each drug was tested
in triplicate and parasite growth compared to control wells
(which consituted 100% parasite growth). After 24 h incubation
at 37 °C, 0.5 µCi of hypoxanthine was added to each well.
Cultures were incubated for a further 24 h before they were
harvested onto filtermats, dried for 1 h at 55 °C, and counted
using a Wallac 1450 Microbeta Trilux liquid scintillation and