P. B. Madrid et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3014–3017
3015
and then to counter-screen them for binding to a panel
of dopaminergic and serotonergic receptors. Since no
structural relationship between Mtb NDH-2 and the
postsynaptic neurological receptors is evident in hu-
mans, our hypothesis is that it should be possible to cre-
ate phenothiazines with enhanced selectivity toward
Mtb growth inhibition and reduced binding to the dopa-
minergic and serotonergic receptors.
linked together to form a dimer or ‘bis’ compound. This
phenomenon has been observed for quinoline antimala-
rials13 and acridine antiprion14 compounds. To explore
this possibility for the phenothiazines, we synthesized
two bis-phenothiazines using an alkyl and an aromatic
linking group (Scheme 3).
The microplate alamar blue assay (MABA) was used
to assess the antitubercular activity of the phenothia-
zines, measured as the Minimum Inhibitory Concen-
tration (MIC) against Mtb strain H37Rv (Table 1).
The pharmacology of the existing phenothiazine drugs
makes it clear that substitutions around the phenothia-
zine ring can lead to pronounced effects on their biolog-
ical activities.7 First, a diphenylamine intermediate was
synthesized by coupling a substituted aniline with
bromobenzene using a Buchwald palladium coupling10
(Scheme 1). The diphenylamines were then cyclized
through reaction with sulfur and catalytic iodide11 under
microwave irradiation, using previously described con-
ditions,12 to give substituted 10H-phenothiazines in
10%–50% yields, with the remaining material recovered
as unreacted starting material. These phenothiazines
were then alkylated with a common basic side chain to
permit direct comparison of effects of the substituted
ring substituents. This route allowed us to synthesize
10 substituted phenothiazine derivatives along with
three compounds, 5a, 5b, and 5c, made from commer-
cially available rings (Table 1).
15
The unsubstituted phenothiazine 5a was significantly
less potent than the analog with the 2-CF3 substitution
found in TPZ. The two compounds with phenyl sub-
stitutions, 5d and 5e, were the most potent, with MICs
of 4.5 and 2.1 lg/mL, respectively. The increased activ-
ity of the phenyl substituted phenothiazine rings sug-
gests that there may be space for additional steric
interaction in the receptor binding pocket in this re-
gion. It is also notable that replacement of the pheno-
thiazine ring with a phenoxazine ring (compound 5b)
and introduction of a heteroatom into the ring system
(compounds 5i and 5j) led to significant losses of
activity.
The compounds with substitutions to the side chain gen-
erally led to a loss in activity relative to TFP, with the
exception of compounds 9f and 9h, with MICs of
4.6 lg/mL and 4.2 lg/mL, respectively. These com-
pounds are the only two with side chains that are both
secondary amines and are expected to be protonated
at the pH of the assay, 7.4. In general, the compounds
with the benzyl substitution (12a–k) and bulkier side
chains (12c, 12d, 12j–k) were less active. Interestingly,
the two bis-phenothiazines, 13 and 14, were both potent
compounds, with MICs of 2.3 lg/mL and 2.0 lg/mL,
respectively.
The existing phenothiazine drugs all have a basic side
chain at the 10-position, so we maintained the distal
nitrogen and added diverse substitutions with both an
alkyl and a benzyl linker (Scheme 2). The unsubstituted
10H-phenothiazine rings were first alkylated with a bro-
mo- or iodo-alkane, then converted to alkyl iodides that
were poised for nucleophilic attack by a primary or a
secondary amine diversity reagent. Amines were chosen
to encompass aliphatic, cyclic aliphatic, aromatic, and
heteroaromatic groups at this position. Also, this set
of amine starting materials gave products consisting of
both secondary and tertiary amines. Overall, yields ran-
ged from 20% to 80%, and thus were acceptable for our
initial screening efforts.
All these compounds were then counter-screened for
binding to three dopamine and three serotonin receptors
at 10 lM using a radioligand displacement assay (Table
1).16 This assay allows us to assess the selectivity toward
Mtb growth inhibition versus binding to the postsynap-
tic receptors. The control compounds, CPZ and TFP,
both demonstrated near maximum binding to all the
receptors except the 5-HT1A receptor at 10 lM concen-
tration. The ring-substitution analogs (5a–m) retained
high binding to most of the receptors with some loss
in affinities to the D1, 5-HT1A, and 5-HT2C receptors.
The most potent of this series, 5d and 5e, had little ten-
dency to bind to the serotonin receptors, but had only a
moderate reduction in affinity for the dopamine recep-
tors. Notably, the two compounds with the greatest
overall reduction in binding to these receptors were
the phenoxazine compound 5b and the 3-morpholino
substituted compound 5m, which were also inactive
against Mtb. The compounds with an alkyl linker and
diversity at the pendant nitrogen (9a–k) retained a bind-
ing profile similar to that of the control drugs, with the
exception of 9k, containing a large diphenyl ether substi-
tution on the nitrogen. These data seem to indicate that
large groups in this position could reduce much of the
binding to the dopamine and serotonin receptors. The
There are precedents for some classes of compounds to
have greater activity when the minimal active units are
Scheme 1. Reagents and conditions: (a) Pd2dba3, X-phos, K3PO4,
toluene, reflux, 20 h. (b) Sulfur (2 equiv), I2 (cat), H2O, lW, 190 °C,
20 min. (c) 3-Chloromethyl-1-methylpiperazine, LHMSD (1 M in
hexanes), toluene, 80 °C, 15 min.