Phenothiazine drugs are well-known antipsychotic drugs. Members of this drug class
include thioridazine (1), fluphenazine (2) and chlorpromazine (3), the first antipsychotic drug on the market, and methylene blue (4) (Figure [1]). Thioridazine (1) was introduced as a treatment for schizophrenia and psychosis, and soon became widely
used.[1] Compared to chlorpromazine, thioridazine results in less pronounced extrapyramidal
side effects.[2]
[3]
Figure 1 Structures of four well-known phenothiazines
A common side effect of antipsychotic drugs is a prolonged QT interval, i.e., the
time from when the cardiac ventricles start to contract until they are finished relaxing,
and this was soon reported to be the case for thioridazine.[4] Therapeutic doses can cause cardiac repolarization abnormalities and prolongation
of QT in a dose-dependent manner.[5]
[6] Cases of the potentially fatal ventricular arrhythmia, Torsades de Pointes, have
been reported. To date, 41 sudden deaths in patients have been associated with treatment
with thioridazine.[5]
,
[7–12] Around the turn of the millennium, the risks for these severe side effects were
found to be higher than for other antipsychotic drugs.[13] Accordingly, branded versions of thioridazine were withdrawn in 2005.[14]
In addition to antipsychotic properties, several members of the phenothiazine class
have significant antimicrobial activity against a wide range of bacteria.[14] However, thioridazine has not been used clinically as an antibacterial agent. Nevertheless,
thioridazine has attracted renewed interest in recent years due to its activity against
multi-drug-resistant and extensively drug-resistant tuberculosis.[15] Particularly interesting are reports of increased sensitivity of drug-resistant
Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA) to thioridazine.[7]
[15]
[16] The suggested mechanism of action is inhibition of bacterial efflux pumps.[17]
Moreover, it has been demonstrated that thioridazine is concentrated in macrophages.
Infections in macrophage-rich tissue (e.g., lung, kidney, spleen and liver) include
tuberculosis. Consequently, lower doses are expected to be needed for the treatment
of such infections, compared to treatment of psychosis.[18]
[19]
[20]
[21]
Furthermore, thioridazine has the ability to suppress the growth of several types
of cancer cells.[22] Recently, studies revealed thioridazine to target cancer stem cells selectively.[23]
[24]
[25] It has also been demonstrated that thioridazine makes multidrug-resistant cancer
cells vulnerable to cytotoxic agents to which they were originally resistant.[26]
The majority of the reports on thioridazine are concerned with the biology of the
commercially available thioridazine, which is the hydrochloride of the racemate. During
the last 30 years, several groups have investigated the properties of the separate
enantiomers, rather than the racemate. Experiments in rat brains revealed that the
(+)-enantiomer has approximately 2.7 times greater affinity than the (–)-enantiomer
for the D2 dopamine receptor, the inhibition of which results in the antipsychotic effect of
thioridazine. The (+)-enantiomer also has 4.5 times higher affinity for the norepinephrine
α1 receptor. In contrast, (–)-thioridazine has 10 times higher affinity for the D1 dopamine receptor.[27]
[28]
The (–)-enantiomer causes significantly less ventricular action potential prolongation
of the rabbit papillary muscle compared to (+)-thioridazine.[29] With its reduced antipsychotic effects and reduced prolongation of APD, (–)-thioridazine
warrants further biological investigation.
There are only a couple of published procedures for the preparation of enantiomers
of thioridazine.[30]
[31]
[32] Bourquin et al. reported the first synthesis of 1, starting from racemic piperidine alcohol 5 (Scheme [1]). Enantiomers of 1 were separated via chiral resolution employing di-p-tolyl-l-tartaric acid as a chiral derivatizing agent, making this a rather inefficient synthesis
of the desired enantiomer.[30]
Scheme 1 Synthesis by Bourquin et al.[30] Reagents and conditions: (i) thionyl chloride, CHCl3, 70 °C, 90% yield; (ii) NaNH2, phenothiazine 7, PhCH3, xylene, 180 °C, 60% yield.
An alternative to this approach starts with optically pure piperidine-2-carboxylic
acid;[33] the synthesis is shown in Scheme [2]. Piperidine-2-carboxylic acid is treated under Eschweiler–Clarke conditions followed
by esterification. The ester is reduced using LiAlH4 and transformed into the corresponding nitrile. The nitrile is hydrolyzed with hydrochloric
acid in methanol and the resulting methyl ester is reduced to the alcohol and treated
with thionyl chloride. The specific rotation of the resulting chloride (–)-6 was compared with the patent literature in order to define the absolute stereochemistry
of (+)-1 and (–)-1 as R and S, respectively.[33] It should also be noted that (–)-5 can be made through chiral resolution, but with tedious and low-yielding nine recrystallizations
to obtain sufficient optical purity. Most of the eight steps are high yielding, but
the overall yield is 8% and the starting material, piperidine-2-carboxylic acid, is
quite expensive and constitutes a limitation for scale up.
Scheme 2 Synthesis of the thioridazine developed by Patrick and Singletary.[33] Reagents and conditions: (i) HCO2H, CH2O, 16 h, reflux, 76%; (ii) AcCl, 3 M HCl, EtOH, 18 h, reflux, 89%; (iii) LiAlH4, Et2O, reflux, 6 h, 81%; (iv) KCN, 18-crown-6-ether, MeCN, Bu3P, 36 h, reflux, 80%; (v) HCl in MeOH, 2 °C, 67%; (vi) LiAlH4, Et2O, reflux, 6 h, 90%; (vii) CHCl3, HCl (gas), Cl2SO, 5 h, 0 °C, 51%; (viii) NaOH, 7, PhCH3, reflux, 3 h, 60% (for final reaction refer to Bourquin et al.[30]).
Another alternative is the procedure reported by Choi et al., in which the menthyl
carbamate of 1 is prepared and the diastereomers are separated by flash chromatography. However,
the carbamate is easily hydrolyzed, which makes the separation step hard to scale
up. LiAlH4 reduction of these carbamates gives the enantiomers of 1.[31] This procedure gives good enantiomeric excess, but the overall yield is poor. In
addition, the production of larger quantities is laborious, due to the bottleneck
of the diastereomer separation step.
Mohammad et al. reported the total syntheses of racemic thioridazine and isotope-labeled
analogues.[34]
[35] The strategy is elegant, but not suited for asymmetric synthesis. Accordingly, we
decided to develop our own total synthesis that can be scaled up to produce larger
volumes of both enantiomers.
Our key step involves the preparation of enantiopure alcohol 5. The retrosynthesis is depicted in Scheme [3]. The piperidine-2-carboxylic acid approach is opportune, as the stereogenic center
is present in the starting material. However, previously we have observed the lability
towards racemization of α-amino acids during elongation.[36] The allyl moiety is easy to handle, and can be oxidized to an aldehyde in a straightforward
manner. For asymmetric synthesis of amines, chiral auxiliaries, for example, the use
of chiral sulfonamides, are excellent choices. It is also known that sulfonamides
can be selectively alkylated in an aqueous environment.[37] After hydrolysis, the sulfonamides can be recovered and reused.[38]
Scheme 3 Retrosynthesis
First, we aimed for the S-enantiomer of 1. Condensation of 5-chloropentanal 9 with (S)-tert-butanesulfinamide, gave the corresponding N-tert-butanesulfinyl aldimine 10 (Scheme [4]). Indium-mediated allylation of (R)-10 in aqueous media, gave (R,S)-11 and (R,R)-11 in close to 10:1 diastereomeric ratio and quantitative yield. The two diastereomers
were separated by flash chromatography to give single diastereomers, and the major
product, (R,S)-11, was used in the next reaction.[39]
Scheme 4 Synthesis of the S-enantiomer of thioridazine. Reagents and conditions: (i) (S)-tert-butanesulfinamide, CuSO4, CH2Cl2, r.t., overnight, 91%; (ii) allyl bromide, NaBr, In, H2O, r.t., overnight, quant; (iii) HCl, 1,4-dioxane, 1 h, r.t.; (iv) K2CO3, DMF, 15 min, 120 °C microwave heating; (v) K2CO3, Boc2O, THF, 0 °C, overnight; 64% over three steps; (vi) OsO4 (2.5wt% in tert-butanol), 1,4-dioxane, 2,6-lutidine, 5 h, r.t. 82%; (vii) LiAlH4 (2.0 M in THF), 50 °C, 18 h, 88%; (viii) SOCl2, CHCl3, reflux, 3 h, quant; (ix) NaOH, 7, PhCH3, reflux, 3 h, 81%.
Acid-mediated hydrolysis of the tert-butanesulfonamide, with a subsequent intramolecular alkylation, best achieved using
microwave heating, yielded the piperidine motif. Next, the amine was protected as
the tert-butyl-carbamate (S)-12, to avoid possible formation of the N-oxide, during the Lemieux–Johnson oxidation. The oxidation worked well, yielding
aldehyde (S)-13. Focusing on the active nitrogen in the phenothiazine ring, reductive amination was
envisaged as an alternative. However, this approach proved unsuccessful as we only
recovered starting material.
The carbamate functionality was hydrolyzed, and the resulting piperidine was methylated
under Eschweiler–Clarke conditions. Focusing on the active nitrogen in the phenothiazine
ring, reductive amination was considered as an alternative. However, this also proved
unsuccessful.
Finally, LiAlH4 reduction of the carbamate (S)-13 gave amino alcohol (S)-5. All data are in accordance with published data. Amino alcohol (S)-5 was transformed into the corresponding chloride (S)-6 and coupled with phenothiazine 7, to give the (S)-enantiomer of thioridazine.[30]
Corresponding reactions starting from (R)-tert-butanesulfinamide gave the (R)-enantiomer of thioridazine.
The optical purity of the target molecule was checked by measurement of the specific
rotation and by chiral HPLC. The specific rotations were found to be the same as reported
for both enantiomers.[30] For the chiral HPLC analysis, the method reported by Schaluder et al. was used.[40] As reported, full separation of the two enantiomers on chiral HPLC was not achieved.
Different chiral columns, different eluent mixtures and different base additives were
tested, but only partial separation was obtained. However, for the scalemic samples
only single peaks were observed. In combination with the specific rotation values,
that were the same as reported in the literature, we therefore conclude that our samples
were of high enantiopurity.
Minimum inhibitory concentrations (MIC) against different strains of seven bacteria
were measured for both enantiomers and the racemate (Table [1]). Thioridazine racemate and the pure enantiomers were found to be relatively active
against the Gram-positive bacteria Mycobacterium samegmatis, Staphylococcus aureus MRSA, Enterococcus faecalis, and Enterococcus faecium, but not effective against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. The compounds are also slightly active against nosocomial strains of Acinetobacter baumannii, which are difficult to treat in hospitals due to their resistance to most antibiotics.
The activities of the enantiomers do not differ significantly from those of the racemic
mixture.
Table 1 Minimum Inhibitory Concentrations (MIC) of Thioridazine (μg/mL) toward a Selected
Set of Important Pathogens
|
Bacteriaa
|
Compounda
|
|
rac
|
+
|
–
|
|
M. smegmatis (n=2)
|
10
|
10
|
10
|
|
E. faecalis (n=4)b
|
20
|
20
|
20
|
|
MRSA (n=4)
|
20–40
|
20–40
|
20–40
|
|
E. faecium (n=4)
|
20
|
20
|
10–20
|
|
A. baumanii (n=4)c
|
80–160
|
80–160
|
40–160
|
|
E. coli (n=3)
|
>1280
|
>1280
|
>1280
|
|
P. aeruginosa (n=2)
|
>1280
|
>1280
|
>1280
|
a The number of tested strains of each species given as n.
b Including one nosocomial VRE.
c Nosocomial strains.
In conclusion, both enantiomers of thioridazine have been prepared in high optical
purity and good overall yield.[41] The route via Lemieux–Johnson oxidation of sulfonamide 11 followed by global reduction to give compound 5 proves to be a good, and cheaper alternative to both enantiomers of thioridazine
1.
Antimicrobial effects of the two enantiomers are comparable to the effects of the
racemate. Of special interest is Mycobacterium smegmatis, as its genus includes harmful pathogens, including Mycobacterium tuberculosis and Mycobacterium leprae. Similar findings were also reported by Poulsen et al.[42]
If the published results by Jensens et al.[29] on prolongation of the rabbit papillary muscle can be translated to human heart
muscle, (S)-1 is potentially a promising target in the fight against pathogens and cancer cells.
However, more investigations on the enantiomers are needed to rule out side effects.
Further biological tests will be carried out in the near future.
