CA2310205A1 - Unknown - Google Patents

Description

Novel N-Substituted Phenothiazines and Their Use as Modulators of Serine Hydrolase Enzymes Background of the Invention Alzheimer's disease (AD) is a common neurodegenerative disorder causing dementia. The incidence of AD increases with age (1). The prevalence of dementia rises from 3o at age 65 years to 47% after age 85 years (1).
The population of the elderly continues to rise and hence incidence of AD is also expected to rise. The frequency of dementia doubles every 5 years after the age of 60 years. In the United States, the annual cost for AD is estimated to be in excess of $60 billion annually (2, 3). With the rise in numbers of elderly individuals, the prevalence of AD is also expected to rise with concomitant rise in the cost for AD.
Development of drugs to delay the progression of AD as well as provide symptomatic treatment of this disorder is thus of paramount importance (l, 2, 3).
In AD there are three major microscopic features that are recognized as the hallmarks of the disease, namely neuritic plaques (NP), neurofibrillary tangles (NFT) and amyloid angiopathy (AA) (4). In addition, there is widespread cell loss, particularly of cholinergic neurons in the brain (5). Loss of cholinergic cells leads to reduction in the levels of the neurotransmitter acetylcholine, its synthesizing enzyme choline acetyltransferase, as well as its deactivating enzyme acetylcholinesterase (AChE) (5, 6).
Reduction of cholinergic neurotransmission leads to some of the symptoms of AD (6).

Although the level of AChE is reduced in AD, the level of the closely related enzyme butyrylcholinesterase (BuChE 3.1.1.8) is increased in AD brains (7). BuChE is found in all the neuropathological lesions associated with AD, namely, NP, NFT and AA (7). Importantly, BuChE is found in NP in brains of patients with AD. BuChE is found in a higher number of plaques in brains of elderly individuals with AD relative to those without AD (8). BuChE in Alzheimer brains requires 10-100 times the concentration of inhibitors to completely inhibit its esterase activity relative to BuChE in normal brains (9). It has been shown that some BuChE inhibitors not only improve cognition in an animal model but also reduce the production of (3-amyloid which is one of the principal constituents of neuritic plaques (10).
From a neuropathology perspective, deposition of amyloid and formation of NP is one of the central mechanisms in the evolution of AD (11, 12). However, amyloid plaques are also found in brains of elderly individuals who do not have dementia (13). It has been suggested that the amyloid plaques in individuals without dementia are "benign" and they become "malignant", causing dementia, when they are transformed into plaques containing degenerated neurites (13). These plaques are called neuritic plaques (NP). The mechanism of transformation from "benign" to "malignant"
plaques is as yet unknown. It has been suggested that BuChE
may play a major role in this transformation based on the observation that BuChE is found predominately in plaques that contain dystrophic neurites and not in plaques without dystrophic neurites (13).

Taken together these observations suggest that in brains of patients with AD there is a significant alteration of the biochemical properties of BuChE that alters its normal regulatory role in the brain thus contributing to the pathology of AD.
Recently, a brain specific serine protease called trypsin IV has been isolated and it is presumed to be involved in APP processing (24). Amyloid precursor protein (APP) is a transmembrane glycoprotein, which possesses a Kunitz-type serine protease inhibitor domain. The APP may be involved in protease regulation in the brain (14, 15).
Of particular importance is the fact that abnormally cleaved APP results in the formation of a 40-42 amino acid residue ~-amyloid protein fragment. This fragment is the main constituent of NP (16).
The proteolytic sites in APP have been studied extensively (18). There are three known proteolytic sites.
The first is the a-secretase site which when cleaved yields a 120 KDa fragment that does not accumulate in amyloid plaques (18). A basic amino acid residue such as arginine at this site is required for cleavage (19). Enzymes that require a basic amino acid residue at the cleavage site of their substrates are serine peptidases, such as trypsin.
The second cleavage site, the y-secretase site, cleaves at lys-28 (also a tryptic-site), which is the last amino acid of the extracellular APP domain (20). The third cleavage site, the ~-secretase site, occurs at the N-terminus (21).
The latter two sites lead to fragments that accumulate in amyloid plaques.

The enzymes that cleave amyloid precursor protein are called "secretases" but they have not been fully identified (22). It has been observed that a basic amino acid residue is required at some of the sites where APP
undergoes proteolytic cleavage (19). Two well-known enzymes that cleave peptides at basic amino acid residue sites are trypsin and carboxypeptidase B (23). Both of these enzymes are mainly recognized as pancreatic enzymes involved in digestion, but trypsin-like serine proteases have been found in the brain and are thought to be involved in APP
processing (24, 25, 26, 27). Interestingly, an enzyme with tryptic-like activity is closely associated with BuChE (28, 29). Recent observations that BuChE considerably enhances tryptic activity under normal circumstances (30, 31) and the observations that BuChE, which is found in high levels in NP, has altered biochemical properties, suggests that there may be a loss of regulation of tryptic activity, and other serine peptidase activity, associated with BuChE. This loss of regulation may play a role in abnormal proteolytic processing of APP. Recent evidence suggests that inhibition of BuChE enhances cognitive performance in rats, and that it promotes non-amyloidogenic processing of amyloid precursor protein (10).
Development of molecules that inhibit the activity of BuChE and/or AChE and simultaneously enhance the activity of serine proteases would not only provide symptomatic treatment of AD but would also lead to discovery of drugs that stop the progression of AD.

Summary of the Invention The present invention provides N-substituted phenothiazines that modulate serine hydrolase activity.
They inhibit activity of BuChE and/or AchE. They preferably have the general structure of the reaction products shown below in Schemes 1 to 5.
The present invention extends to a pharmaceutical composition that comprises an active compound disclosed herein, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable diluent or carriers, for modulating serine hydrolase activity in a mammal, preferably a human. The pharmaceutical composition can be used to treat, inhibit or prevent a pathological condition that is manifested in an abnormal concentration of, and/or activity of, a serine hydrolase enzyme. Among those pathological conditions are Alzheimer's disease; tumours such as brain tumours, for example gliomas; glaucoma; cardiac disease; central nervous system disorders; respiratory infections; gastrointestinal and renal diseases.
Figures 1 to 26 shown inhibition of enzyme activity by the preferred compounds of the invention.
O
H IC-R
I I
\ + I) Et~ ~ N \
\ ~ / R-C-Cl CH2C12 \ ~ / + HCl S wS
phenothiazine an amide Scheme 1: General reaction scheme for Series 1.

A second series of compounds was also prepared involving the reaction of phenothiazine-10-carbonyl chloride (PT-10-COC1) with primary and secondary amines. Scheme 2 shows the general reaction with a primary amine to give an N-substituted phenothiazine urea.
C1 ~NHR
I I
/ N \ CH2Cl2 / N \
+ RNH2 ~ I I + HC1 \ S / \ S /
phenothiazine-10-carbonyl chloride a urea Scheme 2: General reaction scheme for Series 2 The reactions were fast, producing clean, easily-purified products. All of the derivatives were specific inhibitors of BuChE and some were very potent inhibitors.
The compounds in Series 3a (Scheme 3) have a urea-type group (-N-(C=0)-N-) and an amine functionality at the end of the N-substituted chain. The linkage between the two nitrogens was varied using selected diamines.
O O
i C 1 CN H~"w'N R2 / I N \ CH2Clz / N \
+ H2N,~,~~~1 R2 ~ \ I I / + HC 1 \ S / S
Scheme 3: General reaction scheme for Series 3a.
Diamines with two primary amino groups can form both the 1:1 product and the 2:1 product with PT-10-COC1. The general reaction scheme for the 2:1 products (Series 3b) is shown in Scheme 4.
O O
ICCI NH_ ~C
/ ~ N \ CH2Ch \ \
+ H2N,~~~~NH2 \ S / / /
Scheme 4: General reaction scheme for Series 3b.
To synthesize the compounds in Series 4 (Scheme 5), chloroacetyl chloride was first prepared from thionyl chloride and chloroacetic acid. The secondary amine functionality of phenothiazine was then acylated using chloroacetyl chloride to produce N-chloroacetyl phenothiazine. This compound was then reacted with a variety of amines to produce an N-substituted phenothiazine with a methylene group between the amide carbonyl and the amine nitrogen.

g O
C1CH2C02H + SOCK -> C1CH2CC1 + 502 + HCl O
II
N NCH2Cl 101 / \ Et3N / \
C1CH2CC1 + 3.
CH2C12 \ ~ ~ /
S S

O
I I

/ N \
\ S /
Scheme 5: General reaction scheme for the synthesis of Series 4.
Compounds 1 to 17 have the formulae as shown below.

q i NHCH2CH2NH2 ~NHCH2CH2CH2NH2 / \ / \
\ ~ / \ ~ ~ /

CH3 ~ H
CNHCH2CCH2NH2 ~NHCH2CH2N(CH3)2 / I \CH3 / \
\ / \ /
H
~NHCH2CH2N(CHZCH3)2 ~NHCH CH CH N CH
2 2 2 ( 3~2 / \ / \
\ / \

~NHCH2CH2CH2N(CH2CH3)2 ~NHCH CCH N CH
2 I 2 ( 3~2 / \ / \CH3 \ / \ ~ /
n Structures of compounds in Series 3a.

O O

/ \ / \
\ ~ ~ / \ ~ ~ /
O O

/ \ / \
\ / \ /

/ \ CH3/
\ / \

Structures of compounds in Series 3b.

+
~CH2C1 i CHZNH2CH2CH2CH3 / \ / \
\ / \ /

+ ~ +
CCH2NH2CH2CH2CH2CH3 CCH2NH2CH2CH(CH3)2 / \ / \
\ / \ /

~CH2N
/ \
\ /

Structures of compounds in Series 4.

Experimental Melting points were recorded on a Mel-Temp II apparatus and are uncorrected. Infrared spectra were recorded as Nujol mulls on sodium chloride plates using a Nicolet Model 205 FT-IR spectrometer. Peak positions were obtained in ~~Peak Pick" mode. The NMR spectra were determined on a Bruker AC250F spectrometer at The Atlantic Region Magnetic Resonance Centre. Chemical shifts are reported in ppm relative to TMS. Mass spectra were recorded on a CEC 21-110B mass spectrometer at Dalhousie University.
Phenothiazine and phenothiazine-10-carbonyl chloride were purchased from Aldrich and Acros, respectively, and used without further purification. The amines were purified by fractional or simple distillation. All reactions were performed under anhydrous conditions. The reactions were monitored by TLC using plastic-backed silica plates with fluorescent indicator and CHZC12 as developing solvent.
Phenothiazine and phenothiazine-10-carbonyl chloride both have Rf values of 0.61 under these conditions while the products remain close to the origin.
Although the compounds were homogeneous as indicated by TLC, some of the 1H NMR spectra showed small amounts of impurities. Other than recrystallization, no attempt was made to purify the compounds further since the goal was to generate as many compounds as possible for the kinetic studies. In addition, no attempt was made to improve the low yields of the compounds (only a few milligrams were required for testing).

Synthesis of Compounds: Series 3a General Procedure for the Preparation of Compounds 1-3.
PT-10-COC1 (1 g, 4 mmol) was dissolved in CHZC12 (20 mL) and this solution was slowly added through a dropping funnel (over the period of an hour) to a well-stirred solution of the diamine (12 mmol) in CHZC12 (15 mL). The reaction was essentially complete within 5-10 minutes after addition of the PT-10-COC1 solution as monitored by TLC. Any precipitate in the reaction mixture was gravity filtered, characterized by IR and was determined to be either the 2:1 product or the hydrochoride salt of the diamine or a mixture of both. The CHZC12 solution was extracted with 0.1 N NaOH
(2 x 30 mL), washed with distilled H20 (2 x 30 mL), dried with MgS04, filtered and evaporated to dryness on a rotary evaporator.
Compound 1. By the general procedure described above, reaction between PT-10-COC1 (1.00 g, 3.9 mmol) and 1,2-ethanediamine (0.69 g, 12 mmol) gave 1 as an oily residue.
On cooling to -20°C, white crystals formed (0.71g as free base, 540). Characterization by 1H NMR indicated that the compound contained slight impurities. Recrystallization from CHZC12/pentane failed to remove these impurities.
Compound 2. This compound was prepared from PT-10-COC1 (0.97 g, 3.7 mmol) and 1,3-propanediamine (0.94 g, 12.7 mmol) according to the procedure described above. The CH2C12 solution was extracted with 0.1 N NaOH and then 0.1 N HC1.
The aqueous acid layers were combined and made basic by addition of NaOH pellets (20 pellets were required); the solution turned milky white. The basic solution was extracted with CH2C12 (2 x 30 mL). The organic layers were combined, dried with MgS04, gravity filtered and evaporated to dryness on a rotary evaporator to give a gum. Addition of diethyl ether (10 mL) induced the formation of white crystals. Evaporation of the solution yielded 2 (0.82 g as free base, 72%).
Compound 3. By the general procedure described above, reaction between PT-10-COC1 (1.00 g, 3.8 mmol) and 2,2-dimethyl-1,3-propanediamine (1.17 g, 11.5 mmol) yielded 3 as a pale orange, sticky solid. The solid was recrystallized from H20/MeOH and air-dried to give 3 as a white powder (0.21 g as free base, 17 %).
Compound 4. N,N-dimethyl-1,2-ethanediamine (0.33 g, 3.9 mmol) was added dropwise to PT-10-COC1 (1.02 g, 3.9 mmol) in CHZC12 (25 mL) with stirring. A white precipitate had formed after 24 hours of stirring. The precipitate was filtered and rinsed with CH2Clz to give 4 (0.62 g as HC1 salt, 470).
General Procedure for the Preparation of Compounds 5-9.
The diamine (9-12 mmol) in CHZC12 (5 mL) was added through a dropping funnel to a solution of PT-10-COC1 (1.00 g, 3.9 mmol) in CH2C12 (25 mL) with stirring. The reaction was complete after an additional 5-10 minutes of stirring as monitored by TLC. The CHZC12 solution was extracted with 0.1 N NaOH (2 x 50 mL), washed with distilled H20, dried with MgS04, filtered and evaporated to dryness on a rotary evaporator. If the product smelled of amine, it was dissolved in CHzCl2 (25 mL) and extracted with 0.1 N HCl (2 x 30 mL) and then with 0.1 N NaOH (2 x 30 mL). The CHZC12 layer was washed with distilled H20, dried with MgS09, filtered and evaporated to dryness on the rotary evaporator.
If an oil resulted, it was taken up in diethyl ether (10 mL) and concentrated HCl was added dropwise (5-6 drops were 1$
required) to precipitate the product as the hydrochloride salt. The solution was gravity filtered and the product dried in a desiccator.
Compound 5. By the general procedure described above, reaction of PT-10-COCl (1.02 g, 3.9 mmol) with N,N-diethyl-1,2-ethanediamine yielded 5 (0.58 g as HC1 salt, 45 0).
Compound 6. By the general procedure described above, reaction of PT-10-COC1 (1.01 g, 3.9 mmol) with N,N-dimethyl-1,3 propanediamine (0.97 g, 9.5 mmol) yielded 6 as a white powder (0.78 g as free base, 63%).
Compound 7. By the general procedure described above, reaction between PT-10-COC1 (1.00 g, 3.8 mmol) and N,N-diethyl-1,3-propanediamine (1.25 g, 9.6 mmol) yielded a yellow oil. Conversion of the product to the hydrochloride salt gave 7 as a white powder (0.54 g as HC1 salt, 36o).
Compound 8. By the general procedure described above, reaction between PT-10-COCl (1.00 g, 3.8 mmol) and N,N,2,2-tetramethyl-1,3-propanediamine (1.44 g, 11.1 mmol) gave 8 as a white powder (0.14 g as free base, l00).
Compound 9. By the general procedure described above, reaction between PT-10-COCl (1.00 g, 3.8 mmol) and 1-methylpiperazine (1.43 g, 14.3 mmol) gave 9 as a white powder (0.36 g as HCl salt, 260).
Series 3b General Procedure for the Preparation of Compounds 10-11. The diamine (4 mmol) was added through a dropping funnel to a well-stirred solution of PT-10-COC1 (1.00 g, 3.9 mmol). A voluminous white precipitate formed, which was filtered, rinsed with CHzCl2, air-dried and characterized.
Compound 10. By the general procedure described above, reaction between PT-10-COC1 (1.03 g, 3.9 mmol) and 1,2-ethanediamine (0.23 g, 3.8 mmol) gave 10 as a white powder (1.00 g, 1000).
Compound 11. By the general procedure described above, reaction between PT-10-COC1 (1.00 g, 3.8 mmol) and 1,3-propanediamine yielded 11 as a white powder (0.93 g, 930).
Compound 12. 2,2-dimethyl-1,3-diaminopropane (0.24 g, 2.3 mmol) in CHZC12 (5 mL) was added through a dropping funnel to a solution of PT-10-COCl (1.00 g, 3.9 mmol) in CHZC12 (20 mL) with stirring. After 48 hours of stirring, starting material was still present as indicated by TLC. A
white precipitate had formed and was gravity filtered. From the IR spectrum, the precipitate was determined to be the hydrochloride salt of 2,2-dimethyl-1,3-diaminopropane. The reaction mixture was evaporated to dryness on a rotary evaporator to give a white solid (0.62 g). TLC of the solid in CHZC12 showed unreacted PT-10-COC1 (Rf = 0.60) and the presumed 2:1 product (at origin). With 5% MeOH/CHZC12 as the developing solvent, both spots moved: Rf=0.82 for PT-10-COC1 and Rf=0.69 for the presumed product. Based on the TLC results, the solid was subjected to column chromatography using 20 g of silica gel; the PT-10-COCl was eluted with CHZC12. On elution with 5% MeOH/CHZC12, 12 was isolated as a pale pink powder (0.29 g, 23%).
Series 4 Preparation of Compound 13 (10-chloroacetylphenothiazine, PT-10-COCH2C1). Chloroacetyl chloride was prepared by adding thionyl chloride (32 mL, 0.44 mol) through a dropping funnel to chloroacetic acid (50 g, 0.53 mol). The reaction mixture was refluxed for two hours and then distilled using a fractionation column (bpobs=105.0-105.5, bplit=105) . Only 8 mL of chloroacetyl 1~
chloride were collected (10.35 g, 0.092 mol). Phenothiazine (10.00 g, 50 mmol) was dissolved in 200 mL CHZC12 and triethylamine (5.00 g, 50 mmol) was added to the solution.
Chloroacetyl chloride (10.35 g, 92 mmol) in CHZC12 (10 mL) was added through a dropping funnel to the solution. The reaction mixture was refluxed for 24 hours and was monitored by TLC. Disappearance of the PT spot (Rf=0.61) indicated that the reaction was complete. The product had an Rf value of 0.32 in CHZC12. The reaction mixture was extracted with 5o NaHC03 (3 x 50 mL), 5o HCl (3 x 50 mL) and then 5%
Na2S203 ( 3 x 50 mL) . The CHZC12 layer was washed with distilled Hz0 (50 mL), dried with MgS04, gravity filtered and evaporated to dryness to give 8.92 g (650) of the crude product. Recrystallization afforded off-white crystals, which appeared to be homogeneous by TLC and NMR.
General Procedure for the Preparation of Compounds 14-17. The amine (5 mmol) in CH2C12 was added through a dropping funnel to a solution of PT-10-COCHZC1 (0.50 g, 1.9 mmol) in CHZC12 (10 mL). The reaction mixture was refluxed and monitored by TLC frequently. When the reaction was complete, as judged by disappearance of the spot at Rf=0.32, the CHZC12 solution was extracted with 0.1 N NaOH (3 x 30 mL), washed with distilled Hz0 (30 mL), dried with MgS04, gravity filtered and evaporated to dryness on a rotary evaporator. If a solid resulted, it was recrystallized from petroleum ether/CHZC12. If an oil resulted, it was taken up in diethylether (10 mL) and concentrated HC1 was added (4-6 drops were required) to convert the amine product to the HC1 salt, which precipitated from the solution. The solution was gravity filtered and the solid was dried in a desiccator.

Compound 14. By the general procedure described above, reaction between PT-10-COCHZC1 (0.39 g, 1.4 mmol) and n-propylamine (0.21 g, 3.5 mmol) gave 14 as a pink solid (77 mg as HC1 salt, 160). The reaction was complete after refluxing the reaction mixture for 5 hours and stirring for two days. This product needs to be purified and has not been adequately characterized.
Compound 15. By the general procedure described above, reaction of PT-10-COCHZC1 (0.51 g, 1.9 mmol) and n-butylamine (0.41 g, 5.6 mmol) gave 15 as a white powder (60 mg as HCl salt, 90). The reaction was complete after 20 hours of refluxing.
Compound 16. By the general procedure described above, reaction between PT-10-COCHZCl (0.48 g, 1.7 mmol) and isobutylamine (0.38 g, 5.22 mmol) gave 16 as a white powder (62 mg as HC1 salt, 10%). The reaction was complete after refluxing the reaction mixture for 4 hours and stirring for 2 days.
Compound 17. This compound was prepared from PT-10-COCHzCl (0.53 g, 1.9 mmol) and pyrrolidine (0.41 g, 5.7 mmol) according to the procedure described above. The reaction was complete after 45 minutes of refluxing and the isolated product was recrystallized from petroleum ether/CHZC12 ( 0 . 34 g as free base, 60 0 ) .
Biochemical Studies The ability of the compounds to inhibit AChE was evaluated spectrophotometrically using AChE from human erythrocytes and acetylthiocholine as the substrate. BuChE
inhibitory activity studies were carried out similarly using BuChE from human serum and butyrylthiocholine as the substrate. The kinetic studies were based on the rate of hydrolysis of the substrate and the subsequent reaction of thiocholine with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)) to produce the yellow anion of 5-thio-2-nitrobenzoate which absorbs at 412 nm. The enzymatic activities of AChE and BuChE were examined without inhibitor to determine their Km values (Km Michaelis constant, the dissociation constant for the enzyme-substrate complex) and with inhibitor to determine their Ki values (Ki= inhibition constant, the dissociation constant for the enzyme-inhibitor complex).

Table 1: AChE and BuChE Inhibition Results Compound BuChE
Ki (~) 1 Selectivity BuChE AchE (Ki AChE/Ki BuChE) phenothiazine 12 -ethopropazine 0.24 -Series 2 0.56 0.05 -Derivative 8.0 1.3 -2 7.9 -3 1.2 -0.69 0.07 42 6 61 5 0.55 0.02 26 6 47 6 1.92 0.02 174 42 91 7 0.96 0.04 20.0 0.2 21 8 0.59 -10 inc. -11 inc. -12 inc. -14 inc. -15 2.8 16 4.7 -17 4.0 1 A dash (-) indicates insignificant inhibition; "inc." indicates that inhibition studies are incomplete.

Materials and Methods Preparation of Reagents and Enzymes 5,5'-Dithio-bis(2-Nitrobenzoic Acid) (DTNB) Stock In 20 mL of O.1M phosphate buffer (pH 7.0), 0.038 of sodium bicarbonate and 0.0798 of DTNB were combined and mixed.
Buffered 5,5'-Dithio-bis(2-Nitrobenzoic Acid) (DTNB) ~.., ,. ~: ..., 3.6 mL of stock DTNB was combined with 96.4mL of O.1M
phosphate buffer (pH 8.0).
Acetylthiocholine (AcTCH) 0.0868 of AcTCH was dissolved in 20 mL of distilled water to give a stock concentration of l5.OmM. O.lmL of the stock solution in a final volume of 3.OmL gave a concentration of 0.50mM in the cuvette. A number of 50o serial dilutions were performed from the stock solution to produce the substrate concentrations employed in the assay.
Butylthiocholine (BUTCH) 0.09528 of BUTCH was dissolved in 20 mL of distilled water to give a concentration of l5.OmM. O.lmL of the stock solution in a final volume of 3.OmL gave a concentration of 0.50mM in the cuvette. A number of 50% serial dilutions were performed from the stock solution to produce the substrate concentrations employed in the assay.
Human Butylcholinesterase (BuChE) 1.0 mL of 0.005% aqueous gelatin was added to a stock bottle containing 100U of enzyme. Appropriate ratios of stock enzyme solution and 0.005% aqueous gelatin were combined, such that the diluted enzyme solution gave a change in absorbance per minute of approximately 1.00 at the highest concentration of BUTCH (i.e. 0.50mM).

Human Acetylcholinesterase (AChE) 0.02068 of enzyme was combined with 4.0 mL of either 0.0050 aqueous gelatin or 0.50 Triton-X 100, and ground to a slurry with a mortar and pestle. The resulting enzyme solution gave a change in absorbance per minute of approximately 0.300 at the highest concentration of AcTCH (i.e. 0.50mM).
Inhibitor Solutions All inhibitor solutions were made in 50% aqueous acetonitrile with a stock concentration of 5x10-3M.
Kinetic Studies The esterase activity of human serum BuChE and human erythrocyte AChE was studied using a modified Ellman assay (Ellman et al., 1961). In a quartz cuvette of 1-cm path length the following reaction components were combined and mixed, to give a final volume of 3.0 mL: 2.7 mL of buffered DTNB (pH 8.0), 0.1 mL of enzyme (AChE or BuChE) and, 0.1 mL
of either 50% aqueous acetonitrile or inhibitor in 50%
aqueous acetonitrile. The reaction was initiated by the addition of substrate (AcTCH or BuTCH), and was analyzed at room temperature using a Milton-Ray UV-visible spectrophotometer set at 412nm. The change of absorbance was recorded at 5-second intervals for a period of one minute.
The Abs/min values represent the rate of hydrolysis of the substrate by the enzyme.
Determination of Inhibitor Specificity AChE and BuChE were exposed to a number of serial dilutions of each compound (1.7x10-4-1.7x10-9M), at the highest substrate concentration (0.50mM). Inhibition profiles were generated by plotting the rate of substrate hydrolysis (Abs/min) versus the log of the inhibitor concentration.

Generation of Lineweaver-Burk plots Lineweaver-Burk plots were produced by plotting the inverse of the rate (Abs/min) versus the inverse of the substrate concentration. Three separate runs were performed, each employing a different inhibitor concentration (one without inhibitor and two carried out in the presence of different inhibitor concentrations). The inhibitor concentrations used were selected from the inhibition profile described above. Each run consisted of a series of assays in which the concentration of enzyme and inhibitor were held constant while the substrate concentration was varied (i.eØ50mM - 0.0313mM). Km and VmaX values, in addition to the type of inhibition were obtained from the Lineweaver-Burk plots.
Determination of the Inhibition constant (Ki) The strength of the inhibition, the inhibition constant (Ki), was determined by plotting the slope of each of the Lineweaver-Burk lines against their respective inhibitor concentrations. Each Ki value was obtained from the x-intercept of its respective graph. The Ki values provided represent the average of two values.
Series 1 ("amide" series) and series 2 ("urea" series) compounds The PTZ derivatives were designated as belonging to either series 1 (" amide" series ) , or series 2 (" urea"
series), based on the type of product obtained. Series 1 compounds were synthesized by reacting PTZ with a given acid chloride (reaction 1.). Series 2 compounds were synthesized by reacting PTZ-10 carbonyl chloride with a given amine (reaction 2.). Adding the amine dropwise to an excess of PTZ-10 carbonyl chloride produced the 2:1 products of series 2. Reversing the order and adding the 10-carbonyl chloride to an excess of amine produced the corresponding l:l compounds.

l.General reaction scheme for N-substituted phenothiazine amides:
O
H IC-R
I I
/ N \ II Et3N / N \
\ ~ ~ / + R-C-Cl ~ I + HCl CH2C12 \ S /
S
phenothiazine an amide 2. General reaction scheme for N-substituted phenothiazine ureas:

/ \ CH2C12 / \
+ RNH2 ~ I + HCl \ S / \ S /
phenothiazine-10-carbonyl chloride a urea Table 2. Name, number designation and structure of R group for series 1 ("amide" series) derivatives.
Name and number Structure of R group designation of series for series 1("amide"
1("amide" series) series) derivatives derivatives 1. Acetyl O

2.Chloroacetyl 3. Propanoyl 4. Butanoyl ~CH3 5. Isovaleryl O H2CH
~CH3 O
6. Benzoyl Table 3. Name, number designation and structure of R group for series 2 ("urea" series) derivatives.
Name and number Structure of R group for designation of series 2 series 2("urea" series) ("urea" series) derivatives derivatives 7. N-propyl O NCH2CH2CH3 ~H
8. Butyl O NCH2CH2CH2CH3 ~H
9. Isobutyl /CH3 O~/ NCH2C/H
H ~CH3 10. Sec-butyl ~CH2CH3 O NCH
~H ~CH3 11. Tert-butyl O N C (CH3 )3 ~H
12.2-methoxyethyl O N CH2CH20CH3 ~H
13 . Neopentyl O N CH2C(CH3)3 H
N
14. Cyclohexyl O N
~H

Table 3 continued 15. Benzyl ~H
16. Diethyl /CH2CH3 O /N\
\CH2CH3 17. Pyrrolidine O N

18. Piperidine O N~

19. Morpholine O N O

20. N,N-dimethyl O /CH3 ethylene diamine H CH2CH2N /~

21. N,N-diethyl ethylene O /CH2CH3 diamine H CH2CH2N /~

22. N,N-diethyl propyl ,CH3 diamine O H CH2CH2CH2 ~N
\CH3 23. N,N-diethyl propyl ,CH2CH
diamine O H CH2CH2CH2 ~N
\CH2CH

24. Ethylene diamine O NCH2CH2NH2 H
N

Table 3 continued 25. Ethylene diamine O NCH2CH2N
(2:1 product) ~H H

26. 1,3-propyl diamine O NCH2CH2CH2N O
(2:1 product) ~H H

Table 4. Specificity of inhibition displayed by the compounds within series 1("amide" series) and the resulting inhibition constants.
PTZ Enzyme Enzyme Inhibition (Ki) (M) derivative inhibited inhibited constant R-C=O

AChE BuChE AChE BuChE

Propanoyl + + 8.38x10-5 2.58x10-5 Chloroacetyl + + 1. 12x10-4 1.52x10-5 insignificant Benzoyl inhibition + 1.04x10-5 Butanoyl + + 3.88x10-5 2.19x10-5 Iso-valeryl + + * 1.12x10-5 Acetyl + + 1.13x10-4 7.56x10-5 + designates that significant inhibition was observed.
* Enzyme kinetics not pursued due to inhibitor concentration limitations Table 5. Specificity of inhibition displayed by the compounds in series 2("urea" series) and their corresponding inhibition constants.
PTZ derivative Enzyme inhibited Inhibition constant (M) R

AChE BuChE AChE BuChE

n-propyl insignificant + 2.56x10-5 inhibition Neo-pentyl insignificant + 2.04x10-6 inhibition Butyl insignificant + 2.78x10-5 inhibition Iso-butyl insignificant + 1.2x10-5 inhibition Tert-butyl insignificant + 1.06x10-5 inhibition Sec-butyl insignificant + 1.18x10-5 inhibition 2-methoxyethyl insignificant + 3.27x10-5 inhibition Table 5 continued Diethyl insignificant + 9.82x10-' inhibition Pyrrolidine insignificant + 5.6x10-' inhibition Piperidine insignificant + 1.42x10-6 inhibition Cyclohexyl insignificant + 2.98x10-6 inhibition Morpholine insignificant + 6.44x10-6 inhibition Benzyl insignificant + 5.87x10-6 inhibition N,N-dimethyl + + 3.88x10-5 3.63x10-' ethylene diamine N, N-diethyl + + 2 . 62x10-5 5 . 4 9x10-' ethylene diamine N, N-dimethyl + + 1 . 74x10-4 1. 92x10-6 propyl diamine Table 5 continued N, N-diethyl + + 2 . OxlO-5 9 . 56x10-' propyl diamine Ethylene + + 4.51x10-6* 6.97x10-6 diamine (2:1 product) 1,3-propyl + Insignificant 1.1x10-5*

diamine (2:1 inhibition product) +designates that significant inhibition was observed.
*The Ki value represents a single value.
Table.6. PTZ derivatives displaying mixed non-competitive inhibition towards the cholinesterases.
PTZ derivative Enzyme inhibited Mode of inhibition AChE BuChE Mixed non-competitive N,N-dimethyl + + Mixed non-ethylene diamine competitive urea Table 6 continued N,N-diethyl + + Mixed non-propyl diamine competitive urea Acetyl x + Mixed non-competitive Chloroacetyl x + Mixed non-competitive Butanoyl x + Mixed non-competitive Sec-butyl x + Mixed non-competitive Tert-butyl x + Mixed non-competitive Diethyl urea x + Mixed non-competitive Neopentyl urea x + Mixed non-competitive Pyrrolidine urea x + Mixed non-competitive Table 6 continued Piperidine urea x + Mixed non-competitive Cyclohexyl urea x + Mixed non-competitive morpholine urea x + Mixed non-competitive Benzyl urea x + Mixed non-competitive Ethylene diamine x + Mixed non-urea competitive N,N-diethyl x + Mixed non-ethylene diamine competitive urea Ethylene diamine x + Mixed non-urea (2:1 competitive product) Iso-valeryl x + Mixed non-competitive Table 6 continued N,N-dimethyl x + Mixed non-propyl diamine competitive urea Table 7. PTZ derivatives displaying non-competitive inhibition towards the cholinesterases.
PTZ derivative Enzyme inhibited Mode of inhibition AChE BuChE Non-competitive Propanoyl x + Non-competitive Benzoyl x + Non-competitive N-propyl urea x + Non-competitive Butyl urea x + Non-competitive Iso-butyl urea x + Non-competitive 2-methoxyethyl x + Non-competitive urea Acetyl + x Non-competitive Chloroacetyl + x Non-competitive N,N-diethyl + x Non-competitive ethylene diamine urea 1,3-propyl + x Non-competitive diamine urea (2:1 product) Table 8. PTZ derivatives displaying competitive inhibition towards the cholinesterases.
PTZ Enzyme inhibited Mode of derivative inhibition AchE BuChE

Propanoyl + x Competitive N,N-dimethyl + x Competitive propyl diamine urea Table 9. Inhibition constants resulting from the addition of specific functional groups at the Nlo position of phenothiazine.
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Claims (2)

1. N-substituted phenothiazines.

2. A pharmaceutical composition comprising an N-substituted phenothiazine, or a pharmaceutically acceptable salt thereof, for the modulation of an activity of a serine hydrolase.