Identification of A Butyrophenone Analog as a Potential Atypical Antipsychotic Agent: 4-[4-(4-Chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one

Abstract

The synthesis and exploration of novel butyrophenones have led to the identification of a diazepane analog of haloperidol, 4-[4-(4-Chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one (Compound 13) with an interesting multireceptor binding profile. Compound 13 was evaluated for its binding affinities at DA subtype receptors, 5HT subtype receptors, H-1, M-1 receptors and at NET, DAT and SERT transporters. At each of these receptors, compound 13 was equipotent or better than several of the standards currently in use. In in vivo mouse and rat models to evaluate its efficacy and propensity to elicit catalepsy and hence EPS in humans, compound 13 showed similar efficacy as clozapine and did not produce catalepsy at five times its ED₅₀ value.

The introduction of clozapine¹ (Clozaril, 1) as a treatment option in the 1990s has opened the door to the use of atypical antipsychotics and the replacement of the prototypic agent, haloperidol (2) as a drug of choice for the treatment of schizophrenia. The superior therapeutic profile of clozapine and other atypical antipsychotic drugs and the long-term debilitating side-effects² associated with haloperidol treatment have conspired to diminish its use in the clinic. Among several hypotheses put forth to explain the long-term extrapyramidal side effects of haloperidol including tardive dyskinesia, is the fact that haloperidol undergoes biotransformations that lead to the formation of toxic metabolites.^3,4 Specifically, haloperidol has been shown to undergo CYP450-mediated oxidation to form pyridinium metabolites⁵ which can potentially damage dopamine receptors in the nigrostriatum of the brain (Chart 1). Based on this hypothesis, we ^6-9 proposed a drug design strategy that replaces the piperidine ring in haloperidol with bioisosteric equivalents which could not undergo in vivo biotransformation to pyridinium species known to be associated with neuronal toxicity. A similar strategy has more recently appeared in the literature.¹⁰ The outcome of such a strategy was to enhance the pharmacological and side-effect profiles of the resulting agents.

Chart 1.

Our laboratory has previously shown that modification in the piperidine ring of haloperidol results in significant changes in pharmacological activity.^6-7 We also identified new bioisosteric groups of the piperidine ring with binding profiles similar or better than those of haloperidol at the D2 dopamine receptor subtype.^7-9 Several of these compounds are shown in Chart 2, with new binding affinity data at selected receptors reported in Table 1.

Chart 2.

Table 1.

Binding Affinity Constants of Compounds to Selected 5-HT and H-1 Receptors.

Compds	Binding Data of Compounds, Ki ± SEM (nM)						Ki D2/Ki D4 Ratio
	DAD2	DAD4	5HT1A	5HT2A	5HT2C	H-1
Cloz (1)a	130	54	140	8.9	17	1.8	2.4
Hal (2)a	0.89	10	3600	120	4700	440	0.09
4b	253.5±38.9	17.5±2.0	90.9±21.0	109.6±16.0	3552±943	157.6±36.0	2.8
5b	1.6±0.14	5.3±1.0	27.7±8.0	30.9±6.0	872.1±178.0	8780±1625	0.30
6b	2.3±0.28	19.2±2.3	37.6±6.0	12.3±3.0	>10,000	635.4±96.0	0.12
7b	124.4±16.5	176.8±36.0	414.4±101.0	106.0±21.0	1065±244	973.5±207.0	0.70
(+)-8b	51.1±6.0	3.6±0.48	772.3±90.0	75.8±12.0	3598±1162	1467±473	14.2
(-)-8b	489.4±119.4	245.5±29.2	831.5±126.0	241.5±39.0	1252±461	259.6±34.0	2.0

^aBinding data obtained from reference 12b.

^bThe synthesis and/or DA binding data of compounds previously reported in ref. 7 - ⁹

Although compounds 5 and 6 have high affinity for 5HT_1A and 5HT_2A receptors, the demonstration that compound 5 elicits a higher catalepsy than haloperidol dampened our desire to pursue the tropane moiety as a piperidine bioisostere.⁹ It is important to note that the alcoholic function in compounds 5 and 6 contributes significantly to affinity not only at the D2-like receptors but also at the 5HT_1A and 5HT_2A receptors (cf compounds 5, 6 and 7). Interestingly, the OH group does not appear to play a significant role in the binding of the tropane analogs to the 5HT_2C receptor.

The reaction mechanism by which haloperidol is metabolized in vivo to a pyridinium species or BCPP+ has been suggested to begin with dehydration of the tertiary alcoholic function.⁵ A drug design strategy to prevent such a biotransformation is to eliminate the alcoholic function. Compound 3, a haloperidol analog without its alcoholic function, was previously synthesized and evaluated at D2-like receptors.⁷ The results supported the notion that the alcoholic function contributes to affinity but is not an absolute requirement for binding to dopamine receptors. Compound 4 in which the C-OH group was replaced with a nitrogen atom showed a lower binding affinity than haloperidol at the dopamine receptors.⁸ Compounds 5 and 6 were designed on the premise that the tropane moiety could not form a pyridinium ring due to the fact that a double bond could not form at the ring junctions adjacent to the N atom.⁹ In addition, bridging the ring restricts conformational mobility and holds the alcoholic function in a preferred conformation.⁸ Binding evaluation showed that both compounds were as potent or more potent than haloperidol at D2-like receptors. It was further demonstrated that the OH group is in the axial position and this led to the suggestion that haloperidol most likely binds to the dopamine receptors with its OH in the axial position. Comparing 5 and 6 suggests that the chloro group contributes to binding but is also not an absolute requirement. Removal of the OH group from the tropane ring (Compound 7) again decreased affinity but retained sufficient dopamine binding affinity as observed for compound 3. Contraction of the piperidine ring to a pyrrolidine (8) resulted in a decrease in affinity but expansion to a 7-membered homopiperidine ring (9) retained affinity as in haloperidol, thus suggesting that homopiperidine ring is bioisosteric to piperidine ring.⁷ Separation of the enantiomers of compound 8 and their subsequent evaluation at D2-like receptors revealed that (+)-8 was the eutomer.⁶

To further explore these changes in the piperidine moiety of haloperidol in our search for new antipsychotic drugs with atypical pharmacological profiles, we have embarked on SAR studies with modifications primarily in the piperidine ring that could lead to the identification of new agents with clozapine-like binding profiles but without the associated weight gain and new onset type II diabetes.¹¹ Thus, it was desirable not only to evaluate the binding of the compounds at dopamine receptors but also at serotonergic receptors (5HT_1A and 5HT_2A) since binding to these receptors is often associated with therapeutic advantages observed in atypical antipsychotic drugs.¹² In addition, evaluation of the compounds at 5HT_2C and histamine H-1 receptors will be valuable indicators of whether these compounds have the potential to develop the weight gain side effect associated with atypical antipsychotic drugs.¹³ In line with the multiple receptor targeting strategy in the development of new antipsychotic agents, we have focused our design efforts on obtaining agents that have the following receptor binding profiles:

1. Binding to DA D2 receptor with moderate affinity (30 < Ki < 150 nM)

2. Binding to DA D4 receptor with high affinity (Ki < 10 nM)

3. Binding to 5HT_1A and 5HT_2A receptors with high affinity (Ki < 50 nM)

4. Binding to 5HT_2C and H-1 receptors with very low affinity (Ki > 500 nM)

The above criteria (a – d) will also guide the selection of compounds for evaluation in in vivo animal models for efficacy and the propensity of the compounds to produce catalepsy, a condition associated with extrapyramidal side-effects in humans.¹⁴ To achieve the drug design objectives, we have utilized observations from previous SAR studies to design compounds 10 - 14 (Chart 3) as probes for the dopamine, serotonin and histamine H-1 receptor subtypes in an attempt to obtain new prototype drugs.

Chart 3.

Chemistry

Construction of the ethylene-bridged piperazine moiety was initiated using (2S, 5R)-diethyl 2,5-dibromohexanedioate (16) as the starting material (Scheme 1) as previously reported.^15,16 Double alkylation of 4-chlorophenyl aniline with 16 yielded N-chlorophenyl pyrrolidine (17). Amidation of 17 with benzylamine produced the amide, 18 which underwent a ring closure with the remaining ester function to form the bridged piperazine, 19. An attempt to reduce the lactam functions of 19 with LiAlH₄ also resulted in the removal of the 4-chloro group on the phenyl ring to form the di-substituted 3,8-diaza-bicyclo[3.2.1]octane (20). Debenzylation of 20 to form 22 was accomplished by reaction with ethyl chloroformate and the subsequent hydrolytic cleavage of the resulting carbamate, 21. Compound 10 was obtained by alkylation of the secondary amine function of 22 with 4-chloro-4′-fluorobutyrophenone. Target compounds 11 and 12 were obtained by alkylating the corresponding starting materials, 23 and 24 respectively (commercially available from Sigma-Aldrich) with 4-chloro-4′-fluorobutyrophenone (Schemes 2 and 3). Scheme 4 describes the synthesis of target compound 13 in five steps. Initial starting material 4-chloroboronic acid (26) was synthesized from 4-chlorobromobenzene by lithiation with n-butyl lithium and treatment with trimethylborate. The chloroboronic acid was coupled to N-Boc protected 1,4-diazepane (28) under palladium-catalyzed amination condition to form 29. Deprotection of 29 in trifluoroacetic acid delivered amine 30 which was alkylated in the usual way with 4-chloro-4′-fluorobutyrophenone to form 13. An alternative synthesis of compound 13 was also explored in which the 4-chlorophenyl homopiperazine (30) was obtained in one step without the protection and deprotection steps. The reaction involved the coupling of 4-chloroiodobenzene directly with unprotected homopiperazine under CuI catalysis to form compound 30 in excellent yield. The same alkylation step delivered the desired compound 13 in 61% overall yield (Scheme 5).

Scheme 1^a.

^a Reagents: (i) 4-Chloroaniline, DME, KI; (ii) Benzylamine, o-Xylene; (iii) (a) NaOH, EtOH; (b) Ac₂O, NaOAc; (iv) LiAlH₄; (v) ClCO₂Et, Toluene; (vi) KOH, EtOH, NH₂NH₂; (vii) 4-Chloro-4′-fluorobutyrophenone, KI, K₂CO₃, DME.

Scheme 2^a.

^a Reagents: (i) 4-Chloro-4′-fluorobutyrophenone, KI, K₂CO₃, DME.

Scheme 3^a.

^a Reagents: (i) 4-Chloro-4′-fluorobutyrophenone, KI, K₂CO₃, DME.

Scheme 4^a.

^a Reagents: (i) (a) n-BuLi, -78 °C, THF; (b) B(OMe)₃; (ii) t-BuOH, NaOH, (t-BuOCO)₂O; (iii) Cu(OAc)₂, NEt₃, Mol sieves (4Å), rt; (iv) TFA, CH₂Cl₂; (v) 4-Chloro-4′-fluorobutyrophenone, KI, K₂CO₃, DME.

Scheme 5^a.

Alternative Synthetic Scheme for Compound 13

^a Reagents and conditions: (i) CuI, ethylene glycol, K₂CO₃, ⁱPrOH, 80 °C, reflux, 12 h; (ii): KI, K₂CO₃, DME, 4-chloro-4′-fluorobutyrophenone, reflux, 12 h.

8-Methyl-8-aza-bicyclo[3.2.1]octa-3-one (31) served as the starting material for the synthesis of target compound 14 (Scheme 5). Compound 31 was converted to the cyclic lactam (32) by insertion of a nitrogen atom followed by reduction using lithium aluminum hydride to form 33 in very good yield (80%). The free secondary amine in 33 was BOC protected prior to its treatment with ethyl chloroformate to form the carbamate protected amine, 35. Compound 35 was BOC deprotected and then coupled with 4-chloroboronic acid in the presence of copper acetate to yield 37. Decarbamylation of 37 was achieved using KOH in ethylene glycol and the resulting secondary amine was alkylated with 4-chloro-4′-fluorobutyrophenone under the usual alkylating condition to produce the desired compound 14.

Results and Discussion

Our previous observation that bridging the piperidine ring to form the tropane analogs of haloperidol resulted in 7- and 3-fold increases in binding affinity at the D2 and D4 receptors respectively informed the design of target compounds 10 and 14. The rationale was to investigate bridged analogs to probe the effect of conformational restriction on piperazine and homopiperazine equivalents of haloperidol. Compound 10 can also be viewed as an analog of 7 with the carbon at position 4 of the ring replaced by a nitrogen atom. The ethylene bridge in compound 10 holds the piperazine ring in the chair conformation while in compound 11 the ring is held in the boat conformation by a methylene group. Binding affinity data for compounds 10 -14 are reported in Table 2.

Table 2.

Binding Affinity Constants of Compounds to Dopamine, 5HT and Histamine Receptors.

Compds	Binding (Ki±SEM) Data of Synthetic Compounds at Selected Receptors (nM)
	D2	D3	D4	5HT1A	5HT2A	5HT2C	H1
Cloz(1)a	130	240	54	140	8.9	17.0	1.8
Hal (2) a	1.4	2.5	3.3	3600	120	4700	440
10b	155	600	2100	ND	ND	ND	ND
11b	170	220	520	ND	ND	ND	ND
12	98.0±15.3	244.1±106.0	6.53±0.76	30.5±5.0	22.0±4.0	4132±1081	911.8±152.0
13	43.3±13.3	158.8±35.1	6.6±0.6	117.4±32.6	23.6±2.7	1425±207	188.6±16.0
14	178.4±29.2	548.1±246.0	41.8±9.0	2332±470	194.8±53.0	3513±912	1014±206

ND = Not Determined.; Hal = Haloperidol; Cloz = Clozapine

^aData for compounds 1 & 2 were obtained from ref. 12b.

^bData from Pfizer Global Research and Development Laboratories by A. W. Schmidt.

Both compounds 10 and 11 have only moderate to weak binding affinity at the D2-like receptors and do not meet the proposed criteria. Interestingly, compound 12, previously reported by Yevich, J. P. et al.¹⁷ with the 4-chlorophenyl ring in compound 4 replaced by 1,3-diazine moiety, met the four criteria set of interest (Table 3). The authors pharmacologically evaluated compound 12 and its ketone-reduced analog for their binding affinity for DAD2, sigma and cortical α₁ adrenergic receptors but not at the receptors of interest in this study. Thus, its synthesis provides an opportunity to compare the contributions of 4-chlorophenyl moiety in compound 4 with the 1,3-diazine ring in 12 to the binding affinities at the various receptors.

Table 3.

Comparative Binding Affinity Data for Compound 13 and Agents currently in use at Human Receptors and Rat Transporters.

a Receptor	Binding (Ki±SEM) Data of Compd 13 and standard agents at Selected Receptors (nM)b
	Comp 13	bClozapine	bRisperidone	bZiprasidone	bOlanzapine	bQuetiapine	bHaldol
	DA D1	162.7±33.1	290	580	130	52	1300	120
DA D2	43.3±13.3	130	2.2	3.1	20	180	1.4
DAD3	158.8±35.1	240	9.6	7.2	45	320	2.5
DAD4	6.6±0.6	54	8.5	32	50	2200	3.3
5HT1A	117.4±32.6	140	210	2.5	2100	230	3600
5HT2A	23.6±2.7	8.9	0.29	0.39	3.3	220	120
5HT2B	495.2±94.0	NR	NR	NR	NR	NR	NR
5HT2C	1425.0±207	17	10	0.72	10	1400	4700
5HT6	295.9±48.6	11	2000	76	10	1400	6000
Hist H1	188.6±16.0	1.8	19	47	2.8	8.7	440
M1	871.6±75.6	1.8	2800	5100	4.7	100	1600
DAT	1150±133	NR	NR	NR	NR	NR	NR
NET	850.7±175.0	390	28000	48	2000	680	5500
SERT	56.6±6.0	3900	1400	53	>15,000	>18,000	1800

^aHuman cloned receptors were used in all cases except for the NT transporters which are from rats.

^bData for standard drugs were obtained from Schmidt et al., reference 12b.

NR = Not Reported.

Compound 13, which can be viewed as a homopiperazine analog of haloperidol and compound 9, meets all the criteria except for its binding affinity at the 5HT_1A receptor. Compound 14, a bridged analog of compound 13, also showed low binding affinity for the receptors of interest (Table 2) and failed to meet the other stated criteria. Thus, it would appear that bridging does not necessarily result in favorable attributes for these compounds. Based on these observations, compound 13 was selected for further pharmacological evaluation.

Table 3 shows a head to head comparison of the binding affinities of compound 13 and several atypical antipsychotic drugs currently in use at several receptors known to have implications for the therapeutic value of atypical antipsychotic drugs. Compound 13 has moderate affinity for the DA D2 receptor and high affinity at the D4 receptor. The effect of compound 13 on apormophine-treated mice (see later) suggests D2 receptor antagonism and thus, would be expected to demonstrate antipsychotic properties in humans.¹⁸ 5HT_1A receptor activation has been suggested to contribute to the improved activity of certain atypical antipsychotic drugs.^12b,19

Compound 13 has moderate affinity for 5HT_1A (Ki = 117 nM). This binding affinity is higher than that for risperidone and quetiapine but similar to that of clozapine (Ki = 140 nM). Compound 13 also binds with high affinity at the 5HT_2A receptor (Ki = 23.6 nM), a receptor implicated in the therapeutic efficacy of atypical antipsychotic drugs in treating the negative symptoms of schizophrenia and preventing motor disturbances associated with antipsychotic treatment.²⁰ While this affinity is 10-fold better than that of quetiapine, other atypical antipsychotics have higher affinity for this receptor. 5HT₆ receptors are implicated in the amelioration of the cognitive disturbances in schizophrenia and both clozapine and olanzapine have high affinity for this receptor.²¹ While 13 has weak affinity for the 5HT₆ receptor (Ki = 295.8 nM), it is over 13-fold more potent at this receptor than quetiapine and over 6-fold better than risperidone.

Treatment of schizophrenia with atypical antipsychotic drugs has been associated with weight gain and the onset of type II diabetes mellitus. Two receptors, histamine H-1 and 5HT_2C, have been suggested to be involved in this adverse event. Several papers²² have demonstrated that there is significant correlation between affinity for H-1 receptor and weight gain and hence the observation that compound 13 has much lower affinity (Ki = 186 nM) for H1 receptor than risperidone (19 nM), quetiapine (8.7 nM), ziprasidone (47 nM), olanzapine (2.8 nM) and clozapine (1.8 nM) is an indication that compound 13 may have a lower propensity to induce weight gain. In addition, the low affinity of 13 for the 5HT_2C receptor (Ki = 1425 nM) compared to ziprasidone (Ki = 0.72 nM), risperidone (Ki = 10 nM) and olanzapine (Ki = 10 nM) suggests a possible decrease in the propensity of 13 for treatment-emergent weight gain. The observation that 5-HT_2B receptor agonists are associated with increased risk for valvular heart disease²³ will require that antipsychotics that interact with 5HT receptors be evaluated for binding to the 5HT_2B receptor. Thus, it is encouraging to note that compound 13 does not bind appreciably to 5HT_2B receptor (Ki = 495). Also encouraging is the observation that compound 13 lacks affinity for the cholinergic muscarinic M₃ receptor (Ki = 3000 nM), a receptor reported to have a contributing role in new-onset diabetes in schizophrenic patients treated with atypical antipsychotics.^12a

Compound 13 was also evaluated at neurotransmitter uptake transporters. Compound 13 has very weak affinity for both the dopamine (DAT) and norepinephrine (NET) transporters but has similar affinity for the serotonin transporter (SERT) (Ki = 56.6 nM) compared to aripiprazole (Ki = 98 nM).²⁴ This binding profile at the transporters suggests that while it has potential for antidepressant properties, it has a much lower potential to induce cocaine-like adverse properties since it has a very weak binding affinity for DAT.

Animal behavioral studies were initiated to provide preliminary evidence on efficacy and to evaluate the propensity of compound 13 to induce catalepsy. The ability to block apomorphine induced stereotypy is an indication of a drug's in vivo capacity to antagonize dopamine D2 receptors while induction of catalepsy is considered to be an indication of a drug's propensity to produce extrapyramidal symptoms in humans. The ED₅₀ values of compound 13, clozapine and haloperidol are estimated from the dose-response curves in Figs 1–3. The ED₅₀ value of compound 13 in inhibiting apomorphine-induced stereotypy was estimated to be 35.8 μmole/kg compared to clozapine's 42.4 μmole/kg suggesting that compound 13 is slightly more potent at blocking the D2 receptors in vivo than clozapine on a molar basis. This is also consistent with their estimated Ki values at the D2 receptor. The ED₅₀ values comparing compound 13, clozapine and haloperidol is reported in Table 4. Compound 13 and clozapine have ED₅₀s (μmole/kg) that are about 500 times that of haloperidol. The results of these behavioral studies support the radioligand binding data that compound 13 does not bind to D2 receptors as well as haloperidol, but does bind to D2 receptors with about the same affinity as clozapine.

Fig 1.

Fig 3.

Table 4.

Comparison of ED50 values for Apomorphine Test

Test Compound	FW	ED50, mg/kg	95% Confidence Interval, mg/kg	ED50, μmoles/kg
Compound 13	411.3	14.7	13.4 to 16.1	35.8
Clozapine	326.8	13.8	7.57 to 25.3	42.4
Haloperidol	375.9	0.0293	0.0112 to 0.0768	0.0779

Fig 4 depicts plots of mean catalepsy scores for compound 13 with clozapine and haloperidol as controls. The vehicle served as a negative control in the study. The ED₅₀ value for each compound was used as the basis for the dose used for the first set of comparative evaluations. It is clear that haloperidol showed the highest propensity to induce catalepsy while clozapine and compound 13 had no significant difference between them. The extent of the absence of catalepsy was evaluated by increasing the dose of compound 13 up to 5 times the ED₅₀ value at which point it was impossible to retain the drug in solution for intraperitoneal delivery. At 5× the ED₅₀ value, there was no appreciable cataleptogenic activity observed. Taken together, the combined receptor binding profile and the absence of catalepsy suggest that compound 13, a butyrophenone analogue which does not induce catalepsy up to 5× its ED₅₀ value may serve as an atypical antipsychotic agent.

Fig 4.

Experimental

Melting points were determined on a Gallenkamp (UK) apparatus and are uncorrected. NMR spectra were obtained on a Varian 300 MHz Mercury Spectrometer. Elemental analyses were carried out by Atlantic Microlab, Inc., Norcross, GA and are within 0.4% of theory unless otherwise noted. Flash chromatography was performed with Davisil grade 634 silica gel. N,N-Dimethylformamide was distilled from CaSO4 and stored over 4 Ǻ molecular sieves. 4-Chloro-4′-fluorobutyrophenone was obtained from Sigma-Aldrich but was purified by distillation under reduced pressure to a colorless liquid prior to use. Other starting materials were used without further purification.

Synthesis of compound 10

The synthesis of compound 10 followed the methods previously reported by Cignarella et al.¹⁵ and Thompson et. al.¹⁶ as depicted in Scheme 1.

Cis-2,5-dicarboethoxy-1-[4-chlorophenyl]pyrrolidine, 17

To a solution of (2S, 5R)-diethyl 2, 5-dibromo-hexanedioate (16) (10g, 27.8 mmol) in anhydrous DME (80 mL), was added 4-chloroaniline (14.2g, 111.1 mmol) and KI (1g, 6.02 mmol). The resulting mixture was refluxed under N₂ with stirring for 16 h. and allowed to cool to room temperature. Excess DME was removed under vacuum and the residue was triturated in Et₂O (100 mL) to yield a solid. The solid obtained was filtered, washed with Et₂O and the organic phase was treated with 100 mL of ice cold 5 N HCl and H₂O, dried over MgSO₄ and solvent removed in vacuo. The crude compound was purified by chromatography on silica gel (8:2 hexane :EtOAc) to give compound 17 (8.5g, 93.5%) as a mixture of trans-2,5-dicarboethoxy-1-[4-chlorophenyl]pyrrolidine and cis-2,5-dicarboethoxy-1-[4-chlorophenyl]pyrrolidine.

2-Benzylcarbamyl-5-carboethoxy-1-[4-chlorophenyl]pyrrolidine, 18

A mixture of cis- and trans-2,5-dicarboethoxy-1-[4-chlorophenyl]pyrolidine, 17 (8.5g, 26 mmol) was dissolved in 10 mL of o-xylene. Benzylamine (3.61 g, 33.74 mmol) was added to the reaction mixture and refluxed for 18 h. under N₂, cooled and solvent evaporated. The residue was subjected to column chromatography (silica gel) eluting with (1:1) hexane: EtOAc to give compound 18 (4.4 g, 39.8 %). ¹H NMR (300 MHz, CDCl₃) δ 1.19 (t, J = 7.13 Hz, 3H), 2.01-2.45 (m, 4H), 4.11-4.37 (m, 4H), 4.40-4.46 (dd, J = 6.0, 3.2 Hz, 2H), 6.36-6.44 (m, 2H), 7.09-7.24 (m, 7H), 8.52 (m, 1H).

3-Benzyl-8-[4-chlorophenyl]-3,8-diazabicyclo[3.2.1]octane-2,4-dione, 19

The solution of compound 18 (4.0 g, 10.35mmol) in 30 mL of 1N NaOH (75% aq. ethanol) was stirred at room temperature for 2 h. The reaction was quenched with HCl gas to the end point of phenolphthalein and the solid was filtered. The solvent was removed in vacuo and the solid material was re-dissolved in absolute alcohol. The solution was partially removed in vacuo and the residue was treated with Ac₂O (5 mL) and NaOAc (1 g). After refluxing overnight under N₂, the excess Ac₂O was removed and the reaction mixture was quenched with aq. Na₂CO₃, extracted with CH₂Cl₂ (3 × 100 mL) and dried over anhydrous MgSO₄. The solvent was removed in vacuo and separated on a silica gel column (7:3 hexane : EtOAc) to give compound 19 (2.56 g, 56.7%, white solid, mp 163-164 °C). ¹H NMR (270 MHz, CDCl₃) δ 2.04-2.12 (m, 2H), 2.46-2.55 (m, 2H), 4.63-4.66 (dd, J = 5.1, 2.7 Hz, 2H), 4.70 (s, 2H), 6.70-6.82 (m, 4H), 7.03-7.12 (m, 5H).

3-Benzyl-8-phenyl-3, 8-diazabicyclo[3.2.1]octane, 20

To a solution of compound 19 (0.58 g, 1.70 mmol) in anhydrous THF (3 mL), was added slowly, 1 M LiAlH₄ (10.24 mL, 10.24 mmol) and refluxed for 2 days under N₂. The reaction was quenched in ice bath with 10% NaOH (20 mL) and extracted with CH₂Cl₂. The organic phase was separated, dried over MgSO₄ and removed in vacuo. The residue was subjected to column chromatography (silica gel, 9:1 hexane:ethyl acetate) to give compound 20 (0.4 g, 84.6%, white crystals, mp 115-117 °C). ¹H NMR (270 MHz, CDCl₃) δ 1.87-2.08 (m, 4H), 2.51-2.52 (d, J = 2.0 Hz, 4H), 3.41 (s, 2H), 4.13-4.15 (m, 2H), 6.65-6.78 (m, 3H), 7.15-7.30 (m, 7H).

3-Carboethoxy-8-phenyl-3, 8-diazabicyclo[3.2.1]octane, 21

To a solution of compound 20 (0.77 g, 2.8 mmol) in anhydrous toluene (15 mL), was added ethyl chloroformate (1.6 mL, 16.6 mmol) and refluxed for 16 h under N₂. The resulting solution was allowed to cool to RT and excess solvent was removed under vacuum. The resulting residue was subjected to column chromatography (silica gel, 8:2 hexane:ethyl acetate) to give compound 21 (0.65 g; 90.5%) as white crystals, mp 124.4 °C. ¹H NMR (270 MHz, CDCl₃) δ 1.22 (t, J = 8.1 Hz, 3H), 1.79-1.84 (m, 2H), 1.98-2.04 (m, 2H), 3.29 (t, J = 13.5 Hz, 2H), 3.60-3.76 (dd, J = 13.5 Hz, 2H), 4.07-4.18 (m, 4H), 6.70-6.80 (m, 3H), 7.18-7.26 (m, 2H).

8-Phenyl-3,8-diazabicyclo[3.2.1]octane, 22

To a solution of compound 21 (0.73 g, 2.48 mmol) in EtOH (5 mL), was added KOH [3 g in H₂O (3 mL)], EtOH (25 mL) followed by NH₂NH₂ (3 mL). The resulting mixture was refluxed for 16 h. Excess alcohol was removed and the residue was extracted with EtOAc (4 × 50 mL) and H₂O (20 mL) to give compound 22 (0.4 g 86%). ¹H NMR (270 MHz, CDCl₃) δ 1.88-2.07 (m, 4H), 2.51-2.57 (dd, J = 13.5, 2.7 Hz, 2H), 3.19-3.24 (d, J = 13.5 Hz, 2H), 4.09 (br s, 2H), 6.67-6.78 (m, 3H), 7.17-7.24 (m, 2H).

8-Phenyl-3,8-diazabicyclo[3.2.1]octan-1-yl-1-(4-fluorophenyl)butan-1-one, 10

To a mixture of compound 22 (0.56 g, 2.96 mmol), K₂CO₃ (1.4g, 10 mmol) and KI (0.3 g) in DME (10 mL), was added 4-chloro-4-fluorobutyrophenone (2.0 g, 10.01 mmol) and stirred. The mixture was refluxed for 16 h under N₂ atmosphere and the reaction mixture was allowed to cool to room temperature. Excess DME was removed in vacuo and the residue was extracted with CH₂Cl₂ (3 × 50 mL) followed by brine (50 mL). The organic phase was collected and dried over anhydrous MgSO₄ and the solvent evaporated. The resulting crude product was subjected to column chromatography (silica gel, 8:2 EtOAc:MeOH) to give an oily residue (1.21 g, 68.2 %) and subsequently converted to an oxalate salt as a white solid, mp 194.7-195.5 °C. ¹H-NMR (270 MHz, CDCl₃) δ 1.89-1.99 (m, 6H), 2.39 (t, J = 6.8, 2H), 2.47-2.65 (m, 4H), 3.06 (t, J = 7.3, 2H), 4.21 (m, 2H), 6.73-6.85 (m, 3H), 7.17-7.33 (m, 4H), 8.05-8.10 (m, 2H). Anal. (C₂₄ H₂₆CFN₂O₅:H₂O) calcd. C 64.62, H 6.19, N 6.28. Found C 64.66, H 6.11, N 6.14.

4-[5-(4-Chloro-phenyl)-2,5-diaza-bicyclo[2.2.1]hept-2-yl]-1-(4-fluoro-phenyl)-butan-1-one, 11

A mixture of compound 23 (0.5g, 2.4 mmol), 4-chloro-4-fluorobutyrophenone (1.9g, 9.6 mmol) KI (50mg) and K₂CO₃ (138 mg) in DME (10 ml) was refluxed in an atmosphere of N₂ for 60 hours. Excess DME was removed under vacuum and residue was partitioned between CH₂Cl₂ (3 × 75 ml) and brine (50 ml). The organic phase was pooled, dried over anhydrous Na₂SO₄ and the solvent removed in vacuo to afford a residue as the crude product. The crude product was purified by column chromatography using 0 - 20% MeOH in EtOAc to yield compound 11 (755mg, 84.5%), which was subsequently converted to the oxalic acid salt as a white crystal, mp 116.4 -116.8 °C. ¹H-NMR (300 MHz, DMSOd₆) δ 1.86-1.93 (m, 4H), 2.09-2.21 (AB system, J=10.7, 2H), 3.00-3.37 (m, 6H), 3.11 (t, J=6.7, 2H), 3,37-3.58 (AB system, J=9.3, 2H). 4.42 (m, 1H), 4.60 (m, 1H), 6.65-6.68 (Part of AAXX, 2H), 7.32-7.38 (Part of AAXX, 2H), 8.00-8.04 (Part of AAXX, 2H). Anal. C₂₁H₂₂ClFN₂O.1.5C₂H₂O₄ calcd: C 56.73, H 4.96, N 5.52; Found: C 56.49, H 4.91, N 5.42

4-[4-(2-Pyrimidine)piperazin-1-yl]-1-(4-fluorophenyl)butan-1-one, 12

A mixture of 2-(piperazin-1-yl)pyrimidine dihydrochloride (0.5g, 2.1 mmol), 4-chloro-4′-fluorobutyrophenone (1.3 g, 6.3 mmol), KI (25 mg), K₂CO₃ (1.5 g, 10.9 mmol), in DME (10 mL) was refluxed with stirring under N₂ for 12 h. After cooling to room temperature, the mixture was diluted with EtOAc (200 mL) and washed with H₂O. The organic layer was dried (Na₂SO₄) and filtered. The filtrate was concentrated in vacuo to dryness and the residue was purified by chromatography on silica gel to give compound 12 (563 mg, 78%), mp 109 -110 °C. ¹H NMR (CDCl₃): 8.29 (2H, d, J = 4.5 Hz), 8.02 (2H, dd, J = 5.1, 8.7 Hz), 7.13 (2H, t, J = 8.7 Hz), 6.47 (1H, t, J = 4.5 Hz), 3.76 (4H, m), 3.01 (2H, t, J = 7.5 Hz), 2.46 (6H, m), 1.99 (2H, m). Calcd for C₁₈H₂₁FN₄O: C, 65.84; H, 6.45; N, 17.06. Found: C, 65.91; H, 6.51; N 16.86.

4-Chlorophenylboronic Acid, 26

To a solution of 4-bromochlorobenzene, 25 (4 g, 20.9 mmol) in THF (80 mL) was added n-BuLi (22 mL, 1.6 M) at −78 °C under N₂ and stirred for 1h at the same temperature. The reaction mixture was treated with B(OMe)₃ (398.8 ml, 78.3 mmol) at −78 °C, allowed to warm up to room temperature and then stirred overnight. The pH of the mixture was then adjusted with a solution of NaHCO₃ to pH 6.5 and extracted with CH₂Cl₂ (3 × 150 mL). The pooled organic phase was dried (MgSO₄), the solvent was removed in vacuo and the crude product was column chromatographed (silica gel, 7:3, hexane:EtOAc) to yield compound 26 (4-chlorophenyl-boronic acid) (2 g, 61 %) as a white solid. ¹H-NMR (270 MHz, CD₃OD) δ 7.30-7.77 (m, 4H).

1-t-Butyl homopiperazine carboxylate, 28

To a solution of homopiperazine, 27 (10g, 0.1 mol) in H₂O (104 mL) and t-BuOH (119 mL) containing 2.5 N NaOH (16 mL) was added di-tert-butyl dicarbonate (8.55 g, 0.039 mol) at 0 °C for 40 min. The resulting mixture was stirred for 1 h at room temperature and the solution was concentrated in vacuo. The crude material was extracted with CH₂Cl₂ (3 × 100 mL), dried over MgSO₄ and the solvent evaporated. The crude product was subjected to column chromatography (silica gel, 9:1:1 MeOH : CH₂Cl₂ : NH₄OH to give 1-t-Butyl homopiperazine carboxylate, 28 (5 g, 64 %). ¹H-NMR (270 MHz, CD₃OD) δ 1.46 (s, 9H), 1.79 (q, J=5.9, 2H), 2.78-2.87 (m, 4H), 3.41-3.49 (m, 4H).

1-t-Butyl-4-(4-chlorophenyl)homopiperazine carboxylate, 29

The mixture of 4-chlorophenyl-boronic Acid, 26 (1.65 g, 10.54 mmol), 28 (1.05g, 5.27 mmol), Cu(OAc)₂ (1.43 g, 7.87 mmol), triethylamine (0.726 mL) and molecular sieves (4 g) in CH₂Cl₂ (50 mL) was stirred at room temperature under air. The reaction was quenched with a 4 ml of NH₄OH in 4 mL of MeOH. The mixture was filtered through celite and purified by column chromatography using hexane:EtOAc (7:3) as eluent on silica gel to give compound 29 (1.2 g, 73%) ¹H-NMR (300 MHz, CDCl₃) δ 1.35 (s, 4H), 1.42 (s, 5H), 1.91-1.98 (m, 2H), 3.18 (t, 1H, J = 6.2 Hz), 3.29 (t, 1H, J = 6), 3.48-3.7 (m, 6H), 6.58 (d, 2H, J = 8.7 Hz), 7.12 (m, 2H, J = 9 Hz)

1-(4-Chlorophenyl)homopiperazine, 30

Trifluoroacetic acid (5 mL) was slowly added in a drop-wise manner to a solution of compound 29 in CH₂Cl₂ (5 mL) at ambient temperature and the resulting mixture was allowed to stir overnight. The reaction was quenched with aq. NaOH and extracted with EtOAc (3 × 100 mL), washed with NaHCO₃ solution (50 mL) and brine (50 mL). The organic phase was collected and dried over MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography (silica gel, 9:1:1 MeOH: CH₂Cl₂ :NH₄OH to yield 1-(4-chlorophenyl)homopiperazine, 30 (0.720 g, 88%). ¹H-NMR (300 MHz, CDCl₃) δ 1.92-1.98 (m, 2H), 2.86 (t, 2H, J = 5.7), 3.04 (t, J = 5.4 Hz), 3.53 (q, 4H, J = 6.4 Hz), 5.50 (br s, 1H), 6.58 (d, 2H, J = 9), 7.14 (d, 2H, J = 9.2 Hz).

4-[4-(4-Chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one, 13

A mixture of compound 30 (709 mg, 3.4 mmol) in dry DME (15 mL) and K₂CO₃ (1.9 g, 13.9 mmol) was heated for 20 min. after which 4-chloro-4-flourobutyrophenone (2.8 g, 13.9 mmol) was added followed by KI (0.3 g). The reaction mixture was refluxed for 18 h under an atmosphere of N₂. Excess DME was removed in vacuo and the residue was extracted with CH₂Cl₂ (3 × 100 mL) and brine (50 mL). The organic phase was collected and dried over MgSO₄ and solvent removed under vacuum. The crude product was subjected to column chromatography (silica gel, 9:1 EtOAc:MeOH) to yield compound 13 (1.19 g, 87%) as an oil. ¹H-NMR (300 MHz, CDCl₃) δ 1.84-1.95 (m, 4H), 2.53-2.62 (m, 4H), 2.75 (t, 2H, J = 5.1 Hz), 2.93 (t, 2H, J = 7.2 Hz) 3.39-3.48 (m, 4H), 6.58 (d, 2H, J = 9.6 Hz), 7.05-7.13 (m, 4H), 7.96-7.92 (dd, 2H, J = 9, 5.1 Hz). Subsequently, compound 13 was converted to the oxalate salt, crystallized from MeOH/Et₂O as a white solid, mp 146.6-146.4 °C. Anal. (C₂₃H₂₆ClFN₂O₂) calcd. C 59.42, H 5.64, N 6.03 found C 59.27, H 5.70, N 6.05.

Alternative Method for Compound 13

A mixture of homopiperazine (2 g, 20 mmol), CuI (380 mg, 2 mmol), K₂CO₃ (5.5 g, 40 mmol), ethylene glycol (2.4 g, 40 mmol), 1-chloro-4-iodobenzene (4.76 g, 20 mmol) in ⁱPrOH (50 mL) was refluxed for 12 h under N₂. The mixture was diluted with EtOAc (500 mL) and washed with water. The organic phase was dried (Na₂SO₄), and filtered. The filtrate was concentrated in vacuo to dryness followed by chromatography on silica gel (EtOAc then MeOH/EtOAc =10/90) to afford 1-(4-chlorophenyl)-1,4-diazepane (3.8 g, 90%).

A mixture of 1-(4-chlorophenyl)-1,4-diazepane (30) (540 mg, 2.57 mmol), 4-chloro-4-fluorobutyrophenone (2 g, 10.28 mmol), KI (25 mg), K₂CO₃ (880 mg, 6.38 mmol) in DME (20 mL) was refluxed under N₂ for 12 h. The mixture was diluted with EtOAc (500 mL) and washed with H₂O. The organic phase was dried (Na₂SO₄), and filtered. The filtrate was concentrated in vacuo to dryness followed by column chromatography on silica gel (pure EtOAc) to afford 4-[4-(4-chlorophenyl)-1, 4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one, 13. The product was converted to the salt form using ethereal HCl and then crystallized from MeOH/Et₂O to give the pure HCl salt (715 mg, 68%) mp 165-166 °C, ¹H NMR (DMSO): δ 10.48 (1H, brs), 8.04 (2H, dd, J = 5.7, 9.3 Hz), 7.35 (2H, t, J = 8.7 Hz), 7.19 (2H, d, J = 9.3 Hz), 7.76 (2H, d, J = 9.3 Hz), 3.78 (2H, m), 3.42 (4H, m), 3.15 (6H, m), 2.38 (1H, m), 2.15 (1H, m), 2.03 (2H, m). Calcd for C₂₁H₂₅Cl₂FN₂O: C, 61.32; H, 6.13; N, 6.81. Found: C, 61.20; H, 5.98; N, 6.78.

9-Methyl-3,9-diazabicyclo[4.2.1]nonan-4-one, 32

Using the method reported by Michaels and Zaugg,²⁵ conc. H₂SO₄ (37.5 mL) was added drop wise at −20 °C, to a mixture of tropinone, 31 (16.7g, 32 mmol) in anhydrous CHCl₃ (120 mL). This was followed by a slow addition of sodium azide (15.6 g) for 30 minutes while maintaining the temperature below −5 °C. After completing the addition, the reaction mixture was stirred at room temperature for 15 min, and then heated at 50 °C overnight under nitrogen. The reaction mixture was then neutralized with NaHCO₃, extracted with CH₂Cl₂, and the organic layer was collected, dried over Na₂SO₄ and evaporated in vacuo to afford a light yellowish substance as 32 (13.8 g, 78 %); MS: 155 (M+1)⁺, IR (KBr): 3190 (-NH), 1620 (C=O).

3,9-Diazabicyclo[4.2.1]nonane-3,9-dicarboxylic acid 3-tert-butyl ester 9-ethyl ester, 35

To a solution of compound 32 (15.2g, 30 mmol) in anhydrous THF (100 mL), was added very slowly under nitrogen powdered LAH (14.1 g, 8 eq). The mixture was allowed to stir at room temperature overnight before being quenched with MeOH, followed by H₂O, filtered, washed twice with CH₂Cl₂, extracted, dried over Na₂SO₄ and evaporated to yield 9-Methyl-3,9-diazabicyclo[4.2.1]nonane, compound 33 (11 g, 80 %). TLC analysis showed a single spot and was used as such without characterization. Compound 33 (12.0 g, 24 mmol) was dissolved in 15 mL of t-BuOH and added a solution of Boc-anhydride (5.8g, 28mmol) in t-BuOH (10 mL) for 5 min. and the reaction mixture was stirred overnight. The reaction was then neutralized with NaHCO₃, extracted with CH₂Cl₂, dried over Na₂SO₄ and evaporated in vacuo to yield 9-Methyl-3,9-diazabicyclo[4.2.1]nonane-3-carboxylic acid t-butyl ester, 34 (13.8 g, 67 %). Compound 34 (5.8 g, 32 mmol) was dissolved in anhydrous toluene (20 mL) and ethyl chloroformate (3.6 g, 33.3 mmol) was slowly added in a drop-wise manner. The reaction mixture was refluxed overnight and the solvent was evaporated as an azeotrope with alcohol. The resulting product, 3,9-diazabicyclo[4.2.1]nonane-3,9-dicarboxylic acid 3-tert-butyl ester 9-ethyl ester, 35 was dried and used as such without further purification for the next reaction.

3-(4-Chlorophenyl)-3,9-diazabicyclo[4.2.1]nonane-9-dicarboxylic acid ethyl ester, 37

Compound 35 (5.5 g, 18.4 mmol) was dissolved in CF₃COOH (3 mL) and stirred for 4h at room temperature. The reaction mixture was then neutralized with NaHCO₃, extracted with CH₂Cl₂, dried over Na₂SO₄ and evaporated to get a light yellowish oil, 3,9-diazabicyclo[4.2.1]nonane-9-carboxylic acid ethyl ester, 36 (2.85 g, 78 %). Compound 36 (5 g, 25.6 mmol), 4-chlorophenylboronic acid (5.6 g, 35.8 mmol), Cu(OAc)₂ (5.7g, 31.4 mmol) and Et₃N (2 mL) in CH₂Cl₂ (50 mL) with molecular sieves (2 g) was stirred in open air for 48 h. The reaction mixture was then quenched with methanolic NH₃ solution, filtered over celite, extracted with CH₂Cl₂ (100 mL), dried and solvent evaporated under vacuum. The crude residue was subjected to column chromatography (silica gel, 4:1 CH₂Cl₂:MeOH) to give a yellowish compound 37 (4.5 g, 58 %). ¹H NMR (300MHz, CDCl₃): δ 1.20-1.26 (m, 3H), 1.39-1.6 (m, 2H), 1.75-1.95 (m, 2H), 2.0-2.29 (m, 2H), 3.0-3.30 (m, 2H), 3.54-3.68 (m, 1H), 3.70-3.8 (m, 1H), 4.05-4.20 (m, 2H), 426-4.43 (m, 2H), 6.62-6.66 (dd, 2H, J = 7.8, 3.3 Hz), 7.06-7.12 (m, 2H).

3-(4-Chlorophenyl)-3,9-diazabicyclo[4.2.1]nonane, 38

Compound 37 (2.2 g, 7.12 mmol), 50 % aq. KOH (2 ml) in ethylene glycol (2 mL) was heated at 90 °C overnight. The reaction mixture was then extracted with CH₂Cl₂ (2 × 80 mL), dried over Na₂SO₄ and evaporated under vacuum. The crude product was subjected to column chromatography (silica gel, 4:2 CH₂Cl₂:MeOH) to yield compound 38 (0.99 g, 59 %). ¹H NMR (300MHz, CDCl₃): δ 1.32-1.76 (m, 4H), 1.90-2.25 (m, 4H), 3.15-3.35 (m, 4H), 6.63 (d, 2H, J = 9 Hz), 7.09 (d, 2H, J = 9 Hz).

4-[3-(4-Chlorophenyl)-3,9-diazabicyclo[4.2.1]non-9-yl]-1-(4-fluorophenyl)butan-1-one, 14

A mixture of compound 38 (0.45g, 1.9 mmol) and K₂CO₃ (1g, 7.6 mmol) in dry DME (15 mL) was heated at 80 °C for 20 minutes and 4-chloro-4-fluorobutyrophenone (2.2 ml, 7.6 mmol) was added in a drop-wise manner and refluxed overnight under nitrogen. Excess DME was removed in vacuo and the residue was extracted with CH₂Cl₂ (3 × 60 ml). The pooled organic phase was collected and dried over Na₂SO₄ and evaporated under vacuum. The crude product was subjected to column chromatography (silica gel, 4:1 CH₂Cl₂-EtOAc) to yield compound 4 (0.55 g, 68.2 %) as an oil which was converted into the oxalate salt. (White crystalline solid, mp:182-184 °C). ¹H NMR (300 MHz, CDCl₃): δ 1.20 -1.66 (m, 4H), 1.80-2.00 (m, 4H), 2.50-2.75 (m, 2H), 2.90-3.05 (m, 2H), 3.35-3.60 (m, 6H), 6.58 (m, 2H), 7.05-7.15 (m, 4H), 7.91-7.97 (m, 2H), Anal. Calcd. for C₂₅H₂₆N₂O₅ClF: 0.25H₂O: C, 60.61; H, 5.80; N, 5.65. Found C, 60.53; H, 5.75, N, 5.53.

Biology

Receptor Binding Studies

Binding affinities reported in Tables 1 – 3 were conducted by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH-PDSP) unless otherwise stated. Details of the methods and radioligands used for the binding assays were previously reported.²⁶

Apomorphine Induced Climbing Stereotypy

A modified climbing test by Needham et al. was used²⁷ and provides the extent a potential antipsychotic agent inhibits the effect of apomorphine induced stereotypy. Swiss Webster male mice (∼30 g) in groups of 5 were administered an intraperitoneal (IP) injection of 0.3 mL of vehicle (filtered 1% lactic acid) or increasing doses of the dopamine antagonists haloperidol (0.001, 0.01, 0.02, 0.05, 0.1, 0.2 mg/kg), compound 13 (12, 15, 17, 20, 25, 50, 80 mg/kg), and clozapine (3, 10, 20, 30, 60 mg/kg). Animals were then challenged at 30 minutes post injection with 1.5- 1.6 mg/kg of the agonist apomorphine in 0.9% NaCl + 0.1% ascorbic acid, placed in cylindrical wire cages (12 cm in diameter, 14 cm in height), and observed for climbing behavior at 10 and 20 minutes post dose. Climbing behavior was assessed as follows: 3-4 paws on the cage floor = 0 score; 2 and 3 paws on the cage = 1 score; 4 paws on the cage = 2 score. Scores were expressed as mean % climbing inhibition, and plotted in figures 1-3. ED50s in Table 4 were calculated using Graph Pad Prism non-linear regression software with sigmoidal dose-response, variable slope curve-fitting.

Bar Test for Catalepsy

The bar test used by Hoffman and Donovan²⁸ was modified for our use. Male Sprague-Dawley rats (88-207 g) in groups of 5-8 were dosed by IP injection with 1-23 mL/kg of vehicle (filtered 1% lactic acid), haloperidol (0.3 mg/kg), compound 13 (28, 42, 70 mg/kg), or clozapine (30 mg/kg). Catalepsy severity was assessed at time points (15, 30, 45, 60, and 90 min) post injection, by scoring how long the rat maintained both forepaws motionless on a horizontal metal bar (1.1 cm in diameter, 10 cm above the bench top in a box) up to a maximum of 120 seconds. This was repeated 2 more times and the average (n=3 tries) seconds recorded for each rat. The mean of all the average seconds for one dose for one time point (n=5-8 rats) was divided by 5 and a mean catalepsy score assigned and plotted in figure 4 with a maximum possible score of 24.

Fig 2.

Scheme 6^a.

^a Reagents: (i) H₂SO₄, NaN₃; (ii) LiAlH₄, THF; (iii) t-BuOH, (t-BuOCO)₂O; (iv) ClCO₂Et, Toluene; (v) TFA (vi) 4-Chlorophenylboronic Acid, Cu(OAc)₂, Et₃N, CH₂Cl₂, Mol sieves (4Å), RT, 24 – 48 hr; (vii) KOH, Ethylene glycol; (viii) 4-Chloro-4′-fluorobutyrophenone, KI, K₂CO₃, DME.