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The purpose of the study is to characterize modified 5-FU (MFU) and investigate its anticancer activity in liposomal nanoparticles (MFU-Lnps) against colorectal and pancreatic cancer cell lines. Methods: The synthesized MFU was characterized by NMR and elemental analysis and formulated as MFU-Lnps. The particle size, polydispersity index (PDI), and zeta potential were determined using a particle size analyzer. The in-vitro studies were performed on 2D and 3D models of HCT-116 and MiaPaCa-2 cell lines. Cellular uptake of rhodamine (Rho) (cationic dye) alone or Rho-linked-Lnps (Rho-Lnp) was determined by confocal fluorescence microscopy. In-vitro release of MFU from MFU-Lnps was performed using PBS and methanol (3:1) at 37° C under sink conditions. To evaluate the pharmacokinetics (PK) parameters and tissue biodistribution behavior of Lnp, Lnp was loaded with gadolinium-hexanoate (Gad-Hex-Lnps), and the quantified by inductively coupled plasma mass spectrometry (ICP-MS). Results: The 1H NMR peaks at 1.02 ppm (-C-N on 2') and 1.04 ppm (-C-N on 2''), represent the amine bond between 4-NH2 group of 5-FU and tetrahydrofuran acetate. This confirmed that the MFU was successfully synthesized. The elemental analysis of MFU showed 99.5% purity by comparing observed and theoretical values (Observed: carbon (C) = 53.57 %, hydrogen (H)= 5.64 %, and nitrogen (N) =10.44 % and, theoretical: C=53.33%, H=5.59%, and N=10.37 %). The MFU-Lnps hydrodynamic diameter was 124.9 ± 3.2 nm, polydispersity was found to be 0.16 ± 0.005, while entrapment efficiency and zeta potential values were 97.2% and -30.3 ± 12 mV, respectively. The MFU treated-2D and 3D MiaPaCa-2 cultures (IC50 values: 3.9 ± 0.9 µM2D and 6.7±1.1µM3D) was more effective than 5-FU-treated 2D and 3D MiaPaCa-2 cultures (IC50 values: 5.4 ± 0.8 µM2D and 8.5 ± 1.0 µM3D). However, MFU-Lnps treated-MiaPaCa-2 3D-organoid was 4-fold more effective compared with MFU (IC50 values: 9.8 ± 1.4 µMMFU-Lnps versus 42.3 ± 2.9 µMMFU). A similar high cytotoxicity effect of MFU-Lnp was observed when MFU-Lnp (IC50 values: 2.0 ± 0.7 µM2D and 3.4 ±0.8 µM3D) was exposed to HCT-116 2D and 3D cultures compared to MFU (3.1± 0.8µM2D and 6.8 ± 0.9 µM3D). For cellular uptake, we observed an increase in the uptake of Rho-linked-Lnps compared with Rho alone. The MFU release MFU-Lnps exhibited Higuchi release kinetics model based on the coefficient of determination (R2). For PK results, the half-life of Gad-Hex-Lnps (32.5 ± 2.6 min) was significantly high compared to that of Gad-Hex (21.5 ± 0.7 min) and, AUC of Gad-Hex-Lnps (1641 ± 499 µg/L*min) was higher than Gad-Hex (932 ± 256 µg/L*min). The accumulation of Gad-Hex-Lnps in the pancreas was remarkably higher than Gad-Hex. In conclusion, we have demonstrated that MFU-Lnps improves the anticancer efficacy of 5-FU and Lnps delivery system may improve PK parameters and increases the biodistribution of 5-FU. Citation Format: Raviteja Bulusu, Nkafu B. Ndemazie, Esther K. Frimpong, Andriana Inkoom, Joy C. Okoro, Xue Y. Zhu. Characterization and evaluation of modified 5-fluorouracil loaded liposomal nanoparticle on pancreatic and colorectal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 839.

Our previous study has revealed 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one·2HCl (SYA013) 1 as a sigma ligand with moderate selectivity for the sigma-2 receptor. Given the overexpression of sigma receptors in solid tumors and reports of sigma ligands with anticancer activities, we selected 1 for evaluation in several solid tumor cell lines. In addition, we have synthesized new analogs of 1 and now report that several of them bind preferentially at the sigma-2 receptor and have shown inhibition of several cancer cell lines including MDA-MB-231, MDA-MB-486, A549, PC-3, MIA PaCa-2 and Panc-1 cells. In particular, compounds 1 and 12 have demonstrated sub-micromolar activity against the Panc-1 cell line. It has also been observed that several of these compounds demonstrate selective toxicity toward cancer cells, when compared to normal cells.

Several lines of evidence suggest that selective sigma-2 (σ2) ligands might be useful for the treatment of solid tumors. However, very few selective σ2 ligands have been identified. This study was aimed at identifying new selective σ2 receptor ligands using a previously identified agent, SYA 013 as a lead. Four groups, homopiperazine, piperazine, tropane and selected oxime analogs of the homopiperazines were identified, synthesized and subsequently screened at the σ1 and σ2 receptors. The results demonstrate that these scaffolds can be modified to obtain selective σ2 receptor ligands. 1-(5-Chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane, 7 and 3-(4-chlorophenyl)-8-(3-((2-fluorophenyl)thio)propyl)-8-azabicyclo[3.2.1]octan-3-ol, 21 were identified as the highest binding affinity ligands (σ2Ki = 2.2 nM) and (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)-butan-1-one oxime, 22 as a high affinity and the most selective ligand for the σ2 receptor (σ1Ki/σ2Ki = 41.8).

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive and scourging soft tissue tumor worldwide. Its current incidence is 13 per 100,000 US population with a very low 5-years survival rate (5%) and estimated to be the second leading cause of cancer-related death by 2030. Most diagnoses are made at advanced disease states warranting combination chemotherapy. Fluorouracil (5-FU) is an effective drug in treating PDAC; however, rapid degradation and systemic instability remain a drawback to its efficacy. The objective of this study was to chemically modify 5-FU to 1,3 bistetrahydrofuran-2yl-5-FU (MFU) to prolong its systemic stability and enhance the anticancer activity of 5-FU. Method: 5-FU was chemically modified to MFU using tetrahydrofuran-2-yl acetate and 1,8-Diazabicyclo (5.4. 0)undec-7-ene (DBU) in Dimethyl Formamide (DMF). MFU was characterized using nuclear magnetic resonance (NMR) to determine new bond formation, mass spectrometer (MS) for molecular weight determination, high-performance liquid chromatography (HPLC) to determine percent purity, and micro-elemental analysis used to ascertain the presence of elemental composition and purity. Percent conversion of MFU to 5FU was determined using PDAC cells. In vitro anticancer activity of MFU was tested using 2D and 3D MiaPaCa-2 and PANC-1 PDAC cultures and cell viability study performed using the alamar blue assay. Apoptotic and cell cycle studies were performed on MFU treated MiaPaCa-2 and PANC-1 cells using flow cytometry. Results: Molecular weight of synthesized MFU (C12H15FN2O4) was determined to be 270.02 using the Electrospray Ionization (ESI) (MS (M+H)+ = 270.08). While percent purity of MFU was found to be greater than 99.6%, melting point was determined to be 115±2 °C and retention time (RT) was 1.85 minutes. For MFU conversion to 5-FU studies, 4.1% of 25µM and 7.6% of 50µM of MFU incubated MiaPaCa-2 cells was converted to 5-FU over a period of 24 hr showing an incremental production of 5-FU from MFU based on concentration. For in vitro study, half-maximal inhibitory concentration (IC50) of 5-FU treated 2D MiaPaCa-2 culture was 3.4 ±1.1µM while that of MFU treated 2D MiaPaCa-2 culture was 2.1± 0.2 µM. The IC50 value of 5-FU treated 3D MiaPaCa-2 culture was found to be (8.5±1.2µM), while IC50 value for MFU treated 3D MiaPaCa-2 culture was (7.2±1.1µM). Put together, IC50 value for MFU for either 2D or 3D was significantly lower compared with IC50 value 5-FU (P=0.021 (MFU vs 5-FU) for 2D and P=0.04 (MFU vs 5-FU) for 3D). A similar trend was noted when PANC-1 cells were used. Apoptotic and cell cycle studies were similar to that of the 5-FU with most of the cells (54%) in late apoptosis after 48 h of treatment and 64% vs 33% of cells arrested at the G1 and S phase of the cell cycle respectively. Conclusion: The studies demonstrated that MFU may be an alternate approach to improve the delivery, systemic stability, and efficacy of 5-FU in the treatment of PDAC tumors. Citation Format: Nkafu Bechem Ndemazie, Andriana Inkoom, Xue Y. Zhu, Edward Agyare. Optimizing the efficacy of 5-FU as a chemotherapeutic agent in advanced pancreatic ductal adenocarcinoma (PDAC) using MIAPaCa-2 and PANC-1 cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-047.

Diseases of the CNS are often complex and involve multiple receptor systems and thus, the treatment options for these diseases must focus on targeting the multiple receptors implicated in the various disorders. Schizophrenia and depression are examples of such diseases and their pharmacotherapy thus depends on agents which target multiple receptors including the dopamine, serotonin and even cholinergic receptors at the same time. In our previous campaign to find multi-receptor ligands, we have identified the benzothiazole 1a as an initial lead molecule. In the current work, we have expanded the structure affinity relationship (SAFIR) of 1a resulting in the identification of a partially restrained butyrophenone 3j as a potent and selective dual 5-HT1A and 5-HT7 receptor ligand. It is expected that compound 3j may serve as a new lead for further development in our search for newer and novel ligands with the potential to treat diseases of CNS origin.

Several known D2 pharmacophores have been explored as templates for identifying ligands with multiple binding affinities at dopamine and serotonin receptors considered as clinically relevant receptors in the treatment of neuropsychiatric diseases. This approach has resulted in the identification of ligands that target multiple CNS receptors while avoiding others associated with deleterious effects. In particular, compounds 11, 15 and 22 may have potential for further development as antipsychotic agents as they favorably interact with the clinically relevant receptors including D2R, 5-HT1AR, and 5-HT7R. We have also identified the pair of compounds 11 and 10 as high affinity D2R ligands with and without SERT binding affinities, respectively. These differential binding profiles endow the pair with the potential for evaluating SERT contributions to antipsychotic drug activity in animal behavioral models. In addition, compound 11 has no significant affinity for 5-HT2CR and binds only moderately to the H1R, suggesting it may not induce weight gain or sedation when used clinically. Taken together, compound 11 displays an interesting pharmacological profile that necessitates the evaluation of its functional and in vivo effects in animal models which are currently ongoing.

Several lines of evidence suggest that selective sigma-2 (σ(2)) ligands might be useful for the treatment of solid tumors. However, very few selective σ(2) ligands have been identified. This study was aimed at identifying new selective σ(2) receptor ligands using a previously identified agent, SYA 013 as a lead. Four groups, homopiperazine, piperazine, tropane and selected oxime analogs of the homopiperazines were identified, synthesized and subsequently screened at the σ(1) and σ(2) receptors. The results demonstrate that these scaffolds can be modified to obtain selective σ(2) receptor ligands. 1-(5-Chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane, 7 and 3-(4-chlorophenyl)-8-(3-((2-fluorophenyl)thio)propyl)-8-azabicyclo[3.2.1]octan-3-ol, 21 were identified as the highest binding affinity ligands (σ(2)Ki=2.2 nM) and (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)-butan-1-one oxime, 22 as a high affinity and the most selective ligand for the σ(2) receptor (σ(1)Ki/σ(2)Ki = 41.8).

The 3-dimensional quantitative structure-activity relationship (3D-QSAR) molecular modeling technique or comparative molecular field analysis (CoMFA) has been used to design analogs of the natural product cryptolepine (1). Twenty-three compounds with their in vitro biological activities (IC50 values) against Crytococcus neoformans were used to generate the training set database of compounds for the CoMFA studies. The cross-validated q(2), noncross-validated r(2), and partial least squares (PLS) analysis results were used to predict the biological activity of 11 newly designed test set compounds. The best CoMFA model produced a q(2) of 0.815 and an r(2) of 0.976 indicating high statistical significance as a predictive model. The steric and electrostatic contributions from the contour map were interpreted from the color-coded contour plots generated from the PLS model and the active structural components for potency against C. neoformans were determined and validated in the test set compounds. The 3-substituted benzylthio quinolinium salts (4) that make up the test set were synthesized and evaluated based on the predicted activity from the CoMFA model and the results produced a good correlation between the predicted and experimental activity (R=0.82). Thus, CoMFA has served as an effective tool to aid the design of new analogs and in this case, it has aided the identification of compounds equipotent with amphotericin B, the gold standard in antifungal drug design.

5-HT1A and 5-HT7 receptors have been at the center of discussions recently due in part to their major role in the etiology of major central nervous system diseases such as depression, sleep disorders, and schizophrenia. As part of our search to identify dual targeting ligands for these receptors, we have carried out a systematic modification of a selective 5HT7 receptor ligand culminating in the identification of several dual 5-HT1A and 5-HT7 receptor ligands. Compound 16, a butyrophenone derivative of tetrahydroisoquinoline (THIQ), was identified as the most potent agent with low nanomolar binding affinities to both receptors. Interestingly, compound 16 also displayed moderate affinity to other clinically relevant dopamine receptors. Thus, it is anticipated that compound 16 may serve as a lead for further exploitation in our quest to identify new ligands with the potential to treat diseases of CNS origin.

Using haloperidol as a scaffold, new agents were designed to investigate the structural contributions of various groups to binding at CNS receptors associated with atypical antipsychotic pharmacology. It is clear that each pharmacophoric group, the butyrophenone, the piperidine and the 4-chlorophenyl moieties contributes to changes in binding to the receptors of interest. This strategy has resulted in the identification of several new agents, compounds 16, 18, 19, 23, 24 and 25, with binding profiles which satisfy our stated criteria for agents to act as potential atypical antipsychotics. This research demonstrates that haloperidol can serve as a useful lead in the identification and design of new agents that target multiple receptors associated with antipsychotic pharmacology.

Ring-opened benzothieno[3,2-b]quinolinium salts (3) were designed and synthesized with substitution on the thiophene moiety. In vitro screenings were carried out against fungal pathogens including Cryptococcus neoformans, Candida albicans, Candida glabrata, Candida krusei and Aspergillus fumigatus. In all, by replacing the N-methyl group (2) with N-ω-phenylpentyl or ω-cyclohexylpentyl group to form substituted 3-(phenylthio)quinolinium compounds produced remarkable potencies, as high as 300-fold (cf, cryptolepine (1) = 250 μg/mL vs 11 p = 0.8 μg/mL for C. albicans) over the starting tetracyclic parent. In addition, all the N-ω-cyclohexylpentyl analogs produced superior activity against all the microorganisms tested than the N-ω-phenylpentyl substituted compounds. The potential of these compounds to induce toxicity in Vero cells was also investigated and the majority of them showed lower or no cytotoxicity at 10 μg/mL than amphotericin B, the gold standard in antifungal drug development. For instance, the trifluoromethyl substituted analogs (11n-p) have selectivity indices over 2-fold better than those of amphotericin B in C. neoformans. Overall, this ring-opened scafford of benzothienoquinolines, with substitution on the thiophenyl moiety, serves as a new lead for further development.

Several N-substituted quindolines were made to further evaluate the role of N-alkylation on the activity of indoloquinolines as antifungal agents. While N-5 substitution is required for these activities, N-10 alkylation alone leads to inactive products but is tolerated in the presence of N-5 alkyl groups. It was also discovered that bis-quindolines appear to have a more expanded antimicrobial spectrum and lower cytotoxicity than their monomeric counterparts.

An advanced pentacyclic intermediate, amenable to further elaboration into the target molecules simalikalactone D and quassimarin, has been synthesized from (S)-(+)-carvone in 21 steps and with an overall yield of 12 %. The synthesis is efficient, stereocontrolled, enantiospecific, and chirality productive, creating eight new chiral centres in pentacycle, and should provide opportunities for rapid access to simalikalactone D analogues and other bioactive quassinoids. The reaction sequence involves a regioselective bishydroxylmethylation, a stereocontrolled epoxidation, an epoxymethano-bridge formation, a 1,3-sigmatropic rearrangement and an intramolecular Diels-Alder reaction as the key steps.

Previous work from our labs has indicated that a tropane analog of haloperidol with potent D2 binding but designed to avoid the formation of MPP(+)-like metabolites, such as 4-(4-chlorophenyl)-1-(4-(4-fluorophenyl)-4-oxobutyl)pyridin-1-ium (BCPP(+)) still produced catalepsy, suggesting a strong role for the D2 receptor in the production of catalepsy in rats, and hence EPS in humans. This study tested the hypothesis that further modifications of the tropane analog to produce compounds with less potent binding to the D2 receptor than haloperidol, would produce less catalepsy. These tests have now revealed that while haloperidol produced maximum catalepsy, these compounds produced moderate to low levels of catalepsy. Compound 9, with the least binding affinity to the D2R, produced the least catalepsy and highest Minimum Adverse Effective Dose (MAED) of the analogs tested regardless of their affinities at other receptors including the 5-HT1AR. These observations support the hypothesis that moderation of the D2 binding of the tropane analogs could reduce catalepsy potential in rats and consequently EPS in man.

The dopamine D4 receptor has been shown to play key roles in certain CNS pathologies including addiction to cigarette smoking. Thus, selective D4 ligands may be useful in treating some of these conditions. Previous studies in our laboratory have indicated that the piperazine analog of haloperidol exhibits selective and increased affinity to the DAD4 receptor subtype, in comparison to its piperidine analog. This led to further exploration of the piperazine moiety to identify new agents that are selective at the D4 receptor. Compound 27 (KiD4 = 0.84 nM) was the most potent of the compounds tested. However, it only had moderate selectivity for the D4 receptor. Compound 28 (KiD4 = 3.9 nM) while not as potent, was more discriminatory for the D4 receptor subtype. In fact, compound 28 has little or no binding affinity to any of the other four DA receptor subtypes. In addition, of the 23 CNS receptors evaluated, only two, 5HT1AR and 5HT2BR, have binding affinity constants better than 100 nM (Ki < 100 nM). Compound 28 is a potentially useful D4-selective ligand for probing disease treatments involving the D4 receptor, such as assisting smoking cessation, reversing cognitive deficits in schizophrenia and treating erectile dysfunction. Thus, further optimization, functional characterization and evaluation in animal models may be warranted.

Opportunistic infections are devastating to immunocompromised patients. And in especially sub-Saharan Africa where the AIDS epidemic is still raging, the mortality rate was recently as high as 70%. The paucity of anti-opportunistic drugs, the decreasing efficacy and the development of resistance against the azoles and even amphotericin B have stimulated the search for new drugs with new mechanisms of action. In a previous work, we showed that a new chemotype derived from the natural product cryptolepine displayed selective toxicity against opportunistic pathogens with minimal cytotoxicity to normal cells. In this manuscript, we report the design and synthesis of substituted benzylthioquinolinium iodides, evaluated their anti-infective properties and formulated some initial structure-activity relationships around phenyl ring A from the original natural product. The sensitivity of the most potent analog 10l, to selected strains of C. cerevisiae was also evaluated leading to the observation that this scaffold may have a different mode of action from its predecessor, cryptolepine.

The CE ring 15 of the pentacyclic quassinoid skeleton is fabricated from (S)-carvone involving regioselective bishydroxylmethylation, acetonation, stereocontrolled epoxidation and epoxymethano-bridge formation, selective protection, deprotection, and oxidation.

The synthesis and evaluation of several benzothiazole-based compounds are described in an attempt to identify novel dual-acting 5HT(1A) receptor and SERT inhibitors as new antidepressants. Binding affinities at the 5HT(1A) receptor and the serotonin transporter do not appear to be congruent and other areas of the binding sites would need to be explored in order to improve binding simultaneously at both sites. Compounds 20 and 23 show moderate binding affinity at the 5HT(1A) receptor and the SERT site and thus, have the potential to be further explored as dual-acting agents. In addition, compound 20 binds with low affinity to the dopamine transporter (DAT), the norepinephrine transporter (NET) and 5HT(2C) receptor, which are desirable properties as selectivity for SERT (and not DAT or NET) is associated with an absence of cardiovascular side effects.

Structure-activity relationship studies on 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one (SYA 013), a homopiperazine analog of haloperidol has resulted in an understanding of the effect of structural modifications on binding affinity at dopamine and serotonin receptor subtypes. Further exploration, using bioisosteric replacement strategies has led to the identification of several new agents including compounds 7, 8, 11 and 12 which satisfy the initial criteria for further exploration as new antipsychotic agents. In addition, compound 18, a D(3) selective tropanol, has been identified as having the potential for further optimization into a useful drug which may combat neuropsychiatric diseases.

SYA013, a homopiperazine analog of haloperidol, was further evaluated for antipsychotic potential using additional animal models. Previously, SYA013 was tested in mice with an antipsychotic screening model in which it inhibited apomorphine induced climbing behavior, indicating antagonism of the dopaminergic system and the potential for use in the treatment of schizophrenia. In this study, SYA013 was shown to inhibit both d -amphetamine-induced locomotor activity in rats and conditioned avoidance response (CAR) in rats in a dose dependent manner and in the case of CAR, without producing any escape failure responses (EFRs), two tests predictive of antipsychotic action. The selective 5HT 1A antagonist WAY100,635 was used to determine if binding of SYA013 to the 5HT 1A receptor contributed to suppression of CAR. The results indicated that 0.63 mg/kg WAY100,635 did not have a significant effect on the inhibition of CAR by SYA013. Pharmacokinetic parameters in brain and plasma were determined for SYA013. A log brain/plasma concentration ratio at a t max of 1.48 suggests that SYA013 readily crosses the blood brain barrier (BBB). The hypothesis that binding of SYA013 to the 5HT 1A receptor contributed to the lack of significant catalepsy was investigated using the 5HT 1A antagonist WAY100,635. The results of acute and semi-chronic tests suggest that binding to the 5HT 1A receptor alone did not significantly account for the lack of catalepsy. Lack of catalepsy was preserved after the semi-chronic challenge with SYA013. These tests further indicate that SYA013 has a pharmacological profile with the potential for use in the treatment of neuropsychiatric diseases. In addition, the 5HT 1A receptor does not appear to play a significant role in the pharmacological profile of SYA013.

Previous studies on the indoloquinoline alkaloid, cryptolepine (2), revealed that it has antii-nfective properties among other activities. Using Structure-activity relationship (SAR) techniques, several ring-opened analogs of cryptolepine (3-phenylaminopyridinium and 3-phenylaminoquinolinium derivatives) were designed to improve the potency and lower the cytotoxicity shown by several of the precursor agents. Results indicate that these ring-opened analogs constitute new anti-infective agents with over a 100-fold potency and several fold lower cytotoxicity than cryptolepine from which they are derived.

Substitution around 5-methyl benzothieno[3,2-b]quinolinium (2) ring system was explored in order to identify positions of substitution that could improve its antifungal profile. The 3-methoxy (10b) was active against C. albicans, C. neoformans, and A. fumigatus and the 4-chloro (10f) analog showed moderate increases in anti-cryptococcal and anti-aspergillus activities. The effectiveness of 10b and 10f were validated in murine models of candidiasis and cryptococcosis, respectively. The efficacy of 10f in reducing brain cryptococcal infection and its observation in the brain of mice injected with this quaternary compound confirm the capacity of these compounds to cross the blood-brain barrier of mice. Overall, several of the chloro and methoxy substituted compounds showed significant improvements in activity against A. fumigatus, the fungal pathogen prevalent in patients receiving organ transplant. Opening the benzothiophene ring of 2 to form 1-(5-cyclohexylpentyl)-3-(phenylthio)quinolinium compound (3) resulted in the identification of several novel compounds with over 50-fold increases in potency (cf. 2) while retaining low cytotoxicities. Thus, compound 3 constitutes a new scaffold for development of drugs against opportunistic infections.

The CE ring 15 of the pentacyclic quassinoid skeleton is fabricated from (S)-carvone involving regioselective bishydroxylmethylation, acetonation, stereocontrolled epoxidation and epoxymethano-bridge formation, selective protection, deprotection, and oxidation.

The pentacyclic quassinoid skeleton 3 is constructed from (S)-carvone involving a 1,3-sigmatropic rearrangement and an intramolecular Diels–Alder reaction as the key steps.

Arylcycloalkylamines, such as phenyl piperidines and piperazines and their arylalkyl substituents, constitute pharmacophoric groups exemplified in several antipsychotic agents. A review of previous reports indicates that arylalkyl substituents can improve the potency and selectivity of the binding affinity at D(2)-like receptors. In this paper, we explored the contributions of two key pharmacophoric groups, that is, 4'-fluorobutyrophenones and 3-methyl-7-azaindoles, to the potency and selectivity of synthesized agents at D(2)-like receptors. Preliminary observation of binding affinities indicates that there is little predictability of specific effects of the arylalkyl moieties but the composite structure is responsible for selectivity and potency at these receptors.

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. Although compounds 5 and 6 have high affinity for 5HT1A and 5HT2A 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 5HT1A and 5HT2A 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 5HT2C 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 (5HT1A and 5HT2A) since binding to these receptors is often associated with therapeutic advantages observed in atypical antipsychotic drugs.12 In addition, evaluation of the compounds at 5HT2C 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: Binding to DA D2 receptor with moderate affinity (30 < Ki < 150 nM) Binding to DA D4 receptor with high affinity (Ki < 10 nM) Binding to 5HT1A and 5HT2A receptors with high affinity (Ki < 50 nM) Binding to 5HT2C 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

Isosteres of cryptolepine (1) were synthesized and evaluated for their antiinfective activities. Overall, the sulfur isostere, 5-methyl benzothieno[3,2-b]quinolinium salt (5b), was equipotent to 1 and has shown no cytotoxicity at 23.8 microg/mL. Compound 5b was also found to have a broad spectrum of activity. Both the carbon and oxygen isosteres were less potent than cryptolepine. A limited library of 2-substituted analogs of 5b has been synthesized and evaluated in antifungal screens but did not show increase in potency compared to the unsubstituted 5b. Similarly, evaluation of tricyclic benzothieno[3,2-b]pyridines while showing promise in individual screens did not produce an overall increase in potency. Overall, the evaluation of the activities of 5b compared with standard antifungal/anti-protozoal agents suggests that the benzothienoquinoline scaffold could serve as a lead for optimization.

The pentacyclic quassinoid skeleton 3 is constructed from (S)-carvone involving a 1,3-sigmatropic rearrangement and an intramolecular Diels–Alder reaction as the key steps.