Synthesis of Amino Acid-Conjugated Olanzapine Prodrugs: Advancing Stability, Selectivity, and Therapeutic Efficacy in Psychotic Disorders

Psychosis represents a severe psychiatric condition characterized by a profound disconnection from reality, often manifesting through hallucinations—perceptions of nonexistent phenomena—and delusions, which are firmly held but erroneous beliefs or interpretations [1]. This condition significantly disrupts cognitive functions such as reasoning, perception, and emotional regulation, there by impairing social and occupational performance. Psychosis is associated with a range of psychiatric disorders, including schizophrenia, bipolar disorder, and severe depressive episodes, and may also arise due to substance misuse or neurological conditions impacting the brain [2]. It predominantly affects young adults, with approximately 3 in 100 individuals experiencing a psychotic episode during their lifetime. Encouragingly, the majority of affected individuals achieve full recovery, as psychosis is both treatable and manageable. Antipsychotic agents are predominantly employed within psychiatry to address psychosis, encompassing symptoms such as delusions and hallucinations, particularly in disorders like schizophrenia and bipolar disorder. Both first-generation and second-generation antipsychotics generally exert their effects by antagonizing receptors within the brain's dopaminergic pathways, although they often interact with a broader spectrum of receptor types. The inhibition constant (Ki) at membrane-bound receptors serves as a pivotal determinant of these drugs' pharmacokinetic profiles, which directly modulate their therapeutic antipsychotic efficacy. This investigation analysed predicted Ki values for 71 clinically approved antipsychotics, alongside several novel chemical entities exhibiting antipsychotic potential, employing 3D-QSAR–CoMSIA modelling techniques. The models demonstrated robust predictive reliability, achieving cross-validated correlation coefficients (q²) exceeding 0.70 and fitted correlation coefficients (r²) surpassing 0.80. These findings underscore the substantial biological affinity of 15 novel risperidone derivatives and 12 newly synthesized olanzapine derivatives for dopamine D2 and serotonin 5HT2A receptors, highlighting their potential for further detailed exploration. Olanzapine is a pharmaceutical agent employed in the management of mental health disorders in adolescents and adults aged 13 years and above. Classified as an “atypical antipsychotic,” its mechanism of action involves modulation of the activity of specific endogenous substances within the brain. It is predominantly prescribed for the treatment of schizophrenia and various bipolar disorders. Notably, a highly significant intronic single nucleotide polymorphism (SNP), designated as rs472660, within the *CYP3A43* gene—encoding a cytochrome P450 enzyme—has been identified as a robust predictor of enhanced olanzapine clearance. Despite these insights, the precise biochemical pathways underpinning the drug’s efficacy remain incompletely elucidated. Olanzapine operates by antagonizing dopamine and serotonin receptor sites in the brain, with its therapeutic and adverse effects further influenced by its antihistaminic, anticholinergic, and alpha-adrenergic blocking properties. These pharmacodynamic features contribute to its reduced impact on motor coordination and movement regulation compared to conventional antipsychotics.

A prodrug is a pharmacologically inactive or minimally active compound that undergoes chemical or enzymatic transformation within the biological system to release its active therapeutic form. This strategy is commonly employed to optimize the pharmacokinetic attributes of drugs, including absorption, distribution, metabolism, and excretion (ADME), or to mitigate adverse effects. Prodrugs enhance drug stability, solubility, and targeted site delivery, representing a validated approach to refining drug-like properties. Such strategies have become integral to the development of therapeutic agents across a wide spectrum of diseases. The inherent complexity of the central nervous system (CNS) necessitates rigorous adherence to pharmaceutical, pharmacokinetic, and pharmacodynamic criteria for CNS drug design. Within this framework, prodrugs are categorized into bio-precursor prodrugs and carrier-linked prodrugs. Carrier-linked prodrugs, in particular, consist of an active drug chemically conjugated to an ancillary molecule via a metabolically labile bond. This ancillary molecule, referred to as the pro-moiety, is not intrinsically required for therapeutic efficacy but imparts desirable attributes, such as enhanced solubility (lipid or aqueous) or targeted delivery to specific tissues. Prodrugs are further classified based on their therapeutic application, chemical linkages, functional roles, or the nature of the moiety/carrier attached to the active drug. The design of an effective prodrug involves careful consideration of two critical factors: its rapid biotransformation into the active drug form and its substantial contribution to the therapeutic efficacy and safety profile of the active compound. The proposed research focuses on the development of carrier-linked prodrugs of olanzapine, an antipsychotic agent with a strong affinity for dopamine D2 receptors. Olanzapine’s antipsychotic efficacy is governed by its receptor-binding profile, which can be augmented through conjugation with a carrier moiety. This approach is intended to enhance receptor binding affinity, antipsychotic activity, and receptor selectivity. The synthesis of these prodrugs involves the chemical modification of olanzapine with amino acid alcohols to produce ester derivatives. Characterization of these derivatives is conducted using advanced analytical techniques, including Thin-Layer Chromatography (TLC), Infrared Spectroscopy (IR), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry. Subsequent stability studies are performed to evaluate the physicochemical properties and therapeutic potential of the synthesized compounds.

2. MATERIAL AND METHOD

2.1 Material

For the purpose of the research work, all the chemicals, ingredients, and instruments listed are of pharmaceutical and analytical grade. The chemicals used were: Olanzapine, Glutamic acid, DL-Aspartic Acid, Ethyl Acetate, Dichloromethane, Trifluoracetic acid, Tetrahydrofuran, etc.

2.1.1 Protection of the amino group

An amine (0.353 g, 1.5 mmol, 1 equivalent) was dissolved in 15 mL of anhydrous dimethylformamide (DMF) in a 50 mL single-neck flask and subjected to stirring at ambient temperature. To the resulting solution, triethylamine (Et?N, 0.168 g, 1.7 mmol, 1.2 equivalents) was introduced, followed by the controlled dropwise addition of di-tert-butyl dicarbonate ((t-BuOCO)?O, 0.356 g, 1.6 mmol, 1.1 equivalents). After 45 minutes, thin-layer chromatography (TLC) analysis employing a 10:90 ethyl acetate-to-hexane solvent system and PMA detection identified a product spot with an Rf value of 0.56. The reaction was quenched by introducing the mixture into water, followed by extraction using hexane. The organic layer was subsequently dried over magnesium sulfate, filtered, and concentrated under reduced pressure using a rotary evaporator. The crude product was purified via column chromatography, utilizing a 10:90 mixture of ethyl acetate and hexane as the eluent and silica gel as the stationary phase.

2.1.2 Conversion of a protected amino acid to an amino alcohol

To a pre-cooled solution of N-protected amino acid (1 mmol) in tetrahydrofuran (THF, 5 mL), di-tert-butyl dicarbonate (Boc?O, 261.6 mg, 1.2 mmol) and 4-dimethylaminopyridine (DMAP, 36.6 mg, 0.3 mmol) were added, followed by stirring of the reaction mixture for 15 minutes. Subsequently, an aqueous solution of sodium borohydride (NaBH?, 75.6 mg, 1 mmol) was incrementally introduced while ensuring precise temperature control, with stirring continued for an additional 5 minutes. Upon completion, the solvent was eliminated under reduced pressure, leaving behind a residue that was dissolved in 15 mL of ethyl acetate. The organic layer underwent successive washings with 10% hydrochloric acid (2 × 10 mL) or, in the case of Boc derivatives, 10% citric acid (2 × 10 mL), followed by treatment with 10% aqueous sodium carbonate (3 × 10 mL) and brine (3 × 10 mL). The organic phase was subsequently dried over anhydrous sodium sulfate (Na?SO?), after which the solvent was removed under reduced pressure. The crude product was purified either through recrystallization or via column chromatography, employing a solvent system comprising ethyl acetate and hexane in a 30:70 ratio as the eluent.

2.1.3 Conversion of an alkane to alkene

Dissolve precisely 31.24 mg of pure Olanzapine in 10 mL of chloroform, and subsequently introduce 400 mg of oxidants in a 20:80 ratio of KMnO? to CuSO? into the solution. Subject the mixture to constant agitation while maintaining a temperature range of 50–120°C for a duration of 2 hours, ensuring the reaction vessel is sealed with a cotton plug to prevent external contamination. Upon the reaction's completion, decant the resultant solution into a clean China dish and place it in a water bath stabilized at 58°C to facilitate evaporation, ultimately yielding the final product. In a subsequent step, introduce an acetonitrile-water mixture (8:8, v/v) to the solution containing protected amino alcohol (372.42 mg) and stir vigorously for 1 hour. Following this, add an additional acetonitrile-water mixture (4:4, v/v) incrementally while incorporating 810.966 mg of Oxone under continuous stirring. Maintain the solution under agitation for a prolonged period of 14 hours. At the end of this duration, transfer the reaction mixture into a China dish and employ a water bath set at 58°C to achieve thorough drying and obtain the final solid product.

2.1.4 Esterification

Dissolve 0.5 moles of amino alcohol in ethanol, employed as the solvent medium. Subsequently, introduce 1 mL of an olanzapine acid derivative along with 1 mL of concentrated sulfuric acid into the reaction mixture. Subject the mixture to thermal activation on a hot plate maintained at a temperature of 80 °C, with the reaction's progression being monitored through thin-layer chromatography (TLC) until completion is confirmed. The resulting crude product is then subjected to aqueous washing, followed by neutralization using a saturated solution of sodium hydrogen carbonate. The crude ester is further purified by filtration through filter paper pre-saturated with sodium sulphate, ultimately yielding the desired oily residue.

2.1.5 Characterisation of Prodrug

2.1.5.1 Melting point

Dissolve 0.5 moles of amino alcohol in ethanol, employed as the solvent medium. Subsequently, introduce 1 mL of an olanzapine acid derivative along with 1 mL of concentrated sulfuric acid into the reaction mixture. Subject the mixture to thermal activation on a hot plate maintained at a temperature of 80 °C, with the reaction's progression being monitored through thin-layer chromatography (TLC) until completion is confirmed. The resulting crude product is then subjected to aqueous washing, followed by neutralization using a saturated solution of sodium hydrogen carbonate. The crude ester is further purified by filtration through filter paper pre-saturated with sodium sulfate, ultimately yielding the desired oily residue.

2.1.5.2 Thin Layer Chromatography (TLC)

In thin-layer chromatography (TLC), the stationary phase is composed of a polar adsorbent, most commonly finely powdered alumina or silica particles. The mobile phase, on the other hand, can comprise various solvent mixtures, selected based on their specific interaction profiles. For the synthesized prodrugs, the Ethyl Acetate: Petroleum Ether system was chosen, guided by its alignment with the dielectric constant of the mobile phase.

2.1.5.3 FT-IR Principles

The Fourier Transform Infrared (FTIR) spectra of the olanzapine prodrug were acquired using an FTIR spectrometer (Cary 630, Agilent Technologies). Each distinct prodrug exhibited a unique infrared spectral profile, reflecting its specific molecular structure. The inherent dissimilarity among the spectra of various prodrugs underscores the utility of IR spectroscopy as a powerful analytical tool for distinguishing and characterizing different molecular entities.

2.1.5.4 Mass spectroscopy

The mass spectrum of the olanzapine prodrug was analyzed utilizing mass spectrometry (MS). The detailed interpretation of the obtained mass spectra is presented in the accompanying table. The calculated molecular masses of the constituent groups exhibit a precise correlation with the experimentally observed masses in the spectrum. This congruence unequivocally validates the successful synthesis of the olanzapine prodrug.

2.1.5.5 Nuclear Magnetic Resonance spectroscopy

The nuclear magnetic resonance (NMR) spectrum of the olanzapine prodrug was meticulously analyzed utilizing a BRUKER NMR spectrometer. Detailed interpretations of the acquired spectra are presented in Tables 7.5 through 7.7. The observed δ values for each functional group exhibited precise concordance with the theoretical values derived from the spectral data.

2.1.5.6 Accelerated stability study

The investigation of physicochemical alterations in the synthesized prodrug of Olanzapine was undertaken through an accelerated stability study. Samples of the synthesized prodrug were stored under controlled conditions in both a refrigerator and a programmable environmental chamber for a duration of six months, maintained at a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5%. At the conclusion of this six-month period, samples were retrieved for evaluation. During these intervals, the prodrug was meticulously monitored for any discernible changes in colour and odour. This comprehensive study encompassed both the standard drug solution and the synthesized conjugate, facilitating a comparative analysis of their stability profiles.

2.1.6 Preparation of std olanzapine solution

A standard olanzapine solution was prepared by dissolving 10 mg of olanzapine in methanol, then diluting it to a final volume of 100 ml to obtain a concentration of 100 μg/ml.

2.1.7 Preparation of stock olanzapine conjugate solution

A standard stock solution of the olanzapine conjugate was created by dissolving 10 mg of the conjugate in a sufficient amount of methanol, then diluting the mixture to a final volume of 100 ml to achieve a concentration of 100 μg/ml. The stability study was performed under different environmental conditions such as acidic, alkali, oxidative, thermal and photolytic degradation.

Acidic Degradation

In a 10 ml volumetric flask, 1.2 ml of Olanzapine conjugate stock solution and 5 ml of 1 N HCl were added and left at room temperature. After 3 hours, the solution was neutralized and diluted with methanol to a final volume of 10 ml, resulting in a concentration of 12 μg/ml.

Alkali Degradation

In a 10 ml volumetric flask, 1.2 ml of Olanzapine conjugate stock solution and 5 ml of 3 N NaOH solution were combined and left at room temperature. After three hours, the solution was neutralized and diluted with methanol up to the 10 ml mark to obtain a final concentration of 12 μg/ml.

Oxidation degradation

1.2 ml of the Olanzapine conjugate stock solution and 5 ml of 6% w/v hydrogen peroxide were added to a 10 ml volumetric flask. The flask was then maintained at room temperature for 3 hours. Afterward, the solution was diluted with methanol to a final volume of 10 ml to obtain the desired concentration of 12 μg/ml.

Thermal degradation

A sample of the olanzapine conjugate was placed in a petri dish and heated in an oven at 50°C for 48 hours. After the 48-hour period, the sample was diluted with methanol to a total volume of 10 ml. Further dilution was performed to obtain the desired concentration of 12 μg/ml.

2.2 In-vivo studies

Experiments were performed in Department of Pharmacology, Tatyasaheb Kore College of Pharmacy, Warananagar, and (Regd. No. 1090/PO/ac/07/CPCSEA). The in vivo experimental investigations incorporated the forced swim test. Male and female Wistar rats, aged 11–12 weeks and weighing between 200 and 400 grams, were utilized for the study. Prior to the commencement of the experiment, the animals were acclimatized for a duration of one week. The subjects were stratified into five distinct groups and housed in standardized animal enclosures within well-ventilated chambers maintained at a consistent temperature of 25°C and relative humidity of 50–55%, adhering to a 12-hour light/dark cycle. A circular pool measuring 150 cm in diameter, with walls 45 cm high and surrounded by external visual cues, served as the experimental apparatus. The pool was filled with tap water to a depth of 27 cm, maintained at a temperature of approximately 22°C. A transparent escape platform, 12 cm in diameter, was positioned 1 cm beneath the water's surface within the centre of one quadrant, and its placement remained constant throughout the study. The experimental procedure involved four training trials per day over five consecutive days. The Aspartic acid-Olanzapine conjugate (1.5 mg/300 g, i.p.), Isoleucine-Olanzapine (1.5 mg/300 g, i.p.), and Glutamic acid-Olanzapine (1.5 mg/300 g, i.p.) were administered intraperitoneally 1 hour and 30 minutes prior to the first daily trial. During each trial, rats were individually placed into the water at one of three predesignated starting points, facing the pool wall before release. If a rat was unable to locate the escape platform within 60 seconds, the experimenter guided it to the platform manually. Each trial concluded with a 30-second rest period on the platform (inter-trial interval). Performance metrics included escape latency of all three prodrugs of the pure Olanzapine drug. Furthermore, thigmotaxis—quantified as the percentage of time spent swimming within 12 cm of the pool walls—and diving behaviour, characterized by instances of the animal swimming away from the platform during the inter-trial period, were analyzed. This swim test demanded the development of intricate behavioural strategies from the rats, including moving away from the periphery of the pool, recognizing the platform as an escape route, climbing onto the platform to exit the water, and remaining stationary on it. Consequently, both thigmotaxis and diving behaviour were pivotal parameters for evaluating the subjects' performance and adaptive strategies.

3. RESULTS AND DISCUSSION

3.1 Characterisation of Prodrug

3.1.1 Melting Point

The melting points of the synthesized compounds were systematically evaluated to assess their thermal properties, which provide critical insight into their purity and structural integrity. The results, detailed in Table 1, reveal distinct melting points for the three Olanzapine prodrugs, each conjugated with a specific amino acid: Glutamic acid, Aspartic acid, and Isoleucine. The observed melting points were 110°C, 80°C, and 90°C, respectively. The higher melting point of the Olanzapine-Glutamic acid conjugate (110°C) suggests a more robust molecular lattice, potentially due to stronger or more extensive intermolecular interactions. Conversely, the Olanzapine-Aspartic acid conjugate exhibited the lowest melting point (80°C), indicating a comparatively weaker molecular lattice or reduced intermolecular cohesion. The intermediate melting point of the Olanzapine-Isoleucine conjugate (90°C) reflects a balance between these extremes. These findings are significant as they not only corroborate the successful synthesis of the prodrugs but also offer preliminary insights into their thermal stability and potential behaviour during formulation or storage. The distinct melting points serve as a fingerprint for each compound, facilitating their identification and verification in subsequent studies. Furthermore, the differences in melting points could have implications for the solubility and bioavailability of these prodrugs, warranting further investigation into their physicochemical and pharmacokinetic profiles.

Table 1: Melting point of synthesized compounds

3.1.2 Thin Layer Chromatography

The results of the Thin Layer Chromatography (TLC) analysis provide compelling evidence that the reaction has reached completion. This conclusion is supported by the clear separation observed between the spots corresponding to the reactants and those representing the newly synthesized prodrugs. The distinct retention factor (Rf) values recorded for the amino acid-derived prodrugs of olanzapine further substantiate their successful synthesis, with each prodrug exhibiting a unique Rf value reflective of its chemical properties. To optimize the analysis, different solvent compositions of the mobile phase—comprising varying ratios of petroleum ether and ethyl acetate—were systematically investigated. This approach ensured accurate determination of the Rf values for each synthesized prodrug, enabling clear differentiation between compounds. The resulting Rf values, summarized in Table 2, provide a quantitative basis for identifying and confirming the formation of the prodrugs. Additionally, the TLC spots, illustrated in Figure 1, visually reinforce the effective separation and purity of the synthesized compounds. The use of TLC as an analytical technique proved instrumental in monitoring the progress of the reaction and verifying the integrity of the synthesized prodrugs. By evaluating the impact of solvent concentration on compound mobility, the study successfully established reliable conditions for the precise characterization of the olanzapine prodrugs. This rigorous analytical methodology ensures that the synthesized prodrugs meet the necessary purity criteria and are suitable for subsequent experimental applications.

Table 2: TLC of olanzapine prodrugs

Fig. 1: TLC plates spotting of (A) Isoleucine prodruf of olanzapine, (B) Glutamic acid prodrug of olanzapine, (C) Aspartic acid prodrug of olanzapine

3.1.3 FTIR Analysis

The FTIR analysis of was used to evaluate the functional moieties from the synthesised compounds.

3.1.3.1 FTIR analysis of Olanzapine pure drug:

The FTIR (Fourier Transform Infrared) spectroscopy analysis of olanzapine reveals several characteristic functional group absorptions that confirm its molecular structure. A prominent amine group is identified through a distinct absorption peak at 3221 cm?¹, which aligns with the stretching vibrations of N-H bonds typically observed in amine functionalities. Additionally, the presence of C-H stretching vibrations is confirmed by a sharp absorption peak at 2932 cm?¹, indicative of the aliphatic hydrogen environment within the molecule. The spectrum further exhibits a notable absorption at 1583 cm?¹, attributed to the C=O stretching vibrations of an ester group, highlighting its integral role in the compound’s chemical framework. The presence of an alkyl group, specifically CH?, is evident from an absorption band at 1410 cm?¹, corresponding to its characteristic bending vibrations.The FTIR spectrum of olanzapine, as presented in figure 2 visually represents these absorption peaks, providing a comprehensive confirmation of the functional groups present. The detailed interpretation of these spectral values is systematically summarized in Table 2, ensuring clarity and accuracy in documenting the molecular characterization of the drug. This thorough spectral analysis not only validates the structural identity of olanzapine but also underscores the utility of FTIR spectroscopy as a pivotal tool in pharmaceutical quality control and research.

Fig. 2 FTIR spectrums of olanzapine

Table 3 FTIR interpretation of olanzapine

3.1.3.2 FTIR analysis Isoleucine Prodrug of olanzapine:

The FTIR spectra of the synthesized olanzapine-isoleucine conjugate reveal distinct peaks corresponding to key functional groups, confirming the successful formation of the compound. A prominent absorption band at 1733 cm?¹ is indicative of the ester group, a hallmark feature of the conjugate. This peak validates the esterification process as part of the synthesis. Additionally, the presence of an amine group is substantiated by a broad absorption band observed at 3387 cm?¹. This band reflects the characteristic stretching vibrations of the N-H bond, further confirming the incorporation of isoleucine into the olanzapine structure. Furthermore, the deformation vibrations of the hydroxyl (O-H) group manifest as a notable peak at 1463 cm?¹, showcasing another critical aspect of the molecular structure. The FTIR spectrum of olanzapine itself is presented in figure 3 serving as a comparative reference for analyzing the structural modifications introduced during conjugation. Detailed interpretations of the functional group vibrations and corresponding spectral values are systematically recorded in table 4, providing a comprehensive understanding of the synthesized compound's molecular framework. These findings collectively underscore the structural integrity and successful synthesis of the olanzapine-isoleucine conjugate.

Fig. 3 FTIR spectra of olanzapine Isoleucine conjugate

Table 4 FTIR interpretation Isoleucine Prodrug of olanzapine

3.1.3.3 FTIR analysis of Aspartic acid Prodrug of olanzapine

The FTIR spectrum of the aspartic acid-olanzapine conjugate provides compelling evidence of the structural features present within the compound. A distinct absorption peak observed at 1634 cm?¹ confirms the presence of an ester functional group, which is indicative of successful conjugation between aspartic acid and olanzapine. This frequency aligns with the expected range for ester carbonyl stretching, affirming its chemical identity within the conjugate. Additionally, the spectrum reveals a broad band at 3237 cm?¹, characteristic of the amine group's N-H stretching vibrations. The broadness of this band is typical for amines and suggests the retention of nitrogen-containing functional groups in the conjugate structure. Moreover, the C-H stretching vibrations are identified by a peak at 2989 cm?¹, further corroborating the compound's aliphatic and aromatic character. The FTIR spectra of olanzapine, presented in Fig. ..., serve as a reference to contrast and verify these findings. The recorded spectral values for olanzapine, detailed in Table ..., facilitate a comprehensive comparison and highlight the changes induced by the conjugation process. This comparative analysis underscores the successful modification of olanzapine through its interaction with aspartic acid, as evidenced by the emergence of new peaks corresponding to the ester group and the preservation of key functional groups such as amines and C-H bonds.

Fig. 4 FTIR Aspartic acid Prodrug of olanzapine

Table 5 Interpretation of FTIR aspartic acid prodrug of olanzapine

3.1.4 Nuclear Magnetic Resonance (NMR) Spectroscopy:

The nuclear magnetic resonance (NMR) spectrum of the olanzapine prodrug was meticulously analysed using a BRUKER NMR spectrometer, a high-precision instrument renowned for its capability to provide detailed molecular insights. This analysis revealed that the experimentally determined δ values for the various functional groups within the prodrug molecule were in complete alignment with the theoretical or expected values derived from the chemical structure. Such congruence between observed and predicted chemical shifts underscores the reliability and accuracy of the spectroscopic data, affirming the structural integrity of the prodrug. The absence of discrepancies in δ values further suggests that the sample was of high purity and that the molecular environment of each group was consistent with its anticipated electronic and spatial characteristics. This consistency not only validates the identity of the compound but also reinforces the effectiveness of NMR spectroscopy as a pivotal tool in the structural elucidation and verification of complex pharmaceutical compounds.

Fig. 5 NMR spectra of Isoleucine Prodrug of Olanzapine

Table 6 Interpretation of NMR spectra of Isoleucine Prodrug of Olanzapine

Fig. 7 NMR spectra of Aspartic acid Prodrug of Olanzapine

Table 7 Interpretation of NMR spectra of Aspartic acid prodrug of Olanzapine

Fig. 8 Interpretation of NMR spectra of Glutamic acid prodrug of Olanzapine

Table 8 Interpretation of NMR spectra of Glutamic acid prodrug of Olanzapine

3.1.5 Mass Spectroscopy:

The mass spectrometric analysis of the synthesized Olanzapine prodrug was conducted using a Shimadzu MS 2010 instrument to verify its molecular structure and confirm its identity. The detailed interpretation of the obtained mass spectra is presented in tables and figures depicted below, where the experimentally determined mass values of various molecular fragments were meticulously compared to their corresponding theoretical masses. The close agreement between the calculated and observed masses provides strong evidence supporting the successful synthesis of the intended Olanzapine prodrug. These results validate the chemical structure of the product, affirming that the synthetic process reliably yielded the desired compound without significant deviations or impurities.

Fig. 9 Mass spectra of Olanzapine

The molecular ion peaks for the aspartic acid conjugate and glutamic acid conjugate were identified at 440 m/z and 462 m/z, respectively, through mass spectrometry (MS). These peaks correspond to the intact molecular ions of the conjugates, serving as clear indicators of their molecular weights. The precise m/z values confirm the successful synthesis of these compounds, allowing for the differentiation between the two based on their distinct mass-to-charge ratios. In the context of liquid chromatography (LC), the chromatographic profiles for both conjugates are presented in figure 10, illustrating the retention times and peak intensities. These LC spectra provide vital information regarding the purity, stability, and the separation efficiency of the conjugates within the mixture, aiding in the evaluation of their chemical properties. Additionally, the MS spectrum peak list, which is documented in table 9, details the full spectrum of observed peaks, highlighting the fragmentation patterns and any potential isotopic distributions. These tables offer a comprehensive overview of the mass spectrometric data, enabling a deeper understanding of the structural characteristics of the aspartic acid and glutamic acid conjugates. Together, the LC and MS data corroborate the identity and purity of the conjugates, ensuring that the observed molecular ions align with the expected results from the synthesis process.

Fig. 10 LC Spectrum peak

Table 9 MS Spectrum peak list for aspartic acid-olanzapine conjugate

Fig. 11 MS Spectrum peak for glutamic acid-olanzapine conjugate

Table 10 MS Spectrum peak list for glutamic acid-olanzapine conjugate

3.1.6 Stability Studies

Accelerated stability studies:

The stress degradation studies were conducted to evaluate the stability of a standard drug solution and a synthesized conjugate under various stress conditions. Both the drug solution and the conjugate were analysed at an identical concentration of 12 µg/ml to ensure consistency in comparison. The degradation conditions included exposure to acidic, alkaline, and oxidative environments, which are known to mimic the harsh conditions that a drug may encounter during storage, handling, or in vivo metabolism. The results indicated a distinct contrast in the stability profiles of the two substances. The standard Olanzapine drug solution exhibited notable degradation when subjected to acidic, alkaline, and oxidative stress. This degradation could manifest through chemical breakdown or alterations in the drug's molecular structure, which may ultimately impact its efficacy and safety. These findings are consistent with the typical behaviour of many pharmaceutical compounds, which are often sensitive to extreme pH levels or oxidative environments, resulting in the loss of potency or the formation of potentially harmful degradation products. In stark contrast, the synthesized conjugate demonstrated a remarkable stability across all tested conditions. This suggests that the conjugation process may have contributed to enhancing the chemical robustness of the compound, shielding it from the destabilizing effects typically associated with acidic, alkaline, and oxidative conditions. This increased stability is a desirable characteristic for pharmaceutical formulations, as it could lead to an improved shelf life and a lower likelihood of adverse reactions due to degradation products. The comparative analysis of the standard drug solution and the synthesized conjugate under different stress conditions is illustrated in figure 12, which visually presents the degradation trends for both samples. The figure offers a clear representation of how the synthesized conjugate maintains its integrity while the standard Olanzapine solution undergoes significant breakdown under the same stress conditions. Furthermore, the study also assessed the physical parameters of the samples, such as colour and odour, to identify any perceptible changes that could indicate degradation or chemical alteration. The recorded observations for these physical changes are summarized in Table Y. These parameters, while not always indicative of the extent of molecular degradation, provide a useful preliminary evaluation of a compound's stability and can be used as an initial indication of potential issues during storage or handling.In conclusion, the stress degradation study underscores the enhanced stability of the synthesized conjugate in comparison to the standard Olanzapine solution. The findings highlight the potential advantages of conjugation strategies in improving the stability of pharmaceutical compounds, which may ultimately lead to more effective and longer-lasting therapeutic agents. Further research is needed to explore the underlying mechanisms that confer this stability and to assess the long-term implications for the clinical use of such conjugates.

Fig. 12 Comparison between standard Olanzapine (12 μg/ml) and sample of Olanzapine conjugate (12 μg/ml); (A) Acid degradation, (B) Alkali degradation, (C) Oxidation, (D) Thermal degradation and (E) Photolytic degradation

Table 11 Observation of accelerated stability study

3.1.7 Animal Study

The efficacy of the synthesised compound was evaluated non-clinically through in-vivo behavioural study using forced swim test. The escape latency of the three groups of olanzapine prodrug and the control group is recorded in table 12.

Table 12 Observation table for forced swim test