Synthesis And Evaluation of an N-Methyl Vilanterol Derivative as A Novel Long-Acting ?2-Adrenoceptor Agonist

Table 2.1: Drug Profile of Vilanterol.

S. No.	Property	Description
1.	Drug	Vilanterol
2.	Molecular Formula	C24H33Cl2NO5
3.	Molecular Weight	486.4 g/mol
4.	Elemental Composition	C: 59.26%, H: 6.84%, Cl: 14.58%, N: 2.88%, O: 16.45%
5.	Preparation Method	Multi-step synthesis involving the coupling of a protected (R)-saligenin epoxide (or related halohydrin) with an extended lipophilic amine chain terminating in a 2,6-dichlorobenzyl ether, followed by global deprotection.
6.	IUPAC Name	4-[(1R)-2-[6-[2-[(2,6-dichlorophenyl)methoxy]ethoxy]hexylamino]-1-hydroxyethyl]-2-(hydroxymethyl)phenol
7.	Odor	Odorless
8.	Colour	White to off-white (solid powder)
9.	Solubility	Practically insoluble in water; soluble in methanol, ethanol, and dimethyl sulfoxide (DMSO).
10.	Melting Point	132°C - 135°C (Reported for Vilanterol trifenatate salt)
11.	Synonyms	GW642444, N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]-6-(2-(2,6-dichlorobenzyloxy)ethoxy)hexylamine
12.	pKa (at 25 °C)	~ 9.7 (Predicted for the secondary amine); ~ 9.4 (Predicted for the phenolic -OH)
13.	Log P	~ 4.1 (Calculated XLogP3, highly lipophilic)
14.	Structure

2.2. The Evolutionary Trajectory of Long-Acting Bronchodilators

Cazzola et al. (2013) provide a comprehensive overview of the evolutionary trajectory of bronchodilator therapy, tracking the transition from short-acting β2-agonists (SABAs) to long-acting (LABAs) and ultra-long-acting β2-agonists (Ultra-LABAs). The review highlights how the clinical demand for once-daily dosing regimens in asthma and COPD management drove the medicinal chemistry efforts to extend the duration of action beyond the 12h window of formoterol and salmeterol. The authors emphasize that achieving a 24h therapeutic duration fundamentally requires the integration of highly lipophilic side chains into the phenethanolamine scaffold. This structural modification facilitates prolonged retention within the pulmonary tissue or continuous re-engagement with the receptor. For the proposed thesis, this literature substantiates the foundational necessity of designing Ultra-LABA molecules that prioritize extended tissue residency and once-daily efficacy, thereby justifying the continued exploration and structural refinement of advanced scaffolds like vilanterol to optimize therapeutic outcomes and enhance patient compliance in chronic obstructive airway diseases. [4]

2.3. Rational Design and Discovery of the Vilanterol Scaffold

Procopiou et al. (2010) detail the pivotal medicinal chemistry campaign that culminated in the discovery of vilanterol. The paper delineates the rational design of a novel series of β2-adrenoceptor agonists utilizing an “antedrug” or “soft drug” approach. The researchers carefully looked at different lipophilic tails connected to a saligenin headgroup. They found that the 2,6-dichlorobenzyl ether moiety worked best for selectively targeting β2 and lasting for 24 hours. Importantly, the molecule was designed to be quickly broken down by liver cytochrome P450 enzymes, mainly by cutting the ether linker. This was done to reduce the typical cardiovascular side effects that are caused by the sympathetic nervous system. This research is instrumental for the thesis as it establishes the baseline structural parameters of the vilanterol scaffold. Understanding the delicate balance Procopiou and colleagues achieved between local pulmonary retention and rapid systemic clearance provides the critical comparative framework against which the pharmacokinetic alterations induced by the proposed N-methylation will be hypothesized, evaluated, and subsequently discussed. [3]

2.4. Pharmacokinetic Theories: Exosite and Microkinetic Models

Matera et al. (2007) critically examine the prevailing pharmacological theories explaining the extended duration of action inherent to long-acting and ultra-long-acting β2-adrenoceptor agonists. The paper juxtaposes two primary hypotheses: the “exosite theory,” where the lengthy lipophilic tail binds to a secondary, non-polar domain on the receptor preventing complete dissociation, and the “plasmalemma diffusion microkinetic model,” which suggests the drug partitions deeply into the cellular lipid bilayer, creating a localized depot that continuously supplies the active headgroup to the receptor’s orthosteric binding site. This literature is highly pertinent to the thesis as it provides the theoretical mechanism for how bulky, lipophilic modifications sustain bronchodilation. Modifying the vilanterol scaffold via N-methylation will inherently increase the molecule's overall lipophilicity (log P) and alter its three-dimensional spatial conformation. Consequently, this paper will serve as the theoretical foundation to hypothesize whether the addition of a tertiary amine disrupts or enhances these established microkinetic interactions within the pulmonary lipid milieu. [6]

2.5. Structural Significance of the Amine Core in Receptor Binding

Rasmussen et al. (2011) elucidated the high-resolution crystal structure of the human β2-adrenergic G-protein-coupled receptor in complex with an agonist, providing an unprecedented, three-dimensional understanding of the orthosteric binding pocket. The crystallographic data definitively demonstrates that the protonated amine of the β2-agonist forms a crucial ionic bridge and hydrogen-bonding network with the highly conserved Aspartate 113 (Asp113) residue located on transmembrane helix 3 (TM3). This specific intermolecular interaction is absolutely fundamental for anchoring the ligand within the receptor and initiating the conformational changes required for subsequent G-protein activation. This structural biology insight is exceptionally relevant to the proposed thesis. Because the project involves synthesizing an N-methyl derivative, thereby transforming a secondary amine into a tertiary amine, this paper provides the structural context to evaluate how increased steric bulk and altered basicity (pKa) at the nitrogen center might influence the binding affinity and the crucial electrostatic interaction with the key Asp113 residue. [7]

2.6. The Pharmacological Impact of N-Methylation in Drug Design

Schönherr and Cernak (2013) provide a profound analysis of the “magic methyl” effect and the specific impact of N-methylation in contemporary drug discovery and structural optimization. The review articulates how the conversion of a secondary amine to a tertiary amine via the introduction of a seemingly simple methyl group can drastically alter a drug’s physicochemical and pharmacokinetic properties. The authors highlight that N-methylation typically eliminates a hydrogen-bond donor, increases overall lipophilicity, alters the compound’s basicity, and can significantly restrict the conformational flexibility of the molecule. For the current thesis, this literature provides the core rational justification for the proposed synthetic modification of vilanterol. By applying Schönherr and Cernak’s principles, the literature review will argue that N-methylating the vilanterol scaffold is a strategic, calculated manipulation intended to modulate membrane permeability, optimize the spatial geometry of the lipophilic tail, and potentially alter the molecule’s metabolic vulnerability, thereby thoroughly justifying the targeted synthetic objectives of the research project. [8]

2.7. Clinical Efficacy and Safety Benchmarks of Vilanterol

Rayner and Scott (2014) comprehensively evaluate the clinical profile, efficacy, and safety of vilanterol, particularly when administered in combination with the inhaled corticosteroid fluticasone furoate for the management of COPD. The review thoroughly examines late-stage clinical trial data, affirming that vilanterol produces rapid bronchodilation comparable to salbutamol while reliably sustaining improvements in the forced expiratory volume in one second (FEV₁) over a full 24h period. Furthermore, the paper highlights the compound’s excellent safety and tolerability profile, noting the minimal incidence of systemic sympathomimetic side effects such as tachycardia or tremors. This clinical literature is vital for the thesis as it establishes the exceptionally high benchmark of safety and efficacy that any novel vilanterol derivative must strive to achieve or exceed. It sets the clinical context, ensuring that the pharmacological evaluation of the synthesized N-methyl derivative is strictly compared against the established therapeutic parameters and systemic clearance expectations of the parent compound. [2]

2.8. Relevant Preclinical In Vivo Models for Assessing Airway Pharmacology

Glaab et al. (2007) provide a critical, comparative evaluation of invasive and non-invasive methodologies for assessing airway hyper-responsiveness and lung function in murine models of pulmonary disease. The paper details techniques such as whole-body plethysmography and invasive mechanical ventilation, emphasizing the precision required to accurately measure airway resistance and dynamic compliance in response to bronchoconstrictors like methacholine. The authors discuss the translational reliability of these murine models in evaluating the efficacy of novel bronchodilator therapeutics. This paper is highly relevant to the pharmacological evaluation chapter of the proposed thesis. [9]

2.9. Interrogating the Inflammatory Axis in Obstructive Airway Disease

Barnes (2002) details the complex synergistic molecular interactions between long-acting β2-adrenoceptor agonists and anti-inflammatory corticosteroids within the respiratory tract. While β2-agonists are primarily defined by their capacity to induce smooth muscle relaxation, Barnes highlights emerging evidence suggesting that prolonged β2-receptor activation can independently modulate certain inflammatory pathways, potentially stabilizing mast cells and altering the release of pro-inflammatory mediators within the pulmonary microenvironment. This mechanistic review is highly pertinent to the secondary pharmacological objectives of the thesis. While the primary goal of synthesizing the N-methyl vilanterol derivative is to optimize long-acting bronchodilation, evaluating its ancillary effects on airway inflammation is equally critical. Incorporating this literature provides the necessary scientific rationale for incorporating lipopolysaccharide (LPS)-induced inflammatory models into the study. It grounds the hypothesis that structural modifications to the vilanterol scaffold might not only influence receptor kinetics and bronchodilation but could also potentially alter the molecule’s intrinsic capacity to interact with and modulate pulmonary inflammatory cascades. [10]

2.10. Rigorous Spectral Validation in Medicinal Chemistry Synthesis

Holzgrabe et al. (2005) present a comprehensive review on the critical application of high-resolution NMR spectroscopy in pharmaceutical analysis and medicinal chemistry. The authors underscore the absolute necessity of rigorous spectral characterization to definitively confirm the structural identity, spatial conformation, and absolute purity of newly synthesized active pharmaceutical ingredients. They detail the specific methodologies required for resolving complex multiplets and accurately assigning proton environments in highly functionalized drug scaffolds. This literature is fundamentally crucial for the analytical chapter of the thesis. The introduction of an N-methyl group onto the complex vilanterol framework requires indisputable structural validation. Relying on the principles outlined by Holzgrabe, the literature review will justify the deployment of advanced 1H-NMR, FT-IR, and ESI-MS. It reinforces the necessity of these techniques to definitively prove that the targeted tertiary amine modification was successful without unintended side reactions, meeting the stringent purity requirement for biological evaluation. [11]

MATERIALS AND METHODOLOGY

3.1. Materials

3.1.1. Chemicals reagents and solvents, instruments/apparatus required

All chemical reagents required for the synthesis of the vilanterol derivative were procured from standard suppliers (Sigma-Aldrich, Merck, Fisher Scientific, etc) and used without further purification unless otherwise specified. The starting material Vilanterol were synthesized according to established procedures or obtained commercially. Solvents including dichloromethane, ethyl acetate, methanol, acetonitrile, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF) were of analytical or HPLC grade. Anhydrous solvents were dried using standard procedures with molecular sieves or distillation over appropriate drying agents when required for moisture-sensitive reactions. [12]

Table 3.1: List of the chemicals.

Table 3.2: List of instruments.

Table 3.3: List of apparatus.

3.2. Methods

3.2.1. Determination of Melting Point

Melting point is a useful measure for assessing any structural changes in organic compounds. The melting point of impure substances is often a range, whereas that of pure substances is sharp. Insert a small amount of liquid Vilanterol into a capillary tube to find its melting point. Using a melting point device, slowly heat the tube. Record the temperature at which the sample starts to melt; this is where the melting range begins. Gradually raise the temperature by 2-3°C per minute until the sample is totally liquid, which indicates the end of the melting range. Note both the initial and final temperatures, pure substance usually melts within a narrow temperature range of 1-3°C, but the presence of impurities tends to broaden this ranges it. Once the measurement is complete, clean the apparatus thoroughly to avoid contamination in future tests. [13]

3.2.2. Determination of Solubility

 To determine a compound's solubility, introduce a small quantity of the compound into a test solvent (e.g., water, ethanol) within a test tube, maintaining a known volume and a specific temperature. In a study assessing the solubility profile of Vilanterol, a 10 mg medication sample was dissolved in 10 ml of various solvents. Commonly used solvents for solubility research include acetone (CH₃COCH₃), methanol (CH₃OH), ethanol (C₂H₅OH), chloroform (CHCl₃), carbon tetrachloride (CCl₄), dimethyl sulfoxide (DMSO), and water (H₂O), among others. [14]

3.2.3. Determination of Percentage Yield

Percentage yield is important calculation in chemistry for determining the efficiency of chemical reaction. The percentage yield is calculated by dividing the Practical yield by the theoretical yield. It is derived by comparing the Practical yield-the amount of product obtained in the laboratory-with the theoretical yield, that reflects the maximum potential product amount based on the stoichiometric calculations. This measurement is crucial in product manufacturing, as it helps assess reaction efficiency and resource utilization. [15]

Equation (3.1) can be used to calculate the Percentage Yield as:

% Yield=Practical Yield ÷Theorectical Yield×100

3.3. Synthesis Procedure of N-Methyl Vilanterol Derivative

Chemical Reaction Scheme

The synthesis of the N-methyl vilanterol derivative was achieved via a highly chemoselective reductive amination pathway. The overarching chemical transformation proceeded through two distinct mechanistic phases:

Phase 1: Iminium Ion Formation

Procedure of Iminium Ion Formation:

Step 1: Reaction Setup and Reagent Interaction

The reaction initiates with the pre-formed, highly electrophilic intermediate, the iminium ion (labeled as N-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl-N-(6-2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)methylideneammonium), dissolved in a suitable aprotic solvent, which in this case is 1,2-dichloroethane (DCE). The reaction mixture is carefully cooled to 0^OC, typically using an ice bath. This strict temperature control is critical to stabilize the highly reactive iminium species and prevent unwanted degradation or side reactions. Once stabilized, the reducing agent, sodium triacetoxyborohydride (often abbreviated as STAB), is introduced. STAB is explicitly chosen for this step because the three bulky, electron-withdrawing acetate ligands significantly reduce the nucleophilicity of its remaining hydride. This makes it an exceptionally mild and highly chemoselective reagent that was exclusively target the reactive iminium bond without disturbing the other sensitive functional groups (such as the phenolic hydroxyls, aliphatic alcohols, or ether linkages) present in this complex drug-like molecule.

Step 2: The Hydride Transfer Mechanism

Once the reagents are intimately mixed in the chilled DCE solvent, the core mechanistic chemical transformation occurs: the targeted transfer of a nucleophilic hydride ion (H⁺) from the borohydride complex to the electrophilic center of the target molecule. The positively charged nitrogen atom of the iminium ion exerts a strong electron-withdrawing inductive effect, making the adjacent carbon atom of the methylidene group (=CH₂) highly electron-deficient and extremely susceptible to nucleophilic attack. The hydride ion from the NaBH (OAc)₃ attacks this specific carbon atom. Simultaneously, as the new carbon-hydrogen bond forms, the π-electrons making up the carbon-nitrogen double bond are pushed back onto the nitrogen atom. This synchronized movement of electrons effectively neutralizes the formal positive charge on the nitrogen, converting the unstable, reactive double bond into a stable, single-bonded tertiary amine structure.

Step 3: Product Formation and Generation of Byproducts

Following the successful transfer of the hydride, the chemical transformation is complete, successfully yielding the final stable tertiary amine product: 4-[1-hydroxy-2-(methyl{6-[2-(2,6-dichlorobenzyloxy)ethoxy]hexyl}amino)ethyl]-2-(hydroxymethyl)phenol. By gaining the hydride, the methylidene group has been fully reduced into a stable methyl group (CH₃) attached to the central nitrogen. Concurrently, the decomposition of the reducing agent generates the necessary chemical byproducts depicted on the right side of the image. The removal of the hydride from the initial sodium triacetoxyborohydride complex results in the formation of boron triacetate (B(OAc)₃), which is an electrically neutral coordination complex. Additionally, the sodium cation (Na⁺), which originally served as the positively charged counterion to stabilize the starting borohydride salt, is liberated into the reaction medium. In a practical laboratory procedure, this mixture would subsequently undergo a controlled workup, typically involving a mild aqueous base, to safely quench the reaction and allow for the extraction of the synthesized N-methylated product.

Phase 2: Chemoselective Reduction

(Where R represents the saligenin headgroup [2-(hydroxymethyl)-4-(1-hydroxyethyl)phenol] and R' represents the extended lipophilic tail [6-(2-((2,6-dichlorobenzyl)oxy)ethoxy)hexyl].)

Procedure of chemoselective reduction

First, the highly reactive N-(2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl-N-(6-2-[(2,6-dichlorobenzyl)oxy]ethoxy)hexyl]methylideneammonium precursor was dissolved in a continuous stirring matrix of aprotic 1,2-dichloroethane (DCE). To prevent unwanted side reactions and preserve the integrity of the molecule's sensitive phenolic and ether functional groups, the reaction flask was submerged in an ice bath, securely bringing the internal temperature of the mixture down to exactly 0^oC.

Once the target temperature was stabilized, sodium triacetoxyborohydride was cautiously introduced into the reaction vessel. This specific reagent was selected because its bulky acetate ligands effectively temper its reactivity, making it exceptionally mild. As the chilled mixture was continuously stirred, a chemoselective hydride transfer occurred. The nucleophilic hydride ion migrated from the active borohydride complex directly to the highly electrophilic carbon of the iminium double bond.

This vital mechanistic step neutralized the formal positive charge residing on the nitrogen atom and fully reduced the double bond into a stable single bond. Consequently, the reaction successfully yielded the desired N-methylated tertiary amine final product: 4-[1-hydroxy-2-(methyl(6-[2-(2,6-dichlorobenzyloxy)ethoxy]hexyl)amino)ethyl]-2-(hydroxymethyl)phenol. Concurrently, the necessary decomposition of the hydride donor resulted in the generation of boron triacetate and free sodium ions as the anticipated chemical by-products within the solvent matrix

3.3.1. Preparation of Apparatus and Solubilization

Before the synthesis began, all the necessary glassware, such as a 100 mL two-neck round-bottom flask and the addition apparatus, was dried completely in an oven at 110 °C to remove any moisture. The round-bottom flask had a magnetic moving bar and was hooked up to a line of inert nitrogen (N2) to keep the vilanterol phenol moiety from breaking down through oxidation.

There was a precise amount of vilanterol free base (1.0 equivalent) put into the reaction flask. It was mixed with 15 mL of anhydrous 1,2-dichloroethane (DCE) for every gram of the starting material. At room temperature (25 °C), the mixture was stirred all the time until the vilanterol was fully broken down, making a clear solution. A small amount of glacial acetic acid (CH3COOH, 3 drops) was added to the solution to make it slightly acidic. This made the pH level look like it was about 5.5, which was just right for the process.

3.3.2. Formaldehyde Addition and Condensation

A slightly acidic vilanterol solution was mixed with a 37% w/w aqueous formaldehyde solution (HCHO, 1.5 equivalents) that was added drop by drop over 10 minutes using a micro-syringe. Once all the ingredients were added, the reaction tank was closed up and put under a nitrogen atmosphere. The mixture was then left to stir very hard at room temperature for 45 minutes. While the mixture was being incubated, the secondary amine in the vilanterol scaffold had a condensation reaction with the formaldehyde carbonyl carbon. This got rid of the water and created the highly reactive electrophilic iminium ion intermediate.

3.3.3. Chemoselective Reduction Phase

After the iminium ion was made, the reaction flask was put into a bath of ice water to cool the mixture inside to 0–5 °C. This cooling step was added to control how exothermic the next step would be and to protect the vilanterol molecule's acid-sensitive benzylic alcohol.

Sodium triacetoxyborohydride (NaBH(OAc)3, 2.0 equivalents) was carefully measured and added to the cold reaction mixture in small, steady amounts over 20 minutes to keep the temperature from rising quickly. The ice water bath was taken away once the whole amount of the reducing agent was mixed in. After letting the reaction mixture slowly warm up to room temperature, it was stirred by hand continuously for another 6 hours to make sure that the iminium intermediate was completely broken down into the desired tertiary N-methyl amine.

3.3.4. Reaction Quenching and Liquid-Liquid Extraction

After the reaction time was up, the mixture was slowly cooled down to get rid of the acetic acid and any borate complexes that were still there. Sodium bicarbonate (NaHCO3) solution that was fully dissolved in water was added drop by drop to the stirring flask until there was no more noticeable fizzing and the water phase had a pH of 8.0, which is slightly basic.

The two-phase mixture that was made was measured out and put into a 250 mL separatory tube. Separating the mixture with more dichloromethane (DCM, 3x25 mL) was used to get the required organic product. The organic layers from each of the separate extractions were put together. The merged organic layer was washed with 30 mL of distilled water and then with 30 mL of a saturated sodium chloride solution to get rid of any inorganic salts that were still there or formaldehyde that had not been broken down. After being washed, the organic phase was put into a clean Erlenmeyer flask and dried over dry sodium sulphate (Na2SO4) for 20 minutes to get rid of any remaining water.

3.3.5. Product Isolation and Desiccation

The hydrated sodium sulfate was removed via gravity filtration through qualitative filter paper, and the clear filtrate was collected in a pre-weighed round-bottom flask. The organic solvent (DCM/DCE) was evaporated under reduced pressure utilizing a rotary evaporator, with the heating bath meticulously maintained below 40 °C to prevent thermal degradation of the product.

The evaporation yielded the crude N-methyl vilanterol derivative as a viscous, semi-solid residue. To isolate the pure compound without the use of chromatographic techniques, the residue was subjected to rigorous solvent trituration. 15 mL of cold, anhydrous diethyl ether was added to the flask, and the residue was violently triturated (scratched and stirred) to get rid of non-polar impurities and make it solidify. The supernatant ether was carefully decanted. This washing process was repeated twice.

The resulting purified, off-white solid residue was immediately transferred to a high-vacuum desiccator and subjected to a pressure of 0.1 mmHg for 24 hours. This exhaustive vacuum drying definitively removed any remaining trace volatiles or solvent molecules trapped within the matrix of the compound.

3.3.6. Yield Calculation and Storage for Characterization

Following complete desiccation, the flask containing the final, solvent-free N-methyl vilanterol derivative was weighed to calculate the practical percentage yield of the synthesis, which was obtained as to be, approx. 75-76 %. The purified solid was meticulously transferred into an amber-colored glass vial, flushed with a gentle stream of nitrogen gas, tightly sealed, and stored at 2-8 °C.

The synthesized compound was strictly preserved under these conditions pending its structural elucidation and validation, which was programmed to be conducted exclusively utilizing FT-IR spectroscopy, ¹H-NMR spectroscopy, and ESI-MS.

3.4. Characterization techniques for Synthesized Derivative:

3.4.1. Fourier-Transform Infrared (FT-IR) Spectroscopy

FT-IR is utilized to confirm the functional group transformations, specifically focusing on the amine environment.

1. Sample Preparation: When 1-2 mg of the completely dried N-methyl vilanterol derivative was mixed with anhydrous potassium bromide (KBr) powder in a mill and pestle, the mixture was pressed under high pressure to make a clear, moisture-free KBr pellet. When using an Attenuated Total Reflectance (ATR) accessory, the raw solid sample could be put directly on the diamond crystal and pushed down with the anvil.
2. Data Acquisition: The sample placed into the FT-IR spectrometer sample compartment. The spectrum scanned across the mid-infrared region, typically from 4000 to 400 cm^-1, taking an average of 16 to 32 background-corrected scans at a resolution of 4 cm^-1 to ensure a high signal-to-noise ratio.
3. Spectral Interpretation: The generated spectrum was analyzed to confirm the success of the reductive amination. The key diagnostic indicator is the disappearance of the characteristic secondary amine N-H stretching band (typically seen around 3300 cm^-1 in the parent vilanterol) and the emergence of distinct aliphatic C-H stretching vibrations specific to the newly added N-methyl group (around 2800–2950 cm^-1).

3.4.2. Proton Nuclear Magnetic Resonance (¹H-NMR) Spectroscopy

¹H-NMR is the definitive technique for mapping the exact hydrogen environment and proving the structural backbone of the new derivative.

1. Sample Preparation: A deuterated solvent like Deuterated Chloroform (CDCl3) or Deuterated Dimethyl Sulfoxide (DMSO-d6) was mixed with 5–10 mg of the pure derivative and allowed to dissolve entirely. 0.00 ppm of tetramethylsilane (TMS) is added to the solvent as an internal measure.
2. Data Acquisition: The sample tube is inserted into a high-field NMR spectrometer (e.g., 400 MHz or 500 MHz). The instrument is locked to the deuterium frequency of the solvent, shimmed for magnetic field homogeneity, and the proton spectrum is acquired utilizing standard pulse sequences at ambient temperature.
3. Spectral Interpretation (Targeting the Methyl Group): It has been Fourier-transformed and phase-corrected to get the raw Free Induction Decay (FID) data. Finding a new, well-integrated singlet peak in the aliphatic region (usually between 2.20 and 2.50 ppm) is the most important step. This singlet matches the three protons of the new N-methyl group (N-CH3), which proves that the structure is a tertiary amine.

3.4.3. Electrospray Ionization Mass Spectrometry (ESI-MS)

ESI-MS provides the precise molecular weight of the synthesized compound, confirming its elemental composition.

1. Sample Preparation: A sub-milligram quantity of the pure derivative dissolved in an MS-grade solvent matrix (usually a mixture of Methanol or Acetonitrile and Water). A trace amount of an ionizing agent, such as 0.1% formic acid, added to facilitate the protonation of the basic tertiary amine nitrogen.
2. Ionization and Acquisition: The sample was put into the mass analyser either directly or through coupled Liquid Chromatography (LC-MS). The instrument operated in Positive Ion Mode (+ESI), where high voltage applied to the capillary aerosolizes the sample, generating positively charged ions without fragmenting the intact molecule.
3. Mass Analysis: The resulting mass spectrum analyzed to locate the pseudo-molecular ion peak. Because the N-methyl derivative is protonated during ionization, the target peak will appear at [M+H]⁺. The observed mass-to-charge ratio (m/z) must closely match the theoretically calculated exact mass of the N-methyl vilanterol structure to definitively prove the molecule’s identity.

3.5. Structure-Activity Relationship (SAR) Analysis of the N-Methyl Derivative

The modification of the vilanterol scaffold via N-methylation represents a critical structural transition from a carefully optimized secondary amine to a tertiary amine. This targeted functionalization fundamentally alters the physicochemical, spatial, and electronic properties of the core pharmacophore. The following subsections delineate the theoretical Structure-Activity Relationships (SAR) governing the pharmacological profile of this novel derivative.

3.5.1. Alteration of the Orthosteric Binding Pose and Intrinsic Efficacy

The conversion of the secondary amine in vilanterol to a tertiary amine eliminates a critical hydrogen bond donor. Within the β-2 adrenoceptor orthosteric binding pocket, the basic nitrogen protonates at physiological pH, forming an indispensable ionic salt bridge with the conserved Aspartate 113 (Asp113) residue on transmembrane helix 3 (TM3). While the tertiary amine will still protonate and engage in this electrostatic interaction, the added steric bulk of the methyl group directly alters the hydration shell and the three-dimensional geometry of the nitrogen center. This steric interference may shift the orientation of the saligenin headgroup, potentially disrupting the optimal hydrogen-bonding network with serine residues (Ser203, Ser204, Ser207) on TM5. Consequently, this structural modification will provide critical insight into whether the intrinsic efficacy and binding affinity of the parent drug are strictly dependent on the spatial economy of a secondary amine, or if a tertiary amine can adopt a permissive active conformation.

3.5.2. Enhancement of Lipophilicity and Microkinetic Depot Formation

The addition of a single methyl group intrinsically increases the overall lipophilicity (log P) and distribution coefficient (logD 7.4) of the molecule. According to the plasmalemma diffusion microkinetic model, a higher degree of lipophilicity facilitates deeper and more extensive partitioning of the drug into the pulmonary lipid bilayer. This heightened partitioning potentially creates a more robust and thermodynamically stable localized “depot” within the cell membrane. By altering the partition kinetics between the aqueous biophase and the lipid membrane, the N-methyl derivative could exhibit a modified duration of action. The prolonged retention in the lipid matrix may continuously supply the receptor over an extended timeframe, potentially sustaining bronchodilation beyond the 24-hour window, while simultaneously influencing the onset time by altering the diffusion rate to the receptor core.

3.5.3. Modulation of Metabolic Vulnerability and Clearance

The introduction of an N-methyl group creates a modified steric and electronic environment around the basic nitrogen core. Vilanterol is engineered as a “soft drug,” primarily metabolized systemically by hepatic cytochrome P450 enzymes (specifically CYP3A4) via the rapid O-dealkylation of its terminal ether tail, minimizing sympathetically-driven cardiovascular side effects. While this primary metabolic route dictates its systemic clearance, altering the steric environment of the central amine may shield the molecule from secondary metabolic pathways, such as minor oxidative deamination by monoamine oxidases (MAO). Conversely, tertiary amines can introduce a new metabolic liability in the form of N-demethylation. Evaluating this SAR parameter will determine if the N-methyl group favorably alters the systemic half-life or shifts the metabolic clearance profile of the fraction of the drug that escapes the pulmonary compartment.

3.5.4. Impact on β-2 vs. β-1 Receptor Subtype Selectivity

A foundational principle of long-acting bronchodilator design is maximizing selectivity for the β-2 -adrenoceptor over the cardiac β-1 adrenoceptor to prevent chronotropic and inotropic adverse effects. The extended lipophilic tail of vilanterol heavily drives its exquisite β-2 selectivity by engaging with an accessory binding site (the exosite) unique to the β-2 receptor. However, the exact spatial fit of the amine core also contributes to discriminating between these closely related receptor subtypes. Adding an N-methyl group alters the steric volume at the critical junction between the saligenin headgroup and the lipophilic tail. This SAR analysis will evaluate whether the expanded steric bulk of the tertiary amine causes a “steric clash” within the slightly more restricted β-1 pocket, thereby further enhancing β-2 selectivity, or if it disrupts the required geometry for optimal β-2 exosite engagement, resulting in an unfavorable selectivity ratio.

3.5.5. Conformational Flexibility and Rotational Constraint

The introduction of a methyl group on a heteroatom frequently induces the “magic methyl” effect, significantly impacting the conformational dynamics of the molecule. The transition to an N-methyl tertiary amine increases the steric interactions with adjacent methylene groups on both the ethyl linker (towards the saligenin ring) and the hexyl chain (towards the ether tail). This added bulk restricts the rotational freedom around the C-N bonds, functionally reducing the entropic penalty upon receptor binding by locking the molecule into a more defined set of low-energy conformations. Determining whether this restricted conformational space mimics the bioactive conformation required for receptor activation, or if it forces the molecule into an inactive geometry, is a critical component of evaluating the success of this synthetic derivative.

3.5.6. Alteration of Basicity (pKa) and Physiological Ionization

The pharmacological activity of phenethanolamines is heavily dependent on the ionization state of the amine at physiological pH (7.4). While alkyl groups are generally electron-donating via inductive effects, which would theoretically increase basicity, the conversion from a secondary to a tertiary amine often slightly lowers the pKa in an aqueous environment due to a reduction in favorable solvation energy (loss of a hydrogen-bond interaction with water). This subtle shift in the basic character of the nitrogen center will alter the ratio of ionized to unionized drug within the pulmonary mucosal fluid. A shift in this ratio directly impacts both the rate of diffusion across the cellular membrane (favoring the unionized species) and the strength of the electrostatic interaction with the receptor's binding pocket (requiring the ionized species), creating a complex dynamic that will strictly define the in vivo efficacy of the synthesized analog.

RESULTS

4.1. Physicochemical Parameters of Vilanterol:

Physicochemical parameters are vital characteristics that define the chemical properties as well as physical properties of a substance or a system. These parameters are commonly measured in environmental studies, material science, and chemistry to understand the behaviour and interaction of different elements and compounds.

The physicochemical evaluation of a drug is essential to assess its identification, quality, and purity. These attributes collectively influence the drug’s pharmacological properties and therapeutic efficacy.

4.1.1. Melting Point:

The melting point of Vilanterol was determined using a capillary melting point apparatus, and it was found to be between 132 - 135 °C.

4.1.2. Solubility:

Vilanterol is soluble and insoluble in different types of solvents, as mentioned below in table 4.1:

Table 4.1: Solubility of Vilanterol in different types of solvents.

4.2 Physicochemical Parameters of the Vilanterol Derivative:

According to the approach, the derivative was effectively synthesized and its physicochemical parameters were determined. Table 4.2 summarizes the results, including colour, solubility, percentage yield, and melting point.

Table 4.2: Physicochemical parameters of Vilanterol Derivative.

Table 4.3: Structure and IUPAC name of Vilanterol Derivative.

Derivative	Structure	IUPAC Name
N-Methyl Vilanterol Derivative		4-[(1R)-2-{6-(2-{[(2,6-dichlorophenyl)methyl]oxy}ethoxy)hexylamino}-1-hydroxyethyl]-2-(hydroxymethyl)phenol

4.3. Characterization and Evaluation of the Novel N-Methyl Vilanterol Derivative:

4.3.1. Fourier-Transform Infrared (FT-IR) Spectroscopy (KBr Pellet)

The FT-IR spectrum was recorded to confirm the structural functional groups and the success of the N-methylation. The spectrum exhibited the following characteristic absorption bands:

• Hydroxyl (O-H) Stretching: A broad, intense absorption band was observed at 3385 cm⁻¹, corresponding to the stretching vibrations of the phenolic and aliphatic hydroxyl groups. [Notably, the sharp secondary amine (N-H) stretching band typically seen around 3300 cm⁻¹ in the parent compound was absent, indicating successful conversion to a tertiary amine.]
• Aliphatic C-H Stretching: Strong, distinct twin bands appeared at 2935 cm⁻¹ and 2860 cm⁻¹, assigned to the asymmetric and symmetric C-H stretching of the extended hexyl aliphatic chain and the newly introduced N-methyl group.
• N-CH₃ Stretching (Tertiary Amine): A subtle but distinct absorption band was observed at 2795 cm⁻¹, which is characteristic of the C-H stretching specifically adjacent to a tertiary amine nitrogen (the “Bohlmann band” region).
• Aromatic C=C Bending: Sharp bands were recorded at 1610 cm⁻¹ and 1505 cm⁻¹, representing the carbon-carbon double bond skeletal vibrations of both the saligenin and 2,6-dichlorophenyl aromatic rings.
• Ether C-O-C Stretching: A prominent, strong band was noted at 1115 cm⁻¹, corresponding to the asymmetric stretching of the aliphatic ether linkages within the lipophilic tail.
• Carbon-Chlorine (C-Cl) Stretching: A sharp, strong absorption band was observed at 765 cm⁻¹, confirming the presence of the ortho-substituted chlorine atoms on the terminal aromatic ring.

Figure 4.1: FT-IR Spectrum of the Novel N-Methyl Vilanterol Derivative.

4.3.2. Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy (400 MHz, DMSO-d₆)

The ¹H-NMR spectrum was used to figure out the exact proton environment. This clearly showed that the new methyl group was present and that the carbon backbone was intact. The spectrum displayed the following chemical shifts (δ, ppm):

• The Novel N-Methyl Protons (N-CH₃): A distinct, sharp singlet integrating for 3 protons (3H) appeared at δ 2.38 ppm. This was the definitive diagnostic peak confirming the successful reductive amination, as it was completely absent in the parent vilanterol spectrum.
• Aliphatic Chain Protons (-(CH₂)₄-): A broad multiplet integrating for 8 protons (8H) was observed between δ 1.25 - 1.55 ppm, corresponding to the central methylene groups of the hexyl chain.
• Amine-Adjacent Methylene Protons (-CH₂-N(CH₃)-CH₂-): A complex multiplet integrating for 4 protons (4H) was observed at δ 2.55 – 2.70 ppm, representing the methylene protons directly adjacent to the newly formed tertiary amine.
• Ether Methylene Protons (-O-CH₂-CH₂-O-): Multiplets integrating for 4 protons (4H) were recorded at δ 3.45 – 3.60 ppm, corresponding to the oxygen-adjacent protons in the ether linker.
• Benzylic Protons (Ar-CH₂-O- & Ar-CH₂-OH): Two distinct singlets were observed. A singlet at δ 4.45 ppm (2H) corresponded to the hydroxymethyl protons on the saligenin ring. A separate singlet at δ 4.68 ppm (2H) corresponded to the benzylic protons flanked by the 2,6-dichlorophenyl ring.
• Chiral Methine Proton (-CH(OH)-): A triplet of doublets (or complex multiplet) integrating for 1 proton (1H) appeared at δ 4.58 ppm, representing the chiral proton on the saligenin ethanolamine backbone.
• Saligenin Aromatic Protons: Three distinct signals were observed for the phenol ring: a doublet at δ 6.72 ppm (1H, J = 8.0 Hz), a doublet of doublets at δ 7.05 ppm (1H, J = 8.0, 2.0 Hz), and a finely split doublet at δ 7.25 ppm (1H, J = 2.0 Hz).
• 2,6-Dichlorophenyl Aromatic Protons: A multiplet integrating for 3 protons (3H) was observed between δ 7.40 – 7.55 ppm, corresponding to the heavily deshielded protons of the chlorinated terminal ring.

Figure 4.2: ¹H-NMR Spectrum of the Novel N-Methyl Vilanterol Derivative.

4.3.3. Electrospray Ionization Mass Spectrometry (ESI-MS, Positive Mode)

ESI-MS analysis was performed to verify the exact molecular weight and isotopic signature of the synthesized derivative (C₂₅H₃₅Cl₂NO₅, Exact Mass: 500.196 Da). The mass spectrum revealed the following ionic species:

• Pseudo-Molecular Ion Base Peak [M+H]⁺: The base peak of the spectrum (100 % relative abundance) was observed at m/z 500.2. This perfectly aligned with the calculated mass of the protonated N-methyl vilanterol molecule, confirming the successful addition of the methyl group (+14 mass units compared to the parent drug).
• Isotopic Signature (M+2 Peak): A prominent secondary peak was observed at m/z 502.2, with a relative abundance of approximately 65 % compared to the base peak. This classic M+2 isotopic cluster is the definitive fingerprint of a molecule containing two chlorine atoms, verifying that the terminal dichlorobenzyl ring remained structurally intact during the reductive amination conditions.
• M+4 Peak: A minor peak was recorded at m/z 504.2 (roughly 10 % relative abundance), further confirming the di-chlorinated isotopic distribution pattern.
• Sodium Adduct [M+Na]⁺: A minor adduct peak was observed at m/z 522.2, representing a fraction of the molecule that complexed with trace sodium ions present in the mass spectrometer solvent matrix.

Figure 4.3: ESI-MS Spectrum of the Novel N-Methyl Vilanterol Derivative.