Synthetic Strategies for Warfarin and Its Deuterated Analogues: Implications for Anticoagulant Therapy

The heavier, non-radioactive stable isotope of hydrogen that occurs naturally is called deuterium. Deuterium was discovered in 1931 by Harold Urey, who was given the Nobel Prize in 1934 for his discovery. An essential tool for understanding the mechanisms of chemical reactions is the deuterium isotope effect. One proton, one electron, and one neutron make up deuterium, thus doubling its mass without significantly changing its characteristics. The C-D bond, on the other hand, is a little shorter, has less electrical polarizability, and stabilizes surrounding bonds by hyper conjugation (including the developing anti-bonding orbital of a newly forming bond). It has the ability to inhibit van der Waals stabilization and to produce other difficult-to-predict properties like alterations. [1]

1.1 General introduction

The first deuterated molecule patent in the country was issued in the 1970s. Since then, patents for deuterated drugs have increased in frequency.[2] The application of the deuterium isotope effect has become more widespread throughout time and is currently applied in a variety of industries. [3]Research on pharmacokinetics and mechanistic investigations of drug metabolism (PK) are important. Efficacy, acceptability, bioavailability, and safety are also important considerations. Deuterated drug candidates first appeared in the year 2000. In the 1970s, deuterated metabolites were initially examined. The government finally took action after more than 40 years, though. Deutetrabenazine was the first deuterated drug to receive FDA approval. Many publications have discussed this subject. [3]

The first deuterated molecule patent in the country was issued in the 1970s. [3]Since then, therehave been numerous patents on deuterated medicines. The use of the deuterium isotope effect has gained prominence over time and is currently utilized frequently in many different fields. Research on pharmacokinetics and mechanistic investigations of drug metabolism (PK)are also important. Efficacy, acceptability, bioavailability, and safety are also important considerations. Deuterated drug candidates first appeared in the year 2000. In the 1970s, deuterated metabolites were initially examined. [3]. The government finally took action after more than 40 years, though Deutetrabenazine was the first deuterated drug to receive FDA approval. Many publications have discussed this subject.  The advantages and disadvantages of deuterated medicine. [3].

The conversion of a protonated drug into a deuterated form is an efficient way to change the physicochemical characteristics of many medicinal compounds. The interconversion of vitamin K and vitamin K epoxide, as well as vitamin K's function in the carboxylation of a number of clotting cascade proteins, are interfered with by this anticoagulant, which prevents the start of clotting. 1 Warfarin-containing medications have been used to treat and prevent blood clots in the context of cardiac valve replacement, pulmonary embolism, venous thrombosis, and atrial fibrillation.[4]

1.3 Advantages of deuterated warfarin over warfarin

• Because a deuterated form of warfarin breaks down more slowly, it remains in the body for longer and needs fewer doses (in terms of strength & regimen).
• Deuterated warfarin has a much lower rate of metabolism and a prolonged half-life due to the kinetic isotope effect.
• Deuterium warfarin may also lessen toxicity by lowering the production of harmful metabolites.
• In addition, there are fewer drug-drug interactions thanks to the deuterated warfarin's increased stability in the presence of other medications.

1.4 Methods of deuteration

(A) Biodeuteration

Depending on the needed level of deuteration, the biodeuteration of molecules may involve the usage of heavy water (D₂O) in the host organism's growth medium. With the help of the 13C and

Fig.1.1 Methods of deuteration

(B) Chemical Deuteration

Chemical deuteration is the process of preparing a desired molecule by deuterating complete molecules or molecular building blocks by subjecting them to heavy water (deuterium oxide) at high temperatures and pressures while being in the presence of a catalyst. If necessary, molecules can be created using organic chemistry methods from the deuterated building blocks. All of the deuterated substances produced will be analyzed and characterized using the analytical tools at the chemical deuteration facility. Multinuclear NMR spectroscopy (1H, 2H, 13C, and 31P (for phospholipids)) is used to determine purity and calculate the deuteration amount at various chemical sites. The isotopic abundances of the various isotopologues will also be calculated using MS to provide a measurement of the product's overall deuteration level.[5]

1.5 Brief introduction of warfarin Brand

Names-Coumadin, Jantoven

Chemical Formula-C19H16O

Molecular Weight- 308.3279

Absorption-Completely absorbed from the GI tract. The mean Tmax for warfarin sodium tablets is 4 hours

Volume of distribution-0.14 L/kg of Vd was detected. Label, 17,9 Warfarin D's distribution phase lasts 6 to 12 hours. 17 It has been found to cross the placenta and obtain serum concentrations in the fetus that are similar to those in the mother. [5].

Route of elimination:

Warfarin is almost fully eliminated by metabolism, with just a little quantity excreted unaltered. 8 percent of the whole dose is eliminated in the urine, while the remaining 20% is excreted in the faeces.

Pharmacokinetics

? Highly bound to plasma proteins

? Metabolism occurs through liver

? Substrate of CYP450 enzyme

? Excreted in urine and stool

1 Background history of selected drug

Dicoumarol, the first coumarin, was found after which warfarin was synthesized. In 1948, warfarin was widely used for the first time as rat poison. In 1954, the USFDA approved the use of warfarin as a blood clot preventive in people. There are generic variants of the drug warfarin available. With nearly 14 million prescriptions given, it was the 50th most often prescribed medication in the US in 2019. Factor Xa inhibitors have recently become more well-liked as warfarin alternatives because to a decreased risk of side effects. Warfarin, an anticoagulant medication, prevents blood clots and migration. Warfarin was originally developed as a pesticide, but it has since become the most often administered oral anticoagulant in North America (D-Con, Rodex, and others). [5]

1.5.2 Adverse effects

? Osteoporosis, valve and

? Artery calcification,

? Toe syndrome,

Warfarin use has also been related to drug interactions. Warfarin does not really change blood viscosity; instead, it prevents the creation of multiple regulatory and physiologically active versions of various clotting factors, which are dependent on vitamin K.

1.6 Pharmacology Of Warfarin:

A coumarin derivative called warfarin prevents the cyclic interconversion of vitamin K and its 2,3 epoxide, which is how it works as an anticoagulant (vitamin K epoxide). For the carboxylation of glutamate residues to -carboxyglutamates (Gla) on the N-terminal sections of vitamin K-dependent proteins, vitamin K serves as a cofactor. 1–6 only after being - carboxylated by vitamin K are these proteins, which make up the coagulation factors II, VII, IX, and X, physiologically active. Warfarin stimulates the hepatic synthesis of partly decarboxylated proteins with reduced coagulant activity by blocking the vitamin K conversion cycle. the relationship between the vitamin K cycle and the glutamic acid carboxylation of vitamin K-dependent coagulation proteins. Vitamin K1 from dietary sources is changed into vitamin KH2 by a vitamin K reductase that is resistant to warfarin.[5]

Fig.1.2 Mechanism of warfarin

After synthesis, the deuteration must be verified, and then their medicinal qualities should be evaluated. For deuterated warfarin screening, a number of analytical techniques offer useful information. Deuterated warfarin is analyzed using a variety of physicochemical tests, chromatographic procedures, and spectroscopic techniques. Primary confirmation of salt production is provided by physicochemical tests, while information is provided by spectroscopic techniques like IR and ¹H NMR[6].

Structural modifications that take place as deuterated warfarin is formed. Different methods for analysis of the deuterated warfarin are

Depicted in figure 1.3.

Fig 1.3. Method of analysis of deuterated warfarin

When compared to the original warfarin, deuterated warfarin exhibits various physicochemical features. Here, a few physicochemical characteristics for comparing warfarin with deuterated warfarin are discussed.The pH of the solution changes as deuterated warfarin that is water hyphen solubility forms. Therefore, measuring pH is very important. A drug's solubility is crucial to its capacity to dissolve, absorb, and be bioavailable. Since warfarin has a very low water solubility, one of the initial methods for improving the compound's water solubility is deuterated warfarin production. Finding a substance's melting point is another method for measuring the chemical changes. The changes in melting point indicate structural modifications brought on by reaction completion.

1.8. Chromatographic Techniques for Analysis of deuterated warfarin

1.8.1.Thin Layer Chromatography (TLC):

Because of its convenience of use, availability, high sensitivity, and quick separation time, thin layer chromatography (TLC) is a regularly used analytical technique. The basis of TLC is the idea that a compound will have varying affinities for the mobile and stationary phases, which will influence how quickly it migrates. The components will move along the plate at varying speeds because some components have a larger attraction for the extremely polar silica gel (stationary phase), while others will move to the solvent more quickly. TLC analysis can be used to monitor a reaction's progress, separate a mixture, analyze purity, and purify small quantities of a compound on a preparative scale. Individual components emerge because of the separation.

The formula for RF is,

RF= Distance traveled by sample/Distance traveled by solvent

Because they are specific to each compound, the RF value can be used to identify substances. When comparing two substances under identical circumstances, the substance with a higher RF value is less polar because it does not stick to the stationary phase for as long as the polar substance, which would have a lower RF value.[7]

1.9. Spectroscopic Techniques for analysis of deuterated warfarin

1.9.1. Ultra-Violet (UV) Spectroscopy:

A portion of the light energy would be absorbed by the sample when it is exposed to a wavelength that corresponds to the energy of the electronic transitions within the molecule, and the electrons would then be moved to the higher energy state orbital. A spectrometer logs a sample's absorbance at various wavelengths. The term "max" refers to the wavelength at which the sample absorbs the most light. The Beer-Lambert law controls how much light molecules absorb. The Synthesis and Analysis of Deuterated Warfarin 10 is Related by Beer's Law Lambert's law connects the total absorbance to the optical path length, and absorbance to the concentration of the absorbing solute.

1.9.2. Infrared (IR) spectroscopy:  Some infrared light frequencies are absorbed as it passes through a sample of an organic chemical, while other frequencies pass through the sample undisturbed. An infrared spectrum is produced by plotting the absorbance or transmittance against wave number. Since infrared spectroscopy is essentially vibrational spectroscopy, the main benefit of the method to organic chemists is related to the next finding. Different bonds (C-C, C=C, C==C, C-O, C=O, O-H, and N-H) have different Vibrational frequencies, and we may recognize these unique frequencies as an absorption band in the infrared spectrum to determine the presence of these bonds in an organic molecule. Synthesis and Analysis of deuterated warfarin the well-defined IR regions are given in table 1.2.

No two molecules of a different structure have exactly the same infrared spectrum because every type of bond has a unique inherent frequency of vibration and because two of the same type of bonds in two distinct compounds are in two slightly different surroundings. We can determine whether two substances that are believed to be identical are indeed identical by comparing the infrared spectra of the twosubstances.

Finding out a molecule's structural details is a second, more crucial usage of the infrared spectrum. Each type of bond's absorption—N-H, C-H, O-H, C-X, C=O, C-O, C-C, and C=C—is typically only observed in a tiny section of the Vibrational IR area. For each kind of link, a constrained range of absorption can be established.

1.9.3.¹H Nuclear Magnetic Resonance spectroscopy:

NMR, or nuclear magnetic resonance, is the most efficient method for acquiring structural information. The number of atoms of the type being examined that are mechanically distinct is indicated by NMR. When we use NMR spectroscopy to study a molecule, we may note changes in the magnetic properties of the different nuclei that are present and estimate, to a significant extent, where these nuclei are located inside the molecule. It would be simple to calculate the amount of each different type of hydrogen nuclei and the characteristics of each type's immediate environment when hydrogen (proton) nuclei are studied..[9]

Because it has both an electric charge and a mechanical spin, the proton, the nucleus of the Predicting the ¹H NMR spectrum of an organic compound begins with predicting the chemical shift, Positions for the different hydrogen are in the molecule.

Figure 1.4 is a useful chart containing approximate proton classification

Figure.1.4: Approximate chemical shift positions for protons in organic molecules. Introduction to the present work:

Figure.1.5 Structure of deuterated warfarin

Drug profiles of warfarin and deuterated warfarin are given in table no 1.3

Aim of the work: To synthesize the deuterated warfarin and to perform its analysis.

Objective of the work: The objectives of the work are listed below: Optimize the process parameters for synthesis of deuterated warfarin. When compared to protonated warfarin, deuterated warfarin has a reduced toxicity

EXPERIMENTAL WORK

4.2 Chemicals and reagents: Chemicals used during the project work are mentioned in

4.3.1. Method of preparation of warfarin

Step 1.Synthesis of benzalacetone

8.0 g (11.0 moles, 800 cc) of acetone and 4.02 g (400 cc, 4.0 moles) of benzaldehyde were combined with 4 ml (0.22moles) of water in a round bottom flask equipped with a mechanical stirrer. To this,1 ml (0.025moles) sodium hydroxide (10%) was added. The solution was stirred for 3.5 hours. Then the solution was cooled. Completion of the reaction was confirmed by thin layer chromatography. The reaction was quenched using diluted HCl. Ethylacetate was added to this solution and the organic layer was separated and dried over sodium sulphate. Ethylacetate was evaporated to obtain the titled compound.[9] Benzalacetone was synthesized from benzaldehyde and acetone. The scheme of synthesis depicted in fig 4.1

Step 2 Synthesis of Warfarin:

Using a reflux condenser and stirrer connected to a flask, added the 4 hydroxycoumarin (0.308 moles,5.0gm) and benzalacetone (0.0342 moles,5.0gm), 35ml H₂O(1.942moles), and ammonia (0.006moles,0.11ml) all added the same time. The solution was boiled and kept at a constant temperature for 2.5 hours, during which time a thick precipitate formed. Refluxing was continued for an hour with mild agitation. Reduced the temperature of the reaction mixture to room temperature. The product was filtered to separate the solid. [11]. The solution was floated in fresh water until it was completely dry. Hydrochloric acid was used to neutralise the base NaOH (dilute). Using distilled water, the product was washed several times. The final product was isolated and characterized using various spectral, chromatographic studies. Scheme of synthesis is depicted in fig 4.2

Fig 4.2. Synthesis of warfarin

4.3.2Method of preparation of deuterated warfarin

Step1: Synthesis of deuterated salicylaldehyde:

A solution of potassium butoxide (0.20g, 0.2303 moles) in D₂O (4.15g, 0.2303 moles) was prepared and rapidly added to phenol (1.0g, 0.0078 moles). The resultant solution was heated on a water bath between 70ºC and 80ºC. CDCl₃ is added gradually drop by drop over 1.5 hours.Then stirring was further continued for a further an hour. The reaction mixture was allowed to cool down. After cooling 1.74g (0.04 moles) of HCl was added while vigorously swirling the mixture to make it acidic. The separated oil was combined with a sizable amount of Nacl (1.5g). Enough water (D₂O) was added to completely dissolve the ingredients.

The product was taken from the oil and hot water was used to wash it multiple times. The product was recrystallized using ethanol (0.75g, 0.0162moles).[11].

The ratio of salisaldehyde to zinc dust for the preparation of benzaldehyde should be 1:1.2. For each milliliter of salisaldehyde, was added 0.823g (0.0067moles) of zinc. Solution was heated in a water bath to 130°C. Utilizing Whatmann filter paper, the solution was filtered and was allowed to cool. Ethyl acetate was added and the solution was kept overnight. Ethyl acetate was evaporated. And the product was collected.[12]

Step 3: Synthesis of deuterated benzalacetone:

8.0g (11.0 moles, 800 cc) of acetone and 4.02 g (400cc, 4.0 moles) of deuterated benzaldehyde were combined with 4(0.22moles) ml of water in a round bottom flask equipped with a mechanical stirrer. To this, 1 ml sodium hydroxide (10%) was added.The solution was stirred for 3.5hours. Then the solution was cooled. Completion of the reaction was confirmed by thin layer chromatography. The reaction was quenched using diluted HCl. Ethylacetate was added to this solution and the organic layer was separated and dried over sodium sulphate. Ethylacetate was evaporated to obtain the deuterated benzalacetone.

Step 4: Synthesis of deuterated warfarin:

Using a reflux condenser and stirrer connected to a flask, added to the 4 hydroxycoumarin (0.0061moles,1.0gm) and deuterated benzalacetone (0.0068 moles,1.0gm), 7ml  H2O(0.388moles), and ammonia (0.012moles,0.022ml) were added. The solution was boiled and kept at a constant temperature for 2.5 hours, during which time a thick precipitate was formed. Refluxing was continued for an hour with mild agitation. The reaction mixture was allowed to cool to room temperature. The product was filtered to separate the solid. The solution  was floated in fresh water until it was completely dry. Hydrochloric acid was used to neutralise the base NaOH (dilute). Using distilled water, the product was washed several times.Final product was isolated and characterized using various spectral, chromatographic studies.[11] Warfarin was synthesized from benzalacetone and 4- hydroxycoumarin. Scheme of synthesis depicted in fig.4.3.

Fig.4.3. Synthesis of deuterated warfarin

Another method used for benzalacetone synthesis:

Method 1:

In a 100 ml conical flask, benzaldehyde (2.2345 mol, .2 ml) and acetone (2.2324 mol, 2.6 ml) were added. NaOH (2 g) pellets were then added to ethanol (20 ml) and distilled water (20 ml) and thoroughly mixed. Then, this solution was added to the acetone and benzaldehyde combination, and it was continually swirled for 38 minutes. Using distilled water as a subpar solvent, the result was then suction filtered from the filter paper. Before the solvent was removed using suction filtration, the crude product was recrystallized using hot ethyl acetate as the solvent heated to 150oC. On a piece of filter paper placed on a watch glass, the crystals were spread out and cured under a lamp for eight minutes. Yield and melting point information for the.[11]

Method 2:

At room temperature, benzaldehyde (0.02 mol, 2.12 g) was added drop wise to a solution of NaOH (0.05 mol, 2 g) in aqueous ethanol (1:1). Acetone (0.02 mol, 1.17g) was added drop wise and swirled for 30 minutes after continuing to mix for another 10 minutes. The reaction mixture received water (20 ml), which was then filtered. The item was purified by re-crystallizing from ethanol, rinsed with water (20 ml x 3), and then allowed to dry.

Another method used for warfarin synthesis:

Method 1: About 8 hours at 50 °C were used mixing 4-Hydroxycoumarin (1 mmol), benzalacetone (2 mmol), and [brim] BF4 (1 mmol). Ethyl acetate was used to extract the final product 3 after water (5 ml) was added (2.5mL). The organic phase was dried using Na2SO4 anhydride. In order to produce warfarin 3, the solvent was evaporated. Melting point and TLC were used to identify compound 3 after synthesis..[11]

Method 2: In a flask connected with a reflux condenser and a stirrer, the following reagents were combined: 1.5 g of 4-hydroxycoumarin, 1.5 g of benzalacetone, 35 cc of water, and 0.11 cc of ammonia. A sizable precipitate formed after the mixture was heated to a boil and kept at reflux for 2:30 hours. During refluxing, the reaction mixture was rapidly stirred for a further hour before being cooled to room temperature. The solid crude product was removed through filtration, after which it was floated in fresh water and as dryly as possible sucked. The solid crude was dissolved in benzene, refluxed for 45 minutes with stirring, cooled, filtered, rinsed with new benzene while on the filter, and sucked as dry as possible..[11]

Method 3: 4-hydroxycoumarin (1 mmol), benzalacetone (2 mmol), and [brim] Br (1 mmol)  were combined for 5 hours at room temperature. The resultant product 3, which contained water, was then extracted using ethyl acetate (2x5mL). The organic phase was dried using Na₂SO₄ anhydride. Additionally, the solvent was evaporated to get pure warfarin 3.[11]

4.3.3. Determination of physicochemical properties: The general procedures to evaluate the physicochemical properties of warfarin and deuterated warfarin are discussed in below:

4.3.3.1. Determination of pH: The pH of warfarin and deuterated warfarin was determined by using digital pH meter. The pH meter was calibrated with a standard buffer solution (pH 4 and pH 7) before use.

4.3.3.2. Melting point: The capillary containing the sample was placed in the digital melting point apparatus. The temperature was gradually increased at the rate of 20ºC per min, till the temperature reached 90ºC and thereafter at the rate of 10ºC per min. The temperature was noted when the sample starts melting. This was done in triplicate for both samples.

4.3.3.3. Solubility: Solubility was determined as per the Indian Pharmacopoeia (IP). 1 gm of substance was dissolved in Different solvents, such as water, methanol, chloroform, tetrahydrofuran (THF), dimethylsulfoxide (DMSO), etc. The amount of solvent required dissolving warfarin and deuterated warfarin was noted as per solubility criteria of I.P. and U.S.P.

4.3.3.4. Chromatographic Analysis: Chromatographic analysis was done using TLC to get resolution between warfarin and deuterated warfarin.

4.3.3.5. Thin Later Chromatography (TLC): To resolve the situation between warfarin and deuterated warfarin, different movable phases were tried. Because deuterated warfarin is more polar than warfarin, which is a less polar molecule, it was suggested that it should move up the TLC plate slowly or not at all (lower RF value) .A, few representative trials are shown in the table 5.3 of section 5.2.1.

4.3.3.6. Spectroscopic techniques for analysis: Spectral analysis of deuterated warfarin and warfarin was done by using UV spectroscopy, IR spectroscopy, ¹H NMR spectroscopy. The product was characterized partly in-house and partly outsourced to Diya Labs and Dr. M. K. Rangnekar Memorial Drug Testing Laboratory.

4.3.3.6.1. Ultra-Violet spectroscopy: UV spectra of warfarin and deuterated were compared. The procedure for preparation of 10 ppm solutions of deuterated warfarin; warfarin is discussed in section 5.3.1 (A) of this chapter.  The UV Spectra of warfarin (10 ppm), deuterated warfarin (10ppm) in Acetonitrile: water (1:1) was compared in range of 200-400 nm.[8]

4.3.3.6.2. Infra-Red Spectroscopy: Using an electrically powered KBr press model, KBr was combined with warfarin (100%), deuterated warfarin (100%), and a mixture of deuterated warfarin and warfarin (1:1). The manufactured disc containing deuterated warfarin, warfarin, and a combination was utilized to get the IR spectra using a Jasco FT/IR-4100 type A Fourier transform spectrophotometer. There was a 4000-400 cm-1 scanning range. In order to establish the structural changes in deuterated form, IR analyses of warfarin, warfarin deuterated, and combination (50 percent warfarin deuterated by weight) were conducted. Results were seen in the mid-IR (4000-400 cm-1) and fingerprint region of IR (1500-400 cm-1) of the IR. Results of IR are given in section 5.3.2.

4.3.3.6.3. Proton Nuclear Magnetic Resonance (¹H NMR) Spectroscopy: One of the most important methods for interpreting structural data is NMR spectroscopy. In order to conduct analysis at 400 MHz and identify structural changes, deuterated warfarin and warfarin were dissolved in a suitable solvent, such as deuterated dimethyl sulfoxide (DMSO-d6). Warfarin's and deuterated warfarin's spectra were examined for the presence of the methyl (-CH) proton. Warfarin would contain the -CH proton but not deuterated warfarin. Results of ¹H NMR are given in section 5.3.3.

RESULTS AND DISCUSSION

5.1: Evaluation of physicochemical properties: The synthesized batches of deuterated warfarin as well as warfarin were evaluated for Physicochemical properties such as pH, melting point, solubility. The results of tests are shown in table 5.2

pH- Therefore, the pH of deuterated warfarin was found to be 6.5, whereas the pH of warfarin was 6.

Melting point- The melting point of warfarin and deuterated warfarin was found to be in the range of 155-160 ºC and 150-155ºC respectively. The shifts in melting point indicate the changes in structure dueto the completion of reaction.

Solubility- deuterated warfarin was insoluble in water and soluble organic solvents. Both warfarin and deuterated warfarin was moderately soluble in methanol but was insoluble in water. It was found that both the compounds were soluble in polar aprotic solvents, such as tetrahydrofuran and dimethyl sulfoxide.

5.2. Chromatographic analysis of warfarin and deuterated warfarin using TLC: Chromatographic techniques were conducted to obtain resolution between warfarin and deuterated warfarin The results of TLC are discussed in the following sections.

5.2.1. Thin Layer Chromatography (TLC): The affinities of substances for the stationary and mobile phases are the base of TLC, as they are for other chromatographic techniques. Due to the mobility, substances that have a higher affinity for the stationary phase travel slowly while the other substances move more slowly. Between warfarin and deuterated warfarin. To achieve resolution between Deuterated warfarin and warfarin, numerous mobile phases were considered. Few representative trials are shown below in table 5.5.

Comparative spectral analysis was done to check for the absence of any interference .Different spectroscopic techniques, such as Ultra Violet (UV) spectroscopy, Infra-Red (IR) spectroscopy, and Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy were tried. Results of each technique are given in the sections below.

5.3.1. Ultra Violet (UV) spectroscopy: The UV spectra of deuterated warfarin (10 μg/mL), warfarin (10 μg/mL), were compared. The sample preparation of 10μg/mL solutions of, warfarin and deuterated warfarin are given in section 4.3.3.1 (A) of chapter. UV spectra for deuterated warfarin (mg/mL), warfarin (mg/ml) sample mentioned above are depicted in figures 5.7 to 5.8 and results of absorbance are depicted in table 5.6. ACN: Water (1:1) was chosen as diluents since both deuterated warfarin well as warfarin had to be solubilized. UV.[8]

Fig 5.3. UV spectrum of (10μg/mL) Table.5.4 Results of UV analysis

The UV spectra of warfarin and deuterated warfarin were comparable. Deuterated warfarin displayed a lower absorbance than warfarin. 5.3.2 Infra-Red Spectroscopy:  IR spectra of warfarin and deuterated warfarin was recorded and results were observed in(400cm^-1- 4000 cm -1) and fingerprint region between 400cm^-1-1500cm^-1 to confirm the structural changes in deuteration. Results of IR analysis are shown in fig 3 and comparison of bands present in warfarin and deuterated warfarin is shown in fig 5.4 and fig 5.5.

Fig.5.5.IR spectra of deuterated warfarin (10ppm)

It was attempted to detect structural alterations using 1H NMR analysis. Additionally, warfarin rather than deuterated warfarin would show the C-H proton on each carbon. Warfarin and deuterated warfarin's 1H NMR analyses are depicted in fig, respectively. Interpretation of NMR spectrum of deuterated warfarin is given in table no 5.6 and 5.7.

Fig. 5.7.1H NMR spectrum of deuterated Warfarin in DMSO-d6 measured at 400 MH

Methyl protons of carbonyl group (C12) appear as uncoupled singlet at δ 2.22 ppm whereas the C7 proton appears at δ 7.36 ppm (triplet). Donating electron density causing the C6 proton to shift up field to δ 6.98 ppm (doublet) while the C8 proton appears at δ 7.19 ppm (triplet) as stated in figure. While C12 protons appear as δ2.2 (doublet) and C10 protons appear as δ3.2 (singlet).

Fig.5.8. Structure of Warfarin

Fig 5 9.1H NMR spectrum of Warfarin in DMSO-d6 measured at 400 MHZ

Warfarin's deuteration can be distinguished by a distinctive downfield chemical change at 4.8 ppm. When compared to the C7 proton, methyl protons of the carbonyl group (C12) emerge as uncoupled singlets at 2.22 ppm (triplet). The C6 proton shifts up field to 6.98 ppm (doublet) due to the electron density donation, but the C8 proton appears at 7.19 ppm (triplet), as shown in the figure. While C10 protons appear as 3.2 and C12 protons as 2.2 (doublet), respectively (singlet).

Some representative trials of 1H NMR of Warfarin:

Trial 1) ¹ H NMR Of deuterated warfarin

Trial 2).¹H NMR Of deuterated warfarin

CONCLUSION: