Two Steps Non-Enzymatic Synthesis of
Molnupiravir, which is Free from Mutagenic Impurity and Analytical Method
Development for Estimation of Genotoxic Impurity (Hydroxylamine Hydrochloride
Content) by using
RP-HPLC Technique
Sunandana Akkala, Govinda Gopalakrishna Kilaru, Gopi Bandreddy, Suman
Baindla Madhusudhan Gutta*
Vijayasri Organics, IDA Bolloram,
Jinnaram, Medak, Telangana-502325.
*Corresponding Author E-mail:
gopalkilaru@gmail.com
ABSTRACT:
Genotoxic impurity free Molnupiravir was synthesised
by a novel and an elegant non-enzymatic method, which is simple operationally
and ecologically. The genotoxic raw material, Hydroxylamine, was used in the
initial stages of synthesis to get the mutagenic impurity free Molnupiravir. A
new and sensitive HPLC method was developed and validated for the determination
of hydroxylamine in Molnupiravir drug substance according to ICH guidelines.
The HPLC method was developed and optimized on Zorbax SB C18 150×4.6mm, 3.5µm
column with oven temperature maintained at 40°C. 1.0 mL of Orthophosphoric acid
in 1000 mL water was selected as mobile phase in isocratic reverse phase mode.
Chromatographic parameters are flow rate: 0.8 ml/min, wavelength detection: 252
nm, injection volume: 10µl and run time: 20 min. Based on the validation data;
the method was found to be specific, sensitive, accurate and precise. This
method can be used as a good quality control tool for quantification of
hydroxylamine at the low level. The experimental data was deliberated in detail
in this research paper.
KEYWORDS: Molnupiravir, Non-enzymatic, Hydroxylamine, mutagenic
impurity, Synthesis Recovery, RP-HPLC, LOD, LOQ, linearity and accuracy.
INTRODUCTION:
4-Oxime-5`-(2-methylpropionyl) uridine compound known
as molnupiravir (development codes EIDD-2801, MK-4482) is a prodrug of an oral,
biologically active ribonucleoside analog β-d-N4-hydroxycytidine (NHC;
EIDD-1931). The active drug is incorporated into the genome of RNA viruses,
leading to an accumulation of mutations known as viral error catastrophe. As
per the recent studies, EIDD-2801 was shown to have a broad-spectrum antiviral
activity against the pathogens of SARS-CoV-2, MERS-CoV, SARS-CoV and COVID-19. Molnupiravir
may prove to be an effective antiviral drug against different infections.
As oral medications are convenient to use, they can be
used as preventive medications and are also suitable for inpatients as well
outpatients.
Currently, there are limited synthetic routes known
for preparation of the 4-oxime-5`-(2-methylpropionyl) uridine compound.
One of the synthetic routes as suggested in the patent
application WO20191134621, uses uridine as the starting material.
This approach includes first protecting the dihydroxy group, esterification
with isobutyric anhydride, followed by reaction with 1,2,4-triazole. In this
route, uridine used is costly and it gives lower yields thereby rendering the
process inefficient and commercially nonviable at an industrial level.
In Chinese patents, CN112608357A2 and most
patent applications involve the use of enzymes. As known in the art, enzymes
are highly costly materials requiring much longer reaction cycles and
consumption of large quantities of solvents or buffered aqueous medium. Thus,
such approaches are industrially not feasible. In the non-enzymatic synthesis
hydroxylamine, sulphate was used in last stages of API isolation; hence, it
might be difficult to remove the mutagenic impurity in isolated drug substance.
Snead3 and co-workers reported a two-step
concise synthesis of molnupiravir in a total yield of 75% by column
chromatography (Scheme 2). This route was characterized by enzyme-mediated
selective esterification and direct hydroxyamination, but the cost of this method
limited its industrial application. In addition, they provided an enzyme free
synthetic route from cytidine, which was similar to the initially reported
route of synthesis except for the hydroxyamination stage (Scheme 3). This
synthesis provided a relatively higher overall yield (44%) than that of the
initial synthesis, but it also suffered from the hydrolysed impurities in the
final step due to the use of acetonide protection.
Novel route of
synthesis of Molnupiravir:
Accordingly, there remains a need for an improved
process for a synthesis of molnupiravir and related intermediates that can help
to overcome the shortcomings of the processes known in the art. It is required
to fulfil the need for a process for the synthesis of molnupiravir which
involves minimum use of solvents, can be carried out in non-enzymatically, with
lower time cycle and higher yield without requiring additional steps of
purifications to provide an industrially scalable and commercially and ecologically
viable synthetic process. There also remains an unmet need of intermediate
compounds, which can curtail formation of impurities during the process to
provide the compounds with high purity. It is evident that the key to the
achievement of the goal is to find treating the cytidine with hydroxylamine
sulphate in DM water and protecting groups masking the 2’, 3’-hydroxyl groups
as well as being deprotected under mild conditions. Herein, we discovered that
N,N-Dimethylformamide dimethyl acetal (DMF-DMA)4 could serve as an
excellent protecting agent. After protective esterification with Isobutyric
anhydride, then simple deprotection to get the Mutagenic free Molnupiravir API
in high yield5, complying with ICH specification.
General Information:
The materials and reagents, including cytidine,
N,N-dimethylformamide dimethyl acetal (DMF-DMA), hydroxylamine sulfate,
isobutyric anhydride, triethylamine (NEt3), N,N-dimethylaminopyridine
(DMAP),tetrahydrofuran, dichloromethane, benzaldehyde and isopropyl alcohol
were commercially available. 1HNMR, 13C NMR and 31P NMR spectra were determined
on JNM 400MHz, Joel FT NMR Spectrometer instrument in DMSO-d6 with TMS as a
reference. LC-MS, Shimadzu using the electrospray ionization mode. Reactions
were monitored by thin-layer chromatography (TLC) on 25.4×76.2 mm silica gel
plates
Hydroxylamine:
Hydroxylamine and its salts are commonly used as
reducing agents in a myriad of organic and inorganic reactions. It can also act
as antioxidant for fatty acids and used to prepare oximes, an important
functional group in the synthesis of molnupiravir, hydroxylamine sulphate is
used in the preparation of N-Hydroxycytidne6,7 intermediate from cytidine. Hydroxylamine
Sulphate was reported to be mutagenic in the mouse lymphoma tk mutation assay,
with and without metabolic activation but the data do not convincingly meet the
up-to-date criteria for positive results in this assay. It is considered that
hydroxylamine induces tumours via a mode of action with a threshold (i.e.,
hemosiderosis of the spleen). An increase in tumours was observed in male rats
at ≥ 5 ppm or 0.2 mg/kg/day for hemangiosarcomas and females at the high
dose of 80 ppm or 6.2 mg/kg/day (hemangiosarcomas and hemangiomas). By
referring available literature, ICH. M7(R1) –step-2 [8], concluded that the lowest observed adverse effect
level (LOAEL) in the 2-year rat study was 0.2 mg/kg/day8 in males
and according to this NOEL value lifetime PDE of hydroxylamine is 23µg/day. By
considering this PDE information, It was important to develop a sensitive,
rugged and reproducible HPLC method for the determination of genotoxic impurity
in the drug substance as well as formulations.
Experimental procedures: Stage-1:
To a four-neck round bottom flask equipped with an
overhead stirrer, condenser, thermometer socket and a stopper. charged cytidine
(100.00 g, 1.0 eq), hydroxylamine sulphate (41.2 g, 1.5 eq) and distilled water
(500 mL). The mixture was stirred and heated to 70-75°C. After the solution
turned clear, it was stirred for a total of 3 hours at the same temperature (70
şC). TLC monitored the reaction. The heating was turned off, suspension allowed
to slowly cool to ambient temperature 30°C, then cooled to an internal
temperature 0-5°C using an ice-salt bath and stirred for additional 3 hours.
The solids were isolated by filtration through buchner funnel, washed with
ice-cold water (100 mL), and dried under vacuum oven (50 şC) for an overnight
to afford a white crystalline solid with 84% yield (95.0 g), HPLC. 1H NMR (400
MHz, DMSO-D6): δ 6.83 (d,1H), 5.71 (d,1H), 5.59 (dd,1H), 5.37 (d,1H), 5.22
(d, 1H), 4.22 (m, 2H), 3.98 (m, 1H), 3.90 (dd, 2H) ,2.54(m,1H),1.10(S,6H) ppm.
LC-MS, Shimadzu. The Peak obtained at m/z 260.31 in ESI positive indicates the
(M+1) molecular weight of hydroxyl impurity is 259.22.
A four necked RBF flask fitted with a mechanical
stirrer, condenser, thermometer socket and stopper was charged to Stage-1
(50.0g, 19.2mmol), followed by DMF-DMA (27.6, 23.14mmol) and the reaction mass
was stirred at 55-60°C over a period of 6.0hrs under N2 atmosphere. The
progress of reaction was monitored by TLC (mobile phase: 8:2, Methylene
dichloride: Methanol). When the reaction was completed (Stage-1 absent), the
reaction mixture was evaporated under reduced pressure to give the crude
stage-2a, which was used in the next step without purification. The crude
material was dissolved in dichloromethane (350mL) and cooled to 5-10°C.Then
triethylamine (58.0g, 57.27mmol), DMAP (100mg), and Isobutyric anhydride
(45.3g, 28.6mmol) were added to the solution in dichloromethane. The reaction
was stirred for 4hr at 10-15°C under N2 atmosphere. The progress of the
reaction was monitored by TLC (Mobile phase system: 8:2, Methylene dichloride:
Methanol) When the reaction was completed, isopropyl alcohol (300.0ml) was added
to quench the reaction and it was continued under stirring for 3h at that
temperature. The reaction mixture was concentrated to afford the crude product.
That crude material was triturated with isopropyl alcohol (200ml) at 25-30°C
and chilled to 5-10°C.The precipitate was isolated as white solid material. 1H
NMR (500 MHz, CD3OD) δ 6.90(d, 1H), 5.81 (d, 1H), 5.61 (d,1H),
4.28 (d, 2H), 4.13 (t, 1H), 4.09 – 4.05 (m, 2H), 2.63 (multi 1H), 1.17 (dd,
6H).13CNMR (500MHz, CD3OD) δ 176.05, 149.5, 143.39, 129.9,
98.84, 87.82, 80.78, 72.05, 70.01, 63.94, 33.26, 18.85, 18.82.MS (ESI) calculated
forC13H20N3O7 330.12 [M +H]+
was found 330.37.
This HPLC9 method was able to monitor (LOD,
LOQ) by adopting a derivatization method using benzaldehyde. The formation of
Benzaldehyde oxime is schematically explained below.
Derivatization procedure10,11:
Method of analysis:
Chromatographic
purity by HPLC (%):
Instrument:
A High-Performance
Liquid Chromatography equipped with Gradient elution capability, Ultraviolet
detector.
Chemicals and reagents:
Orthophosphoric acid, Benzaldehyde, Methanol,
Acetonitrile and Milli-Q water
Chromatographic
conditions:
Column : Zorbax SB C18 150×4.6mm,
3.5µm
Detector wavelength : 252nm
Injection volume : 10μL
Flow rate : 0.8mL/min
Column temperature : 40°C
Auto sampler temperature : 10°C
Elution mode : Isocratic
Run time : 20.0 min
Preparation of buffer:
Accurately transfer 1.0mL of orthophosphoric acid in
1000mL water, mix well and sonicate.
Preparation of Mobile phase -A:
Buffer: Acetonitrile (60:40)
Preparation of Diluent:
Dissolve 0.2mL of benzaldehyde in a mixture of
acetonitrile and methanol in the ratio of (50:50)
Preparation of
standard stock solution:
Weigh and transfer about 7.2mg of hydroxylamine
standard into a 100mL volumetric flask, add 2.0mL of water and heat the
solution at 40°C for 30minutes, dissolve and make up to the volume with
diluent.
Dilute 1.0mL of above solution into a 50mL volumetric
flask and make up to the volume with diluent.
Preparation of
standard solution:
Pipette out 1.0mL of standard stock solution into a
100 mL volumetric flask and dilute to the volume with diluent.
Preparation of test solution:
Weigh accurately about 25mg of test sample into a 50
mL volumetric flask, add 2.0mL of water and heat the solution at 40°C for 30
minutes, dissolve and make up to the volume with diluent.
Procedure:
Equilibrate the system with mobile phase until stable
baseline is observed. Inject diluent as blank followed by six replicate
standard and test solution in duplicate into the chromatographic system and
record the chromatogram. The
retention time (RT) of derivatized hydroxylamine was 4.3 minutes. The desired
peak is integrate in standard and test solution and derivatized hydroxylamine
is calculated by using below formula.
System suitability criteria:
The system is said to be suitable if,
1. The % of RSD for the peak area of
hydroxylamine obtained from six replicate injections of standard solution
should be not more than 5.0.
Chromatograms:
Figure-1(Blank):
Figure-2(Standard solution):
Figure-3(Test solution):
Calculation:
Calculate the % of hydroxylamine content in test
solution by using the following formula
AT WS 1 1 50
Hydroxyl amine content (ppm) = -------- ×
-------- × -------- × ------- × -------- × 106
AS 100 50 100 WT
Where,
AT =
Average area of hydroxylamine peak from test solution
AS =
Average area of hydroxylamine peak from standard solution.
WS = weight
of the hydroxylamine in the standard solution
WT = weight
of the hydroxylamine in the test solution
Analytical method validation12:
Specificity:
Specificity is the ability of the method to detect and
separate all related substances present in the molnpiravir drug substance.
Molnupiravir drug substance spiked with all known impurities including
hydroxylamine were
injected to confirm any co-elution with a
hydroxylamine derivative peak from any known impurity. There is no interfering
peak at the retention time of the hydroxylamine derivative13,14and15.
The peak is well resolved from all other known impurities.
Chromatograms:
Figure-1 (Blank):
Figure-2 (cytidine impurity):
Figure-3 (Hydroxy impurity):
Figure-5 (Test spike):
Limit of detection and limit of quantification (LOD
and LOQ):
LOD and LOQ concentrations of derivatized
hydroxylamine with respect to Molnupiravir test concentration. LOD is 0.86ppm
i.e. 0.86µg/gm and LOQ is 2.6ppm i.e. 2.6µg/gm. Precision of LOQ is
checked by injecting six replicate injections. Relative standard deviation
(RSD) of peak area of hydroxylamine derivative at LOQ level is observed 1.85%.
Accuracy:
Accuracy of the method was determined by recovery
study. Sample solution was prepared by spiking hydroxylamine at levels of, 50%,
100% and 150% on specification as per test method inject each solution and
calculate the % of recoveries.
Accuracy data:
Table-1:
|
Name of the sample
|
Amount added
|
Amount found
|
%
of recovery
|
|
LOQ level
|
7.24
|
6.91
|
95.5
|
|
50%
|
3.69
|
3.56
|
96.7
|
|
100%
|
7.37
|
7.20
|
97.8
|
|
150%
|
11.06
|
9.90
|
89.6
|
Linearity:
Linearity was checked by preparing solutions at six
concentration levels from 50%, 75%, 100%, 125%, 150% and 200% of specification
level by preparing using hydroxylamine standard solution and each solution was
injected in HPLC.
Linearity was established by using concentration on
X-axis and area on Y-axis and calculation of Statistical value, slope,
intercept, correlation coefficient.
Linearity curve:
Figure-7:
Chromatograms:
Figure-8 (Blank):
Figure-9 (Linearity solutions):
Solution stability:
The solution stability until 24hours of derivatized
hydroxylamine was checked by injecting standard solution. The solution was
prepared freshly before injection and the same was injected after 24hours. The
peak area of derivatized hydroxylamine of freshly prepared standard solution
was observed 1.484 after 12 hours 1.439 and after 24hrs it was 1.457. There is
no significant change in area was observed up to 24hours.
Hence, the solution was stable up to 24hours.
Table -2:
|
Sample name
|
Time
|
Area
|
|
Standard solution
|
Initial
|
1.484
|
|
12 hours
|
1.439
|
|
24 hours
|
1.457
|
Ruggedness study:
The ruggedness of the method was evaluated by
estimating % RSD of the derivatized hydroxylamine standard solution tested by
two different analysts on different days with different HPLC instruments. Such
six different preparations were prepared by each analyst. RSD of content of
hydroxylamine derivative found to be 1.6%
Robustness study:
Robustness of the method was established by analysing
the standard solution and batch analysis with deliberate change in the
parameters like (a) flow rate of the mobile phase ±0.1mL/min (b) column
temperature ±5°C and (c) mobile phase concentration.
Method validation summary:
Table-3
|
S. No.
|
PARAMETER
|
ACCEPTANCE CRITERIA
|
RESULTS
|
|
1.0
|
Specificity
|
Hydroxylamine derivative peak should be well
separated from all known and unknown peaks of Molnupiravir
|
Complies
|
|
2.0
|
Limit of detection (LOD)
|
Report the result
|
0.86 ppm
With respect to test concentration
|
|
3.0
|
Limit of quantification (LOQ)
|
Report the result
|
2.6 ppm
With respect to test concentration
|
|
4.0
|
LOQ precision
|
%RSD for six replicates of LOQ level standard
solutions is NMT:10.0%
|
1.85
|
|
5.0
|
Accuracy
|
Recovery should be between 80% to 120%
|
Complies
|
|
6.0
|
Linearity
|
Correlation: NLT 0.99
|
0.9954
|
|
7.0
|
Solution stability
|
Report the result
|
Solution is stable up to 24hours
|
|
8.0
|
Ruggedness
|
%RSD for results of six test preparations is NMT:5.0%
|
1.6
|
|
9.0
|
Robustness
|
|
Flow 0.7 mL/min
|
%RSD for Results of test preparations in actual
condition and changed condition is NMT:5.0%
|
0.94
|
|
Flow 0.9 mL/min
|
1.43
|
|
Mobile phase ratio + 10%
|
%RSD for Results of test preparations in actual
condition and changed condition is NMT:5.0%
|
1.55
|
|
Mobile phase ratio - 10%
|
1.43
|
|
Column temperature 35°C
|
%RSD for Results of test preparations in actual
condition and changed condition is NMT:5.0%
|
1.19
|
|
Column temperature 45°C
|
0.94
|
CONCLUSION:
We have developed and validated the HPLC method for
the determination of Hydroxylamine content in Molnupiravir drug substance using
a derivatization reaction with RP-HPLC UV detector. The advanced techniques
like LC_MS16,17 and GC-MS18 are not available in all
quality control labs but HPLC19,20,21,22,23,24 with UV25,26
is a common instrument. The results of validation parameters proved that the
method is sensitive, accurate, specific, linear and precise. Hence, this method
can be used in the quality control department for routine low-level
hydroxylamine in pharmaceutical substances27,28,29.
ACKNOWLEDGMENTS:
The authors gratefully acknowledge and thank the
management of Vijayasri Organics Ltd., R&D centre, for valuable support and
encouragement. And also great thankful to Dr. Hariharakrishanan for his
valuable suggestions and support
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DOI: 10.52711/2231-5691.2024.00031