Key features of the article:
- 1. Improved process for the synthesis of Afatinib Dimaleate.
- 2. Identification, synthesis and characterization of one new process impurity by NMR, LC-MS and HPLC.
- 3. Forced degradation studies as per ICH guidelines to have control on process impurities.
- 4. Developed HPLC method which is highly sensitive, specific, selective and robust for analysis.
1. Introduction
Over the years many quinazoline derivatives were reported as epidermal growth factor receptor (EGFR) signal transduction pathway inhibitors and Afatinib Dimaleate [1] is one of them which is powerful, irreversible tyrosine kinase inhibitor of EGFR, with IC50 value (half-maximal inhibitory concentration) of 0.5 nM, exhibits potent anti-tumor activity against non-small cell lung cancer (NSCLC) (https://www.medchemexpress.com). Afatinib [BIBW 2992; N- [4-[(3-chloro-4-fluorophenyl) amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide] is an ATP-competitive anilinoquinazoline derivative harboring a reactive acrylamide group [2,3,4]. Afatinib Dimaleate is approved by the FDA as a first line treatment of patients detected with metastatic non-small cell lung cancer (NSCLC) with common epidermal growth factor receptor (EFGR) mutations as detected by an FDA-approved tests (Gilotrif FDA Label). It was designed to covalently bind and irreversibly block enzymatically active ErbB receptor family members [5].
As per prior art methods very few synthetic procedures are reported for Afatinib Dimaleate and none of process has discussed clearly about the process impurities and their control measures [6]. Impurities in active pharmaceutical ingredient (API) are highly undesirable and in some cases can prove to be harmful to the patient. The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q7 is a guidance for API manufacturers, mentions that impurities to be maintained below set limits [7]. Thus, it is pertinent to identify and characterize the impurities in API to develop suitable process where in their levels can be kept within permissible limits (FDA guidelines for good manufacturing practices for API). The impurity profile study should be carried out for any bulk drug to identify and characterize all the unknown impurities that are present at a level of above 0.05%. A comprehensive study has been undertaken to isolate and characterize these impurities by spectroscopic techniques. This research article describes the improved process for the synthesis of Afatinib Dimaleate, identification, isolation, synthesis and characterization of impurities that are present in the range of 0.08%-0.30% by area percent in the Afatinib Dimaleate.
During the analysis of laboratory batches of Afatinib Dimaleate by high performance liquid chromatography (HPLC) four different and major impurities were observed, one of them was identified as new process impurity, two of them as major degradant impurities and one of them as both process impurity or degradation impurity. The impurities were in the range of 0.08-0.30% along with drug substance. In regulatory terms, the level of impurities in drug substance is quite important for the drug approval and can show a significant impact on the quality and safety of drug. Thus, impurity profiling is the most concerning task in the modern pharmaceutical analysis especially when it comes to oncology drugs [8].
None of processes reported in the prior art has explained the controlled
measures for the listed impurities. Based on these views, our focus was to
develop a highly effective, optimized and efficient process which should have all
the control measures for these impurities, synthesize and characterize the new
impurity and to control the degradation impurities in the drug substance. The
structure of new impurity was presumed based on the liquid chromatograph-mass spectrometer (LC-MS)/MS data and confirmed
its synthesis followed by spectroscopic analysis such as 1H NMR, 13C
NMR, mass and IR. In addition to this, an effective and sensitive HPLC method was
developed to separate and quantify all the related substance of Afatinib
Dimaleate. To our knowledge this is the first study that comprehensive analysis
of the potential impurities and degradation products in Afatinib Dimaleate has
been done, including their synthesis and characterization.
The Afatinib Dimaleate was used from in-house sources as synthesized in Chemical
Research and Development laboratory of Oncogen Pharma (Malaysia) Sdn Bhd. HPLC
grade methanol, acetonitrile, OPA (85%), TEA and other chemical reagents were
purchased from Merck & JT Baker. The solvent N, N-dimethylacetamide from sigma
Aldrich (with appropriate specification). Milli-Q-Purified water by Milli-Q plus
purification system from Millipore (Bradford, USA), was used during experimental
studies. The process used deoxygenated water which was generated by purging
nitrogen gas in the Milli-Q-water for 3 h. All the general chemicals were brought
either from Merck Sdn Bhd., Malaysia or local chemical supplier. Solvents from
Polyscientific Enterprise Sdn. Bhd. Malaysia. IR spectra were recorded with KBr
pellets using Shimadzu FTIR Tracer-100 spectrophotometer, 1HNMR and
13C CNMR were recorded in solvents CDCl3, DMSO-d6 and CD3OD at
300 MHz and 75 MHz respectively using Bruker instrument. All the chemical shift
values are reported in δ units downfield from TMS as internal standard.
Differential scanning calorimetry (DSC) were performed using T. A. instrument
model no. DSCQ20. X-Ray Diffraction pattern (XRD) analysis were performed using
PANalytical instrument model no. Empyrean. Melting point were recorded using
BUCHI melting point apparatus with model no. M-565.
The prior art method [9,10] for the synthesis
of Afatinib Dimaleate involves the series of reaction wherein the compound
4-[(3-chloro-4-fluorphenyl) amino]-6-nitro-7-fluoro quinazoline 5, was
used as a starting material, substitution reaction of 5 with
S-(3)-hydroxy tetrahydrofuran 6, in the presence of catalytical amount of
potassium tert. butoxide resulted in 4-[(3-chloro-4-fluorophenyl)
amino]-6-nitro-7-(S) -(tetrahydrofuran-3-yl) oxy] quinazoline 7, which
on reduction at 6th position of nitro group to resulted in corresponding
amine 8. which reacts with bromo crotonyl chloride to get intermediate
9. Amination reaction of the 9 with dimethylamine affords
Afatinib free base 2. The entire reaction sequence is depicted below Figure 2.
Based the FD data it was easy to identify the possible degradants and their
conditions and thus it became easy to establish their control measure in the
process. The HPLC purity of control sample used for the study was 99.94% with
impurities at about RRT 0.44 (0.01%), 0.50 (0.03%) and 0.81 (0.01%). All the
FD study performed is summarized below as:
The Afatinib Dimaleate was treated with 5% hydrogen peroxide solution for 2 h
at 70 °C. Few unknown peaks were observed at different RRT's with area
percent ranging from 1.6% to 3.17%. During the laboratory development batches,
one impurity at about RRT 0.49 was in concordant with impurity observed during
oxidative degradation. Thus, based on the LC-MS data of that impurity it was
concluded as Afatinib-N-oxide (3.17%) with m/z 524.2 (the actual molecular
weight is 501.4), so it was confirmed as sodiated adduct of Afatinib-N-oxide.
Thus, as per this analysis one of the impurities identified which need to be
controlled in the process was Afatinib-N-Oxide. The HPLC chromatogram after
before and after the oxidative degradation studies is shown in Figure 4:
The Afatinib Dimaleate was treated with 0.5N aqueous sodium hydroxide solution
for 2 h at 70 °C. Different peaks were observed at about RRT 0.56
(11.03%), 0.74 (0.58%), 0.80 (1.51%) and 1.11 (0.20%). Among the list of
impurities, two major impurities at about RRT 0.58 and 0.84 were in concordant
with impurities observed regularly during laboratory development batches. Based
on the LC-MS data of these RRT's, impurity at about RRT 0.56 was observed as
hydroxy impurity with m/z 459.2 (the actual molecular weight is 458.1), so it was
confirmed as protonated mass of hydroxy impurity and impurity at RRT 0.84 was
observed as intermediate-1 with m/z of 375.1 (the actual molecular weight is
374.09), so it was confirmed as protonated mass of intermediate-1.Thus, as per
this analysis two impurities identified which need to be controlled in the
process was hydroxy impurity and intermediate-1. The HPLC chromatogram after
before and after the basic degradation studies is shown in Figure 5:
The Afatinib Dimaleate was treated with 1.0N aqueous Hydrochloric acid solution
for 4 h at 70 °C. Only one peak was observed as major degradant at about
RRT 0.81 (9.20%). Since the impurity at RRT 0.81 was in concordant with the
impurity observed constantly during laboratory development batches, so LC-MS was
not performed. It is evident by the FD studies that intermediate-1 is the major
degradant during both acidic and basic hydrolysis and is also the process
impurity during the reaction. Thus, it needs to be controlled in such a way that
the resulting sample should comply with ICH guidelines with actual limit not more
than 0.10%. The HPLC chromatogram after before and after the acidic degradation
studies is shown in Figure 6:
Based on forced degradation studies, three major impurities were identified in
the process and control measure of those need to be established to comply the
material as per ICH guidelines (all the probable impurities not more than
0.10%). The fourth impurity was new in the process and was identified based on
the LC-MS data of developmental batches, which is explained later.
In a clean and dry glass assembly, charged ethanol (4.5 L),
(S)-N-(3-chloro-4-fluorophenyl)-6-nitro-7-((tetrahydrofuran-3-yl) oxy)
quinazolin-4-amine, 4 (300.0 g, 0.7411 moles) and activated carbon (60 g), heated
the suspension to 60-70 °C and added hydrazine hydrate (470 ml) and after
addition raised the reaction temperature to 70-80 °C, monitored the
reaction on HPLC (reaction time 1.0hr), added hyflo (5 g) in the reaction mass,
stirred for 30-45 mins and then filtered the reaction mass through buchner funnel
under hot conditions. Washed the entire bed with hot ethanol (300 ml).
Concentrated the clear, pale green coloured mother liquor to 80-90% under vacuum
not less than 640 mm Hg at 60-65 °C, added water (3000 ml) to the
distilled residue, slurried the residue at ambient temperature for 30-45 minutes
and filtered the solid. Washed the solid with water (100 ml.). Dried the solid
under vacuum not less than 660 mm Hg at 60-65 °C for 4 hrs. Heated the
dried solid with acetonitrile (2100 ml) at 55-65 °C and then gradually
cooled to ambient temperature and then to 0-10 °C. Stirred the
suspension for 45-60 mins at 0-10 °C. Filtered the solid, washed with
chilled acetonitrile (300 ml.). Dried the wet solid under vacuum not less than 700
mm Hg at 60-65 °C for 10 h (moisture content 0.26% w/w) to afford
255.5g of title compound with HPLC Purity: 99.85%. 1H-NMR (DMSO-d6):
δ 2.10 (m, 1H), 2.35 (m, 1H), 3.81 (dt, 1H), 4.00-3.94 (q, 3H), 5.20 (s,
1H), 5.77 (bs, 2H), 7.18 (s, 1H), 7.43-7.58 (m, 1H), 7.79-7.73 (m, 1H), 8.10 (dd,
1H), 8.57 (s, 1H), 10.33 (s, 1H), 13C-NMR (DMSO-d6): 32.32, 66.49,
71.91, 78.60, 101.23, 102.94, 109.42, 116.36, 116.65, 118.63, 118.87, 123.30,
123.40, 124.48, 135.82, 135.86, 137.47, 139.96, 147.60, 151.28, 152.14, 155.37,
156.08, Mass (M+H):375.0, IR (cm-1): 1627, 1570, 1431, 1215, 1242, 1161,
3317, 856, 2862.
In a clean and dry glass assembly charged N, N-dimethylacetamide
(2000ml), followed by N,N-dimethylcrotonic acid hydrochloride (154.6 g, 0.933
moles) to get suspension. Cooled the reaction mass to -12 to -6 °C and
added thionyl chloride (155.3 g, 1.306 moles) dropwise in the reaction mass
maintaining the reaction temperature. This part is labelled as solution-A.
Dissolved intermediate-1 (250.0 g, 0.6670 moles) in N, N-dimethylacetamide (750 ml)
and labelled as solution-B. Added solution-B in the solution-A at -12 to -6
°C within 20-25 minutes. After addition, immediately monitored the
reaction on HPLC (intermediate-1, NMT: 0.15%). Quenched the reaction mass by
adding aq. triethylamine (250 ml: TEA 100 ml and water 150 ml) dropwise in the
reaction mass. Gradually, raised the reaction temperature to 20-25 °C.
Diluted the reaction mass by adding water (250 ml.). Adjusted the pH of reaction
mass using liquor ammonia (250 ml). Stirred the heterogenous reaction mass for
45-60 mins. Filtered the precipitated solid and washed the solid with water
(250 ml.). Suck dried the solid sufficiently under vacuum not less than 700 mm Hg
and under nitrogen blanketing. Treated the sufficiently suck dried solid
(moisture content NMT 5%) with mixed solvent system [dissolved the solid in
tetrahydrofuran (1250 ml) at 25-30 °C and precipitated the solid by
dropwise addition of water (3125ml) at 25-30 °C, gradually cooled the
reaction mass to ambient temperature and further to 0-10 °C. Filtered
the solid and washed the solid with tetrahydrofuran (50 ml.)]. Dried the solid
under vacuum not less than 700 mm Hg without heating. Yield: 294.9 g (91% on
theoretical basis), HPLC 99.87% with all impurities less than 0.10% by area
percent. 1H-NMR (DMSO-d6): δ 2.15 (s, 1H), 2.35 (m, 1H), 3.10
(d, 2H), 3.79 (td, 1H), 3.94 (q, 1H), 4.01 (d, 2H), 5.29 (d, 1H), 6.60 (d, 1H),
6.80 (dt, 1H), 6.85 (t, 1H), 7.42 (t, 1H), 7.80 (m, 1H), 8.13 (dd, 1H), 8.52 (s,
1H), 8.96 (s, 1H), 9.44 (s, 1H), 9.81 (s, 1H); 13C-NMR (DMSO-d6):
32.32, 45.06, 59.69, 66.51, 71.92, 78.70, 107.92, 108.87, 116.18, 116.46, 122.34,
123.43, 125.72, 127.46, 136.80, 142.08, 148.59, 151.46, 153.10, 153.75, 154.68,
156.68; Mass (M+H): 486.0; IR (cm-1): 1620, 1674, 1577, 1427, 1215, 1249,
1149, 1531, 3317, 817 and 2862. The developed process not only resulted in high
quality Afatinib free base with all the impurities less than 0.10% but also
resulted in novel polymorph [2,9] as designated by the following 2-theta values
as tabulated in Table 1:
In a clean and dry glass assembly charged tetrahydrofuran (2000 ml) and
(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6- yl]-4-(dimethylamino)but-2-enamide, 2 (250.0 g,
0.5145 moles) to get suspension.
Heated the reaction mass to 35-45 °C to get clear solution and added
solution of maleic acid in tetrahydrofuran (122.45g, 1.054 moles in 750 ml
tetrahydrofuran) maintaining the temperature. After precipitation of solid,
gradually cooled the reaction mass to ambient temperature and further to 10-15
°C. Maintained the reaction mass at 10-15 °C for 60-90 mins,
filtered the solid and washed with tetrahydrofuran (250ml). Dried the solid under
vacuum not less than 700 mm Hg at 45-50 °C for 16 h. (m/c 0.45%).
Yield: 328.8g (89% on theoretical basis), HPLC Purity: 99.90%. 1H-NMR (DMSO-d6): δ 2.36
and 2.14 (m, m, 2H), 2.83 (s, 6H), 3.78 (m, 1H), 4.01
and 3.92 (m, m, 5H), 5.32 (m, 1H), 6.14 (s, 4H), 6.80
(m, 2H), 7.28 (s, 1H), 7.44 (t, 1H), 7.78 (m, 1H), 8.09
(m, 1H), 8.59 (s, 1H), 8.96 (s, 1H), 9.76 (s, 1H), 10.03
(bs, 1H); 13C-NMR (DMSO-d6): 32.31, 42.11, 56.84,
66.51, 71.91, 78.93, 107.22, 108.57, 116.30, 116.59,
116.74, 118.58, 118.83, 122.71, 122.80, 123.93, 127.22,
131.59, 132.22, 133.35, 136.31, 136.35, 147.41, 151.80,
153.47, 153.60, 155.02, 156.99, 162.34, 166.92; Mass
(M+H): 486.0; IR (cm-1): 1616, 1681, 1573, 1427,
1492, 1192, 3344, 1249, 1149, 1527, 3317, 817, 1458,
2862 and 1350.
All the impurities discussed in the article is observed during the stage-02
(amidation step). Typical HPLC chromatogram of Afatinib dimaleate with all the
process impurities formed in the process is shown in Figure 8. Detailed
description of impurities observed during the process development studies of
Afatinib dimaleate is discussed below:
During initial developmental batches a particular impurity at RRT 0.93 were
observed constantly. The knowledge about the fragmentation pattern of impurities
could acquire structural information and therefore taken further studies using
MS/MS. Based on the initial characterization by LC-MS, the impurity manifested
protonated molecular mass of m/z 417.11 (M+H) and two daughter ions as m/z 346.06
and m/z 304.05. The entire fragment pattern is shown below with chemical structures in Figure 9:
The acetamide impurity was formed by reaction between acetic acid and
intermediate-1. The speculation of impurity formation with acetic acid was
confirmed by adding catalytic amount of acetic acid in amidation reaction which
led to same impurity at about RRT 0.93. The source of acetic acid was identified
as solvent N, N-dimethylacetamide which was the reaction media for amidation
step. By titrimetric analysis and gas chromatography it was confirmed that
catalytic amount of acetic acid was present in the solvent N,
N-dimethylacetamide. Thus, the content of acetic acid was controlled in N,
N-dimethylacetamide up to a level of 0.002%. However, that catalytical amount
was not reflecting the actual percentage of impurity forming in the reaction
mass. Even by controlling the amount of acetic acid in N, N-dimethylacetamide
acetamide impurity was still observed. Later it was postulated that the N,
N-dimethylacetamide is degrading into acetic acid in the reaction mass under
acidic pH (source of acidity was excess of thionyl chloride) and presence of
moisture (either from raw material or intermediate-1). To prove this postulation,
a reaction was performed with excess quantity of thionyl chloride and elevated
content of acetamide impurity was observed in the isolated solid. Thus, by
controlling the acidity of reaction mass (by using the optimal quantity of
thionyl chloride) and moisture in raw material and intermediate-1, this acetamide
impurity was controlled up to a level of less than 0.04%.
In a clean and dry glass assembly added, dichloromethane (50 ml) followed by
intermediate-1 (5.0 g) and acetic anhydride (1.49g, 0.0145 moles). Heated the
reaction mass to 35-40 °C for 45-60 mins and monitored the reaction mass
on HPLC. Distilled the reaction mass to dryness on rotavapor and slurried the
distilled residue with n-heptane (50 ml). Filtered the solid, washed with
n-heptane (25 ml). Dried the material in oven at vacuum not less than 660 mm Hg
till constant weight. Yield: 3.0 g (60% on w/w basis). HPLC : 98.74%. The HPLC
chromatogram of the prepared acetamide impurity is shown in Figure 10 and was
characterized by NMR (1H and 13C), (Figure 11, Figure 12), Mass (Figure 13)
and IR (Figure 14) as per the structure given below:
1H-NMR (DMSO-d6): NH (20) δ 9.77 (s, 1H), C14-H 9.33
(s, 1H), C16-H 8.81 (s, 1H), C19-H 8.52 (s, 1H), C3-H 8.13 (dd, 1H), C5-H 7.80
(m, 1H), C6-H 7.41 (t, 1H), NH (9) 7.16 (s, 1H), C22-H 5.27 (d, 1H), C23 and
C25-Hb (3.94 (q, 1H), C31-H 3.10 (d, 1H), C26-Ha 2.33 (m, 1H), C29-H and C26-Hb
2.15 (s, 3H and 1H);
13C-NMR (DMSO-d6): C10 (168.68), C27
(156.67), C18 (153.73), C14 (153.1), C1 (151.46), C12 (148.61), C4 (136.74), C17
(127.62), C28 (123.44), C2 (122.34), C5 (118.71), C3 (118.46), C6 (116.46), C11
(116.18), C19 (108.8), C16 (107.92), C22 (78.61), C23 (71.93), C25 (66.5), C29
(32.34); Mass (M+H): 417.11; IR (cm-1): 1624, 1681, 1531,
1577, 1431, 1207, 1027, 1141, 3529, 3298, 817, 2881.
As per the developed analytical method hydroxy impurity elutes at about RRT
0.58. The initial characterization was based on the LC-MS data. The mass
spectrophotometer manifested protonated molecular mass of hydroxy impurity at m/z
459.2 (M+H). The typical mass chromatogram is shown below in Figure 15:
As per the developed analytical method Afatinib-N-oxide elutes at RRT 0.50. The
initial characterization was based on the LC-MS data. The knowledge about the
fragmentation pattern of impurities could acquire structural information and
therefore taken further studies using MS/MS. The mass spectrophotometer
manifested molecular mass of Afatinib-N-oxide at m/z 524.2 (M+Na) The mass
chromatogram is represented in the Figure 16 as below:
A waters HPLC system equipped with alliances 2695 series low pressure quaternary
gradient pump along with photo diode array detector and auto sampler has been
used for the analysis of samples. The data was collected and processed using
waters "Empower 2" software. An Inertsil C18 (150* 4.6 mm, 5-Micron, GL Sciences,
Japan) column was employed for the separation of impurities from Afatinib
Dimaleate. The column eluent was monitored at 254 nm. A simple gradient
reverse-phase HPLC method was optimized for the separation of impurities from
Afatinib Dimaleate active pharmaceutical ingredient where the mobile phase was a
mixture of 2 mmol L-1 ammonium acetate and acetonitrile (composition of
mobile phase 0.02M potassium dihydrogen phosphate and 1.0 g/L
1-octanesulphonicacid sodium salt and acetonitrile). Chromatography was performed
at room temperature using at a flow rate of 1.0 mL min-1. The
chromatographic run time was 40 min. The result was analysed weight/weight (w/w)
with respect to reference standard and all the total impurities were complying
the ICH guidelines. The developed method was validated as per ICH guidelines with
respect to precision, accuracy, linearity, robustness, specificity and system
suitability.
LC-MS/MS analysis has been performed on API 2000, Mass Spectrometer. The
analysis was performed in positive ionization mode with turbo ion spray
interface. The parameters for ion source voltage IS = 5500 V, declustering
potential, DP = 70 V, focusing potential, FP = 400 V, entrance potential, EP = 10
V were set with nebulizer gas as air at a pressure of 40 psi and curtain gas as
nitrogen at a pressure of 25 psi. An Inertsil C18 (150 * 4.6 mm, 5-Micron, GL
Sciences, Japan) column was used for the separation. The mobile phase is a
mixture of 2 mmol L-1 ammonium acetate and acetonitrile with a flow rate of
1.0 mL min -1.
The 1H and 13C NMR experiments were carried out at frequencies of 300
MHz and 75 MHz respectively, in DMSO-d6 at 25 °C temperature on a
Varian-400 FT NMR spectrometer. 1H and 13C chemical shifts are reported
on the d scale in ppm, relative to tetra methyl silane (TMS) δ 0.00 and
CDCl3 at 77.0 ppm in 13C NMR respectively.
Initial studies for the synthesis of Afatinib involves lot of efforts starting
from selection of reducing agents to selection of solvent for amidation,
selection of chlorinating agent for N, N-dimethylcrotonic acid hydrochloride,
appropriate reaction temperature for chlorination and amidation, isolation
procedure for Afatinib free base as it is highly unstable and degrades easily in
the presence of moisture and oxygen. The main challenge was to isolate the
Afatinib free base in highly pure and stable form, its purification (if required)
and stability over the period. For the synthesis of Afatinib free base
2, the entire study was divided in three parts, part-1 was the selection
of solvent and chlorinating agent for the conversion of N, N-dimethylcrotonic
acid hydrochloride to acid chloride, part-2 was the selection of solvent for
amidation and addition mode and part-3 was the isolation of Afatinib free base.
For part-1, based on the nature of reaction, all protic solvents were ruled out,
during exploratory studies solvents such as toluene, ethyl acetate, acetonitrile
and thereof were ruled out, the options left were polar aprotic solvents such as
N, N-dimethylformamide, N-methylpyrrolidone (NMP) and N, N-dimethylacetamide. Out
of these solvents only N, N-dimethylacetamide was feasible and effective, rest
solvents were either charring the reaction or resulting in incomplete conversion.
To select chlorinating agents, wide variety of reagents were available such as
thionyl chloride, oxalyl chloride and thereof but with oxalyl chloride complete
conversion of reaction was never achieved. Thus, based on effectiveness and cost
thionyl chloride was opted as chlorinating reagent. By optimal quantification all
other parameters such as reaction temperature, mode and rate of addition of
chlorinating agent, the complete conversion of N, N-dimethylcrotonic acid
hydrochloride to acid chloride was achieved in 1.0-1.5 h. For part-2, during
exploratory studies it was observed that mixture of solvent is not capable for
complete conversion (from 3 to 2) so single solvent reaction
was finalized and only N, N-dimethylacetamide was used throughout the reaction.
Later the addition pattern was studied whether solution of 3 in acid
chloride reaction mass or vice-versa. Based on exploratory studies, addition of
acid chloride solution to 3 was generating lot of impurities (since acid
chloride is highly unstable) and reaction time was longer (more than 3 h), so the
idea was dropped, and the only feasible option was to add solution of 3
in acid chloride solution. By doing this, as soon as the addition of 3
was completed reaction complies with conversion rate of more than 99.5% and all
the process as well degradation impurities were well within the controllable
limit. For part-3, during the exploratory studies it was observed that quenching
of thionyl chloride with water would be tedious during the scale up, thus it was
suggested to reduce the pH of reaction mass followed by quenching with water. To
follow this, aqueous triethylamine was used instead of water which would not only
reduce the pH but would also quench the reaction mass. Since, 2 is in
its hydrochloride form and to isolate it in pure form as base further pH
adjustment is required. Lot of organic and in-organic bases were explored but
based on the degradation data under basic condition, it was suggested to have
that base which should not facilitate the degradation of 2. Thus, based
on the scientific logic and available data, 10-15% liquor ammonia was used and
after pH adjustment, nitrogen was purged to remove the excess ammonia. This
approach not only avoided the degradation of compound but also made the isolation
of product in pure form with HPLC purity around 99.59% by area percent. Later,
the 2 was treated with tetrahydrofuran and water to get compound with
HPLC purity more than 99.80% and all listed process impurities less than 0.10%.
As per above establishment, synthesis of Afatinib dimaleate was completed in
three steps starting from
(S)-N-(3-Chloro-4-fluorophenyl)-6-nitro-7-((tetrahydro- furan-3-yl) oxy)
quinazolin-4-amine 4. The yield in every step is quantitative. The
entire protocol is depicted in figure-1
Afatinib Dimaleate is a potent aromatase inhibitor drug used in the treatment of
cancer diseases. The present research work describes an improved process wherein
all impurities (known and unknown) are controlled to a level of 0.10%. New HPLC
method was developed for the detection and separation of four process related
impurities from Afatinib Dimaleate. The reported process explains the formation
of new process impurity which needs to be controlled to achieve the material as
per regulatory guidelines. All the four impurities detected using the new HPLC
method and were characterized using LC-MS and NMR data.
2. Experimental
2.1 Chemicals and Reagents
2.2 Prior art method for the synthesis of Afatinib Dimaleate
2.3 Synthetic Process for Afatinib Dimaleate
Synthesis of Afatinib Dimaleate was followed as per the scheme shown below:
2.4 Forced Degradation (FD) studies
2.5 Oxidative Degradation
2.6 Degradation under basic condition
2.7 Degradation under acidic condition
2.8 Synthesis of
N-(3-chloro-4-fluorophenyl)-6-amino-7-[[(3S)-tetrahydro-3-furanyl] oxy]-4-quinazolinamine (Intermediate-1, 3)
2.9 {Synthesis of (E)-N-[4-(3-chloro-4-fluo-roanilino)-7-[(3S)-oxolan-3-yl] oxyquina-zolin-6-yl]-4-(dimethylamino)but-2- enamide (Afatinib free base, 2))
2.10 Synthesis of (Z)-but-2-enedioic
acid;(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide (Afatinib Dimaleate, 1)
2.11 Process impurities and their Structure elucidation
(S)-N-(4-((3-chloro-4-fluorophenyl) amino)-7-((tetrahydrofuran-3-yl)oxy) quinazolin-6-yl) acetamide (acetamide impurity)
2.12 Synthesis of Acetamide Impurity
2.13 High Performance Liquid Chromatography (analytical)
2.14 Mass Spectrometry (LC-MS/MS)
2.15 NMR spectroscopy
3. Results and discussion
4. Conclusion