Impurities in finasteride: Identification, synthesis, characterization and control of potential carry-over impurities from reagents used for the process

Article abstract of DOI:10.14233/ajchem.2014.16603

An assessment of the impurity profile of finasteride and possible carry-over related substances likely to arise during the synthesis of finasteride is described in this article. Impurities in reaction mass were monitored by HPLC, potential impurities isolated with preparative HPLC and structures were substantiated by ¹H NMR, MS and MS-MS. Impurities RRT's were established by HPLC co-injection. Based on the spectral data structure of impurity I and impurity II were characterized as cyclohexyl and phenyl analog of finasteride.

Full text of DOI:10.14233/ajchem.2014.16603

Asian Journal of Chemistry; Vol. 26, No. 14 (2014), 4375-4380
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.16603
Impurities in Finasteride: Identification, Synthesis, Characterization and Control of
Potential Carry-Over Impurities from Reagents Used for the Process
*
SANDEEP MOHANTY , B. PAVAN KUMAR and ARUN CHANDRA KARMAKAR
Process Research and Development, Dr. Reddy's Laboratories Limited, API Plant, Bollaram-III, Plot No's 116, 126C, Survey No.157, S.V.
Co-operative Industrial Estate, IDA Bollaram, Jinnaram Mandal, Medak District, Hyderabad-502 325 India
*Corresponding author: E-mail: sandeepmohonty@drreddys.com
Received: 29 October 2013;
Accepted: 31 December 2013;
Published online: 5 July 2014;
AJC-15479
An assessment of the impurity profile of finasteride and possible carry-over related substances likely to arise during the synthesis of
finasteride is described in this article. Impurities in reaction mass were monitored by HPLC, potential impurities isolated with preparative
HPLC and structures were substantiated by 1H NMR, MS and MS-MS. Impurities RRT's were established by HPLC co-injection. Based
on the spectral data structure of impurity I and impurity II were characterized as cyclohexyl and phenyl analog of finasteride.
Keywords: Synthesis, Preparative isolation, Liquid chromatography, Mass spectrometry, NMR, MS, Characterization.
H
N
O
INTRODUCTION
Finasteride1 (1; Fig. 1) is a synthetic 4-azasteroid compound
and a specific inhibitor of Type II 5α-reductase wherein 5α-
reductase is an intracellular enzyme that converts the androgen
testosterone into 5α-dihydrotestosterone (DHT). The mechanism
of action of finasteride is based on its preferential inhibition
of Type II 5α-reductase through the formation of a stable complex
with the enzyme. Inhibition of Type II 5α-reductase blocks
the peripheral conversion of testosterone to DHT, resulting in
significant decreases in serum and tissue DHT concentrations2.
Finasteride (1) is sold as Propecia and Proscar by Merck & Co,
the former is commonly used for the treatment of male pattern
baldness3 and the latter for prostatic hyperplasia. FDA approved
oral administration of finasteride is only five mg per day. How-
ever, the presence of a trace level related substance can influence
the efficacy and safety of the final product. It is mandatory to
identify, characterize and control any single maximum impu-
rities in an API, if present above the accepted limits of 0.1%4.
To achieve this, understanding of the overall manufacturing
process, quality assessment of raw materials and reagents used
in the process, detection, identification and control of impu-
rities at the source is critical for delivering finasteride of high
quality. In this article, we wish to report two carry-over impurities
likely to arise during the synthesis of finasteride (1). In connec-
tion with our continuing interest in azasteroids, we identified,
synthesized and established a control in specification of DCC
where carry-over of starting material in DCC resulted un-
desired related substances in finasteride (1).
H
H
H
O
N
H
H
(1)
Fig. 1. Structure of finasteride
EXPERIMENTAL
The crude sample of finasteride was obtained from Dr.
Reddy's Laboratories Ltd, Hyderabad, India. The unknown
impurities were isolated by preparative HPLC. The HPLC
grade acetonitrile, tetrahydrofuran, methanol and formic acid
were obtained from the Merck Co., Mumbai, India. The water
used for preparing mobile phase was purified using a Millipore
Milli-Q+ purification system.
HPLC (analytical): A Waters ModelAlliance 2690-sepa-
ration module equipped with a Waters 996-photo diode array
detector was used for the studies. The analysis was carried out
on Novapak, C-18 columns, 250 × 4.6 mm i.e., 4 µM particle
size with a mobile phase consisting of solvent A (water),
solvent B (tetrahydrofuran) and solvent C (acetonitrile) (8:1:1).
Sample was prepared at 1 mg/mL concentration in acetonitrile
and water (1:1, v/v). The injection load was 15 µL. The eluents
were detected at 210 nm at a flow rate of 1.8 mL min-1. The
column temperature was maintained at 55 °C. The data was
recorded using Waters Millennium software.

4376 Mohanty et al.
Asian J. Chem.
HPLC (preparative): AWaters preparative HPLC system
equipped with W600 quaternary solvent delivery module and
Delta prep 2487 dual wavelength UV detector was used. Data
was processed through Waters Empower software.A Reprosil
C18 column (250 × 20 mm, 7 µm particle size) was used for
preparative work. An isocratic method was used with the
mobile phases such as 0.1 % Formic Acid (Aqueous) and
methanol with 60:40 ratio. The flow rate was kept at 20 mL/
min and the column eluent was monitored at 210 nm at ambient
temperature.
scale in ppm, relative to TMS (δ = 0.00) and the 13C chemical
shift values were reported relative to CDCl3 (δ = 77.00 ppm)
as internal standards. Standard pulse sequences provided by
Varian were used for distortion less enhancement by polariza-
tion transfer (DEPT), gradient double quantum filtered correla-
tion spectroscopy (gDQCOSY), gradient hetero nuclear single
quantum coherence spectroscopy (gHSQC), gradient hetro-
nuclear multibond coherence spectroscopy (gHMBC) (J = 8.0
Hz) experiments. Nuclear Overhauser Effect Spectroscopy
(NOESY) experiment was run using a mixing time of 600 ms.
Reported (Scheme-I) finasteride (Fig. 1) manufacturing
process starts with the hydrolysis of 2 with MeOH-NaOH to
give 3. Amidation of 3 in presence of N,N'-Dicyclohexyl-
carbodiimide (DCC) and hydroxybenzotriazole (HOBT) with
t-butylamine gave 4. Ring-A oxidation in presence of 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and N,O-
bis(trimethylsilyl)trifluoroacetamide (BSFTA) in toluene gave
crude finasteride (1b). Purification of crude product with acetic
acid, methanol and water gave pure 1(a). Re-crystallization
of 1(a) in MDC-cyclohexane gave finasteride (1) of desired
polymorphic form. Two unknown impurities were observed
in the HPLC chromatogram of crude 1(b) at about 1.6 and 1.7
RRT's with respect to the parent peak to the extent of 0.1 %
along with known impurities of finasteride, impurity-A (4)
and impurity-C6 (over oxidized impurity).
LC-MS and LC-MS/MS: Analytical LC conditions
described in Fig. 2 were applied for the LC-MS analysis.
Electrospray ionization (ESI) and tandem mass spectrometry
experiments were performed using a triple quadrupole mass
spectrometer (PE Sciex model API 3000). The positive and
negative electrospray data were obtained by switching the
capillary voltage between +5000 and -4500V, respectively. The
electrospray ionization (ESI) was performed by using collision
potential (30V) and nitrogen gas was used to assist nebulisation
in the collision cell for MS-MS studies.
Ultra performance liquid chromatography (UPLC)-
TOF MS: Finasteride and its isolated impurities were directly
infused into the UPLC-TOF-MS instrument. Leucine Encephalin
was used as Lock spray for accurate mass measure. The mass
spectra of impurities were recorded on LCT premier XE,
Waters Micromass Mass Spectrometer.
The process was robust and met predefined quality and
quantity criteria, however product 1(b) has to be purified again
for removal of unknown impurities I7 and II8. No doubt an
additional purification step effectively washes out the impurities
NMR spectroscopy: NMR experiments were performed
on Varian spectrometer operating at 500 MHz in CDCl3 at
1
25 °C. The H chemical shift values were reported on the δ
O
H
N
O
O
OCH3
OH
H
H
H
MDC, MeOH, DCC,
HOBT, t-butylamine
MeOH/NaOH
50-55 oC
H
2
H
H
3
H
H
4
H
O
N
H
O
N
H
O
N
H
H
H
H
Toluene, DDQ,
BSFTA, 90-95oC
H
N
O
H
N
O
H
N
O
MDC,
cyclohexane
H
H
H
Purification,
AcOH-MeOH-water
H
H
H
H
H
H
O
N
H
O
N
H
O
N
H
H
H
H
1(a)
1
1(b)
Scheme-I: Finasteride (1) manufacturing process5
H
N
O
H
N
H
N
H
N
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
O
N
O
N
H
O
N
H
O
N
H
H
H
H
H
Impurity - II
Impurity-A
Impurity-C
Impurity - I
Fig. 2. Impurities of finasteride (1)

Vol. 26, No. 14 (2014)
Impurities in Finasteride 4377
H
N
H
N
O
O
NH2
Toluene, DDQ,
BSFTA, 90-95oC
H
H
(5)
H
H
H
H
O
MDC,DCC,
HOBT,
OH
O
N
H
O
N
H
H
H
(7)
Impurity I
H
H
N
O
H
H
H
N
O
O
N
H
NH2
H
H
Toluene, DDQ,
BSFTA, 90-95oC
H
(3)
(6)
H
H
H
H
MDC,DCC,
HOBT,
O
N
H
O
N
H
H
H
Impurity II
(8)
Scheme-II: Synthesis of Finasteride impurities
from the product but the use of an additional purification step
affects the overall yield as well as an additional unit operation
on a large-scale manufacturing results in poor utilizations of
assets. Impurity profile of a drug substance is critical for its
safety assessment and manufacturing process. Since the levels
of unknown impurities in crude finasteride were above the
acceptable limits, a comprehensive study was carried out by
isolation, characterization and synthesis of these impurities
with the pathway of their formation.
isolation of these impurities. The fractions of impurities were
collected and the organic layer was evaporated by rotavapor
under high vacuum at 45 °C. The remaining aqueous solution
was kept for lyophilization. Finally the impurities were isolated
as fluffy solids.
Structure elucidation of impurity-I: In the positive mass
spectrum, the protonated, [M + H]+ ion was detected at m/z
399.4 (Fig. 4). The molecular ion of impurity-I was found to
be at m/z 398. The mass difference between finasteride and
impurity-I was 26 amu. The positive HR-MS spectrum showed
protonated molecular ion peak at m/z 399.3012 corresponding
to molecular formula C25H39N2O2 [M+ + H]. The molecular
formulae of finasteride (C23H36N2O2) and impurity-I were
compared and found that, the impurity-I has addition of two
carbons and two hydrogen atoms. Further, this can be attributed
to one double bond equivalence.
Amidation of 3 in presence of N,N'-dicyclohexyl-carbo-
diimide and Hydroxybenzotriazole with amine ( 5 & 6) gave
amide ( 7 & 8). Oxidation of Ring A in 7 and 8 in presence
of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and N,O-
bis(trimethylsilyl)trifluoroacetamide in toluene gave impurity
I and II.
RESULTS AND DISCUSSION
The MS/MS data of impurity-I was compared with
finasteride. The impurity-I has also showed two major
fragments at m/z 331 and 317 (Fig. 5). The common fragment
at 317 confirms that the change has taken place on aliphatic
amide group.
Detection of impurity by HPLC: The crude finasteride
was analyzed by HPLC method described in Fig. 3. The
analysis showed presence of two unknown peaks (Fig. 3) at
about 1.6 RRT (impurity-I) and 1.7 RRT (impurity-II).
Isolation of impurity by preparative HPLC: An isocratic
method as discussed in experimental section was used for the
1
The H NMR (500 MHz, CDCl3) spectrum (Fig. 6) of
impurity-I showed two additional protons in aliphatic region
mAU
50
40
30
20
10
0
0
20
40
Fig. 3. HPLC chromatogram of crude Finasteride
60
min

4378 Mohanty et al.
Asian J. Chem.
399.3004
100
110.3265
400.3046
0
160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
m/z
Fig. 4. UPLC-TOF mass spectrum of Impurity-I
399.3004
100
440.3265
441.3307
225.1966
266.2238
300
130.1589
150
798.6033
800
498.3670
391.2849
350 400
442.3345
623.4935648.8922
928.5131
857.5776
850
990.5610
571.2437
0
200
250
450
500
550
600
650
700
750
900
950 1000
m/z
Fig. 5. MS/MS spectrum of Impurity-I
when compared with finasteride. It was observed that the signal
at δ 1.40 ppm corresponding to t-butyl moiety was absent.
This observation was further supported by 13C NMR spectrum
in CDCl3 showed absence of carbon signals at 50.6 and 28.7
ppm of finasteride. The HSQC spectrum showed one methine
and three methylene signals. It was observed that two of the
three methylene groups correspond to two carbons each. The
COSY spectrum showed correlations between the amide proton
at 5.16 ppm and a methane proton at 3.82 ppm, which further
showed correlations with the additional methylene signals. The
NMR observations show that the additional group corresponds
to a cyclohexyl moiety.
Structure elucidation of impurity-II: In the positive
mass spectrum, the protonated, [M + H]+ molecular ion peak
was detected at m/z 393.4 (Fig.7). The molecular ion peak of
impurity-II was found to be at m/z 392. The mass difference
between finasteride and impurity-II was 20 amu. The positive
HR-MS spectrum showed protonated molecular ion at m/z
393.2537 corresponding to molecular formula C25H33N2O2 [M+
+ H]. The molecular formulae of finasteride (C23H36N2O2) and
impurity-II were compared and found that the impurity-II has
two carbons more and less by four hydrogen atoms. Further,
this can be attributed to four additional double bond equi-
valences. The MS/MS data of impurity-II was compared with
finasteride. Finasteride gave two major fragments at m/z 317
and 305. The impurity-II showed one major fragment at m/z
325 (Fig. 8). The fragment of finasteride at m/z 305 showed
20 amu differences with the fragment observed for impurity-
II at m/z 325. This showed the relation between these two
fragments.
These observations from NMR and MS data confirmed
that the structure of impurity-I as t-butyl group of finasteride
substituted by cyclohexyl group.
The 1H NMR (500 MHz, CDCl3) spectrum of impurity-II
showed five protons in aromatic region corresponding to one
phenyl moiety and the absence of protons corresponding to t-
butyl moiety, which was observed at δ 1.40 ppm in finasteride.
This could be due to the substitution of t-butyl moiety by phenyl
group. The amide proton at δ 6.96 ppm showed long range 1H
13C correlations (HMBC) with two quaternary carbon signals
at δ 170.60 ppm and δ 137.91 ppm. The signal at δ 170.60 ppm
corresponds to the carbonyl carbon and the signal at δ 137.91
ppm corresponds to the aromatic carbon. The remaining NMR
signal assignments were comparable with those of finasteride.
These observations from NMR and MS data (Figs. 8 and 9)
confirmed that the structure of impurity-II as t-butyl group of
finasteride substituted by phenyl group.
(c)
(b)
(a)
8
7
6
5
4
3
2
1
0 ppm
1
Fig. 6. Overlay of H NMR data: (a) Finasteride, (b) impurity-I and (C)
impurity-II

Vol. 26, No. 14 (2014)
Impurities in Finasteride 4379
393.2537
100
434.2809
394.2569
435.2836
0
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
480
500
520
540
560
580
m/z
Fig. 7. UPLC-TOF mass spectrum of Impurity-II
393.2537
434.2809
100
394.2569
395.2608
435.2836
436.2879
318.2037
320
391.2791
380
213.0426
200
578.2584
580
238.0225
359.2312
360
559.2618
560
227.0639
220
279.1628
280
339.3331
340
471.2742492.3165
513.2751551.4769
0
180
240
260
300
400
420
440
460
480
500 520 540
m/z
Fig. 8. MS/MS spectrum of Impurity-II
H
N
O
O
O
H
N
2
NH
3
CH
CH
CH
3
3
3
CH
H
CH
CH
H
H
3
3
3
H
H
H
H
H
H
N
H
N
H
O
O
H
H
Finasteride
m/z : 373
m/z : 305
m/z : 317
O
O
O
NH
NH
2
3
NH
CH
H
3
CH
H
3
H
H
H
H
H
H
H
N
H
O
N
H
H
O
H
H
m/z : 331
Impurity-I
m/z : 399
m/z : 317
O
O
NH
2
NH
H
H
H
H
H
H
O
N
H
H
H
Impurity-II
m/z : 393
Fig. 9. Mass fragmentation of impurity-I and impurity II
m/z : 325

4380 Mohanty et al.
Asian J. Chem.
Formation of impurities: Since the compound (4) was a
key intermediate for finasteride (1) hence its quality is critical
for the subsequent stages in the manufacturing process. In this
regard a comprehensive study was carried out for identification
of the two impurities. impurity in reaction mass was isolated
with preparative HPLC and structures were substantiated by
1H NMR, MS and MS-MS. Impurities were synthesized
(Scheme-II), RRT's were established by HPLC co-injection.
Based on the spectral data structure of impurity I and impurity
II were characterized as cyclohexyl and phenyl analog of
finasteride. Noteworthy, to mention that aniline9 and cyclo-
hexyl amine10 are the basic building block for synthesis of
N,N'-Dicyclohexylcarbodiimide (DCC). Hence during our
investigation we focused on the quality of the commercial lots
used in the manufacturing process of finasteride. We envisaged
that if our assumption is true commercial lots of DCC would
show residual impurities 5 and 6. Samples (DCC) were drawn
from plant and subjected to Gas Phase Chromatography.
Interestingly we observed varied percentage of 5 and 6 in the
commercial lots and this was further confirmed by co-injection
of working standard (5 and 6) with DCC.
ACKNOWLEDGEMENTS
The authors thank the management of R&D-CTO and
IPDO, Dr.Reddy's Laboratories Ltd., for supporting this work.
Cooperation from the colleagues from analytical development
is highly appreciated.
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Conclusion
In conclusion, we have isolated and characterized two
process related impurities of finasteride at crude stage.
Structures of these compounds were confirmed based on their
MS and NMR spectral data followed by their independent
synthesis. The identified impurities were synthesized to obtain
sufficient quantities for final confirmation by co-injection in
HPLC.
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