Synthesis and spectral characterization of related substances of vardenafil, an erectile dysfunction drug

Article abstract of DOI:10.1080/00397911.2011.585268

Vardenafil hydrochloride trihydrate (Levitra) is used to treat erectile dysfunction (ED) and is an inhibitor of phosphodiesterase type 5 (PDE-5) enzyme. It maintains higher levels of cyclic guanosine monophosphate (cGMP), relaxes smooth muscles, promotes penile blood flow, and enhances erectile function. During the bulk drug synthesis of vardenafil hydrochloride trihydrate, six related substances (impurities), vardenafil dimer, vardenafil N-oxide, vardenafil glycene, vardenafil oxopiperazine, vardenafil oxoacetic acid, and phenyl vardenafil were identified, and these are reported herein for the first time. The present work describes the synthesis and characterization of these impurities.

Full text of DOI:10.1080/00397911.2011.585268

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Synthesis and Spectral Characterization
of Related Substances of Vardenafil, an
Erectile Dysfunction Drug
Vajrala Venkata Reddy a b , Mandava Venkata Naga Brahmeswara
Rao a , Ghanta Mahesh Reddy a , Khagga Mukkanti b & Ganta
Madhusudhan Reddy a
a Research and Development, Integrated Product Development, Dr.
Reddy's Laboratories Ltd., Qutubullapur, Andhra Pradesh, India
b Center for Pharmaceutical Sciences, Institute of Science and
Technology, Jawaharlal Nehru Technological University, Hyderabad,
Andhra Pradesh, India
Accepted author version posted online: 12 Jan 2012.Published
online: 02 Aug 2012.
To cite this article: Vajrala Venkata Reddy , Mandava Venkata Naga Brahmeswara Rao , Ghanta
Mahesh Reddy , Khagga Mukkanti & Ganta Madhusudhan Reddy (2012): Synthesis and Spectral
Characterization of Related Substances of Vardenafil, an Erectile Dysfunction Drug, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
42:23, 3513-3523
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Synthetic Communications1, 42: 3513–3523, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.585268
SYNTHESIS AND SPECTRAL CHARACTERIZATION OF
RELATED SUBSTANCES OF VARDENAFIL, AN
ERECTILE DYSFUNCTION DRUG
Vajrala Venkata Reddy,1,2 Mandava Venkata Naga
Brahmeswara Rao,1 Ghanta Mahesh Reddy,1
Khagga Mukkanti,2 and Ganta Madhusudhan Reddy1
1Research and Development, Integrated Product Development,
Dr. Reddy’s Laboratories Ltd., Qutubullapur, Andhra Pradesh, India
2Center for Pharmaceutical Sciences, Institute of Science and Technology,
Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh,
India
GRAPHICAL ABSTRACT
Abstract Vardenafil hydrochloride trihydrate (Levitra) is used to treat erectile dysfunction
(ED) and is an inhibitor of phosphodiesterase type 5 (PDE-5) enzyme. It maintains higher
levels of cyclic guanosine monophosphate (cGMP), relaxes smooth muscles, promotes
penile blood flow, and enhances erectile function. During the bulk drug synthesis of varde-
nafil hydrochloride trihydrate, six related substances (impurities), vardenafil dimer, varde-
nafil N-oxide, vardenafil glycene, vardenafil oxopiperazine, vardenafil oxoacetic acid, and
phenyl vardenafil were identified, and these are reported herein for the first time. The
present work describes the synthesis and characterization of these impurities.
Received January 13, 2011.
Address correspondence to Ganta Madhusudhan Reddy, Research and Development, Integrated
Product Development, Innovation plaza, Dr. Reddy’s Laboratories Ltd., Survey Nos. 42, 45, 46, and
54, Bachupally, Qutubullapur, R. R. Dist. 500 072, Andhra Pradesh, India. E-mail: madhusudanrg@
drreddys.com, madhusudanrg@yahoo.co.in
3513

3514
V. V. REDDY ET AL.
Keywords Erectile dysfunction drug; related substances; spectral characterization;
synthesis; vardenafil
INTRODUCTION
Phophodiesterase (V) inhibitors (PDE-5 inhibitors) are an important class of
pharmaceutical compounds, and the first clinical use of these compounds was in
the treatment of male erectile dysfunction (MED).[1] Vardenafil and other ED drugs
inhibit PDE-5 enzyme, which in turn maintains greater levels of cGMP, which
relaxes smooth muscles, promotes penile blood flow, and enhances erectile function.
Vardenafil hydrochloride is designated chemically as 2-[2-ethoxy-5-(4-ethylpipe-
razine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3H-imidao[5,1-f]-[1,2,4]triazine-4-one
monohydrochloride trihydrate (1) and has been demonstrated to be effective in the
treatment of ED.[2,3]
Active pharmaceutical ingredients (APIs) are manufactured by scale-up of syn-
thetic chemical processes. Development of synthetic chemical process can lead to the
generation of unwanted organic impurities. These undesired impurities usually pro-
vide no benefit to patients and can pose risks to patient safety or drug efficacy.[4,5]
Thus, the detection, identification, synthesis, quantification, and control of such
impurities originating in the manufacturing process have become important elements
of drug development to ensure product quality and ultimately patient safety.[6–8]
Figure 1. Structural frame work of vardenafil hydrochloride (1), N-oxide (2), oxopiperazine (3), dimer (4),
vardenafil glycene (5), oxoacetic acid (6), and phenyl vardenafil (7).

IMPURITY PROFILE STUDY OF VARDENAFIL
3515
Regulatory agencies also explicitly regulate the control criteria for these drug-related
substances in APIs and subsequent drug products by providing guidance for the
pharmaceutical industry.[9,10] Pure related substances are required to check analyti-
cal performance characteristics.[10] These impurities are also frequently used to check
the resolution in the analytical method at the time of API despatches. In our manu-
facturing process of vardenafil hydrochloride, we have identified six impurities
(Fig. 1); 2-f2-ethoxy-5-[(4-ethyl-4-hydroxy-4k5-piperazin-1-yl)sulfonyl]phenylg-5-
methyl-7-propylimidazo[5,1-f]-1,2,4-triazin-4-(3H)-one (N-oxide, 2); 2f2-ethoxy-5-
[(3-oxo-1-piperazinyl)sulfonyl]phenylg-5-methyl-7-propylimidazo[5,1-f]-1, 2,4-triazin-
4(3H)-one (oxopiperazine, 3); dimer, 4; N-f[4-ethoxy-3-(5-methyl-4-oxo-7-
propyl-3,4-dihydroimidazo[5,1-f]-1,2,4-triazin-2-yl)phenyl]sulfonylgglycine (glycine,
5);
f[2-(f[4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydroimidazo[5,1-f]-1,2,4-
triazin-2-yl) phenyl]sulfonylgamino)ethyl]aminog(oxo) acetic acid (oxoacetic acid,
6); and 2-[2-ethoxy-5-(4-ethylpiperazine-1-sulfonyl)-phenyl]-5-methyl-7-phenyl-3H-
imidazo[5,1-f]-[1,2,4] triazin-4-one (phenyl vardenafil, 7). Though an increasing num-
ber of publications on analytical methods for the determination of vardenafil and its
active metabolite, N-desethylvardenafil, have been reported,[11–13] a detailed synthetic
procedure has not been reported. In this study, we have up synthesized and character-
ized these related substances without recourse to chromatographic techniques.
RESULTS AND DISCUSSION
Vardenafil hydrochloride trihydrate (1) was synthesized (Scheme 1) by reaction
of imidazolotriazinone 8 with chlorosulfonic acid followed by condensation with N-
ethylpiperazine, and the resulting sulfonamide (1a) was converted to hydrochloride
salt using aqueous hydrochloric acid.[2]
Vardenafil N-oxide impurity was presumably formed by aerial oxidation of
vardenafil, and it was prepared from vardenafil (1a) by treating with per acetic acid
in dichloromethane (Scheme 2). The protonated molecular ion appeared at m=z
505.2. Two doublets at d 7.17 and 8.38 and one multiplet at d 7.86–7.88 are due
Scheme 1. Large-scale synthesis of vardenafil hydrochloride trihydrate (1).

3516
V. V. REDDY ET AL.
Scheme 2. Synthesis of vardenafil N-oxide 2.
to aromatic protons, eight protons appeared at d 3.26–3.43 as a multiplet corre-
sponding to piperazine protons, signals observed at d 1.01, 1.39, 1.57, and 2.64 indi-
cate the presence of methyl protons, and protons at d 1.82–1.91, 2.98, 3.26–3.43, and
4.33 designate the presence of four methylene groups. A deuterium-exchangeable
singlet at d 9.10–10.10 corresponds to amide NH function.
Vardenafil oxopiperazine (3) was prepared from 4-ethoxy-3-(5-methyl-4-
oxo-7-propyl-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-2-yl)-benzenesulfonyl chloride
(9) by the condensation with oxopiperazine (13) in methanol (Scheme 3). The start-
ing material, oxopiperazine (13), was prepared by the condensation of ethylenedia-
mine (10) with ethylchloro acetate (11) followed by cyclization in the presence of
sodium methoxide. The exact reason for the formation of this impurity is not be
known, but acidic conditions favor the formation of this impurity. The protonated
molecular ion appeared at m=z 475.3, and sodium adduct appeared at m=z 497.3.
Two triplets at d 0.92 and 1.32 and one singlet at d 2.48 are due to methyl protons,
and three aromatic protons appeared at d 7.38–7.41 and d 7.92–7.95 as multiplets.
Oxopiperazine protons appeared as multiplets at d 3.22–3.30 and singlet at d 3.52.
Multiplets at d 1.69–1.78, d 4.19–4.24, and one singlet at d 2.83 indicate the presence
of three methylene group protons.
Scheme 3. Synthesis of vardenafil oxopiperazine 3.

IMPURITY PROFILE STUDY OF VARDENAFIL
3517
Scheme 4. Synthesis of vardenafil dimer 4.
Vardenafil dimer 4 was formed due to contamination of piperazine in N-ethyl
piperazine, and it was prepared from 4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-
dihydro-imidazo[5,1-f][1,2,4]triazin-2-yl)benzenesulfonyl chloride (9). Reaction of 9
with piperazine in the presence of triethylamine provided the desired vardenafil
dimer 4 (Scheme 4). The protonated molecular ion appeared at m=z 835.1, and
sodium adduct appeared at m=z 857.1. This compound was identified as dimer 4
based on the spectroscopic data.
This impurity was presumably formed by oxidation and followed by opening
of a six-membered ring in the presence of acidic conditions. Vardenafil glycine 5
was prepared from 4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydro-imidazo[5,1-
f][1,2,4]triazin-2-yl)-benzenesulfonyl chloride (9) by treatment with glycine in the
presence of alkali (Scheme 5). The protonated molecular ion appeared at m=z
450.4, sodium adduct appeared at m=z 472.2, and the spectral data are consistent
with the structure of related substance 5.
The impurity 6 was also formed as a result of oxidation followed by opening of
the six-membered ring in the presence of acidic conditions used for the synthesis of
hydrochloride salt. Vardenafil oxoacetic acid (6) was prepared from 4-ethoxy-3-
(5-methyl-4-oxo-7-propyl-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-2-
yl)-benzenesulfonyl chloride (9) (Scheme 6). Treatment of 9 with ethylenediamine
(10) followed by reaction with ethyloxalyl chloride in the presence of triethylamine
furnished the desired vardenafil oxoacetic acid (6). The protonated molecular ion
appeared at m=z 507, and the sodium adduct appeared at m=z 529.4. Characteristic
deuterium exchangeable signals are observed at d 3.3, d 7.75, d 11.62 corresponding
Scheme 5. Synthesis of vardenafil glycine 5.

3518
V. V. REDDY ET AL.
Scheme 6. Synthesis of vardenafil oxoacetic acid (6).
to two NH protons adjacent to the sulfonyl and carbonyl and one proton from the
carboxylic groups, respectively.
Preparation of intermediate 8 involves condensation of butyryl chloride with
DL-alanine. If butyryl chloride contains traces of benzoyl chloride, it reacts with
DL-alanine to provide phenylvardenafil after several steps. Synthesis of phenyl var-
denafil (7) starts from compound 15 (Scheme 7). Amidrazone 20 is prepared by the
Scheme 7. Synthesis of phenyl vardenafil (7).

IMPURITY PROFILE STUDY OF VARDENAFIL
3519
reaction of hydrazine hydrate with amidine 19. The protonated molecular ion
appeared at m=z 523.1 corresponding to protonated molecular ion, the infrared
and (IR) spectrum showed stretching corresponding to NH and sulfonyl group.
Based on the spectral data, the structure of this substance was assigned as 2-[2-
ethoxy-5-(4-ethylpiperazine-1-sulfonyl)-phenyl]-5-methyl-7-phenyl-3H-imidazo[5,1-
f][1,2,4]triazin-4-one.
EXPERIMENTAL
A gradient reverse-phase liquid chromatographic method was developed for
the quantitative determination of vardenafil hydrochloride trihydrate, process-
related impurities, and intermediates in bulk drugs. The stationary phase used was
a Waters Symmetry shield RP-8, 250 ꢀ 4.6 mm, 5-mm particles. The solution A con-
tains a mixture of 0.025 mM of NaH2PO4 H2O, 1 mL of triethylamine in 1000 mL of
water (pH 7.5)–acetonitrile (85:25, v=v), and solution B contains a mixture of water–
acetonitrile (22:78, v=v). The flow rate was 1.0 mL=min. The high-performance liquid
chromatographic (HPLC) gradient program was set as time in min per % solution B:
0.01=15, 5=15, 37=70, 55=70, 57=15, and 60=15 with a postrun time of 5 min. The
column temperature was maintained at 27 ꢁC, the detection was monitored at a
wavelength of 240 nm, and the injection volume was 10 mL. A mixture of water–
1
acetonitrile (22:78) was used as diluent. The H NMR spectra were recorded at
400 MHz on a Varian Mercury Plus 400-MHz Fourier transform (FT) NMR spec-
trometer. The infrared (IR) spectra were recorded in the solid state as KBr dispersion
medium using a Perkin-Elmer Spectrumone FT IR spectrophotometer. The mass
analyses were performed on AB-4000 Q-trap LC-MS=MS mass spectrometer
MDS SCIEX, Vendor name; Applied Bio systems, Foster City. The solvents and
reagents were used without further purification.
2-f2-Ethoxy-5-[(4-ethyl-4-hydroxy-4k5-piperazin-1-
yl)sulfonyl]phenylg-5-methyl-7-propyl imidazo-[5,1-f]-1,2,4-
triazin-4(3H)-one (N-Oxide, 2)
Per acetic acid (15%, 17 g, 0.04 mol) was added drop wise to a solution of
vardenafil (15 g, 0.03 mol, 1a) in dichloromethane (300 mL) at 0–5 ꢁC. The resulting
solution was stirred for 2 h at 0–5 ꢁC, and then the reaction mixture was allowed to
warm to 20 ꢁC. Water (300 mL) was added to the reaction mass and stirred for
10 min. The organic layer was separated, dried over anhydrous sodium sulfate
(5 g), and concentrated to dryness under vacuum. Solid was isolated from petroleum
ether (150 mL) at 20 ꢁC and dried to a constant weight at 40 ꢁC to yield white solid 2.
Yield 8 g (52%); HPLC purity 99.2%; MS, m=z (EI): 505.2 (Mþ þ H); IR (KBr, cmꢂ1
)
1
=
=
1337 (O S¼O), 3361 (NH), 1699 (C O); H NMR (400 MHz, CDCl3): d 1.01 (t,
J ¼ 8 Hz, 3H), 1.39 (t, J ¼ 7.6 Hz, 3H), 1.57 (t, J ¼ 6.8 Hz, 3H), 1.82–1.91 (m, 2H),
2.64 (s, 3H), 2.98 (t, J ¼ 8 Hz, 2H), 3.26–3.43 (m, 8H), 3.67 (d, J ¼ 12 Hz, 2H),
4.33 (q, J ¼ 4.32 Hz, 2H), 7.17 (d, J ¼ 8.8 Hz, 1H), 7.86–7.88 (m, 1H), 8.38 (d,
J ¼ 2.4 Hz, 1H), 9.10–10.10 (br s, 1H), Anal. calcd. for C23H32N6O5S: C, 54.75; H,
6.39; N, 16.65. Found: C, 54.81; H, 6.37; N, 16.73.

3520
V. V. REDDY ET AL.
2-f2-Ethoxy-5-[(3-oxo-1-piperazinyl)sulfonyl]phenylg-5-methyl-
7-propylimidazo[5,1-f]-1,2,4-triazin-4(3H)-one (Vardenafil
Oxopiperazine, 3)
A solution of ethylchloroacetate (27 g, 0.248 mol) in methanol (50 mL) was
added at 2 ꢁC to a solution of ethylenediamine (80 g, 1.333 mol) in methanol
(150 mL) over a period of 30 min. The reaction mixture was stirred for 15 min,
warmed to 24 ꢁC, and stirred for 90 min. Sodium methoxide (15 g, 0.283 mol) was
added to the reaction mixture and stirred for 5 min at 25–30 ꢁC. The undissolved par-
ticulates were removed by filtration, and the filtrate was distilled off under reduced
pressure at 135 ꢁC to afford oxopiperazine 13.
Compound 9 (5 g, 0.012 mol) was added to a solution of oxopiperazine of 13
(6.09 g, 0.06 mol) in dichloromethane (150 mL) at 25–30 ꢁC and stirred for 30 min.
The reaction mass was washed with water (3 ꢀ 50 mL), and the organic layer was
dried over anhydrous Na2SO4. The organic layer was distilled off under reduced
pressure below 40 ꢁC. The resulted residue was dissolved in MeOH (370 mL), and
the solution was heated to 65 ꢁC. The solution was cooled to 25–30 ꢁC and stirred
for 60 min. The precipitated solid was filtered, washed with methaþnol (20 mL), and
dried at 45 ꢁC for 3 h to afford 5.0 g of 3. MS (EI): m=z 475.3 (M þ H), IR (KBr,
1
cmꢂ1) 1337 (O S¼O), 3361 (NH), 1699 (C O); H NMR (400 MHz, DMSO-d6):
d 0.92 (t, J ¼ 7.2 Hz, 3H), 1.32 (t, J ¼ 6.8 Hz, 3H), 1.69–1.78 (m, 2H), 2.48 (s, 3H),
2.83 (t, J ¼ 8 Hz, 2H), 3.22–3.30 (m, 4H), 3.52 (s, 2H), 4.19–4.24 (m, 2H),
7.38–7.41 (m, 1H), 7.92–7.95 (m, 2H), 8.07 (s, 1H), 11.64 (s, 1H); Anal. calcd. for
C21H26N6O5S: C, 53.15; H, 5.52; N, 17.71. Found: C, 53.18; H, 5.56; N, 17.82.
=
=
Dimer (4)
Piperazine (0.7 g, 0.008 mol) and triethylamine (5 mL, 0.035 mol) were added to
solution of 4-ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydroimidazo[5,1-f]-
a
[1,2,4]triazin-2-yl)benzenesulfonyl chloride (5.6 g, 0.0136 mol, 9) in dichloromethane
(125 mL) at 25–30 ꢁC. The reaction mass was stirred for 30 min at 25–30 ꢁC, and
water (50 mL) was added to the reaction mass and stirred for 30 min. The isolated
solid was filtered, washed with water (5 mL), and dried at 45 ꢁC to get a white solid.
Yield 2.8 g (20%); purity by HPLC: 99%; mp: 300 ꢁC (dec.); MS (EI): m=z 835.1
1
(Mþ þ H), 857.0 (Mþ þ Na); 873.1 (Mþ þ K); H NMR (400 MHz, DMSO-d6): d
0.96 (t, J ¼ 7.2 Hz, 6H), 1.34 (t, J ¼ 7.0 Hz, 6H), 1.75 (m, 4H), 2.55 (s, 6H), 2.82 (t,
J ¼ 7.6 Hz, 4H), 3.00 (s, 8H), 4.25 (q, J ¼ 7.0 Hz, 4H), 7.4 (d, J ¼ 4.6 Hz, 2H), 7.85
(d, J ¼ 2.2 Hz, 2H), 7.9 (s, 2H), 11.61 (s, 2H); IR (KBr, cmꢂ1): 1703 (C O amide),
=
=
3374 (NH amide), 1352 (O S¼O), Anal. calcd. for C38H46N10O8S2: C, 54.66; H,
5.55; N, 16.78. Found: C, 54.69; H, 5.62; N, 16.62.
N-f[4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydroimidazo[5,1-
f]-1,2,4-triazin-2-yl) phenyl]sulfonylgglycene (Vardenafil Glycene, 5)
Glycene (10 g, 0.133 mol) was added to a solution of sodium hydroxide (8.0 g,
0.2 mol) in water (300 mL) at 25–30 ꢁC and stirred for 30 min at 25–30 ꢁC. The
reaction mixture was cooled to 2 ꢁC, and a solution of 4-ethoxy-3-(5-methyl-4-
oxo-7-propyl-3,4-dihydro-imidazo[5,1-f][1,2,4]triazin-2-yl)benzenesulfonyl chloride

IMPURITY PROFILE STUDY OF VARDENAFIL
3521
(10 g, 0.024 mol, 9) in dichloromethane (100 mL) was added and stirred for 60 min.
The reaction mixture was allowed to warm to 20 ꢁC and stirred for 90 min. The
organic and aqueous layers were separated, and the aqueous layer pH was adjusted
to 1.6 using aqueous hydrochloric acid (10 mL). The aqueous layer was extracted
with EtOAc (2 ꢀ 100 mL). The combined organic layer was dried over anhydrous
Na2SO4 and distilled off solvent under vacuum. Water (100 mL) was added to the
crude product, and pH was adjusted to 10 using caustic lye (12 mL). The aqueous
layer was washed with dichloromethane (3 ꢀ 100 mL). The pH of the aqueous layer
was adjusted to 1.5 using hydrochloric acid (28 mL) and stirred for 90 min. The pre-
cipitated solid was filtered, washed with water (25 mL), and dried at 40 ꢁC to afford
1
7.0 g of 5 with 99% purity by HPLC. MS (EI): m=z 450.4 (Mþ þ H); H NMR
(400 MHz, DMSO-d6): d 0.95 (t, J ¼ 7.6 Hz, 3H), 1.33 (t, J ¼ 6.8 Hz, 3H), 1.82
(q, J ¼ 7.6 Hz, 2H), 2.62 (s, 3H), 3.04 (t, J ¼ 9.2 Hz, 2H), 3.60 (m, 2H), 4.21 (t,
J ¼ 7.2 Hz, 2H), 7.37 (d, J ¼ 8.8 Hz, 1H), 7.93–7.99 (m, 2H), 8.10 (m, 1H), 12.35
(s, 1H); IR (KBr, cmꢂ1): 1733 (C O), 3299 (NH amide), 1335 (O S¼O), Anal.
calcd. for C19H23N5O6S: C, 50.77; H, 5.16; N, 15.58. Found: C, 50.75; H, 5.23; N,
15.52.
=
=
f[2-(f[4-Ethoxy-3-(5-methyl-4-oxo-7-propyl-3,4-dihydroimidazo[5,1-
f]-1,2,4-triazin-2-yl) phenyl]sulfonylgamino)ethyl]aminog(oxo)acetic
Acid (Oxoacetic Acid, 6)
A solution of 9 (5 g, 0.012 mol) in dichloromethane (350 mL) was added slowly
at 2 ꢁC to an ethylene diamine (50 mL, 0.747 mol) over a period of 80 min. Reaction
mass was washed with water (2 ꢀ 50 mL). The combined organic layer was dried over
anhydrous Na2SO4. Ethyloxalyl chloride (2.7 g, 0.017 mol) and Et3N (4.1 g,
0.040 mol) were added at 25–30 ꢁC to the organic layer and stirred for 15 min. Sol-
vent was distilled under reduced pressure at below 32 ꢁC. A solution of 1 N NaOH
solution (2.2 g in 55 mL water) and MeOH (140 mL) was added and stirred for
15 min at 25 ꢁC. Solvent was distilled under reduced pressure at below 45 ꢁC, water
(30 mL) was added to the residue, and the solution was washed with dichloro-
methane (2 ꢀ 50 mL). The pH of the aqueous layer was adjusted to 1.2 using diluted
hydrochloric acid; the compound was extracted into EtOAc (50 mL) and stirred for
20 min at 25–30 ꢁC. The precipitated solid was filtered and dried at 45 ꢁC to afford
1
5.0 g of 6. HPLC purity 98%; MS m=z (EI): 507 (Mþ þ H), 529.4 (Mþ þ Na); H
NMR (400 MHz, DMSO-d6): d 0.92 (t, J ¼ 7.2 Hz, 3H), 1.32 (t, J ¼ 6.8 Hz, 3H),
1.69–1.78 (m, 2 H), 2.48–2.51 (m, 5H), 2.82–2.91 (m, 4H), 3.20 (t, J ¼ 6.4 Hz, 2H),
3.3 (br s, 1H), 7.34 (d, J ¼ 9.2 Hz, 1H), 7.75–7.76 (m, 2H), 7.92 (t, J ¼ 10.4 Hz,
1H,), 8.75 (t, J ¼ 12 Hz, 1H), 11.63 (s, 1H); IR (KBr, cmꢂ1): 1692 (C O), 1624
=
=
=
(C O amide), 3325 (NH amide), 1326 (O S¼O), Anal. calcd. for C21H26N6O7S:
C, 49.79; H, 5.17; N 16.59. Found: C, 49.86; H, 5.23; N, 16.48.
2-[2-Ethoxy-5-(4-ethylpiperazine-1-sulfonyl)phenyl]-5-methyl-
7-phenyl-3H-imidazo[5,1-f] [1,2,4]triazin-4-one (Phenyl Vardenafil, 7)
DL-alanine (25 g, 0.28 mol, 15) was added slowly to a solution of sodium
hydroxide (35 g, 0.875 mol) in water (125 mL) at 6 ꢁC and stirred for 10 min. The

3522
V. V. REDDY ET AL.
reaction mass was cooled to 3 ꢁC, and benzoyl chloride (49 mL, 0.422 mol) was
added dropwise at 3 ꢁC and stirred for 60 min. The reaction mass was warmed to
25–30 ꢁC and stirred for 3 h. The pH of the reaction mass was adjusted to 1.8 using
hydrochloric acid (40 mL) and stirred for 25 min. The precipitated solid was filtered,
and the wet solid was purified from dichloromethane. The compound was dried at
45 ꢁC for 3 h (45 g, yield 76%) to give 2-benzoylamino propionic acid (16). A solution
of (N,N-dimethylamino)pyridine (DMAP) (2.0 g, 0.016 mol) and 50 mL pyridine in
toluene (400 mL) was added to compound 16 (45 g, 0.233 mol) in toluene (100 mL)
and stirred for 15 min. Ethyloxalyl chloride (50 mL, 2.68 mol) was added at
25–30 ꢁC for 45 min. The reaction mass was heated to 80 ꢁC and stirred for 3 h.
The reaction mass was cooled to 25–30 ꢁC and washed with water (2 ꢀ 100 mL).
The organic layer was dried over anhydrous Na2SO4 and distilled off under reduced
pressure at below 60 ꢁC to afford 17 (yield 61.3 g, 83%).
A mixture of 2-ethoxybenzamidine hydrochloride (25 g, 0.12 mol) and hydra-
zine hydrate (8.33 g, 0.16 mol) in methanol (100 mL) was stirred for 90 min at
28 ꢁC. Compound 17 was added slowly to the solution and then heated to 65 ꢁC.
The reaction mass was stirred for 90 min at 65 ꢁC. The reaction mass was com-
pletely distilled off under reduced pressure below 60 ꢁC. Solvent was chased with
toluene (100 mL) and distilled off under reduced pressure below 60 ꢁC. A solution
of POCl3 (15 mL) in toluene (250 mL) was added slowly to the residue, at 60 ꢁC the
reaction mass was heated to 80 ꢁC and stirred for 4 h. The solvent was distilled off
under reduced pressure below 65 ꢁC and the residue cooled to 25–30 ꢁC. The resi-
due was portioned between water (250 mL) and dichloromethane (250 mL) and
then pH was adjusted 6.5 using caustic lye. Organic and aqueous layers were
separated, and the aqueous layer was washed with dichloromethane (3 ꢀ 150 mL).
The organic layer was dried over anhydrous Na2SO4 and distilled off under
reduced pressure below 40 ꢁC. The compound was recrystallized from acetonitrile
(75 mL) and filtered at 5 ꢁC. The wet material was dried at 40 ꢁC to afford 18.
Yield 20 g (30%).
Compound 18 (20 g, 0.05 mol) was added to chlorosulfonic acid (40 mL) at
0 ꢁC, stirred for 10 min, and warmed to 25–30 ꢁC. The reaction mass was quenched
with water at 0 ꢁC and extracted with dichloromethane (1 ꢀ 300 mL, 2 ꢀ 100 mL).
The combined organic layer was dried on anhydrous Na2SO4. N-Ethylpiperazine
(13.0 g, 0.11 mol) was added to the mass and stirred for 90 min at 25–30 ꢁC. The reac-
tion was quenched with water (150 mL), and the aqueous layer and organic layer
were separated. The organic layer was washed with water (50 mL). The combined
organic layer was dried over anhydrous Na2SO4, and volatiles were removed in
vacuo to yield 7 (yield: 8.5 g, 30%). MS m=z (EI): 523.1 (Mþ þ H); 1H NMR
(400 MHz, CDCl3): d 1.05 (t, 3H), 1.62 (t, 3H), 2.41 (t, 2H), 2.55–2.60 (m, 4H),
2.75 (s, 3H), 3.00–3.11 (m, 4H), 4.35 (q, 2H), 7.18 (d, 1H), 7.45–7.5 (m, 3H), 7.9
(d, 1H), 8.38 (d, 2H), 8.58 (s, 1H), 9.70 (brs 1H); IR (KBr, cmꢂ1): 1715 (C O),
=
=
3303 (NH), 1351 (O S¼O). Anal. calcd. for C26H30N6O4S: C, 59.75; H, 5.79; N,
16.08; Found: C, 59.83; H, 5.69; N, 16.92. 13C NMR (200 MHz, CDCl3): 11.74,
14.26, 14.47, 45.90, 51.56, 51.69, 65.75, 76.36, 77.00, 77.63, 112.82, 114.77, 118.75,
127.94, 128.12, 128.30, 128.46, 129.38, 130.26, 132.44, 141.12, 142.37, 145.22,
154.89, 157.13, 159.95.

IMPURITY PROFILE STUDY OF VARDENAFIL
3523
ACKNOWLEDGMENTS
The authors are grateful to their colleagues in the Analytical Research
Department of IPDO, Discovery Research, and the management of Dr. Reddy’s
Laboratories Ltd., for providing necessary support.
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