The discovery of tadalafil: A novel and highly selective PDE5 inhibitor. 2: 2,3,6,7,12,12a-Hexahydropyrazino[1′,2′ :1,6]pyrido[3,4-b]indole-1,4-dione analogues

Article abstract of DOI:10.1021/jm0300577

Modification of the hydantoin ring in the previously described lead compound 2a has led to the discovery of compound 12a, tadalafil, a highly potent and highly selective PDE5 inhibitor. The replacement of the hydantoin in compound 2a by a piperazinedione ring led to compound cis-11a which showed similar PDE5 inhibitory potency. Introduction of a 3,4-methylenedioxy substitution on the phenyl ring in position 6 led to a potent PDE5 inhibitor cis-11c with increased cellular potency. Optimization of the chain on the piperazinedione ring led to the identification of the racemic cis-N-methyl derivative 11i. High diastereospecificity for PDE5 inhibition was observed in the piperazinedione series with the cis-(6R,12aR) enantiomer displaying the highest PDE5 inhibitory activity. The piperazinedione 12a, tadalafil (GF196960), has been identified as a highly potent PDE5 inhibitor (IC₅₀ = 5 nM) with high selectivity for PDE5 vs PDE1-4 and PDE6. Compound 12a displays 85-fold greater selectivity vs PDE6 than sildenafil 1. 12a showed profound and long-lasting blood pressure lowering activity (30 mmHg/>7 h) in the spontaneously hypertensive rat model after oral administration (5 mg/kg).

Full text of DOI:10.1021/jm0300577

J . Med. Chem. 2003, 46, 4533-4542
4533
Th e Discover y of Ta d a la fil: A Novel a n d High ly Selective P DE5 In h ibitor . 2:
2,3,6,7,12,12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e
An a logu es
Alain Daugan,* Pascal Grondin, Ce´cile Ruault, Anne-Charlotte Le Monnier de Gouville, Herve´ Coste,
J ean Michel Linget, J orge Kirilovsky, Franc¸ois Hyafil, and Richard Labaudinie`re†
Laboratoire GlaxoSmithKline, Centre de Recherches, 25-27 Avenue du Que´bec, 91951 Les Ulis Cedex, France
Received February 5, 2003
Modification of the hydantoin ring in the previously described lead compound 2a has led to
the discovery of compound 12a , tadalafil, a highly potent and highly selective PDE5 inhibitor.
The replacement of the hydantoin in compound 2a by a piperazinedione ring led to compound
cis-11a which showed similar PDE5 inhibitory potency. Introduction of a 3,4-methylenedioxy
substitution on the phenyl ring in position 6 led to a potent PDE5 inhibitor cis-11c with
increased cellular potency. Optimization of the chain on the piperazinedione ring led to the
identification of the racemic cis-N-methyl derivative 11i. High diastereospecificity for PDE5
inhibition was observed in the piperazinedione series with the cis-(6R,12aR) enantiomer
displaying the highest PDE5 inhibitory activity. The piperazinedione 12a , tadalafil (GF196960),
has been identified as a highly potent PDE5 inhibitor (IC50 ) 5 nM) with high selectivity for
PDE5 vs PDE1-4 and PDE6. Compound 12a displays 85-fold greater selectivity vs PDE6 than
sildenafil 1. 12a showed profound and long-lasting blood pressure lowering activity (30 mmHg/
>7 h) in the spontaneously hypertensive rat model after oral administration (5 mg/kg).
Ch a r t 1
In tr od u ction
Inhibition of cyclic nucleotide phosphodiesterase type
5 (PDE5) as a therapeutic target has received consider-
able attention in recent years, particularly for the
treatment of cardiovascular diseases, e.g., angina, hy-
pertension, and congestive heart failure.1,2 Research in
this area was refocused and stimulated by the demon-
stration of the role of PDE5 in penile erection and the
recognition that PDE5 inhibition would be useful in
treating male erectile dysfunction (MED).3 After these
discoveries, sildenafil 1 (Viagra)4 was approved for the
treatment of MED.5,6 Despite the efficacy of sildenafil,
clinically significant adverse effects have been noted,7
and it has been proposed that some of these side effects
may be due to a lack of selectivity for PDE5.8 Notably,
visual disturbances could stem from inhibition of the
retina-specific PDE enzyme, namely, PDE6, involved in
vision signal transduction. We describe the discovery
of a structurally different series of PDE5 inhibitors that
show greatly enhanced PDE5 selectivity, most signifi-
cantly vs PDE1 and PDE6.
Previously,9 we described the discovery of a series of
hydantoins and the identification of butyl hydantoin 2a
as a potent and selective inhibitor of PDE5 (Chart 1).
This compound was found to be highly potent on PDE5
enzyme, selective versus the other phosphodiesterases,
and capable of increasing intracellular cGMP levels in
rat smooth muscle cells (RSMC assay). However, hy-
dantoin 2a displayed poor hypotensive activity in spon-
taneously hypertensive rats after oral administration
(30 mg/kg). The objective of our research in the early
1990s was to identify a novel chemical series of potent
and selective inhibitors of PDE5, especially vs PDE1 and
PDE6, with improved pharmacokinetic and pharmaco-
logical profiles.
In the present paper, we describe the results we
obtained by modification of the hydantoin ring in
compound 2a to discover piperazinedione 12a (GF
196960, tadalafil), a highly potent, selective, efficacious,
and orally bioavailable inhibitor of PDE5.
Ch em istr y
Racemic compounds bearing structural modifications
of the hydantoin ring were prepared from key interme-
diates 3a ,c (cis or trans), as presented in Scheme 1. The
hydantoin cis-2a was prepared as described in the
previous paper9 by treatment of cis-aminoester 3a with
butyl isocyanate in refluxing 2-butanone. Reduction of
the carbonyl groups of 2a with a large excess of lithium
aluminum hydride in THF according to the literature
procedure10 led to the formation of the imidazolidine
derivative 4. Treatment of cis-3c with a large excess of
methylamine in methanol11 gave the corresponding
amide 5. Reduction of the amide 5 with lithium alumi-
num hydride in refluxing THF, followed by cyclization
* Corresponding author: Phone: +33 1 69 29 61 30. Fax: +33 1 69
07 48 92. E-mail: acd9791@gsk.com.
† Current address: Genome Therapeutics Corporation, 100 Beaver
Street, Waltham, MA 02453-8443.
10.1021/jm0300577 CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

4534 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
Sch em e 1a
a
Conditions: (a) Bu-NCO, 2-butanone, reflux; (b) LiAlH4, THF, 40 °C; (c) Me-NH2, MeOH; (d) triphosgene, CH2Cl2; (e) PhCH2NH2; (f)
(HCHO)n, toluene.
Sch em e 2a
a
Conditions: (a) R1-Ph-CHO, CF3CO2H, CH2Cl2; (b) chloroacetyl chloride, Et3N, CH2Cl2 or NaHCO3, CHCl3; (c) R2-NH, MeOH, reflux.
of the diamine intermediate 6 in the presence of
triphosgene or 1,1′-carbonyldiimidazole,12 led to the
formation of 3-oxo-derivative 7. The benzyl amide 8,
obtained by heating trans-3a with benzylamine, was
cyclized to give imidazolidinone derivative 9 using
paraformaldehyde in refluxing toluene.
Piperazinedione derivatives cis- and trans-11a -p
were prepared via the elaboration of key intermediate
3a -g as illustrated in Scheme 2. The modified Pictet-
Spengler reaction, starting from racemic tryptophan
methyl ester, was used to generate the cis- and trans-
tetrahydro-â-carbolines 3a -g as described in the previ-
ous paper.9 Chloroacetylation of the cis- or trans-â-
carbolines 3a -g, in the presence of triethylamine or
sodium hydrogen carbonate as base, provided, respec-
tively, the cis- and the trans-chloroacetyl derivatives
10a -g in good yield. The piperazinedione derivatives
cis- and trans-11a -p were then obtained by ring closure
of the cis- or trans-chloroacetyl derivatives 10a -g in the
presence of primary amines in refluxing methanol.
Chiral derivatives 12a , 12d , 13-16 and 12b, 12c were
prepared using the same route as shown in Scheme 2
but starting from, respectively, D-(-) and L-(+) tryp-
tophan methyl ester as starting materials (Scheme 3).
The piperazinedione 12a was subjected to chiral HPLC
analysis, and no trace of diastereoisomers 12b-d was
detected, indicating that no racemization occurred dur-
ing the process.
Resu lts a n d Discu ssion
Compounds were evaluated in an in vitro assay for
inhibitory activity against bovine PDE5. Each compound
was evaluated in two steps. The first step was the

Discovery of Tadalafil. Part 2
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4535
Sch em e 3a
a
Conditions: (a) R1-Ph-CHO, CF3CO2H, CH2Cl2; (b) chloroacetyl chloride, NaHCO3, CHCl3; (c) R2-NH2, MeOH, reflux.
Ta ble 1. PDE5 Inhibition and Cellular Activity: Modification of the Hydantoin Ring
PDE5
RSMC
compd
R1
R2
IC50 (µM)a
EC50 (µM)
cis-2a
cis-4
cis-2b
cis-7
trans-2c
trans-9
trans-2d
cis-11a
4-OMe
4-OMe
4-OMe
3,4-OCH2O
4-OMe
4-OMe
3,4-OCH2O
4-OMe
butyl
butyl
0.008
>10
0.7
ndb
1
>10
>10
>10
3
methyl
methyl
benzyl
benzyl
benzyl
butyl
0.010
0.060
0.018
0.060
0.006
0.005
1.5
a
b
IC50 values were reproducible to (25%. Not determined.
determination of the percentage of inhibition at 10 µM
performed in triplicate. For compounds displaying a
percentage of inhibition greater than 50% at 10 µM, the
IC50 was determined from a concentration-response
curve using concentrations of 1 nM, 10 nM, 100 nM, 1
µM, and 10 µM, each tested in duplicate. IC50 values
were reproducible to (25%. Selectivity toward other
PDEs was evaluated using similar assays. Rat PDE5
was not available at the time, but it was known that
there is 94.3% homology between rat and bovine PDE5.13
We therefore made the assumption that our PDE5
inhibitors would exhibit similar potency against the rat
enzyme as against bovine. Selected compounds were
then tested in a cellular assay using atrial natriuretic
factor treated rat aortic smooth muscle cells (RSMC).
This assay is designed to reveal the ability of the tested
compound to increase intracellular cGMP concentration
in cells. In this assay the EC50 values were determined
as the concentration giving half-maximal increase of
cGMP observed with saturating compound concentra-
tion. Finally, spontaneously hypertensive rat model
(SHR) was used to measure blood pressure lowering
ability of the best compounds.
Compounds were initially prepared in racemic form.
Both cis- and trans-isomers were synthesized since
we had previously shown that in the hydantoin series9
the isomers gave equivalent potency of PDE5 inhibi-
tion.
Mod ifica tion of th e Hyd a n toin Rin g. Modification
of the fused hydantoin ring system led to the following
general conclusions (Table 1): Deletion of the two
carbonyl groups (cis-2a vs cis-4) led to a marked
decrease in potency, whereas removal of the lower
carbonyl group (trans-2c vs trans-9) was only margin-
ally deleterious. The analogue lacking the upper car-
bonyl group (cis-2b vs cis-7) also showed slightly
decreased potency (the 4-methoxy substituent on the
pendant phenyl group was essentially equivalent to the
3,4-methylenedioxy group trans-2c vs trans-2d ). As
noted previously,9 there was little difference in potency
between the cis- and trans-hydantoin compounds (cis-
2a vs trans-2a ).

4536 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
Ta ble 2. PDE5 Inhibition and Cellular Activity: SAR on the
Ta ble 3. PDE5 Inhibition and Cellular Activity: SAR on the
Phenyl Ring in C-6 Position
Piperazinedione Ring
PDE5
RSMC
PDE5
RSMC
compd
R1
R2
butyl
IC50 (µM)a EC50 (µM)
compd
cis-2a
R1
IC50 (µM)a
EC50 (µM)
cis-11a
cis-11c
cis-11h
cis-11i
cis-11j
cis-11k
cis-11l
4-OMe
3,4-OCH2O butyl
3,4-OCH2O hydrogen
3,4-OCH2O methyl
4-OMe
4-OMe
0.005
0.005
0.012
0.005
0.041
0.009
0.005
0.006
0.006
0.023
0.003
1.5
0.6
0.6
0.5
0.2
0.5
0.3
0.5
0.5
0.6
0.8
4-OMe
4-OMe
4-OMe
4-OMe
H
3,4-OCH2O
3,4-OCH2O
4-CN
0.008
0.005
0.005
0.070
0.091
0.005
0.138
0.758
0.015
0.026
62%c
0.7
1
1.5
trans-2a
cis-11a
trans-11a
cis-11b
cis-11c
trans-11c
cis-11d
cis-11e
cis-11f
>10
>10
0.6
methyl
ethyl
3,4-OCH2O c.hexyl
3.5
ndb
>10
0.35
ndb
cis-11m 3,4-OCH2O i-propyl
cis-11n
cis-11o
cis-11p
4-OMe
4-Me
3,4-OCH2O benzyl
CH2-c.propyl
CH2-c.propyl
4-Cl
4-Me
3,4-OMe
cis-11g
a
a
b
c
IC50 values were reproducible to (25%.
IC50 values were reproducible to (25%. Not determined.
Inhibition at 10 µM.
%
one series. As shown in Table 2, the cis-diastereoisomers
were found to be significantly more potent than the
corresponding trans-diastereoisomers (cf. cis-11a , -11c
vs trans-11a , -11c). These results are in marked con-
trast to the results previously obtained in the hydantoin
series where the cis- and trans-isomers 2a showed
equivalent PDE5 inhibitory potencies.9
Since it appeared that retention of the two carbonyl
groups was nevertheless beneficial, we decided to ex-
pand the hydantoin ring to the six-membered pipera-
zinedione system. Gratifyingly, the piperazinedione cis-
11a retained the PDE5 inhibitory potency of the
corresponding hydantoin cis-2a , with a similar func-
tional effect on the intracellular cGMP concentration in
ANF-treated rat aortic smooth muscle cells (Table 1).
Mod ifica tion a t th e C-6 P osition . Having shown
that a hydantoin ring can be replaced by a piperazinedi-
one heterocycle, we turned our attention to the explora-
tion of the SAR around the C-6 aryl substituent. A series
of substituted phenyl groups was incorporated at C-6
position and the results are shown in Table 2.
N-Su bstitu tion of th e 1,4-P ip er a zin ed ion e. Re-
taining the best 6-aryl substituents (4-MeO, 3,4-OCH2O,
and 4-Me) and the cis-relative stereochemistry, different
N-substituted piperazinedione derivatives were evalu-
ated for their impact on PDE5 inhibition and functional
activity. Results are shown in Table 3. It is apparent
that a wide range of N-alkyl groups is tolerated on the
piperazinedione ring, similar to what has been observed
in the hydantoin series.9
Substitution on the C-6 aromatic ring with electron-
donating groups generally increased inhibitory potency
compared with the unsubstituted compound 11b. The
best results were obtained with the 4-methoxy (cis-11a )
and 3,4-methylenedioxy substituents (cis-11c) where an
18-fold improvement in PDE5 inhibitory potency was
observed, compared to the unsubstituted analogue 11b.
In contrast, introduction of strong electron-withdrawing
groups such as cyano (cis-11d ) at the 4-position of the
phenyl ring resulted in a marked decrease in PDE5
inhibitory activity. Introduction of the weaker electron-
withdrawing chlorine atom in the para position (cis-11e)
retained the PDE5 inhibitory activity but totally lost
functional activity in the cellular assay. Substitution at
the 4-position with a methyl group (cis-11f) also resulted
in improved potency vs PDE5 compared with the
unsubstituted compound 11b. Disubstitution of the
phenyl ring at positions 3 and 4 by methoxy groups (cis-
11g) led to a complete loss in activity on PDE5.
Interestingly, the conformationally constrained 3,4-
methylenedioxy analogue, cis-11c, retained good PDE5
inhibitory potency, suggesting that 3,4-substitution
needs to be sterically compact.
Absolu te Ster eoch em ica l Requ ir em en t for P DE5
In h ibition . The two pairs of enantiomers 12a ,c and
12b,d were prepared and evaluated for PDE5 inhibi-
tory potency (Table 4). In the cis-isomer series the
(6R,12aR) diastereoisomer 12a was the more potent
PDE5 inhibitor, while in the enantiomeric cis-compound
of (6S,12aS) absolute configuration 12c was inactive
at concentrations up to 10 µM. Of the two trans-
compounds the (6R,12aS) enantiomer 12b was signifi-
cantly more potent than the (6S,12aR) isomer 12d .
Compound 12a was found to be 18-fold more potent than
12b and 1200-fold more potent than 12d on PDE5,
demonstrating the critical stereochemical requirement
at position-6 for a R absolute configuration to display
strong PDE5 inhibitory activities in this piperazinedione
series.
Ta ble 4. PDE5 Inhibition and Absolute Configuration
compd
configuration
PDE5 IC50 (µM)a
12a
12b
12c
12d
(6R,12aR)
(6R,12aS)
(6S,12aS)
(6S,12aR)
0.005
0.090
>10
6
We next investigated the stereochemical require-
ments for PDE5 inhibitory activity in the piperazinedi-
a
IC50 values were reproducible to (25%.

Discovery of Tadalafil. Part 2
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4537
blood pressure was maintained for at least 7 h after oral
administration.
Consistent with the in vivo activity observed, a
pharmacokinetic study (Table 7) demonstrated that
compound 12a has an excellent pharmacokinetic profile
in the rat with a bioavailability of 63% and a low plasma
clearance, indicative of a high metabolic stability.
Con clu sion
The hydantoin lead 2a is a potent and specific PDE5
inhibitor which shows moderate antihypertensive activ-
ity in SHR model. A medicinal chemistry program based
on the hydantoin series has led to the discovery of the
piperazinedione 12a (GF196960), a potent inhibitor of
phosphodiesterase type 5, which shows a high degree
of selectivity for PDE5 vs PDE1-4 and PDE6. Com-
pound 12a displays profound and long-lasting blood
pressure lowering activity in the spontaneously hyper-
tensive rat model after acute treatment. GF196960, 12a
was selected as a clinical development candidate for the
treatment of hypertension and congestive heart failure
and is now approved for the treatment of male erectile
dysfunction (INN, tadalafil; Trademark, Cialis).
F igu r e 1. X-ray Crystal Structure for Compound 12a .
Ta ble 5. PDE5 Inhibition and Cellular Activity: (6R,12aR)
Enantiomers of Selected Piperazinediones
Exp er im en ta l Section
All starting materials were commercially available and used
without further purification. All reactions were carried out
with the use of standard techniques under an inert atmosphere
(Ar or N2). Organic extracts were routinely dried over anhy-
drous Na2SO4. Solvent removal refers to rotary evaporation
under reduced pressure at 30-40 °C. The analytical thin-layer
chromatography (TLC) was carried out on E. Merck 60-F254
precoated silica gel plates and components were usually
visualized using UV light, iodine vapor, or Dragendorff
preparation. Flash column chromatography was performed on
silica gel 60 (E. Merck, 230-400 mesh). Melting points were
determined on a hit-stage Kofler apparatus and are uncor-
rected. Proton NMR (1H NMR) and carbon NMR (13C NMR)
spectra were recorded at ambient temperature on a Bruker
Avance 300 DPX spectrometer using tetramethylsilane as
internal standard, and proton chemical shifts are expressed
in ppm in the indicated solvent. The following abbreviations
are used for multiplicity of NMR signals: s ) singlet, d )
doublet, t ) triplet, q ) quadruplet, dd ) double doublet, m )
multiplet. Mass spectra were determined using a Hewlett-
Packard 5890 and 5971 series. The elemental analyses were
performed by Wolff Laboratories and are within (0.4% of the
theoretical value, unless stated otherwise. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter in a 1-dm
cell in the indicated solvent.
PDE5
RSMC
compd
R1
R2
IC50 (µM)a
EC50 (µM)
12a
13
14
15
16
3,4-OCH2O
3,4-OCH2O
3,4-OCH2O
4-OCH3
methyl
butyl
i-propyl
c.pentyl
hydrogen
0.005
0.003
0.008
0.017
0.011
0.15
1
0.15
0.2
0.3
3,4-OCH2O
a
IC50 values were reproducible to (25%.
The relative stereochemistry of compound 12a was
confirmed by crystal X-ray structure determination as
illustrated in Figure 1.
The stereochemically preferred (6R,12aR) isomers of
a selection of the most potent racemic cis-compounds
were prepared (Table 5). Compound 12a was chosen for
further study based on its potency vs PDE5 and in the
RSMC assay.
P h osp h od iester a se Selectivity. As shown in Table
6, compound 12a is highly selective for PDE5 vs PDE1-
4. More significantly, compound 12a shows considerably
greater selectivity vs PDE6 compared to sildenafil 1.
Furthermore, these two compounds displayed the same
IC50 against both bovine and human recombinant PDE5
(data not shown).
Acu te Effects of Com p ou n d 12a on Blood P r es-
su r e. Compound 12a was evaluated in the conscious
spontaneously hypertensive rat model (SHR), which is
generally considered as a reference model for antihy-
pertensive drug evaluation. As shown in Figure 2,
compound 12a (5 mg/kg) reduced mean blood pressure
by 30 mmHg within the first 30 min, and the fall in
Meth yl 1-(1,3-Ben zod ioxol-5-yl)-2,3,4,9-tetr a h yd r o-1H-
â-ca r bolin e-3-ca r boxyla te (cis- a n d tr a n s-3c). Rep r esen -
ta tive exa m p le: Racemic tryptophan methyl ester (13 g, 59.5
mmol, 1 equiv) and 1,3-benzodioxole-5-carboxaldehyde (9.7 g,
64.66 mmol, 1.08 equiv) were dissolved in CH2Cl2 (300 mL)
and cooled to 0 °C with an ice bath. To this solution was added
dropwise TFA (9 mL), and the mixture was stirred at room
temperature for 24 h. The reaction mixture was then basified
with aqueous NaHCO3 and extracted with CH2Cl2 (3×). The
organic layer was washed with water, brine, dried over Na2-
SO4, filtered, and evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica
gel eluting with CH2Cl2/MeOH, 99/1, to give first the cis-isomer
3c as white crystals (6.5 g, 31%), mp 90-93 °C after recrys-
1
tallization from methanol; H NMR (CDCl3) δ 7.51 (m, 2H),
7.29-7.08 (m, 3H), 6.91-6.75 (m, 3H), 5.94 (s, 2H), 5.16 (s,
1H, C1-H), 3.95 (dd, 1H, J ) 11.2, 4.2 Hz, C3-H), 3.80 (s,
3H), 3.21 (dd, 1H, J ) 15.2, 4.3 Hz, C4-H), 2.99 (dd, 1H, J )
15.1, 11 Hz, C4-H), 2.30 (br s, 1H); 13C NMR (CDCl3) δ 173.8,
148.8, 148.5, 136.8, 135.3, 135.2, 127.8, 122.6, 120.3, 118.9,

4538 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
Ta ble 6. PDE5 Activity and Selectivity of 12a on PDE Isoforms
IC50 (µM)d
IC50 ratio
PDE1/5
IC50 ratio
PDE6/5
compd
12a
sildenafil 1
PDE1a
PDE2b
PDE3a
PDE4b
PDE5a
PDE6c
>10
>10
>10
>10
>10
0.005
0.006
5.1
0.074
>2000
1000
12
1.1
9.2
7.8
180
a
b
d
Bovine aorta PDE. Human recombinant PDE. c Bovine retina PDE. IC50 values were reproducible to (25%.
tr a n s-3h ). The title compounds were prepared from d-tryp-
tophan methyl ester and 1,3-benzodioxole-5-carboxaldehyde to
afford the cis-isomer (1R,3R) 3h as a white solid (17.4 g, 42%),
mp 154-155 °C (2-propanol), and the trans-isomer (1S,3R) 3h
as a white solid (11.4 g, 27%), mp 188-190 °C (CH2Cl2).
(1R,3S)- a n d (1S,3S)-Meth yl 1-(1,3-Ben zod ioxol-5-yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis- a n d
tr a n s-3i). The title compounds were prepared from l-tryp-
tophan methyl ester and 1,3-benzodioxole-5-carboxaldehyde to
afford the cis-isomer (1S,3S) 3i as a white solid (1.31 g, 36%),
mp 154 °C (Et2O/hexane), and the trans-isomer (1R,3S) 3i as
a white solid (1.29 g, 35%), mp 187-189 °C (MeOH).
(1R,3R)- a n d (1S,3R)-Met h yl 1-(4-Met h oxyp h en yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis- a n d
tr a n s-3j). The title compounds were prepared from d-tryp-
tophan methyl ester and 4-methoxybenzaldehyde to afford the
cis-isomer (1R,3R) 3j as a pale yellow solid (3.2 g, 41%), mp
117-119 °C (petroleum ether), and the trans-isomer (1S,3R)
3j as a white solid (2 g, 25%), mp 219-222 °C (CH2Cl2/AcOEt).
cis-2-Bu tyl-5-(4-m eth oxyp h en yl)-2,3,5,6,11,11a -h exa h y-
d r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole (4). To a well-
stirred dispersion of lithium aluminum hydride (0.75 g, 19.9
mmol) in THF (20 mL) and cooled at 0 °C was added dropwise
a solution of butyl hydantoin 2a (1 g, 2.48 mmol) in THF (20
mL). The resulting mixture was then heated at 40 °C for 2 h,
cooled at 0 °C, and water carefully added. The precipitated
salts were removed by filtration and washed thoroughly with
THF. The filtrate was then evaporated to dryness under
reduced pressure, and the crude product was purified by flash
column chromatography on silica gel eluting with CH2Cl2/
MeOH, 95/5, to give 4 as a white solid (0.35 g, 38%) after
crystallization from 2-propanol/H2O, mp 152-154 °C; 1H NMR
(CDCl3) δ 7.53-7.48 (m, 1H), 7.37-7.27 (m, 3H), 7.20-7.05
(m, 3H), 6.88 (d, 2H, J ) 8.6 Hz), 4.5 (s, 1H), 3.81 (s, 3H), 3.62
(d, 1H, J ) 4.9 Hz), 3.36 (d, 1H, J ) 4.9 Hz), 3.24-3.10 (m,
2H), 3.04-2.94 (m, 1H), 2.91-2.72 (m, 2H), 2.62-2.48 (m, 2H),
1.49-1.23 (m, 4H), 0.88 (t, 3H, J ) 7.2 Hz). GCMS m/z 375;
Anal. (C24H29N3O) C, H, N.
F igu r e 2. Acute effects of 12a on blood pressure in SHR
model. Time course of the changes in mean arterial blood
pressure produced by 12a (b) or its vehicule (O) after oral
administration (5 mg/kg) to spontaneously hypertensive rats
(SHR). Statistical analysis carried out at time 1, 4, and 7 h
revealed a highly significant difference between treated and
control group at 1 and 3 h (p < 0.01) and a significant
difference (p < 0.005) at 7 h on blood pressure.
Ta ble 7. Pharmacokinetic Parameters of 12a in Rats
d
compd
Fa (%)
63
Vdb (l/kg)
5.2
Clc (ml/min/kg)
5.1
t1/2 (h)
12a
2.4
a
Oral bioavailability. b Volume of distribution. c Clearance. d Half-
life.
111.6, 109.5, 109.4, 108.9, 101.9, 59 (C1), 57.5 (C3), 52.9, 26.3;
1H-13C HMQC (CDCl3) δH (δC) 5.16 (59, C1), 3.95 (57.5, C3),
followed by the trans-isomer 3c as white crystals (6.4 g, 30%),
mp 170 °C after recrystallization from toluene; 1H NMR
(CDCl3) δ 7.61 (br s, 1H), 7.54 (d, 1H, J ) 7 Hz), 7.31-7.09
(m, 3H), 6.75 (s, 3H), 5.92 (s, 2H), 5.34 (s, 1H, C1-H), 3.98 (t,
1H, J ) 5.8 Hz, C3-H), 3.71 (s, 3H), 3.26 (dd, 1H, J ) 15.4,
5.4 Hz, C4-H), 3.12 (dd, 1H, J ) 15.6, 6.6 Hz, C4-H), 2.35 (br
s, 1H); 13C NMR (CDCl3) δ 174.8, 148.7, 148.1, 133.8, 136.6,
133.9, 127.6, 122.6, 122.4, 120.1, 118.9, 111.6, 109.3, 108.9,
108.8, 101.8, 55.3 (C1), 53.2 (C3), 52.8, 25.2; 1H-13C HMQC
(CDCl3) δH (δC) 5.34 (55.3, C1), 3.98 (53.2, C3).
cis-1-(1,3-Ben zodioxol-5-yl)-N-m eth yl-2,3,4,9-tetr ah ydr o-
1H-p yr id o[3,4-b]in d ole-3-ca r boxa m id e (5). To a suspension
of cis-3c (2 g, 5.7 mmol) in methanol (100 mL) was added a
33% solution of methylamine in ethanol (2.7 mL, 28.5 mmol),
and the mixture was heated at 50 °C in a sealed pressure flask
for 48 h. The solution was evaporated under reduced pressure,
and the crude product was purified by flash column chroma-
tography on silica gel eluting with CH2Cl2/MeOH, 97/3, to give
5 as a white solid (1.7 g, 86%) after crystallization from AcOEt/
The following compounds were prepared using a similar
procedure with appropriate aldehydes.
1
pentane, mp 216-218 °C; H NMR (CDCl3) δ 7.58-7.48 (m,
Eth yl 1-(4-Meth ylp h en yl)-2,3,4,9-tetr a h yd r o-1H-â-ca r -
bolin e-3-ca r boxyla te (cis- a n d tr a n s-3f). The title com-
pounds were prepared from tryptophan ethyl ester and
4-methylbenzaldehyde. The crude residue was purified by flash
column chromatography eluting with CH2Cl2/MeOH, 95/5, to
give first cis-3f as a white solid (2.45 g, 39.8%), mp 148 °C,
followed by trans-3f as a white solid (1.3 g, 21.1%), mp 180
°C.
Met h yl 1-(3,4-Dim et h oxyp h en yl)-2,3,4,9-t et r a h yd r o-
1H-â-ca r bolin e-3-ca r boxyla te (cis- a n d tr a n s-3g). The title
compounds were prepared from racemic tryptophan methyl
ester and 3,4-dimethoxybenzaldehyde. The crude residue was
purified by flash column chromatography eluting with CH2-
Cl2/MeOH, 99/1, to give first cis-3g as a white solid (5.5 g, 64%),
mp 174-176 °C (MeOH), followed by trans-3g as a white solid
(1.5 g, 18%), mp 194-196 °C (toluene/ hexane).
2H), 7.27-7.07 (m, 3H), 6.99 (br s, 1H), 6.84-6.79 (m, 2H),
6.76 (s, 1H), 5.95 (s, 2H), 5.13 (s, 1H), 3.74 (dd, 1H, J ) 11.1,
4.4 Hz), 3.38 (dd, 1H, J ) 15.6, 4.5 Hz), 2.90-2.77 (m, 4H),
1.72 (br s, 1H).
cis-1-[1-(1,3-Ben zod ioxol-5-yl)-2,3,4,9-t et r a h yd r o-1H -
p yr id o[3,4-b]in d ole-3-yl]-N-m eth ylm eth a n a m in e (6). To
a stirred solution of 1 M lithium aluminum hydride in THF
(24 mL, 24 mmol) was added at room temperature a solution
of 5 in dry THF (40 mL), and the mixture was heated to reflux
for 24 h. After cooling at 0 °C, water was carefully added and
the precipitated salts were removed by filtration and washed
thoroughly with THF. The filtrate was then evaporated to
dryness under reduced pressure, and the crude residue was
purified by flash column chromatography on silica gel eluting
with CH2Cl2/MeOH, 80/20, to give 6 as a colorless oil (0.5 g,
37%); 1H NMR (CDCl3) δ 7.6-7.45 (m, 2H), 7.35-7.10 (m, 3H),
6.95-6.75 (m, 3H), 5.95 (s, 2H), 5.1 (s, 1H), 3.35-3.15 (m, 1H),
2.95-2.55 (m, 5H), 2.5 (s, 3H); GCMS m/z 335.
(1R,3R)- a n d (1S,3R)-Meth yl 1-(1,3-Ben zod ioxol-5-yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis- a n d

Discovery of Tadalafil. Part 2
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4539
cis-5-(1,3-Be n zod ioxol-5-yl)-2-m e t h yl-1,2,5,6,11,11a -
h e xa h yd r o-3H -im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ol-3-
on e (7). To a solution of 6 (0.5 g, 1.49 mmol) in CH2Cl2 (20
mL) was added dropwise triphosgene (0.15 g, 0.49 mmol) at 0
°C. The mixture was stirred at the same temperature for 0.5
h and was then poured into ice water. The aqueous layer was
extracted with CH2Cl2 (2 × 25 mL), and the resulting organic
layer was washed with brine, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was purified
by flash column chromatography on silica gel eluting with CH2-
Cl2/MeOH, 99/1, to give 7 as a white solid (0.22 g, 42%) after
crystallization from CH2Cl2/Et2O, mp 309-312 °C; 1H NMR
(CDCl3) δ 7.55-7.45 (m, 2H), 7.22-7.05 (m, 3H), 6.92 (d, 1H,
J ) 7.9 Hz), 6.78-6.73 (m, 2H), 5.89 (s, 2H), 5.46 (s, 1H), 3.97-
3.82 (m, 1H), 3.57 (t, 1H, J ) 7.4 Hz), 3.26-3.12 (m, 2H), 2.87
(dd, 1H, J ) 14.7, 10.7 Hz), 2.79 (s, 3H); GCMS m/z 361; Anal.
(C21H19N3O3) C, H, N.
tr a n s-1-(4-Met h oxyp h en yl)-N-(p h en ylm et h yl)-2,3,4,9-
tetr a h yd r o-1H-p yr id o[3,4-b]in d ole-3-ca r boxa m id e (8). A
solution of trans-3a (4 g, 11.90 mmol) in (phenylmethyl)amine
(5 mL) was heated at 80 °C for 24 h. After cooling at room
temperature, diethyl ether was added and the resulting
precipitate was filtered and recrystallized from ethanol to give
the benzylamide 8 as a white solid (3.8 g, 78%), mp 199 °C;
1H NMR (CDCl3) δ 7.74 (s, 1H), 7.58 (d, 1H, J ) 7 Hz), 7.37-
7.06 (m, 11H), 6.81 (d, 2H, J ) 8.7 Hz), 5.20 (s, 1H), 4.50 (dd,
1H, J ) 14.9, 6.4 Hz), 4.34 (dd, 1H, J ) 14.9, 5.4 Hz), 3.76 (s,
3H), 3.70 (dd, 1H, J ) 9.4, 4.9 Hz), 3.33 (dd, 1H, J ) 16, 5.1
Hz), 2.96 (dd, 1H, J ) 16, 9.4 Hz), 2.17 (br s, 1H).
tr a n s-5-(4-Met h oxyp h en yl)-2-(p h en ylm et h yl)-2,3,5,6,-
11,11a-h exah ydr o-1H-im idazo[1′,5′:1,6]pyr ido[3,4-b]in dol-
1-on e (9). A mixture of 8 (0.41 g, 1 mmol) and paraformal-
dehyde (40 mg) in anhydrous toluene (25 mL) was heated to
reflux with a Dean-Stark apparatus for 48 h. The solvent was
then evaporated under reduced pressure, and the residue was
purified by flash column chromatography on silica gel eluting
with toluene/AcOEt, 80/20, to give 9 as a pale yellow solid (100
mg, 23%) after crystallization from MeOH/H2O, mp 116-120
°C; 1H NMR (CDCl3) δ 7.6-7.5 (m, 2H), 7.4-7 (m, 8H), 6.9 (d,
2H, J ) 8.7 Hz), 6.8 (d, 2H, J ) 8.7 Hz), 5.05 (s, 1H), 4.75 (d,
1H, J ) 14.3 Hz), 4.1-3.95 (m, 2H), 3.85-3.75 (m, 2H), 3.7 (s,
3H), 3.25 (dd, 1H, J ) 16, 5 Hz), 2.95 (dd, J ) 16, 9.5 Hz);
Anal. (C27H25N3O2) C, H, N.
cis-Met h yl 1-(1,3-Ben zod ioxol-5-yl)-2-(ch lor oa cet yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis-10c).
Rep r esen ta tive exa m p le: To a stirred solution of cis-3c (2
g, 5.7 mmol, 1 equiv) and NaHCO3 (0.575 g, 6.89 mmol, 1.2
equiv) in CHCl3 (40 mL) was added dropwise chloroacetyl
chloride (1.09 mL, 13.69 mmol, 2.4 equiv) under ice cooling.
The mixture was then stirred at room temperature under a
nitrogen atmosphere for 1 h. The mixture was diluted with
CH2Cl2, washed with a solution of NaHCO3, brine, dried over
Na2SO4, and evaporated under reduced pressure. The oily
residue was then crystallized from diethyl ether to give cis-
10c as a pale yellow solid (2 g, 82%), mp 215-218 °C; 1H NMR
(CDCl3) δ 7.74 (br s, 1H), 7.46 (d, 1H, J ) 7.1 Hz), 7.34-6.97
(m, 3H), 6.93-6.38 (m, 4H), 5.76 (s, 2H), 4.80 (br s, 1H), 4.36-
3.99 (m, 2H), 3.55 (d, 1H, J ) 16 Hz), 3.06 (m, 4H).
10c). The title compound was obtained from trans-3c as a pale
yellow solid in 76% yield, mp 118 °C (Et2O/hexane).
cis-Meth yl 2-(Ch lor oa cetyl)-1-(3,4-d im eth oxyp h en yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis-10g).
The title compound was obtained from cis-3g as a pale yellow
solid in 87% yield, mp 228 °C (hexane).
(1R,3R)-Meth yl 1-(1,3-Ben zodioxol-5-yl)-2-(ch lor oacetyl)-
2,3,4,9-tetr ah ydr o-1H-â-car bolin e-3-car boxylate (cis-10h ).
The title compound was obtained from cis-3h as a pale yellow
solid in 93% yield, mp 232-234 °C (Et2O).
(1S,3R)-Meth yl 1-(1,3-Ben zodioxol-5-yl)-2-(ch lor oacetyl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (tr a n s-
10h ). The title compound was obtained from trans-3h as a
white solid in 72% yield, mp 209 °C (Et2O).
(1S,3S)-Meth yl 1-(1,3-Ben zodioxol-5-yl)-2-(ch lor oacetyl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis-10i).
The title compound was obtained from cis-3i as a beige solid
in 78% yield, mp 233 °C (Et2O/hexane).
(1R,3S)-Meth yl 1-(1,3-Ben zodioxol-5-yl)-2-(ch lor oacetyl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (tr a n s-
10i). The title compound was obtained from trans-3i as a pale
yellow solid in 72% yield, mp 208 °C (Et2O).
(1R,3R)-Meth yl 2-(Ch lor oa cetyl)-1-(4-m eth oxyp h en yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (cis-10j).
The title compound was obtained from cis-3j as a pale yellow
solid in 78% yield, mp 223-225 °C (Et2O).
6-(1,3-Ben zod ioxol-5-yl)-2-bu tyl-2,3,6,7,12,12a -h exa h y-
d r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-
11c). Rep r esen ta tive exa m p le: A solution of cis-10c (0.6 g,
1.4 mmol, 1 equiv) and butylamine (0.28 mL, 2.8 mmol, 2
equiv) in methanol (25 mL) was heated to reflux under a
nitrogen atmosphere for 16 h. The reaction mixture was cooled
at room temperature and evaporated to dryness under reduced
pressure. The residue was dissolved in CH2Cl2, and the organic
layer was washed with water, dried over Na2SO4, filtered, and
concentrated to dryness. The crude product was then purified
by flash column chromatography eluting with toluene/AcOEt,
80/20, to give cis-11c as a white solid (370 mg, 61%) after
1
recrystallization from MeOH/H2O, mp 241-243 °C; H NMR
(CDCl3) δ 8.02 (s, 1H), 7.58 (m, 1H), 7.29-7.10 (m, 3H), 6.82
(d, 1H, J ) 8.1 Hz), 6.71 (s, 1H), 6.66 (d, 1H, J ) 8.1 Hz), 6.17
(s, 1H), 5.84 (d, 2H, J ) 6.4 Hz), 4.27 (dd, 1H, J ) 11.2, 4.3
Hz), 4.06 (d, 1H, J ) 17.5 Hz), 3.90 (d, 1H, J ) 17.5 Hz), 3.73
(dd, 1H, J ) 16.1, 4.6 Hz), 3.65-3.53 (m, 1H), 3.37-3.28 (m,
1H), 3.20 (dd, 1H, J ) 16.1, 11.6 Hz), 1.61-1.48 (m, 2H), 1.40-
1.26 (m, 2H), 0.94 (t, 3H, J ) 7.2 Hz); Anal. (C25H25N3O4) C,
H, N.
The following compounds were prepared using a similar
procedure with appropriate chloroacetyl derivatives and pri-
mary amines.
6-(1,3-Ben zod ioxol-5-yl)-2-bu tyl-2,3,6,7,12,12a -h exa h y-
dr opyr azin o[1′,2′:1,6]pyr ido[3,4-b]in dole-1,4-dion e (tr a n s-
11c). The title compound was obtained from trans-10c and
butylamine as a white solid in 55% yield, mp 243 °C (toluene);1H
NMR (CDCl3) δ 7.97 (s, 1H), 7.52 (d, 1H, J ) 7.5 Hz), 7.36-
7.09 (m, 3H), 6.95 (s, 1H), 6.79 (s, 1H), 6.70 (s, 2H), 5.92 (s,
2H), 4.32 (dd, 1H, J ) 11.8, 4.2 Hz), 4.12 (d, 1H, J ) 17.7 Hz),
3.96 (d, 1H, J ) 17.7 Hz), 3.61-3.48 (m, 2H), 3.23 (m, 1H),
2.92 (dd, 1H, J ) 15.5, 11.9 Hz), 1.61-1.48 (m, 2H), 1.41-
1.26 (m, 2H), 0.94 (t, 3H, J ) 7.2 Hz); Anal. (C25H25N3O4) C,
H, N.
2-Bu tyl-6-(4-m eth oxyp h en yl)-2,3,6,7,12,12a -h exa h yd r o-
p yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-11a ).
The title compound was obtained from cis-10a and butylamine
as a white solid in 35% yield, mp 157 °C (MeOH); 1H NMR
(CDCl3) δ 7.93 (s, 1H), 7.59 (m, 1H), 7.28-7.12 (m, 5H), 6.75
(d, 2H, J ) 8.7 Hz), 6.21 (s, 1H), 4.29 (dd, 1H, J ) 11.7, 4.3
Hz), 4.07 (d, 1H, J ) 17.3 Hz), 3.89 (d, 1H, J ) 17.3 Hz), 3.80-
3.52 (m, 2H), 3.70 (s, 3H), 3.35-3.15 (m, 2H), 1.66-1.47 (m,
2H), 1.39-1.26 (m, 2H), 0.93 (t, 3H, J ) 7.4 Hz); Anal.
(C25H27N3O3‚0.5H2O) C, H, N.
The following compounds were prepared using a similar
procedure with appropriate tetrahydro-â-carbolines.
cis-Meth yl 2-(Ch lor oacetyl)-1-(4-m eth oxyph en yl)-2,3,4,9-
t et r a h yd r o-1H -â-ca r b olin e-3-ca r b oxyla t e (cis-10a ). The
title compound was obtained from cis-3a as a pale yellow solid
in 75% yield, mp 188 °C (Et2O/hexane).
tr a n s-Meth yl 2-(Ch lor oa cetyl)-1-(4-m eth oxyp h en yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (tr a n s-
10a ). The title compound was obtained from trans-3a as a
beige solid in 75% yield, mp 138-142 °C (Et2O/hexane).
cis-Meth yl 2-(Ch lor oa cetyl)-1-p h en yl-2,3,4,9-tetr a h y-
d r o-1H-â-ca r bolin e-3-ca r boxyla te (cis-10b). The title com-
pound was obtained from cis-3b as a pale yellow solid in 90%
yield, mp 204 °C (hexane).
2-Bu tyl-6-(4-m eth oxyp h en yl)-2,3,6,7,12,12a -h exa h yd r o-
p yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (tr a n s-
11a ). The title compound was obtained from trans-10a and
tr a n s-Met h yl 1-(Ben zod ioxol-5-yl)-2-(ch lor oa cet yl)-
2,3,4,9-tetr a h yd r o-1H-â-ca r bolin e-3-ca r boxyla te (tr a n s-

4540 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
butylamine as a white solid in 75% yield, mp 212-214 °C
(MeOH); 1H NMR (CDCl3) δ 8.28 (s, 1H), 7.64 (m, 1H), 7.41-
7.22 (m, 5H), 7.07 (s, 1H), 6.88 (d, 2H, J ) 8.7 Hz), 4.40 (dd,
1H, J ) 11.9, 4.2 Hz), 4.18 (d, 1H, J ) 17.5 Hz), 4.03 (d, 1H,
J ) 17.5 Hz), 3.85 (s, 3H), 3.67-3.60 (m, 2H), 3.37-3.23 (m,
1H), 3.02 (dd, 1H, J ) 15.2, 11.8 Hz), 1.70-1.56 (m, 2H), 1.50-
1.38 (m, 2H), 1.05 (t, 3H, J ) 7.2 Hz); Anal. (C25H27N3O3) C,
H, N.
2-Bu t yl-6-p h en yl-2,3,6,7,12,12a -h exa h yd r op yr a zin o-
[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-11b). The title
compound was obtained from cis-10b and butylamine as a
white solid in 89% yield, mp 243-245 °C (MeOH/H2O); 1H
NMR (CDCl3) δ 7.94 (s, 1H), 7.58 (m, 1H), 7.34-7.11 (m, 8H),
6.26 (s, 1H), 4.30 (dd, 1H, J ) 11.5, 4.5 Hz), 4.06 (d, 1H, J )
17.4 Hz), 3.90 (d, 1H, J ) 17.4 Hz), 3.75 (dd, 1H, J ) 16, 4.5
Hz), 3.65-3.51 (m, 1H), 3.37-3.19 (m, 2H), 1.62-1.50 (m, 2H),
1.39-1.25 (m, 2H), 0.93 (t, 3H, J ) 7.2 Hz); Anal. (C24H25N3O2)
C, H, N.
11i). The title compound was obtained from cis-10c and a 33%
solution of methylamine in ethanol as a white solid in 94%
yield, mp 253-255 °C (MeOH); 1H NMR (CDCl3) δ 8.11 (s, 1H),
7.60 (m, 1H), 7.28-7.12 (m, 3H), 6.81 (d, 1H, J ) 7.9 Hz), 6.71
(s, 1H), 6.66 (d, 1H, J ) 8.1 Hz), 6.12 (s, 1H), 5.83 (d, 2H, J )
6.6 Hz), 4.28 (dd, 1H, J ) 11.5, 4.3 Hz), 4.08 (d, 1H, J ) 17.5
Hz), 3.90 (d, 1H, J ) 17.5 Hz), 3.77 (dd, 1H, J ) 16.1, 4.3 Hz),
3.20 (dd, 1H, J ) 16.1, 11.5 Hz), 3.02 (s, 3H); Anal. (C22H19N3O4)
C, H, N.
6-(4-Met h oxyp h en yl)-2-m et h yl-2,3,6,7,12,12a -h exa h y-
d r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-
11j). The title compound was obtained from cis-10a and a 33%
solution of methylamine in ethanol, as a white solid in 22%
1
yield, mp 257-263 °C (2-propanol); H NMR (CDCl3) δ 7.98
(s, 1H), 7.64 (m, 1H), 7.33-7.17 (m, 5H), 6.79 (d, 2H, J ) 8.5
Hz), 6.23 (s, 1H), 4.34 (dd, 1H, J ) 11.5, 4.3 Hz), 4.13 (d, 1H,
J ) 17.5 Hz), 3.94 (d, 1H, J ) 17.5 Hz), 3.83 (dd, 1H, J ) 16,
4.5 Hz), 3.75 (s, 3H), 3.27 (dd, 1H, J ) 16, 11.5), 3.06 (s, 3H);
Anal. (C22H21N3O3) C, H, N.
The following piperazinediones cis-11d , -11e, -11f were
obtained by a two-step procedure starting from tetrahydro-â-
carboline: cis-3d , -3e, and -3f, respectively without isolation
of their chloroacetyl derivative.
2-Eth yl-6-(4-m eth oxyp h en yl)-2,3,6,7,12,12a -h exa h yd r o-
p yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-11k ).
The title compound was obtained from cis-10a and a 2 M
solution of ethylamine in methanol as a white solid in 67%
yield, mp 245-255 °C (MeOH);1H NMR (CDCl3) δ 7.95 (s, 1H),
7.64 (m, 1H), 7.32-7.15 (m, 5H), 6.80 (d, 2H, J ) 8.7 Hz), 6.26
(s, 1H), 4.31 (dd, 1H, J ) 11.5, 4.6 Hz), 4.10 (d, 1H, J ) 17.3
Hz), 3.92 (d, 1H, J ) 17.3 Hz), 3.83-3.65 (m, 2H), 3.75 (s, 3H),
3.43-3.19 (m, 2H), 1.21 (t, 3H, J ) 7.3 Hz); Anal. (C23H23N3O3)
C, H, N.
6-(1,3-Be n zod ioxol-5-yl)-2-cycloh e xyl-2,3,6,7,12,12a -
h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d i-
on e (cis-11l). The title compound was obtained from cis-10c
and cyclohexylamine as a white solid in 71% yield, mp 268-
269 °C (MeOH/H2O); 1H NMR (CDCl3) δ 7.93 (s, 1H), 7.56 (m,
1H), 7.28-7.06 (m, 3H), 6.80-6.60 (m, 3H), 6.15 (s, 1H), 5.82
(d, 2H, J ) 6.6 Hz), 4.49-4.31 (m, 1H), 4.24 (dd, 1H, J ) 11.5,
4.5 Hz), 3.95 (d, 1H, J ) 17.3 Hz), 3.82 (d, 1H, J ) 17.3 Hz),
3.69 (dd, 1H, J ) 16, 4.7 Hz), 3.17 (dd, 1H, J ) 16, 11.5 Hz),
1.87-0.95 (m, 11H); Anal. (C27H27N3O4) C, H, N.
2-Bu tyl-6-(4-cya n op h en yl)-2,3,6,7,12,12a -h exa h yd r op y-
r azin o[1′,2′:1,6]pyr ido[3,4-b]in dole-1,4-dion e (cis-11d). The
title compound was obtained from cis-3d with an overall yield
1
of 24.7% as a white solid, mp 246 °C (MeOH/H2O); H NMR
(CDCl3) δ 7.86 (s, 1H), 7.68-7.05 (m, 8H), 6.20 (s, 1H), 4.28
(dd, 1H, J ) 11.3, 4.5 Hz), 4.06 (d, 1H, J ) 17.5 Hz), 3.88 (d,
1H, J ) 17.5 Hz), 3.76 (dd, 1H, J ) 16.2, 4.5 Hz), 3.64-3.47
(m, 1H), 3.36-3.12 (m, 2H), 1.62-1.45 (m, 2H), 1.38-1.22 (m,
2H), 0.91 (t, 3H, J ) 7.2 Hz); Anal. (C25H24N4O2‚1H2O) C, H,
N.
2-Bu t yl-6-(4-ch lor op h en yl)-2,3,6,7,12,12a -h exa h yd r o-
p yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-11e).
The title compound was obtained from cis-3e with an overall
yield of 35% as a white solid, mp 164-166 °C (MeOH/H2O);
1H NMR (CDCl3) δ 7.92 (s, 1H), 7.59 (m, 1H), 7.31-7.13 (m,
7H), 6.21 (s, 1H), 4.29 (dd, 1H, J ) 11.5, 4.6 Hz), 4.07 (d, 1H,
J ) 17.5 Hz), 3.89 (d, 1H, J ) 17.5 Hz), 3.76 (dd, 1H, J ) 16,
4.5 Hz), 3.64-3.52 (m, 1H), 3.39-3.06 (m, 2H), 1.66-1.48 (m,
6-(1,3-Be n zod ioxol-5-yl)-2-isop r op yl-2,3,6,7,12,12a -
h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d i-
on e (cis-11m ). The title compound was obtained from cis-10c
and isopropylamine as a white solid in 65% yield, mp 248-
250 °C (MeOH); 1H NMR (CDCl3) δ 7.93 (s, 1H), 7.59 (m, 1H),
7.35-7.07 (m, 3H), 6.90-6.65 (m, 3H), 6.18 (s, 1H), 5.85 (d,
2H, J ) 5.8 Hz), 4.90-4.79 (m, 1H), 4.25 (dd, 1H, J ) 11.3,
4.5 Hz), 3.95 (d, 1H, J ) 17.3 Hz), 3.85 (d, 1H, J ) 17.3 Hz),
3.73 (dd, 1H, J ) 16.2, 4.7 Hz), 3.21 (dd, 1H, J ) 16, 11.5 Hz),
1.17 (d, 6H, J ) 6.1 Hz); Anal. (C24H23N3O4) C, H, N.
2-Cyclop r op ylm eth yl-6-(4-m eth oxyp h en yl)-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (cis-11n ). The title compound was obtained from cis-
10a and (cyclopropylmethyl)amine as a white solid in 69%
yield, mp 180-185 °C (MeOH); 1H NMR (CDCl3) δ 7.93 (s, 1H),
7.59 (m, 1H), 7.28-7.12 (m, 5H), 6.75 (d, 2H, J ) 8.8 Hz), 6.23
(s, 1H), 4.30 (dd, 1H, J ) 11.5, 4.6 Hz), 4.16 (d, 1H, J ) 17.4
Hz), 4.02 (d, 1H, J ) 17.4 Hz), 3.82-3.56 (m, 2H), 3.71 (s, 3H),
3.29-3.01 (m, 2H), 1.02-0.88 (m, 1H), 0.61-0.51 (m, 2H),
0.31-0.22 (m, 2H); Anal. (C25H25N3O3‚0.5H2O) C, H, N.
2-Cyclop r op ylm et h yl-6-(4-m et h ylp h en yl)-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (cis-11o). The title compound was obtained from cis-
3f with an overall yield of 30% as a white solid, mp 194 °C
2H), 1.41-1.25 (m, 2H), 0.94 (t, 3H, J ) 7.3 Hz); Anal. (C24H24
ClN3O2) C, H, N.
-
2-Bu t yl-6-(4-m et h ylp h en yl)-2,3,6,7,12,12a -h exa h yd r o-
p yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-11f).
The title compound was obtained from cis-3f with an overall
1
yield of 57.7% as a white solid, mp 194 °C (MeOH); H NMR
(CDCl3) δ 7.90 (s, 1H), 7.59 (m, 1H), 7.30-6.98 (m, 7H), 6.22
(s, 1H), 4.29 (dd, 1H, J ) 11.5, 4.5 Hz), 4.06 (d, 1H, J ) 17.3
Hz), 3.88 (d, 1H, J ) 17.3 Hz), 3.74 (dd, 1H, J ) 16, 4.7 Hz),
3.64-3.50 (m, 1H), 3.38-3.17 (m, 2H), 2.25 (s, 3H), 1.66-1.48
(m, 2H), 1.39-1.25 (m, 2H), 0.93 (t, 3H, J ) 7.2 Hz); Anal.
(C25H27N3O2‚0.5H2O) C, H, N.
2-Bu tyl-6-(3,4-d im eth oxyp h en yl)-2,3,6,7,12,12a -h exa h y-
d r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-
11g). The title compound was obtained from cis-10g and
butylamine as a white solid in 75% yield, mp 154-156 °C
(MeOH); 1H NMR (CDCl3) δ 7.96 (s, 1H), 7.58 (m, 1H), 7.29-
7.06 (m, 3H), 6.84 (s, 1H), 6.80 (d, 1H, J ) 8.3 Hz), 6.68 (d,
1H, J ) 8.3 Hz), 6.21 (s, 1H), 4.26 (dd, 1H, J ) 11.5, 4.7 Hz),
4.05 (d, 1H, J ) 17.3 Hz), 3.88 (d, 1H, J ) 17.3 Hz), 3.75 (s,
3H), 3.73 (s, 3H), 3.73-3.66 (m, 1H), 3.60-3.49 (m, 1H), 3.38-
3.13 (m, 2H), 1.57-1.45 (m, 2H), 1.39-1.21 (m, 2H), 0.91 (t,
3H, J ) 7.4 Hz); Anal. (C26H29N3O4) C, H, N.
6-(1,3-Ben zod ioxol-5-yl)-2,3,6,7,12,12a -h exa h yd r op y-
r azin o[1′,2′:1,6]pyr ido[3,4-b]in dole-1,4-dion e (cis-11h ). The
title compound was obtained from cis-10c and a 2 M solution
of ammonia in methanol as a white solid in 50% yield, mp
283-285 °C (MeOH); 1H NMR (CDCl3) δ 7.78 (s, 1H), 7.59 (m,
1H), 7.31-7.12 (m, 3H), 6.89-6.83 (m, 3H), 6.19 (s, 1H), 6.02
(s, 1H), 5.87 (d, 2H, J ) 6.9 Hz), 4.35 (dd, 1H, J ) 11.5, 4.5
Hz), 4.14-3.99 (m, 2H), 3.72 (dd, 1H, J ) 16.2, 4.7 Hz), 3.21
(dd, 1H, J ) 16.2, 11.9 Hz); Anal. (C21H17N3O4) C, H, N.
6-(1,3-Ben zodioxol-5-yl)-2-m eth yl-2,3,6,7,12,12a-h exah y-
d r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (cis-
1
(MeOH/H2O); H NMR (CDCl3) δ 7.93 (s, 1H), 7.59 (m, 1H),
7.32-6.96 (m, 7H), 6.23 (s, 1H), 4.30 (dd, 1H, J ) 11.3, 4.1
Hz), 4.15 (d, 1H, J ) 17.4 Hz), 4.02 (d, 1H, J ) 17.4 Hz), 3.83-
3.54 (m, 2H), 3.29-3.02 (m, 2H), 2.24 (s, 3H), 1.02-0.88 (m,
1H), 0.61-0.51 (m, 2H), 0.31-0.22 (m, 2H); Anal. (C25H25N3O2‚
1.1H2O) C, H, N.
6-(1,3-Ben zod ioxol-5-yl)-2-(p h en ylm et h yl)-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (cis-11p ). The title compound was obtained from cis-
10c and (phenylmethyl)amine as a white solid in 80% yield,
1
mp 285-287 °C (CH2Cl2/hexane); H NMR (CDCl3) δ 7.80 (s,

Discovery of Tadalafil. Part 2
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4541
1H), 7.60 (m, 1H), 7.39-7.08 (m, 8H), 6.86-6.65 (m, 3H), 6.15
(s, 1H), 5.86 (d, 2H, J ) 5.2 Hz), 4.90 (d, 1H, J ) 14.5 Hz),
4.41 (d, 1H, J ) 14.5 Hz), 4.35 (dd, 1H, J ) 11.5, 4.8 Hz), 3.97-
3.74 (m, 3H), 3.26 (dd, 1H, J ) 16, 11.5 Hz); Anal. (C28H23N3O4‚
1H2O) C, H, N.
(d, 1H, J ) 17.3 Hz), 3.72 (dd, 1H, J ) 16, 4.7 Hz), 3.21 (dd,
1H, J ) 16, 11.5 Hz), 1.17 (d, 6H, J ) 6.8 Hz); Anal.
(C24H23N3O4) C, H, N.
(6R,12aR)-6-(1,3-Ben zodioxol-5-yl)-2-cyclopen tyl-2,3,6,7,-
12,12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-
1,4-d ion e (15). The title compound was obtained from cis-
10j and cyclopentylamine as a white solid in 77% yield, mp
210-211 °C (Et2O); [R]D +29° (c ) 1.07; CHCl3); 1H NMR
(CDCl3) δ 8.01 (s, 1H), 7.60 (m, 1H), 7.27-7.12 (m, 5H), 6.74
(d, 2H, J ) 8.9 Hz), 6.22 (s, 1H), 4.98-4.86 (m, 1H), 4.26 (dd,
1H, J ) 11.5, 4.8 Hz), 3.90 (s, 2H), 3.76-3.64 (m, 1H), 3.70 (s,
3H), 3.23 (dd, 1H, J ) 16, 11.5), 1.94-1.42 (m, 8H); Anal.
(C26H27N3O3) C, H, N.
(6R,12aR)-6-(1,3-Ben zodioxol-5-yl)-2,3,6,7,12,12a-h exah y-
d r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-d ion e (16).
The title compound was obtained from cis-10h and a 2 M
solution of ammonia in methanol as a white solid in 31% yield,
mp 285-290 °C (MeOH); [R]D +88° (c ) 0.48; pyridine); 1H
NMR (CDCl3) δ 7.78 (s, 1H), 7.59 (m, 1H), 7.31-7.12 (m, 3H),
6.89-6.83 (m, 3H), 6.19 (s, 1H), 6.02 (s, 1H), 5.87 (d, 2H, J )
6.9 Hz), 4.35 (dd, 1H, J ) 11.5, 4.5 Hz), 4.14-3.99 (m, 2H),
3.72 (dd, 1H, J ) 16.2, 4.7 Hz), 3.21 (dd, 1H, J ) 16.2, 11.9
Hz); Anal. (C21H17N3O4) C, H, N.
(6R,12a R)-6-(1,3-Ben zod ioxol-5-yl)-2-m eth yl-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (12a ). The title compound was obtained from cis-10h
and a 33% solution of methylamine in ethanol as a white solid
in 77% yield, mp 301-303 °C (CH2Cl2/hexane); [R]D +71° (c )
1.13; CHCl3); 1H NMR (CDCl3) δ 8.11 (s, 1H), 7.60 (d, 1H, J )
7.6 Hz), 7.28-7.12 (m, 3H), 6.81 (d, 1H, J ) 7.9 Hz), 6.71 (s,
1H), 6.66 (d, 1H, J ) 8.1 Hz), 6.12 (s, 1H, C6-H), 5.83 (d, 2H,
J ) 6.6 Hz), 4.28 (dd, 1H, J ) 11.5, 4.3 Hz, C12a-H), 4.08 (d,
1H, J ) 17.5 Hz, C3-H), 3.90 (d, 1H, J ) 17.5 Hz, C3-H),
3.77 (dd, 1H, J ) 16.1, 4.3 Hz, C12-H), 3.20 (dd, 1H, J ) 16.1,
11.5 Hz, C12-H), 3.02 (s, 3H); 13C NMR (CDCl3) δ 167.5, 167.1,
148.5, 147.8, 137.2, 136, 133.5, 126.8, 123.2, 121.4, 120.8,
119.3, 111.9, 108.9, 108.1, 107.2, 101.8, 57.4 (C6), 56.9 (C12a),
52.8, 34.3, 24.5; 1H-13C HMQC (CDCl3) δH (δC) 6.12 (57.4, C6),
4.28 (56.9, C12a); Anal. (C22H19N3O4) C, H, N.
(6R,12a S)-6-(1,3-Ben zod ioxol-5-yl)-2-m eth yl-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (12b). The title compound was obtained from trans-
10i and a 33% solution of methylamine in ethanol as a white
solid in 81% yield, mp 287-289 °C (toluene); [R]D -293° (c )
P h osp h od iester a se P r ep a r a tion s. PDE1, -3, and -5 were
purified from bovine aorta as previously described in the
literature.14 Bovine PDE6 (ROS-PDE) was supplied by Dr.
N. Virmaux (Inserm U338, Strasbourg, France). Human
recombinant PDE2 and 4 were provided from Drs. Vince Florio
and Tim Martins (ICOS Corporation, Bothell, WA).
1
1.28; CHCl3); H NMR (CDCl3) δ 8.06 (s, 1H), 7.62 (m, 1H),
7.45-7.23 (m, 3H), 7.07 (s, 1H), 6.90 (s, 1H), 6.80 (s, 2H), 6.02
(s, 2H), 4.44 (dd, 1H, J ) 11.8, 4.1 Hz), 4.23 (d, 1H, J ) 17.7
Hz), 4.08 (d, 1H, J ) 17.7 Hz), 3.64 (dd, 1H, J ) 15.5, 4.2 Hz),
3.13-2.99 (m, 1H), 3.09 (s, 3H); Anal. (C22H19N3O4‚0.25
toluene) C, H, N.
P h osp h od iester a se Assa ys. The PDE assay14,15 was based
on the use of Multiscreen plates (Millipore) and a vacuum
manifold (Millipore). In such plates, both the reaction and the
subsequent separation between substrates and products can
be achieved. The assay (100 µL) contained 50 mM Tris pH 7.5,
5 mM Mg acetate, 1 mM EGTA, and 250 µg/mL snake venom
nucleotidase. Fifty nanomolar [8-3H]-cGMP (15 Ci/mmol; Am-
ersham) or [8-3H]-cGMP (25 Ci/mmol; Amersham) was added.
Reactions were started by the addition of 25 µL of the diluted
enzyme preparation. The assays were incubated for 30 min
at 30 °C. Microcolumns were prepared by aliquoting 300 µL
per well of QAE Sephadex previously swollen for 2 h in water
(12 mL/g). At the end of the incubation, the total volume of
the assays was loaded on microcolumn plate by filtration. The
elution of free radioactivity was obtained by 200 µL of water
from which 50 µL aliquots were analyzed by scintillation
counting.
In this PDE assay, the substrate concentration never
exceeded 30% of the Km of the enzyme tested. Under such
conditions, the IC50 obtained for any given compound closely
corresponded to the Ki for such compound. In addition, all
enzymes studies were performed under conditions of initial
velocity (maximal substrate hydrolysis of 10-15%). Stock
solutions of PDE inhibitors were prepared in dimethyl sulfox-
ide, and the final solvent concentration in each assay was 2%
(v/ v).
Deter m in a tion of cGMP Accu m u la tion in RSMC. Rat
aortic smooth muscle cells (RSMC) were prepared according
to Chamley.16 Cells were cultured in Dubelcco’s modified Eagle
medium (GIBCO) containing 10% fetal calf serum, 1%
glutamine, and 1% penicillin-streptomycin at 37 °C in a 95%
air-5% CO2 humidified atmosphere.
Cells were seeded in 24-well culture dishes at a density of
(2-5) × 104 cells/well. Experiments were performed after 3-5
days in culture when cells reached confluence. Media were
aspired and replaced with 0.5 mL of PBS containing the PDE
inhibitor. After 30 min at 37 °C, soluble or particular guanylate
cyclase was stimulated by addition of atrial natriuretic factor
(0.1 µM) for 10 min at 37 °C. At the end of the incubation, the
medium was removed and stored at -20 °C for extracellular
cyclic nucleotides determinations. Intracellular cyclic nucle-
otides were extracted by two ethanolic (65%) washes at 4 °C
for 5 min. The ethanolic extracts were pooled, evaporated to
dryness using a speed-Vac system, and stored at -20 °C. cGMP
was measured by scintillation proximity immunoassay (Am-
(6S,12a S)-6-(1,3-Ben zod ioxol-5-yl)-2-m eth yl-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (12c). The title compound was obtained from cis-10i
and a 33% solution of methylamine in ethanol as a white solid
in 71% yield, mp 283-287 °C (2-propanol); [R]D -71° (c ) 1.02;
1
CHCl3); H NMR (CDCl3) δ 8.11 (s, 1H), 7.60 (m, 1H), 7.28-
7.12 (m, 3H), 6.81 (d, 1H, J ) 7.9 Hz), 6.71 (s, 1H), 6.66 (d,
1H, J ) 8.1 Hz), 6.12 (s, 1H), 5.83 (d, 2H), 4.28 (dd, 1H, J )
11.5, 4.3 Hz), 4.08 (d, 1H, J ) 17.5 Hz), 3.90 (d, 1H, J ) 17.5
Hz), 3.77 (dd, 1H, J ) 16.1, 4.3 Hz), 3.20 (dd, 1H, J ) 16.1,
11.5 Hz), 3.02 (s, 3H); Anal. (C22H19N3O4) C, H, N.
(6S,12a R)-6-(1,3-Ben zod ioxol-5-yl)-2-m eth yl-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (12d ). The title compound was obtained from trans-
10h and a 33% solution of methylamine in ethanol as a white
solid in 70% yield, mp 285-287 °C (toluene); [R]D +297° (c )
1
1.21; CHCl3); H NMR (CDCl3) δ 8.06 (s, 1H), 7.62 (m, 1H),
7.45-7.23 (m, 3H), 7.07 (s, 1H), 6.90 (s, 1H), 6.80 (s, 2H), 6.02
(s, 2H), 4.44 (dd, 1H, J ) 11.8, 4.1 Hz), 4.23 (d, 1H, J ) 17.7
Hz), 4.08 (d, 1H, J ) 17.7 Hz), 3.64 (dd, 1H, J ) 15.5, 4.2 Hz),
3.13-2.99 (m, 1H), 3.09 (s, 3H); Anal. (C22H19N3O4‚0.3 toluene)
C, H, N.
(6R,12a R)-6-(1,3-Ben zod ioxol-5-yl)-2-b u t yl-2,3,6,7,12,-
12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-1,4-
d ion e (13). The title compound was obtained from cis-10h and
butylamine as a white solid in 57% yield, mp 209-210 °C
(toluene/hexane); [R]D +50° (c ) 0.53; CHCl3); 1H NMR (CDCl3)
δ 8.02 (s, 1H), 7.58 (m, 1H), 7.29-7.10 (m, 3H), 6.82 (d, 1H, J
) 8.1 Hz), 6.71 (s, 1H), 6.66 (d, 1H, J ) 8.1 Hz), 6.17 (s, 1H),
5.84 (d, 2H, J ) 6.4 Hz), 4.27 (dd, 1H, J ) 11.2, 4.3 Hz), 4.06
(d, 1H, J ) 17.5 Hz), 3.90 (d, 1H, J ) 17.5 Hz), 3.73 (dd, 1H,
J ) 16.1, 4.6 Hz), 3.65-3.53 (m, 1H), 3.37-3.28 (m, 1H), 3.20
(dd, 1H, J ) 16.1, 11.6 Hz), 1.61-1.48 (m, 2H), 1.40-1.26 (m,
2H), 0.94 (t, 3H, J ) 7.2 Hz); Anal. (C25H25N3O4) C, H, N.
(6R,12a R)-6-(1,3-Ben zod ioxol-5-yl)-2-isop r op yl-2,3,6,7,-
12,12a -h exa h yd r op yr a zin o[1′,2′:1,6]p yr id o[3,4-b]in d ole-
1,4-d ion e (14). The title compound was obtained from cis-
10h and isopropylamine as a white solid in 69% yield, mp 290-
293 °C (MeOH); [R]D +52° (c ) 1.14; CHCl3); 1H NMR (CDCl3)
δ 8.04 (s, 1H), 7.59 (m, 1H), 7.32-7.09 (m, 3H), 6.88-6.65 (m,
3H), 6.17 (s, 1H), 5.84 (d, 2H, J ) 4.5 Hz), 4.93-4.79 (m, 1H),
4.25 (dd, 1H, J ) 11.4, 4.5 Hz), 3.96 (d, 1H, J ) 17.3 Hz), 3.85

4542 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
a Potent and Selective Inhibitor of Type 5 cGMP Phosphodi-
esterase with Utility for the Treatment of Male Erectile Dys-
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ersham). In all cases, any given treatment with effectors was
performed in triplicate wells. Stock solutions of PDE inhibitors
were made in dimethyl sulfoxide. In the assays, the final
concentration of dimethyl sulfoxide never exceeded 0.1%
(v/v).
Effect on Blood P r essu r e of Con sciou s SHR After
Acu te Tr ea tm en t. The experiments were performed in
spontaneously hypertensive rats (SHR, Charles River, France)
weighting 340-380 g. The day before experiment, the left
carotid artery was catheterized under pentobarbital anesthe-
sia. On the day of experiment, the catheter was connected to
pressure transducers for blood pressure measurement. After
an equilibration period of ca. 30 min, the product was dissolved
in a mixture of 5% (v/v) DMF in olive oil. The concentration
was adjusted in order to administer the compound per os in a
volume of 1 mL. Arterial blood pressure was monitored
continuously over 7 h.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the following people for their contributions: San-
drine Martin, Didier Chevret, Florence Loriolle, Vale´rie
Baudet, Florence Blandel, Anne-Benedicte Boullay,
Vale´rie Boullay, and Sean Lynn.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data for compound 12a . This material is available free
of charge via the Internet at http://pubs.acs.org.
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