The discovery of tadalafil: A novel and highly selective PDE5 inhibitor. 1: 5,6,11,11a-Tetrahydro-1H-imidazo[1′,5′ :1,6]pyrido[3,4-b]indole-1,3(2H)-dione analogues

Article abstract of DOI:10.1021/jm030056e

Starting from ethyl β-carboline-3-carboxylate (β-CCE), 1, a modest inhibitor of type 5 phosphodiesterase (PDE5), a series of functionalized tetrahydro-β-carboline derivatives has been identified as a novel chemical class of potent and selective PDE5 inhibitors. Optimization of the side chain on the hydantoin ring of initial lead compound 2 and of the aromatic ring on position 5 led to the identification of compound 6e, a highly potent and selective PDE5 inhibitor, with greater selectivity for PDE5 vs PDE1-4 than sildenafil. Compound 6e demonstrated a long-lasting and significant blood pressure lowering effect after iv administration in the spontaneously hypertensive rat model but showed only moderate oral in vivo efficacy.

Full text of DOI:10.1021/jm030056e

J . Med. Chem. 2003, 46, 4525-4532
4525
Th e Discover y of Ta d a la fil: A Novel a n d High ly Selective P DE5 In h ibitor . 1:
5,6,11,11a -Tetr a h yd r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e
An a logu es
Alain Daugan,* Pascal Grondin, Ce´cile Ruault, Anne-Charlotte Le Monnier de Gouville, Herve´ Coste,
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
Starting from ethyl â-carboline-3-carboxylate (â-CCE), 1, a modest inhibitor of type 5
phosphodiesterase (PDE5), a series of functionalized tetrahydro-â-carboline derivatives has
been identified as a novel chemical class of potent and selective PDE5 inhibitors. Optimization
of the side chain on the hydantoin ring of initial lead compound 2 and of the aromatic ring on
position 5 led to the identification of compound 6e, a highly potent and selective PDE5 inhibitor,
with greater selectivity for PDE5 vs PDE1-4 than sildenafil. Compound 6e demonstrated a
long-lasting and significant blood pressure lowering effect after iv administration in the
spontaneously hypertensive rat model but showed only moderate oral in vivo efficacy.
Ch a r t 1
In tr od u ction
Cyclic nucleotide phosphodiesterases (PDEs) are en-
zymes that catalyze the hydrolysis of cyclic nucleotides
cGMP and cAMP into their respective 5′ nucleoside
monophosphates. To date, the phosphodiesterase su-
perfamily has been classified into 11 different isoforms
(PDE1-11) according to their primary protein and
cDNA sequences, their specificities toward hydrolysis
of cyclic AMP or cyclic GMP, their mechanisms of
regulation, and their sensitivities to various pharma-
cological agents.1-4 Phosphodiesterase type 5, (PDE5),
one member of the superfamily of cyclic nucleotide hy-
drolyzing enzymes that specifically cleaves cyclic gua-
nosine monophosphate (cGMP), is distributed through-
out vascular smooth muscle tissue and to a lesser extent
in the lung, kidney, and platelets. Inhibition of PDE5
as a therapeutic target has received considerable at-
tention over the years, particularly for the treatment
of cardiovascular diseases, e.g., angina, hypertension,
and congestive heart failure.5,6 More recently, it was
demonstrated that PDE5 plays a critical role in the
mechanism of penile erection and that PDE5 inhibition
would be of therapeutic utility in treating male erectile
dysfunction (MED).7 After these discoveries, sildenafil
(Viagra) was approved for the treatment of MED.8,9
The primary objective of our research effort in the
early 1990s was to identify chemically novel, selective
(PDE5 vs other PDEs), and orally active PDE5 inhibi-
tors for the treatment of hypertension and congestive
heart failure. â-Carbolines had been previously found
to increase basal level of cGMP in rat cerebellum10 and
to induce a concentration-dependent relaxation of rat
aortic rings precontracted with KCl.11 Furthermore,
â-carbolines were also reported to inhibit crude rat
aortic cyclic nucleotide phosphodiesterase activity.11 In
fact, in our hands, ethyl â-carboline-3-carboxylate (â-
CCE), compound 1, displayed modest inhibitory activity
toward PDE5 (IC50 ) 0.8 µM). The â-carboline scaffold
was then used as a basis for substructure searching in
our internal database to find novel type 5 phosphodi-
esterase inhibitors (Chart 1). The hydantoin derivative
2 was identified as a promising PDE5 inhibitor, with a
selectivity comparable to zaprinast 3, the reference
PDE5 inhibitor at that time (see Table 5).
In the present paper, we describe the discovery and
optimization of a novel series of highly potent and
selective inhibitors of PDE5 via a program of medicinal
chemistry based upon hydantoin 2.
Ch em istr y
The general synthesis of hydantoin compounds 6 is
illustrated in Scheme 1. Racemic tryptophan methyl or
ethyl ester and appropriate aldehydes 4 were subjected
to a modified Pictet-Spengler reaction. Since we ini-
tially desired access to both the cis- and trans-isomers
5, the reaction was carried out under nonstereospecific
conditions. The cis- and trans-1,3-disubstituted tetrahy-
dro-â-carbolines 5, which can be separated by flash
chromatography or crystallization, reacted with ap-
propriate, commercially available isocyanates to afford
* 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/jm030056e CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

4526 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
Sch em e 1a
a
Conditions: (a) R ) Me(Et), CF3CO2H, CH2Cl2; (b) R ) H, dil. H2SO4; (c) R3-NdCdO, 2-butanone, reflux.
Sch em e 2a
a
Conditions: (a) Cl-(CH2)2-NCO, 2-butanone, reflux; (b) Me2NH·HCl, K2CO3, DMF; (c) R1R2N(CH2)nNH2, CDI, THF; (d) THF, reflux.
the desired cis- and trans-hydantoins 6, respectively.
The N-alkyl indole derivatives 7 were prepared using a
similar synthetic pathway as described in Scheme 1 but
starting from racemic N-methyl tryptophan.12
pathways were explored as illustrated in Scheme 2. The
trans-1,3-disubstituted tetrahydro-â-carboline 5e re-
acted with 2-chloroethyl isocyanate to give the corre-
sponding hydantoin intermediate 10a as the sole prod-
uct. Subsequent attempts to substitute the halide 10a
with various secondary amines using different experi-
mental conditions either failed or led to the desired
compound 9a in a very low yield. To improve general
access to polar compounds, a second approach was used.
Primary amines were treated with carbonyl diimidazole
in THF and the resulting imidazolide intermediates
were reacted with trans-1,3-disubstituted tetrahydro-
â-carboline 5e to give the intermediate ureas 11a -d
which underwent intramolecular cyclization in refluxing
THF to produce the hydantoins 9a -d in good yields.
The assignment of cis/trans-stereochemistry for tet-
rahydro-â-carboline 5 was based on a detailed study of
1
the H and 13C NMR spectroscopy and by comparison
with the literature data, well-established by Cook.13,14
Thus, the signals for C-1 and C-3 in the trans-isomer
appeared at higher field in the carbon spectrum than
the analogous carbons of the corresponding cis-isomer,
probably due to the 1,3-interactions present in the trans-
isomer. Moreover, the NMR signals for the proton at
C-1 is more shielded in the cis-isomer compared to the
trans-isomer. A correlation exists between Rf value on
TLC and the stereochemistry of the 1,3-disubstituted
tetrahydro-â-carbolines as described in the literature.14
The cis-isomer 5 is systematically less polar than trans-
5, as indicated by the order of elution during the
purification on silica gel (toluene/ethyl acetate or dichlo-
romethane/methanol as eluent). However, in the hy-
dantoin series 6, the polarity is reversed, the cis-isomer
becomes more polar than the trans-isomer.
Resu lts a n d Discu ssion
Compounds were evaluated for inhibitory activity
against bovine PDE5. Each compound was evaluated
in two steps. The first step was the 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
For the synthesis of analogues bearing a polar chain
on the hydantoin ring 9a -d , two different synthetic

Discovery of Tadalafil. Part 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4527
Ta ble 1. PDE5 Inhibitory Potency of C-5 Aromatic or
Ta ble 2. PDE5 Inhibition and Cellular Activity of Substituted
Heterocyclic Derivatives
Phenyl Analogues
PDE5
compd
R2
IC50 (µM)a
PDE5
RSMC
2
4-pyridinyl
3-pyridinyl
3-thienyl
3-furanyl
phenyl
0.30
0.09
0.03
0.10
0.06
cis-6a
cis-6b
cis-6c
cis-6d
compd
R
IC50 (µM)a
EC50 (µM)
cis-6d
trans-6d
cis-6e
trans-6e
cis-6f
trans-6f
cis-6g
trans-6g
cis-6h
trans-6h
trans-6i
cis-7
H
H
0.060
0.020
0.008
0.005
0.050
0.050
0.050
0.020
0.9
5
2
0.7
1
4-OMe
4-OMe
3-Cl
3-Cl
4-Cl
a
IC50 values were reproducible to (25%.
4
>10
>10
1.5
ndb
ndb
ndb
ndb
ndb
µM, each tested in duplicate. IC50 values were reproduc-
ible to (25%. Selectivity toward other PDEs was evalu-
ated 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 PDE5.15 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 concentration. Fi-
nally, spontaneously hypertensive rat model (SHR) was
used to measure blood pressure lowering ability of the
best compounds.
4-Cl
4-CN
4-CN
2-OMe
4-OMe
4-OMe
0.3
1
>10
trans-7
2
a
b
IC50 values were reproducible to (25%. Not determined.
Ta ble 3. PDE5 Inhibition and Cellular Activity of
N-Substituted Hydantoins
Mod ifica tion a t th e C-5 P osition . In an attempt
to improve the potency and selectivity of the hydantoin
lead 2, we explored first the possibility of replacing the
pendant pyridine ring of 2 with aromatic or other
heteroaromatic systems (Table 1). Substitution with a
3-pyridinyl 6a or with a five-membered heterocycle such
as a thienyl 6b or a furanyl ring 6c showed improve-
ment in potency compared to the initial hit 2. More
interestingly, the introduction of a phenyl ring 6d led
to very encouraging in vitro results.
PDE5
RSMC
compd
cis-6e
trans-6e
trans-6j
cis-6k
cis-6l
trans-6l
cis-6m
R3
butyl
IC50 (µM)a
EC50 (µM)
0.008
0.005
0.020
0.010
0.010
0.007
0.004
0.018
0.007
0.003
0.7
1
3
butyl
hydrogen
methyl
ethyl
1
0.4
1.5
0.15
>10
0.3
0.1
ethyl
benzyl
benzyl
c.hexyl
c.hexyl
trans-6m
cis-6n
trans-6n
SAR on th e P h en yl Rin g in C-5 P osition . Having
shown that a phenyl ring could advantageously replace
the pyridinyl moiety in compound 2, a study of the
structure-activity relationships around the phenyl
group of compound 6d was carried out (Table 2).
Substitution at the 4-position on the phenyl ring with
a methoxy group 6e gave an increase in potency of
PDE5 inhibition. Analogues with a chlorine atom in the
3- (6f) or the 4-position (6g) gave no improvement of in
vitro activity compared with compound 6d . Introduction
of an electron-withdrawing substituent in the 4-position
of the phenyl ring, such as cyano, had a deleterious
effect on potency (Table 2, 6d vs 6h ). Introduction of a
methoxy group in the 2-position resulted in loss of
activity (6i), possibly suggesting that the presence of a
2-substituent prevents the optimal orientation of the
phenyl ring.
a
IC50 values were reproducible to (25%.
reospecificity for PDE5 inhibition in this series (Table
2, cis-6e, -6f vs trans-6e, -6f).
Alkylation of the indole nitrogen atom led to a drastic
fall in PDE5 inhibitory potency (cf. 6e vs cis- or trans-
7).
N-Su bstitu tion of th e Hyd a n toin Rin g. Given the
increased potency of compounds bearing a p-methoxy
substituent on the phenyl ring, this substitution was
retained while we modified the chain on the hydantoin
ring. The presence or the nature of the alkyl side-chain
group on the hydantoin ring does not seem to signifi-
cantly influence the potency (Table 3, 6e vs 6j-l).
Moreover, the introduction of a benzyl group 6m or a
cycloalkyl group such as cyclohexyl ring 6n gave com-
pounds equipotent with butyl hydantoin 6e (Table 3).
The PDE5 enzyme appears to be tolerant of a wide
Comparison of the activities of cis- and trans-
compounds showed that there was no clear diaste-

4528 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
F igu r e 1. Effect of trans-6e on Blood Pressure after iv (a) and po (b) Administration in SHR Model. 1a : Time course of the
changes in mean arterial blood pressure produced by trans-6e (b) or its vehicule (O) after iv administration (10 mg/kg) to
spontaneously hypertensive rats (SHR). 1b: Time course of the changes in mean arterial blood pressure produced by trans-6e
(b) or its vehicule (O) after po administration (30 mg/kg) to spontaneously hypertensive rats (SHR).
Ta ble 4. PDE5 Inhibition and Cellular Activity of Hydantoins
Ta ble 5. PDE5 Activity and Selectivity of Hydantoins on PDE
Substituted with Basic Chains
Isoforms
IC50 (µM)c IC50 (µM)
compd
lead-2
Zaprinast-3
trans-6e
cis-6e
cis-6m
cis-6n
sildenafil
PDE5a
PDE1a
PDE2b PDE3a PDE4b
0.3
0.2
2
2.6
ndd
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.005
0.008
0.004
0.007
0.006
>10
>10
>10
>10
1.1
9.2
7.8
PDE5
RSMC
a
b
Bovine aorta PDE. Human recombinant PDE. c IC50 values
d
were reproducible to (25%. Not determined.
compd
n
R4
NMe2
3-pyridinyl
2-pyridinyl
pyrrolidinyl
IC50 (µM)a
EC50 (µM)
trans-9a
trans-9b
trans-9c
trans-9d
2
1
2
2
0.030
0.020
0.006
0.100
3.5
5.5
1.5
10
pressure was observed after oral dosing (30 mg/kg,
Figure 1b), probably reflecting poor oral absorption of
trans-6e.
a
IC50 values were reproducible to (25%.
Con clu sion
range of substituents on the hydantoin nitrogen. How-
ever, in the RSMC test, the benzyl cis-6m and the
cyclohexyl 6n compounds showed a significant increase
in potency. Surprisingly, the trans-benzyl analogue
trans-6m was completely inactive at concentrations up
to 10 µM, despite an only 5-fold decrease in PDE5
inhibition.
Incorporation of basic groups into the hydantoin side
chain 9a -d led to somewhat decreased PDE5 inhibitory
potency in both enzymatic and cellular assays (Table
4).
P h osp h od iester a se Selectivity. To further evalu-
ate the interest of this new class of PDE5 inhibitors,
representative compounds were tested against other
PDE isoforms. As shown in Table 5, all compounds
tested in this series have high selectivity vs PDE1-
PDE4 and much greater selectivity than sildenafil on
PDE1, PDE3, and PDE4.
Acu te Effects of Com p ou n d tr a n s-6e on Blood
P r essu r e. On the basis of the PDE5 inhibitory potency,
isoform selectivity, and cellular potency (RSMC test),
several compounds were evaluated in the conscious
spontaneously hypertensive rat model (SHR) for anti-
hypertensive activity. Compound trans-6e was repre-
sentative of this chemical series. A significant (-25
mmHg) and long-lasting blood pressure lowering effect
(>7 h) was observed following iv administration (10 mg/
kg, Figure 1a). In contrast, only a modest effect on blood
In this paper, we described the discovery of 5,6,11,-
11a-tetrahydro-1H-imidazo[1′,5′:1,6]pyrido[3,4-b]indole-
1,3(2H)-dione analogues as a novel class of highly potent
and selective PDE5 inhibitors. The best inhibitors in
this series demonstrated high selectivity for PDE5 vs
PDE1-4 when compared to sildenafil. However, a
representative compound showed only moderate oral
efficacy in conscious spontaneously hypertensive rats
(SHR), probably due to limited oral bioavailability.
Nevertheless, this new series of hydantoin compounds
represents an attractive starting point for the design
and synthesis of selective and potent PDE5 inhibitors
with more desirable pharmacokinetic and pharmaco-
logical profiles (see Part 2).
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

Discovery of Tadalafil. Part 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4529
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. The elemental analysis were performed by Wolff
Laboratories and are within (0.4% of the theoretical value,
unless stated otherwise.
tryptophan methyl ester and 3-chlorobenzaldehyde. The crude
residue was purified by flash column chromatography eluting
with CH2Cl2/MeOH, 99/1, to give the mixture of cis- and trans-
5f (4.4 g, 79%), mp 150-160 °C.
Met h yl 1-(4-Ch lor op 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-5g). The title
compounds were prepared from racemic tryptophan methyl
ester and 4-chlorobenzaldehyde. The crude residue was puri-
fied by flash column chromatography eluting with toluene/
AcOEt, 90/10, to give first cis-5g as a white solid (3.6 g, 54%),
mp 208-209 °C, followed by trans-5g as a white solid (0.56 g,
9%), mp 108-109 °C.
Eth yl 1-(4-Cya n op 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-5h ). The title com-
pounds were prepared from racemic tryptophan ethyl ester and
4-cyanobenzaldehyde. The crude residue was purified by flash
column chromatography eluting with toluene/AcOEt, 80/20, to
give first cis-5h as a white solid (3.47 g, 46%), mp 200 °C,
followed by trans-5h as a white solid (1.54 g, 20.5%), mp 156
°C.
Meth yl 1-(2-Meth oxyp h en yl)-2,3,4,9-tetr a h yd r o-1H-â-
ca r bolin e-3-ca r boxyla t e (cis- a n d tr a n s-5i). The title
compounds were prepared from racemic tryptophan methyl
ester and 2-methoxybenzaldehyde. The crude residue was
purified by flash column chromatography eluting with CH2-
Cl2/MeOH, 99/1, to give first cis-5i as a white solid (7.89 g,
40%), mp 195 °C, followed by trans-5i as a white solid (2.01 g,
10%), mp 180 °C.
1-(4-Meth oxyp h en yl)-9-m eth yl-2,3,4,9-tetr a h yd r o-1H-
â-ca r bolin e-3-ca r boxylic Acid a s a Mixtu r e of Isom er s
(cis- a n d tr a n s-8). 4-Methoxybenzaldehyde (0.68 g, 5 mmol,
1.1 equiv) was added to a stirred solution of N-methyl
tryptophan (1 g, 4.58 mmol) in water (13 mL). A 1 N solution
of H2SO4 (4.6 mL) was added, and the mixture was heated at
50 °C for 16 h. The mixture was then cooled at room
temperature, and the precipitated product was collected by
filtration, washed with water, and dried to give the mixture
of cis- and trans-8 as a beige solid (1.14 g, 74%), mp >230 °C.
5-(4-Meth oxyp h en yl)-2-(p h en ylm eth yl)-5,6,11,11a -tet-
r a h yd r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-
d ion e (tr a n s-6m ). Representative example: To a stirred
solution of tetrahydro-â-carboline trans-5e (369 mg, 1.1 mmol,
1 equiv) in methylethyl ketone (10 mL) was added benzyl
isocyanate (135 µL, 1.1 mmol, 1 equiv), and the mixture was
stirred at reflux for 16 h under a nitrogen atmosphere. The
solvent was evaporated under reduced pressure, and the
residue was purified by flash column chromatography eluting
with cyclohexane/AcOEt, 80/20, to give trans-6m as a white
solid (300 mg, 62.5%) after crystallization from 2-propanol, mp
208-212 °C; 1H NMR (CDCl3) δ 7.84 (s, 1H), 7.53 (d, 1H, J )
7 Hz), 7.43-7.10 (m, 10H), 6.83 (d, 2H, J ) 8.7 Hz), 6.25 (s,
1H, C5-H), 4.70 (d, 1H, J ) 14.6 Hz), 4.60 (d, 1H, J ) 14.6
Hz), 4.29 (dd, 1H, J ) 11, 5.6 Hz, C11a-H), 3.76 (s, 3H), 3.46
(dd, 1H, J ) 15.3, 5.5 Hz), 2.82 (dd, 1H, J ) 15.3, 11.1 Hz);
13C NMR (CDCl3) δ 173.1, 160.6, 155.2, 137.2, 136.7, 131.9,
131.4, 130.2, 129.4, 129.3, 128.6, 126.8, 123.5, 120.8, 119.1,
115.1, 111.9, 108.7, 56, 53.9 (C11a), 52.2 (C5), 43, 24.1; 1H-13C
HMQC (CDCl3) δH (δC) 6.25 (52.2, C5), 4.29 (53.9, C11a); Anal.
(C27H23N3O3) C, H, N.
Meth yl 1-(4-Meth 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-5e). Representa-
tive example: Racemic tryptophan methyl ester (10.76 g, 49.35
mmol, 1 equiv) and 4-methoxybenzaldehyde (7.39 g, 54 mmol,
1.1 equiv) were dissolved in CH2Cl2 (70 mL) and cooled to 0
°C with an ice bath. To this solution was added dropwise TFA
(7.6 mL), and the mixture was stirred at room temperature
for 4 days. The reaction mixture was then basified with
aqueous NaHCO3 and extracted with CH2Cl2 (3×). The organic
layer was washed with water, brine, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was purified by flash column chromatography on silica gel
eluting with toluene/AcOEt, 70/30, to give first the cis-isomer
5e as a white solid (4.15 g, 25%), mp 132 °C, followed by the
trans-isomer 5e as a white solid (6.09 g, 37%), mp 202 °C. cis-
5e: 1H NMR (CDCl3) δ 7.58-7.37 (m, 2H), 7.32-7.02 (m, 5H),
6.84 (d, 2H, J ) 8.3 Hz), 5.14 (s, 1H, C1-H), 3.91 (dd, 1H, J )
11.3, 4.3 Hz, C3-H), 3.76 (br s, 6H), 3.16 (dd, 1H, J ) 15.2, 4.3
Hz, C4-H), 2.96 (dd, 1H, J ) 15.2, 11.2 Hz, C4-H), 2.20 (br s,
1H); 13C NMR (CDCl3) δ 173.8, 160.5, 136.8, 135.5, 133.2,
130.5, 127.8, 122.6, 120.3, 118.9, 114.9, 111.6, 109.5, 58.7 (C1),
57.6 (C3), 56, 52.9, 26.3; 1H-13C HMQC (CDCl3) δH (δC) 5.14
(58.7, C1), 3.91 (57.6, C3); trans-5e: 1H NMR (CDCl3) δ 7.73
(br s, 1H), 7.61 (d, 1H, J ) 7 Hz), 7.44-7.16 (m, 5H), 6.91 (d,
2H, J ) 8.7 Hz), 5.41 (s, 1H, C1-H), 4.14-4.0 (m, 1H, C3-H),
3.84 (s, 3H), 3.77 (s, 3H), 3.32 (dd, 1H, J ) 15.4, 5.4 Hz, C4-H),
3.15 (dd, 1H, J ) 15.5, 6.8 Hz, C4-H), 2.53 (br s, 1H); 13C NMR
(CDCl3) δ 174.7, 160.1, 136.8, 134.5, 134.1, 130.3, 127.6, 122.6,
120.2, 118.9, 114.7, 111.6, 108.9, 56, 54.9 (C1), 53.2 (C3), 52.8,
25.2; 1H-13C HMQC (CDCl3) δH (δC) 5.41 (54.9, C1), 4.14 (53.2,
C3).
The following compounds were prepared using a similar
procedure with appropriate aldehydes.
Eth yl 1-(3-P yr idin yl)-2,3,4,9-tetr ah ydr o-1H-â-car bolin e-
3-ca r boxyla te (cis- a n d tr a n s-5a ). The title compounds were
prepared from racemic tryptophan ethyl ester and 3-pyridin-
ecarboxaldehyde. The crude residue was purified by flash
column chromatography eluting with CH2Cl2/MeOH, 95/5, to
give first cis-5a as a white solid (0.5 g, 13%), mp 230-232 °C,
followed by trans-5a as a white solid (0.3 g, 7.9%), mp 210-
214 °C.
Eth yl 1-(3-Th ien 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-5b). The title compounds were
prepared from racemic tryptophan ethyl ester and 3-thiophen-
ecarboxaldehyde. The crude residue was purified by flash
column chromatography eluting with toluene/AcOEt, 70/30, to
give first cis-5b as a white solid (2.3 g, 51%), mp 130 °C,
followed by trans-5b as a white solid (0.45 g, 10%), mp 182-
184 °C.
Meth yl 1-(3-Fu r an yl)-2,3,4,9-tetr ah ydr o-1H-â-car bolin e-
3-ca r boxyla te a s a Mixtu r e of Isom er s (cis- a n d tr a n s-
5c). The title compound was prepared from racemic tryptophan
methyl ester and 3-furancarboxaldehyde. The crude residue
was purified by flash column chromatography eluting with
CH2Cl2/MeOH, 98/2, to give the mixture of cis- and trans-5c
(3 g, 51%), mp 130 °C.
Eth yl 1-(P 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-5d ). The title compounds were
prepared from racemic tryptophan ethyl ester and benzalde-
hyde. The crude residue was purified by flash column chro-
matography eluting with CH2Cl2/MeOH, 99/1, to give first cis-
5d as a white solid (0.9 g, 34%), mp 153-155 °C, followed by
trans-5d as a white solid (0.55 g, 20%), mp 180-182 °C.
Met h yl 1-(3-Ch lor op 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 a s a Mixtu r e of Isom er s (cis-
a n d tr a n s-5f). The title compound was prepared from racemic
The following compounds were prepared using a similar
procedure starting from appropriate tetrahydro-â-carbolines
and isocyanates.
5-(4-Meth oxyp h en yl)-2-(p h en ylm eth yl)-5,6,11,11a -tet-
r a h yd r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-
d ion e (cis-6m ). The title compound was prepared from
tetrahydro-â-carboline cis-5e and benzyl isocyanate. The crude
residue was purified by flash column chromatography eluting
with cyclohexane/AcOEt, 80/20, to give cis-6m as a pale yellow
solid (630 mg, 46.6%) after crystallization from EtOH, mp
240-243 °C; 1H NMR (CDCl3) δ 7.62-7.53 (m, 2H), 7.41-7.11
(m, 10H), 6.84 (d, 2H, J ) 8.6 Hz), 5.73 (s, 1H, C5-H), 4.68 (d,
1H, J ) 14.5 Hz), 4.55 (d, 1H, J ) 14.5 Hz), 4.34 (dd, 1H, J )
11.5, 4.5 Hz, C11a-H), 3.76 (s, 3H), 3.49 (dd, 1H, J ) 14.9, 4.7
Hz), 3.05 (dd, 1H, J ) 14.9, 11.3 Hz); 13C NMR (CDCl3) δ 172.1,

4530 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
160.3, 155.2, 137.4, 136.8, 134.4, 131.1, 129.8, 129.5, 129.3,
128.6, 126.9, 123.4, 120.8, 119.1, 114.9, 111.9, 107.6, 58.8 (C11a),
56.9 (C5), 55.8, 42.9, 23.1; 1H-13C HMQC (CDCl3) δH (δC) 5.73
(56.9, C5), 4.34 (58.8, C11a); Anal. (C27H23N3O3) C, H, N.
2-Bu tyl-5-(4-m eth oxyph en yl)-5,6,11,11a-tetr ah ydr o-1H-
im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-1,3(2H)-dion e (tr a n s-
6e). The title compound was prepared from tetrahydro-â-
carboline trans-5e and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with CH2-
Cl2/MeOH, 99/1, to give trans-6e as a white solid (520 mg, 49%)
after crystallization from EtOH/H2O, mp 173-174 °C; 1H NMR
(CDCl3) δ 7.80 (s, 1H), 7.55 (d, 1H, J ) 7 Hz), 7.35-7.10 (m,
5H), 6.85 (d, 2H, J ) 8.7 Hz), 6.25 (s, 1H, C5-H), 4.30 (dd, 1H,
J ) 11, 5.6 Hz, C11a-H), 3.75 (s, 3H), 3.60-3.40 (m, 3H), 2.85
(dd, 1H, J ) 15.3, 11.1 Hz), 1.70-1.55 (m, 2H), 1.45-1.25 (m,
2H), 0.95 (t, 3H, J ) 7.4 Hz); Anal. (C24H25N3O3) C, H, N.
2-Bu t yl-5-(3-p yr id in yl)-5,6,11,11a -t et r a h yd r o-1H -im i-
d a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-6a ).
The title compound was prepared from tetrahydro-â-carboline
cis-5a and butyl isocyanate. The crude residue was purified
by crystallization from 2-propanol to give cis-6a as a white
solid (350 mg, 78%), mp 257-263 °C; 1H NMR (CDCl3) δ 10.27
(s, 1H), 8.44 (d, 1H, J ) 3.8 Hz), 8.38 (s, 1H), 7.60 (d, 1H, J )
6.6 Hz), 7.48 (d, 1H, J ) 8.1 Hz), 7.32 (d, 1H, J ) 6.4 Hz),
7.25-7.10 (m, 3H), 5.85 (s, 1H), 4.39 (dd, 1H, J ) 11.5, 4.5
Hz), 3.60-3.37 (m, 3H), 3.07 (dd, 1H, J ) 14.8, 11.3 Hz), 1.61-
1.49 (m, 2H), 1.35-1.21 (m, 2H), 0.89 (t, 3H, J ) 7.3 Hz); Anal.
(C22H22N4O2) C, H, N.
2-Bu tyl-5-(3-th ien yl)-5,6,11,11a-tetr ah ydr o-1H-im idazo-
[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H )-d ion e (cis-6b). The
title compound was prepared from tetrahydro-â-carboline cis-
5b and butyl isocyanate. The crude residue was purified by
flash column chromatography eluting with toluene/AcOEt, 80/
20, to give cis-6b as a white solid (250 mg, 22%) after
crystallization from 2-propanol, mp 219-221 °C; 1H NMR
(CDCl3) δ 7.63 (s, 1H), 7.55 (d, 1H, J ) 8.3 Hz), 7.38 (s, 1H),
7.28-7.10 (m, 4H), 6.86 (d, 1H, J ) 6.2 Hz), 5.97 (s, 1H), 4.34
(dd, 1H, J ) 11.3, 4.6 Hz), 3.56-3.40 (m, 3H), 3.05 (dd, 1H, J
) 14.8, 11.3 Hz), 1.63-1.50 (m, 2H), 1.37-1.24 (m, 2H), 0.90
(t, 3H, J ) 7.2 Hz); Anal. (C21H21N3O2S) C, H, N.
2-Bu t yl-5-(3-ch lor op h en yl)-5,6,11,11a -t et r a h yd r o-1H -
im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-
6f a n d tr a n s-6f). The title compounds were prepared from
tetrahydro-â-carboline cis- and trans-5f and butyl isocyanate.
The crude residue was purified by flash column chromatog-
raphy eluting with toluene/AcOEt, 90/10, to give first trans-
6f as a white solid (500 mg, 28%) after crystallization from
1
EtOH, mp 207-209 °C; H NMR (CDCl3) δ 7.82 (s, 1H), 7.52
(d, 1H, J ) 7.3 Hz), 7.27-7.11 (m, 7H), 6.22 (s, 1H), 4.24 (dd,
1H, J ) 11, 5.5 Hz), 3.50-3.41 (m, 3H), 2.82 (dd, 1H, J ) 15.5,
11.1 Hz), 1.63-1.48 (m, 2H), 1.37-1.19 (m, 2H), 0.87 (t, 3H, J
) 7.4 Hz); Anal. (C23H22ClN3O2) C, H, N; followed by cis-6f as
a white solid (500 mg, 28%) after crystallization from Et2O/
cyclohexane, mp 215-217 °C; 1H NMR (CDCl3) δ 7.60 (s, 1H),
7.55 (d, 1H, J ) 8.7 Hz), 7.27-7.12 (m, 7H), 5.74 (s, 1H), 4.34
(dd, 1H, J ) 11.3, 4.5 Hz), 3.54-3.36 (m, 3H), 3.03 (dd, 1H, J
) 15.1, 11.5 Hz), 1.62-1.50 (m, 2H), 1.34-1.21 (m, 2H), 0.88
(t, 3H, J ) 7.3 Hz); Anal. (C23H22ClN3O2) C, H, N.
2-Bu tyl-5-(3-fu r an yl)-5,6,11,11a-tetr ah ydr o-1H-im idazo-
[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-6c). The
title compound was prepared from tetrahydro-â-carboline cis-
and trans-5c and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with toluene/
AcOEt, 80/20, to give cis-6c as a white solid (160 mg, 13%)
2-Bu t yl-5-(4-ch lor op h en yl)-5,6,11,11a -t et r a h yd r o-1H -
im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-
6g). The title compound was prepared from tetrahydro-â-
carboline cis-5g and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with toluene/
AcOEt, 85/15, to give cis-6g as a pale yellow solid (340 mg,
1
after crystallization from toluene, mp 155-160 °C; H NMR
(CDCl3) δ 7.69 (s, 1H), 7.61 (s, 1H), 7.56 (d, 1H, J ) 8.5 Hz),
7.37 (s, 1H), 7.26-7.13 (m, 3H), 6.18 (s, 1H), 5.88 (s, 1H), 4.30
(dd, 1H, J ) 11.5, 4.7 Hz), 3.55-3.43 (m, 3H), 2.96 (dd, 1H, J
) 15.1, 11.5 Hz), 1.65-1.50 (m, 2H), 1.40-1.25 (m, 2H), 0.91
(t, 3H, J ) 7.4 Hz); Anal. (C21H21N3O3) C, H, N.
1
57%) after crystallization from MeOH, mp 252 °C; H NMR
(CDCl3) δ 7.58 (s, 1H), 7.56 (s, 1H), 7.36-7.14 (m, 7H), 5.79
(s, 1H), 4.36 (dd, 1H, J ) 11.3, 4.6 Hz), 3.59-3.38 (m, 3H),
3.04 (dd, 1H, J ) 14.9, 11.3 Hz), 1.64-1.51 (m, 2H), 1.38-
1.23 (m, 2H), 0.90 (t, 3H, J ) 7.4 Hz); Anal. (C23H22ClN3O2)
C, H, N.
2-Bu t yl-5-p h en yl-5,6,11,11a -t et r a h yd r o-1H -im id a zo-
[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-6d ). The
title compound was prepared from tetrahydro-â-carboline cis-
5d and butyl isocyanate. The crude residue was purified by
crystallization from toluene to give cis-6d as a white solid (150
2-Bu t yl-5-(4-ch lor op h en yl)-5,6,11,11a -t et r a h yd r o-1H -
im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-1,3(2H)-dion e (tr a n s-
6g). The title compound was prepared from tetrahydro-â-
carboline trans-5g and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with toluene/
AcOEt, 85/15, to give trans-6g as a pale yellow solid (260 mg,
1
mg, 33%), mp 225-227 °C; H NMR (CDCl3) δ 7.50 (s, 1H),
7.48 (s, 1H), 7.34-7.01 (m, 8H), 5.72 (s, 1H), 4.30 (dd, 1H, J )
11.3, 4.5 Hz), 3.50-3.28 (m, 3H), 2.97 (dd, 1H, J ) 14.1, 11.5
Hz), 1.55-1.41 (m, 2H), 1.28-1.13 (m, 2H), 0.82 (t, 3H, J )
7.1 Hz); Anal. (C23H23N3O2) C, H, N.
1
62%) after crystallization from MeOH, mp 174 °C; H NMR
(CDCl3) δ 7.75 (s, 1H), 7.52 (d, 1H, J ) 7.3 Hz), 7.35-7.12 (m,
7H), 6.25 (s, 1H), 4.22 (dd, 1H, J ) 11.1, 5.6 Hz), 3.56-3.39
(m, 3H), 2.84 (dd, 1H, J ) 15.3, 11 Hz), 1.63-1.49 (m, 2H),
2-Bu t yl-5-p h en yl-5,6,11,11a -t et r a h yd r o-1H -im id a zo-
[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (tr a n s-6d ). The
title compound was prepared from tetrahydro-â-carboline
trans-5d and butyl isocyanate. The crude residue was purified
by flash column chromatography eluting with cyclohexane/
AcOEt, 80/20, to give trans-6d as a white solid (130 mg, 20%)
after crystallization from 2-propanol, mp 240-243 °C; 1H NMR
(CDCl3) δ 7.73 (s, 1H), 7.47 (d, 1H, J ) 7.6 Hz), 7.30-7.06 (m,
8H), 6.22 (s, 1H), 4.20 (dd, 1H, J ) 11.2, 5.7 Hz), 3.50-3.35
(m, 3H), 2.80 (dd, 1H, J ) 15.3, 11.5 Hz), 1.56-1.43 (m, 2H),
1.30-1.14 (m, 2H), 0.83 (t, 3H, J ) 7.3 Hz); Anal. (C23H23N3O2)
C, H, N.
2-Bu tyl-5-(4-m eth oxyph en yl)-5,6,11,11a-tetr ah ydr o-1H-
im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-
6e). The title compound was prepared from tetrahydro-â-
carboline cis-5e and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with CH2-
Cl2/MeOH, 99/1, to give cis-6e as a white solid (280 mg, 63%)
after crystallization from MeOH, mp 220-225 °C; 1H NMR
(CDCl3) δ 7.71-7.58 (m, 2H), 7.30-7.17 (m, 5H), 6.91 (d, 2H,
J ) 8.6 Hz), 5.82 (s, 1H, C5-H), 4.40 (dd, 1H, J ) 11.3, 4.5
Hz, C11a-H), 3.82 (s, 3H), 3.61-3.42 (m, 3H), 3.08 (dd, 1H, J )
14.9, 11.3 Hz), 1.70-1.55 (m, 2H), 1.42-1.27 (m, 2H), 0.94 (t,
3H, J ) 7.4 Hz); Anal. (C24H25N3O3) C, H, N.
1.36-1.21 (m, 2H), 0.88 (t, 3H, J ) 7.4 Hz); Anal. (C23H22
ClN3O2) C, H, N.
-
2-Bu t yl-5-(4-cya n op h en yl)-5,6,11,11a -t et r a h yd r o-1H -
im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-
6h ). The title compound was prepared from tetrahydro-â-
carboline cis-5h and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with toluene/
AcOEt, 80/20, to give cis-6h as a white solid (530 mg, 92%)
after crystallization from 2-propanol, mp 260 °C; 1H NMR
(CDCl3) δ 7.71-7.53 (m, 4H), 7.42 (d, 2H, J ) 7.9 Hz), 7.32-
7.11 (m, 3H), 5.86 (s, 1H), 4.38 (dd, 1H, J ) 11.3, 4.6 Hz), 3.61-
3.38 (m, 3H), 3.06 (dd, 1H, J ) 14.8, 11.7 Hz), 1.65-1.49 (m,
2H), 1.36-1.19 (m, 2H), 0.90 (t, 3H, J ) 7.5 Hz); Anal.
(C24H22N4O2) C, H, N.
2-Bu t yl-5-(4-cya n op h en yl)-5,6,11,11a -t et r a h yd r o-1H -
im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-1,3(2H)-dion e (tr a n s-
6h ). The title compound was prepared from tetrahydro-â-
carboline trans-5h and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with CH2-
Cl2/MeOH, 99/1, to give trans-6h as a white solid (400 mg,
72.7%) after crystallization from Et2O/cyclohexane, mp 158 °C;
1H NMR (CDCl3) δ 7.87 (s, 1H), 7.63 (d, 2H, J ) 8.1 Hz), 7.56

Discovery of Tadalafil. Part 1
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4531
(d, 1H, J ) 7.5 Hz), 7.48 (d, 2H, J ) 8.3 Hz), 7.35-7.14 (m,
3H), 6.36 (s, 1H), 4.24 (dd, 1H, J ) 11.1, 5.7 Hz), 3.57-3.44
(m, 3H), 2.90 (dd, 1H, J ) 15.5, 11.2 Hz), 1.67-1.53 (m, 2H),
1.39-1.26 (m, 2H), 0.92 (t, 3H, J ) 7.4 Hz); Anal. (C24H22N4O2)
C, H, N.
2-Bu tyl-5-(2-m eth oxyph en yl)-5,6,11,11a-tetr ah ydr o-1H-
im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-1,3(2H)-dion e (tr a n s-
6i). The title compound was prepared from tetrahydro-â-
carboline trans-5i and butyl isocyanate. The crude residue was
purified by flash column chromatography eluting with toluene/
AcOEt, 70/30, to give trans-6i as a pale yellow solid (480 mg,
80%) after crystallization from 2-propanol, mp 181 °C; 1H NMR
(CDCl3) δ 8.39 (s, 1H), 7.49 (d, 1H, J ) 7.4 Hz), 7.33-6.92 (m,
7H), 6.60 (s, 1H), 4.59 (dd, 1H, J ) 11, 5.5 Hz), 3.98 (s, 3H),
3.62-3.43 (m, 3H), 2.84 (dd, 1H, J ) 15.1, 11 Hz), 1.71-1.59
(m, 2H), 1.41-1.29 (m, 2H), 0.93 (t, 3H, J ) 7.4 Hz); Anal.
(C24H25N3O3) C, H, N.
1H), 4.36 (dd, 1H, J ) 11.3, 4.5 Hz), 3.78 (s, 3H), 3.60-3.41
(m, 3H), 3.05 (dd, 1H, J ) 15, 11.5 Hz), 1.21 (t, 3H, J ) 7.3
Hz); Anal. (C22H21N3O3) C, H, N.
2-Cycloh exyl-5-(4-m eth oxyp h en yl)-5,6,11,11a -tetr a h y-
d r o-1H -im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H )-d i-
on e (cis-6n a n d tr a n s-6n ). The title compounds were pre-
pared from tetrahydro-â-carboline cis- and trans-5e and
cyclohexyl isocyanate. The crude residue was purified by flash
column chromatography eluting with cyclohexane/AcOEt, 70/
30, to give first trans-6n as a white solid (350 mg, 18.3%) after
crystallization from 2-propanol, mp 265-269 °C; 1H NMR
(CDCl3) δ 8.05 (s, 1H), 7.78 (d, 1H, J ) 7.4 Hz), 7.56-7.36 (m,
5H), 7.08 (d, 2H, J ) 8.7 Hz), 6.46 (s, 1H), 4.44 (dd, 1H, J )
10.9, 5.4 Hz), 4.18-4.09 (m, 1H), 3.99 (s, 3H), 3.70 (dd, 1H, J
) 15.2, 5.4 Hz), 3.07 (dd, 1H, J ) 15.3, 11.1 Hz), 2.5-1.4 (m,
10H); Anal. (C26H27N3O3) C, H, N; followed by cis-6n as a white
solid (650 mg, 34.1%) after crystallization from EtOH, mp
250-260 °C; 1H NMR (CDCl3) δ 7.65-7.54 (m, 2H), 7.35-7.17
(m, 5H), 6.91 (d, 2H, J ) 8.7 Hz), 5.80 (s, 1H), 4.31 (dd, 1H, J
) 11.3, 4.5 Hz), 3.97-3.86 (m, 1H), 3.83 (s, 3H), 3.52 (dd, 1H,
J ) 15, 4.5 Hz), 3.07 (dd, 1H, J ) 14.9, 11.3 Hz), 2.27-1.19
(m, 10H); Anal. (C26H27N3O3) C, H, N.
2-Bu t yl-5-(4-m et h oxyp h en yl)-9-m et h yl-5,6,11,11a -t et -
r a h yd r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-
d ion e (cis-7 a n d tr a n s-7). The title compounds were pre-
pared from tetrahydro-â-carboline carboxylic acid cis- and
trans-8 and butyl isocyanate. The crude residue was purified
by flash column chromatography eluting with cyclohexane/
AcOEt, 80/20, to give first trans-7 as a white solid (420 mg,
33.8%) after crystallization from diisopropyl ether, mp 149-
151 °C; 1H NMR (CDCl3) δ 7.57 (d, 1H, J ) 7.7 Hz), 7.30-
7.15 (m, 5H), 6.85 (d, 2H, J ) 8.8 Hz), 6.36 (s, 1H), 4.21 (dd,
1H, J ) 11.2, 5.9 Hz), 3.78 (s, 3H), 3.57-3.44 (m, 3H), 3.34 (s,
3H), 2.90 (dd, 1H, J ) 15.3, 11.2 Hz), 1.64-1.55 (m, 2H), 1.38-
1.25 (m, 2H), 0.91 (t, 3H, J ) 7.2 Hz); Anal. (C25H27N3O3) C,
H, N; followed by cis-7 as a white solid (250 mg, 20.1%) after
5-(4-Meth oxyp h en yl)-2-(3-p yr id in ylm eth yl)-5,6,11,11a -
tetr a h yd r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3-
(2H)-d ion e (tr a n s-9b). Representative example: To a solu-
tion of carbonyl diimidazole (0.28 g, 1.72 mmol, 1.16 equiv) in
dry THF (5 mL) was added dropwise (3-pyridinylmethyl)amine
(0.18 g, 1.66 mmol, 1.13 equiv), and the reaction mixture was
stirred at room temperature. After 30 min, a solution of
tetrahydro-â-carboline trans-5e (0.5 g, 1.49 mmol, 1 equiv) in
THF (10 mL) was added, and the mixture was stirred at reflux
under a nitrogen atmosphere overnight. The solvent was
removed under reduced pressure, and the residue was dis-
solved in CH2Cl2. The organic layer was washed with water
and brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by flash column chroma-
tography eluting with CH2Cl2/MeOH, 99/1, to give trans-9b
as a white solid (470 mg, 72.2%) after crystallization from
1
crystallization from 2-propanol, mp 249 °C; H NMR (CDCl3)
δ 7.58 (d, 1H, J ) 7.5 Hz), 7.28-7.10 (m, 5H), 6.81 (d, 2H, J
) 8.7 Hz), 5.90 (s, 1H), 4.30 (dd, 1H, J ) 11.4, 4.3 Hz), 3.77 (s,
3H), 3.58-3.36 (m, 3H), 3.32 (s, 3H), 3.05 (dd, 1H, J ) 14.7,
11.5 Hz), 1.61-1.48 (m, 2H), 1.35-1.22 (m, 2H), 0.89 (t, 3H, J
) 7.3 Hz); Anal. (C25H27N3O3) C, H, N.
5-(4-Meth oxyph en yl)-5,6,11,11a-tetr ah ydr o-1H-im idazo-
[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (tr a n s-6j). The
title compound was prepared from tetrahydro-â-carboline
trans-5e and potassium cyanate as previously described.16 The
crude residue was purified by flash column chromatography
eluting with CH2Cl2/MeOH, 96/4, to give trans-6j as a pale
yellow solid (80 mg, 4%) after crystallization from MeOH, mp
300-310 °C; 1H NMR (CDCl3) δ 7.76 (s, 1H), 7.56 (d, 1H, J )
7.4 Hz), 7.34-7.14 (m, 6H), 6.86 (d, 2H, J ) 8.6 Hz), 6.26 (s,
1H), 4.37 (dd, 1H, J ) 11, 5.5 Hz), 3.79 (s, 3H), 3.48 (dd, 1H,
J ) 15.2, 5.6 Hz), 2.98 (dd, 1H, J ) 15.3, 11.2 Hz); Anal.
(C20H17N3O3) C, H, N.
5-(4-Meth oxyp h en yl)-2-m eth yl-5,6,11,11a -tetr a h yd r o-
1H-im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-1,3(2H)-dion e (cis-
6k ). The title compound was prepared from tetrahydro-â-
carboline cis-5e and methyl isocyanate. The crude residue was
purified by crystallization from EtOH to give cis-6k as a white
solid (220 mg, 39.3%), mp 233-240 °C; 1H NMR (CDCl3) δ 7.68
(s, 1H), 7.60 (d, 1H, J ) 7.4 Hz), 7.30-7.17 (m, 5H), 6.90 (d,
2H, J ) 8.7 Hz), 5.82 (s, 1H), 4.41 (dd, 1H, J ) 11.3, 4.5 Hz),
3.82 (s, 3H), 3.55 (dd, 1H, J ) 15.1, 4.7 Hz), 3.09 (dd, 1H, J )
15, 11.5 Hz), 3.02 (s, 3H); Anal. (C21H19N3O3) C, H, N.
1
EtOH, mp 160-165 °C; H NMR (CDCl3) δ 8.64 (s, 1H), 8.50
(d, 1H, J ) 4.9 Hz), 8.02 (s, 1H), 7.75 (d, 1H, J ) 8 Hz), 7.54
(d, 1H, J ) 7.1 Hz), 7.31-7.10 (m, 6H), 6.84 (d, 2H, J ) 8.7
Hz), 6.25 (s, 1H), 4.72 (d, 1H, J ) 14.7 Hz), 4.63 (d, 1H, J )
14.7 Hz), 4.33 (dd, 1H, J ) 11.1, 5.4 Hz), 3.76 (s, 3H), 3.49
(dd, 1H, J ) 15.3, 5.5 Hz), 2.84 (dd, 1H, J ) 15.3, 11.1 Hz);
Anal. (C26H22N4O3) C, H, N.
The following compounds were prepared using a similar
procedure with appropriate primary amines.
2-[2-(Dim eth yla m in o)eth yl]-5-(4-m eth oxyp h en yl)-5,6,-
11,11a-tetr ah ydr o-1H-im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-
1,3(2H)-d ion e (tr a n s-9a ). The title compound was prepared
from tetrahydro-â-carboline trans-5e and N,N-dimethyl-1,2-
ethanediamine. The crude residue was purified by flash
column chromatography eluting with CH2Cl2/MeOH, 92/8, to
give trans-9a as a white solid (400 mg, 64.5%) after crystal-
1
lization from MeOH, mp 145 °C; H NMR (CDCl3) δ 10.09 (s,
1H), 7.42-7.16 (m, 6H), 6.86 (d, 2H, J ) 8.7 Hz), 6.59 (s, 1H),
4.14 (dd, 1H, J ) 11, 6.2 Hz), 3.84 (s, 3H), 3.80-3.70 (m, 2H),
2.99-2.82 (m, 2H), 2.61-2.50 (m, 1H), 2.14 (s, 6H), 1.76-1.61
(m, 1H); Anal. (C24H26N4O3) C, H, N.
5-(4-Met h oxyp h en yl)-2-[2-(2-p yr id in yl)et h yl]-5,6,11,-
11a -tetr a h yd r o-1H-im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-
1,3(2H)-d ion e (tr a n s-9c). The title compound was prepared
from tetrahydro-â-carboline trans-5e and [2-(2-pyridinyl)ethyl]-
amine. The crude residue was purified by flash column
chromatography eluting with CH2Cl2/MeOH, 99/1, to give
trans-9c as a white solid (350 mg, 52.2%) after crystallization
from EtOH/H2O, mp 140-143 °C; 1H NMR (CDCl3) δ 8.40 (d,
1H, J ) 4.2 Hz), 7.90 (s, 1H), 7.60-7.47 (m, 2H), 7.30-7.06
(m, 7H), 6.84 (d, 2H, J ) 8.8 Hz), 6.23 (s, 1H), 4.24 (dd, 1H, J
) 10.9, 5.5 Hz), 3.97-3.86 (m, 2H), 3.78 (s, 3H), 3.44 (dd, 1H,
J ) 15.3, 5.5 Hz), 3.11 (t, 2H, J ) 6.9 Hz), 2.82 (dd, 1H, J )
15.3, 10.9 Hz); Anal. (C27H24N4O3) C, H, N.
2-Eth yl-5-(4-m eth oxyp h en yl)-5,6,11,11a -tetr a h ydr o-1H-
im id a zo[1′,5′:1,6]p yr id o[3,4-b]in d ole-1,3(2H)-d ion e (cis-
6l a n d tr a n s-6l). The title compounds were prepared from
tetrahydro-â-carboline cis- and trans-5e and ethyl isocyanate.
The crude residue was purified by flash column chromatog-
raphy eluting with cyclohexane/AcOEt, 70/30, to give first
trans-6l as a white solid (270 mg, 12.1%) after crystallization
1
from 2-propanol, mp 245-248 °C; H NMR (CDCl3) δ 7.93 (s,
1H), 7.61 (d, 1H, J ) 7.4 Hz), 7.36-7.18 (m, 5H), 6.90 (d, 2H,
J ) 8.6 Hz), 6.31 (s, 1H), 4.33 (dd, 1H, J ) 11.1, 5.6 Hz), 3.83
(s, 3H), 3.69-3.49 (m, 3H), 2.93 (dd, 1H, J ) 15.3, 11 Hz),
1.27 (t, 3H, J ) 7.2 Hz); Anal. (C22H21N3O3) C, H, N; followed
by cis-6l as a white solid (1320 mg, 59.1%) after crystallization
from EtOH, mp 210-220 °C; 1H NMR (CDCl3) δ 7.65-7.54
(m, 2H), 7.30-7.10 (m, 5H), 6.85 (d, 2H, J ) 8.7 Hz), 5.75 (s,
5-(4-Met h oxyp h en yl)-2-[2-(1-p yr r olid in yl)et h yl]-5,6,-
11,11a-tetr ah ydr o-1H-im idazo[1′,5′:1,6]pyr ido[3,4-b]in dole-

4532 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Daugan et al.
1,3(2H)-d ion e (tr a n s-9d ). The title compound was prepared
from tetrahydro-â-carboline trans-5e and [2-(1-pyrrolidinyl)-
ethyl]amine. The crude residue was purified by flash column
chromatography eluting with CH2Cl2/MeOH, 95/5, to give
trans-9d as a white solid (340 mg, 64.4%) after crystallization
in olive oil for p.o. administration and in a mixture of DMF/
tetraglycol/glucose (30/25/45%) for i.v. administration. 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: Nath-
alie Faudon, Sandrine Martin, Didier Chevret, Florence
Loriolle, Vale´rie Baudet, Florence Blandel, Anne-Bene-
dicte Boullay and Vale´rie Boullay.
1
from EtOH/H2O, mp 126-130 °C; H NMR (CDCl3) δ 9.91 (s,
1H), 7.43-7.09 (m, 6H), 6.86 (d, 2H, J ) 8.7 Hz), 6.51 (s, 1H),
4.12 (dd, 1H, J ) 10.9, 6 Hz), 3.81 (s, 3H), 3.74 (t, 2H, J ) 5.8
Hz), 3.12-2.88 (m, 2H), 2.79-2.54 (m, 5H), 1.60-1.29 (m, 5H);
Anal. (C26H28N4O3) C, H, N.
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.17 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).
Refer en ces
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P h osp h od iester a se a ssa ys. The PDE assay17,18 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]-cAMP (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).
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3-Carboxylate and Diazepam on Cerebellar Cyclic GMP Content
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Deter m in a tion of cGMP Accu m u la tion in RSMC. Rat
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to Chamley.19 Cells were cultured in Dubelcco’s modified Eagle
medium (GIBCO) containing 10% fetal calf serum, 1%
glutamine and 1% penicilin-streptomycin at 37 °C in a 95%
air-5% CO2 humidified atmosphere.
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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, 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 nucleotides
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 (Amer-
sham). 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).
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Tetrahydro-â-Carbolines by Carbon-13 Spectroscopy. J . Am.
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Effect on Blood P r essu r e of Con sciou s SHR. 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 anesthesia. 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 compound was dissolved in a mixture of 5% (v/v) DMF
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J M030056E