Synthesis and biological activities of novel β-carbolines as PDE5 inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)01036-3

A series of N²-furoyl and N²pyrimidinyl β-carbolines was discovered to possess potent inhibitory activity against PDE5. During the synthesis we developed a tandem resin quenching protocol, which allowed us to synthesize large number

Full text of DOI:10.1016/S0960-894X(02)01036-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 761–765
Synthesis and Biological Activities of Novel ꢀ-Carbolines
as PDE5 Inhibitors
Zhihua Sui,* Jihua Guan, Mark J. Macielag, Weiqin Jiang, Yuhong Qiu,
Patricia Kraft, Sheela Bhattacharjee, T. MatthewJohn, Elizabeth Craig,
Donna Haynes-Johnson and Joanna Clancy
Johnson & Johnson Pharmaceutical Research & Development L.L.C., 1000 Route 202, Raritan, NJ 08869, USA
Received 17 May 2002; accepted 3 October 2002
Abstract—A series of N2-furoyl and N2pyrimidinyl b-carbolines was discovered to possess potent inhibitory activity against PDE5.
During the synthesis we developed a tandem resin quenching protocol, which allowed us to synthesize large number of target
compounds in a rapid fashion. Representative compounds exhibit superior selectivity to sildenafil versus other isozymes of PDEs,
and demonstrated in vivo efficacy in increasing introcavernosal pressure in dogs.
# 2003 Elsevier Science Ltd. All rights reserved.
Cyclic nucleotides (cAMP and cGMP) are important
secondary messengers which control many physiological
processes. The levels of intracellular cyclic nucleotides
are determined by the activities of cyclases that synthe-
size them and phosphodiesterases (PDEs) that degrade
them. PDEs play critical roles in modulating the levels
of cyclic nucleotide, their duration of action and ulti-
mately the physiological outcome. To date, the 21
mammalian PDE genes which have been cloned are
classified into 11 families according to sequence homol-
ogy and biochemical properties.1À3 These families are:
PDE1, Ca2+/calmodulin dependent; PDE2, cGMP-sti-
mulated; PDE3, cGMP-inhibited; PDE4, cAMP-specific
and rolipram-sensitive; PDE5, cGMP-specific; PDE6,
photoreceptor cGMP-specific; PDE7, cAMP-specific
and rolipram-insensitive; PDE8, cAMP-specific and
IBMX-insensitive; PDE9, cGMP-specific; PDE10 and
PDE11, hydrolyzing both cAMP and cGMP. Among
the 11 families, PDE5, initially discovered in lung tis-
sues, was the first recognized cGMP specific subclass.
Subsequent studies revealed that it is the major isozyme
of PDEs in Corpus cavernosum in penis and plays an
important role in penile erection.4,5 Many potent PDE5
inhibitors have been reported in the literature as vaso-
dialators.6 The introduction of sildenafil,7 a PDE5
inhibitor, as an agent for the treatment of male erectile
dysfunction (MED) in 1998 has raised the public
awareness of MED, and generated significant interest in
the discovery of more selective PDE5 inhibitors. The
results of this increased interest have begun to emerge in
the literature.8À12 As part of our on-going efforts to
identify novel PDE5 inhibitors that have improved
selectivity versus PDE1 and PDE6,13À15 we became
interested in compounds structurally distinct from sil-
denafil. Among the many structures reported in the lit-
erature, b-carbolines such as structure A drewour
attention.16 b-Carbolines are readily accessible and well
suited for parallel synthesis. To avoid the potential
metabolic liability of the olefins in A, we decided to
replace the alkene or the acrylate moieties in structure A
with a series of heterocycles as indicated in structures B
and C. We envisioned that the nitrogen atom in the
pyrimidine and pyridine of C would mimic the amide
moiety in structure A.
The b-carboline precursors
through Pictet–Spengler reactions of tryptamine 1 and
3
were synthesized
*Corresponding author. Tel.:+1-908-704-5778; fax:+1-908-526-6469;
e-mail: zsui@prius.jnj.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01036-3

762
Z. Sui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 761–765
benzaldehydes 2 in accord with the known procedures
as shown in Scheme 1. The acyl analogues 5 were syn-
thesized through N-acylation of 3 with appropriate acid
chlorides 4 either by conventional solution reactions or
by a tandem resin quenching protocol in a parallel fash-
ion with dichloromethane as solvent. In the parallel
synthesis, piperidinomethyl polystyrene resin was used as
an acid scavenger and excess acid chloride was used dur-
ing the acylation. The reaction mixture was then treated
with a primary amine resin, tris-(2-aminoethyl)-amine
polystyrene to react with any excess acid chloride. Simple
filtration removed most of the impurities, and recrystalli-
zation where necessary gave products in high purity. This
allowed us to synthesize target compounds of high purity
in a rapid fashion without chromatographic purification.
corresponding boronic acid or stannane under standard
Suzuki or Stille conditions (Scheme 4). All compounds
were characterized by conventional analytical methods
such as MS, NMR and elemental analysis.
We first studied the acyl analogues where we utilized the
cluster analysis concept and knowledge of carboline
PDE5 inhibitor SAR to select the following 10 different
substituents for the initial set of R1: 4-NO2, 4-OCH3,
4-CN, 4-CH3, 3,4-Cl2, 3,4-(OCH3)2, 3,4-(CH3)2, 3,5-
(CH3)2, 4-Cl-3-CF3 and 3,4-OCH2O-. We then selected
a set of N-acyl groups with a wide variety of structures
resembling the acyl moiety described in structure B (Fig.
1). The initial selection of the acyl groups was primarily
based on the commercial availability.
The pyrimidine analogues were synthesized by reacting
2-chloropyrimdines 6 with 3 in the presence of Hunig’s
base in DMF at 120 ꢀC. When employing temperature-
sensitive substrates, the reaction temperature can be
lowered to 60 ꢀC by pre-heating the chloropyrimidine
with KF (Scheme 2). This modification was especially
useful in the synthesis of the pure enantiomers.
2-Chloropyrimidines were synthesized from substituted
malondialdehydes 8 by treatment with methylurea, fol-
lowed by reaction with chlorinating reagents such as
phosphorous pentachloride and phosphorus oxychloride
as shown in Scheme 3. When R2 substituent is basic (e.g.,
pyridine and imidazole) or contains a basic functional
group, the chlorination reaction is sluggish due to the
precipitation of the hydrochloride salt. In these cases, the
target compounds were synthesized by palladium-medi-
ated couplings of a bromopyrimidinyl precursor with a
A large number of analogues were rapidly synthesized
by a combination of conventional methods and the par-
allel protocol previously described. A quick survey of the
SAR of these analogues in a PDE5 inhibition study
revealed that a wide range of acyl groups are tolerated.
Interestingly, among the R1 selected here, only com-
pounds bearing a 3,4-methylenedioxy were active against
PDE5 in the inhibition assays. We decided to conduct
more in-depth SAR studies on structures with methyle-
nedioxy as R1 and a1, the 5-arylfuroyl, as the acyl moiety.
As illustrated in Figure 2, replacing NH in the indole
moiety with S abolished the activity (12b vs 13), indicating
that the indole proton is essential to the biological activity.
Eliminating the carbonyl group decreased the activity (12a
vs 14), indicating the importance of the amide structure.
We then decided to choose N-phenylfuroyl b-carboline
as pharmacophore for further studies. Table 1 sum-
marizes the SAR study on the phenyl ring in the phe-
nylfuroyl moiety of structure 12 where the heterocycle is
a 2,5-disubstituted furan.
Biological studies showed that both electron-donating
and electron-withdrawing groups are tolerated for
PDE5 inhibition. The positions of the substituents do
not significantly influence the potency. We took advan-
tage of this observation to introduce polar functional
groups to improve the solubility of this series. Indeed,
Scheme 1.
Scheme 4.
Scheme 2.
Figure 1. Representative N-acyl groups.
Scheme 3.

Z. Sui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 761–765
763
Figure 3. Examples of R1 contributions.
Table 2 summarizes the SAR of phenylpyrimidinyl moi-
ety. Even though both electron-donating groups such as
methoxy and hydroxy and electron-withdrawing groups
such as nitro are tolerated, lipophilic groups such as
methyl seem to be disfavored. In addition, an attempt to
increase the aqueous solubility through the introduction
of a basic appendage (15g) was not successful.
Figure 2. Pharmacophore identification.
Table 1. PDE5 Inhibition by phenyl-furoyl b-carbolines17
Compd
R2
Ki, nM (SD)a
12a
12b
12c
12d
12e
3-CF3
3-NO2
4-NO2
2-NO2
H
3-NH2
126 (Æ73)
130 (Æ30)
77.5 (Æ16)
219 (Æ30)
ꢁ750
In an effort to improve the aqueous solubility without
increasing the size of the molecules, we synthesized
pyridine and imidazole analogues such as 17 and 18. To
our delight, these modifications not only increased the
solubility to facilitate the in vivo studies, but also
increased the potency. As shown in Figure 4, replacing
phenyl with dimethylimidazole significantly increased
the potency. Replacing both the phenyl and pyrimidinyl
with pyridinyl also significantly increased the potency
against PDE5. The potency of 18 is comparable to that
of sildenafil.
12f
179 (Æ52)
123 (Æ18)
145 (Æ34)
64.2 (Æ8.6)
50.6 (Æ22)
ꢁ95
12g
12h
12i
12j
12k
12l
4-NH2
4-Cl
4-MeCONH–
3-MeCONH–
3-HOOC(CH2)3CONH–
4-HOOC(CH2)3CONH–
ꢁ100
Sildenafil
1.98 (Æ0.3)
aValues are means of three experiments except 12e, 12k, and 12k where
IC50 was determined instead of Ki.
analogues with acidic functional groups such as 12k and
12l showed potency comparable to the aniline pre-
cursors (12f and 12g). Furthermore, replacing methyle-
nedioxyphenyl with dihydrobenzofuran at R1 position
does not significantly decrease the potency against
PDE5. When we introduced basic functional groups in
R2 region of the molecule to improve solubility, we
identified more potent analogues such as 12q and 12r.
During SAR studies, we discovered that alkyl substitu-
tion on the indole nitrogen with alkyl groups abolished
the activity (not shown). On the other hand, introduction
Table 2. PDE5 inhibition by phenylpyrimidyl-b-carbolines
Compd
Structure
R2
Ki, nM (SD)a
8a
8b
8c
8d
8e
8f
8g
I
I
I
I
I
II
II
II
4-OMe
3,4-(OMe)2
2-NO2, 4-SO2Me
4-Me
4-Cl
4-OMe
4-O(CH2)2pyrrolidine
4-OH
246 (Æ41)
62.8 (Æ8.2)
132 (Æ18)
543 (Æ10)
126 (Æ38)
189 (Æ94)
>1000
In the pyrimidine series, similar SAR was observed in
the R1 region. Among many substituents tested, only
bicyclic moieties such as 3,4-methylenedioxy showed
activity in the PDE5 inhibition assay. Neither mono-
substituted nor simple disubstituted phenyl groups,
including electron donating, electron withdrawing,
lipopholic and functional groups with hydrogen bond-
ing abilities, showed any inhibition. For example, when
R1 is dimethoxy (16) in place of methylenedioxy (15a)
the activity is abolished. Interestingly, 15f showed
potency similar to 15a, indicating that bicyclic structure
is required in this region (Fig. 3).
8h
Sildenafil
154 (Æ17)
1.87
aValues are means of three experiments.
Figure 4. More soluble analogues.

764
Z. Sui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 761–765
of a carbonyl group at 4-position of the b-carboline sig-
nificantly improved the potency against PDE5 (Fig. 5).
Compound 19 has a Ki comparable to that of sildenafil
versus PDE5. The carbonyl group can serve as a hydro-
gen-bonding acceptor as well as increase the acidity of
the indole nitrogen. If the carbonyl group contribution
to the potency increase is due to hydrogen-bonding, one
would expect that the hydroxy analogue 20 should pos-
sess at least comparable potency to the parent compound
15b since hydroxy group can serve as both a hydrogen-
bond acceptor and donor. The fact that 20 (tested as mix-
ture of diastereomers) did not showpotent activity con-
firms our earlier observation that the ability of the indole
proton as hydrogen-bonding donor may play a critical
role. It is unclear to us at this point why the unsubstituted
analogue 15b is more potent than the hydroxy analogue 20.
Figure 6. Influence of the chiral center.
To determine the selectivity of the potent analogues, the
inhibition of a panel of PDEs by our compounds was
studied in comparison with sildenafil (Table 3). Com-
pound 12r has a comparable potency to sildenafil but
much better selectivity versus other PDE isozymes,
especially versus PDE1, which might contribute to the
cardiovascular side effects of sidenafil, and PDE6 which
is responsible for the visual disturbance experienced by
patients using sildenafil. Compound 17 also demon-
strated better selectivity than sildenafil versus PDE1-6.
To determine the influence of the chiral center on the
biological activity, compound 12m was separated with
chiral HPLC into the two enantiomers, 12n (R) and 12p
(S). The stereochemistry was proved independently by
N-acylation of the 2-position-unsubstituted enantiopure
b-carboline. As shown in Figure 6, only the R-enantio-
mer is active while the S-enantiomer is inactive. This
confirmed the importance of the chiral center to PDE5
inhibition. In line with this observation, in the pyr-
imidine series, the R-enantiomer of 15b is also more
potent than the racemate.
Figure 7. Efficacy in increasing intracavernosal pressure in dogs by the
iv route.18
During the in vivo studies in dogs (Fig. 7), a compound
21 (Ki=18 nM, 4-methoxy in place of 3,4-dimethoxy in
19) showed efficacy with an EC50 of 173 mg/mL by the
intravenous route. Sildenafil has an EC50 of 5.1 mg/mL.
The efficacy difference reflects in part the potency dif-
ference of the two compounds. More potent analogues
such as the R-enantiomer of 18 may demonstrate better
efficacy in this model.
In summary, we have identified a series of novel b-car-
boline PDE5 inhibitors by parallel synthesis utilizing a
tandem resin quenching protocol and intuitive design.
Representative compounds demonstrated better selec-
tivity versus other isozymes of PDEs (PDE1–6). Sys-
tematic SAR studies identified the essential structural
features for PDE5 inhibition. This information was
crucial for the identification of more potent PDE5
inhibitors. Representative compounds showed in vivo
efficacy in dogs by the intravenous route.
Figure 5. Importance of the indole proton.
Table 3. Selectivity studies versus other isozymes of PDEsa
Compd
PDE5, Ki (nM)
PDE1/PDE5
PDE2/PDE5
PDE3/PDE5
PDE4/PDE5
PDE6/PDE5
12q
12r
17
19
32.3
8.11
24
8.86
1.87
>3000
2963
>4000
674
308
>10,000
>4000
23,000
4000
>3000
>10,000
>4000
>24,000
7000
180
>10,000
>4000
161
32
119
187
4.0
Sildenafil
120
3,000
4.0
aValues are means of three experiments; Source of enzymes are from the following human tissues: PDE1, heart; PDE2, corpus cavernosum; PDE3,
platelets; PDE4, skeleton muscle; PDE5, corpus cavernosum; PDE6, retina cone (PDE6 from retina rod showed slightly higher ratio vs PDE5).

Z. Sui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 761–765
765
References and Notes
according to the protocol described by: Boolell, M.; Allen, M.
J.; Ballard, S. A.; Geo-Attee, S.; Muirhead, G. J.; Naylor, A.
M.; Osterloh, I. H.; Gingell, C. Intl. J. Impotence Res. 1996, 8,
47, with minor modifications. The PDE5 activity was deter-
mined in a two-step assay described by Thompson and
Appleman in Biochemistry 1971, 10, 311, with adaptation. The
Ki values for the compounds were determined as follows.
Stock solution of the compounds were prepared in 100%
DMSO, diluted in 100% DMSO to the appropriate con-
centrations, and added to the assay buffer to give a final con-
centration of 2% DMSO. The amount of enzyme used in each
reaction was such that the hydrolysis of substrates did not
exceeded 15% so that the amount of product increased line-
arly with time. A 30 nM substrate concentration ([S]ꢂKm)
was used such that IC50 values approximate the Ki values.
18. This study was adapted from Carter, A. J.; Ballard, S. A.;
Naylor, A. M. J. Urol. 1998, 160, 212, with modifications.
Male beagles were anesthetized and the anesthesia was main-
tained throughout the course of the experiment by pento-
barbital infusion. Arterial blood pressure was monitored. The
pelvic nerve was exposed and hooked to a bipolar electrode.
Penis was denuded and a 19-gauge needle attached to a pres-
sure transducer via a catheter was inserted into the corpus
cavernosum to record intracavernosal pressure (ICP). Control
ICP responses were generated by electrical stimulation to the
pelvic nerve at appropriate current output and frequency set-
ting that increased the ICP to about 30% of systolic pressure.
After the control responses were established, ascending con-
centrations of test compound were given intravenously at
40-min intervals. The effect on ICP was evaluated at 15 min
after each dosing by electrical stimulation to the pelvic nerve
at the same setting. To determine the effect of the com-
pound, the area under the curve (AUC) for ICP at each
stimulation was computed and the control response AUC
was subtracted. At the end of each experiment, 300 mg/kg
sildenafil was given intravenously. This dose was shown to
induce a maximal response in our hands and thus designated
as 100%.
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