Tetrahydropyrroloquinolinone type dual inhibitors of aromatase/aldosterone synthase as a novel strategy for breast cancer patients with elevated cardiovascular risks

Article abstract of DOI:10.1021/jm301408t

The application of aromatase inhibitors to postmenopausal breast cancer patients increases the risk of cardiovascular diseases (CVD), which is believed to be caused by the abnormally high concentrations of aldosterone as a consequence of the estrogen deficiency. Dual inhibitors of aromatase (CYP19) and aldosterone synthase (CYP11B2) are therefore proposed as a novel strategy for the adjuvant therapy to reduce the CVD risk for these patients. By combining decisive structural features of CYP11B2 and CYP19 inhibitors into a common template, a series of pyridinylmethyl substituted 1,2,5,6-tetrahydro-pyrrolo[3, 2,1-ij]quinolin-4-ones were designed and synthesized. Interestingly, the substituents on the methylene bridge showed strong influences on the inhibitory activities leading to opposite effects, that is, a given substituent showed an increase in inhibition of one enzyme, while it led to a decrease for the other enzyme. The compromise of this conflict led to compounds 3j, 3k, 3n, and 3p as potent and selective dual inhibitors of CYP19 and CYP11B2, especially compound 3p, which exhibited IC₅₀ values of 32 and 41 nM for CYP19 and CYP11B2, respectively, and a high selectivity toward CYP17 and CYP11B1. This compound is considered as a candidate for further evaluation in vivo.

Full text of DOI:10.1021/jm301408t

Article
pubs.acs.org/jmc
Tetrahydropyrroloquinolinone Type Dual Inhibitors
of Aromatase/Aldosterone Synthase as a Novel Strategy
for Breast Cancer Patients with Elevated Cardiovascular Risks
Lina Yin,†,‡ Qingzhong Hu,† and Rolf W. Hartmann*,†
†Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Campus C2-3, D-66123 Saarbrucken, Germany
̈
‡ElexoPharm GmbH, Campus A1, D-66123 Saarbrucken, Germany
̈
S
* Supporting Information
ABSTRACT: The application of aromatase inhibitors to postmenopausal breast cancer patients increases
the risk of cardiovascular diseases (CVD), which is believed to be caused by the abnormally high concen-
trations of aldosterone as a consequence of the estrogen deficiency. Dual inhibitors of aromatase (CYP19)
and aldosterone synthase (CYP11B2) are therefore proposed as a novel strategy for the adjuvant therapy to
reduce the CVD risk for these patients. By combining decisive structural features of CYP11B2 and CYP19
inhibitors into a common template, a series of pyridinylmethyl substituted 1,2,5,6-tetrahydro-pyrrolo
[3,2,1-ij]quinolin-4-ones were designed and synthesized. Interestingly, the substituents on the methylene
bridge showed strong influences on the inhibitory activities leading to opposite effects, that is, a given substituent showed an increase
in inhibition of one enzyme, while it led to a decrease for the other enzyme. The compromise of this conflict led to compounds 3j,
3k, 3n, and 3p as potent and selective dual inhibitors of CYP19 and CYP11B2, especially compound 3p, which exhibited IC50 values
of 32 and 41 nM for CYP19 and CYP11B2, respectively, and a high selectivity toward CYP17 and CYP11B1. This compound is
considered as a candidate for further evaluation in vivo.
of the renin−angiotensin−aldosterone system (RAAS) such as
renin, angiotensin II, angiotensin converting enzyme, and angio-
tensin type 1 receptor, which further elevate the aldosterone
biosynthesis.9 The abnormally high concentrations of aldoste-
rone cause retention of sodium and water, leading to an increase
of blood volume and the subsequent elevation of blood
pressure.10 It also promotes the influx of calcium into vascular
smooth muscle cells11 and the expression of adrenomedullin and
regulator of G protein signaling-2,12 leading to vasoconstriction,
which together with the increase of blood volume results in
chronic hypertension. Moreover, excessive aldosterone acts as a
pro-inflammation factor13 and induces the production of reactive
oxygen species (ROS).14 Inflammation and ROS formation lead
to cardiac myocyte necrosis, collagen synthesis, and fibroblast
proliferation, thus resulting in cardiac and vascular fibrosis and an
increase in myocardial stiffness.15 Subsequently, cardiac hyper-
trophy and ventricular remodelling occur as further structural
deterioration with functional degradation proceeds.16 The ventric-
ular remodeling causes diastolic dysfunction, diminishes con-
tractile capability, reduces the stroke volume, and ultimately results
in heart failure, often leading to sudden death.
INTRODUCTION
■
Estrogens not only act physiologically as important sexual hor-
mones to promote the development of female secondary sexual
characteristics and to maintain the functions of the reproductive
system but also exhibit dichotomous impacts on human health
under some pathological circumstances. On the one hand,
estrogens stimulate the proliferation of breast cancer (BC) cells
that are estrogen receptor (ER) or progesterone receptor (PgR)
positive.1 Estrogen deprivation is therefore a rational treatment
for BC. Two approaches have been implemented in clinic: selec-
tive estrogen receptor modulators2 and aromatase (CYP19)
inhibitors, such as letrozole and vorozole (Chart 1). Since CYP19
inhibitors show fewer side effects especially in long-term applica-
tion, they are first choice nowadays as adjuvant therapeutics for
postmenopausal breast cancer patients. On the other hand, estrogen
deficiency is correlated with cardiovascular disease (CVD), which
has been demonstrated by the fact that the incidence of CVD in
postmenopausal women is triple that of premenopausal women at
the same age.3 Estrogens have been proven to exhibit protective
effects on heart4 and kidney.5 The administration of estrogen pre-
vents heart failure after myocardial infarction6 and attenuates ventric-
ular hypertrophy and remodeling.7 As for BC patients, menopause
and the application of CYP19 inhibitors decrease plasma estro-
gen concentrations to undetectable levels,8 which leads to an even
higher risk of CVD.
Aldosterone synthase (CYP11B2) is the crucial enzyme catalyz-
ing the conversion of 11-deoxycorticosterone to aldosterone. Its
inhibition, leading to a reduction of aldosterone levels, would be
beneficial for postmenopausal BC patients under CYP19 inhibitor
treatment. Instead of a combination therapy with inhibitors of
It is believed that the high CVD risk resulting from estrogen
depletion is caused to a great extent by an increase of the aldo-
sterone levels. The estrogen deficiency not only directly pro-
motes aldosterone secretion but also upregulates other components
Received: September 27, 2012
Published: January 3, 2013
© 2013 American Chemical Society
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Chart 1. Design Concept of Dual CYP19/CYP11B2 Inhibitors
CYP11B2 and CYP19, dual inhibitors of both enzymes would be
advantageous for a better compliance and for decreasing side
effects. Besides, application of this kind of dual-target-directed
agent17 could also avoid drug−drug interactions often observed
after the administration of two drugs in combination.
Accordingly, we would like to develop dual inhibitors of both
enzymes as an innovative approach for the treatment of BC.
Important structural features of known CYP11B2 and CYP19
inhibitors were combined into a common template and a series of
pyridinylmethyl-substituted 1,2,5,6-tetrahydro-pyrrolo[3,2,1-ij]-
quinolin-4-ones, 3a−3q, 4a, 4b, 5a, 5b, 6a, 6b, 7a, and 7b, were
synthesized. The inhibition of CYP11B2 and CYP19 was
determined in comparison with fadrozole (Chart 1), which is a
potent, but unselective CYP19 inhibitor showing also inhibition
of 11β-hydroxylase (CYP11B1) and CYP11B2. For safety reasons,
the selectivity of the new compounds against CYP11B1 and
17α-hydroxylase-17,20-lyase (CYP17), which are the crucial enzymes
in the biosyntheses of glucocorticoids and androgens, respectively,
were also determined.
triazole providing the sp2 hybrid N, a methylene linker between
the N-containing heterocycle and hydrophobic core, and two
hydrophobic aryls. Both compounds showed potent inhibition of
CYP19 and no inhibition of CYP11B2. In contrast, fadrozole
(Chart 1), in which one aryl was replaced by alkyl fused onto the
imidazole, was not selective toward CYP11B1 and CYP11B2
(IC50 of 6.3 and 0.8 nM, respectively). Hence, the scaffold of
arylmethyl-substituted N-containing heterocycle was considered
to be crucial for a strong CYP19 inhibition, whereas the sub-
stituents on the methylene bridge could be important for
the inhibition of CYP11B enzymes. Based on this scaffold,
introduction of structural features from CYP11B2 inhibitors
as aryl and N-containing heterocycle as well as suitable substit-
uents onto the methylene bridge could acquire the desired inhibi-
tion of CYP11B2 and selectivity over CYP11B1. To achieve
this selectivity is challenging because the homology between
CYP11B2 and CYP11B1 is as high as 93%. Our efforts to develop
potent and selective CYP11B221 and CYP11B120 inhibitors had
successfully led to the reference compounds ref 121h and ref 220d
(IC50(CYP11B2) of 1.1 and 24 nM, respectively) with 1,2,5,6-
tetrahydro-pyrrolo[3,2,1-ij]quinolin-4-one as hydrophobic core.
This core was considered to be important for a strong CYP11B2
inhibition and thus introduced into the scaffold to replace the
original aryl. Moreover, reference compound ref 2 was actually
an even stronger inhibitor of CYP11B1 (IC50(CYP11B1) =
2.2 nM), which is rendered by the imidazole moiety and cyclo-
propyl on the methylene bridge. According to our experience
that pyridyl can cast different selectivity profiles from imdazole
among CYP enzymes,19d pyridyl adopted from reference com-
pounds ref 1 was exploited as N-containing heterocycle to
improve selectivity over CYP11B1. Since groups on the methy-
lene bridge also influenced the inhibitory profiles significantly,
various substituents were furnished, leading to a series of pyridinyl-
methyl-substituted 1,2,5,6-tetrahydro-pyrrolo[3,2,1-ij]quinolin-4-
ones (Chart 1). When choosing substituents, three features were
taken into consideration, bulkiness and electrostastic and H-bond
forming properties.
DESIGN CONCEPT FOR DUAL INHIBITORS
■
All cytochrome P450s are cysteinato-heme enzymes, and the iron
in the protoporphyrine acts as the reactive center to activate
oxygen, which is required for the oxidation of the substrates.
Therefore, competitive inhibition via coordination of this Fe by
sp2 hybrid nitrogens is a common mechanism for CYP enzyme
inhibitors. First identified for CYP19 inhibitors,18a it was
subsequently applied for inhibitors of CYP17,19 CYP11B1,20
and CYP11B2.21 Despite a poor sequence identity of less than
20% across the whole CYP superfamily, CYP proteins share
similar folding configurations and conserve substrate binding
pockets. This makes it difficult to achieve selectivity but could be
advantageous to develop dual inhibitors. In this study, a simple
design strategy was applied: the combination of important struc-
tural features of selective CYP19 and CYP11B2 inhibitors into
one molecule. Plenty of CYP19 inhibitors18 have been designed
and synthesized with letrozole and vorozole (Chart 1) as the
most prominent representatives. It is obvious that three common
structural features can be found in these two compounds: a
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a
Scheme 1
a
Reagents and conditions: (i) Nicotinoyl or isonicotinoyl chloride hydrochloride, AlCl3, 140 °C; (ii) method A, RMgX, THF, −78 °C to room
temp; (iii) method B, Et3SiH, TFA, CF3SO3H, CH2Cl2, 0 °C to room temp; (iv) CH3PPh3Br, n-BuLi, THF, −78 °C to room temp; (v) Pd/C, H2,
MeOH, room temp; (vi) NaBH4, MeOH, 0 °C.
Table 1. Inhibition of CYP19, CYP11B2, CYP11B1, and CYP17 by 3-Pyridyl Compounds 4a−7a and 3a−3e
a
a
b
g
IC50, nM
IC50, nM
%Inhib
c
d
e
f
compd
4a
R
CYP19
CYP11B2
CYP11B1
SF
CYP17
OH
H
>5000
3340
>5000
74
750
16
<0.1
0.2
i
<3
<3
<3
i
5a
6a
CH2
>5000
>5000
2130
468
41
0.1
7a
Me
37
4
0.1
3a
Ph
132
68
0.5
3b
2-MeOPh
3-MePh
3-ClPh
4-FPh
3660
>5000
>5000
>5000
587
2270
1220
36
<0.3
<0.2
<0.1
0.1
i
3c
>5000
1350
i
3d
i
3e
140
78
i
h
ref 1
ref 2
fadrozole
letrozole
18%
1.1
715
2.2
650
6
228
41
24
0.1
7.9
1.8
41
<3
7
0.8
6.3
36
1420
2620
a
b
Mean value of at least three experiments, relative standard deviation less than 25%. Mean value of at least two experiments, standard deviation less
c
than 10%; inhibitor concentration, 500 nM. Human placental CYP19; substrate androstenedione, 500 nM; inhibitor concentration, 500 nM.
d
e
Hamster fibroblasts expressing human CYP11B2; substrate deoxycorticosterone, 100 nM. Hamster fibroblasts expressing human CYP11B1;
f
g
substrate deoxycorticosterone, 100 nM. SF = IC50(CYP11B1)/IC50(CYP11B2). Escherichia coli expressing human CYP17; substrate progesterone,
25 μM; inhibitor concentration, 2.0 μM; ketoconazole, IC50 = 3.5 μM. Inhibitor concentration 500 nM. Not determined.
h
i
RESULTS AND DISCUSSION
Chemistry. The preparation of compounds 3a−3q, 4a, 4b,
5a, 5b, 6a, 6b, 7a, and 7b is illustrated in Scheme 1. The 1,2,5,
6-tetrahydro-pyrrolo[3,2,1-ij]quinolin-4-one core synthesized
■
according to previous reports20d,21h was converted into the
pyridinyl ketones 1a (3-py) and 1b (4-py) via Friedel−Crafts
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Table 2. Inhibition of CYP19, CYP11B2, CYP11B1, and CYP17 by 4-Pyridyl Compounds 2h, 4b−7b, and 3f−3q
a
a
b
IC50, nM
IC50, nM
%Inhib
c
d
e
f
g
compd
4b
R
CYP19
CYP11B2
CYP11B1
SF
CYP17
OH
H
>5000
2130
565
1500
889
74
563
204
96
347
224
0.6
1.1
<3
8
5b
6b
CH2
1230
140
13
17
i
7b
Me
74
1.9
1.9
3f
i-Pr
750
1290
85
1420
>5000
745
i
3g
c-Hex
>4
8.8
14
2.2
18
i
2h
Ph
105
880
81
43
i
3h
Ph, OH
2-MeOPh
3-MeOPh
4-MeOPh
4-MeOPh
4-MeOPh
3-FPh
390
128
139
52
5399
276
3i
33
50
53
i
3j
59
2539
810
3k
124
273
92
16
37
(+)-3k
(−)-3k
3l
46
1688
511
74
6.9
i
74
88
532
6.0
1.6
i
3m
4-FPh
116
19
56
88
55
58
60
46
i
3n
3-ClPh
4-ClPh
3-CH3Ph
3,5-diCF3Ph
51
646
13
2.9
33
3o
55
32
92
3p
32
41
1336
>5000
715
3q
246
1580
1.1
24
>3
h
ref 1
ref 2
fadrozole
letrozole
18%
650
6
228
41
2.2
0.1
41
<3
7
0.8
1420
6.3
7.9
1.8
36
2620
a
b
Mean value of at least three experiments, relative standard deviation less than 25%. Mean value of at least two experiments, standard deviation less
c
than 10%; inhibitor concentration, 500 nM. Human placental CYP19; substrate androstenedione, 500 nM; inhibitor concentration, 500 nM.
d
e
Hamster fibroblasts expressing human CYP11B2; substrate deoxycorticosterone, 100 nM. Hamster fibroblasts expressing human CYP11B1;
f
g
substrate deoxycorticosterone, 100 nM. SF = IC50(CYP11B1)/IC50(CYP11B2). E. coli expressing human CYP17; substrate progesterone, 25 μM;
inhibitor concentration, 2.0 μM; ketoconazole, IC50 = 3.5 μM. Inhibitor concentration 500 nM. Not determined.
h
i
acylation with the corresponding nicotinoyl or isonicotinoyl
chlorides in the solid phase reactions. The ketones were trans-
formed into alcohols 2a−2q with various Grignard reagents.
After the removal of OH groups using triethylsilane under acidic
conditions, compounds 3a−3q with various substituents at the
methylene bridge were obtained. Reduction of the ketones by
sodium borohydride followed by triethylsilane treatment led to
nonsubstituted analogues 5a and 5b. The methylidene group was
introduced onto the methylene bridge via Wittig reaction leading
to compounds 6a and 6b, which were subsequently saturated by
hydrogenation to yield 7a and 7b.
Biology. Inhibition of Human CYP19, CYP11B2, and CYP11B1.
The synthesized compounds were investigated for their inhibi-
tory activities against CYP19, using microsomal placenta pre-
parations,22a as well as against CYP11B1 and CYP11B2, using
V79 MZh cells expressing the corresponding enzymes.22b,c IC50
values are presented in comparison to reference compounds
fadrozole and letrozol.
with IC50 values of 74 and 37 nM, respectively. Introduction of an
unsaturated methylidene group (6a) decreased the inhibitory
activity to 468 nM, probably due to the reduction of confor-
mational flexibility. The loss of potency rendered by OH (4a)
leads to the assumption that hydrophilicity is not tolerated in this
area. Moreover, an interesting SAR was observed for compounds
3a−3e. When substituted with a phenyl (3a), the compound was
potent (IC50 = 132 nM). However, further introduction of methoxyl
(3b), methyl (3c), and chloro (3d) substituents into the phenyl led
to a total loss of inhibitory potency (IC50 > 5000 nM). Because the
reduction of activity was less pronounced with the small fluorine
substituent (3e, IC50 = 587 nM), it can be assumed that these effects
are caused by steric restrictions. Interestingly, all the 3-pyridyl
analogues showed preference for CYP11B1 with selectivity factors
(SF = IC50(CYP11B1)/IC50(CYP11B2)) less than 0.1.
As for 4-pyridyl analogues, much more potent inhibitors of
CYP19 and CYP11B2 were obtained (Table 2). When alkyl
groups were introduced onto the methylene bridge, completely
oppositional SARs were observed for the inhibition of CYP19
and CYP11B2. As the bulkiness of the substituents increased
from H (5b) to methyl (7b), isopropyl (3f), and cyclohexyl (3g),
the inhibitory potency toward CYP19 increased from 2130 to
1500, 889, and 74 nM, while a reduction of CYP11B2 inhibition
from 204 (5b, H) to 1290 nM (3g, cyclohexyl) was observed.
Although potent inhibitors for CYP19 or CYP11B2 were identified,
The 3-pyridyl compounds 4a, 5a, 6a, 7a, and 3a−3d showed
weak to no inhibition of CYP19 regardless of the substituents at
the methylene bridge (Table 1). An exception was compound 3e
with 4-F Ph at the methylene bridge exhibiting potent inhibition
with an IC50 value of 140 nM. In contrast, some compounds were
much more potent toward CYP11B2, namely, compounds with
no (5a) or methyl (7a) substituents on the methylene bridge,
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Figure 1. The binding of (A) R- and (B) S-enantiomers of 3k in CYP11B2 homology model.
respectively, no potent dual inhibitor could be identified. More-
over, these compounds exhibited no or little selectivity over
CYP11B1 with SFs ranging from 1 to 4 with the exception of the
methylidene compound 6b (SF = 13). This compound also
exhibited a strong inhibition of CYP11B2 (IC50 = 96 nM) and a
moderate inhibition of CYP19 (IC50 = 565 nM). Similarly, as
observed in the 3-pyridinyl class, OH on the methylene bridge
(4b) significantly decreased the inhibition for both enzymes.
In contrast, introduction of substituted phenyl moieties
resulted in strong inhibition of CYP19 with IC50 values between
19 and 124 nM. Interestingly, the phenyl group also rendered
potent CYP11B2 inhibition with IC50 values ranging from 32 to
139 nM. This is in contrast to the low inhibition of analogues
with bulky alkyl groups, such as isopropyl (3f, IC50 = 750 nM)
and cyclohexyl (3g, IC50 = 1290 nM). This finding indicates that
the hydrophobic pocket in CYP11B2 might be rather flat so that
the isopropyl or cyclohexyl group does not fit in well. Possible
π−π interactions between phenyl and some amino acid residues
in the pocket might also be responsible for this difference.
Opposing effects on the inhibition of CYP19 and CYP11B2 were
observed for the H-bond acceptors (OMe and F) on the phenyl
ring. Regarding CYP19, the 2- and 3-methoxy-substituted com-
pounds, 3i and 3j, showed increased inhibition compared with
the nonsubstituted 2h (IC50 = 105 nM) with IC50 values of 81
and 59 nM, respectively, whereas the 4-MeO analogue 3k exhib-
ited a slightly decreased inhibition (IC50 = 124 nM). In contrast,
compounds 3i and 3j showed a reduced inhibition of CYP11B2
(IC50 values around 130 nM) compared with 2h (IC50 = 85 nM),
while compound 3k was more potent (IC50 = 52 nM). Similar
observations were also found for the F analogues 3l and 3m.
Nonetheless, a chloro (3n and 3o) and methyl (3p) substitution
on the phenyl moiety increased the inhibitory potency toward
both enzymes regardless of the substitution positions, leading
to potent dual inhibitors (IC50 values below 50 nM for both
enzymes). Although 4-ClPh-substituted compound 3o exhibited
poor selectivity (SF = 2.9) similar to 4-FPh analogue 3m
(SF = 1.6) toward CYP11B1, compounds 3n (3-ClPh) and 3p
(3-CH3Ph) showed good selectivity with SFs of 13 and 33,
respectively. Especially compound 3p turned out to be superior
to the reference compounds fadrozle and letrozole considering
the inhibitory potency toward CYP19 and CYP11B2, as well as
the selectivity regarding CYP11B1. It is interesting to observe
that the compounds with the 3-substituted Ph moiety were more
selective than the corresponding 4-substituted Ph analogues
(for comparison, 3j and 3k, 3l and 3m, and 3n and 3o). Also,
3-substituted Ph compounds were more potent toward CYP19,
whereas 4-substituted Ph derivatives inhibited CYP11B2 in a
stronger way. Furthermore, the loss of CYP11B2 inhibition for
compound 3q, which was substituted with two trifluoromethyl
groups in the m-position, indicated that the binding pocket is
very narrow.
The potent (IC50(CYP19) = 124 nM and IC50(CYP11B2) =
52 nM) and selective (SF = 16) dual inhibitor 3k was resolved
into pure enantiomers to examine the influence of the chiral
center on biological activity. One enantiomer, (+)-3k, showed
similar CYP11B2 inhibition (IC50 = 46 vs 52 nM) and enhanced
selectivity (SF = 37 vs 16); however, the CYP19 inhibitory
activity was reduced (IC50 = 273 vs 124 nM). Contrarily, the
other enantiomer, (−)-3k, exhibited a little stronger inhibition of
CYP19 (IC50 = 92 vs 124 nM); but CYP11B2 inhibition (IC50 =
74 vs 52 nM) and selectivity (SF = 7 vs 16) were decreased.
Since the aim is to obtain potent and selective dual inhibition, the
racemate is considered to be advantageous. The modest potency
difference between enantiomers toward CYP19 and minor
difference toward CYP11B2 indicate that the binding sites of
both enzymes are relatively promiscuous, which is supported by
the docking results.
Selectivity over Human CYP17. The inhibition of CYP17 by
the synthesized compounds was evaluated due to its important
role in the biosynthesis of androgens. The percent inhibi-
tion values were obtained using the 90000g sediment of E. coli
coexpressing human CYP17 and cytochrome P450 reductase at
an inhibitor concentration of 2 μM.22d,e The potent dual inhibi-
tors 2h and 3i−3p showed weak inhibition (around 50% at
2 μM) toward CYP17, which is tolerable considering their strong
potency against CYP19 and CYP11B2 with IC50 values ranging
from 19 to 139 nM.
Docking Studies. For better understanding of the binding of
these dual inhibitors into their target enzymes, especially the
tolerance of both enantiomers, the R- and S-enantiomers of 3k
were docked into the CYP19 crystal (PDB ID 3EQM) and
CYP11B2 homology model.21b Since the active site in CYP19
was relatively capacious to accommodate both enantiomers, the
three aromatic moieties, that is, tetrahydropyrroloquinolinone,
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phenyl, and pyridyl occupied the same pockets with only minor
shift in position if comparing both enantiomers (Figure S1, see
Supporting Information). As for CYP11B2, when R-enantiomer
fit into the binding pocket, the pyridyl group coordinated to the
heme iron with its sp2 hybrid N in a nearly perpendicular manner
(Figure 1A). It also bolstered the other two aromatic rings which
twisted into an “L” shape and were spatially plumb to each other.
The tetrahydropyrroloquinolinone moiety leaned on I-helix and
oriented to the N-terminus of I-helix; while the phenyl group
directed to B−B′ loop. The S-enantiomer presented a similar
binding mode except that the tetrahydropyrroloquinolinone and
phenyl moieties exchanged their locations (Figure 1B). Since
these enantiomers were more potent toward CYP11B2 with less
difference in inhibitory potency, the binding of CYP11B2 was
further analyzed. Scrutinizing the binding pocket revealed that
two amino acid residues Tyr456 and Leu198 delimiting the
boundary obstructed the aromatic rings that leaned on I-helix
from orientating toward the C-terminus of I-helix (Figure 2C).
positions as the only major difference. Moreover, Trp231, the
ceiling of this region, and Asp288 can form hydrogen bonds with
the carbonyl of tetrahydropyrroloquinolinone or methoxyl on
the phenyl group (Figure 1). Furthermore, the aromatic rings
pointing to the B−B′ loop actually wedged in a narrow planar
cleft confined by Phe101, Leu102, and Phe458 (Figure 2A).
These two phenylalanines formed π−π interactions with the
aromatic rings in parallel or perpendicular manners, respectively
(Figure 1). The narrow planar feature of the pocket and the
possible π−π interactions are the reasons why the inhibitory
potency dove along with the increase of substituent bulkiness
when alkyl groups were furnished on the methylene bridge,
whereas phenyl rendered a much more potent inhibition although
it was bulky as well. This pocket was also rather shallow with the
bottom defined by amino acid residues on the B−B′ loop such as
Val88, Trp87, Pro86, Glu85, and Leu84 (Figure2B). Among them,
the backbones of Glu85 and Pro86 were accessible and therefore
could form hydrogen bonds with carbonyl, MeO, F, or OH on the
corresponding aromatic rings, especially the ones at para-position
(Figure 1). This could also be one of the factors responsible for
the 4-substituted phenyl analogues being more potent than 3- or
2-substituted ones toward CYP11B2.
CONCLUSION
■
Estrogen deficiency has been observed to be closely correlated
with CVD, which was mediated to a large extent by abnormally
high concentrations of aldosterone. The application of CYP19
inhibitors to postmenopausal BC patients reduces estrogens
to undetectable levels in plasma. However, this therapy also
increases the risk of CVD strongly. Accordingly, the rationale to
develop dual inhibitors of CYP19 and CYP11B2 for an improved
BC therapy is obvious. With such dual inhibitors as adjuvant
therapy, the overall survival and life quality of BC patients would
be further improved.
By combining the 1,2,5,6-tetrahydro-pyrrolo[3,2,1-ij]-
quinolin-4-one core from CYP11B2 inhibitors with structural
elements derived from CYP19 inhibitors, a series of potent dual
inhibitors were designed and synthesized. The substituents on
the methylene bridge showed significant influence on the inhib-
itory activity in the 4-pyridyl series. Opposite SARs were
commonly observed that modifications increasing the inhibition
of one enzyme always reduced that of the other. The compromise
of these conflicts finally led to compounds 3j, 3l, 3n, and 3p as
potent and selective dual inhibitors of CYP19 and CYP11B2.
These inhibition profiles together with reasonable selectivity
toward CYP17 and CYP11B1 make these compounds ideal
candidates for further evaluation in vivo and the proof of concept,
especially compound 3p, which exhibited IC50 values of 32 and
41 nM for CYP19 and CYP11B2, respectively, and a SF of 33
over CYP11B1.
Since three series of substituted methylene tetrahydropyrro-
loquinolinones, which differed predominantly in N-containing
heterocycles (i.e., 1-imidazoles,20d 3- and 4-pyridines), have been
designed, synthesized, and tested as CYP enzyme inhibitors, their
inhibition profiles among these enzymes were compared and
analyzed as shown in Table 3. It is apparent that all compounds
showed no inhibition toward CYP17, while for the other three
enzymes, situations were complicated but also interesting. When
the alkyl groups were substituted at the methylene bridge, 1-Im
and 3-Py compounds exhibited weak to no inhibition toward
CYP19, whereas very potent inhibition of CYP11Bs. Since the
inhibition of CYP11B1 is always stronger than that of CYP11B2,
selective CYP11B1 inhibitors were thus identified. As for 4-Py
Figure 2. The confines of the binding pocket in CYP11B2. (A) the
narrow planar cleft near B−B′ loop (view facing I-helix); (B) the bottom
of the cleft (side view); (C) obstruction in the C-terminal direction of
I-helix (view facing B−B′ loop).
This obstruction forced both enantiomers to adopt consenta-
neous binding modes with the interchange of two aromatic ring
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determined by TLC analysis on Alugram SIL G/UV254 (Macherey-
Nagel). Reagents and solvents were used as obtained from commercial
suppliers without further purification.
Table 3. Inhibitory Tendency of N-Heterocycle Substituted
Methylene Tetrahydropyrroloquinolinones
a
Resolution of Racemate. Enantiomers were separated via prepara-
tive HPLC on a Agilent Technologies 1200 series system (quaternary
pump, MWD, fraction collector) using a NucleoCel Delta column,
0.8 cm × 25 cm. Retention times (tR) were determined with an analytical
column using 0.3 mL/min and UV detection (254 nm). The ee values
were measured on a Merck-Hitachi LaChrome D-7000 System (iso-
cratic pump L7100, diode array detector L7455, autosampler L-7200)
using a Chiracel OD-H column, 0.46 cm ×25 cm.
8-(Pyridin-3-ylcarbonyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (1a). Starting materials 1,2,5,6-tetrahydro-4H-pyrrolo-
[3,2,1-ij]quinolin-4-one (0.50 g, 2.89 mmol), nicotinoyl chloride hydro-
chloride (0.77 g, 4.33 mmol), and AlCl3 (2.60 g,19.5 mmol) were melted
at 140 °C for 4 h followed by being cooled to 0 °C. A mixture of
ice/water was added to decompose the excessive AlCl3, and the resulting
mixture was stirred at ambient temperature for 1 h. Extraction with
CHCl3 (3 × 20 mL) gave the organic layers, which were combined and
dried over MgSO4. After removal of solvent in vacuo, the residue was
purified by flash chromatography column on silica gel (methanol/
dichloromethane, 1/100, Rf = 0.07) to give yellow solids (0.69 g, 86%),
mp 135−137 °C. 1H NMR (500 MHz, CDCl3): δ 2.72 (t, J = 7.8 Hz,
2H), 3.03 (t, J = 7.8 Hz, 2H), 3.25 (t, J = 8.5 Hz, 2H), 4.16 (t, J = 8.5 Hz,
2H), 7.45 (ddd, J = 0.8, 4.9, 7.9 Hz, 1H), 7.54 (s, 1H), 7.59 (s, 1H), 8.08
(dt, J = 2.0, 7.9 Hz, 1H), 8.80 (dd, J = 1.7, 4.7 Hz, 1H), 8.94 (dd, J = 0.8,
2.2 Hz, 1H). MS (ESI) m/z = 279 [M + H]+.
b
Het =
R =
1-Im
3-Py
4-Py
c
d
e
alkyl subst. Ph alkyl subst. Ph
alkyl
subst. Ph
CYP19
W
VP
VP
VP
N
N
W or P
N or M
N to VP
VP to N
P to N
N
VP
VP
W
CYP11B2
CYP11B1
CYP17
VP
EP
N
VP
EP
N
VP or N
N
N
a
Only general trends were considered, outliers were not mentioned.
N no inhibition, IC50 >1000 nM; W indicates weak inhibition, 1000 nM ≥
IC50 >500 nM; M indicates moderate inhibition, 500 nM ≥ IC50 >250 nM;
P indicates potent inhibition, 250 nM ≥ IC50 >100 nM; VP indicates very
potent inhibition, 100 nM ≥ IC50 > 20 nM; EP indicates extremely potent
b
c
inhibition, 20 nM ≥ IC50. Data adopted from ref 20d. Number of
d
examples is limited. inhibition varied dramatically to subtle differ-
e
ences in structure, extremely sensitive to alternation of substituents. Inhibi-
tion is sensitive to the bulkiness of alkyls and ranges very widely.
Trends along the bulkiness elevation are presented in the table.
with an alkyl substitution, the inhibition of all three enzymes
altered gradually according to the bulkiness of the alkyl, ranging
from no inhibition to high potency. As discussed above, the
effects rendered by a given modification on the inhibition of
CYP11Bs are usually opposite to those on CYP19 inhibition. In
contrast, compounds with substituted phenyl at the methylene
bridge acted in different manners. 1-Im analogues showed very
potent inhibition of all CYP enzymes except CYP17; while 4-Py
compounds are very potent inhibitors of CYP19 and CYP11B2.
The inhibitory profiles of 3-Py compounds are extremely
sensitive to the subtle alternation of the substituents on phenyl
moiety, dramatic deviations of inhibition are therefore observed
for CYP19, CYP11B1, and CYP11B2. Consistent with our previ-
ous findings,19d 1-Im and 3- or 4-Py rendered distinct inhibition
profiles among steroidogenic CYP enzymes, which could be the
results of differences in size, interaction angle, and electron
density on the sp2 hybrid N.
8-(Pyridin-4-ylcarbonyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (1b). Starting materials 1,2,5,6-tetrahydro-4H-pyrrolo-
[3,2,1-ij]quinolin-4-one (1.00 g, 5.77 mmol), isonicotinoyl chloride
hydrochloride (1.54 g, 8.65 mmol), and AlCl3 (3.85 g, 28.9 mmol) were
melted at 140 °C for 4 h followed by being cooled to 0 °C. A mixture of
ice/water was added to decompose the excessive AlCl3, and the resulting
mixture was stirred at ambient temperature for 1 h. Extraction with
CHCl3 (3 × 20 mL) gave the organic layers, which were combined and
dried over MgSO4. After removal of solvent in vacuo, the residue was
purified by flash chromatography column on silica gel (methanol/
dichloromethane, 1/100, Rf = 0.05) to give yellow solids (1.15 g, 72%),
mp 171−173 °C. 1H NMR (500 MHz, CDCl3): δ 2.74 (t, J = 7.8 Hz,
2H), 3.03 (t, J = 7.9 Hz, 2H), 3.24 (t, J = 8.5 Hz, 2H), 4.16 (t, J = 8.5 Hz,
2H), 7.52 (dd, J = 1.6, 4.4 Hz, 2H), 7.53 (s, 1H), 7.58 (s, 1H), 8.80 (dd,
J = 1.6, 4.4 Hz, 2H). MS (ESI) m/z = 279 [M + H]+.
Method A: Grignard Reaction. To a solution of a ketone (1.0 equiv)
in anhydrous THF was added dropwise a solution of an appropriate
Grignard reagent (2.0−3.0 equiv) under an atmosphere of nitrogen
at −78 °C. The reaction was stirred at the same temperature for another
1 h, and subsequently warmed to room temperature. After being stirred
for 15 h, the reaction was quenched with saturated aqueous NHCl4
(5 mL), extracted with ethyl acetate (3 × 10 mL), dried over MgSO4,
and concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (methanol/dichloromethane, 0 to 1:30)
to yield the corresponding alcohol.
EXPERIMENTAL SECTION
■
Biology. CYP19 Preparation and Assay. Human CYP19 was
obtained from microsomal preparations of human placenta, and the
assay was performed using the 3H2O-method as described and
[1β-3H]androstenedione as substrate.22a
8-[Hydroxy(phenyl)pyridin-4-ylmethyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (2h). The title compound was obtained
according to Method A using 1b (200 mg, 0.72 mmol) and phenyl-
magnesium bromide (1 M in THF, 2.16 mL, 2.16 mmol) in anhydrous
THF (5 mL) as pale yellow solids (120 mg, 47%), mp 196−197 °C. 1H
NMR (500 MHz, CDCl3): δ 2.63 (t, J = 7.7 Hz, 2H), 2.88 (t, J = 7.7 Hz,
2H), 3.11 (t, J = 8.3 Hz, 2H), 3.53 (s, br, 1H), 4.04 (t, J = 8.3 Hz, 2H),
6.88 (s, 1H), 6.93 (s, 1H), 7.25−7.33 (m, 7H), 8.49 (d, J = 3.2 Hz, 2H).
13C NMR (125 MHz, CDCl3): δ 24.4, 27.7, 31.5, 45.4, 81.2, 119.5,
Inhibition of CYP11B1 and CYP11B2. V79MZh cells expressing
human CYP11B1 or CYP11B2 were incubated with [1,2-3H]-11-deoxy-
corticosterone as substrate and the inhibitor at different concentrations.
The assay was performed as previously described.22b,c
CYP17 Preparation and Assay. The inhibition of CYP17 was deter-
mined using the 90000g sediment of E. coli coexpressing human CYP17
and cytochrome P450 reductase22d with progesterone as substrate and
NADPH as cofactor.22e
Chemistry. General Method. Melting points were determined on a
Mettler FP1 melting point apparatus and are uncorrected. 1H NMR and
13C NMR spectra were measured on a Bruker DRX-500 (500 MHz).
Chemical shifts are given in parts per million (ppm), and TMS was used
as an internal standard for spectra obtained. All coupling constants (J)
are given in Hz. ESI (electrospray ionization) mass spectra were deter-
mined on a TSQ quantum (Thermo Electron Corporation) instrument.
The purities of the final compounds were controlled by Surveyor-LC-
system. Purities were greater than 95%. Column chromatography was
performed using silica-gel 60 (50−200 μm), and reaction progress was
122.6, 123.1, 125.3, 127.7, 127.8, 128.2, 128.7, 140.9, 141.4, 145.7, 149.4,
155.6, 167.6. MS (ESI) m/z = 357 [M + H]+.
Method B: Triethylsilane Reduction. To an alcohol (1.0 equiv) in
anhydrous dichloromethane was added in sequence by syringe trifluo-
roacetic acid (10 equiv), triethylsilane (3.0 equiv), and trifluorometh-
anesulfonic acid (0.1 equivalent) under an atmosphere of nitrogen at
0 °C. The resulting solution was stirred at room temperature for 18−48 h.
Afterward, the reaction mixture was separated, and the queous layer was
extracted with dichloromethane (2 × 10 mL). The combined organic
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layers were washed with aqueous NaHCO3 and brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by flash
chromatograph on silica gel (methanol/dichloromethane, 0 to 1:40) to
yield the corresponding dehydroxyl product.
8-(Pyridin-3-ylmethyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (5a). The title compound was obtained according to
Method B using 4a (70 mg, 0.25 mmol), trifluoroacetic acid (0.19 mL,
2.50 mmol), triethylsilane (0.12 mL, 0.75 mmol), and trifluorometh-
anesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2 (5 mL) as white
solids (44 mg, 67%), mp 108−110 °C. 1H NMR (500 MHz, CDCl3): δ
2.65 (t, J = 7.7 Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 3.14 (t, J = 8.5 Hz, 2H),
3.90 (s, 2H), 4.06 (t, J = 8.5 Hz, 2H), 6.80 (s, 1H), 6.88 (s, 1H), 7.20 (dd,
J = 4.8, 7.7 Hz, 1H), 8.46 (d, J = 7.8 Hz, 1H), 8.46 (dd, J = 1.1, 7.8 Hz, 1H),
8.49 (d, J = 1.3 Hz, 1H). MS (ESI) m/z = 265 [M + H]+.
8-[Phenyl(pyridin-3-yl)methyl]-1,2,5,6-tetrahydro-4H-pyrrolo-
[3,2,1-ij]quinolin-4-one (3a). The title compound was obtained
according to Method B using 2a (60 mg, 0.17 mmol), trifluoroacetic
acid (0.13 mL, 1.68 mmol), triethylsilane (82 μL, 0.51 mmol), and
trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(3 mL) as pale yellow solids (46 mg, 81%), mp 142−144 °C. 1H NMR
(500 MHz, CDCl3): δ 2.65 (t, J = 7.7 Hz, 2H), 2.89 (t, J = 7.7 Hz, 2H),
3.12 (t, J = 8.4 Hz, 2H), 4.06 (t, J = 8.4 Hz, 2H), 5.49 (s, 1H), 6.74 (s,
1H), 6.80 (s, 1H), 7.10 (m, 2H), 7.22 (dd, J = 4.7, 7.9 Hz, 1H), 7.24 (m,
1H), 7.31 (m, 2H), 7.40 (dt, J = 1.7, 7.9 Hz, 1H), 8.42 (d, J = 2.0 Hz,
1H), 8.48 (dd, J = 1.4, 4.7 Hz, 1H). MS (ESI) m/z = 341 [M + H]+.
8-[(2-Methoxyphenyl)(pyridin-3-yl)methyl]-1,2,5,6-tetrahydro-
4H-pyrrolo[3,2,1-ij]quinolin-4-one (3b). The title compound was
obtained according to Method B using 2b (57 mg, 0.15 mmol), trifluo-
roacetic acid (0.11 mL, 1.48 mmol), triethylsilane (71 μL, 0.44 mmol),
and trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous
CH2Cl2 (4 mL) as pale yellow solids (31 mg, 56%), mp 65−68 °C. 1H
NMR (500 MHz, CDCl3): δ 2.65 (t, J = 7.7 Hz, 2H), 2.89 (t, J = 7.7 Hz,
2H), 3.12 (t, J = 8.5 Hz, 2H), 3.74 (s, 3H), 4.06 (t, J = 8.5 Hz, 2H), 5.85
(s, 1H), 6.71 (s, 1H), 6.78 (s, 1H), 6.84−6.91 (m, 3H), 7.20 (dd, J = 4.8,
7.8 Hz, 1H), 7.25 (m, 1H), 7.36 (dt, J = 1.7, 7.8 Hz, 1H), 8.38 (d, J = 2.1 Hz,
1H), 8.45 (dd, J = 1.5, 4.8 Hz, 1H). MS (ESI) m/z = 371 [M + H]+.
8-[(3-Methylphenyl)(pyridin-3-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3c). The title compound was obtained
according to Method B using 2c (62 mg, 0.17 mmol), trifluoroacetic acid
(0.13 mL, 1.67 mmol), triethylsilane (0.81 μL, 0.50 mmol), and tri-
fluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(4 mL) as pale yellow solids (30 mg, 50%), mp 164−166 °C. 1H NMR
(500 MHz, CDCl3): δ 2.31 (s, 3H), 2.65 (t, J = 7.7 Hz, 2H), 2.90 (t, J =
7.7 Hz, 2H), 3.12 (t, J = 8.4 Hz, 2H), 4.06 (t, J = 8.4 Hz, 2H), 5.44 (s,
1H), 6.73 (s, 1H), 6.80 (s, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.92 (s, 1H),
7.06 (d, J = 7.5 Hz, 1H), 7.18−7.23 (m, 2H), 7.40 (dd, J = 1.8, 7.9 Hz,
1H), 8.41 (d, J = 1.6 Hz, 1H), 8.47 (dd, J = 1.2, 4.7 Hz, 1H). MS (ESI)
m/z = 355 [M + H]+.
8-[(3-Chlorophenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3d). The title compound was obtained
according to Method B using 2d (72 mg, 0.18 mmol), trifluoroacetic
acid (0.14 mL, 1.84 mmol), triethylsilane (0.89 μL, 0.55 mmol), and
trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(4 mL) as to give pale yellow solids (36 mg, 53%), mp 129−131 °C. 1H
NMR (500 MHz, CDCl3): δ 2.68 (t, J = 7.7 Hz, 2H), 2.93 (t, J = 7.7 Hz,
2H), 3.16 (t, J = 8.4 Hz, 2H), 4.10 (t, J = 8.4 Hz, 2H), 5.48 (s, 1H), 6.73
(s, 1H), 6.80 (s, 1H), 7.01 (m, 1H), 7.10 (s, 1H), 7.27 (m, 3H), 7.41 (d,
J = 7.9 Hz, 1H), 8.43 (d, J = 1.9 Hz, 1H), 8.52 (dd, J = 1.3, 4.7 Hz, 1H).
MS (ESI) m/z = 375 [M + H]+.
8-[(4-Fluorophenyl)(pyridin-3-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3e). The title compound was obtained
according to Method B using 2e (90 mg, 0.24 mmol), trifluoroacetic acid
(0.18 mL, 2.40 mmol), triethylsilane (0.12 mL, 0.72 mmol), and
trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(5 mL) as pale yellow solids (50 mg, 58%), mp 175−177 °C. 1H NMR
(500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H),
3.13 (t, J = 8.4 Hz, 2H), 4.07 (t, J = 8.4 Hz, 2H), 5.47 (s, 1H), 6.71 (s,
1H), 6.78 (s, 1H), 7.00 (m, 2H), 7.05 (m, 2H), 7.24 (dd, J = 4.8, 7.9 Hz,
1H), 7.38 (dt, J = 1.7, 7.9 Hz, 1H), 8.40 (d, J = 1.0 Hz, 1H), 8.49 (dd, J =
1.4, 4.7 Hz, 1H). MS (ESI) m/z = 359 [M + H]+.
8-(Pyridin-4-ylmethyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (5b). The title compound was obtained according to
Method B using 4b (125 mg, 0.45 mmol), trifluoroacetic acid (0.34 mL,
4.46 mmol), triethylsilane (0.22 mL, 1.34 mmol), and trifluorometha-
nesulfonic acid (4 μL, 0.04 mmol) in anhydrous CH2Cl2 (5 mL) as pale
1
yellow solids (65 mg, 55%), mp 199−200 °C. H NMR (500 MHz,
CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.93 (t, J = 7.7 Hz, 2H), 3.15 (t, J =
8.5 Hz, 2H), 3.89 (s, 2H), 4.07 (t, J = 8.5 Hz, 2H), 6.80 (s, 1H), 6.88 (s,
1H), 7.09 (dd, J = 1.4, 4.5 Hz, 2H), 8.50 (dd, J = 1.4, 4.5 Hz, 2H). MS
(ESI) m/z = 265 [M + H]+.
8-(2-Methyl-1-pyridin-4-ylpropyl)-1,2,5,6-tetrahydro-4H-pyrrolo-
[3,2,1-ij]quinolin-4-one (3f). The title compound was obtained
according to Method B using 2f (167 mg, 0.52 mmol), trifluoroacetic
acid (0.40 mL, 5.18 mmol), triethylsilane (0.25 mL, 1.55 mmol), and
trifluoromethanesulfonic acid (5 μL, 0.05 mmol) in anhydrous CH2Cl2
1
(8 mL) as white solids (105 mg, 66%), mp 137−139 °C. H NMR
(500 MHz, CDCl3): δ 0.88 (q, J = 6.5 Hz, 6H), 2.40−2.47 (m, 1H), 2.64
(t, J = 7.7 Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 3.14 (t, J = 8.4 Hz, 2H), 3.32
(d, J = 10.9 Hz, 1H), 4.04 (t, J = 8.4 Hz, 2H), 6.86 (s, 1H), 6.96 (s, 1H),
7.18 (dd, J = 1.4, 4.6 Hz, 2H), 8.47 (dd, J = 1.4, 4.6 Hz, 2H). MS (ESI)
m/z = 307 [M + H]+.
8-[Cyclohexyl(pyridin-3-yl)methyl]-1,2,5,6-tetrahydro-4H-pyrrolo-
[3,2,1-ij]quinolin-4-one (3g). The title compound was obtained
according to Method B using 2g (150 mg, 0.41 mmol), trifluoroacetic
acid (0.32 mL, 4.14 mmol), triethylsilane (0.20 mL, 1.24 mmol), and
trifluoromethanesulfonic acid (4 μL, 0.04 mmol) in anhydrous CH2Cl2
(8 mL) as white crystals (80 mg, 54%), mp 83−85 °C. 1H NMR (500
MHz, CDCl3): δ 0.86 (m, 2H), 1.14−1.26 (m, 3H), 1.62−1.70 (m, 5H),
2.05 (m, 1H), 2.64 (t, J = 7.7 Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 3.14 (t, J =
8.4 Hz, 2H), 3.40 (d, J = 10.9 Hz, 1H), 4.04 (t, J = 8.4 Hz, 2H), 6.85 (s,
1H), 6.95 (s, 1H), 7.17 (dd, J = 1.4, 4.6 Hz, 2H), 8.46 (dd, J = 1.4, 4.6 Hz,
2H). MS (ESI) m/z = 347 [M + H]+.
8-[Phenyl(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-pyrrolo-
[3,2,1-ij]quinolin-4-one (3h). The title compound was obtained
according to Method B using 2h (70 mg, 0.20 mmol), trifluoroacetic
acid (0.15 mL, 1.96 mmol), triethylsilane (95 μL, 0.59 mmol), and tri-
fluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(5 mL) as pale yellow solids (48 mg, 72%), mp 168−169 °C. 1H NMR
(500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H),
3.13 (t, J = 8.4 Hz, 2H), 4.07 (t, J = 8.4 Hz, 2H), 5.43 (s, 1H), 6.73 (s,
1H), 6.79 (s, 1H), 7.04 (d, J = 5.3 Hz, 2H), 7.08 (d, J = 7.2 Hz, 2H), 7.26
(m, 1H), 7.32 (t, J = 7.4 Hz, 2H), 8.52 (d, J = 5.3 Hz, 2H). MS (ESI)
m/z = 341 [M + H]+.
8-[(2-Methoxyphenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-
4H-pyrrolo[3,2,1-ij]quinolin-4-one (3i). The title compound was
obtained according to Method B using 2i (77 mg, 0.20 mmol), tri-
fluoroacetic acid (0.15 mL, 1.99 mmol), triethylsilane (97 μL, 0.60 mmol),
and trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous
CH2Cl2 (4 mL) as pale yellow solids (55 mg, 75%), mp 70−72 °C. 1H
NMR (500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz,
2H), 3.12 (t, J = 8.5 Hz, 2H), 3.73 (s, 3H), 4.07 (t, J = 8.5 Hz, 2H), 5.80 (s,
1H), 6.71 (s, 1H), 6.78 (s, 1H), 6.83 (m, 1H), 6.90 (m, 2H), 7.00 (d, J =
4.6 Hz, 2H), 7.26 (m, 1H), 8.48 (d, J = 4.6 Hz, 2H). MS (ESI) m/z = 371
[M + H]+.
8-[(3-Methoxyphenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-
4H-pyrrolo[3,2,1-ij]quinolin-4-one (3j). The title compound was
obtained according to Method B using 2j (102 mg, 0.26 mmol), tri-
fluoroacetic acid (0.20 mL, 2.64 mmol), triethylsilane (0.13 mL, 0.79 mmol),
and trifluoromethanesulfonic acid (2 μL, 0.03 mmol) in anhydrous CH2Cl2
(8 mL) as pale yellow solids (48 mg, 50%), mp 148−150 °C. 1H NMR (500
MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H), 3.12 (t, J =
8.4 Hz, 2H), 4.06 (t, J = 8.4 Hz, 2H), 5.39 (s, 1H), 6.63 (m, 1H), 6.68 (d, J =
7.7 Hz, 1H), 6.72 (s, 1H), 6.80 (m, 2H), 7.04 (d, J = 4.6 Hz, 2H), 7.24 (t, J =
8.0 Hz, 1H), 8.51 (d, J = 4.6 Hz, 2H). MS (ESI) m/z = 371 [M + H]+.
8-[(4-Methoxyphenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-
4H-pyrrolo[3,2,1-ij]quinolin-4-one (3k). The title compound was ob-
tained according to Method B using 2k (102 mg, 0.26 mmol), tri-
fluoroacetic acid (0.20 mL, 2.64 mmol), triethylsilane (0.13 mL, 0.79 mmol),
and trifluoromethanesulfonic acid (2 μL, 0.03 mmol) in anhydrous CH2Cl2
1
(8 mL) as pale yellow solids (83 mg, 86%), mp 71−73 °C. H NMR
467
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(500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H),
3.12 (t, J = 8.4 Hz, 2H), 3.80 (s, 3H), 4.06 (t, J = 8.4 Hz, 2H), 5.38
(s, 1H), 6.71 (s, 1H), 6.78 (s, 1H), 6.85 (d, J = 8.7 Hz, 2H), 7.00 (d, J =
8.7 Hz, 2H), 7.03 (d, J = 4.7 Hz, 2H), 8.51 (d, J = 4.7 Hz, 2H). MS (ESI)
m/z = 371 [M + H]+. The racemate 3k was separated by preparative
5.55 (s, 1H), 6.68 (s, 1H), 6.75 (s, 1H), 7.00 (d, J = 4.6 Hz, 2H), 7.54
(s, 2H), 7.80 (s, 1H), 8.58 (d, J = 4.6 Hz, 2H). MS (ESI) m/z = 477
[M + H]+.
8-(1-Pyridin-3-ylvinyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (6a). To a suspension of methyltriphenylphosphonium
bromide (564 mg, 1.58 mmol) in anhydrous THF (25 mL) was added
n-BuLi (2.5 M in hexanes, 0.58 mL, 1.44 mmol) slowly under an
atmosphere of nitrogen at −78 °C. The reaction mixture was stirred at
room temperature for 1 h, and then cooled to −78 °C again followed by
addition of a solution of 1a (200 mg, 0.72 mmol) in THF (5 mL). The
resulting mixture was stirred at ambient temperature for 5 h and
subsequently quenched with aqueous NH4Cl, extracted with ethyl
acetate (3 × 10 mL), dried over MgSO4, and concentrated in vacuo. The
crude product was purified by flash chromatography column on silica
gel (methanol/dichloromethane, 0 to 1:70) to give off-white solids
HPLC on chiral stationary phase (NucleoCel Delta column, 0.8 cm ×
20
25 cm, 50% hexane/isopropanol, 1.0 mL/min) to yield (+)-3k ([α]D
=
+5.5 (c = 1.0, DMSO), 100% ee and tR = 32.1 min by analytical HPLC, and
(−)-3k ([α]D20= −4.8 (c = 1.0, DMSO), 90.3% ee and tR = 38.2 min by
analytical HPLC.
8-[(3-Fluorophenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3l). The title compound was obtained
according to Method B using 2l (96 mg, 0.26 mmol), trifluoroacetic acid
(0.20 mL, 2.56 mmol), triethylsilane (0.13 mL, 0.78 mmol), and
trifluoromethanesulfonic acid (2 μL, 0.03 mmol) in anhydrous CH2Cl2
(6 mL) as pale yellow solids (40 mg, 43%), mp 162−165 °C. 1H NMR
(500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.91 (t, J = 7.7 Hz, 2H),
3.14 (t, J = 8.4 Hz, 2H), 4.07 (t, J = 8.4 Hz, 2H), 5.42 (s, 1H), 6.71 (s,
1H), 6.78 (m, 2H), 6.88 (d, J = 7.7 Hz, 1H), 6.96 (td, J = 2.2, 8.3 Hz,
1H), 7.03 (d, J = 4.6 Hz, 2H), 7.29 (m, 1H), 8.53 (d, J = 4.6 Hz, 2H). MS
(ESI) m/z = 359 [M + H]+.
1
(117 mg, 59%), mp 97−99 °C. H NMR (500 MHz, CDCl3): δ 2.67
(t, J = 7.7 Hz, 2H), 2.95 (t, J = 7.7 Hz, 2H), 3.17 (t, J = 8.5 Hz, 2H), 4.10
(t, J = 8.5 Hz, 2H), 5.40 (s, 1H), 5.48 (s, 1H), 6.95 (s, 1H), 7.03 (s, 1H),
7.27 (dd, J = 4.9, 7.9 Hz, 1H), 7.61 (dt, J = 2.1, 7.9 Hz, 1H), 8.56 (dd, 3J =
1.6, 4.8 Hz, 1H), 8.60 (d, J = 1.6 Hz, 1H). MS (ESI) m/z = 277
[M + H]+.
8-(1-Pyridin-4-ylvinyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (6b). To a suspension of methyltriphenylphosphonium
bromide (564 mg, 1.58 mmol) in anhydrous THF (25 mL) was added
n-BuLi (2.5 M in hexanes, 0.58 mL, 1.44 mmol) slowly under an
atmosphere of nitrogen at −78 °C. The reaction mixture was stirred at
room temperature for 1 h and then cooled to −78 °C again followed by
addition of a solution of 1b (200 mg, 0.72 mmol) in THF (5 mL). The
resulting mixture was stirred at ambient temperature for 5 h and
subsequently quenched with aqueous NH4Cl, extracted with ethyl
acetate (3 × 10 mL), dried over MgSO4, and concentrated in vacuo. The
crude product was purified by flash chromatography column on silica gel
(methanol/dichloromethane, 0 to 1:50) to give pale yellow semisolids
(115 mg, 58%). 1H NMR (500 MHz, CDCl3): δ 2.69 (t, J = 7.7 Hz, 2H),
2.95 (t, J = 7.7 Hz, 2H), 3.18 (t, J = 8.5 Hz, 2H), 4.10 (t, J = 8.5 Hz, 2H),
5.51 (d, J = 8.1 Hz, 2H), 6.93 (s, 1H), 7.00 (s, 1H), 7.23 (dd, J = 1.6, 4.5 Hz,
2H), 8.58 (dd, J = 1.5, 4.6 Hz, 2H). MS (ESI) m/z = 277 [M + H]+.
8-(1-Pyridin-3-ylethyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (7a). To a solution of 6a (59 mg, 0.21 mmol) in
methanol (8 mL) was added 5% Pd/C (9 mg). Subsequently, the
reaction was stirred under a hydrogen atmosphere (maintained with
balloons) for 10 h followed by filtration through a Celite cake and
concentration in vacuo. The crude product was purified by flash chro-
matography column on silica gel (methanol/dichloromethane, 0 to
8-[(4-Fluorophenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3m). The title compound was
obtained according to Method B using 2m (65 mg, 0.17 mmol), tri-
fluoroacetic acid (0.13 mL, 1.74 mmol), triethylsilane (80 μL, 0.52 mmol),
and trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous
CH2Cl2 (5 mL) as pale yellow solids (45 mg, 74%), mp 155−157 °C. 1H
NMR (500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz,
2H), 3.13 (t, J = 8.5 Hz, 2H), 4.07 (t, J = 8.5 Hz, 2H), 5.41 (s, 1H), 6.70 (s,
1H), 6.77 (s, 1H), 6.99−7.06 (m, 6H), 8.53 (dd, J = 1.6, 4.5 Hz, 2H). MS
(ESI) m/z = 359 [M + H]+.
8-[(3-Chlorophenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3n). The title compound was obtained
according to Method B using 2n (85 mg, 0.22 mmol), trifluoroacetic
acid (0.17 mL, 2.20 mmol), triethylsilane (0.11 mL, 0.65 mmol), and
trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(5 mL) as pale yellow solids (42 mg, 51%), mp 163−165 °C. 1H NMR
(500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.91 (t, J = 7.7 Hz, 2H),
3.14 (t, J = 8.4 Hz, 2H), 4.08 (t, J = 8.4 Hz, 2H), 5.40 (s, 1H), 6.70 (s,
1H), 6.77 (s, 1H), 6.98 (m, 1H), 7.02 (d, J = 4.6 Hz, 2H), 7.07 (s, 1H),
7.25 (m, 2H), 8.54 (d, J = 4.6 Hz, 2H). MS (ESI) m/z = 375 M+.
8-[(4-Chlorophenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3o). The title compound was obtained
according to Method B using 2o (95 mg, 0.24 mmol), trifluoroacetic
acid (0.19 mL, 2.43 mmol), triethylsilane (0.12 mL, 0.73 mmol), and
trifluoromethanesulfonic acid (3 μL, 0.02 mmol) in anhydrous CH2Cl2
(5 mL) as pale yellow solids (56 mg, 62%), mp 136−138 °C. 1H NMR
(500 MHz, CDCl3): δ 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J = 7.7 Hz, 2H),
3.13 (t, J = 8.4 Hz, 2H), 4.07 (t, J = 8.4 Hz, 2H), 5.40 (s, 1H), 6.69
(s, 1H), 6.76 (s, 1H), 7.02 (m, 4H), 7.28 (d, J = 8.5 Hz, 2H), 8.53 (d, J =
4.6 Hz, 2H). MS (ESI) m/z = 375 M+.
8-[(3-Methylphenyl)(pyridin-4-yl)methyl]-1,2,5,6-tetrahydro-4H-
pyrrolo[3,2,1-ij]quinolin-4-one (3p). The title compound was obtained
according to Method B using 2p (65 mg, 0.18 mmol), trifluoroacetic
acid (0.14 mL, 1.80 mmol), triethylsilane (0.85 μL, 0.53 mmol), and
trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in anhydrous CH2Cl2
(4 mL) as pale yellow solids (32 mg, 50%), mp 180−182 °C. 1H NMR
(500 MHz, CDCl3): δ 2.31 (s, 3H), 2.66 (t, J = 7.7 Hz, 2H), 2.90 (t, J =
7.7 Hz, 2H), 3.13 (t, J = 8.4 Hz, 2H), 4.07 (t, J = 8.4 Hz, 2H), 5.39 (s,
1H), 6.72 (s, 1H), 6.79 (s, 1H), 6.87 (d, J = 7.7 Hz, 1H), 6.91 (s, 1H),
7.04 (d, J = 4.6 Hz, 2H), 7.07 (d, J = 7.5 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H),
8.51 (d, J = 4.6 Hz, 2H). MS (ESI) m/z = 355 [M + H]+.
8-[((3,5-Bis(trifluoromethyl)phenyl)(pyridin-4-yl)methyl]-1,2,5,6-
tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one (3q). The title com-
pound was obtained according to Method B using 2q (95 mg, 0.19 mmol),
trifluoroacetic acid (0.15 mL, 1.93 mmol), triethylsilane (0.93 μL,
0.58 mmol), and trifluoromethanesulfonic acid (2 μL, 0.02 mmol) in
anhydrous CH2Cl2 (6 mL) as pale yellow solids (38 mg, 42%), mp
182−184 °C. 1H NMR (500 MHz, CDCl3): δ 2.68 (t, J = 7.7 Hz, 2H),
2.92 (t, J = 7.7 Hz, 2H), 3.15 (t, J = 8.4 Hz, 2H), 4.09 (t, J = 8.4 Hz, 2H),
1
1:50) to give white solids (36 mg, 62%), mp 100−102 °C. H NMR
(500 MHz, CDCl3): δ 1.64 (d, J = 7.2 Hz, 3H), 2.65 (t, J = 7.8 Hz, 2H),
2.92 (t, J = 7.8 Hz, 2H), 3.14 (t, J = 8.4 Hz, 2H), 4.06 (t, J = 8.4 Hz, 2H),
4.11 (q, J = 7.2 Hz, 1H), 6.83 (s, 1H), 6.91 (s, 1H), 7.21 (dd, J = 4.7,
7.9 Hz, 1H), 7.49 (dt, J = 1.8, 7.9 Hz, 1H), 8.44 (dd, J = 1.5, 4.7 Hz, 1H),
8.51 (d, J = 2.2 Hz, 1H). MS (ESI) m/z = 279 [M + H]+.
8-(1-Pyridin-4-ylethyl)-1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]-
quinolin-4-one (7b). To a solution of 6b (115 mg, 0.38 mmol) in
methanol (8 mL) was added 5% Pd/C (16 mg). Subsequently, the
reaction was stirred under a hydrogen atmosphere (maintained with
balloons) for 10 h followed by filtration through a Celite cake and con-
centration in vacuo. The crude product was purified by flash chro-
matography column on silica gel (methanol/dichloromethane, 0 to
1:50) to give off-white solids (84 mg, 79%), mp 103−105 °C. 1H NMR
(500 MHz, CDCl3): δ 1.61 (d, J = 7.2 Hz, 3H), 2.66 (t, J = 7.8 Hz, 2H),
2.92 (t, J = 7.8 Hz, 2H), 3.14 (t, J = 8.4 Hz, 2H), 4.06 (m, 3H), 6.81 (s,
1H), 6.90 (s, 1H), 7.13 (dd, J = 1.3, 4.8 Hz, 2H), 8.50 (dd, J = 1.3, 4.8 Hz,
2H). MS (ESI) m/z = 279 [M + H]+.
Docking Study. Crystal Processing. Hydrogens and partial charges
were added via Protonate3D application in MOE 2008.
Ligands. Compounds were built and energy minimized in the
MMFF94s force field with MOE 2008. No conformation search was
performed, because the docking software GOLD automatically
generates them.
Docking. The automatic active-site detection was switched on, while
heme iron was selected as active-site origin and the radius of active site
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Journal of Medicinal Chemistry
Article
was set to 19 Å. A distance constraint of 1.9−2.5 Å between the sp2
hybrid N and the iron was set as well. Ligand was docked in 50
independent genetic algorithm (GA) iterations for each of the three
GOLD-docking runs. Moreover, the goldscore.p450_pdb parameters
were exploited, and the genetic algorithm default parameters were set.
The resulting poses were subsequently ranked according to fitness and
further evaluated with LigX module in MOE 2008.
treatment prevents development of heart failure post-myocardial
infarction in the rat. Basic Res. Cardiol. 2007, 102, 9−18.
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system and gender differences in the cardiovascular system. Cardiovasc.
Res. 2002, 53, 672−677. (b) Roesch, D. M.; Tian, Y.; Zheng, W.; Shi, M.;
Verbalis, J. G.; Sandberg, K. Estradiol attenuates angiotensin-induced
aldosterone secretion in ovariectomized rats. Endocrinology 2000, 141,
4629−4636. (c) Chappell, M. C.; Gallagher, P. E.; Averill, D. B.;
Ferrario, C. M.; Brosnihan, K. B. Estrogen or the AT1 antagonist
olmesartan reverses the development of profound hypertension in the
congenic mRen2.Lewis rat. Hypertension 2003, 42, 781−786.
(10) Duprez, D. A. Aldosterone and the vasculature: Mechanisms
mediating resistant hypertension. J. Clin. Hypertens. 2007, 9, 13−18.
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Aldosterone induces contraction of the resistance arteries in man.
Atherosclerosis 2003, 166, 345−349.
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́ ́ ́
genes in cardiomyocytes: Clues to cardiac remodeling? Endocrinology
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(13) Joffe, H. V.; Adler, G. K. Effect of aldosterone and
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ASSOCIATED CONTENT
* Supporting Information
■
S
The experimental details and characterization of compounds 4a,
4b, and intermediates 2a−2g and 2i−2q, the 13C NMR and
HPLC purities of all final compounds, and docking results of
both enantiomers of compound 3k in CYP19. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +(49) 681 302 70300. Fax: +(49) 681 302 70308.
E-mail: rolf.hartmann@helmholtz-hzi.de. Homepage: http://
www.helmholtz-hzi.de/?id=3897.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Jorg Haupenthal, Dr. Christina Zimmer,
̈
Jeannine Jung, and Jannine Ludwig for their help in performing
the in vitro test, as well as Dr. Stefan Boettcher, Frauke Maurer,
and Michael Zender for chiral separation and ee determination.
The authors also appreciate Professor Hermans (University of
Maastricht, The Netherlands) for providing us with V79MZh11B1
cells expressing human CYP11B1.
(15) (a) Leibovitz, E.; Ebrahimian, T.; Paradis, P.; Schiffrin, E. L.
Aldosterone induces arterial stiffness in absence of oxidative stress and
endothelial dysfunction. J. Hypertens. 2009, 27, 2192−2200. (b) Weber,
K. T.; Brilla, C. G.; Campbell, S. E.; Guarda, E.; Zhou, G.; Sriram, K.
Myocardial fibrosis: Role of angiotensin II and aldosterone. Basic Res.
Cardiol. 1993, 88, 107−124.
(16) Qin, W.; Rudolph, A. E.; Bond, B. R.; Rocha, R.; Blomme, E. A. G.;
Goellner, J. J.; Funder, J. W.; McMahon, E. G. Transgenic model of
aldosterone-driven cardiac hypertrophy and heart failure. Circ. Res.
2003, 93, 69−76.
ABBREVIATIONS USED:
■
BC, breast cancer; CYP, cytochrome P450; ER, estrogen receptor;
PgR, progesterone receptor; CYP19, aromatase, estrogen
synthase; CVD, cardiovascular disease; CYP11B2, aldosterone
synthase; ROS, reactive oxygen species; CYP17, 17α-hydroxylase-
17,20-lyase; CYP11B1, 11β-hydroxylase; DMA, N,N-dimethyl-
acetamide; NBS, N-bromosuccinimide; SF, selectivity factor =
IC50(CYP11B1)/IC50(CYP11B2)
(17) (a) Morphy, R.; Rankovic, Z. Designed multiple ligands. An
emerging drug discovery paradigm. J. Med. Chem. 2005, 48, 6523−6543.
(b) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.;
Recanatini, M.; Melchiorre, C. Multi-target-directed ligands to combat
neurodegenerative diseases. J. Med. Chem. 2008, 51, 347−372.
(c) Morphy, R. Selectively nonselective kinase inhibition: Striking the
right balance. J. Med. Chem. 2010, 53, 1413−1437.
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