Design, Synthesis, and Biological Evaluation of Novel Tetrahydroprotoberberine Derivatives (THPBs) as Selective α1A-Adrenoceptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.6b01217

A novel series of tetrahydroprotoberberine derivatives (THPBs) were designed, synthesized, and evaluated as selective α_1A-adrenergic receptors (AR) antagonists for the treatment of benign prostatic hyperplasia. On the basis of the pharmacophore model of the marketed drug silodosin, THPBs were modified by introducing an indole segment into their core scaffolds. In calcium assays, 7 out of 32 compounds displayed excellent antagonistic activities against α_1A-ARs, with IC₅₀ less than 250 nM. Among them, compound (S)-27 had the most potent biological activity; its IC₅₀ toward α_1A-AR was 12.8 ± 2.2 nM, which is 781 and 20 times more selective than that toward α_1B- and α_1D-AR, respectively. In the functional assay using isolated rat tissues, compound (S)-27 inhibited norepinephrine-induced urethra smooth muscle contraction potently (IC₅₀ = 0.5 ± 0.3 nM), without inhibiting the aortic contraction (IC₅₀ > 1000 nM), displaying a better tissue selectivity than the marketed drug silodosin. Additional results of preliminary safety studies (acute toxicity and hERG inhibition) and pharmacokinetics studies indicated the potential druggability for compound (S)-27 which is a promising lead for the development of selective α_1A-AR antagonists for the treatment of BPH.

Full text of DOI:10.1021/acs.jmedchem.6b01217

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Article
Design, Synthesis and Biological Evaluation
of Novel Tetrahydroprotoberberine Derivatives
(THPBs) as Selective #1A-Adrenoceptor Antagonists
Diliang Guo, Jing Li, Henry Lin, Yu Zhou, Ying Chen, Fei Zhao, Haifeng Sun, Dan Zhang,
Honglin Li, Brian K. Shoichet, Lei Shan, Weidong Zhang, Xin Xie, Hualiang Jiang, and Hong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01217 • Publication Date (Web): 06 Oct 2016
Downloaded from http://pubs.acs.org on October 8, 2016
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Design, Synthesis and Biological Evaluation of Novel
Tetrahydroprotoberberine Derivatives (THPBs) as Selective
α ꢀAdrenoceptor Antagonists
1A
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a,
†
a,
†
b,
†
a,
†
a
a
e
Diliang Guo, Jing Li, Henry Lin, Yu Zhou, Ying Chen, Fei Zhao,
a
a
c
b,d
Haifeng Sun, Dan Zhang, Honglin Li, Brian K. Shoichet, Lei Shan,
e
a,
a,
a,
Weidong Zhang, Xin Xie, * Hualiang Jiang, * Hong Liu *
a
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China.
b
Department of Pharmaceutical Chemistry, University of California, San Francisco,
California, USA.
c
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New
Drug Design, School of Pharmacy, East China University of Science and Technology,
Shanghai 200237, China.
d
Department of Pharmacology, University of North Carolina Chapel Hill School of
Medicine, Chapel Hill, North Carolina, USA.
e
Department of Natural Product Chemistry, School of Pharmacy, Second Military
Medical University, 325 Guohe Road, Shanghai 200433, China.
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Abstract
A novel series of tetrahydroprotoberberine derivatives (THPBs) were designed,
synthesized and evaluated as selective α1Aꢀadrenergic receptors (AR) antagonists for
the treatment of benign prostatic hyperplasia. Based on the pharmacophore model of
the marketed drug silodosin, THPBs were modified by introducing an indole segment
into their core scaffolds. In calcium assays, 7 out of 32 compounds displayed
excellent antagonistic activities against α1AꢀARs, with IC50s less than 250 nM. Among
them, compound (S)ꢀ27 had the most potent biological activity; its IC50 toward
α1AꢀAR was 12.8 ± 2.2 nM, which is 781 and 20 times more selective than that toward
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α
1Bꢀ and α1DꢀAR, respectively. In the functional assay using isolated rat tissues,
compound (S)ꢀ27 inhibited norepinephrineꢀinduced urethra smooth muscle
contraction potently (IC50 = 0.5 ± 0.3 nM), without inhibiting the aortic contraction
(IC50 > 1000 nM), displaying a better tissue selectivity than the marketed drug
silodosin. Additional results of preliminary safety studies (acute toxicity and hERG
inhibition) and pharmacokinetics studies indicated the potential druggability for
compound (S)ꢀ27 which is a promising lead for the development of selective α1AꢀAR
antagonists for the treatment of BPH.
1
. Introduction
Benign prostatic hyperplasia (BPH) is a benign increase in the size of the prostate
that leads to urinary hesitancy, frequent urination, dysuria and increased risk of lower
1
,2
urinary tract symptoms (LUTS). An estimated 50% of men have histological
evidence of BPH by the age of 50 years and that number increases to 75% by the age
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of 80 years. As life expectancy rises, so does the occurrence of BPH. There are two
components of BPH/LUTS, namely increased size and elevated muscle tone of the
gland. Therefore, medications are also divided into two categories: those that decrease
the gland size and those that relax the urethra smooth muscle.
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Figure 1. Marketed α1AꢀAR selective antagonists, tamsulosin and silodosin.
α1ꢀAdrenergic receptors (α ꢀadrenoceptors, α1ꢀARs) belong to the
G
1
proteinꢀcoupled receptor (GPCR) superfamily, and regulate the contraction of smooth
muscle by activating phospholipase C, followed by the increase of intracellular
4,5
6ꢀ8
calcium levels.
1AꢀAR, expressed mainly in the prostate, bladder and urethra, is considered to play a
major role in regulating prostatic muscle contraction, while α ꢀAR has a minor
1
α ꢀARs are divided into three subtypes, α1A, α1B and α1DꢀAR. The
α
1
D
4
,9
contribution. On the other hand, α1BꢀAR, expressed predominantly in the heart and
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vascular smooth muscle, is considered a drug target for treating hypertension.
α
1A
antagonists are used as antiꢀBPH agents; however, antagonism of the α1B subtype will
12,13
lead to cardiovascular side effects, such as hypotension.
The α1D subtype is
predominant and functional in human epicardial coronary arteries, and its inhibition
14
might result in coronary vasodilation. In the last two decades, α1AꢀAR selective
antagonists, such as the marketed drugs tamsulosin (1) and silodosin (2), have been
developed to treat BPH/LUTS (Figure 1). These agents have relatively high α1AꢀAR
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subtype selectivity and effectively relieve the symptoms of BPH, with reduced side
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effects.
However, the cardiovascular adverse effects can still be observed and
impact blood pressure. Clinical trial results showed that tamsulosin treatment results
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in a significant decrease in mean systolic blood pressure. Marks et al reported the
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incidence of orthostatic hypotension caused by silodosin is 2.6%. Recently,
increasing efforts have been made to identify novel small molecule α1AꢀAR selective
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1ꢀ32
antagonists based on the scaffolds of the molecules mentioned above.
Table 1. The predicted target of THPB analogs by SEA, ChemMapper and calcium
assay evaluation
Predicted
Target
Similarity
Calcium Assay
a
Compd.
Structure
Eꢀvalue
3.75eꢀ8
ꢀ
b
Score
(IC50)
3
4
α
1AꢀAR
1AꢀAR
1.9
5.4 ꢁM
4
1.2% inhibition
@ 10 ꢁM
α
ꢀ
5
α
1AꢀAR
1AꢀAR
ꢀ
ꢀ
ꢀ
7.6 ꢁM
462 nM
6
α
ꢀ
a
b
SEA Eꢀvalues against all 246 MDDR activity classes. ChemMapper similarity score against the
ChEMBL database.
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As a rapid complementary approach to experimental methods, in silico target
prediction methods had been approved to identify the pharmacological effects of a
33,34
drug.
Tetrahydroprotoberberines (THPBs) extracted from the Chinese herb
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Corydalis ambigua, are important and intriguing scaffolds. Herein, to find their
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potential targets, we have used the similarity ensemble approach (SEA) and
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ChemMapper
to predict their potential targets.
Through comparison of the
results derived from the above two methods, adrenergic receptor is an interesting
unreported target for these compounds, which were therefore selected for further
biological evaluation. The calcium assay results indicated that THPBs presented
moderate antagonism against α1AꢀAR, with subꢀmicromolar affinities (Table 1,
compounds 3ꢀ6).
i 1
Figure 2. THPB analogs and their binding affinities (pK ) on α ꢀAR.
With the above results in mind, a literature research was carried out and evidence
that some THPBs including tetrahydroberberines (THB, 7), lꢀtetrahydropalmatine
(
lꢀTHP, 4), lꢀ stepholidine (lꢀSPD, 8) and lꢀchlorosoulerine (lꢀCSL, 9), were observed
to possess weak α ꢀAR binding affinity and to block phenylephrineꢀinduced
vasocontraction (Figure 2).
1
35
Based on these findings, we believed that THPB derivatives should be explored
as a novel class of α ꢀAR antagonists for the treatment of BPH/LUTS. Therefore, we
1
A
report the design and synthesis of a series of novel indoleꢀcontaining THPB
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derivatives as selective α1AꢀAR antagonists, as well as the detailed structureꢀactivity
relationship analysis, in vitro and in vivo biological evaluation, preliminary toxicity
and pharmacokinetic studies.
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. Chemistry
.1 Design of Target Compounds
In addition to a chemical similarity approach, pharmacophore models and
quantitative structure–activity relationship (QSAR) studies have been used frequently
to design novel α1AꢀAR antagonists in recent years. Meaningful pharmacophore and
QSAR models have been established, and several key components of pharmacophores
were identified in various scaffolds, including the marketed drugs tamsulosin and
36ꢀ40
silodosin.
To design our compounds, silodosin was treated as a model molecule. Interestingly,
we found that THB has a pharmacophore similar to silodosin. The positive ionizable
(
PI) nitrogens in the center of both molecules are probably crucial for recognition
between the compound and the receptor. In addition, the two aromatic ring segments
Ar) at both ends of the two compounds represent evidence of hydrophobic interaction.
The alkoxy groups on the aromatic rings are potential hydrogen bond sites (HBS) that
form interactions with α ꢀAR. Note that both molecules have an chiral carbon located
next to the PI nitrogen. These common pharmacophore patterns of the two molecules
(
1
provide a reasonable explanation for the interaction between THB and α
3).
1
ꢀAR (Figure
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Figure 3. Similar pharmacophore models of silodosin and THB.
Based on these findings, we hypothesized that THB could be modified into
1AꢀAR antagonists with higher affinity and selectivity. In our previous studies, a class
α
of indoleꢀcontaining polycyclic compounds obtained by the gold catalyzed cascade
41
reaction was identified as selective α1AꢀAR blockers with moderate activities. Other
indoleꢀbased scaffolds have also been reported to exhibit good bioactivities toward
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ARs.
Thus, an indole fragment was introduced into THB on its Dꢀring, such that a
series of indoleꢀcontaining THB derivatives were designed and synthesized (Figure 4).
All synthesized compounds (13ꢀ44) were further evaluated for their biological
activities using calcium mobilization assays. In addition, chiral compounds (S)ꢀ18,
(
S)ꢀ27 and (R)ꢀ27 were also synthesized to explore the influence of the molecular
configuration (Scheme 2).
Figure 4. The structural optimization for THB
.2 Synthetic Procedures of Target Compounds
2
The designed compounds were synthesized via the procedures shown in Schemes
ꢀ3. Condensation of commercially available phenylethanamines and indoleꢀ3ꢀacetic
1
acids generated amides 10aꢀu, which were further cyclized under the presence of
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phosphoryltrichloride (POCl
the procedures of the Bischler–Napieralski reaction. Reduction of the resulting imines
1aꢀu with sodium borohydride produced the key amine intermediates 12aꢀu.
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Cyclization of amines 12aꢀu via the Pictect–Spengler reaction with various aldehydes
resulted in the target products 13ꢀ35 (Scheme 1). Further deprotection of groups of
products 32, 34 and 35 gave compounds 36ꢀ38, respectively. Compounds 39ꢀ44 were
synthesized by substitution reactions of 37 and 38 with different alkyl bromides
(
Scheme 2).
Additionally, chiral compounds (S)ꢀ18, (S)ꢀ27 and (R)ꢀ27 were prepared
according to the procedure outlined in Scheme 3. Asymmetric hydrogenation of
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imines 11b and 11d, catalyzed by a chiral Ruꢀ(II) complex (Noyori’s catalyst)
produced chiral amines (S)ꢀ12b, d and (R)ꢀ12d, followed by cyclization with
formaldehyde to give (S)ꢀ18, (S)ꢀ27 and (R)ꢀ27. All target compounds were
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characterized by HꢀNMR, CꢀNMR and MS (see Experimental Section).
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a
Scheme 1. Synthesis of compounds 13ꢀ35.
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a
3 2 2 3 3 4 7
Reagents and conditions: (a) EDCI, Et N, CH Cl , rt, 8 h; (b) POCl , CH CN, reflux; (c) NaBH , methanol, rt, 8 h; (d) HCOOH, R CHO, 25–90°C, 2 h.
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Scheme 2. Synthesis of compounds 36ꢀ44.
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Reagents and conditions: (a) H
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Scheme 3. Synthesis of compounds (S)ꢀ18, (S)ꢀ27 and (R)ꢀ27.
a
6 3
Reagents and conditions: (g) (R,R)ꢀNoyori’s catalyst, HCOONa, AgSbF , La(OTf) ,
CTAB, H O, 40 °C, 12 h; (h) (S,S)ꢀNoyori’s catalyst, HCOONa, AgSbF , La(OTf) ,
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CTAB, H O, 40 °C, 12 h; (i) HCOOH, 40% HCHO, 25–90 °C, 2 h.
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Results and Discussion
.1 Chemistry
3
On the basis of the structure features of THB, an indole fragment was introduced on its
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Dꢀring, and 32 new indoleꢀcontaining compounds were designed and synthesized. Their
synthetic routes and chemical structures (13ꢀ44) are shown in Scheme 1. In addition, chiral
compounds (S)ꢀ18, (S)ꢀ27 and (R)ꢀ27 were also synthesized to explore the influence of
their configuration (Scheme 2). The details of the synthetic procedures and structural
characterizations are described in the Experimental Section.
3.2 StructureꢀActivity Relationship for All Compounds
All target compounds (13ꢀ44, (S)ꢀ18, (S)ꢀ27 and (R)ꢀ27) were evaluated for their
1
biological activities toward α ꢀAR using calcium mobilization assays. The initial screening
was carried out at a concentration of 10 ꢁM for each compound, and compounds that
displayed >80% inhibition were further evaluated for their IC s. The results are
5
0
summarized in Table 2, and the details of the bioassay procedures are described in the
Experimental Section. As shown in Table 2, twenty compounds demonstrated good
inhibitory activities, with >80% inhibition at a concentration of 10 uM for each compound.
Further analysis showed that introducing substituents (alkyl or aryl groups) into the R7
position was detrimental to the inhibitory potency when R1 and R2 were replaced by a
methoxyl group (see compounds 13ꢀ17, as shown in Table 2 and Scheme 1), respectively.
However, the introduction of a methylenedioxy group into the R1 and R2 position of the
scaffold of compound 13 afforded the new compound 18, which displayed an excellent
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inhibitory effect toward α1AꢀAR, its IC50 value was 125.0 ± 20.4 nM. Based on these
positive results, several bulky alkyl and aryl substituents were introduced into R7 position
of the scaffold of 18 to form compounds 19ꢀ22; unfortunately, the introduction of these
bulky groups caused a substantial loss in inhibitory activity, which suggested that the
bulky steric hindrance at R7 position might decrease their activity. Thereafter, we
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
attempted to remove the R2 group (ꢀOCH ) from the scaffold of compound 13 to prepare
compound 23, and the bioassay showed a moderate inhibitory potency against α1AꢀAR,
with an IC of 552.3 ± 67.9 nM. Based on this finding, we further substituted R7 position
50
with methyl, cyclopropyl and 4ꢀmethoxyphenyl to form compounds 24ꢀ26, which have
hardly any inhibitory potency against α1AꢀAR.
a
Table 2. Inhibition ratio and IC50 values of all synthesized compounds for α1AꢀAR.
b
IR
IC50 ( Means ± SEM) on calcium assays, nM
Selectivity
Compd.
(10 ꢁM)
α1A
α1B
α1D
1B 1A
α /α
1D 1A
α /α
1
1
1
1
1
1
1
3
4
5
6
7
8
9
60%
72%
0%
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0%
/
/
/
/
21%
100%
82%
0%
/
/
/
/
1
25.0±20.4
> 10000
> 10000
>80.0
>80.0
> 10000
/
/
/
/
/
/
/
/
/
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3
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5
6
0%
/
/
/
/
/
0%
/
/
/
/
/
100%
90%
31%
80%
99%
100%
100%
100%
100%
100%
100%
100%
97%
100%
56%
0%
552.3±67.9
8669±625.6
/
/
/
/
/
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
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49
50
51
52
53
54
55
56
57
58
59
60
/
/
/
/
/
/
/
/
> 10000
17.6±2.1
202.3±32.5
594.0±147.0
2057±183.0
253.8±32.6
2880±77.5
1474±138.8
124.7±7.1
> 10000
402.9±53.6
/
/
/
/
/
27
> 10000
4038±1725
>568.2
229.4
2
8
> 10000
> 10000
>19.4
>19.4
29
> 10000
> 10000
>16.8
>16.8
3
0
/
/
/
/
31
> 10000
8392±526.4
>39.4
33.1
3
2
/
/
/
/
33
/
/
/
/
3
4
7928±250.3
640.6±95.7
53.6
5.1
35
/
/
/
/
3
6
9170±1133
1456±68.6
22.8
3.6
37
/
/
/
/
3
8
/
/
/
/
/
39
75%
100%
98%
/
/
/
/
/
4
0
225.3±35.2
2286±429.3
> 10000
/
> 10000
/
>44.4
/
>44.4
/
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
4
2
3
4
17%
/
/
/
/
/
21%
/
/
/
/
/
63%
/
/
/
/
/
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
S)ꢀ18
(S)ꢀ27
R)ꢀ27
99%
57.4±10.2
12.8±2.2
3187±149.4
2.2±0.3
1.8±0.1
> 10000
> 10000
/
> 10000
250.7±40.7
/
>174.0
>780.6
/
>174.0
19.6
/
100%
100%
100%
100%
(
c
tamsulosin
4.8±0.9
116.0±12.5
1.4±0.2
6.3±1.0
2.2
0.6
c
silodosin
66.0
3.6
a
The initial screening was carried out at a concentration of 10 ꢁM for each compound and IC50 were
measured for compounds that displayed >80% inhibition of α1AꢀAR, and “/” means that no experiment
b
c
was conducted. IR represents inhibition ratio. The reference drug.
On the basis of above results, we held the methylenedioxy group at R1 and R2
position on the molecular scaffold of compounds 19ꢀ22, and further explored the influence
of different substituents on the indole ring on inhibitory potency of α1AꢀAR, namely that
introducing different substituents into R3, R4, R5 and R6 position, respectively. The
results demonstrated that introducing a methoxy group into the R4 position of the indole
ring can produce the highest inhibitory activity against α1AꢀAR, with an IC50 of 17.6 ± 2.1
nM (27). However, moving this methoxy group from R4 to R3 or R5 position of the indole
ring brought about a decrease in inhibitory activity against α1AꢀAR (28 and 29). The
introduction of a fluorine atom (30 and 31) and an electronꢀdonating group (32 and 33)
into R4, R5 or R6 position of the scaffold of the indole could not increase the inhibitory
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potency. It follows that substituting R4 with a methoxy group was favorable to retain the
inhibitory effects against α1AꢀAR. Therefore, in the subsequent structural modification, we
kept a methoxy group on the R4 position of indole ring and further investigated the
influences of different R1 and R2 substituents (34, 35, 37ꢀ44). The results displayed that a
methoxy group in the R1 position is important to maintain the inhibitory potency of the
compounds toward α1AꢀAR (34, 40). Removal of the methoxy group from the R1 position
almost completely ablated their activities (38, 42ꢀ44).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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39
40
41
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45
46
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49
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52
53
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55
56
57
58
59
60
For some natural products, such as lꢀSPD, the Rꢀconfiguration exhibited worse
50ꢀ52
α1AꢀAR antagonistic activity compared to its Sꢀconfigured counterparts.
Therefore, we
also synthesized the chiral compounds (S)ꢀ18, (S)ꢀ27 and (R)ꢀ27, and further evaluated
their biological activities toward α1AꢀAR. The results showed that the Sꢀconfiguration is an
important determinant of the α1AꢀAR inhibitory activity. As shown in Table 2, the
Sꢀconfigured enantiomer (S)ꢀ18 (IC50 = 57.4 ± 10.2 nM) offered almost twoꢀfold higher
potency than the racemate 18 (IC50 = 125.0 ± 20.4 nM). The Sꢀconfigured enantiomer
(S)ꢀ27 exhibited the most potent α1AꢀAR antagonistic activity; its IC50 reached 12.8 ± 2.2
nM, which was much more effective than its racemate (27, IC50 = 17.6±2.1 nM) and
Rꢀconfigured compound ((S)ꢀ27, IC50 = 3187±149.4 nM). These data demonstrated that the
stereochemical configuration has an important influence on the α ꢀAR antagonistic
1
A
activity of these compounds.
.2 Evaluation of α ꢀAR Subtype Selectivity for Selected Compounds
3
1
Previous studies indicated that the α1B subtype is found widely in vascular smooth
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2
2
2
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3
muscle, and blocking it can cause orthostatic hypotension. The α1D subtype is
predominant and functional in human epicardial coronary arteries, and its inhibition might
54
mediate coronary vasodilation. Therefore, to characterize α ꢀAR subtype selectivity,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
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9
0
1
representative compounds (18, 27ꢀ29, 31, 33, 35, 40, (S)ꢀ18 and (S)ꢀ27) were selected to
determine their selectivities using a calcium mobilization assay. The results are
summarized in Table 2. In general, most of these compounds displayed moderate to high
antagonist activity toward α1AꢀAR. However, none of them displayed a significant
inhibitory effect toward α ꢀAR. Only a few compounds had measurable antagonist
1B
activity on α1DꢀAR. All compounds displayed much better α1AꢀAR selectivity compared
with the reference drug tamsulosin. Although compounds 27 and (S)ꢀ27 showed slightly
less potent antagonistic activity than silodosin against α1AꢀAR, both demonstrated much
higher selectivities than silodosin and tamsulosin. (S)ꢀ27 showed higher selectivity for
α ꢀAR (IC = 12.8 nM) compared with α ꢀAR (IC >10 ꢁM, α /α > 780) and α ꢀAR
1
A
50
1B
50
1B 1A
1D
(
IC50 = 250.7 nM, α1D/α1A =19.6), which was much better than tamsulosin (α1B/α1A = 2.2,
α1D/α1A = 0.6) and silodosin (α1B/α1A = 66.0, α1D/α1A = 3.6).
3.3 Functional Assay in Isolated Rat Tissues
It is desirable to develop α
1
ꢀAR antagonists that can selectively suppress the tone of
the lower urinary tract, without vascular effects, to treat urinary outlet obstruction
54
problems in patients with BPH. Therefore, we selected the most effective compounds, 27
and (S)ꢀ27, and evaluated their antagonist effect on smooth muscle contraction of rat
urethras and aortas. As shown in Table 3, both 27 and (S)ꢀ27 showed strong
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antiꢀcontractile activity on urethra smooth muscle stimulated with norepinephrine.
Compound (S)ꢀ27 (IC50 = 0.5±0.3 nM) was slightly more potent than the marketed drug
silodosin (IC = 0.8±0.03 nM). Encouragingly, neither compound had a significant effect
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
50
on norepinephrineꢀinduced contraction of aortic smooth muscle (IC50 >1000 nM), while
the control compound silodosin potently inhibited aortic contraction (IC50 = 90.1±7.6 nM).
The uroselectivity of compound (S)ꢀ27 was better than silodosin, indicating that (S)ꢀ27
might have fewer vascular side effects than silodosin.
Table 3. Antagonist effect on smooth muscle contraction of rat urethra and aorta.
IC50 (nM)
Selectivity
Urethra/ Aorta
>22.02
Compd.
Urethra
45.4±8.8
0.5±0.3
Aorta
>1000
2
7
(
S)ꢀ27
>1000
>2083
a
silodosin
0.8±0.03
90.1±7.6
112.6
a
The reference drug.
3.4 Micturition Behavior in BPH Model Rats.
The BPH model rats have higher micturition frequency (Figure 5A) and lower mean
voided volume (Figure 5B) than the sham rats. In our studies, we found that silodosin can
doseꢀdependently reduce the urinary frequency and increase the voided volume (Figure 5A
and 5B). Our compounds 27 and (S)ꢀ27 can also offer improved effects in the micturition
behavior of BHP rats. The minimal effective dose of reducing micturition frequency is 10
mg/kg and 3 mg/kg (Figure 5A), respectively; and the minimal effective dose of increasing
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6
the mean voided volume is 3 mg/kg (Figure 5B). The most optimal dose of 27 and (S)ꢀ27
in this study is 10 mg/kg, which led the maximum the urinary frequency reduction and
voided volume increase. Although compounds (S)ꢀ27 shows a slightly weaker activity in
BPH rats than silodosin, it can effectively alleviate voiding symptoms of BPH rats, which
is worthy of further investigation.
0
1
2
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4
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0
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0
1
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3
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Effects of 27 and (S)ꢀ27 on the micturition parameters in BPH rats. (A)
Micturition frequency and (B) mean voided volume were measured in a metabolic cage.
#
##
*
**
***
P < 0.001, versus sham control. P < 0.05, P < 0.01, P < 0.001 versus vehicle
control.
3.5 Safety Evaluation: Inhibitory Potency against hERG Potassium Ion Channel
In view of excellent biological activities of compound (S)ꢀ27, we further evaluated its
hERG potassium ion channel inhibition profiles. As shown in Table 4, these results
displayed that (S)ꢀ27 and its racemic compound 27 have lower hERG potassium ion
channel inhibition than the marketed drug silodosin and dofetilide.
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Table 4. Inhibition assay of the hERG potassium ion channel
hERG inhibition
Compound
IC50 (ꢁM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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44
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49
50
51
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55
56
57
58
59
60
2
7
1
6.5
3.2
(
S)ꢀ27
1
a
silodosin
8
.2
a
dofetilide
0
.2
a
The reference drug
3
.6 Safety Evaluation: Acute Toxicity for Compound (S)ꢀ27
We performed acute toxicity tests in Kunming mice. Compound (S)ꢀ27 was given
orally in a singleꢀdosing experiment at 500 mg/kg. The animals were closely monitored,
and no animal died within 7 days after treatment. Body weights were not affected and the
animals had shining fur. In addition, the behavior of the mice was unaffected by the
singleꢀdose administration of (S)ꢀ27 at 500 mg/kg.
3
.7 Preliminary Pharmacokinetic Evaluation for Compound (S)ꢀ27.
Compound (S)ꢀ27 was further evaluated for its preliminary pharmacokinetic profile in
rat. The results showed that compound (S)ꢀ27 had a good pharmacokinetic profile, with
0.9% oral bioavailability, an AUC0ꢀt (area under the plasma concentrationꢀtime curve
6
from zero (0) hours to time (t)) of 2274 ng/mL*h, and had a good half life (Table 5). We
evaluated the plasma protein binding of this compound in rat. The results showed that
compound (S)ꢀ27 present a high plasma protein binding, which is about 97%.
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Preliminary pharmacokinetic parameters for compound (S)ꢀ27.
Dose
T
max
C
max
AUC0ꢀt
AUC0ꢀ∞
MRT
h
t
1/2
CLz
F
mg/kg
h
ng/mL ng/mL*h ng/mL*h
h
L/h/kg
%
0
1
2
3
4
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6
7
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9
0
1
2
3
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5
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7
8
9
0
1
2
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5
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8
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1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
a
ig
20
10
1.0
684
855
2274
1867
2277
1877
2.52 1.18
1.79 0.98
/
60.9
/
b
iv
0.25
5.79
a
b
Intragastric administration (oral gavage). Intravenous injection.
. Conclusions
A class of novel indoleꢀcontaining THPB derivatives were designed and synthesized,
4
and their antagonistic activities against α1AꢀARs were evaluated using calcium
mobilization assays. Among them, seven compounds displayed excellent antagonistic
activities against α1AꢀARs, with IC50s < 250 nM. Compound (S)ꢀ27 showed the best
biological activity, with an IC50 of 12.8 nM. More importantly, (S)ꢀ27 showed less
inhibition against α1Bꢀ and α1DꢀARs and excellent selectivity towards α1AꢀAR, which is
superior to silodosin. Compound (S)ꢀ27 potently inhibited norepinephrineꢀinduced urethra
smooth muscle contraction without inhibiting the aortic contraction, displaying better
uroselectivity than the control drug silodosin. Additionally, compound (S)ꢀ27 has lower
hERG potassium ion channel inhibition than the marketed drug silodosin and dofetilide.
Preliminary pharmacokinetics studies in rats indicated that compound (S)ꢀ27 has a good
pharmacokinetic profile. In summary, on the basis of the excellent antagonistic activities
and selectivities against α ꢀARs, these novel indoleꢀcontaining THPB derivatives,
1
A
especially compound (S)ꢀ27, have promising potential as candidate selective α1AꢀAR
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antagonist drugs for the treatment of BPH.
5
. Experimental Section
.1 Chemistry. Chemicals and solvents were purchased from commercial sources and
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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40
41
42
43
44
45
46
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49
50
51
52
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57
58
59
60
used without further purification. Analytical thin layer chromatography (TLC) was HSGF
54 (0.15ꢀ0.2 mm thickness, YantaiHuiyou Company, China). Column chromatography
2
was performed with CombiFlash® Companion system (Teledyne Isco, Inc.). All target
1
products were characterized by H NMR and LCꢀMS (ESI), and some products were also
13
1
characterized by C NMR. H NMR spectra were recorded on a Brucker AMX 300 or 400
13
MHz instrument (TMS as IS). C NMR spectra were recorded on a Brucker AMX 100
MHz instrument (TMS as IS). Chemical shifts were reported in parts per million (ppm).
Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). In addition, the purity of all tested compound was
determined in the system condition of CH OH/H O which CH OH gradient changed from
3
2
3
70%(v/v) to 85%(v/v) by Agilent 1260 with binary pump, photodiode array detector
(DAD), Agilent Eclipse XDBꢀC18 (4.6×150mm, 5 ꢁm particle size). The percentage of
purity of all products were more than 96%.
General Synthetic Procedures for the Target Compounds 13ꢀ32 and 34ꢀ36
(Compound 13 as the example)
2ꢀ(3,4ꢀdimethoxyphenyl)ethanꢀ1ꢀamine (1.5 g, 8.562 mmol), 2ꢀ(1Hꢀindolꢀ3ꢀyl)acetic
acid (1.5 g, 8.276 mmol, 1 equiv) and 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hydrochloride (2.8 g, 14.583 mmol, 1.7 equiv) were dissolved in 30 mL of
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dichloromethane. Triethylamine (2.36 mL, 17 mmol, 2 equiv) was added dropwise to the
solution and the mixture was stirred for 12 h at rt. The reaction mixture diluted with water.
The organic layer was separated and washed with water (30 mL), dried (Na SO ). The
0
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0
2
4
combined organic phase was evaporated under reduced pressure to get the crude product
+
(
10a) and used for the next step without further purification. ESIꢀMS m/z: 339 [M+H] .
Nꢀ(3,4ꢀdimethoxyphenethyl)ꢀ2ꢀ(1Hꢀindolꢀ3ꢀyl)acetamide (10a) (2.1 g, 6.206 mmol)
3
was dissolved in 30 mL of acetonitrile and added POCl (2.1 mL, 13.2 mmol, 2 equiv).
The solution was heated to reflux under argon for 1.5 h. The solvents were evaporated
under reduced pressure. The pH of the mixture was adjusted to alkalinity with the addition
3
of saturated NaHCO . The organic layer was separated and washed with water. The
combined organic phase was evaporated under reduced pressure to get the crude product
+
(
11a) and used for the next step without further purification. ESIꢀMS m/z: 321 [M+H] .
The intermediate 11a was dissolved in 20 mL of methanol, and NaBH (2.5 g, 66
4
mmol, 10 equiv) was added in batches at 0 °C. The mixture was stirred for 10 h at rt. The
reaction mixture was quenched with water and extracted with ethylacetate. The organic
layer was washed with satd brine, and the combined organic phase was evaporated under
reduced pressure to get the crude product, which was purified by flash chromatography on
1
silica gel to get key intermediate 12a (1.5 g, 4.658 mmol, 80% over two steps). H NMR
(CDCl
3
, 400 MHz): δ 8.66 (br, 1H), 7.69ꢀ7.67 (m, 1H), 7.34ꢀ7.26 (m, 1H), 7.23ꢀ7.11 (m,
2H), 7.17ꢀ7.12 (m, 1H), 7.00ꢀ6.99 (m, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 4.32ꢀ4.28 (m, 1H),
3.87 (s, 3H), 3.85 (s, 3H), 3.45ꢀ3.39 (m, 2H), 3.23ꢀ3.19 (m, 1H), 3.13ꢀ3.05 (m, 1H),
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+
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.91ꢀ2.85 (m, 1H), 2.79ꢀ2.73 (m, 1H), 2.23 (br, 1H). ESIꢀMS m/z: 323 [M+H] .
The key intermediate 12a (1.5g, 4.658 mmol), 5 mL of formaldehyde and 1 mL of
formic acid was dissolved in 30 mL of acetonitrile. The mixture was stirred for 2 h at
0~90 °C. The pH of the mixture was adjusted to alkalinity with the addition of satd
NaHCO . The organic layer was separated and washed with water. The combined organic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
3
phase was evaporated under reduced pressure and then chromatographed on silica gel to
1
give the target product 13 (1.28 g, 82%). H NMR (CD
3
OD, 400 MHz): δ 7.47ꢀ7.41 (m,
2
H), 7.16ꢀ7.10 (m, 1H), 7.07ꢀ7.02 (m, 1H), 6.90ꢀ6.88 (m, 1H), 6.69 (s, 1H), 5.45 (m, 1H),
4.20ꢀ4.15 (m, 1H), 3.97ꢀ3.90 (m, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.67ꢀ3.63 (m, 1H),
13
3
.43ꢀ3.37 (m, 1H), 3.22ꢀ3.19 (m, 1H), 3.07ꢀ2.96 (m, 1H), 2.82ꢀ2.62 (m, 2H). C NMR
(125 MHz, CDCl
3
) δ 147.68, 147.65, 136.48, 133.65, 130.67, 127.34, 126.83, 121.06,
1
19.46, 118.11, 112.11, 110.20, 110.16, 108.24, 65.75, 59.77, 56.25, 55.87, 51.70, 51.58,
+
+
29.58, 29.47. ESIꢀMS m/z: 335 [M+H] . EIꢀHRMS m/z calcd C H N O (M ) 334.1681,
21
22
2
2
found 334.1674.
2,3ꢀDimethoxyꢀ8ꢀisobutylꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine
(
14). This compound was prepared by replacement of formaldehyde with 3ꢀmethylbutanal
1
using a similar synthetic procedure of product 13. H NMR (CDCl
3
, 400 MHz): δ 7.81 (br,
1
H), 7.50ꢀ7.48 (m, 1H), 7.36ꢀ7.32 (m, 1H), 7.20ꢀ7.08 (m, 2H), 6.72 (s, 1H), 6.66 (s, 1H),
4.30ꢀ4.25 (m, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.86ꢀ3.84 (m, 1H), 3.24ꢀ3.10 (m, 2H),
.92ꢀ2.88 (m, 2H), 2.82ꢀ2.70 (m, 2H), 1.93ꢀ1.83 (m, 1H), 1.55ꢀ1.46 (m, 1H), 1.30ꢀ1.26 (m,
2
1
3
3
1H), 1.02ꢀ0.98 (m, 6H). C NMR (125 MHz, CDCl ) δ 147.18, 146.73, 135.52, 126.61,
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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21.07, 118.92, 117.51, 111.22, 110.24, 109.18, 68.56, 55.57, 55.44, 51.13, 46.26, 43.78,
+
2
9.15, 25.77, 24.71, 22.65, 21.91. ESIꢀMS m/z: 391 [M+H] . EIꢀHRMS m/z calcd
+
C H N O (M ) 390.2307, found 390.2296.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
25 30
2
2
2,3ꢀDimethoxyꢀ8ꢀphenylꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine
(15). This compound was prepared by replacement of formaldehyde with benzaldehyde
1
using a similar synthetic procedure of product 13. H NMR (CDCl
3
, 400 MHz): δ
7.61ꢀ7.57 (m, 1H), 7.48ꢀ7.44 (m, 2H), 7.41ꢀ7.34 (m, 3H), 7.25ꢀ7.15 (m, 2H), 7.13ꢀ7.07 (m,
H), 6.92 (s, 1H), 6.60 (s, 1H), 4.61 (s, 1H), 4.15ꢀ4.08 (m, 1H), 3.94 (s, 3H), 3.86 (s, 3H),
2
1
3
3.51ꢀ3.45 (m, 1H), 3.07ꢀ3.02 (m, 1H), 2.95ꢀ2.86 (m, 2H), 2.54ꢀ2.42 (m, 2H). C NMR
(125 MHz, CDCl ) δ 146.46, 140.44, 136.31, 134.04, 129.48, 127.95, 127.86, 127.20,
3
126.34, 126.11, 120.50, 118.38, 117.21, 110.16, 109.84, 107.99, 107.38, 66.20, 59.34,
+
55.16, 54.84, 47.43, 28.80, 28.72. ESIꢀMS m/z: 411 [M+H] . EIꢀHRMS calcd
+
C H N O (M ) 410.1994, found 410.1989.
27
26
2
2
2
,3ꢀDimethoxyꢀ8ꢀ(4ꢀfluorophenyl)ꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]qui
nolizine (16). This compound was prepared by replacement of formaldehyde with
1
4
ꢀfluorobenzaldehyde using a similar synthetic procedure of product 13. H NMR (CDCl
3
,
400 MHz): δ 7.61ꢀ7.58 (m, 1H), 7.45ꢀ7.40 (m, 1H), 7.25ꢀ7.18 (m, 2H), 7.15ꢀ7.03 (m, 4H),
.91 (s, 1H), 6.60 (s, 1H), 4.61 (s, 1H), 4.02ꢀ3.98 (m, 1H), 3.94 (s, 3H), 3.86 (s, 3H),
6
1
3
3.50ꢀ3.44 (m, 1H), 3.04ꢀ2.98 (m, 1H), 2.94ꢀ2.85 (m, 2H), 2.56ꢀ2.41 (m, 2H). C NMR
(125 MHz, CDCl ) δ 162.84, 160.91, 147.54, 147.48, 137.29, 136.75, 133.51, 131.82,
3
131.24, 131.18, 126.95, 126.09, 121.31, 118.81, 118.32, 115.09, 114.92, 112.80, 111.52,
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+
1
10.57, 108.14, 62.59, 56.04, 55.89, 51.11, 46.85, 29.83, 27.99. ESIꢀMS m/z: 429 [M+H] .
+
EIꢀHRMS calcd C27
H
25FN
2
O
2
(M ) 428.1900, found 428.1897.
2
,3ꢀDimethoxyꢀ8ꢀ(4ꢀmethoxyphenyl)ꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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39
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
quinolizine (17). This compound was prepared by replacement of formaldehyde with
1
4
ꢀmethoxybenzaldehyde using a similar synthetic procedure of product 13. H NMR
(
CDCl
(m, 1H), 7.15ꢀ7.10 (m, 2H), 6.93ꢀ6.90 (m, 3H), 6.61 (s, 1H), 4.57 (s, 1H), 4.01ꢀ3.98 (m,
H), 3.96 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H), 3.51ꢀ3.45 (m, 1H), 3.10ꢀ3.06 (m, 1H),
3
, 400 MHz): δ 7.62ꢀ7.59 (m, 1H), 7.38ꢀ7.36 (m, 1H), 7.28ꢀ7.25 (m, 1H), 7.21ꢀ7.18
1
1
3
2.96ꢀ2.86 (m, 2H), 2.56ꢀ2.40 (m, 2H). C NMR (125 MHz, CDCl
3
) δ 159.08, 147.01,
135.80, 129.67, 126.69, 121.01, 118.91, 117.73, 113.70, 110.70, 110.35, 108.52, 107.89,
+
66.06, 59.99, 55.70, 55.38, 54.82, 47.69, 29.24. ESIꢀMS m/z: 441 [M+H] . EIꢀHRMS
+
calcd C28
H
28
N
2
O
3
(M ) 440.2100, found 453.2451.
2,3ꢀMethylenedioxyꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine (18).
This compound was prepared by replacement of 2ꢀ(3,4ꢀdimethoxyphenyl)ethanꢀ1ꢀamine
with 2ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine using a similar synthetic procedure of
1
product 13. H NMR (CD
3
OD, 400 MHz): δ7.42ꢀ7.39 (m, 1H), 7.27ꢀ7.17 (m, 2H),
7.08ꢀ7.00 (m, 1H), 6.83 (s, 1H), 6.57 (s, 1H), 5.89 (s, 2H), 5.45 (s, 1H), 4.20ꢀ4.03 (m, 2H),
13
3
.74ꢀ3.64 (m, 2H), 3.22ꢀ3.04 (m, 2H), 2.77ꢀ2.62 (m, 2H). C NMR (125 MHz, DMSOꢀd6
)
δ 145.72, 145.44, 136.00, 133.07, 131.40, 127.47, 126.84, 120.62, 119.02, 117.59, 109.70,
08.03, 107.65, 105.99, 100.54, 65.26, 59.48, 54.89, 51.10, 50.84, 29.38, 29.17. ESIꢀMS
1
+
+
18 2 2
m/z: 319 [M+H] . EIꢀHRMS calcd C20H N O (M ) 318.1368, found 318.1366.
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(
S)ꢀ2,3ꢀMethylenedioxyꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine
Sꢀ18). This compound was prepared by replacement of 2ꢀ(3,4ꢀdimethoxyphenyl)ethanꢀ
(
1
ꢀamine with 2ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine using a similar synthetic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
procedure of product 13. H NMR (CD
3
OD, 400 MHz): δ7.60ꢀ7.57 (m, 1H), 7.42ꢀ7.39 (m,
1
H), 7.15ꢀ7.12 (m, 1H), 7.08ꢀ7.03 (m, 1H), 6.87 (s, 1H), 6.60 (s, 1H), 5.90 (s, 2H), 5.48 (s,
H), 4.30ꢀ4.25 (m, 2H), 3.82ꢀ3.72 (m, 2H), 3.42ꢀ3.29 (m, 2H), 2.80ꢀ2.66 (m, 2H). ESIꢀMS
1
+
13
m/z: 319 [M+H] . C NMR (125 MHz, DMSOꢀ
6
d ) δ 145.72, 145.44, 136.00, 133.07,
1
31.40, 127.47, 126.84, 120.62, 119.02, 117.59, 109.70, 108.03, 107.65, 105.99, 100.54,
+
65.26, 59.48, 54.89, 51.10, 50.84, 29.38, 29.17. ESIꢀMS m/z: 319 [M+H] . EIꢀHRMS
+
calcd C20
H
18
N
2
O
2
(M ) 318.1368, found 318.1360.
2,3ꢀMethylenedioxyꢀ8ꢀethylꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizi
ne (19). This compound was prepared by replacement with
2ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine and propionaldehyde using a similar synthetic
1
procedure of product 13. H NMR (CDCl
3
, 400 MHz): δ 7.88 (br, 1H), 7.49ꢀ7.45 (m, 1H),
7.34ꢀ7.31 (m, 1H), 7.19ꢀ7.07 (m, 2H), 6.68 (s, 1H), 6.62 (s, 1H), 5.93 (s, 2H), 4.28ꢀ4.23
m, 1H), 3.70ꢀ3.65 (m, 1H), 3.21ꢀ3.07 (m, 2H), 2.95ꢀ2.70 (m, 4H), 1.93ꢀ1.75 (m, 2H),
(
13
1.19ꢀ1.12 (m, 3H). C NMR (125 MHz, CDCl
3
) δ 145.55, 145.12, 135.10, 136.00, 132.50,
1
26.55, 121.01, 118.85, 117.52, 110.19, 108.22, 106.71, 106.18, 100.17, 62.24, 51.73,
+
46.15, 29.68, 27.51, 26.03, 17.92, 11.42. ESIꢀMS m/z: 347 [M+H] . EIꢀHRMS calcd
+
C
22
H
22
N
O
2 2
(M ) 346.1681, found 346.1672.
2,3ꢀMethylenedioxyꢀ8ꢀisobutylꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinol
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izine
(20).
This
compound
was
prepared
by
replacement
with
2ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine and 3ꢀmethylbutanal using a similar synthetic
1
procedure of product 13. H NMR (CDCl , 400 MHz): δ 7.85 (br, 1H), 7.49ꢀ7.46 (m, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
7
.34ꢀ7.31 (m, 1H), 7.20ꢀ7.08 (m, 2H), 6.69 (s, 1H), 6.63 (s, 1H), 5.93 (s, 2H), 4.23ꢀ4.14
(m, 1H), 3.87ꢀ3.82 (m, 1H), 3.21ꢀ3.09 (m, 2H), 2.89ꢀ2.85 (m, 2H), 2.78ꢀ2.69 (m, 2H),
13
1.83ꢀ1.75 (m, 1H), 1.31ꢀ1.22 (m, 2H), 1.07ꢀ1.03 (m, 6H). C NMR (125 MHz, DMSOꢀ
d
6
)
δ 145.49, 145.04, 136.41, 136.00, 133.37, 126.68, 120.36, 118.12, 117.44, 110.85, 108.35,
06.86, 105.10, 100.40, 58.30, 51.04, 45.78, 43.86, 29.92, 25.34, 24.74, 23.56, 21.83.
1
+
+
ESIꢀMS m/z: 375 [M+H] . EIꢀHRMS calcd C24
,3ꢀMethylenedioxyꢀ8ꢀ(4ꢀfluorophenyl)ꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀ
g]quinolizine (21). This compound was prepared by replacement with
26 2 2
H N O (M ) 374.1994, found 374.1992.
2
2ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine and 4ꢀfluorobenzaldehyde using a similar
1
synthetic procedure of product 13. H NMR (CDCl , 400 MHz): δ7.72 (br, 1H), 7.58ꢀ7.55
3
(m, 1H), 7.30ꢀ7.27 (m, 1H), 7.22ꢀ7.14 (m, 4H), 7.01ꢀ6.94 (m, 2H), 6.69 (s, 1H), 6.56 (s,
1H), 5.89 (s, 2H), 5.20ꢀ5.18 (m, 1H), 4.16ꢀ4.11 (m, 1H), 3.27ꢀ3.21 (m, 2H), 2.97ꢀ3.85 (m,
13
3H), 2.72ꢀ2.65 (m, 1H). C NMR (125 MHz, CDCl
3
) δ 145.51, 145.42, 135.89, 131.96,
131.45, 126.60, 126.46, 121.49, 119.07, 117.80, 114.62, 114.45, 110.45, 109.14, 108.01,
+
105.83, 100.22, 62.53, 51.35, 46.84, 29.37, 27.88. ESIꢀMS m/z: 413 [M+H] . EIꢀHRMS
+
calcd C26
H
21FN
2
O
2
(M ) 412.1587, found 412.1581.
2,3ꢀMethylenedioxyꢀ8ꢀ(4ꢀmethoxyphenyl)ꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2
,3ꢀg]quinolizine (22). This compound was prepared by replacement with
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2
2
2
2
2
2
3
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5
5
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6
2
ꢀ(benzo[d][1,3]dioxolꢀ5ꢀyl)ethanꢀ1ꢀamine and 4ꢀmethoxybenzaldehyde using a similar
1
synthetic procedure of product 13. H NMR (CDCl
3
, 400 MHz): δ 7.78 (br, 1H), 7.58ꢀ7.55
(m, 1H), 7.26ꢀ7.24 (m, 1H), 7.19ꢀ7.10 (m, 4H), 6.83ꢀ6.80 (m, 2H), 6.69 (s, 1H), 6.56 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
1
H), 5.89 (s, 2H), 5.06 (s, 1H), 4.16ꢀ4.09 (m, 1H), 3.76 (s, 3H), 3.26ꢀ3.13 (m, 2H),
1
3
.97ꢀ2.88 (m, 1H), 2.85ꢀ2.78 (m, 2H), 2.67ꢀ2.61 (m, 1H). C NMR (125 MHz, CDCl
3
) δ
58.81, 145.39, 135.88, 132.64, 130.12, 126.79, 126.53, 121.27, 118.91, 117.70, 113.02,
110.41, 108.94, 107.95, 105.82, 100.18, 62.71, 54.79, 51.48, 47.00, 29.43, 28.30.ESIꢀMS
+
+
m/z: 425 [M+H] . EIꢀHRMS calcd C H N O (M ) 424.1787, found 424.1777.
27
24
2
3
3ꢀMethoxyꢀ5,8,14,14aꢀ tetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine (23). This
compound was prepared by replacement of 2ꢀ(3,4ꢀdimethoxyphenyl)ethanꢀ1ꢀamine with
1
2ꢀ(4ꢀmethoxyphenyl)ethanꢀ1ꢀamine using a similar synthetic procedure of product 13. H
3
NMR (CDCl , 400 MHz): δ7.49ꢀ7.46 (m, 1H), 7.42ꢀ7.39 (m, 1H), 7.29ꢀ7.13 (m, 3H),
6.84ꢀ6.80 (m, 1H), 6.70 (s, 1H), 5.48ꢀ5.38 (m, 1H), 3.97ꢀ3.88 (m, 1H), 3.83 (s, 3H),
13
3
.72ꢀ3.61 (m, 1H), 3.37ꢀ3.19 (m, 2H), 3.05ꢀ3.01 (m, 1H), 2.79ꢀ2.68 (m, 3H). C NMR
(125 MHz, CDCl
3
) δ 157.44, 135.83, 134.94, 131.57, 129.48, 126.89, 121.06, 119.34,
117.56, 112.58, 112.19, 108.68, 108.59, 66.14, 59.08, 54.77, 51.13, 50.86, 29.22, 28.65.
+
+
ESIꢀMS m/z: 305 [M+H] . EIꢀHRMS calcd C20
20 2
H N O(M ) 304.1576, found 304.1571.
3ꢀMethoxyꢀ8ꢀmethylꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine (24).
This compound was prepared by replacement with 2ꢀ(4ꢀmethoxyphenyl)ethanꢀ1ꢀamine and
1
acetaldehyde using a similar synthetic procedure of product 13. H NMR (CDCl
3
, 400
MHz): δ 7.50ꢀ7.47 (m, 1H), 7.37ꢀ7.28 (m, 1H), 7.18ꢀ7.04 (m, 3H), 6.80ꢀ6.78 (m, 1H), 6.66
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Page 29 of 54
Journal of Medicinal Chemistry
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(
s, 1H), 3.96ꢀ3.90 (m, 1H), 3.89ꢀ3.85 (m, 1H), 3.80 (s, 3H), 3.20ꢀ3.12 (m, 3H), 2.85ꢀ2.63
13
(m, 3H), 1.30ꢀ1.25 (m, 3H). C NMR (125 MHz, CDCl
3
) δ157.87, 136.06, 135.44, 131.67,
1
5
27.54, 121.50, 119.36, 118.07, 113.37, 112.22, 110.72 108.24, 107.28, 60.08, 55.54,
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+
5.24, 53.43, 51.44, 46.78, 30.36, 28.27. ESIꢀMS m/z: 319 [M+H] . ESIꢀHRMS calcd
+
C
21
H
22
N
2
O (M+H ) 319.1805, found 319.1804.
3
ꢀMethoxyꢀ8ꢀcyclopropylꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quinolizine
(25). This compound was prepared by replacement with
2
ꢀ(4ꢀmethoxyphenyl)ethanꢀ1ꢀamine and cyclopropanecarbaldehyde using a similar
1
synthetic procedure of product 13. H NMR (CDCl
3
, 400 MHz): δ 8.21 (br, 1H), 7.52ꢀ7.47
(m, 1H), 7.38ꢀ7.32 (m, 1H), 7.26ꢀ7.23 (m, 1H), 7.19ꢀ7.09 (m, 2H), 6.82ꢀ6.79 (m, 1H), 6.69
(s, 1H), 3.92ꢀ3.85 (m, 2H), 3.82 (s, 3H), 3.46ꢀ3.38 (m, 1H),3.21ꢀ3.12 (m, 1H), 3.07ꢀ2.98
13
(m, 1H), 2.83ꢀ2.61 (m, 3H), 1.27ꢀ1.13 (m, 1H), 0.95ꢀ0.92 (m, 2H), 0.67ꢀ0.63 (m, 2H). C
NMR (125 MHz, CDCl ) δ 157.71, 136.42, 135.95, 127.92, 126.88, 120.93, 118.61,
3
1
18.01, 113.33, 112.60, 111.41, 106.42, 64.33, 55.43, 52.62, 47.29, 30.67, 28.99,13.63,
+
+
5.16, 2.05. ESIꢀMS m/z: 345 [M+H] . EIꢀHRMS calcd C23
44.1871.
3ꢀMethoxyꢀ8ꢀ(4ꢀmethoxyphenyl)ꢀ5,8,14,14aꢀtetrahydroꢀ6Hꢀbenzo[a]indolo[2,3ꢀg]quin
olizine (26). This compound was prepared by replacement with
24 2
H N O (M ) 344.1889, found
3
2ꢀ(4ꢀmethoxyphenyl)ethanꢀ1ꢀamine and 4ꢀmethoxybenzaldehyde using a similar synthetic
1
procedure of product 13. H NMR (CDCl
3
, 400 MHz): δ 7.80 (br, 1H), 7.57ꢀ7.54 (m, 1H),
7.37ꢀ7.35 (m, 1H), 7.27ꢀ7.23 (m, 1H), 7.18ꢀ7.09 (m, 4H), 6.83ꢀ6.78 (m, 2H), 6.76ꢀ6.72 (m,
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