Design, Synthesis, and Biological Evaluation of Indoline and Indole Derivatives as Potent and Selective α1A-Adrenoceptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.5b02023

A series of indoline and indole derivatives were designed, synthesized, and evaluated as selective α_1A-adrenergic receptor (α_1A-AR) antagonists for the treatment of benign prostatic hyperplasia (BPH). In this study, two highly selective and potent α_1A-AR antagonists, compounds (R)-14r (IC₅₀ = 2.7 nM, α_1B/α_1A = 640.1, α_1D/α_1A = 408.2) and (R)-23l (IC₅₀ = 1.9 nM, α_1B/α_1A = 1506, α_1D/α_1A = 249.6), which exhibited similar activities and better selectivities in cell-based calcium assays as compared with the marketed drug silodosin (IC₅₀ = 1.9 nM, α_1B/α_1A = 285.9, α_1D/α_1A = 14.4), were identified. In the functional assays with isolated rat tissues, compounds (R)-14r and (R)-23l also showed high potency and uroselectivity. Most importantly, (R)-14r and (R)-23l can significantly decrease the micturition frequency and increase the mean voided volume of the BPH rats in a dose-dependent manner, making them worthy of further investigation for the development of anti-BPH agents.

Full text of DOI:10.1021/acs.jmedchem.5b02023

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Article
Design, Synthesis and Biological Evaluation of Indoline and Indole
Derivatives as Potent and Selective #1A-Adrenoceptor Antagonists
Fei Zhao, Jing Li, Ying Chen, Yanxin Tian, Chenglin Wu,
Yanan Xie, Yu Zhou, Jiang Wang, Xin Xie, and Hong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b02023 • Publication Date (Web): 31 Mar 2016
Downloaded from http://pubs.acs.org on April 1, 2016
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Design, Synthesis and Biological Evaluation of
Indoline and Indole Derivatives as Potent and
Selective α ꢀAdrenoceptor Antagonists
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A
†
,||
‡,||
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Fei Zhao, Jing Li, Ying Chen, Yanxin Tian, Chenglin Wu, Yanan Xie, Yu Zhou, Jiang
‡
,‡
,‡
Wang, Xin Xie,* Hong Liu*
†
Antibiotics Research and Reꢀevaluation Key Laboratory of Sichuan Province, Sichuan
Industrial Institute of Antibiotics, Chengdu University, 168 Hua Guan Road, Chengdu
610052, People’s Republic of China
‡
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, People’s Republic of
China
Abstract: A series of indoline and indole derivatives were designed, synthesized and
evaluated as selective α1Aꢀadrenergic receptor (α1AꢀAR) antagonists for the treatment of
benign prostatic hyperplasia (BPH). In this study, two highly selective and potent α ꢀAR
1A
antagonists, compounds (R)ꢀ14r (IC50 = 2.7 nM, α1B/α1A = 640.1, α1D/α1A = 408.2) and
R)ꢀ23l (IC50 = 1.9 nM, α1B/α1A = 1506, α1D/α1A = 249.6), which exhibited similar activities
(
and better selectivities in cellꢀbased calcium assays as compared with the marketed drug
silodosin (IC50 = 1.9 nM, α1B/α1A = 285.9, α1D/α1A = 14.4), were identified. In the functional
assays with isolated rat tissues, compounds (R)ꢀ14r and (R)ꢀ23l also showed high potency
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and uroselectivity. Most importantly, (R)ꢀ14r and (R)ꢀ23l can significantly decrease the
micturition frequency and increase the mean voided volume of the BPH rats in a
doseꢀdependent manner, making them worthy of further investigation for the development of
antiꢀBPH agents.
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Introduction
Benign prostatic hyperplasia (BPH) is one of the most common diseases of middle and old
1ꢀ3
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aged men. It is also the main cause of lower urinary tract symptoms (LUTS) in males.
LUTS can be categorized into filling or irritative symptoms (increased frequency of urination,
increased urgency of urination, painful urination, and excessive passage of urine at night) and
voiding or obstructive symptoms (poor stream, hesitancy, terminal dribbling, incomplete
5
voiding, and overflow incontinence). The irritative and obstructive symptoms seriously
6ꢀ8
affect the health and quality of life of patients. Medical therapy and surgery are two
common treatments for LUTS associated with BPH, and medical therapy is the first line
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treatment for most patients.
Among the available medical therapies, αꢀadrenergic receptor
(αꢀAR) antagonists are an important class of drugs that can relieve the obstructive symptoms
16ꢀ20
of BPH via relaxing the smooth muscle tone of the lower urinary tract.
The αꢀARs are members of the G proteinꢀcoupled receptor (GPCR) superfamily, α
are the two subtypes of αꢀARs. In particular, three distinct α ꢀAR subtypes, namely α1A, α1B
and α , have been characterized by molecular cloning and functional studies,
1 2
and α
1
2
1ꢀ24
and they
1
D
25ꢀ27
are different in tissue distribution and biological functions.
The α1AꢀAR is predominantly
and blockade of this subtype
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expressed in the bladder neck, prostate and prostatic urethra,
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can alleviate BPH symptoms by relaxation of the lower urinary tract.
The α1B subtype is
extensively expressed in vascular smooth muscle, and antagonism of this receptor can lead to
cardiovascular side effects such as orthostatic hypotension, tachycardia, arrhythmia and
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dizziness.
The α ꢀAR is widely found in the bladder detrusor and epicardial coronary
1D
arteries, thus α1DꢀAR blockers can relieve voiding symptoms but at the risk of causing
35ꢀ37
coronary vasodilation.
Collectively, antagonists with better selectivities for α1AꢀAR over
α
1BꢀAR and α1DꢀAR may alleviate the symptoms associated with BPH efficiently without
causing significant cardiovascular side effects.
To date, three generations of αꢀAR antagonists have been developed, and seven αꢀAR
antagonists have been approved by US Food and Drug Administration for the treatment of
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BPH. The first generation is nonselective αꢀAR antagonists such as phenoxybenzamine; the
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second generation is nonselective α
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ꢀAR antagonists such as prazosin, terazosin,
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doxazosin and alfuzosin; the third generation is selective α1AꢀAR antagonists such as
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tamsulosin
and silodosin.
Compared with the first and second generation antagonists,
the third generation antagonists improve the BPH symptoms with minimal cardiovascular
side effects, due to their selective blockade of the α1AꢀAR present in the lower urinary tract
but avoiding the antagonism of the receptors in vascular smooth muscle. It is worth noting
that silodosin exhibits unprecedented selectivities for α1AꢀAR over both α1BꢀAR and α1DꢀAR
among the currently marketed drugs.
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Figure 1. Marketed αꢀAR antagonists.
Results and Discussion
Design of the target compounds. Although silodosin is a marketed drug used for the
treatment of BPH, there is very limited report on the structureꢀactivity relationship of
silodosin. Besides, silodosin is still associated with cardiovascular side effects such as
orthostatic hypotension and dizziness. Therefore, structural modification of silodosin was
carried out in this paper to improve its selectivity to reduce cardiovascular side effects. We
hypothesized that the R substituents on the benzene ring might modulate the antagonist
activities and subtype selectivities of the compounds (Figure 2). Therefore, we designed
indoline derivatives (14) of silodosin to evaluate the influence of the R substituents on the
antagonist activities and subtype selectivities. We also designed indole derivatives (23) using
a scaffold hopping strategy based on the structure of silodosin in order to identify more
potent and selective α1AꢀAR antagonists. In this paper, we report the design, synthesis and
biological evaluation of indoline and indole derivatives as potent and selective α1AꢀAR
antagonists.
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Figure 2. Design and modification strategies of the target compounds.
Synthesis procedures of the target compounds. The synthetic approach for the
preparation of the key intermediates 9 and 10 is outlined in Scheme 1. Nucleophilic
substitution of indoline with 3ꢀbromoꢀ1ꢀpropanol in the presence of K CO provided
2
3
compound 2 in 85% yield, which was acylated with benzoyl chloride to give intermediate 3.
Intermediate 3 was subjected to VilsmeierꢀHaack reaction to yield the aldehyde 4, which was
condensed with CH CH NO to form compound 5. Reduction of 5 with sodium borohydride
3
2
2
produced compound 6 in high yield, and subsequent formylation of 6 in the presence of DMF
and POCl produced the aldehyde 7. Condensation of 7 with hydroxylamine hydrochloride
3
gave intermediate 8. Hydrogenation of 8 provided the key intermediate 9 in 93% yield, and
the resolution of 9 using Lꢀ(+)ꢀtartaric acid produced the key intermediate 10.
a
Scheme 1. Synthesis of the key intermediates 9 and 10
a
o
Reagents and conditions: (a) 3ꢀbromoꢀ1ꢀpropanol, K
2
CO
3 3
, CH CN, 90 C, 12 h; (b) benzoyl
o
o
o
o
chloride, NEt
3
, DCM, 0 Cꢀ25 C, 8 h; (c) DMF, POCl
3
, DCE, 0 Cꢀ80 C, 2 h; (d)
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o
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CH
DMF, POCl
h) Pd/C, H
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CH
2
NO
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, CH
3
COONH
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, CH
3
COOH, 80 C, 4 h; (e) NaBH
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, MeOH, 0 Cꢀ25 C, 1 h; (f)
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, 0 Cꢀ80 C, 2 h; (g) hydroxylamine hydrochloride, Py, Ac
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O, THF, 70 C, 36 h;
o
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, MeOH:THF = 1:1, 40 C, 48 h; (i) Lꢀ(+)ꢀtartaric acid, Acetone:H O = 1:1, 48 h.
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The desired products 14aꢀ14t and (R)ꢀ14r were synthesized according to the pathway
shown in Scheme 2. Reductive amination of key intermediates 9 or 10 with compound 11
produced compound 12, which was hydrolyzed by NaOH to give compound 13. A further
hydrolysis of the cyano group of 13 afforded the desired products 14aꢀ14t and (R)ꢀ14r. In
particular, compound (S)ꢀ14r was prepared by the resolution of racemic 14r.
a
Scheme 2. Synthesis of the target compounds 14aꢀ14t, (R)ꢀ14r and (S)ꢀ14r
a
Reagents and conditions: (j) NaBH(OAc) , DCE, rt, 12 h; (k) 1 mol/L NaOH, MeOH, rt, 2 h;
3
2 2
(l) 5 mol/L NaOH, 30% H O , DMSO, 48 h.
The synthetic method of the key intermediate 17 is shown in Scheme 3. Oxidation of 7
with 2, 3ꢀDichloroꢀ5, 6ꢀdicyanoꢀ1, 4ꢀbenzoquinone (DDQ) gave compound 15 in 90% yield,
which was condensed with hydroxylamine hydrochloride to produce compound 16.
Hydrogenation of 16 provided the key intermediate 17.
a
Scheme 3. Synthesis of the key intermediate 17
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a
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Reagents and conditions: (m) DDQ, EA, 25 C, 2 h；(n) hydroxylamine hydrochloride, Py,
o
o
2 2
Ac O, THF, 70 C, 36 h; (o) Pd/C, H , MeOH:THF = 1:1, 40 C, 48 h.
The synthetic route of the key intermediate 20 is described in Scheme 4. Protection of the
amino group of 10 with Boc O gave compound 18 in quantitative yield, and subsequent
2
oxidation of 18 with DDQ provided compound 19. Deprotection of 19 using CF COOH
3
yielded the key intermediate 20.
a
Scheme 4. Synthesis of the key intermediate 20
a
o
o
2 3
Reagents and conditions: (p) (Boc) O, NEt , DCM, 25 C, 10 h; (q) DDQ, EA, 25 C, 2 h; (r)
o
CF
3
COOH, DCM, 25 C, 10 h.
The desired products 23aꢀ23m and (R)ꢀ23l were synthesized according to the pathway
shown in Scheme 5. Reductive amination of the key intermediate 17 or 20 with compound 11
produced intermediate 21, which was hydrolyzed by NaOH to give intermediate 22. A further
hydrolysis of the cyano group of 22 afforded the desired products 23aꢀ23m and (R)ꢀ23l. In
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particular, compound (S)ꢀ23l was prepared by the resolution of racemic 23l.
a
Scheme 5. Synthesis of the target compounds 23aꢀ23m, (R)ꢀ23l and (S)ꢀ23l
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(
u) 5 mol/L NaOH, 30% H O , DMSO, 48 h.
2 2
Biological evaluation
Calcium assays. The antagonist activities of the indoline compounds were evaluated using
calcium mobilization assays in HEK293 cells expressing α1A, α1B or α1DꢀAR, the marketed
drug silodosin was used as the positive control (Table 1). Compared with silodosin (IC50 = 1.9
nM, α1B/α1A = 285.9, α1D/α1A = 14.4), compounds 14aꢀ14c with two substituents such as
2
ꢀBrꢀ4ꢀF, 3, 4ꢀOCH O and 2, 6ꢀdiꢀMe and compounds 14dꢀ14e with three substituents such
2
as 2, 3, 6ꢀtriꢀMe and 2, 4, 6ꢀtriꢀMe on the benzene ring showed moderate potency for α1A
IC50 >20 nM) and moderate subtype selectivities over α1B (14<α1B/α1A<150). We were
pleased to find that compounds 14fꢀ14j possessing hydrophobic groups such as Me, Et, OMe,
OCH CF , and OBn at the C2 position exhibited improved activities (3.2 nM<IC50<25.6 nM)
(
2
3
and selectivities (74<α /α <417) on the whole. Among them, compounds 14h and 14i
1B 1A
displayed the most potent activities with IC50 values of 4.8 nM and 3.2 nM, respectively.
Although compounds 14h and 14i showed almost equal activities as compared with silodosin,
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the subtype selectivities of compounds 14h (α1B/α1A = 126) and 14i (α1B/α1A = 83.4) were
unsatisfactory. Therefore, further optimization was carried out to improve the selectivities
while maintaining the activities of compounds 14h and 14i. Various substituents were
introduced on the benzene ring of 14h to produce compounds 14kꢀ14p. Compound 14k with
a 5ꢀF group and compound 14o with a 4ꢀMe group both showed a substantial loss of the
selectivities for α1AꢀAR over α1B, while compounds 14l (4ꢀF), 14m (4ꢀCl), 14n (4ꢀBr)
exhibited more than 2ꢀfold increase in selectivity, and the introduction of the bulky ethyl
group (14p) at the C4 position of 14h led to a sharp reduction in both activity and selectivity.
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Among the compounds obtained by the structural optimization of 14h, compound 14n (IC =
50
2.9 nM, α1B/α1A = 294.5) turned out to be the most potent molecule which displayed slightly
higher selectivity but slightly weaker activity than silodosin. Subsequently, the structure
optimization of 14i was conducted and different groups were introduced on the benzene ring
of 14i to produce compounds 14qꢀ14t. Compound 14q possessing a 4ꢀF group and compound
14s processing a 5ꢀBr group both suffered a significant decrease in subtype selectivity for
α1AꢀAR over α1B, compound 14t bearing a 5ꢀEt group showed a sharp reduction in potency.
To our delight, compound 14r with a 5ꢀCl group exhibited a considerable improvement in
selectivity for α1AꢀAR over both α1BꢀAR and α1DꢀAR while maintaining antagonist activity. It
is worth noting that compound 14r (IC50 = 3.2 nM, α1B/α1A = 371.4, α1D/α1A = 126.3)
displayed higher selectivities for α ꢀAR over α ꢀAR and α ꢀAR than silodosin.
1A
1B
1D
Furthermore, (R)ꢀ14r and (S)ꢀ14r were synthesized to evaluate the effect of the enantiomers
on the activities and selectivities. As a result, (R)ꢀ14r showed much better activities and
selectivities than (S)ꢀ14r. This indicates that R configuration was preferred for receptor
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function. (R)ꢀ14r (IC50 = 2.7 nM, α1B/α1A = 640.1, α1D/α1A = 408.2) was regarded as the most
potent and selective α1AꢀAR antagonist among all the indoline compounds.
Table 1. Antagonist activities and subtype selectivities of indoline compounds
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IC50 on calcium assays
Selectivity
Compound
14a
R
nM (Mean±SEM)
α
1A
α
1B
α
1D
α
1B/α1A
α1D/α1A
2ꢀBr, 4ꢀF
70.8±15.6
27.8±5.0
21.8±4.0
1361±323.2 436.2±59.6
2768±535.2 5208±1649
3266±1090 1621±356.4
19.2
7.3
1
4b
14c
4d
14e
4f
14g
4h
14i
4j
14k
4l
14m
3, 4ꢀOCH
2
O
99.5
187.1
74.2
3.1
2, 6ꢀdiꢀMe
2, 3, 6ꢀtriꢀMe
2, 4, 6ꢀtriꢀMe
2ꢀMe
149.5
14.8
1
324.2±71.1 4807±265.0 996.1±258.3
3139±118.5 1554±357.2
5992±1246 37.4±18.7
58.0±3.7
14.4±7.6
25.6±12.4
4.8±0.9
3.2±0.4
8.6±0.1
2.9±0.5
4.6±0.7
8.1±0.1
54.1
26.8
2.6
1
416.1
2ꢀEt
5320±1398 457.5±268.7 207.8
17.9
3.3
1
2ꢀOMe
610.2±167.8
263.9±9.1
15.8±2.4
13.4±3.8
1.7±0.3
0.4±0.1
49.6±8.7
126.0
83.4
2ꢀOCH
2
CF
3
4.2
1
2ꢀOBn
640.6±165.6
168.9±5.5
74.7
0.2
2ꢀOMe, 5ꢀF
2ꢀOMe, 4ꢀF
2ꢀOMe, 4ꢀCl
57.5
0.1
1
1284±261.7
280.0
265.4
10.8
24.2
2162±297.6 196.8±13.2
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1
4n
4o
2ꢀOMe, 4ꢀBr
2ꢀOMe, 4ꢀMe
2ꢀOMe, 4ꢀEt
2.9±0.2
7.6±1.7
57.0±8.6
4.0±0.7
3.2±0.6
2.7±0.5
866.1±65.0
617.6±53.6
274.0±51.2
60.6±9.1
294.5
81.3
39.5
40.0
93.2
8.0
1
1
1
4p
4q
2248±157.9 883.2±85.6
158.5±19.0 143.9±25.4
15.5
36.3
126.3
0.1
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
2ꢀOCH
2
CF
3
, 4ꢀF
1
4r
4s
2ꢀOCH
2
CF
3
, 5ꢀCl
1206±177.4 410.2±116.1 371.4
1
2ꢀOCH
2
CF
3
, 5ꢀBr
32.7±1.7
0.3±0.07
3464±1267
1107±5.3
12.2
222.2
640.1
46.9
1
4t
R)ꢀ14r
S)ꢀ14r
Silodosin
2ꢀOCH CF , 5ꢀEt 38.20±13.16 8488±6532
90.70
408.2
7.2
2
3
(
ꢀ
ꢀ
ꢀ
2.7±1.4
63.1±12.8
1.9±0.3
1736±189.3
(
2959±341.5 457.1±60.8
541.4±242.8 27.3±26.2
285.9
14.4
The activities and selectivities of indole compounds were also evaluated. As shown in
Table 2, compound 23a with a 2ꢀBrꢀ4ꢀF group on the benzene ring exhibited similar high
α
1AꢀAR potency (IC50 = 2.7 nM) and moderate selectivity for α1AꢀAR over α1BꢀAR (α1B/α1A
=
1
60.6). Compound 23b with a 2ꢀEt group displayed good selectivity (α1B/α1A = 208.9), but
low potency (IC = 20.7 nM). Compounds 23cꢀ23f bearing hydrophobic groups such as OMe,
5
0
2 3
OEt, OBn, OCH CF at the C2 position showed promising potency (1.9 nM<IC50<4.4 nM)
but low selectivities (α1B/α1A<100). Among them, Compounds 23c and 23f were found to be
the most potent molecules in terms of activity and selectivity. Subsequently, various groups
were introduced on the benzene ring of 23c and 23f in order to improve their selectivities for
α ꢀAR over α ꢀAR. The introduction of a 4ꢀCl (23i) or 4ꢀBr (23j) group on the benzene
1
A
1B
ring of 23c caused a slight reduction in selectivity, while the introduction of a 5ꢀF (23g) or
4
ꢀF (23h) group leaded to a significant increase in selectivity. However, the selectivities of
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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
2
3g and 23h were still lower than silodosin. To identify highly selective α1AꢀAR antagonists,
the structural modification of 23f was carried out. The introduction of a 5ꢀCl (23k) or 5ꢀMe
23l) group on the benzene ring of 23f resulted in a great improvement in selectivity while
(
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
maintaining the potency, and the introduction of the bulky 5ꢀEt group (23m) on the benzene
ring of 23f led to a sharp reduction in potency. Remarkably, compound 23l (IC50 = 2.2 nM,
α
1B/α1A = 998.6, α1D/α1A = 611.4) displayed comparable potency and higher selectivity than
silodosin. Further research revealed that (R)ꢀ23l showed much better activities and
selectivities than (S)ꢀ23l. (R)ꢀ23l (IC50 = 1.9 nM, α1B/α1A = 1506, α1D/α1A = 249.6) was
regarded as the most potent and selective α ꢀAR antagonist among all the indole compounds.
1A
Compared with silodosin, (R)ꢀ23l exhibited equal potency for α1AꢀAR, while the selectivity
for α1AꢀAR over α1BꢀAR is 5 times that of silodosin.
Table 2. Antagonist activities and subtype selectivities of indole compounds
IC50 on calcium assays
Selectivity
nM (Mean±SEM)
Compound
R
α
1A
α
1B
α
1D
α
1B/α1A
α1D/α1A
2
3a
3b
2ꢀBr, 4ꢀF
2ꢀEt
2.7±0.5
20.7±1.8
4.4±0.5
1.9±0.3
980.5±182.5
4320±567.9
262.9±33.4
35.6±5.1
21.7±1.3
0.4±0.1
0.4±0.1
2.6±0.6
160.6
208.9
59.2
8.1
0.02
0.09
1.4
2
2
3c
2ꢀOMe
2ꢀOEt
2
3d
18.7
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2
3e
3f
2ꢀOBn
2.7±0.04
1.9±0.4
130.6±29.4
180.1±17.3
70.9±7.3
0.1±0.001
15.9±4.0
0.4±0.08
10.6±2.6
31.6±4.5
162.2±18.8
94.0±30.8
47.7
92.7
0.04
8.2
2
2ꢀOCH CF
2
3
2
3g
3h
2ꢀOMe, 5ꢀF
2ꢀOMe, 4ꢀF
2ꢀOMe, 4ꢀCl
2ꢀOMe, 4ꢀBr
0.3±0.1
221.4
117.7
54.2
1.3
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
2
8.0±1.5
936.6±213.8
835.9±76.4
169.6±30.2
287.1±39.6
1.3
2
3i
3j
3k
3l
3m
R)ꢀ23l
15.4±0.4
3.7±0.6
2.0
2
46.0
44.0
74.1
611.4
221.9
249.6
8.2
2
2ꢀOCH CF , 5ꢀCl
1.3±1.0
227.7
998.6
443.5
1506
2
3
2
2ꢀOCH
2
CF
3
, 5ꢀMe
, 5ꢀEt
2.2±0.6
2197±463.9 1345±213.7
7583±587.8 3794±641.8
2862±846.7 474.3±156.4
2
2ꢀOCH
2
CF
3
17.1±3.3
1.9±0.4
(
ꢀ
ꢀ
ꢀ
(S)ꢀ23l
453.9±106.1
1.9±0.3
>10000
3717±210.8 >22.0
27.3±26.2 285.9
Silodosin
541.4±242.8
14.4
Functional assays with isolated rat tissues. The effects of compounds (R)ꢀ14r and
R)ꢀ23l, which displayed optimal potency and selectivities on α1AꢀAR, on the
(
noradrenalineꢀinduced contraction of isolated rat urethras and aortas were tested. The results
of three separate experiments are summarized in Table 3. Compounds (R)ꢀ14r and (R)ꢀ23l
inhibited the contraction of urethra smooth muscle with IC50 values of 30.1 nM and 76.8 nM,
respectively. Compared with silodosin, compound (R)ꢀ14r showed a similar potency while
compound (R)ꢀ23l exhibited slightly weaker potency on isolated urethra tissue. In addition,
compounds (R)ꢀ14r and (R)ꢀ23l displayed 8.8ꢀfold and 3.9ꢀfold tissue selectivities,
respectively. Both of them displayed higher uroselectivity than silodosin, which is very
promising for the reduction of cardiovascular side effects.
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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
Table 3. Functional studies of compounds (R)ꢀ14r and (R)ꢀ23l on isolated rat tissues
IC50 (nM)
Urethra
Uroselectivity
Compound
Aorta
263.6±22.4
297.8±46.0
26.6±3.0
Urethra/Aorta
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
(R)ꢀ14r
30.1±9.3
76.8±24.7
17.3±4.6
8.8
3.9
1.5
(R)ꢀ23l
Silodosin
Micturition behavior in BPH model rats. Similar to BPH patients, the BPH model rats
have significantly increased micturition frequency (Fig.1A) and significantly reduced mean
voided volume (Fig.1B) as compared with the sham rats. Silodosin significantly reduced the
urinary frequency and increased the mean voided volume in a doseꢀdependent manner, with a
minimal effective dose of 3 mg/kg (Fig.1A) and 1 mg/kg (Fig.1B), respectively. Both (R)ꢀ14r
and (R)ꢀ23l showed beneficial effects in the micturition behavior of BPH rats, with the
minimal effective dose of 10 mg/kg and 3 mg/kg (Fig.1A), respectively, in reducing
micturition frequency; and minimal effective dose of 3 mg/kg and 10 mg/kg (Fig.1B),
respectively, in increasing the mean voided volume. Although compounds (R)ꢀ14r and
(R)ꢀ23l showed slightly weaker potency in BPH rats as compared with slodosin, they can
significantly alleviate voiding symptoms of BPH rats, which makes them worthy of further
investigation for the development of antiꢀBPH agents.
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. Effects of (R)ꢀ14r and (R)ꢀ23l on the micturition parameters in BPH rats.
Micturition frequency (A) and mean voided volume (B) were measured in a metabolic cage.
###
*
**
P < 0.001, versus sham control. P < 0.05， P < 0.01 versus vehicle control.
Preliminary pharmacokinetic evaluation of compounds (R)ꢀ14r and (R)ꢀ23l. The
pharmacokinetic properties of (R)ꢀ14r and (R)ꢀ23l were evaluated in rats after intravenous
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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
(
10 mg/kg) and oral administration (20 mg/kg) (Table 4). Compound (R)ꢀ14r given orally at
2
0 mg/kg dosage displayed a Tmax of 4 h, a Cmax of 152.3 ng/mL, an AUC0ꢀ∞ of 1702.7
ng/mL*h, a mean retention time (MRT) of 6.22 h, and a t1/2 of 4.67 h. Compound (R)ꢀ14r
showed good exposure and oral bioavailability (F = 24.3%). Compound (R)ꢀ23l given orally
at 20 mg/kg dosage exhibited a Tmax of 0.5 h, a Cmax of 108.6 ng/mL, an AUC0ꢀ∞ of 613.2
ng/mL*h, a MRT of 3.54 h, and a t1/2 of 1.22 h, indicating the relatively low exposure and
oral bioavailability (F = 7.0%) of compound (R)ꢀ23l.
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
Table 4. Pharmacokinetic parameters of compounds (R)ꢀ14r and (R)ꢀ23l (po and iv) in rats.
Dose
mg/kg
20
T
max
C
max
AUC0ꢀ∞
MRT
h
t
1/2
CLz
F
%
Compound
h
ng/mL ng/mL*h
h
L/h/kg
po
4
152.3
1702.7
3506.4
613.2
6.22 4.67
2.34 1.63
3.54 1.22
/
2.85
/
24.3
/
(R)ꢀ14r
(R)ꢀ23l
iv
po
iv
10
0.083 3004.7
0.5 108.6
0.083 2425.1
20
7.0
/
10
4396.0
2.94 1.10 2.275
Rat plasma protein binding assays of compounds (R)ꢀ14r and (R)ꢀ23l. The rat plasma
protein binding (PPB) rate of the tested compounds was evaluated at the concentration of 2
µM using equilibrium dialysis. As shown in Table 5, compounds (R)ꢀ14r and (R)ꢀ23l
displayed PPB rate of 83.52% and 62.25%, respectively. They both showed much higher PPB
rate than silodosin (38.73%), this may led to their lower effective concentration in target
tissue and weaker potency in BPH rats. We speculate the high PPB rate as well as
unsatisfactory pharmacokinetic profiles of (R)ꢀ14r and (R)ꢀ23l are responsible for their
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slightly weaker potency in BPH rats as compared with silodosin, further optimization of
(R)ꢀ14r and (R)ꢀ23l aiming to improve their pharmacokinetic profiles and lower their PPB
rate to improve their potency in vivo is undergoing in our laboratory.
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
Table 5. Rat plasma protein binding rate of compounds (R)ꢀ14r and (R)ꢀ23l.
Rat plasma protein
Compound
binding rate (%)
(
R)ꢀ14r
83.52±0.38
62.25±0.97
38.73±1.90
98.48±0.01
(
R)ꢀ23l
Silodosin
Warfarin (positive control)
Conclusion
In this paper, we have described the design, synthesis, and biological evaluation of indoline
and indole derivatives as selective α1AꢀAR antagonists for the treatment of BPH.
Structureꢀactivity relationship (SAR) exploration led to the identification of two highly
selective and potent α1AꢀAR antagonists (R)ꢀ14r (IC50 = 2.7 nM, α1B/α1A = 640.1, α1D/α1A
=
4
08.2) and (R)ꢀ23l (IC = 1.9 nM, α /α = 1506, α /α = 249.6). Compared with
50
1B 1A
1D 1A
silodosin (IC50 = 1.9 nM, α1B/α1A = 285.9, α1D/α1A = 14.4), both compounds displayed similar
activities and better selectivities in calcium assays. In addition, compounds (R)ꢀ14r and
(R)ꢀ23l showed high potency and uroselectivity in the isolated rat tissues. More importantly,
both compounds can significantly decrease the micturition frequency and increase the mean
voided volume of the BPH rats in a doseꢀdependent manner. These results indicate that
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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
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5
5
5
5
5
5
5
5
6
compounds (R)ꢀ14r and (R)ꢀ23l are worthy of further investigation for the development of
antiꢀBPH agents.
Experimental section
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
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1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chemistry. Chemicals and solvents were purchased from commercial sources (Alfa, Acros,
Sigmaꢀaldrich and Shanghai Chemical Reagent Company), and used without further
purification. Analytical thin layer chromatography (TLC) was HSGF 254 (0.15ꢀ0.2 mm
thickness, YantaiHuiyou Company, China). Column chromatography was performed with
CombiFlash® Companion system (Teledyne Isco, Inc.). Nuclear magnetic resonance spectra
were recorded on a Brucker AMXꢀ400 or 500 MHz instrument (TMS as IS). Chemical shifts
were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton
coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broad (br). Low and highꢀresolution mass spectra (LRMS and HRMS) were
measured on Finnigan MAT 95 spectrometer. All target compounds were confirmed with
over 95% purity which were determined by Agilentꢀ1100 HPLC with binary pump,
photodiode array detector (DAD), using Agilent ExtendꢀC18 column (150×4.6 mm, 5 µm),
3 2 2
CH OH/H O (0.1% diethylamine was added in H O) = 70/30 (v/v) at 0.6 mL/min, and
calculated the peak areas at 254 nM. The enantiomeric excess (ee) of compound 10 was
identified by Agilentꢀ1100 HPLC, and CHIRALPAK ADꢀRH column (0.46 cm × 15 cm, 5
µm) was used with elution phase H O/CH CN (0.1% diethylamine was added in CH CN),
2
3
3
20/80 (v/v) at 1.0 mL/min. The enantiomeric excess (ee) of compounds 20, (R)ꢀ14r, (S)ꢀ14r,
(R)ꢀ23l and (S)ꢀ23l were identified by SHIMADZU LCꢀ20AD with photodiode array detector
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(
DAD), and CHIRALPAK ADꢀH column (0.46 cm × 25 cm, 5 µm) was used with elution
phase ethanol/hexane (0.1% diethylamine was added in hexane), 20/80 (v/v) at 1.0 mL/min.
The ee (%) values were calculated by peak areas at 254 nM.
10
11
12
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3
ꢀ(indolinꢀ1ꢀyl)propanꢀ1ꢀol (2). To a solution of indoline (10.0 g, 83.92 mmol) and
ꢀbromoꢀ1ꢀpropanol (9.5 ml, 105.05 mmol) in CH CN (300 ml) was added K CO (23.3 g,
68.58 mmol), the resulting mixture was heated to reflux for 12 h. Then the reaction mixture
3
1
3
2
3
was cooled to room temperature and filtered. The filtrate was concentrated, and the residue
1
obtained was purified by flash chromatography to give 2 (12.7 g, 85.4%) as yellow oil. H
NMR (500 MHz, CDCl ) δ 7.17ꢀ7.09 (m, 2H), 6.77ꢀ6.70 (m,1H), 6.61 (d, J = 7.7 Hz, 1H),
3
3
.81 (t, J = 6.0 Hz, 2H), 3.37 (t, J = 8.2 Hz, 2H), 3.22 (t, J = 6.7 Hz, 2H), 2.99 (t, J = 8.2 Hz,
13
2
H), 1.93ꢀ1.86 (m, 2H); C NMR (126 MHz, CDCl
3
) δ 152.53, 130.25, 127.30, 124.47,
+
1
18.22, 107.70,61.66, 53.67, 47.79, 29.87, 28.57; ESIꢀMS m/z: 178 [M+H] .
3ꢀ(indolinꢀ1ꢀyl)propyl benzoate (3). Benzoyl chloride (9.15 ml, 78.82 mmol) was added
dropwise to a solution of 2 (12.7 g, 71.65 mmol) and NEt (19.92 ml, 143.31 mmol) in
3
o
anhydrous DCM (250 ml) at 0 C under nitrogen, the mixture was stirred for 8 h at room
2 4
temperature. Then the resulting mixture was washed with water and brine, dried over Na SO ,
and concentrated. The residue was purified by flash chromatography to give 3 (19.0 g,
1
9
4.3%). H NMR (500 MHz, CDCl
3
) δ 8.19ꢀ8.12 (m, 2H), 7.66ꢀ7.60 (m,1H), 7.55ꢀ7.49 (m,
2H), 7.19ꢀ7.10 (m,2H), 6.79ꢀ6.69 (m, 1H), 6.59 (d, J = 6.5 Hz, 1H), 4.53 (t, J = 5.6 Hz, 2H),
.44 (t, J = 8.3 Hz, 2H), 3.31 (t, J = 7.0 Hz, 2H), 3.04 (t, J = 8.2 Hz, 2H), 2.19ꢀ2.10 (m,2H);
3
1
3
C NMR (126 MHz, CDCl
3
) δ 166.45, 152.28, 132.91, 130.24, 129.94, 129.49, 128.36,
+
1
27.28, 124.41, 117.71, 106.93, 62.83, 53.23, 46.10, 28.53, 26.78; ESIꢀMS m/z: 282 [M+H] .
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ꢀ(5ꢀformylindolinꢀ1ꢀyl)propyl benzoate (4). Phosphorus oxychloride (18.55 ml, 202.60
mmol) was added dropwise to a solution of DMF (15.62 ml, 202.60 mmol) in anhydrous 1,
o
2
ꢀdichloroethane (50 ml) at 0 C under nitrogen. The reaction mixture was stirred for 1 h,
0
1
2
3
4
5
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7
8
9
0
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7
8
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7
8
9
0
then a solution of 3 (19.0 g, 67.53 mmol) in 1, 2ꢀdichloroethane (200 ml) was added dropwise.
o
After addition, the mixture was heated to 80 C for 2 h. Then the reaction mixture was
allowed to cool and poured into a vigorously stirred and iceꢀcold saturated aqueous NaHCO
3
solution (400 ml). The organic layer was separated, dried and concentrated, and the residue
1
was purified by flash chromatography to give 4 (16.8 g, 80.4%). H NMR (500 MHz, CDCl
3
)
δ 9.61 (s, 1H), 8.01 (d, J = 7.8 Hz, 2H), 7.57ꢀ7.52 (m, 1H), 7.51ꢀ7.47 (m, 2H), 7.45ꢀ7.39
m,2H), 6.36 (d, J = 8.5 Hz, 1H), 4.38 (t, J = 6.2 Hz, 2H), 3.59 (t, J = 8.6 Hz, 2H), 3.37 (t, J =
(
13
6.9 Hz, 2H), 3.00 (t, J = 8.6 Hz, 2H), 2.13ꢀ1.95 (m, 2H); C NMR (126 MHz, CDCl
3
) δ
1
89.79, 166.37, 157.12, 134.18, 133.08, 130.18, 129.96, 129.44, 128.41, 126.68, 124.51,
+
104.22, 62.38, 52.24, 43.94, 27.16, 26.39; ESIꢀMS m/z: 310 [M+H] .
3
ꢀ(5ꢀ(2ꢀnitropropꢀ1ꢀenꢀ1ꢀyl)indolinꢀ1ꢀyl)propyl benzoate (5). CH COONH (10.47 g,
3 4
135.76 mmol) and CH
3
COOH (100 ml, 1.75 mol) were added to a solution of 4 (16.8 g,
NO (200 ml, 2.78 mol), the resulting mixture was heated to reflux
for 4 h and then concentrated. The residue was dissolved in EA, the resulting mixture was
washed with saturated aqueous NaHCO solution, dried over Na SO and concentrated. Then
5
4.31 mmol) in CH
3
CH
2
2
3
2
4
the residue was suspended in isopropanol, and the mixture was stirred overnight at room
temperature. The red solids of 5 (13.9 g, 69.9%) which had precipitated out were obtained by
1
filtration. H NMR (500 MHz, CDCl
3
) δ 8.09ꢀ8.00 (m, 3H), 7.61ꢀ7.54 (m,1H), 7.49ꢀ7.42 (m,
2
H), 7.24ꢀ7.17 (m, 2H), 6.44 (d, J = 8.1 Hz, 1H), 4.43 (t, J = 6.2 Hz, 2H), 3.58 (t, J = 8.5 Hz,
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H), 3.37 (t, J = 6.9 Hz, 2H), 3.05 (t, J = 8.5 Hz, 2H), 2.47 (s, 3H), 2.15ꢀ2.05 (m, 2H); C
NMR (126 MHz, CDCl ) δ 166.56,154.25, 142.40, 135.55, 133.18, 132.98, 130.58, 130.14,
29.58, 128.52, 126.75, 120.90, 105.73, 62.58, 52.55, 44.57, 27.88, 26.65, 14.53; ESIꢀMS
3
1
10
11
12
13
14
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17
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
m/z: 367 [M+H] .
ꢀ(5ꢀ(2ꢀnitropropyl)indolinꢀ1ꢀyl)propyl benzoate (6). To a solution of 5 (13.9 g, 37.94
mmol) in DCM (50 ml) and MeOH (200 ml) in ice bath was added NaBH (14.35 g, 379.36
mmol) by portion. After addition, the mixture was allowed to stir at room temperature for 1 h,
then concentrated. EA and saturated solution of NH Cl was added to the residue obtained, the
organic layer was separated and washed with brine, dried over Na SO , and concentrated.
3
4
4
2
4
1
The residue was purified by flash chromatography to afford 6 (12.85 g, 91.9%). H NMR
(500 MHz, CDCl ) δ 8.08ꢀ8.02 (m, 2H), 7.61ꢀ7.55 (m, 1H), 7.49ꢀ7.43 (m, 2H), 6.86 (s, 1H),
.83 (d, J = 8.0 Hz, 1H), 6.40 (d, J = 7.9 Hz, 1H), 4.76ꢀ4.64 (m, 1H), 4.45 (t, J = 6.3 Hz, 2H),
3
6
3.38 (t, J = 8.3 Hz, 2H), 3.25ꢀ3.15 (m, 3H), 2.94 (t, J = 8.3 Hz, 2H), 2.88 (dd, J = 14.0, 7.0
13
Hz, 1H), 2.12ꢀ2.03 (m, 2H), 1.51 (d, J = 6.6 Hz, 3H); C NMR (126 MHz, CDCl ) δ 166.68,
3
151.94, 133.11, 130.75, 130.37, 129.65, 128.53, 128.17, 125.27, 124.51, 106.83, 85.08, 62.94,
+
5
3.47, 46.20, 41.00, 28.55, 26.98, 18.73; ESIꢀMS m/z: 369 [M+H] .
3ꢀ(7ꢀformylꢀ5ꢀ(2ꢀnitropropyl)indolinꢀ1ꢀyl)propyl benzoate (7). Phosphorus oxychloride
(
9.58 ml, 104.63 mmol) was added dropwise to anhydrous DMF (50 ml, 648.48 mmol) at 0
o
C under nitrogen. The resulting mixture was stirred for 1 h, then a solution of 6 (12.85 g,
3
4.88 mmol) in DMF (50 ml, 648.48 mmol) was added dropwise. After addition, the mixture
o
was heated to 80 C for 2 h. Then the reaction mixture was allowed to cool and poured into a
vigorously stirred and iceꢀcold saturated aqueous NaHCO
3
solution (400 ml). The mixture
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6
was extracted with ethyl acetate. The organic extracts were washed with brine, dried over
Na
2
4
SO , and concentrated. The residue was purified by flash chromatography to give 7 (12.0
1
g, 86.8%) as yellow solid. H NMR (400 MHz, CDCl
3
) δ 9.93 (s, 1H), 8.08ꢀ8.01 (m, 2H),
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
7
1
8
.59ꢀ7.52 (m, 1H), 7.48ꢀ7.40 (m, 2H), 7.21 (s, 1H), 6.92 (d, J = 1.7 Hz, 1H), 4.78ꢀ4.64 (m,
H), 4.40 (t, J = 6.3 Hz, 2H), 3.71ꢀ3.57 (m,4H), 3.17 (dd, J = 14.2, 7.7 Hz, 1H), 3.02 (t, J =
1
3
.8 Hz, 2H), 2.90 (dd, J = 14.2, 6.4 Hz, 1H), 2.15ꢀ2.04 (m, 2H), 1.53 (d, J = 6.6 Hz, 3H); C
) δ 189.41, 166.62, 152.37, 134.24, 133.11, 131.48, 130.14, 129.86,
29.66, 128.50, 123.88, 118.00, 84.60, 62.76, 54.70, 49.95, 40.29, 27.78, 26.86, 18.82;
NMR (126 MHz, CDCl
3
1
+
ESIꢀMS m/z: 397 [M+H] .
ꢀ(7ꢀcyanoꢀ5ꢀ(2ꢀnitropropyl)indolinꢀ1ꢀyl)propyl
3
benzoate
(8).
Hydroxylamine
hydrochloride (4.21 g, 60.54 mmol) and pyridine (9.74 ml, 121.08 mmol) were added to a
solution of 7 (12.0 g, 30.27 mmol) in anhydrous THF (150 ml), the mixture was stirred for 12
h under reflux. Then acetic anhydride (14.21 ml, 151.35 mmol) was added slowly and the
resulting mixture was stirred for 24 h under reflux. Water was added and the mixture was
extracted with EA, the organic layer was washed sequentially with 1 mol/L HCl, saturated
aqueous NaHCO
3
solution and brine, and dried with Na
2
SO
4
. The solid obtained by
1
concentration was wash with MeOH to give 8 (10.5 g, 88.2%) as yellow solid. H NMR (500
3
MHz, CDCl ) δ 8.09ꢀ8.00 (m, 2H), 7.58ꢀ7.52 (m, 1H), 7.46ꢀ7.39 (m, 2H), 6.92 (s, 1H), 6.88
(d, J = 0.9 Hz, 1H), 4.7ꢀ4.62 (m, 1H), 4.46 (t, J = 6.3 Hz, 2H), 3.75 (t, J = 7.2 Hz,2H), 3.60 (t,
J = 8.7 Hz, 2H), 3.10 (dd, J = 14.3, 7.9 Hz, 1H), 2.95 (t, J = 8.7 Hz, 2H), 2.84 (dd, J = 14.3,
13
6.2 Hz, 1H), 2.20ꢀ2.10 (m, 2H), 1.52 (d, J = 6.6 Hz, 3H); C NMR (126 MHz, CDCl
3
) δ
166.68, 152.33, 133.28, 133.10, 131.87, 130.16, 129.73, 129.01, 128.45, 123.92, 119.15,
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7.94, 84.47, 62.59, 53.37, 45.11, 40.03, 27.30, 27.20, 18.84; ESIꢀMS m/z: 394 [M+H] .
3
ꢀ(5ꢀ(2ꢀaminopropyl)ꢀ7ꢀcyanoindolinꢀ1ꢀyl)propyl benzoate (9). A solution of 8 (10.5 g,
2
6.69 mmol) in MeOH (100 ml) and THF (100 ml) was treated with 10% Pd/C catalyst (1.0
10
11
12
13
14
15
16
17
18
19
20
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
g), the mixture was stirred for 48 h at 40 C under hydrogen. After the removal of the catalyst
1
by filtration, the filtrate was concentrated to afford 9 (9.0 g, 92.8%) as pale yellow oil. H
NMR (400 MHz, MeOD) δ 8.06ꢀ8.00 (m, 2H), 7.63ꢀ7.56 (m, 1H), 7.49ꢀ7.42 (m, 2H), 7.02 (d,
J = 1.5 Hz, 1H), 6.95 (s, 1H), 4.45 (t, J = 6.2 Hz, 2H), 3.76 (t, J = 7.1 Hz,2H), 3.61 (t, J = 8.7
Hz, 2H), 3.06ꢀ2.90 (m, 3H), 2.54ꢀ2.38 (m, 2H), 2.21ꢀ2.08 (m, 2H), 1.06 (d, J = 6.4 Hz, 3H);
1
3
C NMR (126 MHz, MeOD) δ 168.02, 153.13, 134.60, 134.20, 132.32, 131.32, 130.95,
1
30.63, 129.50, 120.56, 88.48, 63.97, 54.14, 49.27, 46.36, 45.34, 28.16, 27.86, 22.45;
+
ESIꢀMS m/z: 364 [M+H] .
(R)ꢀ3ꢀ(5ꢀ(2ꢀaminopropyl)ꢀ7ꢀcyanoindolinꢀ1ꢀyl)propyl benzoate (10). An aqueous
solution of Lꢀ(+)ꢀtartaric acid (1.15 g, 7.66 mmol) in water (30 ml) was added dropwise to a
solution of 9 (5 g, 13.76 mmol) in acetone (30 ml) over 1 h. The resulting mixture was stirred
for 48 h, the white solids which had precipitated out were obtained by filtration. The white
solids were further recrystallized with a solvent mixture of acetone and water (acetone/water
1
= 1/1) to give pure Lꢀ(+)ꢀtartaric acid salt of 10 (2.05 g). H NMR (400 MHz, DMSO + D2O)
δ 7.96 (d, J = 7.3 Hz, 2H), 7.66ꢀ7.58 (m,1H), 7.51ꢀ7.42 (m, 2H), 7.07 (s, 1H), 7.00 (s, 1H),
4.35 (t, J = 6.0 Hz, 2H), 4.07 (s, 2H), 3.66 (t, J = 7.1 Hz, 2H), 3.56 (t, J = 8.7 Hz, 2H),
3
.32ꢀ3.20 (m, 1H), 2.91 (t, J = 8.6 Hz, 2H), 2.80 (dd, J = 13.5, 5.2 Hz, 1H), 2.52 (dd, J = 13.5,
+
5.4 Hz, 1H), 2.09ꢀ1.99 (m, 2H), 1.08 (d, J = 6.5 Hz, 3H); ESIꢀMS m/z: 364 [M+H] . The
Lꢀ(+)ꢀtartaric acid salt of 10 (2.05 g) was dissolved in water (50 ml), and saturated aqueous
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2
2
2
2
2
2
3
3
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3
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3
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Na
2
CO
3
solution was added to adjust the pH to 10. Then the mixture was extracted with
DCM, the organic extracts were dried and concentrated to yield 10 (1.25 g, 25%) as pale
1
yellow oil. ee > 99.9%. H NMR (400 MHz, MeOD) δ 8.08ꢀ8.01 (m, 2H), 7.65ꢀ7.56 (m, 1H),
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
7
3
=
.51ꢀ7.43 (m, 2H), 7.05 (s, 1H), 6.97 (s, 1H), 4.48 (t, J = 6.1 Hz, 2H), 3.80 (t, J = 7.1 Hz,2H),
.64 (t, J = 8.7 Hz, 2H), 3.07ꢀ2.92 (m, 3H), 2.55ꢀ2.40 (m, 2H), 2.22ꢀ2.08 (m, 2H), 1.07 (d, J
1
3
6.4 Hz, 3H); C NMR (126 MHz, MeOD) δ 167.99, 153.11, 134.58, 134.18, 132.32,
131.34, 130.95, 130.63, 129.53, 129.50, 120.57, 88.51, 63.97, 54.15, 49.26, 46.38, 45.41,
+
28.17, 27.88, 22.53; ESIꢀMS m/z: 364 [M+H] .
3
ꢀ(7ꢀformylꢀ5ꢀ(2ꢀnitropropyl)ꢀ1Hꢀindolꢀ1ꢀyl)propyl benzoate (15). To a solution of 7
6.0 g, 15.13 mmol) in EA (200 ml) was added DDQ (5.2 g, 22.91 mmol). After addition, the
mixture was stirred at room temperature for 2 h. Then the reaction mixture was washed with
(
saturated aqueous Na
2
CO
3
solution. The organic layer was dried and concentrated, the
1
residue was purified by flash chromatography to afford 15 (5.4 g, 90%) as yellow solid. H
NMR (500 MHz, CDCl ) δ 10.04 (s, 1H), 8.06ꢀ7.96 (m,2H), 7.67 (d, J = 1.5 Hz, 1H),
3
7.60ꢀ7.53 (m,1H), 7.48ꢀ7.38 (m, 3H), 7.16 (d, J = 3.2 Hz, 1H), 6.55 (d, J = 3.2 Hz, 1H),
4.89ꢀ4.71 (m, 3H), 4.24 (t, J = 6.1 Hz, 2H), 3.45 (dd, J = 14.2, 7.7 Hz, 1H), 3.16 (dd, J = 14.2,
1
3
6.5 Hz, 1H), 2.24ꢀ2.13 (m, 2H), 1.58 (t, J = 10.0 Hz, 3H); C NMR (126 MHz, CDCl
3
) δ
191.53, 166.50, 133.12, 133.05, 132.58, 132.53, 131.66, 130.10, 129.66, 128.74, 128.48,
+
126.17, 122.88, 102.97, 84.77, 62.10, 48.11, 40.63, 30.48, 18.95; ESIꢀMS m/z: 395 [M+H] .
ꢀ(7ꢀcyanoꢀ5ꢀ(2ꢀnitropropyl)ꢀ1Hꢀindolꢀ1ꢀyl)propyl benzoate (16). Compound 16 (4.7 g,
3
87%) was obtained as yellow solid following the similar procedure carried out for compound
1
8
. H NMR (400 MHz, CDCl
3
) δ 8.02 (d, J = 7.8 Hz, 2H), 7.62 (s, 1H), 7.61ꢀ7.55 (m, 1H),
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.49ꢀ7.41 (m,2H), 7.35 (s, 1H), 7.18 (d, J = 3.1 Hz, 1H), 6.52 (d, J = 3.1 Hz, 1H), 4.87ꢀ4.76
(m,1H), 4.66 (t, J = 6.8 Hz, 2H), 4.36 (t, J = 6.0 Hz, 2H), 3.39 (dd, J = 14.2, 7.9 Hz, 1H),
13
3.11 (dd, J = 14.2, 6.2 Hz, 1H), 2.46ꢀ2.34 (m, 2H), 1.58 (d, J = 6.7 Hz, 3H); C NMR (126
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
MHz, CDCl ) δ 166.54, 133.34, 133.27, 131.69, 131.13, 129.98, 129.76, 129.09, 128.55,
3
127.05, 126.76, 118.30, 102.74, 94.06, 84.73, 61.84, 44.65, 40.62, 30.57, 19.03; ESIꢀMS m/z:
+
3
92 [M+H] .
3ꢀ(5ꢀ(2ꢀaminopropyl)ꢀ7ꢀcyanoꢀ1Hꢀindolꢀ1ꢀyl)propyl benzoate (17). Compound 17 (4.0 g,
92%) was obtained as yellow oil following the similar procedure carried out for compound 9.
1
H NMR (400 MHz, MeOD) δ 7.92ꢀ7.87 (m, 2H), 7.67 (d, J = 1.5 Hz, 1H), 7.59ꢀ7.54 (m,
1H), 7.44ꢀ7.39 (m, 2H), 7.38 (d, J = 1.5 Hz, 1H), 7.35 (d, J = 3.2 Hz, 1H), 6.54 (d, J = 3.2 Hz,
1H), 4.66 (t, J = 6.9 Hz, 2H), 4.33 (t, J = 5.9 Hz, 2H), 3.11 (dd, J = 13.4, 6.5 Hz, 1H),
13
2
.77ꢀ2.63 (m, 2H), 2.39ꢀ2.29 (m, 2H), 1.09 (d, J = 6.4 Hz, 3H); C NMR (126 MHz, MeOD)
δ 167.83, 134.19, 132.63, 132.47, 131.75, 131.07, 130.57, 130.38, 129.43, 128.23, 119.70,
+
1
03.31, 94.14, 63.50, 49.66, 45.91, 45.53, 31.41, 22.52; ESIꢀMS m/z: 362 [M+H] .
(R
)ꢀ3ꢀ(5ꢀ(2ꢀ((tertꢀbutoxycarbonyl)amino)propyl)ꢀ7ꢀcyanoindolinꢀ1ꢀyl)propyl benzoate
18). To a solution of 10 (1.0 g, 2.75 mmol) and NEt (0.8 ml, 5.76 mmol) in DCM (30 ml)
was added diꢀtertꢀbutyl dicarbonate (0.72 g, 3.30 mmol), and the mixture was stirred at room
temperature for 10 h. Then the reaction mixture was washed with water, dried with Na SO
(
3
2
4
and concentrated. The residue was purified by flash chromatography to give 18 (1.26 g, 99%)
1
as pale yellow oil. H NMR (400 MHz, CDCl
3
+ D2O) δ 8.06 (d, J = 7.7 Hz, 2H), 7.58ꢀ7.51
(m, 1H), 7.47ꢀ7.39 (m, 2H), 6.98 (s, 1H), 6.92 (s, 1H), 4.47 (t, J = 6.3 Hz, 2H), 3.80ꢀ3.71 (m,
3H), 3.58 (t, J = 8.7 Hz, 2H), 2.95 (t, J = 8.7 Hz, 2H), 2.72ꢀ2.60 (m, 1H), 2.47 (dd, J = 13.6,
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.3 Hz, 1H), 2.22ꢀ2.11 (m, 2H), 1.42 (s, 9H), 1.06 (d, J = 6.6 Hz, 3H); ESIꢀMS m/z:
+
4
64[M+H] .
(R)ꢀ3ꢀ(5ꢀ(2ꢀ((tertꢀbutoxycarbonyl)amino)propyl)ꢀ7ꢀcyanoꢀ1Hꢀindolꢀ1ꢀyl)propyl
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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9
0
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8
9
0
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2
3
4
5
6
7
8
9
0
benzoate (19). Compound 19 (0.92 g, 73%) was obtained as pale yellow oil following the
1
similar procedure carried out for compound 15. H NMR (400 MHz, CDCl
3
+ D2O) δ 8.02 (d,
J = 7.6 Hz, 2H), 7.64 (s, 1H), 7.61ꢀ7.52 (m, 1H), 7.48ꢀ7.41 (m, 2H), 7.37 (s, 1H), 7.15 (d, J =
.9 Hz, 1H), 6.51 (d, J = 3.1 Hz, 1H), 4.66 (t, J = 6.6 Hz, 2H), 4.45ꢀ4.30 (m, 3H), 2.96ꢀ2.87
2
(m, 1H), 2.75 (dd, J = 13.6, 7.2 Hz, 1H), 2.47ꢀ2.32 (m, 2H), 1.42 (s, 9H), 1.09 (d, J = 6.6 Hz,
+
3
H); ESIꢀMS m/z: 462[M+H] .
(R
)ꢀ3ꢀ(5ꢀ(2ꢀaminopropyl)ꢀ7ꢀcyanoꢀ1Hꢀindolꢀ1ꢀyl)propyl benzoate (20). To a solution of
19 (0.92 g, 1.99 mmol) in DCM (20 ml) was added CF COOH (2.0 ml, 26.93 mmol), the
mixture was stirred at room temperature for 10 h. The reaction mixture was extracted with
3
water, the organic extracts were washed sequentially with saturated aqueous NaHCO
3
1
solution and rine, dried with Na SO , and concentrated to give 20 (0.68 g, 94%). H NMR
2
4
(400 MHz, MeOD) δ 7.94ꢀ7.89 (m, 2H), 7.70 (d, J = 1.6 Hz, 1H), 7.63ꢀ7.56 (m, 1H),
.47ꢀ7.40 (m, 3H), 7.39 (d, J = 3.2 Hz, 1H), 6.57 (d, J = 3.2 Hz, 1H), 4.72 (t, J = 6.9 Hz, 2H),
7
4.37 (t, J = 5.9 Hz, 2H), 3.21ꢀ3.03 (m, 1H), 2.81ꢀ2.63 (m, 2H), 2.45ꢀ2.31 (m, 2H), 1.10 (d, J
13
=
6.4 Hz, 3H); C NMR (126 MHz, MeOD) δ 167.82, 134.17, 132.62, 132.48, 131.76,
131.10, 130.57, 130.38, 129.42, 128.22, 119.70, 103.31, 94.16, 63.48, 49.65, 45.94, 45.53,
+
31.42, 22.55; ESIꢀMS m/z: 362[M+H] .
5ꢀ(2ꢀ((2ꢀ(2ꢀbromoꢀ4ꢀfluorophenoxy)ethyl)amino)propyl)ꢀ1ꢀ(3ꢀhydroxypropyl)indolineꢀ
7ꢀcarboxamide (14a). To
a
solution of
9
(200 mg, 550 µmol) and
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ꢀ(2ꢀbromoꢀ4ꢀfluorophenoxy)acetaldehyde (205 mg, 880 µmol) in 1,2ꢀdichloroethane (6.0 ml)
3
was added NaBH(OAc) (187 mg, 880 µmol), the resulting mixture was stirred at room
temperature for 12 h. Then the reaction mixture was purified by flash chromatography to give
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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55
56
57
58
59
60
+
1
2a (R = 2ꢀBr, 4ꢀF) (271.5 mg, 85%). ESIꢀMS m/z: 580:582 = 1:1 [M+H] . A solution of 12a
(
R = 2ꢀBr, 4ꢀF) (271.5 mg, 468 µmol) in MeOH (5.0 ml) was treated with 1 mol/L NaOH (1.0
ml). Two hours later, the reaction mixture was extracted with EA, the organic extracts were
dried and concentrated to give 13a (R = 2ꢀBr, 4ꢀF) (220.6 mg, 99%). ESIꢀMS m/z: 476:478 =
+
1
:1 [M+H] . To a solution of 13a (R = 2ꢀBr, 4ꢀF) (220.6 mg, 463 µmol) in DMSO (5.0 ml) in
ice bath was added 5 mol/L NaOH (280 µl) and 30% H O (200 µl), the mixture was warmed
2
2
to room temperature and stirred for 24 h. Then water was added and resulting mixture was
extracted with EA, the organic extracts were washed with brine, dried with Na SO , and
2
4
concentrated. The residue was purified by flash chromatography to afford 14a (217.0 mg,
1
95%) as pale yellow oil. H NMR (400 MHz, CDCl
3
) δ 7.21 (dd, J = 7.8, 3.0 Hz, 1H), 7.17 (s,
1
H), 7.00 (s, 1H), 6.99ꢀ6.88 (m, 2H), 6.80ꢀ6.76 (m, 2H), 4.12ꢀ3.93 (m, 2H), 3.69 (t, J = 5.6
Hz, 2H), 3.40ꢀ3.32 (m, 2H), 3.18ꢀ3.10 (m, 2H), 2.98ꢀ2.90 (m, 5H), 2.66ꢀ2.51 (m, 2H),
+
1
.81ꢀ1.71 (m, 2H), 1.07 (d, J = 6.2 Hz, 3H); ESIꢀMS m/z: 494:496 = 1:1 [M+H] .
+
ESIꢀHRMS calcd for C23
H
30BrFN
3
O
3
[M+H] 494.1455, found 494.1450. Purity: 96.8%.
Compounds 14bꢀ14t were prepared following the similar procedure carried out for 14a.
5ꢀ(2ꢀ((2ꢀ(benzo[
d
][1,3]dioxolꢀ5ꢀyloxy)ethyl)amino)propyl)ꢀ1ꢀ(3ꢀhydroxypropyl)indolin
1
eꢀ7ꢀcarboxamide (14b). Pale yellow oil (194.2 mg, yield 95%). H NMR (400 MHz, CDCl
3
)
δ 7.17 (s, 1H), 7.00 (s, 1H), 6.86 (s, 1H), 6.65 (d, J = 8.5 Hz, 1H), 6.56 (s, 1H), 6.38 (d, J =
2
.5 Hz, 1H), 6.23 (dd, J = 8.5, 2.5 Hz, 1H), 5.88 (s, 2H), 4.01ꢀ3.89 (m, 2H), 3.71 (t, J = 5.6
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Hz, 2H), 3.40 (t, J = 8.5 Hz, 2H), 3.17 (t, J = 6.8 Hz, 2H), 3.00ꢀ2.85 (m, 5H), 2.64 (dd, J =
13.5, 6.9 Hz, 1H), 2.55 (dd, J = 13.6, 6.5 Hz, 1H), 1.83ꢀ1.73 (m, 2H), 1.07 (d, J = 6.2 Hz, 3H);
+
+
ESIꢀMS m/z: 442 [M+H] . ESIꢀHRMS calcd for C24
32 3 5
H N O [M+H] 442.2342, found
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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7
8
9
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2
3
4
5
6
7
8
9
0
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4
5
6
7
8
9
0
442.2344. Purity: 97.1%.
5ꢀ(2ꢀ((2ꢀ(2,6ꢀdimethylphenoxy)ethyl)amino)propyl)ꢀ1ꢀ(3ꢀhydroxypropyl)indolineꢀ7ꢀca
1
rboxamide (14c). Pale yellow oil (177.3 mg, yield 90%). H NMR (400 MHz, CDCl3) δ 7.27
(s, 1H), 7.16 (s, 1H), 6.96 (s, 1H), 6.95ꢀ6.87 (m, 3H), 6.87ꢀ6.80 (m, 1H), 3.93ꢀ3.82 (m, 2H),
3
2
3
.65ꢀ3.53 (m, 2H), 3.29 (t, J = 8.4 Hz, 2H), 3.15ꢀ2.97 (m, 5H), 2.87 (t, J = 7.9 Hz, 2H),
.83ꢀ2.70 (m, 1H), 2.64ꢀ2.53 (m, 1H), 2.15 (s, 6H), 1.69ꢀ1.66 (m, 2H), 1.11 (d, J = 6.0 Hz,
+
+
36 3 3
H); ESIꢀMS m/z: 426 [M+H] . ESIꢀHRMS calcd for C25H N O [M+H] 426.2757, found
426.2750. Purity: 96.3%.
1ꢀ(3ꢀhydroxypropyl)ꢀ5ꢀ(2ꢀ((2ꢀ(2,3,6ꢀtrimethylphenoxy)ethyl)amino)propyl)indolineꢀ7ꢀ
1
carboxamide (14d). Pale yellow oil (187.2 mg, yield 92%). H NMR (400 MHz, CDCl
3
) δ
7
.19 (s, 1H), 7.02 (s, 1H), 6.97 (s, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H),
6.72 (s, 1H), 3.88ꢀ3.76 (m, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.37 (t, J = 8.5 Hz, 2H), 3.14 (t, J =
.8 Hz, 2H), 3.07ꢀ3.02 (m, 1H), 3.00ꢀ2.91 (m, 4H), 2.69 (dd, J = 13.5, 6.7 Hz, 1H), 2.58 (dd,
J = 13.5, 6.5 Hz, 1H), 2.19 (s, 3H), 2.16 (s, 3H), 2.11 (s, 3H), 1.79ꢀ1.70 (m, 2H), 1.09 (d, J =
6
+
+
6
.3 Hz, 3H); ESIꢀMS m/z: 440 [M+H] . ESIꢀHRMS calcd for C26
found 440.2906. Purity: 96.8%.
ꢀ(3ꢀhydroxypropyl)ꢀ5ꢀ(2ꢀ((2ꢀ(2,4,6ꢀtrimethylphenoxy)ethyl)amino)propyl)indolineꢀ7ꢀ
38 3 3
H N O [M+H] 440.2913,
1
o
1
carboxamide (14e). Pale yellow solid (189.3 mg, yield 93%), mp 105ꢀ106 C. H NMR (400
MHz, CDCl
3
) δ 7.19 (s, 1H), 7.02 (s, 1H), 7.01ꢀ6.98 (m, 1H), 6.78 (s, 2H), 6.70 (s, 1H),
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3.90ꢀ3.80 (m, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.37 (t, J = 8.5 Hz, 2H), 3.14 (t, J = 6.8 Hz, 2H),
3.07ꢀ3.02 (m, 1H), 3.01ꢀ2.93 (m, 4H), 2.71 (dd, J = 13.5, 6.7 Hz, 1H), 2.63ꢀ2.56 (m, 1H),
2.20 (s, 3H), 2.16 (s, 6H), 1.79ꢀ1.69 (m, 2H), 1.10 (d, J = 6.3 Hz, 3H); ESIꢀMS m/z: 440
10
11
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13
14
15
16
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19
20
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45
46
47
48
49
50
51
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53
54
55
56
57
58
59
60
+
+
[
M+H] . ESIꢀHRMS calcd for C H N O [M+H] 440.2913, found 440.2909. Purity:
26 38 3 3
9
6.3%.
1
ꢀ(3ꢀhydroxypropyl)ꢀ5ꢀ(2ꢀ((2ꢀ(oꢀtolyloxy)ethyl)amino)propyl)indolineꢀ7ꢀcarboxamide
1
(
14f). Pale yellow oil (167.7 mg, yield 88%). H NMR (400 MHz, CDCl
3
) δ 7.17 (s, 1H),
7
4
3
.13ꢀ7.01 (m, 3H), 6.98 (s, 1H), 6.90 (s, 1H), 6.84ꢀ6.79 (m, 1H), 6.75 (d, J = 8.1 Hz, 1H),
.09ꢀ3.97 (m, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.39ꢀ3.31 (m, 2H), 3.14 (t, J = 6.8 Hz, 2H),
.11ꢀ3.04 (m, 1H), 3.03ꢀ2.94 (m, 2H), 2.90 (t, J = 8.8 Hz, 2H), 2.70ꢀ2.55 (m, 2H), 2.03 (s,
13
3H), 1.82ꢀ1.69 (m, 2H), 1.11 (d, J = 6.2 Hz, 3H); C NMR (126 MHz, CDCl
56.73, 149.99, 133.97, 130.61, 130.22, 128.25, 127.94, 126.82, 126.62, 120.51, 118.42,
110.98, 67.03, 59.44, 54.20, 53.62, 50.94, 46.26, 42.75, 31.05, 28.28, 20.05, 16.15; ESIꢀMS
3
) δ 171.58,
1
+
+
m/z: 412 [M+H] . ESIꢀHRMS calcd for C H N O [M+H] 412.2600, found 412.2609.
2
4
34
3
3
Purity: 95.1%.
5
ꢀ(2ꢀ((2ꢀ(2ꢀethylphenoxy)ethyl)amino)propyl)ꢀ1ꢀ(3ꢀhydroxypropyl)indolineꢀ7ꢀcarboxa
1
mide (14g). Pale yellow oil (187.2 mg, yield 95%). H NMR (400 MHz, CDCl
3
) δ 7.17 (s,
1
H), 7.15ꢀ7.07 (m, 2H), 7.06ꢀ6.97 (m, 2H), 6.90 (s, 1H), 6.89ꢀ6.83 (m, 1H), 6.78 (d, J = 7.9
Hz, 1H), 4.07ꢀ3.95 (m, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.36 (t, J = 8.6 Hz, 2H), 3.14 (t, J = 6.7
Hz, 2H), 3.08ꢀ3.01 (m, 1H), 2.99ꢀ2.89 (m, 4H), 2.63 (dd, J = 13.4, 7.1 Hz, 1H), 2.55 (dd, J =
13.6, 6.3 Hz, 1H), 2.47 (q, J = 7.5 Hz, 2H), 1.82ꢀ1.69 (m, 2H), 1.12ꢀ1.04 (m, J = 10.0, 5.0 Hz,
13
6
H); C NMR (101 MHz, CDCl
3
) δ 171.68, 156.36, 149.94, 133.93, 132.55, 130.16, 128.97,
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