Structure-Based Design of Potent Nicotinamide Phosphoribosyltransferase Inhibitors with Promising in Vitro and in Vivo Antitumor Activities

Article abstract of DOI:10.1021/acs.jmedchem.6b00324

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) has the potential to directly limit NAD production in cancer cells and is an effective strategy for cancer treatment. Using a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of NAMPT. Several designed compounds showed promising antiproliferative activities in vitro. (E)-N-(5-((4-(((2-(1H-Indol-3-yl)ethyl)(isopropyl)amino)methyl)phenyl)amino)pentyl)-3-(pyridin-3-yl)acrylamide, 30, bearing an indole moiety, has an IC₅₀ of 25.3 nM for binding to the NAMPT protein and demonstrated promising inhibitory activities in the nanomolar range against several cancer cell lines (MCF-7 GI₅₀ = 0.13 nM; MDA-MB-231 GI₅₀ = 0.15 nM). Triple-negative breast cancer is the most malignant subtype of breast cancer with no effective targeted treatments currently available. Significant antitumor efficacy of compound 30 was achieved (TGI was 73.8%) in an orthotopic MDA-MB-231 triple-negative breast cancer xenograft tumor model. This paper reports promising lead molecules for the inhibition of NAMPT which could serve as a basis for further investigation.

Full text of DOI:10.1021/acs.jmedchem.6b00324

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Article
Structure-Based Design of Potent Nicotinamide Phosphoribosyltransferase
Inhibitors with Promising in Vitro and in Vivo Antitumor Activities
Jinhong Bai, Chenzhong Liao, Yanghan Liu, Xiaochu Qin, Jiaxuan Chen,
Yatao Qiu, Dongguang Qin, Zheng Li, Zheng-Chao Tu, and Sheng Jiang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00324 • Publication Date (Web): 25 May 2016
Downloaded from http://pubs.acs.org on May 26, 2016
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Structure-Based
Design
of
Potent
Nicotinamide
Phosphoribosyltransferase Inhibitors with Promising in Vitro and in
Vivo Antitumor Activities
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a, §
b, §
a
a
a
Jinhong Bai, Chenzhong Liao, Yanghan Liu, Xiaochu Qin, Jiaxuan Chen,
a
a,d
c,*
a,*
a,*
Yatao Qiu, Dongguang Qin, Zheng Li, ZhengꢀChao Tu, Sheng Jiang
a
Laboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health,
Chinese Academy of Sciences, Guangzhou 510530, China
b
School of Medical Engineering, Hefei University of Technology, Hefei, Anhui
30009, China
2
c
The Houston Methodist Research Institute, Houston, Texas 77030, USA
d
ABA Chemicals Corporation, Shanghai 200063, China
*
Corresponding
author:
jiang_sheng@gibh.ac.cn
(S.
Jiang);
or
tu_zhengchao@gibh.ac.cn (Z. Tu) or zli@HoustonMethodist.org (Z. Li).
Abstract
Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) has the potential to
directly limit NAD production in cancer cells and is an effective strategy for cancer
treatment. Using a structureꢀbased strategy, we have designed a new class of potent
smallꢀmolecule inhibitors of NAMPT. Several designed compounds showed
promising
antiproliferative
E)ꢀNꢀ(5ꢀ((4ꢀ(((2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)(isopropyl)amino)methyl)phenyl)amino)pentyl
ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylamide, 30, bearing an indole moiety has an IC50 of 25.3 nM for
activities
in
vitro.
(
)
binding to the NAMPT protein, and demonstrated promising inhibitory activities in
the nanomolar range against several cancer cell lines (MCFꢀ7 GI50 = 0.13 nM;
MDAꢀMBꢀ231 GI50 = 0.15 nM). Tripleꢀnegative breast cancer is the most malignant
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subtype of breast cancer with no effective targeted treatments currently available.
Significant antitumor efficacy of compound 30 was achieved (TGI was 73.8%) in an
orthotopic MDAꢀMBꢀ231 tripleꢀnegative breast cancer xenograft tumor model. This
paper reports promising lead molecules for the inhibition of NAMPT which could
serve as a basis for further investigation.
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Introduction
Nicotinamide phosphoribosyltransferase (NAMPT), also known as preꢀBꢀcell
colonyꢀenhancing factor (PBEF) or visfatin, was originally found in peripheral
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1
lymphocytes and identified as a secreted growth factor for early B cells. NAMPT is a
rate limiting enzyme that converts nicotinamide (NAM) to nicotinamide
mononucleotide (NMN) in the biosynthesis of nicotinamide adenine dinucleotide
(
NAD), which is a crucial cofactor or substrate for a wide range of metabolic
2ꢀ4
enzymes.
This NAMꢀdependent process of NMN formation enables the efficient
intracellular recycling of NAM, which is produced by NADꢀconsuming enzymes such
5,6
as sirtuins and polyꢀADPꢀribose polymerases (PARPs). Proper maintenance of
NAD levels in cells is known to be crucial to sustaining the energy required for
7ꢀ10
numerous physiologic processes related to cell metabolism.
Due to their
uncontrolled growth and proliferation, cancer cells have increased energy demands for
essential nutrients. They have a higher rate of NAD consumption than normal cells,
which may make them more susceptible to NAMPT inhibition in comparison with
nonꢀcancerous cells. Thus, NAMPT is overꢀexpressed in a number of cancer types and
efficiently inhibition of NAMPT enzyme activity causes a direct inhibition of NAD
11,12
production in cancer cells which leads to loss of ATP and ultimately cell death.
Normal cells on the other hand are protected from NAMPT inhibition as they have
access
to
an
alternative
pathway,
catalyzed
by
nicotinic
acid
13,14
phosphoribosyltransferase (NAPRT) for NAD synthesis from nicotinic acid (NA).
Many cancer cell lines however are deficient in NAPRT activity and are therefore
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unable to convert NA to NAD.
Consequently, NAMPT inhibition has emerged as a
promising and novel strategy for selective inhibition of cancer cell growth and is a
16, 17
promising target for cancer treatment.
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Two small molecule inhibitors of NAMPT, (E)ꢀNꢀ(4ꢀ(1ꢀbenzoylpiperidinꢀ
1
8
4
ꢀyl)butyl)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylamide (1, APOꢀ866, FK866)
and (E)ꢀ1ꢀ(6ꢀ
1
9
(
4ꢀChlorophenoxy)hexyl)ꢀ2ꢀcyanoꢀ3ꢀ(pyridinꢀ4ꢀyl)ꢀ guanidine (2, CHS828) are
currently in phase II clinical trials (Figure 1). Although they potently inhibit NAMPT
and/or NAD synthesis in cancer cells and thereby induce cell death, both compounds
failed to demonstrate sustained efficacy in cancer patients due to doseꢀlimiting
toxicity, while thrombocytopenia and gastrointestinal toxicity are the most commonly
20,21
encountered adverse events for them.
To date, a few other NAMPT inhibitors
have been reported and shown potent in vitro antiproliferative activity in a spectrum
1
7,22,23
of cancer cell lines (Figure 1).
Specific and potent NAMPT inhibitor with
reduced side effects and improved pharmacokinetic properties is highly desirable.
Here, we report the identification of a new series of NAMPT inhibitors using in silico
rational design and structure activity analysis (SAR). The lead inhibitor demonstrated
more potent NAMPT enzyme inhibitory effect in comparison to 1, as well as
promising in vitro anticancer activities against several cancer cell lines and good
therapeutic effects in a human breast carcinoma (MDAꢀMBꢀ231) xenograft model.
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N
Cl
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O
O
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N
1 (FK866/APO866)
2 (CHS828)
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O
N
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S
H
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O
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H
HN
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S
O O
F
H
Br
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S
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O
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N
O
H
H
N
N
N
N
H
H
S
N
O O
7
8
9
Figure 1. Reported NAMPT inhibitors.
Results and Discussion
A New Chemical Scaffold to Target NAMPT
The Xꢀray crystal structure of the enzyme NAMPT reveals that it functions as a
homodimer with a head to tail interaction, and has two active sites accessible only
through a long, narrow and predominantly hydrophobic tunnel (15 Å × 6 Å) at the
interface between the monomers (Figure 2A). The crystal structure of NAMPT bound
1
2
to 1 (PDB codes 2GVJ and 4O1D ) shows that its pyridine moiety is sandwiched
between the side chains of Phe193 and Tyr18, and the main binding interaction is a
24ꢀ26
πꢀπ offset stacking.
The oxygen atom and the nitrogen atom of the amide bond in
1 form two hydrogen bonds with the hydroxyl group of the Ser 275 side chain and a
water molecule, respectively. This water molecule, as a hydrogen bond bridge, further
forms three hydrogen bonds with Asp219 and Val242. Analysis of the coꢀcrystal
structure of 1 complexed with NAMPT showed that the tail group plays an important
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role because of its ability to establish hydrophobic interactions with a hydrophobic
cleft, which can accommodate a larger group than the benzoylpiperidine ring.
Superimposition of two published coꢀcrystal structures of 1 complexed with NAMPT
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(
PDB codes: 2GVJ and 4O1D ), reveals that 1 has two different conformations in the
binding tunnel in these two crystal structures. The 3ꢀ(pyridinꢀ3ꢀyl)acrylamide
moieties are almost identical, but the tail part of 1, i.e., the benzoylpiperidine segment,
has different orientations in the two structures (Figure 2B). This is also the case when
3
2
bound to the protein (Figure 2B): in the crystal structure of 4O12 , different
conformations of 2 can be observed: the chlorobenzene ring of 2 can interacts with the
protein in two different ways (Figure 2B). Based on this structural information, we
speculated that if we retain the 3ꢀ(pyridinꢀ3ꢀyl)acrylamide part of 1 and modify the
linker and tail group and extend the length between the cap group and the tail group,
they may occupy the two locations simultaneously as does the tail of 2, and this may
increase the binding affinity to NAMPT. Additionally, the Tyr188 residue in the rim
of the binding tunnel of NAMPT is exposed to solvent, and inhibitors which have πꢀπ
stacking interaction with Tyr188 are also expected to show an increased binding
affinity. Based on this strategy, we designed and synthesized compound 10 as a new
template, in which the 3ꢀ(pyridinꢀ3ꢀyl)acrylamide part of 1 was retained, and the alkyl
chain linker was replaced by a linker containing a more rigid aromatic ring. The
benzoylpiperidine ring in the tail part in 1 was replaced by an indole moiety, which is
connected with a flexible chain to the benzene ring. It was expected that this indole
moiety in the designed NAMPT inhibitors could occupy the two locations of the rim
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in the binding tube of NAMPT simultaneously, or at least, it could be located where it
could have a πꢀπ stacking interaction with Tyr188 of NAMPT. Accordingly, we
designed and synthesized compound 10 as a template for further optimization (Figure
10
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3
). Compound 10 had an IC value of 702 nM to NAMPT, and is 22 times less potent
50
than 1 (IC50 = 32.0 nM) (Table 1).
Figure 2. (A) The binding mode of 1 (PDB code: 4O1D). (B) The tail groups of
NAMPT inhibitors may have different orientations in the active site. Yellow, 1 (PDB
code: 4O1D); magenta, 1 (PDB code: 2GVJ); green, 2(PDB code: 4O12).
The nitrogen substituent of compound 10 might have considerable impact on the
efficacy against NAMPT of the related compounds. To assess the contribution of the
isopropyl group to the efficacy, we first synthesized compound 11 in which this group
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is replaced by a hydrogen atom. Compound 11 is half as potent as 10, confirming the
importance of the isopropyl group, which can easily be understood as the binding tube
is hydrophobic. Therefore, introduction of increasingly hydrophobic groups into this
nitrogen of 10 would improve the van der Waals interactions. Accordingly, we
incorporated a series of hydrophobic groups such as methyl, ethyl, propyl,
hydroxyethyl, phenyl or tꢀBoc groups at this position, as shown in Table 1. Among
these compounds, 14 with hydroxyethyl at this position had an IC50 value of 181 nM
against NAMPT in the enzyme inhibition assay, and was therefore about 3.8 times
more potent than the original compound 10. Compared with the isopropyl group, the
hydroxyethyl group in 14 is less hydrophobic, and its favorable binding affinity can
be explained by its docking pose in which its two carbons have hydrophobic
interactions with Val242 and Tyr188, while the hydroxyl group forms a hydrogen
bond with Arg191. However, compound 14 was equally potent as compound 10 in
cell inhibition assay (see Table 1). We assumed that compound 14 was very
hydrophilic, which makes it difficult to across the cell membrane.
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Cap group
Tail group
Linker
H
N
N
N
O
10
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O
Connecting
unit
1
O
N
R
N
n
H
N
N
H
1
1
1
1
1
0: R = -CH(CH ) , n = 3; 11: R = -H; n = 3;
2: R = -CH CH , n = 3;
4: R = -CH CH OH, n = 3; 15: R = -CH Ph, n = 3;
6: R = -CH , n = 3;
8: R = -CH(CH ) , n = 1; 19: R = -CH(CH ) , n = 5;
20: R = -CH(CH ) , n = 7.
3 2
13: R = -CH CH CH , n = 3;
2 2 3
2
3
2
2
2
17: R = Boc, n = 3;
3
3
2
3 2
3
2
Figure 3. Chemical structures of newly designed NAMPT inhibitors.
Table 1. In Vitro NAMPT Enzyme and MDAꢀMBꢀ231 Inhibitory Activities of
a
Compounds 1, 10ꢀ20.
NAMPT
(IC50 (nM))
32.0±2.50
702±100.4
MDAꢀMBꢀ231
(GI50 (nM))
0.78±0.04
Compd
1
1
0
30.5±3.39
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1410±45.47
873±233.4
544±55.3
358±40.91
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76.4±7.42
36.0±3.38
181±10.0
31.4±1.76
269±26.15
1
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6
1000±59.42
780±69.86
715±67.56
213±17.77
66.9±7.0
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123±14.85
134±14.59
0.89±0.00
0.84±0.16
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18
1
2
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52.7±1.2
a
1
was used as the positive control. Values are means of three experiments. The
incubation time for MDAꢀMBꢀ231 inhibitory activities is 72 h.
Having identified isopropyl as a suitable substituent group on the nitrogen for
effective interaction with NAMPT, we sought to determine the optimal length (n) of
the alkyl chain. We designed and synthesized compounds 18-20, in which n = 1, 5,
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and 7 carbon atoms, respectively. Compound 18, an analog with a single carbon atom
in the side chain, was 3 times as potent against NAMPT as the compound 10. When
the alkyl chain was extended to n = 5, the resulting compound 19 had an IC50 value of
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6.9 nM, which was about 10 times as potent as the compound 10. Compound 20 with
seven methylene spacers, had IC50 value of 52.7 nM and was approximately
equivalent in activity to compound 19. Molecular modeling indicated that the benzene
ring in the linker of 20 was positioned in the external rim of the binding tube,
indicating that in this binding tube a flexible alkyl chain is accommodated better than
a chain with a bulkier benzene ring.
To further study the SAR of compound 19 and explore the effect of the tail group
on the enzymatic inhibitory activities, we designed and synthesized compounds 21-29
(Table 2). Based on the binding mode of compound 19 to NAMPT, we assumed that
replacement of the indole moiety of 19 with a phenyl ring would attenuate the affinity
because the indole ring enjoys a hydrophobic interaction with the protein.
Replacement of the indole ring by a phenyl had a significant impact on the
effectiveness of the compounds against NAMPT; compound 21 was found to be
almost 4 times less potent than compound 19. It was observed that different electron
withdrawing functional groups at the metaꢀ or paraꢀposition of the phenyl ring
decreased the inhibitory activity against NAMPT markedly (26, 27). For instance,
when a trifluoromethyl group was introduced at the metaꢀposition of compound 21,
the resulting compound (26) was 1.3 times less potent than 21. Conversely, when
electron donating functional groups such as methyl and methoxyl were introduced at
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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
4
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4
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5
5
5
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5
5
5
6
the paraꢀposition of compound 21 (22 and 28 respectively), their inhibitory activities
against NAMPT were significantly increased in comparison to that of compound 21.
Compound 28 with a methoxyl group at the paraꢀposition had an IC50 value of 54.8
nM against NAMPT, and thus was about 5.2 times more potent than compound 21.
Interestingly, replacement of the phenyl ring with a thiophene group (29) increased
the inhibitory activity, suggesting that tail group might be a feasible position for
further optimization.
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
a
Table 2. In Vitro NAMPT Enzyme Inhibitory Activities of Compounds 21 – 30.
O
X
N
N
H
R
N
NAMPT
(IC50, nM)
32.0±2.50
Compd
1
21: R =
2
85±14.5
X = CH2
22: R=
99.6±11.6
X = CH2
23: R =
1
49±19.9
F
X = CH2
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2
4: R =
82.5±12.0
X = CH2
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
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25: R =
1
20±8.4
N
H
X = CH2
F3C
26: R =
3
81±18.6
X = CH2
2
7: R =
F3C
362±28.6
X = CH2
2
8: R =
O
54.8±6.20
195±19.0
X = CH2
29: R =
S
X = CH2
2
5.3±2.10
30: R=
X = NHCH2
HN
a
1 was used as the positive control. Values are means of three experiments.
Based on the binding mode of compound 19 to NAMPT, we assumed that
incorporation of a nitrogen atom into the linker could increase the affinity to the
protein via hydrophobic interactions. When the nitrogen atom was inserted into the
linker, the resulting compound 30 exhibited slightly better binding affinity for Nampt
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1
1
2
2
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2
2
2
2
2
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3
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than 1 (Table 2). A docking study of compound 30, a very flexible compound, led to
several possible docking poses. One pose (Figure 4) indicated that the isopropyl group
can occupy the position of the phenyl ring of 1 as in the conformation of crystal
structure of 4O1D, and the indole ring can occupy the position of the phenyl ring of 1
as in the conformation of crystal structure of 2GVJ. 1 at least has two different
conformations when complexed with NAMPT and the tail of 1 can have two different
hydrophobic interactions in the tunnel. Compound 30 may be able to have these two
hydrophobic interactions similar to the two conformations of complexed 1 in NAMPT.
Another docking pose showed that the indole ring has a πꢀπ stacking interaction with
Tyr188. Either of these two docking poses may explain why these two compounds
have good binding affinity against NAMPT. In addition, we synthesized compound
S1 with a six methylene linker and showed that it was less potent than the compound
30 (supporting information), which demonstrated that NHꢀcontaining linker present in
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
3
0 was preferred.
Figure 4. Green: one docking pose of compound 30; Yellow: the binding pose of 1
(PDB code: 4O1D); Pink: the binding pose of 1 (PDB code: 2GVJ).
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We further investigated the antiproliferative activities of these derivatives with
Hela (human cervical cancer cell line), MCF7 (breast cancer cell line), H1975 (human
nonꢀsmallꢀcell lung cancer cell line), U937 (human histiocytic leukemia cell line) and
MDAꢀMBꢀ231（human breast adenocarcinoma cell line）with 1 and taxol as the
positive controls. As shown in Table 3, compounds 19, 20, 25, 27, 28 and 30 have
GI50 values in the nanomolar range against human cancer cell lines. It was remarkable
that compound 30, especially in view of its higher NAMPT inhibitory activities, had
anticancer activity against several cancer cell lines which was much better than that of
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
1
. In contrast, compounds 11, 15, 17 and 18, weaker inhibitors of NAMPT, show
much reduced activity against these cell lines. The results of the enzyme and
cellꢀbased assays thus supported for NAMPT a correlation between the in vitro
inhibitory activity and cellular cytotoxicity. Compound 30 was therefore further
examined in in vivo pharmacokinetic (PK) studies in rats and in a xenograft efficacy
experiment to evaluate its antiꢀtumor effects.
Table 3. Antiproliferative Activities of Compounds 10-30 against Five Different
aꢀc
Cancer Cell Lines.
GI (nM)
5
0
Compd
Hela
MCF7
H1975
U937
MDAꢀMBꢀ231
0.78±0.04
1
1.34±0.20
90.7±7.12
0.29±0.04
22.4±1.34
3.95±0.14
79.7±6.90
0.36±0.06
24.2±1.63
1
0
30.5±3.39
11
405±40.97 86.9±6.46 963±84.34 187±12.36
358±33.91
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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
1
2
200±16.02 41.0±3.97 314±15.59
48.3±2.84
76.4±7.42
36.0±3.38
31.4±1.76
269±22.15
84.7±7.08
123±11.85
134±4.59
0.89±0.00
0.84±0.16
10.1±1.31
7.19±0.67
13.6±1.29
12.6±0.26
3.03±0.32
6.92±0.38
2.40±0.32
2.86±0.32
13
14
55.7±5.18
69.4±5.95
379±24.95
23.8±1.89 109±12.18
28.1±2.12
18.3±2.35
85.2±4.66
69.1±5.85
54.3±1.67
77.9±5.81
0.61±0.09
0.75±0.11
6.21±0.14
8.73±0.72
7.67±0.88
11.1±1.41
1.58±0.12
5.10±0.65
1.57±0.37
2.12±0.33
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
18.1±1.69
101±6.29
107±3.54
514±3.82
1
1
1
1
1
5
6
7
8
9
250±17.23 75.4±6.43 224±19.30
288±14.03 85.5±8.55 399±18.73
173±15.84 75.1±6.49
335±9.83
4.20±0.45
4.20±0.39
61.3±6.07
39.2±2.87
34.1±1.77
49.0±2.09
13.3±1.52
26.2±2.38
9.54±0.97
9.87±0.39
3.23±0.43
2.64±0.28
19.6±1.83
26.6±1.47
20.9±2.97
40.1±2.42
7.93±0.74
15.2±1.98
8.98±0.98
6.95±0.23
0.63±0.05
0.74±0.08
7.18±0.22
5.53±0.42
7.37±0.89
6.37±0.28
1.67±0.57
2.41±0.24
1.60±0.19
1.38±0.32
20
21
2
2
2
2
2
2
3
4
5
6
27
28
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29
43.7±2.10 8.47±0.55
71.8±7.05
1.61±0.27
14.8±1.18
23.6±2.05
3
0
0.50±0.02 0.13±0.02
0.25±0.03
2.12±0.35
0.15±0.05
8.67±0.84
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Taxol
2.34±0.24
4.38±0.40
5.77±0.98
a
b
Taxol and 1 were used as the positive control. Inhibition of cell growth by the listed
compounds was determined by using CCKꢀ8 assay.
To validate the target specificity at the cellular level, the compound 30 was evaluated
for its ability in reducing the cellular NAD levels. After incubation with human
MCFꢀ7 cells for 24 hours, the compound 30 decreased the cellular NAD level
significantly. The IC50 for compound 30 reducing NAD level was 0.155 nM (Figure
5
).
compound 30 inhibit NAD production
100
80
60
40
20
0
-11.0
-10.5
-10.0
-9.5
-9.0
-8.5
C(M)
Compound 30
1.550e-010
IC50
Figure 5. The NAD level response curve of the compound 30 on MCFꢀ7 cells after 24
hours treatment.
As summarized in Table 4, compound 30 demonstrated good PK properties in rats.
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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
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
After an IV administration of a dose of 5 mg/kg, it showed good plasma clearance and
a high volume of distribution. The Cmax was about 500 times greater than the
corresponding cell IC50 value and compound 30 exhibited halfꢀlife of 1.55 h, longer
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
23
than that of 1, which is approximately 20 min .
Table 4. Pharmacokinetic Parameters of Compound 30 in Rats (n = 4)
route
C
max
T
max
AUC0ꢀ∞
T
1/2
CL
F
(ug/L)
(h)
(ug/L*h) (h)
(L/h/kg)
IV
5 mg/kg)
PO
25 mg/kg)
Values are the average of three runs. Vehicle: 2% DMSO, 4% EtOH, 4% castor oil
3790.6 0.033
1709.4
1.55
2.94
(
107.6
2.0
373.9
2.37
68.13
4.4%
(
a
and 90% doubly distilled H O. CL, clearance; T , halfꢀlife; C maximum
2
1/2
max,
concentration; Tmax, time of maximum concentration; AUC0−∞, area under the plasma
concentration time curve; F, oral bioavailability.
On the basis of the in vivo pharmacokinetic data, we next evaluated the antitumor
efficacy of 30 in the MDAꢀMBꢀ231 xenograft tumor model (Figure 6). The human
breast adenocarcinoma cell line MDAꢀMBꢀ231 has the tripleꢀnegative breast cancer
phenotype, which is the most malignant type that poises worst prognosis in patients
with no specific treatment. Our data showed that compound 30 at 15 mg/kg
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effectively inhibits tumor growth, with a tumor growth inhibition (TGI) value of
7
3.8% (p < 0.01). During the treatment study, the mouse body weight was measured
as shown in Figure S1 (Supporting Information). The result suggested the compound
0 at the current dose is wellꢀtolerated, with no mortality or significant loss of body
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
weight observed during treatment.
Control
4000
Compound 30
3000
2000
1000
0
0
3
6
9
12 15 18 21 24 27 30
Day
Figure 6. Growth curve of the MDAꢀMBꢀ231 breast cancer xenograft in nude mice
treated with i.v. injections of vehicle alone or compound 30 (15 mg/kg) 4 days a week.
(p < 0.01). Data are expressed as the mean tumor volume ± SE of the animals in each
treatment group.
Chemistry
Compounds 10ꢀ17 were prepared according to the general procedures shown in
Scheme 1. Tryptamine 31 was reacted with methyl 4ꢀformylcinnamate 36 via
reductive amination to generate substituted methyl 4ꢀaminomethyl cinnamates (32).
Compound 32 was protected with Boc group to give 33, which was reacted with
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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
LiAlH
4
to furnish the alcohol 34 in 60% yield. Subsequent transformation of the
alcohol 34 into the amine 35 involved a threeꢀstep sequence: (i) mesylation of alcohol
4, (ii) formation of the azide, and (iii) reduction using PPh (73% yield).
Condensation of (E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid 37 with amine 35 in the presence of
ꢀEthylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDCI) gave compound 17, which
3
3
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
was further deprotected to give intermediate 11 in 85% yield. Subsequent coupling
with alkyl halides gave 10 and 12-16 in 42ꢀ80% yields.
O
O
H
NH2
N
a
b
HN
HN
O
3
1
32
Boc
O
Boc
OH
c
d
N
N
HN
HN
HN
33
34
O
Boc
OH
N
e
Boc
N
H
f
N
N
35
HN
17
O
O
N
R
N
N
H
N
g
H
H
N
N
HN
HN
11
1
0: R = -CH(CH3);
12: R = -CH CH ;
2 3
13: R = -CH
5: R = -CH
2
2
CH
Ph;
2
CH
3
; 14: R = -CH
2 2
CH OH;
1
16: R = -CH ;
3
Scheme 1. General procedure for the synthesis of compounds 10-17. Reagents and
conditions: (a) methyl 4ꢀformylcinnamate (36), NaBH CN, AcOH, MeOH, rt, 65%. (b)
Boc) O, triethylamine, dioxane, H O, rt, 85%. (c) LiAlH , THF, 0 °C ꢀ rt, 60%. (d) (i)
3
(
2
2
4
MsCl, triethylamine, DCM, 0 °C. (ii) NaN , DMF, 80 °C. (iii) PPh , H O, THF, 60 °C,
3
3
2
73% in 3 steps. (e) (E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid (37), EDCI, hydroxybenzotriazole
HOBt), triethylamine, DMF, rt, 86%. (f) 3M HCl, EtOAc, rt, 90%. (g) RꢀX, K CO
DMF, 80 °C, 42%ꢀ80%.
(
2
3
,
Compounds 18ꢀ20 were synthesized by using the procedure described in Scheme 2.
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Tryptamine (31) was reacted with aldehydes 39-41 via reductive amination to afford
the intermediates 42-44 in 42ꢀ60% yield. Coupling of which with 2ꢀiodopropane
afforded 45ꢀ47 was achieved in 50ꢀ70% yields, which were further reacted with
LiAlH4 to furnish the alcohols 48ꢀ50 in 63%ꢀ77% yields. Subsequently,
transformation of alcohols 48ꢀ50 into amines 51ꢀ53 was achieved using similar
methods, involving a threeꢀstep sequence: (i) mesylation of alcohols, (ii) formation of
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
the azide, and (iii) reduction using PPh
3
(49ꢀ70% yields). Condensation of
(E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid 37 with amines 51ꢀ53 in the presence of EDCI gave
the final products 18ꢀ20 in 59ꢀ80% yields, respectively.
OEt
OEt
H
N
n
O
NH2
n
N
O
a
b
HN
HN
HN
4
2: n = 0
3: n = 4
31
4
45: n = 0
46: n = 4
44: n = 6
47: n = 6
NH2
OH
d
n
n
c
N
N
e
HN
HN
4
4
5
8: n = 1
9: n = 5
0: n = 7
51: n = 1
52: n = 5
53: n = 7
O
N
OEt
n
H
N
n
O
N
OHC
HN
3
9: n = 0
40: n = 4
1: n = 6
1
8: n = 1
19: n = 5
0: n = 7
4
2
Scheme 2. Synthesis of compounds 18-20. Reagents and conditions: (a) aldehyde
9-41, NaBH CN, AcOH, MeOH, rt, 42%ꢀ60%. (b) 2ꢀiodopropane, K CO
triethylamine, CH CN, 80 °C, 50ꢀ70%. (c) LiAlH , THF, 0 °C ꢀ rt, 63%ꢀ77%. (d) (i)
MsCl, triethylamine, DCM, 0 °C. (ii) NaN , DMF, 80 °C. (iii) PPh , H O, THF, 60 °C,
3
3
2
3
,
3
4
3
3
2
49ꢀ70% in 3 steps. (e) (E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid (37), EDCI, HOBt,
triethylamine, DMF, rt, 59ꢀ80%.
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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
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To study the SAR of compound 19, a new route was developed for synthesis of
analogs 21ꢀ29. The introduction of the aldehyde group selectively into the 4ꢀposition
of methyl 5ꢀphenylpentanoate (54) was achieved by a Duff reaction to give 55 in 32%
yield, which was further allowed to react with methyl orthoformate affording
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
compound 56 in 56% yield. Reduction of the ester group in the presence of LiAlH
4
provided compound 57 in high yields. Using similar methods, alcohol 57 was
transformed into the amine (58) in 35% yield, and this was allowed to react with
(E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid (37) in the presence of EDCI giving 59 in 88% yield.
Deprotection of 59, reductive amination with different amines and finally, coupling
with 2ꢀiodopropane gave compounds 21-29 in 60ꢀ86% yield.
O
O
O
O
O
b
c
a
O
3
3
3
O
OHC
O
54
55
56
O
NH2
N
N
OH
d
e
3
3
3
H
O
O
O
O
O
O
5
8
59
57
O
O
f
N
N
g
N
N
3
H
3
H
O
R
N
H
6
0
21: R = Bn; 22:R = 4-Me-Bn; 23:R = 4-F-Bn;
24: R = Ph; 25:R = 1H-indole-2-CH3-3-yl;
26: R = 3-CF
3 3
-Bn; 27: R = 4-CF -Bn;
28: R = 4-MeO-Bn; 29: R = thiophen-3-yl.
Scheme 3. Synthesis of compounds 21-29. Reagents and conditions: (a)
hexamethylenetetramine, TFA, reflux, 32%; (b) (MeO) CH, TsOH, MeOH, reflux,
6%; (c) LAH, THF, 0 C, 73%; (d) (i) MsCl, triethylamine, DCM, 0 °C. (ii) NaN
DMF, 80 °C. (iii) PPh , H O, THF, 60 °C, 35% in 3 steps. (e)
3
o
5
3
,
3
2
(E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid (37), EDCI, HOBt, TEA, DMF, 88% (f) 6M HCl,
THF, rt, 90%; (g) (i) different amines, NaBH , MeOH, rt; (ii) 2ꢀiodopropane, K CO ,
4
2
3
o
TEA, 60 C, 60ꢀ86% in two steps.
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The synthesis of compound 30 is illustrated in scheme 4. Tryptamine (31) was
reacted with 4ꢀnitrobenzaldehyde via reductive amination to provide compound 61,
which was reacted with 2ꢀiodopropane to generate compound 62. The nitro group of
compound (62) was reduced by SnCl .2H O to obtain the amine 63 in 58% yield,
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
2
which was coupled with tertꢀbutyl (5ꢀiodopentyl) carbamate (38) to furnish the
intermediate 64. Deprotection of 64 and coupling with (E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid
(37) in the presence of EDCI gave 30 in 45% yield.
NO2
NO2
H
NH2
a
b
N
N
HN
HN
HN
31
61
62
H
N
NH2
NHBoc
3
c
N
d
e
N
HN
HN
63
64
N
H
H
N
H
N
NH2
N
3
N
N
f
O
HN
HN
65
30
Scheme 4. Synthesis of compound 30. Reagents and conditions: (a)
ꢀnitrobenzaldehyde, NaBH CN, AcOH, MeOH, rt, 70%. (b) 2ꢀiodopropane, DIPEA,
CH CN, 80 °C, 48 h, 93%. (c) SnCl .2H O, EtOH, rt, 58%. (d) tertꢀbutyl
4
3
3
2
2
o
(
5ꢀiodopentyl) carbamate (38), K CO , MeCN, 50 C, 43%. (e) 3M HCl, THF, rt, 82%.
2 3
(f) (E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid (37), EDCI, HOBt, triethylamine, DMF, rt, 45%.
Conclusion
We have designed and synthesized a series of novel NAMPT inhibitors using a
structureꢀbased design strategy based upon the crystal structure of NAMPT
complexed with 1. Structureꢀactivity relationships were thoroughly studied by
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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
changing the length of the linker and the flexibility of the surface tail group. We
identified a representative lead compound 30, which inhibits NAMPT with an IC50
value of 25.3 nM and shows promising in vitro anticancer activities against several
cancer cell lines. It also exhibited significant antitumor efficacy in the MDAꢀMBꢀ231
xenograft tumor model, which is an aggressive tripleꢀnegative breast cancer
phenotype with no specific treatment so far. In addition, compound 30 showed an
excellent halfꢀlife (1.55 h), much longer than that of 1 (20 min). These results make it
clear that compound 30 is a promising NAMPT inhibitor well suited to further
evaluations as a new anticancer agent.
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
Experimental Section
General. Reagents and solvents were used as purchased without further purification.
The progress of all reactions was monitored by TLC using EtOAc/nꢀhexane or
DCM/MeOH as a solvent system, and spots were visualized by irradiation with
ultraviolet light (254 nm). Flash chromatography was performed using silica gel
1
13
(
300−400 mesh). H NMR and C NMR spectra were recorded on Bruker Avance
ARXꢀ400 (or Bruker Avance ARXꢀ500). The low or high resolution of ESIMS was
recorded on an Agilent 1200 HPLCꢀMSD mass spectrometer or Applied Biosystems
QꢀSTAR Elite ESIꢀLCꢀMS/MS mass spectrometer, respectively. Anhydrous solvents
were obtained as follows: THF by distillation from sodium and benzophenone;
CH Cl , toluene and DMF from CaH . All other solvents were reagent grade. All
2
2
2
moisture sensitive reactions were carried out in a flame dried flask under an argon
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atmosphere. The purity of the final compounds was determined in an Agilent 1260
series HPLC system using the following conditions: Cꢀ18 column (DicKma, 4.6 mm
×
250 mm) with the solvent system (elution conditions: mobile phase A consisting of
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
MeOH; mobile phase B consisting of H O containing 0.1% ammonia), with
2
monitoring between 190 and 800 nm. A flow rate of 1.0 mL/min was used. The
R
retention time was reported as t (min). The purity of final compounds is >95%.
(E)-N-(3-(4-(((2-(1H-indol-3-yl)ethyl)(isopropyl)amino)methyl)phenyl)propyl)-3-(
pyridin-3-yl)acrylamide (10)
To a stirred solution of compound 11 (22 mg, 0.05 mmol) in DMF (2 mL) was added
potassium carbonate (14 mg, 0.1 mmol) and 2ꢀiodopropane (10.2 mg, 0.06 mmol), the
mixture was stirred for 8 h at room temperature. DMF was evaporated under reduced
pressure. The residue was extracted with ethyl acetate (4 mL × 3). The combined
organic layers were washed with saturated aqueous NaHCO solution and brine, dried
3
2 4
over anhydrous Na SO , filtered and concentrated under reduced pressure. The
resulting mixture was purified by silica column chromatography and eluted with
1
petroleum ether/EtOAc (2:1) to give 10 (12 mg, 52%) as a yellow solid. H NMR
(400 MHz, CDCl
d, J = 8.1 Hz, 1H), 7.59 (d, J = 15.8 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.31 (t, J = 7.9
Hz, 4H), 7.19 ꢀ 7.00 (m, 4H), 6.92 (s, 1H), 6.37 (d, J = 15.6 Hz, 1H), 5.63 (br, 1H),
3
): δ 8.72 (s, 1H), 8.56 (d, J = 4.7 Hz, 1H), 8.16 ꢀ 8.01 (m, 1H), 7.75
(
3
2
.63 (s, 2H), 3.42 (q, J = 6.9 Hz, 2H), 3.11 ꢀ 3.00 (m, 1H), 2.89 ꢀ 2.79 (m, 2H), 2.78 ꢀ
13
.61 (m, 4H), 1.92 (t, J = 7.3 Hz, 2H), 1.05 (d, J = 6.5 Hz, 6H) ppm. C NMR (125
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8
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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
MHz, CDCl
3
): δ 165.1, 150.3, 149.1, 137.3, 136.2, 134.3, 130.6, 128.8, 128.1, 127.6,
1
2
26.4, 123.6, 122.7, 121.7, 121.5, 118.9, 118.8, 110.9, 53.9, 50.5, 39.5, 32.9, 31.1,
+
5.1, 18.2 ppm. HRMS (ESI): m/z [M + H] calculated for C31
H
37
N
4
O, 481.2962;
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
found, 481.2966. HPLC purity: 98.6%.
(E)-N-(3-(4-((2-(1H-indol-3-yl)ethylamino)methyl)phenyl)propyl)-3-(pyridin-3-yl
)acrylamide (11)
To a stirred solution of compound 17 (38 mg, 0.07 mmol) in ethyl acetate (2 mL) was
added 3 M HCl (2 mL), the mixture was stirred for 8 h at room temperature. The
reaction mixture was quenched by 2 M NaOH (3 mL), extracted with ethyl acetate (6
mL × 3). Then the combined organic layers were washed with saturated NaHCO
3
solution and brine, dried over Na SO (anhydrous), filtered and concentrated. The
2
4
resulting mixture was purified by silica column chromatography and eluted with
1
petroleum ether/EtOAc (1:1) to give 11 (28 mg, 90%) as a yellow solid. H NMR
(
400 MHz, CD
3
OD): δ 8.67 (s, 1H), 8.49 (d, J = 4.9 Hz, 1H), 8.01 (dd, J = 8.2, 1.9 Hz,
H), 7.63 ꢀ 7.21 (m, 8H), 7.16 ꢀ 6.93 (m, 3H), 6.73 (d, J = 15.9 Hz, 1H), 4.07 (s, 2H),
.21 (t, J = 7.6 Hz, 2H), 3.12 (t, J = 7.5 Hz, 2H), 2.69 (t, J = 7.7 Hz, 2H), 1.98 ꢀ 1.76
1
3
13
(
m, 4H) ppm. C NMR (125 MHz, CD
37.4, 136.2, 132.9, 131.6, 130.8, 130.2, 128.2, 125.5, 124.8, 124.0, 122.7, 119.9,
18.9, 112.50 , 110.8, 52.2, 40.1, 33.8, 32.0, 24.0, 23.7 ppm. HRMS (ESI): m/z [M +
3
OD): δ 167.7, 150.7, 149.7, 144.1, 138.3,
1
1
+
31 4
H] calculated for C28H N O, 439.2492; found, 439.2493. HPLC purity: 98.8%.
(E)-N-(3-(4-(((2-(1H-indol-3-yl)ethyl)(ethyl)amino)methyl)phenyl)propyl)-3-(pyri
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din-3-yl)acrylamide (12)
The procedure was the same as described above for the synthesis of 10. Compound 12
1
was obtained as a white solid (19 mg, 82%). H NMR (400 MHz, CD
3
OD): δ 8.68 (s,
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
1
H), 8.49 (d, J = 3.2 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 15.8 Hz, 1H), 7.44
dd, J = 7.9, 5.2 Hz, 1H), 7.41 ꢀ 7.20 (m, 6H), 7.06 (d, J = 6.1 Hz, 2H), 6.97 (t, J = 7.5
Hz, 1H), 6.74 (d, J = 15.8 Hz, 1H), 4.05 (s, 2H), 3.34 ꢀ 3.31 (m, 2H), 3.14 ꢀ 2.93 (m,
(
13
6
H), 2.71 (t, J = 7.7 Hz, 2H), 1.94 ꢀ 1.85 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H) ppm. C
NMR (125 MHz, CD OD): δ 167.7, 150.7, 149.7, 143.9, 138.2, 137.4, 136.2, 132.9,
31.6, 130.0, 128.3, 125.5, 124.8, 123.7, 122.6, 119.8, 119.0, 112.4, 111.6, 58.1, 53.8,
3
1
4
+
0.2, 33.9, 32.1, 22.1, 10.3 ppm. HRMS (ESI): m/z [M + H] calculated for
30 35 4
C H N
O, 467.2805; found, 467.2806. HPLC purity: 97.3%.
(E)-N-(3-(4-(((2-(1H-indol-3-yl)ethyl)(propyl)amino)methyl)phenyl)propyl)-3-(py
ridin-3-yl)acrylamide (13)
The procedure was the same as described above for the synthesis of 10. Compound 13
1
was obtained as a white solid (12 mg, 50%). H NMR (400 MHz, CD
3
OD): δ 8.70 (s,
1H), 8.50 (d, J = 4.4 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 16.0 Hz, 1H), 7.45
dd, J = 7.9, 5.1 Hz, 1H), 7.33 (dd, J = 21.4, 7.8 Hz, 4H), 7.22 (d, J = 7.6 Hz, 2H),
7.10 ꢀ 6.90 (m, 3H), 6.72 (d, J = 16.0 Hz, 1H), 3.81 (s, 2H), 3.35 ꢀ 3.31 (m, 2H), 3.01
(
ꢀ
2.93 (m, 2H), 2.89 ꢀ 2.85 (m, 2H), 2.72 ꢀ 2.64 (m, 4H), 1.93 ꢀ 1.86 (m, 2H), 1.67 ꢀ
13
1
1
1
3
.59 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H) ppm. C NMR (125 MHz, CD OD): δ 166.3,
49.2, 148.2, 141.1, 136.7, 135.9, 134.8, 131.5, 129.5, 129.4, 128.1, 127.1, 124.1,
23.4, 121.8, 120.8, 118.1, 117.7, 111.9, 110.8, 57.6, 55.3, 53.7, 38.8, 32.4, 30.7, 21.6,
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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
+
1
8.9, 10.6 ppm. HRMS (ESI): m/z [M + H] calculated for C31
H
37
N
4
O, 481.2962;
found, 481.2963. HPLC purity: 96.4%.
(
E)-N-(3-(4-(((2-(1H-indol-3-yl)ethyl)(2-hydroxyethyl)amino)methyl)phenyl)pr
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
opyl)-3-(pyridin-3-yl)acrylamide (14)
The procedure was the same as described above for the synthesis of 10. Compound 14
1
was obtained as a yellow solid (18 mg, 75%). H NMR (400 MHz, CDCl ): δ 8.72 (s,
3
1
H), 8.55 (d, J = 4.4 Hz, 1H), 8.34 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 15.6
Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.36 ꢀ 7.23 (m, 2H), 7.21 ꢀ 7.02 (m, 6H), 6.89 (s,
H), 6.41 (d, J = 15.6 Hz, 1H), 5.89 (br, 1H), 3.69 (s, 2H), 3.59 ꢀ 3.49 (m, 2H), 3.40
1
(q, J = 6.0 Hz, 2H), 2.94 (t, J = 7.0 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.74 (t, J = 5.4
13
Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.93 ꢀ 1.87 (m, 2H) ppm. C NMR (125 MHz,
CDCl
3
): δ 165.2, 150.2, 149.0, 140.2, 137.2, 136.3, 134.4, 130.7, 129.2, 128.4, 127.4,
1
23.7, 122.9, 122.8, 121.8, 121.6, 119.1, 118.6, 113.9, 111.2, 58.5, 58.2, 55.3, 54.0,
+
39.4, 32.9, 30.9, 23.1 ppm. HRMS (ESI): m/z [M + H] calculated for C H N O ,
30
35
4
2
4
83.2755; found, 483.2756. HPLC purity : 97.5% .
(
E)-N-(3-(4-(((2-(1H-indol-3-yl)ethyl)(benzyl)amino)methyl)phenyl)propyl)-3-(py
ridin-3-yl)acrylamide (15)
The procedure was the same as described above for the synthesis of 10. Compound 15
1
was obtained as a yellow solid (22 mg, 85%). H NMR (400 MHz, CD
3
OD): δ 8.68 (s,
1
ꢀ
H), 8.48 (d, J = 4.4 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 15.6 Hz, 1H), 7.43
7.39 (m, 1H), 7.37 ꢀ 7.09 (m, 11H), 7.02 (t, J = 7.6 Hz, 1H), 6.93 ꢀ 6.81 (m, 2H),
6
.70 (d, J = 15.6 Hz, 1H), 3.64 (s, 2H), 3.60 (s, 2H), 3.41 ꢀ 3.31 (m, 2H), 2.91 (t, J =
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3
7
.6 Hz, 2H), 2.73 ꢀ 2.64 (m, 4H), 1.91 ꢀ 1.84 (m, 2H) ppm. C NMR (125 MHz,
CD OD): δ 167.7, 150.7, 149.6, 141.5, 140.9, 138.4, 138.1, 137.4, 136.2, 132.9, 130.2,
3
1
30.1, 129.3, 129.2, 128.8, 127.9, 125.5, 124.8, 123.0, 122.1, 119.4, 119.3, 114.4,
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
+
1
12.1, 59.4, 59.1, 55.2, 40.3, 33.9, 32.2, 23.9 ppm. HRMS (ESI): m/z [M + H]
calculated for C35 O, 529.2962; found, 529.2964. HPLC purity: 96.8%.
37 4
H N
(E)-N-(3-(4-(((2-(1H-indol-3-yl)ethyl)(methyl)amino)methyl)phenyl)propyl)-3-(py
ridin-3-yl)acrylamide (16)
The procedure was the same as described above for the synthesis of 10. Compound
1
16 was obtained as a yellow solid (157 mg, 75%). H NMR (400 MHz, CD
3
OD): δ
8
.69 (s, 1H), 8.49 (d, J = 4.9 Hz, 1H), 8.00 (dd, J = 8.1, 2.0 Hz, 1H), 7.53 (d, J = 15.8
Hz, 1H), 7.48 ꢀ 7.37 (m, 2H), 7.34 ꢀ 7.14 (m, 5H), 7.12 ꢀ 6.86 (m, 3H), 6.71 (d, J =
1
5.8 Hz, 1H), 3.60 (s, 2H), 3.42 ꢀ 3.31 (m, 2H), 2.96 (dd, J = 10.3, 6.1 Hz, 2H), 2.74 ꢀ
13
2
3
.66 (m, 4H), 2.33 (s, 3H), 1.92 ꢀ 1.85 (m, 2H) ppm. C NMR (125 MHz, CD OD): δ
1
70.2, 153.2, 152.2, 144.8, 140.7, 139.9, 138.7, 138.6, 135.4, 133.5, 131.9, 131.2,
1
28.0, 127.3, 125.6, 124.8, 122.0, 121.8, 116.2, 114.7, 65.1, 61.3, 44.9, 42.8, 36.4,
+
3
4.7, 26.2 ppm. HRMS (ESI): m/z [M + H] calculated for C H N O, 453.2649;
2
9
33
4
found, 453.2649. HPLC purity: 96.3%.
(E)-tert-butyl
2-(1H-indol-3-yl)ethyl(4-(3-(3-(pyridin-3-yl)acrylamido)propyl)
benzyl)carbamate (17)
To a mixture of (E)ꢀ3ꢀ(pyridinꢀ3ꢀyl)acrylic acid (18 mg, 0.12 mmol) in DMF (2 mL)
was added EDCI (38 mg, 0.2 mmol), HOBt (20 mg, 0.15 mmol)，triethylamine (0.03
mL, 0.2 mmol) and compound 35 (41 mg, 0.1 mmol), the reaction mixture was stirred
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