Design and Synthesis of New 9-Substituted Norharmane Derivatives as Potential Sirt5 Inhibitors

Article abstract of DOI:10.1002/jhet.2732

Sirt5 is a potential new drug target for the treatment of cancer, Alzheimer's disease, and Parkinson's disease. Given that norharmane is an important chemical synthon for some biologically important compounds and 9-substituted norharmane derivatives containing a negatively charged carboxyl group may accord with the characteristic of potential Sirt5 inhibitors, a series of novel 9-substituted norharmane derivatives were synthesized. The chemical structures and purities of all the target compounds were characterized by ¹H NMR, ¹³C NMR, MS, and HPLC. By in vitro SIRT5 inhibitory assays, three compounds (1a, 3a, and 3b) show over 30% inhibition ratios at concentration of 100 μM, and the most active compound 3b has 35% and 52% inhibition ratios at 30 μM and 100 μM, respectively. Docking analysis showed that compound 3b is likely to fit very well on the substrate binding site of Sirt5, and hence, we believe that compound 3b can serve as a lead compound for further efforts to develop specific Sirt5 inhibitors.

Full text of DOI:10.1002/jhet.2732

Month 2016
Design and Synthesis of New 9-Substituted Norharmane Derivatives as
Potential Sirt5 Inhibitors
Ling-Ling Yang,* Yan-Ying He, Quan-Long Chen, Shan Qian, and Zhou-Yu Wang*
College of Food and Bioengineering, Xihua University, Sichuan 610039, China
*
E-mail: yangll0808@sina.com; zhouyuwang77@gmail.com
Additional Supporting Information may be found in the online version of this article.
Received April 3, 2016
DOI 10.1002/jhet.2732
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
Sirt5 is a potential new drug target for the treatment of cancer, Alzheimer’s disease, and Parkinson’s dis-
ease. Given that norharmane is an important chemical synthon for some biologically important compounds
and 9-substituted norharmane derivatives containing a negatively charged carboxyl group may accord with
the characteristic of potential Sirt5 inhibitors, a series of novel 9-substituted norharmane derivatives were
1
synthesized. The chemical structures and purities of all the target compounds were characterized by H
1
3
NMR, C NMR, MS, and HPLC. By in vitro SIRT5 inhibitory assays, three compounds (1a, 3a, and 3b)
show over 30% inhibition ratios at concentration of 100 μM, and the most active compound 3b has 35%
and 52% inhibition ratios at 30 μM and 100 μM, respectively. Docking analysis showed that compound
3
b is likely to fit very well on the substrate binding site of Sirt5, and hence, we believe that compound 3b
can serve as a lead compound for further efforts to develop specific Sirt5 inhibitors.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
By analyzing the crystal structure of Sirt5 in complex
with its substrate, we found that there are three important
features for substrate binding pocket, which would be
useful in the design of new Sirt5 inhibitors (Fig. 2) [12].
First, three hydrophobic residues Leu227, Val254, and
Phe223 form a triangle-shaped entrance for the pocket,
which suggests that the compounds having triangle-
shaped motif that can fit with this entrance could be
potential inhibitors of Sirt5 [13]. Second, there are two
specific non-hydrophobic residues, Arg105 and Tyr102,
in the deep end of substrate binding pocket, which can
interact with the carboxyl of the substrate by hydrogen-
bonding and electrostatic interactions, indicating that some
negatively charged groups, such as carboxyl and tetrazole,
and some high polar groups, such as sulfonamide and
isoxazol-3-ol, could be preferred chemical moieties to
occupy this kind of pocket [14,15]. Third, the gate keeper
residue Phe223 in loop S of Sirt5 may restrict large
fragments to go into the deep end of the binding pocket,
suggesting that it is better to use some rigid, linear chemical
moieties or small aromatic rings as linkers to introduce the
essential pharmacophore features into the deep end.
Sirtuins (Sirts) are known as a class of protein deacetylases,
which carry out the deacetylation by using nicotinamide
adenine dinucleotide as a cofactor. On the basis of sequence
similarity, Sirts can be grouped into seven isoforms in
mammals, Sirt1 to Sirt7 [1,2]. Among the seven Sirts, Sirt5
is a very interesting member, which only has weak
deacetylation but has robust desuccinylation, demalonylation,
and deglutarylation activities in vitro and in vivo [3–5].
Because these modifications by Sirt5 cover a broad range of
pivotal protein substrates involved in cellular metabolism
and metabolic energy homeostasis, aberrant activity of Sirt5
was considered to be a very critical factor for many human
diseases, for example, cancer, Alzheimer’s disease, and
Parkinson’s disease [6–8]. Therefore, Sirt5 may represent a
fascinating drug target, and its inhibitors could be potential
new treatments for associated diseases. However, up to now,
only a few small-molecule Sirt5 inhibitors have been reported
(
Fig. 1), [9–11] and most of these compounds were initially
investigated as inhibitors of other isoforms rather than Sirt5,
except compound I. Thus, it is highly needed to develop novel
Sirt5 inhibitors for characterizing the physiological function
and therapeutical potentials.
Norharmane and its derivatives are found to possess a
variety of biological effects including anticancer and
©
2016 Wiley Periodicals, Inc.

L.-L. Yang, Y.-Y. He, Q.-L. Chen, S. Qian, and Z.-Y. Wang
Vol 000
Figure 1. Known Sirt5 inhibitors and their activities.
Figure 3. Schematic of structural modifications of 9-substituted
norharmanes.
Figure 2. The features of substrate binding pocket of Sirt5 protein. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
RESULTS AND DISCUSSION
Chemistry.
The novel 9-substituted norharmane
antimicrobial activities, and hence, norharmane is consid-
ered to be an important chemical synthon for some biolog-
ically important compounds [16–18]. Specifically, the
derivatives shown in Tables 1 and 2 were synthesized by
the synthetic routes outlined in Scheme 1 (1a–c, 2a–c,
and 3a–c) and Scheme 2 (4a–f). Briefly, all of the target
compounds were prepared from the commercially
available norharmane (7), which reacted with 2-(tert-
butoxycarbonylamino)ethyl-4-methylbenzenesulfonate (6)
and ethyl 2-bromoacetate (10) to afford the compounds 8
and 11 in the presence of NaH. Then, the key
intermediates 9 and 12 were obtained by the reaction of
de-protection of amino and hydrolyzation of ester bond,
respectively. Subsequently, target compounds 1a–c were
acquired through a two-step reductive amination reaction
9
-substituted norharmane derivatives seem to have a good
match with the triangle-shaped entrance of the binding
pocket of Sirt5. Thus, we thought that incorporation of a
negatively charged carboxyl group to the terminal of
substituents at the 9-position of norharmane by a rigid, lin-
ear-like linker with length of 6–8 atoms (Fig. 3) may make
those derivatives to be potential Sirt5 inhibitors.
In order to examine our ideas, we first performed some
chemical synthesis using the norharmane as raw material
to obtain new 9-substituted norharmane derivatives. Then,
the target compounds were tested by in vitro Sirt5
inhibitory assays. Molecular docking analysis was finally
used to predict the possible binding modes of the most
potent norharmane derivative.
between intermediate
9
with various benzene
formaldehydes. The compounds 2a and 2b were obtained
by the hydrolysis reaction following the condensation of
distinct benzoic acids with 9 in the presence of 1.0 equiv
of 1-hydroxybenzotriazole (HOBT), 1.0 equiv of 1-(3-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design and Synthesis of New 9-Substituted Norharmane Derivatives as Potential
Sirt5 Inhibitors
Table 1
The Sirt 5 inhibitory activities of 1a, 1b, 2a, 2b, 3a, 3b, 4a, and 4b.
a
%
Inhibition (Sirtuin5)
Compound
Structure
10 μM
30 μM
100 μM
1a
1b
2a
2b
3a
9
24
32
10
19
22
15
25
36
4
6
11
13
19
24
3
4
4
b
a
b
19
35
15
52
21
9
6
17
25
76
Nicotinamide
—
—
a
Each compound was tested twice; the data are presented as average values.
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L.-L. Yang, Y.-Y. He, Q.-L. Chen, S. Qian, and Z.-Y. Wang
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Table 2
The Sirt5 inhibitory activities of 1c, 2c, 3c, and 4c–f.
a
%
Inhibition (Sirtuin5)
Compound
Structure
10 μM
30 μM
100 μM
10
1c
4
8
2
c
0
0
0
1
7
7
7
3
4
c
c
10
8
4
4
d
e
6
8
10
10
12
18
4
f
10
15
24
76
Nicotinamide
Each compound was tested twice; the data are presented as average values.
—
—
a
(
(
(
dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride
EDCI), and 1.5 equiv of N,N-diisopropylethylamine
DIEA), while compound 2c was obtained firsthand
different isocyanates at refluxing CH Cl and hydrolysis
2 2
reaction of the esters in high yields [19]. The series of final
compounds 4a–f were synthesized using the same method
as that for compounds 2a or 2c with yields ranging from
48% to quantitative.
through the condensation reaction. Similarly, 3a and 3b
were acquired by the treatment of intermediate 9 with
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design and Synthesis of New 9-Substituted Norharmane Derivatives as Potential
Sirt5 Inhibitors
Scheme 1. Synthetic routes for the target compounds 1a–c, 2a–c, and 3a–c.
Scheme 2. Synthetic routes for the target compounds 4a–f.
Biological activity. The inhibitory activities of target
compounds 1a, 1b, 2a, 2b, 3a, 3b, 4a, and 4b against
Sirt5 were evaluated using in vitro enzymatic inhibition
assays through BPS Bioscience (San Diego, CA). All of
these compounds were tested at concentrations of 10,
N-benzylpropionamide, are shown in Table 1. We can
see from Table 1 that these compounds exhibited
distinct inhibitory activities against Sirt5. Compounds
1a, 3a, and 3b have over 30% inhibition ratios against
Sirt5 at 100 μM. The most potent compound 3b shows
35% and 52% inhibition ratios at 30 and 100 μM,
respectively. These results indicated that compounds
containing rigid link bridges like 1-phenyl-3-propylurea
may possess high inhibitory activities against Sirt5.
Subsequently, to further examine the importance of the
3
0, and 100 μM, and nicotinamide was used as the
positive control [20]. The Sirt5 inhibitory activities of
these novel 9-substituted norharmane derivatives with
different link bridges, including N-benzylpropan-1-
amine, N-propylbenzamide, 1-phenyl-3-propylurea, and
Journal of Heterocyclic Chemistry
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L.-L. Yang, Y.-Y. He, Q.-L. Chen, S. Qian, and Z.-Y. Wang
Vol 000
Figure 4. (a) The predicted binding mode of compound 3b with Sirt5; (b) the binding mode of compound 3b in comparison with the substrate of Sirt5.
Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
[
carboxyl group at the end of substituent, we synthesized
compounds 1c, 2c, 3c, and 4c–f without the carboxyl
group and evaluated their Sirt5 inhibitory activities. As
shown in Table 2, the compounds without carboxyl groups
only have low or no inhibitory activities against Sirt5.
rigid 1-phenyl-3-propylurea as link bridge exhibited most
potent inhibitory activities, which has 35% and 52%
inhibition ratios at 30 and 100μM, respectively. Finally,
compound 3b was observed by molecular docking to have
good fit with the substrate binding site of Sirt5, especially
have a very similar binding mode with Sirt5 substrate, and
hence, we believe that compound 3b can serve as a lead
compound for further development of specific Sirt5 inhibitors.
Molecular docking.
We further used molecular
docking to analyze the possible binding mode of
compound 3b. The AutoDock Vina docking program was
used here, and details about the docking method were
given in the Experimental section. A total of six docking
poses were generated for compound 3b, and all of them
have very similar binding modes, indicating those may be
true binding modes. As shown in Figure 4a, the carboxyl
group of compound 3b forms ionic bonding interactions
with Arg105 as well as tight hydrogen-bonding interactions
with Arg105 and Tyr102, and the urea motif of compound
EXPERIMENTAL
Chemistry methods.
Unless otherwise noted, all
starting materials, reagents, and solvents were purchased
from commercial vendors and used as supplied without
further purification. Analytical thin layer chromatography
(TLC), performed on Qingdao Haiyang silica gel F-254,
was used to monitor all of the reactions. Column
chromatography, performed on Qingdao Haiyang silica
gel 60 (300–400mesh), was used to purify the
intermediates and target compounds. All of the
compound spots were visualized by UV light (254nm).
3b seems to be parallel with Phe223 which may have polar–
pi interactions between them. By overlapping the docking
pose of compound 3b with the co-crystal substrate, we can
see that compound 3b actually has a similar binding mode
with that of Sirt5 substrate; for example, they both have
interactions with Arg105 and Tyr102, and they also have
good fit with the triangle-shaped entrance of the binding
pocket formed by Phe223, Leu227, and Val254 (Fig. 4b).
1
13
H NMR and C NMR spectra were recorded on a
Bruker AVANCE-III 400MHz, respectively. Chemical
shifts were expressed in parts per million (ppm, δ) relative to
tetramethylsilane as an internal standard. Proton coupling
patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), and broad (br). Low-resolution
and high-resolution mass spectral (MS) data were acquired
on an Agilent 1100 series LC-MS instrument with UV
detection at 254nm. HPLC analysis was conducted for all
compounds listed in the tables on an UltiMate 3000 HPLC
CONCLUDING REMARKS
In this study, by analyzing the complex structure of Sirt5
with its substrate, we hypothesized that 9-substituted
norharmane derivatives with a negatively charged carboxyl
in the tail end of substituent may be potential Sirt5 inhibi-
tors. A series of novel 9-substituted norharmane deriva-
tives were then synthesized starting from the
commercially available norharmane, followed by in vitro
Sirt5 inhibitory assays to evaluate the inhibitory activities
of these derivatives. The compound 4-(3-(2-(9H-pyrido
1
13
system (Dionex, USA). The H NMR, C NMR, HRMS
spectra and HPLC chromatograms of target compounds
were given in Supporting Information.
2-(9H-Pyrido[3,4-b]indol-9-yl)ethan-1-amine (9).
A mix-
ture of tert-butyl (2-hydroxyethyl)carbamate (5, 5.00g,
[3,4-b]indol-9-yl)ethyl)ureido)benzoic acid (3b) bearing a
31.1mmol) and TsCl (11.88g, 62.2mmol) in pyridine
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design and Synthesis of New 9-Substituted Norharmane Derivatives as Potential
Sirt5 Inhibitors
(
20mL) was stirred at ambient temperature for 12h. Upon
propyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.0
equiv), and N,N-diisopropylethylamine (1.5 equiv) in
tetrahydrofuran was stirred at room temperature for 0.5 h.
Next, the intermediate 9 (1.0 equiv) was added to the
mixture, which was heated to reflux for 12h. Then, the
mixture was cooled to room temperature, and amount of
water was added. The precipitate was filtered, washed
with ice water and tetrahydrofuran, and purified by
column chromatography (eluent gradient CH Cl /MeOH)
completion of the reaction as determined by TLC, the
solvent was removed in vacuo, and the crude mixture was
extracted with ethyl acetate (3×200mL). Then, the
combined organic layers were washed with brine, dried,
and concentrated. The residue was purified by column
chromatography (eluent gradient PE/EA=3/1) to give the
intermediate
2-((tert-butoxycarbonyl)amino)ethyl
4-
methylbenzenesulfonate (6,4.89 g, yield 50%) as a white solid.
Norharmane (7, 0.50 g, 2.98mmol) and NaH (0.24 g,
2
2
to afford the products ethyl 3-((2-(9H-pyrido[3,4-b]indol-
9-yl)ethyl)carbamoyl)benzoate, ethyl 4-((2-(9H-pyrido
5
2
(
.96mmol) in DMF (10 mL) was stirred at 45°C for 2 h, then
-((tert-butoxycarbonyl)amino)ethyl 4-methylbenzenesulfonate
6,2.81 g,8.94mmol) was slowly added to the mixture, and ad-
[3,4-b]indol-9-yl)ethyl)carbamoyl)benzoate,
and
compound 2c. Then, the two compounds, which contain
ditional stirring at room temperature was continued for 16 h.
When TLC indicated complete consumption of compound 7,
the reaction mixture was treated with ice water (200mL) and
extracted with EtOAc (3 ×150 mL). The combined extracts
were washed with brine, dried, and concentrated. The residue
obtained was purified by column chromatography (eluent gradi-
ent CH Cl /MeOH = 30/1) to give tert-butyl(2-(9H-pyrido[3,4-
an ester, were undergone hydrolysis by NaOH in the
solution of EtOH/H O (1:1) at 85°C for 1.5 h to give the
2
target compounds 2a and 2b in high yields.
General procedure for the preparation of 3a–c.
A
solution of substituted anilines (1.0 equiv) dissolved in a
spot of CH Cl was slowly dripped into a stirred solution
2
2
of triphosgene (0.33 equiv) in CH Cl2 by using a
2
2
2
b]indol-9-yl)ethyl)carbamate (8, 0.74 g, yield 77%) as a light
yellow solid. H NMR (400 MHz, DMSO): δ 8.92 (s, 1H,
constant-pressure dropping funnel. The catalytic quantity
1
of Et N was then added slowly to the reaction mixture
3
ArH), 8.49 (d, 1H, J= 5.2Hz,ArH), 8.17 (d, 1H, J=8.0Hz,
ArH), 7.98 (d, 1H, J= 5.2Hz, ArH), 7.62 (t, 1H, J=7.6Hz,
ArH), 7.55 (d, 1H, J= 8.0Hz, ArH), 7.33 (t, 1H, J=7.6Hz,
ArH), 4.65 (s, br, 1H, NH), 4.59 (t, 2H, J=5.6Hz, N–CH2),
after the anilines were added, and the mixture was
continued to stir for 0.5 h. After evaporation of the
solvent, the residue was taken up in CH Cl , and
2
2
intermediate 9 (1.0 equiv) was added directly to the
residue. The reaction mixture was stirred at reflux for 2 h,
and the solvent was subsequently removed in vacuo. The
residue was extracted with ethyl acetate twice, and the
combined organic layers were dried over MgSO4 and
concentrated. The crude product was purified by column
chromatography (eluent gradient CH Cl /MeOH) to
3
.62 (q, 2H, J=6.0Hz, NH–CH ), 1.45 (s, 9H, 3CH ) ppm.
2 3
To
a
stirring solution of intermediate
8
(
0.21g,0.66mmol) in CH Cl (40 mL) was added
2
2
trifluoroacetic acid (1.60 mL) at ambient temperature. The
reaction mixture was stirred at room temperature for 5 h.
After completion (monitored by TLC), the solvent was re-
moved in vacuo, the crude residue was treated with 150mL
of ice water, and the pH was adjusted to >7 with saturated
2
2
afford the ureasethyl 3-(3-(2-(9H-pyrido[3,4-b]indol-9-yl)
ethyl)ureido)benzoate, ethyl 4-(3-(2-(9H-pyrido[3,4-b]
indol-9-yl)ethyl)ureido)benzoate, and 3c in high yields.
Similarly, the two compounds, which contain an ester,
were undergone hydrolysis by NaOH in the solution of
NaHCO . The water solution was treated with EtOAc
3
(
3 × 150mL). The combined extracts were washed with
brine, dried, and concentrated. The residue was not purified
to give 2-(9H-pyrido[3,4-b]indol-9-yl)ethan-1-amine (9,
EtOH/H O (1:1) at 100°C for 2h to give the target
2
0
.44g, yield 86%) as a light yellow solid.
compounds 3a and 3b in yields 81% and 88%, respectively.
General procedure for the preparation of 1a–c. Catalytic
3-(((2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)amino)methyl)
benzoic acid (1a). Yield: 54%; HPLC purity: 98.58%. H
1
amount of molecular sieve and trifluoroacetic acid was
added to the solution of 2-(9H-pyrido[3,4-b]indol-9-yl)
ethan-1-amine (9, 1.0 equiv), benzene formaldehydes (1.2
equiv), and hantzschester (1.2 equiv) in CH Cl at room
NMR (400 MHz, DMSO): δ 13.00 (s, 1H, COOH), 9.08
(s, 1H, ArH), 8.37 (d, 1H, J=4.8Hz, ArH), 8.28 (d, 1H,
J=4.8Hz, ArH), 8.27 (d, 1H, J=8.0Hz, ArH), 8.13 (d,
1H, J=5.2Hz, ArH), 7.90 (s, 1H, ArH), 7.78 (d, 1H,
J=7.6Hz, ArH), 7.74 (d, 1H, J=8.4Hz, ArH), 7.42 (d,
1H, J=7.6Hz, ArH), 7.35 (t, 1H, J=7.6Hz, ArH), 7.28 (t,
2
2
temperature, and the reaction was warmed to 40°C and
reacted for 10h. After completion (monitored by TLC),
the reaction was filtered, and the crude residue was
obtained by concentrating the filtrate in vacuo. Finally, the
crude residue was purified by column chromatography
1H, J=7.6Hz, ArH), 4.60 (t, 2H, J=6.4Hz, N–CH ), 3.77
2
(s, 2H, Ph–CH ), 2.97 (t, 2H, J=6.4Hz, NH–CH ) ppm.
2
2
+
(
eluent gradient CH Cl /MeOH) to give the target
HRMS: m/z calcd for C H N O [M+H] 346.1550,
2
2
21 20 3 2
compounds in 54–63% yield.
found 346.1534.
General procedure for the preparation of 2a–c.
mixture of substituted benzoic acids (1.0 equiv), 1-
hydroxybenzotriazole (1.0 equiv), 1-(3-(dimethylamino)
A
4-(((2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)amino)methyl)benzoic
acid (1b). Yield: 58%; HPLC purity: 96.63%. H NMR
(400MHz, DMSO): δ 12.98 (s, 1H, COOH), 9.11 (s, 1H,
1
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L.-L. Yang, Y.-Y. He, Q.-L. Chen, S. Qian, and Z.-Y. Wang
Vol 000
ArH), 8.45 (d, 1H, J=5.2Hz, ArH), 8.32 (d, 1H, J=8.0Hz,
3-(3-(2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)ureido)benzoic
ArH), 8.21 (d, 1H, J=5.2Hz, ArH), 7.98 (d, 2H, J=8.4Hz,
PhH), 7.77 (d, 1H, J=8.4Hz, ArH), 7.67 (t, 1H, J=7.2Hz,
ArH), 7.57 (d, 2H, J= 8.0Hz, PhH), 7.35 (t, 1H, J=7.2Hz,
acid (3a).
Yield: 72% for two steps; HPLC purity:
1
98.28%. H NMR (400MHz, DMSO): δ 12.81 (s, br, 1H,
COOH), 9.07 (s, 1H, ArH), 8.78 (s, 1H, Ph-NH), 8.38 (d,
1H, J = 5.2 Hz, ArH), 8.28 (d, 1H, J= 7.6 Hz, ArH),8.14
(d, 1H, J = 4.2 Hz, ArH), 8.03 (s, 1H, ArH), 7.75 (d, 1H,
J = 8.4 Hz, ArH), 7.63–7.56 (m, 2H, ArH), 7.49 (d, 1H,
J = 7.6 Hz, ArH), 7.35–7.27 (m, 2H, ArH), 6.31 (t, 1H,
J = 5.2 Hz, CH –NH), 4.61 (t, 2H, J= 6.4 Hz, N–CH ),
ArH), 4.80 (t, 2H, J=6.8Hz, N–CH ), 4.26 (s, 2H, Ph–
2
13
CH ), 3.45 (t, 2H, J=6.4Hz, NH–CH ) ppm. C NMR
2
2
(100MHz, DMSO): δ 167.8, 167.6, 144.7, 142.1, 138.2,
137.2, 132.5, 131.7, 129.8, 129.2, 128.7, 127.7, 122.6,
1
21.0, 120.5, 115.3, 110.8, 46.3, 42.6ppm. HRMS: m/z
2
2
+
calcd for C H N O [M+H] 346.1549, found 346.1533.
3.56 (q, 2H, J= 6.0 Hz, NH–CH ) ppm. HRMS: m/z calcd
21
20
3
2
2
+
N-Benzyl-2-(9H-pyrido[3,4-b]indol-9-yl)ethan-1-amine
for C H N O [M + H] 375.1452, found 375.1451.
21 19 4 3
1
(
1c).
Yield: 63%; HPLC purity: 98%. H NMR
4-(3-(2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)ureido)benzoic
acid (3b). Yield: 78% for two steps; HPLC purity:
(
400 MHz, CDCl ): δ 9.08 (s, 1H, ArH), 8.41 (d, 1H,
3
1
J =5.2 Hz, ArH), 8.26 (d, 1H, J =8.0Hz, ArH), 8.08 (d,
99.32%. H NMR (400MHz, DMSO): δ 12.80 (s, br, 1H,
COOH), 9.07 (s, 1H, ArH), 8.96 (s, 1H, Ph-NH), 8.38 (d,
1H, J=4.8Hz, ArH), 8.28 (d, 1H, J= 7.2 Hz, ArH), 8.15
(d, 1H, J= 4.2 Hz, ArH), 7.81 (d, 2H, J= 7.6 Hz, ArH),
7.75 (d, 1H, J=8.0Hz,), 7.61 (t, 1H, J=6.8Hz,), 7.46 (d,
2H, J=7.2Hz, ArH), 7.29 (t, 1H, J=7.2Hz,), 6.43 (t, 1H,
J=5.2Hz, CH -NH), 4.61 (t, 2H, J=6.4Hz, N–CH ),
1
1
H, J= 5.2Hz, ArH), 7.84–7.87 (m, 4H, ArH), 7.66 (t,
H, J = 7.2Hz, ArH), 7.49 (d, 2H, J = 8.0Hz, ArH), 7.35
(t, 1H, J= 7.2Hz, ArH), 4.78 (t, 2H, J= 6.8Hz, ArH),
4
2
.23 (s, 2H, N–CH ), 3.42 (t, 2H, J = 6.4Hz, Ph–CH ),
2 2
1
3
.61 (m, 1H, NH–CH ) ppm. C NMR (100MHz,
2
DMSO): δ 167.3, 141.1, 140.3, 137.3, 135.5, 131.7,
2
2
1
29.2, 128.7, 127.6, 127.3, 122.2, 121.1, 114.8, 109.4,
3.56 (q, 2H, J=6.0Hz, NH–CH ) ppm. HRMS: m/z calcd
2
+
+
47.2, 43.3ppm. LC-MS: m/z 302.2 [M+ H] .
for C H N O [M+H] 375.1452, found 375.1450.
21 19 4 3
3
-((2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)carbamoyl)benzoic
1-(2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)-3-phenylurea (3c).
Yield: 78%; HPLC purity: 98.88%. H NMR (400MHz,
1
acid (2a).
Yield: 53% for two steps; HPLC purity:
8.35%. H NMR (400 MHz, DMSO): δ 13.02 (s, 1H,
COOH), 9.03 (s, 1H, ArH), 8.84 (t, 1H, J = 6.0 Hz, NH),
.35 (d, 1H, J= 5.2Hz, ArH), 8.26 (d, 2H, J= 4.4 Hz,
1
9
CDCl ): δ 8.81 (s, 1H, Ph-NH), 8.25 (d, 1H, J =5.2 Hz,
3
ArH), 8.12 (d, 1H, J = 7.6 Hz,), 7.86 (d, 1H, J =5.2 Hz,
ArH), 7.62–7.55 (m, 2H, ArH), 7.31 (t, 1H, J =7.2 Hz,
ArH), 7.20 (t, 2H, J = 7.6 Hz, ArH), 7.10–7.00 (m, 4H,
8
ArH), 8.12 (d, 1H, J =5.2 Hz, ArH), 8.03 (d, 1H,
J =8.0 Hz, ArH), 7.85 (d, 1H, J= 8.0Hz, ArH), 7.72 (d,
ArH), 5.26 (t, 1H, J= 6.0 Hz, CH –NH), 4.53 (t, 2H,
2
1
1
3
H, J= 8.4Hz, ArH), 7.58–7.50 (m, 2H, ArH), 7.26 (t,
H, J = 7.6Hz, ArH), 4.71 (t, 2H, J= 6.0Hz, N–CH2),
J = 6.0 Hz, N–CH ), 3.66 (q, 2H, J = 6.0 Hz, NH–CH )
2
2
13
ppm. C NMR (100 MHz, DMSO): δ 156.0, 141.5,
140.8, 138.6, 136.8, 132.8, 129.1, 128.9, 127.9, 122.5,
121.7, 120.9, 120.1, 118.4, 115.1, 110.6, 43.1, 39.0ppm.
.74 (q, 2H, J = 6.0 Hz, NH–CH ) ppm. HRMS: m/z calcd
2
+
for C H N O [M +H] 360.1343, found 360.1332.
21 18 3 3
+
4
-((2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)carbamoyl)benzoic
LC-MS: m/z 331.1 [M + H] .
acid (2b). Yield: 66% for two steps; HPLC purity: 100%.
H NMR (400 MHz, DMSO): δ 13.20 (s, br, 1H, COOH),
2-(9H-Pyrido[3,4-b]indol-9-yl)acetic acid (12). A mixture
of norharmane (7, 0.50g, 2.98mmol) and NaH (0.24g,
5.96mmol) in DMF (10mL) was stirred at ambient temper-
ature for 2h. Subsequently, ethyl 2-bromoacetate (2,
0.49mL, 4.46mmol) was slowly added to the mixture and
additional stirring at room temperature was continued for an-
other 5h. Upon completion of the reaction as determined by
TLC, The reaction mixture was then treated with ice water
(250mL) and extracted with EtOAc (3×150mL). The com-
bined extracts were washed with brine, dried, and concen-
trated. The residue obtained was purified by column
chromatography (eluent gradient CH Cl /MeOH=60/1) to
1
9
1
.15 (s, 1H, ArH), 8.81 (t, 1H, J = 5.6Hz, NH), 8.42 (d,
H, J = 5.2Hz, ArH), 8.29 (d, 1H, J= 5.2Hz, ArH), 7.92
(d, 2H, J =6.8 Hz, ArH), 7.79 (d, 1H, J= 4.4Hz, ArH),
7
.69–7.61 (m, 3H, ArH), 7.31 (t, 1H, J = 7.6Hz, ArH),
4
.75 (t, 2H, J = 6.4Hz, N–CH ), 3.76 (q, 2H, J= 6.0 Hz,
2
1
3
NH–CH ) ppm. C NMR (100MHz, DMSO): δ 167.2,
2
1
1
3
3
66.6, 142.2, 138.4, 136.8, 136.7, 133.4, 131.3, 129.6,
29.2, 127.7, 122.8, 120.7, 120.5, 115.7, 110.7, 42.6,
+
9.2ppm. HRMS: m/z calcd for C H N O [M+ H]
2
1 18 3 3
60.1344, found 360.1333.
2
2
N-(2-(9H-Pyrido[3,4-b]indol-9-yl)ethyl)benzamide (2c).
Yield: 84%; HPLC purity: 98.16%. H NMR (400MHz,
provide ethyl 2-(9H-pyrido[3,4-b]indol-9-yl)acetate(11,
yield 54%) as a light yellow solid. H NMR (400MHz,
1
1
CDCl ): δ 8.93 (s, 1H, ArH), 8.38 (t, 1H, J=5.2Hz, NH),
DMSO): δ 8.85 (s, 1H, ArH), 8.51 (d, 1H, J=9.6Hz,
ArH), 8.17 (d, 1H, J=7.6Hz, ArH), 7.99 (d, 1H,
J=5.2Hz, ArH), 7.63 (t, 1H, J=7.6Hz, ArH), 7.42 (d, 1H,
J=8.4Hz, ArH), 7.35 (t, 1H, J=7.6Hz, ArH), 5.10 (s, 2H,
N–CH ), 4.24 (q, 2H, J=7.2Hz, OCH ), 1.26 (t, 3H,
3
8
.16 (d, 1H, J=7.6Hz, ArH), 7.96 (d, 1H, J=5.2Hz,
ArH), 7.62–7.56 (m, 4H,Ar H),7.47 (t, 1H, J=7.2Hz,
ArH), 7.38–7.30 (m, 3H, ArH), 6.69 (t, 1H, J=5.2Hz,
ArH), 4.72 (t, 2H, J=6.0Hz, N–CH ), 3.95 (q, 2H,
2
2
2
+
J=6.0Hz, NH–CH ) ppm. LC-MS: m/z 316.1 [M+H] .
J=6.8Hz, CH ) ppm.
2
3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Design and Synthesis of New 9-Substituted Norharmane Derivatives as Potential
Sirt5 Inhibitors
A mixture of ethyl 2-(9H-pyrido[3,4-b]indol-9-yl)ace-
tate (11, 0.20g, 0.79mmol) and NaOH (0.10g, 2.37mmol)
N-(4-Chlorobenzyl)-2-(9H-pyrido[3,4-b]indol-9-yl)acetamide
(4e). The title compound was prepared from intermediate
12 and (4-chlorophenyl)methanamine using the same
was reacted for 2h in the solution of EtOH/H O (5 mL/
2
5
mL) at 100°C. After evaporation of the organic solvent,
method of compound 2c, yield: 69%; HPLC purity:
1
the residue was treated with 150mL of ice water, and the
PH was adjusted to 6–7 with diluted HCl. At this time, a
white solid was formed and collected by filtration. The
crude product was dried in a vacuum oven to give the title
compound 12 (0.16 g, yield 90%).
97.52%. H NMR (400MHz, CDCl ): δ 8.70 (s, 1H, NH),
3
8.34–8.30 (m, 2H, ArH), 8.19 (s, 1H, ArH), 8.08 (d, 1H,
J=8.0Hz, ArH), 7.83 (d, 1H, J=5.2Hz, ArH), 7.63 (t, 1H,
J=7.6Hz, ArH), 7.44 (t, 2H, J=8.0Hz, ArH), 7.35 (t, 1H,
J=7.6Hz, ArH), 7.13 (q, 1H, J=4.8Hz, J= 2.8 Hz, ArH),
3
-((2-(9H-Pyrido[3,4-b]indol-9-yl)acetamido)methyl)benzoic
6.84 (t, 1H, J=6.0Hz, ArH), 5.02 (s, 2H, N–CH ), 4.37 (d,
2
13
acid (4a). The title compound was prepared from inter-
mediate 12 and ethyl 3-(aminomethyl)benzoate using the
2H, J=6.0Hz, Ph–CH ) ppm. C NMR (100MHz,
2
CDCl ): δ 167.3, 141.1, 140.3, 136.4, 135.6, 133.4, 131.6,
3
same method of compound 2a, yield: 48%; HPLC purity:
129.3, 129.2, 128.8, 128.7, 122.3, 121.7, 121.2, 114.8,
1
9
8.68%. H NMR (400MHz, DMSO): δ 12.89 (s, br, 1H,
109.4, 47.2, 42.6 ppm. HRMS: m/z calcd for C H N O
2
0 17 3
+
COOH), 9.00 (s, 1H, ArH), 8.94 (t, 1H, J= 5.6 Hz, NH),
.40 (d, 1H, J=4.8Hz, ArH), 8.28 (d, 1H, J=7.6Hz,
ArH), 8.14 (d, 1H, J=5.2Hz, ArH), 7.91 (s, 1H, ArH),
[M+H] 350.1055, found 350.1043.
8
N-(Pyridin-3-ylmethyl)-2-(9H-pyrido[3,4-b]indol-9-yl)acet-
amide (4f). The title compound was prepared from inter-
mediate 12 and pyridin-3-ylmethanamine using the same
7
7
7
2
.84 (d, 1H, J= 7.6 Hz, ArH), 7.66–7.59 (m, 2H, ArH),
.52 (d, 1H, J=7.6Hz, ArH), 7.45 (t, 1H, J=7.6Hz, ArH),
method of compound 2c, yield: 71%; HPLC purity:
1
.31 (t, 1H, J=7.6Hz, ArH), 5.26 (s, 2H, N–CH ), 4.40 (d,
99.84%. H NMR (400 MHz, CDCl ): δ 8.80 (s, 1H,
2
3
13
H, J=6.0Hz, Ph–CH ) ppm. C NMR (100MHz,
NH), 8.50 (d, 1H, J= 5.2 Hz, ArH), 8.39 (d, 1H,
J = 4.8 Hz, ArH), 8.28 (s, 1H, ArH), 8.14 (d, 1H,
J = 8.0 Hz, ArH), 7.92 (d, 1H, J = 5.2 Hz, ArH), 7.65 (t,
1H, J =7.6 Hz, ArH), 7.46–7.43 (m, 2H, ArH), 7.38 (t,
1H, J= 8.0 Hz, ArH), 7.16 (q, 1H, J = 4.8 Hz, J = 2.8Hz,
2
DMSO): δ 167.8, 167.6, 141.8, 140.1, 139.2, 137.3, 133.3,
132.2, 131.5, 129.1, 128.8, 128.6, 128.4, 128.0, 122.3,
1
21.1, 120.2, 115.0, 110.6, 46.2, 42.6ppm. HRMS: m/z
+
calcd for C H N O [M+H] 360.1345, found 360.1334.
21 18 3 3
4
-((2-(9H-Pyrido[3,4-b]indol-9-yl)acetamido)methyl)benzoic
ArH), 6.42 (s, br, 1H, ArH), 5.10 (s, 2H, N–CH ), 4.41
2
13
acid (4b). The title compound was prepared from inter-
mediate 12 and ethyl 4-(aminomethyl)benzoate using the
(d, 2H, J =6.0 Hz, Ph–CH ) ppm. C NMR (100MHz,
2
CDCl ): δ 167.6, 148.8, 148.7, 141.1, 140.1, 136.4,
3
same method of compound 2a, yield: 51%; HPLC purity:
135.3, 133.3, 131.5, 129.3, 123.5, 122.2, 121.6, 121.1,
114.7, 109.3, 47.1, 40.8ppm. LC-MS: m/z 317.1 [M +H] .
1
+
9
6.34%. H NMR (400MHz, DMSO): δ 12.87 (s, br, 1H,
COOH), 9.08 (s, 1H, ArH), 8.96 (t, 1H, J = 6.0 Hz, NH),
.44 (d, 1H, J=5.2Hz, ArH), 8.32 (d, 1H, J=8.0Hz,
Molecular docking methods.
The AutoDock Vina
8
program was used to predict the binding mode of
compound 3b to Sirt5. The crystal structure of Sirt5 in
complex with succinyl-lysine substrate (PDB entry:
3RIY) was used as the docking template. Gasteiger–
Marsili charges were added to the models of protein and
compounds. The grid center were set as the center of
substrate (x, y, z= À8.1, 4.5, À10.5), and the size was
25Å ×25Å × 25Å, which encompassed the entire
substrate binding site. The numbers of docking poses
were set as 20. The other parameters for Vina were set as
default.
ArH), 8.24 (d, 1H, J=5.2Hz, ArH), 7.89 (d, 2H,
J= 8.0 Hz, ArH), 7.70–7.63 (m, 2H, ArH), 7.39 (d, 2H,
J=8.0Hz, ArH), 7.34 (t, 1H, J= 6.8 Hz, ArH), 5.30 (s, 2H,
13
N–CH ), 4.40 (d, 2H, J=5.6Hz, Ph–CH ) ppm. C NMR
2
2
(100MHz, DMSO): δ 167.8, 167.6, 144.7, 142.1, 138.2,
1
37.2, 132.5, 131.7, 129.8, 129.2, 128.7, 127.7, 122.6,
1
21.0, 120.5, 115.3, 110.8, 46.3, 42.6ppm. HRMS: m/z
+
calcd for C H N O [M+H] 360.1343, found 360.1332.
21 18 3 3
N-Benzyl-2-(9H-pyrido[3,4-b]indol-9-yl)acetamide (4c).
The title compound was prepared from intermediate 12
and phenylmethanamine using the same method of com-
1
pound 2c, yield: 66%; HPLC purity: 100%. H NMR
Acknowledgments. This work was supported by Chunhui of
Ministry of Education Project (no. Z2015120) and Talent
Introduction Key Project of Xihua University (no. z1410526).
(
400 MHz, CDCl ): δ 8.91 (s, 1H, NH), 8.55 (d, 1H,
3
J =8.4 Hz, ArH), 8.19 (d, 1H, J =7.6 Hz, ArH), 7.99 (d,
H, J = 5.2Hz, ArH), 7.67 (t, 1H, J = 7.6Hz, ArH), 7.49
d, 1H, J =8.4 Hz, ArH), 7.40 (t, 1H, J= 7.6Hz, ArH),
1
(
7
2
.70–7.08 (m, 5H, ArH), 6.04 (s, br, 1H, ArH), 5.11 (s,
REFERENCES AND NOTES
13
H, N–CH ), 4.44 (d, 2H, J = 4.0Hz, Ph–CH ) ppm.
C
2
2
NMR (100 MHz, CDCl ): δ 167.3, 141.1, 140.3, 137.3,
[1] Schemies, J.; Uciechowska, U.; Sippl, W.; Jung, M. Med Res
Rev 2010, 30, 861.
3
1
36.5, 131.7, 129.2, 128.7, 127.6, 127.3, 122.2, 121.7,
[2] Jing, H.; Lin, H. Chem Rev 2015, 115, 2350.
1
21.1, 114.8, 109.4, 47.2, 43.4 ppm. HRMS: m/z calcd
[3] Roessler, C.; Tuting, C.; Meleshin, M.; Steegborn, C.;
+
for C H N O [M+ H] 316.1444, found 316.1434.
Schutkowski, M. J Med Chem 2015, 58, 7217.
20 18 3
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

L.-L. Yang, Y.-Y. He, Q.-L. Chen, S. Qian, and Z.-Y. Wang
Vol 000
[
4] Park, J.; Chen, Y.; Tishkoff, D. X.; Peng, C.; Tan, M.; Dai, L.;
[12] Roessler, C.; Nowak, T.; Pannek, M.; Gertz, M.; Nguyen, G.
T.; Scharfe, M.; Born, I.; Sippl, W.; Steegborn, C.; Schutkowski, M.
Angew Chem Int Ed Engl 2014, 53, 10728.
Xie, Z.; Zhang, Y.; Zwaans, B. M.; Skinner, M. E.; Lombard, D. B.; Zhao,
Y. Mol Cell 2013, 50, 919.
[
5] Tan, M.; Peng, C.; Anderson, K. A.; Chhoy, P.; Xie, Z.; Dai,
[13] Jackson, M. D.; Schmidt, M. T.; Oppenheimer, N. J.; Denu, J.
M. J Biol Chem 2003, 278, 50985.
[14] Gertz, M.; Steegborn, C. Biochim Biophys Acta 2010, 1804,
1658.
[15] Du, J.; Zhou, Y.; Su, X.; Yu, J. J.; Khan, S.; Jiang, H.;
Kim, J.; Woo, J.; Kim, J. H.; Choi, B. H.; He, B.; Chen, W.; Zhang,
S.; Cerione, R. A.; Auwerx, J.; Hao, Q.; Lin, H. Science 2011, 334,
806.
L.; Park, J.; Chen, Y.; Huang, H.; Zhang, Y.; Ro, J.; Wagner, G. R.;
Green, M. F.; Madsen, A. S.; Schmiesing, J.; Peterson, B. S.; Xu, G.;
Ilkayeva, O. R.; Muehlbauer, M. J.; Braulke, T.; Muhlhausen, C.; Backos,
D. S.; Olsen, C. A.; McGuire, P. J.; Pletcher, S. D.; Lombard, D. B.;
Hirschey, M. D.; Zhao, Y. Cell Metab 2014, 19, 605.
[6] Parihar, P.; Solanki, I.; Mansuri, M. L.; Parihar, M. S. Exp
Gerontol 2015, 61, 130.
[
7] Lai, C. C.; Lin, P. M.; Lin, S. F.; Hsu, C. H.; Lin, H. C.; Hu,
[16] Itakura, S.; Kawabata, S.; Tanaka, H.; Enoki, A. J Insect Sci
2008, 8, 13.
M. L.; Hsu, C. M.; Yang, M. Y. Tumour Biol 2013, 34, 1847.
[
8] Liu, L.; Peritore, C.; Ginsberg, J.; Shih, J.; Arun, S.; Donmez,
G. Behav Brain Res 2015, 281, 215.
9] Maurer, B.; Rumpf, T.; Scharfe, M.; Stolfa, D. A.; Schmitt, M. L.;
He, W.; Verdin, E.; Sippl, W.; Jung, M. ACS Med Chem Lett 2012, 3, 1050.
10] Suenkel, B.; Fischer, F.; Steegborn, C. Bioorg Med Chem Lett
013, 23, 143.
11] Schuetz, A.; Min, J.; Antoshenko, T.; Wang, C. L.; Allali-
[17] Umezawa, K.; Shirai, A.; Matsushima, T.; Sugimura, T. Proc
Natl Acad Sci U S A 1978, 75, 928.
[
[18] Moloudizargari, M.; Mikaili, P.; Aghajanshakeri, S.; Asghari,
M. H.; Shayegh, J. Pharmacogn Rev 2013, 7, 199.
[19] Yang, L. L.; Li, G. B.; Yan, H. X.; Sun, Q. Z.; Ma, S.; Ji,
P.; Wang, Z. R.; Feng, S.; Zou, J.; Yang, S. Y. Eur J Med Chem
2012, 56, 30.
[
2
[
Hassani, A.; Dong, A.; Loppnau, P.; Vedadi, M.; Bochkarev, A.;
Sternglanz, R.; Plotnikov, A. N. Structure 2007, 15, 377.
[20] Fischer, F.; Gertz, M.; Suenkel, B.; Lakshminarasimhan, M.;
Schutkowski, M.; Steegborn, C. PLoS One 2012, 7e45098.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet