Triazolyl tryptoline derivatives as β-secretase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.043

Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The tryptoline was linked with different side chains by 1

Full text of DOI:10.1016/j.bmcl.2010.09.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6572–6576
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triazolyl tryptoline derivatives as b-secretase inhibitors
Jutamas Jiaranaikulwanitch a, Chantana Boonyarat b, Valery V. Fokin c, Opa Vajragupta a,
⇑
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhya Road, Bangkok 10400, Thailand
b Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
c Department of Chemistry, The Scripps Research Institute, 10500 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 May 2010
Revised 26 August 2010
Accepted 8 September 2010
Available online 22 September 2010
Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the
Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The
tryptoline was linked with different side chains by 1,2,3-triazole ring readily synthesized by catalytic
azide-alkyne cycloaddition reactions. Twenty two triazolyl tryptoline derivatives were synthesized and
screened for the inhibitory action against BACE1. JJCA-140 was the most potent inhibitor (IC50 = 1.49
and was 100 times more selective for BACE1 than for Cat-D.
lM)
Keywords:
BACE1
Ó 2010 Elsevier Ltd. All rights reserved.
BACE1 inhibitor
JJCA-140
Tryptoline
Docking
Binding mode
Enzyme assay
Cathepsin-D
Alzheimer’s disease (AD) is a common neuro-degenerative
disorder which affects 20–30 million individuals worldwide.1 The
patients’ cognitive function slowly declines, leading to end-stage
disease and death with-in 9 years after the diagnosis.1,2 The depo-
sition of aggregated b-amyloid peptides (Ab40,42) as plaques in
brain is a hallmark of AD. Inhibition of the formation of amyloid
plaques has been targeted in the new drug development. b-Secre-
4.0)14 and pharmacokinetic and toxicity filtering (Discovery Studio,
Accelrys).15 The Chemiebase covers the herbs in Thai Traditional
Pharmacopoeias and the herbs used by the local practitioners for
the preparation of traditional medicines. The flora in the database
ranges from common to scarce and some plants are in danger of
extinction in Thailand. Most of the plants are also common in
neighboring countries or countries in other part of the world with
the same climate. All identified compounds from the herbs
reported under the same botanical names were collected and com-
piled. Currently, there are 2048 compounds in the database. Based
on Thai traditional knowledge and the reduced search space, this
database considered a knowledge-based database, and virtual
screening of a knowledge-based database or focus library is recog-
nized as an efficient strategy for lead identification. This database
was selected on the basis of scaffold diversity of the natural com-
pounds it contains. The protein template was constructed from
two crystal structures of BACE1 bound to inhibitors (Protein Data
Bank code: 2IRZ16 and 1FKN17). In order to prepare the target pro-
tein as a template, the bound ligands (I02 and OM99-2) and crys-
tallographic water were removed. The unbound proteins were
superimposed (SwissPdb-Viewer)18 to reconstruct the missing
atoms (Leu152 and Ser173) in 2IRZ. The chain between residues
152 and 173 from 1FKN was cut and inserted in 2IRZ in the missing
region to form a new template 2IRZ-F in a reasonable conforma-
tion. Hydrogens and Gasteiger charges were added to the fixed
protein template, 2IRZ-F, by using AutoDockTools (ADT). During
final preparations nonpolar hydrogens were merged. Grid maps
tase (BACE1) and
c-secretase are the key enzymes to generate
these peptides from amyloid precursor protein (APP). The cleavage
of APP by BACE1 is the initial step in Ab formation; also, BACE1-
knockout mice are deficient in Ab production with no compensa-
tory mechanism. Thus, inhibition of BACE1 activity becomes the
promising target is an attractive target for AD drug development.2,3
BACE1 inhibitor was studied for more than a decade. Most of
them were developed from nonhydrolyzable hydroxyethylene
dipeptide isostere,4–6 high-throughput screening (HTS),7,8 and
fragment-base screening.9–11 In this study, new core structures of
BACE1 inhibitors were identified via virtual screening of the Thai
medicinal database. Ochrolifuanine E and its tryptoline core have
not previously been described pharmacophores for BACE1 inhibi-
tion, and this discovery represents another direction in the design
of BACE1 inhibitors.
Thai medicinal database (Chemiebase12,13) compounds were
the source of various scaffolds for virtual screening (AutoDock
⇑
Corresponding author. Tel.: +66 2 6448677; fax: +66 2 6448695.
E-mail address: pyovj@mahidol.ac.th (O. Vajragupta).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.043

J. Jiaranaikulwanitch et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6572–6576
6573
for each atom type in the ligands were set due to the centered on
dimension 29.557, 40.506, 3.896 with 80 ꢀ 100 ꢀ 80 Å and 0.375 Å
spacing between grids points were computed using AutoGrid 4.0.
All ligands were generated and optimized with ChemDraw Ultra
9.0 and Chem3D Ultra 9.0. Gasteiger charge was assigned, nonpolar
hydrogen was merged, aromatic carbons identified, and lastly, the
rigid root and rotatable bonds were defined.
2047 compounds from Thai medicinal data base
Docking to BACE1 template (2IRZ-F)
Ranking by the lowest energy
Energy cut-off = -10.00 kcal/mol
The constructed BACE1 template was validated by redocking
with the native ligands, I02, I03 and 5HA (PDB code: 2IRZ, 2ISO
and 2B8L). AutoDock 4.0 was employed to perform the docking cal-
culation. The 3D configuration of I02, I03 and 5HA obtained from
docking, or docked pose were compared with the crystallographic
pose. Ligands docked in the binding pocket in the same configura-
tion as found in crystal structure with RMSD <2.0 Å (Supplemen-
tary data). The redocking result or validation indicated that the
prepared BACE1 template (2IRZ-F) is a good model for virtual
screening.
40 compounds
Lowest binding energy
LE < -0.3
Molecular weight < 500
Log P < 5
% member in highest cluster > 50
For virtual screening, after each 100 docking runs per compound,
the conformations that had the lowest binding energy to BACE1
were clustered and ranked. Each cluster consisted of conformers
that had similar 3D structures (RMSD <2 Å). Fourty compounds that
had the binding energy lower than ꢁ10 kcal/mol were selected
7 compounds
Figure 1. The procedure in virtual screening.
Table 1
Selected hit compounds from virtual screening
Compound
Structure
MW
Log P
Binding energy in highest
clustering (kcal/mol)
% member in
highest cluster
LE
N
H
HN
HN
HN
HN
JV5-40
438.61
4.06
ꢁ13.45
ꢁ13.41
86
85
ꢁ0.41
NH
N
H
JV5-42
436.59
421.44
349.40
3.79
3.30
ꢁ0.41
0.34
NH
HO
HO
JV2-697
JV4-345
ꢁ10.39
ꢁ10.36
63
77
N
OH
HO
OH
HO
O
O
N
NH
O
ꢁ0.20
ꢁ0.43
HO
S
NH2
HO
HO
JV2-695
421.44
3.30
ꢁ10.33
68
ꢁ0.33
N
OH
HO
OH
HO
O
NH2
NH2
O
O
NH
JV4-346
JV2-326
349.40
365.40
ꢁ0.20
ꢁ0.58
ꢁ10.26
ꢁ10.22
86
74
ꢁ0.43
ꢁ0.41
N
S
HO
O
O
O
NH
N
S
OH
OH

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J. Jiaranaikulwanitch et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6572–6576
Table 2
Binding energy, pharmacokinetic and toxicity of selected core structure tryptoline
Asp32
Asp228
NH
S1
Properties
JV5-40
Tryptoline
H
N
Binding energy (kcal/mol) in highest clustering
BBB levela
ꢁ13.45
ꢁ7.42
N N
0
2
N
R
Absorption levelb
Solubility levelc
1
0
0
2
0
3
0
1
S2
CYP2D6d
PPB levele
S1'
Hepatotoxicityf
1
0
Probability of mutagenicity
0
0.001
701,500
Figure 3. The expected interaction of the designed structure.
Rat oral LD50
(lg/kg)
235.5
a
b
c
d
e
f
Blood–brain barrier penetration, 0 = very high, 2 = medium.
Absorption level, 0 = good, 1 = moderate.
Aqueous solubility, 0 = extremely low, 3 = good.
Cytochrome P450 2D6, 0 = non-inhibitor.
Plasma protein binding, 1 = binding is >90%, 2 = binding is >95%.
Hepatotoxicity, 0 = nontoxic, 1 = toxic.
uration as the side chain of S-compound can access to the S2 pock-
et better than R-compound. Therefore, (S)-3-(azidomethyl)-2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole was selected to be a new core
structure for BACE1 inhibitor. The expected interaction of the de-
signed structure was showed in Figure 3. The Cu(I)-catalyzed
[3+2]azide-alkyne cycloaddition reaction is the method to connect
the side chain (R) to the core nucleus due to the high yield and
purity.
The designed compounds with various Rs were docked to the
BACE1 template. Among 105 Rs including 29 alicyclics, 34 aromat-
ics, 22 heterocyclics, 19 bicyclic of aromatics and heterocyclics and
1 tricyclic (Supplementary data), the top 22 compounds giving
good docking results in binding energy and % member in the high-
est cluster were proceeded to the synthesis in 96 well plate for
screening activity. The (S)-3-methyl tryptoline azide was prepared
to react with 22 different alkynes (Fig. 4).
(Supplementary data). The 40 top ranked compounds were filtered
by five parameters: binding energy, ligand efficiency19–22 (<ꢁ0.3),
molecular weight (<500), Log P (<5)23 and % member in highest clus-
ter (>50), resulting in seven hit compounds (Fig. 1 and Table 1). Phar-
macokinetic and toxicity property of these compounds were
evaluated in silico. Ochrolifuanine E found in the local plant Dyera
costulata, the bark and leaves of D. costulata have been used in folk
medicine for treatment for fever by traditional doctors in the South
of Thailand.24,25 The hit compound JV5-40, ochrolifuanine E was
found to be most promising hit compound because it had the good
binding (ꢁ13.45 kcal/mol) and the good pharmacokinetic proper-
ties: blood–brain barrier penetration (BBB) level = 0, absorption le-
vel = 1 and CYP2D6 = 0 (Table 2). While either other compounds had
higher binding energy, no interaction with catalytic site Asp32 or
Asp228 or low blood–brain barrier penetration property. However,
the predicted toxicity of this compound was considerably high (LD50
The general synthetic scheme for the preparation of azide is
shown in Scheme 1 (Supplementary data). L-1,2,3,4-Tetrahydro
norharman-3-carboxylic acid 1 was esterified to yield methyl ester
2 (4.68 g, 88%). Reduction of 2 with sodium borohydride gave (S)-
(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-yl) methanol 3 (3.49
g, 85%). The hydroxy group of 3 was converted to the nosylate,
which was treated at 70 °C with sodium azide to give (S)-3-(azido-
methyl)-2-(4-nitrophenyl sulfonyl)-2,3,4,9-tetrahydro-1H-pyrido-
[3,4-b]indole 4 (2.76 g, 64%). After the removal of the nosyl group
of 235.5 lg/kg). The binding mode of ochrolifuanine E from docking
consists of two hydrogen bondings with Asp32 and Gly230, the
structure locates in pockets S2 and S10 of the substrate binding site
resulting in Van der Waals interaction. Tryptoline (2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole) part not only formed hydrogen bond
with Asp32, the residue in the catalytic site (Fig. 2) but also gave bet-
ter results in all parameters except the binding energy and BBB pen-
etration when compared to ochrolifuanine E, (Table 2). Therefore,
tryptoline was selected as the core nucleus for further design as
the binding energy can be modified by the side chain. After trypto-
line was docked with BACE1 template, phenyl ring could access
the hydrophobic S1 pocket, and polar hydrogen NH at position 2
was hydrogen bonded with O@C of Asp228.
In order to locate the side chain in the S2 pocket, the side chain
substitution was changed from C1 to C3. The 3-substituted trypto-
line derivative in both R and S-configurations were docked with
BACE1 template. The S-configuration at C3 position showed better
binding energy and % member in the highest cluster than R-config-
O
CN
O
O
NH2
O
O
A2
A1
A3
A4
O
O
N
N
NH
A7
O
A6
A8
A5
HO
O
OH
A11
OH
A12
A10
A9
OH
S
A13
OH
A15
A14
A18
A16
F
S1
N
N
O
S
S
Asp32
Gly230
F
H+
N
A19
A17
A20
+
NH2
H
S
NH
HN
OH
A22
Figure 4. Alkynes used to synthesize the library.
S1'
S2
A21
Figure 2. The binding mode of ochrolifuanine E.

J. Jiaranaikulwanitch et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6572–6576
6575
H
N
H
N
H
N
b
a
NH
NH
NH
O
OH
OH
O
O
1
2
3
4
c
H
H
N
H
N
Ns
N
d
e
NH
N
NH
N
N
N3
N3
N
R
6
5
Scheme 1. Synthesis of screening compounds. Reagents and conditions: (a) H2SO4,
methanol, reflux, 18 h; (b) NaBH4, ethanol, THF, reflux, 18 h; (c) (i) 4-nitro-
benzenesulfonyl chloride, TEA, CH2Cl2, rt, 4 h; (ii) NaN3, DMF, 70 °C, 6 h; (d) K2CO3,
thiophenol, DMF, 50 °C, 2 h; (e) alkyne, CuSO4ꢂ5H2O, sodium ascorbate, DMSO, rt,
24 h.
with thiophenol and potassium carbonate, (S)-3-methyl tryptoline
azide 5 (0.86 g, 57%) was obtained. Azides reacted in a 1:1 ratio with
22 alkynes to give the final triazole 6. The final reactions were run in
microtiter plates, Cu(I) was generated in situ using a combination of
CuSO4 and sodium ascorbate.26 After the completion, reactions mix-
tures were diluted with 50 mM sodium acetate (pH 4.5) buffer to
give a 25 mM stock solutions of screening candidates based on the
amount of azides used in the cycloaddition reactions, and with the
production of product more than 80% detected by LC–MS.
The 22 crude compounds were screened at 5
BACE1 (n = 3) (Fig. 5). Compound JJCA-14027 corresponding to al-
kyne A18, showed inhibitory activity more than 50% at 5 M,
lM against the
l
was resynthesized on a larger scale for complete characterization
(melting point, FTIR, 1H NMR, 13C NMR, MS, and LC–MS). JJCA-
140 was synthesized from azide 5 reacting with 2-ethynyl-6-meth-
oxy-napthalene in t-BuOH/H2O/EtOH. Cu(I) was generated in situ
as described above, using a combination of 5 mol % CuSO4 and
20 mol % sodium ascorbate. The pure compound was assayed for
its activity against the BACE1 and Cat-D. BACE1 and Cat-D inhibi-
tory activity of the compound were determined by enzymatic as-
say using a recombinant human BACE1 with FRET (fluorescence
resonance energy transfer) substrate from PanVeraÒ and Cat-D
with FRET substrate from SensoLyteÒ. b-Secretase inhibitor IV (Cal-
biochem) and pepstatin A were the positive controls to evaluate
the potency for BACE1 and Cat-D, respectively. Compound JJCA-
Figure 6. The binding modes of JJCA-140 (green) with BACE1.
In this study, Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition
reaction was used to synthesize a focused library from 22 different
alkynes and an azide-containing pharmacophore. The compound
JJCA-140 showed low micromolar inhibitory activity against BACE1
and 100 times more selective to BACE1 than Cathepsin-D in enzy-
matic assay. Tryptoline was the important part responsible for the
binding to the catalytic site Asp32 and Asp228. Triazole was crucial
for increasing the activity via S2 pocket interactions. The alkyne
substitutent, methoxy naphthalene, appears to reach the S2 pocket
thereby contributing to the increased activity.
140 showed potent inhibitory activity with BACE1 (IC50 = 1.49
and more selective with BACE1 than Cat-D (IC50 >100 M).
lM)
l
The binding mode of JJCA-140 to BACE1 was shown in Fig 6. The
phenyl group of tryptoline fitted with hydrophobic S1 pocket
(Leu30, Tyr71, Phe108, and Trp115). Two polar hydrogens of tryp-
toline are involved in hydrogen bonding interactions in the S1
domain, that is, NH to Asp32 and Asp228. The alkyne part was de-
signed to increase activity by accessing S2 sites (Gln73, Thr232 and
Lys321) and providing dipole–dipole interaction. Triazole part has
dipole–dipole interaction with Thr231 and Arg235 in S2 pocket.
Acknowledgments
This work was funded by Thailand Research Fund (TRF) through
the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0209/
2548) and the Commission of Higher Education Thailand (CHE-
RG 2551).We thank Drs. Larissa Krasnova, Suresh Pitram and Gian
Cesare Tron for valuable suggestions and discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.043.
References and notes
1. Heneka, M. T.; O’Banion, M. K. J. Neuroimmunol. 2007, 184, 69.
2. Citron, M. Neuroscience 2004, 5, 677.
Figure 5. Assays of the library members at 5
lM against BACE1.
3. Findeis, M. A. Pharmacol. Ther. 2007, 116, 266.

6576
J. Jiaranaikulwanitch et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6572–6576
4. Stanton, M. G.; Stauffer, S. R.; Gregro, A. R.; Steinbeiser, M.; Nantermet, P.;
Sankaranarayanan, S.; Price, E. A.; Wu, G.; Crouthamel, M. C.; Ellis, J.; Lai, M. T.;
Espeseth, A. S.; Shi, X. P.; Jin, L.; Colussi, D.; Pietrak, B.; Huang, Q.; Xu, M.;
Simon, A. J.; Graham, S. L.; Vacca, J. P.; Selnick, H. J. Med. Chem. 2007, 50, 3431.
5. Ghosh, A. K.; Kumaragurubaran, N.; Hong, L.; Kulkarni, S. S.; Xu, X.; Chang, W.;
Weerasena, V.; Turner, R.; Koelsch, G.; Bilcer, G.; Tang, J. J. Med. Chem. 2007, 50,
2399.
6. Hom, R. K.; Gailunas, A. F.; Mamo, S.; Fang, L. Y.; Tung, J. S.; Walker, D. E.; Davis,
D.; Thorsett, E. D.; Jewett, N. E.; Moon, J. B.; John, V. J. Med. Chem. 2004, 47, 158.
7. Malamas, M. S.; Erdei, J.; Gunawan, I.; Turner, J.; Hu, Y.; Wagner, E.; Fan, K.;
Chopra, R.; Olland, A.; Bard, J.; Jacobsen, S.; Magold, R. L.; Pangalos, M.;
Robichaud, A. J. J. Med. Chem. 2010, 53, 1146.
8. Cole, D. C.; Manas, E. S.; Stock, J. R.; Condon, J. S.; Jennings, L. D.; Aulabaugh, A.;
Chopra, R.; Cowling, R.; Ellingboe, J. W.; Fan, K. Y.; Harrison, B. L.; Hu, Y.;
Jacobsen, S.; Jin, G.; Lin, L.; Lovering, F. E.; Malamas, M. S.; Stahl, M. L.; Strand, J.;
Sukhdeo, M. N; Svenson, K.; Turner, M. J.; Wagner, E.; Wu, J.; Zhou, P.; Bard, J. J.
Med. Chem. 2006, 49, 6158.
16. Rajapakse, H. A.; Nantermet, P. G.; Selnick, H. G.; Munshi, S.; McGaughey, G. B.;
Lindsley, S. R.; Young, M. B.; Lai, M. T.; Espeseth, A. S.; Shi, X. P.; Colussi, D.;
Pietrak, B.; Crouthamel, M. C.; Tugusheva, K.; Huang, Q.; Xu, M.; Simon, A. J.;
Kuo, L.; Hazuda, D. J.; Graham, S. J. Med. Chem. 2006, 49, 7270.
17. Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.; Zhang, X. C.;
Tang, J. Science 2000, 290, 150.
18. Guex, N.; Diemand, A.; Peitsch, M. C. Trends Biochem. Sci. 1999, 24, 364.
19. Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 9997.
20. Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Discovery Today 2004, 9, 430.
21. Abad-Zapatero, C.; Metz, J. T. Drug Discovery Today 2005, 10, 464.
22. Hetényi, C.; Maran, U.; García-Sosa, A. T.; Karelson, M. Bioinformatics 2007, 23,
2678.
23. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46, 3.
24. Reanmongkol, W.; Subhadhirasakul, S.; Pairat, C.; Poungsawai, C.; Choochare,
W.; Songklanakarin, J. Sci. Technol. 2002, 24, 227.
9. Godemann, R.; Madden, J.; Krämer, J.; Smith, M.; Fritz, U.; Hesterkamp, T.;
Barker, J.; Höppner, S.; Hallett, D.; Cesura, A.; Ebneth, A.; Kemp, J. Biochemistry
2009, 48, 10743.
25. Mirand, C.; Men-Olivier, L.; Men, J. L.; Delaude, C. Phytochemistry 1983, 22, 577.
26. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
10. Yang, W.; Fucini, R. V.; Fahr, B. T.; Randal, M.; Lind, K. E.; Lam, M. B.; Lu, W.; Lu,
Y.; Cary, D. R.; Romanowski, M. J.; Colussi, D.; Pietrak, B.; Allison, T. J.; Munshi,
S. K.; Penny, D. M.; Pham, P.; Sun, J.; Thomas, A. E.; Wilkinson, J. M.; Jacobs, J.
W.; McDowell, R. S.; Ballinger, M. D. Biochemistry 2009, 48, 4488.
11. Congreve, M.; Aharony, D.; Albert, J.; Callaghan, O.; Campbell, J.; Carr, R. A. E.;
Chessari, G.; Cowan, S.; Edwards, P. D.; Frederickson, M.; McMenamin, R.;
Murray, C. W.; Patel, S.; Wallis, N. J. Med. Chem. 2007, 50, 1124.
12. Sangma, C.; Chuakheaw, D.; Jonkon, N.; Gadavanij, S. Curr. Pharm. Des. 2010, 16,
1753.
13. The structural database of chemical found in Thai medicinal plants. Available
at: http://chemiebase.ku.ac.th.
14. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
27. Melting point, IR, 1H, and 13C NMR, mass, and LC–MS determination. For JJCA-
140,
(S)-3-((4-(6-methoxynaphthalen-2-yl)-1H-1,2,3-triazol-1-yl)methyl)-
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as a light brown solid: mp 232–
234 °C; FTIR (KBr) (cmꢁ1): 3421.55 (pyrrole N–H, st.), 3302.33 (N–H, st.),
33109.19 (aromatic C–H, st.), 1613.26, 1481.43 (aromatic C–C, st.), 1266.38 (C–
O–C, st.), 1215.99 (C–N, st), 1029.17 (C–O–C, st.), 743.52 (aromatic C–H,
bending); 1H NMR (300 MHz, DMSO-d6) 10.70 (s, 1H, NH), 8.67 (s, 1H, H5jj),
8.33 (s, 1H, H1j), 7.96 (db, 1H, H5j), 7.88 (db, 2H, H3j, H4j) 7.34–7.32 (m, 2H,
H7j, H8j), 7.26 (db, 1H, H8), 7.18 (db, 1H, H5), 6.99 (t, 1H, H7), 6.91 (t, 1H, H6),
4.58 (s, 2H, H1), 3.88 (s, 2H, H4), 3.88 (s, 3H, CH3), 2.66 (db, 1H, CH2), 2.49 (db,
1H, CH2); 13C NMR (300 MHz, DMSO-d6); 157.53, 146.45, 135.90, 133.94,
129.60, 128.66, 127.45, 127.10, 126.20, 124.24, 123.49, 122.08, 120.48, 119.16,
118.36, 117.20, 110.95, 106.17, 106.02, 55.31, 54.16, 53.50, 41.96, 25.48; MS m/
z 411.4 [MꢁH], 410.3 [M]; LC–MS m/z 410.1896 [MꢁH].
15. O’Brien, S. E.; de Groot, M. J. J. Med. Chem. 2005, 48, 1287.