A direct synthesis of 5,6-dihydroindolo[2,1-a]isoquinolines that exhibit immunosuppressive activity

Article abstract of DOI:10.1016/j.bmcl.2009.08.057

Dihydroindolo[2,1-a]isoquinolines were synthesized from tetrahydroisoquinolines and α-fluoroaldehydes by a novel two-step procedure. These compounds exhibited significant immunosuppressive activity against IL-2, IL-10 and IFN-γ.

Full text of DOI:10.1016/j.bmcl.2009.08.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5539–5542
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A direct synthesis of 5,6-dihydroindolo[2,1-a]isoquinolines that exhibit
immunosuppressive activity
a
b
b
George A. Kraus a, , Vinayak Gupta , Marian Kohut , Navrozedeep Singh
*
a Department of Chemistry, Iowa State University, Ames, IA 50011, USA
b Immunobiology Program, Iowa State University, Ames, IA 50011, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Dihydroindolo[2,1-a]isoquinolines were synthesized from tetrahydroisoquinolines and
des by a novel two-step procedure. These compounds exhibited significant immunosuppressive activity
against IL-2, IL-10 and IFN-
a-fluoroaldehy-
Received 21 May 2009
Revised 13 August 2009
Accepted 14 August 2009
Available online 20 August 2009
c
.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Immunosuppressive
Indoloisoquinoline
Phosphazine
Condensation
The indolo[2,1-a]isoquinolines 1 represent a growing class of
natural and synthetic compounds with useful biological activity.
A sub-class whose members contain a quaternary ammonium salt
is represented by mangochinine (2a),1 cryptaustoline (2b),2 and O-
methylcryptaustoline (2c)3 and is shown in Figure 1. Certain indo-
lo[2,1-a]isoquinolines have been reported to inhibit the growth of
human mammary carcinoma cells,4 to treat multiple sclerosis,5 and
to exhibit antiviral activity.6 Compound 3 strongly inhibited tubu-
lin polymerization.7 To the best of our knowledge, there have been
no reports that indolo[2,1-a]isoquinolines exhibit immunosup-
pressive activity. We report herein that compounds synthesized
by our novel two-step procedure exhibit immunosuppressive
one, the yields were modest. Kametani reported the synthesis of
an indolo[2,1-a]isoquinoline via an intramolecular benzyne reac-
tion.11 Several groups reported intramolecular radical cyclizations
onto indoles 7 to form the indolo[2,1-a]isoquinoline ring sys-
tem.12,13 The radicals were generated using either trialkyltin hy-
drides or trialkylgermanium hydrides. This pathway is flexible
with regard to substitution on either the indole or the bromoben-
zene ring.
Our approach to the synthesis of indolo[2,1-a]isoquinolines is
depicted in Scheme 2. This approach involves the preparation of
aldehyde 8 by the coupling of 9 and 10 followed by a base-induced
cyclization to generate the indolo[2,1-a]isoquinoline system. Since
tetrahydroisoquinolines are readily available14 and several 2-flu-
orobenzaldehydes are commercially available, this approach has
the potential to be a very flexible one. This synthetic strategy is dis-
tinctly different from the four general synthetic routes to indo-
lo[2,1-a]isoquinolines described above. It may also be noted that
recently, De Koning reported the deprotonation and cyclization
of N-benzyl pyrroles using t-BuOK to form related heterocyclic
systems.15
activity against IL-2, IL-10 and IFNc.
Scheme 1 depicts four versatile methods for the synthesis of
indolo[2,1-a]isoquinolines that have been reported. Orito reported
the cyclization of 1-bromobenzyl-5,6-dihydroisoquinolines 4 by
the nucleophilic addition of the dihydroisoquinoline nitrogen atom
to the bromobenzyl moiety.8 He constructed several analogs with
different patterns of oxygenation. Lautens and co-workers reported
an innovative palladium-catalyzed tandem reaction sequence
starting from a N-(2-bromoethyl)indole 5 and an aryl iodide.9
Importantly, this sequence can accommodate both electron-with-
drawing and electron-donating groups on the aromatic ring. Saa
and co-workers reported the synthesis of 1 from 3,4-dihydroiso-
quinolines 6 and benzyne.10 Although this pathway is a direct
In order to test the concept, we treated 2-fluorobenzaldehyde
(10a) with anhydrous potassium carbonate and tetrahydroisoquin-
oline in DMF to generate 8a in 48% yield.16 Cyclizationof aldehyde 8a
was attempted using t-BuOK, LDA, Li-TMP, KH, and P4-t-Bu. Only P4-
t-Bu (Scheme 3), a sterically hindered phosphazine base developed
by Schwesinger,17 generated the desired tetracyclic product 1a.
When aldehyde 8a was treated with P4-t-Bu at 80 °C for 2 h, indo-
lo[2,1-a]isoquinoline 1a was produced in 35% isolated yield.18
* Corresponding author. Tel. +1 515 294 7794; fax: +1 515 294 0105.
E-mail address: gakraus@iastate.edu (G.A. Kraus).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.057

5540
G. A. Kraus et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5539–5542
6
MeO
R1O
HO
Bu
R5
R1
R2
Me
N
N
N
R4
2
9
OMe
OR2
OH
12
R3
OHC
1
2a: R1 = R2 = H
3
2b: R1 = H, R2 = Me
2c: R1 = R2 = Me
Figure 1.
R1
R2
R1
R5
N
N
Br
R2
Br
R4
R5
R4
R3
4
7
R5
R3
R1
R2
N
R4
R3
1
Br
+
R5
R5
R4
R3
R4
R3
R1
R2
R1
R2
I
N
+
NH
5
6
Scheme 1.
R5
R1
R2
R1
R2
R5
R4
R3
R5
F
R1
N
OHC
R4
R3
N
+
R4
R3
NH
OHC
R2
1a-j
8a-j
9
10
Scheme 2.
With a successful two-step synthesis of dihydroindolo[2,1-
a]isoquinolines, we generated a number of related compounds
from commercially available tetrahydroisoquinolines and 2-fluoro-
benzaldehydes. The results of this effort are shown in Table 1.
Compound 1f could be used in a direct synthesis of O-methyl-
cryptaustoline 3c as shown in Scheme 4. The reduction of com-
pound 1f using sodium cyanoborohydride in acetic acid at
ambient temperature afforded the tetrahydro compound 111
which on treating with methanol containing excess methyl iodide
over 48 h afforded 2c in 73% overall yield from 16. The NMR and
melting point of our synthetic compound were identical to that
of the literature19 compound.
We next evaluated compounds 1a–j for their ability to modu-
late immune response. Spleen cells obtained from influenza
virus-infected mice were cultured in vitro with compounds 1a–j
and influenza virus. Addition of virus to cultures significantly in-
creases cytokine production. The virus-induced change was ob-
served in the comparison between vehicle treatment and
vehicle + virus treatment in Figures 2a–c. With respect to the cyto-
kine interleukin-10 (IL-10), viral infection may induce secretion
from T lymphocytes as well as monocytes and macrophages. IL-
10 often acts to limit inflammation. Each compound (1a–j) sup-
pressed the production of IL-10 (Fig. 2a). In Figure 2b, influenza
virus-induced Interferon-c (IFNc) results are shown. IFNc is typi-
N
P
N
N
P
N
P
N
K2CO3,
F
P4-t-Bu,
C6H6, reflux
N
N
N
N
N
DMF, reflux
N
+
NH
N
P
N
OHC
N
N
OHC
9a
1a
10a
8a
P4-t-Bu
Scheme 3.

G. A. Kraus et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5539–5542
5541
Table 1
Synthesis of 5,6-dihydroindolo[2,1-a]isoquinolines
R1
R5
R5
R5
K2CO3,
DMF, reflux
R1
R2
R1
R2
P4-t-Bu,
C6H6, reflux
F
R4
R3
R4
R3
N
N
R4
R3
R2
+
NH
OHC
OHC
1a - 1j
10
8a - 8j
9
S.No
R1
R2
R3
R4
R5
Yield of 8a–j (%)
Yield of 1a–j (%)
a
b
c
d
e
f
g
h
i
H
H
H
OMe
OMe
OMe
H
OMe
H
H
H
H
OMe
OMe
OMe
H
OMe
H
OMe
H
H
OMe
H
H
OMe
CF3
CF3
H
H
H
OMe
H
H
OMe
H
H
CF3
CF3
H
OMe
H
H
OMe
H
H
H
H
H
48
42
48
48
37
40
60
66
52
53
35
52
38
25
32
39
44
35
42
39
j
OMe
H
MeO
MeO
Me
I-
MeO
MeO
MeO
N
N
N
NaBH3CN,
AcOH
MeI, MeOH
OMe
OMe
OMe
OMe
OMe
OMe
MeO
11
Scheme 4.
1f
2c
Interleukin-10
Interleukin-2
70
300
300
250
200
150
100
50
60
50
40
30
20
10
0
250
200
150
100
50
0
0
1a 1b 1c 1d 1e 1f 1g 1h 1i
1j veh veh+
1a 1b 1c 1d 1e 1f 1g 1h 1i
1j veh veh+
virus
In vitro treatment condition
In vitro treatment condition
Figure 2c. Suppression of IL-2 by 1a–j.
Figure 2a. Suppression of IL-10 by 1a–j.
cally secreted by cytotoxic T lymphocytes, and exhibits anti-viral
activity, but prolonged release may result in immunopathology.
Interferon-γ
Again, every compound (1a–j) inhibited production of IFN
leukin-2 (IL-2) is produced primarily by T-helper cells during influ-
enza infection and plays an important role in activating
c. Inter-
400
300
200
100
0
400
300
200
100
0
T
lymphocytes. Compounds 1a–j also significantly reduced IL-2
production.
All compounds were then evaluated for their impact on cell via-
bility, as reduced cell viability may limit cytokine production.
Spleen cells typically do not proliferate in culture without stimu-
lus, and in these experiments, no stimulus was added. Therefore,
these results likely reflect cell viability rather than proliferation.
Each of the compounds was incubated with spleen cells obtained
from non-infected mice for 24, 48 and 72 h. Compounds 1a–d, 1f,
and 1h significantly reduced viability at all three times points as
compared to the vehicle control (Fig. 3). Compounds 1i and 1j re-
duced viability at 24 h, but not after 48 or 72 h, however, there
1a 1b 1c 1d 1e 1f 1g 1h 1i
1j veh veh+
virus
In vitro treatment condition
Figure 2b. Suppresion of IFN
c
by 1a–j.

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G. A. Kraus et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5539–5542
11. Kametani, T.; Ogasawara, K. J. Chem. Soc. C 1967, 2208.
Cell Viability
1400
1200
1000
800
600
400
200
0
12. Allin, S. M.; Bowman, W. R.; Elsegood, M. R. J.; McKee, V.; Karim, R.; Rahman, S.
S. Tetrahedron 2005, 61, 2689.
13. Yasuda, S.; Hirasawa, T.; Yoshida, S.; Hanaoka, M. Chem. Pharm. Bull. 1989, 37,
1682.
14. Youn, S. W. Org. Prep. Proced. Int. 2006, 38, 505.
15. Loetter, A. N. C.; Pathak, R.; Sello, T. S.; Fernandes, M. A.; van Otterlo, W. A. L.;
de Koning, C. B. Tetrahedron 2007, 63, 2263.
24h
48h
72h
16. Representative procedure A: for the preparation of 2-[3,4-dihydroisoquinolin-
2(1H)-yl]benzaldehyde 8a: To
a solution of 1,2,3,4-tetrahydroisoquinoline
(0.56 g, 4.2 mmol) in dry DMF (6 mL), dry K2CO3 (0.58 g, 4.2 mmol) was added
followed by solution of 2-fluorobenzaldehyde (0.50 g, 4.0 mmol) in DMF at rt.
Reaction mixture was heated to reflux for 20 h. After the completion of
reaction, reaction mixture was cooled to rt, diluted with water and extracted
with ethyl acetate (three times). Organic layer was then washed with water,
brine and dried over MgSO4. Excess solvent was evaporated in vacuo to obtain
crude product. Crude product was subjected to column purification using 5%
ethyl acetate: petroleum ether to obtain pure product as yellow liquid (48%
yield).1H NMR (400 MHz, CDCl3) d 3.07 (t, J = 5.6 Hz, 2H), 3.46 (t, J = 5.6 Hz, 2H),
4.34 (s, 2H), 7.10–7.13 (m, 2H), 7.19–7.22 (m, 4H), 7.54 (dt, J = 7.8 Hz, J = 2 Hz,
1H), 7.86 (dd, J = 7.6 Hz, J = 1.6 Hz, 1H), 10.34 (s, 1H); 13C NMR (100 MHz,
CDCl3) d: 29.1, 53.6, 54.8, 119.0, 122.3, 126.1, 126.4, 126.6, 128.6, 129.0, 130.0,
134.1, 134.2, 134.9, 155.2, 191.3; MS (m/z): 237, 149, 125, 123, 95, 83, 69, 55;
HRMS: calcd for C16H15NO: 237.1154, found 237.1156.
1a 1b
1c
1d 1e
1f
1g 1h
In vitro treatment condition
1i
1j veh
Figure 3. Cell viability data of compounds 1a–j.
was a large degree of variability in the response with spleen cells
from some mice showing a large increase in viability whereas
spleen cells from other mice showed a decrease in viability. Com-
pound 1g did not significantly reduce viability as compared to
vehicle at any time point. In Figure 2a, it is apparent that adding
virus induces production of each cytokine as compared to the con-
trol wells without virus [vehicle compared to vehicle (DMSO) +
virus]. Each chemical compound (1a–j) when added to the well
with virus resulted in a significant (p <0.05) decline of cytokine
17. Kraus, G. A.; Zhang, N. Tetrahedron Lett. 2002, 43, 9597; Kraus, G. A.; Zhang, N.;
Verkade, J. G.; Nagarajan, M.; Kisanga, P. B. Org. Lett. 2000, 2, 2409.
18. Representative procedure B: for the preparation of 5,6-dihydroindolo[2,1-
a]isoquinoline 1a: to a solution of aldehyde (0.10 g, 0.42 mmol) in freshly
distilled dry benzene (25 mL), P4-t-Bu solution (0.46 mL, 0.46 mmol) was
added at rt and reaction mixture was heated to reflux with monitoring (2 h).
After the completion of reaction, benzene was partially evaporated and
reaction mixture was purified by column purification using 3% ethyl acetate:
petroleum ether to obtain pure product as Pale green solid (yield 35%).mp:
165–167 °C; 1H NMR (400 MHz, CDCl3)
d 3.21 (t, J = 6.8 Hz, 2H), 4.27 (t,
J = 6.4 Hz, 2H), 6.89 (s, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.20–7.36 (m, 5H), 7.65 (d,
J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) d: 29.4, 40.3,
96.6, 109.1, 115.5, 120.0, 120.9, 121.8, 124.5, 127.4, 128.6, 128.5, 128.9, 132.3,
135.8, 143.7; MS (m/z): 219, 109, 108; HRMS: calcd for C16H13N: 219.1048,
found 219.1052.
produced (Fig. 2a, IL-10; Fig. 2b, IFNc, and Fig. 2c, IL-2). In contrast
to the cytokine production, not all compounds reduced viability.
Instead, compounds 1a–f, and 1h reduced viability as compared
to vehicle control whereas 1g, 1i, and 1j were not different from
vehicle control. However, it is important to note that the effects
of compounds 1g, 1i, and 1j were variable. In some mice these
compounds reduced viability whereas in other mice these com-
pounds actually increased viability as assessed with Cell Titer
reagent.
In conclusion, this methodology constitutes a novel and direct
route to dihydroindolo[2,1-a]isoquinolines. The route is flexible
with respect to functionality and can be scaled up to prepare gram
quantities of dihydroindolo[2,1-a]isoquinolines. These compounds
exhibited significant immunosuppressive activity against IL-2, IL-
19. Elliott, I. W., Jr. J. Org. Chem. 1982, 47(27), 5398.
20. Procedure to evaluate immunomodulation: spleen cells were obtained from mice
infected with a mouse-adapted influenza virus (A/PR/8/34-H1N1) seven days
prior to cell collection. Mice were infected with a dose of virus resulting in <5%
mortality. Single cell suspensions were prepared from the spleens and red
blood cells were lysed with ammonium chloride. After washing with Hanks
Balanced Salt Solution to remove remaining ammonium chloride, the cells
were then adjusted to a concentration of 5 ꢀ 106 cell/ml in AIM-V cell-culture
medium (Invitrogen, Carlsbad CA). Cells were plated in 24 well plates. Control
wells contained either no influenza virus (no immune stimulation), vehicle
with no virus, virus (immune stimulation with influenza A/PR/8/34 virus conc.
of 10 HAU/ml), or vehicle + virus. Test wells contained one each of the chemical
compounds (1a–j) at a final concentration of 0.5 mg/ml performed in triplicate.
Cells were incubated at 37 °C, 5% CO2, in a humidified atmosphere, and cell
supernatant were collected 48–96 h later. Cell supernatants were collected at
10 and IFN-c, and the majority of the compounds reduced cell via-
bility with the exception of 1g, 1i, and 1j.20
48 h for the cytokines interleukin-2 and interferon-
cytokine interleukin-10. ELISA kits were used to analyze cytokines (BD
Biosciences). Briefly, immunosorbent 96 wells plates were coated with
c, and at 96 h for the
a
Acknowledgment
monoclonal capture antibody specific for each cytokine and incubated
overnight at 4 °C. Assay diluent (PBS) containing 10% fetal bovine serum was
used to block plates. After washing, appropriate cell culture samples and
cytokine standards were added to the plate, followed by 2 h incubation at room
temperature. Plates were again washed, and a second biotinylated detection
antibody as well as avidin-horseradish peroxidase conjugate (enzyme reagent)
was added. After another 1 h incubation at room temperature, plates were
washed again and TMB substrate containing 3,30,5,50-tetramethylbenzidine
(TMB) and hydrogen peroxide (H2O2) was added to each well. Following
30 min incubation, the absorbance was read on a Fluostar microplate reader. A
standard curve was created from the known standards and the cytokine
protein concentration was calculated by software using a four parameter curve
fit.
We thank the Department of Chemistry at Iowa State University
for partial support of Vinayak Gupta.
References and notes
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10. Atanes, N.; Guitian, E.; Saa, C.; Castedo, L.; Saa, J. M. Tetrahedron Lett. 1987, 28, 817.
A cell viability study of these compounds was also tested in spleen cells from
non-infected mice. Briefly, mouse spleen cells at
a concentration of
5 ꢀ 106 cell/ml in AIM-V medium were cultured in 96-well plates. Each well
contained a volume of 250
l
l (1.25 ꢀ 105 cells/well). The cells were incubated
with each of the compounds for 24, 48 or 72 h at 37 °C, 5% CO2, in a humidified
atmosphere. At the appropriate time point (24, 48 or 72 h), 20 ll volume of cell
titer reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] plus phenazine ethosulfate)
(Promega, Madison, WI) was added to each well. Plates were then returned to
the incubator for 3 h. After this time, the plates were read on a Fluostar
microplate reader at an absorbance of 490 nm.