Microwave assisted Westphal condensation and its application to synthesis of sempervirine and related compounds

Article abstract of DOI:10.1016/j.tetlet.2012.11.059

A concise synthesis of a potent lead in anticancer therapeutics, sempervirine, was achieved by one pot Westphal condensation, ester hydrolysis, and decarboxylation under microwave irradiation. The method was extended to the synthesis of several similar he

Full text of DOI:10.1016/j.tetlet.2012.11.059

Tetrahedron Letters 54 (2013) 487–490
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Microwave assisted Westphal condensation and its application to synthesis
of sempervirine and related compounds
T. S. Chinta Rao a,b, Sanjay Saha a,b, Gajendra B. Raolji a, Balaram Patro a, , Prabhaker Risbood c,
⇑
Michael J. Difilippantonio c, Joseph E. Tomaszewski c, Sanjay V. Malhotra d,
⇑
a GVK Biosciences Pvt. Ltd. Medicinal Chemistry Division, Nacharam, Hyderabad, India
b Department of Chemistry, JNT University, Kukatpally, Hyderabad, India
c Division of Cancer Treatment and Diagnosis;Center for Cancer Research;National Cancer Institute;National Institutes of Health;Bethesda, MD, USA
d Laboratory of Synthetic Chemistry, SAIC-Frederick Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A concise synthesis of a potent lead in anticancer therapeutics, sempervirine, was achieved by one pot
Westphal condensation, ester hydrolysis, and decarboxylation under microwave irradiation. The method
was extended to the synthesis of several similar heterocycles.
Received 7 September 2012
Revised 15 November 2012
Accepted 18 November 2012
Available online 29 November 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Sempervirine
Westphal condensation
Microwave assisted
DNA intercalator
Antiproliferative agent
Sempervirine, an alkaloid isolated1a,b from the roots of Gelsemi-
um sempervirens, is known as an antiproliferative agent both
in vitro2,3 and in vivo.4,5 Earlier, in a high throughput screening
(HTS) campaign of natural products, sempervirine was discovered
as a MDM2 E3 ubiquitin ligase inhibitor.6,7 Sempervirine is known
to stabilize p53 tumor suppressor protein levels8 by blocking its
proteasomal degradation9 via a ubiquitin-dependent pathway. It
inhibits both murine double minutes-2 (MDM2) dependent p53
ubiquitinylation and MDM2 auto-ubiquitinylation.10 Thus cancer
cells carrying wild-type p53 when treated with this compound in-
duce stabilization of p53 leading to apoptosis.10Sempervirine is
also known to intercalate DNA, and inhibits DNA topoisomerase
I;11therefore, it is a potential lead in anticancer therapeutics.
The structure of sempervirine was reported by Woodward1c and
Stevens1d as a resonance hybrid of 5A and B (Fig. 1a). A concise syn-
thesis of sempervirine methochloride was reported by Woodward
et al. (Fig. 1b),12 but the application of their method to the actual
synthesis of sempervirine never appeared in the literature. Though
many routes to the synthesis of sempervirine are known,13–18 all
but one route by Mattingly16 involve a long synthetic sequence,
while all reported methods suffer from low overall yield, and use
starting materials which are difficult to prepare.
C
N
N
A
B
D
N
E
N
5B
5A
Figure 1a. Structure of semervirine.1c,d
CHOiPr
+
N
O
N
Li
N
N
Figure 1b. Synthesis of sempervirine methochloride.12
We embarked on the synthesis of sempervirine, required by us
in gram quantities for biological evaluation, following the route
13
´
described by Lipinska. We started with 3-(methylthio)-1,2,4-tri-
azine, which was converted to 3-(1-(phenylsulfonyl)-1H-indol-2-
yl)-5,6,7,8-tetrahydroisoquinoline in 7 steps in 1.5% overall yield.
However, construction of C ring via Gribble’s15 C-lithiation strategy
was not successful. The condensation of 1-methyl-2-ethoxycar-
bonylmethyl-9H-pyrido [3, 4–b] indolinium bromide
2 with
⇑
Corresponding authors. Tel.: +91 40 66281666; fax: +91 40 66281505.
E-mail addresses: balaram.patro@gvkbio.com (B. Patro), malhotrasa@mail.
cyclohexan-1,2-dione D1 as described by Pottsand Mattingly16
(Scheme 1), afforded the cyclized compound 3 in ꢀ10 % yield.
nih.gov (S.V. Malhotra).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.11.059

488
T. S. Chinta Rao et al. / Tetrahedron Letters 54 (2013) 487–490
O
Br
N
O
OMe
b
Br
a
Br
NaOMe
+
N
N
N
N
H
OEt
OMe
N
H
1
N
H
2
N
H
3a
O
O
3
O
Br
N
NO3
OH
N
N
c
N
N
N
H
6
H
5B
4
reflux, 18 h, 90%; b) 1,2-cyclohexanedione, microwave, 95 °C,
Reagents and Conditions: a) ethyl bromoacetate, acetone,
1 h, 53 %; c) i)MeOH, 6N HCl, 2h, ii) MeOH, NaNO ,
2h, 78 %;
3
Scheme 1. Synthesis of sempervirine via microwave heating, and its conversion to the nitrate salt.
Table 1
Entry
Base
Equiv
Heating
Temperature
Time
3a
3aa
4a
5a
1
2
3
4
5
6
7
NaOAc20
NaOMe
NaOMe
NaOMe
NaOMe
NaOMe
NaOMe
3 equiv
2.5 equiv
3 equiv
3 equiv
3 equiv
3 equiv
4 equiv
Oil bath
Oil bath
60 °C
70 °C
80 °C
80 °C
95 °C
95 °C
95 °C
24 h
16 h
—
—
—
Nf
—
Nf
12%
30%
15%
30%
10%
Nf
60%
Nf
Nf
Nf
Nf
Nf
lwave
lwave
lwave
lwave
lwave
10 min
30 min
40 min
60 min
60 min
13%
14%
15%
20%
Nf
10%
14%
30%
45%
75%
Nf: not formed.
a
The reported numbers are percentage values from LCMS spectra, not the isolated yield.
Br
The decarboxylation of 4 at 250 °C resulted in decomposition, and
hence no product could be isolated. During further investigations
on the synthesis of sempervirine by Westphal condensation reac-
tion,19 the idea of application of microwave technology was con-
ceived by us. Sempervirine (5B) was obtained by the reaction of
quaternary compound 221 with 1,2-cyclohexanedione under
microwave heating, accomplishing three sequential steps in one
pot (Scheme 1).This methodology was also applied to other sub-
strates, which has allowed us to gain access to compounds similar
to sempervirine. The results of our initial investigations on the
Westphal condensation reaction are summarized in Table 1.
When a mixture of 2 and 1, 2-cyclohexanedione was heated in
methanol in the presence of sodium methoxide, formation of the
condensation product 3a (major) and cyclized ester 3 (minor) (en-
try 2) was observed. On further heating, there was slow conversion
of 3a to 3, but 3a remained as the major product even after 72 h
owing to the preference for the s-trans conformation. In order to
enhance the rate of cyclization, it was decided to carry out the con-
densation reaction under microwave heating. We were absolutely
delighted to see the dramatic impact microwave heating had on
the course of the reaction (entry 3). A mixture of 3, 4, and 5B (sem-
pervirine) was formed, with no detectable quantity of 3a. Further
optimization of the reaction conditions was done by varying the
reaction temperature, time, and the quantity of base.
N
S
N
N
N
N
N
N
H
F2
F3
F1
F5
F4
CO2Et
N
MeO
N
N
N
F7
F9
F6
F8
O
O
O
O
Ph
O
MeO
N
Ph
N
H
O
D1
D3
D2
F10
Figure 2. Heterocycle and diketone building blocks.
mation of the nitrate salt was also confirmed by overlapping IR
spectra of an authentic sample.
In order to explore the scope of this methodology several five-
and six-membered heterocycles (F1–10) were (Fig. 2) reacted with
ethyl bromoacetate in ethanol or acetone to afford the quaternized
compounds (Q1–10) in 70–90% yield (Scheme 2).21 The Westphal
condensation reactions were carried out by heating a mixture of
quaternized compound Qi (1 mmol), diketone Dj (1.2 equiv), and
sodium methoxide (2.5–4 equiv) in methanol between 95 °C and
105 °C for an hour.24 1–3 runs were done for each substrate,
varying the quantity of base (2.5–4 equiv) and/or temperature
(95–105 °C) and/or the dilution.
With an increase in the reaction temperature from 80 °C to
95 °C and a concomitant increase in reaction time, a gradual
improvement in conversion of the reaction intermediates to sem-
pervirine 5B was observed (entry 3 to 7). The best conversion
was obtained by heating the substrates in methanol with 4 equiv
of sodium methoxide for 60 minutes at 95 °C (entry 7). Sempervi-
rine 5B was isolated in 48% overall yield from harmane 1.22 It was
further transformed into nitrate via its hydrochloride salt.23 For-
In most of the reactions carried out at 95 °C, the acid Aij was
formed as the minor product. In one of the cases, when a purified

T. S. Chinta Rao et al. / Tetrahedron Letters 54 (2013) 487–490
489
Table 2
O
b
a
O
R
+
Br
R
N
N
Entry
Base
P22 (%)
OEt
O
1
2
3
4
5
6
7
Triethylamine
DBU
Pyridine
Morpholine
n-Butylamine
N-Methylpiperazine
Tri-n-Butylamine
36
46
40
25
Nil
16
50
Dj
Fi
Qi
O
R
O
Br
Br
Br
N
R
N
R
N
R
OMe
OH
Eij
Aij
Pij
R
R
i
j
=1 to 10; =1 to 3
in >95 % chromatographic purity and were characterized by 1H
NMR and mass spectra.
Reagents and Conditions: a) ethyl bromoacetate, acetone/EtOH,
reflux, 18 h, 70-90%; b) NaOMe, µwave, 95 °C, 1 h;
In order to explore the Westphal condensation reaction in the
presence of various organic bases, 1-(2-ethoxy-2-oxoethyl)-
2-methylpyridinium bromide Q2 and benzil D2 were chosen as
substrates. The reactions were carried out using Q2 (1 mmol),
benzil (1.2 mmol) and base (4 equiv) at 95 °C/1 h using methanol
as solvent. The results are summarized in Table 2.
Scheme 2. Synthesis of sempervirine and its analogues.
Br
N
N
N
Ph
The results indicated that the condensation reaction worked
quite well in the presence of tertiary bases: triethylamine,
tri-n-butylamine, DBU, and pyridine; whereas N-methylpiperazine
and morpholine were not so effective. Most importantly, no acid
derivative was detected in the reaction mixtures, indicating occur-
rence of spontaneous decarboxylation under microwave heating.
The effect of solvent on Westphal condensation reaction was
also explored using 1-(2-ethoxy-2-oxoethyl)-2-methylpyridinium
bromide (Q2, 1 mmol) and cyclohexane-1,2-dione (D1, 1.2 eq) as
substrates. The reactions were carried out at 95 °C/1 h using four
solvents: ethanol, acetonitrile, 1,4-dioxane, and N,N-dimethyl-
formamide. The reactions in acetonitrile and ethanol afforded the
desired product P21 (Fig. 3) in 44 % and 46 % yield respectively.
The reactions in DMF and dioxane were not clean (TLC) and hence
the product was not isolated.
In conclusion, we have synthesized sempervirine by Westphal
condensation reaction under microwave irradiation. We have also
synthesized several known as well as new quinolizinium and
pyridinium compounds by the same methodology. The usefulness
of this one-pot method lies in the fact that the starting materials
are very easy to prepare, and the method can be applied to a vari-
ety of starting materials bearing different functional groups. Ini-
tially, all the products were synthesized using sodium methoxide
as the base. Further investigations revealed that tertiary amines
like DBU, triethylamine, and di-tert-butylamines were superior
compared to sodium methoxide and afforded cleaner reactions
with improved yields.
R1
N
N
S
P11: R1= H (52%)
P31 (32%)
Ph
P12 (42%)
1
P101
:
R = OMe (32%)
R3
R3
Ph
Br
N
Ph
Ph
N
R4
N
S
R4
Ph
P32 (28%)
Br
Br
21: R3=R4=H (36%)
P
P
22: R3=R4=H (48%)
52: R3=H, R4=Br (36%)
P
P
51: R3=H, R4=Br (55%)
N
P
P
61: R3=H, R4=OMe (30%) P62: R3=H, R4=OMe (58%)
71: R3=CH3, R4=H (28%) P72: R3=CH3, R4=H (36%)
Br
P73 (35%)
Br
N
Br
N
HO2C
Ph
Ph
N
N
N
Br
P41 (42%)
P42 (40%)
P91 (50%)
HO2C
Ph
Ph
Br
N
N
P82 (42%)
Ph
Br
P92 (32%)
Ph
Figure 3. Compounds prepared by Westphal condensation. The number in brackets
indicate isolated yield.
sample of 11-carboxy-5-methyl-7,8,9,10-tetrahydro-5H-benzo
[4,5]imidazo [1,2-b]isoquinolin-12-ylium bromide (A41) (Obtained
from the reaction between Q4 and D1) was heated in methanol in a
microwave reactor at 100 °C for 30 minutes, no decarboxylation
was observed. However, spontaneous decarboxylation occurred
when the condensation reaction of Q4 and D1 was done at 105 °C
with two fold increase in dilution. This suggests that the decarbox-
ylation is base mediated and is also favored under higher dilution.
In all cases, the required products were purified by column chro-
matography over silica-gel. The compounds prepared are shown
in Figure 3.
In another case, cyclization of 5-bromo-1-(2-ethoxy-2-oxoeth-
yl)-2-methylpyridinium bromide Q5 with cyclohexane-1,2-dione
D1 and with benzil D2 resulted in the formation of side products
by substitution of bromo group with methoxy group. The forma-
tion of this side product was eliminated by using a lesser quantity
(2 equiv) of sodium methoxide.
Acknowledgments
This project has been funded in whole or in part with Federal
Funds from the National Cancer Institute, National Institutes of
Health, under Contract No. HHSN261200800001E. The content of
this publication does not necessarily reflect the views or policies
of the Department of Health and Human Services, nor does men-
tion of trade names, commercial products, or organizations imply
endorsement by the U.S. Government. T.S.C.R., S.S., B.P,. and
G.B.R. sincerely thank GVK Biosciences Private Limited for financial
support and encouragement. Support from analytical department
is also acknowledged.
Supplementary data
Supplementary data (1H, 13C NMR and analytical data for
Sempervirine free base 5 and nitrate salt 6 is available. For all other
compounds 1H NMR scans are available. LCMS scans of Table 1
To summarize, the products Pij were obtained in modest yields
ranging between 28 % and 58 %. All the compounds were obtained

490
T. S. Chinta Rao et al. / Tetrahedron Letters 54 (2013) 487–490
mixture was refluxed for 16 h. The progress of the reaction was monitored by
TLC. The reaction mixture was cooled and concentrated under reduced
pressure. The residue was crystallized in acetone (ꢀ100 ml) to give the
desired quaternary ammonium salt.
entries are also available) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
11.059.
22. Preparation of sempervirine (5): To a stirred solution of 2 (6.0 g, 17.2 mmol) in
methanol, 1,2-cyclohexanedione (2.5 g, 22.3 mM) and sodium methoxide
(3.6 g, 66.9 mmol) were added at room temperature. The resulting reaction
mixture was stirred in CEM-Microwave (Power-150 W, Pressure-250 psi) at
95 °C for 1 h after which it was cooled and acidified with dilute acetic acid. The
mixture was concentrated, and the obtained residue was purified by flash
column chromatography over silica-gel (230–400 mesh) using 0–10% MeOH in
DCM as eluent, to afford 2.4 g (53% yield) of as yellow solid. 1HNMR (DMSO-d6,
400 MHz): d 1.90 (m, 4H), 3.01 (m, 2H), 3.17 (m, 2H), 7.43 (t, 1H, J = 10 Hz),
7.67 (t, 1H, J = 10 Hz), 7.83 (d, 1H, J = 10.8 Hz), 8.38 (1H, d, J = 10.8 Hz), 8.68
(1H, d, J = 9.2 Hz), 8.76 (bs, 1H), 8.86 (1H, d, J = 9.2 Hz), 13.17 (bs, 1H). 13C NMR
(CD3OD, 100 MHz) d: 22.7, 22.8, 27.4, 30.4, 113.5, 116.9, 120.4, 122.5, 122.2,
123.0, 127.1, 130.3, 130.9, 132.2135.0, 135.9, 142.5, 151.4; MS (m/z): 273.18
(100%), 274.19 (13%); mp: 270–271 °C [Lit: 258–260 °C]14; UV kmax(MeOH):
388, 337, 295, 242 nm.
References and notes
1. (a) Prelog, V. Helv. Chim. Acta 1948, 31, 588–593; (b) Schwarz, H.; Marion, L.
Can. J. Chem. 1953, 31, 958–975; (c) Woodward, R. B.; Witkop, B. J. Am. Chem.
Soc. 1949, 71, 379; (d) Bentley, R.; Stevens, T. S. Nature 1949, 141–142.
2. Beljanski, M.; Beljanski, M. S. IRCS Med. Sci. 1984, 12, 587–588.
3. Beljanski, M.; Beljanski, M. S. Exp. Cell Biol. 1982, 50, 79–87.
4. Bassleer, R.; Clermont, D.; Marnette, J. M.; Caprasse, M.; Tits, M.; Angenot, L.
Ann. Pharm. Fr. 1985, 43, 83–88.
5. Beljanski, M.; Bugiel, J. Fr. Demande. FR 2419725, 1979.
6. Honda, R.; Tanaka, H.; Yasuda, H. FEBS lett. 1997, 420, 25–27.
7. Sasiela, C. A.; Stewart, D. H.; Kitagaki, J.; Safiran, Y. J.; Yang, Y.; Weissman, A.;
Oberoi, P.; Davydov, I. V., et al J. Biomol. Screening 2008, 13, 229–237.
8. Matlashewski, G.; Lamb, P.; Pim, D.; Peacock, J.; Crawford, L.; Benchimol, S.
EMBO J. 1984, 3, 3257–3262.
23. Preparation of sempervirine nitrate (6): To a suspension of (1.5 g, 5.5 mmol) in
methanol (10 ml), 6 N HCl (7.0 ml) was added at room temperature. The
resulting clear solution was stirred for 2 h, and then concentrated to dryness.
The residue was dissolved in methanol (10 ml); a solution of sodium nitrate
(10 ml) was added to it. A solid precipitated out after some time. It was stirred
for 2 h and filtered. The solid was washed with water, and air dried to yield
1.4 g of sempervirine nitrate as greenish yellow solid. 1HNMR (DMSO-d6,
400 MHz): d 1.90 (m, 4H), 3.02 (m, 2H), 3.18 (m, 2H), 7.44 (t, 1H, J = 10 Hz),
7.68 (t, 1H, J = 9.6 Hz), 7.83 (d, 1H, J = 10.8 Hz), 8.39 (1H, d, J = 10.8 Hz), 8.69
(1H, d, J = 8.4 Hz), 8.86 (1H, d, J = 9.2 Hz), 9.27 (1H, bs), 13.17 (1H, bs). MS (m/
z): 273.05 (100%), 274.06 (13%). HRMS: 273.1370 which corresponds to
[C19H17N2]+; IR (cmÀ1): 3400, 2937, 1646, 1632, 1524, 1469, 1383. mp: 280.5–
9. Peters, J. M.; Franke, W. W.; Kleinschmidt, J. A. J. Biol. Chem. 1994, 269, 7709–
7718.
10. Dickens, P.; Fitzgerald, R.; Fischer, P. M. Semin. Cancer Biol. 2010, 20, 10–18.
11. Zhizhen, Z.; Ping, W.; Wei, Y.; Shiyou, L. Plantamedica 2008, 74, 1818–1822.
12. Woodward, R. B.; McLamore, W. M. J. Am. Chem. Soc. 1949, 71, 379–380.
13. (a) Lipin´ ska, T. M. Tetrahedron 2006, 62, 5736–5747; (b) Lipin´ ska, T. M.;
Rykowski, A. Synth. Commun. 1996, 26, 4409–4414.
14. Chatterjee, A.; Sahu, A.; Saha, M.; Bannerji, J. Monatsh. Chem. 1996, 127, 1259–
1262.
15. (a) Gribble, G. W.; Barden, T. C.; Johnson, D. A. Tetrahedron 1988, 44, 3195–
3202; (b) Gribble, G. W.; Johnson, D. A. Tetrahedron Lett. 1987, 28, 5259–
5262.
16. Potts, K. T.; Mattingly, G. S. J. Org. Chem. 1968, 33, 3985–3987.
17. Ban, Y.; Seo, M. Tetrahedron 1961, 16, 11–15.
18. Swan, G. A. J. Chem. Soc. 1958, 2038–2044.
19. Westphal, O.; Jahn, K.; Heffe, W. Arch. Pharm. 1961, 294, 37–45.
20. One reaction was done following the conditions described in the following
reference: Matiaa, M. P.; Ezquerrab, J.; Garcia-Navfoa, J. L.; Vaqueroa, J. J.;
Alvarez-Builla, J. Tetrahedron Lett. 1991, 32, 7575–7578.
282.1 °C [Lit: 267 °C, decomp]18 13C NMR (DMSO-d6, 100 MHz) d: 21.1, 21.1,
;
25.7, 28.7, 112.5, 115.7, 118.9, 120.4, 120.7, 121.4, 121.5, 125.7, 128.6, 129.2,
130.0, 132.7, 134.5, 140.4, 148.9.
24. General procedure: To a solution of quanternary ammonium bromide derivative
(1.0 mmol) in methanol, the diketo compound (1.2 mmol, 1.2 equiv) and
sodium methoxide (4.0 mmol, 4.0 equiv) were added at room temperature. The
resulting reaction mixture was heated in CEM-Microwave (Explorer) between
95 °C and 105 °C (Power 150 W and Pressure 250 psi) temperature for 50 min.
The reaction mixture was acidified with acetic acid and concentrated under
reduced pressure. The crude product was purified by column chromatography
over a silica-gel eluting with a mixture of methanol and dichloroemethane.
21. General procedure for quaternization: To
a solution of the heterocycle
(10.0 mmol) in ethanol (20 ml) or in acetone (20 mL), ethyl bromoacetate
(15.0 mmol, 1.5 equiv) was added at room temperature. The resulting reaction