Facile synthesis of new 4-aza-podophyllotoxin analogs via microwave-assisted multi-component reactions and evaluation of their cytotoxic activity

Article abstract of DOI:10.1016/j.bmcl.2011.09.082

A series of new 4-aza-podophyllotoxin analogs containing thiazole unit were synthesized via multi-component reactions of aldehydes, tetronic acid and 2-methylbenzo[d]thiazol-5-amine under microwave irradiation. The method not only provides a valuable tool

Full text of DOI:10.1016/j.bmcl.2011.09.082

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7119–7123
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Facile synthesis of new 4-aza-podophyllotoxin analogs via
microwave-assisted multi-component reactions and evaluation
of their cytotoxic activity
Feng Shi a, , Xiao-Ning Zeng b, , Ge Zhang a, Ning Ma a, Bo Jiang a, Shujiang Tu a,
⇑
a School of Chemistry and Chemical Engineering, Xuzhou Normal University, Xuzhou 221116, PR China
b Clinical Research Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 4-aza-podophyllotoxin analogs containing thiazole unit were synthesized via multi-com-
ponent reactions of aldehydes, tetronic acid and 2-methylbenzo[d]thiazol-5-amine under microwave irra-
diation. The method not only provides a valuable tool in design and synthesis of new 4-aza-podophyllotoxin
analogs but also has the advantages of atom-economy, environmental-friendliness, good yields and oper-
ational simplicity. More importantly, the preliminary evaluation on the cytotoxic activity of this type of new
4-aza-podophyllotoxin analogs has resulted in the finding of several compounds with potent and effica-
cious cytotoxicity to three carcinoma cell lines M14, MCF7 and SW1116.
Received 4 August 2011
Revised 18 September 2011
Accepted 20 September 2011
Available online 29 September 2011
Keywords:
Podophyllotoxin analogs
Synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
Microwave irradiation
Multi-component reaction
Cytotoxicity
Cancer has been proven to be the second most common cause of
death after cardiovascular disease in the developed world.1 Since
its glucopyranoside derivative was recognized as a potent antitu-
mor factor, the aryltetralin lactone (À)-podophyllotoxin (Fig. 1)
occupies a unique position among lignan natural products.2 How-
ever, podophyllotoxin analogs, the cytotoxic activity of which is
based on its ability to inhibit the microtubule assembly during cell
division, were mostly unsuccessful and complicated by side effects
when it was used for treatment of human neoplasia. Thus, it is
often used as a starting material for structural modification or
synthesis of antitumour drugs, such as etoposide (VP-16-213)
and teniposide (VM-26) (Fig. 1), which have fewer side effects.3–6
In the aim of obtaining other podophyllotoxin analogs with little
side effects, great attention has been paid to the synthesis of 4-aza-
podophyllotoxin (Fig. 2) and its analogs. For instance, 4-aza-podo-
phyllotoxin analogs I have been prepared by Itokawa,7a Takeya7b
and Tratrat7c groups. Besides, great efforts have been carried out
in our group on the one-pot synthesis of 4-aza-podophyllotoxin
analogs II and III (Fig. 2)7d,e to provide candidate compounds for
bioassay.
podophyllotoxin skeleton has not attracted enough interest so
far. Thiazole ring system, which is a core structure in various syn-
thetic pharmaceuticals, displays a broad spectrum of biological
activities such as cytotoxic, antioxidant, antibacterial and anti-
fungal properties.8–15 It is promising that 4-aza-podophyllotoxin
analogs embedded with a thiazole ring may exhibit improved or
novel bioactivities. Therefore, we tentatively introduce a thiazole
unit to the skeleton of 4-aza-podophyllotoxin on the chance of
achieving its derivates with more potent pharmaceutical activity
but less toxicity.
In view of the pharmaceutical significance of 4-aza-podophyllo-
toxin analogs and as an continuation of our efforts on the synthesis
of bioactive heterocycles via microwave-assisted multi-component
reactions,8,16 we herein report an efficient synthesis of new
4-aza-podophyllotoxin analogs containing thiazole skeletons 4
via microwave-assisted three-component reactions of aromatic
aldehydes 1, tetronic acid 2 and 2-methylbenzo[d]thiazol-5-amine
3 (Scheme 1), and the evaluation on their cytotoxic activity.
The synthesis of ( )-dihydrofuro[3,4-b]thiazolo[4,5-f]quinolin
4b via the three-component reaction of 4-chlorophenyl aldehyde
1b (1 mmol), tetronic acid 2 (1 mmol) and 2-methylbenzo[d]thia-
zol-5-amine 3 (1 mmol) under microwave irradiation (MW) was
employed as a model reaction to optimize the reaction conditions.
This reaction was investigated in different organic solvents,
containing glacial acetic acid (HOAc), glycol, EtOH, water and
N,N-dimethylformamide (DMF) at 100 °C (Table 1, entries 1–5).
The results showed the reaction in HOAc gave the highest yield
In spite of the much attention concentrated on the synthesis
or structure modifications of 4-aza-podophyllotoxins, the
introduction of a sulfur-contained heterocyclic unit to the 4-aza-
⇑
Corresponding author. Tel./fax: +86 516 83500065.
E-mail address: laotu@xznu.edu.cn (S. Tu).
These authors contributed equally to the work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.082

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F. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7119–7123
OH
OH
H3CO
OCH3
H3CO
OCH3
O
O
H
H
OCH3
OCH3
O
O
O
O
H
H
H3CO
O
O
OH
O
O
O
H
O
O
O
O
H
OH
O
OH
O
O
H
OH
OH
S
O
(-)-podophyllotoxin
VM-26
VP-16-213
Figure 1. Structures of podophyllotoxin, VP-16-213 and VM-26.
R4
OCH3
OCH3
R'
R
R5
R3
H3CO
O
O
O
O
O
R2
R1
O
O
O
O
O
N
N
N
H
N
H
H
H
4-aza-podophyllotoxin
I
II
III
Figure 2. Structures of 4-aza-podophyllotoxin analogs.
(entry 5). Then, the same reaction was carried out in the range of
80–140 °C with the increment of 10 °C each time in HOAc. When
the temperature was increased from 80 to 120 °C, the yield of
product 4b was improved from 56% to 82% (entries 5–9). However,
the yield leveled off when the temperature was further increased
to 130 and 140 °C (entries 10 and 11). Apparently, the best temper-
ature for the reaction is 120 °C.
Under these optimized reaction conditions [HOAc, 120 °C], a
variety of substituted aromatic and heteroaromatic aldehydes as
reactants were applied to this reaction so as to demonstrate the
efficiency and applicability of the methodology. The results are
summarized in Table 2. Both aromatic aldehydes bearing either
Ar
N
N
O
O
ArCHO
1
+
S
S
HOAc
MW
O
O
N
H
4
H2N
O
2
3
Scheme 1. Synthesis of 4-aza-podophyllotoxin analogs.
Table 1
Optimization of solvent and temperature for the synthesis of 4ba
Table 2
Synthesis of 4 under microwave irradiationa
Cl
Cl
Ar
N
N
O
O
N
O
S
S
N
S
HOAc
O
Ar
+
Solvent
MW
+
+
S
O
o
O
+
O
120 C, MW
CHO
O
N
H
H2N
O
O
H2N
CHO
1b
N
H
4b
2
3
1
2
4
3
Entry Product Ar
Time (min) Yield (%)b mp (°C)
Entry
Solvent
T (°C)
Time (min)
Yield (%)b
1
2
3
4
5
6
7
8
9
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-FC6H4
8
8
8
7
7
10
8
8
8
7
80
82
83
78
82
79
80
79
81
77
73
70
278–280
278–281
289–291
296–298
288–290
297–298
295–297
168–171
293–295
280–282
224–226
283–285
1
2
3
4
5
6
7
8
9
Glycol
EtOH
DMF
100
100
100
100
100
80
8
8
8
8
8
8
8
8
8
8
8
55
63
45
43
74
56
68
78
82
80
79
4-ClC6H4
4-BrC6H4
3-NO2C6H4
4-NO2C6H4
C6H5
4-CH3C6H4
4-OCH3C6H3
2,4-Cl2C6H3
Water
HOAc
HOAc
HOAc
HOAc
HOAc
HOAc
HOAc
90
110
120
130
140
10
11
12
4j
4k
4l
4-OH-3-NO2C6H3
3,4,5-(OCH3)3C6H2 10
Thien-2-yl 10
10
11
a
a
All the reactions were carried out in 1 mmol scale in 2 mL solvent under MW at
All the reactions were carried out in 1 mmol scale in 2 mL HOAc at 120 °C under
the initial power of 100 W and the maximum power of 150 W, and the ratio of 1b/2/
3 was 1/1/1.
MW at the initial power of 100 W and the maximum power of 150 W, and the ratio
of 1/2/3 was 1/1/1.
b
b
Isolated yields.
Isolated yields.

F. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7119–7123
7121
Figure 3. The structure of 4c.
NH2
N
S
Ar
O
O
N
O
O
-H2O
S
H
C
O
O
3
ArCHO
O
O
2
Ar
O
1
NH
5
6
Ar
O
N
S
O
Ar
N
Ar
N
O
O
S
S
-H2O
O
O
O
N
H
NH2
N
H
HO
7
4
8
Scheme 2. The proposed reaction mechanism.
electron-donating groups (such as alkoxy and methyl) or electron-
withdrawing groups (such as nitro or halide) and heterocyclic thi-
ophene-2-carbaldehyde (entry 12) showed high reactivity and
afforded good yields of the 4-aza-podophyllotoxin analogs 4.
The structures of all the products 4 were unambiguously char-
acterized by IR, 1H NMR, and HRMS (ESI).17 Take compound 4c for
example, in the 1H NMR spectrum, a singlet at 10.31 ppm was
identified as the proton of the amino group, and the two doublets
at 7.10 and 7.89 ppm with coupling constants of 8.4 Hz were as-
signed to the two aromatic protons of the benzo[d]thiazol group,
which was verified by a singlet of 3H at 2.71 ppm designated as
the methyl group. The two doublets at 7.16 and 7.39 ppm with a
coupling constant of 8.4 Hz were identified as the four protons in
the 4-bromophenyl group and another two doublets at 4.91 and
5.01 ppm with a coupling constant of 16.0 Hz were designated as
the two methylene protons in the furan ring. At 5.56 ppm, a singlet
was assigned to the tertiary proton in the pyridine ring. The IR
spectrum of compound 4c showed strong absorbance at 3291 cm
The possible reaction mechanism is shown in Scheme 2. In the
initial step, the Knoevenagel condensation between aldehydes 1
and tetronic acid 2 afforded an intermediate 5. Then, the Michael
addition of 2-methylbenzo[d]thiazol-5-amine 3 to intermediate 5
furnished another open-chain intermediate 7, which gave rise to fi-
nal products 4 upon intramolecular cyclization and dehydration.
In order to survey the possible bioactivity of this class of novel
compounds, some selected 4-aza-podophyllotoxin analogs 4 were
subject to the test of in vitro cytotoxicity to malignant melanin car-
cinoma cell line M14, mammary carcinoma cell line MCF7 and co-
lon carcinoma cell line SW1116 (Table 3).
In general, most of the tested compounds exhibited moderate to
strong cytotoxicity to the three carcinoma cell lines. Among the
tested compounds, 4-aza-podophyllotoxin analog 4j showed the
most potent cytotoxicity to malignant melanin carcinoma cell line
M14 with IC50 value of 33.9
and compound 4g possessed the strongest cytotoxicity to mam-
mary carcinoma cell line MCF7 with IC50 value of 25.68 g/mL (en-
lg/mL (Table 3, entry 8, column 3),
l
À1
due to the presence of NH on the pyridine ring and a sharp
try 5, column 4), while compound 4d was the most powerful one to
inhibit the growth of colon carcinoma SW1116 cells with IC50 value
absorption at 1721 cmÀ1 suggested the existence of the C@O
group. Besides, the structure of 4c was further established by an
X-ray crystallographic analysis (Fig. 3).18
of 25.98
lg/mL (entry 2, column 5). It is worth-noting that
4-aza-podophyllotoxin analog 4j exhibited remarkable cytotoxic

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F. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7119–7123
Table 3
Table 3 (continued)
Cytotoxicity of new 4-aza-podophyllotoxin analogs 4
Entry
Compounds 4
IC50 to carcinoma cellsa
M14 cells MCF7 cells SW1116 cells
(lg/mL)
Entry
Compounds 4
IC50 to carcinoma cellsa
M14 cells MCF7 cells SW1116 cells
(lg/mL)
OH
Br
O2N
N
O
O
N
S
8
33.9
56
69.4
O
O
S
1
167.33
748.38
257.41
N
H
N
H
4j
4c
OCH3
O2N
H3CO
OCH3
N
N
O
O
O
O
S
S
9
>1000
685.74
383.18
2
77.93
151.46
25.98
N
H
N
H
4k
4d
NO2
a
The IC50 value corresponded to the compound concentration causing 50%
mortality in carcinoma cells.
b
Not tested.
N
O
O
S
3
>1000
>1000
>1000
activities to all three carcinoma cell lines with IC50 value of 33.9, 56
N
H
and 69.4 lg/mL, respectively (entry 8). It is interesting that both of
compounds 4d and 4j have a nitro group in the 3-position of their
aromatic rings, which illustrates that 3-nitro group may be helpful
to increase their cytotoxicity. Furthermore, compounds 4g has a 4-
methyl group in its aromatic ring, indicating alkyl group in the 4-
position of aromatic ring may also be beneficial to the cytotoxicity.
Therefore, future structural modifications of these 4-aza-podophyl-
lotoxin analogs may focus on introducing 3-nitro and 4-alkyl
groups to the skeletons.
4e
N
O
O
S
4
>1000
>1000
155.74
N
H
Therefore, these 4-aza-podophyllotoxin analogs 4 with moder-
ate to strong cytotoxicity may become promising antitumor drug
candidates after further structural modifications and biological
investigations.
4f
In conclusion, an efficient microwave-assisted multi-compo-
nent reaction has been developed to constitute the 4-aza-podo-
phyllotoxin skeleton embedded with a thiazole ring system. This
approach not only provides a valuable tool in design and synthesis
of new 4-aza-podophyllotoxin analogs, but also has the advantages
of atom-economy, environmental-friendliness, good yields and
operational simplicity. More importantly, the preliminary evalua-
tion on the cytotoxic activity of this type of new 4-aza-podophyl-
lotoxin analogs has resulted in the finding of several compounds
with potent and efficacious cytotoxicity to three carcinoma cell
lines M14, MCF7 and SW1116, which indicates that this novel class
of 4-aza-podophyllotoxin analogs may find their medicinal appli-
cations after further structural modulation and biological studies.
N
N
N
O
O
S
S
S
5
224.64
25.68
121.83
N
H
4g
OCH3
O
O
b
6
508.11
—
>1000
N
H
Acknowledgments
4h
Cl
We are grateful for financial supports by Natural Science Foun-
dation of China (No. 21002083 and 21072163), the Natural Science
Foundation (09KJB150011) and Qing Lan Project (08QL001) of
Jiangsu Education Committee.
Cl
O
b
7
>1000
539.25
—
O
Supplementary data
N
H
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.082.
4i

F. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7119–7123
7123
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17. The details on the experimental procedures and the spectroscopic data of the
synthesized compounds are available in the supporting information.
18. CCDC 843782 for compound 4c. The single-crystal growth was carried out in
N,N-dimethylformamide at room temperature. X-ray crystallographic analysis
was performed with a Siemens SMART CCD and a Semens P4 diffractometer.
Crystal data for 4c: C22H20BrN3O3S, yellow, crystal dimension 0.31 Â 0.13 Â
0.07 mm, triclinic, space group P-1, a = 5.6549(6) Å, b = 11.2821(14) Å,
c = 17.5553(18) Å,
a
= 82.760(2)°, b = 86.367(2)o,
c
= 75.5730(10)o, V =
1075.5(2) Å3, Mr = 486.38, Z = 2, Dc = 1.502 g/cm3, k = 0.71073 Å,
l (Mo
K ) = 2.037 mm-1, F(000) = 496, R1 = 0.1440, wR2 = 0.2452.
a