Praveen Kumar Reddy Nagireddy1, Vamsi Krishna Kommalapati1, Vagolu Siva Krishna2, Dharmarajan Sriram2, Anjana Devi Tangutur1,3, Srinivas Kantevari1,3. 1. Fluoro and Agrochemicals Division and Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India. 2. Medicinal Chemistry and Antimycobacterial Research Laboratory, Pharmacy Group, Birla Institute of Technology & Science Pilani, Hyderabad Campus, Hyderabad 500078, Telangana, India. 3. Academy of Scientific and Innovative Research (AcSIR), Chennai 600113, Tamil Nadu, India.
Abstract
Noscapine, a phthalide isoquinoline alkaloid isolated from the opium poppy Papaver somniferum, is traditionally being used as an anticough drug. With a safe in vitro toxicological profile, noscapine and its analogues have been explored to show microtubule-regulating properties and anticancer activity against various mammalian cancer cell lines. Since then, our group and other research groups worldwide are working on developing new noscapinoids to tap its potential as the leading drug molecule. With our continuing efforts, we herein present synthesis and anticancer evaluation of a series of imidazothiazole-coupled noscapinoids 7a-o and 11a-o. Natural α-noscapine was N-demethylated to nornoscapine 4 and then reacted with 4-(chloromethyl) thiazole-2-amine. The resultant noscapinoid 5 was coupled with various bromomethyl ketones 10a-o to give N-imidazothiazolyl noscapinoids 7a-o in very good yields. Similarly, natural α-noscapine 1 was O-demethylated using sodium azide/sodium iodide, reacted with 4-(chloromethyl)thiazole-2-amine, and coupled with bromomethyl ketones 10a-o to result in O-imidazothiazolyl noscapinoids 11a-o. All the new analogues 7a-o and 11a-o were fully characterized by their NMR and mass spectral analysis. In vitro cytotoxicity assay was performed for compounds 5, 7a-o, 9, and 11a-o against four different cancer cell lines: HeLa (cervical), MIA PaCa-2 (pancreatic), SK-N-SH (neuroblastoma), and DU145 (prostate cancer). Among these conjugates, 5, 7a, 9, 11b, 11c, 11e, and 11o showed potent cytotoxicity with low IC50 values. Further, flow cytometry analysis revealed that MIA PaCa-2 cells treated with these compounds induced cell cycle G2/M-phase arrest. In addition, Western blot analysis revealed that the cells treated with these conjugates accumulate tubulin in the soluble fraction and also elevate cyclin-B1 protein expression levels. Moreover, the conjugates also increased the expression of caspase-3 and PARP levels which is indicative of apoptotic cell death. In silico molecular docking studies showed several noncovalent interactions like van der Waals and hydrogen-bonding with tubulin protein and with good binding energy. The results indicated that these noscapine analogues may serve as novel compounds that can possibly inhibit tubulin protein and can be considered for further optimization as a clinical candidate for treating pancreatic cancer.
Noscapine, a phthalide isoquinoline alkaloid isolated from the opium poppyPapaver somniferum, is traditionally being used as an anticough drug. With a safe in vitro toxicological profile, noscapine and its analogues have been explored to show microtubule-regulating properties and anticancer activity against various mammaliancancer cell lines. Since then, our group and other research groups worldwide are working on developing new noscapinoids to tap its potential as the leading drug molecule. With our continuing efforts, we herein present synthesis and anticancer evaluation of a series of imidazothiazole-coupled noscapinoids 7a-o and 11a-o. Natural α-noscapine was N-demethylated to nornoscapine 4 and then reacted with 4-(chloromethyl) thiazole-2-amine. The resultant noscapinoid 5 was coupled with various bromomethyl ketones 10a-o to give N-imidazothiazolyl noscapinoids 7a-o in very good yields. Similarly, natural α-noscapine 1 was O-demethylated using sodium azide/sodium iodide, reacted with 4-(chloromethyl)thiazole-2-amine, and coupled with bromomethyl ketones 10a-o to result in O-imidazothiazolyl noscapinoids 11a-o. All the new analogues 7a-o and 11a-o were fully characterized by their NMR and mass spectral analysis. In vitro cytotoxicity assay was performed for compounds 5, 7a-o, 9, and 11a-o against four different cancer cell lines: HeLa (cervical), MIA PaCa-2 (pancreatic), SK-N-SH (neuroblastoma), and DU145 (prostate cancer). Among these conjugates, 5, 7a, 9, 11b, 11c, 11e, and 11o showed potent cytotoxicity with low IC50 values. Further, flow cytometry analysis revealed that MIA PaCa-2 cells treated with these compounds induced cell cycle G2/M-phase arrest. In addition, Western blot analysis revealed that the cells treated with these conjugates accumulate tubulin in the soluble fraction and also elevate cyclin-B1 protein expression levels. Moreover, the conjugates also increased the expression of caspase-3 and PARP levels which is indicative of apoptotic cell death. In silico molecular docking studies showed several noncovalent interactions like van der Waals and hydrogen-bonding with tubulin protein and with good binding energy. The results indicated that these noscapine analogues may serve as novel compounds that can possibly inhibit tubulin protein and can be considered for further optimization as a clinical candidate for treating pancreatic cancer.
Of the various cancers
existing globally, pancreatic cancer (PC)
remains very lethal and accounts for about 8% of the mortality rate
in cancerpatients.[1] Even though tremendous
efforts have been taken to diagnose, identify, and find suitable drugs
to treat PC, the disease still largely remains an enigma to biologists
and medicinal chemists. Estimates by American cancer society have
shown that PC may become the second leading cause of death by 2030.[2,3] The diagnosis of PC is difficult with symptoms usually seen at a
later stage, making drug therapies widely useless in curing the disease.
Even though surgery has been in existence for a long time, the rate
of relapse and recurrence of this cancer is very high, making it a
potentially noncurable disease.[4−6] In addition to the already existing
problems, PC has also been established to be immune-quiescent, rendering
immunotherapy a major failure. The lack of understanding of the cancer
progression coupled with metastasis has created a huge void in the
process of effective drug discovery and development, creating a serious
challenge to medicinal chemists to effectively utilize the available
database to generate potential chemotherapeutic agents.[7,8]Among the different naturally occurring products, noscapine
is
one such molecule that was found to be effective as an anticancer
agent. Noscapine 1,[9,10] previously known as
narcotine, is a natural phthalide isoquinoline alkaloid constituting
7% in opium alkaloids (Figure ). Noscapine displays antitussive properties and also exhibits
a good safety profile. Studies have shown that noscapine possesses
anticancer properties with tubulin disruption. Noscapine is a well-known
apoptotic trigger in many cancer cell lines via different pathways.[11] Exhaustive research has led to the development
of many noscapinoids which are more effective than parent noscapine
as potent anticancer agents.[12−15]
Figure 1
Natural α-noscapine and its congeners as potential
chemotherapeutic
cancer agents.
Natural α-noscapine and its congeners as potential
chemotherapeutic
cancer agents.Structural diversity on this scaffold
has generated considerable
interest in recent years with modifications being done at 1-, 7-,
6′-, and 9′ positions of noscapine. Halogen substitutions
at 9′-position yielded improved activity while deletion of
the lactone ring at 1-position and replacement of the same with cyclic
ethers are favorable while retaining the activity. O-substituted and
7-amino noscapinoids demonstrated S-phase arrest of the cell cycle
along with G2/M phase arrest. These interesting attributes
have attracted many researchers to work on noscapine for the development
of potent anticancer molecules.[16−19]Imidazo[2,1-b]thiazole, a
fused heterocycle, has
been established to exert a wide spectrum of biological activities.[20] The presence of this scaffold as a core unit
in antihelminth and immune-modulatory drug Levamisole (I, Figure )[21] caught the eyes of many medicinal chemists.
In the recent years, this scaffold has been exploited for its potency
against various other diseases such as cystic fibrosis as an antiviral
agent, sirtuin activators, cardio-depressants, and antitumor agents.[22] Guanyl hydrazone-containing derivatives of imidazothiazole
(III)[23] displayed potent antiproliferative
activity and is considered a promising lead in further development
of molecules with this core skeleton. Tubulin polymerization inhibition
was prominently observed in hybrids containing an imidazo[2,1-b] thiazole scaffold,[24−28] cementing the fact that this moiety can be exploited for drug discovery
and development to generate drug molecules in the future. 2-Aryl benzo[d]imidazo[2,1-b] thiazole derivative (II, YM-201627) is another example of an imidazothiazole molecule
with antitumor properties against solid tumors.[29] A phase III clinical trial compound (IV, AC220)
for combating FMS-like tyrosine kinase-3 is another example of an
imidazothiazole-containing molecule with nanomolar potency.[30]
Figure 2
Cytotoxic natural products with an imidazothiazole (red)
pharmacophore.
Cytotoxic natural products with an imidazothiazole (red)
pharmacophore.Our previous efforts on developing
a novel noscapinoid as a potent
anticancer molecule yielded potent analogues at 7- and 9-positions
with varying substitutions.[31,32] In continuation of
our work on the development of novel analogues of noscapine, we now
present in this paper, imidazothiazole-hybridized noscapine analogues
as potent anticancer agents designed as shown in Figure .
Figure 3
Design strategy for new
imidazothiazole-type α-noscapine
congeners.
Design strategy for new
imidazothiazole-type α-noscapine
congeners.
Results and Discussion
Chemistry
α-Noscapine
structurally consists of
two major constituents (isoquinoline and phthalide ring systems) connected
with a sensitive C–C bond which is labile to strong acids and
bases. Therefore, the synthesis of noscapine analogues is always challenging.
In the present work, we have optimized the reaction conditions for
the synthesis of noscapinoids without affecting the sensitive C–C
bond. The synthetic route for preparing new noscapinoids 7a–o and 11a–o is depicted in Scheme .
Scheme 1
Synthesis of Noscapine–Imidazothiazole
Analogues
Reaction Conditions: (i) (a) m-CPBA, dichloromethane; (b) 2 N HCl; (c) FeSO4·7H2O; (ii) NaN3, NaI, DMF, 140 °C,
4 h, 65%, (iii) 4-(chloromethyl)thiazole-2-amine, K2CO3, KI, acetone, rt, 4 h, 60%, (iv) 4-(chloromethyl)thiazole-2-amine,
K2CO3, KI, acetone, reflux, 4 h, 60%, (v) 10a–o, 2-propanol, reflux, 12 h, 60–80%.
Synthesis of Noscapine–Imidazothiazole
Analogues
Reaction Conditions: (i) (a) m-CPBA, dichloromethane; (b) 2 N HCl; (c) FeSO4·7H2O; (ii) NaN3, NaI, DMF, 140 °C,
4 h, 65%, (iii) 4-(chloromethyl)thiazole-2-amine, K2CO3, KI, acetone, rt, 4 h, 60%, (iv) 4-(chloromethyl)thiazole-2-amine,
K2CO3, KI, acetone, reflux, 4 h, 60%, (v) 10a–o, 2-propanol, reflux, 12 h, 60–80%.Commercial natural α-noscapine (1) was treated
with meta-chloroperbenzoic acid (m-CPBA) and acidified to give N-oxide hydrochloride salt 6.[12] The salt 6 was further
reacted with FeSO4·7H2O (modified nonclassical
Polonovski reaction conditions) to yield 48% of nornoscapine 4.[12,13,31] The free amine of noscapine was reacted with 4-(chloromethyl)-thiazol-2-amine
in the presence of K2CO3 and KI in acetone for
4h to yield the thiazole amine-coupled noscapinoid 5.
Further, 5 was reacted with substituted aryl/heteroaryl
α-bromomethyl ketones 10a–o (prepared by
refluxing appropriate acetophenones/2-acetyl thiophenes with oxone
and NH4Br in methanol for 1 h). After completion, water
was added and partitioned between water and dichloromethane. The organic
layer collected was removed in vacuum, and the residue was flash-chromatographed
over silica gel pretreated with triethyl amine using hexane/ethyl
acetate (7:3) as an eluent to give the desired imidazothiazolyl noscapinoids 7a–o in excellent yields (60–80%). All the products
were fully characterized by IR, 1H &13C
NMR, and mass (ESI and HRMS) spectral data (Supporting Information). For example, 1H NMR spectrum of 7f exhibited imidazothiazole characteristic protons as singlets
at δ 7.72 (1H), δ 6.55 (1H). The aromatic protons appeared
at δ 7.84 (2H) as a doublet, δ 6.98–6.92 (3H) as
a multiplet, δ 6.36 (1H) as a singlet, and δ 6.14 (1H)
as a doublet of the doublet. Characteristic C–C bridged protons
of noscapine appeared at δ 5.73 (1H) as a doublet of the doublet
and δ 4.60 (1H) as a doublet, methylenedioxy protons appeared
at δ 5.96 (2H) as a singlet, one methoxy group of noscapine
and one proton from N–CH2 group linking imidazothiazole
appeared at δ 4.08–4.03 (4H) as a multiplet, three other
methoxy groups of noscapine appeared at δ 4.01 (3H), δ
3.85 (3H), and δ 3.82 (3H) as singlets, and other N–CH2 proton appeared at δ 3.90 (1H) as a doublet. Aliphatic
protons of the isoquinoline ring appeared at δ 2.63–2.53
(1H), δ 2.48–2.32 (2H), and δ 2.15–2.06
(1H) as multiplets. HRMS of 7f, appeared at m/z 642.18898 for C34H31N3O8S [M + H]+, confirmed the molecular
formula and the structure.Natural noscapine 1 was O-demethylated
with NaN3 and NaI in dimethylformamide (DMF) at 135–140
°C to give (S)-7-hydroxy-6-methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]dioxolo-[4,5-g] isoquinolin-5-yl)isobenzofuran-1(3H)-one
(8, Nos-OH) in 78% yield.[33] With Nos-OH in hand, we next reacted with 4-(chloromethyl)thiazol-2-amine
in acetone using K2CO3 and KI at reflux for
4 h to yield (S)-7-((2-aminothiazol-4-yl)methoxy)-6-methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one
(9). Noscapinoid 9 was further converted
to 11a–o by reacting with substituted aryl/hetero-aryl
α-bromomethyl ketones 10a–o in 2-propanol
at reflux for 12 h. Post completion, the reaction mixture was partitioned
between water and dichloromethane, the organic layer was collected,
removed in vacuum, and flash chromatographed over silica gel with
petroleum ether/ethyl acetate (7:3) to give desired O-imidazothiazole-coupled noscapinoids 11a–o in
excellent yields (Table ). All the products 8, 9, and 11a–o were fully characterized by IR, 1H & 13C NMR, and mass (ESI and HRMS) spectral data (Supporting Information). For example, 1H NMR spectrum
of 11f exhibited imidazothiazole-characteristic protons
as singlets at δ 8.19 (1H) and δ 6.78 (1H). The aromatic
protons appeared at δ 7.81 (2H), δ 6.95 (2H), δ
6.90 (1H), and δ 6.01 (1H) as doublets and δ 6.28 (1H)
as a singlet. Characteristic C–C bridged protons of noscapine
appeared at δ 5.62 (1H) and δ 4.41 (1H) as doublets, methylenedioxy
protons appeared at δ 5.94 (2H) as a doublet of the doublet,
O–CH2 group linking imidazothiazole appeared at
δ 5.49 (2H) as a doublet of the doublet, and three methoxy groups
of noscapine appeared at δ 4.05 (3H), δ 3.85 (3H), and
δ 3.85 (3H) as singlets. The characteristic N–CH3 group of noscapine and one of the aliphatic protons of the
isoquinoline ring appeared at δ 2.54–2.47 (4H) as a multiplet
and remaining aliphatic protons of the isoquinoline ring appeared
at δ 2.34–2.25 (2H) δ 1.75–1.67 (1H) as
multiplets. HRMS of 11f at m/z 642.18924 for C34H31N3O8S [M + H]+ confirmed the molecular formula
and the structure.
Table 1
Synthesis of Noscapine Imidazothiazole
Derivatives 7a–o and 11a–o
Isolated yield.
Isolated yield.
Biology
In Vitro Antitumor Activity of the Noscapine Conjugates
All the synthesized noscapine conjugates 5, 7a–o, 9, and 11a–o was screened for
their in vitro cytotoxicity against four tumor cell lines: DU-145
(prostate), MCF-7 (breast), SK-N-SH (neuroblastoma), and MIAPaCa-2
(pancreatic) employing SRB assay. Noscapine (1), 9-bromo
noscapine (2c), NOS-NH (4), and NOS-OH (8) were used as standards for this assay. These conjugates
were evaluated in a five dose screening (0.01, 0.1, 1.0, 10, 100 μM)
protocol set by NCI-60 cell screen to examine their potency. Among
the 32 conjugates, seven compounds (5, 7a, 9, 11b, 11c, 11e, and 11o) were found to be active against the tested
cancer cell lines with different IC50 values represented
in Table .
Table 2
Inhibitory Concentrations (IC50 in μM)a of Noscapinoids 5, 7a–o, 9, and 11a–ob
compounds
DU145
MCF7
SK-N-SH
MIAPaCa-2
5
22.2 ± 1.1
32.7 ± 1.5
24 ± 2.1
2.7 ± 1.2
7a
21.2 ± 1.1
37.4 ± 2.1
33.1 ± 0.5
4.2 ± 0.6
7b
54.4 ± 0.16
55.8 ± 2.4
66.8 ± 3.3
56.8 ± 2.1
7c
53.7 ± 2.7
59.1 ± 2.5
81.6 ± 1.5
53.4 ± 1.6
7d
51.6 ± 2.4
54.5 ± 2.2
86.6 ± 0.3
42.7 ± 1.1
7e
46.9 ± 2.2
54.8 ± 3.2
83.7 ± 6.2
46.9 ± 2.9
7f
46 ± 0.2
58.3 ± 1
94.4 ± 2.8
50.1 ± 3.6
7g
56.9 ± 2.3
54.4 ± 0.5
98.4 ± 4.6
53.4 ± 5.3
7h
50.5 ± 0.74
56.5 ± 2.7
87.7 ± 0.75
54.9 ± 4.7
7i
56.6 ± 1
59.1 ± 1
98.8 ± 2.8
50 ± 2.5
7j
47.5 ± 0.3
55.1 ± 0.6
74.1 ± 0.7
48.6 ± 0.6
7k
50.5 ± 1.4
54.7 ± 0.1
98.6 ± 4.5
46.9 ± 2
7l
50.7 ± 2.9
57.2 ± 2.3
>100
45.8 ± 1.6
7m
48 ± 2.2
54.4 ± 2
98.7 ± 6.6
48.7 ± 4.7
7n
54.3 ± 2.8
57.3 ± 1.8
65.3 ± 4.5
50.3 ± 6.1
7o
55.7 ± 2.6
58.6 ± 2.9
94.9 ± 3.3
44.9 ± 0.4
9
21.3 ± 0.9
27.9 ± 0.8
21.2 ± 1.4
7.3 ± 0.7
11a
55.8 ± 2.8
57.5 ± 4.2
84.7 ± 1.7
48 ± 5.5
11b
21.8 ± 1.4
37.5 ± 1.4
33.2 ± 3.4
3.9 ± 0.6
11c
20.5 ± 1.7
32.2 ± 2.1
50.5 ± 2.2
4.2 ± 1.4
11d
50 ± 0.8
64.6 ± 2.1
94.2 ± 2
44.7 ± 1.9
11e
18.7 ± 0.5
33.8 ± 1.8
48.3 ± 2.3
6.9 ± 1.4
11f
45.9 ± 1.5
55.8 ± 1.7
69.3 ± 3
44 ± 3.9
11g
54.9 ± 1.3
54.6 ± 2.8
72.9 ± 1.2
42.6 ± 3.7
11h
51 ± 3.3
58.2 ± 1.2
70 ± 0.9
45.3 ± 4.2
11i
54.6 ± 3.1
57.4 ± 0.7
61.2 ± 4.5
43.1 ± 2.7
11j
47.2 ± 0.5
53.9 ± 0.4
63.4 ± 5.9
40.8 ± 0.7
11k
56.4 ± 1.3
53.4 ± 3.3
85 ± 6.3
46.1 ± 0.4
11l
45.6 ± 0.4
56.1 ± 1.1
82.7 ± 2.1
42.1 ± 1.9
11m
59.5 ± 2.1
53.8 ± 1.4
87.7 ± 2.2
48.7 ± 3.9
11n
47.3 ± 0.8
62.6 ± 1.3
88.3 ± 2.1
46.4 ± 1.5
11o
17.1 ± 1.1
35.4 ± 2.3
47.2 ± 3.1
3.6 ± 1.3
1
98 ± 2.1
26 ± 1.4
97 ± 3.1
98 ± 1.4
2c
1.8 ± 0.5
2.6 ± 0.6
10.2 ± 1.6
1.6 ± 0.5
4
>100
>100
>100
>100
8
23.4 ± 2.1
12.4 ± 1.6
24.4 ± 2.4
9.1 ± 1.4
IC50 = concentration
of the compound to inhibit proliferation of tumor cells by 50%.
Presented data is the mean ±
SEM from the dose–response curves of three independent experiments.
IC50 = concentration
of the compound to inhibit proliferation of tumor cells by 50%.Presented data is the mean ±
SEM from the dose–response curves of three independent experiments.To our surprise, the intermediate
molecules 5 (IC50 = 2.7 ± 1.2 μM)
and 9 (IC50 = 7.3 ± 0.7 μM) with
a thiazole amine substitution coupled
via nitrogen at 7th position and oxygen respectively were found to
be effective in the total study. It may be attributed to the presence
of heterocyclic thiazole and the free amine which can form hydrogen
bonding with the tubulin protein. Among the noscapine N-derived imidazothiazoles
(7a–o), most compounds displayed moderate to poor
activity (IC50 range—4.2 to >100 μM) on
all
the tested cell lines. The synthesized derivatives were selectively
effective on the PC cell line, MIAPaCa-2, indicating that these imidazothiazole
derivatives can be improvised further to develop potent PC-targeting
lead molecules. N-derived imidazothiazole, 7a with the
para-fluoro substitution was observed to be potent with IC50 of 4.2 ± 0.6 μM on the PC cell line, MIAPaCa-2. Interestingly,
change in the substitution from fluorine to chlorine (7b, IC50 = 56.8 ± 2.1 μM) resulted in a drastic
loss of activity against MIAPaCa-2 cancer cells. No appreciable activity
was observed with other N-derived imidazothiazoles 7a–o on the PC cell line. On the other hand, O-derived noscapine imidazothiazole
compounds (11a–o, IC50 = 3.6–88
μM) exhibited some interesting anticancer profiles. Compound 11o (IC50 = 3.6 ± 1.3 μM) displayed
the most potent anticancer activity among the O-derived imidazothiazole
noscapinoids. The presence of thiophene may be a contributing factor
for this potency while the N-derived counterpart 7o was
not as effective. An important observation from the data of compounds 11o (IC50 = 3.6 ± 1.3 μM) and 11m (IC50 = 48.7 ± 3.9 μM) is that even
though the presence of halogen played an important role in the anticancer
activity, the presence of an electron-donating methyl group has played
a pivotal role in deciding the level of potency as seen in 11o. Derivative 11b (IC50 = 3.9 ± 0.6 μM)
also displayed good potency against PC cell lines, indicating the
importance of halogens in imparting good anticancer activity to the
molecule. A surprising finding from the study is the display of a
good anticancer profile by compound 11c (IC50 = 4.2 ± 1.4 μM) which has a para-bromo
substitution again emphasizing the importance of halogens in imparting
cytotoxicity to the molecule. The presence of bromine in compounds 11o and 11c establishes the fact that imidazothiazole
unit-linked via O-noscapine can yield better anticancer
profiles than their counterparts N-noscapinoids 7c and 7o. Compound 11e (IC50 = 6.9 ± 1.4 μM) was also found to show good cell
proliferation inhibition as compared to other molecules within the
same series. The presence of the methoxy group may be said to be favorable
for anticancer activity while it may not be much effective for N-derived
imidazothiazole noscapinoid 7e.An overall assessment
can be made from the in vitro evaluation
of anticancer activity that imidazothiazoles coupled to noscapine
via O-linkage are much more effective than N-linkage. Four of the
15 O-linked imidazothiazole noscapinoids (11b, 11c, 11e, and 11o) proved to be
effective against the PC cell line while only one (7a) among the N-coupled imidazothiazole noscapine derivatives was observed
to have anticancer activity. Also, intermediate compound 5 is a promising candidate for further lead development of noscapine
derivatives as anticancer molecules.It is known that noscapine
and its congeners do not reduce crucial
functions of the microtubule, thereby leading to less toxicity.[34] Previous reports also suggest that noscapine
decreased cell viability selectively in malignant cells in a time-
and dose-dependent manner but not in noncancerous cells, indicating
that noscapine possesses selective antitumor activity against cancer
cells.[35] Corroborating with previous studies,
our observations revealed that these noscapine analogues 5, 7a, 9, 11b, 11c, 11e, and 11o exhibited low activity in
normal Chinese hamster ovary cells (CHO) even at 100 μM concentration,
suggesting that these noscapine analogues can serve as better chemotherapeutic
agents from the pharmacological point of view with low side effects.
Morphological Observations upon Treatment with Noscapine Conjugates
The test compounds 5, 7a, 9, 11b, 11c, 11e, and 11o and the standard reference compounds 1, 2c, 4, and 8 were administered to
MIAPaCa-2 cells at a concentration of 10 μM for 24 h to observe
the morphological changes using an inverted microscope. The control
cells appeared flattened with a polygonal morphology while the positive
controls 1, 2c, 4, and 8 exhibited extensive clustering and aggregation of cells,
inducing the retraction of cellular protrusions and cell rounding
similar to mitotic arrest, a characteristic feature of G2/M arrest. Cells treated with compounds 2c, 5b, 7a, 8, 9, 11c, 11e, and 11o became more rounded at 24h
time point when compared to cells treated with 1, 4, and 11b where the rounding effect of the cells
was not that prominent. This indicates that noscapine analogues 2c, 8, 5, 7a, 9, 11c, 11e, and 11o are possibly more potent in inducing G2/M arrest when
compared to the standard compounds (Figure ), which was further confirmed by FACS analysis.
Figure 4
Effect
of noscapine analogues 5, 7a, 9, 11b, 11c, 11e, 11o, and standard reference compounds 1, 4, 8, and 2c on the morphology of
MIAPaCa-2 cells. MIA PaCa-2 cells were treated either in the absence
(control) or presence of 10 μM noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o for 24 h. Morphological
changes were assessed by an Olympus CKX41 inverted microscope. Data
are representative of three independent experiments. Scale bar = 50
μm.
Effect
of noscapine analogues 5, 7a, 9, 11b, 11c, 11e, 11o, and standard reference compounds 1, 4, 8, and 2c on the morphology of
MIAPaCa-2 cells. MIA PaCa-2 cells were treated either in the absence
(control) or presence of 10 μM noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o for 24 h. Morphological
changes were assessed by an Olympus CKX41 inverted microscope. Data
are representative of three independent experiments. Scale bar = 50
μm.
Effect of Noscapine Conjugates
on Clonogenic Cell Survival Assay
The clonogenic or the colony-forming
assay tests the ability of
the cells to reproduce and multiply in the presence of drugs or test
compounds upon treatment. The effect of the selected noscapinoids
(5, 7a, 9, 11b, 11c, 11e, and 11o) were
determined for their inhibition of colony formation in MIAPaCa-2 cell
line by treating the cells with 10 μM of the test compounds
for 24 h after which the media was replenished with fresh media, and
the cells were further incubated for 10 days.Among the reference
compounds 1, 2c, 4, and 8, compounds 2c and 8 demonstrated
a decrease in colony formation, while not much difference was detected
with 1 and 4 in comparison with the control.
Compound 11o was found to inhibit the colony formation
in pancreatic cells effectively which can be attributed to the presence
of the heterocycle in the molecule. Compounds 11e and 11c also displayed almost equal colony inhibition which again
emphasizes the presence of halogens in the molecule for effective
anticancer activity. The number of colonies observed after the treatment
with noscapine analogues 11b, 11c, 11e, and 11o decreased significantly compared
to control (Figure B), indicating that O-derived imidazothiazole noscapinoids are better
than N-derived imidazothiazole noscapinoids. It was observed that
the survival fraction decreased for compounds 5, 7a, 9, 11b, 11c, 11e, and 11o-treated MIAPaca-2 cells while not
much difference was detected with 4 and 1 which is similar to the observed decrease in the number of colonies
(Figure C).
Figure 5
Effect of noscapine
conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o on clonogenicity. (A) MIAPaca-2 cells were treated in
the presence or absence of 10 μM noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o. Photographs of
colonies were taken with Gel Doc XR System. (B) Number of colonies
was counted with the help of clonocounter software. (C) Number of
colonies was determined, and the surviving fraction was calculated
by dividing the number of colonies formed after the treatment by the
number of cells seeded × PE (Plating efficiency), where PE= (number
of colonies formed/number of cells seeded) × 100. Results represent
mean values ± S.D. (***P < 0.001 analyzed
by Tukey post hoc analysis after one-way ANOVA).
Effect of noscapine
conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o on clonogenicity. (A) MIAPaca-2 cells were treated in
the presence or absence of 10 μM noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o. Photographs of
colonies were taken with Gel Doc XR System. (B) Number of colonies
was counted with the help of clonocounter software. (C) Number of
colonies was determined, and the surviving fraction was calculated
by dividing the number of colonies formed after the treatment by the
number of cells seeded × PE (Plating efficiency), where PE= (number
of colonies formed/number of cells seeded) × 100. Results represent
mean values ± S.D. (***P < 0.001 analyzed
by Tukey post hoc analysis after one-way ANOVA).
Effect of Noscapine Conjugates on Tubulin Polymerization
The dynamic equilibrium between polymerization and depolymerization
of tubulin into dimers and free tubulin, respectively, is targeted
by many tubulin polymerization inhibitors in order to disrupt mitosis
and cell proliferation.[36] Keeping in view
this key aspect, we evaluated tubulin levels in MIAPaCa-2 cells following
the treatment with 10 μM of 5, 7a, 9, 11b, 11c, 11e, and 11o for 24 h. In addition, cells were treated with 1, 2c, 4, and 8 as positive
controls and dimethyl sulfoxide (DMSO) as a negative control. Western
blot analysis revealed that cells treated with 2c and 8 showed a remarkable shift in tubulin protein levels, wherein
the protein was found to be more in the soluble fraction, indicating
that these compounds are tubulin depolymerizing agents. Earlier studies
established that 1 possesses very low antimitotic activity
with no observable detection of a change in the soluble and insoluble
tubulin fraction.[9] Our study, similar to
previous reports, revealed that 1 and 4 did
not show any detectable change in the soluble and insoluble tubulin
fractions. Similar to 2c and 8, it was found
that the cells treated with compounds 5, 7a, 9, 11b, 11c, 11e, and 11o showed accumulation of tubulin in the soluble
fraction and the tubulin protein amount in insoluble fractions was
more or less the same as in control/DMSO-treated cells. Therefore,
these results suggest that 5, 7a, 9, 11b, 11c, 11e, and 11o are likely to act as microtubule-destabilizing agents
(Figure ). Hence,
increased tubulin in the soluble fraction of cells treated with these
conjugates corroborates with the inhibition of the tubulin assembly
and arrested cells in the G2/M phase.
Figure 6
Effect of noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o on soluble and
insoluble tubulin. (A) Tubulin distribution
in insoluble vs soluble portions analyzed by immunoblotting in treated
MIAPaca-2 cells. The cells were treated with 10 μM of noscapine
conjugates and 1, 2c, 4, and 8 for 24 h. The fractions containing soluble and insoluble
tubulin were collected and separated by SDS-PAGE. Tubulin was detected
by Western blot analysis using β-tubulin antibody. (B,C) Relative
levels of insoluble tubulin to soluble tubulin were determined densitometrically
with the help of ImageJ software. Results represent mean values ±
S.D. (***P < 0.001 analyzed by Tukey post hoc
analysis after one-way ANOVA).
Effect of noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o on soluble and
insoluble tubulin. (A) Tubulin distribution
in insoluble vs soluble portions analyzed by immunoblotting in treated
MIAPaca-2 cells. The cells were treated with 10 μM of noscapine
conjugates and 1, 2c, 4, and 8 for 24 h. The fractions containing soluble and insoluble
tubulin were collected and separated by SDS-PAGE. Tubulin was detected
by Western blot analysis using β-tubulin antibody. (B,C) Relative
levels of insoluble tubulin to soluble tubulin were determined densitometrically
with the help of ImageJ software. Results represent mean values ±
S.D. (***P < 0.001 analyzed by Tukey post hoc
analysis after one-way ANOVA).
Effect of Noscapine Conjugates on Cell-Cycle Progression
The effect of compounds 5, 7a, 9, 11b, 11c, 11e, and 11o on cell cycle progression in MIAPaCa-2 cells (Figure ) was determined
by flow cytometry. The tested conjugates exhibited significant G2/M arrest compared to the reference compounds 1, 2c, 4, and 8. Reference
compound 8 was not particularly effective in displaying
the cell cycle arrest when compared to other standard references.
Noscapinoid 5 with thiazole-amine functionality proved
again to be the most potent derivative in arresting the cell cycle
at the G2/M phase with 76.94% of the total cell population
trapped in G2/M phase. N-derived imidazothiazole noscapinoid 7a displayed 56.33% arrest of the cell cycle, which can be
attributed to the presence of fluorine in the compound. Among the
O-derived imidazothiazole noscapinoids, 11e exhibited cell cycle G2/M-phase arrest with ∼43% cells which
may be due to the presence of an electron-donating meta-methoxy substitution. The O-coupled imidazothiazole counterpart 9 also caused 42.6% arrest of cells in the G2/M
phase but not as potent as its N-derived imidazothiazole 5. Compounds 11b, 11c, and 11o were found to induce cell cycle G2/M-phase arrest moderately with 36.23, 33.05, and 39.07% of cells compared to the
DMSO-treated cells (control, 25.68%). The study provides an insight
into the synthesized compound intermediate 5 free thiazoleamine which is much more effective in inducing G2/M arrest
than the cyclized imidazothiazole compounds.
Figure 7
Noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o cause G2/M arrest in MIA Paca-2 cells. Cells
were treated for 24 h in the absence (control) and presence of noscapine
conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o (10 μM) and 1, 2c, 4, and 8 (10 μM) as positive controls. Cell cycle
distribution was analyzed by flow cytometry after staining with PI.
Cell cycle distribution is expressed in the form of histograms, as
the percentage of cells in each cell cycle phase of the MIA Paca-2
cells.
Noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o cause G2/M arrest in MIA Paca-2 cells. Cells
were treated for 24 h in the absence (control) and presence of noscapine
conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o (10 μM) and 1, 2c, 4, and 8 (10 μM) as positive controls. Cell cycle
distribution was analyzed by flow cytometry after staining with PI.
Cell cycle distribution is expressed in the form of histograms, as
the percentage of cells in each cell cycle phase of the MIA Paca-2
cells.
Effect of Noscapine Conjugates
on Cell Cycle Proteins
The progression of the eukaryotic
cell cycle is regulated by the
activation of cyclin-dependent kinases (CDKs) sequentially, which
depends upon their association with regulatory cyclins. A complex
between CDK 1 and cyclin B1 is important for entry into mitosis in
most organisms.[37−39] Cyclin B1, often deregulated in tumors, is elevated
before the cells enter the M phase prematurely causing the loss of
cell division control, thereby leading to apoptosis. So, we also investigated
the expression level of cyclin B1 and its partner CDK1. The MiaPaCa-2
cells were exposed to 5, 7a, 9, 11b, 11c, 11e, and 11o for 24 h and were then evaluated by western blotting.
As shown in Figure , there is a marked increase in cyclin B1 and CDK1 protein levels
for all the compounds as compared with the control sample. Surprisingly, 11o, the most potent imidazothiazole noscapinoid did not show
an effective increase in either CDK1 or cyclin B1 when compared to
control along with the standard references 2c and 4. While intermediates 5 and 9 displayed
a marked increase in the kinase levels, they showed a moderate increase
in cyclin B1 levels, nevertheless emphasizing an increase in the cell
cycle arrest in the G2/M phase. Only compound 11e (3-methoxyphenyl) exhibited a potent increase in both cyclin B1
and CDK1 levels among the tested imidazothiazole compounds. It is
interesting to observe that natural noscapine 1 and 7-demethylated
noscapine 8 have proven to remarkably increase the levels
of CDK1 when compared to control but failed to show their effect on
cyclin B1 with no comparable increment in the protein levels. This
result can be corroborated from FACS where 1 did not
show any morphological changes. It can be hypothesized that the presence
of the electron-donating methoxy group is much more favorable than
electron-withdrawing halogens as evident from the results. The above
results show that noscapine analogues 5, 7a, 9, 11b, 11c, 11e, and 11o induce cell cycle G2/M-phase arrest involving cell-cycle regulators cyclin B1 and CDK1 (Figure ).
Figure 8
Effect of noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o on G2/M arrest and apoptosis in MIA PaCa-2
cells. (A) Cells were treated with 10 μM of noscapine conjugates
for 24 h. The lysates containing total protein were collected and
separated by SDS-PAGE. Cyclin B1, CDK1, Cleaved Caspase 3, PARP, and
β-actin expression were detected by Western blot analysis using
specific antibodies. β-Actin is used as a loading control. (B)
Relative levels of cyclin B1. (C) Cleaved PARP. (D) CDK1 was measured
densitometrically using ImageJ software. Results represent mean values ±
S.D. (***P < 0.001 analyzed by Tukey post hoc
analysis after one-way ANOVA).
Effect of noscapine conjugates 5, 7a, 9, 11b, 11c, 11e, and 11o on G2/M arrest and apoptosis in MIA PaCa-2
cells. (A) Cells were treated with 10 μM of noscapine conjugates
for 24 h. The lysates containing total protein were collected and
separated by SDS-PAGE. Cyclin B1, CDK1, Cleaved Caspase 3, PARP, and
β-actin expression were detected by Western blot analysis using
specific antibodies. β-Actin is used as a loading control. (B)
Relative levels of cyclin B1. (C) Cleaved PARP. (D) CDK1 was measured
densitometrically using ImageJ software. Results represent mean values ±
S.D. (***P < 0.001 analyzed by Tukey post hoc
analysis after one-way ANOVA).
Effect of Noscapine Conjugates on Apoptosis
The caspase
activation plays a pivotal role in the process of apoptosis or programmed
cell death.[40] Caspases synthesized as inactive
proenzymes are activated by specific proteolytic cleavage reactions.
Caspases-2, -8, -9, and -10 are usually activated first in the process
of programmed cell death and thereby termed as initiator caspases,
which in turn activate effector caspases, especially, caspase-3.[41] As shown in Figure , all the tested compounds 5, 7a, 9, 11b, 11c, 11e, and 11o induced proteolytic cleavage
of caspase-3 except 4 and 1. We observed
that the DNA repair enzyme PARP cleavage was detectable after 24 h
from 116 kDa to an inactive 89 kDa form on treatment with compounds 5, 7a, 9, 11b, 11c, 11e, and 11o except 4 and 1. These obseravtaions are in agreement with the
cleavage of caspase-3. Altogether, these results show that noscapine
conjugates induce caspase-dependent apoptosis in MIAPaCa-2 cells.
Molecular Modelling
Molecular docking was performed
on tubulin protein (PDB ID: 1SA0) to better understand
the binding modes of the compounds in silico. It was observed from
the results that the noscapine conjugates showed good binding energy
to the target protein compared to noscapine. The docking results were
drawn based on the docking score, hydrogen bonding, and van der Waals
interactions of the ligand with the enzyme (Table ). Based on the docking results (Figure ), all the test compounds 5, 9, 11b, 11c, 11e, and 11o barring 7a showed hydrogen
bonding with the binding pocket residues while no bonds were visible
in case of compound 7a. The anticancer property of the
active compounds could be due to the inhibition of tubulin protein.
Compound 5 (N-derived thiazole amine noscapinoid) displayed
hydrogen bonding with LYS254 via 6-methoxy substitution on the phthalide
ring while the 7-methoxy group did not display any hydrogen bonds
with the protein. O-derived thiazole amine noscapinoid (9) exhibited three hydrogen bonds: GLN11 bonding with 1′-O
on the methylenedioxy group, THR179 bonding with nitrogen on the isoquinoline
ring, and ALA317 showing the third hydrogen bond with hydrogen on
the free amine of the thiazole ring. Noscapinoid 11b with
a 4-chlorophenyl substitution bonded with a tubulin protein via two
hydrogen bonds, one at LYS254 bonding to nitrogen on the imidazothiazole
ring while the second hydrogen bond was observed at THR179 with nitrogen
on the isoquinoline moiety. Compound 11c (4-bromophenyl)
was found to show only one hydrogen bond, unlike its chlorine counterpart.
The hydrogen bond was observed between LYS254 and 6-methoxy group
on the phthalide ring of noscapine. It is surprising to observe the
difference in binding as one would expect a similar binding of imidazothiazolenitrogen to bond to LYS254 in both cases (compound 11b and 11c) which evidently is not the case. 6-Methoxy
group of the phthalide ring was again noticed to bond with SER178
and THR353 in case of compound 11e. It is interesting
to note that the methoxy groups present elsewhere on the molecule
did not participate in the bonding with the tubulin protein. Compound 11o (4-methyl-5-bromo thiophene) followed the same suit as
other compounds wherein the 6-methoxy substitution on the phthalide
ring once again displayed hydrogen bonding with ASN101 of tubulin
protein. From the docking results, it can be summarized that the presence
of an electron-donating 6-methoxy group on the phthalide ring is essential
for the anticancer property of the molecule irrespective of the substitution
present on either fragment of the molecule.
Table 3
Docking Scores of Noscapine and Its
Bioactive Analogues
s. no.
compound
XP G score (kcal/mol)
1
noscapine
–5.627
2
5
–6.277
3
7a
–6.656
4
9
–7.836
5
11b
–5.902
6
11c
–5.010
7
11e
–6.212
8
11o
–5.448
Figure 9
Binding modes of compounds 5 (a), 7a (b), 9 (c), 11b (d), 11c (e), 11e (f), 11o (g), and noscapine (h).
Binding modes of compounds 5 (a), 7a (b), 9 (c), 11b (d), 11c (e), 11e (f), 11o (g), and noscapine (h).
Conclusions
A
series of noscapine–imidazothiazole conjugates 7a–o and 11a–o linked at 5′-N and 7-O positions,
respectively, were synthesized and evaluated as anticancer agents.
Among the synthesized set of noscapinoids, it was interesting to note
that initial intermediate compounds 5 and 9 were found to be active. Among which compound 5, 5′-N
derived imidazothiazole was found to be the most potent among the
series. Compounds 7a, 9, 11b, 11c, 11e, and 11o were found
to be potent in the cytotoxicity studies, and the same was corroborated
with the molecular modeling studies where the binding energies were
found to be in accordance with the activities observed. Cell cycle
analysis revealed that these molecules were active in the G2/M phase of the cell cycle via induction of apoptosis, inhibiting
caspase-3 and increasing the levels of cyclin-B1 and CDK-1.
Experimental
Section
General Information
Reagents and
all solvents used
were analytically pure. Air-sensitive reagents were transferred by
a syringe or double-ended needle. Evaporation of solvents was performed
at reduced pressure by using a heidolph rotary evaporator. TLC (precoated
silica plates and visualizing under UV light) is used to monitor progress
of the reactions. 1H and 13C NMR spectra of
samples in CDCl3 were recorded on an AVANCE-300, 400, 500
MHz spectrometer. Chemical shifts presented are relative to an internal
standard TMS (δ = 0.0). Spin multiplicities are described as
s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet),
or m (multiplet). Coupling constants are reported in hertz (Hz). Mass
spectra were recorded in ESI conditions at 70 eV on an LC-MSD (Agilent
technologies) spectrometer. All high-resolution spectra were recorded
on the QSTAR XL hybrid MS/MS system (Applied Bio systems/MDS sciex,
Foster
city, USA), equipped with an ESI source (CSIR-IICT, Hyderabad). Column
chromatography was performed on silica gel (60–120 mesh) supplied
by Acme Chemical Co., India. TLC was performed on Merck 60 F-254 silica
gel plates. Commercially available anhydrous solvents dichloromethane,
methanol, acetone, and ethyl acetate were used as such. Natural α-noscapine
was procured from Sigma-Aldrich.
Natural α-noscapine 1 (1.0 g, 2.42
mmol) was demethylated by following the procedure developed in our
lab[31] to give 4 (0.46 g, 48%)
as a white solid. Mp 171–172 °C (lit.[31] mp 170 °C). The NMR and mass spectral data of 4 is fully in agreement with the reported data.[31]
To a solution of nornoscapine 4 (1.0 g, 2.50 mmol) in acetone (10 mL) were added anhydrous
potassium carbonate (0.69 g, 5.0 mmol), potassium iodide (0.83 g,
5.0 mmol), and 4-(chloromethyl) thiazol-2-amine (0.37 g, 3.75 mmol)
and stirred at room temperature. After 4 h, the reaction mixture was
filtered, and the filtrate was evaporated in vacuum with the aid of
a rotary evaporator. Water (5 mL) was then added and extracted with
dichloromethane (2 × 10 mL). The combined organic fractions were
dried with anhydrous Na2SO4 and then concentrated.
The residue was purified by column chromatography on a triethyl amine-treated
silica gel column with hexane/ethyl acetate (7:3) as an eluent to
give 5 as a white solid product. Yield: 70% (0.90 g);
mp 224–226 °C; 1H NMR (500 MHz, CDCl3): δ 6.99 (d, J = 8.24 Hz, 1H), 6.34 (s, 1H),
6.32 (s, 1H), 6.25 (d, J = 8.24 Hz, 1H), 5.92 (s,
2H), 5.62 (d, J = 4.12 Hz, 1H), 5.07 (br s, 2H),
4.66 (d, J = 4.12 Hz, 1H), 4.08 (s, 3H), 3.96 (s,
3H), 3.86 (s, 3H), 3.83 (d, J = 14.49 Hz, 1H), 3.72
(d, J = 14.49 Hz, 1H), 2.71–2.63 (m, 1H),
2.57–2.46 (m, 2H), 2.10–2.02 (m, 1H); 13C
NMR (100 MHz, CDCl3): δ 168.1, 167.2, 152.1, 150.2,
148.4, 147.7, 141.4, 140.5, 133.9, 131.6, 119.7, 118.3, 117.8, 116.8,
105.4, 102.4, 100.6, 81.7, 62.3, 59.3, 58.8, 56.8, 56.7, 46.0, 26.6;
IR (KBr): 3442, 3287, 3182, 2940, 1753, 1616, 1523, 1498, 1269, 1115,
1041, 971, 890, 726, 655 cm–1; MS (ESI) m/z: 512 [M + H]+; HRMS (ESI):
calcd for C25H25N3O7S
[M + H]+, 512.14860; found, 512.14642.
General Procedure
for the Synthesis of N-Imidazothiazole
Noscapine Derivatives (7a–o)
To the solution
of (S)-3-((R)-6-((2-aminothiazol-4-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one 5 (0.25 g, 0.46 mmol) in propan-2-ol
(5 mL) was added substituted α-bromo acetophenones/2-(2-Bromo
acetyl)thiophenes (10a–o) (0.56 mmol) and stirred
at reflux for 12 h. The solvent was evaporated under vacuum, and the
solid residue was treated with water (5 mL) and extracted with dichloromethane
(2 × 10 mL). The combined organic layer was separated, washed
with water, dried over anhydrous Na2SO4, and
evaporated in vacuum with the aid of a rotary evaporator. The residue
thus obtained was chromatographed over a triethyl amine-treated silica
gel column eluted with hexane/ethyl acetate (7:3) to yield 7a–o as solid products.
Following the reported procedure,[33] compound 8 was prepared by heating
noscapine 1 (2.0 g, 4.84 mmol), sodium azide (0.63 g,
9.68 mmol), and
sodium iodide (0.36 g, 2.42 mmol) in anhydrous DMF (5.0 mL) at 140
°C for 4 h. The mixture was concentrated under reduced pressure,
residue thus obtained was dissolved in EtOAc (50 mL), solid particles
was filtered through Celite, and the filtrate was diluted with EtOAc
(50 mL) followed by washing with water (2 × 25 mL) and brine
(2 × 25 mL). The combined organic layer was separated, dried
over anhydrous Na2SO4, and evaporated to give
a crude product which was crystallized from methanol. The product 8 was isolated as an off-white solid (78% yield). Mp 142–143
°C (lit.[33] mp 142–144 °C).
The NMR and mass spectral data of 4 is fully in agreement
with the reported data.[31]
A mixture of 8 (1.0 g, 2.50
mmol), potassium carbonate (0.69 g, 5.0 mmol), potassium iodide (0.83
g, 5.0 mmol), and 4-(chloromethyl)thiazol-2-amine (0.37 g, 3.75 mmol)
in acetone (10 mL) was stirred at reflux for 4 h. The reaction mixture
was filtered, the filtrate was evaporated under vacuum, and water
(5 mL) and dichloromethane (2 × 10 mL) was added. The separated
organic layer was washed with water, dried with anhydrous Na2SO4, and concentrated. The residue was further chromatographed
over a triethyl amine-treated silica gel column eluted with hexane/ethyl
acetate (6:4) to yield 9 as a yellow solid product. Yield:
62% (0.80 g); mp 74–76 °C; 1H NMR (400 MHz,
CDCl3): δ 6.94 (d, J = 8.31 Hz,
1H), 6.80 (s, 1H), 6.30 (s, 1H), 6.04 (d, J = 8.31
Hz, 1H), 5.93 (dd, J = 1.34, 3.42 Hz, 2H), 5.58 (d, J = 4.15 Hz, 1H), 5.28–5.15 (m, 4H), 4.40 (d, J = 4.15 Hz, 1H), 4.03 (s, 3H), 3.83 (s, 3H), 2.61–2.53
(m, 4H), 2.39–2.29 (m, 2H), 1.92–1.83 (m, 1H); 13C NMR (100 MHz, CDCl3): δ 168.2, 152.2,
148.2, 147.9, 145.8, 140.8, 140.2, 133.8, 132.0, 120.8, 118.2, 117.8,
117.7, 116.8, 106.6, 102.2, 100.6, 81.7, 71.4, 60.6, 59.3, 56.7, 49.4,
46.2, 27.9; IR (KBr): 3422, 2929, 1752, 1620, 1492, 1380, 1209, 1035,
934, 712 cm–1; MS (ESI) m/z: 512 [M + H]+; HRMS (ESI): calcd for C25H25N3O7S [M + H]+, 512.14860; found, 512.14799.
General Procedure for the
Synthesis of O-Imidazothiazole
Noscapine Derivatives (11a–o)
To the
solution of (S)-7-((2-aminothiazol-4-yl)methoxy)-6-methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one 9 (0.25 g, 0.46 mmol) in propan-2-ol (5 mL) was added substituted
α-bromo acetophenones/2-(2-bromo acetyl)thiophenes (10a–o) (0.56 mmol) and stirred at reflux for 12 h. The residue obtained
after solvent evaporation was treated with water (5 mL) and dichloromethane
(2 × 10 mL). The organic layer was separated, washed with water,
dried with anhydrous Na2SO4, and concentrated
under vacuum. The residue thus obtained was chromatographed over a
triethyl amine-treated silica gel column eluted with hexane/ethyl
acetate (7:3) to yield 11a–o as solid products.
The cell lines, MIAPaCa-2, DU145, HeLa,
and SK-N-SH purchased from ATTC (Rockville, MD, USA), were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma
St. Louis, Mo), supplemented with 10% FBS (Invitrogen, Karlsruhe,
Germany) and Penstrep (Invitrogen). The cell lines were then incubated
at 37 °C in a humidified 5% CO2 incubator.
Cell
Proliferation Assay
Cells were seeded in 96-well
microplates at 1 × 104 cells per well and kept for
overnight incubation. After treating the cells with the compounds
for the required time period of 24 or 48 h, the cells were fixed in
10% trichloroacetic acid for 1 h at 4 °C and washed four
times in distilled water. The cells were then stained with 0.05% SRB
at 100 μL per well for 30 min at room temperature. After the
incubation, the cells were washed four times in 0.1% acetic acid.
The stained cells were lysed in 10 mM Tris-buffer, and the optical
density was measured at 510 nm.
Cell Cycle Analysis
Human PC cells (MIA PaCa-2) in
60 mm plates were incubated in the presence of the noscapine compounds
for 24 h. Cells were collected with trypsin ethylenediaminetetraacetic
acid and fixed with 70% ice-cold ethanol. The cells were stained with
propidium iodide solution (1× propidium iodide (PI), 5 μL/mL
of RNase A, and 50 μL/mL of Triton X-100) for 30 min. The DNA
content of 10 000 events was measured by flow cytometry (Beckman
coulter CytoFLEX).
Measurement of Insoluble and Soluble Tubulin
1 ×
105 MiaPaCa-2 cells were seeded in 12-well plates and were
treated with different concentrations of noscapine conjugates for
24 h. Soluble and insoluble tubulin fractions were collected subsequently.
The soluble tubulin fractions were collected by using 200 μLpre-warmed
lysis buffer [1 mM MgCl2, 1 mM EGTA, 80 mM Pipes-KOH (pH
6.8), 10% glycerol, 0.2% Triton X-100, and 0.1% protease inhibitor
cocktail (Sigma-Aldrich)]. Lysis buffer was removed gently and mixed
with 100 μL of 5× Laemmli’s sample buffer (0.01%
bromophenol blue, 180 mM Tris-Cl pH 6.8, 7.5% β-mercaptoethanol,
15% glycerol and 6% SDS). Samples were heated to 95 °C for 3
min. To collect the insoluble tubulin fraction in the remaining cells,
200 μL of 1× Laemmli’s sample buffer was added in
each well and collected and the samples were heated to 95 °C
for 3 min. Equal volumes of the samples were resolved using SDS-polyacrylamide
gel (10%) and transferred to the poly(vinylidene difluoride) (PVDF)
membrane. Blots were then incubated with primary antibodies against
α-tubulin (Sigma) overnight at 4 °C, and membranes were
next incubated with peroxidase-labeled secondary antibodies (Santa
Cruz Biotechnology, Texas, United States) for 1 h. Membranes were
visualized using an enhanced G-BOX (Syngene, USA).
Clonogenic
Assay
For the clonogenic assay, 1 ×
103 cells were seeded in 6-well plates and incubated for
24 h in the cells were treated with 10 μM noscapine analogues 5, 7a, 9, 11b, 11c, 11e, and 11o along with the
reference compounds 1, 2c, 4, and 8 for 24 h. Later, the media was replaced with
that fresh media, and the cells were grown for an additional 10 days.
Colonies were washed in PBS, fixed with 70% ethanol (30 min, RT),
and stained with EtBr solution (10 μg/mL).
Western Blot
Analysis
MiaPaCa-2 cells were incubated
in the presence of noscapine analogues, and the total cell lysates
were obtained by using the Laemmli sample buffer. Equal volumes of
the protein lysate were resolved using SDS-polyacrylamide gel (10%)
and transferred to the PVDF membrane. The membrane was blocked for
1 h at room temperature in TBS with 0.1% Tween20 (TBST) containing
5% (w/v) nonfat dry milk (Santa Cruz Biotechnology). After 5 min of
TBST wash, the membrane was incubated with primary antibodies against
Caspase-3 (C8487), CDK1 (SAB4500050), cyclin-B1 (SAB4503501), PARP
(#9542) β-actin (Sigma) at 4 °C overnight. The blots were
then incubated with peroxidase-labeled secondary antibodies (Santa
Cruz Biotechnology) for 1 h at room temperature. The membranes were
washed with TBST and then visualized using the G-BOX (Syngene, USA).
The protein expression was normalized relative to the control gene
and actin expression.
In Silico Molecular Docking
In silico
analysis were
performed in a Dell Precision T7610 workstation (8 processors; 8 GB
RAM; ZOTAC 3GB graphics; Maestro 9.8, Schrodinger, New York, U.S.A)
running on Redhat 6.1 Linux environment.The structure of the
ligand was drawn in Chemdraw Ultra 6.0. The 3D coordinate file of
the target protein was retrieved from the protein data bank (PDB).
Molecular docking studies were performed against tubulin PDB ID: 1SA0. The protein was
prepared with the help of Protein Preparation Wizard of Schrödinger
Suite 9.8. The prepared protein was optimized and minimized using
algorithm OPLS_2005 (optimized potential for liquid simulations) force
field, and the grid was generated using the Glide Grid Generation
panel in Glide. The known inhibitor noscapine and test compounds were
energy minimized using the LigPrep module. The minimized test compounds
were docked using Glide XP docking calculations. The XP Glide scoring
function was used to get the best-ranked compounds, and the specific
interactions like H-bonds and van der Waals were analyzed using a
XP visualizer in the Glide module.[42,43]
Authors: Richard A Friesner; Robert B Murphy; Matthew P Repasky; Leah L Frye; Jeremy R Greenwood; Thomas A Halgren; Paul C Sanschagrin; Daniel T Mainz Journal: J Med Chem Date: 2006-10-19 Impact factor: 7.446
Authors: Aaron J Debono; Sarah J Mistry; Jinhan Xie; Divya Muthiah; Jackson Phillips; Sabatino Ventura; Richard Callaghan; Colin W Pouton; Ben Capuano; Peter J Scammells Journal: ChemMedChem Date: 2013-12-11 Impact factor: 3.466
Authors: Aaron J DeBono; Jin Han Xie; Sabatino Ventura; Colin W Pouton; Ben Capuano; Peter J Scammells Journal: ChemMedChem Date: 2012-10-10 Impact factor: 3.466
Authors: Dong Qian; Zipeng Lu; Qingcheng Xu; Pengfei Wu; Lei Tian; Liangtao Zhao; Baobao Cai; Jie Yin; Yang Wu; Kevin F Staveley-O'Carroll; Kuirong Jiang; Yi Miao; Guangfu Li Journal: Cancer Lett Date: 2017-03-21 Impact factor: 8.679
Authors: Jacques Ferlay; Isabelle Soerjomataram; Rajesh Dikshit; Sultan Eser; Colin Mathers; Marise Rebelo; Donald Maxwell Parkin; David Forman; Freddie Bray Journal: Int J Cancer Date: 2014-10-09 Impact factor: 7.396
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