Imidazo[2,1-b]thiazole-Coupled Natural Noscapine Derivatives as Anticancer Agents

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.

Introduction

Of the various cancers existing globally, pancreatic cancer (PC) remains very lethal and accounts for about 8% of the mortality rate in cancer patients.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 1). 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.

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 2)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.

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 3.

Figure 3.

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 1.

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%.

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 1). 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.

^aIsolated 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 2.

Table 2. Inhibitory Concentrations (IC₅₀ in μM)^a of Noscapinoids 5, 7a–o, 9, and 11a–o^b.

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

^aIC₅₀ = concentration of the compound to inhibit proliferation of tumor cells by 50%.

^bPresented 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 4), 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 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 5B), 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 5C).

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 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 6). 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 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 7) 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 thiazole amine 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.

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 8, 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 8).

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 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 8, 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 3). Based on the docking results (Figure 9), 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 imidazothiazole nitrogen 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).

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

Chemistry

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.

(S)-6,7-Dimethoxy-3-((R)-4-methoxy-5,6,7,8tetrahydro[1,3]dioxolo[4,5-g]isoquinolin-5yl)isobenzofuran-1(3H)-one (4)

Natural α-noscapine 1 (1.0 g, 2.42 mmol) was demethylated by following the procedure developed in our lab³¹ 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

(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)

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.

(S)-3-((R)-6-((6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7a)

Yellow solid; yield: 64%; mp 112–114 °C; ¹H NMR (CDCl₃, 500 MHz): δ 7.93–7.88 (m, 2H), 7.79 (s, 1H), 7.14–7.08 (m, 2H), 6.94 (d, J = 8.24 Hz, 1H), 6.57 (s, 1H), 6.36 (s, 1H), 6.11 (d, J = 8.24 Hz, 1H), 5.97 (d, J = 1.24 Hz, 2H), 5.76 (d, J = 4.42 Hz, 1H), 4.62 (d, J = 4.42 Hz, 1H), 4.10–4.06 (m, 4H), 4.00 (s, 3H), 3.90 (d, J = 14.34 Hz, 1H), 3.82 (s, 3H), 2.61–2.52 (m, 1H), 2.41–2.34 (m, 1H), 2.29–2.22 (m, 1H), 2.11–2.02 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.0, 163.1 (d, J_C–F = 245.22 Hz), 152.4, 149.7, 148.8, 148.1, 146.5, 140.5, 134.0, 130.9 (d, J_C–F = 9.99 Hz), 130.5, 130.4 (d, J_C–F = 2.72 Hz), 129.1, 127.0 (d, J_C–F = 7.26 Hz), 119.3, 118.4, 117.5, 115.4, 115.4 (d, J_C–F = 21.79 Hz), 109.6, 107.7, 102.5, 100.8, 80.6, 62.3, 59.4, 59.3, 56.6, 53.2, 44.1, 24.6; IR (KBr): 3415, 3103, 2927, 1756, 1619, 1471, 1267, 1217, 1035, 838, 738, 570 cm^–1; MS (ESI) m/z: 630 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₈FN₃O₇S [M + H]⁺, 630.17048; found, 630.16949.

(S)-3-((R)-6-((6-(4-Chlorophenyl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7b)

White solid; yield: 68%; mp 120–122 °C; ¹H NMR (500 MHz, CDCl₃): δ 7.88 (d, J = 8.54 Hz, 2H), 7.84 (s, 1H), 7.38 (d, J = 8.54 Hz, 2H), 6.94 (d, J = 8.24 Hz, 1H), 6.58 (s, 1H), 6.36 (s, 1H), 6.10 (d, J = 8.24 Hz, 1H), 5.98–5.96 (m, 2H), 5.77 (d, J = 4.42 Hz, 1H), 4.61 (d, J = 4.42 Hz, 1H), 4.10–4.05 (m, 4H), 4.01 (s, 3H), 3.90 (d, J = 14.19 Hz, 1H), 3.82 (s, 3H), 2.61–2.53 (m, 1H), 2.40–2.33 (m, 1H), 2.29–2.21 (m, 1H), 2.10–2.03 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.4, 149.8, 148.8, 148.1, 146.2, 140.5, 140.4, 134.0, 132.7, 132.6, 130.5, 129.1, 128.6, 126.6, 119.3, 118.3, 117.5, 115.4, 109.8, 108.2, 102.5, 100.8, 80.5, 62.4, 59.4, 59.3, 56.6, 53.2, 44.0, 24.5; IR (KBr): 3416, 3102, 2926, 1756, 1620, 1470, 1267, 1214, 1037, 934, 831, 737, 505 cm^–1; MS (ESI) m/z: 646 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₈ClN₃O₇S [M + H]⁺: 646.14093; found, 646.14041.

(S)-3-((R)-6-((6-(4-Bromophenyl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7c)

Off-white solid; yield: 68%; mp 128–130 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.85 (s, 1H), 7.82 (d, J = 8.55 Hz, 2H), 7.54 (d, J = 8.55 Hz, 2H), 6.94 (d, J = 8.31 Hz, 1H), 6.58 (s, 1H), 6.36 (s, 1H), 6.10 (dd, J = 0.61, 8.31 Hz, 1H), 5.97 (dd, J = 1.34, 2.56 Hz, 2H), 5.77 (dd, J = 0.61, 4.40 Hz, 1H), 4.62 (d, J = 4.40 Hz, 1H), 4.10–4.05 (m, 4H), 4.01 (s, 3H), 3.90 (d, J = 14.90 Hz, 1H), 3.82 (s, 3H), 2.61–2.52 (m, 1H), 2.41–2.34 (m, 1H), 2.29–2.19 (m, 1H), 2.11–2.02 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.4, 149.9, 148.8, 148.2, 146.3, 140.5, 140.5, 134.0, 133.1, 131.6, 130.5, 129.1, 127.0, 120.8, 119.3, 118.4, 117.5, 115.5, 109.9, 108.3, 102.6, 100.8, 80.6, 62.4, 59.4, 59.4, 56.7, 53.2, 44.1, 24.6; IR(KBr): 3416, 2926, 1755, 1626, 1469, 1214, 1036, 826, 739 cm^–1; MS (ESI) m/z: 690 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₈BrN₃O₇S [M + H]⁺, 690.09041; found, 690.08927.

(S)-3-((R)-6-((6-(3,4-Dichlorophenyl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1 (3H)-one (7d)

Pale yellow solid; yield: 72%; mp 84–86 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.09 (d, J = 1.95 Hz, 1H), 7.90 (s, 1H), 7.81 (dd, J = 1.95, 8.31 Hz, 1H), 7.48 (d, J = 8.31 Hz, 1H), 6.94 (d, J = 8.31 Hz, 1H), 6.60 (s, 1H), 6.36 (s, 1H), 6.09 (d, J = 8.31 Hz, 1H), 5.97 (dd, J = 1.34, 2.93 Hz, 2H), 5.79 (d, J = 4.40 Hz, 1H), 4.61 (d, J = 4.40 Hz, 1H), 4.11–4.06 (m, 4H), 4.02 (s, 3H), 3.90 (d, J = 14.06 Hz, 1H), 3.82 (s, 3H), 2.63–2.52 (m, 1H), 2.38–2.30 (m, 1H), 2.22–2.13 (m, 1H), 2.09–2.000 (m, 1H); ¹³C NMR (CDCl₃, 100 MHz): 168.2, 152.4, 150.0, 148.8, 148.2, 145.1, 140.5, 140.4, 134.3, 134.0, 132.6, 130.5, 130.4, 129.1, 127.0, 124.7, 119.3, 118.4, 117.5, 115.3, 110.2, 108.9, 102.6, 100.8, 80.4, 62.4, 59.5, 59.3, 56.7, 53.1, 43.8, 24.3; IR (KBr): 3415, 2935, 1756, 1619, 1469, 1386, 1267, 1213, 1035, 819, 735, 526 cm^–1; MS (ESI) m/z: 680 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₇Cl₂N₃O₇S [M + H]⁺, 680.10195; found, 680.10101.

(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-((6-(3-methoxyphenyl)imidazo[2,1-b]thiazol-2-yl)methyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one (7e)

Yellow solid; yield: 62%; mp 122–124 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.82 (s, 1H), 7.52 (dd, J = 1.58, 2.56 Hz 1H), 7.48 (dt, J = 1.10, 7.94 Hz, 1H), 7.31 (t, J = 7.94 Hz, 1H), 6.94 (d, J = 8.31 Hz, 1H), 6.83 (dd, J = 2.56, 7.94 Hz, 1H), 6.59 (s, 1H), 6.36 (s, 1H), 6.14 (d, J = 8.31 Hz, 1H), 5.96 (s, 2H), 5.72 (d, J = 4.40 Hz, 1H), 4.60 (d, J = 4.40 Hz, 1H), 4.08–4.03 (m, 4H), 4.00 (s, 3H), 3.94–3.88 (m, 4H), 3.81 (s, 3H), 2.68–2.54 (m, 1H), 2.49–2.29 (m, 2H), 2.16–2.04 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.0, 159.8, 152.3, 149.6, 148.7, 148.0, 147.2, 140.5, 140.5, 135.5, 133.9, 130.3, 129.4, 129.0, 119.2, 118.3, 117.7, 117.5, 115.5, 113.6, 109.9, 109.7, 108.0, 102.5, 100.7, 80.6, 62.3, 59.4, 59.2, 56.6, 55.3, 53.0, 44.1, 24.5; IR (KBr): 3422, 2934, 2836, 1757, 1617, 1497, 1476, 1268, 1215, 1039, 931, 850, 789, 729, 661, 458 cm^–1; MS (ESI) m/z: 642 [M + H]⁺, HRMS (ESI): calcd for C₃₄H₃₁N₃O₈S [M + H]⁺, 642.19046; found, 642.19423.

(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-((6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-2-yl) methyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one (7f)

Yellow solid; yield: 64%; mp 103–105 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.84 (d, J = 8.80 Hz, 2H), 7.72 (s, 1H), 6.98–6.92 (m, 3H), 6.55 (s, 1H), 6.36 (s, 1H), 6.14 (dd, J = 0.61, 8.31 Hz, 1H), 5.96 (s, 2H), 5.73 (dd, J = 0.61, 4.40 Hz, 1H), 4.60 (d, J = 4.40 Hz, 1H), 4.08–4.03 (m, 4H), 4.01 (s, 3H), 3.90 (d, J = 14.30 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 2.63–2.53 (m, 1H), 2.48–2.32 (m, 2H), 2.15–2.06 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 158.8, 152.3, 149.5, 148.7, 148.0, 147.2, 140.5, 140.5, 134.0, 130.5, 129.0, 126.9, 126.5, 119.2, 118.4, 117.5, 115.5, 113.9, 109.2, 106.8, 102.5, 100.7, 80.7, 62.3, 59.4, 59.2, 56.6, 55.2, 53.1, 44.2, 24.7; IR (KBr): 3385, 2929, 1756, 1616, 1469, 1264, 1173, 1033, 932, 832, 742, 705, 524 cm^–1; MS (ESI) m/z: 642 [M + H]⁺, HRMS (ESI): calcd for C₃₄H₃₁N₃O₈S [M + H]⁺, 642.19046; found, 642.18898.

(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-((6-(3-(trifluoromethyl)phenyl)imidazo[2,1-b]thiazol-2-yl)methyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one (7g)

Pale yellow solid; yield: 62%; mp 145–147 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.23 (s, 1H), 8.14 (d, J = 7.09 Hz, 1H), 7.94 (s, 1H), 7.57–7.49 (m, 2H), 6.94 (d, J = 8.31 Hz, 1H), 6.61 (s, 1H), 6.36 (s, H), 6.10 (d, J = 8.31 Hz, 1H), 5.99–5.95 (m, 2H), 5.57 (d, J = 4.27 Hz, 1H), 4.62 (d, J = 4.27 Hz, 1H), 4.10–4.03 (m, 4H), 4.00 (s, 3H), 3.92 (d, J = 14.30 Hz, 1H), 3.81 (s, 3H), 2.67–2.53 (m, 1H), 2.44–2.34 (m, 1H), 2.29–2.19 (m, 1H), 2.11–2.01 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.1, 152.4, 150.0, 148.8, 148.1, 145.8, 140.5, 140.4, 135.0, 134.0, 130.9 (d, J_C–F = 32.69 Hz), 130.4, 129.1, 128.9, 128.5, 125.3 (d, J_C–F = 272.4 Hz), 123.5 (d, J_C–F = 3.63 Hz), 122.0 (d, J_C–F = 3.63 Hz), 119.2, 118.3, 117.5, 115.3, 110.2, 108.7, 102.6, 100.8, 80.4, 62.3, 59.4, 59.2, 56.6, 53.1, 43.9, 24.1; IR (KBr): 3421, 3104, 2936, 1758, 1619, 1497, 1476, 1333, 1268, 1214, 1165, 1120, 1038, 930, 808, 730, 699, 659, 447 cm^–1; MS (ESI) m/z: 680 [M + H]⁺; HRMS (ESI): calcd for C₃₄H₂₈F₃N₃O₇S [M + H]⁺, 680.16728; found, 680.17129.

4-(2-(((R)-5-((S)-4,5-Dimethoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-7,8-dihydro-[1,3]dioxolo[4,5-g]isoquinolin-6(5H)-yl)methyl)imidazo[2,1-b]thiazol-6-yl)benzonitrile (7h)

Yellow solid; yield: 64%; mp 138–140 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.09 (d, J = 8.54 Hz, 1H), 8.02 (s, 1H), 7.70 (d, J = 8.54 Hz, 1H), 6.95 (d, J = 8.24 Hz, 1H), 6.61 (s, 1H), 6.37 (s, 1H), 6.07 (d, J = 8.24 Hz, 1H), 5.98 (dd, J = 1.37, 4.27 Hz, 2H), 5.82 (d, J = 4.27 Hz, 1H), 4.63 (d, J = 4.27 Hz, 1H), 4.12–4.08 (m, 4H), 4.00 (s, 3H), 3.90 (d, J = 14.19 Hz, 1H), 3.83 (s, 3H), 2.60–2.52 (m, 1H), 2.33–2.26 (m, 1H), 2.14–1.99 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 168.2, 152.3, 150.1, 148.8, 148.0, 145.2, 140.3, 140.2, 138.6, 133.9, 132.2, 130.3, 129.0, 125.6, 119.3, 119.2, 118.4, 118.4, 117.6, 115.0, 110.6, 110.1, 109.7, 102.5, 100.8, 80.2, 62.2, 59.4, 59.3, 56.5, 53.1, 43.6, 24.0; IR (KBr): 3402, 3103, 2926, 2222, 1755, 1608, 1497, 1473, 1266, 1214, 1037, 932, 844, 750, 548 cm^–1; MS (ESI) m/z: 637 [M + H]⁺; HRMS (ESI): calcd for C₃₄H₂₈N₄O₇S [M + H]⁺, 637.17515; found, 637.17441.

(S)-6,7-Dimethoxy-3-((R)-4-methoxy-6-((6-(naphthalen-2-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one (7i)

Light yellow solid; yield: 65%; mp 136–138 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.45 (s, 1H), 8.02 (dd, J = 1.58, 8.55 Hz, 1H), 7.97–7.87 (m, 3H), 7.82 (d, J = 7.58 Hz, 2H), 6.94 (d, J = 8.31 Hz, 1H), 6.60 (s, 1H), 6.37 (s, 1H), 6.15 (d, J = 8.31 Hz, 1H), 5.96 (s, 2H), 5.75 (d, J = 4.40 Hz, 1H), 4.63 (d, J = 4.40 Hz, 1H), 4.12–4.06 (m, 4H), 4.00 (s, 3H), 3.96 (d, J = 5.95 Hz, 1H), 3.79 (s, 3H), 2.67–2.55 (m, 1H), 2.50–2.32 (m, 2H), 2.16–2.03 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.3, 149.9, 148.0, 147.0, 143.7, 140.5, 133.9, 133.6, 132.6, 130.3, 128.0, 127.5, 125.9, 125.4, 123.6, 118.3, 117.5, 115.4, 112.5, 109.8, 109.1, 108.4, 102.5, 102.3, 100.7, 80.5, 62.3, 59.3, 59.2, 56.5, 53.0, 44.0, 22.5; IR (KBr): 3419, 2927, 1756, 1622, 1470, 1268, 1213, 1036, 820, 754, 475 cm^–1; MS (ESI) m/z: 662 [M + H]⁺; HRMS (ESI): calcd for C₃₇H₃₁N₃O₇S [M + H]⁺, 662.19555; found, 662.19449.

(S)-3-((R)-6-((6-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyiso Benzofuran-1(3H)-one (7j)

Off-white solid; yield: 62%; mp 121–123 °C; ¹H NMR (500 MHz, CDCl₃): δ 7.68 (s, 1H), 7.44–7.35 (m, 2H), 6.97–6.88 (m, 2H), 6.56 (s, 1H), 6.36 (s, 1H), 6.16 (d, J = 8.31 Hz, 1H), 5.96 (s, 2H), 5.70 (d, J = 4.40 Hz, 1H), 4.59 (d, J = 4.40 Hz, 1H), 4.29 (s, 4H), 4.04 (s, 6H), 3.83 (s, 3H), 3.38 (t, J = 7.38 Hz, 1H), 2.84 (s, 1H), 2.65–2.53 (m, 1H), 2.49–2.42 (m, 1H), 2.19–2.07 (m, 1H), 2.07–1.95 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 152.4, 149.5, 148.8, 148.1, 147.0, 143.5, 142.9, 140.6, 140.6, 134.0, 130.4, 129.1, 127.9, 119.2, 118.6, 118.4, 117.5, 117.3, 115.7, 114.2, 109.4, 107.0, 102.5, 100.8, 80.8, 64.4, 64.3, 62.4, 59.4, 59.2, 56.6, 53.1, 44.4, 24.9; IR (KBr): 3418, 2927, 1757, 1667, 1620, 1497, 1474, 1387, 1266, 1215, 1039, 966, 890, 817, 732, 657 cm^–1; MS (ESI) m/z: 670 [M + H]⁺; HRMS (ESI): calcd for C₃₅H₃₁N₃O₉S [M + H]⁺, 670.18538; found, 670.18462.

(S)-3-((R)-6-((6-([1,1′-Biphenyl]-4-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7k)

Pale yellow solid; Yield: 64%; mp 115–117 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.99 (dd, J = 1.83, 6.71 Hz, 2H), 7.85 (s, 1H), 7.69–7.64 (m, 4H), 7.48–7.43 (m, 2H), 7.36–7.32 (m, 1H), 6.94 (d, J = 8.24 Hz, 1H), 6.59 (s, 1H), 6.36 (s, 1H), 6.14 (d, J = 8.24 Hz, 1H), 5.96 (s, 2H), 5.74 (d, J = 4.42 Hz, 1H), 4.62 (d, J = 4.42 Hz, 1H), 4.08–4.06 (m, 4H), 4.02 (s, 3H), 3.92 (d, J = 14.49 Hz, 1H), 3.82 (s, 3H), 2.63–2.55 (m, 1H), 2.49–2.42 (m, 1H), 2.41–2.34 (m, 1H), 2.15–2.07 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.0, 152.4, 149.8, 148.8, 148.1, 147.0, 140.8, 140.5, 139.7, 134.0, 133.1, 130.5, 129.1, 128.6, 127.1, 127.0, 126.8, 125.7, 119.2, 118.4, 117.5, 115.6, 109.7, 108.0, 102.5, 100.8, 80.7, 62.4, 59.4, 59.3, 56.6, 53.1, 44.3, 24.7; IR (KBr): 3391, 2924, 1756, 1615, 1468, 1266, 1033, 840, 742, 700 cm^–1; MS (ESI) m/z: 688 [M + H]⁺; HRMS (ESI): calcd for C₃₉H₃₃N₃O₇S [M + H]⁺, 688.21120; found, 688.21544.

(S)-3-((R)-6-((6-(5-Chlorothiophen-2-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7l)

Yellow solid; yield: 62%; mp 128–130 °C; ¹H NMR (500 MHz, CDCl₃): δ 7.71 (s, 1H), 7.29 (d, J = 3.91 Hz, 1H), 6.95 (d, J = 8.31 Hz, 1H), 6.89 (d, J = 3.91 Hz, 1H), 6.57 (s, 1H), 6.36 (s, 1H), 6.12 (dd, J = 0.73, 8.31 Hz, 1H), 5.97 (dd, J = 1.34, 2.20 Hz, 2H), 5.76 (dd, J = 0.73, 4.40 Hz, 1H), 4.59 (d, J = 4.40 Hz, 1H), 4.08 (s, 3H), 4.03–4.00 (m, 4H), 3.88 (d, J = 14.55 Hz, 1H), 3.84 (s, 3H), 2.63–2.53 (m, 1H), 2.39–2.32 (m, 1H), 2.27–2.18 (m, 1H), 2.12–2.04 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 152.3, 149.5, 148.8, 148.0, 141.4, 140.5, 140.4, 136.5, 134.0, 130.3, 129.0, 127.9, 126.8, 121.8, 119.2, 118.4, 117.5, 115.3, 109.9, 107.4, 102.6, 100.0, 81.5, 62.3, 59.4, 59.3, 56.6, 53.0, 43.8, 24.2; IR (KBr): 3419, 3100, 2932, 1756, 1620, 1469, 1267, 1213, 1036, 801, 718 cm^–1; MS (ESI) m/z: 652 [M + H]⁺; HRMS (ESI): calcd for C₃₁H₂₆ClN₃O₇S₂ [M + H]⁺: 652.09735; found, 652.09652.

(S)-3-((R)-6-((6-(5-Bromothiophen-2-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7m)

Yellow solid; yield: 68%; mp 198–200 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.72 (s, 1H), 7.28 (d, J = 3.91 Hz, 1H), 7.02 (d, J = 3.91 Hz, 1H), 6.95 (d, J = 8.31 Hz, 1H), 6.57 (s, 1H), 6.36 (s, 1H), 6.12 (dd, J = 0.48, 8.31 Hz, 1H), 5.97 (dd, J = 0.48, 1.92 Hz, 2H), 5.76 (dd, J = 0.48, 4.40 Hz, 1H), 4.59 (d, J = 4.40 Hz, 1H), 4.10–4.04 (m, 4H), 4.01 (s, 3H), 3.91–3.82 (m, 4H), 2.63–2.53 (m, 1H), 2.41–2.31 (m, 1H), 2.28–2.19 (m, 1H), 2.13–2.03 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 152.3, 149.5, 148.7, 147.9, 141.3, 140.4, 140.3, 139.4, 133.9, 130.5, 130.3, 128.9, 122.7, 119.1, 118.3, 117.5, 115.3, 110.2, 109.9, 107.4, 102.5, 100.7, 80.4, 62.2, 59.4, 59.2, 56.5, 52.9, 43.7, 24.1; IR (KBr): 3446, 3144, 2936, 1755, 1621, 1497, 1472, 1259, 1212, 1041, 972, 925, 799, 715 cm^–1; MS (ESI) m/z: 696 [M + H]⁺; HRMS (ESI): calcd for C₃₁H₂₆BrN₃O₇S₂ [M + H]⁺, 696.04683; found, 696.04595.

(S)-3-((R)-6-((6-(2,5-Dichlorothiophen-3-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7n)

Brick red solid; yield: 72%; mp 107–109 °C; ¹H NMR (500 MHz, CDCl₃): δ 7.89 (s, 1H), 7.41 (s, 1H), 6.95 (d, J = 8.24 Hz, 1H), 6.67 (s, 1H), 6.36 (s, 1H), 6.12 (dd, J = 0.61, 8.24 Hz, 1H), 5.96–5.95 (m, 2H), 5.68 (dd, J = 0.61, 4.42 Hz, 1H), 4.63 (d, J = 4.42 Hz, 1H), 4.10 (d, J = 14.34 Hz, 1H), 4.05 (s, 3H), 4.03 (s, 3H), 3.95 (d, J = 14.34 Hz, 1H), 3.85 (s, 3H), 2.62–2.50 (m, 3H), 2.18–2.09 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 167.8, 152.3, 149.2, 148.8, 147.9, 140.6, 140.6, 140.4, 139.9, 133.9, 132.1, 130.6, 129.2, 126.7, 126.0, 119.6, 119.2, 118.4, 117.5, 115.6, 110.0, 109.7, 102.5, 100.8, 81.0, 62.2, 59.3, 59.1, 56.6, 53.4, 45.3, 25.7; IR (KBr): 3422, 3101, 2932, 1758, 1620, 1474, 1265, 1213, 1035, 812, 728, 660, 557, 480 cm^–1; MS (ESI) m/z: 686 [M + H]⁺; HRMS (ESI): calcd for C₃₁H₂₅Cl₂N₃O₇S₂ [M + H]⁺, 686.05837; found, 686.05726.

(S)-3-((R)-6-((6-(5-Bromo-4-methylthiophen-2-yl)imidazo[2,1-b]thiazol-2-yl)methyl)-4-methoxy-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one (7o)

Pale yellow solid; Yield: 72%; mp 102–14 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.65 (s, 1H), 7.19 (s, 1H), 6.96 (d, J = 8.31 Hz, 1H), 6.57 (s, 1H), 6.36 (s, 1H), 6.13 (dd, J = 0.68, 8.31 Hz, 1H), 5.96 (s, 2H), 5.73 (dd, J = 0.68, 4.40 Hz, 1H), 4.59 (d, J = 4.40 Hz, 1H), 4.10–4.06 (m, 4H), 4.04 (s, 3H), 3.90–3.83 (m, 4H), 2.62–2.52 (m, 1H), 2.43–2.25 (m, 2H), 2.23 (s, 3H), 2.14–2.02 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 152.4, 149.6, 148.8, 148.0, 141.6, 140.5, 140.5, 137.7, 137.0, 134.0, 130.4, 129.0, 124.5, 119.2, 118.4, 117.5, 115.5, 109.9, 107.6, 107.1, 102.5, 100.8, 80.6, 62.3, 59.4, 59.3, 56.6, 53.1, 44.2, 24.6, 15.2; IR (KBr): 3448, 2928, 1757, 1621, 1497, 1472, 1268, 1214, 1038, 933, 819, 715 cm^–1; MS (ESI) m/z: 710 [M + H]⁺; HRMS (ESI): calcd for C₃₂H₂₈BrN₃O₇S₂ [M + H]⁺, 710.06248; found, 710.06417.

(S)-7-Hydroxy-6-methoxy-3-((R)-4-methoxy-6-methyl5,6,7,8-tetrahydro[1,3]dioxolo-[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one (8)

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

(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)

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.

(S)-7-((6-(4-Fluorophenyl)imidazo[2,1-b]thiazol-3-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 (11a)

Yellow solid; yield: 64%; mp 114–116 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.30 (s, 1H), 7.88–7.82 (m, 2H), 7.13–7.05 (m, 2H), 6.92 (d, J = 8.31 Hz, 1H), 6.81 (s, 1H), 6.28 (s, 1H), 6.02 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.34, 5.13 Hz, 2H), 5.62 (d, J = 4.15 Hz, 1H), 5.46 (dd, J = 12.34, 24.20 Hz, 2H), 4.40 (d, J = 4.15 Hz, 1H), 4.05 (s, 3H), 3.76 (s, 3H), 2.54–2.44 (m, 4H), 2.35–2.24 (m, 2H), 1.77–1.66 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.1, 163.0 (d, J_C–F = 245.22 Hz), 152.3, 149.2, 148.3, 146.3, 143.7, 140.8, 140.2, 133.9, 132.0, 130.5 (d, J_C–F = 2.72 Hz), 127.8, 126.6 (d, J_C–F = 8.17 Hz), 120.8, 118.9, 117.7, 116.6, 115.4 (d, J_C–F = 20.88 Hz), 112.2, 108.4, 102.2, 100.7, 82.0, 66.7, 60.6, 59.3, 56.4, 50.0, 46.3, 28.0; IR (KBr): 3416, 3134, 2937, 1754, 1619, 1545, 1472, 1269, 1214, 1086, 1038, 936, 839, 740, 574, 520 cm^–1; MS (ESI) m/z: 630 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₈FN₃O₇S [M + H]⁺, 630.17048; found, 630.16943.

(S)-7-((6-(4-Chlorophenyl)imidazo[2,1-b]thiazol-3-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 (11b)

Pale yellow solid; yield: 62%; mp 113–115 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.35 (s, 1H), 7.82 (d, J = 8.54 Hz, 2H), 7.37 (d, J = 8.54 Hz, 2H), 6.92 (d, J = 8.24 Hz, 1H), 6.82 (s, 1H), 6.28 (s, 1H), 6.03 (d, J = 8.24 Hz, 1H), 5.94 (dd, J = 1.37, 6.40 Hz, 2H), 5.63 (d, J = 4.12 Hz, 1H), 5.47 (dd, J = 12.35, 29.44 Hz, 2H), 4.41 (d, J = 4.12 Hz, 1H), 4.05 (s, 3H), 3.76 (s, 3H), 2.54–2.46 (m, 4H), 2.36–2.25 (m, 2H), 1.77–1.67 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.2, 152.3, 149.4, 148.4, 146.2, 143.7, 140.8, 140.3, 133.9, 132.9, 132.6, 128.7, 127.8, 126.3, 120.9, 118.9, 117.7, 116.6, 112.5, 108.9, 102.2, 100.7, 82.0, 66.7, 60.7, 59.4, 56.5, 50.0, 46.4, 28.0; IR (KBR): 3418, 3131, 2931, 1754, 1619, 1469, 1375, 1268, 1207, 1085, 1038, 936, 828, 739, 508 cm^–1; MS (ESI) m/z: 646 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₈ClN₃O₇S [M + H]⁺, 646.14093; found, 646.14025.

(S)-7-((6-(4-Bromophenyl)imidazo[2,1-b]thiazol-3-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 (11c)

Pale yellow solid; yield: 62%; mp 113–115 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.37 (s, 1H), 7.76 (d, J = 8.54 Hz, 2H), 7.52 (d, J = 8.54 Hz, 2H), 6.92 (d, J = 8.39 Hz, 1H), 6.82 (s, 1H), 6.28 (s, 1H), 6.03 (d, J = 8.39 Hz, 1H), 5.94 (dd, J = 1.37, 6.40 Hz, 2H), 5.62 (d, J = 4.12 Hz, 1H), 5.47 (dd, J = 12.20, 27.77 Hz, 2H), 4.41 (d, J = 4.12 Hz, 1H), 4.05 (s, 3H), 3.76 (s, 3H), 2.54–2.46 (m, 4H), 2.35–2.25 (m, 2H), 1.77–1.68 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.2, 152.3, 149.4, 148.3, 146.1, 143.7, 140.8, 140.2, 133.9, 133.3, 132.0, 131.6, 127.8, 126.6, 120.8, 118.9, 117.7, 116.6, 112.5, 109.0, 102.2, 100.7, 82.0, 66.7, 60.7, 59.3, 56.4, 50.0, 46.4, 28.0; IR (KBR): 3421, 2935, 2795, 1754, 1619, 1470, 1379, 1269, 1207, 1084, 1038, 1007, 936, 829, 737 cm^–1; MS (ESI) m/z: 689 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₈BrN₃O₇S [M + H]⁺, 690.09041; found, 690.08906.

(S)-7-((6-(3,4-Dichlorophenyl)imidazo[2,1-b]thiazol-3-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 (11d)

Yellow solid; yield: 62%; mp 117–119 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.40 (s, 1H), 8.00 (d, J = 2.07 Hz, 1H), 7.70 (dd, J = 2.07, 8.31 Hz, 1H), 7.45 (d, J = 8.31 Hz, 1H), 6.93 (d, J = 8.31 Hz, 1H), 6.84 (s, 1H), 6.29 (s, 1H), 6.04 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.34, 5.38 Hz, 2H), 5.63 (d, J = 4.03 Hz, 1H), 5.46 (dd, J = 12.47, 22.00 Hz, 2H), 4.41 (d, J = 4.03 Hz, 1H), 4.06 (s, 3H), 3.77 (s, 3H), 2.55–2.46 (m, 4H), 2.36–2.25 (m, 2H), 1.78–1.68 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.2, 152.3, 149.6, 148.4, 144.9, 143.7, 140.8, 140.2, 134.4, 133.9, 132.6, 130.4, 127.8, 126.7, 124.2, 120.9, 119.0, 117.7, 116.6, 112.7, 109.5, 102.2, 100.8, 82.0, 66.7, 60.7, 59.3, 56.4, 50.0, 46.4, 28.0; IR (KBr): 3414, 3130, 2932, 1753, 1612, 1466, 1381, 1268, 1035, 887, 818, 738 cm^–1; MS (ESI) m/z: 680 [M + H]⁺; HRMS (ESI): calcd for C₃₃H₂₇Cl₂N₃O₇S [M + H]⁺, 680.10195; found, 680.10087.

(S)-6-Methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]iso quinolin-5-yl)-7-((6-(3-methoxyphenyl)imidazo[2,1-b]thiazol-3-yl)methoxy)isobenzofuran-1(3H)-one (11e)

Pale yellow solid; yield: 65%; mp 90–92 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.31 (s, 1H), 7.49–7.42 (m, 2H), 7.31 (t, J = 7.94 Hz, 1H), 6.92 (d, J = 8.31 Hz, 1H), 6.86–6.80 (m, 1H), 6.29 (s, 1H), 6.09 (d, J = 8.31 Hz, 1H), 5.93 (dd, J = 1.22, 4.76 Hz, 2H), 5.65 (d, J = 4.03 Hz, 1H), 5.48 (q, J = 29.29 Hz, 2H), 4.45 (d, J = 4.03 Hz, 1H), 4.01 (s, 3H), 3.89 (s, 3H), 3.75 (s, 3H), 2.56–2.52 (m, 4H), 2.39–2.30 (m, 2H), 2.08–1.99 (m, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 168.1, 161.0, 152.1, 147.6, 146.4, 143.7, 141.3, 138.9, 138.4, 134.6, 130.8, 128.8, 125.9, 122.4, 119.9, 118.2, 117.7, 117.6, 117.4, 116.6, 100.8, 81.8, 64.5, 64.1, 62.2, 60.9, 59.5, 56.7, 49.5, 45.9, 22.8; IR (KBr): 3412, 2929, 1755, 1618, 1476, 1379, 1270, 1212, 1039, 934, 812, 787 cm^–1; MS (ESI) m/z: 642 [M + H]⁺; HRMS (ESI): calcd for C₃₄H₃₁N₃O₈S [M + H]⁺, 642.19046; found, 642.18975.

(S)-6-Methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]iso quinolin-5-yl)-7-((6-(4-methoxyphenyl)imidazo[2,1-b]thiazol-3-yl)methoxy)isobenzofuran-1(3H)-one (11f)

Off-white solid; yield: 65%; mp 120–122 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.19 (s, 1H), 7.81 (d, J = 8.85 Hz, 2H), 6.95 (d, J = 8,85 Hz, 2H), 6.90 (d, J = 8.24 Hz, 1H), 6.78 (s, 1H), 6.28 (s, 1H), 6.01 (d, J = 8.24 Hz, 1H), 5.94 (dd, J = 1.37, 5.79 Hz, 2H), 5.62 (d, J = 4.12 Hz, 1H), 5.49 (dd, J = 12.35, 43.02 Hz, 2H), 4.41 (d, J = 4.12 Hz, 1H), 4.05 (s, 3H), 3.85 (s, 3H), 3.74 (s, 3H), 2.54–2.47 (m, 4H), 2.34–2.25 (m, 1H), 1.75–1.67 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.2, 158.9, 152.4, 149.1, 148.4, 147.2, 143.8, 140.8, 14.3, 134.0, 132.1, 127.8, 127.2, 126.3, 120.9, 118.8, 117.7, 116.7, 114.0, 112.1, 107.6, 102.3, 100.7, 82.0, 66.9, 60.7, 59.4, 56.5, 55.2, 50.1, 46.4, 28.1; IR (KBr): 3415, 3135, 2936, 2842, 2795, 1754, 1617, 1470, 1269, 1034, 935, 834, 740, 705, 578, 523 cm^–1; MS (ESI) m/z: 642 [M + H]⁺; HRMS (ESI): calcd for C₃₄H₃₁N₃O₈S [M + H]⁺, 642.19046; found, 642.18924.

(S)-6-Methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]iso quinolin-5-yl)-7-((6-(3-(trifluoromethyl)phenyl)imidazo[2,1-b]thiazol-3-yl)methoxy)iso Benzofuran-1(3H)-one (11g)

Yellow solid; yield: 60%; mp 97–99 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.44 (s, 1H), 8.15 (s, 1H), 8.08–8.04 (m, 1H), 7.53–7.50 (m, 2H), 6.94 (d, J = 8.31 Hz, 1H), 6.85 (s, 1H), 6.29 (s, 1H), 6.06 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.34, 5.13 Hz, 2H), 5.64 (d, J = 4.15 Hz, 1H), 5.48 (dd, J = 12.34, 28.85 Hz, 2H), 4.42 (d, J = 4.15 Hz, 1H), 4.05 (s, 3H), 3.78 (s, 3H), 2.57–2.48 (m, 4H), 2.37–2.26 (m, 2H), 1.81–1.70 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.3, 149.5, 148.3, 145.7, 143.6, 140.7, 140.2, 135.1, 133.9, 131.9, 130.9 (d, J_C–F = 32.27 Hz), 128.9, 128.0, 127.8, 125.5 (d, J_C–F = 272.88 Hz), 123.4 (d, J_C–F = 3.66 Hz), 121.7 (d, J_C–F = 3.66 Hz), 120.7, 119.0, 117.7, 116.4, 112.7, 109.4, 102.2, 100.7, 81.9, 66.7, 60.6, 59.3, 56.3, 49.9, 46.2, 27.8; IR (KBr): 3447, 3133, 2941, 2798, 1755, 1619, 1494, 1335, 1270, 1165, 1122, 1038, 892, 808, 696, 659 cm^–1; MS (ESI) m/z: 680 [M + H]⁺; HRMS (ESI): calcd for C₃₄H₂₈F₃N₃O₇S [M + H]⁺, 680.16728; found, 680.16641.

4-(3-((((S)-5-Methoxy-1-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g] Isoquinolin-5-yl)-3-oxo-1,3-dihydroisobenzofuran-4-yl)oxy)methyl)imidazo[2,1-b]thiazol-6-yl)benzonitrile (11h)

Yellow solid; yield: 72%; mp 144–146 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.60 (s, 1H), 7.99 (d, J = 8.55 Hz, 2H), 7.68 (d, J = 8.35 Hz, 2H), 6.95 (d, J = 8.31 Hz, 1H), 6.87 (s, 1H), 6.29 (s, 1H), 6.06 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.34, 5.86 Hz, 2H), 5.63 (d, J = 4.15 Hz, 1H), 5.44 (s, 2H), 4.41 (d, J = 4.15 Hz, 1H), 4.05 (s, 3H), 3.80 (s, 3H), 2.54–2.45 (m, 4H), 2.37–2.25 (m, 2H), 1.80–1.69 (m, 1H); ¹³C NMR (75 MHz, CDCl₃): δ 168.2, 152.3, 149.9, 148.4, 145.3, 143.6, 140.9, 140.2, 138.8, 133.9, 132.4, 131.9, 127.8, 125.3, 120.9, 119.2, 119.1, 117.8, 116.5, 113.1, 112.8, 109.9, 102.2, 100.7, 82.0, 66.6, 60.6, 59.3, 56.5, 50.0, 46.3, 27.9; IR (KBr): 3426, 3130, 2932, 2796, 2221, 1753, 1610, 1470, 1268, 1037, 935, 842, 750, 584 cm^–1; MS (ESI) m/z: 637 [M + H]⁺; HRMS (ESI): calcd for C₃₄H₂₈N₄O₇S [M + H]⁺, 637.17515; found, 637.17423.

(S)-6-Methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]iso quinolin-5-yl)-7-((6-(naphthalen-2-yl)imidazo[2,1-b]thiazol-3-yl)methoxy)isobenzofuran-1(3H)-one (11i)

Pale yellow solid; yield: 68%; mp 217–219 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.43 (s, 1H), 8.41 (s, 1H), 7.96 (dd, J = 1.71, 8.43 Hz, 1H), 7.91–7.85 (m, J = 1.71, 8.43 Hz, 2H), 7.84–7.80 (m, 1H), 7.50–7.41 (m, 2H), 6.91 (d, J = 8.31 Hz, 1H), 6.82 (s, 1H), 6.28 (s, 1H), 6.01 (d, J = 8.31 Hz, 1H), 5.93 (dd, J = 1.34, 5.13 Hz, 2H), 5.63 (d, J = 4.15, 1H), 5.52 (dd, J = 12.34, 35.09 Hz, 2H), 4.41 (d, J = 4.15 Hz, 1H), 4.06 (s, 3H), 3.75 (s, 3H), 2.55–2.47 (m, 4H), 2.37–2.25 (m, 2H), 1.76–1.67 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.2, 152.3, 149.5, 148.3, 147.2, 143.7, 140.7, 140.2, 133.9, 133.6, 132.7, 132.1, 131.6, 128.1, 128.1, 127.8, 127.5, 126.0, 125.4, 123.5, 123.3, 120.8, 118.8, 117.6, 116.6, 112.4, 109.1, 102.2, 100.7, 82.0, 66.8, 60.6, 59.3, 56.4, 50.1, 46.4, 28.0; IR (KBr): 3422, 3125, 2938, 2789, 1750, 1622, 1475, 1379, 1269, 1036, 1006, 934, 819, 752, 475 cm^–1; MS (ESI) m/z: 662 [M + H]⁺; HRMS (ESI): calcd for C₃₇H₃₁N₃O₇S [M + H]⁺, 662.19555; found, 662.19454.

(S)-7-((6-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)imidazo[2,1-b]thiazol-3-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 (11j)

Yellow solid; yield: 65%; mp 123–125 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.13 (s, 1H), 7.40–7.34 (m, 2H), 6.89 (q, J = 8.31 Hz, 2H), 6.78 (s, 1H), 6.28 (s, 1H), 6.01 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.34, 4.64 Hz, 2H), 5.62 (d, J = 4.15 Hz, 1H), 5.48 (dd, J = 12.47, 37.16 Hz, 2H), 4.41 (d, J = 4.15 Hz, 1H), 4.28 (s, 4H), 4.05 (s, 3H), 3.73 (s, 3H), 2.56–2.47 (m, 4H), 2.35–2.25 (m, 2H), 1.77–1.65 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.4, 149.0, 148.3, 140.9, 143.8, 143.5, 142.8, 140.7, 140.2, 133.9, 131.9, 128.0, 127.8, 120.7, 118.8, 118.3, 117.7, 117.2, 116.4, 113.9, 112.1, 107.7, 102.2, 100.7, 81.9, 66.9, 64.3, 64.2, 60.7, 59.3, 56.4, 49.9, 46.2, 27.8; IR (KBr): 3417, 3135, 2927, 1754, 1617, 1488, 1376, 1271, 1038, 932, 891, 816, 741 cm^–1; MS (ESI) m/z: 670 [M + H]⁺; HRMS (ESI): calcd for C₃₅H₃₁N₃O₉S [M + H]⁺, 670.18538; found, 670.18461.

(S)-7-((6-([1,1′-Biphenyl]-4-yl)imidazo[2,1-b]thiazol-3-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 (11k)

Pale yellow solid; yield: 60%; mp 103–105 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.37 (s, 1H), 7.98–7.94 (m, 2H), 7.68–7.24 (m, 4H), 7.48–7.41 (m, 2H), 7.38–7.31 (m, 1H), 6.91 (d, J = 8.31 Hz, 1H), 6.81 (s, 1H), 6.28 (s, 1H), 6.02 (d, J = 8.31 Hz, 1H), 5.93 (dd, J = 1.34, 5.13 Hz, 2H), 5.63 (d, J = 4.15 Hz, 1H), 5.49 (dd, J = 12.49, 32.64 Hz, 2H), 4.41 (d, J = 4.15 Hz, 1H), 4.05 (s, 3H), 3.76 (s, 3H), 2.57–2.46 (m, 4H), 2.37–2.24 (m, 2H), 1.78–1.66 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.3, 149.3, 148.4, 146.9, 143.7, 140.2, 139.6, 139.3, 133.3, 131.9, 128.6, 127.8, 127.2, 127.0, 126.7, 125.4, 120.7, 120.6, 118.9, 117.7, 112.3, 108.7, 102.2, 100.7, 81.9, 66.8, 60.7, 59.3, 56.4, 49.9, 46.2, 27.8; IR (KBr): 3418, 2935, 2795, 1753, 1671, 1618, 1471, 1379, 1268, 1207, 1084, 1036, 934, 842, 738, 697 cm^–1; MS (ESI) m/z: 688 [M + H]⁺; HRMS (ESI): calcd for C₃₉H₃₃N₃O₇S [M + H]⁺, 688.21120; found, 688.21008.

(S)-7-((6-(5-Chlorothiophen-2-yl)imidazo[2,1-b]thiazol-3-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 (11l)

Yellow solid; yield: 69%; mp 108–110 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.19 (s, 1H), 7.14 (d, J = 3.66 Hz, 1H), 6.93 (d, J = 8.31 Hz, 1H), 6.86 (d, J = 3.66 Hz, 1H), 6.82 (s, 1H), 6.29 (s, 1H), 6.04 (d, J = 8.31 Hz, 1H), 5.94 (d, J = 4.03 Hz, 2H), 5.63 (d, J = 3.66 Hz, 1H), 5.45 (dd, J = 12.22, 32.03 Hz, 2H), 4.42 (d, J = 3.66 Hz, 1H), 4.05 (s, 3H), 3.78 (s, 3H), 2.58–2.46 (m, 4H), 2.37–2.24 (m, 2H), 1.79–1.67 (m, 1H);¹³C NMR (CDCl₃, 100 MHz): δ 168.2, 152.3, 149.3, 148.4, 143.7, 141.4, 140.8, 140.3, 136.6, 134.0, 132.0, 128.0, 127.8, 126.7, 121.4, 120.9, 119.0, 117.7, 116.6, 112.7, 108.1, 102.3, 100.8, 82.0, 66.8, 60.7, 59.4, 56.5, 50.0, 46.3, 28.0; IR (KBr): 3412, 2929, 2796, 1754, 1659, 1619, 1496, 1471, 1270, 1209, 1037, 1009, 934, 793 cm^–1; MS (ESI) m/z: 652 [M + H]⁺; HRMS (ESI): calcd for C₃₁H₂₆ClN₃O₇S₂ [M + H]⁺, 652.09735; found, 652.09626.

(S)-7-((6-(5-Bromothiophen-2-yl)imidazo[2,1-b]thiazol-3-yl)methoxy)-6-methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)iso Benzofuran-1(3H)-one (11m)

Yellow solid; yield: 74%; mp 104–106 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.19 (s, 1H), 7.12 (d, J = 3.66 Hz, 1H), 6.99 (d, J = 3.66 Hz, 1H), 6.92 (d, J = 8.31 Hz, 1H), 6.82 (s, 1H), 6.29 (s, 1H), 6.03 (d, J = 8.31 Hz, 1H), 5.94 (d, J = 4.15 Hz, 2H), 5.62 (d, J = 4.15 Hz, 1H), 5.45 (dd, J = 1.22, 33.01 Hz, 2H), 4.41 (d, J = 4.15 Hz, 1H), 4.05 (s, 3H), 3.77 (s, 3H), 2.56–2.46 (m, 4H), 2.36–2.25 (m, 2H), 1.77–1.66 (m, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 168.1, 152.3, 149.3, 148.4, 143.6, 141.3, 140.7, 140.2, 139.5, 133.9, 131.9, 130.4, 127.7, 122.4, 120.8, 119.0, 117.7, 116.4, 112.7, 110.3, 108.2, 102.2, 100.7, 82.0, 66.7, 60.7, 59.3, 56.4, 50.0, 46.2, 27.9; IR (KBr): 3426, 3113, 2929, 2796, 1753, 1619, 1470, 1269, 1208, 1037, 934, 793, 716 cm^–1; MS (ESI) m/z: 698 [M + H]⁺; HRMS (ESI): calcd for C₃₁H₂₆BrN₃O₇S₂ [M + H]⁺, 698.04249; found, 698.04907.

(S)-7-((6-(2,5-Dichlorothiophen-3-yl)imidazo[2,1-b]thiazol-3-yl)methoxy)-6-methoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)iso Benzofuran-1(3H)-one (11n)

Pale yellow solid; yield: 69%; mp 108–110 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.44 (s, 1H), 7.42 (s, 1H), 6.92 (d, J = 8.31 Hz, 1H), 6.89 (s, 1H), 6.29 (s, 1H), 6.03 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.46, 5.01 Hz, 2H), 5.62 (d, J = 4.15 Hz, 1H), 5.52 (dd, J = 0.61, 12.22 Hz, 1H), 5.43 (dd, J = 0.61, 12.22 Hz, 1H), 4.41 (d, J = 4.15 Hz, 1H), 4.06 (s, 3H), 3.75 (s, 3H), 2.56–2.48 (m, 4H), 2.35–2.26 (m, 2H), 1.80–1.72 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.4, 148.6, 148.4, 143.8, 140.7, 140.2, 139.9, 134.0, 132.3, 132.1, 127.9, 126.6, 126.0, 120.8, 119.5, 118.9, 117.6, 116.7, 113.1, 110.9, 102.2, 100.7, 82.1, 66.8, 60.7, 59.4, 56.5, 50.1, 46.4, 28.1; IR (KBr): 3419, 3103, 2936, 2796, 1755, 1618, 1479, 1269, 1037, 935, 837, 735, 559, 478 cm^–1; MS (ESI) m/z: 686 [M + H]⁺; HRMS (ESI): calcd for C₃₁H₂₅Cl₂N₃O₇S₂ [M + H]⁺, 686.05837; found, 686.05728.

(S)-7-((6-(5-Bromo-4-methylthiophen-2-yl)imidazo[2,1-b]thiazol-3-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 (11o)

Yellow solid; yield: 62%; mp 128–130 °C; ¹H NMR (500 MHz, CDCl₃): δ 8.14 (s, 1H), 7.07 (s, 1H), 6.92 (d, J = 8.31 Hz, 1H), 6.81 (s, 1H), 6.29 (s, 1H), 6.04 (d, J = 8.31 Hz, 1H), 5.94 (dd, J = 1.37, 5.95 Hz, 2H), 5.63 (d, J = 4.12 Hz, 1H), 5.45 (q, J = 12.20 Hz, 2H), 4.42 (d, J = 4.12 Hz, 1H), 4.05 (s, 3H), 3.77 (s, 3H), 2.57–2.48 (m, 4H), 2.36–2.27 (m, 2H), 2.20 (s, 3H), 1.79–1.68 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 168.1, 152.3, 149.3, 148.5, 143.7, 141.4, 140.6, 140.2, 137.6, 137.1, 133.9, 131.8, 127.7, 124.1, 120.7, 119.0, 117.7, 116.2, 112.6, 107.9, 107.5, 102.3, 100.7, 81.9, 66.8, 60.7, 59.3, 56.4, 49.8, 46.0, 22.6, 15.2; IR (KBr): 3427, 2937, 2796, 1755, 1620, 1474, 1379, 1269, 1210, 1038, 935, 891, 816, 715 cm^–1; MS (ESI) m/z: 710 [M + H]⁺; HRMS (ESI): calcd for C₃₂H₂₈BrN₃O₇S₂ [M + H]⁺, 710.06248; found, 710.06420.

Biology

Cell Culture

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

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b02789.

• Copies of ¹H, ¹³C NMR, and mass (HR-MS) spectra of the final molecules and flow cytometry analysis (PDF)

Author Contributions

S.K. and P.K.R.N. designed and synthesized the novel compounds. A.D.T. and V.K.K. performed biological experiments. D.S. and V.S.K. performed the molecular modeling experiments. S.K. and A.D.T. wrote the paper, and all authors reviewed and edited their part.

The authors declare no competing financial interest.