First noscapine glycoconjugates inspired by click chemistry †

A number of novel 7-O -noscapine glycoconjugates have been synthesized starting from noscapine, an alkaloid found in the opium plant, via two successive steps. The ﬁ rst step is a selective 7-O - demethylation of noscapine and the next is a subsequent prop-argylation which a ﬀ ords 7-O -propargyl noscapine ( 3 ) in good yield. The structure was con ﬁ rmed by extensive spectroscopic data including single crystal X-ray data. The 1,3-dipolar cycloaddition of the developed noscapine derivative 3 with glycosyl azides 6a – m was investigated to give the triazole-linked second-generation noscapine analogs in their glycoconjugate forms ( 8a – m ) to augment the therapeutic e ﬃ cacy of noscapine.


Introduction
Natural products and their derivatives are now well established biologically relevant moieties and participate in critical roles in modern drug discovery and development. 1Alkaloids obtained from nature are the most potent and pharmaceutically interesting scaffolds. 2One member of this group, noscapine ('a phthalideisoquinoline alkaloid'), has a benzofuranone ring attached to the hetero ring of isoquinoline.Noscapine is available in about 7% abundance during opium harvesting. 3It has been used as an antitussive agent for several decades because of its favourable toxicity prole.Recently, it was found to bind tubulin and alter its conformation and properties, and alter microtubule dynamics. 4,5Additionally, noscapine has also shown the successful inhibition of various neoplasms in vitro as well as in vivo, such as leukaemia and lymphoma, [6][7][8] along with melanoma, 9 ovarian cancer, 10 gliomas, 11 and breast, 12 lung 13 and colon 14 cancers.Recently, Joshi et al. have assessed the mechanistic path of this anticancer effect aer performing several studies where they found that noscapine can perturb tubulin dynamics. 15Recent literature has revealed that chemical modications at its 7-position via selective demethylation on the benzofuranone ring system has been achieved and showed that the O-alkylated derivatives, including the 7hydroxyl compounds, were 100-fold more effective than the parent noscapine. 16,17This strongly suggests that the presence and modication of the benzofuranone ring in the parent molecule has a signicant impact on its biological activity (Fig. 1).
Carbohydrates and their diverse saccharide forms (mono to poly) always attract synthetic chemists for their utilization in medicinal chemistry because their use yields effective control over biological functions. 18Additionally, the multivalent nature of carbohydrate molecules is frequently used to enhance their affinities for targets in different biological processes, such as the binding of bacteria, bacterial toxins, galectins and other lectins. 19Although the carbohydrates alone demonstrate no therapeutic action, their presence in synthetic and naturally occurring molecules creates a prominent change in their physical, chemical and biological properties.This also inuences the biological activity of most of the drugs which incorporate them. 20g. 1 Structure of noscapine (1a) and its potent biologically active 7-O-analogs (1b, 1c), against tubulin polymerization.
The Cu(I)-catalysed click reaction 21,22 is a precise tool for the joining of two dissimilar moieties having azide and terminal alkyne functionalities and has emerged as an important strategy for the discovery and optimization of leads.4][25][26][27][28] Based upon this impetus and with our previous experience, 29-32 herein we have successfully incorporated a terminal alkyne functionality in naturally occurring a-noscapine at its C-7 position.This strategy afforded novel 7-O-analogs which were further utilized for developing second-generation noscapine derivatives in their glycoconjugate forms using Cu(I)-catalyzed click chemistry.We hope this will satisfy the increasing demand for more potent analogs of this molecule to modulate microtubules more effectively.

Results and discussion
Our strategy started with the demethylation of the parent compound noscapine.Sodium azide and sodium iodide in dimethylformamide (DMF) were used to cleave the methyl group selectively at position 7 of the benzofuranone ring.17b Briey, noscapine was dissolved in anhydrous DMF along with sodium azide and sodium iodide followed by stirring at 140 C for 4 h to obtain 7-hydroxy noscapine 2 (Scheme 1).Compound 2 was then propargylated at its hydroxyl moiety using K 2 CO 3 in reuxing acetone at 80 C to afford 7-O-propargylated noscapine 3 in 75% yield (Scheme 1).Surprisingly, this reaction did not occur in DMF at room temperature using the same base.Compound 3 served as a scaffold to synthesize various C-7-modied derivatives of noscapine 8a-m in their glycoconjugate form.The structures of the new C-7 analogs of noscapine 3 were deduced from their extensive spectral studies (IR, NMR, and MS).Single crystal X-ray analysis of compound 3 conrmed the selective demethylation of the parent molecule at the C-7 position.
The 1 H NMR spectrum of compound 3 exhibited one singlet signal at d 2.62, merged with the 3 protons of N-Me, which was assigned to the acetylene proton.Shiing of the ortho-coupled aromatic protons from d 5.11 (d, J ¼ 8.4 Hz) to 6.10 (d, J ¼ 8.4 Hz) for C-9 and from d 6.44 (d, J ¼ 8.4 Hz) to 6.96 (d, J ¼ 8.4 Hz) for C-10 also conrmed the substitution at the 7-hydroxy group.In addition to other signals, the appearance of a multiplet at d 5.05 attributed to OCH 2 nally conrmed the addition of the propargyl group, leading to the formation of compound 3.In the 13 C NMR, two new resonances were observed at d 81.9 and d 75.4 which were assigned to both acetylene carbons.The molecular structure of compound 3 was also conrmed by single crystal X-ray analysis (Fig. 2

, see ESI Table 1 †).
Once we achieved the second generation (C-7) noscapine analogue 3, having one terminal alkyne, we attempted the synthesis of various sugar azides for glycoconjugation of the novel noscapine derivative.We prepared sugar azides with the economical and readily available monosaccharides, i.e.D- glucose, D-galactose, D-xylose and a disaccharide, lactose, which, aer processing through a number of high-yielding steps involving protections and diverse modications, afforded deoxy-azido sugars 6a-j in good yields. 33The sugar azides 6k-m were synthesised via a substitution reaction on the orthogonally protected carbohydrate with epichlorohydrin in the presence of NaH in dry DMF at 0 C-r.t., which afforded a diastereoisomeric mixture of glycosyl epoxides 5k-m.These epoxides, on reaction with NaN 3 and NH 4 Cl in EtOH/H 2 O at 65 C, afforded their respective O-substituted glycosyl azido alcohols 6k-m (Scheme 2).All the developed azidosugars 6a-m underwent glycoconjugation using compound 3 via the copper-catalyzed azide-alkyne click reaction.Generally, copper-catalyzed azidealkyne click reactions require the presence of Cu(I) species which may be provided directly or in situ depending on the catalyst.Hence, we carried out the reaction using both methods, rst using CuI/DIPEA in dichloromethane and then CuSO 4 -$5H 2 O/sodium ascorbate in aqueous medium.We preferred the former reaction system due to its better yield and shorter reaction time (Scheme 3).Hence, the click reaction of deoxyazido sugar 6a (0.19 mmol) with 3 (0.16 mmol) in the presence of CuI (0.08 mmol) and DIPEA (0.16 mmol) was carried out in anhydrous CH 2 Cl 2 under argon atmosphere at ambient temperature to afford 7-O-noscapine triazolyl glycoconjugate 8a regioselectively in 95% yield.The regioisomeric nature of compound 8a was established based on its spectroscopic data  Further, having established the reaction conditions for the regioselective cycloaddition of the 7-O-propargyl noscapine 3, we explored the scope of other sugar azides in this cycloaddition and prepared a library of 7-O-noscapine triazolyl glycoconjugates 8b-m in efficient yields (Table 1).Using extensive spectral studies (IR, MS, 1 H, and 13 C NMR), the structures of all the developed noscapine glycoconjugates 8a-m were elucidated.

Weak interactions in compound 3 and their biological importance via stabilisation of geometrical conformations
Noncovalent inter-and intramolecular interactions play a subtle role in molecular recognition and conformational stabilization within the crystal lattice for biological assays. 34,35herefore, it is important to quantify the various interactions within the molecules in the crystal structures.Compound 3 is rich in C-H donors and O, p acceptors.In the isoquinoline ring, the N-methyl hydrogens, methylene hydrogens and also the acetylene acidic hydrogen act as a donor whenever oxygen atoms and the p-electron ring system act as acceptors.Intramolecular and intermolecular CH/O and CH/p interactions stabilize the geometry of the molecule and show their effects in the relative changes in the geometrical conformations of compound 3.
These weak interactions generate a number of six member ring systems which were known for their crucial role in biological activities. 35Intramolecular interactions have been shown with two six member ring along with a CH/p ring system.
Out of various types of intramolecular interactions (Fig. 3), three of them that cause conformational changes have been presented.A CH/O (I) interaction between the N-methyl hydrogens and furanone ring oxygen, with a measured distance of 2.538 Å, and a CH/p (III) interaction between the methylene hydrogen of quinoline and aromatic system fused with the  lactone ring, with a measured distance of 2.977 Å, are attempting to place both of the fused ring systems in parallel planes, but the repulsion between the oxygen lone pairs of both fused ring systems pushes them to their maximum distances and overcomes the effect of a possible p/p interaction between both of the benzene rings.One of the CH/O (II) interactions, with a measured distance of 2.37 Å, generates a sixmembered ring system.All these weak interactions conrm the efficacy of the developed molecules in a biological system due to the presence of a number of interacting sites, which create the effect of interacting with problematic enzymes and proteins to reduce their activities during clinical treatment. 36Also, intermolecular interactions within the crystal packing have an effect on geometrical conformations and form dimeric structures.The dimeric structures (IV, V, VI) appeared in three forms depending on the type of interactions and the positions of their sites (Fig. 4).
Substitution at the C-7 position in the parent noscapine scaffold creates new interaction sites, such as CH/O, with measured distances of 2.503 Å (IV) and 2.587 Å (VI), between the acetylene and methylene hydrogens of the adjoining part and the oxygen of the parent molecule.An intermolecular CH/p (2.503 Å, V) interaction has effects on the conformations in the crystal packing.Thus, the creation of new binding sites in noscapine C-7 analog 3 is evidenced for the well-known potency towards modulating tublin polymerization.Furthermore, because of the multivalent nature of carbohydrates, 18 their introduction to noscapine is envisaged to provide more binding sites and could result in increased efficacy; however, continued efforts are required for the conclusive investigation to this end.

Conclusion
In conclusion, a number of sugar azides were prepared and further subjected to a Cu(I)-catalyzed azide alkyne cycloaddition reaction (click) with 7-O-propargylated noscapine.We have developed thirteen second generation noscapine triazolyl glycoconjugates at the C-7 position in good to excellent yields.Also, the role of weak interactions has been correlated with the biological action of noscapine analogs.The methodology is efficient for the preparation of modied conjugates of noscapine to improve its therapeutic efficacy and its pharmacological properties.Further research into the development of noscapine glycoconjugates as potential anti-cancer agents is in progress in our laboratory.

General methods
All of the reactions were performed in anhydrous solvents (where required) under an argon atmosphere in oven dried glassware at 100 C. All reagents and solvents were of pure analytical grade.Thin-layer chromatography (TLC) was performed on 60 F 254 silica gel, pre-coated on aluminum plates, and revealed with either a UV lamp (l max ¼ 254 nm), a specic colour reagent (Draggendorff reagent or iodine vapour) or by spraying with methanolic-H 2 SO 4 solution and subsequent charring by heating at 100 C. 1 H and 13 C NMR were recorded at 300 and 75 MHz, respectively.Chemical shis are given in ppm downeld from internal TMS; J values are in Hz.The high resolution mass spectrometry (HRMS) was carried out using electrospray ionization mass spectrometry.The infrared spectra were recorded as Nujol mulls on KBr plates.Single-crystal X-ray data were collected on an Xcalibur Eos (Oxford) CCDdiffractometer.
General procedure for synthesis of sugar azides (6a-g).The compounds 6a-g were prepared from readily available carbohydrates (D-glucose, D-galactose, and D-ribose etc.) using standard protection and modication methodologies. 33eneral procedure for the synthesis of glycosyl epoxides (5km).A solution of orthogonally protected sugar 4k-m having one free hydroxyl group (1.0 mmol) in anhydrous DMF was cooled to 0 C and sodium hydride (2.0 equiv.) was added portion-wise.The reaction mixture was stirred at 0 C under argon atmosphere for 20 minutes.Epichlorohydrin (1.2 mmol) was added at 0 C and allowed to stir for 12 hour at room temperature.Upon completion of the reaction, the remaining sodium hydride was quenched with water; the solvent was removed under reduced pressure followed by extraction with ethyl acetate.The combined organic layer was washed with brine solution, dried over anhydrous Na 2 SO 4 , ltered and concentrated under reduced pressure to get the crude product.Purication using ash chromatography (ethyl acetate/n-hexane) afforded the desired glycosyl epoxide 5k-m.
General procedure for the synthesis of glycosyl azido alcohols 6k-m.A solution of glycosyl epoxide 5k-m in EtOH-H 2 O (1 : 1) was treated with NaN 3 and NH 4 Cl at 65 C for 8 h.Upon completion of the reaction, the solvent was removed under reduced pressure, and extracted with ethyl acetate and water.The organic layer was dried over anhydrous Na 2 SO 4 , ltered, and concentrated under vacuum, followed by ash chromatography (ethyl acetate/hexane) affording the desired glycosyl azido alcohol 6k-m in good yield.
General procedure for 7-O-propargyl noscapine 3. To a stirring solution of compound 2 (1.0 g, 2.5 mmol) in dry acetone (25 mL), propargyl bromide (0.291 mL, 3.2 mmol) and K 2 CO 3 (690 mg, 5.0 mmol) were added at room temperature.The reaction was tted with a water condenser and reuxed at 80 C under inert conditions for 12 h.Aer completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo, extracted with CH 2 Cl 2 (2 Â 50 mL) and washed with H 2 O (10 mL).The organic layer was separated and dried over anhydrous Na 2 SO 4 , and the solvent evaporated under reduced pressure followed by purication (ash column chromatography using gradient mixtures of n-hexane/ethyl acetate) to afford compound 3 as a yellowish solid (819 mg, yield 75% General procedure for the synthesis of noscapine glycoconjugates (8a-m) Noscapine glycoconjugate 8a.To a stirring solution of compound 3 (70 mg, 0.16 mmol) and azido-sugar 6a (71 mg, 0.19 mmol) in anhydrous CH 2 Cl 2 (10 mL), CuI (15 mg, 0.08 mmol) and DIPEA (0.027 ml, 0.16 mmol) were added and stirring was continued at room temperature for 14 h under argon atmosphere.Aer completion of the reaction (monitored by TLC), the reaction mixture was concentrated in vacuo to obtain a crude residue which was puried using silica gel (230-400 mesh) column chromatography (ethyl acetate/n-hexane) to afford the desired noscapine glycoconjugate 8a as a brown solid (124 mg, yield 95%); R f ¼ 0.

Fig. 2 Scheme 3 Table 1
Fig. 2 Molecular structure of 3. Thermal ellipsoids of C, N, and O are set at 40% probability.

a
Molar ratios: deoxy-azido sugar (1.0 equiv.),7-O-propargylated noscapine (1.0 equiv.),CuI (0.5 equiv.)and DIPEA (1.0 equiv.).b Noscapine glycoconjugates.c Isolated yield by column chromatography (SiO 2 ).(IR,MS, 1 H NMR and13  C NMR) and the purity (evidenced by HRMS) is in close agreement with calculated values.In the 1 H NMR spectrum, two doublets and one singlet of the aromatic protons resonated at d 6.95 (d, J ¼ 8.4 Hz), 6.07 (d, J ¼ 8.4 Hz) and d 6.30, along with a triazolyl proton singlet observed at d 8.25.The anomeric proton of the glucopyranose sugar resonated as a doublet at d 5.86 (J ¼ 9.6 Hz), while four other sugar protons, along with one noscapine and two oxymethylene protons, appeared at their usual chemical shi values, i.e. between d 5.60-5.22.Two singlets of methyl protons appeared at d 4.03, 3.84 and were established as the methoxy signals present at the aromatic rings of noscapine and another singlet at d 2.54 was established as the N-Me protons of the hetero carbon ring.The twelve protons of the acetyl moieties on the sugar scaffold were observed as four singlets having three protons each at d 2.10, 2.07, 2.04 and 1.85.A total of seven remaining protons of noscapine were assigned at d 5.93 (s, 2H), 4.40 (d, J ¼ 3.9 Hz, 1H), 2.33 (m, 2H), one merged with the acetyl protons and the last one with the N-methyl protons.One of the remaining sugar protons in compound 8a resonated at d 4.28 (dd, J ¼ 4.8 & 12.6 Hz) and the next one appeared as a multiplet at d 4.16, which conrms the structure.

Fig. 3
Fig. 3 Intramolecular CH/O and CH/p interactions.Weak interactions are represented by broken light green lines.Carbon atoms are colored brown, hydrogen atoms green, oxygen atoms red, and nitrogen atoms blue.

Fig. 4
Fig. 4 Intermolecular CH/O and CH/p interactions.Weak interactions are represented by broken light green lines.Carbon atoms are brown, hydrogen atoms green, oxygen atoms red, and nitrogen atoms blue.