Synthesis and click reaction of tubulin polymerization inhibitor 9-azido-α-noscapine

Article abstract of DOI:10.1007/s11164-016-2773-7

Abstract: An efficient protocol for the synthesis of tubulin polymerization inhibitor, 9-azido-α-noscapine 2h from 9-amino-α-noscapine 2g is developed using mild reaction conditions (t-butyl nitrite/trimethylsilyl azide in acetonitrile at room temperature). Operational simplicity, high product yield without formation of any side products are the advantages of this protocol. Further copper catalyzed click reactions of 9-azido-α-noscapine 2h with alkynes 6a–f resulted 9-triazolyl noscapinoids 7a–f resulted in excellent yields. Graphical Abstract: Developed an amicable protocol for the synthesis of 9-azido-α-noscapine from 9-amino-α-noscapineunder mild reaction conditions; was further derivatized to triazoles using click chemistry.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s11164-016-2773-7

Res Chem Intermed
DOI 10.1007/s11164-016-2773-7
Synthesis and click reaction of tubulin polymerization
inhibitor 9-azido-a-noscapine
Naresh Kumar Manchukonda1 Praveen Kumar Reddy Nagireddy1
•
•
Balasubramanian Sridhar2 Srinivas Kantevari1,3
•
Received: 8 August 2016 / Accepted: 8 October 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract An efficient protocol for the synthesis of tubulin polymerization inhibitor,
9-azido-a-noscapine 2h from 9-amino-a-noscapine 2g is developed using mild reaction
conditions (t-butyl nitrite/trimethylsilyl azide in acetonitrile at room temperature). Opera-
tional simplicity, high product yield without formation of any side products are the advan-
tages of this protocol. Further copper catalyzed click reactions of 9-azido-a-noscapine 2h
with alkynes 6a–f resulted 9-triazolyl noscapinoids 7a–f resulted in excellent yields.
Graphical Abstract Developed an amicable protocol for the synthesis of 9-azido-a-
noscapine from 9-amino-a-noscapineunder mild reaction conditions; was further
derivatized to triazoles using click chemistry.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-016-2773-
7) contains supplementary material, which is available to authorized users.
&
Srinivas Kantevari
kantevari@yahoo.com; kantevari@gmail.com
1
2
3
Organic Chemistry Division-II (C P C Division), CSIR-Indian Institute of Chemical
Technology, Hyderabad, Telangana 500007, India
Laboratory of X-ray Crystallography, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India
Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500007, India
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N. K. Manchukonda et al.
Keywords Noscapine Á Heterocycle Á Tubulin Á Inhibitor Á Click chemistry Á Azide Á
Alkaloid
Introduction
Natural products were and are playing vital roles in drug discovery and development
[1–3]. Several blockbuster and life saving drugs currently on the market are either
from natural sources or generated through manifestation of the structures of natural
products [4–6]. Alkaloids are one such class of natural products that contribute
significantly in identifying pharmaceutically relevant scaffolds [7–9]. Among
several natural alkaloids, a-noscapine 1, (also known as Narcotine, Anarcotine and
Nospen) with benzyl isoquinoline architecture attracted attention of several groups
worldwide [10–14]. It is isolated from plants of the Papaveraceae family at about
7 % during opium harvesting [15–17]. It has been commonly used for several
decades as an oral antitussive agent without any side effects [17]. With a favourable
toxicity profile, Zhou et al. [18] and Ye et al. [19] repurposed a-noscapine as a
tubulin-binding anticancer agent that arrested cancer cells in mitosis and induced
apoptosis. Unlike the other tubulin-targeting agents, a-noscapine binds at the
colchicine binding site and does not alter the steady state monomer/polymer ratio of
tubulin [18, 19]. In addition, a-noscapine has some other advantageous properties
[20] as lead molecule: (1) it has favourable pharmacokinetics (clearance in 6–10 h);
(2) it retains activity against the epothilone-resistant cell line (1A9/A8) and
paclitaxel-resistant cell lines (1A9/PTX10, 1A9/PTX22); and (3) it is free from
immunological and neurological toxicities [10–17].
To enhance anticancer activity of a-noscapine, further efforts were made to add
various functional substituents on the noscapine core [12]. For instance, 9-substi-
tuted noscapine analogues (9-bromo, 9-fluoro, 9-chloro, 9-iodo, 9-nitro, 9-azido,
9-amino and 9-aryl analogues (Fig. 1) [21–25] exhibited higher anticancer activity
than the parent a-noscapine without significant toxicity. Among them, 9-bromo-a-
noscapine 2a (EM011) has been recently introduced into phase I/II clinical trials
against non-Hodgkin’s lymphoma and chronic lymphocytic leukemia [26–28].
Similarly, 9-nitro-a-noscapine 2f was active against T cell lymphoma cells and
drug-resistant ovarian cancer [29]. Our group also synthesized 9-amino (2g, Fig. 1),
9-aryl (2e) and other a-noscapine derivatives, and they were found to possess
improved tubulin binding anticancer activity [30–34].
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Synthesis and click reaction of tubulin polymerization…
Fig. 1 Structures of natural a-Noscapine 1 and its potent bioactive 9-substituted analogs 2a–g
Recently, 9-azido-a-noscapine (2h) [(S)-3-((R)-9-azido-4-methoxy-6-methyl-
5,6,7,8-tetra hydro-[1, 3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofu-
ran-(3H)-one] was identified as a potential anticancer clinical agent without any
significant toxicity [35]. The IC50 value of 2h is 2.6 lM against human acute
lymphoblastic leukemia cells (CEM). Computational methods predicted that 2h
binds to tubulin at a site overlapping with the colchicine-binding site and possesses
better antitumor activity than the parent compound 1 [35, 36]. However, further
development of 2h as an anticancer lead molecule is hampered due to non-
availability of a suitable procedure for its preparation [12]. In view of the increased
utility of 9-azido-a-noscapine in anticancer evaluations, it is desirable to develop an
efficient method for the synthesis of 2h.
In our programme [30–34] on the synthesis of natural products and its analogues
for bio-evaluations, we herein report our efforts on development of an amicable
procedure for the preparation of 9-azido-a-noscapine 2h from natural a-noscapine.
Further, the azide 2h thus obtained reacted with alkyl/aryl alkynes to give respective
1,2,3-triazole analogs using copper catalyzed click chemistry.
Results and discussion
Previously, synthesis of 9-azido-a-noscapine 2h was reported via a two step route
involving aromatic bromination of a-noscapine using aqueous HBr/Br2–H2O
followed by azidation using NaN3 and NaI in DMF [25, 26]. To the best of our
knowledge, this is the only method available in the literature [25, 26] for the
synthesis of 2h. Taking it as a starting point, 9-bromo-a-noscapine 2a required was
prepared in excellent yield (90 %) from commercially available natural a-noscapine
1 using bromine water in 48 % aqueous HBr [33]. 9-Bromo-a-noscapine 2a thus
1
obtained was fully characterized by IR, H and 13C NMR and Mass (ESI and
HRMS) spectra data [33]. Following the sequencing, conversion of 2a with sodium
azide/sodium iodide was attempted in DMF heating at 80–85 °C for 15 h. Contrary
to our expectation, the reaction did not proceed in our hands and after a prolonged
reaction time (48 h) produced 9-azido-a-noscapine 2h only in a trace amount
(*1 %). Repeated reactions under modified conditions; changing the mole ratio of
sodium azide/sodium iodide, temperature, time and solvent did not give a fruitful
result.
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N. K. Manchukonda et al.
Further, we replaced sodium azide with trimethylsilyl azide, tosyl azide,
tributyltin azide, triethyl ammonium azide, tetrabutyl ammonium azide, but this did
not yield the desired product. Reaction of 2a with 2.0 equiv. of sodium azide and
heating at elevated temperatures (135–140 °C) for 4 h, we noticed a sluggish
reaction mixture containing several compounds (Scheme 1). Silica gel column
chromatography led to separation and identification of a major amount as
O-demethylated compound 3 (65 %) and minor amounts of 4 (5 %) and regenerated
a-noscapine 1 (4 %). Another product, Opianic acid 5, was identified as generated
though cleavage of sensitive C–C bond between two heterocyclic lobes, isoquino-
line and isobenzofuran-(3H)-one. Such sodium azide mediated regioselective
O-demethylation of a-noscapine at elevated temperature is consistent with the
literature [37, 38].
We next considered copper catalyzed Ullmann type conversion as an alternate
method for the preparation of 2h. Here, the reaction was carried out using CuI,
NaN3 and proline as an additive ligand [39, 40]. However this method also suffered
from drawbacks of a low reaction rate and did not yield the desired azide. At
elevated reaction temperatures (135–140 °C) 9-bromo-a-noscapine 2a reacted with
sodium azide (2.0 equiv), in the presence of CuI (2.0 equiv), and proline (4.0 equiv)
in DMSO for 3.0 h gave 9-amino-a-noscapine 2g in 62 % yield [33]. The protocol
did not give any O-demethylated compound 3 or 4 or 9-azido-a-noscapine 2h and
also did not affect the sensitive C–C bond between the two heterocyclic units. Here,
15 % of noscapine 1 was recovered.
A report from Anderson et al. [41] indicated that proline is not a suitable ligand
in copper catalyzed conversion of bromo aromatics to azides. The conversion is best
achieved using CuI/N,N0-dimethyl ethylene diamine. With this clue, CuI catalyzed
conversion of 9-bromo noscapine 2a to azide 2h was carried out with sodium azide
and N,N0-dimethylethylene diamine as a ligand in ethanol: water (7:3) at 100 °C.
However, the result was unchanged, to yield 9-amino noscapine 2g along with re-
Scheme 1 Attempted synthesis of 9-azido-a-noscapine 2h
123

Synthesis and click reaction of tubulin polymerization…
(I)
(II)
(III)
(IV)
Fig. 2 Time dependent positive ion ESI mass spectra (Top) of the CuI catalyzed reaction of 2a with
sodium azide. Panels I: 1 h, II: 3 h, III: 5 h and IV: 8 h; and schematic representation (Bottom) of
various intermediates identified in the ESI mass spectra. Peak representations are A: m/z: 414 (1 ? H)?;
B: m/z: 429 (2 g ? H)?; C: m/z: 455 (2 h ? H)?; D: m/z: 492 (2a ? H)?; E: m/z: 516 (2a ? Na)?; F: m/
z: 427 (2i ? H)?; G: m/z: 443 (2j ? H)?; H: m/z: 436 (1 ? Na)?; I: m/z: 492 (2 g ? Na)?
generated noscapine 1. Only trace amounts (*3 %) of 9-azidonospine 2h was
formed. In order to get mechanistic insight into this CuI catalyzed reaction, a time
dependentd ESI-mass spectral analysis was carried out by drawing the samples of
123

N. K. Manchukonda et al.
reaction mixture at 1, 3, 5 and 8 h time intervals. From the ESI- mass spectral
analysis (panels I–IV, Fig. 2) it is evident that formation of azide 2h [m/z:
455(M ? H)] is taking place within 1 h (Panel I, Fig. 2), along with de-brominated
product, noscapine 1 [m/z: 414 (M ? H)] and amine 2g [m/z: 429 (M ? H)]. As the
time progressed, formation and dissociation/reduction of azide was taking place
simultaneously through aryl nitrene [m/z: 427 (M ? H)] and aryl nitroso [m/z: 443
(M ? H)] intermediates. After 8 h, as evident from mass spectra (panel IV, Fig. 3),
only two significant compounds, 9-amino noscapine 2g (45 %) and de-brominated
product noscapine 1 (38 %) were isolated. The study clearly revealed that 9-azido
noscapine thus formed in the reaction mixture is simultaneously undergoing
dissociation and reduction to form 9-amino noscapine 2g and de-brominated
product, noscapine 1.
A literature search indicated that compared to alkyl azides, synthesis of aryl
azides relies on limited selection of transformations [42–46]. The most common one
is from aryl amine via diazonium salts [44]. This transformation might be
problematic due to sensitivity of the C–C bond between two heterocyclic units and
functional groups. The alternate Wong’s (TfN3) methodology [45] of converting
aryl amines to azides uses excess Triflic anhydride and is also likely to alter
noscapine architecture. Later methods of Das et al. (tert-butyl nitrite/excess NaN3)
[47] and Barral et al. (tert-butyl nitrite/TMSN3) [48] are attractive for the desired
conversion. Given the complexity in noscapine architecture, tert-butyl nitrite/
TMSN3 was envisaged as a choice for exploration in our study due to its
applicability under mild reaction conditions [49].
Fig. 3 ORTEP representation of 9-azido a-noscapine 2h with thermal displacement ellipsoids drawn at
the 30 % probability level and H atoms are represented by circles of arbitrary radii
123

Synthesis and click reaction of tubulin polymerization…
Having 9-amino-a-noscapine 2g in hand, we next reacted with tert-butyl nitrite/
TMSN3 (Scheme 2). Initially, 2g (1.0 equiv.) was reacted with tert-butyl nitrite (1.0
equiv.) TMSN3 (1.0 equiv.) stirring at 25 °C in acetonitrile for 24 h to give 46 % of
9-azido noscapine 2h. After a series of reactions, the optimum yield was obtained
reacting 2g with tert-butyl nitrite (2.0 equiv) and TMSN3 (2.0 equiv.) in acetonitrile
at 40 °C for 36 h to give azide 2h. The crude reaction mixture was purified over
silica gel column chromatography to give azide 2h in 74 % yield with[95 % HPLC
purity. Azide 2h was fully characterized by IR, 1H and 13C NMR and Mass (ESI and
HRMS) spectral data. Single crystal X-ray analysis of 2h unambiguously confirmed
the structure (Fig. 3).
Further, to enhance the scope of 9-azido-a-noscapine 2h and to develop potent
antiproliferative agents, we explored copper catalyzed click chemistry [50–52] and
reacted with various alkyl/aryl alkynes 6a–f (Scheme 3). For example, azide 2h
(1 mmol) was reacted with propagyl alcohol 6a in the presence of copper sulphate
(0.20 mmol) and sodium ascorbate (0.20 mmol) in tert-butanol and water (10 mL,
1:1, v/v) for 6 h to produce triazolyl noscapine 7a in a 70 % yield. Similarly, all
other alkynes 6b–f reacted with azide 2h to give 9-triazolyl noscapinoids 7b–f in
1
excellent yields. All the products 7a–f obtained were fully characterized by IR, H
and 13C NMR and mass (ESI and HRMS) spectral data.
Conclusion
In summary, we have developed an efficient and amicable protocol for the synthesis
of 9-azido-a-noscapine 2h from 9-amino-a-noscapine 2g in 74 % yield using a
relatively non-explosive reagent system, tert-butyl nitrite/TMSN3. In view of the
potential use of 9-azido-a-noscapine 2h as a tubulin binding anticancer agent, the
procedure developed here is highly useful for the preparation of required quantities
of 2h with excellent purity. The azide 2h was further reacted with various alkynes
6a–f utilizing copper catalyzed click chemistry to give 9-triazolyl noscapinoids 7a–f
in excellent yields. Antiproliferative evaluation and potential tubulin polymerization
inhibition of 9-azido-a-noscapine 2h and its triazole analogues 7a–f are in progress
and will be reported later as a separate study.
Scheme 2 Synthesis of 9-azido-nospaine 2h
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N. K. Manchukonda et al.
Scheme 3 Click synthesis of 9-traizolyl a-noscapinoids 7a–f. Reaction time, % yield of the product
Experimental
General remarks
Air-sensitive reagents were transferred by syringe or double-ended needle.
Evaporation of solvents was performed at reduced pressure on a Buchi rotary
evaporator. Melting points were determined on a Fischer-Johns melting point
123

Synthesis and click reaction of tubulin polymerization…
1
apparatus and are uncorrected. H and 13C NMR spectra of samples in CDCl3 and
DMSO-d6 were recorded on a Bruker UXNMR FT-300 MHz (Avance) spectrom-
eter and Varian FT-500 MHz (Inova). Chemical shifts reported are relative to an
internal standard TMS (d = 0.0). Mass spectra were recorded in ESI or EI
conditions at 70 eV on LC-MSD (Agilent technologies) spectrometers. All high-
resolution spectra were recorded on a QSTAR XL hybrid MS/MS system (Applied
Bio systems/MDS sciex, Foster City, USA), equipped with an ESI source (IICT,
Hyderabad). All the reactions were monitored by TLC (Precoated silica plates and
visualizing under UV light) using Merck 60 F-254 silica gel plates. Column
chromatography was performed on silica gel (60–120 mesh) supplied by Acme
Chemical Co., India. Analytical HPLC was performed on an 1260 infinity model of
Agilent technologies using a C18 column, gradient elution with acetonitrile: water.
Commercially available anhydrous solvents were used as such without further
purification. Noscapine, TMS-azide, and t-butyl nitrite were purchased from Sigma-
Aldrich and used as such without any further purification.
Synthesis of (S)-3-((R)-9-azido-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]-
dioxolo[4,5-g] isoquinolin-5-yl)-6,7-dimethoxyiso benzo furan-1(3H)-one
(2h) To a stirred solution of 9-amino-a-noscapine 2g (2.0 g, 4.66 mol) in dry
acetonitrile (15 mL), t-butyl nitrite (1.10 mL, 9.33 mol) and trimethylsilyl azide
(1.22 mL, 9.33 mol) were sequentially added at 0 °C under nitrogen. The reaction
mixture was brought to 40 °C and vigorously stirred for 36 h (monitored by TLC).
It was quenched with water (10 mL), extracted with dichloromethane (3 9 25 mL),
combined organic layers were washed with brine, dried over anhydrous Na2SO4 and
concentrated at reduced pressure. Crude azide thus obtained was purified over silica
gel column chromatography eluted with EtOAc : Hexane (40:60) to give pure azide
1
2h (1.56 g, 74 %). White solid, mp 116 °C; [a]2D5: -102.3(c = 1, CH2Cl2). H
NMR (300 MHz, CDCl3,): d 7.03(d, J = 8.2 Hz, 1H), 6.27(d, J = 8.0 Hz, 1H),
6.01 (d, J = 1.6 Hz, 2H), 5.51(d, J = 4.4 Hz, 1H), 4.31(d, J = 4.4 Hz, 1H), 4.09
(s, 3H), 3.97(s, 3H), 3.88(s, 3H), 2.69–2.61(m, 1H), 2.55–2.45(m, 4H),
2.40–2.32(m, 1H), 2.39–2.32(m, 1H). 13C NMR (75 MHz, CDCl3): d 167.9,
152.1, 147.6, 141.0, 140.9, 137.8, 134.6, 123.8, 119.6, 118.1, 118.0, 117.4, 113.8,
101.5, 81.3, 62.2, 60.6, 59.4, 56.6, 48.4, 45.4, 21.6. MS (ESI): m/z 455(M ? H)?,
427(M - N2)?. HRMS (ESI): Calcd for C22H23N4O7 (M ? H)?: 455.15613,
found: 455.15600.
General procedure for the synthesis of 9-triazolyl noscapinoids 7a–f
To a stirred solution of 9-azido-a-noscapine 2h (1.0 mmol), alkynes 6a–f
(1.5 mmol) in tert-butanol and water (1:1, v/v, 10 mL), copper sulphate pentahy-
drate (0.20 mmol) and sodium ascorbate (0.20 mmol) were added and stirred at
room temperature for an appropriate time. After completion of reaction (TLC), to
the reaction mixture was added water (5 mL), extracted with dichloromethane
(3 9 25 mL), combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated at reduced pressure. Crude residue thus
123

N. K. Manchukonda et al.
obtained was purified over silica gel column chromatography to give pure
9-triazolyl noscapinoids 7a–f.
(S)-3-((R)-9-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-4-methoxy-6-methyl-5,6,
7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-
1(3H)-one (7a) Colourless solid; yield: 70 %; mp 149 °C; [a]2D5: -103.3(c = 1,
CH2Cl2). IR (KBr): 3418, 3448, 2925, 2797, 1758, 1618, 1497, 1461, 1217, 1034,
969, 904, 821 cm-1 1H NMR (CDCl3, 300 MHz): d 7.71(s, 1H), 7.11(d,
.
J = 8.3 Hz, 1H), 6.51(d, J = 8.3 Hz, 1H), 6.01(d, J = 11.3 Hz, 2H), 5.50(d,
J = 4.5 Hz, 1H), 4.89(s, 2H),4.39(d, J = 4.5 Hz, 1H), 4.09(s, 3H), 4.06(s, 3H),
3.90(s, 3H), 2.76–2.69(m, 1H), 2.52(s, 3H), 2.42–2.32(m, 1H), 2.29–2.20(m,
1H),1.90–1.79(m, 1H). 13C NMR (CDCl3, 75 MHz): 168.0, 152.4, 147.7, 147.3,
145.4, 143.5, 141.5, 141.0, 134.2, 128.3, 124.1, 118.4, 118.2, 117.6, 102.1, 81.3,
62.3, 60.8, 59.5, 56.8, 56.5, 48.2, 45.3, 22.3. MS (ESI): m/z 511 (M ? H)?. HRMS
(ESI) Calcd for C25H27N4O8 (M ? H)?: 511.18234, found: 511.17816.
(S)-3-((R)-9-(4-cyclopropyl-1H-1,2,3-triazol-1-yl)-4-methoxy-6-methyl-5,6,7,8-te-
trahydro-[1,3] dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-
one (7b) Colourless solid; yield: 67 %; mp 174 °C; [a]2D5: -136.0 (c = 1,
CH2Cl2). IR (KBr): 3139, 2926, 2802, 1764, 1617, 1499, 1457, 1270, 1084, 1033,
966, 936, 816, 664, 436 cm-1. 1H NMR (300 MHz, CDCl3): d 7.41(s, 1H), 7.09 (d,
J = 8.3 Hz, 2H), 6.47(d, J = 8.3 Hz, 1H), 6.03(d, J = 1.3 Hz, 1H), 5.99(d,
J = 1.3, 1H), 5.49(d, J = 4.3 Hz, 1H), 4.38(d, J = 4.3 Hz, 1H), 4.10 (s, 3H),
4.07(s, 3H), 3.89(s, 3H), 2.74–2.64(m, 1H), 2.52(s, 3H), 2.39–2.28(m, 1H),
2.26–2.13(m, 1H), 1.89–1.75(m, 1H), 1.03–0.92(m, 4H). 13C NMR (75 MHz,
CDCl3): d 167.9, 152.3, 149.7, 147.6, 143.3, 141.3, 141.0, 134.1, 128.5, 122.0,
119.6, 118.4, 117.6, 112.5, 101.9, 81.4, 62.2, 60.8, 59.4, 56.7, 48.4, 45.5, 29.6, 22.5,
7.8, 6.6. MS (ESI): m/z 521 (M ? H)?. HRMS (ESI): Calcd for C27H29N4O7
(M ? H)?: 521.20308, found: 521.19946.
(S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-9-(4-(thiophen-3-yl)-1H-1,2,3-tri-
azol-1-yl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-
1(3H)-one (7c) Pale yellow solid; yield: 76 %; mp 111 °C; [a]2D5: -97.3(c = 1,
CH2Cl2). IR (KBr): 3422, 3103, 2934, 1757, 1620, 1497, 1457, 1268, 1033, 935,
1
788, 622 cm-1. H NMR (300 MHz, CDCl3): d 7.84(s, 1H), 7.76(d, J = 3.0 Hz,
1H), 7.51(dd, J = 1.5&5.2 Hz, 1H), 7.42(dd, J = 3.0&5.2 Hz, 1H), 7.13 (d,
J = 7.5 Hz, 1H), 6.54(d, J = 8.3 Hz, 1H), 6.06(d, J = 1.5, 1H), 6.01(d,
J = 1.5 Hz, 1H), 5.50(d, J = 4.5 Hz, 1H), 4.39(d, J = 4.5 Hz, 1H), 4.10(s, 3H),
4.08(s, 3H), 3.91(s,3H), 2.84–2.66(m, 1H), 2.54(s, 3H), 2.46–2.20(m, 2H),
2.01–1.81(m, 1H). 13C NMR (75 MHz, CDCl3): d 167.9, 152.4, 147.7, 143.6,
143.4, 141.5, 141.1, 134.1, 131.3, 128.4, 126.4, 125.7, 121.6, 121.4, 119.5, 118.5,
118.3, 117.6, 112.2, 102.0, 81.3, 62.2, 60.9, 59.4, 56.8, 48.2, 45.3, 22.4. MS (ESI):
m/z 563 (M ? H)?. HRMS (ESI): Calcd for C28H27N4O7(M ? H)?: 563.19950,
found: 563.15679.
(S)-6,7-dimethoxy-3-((R)-4-methoxy-9-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-
6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-
123

Synthesis and click reaction of tubulin polymerization…
one (7d) Colourless solid; yield: 80 %; mp 157 °C; [a]2D5: -115.1(c = 1, CH2Cl2).
IR (KBr): 2936, 2836, 1761, 1618, 1587, 1797, 1468, 1276, 1033, 781, 694 cm-1
.
1H NMR (500 MHz, CDCl3): d 7.94(s, 1H), 7.52(s, 1H), 7.42(d, J = 7.4 Hz, 1H),
7.35(t, J = 7.9 Hz, 1H), 7.12(d, J = 8.2 Hz, 1H), 6.92(d, J = 7.4 Hz, 1H), 6.53(d,
J = 8.0 Hz, 1H), 6.03(d, J = 19.6 Hz, 2H), 5.50(d, J = 4.2 Hz, 1H), 4.40(d,
J = 4.2 Hz, 1H), 4.10(s, 3H), 4.08(s, 3H), 3.91(s, 3H), 3.89(s, 3H), 2.78–2.69(m,
1H), 2.54(s, 3H), 2.42–2.25(m, 2H), 2.01–1.87(m, 1H). 13C NMR (75 MHz,
CDCl3): d 167.9,159.9, 152.3, 147.6, 147.2, 143.3, 141.5, 141.0, 134.1, 131.4,
129.8, 128.3, 122.0, 119.5, 118.5, 118.3, 118.1, 117.6, 114.3, 112.2, 110.8, 102.0,
81.3, 62.3, 60.8, 59.4, 56.7, 55.3, 48.3, 45.3, 22.5. MS (ESI) m/z 587(M ? H)?.
HRMS(ESI) Calcd for C31H31N4O8 (M ? H)?: 587.21364, found: 587.21049.
(S)-3-((R)-9-(4-(1-hydroxycyclopentyl)-1H-1,2,3-triazol-1-yl)-4-methoxy-6-
methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxy-
isobenzofuran-1(3H)-one (7e) Colourless solid; yield: 78 %; mp 144 °C;
[a]2D5: -147.2(c = 1, CH2Cl2). IR (KBr): 3510, 2927, 2804, 1715, 1500, 1458,
1
1275, 1033, 964, 902, 820 cm-1. H NMR (CDCl3, 300 MHz): d 7.64(s, 1H),
7.10(d, J = 8.3 Hz, 1H), 6.48(d, J = 8.3, 1H), 6.03(d, J = 1.1 Hz, 1H), 5.99(d,
J = 1.1 Hz, 1H), 5.51(d, J = 4.5 Hz, 1H), 4.40(d, J = 4.5 Hz, 1H), 4.09(s, 3H),
4.06(s, 3H), 3.89(s, 3H), 2.77–2.65(m, 1H), 2.53(s, 3H), 2.41–1.75(m, 11H). 13C
NMR (CDCl3, 75 MHz): d 167.9, 153.8, 152.3, 147.6, 143.3, 141.3, 140.9,
134.1, 128.4, 122.0, 119.5, 118.3, 118.2, 117.5, 112.3, 101.9, 81.3, 78.9, 62.2,
60.7, 59.4, 56.7, 48.4, 45.5, 41.2, 23.5, 22.6. MS (ESI): m/z 565 (M ? H)?.
HRMS (ESI) Calcd for C29H33N4O8 (M ? H)?: 565.22929, found: 565.22622.
(S)-3-((R)-9-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)-4-methoxy-6-methyl-
5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofu-
ran-1(3H)-one (7f) Colourless solid; yield: 62 %; mp 91 °C; [a]2D5: -77.3(c = 1,
CH2Cl2). IR (KBr): 3422, 2929, 2854, 1759, 1620, 1498, 1457, 1269, 1034, 969,
935, 818, 727 cm-1 1H NMR (CDCl3, 300 MHz) d 7.63(s, 1H), 7.10 (d,
.
J = 8.3 Hz, 1H), 6.50(d, J = 8.3 Hz, 1H), 6.01(d, J = 11.5 Hz, 2H), 5.52 (d,
J = 3.9 Hz, 1H), 4.41(d, J = 3.9 Hz, 1H), 4.09(s,3H), 4.05(s, 3H), 3.90 (s, 3H),
2.79–2.66(m, 1H), 2.53(s, 3H), 2.44–0.79(m, 13H). 13C NMR (75 MHz, CDCl3) d
167.9, 155.4, 152.4, 147.6, 143.4, 141.3, 141.0, 134.1, 128.3, 122.0, 118.4, 117.6,
112.4, 118.1, 119.5, 102.0, 81.3, 69.6, 62.2, 60.8, 59.4, 56.7, 48.3, 45.3, 38.0, 25.3,
22.5, 21.9. MS (ESI) m/z 579(M ? H)?. HRMS(ESI) Calcd for C30H35N4O8Na
(M ? H)?: 579.24494, found: 579.24246.
Single crystal X-ray analysis
X-ray data for the azide 2h were collected at room temperature using a Bruker
Smart Apex CCD diffractometer with graphite monochromated MoKa radiation
˚
(k = 0.71073 A) with the x-scan method. Preliminary lattice parameters and
orientation matrices were obtained from four sets of frames. Integration and scaling
of intensity data were accomplished using the SAINT program [53]. The structure
was solved by direct methods using SHELXS97 and refinement was carried out by
123

N. K. Manchukonda et al.
the full-matrix least-squares technique using SHELXL97 [54]. Anisotropic
displacement parameters were included for all non-hydrogen atoms.
Crystal data for 2h: C22H22N4O7, M = 454.44, colourless plate, 0.18 9 0.17 9
0.07 mm3, orthorhombic, space group P212121 (No. 19), a = 11.5800(8),
3
b = 12.2181(9), c = 14.9647(11) A, V = 2117.3(3) A , Z = 4, Dc = 1.426 g/
˚
˚
cm3, F000 = 952, CCD Area Detector, MoKa radiation, k = 0.71073 A,
˚
T = 294(2)K, 2hmax = 50.0°, 20173 reflections collected, 2121 unique
(Rint = 0.0193). Final GooF = 1.046, R1 = 0.0282, wR2 = 0.0765, R indices
based on 2021 reflections with I [ 2r(I) (refinement on F2), 302 parameters, 0
restraint, l = 0.108 mm-1. CCDC 944742 contains supplementary Crystallo-
graphic data for the structure.
Acknowledgments Authors thank the Department of Biotechnology, Ministry of Science and
Technology, India for financial support (BT/Indo-Aus/07/06/2013). NKM and PRN are thankful to
CSIR and UGC, respectively, for senior research fellowships.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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