Rationally designed molecules for resurgence of cyanide mitigated cytochrome c oxidase activity

Article abstract of DOI:10.1016/j.bioorg.2018.10.045

Cytochrome c oxidase (CcOX) containing binuclear heme a₃-Cu B centre (BNC) mechanises the process of electron transfer in the last phase of cellular respiration. The molecular modelling based structural analysis of CcOX – heme a₃-Cu B complex was performed and the disturbance to this complex under cyanide poisoning conditions was investigated. Taking into consideration the results of molecular docking studies, new chemical entities were developed for clipping cyanide from the enzyme and restoring its normal function. It was found that the molecules obtained by combining syringaldehyde, oxindole and chrysin moieties bearing propyl/butyl spacing groups occupy the BNC region and effectively remove cyanide bound to the enzyme. The binding constant of compound 2 with CN^– was 2.3 × 10⁵ M^?1 and its ED₅₀ for restoring the cyanide bound CcOX activity in 10 min was 16 μM. The compound interacted with CN^– over the pH range 5–10. The comparison of the loss of enzymatic activity in the presence of CN^– and resumption of enzymatic activity by compound 2 mediated removal of CN^– indicated the efficacy of the compound as antidote of cyanide.

Full text of DOI:10.1016/j.bioorg.2018.10.045

Accepted Manuscript
Rationally Designed Molecules for Resurgence of Cyanide Mitigated Cyto-
chrome c Oxidase Activity
Harpreet Kaur, Palwinder Singh
PII:
S0045-2068(18)30932-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.045
YBIOO 2579
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
25 August 2018
19 October 2018
22 October 2018
Please cite this article as: H. Kaur, P. Singh, Rationally Designed Molecules for Resurgence of Cyanide Mitigated
Cytochrome c Oxidase Activity, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.045
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Rationally Designed Molecules for Resurgence of Cyanide Mitigated
Cytochrome c Oxidase Activity
Harpreet Kaur, Palwinder Singh*
Department of Chemistry, UGC Sponsored Centre for Advanced Studies, Guru Nanak Dev
University, Amritsar-143005. India
e-mail: palwinder_singh_2000@yahoo.com
Abstract: Cytochrome c oxidase (CcOX) containing binuclear heme a3-Cu B centre (BNC)
mechanises the process of electron transfer in the last phase of cellular respiration. The
molecular modelling based structural analysis of CcOX – heme a3-Cu B complex was
performed and the disturbance to this complex under cyanide poisoning conditions was
investigated. Taking into consideration the results of molecular docking studies, new
chemical entities were developed for clipping cyanide from the enzyme and restoring its
normal function. It was found that the molecules obtained by combining syringaldehyde,
oxindole and chrysin moieties bearing propyl / butyl spacing groups occupy the BNC region
and effectively remove cyanide bound to the enzyme. The binding constant of compound 2
with CN- was 2.3 x 105 M-1 and its ED50 for restoring the cyanide bound CcOX activity in 10
min was 16 µM. The compound interacted with CN- over the pH range 5–10. The comparison
of the loss of enzymatic activity in the presence of CN- and resumption of enzymatic activity
by compound 2 mediated removal of CN- indicated the efficacy of the compound as antidote
of cyanide.
Keywords: cytochrome c oxidase; cyanide; molecular modelling; design of molecules
1

Introduction
Cytochrome c oxidase (CcOX) is the last enzyme in the respiratory chain that takes electrons
from cytochrome c (cyt c), oxidizing Fe2+ to Fe3+, and subsequently reduces molecular
oxygen to water and ultimately generates ATP. The enzyme is the life line of the living
organisms; either the shortage of oxygen or the blockage of oxygen binding site of the
enzyme leads to chemical asphyxiation of cells causing hypoxia that may prove lethal.
Mechanistically, the working of the enzyme involves the formation of Cyt c-CcOX complex
through the interaction between positively charged lysine residues on Cyt c with negatively
charged residue (Glu, Asp, Tyr) on CcOX subunit-II. Active site of CcOX contains binuclear
heme a3-Cu B center (BNC) with Fe-Cu separation of 4.58 Å. Cu is coordinated to H290,
H291 and H240 (Figure 1A). The imidazol ring of H240 is covalently linked to Y244 and it
restricts the movements of its phenol group towards Fe site. Cu ion acts as an electron storage
site and stabilizes the Fe2+–O2 adduct [1-11].
Figure 1. O2 Binding site of CcOX showing: (A) Cu bounded to H290, H291, H240 and
placed 4.58 Å from Fe and (B) cyanide ion (shown in grey-blue) is placed between Cu and Fe
interacting with both the metal ions.
2

The cyanide mediated disturbance of the above mentioned terminal step of the cellular
respiration owing to the involvement of about 1.5 million tonnes of cyanide annually in gold
mining, electroplating and metallurgical process, manufacturing of perspex, nylon, acrylic
plastics, nitriles, methionine and other amino acids in the animal feed industry [12-20] has far
reaching physiological consequences. Cyanide targets CcOX and its toxicity [21-23] arises
when this anion is sandwiched between Fe2+ and Cu+ of CcOX (Figure 1B) that results in the
breakage of electron chain. The interaction of cyanide with Cu+ displaces H290 and the new
complex competes with the molecular oxygen for binding to the catalytic site and leads to
oxidative stress and limits oxidative metabolism in the body. The moment cyanide level
exceeds 1.9 µM, it proves lethal [24-28]. As a remedial measure; in addition to the available
reports on cyanide sensors [29-36] and cyanide scavengers [37-52] including
hydroxocobalamin, dicyanocobalt (III) porphyrins, vitamin B12 analogues and hexahydrated
dichlorides of cobalt (II); compound 1 (Chart 1) was also identified to remove cyanide from
aqueous medium and human blood serum [53, 54]. Since chemical capturing of CN- and its
disposal as safe metabolite/s is desirable to get rid of cyanide poisoning, compound 1
removed cyanide through its more prevalent keto- form at the phenolic moiety and disposed it
of in the form of COOH. Supporting the experimental results, the molecular docking of the
keto-form of compound 1 in the oxygen/cyanide binding site of CcOX showed the approach
of keto- group to cyanide at a distance 4.44 Å (Figure 2A) whereas the phenolic group in its
enol- form (Figure 2B) stayed away from the CN-. The molecular dynamic studies also
indicated that the keto- tautomer of 1 interacted with CN- maximally through its keto- group
present at the phenyl ring.
In order to clip cyanide from CcOX more effectively, it was envisaged that the molecule
must possess more number of C=O and OH groups so that the interactions with CN- are
increased. Adopting this hypothesis, we combined chrysin and compound 1 through a spacer
3

group to design compounds 2–5 expecting that oxindole and chrysin moiety may encircle the
cyanide bound site of CcOX. The molecular docking of compounds 2–5 in the cyanide bound
pocket of CcOX were compared with that of compound 1 and chrysin. It was apparent from
the docking poses that the polar groups of the keto- as well as the enol- form of compound 2
(Figure 2C, 2D) were enveloping the BNC of the enzyme complex and hence it may prove as
an efficient scavenger of CN- in comparison to its precursors- compound 1 (Figure 2A) and
chrysin (Figure S1). Since the non-toxicity of compound 1 has already been checked, its
association with another biologically acceptable and cyanide captivating moiety chrysin may
provide a new chemical entity useful as antidote of cyanide poisoning. The docking poses
and interactions of compound 3 in the active site pocket of the enzyme (Figure S2) were also
similar to those shown by compound 2. However, with the increase in the length of the spacer
group between chrysin and oxindole moieties, the polar groups of the resulting compounds 4
and 5 explicitly stay away from the cyanide bound BNC of the enzyme (Figure S3, S4).
Chart 1. Cyanide scavengers reported recently (1) and newly designed molecules 2-5.
4

Figure 2. Compound 1 (A) keto- form and (B) enol- form docked in the oxygen binding
pocket of CcOX (PDB ID 3AG4). Magenta lines show distance in Å of chrysin from cyanide
(CYN520) and heme 516. Compound 2 (C) enol- form and (D) keto- form docked in CcOX.
Distances of the polar groups of the molecule from cyanide and heme are represented in
dotted lines. Docking of both E- and Z- isomers was tried and the molecule takes same
geometry in the binding site of the enzyme.
Results and Discussion
Chemistry. The synthesis of the desired compounds was accomplished as per the protocol of
Scheme 1. Compound 6, obtained by the methylation of syringaldehyde, was made to react
o
with oxindole by heating at 145 C and thereby compound 7 was obtained (Scheme 1).
Compound 7 was procured as E/Z isomers in the ratio 3:1. Compound 7 was treated with
equivalent amount of 1,3-dibromopropane/1,4-dibromobutane/1,5-dibromopentane/1,6-
dibromohexane in the presence of NaH in dimethylformamide and products 8–11 were
5

procured. Reaction of compounds 8–11 with chrysin in the presence of K2CO3 in
dimethylformamide gave respective compounds 14–17. Selective demethylation of
compound 14 – 17 by using anhydrous AlCl3 in dry dichloromethane led to the formation of
6

desired compounds 2 – 5 (Scheme 1). Compounds 2 – 5 were obtained as inseparable mixture
of E- and Z- isomers in the ratio 3:1 (1H NMR spectrum). Compounds 12 and 13 were
synthesized by the selective demethylation of 8 and 9 and these two compounds were
included for making comparative studies. Methylation at the OH group of chrysin unit of
compound 14 provided compound 18.
Removal of cyanide from CcOX
Before proceeding to the enzyme immunoassays for the removal of CcOX bound cyanide,
the selective interaction of compounds 2 and 3 with cyanide was ascertained. The relative
change in the absorbance at 368 nm and 529 nm of the UV-vis spectra of solutions (10×10-6
M, DMSO:H2O (1:9)) of compound 1, 2, 3, 5 and 12 with equivalent amount of cyanide
indicated that compounds 2 and 3 were more responsive to cyanide (Figure 3A, Figure S105).
It seems that the length of the spacer group as in compound 5 may affect the conformation of
the molecule and hence its interaction with CN-. Addition of CN- to 5 x 10-5 M solution of
chrysin in DMSO:H2O (1:9) exhibited changes in the UV-vis absorbance at 315 and 368 nm
only. Both compounds 2 and 3 displayed selective binding with cyanide (Figure 3B).
Stochiometry 1:2 and Ka 2.3x105 M-1 was observed for 2 and CN- (supporting information)
Figure 3. (A) Change in the absorbance at 368 and 529 nm of compounds 1, 2, 3, 5 and 12 in
the presence of CN- (315 and 368 nm for chrysin). (B) Change in the absorbance of
compound 2 at 529 nm in the presence of different anions showing selectivity and
competitive binding with CN-. (C) Response of compound 2 (change in UV-vis absorbance)
to CN- at different pH.
7

and this compound interacted with CN- over pH range 5 – 10 though the effect was more
pronounced at pH 9 – 10 (Figure 3C).
Cyt c-Fe2+ was oxidized to Fe3+ in the presence of CcOX and hence the absorbance
intensity at 550 nm was reduced (Figure 4A). Sharp oxidative activity of CcOX attaining the
saturation stage within 6 min was observed (Figure 4B). Equally fast was the diminishing in
CcOX activity in the presence of cyanide and it gets lost in 8-9 min (Figure 4C, 4D).
Apparently, the blockage of CcOX by cyanide is a fast process and hence need to have
immediate and efficient remedial measures in case of cyanide poisoning. The stepwise
Figure 4. Working of the compound with cytochrome c oxidase: (A) UV-visible absorbance
of 5.5 µM cyt c in assay buffer was monitored at wavelength 550 nm on addition of CcOX
(50 µL, diluted in enzyme buffer). Decrease in absorbance was due to oxidation of Fe2+ to
Fe3+ of cyt c by CcOX. (B) Activity of CcOX as a function of time. (C) Change in
absorbance at 550 nm of cyt c on addition of CcOX solution along with 0 – 3 μM CN-
showing decrease in enzymatic activity of CcOX in the presence of cyanide. (D) decrease in
the activity of CcOX as a function of time. (E) Absorbance intensity of solution containing
cyt c, Ccox, CN- was decreased on addition of compound 2 (1 µM to 25 µM) indicating
removal of cyanide from CcOX by the compound. (F) Revival of CcOX activity by
compound 1, 2, 5 and sodium pyruvate as a function of time.
8

addition of compound 2 (1 µM to 25 µM) to the above solution of cyt c – CcOX – CN-
decreased the absorbance intensity at 550 nm (Figure 4E). Apparently, 25 µM of compound 2
was sufficient to remove 3 µM cyanide from CcOX and the later oxidized cyt c-Fe2+ to Fe3+
resulting in the decrease in absorbance intensity at 550 nm. Interestingly, the resurgence of
CcOX activity after the removal of CN- by compound 2 was much fast as compared to that of
compound 1, 5 and the standard sodium pyruvate [55] (Figure 4F). The ED50 of compound 2
for recovering cyanide affected CcOX activity was 16 µM that was better than that of
compound 1 and 5 (Table 1, Figure S108). Therefore, as per the design of the molecules,
compound 2 seems an effective scavenger of CN- capable to remove the anion from CcOX.
In parallel to the results of molecular docking studies, compound 5 was less effective in the
removal of CN- from CcOX (Figure 4F).
Table 1. ED50 of compounds 1, 2 and 5 for revival of CcOX activity.
compound
ED50 (µM)
30±3
1
2
5
16±2
27±3
Removal of cyanide from the blood serum. The results of removal of cyanide from CcOX
by compound 2 were supplemented by a physical experiment. Proteinaceous blood sample
was diluted in distilled water - acetone and taken in two vials marked i and ii (Figure 5).
Equimolar solution of cyanide in acetone-water was added to the two vials. Compound 2 was
added to vial (ii) and the solution turned red. The solutions of vial (i) and vial (ii) were
extracted with ethyl acetate. The aqueous part obtained from the two solutions was treated
with compound 2. Addition of compound 2 to the aqueous part of vial (i) turned it red
whereas compound 2 did not affect aqueous solution of vial (ii) indicating that all the cyanide
was bound to the compound and extracted with ethyl acetate. These observations clearly
9

indicated that compound 2 possesses the ability to capture cyanide present in the blood
serum.
Figure 5. Schematic reperentation of the experiment showing extraction of cyanide from the
human blood with the help of compound 2.
1H NMR titration experiment for confirming the probable mode of action of the
compound. In consonance with the design, the part of the molecule 2 that is responsible for
the interaction with CN- was confirmed with the help of 1H NMR spectra of the solution of
compound and cyanide. The 1H resonance assignments of the protons of compound 2 (keto- /
enol- form) are depicted in Fig. 6A; the OH signals of H-19 and H-29 were assigned at δ 5.86
and 12.69, respectively (Figure 6A). The incremental addition of TBACN (0.5 equiv at each
1
step) to the CDCl3 solution of compound 2 was made and H NMR spectra were recorded
after each addition. The NMR signal corresponding to H-19 of compound 2 was finished
when 1 equiv of cyanide was added (Figure 6B). The signal due to H-29 was disappeared
after the addition of 3.5 equiv of cyanide. Therefore, the OH groups of compound 2 were
probably responsible for capturing CN-. Apparently, under basic pH conditions, more of the
keto- form may get formed and capturing of CN- is more effective. The participation of OH
groups in capturing cyanide was also supported by the fact that compound 18, not having free
OH group, was unresponsive to CN-. The recording of 1H NMR in CDCl3 by using TBACN
also ruled out the possibility of deprotonation at the OH groups under the influence of
hydroxide ion generated from NaCN in water.
10

A
B
Figure 5. (A) 1H NMR spectrum of compound 2. (B) Parts of 1H NMR spectra of compound
2 (8.0 × 10-3 M, 600 µL CDCl3) after incremental addition of TBACN (0.5 equiv CN- of 4 ×
10-1 M in 100 µL CHCl3). Peak at δ 5.89-5.99 disappear when 1 equiv CN- was added
whereas peak at δ 12.70-12.71 disappear with 3.5 equiv CN-.
Conclusions
The molecular modelling based scrutiny of the catalytic mode of the enzyme paved the
way for the rational design of new chemical entities. The modification of one of the previous
molecules was made on the basis of the results of molecular docking studies and compound 2
11

was found to be highly efficacious in the removal of CN- bound to CcOX. The ED50 of
compound 2 for reversal of CcOX activity was 16 µM and as per its design, it exhibited 1:2
stoichiometry with CN-. This compound was not toxic to the normal cells (NCI results not
shown here) and it restored the oxidizing activity of cyanide bound CcOX within 10 min.
Further studies on compound 2 by using animal models are underway.
Experimental Section
1H and 13C NMR spectra were recorded on JEOL 400 MHz and Bruker 500 MHz NMR
spectrometer, respectively using CDCl3 as solvent. Chemical shifts are given in ppm with
TMS as internal reference. Mass spectra were recorded on Bruker micrOTOF-Q II Mass
spectrometer. Reactions were monitored by thin layer chromatography (TLC) on glass plates
coated with silica gel GF-254. Column chromatography was performed with 60-120 mesh
silica. IR and UV-vis spectral data were recorded on FTIR Agilent CARY 630 and BIOTEK
Synergy H1 Hybrid Reader instruments, respectively.
Synthesis of compound 2
To the solution of compound 10 (100 mg, 0.16 mmol) in dry dichloromethane was added
anhydrous AlCl3 (34 mg, 0.26 mmol) under nitrogen atmosphere and the reaction mixture
was stirred at room temperature for 2h. The reaction was quenched with cold water and
extracted with DCM (4 × 25 mL). The organic layer was washed with brine, dried over
Na2SO4 and evaporated under vacuum. The crude product was purified by column
chromatography using ethyl acetate/hexane (7:3) as eluent to procure a mixture of E- and Z-
o
isomers in the ration 3:1 (1H NMR spectrum). Yellow solid (50%), mp 175 C, IR: ῡ (cm-1):
3391 (OH), 1654 (C=O), δH (500 MHz, CDCl3): 2.25-2.29 (m, 4H, CH2(major, minor)), 3.90 (s,
6H, OCH3(major)), 3.95 (s, 6H, OCH3(minor)), 4.01-4.05 (m, 4H, CH2(major, minor)), 4.11-4.13 (m,
4H, CH2(major, minor)), 5.89 (s, 1H, OH(major)), 5.99 (s, 1H, OH(minor)), 6.32 (d, J = 2.26 Hz, 1H,
CH(minor)), 6.34 (d, J = 2.26 Hz, 1H, CH(major)), 6.43 (d, J = 2.26 Hz, 1H, CH(minor)), 6.49 (d, J
12

= 2.26 Hz, 1H, CH(major)), 6.64 (s, 1H, CH(minor)), 6.66 (s, 1H, CH(major)), 6.86 (d, J = 7.97 Hz,
1H, ArH(minor)), 6.89-6.92 (m, 1H, ArH(major)), 6.95 (s, 2H, ArH(major)), 7.05 (t, J = 7.41 Hz,
1H, ArH(minor)), 7.22 (t, J = 7.41 Hz, 2H, ArH(major, minor)), 7.43 (s, 1H, bridged H(minor)), 7.49-
7.56 (m, 1H, ArH), 7.77 (s, 1H, bridged H(major)), 7.84 (d, J = 2.64 Hz, 1H, CH(minor)), 7.86 (s,
2H, CH(major)), 7.88 (d, J = 7.12 Hz, 1H, CH(major)), 12.70 (s, 1H, OH(minor)), 12.71 (s, 1H,
OH(major)). δC (normal/DEPT-135; CDCl3): 27.43 (CH2), 36.86 (CH2), 56.42
(OCH3(major)),56.45 (OCH3(minor)), 65.94 (CH2(major)), 66.03 (CH2(minor)), 93.06 (CH(major), 93.11
(CH(minor)), 98.67 (CH(major)), 98.70 (CH(minor)), 105.84 (CH(minor)), 105.89 (CH(major)), 105.89
(CH), 106.78 (CH), 107.81 (CH), 108.35 (CH(major)), 109.99 (CH(minor)), 118.54 (CH), 121.49
(CH), 121.61 (CH), 122.74 (CH), 123.41 (CH), 125.77 (C), 125.91 (C), 126.29 (C), 128.23
(CH),129.09 (CH), 129.55 (CH), 131.82 (C), 131.86 (C), 136.56 (CH), 137.85 (CH), 143.35
(C), 147.06(C), 157.71 (C), 157.78 (C), 162.15 (C), 164.65 (C), 165. 70 (C), 166.51 (C=O),
168.83 (C), 182.43 (C=O(minor)), 182.49 (C=O(major)). HRMS (ESI) m/z for C35H29O8N
[M+H]+ calcd 592.1965, found 592.2076.
Synthesis of compound 3
Anhydrous AlCl3 (35 mg, 0.26 mmol) was added to the solution of compound 11 (100 mg,
0.16 mmol) in dry dichloromethane under nitrogen atmosphere and the reaction mixture was
stirred at room temperature for 2h. The reaction was quenched with cold water and extracted
with DCM (4 × 25 mL). The organic layer was washed with brine, dried over Na2SO4 and
evaporated under vacuum. The crude product was purified by chromatography using ethyl
acetate/hexane (7:3) as eluent. Yellow solid (60%), mp 161 oC, IR: ῡ (cm-1): 3421 (OH), 1654
(C=O), δH (500 MHz, CDCl3): 1.94 (m, 6H, CH2(major)), 3.90 (s, 12H, OCH3(major, minor)), 4.00
(m, 2H, CH2(major)), 4.10 (m, 8H, CH2(minor)), 5.86 (s, 1H, OH(major)), 5.96 (s, 1H, OH(minor)),
6.30 (s, 1H, CH(minor)), 6.34 (s, 1H, CH(major)), 6.45 (s, 1H, CH(minor)), 6.48 (s, 1H, CH(major)),
6.85 (d, J = 7.37 Hz, 1H, ArH(minor)), 6.89 (d, J = 3.15 Hz, 1H, ArH(major)), 6.91 (t, J = 3.09
13

Hz, 1H, ArH(major)), 6.94 (s, 2H, ArH(major)), 7.06 (t, J = 7.90 Hz, 1H, ArH(minor)), 7.42 (s, 1H,
bridged H(minor)), 7.51-7.56 (m, 5H, ArH), 7.77 (s, 1H, bridged H(major)), 7.84 (d, J=7.97 Hz,
2H, CH(major, minor)), 7.87 (d, J=7.97 Hz, 2H, CH(minor)), 7.92 (s, 1H, CH(minor)), 12.68 (s, 1H,
OH(minor)), 12.69 (s, 1H, OH(major)). δC (normal/DEPT-135; CDCl3): 24.14 (CH2), 26.33 (CH2),
39.32 (CH2(minor)), 39.47 (CH2(major)), 56.44 (OCH3(major)), 56.51(OCH3(minor)), 67.85
(CH2(major)), 67.92 (CH2(minor)), 93.05 (CH), 98.66 (CH(minor)), 98.68 (CH(major)), 105.73,
105.87, 106.76 (CH), 107.99 (CH), 108.50 (CH), 110.04 (CH), 118.53 (CH), 121.55 (CH),
121.67 (CH), 122.75 (CH), 123.50 (CH), 125.80 (C), 125.95 (C), 126.29 (C), 128.15 (CH),
129.08 (CH), 129.47 (CH), 131.34 (CH), 137.82 (CH), 136.52 (C), 137.82 (CH), 138.03
(CH), 143.32 (C), 146.60 (C), 147.04 (C), 157.81 (C), 162.14 (C(minor)), 163.95 (C(major)),
164.92 (C), 166.37 (C=O(minor)), 168.78 (C=O(major)), 182.46 (C=O). HRMS (ESI) m/z for
C36H31O8N [M+H]+ calcd 606.2122, found 606.2391.
Synthesis of compound 4
To the solution of compound 16 (100 mg, 0.16 mmol) in dry dichloromethane (DCM),
anhydrous AlCl3 (34 mg, 0.26 mmol) was added under nitrogen atmosphere and the reaction
mixture was stirred at room temperature for 2h. The reaction was quenched with cold water
and extracted with DCM (4 × 25 mL). The organic layer was washed with brine, dried over
Na2SO4 and evaporated under vacuum. The crude product was purified by column
chromatography using ethyl acetate/hexane (7:3) as eluent to procure a mixture of E- and Z-
o
isomers in the ratio 3:1. Yellow solid (30%), mp 180 C, δH (500 MHz, CDCl3+ DMSO-d6):
1.73-1.82 (m, 4H, CH2(major, minot)), 1.90-2.00 (m, 4H, CH2(major, minor)), 2.58-2.65 (m, 4H,
CH2(major, minor)), 3.88 (s, 6H, OCH3(major)), 3.96 (s, 6H, OCH3 (minor)), 4.07 (m, 4H, CH2(major,
minor)), 4.81-5.15 (m, 4H, CH2(major, minor)), 5.27-5.33 (br, 1H, OH), 6.32 (s, 1H, CH), 6.47 (s,
1H, CH), 6.63 (s, 1H, CH), 6.92-6.95 (m, 2H, ArH), 7.06-7.11 (m, 1H, ArH), 7.26-7.28 (m,
1H, ArH), 7.50 (s, 1H, bridged H), 7.74 (m, 1H, ArH), 7.91-8.00 (m, 5H, ArH), 12.69 (s, 1H,
14

OH). δC (normal/DEPT-135; CDCl3): 22.38 (CH2), 23.16 (CH2), 26.67 (CH2), 27.10 (CH2),
28.36 (CH2), 31.31 (CH2), 32.21 (CH2), 56.29 (OCH3(major)), 56.33 (OCH3(minor)), 68.22
(CH2(minor)), 68.50 (CH2(major)), 92.99 (CH(major), 94.32 (CH(minor), 98.51 (CH(major)), 98.69
(CH(minor)), 105.51 (C), 105.68 (CH(minor)), 106.80 (CH(major)), 107.14 (CH), 107.30 (CH),
108.34 (CH), 109.91 (CH), 110.50 (CH), 118.02 (CH), 121.26 (CH), 121.47 (CH), 122.45
(CH), 126.25 (CH), 126.44 (C), 129.07 (CH), 129.26 (CH), 131.18 (C), 131.69 (C), 137.85
(CH), 147.38 (C), 157.77 (C), 161.92(C), 162.44 (C), 163.61 (C), 164.45 (C), 167.60 (C=O),
182.33 (C=O).
Synthesis of compound 5
To the solution of compound 17 (100 mg, 0.16 mmol) in dry dichloromethane was added
anhydrous AlCl3 (34 mg, 0.26 mmol) under nitrogen atmosphere and the reaction mixture
was stirred at room temperature for 2h. The reaction was quenched with cold water and
extracted with DCM (4 × 25 mL). The organic layer was washed with brine, dried over
Na2SO4 and evaporated under vacuum. The crude product was purified by column
chromatography using ethyl acetate/hexane (7:3) as eluent to procure a mixture of E- and Z-
isomers in the ratio 3:1. Yellow solid (30%), δH (500 MHz, CDCl3+ DMSO-d6): 1.14-1.29
(m, 8H, CH2(major, minor)), 1.42-1.49 (m, 4H, CH2(major, minor)), 1.67-1.82 (m, 4H, CH2(major, minor)),
3.88-4.03 (s, 6H, OCH3(major, minor)), 4.68-4.82 (m, 4H, CH2(major, minor)), 6.33 (s, 1H, CH(minor)),
6.47 (s, 1H, CH(minor)), 6.63 (s, 1H, CH(minor)), 6.65 (s, 1H, CH(major)), 6.93-6.97 (m, 2H, ArH),
7.11 (m, 1H, ArH), 7.26 (m, 1H, ArH), 7.38-7.39 (m, 1H, ArH), 7.53 (m, 4H, ArH), 7.65-
7.72 (m, 1H, ArH), 7.87-7.89 (m, 3H, ArH), 10.18 (OH), 12.69 (OH). δC (normal/DEPT-135;
CDCl3): 25.57 (CH2), 25.70 (CH2), 26.43 (CH2), 28.66 (CH2), 28.53 (CH2), 32.24 (CH2),
32.32 (CH2), 34.84 (CH2), 35.31 (CH2), 56.38 (OCH3(major)), 68.47 (CH2(minor)), 70.37
(CH2(major)), 92.97 (CH(major), 94.28 (CH(minor)), 98.60 (CH(major)), 98.62 (CH(minor)), 104.74 (C),
105.47 (CH(minor)), 105.62 (CH(major)), 107.03 (CH), 107.15 (CH), 110.18 (CH), 120.18 (CH),
15

120.97 (CH), 123.41 (CH), 125.66 (C), 126.16 (C), 129.26 (CH), 129.60 (CH), 131.33 (C),
131.77 (C), 137.55 (CH), 147.38 (C), 157.89 (C), 161.89 (C), 162.02 (C), 163.61 (C), 164.45
(C), 167.60 (C=O), 182.27 (C=O). HRMS (ESI) m/z for C38H35O8N [M+H]+ calcd 634.2435,
found 634.2852.
Synthesis of 3,4,5-trimethoxybenzaldehyde (6)
To the stirred solution of syringaldehyde (4 g, 21.9 mmol) in DMF (50 mL); K2CO3 (4.54 g,
32.92 mmol), CH3I (3.73 g, 26.34 mmol) and KI (catalytic amount) were added. The reaction
was allowed to stir overnight at room temperature. After the completion of reaction, it was
quenched by adding water and extracted with ethyl acetate. The organic layer was separated,
dried over Na2SO4, concentrated under vacuum to procure pure product 6, creamish white
o
solid (90%), mp 75-76 C, δH (400 MHz; CDCl3): 3.94 (s, 9H, OCH3), 7.13 (s, 2H, ArH),
9.87 (s, 1H, CHO); δC (normal/DEPT-135; CDCl3): 56.26 (OCH3), 61.01 (OCH3), 106.66
(CH), 131.64 (C), 143.49 (C), 153.57 (C), 191.10 (C=O). HRMS (ESI) m/z for C10H12O4
[M+H]+ calcd 197.0808, found 197.0727.
Synthesis of (E,Z)-3-(3,4,5-trimethoxybenzylidene)indolin-2-one (7)
3,4,5-Trimethoxybenzaldehyde (2 g, 10.20 mmol) and oxindole (1.35 g, 13.58 mmol) were
heated at 145 oC for 1h. The reaction mass was purified by column chromatography to obtain
compound 7 as mixture of E- and Z- isomers (3:1, 1H NMR), yellow solid (70%), mp 156 oC.
δH (500 MHz, CDCl3): 3.90 (s, 6H, OCH3(major)), 3.96 (s, 3H, OCH3(major)), 3.96 (s, 3H,
OCH3(minor)), 3.99 (s, 6H, OCH3(minor)), 6.89 (d, J = 7.92 Hz, 1H, ArH(minor)), 6.91 (d, J = 2.65
Hz, 1H, ArH(major)), 6.94 (t, J = 2.38 Hz, 1H, ArH(major)), 6.95 (s, 2H, ArH(major)), 7.06 (t, J =
7.90 Hz, 1H, ArH(minor)), 7.25 (t, J = 7.38 Hz, 2H, ArH(major, minor)), 7.50 (s, 1H, bridged
H(minor)), 7.54 (d, J = 7.60 Hz, 1H, ArH(minor)), 7.78 (s, 1H, bridged H(major)), 7.81 (d, J = 7.97
Hz, 1H, CH(major)), 7.84 (s, 2H, CH(minor)), 8.13 (br, 1H, NH(minor)), 8.26 (br, 1H, NH(major)). δC
(normal/DEPT-135; CDCl3): 56.27 (OCH3), 61.05 (OCH3), 106.87 (CH), 109.85 (CH),
16

110.16 (CH), 119.05 (CH), 121.72 (CH), 121.81 (CH), 123.18 (CH), 126.71 (C), 128.70
(CH), 129.82 (CH), 130.5 (C), 137.62 (CH), 139.57 (C), 141.53 (C), 152.79 (C), 153.31 (C),
169.52 (C=O). HRMS (ESI) m/z for C18H17O4N [M+H]+ calcd 312.1230, found 312.1182.
Synthesis of compound 8
A mixture of (E,Z)-3-(3,4,5-trimethoxybenzylidene) indolin-2-one (500 mg, 1.6 mmol) was
treated with 1,3-dibromopropane (486.6 mg, 2.4 mmol) in the presence of NaH (96.4 mg, 2.4
o
mmol) in dimethylformamide at 60 C for 2h. The crude product was purified by column
chromatography using ethyl acetate/hexane (1:9) as eluent. Yellow thick oil (80%), δH (400
MHz, CDCl3): 2.26-2.33 (m, 2H, CH2), 3.45-3.49 (m, 2H, CH2), 3.82-3.87 (m, 2H, CH2),
3.93 (s, 4H, OCH3), 3.97 (s, 5H, OCH3), 6.94 (d, J = 7.82 Hz, 1H, ArH), 7.06 (t, J = 7.53 Hz,
1H, ArH), 7.27-7.31 (m, 1H, ArH), 7.46 (s, 1H, bridged H), 7.53 (d, J = 6.99 Hz, 1H, ArH),
7.82 (s, 2H, ArH). δC (normal/DEPT-135; CDCl3): 30.53 (CH2(minor)), 30.68 (CH2(major)), 30.91
(CH2(minor)), 38.46 (CH2(major)), 38.54 (CH2(minor)), 56.22 (OCH3(minor)), 56.30(OCH3(major)),
60.98 (OCH3(major)), 61.00 (OCH3(minor)), 106.61 (CH), 108.00 (CH(major)), 108.47 (CH(minor)),
109.81 (CH(minor)), 109.90 (CH(major)), 118.82 (CH), 121.73 (CH), 124.64 (C), 124.71 (C),
126.21 (C), 128.68 (CH), 129.82 (CH), 137.57 (CH(minor)), 137.64 (CH(major)), 140.51 (C),
141.13 (C), 152.69 (C), 153.24 (C), 166.18 (C=O), 168.61 (C=O).
Synthesis of compound 9
The compound 7 (500 mg, 1.6 mmol) was treated with 1,4-dibromobutane (520.5 mg, 2.4
o
mmol) in the presence of NaH (96.4 mg, 2.4 mmol) in dimethylformamide heated at 60 C
for 2h. The crude product was purified by column chromatography using ethyl acetate/hexane
(1:9) as eluent. Yellow thick oil (80%), δH (500 MHz, CDCl3): 1.91-2.00 (m, 6H, CH2(major)),
3.47-3.51 (m, 2H, CH2(major)), 3.84-3.87 (m, 8H, CH2(minor)), 3.89 (s, 6H, OCH3(minor)), 3.95 (s,
3H, OCH3(major)), 3.95 (s, 3H, OCH3(minor)), 3.99 (s, 6H, OCH3(major)), 6.86 (d, J = 7.82 Hz, 1H,
ArH(major)), 6.89 (d, J = 8.01 Hz, 1H, ArH(minor)), 6.92 (s, 1H, ArH(minor)), 6.927-6.95 (t, J =
17

7.63 Hz, 1H, ArH(minor)), 7.06-7.09 (t, J = 7.63 Hz, 1H, ArH(major)), 7.29-7.31 (t, J = 7.42 Hz,
2H, ArH), 7.47 (s, 1H, bridged H(major)), 7.55 (d, J = 7.32 Hz, 1H, ArH(major)), 7.79 (s, 1H,
bridged H(minor)), 7.81 (d, J = 7.75 Hz, 1H, ArH(minor)), 7.84 (s, 2H, ArH(major)). δC
(normal/DEPT-135; CDCl3): 26.16 (CH2(minor)), 26.23 (CH2(major)), 29.79 (CH2(minor)), 29.87
(CH2(major)), 33.09 (CH2), 38.80 (CH2(minor)), 38.90 (CH2(major)), 56.27 (OCH3(minor)), 56.36
(OCH3(major)), 60.96 (OCH3(major)), 61.04 (OCH3(minor)), 106.79 (CH), 108.02 (CH), 108.49
(CH), 110.10 (C), 118.82 (CH), 121.61 (CH), 121.74 (CH), 123.06 (CH), 124.77 (C), 124.88
(C), 126.37 (C), 128.60 (CH), 129.37 (C), 129.73 (CH), 137.47 (CH), 139.45 (C), 140.68 (C),
141.17 (C), 143.43 (C), 152.74 (C), 153.30 (C), 166.16 (C=O), 168.52 (C=O).
Synthesis of compound 10
Compound 7 (500 mg, 1.6 mmol) was treated with 1,5-dibromopentane (552 mg, 2.4 mmol)
o
in the presence of NaH (96.4 mg, 2.4 mmol) in dimethylformamide at 60 C for 2h. The
crude product was purified by column chromatography using ethyl acetate-hexane (1:9) as
1
eluent to obtain compound 10 as mixture of E- and Z- isomers (3:1, H NMR). Yellow thick
oil (80%), δH (500 MHz, CDCl3): 1.51-1.58 (m, 4H, CH2(major, minor)), 1.71-1.78 (m, 4H,
CH2(major, minor)), 1.86-1.96 (m, 4H CH2(major, minor)), 3.38-3.44 (m, 4H, CH2(major, minor)), 3.78-
3.81 (m, 4H, CH2(major, minor)), 3.87 (s, 6H, OCH3(major)), 3.93 (s, 6H, OCH3(minor)), 3.93 (s, 3H,
OCH3(minor)), 3.97 (s, 3H, OCH3(major)), 6.82 (d, J = 7.70 Hz, 1H, ArH(minor)), 6.85 (d, J = 7.70
Hz, 1H, ArH(major)), 6.90 (s, 2H, ArH(major)), 6.91 (t, J = 7.38 Hz, 1H, ArH(major)), 7.057 (t, J =
7.38 Hz, 1H, ArH(minor)), 7.26 (t, J = 5.73 Hz, 1H, ArH(major, minor)), 7.45 (s, 1H, bridged
H(minor)), 7.53 (d, J = 7.54 Hz, 1H, ArH(minor)), 7.77 (s, 1H, bridged H(major)), 7.79 (d, J = 7.54
Hz, 1H, ArH(major)), 7.83 (s, 2H, ArH(minor)). δC (normal/DEPT-135; CDCl3): 25.53
(CH2(minor)), 25.57 (CH2(major)), 26.80 (CH2(major)), 26.82 (CH2(minor)), 32.34 (CH2(minor)), 32.37
(CH2(major)), 33.36 (CH2(major, minor)), 39.57 (CH2(minor)), 39.68 (CH2(major)), 56.26 (OCH3(major)),
18

56.36 (OCH3(minor)), 60.95 (OCH3(minor)), 61.03 (OCH3(major)), 106.80 (CH), 108.00 (CH(major)),
108.47 (CH(major)), 109.81 (CH(minor)), 110.08 (CH(major)), 118.79 (CH), 121.36 (C), 121.53
(CH), 121.64 (CH), 123.03 (CH), 124.77 (C), 125.00 (C), 126.46 (C), 128.54 (CH), 129.42
(C), 129.68 (CH), 130.20 (C), 137.33 (CH(minor)), 137.35 (CH(major)), 139.40 (C), 140.62 (C),
141.33 (C), 143.55 (C), 152.73 (C), 153.25 (C), 166.11 (C=O(minor)), 168.46 (C=O(major)).
Synthesis of compound 11
A mixture of 7 (500 mg, 1.6 mmol) and 1,6-dibromohexane (588.4 mg, 2.4 mmol) in
dimethylformamide was heated at 60 oC for 2h in the presence of NaH (96.4 mg, 2.4 mmol).
The crude product was purified by column chromatography using ethyl acetate-hexane (1:9)
1
as eluent to obtain compound 11 as mixture of E- and Z- isomers (3:1, H NMR). Yellow
thick oil (70%), δH (500 MHz, CDCl3): 1.38-1.46 (m, 4H, CH2(major, minor)), 1.47-1.53 (m, 4H,
CH2(major, minor)), 1.70-1.76 (m, 4H, CH2(major, minor), 1.82-1.89 (m, 4H, CH2(major, minor)), 3.37-
3.41 (m, 4H, CH2(major, minor)), 3.77-3.80 (m, 4H, CH2(major, minor)), 3.87 (s, 6H, OCH3(major)),
3.93 (s, 6H, OCH3(minor)), 3.93 (s, 3H, OCH3(minor)), 3.97 (s, 3H, OCH3(major)), 6.82 (d, J= 7.75
Hz, 1H, ArH(minor)), 6.85 (d, J= 7.75 Hz, 1H, ArH(major)), 6.90 (s, 2H, ArH(major)), 6.90 (t, J=
7.55 Hz, 1H, ArH(major)), 7.05 (t, J = 7.57 Hz, 1H, ArH(minor)), 7.26 (t, J = 7.78 Hz, 2H,
ArH(major + minor)), 7.45 (s, 1H, bridged H(minor)), 7.53 (d, J = 7.50 Hz, 1H, ArH(minor)), 7.77 (s,
1H, bridged H(major)), 7.80 (d, J = 7.72 Hz, 1H, ArH(major)), 7.83 (s, 2H, ArH(minor)). δC
(normal/DEPT-135; CDCl3): 26.15 (CH2), 27.48 (CH2), 27.86 (CH2(major)), 27.88 (CH2(minor)),
32.63 (CH2), 33.72 (CH2(minor)), 33.75 (CH2(major)), 39.73 (CH2(minor)), 39.84 (CH2(major)), 56.26
(OCH3(major)), 56.36 (OCH3(minor)), 60.98 (OCH3(minor)), 61.00 (OCH3(major)), 106.74 (CH),
108.05 (CH(major)), 108.52 (CH(minor)), 110.03 (CH), 118.82 (CH), 121.49 (CH), 121.61 (CH),
123.02 (CH), 124.77 (C), 125.08 (C), 126.52 (C), 128.54 (CH), 129.45 (C), 129.67 (CH),
130.24 (C), 137.30 (CH), 139.33 (C), 140.56 (C), 141.42 (C), 143.67(C), 152.73 (C), 153.28
(C), 166.11 (C=O(minor)), 168.48 (C=O(major)).
19

Synthesis of compound 12
The solution of compound 8 (100 mg, 0.23 mmol) in dry dichloromethane with addition of
anhydrous AlCl3 (49.55 mg, 0.37 mmol) under nitrogen atmosphere was stirred at room
temperature for 2h. The reaction mass was purified by column chromatography using ethyl
acetate/hexane (4:6) as eluent. Yellow thick oil (50%), δH (500 MHz, CDCl3): 2.27-2.32 (m,
4H, CH2(major, minor)), 3.46-3.49 (m, 4H, CH2(major, minor)), 3.91 (s, 6H, OCH3(major, minor), 3.93-
3.96 (m, 4H, CH2(major, minor)), 3.97 (s, 3H, OCH3(minor)), 4.01 (s, 3H, OCH3(major)), 5.85 (s, 1H,
OH(major)), 5.98 (s, 1H, OH(minor)), 6.91-6.92 (d, J = 7.65 Hz, 1H, ArH(major)), 6.94-6.956 (t, J =
7.87 Hz, 1H, ArH(major)), 6.95 (s, 2H, ArH(major)), 6.96 (d, J=7.87 Hz 1H, ArH
(minor)), 7.07 (t, J = 7.21 Hz, 1H, ArH(minor)), 7.28 (t, J = 7.82 Hz, 2H, ArH(major, minor)), 7.45 (s,
1H, bridged H(minor)), 7.52 (d, J = 7.20 Hz, 1H, ArH(minor)), 7.77 (s, 1H, bridged H(major)), 7.83
(d, J = 7.62 Hz, 1H, ArH(major)), 7.93 (s, 2H, ArH(minor)). δC (normal/DEPT-135; CDCl3): 30.51
(CH2(major)), 30.68 (CH2(minor)), 30.95 (CH2(major)), 31.00 (CH2(minor)), 38.49 (CH2(minor)), 38.56
(CH2(major)), 56.45 (OCH3(major)), 56.53 (OCH3(minor)), 106.74 (CH(minor)), 107.94 (CH(minor)),
108.44 (CH(major)), 110.04 (CH(major)), 118.54 (CH(minor)), 121.55 (CH(major)), 121.66 (CH(minor)),
122.70 (C(major)),123.35 (C(minor)), 124.95 (C(minor)), 125.24 (C(major)), 125.77 (C(minor)), 125.93
(C(major)), 128.26 (CH(minor)),129.56 (CH(major)), 136.54 (C(major)), 137.89 (C(minor)), 138.20
(CH), 140.90 (C(minor)), 143.25 (C(major)), 146.63 (C(minor)), 147.05 (C(major)), 166.168.83
(C=O(minor)), 168.78 (C=O(major)).
Synthesis of compound 13
Anhydrous AlCl3 (48 mg, 0.35 mmol) was added to the solution of compound 9 (100 mg,
0.22 mmol) in dry dichloromethane under nitrogen atmosphere and the reaction mixture was
stirred at room temperature for 2h. The reaction mass was purified by column
chromatography using ethyl acetate/hexane (4:6) as eluent. Yellow thick oil (50%), δH (500
MHz, CDCl3): 1.90-1.96 (m, 8H, CH2(major, minor)), 3.47-3.50 (m, 4H, CH2(major, minor)), 3.60-
20

3.63 (m, 4H, CH2(major, minor)), 3.82-3.86 (m, 4H, CH2(major, minor)), 3.91 (s, 6H, OCH3(major)),
4.01 (s, 6H, OCH3(minor)), 5.86 (s, 1H, OH(major)), 5.98 (s, 1H, OH(minor)), 6.86 (d, J = 7.87 Hz,
1H, ArH(minor)), 6.89 (d, J = 7.87 Hz, 1H, ArH(major)), 6.92 (t, J = 7.87 Hz, 1H, ArH(major)), 6.95
(s, 2H, ArH(major)), 7.06 (t, J = 7.31 Hz, 1H, ArH(minor)), 7.25 (t, J = 7.54 Hz 2H, ArH(major,
minor)), 7.44 (s, 1H, bridged H(minor)), 7.51 (d, J = 7.81 Hz, 1H, ArH(minor)), 7.77 (s, 1H, bridged
H(major)), 7.84 (d, J = 7.81 Hz, 1H, ArH(major)), 7.93 (s, 2H, ArH(minor)). δC (normal/DEPT-135;
CDCl3): 26.14 (CH2(major)), 26.25 (CH2(minor)), 29.74 (CH2(major)), 29.85 (CH2(minor)), 33.20
(CH2), 38.78 (CH2(minor)), 38.85 (CH2(major)), 56.43 (OCH3(major)), 56.50 (OCH3(minor)), 106.67
(CH(minor)), 107.96 (CH(minor)), 108.48 (CH(major)), 109.97 (CH(major)), 118.52 (CH(minor)),
121.55 (CH(major)), 121.66 (CH(minor)), 122.70 (CH(major)), 125.31 (C(major)), 125.93 (C(minor)),
128.16 (CH(major)), 129.47 (CH(major)), 136.42 (CH), 138.05 (CH), 143.21 (C), 146.58 (C),
146.99 (C), 166.32 (C=O(minor)), 168.70 (C=O(major)).
Synthesis of compound 14
The solution of compound 8 (250 mg, 0.57 mmol) and chrysin (146.98 mg, 0.57 mmol) in
o
dimethylformamide was heated at 60 C for 6h in the presence of K2CO3 (119.9 mg, 0.84
mmol). The crude product was column chromatography using ethyl acetate/hexane (5:5) as
eluent to procure a mixture of E- and Z- isomer in the ration 3:1. Yellow solid (60%), mp 182
oC, δH (500 MHz, CDCl3): 2.25-2.28 (m, 4H, CH2(major, minor), 3.87 (s, 6H, OCH3(major, minor)),
3.91 (s, 6H, OCH3(minor)), 3.93 (s, 6H, OCH3(major)), 4.01-4.04 (m, 4H, CH2(major, minor)), 4.10-
4.13 (m, 4H, CH2(major, minor)), 6.33 (d, J = 1.90 Hz, 1H, CH(minor)), 6.35 (d, J = 1.90 Hz, 1H,
CH(major)), 6.45 (d, J = 1.90 Hz, 1H, CH(minor)), 6.48 (d, J = 1.84 Hz, 1H, CH(major)), 6.65 (s,
1H, CH(minor)), 6.66 (s, 1H, CH(major)), 6.87 (d, J = 7.86 Hz, 1H, ArH(major)), 6.90 (t, J = 6.52
Hz, 1H, ArH (major)), 6.91 (s, 2H, ArH(major)), 7.04 (t, J = 8.06 Hz, 1H, CH(minor)), 7.24 (t, J =
7.58 Hz, 2H, ArH (major, minor)), 7.45 (s, 1H, bridged H(minor)), 7.50-7.56 (m, 4H, ArH), 7.77 (s,
1H, bridged H (major)), 7.80 (s, 2H, ArH(minor)), 7.85-788 (m, 3H, ArH), 12.70 (s, 1H,
21

OH(minor)), 12.70 (s, 1H, OH(major)). δC (normal/DEPT-135; CDCl3): 27.42 (CH2), 36.81
(CH2(minor)), 36.88 (CH2(major)), 56.27 (OCH3), 61.03 (OCH3), 65.91 (CH2(major)), 65.95
(CH2(minor)), 93.03 (CH(major)), 93.08 (CH(minor)), 98.66 (CH(minor)), 98.70 (CH(major)), 105.78
(C), 105.88 (CH(minor)), 105.91 (CH(major)), 106.74 (CH(minor)), 107.89 (CH), 108.37 (CH),
109.99 (CH(major)), 118.82 (CH(minor)), 121.29 (C), 121.69 (CH), 121.82 (CH), 123.07 (CH),
124.71 (C), 124.82 (C), 126.29 (CH), 128.68 (CH), 129.09 (CH), 129.35 (CH), 129.82 (CH),
131.30 (C), 131.85 (CH), 137.53 (CH), 137.58 (CH), 139.40(C), 140.60 (C), 141.27 (C),
143.51 (C), 152.69 (C), 153.29 (C), 157.73 (C), 157.79 (C), 162.15 (C), 162.17 (C), 163.95
(C), 164.01 (C), 164.63 (C), 164.67 (C), 166.34 (C=O), 168.65 (C=O), 182.42 (C=O), 182.48
(C=O). HRMS (ESI) m/z for C36H31O8N [M+H]+ calcd 606.2122, found 606.6130.
Synthesis of compound 15
The solution of compound 9 (250 mg, 0.56 mmol) and chrysin (142.4 mg, 0.56 mmol) in
o
dimethylformamide was heated at 60 C for 2h in the presence of K2CO3 (116.195 mg, 0.84
mmol). The crude product was purified by column chromatography using ethyl
acetate/hexane (5:5) as eluent to procure E- and Z- isomers in the ration 3:1. Yellow solid
o
(60%), mp 177 C, δH (500 MHz, CDCl3): 1.92-1.94 (m, 8H, CH2(major, minor)), 3.87 (s, 6H,
OCH3(minor)), 3.88-3.91 (m, 4H, CH2(major, minor)), 3.92 (s, 3H, OCH3(major)), 3.93 (s, 3H,
OCH3(minor)), 3.96 (s, 6H, OCH3(major)), 4.08-4.12 (m, 4H, CH2(major, minor)), 6.31 (d, J = 2.20
Hz,1H, CH(major)), 6.34 (d, J = 2.20 Hz, 1H, CH(minor)), 6.45 (d, J = 2.20 Hz,1H, CH(major)),
6.49 (d, J = 2.20 Hz, 1H, CH(minor)), 6.65 (s, 1H, CH(major)), 6.66 (s, 1H, CH(minor)), 6.85 (d, J =
7.15 Hz, 1H, ArH(major)), 6.90 (s, 2H, ArH(major)), 6.93 (d, J = 7.57 Hz, 1H, ArH(minor)), 7.06 (t,
J = 8.06 Hz, 1H, ArH(minor)), 7.27 (t, J = 8.01 Hz, 2H, ArH(major, minor)), 7.44 (s, 1H, bridged
H(major)), 7.49-7.56 (m, 4H, ArH), 7.78 (s, 1H, bridged H(minor)), 7.80 (d, J = 7.89 Hz, 1H,
CH(minor)), 7.82 (s, 2H, CH(major)), 7.86-7.89 (m, 2H, ArH), 12.68 (s, 1H, OH(major)), 12.69 (s,
1H, OH(minor)). δC (normal/DEPT-135; CDCl3): 24.15 (CH2), 26.35 (CH2), 39.34 (CH2(major)),
22

39.40 (CH2(minor)), 56.26 (OCH3(major)), 56.35 (OCH3(minor)), 60.95 (OCH3(major)), 61.03
(OCH3(minor)), 67.85 (CH2(major)), 65.94 (CH2(minor)), 93.07 (CH), 98.63 (CH(major)), 98.67
(CH(minor)), 105.72 (CH(minor)), 105.80 (CH(major)), 106.82 (CH), 108.04 (CH(major)), 108.05
(CH(minor)), 110.10 (CH), 118.81 (CH), 121.62 (CH), 121.74 (CH), 123.07 (CH), 124.79 (C),
124.91 (C), 126.29 (C), 128.58 (C), 129.07 (CH), 129.72 (CH), 131.36 (C), 137.44
(CH(major)), 137.48 (CH(minor)), 139.46 (C), 140.66 (C), 144.21 (C), 152.72 (C), 153.29 (C),
157.82 (C), 162.18 (C), 163.96 (C), 164.92 (C), 166.23 (C=O(major)), 168.52 (C=O(minor)),
182.45 (C=O). HRMS (ESI) m/z for C37H33O8N [M+H]+ calcd 620.2279, found 620.6404.
Synthesis of compound 16
Compound 10 (250 mg, 0.54 mmol) was treated with chrysin (138.02 mg, 0.54mmol) in the
o
presence of K2CO3 (112.9 mg, 0.81mmol) in dimethylformamide at 60 C for 6h. The crude
product was purified by column chromatography using ethyl acetate/hexane (5:5) as eluent.
o
Yellow solid (60%), mp 120 C, δH (500 MHz, CDCl3): 1.58-1.60 (m, 8H, CH2(major, minor)),
1.80-1.83 (m, 4H, CH2(major, minor)), 1.84-1.92 (m, 4H, CH2(major, minor)), 3.86 (s, 6H,
OCH3(major)), 3.92 (s, 3H, OCH3(minor)), 3.93 (s, 6H, OCH3(minor)), 3.96 (s, 3H, OCH3(major)),
4.01-4.05 (m, 4H, CH2(major, minor)), 6.32 (s, 1H, ArH(minor)), 6.34 (s, 1H, ArH(major)), 6.45 (s,
1H, ArH(minor)), 6.48 (s, 1H, ArH(major)), 6.63 (s, 1H, ArH(minor)), 6.65 (s, 1H, ArH(major)), 6.84
(d, J = 7.85 Hz, 1H, ArH(minor)), 6.87 (d, J = 7.85 Hz, 1H, ArH(major)), 6.91 (t, J = 7.59 Hz, 1H,
CH(major)), 6.90 (s, 2H, ArH(major)), 7.04 (t, J = 7.59 Hz, 1H, CH(minor)), 7.27 (t, J = 7.81 Hz,
2H, ArH(major, minor)), 7.45 (s, 1H, bridged H(minor)), 7.50-7.54 (m, 4H, ArH), 77.78 (s, 1H,
bridged H (major)), 7.80 (d, J = 7.81 Hz, 1H, ArH(major)), 7.82 (s, 2H, ArH(minor)), 7.87-7.88 (m,
2H, ArH), 12.68 (s, 1H, OH(major)), 12.74 (s, 1H, OH(minor)). δC (normal/DEPT-135; CDCl3):
23.43 (CH2(minor)), 23.45(CH2(major)), 27.34 (CH2), 28.63 (CH2(major)), 28.68 (CH2(minor)), 39.72
(CH2(minor)), 39.80 (CH2(major)), 56.28 (OCH3), 60.97 (OCH3), 61.05 (OCH3), 68.30
(CH2(major)), 68.37 (CH2(minor)), 93.06 (CH(major)), 93.10 (CH(minor)), 98.68 (CH(minor)), 99.61
23

(CH(major)), 105.69 (C), 105.89 (CH), 106.84 (CH), 108.05 (CH(major)), 108.53 (CH(minor)),
110.12 (CH(major)), 118.81 (CH(minor)), 121.40 (C), 121.57 (CH), 121.68 (CH), 123.07 (CH),
124.80 (C), 125.03 (C), 126.31 (CH), 126.47 (C), 128.56 (CH), 129.09 (CH), 129.42 (C),
129.70 (CH), 130.19 (C), 131.41 (C), 131.81 (CH), 137.40 (CH), 137.44 (CH), 139.46 (C),
141.39 (C), 141.27 (C), 143.63 (C), 152.75 (C), 153.31 (C), 157.84 (C), 162.18 (C), 163.98
(C), 165.08 (C), 166.19 (C=O(minor)), 168.57 (C=O(major)), 182.47 (C=O). HRMS (ESI) m/z for
C38H35O8N [M+H]+calcd 634.2435, found 634.2533.
Synthesis of compound 17
Compound 11 (250 mg, 0.53 mmol) was treated with chrysin (133.96 mg, 0.53 mmol) in the
presence of K2CO3 (107.9 mg, 0.78 mmol) in dimethylformamide at 60 oC for 6h. The crude
product was purified by column chromatography using ethyl acetate/hexane (5:5) as eluent.
o
Yellow solid (50%), mp 105 C , δH (500 MHz, CDCl3): 1.47-1.55 (m, 4H, CH2), 1.75-1.78
(m, 4H, CH2), 1.80-1.84 (m, 4H, CH2), 3.08-3.83 (m, 4H, CH2(major, minor), 3.86 (s, 6H,
OCH3(major)), 3.92 (s, 3H, OCH3(minor)), 3.93 (s, 6H, OCH3(minor)), 3.93 (s, 6H, OCH3(major)),
4.00-4.04 (m, 2H, CH2(major)), 4.01-4.04 (m, 2H, CH2( minor)), 6.33 (s, 1H, ArH(minor)), 6.35 (s,
1H, ArH(major)), 6.46 (s, 1H, ArH(minor)), 6.48 (s, 1H, ArH(major)), 6.65 (s, 1H, CH), 6.83 (d, J =
7.83 Hz, 1H, ArH(minor)), 6.86 (d, J = 8.01 Hz, 1H, ArH(minor)), 6.88 (s, 2H, ArH(major)), 6.90 (t,
J = 7.72 Hz, 1H, CH(major)), 7.04 (t, J = 7.59 Hz, 1H, CH(minor)), 7.27 (t, J = 7.58 Hz, 2H,
ArH(major, minor)), 7.44 (s, 1H, bridged H(minor)), 7.50-7.53 (m, 4H, ArH), 7.76 (s, 1H, bridged H
(major)), 7.83 (s, 2H, ArH(minor)), 7.79 (d, J = 7.77 Hz, 1H, ArH(major)), 7.87-78 (m, 2H,
ArH(minor)), 12.69 (s, 1H, OH). δC (normal/DEPT-135; CDCl3): 25.74 (CH2(major)), 25.76
(CH2(major)), 26.62 (CH2(minor)), 26.67 (CH2(major)), 27.55 (CH2), 28.81 (CH2(major)), 28.82
(CH2(minor)), 39.78 (CH2(minor)), 39.83 (CH2(major)), 56.24 (OCH3), 56.35 (OCH3), 60.90
(OCH3), 61.03 (OCH3), 68.47 (CH2(major)), 68.49 (CH2(minor)), 93.09 (CH(major)), 93.11
(CH(minor)), 98.60 (CH(minor)), 98.63 (CH(major)), 105.63 (C), 105.86 (CH(minor)), 106.71
24

(CH(minor)), 108.06 (CH), 108.54 (CH), 110.02 (CH(major)), 118.78 (CH(minor)), 121.36 (C),
121.49 (CH), 121.62 (CH), 123.01 (CH), 124.77 (C), 125.07 (C), 126.29 (CH),126.49 (C),
128.54 (CH), 129.45 (CH), 129.67 (CH), 130.21 (CH), 131.38 (C), 131.80 (CH), 137.31
(CH),139.30(C), 140.53 (C), 141.43 (C), 152.71 (C), 153.26 (C), 157.80 (C), 162.14 (C),
163.92(C), 165.12 (C), 166.13 (C=O), 168.49 (C=O), 182.46 (C=O). HRMS (ESI) m/z for
C39H37O8N [M+H]+ calcd 648.2547, found 648.2796.
Synthesis of compound 18
To the stirred solution of compound 14 (100 mg, 0.165 mmol) in acetonitrile, K2CO3 (27.36
mg, 0.198 mmol), CH3I (30 mg, 0.198 mmol) and KI (catalytic amount) were added. The
reaction was allowed to stir for 6h at room temperature. After the completion of reaction, it
was quenched by adding water and extracted with ethyl acetate. The organic layer was
separated and dried over Na2SO4. The crude product was purified by column chromatography
using ethyl acetate/hexane (8:2) as eluent to procure compound 18 as mixture of E- and Z-
isomers in the ratio 3:1,yellow solid (60%), mp 160 oC, δH (500 MHz, CDCl3): 2.27-2.31 (m,
2H, CH2(major)), 2.27-2.31 (m, 2H, CH2(minor)), 3.86 (s, 6H, OCH3(major)), 3.90 (s, 6H,
OCH3(minor)), 3.932 (s, 3H, OCH3(major, minor)), 3.938 (s, 3H, OCH3(major)), 3.94 (s, 3H,
OCH3(minor)), 4.03-4.05 (m, 4H, CH2(major, minor)), 4.15-4.17 (m, 4H, CH2(major, minor)), 6.32 (d, J
= 1.65 Hz, 1H, ArH(minor)), 6.36 (d, J = 1.77 Hz, 1H, ArH(major)), 6.51 (d, J = 1.85 Hz, 1H,
ArH(minor)), 6.44 (d, J=1.85 Hz, 1H, ArH(major)), 6.66 (s, 1H, ArH(minor)), 6.67 (s, 1H,
ArH(major)), 6.86 (d, J = 7.85 Hz, 1H, ArH(minor)), 6.90 (s, 2H, ArH(major)), 6.93 (t, J = 6.90 Hz,
1H, ArH(major)), 7.04 (t, J = 7.33 Hz, 1H, CH(minor)), 7.22 (t, J = 7.58 Hz, 2H, ArH(major, minor)),
7.46 (s, 1H, bridged H(minor)), 7.47-7.51 (m, 3H, ArH), 7.53 (d, J = 7.57 Hz, 1H, ArH(minor)),
7.77 (s, 1H, bridged H(major)), 7.80 (s, 2H, ArH(minor)), 7.85-7.86 (m, 3H, ArH). δC
(normal/DEPT-135; CDCl3): 27.48 (CH2(major)), 27.48 (CH2(minor)), 39.92 (CH2(minor)), 36.99
(CH2(major)), 56.31 (OCH3(major)), 56.34 (OCH3(minor)), 56.45 (OCH3(major, minor)), 60.97
25

(OCH3(major)), 61.06 (OCH3(minor)), 65.97 (CH2(major, minor)), 93.57 (CH(major, minor)), 96.45
(CH(minor)), 96.50 (CH(major)), 106.71 (CH), 107.94 (CH), 108.41 (CH), 109.09 (CH), 109.48
(CH), 110.15 (CH(major)), 118.82 (CH), 121.38 (C), 121.76 (CH), 121.88 (CH), 123.11 (CH),
124.79 (C), 126.00 (CH(major)), 126.30 (CH(minor)), 128.67 (CH), 128.96 (CH),129.31 (CH),
129.81 (CH), 130.06 (CH), 131.22 (C), 131.57 (CH), 137.65 (CH), 139.61 (C), 141.30 (C),
143.52 (C), 152.71 (C), 153.26 (C), 159.90 (C), 160.77 (C), 161.01(C), 163.14 (C), 168.73
(C=O), 177.63 (C=O).
UV-Vis titration of chrysin with CN-
Stock solution of chrysin (5× 10-3M) was diluted to 50 µM concentration by using DMSO–
H2O, 1:9 v/v. Stock solution of NaCN (1×10-3 M, water) was prepared. The absorbance of
solution was noted which shows absorption maxima at 315 nm. Incremental addition (10 μL,
1 equiv) of cyanide solution to the compound solution was made. The absorption intensity at
315 nm gets decreased and the intensity of new band at 368 nm goes up.
UV-Vis titration of compounds with CN-
Stock solution of the compounds (1×10-3 M) in DMSO-H2O (1:9) was diluted to 10 μM
concentration by using H2O. Stock solution of NaCN (1×10-3 M, water) was prepared. The
absorbance of compound solution was noted which shows absorption maxima at 368 nm.
Incremental addition (10 μL) of cyanide solution to the compound solution was made. The
absorption intensity at 368 nm gets decreased and the intensity of new band at 529 nm goes
up. UV-vis spectra of the compounds were also recorded in CHCl3 taking TBACN as the
source of CN- (supporting information).
Selectivity and Competitive binding of CN- with compound 2, 3
-
-
2-
Stock solutions of CN-, Br-, F-, AcO-, H2PO4 , HCO3 , CO3 , Cl-, I- (10-3 M in water) were
prepared. 200 μL (20 equiv) of each anion was added to the compound solution one by one
26

and UV-vis spectrum was recorded. Decrease in the absorption band at 368 nm and
emergence of new band was observed only in the case of cyanide solution.
Working of the compound with cytochrome c oxidase
The preparation of assay buffer, enzyme buffer and other reagents was as per the protocol
available with the assay kit [56]. The absorbance of cyt c (5.5 µM) at 550 nm was noted. To
the solution of cyt c (5.5 µM having 25 µL cyt c and 975 µL assay buffer), 0.5 µL DTT (0.1
M) was added and kept for 15 min. Incremental addition (10 µL to 50 µL) of CcOX (5µL
CcOX diluted with 45 µL enzyme dilution buffer) was made that led to diminishing of
absorbance intensity at 550 nm. Addition of cyanide (1 μM – 3 μM) to the solution of CcOX
was followed by the transfer of each of this solution (CcOX – 1 μM CN-, CcOX – 2 μM CN-,
CcOX – 3 μM CN-) to the vial containing cyt c and absorbance at 550 nm was increased. In
order to check the affinity of compound for cyanide bound to cyt c oxidase, stepwise addition
of compound 2 (1 μM to 25 µM) was made to the solution containing CcOX – 3 μM CN- and
cyt c. The absorbance intensity at 550 nm gets reduced.
Removal of cyanide from aqueous medium
Blood from the healthy subject was taken and the protocol was approved by the institute
ethical committee for the use of human subjects. Cyanide (10 µL, 1×10-1 M) was added to
each of the two vials containing 1 mL blood serum diluted in acetone-water. Compound 2
(100 µL, 1×10-3 M) in acetone – water (1:9, 1 mL) was added to vial ii. The solutions of two
vials were extracted with ethyl acetate (4 x 25 mL). Addition of compound 2 (100 μL) to the
aqueous part (obtained after extraction with ethyl acetate) of vial ii did not change the colour
of the solution indicating absence of cyanide whereas the aqueous part of vial i responded to
compound 2.
Molecular modelling studies
27

The 3-D crystal structure of cytochrome c oxidase (CcOX) and in complex with cyanide
(PDB ID 1OCC and 3AG4 respectively) were downloaded from protein data bank
(www.rcsb.org) and used for the docking studies. The compounds were docked in active site
of CcOX in complex with cyanide using Schrodinger (Schrödinger Release 2015-4: Maestro,
version 10.0, Schrödinger, LLC, New York, NY, 2014) software package and following the
protein preparation, ligand preparation, receptor grid generation steps for docking as
described in one of the previous reports [57].
AUTHOR INFORMATION
Corresponding Author
Palwinder Singh, Department of Chemistry, Guru Nanak Dev University, Amritsar-143005.
India; e-mail: palwinder_singh_2000@yahoo.com
ACKNOWLEDGMENTS
Financial assistance from SERB-DST, New Delhi is gratefully acknowledged. HK thanks
UGC for fellowship under UPE programme. UGC, New Delhi is gratefully acknowledged for
grant to GNDU under University with Potential for Excellence Programme.
ABBREVIATIONS
CcOx, cytochrome c oxidase; ED50, cyto c, cytochrome c; BNC, binuclear centre; TBACN,
tetrabutyl ammonium chloride; ED50, effective dose causing 50% effect; ACN, acetonitrile;
TLC, thin layer chromatography; DMSO, dimethyl sulphoxide; Ka, association constant.
ASSOCIATED CONTENT
Supporting information
1
Experimental data, H and 13C NMR spectra, mass spectra, IR spectra and molecular docking
data (PDF)
28

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