4-Aminoquinoline-ferrocenyl-chalcone conjugates: Synthesis and anti-plasmodial evaluation

Accepted Manuscript 4-Aminoquinoline-ferrocenyl-chalcone conjugates: Synthesis and anti-plasmodial evaluation Amandeep Singh, Jiri Gut, Philip J. Rosenthal, Vipan Kumar PII:

S0223-5234(16)30774-7

DOI:

10.1016/j.ejmech.2016.09.044

Reference:

EJMECH 8910

To appear in:

European Journal of Medicinal Chemistry

Received Date: 29 July 2016 Revised Date:

13 September 2016

Accepted Date: 14 September 2016

Please cite this article as: A. Singh, J. Gut, P.J. Rosenthal, V. Kumar, 4-Aminoquinoline-ferrocenylchalcone conjugates: Synthesis and anti-plasmodial evaluation, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.044. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

4-Aminoquinoline-ferrocenyl-chalcone conjugates: Synthesis and AntiPlasmodial Evaluation Amandeep Singh,a Jiri Gut,b Philip J. Rosenthal,b Vipan Kumar, a* Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, Punjab, India.

b

Department of Medicine, University of California, San Francisco, CA, USA

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N N N

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Cl

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a

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Fe

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Most potent conjugate IC50 = 0.37 µM (W2 strain)

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Synthesis and anti-plasmodial evaluation of 1H-1,2,3-triazole-tethered 4-aminoquinoline ferrocenenylchalcone conjugates with the most potent and non-cytotoxic conjugate exhibiting an IC50 of 0.37 µM against CQ-R W2 strain of P. falciparum.

ACCEPTED MANUSCRIPT

4-Aminoquinoline-ferrocenyl-chalcone conjugates: Synthesis and AntiPlasmodial Evaluation Amandeep Singh,a Jiri Gut,b Philip J. Rosenthal,b Vipan Kumar a* Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, Punjab, India b Department of Medicine, University of California, San Francisco, CA, USA

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a

Abstract: A series of aliphatic and aromatic substituted 1H-1,2,3-triazole-tethered 4-aminoquinoline-ferrocenylchalcone conjugates has been synthesized and evaluated for anti-plasmodial activity. The conjugates with flexible aliphatic (aminoethanol or aminopropanol) substituents on the

SC

quinoline ring showed better anti-plasmodial activities compared to those with cyclic (piperazine or aminophenol) substituents. The conjugate 17j was the most potent and non-cytotoxic, with an IC50

Keywords:

4-amino-quinoline-ferrocenylchalcone,

activities, Structure-activity relationship, 1.

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value of 0.37 µM against the chloroquine-resistant W2 strain of Plasmodium falciparum.

Introduction

1,3-dipolar

cycloaddition,

antiplasmodial

Plasmodium falciparum, the causative organism for the most severe form of malaria,

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poses significant global health and socio-economic burdens.[1] The disease remains a major cause of illness and deaths in tropical and sub-tropical countries including Asia, Africa and South America.[2] According to the World Health Organization, an estimated 3.3 billion people are at risk of being infected with malaria while 1.2 billion

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are at high risk.[3] In 2015, 214 million cases of malaria were estimated world-wide, with 438,000 deaths.[4] Chloroquine (CQ) was the most accessible, effective and safe

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drug in the history of malaria chemotherapy.[5-7] The drug was used for many years as the principle treatment of faciparum malaria, and it is still the drug of choice for the treatment of uncomplicated malaria caused by Plasmodium vivax and other species.[8] The development of CQ-resistance in P. falciparum[9] paved the way for its replacement with artemisinin-based combination therapy (ACT).[10] However, resistance has been reported to both

artemisinins and key partner drugs in

southeast Asia, providing impetus for the development of new anti-plasmodial agents.[11-14]

ACCEPTED MANUSCRIPT Among strategies for new anti-plasmodial drug development is 4aminoquinoline hybridization. Quinoline-hybridization is an extension of the concept of combination therapy, combining two pharmacophoric groups via a covalent bond to create a single entity.[15] The success of quinoline-hybridization in malaria chemotherapy is exemplified by ferroquine (7-chloroquinoline-ferrocene). The

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organometallic conjugate ferroquine has shown activity against CQ-sensitive and CQresistant strains of P. falciparum[16-19] and has recently completed phase IIb clinical trials.[20] Ferroquine has been suggested to generate oxidative stress due to redox properties of the ferrocene (Fe2+)/ferrocenium (Fe3+) system,[21-24] creating a

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reactive oxygen species through a Fenton-like reaction in the digestive vacuole of the parasite.[25-30] The high anti-plasmodial potency of ferroquine can be attributed

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because of its higher fraction of neutral and monoprotonated form compared to chloroquine and non-covalent interactions with hemozoin because of the presence of a ferrocene core.[31]1,2,4-trioxane-ferrocene conjugates had IC50 values ranging from 7.2-30.2 nM against the 3D7 strain of P. falciparum.[32] A carbosilane congener of ferroquine exhibited better activity than ferroquine against the NF54 strain of P.

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falciparum.[33]

Chalcones represent a key structural motif, belonging to the flavonoid class of natural products and exhibiting a myriad of pharmacological properties.[34-35]The discovery of the promising antiplasmodial activity of Licochalcone A from Chinese licorice

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root prompted researchers to study the anti-plasmodial efficacies of variedly functionalized synthetic chalcones.[36] Structure-activity relationship (SAR) studies confirmed the

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presence of α,β-unsaturated ketone linkers and the E-configuration as critical pre-requisites for good antiplasmodial activity.[37-38] The antiplasmodial activity of chalcones is potentially augmented by their ability to act as inhibitors of plasmodial aspartate proteases,[39] cysteine proteases[40] and cyclin-dependent protein kinases[41] along with their

inhibition of new permeation pathways induced by the parasite in erythrocytic

membranes.[42] In continuation with our interest in the synthesis of biologically relevant molecular conjugates,[43] the present manuscript describes the synthesis of a series of 1H1,2,3-triazole linked 4-aminoquinoline-ferrocenyl-chalcone conjugates as depicted in Figure 1 and evaluation of their antiplasmodial activity. 1H-1,2,3-triazole core was

ACCEPTED MANUSCRIPT introduced as a linker due to its stability under basic, acidic, reductive and oxidative conditions, as well as favourable properties including high dipole moment, hydrogen bonding, and rigidity in binding with bio-molecular targets.[44] The nature of the substituent as well as the length of the alkyl chain between the two pharmacophores (7-chloroquinoline and ferrocenylchalcone) was altered to study the SAR of the

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synthesized conjugates. 2. Results and Discussion:

The precursor O-alkylazido-ferrocenyl chalcone 4, was prepared by a recently reported protocol,[43] involving an initial alkylation of 4-hydroxy-acetophenone 1 using dibromo-

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alkanes and subsequent reaction with sodium azide in dry DMF to afford O-alkylazidoacetophenones 3. Aldol-condensation of 3 with ferrocene-carboxaldehyde afforded the

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desired O-alkylazido-ferrocenyl chalcones 4 in good yields. For the synthesis of desired piperazine-linked 4-aminoquinoline-ferrocenylchalcone conjugates 8, 4,7-dichloroquinoline 5 was initially reacted with piperazine to yield 7-chloro-4-piperazinequinoline6 [45] which was N-propargylated to afford precursor 7. Cu-promoted azide-alkyne cycloaddition of 4 with 7 resulted in the formation of desired 8 in good to excellent yields (Scheme 2).

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Structures to the conjugates were assigned on the basis of spectral studies and analytical evidence. For example, compound 8a exhibited a molecular ion peak at 686.1849 in its high resolution mass spectrum (HRMS). Its 1H NMR spectrum showed the presence of a singlet at δ 4.18 corresponding to 5H (cyclopentadiene ring of ferrocene) along with singlets at δ 4.50

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(4H) and δ 4.59 (2H) due to the presence of ferrocene ring and methylene protons. The presence of two singlets at δ 2.86 (4H) and 3.29 (4H) corresponding to the piperazine ring

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protons and a characteristic singlet at δ 8.07 (1H) corresponding to the triazole ring proton supported the assigned structure. The presence of characteristic peaks at δ 49.7 and 51.8 ppm corresponding to the piperazine ring and an α,β-unsaturated carbonyl carbon at δ 187.8 in 13C NMR spectrum further corroborated the assigned structure. Another precursor, (7-Chloro-quinolin-4-yl)-(4-prop-2-ynyloxy-phenyl)-amine

12, was prepared via an initial propargylation of 4-nitrophenol 9 followed by its reduction with iron powder in ethanol:water (90:10) mixture to yield 4-Prop-2ynyloxy-phenylamine 11.[46] Heating a mixture of 11 with 4,7-dichloroquinoline 5 in refluxing ethanol yielded 12. Cu-promoted click chemistry of 12 with O-alkylazidoferrocenyl chalcones 4 led to the isolation of crude solids, which were purified on

ACCEPTED MANUSCRIPT silica gel (60:120) using CHCl3:methanol (95:5) mixture as eluent, to yield 13 in good to excellent yields (Scheme 3).The structure of the compound was assigned on the basis of spectral studies and analytical evidence. For example, compound 13a exhibited a molecular ion peak at 709.1543 in its high resolution mass spectrum (HRMS). Its

1

H NMR spectrum showed the presence of a singlet at δ 4.17

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corresponding to 5H (cyclopentadiene ring of ferrocene) along with singlets at δ 4.49 (2H) and δ 4.58 (2H) due to the presence of ferrocene ring protons. The appearance of a doublet at δ 7.75 (1H, J=15.3Hz) confirmed the presence of the trans-olefinic proton of ferrocenylchalcone, along with a characteristic singlet at δ 7.85 (1H)

69.0, 69.7, 71.4 and 79.1 in the

13

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corresponding to the triazole ring proton.The appearance of absorption peaks at δ C NMR spectrum corresponding to ferrocene ring

supported the assigned structure.

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carbons and the presence of an α,β-unsaturated carbonyl carbon at δ 188.1

The developed protocol was extended to synthesize a series of amino-alcohol tethered 4-aminoquinoline-ferrocenylchalcone conjugates 17. The protocol involved initial treatment of 4,7-dichloroquinoline 5 with aminoethanol/aminopropanol, with subsequent

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propargylation to result in the formation of 16. Huisgen’s azide-alkyne cycloaddition of 16 with 4 gave the desired amino-alcohol-tethered-7-chloroquinoline-ferrocenylchalcone conjugates 17 in excellent yields (Scheme 4). The structures of the conjugates were assigned on the basis of spectral data and analytical evidence. For example, compound 17e exhibited

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a molecular ion peak at 717.2129 in its high resolution mass spectrum (HRMS). Its 1H NMR spectrum showed the presence of multiplets at δ 1.43 (2H) and 1.54 (2H) along with triplets

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at δ 1.80 (2H), 1.94 (2H), and singlets at δ 3.54 (2H) and 4.70 (2H) due to the presence of nhexyl chain protons. The appearance of doublets at δ 6.94 (2H) and 8.05 (2H) confirmed the presence of aromatic ring protons, and a characteristic singlet at δ 7.51 (1H) corresponded to the triazole ring protons.The appearance of characteristic peaks corresponding to the ferrocenyl ring and theα,β- unsaturated carbonyl carbon confirmed the assigned structure. The synthesized 4-aminoquinoline-ferrocenylchalcone conjugates 8, 13 and 17 were evaluated for their antiplasmodial activities against the chloroquine-resistant W2 strain of P. falciparum (Table 1). All of the compounds showed good antiplasmodial activity with IC50 values ranging from 0.37-5.08 µM. Inspection of the data revealed an interesting SAR, with activities largely dependent upon the nature of

ACCEPTED MANUSCRIPT substituents introduced on the quinoline ring and on the length of the alkyl chain introduced as a linker between two pharmacophores. In the case of piperazine-linked conjugates 8a-8f, the compounds exhibited IC50 values in the range of 2.55-5.08 µM. Conjugate 8a (n=2) was most potent in this series, while activity seems to be independent upon the length of the alkyl chain introduced. Replacement of the

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piperazine ring with 4-aminophenol did not improve antiplasmodial efficacy except in the cases of 13e (n= 6) and 13f (n=8), with IC50s of 2.40 and 1.16 µM respectively, indicating an improvement in antiplasmodial activity with increasing alkyl chain length. Introduction of amino-alcohols as substituents on the quinoline core

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improved the activity profiles, as evident for conjugates 17a-l. Among amino-ethanolsubstituted scaffolds 17a-f, the conjugates showed a decrease in activity with

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increase in chain length, as evident for compounds 17a (n=2, IC50= 0.95 µM) and 17d (n=5, IC50= 2.92 µM), while a further increase in chain length improved the activity, as shown for conjugates 17e and 17f. Similar comparison among amino-propanolsubstituted compounds 17g-l revealed the conjugates to be the most potent among the tested compounds, with activities largely independent of the length of alkyl chain

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linker. Conjugate 17j, with an optimum combination of amino-propanol as substituent and an n-pentyl chain linker, was the most potent of the tested compounds, with an IC50 of 0.37 µM. Comparing the anti-plasmodial activities of the present conjugates with amide-tethered similar scaffolds reported earlier by our

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group revealed the preference for an amide functionality over the 1H-1,2,3-triazole linker with the most potent 4-aminoquinoline-ferrocenylchalcone conjugate of the

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series exhibiting an IC50 of 0.15 µM.[43e] Cytotoxicity of the most potent conjugates viz. 17g-k was determined against mammalian HeLa cells in order to ascertain whether the observed activities are due to their antiplasmodial efficacy or cytotoxicity (Table 2). As evident, the synthesized conjugates exhibited IC50 values in the range of 16.12-23.34 µM while doxorubicin displayed an IC50 of 8.3 µM suggestive of the fact that these conjugates can act as starting points for the synthesis of new pharmacological templates against P. falciparum.

ACCEPTED MANUSCRIPT

3. Conclusion In conclusion, a series of 1H-1,2,3-triazole tethered 7-chloroquinoline-ferrocenyl chalcones with varied substituents on the quinoline core was prepared and evaluated

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for activity against P. falciparum. The presence of a flexible chain as a substitutent on the quinoline core improved antiplasmodial activity compared to the presence of cyclic piperazine/aminophenol functionalities. The conjugates with amino alcohols (amino-propanol) as substituents were non-cytotoxic and had activities in the sub-

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micromolar range (0.37-1.78 µM), and can be seen as templates for further design of

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new antiplasmodial scaffolds.

4. Experimental Section General Information

Melting points were determined by an open capillary using a Veego Precision Digital

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Melting Point apparatus (MP-D) and are uncorrected. 1H NMR spectra were recorded in CDCl3 with a BRUKER AVANCE II (500 MHz) spectrometer using TMS as internal standard. Chemical shift values are expressed as parts per million downfield from TMS and J values are in hertz. Splitting patterns are indicated as s: singlet, d: doublet,

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t: triplet, m: multiplet, dd: double doublet, ddd: doublet of a doublet of a doublet, and br: broad peak. 13C NMR spectra were recorded in CDCl3 with a BRUKER AVANCE

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II (125 MHz) using TMS as internal standard. Mass spectra were recorded on a BRUCKER high resolution mass spectrometer (micrOTOF-QII). Elemental analyses were

performed

on

a

Heraus

CHN-O-Rapid

Elemental

Analyzer.

Column

chromatography was performed on a silica gel (60–120 mesh) using ethyl chloroform:hexane mixture as eluent. 4.1 Procedure for synthesis of 1H-1,2,3-triazole-tethered piprazine-linked 4aminoquinoline-ferrocenylchalcone conjugates (8a-f):To a stirred solution of oalkylazido

chalcones

4(1

mmol)

and

N-propargylated

7-chloro-4-

piprazinequinoline7(1 mmol) in an ethanol−water (90:10) mixture was added copper

ACCEPTED MANUSCRIPT sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol).The reaction mixture was allowed to stir at room temperature for 7−10 h, and the progress was monitored using TLC. After the reaction was complete, water (20 mL) was added, and the reaction mixture was extracted twice with dichloromethane (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated

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under reduced pressure to yield a crude product, which was purified via column chromatography using 10:90 (methanol:chloroform) mixture. 4.1.1

1-[4-(2-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-[1,2,3]triazol-1-yl}-

ethoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (8a): Yield 74%. Dark red solid,

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m.p: 150-151 oC; 1H NMR (CDCl3, 500MHz): 2.86 (s, 4H, piprazine-H), 3.29 (s, 4H, piprazine-H), 3.89 (s, 2H, H9), 4.18 (s, 5H, H1), 4.49-4.51 (m, 4H, H2+H7), 4.59 (s, 2H,

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H3), 4.84 (t, J=4.9 Hz, 2H, H6), 6.84 (d, J=4.8 Hz, 1H, Ar-H), 6.96 (d, J=8.5 Hz, 2H, Ar-H), 7.10 (d, J=15.2 Hz, 1H, H5), 7.43 (d, J=7.8 Hz, 1H, Ar-H), 7.74 (d, J=15.3 Hz, 1H, H4), 7.81 (s, 1H, H8), 7.92 (d, J=8.9 Hz, 1H, Ar-H), 7.99 (d, J=8.5 Hz, 2H, Ar-H), 8.07 (s, 1H, Ar-H), 8.72 (d, J=5.0 Hz, 1H, Ar-H).

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C NMR (CDCl3, 125MHz):49.7, 51.8, 52.6, 53.0,

66.4, 68.9, 69.7, 71.3, 79.2, 99.9, 108.9, 114.1, 118.5, 121.7, 124.2, 125.1, 126.2, 128.6, 130.6, 132.4, 135.1, 146.3, 149.8, 151.6, 151.8, 161.1, 187.8. HRMS calcd for

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C37H35ClFeN6O2 [M]+ 686.1859,found 686.1868.Anal Calcd(%) for: C,64.69; H,5.13; N, 12.23. found: C,64.62; H,5.19; N,12.30.

1-[4-(3-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-[1,2,3]triazol-1-yl}-

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4.1.2

propoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (8b): Yield 80%. Dark red solid,

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m.p: 145-146 oC; 1H NMR (CDCl3, 500MHz): 2.06-2.09 (m, 2H, H7), 2.83 (s, 4H, piprazine-H), 3.27 (s, 4H, piprazine-H),3.85 (s, 2H, H10), 4.00 (t, J=5.3 Hz, 2H, H8), 4.16 (s, 5H, H1), 4.49 (s, 2H, H2), 4.60-4.66 (m, 4H, H3+H6), 6.82 (d, J=4.6 Hz, 1H, Ar-H), 6.92 (d, J=8.4 Hz, 2H, Ar-H), 7.13 (d, J=15.1 Hz, 1H, H5), 7.41 (d, J=7.6 Hz, 1H, Ar-H), 7.75 (d, J=15.2 Hz, 1H, H4), 7.81 (s, 1H, H8), 7.91 (d, J=8.9 Hz, 1H, Ar-H), 7.97 (d, J=8.4 Hz, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.77 (d, J=4.9 Hz, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 28.0, 49.5, 51.6, 52.7, 64.2, 64.3, 68.9, 69.7, 71.3, 79.3, 99.5, 108.4, 114.4, 118.3, 121.5, 124.6, 125.3, 126.5, 128.4, 130.4, 132.6, 135.3, 146.6, 149.7, 151.5, 151.8, 161.4, 187.7. HRMScalcd for C38H37ClFeN6O2 [M]+ 700.2016,found 700.2028.Anal Calcd(%) for: C,65.10; H,5.32; N, 11.99. found: C,65.14; H,5.29; N, 11.83.

ACCEPTED MANUSCRIPT 4.1.3

1-[4-(4-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-[1,2,3]triazol-1-yl}-

butoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (8c): Yield 79%. Dark red solid, m.p: 140-141 oC; 1H NMR (CDCl3, 500MHz):1.87-1.91 (m, 2H, H8), 2.16-2.21(m, 2H, H7), 2.92 (s, 4H, piprazine-H), 3.32 (s, 4H, piprazine-H), 3.92(s, 2H, H11), 4.09 (t, J=5.7

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Hz, 2H, H9), 4.19 (s, 5H, H1), 4.49-4.50 (m, 4H, H2+H6), 4.60 (s, 2H, H3), 6.85 (d, J=4.5 Hz, 1H, Ar-H), 6.95 (d, J=8.5 Hz, 2H, Ar-H), 7.14 (d, J=15.1 Hz, 1H, H5), 7.42 (d, J=7.5 Hz, 1H, Ar-H), 7.76 (d, J=15.1 Hz, 1H, H4), 7.82 (s, 1H, H10), 7.94 (d, J=8.6 Hz, 1H, Ar-H), 7.98 (d, J=8.5 Hz, 2H, Ar-H), 8.07 (s, 1H, Ar-H), 8.72 (d, J=4.0 Hz, 1H, Ar-H).

13

C NMR

SC

(CDCl3, 125MHz): 26.1, 27.1, 29.6, 31.6, 50.0, 52.9, 67.1, 68.9, 69.7, 71.2, 79.4, 99.4, 108.9, 114.1, 118.8, 121.6, 124.0, 125.0, 126.3, 128.3, 130.5, 132.4, 135.2, 146.4, calcd for C39H39ClFeN6O2 [M]+

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149.5, 151.6, 151.7, 161.5, 187.9. HRMS

714.2176,found 714.2188.Anal Calcd(%) for: C,65.51; H,5.50; N, 11.75. found: C,65.54; H,5.59; N,11.83.

4.1.4

1-[4-(5-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-[1,2,3]triazol-1-yl}-

pentyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (8d): Yield 84%. Dark red solid,

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m.p: 139-140 oC; 1H NMR (CDCl3, 500MHz):1.54-1.56 (m, 2H, H8), 1.85-1.87 (m, 2H, H9), 2.00-2.03 (m, 2H, H7), 2.95 (s, 4H, piprazine-H), 3.37 (s, 4H, piprazine-H), 4.02 (s, 2H, H12), 4.09 (t, J=5.4 Hz, 2H, H10), 4.18 (s, 5H, H1), 4.49-4.51 (m, 4H, H2+H6), 4.62 (s,

EP

2H, H3), 6.82 (d, J=4.1 Hz, 1H, Ar-H), 6.92 (d, J=8.4 Hz, 2H, Ar-H), 7.16 (d, J=15.3 Hz, 1H, H5), 7.46 (d, J=7.4 Hz, 1H, Ar-H), 7.74 (d, J=15.3 Hz, 1H, H4), 7.84 (s, 1H, H11), 7.93

AC C

(d, J=8.4 Hz, 1H, Ar-H), 7.83 (d, J=8.4 Hz, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.74 (d, J=4.5 Hz, 1H, Ar-H). 13C NMR (CDCl3, 125MHz):23.1, 26.3, 27.5, 29.2, 31.7, 50.1, 52.6, 67.3, 68.5, 69.5, 71.4, 79.2, 99.5, 108.6, 114.5, 118.6, 121.4, 124.4, 125.5, 126.6, 128.2, 130.3, 132.6, 135.6, 146.6, 149.3, 151.3, 151.5, 161.6, 187.7. HRMS

calcd for

C40H41ClFeN6O2 [M]+ 728.2329,found 728.2338.Anal Calcd(%) for: C,65.89; H,5.67; N, 11.53. found: C,65.81; H,5.62; N,11.63.

4.1.5

1-[4-(6-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-[1,2,3]triazol-1-yl}-

hexyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (8e): Yield 74%. Dark red solid, m.p: 135-136 oC; 1H NMR (CDCl3, 500MHz):1.39-1.43 (m, 2H, H9), 1.51-1.55(m, 2H,

ACCEPTED MANUSCRIPT H8), 1.79-1.82(m, 2H, H10), 1.92-1.96 (m, 2H, H7), 2.92 (s, 4H, piprazine-H), 3.35 (s, 4H, piprazine-H), 4.03 (s, 2H, H13), 4.05 (t, J=5.2 Hz, 2H, H11), 4.17 (s, 5H, H1), 4.47-4.49 (m, 4H, H2+H6), 4.64 (s, 2H, H3), 6.84 (d, J=4.2 Hz, 1H, Ar-H), 6.95 (d, J=8.6 Hz, 2H, ArH), 7.14 (d, J=15.2 Hz, 1H, H5), 7.44(d, J=7.3 Hz, 1H, Ar-H), 7.71 (d, J=15.2 Hz, 1H, H4), 7.82 (s, 1H, H12), 7.85 (d, J=8.4 Hz, 1H, Ar-H), 7.88 (d, J=8.4 Hz, 2H, Ar-H), 8.03 (s, 1H,

RI PT

Ar-H), 8.72 (d, J=5.1 Hz, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 22.5, 23.1, 26.5, 27.8, 29.6, 31.4, 50.8, 52.9, 67.6, 68.4, 69.7, 71.8, 79.1, 99.1, 108.4, 114.7, 118.2, 121.2, 124.7, 125.9, 126.2, 128.5, 130.6, 132.7, 135.7, 146.3, 149.8, 151.4, 151.6, 161.6, 187.9. HRMS calcd for C41H43ClFeN6O2 [M]+ 743.2485,found 743.2498.Anal Calcd(%)

1-[4-(8-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-[1,2,3]triazol-1-yl}-

M AN U

4.1.6

SC

for: C,66.27; H,5.83; N, 11.31. found: C,66.32; H,5.94; N,11.43.

octyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (8f): Yield 77%. Dark red solid, m.p: 120-121 oC; 1H NMR (CDCl3, 500MHz):1.39-1.42 (m, 4H, H9+H10), 1.43-1.45 (m, 2H, H11), 1.46-1.47 (m, 2H, H8), 1.80-1.82 (m, 2H, H12), 1.95-2.00 (m, 2H, H7), 2.98 (s, 4H, piprazine-H), 3.38 (s, 4H, piprazine-H), 3.98 (s, 2H, H15), 4.03 (t, J=5.8 Hz, 2H, H13), 4.18 (s, 5H, H1), 4.38 (t, J=6.2 Hz. 2H, H6), 4.48 (s, 2H, H2), 4.60 (s, 2H, H3), 6.85 (d,

TE D

J=4.2 Hz, 1H, Ar-H), 6.96 (d, J=8.2 Hz, 2H, Ar-H), 7.18 (d, J=15.3 Hz, 1H, H5), 7.48 (d, J=7.3 Hz, 1H, Ar-H), 7.77 (d, J=15.3 Hz, 1H, H4), 7.89 (s, 1H, H15), 7.93 (d, J=8.2 Hz, 1H, Ar-H), 7.87 (d, J=8.2 Hz, 2H, Ar-H), 8.38 (s, 1H, Ar-H), 8.78 (d, J=4.7 Hz, 1H, Ar-H). 13C

EP

NMR (CDCl3, 125MHz):22.6, 25.8, 26.4, 28.8, 29.0, 29.6, 30.2, 31.6, 50.4, 52.4, 68.0, 68.9, 69.7, 71.1, 79.4, 99.5, 108.2, 114.5, 118.5, 121.1, 124.3, 125.8, 126.1, 128.3, calcd for

AC C

130.8, 132.3, 135.4, 146.5, 149.5, 151.6, 151.7, 161.9, 187.3. HRMS

C43H47ClFeN6O2 [M]+ 770.2798,found 770.2788.Anal Calcd(%) for: C,66.97; H,6.14; N, 10.90. found: C,66.88; H, 6.23; N,10.83. 4.2 Procedure for synthesis of 1H-1,2,3-triazole-tethered 7-Chloro-quinolin-4-yl)-(4prop-2-ynyloxy-phenyl)-amine ferrocenylchalcone conjugates (13a-f): To a stirred solution of O-alkylazide ferrocenyl chalcone 4 (1 mmol) ,and 7-Chloro-quinolin-4-yl)(4-prop-2-ynyloxy-phenyl)-amine 12 (1 mmol) in an ethanol:water(90:10) mixture was added copper sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol). The reaction mixture was allowed to stir at room temperature for 10−11 h, and the progress was monitored using TLC. After the reaction was complete, water (20 mL)

ACCEPTED MANUSCRIPT was added, and the reaction mixture was extracted twice with dichloromethane (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield a crude product, which was purified via column chromatography using a 10:90 (methanol:chloroform) mixture.

RI PT

4.2.1 1-[4-(2-{4-[4-(7-Chloro-quinolin-4-ylamino)-phenoxymethyl]-[1,2,3]triazol-1-yl}ethoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (13a): Yield 90%. Dark red solid, m.p: 170-171 oC; 1H NMR (CDCl3, 500MHz): 4.17 (s, 5H, H1), 4.49 (s, 4H, H2+H6), 4.58 (s, 2H, H3), 4.83 (s, 2H, H7), 5.24 (s, 2H, H9), 6.65 (s, 1H, Ar-H), 6.89 (d, J=8.0 Hz, 2H,

SC

Ar-H), 7.02 (d, J=7.3 Hz, 2H, Ar-H), 7.10 (d, J=15.3 Hz, 1H, H4), 7.21 (d, J=6.9 Hz, 2H, Ar-H), 7.44 (d, J=6.9 Hz, 1H, Ar-H), 7.75 (d, J=15.3 Hz, 1H, H5), 7.85 (s, 1H, H8), 7.94 (d, 13

C NMR (CDCl3,

M AN U

J=8.0 Hz, 2H, Ar-H), 8.02-8.05 (m, 2H, Ar-H), 8.44 (s, 1H, Ar-H).

125MHz): 49.8, 62.1, 66.5, 69.0, 69.7, 71.4, 79.1, 101.2, 114.2, 115.9, 117.4, 118.5, 121.7, 124.0, 125.9, 126.1, 128.0, 130.6, 132.3, 132.4, 135.5, 144.2, 146.5, 149.5, 150.9, 151.0, 156.1, 161.1, 188.1. HRMS

calcd for C39H32ClFeN5O2 [M]+

709.1543,found 709.1532.Anal Calcd(%) for: C,66.97; H,4.54; N, 9.88. found: C,66.88;

TE D

H, 4.63; N,9.75.

4.2.2 1-[4-(3-{4-[4-(7-Chloro-quinolin-4-ylamino)-phenoxymethyl]-[1,2,3]triazol-1-yl}propoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (13b): Yield 89%. Dark red solid,

EP

m.p: 167-168 oC1H NMR (CDCl3, 500MHz): 2.04-2.06 (m, 2H, H7), 4.15 (s, 5H, H1), 4.47 (s, 4H, H2+H6), 4.56 (s, 2H, H3), 4.83 (s, 2H, H8), 5.25 (s, 2H, H10), 6.64 (s, 1H, Ar-H),

AC C

6.88 (d, J=8.1 Hz, 2H, Ar-H), 7.04 (d, J=7.2 Hz, 2H, Ar-H), 7.11 (d, J=15.2 Hz, 1H, H4), 7.22 (d, J=7.0 Hz, 2H, Ar-H), 7.46 (d, J=6.9 Hz, 1H, Ar-H), 7.76 (d, J=15.2 Hz, 1H, H5), 7.83 (s, 1H, H9), 7.94 (d, J=8.1 Hz, 2H, Ar-H), 8.03-8.06 (m, 2H, Ar-H), 8.42 (s, 1H, ArH).

13

C NMR (CDCl3, 125MHz) 27.8, 49.4, 62.4, 66.6, 69.2, 69.5, 71.2, 79.5, 101.4,

114.6, 115.6, 117.6, 118.7, 121.8, 124.5, 125.6, 126.3, 128.6, 130.7, 132.5, 132.6, 135.5, 144.6, 146.7, 149.8, 150.8, 150.9, 156.4, 161.3, 188.2. HRMS

calcd for

C40H34ClFeN5O3 [M]+ 723.1700,found 723.1712.Anal Calcd(%) for: C,66.35; H,4.73; N, 9.67. found: C,66.45; H, 4.63; N,9.75.

ACCEPTED MANUSCRIPT 4.2.3 1-[4-(4-{4-[4-(7-Chloro-quinolin-4-ylamino)-phenoxymethyl]-[1,2,3]triazol-1-yl}butoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (13c): Yield 93%. Dark red solid, m.p: 159-160 oC 1H NMR (CDCl3, 500MHz):1.83-1.85(m, 2H, H8), 2.13-2.17(m, 2H, H7), 4.14 (s, 5H, H1), 4.45 (s, 4H, H2+H6), 4.57 (s, 2H, H3), 4.85 (s, 2H, H9), 5.27 (s, 2H, H11), 6.65 (s, 1H, Ar-H), 6.84 (d, J=8.2 Hz, 2H, Ar-H), 7.03 (d, J=7.1 Hz, 2H, Ar-H), 7.12 (d,

RI PT

J=15.3 Hz, 1H, H4), 7.25 (d, J=7.1 Hz, 2H, Ar-H), 7.48 (d, J=6.8 Hz, 1H, Ar-H), 7.72 (d, J=15.3 Hz, 1H, H5), 7.86 (s, 1H, H10), 7.96 (d, J=8.2 Hz, 2H, Ar-H), 8.05-8.08 (m, 2H, ArH), 8.45 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz) 26.6, 27.6,49.4, 62.8, 66.4, 69.6, 69.9, 71.8, 79.8, 101.8, 114.9, 115.8, 117.8, 118.4, 121.4, 124.9, 125.8, 126.8, 128.9, 130.6,

SC

132.4, 132.8, 135.4, 144.4, 146.6, 149.5, 150.8, 151.1, 156.3, 161.5, 188.6. HRMS calcd for C41H36ClFeN5O3 [M]+ 737.1856,found 737.1862.Anal Calcd(%) for: C,66.72;

M AN U

H,4.92; N, 9.49. found: C,66.65; H, 4.83; N,9.55.

4.2.4 1-[4-(5-{4-[4-(7-Chloro-quinolin-4-ylamino)-phenoxymethyl]-[1,2,3]triazol-1-yl}pentyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (13d): Yield 90%. Dark red solid, m.p: 160-161 oC 1H NMR (CDCl3, 500MHz):1.51-1.54 (m, 2H, H8), 1.85-1.85 (m, 2H, H9), 2.01-2.03 (m, 2H, H7), 4.14 (s, 5H, H1), 4.47 (s, 4H, H2+H6), 4.57 (s, 2H, H3),

TE D

4.86 (s, 2H, H10), 5.27 (s, 2H, H12), 6.67 (s, 1H, Ar-H), 6.87 (d, J=8.0 Hz, 2H, Ar-H), 7.04 (d, J=7.3 Hz, 2H, Ar-H), 7.11 (d, J=15.3 Hz, 1H, H4), 7.22 (d, J=6.9 Hz, 2H, Ar-H), 7.41 (d, J=6.9 Hz, 1H, Ar-H), 7.73 (d, J=15.3 Hz, 1H, H5), 7.83 (s, 1H, H8), 7.93 (d, J=8.0 Hz, 2H,

EP

Ar-H), 8.04-8.07 (m, 2H, Ar-H), 8.46 (s, 1H, Ar-H).

13

C NMR (CDCl3, 125MHz) 23.1,

26.3, 27.5, 49.6, 61.8, 65.3, 69.9, 69.2, 71.5, 78.9, 101.6, 114.7, 116.0, 117.5, 117.8,

AC C

121.2, 123.8, 126.0, 126.5, 127.7, 130.1, 132.2, 132.7, 135.9, 144.9, 146.8, 148.8, 150.9, 151.1, 156.4, 161.6, 188.5. HRMS

calcd for C42H38ClFeN5O3 [M]+

751.2013,found 751.2022.Anal Calcd(%) for: C,67.07; H,5.09; N, 9.31. found: C,67.15; H, 5.13; N,9.45.

4.2.5 1-[4-(6-{4-[4-(7-Chloro-quinolin-4-ylamino)-phenoxymethyl]-[1,2,3]triazol-1-yl}hexyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (13e): Yield 91%. Dark red solid, m.p: 150-152 oC;

1

H NMR (CDCl3, 500MHz):1.35-1.38 (m, 2H, H9), 1.53-1.57(m, 2H,

H8), 1.77-1.80(m, 2H, H10), 1.94-1.96 (m, 2H, H7), 4.15 (s, 5H, H1), 4.45 (s, 4H, H2+H6), 4.54 (s, 2H, H3), 4.84 (s, 2H, H11), 5.23 (s, 2H, H13), 6.66 (s, 1H, Ar-H), 6.88 (d, J=8.1 Hz,

ACCEPTED MANUSCRIPT 2H, Ar-H), 7.03 (d, J=7.2 Hz, 2H, Ar-H), 7.14 (d, J=15.2 Hz, 1H, H4), 7.26 (d, J=7.0 Hz, 2H, Ar-H), 7.45 (d, J=6.9 Hz, 1H, Ar-H), 7.75 (d, J=15.3 Hz, 1H, H5), 7.81 (s, 1H, H12), 7.91 (d, J=8.0 Hz, 2H, Ar-H), 8.03-8.06 (m, 2H, Ar-H), 8.44 (s, 1H, Ar-H).

13

C NMR

(CDCl3, 125MHz): 22.3, 23.4, 26.4, 27.7, 49.7, 61.7, 65.5, 69.5, 69.6, 71.8, 78.7, 101.8, 114.5, 116.7, 117.4, 117.7, 121.6, 123.5, 126.5, 126.8, 127.6, 130.6, 132.4, 132.5, calcd for

RI PT

135.7, 144.8, 146.6, 148.7, 150.8, 151.3, 156.2, 161.5, 188.7. HRMS

C43H40ClFeN5O3 [M]+ 765.2169,found 765.2175.Anal Calcd(%) for: C,67.41; H,5.26; N, 9.14. found: C,67.55; H, 5.17; N,9.25.

SC

4.2.6 1-[4-(8-{4-[4-(7-Chloro-quinolin-4-ylamino)-phenoxymethyl]-[1,2,3]triazol-1-yl}octyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (13f): Yield 90%. Dark red solid,

M AN U

m.p: 139-141 oC 1H NMR (CDCl3, 500MHz):1.40-1.43 (m, 4H, H9+H10), 1.45-1.47 (m, 2H, H11), 1.48-1.49 (m, 2H, H8), 1.84-1.86 (m, 2H, H12), 1.93-1.95 (m, 2H, H7), 4.16 (s, 5H, H1), 4.47 (s, 4H, H2+H6), 4.53 (s, 2H, H3), 4.84 (s, 2H, H13), 5.26 (s, 2H, H15), 6.65 (s, 1H, Ar-H), 6.87 (d, J=8.0 Hz, 2H, Ar-H), 7.05 (d, J=7.1 Hz, 2H, Ar-H), 7.13 (d, J=15.3 Hz, 1H, H4), 7.24 (d, J=7.0 Hz, 2H, Ar-H), 7.41 (d, J=6.7 Hz, 1H, Ar-H), 7.76 (d, J=15.3 Hz,

1H, Ar-H).

13

TE D

1H, H5), 7.83 (s, 1H, H14), 7.91 (d, J=8.0 Hz, 2H, Ar-H), 8.01-8.03 (m, 2H, Ar-H), 8.41 (s, C NMR (CDCl3, 125MHz): 22.1, 22.7, 24.4, 25.4, 27.8, 49.2, 61.2, 65.9,

69.4, 69.8, 71.4, 78.3, 101.3, 114.1, 116.4, 117.7, 117.8, 122.3, 123.8, 126.5, 126.8, 127.2, 130.9, 132.2, 132.6, 135.2, 144.4, 145.6, 148.3, 151.1, 151.6, 154.2, 156.1,

EP

161.7, 188.1. HRMS calcd for C45H44ClFeN5O3 [M]+ 793.2482,found 793.2475.Anal

AC C

Calcd(%) for: C,66.06; H,5.58; N, 8.82. found: C,66.15; H, 5.67; N,8.91.

4.3 Procedure for synthesis of 1H-1,2,3-triazole-tethered 4-aminoquinolineferrocenylchalcone conjugates-ferrocenylchalcone conjugates (17a-l): To a stirred solution of O-alkylazide ferrocenyl chalcone 4 (1 mmol) ,and 7-chloro-N-(2-(propzynyloxy)ethyl)quinolin-4-amine 16 (1 mmol) in an ethanol:water (90:10) mixture was added copper sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol). The reaction mixture was allowed to stir at room temperature for 10−12 hrs, and the progress was monitored using TLC. After the reaction was complete, water (20 mL) was added, and the reaction mixture was extracted twice with dichloromethane (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and

ACCEPTED MANUSCRIPT concentrated under reduced pressure to yield a crude product, which was purified via column chromatography using a 10:90 (methanol:chloroform) mixture. 4.3.1

1-[4-(2-{4-[2-(7-Chloro-quinolin-4-ylamino)-ethoxymethyl]-[1,2,3]triazol-1-yl}-

ethoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17a): Yield 82%. Dark red solid, m.p: 160-161 oC; 1H NMR (CDCl3, 500MHz): 3.54 (s, 2H, H11), 3.94 (s, 2H, H10), 4.02 (s,

RI PT

2H, H9), 4.18 (s, 5H, H1), 4.36 (t, J= 4.3 Hz, 2H, H6), 4.48 (s, 2H, H2), 4.60 (s, 2H, H3), 4.75 (s, 2H, H7), 6.40 (s, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 6.94 (d, J=8.4 Hz, 2H, Ar-H), 7.15 (d, J=15.3 Hz, 1H, H4), 7.40-7.51 (m, 2H, Ar-H+H8), 7.74 (d, J= 15.3Hz, 1H, H5), 8.008.07 (m, 3H, Ar-H), 8.45 (s, 1H, Ar-H).

13

C NMR (CDCl3, 125MHz): 30.1, 43.2, 50.3,

SC

64.1, 67.8, 68.9, 69.7, 71.2, 79.4,98.7, 114.1, 116.9, 118.8, 118.9, 122.1, 122.5, 125.8, 126.5, 130.5, 131.3, 136.0, 144.6,145.8, 149.6, 151.3, 162.5, 188.0. HRMS calcd for

M AN U

C35H32ClFeN5O3 [M]+ 661.1543,found 661.1555.Anal Calcd(%) for: C,63.50; H,4.87; N, 10.58. found: C,63.59; H, 4.77; N,10.48.

4.3.2

1-[4-(3-{4-[2-(7-Chloro-quinolin-4-ylamino)-ethoxymethyl]-[1,2,3]triazol-1-yl}-

propoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17b): Yield 82%. Dark red solid, m.p: 153-154 oC; 1H NMR (CDCl3, 500MHz): 2.13-2.16 (m, 2H, H7), 3.52 (s, 2H, H12),

TE D

3.91 (s, 2H, H11), 4.04 (s, 2H, H10), 4.15 (s, 5H, H1), 4.37 (t, J= 4.1 Hz 2H, H6), 4.43 (s, 2H, H2), 4.64 (s, 2H, H3), 4.75 (s, 2H, H8), 6.43 (s, 1H, Ar-H), 6.73 (s, 1H, Ar-H), 6.91 (d, J=8.3 Hz, 2H, Ar-H), 7.12 (d, J=15.2 Hz, 1H, H4), 7.43-7.46 (m, 2H, Ar-H+H9), 7.77 (d,

EP

J=15.2, 1H, H5), 8.00-8.02 (m, 3H, Ar-H), 8.41 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 26.1, 30.4, 43.7, 50.8, 64.4, 67.5, 68.4, 69.3, 71.7, 79.8, 98.4, 114.5, 116.4, 118.4,

AC C

118.4, 122.5, 122.7, 125.5, 126.3, 130.3, 131.7, 136.5, 144.1, 145.9, 149.9, 151.8, 162.9, 188.1. HRMS calcd for C36H34ClFeN5O3 [M]+ 675.1700,found 661.1715.Anal Calcd(%) for: C,63.96; H,5.07; N, 10.36. found: C,63.85; H, 5.17; N,10.48.

4.3.3

1-[4-(4-{4-[2-(7-Chloro-quinolin-4-ylamino)-ethoxymethyl]-[1,2,3]triazol-1-yl}-

butoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17c): Yield 86%. Dark red solid, m.p: 152-153 oC;1H NMR (CDCl3, 500MHz):1.82-1.87(m, 2H, H8), 2.13-2.17(m, 2H, H13), 3.56 (s, 2H, H13), 3.91 (s, 2H, H12), 4.06 (s, 2H, H11), 4.16 (s, 5H, H1), 4.35 (t, J= 4.3 Hz 2H, H6), 4.46 (s, 2H, H2), 4.65 (s, 2H, H3), 4.75 (s, 2H, H9), 6.42 (s, 1H, Ar-H), 6.75 (s, 1H, Ar-H), 6.97 (d, J=8.2 Hz, 2H, Ar-H), 7.12 (d, J=15.3 Hz, 1H, H4), 7.45-7.52 (m, 2H,

ACCEPTED MANUSCRIPT Ar-H+H10), 7.71 (d, J= 15.3Hz, 1H, H5), 8.06-8.08 (m, 3H, Ar-H), 8.41 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz):25.8, 26.3, 30.1, 43.5, 50.6, 64.1, 67.7, 68.6, 69.5, 71.4, 79.7, 98.5, 114.4, 116.6, 118.5, 118.8, 122.4, 122.7, 125.6, 126.2, 130.8, 131.9, 135.6, 144.5, 145.7, 149.8, 151.7, 162.6, 188.3. HRMS calcd for C37H36ClFeN5O3 [M]+ 689.1856,found 689.1865.Anal Calcd(%) for: C,64.40; H,5.26; N, 10.15. found:

4.3.4

RI PT

C,64.35; H, 5.17; N,10.23.

1-[4-(5-{4-[2-(7-Chloro-quinolin-4-ylamino)-ethoxymethyl]-[1,2,3]triazol-1-yl}-

pentyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17d): Yield 90%. Dark red

SC

solid, m.p: 147-148 oC1H NMR (CDCl3, 500MHz):1.43-1.46 (m, 2H, H8), 1.81-1.84 (m, 2H, H9), 2.01-2.04 (m, 2H, H7), 3.52 (s, 2H, H14), 3.89 (s, 2H, H13), 4.06 (s, 2H, H12), 4.13

M AN U

(s, 5H, H1), 4.31 (t, J= 4.3 Hz 2H, H6), 4.42 (s, 2H, H2), 4.62 (s, 2H, H3), 4.71 (s, 2H, H10), 6.45 (s, 1H, Ar-H), 6.71 (s, 1H, Ar-H), 6.95 (d, J=8.2 Hz, 2H, Ar-H), 7.15 (d, J=15.1 Hz, 1H, H4), 7.41-7.46 (m, 2H, Ar-H+H11), 7.71 (d, J= 15.1Hz, 1H, H5), 8.04-8.07 (m, 3H, ArH), 8.46 (s, 1H, Ar-H).

13

C NMR (CDCl3, 125MHz): 23.6, 25.3, 26.6, 30.6, 43.3, 50.3,

64.4, 67.4, 68.5, 69.3, 71.3, 79.8, 98.3, 114.1, 116.4, 118.4, 118.5, 122.2, 122.6, 125.4, 126.6, 130.5, 131.6, 134.2, 144.2, 145.5, 149.7, 151.9, 162.3, 188.7. HRMS calcd for

TE D

C38H38ClFeN5O3 [M]+ 703.2013,found 703.2021.Anal Calcd(%) for: C,64.83; H,5.44; N, 9.95. found: C,64.75; H, 5.37; N,9.83. 4.3.5

1-[4-(6-{4-[2-(7-Chloro-quinolin-4-ylamino)-ethoxymethyl]-[1,2,3]triazol-1-yl}-

EP

hexyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17e): Yield 85%. Dark red solid, m.p: 140-142 oC 1H NMR (CDCl3, 500MHz):1.40-1.46 (m, 2H, H9), 1.53-1.57 (m, 2H,

AC C

H8), 1.78-1.83 (m, 2H, H10), 1.92-1.98 (m, 2H, H7), 3.54 (s, 2H, H15), 3.95 (s, 2H, H14), 4.02 (t,J= 6.2 Hz, 2H, H13), 4.19 (s, 5H, H1), 4.37 (t, J=7.0 Hz, 2H, H6), 4.49 (s, 2H, H2), 4.60 (s, 2H, H3), 4.75 (s, 2H, H11), 6.40 (s, 1H, Ar-H), 6.66-6.70 (s, 1H, Ar-H), 6.94 (d, 2H, 8.4 Hz, Ar-H), 7.15 (d, J= 15.2, 1H, H4), 7.41 (d,J=7.5 Hz, 1H, Ar-H), 7.51 (s, 1H, H12), 7.75 (d, J=15.3 Hz, 1H, H5), 7.99-8.06 (m, 3H, Ar-H), 8.47 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 25.4, 26.1, 28.8, 29.6, 30.1, 43.2, 50.3, 64.1, 67.8, 68.9, 69.7, 71.2, 79.4, 98.7, 114.1, 116.9, 118.8, 118.9, 122.1, 122.5, 125.8, 126.5, 130.5, 131.3, 136.0, 144.6, 145.8, 149.6, 151.3, 162.5, 188.0. HRMS calcd for C39H40ClFeN5O3 [M]+ 717.2169,found 717.2176.Anal Calcd(%) for: C,64.75; H,5.61; N, 9.75. found: C,64.83; H, 5.52; N,9.83.

ACCEPTED MANUSCRIPT 4.3.6

1-[4-(8-{4-[2-(7-Chloro-quinolin-4-ylamino)-ethoxymethyl]-[1,2,3]triazol-1-yl}-

octyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17f): Yield 79%. Dark red solid, m.p: 135-136 oC; 1H NMR (CDCl3, 500MHz):1.40-1.44 (m, 4H, H9+H10), 1.45-1.47 (m, 2H, H11), 1.48-1.50 (m, 2H, H8), 1.83-1.85 (m, 2H, H12), 1.93-1.96 (m, 2H, H7), 3.52 (s, 2H, H17), 3.90 (s, 2H, H16), 4.08 (s, 2H, H15), 4.17 (s, 5H, H1), 4.33 (t, J= 4.2 Hz 2H, H6),

RI PT

4.45 (s, 2H, H2), 4.65 (s, 2H, H3), 4.74 (s, 2H, H13), 6.43 (s, 1H, Ar-H), 6.75 (s, 1H, Ar-H), 6.92 (d, J=8.4 Hz, 2H, Ar-H), 7.13 (d, J=15.1 Hz, 1H, H4), 7.43-7.47 (m, 2H, Ar-H+H14), 7.75 (d, J= 15.1 Hz, 1H, H5), 8.04 (m, 3H, Ar-H), 8.43 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 22.2, 25.5, 26.5, 28.8, 29.2, 29.9, 30.1, 43.5, 49.4, 62.5, 66.4, 69.5, 69.8,

SC

71.7, 79.4, 101.6, 118.8, 121.5, 124.4, 125.5, 126.5, 128.4, 130.6, 132.7, 132.7, 135.8, 144.6, 146.3, 149.8, 150.4, 156.3, 161.4, 188.4. HRMS calcd for C41H44ClFeN5O3 [M]+

M AN U

745.2482,found 745.2476.Anal Calcd(%) for: C,66.00; H,5.94; N, 9.39. found: C,66.12; H, 5.84; N,9.45.

4.3.7 1-[4-(2-{4-[3-(7-Chloro-quinolin-4-ylamino)-propoxymethyl]-[1,2,3]triazol-1-yl}ethoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17g): Yield 74%. Dark red solid, m.p: 142-143 oC; 1H NMR (CDCl3, 500MHz): 1.23-1.25 (m, 2H, H11), 2.43 (s, 2H, H12),

TE D

3.45 (s, 2H, H10), 3.75 (s, 2H, H6), 4.02 (s, 2H, H9), 4.18 (s, 5H, H1), 4.47 (s, 2H, H2), 4.60 (s, 2H, H3), 4.66 (s, 2H, H7), 6.38 (s, 1H, Ar-H), 6.90 (d, J=8.0 Hz, 2H, Ar-H), 7.12 (d, J=15.3 Hz, 1H, H4), 7.28 (s, 2H, Ar-H), 7.54 (s, 1H, H8), 7.73-7.82 (m, 2H, Ar-H+H4), 7.97

EP

(d, J=8.0 Hz, Ar-H), 8.41 (s, 1H, Ar-H).

13

C NMR (CDCl3, 125MHz): 29.1, 42.4, 47.2,

64.2, 64.6, 68.3, 69.3, 71.2, 79.6, 98.4, 114.6, 116.2, 118.2, 122.6, 123.7, 125.2, 130.2,

AC C

131.2, 136.2, 144.1, 146.2, 151.2, 161.2, 188.2. HRMS calcd for C36H34ClFeN5O3 [M]+ 675.1700,found 675.1712.Anal Calcd(%) for: C,63.96; H,5.07; N, 10.36. found: C,63.83; H, 5.14; N, 10.42.

4.3.8 1-[4-(3-{4-[3-(7-Chloro-quinolin-4-ylamino)-propoxymethyl]-[1,2,3]triazol-1-yl}propoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17h): Yield 78%. Dark red solid, m.p: 130-131 oC 1H NMR (CDCl3, 500MHz): 1.26-1.29 (m, 2H, H12), 2.06 (s, 2H, H7), 2.41 (s, 2H, H13), 3.48 (s, 2H, H11), 3.78 (s, 2H, H6), 4.00 (s, 2H, H10), 4.18 (s, 5H, H1), 4.49 (s, 2H, H2), 4.60 (s, 2H, H3), 4.66 (s, 2H, H8), 6.38 (s, 1H, Ar-H), 6.90 (d, J=8.0, 2H, Ar-H), 7.12 (d, J=15.3 Hz, H4), 7.28 (s, 2H, Ar-H), 7.54 (s, 1H, H9), 7.73-7.82 (m, 2H, Ar-

ACCEPTED MANUSCRIPT H+H5), 7.97 (d, J=8.0 Hz, 2H, Ar-H), 8.41 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 28.0, 29.6, 31.5, 42.1, 47.0, 64.2, 64.3, 68.9, 69.7, 71.3, 79.3, 98.1, 114.1, 116.6, 117.2, 118.7, 122.7, 123.0, 125.8, 126.3, 128.2, 130.6, 131.9, 136.3, 144.5, 146.1, 151.7, 161.7, 188.0. HRMS calcd for C37H36ClFeN5O3 [M]+ 689.1856,found 689.1844.Anal

RI PT

Calcd(%) for: C,64.40; H,5.26; N, 10.15. found: C,64.33; H, 5.14; N, 10.22.

4.3.9 1-[4-(4-{4-[3-(7-Chloro-quinolin-4-ylamino)-propoxymethyl]-[1,2,3]triazol-1-yl}butoxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17i): Yield 80%. Dark red solid, m.p: 125-127 oC 1H NMR (CDCl3, 500MHz):1.23-125 (m, 2H, H13), 1.83-1.89 (m, 2H,

SC

H8), 2.14-2.19 (m, 2H, H7), 2.39 (s, 2H, H14), 3.43 (s, 2H, H12), 3.73 (s, 2H, H6), 4.04 (s, 2H, H11), 4.15 (s, 5H, H1), 4.45 (s, 2H, H2), 4.62 (s, 2H, H3), 4.65 (s, 2H, H9), 6.32 (s, 1H,

M AN U

Ar-H), 6.93 (d, J=8.1, 2H, Ar-H), 7.14 (d, J=15.2 Hz, H4), 7.25 (s, 2H, Ar-H), 7.52 (s, 1H, H10), 7.72-7.81 (m, 2H, Ar-H+H5), 7.93 (d, J=8.1 Hz, 2H, Ar-H), 8.43 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 28.0, 29.5, 30.2, 31.2, 42.6, 47.4, 64.8, 64.1, 68.3, 69.9, 71.2, 79.8, 98.4, 114.5, 116.6, 117.6, 118.9, 122.6, 123.3, 125.4, 126.7, 128.3, 130.4, 131.5, 136.7, 144.7, 146.4, 151.7, 161.4, 188.3.

HRMS

calcd for C38H38ClFeN5O3 [M]+

H, 5.34; N, 9.84.

4.3.10

TE D

703.2013,found 703.2024.Anal Calcd(%) for: C,64.83; H,5.44; N, 9.95 found: C,64.73;

1-[4-(5-{4-[3-(7-Chloro-quinolin-4-ylamino)-propoxymethyl]-[1,2,3]triazol-1-

EP

yl}-pentyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17j): Yield 83%. Dark red solid, m.p: 120-121 oC 1H NMR (CDCl3, 500MHz): 1.25-1.27 (m, 2H, H14), 1.53-1.57 (m,

AC C

2H, H8), 1.82-1.85 (m, 2H, H9), 2.01-2.04 (s, 2H, H7), 2.34 (s, 2H, H15), 3.45 (s, 2H, H13), 3.75 (s, 2H, H6), 4.06 (s, 2H, H12), 4.17 (s, 5H, H1), 4.43 (s, 2H, H2), 4.62 (s, 2H, H3), 4.62 (s, 2H, H10), 6.32 (s, 1H, Ar-H), 6.95 (d, J=8.2, 2H, Ar-H), 7.16 (d, J=15.1 Hz, H4), 7.27 (s, 2H, Ar-H), 7.52 (s, 1H, H11), 7.73-7.79 (m, 2H, Ar-H+H5), 7.91 (d, J=8.1 Hz, 2H, Ar-H), 8.46 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 26.3, 28.4, 29.4, 30.5, 31.4, 42.5, 47.4, 64.3, 64.8, 68.4, 69.4, 71.8, 79.4, 98.6, 114.7, 116.8, 117.3, 118.7, 122.9, 123.6, 125.5, 126.7, 128.4, 130.4, 131.6, 136.7, 144.6, 146.5, 151.4, 161.5, 188.4. HRMS calcd for C39H40ClFeN5O3 [M]+ 717.2169,found 717.2154.Anal Calcd(%) for: C,65.23; H,5.61; N, 9.75 found: C,65.33; H, 5.54; N, 9.84.

ACCEPTED MANUSCRIPT 4.3.11

1-[4-(6-{4-[3-(7-Chloro-quinolin-4-ylamino)-propoxymethyl]-[1,2,3]triazol-1-

yl}-hexyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17k): Yield 74%. Dark red solid, m.p: 110-111 oC 1H NMR (CDCl3, 500MHz): 1.25-1.27 (m, 2H, H15), 1.32-137 (m, 2H, H9), 1.52-1.56 (m, 2H, H8), 1.83-1.86 (m, 2H, H10), 2.03-2.05 (s, 2H, H7), 2.36 (s, 2H, H16), 3.42 (s, 2H, H14), 3.78 (s, 2H, H6), 4.08 (s, 2H, H13), 4.14 (s, 5H, H1), 4.46 (s, 2H,

RI PT

H2), 4.66 (s, 2H, H3), 4.66 (s, 2H, H11), 6.31 (s, 1H, Ar-H), 6.98 (d, J=8.0, 2H, Ar-H), 7.14 (d, J=15.3 Hz, H4), 7.24 (s, 2H, Ar-H), 7.55 (s, 1H, H12), 7.75-7.79 (m, 2H, Ar-H+H5), 7.94 (d, J=8.0 Hz, 2H, Ar-H), 8.43 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 25.7, 26.5, 28.7, 29.7, 30.3, 31.7, 42.7, 47.9, 64.7, 64.8, 68.8, 69.6, 71.6, 79.7, 98.8, 114.5, 116.4,

SC

117.3, 118.7, 122.7, 123.7, 125.9, 126.6, 128.4, 130.5, 131.6, 136.5, 144.7, 146.8, 151.5, 161.7, 188.3. HRMS calcd for C40H42ClFeN5O3 [M]+ 731.2326,found

4.3.12

M AN U

731.2334.Anal Calcd(%) for: C,65.62; H,5.78; N, 9.75 found: C,65.73; H, 5.64; N, 9.84.

1-[4-(8-{4-[3-(7-Chloro-quinolin-4-ylamino)-propoxymethyl]-[1,2,3]triazol-1-

yl}-octyloxy)-phenyl]-3-ferrocenyl-1,3-dienyl-propenone (17l): Yield 69%. Dark red solid, m.p: 105-106 oC; 1H NMR (CDCl3, 500MHz): 1.25-1.27 (m, 2H, H17), 1.41-143 (m, 4H, H9+H10), 1.46-1.49 ( m, 2H, H11), 1.51-1.55 (m, 2H, H8), 1.85-1.87 (m, 2H, H8), 2.05-

TE D

2.07 (s, 2H, H7), 2.38 (s, 2H, H18), 3.45 (s, 2H, H16), 3.73 (s, 2H, H6), 4.03 (s, 2H, H15), 4.15 (s, 5H, H1), 4.48 (s, 2H, H2), 4.69 (s, 2H, H3), 4.70 (s, 2H, H13), 6.34 (s, 1H, Ar-H), 6.95 (d, J=8.1 Hz, 2H, Ar-H), 7.17 (d, J=15.3 Hz, H4), 7.27 (s, 2H, Ar-H), 7.58 (s, 1H, H14),

EP

7.73-7.75 (m, 2H, Ar-H+H5), 7.96 (d, J=8.1 Hz, 2H, Ar-H), 8.43 (s, 1H, Ar-H). 13C NMR (CDCl3, 125MHz): 22.5, 25.1, 25.6, 26.4, 28.4, 29.8, 30.6, 31.4, 42.4, 47.6, 64.4, 64.5,

AC C

68.4, 69.3, 71.8, 79.4, 98.5, 114.8, 116.2, 117.5, 118.4, 122.5, 123.8, 125.5, 126.4, 128.8, 130.6, 131.8, 136.4, 144.6, 146.5, 151.8, 161.3, 188.5. HRMS

calcd for

C41H45ClFeN5O3 [M]+ 746.2560,found 746.2554.Anal Calcd(%) for: C,65.91; H,6.07; N, 9.37 found: C,65.83; H, 6.14; N, 9.47. 4.4 Methods for assessment of antiplasmodial activity of test compounds: The W2 strain of P. falciparum was cultured in RPMI-1640 medium with 10% human serum, following standard methods, and parasites were synchronized with 5% Dsorbitol.[47] Beginning at the ring stage, microwell cultures were incubated with different concentrations of compounds for 48 h. The compounds were added from DMSO stocks; the maximum concentration of DMSO used was 0.1%. Controls

ACCEPTED MANUSCRIPT without inhibitors included 0.1% DMSO. After 48 h when control cultures had progressed to new rings, the culture medium was removed, and cultures were incubated for 48 h with 1% formaldehyde in PBS, pH 7.4, at room temperature. Fixed parasites were then transferred to 0.1% Triton X-100 in PBS containing 1 nM YOYO1 dye (Molecular Probes). Parasitemia was determined from dot plots (forward scatter

RI PT

vs. fluorescence) acquired on a FACSort flow cytometer using Cell Quest software (Beckton Dickinson). IC50 values for growth inhibition were determined from plots of percent control parasitemia over inhibitor concentration using the Prism 3.0 program, (GraphPad Software), with data from duplicate experiments fitted by non-linear

4.5 In vitro analysis of cytotoxicity on HeLa cells:

SC

regression.[48]

M AN U

HeLa cells were cultured in 60 mm x 15 mm tissue culture dishes containing 5 mL of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin and streptomycin. Compounds were dissolved in DMSO to 100 µM concentrations. Once cell cultures reached 70% confluency, 5 µL of compound was added to the DMEM in the tissue culture dish for a final concentration of 100 µM. Cells were incubated for

TE D

24 h in a 37 οC CO2 incubator. After 24 h incubation, the media was removed from the HeLa cells and the cells were then washed with 5 mL of 1 x PBS. The cells were then cleaved off of the bottom of the plate via 5-min incubation with 0.5 mL of 0.25% trypsin. Cells were re-suspended in 1mL of 1 x PBS and transferred to a

EP

microcentrifuge tube. 100 µL of trypan blue solution were added to the re-suspended cells and allowed to incubate at room temperature for approximately 10 min. Viable

AC C

and dead cells were visualized and counted with a hemacytometer. IC50 values were determined using GraphPad PRISM.

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ACCEPTED MANUSCRIPT [49]C. Biot, W. Daher, C. M. Ndiaye, P. Melnyk, B. Pradines, N. Chavain, A. Pellet, L. Fraisse, L. Pelinski, C. Jarry, J. Brocard, J. Khalife, I. Forfar-Bares, D. Dive; J. Med. Chem., 49 (2006) 4707

Supporting information

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1H and 13C NMR spectra of representative compounds viz. 8a, 8c, 13a, 17e are provided in the supporting information.

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Acknowledgements

Financial assistance from University Grants Commission (UGC), New Delhi, India, under

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UGC-JRF Fellowship with Ref. No. 23/12/2012(ii)EU-V is gratefully acknowledged (AS). VK acknowledges the financial assistance provided by Science and Engineering Board (SERB), New Delhi under grant no. EMR/2015/001687. Captions:

Figure 1. 1H-1,2,3-triazole linked 4-aminoquinoline-ferrocenyl chalcone conjugates

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Scheme 1: Synthesis of O-alkyl azide ferrocenyl-chalcones

Scheme 2: Synthesis of piperazine-linked 7-chloroquinoline-ferrocenylchalcone conjugates Scheme 3: Synthesis of phenyl linked 7-chloroquinoline-ferrocenylchalcone conjugates

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Scheme 4: Synthesis of amino alcohol linked 7-chloroquinoline-ferrocenylchalcone conjugates

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Table 1:In vitro anti-malarial activity of compounds(8a-f, 13a-f, 17a-l) against W2 strain of P.falciparum Table 2: Cytotoxicity and selectivity index of the conjugates 17g-17k

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Figure 1. 1H-1,2,3-triazole linked 4-aminoquinoline-ferrocenyl chalcone conjugates

Scheme 1: Synthesis of O-alkyl azide ferrocenyl-chalcones

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Scheme 2: Synthesis of piperazine-linked 7-chloroquinoline-ferrocenylchalcone conjugates

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Scheme 3: Synthesis of phenyl linked 7-chloroquinoline-ferrocenylchalcone conjugates

Scheme 4: Synthesis of amino alcohol linked 7-chloroquinoline-ferrocenylchalcone conjugates

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IC50 (µM)

Compounds

IC50 (µM)

Compounds

8a

2.55±0.25

13c

4.25±0.81

17e

0.66±0.02

8b

4.98±0.33

13d

3.72±0.19

17f

0.69±0.17

8c

5.08±0.45

13e

2.40±0.42

17g

0.42±0.02

8d

3.33±0.20

13f

1.16±0.04

17h

0.41±0.17

8e

3.29±0.36

17a

0.95±0.03

17i

0.53±0.04

8f

3.73±0.09

17b

0.91±0.10

17j

0.37±0.03

13a

4.27±0.87

17c

1.34±0.14

17k

0.55±0.05

13b

4.98±0.11

17d

2.92±0.37

17l

1.78±0.07

CQ

0.060

FQ49

0.0081

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IC50 (µM)

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Compounds

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Table 1: In vitro anti-malarial activity of compounds(8a-f, 13a-f, 17a-l) against W2 strain of P.falciparum

Table 2: Cytotoxicity on HeLa cells and selectivity index of the conjugates 17g-17k

IC50 (µM)

Cytotoxicity (µM)

Selectivity Index (SI)

0.42

17.45

41.54

17h

0.41

20.12

49.07

17i

0.53

23.34

43.84

17j

0.37

18.20

49.18

17k

0.55

16.12

29.30

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17g

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Compound

Doxorubicin

8.30

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Synthesis and antiplasmodial evaluation of 4-amino-quinoline-ferrocenylchalcone conjugates
Conjugates with aliphatic substituents exhibited better activities than cyclic counterparts
Most potent and non-cytotoxic conjugate displayed an IC50 value of 0.37 µM W2 strain