Water mediated procedure for preparation of stereoselective oximes as inhibitors of MRCK kinase

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Water mediated procedure for preparation of stereoselective oximes as inhibitors of

MRCK kinase

Manoj Kumar Shrivash, Shilipi Singh, Akhilesh Kumar Shukla, Suaib Luqman, Jyoti

Pandey, Krishna Misra

PII:

S0022-2860(20)31024-3

DOI:

https://doi.org/10.1016/j.molstruc.2020.128699

MOLSTR 128699

Reference:

To appear in: Journal of Molecular Structure

Received Date: 17 April 2020

Revised Date: 3 June 2020

Accepted Date: 11 June 2020

Please cite this article as: M.K. Shrivash, S. Singh, A.K. Shukla, S. Luqman, J. Pandey, K. Misra, Water

mediated procedure for preparation of stereoselective oximes as inhibitors of MRCK kinase, Journal of

Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128699.

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Credit Author Statement

I hereby declare that the manuscript is being sent with the consent of all authors.There is no conflict

of interest amonst the authors.

Krishna Misra

Graphical abstract:

Water Mediated Procedure for Preparation of Stereoselective Oximes as Inhibitors of

MRCK Kinase

Manoj Kumar Shrivash, Shilipi Singh , Akhilesh Kumar Shukla, Suaib Luqman, Jyoti Pandey and

Krishna Misra

MRCK

Water Mediated Procedure for Preparation of Stereoselective Oximes as Inhibitors of

MRCK Kinase

c

Manoj Kumar Shrivash,^abShilipi Singh , Akhilesh Kumar Shukla^b, Suaib Luqman^c, Jyoti

Pandey^band Krishna Misra*^a

^aCentre of Biomedical Research, SGPGIMS, Campus Lucknow, 226014, India.

^bDepartment of Applied Chemistry, Babasaheb Bhimrao Ambedkar University, Lucknow

226025, India

^cCSIR-Central Institute of Medicinal & Aromatic Plants, Lucknow-226015, India.

*Corresponding email: krishnamisra4@gmail.com

ABSTRACT

Stereoselective aldoximes, preferably Z form have been obtained from α-cyano substituted

carbonyl conjugated alkenes. This reaction occurs through Michael addition type reaction

followed by retro-Knoevenagel reaction without transition-metal catalysis via C−C bond

cleavage. These oximes are evaluated against cancer cell lines employing mechanistic study.

Two oximes showed significant cytotoxic activity, which through in silico studies were found to

inhibit MRCK Kinase, responsible for metastatic spread of cancer mortality.

Keywords: Stereoselective synthesis; Z-oximes; C-C bond cleavage, Retro-Knoevenagel

reaction; MRCK kinase; Cancer cell lines

1. Introduction

Non-metallic catalytic C-C cleavage appears impossible because of the high energy block. The

cleavage of carbon-carbon bond is a significant issue in organic chemistry due to its inert nature

[1]. Although, the importance of the C−C bond cleavage has already resulted in different

methodologies for the cleavage of C−C, C=C, and carbon-carbon triple bond [2-4]. The

development of new routes for selective cleavage of C−C bond still remains an important and

challenging goal for the chemists and biologists. The breaking of C-C bond with transition-metal

complex is often studied [5]. In this regard, C-C bonds that are activated followed by

transformation offer an attractive alternative approach in terms of disconnecting atoms, forming

more active linkages, and producing more versatile scaffolds [6]. Stereoselective oximes are

significant intermediates in organic synthesis [7-8]. The oximes can either be dehydrated to

nitriles [9] or converted to amides via an acid catalyzed Beckmann rearrangement [10]. Oximes

are also key to ligands in the formation of mono- and poly metal complexes [11]. Oximes are

used for protection, purification [12] and characterization of carbonyl compounds [13].

Moreover, oximes have been widely used in medicine, industry, and analytical chemistry [14-

18]. The previously reported method for the synthesis of oximes is the reaction of a carbonyl

compound with hydroxylamine hydrochloride in the presence of base (Scheme 1) [19-20].

However, this method has some drawbacks which restrict its further application. Recently, other

studies have been achieved to obtain oximes via the redox methods under oxidation or reduction

conditions [21] e.g .aerobic oxidation of primary amines by using 1,1-diphenyl-2-picryl-hydrazyl

and tungstated alumina [22-25]. Some workers have reported the synthesis of oximes via

oxidative ammoniation of carbonyl compounds [26-27]. However, these methods have more

short-comings such as harsh reaction conditions, toxic metal salts, low yields, which adversely

affect the applications extensively for the synthesis of oximes.

Scheme1: Conventional methods of synthesis of oximes yielding mixture of E & Z isomers and

present method yielding predominantly Z isomer respectively.

The catalytic oxidation of amines is of major importance from both bioorganic and synthetic

processes [28-29]. However, beneficial methods for catalytic oxidation are restricted, because of

the sensitivity of amines. We report herein the first example where alkenes-bridged with

electron-withdrawing groups undergo C-C bond cleavage with the concomitant loss of the

specific group by Michael addition type reaction followed by retro Knoevenagel reaction. This

method uses a weak base, water and methanol thus avoiding the use of DMF or NMP, which are

expensive and difficult to remove from reaction mixture [30].

Our research group has now developed the synthesis of oximes, through direct conversion of

aryl/alkyl cyano ester to aryl/alkyl oximes. The alkene-bridged ethyl cyano aryl acrylate

compounds undergo C-C bond cleavage with the associated loss of the ethyl cyanoacetate group

by Michael addition of hydroxylamine to a benzylidene cyanoacetate followed by a retro

Knoevenagel reaction (1, 3 proton shift) without transition-metal catalysis. Besides this, the

significant advantage of the present method is the formation of stereoselective oximes

(Mainly Z –form) since this form is known to be biologically active (Scheme 1). The utility of

this method is seen not only in the synthesis of stereoselective substituted aryl/alkyl oximes but

other styrene derivatives of commercial utility also by using different compatible nucleophiles.

In recent years, there has been an increasing interest in the application of oximino group for

enhancing the biological activity in a large variety of molecules. In Steroids oximino group is

well known for its anticancer properties [31]. The oxime derivatives of curcumin show strong

antiproliferative activity against some cancer cell lines. Besides, the efficacy of these compounds

in reversing the efflux-mediated resistance developed by cancer cells has also been studied [32].

It is reported that the oxime derivatives of 1, 4-naphthoquinone exhibited higher cytotoxic

activity against cancer cells MDA-MB-231 and (HSF), but lower cytotoxic activity against

normal cell [33]. 5-Nitro, 5- flouro-indirubin-3-oxime showed antiproliferative activity against

human cancer cell line [34]. α, β- unsaturated oximes have exhibited high antiproliferative

activity against (Panc-1), (A-549), (PaCa-2) and (PC-3) cell lines [35] Quinazolinone was

inactive against nasopharyngeal carcinoma (NPC-TW01), lung carcinoma and leukemia while

quanozolinone oxime and quanozolinone methyl oxime exhibited more potent antiproliferative

activity [36]. The two structurally related protein kinase families, the Rho kinases (ROCK) and

themyotonic dystrophy kinase-related Cdc-binding kinases (MRCK) are required for migration

and invasion of cancer cells [37]. These regulate the assembly and organization of the actin

cytoskeleton in all eukaryotic cells and have been the center of much attention in cancerous

growth and metastatic [38]. It has been reported that MRCK inhibition was adequate to decrease

invasion by squamous cell carcinoma cells [39-41].

Simultaneous targeting of these two kinase families is likely to block the migration and invasion

of metastatic cancers. If inhibition of ROCK and MRCK kinases has a greater effect in

preventing cancer spread motility and local invasion, highly selective small-molecules of oximes

may act as inhibitors, thus proving useful for prevention of tumor invasion and metastasis. We

have designed and prepared eighteen Z-oximes, out of which two have shown high activity

against all the nine different cancer cell lines.

2. Results and discussion

2.1 Synthesis of Z oximes (2a-2v)

In continuation of our materials research, we focused on the study of 2-Cyano-3-N-

hydroxylamino-3-phenyl ethyl propionate (2). Methanolic solution of a benzylidene cyanoacetate

(1) was treated with hydroxylamine hydrochloride in aqueous sodium carbonate solution

(Scheme 3; Table 1, entry 7) to synthesize (2). Unexpectedly, benzyl oxime 2a was formed

because of the loss of the ethyl cyanoacetate moiety (Scheme 3). This structure was confirmed

1

13

by using IR spectra, H NMR, C NMR spectrum. The other expected products as shown in

(Scheme 2) were also not detected. A preliminary examination of the impact of solvents on

reactions shows that the oxime (2a) is obtained as the major product with most of the alcoholic

solvents used. Perhaps surprisingly, using water (Table 1, entry 7) it was obtained as the major

product. With an increase in reaction temperature to 100 °C, for 1h only (2a) was isolated. A

chemical reaction involving loss of ethyl cyanoacetate group by C−C bond cleavage, by the

Michael type addition of hydroxylamine hydrochloride has not been reported previously.

Another example of ethyl cyanoacetate group loss was observed when aqueous hydroxylamine

hydrochloride was reacted with other similar substrates (Scheme 4-5).The screening of different

solvents revealed that the solvent plays an important role in this reaction. Particularly, methanol:

water (9:1) was the only efficient solvent for the formation of oximes, while no reaction occurred

in other solvents (Table 1, entries, 1–6). Moreover, the effect of the reaction temperature was

also examined, and it was found that a temperature of 100 °C was optimal for this reaction

(Table 1, entries, and 7–12). Although methanol: water (9:1) was the best choice for this

reaction, other solvents such as ethanol: water and DMSO: water were also effective, however

affording the products with slightly reduced yields (Table 1, entries 8–12).

Scheme 2: Formation of oxime due to C-C bond cleavage.

Table 1: Optimization of the reaction conditions

Scheme 3: Unexpected formation of benzyl oxime (2a)

Scheme 4: Benzyl oxime (2a) from benzylidene malononitrile.

The Screening of various bases as catalysts (Table 2, entries,1-7) show that these play an

important role in this reaction. With the increase of pKa value yield of (2a) decreased (Table 2,

entries, 1-7) therefore sodium carbonate and potassium carbonate gave better conversion of

stereoselective oximes. Further optimization of the results showed that optimum amount of base

i.e. 1.1 equivalent gave (Table 2, entries 1–7) excellent yield. Additionally, the reaction was less

efficient while proceeding in only water (Table 1, entry 12). The scope of reaction was assessed

by using other substrates under similar conditions (Scheme 4-5) to yield corresponding

stereoselective oximes (Z-isomer) followed by carbon–carbon bond cleavage. With the optimized

conditions in hand, we turned our attention to the scope of the reaction. As summarized in (Table

3), a variety of ethyl cyanoaryl acrylates were converted to corresponding oximes. The reaction

could be successfully applied to a range of different substituted substrates yielding the

corresponding oximes in almost excellent yields. Ethyl cyanoaryl acrylates with either electron

donating or electron-withdrawing groups on the benzene ring smoothly generated the

corresponding products, however in varying yields Electronic effects play an important role, as

electron-withdrawing substituents (Table 3, 2p) on the benzene ring favored the transformation.

Meanwhile, electron-donating substituents (Table 3, entries 2b–2h, and 2n, 2o) hindered the

transformation. This is evident from the mechanism given in (Scheme 1), since electron-

withdrawing groups will facilitate nucleophilic attack at α-position. The ethyl (E)-2-cyano-3-(4-

methoxyphenyl) acrylate substrate gave the two isomeric products, i.e. E isomer oxime and Z

1

isomer oxime in 90:10 ratio. Both isomers were confirmed by melting points and HNMR

Spectrum.

Scheme 5: Benzyl oxime (2a) from dimethyl-2-benzylidene malonate.

Table 2: Impact of base on yield of benzyl oxime.

Additionally, the reaction conditions were also compatible with fluoro, chloro, and bromo

substituents (Table 3, entries 2j–2l). The yield was excellent in case of fluorine being in the para

position of benzene followed by bromine in the same position. With chlorine at either ortho or

meta positions (Table 3, entries 2k and 2l), the yield was slightly lower than fluoro /bromo

derivatives. This may be due, in part, to steric hindrance. Ethyl (E-2-cyano-3-(naphthalen-1-yl)

acrylate could also be successfully converted into the corresponding oximes in good yields

(Table 3, 2i). Moreover, we also conducted the reaction with heterocyclic substrates under the

optimized reaction conditions (Table 3, 2s), with good yields.

Scheme 6: Control experiment.

Table 3: Substrate scope of Z oximes.

Scheme 7: a plausible mechanism is proposed for the synthesis of benzyl oxime (2a).

Surprisingly, in the case of ethyl (E)-2-cyano-3-(1H-indole-3yl) acrylate, a heterocyclic

derivative an equivalent yield of oxime was obtained (Table 3, 2s). However, the yield in the

case of aliphatic oximes was quite low (See table 3; 2t, 2u, 2v). To demonstrate the scope and

efficiency of the present method, this mild system was then extended for the synthesis of

aliphatic oximes. However, we found that the heptyl substrate (Table 3, 2r) did react and gave

very low conversion and amount of yield. To check the viability of ethyl cyanoacetate as leaving

group, we also replaced it with malononitrile and dimethylmalonate groups. Using optimized

reaction conditions we found that both these groups gave comparable yields of benzyl oxime

(Scheme 4-5). To assess the effect of electron-withdrawing group we used one EWG in place of

two and observed that C-C cleavage did not take place and no oxime was formed. The role of

water was also assessed and the exclusion of water also resulted in the non-formation of oxime.

Experiment with ethyl cinnamate which has only one EWG when reacted with aq. hydroxyl

amine in methanol at 100 °C for 1h we did not find benzyl oxime (2a), rather N-

hydroxycinnamamide was formed, therefore one electron withdrawing group did not leave from

the substrate as shown in (Scheme 6). The presence of two EWG i.e cyano and ester groups

gave the oxime as shown in (Scheme 7). Based on the above control experiments (,a plausible

mechanism is proposed for the synthesis of 2a (Scheme 7). Firstly, the reaction of ethyl (E)-2-

cyano-3-phenylacrylate with hydroxylamine catalyzed by Na₂CO₃give a Michael addition

product as an intermediate. Then a retro Knoevenagel reaction is taken place through attacked

base on intermediate (1, 3 proton shift) and subsequent carbon-carbon bond cleavage to give the

final product 2a. The mechanism shown explains the formation of Z-isomer preferentially since

the heavier leaving group orients the preferable configuration (scheme 7).

3. Biological activities

3.1 Anticancer activity of synthetic oximes (MTT Assay)

Among all the twenty two synthesized compounds, 2g (2, 3, 4-trimethoxy benzyl oxime) showed

maximum growth inhibition in all the tested cancer cell lines (range of IC₅₀9.42 ± 0.39-18.75 ±

3.39 µg/mL) but it also showed moderate toxicity for normal cell line (L-132) with an IC₅₀value

of 44.26 ± 0.39 µg/mL (Table 4). Compound 2d (p-methoxy benzyl oxime) showed toxicity

against all the tested cancer cell lines (range of IC₅₀8.63 ± 0.15-23.84 ± 0.50 µg/mL) as well as

in normal cell line (IC₅₀37.84 ± 1.27 µg/mL). Compound 2i inhibits the growth of skin, lung and

breast cancer cell lines (12.25 ± 0.40-24.49 ± 1.27 µg/mL) but slightly altered the growth of the

squamous carcinoma and hepatic carcinoma with the percent inhibition of 43.23 ± 1.03 and

47.58 ± 3.68 respectively at the highest tested concentration (50µg/mL). It also showed toxicity

against normal cell line (L-132) with an IC₅₀value of 28.69 ± 0.86 µg/mL whereas compound 2l

and 2k selectively showed toxicity against the skin carcinoma (IC₅₀40.80 ± 1.55 µg/mL) and

breast carcinoma (IC₅₀38.84 ± 0.70 µg/mL, (Table 4, Fig 1). Rest of the compounds altered the

growth of cells feebly with the percent inhibition ranged from 14.31 ± 0.95-48.57 ± 5.47.

Table 4: IC₅₀value of synthesized compound for tested cell line.

Figure 1: Percent cytotoxicity of synthetic oximes (2b-2p) vs standard drug Tamoxifen.

3.2 Docking study of bioactive oximes against MRCK protein

The oxime moiety of the paramethoxy benzaldehyde oxime (2d) ligands provides binding site

opening interacting with MRCK around the Gln 144, Val 35, Asp 32 and oxime moiety of tri

methoxy benzaldehyde oxime (2g) interacted with MRCK Gln 144, Val 35, His 395, Lys 109,

Gln 70. As well as binding energy of 2d, 2g respected -5.1 and -5.9 (Fig. 2). 2g ligand interacted

with MRCK kinase with good binding energy compared with 2d and both ligand are inhibited

cell invasion pathways of metastatic stage.

Figure 2: (A) Interaction of 2d with MRCK Kinase (3D); (B) Interaction of 2d with MRCK

Kinase (2D); (C) Interaction of 2g with MRCK Kinase (3D); (D) Interaction of 2g with MRCK

Kinase (2D).

3.3 Cell cycle analysis

Compound 2g and 2d at the inhibitory concentration (IC₅₀) and double the inhibitory

concentration IC₅₀for 24 h altered the phases of the cell cycle. In A431 cell line, the compound

2g slightly increased the apoptotic cells at both the tested concentration. Compound 2g reduced

the G0/G1 phase up to 1.14 fold and increased the number of cells in S phase at both the

concentration 10.24µg (1.1fold) and 20.48 (1.6 fold). There is a non-significant alteration was

observed in the G2/M phase, whereas compound 2d increased 1.1 and 1.2 fold number of cells in

the G2/M phase at both the tested concentration, respectively. Compound 2d at the concentration

of 9.11 µg decreased the cells in S phase, but at the concentration of 18.22µg, it increased the 1.2

fold number of cells (Fig 3). The reduction of cells in G0/G1 phase was observed at both the

concentration up to 1.3 fold. The compound 2d showed up to 4.3 fold increased number of

apoptotic cells in comparison to control. In NCIH-460 cell line, the number of apoptotic cells

was increased 1.4 and 3.1fold at both the tested concentration respectively after the treatment

with compound 2g. Simultaneously, compound 2g reduced the number of cells in G0/G1, S, and

G2/M phase up to 2.4 fold.

Similarly, Compound 2d increased the apoptotic cells at the both tested concentrations 8.63 µg

(3.2 fold) and 17.26µg (4.4 fold) and decreased the number of cells in G0/G1, S and G2/M phase

up to 1.5 fold (Fig 4). From the results, we can interpret that the compound 2g inhibit the cell

proliferation of A431 cells by arresting the cell cycle in S phase, whereas in NCIH-460 cells by

increasing apoptosis. The compound 2d altered the cell growth of A431 cells by inducing

apoptosis and G2/M phase arrest, but in NCIH-460 cells, the cell proliferation was inhibited by

inducing apoptosis.

3.4 Apoptosis assay (Annexin-V-FITC)

Both the compound showed apoptosis in NCIH-460 cell line; therefore, we further performed the

annexin-V assay to confirm the apoptosis at the two concentrations IC₅₀and Double of IC₅₀.

Compound 2g showed 0.7% and 8.25% late apoptosis, 1.8 % and 3.6 % necrosis at both the

tested concentration, respectively (Fig 5). Compound 2d showed 3% and 19.3% late apoptosis,

1.4 % and 2.6 % necrosis at both the tested concentration respectively.

3.5 Reactive oxygen species (ROS) estimation

Both the tested compounds increased the apoptosis in NCIH-460 cell line, and as earlier reports

suggested that the ROS are involved in the apoptotic pathway46. Therefore, we further evaluate

the ROS inducing effect in NCIH-460 cell line at the two concentrations IC₅₀and Double of IC₅₀.

The results showed that the compound 2g induced ROS formation (1.05 fold) only at the Double

of IC₅₀(17.26µg) whereas the compound 2d increased the ROS formation 1.2 fold and 1.6 fold at

both the tested concentration respectively in comparison to control (Fig 6). The results indicated

that the apoptotic potential of compound 2d might be due to ROS production.

3.6 Mitochondrial membrane potential estimation

The mitochondrial membrane potential is also associated with the apoptotic pathway; therefore

we also evaluated the effect on mitochondrial potential by using the rhodamine dye in NCIH-460

cell line at the two concentrations IC₅₀and Double of IC₅₀. The data showed that both the

compounds 2g and 2d did not alter the mitochondrial membrane potential significantly (Fig 7).

The finding suggests that the compounds did not use mitochondria-mediated apoptosis pathway

for cell growth inhibition.

Figure 3: The effect of compound 2g and 2d in the A431 cell line on different phases of the cell

cycle. The cell cycle analysis was performed by using propidium iodide (PI) staining. The

pictorial graphs and percent population graphs are presented, and the pictorial graphs are one

representative example of two experiments.

Figure 4: The effect of compound 2g and 2d in NCIH-460 cell line on different phases of the cell

cycle. The cell cycle analysis was performed by using propidium iodide (PI) staining. The

pictorial graphs and percent population graphs are presented, and the pictorial graphs are one

representative example of two experiments.

Figure 5: The apoptotic effect of compound 2g and 2d in NCIH-460 cell line. The apoptosis

assay was performed by using Annexin-V-FITC staining. The pictorial graphs and percent

population graphs are presented, and the pictorial graphs are one representative example of two

experiments.

Figure 6: The ROS generation effect of compound 2g and 2d in NCIH-460 cell line. The reactive

oxygen species (ROS) assay was performed by using DCFDA dye. The pictorial graphs and

FITC mean graphs are presented, and the pictorial graphs are one representative example of

two experiments.

Figure 7: The effect of compound 2g and 2d on mitochondrial membrane potential in NCIH-460

cell line. The mitochondrial membrane potential (MMP) assay was performed by using

Rhodamine 123 dye. The pictorial graphs and FITC mean graphs are presented, and the

pictorial graphs are one representative example of two experiments.

4.Experimental section

4.1 General

All chemicals used in these syntheses were purchased from Sigma-Aldrich, Fisher Scientific,

Avra chemicals and used without further purification. Unless otherwise stated, all reactions were

carried out without inert atmosphere of nitrogen, NMR spectra were recorded in CDCl₃, DMSO-

1

D₆with a Bruker Avance III spectrometer operating at 400 MHz and 800MHz, for H spectra

and 101 MHz and 201 MHz for ¹³C spectra. ¹H and ¹³C NMR spectra were referenced relative to

13

TMS (δ = 0.00 ppm) C NMR spectra were proton-decoupled. Chemical shifts (δ) are reported

in ppm and coupling constants in Hz. Electro spray ionization (ESI) mass spectrometry (MS)

experiments were performed on Agilent Technologies 6530Accurate-Mass QTOF LC/MS.

4.2. Chemistry

4.2.1 General Procedure for the Preparation of α, β-unsaturated Compounds (1a-1v).

A mixture of substituted aldehyde (10 mmol), ethyl cyanoacetate or malononitrile (10.1

mmol) and piperidine (20 mol %) in 25 ml ethanol was reflux at 80-85 °C for 4-8h and room

temperature for 8-24h. The progress of the reaction was monitored by TLC. After completion of

reaction, as monitored by TLC, the reaction mixture was directly charged onto silica gel column

and eluted with a mixture of ethyl acetate: n-hexane to afford pure olefin. All the products were

prepared by using the same procedure. The olefinic products thus obtained were characterized by

comparison of their ¹H NMR, ¹³C NMR spectroscopy and mass spectroscopic.

4.2.2 General procedure for preparation of stereoselective oximes (2a-2v).

To the stirred sodium carbonate (632 mg, 5.9 mmol), hydroxylamine hydrochloride (414mg,

5.9 mmol), water (2.5 mL), and the solution of substituted Ethyl (E)-2-cyano-3-phenylacrylate

(4.9 mmol), methanol (22.5 mL) was added in the reaction mixture and refluxed for 1h. Reaction

monitored with TLC, after completion of reaction, the mixture was concentrated under reduced

pressure and the crude compound purified with column chromatography elution ingredients

hexane: ethyl acetate.

4.2.2.1 (Z)-benzaldehyde oxime (2a). Isolation condition: Hexane: ethyl acetate (95:5), Yellow

gel (556mg, 92%) ¹H NMR (400 MHz, CDCl₃) δ 8.76 (s, 1H), 8.17 (s, 1H), 7.60 – 7.54 (m, 2H),

7.41 – 7.36 (m, 3H).¹³C NMR (101 MHz, CDCl₃) δ 150.42, 131.92, 130.13, 128.83, 127.08,

77.06, HRMS (ESI): Calculated for C₇H₇NO [M+H]⁺: 122.0528, found 122.0600.

4.2.2.2 (Z)-4-hydroxybenzaldehyde oxime (2b). Isolation condition : Hexane: ethyl acetate

1

(8:2), White solid (450mg, 83.9%), H NMR (800 MHz, DMSOD₆) δ 10.83 (s, 1H), 9.73 (s,

13

1H), 7.99 (s, 1H), 7.39 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H). C NMR (201 MHz,

DMSOD₆) δ 159.00, 148.34, 128.41, 124.48, 116.00, 40.24, 40.14, 40.03, 39.93, 39.82, 39.72,

39.62, 38.02, HRMS (ESI): Calculated for C₇H₇NO₂[M+H]⁺: 138.0477, found 138.0532.

4.2.2.3 (Z)-3,4-dihydroxybenzaldehyde oxime (2c). Isolation condition : Hexane: ethyl acetate

1

(5:5), White solid (401mg, 77%), H NMR (400 MHz, DMSOD₆) δ 10.71 (s, 1H), 9.15 (s, 1H),

9.03 (s, 1H), 7.82 (d, J = 24.7 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 6.74 (dd, J = 8.2, 1.9 Hz, 1H),

13

6.66 (d, J = 8.1 Hz, 1H). C NMR (101 MHz, DMSOD₆) δ 148.59, 147.40, 145.93, 124.91,

119.71, 115.98, 113.04, 40.52, 40.31, 40.11, 39.90, 39.69, 39.48, 39.27, HRMS (ESI):

Calculated for C₇H₇NO₃[M+H]⁺: 154.0426, found 154.0459.

4.2.2.4 (Z)- 4-methoxybenzaldehyde oxime (2d).Isolation condition: Hexane: ethyl acetate (8:2),

1

Pale yellow semi solid (550mg, 85.6%), H NMR (400 MHz, CDCl₃) δ 9.25 (s, 1H), 8.11 (s,

13

1H), 7.53 – 7.48 (m, 2H), 6.92 – 6.87 (m, 2H), 3.81 (s, 3H) C NMR (101 MHz, CDCl₃) δ

161.08, 150.02, 128.59, 124.59, 114.30, 77.09, 55.36, HRMS (ESI): Calculated for C₈H₉NO₂

[M+H]⁺: 152.0633, found 152.0667.

4.2.2.5(Z)-3-methylbenzaldehyde oxime (2e).Isolation condition, Hexane: ethyl acetate (9:1),

1

Transparent liquid (560mg, 89%), H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 13.2 Hz, 1H), 8.43

(s, 1H), 7.68 – 7.64 (m, 1H), 7.28 (td, J = 7.4, 1.4 Hz, 1H), 7.24 – 7.17 (m, 2H), 2.43 (s, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 149.30, 136.84, 130.87, 130.22, 129.88, 126.72, 126.27, 77.36,

77.05, 76.73, 19.77, HRMS (ESI): Calculated for C₈H₉NO [M+H]⁺: 136.0685, found 136.0758.

4.2.2.6 (Z)-3,4-dimethoxybenzaldehyde oxime (2f). Isolation condition, Hexane: ethyl acetate

(8:2), White solid (598mg, 86%),¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 8.11 (s, 1H), 7.24

(d, J = 1.9 Hz, 1H), 7.05 (dd, J = 8.3, 1.9 Hz, 1H), 6.89 – 6.86 (m, 1H), 3.92 (s, 6H). ¹³C NMR

(101 MHz, CDCl₃) δ 150.82, 150.24, 149.30, 124.82, 121.73, 110.76, 107.99, 77.39, 77.07,

76.75, 55.94, 55.89, HRMS (ESI): Calculated for C₉H₁₁NO₃[M+H]⁺: 182.0739, found 182.0802.

4.2.2.7 (Z)- 2,3,4-trimethoxybenzaldehyde oxime (2g). Isolation condition, Hexane: ethyl

1

acetate (7.5:2.5), White crystalline solid (635mg, 88%), H NMR (400 MHz, CDCl₃) δ 8.29 (s,

1H), 7.35 (d, J = 8.8 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H).

¹³C NMR (101 MHz, CDCl₃) δ 155.29, 152.69, 146.29, 142.17, 121.45, 118.63, 107.88, 77.05,

61.72, 60.95, 56.08, HRMS (ESI): Calculated for C₁₀H₁₃NO₄[M+H]⁺: 212.0845, found

212.0917.

4.2.2.8 (Z)-4-hydroxy-3-methoxybenzaldehyde oxime (2h). Isolation condition, Hexane: ethyl

acetate (7:3), pale yellow solid (550mg, 85%),¹H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 9.36

(s, 1H), 8.00 (s, 1H), 7.17 (d, J = 1.5 Hz, 1H), 6.98 (dd, J = 8.1, 1.6 Hz, 1H), 6.79 (d, J = 8.1 Hz,

1H), 3.78 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 148.50, 148.42, 148.26, 124.88, 120.93,

115.89, 109.59, 55.89, 39.93, HRMS (ESI): Calculated for C₈H₉NO₃[M+H]⁺: 168.0582, found

168.0627.

4.2.2.9 (Z)-1-naphthaldehyde oxime (2i). Isolation condition, Hexane: ethyl acetate (8.5:1.5),

Light green solid (600mg, 88%), ¹H NMR (800 MHz, DMSO) δ 11.49 (s, 1H), 8.79 (s, 1H), 8.67

(d, J = 8.4 Hz, 1H), 7.96 (t, J = 9.2 Hz, 2H), 7.80 (d, J = 7.0 Hz, 1H), 7.57 (ddd, J = 23.4, 15.1,

7.4 Hz, 3H). ¹³C NMR (201 MHz, DMSO) δ 148.31, 133.49, 130.01, 129.73, 128.79, 128.66,

127.01, 126.93, 126.20, 125.56, 124.75, 39.62, 39.51, 39.40, HRMS (ESI): Calculated for

C₁₁H₉NO [M+H]⁺: 172.0684, found 172.0745.

4.2.2.10 (Z)-4-fluorobenzaldehyde oxime (2j). Isolation condition, Hexane: ethyl acetate (9:1),

1

White crystalline solid (580mg, 92%), H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 7.82 (s, 1H),

7.60 – 7.53 (m, 2H), 7.12 – 7.04 (m, 2H) , ¹³C NMR (101 MHz, CDCl₃) δ 165.06, 162.57,

149.30, 128.94, 128.86, 128.16, 116.09, 115.87, 77.35, 77.03, 76.71, HRMS (ESI): Calculated

for C₇H₆FNO [M+H]⁺: 140.0433, found 140.0490.

4.2.2.11(Z)-2-chlorobenzaldehyde oxime (2k). Isolation condition, Hexane:ethyl acetate (9:1),

White crystalline solid( 580mg, 88%),¹H NMR (400 MHz, CDCl₃) δ 8.58 (s, 1H), 8.31 – 8.22

(m, 1H), 7.82 (dd, J = 7.7, 1.8 Hz, 1H), 7.41 – 7.38 (m, 1H), 7.32 (ddd, J = 9.8, 5.7, 2.0 Hz, 1H),

13

7.29 – 7.24 (m, 1H). C NMR (101 MHz, CDCl₃) δ 147.60, 133.97, 132.03, 131.01, 129.95,

129.81, 129.77, 127.16, 127.03, 126.55, 77.34, 77.03, 76.71, HRMS (ESI): Calculated for

C₇H₆ClNO [M+H]⁺: 156.0138, found 156.0192.

4.2.2.12 (Z)-3-chlorobenzaldehyde oxime (2l). Isolation condition, Hexane:ethyl acetate (9:1),

White crystalline solid( 581mg, 88%),¹H NMR (400 MHz, CDCl₃) δ 8.18 (d, J = 1.7 Hz, 1H),

8.10 (s, 1H), 7.59 (q, J = 1.6 Hz, 1H), 7.44 (dt, J = 7.3, 1.3 Hz, 1H), 7.36 (dt, J = 3.5, 1.5 Hz,

1H), 7.34 – 7.29 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 149.19, 134.87, 133.75, 130.04,

126.85, 125.27, 77.35, 77.03, 76.71, HRMS (ESI): Calculated for C₇H₆ClNO [M+H]⁺: 156.0138,

found 156.0200.

4.2.2.13(Z)-4-bromobenzaldehyde oxime (2m). Isolation condition, Hexane: ethyl acetate (9:1),

White crystalline solid(480mg, 90%),¹H NMR (400 MHz, CDCl₃) δ 8.27 (s, 1H), 8.10 (s, 1H),

7.55 – 7.49 (m, 2H), 7.46 – 7.39 (m, 2H).¹³C NMR (101 MHz, CDCl₃) δ 149.43, 132.06, 130.87,

128.46, 124.32, 77.36, 77.04, 76.72, HRMS (ESI): Calculated for C₇H₆BrNO [M+H]⁺: 199.9633,

found 199.9679

4.2.2.14 (Z)-O-(4-((hydroxyimino)methyl)-3-methoxyphenyl) dimethyl carbamothioate (2n).

Isolation condition, Hexane: ethyl acetate (7:3), White crystalline solid(840mg, 90.71%),¹H

NMR (400 MHz, CDCl₃) δ 8.71 (s, 1H), 8.12 (s, 1H), 7.28 (d, J = 1.7 Hz, 1H), 7.11 (dd, J = 8.2,

13

1.8 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H), 3.84 (s, 3H), 3.45 (s, 3H), 3.35 (s, 3H). C NMR (101

MHz, CDCl₃) δ 187.43, 151.90, 149.78, 144.31, 130.78, 124.33, 120.59, 109.87, 77.45, 77.14,

76.82, 56.07, 43.43, 38.84, HRMS (ESI): Calculated for C₁₁H₁₄N₂O₃S [M+H]⁺: 255.0725, found

255.0798.

4.2.2.15(Z)-4-(dimethylamino)benzaldehyde oxime (2o). Isolation condition, Hexane: ethyl

acetate (9:1), Light yellow crystalline solid(500mg, 86%),¹H NMR (400 MHz, CDCl₃) δ 8.79 (s,

1H), 8.06 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 2.99 (s, 6H). ¹³C NMR (101

MHz, CDCl₃) δ 151.56, 150.45, 128.34, 119.64, 111.95, 77.08, 40.25, HRMS (ESI): Calculated

for C₉H₁₂N₂O [M+H]⁺: 165.0950, found 165.1008.

4.2.2.16(Z)-2-nitrobenzaldehyde oxime (2p). Isolation condition, Hexane: ethyl acetate (8:2),

Light yellow crystalline solid(598mg, 85%),¹H NMR (400 MHz, CDCl₃) δ 8.69 (s, 1H), 8.12 (s,

1H), 8.07 (dd, J = 8.2, 1.1 Hz, 1H), 7.92 (dd, J = 7.8, 1.5 Hz, 1H), 7.69 – 7.62 (m, 1H), 7.56

13

(ddd, J = 8.2, 7.6, 1.5 Hz, 1H). C NMR (101 MHz, CDCl₃) δ 147.08, 133.58, 130.44, 128.83,

127.16, 124.88, 77.35, 77.03, 76.72, HRMS (ESI): Calculated for C₇H₆N₂O₃[M+H]⁺: 167.0378,

found 167.0448.

4.2.2.17(Z)-4-nitrobenzaldehyde oxime (2q). Isolation condition, Hexane: ethyl acetate (8:2),

Light yellow crystalline solid (571mg, 85%),¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 8.6 Hz,

13

2H), 8.23 (s, 1H), 7.98 (s, 1H), 7.77 (d, J = 8.6 Hz, 2H). C NMR (101 MHz, CDCl₃) δ 148.51,

148.40, 138.16, 127.68, 124.08, 77.35, 77.03, 76.72, HRMS (ESI): Calculated for C₇H₆N₂O₃

[M+H]⁺: 167.0378, found 167.0449.

4.2.2.18(Z)-4-((hydroxyimino)methyl)benzonitrile (2r). Isolation condition, Hexane: ethyl

1

acetate (8.5:1.5), Bright needle crystalline solid (574mg, 89%), H NMR (400 MHz, DMSO) δ

13

11.74 (s, 1H), 8.24 (s, 1H), 7.86 (d, J = 6.7 Hz, 2H), 7.78 (d, J = 1.7 Hz, 2H). C NMR (101

MHz, DMSO) δ 147.59, 138.08, 133.14, 127.48, 119.17, 111.84, 40.59, 40.38, 40.17, 39.96,

39.75, 39.54, 39.33, HRMS (ESI): Calculated for C₇H₆N₂O₃[M+H]⁺: 147.0480, found 147.0514.

4.2.2.19(Z)-1H-indole-3-carbaldehyde oxime (2s). Isolation condition, Hexane: ethyl acetate

1

(7:3), Light yellow solid (359mg, 85%), H NMR (400 MHz, DMSO) δ 11.40 (s, 1H), 10.52 (s,

1H), 8.29 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.63 (d, J = 2.7 Hz, 1H), 7.45 – 7.41 (m, 1H), 7.20 –

7.15 (m, 1H), 7.13 – 7.07 (m, 1H). ¹³C NMR (101 MHz, DMSO) δ 145.09, 137.35, 128.85,

124.71, 122.75, 121.94, 120.50, 112.24, 110.09, 40.59, 40.38, 40.17, 39.97, 39.76, 39.55, 39.34,

HRMS (ESI): Calculated for C₉H₈N₂O [M+H]⁺: 161.0637, found 161.0687.

4.2.2.20 (Z)-heptanal oxime (2t). Isolation condition, Hexane: ethyl acetate (9:1), Liquid

(400mg, 46%),¹H NMR (400 MHz, CDCl₃) δ 7.95 (s, 1H), 6.72 (t, J = 5.5 Hz, 1H), 2.38 (td, J =

7.5, 5.5 Hz, 2H), 1.54 – 1.44 (m, 2H), 1.39 – 1.25 (m, 6H), 0.92 – 0.87 (m, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 153.02, 77.34, 77.23, 77.03, 76.71, 31.51, 29.04, 26.02, 24.97, 22.53, 14.03,

HRMS (ESI): Calculated for C₇H₁₅NO [M+H]⁺: 130.1154, found 130.1187.

4.2.2.21(Z)-Octanal oxime (2u):¹H NMR (400 MHz, CDCl₃) δ 9.92 (s, 1H), 9.92 (s, 1H), 7.75

(d, J = 9.5 Hz, 1H), 7.75 (d, J = 9.5 Hz, 1H), 6.20 – 5.98 (m, 2H), 6.19 – 5.98 (m, 2H), 2.16 (dd,

J = 14.2, 7.2 Hz, 2H), 1.47 – 1.36 (m, 2H), 1.35 – 1.20 (m, 6H), 0.89 (t, J = 6.8 Hz, 3H).¹³C

NMR (101 MHz, CDCl₃) δ 151.82, 143.02, 123.63, 77.41, 77.09, 76.77, 32.93, 32.78, 31.63,

28.81, 28.61, 28.46, 14.03.

4.2.2.22(Z)-2-Methyl prapanal oxime (2v): ¹H NMR (400 MHz, CDCl₃) δ 7.47 (s, 1H), 3.36 (d,

J = 2.8 Hz, 1H), 2.01 – 1.90 (m, 1H), 1.17 (d, J = 17.1 Hz, 6H).¹³C NMR (101 MHz, CDCl₃) δ

158.22, 81.99, 77.34, 77.02, 76.71, 41.67, 29.30, 24.30, 22.60, 22.49, 16.59.

5. Biological and computational study

5.1. In silico study

5.1.1. Molecular docking studies of 2d (p-Methoxy benzyl oxime) and 2g (2,3,4-

Trimethoxybenzyl oxime)

PDB structure of MRCK kinase was downloaded from PDB (ID: 5OTE) isolated from

Escherichia coli. The obtained structure of the protein is made of 419 amino acid and the active

site

of

the

enzyme

was

determined

using

an

online

server

CASTp

http://sts.bioe.uic.edu/castp/index.html?j_5d6259eb0df35. Active site of the enzyme was

recognized with surface area of 573.84 and volume of 546.66. The 3D structure of the 2d and 2g

were drawn and validated using ChemBioDrwa (3D). Windows based docking tool Autodock

4.2.6 was used for docking study. Discovery studio visualizer was used to analyze the molecular

interactions between docked molecules with MRCK Kinase. Autodock 4.2.6 tool used for

molecular docking study. Docking analysis was initiated with the blind docking followed by site

specific docking to find out the best-fit protein-ligand docked model for the Tamoxifen and 2d

and 2g. All water molecules of MRCK kinases were removed and polar hydrogen atoms and

Gasteiger charges were added. Interaction between MRCK Kinase protein and compound 2d and

2g are respected GLN (144), VAL (35), ASP(32) and VAL (35), GLN (144), HIS (395), LYS

(109), GLN (70) (Figure 2.).

5.1.2 Invitro study

5.1.3. Experimental

Nine cancer cell lines were used for testing the prepared oximes viz. A431 (human

epidermoid carcinoma cell line); A549 (adenocarcinomic human alveolar basal epithelial cells);

L-132 (Human lung cells carcinoma).; NCI-H 460 (non-small-cell lung cancer cell lines); NCI-

H520 (non-small cell lung carcinoma cell lines); PC3 (PC-3) is a human prostate cancer cell line;

HepG2 (human liver cancer cell line); MDA-MB-231 (epithelial, human breast cancer cell

line);.MCF-7 (b The RPMI & DMEM media, trypsin, antibiotic-antimycotic (Ab/Am) solution,

Sodium bicarbonate, HEPES and MTT dye were purchased from Sigma-Aldrich, India. The fetal

bovine serum was obtained from Gibco-BRL, India. The 96-well plates, T-25 and T-75 flasks

were procured from Nunc, Thermo-Fisher Scientific, and India. DMSO and ethanol were

purchased from Merck Pvt. Ltd., India.

The compounds were tested against a set of of cancer cell lines including MCF-7, A431, A549,

PC-3, HepG2, MDAMB-231, L-132, NCIH-520, NCIH-460 procured from the National Centre

for Cell Science (NCCS), Pune, India. The antiproliferative potential of the synthesized

compounds was evaluated by employing MTT assay and the experiments were performed by

following the method reported previously[42]. In brief, the cells were grown in a CO₂incubator

at 37°C and 85% humidity in media supplemented with 1 % Ab/Am and 10 % FBS.

Experimentally, the cells were seeded in a 96-well flat bottom plate and incubated for 24h. After

incubation, the cells were treated with compounds at different concentrations (0.4-50µg/mL) and

further incubated for 24 h. The MTT dye was added and then plate was incubated for 4 h at

37°C in dark. After that, the dye was removed and DMSO was added to dissolve the formazan

crystals followed by recording of absorbance at 570nm. Tamoxifen was used as a standard. The

percent cytotoxicity and inhibitory concentration (IC₅₀) was calculated as reported previously

[43] (Table 4, Fig 1). reast cancer cell line).

5.1.3. Cell cycle analysis

Cell cycle analysis was performed to check the effect of compound (2g and 2d) in a

different phase of the cell cycle in. The cell lines A431 and NCIH-460 were treated with the

compound 2g and 2d at IC₅₀and double of IC₅₀concentrations. The PI staining method was used

as reported previously [44]. The cells (A431 and NCIH-460) were seeded in 6 well plates and

incubated for 24h. After incubation, the cells were treated with the compounds for 24h at 37°C.

After that, the cells were collected and fixed in 70% ice-cold ethanol. Afterward, the cells were

re-suspended in PBS and add DNA extraction buffer and further incubated for 5 min. After that,

cells were centrifuged and re-suspended in the buffer and incubated with propidium iodide and

RNAse A for 30 min in the dark at room temperature. The samples were analyzed by LSRII

Flow-Cytometer (BD Biosciences).

5.1.4.Annexin V-FITC assay

The apoptosis assay was performed as per the reported protocol [45]. NCIH-460 cells were

seeded in 6 well plates and incubated for 24h. The cells were treated with compounds 2g and 2d

at different concentration (IC₅₀and double of IC₅₀) for 24h. After incubation, the cells were

collected and washed with ice-cold PBS centrifuged for 5 min at 5000 rpm. The pellet was

dissolved in 1x binding buffer, and the dyes FITC-annexin V and propidium iodide were added

as per the manufacture protocol of BD Bioscience kit manual. The samples were incubated for

15 min, and after that analyzed by the LSRII Flow-Cytometer (BD Biosciences). The data were

analyzed as reported earlier protocol [46].

5.1.5. Reactive oxygen species (ROS) assay

The ROS formation was determined by using DCFDA dye as per the protocol of

Hamidullah et al., 2015. The NCIH-460 cells were seeded in 6 well plates for 24h. The cells

were treated with the compound 2g and 2d at different concentration (IC₅₀and double of IC₅₀)

for 24h. After incubation, the cells were collected and further incubate with DCFDA dye at room

temperature for 30 min in the dark. After that, the cells were centrifuged at 5000 rpm and re-

suspended in PBS. The samples were analyzed by using LSRII Flow-Cytometer (BD

Biosciences).

5.1.6. Mitochondrial membrane potential

The mitochondrial membrane potential was measured using rhodamine 123 as per the

method of Yu et al., 2015. The NCIH-460 cells were seeded in 6 well plates and incubated for 24

h. Different concentrations (IC₅₀and double of IC₅₀) of 2g and 2d were added and further

incubated for 24h. After incubation, the cells were collected and treated with rhodamine dye at

37°C in the dark for 30 min. The cells were washed with PBS and re-suspended in PBS. The

samples were analyzed by LSRII Flow-Cytometer (BD Biosciences).

6. Conclusion

An unexpected mild base catalyzed Michael type nucleophilic addition reaction followed with

Retro-Knoevenagel reaction with aqueous hydroxylamine involving C-C bond cleavage in case

of α -cyano substituted carbonyl conjugated and other similarly activated alkenes has been

exploited for the formation of stereoselective aryl/alkyl oximes. The new procedure involves 1,

3 proton shift, Michael addition type reaction, followed by carbon-carbon bond cleavage

resulting in elimination of ethyl cyanoacetate/ malononitrile/ dimethyl malonate. The yields of

oximes were in the order heterocyclic>aryl>alkyl. Our new greener strategy presents one step

and cost effective preparation of predominantly Z -oximes. Moreover, utility of carbon-carbon

bond cleavage in the reaction, offered a great advantage in allowing water for the Michael

addition to afford the corresponding oximes.

Out of the twenty two prepared Z-oximes two viz. p-Methoxy benzyl oxime and 2,3,4-

Trimethoxy benzyl oxime were found to be cytotoxic in all the nine cancer cell lines used. The

cell lines used were A431 (human epidermoid carcinoma cell line); A549 (adenocarcinomic

human alveolar basal epithelial cells); L-132 (Human lung cells carcinoma).; NCI-H 460 (non-

small-cell lung cancer cell lines); NCI-H520 (non-small cell lung carcinoma cell lines); PC3

(PC-3) is a human prostate cancer cell line; HepG2 (human liver cancer cell line); MDA-MB-

231 ( epithelial, human breast cancer cell line);. MCF-7 (breast cancer cell line). MIT, Cell cycle,

Apoptotic, Reactive oxygen and Mitochondrial membrane assays were performed to evaluate the

anticancer activity .These two oximes were found to inhibit MRCK Kinase, responsible for

metastatic spread of cancer mortality.

Acknowledgements

This work was financially supported by the National Academy of Science India, (Senior Scientist

Grant), Allahabad.

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Table 1: Optimization of the reaction conditions

Entry

1.

Base

Time(h)

Solvent

MeOH

Temp. ( ^°C)

Yield (%)^b

Na₂CO₃

1

4

60

70

-

2.

EtOH

3.

THF

70

-

5.

DMF

100

rt

-

6.

DMSO

-

7.

MeOH/Water^a

EtOH / Water^a

THF/ Water^a

DMSO/Water^a

Water

92^b

86

56

67

45

90

8.

9.

10.

11.

12.

MeOH/Water^a

Note: a Solvent / water (9:1), b Isolated yield.

Table 2: Impact of base on yield of benzyl oxime.

Entry

Base

Time

(h)

Temperature (°C Solvent:water

Yield (%)

)

(9:1)

1.

2.

3.

DABCO

TEA

1

100

MeOH : Water

68

66

90

100

MeOH : Water

K₂CO₃

4.

5.

6.

7.

Na₂CO₃

DBU

1

100

MeOH : Water

92

88

75

74

NaOH

KOH

Note: Reaction condition: Ethyl (E)-2-cyano-3-phenyl (4.9 mmol), hydoxylamine hydrochloride

(5.97 mmol), Base (5.97 mmol), 100 °C, 1h.

Table 3: Substrate scope of oximes.

General reaction conditions: ethyl (E)-2-cyano-3-aryl/alkyl acrylate (4.9 mmol),

hydoxylamine hydrochloride (5.97 mmol), sodium carbonate (5.97 mmol), 100 °C, 1h.

Table 4: IC₅₀value of synthesized compound for tested cell line.

Entry A-431

A-549

L-132

NCIH-460 NCIH-520 PC-3

HepG-2

MDA-MB- MCF-7

231

2b

2c

-

9.11±1.54 22.83±1.5 37.84±1.2 8.63±0.15 19.18±2.9 15.13±2.0 21.02±1.7 23.84±0.5 21.34±1.5

2d

2e

2f

2

7

9

2

5

0

1

-

10.24±0.9 12.53±0.9 44.26±0.3 9.42±0.39 16.15±0.6 14.94±0.9 16.44±0.2 17.66±0.6 18.75±3.3

2g

2h

0

6

9

1

9

7

9

-

12.25±0.4 24.49±1.2 28.69±0.8 22.29±1.0

-

18.39±2.1

0

-

21.76±0.9 23.09±3.7

2i

2j

0

7

6

8

4

3

-

40.80±1.5

5

-

2k

-

38.84±0.7

0

-

2l

2m

2n

2o

2p

2q

2r

-

Article Doi

Water mediated procedure for preparation of stereoselective oximes as inhibitors of MRCK kinase

DOI: 10.1016/j.molstruc.2020.128699

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Article abstract of DOI:10.1016/j.molstruc.2020.128699

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