Büchi's model based analysis of local anesthetic action in procaine hydrochloride: Vibrational spectroscopic approach

Accepted Manuscript Büchi's model based analysis of local anesthetic action in procaine hydrochloride: Vibrational spectroscopic approach

Y. Sheeba Sherlin, T. Vijayakumar, J. Binoy, S.D.D. Roy, V.S. Jayakumar PII: DOI: Reference:

S1386-1425(18)30660-7 doi:10.1016/j.saa.2018.07.007 SAA 16276

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

13 May 2018 4 July 2018 5 July 2018

Please cite this article as: Y. Sheeba Sherlin, T. Vijayakumar, J. Binoy, S.D.D. Roy, V.S. Jayakumar , Büchi's model based analysis of local anesthetic action in procaine hydrochloride: Vibrational spectroscopic approach. Saa (2018), doi:10.1016/ j.saa.2018.07.007

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ACCEPTED MANUSCRIPT Büchi’s Model based Analysis of Local Anesthetic Action in Procaine Hydrochloride: Vibrational Spectroscopic Approach Y. Sheeba Sherlina,b,1, T.Vijayakumarc, J.Binoyd, S.D.D. Roya,b and V.S. Jayakumare,* a

Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli-627012, Tamil Nadu, India.

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Abstract

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Department of Physics, Nesamony Memorial Christian College, Marthandam-629165, Tamil Nadu, India. c Department of Physics and Nanotechnology, SRM University, Kattankulathur, Kancheepuram-603203, India. d Department of Physics, Government College for Women, Thiruvananthapuram-695014. Kerala, India. e Mar Baselios Institute of Technology, Anchal- 691306, Kerala, India. * Corresponding author: [email protected]

The drug action of ester type local anesthetic (LA) procaine hydrochloride (PRC HCl) is activated

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by blocking Na+ ion flow when it binds to the ion channel in the ligand gated sodium ion channel protein. Buchi’s model, explains binding action of ester type LA drug with receptor in terms of

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charge transfer, dipole-dipole, hydrogen bonding and van der Waals interactions through lipophilic, ester and hydrophilic moieties. The present work investigates molecular structural and

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vibrational spectral features of para amino benzoate group, ester part and tertiary amino group respectively belonging to lipophilic, ester and hydrophilic moieties, accountable for the binding of

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drug to sodium channel. The electron transport mechanism through the ring responsible for structural deviation from benzenoid to quinonoid form and consequent dipolar nature of carbonyl

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group have been investigated, based on the analysis of XRD, DFT computed molecular structure, 8a ring mode and NBO charges. The characteristic UV absorption peaks and vibrational marker

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bands of LA drugs have been identified and the charge transfer interaction responsible for lipophilic binding has been investigated. The blocking of Na+ in the ion channel has been probed using attractive and repulsive energy profile. The molecular polarizability has been computed to substantiate the correlation between the structure activity relationship of LA drug molecule and molecular polarizability. The low toxicity of PRC HCl was evaluated using in vitro cytotoxicity study, confirming it as a potential short acting local anesthetic. Keywords:

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FT-Raman, DFT, Büchi’s model, Quinonoid structure, Local anesthetic spectral bands, Cytotoxicity.

Permanent Address: Department of Physics, Scott Christian College (Autonomous), Nagercoil-629003, Tamil Nadu, India.

ACCEPTED MANUSCRIPT 1. Introduction Local anesthetics (LA) are amphiphile molecules extensively used to avoid the sense of pain during medical and dental procedures by binding to and inactivating ion channels of nerve membrane [1]. LA penetrates the nerve membrane (phospholipid) as uncharged molecules and interacts with the receptor in their cationic form [2]. The three principal components of LA molecules are lipophilic aromatic ring, intermediate ester or amide chain and hydrophilic terminal

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amine. Each of these components contributes distinct characteristics to the anesthetics viz, protein

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binding, chemical linkage, lipid solubility and dissociation which are respectively correlated to the

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affinity for plasma proteins, metabolism, time of onset and potency of the drug action [3]. Procaine hydrochloride (PRC HCl), also called Novocaine, is a benzoic acid derivative (ester type) with LA

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activity.

The Procaine hydrochloride molecules which are being metabolized in the plasma by the

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enzyme pseudo-cholinesterase through hydrolysis into p-amino-benzoic acid (pABA), a B vitamin, and diethyl amino ethanol (DEAE), a precursor to the B vitamin choline, function in the

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body as a true vitamin molecule at the cellular level effecting cell membrane [4]. PRC HCl is also used as an active ingredient of an anti-aging drug, GH3 (Gerovital H3), and in a few anti-aging

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creams, because of the unique ability of the molecule to transfer the nutrients that are essential to initiate repairing of the damaged membrane of diseased cells and provide recovery for the cell life

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extension [5]. PRC HCl, being a local anesthetic, has been reported as inhibitors of DNA methylation, causing demethylation and reactivation of methylation-silenced genes,[6,7] which

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suggests that procaine and its derivatives may have a potential use for preventing the development of human cancer cells. In addition PRC HCl has also been of much more recent interest due to its anti-inflammatory,

anti-rheumatic,

anti-oxidative

and

anti-spasmodic

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anti-depressive,

pharmaceutical features [8]. Vibrational spectroscopy, being a non-invasive, rapid and high spatial resolution acquisition technique, has become a powerful spectroscopic tool that can provide molecular fingerprint information on biochemical components of complex biological systems [9-11]. The ultimate goal of this investigation is to elucidate the molecular features responsible for the drug action of PRC HCl from its vibrational spectra based on the three principal components of ester type LA which are activated in the binding site through charge transfer interaction.

ACCEPTED MANUSCRIPT 2. Materials and Methods Experimental The LA drug PRC HCl, 2-diethylaminoethyl 4-aminobenzoate hydrochloride (Fig.1), purchased from Aldrich (99.9 %) was used without further purification. A BRUKER RFS 27: FTRaman spectrometer using an Nd:YAG laser at 1064 nm as the excitation source with a resolution of 2 cm-1, was used to measure the NIR-FT Raman spectrum in the region 4000-50 cm-1. The FTIR

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spectrum of PRC HCl in the region 4000-400cm-1 was recorded using a Perkin-Elmer GX FT-IR

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spectrometer with the sample in KBr matrix having a resolution of 4 cm-1. The UV-Vis absorption

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spectrum of PRC HCl was measured using Cary 5000 UV-Vis-NIR spectrophotometer (200-600

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nm).

Fig. 1 Optimized molecular structure of PRC HCl

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Cytotoxic Protocol The cytotoxicity of ligand PRC HCl towards L929 fibroblast cells were used to study its

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cell viability. The cell lines were cultured in DMEM media supplemented with 10% fetal bovine serum, L-glutamine, sodium bicarbonate and penicillin-streptomycin in a humidified 5% CO2 incubator maintained at 37ºC. Before doing the MTT (Methylthiazolyl diphenyl-tetrazolium bromide) assay the cells were seeded in a 96 well tissue culture plate at a density of 5x10 4 cells/well, cultured for 24 hours and then treated with various dosages of PRC HCl (100 µg, 50 µg, 25 µg, 12.5 µg, 6.25 µg in 500 µL of 5% DMEM) and incubated. After 24 hours 30 µL of reconstituted MTT was added to all wells and further incubated at 37ºC for 4 hours following which the supernatant was discarded and 100 µL of MTT solubilization solution dimethyl

ACCEPTED MANUSCRIPT sulfoxide was added and mixed gently. The plates were read at 540 nm using ELISA [12] microplate reader (ERBA, GERMANY). Computational Details The molecular structural optimization of PRC HCl (Table.1) was performed with the Gaussian 09W program package [13] using DFT method with B3LYP functional using 6311++G(d,p) basis set. The calculated harmonic vibrational wavenumbers were uniformly scaled

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down [14] by 0.9679 to account for systematic errors owing to harmonic approximation [15].

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using Auto Dock Tools (ADT) Version 1.5.6 revision 30 [16].

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Optimized structure of PRC HCl was docked with pentameric ligand gated ion channel (PLGIC),

For the analysis of characteristic Local Anesthetic Spectral Peaks (LASP) in UV Visible

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spectra, the simulation of electronic absorption spectra, have been performed using time dependent (TD) DFT at B3LYP/6-31G(d,p) level. Since the computations over estimates the upward vertical

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electronic transition energies, the electronic transition energy has been corrected, the correlation factor being equal to 0.844 [17]. The vibrational modes are assigned on the basis of potential

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energy distribution (PED) analysis using VEDA4 program [18,19]. The experimental and calculated Raman and IR spectra of PRC HCl (Fig. 2 and 3) are compared with the computed

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spectra, to reach reliable vibrational assignments (Table.2).

Fig. 2 Combined (a) experimental and (b) theoretical Raman spectrum of PRC HCl

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Fig. 3 Combined (a) experimental and (b) theoretical IR spectra of PRC HCl

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The X-ray diffraction (XRD) data of PRC HCl has been reported [20,21] which shows that the procaine hydrochloride molecules in the crystalline network are arranged in layers with the

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phenyl ring stacked in herringbone array alternating with layers of chloride ions and the structural

3. Results and discussion

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parameters has been used for the detailed molecular structural and vibrational analysis.

The local anesthetic action of a drug is based on the interaction of specific moieties of drug

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with PLGIC protein. The action is initiated by blocking Na+ ion flow through the channel due to repulsion from drug in cationic form, when drug is bound to the channel moiety (Fig.4). The

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binding is molecular structure dependent and takes place through the characteristic interactions of specific moieties. According to Büchi’s model [22], charge transfer and dipole-dipole interactions

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occur respectively through lipophilic and ester moieties, while hydrogen bonding (HB) and van der Waals interaction takes place through hydrophilic moieties (Fig.5). The molecular vibrational analysis aims to explore characteristic structural features of local anesthetic PRC HCl, based on Büchi’s model and their role in protein binding, responsible for ligand gated action in Na+ channel.

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Fig. 4 Local anesthetic action

Fig. 5 Büchi’s Model for the binding mechanism of ester type LA, PRC HCl with receptor

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Expt.a

Calc.

Bond angle

Expt.a

Calc.

Dihedral angle

Expt.a

Calc.

C1 - C2

1.396

1.402

C1-C2-C3

121.8

120.8

C1-C2-C3-C4

2.87

0.097

C2 - C3

1.370

1.385

C2-C3-C4

121.1

120.6

C2-C3-C4-C5

-4.94

-0.243

C3 - C4

1.404

1.407

C2-C1-C6

116.7

118.5

C3-C4-C5-C6

4.31

0.121

C4 - C5

1.407

1.407

C3- C4-C5

117.0

118.5

C5-C6-C1-C7

177.4

179.9

C5 - C6

1.355

1.383

C6-C1-C7

119.5

118.3

C6-C1-C7-O8

-172.6

-176.7

C6 - C1

1.410

1.403

C1-C7-O8

112.8

112.8

C2-C3-C4-N12

179.3

177.8

C1- C7

1.455

1.475

C7-O8-C9

C7 - O8

1.356

1.367

O8-C9-C10

O8 - C9

1.428

1.440

C10 - N23

1.505

1.499

C7 - O11

1.204

1.211

C4- N12

1.359

N12 - H13 N12 - H14

115.5

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C6-C5-C4-N12

172.3

177.7

109.5

109.5

C6-C1-C7-O11

7.011

2.862

C9-C10-N23

116.1

117.3

C1-C7-O8-C9

178.6

178.6

C3-C4-N12

121.1

120.7

O11-C7-O8-C9

1.028

1.718

C5-C4-N12

121.7

120.6

C7-O8-C9-C10

172.7

175.4

1.381

C10-N23-C24

112.1

112.1

O8-C9-C10-N23

70.23

68.62

0.714

1.008

N23-C24C25

112.2

113.6

C9-C10-N23-C31

-68.81

-75.39

0.805

1.007

C10-N23-C31

114.5

115.7

C9-C10-N23-C24

61.13

56.04

N23- C24

1.512

1.502

N23-C31-C32

112.2

113.4

C10-N23-C31-C32

-55.31

-55.36

N23 -C31

1.508

1.503

C1-C7-O11

126.5

125.3

C10-N23-C24-C25

158.5

163.1

N23 - H38

0.863

1.011

O11-C7-O8

120.6

121.8

C9-C10-N23-H38

177.2

169.1

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116.7

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Bond length

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Table 1. Experimental [20] and optimized bond lengths (Å), bond angles (o) and dihedral angles (o) of PRC HCl

Taken from Ref [20]

3.1. Structural and Vibrational Analysis

ACCEPTED MANUSCRIPT Since Büchi’s model explains the LA action in ester type drug taking place through specific moieties i.e. lipophilic, hydrophilic and ester moieties of the compounds, the investigation of structural features responsible for LA action in PRC HCl can be explored using the vibrational spectral analysis, of para amino benzoic acid ring (pABA ring) belonging to lipophilic moiety, tertiary amino group pertaining to hydrophilic moiety and ester moiety, supported by detailed molecular structural analysis.

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Table 2. The measured and calculated [B3LYP/6-311++G(d,p) level] wavenumbers and assignments of PRC HCl (harmonic frequencies (cm−1), infrared (νIR) intensities (km mol−1), Raman (νRaman) scattering activities (Å4 amu−1)) Cal

IR

Raman

3576

3353 (vs) 3315 (vs)

3316 (w)

3474

3209(s)

3208 (m)

3103

3077 (w)

3075 (m)

3094

3048 (w)

3054 (m)

Ring mode 20a (95)

3015 (w)

Ring mode 20b (90)

2974 (m)

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3010

2956 (m)

2940

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2956

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asymN12-H13(50)+ asym N12-H14(50) symN12-H13(50)+ sym N12-H14(50) Ring mode 2* (96)

Ring mode 7b (92)

2981 (s)

C31-H33(16)+C32-H35(10)+C32H37(50)

2957 (m)

C9-H19(18)+C9-H20(67)

2944 (m)

C24-H26(44)+C24-H27(18)+C25H28(10)+C25-H30(19)

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3002

2979

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M 3008(w)

3061

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3062

2992

PED (%)

2936(m)

C10-H21(78)+C10-H22(12)

2903(m)

C9-H19(73)+C9-H20(13) 2890 (s)

C32-H35(36)+ C32-H36(29)+C32H37(34)

2776

2773 (m) 2588 (vs) 2500 (s)

2780 (w) 2583 (w) 2496 (w)

N23-H38(88)

1701

1699 (vvs)

1692 (s)

O11-C7(85)

1610

1641 (vs)

1644 (vw)

-N12-H14(73)

1592

1606 (vs)

1602 (s)

Ring mode 8a (52)+H13-N12-H14(19)

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1574 (m)

1573 (vw)

Ring mode 8b (71)

1496

1519 (w)

1520 (vw)

N12-C4(11)+ Ring mode 19a (79)

1473

1478 (w)

1478 (vw)

H36-C32-H37(38)

1460

1464 (m)

1464 (s)

H19-C9-H20(54)

1455 (w)

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1456

H28-C25-H29(10)+H28-C25H29(10)+H33-C31-H34(10)+H35-C32H37(31) H35-C32-H37(13)+C10-C9-N23H21(27)+C10-C9-N23-H22(29)

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1453 (m)

1435 (m)

1422

1401 (w)

1380

1385 (m)

1385 (w)

1365

1363 (m)

1369 (w)

1319

1345 (s)

1291

1308 (s)

1237

1269 (vvs)

1165

1171 (s)

1155

1135 (m)

1135 (w)

Ring mode 9a (68)

1114

1113 (vs)

1114 (w)

Ring mode 18b (62)

1066 (m)

1069 (m)

C32-H35-C31-H36(26)

1049 (m)

1041 (m)

N23-C10(11)+H13-N12-C4(12)

1017 (m)

1018 (vw)

N23-C31(19)+C31-C32(16)

1005 (m)

1009 (vw)

C24-C25(35)+O8-C9(13)+N23-C24(15)

943

949 (vw)

Ring mode17a (84)

889

866 (s)

N23-C10(12)+,N23-C31(16)+H19-C9O8-C7(11)

1020 1003

Ring mode 19b (46)

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C32-H36-H37-H35(55)

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1307 (w)

1263 (vs)

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CE

1036

AC

1061

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1434

1163(m)

H21-C10-N23(26)+H26-C24N23(10)+H26-C24-N23(10)+H33-C31N23(16)+H19-C9-O8-C7(11) Ring mode 14 (53)+H13-N12-C4(14) Ring mode 3 (74) C1-C7(29)+O8-C7(10)+ H20-C9O8(17)+  C10-N23-H38 (10) C24-C25-N23-H26(10)+ C24-C25-N23H27(13)+C25-H28-C24-C29(17)

847

852 (m)

846 (s)

C9-C10(11)+O8-C9(12)

824

823 (w)

824 (s)

Ring mode 17b (38)

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770 (vs)

768 (vw)

C24-C25-N23-H26(16)+ C24-C25-N23H27(11)+C31-C32-N23-H33(11)

756

701 (m)

718 (vw)

Ring mode10b (34) +O11-C1-O8-C7(53)

683

654 (m)

642 (w)

Ring mode10a (45)+O11-C1-O8-C7(20)

631

637 (m)

610

608 (m)

609 (w)

Ring mode 6a (39)+O8-C7-O11(18)

558

567 (m)

571 (m)

C9-C10-N23(22)

497

507 (w)

498 (vw)

Ring mode 4 (80)

483

496 (w)

409

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C1-C7-O8(36)+C6-C1-C7(12)

470 (w)

472 (w)

402 (w)

416 (w)

C25-C24-N23(13)+N23-C10-Cl39-C31(19)

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459

Ring mode 6b (63)

Ring mode 16a (78)

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C10-N23-C31-C32(17)+C1-C7-O8C9(14)+C1-C7-O8-C9(14)+C6-C5-C4N12(18) Wilson numbering scheme has been used for ring modes,-Stretching,-in-plane bending, -outof-plane bending, -torsion, asym-asymmetric, sym-symmetric, vvs-very very strong, vs-very strong, s-strong, m-medium, w-weak, vw-very weak 116 (m) 101 (m)

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124

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3.1.1. Benzenoid vs Quinonoid Structure of pABA ring in Lipophilic moiety The analysis of geometric parameters of PRC HCl reveals that para amino benzoate group

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is found to exist in quinonoid resonance structure which is one of the tautomeric forms of benzene [23]. The origin of quinonoid form is found to arise from the electron donation of lone pair of

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electrons by the nitrogen of primary amine, inducing electron transfer through the ring (Fig.6), resulting in partial double bond character of C4-N12 and C1-C7 bonds, in addition to the double bond character of C2-C3 and C5-C6 bonds in the ring. It has also been observed that the transfer of electron from the nitrogen atom to the ring imposes an excess of negative charge at the ortho and para positions to amino group and is found to make the para disubstituted ring electron rich between ortho and meta position, ideal for charge transfer interaction.

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Fig. 6 Resonance Structure of para Amino Benzoate group The experimental C4-N12 bond length is measured to be 1.35 Å which is calculated as 1.38 Å and is lower than the normal C-N bond distance of 1.47 Å [24] that confirms its partial double

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bond character. Similar bond shortening can be observed for C1-C7 whose computed bond distance is 1.47 Å which is lower than the normal C-C bond distance of, 1.54 Å [25], as expected. The

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electron transport mechanism can be confirmed by the electron deficiency at meta position, which can be confirmed by the computed NBO charges (Fig.7). The double bond character of C2-C3 and

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C5-C6 within the ring, evident from the computed bond distance of 1.38 Å which is lower than other C-C bond length 1.40 Å of the ring, confirms its quinonoid nature and is supported by the

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corresponding experimental values of 1.355-1.37 Å, for double bonds and 1.40-1.41 Å, for other C-C bonds.

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The spectral consequence of the increase of C-N double bond character due to electronic transport through the ring leading to quinonoid form of the ring is the higher stretching frequency

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of C4-N12 bond, whose values are 1519 and 1520 cm-1 in IR and in Raman respectively as medium bands, supported by DFT computed frequency of 1496 cm-1. This band position is higher compared

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to normal aromatic C-N stretching region, 1342-1266 cm-1[26] and non-aromatic C-N stretching frequencies arising from the vibrations of N23-C10, N23-C24 and N23-C31, whose respective values in IR are 1049, 1017 and 1005 cm-1. The corresponding Raman bands can be found at 1041, 1018 and 1009 cm-1respectively, where the computed band positions are 1036, 1020 and 1003 cm-1 respectively.

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Fig.7 NBO Atomic Charges of PRC HCl

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Another spectral aftereffect is the increase of C1-C7 stretching frequency to 1237 cm-1, as predicted by DFT, attached to the ring, compared to normal aromatic C-C stretching region of

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1225-1075 cm-1 [27]. The corresponding strong IR and Raman bands are observed at 1269 cm-1 and 1263 cm-1 respectively, where the computed band can be found at lower wavenumber i.e. 1237

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cm-1, justifying the underestimation of orbital delocalization over C-C moiety. The aromatic ring vibrations of PRC HCl are analyzed based on the eigen vector

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distribution of the vibrational modes of disubstituted phenyl ring, in Wilson notation [28]. The stretching vibrations of C2-C3 and C5-C6 bonds are found to have greatest contribution to tangential

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C-C stretching mode 8a of benzene ring (Fig.8(a)) and hence, this mode can be used to analyze the quinonoid form of the ring, which is characterized by the partial double bond character of C 2-

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C3 and C5-C6. The vibrational spectral justification of quinonoid form of ring in PRC HCl has been made by analyzing 8a mode, which includes greatest contribution of C2-C3 and C5-C6 vibrations (Fig. 8(b)), according to PED. On comparison of computed wavenumbers, PRC HCl is found to execute 8a vibration with higher frequency i.e. 1591 cm-1, compared to that of benzene i.e. 1579 cm-1, confirming the double bond character of C2-C3 and C5-C6. The C-C stretching mode 8a manifests as a very strong band at 1606 cm-1 in IR and at 1605 cm-1 in Raman.

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Fig. 8 8a Mode of (a) Benzene and (b) PRC HCl 3.1.2. Ester moiety

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The atomic charges of ortho (C3= -0.249e and C5= -0.227e) and para (C1= -0.207e) carbon atoms present in the phenyl ring are considerably negative due to the resonance arising from the

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involvement of lone pair electron present in the nitrogen N12 and is found to cause extensive charge delocalization of lipophilic aromatic ring with the ester linkage, making C7 more positive and O11

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more negative (Fig.6). This is found to increase the dipolar or ionic nature of C=O moiety, inducing electron withdrawing effect of carbonyl group, having pronounced double bond character, obvious

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from C=O bond length ~1.2 Å and this can be regarded as typical molecular characteristic to act as local anesthetic [29]. The unappreciable lowering of stretching frequency of C=O to ~1700 cmis perceptible from the slight conjugation with C7-O8 bond, evident from the lower bond length

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of C7-O8 bond(1.37 Å), compared to that of C9-O8 (1.44 Å ).

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3.1.3. Hydrophilic Moiety

The XRD data shows that the tertiary amino nitrogen is hydrogen-bonded to a chloride ion

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with H∙∙∙Cl distance equal to 2.218 Å, while the other hydrogen bonding through the amino group of the ring is found to be weaker which is obvious from the longer H∙∙∙Cl distance equal to 2.646

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/ 2.693 Å. The weaker N-H∙∙∙Cl interactions can be viewed as the less polar or weaker N-H bonding, attributed to the electron deficiency of nitrogen, arising from the transfer of nitrogen lone pair to the ring responsible for the electron transport, through the ring. The characteristic spectral feature of tertiary amine is the strong multiband absorption in the region 2700- 2330 cm-1[30] and in PRC HCl, where amino group exists as (R3)-N-H∙∙∙X, three major IR bands can be observed at 2773, 2588 and 2500 cm-1. The former band can be assigned as NH stretching, which has been computationally supported only on incorporating solvent effect, based on PCM [31] and instead of the band expected around 2540 cm-1, a doublet is found which

ACCEPTED MANUSCRIPT indicates presence of Fermi resonance, arising due to the possible interaction of NH stretching fundamental with the overtone of strong band at 1269 cm-1, due to NH bending. The tertiary amine is found to possess the electron donating character of three ethyl groups increasing the electron density at nitrogen. The electron donating behavior of nitrogen is found to cause the weakening of three C-N bonds, evident from the increase of C-N bond length than normal value of 1.469Å and decrease of vibrational spectral C-N stretching wavenumber from the normal

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range 1342-1266 cm-1. The DFT computation shows that the bond length of N23-C10,N23-C24 and

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N23-C31 are respectively equal to1.499, 1.502 and 1.503 Å and the IR stretching wavenumbers of

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the above three bonds are respectively equal to 1049, 1017 and 1005 cm-1, justifying the above argument. Also the increase of electron density at nitrogen make it ideal hydrogen bond donor

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responsible for the characteristic electrostatic interaction of LA action through N-H∙∙∙X interaction. 3.2. Molecular Docking

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3.2.1. Lipophilic Interaction

Molecular docking reveals that PRC HCl is found to bind with PLGIC protein having pdb

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id: 3EHZ [32], with binding energy equal to -5.71 kcal/mol. It was observed that the molecule is preferred to interact with ion channel (Fig.9 and 10) via intermolecular hydrogen bonding of NH2

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group from lipophilic moiety, with the ester group of proline-67, glutamic acid-68 and isoleucine70 residues to form N-H∙∙∙O interaction and the corresponding H∙∙∙O distances are 2.21, 2.25 and

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2.84 Å respectively.

PRC HCl in the binding pocket is further stabilized by the non-covalent π-cation interaction

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with isoleucine-72 residue of the PLGIC protein, with binding distance equal to 2.88 Å. This πcation binding can be modeled as monopole-quadrupole interaction between H+ and electron rich

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phenyl ring and the lowest non-zero multipole moment of the benzene ring is the quadrupole moment which has been influenced by the polarization effect [33]. Since the resonance participation by the substituent does not contribute substantially to the cation-π binding [34] and the dominant role of the inductive effect of the functional group attached to the ring [35] reveals that the electron donating amino group enhances the π- cation interaction. Thus, the interaction is found to be enhanced by polarization due to the electron donation from amino group into the π system, with negligible role of resonance structure, deviating from benzenoid to quinonoid form.

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Fig. 9 Docked conformation of procaine cation in the binding site of 3EHZ

Fig. 10 Non-bonded and electrostatic interactions of procaine ligand and sodium channel receptor

ACCEPTED MANUSCRIPT To analyse the interaction of lipophilic moiety with receptor, the complex constituted by PRC HCl and the isoleucine, the residue which is in close proximity with phenyl ring in the binding pocket, has been simulated using DFT. The HOMO LUMO analysis (Fig.11) shows that the charge transfer occurs between the phenyl ring of PRC HCl and isoleucine which points to the binding

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through charge transfer interaction in lipophilic moiety, as predicted by Büchi’s model.

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Fig.11 (a) HOMO and (b) LUMO plot of PRC HCl with Isoleucine residue. 3.2.2. Dipole-Dipole Interaction

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The dipolar nature of C=O, as revealed by vibrational analysis shows the absence of hydrogen bond formation of carbonyl group. Docking calculation does not show any sign of the

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interaction of carbonyl group with nearby residue and the absence of dipole-dipole interaction can be justified by the absence of any nearby dipole, in the binding pocket. C-O group in ester linkage

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interact with the residue tryptophan-71 through C-H∙∙∙O hydrogen bonding, with H∙∙∙O distance equal to 3.0 Å.

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3.2.3. HB and Van der Waals interaction The tertiary amine of PRC HCl interacts with PLGIC through both HB and van der Waals

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interaction and the NH+ group of tertiary amine interacts with the oxygen atom of glutamic acid66 through salt bridge interaction which is the combination of two non-covalent interactions such as electrostatic interaction and N-H∙∙∙O hydrogen bonding, with interaction distance (H∙∙∙O) equal to 1.73 Å. The compound in PLGIC has been further stabilized by the van der Waals interactions with the residues in the hydrophilic moiety. 3.3. Blocking of Na+ in Ion Channel Since the blocking of Na+ in the ion channel of PLGIC protein occurs when drug is bound to the channel moiety in cationic form and the drug cation-Na+ interaction can be well explored by comparing the energy profile of the complex formed by Na+ with PRC HCl and that with PRC H+.

ACCEPTED MANUSCRIPT The energy profile has been generated by computing the energies of the complexes at B3LYP/6311++G(d,p) level (Fig.12), when Na+ approaches the drug. When drug is in the form of PRC HCl, it will give rise to attractive energy profile and the repulsive profile is created when drug is in cationic form i.e. PRC H+. The energy computation, for each 0.1Å change of Na∙∙∙Cl distance in PRC HCl-Na complex and for the similar change of Na∙∙∙H distance in

PRC H+-Na complex has

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been incorporated in the attractive and repulsive profile respectively.

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Fig.12 Variation of energy of sodium-drug complex, with Na∙∙∙H distance (drug in cationic form) and Na∙∙∙Cl distance (neutral drug molecule)

The analysis of attractive profile shows that the energy profile gives minimum at 2.4 Å and is found close to the equilibrium Na-Cl bond distance of 2.38 Å, computed at the same level of theory. The repulsive profile gives minimum H∙∙∙Na distance at 2.9 Å and is much higher than the Na-H bond distance of 1.887 Å, which justifies the strong repulsion of Na+ by the drug, in cationic form. The Na+ repulsion can be further substantiated by computing electrostatic potential mapping of PRC HCl (Fig.13), in which the red and blue refers to the electron rich and electron deficient regions respectively and the tertiary amino group is a nucleophilic region. The cationic form of the

ACCEPTED MANUSCRIPT drug, obtained by the removal of Cl from the tertiary amine, is found to hold electrophilic region

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around tertiary amine which is responsible for the repulsion of sodium.

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Fig.13 ESP mapped surface of (a) PRC HCl and (b) its cationic form (PRC H+) 3.4. Characteristic LASP in UV

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The characteristic absorption spectra (Fig. 14(a)) of ester type LA should resemble those of benzoate or aromatic carbonyls, where the former group has been reported to generate weak

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band at 380-310 nm and the strong band in the region 310-270 nm which has been found to originate from benzene ring [36]. The weak band has been found to arise from n→π* promotion

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of the carbonyl group and the strong band belongs to π→π* transition of the ring as evident from TD DFT results and the band positions are 332 and 307 nm respectively. Another band arises due

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to charge transfer from the π orbital of the ring to the π*orbital of the carbonyl chromophore, which can be observed at 233 nm. The computed UV absorption spectrum with wavelength scaled is

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given in Fig. 14(b) and the excitation with appreciable oscillator strength is shown as vertical lines. The theoretical UV absorption peaks are 317.4, 316.9 and 222.2 nm which are found to correlate

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233 nm.

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well with the corresponding experimental absorption peaks, respectively equal to 332, 307 and

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Fig.14 (a) Experimental (b) Theoretical UV-Vis spectrum of PRC HCl 3.5. Characteristic IR and Raman Marker Bands

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According to Büchi’s model, ester type LA gets bound to the receptor by means of essential physical interactions [22,37] namely, charge transfer, hydrogen bonding, dipole-dipole attraction

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and van der Waals interaction (Fig. 5). These interactions are provided by the electron donor moiety present in the aromatic ring and the dipolar nature of carbonyl moiety making the drug

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bound to the receptor through charge transfer and dipole-dipole interaction. Also, the ‘onium’ ions [38] present in tertiary amino part promoting the binding of drug with channel receptor effectively

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and efficiently through electrostatic interactions. The vibrational modes of these functional moieties responsible for the anesthetic activity of ester type LA can be considered as the IR and

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Raman Marker Bands [39] for the identification of ester type LA. The Marker bands of PRC HCl correspond to ring C=C stretching ca 1600 cm-1 (8a mode), C=O stretching vibrations ca 1695 cmand C-O stretching vibrations ca 1260 cm-1 are strongly and simultaneously active in both IR and

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1

Raman spectra as observed in other ester type local anesthetics viz, benzocaine, 2-chloroprocaine, tetracaine and oxybuprocaine [40-43]. 3.6. Molecular polarizability and local anesthetic activity Molecular polarizability has been considered to have a significant role in exploring the molecular physio-chemical properties and its biological activities of drug molecules. Highly polarizable drug molecules can possibly have strong attractions with the receptor through van der Waals interaction and contribute a major role in the action of LA. Based on the present analysis, the binding affinity of local anesthetics could be correlated with the mean polarizability, deduced

ACCEPTED MANUSCRIPT using quantum chemical computations, along with that derived from quantitative structure-activity relationship (QSAR) [37,44-46]. Using ab initio method, the mean polarizability   [47] has been computed based on the equation,    ( xx   yy   zz ) 3 and the calculated mean polarizability for procaine, procaine cation and procaine hydrochloride molecules are 19.71×10-24 e.s.u., 21.53×10-24 e.s.u., and

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21.06×10-24 e.s.u., respectively. The mean polarizability pertaining to QSAR studies has also been

2

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4       A  N A 

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determined using the equation proposed by Miller and Savchik [48-50]:

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where  A is the atomic hybrid component and N is the total number of electrons. The molecular polarizability, evaluated using Miller method for procaine, procaine cation and procaine

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hydrochloride molecules are found to be 20.54×10-24 e.s.u., 20.90×10-24 e.s.u., 23.14×10-24 e.s.u., respectively. The ab initio method at HF/6-311G(d,p) level of theory also gives a mean polarizability which is in better agreement with the Miller method for different procaine analogues,

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which substantiates the close relation between molecular polarizability and efficacy linked to

3.7. Cytotoxic activity

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SAR of the LA drug molecule.

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Local anesthetics are generally considered to be safe for regional anesthesia, postoperative pain relief and in biopsies. However, it is reported that LA may cause local tissue toxicity and

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incidental neuronal damage [51,52]. In the present study, the in vitro cytotoxicity of PRC HCl was determined using MTT assay along with Lethal Dose (LD50) value. The cytotoxicity assay of PRC

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HCl against L929 fibroblast cell line was evaluated and the percentage of cell viability derived from the experimental data is given in Table.3. The concentration-dependent morphologic changes induced by treatment with PRC HCL in L929 fibroblast cells cultured in DMEM using inverted phase contrast microscope is given in Fig.15. From these results, it is notable that the cell viability of L929 fibroblast cells declined and cytotoxicity moderately increased in a concentrationdependent manner after treatment with PRC HCl. The LD50 value of PRC HCl is found to be 133.724μg/mL which indicates that PRC HCl provides a significant protection against the lethal effects. As PRC HCl is a typical short acting ester type local anesthetic with its low toxicity, it has been used to reduce the pain of intramuscular injection of penicillin and also in dentistry.

ACCEPTED MANUSCRIPT Table.3. Percentage of viability for PRC HCl in different concentrations Sample Concentration (µg/mL) Average OD Percentage Viability Control

0.9055

100

6.25

0.8557

94.504

12.5

0.8212

25

0.7629

50

0.6243

100

0.5856

90.6902

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84.2518

64.6751

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68.9417

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4. Conclusions

cells

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Fig.15(a-f) Concentration-dependent morphologic changes with PRC HCL in L929 fibroblast

The molecular structural features responsible for LA action of PRC HCl have been investigated, based on the Büchi’s model of binding of drug to the sodium channel in PLGIC protein. The vibrational spectral characterization provides insight into the electronic transport through the ring, which shows that the phenyl ring of lipophilic moiety has been found to deviate from benzenoid to quinonoid form, substantiated by the higher vibrational wavenumber of 8a mode of the ring and the electron deficiency at meta position, as revealed by computed NBO charges. The dipolar or ionic nature of carbonyl group for local anesthetic action has been predicted, which is, arising as a consequence of electron transport through the ring. The molecular

ACCEPTED MANUSCRIPT docking shows that the interaction of lipophilic moiety with protein takes place through π-cation interaction, and that of ester and hydrophilic moieties takes place through hydrogen bonding. The characteristic UV absorption peaks of local anesthetics have been observed for PRC HCl in the expected wavelength range, attributed to n→π* promotion of the carbonyl group, π→π* transition of the ring and charge transfer band from ring to carbonyl group. The characteristic IR and Raman marker bands of ester type LA, PRC HCl are explored based on the functional moieties responsible

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for LA activity, along with spectral signatures of other ester type LA. The molecular polarizability

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computed to analyze the structure activity linkage substantiates the close relation between

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molecular polarizability and efficacy linked with SAR of the LA drug molecule and it is found that the mean polarizability of procaine analogues determined using Miller method correlates well with

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the ab initio methods. The blocking of Na+ in the ion channel has been probed using attractive and repulsive energy profile. The in vitro cytotoxicity shows that the LD50 value of PRC HCl to be

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133.724 μg/mL which indicates that PRC HCl provides a significant protection against the lethal effects, making PRC HCl a good short acting ester type local anesthetic.

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Acknowledgements

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The author Y.Sheeba Sherlin thanks the University Grants Commission (UGC), India for conferring a Teacher Fellowship under Faculty Development Programme leading to Ph.D. The

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author T.Vijayakumar acknowledges the Department of Space, Government of India for RESPOND project and SRM University, Chennai for selective excellence initiative. The author

[1]

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FIST scheme.

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J.Binoy, extends sincere thanks to DST, Govt of India for instrumental support, through DST-

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Graphical abstract