Virtual and experimental high throughput screening of substituted hydrazones on β-Tubulin polymerization

Bioorganic Chemistry 114 (2021) 105094

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Virtual and experimental high throughput screening of substituted

hydrazones on β-Tubulin polymerization

,

Janet Sabina Xavier^a, Karthikeyan Jayabalan^{a *}, V. Ragavendran^b, Muthu Tamizh Manoharan^c,

A. Nityananda Shetty^d

^aDepartment of Chemistry, Sathyabama Institute of Science and Technology, Chennai 600119, India

^bDepartment of Physics, Sri Chandrasekharendra Saraswathi Viswa Mahavidyalaya, Kanchipuram 631561, India

^cDepartment of Chemistry, Siddha Central Research Institute, Central Council for Research in Siddha, Arumbakkam, Chennai 600106, India

^dDepartment of Chemistry, National Institute of Technology Karnataka, Mangalore 575025, India

A R T I C L E I N F O

A B S T R A C T

Keywords:

Microtubule targeting agents that disrupt the dynamic functioning of the mitotic spindle are some of the best

chemotherapeutic agents. Interruption of microtubule dynamics through polymerization or depolymerization

causes cell arrest leading to apoptosis. We report a novel class of aroylhydrazones with anticancer properties.

Tubulin inhibition studies were performed using both computational and biological methods. Docking and

pharmacophore mapping showed efﬁcient binding between the ligands and the protein. Tubulin inhibition assay

showed the aroylhydrazones to be inhibitors of tubulin polymerization. DFT studies explains the geometrical and

electronic properties of the compounds. Furthermore, anticancer studies using lung and liver cancer cell lines

gave low IC₅₀values with the methyl substituted hydrazone MH-2 being the most potent. (IC₅₀of 0.0896 and

0.1040 µM respectively). The methyl group is responsible for the effective binding to the protein. Thus, a new

class of tubulin binding agents have been identiﬁed as potential agents in cancer therapy.

Anticancer agents

β-tubulin

Computational Chemistry

DFT studies

Hydrazones

Molecular docking

1. Introduction

apoptosis. These agents target four sites in the β-tubulin subunit –

colchicine, vinblastine, taxol and laulimalide. Colchicine and vinblas-

Microtubules consisting of

α

- and β-tubulin heterodimers are key

tine inhibit polymerization whilst taxol and laulimalide facilitate it

[8–10]. Combination chemotherapy including a wide range of micro-

tubule binding agents such as vinca alkaloids, taxanes, etc have been

used for different types of malignancies [11–13]. However the

complexity of their synthesis, toxicity and multi-drug resistance have

only succeeded in hampering the effectiveness of such treatments

[14–16]. Even now, extensive research and clinical trials are being

carried out to identify microtubule targeted agents such as vinca alka-

loids, taxanes, colchicine and their structural analogues which will in-

ﬂuence microtubule dynamics and act as potential chemotherapeutic

agents.

components of several important physiological processes such as

mitosis, cell signalling, motility regulation, intracellular transport, etc.

[1–3] The function of microtubules is strongly associated with their

stability and the dynamic character of the tubulin-microtubule equilib-

rium. Microtubules are also composed of multiple isotypes for both α-

and β-tubulin which are characterised by speciﬁc tissue and develop-

mental distribution. The highly diverse C-terminals of the various iso-

types are centres of post translational modiﬁcations which have been

reported for a variety of cancers [4–6]. Changes in the tubulin isotypes

have been shown to affect the dynamics of microtubule assembly [7]. It

is perhaps not surprising that much of the work reported has been on

β-tubulin isotypes since tubulin binding agents speciﬁcally target

β-tubulin protein during chemotherapy. Due to this, microtubules have

become validated targets for cancer chemotherapy. Drugs that interfere

with microtubule functions suppress microtubule dynamics, inhibit the

proliferation of cancer cells leading to mitotic arrest and thereby

We had reported the design and synthesis of a novel series of substituted

chalcones synthesized from the precursor p-[N, N-bis(2-chloroethyl) amino]

benzaldehyde. The chalcones were found to be potent inhibitors of tubulin

polymerization and exhibited very low half maximal inhibitory concentra-

tions (IC₅₀) in the micromolar range [17]. As a continuation of the work, a

new series comprising of aroylhydrazones synthesized from the same

* Corresponding author.

E-mail address: karthikeyan.chemistry@sathyabama.ac.in (K. Jayabalan).

https://doi.org/10.1016/j.bioorg.2021.105094

Received 27 January 2021; Received in revised form 2 June 2021; Accepted 10 June 2021

Available online 13 June 2021

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

nitrogen mustard is reported here. p-[N, N-bis(2-chloroethyl) amino]

benzaldehyde is a potential anticancer agent and is known to be active

against multiple cancer cell lines [18]. Hydrazones, either acyl or aroyl, are

well known for their versatile chemical and biological applications. The

presence of an sp²hybridized nitrogen bonded to an sp³hybridized nitrogen

atom makes them good ligands as well and they are known to form com-

plexes with metals such as nickel, cobtalt, copper, ruthenium, etc. [19–20]

Hydrazones are well known for their antitubercular, antiinﬂammatory,

antibacterial, antifungal, antioxidative, analgesic, anticonvulsant, anti-

tumor, anti-HIV and antimicrobial activity [21–27]. Hence we expect the

hydrazones synthesized from such a promising precursor to show enhanced

anticancer activity through synergistic effect.

chloroethyl) amino] benzaldehyde (1) with the appropriately

substituted benzhydrazide (2a-d) to yield the respective hydrazones

(Scheme 1). The structures of the hydrazones were characterized by UV,

FT-IR, ESI-MS, ¹H and ¹³C NMR spectral methods and single crystal XRD

studies.

2.2. Spectral measurements

2.2.1. Electronic spectra

The UV–Visible absorption spectra of the four hydrazones were

recorded in the region between 200 and 800 nm using ethanol as the

solvent. Generally, hydrazones and their derivatives that bear alkyl or

acyl groups have simple ultraviolet absorption spectra, characterized by

two maxima, one in the region of 220–250 nm, the other at longer wave

lengths of 270–400 nm [35–36]. Accordingly, all the four hydrazones

Four hydrazones based on the results of computational studies were

selected for synthesis and further biological study. Aroylhydrazone with

the methyl substituent was obviously the ﬁrst choice due to the presence

of the methyl group. The methyl group has been known to dramatically

increase the afﬁnity of the ligand with the protein binding site. The

magic methyl effect, a quaint term, has been introduced for the late-

stage functionalization to accelerate drug discovery [28,29]. The ad-

vantages of the humble methyl group are manifold in that its presence

enables the lead molecule to ﬁt snugly in the protein active site, in-

creases solubility, boosts potency, etc. [30] Late-stage functionalization

using a variety of transition metal catalysts including manganese, co-

balt, platinum, etc have also been reported [28,31–34]. The other

hydrazones were selected based on their binding afﬁnity with the pro-

tein receptor.

show two well-deﬁned bands pertaining to the

tions. The * transitions are observed in the range 200 to 250 nm

which is due to the benzoyl chromophore. The n→ * transition is

observed as a broad band between 320 and 400 nm. For all the four

compounds, the * transitions appear as a weak band when

π→π* and n→π* transi-

π→π

π

π→π

compared to the more intense and stronger n→π* transition. The ab-

sorption bands of the hydrazones almost overlap each other at the ab-

sorption maxima. The UV–Visible spectra of the four hydrazones are

given in Fig. 2.1.

2.2.2. FT-IR spectra

The newly synthesized compounds were ﬁrst approached from a

theoretical angle using computational techniques. Molecular docking

has become an integral part of drug design and discovery and we have

employed a mechanistic approach to study the non-covalent interactions

between the ligands and the protein. Pharmacophore mapping was

simultaneously done to identify signiﬁcant pharmacophoric features

present in this family of hydrazones. Density functional theory studies

were performed to study the geometrical and electronic properties of the

hydrazones. The theoretical studies were further substantiated with in

vitro anticancer studies using lung and liver cancer cell lines. Tubulin

polymerization assay was also carried out conﬁrming the results of the

molecular docking technique performed on the hydrazones.

The infra-red spectra of the hydrazones were recorded in the range of

4000 to 400 cm^{ꢀ 1}. The stretching frequency of the amide carbonyl

–

(C O) and azomethine (C N) groups lies in the range of 1600–1700

–

cm^{ꢀ 1}for all the hydrazones. In SH-1, the carbonyl frequency is observed

at 1600 cm^{ꢀ 1}whilst MH-2 shows an intense band around 1650 cm^{ꢀ 1}

.

BH-3 and CH-4 show the carbonyl stretching as sharp peaks at 1620 and

1630 cm^{ꢀ 1}respectively. The amide N H stretching can be observed at

–

3200 cm^{ꢀ 1}for the compounds. The amide N H bending can also be

–

observed between 1520 and 1550 cm^{ꢀ 1}. The C Br and C Cl stretching

–

frequency is seen as a strong, sharp peak at 550 and 840 cm^{ꢀ 1}for BH-3

–

and CH-4 respectively. The aromatic C H stretching is observed

around 3000–3100 cm^{ꢀ 1}and the C C stretches appear as multiple

–

bands between 1350 and 1520 cm^{ꢀ 1}. The strong band at ~ 1645 cm^{ꢀ 1}

2. Results and discussion

and the following overlapping medium intensity bands at ~ 1615 cm^{ꢀ 1}

–

are assigned to the C O and C N stretches respectively [37].

2.1. Synthesis of hydrazones

2.2.3. NMR spectra

The hydrazones were synthesized by treating p-[N, N-bis(2-

The ¹H and ¹³C NMR spectra were recorded using DMSO‑d₆as the

Scheme 1. Synthesis of the hydrazones.

2

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

2

1.8

1.6

1.4

1.2

1

present in the crystal system. The crystallographic data and unit cell

dimensions are provided in Table 1. The packing fraction and intermo-

lecular interactions are presented in the SI Figure S1. Bond lengths and

angles are given in Table S1.

SH-1

MH-2

BH-3

CH-4

2.4. Computational studies

2.4.1. Molecular docking

0.8

0.6

0.4

0.2

In silico studies are perhaps some of the best used methods today for

their ease, simplicity and the accurate results provided. Keeping this in

mind, the four hydrazones were subject to an integrated model in

computational studies such as molecular docking, pharmacophore

mapping and density functional theory studies. Molecular docking was

performed using the Accelrys Discovery Studio software and the

LigandFit protocol was employed to study the binding interactions of the

four hydrazones with the chosen protein β-tubulin. The crystal structure

of the protein (1SA1) was downloaded from the RCSB Protein Data Bank

website (www.rcsb.org) and used. The docking simulations emphasised

the effectiveness of the ligand–protein interactions. Various ligand poses

were analysed and the one with the highest dock scores was selected as

the binding pose. All non-covalent interactions were analysed.

0

200

300

400

500

600

700

800

Wavelength

Fig. 2.1. UV–Visible spectra of the hydrazones.

solvent. The aromatic protons of the nitrogen mustard moiety appear as

doublets in the region of 6.8 and 7.9 ppm with A₂X₂pattern for all the

hydrazones. The azomethine and amide protons appear as singlets

around 8.3 and 11.6 ppm respectively. In SH-1, the protons H2 and H2^′

appear as doublets at 7.1 ppm and the neighbouring H3 and H3^′protons

as multiplets between 7.5 and 7.6 ppm. In MH-2, the methyl proton

appeared as a singlet at 2.1 ppm. The aromatic protons H2 and H2^′in the

nitrogen mustard aromatic ring are split by the neighbouring H3 and H3^′

protons and vice versa to give two doublets at 7.31 and 7.81 respec-

tively. In BH-3, the H2, H2^′and H3, H3^′protons appear at 7.5 and 7.8

ppm respectively. In CH-4, the H2, H2^′and H3, H3^′protons appear at

7.9 and 7.6 ppm respectively. Presence of the methyl group in MH-2 has

given an additional singlet at 2.37 ppm. The methylene protons of the

bis(chloroethyl)amino groups appear in the range of 3.7–3.8 ppm. In the

¹³C NMR spectrum, the azomethine carbon and amide carbonyl appear

between 161.60 and 163.60 ppm for the four hydrazones. The aliphatic

carbons of the ClCH₂CH₂N group appeared around 40–41 and 51–52

ppm. The ¹H and ¹³C spectra of the four hydrazones are given in the

Supporting Information (SI) ﬁle.

Hydrazones are known to exhibit the amide-iminol tautomerism.

They tend to exist in the amide form in the solid state and in equilibrium

in the solution state. The equilibrium of the two tautomers depends on

the substituents present in the molecule. In the absence of water, the

amide tautomer predominates but high polarity solvents may shift the

equilibrium to the iminol side [39]. Docking simulations generated both

the tautomers for the four hydrazones and their interactions with the

protein receptor were studied. It was found that for all the compounds,

the iminol form tended to bond more favourably with the receptor than

the amide form. The dock score of the iminol form was found to be

consistently more than that of the amide for all the hydrazones. The

binding energies of both the forms however differ very little from each

other indicating the stability of the binding afﬁnity of the hydrazones in

general, with the protein. Both the amide and iminol forms of the four

hydrazones have a negative binding energy value indicating that the

ligands were bound spontaneously to the active site of the protein re-

ceptor. The high negative value is also an indication of the effective

lowering of the overall energy of the complex. The effectiveness of the

binding interactions of the iminol-protein complex is probably due to

the way the iminol form ﬁts into the active site of the protein. The

iminiol form is conformationally more ﬂexible and as a result, ﬁts snugly

into the binding pocket thus strengthening the interactions with the

surrounding amino acid residues. The amide forms however, as can be

seen in Fig. 2.3, project out of the active site and hence lose many of

their possible non-covalent interactions with the surrounding amino

acids. This could be the reason why the iminol form is able to generate a

much higher dock score than the corresponding amide form.

2.3. Single crystal X-ray diffraction studies

The crystal structure (ORTEP diagram) of the four hydrazones along

with the atom-numbering scheme is given in Fig. 2.2. The aldehyde

condenses with the appropriate hydrazide producing the hydrazone

which crystallized in the space group of P c a 2₁, P 2₁/c, P 2₁/c and I 41/

a respectively with Z = 4 for SH-1, MH-2 and BH-3 and Z = 16 for CH-4.

–

The new azomethine, that is the C(8) N(2) bond whose bond length of

1.270(5), 1.271(3), 1.275(7), 1.275(4) Å in the four hydrazones

respectively are similar to that of a Csp²= N double bond length of 1.278

(8) Å conﬁrming the amide tautomeric form and that the molecule

When the individual hydrazones are studied, the 2D ligand interac-

tion diagram in Fig. 2.4 shows that the iminol form of SH-1 is completely

surrounded by amino acids conﬁrming its ﬁt inside the binding pocket.

The two aromatic rings are involved in pi-alkyl interactions with

Cysβ12. Both the iminol hydrogen and nitrogen are involved in

hydrogen bonding with Asnβ206 with the oxygen acting as the hydrogen

donor and the nitrogen acting as a hydrogen acceptor to the amide ox-

ygen and amide nitrogen present in the amino acid. The hydrogen bond

distances of 1.78145 and 2.88639 Å respectively conﬁrm the strength of

the bonding. The amide form of SH-1 is also involved in multiple

hydrogen bonding with the surrounding amino acids speciﬁcally

Glnβ11, Aspβ179 and Tyrβ224. The keto oxygen and both the nitrogens

of the hydrazone moiety are involved in hydrogen bonding. The

hydrazone also shows pi-pi stacking with Tyrβ224 at a distance of 5.32

–

adopts trans conformation with respect to the C N bond with torsion

angles of ꢀ 168.01^◦, ꢀ 175.87^◦, ꢀ 176.90^◦and ꢀ 179.33^◦

– – –

N N C8) respectively. The average angles of the hydrazone be-

(C7

◦

C are 122 , 120 and 116^◦

– –

C

– – – –

tween N

C

O, O

C

C and N

respectively which indicate that the position C7 atom has nearly trigonal

planar geometry and that the three bond angles around C7 atom are not

◦

– –

C O bond angle is consider-

equal to 120 each. It is seen that the N

– –

C C angle which decreases the repulsion between

ably greater than N

the lone pairs present on nitrogen and oxygen atoms. The central part of

the molecule adopts a completely extended double bonded conforma-

tion. It can be conﬁrmed by the C7-O1 bond length (1.225(5) Å) which is

–

considerably longer than the standard C O bond length 1.21 Å and N-

–

C7 bond length (1.341(5) Å.). This is shorter than the standard N

C

Å. The amide N H of SH-1 is also involved in bifurcated hydrogen

–

single bond length (1.47 A). The molecular pattern is characterized by

bonding with the phenolic OH of Tyrβ224 and the carboxylic C O of

three different graph-set motifs [38] viz. R²₂(5), R₂²(6), and R₂²(7) type,

Aspβ179. Studies show that bifurcated hydrogen bonding may not be as

–

and these motifs occur due to N H… O and C H… O hydrogen bonds

effective as simple two centred hydrogen bonding [40,41]. The other

3

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Fig. 2.2. ORTEP diagram of the four hydrazones.

two hydrogen bonds are also three centred bifurcated bonds this time

Conventional two centred hydrogen bonding can be seen to occur be-

–

involving the amide N H of Glnβ11 with the keto oxygen and imine

tween the amide O and N H of Asnβ206 and the iminol hydrogen and

nitrogen of the hydrazone. Both the bifurcated bonds effectively reduce

the strength of the binding interactions between the ligand and the

protein. However, the aromatic ring of the hydrazone and the aromatic

ring in Tyrβ224 are involved in an attractive and stabilizing non-

covalent pi-pi stacking interaction.

the nitrogen of MH-2 with a bond distance of 1.73999 and 3.05664 Å

respectively. The ligand protein binding is further stabilized by a host of

non-covalent interactions such as van der Waals interactions, pi-pi

stacking and pi-alkyl interactions between various amino acid residues

and MH-2. In the amide tautomer of MH-2, the aromatic ring along with

its methyl substituent projects right out of the binding pocket of

β-tubulin. Hence it is not involved in any signiﬁcant non-covalent

interaction with the amino acid residues except for a weak alkyl

The methyl substituted hydrazone MH-2 also displays the amide-

iminol tautomerism and its iminol form is shown to have a dock score

of 51.361 as compared to the 29.134 of the amide tautomer.

4

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

distance of 2.44362 Å.

Table 1

Crystallographic data and unit cell dimensions of the hydrazones.

In the iminol form of the bromo substituted hydrazone BH-3, it can

be seen once again that there are multiple interactions that stabilize the

ligand-receptor complex. A three centred hydrogen bond is observed

between the iminol hydrogen (acceptor) and the phenolic OH of

Tyrβ224 and the oxygen of the carboxylic acid moiety of Aspβ179 with a

bond distance of 2.65655 and 1.8366 Å respectively. Signiﬁcant halogen

bonding can also be observed in the interaction between the bromine

and the amide oxygen of Asnβ228. This stabilising interaction is also

coupled with a pi-sulphur interaction between the aromatic ring of BH-3

and the thiol moiety of Cysβ12. Both these interactions are known to

stabilize the ligand-receptor complex [42,43]. Tyrβ224 is also involved

in pi-pi stacking with the same ring and other pi alkyl interactions can be

seen in Fig. 2.4. The amide tautomer of BH-3 exhibits some interesting

non-covalent interactions as well. Two bifurcated bonds can be seen.

One is between the amide oxygen and imine nitrogen of BH-3 with the

Parameters

SH-1

MH-2

BH-3

CH-4

CCDC

1,862,977

C₁₈H₁₉Cl₂N₃

O

1,862,978

1,904,640

1,862,979

Molecular

formula

C₁₈H₁₈Br Cl₂

C₁₈H₁₈Cl₃

C₁₉H₂₁Cl₂

N₃

O

N₃

O

N₃O

Formula

364.26

443.15

398.70

378.29

weight

Temperature

Wavelength

Crystal

293 K

296 K

0.71073 A

Orthorhombic

0.71073 A

Monoclinic

0.71073 A

Monoclinic

0.71073 A

Tetragonal

system

Space group

Point group

Unit cell

dimensions

P c a 2₁

mm2

P 2₁/c

I 41/a

2/m

4/m

a = 9.8170(6)

Å

a ¼ 22.7142

(12) Å

a ¼ 22.401

(4) Å

a ¼ 22.5844

(8) Å b ¼

22.5844(8) Å

c ¼ 15.3874

(5) Å

b = 19.2892

(12) Å

b ¼ 9.4879

(5) Å

b ¼ 9.4835

(18) Å

c = 9.4610(6)

Å

c ¼ 8.6964

(3) Å

c ¼ 8.6793

(12) Å

–

amide N H of Asnβ101 at a bond distance of 1.80241 and 2.71426 Å

α

= β = γ =

–

respectively. The other is between the amide N H of BH-3 with both

α

= β = γ =

α

= γ = 90^o

α

= γ = 90⁰

90⁰

β = 93.2618

β = 93.131

(7)

the oxygens of the carboxylic acid moiety of Gluβ71 with bond distances

of 2.45579 and 2.46863 Å respectively. A long range halogen bond is

also observed though not with the bromine as expected but with the

chlorine of the nitrogen mustard group and Asnβ101. This is probably

because the aromatic ring with bromine is completely outside the

binding pocket and hence is not available for any type of interaction

with the amino acid residues in the active site.

(17)^o

Z, Z’,

4,0

16,0

Calculated

density

1.350 g cm^{ꢀ 3}

1.573 g cm^{ꢀ 3}

1.438 g cm^{ꢀ 3}

1.281 g cm^{ꢀ 3}

R –Factor (%)

F(0 0 0)

3.85

3.13

5.62

6.94

760.0

896

824.0

3168.0

24.986

26,26,18

R

Theta (max)

h , k, l max

Reﬂections

collected/

unique

24.996

24.999

26,11,10

R

24.998

26,11,10

R

11,22,11

R(Reﬂection)

= 0.0385

(2746)

In the chloro substituted hydrazone CH-4, the iminol form ﬁts

completely inside the active site and is involved in various stabilizing

interactions. Conventional hydrogen bonding is observed between the

iminol hydrogen and the amide oxygen of Asnβ206 and between the

(Reﬂection)

= 0.0313

(2462)

wR2

(Reﬂection)

= 0.0562

(1335)

wR2

(Reﬂection)

= 0.0694

(2205)

wR2

(Reﬂections)

= 0.0969

(3155)

wR2

–

iminol nitrogen with the amide N H of the same amino acid with a

(Reﬂections)

= 0.0716

(3295)

(Reﬂections)

= 0.1565

(3241)

(Reﬂections)

= 0.2180

(3461)

bond distance of 1.78754 and 2.36069 Å respectively. pi-pi stacking can

be observed between Tyrβ224 and ring A whilst pi-sulphur interactions

can be seen between ring A and the thiol group of Cysβ12. The amide

tautomer of CH-4 also ﬁts better inside the active site like SH-1. A

bifurcated hydrogen bond is observed between the amide oxygen acting

as acceptor to both the amide hydrogens of Asnβ101 with a bond dis-

–

tance of 2.46379 and 1.59036 Å. The amide N H of CH-4 shows a two

centred hydrogen bond with the amide oxygen of Gln β11 with a bond

distance of 2.65206 Å.

Of the four amide tautomers, SH-1 gives the best dock score of 41.88

followed by 39.769 of CH-4. Both these tautomers ﬁt better inside the

active site which could be the reason for their ligand–protein complex

stability. However, all the four iminol tautomers lie almost in an iden-

tical fashion inside the active site and hence give similar stabilizing

interactions with various amino acid residues inside the active site. Of

the four iminol forms, MH-2 is found to have the highest dock score

closely followed by CH-4. The key amino acid residues involved in non-

covalent interactions are Glnβ11, Cysβ12, Gluβ71, Asnβ101, Aspβ179,

Asnβ206, Tyrβ224 and Asnβ228. The dock scores and H-bond in-

teractions can be viewed in Table 2. The binding interactions of the four

hydrazones with the protein receptor can be seen in Fig. 2.4.

The hydrophobicity surface created around the active site and the

two tautomeric forms are clearly seen in Fig. 2.5. The hydrophilic region

(coloured blue) is at the edge of the active site and it becomes more

hydrophobic (red) as we go deeper into the pocket. The amide forms are

all found at the edge of the active site. They do not penetrate the hy-

drophobic region of the active site while the iminol forms ﬁt completely

inside the active site. The nitrogen mustard moiety, -N(CH₂CH₂Cl)₂of

the hydrazones and the substituents– methyl, bromine and chlorine are

all projected at the hydrophilic edge of the surface. The hydrazone

moiety and the iminol OH are buried deep inside the binding site. Re-

ceptor surfaces such as aromatic, hydrogen bond, interpolated charges,

ionizability and SAS can be found in Figures S2 - S6 in the SI.

Fig. 2.3. The binding site sphere of the four hydrazones in the (A) iminol and

(B) amide forms.

interaction with Alaβ99. As in SH-1, bifurcated hydrogen bonding is

–

observed between the common donor N H of Asnβ101 and the two

hydrogen bond acceptors, the amide oxygen and imine nitrogen of MH-

–

2. The amide N H of MH-2 is however involved in conventional

2.4.2. Pharmacophore mapping

hydrogen bonding with the carboxylic oxygen of Gluβ71 with a bond

The HipHop model was used in the Common Feature Pharmacophore

5

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Fig. 2.4. Binding interactions of the four hydrazones with β-tubulin (left). 2D Ligand interaction diagram of the hydrazones with signiﬁcant amino acid resi-

dues (right).

Generation to determine the molecular framework that is responsible for

the hydrazones’ biological activity. Ten pharmacophore models were

generated with alignment scores ranging from 138.286 to 128.686. A

ﬁve point pharmacophore was generated (R- Ring aromatic, H –

Hydrophobic group, A – H-bond acceptor, D – H-bond donor and Z –

Hydrophobic aliphatic) with a maximum ﬁt of 4. Among the four

hydrazones, both the iminol and amide forms had similar pharmaco-

phore features. However, the iminol forms had greater ﬁt value than the

6

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Fig. 2.4. (continued).

amide forms conﬁrming the docking results discussed above. The ring

aromatic feature was ring B (orange), the hydrophobic groups were the

two chlorine atoms (cyan) of the nitrogen mustard moiety. The iminol

oxygen and the amide group (magenta) constituted the hydrogen bond

donor. Fig. 2.6 shows the pharmacophore model for both the amide and

iminol forms. Figure S7 in SI gives the pharmacophore features for the

four hydrazones in both amide and iminol forms.The pharmacophore

features generated by the HipHop model are given in Table S2.

hydrazones along with numbering of atoms are shown in Fig. 2.7. The

optimized parameters (bond lengths and bond angles) for the four

hydrazones under study are tabulated in Tables S3 and S4. The global

minimum energy obtained by DFT structure optimization procedure was

found to be ꢀ 1857.498750 Hartrees, ꢀ 4431.044775 Hartrees,

ꢀ 2317.124303 Hartrees and ꢀ 1975.475075 Hartrees for the four

hydrazones respectively. The calculated vibrational wavenumbers using

optimized geometry were positive. The optimized structures along with

the optimized parameters (bond lengths and bond angles) obtained from

DFT calculations matches well with the experimentally obtained single

crystal XRD pattern. Thus the accuracy of DFT in elucidating the mo-

lecular structure of large compounds was proved yet again in this study.

2.4.3. Density functional Theory studies

2.4.3.1. Molecular geometry. The optimized structures of the four

7

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Fig. 2.4. (continued).

Table 2

–

Dock score, binding energy, ﬁt value and H bond interactions of the hydrazones.

Compound

Dock score

Lig score 1

Lig score 2

H-interaction

H-bond distance

(Å)

Binding energy

(kcal/mol)

Fit Value

(Max ﬁt ¼ 4)

SH-1

41.88

2.98

4.28

Glnβ11

2.9323

2.6858

1.9851

3.0613

2.8864

1.7814

1.8401

2.6494

1.7400

3.0566

1.8024

2.7143

2.4558

2.4686

2.6566

1.8366

2.4638

1.5904

2.6521

1.7875

2.3607

ꢀ 11401233.25

2.3448

(amide)

Glnβ11

Aspβ179

Tyrβ224

Asnβ206

Asnβ101

Asnβ206

Asnβ101

Gluβ71

SH-1

47.94

2.51

5.08

ꢀ 11401218.81

ꢀ 11401225.75

ꢀ 11401219.63

ꢀ 11401231.13

3.6543

2.3073

3.8408

2.1442

(iminol)

MH-2

29.134

51.361

38.02

ꢀ 999.9

3.78

ꢀ 999.9

5.55

(amide)

MH-2

(iminol)

BH-3

ꢀ 999.9

(amide)

Gluβ71

BH-3

47.35

3.04

4.73

Tyrβ224

Aspβ179

Asnβ101

Gln β11

Asnβ206

ꢀ 11401223.56

ꢀ 11401231.81

3.9995

2.2216

(iminol)

CH-4

39.769

ꢀ 999.9

(amide)

CH-4

48.536

3.62

5.6

ꢀ 11401223.44

3.7796

(iminol)

8

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

–

In SH-1, the C C bond lengths of C9-C11 (1.40 Å), C11-C12 (1.504

Å), C13-C14 (1.457 Å), C17-C19 (1.413 Å), C19-C20 (1.408 Å), C24-C27

(1.526 Å) and C30-C33 (1.524 Å) were found to be elongated from their

–

default value of 1.38 Å. The calculated C H bond length of C13-H42

(1.099 Å), C24-H25 (1.10 Å) and C30-H31 (1.110 Å) were found to be

elongated from the default value of 1.08 Å. This may be due to their

presence in the vicinity of electronegative oxygen (O39), nitrogen (N36,

–

N37, N38) and chlorine (Cl40, Cl41) atoms [44,45]. Further, the C

N

bond lengths C19-N36 (1.401 Å), C24-N36 (1.455 Å) and C30-N36

(1.46 Å) of atoms present near the chlorines were found to be elon-

–

gated from the other C N bonds. This shows the highly electronegative

character of the chlorine (Cl40 and Cl41) atoms.

–

In MH-2, the C C bond lengths of C5-C24 (1.508 Å), C10-C11

(1.502 Å), C12-C14 (1.457 Å), C14-C15 (1.403 Å), C17-C19 (1.415 Å),

C19-C20 (1.412 Å), C28-C47 (1.546 Å), C28-C50 (1.537 Å), C31-C47

(1.521 Å) and C34-C37 (1.530 Å) were extended from their default

–

value of 1.38 Å. The calculated C H bond length of C24-H26 (1.092 Å),

C24-H27 (1.091 Å), C28-H29 (1.091 Å), C47-H8 (1.093 Å), C47-H49

(1.094 Å) and C50-H51 (1.092 Å) had also deviated from the default

value of 1.08 Å. This may be due to the electronegative oxygen (O39),

nitrogen (N36, N37, N38) and chlorine (Cl40, Cl41) atoms [44,45].

–

Further, the C N bond lengths C28-N45 (1.478 Å) and C34-N45 (1.458

Å) present near the chlorine atoms were found to be elongated from the

–

other C N bonds. This shows the highly electronegative character of

Fig. 2.5. The hydrophobicity surface created around the four hydrazones in the

the chlorine (Cl40 and Cl41) atoms.

(A) iminol and (B) amide forms.

–

In BH-3, the C C bond lengths of C11-C12 (1.495 Å), C13-C15

(1.461 Å), C20-C21 (1.399 Å), C25-C28 (1.517 Å) and C31-C34

(1.506 Å) had deviated from their default value of 1.38 Å again due to

Fig. 2.6. Pharmacophore features of the two tautomeric forms of the hydrazones. Hydrophobic group (cyan), Ring aromatic (orange) and H-bond donor (magenta).

(For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 2.7. Optimized structures of the hydrazones.

9

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

the electronegative oxygen (O42), nitrogen (N39, N40, N41), bromine

three hydrazones, the HOMO was spread throughout the molecule

except over the nitrogen mustard aromatic ring.

–

(Br31) and chlorine (Cl37, Cl38) atoms [44,45]. Further, the C N bond

lengths C25-N41 (1.455 Å), C31-N41 (1.463 Å) and C20-N41 (1.393 Å)

which are present near the chlorine atoms were elongated from the other

2.4.3.3. Natural bond orbital analysis. The intramolecular interactions,

delocalization of electrons and stabilization energy of the four hydra-

zones were investigated by performing NBO analysis using NBO 5.1

program as implemented in the Gaussian 09 W package at the DFT-

B3LYP/6-311G** level. The second order perturbation approach was

used to deduce the energy arising from the various hyperconjugative

interactions [48]. E⁽²⁾gave large values indicating electron donating

tendency and hence the greater extent of conjugation within the system.

The second order lowering E⁽²⁾was used to estimate the strength of

the delocalization.

–

C

N bonds. This shows the highly electronegative character of the

–

chlorine (Cl37 and Cl38) atoms. All the calculated C H bond length of

BH-3 lies in the range 0.93 Å-0.97 Å, which differs from their default

value of 1.08 Å. This contraction in wavelength conﬁrms the electron

withdrawing nature of bromine.

–

In CH-4, the C C bond lengths of C1-C10 (1.40 Å), C10-C11 (1.504

Å), C12-C14 (1.457 Å), C14-C15 (1.403 Å), C17-C19 (1.416 Å), C19-C20

(1.411 Å), C30-C33 (1.531 Å) and C24-C27 (1.528 Å) were elongated

–

from their default value of 1.38 Å. The calculated C H bond length of

C12-H13 (1.099 Å), C24-H25 (1.091 Å), C30-H32 (1.091 Å) and C24-

H26 (1.096 Å) were found to be elongated from the default value of

1.08 Å. This may be due to their presence in the vicinity of electro-

negative oxygen (O42), nitrogen (N39, N40, N41) and chlorine (Cl36,

E⁽²⁾= ΔE_ij= qi F(i, j)²/E_jꢀ E_i

(1)

Where E⁽²⁾is the stabilization energy, q_iis the donor orbital occupancy,

E_iand E_jare the diagonal elements and F(i,j) is the off diagonal NBO

Fock matrix element reported [47].

–

Cl37, Cl38) atoms [44,45]. Further, the C N bond lengths C24-N41

(1.454 Å) and C30-N41 (1.458 Å) which are present near the chlorine

The important electron donor acceptor interactions are given in

Tables 4–7 for the four hydrazones. In SH-1, the electron transfer was

–

atoms were found to be elongated from the other C N bonds. This

shows the highly electronegative character of the chlorine (Cl40 and

Cl41) atoms. These results conﬁrm the effect of substituents in SH-1

leading to signiﬁcant alterations in the structural parameters.

predominantly observed from the π-antibonding orbitals and lone pairs

of nitrogen, oxygen and chlorine (N36, N37, N38, O39, Cl40 and Cl41)

atoms. The comparatively lower E⁽²⁾values of 3.59, 4.19, 3.86 and 4.01

kcal/mol for the chlorine atoms (Cl40 and Cl41) indicate that they

transfer fewer electrons into its vicinity than N38 and O39 which is

evident from the large E⁽²⁾values of 44.03, 27.40 and 28.24 kcal/mol. A

2.4.3.2. Frontier molecular orbital analysis. The excitation energy of a

molecule can be calculated by determining the energy difference be-

tween the HOMO and the LUMO. This is a very good indicator of elec-

tronic transition absorption in molecular systems [44,45]. The structural

and physical properties of molecules along with their reactivity can be

better understood by studying the HOMO and LUMO levels. Red and

green colours are used to represent the positive and negative phases

respectively. B3LYP/6-311G** method was used to calculate the HOMO

and LUMO energies, energy gap and other chemical parameters. These

are given in Table 3. LP-LP and LP-bond pair type interactions were

predominant in the four hydrazones due to the HOMO-LUMO orbital

interactions. This was found to be consistent with the molecular orbital

theory [44,46,47]. The HOMO-LUMO plot of the four hydrazones are

presented in Fig. 2.8.

strong hyperconjugative interaction observed due to

π

(C14-C22)—

π

*

(C15-C17),

π

(C15-C17)—

π

*(C19-C20)),

*(C13-N37)—

*(C12-O39), LP(1) N38—

*(C12-N38) and LP(3) Cl40—

π

(C19-C20)—

π

*(C14-C22),

(C9-C11)—

π

*(C12-O39),

π

*(C14-C22),

π*(C19-C20)—

(C15-C17), LP(1) N38—

π

*(C13-N37), LP(2)

O39—

σ

σ*(C30-C33) was found to be

22.30, 21.45, 24.26, 102.71, 86.83, 259.95, 44.03, 27.40, 28.24 and

4.19 kcal/mol respectively which gives additional stability to the

molecule.

In MH-2, the π-antibonding orbitals and lone pairs of nitrogen, ox-

ygen and chlorine (N42, N44, N45, O46, Cl40 and Cl41) atoms were

found to be responsible for the electron transfer. Speciﬁcally, the atoms

N42, N45 and O46 transfer more number of electrons into its vicinity as

is evident from the large E⁽²⁾values 44.21, 27.63, 40.34 and 28.29 Kcal/

mol in comparison with the chlorine atoms (N44, Cl40 and Cl41) with

lesser E⁽²⁾values of 8.10, 3.60 and 3.49 kcal/mol. Strong hyper-

For SH-1, a total of 166 occupied orbitals and 870 virtual orbitals, for

MH-2, 107 occupied orbitals and 545 virtual orbitals, for BH-3, 112

occupied orbitals and 488 virtual orbitals and for CH-4, 103 occupied

orbitals and 479 virtual orbitals were observed. Based on their optimised

geometry, molecular orbital analysis of the four hydrazones showed that

they were composed mostly of p type atomic orbitals. The eventual

charge transfer taking place within the molecules can be clearly

explained by the HOMO-LUMO energy gap. Also, BH-3 (3.841 eV) has a

lesser energy gap than the other three hydrazones indicating its

enhanced reactivity when compared to the other structures. The HOMO-

LUMO plot also shows that the LUMO which was spread throughout the

molecules except for the nitrogen mustard functionality was identical

for all the four hydrazones. However, the HOMO was different for CH-4

where it was found to be spread throughout the molecule. For the other

conjugative interactions observed due to

π

(C1-C10)—

*(C14-C22),

(C6-C8)— *(C3-C5),

*(C14-C22), LP(1) (N42)—

*(C12-N44), LP(1) (N45)— *(C19-C20) and LP

π

*(C11-O46),

(C19-C20)—

(C14-

*(C11-

π

(C12-N44)—

*(C15-C17),

π

*(C14-C22),

(C3-C5)—

(C19-C20)

π

(C19-C20)—

π

*(C1-C10),

π

C22)

π

*(C15-C17),

π

O46), LP(1) (N42)—

(2) (O46)—

π

σ

*(C11-N42) was found to be 119.66, 90.34, 252.47,

169.95, 22.76, 22.65, 21.94, 24.54, 44.21, 27.63, 40.34 and 28.29 kcal/

mol respectively. This helps in stabilising the molecule as a whole.

In BH-3, the electron transfer was observed primarily from the

π

-antibonding orbitals and lone pairs of nitrogen, oxygen, chlorine and

bromine (N39, N40, N41, O42, Cl37, Cl38 and Br1) atoms. As in SH-1

and MH-2, the nitrogen atoms N39, N41, oxygen atom O42 and bromine

atom Br1 transfer more electrons into its vicinity as can be seen from the

large E⁽²⁾values of 44.36, 42.25, 28.11 and 10.02 kcal/mol. The

contribution of the chlorine atoms (Cl38 and N40) however is lesser

which can be deduced from the lower E⁽²⁾values of 4.03 and 7.18 kcal/

Table 3

HOMO, LUMO energies and energy gap of the hydrazones.

Parameters

SH-1

MH-2

BH-3

CH-4

HOMO energy

ꢀ 5.536 eV

ꢀ 1.60 eV

3.936 eV

5.536 eV

1.60 eV

3.568 eV

1.968 eV

0.508

ꢀ 5.451 eV

ꢀ 1.541 eV

3.909 eV

5.450 eV

1.541 eV

3.496 eV

1.955 eV

0.521

ꢀ 5.660 eV

ꢀ 1.819 eV

3.841 eV

5.660 eV

1.819 eV

3.739 eV

1.921 eV

0.519

ꢀ 5.664 eV

ꢀ 1.816 eV

3.848 eV

5.664 eV

1.816 eV

3.739 eV

1.924 eV

0.5116

mol. Strong hyperconjugative interactions observed due to

*(C2-C4), *(C6-C7)— *(C9-C11), *(C9-C11)— *(C12-O42),

C4)— *(C6-C7), (C18-C20)— *(C15-C16), LP(1) N39— *(C12-O42),

LP(1) N39— *(C13-N40), LP(1) N41— *(C18-C20) and LP(2) O42—

π

*(C6-C7)—

LUMO energy

π

(C2-

Energy gap

π

Ionization potential(I)

Electron Afﬁnity(A)

π

Electronegativity(χ)

σ

*(C12-N39) were found to be 145.09, 282.79, 87.46, 22.62, 24.68,

Pauling Hardness(ƞ)

Global softness(

Chemical potential(

Global electrophilicity(

44.36, 26.94, 42.25 and 28.11 Kcal/mol respectively giving stability to

the molecule.

σ

)

μ)

ꢀ 3.568

ꢀ 3.739

ꢀ 3.496

ω

)

3.234

3.640

3.635

3.126

CH-4 also shows that predominant electron transfer is from the

10

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Fig. 2.8. HOMO and LUMO plot of the four hydrazones.

π

-antibonding orbitals and lone pairs of nitrogen, oxygen and chlorine

charge on C12(0.429 e), C13(0.161 e), C19(0.216 e). Atoms C27

(ꢀ 0.225 e) and C33(ꢀ 0.211 e), attached to chlorine (Cl41, Cl40) atoms

show greater negative charge when compared to all the other carbon

atoms in the molecule. N36 shows the highest electronegativity with a

value of ꢀ 0.444 e and C12 shows the largest electropositivity with a

value of 0.429 e. This is a direct indication of the extensive charge

delocalization present in the molecules [50,51]. Positive charge is

localized on the hydrogen atoms. Hydrogen atoms H2, H4, H6, H8, H16,

H18, H21, H23 and H42 show lesser positive charge (≈0.06–0.08 e),

than the hydrogen atoms H43, H25, H26, H28, H29, H31, H32, H34 and

H35 (≈0.1–0.22 e) since the latter are attached to the nitrogen and

chlorine atoms. This conﬁrms the high electronegativity of both the

nitrogen and the chlorine atoms.

(N39, N40, N41, O42, Cl36, Cl37 and Cl38) atoms. The large E⁽²⁾values

of 44.23, 42.26, 28.11 and 12.73 kcal/mol of the atoms N39, N41, O42

and Cl36 indicate the greater number of electrons that they have

transferred when compared to the atoms Cl37 and N40 which have

lower E⁽²⁾values of 4.03 and 8.08 kcal/mol. A strong hyperconjugative

interaction observed due to

(C6-C8), *(C12-N40)— *(C14-C22),

(C6-C8)— *(C3-C5), (C14-C22)—

(C14-C22), LP(1) N39— *(C11-O42), LP(1) N39—

N41— *(C19-C20) and LP(2) O42—

π

*(C3-C5)—

π

*(C1-C10),

π

*(C3-C5)—

π

*

π

*(C1-C10)—

π

*(C11-O42),

π

π*(C15-C17),

π

(C19-C20)—

π

*

π

*(C12-N40), LP(1)

π

σ

*(C11-N39) was found to be

258.22, 139.58, 88.06, 85.44, 22.76, 22.34, 24.68, 44.23, 26.93, 42.26

and 28.11 Kcal/mol respectively which is also a contributing factor to

the stability of the molecule.

Presence of large electronegative atoms (O46, N42, N44, N45) in CH-

2 creates greater positive charge on C11(0.444 e), C19(0.274 e) and C50

(0.261 e). Atoms C24(-0.256 e), C31(-0.291 e), C37(-0.268 e) and C47

(-0.172e) in the neighbourhood of chlorine (Cl40, Cl41) show higher

negative charge when compared to the other carbon atoms present. N45

shows the largest electronegativity (-0.463 e) whilst C11 shows the

largest electropositivity (0.444 e). This indicates the extensive charge

2.4.3.4. Mulliken population analysis. The Mulliken population analysis

of the four hydrazones was performed at DFT-B3LYP/6-311G** level to

obtain the values of the atomic charges [49]. Table 8 lists the Mulliken

atomic charges of the four compounds. Presence of highly electronega-

tive atoms such as O39, N38, N37, N36 in SH-1 creates greater positive

11

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Table 4

Table 5

Second Order Perturbation Theory Analysis of Fock Matrix in NBO Basis of SH-1.

Second Order Perturbation Theory Analysis of Fock Matrix in NBO Basis of MH-

2.

Donor NBO

Aceptor NBO (j)

E(2)

E(j)-E(i)

a.u.

F(i,j)

a.u.

Kcal/mol

Donor NBO (i)

Acceptor NBO (j)

E(2)

E(j)—E(i)

F(i,j)

a.u.

Kcal/mol

a.u.

π

(C1-C3)

π

*(C5-C7)

21.52

20.65

18.68

21.17

19.44

19.59

15.20

8.58

0.28

0.29

0.28

0.29

0.30

0.37

0.26

0.28

0.27

0.29

0.28

0.29

0.62

0.76

0.32

0.29

0.66

0.67

0.02

0.01

0.63

0.72

0.67

0.72

0.67

0.87

0.88

0.97

1.07

0.070

0.068

0.067

0.070

0.068

0.067

0.062

0.055

0.069

0.072

0.064

0.062

0.071

0.075

0.061

0.053

0.071

0.107

0.082

0.102

0.125

0.068

0.067

0.079

0.049

0.045

0.047

0.046

0.055

0.053

0.058

0.062

(C1-C3)

*(C9-C11)

*(C1-C3)

π

(C1-C10)

(C3-C5)

π

*(C3-C5)

18.57

19.77

15.91

22.76

17.71

19.62

22.65

8.65

0.29

0.30

0.28

0.29

0.28

0.29

0.37

0.26

0.29

0.27

0.29

0.28

0.29

0.60

0.32

0.29

0.76

0.27

0.60

0.66

0.67

0.02

0.01

0.02

0.61

0.69

0.72

0.066

0.069

0.063

0.072

0.065

0.067

0.072

0.055

0.069

0.072

0.063

0.062

0.071

0.076

0.060

0.056

0.107

0.082

0.079

0.071

0.097

0.060

0.063

0.102

0.125

0.069

0.067

0.082

0.080

0.048

0.044

0.045

(C5-C7)

*(C6-C8)

(C5-C7)

*(C9-C11)

*(C1-C3)

*(C11-O46)

*(C1-C10)

*(C6-C8)

(C9-C11)

(C13-N37)

(C14-C22)

(C15-C17)

(C19-C20)

*(C5-C7)

(C3-C5)

*(C12-O39)

*(C14-C22)

*(C13-N37)

*(C15-C17)

*(C19-C20)

*(C14-C22)

*(C19-C20)

*(C14-C22)

*(C15-C17)

(C6-C8)

*(C1-C10)

*(C3-C5)

(C6-C8)

20.70

22.30

18.68

15.77

21.45

24.26

15.75

5.67

(C14-N44)

(C14-C22)

(C15-C17)

(C19-C20)

*(C14-C22)

*(C12-N44)

*(C15-C17)

*(C19-C20)

*(C14-C22)

*(C19-C20)

*(C14-C22)

*(C15-C17)

20.82

21.94

17.85

15.44

21.38

24.54

14.85

6.57

σ

(C24-H25)

σ

*(C27-Cl41)

*(C13-H42)

*(N38-H43)

LP(1) N37

LP(1) N38

LP(2) O39

10.07

8.09

σ(C47-H49)

σ

*(C31-Cl40)

LP(1) (N42)

LP(1) (N44)

LP(1) (N45)

LP(2) (O46)

π

*(C11-O46)

*(C12-N44)

44.21

27.63

10.12

8.10

π

*(C12-O39)

*(C13-N37)

44.03

27.40

19.12

28.24

102.71

86.83

259.95

4.27

π

σ

*(C12-H13)

*(N42-H43)

σ

*(C11-C12)

*(C12-N38)

π

*(C19-C20)

40.34

6.71

π*(C9-C11)

π*(C13-N37)

π*(C19-C20)

π

*(C12-O39)

*(C14-C22)

*(C15-C17)

σ

*(C28-C47)

*(C34-C37)

*(C10-C11)

*(C11-N42)

7.28

18.96

28.29

119.66

90.34

252.47

169.95

4.15

LP(1) N36

LP(2) Cl40

LP(3) Cl40

LP(2) Cl41

LP(3) Cl41

σ

*(C30-H31)

*(C33-H34)

*(C30-C33)

*(C27-H28)

*(C24-C27)

*(C30-N36)

*(C24-N36)

*(C19-N36)

*(C19-C20)

3.59

π

(C1-C10)

π

*(C11-O46)

*(C14-C22)

*(C15-C17)

4.19

(C12-N44)

(C19-C20)

3.86

4.01

σ

(C33-H35)

(C27-H29)

(C24-H25)

4.33

LP(1) (N45)

LP(3)Cl 40

LP(2)Cl 41

σ

*(C28-C50)

*(C31-C47)

*(C37-H39)

4.05

3.60

4.31

3.49

(C22-H23)

4.58

Table 6

delocalization in the molecule [50,51]. Hydrogen atoms H2, H4, H7,

H13 and H23 show lesser positive charge (≈0.06–0.09 e), while H9,

H16, H18, H21, H25, H26, H27, H29, H30, H32, H33, H35, H36, H38,

H39, H43, H48 and H49 which are present in the methyl group and

those attached to nitrogen and chlorine atoms show more positive

charge (≈0.1–0.22 e) thus conﬁrming the large electronegativity of the

nitrogen and chlorine atoms.

Second Order Perturbation Theory Analysis of Fock Matrix in NBO Basis of BH-

3.

Donor NBO (i)

Acceptor NBO (j)

E(2)

E(j)—E(i)

F(i,j)

a.u.

Kcal/mol

a.u.

σ

(C2-C4)

σ

*(Br1-C6)

5.50

0.79

0.27

0.28

0.31

0.30

0.80

0.29

0.27

0.31

0.26

0.27

0.28

0.29

0.28

0.62

0.30

0.32

0.29

0.76

0.28

0.60

0.65

0.67

0.72

0.02

0.01

0.02

0.059

0.070

0.067

0.068

0.059

0.069

0.068

0.062

0.069

0.063

0.072

0.076

0.060

0.061

0.072

0.053

0.054

0.107

0.082

0.079

0.071

0.100

0.063

0.064

0.102

0.125

0.048

0.080

0.085

0.069

π

(C2-C4)

(C6-C7)

π

*(C6-C7)

*(C9-C11)

*(C2-C4)

*(C9-C11)

22.62

20.08

18.18

19.02

5.45

BH-3 analysis shows that the large electronegative atoms O42, N39,

N40, N41 create greater positive charge on C12(0.428 e), C13(0.166 e)

and C20(0.205 e). The atoms C11(-0.204 e), C15(ꢀ 0.187 e), C28

(ꢀ 0.266 e) and C34(ꢀ 0.220 e) which are found in the vicinity of

bromine (Br1) and chlorine (Cl38, Cl37) show more negative charge

when compared to the other carbons present in the molecule. N41 shows

the largest electronegativity (-0.439 e) whilst C12 shows the largest

electropositivity (0.428 e) thus conﬁrming the extensive charge delo-

calization in the molecule [50,51]. Positive charge is localized on the

hydrogen atoms. Hydrogen atoms H3, H5, H8, H10, H14, H17, H19,

H22 and H24 shows lesser positive charge (≈0.06–0.09 e) than the

hydrogen atoms H43, H26, H27, H29, H30, H32, H33, H35 and H36

(≈0.1–0.22 e) which are attached to the nitrogen and chlorine atoms.

This once again conﬁrms the large electronegativity of the nitrogen and

chlorine atoms.

σ

(C7-C9)

σ*(Br1-C6)

π

(C9-C11)

(C15-C16)

(C18-C20)

(C21-C23)

π

*(C2-C4)

19.81

20.97

15.15

20.97

17.76

22.33

24.68

14.65

15.24

21.71

5.75

*(C6-C7)

*(C12-O42)

*(C13-N40)

*(C18-C20)

*(C21-C23)

*(C15-C16)

*(C21-C23)

*(C15-C16)

*(C18-C20)

σ(C31-H33)

σ

*(C34-Cl38)

LP(3) Br1

LP(1) N39

LP(1) N40

LP(1) N41

LP(2) O42

LP(2) Cl38

π

*(C6-C7)

10.02

44.36

26.94

10.07

8.08

*(C12-O42)

*(C13-N40)

σ

*(N13-H14)

*(N39-H43)

The analysis of CH-4 indicates that the presence of large electro-

negative atoms such as O42, N39, N40, N41 creates a greater positive

charge on C11(0.435 e), C12(0.162 e) and C19(0.204 e). The atoms C5

(ꢀ 0.267 e), C10(-0.208 e), C27(ꢀ 0.217 e) and C33(ꢀ 0.265 e) which are

in the vicinity of the chlorine (Cl36, Cl38, Cl37) atoms show greater

negative charge when compared to the other carbon atoms present in

the molecule. N41 shows the highest electronegativity (ꢀ 0.437 e) and

C11 shows the highest electropositivity (0.435 e) thus proving the

extensive charge delocalization in the molecule [50,51]. The hydrogen

atoms H2, H4, H7, H9, H13, H16, H18, H21 and H23 show lesser

π

*(C18-C20)

42.25

7.18

σ

*(C25-C28)

*(C31-C34)

*(C11-C12)

*(C12-N39)

*(C34-H36)

7.50

19.33

28.11

4.03

π*(C6-C7)

π*(C9-C11)

π

*(C2-C4)

145.09

282.79

87.46

*(C9-C11)

*(C12-O42)

12

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

unsubstituted rings which are better than those with electron with-

drawing substituents. Possibly, a greater number of hydrazone de-

rivatives with different substituents must be studied before a deﬁnitive

conclusion can be reached. Micro molar IC₅₀values such as can be seen

for the four hydrazones are a good indication of their potency and ac-

tivity against different types of cancer cell lines such as lung and liver

cancers. Hence, these novel hydrazones can be considered as potential

leads in cancer therapy comparable to those reported in literature

[23,53]. The IC₅₀values and dose response graph are provided in

Table 9 and Fig. 2.9 respectively.

Table 7

Second Order Perturbation Theory Analysis of Fock Matrix in NBO Basis of CH-

4.

Donor NBO (i)

Acceptor NBO (j)

E(2)

E(j)-E(i)

a.u.

F(i,j)

a.u.

Kcal/mol

σ

(C1-C3)

σ

*(C5-Cl36)

5.27

0.85

0.27

0.29

0.31

0.30

0.31

0.84

0.28

0.27

0.37

0.26

0.28

0.27

0.29

0.28

0.29

0.32

0.29

0.76

0.28

0.60

0.65

0.67

0.88

0.33

0.72

0.02

0.01

0.02

0.060

0.068

0.069

0.062

0.069

0.067

0.060

0.067

0.070

0.055

0.069

0.072

0.063

0.061

0.072

0.076

0.060

0.107

0.082

0.079

0.071

0.100

0.064

0.063

0.102

0.125

0.055

0.063

0.048

0.068

0.085

0.080

0.067

π

(C1-C10)

(C3-C5)

π

*(C3-C5)

*(C6-C8)

*(C11-O42)

*(C1-C10)

*(C6-C8)

20.88

19.97

15.19

19.25

17.96

5.33

σ

(C6-C8)

σ*(C5-Cl36)

π

(C6-C8)

π

*(C1-C10)

*(C3-C5)

19.84

22.76

8.54

2.5.2. In vitro tubulin polymerization assay

(C6-C8)

Molecular docking studies showed the effective binding of the

hydrazones to β-tubulin in both their tautomeric forms. To explore this

further and determine the type and extent of protein inhibition, in vitro

tubulin polymerization assay was performed. The change in optical

density was calculated based on the measurement of absorbance at 340

nm of polymerized tubulin every ﬁve minutes for thirty minutes. All the

compounds showed lesser absorbance than the control indicating that

they inhibited the polymerization of tubulin. Thus, these four hydra-

zones can be seen to act as tubulin binding agents through depolymer-

ization. MH-2 showed the best result in this assay conﬁrming the

theoretical studies performed. Presence of the methyl group [54] seems

to have a highly inhibitory effect on β-tubulin making MH-2 a potent

therapeutic agent in cancer study. The other three hydrazones show a

lesser inhibitory effect on the polymerization of tubulin when compared

to MH-2. The percentage cell viability graph can be viewed in Fig. 2.10.

(C12-N40)

(C14-C22)

(15-C17)

(C15-C17)

(C19-C20)

*(C14-C22)

*(C12-N40)

*(C15-C17)

*(C19-C20)

*(C14-C22)

*(C19-C20)

*(C14-C22)

*(C15-C17)

*(C11-O42)

*(C12-N40)

20.99

22.34

17.77

15.24

21.70

24.68

14.65

44.23

26.93

10.07

8.08

LP(1) N39

LP(1) N40

LP(1) N41

LP(2) O42

LP(2) Cl36

LP(3) Cl37

σ

*(C12-H13)

*(N39-H43)

π

*(C19-C20)

42.26

7.48

σ

*(C24-C27)

*(C30-C33)

*(C10-C11)

*(C11-N39)

*(C3-C5)

7.19

19.34

28.11

4.32

*(C5-C6)

4.30

3. Experimental

π

*(C3-C5)

12.73

4.03

σ

*(C27-H29)

π

*(C1-C10)

π

*(C11-O42)

*(C1-C10)

*(C6-C8)

85.44

258.22

139.58

88.06

All the chemicals and reagents used were purchased from Sigma

Aldrich and of analaR grade. The nitrogen mustard precursor was pre-

pared according to reported procedure.¹⁸The progress of the reaction

and purity of the products were monitored by thin layer

chromatography.

*(C3-C5)

*(C12-N40)

*(C14-C22)

positive (≈0.06–0.09 e) charge than the hydrogen atoms H25, H26,

H28, H29, H31, H32, H34, H35 and H43 (≈0.1–0.22 e) since the latter

are attached to the nitrogen and chlorine atoms conﬁrming the large

electronegativity of the nitrogen and chlorine atoms.

3.1. Measurements

Melting points of the four hydrazones were determined using a

melting point apparatus. Aluminium TLC plates were used for thin layer

chromatography. The UV–visible spectra for the title compounds were

taken using the Specord 210 UV–VIS spectrophotometer with ethanol as

the solvent in the range 200–800 nm. FT-IR spectra was recorded from

4000 to 400 cm^{ꢀ 1}using Shimadzu DR 8001 series FT-IR instrument with

KBr pellets. A BRUKER AV III 500 MHz FT NMR spectrometer was used

for ¹H and ¹³C NMR spectra with DMSO‑d₆as the solvent at 500 MHz

and 125 MHz respectively. Thermo Scientiﬁc Orbitrap Elite Mass spec-

trometer was used for mass spectral measurements.

2.5. Biological activity

2.5.1. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazoliumbromide) assay

The four hydrazones were tested for their potency against the A549

lung cancer and HepG2 liver cancer cell lines using the MTT assay. The

IC₅₀values were determined using GraphPad Prism 9.1.1. All the com-

pounds displayed signiﬁcant activity showing micro molar IC₅₀values

against the two cancer cells. Of the four hydrazones, MH-2 was found to

give the best results with an IC₅₀value of 0.0896 and 0.1040 µM against

A549 and HepG2 respectively. The electron donating capacity of the

alkyl group seems to have enhanced its activity substantially when

compared to the other three compounds. SH-1 has shown very good

activity against A549 and HepG2 after MH-2 showing that unsubstituted

hydrazones show better activity than compounds containing electron

withdrawing substituents. Both BH-3 and CH-4 display comparable

activity against both the cancer cells with the BH-3 showing slightly

better IC₅₀values than the chloro counterpart. The fact that the methyl

substituted hydrazone MH-2 has given the lowest IC₅₀value is not

surprising since the presence of the methyl group has been known to

enhance potency in various drug molecules. Vitaku et al have reported

the presence of methyl groups in more than 73% of the top 200 small

molecule drugs that are currently being sold which reafﬁrms the

importance of the methyl group in a drug molecule. [52] From the four

compounds studied, it can be seen that aromatic rings with electron

donating substituents show better potency against cancer cells than

3.2. Single crystal X-ray diffraction studies

3.2.1. Unit cell determination

A Bruker AXS X8 KAPPA APEX II single crystal CCD diffractometer

was used for the determination of crystal structure. It was equipped with

a Mo(Kα) (λ = 0.7107 Å) radiation source. The goniometer attached to

the diffractometer was a four circle one with φ, χ, ω and 2θ axes through

which the crystal is rotated. Four crystal specimens of the title com-

pounds were cut and mounted on a glass ﬁbre using cyanoacrylate. The

diffracted intensities from 36 frames were collected for the determina-

tion of unit cell parameters. The intensities were measured in three

different crystallographic zones by using the method of difference vec-

tors. Data collection was done at 293 K using ω-φ scan modes.

3.2.2. Structure solution and reﬁnement

SHELXS 97 [55] was used to solve the structures of all the hydra-

zones. The positions of all non-hydrogen atoms were revealed and

13

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Table 8

Mulliken atomic charge analysis of the hydrazones using DFT/B3LYP/6-31G(d,p).

S. No.

SH-1

MH-2

BH-3

CH-4

Atoms

Charges (e)

Atoms

Charges (e)

Atoms

Charges (e)

Atoms

Charges (e)

1.

C1

ꢀ 0.003

0.092

Br1

C2

0.024

C1

ꢀ 0.021

0.071

C1

ꢀ 0.071

0.087

2.

H2

0.004

H2

3.

C3

ꢀ 0.077

0.086

H3

0.098

C3

0.059

C3

ꢀ 0.075

0.086

4.

H4

C4

0.016

H4

0.105

H4

5.

C5

ꢀ 0.063

0.084

H5

0.101

C5

ꢀ 0.267

0.054

C5

ꢀ 0.091

ꢀ 0.077

0.090

6.

H6

C6

ꢀ 0.210

0.014

C6

7.

C7

ꢀ 0.084

0.083

C7

H7

0.111

H7

8.

H8

0.097

C8

0.002

C8

0.030

9.

C9

ꢀ 0.031

0.070

C9

ꢀ 0.026

0.074

H9

0.098

H9

0.111

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

H10

C11

C12

C13

C14

C15

H16

C17

H18

C19

C20

H21

C22

H23

C24

H25

H26

C27

H28

H29

C30

H31

H32

C33

H34

H35

N36

N37

N38

O39

Cl40

Cl41

H42

H43

H10

C11

C12

C13

H14

C15

C16

H17

C18

H19

C20

C21

H22

C23

H24

C25

H26

H27

C28

H29

H30

C31

H32

H33

C34

H35

H36

Cl37

Cl38

N39

N40

N41

O42

H43

C10

C11

C12

H13

C14

C15

H16

C17

H18

C19

C20

H21

C22

H23

C24

H25

H26

C27

H28

H29

C30

H31

H32

C33

H34

H35

Cl36

Cl37

Cl38

N39

N40

N41

O42

H43

ꢀ 0.208

0.435

C10

C11

C12

H13

C14

C15

H16

C17

H18

C19

C20

H21

C22

H23

C24

H25

H26

H27

C28

H29

H30

C31

H32

H33

C34

H35

H36

C37

H38

H39

Cl40

Cl41

N42

H43

N44

N45

O46

C47

H48

H49

C50

H51

H52

0.179

ꢀ 0.210

0.429

ꢀ 0.204

0.428

0.444

0.162

0.149

0.161

0.166

0.052

ꢀ 0.185

0.002

0.052

ꢀ 0.186

0.005

ꢀ 0.164

ꢀ 0.038

0.109

ꢀ 0.187

ꢀ 0.040

0.071

0.084

0.091

ꢀ 0.113

0.090

ꢀ 0.106

0.089

ꢀ 0.115

0.101

ꢀ 0.096

0.095

0.216

0.204

0.274

ꢀ 0.080

0.104

0.205

ꢀ 0.101

0.097

ꢀ 0.151

0.105

ꢀ 0.110

0.089

ꢀ 0.043

0.070

ꢀ 0.038

0.072

ꢀ 0.071

0.081

0.019

ꢀ 0.113

0.134

0.090

ꢀ 0.124

0.141

ꢀ 0.256

0.129

ꢀ 0.099

0.153

0.131

0.137

0.120

ꢀ 0.225

0.171

0.146

ꢀ 0.217

0.161

0.111

ꢀ 0.266

0.173

ꢀ 0.086

0.153

0.160

0.169

ꢀ 0.112

0.123

0.161

ꢀ 0.097

0.154

0.122

ꢀ 0.122

0.142

ꢀ 0.291

0.170

0.131

0.145

ꢀ 0.211

0.169

0.137

ꢀ 0.265

0.172

0.178

ꢀ 0.220

0.161

ꢀ 0.124

0.152

0.158

0.160

ꢀ 0.444

ꢀ 0.171

ꢀ 0.305

ꢀ 0.349

ꢀ 0.111

ꢀ 0.102

0.060

0.170

ꢀ 0.068

ꢀ 0.110

ꢀ 0.111

ꢀ 0.298

ꢀ 0.172

ꢀ 0.437

ꢀ 0.341

0.218

0.150

ꢀ 0.113

ꢀ 0.109

ꢀ 0.297

ꢀ 0.167

ꢀ 0.439

ꢀ 0.342

0.221

ꢀ 0.268

0.174

0.171

ꢀ 0.125

ꢀ 0.116

ꢀ 0.317

0.213

0.222

ꢀ 0.162

ꢀ 0.463

ꢀ 0.332

ꢀ 0.172

0.138

0.127

0.261

0.121

0.113

asymmetry calculations were calculated using PARST 97. The thermal

ellipsoid plot and packing were determined by ORTEP and PLATON

respectively. PLATON was also used to create non-bonded interacted

graphics. The bond distances and bond angles can be viewed at the

Cambridge Crystallographic Data website (www.ccdc.co.uk).

Table 9

IC₅₀values of the four hydrazones against A549 and HepG2.

Compund

IC₅₀(µM)

A549

HepG2

SH-1

MH-2

BH-3

CH-4

0.2158 ± 0.0053

0.0896 ±0.0029

0.3137±0.0045

0.3248±0.0042

0.3954 ± 0.0031

0.1040 ±0.0039

0.4172 ±0.0046

0.4424 ± 0.0063

3.3. Computational studies

3.3.1. Molecular docking

Values are mean ± SD triplicate assays.

Computer aided drug design and development plays an essential role

in the discovery of therapeutically important molecules for cancer

therapy [56]. In the present study, an integrated model of molecular

docking and pharmacophore mapping has been used to determine the

therapeutic efﬁciency of the title compounds. β-tubulin was identiﬁed as

the protein receptor and its 3D crystal structure (1SA1) was downloaded

from the RCSB protein data bank website (www.rcsb.org) and used.

reﬁned on F²by using a full matrix least squares procedure using

SHELXL 97. Anisotropic reﬁnement of non-hydrogen atoms was done

following which the hydrogen atoms were allowed to ride over their

parent atoms. The ﬁnal reﬁnement cycle converged to their respective

R₁and wR₂values for the observed reﬂections. Least squares planes and

14

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

Fig. 2.9. Dose response graph of the four hydrazones against A549 and HepG2 cancer cell lines.

title compounds using NBO 5.1 program [60] as implemented in the

Gaussian 09 package at DFT/B3LYP level. The second order perturba-

tion approach was utilized to deduce hyperconjugation and interaction

energy within the molecules [61–63].

0.5

0.4

0.3

0.2

0.1

3.4. Synthesis of hydrazones

3.4.1. Synthesis of N’-(4-(bis(2-chloroethyl)amino)benzylidene)

benzhydrazide (SH-1)

Control

SH-1

MH-2

BH-3

CH-4

To a solution of p-[N, N-bis(2-chloroethyl)amino]benzaldehyde 1 (1

mmol) in ethanol, benzhydrazide 2a (1 mmol) in ethanol was added. To

this mixture, three drops of conc. HCl was added and the resulting so-

lution was reﬂuxed for 3 h. The progress of the reaction was monitored

by TLC until the reactants were completely consumed. Upon cooling to

room temperature, the product precipitated out and was collected. It

was then ﬁltered, washed and recrystallized from ethanol to give red

needle like crystals. Yield (82%), m.p. 130 ^◦C; ¹H NMR (500 MHz;

DMSO‑d₆;) δ = 3.73 – 3.84 (m, J = 3.9 Hz, 8H), 6.84 (d, J = 8.6 Hz, 2H),

7.49 – 7.55 (m, 2H), 7.54 – 7.61 (m, 3H), 7.90 (d, J = 7.1 Hz, 2H), 8.33

(s, 1H), 11.60 ppm (s, 1H) ;¹³C NMR (125 MHz; DMSO- d₆) δ = 41.0 (C-

12, 12^′), 51.9 (C-11, 11^′), 111.8 (C-9, 9^′), 122.7 (C-7), 127.5 (C-8, 8^′),

0.0

0

5

10

15

20

25

30

Minutes

Fig. 2.10. Absorbance of the hydrazones at 340 nm in the tubulin polymeri-

zation inhibition assay.

128.4 (C-2, 2^′), 128.8 (C-3, 3^′), 131.5 (C-1), 133.8 (C-4), 148.0 (C-10),

–

148.2 (C-6), 162.8 ppm (C-5); FT-IR (KBr) ν_max= 1600 (C O), 3200

–

Accelrys Discovery Studio software was employed for the computational

work with the LigandFit protocol being used for the docking studies. The

title compounds were docked into the active site of the protein receptor

and their binding interactions were analysed with respect to non-

covalent interactions. The binding energies of the ligand–protein com-

plex was also determined. Pharmacophore mapping was done to identify

essential features of the molecule responsible for the biological activity.

Common Feature Pharmacophore Generation comprising of the HipHop

model was used for the mapping process. All the four hydrazones obey

Lipinski’s rule of 5 and could therefore be viable options for cancer

treatment.

–

(amide N H stretching), 1550 (amide N H bending), 800 (C-Cl),

1

–

3000–3100 (aromatic C H stretching), 1400–1500 cm- (C C); UV/

–

Vis (ethanol): λ_max

(ε

) 220, 348 nm (45454.55, 28735.63 dm³mol^{ꢀ 1}

cm^{ꢀ 1}); ESI-MS: m/z 364[M + 1]⁺; elemental analysis Calcd (%) for

C

₁₈H₁₉C_l2N₃O: C 59.35, H 5.26, Cl 19.46, N 11.54, O 4.39; found: C

59.37, H 5.27, Cl 19.42, N 11.52, O 4.42.

3.4.2. Synthesis of N’-(4-(bis(2-chloroethyl) amino) benzylidene)-4-

methyl benzhydrazide (MH-2)

The method described above was followed for MH-2. MH-2 was

synthesized from 1 and p-toluic hydrazide 2b (1 mmol). The product

formed was ﬁltered, washed and recrystallized in ethanol to give red

needle like crystals. Yield (87%) m.p. 210 ^◦C; ¹H NMR (500 MHz;

DMSO‑d₆;) δ = 2.3 (3H, t, CH₃), 3.7–3 0.8 (8H, m, J = 13.5 Hz, H-11, 11^′,

12, 12^′), 6.8 (2H, d, J = 8.5 Hz, H-9, 9^′), 7.3 (2H, d, J = 8 Hz, H-3, 3^′), 7.5

(2H, d, J = 8.5 Hz, H-8, 8^′), 7.8 (2H, d, J = 8 Hz, H-2, 2^′), 8.3 (1H, s, CH

= N), 11.5 ppm (1H, s, CO-NH); ¹³C NMR (125 MHz; DMSO- d₆) δ = 21.0

(C-4^′), 40.1 (C-12, 12^′), 51.8 (C-11, 11^′), 111.8 (C-9, 9^′), 122.8 (C-7),

3.3.2. Density Functional Theory studies

Quantum mechanical calculations were performed by applying the

DFT method using the Gaussian 09 program suite [57] at the Becke-3-

Lee-Yang-Par (B3LYP) level [58–59] combined with the standard 6-

311G(d,p) basis set for the hydrazones. The parameters were allowed

to relax during the optimization procedure in order to obtain stable

structures with minimization energy. The structure optimization pro-

cedure was used to ascertain the global minimum energy of the hydra-

zones. Natural bonding orbital (NBO) analysis was performed on the

127.5 (C-2, 2^′), 128.7 (C-8, 8^′), 128.9 (C-3, 3^′), 130.8 (C-1), 141.4 (C-4),

–

147.9 (C-6, 10), 162.5 ppm (C-5); FT-IR (KBr) ν_max= 1650 (C O), 3200

–

(amide N H stretching), 1350–1520 (aromatic C C), 840 cm^{ꢀ 1}(C-Cl).

–

15

J.S. Xavier et al.

Bioorganic Chemistry 114 (2021) 105094

UV/Vis (ethanol): λ_max

(

ε

) 232, 345 nm (43103.00, 28985.51 dm³mol^{ꢀ 1}

3.5.2. In vitro tubulin polymerization assay

cm^{ꢀ 1}); ESI-MS: m/z 378.05 [M + 1]⁺; elemental analysis Calcd (%) for

C₁₉H₂₁C_l2N₃O: C, 60.33; H, 5.60; Cl, 18.74; N, 11.11; O, 4.23. Found: C,

60.31; H, 5.62; Cl, 18.72; N, 11.13; O, 4.22.

Molecular docking of the four compounds with β-tubulin was sub-

stantiated by performing an in vitro tubulin inhibition assay using a

commercial tubulin polymerization assay kit (porcine tubulin and

ﬂuorescence based), Cytoskeleton Inc. The manufacturer’s protocol was

used to perform the assay. Puriﬁed tubulin (4 mg/mL) in G-PEM buffer

(80 mM PIPES pH 6.9, 2mMMgCl₂and 0.5 mM EGTA plus 1 mM GTP)

was incubated with the four compounds in pre-warmed plates. The in-

hibition of tubulin polymerization was determined based on a time

dependant increase in ﬂuorescence during polymerization. Polymerized

tubulin shows absorbance at 340 nm. If test compounds inhibited the

polymerization, then the absorbance would correspondingly decrease in

comparison with the control. Hence ﬂuorescence changes for each

compound were recorded at 340 nm using a spectrophotometer at 37 ^◦C

at 5 min intervals.

3.4.3. Synthesis of N’-(4-(bis(2-chloroethyl)amino)benzylidene)-4-

bromobenzohydrazide (BH-3)

To a solution of p- [N, N-bis(2-chloroethyl) amino] benzaldehyde 1

(1 mmol) in two to three ml of DMF was added one mmol of 4-bromo-

benzohydrazide 2c in DMF. The mixture was then magnetically stirred

at 50 ^◦C for three hours. The solution was cooled to room temperature

and poured into crushed ice in a beaker containing one drop of conc.

H₂SO₄. The precipitate obtained was ﬁltered, dried and recrystallized

with DMF to give white needle shaped crystals. Yield (68%) m.p. 175 ^◦C;

¹H NMR (500 MHz; DMSO‑d₆;) δ = 3.7 – 3.8 (8H, m, H-11, 11^′, 12, 12^′),

6.8 (2H, d, J = 9 Hz, H-9, 9^′), 7.5 (2H, d, J = 7.5 Hz, H-3, 3^′), 7.5 (2H, d,

J = 9 Hz, H-8, 8^′), 7.9 (2H, d, J = 7.5 Hz, H-2, 2^′), 8.3 (1H, s, CH = N),

11.5 ppm (1H, s, CO-NH); ¹³C NMR (125 MHz; DMSO- d₆) δ = 41.5 (C-

12, 12^′), 52.3 (C-11, 11^′), 112.3 (C-9, 9^′), 123.2 (C-7), 127.9 (C-2, 2^′),

4. Conclusion

A new class of hydrazone derivatives were designed and synthesized

from a nitrogen mustard precursor to identify potential microtubule

targeted agents. A simple condensation reaction was carried out to

synthesize and crystallize the four aroylhydrazones. They were analyzed

by single crystal XRD studies and identiﬁed to have the P c a 2₁, P 2₁/c

and I 41/a space group respectively. Molecular docking showed that the

iminol forms of the four hydrazones bind most effectively into the active

site of β-tubulin through several signiﬁcant non-covalent interactions

with important amino acid residues. Pharmacophore mapping brings

out both the iminol and amide tautomers as effective pharmacophores

highlighting the features responsible for biological activity. DFT studies

have been used as an effective tool to determine the geometrical and

electronic properties of the compounds. The anticancer studies per-

formed highlight this novel series of hydrazones as effective agents in

anticancer therapy. The four hydrazones had IC₅₀values in the micro-

molar range for both the lung and liver cancer cell lines. MH-2 was

found to be the most potent with an IC₅₀value of 0.0896 and 0.1040 µM

against A549 and HepG2 respectively. This could be attributed to the

presence of the methyl group which is known to increase the binding

afﬁnity of the ligand and selectivity thereby enhancing its potency as a

drug molecule. All the four hydrazones were found to inhibit the poly-

128.8 (C-8, 8^′), 129.2 (C-3, 3^′), 131.9 (C-1), 134.2 (C-4), 148.7 (C-10),

–

148.4 (C-6), 163.2 ppm (C-5); FT-IR (KBr) ν_max= 1620 (C O), 3200

–

(amide N H stretching), 1550 (amide N H bending), 800 (C Cl), 550

1

–

(C-Br), 3000–3100 (aromatic C H stretching), 1400–1500 cm- (C C);

UV/Vis (ethanol): λ_max

(

ε

) 240, 345 nm (41666.67, 28985.51 dm³mol^{ꢀ 1}

cm^{ꢀ 1}); ESI-MS: m/z 441.9 [M + 1]⁺; elemental analysis Calcd (%) for

C

₁₈H₁₈BrCl₂N₃O: C, 48.78; H, 4.09; Br, 18.03; Cl, 16.00; N, 9.48; O, 3.61

Found: C, 48.81; H, 4.10; Br, 18.01; Cl, 16.03; N, 9.45; O, 3.60.

3.4.4. Synthesis of N’-(4-(bis(2-chloroethyl) amino) benzylidene)-4-chloro

benzohydrazide (CH-4)

The method described above was followed for CH-4. CH-4 was

synthesized from 1 and 4-chlorobenzohydrazide 2d (1 mmol). The

precipitate obtained was ﬁltered, dried and recrystallized with DMF to

give white needle shaped crystals. Yield (74%) m.p. 175 ^◦C; ¹H NMR

(500 MHz; DMSO‑d₆;) δ = 3.7–3.8 (8H, m, H-11, 11^′, 12, 12^′), 6.8 (2H, d,

J = 9 Hz, H-9, 9^′), 7.5 (2H, d, J = 9 Hz, H-3, 3^′), 7.6 (2H, d, J = 8.5 Hz, H-

8, 8^′), 7.9 (2H, d, J = 8.5 Hz, H-2, 2^′), 8.3 (1H, s, CH = N), 11.6 ppm (1H,

s, CO-NH); ¹³C NMR (125 MHz; DMSO- d₆) δ = 40.9 (C-4), 51.8 (C-11,

11^′), 111.8 (C-9, 9^′), 122.6 (C-7), 128.5 (C-2, 2^′), 128.8 (C-8, 8^′), 129.4

(C-3, 3^′), 132.4 (C-1), 136.3 (C-4), 148.1 (C-10), 148.5 (C-6), 161.80

merization of α- and β-tubulin heterodimers into microtubules through

–

max

ppm (C-5); FT-IR (KBr) ν_max= 1630 (C O), 3200 (amide N

H

the tubulin inhibition assay. The studies pave the way for the rational

design of tubulin inhibiting microtubule targeted agents.

–

stretching), 1350 and 1520 (C C), 840 (C-Cl); UV/Vis (ethanol): λ

(

ε

) 240, 345 nm (41666.67, 28985.51 dm³mol^{ꢀ 1}cm^{ꢀ 1}); ESI-MS: m/z

397.9 [M + 1]⁺; elemental analysis Calcd (%) for C₁₈H₁₈C_l3N₃O: C,

54.22; H, 4.55; Cl, 26.67; N, 10.54; O, 4.01 Found C, 54.24; H, 4.56Cl,

26.64; N, 10.53; O, 4.03.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

3.5. Biological activity

3.5.1. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

Appendix A. Supplementary material

diphenyltetrazoliumbromide) assay

The potency of the four hydrazones on human lung cancer cells

(A549) and human liver (hepatocellular carcinoma) cancer cells

(HepG2) was determined using the MTT cell viability assay. The cancer

cells and the title compounds were dissolved in DMSO and prepared in

96-well plates with a ﬁnal volume of 100 µL per well. They were placed

in a humidiﬁed incubator containing 5% CO₂and 95% air at a tem-

perature of 37 ^◦C. The medium was regularly examined and changed

twice on a weekly basis. Then 10 µL MTT solution was added to the cells

to achieve a ﬁnal concentration of 0.45 mg mL^{ꢀ 1}and incubated. Once

the purple formazan crystals were formed, they were dissolved in

DMSO. Their absorbance was then recorded at 570 nm. The inhibition

percentage was determined for each compound. A negative control with

the untreated cells was also analysed. The IC₅₀values were determined

by non-linear regression analysis using the log[inhibitor] vs. response –

variable slope equation in GraphPad Prism 9.1.1.

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.105094.

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Virtual and experimental high throughput screening of substituted hydrazones on β-Tubulin polymerization

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