Design and development of Tetrahydro-Quinoline derivatives as dual mTOR-C1/C2 inhibitors for the treatment of lung cancer

Article abstract of DOI:10.1016/j.bioorg.2020.104501

Lung cancer is one of the most prevailed cancer worldwide. Many genes get mutated in lung cancer but the involvement of EGFR, KRAS, PTEN and PIK3CA are more common. Unavailability of potent drugs and resistance to the available drugs are major concern in

Full text of DOI:10.1016/j.bioorg.2020.104501

Journal Pre-proofs
Design and Development of Tetrahydro-Quinoline Derivatives as Dual
mTOR-C1/C2 Inhibitors for the Treatment of Lung Cancer
Udit J. Chaube, Rakesh Rawal, Abhishek B. Jha, Bhavesh Variya, Hardik G.
Bhatt
PII:
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https://doi.org/10.1016/j.bioorg.2020.104501
YBIOO 104501
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Bioorganic Chemistry
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20 November 2020
Please cite this article as: U.J. Chaube, R. Rawal, A.B. Jha, B. Variya, H.G. Bhatt, Design and Development of
Tetrahydro-Quinoline Derivatives as Dual mTOR-C1/C2 Inhibitors for the Treatment of Lung Cancer,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104501
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Design and Development of Tetrahydro-Quinoline Derivatives as Dual mTOR-C1/C2 Inhibitors for
the Treatment of Lung Cancer
1
2
3
3
1
Udit J. Chaube , Rakesh Rawal , Abhishek B. Jha , Bhavesh Variya , Hardik G. Bhatt *
1
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, Ahmedabad 382 481. India.
2
Department of Life Sciences, Gujarat University, Ahmedabad 380 009. India.
3
Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad 382 481. India.

*
Address for correspondence: Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, S.G. Highway,
Chharodi, Ahmedabad 382 481. India.
Phone no. +91 79 71652727; Fax no. +91 2717 241916; Email Id: hardikbhatt23@hotmail.com

Abstract
Lung cancer is one of the most prevailed cancer worldwide. Many genes get mutated in lung cancer but the involvement of EGFR,
KRAS, PTEN and PIK3CA are more common. Unavailability of potent drugs and resistance to the available drugs are major concern
in the treatment of lung cancer. In the present research, mTOR was selected as an important alternative target for the treatment of lung
cancer which involves the PI3K/AKT/mTOR pathway. We studied binding interactions of AZD-2014 with the mTOR protein to identify
important interactions required to design potent mTOR inhibitors which was supported by QSAR studies. Pharmacophore based virtual
screening studies provided core scaffold, THQ. Based on molecular docking interactions, 31 THQ derivatives were synthesized and
characterized. All compounds were screened for cellular mTOR enzyme assay along with antiproliferative activity against the panel of
cancerous cell lines, from which 6 compounds were further screened for colony forming assay. Two most potent compounds, HB-UC-
1
and HB-UC-5, were further screened for flow cytometry analysis, gene expression study and western blot analysis. Gene expression
study revealed the efficiency of compound HB-UC-1 against both mTORC1 and mTORC2 by affecting downstream regulators of
mTORC1 (E BP , eIF EBP ) and mTORC2 (PCK1), respectively. In western blot analysis, both compounds, inhibited phosphorylation
4
4
4
1
of AKT S473 which proved the efficiency these compounds against the mTORC2. These two compounds were further screened for in-
vivo biological evaluation. Both compounds increased lifespan of cancer-bearing animals with improvement in mean survival time.
Further, in bezopyrene induced lung cancer animal model, both compounds showed effectiveness through the biochemical parameters
and histopathological evaluation of the lung tissue. In future, potent hit compound from this series could be modified to develop lead
mTOR inhibitors for the treatment of lung cancer.
Keywords: mTOR, Lung Cancer, Pharmacophore, QSAR, X-ray Crystallography, Tetrahydroquinoline

Introduction
Recent cancer statistics reveal that the lung cancer is one of the most prevailed cancer worldwide. It is mainly classified as small cell
lung cancer (SCLC) and non-small cell lung cancer (NSCLC); among which, cases of NSCLC (85%) are more as compared to SCLC.
[1]
NSCLC is further classified as adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). Many genetic
mutations take place in lung cancer, but KRAS, EGFR, PIK3CA and PTEN are most common. [2-4] KRAS and EGFR mutations are
found in AC, [1,3] while PIK3CA and PTEN mutations occur in the case of SCC. [5] No drugs are available in the market which directly
acts on KRAS [1] and this problem can be solved by targeting the pathway associated with KRAS mutant in NSCLC viz. RAS/RAF/MEK
or PI3K/AKT/mTOR pathway. Selumetinib and BEZ-235 are available in the market for targeting the RAS/RAF/MEK and
PI3K/AKT/mTOR pathway, respectively. Similarly in the case of EGFR-mutant AC, drugs available in market are Gefitinib and
Erlotinib. [3] Major problem associated with these drugs is resistance [4,5] which results into decrease in therapeutic efficacy. As per the
available literature, it is well understood that PI3K/AKT/mTOR pathway is altered in lung cancer [6] and discovery of new inhibitors
against any target of this pathway could be helpful for the treatment of lung cancer. Hence in the present research work, mTOR was
selected as the choice of target as it is downstream to PI3K/AKT and its inhibition may completely block the pathway.
mTOR (Mammalian/Mechanistic Target of Rapamycin) consists of two subunits viz. mTORC1 (mTOR Complex-1) and mTORC2
(mTOR Complex- 2) among which mTORC-1 is considered as rapamycin sensitive target while on the other hand, mTORC2 is
considered as rapamycin insensitive target.[7,8] Most of the rapalogues (Rapamycin analogue) act on mTORC-1 via FKBP12 and
allosterically inhibit its activity [9-11] while on the other side, these compounds are unable to inhibit the mTORC-2. mTORC-1 consists
of Raptor, Deptor, mLST8, and PRAS-40.[12] Excessive mTORC-1 activity results into the phosphorylation of the S6K which is
downstream to the mTORC-1 and results into the uncontrolled protein synthesis, cell proliferation and angiogenesis. Similarly,
eIF4EBP1 is also present in downstream to the mTORC1, where mTORC-1 acts as negative regulator of eIF4EBP1, resulting into
[6]
decrease level of eIF4EBP1 which further results in increase level of protein synthesis and uncontrolled cell proliferation . While on
the other side, mTORC-2 consists of Rictor-mSin-1 complex instead of Raptor which is the only difference between the mTORC-1 and

mTORC-2. [12] This Rictor-mSin-1 complex generates steric hindrance to the Rapamycin-FKBP12 complex which results into the
insensitivity of mTORC-2 protein toward the rapamycin and rapalogues.[13] In the present research work, efforts were made to design
and synthesize small molecules which inhibit both, mTORC-1 and mTORC-2 to avoid problems arises due to resistance and could be
further explored in the treatment of lung cancer.
Results and Discussion
Design of Compounds
As per the available literature, it is well understood that PI3K/AKT/mTOR pathway is altered in lung cancer and discovery of new
inhibitors against any target on this pathway might be helpful for the treatment of lung cancer. Therefore in the present research work,
efforts were made to discover novel compounds that inhibited mTOR and could be used to treat lung cancer. Effectiveness of designed
compound depends upon how precisely it binds with important amino acids of target receptor and orient itself in binding pocket, so to
inhibit target protein. To start with design of novel inhibitors, we studied binding pocket of mTOR and also performed conventional
ligand based and structure based computational approaches.
Binding pocket of mTOR (PDB ID:4JT6) in complex with the PI-103 (as shown in Figure 1(A)) consisted of many important amino
acids which included hydrophobic amino acids viz. Leu2185, Ile2237, Val2240, Ile2356; polar amino acid Tyr2225 and charged amino
acid Asp2357. Among all these amino acids, Asp2195 and Tyr2225 were present in inner binding pocket and formed two hydrogen
bonds with the phenolic group of co-crystallized ligand PI-103, while Val2240 of hinge region was one of the crucial amino acid
responsible for high affinity of compound PI-103 with the mTOR protein. As our research is mainly focused on the lung cancer therapy
via mTOR inhibition; hence, AZD-2014 was selected to study in detail for the designing process as currently, it is in clinical trial for
the treatment of lung cancer (as mTOR inhibitor). The binding mode of AZD-2014 with mTOR protein depicted in Figure 1(B). The

AZD-2014 was discovered by modifications from AZD-8055 (as shown in Figure 1(C)). This modification was carried out to reduce its
residence time in the hepatocyte which may improve the pharmacokinetics and lower the risk of liver toxicity. For the accomplishment
of this objective, benzyl alcohol of AZD-8055 was replaced by benzyl amide which dramatically improved aqueous solubility, but
resulted into slight decline in the potency. One of the reason of the reduction in the potency of the compound AZD-2014 was its inability
to form hydrogen bonds with the Tyr2225 and Asp2195 as depicted in Figure 1(B) which is seen in the case of standard co-crystallized
ligand PI-103. Oxygen of the morpholine ring substituted on the AZD-2014 formed the hydrogen bond with the hinge region (Val2240)
of the mTOR protein and potentially inhibited both mTORC1 and mTORC2. It also showed selectivity against the mTOR as compared
to the other isoforms of PI3K. The detailed literature review of AZD-2014 revealed that the amide group is responsible for the solubility
and less residence time of this compound in the hepatocyte. Detailed study of target protein provided deep insight about important
interactions and their effects on activity.
Detailed study on binding mode of compounds PI-103 and AZD-8055 with the mTOR protein concluded that interactions with specific
amino acids like Tyr2225, Asp2195 and Val2240 are essential to produce potent inhibitory effect. Apart from this, in compound AZD-
2
014, amide linkage played an important role for the improvement of the solubility and reduction in the liver toxicity. Hence, according
to this fact, two amide groups were kept at R and R in design of proposed derivatives as depicted in the Figure 1(C). Additionally, it
1
2
was also observed that the oxygen of the morpholine ring substituted on the AZD-2014 was essential for the binding interaction with
the hinge region (Val2240) of the mTOR protein. Thus, in many of final derivatives, halogen substitutions were introduced which
revealed similar binding interactions with hinge region (Val2240) of the mTOR protein.
To validate the hypothesis of design of molecules based upon AZD-2014 for the identification of the functional groups required for the
mTOR inhibition, 3D-QSAR studies (CoMFA and CoMSIA) were performed on 50 pyridoaminotropanes/tetrahydroquinazoline
derivatives and 28 benzoxazepine derivatives which are already reported as mTOR inhibitors. The results are already published,[14,15]
but for better understanding of designed molecules, brief about both studies are provided. In case of
pyridoaminotropanes/tetrahydroquinazoline derivatives, three diverse alignment methods viz. docking based, distil based &

pharmacophore based alignments were used with distill method was found to be the best method as it gave the good statistical results in
the form of leave-one-out cross-validated coefficients (q ), conventional coefficient (r ) and predicted correlation coefficient (r2pred). In
2
2
2
2
2
case of CoMFA, q , r and r
were found to be 0.664, 0.992, and 0.652, respectively. While in case of CoMSIA, 25 different models
pred
were produced among which the amalgamation of steric, electrostatic & hydrophobic i.e. SEH and steric, electrostatic, hydrophobic,
2
2
donor & acceptor, i.e. SEHDA were found as best amalgamation. In case of CoMSIA (SEH) the statistical values in terms of q , r and
r2
were found to be 0.664, 0.992, and 0.652, respectively. Similarly, in case of CoMSIA (SEHDA) the values of q , r and r
2
2
2
were
pred
pred
found to be 0.739, 0.976, and 0.779, respectively. Furthermore, with the aid of these CoMFA and CoMSIA models various contour
maps were generated. These generated contour maps were validated by the molecular docking study. Based upon the analysis of contour
maps, important features of pyridoaminotropanes/tetrahydroquinazoline were identified which were responsible for the mTOR
inhibition. This included substitutions favorable with bulky groups, electropositive/electronegative groups, proton donor atoms and
hydrophilic groups. Similar to the earlier study, 28 benzoxazepine derivatives were also explored for the CoMFA and CoMSIA analysis.
Here also distil alignment was found to be the best alignment among all these three alignments as it provided good statistical results in
2
2
the form of Leave One Out (LOO), cross validated coefficients (q ), conventional coefficient (r ) and predicted correlation coefficient
r2 ). In case of CoMFA; q , r and r
2
2
2
values were found to be 0.615, 0.990 and 0.930, respectively. While in the case of CoMSIA;
pred
(
pred
2
2
2
q , r and r
values were found to be 0.748, 0.986 and 0.933, respectively. Furthermore, generated contour maps were validated by
pred
the molecular docking study where specific positions of benzoxazepine scaffold was found to be favorable for bulky groups, for
hydrogen bond acceptor groups, for electron donating groups, hydrogen bond acceptor atom, and hydrophobic functional groups.
Overall, with the aid of this 3D-QSAR studies, important structural features required for the mTOR inhibition were identified and
incorporated in designed of our tetrahydroquinoline derivatives.
The study gave the critical insights of functional groups required for the mTOR inhibition as depicted in Figure 1(D). Outcome of 3D-
QSAR study proved presence of substitutions along with importance of amide substitution for the mTOR inhibition. In this 3D-QSAR
studies, distil alignment was found to be the best alignment method as it gave good statistical results with good contour maps orientations.

Contours maps validated by molecular docking study revealed that R position of tetrahydroquinoline substituted with the hydrogen
1
bond acceptor atom, polar functional groups, or combinations of electropositive or electronegative groups might be useful for the mTOR
inhibition. Similarly, R position of tetrahydroquinoline substituted with the bulky functional group, hydrogen bond acceptor atom, and
2
hydrophobic functional groups might be useful for the mTOR inhibition. Thus, with the combination of ligand based approaches like
CoMFA and CoMSIA and structure based approach like molecular dynamics and simulations assisted molecular docking study,
important features required for mTOR inhibition were identified and incorporated in the THQ scaffold, finalized from pharmacophore
based virtual screening method.
Simultaneously, pharmacophore modelling followed by virtual screening were used for identification of core scaffold. six potent mTOR
inhibitors viz. Torin-1, Torin-2, AZD-2014, WYE-354, WYE-687, and WAY-600 reported as mTOR inhibitors (shown in supporting
information material as Figure SF-1) were selected from the literature. Selection criteria was mainly emphasized on the in-vitro potency
and pharmacokinetic profile of the compounds. With the help of these six mTOR inhibitors; total four pharmacophore models were
generated (shown in supporting information material as Table ST-1) in which model_1 with highest DMEAN score was selected and all
six drug like molecules contributed for its generation. The best pharmacophore model contained 4 features viz. two hydrophobic features
(HY_1 and HY_2), one acceptor atom (AA_1) and one acceptor site (AS_1) of the receptor protein inhibitors (shown in supporting
information material as Figure SF-2). This model was further validated with two well-known statistical methods viz. Receiver Operating
Characteristics (ROC) (shown in supporting information material as Figure SF-3) and Gunner Henry (GH) scoring methods (shown in
supporting information material as Table ST-2). The best and validated pharmacophore model was used for virtual screening process
thorough NCI database. Virtual screening provided thousands of compounds and after necessary filtering, it was concluded that
compounds with THQ as a core scaffold showed best results. (Details of pharmacophore based virtual screening are provided in
supporting information file).
Overall in the design process of the novel compounds; pharmacophore based virtual screening [16] was carried out for the identification
of the core scaffold while knowledge based drug design approach to study target protein and 3D-QSAR studies [17] were carried out for

the identification of the substituents required for mTOR inhibition. With the aid of these approach, several hundreds of
tetrahydroquinoline (THQ) derivatives were designed. Further, these designed THQ derivatives were filtered through the molecular
dynamics assisted molecular docking study which resulted into 31 THQ derivatives showing comparable docking score as that of the
co-crystalized ligand (PI-103) of mTOR protein along with good binding affinity. As per the molecular docking study, compound HB-
UC-1 was found to be most effective mTOR inhibitor as it showed all necessary interactions with the mTOR protein as shown in
supporting information.
Docking of Designed Compounds
Tetrahydroquinoline was selected as core scaffold based upon the pharmacophore based virtual screening and in-silico ADMET studies
(shown in supporting information material as Table ST-3 and ST-4 and additional details of selection of core scaffold is given in the
supporting information material). Initially tetrahydroquinoline as core scaffold showed very less docking score compared to the standard
PI-103 (shown in supporting information material as Table ST-4). Although, incorporation of the required substituents, on the basis of
study of target receptor and QSAR study, to the tetrahydroquinoline scaffold drastically increased the docking score of designed
compounds which was comparable with the standard co-crystalized ligand, PI-103 (showed in supporting information material as Table
ST-5). All designed compounds were docked [18-20] on the mTOR protein (4JT6) to select few which formed important interactions with
target protein and could be proved as potent mTOR inhibitor. Compounds HB-UC-1, HB-UC-3 and HB-UC-11 from series-I showed
good docking score with protein as (depicted in supporting information material as Table ST-5). Similarly in the case of series-II,
compounds HB-UC-15, HB-UC-17 and HB-UC-21 showed good docking score. Likewise in series III, docking results revealed that the
compounds HB-UC-31, HB-UC-33 and HB-UC-37 were topmost compounds in terms of the docking score. Overall, compound HB-
UC-1 was found to best compounds as it showed all important binding interactions with the crucial amino acid including Val2240 which
is present in hinge region of the mTOR protein. Additionally, compound HB-UC-1 showed binding interaction with the Asp2195 and
Tyr2225 which is similar to the binding interactions of compound PI-103 with the mTOR protein (shown in supporting information
material as Figure SF-4A and 4B). One important observation was revealed in this docking study that these topmost molecules; HB-

UC-1, HB-UC-3, HB-UC-15, HB-UC-17, HB-UC-31, HB-UC-33 consisted of electron withdrawing groups at R and R position of
1
2
THQ ring which clearly indicated that these electron withdrawing groups are playing important role in the binding with the mTOR
protein.
In-Silico ADMET and Toxicity Prediction
OSIRIS property explorer is generally used for the prediction of toxicity in the form of mutagenic, tumorigenic, irritant and reproductive
effect. Additionally, it also provides the information about the physicochemical properties of designed molecules in the form of clogP
(log C octanol/log C water), TPSA (The Polar Surface Area), solubility, molecular weight, drug score and drug likeness. admetSAR is
also widely used for the prediction of Adsorption, Distribution, Metabolism and Excretion along with the acute oral toxicity in the form
of class-I chemicals (LD ≤ 50mg/kg) , class-II chemicals (500mg/kg > LD > 50mg/kg) , class- III chemicals (5000mg/kg > LD 0 >
5
0
50
5
5
00mg/kg) and class-IV chemicals (LD > 5000mg/kg). It also gives an idea about the carcinogenicity of the chemical. In the present
50
study, both the online tools were used for ADMET property determination. Top 31 newly designed THQ derivatives were further used
for the ADMET prediction with the help of OSIRIS Property Explorer and admetSAR. Most of the marketed drugs having aqueous
solubility in between -6 to 0. Hence the compounds which showed aqueous solubility in between these value considered as a good
candidate for absorption and distribution. Among these 31 designed compounds, Series-III compounds revealed good aqueous solubility
as compared to series-I and series-II compounds. While in case of series-I and series-II the aqueous solubility was acceptable as most
of the compounds shown solubility around -6. In case of TPSA, most of the designed molecules were far away from the TPSA value of
standard drug AZD-8055. Although some of the designed compounds HB-UC-1, HB-UC-15 and HB-UC-31 showed TPSA value close
to AZD-8055. Among the 31 designed compounds, most of the compounds from all three series showed a clogP value less than 5 (data
are provided in supporting information material tables ST-6A, ST-6B and ST-6C). From series-I, compounds like HB-UC-1, HB-UC-
5
, HB-UC-6, HB-UC-11, HB-UC-13 and HB-UC-14 showed clogP less than 5 indicating that they might get absorbed properly as
shown in supporting information material table ST-6A. Similarly, from series-II compounds, HB-UC-15, HB-UC-18, HB-UC-19, HB-
UC-20, HB-UC-24 and HB-UC-25 showed clogP values less than 5 (data are provided in supporting information material table ST-6B).

From series-III compounds, HB-UC-31, HB-UC-34, HB-UC-36. HB-UC-39 and HB-UC-41 showed clogP values less than 5 (as shown
in supporting information material table ST-6C). In addition to that, maximum number of designed compounds were found to be non-
mutagenic, non-tumorigenic, non-irritant and no bad effects on reproductive system except compounds of series-III (shown in supporting
information material table ST-6C). All newly designed compounds of series-III revealed irritant effect (shown in supporting information
material table ST-6C). In addition to that, compounds from series I and II indicated some toxic effect viz. HB-UC-14, HB-UC-19 and
HB-UC-25. Similarly in the case of admetSAR, most of the compounds from all the series showed Human Intestinal Absorption (HIA)
along with the Blood Brain Barrier (BBB) absorption (shown in supporting information material table ST-7). Some of the designed
compounds showed Caco-2 permeability indicated that these compounds may get orally absorbed in the intestine. All designed
compounds were found to be non-carcinogen and showed distribution in the mitochondria and fell in the category of class-III chemicals
(
500mg/kg > LD <5000mg/kg) (shown in supporting information material table ST-7) except compound HB-UC-14 which fell in the
50
category of class-II chemical (500mg/kg > LD > 50mg/kg).
5
0
Chemistry
Based upon the ligand based and structure based approach, 31 tetrahydroquinoline (THQ) derivatives were designed and synthesized
with the help of suitable synthetic scheme as depicted in Figure 2.
THQ was treated with the 9-flurenemethoxycarbonyl (Fmoc) for the protection at nitrogen of THQ which resulted into selective nitration
th
at 6 position of the THQ when treated with potassium nitrate and sulfuric acid. Normally during this nitration reaction, electrophile
th
th
th
attack of nitro group could take place at 6 and 8 of THQ. Due to protection with Fmoc, electrophilic attack at 8 position of THQ get
th
[21]
hindered and thus selective nitration was occurred at 6 position of THQ. After the nitration of Fmoc protected THQ, it was reacted
with pyrrolidine for the deprotection followed by the reaction with three different acid chlorides viz. 4-chlorobenzoyl chloride, benzoyl
chloride and 2-ethylbutanoyl chloride showed as R in the synthetic scheme. Further, this compound is treated with the ammonium
1

chloride and zinc which resulted into the reduction of nitro group into amine. This primary amine was reacted with the different acid
chlorides and sulfonyl chlorides (showed as R ) which resulted into the synthesis of 31 novel desired THQ derivatives. Further, all
2
1
13
synthesized derivatives were characterized by H & C NMR, Mass analysis, and FTIR while the purity was checked by HPLC. In
addition to this, structural confirmation was carried out with the help of XRD analysis. Compound HB-UC-25 was taken as the
representative compound for the single crystal development and XRD analysis proved synthesis of desired compound.
1
13
The chemical structures of all compounds were confirmed by FTIR, H & C NMR and mass spectral analysis. Single crystal XRD
finally proved confirmation of series. The purity of the compounds were determined from HPLC analysis and all compounds are having
>
95% purity. All intermediates along with the final 31 derivatives showed molecular ion peak in mass spectra along with typical halogen
pattern in few. Additionally, all intermediates along with the final derivatives were confirmed with FTIR. All final derivatives showed
-1
-1
single peak of secondary amine (-NH) at 3300-3390 cm along with the carbonyl groups (-C=O) at 1500-1650 cm which confirmed
1
the formation of the amide bond. Likewise, H NMR showed D O exchangeable amide bond proton (-NHCO-) peak around 9-11 ppm
2
1
(
δ value). H NMR also revealed that the signals in between 1 to 4 ppm (δ value) corresponded to the hydrogen attached to the aliphatic
carbons of the THQ. Additionally, some aliphatic side chain compounds like HB-UC-6, HB-UC-14, HB-UC-20, HB-UC-36, and HB-
1
3
UC-41 showed peaks of proton in same region of THQ, i.e. 1 to 4 ppm (δ value). C NMR analysis was carried out to confirm the
1
3
number of carbon atoms of the THQ derivatives. The peaks around 160-178 ppm (δ value) of C NMR indicated the number of carbonyl
carbons. Peaks in between 20-60 ppm (δ value) indicated presence of aliphatic carbons of THQ. In addition to this, aliphatic side chains
were present in some compounds as mentioned earlier which indicated the peaks in the same aliphatic region of THQ viz. 20-60 ppm
(δ value) (Various spectra and HPLC data of final THQ derivatives are given in the supporting information material). The final structural
confirmation was carried out by XRD analysis. Compound HB-UC-25 was used as a representative compound for the single crystal
development and for XRD analysis, which gave the exact synthesized structure of the desired compound HB-UC-25 with CCDC number
1
874590 (Details are given in supporting information material as Figure SF-5 and Table ST-6).
mTOR Enzyme Assay and Structure Activity Relationship Study

All synthesized compounds were evaluated for in-vitro mTOR enzyme assay [22,23] to prove their potency. Measurement of cellular level
of mTOR in A549 cell line indicated that the maximum number of compounds of series-I were found to be potent (as shown in Table
1
). In case of series-I compounds, HB-UC-1 (IC = 3.17±0.19 µM) and HB-UC-5 (IC = 3.42±0.34 µM) were found to be most potent
50 50
among all the other compounds of series-I. The mTOR inhibitory activity of these compounds were found close to the standard AZD-
055 (IC = 1.5±0.31µM) which is dual mTORC1 and mTORC2 inhibitor. While on the other hand, HB-UC-1 and HB-UC-5 were
8
5
0
found to be more potent than standard mTORC1 inhibitor viz. Everolimus (IC = 7.5 µM) revealing the importance of the small
5
0
molecule dual mTORC1 and mTORC2 inhibitor.
In case of mTOR enzyme assay, the study proved the importance of the balanced polarity and hydrophobicity at R position of THQ.
1
As soon as the chlorobenzene group at R was replaced by a simple benzene ring, it resulted in a drastic reduction in the potency of the
1
compounds. This proved that chloro substitution on phenyl ring at R position imparted polarity to the compound and is important for
1
activity. Similarly, aliphatic side chain in series-III at R was not found to be effective for the mTOR inhibition. These results validated
1
the outcome of 3D-QSAR where our hypothesis indicated the importance of the polar functional group, hydrophobicity and hydrogen
bond acceptor for the mTOR inhibition. Though there was a hydrophobic group at R in series-II and series-III, a reduction in the polarity
1
was observed due to the absence of chloro substitution on the ring or aliphatic side chain. In the case of the series-I, the polar functional
groups along with the hydrophobic groups at R position played an important role. Compounds HB-UC-1 (IC = 3.17±0.19 µM) and
2
50
HB-UC-5 (IC = 3.42±0.34 µM) were found as most potent among all the compounds of series-I and they explained the importance of
5
0
polarity due to the nitro and amino group at this position. In addition to this, R postion of compounds HB-UC-1 and HB-UC-5 consisted
2
of the phenyl ring in the form of aminophenyl and nitrophenyl rings, respectively, which imparted proper hydrophobic and hydrophilic
balance which is also required for the activity.
This hypothesis also proved right in case of the compounds HB-UC-4 (IC = 13.09±0.23 µM) and HB-UC-7 (IC = 15.75±0.51 µM)
5
0
50
where hydrogen and methyl groups were presented on benzoyl group at R position, respectively, which resulted in the decrease in the
2
activity of these compounds.

Interestingly, it came to our notice that in the compounds HB-UC-9 (IC =18.24±0.45 µM) and HB-UC-10 (IC = 14.74±0.38 µM),
5
0
50
the increased in bulkiness at R position above the optimum level decrased the activity. This outcome proves that the only increase in
2
the bulkiness or polarity alone will not increase the activity. Proper balance of the hydrophobicity and hydrophiclity is require. The
bulkier groups are require at R position with appropriate polarity, but in case it goes beyond the optimum level, the activity decrease.
2
This hypothesis is proved by the compounds HB-UC-2, HB-UC-3 & HB-UC-10. In case of these compounds, the order of potency was
found to be HB-UC-3 (IC = 4.77±0.18 μM) > HB-UC-2 (IC = 11.06±0.34 μM) > HB-UC-10 (IC = 14.74±0.38 μM). This difference
5
0
50
50
was observed due to the presence of different halogens at R position. In case of compound HB-UC-3, bromine is present at the R
2
2
position in the form of bromophenyl which is optimum for balancing the hydrophobic and polar property. As soon as the polarity at R2
position increases above the optimum level, the activity decreases as it was observed in case of compounds HB-UC-2 and HB-UC-10
where chlorophenyl and flurophenyl were present, respectively.
During the designing part, bioisosteric replacement of a carbonyl group (C=O) was carried out by the sulfonyl group (O=S=O) which
again resulted in decreased activity. This was validated by the compounds HB-UC-2 (IC = 11.06±0.34 µM) and HB-UC-13 (IC =
5
0
50
>
25 µM) where compound HB-UC-2 consisted of a carbonyl group (C=O) while HB-UC-13 consisted of sulfonyl group (O=S=O) at
R position.
2
In-Vitro Antiproliferative MTT Assay and Structure Activity Relationship Study
All synthesized compounds were screened against panel of four proliferative cell lines by MTT assay [24,25] along with four standards;
Everolimus, Cisplatin, AZD-8055 and PI-103 as shown in Table 1. Among all synthesized compounds, most of the compounds were
found to be potent against these panel of cancer cell lines. In case of Lung Cancer (A549) cell line, the maximum number of compounds
were found to be effective. Compounds like HB-UC-1 (IC = 0.06±0.27 µM), HB-UC-3 (IC = 0.657±0.39 µM), HB-UC-5 (IC =
5
0
50
50
0
.224±0.45 µM) and HB-UC-6 (IC = 0.158±0.38 µM) were found to be most potent compounds among all the synthesized compounds.
50
The compound HB-UC-1 was found several fold more potent than the standard compounds viz. Everolimus (IC = 6.09±0.21 µM),
5
0

Cisplatin (IC = 3.15±0.19 µM), AZD-8055 (IC = 8.17±0.25 µM) and PI-103 (IC = 1.65±0.34 µM); while compounds HB-UC-3
5
0
50
50
(
IC = 0.657±0.39 µM), HB-UC-5 (IC = 0.224±0.45 µM) and HB-UC-6 (IC = 0.158±0.38 µM) were found potent than the standard
50 50 50
compounds. In addition to that, these compounds were found to be non-toxic on the Vero cell line as these compounds did not show any
significant inhibition (at least 50%) of the Vero cell line at 25µM.[16] In case of stomach cancer (AGS) cell line; two compounds HB-
UC-1 (IC = 16.35±0.21 µM) and HB-UC-10 (IC = 23.06±0.21µM) were found to be most effective, while all standards Everolimus
5
0
50
(
IC = 9.69± 0.29 µM), Cispaltin (IC = 6.81±0.27 µM), AZD-8055 (IC = 5.73±0.54 µM) and PI-103 (IC = 1.46±0.22 µM) showed
50 50 50 50
good antiproliferative activity against stomach cancer cell line. In case of colon cancer cell line (HCT-15), compounds HB-UC-14 (IC50
7.37±0.64 µM), HB-UC-15 (IC = 11.08±0.08 µM), HB-UC-31 (IC = 10.04±0.13 µM), and HB-UC-37 (IC = 12.24±0.12 µM)
=
5
0
50
50
were found to be effective. These compounds too were found to be non-toxic on the Vero cell line. In case of skin cancer cell line
B16F1), compounds like HB-UC-4 (IC = 24.18±0.14 µM), HB-UC-14 (IC = 3.16±0.21 µM) and HB-UC-33 (IC = 17.41±0.23
(
5
0
50
50
µM) were found to be effective. These compounds too were found to be non-toxic on the Vero cell line. Overall, six compounds; HB-
UC-1, HB-UC-3, HB-UC-4, HB-UC-5, HB-UC-6 and HB-UC-9 were found to be most potent on the lung cancer cell line (A549) and
were further used for various in vitro biological assays.
The present research work mainly deals with the treatment of lung cancer, hence efforts were made for description of the structure
activity relationship of synthesized compounds with respect to MTT assay on lung cancer cell line.
In case of lung cancer cell line (A549), electronegative group at R position was found to be effective for the antiproliferative activity.
1
This was proved by the compounds of series-I where chlorobenzene group was present at R position making this series of compounds
1
effective as compared to the compounds of series-II and series-III where electronegative atoms were absent. Interestingly, in case of
series-II, antiproliferative activity of compounds decreased drastically which proved the importance of polar functional group at R and
1
this results are in line with the outcome of 3D-QSAR study. In case of series-III, increased in the hydrophobicity at R due to the alkyl
1
side chain resulted in decreased antiproliferative activity against the lung cancer cell line (A549). Similar to the R position, R was also
1
2
found to be favorable for the combination of hydrophobic group and polar functional group. This statements were proved by the

compounds HB-UC-1 (IC = 0.06±0.27 µM), HB-UC-4 (IC = 0.971±0.16) and HB-UC-5 (IC = 0.224±0.45), where compounds HB-
5
0
50
50
UC-1 and HB-UC-5 consisted of the nitro (-NO ) and amino (-NH ) groups, respectively making R position polar in nature compared
2
2
2
to the compound HB-UC-4 where there was no polar functional group at R . Additional validation to this statement was observed from
2
the examples of the few other compounds viz. HB-UC-4 (IC = 0.971±0.16) and HB-UC-7 (IC = 3.730±0.54) where HB-UC-4
5
0
50
consisted of simple benzene ring at R while on the other hand compound HB-UC-7 consisted of 4-methylbenzene which resulted into
2
decreased in the polarity of compound HB-UC-7 compared to the HB-UC-4. During this enumeration of SAR, one interesting fact was
observed in case of series-I that an increase in bulkiness without any proper polar functional groups at R position decreased the activity
2
against lung cancer cell line (A549). This was validated with an examples of the compounds viz. HB-UC-7 (IC = 3.730±0.54) and
5
0
HB-UC-9 (IC = 3.280±0.39) where bulky groups like methyl (-CH ) and (3,5-diCF ) were present while in case of compound HB-
5
0
3
3
UC-1 (IC = 0.06±0.27) and HB-UC-5 (IC = 0.224±0.45), the bulkiness at R was found to be appropriate as compared to the
5
0
50
2
compound HB-UC-7 and HB-UC-9 as these compounds HB-UC-1 and HB-UC-5 consisted of polar functional groups like nitro and
amino, respectively .
Colony Forming Assay
Colony forming assay [26,27] is usually performed for observing the effect of the compounds on the uncontrolled cell proliferation of the
cells. In lieu with this, six THQ derivatives viz. HB-UC-1, HB-UC-2, HB-UC-3, HB-UC-4, HB-UC-5 and HB-UC-9 which showed
promising results against the lung cancer line (A549) were further taken for the colony forming assay. Three different concentrations of
each compound i.e. 6.25 µM, 12.5 µM and 25 µM were used and it was observed that the compounds HB-UC-1 and HB-UC-5 showed
a significant decrease (p<0.05) in the colonies at 12.5 µM and 25 µM as compared to the DMSO control (results are shown in supporting
information material as Figure SF-6A). The effect of one of the representative compound, HB-UC-1 on the colony formation is (depicted
in supporting information material as Figure SF-6B), where it was observed that the number of colonies decreased depending on the
concentration. Interestingly, compounds like HB-UC-3, HB-UC-4, HB-UC-6 and HB-UC-9 which showed good IC values against
5
0
A549 cell line in MTT assay did not show any significant decrease in the colonies when three different concentrations of the compounds

were kept up to ten days in the contact of the A549 cell line. This experiment proved that the compounds HB-UC-3, HB-UC-4, HB-
UC-6 and HB-UC-9 indicated promising IC values in MTT assay when kept for 48 hours; but did not stop the uncontrolled cell growth
5
0
of the A549 cells when a sufficient amount of media along with the nutrients were provided to the cells up to 10 days. Thus, only two
compounds HB-UC-1 and HB-UC-5 were further used for screening. During this study it was observed that the compounds HB-UC-1
and HB-UC-5 showed a significant decrease (p<0.05) in the colonies at 12.5 µM and 25 µM compared to the DMSO control as shown
in supporting information material. Thus, only two compounds HB-UC-1 and HB-UC-5 were further used for screening.
Cell Apoptosis Assay
In the present research work for determination of apoptosis - FACS (Fluorescence-Activated Cell Sorting) analysis was carried with the
aid of flow cytometry [28,29]. For this study, compounds HB-UC-1 and HB-UC-5 (two concentrations of each; 3 and 6 µM) along with
the standard Everolimus (6 µM) were selected, as both compounds showed promising activity in enzyme, MTT and colony forming
assay. This analysis revealed the apoptotic properties of Everolimus, HB-UC-1 and HB-UC-5 as compared to DMSO as control (Shown
in Figure 3(A) to 3(F)). In case of Everolimus (6 µM) and the compound HB-UC-1 (6 µM), 40.09 % and 53 .97 % of the cells were
found in late apoptotic phase, respectively. This percentage of late apoptosis of compound HB-UC-1 decreased at 3 µM. Similarly, in
the case of the compound HB-UC-5 (6 µM), 36.13% of the cells were found in late apoptosis which was less as compared to the standard,
Everolimus. This study also concluded that both these compounds were non-necrotic, as a negligible amount of cells were found to be
in necrotic phase. Overall, this analysis demonstrated the apoptotic properties of compound HB-UC-1 and HB-UC-5. Moreover this
study also indicated that both the compounds HB-UC-1 and HB-UC-5 were apoptosis inducer but the compound HB-UC-1 at 6 µM was
one of the best apoptotic inducers compared to Everolimus.
Gene Expression Study
For gene expression study [30,31], isolated RNA samples were selected as they remained intact after the treatment of compound HB-UC-1
(
0.06 µM) and HB-UC-5 (0.11 µM), at their IC concentration as shown in Figure 4 (A). The cDNA form of the cells treated with the
50
compounds at their IC and control was used as a template for further gene amplification with the use of gene-specific primer E4BP4.
5
0

Among these two compounds, HB-UC-1 showed some promising effect as it reduced the expression level of the E4BP4 as shown in
Figure 4(B) and 4(C) which indicated that the compound HB-UC-1 was inhibiting the mTORC1 expression. Although it is well known
that the E4BP4 gene is involved in many other pathways like MEK/ERK, PKA/PTH/FSK, GR/CREB pathways, hence it was difficult
to conclude that the compound is inhibiting the mTORC1. Hence, to overcome the above-mentioned problem of E4BP4 which is not a
specific downstream regulator of mTORC1; a primer of specific downstream regulators of mTORC1 and mTORC2 were used viz.
eIF4EBP1 and PCK1, respectively. eIF4EBP1 had both antioncogenic and proapoptotic properties and hence it was involved in the
normal cellular process.
Usually, the expression of eIF4EBP1 was decreased due to increased expression of mTORC1 as it was a natural inhibitor of eIF4EBP1.
Hence, if there was an inhibition of mTORC1, it could lead to increased expression level of the eIF4EBP1 which is good for the normal
cellular process. The cDNA of cells, treated with the HB-UC-1 were taken and amplified through eIF4EBP1 primers and it was observed
that the expression level of the eIF4EBP1 was increased as shown in Figure 4(D) and 4(E) which indicated that the compound HB-UC-1
inhibited the mTORC1. Similarly, PCK1 was presented downstream to the protein mTORC2. PCK1 was responsible for the
augmentation of the gluconeogenesis which consequently lead to uncontrolled cell proliferation. Hence, inhibition of the mTORC2
resulted in decreased expression of the PCK1 as shown in Figure 4(D) and 4(F). Compound HB-UC-1 increased the expression level of
eIF4EBP1 which proved that this compound inhibited the mTORC1 efficiently. Similarly, compound HB-UC-1 completely decreased
the expression level of PCK1 which demonstrated the effectiveness of this compound as mTORC2 inhibitor. Overall, gene expression
study revealed that the compound HB-UC-1 was found as effective mTOR inhibitor (both mTORC-1 and mTORC-2).
Western Blot Analysis
Gene expression study clearly revealed the mTORC1 and mTORC2 inhibitory activity of compound HB-UC-1. Additionally to know
the indirect mTORC1 inhibition via AKT and direct mTORC2 inhibition by compounds HB-UC-1 and HB-UC-5, western blot analysis
[32,33]
was carried out and depicted in Figure 4(G) and 4(H). AKT is serine/threonine protein kinase which is a downstream regulator of

mTORC2 protein. Hence, inhibition of the phosphorylation of AKT S473 gave an idea about the mTORC2 inhibition. pAKT directly
inhibited downstream regulating protein viz. Glycogen Synthase Kinase 3 (GSK3), Forehead box (FOXO) which consequently resulted
into suppression of apoptotic signals and lead to uncontrolled cell proliferation. In addition to this, AKT indirectly activated the
mTORC1, which further resulted into the cell proliferation. This indirect activation of mTORC1 usually took place through the
phosphorylation of the TSC1/2 which resulted into the increment in the activity of mTORC1; hence, inhibition of phosphorylation of
AKT indirectly decreased the activity of mTORC1. Both these compounds were evaluated at four different concentration ranging from
0
.1 µM to 5 µM. These compounds showed significant inhibition of pAKT S473 at mentioned concentrations compared to the control
(0 µM) as these compounds inhibited the downstream protein regulator of mTORC2 (pAKT S473) in the lung cancer cell line (A549).
These compounds block the phosphorylation of the AKT at S473 site depending on the concentration as shown in Figure 4(G) and 4(H).
This inhibition of the phosphorylation of AKT at S473 site also proved that these compounds HB-UC-1 and HB-UC-5 were useful for
the prevention of feedback activation of AKT in the lung cancer cell line (A549) which consequently lead to activation of mTORC1.
Hence, inhibition of pAKT proved the efficiency of compounds HB-UC-1 and HB-UC-5 as a direct mTORC2 inhibitor and indirect
mTORC1 inhibitor.
Reported rapalogues unable to inhibit the mTORC2 due to their structural arrangements which were different from the mTORC1.
mTORC-2 consist of Rictor-mSin-1 complex instead of Raptor which was the only difference between the mTORC-1 and mTORC-2.
These Rictor-mSin-1 complex generated steric hindrance to the Rapamycin-FKBP12 complex which resulted into the insensitivity of
mTORC-2 protein toward the rapamycin and rapalogs. In the present research work, efforts were made to design and synthesize small
molecules which were not repelled by the steric hindrance of the Rictor-mSin-1 of mTORC2 complex. The western blot analysis of
compound HB-UC-1 and HB-UC-5 showed that these compounds HB-UC-1 and HB-UC-5 inhibited the phosphorylation of AKT S473
which proved the efficiency of this compounds as mTORC2 inhibitors. It also ascertained that these compounds prevent the indirect
activation of mTORC1 via AKT. Hence, this study demonstrated that the compounds HB-UC-1 and HB-UC-5 were inhibited both
mTORC1 and mTORC2 which is a major advantage of these compounds over the rapalogs.

In-Vivo Biological Evaluation
Detailed in-vitro biological evaluation of novel THQ derivatives concluded compounds HB-UC-1 and HB-UC-5 as most effective and
potent against the lung cancer through inhibition of mTOR enzyme. Based upon this evaluations, compounds HB-UC-1 and HB-UC-5
were further selected to evaluate their efficacy on life span and lung cancer animal models. In case of lung cancer animal model,
Everolimus was selected as a standard drug for the comparison.[34-35]
Lifespan Animal Model
For the initial evaluation, a study was performed using the compounds HB-UC-1 and HB-UC-5 to evaluate the effect on the life span of
the cancer-bearing animals.[36] P388D1 leukemia cells were given intraperitoneally to the mice for the induction of the cancer and
observation was made for the 30 days where throughout the experiment, deaths of animals were recorded. Life span study clearly
th
revealed that on the 15 day, all animals in the diseased control group died as shown in Table 2 (also expressed as supporting information
nd
material Figure SF-7). While in case of compounds HB-UC-1 and HB-UC-5, animals were survived on and after 22 day as shown in
(supporting information material as Figure SF-7) and Table 2, respectively. In the case of compound HB-UC-1, the mean survival time
of the animals increased with 83% survival rate as compared to the diseased control group as shown in Table 2. While with HB-UC-5,
the mean survival time of the animals increased compared to the disease control group but the percent rate of survival was observed to
be only 16% as shown in Table 2.
In conclusion, the compound HB-UC-1 was found to be very much effective and safe with the mentioned dose. Additionally, it increased
the lifespan of the animals with a good survival rate. The compound HB-UC-5 showed some promising result by increasing the lifespan
of the animals but the percentage of survival was found to be less in this treatment group compared to the HB-UC-1 group. Therefore
further these two molecules were taken for in-vivo analysis in the lung cancer animal model.

Lung Cancer Animal Model
After the accomplishment of efficacy in lifespan animal model, compounds HB-UC-1 and HB-UC-5 were assessed against the
benzo[a]pyrene induced lung cancer animal model.[37-39] (Mechanism of induction of lung cancer through the Benzo(a)pyrene is provided
as supporting information material Figure SF-8.) Various parameters like body weight and lung weight, serum biomarkers and
histopathological studies were determined in detail to prove effectiveness of synthesized compounds.
Initially, lung cancer was induced in mice with the help of benzo[a]pyrene which is a nicotine analogue. Benzo[a]Pyrene (50mg/kg body
weight) was dissolved in olive oil and given twice a week for four consecutive weeks. Standard drug, Everolimus & two drugs, HB-
th
UC-1 and HB-UC-5 were given thrice a week to the animals after the induction of lung cancer i.e. after the 8 week. The treatment
th
th
th
started in the 9 week and continued up to the 16 week. It was observed that on the 16 week, body weight of disease control animal
declined significantly (P<0.05) compared to the normal control. All the animals were euthanized by cervical dislocation after the 16th
week with the help of ether anesthesia. Lungs of all animals were removed and weighed. Lung weight was increased significantly
(P<0.05) compared to the normal control group. This decreased in the body weight occurred due to the weaker immunity which resulted
due to the induction of the lung cancer while increment in the weight of lungs clearly indicated occurrence of the tumor in the lungs.
Body Weight and Lung Weight Analysis
Usually, an induction of lung cancer resulted in reduction of the body weight while on the other hand, it increased lung weight due to
th
the formation of the tumor. In line with this, in the present research, it was observed in diseased control group that after the 5 week,
there was a slight decline in the body weight of mice which consequently declined each week. After the completion of the experiment,
th
on the 16 week, it was observed that the body weight of disease control animal declined significantly (P<0.05) compared to the normal
control, as shown in Table 3. On the other hand, in the case of treatment group, recovery in body weight was observed which is shown
in Table 3. Group of animals treated with both compounds HB-UC-1 and HB-UC-5 showed significant recovery in the body weight
with compound HB-UC-1 showed significant (p < 0.05) effect and was found to be more effective along with the standard drug

Everolimus as shown in Table 3. The same pattern was observed in the lung weight which increased significantly (p < 0.05) in diseased
control group due to the tumor formation in the lung. While in the treatment group, the lung weight decreased significantly (p < 0.05)
which was primarily indicated the reduction in the tumors. Overall, Everolimus, HB-UC-1 and HB-UC-5 were found to be effective as
a significant (p < 0.05) difference in the lung weight in the treatment groups were observed compared to the diseased control group.
Serum Biomarkers
An abrupt increment in the pyruvate in the serum indicated the uncontrolled cell growth compared to the normal which consequently
happened due to increase in the lactate dehydrogenase (LDH). In the present research work, statistical results and its graphical
representation, as shown in Figure 5 (A), indicated that the LDH level significantly increased (P < 0.05) in disease control group as
compared to normal control (NC) group which confirmed the tumor formation in the mice. In the treatment groups, the LDH level was
found to be significantly less (P < 0.05) than the diseased control (DC) group which indicated the effectiveness of the treatment.
Similar to LDH level, the statistical results of GGT level along with its graphical representation (Figure 5(B)) indicated a significant (P
<
0.05) increase in the GGT level in the diseased control group compared to the normal control group. Although, the treatment group
indicated a significant reduction in the GGT level (P < 0.05). Everolimus and HB-UC-1 treated groups showed the GGT level almost
near to the normal control group.
Overall, Serum biomarkers viz. Lactate Dehydrogenase (LDH), Gamma Glutamyl Transferase (GGT) increased significantly in the
disease control group indicated the induction of tumor while in case of treatment group the level of Lactate Dehydrogenase (LDH),
Gamma Glutamyl Transferase (GGT) decreased significantly indicated effectiveness of these two compounds HB-UC-1 and HB-UC-5
against the lung cancer.
Biomarkers of Tissue Homogenate

Malondialdehyde (MDA) is a product of the lipid peroxidation which occurred due to the reaction between the free radical and lipid.
This MDA is responsible for the DNA base pair mutation which consequently leads to the uncontrolled cell proliferation. A significant
increase in level of MDA (P < 0.05) in the diseased control group was observed as compared to the normal control group which indicated
about lipid peroxidation (as shown in Figure 5(C)). But on the other hand, in the treatment groups, significant (P < 0.05) reduction in
MDA level was observed. HB-UC-1 and HB-UC-5 were found to be as effective as the standard drug Everolimus as it reduced the MDA
level significantly (P < 0.05).
Glutathione is considered as one of the most important antioxidants of the human body. If there is an increase in the free radicles
formation in the body, Glutathione helps in the reduction of the oxidative stress which is usually generated due to this free radicle
formation. But as there is an enormous amount of free radicles like in carcinogenesis, it could lead to a reduction in the level of
Glutathione. The level of Glutathione when significantly reduced (P < 0.05) in the diseased control group compared to the normal
control group indicated the enormous free radical generation due to the cancer induction as shown in Figure 5 (D). But in the case of
treatment groups, viz. Everolimus and HB-UC-1, the level of glutathione was increased indicating the potency of these two compounds
as a free radical scavenger which helped to maintain the glutathione level at normal. Compound HB-UC-5 also showed some antioxidant
property as in this group, a higher level of glutathione was observed compared to the diseased control group.
Catalase is one of the important cellular antioxidants which reduces the cellular damage due to the oxidative stress. Results of the
catalase activity is shown in Figure 5 (E) which indicated that the level of hydrogen peroxide consumption decreased in the disease
control group compared to the normal control due to the decrease in the catalase enzyme activity. This clearly indicated the presence of
oxidative stress in the disease control group compared to the normal control group. But this catalase activity was significantly (P <
0
.05) increased in the Everolimus treatment group which indicated the very high consumption of peroxide free radicals. Similarly, HB-
UC-1 and HB-UC-5 also showed some antioxidant activity as there was an increment in the catalase activity and more consumption of
hydrogen peroxide free radicals.

Overall, a significant level of Malondialdehyde level was increased (P < 0.05) in the diseased control group compared to the normal
control group which indicated about lipid peroxidation. But on the other hand, in the treatment groups, significant (P < 0.05) reduction
of the Malondialdehyde level was observed. This outcome clearly indicated that the compounds HB-UC-1 and HB-UC-5 reduced the
lipid peroxidation effectively. A reduction in the level of Glutathione in diseased control group proved that the enormous amount of free
radicles were generated in it. However treatment group indicated the significant increment of the glutathione level which established the
antioxidant properties of compound HB-UC-1 and HB-UC-5. Decreased in the catalase activity in the disease control group indicated
the production of free radicles. Generation of these free radicles occurred due to the induction of the lung cancer. Nevertheless treatment
with the compounds HB-UC-1 and HB-UC-5 showed higher level of the catalase activity compared to the diseased control group
confirmed the free radicle scavenging properties of the these compounds which consequently helped for the treatment of the lung cancer.
Histopathological Study
Histopathological evaluation of lung tissue was carried out to access the induction of tumor and effectiveness of the synthesized
compounds along with the standard drug Everolimus. Lungs’ photographs and their histopathological evaluation is depicted in Figure
5
(F) and 5(G), respectively. This study proved the effect of compound HB-UC-1 and HB-UC-5 as it indicated the difference in
morphology of lung tissue between the normal control (NC), disease control (DC) and treatment groups. Lung section of a mice from
the normal control group indicated the proper structure of alveoli. In addition to this, it also showed the uniformity in the nuclei. In the
case of disease control group, the hyperchromatic nuclei were found on the walls of alveolar cells and an enormous amount of alveolar
damage was observed which is shown in Figure 5(G). While treatment groups which involved the treatment of Everolimus, HB-UC-1,
HB-UC-5 showed very less damage to the alveoli. Additionally, a few hyperchromatic nuclei observed in these sections proved the
efficacy of the compound HB-UC-1 and HB-UC-5.
Overall, measurement of the various biochemical parameters like Lactate Dehydrogenase (LDH), Gamma Glutamyl Transferase (GGT),
Catalase (CAT), Malondialdehyde (MDA) along with the histopathological evaluation of the lungs of the mice revealed that both the

compounds HB-UC-1 and HB-UC-5 cured the lung cancer up to some extent in the mice as compared to the standard molecule viz.
Everolimus.
Conclusion
The research work was started with understanding of target receptor for binding interactions which gave deep insights for incorporation
of substitutions in designed molecules which was supported by 3D-QSAR studies. It was followed by rationale drug design process by
pharmacophore based virtual screening to conclude THQ as core scaffold. Finally, based upon molecular docking studies, 31 novel
tetrahydroquinoline derivatives were designed and synthesized. The structures of these synthesized tetrahydroquinoline derivatives
1
13
confirmed by FTIR, H & C NMR and Mass. Finally, XRD analysis proved designed series. HPLC analysis gave idea about purity of
synthesized compounds which was >95% for all compounds. To evaluate synthesized compounds for their anticancer potential, various
in-vitro and in-vivo analysis was performed. In-vitro biological evaluation of these 31 derivatives was carried out through mTOR enzyme
assay and MTT assay on various cancer cell lines which gave six compounds as potential target for further studies. Colony forming
assay further helped to narrow down on two compounds, HB-UC-1 & HB-UC-5, for flow cytometry analysis, gene expression study
and western blot analysis. Both compounds showed their potency in all studies. From results of all in-vitro analysis, both compounds
were further investigated for in-vivo biological evaluation. Both molecules, HB-UC-1 and HB-UC-5, showed promising results in the
life span animal model and benzopyrene induced lung cancer animal model. These designed molecules could be further explore in future
as HITs to develop novel Lead molecule for the treatment of Lung cancer.
Experimental Section
Material and Methods
For the synthesis of these designed THQ derivatives suitable synthetic scheme was designed which is depicted in Figure 2. Borosilicate
glassware and dry solvents were utilized for the synthesis of all the intermediates along with the final desired compounds. There were
certain steps in the synthetic scheme where heating and stirring were required which was done with the aid of REMI Rota-mantle.

Solvents were evaporated with the help of BUCHI type rotary vacuum evaporator. Synthesized products were dried under the IR lamp
for an appropriate time duration. After drying all synthesized compounds were taken for meting point determination with the help of
VEEGO corporation melting point apparatus and are uncorrected. All the thirty one tetrahydroquinoline derivatives were purified using
column chromatography and further there purity were confirmed with the help of JASCO HPLC instrument and all compounds gave
>
95% purity. These all tetrahydroquinoline derivatives were characterized with the help of Infrared Spectroscopy (JASCO 6100), Mass
1
13
Spectroscopy (WATERS Mass Spectrometer), H NMR and C NMR (BRUKER AVANCE-II). For the accurate confirmation and
validation of spectral data; X-ray crystallography of one of the compound HB-UC-25 of the series was carried out with the help of
RIGAKU SCX mini diffractometer.
Experimental Procedure and Spectral Characterization of Intermediates
Synthesis of (9H-fluoren-9-yl) methyl-3,4-dihydroquinoline-1(2H)-carboxylate (3):
THQ (1) (3.75 mmol, 0.47 mL) and Fmoc (2) (4.125 mmol, 1.06 gm) were dissolved in the dichloromethane with continued stirring at
0
0
°C. Dropwise addition of trimethylamine (4.125 mmol, 0.5mL) was carried out to above reaction mixture with continuous stirring at
°C. Allow reaction mixture to stir at room temperature for 24 hours. Solvent was evaporated and the desired product was dissolved in
the ethyl acetate. This product was extracted with the dilute HCl to remove the excess trimethylamine and Fmoc. Finally washing was
done with the brine solution and ethyl acetate was evaporated to get the desired white colored product. Yield: 69 %; mp: 88-90°C; IR
-1
(
KBr, γmax cm ): 1577 (C=O), 1710 (C=C), 2360 (C-N), 2942 (C-H aliphatic), 3034 (C-H aromatic); MS (EI) m/z calculated for
C H NO 356 (M+1), found 356 (M+1).
2
4
21
2
Synthesis of (9H-fluoren-9-yl) methyl 6-nitro-3,4-dihydroquinoline-1(2H)-carboxylate (4):
Nitrating mixture was made with the help of KNO (1000 mmol, 0.142 gm) and H SO (1000 mmol, 0.074 mL) and allow to stir at 0°C
3
2
4
for 10 to 15 minutes. Addition of dichloromethane was carried out to above reaction mixture at 0°C and again allowed to stir above

reaction mixture for 15 minutes. (9H-fluoren-9-yl)methyl-3,4-dihydroquinoline-1(2H)-carboxylate (3) (1000 mmol, 0.5 gm) was
dissolved in dichloromethane and added dropwise to above reaction mixture while temperature was maintained at 0°C. Continued
stirring of reaction mixture for 2 hours and 30 minutes at RT. Poured reaction mixture to the crushed ice and product was extracted with
dichloromethane followed by washing with brine resulted into yellow color product. Yield: 96 %; mp: 140-142°C; IR (KBr, γmax cm-
1)
: 1585(C=O), 1706(C=C), 2350(C-N), 2944(C-H aliphatic), 3098(C-H aromatic); MS (EI) m/z calculated for C H N O 401 (M+1),
24 20 2 2
found 418 (M+18).
Synthesis of 6-nitro tetrahydroquinoline (5):
Compound 4 (1000 mmol, 3 gm) was dissolved in dichloromethane. Pyrrolidine (3000 mmol, 1.8 mL) was added dropwise to above
reaction mixture and allowed to stir the reaction mixture for 15 to 30 minutes. Poured mixture to crushed ice and desired product was
extracted with dichloromethane. Finally the product was washed with the brine and dried over sodium sulfate resulted into to the dark
-1
yellow color product. Yield: 85 %; mp: 160-162°C; IR (KBr, γmax cm ): 1609 (C=C), 2359 (C-N), 2962 (C-H aliphatic), 3063 (C-H
aromatic); MS (EI) m/z calculated for C H N O 179 (M+1), found 179.4 (M+1).
9
10
2
2
Synthesis of (4-chlorophenyl)(6-nitro-3,4-dihydroquinolin-1(2H)-yl)methanone (7a):
Compound 5 (1000 mmol, 0.2 gm) was dissolved in dichloromethane and dropwise addition of trimethylamine (1000 mmol, 0.16 mL)
was carried out to above reaction mixture with continues stirring. The reaction mixture was allowed to stir for 30 minutes followed by
the addition of 4-chloro benzoyl chloride (6a) (1000 mmol, 0.14 mL). This reaction mixture was kept on stirring at room temperature
for 24 hours consequently this mixture was added to the crushed ice and extracted with the dichloromethane. Further it was washed with
the bicarbonate solution, brine and dried over sodium sulfate resulted into the light yellow color powder. Yield: 84 %; mp: 170-173°C;
-1
IR (KBr, γmax cm ): 1667(C=O), 1786(C=C), 2360(C-N), 2954(C-H aliphatic), 3095(C-H aromatic); MS (EI) m/z calculated for
C H ClN O 317 (M+1), found 317 (M+1) and 319.3 for chloro pattern.
1
6
13
2
3
Synthesis of (6-nitro-3,4-dihydroquinolin-1(2H)-yl)(phenyl)methanone (7b):

Procedure of synthesis of this compound 7b remained same as that of the synthesis of the compound 7a only difference was that the
-1
benzoyl chloride (6b) was used instead of 4-chloro benzoyl chloride (6a). Yield: 86 %; mp: 168-170°C; IR (KBr, γmax cm ):
1
2
665(C=O), 1784(C=C), 2356(C-N), 2953(C-H aliphatic), 3015(C-H aromatic); MS (EI) m/z calculated for C H ClN O 283, found
16 14 2 3
83 (M+1).
-ethyl-1-(6-nitro-3,4-dihydroquinolin-1(2H)-yl)butan-1-one (7c):
2
Procedure of synthesis of this compound 7c remained same as that of the synthesis of the compound 7a only difference was that the
aliphatic 2-ethyl butanoyl chloride (6c) was used instead of 4-chloro benzoyl chloride (6a).
Synthesis of (6-amino-3,4-dihydroquinolin-1(2H)-yl)(4-chlorophenyl)methanone (8a):
The mixture of ammonium chloride (7000 mmol, 0.5 gm) was prepared in water: methanol (3:7). Simultaneously the compound 7a
(1000 mmol, 0.3 gm) was added to suitable quantity of methanol followed by the addition of the zinc dust (10000 mmol, 0.43 gm). The
above mixture of ammonium chloride was poured into this reaction mixture of compound and zinc dust. This reaction mixture was
allowed to stir at 60 °C for 6 hours resulted into the reduction of nitro group to amine. Further methanol was evaporated and the mixture
was dissolved in the ethyl acetate and extracted with bicarbonate. The compound was dried over the sodium sulfate. Yield: 80 %; mp:
-1
1
90-193°C; IR (KBr, γmax cm ): 1552(C=O), 1600(C=C), 2360(C-N), 2931(C-H aliphatic), 3361(C-H aromatic); MS (EI) m/z
calculated for C H ClN O 287 (M+1), found 287.5 (M+1) and 289.5 for chloro pattern.
1
6
15
2
Synthesis of (6-amino-3,4-dihydroquinolin-1(2H)-yl)(phenyl)methanone (8b):
Procedure of synthesis of this compound remained same as that of the synthesis of the compound 8a, only difference was that the 7b
-1
was used instead of 7a. Yield: 79 %; mp: 189-192°C; IR (KBr, γmax cm-1): IR (KBr, γmax cm ): 1550(C=O), 1601(C=C), 2360(C-
N), 2935(C-H aliphatic), 3363(-NH ), 3366(C-H aromatic); MS (EI) m/z for C H N O 251, found 251 (M+1).
2
16 16
2
1
-(6-amino-3,4-dihydroquinolin-1(2H)-yl)-2-ethylbutan-1-one (8c):

Procedure of synthesis of this compound 8c remained same as that of the synthesis of the compound 8a, only difference was that the
compound 7c was used instead of 7a.
Synthesis and Spectral Characterization of Final Derivatives (Series-I):
Synthesis of (N-(1-(4-chlorobenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-4-nitrobenzamide: (HB-UC-1) (9d):
Compound 8a (1.7 mmol, 0.5 gm) was dissolved in the dichloromethane. Dropwise trimethylamine (4.9 mmol, 0.46 mL) was added to
above reaction mixture and allowed to stir for 30 minutes at room temperature. 4-nitro benzoyl chloride (1.6 mmol, 0.3 gm) was added
to above reaction mixture and stirring was continued for 1 hour. Reaction mixture was transferred to the crushed ice and extracted with
dichloromethane dried over the sodium sulfate and passed through the column for the purification resulted into yellow colored
-1
compound. Dark yellow Solid Yield: 65.78 %; mp: 206-208°C; IR (KBr, γmax cm ): 843 (C-Cl), 1530 (-NO ), 1599 (C=O), 1673
2
1
(
C=C), 2360 (C-N), 2951 (C-H aliphatic), 3015 (C-H aromatic), 3319 (-NH); H NMR (400 MHz, DMSO-d6) δ PPM: 1.95-1.98 (m,
2
2
1
1
H, -CH ), 2.82 (t, J = 5.6 Hz, 2H, -CH ), 3.76 (m, 2H, -CH ), 6.74 (s, 1H, Ar-CH), 7.26 (d, J = 8 Hz, 1H, Ar-CH), 7.35 (d, J = 8 Hz,
2 2 2
H Ar-CH), 7.42 (d, J = 8 Hz, 2H, Ar-CH), 7.71 (s, 1H, Ar-CH), 8.14 (d, J = 8.4 Hz, 2H, Ar-CH), 8.35 (d, J = 8.4 Hz, 2H, Ar-CH),
1
3
0.49 (s, 1H, NH-C=O); C NMR (100 MHz, DMSO-d6) δ PPM: 23.48, 26.48, 44.26, 117.74, 120.16, 123.53, 125.14, 128.23(2C),
28.43(2C), 129.09(2C), 129.50(2C), 130.09, 131.91, 134.52, 135.09, 140.47, 149.08, 163.50, 167.97; MS (EI) m/z calculated for
C H ClN O 436 for (M+1); found 436.4 (M+1), 438.4 (for chloro pattern); HPLC Purity: 97.58%.
2
3
18
3
4
Synthesis of 4-chloro-N-(1-(4-chlorobenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)benzamide (HB-UC-2) (9e):
The procedure remained same as that of the compound 9d, only instead of 4-nitro benzoyl chloride; 4-chloro benzoyl chloride (1.7
-1
mmol, 0.23 mL) was added. Pale yellow Solid Yield: 67.56 %; mp: 230-232°C; IR (KBr, γmax cm ): 842 (C-Cl), 1599 (C=O), 1662
1
(
C=C), 2360 (C-N), 2934(C-H aliphatic), 3071 (C-H aromatic), 3340 (-NH); H NMR (400 MHz, DMSO-d6) δ PPM: 1.95-1.98 (m,
2
H,-CH ), 2.80-2.83 (m, 2H,-CH ), 3.75-3.77 (m, 2H, -CH ), 6.71 (m, 1H, Ar-CH), 7.26-7.28 (m, 1H, Ar-CH), 7.35 (d, 2H, J = 8 Hz,
2 2 2
Ar-CH), 7.41 (d, 2H, J = 8 Hz, Ar-CH), 7.56-7.58 (m, 2H, Ar-CH), 7.61 (m, 2H, Ar-CH), 7.71 (s, 1H, Ar-CH), 10.29 (s, 1H, NH-C=O);