Design, synthesis and biological evaluation of new series of hexahydroquinoline and fused quinoline derivatives as potent inhibitors of wild-type EGFR and mutant EGFR (L858R and T790M)

Design, synthesis and biological evaluation of new series of

hexahydroquinoline and fused quinoline derivatives as potent

inhibitors of wild-type EGFR and mutant EGFR (L858R and

T790M)

a,b

a

a,

Mennatallah A. Shaheen , Ali A. El-Emam , Nadia S. El-Gohary *

a

Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University, New Damietta, Egypt

b

^Corresponding author: dr.nadiaelgohary@yahoo.com, el_gohary@mans.edu.eg

ABSTRACT: New series of hexahydroquinoline and fused quinoline derivatives were designed

and synthesized. The thirty seven new compounds were screened for in vitro antitumor activity

over HepG2, HCT-116 and MCF-7 cancer cell lines. Results indicated that compounds 2e, 2h,

5

b, 5c, 6a, 7d and 9b have the strongest potency over the three cancer cells, and they were

further screened for in vitro antitumor activity over NSCLC cell lines (A431 and H1975), as well

as cytotoxicity against WI38 and WISH normal cell lines. Results revealed that 7d potently

inhibited the growth of H1975 cells harboring EGFR^T⁷⁹⁰^Mmutation (IC = 1.32±0.2 µM) over

5

0

A431 cells overexpressing EGFR^W^T(IC = 4.96±0.3 µM). Moreover, the seven compounds

5

0

displayed low cytotoxicity against the tested normal cells. The seven potent antitumor

WT

compounds were examined for their ability to inhibit the activity of EGFR . The attained data

manifested that 7d has remarkable EGFR^W^Tinhibitory activity (IC₅₀= 0.083±0.002 M)

compared to erlotinib (IC₅₀= 0.067±0.002 M). Compound 7d was further studied for its

enzymatic inhibitory activity against other eight human kinases, and it displayed outstanding

inhibitory activity against EGFR^L⁸⁵⁸^Rand EGFR^T⁷⁹⁰^Mmutants (IC = 0.053±0.002, 0.026±0.001

5

0



M, respectively), as well as JAK3 (IC = 0.069±0.003 M). Analysis of cell cycle evidenced

5

0

that 7d induces cell cycle arrest in G2/M and pre-G1 phases in the tested cancer cells. In

addition, cancer cell death induced by 7d was evidenced to take place via apoptosis supported by

elevated Bax/Bcl-2 ratio in the tested cancer cells. Moreover, docking results confirmed the good

binding interactions of 7d with EGFR , EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3, which came in

agreement with the results of in vitro enzyme assay. Further, 7d is predicted to have good oral

absorption, good drug-likeness properties and low toxicity risks in human.

WT

Keywords: Hexahydroquinolines, Fused quinolines, Antitumor, EGFR inhibition, Cell cycle

analysis, Apoptosis, Computational studies.

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1

. Introduction

Cancer is a hazardous disease affecting human in developed and developing countries [1].

Currently, treatment strategies include surgery [2], chemotherapy [3] and radiotherapy [4].

Despite the extensive research and rapid progress in cancer treatment, there is an urgent need for

new therapies, and it is important to recognize new agents and new targets for cancer treatment.

Receptor tyrosine kinases play a substantial role in signal transduction pathways that control cell

division and differentiation. Epidermal growth factor receptor tyrosine kinase (EGFR-TK)

belongs to a family of receptor tyrosine kinases that includes three other members (erbB2/HER-

2

, erbB3/HER-3 and erbB4/HER-4) [5,6].

EGFR-TK is an essential enzyme for intracellular signaling and cell transformation, and

anomalous expression of this enzyme was reported in a variety of human malignancies,

including non-small cell lung cancer (NSCLC) [7], hepatocellular [8], colorectal [9] and breast

[10] cancers. As such, inhibition of EGFR-TK provides a rational basis for a class of targeted

therapies, EGFR-TK inhibitors (EGFR-TKIs). EGFR-TKIs compete with ATP for ATP-binding

site on EGFR. The ATP-binding site (Fig. 1) has the following characteristic features: Adenine

binding region that contains two principal hydrogen bonds formed through interaction of N1 and

N6 of the adenine ring with the corresponding amino acids. Lots of EGFR-TKIs use one of the

two hydrogen bonds. Sugar pocket that represents a hydrophilic region. Hydrophobic binding

regions and channels, though not used by ATP but play an important role in binding affinity and

inhibitor selectivity. Phosphate binding region which is largely solvent exposed, and can be used

for improving inhibitor selectivity [11].

In the last few years, the oncogenic role of EGFR has been more finely characterized due to

the improved understanding of the mechanisms of receptor activation, the finding of somatic

mutations of the receptor as well as mutations in the components of the signaling pathway, and

in a great measure due to the excellent success of anti-EGFR therapies in clinic [12]. First-

generation EGFR-TKIs such as gefitinib [13] and erlotinib [14] (Fig. 2) were proved to be

effective in treatment of NSCLC; however, drug resistance rapidly develops. Hence, there is still

an urgent need for effective clinical medicines that could overcome drug resistance in patients

with advanced NSCLC harboring EGFR-mutations

Studies revealed that T790M ‘gatekeeper’ mutation in EGFR was the most common

mechanism of resistance to the first-generation EGFR-TKIs [15,16]. Also, L858R mutation was

reported as another mechanism of resistance to first-generation EGFR-TKIs [17]. To overcome

resistance to first-generation EGFR-TKIs, the second-generation EGFR-TKIs (e.g., pelitinib

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2 -

[18] and neratinib [19]) (Fig 2) were developed; however, acquired resistance to these agents

invariably develops. Studies proved that T790M mutation in EGFR gene was also a common

mechanism of resistance to the second-generation EGFR-TKIs [20]. Accordingly, third-

generation EGFR-TKIs (e.g., osimertinib [21] and olmutinib [22]) (Fig. 2) were developed to

overcome resistance to first- and second-generations EGFR-TKIs, and they enabled the

irreversible binding to Cys797 residue in the ATP-binding site. In particular, osimertinib showed

great selectivity toward the drug-resistant mutant EGFR^T⁷⁹⁰^Mover the wild-type EGFR

EGFR ), which made it an efficacious medical regimen for patients harboring EGFR^T⁷⁹⁰^M

WT

(

mutation [20,23-25]. Because T790M [15,16] and L858R [17] are the frequent mutations that

lead to resistance to EGFR-TKIs, we found it interesting to develop new drugs targeting

EGFR^L⁸⁵⁸^Rand EGFR^T⁷⁹⁰^Mmutants to overcome resistance to first- and second-generation

EGFR-TKIs in patients with advanced NSCLC harboring EGFR-mutations.

2

. Rational and design

Quinoline is an important scaffold for a variety of anticancer agents that target a diverse

range of receptor tyrosine kinases. The most widely studied of these receptors is EGFR-TK.

Pelitinib (Figs. 2 and 3), a quinoline-3-carbonitrile derivative, is an anticancer agent with

irreversible EGFR-TK inhibitory activity [18]. Furthermore, neratinib (Figs. 2 and 3) is an

EGFR-TK inhibitor that could be used as an adjuvant therapy in patients with early stage breast

cancer in which HER2 is overexpressed [19]. Moreover, quinoline-3-carbonitriles A [26] (Figs.

2

and 3) and B [26] (Fig. 2) displayed potent antitumor activity against human epidermoid

(A431), breast (SKBR3) and colon (SW620) cancer cells, as well as potent EGFR-TK inhibitory

activity. The quinoline-3-carboxamide derivative C [27] (Figs. 2 and 3) was found to elicit

outstanding antitumor activity against MCF-7 cancer cells through targeting EGFR-TK. Further,

the hexahydroquinoline-3-carboxylate D [28] (Figs. 2 and 3) was described as promising

antitumor agent against human alveolar basal epithelial (A549) and small cell lung (H446)

cancer cells through targeting EGFR-TK. All these quinoline derivatives were proved to act via

competing with ATP for binding at the catalytic domain of EGFR-TK, and they share the

essential pharmacophoric features required for EGFR-TK inhibition, including quinoline or

hexahydroquinoline as a central heteroaromatic ring (occupies the adenine binding region),

cyano, carboxamide or ester substitution for H-bond formation with the ATP-binding site,

hydrophobic head (occupies the hydrophobic binding region I) and hydrophobic tail (occupies

the hydrophobic binding region II). Taking these literature findings into consideration, and as an

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3 -

attempt to prepare new antitumor EGFR-TKIs, the hexahydroquinoline scaffold was introduced

as a central heteroaromatic ring similarly to the hexahydroquinoline EGFR-TKI D, and the

cyano group or carboxylic (bioisostere of carboxamide) was retained at 3-postion. Further, the

hydrophobic head at 4-position was replaced with various hydrophobic aromatic moieties

(substituted with halogens as lipophilic electron-withdrawing groups); in addition, two methyl

substituents were introduced at 7-position of hexahydroquinoline scaffold as a hydrophobic tail

in order to prepare three new series of hexahydroquinolines 2a-j, 3a-c and 4a-h that could target

EGFR-TK (Fig. 3).

Furthermore, the bioactive pyrimidine E [29] (Figs. 4 and 5) possessed potent antitumor

activity against the NSCLC cells (H3255, A431, HCC827, H1975 and PC-9) through targeting

EGFR^W^Tand EGFR^L⁸⁵⁸^R. Additionally, the pyrimidines F [30] (Figs. 4 and 5) and G [30] (Fig.

4

) were established to display prominent antitumor activity against breast (MCF-7), colon

(HCT-116) and human alveolar basal epithelial (A549) cancer cells through targeting EGFR-

TK. Besides, several benzylideneamine derivatives H [31] (Figs. 4 and 5) were described as

promising antitumor agents against human kidney epithelial (293T) cancer cells and EGFR-

TKIs. Moreover, the phenylthiourea derivatives I [32] (Figs. 4 and 5) were proved to exhibit

outstanding antitumor efficacy over MCF-7 cancer cells through targeting EGFR-TK.

Furthermore, [1,2,4]triazole J [33] (Figs. 4 and 5) displayed interesting antitumor efficacy over

HepG2, MCF-7 and HCT-116 cancer cells, as well as potent EGFR-TK inhibitory activity,

whereas the [1,2,4]triazole derivatives K [34] (Figs. 4 and 5) were established as promising

antitumor agents against A549 and small cell lung (H446) cancer cells via targeting EGFR-TK.

Inspired by these findings and as an attempt to study the effect of replacement of the bicyclic

quinoline scaffold in the small-molecule inhibitors with large tricyclic or tetracyclic scaffolds on

the antitumor and EGFR-TK inhibitory activities, the concept of molecular hybridization was

followed to prepare new series of fused quinoline derivatives. Molecular hybridization involves

the combination of different pharmacophoric moieties with pertinent biological activities to

obtain new hybrid compounds with improved activity, selectivity and safety [35,36].

Accordingly, new series of tetrahydropyrimidoquinolines 5a-f, hexahydropyrimidoquinolines

6

a,b, and tetrahydro[1,2,4]triazolopyrimidoquinolines 9a,b were designed through incorporation

of pyrimidine and [1,2,4]triazole rings into the hexahydroquinoline scaffold (Fig. 5). Further, the

manipulation strategy embraced the incorporation of benzylideneamine and phenylthiourea

moieties into the hexahydropyrimidoquinoline scaffold (series 7a-d and 8a,b, respectively) (Fig.

5

) in order to verify the importance of these moieties for the antitumor and EGFR-TK inhibitory

activities.

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4 -

The designed new analogs were prepared and screened for antitumor and EGFR-TK

inhibitory activities. However, inhibition of EGFR-TK activity is not sufficient to eradicate

tumors, meaning that a single-target chemotherapeutic agent is not enough to treat the

symptoms. Since single-targeted chemotherapeutics have limited antitumor activity, researchers

have done their best to find out other better strategies for cancer remedy, for instance, the multi-

target drug design strategy, which is based on the design and synthesis of several bioactive

molecules that direct multiple targets to induce different alterations of cellular function [37-39].

Multitargeting chemotherapeutics have the potential to demonstrate additive or synergistic

effects compared to the single-targeted ones; therefore, they attained an increasing interest in

combating resistant cancer cells [40].

Structure-activity

relationship

study

revealed

that

the

4-imino-3-(4-

(dimethylamino)benzylidene)amino)hexahydropyrimidoquinoline derivative 7d has the most

potent antitumor and EGFR^W^Tinhibitory activities, the promising potency of 7d might be

attributed to presence of 4-(dimethylamino)benzylidene)amino moiety that offers an extra

hydrophobic site, and an extra hydrogen bond acceptor site for binding interactions with the

target. Additionally, compound 7d was evaluated for its ability to inhibit other eight enzymes

from different classes of human tyrosine kinases (EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^M, TYK2, JAK3, BTK,

VEGFR-2, mTOR and HER-4) to ensure its multi-targeted activity, and hence good curative

effect. Also, its capability to induce cell cycle arrest and cell apoptosis, as well as its effect on

Bax/Bcl-2 ratio in HepG2, HCT-116 and MCF-7 cancer cells were assessed. Further, molecular

docking studies utilizing the crystal structures of EGFR , EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3

were performed to investigate the possible binding patterns of the potent compound 7d with

these target kinases.

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3

. Results and discussion

3

.1. Chemistry

A general approach to synthesize the designed compounds is outlined in Schemes 1-3. The

arylidenemalononitriles 1a-j [41-48] were synthesized via reaction of araldehydes with

malononitrile in ethanol [41,48]. In Scheme 1, 2-amino-4-aryl-7,7-dimethyl-5-oxo-5,6,7,8-

tetrahydroquinoline-3-carbonitriles 2a-j were prepared via reaction of arylidinemalononitriles

1

a-j with dimedone in refluxing ethanol in presence of ammonium acetate adopting the literature

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5 -

1

13

procedure [49]. The structures of 2a-j were confirmed on the basis of IR, H NMR, C NMR,

mass spectra and X-ray crystallography (Fig. 6).

Referring to Scheme 2, heating ortho aminonitriles 2a, 2b and 2f with sulfuric acid 70% at

7

0 °C for 1-2 h afforded the corresponding ortho aminocarboxylic acid derivatives 3a-c.

Refluxing ortho aminonitriles 2a-d, 2f-h and 2j with excess triethyl orthoformate afforded the

corresponding ethyl formimidates 4a-h following the literature procedure [49,50]. Compounds

4

a-e and 4h were used as precursors for the synthesis of new pyrimidoquinoline derivatives.

Adopting the reported method [49,50], compounds 4a-e and 4h were refluxed with ammonia

solution 35% in ethanol to give the corresponding 4-aminopyrimidoquinoline derivatives 5a-f.

Furthermore, the hexahydropyrimidoquinolines 6a,b were synthesized via reaction of 4a,b with

hydrazine hydrate in ethanol at room temperature according to the reported procedure [51]. With

respect to Scheme 3, the 3-amino-4-iminohexahydropyrimidoquinoline 6a was allowed to react

with benzaldehyde derivatives in refluxing ethanol in presence of a catalytic amount of

piperidine

benzylidene)amino)hexahydropyrimidoquinolines 7a-d. The 3-(4-substituted phenyl)-1-

substituted hexahydropyrimidoquinolin-3-yl)thioureas 8a,b were synthesized via refluxing 3-

to

produce

the

corresponding

3-(4-(un)substituted

(

amino-4-iminohexahydropyrimidoquinoline 6a with the appropriate phenyl isothiocyanate in

ethanol. Upon heating 3-amino-4-iminohexahydropyrimidoquinolines 6a,b with triethyl

orthoformate, the corresponding tetrahydrotriazolopyrimidoquinolines 9a,b were attained. The

structures of the new compounds were ascertained by spectral and elemental analyses.

3

.2. Biology

3

.2.1. Cytotoxicity testing

3

.2.1.1. In vitro antitumor testing

MTT assay was followed to assess the antitumor activity of the thirty seven new

derivatives over hepatocellular (HepG2), colorectal (HCT-116) and breast (MCF-7) cancer cell

lines [52-54], utilizing doxorubicin, erlotinib and osimertinib as reference drugs. Doxorubicin is

one of the most effective chemotherapeutic agents against several types of human cancers,

including HepG2 [55], HCT-116 [56] and MCF-7 [55] cancers. Two different mechanisms of

action have been proposed for doxorubicin, (i) intercalation of DNA and inhibition of

topoisomerase II leading to changes in chromatin structure, (ii) generation of free radicals and

oxidative damage to biomolecules [57]. However, doxorubicin resistance is a serious problem

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6 -

and several mechanisms such as reduction of drug uptake, activation of drug detoxification,

increased drug efflux and DNA repair capacity, and deflecting apoptotic pathway [58,59] have

been proposed.

Concentrations of compounds that induce 50% inhibition of cell viability (IC , µM) were

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0

determined (Table 1). Results showed that 2e, 2h, 5b, 5c, 6a, 7d and 9b are the most potent

members against all selected cancer cells (IC₅₀= 2.91-10.35 µM) in comparison to the

doxorubicin (IC = 4.17-5.23 µM), erlotinib (IC = 4.16-11.21 µM), and osimertinib (IC =

5

0

50

8

.44-9.72 µM). Also, compounds 4d, 4h and 8b were proved to have strong activity against

HCT-116 cells (IC = 3.85-9.82 µM). Further, compounds 4g and 7b manifested strong activity

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0

against HepG2 cells (IC₅₀= 10.17±0.9 and 9.50±0.8 µM, respectively). Additionally, 4g

exhibited high potency over MCF-7 cells (IC₅₀= 7.92 M). On the other hand, 2f and 5d

demonstrated moderate efficacy over the three cancer cells (IC = 13.64-20.61 M), whereas 8b

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0

was proved to have moderate activity over HepG2 and MCF-7 cells (IC₅₀= 14.49 and

 M, respectively). Furthermore, compounds 4b, 4g, 7a and 7b were found to be



moderately active over HCT-116 cells (IC₅₀= 12.10-19.32 M). The rest of the tested

compounds displayed moderate to weak activity over the three cancer cells.

3

.2.1.1.1. Analysis of structure-activity relationship

Concerning the hexahydroquinolines 2a-j

With regard to analogs carrying substituted phenyl moiety at 4-position of

hexahydroquinoline ring 2a-g, it is evident that presence of 2,4-difluorophenyl moiety at 4-

position of hexahydroquinoline ring led to promising antitumor potency over the three cancer

cells (compound 2e). Replacement of 2,4-difluorophenyl moiety with 2,6-difluorophenyl resulted

in decreased activity over the three cancer cells (compound 2f versus 2e), and this might be due

to the steric hindrance caused by the 2,6-difluoro substituents on the phenyl moiety in 2f.

Replacement of 2,6-difluorophenyl moiety in 2f with 2-chloro-6-fluorophenyl resulted in

decreased activity over the three cancer cells (compound 2d), and this might be attributed to the

increased lipophilicity of 2d (LogP = 2.32) compared to 2f (LogP = 1.80). Replacing 2-chloro-6-

fluorophenyl moiety in 2d with 2,6-dichlorophenyl resulted in decreased potency over the three

cancer cells (compound 2c versus 2d), and this might be attributed to the increased lipophilicity

of 2c (LogP = 2.83) compared to 2d (LogP = 2.32). Replacing 2,6-dichlorophenyl moiety in 2c

with 2,3-dichlorophenyl resulted in decreased potency over all cancer cells (compound 2a versus

2

c), while its replacement with 3,4-dichlorophenyl resulted in improved activity over HCT-116

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7 -

and MCF-7 cells, and decreased activity over HepG2 (compound 2b versus 2c). Further,

replacement of 2,4-difluorophenyl moiety in 2e with biphenyl led to weak activity over the three

cancer cells (compound 2g). On the other hand, presence of [1,3]benzodioxol-4-yl moiety at 4-

position of hexahydroquinoline resulted in high potency over the three cancer cells (compound

2

h). Replacement of [1,3]benzodioxol-4-yl moiety with naphthalen-2-yl led to weak activity over

all cancer cells (compound 2i versus 2h). Contrariwise, replacement of naphthalen-2-yl moiety in

2

i with anthracen-9-yl resulted in improved potency over all cancer cells (compound 2j versus

i).

Concerning the 2-aminohexahydroquinoline-3-carboxylic acid analogs 3a-c

Presence of 3,4-dichlorophenyl moiety at 4-position of hexahydroquinoline-3-carboxylic

acid led to moderate activity over HepG2, and weak activity over HCT-116 and MCF-7 cells

(compound 3b). Replacement of 3,4-dichlorophenyl moiety in 3b with 2,3-dichlorophenyl or

2

,6-difluorophenyl resulted in weak or no activity over all cancer cells (compounds 3a and 3c,

respectively).

Considering the structures of the ethyl formimidates 4a-h

With respect to the analogs carrying substituted phenyl moiety at 4-position of

hexahydroquinoline ring 4a-f, it is obvious that presence of 2-chloro-6-fluorophenyl moiety at 4-

position of hexahydroquinoline ring led to the highest activity over the three cancer cells

(compound 4d). Replacing 2-chloro-6-fluorophenyl moiety in 4d with 2,6-dichlorophenyl or

2

,6-difluorophenyl resulted in decreased activity over all tested cancer cells (compounds 4c and

e, respectively). Replacing 2,6-dichlorophenyl moiety in 4c with 2,3-dichlorophenyl

4

counterpart resulted in mild increase in activity over all cancer cells (compound 4a versus 4c),

while its replacement with 3,4-dichlorophenyl resulted in apparent increase in activity over all

selected cancer cells (compound 4b versus 4c), and we can conclude that the activity of the

dichlorophenyl analogs 4a-c is influenced by the positions of the two chloro substituents,

whereas the decreased activity in 4c compared to 4a and 4b might be due to the steric

hinderance caused by the 2,6-dichloro substituents in 4c. On the other hand, the N-(4-((1,1'-

biphenyl)-4-yl)hexahydroquinolin-2-yl)formimidate 4f showed the lowest activity amongst all

tested N-(4-(substituted phenyl)hexahydroquinolin-2-yl)formimidates, and this might be

attributed to the increased lipophilicity of 4f (LogP = 4.64) in comparison to 4a-e (LogP = 2.60-

4

.13). From another point of view, presence of the bicyclic [1,3]benzodioxol-4-yl moiety at 4-

position of hexahydroquinoline ring led to eminent potency over all selected cancer cells

compound 4g). Replacement of [1,3]benzodioxol-4-yl in 4g with anthracen-9-yl resulted in

(

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8 -

mild increase in activity over HCT-116, and mild decrease in activity over HepG2 and MCF-7

cells (compound 4h versus 4g).

With respect to the hexahydropyrimidoquinolines 5a-f

Presence of 3,4-dichlorophenyl moiety at 5-position of hexahydroquinoline ring led to high

potency over the three cancer cells (compound 5b). Replacement of 3,4-dichlorophenyl moiety

in 5b with 2,3-dichlorophenyl resulted in weak activity over the three cancer cells (compound

5

1

5

a), while its replacement with 2,6-dichlorophenyl led to mild increase in activity over HCT-

16, and mild decrease in activity over HepG2 and MCF-7 cancer cells (compound 5c versus

b). Replacement of 2,6-dichlorophenyl moiety with 2,6-difluorophenyl resulted in decreased

activity over all cancer cells (compound 5e versus 5c), and this might be attributed to the

decreased lipophilicity of 5e (LogP = 2.70) compared to 5c (LogP = 3.73). Replacement of 3,4-

dichlorophenyl moiety in 5b with the polycyclic anthracen-9-yl resulted in decreased activity

over the three cancer cells (compound 5f), and this might be attributed to the bulkiness of the

anthracene moiety.

Referring to the hexahydropyrimidoquinolines 6a,b

It is obvious that presence of 2,3-dichlorophenyl moiety at 5-position of

hexahydropyrimidoquinoline nucleus led to high potency over the three cancer cells (compound

6

a), while its replacement with 3,4-dichlorophenyl led to weak activity over all cancer cells

(compound 6b versus 6a).

Considering the structures of the hexahydropyrimidoquinoline derivatives 7a-d

Presence of unsubstituted benzylideneamino moiety at

hexahydropyrimidoquinoline scaffold led to moderate activity over the three cancer cells

compound 7a). Replacement of unsubstituted benzylideneamino moiety in 7a with 4-

3-position

of

(

chlorobenzylideneamino led to increased activity over HepG2 and HCT-116 cells, and decreased

activity over MCF-7 cells (compound 7b). Replacement of 4-chlorobenzylideneamino moiety in

7

b with 4-bromobenzylideneamino led to apparent decrease in activity over the three cancer

cells (compound 7c), and this might be attributed to the increased lipophilicity of 7c (LogP =

.78) compared to 7b (LogP = 4.65). Contrariwise, replacement of 4-chlorobenzylidene moiety

4

in 7b with 4-(dimethylamino)benzylidene led to compound 7d with close potency to

doxorubicin and higher potency than erlotinib and osimertinib over the three cancer cells, and

this confirms that presence of phenyl substituted with electron-donating group in 7d led to

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9 -

higher potency than the unsubstituted phenyl (compound 7a) or phenyl substituted with electron-

withdrawing group (compounds 7b,c).

Regarding the activity of 1-(substituted hexahydropyrimidoquinolin-3-yl)-3-(4-substituted

phenyl)thiourea derivatives 8a,b

Presence of 3-(4-chlorophenyl)thiourea moiety at 3-position of hexahydropyrimidoquinoline

nucleus led to moderate activity over the three cancer cells (compound 8a). Replacement of 3-

(4-chlorophenyl)thiourea moiety in 8a with 3-(4-methoxyphenyl)thiourea led to evident increase

in activity over all cancer cells (compound 8b versus 8a), and this confirms that presence of

phenyl substituted with electron-donating group improves the antitumor potency.

Referring to the tetrahydrotriazolopyrimidoquinolines 9a,b

Presence

of

2,3-dichlorophenyl

moiety

at

12-position

of

tetrahydrotriazolopyrimidoquinoline nucleus led to moderate activity over HepG2 and HCT-116

cells, and weak activity over MCF-7 cells (compound 9a). Replacement of 2,3-dichlorophenyl

moiety in 9a with 3,4-dichlorophenyl led to high potency over the three cancer cells (compound

9

b versus 9a).

3

.2.1.2. Cytotoxicity testing against NSCLC cell lines that overexpress EGFR^W^T(A431) and

mutant EGFR^T⁷⁹⁰^M(H1975), as well as normal cell lines (WI38 and WISH)

Cytotoxicity of compounds 2e, 2h, 5b, 5c, 6a, 7d and 9b against NSCLC cell lines (A431,

overexpressing EGFR ) and (H1975, harboring mutant EGFR^T⁷⁹⁰^M), as well as lung fibroblast

WT

(WI38) and amnion epithelial (WISH) normal cell lines was assessed adopting MTT assay [52-

5

4]. IC values (µM) were determined, and results are compared to doxorubicin, erlotinib and

5

0

osimertinib (Table 2). Compound 7d exhibited potent activity against H1975 cells (IC₅₀

=

1

.32±0.2 µM) with 3.8-fold selectivity over A431 cells (IC = 4.96±0.3 µM). The potency of 7d

5

0

over A431 cells was proved to be higher that of osimertinib (IC = 5.63±0.3 µM), whereas its

5

0

potency over H1975 was half that of osimertinib (IC = 0.69±0.1 µM), and 6.45 times higher

5

0

than that of erlotinib (IC = 8.52±0.5 µM). Similarly, compounds 2e, 2h and 5c were proved to

5

0

be more selective toward H1975 cells (IC = 7.13±0.6, 3.89±0.4 and 4.46±0.4 µM, respectively)

5

0

compared to A431 cells (IC = 9.82±1.0, 5.78±0.5 and 9.10±1.1 µM, respectively). On contrary,

5

0

compounds 5b, 6a and 9b showed higher activity against A431 cells (7.89±0.6, 10.62±1.5 and

.67±1.3 µM, respectively) over H1975 cells (8.75±0.6, 12.31±1.7 and 10.11±1.3 µM,

9

respectively). Referring to results of cytotoxicity against normal cells (Table 2), the seven tested

compounds showed lower cytotoxicity against WI38 (IC = 39.26-86.42 µM) and WISH (IC =

5

0

50

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10 -

1

9.78-63.37 µM) normal cells than doxorubicin (IC₅₀= 6.72±0.5 and 3.18±0.2 µM,

respectively). In addition, the selectivity index (SI) values of 2e, 2h, 5b, 5c, 6a, 7d and 9b

toward HepG2, HCT-116, MCF-7, A431 and H1975 cancer cells versus WI38 and WISH normal

cell s were calculated (Table 3). The SI values more than 2 are considered as high selectivity

[60,61]. Results indicated that the seven compounds have high SI values toward the five cancer

cells versus the two normal cells (SI = 2.09-48.69), and they are expected to be safe antitumor

agents. In addition, compound 7d displayed the highest SI values toward all tested cancer cells

(

except HCT-116) with the highest SI values toward H1975 NSCLC cells harboring EGFR^T⁷⁹⁰^M

mutation (SI = 48.69 and 35.14) versus WI38 and WISH normal cells, respectively.

3

.2.2. Mechanistic studies

3

.2.2.1. Tyrosine kinase assay

The potent antitumor agents in the current study were subjected to further studies to

investigate their potential mechanism of action. EGFR^W^Tinhibitory activity of the potent

antitumor compounds 2e, 2h, 5b, 5c, 6a, 7d and 9b was studied [62], and results are displayed as

%

inhibition of EGFR^W^T(at 10 µM) (Fig. 7) and IC (M) (Table 4), and they are compared to

5

0

erlotinib and osimertinib as reference drugs. Compound 7d demonstrated the highest EGFR^W^T

inhibitory activity (IC = 0.083±0.002 M) close to that of erlotinib (IC = 0.067±0.002 M)

5

0

50

and about 6 times higher than that of osimertinib (IC = 0.48±0.009 µM), and this result comes

5

0

in accordance with the antitumor screening results. In addition, compounds 2e, 2h and 5b

displayed promising EGFR^W^Tinhibitory activity (IC₅₀= 0.153±0.003, 0.129±0.003 and

0

.143±0.003 M, respectively). Additionally, compounds 5c, 6a and 9b exhibited good EGFR^W^T

inhibitory activity (IC₅₀= 0.216±0.005, 0.347±0.007 and 0.459±0.009 M, respectively).

Further, compound 7d was assessed for its inhibitory activity against EGFR^L⁸⁵⁸^Rand EGFR^T⁷⁹⁰^M

mutants [62]. Results are displayed as % inhibition (at 10 µM) (Fig. 8) and IC (M) (Table 5),

5

0

and compared to erlotinib and osimertinib as reference drugs. Compound 7d displayed potent

inhibitory activity against the drug-sensitive EGFR^L⁸⁵⁸^Rand the drug-resistant EGFR^T⁷⁹⁰^M(IC₅₀

=

0.053±0.002 and 0.026±0.001 M, respectively) compared to that of erlotinib (IC₅₀=

0

.045±0.001 and 0.443±0.002 M, respectively) and osimertinib (IC₅₀= 0.025±0.001 and

.022±0.001 M, respectively). Actually, the activity of 7d was close to that of erlotinib over

EGFR^L⁸⁵⁸^Rand 17 times higher than that of erlotinib over EGFR^T⁷⁹⁰^M. Additionally, 7d

displayed similar activity to that of osimertinib over EGFR^T⁷⁹⁰^Mand half the activity of

-

11 -

osimertinib over EGFR^L⁸⁵⁸^R, and we can conclude that 7d has high selectivity toward mutant

forms of EGFR over the wild-type form, and hence it might overcome resistance to EGFR-TKIs

that develops due to L858R and T790M mutations of EGFR gene.

Compound 7d was subjected to further studies against other six different human kinases

containing SH2 domain (TYK2) or both SH2 and SH3 domains (JAK3 and BTK) and other

human protein or lipid kinases (VEGFR-2, mTOR and HER-4) adopting the kinase profile assay

[62], and using erlotinib as a reference drug. Results are expressed as % inhibition (at 10 µM)

(

Fig. 8) and IC (M) (Table 5). Compound 7d exhibited potent enzymatic inhibitory activity

5

0

against JAK3 (IC = 0.069±0.003 M) which was proved to be 2 times higher than that of

5

0

erlotinib (IC₅₀= 0.146±0.003 M). In addition, it showed promising enzymatic inhibitory

activity against the other tested human kinases (IC₅₀= 0.089-0.298 M). Taking together,

compound 7d is considered to be a promising multi-kinase inhibitor (with the highest selectivity

toward EGFR^T⁷⁹⁰^Mmutant) that might prevent drug resistance by interfering with intracellular

signaling pathways or by inhibiting mutated binding sites or genes, and hence it could be used as

a promising candidate compound for treatment of patients with advanced NSCLC harboring

EGFR^T⁷⁹⁰^Mmutation.

3

.2.2.1.1. Analysis of structure-activity relationship

EGFR^W^T

dimethylamino)benzylidene)amino)hexahydropyrimidoquinoline 7d demonstrated the highest

EGFR^W^Tinhibitory activity (IC = 0.083±0.002 M), the potent inhibitory activity of 7d might

With

respect

to

inhibitory

activity,

the

3-((4-

(

5

0

be related to presence of 4-(dimethylamino)benzylidene moiety that carries an extra site for

hydrophobic interaction as well as an extra hydrogen bond acceptor site. On the other hand, the

4

-amino-5-(3,4-dichlorophenyl)tetrahydropyrimidoquinoline 5b showed good inhibitory activity

(

IC = 0.143±0.003 µM). Replacing 3,4-dichlorophenyl moiety in 5b with 2,6-dichlorophenyl

5

0

resulted in decreased inhibitory activity (compound 5c, IC = 0.216±0.005 µM), the decreased

5

0

activity of 5c in comparison to 5b might be due to the steric hinderence caused by the 2,6-

dichloro substituents on the phenyl moiety in 5c. Replacing 4-aminotetrahydropyrimidoquinoline

in 5b,c with 3-amino-4-iminohexahydropyrimidoquinoline resulted in obvious decrease in

activity (compound 6a, IC = 0.347±0.007 µM), and this might be attributed to the possibility of

5

0

intramolecular hydrogen bonding interaction between 3-amino and 4-imino groups in 6a that

WT

hinders its bonding interaction with EGFR . Further, replacing hexahydropyrimidoquinoline

scaffold in 7d with hexahydroquinoline led to decreased activity (compounds 2e and 2h, IC =

5

0

-

12 -

0

.153±0.003 and 0.129±0.003 µM, respectively). Comparing the activity of compounds 2e and

h, it is evident that presence of 3,4-difluorophenyl moiety at 4-position of hexahydroquinoline

2

led to good EGFR^W^Tinhibitory activity (compound 2e, IC = 0.153±0.003 µM). Replacing 3,4-

5

0

difluorophenyl moiety in 2e with [1,3]benzodioxol-4-yl led to increased potency (compound 2h,

IC = 0.129±0.003 µM), and this might be attributed to presence of two extra hydrogen bond

5

0

acceptor sites in 2h. On contrary, replacing the hexahydropyrimidoquinoline scaffold in 7d with

tetrahydrotriazolopyrimidoquinoline resulted in obvious decrease in activity (compound 9b, IC₅₀

=

0.459±0.009 µM), and this might be attributed to the decreased number of hydrogen bond

acceptor and donor sites in 9b as well as the absence of the extra hydrophobic site present in 7d.

3

.2.2.2. Analysis of cell cycle by flow cytometry assay

Several studies evidenced that EGFR-TKIs are capable of inducing cell cycle arrest and

cellular apoptosis [63-66]. To further understand the antitumor mechanism of compound 7d, its

effects on cell cycle progression in HepG2, HCT-116 and MCF-7 cells were examined (at the

IC of the corresponding cell line) [67-69]. Analysis of cell cycle in HepG2 showed that 7d

5

0

decreased % cells in G0/G1 phase (from 51.88% to 36.22%), and increased % cells in S phase

from 34.03% to 38.12%), G2/M phase (from 14.09% to 25.66%) and pre-G1 phase (from 1.44%

(

to 17.85%) (Figs. 9 and 10). Analysis of cell cycle in HCT-116 illustrated that 7d decreased %

cells in G0/G1 phase (from 48.52% to 34.05%) and S phase (from 35.61% to 27.44%), and

increased % cells in G2/M phase (from 15.87% to 38.51%) and pre-G1 phase (from 1.19% to

2

2.49%) (Figs. 9 and 10). Regarding results of cell cycle analysis in MCF-7, 7d decreased %

cells in G0/G1 phase (from 56.14% to 28.99%) and S phase (from 32.91% to 29.41%), and

increased % cells in G2/M phase (from 10.95% to 41.6%) and pre-G1 phase (from 1.78% to

3

2.11%) (Figs. 9 and 10). From the attained results, we can conclude that 7d demonstrated

considerable elevation in cell population of the three tested cancer cells in G2/M phase but at

different intensities, and induced cellular apoptosis in pre-G1 phase (Figs. 9 and 10), and

accordingly stopped the mitotic cycle.

3

.2.2.3. Induction of cellular apoptosis

Cancer is one of the diseases where little cellular apoptosis develops leading to growth of

malignant cells that will not die. Apoptosis is a well-known target in cancer therapeutics [70].

The mechanism of apoptosis is complex and involves different pathways [70]. Therefore,

compound 7d was examined for its capability to induce apoptosis in HepG2, HCT-116 and

-

13 -

MCF-7 cancer cells adopting the flow cytometry assay [67-69]. The flow cytometry assay results

of cells pigmented with propidium iodide (PI) and annexin V-fluorescein isothiocyanate

(annexin V-FITC) are shown in Fig. 11. The annexin V/PI double staining disclosed that after 24

h of exposure, 7d (at the IC of the corresponding cell line) induced early and late apoptosis in

5

0

the three cancer cells compared to the untreated cells. Compound 7d induced higher percentage

of total apoptosis in MCF-7 cells compared to HepG2 and HCT-116 cells (Figs. 11 and 12). On

contrary, it induced weak necrosis in the three treated cancer cells (Figs. 11 and 12) affirming

that cell death induced by 7d takes place mainly via apoptosis.

3

.2.2.4. Analysis of levels of Bax and Bcl-2 proteins

Bcl-2 (B-cell lymphoma 2) family members are significant regulators of apoptosis that can

be classified into three groups of proteins [71]. The first group protects against apoptosis (Bcl-2

itself) [71]. The second group is represented by Bax (Bcl-2 associated protein X) and Bak (Bcl-2

antagonist), which are the key activators of the apoptosis machinery in response to cellular stress

stimuli [71]. The third group is comprised of different categories of proteins such as Bid (BH3

interacting death domain), Bim (Bcl-2 interacting mediator) and others [71]. Overexpression of

the antiapoptotic protein Bcl-2 leads to antagonism of apoptosis and development of resistance

of tumor cells to common chemotherapeutic agents. Thus, development of new drugs that can

inhibit the antiapoptotic action of Bcl-2 is a very attractive strategy in the design of anticancer

agents [72]. The effect of compound 7d on the expression levels of Bax and Bcl-2 proteins was

determined after treatment of HepG2, HCT-116 and MCF-7 cancer cells with compound 7d (at

1

0 µM) [72] (Fig. 13). Compound 7d showed 5.71, 8.15 and 16.51-fold up-regulation in the

level of the proapoptotic protein Bax in HepG2, HCT-116 and MCF-7 cells, respectively

compared to the untreated cells, while it markedly down-regulated the level of the antiapoptotic

protein Bcl-2 in HepG2, HCT-116 and MCF-7 cells up to 0.72, 0.53 and 0.31-fold compared to

the untreated cells. These findings proved that induction of apoptosis by the active compound 7d

was achieved through up-regulation of Bax and down-regulation of Bcl-2.

4

. Computational studies

.1. Binding mode of compound 7d to EGFR , EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3

WT

4

In order to provide a deep insight into the binding mode of compound 7d (potent EGFR^W^T,

EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3 inhibitor) to EGFR , EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3, a

WT

-

14 -

study based on the crystal structures of EGFR^W^T(PDB code: 3W2S) [73], EGFR^L⁸⁵⁸^R(PDB

code: 4LQM) [74], EGFR^T⁷⁹⁰^M(PDB code: 2JIV) [75] and JAK3 (PDB code: 5TTV) [76] was

accomplished applying “molecular operating environment (MOE) version 2014.0901” Chemical

Computing Group Inc. software [77]. Results of docking of 7d into the ATP-binding pocket of

EGFR^W^Tillustrated that it interacts with EGFR^W^Tthrough two hydrogen bonding interactions,

one formed between NH of pyrimidoquinoline and Asp800, and the second one formed between

oxygen atom of C=O and Ser720, as well as one arene-H interaction between the phenyl of

benzylidene moiety and Leu844 (Fig. 14). Results of docking of 7d into EGFR^L⁸⁵⁸^Rrevealed that

it forms one hydrogen bonding interaction between the Cl at 3-position of phenyl ring and

Met793, as well as two arene-H interactions, one formed between the phenyl of benzylidene

moiety and Leu718, and the second one formed between the 5-phenyl moiety and Val726 (Fig.

1

5). Docking results of 7d into EGFR^T⁷⁹⁰^Mshowed that it forms two hydrogen bonding

interactions through N1 and N3 of pyrimidoquinoline with Met793, as well as one arene-H

interaction between the phenyl of benzylidene moiety and Gly796 (Fig. 16). Further, docking

results of 7d into the binding site of JAK3 indicated that it binds to JAK3 through two hydrogen

bonding interactions, one formed between the C=NH group and Arg911, and the second one

formed between N1 and Lys830, as well as one arene-H interaction between the 5-phenyl moiety

and Arg953 (Fig. 17). Summary of residues involved in arene-H interaction, number of hydrogen

bonds, residues involved in hydrogen bonding interaction and docking interaction energy

kcal/mol) of compound 7d with EGFR , EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3 is presented in

WT

(

Table 6. These results came in agreement with the results of in vitro enzyme assay, and gave a

preliminary conception of the binding mode of the new analogs.

4

.2. In silico studies

In silico studies have a significant rule in discovery of new drugs, since they are of great

importance in predicting the physicochemical properties, pharmacokinetics, and toxicity of drugs

of biological interest [78]. For the new compounds, Lipinski’s rule [79] and Veber’s parameters

[80] were analyzed using Molinspiration software [81]. Further, ADME properties were

predicted using PreADMET software [82]. Likewise, toxicity risks in human and drug-likeness

were prophesied applying Osiris property explorer software [83].

-

15 -

4

.2.1. Molinspiration calculations

Lipinski's rule helps to predict whether a biologically active compound is likely to have the

chemical and physical properties that ensure good oral absorption or not [79]. Likewise,

topological polar surface area (TPSA) and number of rotatable bonds (Nrotb) affect the extent of

drug absorption [80]. Molinspiration software [81] was applied for analysis of TPSA, Nrotb and

Lipinski’s rule parameters of the new compounds. Results (Table 7) illustrated that all of the new

compounds agreed with the adequate criteria of TPSA, Nrotb and Lipinski’s rule with zero or

one violation of Lipinski’s rule, hence they are predicted to have good oral absorption.

4

.2.2. ADME prediction

PreADMET software [82] was utilized to analyze the ADME characteristics of the new

compounds. Blood-brain barrier (BBB) penetration, plasma protein binding (%PPB) and human

intestinal absorption (%HIA) were predicted, and results are listed in Table 8. It was reported

that drugs with BBB (C_b_r_a_i_n/C_b_l_o_o_d) values more than 1 are predicted to be CNS-active candidates,

whereas those with BBB (C_b_r_a_i_n/C_b_l_o_o_d) values less than 1 are foreseen to be CNS-inactive

candidates [84]. Compounds 2a, 2c, 5a, 5c, 6a,b, 7a-d, 8a,b and 9a are foreseen to be CNS-

active candidates (BBB penetration = 1.05-6.24). On contrary, the remaining compounds are

foreseen to be CNS-inactive candidates (BBB penetration = 0.05-0.89). Additionally, all of the

analyzed compounds are predicted to display excellent plasma protein binding (%PPB = 62.55-

1

00.00%), and excellent human intestinal absorption (%HIA = 88.79-97.22%).

4

.2.3. Prediction of toxicity risks in human and drug-likeness

The new derivatives were analyzed for prediction of various toxicity risks in human

(tumorigenicity, irritancy and mutagenicity) and drug-likeness using Osiris software [83].

Results (Table 9) manifested that all of the analyzed compounds are expected to be non-

tumorigenic (except 2j, 4h and 5f), non-irritating (except 2j, 4h and 5f) and non-mutagenic

(except 2j, 4h and 5f).

Drug-likeness rules are used for studying the drug-like properties of a molecule. A positive

value indicates that the compound has fragments which are frequently present in commercial

drugs [85]. Results (Table 9) showed that 3a,b, 5a-c, 6a,b, 7a-d, 8a,b and 9a,b have acceptable

drug-likeness values.

-

16 -

5

. Conclusion

Results of antitumor assay assured that compounds 2e, 2h, 5b, 5c, 6a, 7d and 9b are the

most potent members over HepG2, HCT-116 and MCF-7 cancer cells (IC = 2.91-10.13 µM).

5

0

Moreover, compound 7d exhibited enhanced inhibitory potency over H1975 NSCLC cells

harboring mutated EGFR^T⁷⁹⁰^M(IC = 1.32±0.2 µM) over A431 cells overexpressing EGFR

WT

5

0

(

IC = 4.96±0.3 µM), and hence 7d is considered to be a promising and selective candidate for

50

effective treatment of patients with EGFR^T⁷⁹⁰^M-mutated NSCLC cell lines. In addition, the seven

active antitumor compounds displayed low cytotoxicity over WI38 and WISH normal cells (IC₅₀

=

19.78-86.42 µM), and they might be utilized as potent and safe antitumor agents. Results of

EGFR^W^Tinhibitory activity revealed that 7d has the strongest inhibitory activity (IC₅₀

.083±0.002 M). Further, compound 7d showed potent inhibitory activity against EGFR^L⁸⁵⁸^R

and EGFR^T⁷⁹⁰^Mmutants (IC = 0.053±0.002 and 0.026±0.001 M, respectively), and hence it

=

0

5

0

might overcome resistance to EGFR-TKIs. Additionally, 7d exhibited outstanding inhibitory

activity against JAK3 (IC = 0.069±0.003 M) which was proved to be 2-fold higher than that

5

0

of erlotinib (IC = 0.146±0.005 M). Analysis of cell cycle demonstrated that 7d induces cell

5

0

cycle arrest in G2/M and pre-G1 phases in HepG2, HCT-116 and MCF-7 cancer cells

Furthermore, death of cancer cells induced by 7d was affirmed to occur mainly via apoptosis

supported by elevation of Bax/Bcl-2 ratio. Docking results affirmed the precise fit of 7d into the

binding site of EGFR , EGFR^L⁸⁵⁸^R, EGFR^T⁷⁹⁰^Mand JAK3 enzymes. Moreover, results of

Molinspiration and Osiris calculations proved that 7d is predicted to have good oral absorption,

good drug-likeness properties and low toxicity risks in human. An overview on the attained

results emphasized that 7d is a new potent multi-kinase inhibitor with promising antitumor

activity against H1975 NSCLC cells harboring EGFR^T⁷⁹⁰^Mmutation. These advantages together

with the low cytotoxicity of 7d against normal cells indicate that 7d is considered to be an

auspicious lead compound for future evolution of new more potent and more selective antitumor

agents against resistant NSCLC cells harboring EGFR mutations.

WT

.

6

. Experimental

6

.1. Chemistry

Stuart melting point (SMP30) apparatus was utilized for determining the melting points



1

(

°C). IR spectra were recorded in KBr disc on Unicam SP 1000 IR spectrometer (ν in cm ). H

-

17 -

1

3

and C NMR spectra were recorded on Bruker Avance III HD FT-high resolution (400 MHz)

and (500 MHz). Chemical shifts are expressed in  ppm with reference to tetramethylsilane.

Mass spectra were recorded on Triple Quad Tandem spectrometer (ESI) at 4.0 and 3.5 kV for

positive and negative ion modes, respectively, and on direct probe controller inlet part of thermo

scientific ISQ single quadrupole GC-MS spectrometer (ISQ LT) utilizing X-calibur software,

Regional Center of Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr city,

Cairo, Egypt. Elemental analyses (% C, H, N) were achieved, and they were in harmony with the

predicted structures within ±0.4% of the calculated values. Reaction times were monitored by

TLC plates (Silica gel 60 F254), and spots were visualized by UV (366 nm). Hexane/ethyl

acetate (9:1) was used for elution. The arylidenemalononitriles 1a-j were synthesized adopting

the previously reported procedure [41,48].

6

.1.1. Synthesis of 2-amino-4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitriles 2a-j

A mixture of dimedone (0.7 g, 0.005 mol), ammonium acetate (1.93 g, 0.025 mol) and

arylidenemalononitrile 1a–j (0.005 mol) in absolute ethanol (10 mL) was refluxed for 5-7 h. The

precipitate formed was filtered, dried and crystallized from ethanol to yield the

hexahydroquinoline derivatives 2a-j.

6

.1.1.1. 2-Amino-4-(2,3-dichlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2a)

Pale yellow powder, yield 85%, m.p. 253-255 °C. IR: 3445, 3323 (NH ); 3204 (NH); 2191

2

1

(

C≡N); 1663 (C=O). H NMR (500 MHz, DMSO-d ):  0.97 (s, 3H, CH ), 1.03 (s, 3H, CH ),

6

3

2

1

.07 (d, J = 16.5 Hz, 1H, CH), 2.24 (d, J = 16.0 Hz, 1H, CH), 2.50-2.52 (m, 2H, CH ), 4.77 (s,

2

H, C -H), 7.12 (s, 2H, NH , D O-exchangeable), 7.15 (d, J = 8.0 Hz, 1H, Ar-H), 7.29 (t, J = 7.5

4

2

1

3

Hz, 1H, Ar-H), 7.45-7.47 (dd, J = 1.5, 1.5 Hz, 1H, Ar-H). C NMR: δ 26.9, 28.3, 31.8, 33.7,

9.9, 56.3, 111.6, 119.1, 128.3, 128.5, 128.8, 130.2, 131.8, 144.4, 158.7, 163.3, 195.7. ESI-MS,

4

+

m/z: 362.10 [M+H] , 360.30 [M-H] . Anal. Calcd. (Found) for C H Cl N O (361.07): C, 59.68

1

8

17

2

3

(59.35); H, 4.73 (4.44); N, 11.60 (11.92).

6

.1.1.2. 2-Amino-4-(3,4-dichlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2b) [86]

White crystals, yield 85%, m.p. 243-245 °C [86]. IR: 3531, 3473 (NH ); 3327 (NH); 2187

2

1

(

C≡N); 1687 (C=O). H NMR (500 MHz, DMSO-d ):  0.99 (s, 3H, CH ), 1.04 (s, 3H, CH ),

6

3

2

.06 (d, J = 16.0 Hz, 1H, CH), 2.28 (d, J = 16.0 Hz, 1H, CH), 2.36 (d, J = 20.0 Hz, 1H, CH),

18 -

-

2

.54 (d, J = 20.0 Hz, 1H, CH), 5.20 (s, 1H, C -H), 7.14 (s, 2H, NH , D O-exchangeable), 7.25 (t,

4 2 2

J = 8.0 Hz, 1H, Ar-H), 7.34-7.36 (dd, J = 0.8, 0.8 Hz, 1H, Ar-H), 7.42-7.45 (dd, J = 1.2, 1.2 Hz,

1

3

1

H, Ar-H). C NMR: δ 24.1, 27.2, 35.9, 45.8, 53.7, 53.9, 106.5, 113.5, 123.9, 124.0, 125.3,

+

30.2, 130.7, 132.0, 153.9, 158.5, 191.4. ESI-MS, m/z: 362.10 [M+H] , 360.30 [M-H] . Anal.

Calcd. (Found) for C H Cl N O (361.07): C, 59.68 (59.92); H, 4.73 (4.96); N, 11.60 (11.22).

1

8

17

2

3

6

.1.1.3. 2-Amino-4-(2,6-dichlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2c) [87]

Pale yellow powder, yield 85%, m.p. 240-242 °C [87]. IR: 3473, 3323 (NH ); 3175 (NH); 2189

2

1

(

C≡N); 1684 (C=O). H NMR (500 MHz, DMSO-d ):  0.98 (s, 3H, CH ), 1.04 (s, 3H, CH ),

6

3

2

.06-2.07 (m, 1H, CH), 2.25-2.34 (m, 1H, CH), 2.34-2.38 (m, 1H, CH), 2.52 (m, 1H, CH), 5.19

(s, 1H, C -H), 7.11 (s, 2H, NH , D O-exchangeable), 7.23 (t, J = 7.5 Hz, 1H, Ar-H), 7.33-7.35

4 2 2

1

3

(

dd, J = 1.5, 1.5 Hz, 1H, Ar-H), 7.43-7.45 (dd, J = 1.5, 1.0 Hz, 1H, Ar-H). C NMR: δ 26.9,

1.6, 32.6, 41.3, 49.9, 56.0, 110.0, 119.0, 128.5, 129.0, 134.1, 135.8, 159.4, 163.8, 195.7. ESI-

3

+

MS, m/z: 362.10 [M+H] , 360.40 [M-H] . Anal. Calcd. (Found) for C H Cl N O (361.07): C,

1

8

17

2

3

5

9.68 (59.31); H, 4.73 (4.65); N, 11.60 (11.42).

6

.1.1.4.

2-Amino-4-(2-chloro-6-fluorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinoline-3-carbonitrile (2d)

White powder, yield 72%, m.p. 197-199 °C. IR: 3415, 3331 (NH ); 3212 (NH); 2197 (C≡N);

2

1

683 (C=O). H NMR (500 MHz, DMSO-d ):  0.93 (s, 3H, CH ), 1.04 (s, 3H, CH ), 2.06 (d, J

6

3

=

16.0 Hz, 1H, CH), 2.26 (d, J = 16.5 Hz, 1H, CH), 2.35 (d, J = 18.0 Hz, 1H, CH), 2.55 (d, J =

1

8.0 Hz, 1H, CH), 4.87 (s, 1H, C -H), 7.12 (s, 2H, NH , D O-exchangeable), 7.13-7.25 (m, 3H,

4 2 2

1

3

Ar-H). C NMR: δ 26.3, 28.6, 29.5, 31.7, 49.8, 56.0, 114.7, 115.1, 119.2, 125.2, 125.5 (d, J =

3

.5 Hz), 129.2 (d, J = 9.2 Hz), 133.6 (d, J = 4.1 Hz), 158.2, 159.3, 163.5, 195.7. ESI-MS, m/z:

+

46.10 [M+H] . Anal. Calcd. (Found) for C H ClFN O (345.10): C, 62.52 (62.34); H, 4.96

1

8

17

3

(4.69); N, 12.15 (12.35).

6

.1.1.5. 2-Amino-4-(2,4-difluorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2e)

Pale green powder, yield 70%, m.p. 187-189 °C. IR: 3424, 3333 (NH ); 3227 (NH); 2179 (C≡N);

2

1

685 (C=O). H NMR (500 MHz, DMSO-d ):  0.91 (s, 3H, CH ), 1.03 (s, 3H, CH ), 2.06 (d, J

6

3

=

16.0 Hz, 1H, CH), 2.28 (d, J = 16.0 Hz, 1H, CH), 2.34 (d, J = 17.5 Hz, 1H, CH), 2.54-2.58 (dd,

J = 1.5, 1.0 Hz, 1H, CH), 4.62 (s, 1H, C -H), 7.01 (t, J = 8.5 Hz, 2H, Ar-H), 7.12 (s, 2H, NH ,

4

2

1

3

D O-exchangeable), 7.26-7.32 (m, 1H, Ar-H). C NMR: δ 25.0, 26.0, 28.7, 31.8, 49.8, 54.3,

2

1

10.2, 111.7 (d, J = 25.0 Hz), 119.1 (t, J = 15.5 Hz), 119.5, 129.1 (t, J = 9.5 Hz), 159.3, 159.7 (d,

-

19 -

+

J = 9.5 Hz), 161.7 (d, J = 10.0 Hz), 163.5, 195.7. EI-MS, m/z: 329.30 (2.19, M ), 83.10 (100.00).

Anal. Calcd. (Found) for C H F N O (329.35): C, 65.64 (65.35); H, 5.20 (5.46); N, 12.76

1

8

17

2

3

(12.43).

6

.1.1.6. 2-Amino-4-(2,6-difluorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2f)

White crystals, yield 70%, m.p. 255-257 °C. IR: 3408, 3333 (NH ); 3217 (NH); 2199 (C≡N);

2

1

685 (C=O). H NMR (500 MHz, DMSO-d ):  0.91 (s, 3H, CH ), 1.03 (s, 3H, CH ), 2.06 (d, J

6

3

=

16.5 Hz, 1H, CH), 2.26-2.36 (m, 2H, CH ), 2.57 (d, J = 17.5 Hz, 1H, CH), 4.62 (s, 1H, C -H),

2 4

7

.01 (t, J = 16.0 Hz, 2H, Ar-H), 7.13 (s, 2H, NH , D O-exchangeable), 7.25-7.31 (m, 1H, Ar-H).

2 2

¹³C NMR: δ 25.0, 26.0, 28.7, 31.8, 49.8, 54.4, 110.2, 111.7 (d, J = 25.0 Hz), 119.1 (t, J = 15.5

Hz), 119.5, 129.1 (t, J = 10.8 Hz), 159.3, 159.6 (d, J = 7.4 Hz), 161.6 (d, J = 7.1 Hz), 163.5,

+

1

95.7. ESI-MS, m/z: 328.40 [M-H] . Anal. Calcd. (Found) for C H F N O (329.13): C, 65.64

1

8

17

2

3

(65.34); H, 5.20 (5.41); N, 12.76 (12.43).

6

.1.1.7. 2-Amino-4-((1,1'-biphenyl)-4-yl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2g)

White crystals, yield 90%, m.p. 242-244 °C. IR: 3384, 3318 (NH ); 3208 (NH); 2193 (C≡N);

2

1

682 (C=O). H NMR (500 MHz, DMSO-d ):  0.97 (s, 3H, CH ), 1.04 (s, 3H, CH ), 2.12 (d, J

6

3

=

16.0 Hz, 1H, CH), 2.26 (d, J = 15.5 Hz, 1H, CH), 2.53 (s, 2H, CH ), 4.21 (s, 1H, C -H), 7.05

2 4

(

s, 2H, NH , D O-exchangeable), 7.22 (d, J = 8.5 Hz, 2H, Ar-H), 7.33 (t, J = 7.5 Hz, 1H, Ar-H),

2 2

7

.44 (t, J = 7.5 Hz, 2H, Ar-H), 7.58 (d, J = 8.5 Hz, 2H, Ar-H), 7.62 (d, J = 7.5 Hz, 2H, Ar-H).

¹³C NMR: δ 26.9, 28.4, 31.9, 35.3, 50.0, 58.1, 112.6, 119.8, 126.6, 126.7, 127.3, 127.8, 128.9,

+

1

38.5, 139.9, 144.0, 158.5, 162.5, 195.8. ESI-MS, m/z: 367.70 [M-H] . Anal. Calcd. (Found) for

C H N O (369.18): C, 78.02 (78.32); H, 6.27 (6.40); N, 11.37 (11.64).

2

4

23

3

6

3

.1.1.8. 2-Amino-4-([1,3]benzodioxol-4-yl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-

-carbonitrile (2h)

Greyish green crystals, yield 65%, m.p. 231-233 °C. IR: 3392, 3330 (NH ); 3216 (NH); 2190

2

1

(

C≡N); 1686 (C=O). H NMR (500 MHz, DMSO-d ):  0.94 (s, 3H, CH ), 1.03 (s, 3H, CH ),

6

3

2

6

.06 (d, J = 15.5 Hz, 1H, CH), 2.26 (d, J = 15.5 Hz, 1H, CH), 2.39 (d, J = 17.0 Hz, 1H, CH),

.50-2.58 (m, 1H, CH), 4.24 (s, 1H, C -H), 5.83 (s, 1H, Dioxole-H), 6.02 (s, 1H, Dioxole-H),

4

1

3

.58-6.62 (m, 1H, Ar-H), 6.70-6.74 (m, 2H, Ar-H), 6.98 (s, 2H, NH , D O-exchangeable). C

2

NMR: δ 26.3, 28.7, 31.8, 32.1, 50.0, 57.6, 100.7, 107.1, 110.9, 119.7, 121.3, 121.5, 126.2, 144.5,

+

1

47.1, 150.2, 163.0, 195.7. EI-MS, m/z: 338.18 (100.00, M +1), 337.25 (63.10, M ). Anal.

Calcd. (Found) for C H N O (337.38): C, 67.64 (67.41); H, 5.68 (5.48); N, 12.46 (12.22).

1

9

19

3

-

20 -

6

.1.1.9.

2-Amino-7,7-dimethyl-4-(naphthalen-2-yl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carbonitrile (2i) [88]

White powder, yield 75%, m.p. 255-257 °C [88]. IR: 3348, 3312 (NH ); 3172 (NH); 2190

2

1

(

C≡N); 1684 (C=O). H NMR (500 MHz, DMSO-d ):  0.95 (s, 3H, CH ), 1.03 (s, 3H, CH ),

6

3

2

.07 (d, J = 16.5 Hz, 1H, CH), 2.25 (d, J = 16.0 Hz, 1H, CH), 2.54 (d, J = 4.5 Hz, 2H, CH ), 4.35

2

(

s, 1H, C -H), 7.07 (s, 2H, NH , D O-exchangeable), 7.26-7.28 (dd, J = 1.5, 2.0 Hz, 1H, Ar-H),

4 2 2

7

.44-7.49 (m, 2H, Ar-H), 7.66 (s, 1H, Ar-H), 7.83-7.85 (m, 2H, Ar-H), 7.88 (d, J = 7.5 Hz, 1H,

1

3

Ar-H). C NMR: δ 26.7, 28.4, 31.8, 35.9, 50.0, 58.1, 112.5, 119.8, 125.5, 125.6, 125.7, 126.2,

1

27.5, 127.7, 128.1, 132.0, 132.8, 142.1, 158.5, 162.6, 195.8. ESI-MS, m/z: 344.30 [M+H]⁺,

+

3

41.90 [M-H] . Anal. Calcd. (Found) for C H N O (343.17): C, 76.94 (76.72); H, 6.16 (6.34);

2

21

3

N, 12.24 (12.52).

6

.1.1.10.

carbonitrile (2j)

Dark orange crystals, yield 56%, m.p. 227-228 °C. H NMR (500 MHz, DMSO-d ):  0.86 (s,

2-Amino-4-(anthracen-9-yl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

1

6

3

H, CH ), 1.02 (s, 3H, CH ), 1.88 (d, J = 16.0 Hz, 1H, CH), 2.13 (d, J = 16.0 Hz, 1H, CH), 2.53

3 3

(

d, J = 17.5 Hz, 1H, CH), 2.62 (d, J = 18.0 Hz, 1H, CH), 5.98 (s, 1H, C -H), 7.05 (s, 2H, NH ,

4 2

D O-exchangeable), 7.41-7.46 (m, 2H, Ar-H), 7.57-7.70 (m, 2H, Ar-H), 8.06-8.09 (m, 3H, Ar-

2

1

3

H), 8.52 (s, 1H, Ar-H), 8.65 (d, J = 9.0 Hz, 1H, Ar-H). C NMR: δ 27.5, 28.5, 29.4, 31.5, 50.0,

8.3, 113.9, 119.3, 124.5, 126.0, 126.8, 128.8, 129.8, 130.8, 131.5, 134.6, 158.1, 162.0, 195.7.

5

+

EI-MS, m/z: 395.19 (18.65, M +2), 394.27 (42.81, M +1), 393.38 (49.91, M ), 83.20 (100.00).

Anal. Calcd. (Found) for C H N O (393.49): C, 79.36 (79.66); H, 5.89 (5.79); N, 10.68

2

6

23

3

(10.29).

6

.1.2. Synthesis of 2-amino-7,7-dimethyl-5-oxo-4-(substituted phenyl)-1,4,5,6,7,8-

hexahydroquinoline-3-carboxylic acids 3a-c

The appropriate ortho aminonitrile 2a, 2b and 2f (0.002 mol) was heated with sulfuric acid

7

0% (6 mL) at 70 °C for 1-2 h. The reaction mixture was cooled and neutralized with 10%

NaOH. The precipitated solid was filtered and crystallized from ethanol/water (1:1) to afford the

corresponding ortho aminocarboxylic acids 3a-c.

6

.1.2.1. 2-Amino-4-(2,3-dichlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carboxylic acid (3a)

White powder, yield 60%, m.p. 192-194 °C. IR: 3452 (COOH); 3216, 3123 (NH , NH); 1692

2

1

(

COOH); 1640 (C=O). H NMR (400 MHz, DMSO-d ):  0.99 (s, 3H, CH ), 1.07 (s, 3H, CH ),

6

3

-

21 -

2

.16-2.27 (dd, J = 16.0, 16.0 Hz, 2H, CH ), 2.44 (d, J = 29.6 Hz, 2H, CH ), 2.95 (s, 1H, NH,

2 2

D O-exchangeable), 4.17 (s, 1H, C -H), 7.12 (d, J = 7.2 Hz, 1H, Ar-H), 7.37 (s, 1H, Ar-H), 7.57

2

4

1

3

(

d, J = 7.6 Hz, 1H, Ar-H), 10.21 (s, 1H, COOH, D O-exchangeable). C NMR: δ 28.9, 32.9,

2

3

3.4, 38.2, 50.4, 94.0, 112.0, 127.3, 129.2, 129.6, 131.3, 131.5, 144.7, 153.8, 170.4, 180.7, 195.0.

+

EI-MS, m/z: 381.31 (35.12, M ), 353.28 (100.00). Anal. Calcd. (Found) for C H Cl N O

1

8

18

2

3

(381.25): C, 56.71 (56.49); H, 4.76 (4.44); N, 7.35 (7.72).

6

.1.2.2. 2-Amino-4-(3,4-dichlorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carboxylic acid (3b)

White powder, yield 65%, m.p. 207-209 °C. IR: 3449 (COOH); 3218, 3124 (NH , NH); 1693

2

1

(

COOH); 1641 (C=O). H NMR (500 MHz, DMSO-d ):  0.96 (s, 3H, CH ), 1.04 (s, 3H, CH ),

6

3

2

1

.17-2.24 (m, 2H, CH ), 2.41-2.49 (m, 2H, CH ), 2.96 (s, 1H, NH, D O-exchangeable), 4.14 (s,

2 2 2

H, C -H), 7.09 (d, J = 7.5 Hz, 1H, Ar-H), 7.34 (s, 1H, Ar-H), 7.55 (d, J = 8.0 Hz, 1H, Ar-H),

4

1

3

0.20 (s, 1H, COOH, D O-exchangeable). C NMR: δ 28.5, 32.4, 32.9, 37.7, 49.9, 78.0, 111.5,

2

26.8, 128.7, 128.8, 130.8, 131.0, 144.2, 153.3, 170.0, 173.1, 194.5. Anal. Calcd. (Found) for

C H Cl N O (381.25): C, 56.71 (56.43); H, 4.76 (4.39); N, 7.35 (7.55).

1

8

18

2

3

6

.1.2.3. 2-Amino-4-(2,6-difluorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-

carboxylic acid (3c)

Pale green powder, yield 62%, m.p. 245-246 °C. IR: 3453 (COOH); 3220 (NH); 3160, 3126

1

(

NH ); 1710 (COOH); 1630 (C=O). H NMR (500 MHz, DMSO-d ):  0.88 (s, 3H, CH ), 1.01

2

6

3

(

s, 3H, CH ), 2.01 (d, J = 16.0 Hz, 1H, CH), 2.16 (d, J = 16.0 Hz, 1H, CH), 2.25-2.30 (m, 2H,

3

CH ), 3.08 (s, 1H, NH, D O-exchangeable), 4.48 (s, 1H, C -H), 6.98 (s, 2H, Ar-H), 7.25 (s, 1H,

2

4

1

3

Ar-H), 10.19 (s, 1H, COOH, D O-exchangeable). C NMR: δ 26.5, 28.7, 32.2, 35.6, 50.1, 78.0,

2

1

8

08.5, 111.6 (d, J = 25.0 Hz), 119.7 (t, J = 17.9 Hz), 128.6 (t, J = 9.5 Hz), 153.0, 159.6 (d, J =

+

.4 Hz), 161.5 (d, J = 9.5 Hz), 169.3, 194.3. EI-MS, m/z: 348.23 (28.79, M ), 231.30 (100.00).

Anal. Calcd. (Found) for C H F N O (348.35): C, 62.06 (62.41); H, 5.21 (5.50); N, 8.04

1

8

18

2

3

(8.42).

6

.1.3.

Synthesis

of

ethyl

N-(4-aryl-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinolin-2-yl)formimidates 4a-h

A solution of ortho aminonitrile 2a-d, 2f-h and 2j (0.001 mol) in triethyl orthoformate (10

mL) was refluxed for 7-10 h. Excess triethyl orthoformate was evaporated under reduced

pressure and the solid attained was crystallized from ethanol to produce the ethyl formimidates

4

a-h.

-

22 -

6

.1.3.1.

hexahydroquinolin-2-yl)formimidate (4a)

Pink crystals, yield 60%, m.p. 204-206 °C. H NMR (500 MHz, DMSO-d ):  0.99 (s, 3H, CH ),

Ethyl

N-(4-(2,3-dichlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

1

6

3

1

.04 (s, 3H, CH ), 1.28 (t, J = 7.5 Hz, 3H, OCH CH ), 2.12 (d, J = 16.5 Hz, 1H, CH), 2.25 (d, J

3 2 3

=

16.0 Hz, 1H, CH), 2.58 (s, 2H, CH ), 4.27-4.30 (q, J = 7.5 Hz, 2H, OCH CH ), 5.00 (s, 1H,

2 2 3

C -H), 7.28 (d, J = 7.5 Hz, 1H, Ar-H), 7.34 (t, J = 7.5 Hz, 1H, Ar-H), 7.53-7.54 (dd, J = 1.5, 1.5

4

1

3

Hz, 1H, Ar-H), 8.56 (s, 1H, N=CH). C NMR: δ 14.1, 27.1, 28.2, 31.9, 33.3, 49.9, 64.2, 80.4,

1

4

10.7, 116.6, 128.6, 129.5, 129.6, 130.5, 132.1, 156.6, 162.3, 163.3, 163.8, 195.7. EI-MS, m/z:

+

22.22 (42.81, M +4), 420.21 (18.88, M +2), 418.25 (29.43, M ), 83.13 (100.00). Anal. Calcd.

(

Found) for C H Cl N O (418.32): C, 60.30 (60.54); H, 5.06 (5.39); N, 10.05 (10.44).

2

1

21

2

3

2

6

.1.3.2.

Ethyl

N-(4-(3,4-dichlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinolin-2-yl)formimidate (4b)

Pink crystals, yield 63%, m.p. 205-207 °C. H NMR (500 MHz, DMSO-d ):  0.98 (s, 3H, CH ),

1

6

3

1

.04 (s, 3H, CH ), 1.28 (t, J = 7.0 Hz, 3H, OCH CH ), 2.11 (d, J = 16.0 Hz, 1H, CH), 2.25 (d, J

3 2 3

=

16.5 Hz, 1H, CH), 2.56 (s, 2H, CH ), 4.28-4.29 (q, J = 7.0 Hz, 2H, OCH CH ), 4.89 (s, 1H, C -

2 2 3 4

H), 7.33 (d, J = 8.0 Hz, 1H, Ar-H), 7.40 (d, J = 7.5 Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 8.55 (s,

1

H, N=CH). Anal. Calcd. (Found) for C H Cl N O (418.32): C, 60.30 (60.19); H, 5.06 (5.36);

21 21 2 3 2

N, 10.05 (10.31).

6

.1.3.3.

Ethyl

N-(4-(2,6-dichlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinolin-2-yl)formimidate (4c)

1

White crystals, yield 72%, m.p. 215-216 °C. H NMR (500 MHz, DMSO-d ):  0.99 (s, 3H,

6

CH ), 1.04 (s, 3H, CH ), 1.28 (t, J = 7.0 Hz, 3H, OCH CH ), 2.09 (d, J = 15.5 Hz, 1H, CH),

3

2

3

2

.24-2.28 (dd, J = 5.0, 5.5 Hz, 1H, CH), 2.45 (d, J = 17.5 Hz, 1H, CH), 2.50 (d, J = 18.0 Hz, 1H,

CH), 4.28-4.32 (q, J = 7.0 Hz, 2H, OCH CH ), 5.41 (s, 1H, C -H), 7.28-7.33 (m, 1H, Ar-H),

2

3

4

1

3

7

2

1

6

.38-7.45 (m, 1H, Ar-H), 7.49-7.52 (m, 1H, Ar-H), 8.57 (s, 1H, N=CH). C NMR: δ 14.3, 27.6,

8.7, 32.4, 37.2, 50.4, 64.7, 80.8, 111.2, 117.1, 128.9, 129.9, 131.0, 132.5, 157.1, 162.8, 164.3,

96.3. Anal. Calcd. (Found) for C H Cl N O (418.32): C, 60.30 (60.50); H, 5.06 (5.41); N,

2

1

21

2

3

2

0.05 (10.32).

.1.3.4. Ethyl

N-(4-(2-chloro-6-fluorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinolin-2-yl)formimidate (4d)

Pink crystals, yield 63%, m.p. 196-197 °C. IR: 3448 (NH); 2209 (C≡N); 1668 (C=O). H NMR

500 MHz, DMSO-d ):  0.95 (s, 3H, CH ), 1.04 (s, 3H, CH ), 1.29 (t, J = 7.0 Hz, 3H,

1

(

6

3

OCH CH ), 2.08-2.12 (d, J = 16.5 Hz, 1H, CH), 2.28 (d, J = 15.5 Hz, 1H, CH), 2.43-2.46 (m,

2

3

-

23 -

1

H, CH), 2.59 (d, J = 17.5 Hz, 1H, CH), 4.28-4.32 (q, J = 7.0 Hz, 2H, OCH CH ), 5.08 (s, 1H,

2 3

1

3

C -H), 7.18-7.36 (m, 3H, Ar-H), 8.58 (s, 1H, N=CH). C NMR: δ 13.8, 26.3, 28.5, 31.1, 31.8,

4

1

9.8, 56.0, 64.2, 78.2, 108.2, 109.8, 114.8 (d, 100.0 Hz), 116.7, 126.1 (d, 120.0 Hz), 130.0 (d,

0.7 Hz), 133.9, 156.2-157.8 (m), 162.2, 163.9 (d, 11.0 Hz), 195.7. EI-MS, m/z: 404.32 (1.89,

+

M +2), 401.69 (5.32, M ), 55.13 (100.00). Anal. Calcd. (Found) for C H ClFN O (401.87): C,

2

1

21

3

2

6

2.76 (62.42); H, 5.27 (5.36); N, 10.46 (10.30).

.1.3.5. Ethyl N-(3-cyano-4-(2,6-difluorophenyl)-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

6

hexahydroquinolin-2-yl)formimidate (4e)

White powder, yield 59%, m.p. 192-193 °C. IR: 3392 (NH); 2190 (C≡N); 1668 (C=O). H NMR

500 MHz, DMSO-d ):  0.93 (s, 3H, CH ), 1.05 (s, 3H, CH ), 1.29 (t, J = 7.5 Hz, 3H,

1

(

6

3

OCH CH ), 2.10 (d, J = 16.5 Hz, 1H, CH), 2.29 (d, J = 16.5 Hz, 1H, CH), 2.46 (d, J = 18.0 Hz,

2

3

1

H, CH), 2.60 (d, J = 18.0 Hz, 1H, CH), 4.28-4.32 (q, J = 7.5 Hz, 2H, OCH CH ), 4.85 (s, 1H,

2 3

1

3

C -H), 7.07 (t, J = 9.0 Hz, 2H, Ar-H), 7.33-7.39 (m, 1H, Ar-H), 8.56 (s, 1H, N=CH). C NMR: δ

4

1

3.8, 26.2, 28.5, 30.7, 31.9, 49.8, 64.3, 78.8, 109.3, 111.9 (d, J = 23.9 Hz), 116.9, 117.2 (t, J =

6.8 Hz), 130.0 (t, J = 10.8 Hz), 157.1, 159.7, 161.7, 162.2, 163.9, 195.7. EI-MS, m/z: 387.10

+

(

1.78, M +2), 386.29 (7.45, M +1), 385.25 (32.80, M ), 383.26 (100.00). Anal. Calcd. (Found)

for C H F N O (385.41): C, 65.44 (65.12); H, 5.49 (5.26); N, 10.90 (10.61).

2

1

21

2

3

2

6

.1.3.6.

Ethyl

N-(4-((1,1'-biphenyl)-4-yl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinolin-2-yl)formimidate (4f)

1

Yellow crystals, yield 65%, m.p. 180-182 °C. H NMR (500 MHz, DMSO-d ):  0.99 (s, 3H,

6

CH ), 1.05 (s, 3H, CH ), 1.29 (t, J = 6.5 Hz, 3H, OCH CH ), 2.16 (d, J = 16.0 Hz, 1H, CH), 2.27

3

2

3

(

d, J = 16.5 Hz, 1H, CH), 2.60 (s, 2H, CH ), 4.28-4.32 (q, J = 7.0 Hz, 2H, OCH CH ), 4.46 (s,

2 2 3

1

8

1

4

H, C -H), 7.31-7.36 (m, 3H, Ar-H), 7.45 (t, J = 8.0 Hz, 2H, Ar-H), 7.61-7.64 (m, 4H, Ar-H),

4

1

3

.56 (s, 1H, N=CH). C NMR: δ 13.9, 27.1, 28.2, 31.8, 31.9, 50.0, 64.1, 82.3, 111.5, 117.3,

26.7, 126.9, 127.5, 128.4, 129.0, 139.1, 139.7, 142.0, 156.1, 162.0, 163.1, 195.8. EI-MS, m/z:

+

28.39 (4.07, M +2), 427.32 (21.08, M +1), 426.32 (100.00, M ). Anal. Calcd. (Found) for

C H N O (425.53): C, 76.21 (76.50); H, 6.40 (6.20); N, 9.87 (9.50).

2

7

27

3

2

6

.1.3.7.

Ethyl

N-(4-([1,3]benzodioxol-4-yl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-

hexahydroquinolin-2-yl)formimidate (4g)

1

White crystals, yield 60%, m.p. 220-221 °C. H NMR (500 MHz, DMSO-d ):  0.96 (s, 3H,

6

CH ), 1.04 (s, 3H, CH ), 1.28 (t, J = 7.5 Hz, 3H, OCH CH ), 2.11 (d, J = 15.5 Hz, 1H, CH), 2.26

3

2

3

(

d, J = 16.5 Hz, 1H, CH), 2.58 (d, J = 17.5 Hz, 2H, CH ), 4.27-4.31 (q, J = 7.5 Hz, 2H,

2

OCH CH ), 4.47 (s, 1H, C -H), 5.86 (s, 1H, Dioxole-H), 6.05 (s, 1H, Dioxole-H), 6.69-6.71 (dd,

2

3

4

-

24 -

J = 2.0, 1.5 Hz, 1H, Ar-H), 6.78-6.80 (m, 2H, Ar-H), 8.52 (s, 1H, N=CH). Anal. Calcd. (Found)

for C H N O (393.44): C, 67.16 (67.39); H, 5.89 (5.51); N, 10.68 (10.42).

2

23

3

4

6

2

.1.3.8. Ethyl N-(4-(anthracen-9-yl)-3-cyano-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinolin-

-yl)formimidate (4h)

1

Orange crystals, yield 80%, m.p. 250-252 °C. H NMR (500 MHz, DMSO-d ):  0.87 (s, 3H,

6

CH ), 1.04 (s, 3H, CH ), 1.27 (t, J = 7.5 Hz, 3H, OCH CH ), 1.92 (d, J = 16.0 Hz, 1H, CH), 2.14

3

2

3

(

d, J = 16.5 Hz, 1H, CH), 2.63-2.65 (dd, J = 9.0, 9.0 Hz, 2H, CH ), 4.26-4.31 (q, J = 7.5 Hz, 2H,

2

OCH CH ), 6.24 (s, 1H, C -H), 7.45-7.47 (m, 2H, Ar-H), 7.53-7.61 (m, 2H, Ar-H), 8.09-8.13

2

3

4

1

3

(

m, 3H, Ar-H), 8.58 (s, 1H, N=CH), 8.67-8.71 (m, 2H, Ar-H). C NMR: δ 14.1, 26.9, 28.4, 31.6,

3

1

5.5, 50.0, 64.2, 82.7, 113.1, 116.8, 124.5, 124.6, 125.1, 128.9, 129.1, 130.9, 131.5, 132.6,

+

55.7, 161.9, 162.5, 195.6. EI-MS, m/z: 451.33 (5.32, M +2), 450.36 (20.40, M +1), 449.50

+

(

20.40, M ), 83.08 (100.00). Anal. Calcd. (Found) for C H N O (449.55): C, 77.48 (77.22); H,

2

9

27

3

2

6

.05 (6.32); N, 9.35 (9.63).

6

.1.4. Synthesis of 4-amino-5-aryl-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-b]quinolin-

(7H)-ones 5a-f

A mixture of compound 4a-e and 4h (0.005 mol) and ammonia solution 35% (10 mL) in

absolute ethanol (15 mL) was refluxed for 10-12 h. The solvent was evaporated under reduced

pressure, and the obtained solid was triturated with ice-water, filtered, washed with water, dried

and crystallized from ethanol to yield the pyrimidoquinolines 5a-f.

6

.1.4.1.

4-Amino-5-(2,3-dichlorophenyl)-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-

b]quinolin-6(7H)-one (5a)

1

White crystals, yield 89%, m.p. 227-228 °C. IR: 3478, 3300 (NH ); 3152 (NH); 1659 (C=O). H

2

NMR (500 MHz, DMSO-d ):  0.91 (s, 3H, CH ), 1.05 (s, 3H, CH ), 2.08 (d, J = 16.0 Hz, 1H,

6

3

CH), 2.30 (d, J = 16.5 Hz, 1H, CH), 2.42-2.49 (m, 1H, CH), 2.66 (d, J = 17.5 Hz, 1H, CH), 5.20

(s, 1H, C -H), 6.36 (s, 1H, NH, D O-exchangeable), 7.02 (t, J = 9.0 Hz, 2H, Ar-H), 7.26-7.32

5 2

1

3

(

m, 1H, Ar-H), 8.11 (s, 1H, Pyrimidine-H). C NMR: δ 22.7, 26.1, 28.7, 31.9, 49.9, 94.3, 110.6,

17.0, 117.2, 129.5, 129.6, 129.7, 156.6, 159.7, 161.6, 162.8, 165.3, 195.7. EI-MS, m/z: 393.10

1

+

(

1.52, M +4), 391.10 (7.49, M +2), 389.00 (11.43, M ), 167.90 (100.00). Anal. Calcd. (Found)

for C H Cl N O (389.28): C, 58.62 (58.44); H, 4.66 (4.33); N, 14.39 (14.65).

1

9

18

2

4

-

25 -

6

.1.4.2.

4-Amino-5-(3,4-dichlorophenyl)-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-

b]quinolin-6(7H)-one (5b)

1

White crystals, yield 92%, m.p. 232-233 °C. IR: 3478, 3313 (NH ); 3107 (NH); 1657 (C=O). H

2

NMR (500 MHz, DMSO-d ):  0.89 (s, 3H, CH ), 1.03 (s, 3H, CH ), 2.12 (d, J = 15.5 Hz, 1H,

6

3

CH), 2.31 (d, J = 16.0 Hz, 1H, CH), 2.55-2.65 (dd, J = 16.5, 16.0 Hz, 2H, CH ), 4.92 (s, 1H, C -

2

5

H), 7.05 (s, 2H, NH , D O-exchangeable), 7.15 (d, J = 6.0 Hz, 1H, Ar-H), 7.50 (d, J = 4.0 Hz,

2

1

3

1

5

H, Ar-H), 7.72 (s, 1H, Ar-H), 8.09 (s, 1H, Pyrimidine-H). C NMR: δ 26.9, 29.0, 31.8, 32.4,

0.4, 96.3, 113.9, 128.5, 129.8, 130.8, 130.9, 142.5, 144.3, 157.2, 161.5, 163.1, 164.9, 196.3. EI-

+

MS, m/z: 390.60 (16.60, M +2), 388.87 (29.82, M ), 167.51 (100.00). Anal. Calcd. (Found) for

C H Cl N O (389.28): C, 58.62 (58.46); H, 4.66 (4.39); N, 14.39 (14.79).

1

9

18

2

4

6

.1.4.3.

4-Amino-5-(2,6-dichlorophenyl)-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-

b]quinolin-6(7H)-one (5c)

1

White crystals, yield 80%, m.p. 227-228 °C. IR: 3467, 3385 (NH ); 3164 (NH); 1657 (C=O). H

2

NMR (500 MHz, DMSO-d ):  0.97 (s, 3H, CH ), 1.05 (s, 3H, CH ), 2.06 (d, J = 16.0 Hz, 1H,

6

3

CH), 2.27 (d, J = 16.0 Hz, 1H, CH), 2.51 (d, J = 14.0 Hz, 1H, CH), 2.62 (d, J = 18.0 Hz, 1H,

CH), 5.51 (s, 1H, C -H), 6.15 (s, 1H, NH, D O-exchangeable), 7.28 (t, J = 8.0 Hz, 1H, Ar-H),

5

2

1

3

7

2

1

6

.35-7.36 (m, 1H, Ar-H), 7.52-7.54 (m, 1H, Ar-H), 8.12 (s, 1H, Pyrimidine-H). C NMR: δ

6.5, 28.6, 31.0, 31.6, 50.0, 93.9, 110.2, 128.6, 131.0, 134.5, 136.0, 156.8, 161.9, 162.5, 165.4,

95.7. Anal. Calcd. (Found) for C H Cl N O (389.28): C, 58.62 (58.32); H, 4.66 (4.29); N,

1

9

18

2

4

4.39 (14.67).

.1.4.4. 4-Amino-5-(2-chloro-6-fluorophenyl)-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-

b]quinolin-6(7H)-one (5d)

1

White crystals, yield 79%, m.p. 254-257 °C. IR: 3468, 3387 (NH ); 3157 (NH); 1658 (C=O). H

2

NMR (500 MHz, DMSO-d ):  0.93 (s, 3H, CH ), 1.05 (s, 3H, CH ), 2.08 (d, J = 16.5 Hz, 1H,

6

3

CH), 2.29 (d, J = 16.0 Hz, 1H, CH), 2.52 (d, J = 14.0 Hz, 1H, CH), 2.65 (d, J = 18.0 Hz, 1H,

CH), 5.28 (s, 1H, C -H), 6.23 (s, 1H, NH, D O-exchangeable), 7.17 (s, 1H, Ar-H), 7.28-7.29 (m,

5

2

1

3

2

H, Ar-H), 8.12 (s, 1H, Pyrimidine-H). C NMR: δ 26.3, 28.6, 30.7, 31.8, 49.9, 94.2, 110.6 (d, J

15.9 Hz), 115.1 (d, J = 24.0 Hz), 125.7, 126.2, 128.7, 129.9 (d, J = 10.3 Hz), 133.7, 156.7,

=

+

1

61.6, 162.6, 165.2, 195.7. EI-MS, m/z: 375.22 (12.91, M +2), 374.25 (9.87, M +1), 373.20

+

(

38.92, M ), 338.23 (100.00). Anal. Calcd. (Found) for C H ClFN O (372.83): C, 61.21

1

9

18

4

(61.59); H, 4.87 (4.52); N, 15.03 (15.42).

-

26 -

6

.1.4.5.

4-Amino-5-(2,6-difluorophenyl)-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-

b]quinolin-6(7H)-one (5e)

1

White crystals, yield 86%, m.p. 275-277 °C. IR: 3445, 3332 (NH ); 3134 (NH); 1659 (C=O). H

2

NMR (400 MHz, DMSO-d ):  0.95 (s, 3H, CH ), 1.07 (s, 3H, CH ), 2.10 (d, J = 16 Hz, 1H,

6

3

CH), 2.30 (d, J = 16.0 Hz, 1H, CH), 2.54-2.69 (m, 2H, CH ), 5.22 (s, 1H, C -H), 6.58 (s, 1H,

2

5

NH), 7.32 (t, J = 8.0 Hz, 1H, Ar-H), 7.51 (d, J = 8.0 Hz, 1H, Ar-H), 7.70 (d, J = 7.2 Hz, 1H, Ar-

1

3

H), 8.12 (s, 1H, Pyrimidine-H). C NMR: δ 27.0, 29.1, 32.2, 33.7, 50.4, 95.3, 111.9 (d, J = 25.0

Hz), 128.2 (d, J = 11.0 Hz), 129.8, 130.8 (d, J = 15.0 Hz), 132.5, 132.9, 141.3, 157.2 (d, J = 7.4

+

Hz), 161.9 (d, J = 7.1 Hz), 163.1, 163.2, 165.3, 196.2. EI-MS, m/z: 357.17 (1.46, M +1), 356.21

+

(

7.99, M ), 55.15 (100.00). Anal. Calcd. (Found) for C H F N O (356.14): C, 64.04 (64.41); H,

1

9

18

2

4

5

.09 (5.43); N, 15.72 (15.40).

6

.1.4.6. 4-Amino-5-(anthracen-9-yl)-8,8-dimethyl-5,8,9,10-tetrahydropyrimido[4,5-b]quinolin-

(7H)-one (5f)

1

Yellow crystals, yield 83%, m.p. 234-236 °C. H NMR (500 MHz, DMSO-d ):  0.80 (s, 3H,

6

CH ), 1.05 (s, 3H, CH ), 1.93 (d, J = 16.5 Hz, 1H, CH), 2.17 (d, J = 16.5 Hz, 1H, CH), 2.63-2.74

3

(

dd, J = 17.9, 17.5 Hz, 2H, CH ), 6.46 (s, 1H, C -H), 7.40-7.42 (m, 2H, Ar-H), 7.59 (t, J = 8.0

2 5

Hz, 1H, Ar-H), 7.72 (t, J = 8.0 Hz, 1H, Ar-H), 8.04-8.08 (m, 3H, Ar-H), 8.15 (d, J = 8.0 Hz, 1H,

1

3

Ar-H), 8.60 (s, 1H, Pyrimidine-H), 9.12 (d, J = 9.0 Hz, 1H, Ar-H). C NMR: δ 26.5, 28.5, 28.7,

3

1

1.6, 50.1, 98.0, 114.1, 122.8, 124.1, 125.2, 126.8, 127.3, 128.6, 130.6, 132.3, 134.6, 156.4,

+

60.8, 163.4, 195.9. EI-MS, m/z: 421.21 (59.01, M +1), 419.71 (33.16, M ), 167.51 (100.00).

Anal. Calcd. (Found) for C H N O (420.52): C, 77.12 (77.43); H, 5.75 (5.40); N, 13.32

2

7

24

4

(13.70).

6

.1.5. Synthesis of 3-amino-4-imino-8,8-dimethyl-5-(substituted phenyl)-4,5,7,8,9,10-

hexahydropyrimido[4,5-b]quinolin-6(3H)-ones 6a,b

Hydrazine hydrate 99% (5 mL, excess) in ethanol (10 mL) was added dropwise with

o

vigorous stirring to a cold suspension (5-10 C) of compound 4a,b (0.005 mol) in ethanol (5

mL). The reaction mixture was stirred at room temperature for 3-5 h. The precipitated solid was

filtered and crystallized from ethanol/water (3:1) to produce the pyrimidoquinolines 6a,b.

6

.1.5.1.

3-Amino-5-(2,3-dichlorophenyl)-4-imino-8,8-dimethyl-4,5,7,8,9,10-

hexahydropyrimido[4,5-b]quinolin-6(3H)-one (6a)

1

White powder, yield 96%, m.p. 206-207 °C. IR: 3304, 3285 (NH ); 3196 (NH); 1655 (C=O). H

2

NMR (500 MHz, DMSO-d ):  0.90 (s, 3H, CH ), 1.03 (s, 3H, CH ), 2.05 (d, J = 16.5 Hz, 1H,

6

3

-

27 -

CH), 2.27 (d, J = 16.0 Hz, 1H, CH), 2.51 (d, J = 18.0 Hz, 1H, CH), 2.61 (d, J = 18.0 Hz, 1H,

CH), 5.08 (s, 1H, C -H), 5.66 (s, 2H, NH , D O-exchangeable), 6.56 (s, 1H, NH, D O-

5

2

exchangeable), 6.73 (s, 1H, NH, D O-exchangeable), 7.28-7.45 (m, 3H, Ar-H), 8.06 (s, 1H,

2

1

3

Pyrimidine-H). C NMR: δ 26.5, 28.6, 31.8, 32.9, 50.0, 98.9, 112.1, 127.2, 128.2, 129.2, 130.7,

+

1

31.9, 142.3, 150.3, 152.1, 155.0, 164.1, 195.8. EI-MS, m/z: 408.00 (1.31, M +4), 406.00 (1.90,

+

M +2), 404.00 (3.05, M ), 77.10 (100.00). Anal. Calcd. (Found) for C H Cl N O (404.30): C,

1

9

19

2

5

6.45 (56.80); H, 4.74 (4.41); N, 17.32 (17.60).

.1.5.2. 3-Amino-5-(3,4-dichlorophenyl)-4-imino-8,8-dimethyl-4,5,7,8,9,10-

6

hexahydropyrimido[4,5-b]quinolin-6(3H)-one (6b)

1

White powder, yield 90%, m.p. 200-202 °C. IR: 3303, 3275 (NH ); 3197 (NH); 1654 (C=O). H

2

NMR (500 MHz, DMSO-d ):  0.91 (s, 3H, CH ), 1.03 (s, 3H, CH ), 2.10 (d, J = 16.5 Hz, 1H,

6

3

CH), 2.28 (d, J = 16.0 Hz, 1H, CH), 2.57-2.63 (m, 2H, CH ), 4.77 (s, 1H, C -H), 5.64 (s, 2H,

2

5

NH , D O-exchangeable), 6.81 (s, 1H, NH, D O-exchangeable), 7.11-7.49 (m, 3H, Ar-H), 7.76

2

1

3

(

s, 1H, NH, D O-exchangeable), 8.04 (s, 1H, Pyrimidine-H). C NMR: δ 26.9, 29.1, 32.3, 39.4,

2

5

0.5, 90.4, 111.9, 124.1, 127.8, 129.2, 134.2, 136.4, 137.8, 150.2, 153.3, 155.5, 164.5, 196.3. EI-

+

MS, m/z: 404.86 (16.64, M +1), 379.68 (100.00). Anal. Calcd. (Found) for C H Cl N O

1

9

19

2

5

(404.30): C, 56.45 (56.71); H, 4.74 (4.39); N, 17.32 (17.59).

6

.1.6. Synthesis of 5-(2,3-dichlorophenyl)-4-imino-8,8-dimethyl-3-(substituted

benzylideneamino)-4,5,7,8,9,10-hexahydropyrimido[4,5-b]quinolin-6(3H)-ones 7a-d

A mixture of compound 6a (2.02 g, 0.005 mol), the appropriate benzaldehyde (0.005 mol)

and a catalytic amount of piperidine (0.5 mL) in ethanol (10 mL) was refluxed for 6-8 h. The

mixture was concentrated, and the precipitated solid was filtered, washed several times with cold

ethanol and crystallized from ethanol to yield the pyrimidoquinolines 7a-d.

6

.1.6.1.

hexahydropyrimido[4,5-b]quinolin-6(3H)-one (7a)

Dark yellow crystals, yield 85%, m.p. 226-227 °C. H NMR (500 MHz, DMSO-d ):  0.94 (s,

3-(Benzylideneamino)-5-(2,3-dichlorophenyl)-4-imino-8,8-dimethyl-4,5,7,8,9,10-

1

6

3

H, CH ), 1.06 (s, 3H, CH ), 2.11 (d, J = 16.5 Hz, 1H, CH), 2.33 (d, J = 16.0 Hz, 1H, CH), 2.54

3 3

(

d, J = 18.0 Hz, 1H, CH), 2.68 (d, J = 18.0 Hz, 1H, CH), 5.79 (s, 1H, C -H), 7.27 (d, J = 6.0 Hz,

5

1

H, Ar-H), 7.39-7.46 (m, 4H, Ar-H), 7.71 (s, 3H, Ar-H), 8.12 (s, 1H, Pyrimidine-H), 8.34 (s, 1H,

1

3

N=CH), 10.56 (s, 1H, NH, D O-exchangeable). C NMR: δ 26.4, 28.7, 31.8, 32.1, 49.9, 96.0,

2

1

10.9, 126.6, 126.7, 128.9, 129.2, 129.8, 132.6, 133.4, 134.2, 134.9, 137.5, 144.7, 156.7, 158.4,

-

28 -

+

1

62.5, 164.5, 195.8. EI-MS, m/z: 492.19 (31.26, M ), 222.00 (100.00). Anal. Calcd. (Found) for

C H Cl N O (492.40): C, 63.42 (63.09); H, 4.71 (4.31); N, 14.22 (14.60).

2

6

23

2

5

6

4

.1.6.2.

3-((4-Chlorobenzylidene)amino)-5-(2,3-dichlorophenyl)-4-imino-8,8-dimethyl-

,5,7,8,9,10-hexahydropyrimido[4,5-b]quinolin-6(3H)-one (7b)

1

Yellow crystals, yield 82%, m.p. 224-226 °C. H NMR (500 MHz, DMSO-d ):  0.89 (s, 3H,

6

CH ), 1.05 (s, 3H, CH ), 2.18 (d, J = 16.0 Hz, 1H, CH), 2.34 (d, J = 16.0 Hz, 1H, CH), 2.58-2.67

3

(

dd, J = 16.0, 16.0 Hz, 2H, CH ), 5.76 (s, 1H, C -H), 7.11 (d, J = 8.0 Hz, 1H, Ar-H), 7.47-7.52

2 5

(m, 4H, Ar-H), 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 8.18 (s, 1H, Pyrimidine-H), 8.38 (s, 1H, N=CH),

1

3

1

1.18 (s, 1H, NH, D O-exchangeable). C NMR: δ 26.5, 28.5, 31.0, 32.0, 49.9, 97.2, 113.6,

2

1

27.9, 128.3, 129.0, 129.4, 130.0, 130.6, 130.8, 133.2, 134.2, 143.6, 144.4, 156.8, 158.3, 162.5,

64.3, 196.0. Anal. Calcd. (Found) for C H Cl N O (525.09): C, 59.27 (59.52); H, 4.21 (4.61);

1

2

6

22

3

5

N, 13.29 (13.60).

6

4

.1.6.3.

3-((4-Bromobenzylidene)amino)-5-(2,3-dichlorophenyl)-4-imino-8,8-dimethyl-

,5,7,8,9,10-hexahydropyrimido[4,5-b]quinolin-6(3H)-one (7c)

1

Yellow crystals, yield 78%, m.p. 293-295 °C. H NMR (500 MHz, DMSO-d ):  0.94 (s, 3H,

6

CH ), 1.06 (s, 3H, CH ), 2.13 (d, J = 16.0 Hz, 1H, CH), 2.32 (d, J = 15.5 Hz, 1H, CH), 2.57 (d, J

3

=

18.0 Hz, 1H, CH), 2.69 (d, J = 18.0 Hz, 1H, CH), 5.83 (s, 1H, C -H), 7.22 (t, J = 8.5 Hz, 1H,

5

Ar-H), 7.45 (d, J = 7.0 Hz, 1H, Ar-H), 7.58-7.69 (m, 5H, Ar-H), 8.09 (s, 1H, Pyrimidine-H),

.36 (s, 1H, N=CH), 10.57 (s, 1H, NH, D O-exchangeable), 12.19 (s, 1H, NH, D O-

8

2

1

3

exchangeable). C NMR: δ 26.5, 28.6, 31.8, 33.0, 49.9, 96.3, 111.1, 122.9, 127.4, 128.5, 129.5,

1

5

30.4, 131.9, 132.2, 132.7, 133.5, 141.0, 143.4, 156.8, 158.3, 162.5, 164.7, 195.8. EI-MS, m/z:

+

70.39 (31.68, M -1), 316.80 (100.00). Anal. Calcd. (Found) for C H BrCl N O (571.30): C,

2

6

22

2

5

4.66 (54.45); H, 3.88 (3.61); N, 12.26 (12.60).

6

4

.1.6.4. 5-(2,3-Dichlorophenyl)-4-imino-3-((4-(dimethylamino)benzylidene)amino)-8,8-dimethyl-

,5,7,8,9,10-hexahydropyrimido[4,5-b]quinolin-6(3H)-one (7d)

1

Dark yellow crystals, yield 70%, m.p. 302-304 °C. H NMR (500 MHz, DMSO-d ):  0.94 (s,

6

3

H, CH ), 1.06 (s, 3H, CH ), 2.12 (d, J = 16.5 Hz, 1H, CH), 2.32 (d, J = 16.5 Hz, 1H, CH), 2.56

3 3

(

d, J = 18.0 Hz, 1H, CH), 2.68 (d, J = 18.0 Hz, 1H, CH), 2.95 (s, 6H, N(CH ) ), 5.89 (s, 1H, C -

3 2 5

H), 6.75 (d, J = 9.0 Hz, 2H, Ar-H), 7.18-7.21 (t, J = 8.0 Hz, 1H, Ar-H), 7.44 (d, J = 7.0 Hz, 1H,

Ar-H), 7.51 (d, J = 8.0 Hz, 2H, Ar-H), 7.68 (d, J = 7.5 Hz, 1H, Ar-H), 7.94 (s, 1H, Pyrimidine-

1

3

H), 8.28 (s, 1H, N=CH), 10.21 (s, 1H, NH, D O-exchangeable). C NMR: δ 26.5, 28.7, 31.8,

2

3

1

3.2, 42.7, 50.0, 95.3, 111.6, 111.9, 121.4, 127.3, 128.1, 129.0, 129.4, 130.5, 132.6, 141.3, 145.7,

+

51.3, 156.6, 158.3, 162.4, 164.7, 195.8. EI-MS, m/z: 535.84 (13.34, M +1), 533.78 (3.90, M -

-

29 -

Article Doi

Design, synthesis and biological evaluation of new series of hexahydroquinoline and fused quinoline derivatives as potent inhibitors of wild-type EGFR and mutant EGFR (L858R and T790M)

DOI: 10.1016/j.bioorg.2020.104274

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Article abstract of DOI:10.1016/j.bioorg.2020.104274

Full text of DOI:10.1016/j.bioorg.2020.104274

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