A Schmidt rearrangement-mediated synthesis of novel tetrahydro-benzo[1,4]diazepin-5-ones as potential anticancer and antiprotozoal agents

Article abstract of DOI:10.1016/j.ejmech.2017.10.024

Novel tetrahydro-5H-benzo[e][1,4]diazepin-5-ones, several of them, containing the quinoline pharmacophore, were synthesized via a Schmidt rearrangement from their corresponding 1,2,3,4-tetrahydro-4-quinolones mediated by the NaN₃/H₂SO₄ reaction conditions. Twelve of the obtained compounds were in vitro screened by the US National Cancer Institute (NCI) for their ability to inhibit 60 different human tumor cell lines, where compound 24a presented a remarkable activity against 58 of the 60 cancer cell lines, with the most important GI₅₀ values ranging from 0.047 to 8.16 μM and LC₅₀ values ranging from 9.4 to > 100 μM. Additionally, some of them were evaluated as antimalarial, antitrypanosomal and antileishmanial agents. The best antimalarial response was observed for compound 22g with an EC₅₀ = 13.61 μg/mL for Plasmodium falciparum, while compound 24d exhibited high activity against Trypanosoma cruzi. and Leishmania (V) panamensis with EC₅₀ = 2.78 μg/mL and 3.35 μg/mL respectively.

Full text of DOI:10.1016/j.ejmech.2017.10.024

Accepted Manuscript
A Schmidt rearrangement-mediated synthesis of novel tetrahydro-
benzo[1,4]diazepin-5-ones as potential anticancer and antiprotozoal agents
Daniel Insuasty, Sara M. Robledo, Iván D. Vélez, Paola Cuervo, Braulio Insuasty,
Jairo Quiroga, Manuel Nogueras, Justo Cobo, Rodrigo Abonia
PII:
S0223-5234(17)30816-4
10.1016/j.ejmech.2017.10.024
EJMECH 9816
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 June 2017
Revised Date: 9 October 2017
Accepted Date: 9 October 2017
Please cite this article as: D. Insuasty, S.M. Robledo, Ivá.D. Vélez, P. Cuervo, B. Insuasty, J. Quiroga,
M. Nogueras, J. Cobo, R. Abonia, A Schmidt rearrangement-mediated synthesis of novel tetrahydro-
benzo[1,4]diazepin-5-ones as potential anticancer and antiprotozoal agents, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.024.
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
A Schmidt rearrangement-mediated synthesis of novel tetrahydro-benzo[1,4]diazepin-5-
ones as potential anticancer and antiprotozoal agents
a
b
b
c
a
Daniel Insuasty , Sara M. Robledo , Iván D. Vélez , Paola Cuervo , Braulio Insuasty , Jairo
a
d
d
a
Quiroga , Manuel Nogueras , Justo Cobo , Rodrigo Abonia *
a
Research Group of Heterocyclic Compounds, Department of Chemistry, Universidad del Valle,
A. A. 25360 Cali, Colombia
b
PECET, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia,
PO Box 1226, Medellín, Colombia
c
Group of Studies in Synthesis and Applications of Heterocyclic Compounds, Department of
Chemistry, Universidad Nacional de Colombia
d
Department of Inorganic and Organic Chemistry, Universidad de Jaén, 23071 Jaén, Spain
*
rodrigo.abonia@correounivalle.edu.co; Tel: +(57)-2-3393248.

ACCEPTED MANUSCRIPT

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A Schmidt rearrangement-mediated synthesis of novel tetrahydro-benzo[1,4]diazepin-5-
ones as potential anticancer and antiprotozoal agents
Corresponding Author: Rodrigo Abonia, Ph.D.
Corresponding Author's Institution: Universidad del Valle
First Author: Daniel Insuasty, B.Sc.
Order of Authors: Daniel Insuasty, B.Sc.; Sara M. Robledo, Ph.D.; Iván D. Vélez, Ph.D.; Paola
Cuervo, Ph.D.; Braulio Insuasty, Ph.D.; Jairo Quiroga, Ph.D.; Manuel Nogueras, Ph.D.; Justo
Cobo, Ph.D.; Rodrigo Abonia, Ph.D.
Abstract
Novel tetrahydro-5H-benzo[e][1,4]diazepin-5-ones, several of them, containing the quinoline
pharmacophore, were synthesized via a Schmidt rearrangement from their corresponding 1,2,3,4-
3 2 4
tetrahydro-4-quinolones mediated by the NaN /H SO reaction conditions. Twelve of the
obtained compounds were in vitro screened by the US National Cancer Institute (NCI) for their
ability to inhibit 60 different human tumor cell lines, where compound 24a presented a
remarkable activity against 58 of the 60 cancer cell lines, with the most important GI50 values
ranging from 0.047 to 8.16 µM and LC50 values ranging from 9.4 to > 100 µM. Additionally,
some of them were evaluated as antimalarial, antitrypanosomal and antileishmanial agents. The
best antimalarial response was observed for compound 22g with an EC50 = 13.61 µg/mL for
Plasmodium falciparum, while compound 24d exhibited high activity against Trypanosoma
cruzi. and Leishmania (V) panamensis with EC50 = 2.78 µg/mL and 3.35 µg/mL respectively.

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Keywords: Chalcones, quinolines, Schmidt rearrangement, benzo[1,4]diazepin-5-ones,
anticancer activity, antiprotozoal activity
Highlights
•
•
•
New series of tetrahydro-5H-benzo[e][1,4]diazepin-5-ones were synthesized mediated by a
Schmidt rearrangement.
Twelve of the obtained compounds were in vitro screened by the (US-NCI) for their ability to
inhibit 60 different human tumor cell lines.
Compound 24a showed an outstanding anticancer activity with a GI50 range of 0.047 - 8.16
µM.
•
•
Compound 22g was the most active against P. falciparum.
Compound 24d exhibited high activity against T. cruzi. and L. (V) panamensis.
*
Corresponding author: Dr. Rodrigo Abonia, Facultad de Ciencias Naturales y Exactas,
Departamento de Química, Universidad del Valle, Santiago de Cali, Colombia.
E-mail: rodrigo.abonia@correounivalle.edu.co
FAX: +57-2-3393248.

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1
Introduction
Benzodiazepines [1] and their oxo-devivatives have intensely been investigated over a number of
decades in medicinal and pharmaceutical chemistry, because they have displayed important
biological and pharmacological activities as anxiolytic [2], anticonvulsant [3], sedative [4],
muscle relaxant [5], anticancer [6], antiviral [7], antimicrobial and antioxidant agents [8] and
antiprotozoal, among others [9]. Some examples of benzodiazepine derivatives with such
activities are displayed in Figure 1: thus, compounds 1 showed high selective and potent
antiparasitic activities against P. falciparum [9], compound 2 exhibited significant selective
binding affinity toward cholecystokinin CCK1 receptors [10], and in addition, we have recently
reported pyrazolo[3,4-b][1,4]diazepines
3
with antimalarial, antitrypanosomal and
antileishmanial activity [11].
On the other hand, quinolines and their oxo-derivatives have been widely studied due to their
presence in numerous natural products and structurally diverse synthetic derivatives [12]. In
medicinal chemistry, quinoline derivatives have shown broad spectrum of biological activities as
antimalarial and antimicrobial [13] (see compound 4 [8] in Fig. 1), as well as anticancer [14]
agents (see compounds 5 and 6 [15] in Fig. 1).

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Figure 1. Examples of quinoline- and diazepine-derivatives as antiproliferative agents.
The benzodiazepine derivatives can be prepared via a Schmidt rearrangement from 1,2,3,4-
tetrahydroquinolin-4-ones, which was firstly reported by Ittyerah and Mann [16] affording a
mixture of their corresponding ring-expanded lactams (i.e. tetrahydrobenzo[1,4]diazepin-5-ones
and tetrahydrobenzo[1,5]diazepin-2-ones), through migration of an alkyl or an aryl group,
respectively. The Schmidt rearrangement is a well-known type of reaction that involves the
addition of hydrazoic acid to a carbonyl compound to cause insertion of a nitrogen atom into a
C-C bond of a chain or ring. Depending on the starting material (i.e. carboxylic acids, aldehydes
o ketones), this rearrangement could furnishes amines, nitriles o amides, respectively [17]. The
scope of this reaction is going on explored in depth, so that it still represents an open reaction for
synthetic purposes [18].
Accordingly, the synthesis of new hybrid molecules containing two or more biologically active
fragments in their structures is an important topic in the fields of medicinal and pharmaceutical
chemistry. We are here describing the synthesis of new tetrahydro-5H-benzo[e][1,4]diazepin-5-
ones containing, in most cases, the quinoline pharmacophore in their structures. These new
hybrid derivatives were prepared starting from their corresponding tetrahydroquinolin-4-one
derivatives and mediated by NaN /H SO via a Schmidt rearrangement. These hybrids were
3
2
4

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tested against a panel of 60 human cancer cell lines in the Developmental Therapeutics Program
(DTP) at the National Cancer Institute (NCI) and also tested for their antimalarial,
antileishmanial and antitrypanozomal activities.
2
Results and discussion
2
.1 Chemistry
A series of tetrahydroquinolin-4-ones were prepared to test the Schmidt rearrangement in order
to obtain their corresponding benzodiazepine derivatives [16b,17]. For that, we initially planned
the synthesis of a series of 2-aminochalcones 11-13 as precursors of tetrahydroquinolin-4-ones
1
4-16 after the corresponding cyclization (Scheme 1).
Scheme 1. General procedure for the synthesis of the key tetrahydroquinolin-4-one
intermediates 14-16.
Firstly, the starting 2-aminochalcones 11-13 were readily obtained via a Claisen-Schmidt
reaction in 72-95% yield by heating alcoholic solutions of equimolar amounts of o-

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aminoacetophenones 7 and the corresponding aldehydes 8-10, promoted by NaOH (see
Experimental Section) [19]. The non-commercial quinoline-aldehydes 9-10 had to be prepared
(40-85% yield) following our previously reported methodology [20], which involves a Meth-
Cohn type reaction [21], (Scheme 2).
Scheme 2. General methodology for the synthesis of the non-commercial quinoline-aldehydes 9-
1
0.
Chalcones 11 were previously reported [19], and the structures of the new 2-aminochalcones 12-
3 confirmed by IR, NMR, and mass spectrometry. Taking as a model the 2-aminochalcone 12a
to illustrate the main spectroscopic features for this series: thus, its IR spectrum shows two broad
1
-1
-1
absorption bands at 3381 cm and 3284 cm assigned to the NHC=O and NH
2
functionalities
-1
(
NHC=O band is overlapped with the NH antiymmetric stretching band at 3381 cm ), as well
2
-1
-1
as, two absorption bands at 1660 cm and 1645 cm corresponding to conjugated C=O
stretching in the lactam and ketone functionalities, respectively. The most relevant signals in the
1
H NMR spectrum of compound 12a correspond to two doublets at 7.72 and 8.36 ppm,
3
integrating each one for 1H with coupling constant J = 15.5 Hz assigned to the vinylic protons
α β
(H and H , Scheme 1), which confirms the E configuration for the carbon–carbon double bond

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of these chalcones. The molecular ion peak (m/z = 290) in the mass spectrum, also agrees with
the structure of chalcone 12a.
Subsequently, the intramolecular cyclization of chalcones 11-13 was carried out mediated by
®
Amberlyst -15 [19], that afforded the corresponding tetrahydroquinolin-4-ones 14-16 in 58-94%
yield, as described in Scheme 1 (see Experimental Section). Characterization of quinolin-4-ones
1
4 was previously reported [19] and the new 1,2,3,4-tetrahydroquinolin-4-ones 15-16 were
characterized by means of their FT-IR, NMR, and mass spectra. Taking now compound 15a as a
model to illustrate the main spectroscopic features for this series: In its IR spectrum it can be
−
1
observed a broad absorption band at 3319 cm associated to the stretching vibration of the N-H
1
bond overlapped with the H-NC=O band. The main feature of the H NMR spectrum of 15a
correspond to two double-doublets of the methylene protons (H /H ) in C-3 at 2.70/2.88 ppm
A
B
2
3
3
(
JAB = 16.1 Hz, J = 3.8 Hz and J = 11.1 Hz ppm), and a double-doublet of the stereogenic
AX BX
3
3
proton H in C-2 at 4.78 ppm ( J = 11.1 Hz and J = 4.5 Hz), confirming the generation of
X
BX
AX
an ABX spin system associated with the dihydroquinolin-4-one ring formed.
Once the key intermediates tetrahydroquinolin-4-ones 14-16 were obtained, compound 14a was
used in order to optimized reaction conditions to get the benzodiazepines 20 mediated by a
Schmidt rearrangement. The best conditions were obtained when tetrahydroquinolin-4-one 14a
(
0.29 g, 1.0 mmol) was dissolved in dichloromethane (5 mL) and conc. H SO (70 µL) was
2 4
added under stirring, and to the intense red solution obtained, sodium azide (3.0 mmol) was
added over a period of 30 min and the stirring was continued for 3 h at room temperature. The
1
reaction mixture afforded a solid residue that was easily isolated by filtration, but the H-NMR
analysis confirmed the formation of two new products corresponding to the expected 5-oxo-

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benzo[1,4]-diazepine 20a and 2-oxo-benzo[1,5]diazepine 21a derivatives in a 90:10 ratio,
respectively, Scheme 3.
Scheme 3. Optimized reaction conditions to diazepine derivatives 20a/21a from quinolin-4-one
1
4a via Schmidt rearrangement.
The regioisomers 20a and 21a were separated by column chromatography using silica gel and
DCM/MeOH (30:1) as eluent (See Experimental Section), and afterwards unambiguously
characterized. The above established procedure was extended to the remaining
tetrahydroquinolin-4-ones 14b-d, 15 and 16. Reactions proceeded with the same behavior and in
all cases, [1,4]- regioisomers 20, 22 and 24 corresponded to the major components of the
1
reaction mixtures (determined by H-NMR), Table 1. In some cases, simple purification of the
major derivatives was achieved by recrystallization from acetonitrile.
Table 1. Synthesis of the novel and diversely substituted tetrahydro-benzo[1,4]diazepin-5-ones
2
0, 22 and 24, via Schmidt rearrangement of the tetrahydroquinolin-4-ones 14, 15 and 16,
respectively.

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Structures of the new compounds 20, 22 and 24 were completely established by spectral data
1
13
(
IR, H, C NMR, DEPT-135, HSQC, HMBC and NOESY experiments, mass spectrometry and
microanalysis) and are summarized in the experimental section. The [1,5]- regioisomers 21, 23
and 25 were formed in less than 12% ratio with R values very close to those for 20, 22 and 24.
f
Because of that, in most cases they could not be isolated with enough purity for characterization.
1
Hence, their presence were determined by H-NMR from their crude reaction mixtures.
Particularly, product 24d and its minority regioisomer 25d formed in a 93:7 ratio, respectively,
represent one of the few cases where it was possible to separate them, as it can be observed in
1
their corresponding H-NMR spectra in Figure 2.

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1
Figure 2. Comparison of the H NMR spectra of the crude and the isolated benzodiazepines
(24/25)d. (a) crude mixture, (b) pure 5-oxo-[1,4]-derivative 24d and (c) pure 2-oxo-[1,5]-
derivative 25d. Integ: integral.
Taking this new compound 24d as a model to illustrate the main spectroscopic features for the
series of the major benzo[1,4]diazepin-5-ones: thus, in its IR spectrum it is observed a broad
−
1
absorption band at 3180 cm associated to the both overlapped –NH and NH-C=O
−
1
functionalities and an absorption band at 1647 cm assigned to the 5-C=O lactamic group. In the
1
H NMR spectrum the signals for the protons on the diastereotopic center C-3 appears as a
multiplet at 3.66-3.73 ppm, the H-2 proton is observed as a double-doublet at 5.18 ppm, while
the 4-NH-C=O proton appears as a broad triplet at 6.46 ppm. The 1-NH proton is not observed
due to a rapid chemical proton exchange. The fact that 4-NH-C=O couples with H-3 protons
(appearing as a triplet), support that such structure effectively corresponds to the 5-oxo-[1,4]-
derivative 24d and not to its 2-oxo-[1,5]-regioisomer 25d, as shown in Fig. 2. Additionally,

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single crystals of compound 24d suitable for X-ray diffraction were grown from a solution of
DMF at ambient temperature, Figure 3. (See also Supplementary Material). As expected, the
crystallographic molecular structure confirmed the formation of the 5-oxo-[1,4]-derivative 24d
without any ambiguity.
Figure 3. Displacement ellipsoid plots (50% probability level) of the asymmetric unit for the
racemic 24d•chloroform solvate (Mercury 3.8).
In the same way, the IR spectrum of compound 25d showed a broad absorption band at 3204
−
1
cm associated to the both overlapped –NH and NH-C=O functionalities and an absorption band
−
1
1
at 1668 cm assigned to the 2-C=O lactamic group. In the H NMR spectrum for compound
2
5d, two double-doublets of the methylene protons (H /H ) in C-3 at 2.95/3.06 ppm are
A
B
observed, the H-4 proton is observed as a double-doublet at 5.45 ppm, while the 1-NH-C=O
proton appears as a broad singlet at 7.59 ppm. The 5-NH proton is not observed for the same
reason that 1-NH in 24d. In this case the 1-NH-C=O appears uncoupled supporting the 2-oxo-
[1,5]-structure proposed for this regioisomer, as shown in Fig. 2.

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According to the general mechanistic sequence proposed for this type of rearrangement [18],
products 20, 22 and 24 along with their minority regioisomers 21, 23 and 25, respectively, could
be formed as shown in Scheme 4.
Scheme 4. Proposed mechanistic sequence for the Schmidt rearrangement of the
tetrahydroquinolin-4-ones 14, 15 and 16.
As shown in Scheme 4, rearrangement proceed through two competing carbon atom migrations
toward the exocyclic nitrogen atom (i.e. via A or B in the key species 29). Migration of the CH2
3
sp carbon atom (via A) should afford the main products 20, 22 and 24. Contrary, migration of
2
the aromatic =C sp carbon atom (via B) affords the minority products 21, 23 and 25. The
3
2
,
relative higher stability of the sp carbenium ion against its analog sp which are involved in the
migration process [16b,17b], as well as, the relative higher stability of the species 30-A (by its
resonance with the Ar ring) in comparison with 30-B are in agreement with the significant
preference for the formation of the [1,4]- regioisomers 20, 22 and 24 against their [1,5]-
regioisomers 21, 23 and 25, respectively.

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2
.2 Anticancer activity
As a preliminary screening, structures of all new compounds 15, 16, 20, 22 and 24 were
submitted to the Developmental Therapeutics Program (DTP) at National Cancer Institute (NCI),
Bethesda, MD (USA), for evaluation of their anticancer activity against different human cell
lines and the output from the single dose screening was analyzed by the COMPARE program
[22] (data not shown).
The two-stage screening process started with a primary in vitro evaluation of twelve of the
submitted compounds (i.e. 15a (NSC: 787542/1), 15h (NSC: 787546/1), 22a (NSC: 787544/1),
2
2c (NSC: 787543/1), 22d (NSC: 784187/1), 22f (NSC: 784190/1), 22h (NSC: 787547/1), 22i
(NSC: 787548/1), 22j (NSC: 787541/1), 24a (NSC: 784186/1), 24b (NSC: 787545/1) and 24d
(NSC: 787540/1)) selected by the Drug Evaluation Branch of the (NCI), against 60 human tumor
cell lines at a single dose of 10 µM. The 60 cell lines panel is derived from nine different human
cancer types: i.e. Leukemia, Lung, Melanoma, Colon, CNS, Ovary, Renal, Breast and Prostate.
Results for each compound were reported as a mean graph of the growth percent (GP) of the
treated cells and the obtained results are illustrated in Table 2. The data indicated a selective
influence of some compounds on proliferation of a number of cancer cells with broad-spectrum
of activity. Compound 24a was found to be highly active on MDA-MB-435 (Melanoma) with
cell promotion of -63.77% followed by SF-539 (CNS Cancer) with cell promotion of -25.50%
and M14 (Melanoma) with cell promotion of -23.13%. Compounds (15h, 24d) and 22h showing
moderate activity were highly active on SR (Leukemia) and MCF7 (Breast Cancer) with GP of

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(11.14, 18.12) and 33.37, respectively. While rest of the compounds showed less activity with an
average growth percent of 46.44 - 94.26.
Table 2. Range and mean growth % of NCI human cancer cell lines treated with selected
compounds at one-dose of 10 µM.
Mean
growth
Growth % of most
sensitive cell line
Compound
NSC code
Most sensitive cell line
1
1
2
5a
5h
2a
NSC: 787542/1
NSC: 787546/1
NSC: 787544/1
NSC: 787543/1
NSC: 784187/1
100.27
94.15
T-47D (Breast)
SR (Leukemia)
UO-31 (Renal)
UO-31 (Renal)
HT29 (Colon)
94.26
11.14
76.84
84.35
81.31
99.41
2
2c
2d
2f
2h
103.92
100.90
2
NCI-H522 (Non-Small Cell
Lung)
2
NSC: 784190/1
97.77
63.77
2
NSC: 787547/1
NSC: 787548/1
NSC: 787541/1
92.98
98.50
95.87
MCF7 (Breast)
MOLT-4 (Leukemia)
SR (Leukemia)
33.37
46.44
59.75
2
2i
2
2j
SF-539 (CNS)
M14 (Melanoma)
MDA-MB-435 (Melanoma)
NCI-H522 (Non-Small Cell
Lung)
-25.50
-23.13
-63.77
2
4a
NSC:784186/1
20.27
2
2
4b
4d
NSC: 787545/1
NSC: 787540/1
96.80
58.92
77.36
18.12
SR (Leukemia)
Subsequently, due to compound 24a displayed considerable antiproliferative activity, it was
selected for an advanced assay against the full 60 human cell lines panel at five concentrations at
1
0-fold dilution (i.e. 100, 10, 1.0, 0.1 and 0.001 µM) for a 48h protocol in which sulforhodamide
B (SRB) protein assay was used to estimate cell growth according to protocols described
elsewhere [23,24]. The results were expressed as follows according to previously published
protocols: GI , which is the molar concentration of the compounds required to inhibit 50% of
5
0
the growth of cell lines (relative to untreated cells), and LC , which is a parameter of
5
0
cytotoxicity that reflects the molar concentration needed to kill 50% of the cells [25]. Compound

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2
4a exhibited potent anticancer activity against all the 60 tested cancer cell lines with GI50 values
between 0.047 - 8.16 µM (Table 3). The best results were recorded on the Leukemia cell lines
with values between 0.047 to 3.05 ꢀM, where the best activity was shown against the HL–
6
0(TB) cancer cell line with GI50 = 0.047 µM and LC50 = 65.4 µM, better than
Adriamycin/Doxorubicin (NSC:123127 code) (GI = 0.12 µM and LC = 89.33 µM), the NCI
5
0
50
standard drug for this type of analysis. Also, showing significant activity against the cell line
MDA–MB–435 (Melanoma) with GI50 = 0.34 µM and LC50 = 7.6 µM followed by the cell line
A498 (Renal) with GI50 = 0.39 µM and LC50 > 100 µM and K–562 (Leukemia) with GI50 = 0.84
µM and LC > 100 M. Their LC and GI values reflect that this compound should be a lead
5
0
50
50
structure for the development of a potential antitumor agent.
Table 3. In vitro cytotoxic activities of compound 24a expressed as growth inhibition and lethal
a
concentration of cancer cell lines and compared with the standard drug Doxorubicin.
Compound
Doxorubicin
Compound
Doxorubicin
2
4a
d
24a
d
Panel cell line
Panel cell line
100 µM
100 µM
b
c
b
c
GI50
Leukemia
2.95
LC50
GI50
LC50
GI50
LC50
GI50
LC50
Non–small cell lung
CCRF–CEM
HL–60(TB)
K–562
>100
65.4
0.08
0.12
0.19
0.03
0.08
0.03
100.00
89.33
A549/ATCC
EKVX
3.64
4.76
2.99
1.92
3.13
5.16
4.50
3.11
2.39
53.0
>100
76.7
0.06
0.41
0.07
0.10
0.05
0.15
0.54
0.02
0.03
100.00
47.97
67.61
42.27
6.40
0.047
0.84
3.05
2.86
1.26
>100
>100
>100
>100
100.00
100.00
100.00
100.00
HOP–62
MOLT–4
RPMI–8226
SR
HOP–92
>100
>100
>100
>100
51.1
NCI–H226
NCI–H23
NCI–H322M
NCI–H460
NCI–H522
13.15
67.76
51.29
2.80
Colon
COLO 205
HCC–2998
HCT–116
HCT–15
HT29
2.19
3.58
1.84
1.92
3.21
3.30
23.0
>100
44.7
48.6
67.7
>100
0.18
0.26
0.08
6.46
0.12
0.27
4.33
21.68
54.58
100.00
67.45
92.68
>100
CNS
SF–268
SF–295
SF–539
4.14
1.40
2.29
>100
52.4
44.9
0.10
0.10
0.12
30.48
69.98
27.23
KM12

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SW–620
2.19
>100
0.09
58.61
SNB–19
SNB–75
U251
6.13
1.81
3.03
>100
48.3
50.1
0.04
0.07
0.04
49.77
3.30
Melanoma
2.90
LOX IMVI
MALME–3M
M14
47.3
92.0
92.2
7.6
0.07
0.12
0.18
0.25
0.21
0.08
0.14
0.12
50.35
3.97
4.05
9.57
15.92
0.49
8.15
0.74
30.62
3.58
Ovarian
1.54
IGROV1
OVCAR–3
OVCAR–4
OVCAR–5
OVCAR–8
NCI/ADR–RES
SK–OV–3
4.49
1.31
5.38
6.42
3.45
1.74
2.38
98.5
9.4
0.17
0.39
0.37
0.41
0.10
7.16
0.22
100.00
84.33
MDA–MB–435
SK–MEL–2
SK–MEL–28
SK–MEL–5
UACC–257
0.34
4.79
5.89
2.34
8.16
>100
86.1
>100
>100
>100
97.0
>100
74.30
100.00
43.25
>100
>100
100.00
100.00
Renal
7
86–0
2.84
0.39
3.92
2.83
2.50
3.34
4.93
2.41
4.65
>100
5.95
>100
75.4
90.6
73.8
67.2
0.13
0.10
0.08
0.95
0.10
0.07
0.38
0.49
51.64
1.90
Breast
A498
ACHN
CAKI-1
RXF 393
SN12C
TK–10
UO–31
MCF7
1.73
3.83
3.21
5.22
5.32
1.61
>100
90.3
0.04
0.51
0.33
0.23
0.06
0.05
64.12
34.75
85.70
21.33
85.70
2.52
100.00 MDA–MB–231/ATCC
100.00
4.69
HS 578T
BT–549
>100
>100
>100
>100
72.44
89.95
26.18
T–47D
MDA–MB–468
Prostate
3.06
3.16
PC–3
66.9
0.32
0.11
87.10
DU–145
>100
100.00
a
b
Data obtained from NCI’s in vitro disease–oriented human cancer cell lines screen in µM. GI50 was the drug
concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells
c
during the drug incubation. Determined at five concentration levels (100, 10, 1.0, 0.1, and 0.01 µM). LC50 is a
d
parameter of cytotoxicity that reflects the molar concentration needed to kill 50% of the cells. The values of
activity against human cancer cell lines displayed by Adriamycin (Doxorubicin) correspond to the reported by NCI
at highest concentration of 100 µM. Please visit: https://dtp.cancer.gov/dtpstandard/cancerscreeningdata/index.jsp.
The most active compounds were highlighted in grey.
2
.3 Cytotoxic activity in human U–937 macrophages
Encouraged by our previous results [26], the selected quinoline-based compounds 22a,c,e,f-h
and 24d,g were tested first for toxicity on U–937 human cells at four serial dilution
concentrations (200, 50, 12.5 and 3.125 µg/mL) to determine their 50% Lethal Concentration
(
LC ) (Table 4). Compounds 22a,c,g,h and 24d exhibiting LC <100 µg/mL were considered as
50 50
potentially cytotoxic, compound 22e was mildly cytotoxic (LC50 > 100 < 200 µg/mL), while
compounds 22f and 24g showed a LC > 200 µg/mL and therefore classified as potentially non-
5
0

ACCEPTED MANUSCRIPT
cytotoxic. It is worth mention that the reference drugs amphotericin B showed a LC50 = 36.0
µg/mL (potentially cytotoxic), while benznidazole had a LC = 180.0 µg/mL (mildly cytotoxic),
5
0
in comparison with compounds 22f and 24g.
Table 4. In vitro cytotoxic activity for the selected novel compounds 22a,c,e,f-h and 24d,g
a
Compound
LC50
36.3 ± 1.7
52.8 ± 5.3
192.3 ± 13.0
>200
Compound
LC50
22a
22c
22e
22g
22h
24d
24g
57.0 ± 10.8
8.3 ± 1.8
1.0 ± 0.1
>200
2
2f
b
c
Amph
36.0 ± 7.5
Benz
180 ± 37
a
Data represent the mean value ± standard deviation in µg/mL. LC50: Lethal concentration 50% of promonocitic
human cell U–937. Amph: Amphotericin B (antileishmanial drug control). Benz: Benznidazole (antitrypanosomal
drug control).
b
c
2
.4 Antiplasmodial activity
The selected compounds 22a,c,e,f-h and 24d,g compounds were screened in vitro for
antiplasmodial activity on asynchronic cultures of P. falciparum (3D7 strain, sensitive to
chloroquine) at four serial diluted concentration (100, 25, 6.25 and 1.56 µg/mL) to determine the
Effective Concentration 50 (EC ) (See Table 5). Compound 22g was the most active with EC
50
50
=
13.61 µg/mL, compounds 22d and 24g showed moderate activity with values of EC50 = 43.18
and 50.84 µg/mL, respectively, while compounds 22a,c,f,h and 24d showed less activity. It is
worth mention that compound 22g showed a better EC50 than the reference drug CQ (EC50
8.90). This finding suggest that compound 22g have potential as antimalarial drug, therefore it
=
1
could be further studied in vivo for drug development in malaria therapy. Additionally, a raw
SAR analysis shows that substituents in the quinoline aryl-fused ring of compounds 22 improved
relatively the activity in comparison with the unsubstituted derivative 22a (i.e. their EC50 are

ACCEPTED MANUSCRIPT
lower than 64.70 afforded by 22a). Moreover, the 6-Cl substituent (i.e. 22e, EC50 = 43.18) was
more favored than its 7-Cl analog (i.e. 22f, EC = 59.32), although, the 6-Br substituent in 22g
5
0
was the most favored position showing the lower EC of the series. The blocking of the NHCO
5
0
functionality diminished significantly the activity as happens with 24g (EC50 = 50.84) in
comparison with its analog 22g (EC = 13.61).
5
0
Table 5. In vitro antimalarial screening for the selected novel compounds 22a,c,e,f-h and 24d,g
a
Compound
EC50
Compound
EC50
13.61 ± 0.56
>20
2
2a
64.70 ± 13.59
60.87 ± 0.87
43.18 ± 4.45
59.32 +± 12.59
22g
22h
24d
24g
2
2c
2e
2
>20
2
2f
50.84 ± 8.89
b
CQ
18.90 ± 1.70
a
Data represent the mean value ± standard deviation in µg/mL. EC : Effective Concentration 50: The exact amount
5
0
b
of compounds that decreases 50% of intracellular parasite. CQ: Chloroquine diphosphate salt (antimalarial
control).
2
.5 Antitrypanosomal activity
Antitrypanosomal activity was screened against intracellular amastigotes of Trypanosoma cruzi
Tulahuen strain) at four serial dilutions. The activity of the selected compounds 22a,c,e,f and
4d (in terms of EC50) is summarized in Table 6. Compound 24d was highly active against T.
cruzi with EC50 = 2.68 µg/mL, showing better activity than the drug control benznidazole (EC50
10.7 µg/mL). This result suggests that compound 24d have potential as antitrypanosomal drug
and therefore could be a hit for in vivo studies to validate its potential as lead compound. The
raw SAR analysis shows that a methyl group in the quinoline aryl-fused ring (i.e. 22c, EC50
6.29) improved slightly the relative activity in comparison with the unsubstituted derivative 22a
EC50 = 48.79), but the presence of a chlorine atom diminished it (i.e. 22e, EC50 = 122.77 and
2f, EC50 = 86.01). The 7-Cl substituent in 22f is relatively more favored than its 6-Cl analog
(
2
=
=
4
(
2

ACCEPTED MANUSCRIPT
2
2e. Apparently, the blocking of the NHCO functionality along with an 8-methyl group in
structure 24d greatly improved its relative activity (i.e. EC50 = 2.68) in comparison with the
unsubstituted derivative 22a (EC = 48.79).
5
0
Table 6. In vitro testing of antitrypanosomal activity for the selected novel compounds 22a,c,e,f
and 24d
a
Compound
EC50
Compound
EC50
2
2
2a
2c
48.79 ± 5.53
46.29 ± 1.28
22f
24d
86.01 ± 15.10
2.68 ± 0.52
b
22e
122.77 ± 12.03
Benz
10.7 ± 2.0
a
Data represent the mean value ± standard deviation in µg/mL. EC : Effective Concentration 50: The exact amount
5
0
b
of compounds that decreases 50% of intracellular T. cruzi amastigotes. Benz: Benznidazole (antitrypanosomal drug
control).
2
.6 Antileishmanial activity
The selected compounds 22a,c,e,f-h and 24d,g were screened for antileishmanial activity in
intracellular amastigotes of Leishmania (V) panamensis (MHOM/CO/87/UA140–EpiR–GFP
strain). The EC50 was determined after exposure to four concentrations (Table 7), as done with
antiplasmodial and antitrypanosomal activities. Compounds 22c,e,g,h and 24d evidenced good
activity against Leishmania amastigotes with EC varying from 3.35 to 22.92 µg/mL. For active
5
0
compounds EC ≤ 25 µg/mL. The best antileishmanial activity was displayed by compound 24d
5
0
which is active at 3.35 µg/mL, while compound 24g presented moderate activity with EC50
=
3
4.19 µg/mL. Moreover, the raw SAR analysis shows that substituents in the quinoline aryl-
fused ring of compounds 22 improved the relative activity in comparison with the unsubstituted
derivative 22a. In this case, the presence of a new fused-phenyl ring was the most favored
substitution (i.e. 22h, EC = 4.98). The 7-Cl substituent in 22f is relatively more favored than its
5
0

ACCEPTED MANUSCRIPT
6
-Cl analog 22e. Finally, the blocking of the NHCO functionality along with an 8-methyl group
in structure 24d greatly improved its relative activity (i.e. EC = 3.35) in comparison with the
5
0
unsubstituted derivative 22a (EC = 110.22).
5
0
Table 7. In vitro antileishmanial activity for the selected novel compounds 22a,c,e,f-h and 24d,g
a
Compound
EC50
Compound
EC50
2
2
2
2a
2c
2e
110.22 ± 18.48
22.92 ± 3.95
19.44 ± 1.15
77.92 ± 11.29
0.05 ± 0.01
22g
22h
24d
24g
15.26 ± 1.46
4.98 ± 0.28
3.35 ± 0.33
34.19 ± 3.24
2
2f
b
Amph
a
Data represent the mean value ± standard deviation in µg/mL. EC50: Effective Concentration 50: The exact amount
of compound that decreases 50% of intracellular parasite growth. Amph: Amphotericin B (antileishmanial drug
b
control).
3
Conclusions
In summary, we have established a general and straightforward sequence for the Schmidt
rearrangement-mediated synthesis of novel tetrahydro-benzo[1,4]diazepin-5-one derivatives 20,
2
2 and 24, starting from the 2-aminochalcones 11-13 as readily access precursors. The key
Schmidt rearrangements proceeded regioselectively favoring the formation of the 5-oxo-[1,4]-
regioisomers against their competing 2-oxo-[1,5]-ones. Several of the obtained compounds
showed remarkable anticancer activity against 60 human tumor cell lines at one- and five-dose
tests performed by the US-NCI, as well as, outstanding antiprotozoal (i.e. antimalarial,
antitrypanosomal and antileishmanial) activity against Plasmodium falciparum, Trypanosoma
cruzi and Leishmania (V) panamensis strains, respectively.
4
. Experimental

ACCEPTED MANUSCRIPT
4
.1 General
Melting points were measured using a Stuart SMP3 melting point apparatus and are
uncorrected. IR spectra were recorded on a Shimadzu IRAffinity-1 Spectrophotometer in KBr
1
13
disks. H and C NMR spectra were recorded on a Bruker Avance 400 spectrophotometer
operating at 400 MHz and 100 MHz respectively, using DMSO-d and CDCl as solvents and
6
3
tetramethylsilane as internal standard. Mass spectra were run on a SHIMADZU-GCMS 2010-DI-
010 spectrometer (equipped with a direct inlet probe) operating at 70 eV. Microanalyses were
2
performed on an Agilent CHNS elemental analyzer and the values are within ± 0.4% of the
theoretical values. TLC analyses were performed on silica gel aluminum plates (Merck 60 F )
2
54
and spots visualized with ultra-violet irradiation. The starting aryl aldehydes 8, o-
aminoacetophenones 7, the precursors and reagents for the synthesis of the quinoline-aldehydes
9
and 10, and the required solvents were purchased from Sigma-Aldrich and Merck (analytical
grade reagent), and were used without further purification.
4
4
.2 Chemistry
.2.1 General procedure for the synthesis of the novel 2-aminochalcones 12-13
A mixture of o-aminoacetophenone 7a (1.2 mmol), the corresponding quinoline-aldehydes 9-10
1.0 mmol) and 20% aq NaOH (0.5 mL) in MeOH (10 mL) was heated for 8-12 h. until the
(
starting aldehydes 9-10 were not detected by TLC. The resulting precipitate was collected by
filtration under vacuum and washed with MeOH and water, to afford chalcones 12-13. No
further purification was required.

ACCEPTED MANUSCRIPT
4
.2.1.1. (E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)quinolin-2(1H)-one 12a. Yellow solid;
-1
8
9% yield; m.p. 260–261 °C. FTIR (ATR) ν (cm ): [3381, 3284] (NH , NHC=O), [1660, 1645]
2
1
(
2 x C=O), 1610, 1645. H NMR (400 MHz, DMSO-d ) δ ppm 6.60 (t, J = 7.0 Hz, 1H), 6.81 (dd,
6
J = 8.4, 1.0 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H),
.39 (s, 2H, NH ), 7.54 (t, J = 7.7 Hz, 1H), 7.71 (d, J = 1.2 Hz, 1H), 7.72 (d, J = 15.7 Hz, 1H,
CH), 7.93 (dd, J = 8.3, 1.3 Hz, 1H), 8.36 (d, J = 15.7 Hz, 1H, =CH), 8.54 (s, 1H, Quin-H),
7
2
=
1
3
1
1
2.02 (s, 1H, NHC=O). C NMR (101 MHz, DMSO-d ) δ ppm 114.5, 115.5, 117.0, 117.5,
6
19.2, 122.2, 125.3, 126.3, 128.5, 130.9, 131.4, 134.3, 136.9, 138.8, 140.4, 152.0, 161.0, 190.7.
+
EI MS (70 eV) m/z (%): 290 (M , 80), 170 (51), 158 (100), 146 (34).
4
.2.1.2.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-6-methylquinolin-2(1H)-one
12c.
-1
Yellow solid; 79% yield; m.p. 255-256 °C. FTIR (ATR) ν (cm ): [3317, 3236] (NH2, NHC=O),
1
[
1645, 1606] (2 x C=O), 1581, 1467. H NMR (400 MHz, DMSO-d ) δ ppm 2.34 (s, 3H, Quin-
6
CH ), 6.60 (t, J = 7.3 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.3
3
Hz, 1H), 7.37 (d, J = 6.3 Hz, 1H), 7.38 (s, 2H, NH ), 7.48 (s, 1H, Quin-H), 7.70 (d, J = 15.5 Hz,
2
1
H, =CH), 7.91 (d, J = 7.8 Hz, 1H), 8.37 (d, J = 15.5 Hz, 1H, =CH), 8.45 (s, 1H, Quin-H), 11.95
1
3
(
s, 1H, NHC=O). C NMR (101 MHz, DMSO-d
6
) δ ppm 20.3, 114.5, 115.0, 117.0, 117.5, 119.1,
1
25.2, 126.2, 127.8, 130.9, 131.2, 132.8, 134.2, 136.8, 137.1, 140.4, 152.0, 160.9, 190.7. EI MS
+
(
70 eV) m/z (%): 304 (M , 100), 184 (50), 173 (90) 146 (22), 120 (17).
4
.2.1.3.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-8-methylquinolin-2(1H)-one
12d.
-1
Yellow solid; 81% yield; m.p. 261–262 °C. FTIR (ATR) ν (cm ): [3377, 3284] (NH ), 3178
2
1
(
NHC=O), [1661, 1645] (2 x C=O), 1610, 1673. H NMR (400 MHz, DMSO-d
6
) δ ppm 2.49 (s,
3
H, Quin-CH ), 6.65 (t, J = 7.0 Hz, 1H), 6.85 (dd, J = 8.3, 0.9 Hz, 1H), 6.94 (s, 2H, NH ), 7.04
3 2

ACCEPTED MANUSCRIPT
(t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 7.1 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H),
7
1
1
1
.75 (d, J = 15.5 Hz, 1H, =CH), 7.90 (dd, J = 8.1, 1.3 Hz, 1H), 8.26 (s, 1H, Quin-H), 8.29 (d, J =
1
3
5.5 Hz, 1H, =CH), NHC=O not observed. C NMR (101 MHz, DMSO-d ) δ ppm 16.1, 114.1,
6
16.5, 118.2, 119.0, 120.0, 124.0, 125.0 (x 2), 125.2, 125.7, 130.14, 131.0, 133.0, 137.5, 139.2,
+
50.9, 162.3, 191.1. EI MS (70 eV) m/z (%): 304 (M , 42), 173 (100), 146 (38).
4
.2.1.4.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-6-chloroquinolin-2(1H)-one
12e.
-1
Yellow solid; 88% yield; m.p. 251-252 °C. FTIR (ATR) ν (cm ): [3452, 3433, 3321] (NH ,
2
1
NHC=O), [1660, 1641] (2 x C=O), 1657, 1635. H NMR (400 MHz, DMSO-d ) δ ppm 6.59 (t, J
6
=
7.5 Hz, 1H), 6.81 (dd, J = 8.4, 1.0 Hz, 1H), 7.27 (t, J = 6.9 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H),
7
.41 (s, 2H, NH ), 7.51 (dd, J = 8.8, 2.4 Hz, 1H), 7.68 (d, J = 15.4 Hz, 1H, =CH), 7.73 (d, J = 2.4
2
Hz, 1H), 7.90 (dd, J = 8.4, 1.3 Hz, 1H), 8.37 (d, J = 15.4 Hz, 1H, =CH), 8.42 (s, 1H, Quin-H),
13
NHC=O not observed. C NMR (101 MHz, DMSO-d
6
) δ ppm 114.5, 117.0, 117.5, 118.2, 120.4,
1
25.3, 125.7, 127.0, 127.2, 130.8, 130.9, 134.3, 137.2, 138.9, 152.1, 161.9, 173.8, 190.7. EI MS
+
(
70 eV) m/z (%): 326/324 (M , 25/71), 325/323 (45/100), 193 (15).
4
.2.1.5.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-7-chloroquinolin-2(1H)-one
12f.
-1
Yellow solid; 91% yield; m.p. 246–247°C. FTIR (ATR) ν (cm ): [3363, 3286] (NH
2
), 3214
1
(
NHC=O), [1662, 1645] (2 x C=O), 1616, 1571. H NMR (400 MHz, DMSO-d ) δ ppm 6.59 (t,
6
J = 7.0 Hz, 1H). 6.79 (dd, J = 8.4, 1.0 Hz, 1H), 7.00 (dd, J = 8.4, 2.1 Hz, 1H), 7.26 (t, J = 7.7 Hz,
1
1
H), 7.30 (d, J = 2.1 Hz, 1H), 7.34 (s, 2H, NH ), 7.56 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 15.4 Hz,
2
H, =CH), 7.92 (dd, J = 8.3, 1.3 Hz, 1H), 8.27 (s, 1H, Quin-H), 8.44 (d, J = 15.4 Hz, 1H, =CH),
13
NHC=O not observed. C NMR (101 MHz, DMSO-d ) δ ppm 114.4, 116.9, 117.8, 118.1, 118.7,
6

ACCEPTED MANUSCRIPT
1
19.8, 124.2, 125.5, 129.7, 130.9, 134.0, 134.4, 138.6, 139.5, 146.0, 151.9, 165.3, 191.2. EI MS
+
(
70 eV) m/z (%): 326/324 (M , 36/100), 204 (54), 193 (73), 146 (54), 132 (50).
4
.2.1.6.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-6-bromoquinolin-2(1H)-one
12g.
-1
Yellow solid; 95% yield; m.p. 249–250 °C. FTIR (ATR) ν (cm ): [3392, 3295] (NH NHC=O,
2
1
[
1653, 1630] (C=O), 1624, 1646. H NMR (400 MHz, DMSO-d
6
) δ ppm 6.63 (t, J = 7.5 Hz, 1H),
6
7
=
.84 (d, J = 8.4 Hz, 1H), 7.27–7.35 (m, 2H), 7.42 (s, 2H, NH ), 7.69 (d, J = 15.5 Hz, 1H, =CH),
2
.71 (dd, J = 8.7, 2.0 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.93 (s, 1H), 8.37 (d, J = 15.5 Hz, 1H,
1
3
CH), 8.50 (s, 1H, Quin-H), 12.17 (s, 1H, NHC=O). C NMR (101 MHz, DMSO-d ) δ ppm
6
1
1
2
14.3, 115.0, 117.6, 117.7, 117.9, 121.3, 126.6, 127.9, 130.8, 131.4, 134.3, 134.9, 136.9, 138.2,
+
39.6, 152.6, 161.2, 191.0. EI MS (70 eV) m/z (%): 370/368 (M , 93/100), 250/248 (27/28),
38/236 (51/52).
4
.2.1.7.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)benzo[h]quinolin-2(1H)-one
12h.
-1
Yellow solid; 72% yield; m.p. ˃300 °C. FTIR (ATR) ν (cm ): [3406, 3307] (NH ), 3159 (NH),
2
1
[
1651, 1627] (2 x C=O), 1608, 1573. H NMR (400 MHz, DMSO-d ) δ ppm 6.65 (t, J = 7.5 Hz,
6
1
H). 6.85 (d, J = 8.4 Hz, 1H), 7.42 (s, 2H, NH
2
), 7.61–7.77 (m, 5H), 7.83 (d, J = 15.5 Hz, 1H,
=
=
CH), 7.99 (d, J = 7.7 Hz, 2H), 8.42 (d, J = 15.5 Hz, 1H, =CH), 8.68 (s, 1H, Quin-H), 8.95 (d, J
1
3
8.0 Hz, 1H), NHC=O not observed. C NMR (101 MHz, DMSO-d ) δ ppm 115.0, 116.1,
6
1
1
17.3, 117.5, 118.1, 122.3, 123.0, 123.3, 125.3, 126.0, 127.1, 128.9, 129.0, 131.4, 132.7, 134.7,
+
34.8, 137.6, 141.5, 152.5, 162.6, 191.3. EI MS (70 eV) m/z (%): 340 (M , 34), 146 (100), 120
(28).
4
.2.1.8. (E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-1-butylquinolin-2(1H)-one 12i. Yellow
-1
2
solid; 83% yield; m.p. 115–116 °C. FTIR (ATR) ν (cm ): [3384, 3292] (NH ), [1643, 1608] (2 x

ACCEPTED MANUSCRIPT
1
C=O), 1564, 1537. H NMR (400 MHz, CDCl
3
) δ ppm 1.06 (t, J = 7.3 Hz, 3H, CH
3
), 1.50–1.63
(
m, 2H, CH ), 1.74–1.86 (m, 2H, CH ), 4.30–4.46 (m, 2H, NCH ), 6.42 (s, 2H, NH ), 6.66–6.76
2
2
2
2
(m, 2H), 7.21–7.33 (m, 2H), 7.39 (d, J = 8.6 Hz, 1H), 7.58–7.69 (m, 2H), 7.73 (d, J = 15.3 Hz,
13
1
H, =CH), 7.95 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 8.61 (d, J = 15.3 Hz, 1H, =CH). C NMR (101
MHz, CDCl ) δ ppm 13.9, 20.4, 29.6, 42.7, 114.2, 116.0, 117.1, 119.2, 120.6, 122.3, 126.4,
3
1
27.2, 129.7, 131.6, 131.7, 134.4, 138.5, 139.0, 141.3, 151.1, 160.8, 192.5. EI MS (70 eV) m/z
+
(
%): 346 (M , 100), 289 (36), 226 (37) 214 (63), 170 (63), 159 (45), 146 (24).
4
.2.1.9.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-1-butyl-7-chloroquinolin-2(1H)-one
-1
1
2j. Yellow solid; 92% yield; m.p. 195–196 °C. FTIR (ATR) ν (cm ): [3404, 3304] (NH
2
,
1
NHC=O), [1647, 1606] (2 x C=O), 1575, 1543. H NMR (400 MHz, CDCl ) δ ppm 1.15 (t, J =
3
7
.4 Hz, 3H, CH
NCH ), 6.51 (s, 2H, NH
3
m, 3H), 8.14 (d, J = 15.6 Hz, 1H, =CH), 8.22 (s, 1H, Quin-H). C NMR (101 MHz, CDCl ) δ
3
), 1.56–1.81 (m, 2H, CH
2 2
), 1.94–2.10 (m, 2H, CH ), 4.69 (t, J = 6.5 Hz, 2H,
2
2
), 6.78–6.86 (m, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.79 (s, 2H), 7.93–8.05
1
3
(
ppm 14.4, 20.1, 31.7, 66.7, 106.6, 116.3, 117.8, 119.6, 121.0, 122.8, 125.9, 126.7, 128.7, 131.5,
+
1
3
1
34.7, 138.3, 138.7, 142.7, 151.5, 156.7, 159.4, 192.4. EI MS (70 eV) m/z (%): 382/380 (M ,
2/89), 325/323 (12/33), 262/260 (12/42), 250/248 (38/100), 234/232 (12/33), 209/204 (31/85),
95/193 (17/55), 146 (54), 120 (63).
4
.2.1.10.
(E)-3-(3-(2-Aminophenyl)-3-oxoprop-1-en-1-yl)-6-bromo-1-butylquinolin-2(1H)-one
-1
1
2k. Yellow solid; 85% yield; m.p. 175–176 °C. FTIR (ATR) ν (cm ): [3415, 3290] (NH ),
2
1
[
1647, 1610] (2 x C=O), 1571, 1558. H NMR (400 MHz, CDCl ) δ ppm 1.05 (t, J = 7.3 Hz, 3H,
3
CH ), 1.46–1.62 (m, 2H, CH ), 1.68–1.85 (m, 2H, CH ), 4.24–4.42 (m, 2H, NCH ), 6.42 (s, 2H,
3
2
2
2
2
NH ), 6.66–6.77 (m, 2H), 7.22–7.35 (m, 2H), 7.69 (d, J = 15.2 Hz, 1H, =CH), 7.68–7.71 (m,

ACCEPTED MANUSCRIPT
1
H), 7.79 (d, J = 2.0 Hz, 1H), 7.85 (s, 1H, Quin-H), 8.00 (d, J = 8.1 Hz, 1H ), 8.60 (d, J = 15.2
13
Hz, 1H, =CH). C NMR (101 MHz, CDCl ) δ ppm 13.9, 20.4, 29.6, 42.9, 115.0, 115.9, 116.0,
3
1
1
2
17.2, 119.1, 122.1, 127.6, 128.0, 131.6, 131.7, 134.2, 134.5, 137.8, 137.9, 139.6, 151.1, 160.4,
+
92.3. EI MS (70 eV) m/z (%): 426/424 (M , 99/100), 369/367 (35/37), 294/292 (54/47),
78/276 (28/30), 250/247 (57/55), 169 (30), 146 (68), 120 (92).
4
7
1
1
7
1
=
.2.1.11. (E)-1-(2-Aminophenyl)-3-(2-butoxyquinolin-3-yl)prop-2-en-1-one 13a. Yellow solid;
-1
2
3% yield; m.p. 120–121 °C. FTIR (ATR) ν (cm ): [3452, 3396] (NH ), 1643 (C=O), 1579,
1
645. H NMR (400 MHz, CDCl ) δ ppm 1.08 (t, J = 7.4 Hz, 3H, CH ), 1.49–1.76 (m, 2H, CH ),
3
3
2
.82–2.11 (m, 2H, CH ), 4.64 (t, J = 6.5 Hz, 2H, OCH ), 6.42 (s, 2H, NH ), 6.79–6.70 (m, 2H),
2
2
2
.34 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.79 (d, J = 8.0 Hz,
H), 7.86 (d, J = 8.3 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 15.6 Hz, 1H, =CH), 8.08 (d, J
1
3
3
15.6 Hz, 1H, =CH), 8.26 (s, 1H, Quin-H). C NMR (101 MHz, CDCl ) δ ppm 14.0, 19.7,
3
1
1.2, 66.5, 115.9, 117.4, 119.2, 120.7, 124.5, 125.0, 126.4, 127.0, 127.9, 130.5, 131.1, 134.4,
+
37.7, 139.2, 146.7, 151.2, 160.2, 192.0. EI MS (70 eV) m/z (%): 346 (M , 25), 289 (32), 226
(37), 159 (27), 146 (100), 120 (39).
4
.2.1.12. (E)-1-(2-Aminophenyl)-3-(2-butoxy-6-methoxyquinolin-3-yl)prop-2-en-1-one 13b.
-1
2
Yellow solid; 78% yield; m.p. 140–141 °C. FTIR (ATR) ν (cm ): [3421, 3311] (NH ), 1629
1
(
(
(
(
C=O), 1620, 1560. H NMR (400 MHz, CDCl ) δ ppm 1.08 (t, J = 7.4 Hz, 3H, CH ), 1.56–1.71
3
3
m, 2H, CH ), 1.87–2.03 (m, 2H, CH ), 3.94 (s, 3H, OCH ), 4.60 (t, J = 6.5 Hz, 2H, OCH ), 6.42
2
2
3
2
s, 1H, NH ), 6.78–6.70 (m, 2H), 7.10 (s, 2H), 7.27–7.39 (m, 2H), 7.76 (d, J = 9.1 Hz, 1H), 7.93
2
d, J = 8.5 Hz, 1H ), 7.94 (d, J = 15.5 Hz, 1H, =CH), 8.08 (d, J = 15.5 Hz, 1H, =CH), 8.16 (s, 1H,
1
3
Quin-H). C NMR (101 MHz, CDCl ) δ ppm 14.1, 19.8, 31.3, 55.7, 66.3, 106.3, 115.9, 117.4,
3
1
19.2, 120.6, 122.4, 125.6, 126.4, 128.3, 131.1, 134.4, 137.9, 138.3, 142.3, 151.2, 156.4, 159.0,

ACCEPTED MANUSCRIPT
+
1
1
4
92.0. EI MS (70 eV) m/z (%): 376 (M , 35), 319 (28), 256 (19), 189 (48), 175 (43), 146 (100),
20 (67).
.2.1.13.
(E)-1-(2-Aminophenyl)-3-(2-butoxy-8-methylquinolin-3-yl)prop-2-en-1-one
13d.
-1
Yellow solid; 84% yield; m.p. 108–109 °C. FTIR (ATR) ν (cm ): [3466, 3332] (NH ), 1641
2
1
(
C=O), 1612, 1571. H NMR (400 MHz, CDCl
3
) δ ppm 1.09 (t, J = 7.4 Hz, 3H, CH
), 2.73 (s, 3H, Quin-CH ), 4.66 (t, J = 6.5 Hz, 2H, OCH
.43 (s, 2H, NH ), 6.75 (d, J = 8.1 Hz, 1H), 6.77 (t, J = 6.3 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H),
3
), 1.48–1.76
(m, 2H, CH
2
), 1.87–2.12 (m, 2H, CH
2
3
2
),
6
7
1
2
.35 (t, J = 9.0 Hz, 1H), 7.53 (d, J = 7.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.4 Hz,
13
H), 7.96 (d, J = 15.6 Hz, 1H, =CH), 8.10 (d, J = 15.6 Hz, 1H, =CH), 8.23 (s, 1H, Quin-H). C
NMR (101 MHz, CDCl ) δ ppm 14.1, 17.7, 19.8, 31.2, 66.3, 115.9, 117.4, 119.3, 120.1, 124.1,
3
1
24.8, 125.8, 126.2, 130.7, 131.1, 134.3, 135.2, 137.9, 139.6, 145.5, 151.1, 159.2, 192.1. EI MS
+
(
70 eV) m/z (%): 360 (M , 33), 303 (33), 173 (27), 146 (100), 120 (42).
4
.2.1.14.
(E)-1-(2-Aminophenyl)-3-(6-bromo-2-butoxyquinolin-3-yl)prop-2-en-1-one
13g.
-1
Yellow solid; 82% yield; m.p. 150–151 °C. FTIR (ATR) ν (cm ): [3417, 3307] (NH ), 1633
2
1
(
C=O), 1560, 1529. H NMR (400 MHz, CDCl ) δ ppm 1.07 (t, J = 7.4 Hz, 3H, CH ). 1.55–1.71
3
3
(
m, 2H, CH
2
), 1.88–2.03 (m, 2H, CH
2
), 4.61 (t, J = 6.5 Hz, 2H, OCH
2
2
), 6.43 (s, 2H, NH ), 6.70–
6
.81 (m, 2H), 7.34 (t, J = 7.7 Hz, 1H), 7.71 (s, 2H ), 7.86–7.94 (m, 3H, =CH), 8.06 (d, J = 15.6
1
3
Hz, 1H, =CH), 8.14 (s, 1H, Quin-H). C NMR (101 MHz, CDCl
3
) δ ppm 14.0, 19.7, 31.2, 66.7,
1
1
15.9, 117.4, 117.6, 119.0, 121.6, 126.2, 127.1, 128.7, 129.8, 131.1, 133.6, 134.5, 137.1, 137.8,
+
45.3, 151.2, 160.4, 191.7. EI MS (70 eV) m/z (%): 426/424 (M , 10/10), 368/367 (10/10), 146
(100), 120 (25).
4
2
.2.2 General procedure for the synthesis of the novel 2,3-dihydro-[2,3'-biquinoline]-
',4(1H,1'H)-diones 15 and 2'-butoxy-2,3-dihydro-[2,3'-biquinolin]-4(1H)-ones 16