The synthesis and biological evaluation of new DNA-directed alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates containing a urea linker

Article abstract of DOI:10.1016/j.ejmech.2014.06.066

We synthesized a series of phenyl N-mustard-4-anilinoquinoline conjugates to study their antitumorigenic effects. These agents were prepared by the condensation of 4-[N,N-bis(2-chloroethyl)amino]phenyl isocyanate with 6-amino-4-methylamino or 4-anilinoquinolines. The structure-activity relationship (SAR) studies revealed that the C2-methylquinoline derivatives (18a-o) were generally more cytotoxic than the C2-phenylquinoline conjugates (23a-d) in inhibiting the cell growth of various human tumor cell lines in vitro. However, the methylamino or aniline substituents at C4 of quinoline did not influence the cytotoxic effects. The title conjugates were capable of inducing DNA cross-linking and promoting cell-cycle arrest at the G2/M phase. This study demonstrates that phenyl N-mustard-4-anilinoquinoline conjugates are generally more potent than phenyl N-mustard-4-anilinoquinazoline conjugates against the cell growth of various tumor cell-lines.

Full text of DOI:10.1016/j.ejmech.2014.06.066

European Journal of Medicinal Chemistry 83 (2014) 695e708
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesis and biological evaluation of new DNA-directed
alkylating agents, phenyl N-mustard-4-anilinoquinoline conjugates
containing a urea linker
,
,
,
Bhavin Marvania a b, Rajesh Kakadiya a b, Wilson Christian a b, Tai-Lin Chen a,
Ming-Hsi Wu a, Sharda Suman a, Kiran Tala a, Te-Chang Lee a *, Anamik Shah b,
,
, c, **
Tsann-Long Su a
a Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
b Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
c Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a series of phenyl N-mustard-4-anilinoquinoline conjugates to study their anti-
tumorigenic effects. These agents were prepared by the condensation of 4-[N,N-bis(2-chloroethyl)amino]
phenyl isocyanate with 6-amino-4-methylamino or 4-anilinoquinolines. The structureeactivity rela-
tionship (SAR) studies revealed that the C2-methylquinoline derivatives (18aeo) were generally more
cytotoxic than the C2-phenylquinoline conjugates (23aed) in inhibiting the cell growth of various hu-
man tumor cell lines in vitro. However, the methylamino or aniline substituents at C4 of quinoline did
not influence the cytotoxic effects. The title conjugates were capable of inducing DNA cross-linking and
promoting cell-cycle arrest at the G2/M phase. This study demonstrates that phenyl N-mustard-4-
anilinoquinoline conjugates are generally more potent than phenyl N-mustard-4-anilinoquinazoline
conjugates against the cell growth of various tumor cell-lines.
Received 19 February 2014
Received in revised form
3 June 2014
Accepted 28 June 2014
Available online 28 June 2014
Keywords:
DNA-directed alkylating agents
Phenyl N-mustard
Quinolines
© 2014 Elsevier Masson SAS. All rights reserved.
DNA cross-linking
Cell cycle
Apoptosis
1. Introduction
linking agents that possess high efficacy, chemical stability, and low
toxicity. First-generation DNA-directed cross-linking agents were
Progress in the development of DNA cross-linking agents has
lagged behind that of other chemotherapeutic agents because these
derivatives have several drawbacks, including a low specificity for
targeting the DNA of tumor cells, a high chemical reactivity,
induced bone-marrow toxicity, and an eventual loss in activity due
to cellular DNA repair mechanisms [1e4]. To overcome these gen-
eral drawbacks of cross-linking agents, one strategy is to design a
prodrug (e.g., cyclophosphamide or ifosfamide) or DNA-directed
alkylating agents by linking the alkylating pharmacophore
(warhead) with DNA-tropic molecules (carrier), such as a DNA
minor groove binding agent (e.g., distamycin A) [4]. Previously, we
focused on designing and synthesizing new DNA-directed cross-
synthesized with alkyl N-mustard-9-anilinoacridine conjugates via
an alkoxy or methyl linker [5e7]. Although these conjugates (e.g.,
BO-0742, 1, Fig. 1) exhibited potent antitumor effects in a human
breast adenocarcinoma MX-1 xenograft model [5], they were
chemically unstable and demonstrated a poor pharmacokinetic
(PK) profile. To improve the PK profile of these first-generation
compounds, we synthesized phenyl N-mustard-9-anilinoacridine
conjugates containing a urea, carbamate, or carbonate linker (e.g.,
BO-1051, 2, Fig. 1), which demonstrated potent antitumor activities
in various human tumor xenografts generated from human breast
MX-1, colon cancer HCT 116, and ovarian cancer SK-VO4 cells [8,9].
Indeed, the PK profiles of these conjugates were significantly
improved, and they were shown to possess potent in vitro cytotoxic
and antitumor activities in the human tumor xenograft models
tested. The linkers used for synthesizing DNA-directed cross-link-
ing agents have previously been used for preparing antibody-
directed enzyme prodrug therapy (ADEPT) [10], gene-directed
enzyme prodrug therapy (GDEPT) [11], or melanocyte-directed
enzyme prodrug therapy (MDEPT). Thus, these linkers are able to
* Corresponding author.
** Corresponding author. Institute of Biomedical Sciences, Academia Sinica, Taipei
11529, Taiwan.
E-mail addresses: bmtcl@ibms.sinica.edu.tw (T.-C. Lee), tlsu@ibms.sinica.edu.tw
(T.-L. Su).
http://dx.doi.org/10.1016/j.ejmech.2014.06.066
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
Fig. 1. Structures of several DNA-directed alkylating agents.
reduce the chemical reactivity of the reactive N-mustard moiety
[12e14].
lines and were antitumorigenic in a xenograft model. Unfortu-
nately, these derivatives generally have poor solubility, which
confounds evaluation of their efficacy in animal models. However,
we hypothesized that replacement of the quinazoline moiety in
compound 8 with a quinoline molecule might increase the water
solubility of the compound. Therefore, in the current study, we
synthesized a series of phenyl N-mustard-4-anilinoquinoline con-
jugates, in which the phenyl N-mustard pharmacophore was linked
to C6 of the 4-anilino-2-methyl- or 4-anilino-2-phenylquinolines
via a urea linker. These results demonstrated that the newly syn-
thesized conjugates were more cytotoxic than the corresponding
phenyl N-mustard-4-anilinoquinazoline derivatives and also
showed improved solubility.
Apart from 9-anilinoacridine, quinolines and quinazolines have
also been used as carriers for constructing DNA-directed cross-
linking agents. For example, Gravatt et al. synthesized a series of 4-
anilinoquinoline-linked aniline mustard conjugates (e.g., 3) for
antitumor studies [15], and these compounds demonstrated at
least 10-fold greater cytotoxicity than the corresponding aniline
mustard itself. Recently, we synthesized phenyl N-mustard-quin-
oline conjugates (4) bearing a urea or hydrazinecarboxamide linker
for antitumor evaluation, and these conjugates exhibited potent
therapeutic efficacy in various human tumor xenograft models [16].
We also demonstrated that a urea or hydrazinecarboxamide linker
was able to lower the reactivity of the reactive N-mustard moiety,
resulting in a longer half-life in rat plasma. Another category of
DNA-directed alkylating agents includes the phenyl N-mustard-
quinazoline conjugates, in which the DNA alkylating moiety is
either linked at the C4 or C6 position in the quinazoline ring. Pra-
kash et al. synthesized quinazoline derivatives (e.g., 5) bearing a
phenyl N-mustard residue at C4 via an alkylamino spacer, and it
was shown that compound 5 was not a strong DNA intercalator but
may bind weakly at the major groove site [17]. DNA-directed
alkylating agents synthesized by introducing a DNA alkylating
moiety (e.g., methyltriazene or N-nitrosourea) to C6 of the quina-
zoline ring have also been reported. The triazene derivative 6
(SMA41) was designed as a “combi-molecule” and was shown to
generate a free aminoquinazoline (capable of blocking epidermal
growth factor receptor tyrosine kinase [EGFR TK]) and methyl-
diazonium species (DNA alkylator) in cell culture medium [18]. By
contrast, the nitrosourea derivative 7 demonstrated a weaker
inhibitory effect on the EGFR TK compared to other triazene de-
rivatives, resulting in a weaker antiproliferative effect [19]. More
2. Results and discussion
2.1. Chemistry
The method used for the preparation of N-mustard-4-anilino-2-
methylquinoline conjugates (18aeo) containing a urea linker is
outlined in Scheme 1. The reaction of 4-nitroaniline 9 with ethyl-3-
ethoxybut-2-enoate 10 in 2, 6-di-tert-butylphenol in the presence
of H2SO4 was heated at 250 ꢀC to produce 2-methyl-6-
nitroquinolin-4-ol 11 according to a previously described proce-
dure [21]. Treatment of compound 11 with POCl3 yielded 4-chloro-
2-methyl-6-nitroquinoline 12, which was then reacted with
methylamine hydrochloride 13a in phenol at 120 ꢀC according to
procedures found in the literature, to yield 2-methyl-4-
methylamino-6-nitroquinolines 14a [22]. The 4-anilino-6-
nitroquinoline derivatives (14beo) were synthesized by treating
compound 12 with various substituted anilines (13beo) in iso-
propanol (IPA) in the presence of concentrated HCl under refluxing
conditions, as previously described [23]. The nitro group in 14aeo
was reduced in aqueous ethanol using acetic acid and iron powder
as the reducing agent to adequately yield the corresponding 6-
aminoquinoline derivatives (15aeo). Condensation of 15aeo with
the known 4-[N,N-bis(2-chloroethyl)amino]phenylisocyanate 17
recently, we synthesized
a series of phenyl N-mustard-4-
anilinoquinazoline conjugates via a urea linker (e.g., compound
8), in which the phenyl N-mustard pharmacophore was linked to
C6 of the quinazoline ring [20]. We reported that these conjugates
were moderately cytotoxic in vitro in a panel of human tumor cell

B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
697
Scheme 1. Reaction conditions: (a) sulfuric acid/2, 6-di-tert-butylphenol, 250 ꢀC; (b) POCl3, reflux; (c) RNH2/phenol, 170 ꢀC; (d) RNH2/iso-propanol, reflux; (e) Fe/AcOH/EtOH/H2O,
reflux; (f) triphosgene/Et3N/THF, room temperature; (g) Et3N/DMF, room temperature.
[24] (freshly prepared from N,N-bis(2-chloroethyl)benzene-1,4-
diamine hydrochloride 16 [12]) in anhydrous DMF in the pres-
ence of triethylamine (TEA) at room temperature gave the desired
phenyl N-mustard-4-methylamino or anilinoquinoline conjugates
containing a urea linker (18aeo). The physical data and yields of the
final products are listed in Table 1.
The synthesis of N-mustard-4-anilino-2-phenylquinoline con-
jugates (23aed) is shown in Scheme 2. Commercially available 5-
Table 1
The in vitro cell growth inhibition mediated by 4-anilinoquinoline-6-N-mustard uredo derivatives in human lymphoblastic leukemia (CCRF-CEM and CCRF-CEM/VBL), colon
cancer (PC3), and lung cancer (H1299 and H460) cells.
Sr. no.
R1
R2
Cell growth inhibition (IC50, m
M)a
CCRF-CEM
CCRF-CEM/VBLb
PC3
2.51 0.44
H460
H1299
HCT-116
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18n
18o
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph
Me
0.39 0.09
0.44 0.09
0.70 0.18
0.42 0.07
0.59 0.02
0.73 0.12
0.46 0.02
0.52 0.18
0.54 0.22
0.40 0.17
0.39 0.14
0.59 0.08
0.47 0.10
0.28 0.01
0.80 0.14
2.07 0.06
2.72 0.13
1.84 0.02
0.79 0.07
0.0029 0.0002
2.45 0.65
>100 [>256ꢁ]c
9.37 1.92
1.37 0.10
0.86 0.16
1.01 0.28
1.03 0.07
0.84 0.14
1.18 0.15
0.91 0.29
1.40 0.11
0.82 0.26
0.92 0.05
0.85 0.17
0.70 0.22
1.83 0.03
3.74 0.27
2.26 0.34
7.81 0.44
2.09 0.75
1.89 0.76
e
5.40 4.68
1.73 0.65
1.09 0.06
2.37 0.83
0.88 0.11
1.31 0.21
0.77 0.11
1.35 0.12
1.07 0.21
1.28 0.21
1.05 0.12
1.48 0.43
1.13 0.10
1.32 0.11
2.54 0.26
2.35 0.39
5.39 0.35
1.42 0.04
2.13 0.09
e
1.28 0.25
0.83 0.05
0.74 0.10
1.06 0.12
0.56 0.08
0.77 0.21
0.49 0.15
0.80 0.06
0.83 0.10
0.6 3 0.16
0.49 0.08
0.62 0.11
0.59 0.01
0.62 0.10
1.81 0.63
3.69 0.19
3.58 1.09
1.80 0.56
1.35 0.39
e
Ph
11.51 1.20 [26.15ꢁ]
9.58 1.31 [13.68ꢁ]
11.0 0.79 [26.19ꢁ]
7.56 4.61 [12.81ꢁ]
8.18 1.43 [11.21ꢁ]
5.19 0.84 [11.28ꢁ]
9.53 0.96 [18.33ꢁ]
9.83 0.51 [18.20ꢁ]
10.18 2.87 [25.45ꢁ]
5.85 1.41 [15.00ꢁ]
5.96 0.98 [10.10ꢁ]
4.49 1.18 [9.55ꢁ]
4.22 0.20 [15.07ꢁ]
>100 [>125ꢁ]
1.49 0.12
1.49 0.31
7.16 0.69
1.27 0.11
1.36 0.29
1.41 0.06
1.75 0.20
1.78 0.58
1.32 0.16
1.29 0.27
1.42 0.24
1.17 0.12
2.38 0.13
3.46 0.78
2.13 0.15
8.86 0.22
1.23 0.30
2.21 0.09
30-F-Ph
40-F-Ph
30-Cl-Ph
40-Cl-Ph
30-Br-Ph
40-Br-Ph
30-Cl, 40-F-Ph
30-Br, 40-F-Ph
30-Me-Ph
30-CF3-Ph
30-Ethynyl-Ph
30-MeO-Ph
30,40,50-tri-MeO-Ph
30-Cl-Ph
23a
4.55 0.65 [2.19ꢁ]
7.07 1.67 [2.59ꢁ]
4.15 0.60 [2.25ꢁ]
2.71 0.76 [3.43ꢁ]
0.87 0.0043 [300ꢁ]
3.40 0.99
23b
23c
23d
Vinblastine
Cisplatin
Ph
Ph
Ph
e
30-Cl-40-F-Ph
30-CH3-Ph
30-OCH3-Ph
e
e
e
e
15.51 2.00
3.13 0.06
11.42 3.59
1.96 0.67
a
Cell growth inhibition was determined using the alamarBlue assay. Data represent the mean STDEV of three to six independent experiments for each compound.
CCRF-CEM/VBL is a sub cell line that is 300-fold more resistant to vinblastine when compared to the IC50 of the CCRF-CEM parental line.
The numbers in the brackets represent the fold change in cross-resistance determined by comparison with the corresponding IC50 of the parental cell line.
b
c

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B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
nitroanthranilic acid 19 was reacted with acetophenone in POCl3
under refluxing conditions, according to a previously described
procedure, to give 4-chloro-6-nitro-2-phenylquinoline 20 [25].
Treatment of compound 20 with various substituted anilines in
isopropanol under refluxing conditions gave 4-anilinoquinolines
21. Following the same reduction conditions (Fe/AcOH/EtOH/
H2O), compound 21 was converted into the corresponding 6-
aminoquinolines 22, and the condensation of freshly prepared
compound 18 led to a fair-to-good yield of the desired 23aeo
conjugates.
Upon comparing the effects of the substituent (F, Cl, Me, or MeO
functionality) on the C4-anilino ring between the newly synthe-
sized N-mustard-4-anilinoquinoline conjugates and the corre-
sponding N-mustard-4-anilinoquinazoline conjugate
8
[20]
previously synthesized in our laboratory, the potency of the later
derivative was more affected by these substituents. Moreover, the
newly synthesized N-mustard-4-anilinoquinoline derivatives were
more cytotoxic than the corresponding quinazoline analogues in
inhibiting the human solid tumor cell lines.
We also studied the cytotoxicity of the newly synthesized
compounds in the vinblastine-resistant lymphoblastic leukemia
sub-cell line (CCRF-CEM/VBL), a multidrug-resistant (MDR) cell line
that is approximately 300-fold more resistant to vinblastine
compared to the parental line. These experiments sought to eval-
uate whether the newly synthesized conjugates would affect
vincristine MDR. The results showed that the 2-phenylquinoline
conjugates (23aed) demonstrated little cross-resistance to
vincristine, whereas the 2-methyl substituted derivatives (18aeo)
were somewhat cross-resistant to vinblastine. However, com-
pounds 18a and 18o showed high cross-resistance to vincristine
among the tested compounds.
2.2. In vitro cytotoxicity
We evaluated the cytotoxicity of the newly synthesized com-
pounds using a panel of human cancer cell lines, including
lymphoblastic leukemia cells (CCRF-CEM) and the vincristine
resistant sub-cell line (CCRF-CEM/VBL), prostate cancer cells (PC3),
lung cancer cells (H1299 and H460), and colon cancer cells (HCT-
116). Cisplatin was used for comparison. The structureeactivity
relationships (SARs) of these derivatives were assessed based on
their cytotoxicities against CCRF-CEM cells in culture, as shown in
Table 1. All compounds in the series of 2-methylquinoline de-
rivatives containing either a 4-methylamino (18a) or 4-anilino
substituent (18beo) demonstrated equivalent cytotoxicity in the
CCRF-CEM cell line. This finding indicates that the size (methyl-
amino or anilino) and the physical property of the substituent (e.g.,
F, Br, Cl, Me, or MeO functionality) on the anilino ring at the C4
position of the quinoline have no apparent effect on cytotoxicity.
Similarly, the substituent on the 4-anilino ring of 2-
phenylquinoline derivatives (23aed) did not significantly alter
their cytotoxicity. However, because only 4 compounds were syn-
thesized, no firm conclusions can be made at this point. Among
these derivatives, compounds having a MeO substituent on the C30
of the 4-anilino ring showed better cytotoxic effects, and in general,
2-methylquinoline derivatives (18aeo) were more cytotoxic than
2-phenylquinoline derivatives (23aed).
2.3. DNA cross-linking study
We next used an alkaline agarose gel shift assay to determine
whether the newly synthesized compounds could produce DNA
cross-links. pEGFP-N1 DNA was treated with compounds 18e, 18g,
18k, and 18n at various concentrations (1, 10, and 20 mM) and then
linearized by restriction enzyme digestion. As shown in Fig. 2,
compounds 18k and 18n efficiently formed DNA interstrand cross-
links at the concentration of 20 mM. We also compared the cross-
linking capacity of the newly synthesized compounds by SAR and
found that substitution on the 4-anilinoquinoline with an electron-
donating functional group revealed stronger cross-linking
compared to substitution with an electron-withdrawing func-
tional group. These results suggest that the DNA cross-linking ca-
pabilities of the tested compounds correlate with their in vitro
cytotoxicity and represent the most likely mechanisms by which
they inhibit cancer cell growth.
To a certain degree, the 2-methylquinoline derivatives (18aeo)
demonstrated greater antiproliferative effects compared to the 2-
phenylquinoline derivatives (23aed) in solid tumor cell lines (e.g.,
PC3, H1299 and H460, and HCT-116). Interestingly, compound 18a
was more cytotoxic than 18k, and both compounds were approxi-
mately equal in potency in the CCRF-CEM cell line. Among the
compounds examined,18e, 18g, 18k, and 18n demonstrated a broad
spectrum of in vitro antitumor effects, and in most cases, the newly
synthesized conjugates were more cytotoxic than cisplatin.
2.4. Cell cycle inhibition
We next investigated the effects of the 18g compound on cell
cycle progression in lung cancer H460 cells. H460 cells were treated
with 18g at concentrations of 0.25, 0.5, 1, and 2 mM for 24 h, and the
Scheme 2. Reaction conditions: (a) acetophenone/POCl3, reflux; (b) RNH2/iso-propanol, reflux; (c) Pd/C/H2, MeOH or SnCl2/MeOH; (d) Et3N in DMF.

B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
699
Fig. 2. A representative DNA cross-linking gel shift assay is shown for the 18e, 18g, 18k, and 18n compounds at the indicated concentrations. The control lane shows single-stranded
DNA (SS), while the cross-linking (CL) shown in all tested lanes represents double-stranded DNA. Melphalan (1 and 10 M) was used as a positive control.
cell cycle distributions were then analyzed by flow cytometry. As
shown in Fig. 3, there was a dose-dependent decrease in G1 phase
cells and an increase in G2/M phase cells after 24-h treatment with
compound 18g. However, at a 2 mM dose (approximately 1.5-times
the IC50 dose), the sub-G1 population was increased, indicating
irreversible cell damage and initiation of apoptotic cell death.
molecules. SAR analysis demonstrated that the C2-methyl conju-
gates were more cytotoxic than the C2-phenyl derivatives, although
the former derivatives demonstrated slightly increased multidrug
resistance to vinblastine. We also showed that the substituent(s) on
the 4-anilino ring did not affect the cytotoxicity of the conjugates.
Furthermore, the newly synthesized conjugates were more cyto-
toxic to tumor cell lines and demonstrated improved solubility
when compared to the corresponding phenyl N-mustard-quina-
zoline conjugates [20] previously synthesized in our laboratory. In
addition, we compared the potency of the newly synthesized
compounds to that of phenyl N-mustard-quinoline conjugates, in
which the phenyl N-mustard residue is attached to C4 of the
quinoline ring [16], and these data showed that both series of de-
rivatives were equally cytotoxic. Finally, we demonstrated that all
the derivatives were able to induce DNA damage by DNA cross-
linking, indicating that the location of the N-mustard pharmaco-
phore on the quinoline or quinazoline ring (C4 or C6) was not
critical for their DNA cross-linking or antitumor activities.
2.5. Induction of apoptosis
Apoptosis can be triggered by DNA damage or disturbances in
the cell cycle. Therefore, we performed Annexin V staining to
determine whether 18g could induce apoptosis and to distinguish
this process from necrosis. H460 cells were treated with 2 mM 18g
for 24, 48, or 72 h and then stained with Annexin V-FITC and
propidium iodide (PI) and analyzed by flow cytometry. The results
are shown in Fig. 4. Treatments with 18g resulted in a significant
time-dependent increase in the percentage of Annexin Vþ cells;
after 24, 48 and 72 h, the frequencies of Annexin Vþ cells were
20.4%, 33.0%, and 69.2%, respectively. Thus, these results indicate
that the cytotoxic effects of 18g in H460 cells were due to its ability
to induce apoptosis.
4. Experimental section
4.1. General materials and methods
3. Conclusion
Chemical materials, solvents, and reagents were reagent grade
In the current study, we introduced a phenyl N-mustard phar-
macophore to position C6 of 4-methylamino- or 4-anilinoquinoline
and were used without purification unless otherwise noted. The
melting points were recorded using
a Fargo melting point
Fig. 3. Cell cycle inhibition in human H460 lung cancer cells after treatment with 18g. The cells were incubated with 18g (0.25, 0.5, 1, and 2
was analyzed by flow cytometry. The data are presented as the average of 3 independent experiments.
mM) for 24 h, and cell cycle progression

700
B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
Fig. 4. Treatment with 18g increased the percentage of Annexin V þ H460 cells in a time-dependent manner. H460 cells were treated with either 2
mM 18g for 24, 48 or 72 h or with
5
m
M cisplatin for 72 h. At the end of treatment, the cells were analyzed for early apoptosis by Annexin V staining, and the percentages of Annexin Vþ cells are shown.
apparatus and are shown as uncorrected values. Column chro-
matography was performed using a silica gel G60 (70e230 mesh,
ASTM; Merck and 230e400 mesh, Darmstadt, Germany). Reaction
progress was monitored using analytical thin-layer chromatog-
raphy (TLC) on 0.25-mm Merck F-254 silica gel glass plates.
Visualization was achieved by UV light (254 nm). 1H NMR spectra
were recorded with a Bruker AVANCE 600 DRX and a 400 MHz
spectrometer; chemical shifts were reported in parts per million
4.1.2. 4-Chloro-2-methyl-6-nitroquinoline (12)
2-Methyl-6-nitroquinolin-4-ol 11 (6.10 g, 30.0 mmol) was added
portionwise into POCl3 (50 mL) at room temperature. The reaction
mixture was then slowly heated at reflux for 1 h. After completion
of the reaction, the excess POCl3 was distilled by vacuum distilla-
tion. The residue was poured into ice cold water and neutralized by
adding ammonia solution. The precipitate was collected by filtra-
tion and washed successfully with water and hexane, then dried to
give 12, 5.7 g (86%); mp 145e146 ꢀC (Lit. mp 146e148) [26]; 1H
(d) using tetramethylsilane as the internal standard with coupling
constants (J) reported in hertz (Hz). The peak shapes were denoted
as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
dd, double doublet; and brs, broad singlet. Elemental analyses
were performed on a Heraeus CHNeO Rapid analyzer. High per-
formance liquid chromatography (HPLC) analysis was performed
to assess the purity of synthesized compounds, and the results
were recorded on a Hitachi D-2000 Elite instrument with the
following specifications: column, Mightysil RP-18 GP 250-4.6
NMR (DMSO-d6) d: 2.72 (3H, s, Me), 7.92 (1H, s, ArH), 8.17 (1H, d,
J ¼ 9.2 Hz, ArH), 8.49 (1H, dd, J ¼ 2.4 Hz, J ¼ 9.2 Hz, ArH), 8.88 (1H, d,
J ¼ 2.4 Hz, ArH).
4.1.3. 4-Chloro-6-nitro-2-phenylquinoline (20)
2-Amino-5-nitro-benzoic acid 19 (40 g, 220 mmol) was sus-
pended in (400 mL, 3.2 mol) at room temperature. Acetophenone
(26.46 g, 220 mmol) was added dropwise and the resulting mixture
was heated at 90 ꢀC for 5 h. After completion of the reaction, POCl3
was evaporated and resulting residue was then poured into a
mixture of ice, ammonia and chloroform mixture (10:4:4 w/v). The
reaction mass was stirred at room temperature for overnight.
Chloroform layer was then separated and washed with brine, dried
over Na2SO4, and evaporated under vacuum to give 20 as a yellow
powder. Yield: 52.4 g (84%); mp 161e162 ꢀC (Lit. mp 168e169) [27];
(5
m
L) mobile phase, MeCN/THF (70:30 v/v); flow rate, 1 mL/min;
injected sample 10
m
L, column temp, 27 ꢀC; and wavelength,
254 nm. The purity of all compounds was >95% based on analytical
HPLC.
4.1.1. 2-Methyl-6-nitroquinolin-4-ol (11)
A mixture of 4-nitroaniline 9 (20.0 g, 145.0 mmol), ethyl-3-
ethoxybut-2-enoate 10 (60.0 g, 437.0 mmol) in 2, 6-ditertbutyl
phenol (250 mL) containing catalytic amount of concentrated sul-
furic acid (two drops) was stirred at room temperature. The reac-
tion vessel was equipped with a short distillation apparatus to
remove the ethanol produced during the reaction. The reaction
mixture was heated up to reflux temperature for 30 min. After
completion of the reaction, the reaction mixture was cooled down
to room temperature. The solid appeared was collected by filtration
and washed with hexane and toluene, then dried to give 11, 18.8 g
1H NMR (400 MHz, DMSO-d6)
d: 7.26 (1H, s, ArH), 7.57e7.55 (3H, m,
ArH ꢁ 3), 8.20e8.21 (2H, d, J ¼ 2.32 Hz, ArH ꢁ 2), 8.29e8.31 (1H, d,
J ¼ 9.2 Hz, ArH), 8.55e8.52 (1H, dd, J ¼ 2.5 and 9.2 Hz, ArH), 9.18
(1H, s, ArH).
4.1.4. N,2-dimethyl-6-nitroquinolin-4-amine (14a)
A mixture of 4-chloro-2-methyl-6-nitroquinoline 12 (2.00 g,
9.0 mmol), methylamine hydrochloride 13a (1.21 g, 18.0 mmol) and
phenol (3 g) was heated up to 170 ꢀC for 5 h. After the completion of
reaction, the reaction mixture was poured into 10% aqueous NaOH
solution. The solid separated was collected by filtration and
collected solid was washed with water. The crude product obtained
was purified by chromatography on silica gel column using
(64%); mp > 300 ꢀC; 1H NMR (DMSO-d6)
d: 2.38 (3H, s, Me), 6.06
(1H, s, ArH), 7.65 (1H, d, J ¼ 9.2 Hz, ArH), 8.37 (1H, dd, J ¼ 2.6 Hz,
J ¼ 9.2 Hz, ArH), 8.78 (1H, d, J ¼ 2.6 Hz, ArH), 12.08 (1H, s,
exchangeable, NH).

B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
701
chloroform and methanol (100:2 v/v) as the eluent. The fractions
containing main product were combined and evaporated to dry-
ness afforded compound 14a, which was further recrystallization
from EtOH. Yield was 1.20 g (62%); mp 145e146 ꢀC; 1H NMR
(1H, s, ArH), 7.54e7.55 (2H, m, 2 ꢁ ArH), 7.64e7.66 (2H, m, 2 ꢁ ArH),
8.33 (1H, d, J ¼ 9.3 Hz, ArH), 8.70 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.3 Hz, ArH),
9.78 (1H, d, J ¼ 2.2 Hz, ArH), 11.40 (1H, s, exchangeable, NH); 13C
NMR (500 MHz, DMSO-d6) d: 19.8, 101.6, 115.8, 121.0, 121.5, 127.1,
(DMSO-d6)
d
: 2.50 (3H, s, Me), 2.90 (3H, d, J ¼ 4.7 Hz, Me), 6.44 (1H,
127.2, 129.9, 131.7, 135.8, 138.5, 141.5, 144.6, 154.9, 157.2.
s, ArH), 7.79 (1H, d, J ¼ 7.4 Hz, ArH), 7.90e7.91 (1H, m, exchange-
able, NH), 8.25 (1H, dd, J ¼ 1.9 Hz, J ¼ 7.4 Hz, ArH), 9.21 (1H, d,
4.1.10. N-(3-bromophenyl)-2-methyl-6-nitroquinolin-4-amine
(14g)
J ¼ 1.9 Hz, ArH); 13C NMR (500 MHz, DMSO-d6)
d: 25.4, 30.7, 99.2,
116.1, 119.5, 122.3, 129.6, 142.2, 150.9, 152.4, 162.9.
Compound 14g was synthesized from compound 12 (2.89 g,
13.0 mmol), 3-bromoaniline 13g (2.68 g, 16.0 mmol) and IPA
(40 mL) containing HCl (2 drops): Yield 4.30 g (93%); mp > 300 ꢀC;
4.1.5. N-phenyl-2-methyl-6-nitroquinolin-4-amine (14b)
To a solution of 4-chloro-2-methyl-6-nitroquinoline 12 (2.67 g,
12.0 mmol) and aniline 13b (1.34 g, 14.4 mmol) in 30 mL of iso-
propanol (IPA) was added HCl (2 drops) at room temperature. The
reaction mixture was then heated at reflux temperature for 3 h.
Resulting solution was cooled to room temperature. Solid separated
was collected by filtration and washed with IPA. The crude product
obtained was crystallized from EtOH/CH2C2 to give 14b, 1.91 g
1H NMR (DMSO-d6)
d: 2.68 (3H, s, Me), 6.91 (1H, s, ArH), 7.54e7.67
(2H, m, 2 ꢁ ArH), 7.64e7.66 (1H, m, ArH), 7.73e7.74 (1H, m, ArH),
8.30 (1H, d, J ¼ 9.3 Hz, ArH), 8.71 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH),
9.76 (1H, d, J ¼ 2.3 Hz, ArH), 11.35 (1H, s, exchangeable, NH); 13C
NMR (500 MHz, DMSO-d6) d: 19.9, 101.8, 115.8, 121.0, 121.5, 122.3,
124.2, 127.2, 127.2, 127.9, 130.3, 131.7, 138.5, 141.5, 144.6, 154.8,157.3.
(57%); mp > 300 ꢀC; 1H NMR (DMSO-d6)
d
: 2.65 (3H, s, Me), 6.81
4.1.11. N-(4-bromophenyl)-2-methyl-6-nitroquinolin-4-amine
(14h)
Compound 14h was synthesized from compound 12 (2.00 g,
9.0 mmol), 4-bromoaniline 13h (1.85 g, 11.0 mmol) and IPA (30 mL)
containing HCl (2 drops): Yield 3.00 g (93%); mp > 300 ꢀC; 1H NMR
(1H, s, ArH), 7.44e7.51 (3H, m, 3 ꢁ ArH), 7.58e7.62 (2H, m, 2 ꢁ ArH),
8.26 (1H, d, J ¼ 9.3 Hz, ArH), 8.70 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH),
9.78 (1H, d, J ¼ 2.3 Hz, ArH), 11.30 (1H, s, exchangeable, NH); 13C
NMR (500 MHz, DMSO-d6) d: 19.9, 101.3, 115.7, 121.0, 121.5, 125.1,
127.1, 127.6, 130.0, 136.7, 141.7, 144.6, 154.9, 159.0.
By following the same procedure the following compound were
synthesized.
(DMSO-d6) d: 2.62 (3H, s, Me), 6.90 (1H, s, ArH), 7.45e7.48 (2H, m,
2 ꢁ ArH), 7.76e7.79 (2H, m, 2 ꢁ ArH), 8.26 (1H, d, J ¼ 9.3 Hz, ArH),
8.71 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH), 9.76 (1H, d, J ¼ 2.3 Hz, ArH),
11.30 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d:
4.1.6. N-(3-fluorophenyl)-2-methyl-6-nitroquinolin-4-amine (14c)
Compound 14c was synthesized from compound 12 (2.89 g,
13.0 mmol), 3-fluoroaniline 13c (1.73 g, 16.0 mmol) and IPA (40 mL)
containing HCl (1-2 drops): Yield 3.71 g (95%); mp > 300 ꢀC; 1H
19.8, 101.6, 115.8, 120.0, 121.1, 121.5, 127.2, 127.3, 132.8, 136.2, 141.5,
144.6, 145.0, 154.7, 157.1.
4.1.12. N-(3-chloro-4-fluorophenyl)-2-methyl-6-nitroquinolin-4-
amine (14i)
NMR (DMSO-d6) d: 2.69 (3H, s, Me), 6.95 (1H, s, ArH), 7.27e7.32 (1H,
m, ArH), 7.36e7.44 (2H, m, 2 ꢁ ArH), 7.62e7.66 (1H, m, ArH), 8.33
(1H, d, J ¼ 9.3 Hz, ArH), 8.71 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH), 9.79
(1H, d, J ¼ 2.3 Hz, ArH), 11.04 (1H, s, exchangeable, NH); 13C NMR
Compound 14i was synthesized from compound 12 (2.00 g,
9.0 mmol), 3-chloro-4-fluoroaniline 13i (1.57 g, 11.0 mmol) and IPA
(30 mL) containing HCl (2 drops): Yield 2.84 g (95%); mp > 300 ꢀC;
(500 MHz, DMSO-d6)
d
: 19.9, 101.9, 112.3, 112.5, 114.4, 115.8, 121.2,
1H NMR (DMSO-d6)
d: 2.67 (3H, s, Me), 6.87 (1H, s, ArH), 7.52e7.56
121.6, 127.2, 131.7, 138.5, 141.5, 144.7, 154.8, 157.3, 161.5, 163.5.
(1H, m, ArH), 7.63e7.67 (1H, m, ArH), 7.77e7.80 (1H, m, ArH), 8.32
(1H, d, J ¼ 9.3 Hz, ArH), 8.71 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH), 9.77
(1H, d, J ¼ 2.3 Hz, ArH), 11.40 (1H, s, exchangeable, NH); 13C NMR
4.1.7. N-(4-fluorophenyl)-2-methyl-6-nitroquinolin-4-amine (14d)
Compound 14d was synthesized from compound 12 (2.00 g,
9.0 mmol), 4-fluoroaniline 13d (1.20 g, 11.0 mmol) and IPA (30 mL)
containing HCl (2 drops): Yield 2.61 g (96%); mp > 300 ꢀC; 1H NMR
(500 MHz, DMSO-d6) d: 19.8, 61.9, 101.8, 115.6, 118.1, 118.2, 120.5,
120.7, 120.9,121.6, 126.6,127.3,127.9, 134.0,141.4, 144.7,155.3,157.3.
(DMSO-d6)
d
: 2.64 (3H, s, Me), 6.76 (1H, s, ArH), 7.42e7.47 (2H, m,
4.1.13. N-(3-bromo-4-fluorophenyl)-2-methyl-6-nitroquinolin-4-
amine (14j)
2 ꢁ ArH), 7.51e7.56 (2H, m, 2 ꢁ ArH), 8.23 (1H, d, J ¼ 9.2 Hz, ArH),
8.70 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.2 Hz, ArH), 9.75 (1H, d, J ¼ 2.2 Hz, ArH),
Compound 14j was synthesized from compound 12 (2.00 g,
9.0 mmol), 3-bromo-4-fluoroaniline 13j (2.05 g, 11.0 mmol) and IPA
(30 mL) containing HCl (2 drops): Yield 3.21 g (95%); mp > 300 ꢀC;
11.26 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d:
19.9, 61.9, 101.3, 115.6, 116.8, 117.0, 120.9, 121.5, 127.2, 127.7, 127.8,
133.0, 141.5, 144.6, 155.3, 157.0.
1H NMR (DMSO-d6)
d: 2.67 (3H, s, Me), 6.85 (1H, s, ArH), 7.55e7.63
(2H, m, 2 ꢁ ArH), 7.87e7.89 (1H, m, ArH), 8.31 (1H, d, J ¼ 9.3 Hz,
ArH), 8.71 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH), 9.76 (1H, d, J ¼ 2.3 Hz,
ArH), 11.37 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-
d6) d: 19.8, 101.8, 108.9, 115.6, 117.8, 118.0, 121.0, 121.5, 127.1, 127.2,
130.6, 134.3, 141.4, 144.6, 155.2, 156.3, 157.3, 158.2.
4.1.8. N-(3-chlorophenyl)-2-methyl-6-nitroquinolin-4-amine (14e)
Compound 14e was synthesized from compound 12 (1.55 g,
7.0 mmol), 3-chloroaniline 13e (1.07 g, 8.4 mmol) and IPA (30 mL)
containing HCl (2 drops): Yield 2.00 g (92%); mp > 300 ꢀC; 1H NMR
(DMSO-d6) d: 2.69 (3H, s, Me), 6.92 (1H, s, ArH), 7.49e7.53 (2H, m,
2 ꢁ ArH), 7.60e7.64 (2H, m, 2 ꢁ ArH), 8.31 (1H, d, J ¼ 9.2 Hz, ArH),
4.1.14. N-(3-methylphenyl)-2-methyl-6-nitroquinolin-4-amine
(14k)
Compound 14k was synthesized from compound 12 (2.67 g,
12.0 mmol), m-toluidine 13k (1.54 g, 14.0 mmol) and IPA (30 mL)
containing HCl (2 drops): Yield 2.84 g (81%); mp 296e297 ꢀC; 1H
8.71 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.2 Hz, ArH), 9.78 (1H, d, J ¼ 2.2 Hz, ArH),
11.39 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d:
19.1, 101.9, 115.8, 121.1, 121.5, 123.8, 125.1, 127.2, 127.4, 131.5, 134.0,
138.4, 141.5, 144.6, 154.8, 157.3.
NMR (DMSO-d6) d: 2.40 (3H, s, Me), 2.65 (3H, s, Me), 6.79 (1H, s,
4.1.9. N-(4-chlorophenyl)-2-methyl-6-nitroquinolin-4-amine (14f)
Compound 14f was synthesized from 4-chloro-2-methyl-6-
nitroquinoline 12 (2.00 g, 9.0 mmol), 4-chloroaniline 13f (1.38 g,
11.0 mmol) and IPA (30 mL) containing HCl (2 drops): Yield 2.73 g
ArH), 7.26e7.30 (3H, m, 3 ꢁ ArH), 7.45e7.49 (1H, m, ArH), 8.30 (1H,
d, J ¼ 9.3 Hz, ArH), 8.69 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH), 9.78 (1H,
d, J ¼ 2.3 Hz, ArH), 11.30 (1H, s, exchangeable, NH); 13C NMR
(500 MHz, DMSO-d6) d: 19.7, 24.5, 100.5, 103.7, 117.3, 119.5, 120.4,
(97%); mp > 300 ꢀC; 1H NMR (DMSO-d6)
d: 2.67 (3H, s, Me), 6.87
120.5, 120.9, 121.7, 129.1, 139.6, 142.3, 144.9, 145.4, 151.5, 156.4.

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B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
4.1.15. N-(3-(trifluoromethyl)phenyl)-2-methyl-6-nitroquinolin-4-
amine (14l)
(60 mL) containing HCl (4 drops): Yield 5.2
g (95%); mp
284e285 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d: 7.21 (1H, s, ArH), 7.63
Compound 14l was synthesized from compound 12 (2.00 g,
9.0 mmol), 3-(trifluoromethyl)aniline 13l (1.74 g,11.0 mmol) and IPA
(30 mL) containing HCl (2 drops): Yield 1.80 g (58%); mp > 300 ꢀC; 1H
(5H, s, ArH), 7.82e7.84 (1H, d, J ¼ 5.8 Hz, ArH), 8.01 (2H, d,
J ¼ 6.9 Hz, ArH), 8.45e8.47 (1H, d, J ¼ 7.8 Hz, ArH), 8.69e8.71 (1H, d,
J ¼ 8.4 Hz, ArH), 9.76 (1H, s, ArH), 11.22 (1H, s, exchangeable, NH);
NMR (DMSO-d6)
d
: 2.68 (3H, s, Me), 6.96 (1H, s, ArH), 7.81e7.83 (2H,
13C NMR (500 MHz, DMSO-d6)
d: 107.4, 114.3, 117.7, 117.8, 118.0,
m, 2 ꢁ ArH), 7.85e7.86 (2H, m, 2 ꢁ ArH), 8.30 (1H, d, J ¼ 9.2 Hz, ArH),
118.2, 121.5, 121.8, 127.3, 128.7, 129.1, 132.1, 139.1, 139.5, 143.8, 144.7,
8.71 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.2 Hz, ArH), 9.76 (1H, d, J ¼ 2.2 Hz, ArH),
148.8, 153.2, 162.7; ESI-HRMS calcd for
C22H13ClFN3O2 m/z
11.41 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d:
394.0759 (MþH), found 394.0753 (MþH).
19.8, 101.8, 115.9, 120.4, 121.6, 121.9, 122.6, 123.9, 124.8, 127.2, 129.0,
130.7, 130.9, 131.2, 137.9, 141.5, 144.7, 154.8, 157.5.
4.1.21. N-(m-tolyl)-6-nitro-2-phenylquinolin-4-amine (21c)
Compound 21c was synthesized from compound 20 (4 g,
14 mmol), m-toluidine 13k (1.5 g, 14 mmol) and IPA (60 mL) con-
taining HCl (4 drops): Yield 4.7 g (95%) mp 271e272; 1H NMR
4.1.16. N-(3-ethynylphenyl)-2-methyl-6-nitroquinolin-4-amine
(14m)
Compound 14m was synthesized from compound 12 (2.67 g,
12.0 mmol), 3-ethynylaniline 13m (1.5 g, 13.0 mmol) and IPA
(30 mL) containing HCl (2 drops): Yield 2.80 g (77%); mp
(400 MHz, DMSO-d6) d: 2.40 (3H, s, Me), 7.03 (1H, s, ArH), 7.26e7.28
(1H, m, ArH), 7.40e7.42 (2H, m, ArH), 7.47e7.50 (1H, m, ArH),
7.61e7.68 (3H, m, ArH), 7.94e7.96 (2H, m, ArH), 8.64e8.66 (1H, m,
ArH), 8.73e8.74 (1H, m, ArH), 9.88 (1H, s, ArH), 11.21 (1H, s,
278e279 ꢀC; 1H NMR (DMSO-d6)
d: 2.67 (3H, s, Me), 4.36 (1H, s,
CH), 6.85 (1H, s, ArH), 7.54e7.63 (4H, m, 4 ꢁ ArH), 8.30 (1H, d,
J ¼ 9.3 Hz, ArH), 8.71 (1H, dd, J ¼ 2.3 Hz, J ¼ 9.3 Hz, ArH), 9.78 (1H, d,
J ¼ 2.3 Hz, ArH), 11.35 (1H, s, exchangeable, NH); 13C NMR
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 21.00, 100.0,
116.1, 117.9, 118.2, 121.0, 122.0, 125.5, 127.4, 128.4, 128.7, 129.3, 129.9,
132.3, 136.7, 139.7, 144.7, 148.5, 153.9, 163.3; ESI-HRMS calcd for
(500 MHz, DMSO-d6)
d
: 19.9, 82.0, 82.5, 101.6, 115.8, 121.0, 121.5,
C22H17N3O2 m/z 356.1399 (MþH), found 356.1389 (MþH).
123.3, 125.9, 127.1, 128.1, 130.4, 130.7, 137.2, 141.6, 144.6, 154.8, 157.2.
4.1.22. N-(3-methoxyphenyl)-6-nitro-2-phenylquinolin-4-amine
(21d)
Compound 21d was synthesized from compound 20 (4 g,
14 mmol), m-anisidine 13n (1.72 g, 14 mmol) and IPA (60 mL)
containing HCl (4 drops): Yield 4.2 g (82%) mp 257e258 ꢀC; 1H NMR
4.1.17. N-(3-methoxyphenyl)-2-methyl-6-nitroquinolin-4-amine
(14n)
Compound 14n was synthesized from compound 12 (2.00 g,
9.0 mmol), 3-methoxyaniline 13n (1.33 g, 11.0 mmol) and IPA
(30 mL) containing HCl (2 drops): Yield 2.23 g (80%); mp
(400 MHz, DMSO-d6)
d
: 3.82 (3H, s, OMe), 7.02 (1H, d, J ¼ 8.3 Hz,
275e276 ꢀC; 1H NMR (DMSO-d6)
d: 2.65 (3H, s, Me), 3.82 (3H, s,
ArH), 7.14e7.15 (2H, d, J ¼ 2.6 Hz, ArH), 7.18 (1H, d, J ¼ 7.8 Hz, ArH),
7.49e7.52 (1H, t, J ¼ 8 Hz, ArH), 7.63e7.69 (3H, m, ArH), 7.94 (2H, d,
J ¼ 7.1 Hz, ArH), 8.51 (1H, d, J ¼ 9.2 Hz, ArH), 8.75 (1H, d, J ¼ 8.6 Hz,
ArH), 9.83 (1H, s, ArH), 11.33 (1H, s, NH exchangeable); 13C NMR
Me), 6.87 (1H, s, ArH), 7.01e7.07 (3H, m, 3 ꢁ ArH), 7.48e7.52 (1H, m,
ArH), 8.23 (1H, d, J ¼ 9.3 Hz, ArH), 8.70 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.3 Hz,
ArH), 9.74 (1H, d, J ¼ 2.2 Hz, ArH), 11.22 (1H, s, exchangeable, NH);
13C NMR (500 MHz, DMSO-d6)
d: 19.6, 52.2, 103.4, 115.7, 118.8, 121.8,
(500 MHz, DMSO-d6) d: 55.8, 99.7, 107.4, 107.9, 115.7, 117.9, 118.8,
122.0, 128.6, 129.1, 131.0, 132.4,137.9, 139.4,141.4, 144.7, 155.3, 157.3.
122.8,127.3, 127.8,129.0,131.1, 134.7,139.5,143.0,144.4, 146.5,154.1,
159.6, 163.2; ESI-HRMS calcd for C22H17N3O3 m/z 372.1348 (MþH),
found 372.1343 (MþH).
4.1.18. N-(3,4,5-trimethoxyphenyl)-2-methyl-6-nitroquinolin-4-
amine (14o)
Compound 14o was synthesized from compound 12 (2.00 g,
9.0 mmol), 3,4,5-trimethoxyaniline 13o (1.97 g, 11.0 mmol) and IPA
(30 mL) containing HCl (2 drops): Yield 2.20 g (66%); mp
4.1.23. N4,2-methylquinolin-4,6-diamine (15a)
A mixture of N,2-dimethyl-6-nitroquinolin-4-amine (14a, 1.08 g,
5.0 mmol) and iron powder (2.75 g, 50.0 mmol) in aqueous ethanol
(200 mL, 70% v/v) containing acetic acid (3 mL, 50 mmol) was
heated under reflux for 2 h. After the completion of reaction, the
reaction mixture was cooled to room temperature and basified by
addition of aqueous ammonia solution (20 mL). The insoluble
material was removed by filtration through a pad of Celite and the
filtrate was extracted with chloroform. The organic phase was dried
over anhydrous Na2SO4 and evaporated to dryness. The crude
product obtained was recrystallize in ethanol to give 15a,
293e294 ꢀC; 1H NMR (DMSO-d6)
d: 2.67 (3H, s, Me), 3.74 (3H, s,
Me), 3.81 (6H, s, 2 ꢁ Me), 6.80 (2H, s, 2 ꢁ ArH), 6.91 (1H, s, ArH),
8.26 (1H, d, J ¼ 9.3 Hz, ArH), 8.69 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.3 Hz, ArH),
9.74 (1H, d, J ¼ 2.2 Hz, ArH), 11.20 (1H, s, exchangeable, NH); 13C
NMR (500 MHz, DMSO-d6) d: 20.0, 56.1, 60.1, 61.9,101.8,102.9,115.6,
120.8, 121.7, 127.0, 132.4, 136.5, 141.8, 144.5, 153.6, 154.9, 157.0.
4.1.19. N-(3-chlorophenyl)-6-nitro-2-phenylquinolin-4-amine
(21a)
0.47 g (51%); mp 114e115 ꢀC; 1H NMR (DMSO-d6)
d: 2.37 (3H, s, Me),
Compound 21a was synthesized from compound 20 (4 g,
14 mmol), 3-chloroaniline 13e (1.78 g, 14 mmol) and IPA (60 mL)
containing HCl (4 drops): Yield 5 g (96%) mp > 300 ꢀC; 1H NMR
2.81 (3H, d, J ¼ 4.7 Hz, Me), 5.07 (2H, s, exchangeable, NH2), 6.10
(1H, s, ArH), 6.51e6.52 (1H, m, exchangeable, NH), 6.92 (1H, d,
J ¼ 2.3 Hz, ArH), 6.96 (1H, dd, J ¼ 2.3 Hz, J ¼ 8.8 Hz, ArH), 7.41 (1H, d,
(400 MHz, DMSO-d6)
d: 7.14 (1H, s, ArH), 7.52e7.70 (7H, m, ArH),
J ¼ 8.8 Hz, ArH); 13C NMR (500 MHz, DMSO-d6)
d: 19.9, 27.2, 103.4,
7.84e7.87 (2H, m, ArH), 8.27 (1H, d, J ¼ 9.2 Hz), 8.78e8.81 (1H, dd,
118.8, 121.8, 122.0, 131.0, 137.9, 141.4, 155.3, 157.3.
By following the same procedure the following compound were
synthesized.
ArH, J ¼ 2.3 Hz and 9.3 Hz), 9.61 (1H, s, ArH), 11.25 (1H, s,
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 102.6, 114.9,
116.4, 117.2, 117.8, 120.9, 123.3, 128.1, 128.6, 129.2, 131.5, 132.1, 134.0,
139.5, 143.8, 144.7, 153.2, 162.8; ESI-HRMS calcd for C22H13ClFN3O2
m/z 376.0853 (MþH), found 376.0857 (MþH).
4.1.24. N4-phenyl-2-methylquinolin-4,6-diamine (15b)
Compound 15b was synthesized from N-phenyl-2-methyl-6-
nitroquinolin-4-amine (14b, 1.67 g, 6.0 mmol) and iron (3.30 g,
60.0 mmol) in aqueous ethanol (400 mL, 70% v/v) containing acetic
acid (3.6 mL, 60.0 mmol): Yield 0.98 g (65%); mp 83e84 ꢀC; 1H NMR
4.1.20. N-(3-chloro-4-fluorophenyl)-6-nitro-2-phenylquinolin-4-
amine (21b)
Compound 21b was synthesized from compound 20 (4 g,
14 mmol), 3-chloro-4-flouro aniline 13i (2.0 g, 14 mmol) and IPA
(DMSO-d6)
d: 2.36 (3H, s, Me), 5.25 (2H, s, exchangeable, NH2), 6.79
(1H, s, ArH), 6.99e7.02 (1H, m, ArH), 7.05e7.07 (1H, m, ArH),

B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
703
7.13e7.14 (1H, m, ArH), 7.26e7.28 (2H, m, 2 ꢁ ArH), 7.32e7.36 (2H,
m, 2 ꢁ ArH), 8.32 (1H, s, exchangeable, NH); 13C NMR (500 MHz,
d6) d: 19.6, 100.6, 105.0, 117.9, 121.0, 121.0, 121.6, 122.0, 123.7, 129.2,
131.0, 142.5, 144.1, 144.4, 145.4, 153.1.
DMSO-d6) d: 20.0, 103.6, 116.4, 118.2, 121.5, 121.6, 122.8, 128.6, 131.2,
4.1.30. N4-(4-bromophenyl)-2-methylquinolin-4,6-diamine (15h)
Compound 15h was synthesized from N-(4-bromophenyl)-2-
methyl-6-nitroquinolin-4-amine (14h, 2.14 g, 6.0 mmol) and iron
(3.30 g, 60.0 mmol) in aqueous ethanol (500 mL, 70% v/v) con-
taining acetic acid (3.6 mL, 60.0 mmol): Yield 0.99 g (51%); mp
139.9, 142.8, 144.9, 145.2, 155.6.
4.1.25. N4-(3-fluorophenyl)-2-methylquinolin-4,6-diamine (15c)
Compound 15c was synthesized from N-(3-fluorophenyl)-2-
methyl-6-nitroquinolin-4-amine (14c, 2.69 g, 9.0 mmol) and iron
(4.95 g, 90.0 mmol) in aqueous ethanol (500 mL, 70% v/v) con-
taining acetic acid (5.4 mL, 90.0 mmol): Yield 1.50 g (62%); mp
120e121 ꢀC; 1H NMR (DMSO-d6)
d: 2.38 (3H, s, Me), 5.30 (2H, s,
exchangeable, NH2), 6.84 (1H, s, ArH), 7.06e7.08 (2H, m, 2 ꢁ ArH),
7.20e7.22 (2H, m, 2 ꢁ ArH), 7.45e7.49 (2H, m, 2 ꢁ ArH), 7.52e7.54
(1H, m, ArH), 8.46 (1H, s, exchangeable, NH); 13C NMR (500 MHz,
DMSO-d6) d: 24.5, 100.7, 104.0, 112.8, 120.7, 120.9, 121.8, 129.1, 131.9,
132.3, 141.7, 142.3, 144.6, 145.3, 153.1.
140e141 ꢀC; 1H NMR (DMSO-d6)
d: 2.55 (3H, s, Me), 5.94 (2H, s,
exchangeable, NH2), 6.76 (1H, s, ArH), 7.16e7.20 (1H, m, ArH),
7.28e7.39 (4H, m, 4 ꢁ ArH), 7.53e7.59 (1H, m, ArH), 7.72e7.75 (1H,
m, ArH), 10.06 (1H, s, exchangeable, NH); 13C NMR (500 MHz,
DMSO-d6) d: 19.5, 100.2, 100.7, 111.6, 112.9, 113.1, 118.6, 120.6, 124.0,
130.7, 131.4, 139.9, 140.0, 148.0, 149.9, 151.7, 161.6, 163.6.
4.1.31. N4-(3-chloro-4-fluorophenyl)-2-methylquinolin-4,6-
diamine (15i)
4.1.26. N4-(4-fluorophenyl)-2-methylquinolin-4,6-diamine (15d)
Compound 15d was synthesized from N-(4-fluorophenyl)-2-
methyl-6-nitroquinolin-4-amine (14d, 1.50 g, 5.0 mmol) and iron
(2.77 g, 50.0 mmol) in aqueous ethanol (300 mL, 70% v/v) con-
taining acetic acid (3.0 mL, 50.0 mmol): Yield 0.70 g (52%); mp
Compound 15i was synthesized from N-(3-chloro-4-
fluorophenyl)-2-methyl-6-nitroquinolin-4-amine (14i, 2.32 g,
7.0 mmol) and iron (3.85 g, 70.0 mmol) in aqueous ethanol (500 mL,
70% v/v) containing acetic acid (4.2 mL, 70.0 mmol): Yield 1.12 g
(51%); mp 124e125 ꢀC; 1H NMR (DMSO-d6)
d: 2.38 (3H, s, Me), 5.32
114e115 ꢀC; 1H NMR (DMSO-d6)
d
: 2.34 (3H, s, Me), 5.25 (2H, s,
(2H, s, exchangeable, NH2), 6.77 (1H, s, ArH), 7.06e7.08 (2H, m,
2 ꢁ ArH), 7.22e7.26 (1H, m, ArH), 7.34e7.39 (2H, m, 2 ꢁ ArH),
7.52e7.54 (1H, m, ArH), 8.44 (1H, s, exchangeable, NH); 13C NMR
(500 MHz, DMSO-d6) d: 19.7, 100.5, 103.7, 117.3, 119.5, 120.4, 120.5,
120.9, 121.7, 129.1, 139.6, 142.3, 144.9, 145.4, 151.5, 156.4.
exchangeable, NH2), 6.64 (1H, s, ArH), 7.04e7.07 (1H, m, ArH),
7.12e7.13 (1H, m, ArH), 7.16e7.21 (2H, m, 2 ꢁ ArH), 7.27e7.31 (2H,
m, 2 ꢁ ArH), 7.50e7.52 (1H, m, ArH), 8.31 (1H, s, exchangeable, NH);
13C NMR (500 MHz, DMSO-d6)
d
: 24.6, 100.7, 102.0, 115.7, 115.9,
120.0, 120.8, 123.3, 123.39, 129.0, 138.0, 142.2, 145.2, 146.0, 153.2,
156.9, 158.8.
4.1.32. N4-(3-bromo-4-fluorophenyl)-2-methylquinolin-4,6-
diamine (15j)
4.1.27. N4-(3-chlorophenyl)-2-methylquinolin-4,6-diamine (15e)
Compound 15e was synthesized from N-(3-chlorophenyl)-2-
methyl-6-nitroquinolin-4-amine (14e, 1.88 g, 6.0 mmol) and iron
(3.30 g, 60.0 mmol) in aqueous ethanol (300 mL, 70% v/v) con-
taining acetic acid (3.6 mL, 60.0 mmol): Yield 0.91 g (54%); mp
Compound 15j was synthesized from N-(3-bromo-4-
fluorophenyl)-2-methyl-6-nitroquinolin-4-amine (14j, 2.25 g,
6.0 mmol) and iron (3.30 g, 60.0 mmol) in aqueous ethanol
(500 mL, 70% v/v) containing acetic acid (3.6 mL, 60.0 mmol): Yield
1.4 g (68%); mp 129e130 ꢀC; 1H NMR (DMSO-d6)
d 2.38 (3H, s, Me),
119e120 ꢀC; 1H NMR (DMSO-d6)
d
: 2.41 (3H, s, Me), 5.33 (2H, s,
5.31 (2H, s, exchangeable, NH2), 6.75 (1H, s, ArH), 7.06e7.09 (2H, m,
2 ꢁ ArH), 7.28e7.36 (2H, m, 2 ꢁ ArH), 7.49e7.54 (2H, m, 2 ꢁ ArH),
exchangeable, NH2), 6.89 (1H, s, ArH), 6.97e6.99 (1H, m, ArH),
7.06e7.09 (2H, m, 2 ꢁ ArH), 7.19e7.24 (2H, m, 2 ꢁ ArH), 7.30e7.34
(1H, m, ArH), 7.53e7.55 (1H, m, ArH), 8.51 (1H, s, exchangeable,
8.44 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d:
24.5, 100.6, 103.6, 108.0, 116.9, 117.1, 120.5, 120.9, 121.2, 124.6, 129.1,
139.8, 142.3, 145.0, 145.4, 153.1, 154.5.
NH); 13C NMR (500 MHz, DMSO-d6)
d: 19.2, 100.7, 102.9, 117.8, 120.1,
121.8, 123.5, 123.8, 124.4, 124.8, 126.0, 130.7, 133.8, 139.6, 147.8,
149.8, 150.7, 151.6, 152.3.
4.1.33. N4-(3-methylphenyl)-2-methylquinolin-4,6-diamine (15k)
Compound 15k was synthesized from N-(3-methylphenyl)-2-
methyl-6-nitroquinolin-4-amine (14k, 2.34 g, 8.0 mmol) and iron
(4.40 g, 80.0 mmol) in aqueous ethanol (400 mL, 70% v/v) con-
taining acetic acid (4.8 mL, 80.0 mmol): Yield 1.60 g (60%); mp
4.1.28. N4-(4-chlorophenyl)-2-methylquinolin-4,6-diamine (15f)
Compound 15f was synthesized from N-(4-chlorophenyl)-2-
methyl-6-nitroquinolin-4-amine (14f, 2.19 g, 7.0 mmol) and iron
(3.85 g, 70.0 mmol) in aqueous ethanol (500 mL, 70% v/v) con-
taining acetic acid (4.2 mL, 70.0 mmol): Yield 1.20 g (61%); mp
87e88 ꢀC; 1H NMR (DMSO-d6)
d 2.29 (3H, s, Me), 2.36 (3H, s, Me),
5.24 (2H, s, exchangeable, NH2), 6.76 (1H, s, ArH), 6.82e6.84 (1H, m,
ArH), 7.04e7.08 (3H, m, 3 ꢁ ArH), 7.13e7.14 (1H, m, ArH), 7.20e7.24
(1H, m, ArH), 7.50e7.52 (1H, m, ArH), 8.25 (1H, s, exchangeable,
125e126 ꢀC; 1H NMR (DMSO-d6)
d: 2.38 (3H, s, Me), 5.30 (2H, s,
exchangeable, NH2), 6.82 (1H, s, ArH), 7.06e7.09 (2H, m, 2 ꢁ ArH),
7.25e7.28 (2H, m, 2 ꢁ ArH), 7.35e7.37 (2H, m, 2 ꢁ ArH), 7.52e7.54
(1H, m, ArH), 8.45 (1H, s, exchangeable, NH); 13C NMR (500 MHz,
NH); 13C NMR (500 MHz, DMSO-d6)
d: 21.1, 24.6, 100.8, 102.8, 117.9,
120.3, 120.8, 121.4, 122.9, 129.0, 138.4, 141.8, 142.3, 145.1, 145.5,
153.1.
DMSO-d6) d: 24.5, 100.7, 103.8, 120.6, 120.9, 121.5, 124.1, 124.7, 125.0,
129.0, 129.1, 129.4, 141.2, 142.4, 144.7, 145.3, 153.1.
4.1.34. N4-(3-(trifluoromethyl)phenyl)-2-methylquinolin-4,6-
diamine (15l)
4.1.29. N4-(3-bromophenyl)-2-methylquinolin-4,6-diamine (15g)
Compound 15g was synthesized from N-(3-bromophenyl)-2-
methyl-6-nitroquinolin-4-amine (14g, 1.43 g, 4.0 mmol) and iron
(2.20 g, 40.0 mmol) in aqueous ethanol (300 mL, 70% v/v) con-
taining acetic acid (2.4 mL, 40.0 mmol): Yield 0.6 g (45%); mp
Compound 15l was synthesized from N-(3-(trifluoromethyl)
phenyl)-2-methyl-6-nitroquinolin-4-amine (14l, 1.56 g, 4.5 mmol)
and iron (2.47 g, 45.0 mmol) in aqueous ethanol (400 mL, 70% v/v)
containing acetic acid (2.7 mL, 45.0 mmol): Yield 0.93 g (65%); mp
117e119 ꢀC; 1H NMR (DMSO-d6)
d: 2.40 (3H, s, Me), 5.33 (2H, s,
91e92 ꢀC; 1H NMR (DMSO-d6)
d: 2.41 (3H, s, Me), 5.35 (2H, s,
exchangeable, NH2), 6.88 (1H, s, ArH), 7.05e7.12 (3H, m, 3 ꢁ ArH),
7.24e7.26 (2H, m, 2 ꢁ ArH), 7.38e7.39 (1H, m, ArH), 7.53e7.55 (1H,
m, ArH), 8.50 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-
exchangeable, NH2), 6.92 (1H, s, ArH), 7.08e7.11 (2H, m, 2 ꢁ ArH),
7.25e7.26 (1H, m, ArH), 7.51e7.57 (4H, m, 4 ꢁ ArH), 8.67 (1H, s,
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 24.5, 100.6,

704
B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
105.1, 115.4, 117.3, 121.1, 122.3, 123.1, 125.3, 127.4, 129.2, 129.9, 130.2,
142.5, 144.1, 145.5, 153.1.
4.1.39. N4-(3-chloro-4-fluorophenyl)-2-phenylquinoline-4,6-
diamine (22b)
Compound 22b was synthesized from N-(3-chloro-4-
fluorophenyl)-6-nitro-2-phenylquinolin-4-amine (21b, 5.9 g,
15 mmol) and SnCl2$2H2O (17.1 g, 76 mmol) in methanol (75 mL):
4.1.35. N4-(3-ethynylphenyl)-2-methylquinolin-4,6-diamine (15m)
Compound 15m was synthesized from N-(3-ethynylphenyl)-2-
methyl-6-nitroquinolin-4-amine (14m, 2.12 g, 7.0 mmol) and iron
(3.85 g, 70.0 mmol) in aqueous ethanol (400 mL, 70% v/v) con-
taining acetic acid (4.2 mL, 70.0 mmol): Yield 1.60 g (83%); mp
Yield 4.2 g (76%); mp 237e238 ꢀC; 1H NMR (400 MHz, DMSOed6)
d:
5.61 (2H, s, NH2 exchangeable), 7.13 (1H, s, ArH), 7.17e7.19 (1H, d,
J ¼ 7.5 Hz, ArH), 7.37e7.42 (4H, m, ArH), 7.44e7.49 (3H, m, ArH),
7.72e7.74 (1H, d, J ¼ 8.8 Hz, ArH), 7.97e7.99 (2H, d, J ¼ 7.4 Hz, ArH),
93e94 ꢀC; 1H NMR (DMSO-d6)
d: 2.39 (3H, s, Me), 4.16 (1H, s, CH),
8.73 (1H, s, NH, exchangeable); 13C NMR (500 MHz, DMSO-d6)
d:
5.31 (2H, s, exchangeable, NH2), 6.83 (1H, s, ArH), 7.06e7.08 (3H, m,
3 ꢁ ArH), 7.30e7.35 (3H, m, 3 ꢁ ArH), 7.52e7.55 (1H, m, ArH), 8.43
104.5, 115.2, 116.4, 118.4, 123.2, 124.6, 127.1, 128.6, 129.1, 131.5, 132.1,
134.0, 135.1, 139.3, 139.6, 144.7, 147.4, 153.5; ESI-HRMS calcd for
(1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 24.6,
C21H15ClFN3 m/z 364.1017 (MþH), found 364.1012 (MþH).
80.5, 83.5, 100.7, 104.2, 120.4, 120.8, 121.0, 122.5, 122.7, 124.2, 124.9,
129.1, 129.6, 142.4, 144.7, 145.3, 153.1.
4.1.40. 2-Phenyl-N4-(m-tolyl)quinoline-4,6-diamine (22c)
Compound 22c was synthesized from 6-nitro-2-phenyl-N-(m-
tolyl)quinolin-4-amine (21c, 2.0 g, 5.6 mmol) and stannous chlo-
ride dihydrate (6 g, 26.5 mmol)in methanol (50 mL): Yield
4.1.36. N4-(3-methoxyphenyl)-2-methylquinolin-4,6-diamine (15n)
Compound 15n was synthesized from N-(3-methoxyphenyl)-2-
methyl-6-nitroquinolin-4-amine (14n, 1.85 g, 6.0 mmol) and iron
(3.30 g, 60.0 mmol) in aqueous ethanol (400 mL, 70% v/v) con-
taining acetic acid (3.6 mL, 60.0 mmol): Yield 0.34 g (24%); mp
1.65 g (90%); mp 198e199 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d: 2.38
(3H, s, Me), 6.13 (2H, brs, NH2, exchangeable), 6.86 (1H, s, ArH),
7.17e7.19 (1H, m, ArH), 7.34e7.36 (2H, m, ArH), 7.41e7.45 (2H, m,
ArH), 7.57e7.61 (4H, m, ArH), 7.85e7.87 (2H, m, ArH), 8.25e8.26
(1H, d, J ¼ 9.0 Hz, ArH), 10.41 (1H, s, NH, exchangeable); 13C NMR
88e90 ꢀC; 1H NMR (DMSO-d6)
d: 2.38 (3H, s, Me), 3.74 (3H, s, Me),
5.27 (2H, s, exchangeable, NH2), 6.56e6.59 (1H, m, ArH), 6.82e6.87
(3H, m, 3 ꢁ ArH), 7.06 (1H, dd, J ¼ 2.3 Hz, J ¼ 8.8 Hz, ArH), 7.11 (1H,
d, J ¼ 2.3 Hz, ArH), 7.21e7.25 (1H, m, ArH), 7.52 (1H, d, J ¼ 8.8 Hz,
ArH), 8.33 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
(500 MHz, DMSO-d6) d: 21.0, 97.9, 100.7, 118.8, 121.8, 122.0, 125.4,
127.3, 128.0, 128.2, 129.1, 129.6, 131.2, 132.4, 137.9, 139.4, 148.2,
148.3, 152.3; ESI-HRMS calcd for C22H19N3 m/z 326.1657 (MþH),
found 326.1646 (MþH).
d: 20.0, 56.2, 99.3, 105.5, 108.7, 117.4, 118.2, 121.5, 128.6, 131.2, 139.9,
142.8, 144.9, 145.2, 155.6.
4.1.41. N4-(3-methoxyphenyl)-2-phenylquinoline-4,6-diamine
(22d)
4.1.37. N4-(3,4,5-trimethoxyphenyl)-2-methylquinolin-4,6-diamine
(15o)
Compound 22d was synthesized from N-(3-methoxyphenyl)-6-
nitro-2-phenylquinolin-4-amine (21d, 4.0 g, 10.7 mmol) and stan-
nous chloride dihydrate (12 g, 53 mmol) in methanol (100 mL):
Compound
15o
was
synthesized
from
N-(3,4,5-
(14o,
trimethoxyphenyl)-2-methyl-6-nitroquinolin-4-amine
Yield 3.3 g (90%); mp 173e174 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
1.85 g, 5.0 mmol) and iron (2.75 g, 50.0 mmol) in aqueous ethanol
(400 mL, 70% v/v) containing acetic acid (3.0 mL, 50.0 mmol): Yield
3.75 (3H, s, OMe), 5.54 (2H, brs, NH2, exchangeable), 6.61 (1H, s,
ArH), 6.92e6.97 (2H, m, ArH), 7.16 (2H, m, ArH), 7.27 (1H, m, ArH),
7.38 (1H, m, ArH), 7.45e7.50 (3H, m, ArH), 7.69e7.71 (1H, d,
J ¼ 8.0 Hz, ArH), 7.95e7.96 (2H, m, ArH), 8.56 (1H, s, NH); 13C NMR
0.74 g (44%); mp 192e193 ꢀC; 1H NMR (DMSO-d6)
d: 2.37 (3H, s,
Me), 3.65 (3H, s, Me), 3.74 (6H, s, 2 ꢁ Me), 5.24 (2H, s, exchangeable,
NH2), 6.60 (2H, s, 2 ꢁ ArH), 6.82 (1H, s, ArH), 7.05 (1H, dd, J ¼ 2.3 Hz,
J ¼ 8.9 Hz, ArH), 7.11 (1H, d, J ¼ 2.3 Hz, ArH), 7.50 (1H, d, J ¼ 8.9 Hz,
ArH), 8.21 (1H, s, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
(500 MHz, DMSO-d6) d: 55.0, 99.4, 104.8, 107.7, 110.5, 118.4, 121.5,
126.3,128.3,128.6,130.1,131.2,134.7,139.3,139.7,143.3,147.4,153.5,
160.2; ESI-HRMS calcd for C22H19N3O m/z 342.1606 (MþH), found
342.1600 (MþH).
d: 20.1, 56.9, 60.6, 100.4, 105.3, 118.2, 120.6, 120.8, 129.3, 131.7, 136.1,
137.8, 142.7, 145.2, 155.1, 155.6.
4.1.38. N4-(3-chlorophenyl)-2-phenylquinoline-4,6-diamine (22)
To a solution of N-(3-chlorophenyl)-6-nitro-2-phenylquinolin-
4-amine (21a, 2 g, 5.3 mmol) in methanol (50 mL) was added
SnCl2$2H2O (6 g, 26.5 mmol) and resulting mixture was heated at
refluxed temperature for overnight. The mixture was allowed to
cool at room temperature and concentrated in vacuo to remove
the solvent. The residue was then taken up in 200 mL of 10%
NaHCO3 and extracted with CHCl3 (200 mL). The combined
organic extracts were washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure to obtain
compound 22a as a solid. The crude product was crystallized from
ethanol. Yield 1.65 g (90%); mp 215e216 ꢀC; 1H NMR (400 MHz,
4.1.42. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-methylamino)-
2-methylquinolin-6-yl)urea (18a)
To a solution of N4,2-methylquinolin-4,6-diamine (15a, 0.45 g,
2.4 mmol) in dry DMF (20 mL) containing triethylamine (1.7 mL)
was added a solution of N-mustard-isocyanate 17 (freshly prepared
from 16, 1.84 g, 6.0 mmol and triphosgene, 0.66 g, 2.2 mmol) in dry
DMF (5 mL) at room temperature. After being stirred for 3 h at room
temperature, the solid was filtered and washed with dry DMF. The
filtrate was evaporated to dryness in vacuo. The residue was puri-
fied by column chromatography using CHCl3/MeOH (100:2 v/v) as
an eluent. The fractions containing the main product were com-
bined and evaporated to dryness and the residue was recrystallized
from CHCl3 to give 18a 0.44 g (42%); mp 230e232 ꢀC (dec); 1H NMR
DMSO-d6)
d: 5.60 (2H, s, NH2 exchangeable), 7.10e7.11 (1H, d,
J ¼ 2.1 Hz, ArH), 7.14e7.18 (2H, m, ArH), 7.28e7.31 (1H, m, ArH),
7.34e7.40 (2H, m, ArH), 7.44e7.53 (3H, m, ArH), 7.53 (1H, s, ArH),
7.72 (1H, d, J ¼ 8.8 Hz, ArH), 7.99 (2H, d, J ¼ 7.4, ArH), 8.71 (1H, s,
(DMSO-d6)
d
: 2.62 (3H, s, Me), 3.04 (3H, d, J ¼ 4.7 Hz, Me),
3.67e3.73 (8H, m, 4 ꢁ CH2), 6.60 (1H, s, ArH), 6.72 (2H, d, J ¼ 9.0 Hz,
2 ꢁ ArH), 7.33 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 7.82e7.88 (2H, m,
2 ꢁ ArH), 8.38e8.39 (1H, m, ArH), 8.96e8.97 (1H, m, exchangeable,
NH), 9.10, and 9.44 (each 1H, s, exchangeable, 2 ꢁ NH); 13C NMR
NH exchangeable); 13C NMR (500 MHz, DMSO-d6)
d: 100.2, 102.2,
117.8, 121.7, 122.0, 122.2, 123.8, 126.4, 128.6, 130.4, 131.2, 139.9,
142.8, 144.4, 144.8, 146.4, 151.3; ESI-HRMS calcd for C21H16ClN3 m/z
346.1111 (MþH), found 346.1106 (MþH).
(500 MHz, DMSO-d6) d: 19.6, 30.0, 41.2, 52.4, 97.7,108.6,112.5,116.2,
120.1, 120.3, 125.7, 129.8, 132.9, 138.2, 141.9, 151.7, 152.8, 154.7; ESI-
HRMS calcd for C22H25Cl2N5O m/z 446.1514 (MþH), found 446.1497
(MþH).
By following the same procedure the following compound were
synthesized.

B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
705
By the following procedure he following compound were
synthesized.
ArH), 9.05, 9.45 (each 1H, s, exchangeable, 2 ꢁ NH), 10.28 (1H, brs,
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
: 20.5, 41.2,
d
52.4, 101.4, 108.7, 112.5, 117.7, 120.3, 121.9, 122.6, 123.8, 125.6, 125.8,
129.7, 131.2, 133.8, 138.4, 140.3, 141.9, 151.9, 152.7; ESI-HRMS calcd
for C27H26Cl3N5O m/z 542.1281 (MþH), found 542.1263 (MþH).
4.1.43. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-phenylamino)-
2-methylquinolin-6-yl)urea (18b)
Compound 18b was synthesized from isocyanate 17 (freshly
prepared from 16, 2.14 g, 7.0 mmol and triphosgene, 0.81 g,
2.73 mmol) and N4-phenyl-2-methylquinolin-4,6-diamine (15b,
0.69 g, 2.8 mmol) in dry DMF (30 mL) containing triethylamine
(2.0 mL): Yield 0.56 (40%); mp 271e272 ꢀC (dec); 1H NMR (DMSO-
4.1.47. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((4-
chlorophenyl)amino)-2-methyl-quinolin-6-yl)urea (18f)
Compound 18f was synthesized from isocyanate 17 (freshly
prepared from 16, 2.14 g, 7.0 mmol and triphosgene, 0.69 g,
2.8 mmol) and N4-(4-chlorophenyl)-2-methylquinolin-4,6-diamine
(15f, 0.79 g, 2.8 mmol) in dry DMF (30 mL) containing triethylamine
(1.9 mL): Yield 0.91 (60%); mp 289e290 ꢀC (dec); 1H NMR (DMSO-
d6)
d
: 2.49 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2), 6.70e6.73 (3H,
m, 3 ꢁ ArH), 7.29e7.34 (3H, m, 3 ꢁ ArH), 7.42e7.43 (2H, m,
2 ꢁ ArH), 7.49e7.53 (2H, m, 2 ꢁ ArH), 7.86e7.93 (2H, m, 2 ꢁ ArH),
8.54e8.55 (1H, m, ArH), 9.00, 9.32 (each 1H, s, exchangeable,
2 ꢁ NH), 10.08 (1H, brs, exchangeable, NH); 13C NMR (500 MHz,
d6)
d
: 2.54 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2), 6.72 (2H, d,
J ¼ 9.0 Hz, 2 ꢁ ArH), 6.76 (1H, s, ArH), 7.33 (2H, d, J ¼ 9.0 Hz,
2 ꢁ ArH), 7.45 (2H, d, J ¼ 8.7 Hz, 2 ꢁ ArH), 7.55 (2H, d, J ¼ 8.7 Hz,
2 ꢁ ArH), 7.86e7.92 (2H, m, 2 ꢁ ArH), 8.54e8.55 (1H, m, ArH), 9.01,
9.37 (each 1H, s, exchangeable, 2 ꢁ NH), 10.11 (1H, brs, exchange-
DMSO-d6) d: 20.8, 41.2, 52.4, 100.6, 109.0, 112.5, 117.6, 120.3, 122.3,
124.2, 125.5, 125.8, 129.6, 129.8, 136.4, 138.1, 138.6, 141.9, 152.0,
152.8, 153.2; ESI-HRMS calcd for C27H27Cl2N5O m/z 508.1671
(MþH), found 508.1665 (MþH).
able, NH); 13C NMR (500 MHz, DMSO-d6)
d: 20.8, 41.2, 52.4, 101.1,
108.8, 112.5, 117.7, 120.4, 122.4, 125.6, 125.8, 129.6, 129.7, 137.8,
138.2, 141.9, 151.8, 152.8, 153.4; ESI-HRMS calcd for C27H26Cl3N5O
m/z 542.1281 (MþH), found 542.1267 (MþH).
4.1.44. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
fluorophenyl)amino)-2-methyl-quinolin-6-yl)urea (18c)
Compound 18c was synthesized from isocyanate 17 (freshly
prepared from 16, 1.53 g, 5.0 mmol and triphosgene, 0.56 g,
1.9 mmol) and N4-(3-fluorophenyl)-2-methylquinolin-4,6-diamine
(15c, 0.53 g, 2.0 mmol) in dry DMF(20 mL) containing triethylamine
(1.4 mL): Yield 0.35 (35%); mp 211e212 ꢀC (dec); 1H NMR (DMSO-
4.1.48. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
bromophenyl)amino)-2-methyl-quinolin-6-yl)urea (18g)
Compound 18g was synthesized from isocyanate 17 (freshly
prepared from 16, 1.23 g, 4.0 mmol and triphosgene, 0.46 g,
1.6 mmol) and N4-(3-bromophenyl)-2-methylquinolin-4,6-diamine
(15g, 0.53 g, 1.6 mmol) in dry DMF(20 mL) containing triethylamine
(1.1 mL): Yield 0.44 (48%); mp 179e181 ꢀC (dec); 1H NMR (DMSO-
d6)
d
: 2.57 (3H, s, Me), 3.65e3.72 (8H, m, 4 ꢁ CH2), 6.72 (2H, d,
J ¼ 9.0 Hz, 2 ꢁ ArH), 6.95e6.96 (2H, m, 2 ꢁ ArH), 7.16e7.21 (2H, m,
2 ꢁ ArH), 7.32 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 7.41e7.45 (1H, m, ArH),
7.78e7.79 (2H, m, 2 ꢁ ArH), 8.37e8.38 (1H, m, ArH), 8.65, 8.95 (each
d6)
d
: 2.52 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2), 6.71e6.73 (2H,
1H, s, exchangeable, 2 ꢁ NH), 9.35 (1H, brs, exchangeable, NH); 13
C
m, 2 ꢁ ArH), 6.90 (1H, s, ArH), 7.31e7.37 (5H, m, 5 ꢁ ArH), 7.52e7.53
(1H, m, ArH), 7.76e7.79 (2H, m, 2 ꢁ ArH), 8.35e8.36 (1H, m, ArH),
8.62, 8.91 (each 1H, s, exchangeable, 2 ꢁ NH), 9.35 (1H, brs,
NMR (500 MHz, DMSO-d6) d: 23.1, 41.2, 52.4, 103.3, 108.7, 110.1,
112.5, 117.5, 118.9, 120.5, 124.1, 129.8, 130.9, 131.0, 137.1, 141.9, 142.6,
152.8, 155.2, 161.7, 163.7; ESI-HRMS calcd for C27H26Cl2FN5O m/z
526.4326 (MþH), found 526.1569 (MþH).
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 23.4, 41.2,
52.4, 103.4, 108.6, 112.5, 119.1, 120.2, 120.5, 122.1, 123.9, 125.8, 126.5,
129.8, 131.2, 136.9, 141.9, 142.7, 148.0, 152.8, 155.4; ESI-HRMS calcd
for C27H26BrCl2N5O m/z 586.0776 (MþH), found 586.0751 (MþH).
4.1.45. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((4-
fluorophenyl)amino)-2-methyl-quinolin-6-yl)urea (18d)
Compound 18d was synthesized from isocyanate 17 (freshly
prepared from 16, 1.53 g, 5.0 mmol and triphosgene, 0.56 g,
1.9 mmol) and N4-(4-fluorophenyl)-2-methylquinolin-4,6-diamine
(15d, 0.54 g, 2.0 mmol) in dry DMF (30 mL) containing triethyl-
amine (1.4 mL): Yield 0.45 (43%); mp 251e252 ꢀC (dec); 1H NMR
4.1.49. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((4-
bromophenyl)amino)-2-methyl-quinolin-6-yl)urea (18h)
Compound 18h was synthesized from isocyanate 17 (freshly
prepared from 16, 2.14 g, 7.0 mmol and triphosgene, 0.69 g,
2.8 mmol) and N4-(4-bromophenyl)-2-methylquinolin-4,6-
diamine (15h, 0.92 g, 2.8 mmol) in dry DMF (30 mL) containing
triethylamine (1.9 mL): Yield 0.77 (48%); mp 278e279 ꢀC (dec); 1H
(DMSO-d6)
d
: 2.53 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2), 6.60 (1H,
s, ArH), 6.72 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 7.33 (2H, d, J ¼ 9.0 Hz,
2 ꢁ ArH), 7.36e7.38 (2H, m, 2 ꢁ ArH), 7.45e7.48 (2H, m, 2 ꢁ ArH),
7.86e7.94 (2H, m, 2 ꢁ ArH), 8.55e8.56 (1H, m, ArH), 9.01, 9.35 (each
NMR (DMSO-d6)
d
: 2.55 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2),
6.72 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 6.78 (1H, s, ArH), 7.33 (2H, d,
J ¼ 9.0 Hz, 2 ꢁ ArH), 7.39 (2H, d, J ¼ 8.6 Hz, 2 ꢁ ArH), 7.68 (2H, d,
J ¼ 8.6 Hz, 2 ꢁ ArH), 7.86e7.89 (2H, m, 2 ꢁ ArH), 8.55e8.56 (1H, m,
ArH), 8.96, 9.32 (each 1H, s, exchangeable, 2 ꢁ NH), 10.12 (1H, brs,
1H, s, exchangeable, 2 ꢁ NH), 10.18 (1H, brs, exchangeable, NH); 13
C
NMR (500 MHz, DMSO-d6) d: 20.5, 41.2, 52.4, 100.2, 108.9, 112.5,
116.4, 116.6, 117.3, 120.4, 121.8, 125.7, 126.9, 127.0, 129.7, 134.7, 135.7,
138.2, 141.9, 152.7, 152.8, 153.0, 159.1, 161.0; ESI-HRMS calcd for
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 20.7, 41.2,
C
27H26Cl2FN5O m/z 526.1577 (MþH), found 526.1569 (MþH).
52.4, 101.1, 108.8, 112.5, 117.7, 117.8, 120.3, 122.1, 125.6, 126.1, 129.7,
132.5, 136.1, 138.1, 138.2, 141.9, 151.8, 152.8, 153.3; ESI-HRMS calcd
for C27H26BrCl2N5O m/z 586.0776 (MþH), found 586.0771 (MþH).
4.1.46. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
chlorophenyl)amino)-2-methyl-quinolin-6-yl)urea (18e)
Compound 18e was synthesized from isocyanate 17 (freshly
prepared from 16, 1.23 g, 4.0 mmol and triphosgene, 0.46 g,
1.6 mmol) and N4-(3-chlorophenyl)-2-methylquinolin-4,6-diamine
(15e, 0.45 g, 1.6 mmol) in dry DMF(20 mL) containing triethylamine
(1.1 mL): Yield 0.50 (58%); mp 263e264 ꢀC (dec); 1H NMR (DMSO-
4.1.50. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-chloro-4-
fluorophenyl)amino)- 2-methylquinolin-6-yl)urea (18i)
Compound 18i was synthesized from isocyanate 17 (freshly
prepared from 16, 2.14 g, 7.0 mmol and triphosgene, 0.69 g,
2.8 mmol) and N4-(3-chloro-4-fluorophenyl)-2-methylquinolin-
4,6-diamine (15i, 0.84 g, 2.8 mmol) in dry DMF(30 mL) containing
triethylamine (1.9 mL): Yield 0.92 (59%); mp 207e208 ꢀC (dec); 1H
d6)
d: 2.58 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2), 6.72 (2H, d,
J ¼ 9.0 Hz, 2 ꢁ ArH), 6.81 (1H, s, ArH), 7.33 (2H, d, J ¼ 9.0 Hz,
2 ꢁ ArH), 7.37e7.39 (1H, m, ArH), 7.42e7.44 (1H, m, ArH), 7.52e7.55
(2H, m, 2 ꢁ ArH), 7.90e7.95 (2H, m, 2 ꢁ ArH), 8.58e8.59 (1H, m,
NMR (DMSO-d6)
6.72 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 6.75 (1H, s, ArH), 7.33 (2H, d,
d
: 2.54 (3H, s, Me), 3.68e3.71 (8H, m, 4 ꢁ CH2),

706
B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
J ¼ 9.0 Hz, 2 ꢁ ArH), 7.41e7.45 (1H, m, ArH), 7.52e7.56 (1H, m, ArH),
7.62e7.65 (1H, m, ArH), 7.86e7.87 (2H, m, 2 ꢁ ArH), 8.52e8.53 (1H,
m, ArH), 8.88, 9.24 (each 1H, s, exchangeable, 2 ꢁ NH), 9.95 (1H, brs,
3.1 mmol) and N4-(3-ethynylphenyl)-2-methylquinolin-4,6-
diamine (15m, 0.87 g, 3.2 mmol) in dry DMF (30 mL) containing
triethylamine (1.3 mL): Yield 0.77 (46%); mp 273e274 ꢀC (dec); 1H
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d
: 21.1, 41.2, 52.4,
NMR (DMSO-d6)
d
: 2.57 (3H, s, Me), 3.67e3.72 (8H, m, 4 ꢁ CH2),
101.3, 108.6, 112.5, 117.6, 117.7, 117.8, 120.1, 120.2, 120.4, 122.8, 124.9,
125.4, 126.2, 129.7, 136.3, 136.9, 138.1, 141.9, 151.6, 152.8, 153.6,
153.8, 155.8; ESI-HRMS calcd for C27H25Cl3FN5O m/z 560.1187
(MþH), found 560.1198 (MþH).
4.33 (1H, s, CH), 6.71e6.73 (3H, m, 3 ꢁ ArH), 7.32e7.35 (2H, m,
2 ꢁ ArH), 7.47e7.58 (4H, m, 4 ꢁ ArH), 7.93 (1H, d, J ¼ 9.2 Hz, ArH),
7.98 (1H, dd, J ¼ 1.8 Hz, J ¼ 9.2 Hz, ArH), 8.63 (1H, d, J ¼ 1.8 Hz, ArH),
9.11, 9.51 (each 1H, s, exchangeable, 2 ꢁ NH), 10.51 (1H, brs,
exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 19.6, 41.2,
4.1.51. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-bromo-4-
fluorophenyl)amino) -2-methylquinolin-6-yl)urea (18j)
52.3, 81.8, 82.7, 100.4, 108.9, 109.0, 112.5, 117.2, 120.3, 120.4, 123.1,
125.7, 126.4, 128.0, 129.7, 129.9, 130.2, 133.9, 138.2, 138.8, 141.9,
152.5, 152.7, 153.3; ESI-HRMS calcd for C29H27Cl2N5O m/z 532.1671
(MþH), found 532.1648 (MþH).
Compound 18j was synthesized from isocyanate 17 (freshly
prepared from 16, 2.14 g, 7.0 mmol and triphosgene, 0.69 g,
2.8 mmol) and N4-(3-bromo-4-fluorophenyl)-2-methylquinolin-
4,6-diamine (15j, 0.97 g, 2.8 mmol) in dry DMF(30 mL) containing
triethylamine (1.9 mL): Yield 0.80 (47%); mp 197e198 ꢀC (dec); 1H
4.1.55. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
methoxyphenyl)amino)-2-methylquinolin-6-yl)urea (18n)
Compound 18n was synthesized from isocyanate 17 (freshly
prepared from 16, 0.77 g, 2.5 mmol and triphosgene, 0.29 g,
NMR (DMSO-d6)
d
: 2.52 (3H, s, Me), 3.66e3.72 (8H, m, 4 ꢁ CH2),
6.72 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 6.74 (1H, s, ArH), 7.32 (2H, d,
J ¼ 9.0 Hz, 2 ꢁ ArH), 7.44e7.51 (2H, m, 2 ꢁ ArH), 7.70e7.72 (1H, m,
ArH), 7.83e7.84 (2H, m, 2 ꢁ ArH), 8.47e8.48 (1H, m, ArH), 8.84, 9.17
(each 1H, s, exchangeable, 2 ꢁ NH), 9.75 (1H, brs, exchangeable,
1
mmol) and N4-(3-methoxyphenyl)-2-methylquinolin-4,6-
diamine (15n, 0.28, 1.0 mmol) in dry DMF (15 mL) containing
triethylamine (0.7 mL): Yield 0.29 (54%); mp 215e216 ꢀC (dec); 1H
NH); 13C NMR (500 MHz, DMSO-d6)
d
: 21.7, 41.2, 52.4, 101.6, 108.4,
NMR (DMSO-d6)
d
: 2.58 (3H, s, Me), 3.67e3.71 (8H, m, 4 ꢁ CH2),
108.5,108.6,112.5,117.3, 117.5, 118.0, 120.4,123.9,125.0,128.3,129.7,
137.0, 137.7, 141.9, 150.8, 152.8, 154.1, 154.6, 156.5; ESI-HRMS calcd
for C27H25BrCl2FN5O m/z 604.0682 (MþH), found 604.0676 (MþH).
3.80 (3H, s, Me), 6.71e6.73 (3H, m, 3 ꢁ ArH), 6.95e6.97 (1H, m,
ArH), 7.02e7.04 (2H, m, 2 ꢁ ArH), 7.32e7.35 (2H, m, 2 ꢁ ArH),
7.44e7.48 (1H, m, ArH), 7.92 (1H, d, J ¼ 9.0 Hz, ArH), 7.98 (1H, dd,
J ¼ 1.8 Hz, J ¼ 9.0 Hz, ArH), 8.63 (1H, d, J ¼ 1.8 Hz, ArH), 9.09, 9.47
(each 1H, s, exchangeable, 2 ꢁ NH), 10.50 (1H, brs, exchangeable,
4.1.52. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
methylphenyl)amino)-2-methylquinolin-6-yl)urea (18k)
NH); 13C NMR (500 MHz, DMSO-d6)
d: 19.6, 51.2, 52.3, 55.3, 100.3,
Compound 18k was synthesized from isocyanate 17 (freshly pre-
paredfrom16, 2.14g, 7.0 mmol andtriphosgene, 0.69g, 2.8mmol)and
N4-(3-methylphenyl)-2-methylquinolin-4,6-diamine (15k, 0.74 g,
2.8 mmol) in dry DMF (30 mL) containing triethylamine (2.0 mL):
109.0, 110.7, 112.4, 112.5, 117.0, 117.1, 120.3, 120.4, 126.4, 129.7, 130.5,
133.8, 138.7, 138.9, 141.9, 152.3, 152.7, 153.5, 160.2; ESI-HRMS calcd
for C28H29Cl2N5O2 m/z 538.1777 (MþH), found 538.1753 (MþH).
Yield 0.55 (38%); mp 238e239 ꢀC (dec); 1H NMR (DMSO-d6)
d: 2.38
4.1.56. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3,4,5-
(3H, s, Me), 2.56 (3H, s, Me), 3.67e3.72 (8H, m, 4 ꢁ CH2), 6.64 (1H, s,
ArH), 6.72 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 7.20e7.26 (3H, m, 3 ꢁ ArH), 7.33
(2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 7.41e7.45 (1H, m, ArH), 7.92 (1H, d,
J ¼ 9.1 Hz, ArH), 7.99 (1H, dd, J ¼ 1.7 Hz, J ¼ 9.1 Hz, ArH), 8.60 (1H, d,
J ¼ 1.7 Hz, ArH), 9.12, 9.47 (each 1H, s, exchangeable, 2 ꢁ NH), 10.49
trimethoxyphenyl)amino)- 2-methylquinolin-6-yl)urea (18o)
Compound 18o was synthesized from isocyanate 17 (freshly
prepared from 16, 1.23 g, 4.0 mmol and triphosgene, 0.46 g,
1.6 mmol) and N4-(3,4,5-trimethoxyphenyl)-2-methylquinolin-4,6-
diamine (15o, 0.54 g, 1.6 mmol) in dry DMF (30 mL) containing
triethylamine (1.1 mL): Yield 0.36 (38%); mp 278e279 ꢀC (dec); 1H
(1H, brs, exchangeable, NH); 13C NMR (500 MHz, DMSO-d6)
d: 20.7,
22.4, 41.3, 52.5,101.6,108.7,112.3,117.0,120.4,120.5,125.7,129.7,129.8,
130.5, 133.8, 136.2, 138.4, 142.0, 151.9, 152.6, 153.4, 153.9; ESI-HRMS
calcd for C28H29Cl2N5O m/z 522.1827 (MþH), found 522.1838 (MþH).
NMR (DMSO-d6)
d
: 2.59 (3H, s, Me), 3.67e3.71 (8H, m, 4 ꢁ CH2),
3.72 (3H, s, Me), 3.80 (6H, s, 2 ꢁ Me), 6.72 (2H, d, J ¼ 9.0 Hz,
2 ꢁ ArH), 6.76 (3H, s, 3 ꢁ ArH), 7.33 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 7.91
(1H, d, J ¼ 9.1 Hz, ArH), 7.97 (1H, dd, J ¼ 2.0 Hz, J ¼ 9.1 Hz, ArH), 8.61
(1H, d, J ¼ 2.0 Hz, ArH), 9.07, 9.43 (each 1H, s, exchangeable,
2 ꢁ NH), 10.43 (1H, brs, exchangeable, NH); 13C NMR (500 MHz,
4.1.53. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
(trifluoromethyl)phenyl)amino) -2-methylquinolin-6-yl)urea (18l)
Compound 18l was synthesized from isocyanate 17 (freshly
prepared from 16, 1.84 g, 6.0 mmol and triphosgene, 0.66 g,
2.2 mmol) and N4-(3-(trifluoromethyl)phenyl)-2-methylquinolin-
4,6-diamine (15l, 0.76 g, 2.4 mmol) in dry DMF (30 mL) containing
triethylamine (1.7 mL): Yield 0.63 (46%); mp 252e253 ꢀC (dec); 1H
DMSO-d6) d: 19.5, 41.2, 52.3, 56.0, 60.1, 100.3, 102.9, 109.0, 112.5,
117.0, 120.2, 120.3, 126.4, 129.7, 133.3, 133.7, 136.1, 138.7, 141.9, 152.3,
152.8,153.5,153.7; ESI-HRMS calcd for C30H33Cl2N5O4 m/z 598.1988
(MþH), found 598.1987 (MþH).
NMR (DMSO-d6)
d
: 2.55 (3H, s, Me), 3.68e3.72 (8H, m, 4 ꢁ CH2),
4.1.57. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
chlorophenyl)amino)-2-phenyl-quinolin-6-yl)urea (23a)
6.72 (2H, d, J ¼ 9.0 Hz, 2 ꢁ ArH), 6.87 (1H, s, ArH), 7.33 (2H, d,
J ¼ 9.0 Hz, 2 ꢁ ArH), 7.58e7.60 (1H, m, ArH), 7.68e7.75 (3H, m,
3 ꢁ ArH), 7.86e7.89 (2H, m, 2 ꢁ ArH), 8.31e8.32 (1H, m, ArH), 8.85,
9.22 (each 1H, s, exchangeable, 2 ꢁ NH), 10.16 (1H, brs, exchange-
Compound 23a was synthesized from isocyanate 17 (freshly
prepared from 16, 1.28 g, 4.2 mmol and triphosgene, 0.49 g,
1.62 mmol) and N4-(3-chlorophenyl)-2-phenylquinoline-4,6-
diamine (22a, 0.8 g, 2.3 mmol) in dry DMF (30 mL) containing
triethylamine (1 mL): Yield 0.71 g (51%); mp 158e159 ꢀC; 1H NMR
able, NH); 13C NMR (500 MHz, DMSO-d6)
d: 22.6, 41.2, 52.4, 102.8,
108.6,112.5,118.5,118.8,120.1,120.4,120.7,122.9,124.5,125.1,125.6,
127.2,129.8,129.9,130.1,130.4,130.6,137.4,141.2,141.9,149.0,152.8,
154.8; ESI-HRMS calcd for C28H26Cl2F3N5O m/z 576.1545 (MþH),
found 576.1565 (MþH).
(400 MHz, DMSO-d6)
d
: 3.70 (8H, s, Ali), 6.75e6.77 (2H, d, J ¼ 8.9 Hz,
ArH), 6.98 (1H, s, ArH), 7.36e7.38 (2H, d, J ¼ 8.8 Hz, ArH), 7.65e7.56
(6H, m, ArH), 7.80e7.83 (1H, dd, J ¼ 2.2 and 6.4 Hz, ArH), 7.91e7.93
(2H, d, J ¼ 6.6 Hz, ArH), 8.06e8.09 (1H, m, ArH), 8.33e8.35 (1H, d,
J ¼ 9.4 Hz, ArH), 8.69 (1H, s, ArH), 9.53 (1H, s, NH, exchangeable),
4.1.54. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
ethynylphenyl)amino)-2-methylquinolin-6-yl)urea (18m)
Compound 18m was synthesized from isocyanate 17 (freshly
prepared from 16, 2.45 g, 8.0 mmol and triphosgene, 0.92 g,
9.91 (1H, s, NH, exchangeable), 10.80 (1H, s, NH, exchangeable); 13
C
NMR (500 MHz, DMSO-d6)
d: 41.2, 52.4, 104.4, 112.6, 113.5, 115.8,
117.7, 118.4,122.2,122.6,127.3,127.6,128.8,129.7, 130.8,134.2,135.8,

B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
707
136.4, 140.0, 142.9, 145.8, 146.9, 152.9, 154.1; ESI-HRMS calcd for
32H28Cl3N5O m/z 604.1438 (MþH), found 604.1432 (MþH).
alamarBlue assay [28] after 72 h of incubation using a microplate
spectrophotometer, as previously described. After the addition of
alamarBlue solution, the plates were incubated at 37 ꢀC for 6 h. The
absorbance at 570 and 600 nm was determined using a microplate
reader. The IC50 values were calculated from a doseeresponse curve
at 6 or 7 concentrations of each drug using the CompuSyn software
created by Chou and Martin based on the median-effect principle
and plot [29,30].
C
4.1.58. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-chloro-4-
fluorophenyl)amino) -2-phenylquinolin-6-yl)urea (23b)
Compound 23b was synthesized from isocyanate 17 (freshly
prepared from 16, 4.57 g, 15 mmol and triphosgene, 0.53 g,
1.8 mmol) and N4-(3-chloro-4-fluorophenyl)-2-phenylquinoline-
4,6-diamine (22b, 3 g, 8.2 mmol) in dry DMF (80 mL) containing
triethylamine (3 mL): Yield 2.79 g (54%); mp 172e173 ꢀC; 1H NMR
4.2.2. Alkaline agarose gel shift assay
(400 MHz, DMSO-d6)
d
: 3.71 (8H, s, CH2), 6.72e6.74 (2H, d, J ¼ 8 Hz,
The formation of DNA cross-links was analyzed by alkaline
agarose gel electrophoresis as described previously [31]. Purified
pEGFP-N1 plasmid DNA (1500 ng) was reacted with various con-
ArH), 7.12 (1H, s, ArH), 7.34e7.36 (2H, d, J ¼ 8 Hz, ArH), 7.56e7.59
(5H, m, ArH), 7.74 (1H, s, ArH), 7.92e7.98 (3H, m, ArH), 8.10e8.14
(1H, m, ArH), 8.63 (1H, s, ArH), 9.12 (1H, s, NH, exchangeable), 9.54
(1H, s, NH, exchangeable), 10.31 (1H, s, NH, exchangeable); 13C NMR
centrations (1, 10, and 20 mM) of the tested compounds in 40 mL of
binding buffer (30 mM sodium chloride/10 mM sodium phosphate,
pH 7.4, and 10 mM EDTA). The reaction mixture was incubated at
37 ꢀC for 2 h. Plasmid DNA was linearized by BamH-1 (New England
Biolabs) restriction enzyme digestion for 6 h and purified by
ethanol precipitation. The DNA pellets were dried and washed in
70% ethanol and then dissolved in alkaline buffer (0.5 N NaOH and
10 mM EDTA). Each sample containing 1000 ng DNA was mixed
(500 MHz, DMSO-d6) d: 41.3, 52.6, 100.5, 108.2, 112.5, 117.4, 117.6,
119.8, 119.9, 120.0, 120.6, 121.5, 122.9, 123.5, 126.8, 128.6, 128.9,
129.7, 137.0, 138.9, 139.4, 141.9, 144.9, 147.6, 152.1, 152.8, 154.0,
154.4; ESI-HRMS calcd for C32H27Cl3FN5O m/z 622.1343 (MþH),
found 622.1338 (MþH).
4.1.59. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(2-phenyl-4-(m-
tolylamino)quinolin-6-yl)urea (23c)
with 8
m
L of 2 ꢁ alkaline loading dye and then electrophoresed in
an 0.8% alkaline agarose gel with NaOH-EDTA buffer at 4 ꢀC. Elec-
trophoresis was performed at 18 V for 22 h. DNA was visualized
under UV light after staining the gels with an ethidium bromide
solution.
Compound 23c was synthesized from isocyanate 17 (freshly
prepared from 16, 1.66 g, 5.5 mmol and triphosgene, 0.64 g,
2.1 mmol) and 2-phenyl-N4-(m-tolyl)quinoline-4,6-diamine (22c,
1.0 g, 3 mmol) in dry DMF (35 mL) containing triethylamine
(1.2 mL). Yield 1.23 g (69%); mp 184e185 ꢀC; 1H NMR (400 MHz,
4.2.3. Cell cycle analysis
DMSO-d6)
d
: 2.34 (3H, s, CH3), 3.71 (8H, m, CH2), 6.72e6.74 (2H, d,
The effects of compound 18g on cell cycle progression were
analyzed with a flow cytometer as previously described [20].
Briefly, 1.0 ꢁ 105 H460 cells were seeded in each well of a 6-well
plate and incubated at 37 ꢀC in a 5% CO2 humidified atmosphere
overnight. The cells were then incubated with 18g (0.25, 0.5, 1, or
J ¼ 9.0 Hz, ArH), 6.93e6.95 (1H, d, J ¼ 7.4 Hz, ArH), 7.22e7.23 (2H, d,
J ¼ 5.96 Hz, ArH), 7.30e7.35 (3H, m, ArH), 7.38e7.50 (4H, m, ArH),
7.80e7.81 (1H, dd, J ¼ 2.1 Hz and 9.0 Hz, ArH), 7.89e7.91 (1H, d,
J ¼ 9.0 Hz, ArH), 7.96e7.98 (2H, m, ArH), 8.34e8.35 (1H, m, ArH),
8.57 (1H, s, NH, exchangeable), 8.81 (1H, s, NH, exchangeable), 8.88
2 mM) for 24 h. At the end of treatment, the attached cells were
(1H, s, NH exchangeable); 13C NMR (500 MHz, DMSO-d6)
d
: 21.0,
trypsinized, fixed with ice-cold 70% EtOH, and kept at ꢂ20 ꢀC
41.2, 52.5, 98.7, 108.7, 112.7, 117.6, 120.2, 121.3, 122.1, 125.6, 126.7,
127.8, 128.3, 129.7, 130.0, 131.6, 132.2, 134.4, 137.6, 139.2, 139.5,
141.6, 150.6, 152.8, 153.9; ESI-HRMS calcd for C33H31Cl2N5O m/z
584.1984 (MþH), found 584.1978 (MþH).
overnight. The cells were then stained with 4 mg/mL PI in
phosphate-buffered saline (PBS) containing 0.1 mg/mL RNase A and
1% Triton X-100. The cells were then subjected to flow cytometry
analysis (FACScan flow cytometer; Becton Dickinson, San Jose, CA).
The cell cycle phase distribution based on DNA histograms was
analyzed using ModFit LT 3.0 software (Verity Software House,
Topsham, ME).
4.1.60. 1-(4-(Bis(2-chloroethyl)amino)phenyl)-3-(4-((3-
methoxyphenyl)amino)-2-phenylquinolin-6-yl)urea (23d)
Compound 23d was synthesized from isocyanate 17 (freshly
prepared from 16, 3.1 g, 10 mmol and triphosgene, 1.16 g, 3.9 mmol)
and N4-(3-methoxyphenyl)-2-phenylquinoline-4,6-diamine (22d,
2 g, 5.8 mmol) in dry DMF (50 mL) containing triethylamine (2 mL).
4.2.4. Annexin V-FITC apoptosis analysis
H460 cells were treated with 18g or cisplatin, and the level of
apoptosis induced by these agents was detected by flow cytom-
etry using the Annexin V-FITC Apoptosis Detection Kit (eBio-
sciences, San Diego, CA, USA) according to the manufacturer's
instructions. In the Annexin V flow cytometry assay, cells positive
for Annexin V staining only (bottom right quadrant) and positive
for both Annexin V and PI staining (top right quadrant) represent
early and late apoptotic populations, respectively. Cells positive
for PI staining only (top left quadrant) represent the necrotic
population.
Yield 1.9 g (54%); mp 180e181 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
3.71 (8H, s, CH2), 3.82 (3H, s, CH3), 6.72e6.75 (2H, d, J ¼ 9.2 Hz,
ArH), 7.01e7.03 (1H, d, J ¼ 6.6 Hz, ArH), 7.18 (2H, m, ArH), 7.30e7.33
(2H, m, ArH), 7.38e7.39 (2H, d, J ¼ 8.7 Hz, ArH), 7.43e7.47 (3H, t,
ArH), 7.69 (1H, s, ArH), 7.85 (2H, s, ArH), 7.99e8.01 (1H, d, J ¼ 4 Hz,
ArH), 8.10e8.12 (1H, d, J ¼ 9.7 Hz, ArH), 8.84 (1H, s, NH,
exchangeable), 9.33 (1H, s, NH, exchangeable), 10.12 (1H, s, NH,
exchangeable); 13C NMR (500 MHz, DMSO-d6)
d: 41.2, 52.3, 55.3,
99.1, 104.5, 108.7, 110.6, 112.5, 113.5, 116.9, 119.6, 120.4, 126.8, 127.2,
128.8, 129.2, 130.6, 132.5, 134.1, 136.8, 139.2, 142.0, 145.7, 146.3,
150.7, 152.7, 160.3; ESI-HRMS calcd for C33H31Cl2N5O2 m/z 600.1933
(MþH), found 600.1928 (MþH).
Acknowledgments
We are grateful for the support from the National Science
Council, Taiwan, Republic of China (NSC 99-2320-B-001-014 and
NSC 100-2325-B-001-003) and Academia Sinica, Taipei, Taiwan
(AS-100-TP-B13). The NMR spectra were acquired on a Bruker
400 MHz spectrometer at the Institute of Biological Chemistry, a
Bruker 500 MHz spectrometer at the Institute of Biomedical Sci-
ences, and at the Core Facility for Protein Structural Analysis sup-
ported by the National Core Facility Program for Biotechnology.
4.2. Biological assays
4.2.1. Cytotoxicity assays
The cytotoxicity of the newly synthesized compounds in non-
small cell lung carcinoma H460 and H1299 cells, prostate cancer
PC-3 cells, and colon cancer HCT-116 cells was determined with the

708
B. Marvania et al. / European Journal of Medicinal Chemistry 83 (2014) 695e708
[16] R. Kakadiya, H. Dong, A. Kumar, N. Dodiya, N. Kapuriya, X. Zhang, T.-C. Chou,
Appendix A. Supplementary data
T.-C. Lee, A. Shah, T.-L. Su, Potent DNA-directed alkylating agents. Synthesis
and biological activity of phenyl N-mustard-quinoline conjugates having a
urea or hydrazinecarboximide linker, synthesis and biological activity, Bioorg.
Med. Chem. 18 (2010) 2285e2299.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.066.
[17] A.S. Prakash, A.D. William, T.A. Gourdie, K.K. Valu, P.D. Woodgate,
L.P.G. Wakelin, DNA-directed alkylating ligands as potential antitumor agents:
sequence specificity of alkylation by intercalating aniline mustards,
Biochemistry 29 (1990) 9799e9807.
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