Design and synthesis of novel quinacrine-[1,3]-thiazinan-4-one hybrids for their anti-breast cancer activity

Article abstract of DOI:10.1016/j.ejmech.2017.11.097

In an attempt to develop effective and safe anticancer agents, we designed, synthesized and examined 23 novel quinacrine (QC) derivatives by combining the 9-aminoacridine scaffold and the [1,3]thiazinan-4-ones group. Most of these hybrids showed strong anticancer activities, among which 3-(3-(6-chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-1,3-thiazinan-4-one (25; VR151) effectively killed many different cancer cell types, including eight breast cancer cell lines with different genetic background, two prostate cancer and two lung cancer cell lines. In contrast, compound 25 is less effective against non-cancer cells, suggesting it may be less toxic to humans. Our data showed that cancer cells are arrested in S phase for a prolonged period due to the down-regulation of DNA replication, leading to eventual cell death. We have also shown that the S phase arrest may be resulted by the down-regulation of cyclin A coupled with the continued up-regulation of cyclin E, which coincide with the down-regulation of mTor-S6K and mTor-4EBP1 pathways.

Full text of DOI:10.1016/j.ejmech.2017.11.097

Accepted Manuscript
Design and synthesis of novel quinacrine-[1,3]-thiazinan-4-one hybrids for their anti-
breast cancer activity
V. Raja Solomon, Sheetal Pundir, Hoang-Thanh Le, Hoyun Lee
PII:
S0223-5234(17)31000-0
10.1016/j.ejmech.2017.11.097
EJMECH 9972
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 October 2017
Revised Date: 28 November 2017
Accepted Date: 29 November 2017
Please cite this article as: V.R. Solomon, S. Pundir, H.-T. Le, H. Lee, Design and synthesis of novel
quinacrine-[1,3]-thiazinan-4-one hybrids for their anti-breast cancer activity, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.097.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Anticanceractivity

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Design and synthesis of novel quinacrine-[1,3]-thiazinan-4-one hybrids for
their anti-breast cancer activity
V. Raja Solomon†,‡*, Sheetal Pundir†,‡, Hoang-Thanh Le† and Hoyun Lee†,§,*
†Health Sciences North Research Institute, 41 Ramsey Lake Road, Sudbury, Ontario P3E 5J1,
Canada §Departments of Medicine, the Faculty of Medicine, the University of Ottawa, Ottawa,
Ontario K1H 5M8, Canada. ‡These two authors contributed equally.
* Corresponding author Email:vrajasolomon@gmail.com; hlee@hsnri.ca
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Abstract
In an attempt to develop effective and safe anticancer agents, we designed, synthesized and
examined 23 novel quinacrine (QC) derivatives by combining the 9-aminoacridine scaffold and
the [1,3]thiazinan-4-ones group. Most of these hybrids showed strong anticancer activities,
among
which
3-(3-(6-chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-1,3-
thiazinan-4-one (25; VR151) effectively killed many different cancer cell types, including eight
breast cancer cell lines with different genetic background, two prostate cancer and two lung
cancer cell lines. In contrast, compound 25 is less effective against non-cancer cells, suggesting
it may be less toxic to humans. Our data showed that cancer cells are arrested in S phase for a
prolonged period due to the down-regulation of DNA replication, leading to eventual cell death.
We have also shown that the S phase arrest may be resulted by the down-regulation of cyclin A
coupled with the continued up-regulation of cyclin E, which coincide with the down-regulation
of mTor-S6K and mTor-4EBP1 pathways.
Keywords: 9-aminoacridine, hybrid molecules, anticancer agents, cyclins, S phase arrest,
inhibition of DNA replication.
Abbreviations: DCC, N,N-dicyclohexylcarbodiimide; SRB, sulforhodamine B; QC, Quinacrine,
CQ, chloroquine; CP, cisplatin; EdU, 5-ethynyl-2’-deoxyuridine; HU, hydroxyurea
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1. Introduction
Breast cancer remains the most commonly diagnosed cancer among women as almost a
million new cases are diagnosed globally each year [1]. Early detection, understanding of the
heterogeneity of this disease, and development of targeted therapies have played a major role in
reducing breast cancer mortality rates during the last few decades [2, 3]. Unfortunately, however,
the clinical use of chemotherapeutics is often limited due to undesirable toxic effects [3,4]
underscoring the need of new more effective and safe agents. A promising new approach to
achieve this goal may be combining two or more pharmacophores into a single molecule [5].
There is evidence that a single molecule containing more than one pharmacophore, each with
different mode of action, could be beneficial for the treatment of cancer [6,7]. Compounds
generated by a hybrid approach can not only reduce undesirable side effects [6-8], but also often
provide the opportunity of overcoming drug-resistance [9-11].
The 9-aminoacridine, the main scaffold of quinacrine (QC, Figure 1), is found in many
drugs commonly used for the treatment of malaria. QC has also been reported to treat lupus
erythematosus, rheumatoid arthritis, bronchial asthma and other inflammatory diseases [12, 13].
Other studies showed that QC effectively killed cancer cells by inhibiting topoisomerases, the
NF-kB and Wnt-TCF signaling pathways as well as inducing p53 and p21 tumor suppressors [14-
16]. Satapathy et al. reported that a hybrid nanoparticle of bioactive QC and silver substantially
enhanced the cytotoxicity and reduced angiogenesis in oral cancer stem cells [17].Gomes et al.
showed that novel N-cinnamoyl QC derivatives have substantial anti-proliferative potential
against the MKN-28, Caco-2, MCF7 and HFF-1 cell lines, probably through a bimodal
mechanism of DNA intercalation and interaction with grooves. We previously synthesized hybrid
compounds by linking the main structural unit of the 9-aminoacridine ring system with a five
membered heterocyclic ring system (VR-118, Figure 1), which showed preferential cancer-cell
killing over non-cancer cells [8].
Small heterocyclic molecules have recently been recognized to have great potential for
the discovery of new drug candidates. The [1,3]thiazinan-4-ones group is such a privileged
pharmacophore found in many biologically active compounds. This heterocyclic system is
associated with substantially greater affinity to certain anticancer targets including non-membrane
protein tyrosine phosphatase (SHP-2), JNK-stimulating phosphatase-1 (JSP-1), tumor necrosis
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factor TNF-α, anti-apoptotic biocomplex Bcl-XL-BH3 and integrin [19-21]. Considering these
previous observations, we surmised that the hybridization of the pharmacophores 9-
aminoacridine and [1,3]thiazinan-4-ones could show highly effective anticancer activity. Based
on this postulation, we synthesized hybrid compounds by linking the main structural unit of the 9-
aminoacridine ring system with the [1,3]thiazinan-4-ones group (Figure 1, Table 1), and
examined their cytotoxic effects against three human breast tumor and one matching non-cancer
cell lines (Table 1), and compound 25 (VR151) was further examined against nine other cancer
cell lines and two non-cancer cell lines (Table 2).
2. Results and discussion
2. 1. Chemistry
The target compounds 6-28 were prepared as outlined in Scheme 1. The 6,9-dichloro-2-
methoxyacridine (4) was synthesized according to the protocol described previously, in which N-
arylanthranilic acid (3) was obtained from reaction between 2,4, dichlorobenzoic acid (1) and p-
anisidine (2), followed by condensation with phosphorus oxychloride [22]. The amino
components (5) used in the present study were prepared by aromatic nucleophilic substitution on
6,9-dichloro-2-methoxyacridine with excess of 1,3-diamino propane in neat conditions with the
simple standard workup procedure reported earlier from our laboratory [23]. The quinacrine
derived thiazinan-4-ones were obtained from the appropriate amine (5), substituted aldehyde, and
mercapto propionic acid in the presence of N,N-dicyclohexylcarbodiimide (DCC) in anhydrous
THF at room temperature (Scheme 1, Tables 1). Here DCC was used as a dehydrating agent to
accelerate the intramolecular cyclization, which was rapid and resulted in moderate to high yields
[24].26 After completion of the reaction, which usually takes 1.0 h, desired products were
obtained in excellent yields and purity. Due to the formation of a new stereocenter at C-2
position, the expected enantiomeric (2S/2R) form were not separable by column chromatography.
In the 1H NMR spectra, the signals of the respective protons of the synthesized compounds were
confirmed based on their chemical shifts, multiplicities and coupling constants. The compounds
reported in this study have been thoroughly characterized by elemental analysis and mass spectral
data.
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2.2. Cell killing/anti-proliferative effects of the compounds against cancer and non-cancer
cells
All the compounds synthesized were evaluated for their anti-growth/anti-proliferative
effect against established tumor and non-tumor cell lines, for which compounds were diluted to
achieve at least seven different concentrations ranging from 100 µM to sham control. Following
incubation for 48 h in the presence of a compound, the cells were stained with sulforhodamine B
(SRB) to measure the drug effects, as described previously [6, 8, 23]. The reading of SRB
staining is known to accurately reflect the levels of total cellular macromolecules. The IC50 value
of each compound was calculated with a reference to a standard curve (control cells), which
represents the concentration that results in a 50% decrease in cell growth/proliferation/survival
after 48 h of incubation (Table 1). QC, chloroquine (CQ) and cisplatin (CP) were included in the
experiment for comparison with new compounds. The anti-proliferation/anti-growth effects
measured by the SRB assay agreed well with data obtained by a clonogenic assay, indicating that
the compounds effectively kill cells, not just inhibiting cell growth/proliferation (Figures S1 and
S2).
Among 23 novel hybrid compounds examined (Tables 1), 14 compounds showed IC50
values in the range of 1.21-4.87 µM, 9 compounds 5.19-38.58 µM against MDA-MB468 cells.
Against MDA-MB231 cells, 15 compounds showed IC50 values in the range of 0.77-4.87 µM and
8 compounds 5.36-40.08 µM. Against the MCF7 cell line, 15 compounds showed IC50 values in
the range of 0.69-4.97 µM, and 8 compounds 5.05-39.33 µM. The differences in the IC50 values
may be attributable to a number of factors such as the nature of substitution at the C-2 position of
the [1,3]thiazinan-4-ones ring system and the genetic and biochemical background of the cell
lines.
The structure-activity relationship (SAR) suggests that the modification at the quinacrine lateral
side chain nitrogen atom substantially enhances anti-proliferative activity. The SAR analysis also
suggests that the presence of 4-methyl (7) at the C-2 phenyl ring of the [1,3]thiazinan-4-ones
moiety reduces the anti-proliferative activity in comparison with the unsubstituted phenyl
compound (6). However, the introduction of 2-fluoro substitution (8) improved activity against
MDA-MB468, MDA-MB231 and MCF7 cells. Although the introduction of 2-chloro (12) and 2-
bromo (17) substitutions at the C-2 phenyl ring of the [1,3]thiazinan-4-ones moiety resulted a
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decrease in the anti-proliferative activity in comparison with the 2-fluoro compound (8) on all
breast cancer cell lines examined. These results clearly indicate that a smaller size of the halo of
the group such as a 2-fluoro (8) group is favorable for anticancer activity. The compound having
a 4-fluoro (9), 4-chloro (13) or 4-bromo (18) substitution at the C-2 phenyl ring of the
[1,3]thiazinan-4-ones shows a significant decrease in anti-proliferative activity. The effect of a
nitro substituent on the C-2 phenyl ring was apparent in compounds 21 and 22, among which 2-
nitro compound (21) was more active, as exhibited IC50 values of 1.21, 0.77 and 0.99 µM against
MDA-MB468, MDA-MB231, and MCF7 cells, respectively. The compounds having a 4-
dimethylamino (19) or a 4-diphenylamino (20) group at the C-2 phenyl ring showed
improvement in anti-proliferative effects against MDA-MB468, MDA-MB231 and MCF7 cells,
compared to compounds with a phenyl group (2).
The introduction of a heterocyclic ring system such as 1H-pyrrol-2-yl (23), furan-2-yl
(24), thiophen-2-yl (25), to the C-2 position, as oppose to a phenyl ring, resulted in a substantial
improvement of anti-proliferative effects against MCF7 cells. However, the introduction of
pyridin-4-yl (26) or quinolin-4-yl (27) to the C-2 position, as oppose to a phenyl ring, resulted in
a substantial loss of anti-proliferative effects against MDA-MB468 cells. The replacement of the
C-2 phenyl ring with a cyclohexyl ring (28) at the [1,3]thiazinan-4-ones moiety resulted in a
significant decrease in activity against MDA-MB468, MDA-MB231 and MCF7 cells.
Among this hybrid compound series, the compound 25 was particularly effective, as its
IC50 values were 1.73, 2.80 and 0.69 µM against MDA-MB468, MDA-MB231 and MCF7 cells,
respectively (Table 1). This data demonstrates that the anticancer effect of compound 25 on all
three breast cancer cell lines was 8.4-fold (MDA-MB231) to 37.3-fold (MCF7) more effective
than cisplatin (Table 1). Compound 25 was also substantially more effective than the parental
QC, particularly for MCF7 cells which is 6-fold more effective (IC50 of 0.69 µM vs 4.19 µM).
2.3. Cancer cells, but not non-cancer cells, arrested in S phase in the presence of compound
25
Since this data indicated that compound 25 has significant potential as an anticancer agent, we
extended our study to other cancer cell lines. Data from cell viability assays (Table 2; Figures S1
and S2) showed that the treatment of cells with 1-2 µM of compound 25 (VR151) effectively
killed a wide range of tumor cell lines. Among them, MCF7, BT20 and BT474 mammary
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carcinoma cells were the most sensitive as 1 µM of compound 25 nearly completely killed off the
entire cancer cell population within 96 h post-treatment (Figure S2). The treatment of cells with 2
µM of compound 25 killed most of the cancer cell lines examined, including five other breast
cancer cell lines (SkBr3, CAMA1, MDA-MB453, MDA-MB231 and MDA-MB468), two
prostate cancer cell lines (LNCap and PC-3) and two lung cancer cell lines (A549 and NCI-
H1975). In contrast, the MCF10A and 184B5 non-cancer cell lines required at least 6 µM of
compound 25 to have the same effect (Table 2; Figure S1). Furthermore, data from clonogenic
assays showed that compound 25 is clearly more effective than the parental QC at the same doses
(0.39 µM and 1.5 µM) (Figure S1).
To gain a better understanding of the molecular mechanism about how compound 25
inhibits cell proliferation, we carried out flow cytometry to examine its effects on cell cycle
progression. As shown in Figure 2A, most of the MCF7 and MDA-MB231 cell populations
appeared arrested in S phase in the presence of 2 µM compound 25, along with portion of the cell
population showing sub-G1 DNA content that is typically shown with cells dying by apoptosis.
This conclusion was confirmed by BrdU pulse-labeling, which showed that DNA synthesis was
dramatically down-regulated as early as 6 h and essentially stopped by 48 post-treatment with 2
µM of compound 25 (Figure 2B). In contrast, the cell cycle progression of 184B5 non-cancer
cells was not affected by 2 µM of compound 25 (Figure 2A).
2.4. The treatment of cancer cells with compound 25 resulted in the up-regulation of cyclin
E and down-regulation of cyclin A, leading to the inhibition of DNA replication
Since data in Figure 3 indicated that cancer cells in S phase did not replicate their DNA, we
examined it further by labeling two cancer cell lines and one non-cancer cell line with EdU (5-
ethynyl-2’-deoxyuridine). As shown in Figure 3, approximately 10% of MDA-MB231 and 5% of
MCF7 cells were EdU positive in the presence of 2 µM of compound 25. This is a dramatic
decrease of EdU incorporation into their DNA in the two cancer cell lines in the presence of
compound 25, as 45-63% of sham controls were EdU positive. As expected, the level of EdU
incorporation in the 184B5 cell line was similar between the sham control (55%) and those
treated with 2 µM compound 25 (52%).
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To gain insight into the mechanism of the down-regulation of DNA replication, we
examined the levels of cyclins since they are the main regulators of cell cycle progression [25].
Cyclin D is part of the cell cycle engine that facilitates cell cycle progression in early- to mid-G1.
The Cdk2-cyclin E then takes over the role in late G1, and facilitates cell cycle progression into S
phase. In S phase, the up-regulation of cyclin A and the down-regulation of cyclin E are required
for the replication of both DNA and centrosome [26], while cyclin B is necessary for Cdk1 kinase
function at the G2/M transition [27]. As shown in Figure 3B, the level of cyclin E was
substantially elevated while that of cyclin A was extremely low by 24 h in the presence of
compound 25. On the other hand, the level of cyclin D was similar between sham-treated and
treated with 2 µM compound 25. Together, these data indicate that cell cycle progressed normally
from G1 to S phase; however, the failure of cyclin A induction coupled with the elevated level of
cyclin E in S phase resulted in the suppression of DNA replication. Since cells did not reach to
the G2/M border, the level of cyclin B was still very low.
2.5. Compound 25 did not damage DNA
Since cancer cells arrested in S phase due to the defect in DNA replication, we examined
if it was caused by DNA damage mediated by compound 25. Unlike the X-ray control sample, the
levels of H2A.x phosphorylation on Ser139 were similar between sham controls and those treated
with compound 25. This data indicates that compound 25 did not cause any notable DNA damage
(Figure 4A). We then examined the activation (i.e., phosphorylation) of Chk1 and Chk2, both of
which are relevant to the DNA damage-induced checkpoint control mechanisms [28]. We found
that neither Chk 1 nor Chk2 was activated in the presence of compound 25 (Figure 4B).
Therefore, we have concluded that the S phase arrest in the presence of compound 25 was not
caused by DNA damage.
2.6. The mTor-4EBP1 and mTOR-p70/p85S6K pathways were down-regulated in the
presence of compound 25
Since the parental QC compound is known to inhibit mTor pathway [29], we examined whether
compound 25 also inhibits the mTor pathway. We found that the phosphorylation of mTor on the
Ser2448 residue was down-regulated by 6 h in the presence of 2 µM of compound 25 in MCF7
cells (Figure 5A). Although the treatment of compound 25 did not result in any alteration of S6K
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phosphorylation on Thr389 by 6 h post-treatment, it did markedly down-regulate the
phosphorylation by 24 h post-treatment (Figure 5B). Interestingly, however, phosphorylation of
S6K on Ser371 was not affected under the same conditions. Unlike rapamycin, the
phosphorylation of 4EBP1 on Thr37/Thr46 was substantially down-regulated by 6 h post-
treatment with compound 25 (Figure 5C). Thus, our data showed that the down-regulation of the
two major downstream mTor pathways was sequential: the mTor-4EBP1 was affected early (6 h)
and the mTor-S6K pathway was affected later time (24 h) in the presence of compound 25. In
contrast, the phosphorylation of Akt on Thr308 was up-regulated in the presence of compound 25
(Figure 5D), which is unexpected since the mTor pathway is considered to be the downstream of
the PI3K-Akt pathway [30].32 This point is further discussed below.
The mTor pathway regulates the translation of specific mRNA through the activation of
4EBP1 and p70/p85S6K, its two major downstream sub-pathways. The phosphorylation of
4EBP1 on Thr37 and Thr46 by mTor leads to its dissociation from eIF4E, resulting in the down-
regulation of protein synthesis. The activation of S6K by mTor recruits the 40S ribosomal subunit
to actively translating polysomes on mRNA. Compound 25 appears to inhibit this important
molecular process essential for cell growth and survival. Unlike QC [28, 29], the Akt pathway
was not down-regulated by compound 25. This may indicate that compound 25 functions directly
to mTor, particularly to the mTor-4E-BP1 pathway. It is conceivable that a cell tries (but fails) to
restore the mTor pathway by up-regulating Akt activities through the feedback control
mechanism [30]. Our data is thus consistent with the notion that the levels of certain proteins
including cyclin A are severely down-regulated by the mTor-mediated translational down-
regulation in the presence of compound 25. However, it is currently unclear whether cyclin A is
the direct target of compound 25.
3. Conclusion
Here we describe the design, synthesis, and examination of 23 quinacrine derivatives
generated by the hybridization of the QC core scaffold and [1,3]thiazinan-4-ones in an attempt to
develop effective and safe anticancer agents. Most of the novel 23 hybrid compounds exhibited
substantial anticancer activity against three different human breast cancer cell lines, MDA-
MB468, MDA-MB231 and MCF7. Compound 25 (3-(3-(6-chloro-2-methoxyacridin-9-
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ylamino)propyl)-2-(thiophen-2-yl)-1,3-thiazinan-4-one, VR151) was further characterized as it
was the most active compound among this series. In addition to the three breast cancer cell lines,
1-2 µM of compound 25 effectively killed off all of the cancer cell lines examined including five
other breast cancer cell lines with different genetic background (BT20, BT474, SkBr3, CAMA1
and MDA-MB453), two prostate cancer cell lines (LNCap and PC-3) and two lung cancer cell
lines (A549 and NCI-H1975). Unlike cisplatin and the parental QC, compound 25 showed
preferential killing activity against cancer over non-cancer cells: at least 3-6-fold higher
concentrations of compound 25 were required to kill off the MCF10A and 184B5 non-cancer
cells (i.e., 1-2 µM vs >6 µM). It should also be noted that compound 25 kills cancer cells more
effective than the parental QC at the same doses.
Cancer cells progressed normally from G1 to S phase in the presence of compound 25;
however, they arrested in S phase for a prolonged time, leading to eventual cell death. The S
phase arrest was not due to DNA damage, but because of defect in DNA replication. Our data is
consistent with the notion that the defect in DNA replication is caused by the down-regulation of
cyclin A coupled with the continued up-regulation of cyclin E during S phase. The deregulation
of the cyclins A and E may be due to the down-regulation of mTor-S6K and mTor-4EBP1
pathways.
4. Materials and Methods
General
Melting points (mp) were taken in open capillaries on the Complab melting point apparatus.
Elemental analysis was performed on a Perkin-Elmer 2400 C, H, N analyzer and values were
1
within the acceptable limit (±1.5%) of the calculated values. The H spectra were recorded on a
DPX-500 MHz Bruker FT-NMR spectrometer using CDCl3 and DMSO-d6 as solvent. The
chemical shifts were reported as parts per million (δ ppm) tetramethylsilane (TMS) as an internal
standard. Mass spectra were obtained on a JEOL-SX-102 instrument using fast atom
bombardment (FAB positive). The progress of the reaction was monitored on ready-made silica-
gel plates (Merck) using chloroform-methanol (9:1) as solvent. Iodine was used as a developing
agent or by spraying with the Dragendorff’s reagent. Chromatographic purification was
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performed over a silica gel (100-200 mesh). The residues were obtained recrystallized by the
addition of 80:20 hexane–chloroform. All chemicals and reagents obtained from Aldrich (USA)
were used without further purification.
4-Chloro-2-(phenylamino)benzoic acid (3)
4-Chloro-2-(phenylamino)benzoic acid
was synthesized by the condensation of 2,4,
dichlorobenzoic acid (1) and p-anisidine (2) in the presence of LiNH2 as reported [22].22
(Scheme 1).
6,9-Dichloro-2-methoxyacridine (4)
6,9-Dichloro-2-methoxyacridine was synthesized by the cyclisation of 4-chloro-2-
(phenylamino)benzoic acid with phosporus oxychloride as reported [22] (Scheme 1).
N1-(6-Chloro-2-methoxy-acridin-9-yl)-propane-1,3-diamine (5)
The mixture of 6,9-dichloro-2-methoxyacridine (20.25 mmol), 1,3-diamino propane (1.96 ml,
25.75 mmol) and triethylamine (3.6 ml, 25.75 mmol) was heated slowly to 80 ºC for longer than
1 h while stirring. The temperature was then increased to 130-140 ºC, where it was kept for 6 h
while stirring continuously. The reaction mixture was cooled to room temperature, and then
poured into ice-cold water and filtered. The precipitate was filtered, washed, and recrystallized
using chloroform: methanol (3:1) mixture to obtain as cream-yellow solid. Yield 85%; 1H NMR
(500 MHz, CDCl3): δ 1.81-1.85 (m, 2H, CH2), 2.76-2.79 (m, 2H, CH2), 2.89-2.91 (m, 2H, CH2),
3.25 (br s, 2H, NH2 D2O-exchangeable), 3.87 (s, 3H, OCH3), 6.98 (d, J = 10.0 Hz, 1H, Ar-H),
7.11 (d, J = 5.0 Hz, 1H, Ar-H), 7.18 (d, J = 5.0 Hz, 1H, Ar-H), 7.47 (d, J = 10.0 Hz, 1H, Ar-H),
7.81 (br s, 1H, NH D2O-exchangeable), 7.96 (d, J = 10.0 Hz, 1H, Ar-H), 8.19 (d, J = 10.0 Hz,
1H, Ar-H); 13C NMR (CDCl3): δ 32.25 (2C), 49.74 (2C), 55.65 (2C), 100.67, 114.62, 117.03,
122.67, 122.97, 130.54, 134.11, 146.09, 148.68, 150.75, 155.37; ES-MS m/z 315 (M+, 100), 317
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(M+2, 54); Anal.Calcd for C17H18ClN3O: C, 64.66; H, 5.75; N, 13.31; Found: C, 64.61; H, 5.63;
N, 12.96.
General synthetic procedure for compounds (6-28)
The amino component (1.0 mmol) and aldehyde (2.0 mmol) were stirred in THF under ice-cold
conditions for 5 min, followed by addition of the mercapto propionic acid component (3.0
mmol). After 5 min, DCC (1.2 mmol) was added to the reaction mixture at 0 °C and the reaction
mixture was stirred for an additional 50 min at room temp. DCU was removed by filtration, the
filtrate was concentrated to dryness under reduced pressure, and the residue was taken up in
chloroform. The organic layer was successively washed with 5% aq. sodium hydrogen carbonate
and then finally with brine. The organic layer was dried over sodium sulfate, and the solvent was
removed under reduced pressure to get a crude product that was purified by column
chromatography on silica gel using chloroform: methanol (9:1).
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-phenyl-[1,3]thiazinan-4-one (6)
1
Yellow solid; Yield 75%; mp 146-148 °C; H NMR (500 MHz, CDCl3): δ 1.86-1.89 (m, 2H,
CH2), 2.90-2.97 (m, 4H, CH2), 3.49-3.51 (m, 2H, CH2), 3.74-3.77 (m, 2H, CH2), 4.01 (s, 3H,
OCH3), 5.51 (s, 1H, SCHN), 6.53 (br s, 1H, NH D2O-exchangeable), 7.25-7.28 (m, 3H, Ar-H),
7.35-7.42 (m, 4H, Ar-H), 7.55 (s, 1H, Ar-H), 7.96 (d, J = 10.0 Hz, 1H, Ar-H), 8.04 (s, 1H, Ar-
H), 8.09 (d, J = 10.0 Hz, 2H, Ar-H); 13C NMR (CDCl3): δ 28.53, 34.34, 44.41, 46.07, 55.76,
61.81, 99.51, 115.49, 118.03, 123.47, 124.08, 124.85, 124.90 (2C), 126.47, 128.14, 128.80 (2C),
130.89, 131.38, 132.47, 134.63, 138.67, 150.67, 156.12, 170.71; ES-MS m/z 492 (M+, 100), 494
(M+2, 65); Anal.Calcd for C27H26ClN3O2S: C, 65.91; H, 5.33; N, 8.54; Found: C, 65.89; H, 5.38;
N, 8.50.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-p-tolyl-[1,3]thiazinan-4-one (7)
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1
Orange yellow solid; Yield 78%; mp 171-173 °C; H NMR (500 MHz, CDCl3): δ 1.82-1.86 (m,
2H, CH2), 2.37 (s, 3H, CH3), 2.84-2.95 (m, 4H, CH2), 3.49-3.52 (m, 2H, CH2), 3.69-3.72 (m, 2H,
CH2), 4.01 (s, 3H, OCH3), 5.47 (s, 1H, SCHN), 6.41 (br s, 1H, NH D2O-exchangeable), 7.13 (d,
J = 5.0 Hz, 1H, Ar-H), 7.19 (d, J = 5.0 Hz, 1H, Ar-H), 7.26-7.28 (m, 2H, Ar-H), 7.39 (d, J =
10.0 Hz, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 7.96 (d, J = 10.0 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H),
8.10 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 506 (M+, 100), 508 (M+2, 44); Anal.Calcd for
C28H28ClN3O2S: C, 66.45; H, 5.58; N, 8.30; Found: C, 66.52; H, 5.49; N, 8.36.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-fluoro-phenyl)-[1,3]thiazinan-4-
one (8)
1
Yellow solid; Yield 68%; mp 151-153 °C; H NMR (500 MHz, CDCl3): δ 1.82-1.89 (m, 2H,
CH2), 2.79-2.84 (m, 2H, CH2), 2.89-2.93 (m, 2H, CH2), 2.95-2.99 (m, 2H, CH2), 3.48-3.51 (m,
2H, CH2), 3.99 (s, 3H, OCH3), 5.73 (s, 1H, SCHN), 6.42 (br s, 1H, NH D2O-exchangeable),
7.07-7.24 (m, 3H, Ar-H), 7.28 (d, J = 5.0 Hz, 1H, Ar-H), 7.32 (dd, J1 = 5.0 Hz, J2 = 10.0 Hz, 1H,
Ar-H), 7.36 (d, J = 10.0 Hz, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.94-(d, J = 10.0 Hz, 1H, Ar-H),
8.02 (s, 1H, Ar-H), 8.05 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 510 (M+, 100), 512 (M+2, 44);
Anal.Calcd for C27H25ClFN3O2S: C, 63.58; H, 4.94; N, 8.24; Found: C, 63.62; H, 5.01; N, 8.21.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-fluoro-phenyl)-[1,3]thiazinan-4-
one (9)
1
Orange yellow solid; Yield 75%; mp 121-122 °C; H NMR (500 MHz, CDCl3): δ 1.86-1.90 (m,
2H, CH2), 2.82-7.97 (m, 4H, CH2), 3.51-3.54 (m, 2H, CH2), 3.83-3.91 (m, 2H, CH2), 4.03 (s, 3H,
OCH3), 5.49 (s, 1H, SCHN), 6.46 (br s, 1H, NH D2O-exchangeable), 7.08 (dd, J1 = 5.0 Hz, J2 =
10.0 Hz, 2H, Ar-H), 7.23 (dd, J1= 5.0 Hz, J2 = 10.0 Hz, 2H, Ar-H), 7.30-7.31 (d, J= 5.0 Hz, 1H,
Ar-H), 7.41 (dd, J1= 5.0 Hz, J2 = 10.0 Hz, 1H, Ar-H), 7.55 (d, J = 5.0 Hz, 1H, Ar-H), 7.98 (d, J =
10.0 Hz, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 8.12 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 510 (M+,
100), 512 (M+2, 48); Anal.Calcd for C27H25ClFN3O2S: C, 63.58; H, 4.94; N, 8.24; Found: C,
63.62; H, 4.91; N, 8.27.
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3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,4-difluoro-phenyl)-
[1,3]thiazinan-4-one (10)
Yellow solid; Yield 72%; mp 154-156 °C; 1H NMR (500 MHz, CDCl3): δ 1.82-1.86 (m, 2H,
CH2), 2.77-2.82 (m, 2H, CH2), 2.88-2.92 (m, 2H, CH2), 3.50-3.53 (m, 2H, CH2), 3.76-3.82 (m,
2H, CH2), 4.02 (s, 3H, OCH3), 5.70 (s, 1H, SCHN), 6.33 (br s, 1H, NH D2O-exchangeable),
6.91-6.94 (m, 2H, Ar-H), 7.07 (dd, J1= 5.0 Hz, J2= 10.0 Hz, 1H, Ar-H), 7.28 (d, J= 10.0 Hz,
1H, Ar-H), 7.40 (d, J= 10.0 Hz, 1H, Ar-H), 7.54 (s, 1H, Ar-H), 7.97 (d, J = 10.0 Hz, 1H, Ar-H),
8.06 (s, 1H, Ar-H), 8.11 (d, J = 10.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): δ 25.61, 30.33, 34.99,
44.15, 46.04, 55.06, 55.76, 99.32, 105.52, 105.22, 105.42, 124.70, 124.74, 127.51, 128.12,
131.24, 134.65, 135.81, 146.63, 148.48, 150.31, 156.14, 158.67, 163.94, 164.04, 162.04, 170.54;
ES-MS m/z 528 (M+, 100), 530 (M+2, 52); Anal.Calcd for C27H24ClF2N3O2S: C, 61.42; H, 4.58;
N, 7.96; Found: C, 61.36; H, 4.62; N, 7.92.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-difluoro-phenyl)-
[1,3]thiazinan-4-one (11)
1
Yellow solid; Yield 70%; mp 198-199 °C; H NMR (500 MHz, CDCl3): δ 1.82-1.86 (m, 2H,
CH2), 2.81-2.84 (m, 2H, CH2), 2.95-3.02 (m, 2H, CH2), 3.13-3.17 (m, 2H, CH2), 3.50-3.54 (m,
2H, CH2), 3.99 (s, 3H, OCH3), 5.75 (s, 1H, SCHN), 6.44 (br s, 1H, NH D2O-exchangeable),
6.91-6.98 (m, 3H, Ar-H), 7.24 (d, J = 10.0 Hz, 1H, Ar-H), 7.36 (d, J= 10.0 Hz, 1H, Ar-H), 7.55
(s, 1H, Ar-H), 7.93 (d, J = 10.0 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 8.08 (d, J = 5.0 Hz, 1H, Ar-
H); 13C NMR (CDCl3): δ 23.26, 28.22, 34.48, 44.21, 46.00, 53.43, 55.74, 99.36, 112.36, 112.39,
112.54, 115.74,. 115.78, 118.25, 123.95, 124.74, 124.93, 124.93, 127.99, 130.20, 131.09, 134.53,
148.59, 150.42, 156.08, 161.18, 170.44; ES-MS m/z 528 (M+, 100), 530 (M+2, 54); Anal.Calcd
for C27H24ClF2N3O2S: C, 61.42; H, 4.58; N, 7.96; Found: C, 61.38: H, 4.57; N, 8.01
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-chloro-phenyl)-[1,3]thiazinan-4-
one (12)
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1
Yellow solid; Yield 65%; mp 118-119 °C; H NMR (500 MHz, CDCl3): δ 1.88-1.93 (m, 2H,
CH2), 2.51-2.68 (m, 4H, CH2), 3.59-3.62 (m, 2H, CH2), 3.75-5.78 (m, 2H, CH2), 3.90 (s, 3H,
OCH3), 5.55 (s, 1H, SCHN), 6.64 (br s, 1H, NH D2O-exchangeable), 7.09 (d, J = 10.0 Hz, 2H,
Ar-H), 7.17 (d, J= 5.0 Hz, 1H, Ar-H), 7.28 (d, J = 10.0 Hz, 1H, Ar-H), 7.32 (s, 1H, Ar-H), 7.53
(s, 1H, Ar-H), 7.89 (d, J = 10.0 Hz, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 8.13 (d, J = 10.0 Hz, 1H, Ar-
H); ES-MS m/z 526 (M+, 100), 528 (M+2, 78); Anal.Calcd for C27H25Cl2N3O2S: C, 61.60; H,
4.79; N, 7.98; Found; C, 61.56; H, 4.68; N, 8.03.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-chloro-phenyl)-[1,3]thiazinan-4-
one (13)
1
Orange yellow solid; Yield 67%; mp 150-152 °C; H NMR (500 MHz, CDCl3): δ 1.87-1.92 (m,
2H, CH2), 2.80-2.97 (m, 4H, CH2), 3.50-3.53 (m, 2H, CH2), 3.81-3.84 (m, 2H, CH2), 3.90 (s, 3H,
OCH3), 5.46 (s, 1H, SCHN), 6.39 (br s, 1H, NH D2O-exchangeable), 7.19 (d, J = 10.0 Hz, 2H,
Ar-H), 7.26 (d, J= 5.0 Hz, 1H, Ar-H), 7.37-7.41 (m, 3H, Ar-H), 7.53 (s, 1H, Ar-H), 7.96 (d, J =
13
10.0 Hz, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 8.09 (d, J = 10.0 Hz, 1H, Ar-H); C NMR (CDCl3): δ
21.78, 28.54, 34.26, 44.43, 46.04, 61.25, 99.40, 115.68, 118.61, 124.16 (2C), 124.80, 127.81
(2C), 128.99, 129.46 (2C), 130.94, 131.30, 134.66, 134.78, 137.33 (2C), 148.26, 150.45, 156.14,
170.54; ES-MS m/z 526 (M+, 100), 528 (M+2, 76); Anal.Calcd for C27H25Cl2N3O2S: C, 61.60;
H, 4.79; N, 7.98; Found: C, 61.58; H, 4.81; N, 7.91.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,4-dichloro-phenyl)-
[1,3]thiazinan-4-one (14)
1
Orange yellow solid; Yield 72%; mp 146-148 °C; H NMR (500 MHz, CDCl3): δ 1.94-1.98 (m,
2H, CH2), 2.70-2.77 (m, 2H, CH2), 2.83-2.88 (m, 2H, CH2), 3.00-3.03 (m, 2H, CH2), 3.85-3.88
(m, 2H, CH2), 4.02 (s, 3H, OCH3), 5.74 (s, 1H, SCHN), 6.57 (br s, 1H, NH D2O-exchangeable),
7.06 (d, J = 10.0 Hz, 1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.31 (d, J= 10.0 Hz, 1H, Ar-H), 7.38-7.40
(d, J = 10.0 Hz, 1H, Ar-H), 7.50 (d, J = 5.0 Hz, 1H, Ar-H), 7.98 (d, J = 10.0 Hz, 1H, Ar-H), 8.05
(s, 1H, Ar-H), 8.08 (d, J = 10.0 Hz, 1H, Ar-H); 13C NMR (CDCl3): δ 21.22, 28.37, 34.38, 44.37,
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46.01, 55.75, 58.21, 99.49, 124.22, 124.77 (2C), 124.96 (2C), 126.97 (2C), 127.17 (2C), 130.82,
133.55 (2C), 134.18 (2C), 135.06 (2C), 150.68, 156.16, 170.61; ES-MS m/z 561 (M+, 100), 563
(M+2, 90); Anal.Calcd for C27H24Cl3N3O2S: C, 57.81; H, 4.31; N, 7.49; Found: C, 57.77; H,
4.27; N, 7.55.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2,6-dichloro-phenyl)-
[1,3]thiazinan-4-one (15)
Yellow solid; Yield 65%; mp 124-125 °C; 1H NMR (500 MHz, CDCl3): δ 1.81-1.84 (m, 2H,
CH2), 2.22-2.25 (m, 2H, CH2), 3.00-3.07 (m, 2H, CH2), 3.11-.15 (m, 2H, CH2), 3.98-4.02 (m,
2H, CH2), 4.04 (s, 3H, OCH3), 5.84 (s, 1H, SCHN), 6.33 (br s, 1H, NH D2O-exchangeable), 7.04
(d, J = 10.0 Hz, 1H, Ar-H), 7.23-7.37 (m, 2H, Ar-H), 7.56 (s, 1H, Ar-H), 7.95-7.98 (m, 2H, Ar-
H), 8.02 (d, J= 10.0 Hz, 1H, Ar-H), 8.11 (d, J = 5.0 Hz, 1H, Ar-H), 8.48 (s, 1H, Ar-H); 13C NMR
(CDCl3): δ 24.11, 31.63, 35.37, 42.70, 46.25, 50.23, 55.32, 99.42, 100.21, 123.94, 124.07,
124.49, 124.69, 124.87, 128.79 (2C), 130.44 (2C), 132.13, 132.60, 133.57, 134.61, 136.84,
155.60, 156.15, 157.82, 170.90; ES-MS m/z 561 (M+, 100), 563 (M+2, 78); Anal.Calcd for
C27H24Cl3N3O2S: C, 57.81; H, 4.31; N, 7.49; Found: C, 57.77; H, 4.26; N, 7.52.
2-(2-Chloro-6-fluoro-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-
[1,3]thiazinan-4-one (16)
1
Orange yellow solid; Yield 72%; mp 132-134 °C; H NMR (500 MHz, CDCl3): δ 1.82-1.91 (m,
2H, CH2), 2.98-3.02 (m, 4H, CH2), 3.14-3.16 (m, 2H, CH2), 3.52-3.56 (m, 2H, CH2), 4.03 (s, 3H,
OCH3), 5.92 (s, 1H, SCHN), 6.53 (br s, 1H, NH D2O-exchangeable), 7.04 (m, 2H, Ar-H), 7.26-
7.29 (m, 2H, Ar-H), 7.39 (d, J = 10.0 Hz, 1H, Ar-H), 7.58 (d, J = 5.0 Hz, 1H, Ar-H), 7.97 (d, J =
10.0 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 8.12 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 545 (M+,
100), 547 (M+2, 62); Anal.Calcd for C27H24Cl2FN3O2S: C, 59.56; H, 4.44; N, 7.72; Found: C,
59.63; H, 4.28; N, 7.66.
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2-(2-Bromo-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-[1,3]thiazinan-4-
one (17)
1
Yellow solid; Yield 70%; mp 137-139 °C; H NMR (500 MHz, CDCl3): δ 1.90-1.96 (m, 2H,
CH2), 2.71-2.77 (m, 2H, CH2), 2.88-2.91 (m, 2H, CH2), 3.01-3.04 (m, 2H, CH2), 3.52-3.56 (m,
2H, CH2), 4.03 (s, 3H, OCH3), 5.77 (s, 1H, SCHN), 6.47 (br s, 1H, NH D2O-exchangeable), 7.12
(dd, J1 = 5.0 Hz, J2 = 10.0 Hz, 1H, Ar-H), 7.23 (d, J = 10.0 Hz, 1H, Ar-H), 7.36-7.37 (d, J= 5.0
Hz, 1H, Ar-H), 7.39 (dd, J1= 5.0 Hz, J2 = 10.0 Hz, 1H, Ar-H), 7.42 (d, J = 10.0 Hz, 1H, Ar-H),
7.54-7.55 (d, J = 5.0 Hz, 1H, Ar-H), 7.66 (d, J = 5.0 Hz, 1H, Ar-H), 7.98 (d, J = 10.0 Hz, 1H,
Ar-H), 8.06 (s, 1H, Ar-H), 8.12 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 571 (M+, 100), 573
(M+2, 42); Anal.Calcd for C27H25BrClN3O2S: C, 56.80; H, 4.41; N, 7.36; Found: C, 56.72; H,
4.39; N, 7.29.
2-(4-Bromo-phenyl)-3-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-[1,3]thiazinan-4-
one (18)
Orange yellow solid; Yield 69%; mp 123-124 °C; 1H NMR (500 MHz, CDCl3): δ 1.63-1.78 (m,
2H, CH2), 2.71-2.74 (m, 2H, CH2), 2.80-2.89 (m, 2H, CH2), 2.93-2.97 (m, 2H, CH2), 3.52-3.56
(m, 2H, CH2), 4.02 (s, 3H, OCH3), 5.45 (s, 1H, SCHN), 6.42 (br s, 1H, NH D2O-exchangeable),
7.13 (d, J = 10.0 Hz, 1H, Ar-H), 7.18 (d, J = 5.0 Hz, 1H, Ar-H), 7.30-7.34 (m, 2H, Ar-H), 7.41
(d, J = 10.0 Hz, 1H, Ar-H), 7.53 (d, J = 5.0 Hz, 1H, Ar-H), 7.71 (s, 1H, Ar-H), 7.98 (d, J = 10.0
Hz, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 8.12 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 571 (M+, 100),
573 (M+2, 40); Anal.Calcd for C27H25BrClN3O2S: C, 56.80; H, 4.41; N, 7.36; Found: C, 56.69;
H, 4.50; N, 7.32.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-dimethylamino-phenyl)-
[1,3]thiazinan-4-one (19)
1
Orange yellow solid; Yield 72%; mp 133-134 °C; H NMR (500 MHz, CDCl3): δ 1.86-1.90 (m,
2H, CH2), 2.72-2.76 (m, 2H, CH2), 2.91-2.97 (m, 2H, CH2), 2.98 (s, 3H, N(CH3)2), 3.57-3.61 (m,
2H, CH2), 3.88-3.92 (m, 2H, CH2), 4.04 (s, 3H, OCH3), 5.51 (s, 1H, SCHN), 6.45 (br s, 1H, NH
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D2O-exchangeable), 6.70 (d, J = 10.0 Hz, 2H, Ar-H), 7.13 (d, J = 10.0 Hz, 2H, Ar-H), 7.29 (s,
1H, Ar-H), 7.39 (d, J = 5.0 Hz, 1H, Ar-H), 7.59 (d, J = 5.0 Hz, 1H, Ar-H), 7.97 (d, J = 10.0 Hz,
1H, Ar-H), 8.04 (d, J = 5.0 Hz, 1H, Ar-H), 8.13 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 536
(M+, 100), 538 (M+2, 44); Anal.Calcd for C29H31ClN4O2S: C, 65.09; H, 5.84; N, 10.47; Found:
C, 65.16; H, 5.92; N, 10.43.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-diphenylamino-phenyl)-
[1,3]thiazinan-4-one (20)
Orange yellow solid; Yield 70%; mp 152-154 °C; 1H NMR (500 MHz, CDCl3): δ 1.88-1.92 (m,
2H, CH2), 2.74-2.78 (m, 2H, CH2), 2.93-2.99 (m, 2H, CH2), 2.99 (s, 3H, N(CH3)2), 3.59-3.63 (m,
2H, CH2), 3.90-3.95 (m, 2H, CH2), 4.01 (s, 3H, OCH3), 5.53 (s, 1H, SCHN), 6.47 (br s, 1H, NH
D2O-exchangeable), 6.70 (d, J = 10.0 Hz, 2H, Ar-H), 7.01-7.11 (m, 10H, Ar-H), 7.13 (d, J =
10.0 Hz, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 7.39 (d, J = 5.0 Hz, 1H, Ar-H), 7.59 (d, J = 5.0 Hz, 1H,
Ar-H), 7.97 (d, J = 10.0 Hz, 1H, Ar-H), 8.04 (d, J = 5.0 Hz, 1H, Ar-H), 8.13 (d, J = 10.0 Hz, 1H,
Ar-H); ES-MS m/z 660 (M+, 100), 662 (M+2, 38); Anal.Calcd for C39H35ClN4O2S: C, 71.05; H,
5.35; N, 8.50; Found: C, 71.11; H, 5.41; N, 8.58.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(2-nitro-phenyl)-[1,3]thiazinan-4-
one (21)
1
Orange yellow solid; Yield 67%; mp 137-139 °C; H NMR (500 MHz, CDCl3): δ 1.83-1.93 (m,
2H, CH2), 2.66-2.75 (m, 2H, CH2), 3.52-3.56 (m, 2H, CH2), 3.79-3.83 (m, 2H, CH2), 4.02 (s, 3H,
OCH3), 6.29 (s, 1H, SCHN), 6.52 (br s, 1H, NH D2O-exchangeable), 7.12 (d, J = 10.0 Hz, 1H,
Ar-H), 7.28 (d, J = 10.0 Hz, 1H, Ar-H), 7.37 (d, J = 10.0 Hz, 1H, Ar-H), 7.41 (s, 1H, Ar-H), 7.55
(d, J = 5.0 Hz, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 7.63 (d, J = 10.0 Hz, 1H, Ar-H), 7.67 (d, J = 5.0
Hz, 1H, Ar-H), 7.74 (s, 1H, Ar-H), 7.75 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 538 (M+, 100),
540 (M+2, 44); Anal.Calcd for C27H25ClN4O4S: C, 60.39; H, 4.69; N, 10.43; Found: C, 60.42; H,
4.72; N, 10.48.
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3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-(4-nitro-phenyl)-[1,3]thiazinan-4-
one (22)
1
Orange yellow solid; Yield 67%; mp 137-138 °C; H NMR (500 MHz, CDCl3): δ 1.86-1.93 (m,
2H, CH2), 2.76-2.81 (m, 2H, CH2), 2.91-3.00 (m, 2H, CH2), 3.52-3.56 (m, 2H, CH2), 3.79-3.84
(m, 2H, CH2), 4.02 (s, 3H, OCH3), 5.22 (s, 1H, SCHN), 6.25 (br s, 1H, NH D2O-exchangeable),
7.28 (d, J = 5.0 Hz, 1H, Ar-H), 7.42 (m, 2H, Ar-H), 7.97 (d, J = 10.0 Hz, 1H, Ar-H), 8.05 (s, 1H,
Ar-H), 8.09 (d, J = 10.0 Hz, 2H, Ar-H), 8.25 (d, J = 10.0 Hz, 2H, Ar-H); ES-MS m/z 538 (M+,
100), 540 (M+2, 48); Anal.Calcd for C27H25ClN4O4S: C, 60.39; H, 4.69; N, 10.43; Found: C,
60.41; H, 4.75; N, 10.49.
3-(3-(6-chloro-2-methoxyacridin-9-ylamino)propyl)-2-(1H-pyrrol-2-yl)-1,3-thiazinan-4-one
(23)
Orange Yellow solid; Yield 60%; mp 141-143 °C; 1H NMR (500 MHz, CDCl3): δ 1.87-1.91 (m,
2H, CH2), 2.81-2.83 (m, 2H, CH2), 2.90-2.93 (m, 2H, CH2), 3.13-3.17 (m, 2H, CH2), 3.89-3.93
(m, 2H, CH2), 4.02 (s, 3H, OCH3), 5.51 (s, 1H, SCHN), 6.31 (d, J = 5.0 Hz, 1H, Ar-H), 6.39 (d, J
= 5.0 Hz, 1H, Ar-H), 6.43 (br s, 1H, NH D2O-exchangeable), 6.55 (br s, 1H, NH D2O-
exchangeable), 7.30 (d, J = 5.0 Hz, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.38-7.41 (m, 2H, Ar-H),
7.57 (s, 1H, Ar-H), 7.99 (d, J = 10.0 Hz, 1H, Ar-H), 8.07 (s, 1H, Ar-H), 8.13 (d, J = 10.0 Hz, 1H,
Ar-H); ES-MS m/z 482 (M+, 100), 484 (M+2, 48); Anal.Calcd for C25H25ClN4O3S: C, 62.42; H,
5.24; N, 11.65; Found: C, 62.39; H, 5.21; N, 11.69.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-furan-2-yl-[1,3]thiazinan-4-one
(24)
1
Yellow solid; Yield 60%; mp 146-147 °C; H NMR (500 MHz, CDCl3): δ 1.85-1.89 (m, 2H,
CH2), 2.80-2.84 (m, 2H, CH2), 2.91-2.94 (m, 2H, CH2), 3.12-3.16 (m, 2H, CH2), 3.88-3.92 (m,
2H, CH2), 4.01 (s, 3H, OCH3), 5.49 (s, 1H, SCHN), 6.29-6.30 (d, J = 5.0 Hz, 1H, Ar-H), 6.38 (d,
J = 5.0 Hz, 1H, Ar-H), 6.42 (br s, 1H, NH D2O-exchangeable), 7.29 (d, J = 5.0 Hz, 1H, Ar-H),
7.30 (s, 1H, Ar-H), 7.40-7.43 (m, 2H, Ar-H), 7.56 (s, 1H, Ar-H), 7.98 (d, J = 10.0 Hz, 1H, Ar-
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H), 8.06 (s, 1H, Ar-H), 8.12 (d, J = 10.0 Hz, 1H, Ar-H);ES-MS m/z 482 (M+, 100), 484 (M+2,
48); Anal.Calcd for C25H24ClN3O3S: C, 62.30; H, 5.02; N, 8.72; Found: C, 62.21; H, 5.06; N,
8.69.
3-(3-(6-chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-1,3-thiazinan-4-one
(25)
1
Yellow solid; Yield 70%; mp 128-129 °C; H NMR (500 MHz, CDCl3): δ 1.83-1.87 (m, 2H,
CH2), 2.78-2.82 (m, 2H, CH2), 2.89-2.92 (m, 2H, CH2), 3.10-3.14 (m, 2H, CH2), 3.86-3.90 (m,
2H, CH2), 3.99 (s, 3H, OCH3), 5.47 (s, 1H, SCHN), 6.27 (d, J = 5.0 Hz, 1H, Ar-H), 6.36 (d, J =
5.0 Hz, 1H, Ar-H), 6.40 (br s, 1H, NH D2O-exchangeable), 7.27 (d, J = 5.0 Hz, 1H, Ar-H), 7.28
(s, 1H, Ar-H), 7.38-7.41 (m, 2H, Ar-H), 7.54 (s, 1H, Ar-H), 7.96 (d, J = 10.0 Hz, 1H, Ar-H),
8.04 (s, 1H, Ar-H), 8.10 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 498 (M+, 100), 500 (M+2, 58);
Anal.Calcd for C25H24ClN3O2S2: C, 60.29; H, 4.86; N, 8.44; C, 62.30; H, 5.02; N, 8.72; Found:
C, 60.32; H, 4.75; N, 8.42.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-pyridin-4-yl-[1,3]thiazinan-4-one
(26)
1
Yellow solid; Yield 65%; mp 154-155 °C; H NMR (500 MHz, CDCl3): δ 1.82-1.89 (m, 2H,
CH2), 2.71-2.75 (m, 2H, CH2), 2.80-2.88 (m, 2H, CH2), 2.92-2.96 (m, 2H, CH2), 3.51-3.55 (m,
2H, CH2), 4.02 (s, 3H, OCH3), 5.40 (s, 1H, SCHN), 6.28 (br s, 1H, NH D2O-exchangeable), 7.17
(d, J = 10.0 Hz, 2H, Ar-H), 7.28 (d, J = 10.0 Hz, 1H, Ar-H), 7.41 (d, J = 5.0 Hz, 1H, Ar-H), 7.72
(s, 1H, Ar-H), 7.98 (d, J = 5.0 Hz, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 8.10 (d, J = 10.0 Hz, 1H, Ar-
H), 8.06 (s, 1H, Ar-H), 8.10 (d, J = 10.0 Hz, 1H, Ar-H), 8.66 (d, J = 10.0 Hz, 2H, Ar-H); ES-MS
m/z 494 (M+, 100), 496 (M+2, 38); Anal.Calcd for C26H25ClN4O2S: C, 63.34; H, 5.11; N, 11.36;
Found: C, 63.28; H, 5.17; N, 11.29.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-quinolin-4-yl-[1,3]thiazinan-4-one
1
(27) Yellow solid; Yield 62%; mp 161-163 °C; H NMR (500 MHz, CDCl3): δ 1.42-1.46 (m,
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2H, CH2), 2.75-2.78 (m, 4H, CH2), 3.04-3.07 (m, 2H, CH2), 3.59-3.64 (m, 2H, CH2), 4.03 (s, 3H,
OCH3), 6.17 (s, 1H, SCHN), 6.46 (br s, 1H, NH D2O-exchangeable), 7.11 (d, J = 5.0 Hz, 2H, Ar-
H), 7.40 (d, J = 10.0 Hz, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 7.67 (d, J = 10.0 Hz, 1H, Ar-H), 7.75 (d,
J = 5.0 Hz, 1H, Ar-H), 7.78 (d, J = 10.0 Hz, 1H, Ar-H), 7.81 (d, J = 5.0 Hz, 1H, Ar-H), 7.85 (d, J
= 10.0 Hz, 1H, Ar-H), 7.98 (d, J = 10.0 Hz, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 8.11 (d, J = 10.0 Hz,
1H, Ar-H), 8.21 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 544 (M+, 100), 546 (M+2, 58);
Anal.Calcd for C30H27ClN4O2S: C, 66.35; H, 5.01; N, 10.32; Found: C, 66.39; H, 4.98; N, 10.27.
3-[3-(6-Chloro-2-methoxy-acridin-9-ylamino)-propyl]-2-cyclohexyl-[1,3]thiazinan-4-one
(28)
1
Orange yellow solid; Yield 60%; mp 171-173 °C; H NMR (500 MHz, CDCl3): δ 1.42-1.56 (m,
6H, CH2-cyclohexyl), 1.72-1.77 (m, 4H, CH2-cyclohexyl), 1.83-1.87 (m, 2H, CH2), 2.82-2.85 (m,
1H, CH-cyclohexyl), 2.93-3.01 (m, 4H, CH2), 3.21-3.25 (m, 2H, CH2), 4.04 (s, 3H, OCH3), 4.52
(s, 1H, SCHN), 6.34 (br s, 1H, NH D2O-exchangeable), 7.30-7.32 (d, J = 10.0 Hz, 1H, Ar-H),
7.42 (d, J = 5.0 Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 8.00 (d, J = 5.0 Hz, 1H, Ar-H), 8.08 (s, 1H,
Ar-H), 8.17 (d, J = 10.0 Hz, 1H, Ar-H); ES-MS m/z 498 (M+, 100), 500 (M+2, 52); Anal.Calcd
for C27H32ClN3O2S: C, 65.11; H, 6.48; N, 8.44; Found: C, 65.19; H, 6.41; N, 8.49.
Biological assays
All of the cell lines used were purchased from American Tissue Culture Collection (ATCC)
(Manassas, VA) and cultured according to supplier’s instructions, unless stated otherwise. Cell
line authentication was carried out by Genetica DNA Laboratories (Burlington, NC) using a
short tandem repeat (STR) profiling method (March 2015; July 2015; September 2016)
Reagents
Chloroquine diphosphate (CQ), quinacrine dihydrochloride (QC), and cisplatin (CP) were
purchased from Sigma-Aldrich Canada Ltd (Oakaville, ON, Canada). All the compounds used in
°
the experiments were dissolved in 10-20 mM dimethyl sulfoxide (DMSO) and stored at -20 C
until use. The stock solution was diluted in culture medium (0.1–100 µM) immediately prior to
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use. The final concentration of DMSO in the SRB-based cytotoxicity assays did not exceed
0.1%. To rule out that the DMSO concentration used may affect cell proliferation, culture
medium containing equivalent concentration of DMSO was used as a negative control in all
experiments. In all studies, the concentration of DMSO used did not notably show any anti-
proliferative effect.
SRB assay
Anti-proliferative/anti-growth effects were determined by a Sulphorhodamine B (SRB)-based
protocol.8,23 For a typical screening experiment, 5,000-10,000 cells were inoculated into 100 µl
medium per well of a 96-well microtiter plate as described previously. Briefly, after the
inoculation, the microtiter plate was incubated at 37 °C, 5% CO2, 95% air and 100% relative
humidity for 24 h, prior to addition of experimental drugs. Some of the sample wells were fixed
with 25 µl of 50% tricholoroacetic acid (TCA) as a control of the cell population for each cell
line at the time of adding a drug (Tz). An aliquot of the frozen stock was thawed and diluted to
the desired final maximum test-concentration with complete medium. Two- to ten-fold serial
dilutions were made to provide a total of seven drug concentrations (and a control [C]).
Following addition of drug(s), the culture plate was incubated for additional 48 h. Cells were
fixed in situ by slowly adding 25 µl of ice-cold 50% (w/v) TCA (final concentration, 10% TCA),
and were then incubated for 60 min at 4 °C. The supernatant was discarded, and the plate was
washed five times with tap water, followed by air-dry. 50 µl of SRB solution at 0.4% (w/v) in
1% acetic acid was added to each well, and the plate was incubated for > 30 min at room
temperature. Unbound SRB was removed by five washes with tap water, followed by air-drying.
The cells “stained” with SRB were solubilized with 10 mM trizma base, and the absorbance was
read on an automated plate reader at a wavelength of 515-564 nm. The relative growth rate (%)
was calculated for each of the compound concentrations according to the following formula:
(Ti – Tz)/(C – Tz) × 100
Where Tz, C and Ti denote time zero, control growth and OD for different concentration of
tested compounds, respectively. The IC50 for each compound was obtained from a non-linear
sigmoidal dose-response (variable slope) curve which is fitted by GraphPad Prism v.4.03
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software. Values were calculated for each of these parameters if the level of activity was
reached. However, if the effect was not reached or was exceeded, the value for that parameter
was expressed as greater or less than the maximum or minimum concentration tested [8, 31] .
Western blot assay
Samples treated with compound 25 and sham control were collected at scheduled time points
post-treatment and centrifuged for 5 min at 1100 rpm (AllegraTM X-12 centrifuge, Beckman
Coulter). Cell pellets were washed with PBS by centrifugation for 5 min at 1100 rpm
(AllegraTM X-12 centrifuge), and were then lysed with 100 µl Lysis buffer (150 mmol NaCl, 5
mmol EDTA, 1% Triton X-100, 10 mmol Tris pH 7.4, 1 mmol PMSF, 5 mmol EDTA and 5
mmol protease inhibitors cocktail) by maintaining on ice for 10–15 min, followed by
centrifugation at 1100 rpm (AllegraTM X- 12 centrifuge) for 10 min at 4 ºC. Supernatant was
collected and protein concentration was determined using a BCA protein assay kit (Thermo
Scientific, USA). Cell lysates were diluted with 2 X Laemmli sample buffer, and then boiled at
95–100 °C for 5 min. 30–40 µg protein was resolved by polyacrylamide gel (8% or 10%)
electrophoresis. The resolved proteins were transferred to a PVDF membrane using a semi-dry
gel transfer apparatus (75 min at 24 V), followed by ‘blocking’ with 5% skim milk for 1 h.
Proteins on the membrane were then incubated with primary antibody in 0.1% TBST buffer
containing 5% skim milk for overnight at 4 C. Membrane was washed three times with 0.1%
TBST buffer and incubated with secondary antibody in TBST buffer containing 5% skim milk
for 1 h. Finally, the membrane was washed with TBST buffer thrice, and signals were visualized
on X-ray film using an ECL chemiluminescence kit (Super Signal West pico, Thermo Scientific,
USA). Antibodies used were purchased from Abcam (Canada) or Santa Cruz biotechnology
(Canada) [32].
Clonogenic assay
Cancer cell lines were seeded in 6-well plates at a density of 1000 cells per well. After overnight,
test compounds were added and incubated for 12 h. Then the medium was changed and the cells
were cultivated until cells in control plates formed sufficiently large colonies. Colonies were then
stained with crystal violet staining solution [32].
Acknowledgements
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HL wishes to thank to funders: the Natural Sciences and Engineering Council of Canada
(NSERC), Northern Cancer Research Foundation, Northern Ontario Heritage Fund Corporation
and the Greater Sudbury Business Development Corporation. VRS wishes to acknowledge the
Ontario Ministry of Research and Innovation for postdoctoral fellowship.
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Table 1. Anti-proliferative activity of quinacrine analogs (6-28) against human breast cancer and non-cancer breast cell lines
b,c
IC50
MDA-MB231
Lab
Code
Compounda -R
MDA-MB 468
4.72±0.39
6.97±0.52
2.68±0.11
4.58±0.52
3.79±0.26
3.20±0.11
19.92±0.34
4.43±0.55
3.72±0.31
1.29±0.07
5.19±0.41
38.58±0.34
25.53±0.65
3.56±0.41
1.68±0.38
1.21±0.06
9.41±0.88
4.87±0.05
3.67±0.21
1.73±0.80
21.58±0.21
10.40±0.18
5.25±0.21
28.58±0.25
31.02±0.32
3.96±0.12
MCF7
184B5
9.32±0.25
VR126
VR139
VR127
VR128
VR130
VR131
VR132
VR133
VR134
VR135
VR136
VR137
VR138
VR140
VR141
VR142
VR143
VR150
VR146
VR151
VR147
VR144
VR145
6
7
8
phenyl
2-p-tolyl
4.07±0.02
4.87±0.32
4.11±0.22
5.36±0.31
4.54±0.37
4.41±0.33
10.78±0.54
3.53±0.23
3.07±0.31
7.54±0.72
3.44±0.56
40.08±1.03
37.10±0.98
2.63±0.02
3.32±0.13
0.77±0.11
4.68±0.25
4.56±0.33
3.46±0.02
2.80±1.30
10.78±0.15
7.48±0.31
5.85±0.43
22.52±1.44
23.63±0.23
3.25±0.11
4.79±0.34
5.52±0.23
2.29±0.11
4.97±0.32
3.39±0.45
3.81±0.21
15.35±0.54
3.40±0.32
1.85±0.01
4.41±0.23
4.31±0.22
39.33±0.65
31.31±0.35
3.09±0.21
1.85±0.23
0.99±0.11
7.04±0.21
1.66±0.11
3.57±0.21
0.69±0.41
16.18±0.45
8.94±0.51
5.05±0.22
38.44±0.32
25.77±0.65
4.19±0.14
5.66±0.21
35.58±0.87
28.23±0.58
5.08±0.31
29.27±0.24
69.56±0.58
5.02±0.12
4.28±0.21
3.84±0.21
3.1±0.11
2-fluoro-phenyl
4-fluoro-phenyl
2,4-difluoro-phenyl
2,6-difluoro-phenyl
2-chloro-phenyl
4-chloro-phenyl
2,4-dichloro-phenyl
2,6-dichloro-phenyl
2-chloro-6-fluoro phenyl
2-bromo-phenyl
4-bromo-phenyl
4-dimethylamino-phenyl
4-diphenylamino-phenyl
2-nitro-phenyl
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
15.6±0.28
35.81±0.65
13.19±0.28
3.11±0.21
1.83±0.11
19.51±0.25
12.86±0.36
3.6±0.21
4-nitro-phenyl
2-1H-pyrrol-2-yl
2-furan-2-yl
2-thiophen-2-yl
2-pyridin-4-yl
4.96±0. 24
14.62±0.23
27.1±0.64
7.71±0.24
76.13±0.23
25.54±0.56
4.96±0.16
2-quinolin-4-yl
2-cyclohexyl
CQ
Cisplatin
Quinacrine
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a Chemical structures are shown in Scheme 1. b IC50 values were calculated from sigmoidal dose response curves (variable slope), which were
generated with GraphPad Prism V. 4.02 (GraphPad Software Inc.). c Values are the mean of triplicates of at least two independent experiments.
CQ-Chloroquine diphosphate
Table 2: Summary of cell viability against compound 25 (VR151)
Cell lines
Cell characteristics
VR151 (µM)*
MCF7
BT20
BT474
SkBr3
CAMA1
MDA-MB453
MDA-MB231
MDA-MB468
LNCap
PC-3
A549
NCI-H1975
MCF10A**
184B5**
Mammary adenocarcinoma, ER+
Mammary carcinoma, ER-, IRS-1-/-
Mammary carcinoma, Her2++, p53-
Mammary carcinoma, Her2++, ER-
Mammary carcinoma, PTEN-, ER+,Her2+, c-Myc++
Mammary adenocarcinoma, PTEN-
Mammary carcinoma, ER-, PR_, p53-/-, K-ras mutant
Mammary carcinoma, PTEN-/-, RB1-/-, p53-/-, ER-
Prostate cancer, JAK-1 mutant, androgen dependent,
Prostate cancer, EGFR mutant, androgen independent
Non-small cell lung adenocarcinoma, K-ras mutant
Non-small cell lung cancer, EGFR-, p53-
Non-malignant mammary epithelial cell
Non-malignant mammary epithelial cell
1
1
1
2
2
2
2
2
2
2
2
2
>6
6
* At these VR151 concentrations, cell viability measured by counting number of viable cells was nearly completely inhibited. **Non-malignant
cells were not inhibited at 1-2 µM of VR151; however, they were inhibited at 6 µM or higher concentrations.
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