Multicomponent assembly of 4-aza-podophyllotoxins: A fast entry to highly selective and potent anti-leukemic agents

Article abstract of DOI:10.1016/j.ejmech.2015.10.009

The aim of this study was the synthesis and lead structure selection of a best anti-leukemic agent from a library of aza-podophyllotoxin analogues (APTs). To this end, we report a scalable, modified multicomponent reaction using a "sacrificial" aniline pa

Full text of DOI:10.1016/j.ejmech.2015.10.009

Accepted Manuscript
Multicomponent assembly of 4-aza-podophyllotoxins: A fast entry to highly selective
and potent anti-leukemic agents
Nagalakshmi Jeedimalla, Madison Flint, Lyndsay Smith, Alberto Haces, Dmitriy
Minond, Stéphane P. Roche
PII:
S0223-5234(15)30290-7
10.1016/j.ejmech.2015.10.009
EJMECH 8144
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2015
Revised Date: 30 September 2015
Accepted Date: 6 October 2015
Please cite this article as: N. Jeedimalla, M. Flint, L. Smith, A. Haces, D. Minond, S.P. Roche,
Multicomponent assembly of 4-aza-podophyllotoxins: A fast entry to highly selective and potent anti-
leukemic agents, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.009.
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ACCEPTED MANUSCRIPT
Multicomponent Assembly of 4-Aza-podophyllotoxins: A Fast
Entry to Highly Selective and Potent Anti-Leukemic Agents.
†
†
‡
†
‡║
Nagalakshmi Jeedimalla , Madison Flint , Lyndsay Smith , Alberto Haces , Dmitriy Minond * and
Stéphane P. Roche *
†
†
Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, United States.
‡
Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, United States.
║
Current address: Department of Drug Discovery & Development, Harrison School of Pharmacy, Auburn University, Au-
burn, AL 36849, United States.
ABSTRACT: The aim of this study was the synthesis and lead structure selection of a best anti-leukemic agent from a library of
aza-podophyllotoxin analogues (APTs). To this end, we report a scalable, modified multicomponent reaction using a “sacrificial”
aniline partner as a more general route to rapidly construct the pivotal library of 50 APT analogues. Our preliminary structure activ-
ity relationship studies for anti-leukemic activity also address the innate toxicity of these compounds against non-malignant cells.
As a result, we identified 2 novel compounds 2ca’ and 2jc’ more potent than etoposide 1 (25-60 fold) having high selectivity
against the human THP-1 leukemia cell line and a minimal toxicity (IC50 of 9.3 ± 0.8 and 19.6 ± 1.4 nM respectively) which repre-
sent the best candidates for further pharmacological optimization.
KEYWORDS. Multicomponent reactions; aza-podophyllotoxins; anti-leukemic agents; AML; SAR study; Etoposide.
1
. Introduction.
2. Synthetic strategy to assemble the chemical library.
®
Etoposide 1 (Vepesid ), a synthetic derivative from the natural
product podophyllotoxin (PT), is highly prescribed as an anti-
cancer drug for the treatment of several human malignancies
such as testicular and small-cell lung cancers. More recently,
1
line regimen in the treatment of relapsed acute myelogenous
leukemia (AML). This semisynthetic drug exerts a strong
chemotherapeutic effect by increasing levels of covalent
topoisomerase II-cleaved DNA complexes. Although these
complexes are common intermediates in the DNA strand pas-
sage reaction, higher concentrations of such complexes are
responsible for mutagenic and cell death pathways. On the
other hand, the 4-aza-structural analogues of PT, namely 4-
aza-2,3-didehydropodophyllotoxins (APTs) 2, have also
shown important biological activity as insecticides, anticancer
and vascular-disrupting agents and hold a great potential in
developing novel therapeutics due to their apparent straight-
In multicomponent reactions (MCRs), three or more reagents
are combined to synthesize complex small-molecules contain-
ing structural contributions from each reaction component.
6
1
These one-pot processes are especially useful for the rapid
construction of polycyclic heterocycles, which can be further
decorated to rapidly generate a portfolio of analogues from a
targeted chemotype. Therefore, MCRs are powerful maneu-
vers to rapidly construct large libraries of biologically active
molecules with high degree of complexity while minimizing
was also evaluated and validated as constituent of a third
2
3
7
the number of synthetic operations.
Scheme 1. MCR tactic to the 4-aza-podophyllotoxin chemotype
4,5
forward preparation in comparison to 1.
To increase antitumor efficacy of the APT analogues 2 while
reducing systemic side effects and poor water solubility issues
found in the parent molecules podophyllotoxin and etoposide
1
, site-specific modifications expanding the APT-chemotype’s
chemical space were used to determine the pharmacophores
responsible for the optimal bioactivity. Herein are presented
our efforts to develop a practical multicomponent reaction to
access modified APT analogues 2 for a structure activity rela-
tionship (SAR) study, based on bioassays against leukemia
and pancreas cancer cell lines.
In 2000, Husson and Giorgi-Renault reported the extremely
straightforward synthesis of APTs 2 via a unique 3-component
reaction (Husson-3CR) which entails the condensation of a

ACCEPTED MANUSCRIPT
2
tetronic acid 5 with variously substituted anilines 3 and alde-
hydes 4 (Scheme 1). This unique transformation (Husson-
the nitrogen substitution (R ), as well as numerous possibilities
for positional and functional modifications of the APT chemo-
8
1
3
3
CR) enables the synthesis of podophyllotoxin’s aza-
type on the B-ring (R ) and the E-ring (R ). Recently, scien-
tists at Bayer Cropscience prepared a large library of APTs
(~140 compounds) and reported a lack of synthetic efficiency
using the typical Husson-3CR leading them to advance an
analogues in one-pot while site-specific modifications are
achieved through the modification of anilines or aldehydes
partners utilized. This strategy offers facile modifications of
a-c
d
Scheme 2. Synthesis and biological evaluation of a library of novel 4-aza-podophyllotoxins (APTs) 2xy’
H
N
H
H
N
H
N
H
N
parent structures
F
O
O
HO
N
O
F
HO
O
H
O
O
O
O
O
O
F
O
O
N
HO
O
F
O
O
OH
H
H
O
O
O
O
H
O
O
O
O
H
H
H
H
O
O
H
O
MeO
O
2aa'
OH
bb'
A: 29% yield
EC50 = 1.3 M
OMe
2ca'
A: 5% yield
B: 14% yield
IC50 = 9 nM
MeO
MeO
A: > 70% yield etoposide
IC50 = 13 nM EC50 = 540 nM
O
F
2ae'
A: 26% yield
IC50 = 465 nM
MeO
O
MeO
O
2cc'
2
2bc'
A: 7% yield
B: 21% yield
EC50 = 883 nM
OMe
A: NR
B: 3% yield
EC50 = 17 nM
(
1) MeO
OMe
OH
H
N
H
N
H
N
H
N
H
N
H
N
H
N
F
O
F
MeS
MeS
O
MeS
MeO
MeS
MeS
O
O
O
O
O
F
O
O
O
F
F
F
F
O
H
H
O
O
O
H
H
O
O
F
H
H
F
H
F
F
O
F
F
2ch'
OMe
2da'
A: 20% yield
2dc'
2dh'
MeO
O
F
F
2
cd'
A: 13% yield
IC50 = 202 nM
A: NR
B: 4% yield
EC50 = 17nM
A: 18% yield
B: 49% yield
IC50 = 37 nM
2dd'
2de'
A: 12%yield
B: 39 %yield
IC50 = 1.1 M
A: 30% yield
IC50 = 202 nM
A: 28% yield
IC50 = 470 nM
IC50 = 19 nM
SMe
H
N
SMe
H
N
H
N
H
N
H
N
H
N
H
N
O
O
O
O
O
O
O
O
F
F
O
H
O
O
O
H
H
F
F
H
H
F
H
O
O
F
H
F
MeO
MeO
O
2fh'
F
2fc'
F
2fd'
OMe
OH
F
2ea'
MeO
2eb'
O
A: NR
B: 5% yield
IC50 = 237 nM
A: NR
B: 5% yield
EC50 > 10
2hd'
2hh'
A: 15% yield
B: 25% yield
EC50 = 50 nM
B: 10% yield
IC50 = 14 nM
A: 26% yield
IC50 = 249 nM
A: 77% yield
EC50 > 100
A: 83% yield
EC50 > 100 M
M
M
H
N
H
N
H
N
H
N
H
N
H
N
H
N
O
O
O
O
O
O
O
N
N
F
F
F
F
H
F
H
F
O
O
O
N
O
N
O
N
F
F
O
N
O
H
H
H
F
H
H
F
F
MeO
F
F
F
MeO
MeO
O
2
ga'
2gc'
A: NR
B: 10% yield
2gd'
2id'
OMe
B: 42% yield
IC50 = 6.6
2kd'
F
2ka'
OMe
MeO
A: NR
B: 4% yield
IC50 = 41 nM
O
A: NR
B: 6% yield
IC50 = 247 nM
2gh'
B: 2% yield
IC50 = 122 nM
B: 6% yield
IC50 = 10.2
B: 15% yield
IC50 = 1.1 M
M
M
EC50 = 95 nM
H
N
H
N
H
N
H
N
O
O
O
O
O
O
H
F
H
O
H
F
O
H
MeO
MeO
F
O
OMe 2ja'
A: NR
F
O
F
2jh'
2
jc'
2jd'
c
B: 4% yield
IC50 = 8 nM
B: 27% yield
IC50 = 20 nM
B: 10% yield
IC50 = 40 nM
B: 12% yield
IC50 = 42 nM
a
12
Only novel analogues are presented above.o Conditions. Method A: typical Husson-3CR with an equimolar ratio of tetronic acid 5, aniline 3a-k and ben-
zaldehyde 4a’-h’ under Ar was stirred at 120 C in 2-pentanol (1 mL) for 1 hour; Method B using the sacrificial aniline 3l: aniline 3l (1.0 equiv.) and benzal-
dehyde 4a’-h’ (1.0 equiv.) was stirred under Ar at 120 C in 2-pentanol (1 mL) for 2 hours, followed by addition of tetronic acid 5 (1.0 equiv.) for 10 mins and
finally aniline 3a-k (1.0 equiv.), the resulting mixture was refluxed for an additional 30 mins. Yields refer to pure products 2xy’ isolated after recrystalliza-
tion of crude material in ethanol. Compound 2ja’ was isolated as over-oxidized quinolines and reduced in a second step; overall yield for both steps is re-
o
b
c
d
ported above. The reported IC50 or EC50 values are against the human THP-1 leukemia cell line.

ACCEPTED MANUSCRIPT
9
13
14
improved one-pot procedure. Several other groups also re-
ported their efforts in optimizing the original Husson-3CR,
but a general synthetic method still remains to be found.
ment. Additionally, acute kidney injury was often reported
10
in patients being treated with 1 which makes HEK293 cells a
reasonable choice for this assay. Etoposide 1 was utilized as a
pharmacological assay control. Previous IC50 values reported
3
. Results and discussion.
1
5a
for 1 with THP-1 cells vary widely (e.g. 8.5 µM , 855 nM
3
.1. Chemistry.
15b
)
which suggests that its apparent potency could depend on
Starting a new medicinal chemistry program, we recently re-
visited the Husson-3CR to prepare new structural variations of
APTs 2 in a more efficient manner. Our mechanistic inquiries
lead us to identify an improved protocol in which an external
sacrificial component was added to the typical Husson-3CR
the type of assay or to etoposide’s poor solubility in the assay
media. In our hands, we obtained a similar EC50 value of 540 ±
4
2 nM in the assay with THP-1 cells. Closer examination of
dose response curves revealed differential effect of 1 and test
compound 2ha’ on THP-1 cells as compared to PSN-1 and
1
1
12
(
Schemes 2 & Table 1). In this new process, the electron
deficient ‘sacrificial’ aniline 3l is first engaged with aldehyde
y’ to efficiently generate a reactive imine intermediate (ob-
HEK293 cells (Fig. 1). Overall, compound 2ha’ exhibited
complete response towards THP-1 cells (0-100% viability)
whereas dose response curves for viability of PSN-1 and
HEK293 cells exhibited partial effect (either 50-80% or > 50%
residual viability at highest concentration tested). These data
suggested 2ha’ may have a different set of molecular targets
that affect cell viability of THP-1 cells as compared to PSN-1
and HEK293 cells thus enhancing 2ha’ selectivity as anti-
leukemic agent. Furthermore, a full range response in case of
THP-1 suggests that 2ha’ causes cell death (cytocidal effect),
whereas a partial response in case of PSN-1 and HEK293 cells
suggested that 2 only inhibit cell growth (cytostatic effect) in
the time frame of the viability assay (72 hours). A similar ef-
fect was previously reported for a series of structurally related
4
1
served by H NMR) which then undergo condensation with the
tetronic acid 5 and the desirable aniline partners 3x in a more
orderly fashion to ultimately deliver the APT molecules 2xy’
in higher yields (up to three fold). Our variant of the Husson-
3
CR is highlighted by more sequential protocol in which the
‘
sacrificial’ aniline 3l is extruded during the later stage of con-
densations/dehydration steps and replaced by the desired ani-
line partners 3a-k in the final product. The ‘sacrificial’ aniline
3
l plays therefore a transient role in the cascade mechanism
and is fully regenerated at the end of the reaction. Using this
protocol (Scheme 2, Method B), several compounds 2bc’,
2
ca’, 2dc’, 2dh’ and 2fc’ were synthesized in higher yields (3-
N-hydroxyethyl-4-aza-didehydropodophyllotoxin
deriva-
16
4
9% yields after recrystallization) in comparison to the typical
tives. To test this hypothesis, viability assay were performed
in the format that allows for cells count (Fig. 1). As shown in
Fig. 1, 10 µM of 1 and compound 2ha’ resulted in approxi-
mately 70-50% cell death in case of HEK293 cells (Panel A)
and 100% cell death in case of THP-1 cells (Panel B) which is
in general agreement with CellTiter Glo® viability assay re-
sults (Panels C & D). Etoposide exhibited incomplete cell
death (~75%) in case of CellTiter Glo® viability assay (Panel
D) which could be due to the solubility issues and differences
in compound delivery method (pin tool in case of CellTiter
Glo® viability assay and pipettor in case Trypan Blue assay).
Overall, most APT derivatives 2 from the library may retain a
similar mechanism of action as 1 and act via the same molecu-
lar targets (i.e. topoisomerase II) likely responsible for the
potent anti-leukemic activity.
Husson-3CR (Scheme 2, Method A). More importantly, com-
pounds 2cc’, 2ch’, 2fd’, 2fh’, 2ga’, 2gc’, 2gd’and 2ja’, which
are inaccessible via the typical Husson-3CR, can now be pre-
pared using the ‘sacrificial’ aniline strategy albeit in low
yields and enter the library for biological screening (Table
12
1
). Overall, we were able demonstrate modest improvements
for the MCR, but the practicality of this method enabled us to
1
2
build rapidly a larger library of 34 new APTs 2xy’.
Table 1. Comparison of the Husson-3CR and modified-3CR
Figure 1. Anti-leukemic activity: Comparison of CellTiter Glo®
a
and Trypan Blue viability assay using etoposide 1 and 2ha’.
3
.2. SAR studies.
Having established the optimal reaction conditions to con-
struct the library (50 compounds in total), we extended our
investigation to the structure activity relationship (SAR) stud-
ies (Table 2). To this aim, each APT compound 2 from the
medium-sized library was tested for effect on viability of hu-
man THP-1 leukemia, pancreatic cancer PSN-1 and human
embryonic kidney HEK293 cells and compared with the two
reference compounds 2aa’ and 1 (inset Scheme 2). HEK293
cells are used routinely in drug discovery to ascertain general
cytotoxic profile in the early stage of analogues’ develop-
a
Panel A: HEK293 in Trypan Blue assay; Panel B: THP-1 in
Trypan Blue assay; Panel C: HEK293 in CellTiter Glo® assay;
Panel D: THP-1 in CellTiter Glo® assay.

ACCEPTED MANUSCRIPT
Selective site modifications of the APT chemotype 2 were first
Likely due to similar electronic factors, the aminonaphtyl-
APTs derivatives substituted via a C7-C8 linkage (rows 6 & 7)
are much more potent than the APTs having a C5-C6 linkage
(row 8). Overall, the SAR study suggests the hypothesis that
an electron rich moiety placed at the APTs’ (B-ring) would
greatly enhanced potency against leukemic cancer cell lines.
To confirm these results, the relatively less active APT 2bc’
bearing a free hydroxyl group was etherified to the corre-
sponding MOM-derivative 2lc’ in 25 % yield (Scheme 3). As
expected, APT 2lc’ demonstrated much more potency against
the THP-1 cell line (EC50 = 545 ± 52 nM), activity very simi-
lar to the reference 1 (EC50 = 540 ± 42 nM). Finally, the two
aromatic quinoline derivatives 7ja’ and 7fa’ (IC50 = 316 ± 32
nM and 7.3 ± 0.7 µM respectively) obtained from the modi-
fied-3CR were reduced to the corresponding APTs 2ja’ and
2fa’ which demonstrated extremely high potency (IC50 = 8 ±
0.7 nM and 13 ± 1 nM respectively; Scheme 3). The 3-
dimensional structure of dihydro-quinoline 2fa’ is primarily
accountable for the increase in potency (560 fold) in compari-
son to the parent aromatic quinoline 7fa’.
examined on the E-ring by looking at the electronic nature of
the substituents decorating the ring (Table 2, analysis by col-
umns). These results are mostly in agreement with the previ-
ous reports mentioning that electron donating groups (ethers)
at the 3,4 or 3,4,5-positions of the E-ring (columns 1 & 2)
17
enhanced the biological (antitumoral) activity of the ATPs 2.
Exception being for compounds 2ch’ and 2hd’, all the APT
analogues bearing either no substitution or being pentafluori-
nated on the E-ring (columns 3 & 4) tend to be considerably
less potent against the two cancer cell lines tested in our study.
Furthermore, we decided to correlate these results with the
substitution pattern of the B-ring (Table 2, analysis by rows).
As previously reported, the dioxolane unit on the B-ring is an
effective pharmacophore of the APTs skeleton as demonstrat-
ed by the IC values ranging from 12 ± 1 to 287 ± 24 nM
50
4a
(row 1). More surprisingly, the presence of a thioether at the
C8-position or a free hydroxyl at the C7-position of the B-ring
disturbs entirely the abilities of APTs to inhibit their biological
target (IC50 > 123 nM to 100 µM, rows 4 & 5). Replacing the
free hydroxyl group at C7 (from compounds 2ba’ or 2bc’) by
a difluoromethyl ether or a methyl thioether (rows 2 & 3) en-
hanced drastically the APTs biological activity affording some
of the most active molecules from the present library (e.g .
compounds 2ca’, 2cc’, 2ch’ and 2da’)
a
Scheme 3. Modulations of the APTs pharmacophores
a,b,c
Table 2. SAR study
H
N
8
1
E-ring
decreasing ring electronic density
7
2
3
O
6
4
5
EDG
O
H
MeO
MeO
O
O
OMe
F5
R
B-ring
2aa'
IC50 = 13 nMa
EC50 = 22 nMb
EC50 > 100 Mc
2ac'
2ah'
2ad'
IC50 = 49 nMa IC50 = 103 nMa IC50 = 269 nMa
H
N
EC50 = 180 nMb EC50 = 191 nMb EC50 = 1 Mb
O 7
1
2
3
4
5
6
7
8
EC50 > 50 Mc EC50 = 133 nMc EC50 > 100 Mc
O
6
2
ca'
2cc'
EC50 = 17 nMa
EC50 = 24 nMb EC50 = 88 nMb
EC50 > 100 Mc EC50 > 50 Mc
2ch'
2cd'
H
IC50 = 9 nMa
EC50 = 17 nMa EC50 = 202 nMa
O
7
N
F
EC50 = 40 nMb EC50 = 549 nMb
F
EC50 > 50 Mc EC50 > 100 Mc
a
The reported IC50 values are against the human THP-1 leukemia
2
da'
2dc'
IC50 = 37 nMa
EC50 = 66 nMb EC50 = 57 nMb
2dh'
2dd'
H
IC50 = 19 nMa
IC50 = 1 Ma
EC50 = 2 Mb
IC50 = 470 nMa
EC50 = 745 nMb
cell line.
S
7
N
EC50 > 100 Mc EC50 > 100 Mc EC50 > 100 Mc EC50 > 100 Mc
2
ba'
2bc'
EC50 = 883 nMa
EC50 > 100 Mb
EC50 > 100 Mc
In this collection of 50 APT molecules, 18 compounds have
been identified to inhibit the growth of THP-1 leukemia cells
with IC50 < 50 nM. Moreover, six of them are novel struc-
tures (2ca’, 2cc’, 2ch’, 2da’, 2jc’ and 2hd’) presenting a po-
tent activity against leukemia with IC50 < 20 nM. Figure 2
depicts a comparison of the seven most potent compounds
H
IC50 = 123 nMa
HO
7
N
EC50 = 473 nMb
EC50 > 100 Mc
---
---
12
S
8
H
N
2ea'
EC50 > 100 Ma
EC50 > 100 Mb
EC50 > 100 Mc
---
---
---
(
2ca’, 2cc’, 2da’, 2fa’, 2ha’, 2ja’ and 2jc’) in terms of bioac-
2
ha'
2hc'
IC50 = 11 nMa
EC50 = 17 nMb
2hh'
2hd'
H
N
8
IC50 = 13 nMa
IC50 = 249 nMa IC50 = 17 nMa
tivity and selectivity against three different cell lines (non-
malignant, leukemia and pancreas cancer cells). These com-
pounds are more cytotoxic against leukemia while maintaining
a low toxicity against the non-malignant cells HEK293 (EC50
7
EC50 = 60 nMb
EC50 = 222 nMb EC50 = 24 nMb
EC50 > 50 Mc EC50 > 100 Mc EC50 > 100 Mc EC50 > 500 Mc
H
N
2ga'
2gc'
EC50 = 95 nMa
2gh'
IC50 = 122 nMa
EC50 = 213 nMb EC50 = 121 nMb
EC50 > 100 Mc EC50 > 100 Mc
2gd'
8
IC50 = 41 nMa
EC50 = 71 nMb
EC50 > 100 Mc
IC50 = 247 nMa
EC50 = 968 nMb
EC50 = 3 Mc
7
NC
>
50 µM). 1 remains one of the most selective compound test-
H
2id'
ed to date with 10 fold difference between cancer cell lines.
However some novel compounds such as 2cc’ and 2jc’ also
exert a comparable selectivity (4 and 7 fold respectively). As
shown in Fig. 2 (Panel B), compounds 2cc’ (EC50 = 17 ± 2
nM) and 2jc’ (IC50 = 20 ± 1 nM) have a similar in vitro bio-
logical profile against leukemia cells, but 2jc’ uniquely dis-
plays a full dose response curve of inhibition (100%) when
reaching low micromolar concentrations.
2ic'
2
ih'
2
ia'
N
EC50 = 107 nMa
IC50 = 58 nMa
IC50 = 10.2 Ma
IC50 = 631 nMa
EC50 = 4.5 Mb
EC50 > 100 Mc
6
EC50 = 144 nMb EC50 > 50 Mb
EC50 = 1.6 Mb
EC50 > 50 Mc
5
EC50 > 50 Mc
EC50 > 50 Mc
a
The reported IC50 ob r EC values are against: human THP-1
50
c
leukemia cell line, pancreas cancer PSN-1 cell line and non-
malignant kidney HEK293 cell line.

ACCEPTED MANUSCRIPT
Figure 2. Lead compounds having high potency and selectivity
Figure 3. Best potencies against THP-1 leukemia cells for the
APTs 2ca’, 2da’, 2fa’ and 2ha’
a,b
against THP-1 leukemia cells.
H
N
R
H
H
O
N
N
O
O
H
O
H
O
O
H
MeO
MeO
2
ha'
2fa'
OMe
MeO
2
MeO
MeO
MeO
OMe
OMe
2
2
ca', R= -OCHF
da', R= -SMe
H
H
H
N
F
O
N
N
O
O
O
F
H
O
H
O
O
H
2cc'
MeO
MeO
2ja'
OMe
2jc'
O
O
O
O
Our approach to the APT-library diversity was to maintain a
low toxicity against non-malignant cells and improve the
compounds Absorption, Distribution, Metabolism and Excre-
tion properties (ADME) in comparison to the reference drug
2
0
1
.
For this purpose, specific physicochemical parameters
21
have been calculated: clogP values (clogP < 3-5) to enhance
absorption and cell permeation, topological polar surface area
2
2
2 22
values (60-75 Å < TPSA in Å <140 Å ) to maintain a good
availability and transport of the molecules and finally a direct
correlation was proposed with the numbers of hydrogen-bond
acceptor and donors sites (HBA+HBD < 12) to potentially
increase the number of available binding sites and enhance
2
3
chemical interactions with the cellular biological target. The
calculated physicochemical values for the seven most active
compounds (see Fig. 2) were in optimal range according to the
23
literature. However, these data did not follow a clear trend
2
2
(
3.0 < clogP < 3.8; 57 Å < TPSA < 75 Å ; HBA+HBD <7)
a
EC50 values are reported for HEK293 and PSN-1 cells. Panel A:
which could afford a satisfactory explanation to interpret the
changes in biological activity. Interestingly, by expanding
these calculations to specific series of compounds (C, D and
E-rings constant), a trend can be observed between the biolog-
ical activity (IC50 values against leukemia) and the compounds
lipophilicity. For example, in the series of compounds 2bc’,
b
12
selectivity chart for the 7 best APT compounds of the library.
Panel B: Compound comparison showing high selectivity and
potency.
In addition to compound 2jc’, four other APTs 2ca’, 2da’,
2
lc’, 2dc’, 2cc’, 2hc’ and 2jc’, the lipophilicity increased sub-
2
fa’ and 2ha’ with IC50 < 20 nM are showing a curve of inhi-
bition with full response at sub-micromolar concentrations
Fig. 3). From these bioactivity plots, compound 2ca’ is the
stantially (clogP = 2.0, 2.2, 3.0, 3.1, 3.3 and 3.35) which trans-
lated into an increase in cell permeation and to the observed
bioactivity (corresponding IC50 values of 883, 540, 37, 17, 50
nM). The changes of substituents at C7 from a free-phenol to
various ethers and thioethers resulted in a large improvement
in cytotoxicity which could likely be related to a combination
(
most potent against leukemia as shown by the sharp curve of
activity (Hill slope coefficient) leading to an IC50 = 9.3 ± 0.8
nM and an IC90 < 20 nM. Three other compounds 2da’, 2fa’
and 2ha’ are also showing important activity, with 2ha’ being
the second most potent and selective compound of the series
with an IC50 = 13.0 ± 2.0 nM.
of two factors: an increased in binding ability ( –OCHF and –
2
24
SCH ) and/or a diminished oxidative metabolism of degrada-
tion. Indeed, the increase in lipophilicity resulting from the
3
25
Etoposide 1 exhibits poor physicochemical properties for a
drug, likely causing its modest solubility in water (clogP
=
Lipinski’s rules with a high molecular weight (MW of 588.6
DA > 500 DA) a large number of hydrogen-bond acceptor
sites (HBA(12) > 10) and a topological polar surface area of
ether appendages (e.g. 2bc’ vs 2lc’ and 2cc’) at C7 may favor
a better cell permeation without affecting the binding proper-
ties of the small-molecules. Therefore we could conclude that
lead compounds such as 2ca’ and 2cc’ may present an interest-
ing balance between a relatively high lipophilicity to facilitate
cell permeation (clogP ~ 3.0) while presenting a relatively
1
8
1.16) and absorption. In fact, 1 disobeys three of the five
19
2
2
1
60.83 Å (TPSA > 140 Å ).

ACCEPTED MANUSCRIPT
2
large TPSA (TPSA ~ 70 Å ) to maintain an important binding
affinity to the cellular target.
The authors thank the Department of Chemistry and Biochemistry
of Florida Atlantic University and Torrey Pines Institute for Mo-
lecular Studies for providing financial support.
As shown in this SAR study, both factors of physicochemical
properties and positional pattern of substitution of the APT-
molecules are important to develop novel analogues with high
cytotoxicity. Overall, several pharmacophores have been iden-
tified with the 3,4-dioxolane and the 3,4,5-trimethoxy substitu-
tions of the E-ring and the fused 6,7-cyclopentanyl, 7,8-
phenyl, 7-methylthioether or 7-difluoromethylether on the B-
ring. Single substitution at C5 or C8 were extremely delicate
and generally resulted in largely diminish bioactivity.
ABBREVIATIONS
APT: 4-aza-2,3-didehydropodophyllotoxins; MCR: multicompo-
nent reaction .
5. EXPERIMENTAL SECTION
5.1. Chemistry.
Reactions were performed in flame-dried glassware under a posi-
tive pressure of argon. Yields refer to spectroscopically pure
compounds. Analytical TLC was performed on 0.25 mm glass
backed 60Å F-254 TLC plates (Silicycle, Inc.). The plates were
visualized by exposure to UV light (254 nm) and developed by a
solution of cerium-ammonium-molybdate in water/sulfuric acid
and heat. Flash chromatography was performed using 200-400
mesh silica gels (Silicycle, Inc.). Infrared spectra were recorded
4
. Conclusions.
In summary, we modified the Husson-3CR and create a scala-
ble and practical protocol to synthesize a series of 50 ana-
logues of the etoposide aglycon, namely the 4-aza-
1
2
1
on a Nicolet IS5 FT-IR spectrophotometer. H NMR spectra were
2
,3didehydropodophyllotoxin (APTs 2). This improved Hus-
recorded on a Varian Mercury400 (400 MHz) spectrometer and
are reported in ppm using solvent as an internal standard (DMSO-
d6 at 2.50 ppm). NMR spectra were performed using standard
parameter and data are reported as: (b = broad, s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet; coupling con-
son-3CR enabled the rapid structure-guided optimization of
the APT chemotype providing the basis for our SAR study.
From the designed library of analogues, 18 compounds (of
which 9 are novel structures) demonstrated a greater potency
against the human THP-1 leukemia cell line (IC50 < 50 nM)
than 1 (EC50 = 540 ± 42 nM). Moreover, three potent deriva-
tives 2ca’, 2ha’ and 2jc’ have been identified to be highly
selective against leukemia cells with low nanomolar antipro-
liferative activity (IC50 of 9.3 ± 0.8, 13.0 ± 2.0 and 19.6 ± 1.4
nM respectively) while preserving a low toxicity profile
against the non-malignant cells tested. In addition, the most
potent analogues possess good physicochemical properties and
specific substitution patterns on the B-ring either with an het-
eroatomic ether chain at C7 or a carbocyclic fused appendage
at the C6-C7 or C7-C8 which were therefore identified as im-
portant pharmacological sites. Finally, the synthetic MOM-
derivative 2lc’ displays a comparable anti-leukemic activity
and selectivity to etoposide 1 against leukemia and a lower
toxicity. From the knowledge gained in this study on the APT
tricyclic core, a lead compound 2ca’ may potentially be fur-
ther modified to develop more soluble derivatives as well as
prodrug analogues. Studies along these lines are in progress.
13
stant(s) in Hz, integration). C NMR spectra were recorded on
Varian Mercury400 (100 MHz) spectrometer. Chemical shifts are
reported in ppm, with solvent resonance employed as the internal
standard (DMSO-d6 at 39.5 ppm). Melting points were deter-
mined using Digimelt digital melting point apparatus. The low
resolution mass spectra were performed on Agilent 1200 series
HPLC system/6120 single quadrupole MSD (electrospray ioniza-
tion; ESI) with dual detector (PDA and ELSD). A purity of at
least 95% was obtained for all the compounds by means of chro-
matography, crystallization, or recrystallization. This level of
purity was established by LC/MS on a Agilent 1200 series HPLC
based on the ELSD and UV chromatograms (λ= 360 nm) using a
linear gradient, water + 0.1% formic acid and MeCN + 0.1% for-
mic acid, at 60:40 (0 min) to 0:100 (20 min) and a flow rate of 0.8
mL/min; Retention times are reported for the lead compounds
(
R ). Accurate mass (High resolution HR-MS) was obtained from
t
University of Florida using Agilent 6210 TOF instrument.
Novel aza-podophyllotoxins 2 were synthesized using either the
typical Husson-3CR (method A) or using a modified-3CR enabled
ASSOCIATED CONTENT
12
4f,8b,9,10i
by a sacrificial aniline (method B). Compounds 2aa’
,
4
f
10i
1b,4f,9,10i
4e,8b
9
4f,9
8b,11
4e,4f
*
S
Supporting Information
2ac’ , 2ae’ , 2af’
, 2ag’ , 2ah’ , 2ba’
, 2fa’
and 2kh’
,
4e
10i
4f,10c
10c
10d
10d
Experimental procedures, characterization data, biological profile,
and NMR spectra of all new APT-molecules 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
2ha’ , 2hc’ , 2ia’
, 2ic’
, 2ih’ , 2kc’
were previously reported by others. Only biological activity was
4a
reported for Compound 2ja’ . Compounds 2bc’, 2ca’, 2dc’,
dh’ and 2fc’ have been previously reported and fully character-
2
1
13
11
AUTHOR INFORMATION
Corresponding Author
ized by us ( H, C NMRs, IR, melting points and HRMS).
General procedure A: A round bottom flask was charged under
argon with aniline 3 (1.0 equiv.), aldehyde 4 (1.0 equiv.), and
tetronic acid 5 (1.0 equiv.) in 2-pentanol or ethanol [0.3 M]. Reac-
*sroche2@fau.edu; dzm0032@auburn.edu.
º
º
tion was refluxed (at 125 C or 80 C respectively) for 1 hour, then
the solvent was evaporated under vacuum and the crude product
purified by recrystallization in ethanol or by flash chromatog-
raphy on silica gel.
Author Contributions
The manuscript was written through contributions of AH, DM and
SPR. / All authors have given approval to the final version of the
manuscript.
General procedure B: A two neck round bottom flask under
argon equipped with a condenser was charged with aldehyde 4
Notes
(1.0 equiv.) and 4-chloroaniline 3l (1.1 equiv.) in 2-pentanol [0.3
The authors declare no competing financial interest.
M] and stirred at reflux for two hours. The tetronic acid 5 (1.1
equiv.) in 2-pentanol (minimum amount) was then added at re-
flux. After another 10 mins at reflux, the third component aniline
ACKNOWLEDGMENT
3
(1.0 equiv.) was added neat. The reaction mixture was refluxed

ACCEPTED MANUSCRIPT
for an additional 30 mins. All volatiles were evaporated under
vacuum and the crude product purified by recrystallization in
ethanol or by flash chromatography on silica gel.
Hz, 1H), 6.33 (dd, J = 8.1, 1.6 Hz, 1H), 6.32 (d, J = 1.6 Hz, 1H),
4.92 (d, J = 15.6 Hz, 1H), 4.82 (d, J = 15.6 Hz, 1H), 4.76 (s, 1H),
3.70 (s, 3H); LC-MS (ESI): m/z 326.1 (M+H).
9
-(benzo[d][1,3]dioxol-5-yl)-6,9-dihydro-[1,3]dioxolo[4,5-
6
-(difluoromethoxy)-9-(3,4,5-trimethoxyphenyl)-4,9-
g]furo[3,4-b]quinolin-8(5H)-one (2ac’). Compound 2ac’ was
prepared accordingly to the general procedure A for the one pot
synthesis using 1,3-benzodioxole-5-carbaldehyde 4c’ (150 mg, 1.0
mmol, 1.0 equiv.), 3,4-(methylenedioxy)aniline 3a (137 mg, 1.0
mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0
equiv.) in 2-pentanol (3.3 mL). The crude product was filtered and
washed with cold ethanol (3x 2.5 mL) to obtain compound 2ac’ in
dihydrofuro[3,4-b]quinolin-1(3H)-one 2ca’. Compound 2ca’
was prepared accordingly to the general procedure B for the one
pot sequential synthesis using 4-chloroaniline 3l (72 mg, 0.56
mmol, 1.1 equiv.), 3-difluoromethoxy aniline 3c (81 mg, 0.51
mmol, 1.0 equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (100 mg,
0
1
.51 mmol, 1.0 equiv.) and tetronic acid 5 (56 mg, 0.56 mmol,
.1 equiv.), in 2-pentanol (1.7 mL + 0.4 mL). The crude product
a pure form (171 mg, 0.5 mmol, 49% yield). Light brown solid;
was purified by column chromatography using isocratic solvent
system of 20% acetone in hexanes system to obtain pure com-
pound 2ca’ (30 mg, 0.06 mmol, 14% yield). Compound 2ca’ was
also prepared accordingly to the general procedure A for the one
pot synthesis using the exact same quantities and was purified by
column chromatography using isocratic solvent system of 20%
acetone in hexanes system to obtain pure compound 2ca’ (5 mg,
°
R = 0.29 (acetone/hexanes; 35/65); m.p. > 260 C; IR νmax
f
(
neat): 3224, 3095, 1712, 1642, 1562, 1481, 1248, 1235, 1038,
-
1
1
1
9
1
016, 933, 780 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm):
6
.87 (bs, 1H), 6.83 – 6.75 (m, 1H), 6.73 (s, 1H), 6.65 (dd, J = 7.7,
.5 Hz, 1H), 6.59 (s, 1H), 6.51 (s, 1H), 5.97 – 5.87 (m, 4H), 4.97
(d, J = 15.8 Hz, 1H), 4.83 (s, 1H), 4.83 (d, J = 15.8 Hz, 1H); LC-
MS (ESI): m/z 374.0 (M+Na).
0
.01 mmol, 2% yield). White powder, R = 0.22 (Hex-
f
o
anes/acetone 65 / 35); m.p.>260 C; IR νmax (neat): 3246, 1727,
1
9
-(perfluorophenyl)-6,9-dihydro-[1,3]dioxolo[4,5-g]furo[3,4-
-
1 1
644, 1548, 1492, 1174 cm ; H NMR (400 MHz, DMSO) δ
b]quinolin-8(5H)-one (2ad’). Compound 2ad’ was prepared
accordingly to the general procedure A for the one pot synthesis
using pentafluorobenzaldehyde 4d’ (490 mg, 2.5 mmol, 1.0
equiv.), 3,4-(methylenedioxy)aniline 3a (343 mg, 2.5 mmol, 1.0
equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in eth-
anol (10 mL). The crude product was filtered and washed with
cold ethanol (3x 2.5 mL) to obtain compound 2ad’ (99 mg, 0.3
(ppm):10.13 (s, 1H), 7.19 (s, 1H), 7.18 (t, J = 73.9 Hz, 1H), 6.73
(
3
d, J = 8.1 Hz, 1H), 6.69 (s, 1H), 6.50 (s, 2H), 5.18 – 4.78 (m,
H), 3.70 (s, 6H), 3.60 (s, 3H); C NMR (100 MHz, DMSO-d6)
13
δ (ppm): 171.9, 158.5, 152.8 (2C), 150.0, 142.4, 137.3, 136.1,
1
9
32.1, 121.3, 116.3 (t, J = 278.6 Hz), 113.2, 106.2, 104.9 (2C),
5.7, 65.1, 59.9, 55.8 (2C), 30.7; LC-MS (ESI): R = 5.1 mins,
t
m/z 420.0 (M+H); HR-MS (ESI): m/z Calcd for
mmol, 10% yield). White solid; R = 0.30 (acetone/hexanes;
f
+
°
[C H F NO +H] 420.1253, Found 420.1250 (-0.7 ppm).
3
1
5/65); m.p. >260 C; IR νmax (neat): 3230, 1727, 1650, 1498,
481, 1190, 1040, 1020, 990, 942, 835, 761 cm ; H NMR (400
21 19
2
6
-
1 1
9
-(benzo[d][1,3]dioxol-5-yl)-6-(difluoromethoxy)-4,9-
MHz, DMSO-d ) δ (ppm): 10.12 (bs, 1H), 6.54 (s, 1H), 6.51 (s,
6
dihydrofuro[3,4-b]quinolin-1(3H)-one (2cc’). Compound 2cc’
was prepared accordingly to the general procedure B for the se-
quential one pot synthesis using 4-chloroaniline 3l (140mg, 1.1
mmol, 1.1 equiv.), 1,3-benzodioxole-5-carbaldehyde 4c’ (150 mg,
1
1
4
H), 5.96 (s, 1H), 5.93 (s, 1H), 5.43 (s, 1H), 4.90 (d, J = 16.0 Hz,
H), 4.85 (d, J = 16.0 Hz, 1H); LC-MS (ESI): m/z 398.0 (M+H),
20.0 (M+Na).
1
.0 mmol, 1.0 equiv.), 3-(difluoromethoxy) benzamine 3c (159
mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (110 mg, 1.1 mmol,
.1 equiv.) in 2-pentanol (3.3 mL). The oily crude product was
9
-(4-methoxyphenyl)-6,9-dihydro-[1,3]dioxolo[4,5-g]furo[3,4-
b]quinolin-8(5H)-one (2af’). Compound 2af’ was prepared ac-
cordingly to the general procedure A for the one pot synthesis
using 4-methoxybenzaldehyde 4f’ (340 mg, 2.5 mmol, 1.0
equiv.), 3,4-(methylenedioxy)aniline 3a (343 mg, 2.5 mmol, 1.0
equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in eth-
anol (10 mL). The crude product was filtered and sonicated in
dichloromethane (4 mL) for 15 minutes, followed by centrifuga-
tion at 5,800 rpm for 10 minutes (x3) to obtain pure compound
1
taken in methanol (2.5 mL) and the precipitate was filtered and
washed with cold ethanol (3x 2.5 mL) to obtain pure compound
2
cc’ (11 mg, 0.03 mmol, 3% yield). Off-white crystals; R = 0.41
f
°
(acetone/hexanes; 40/60); m.p. > 260 C; IR νmax (neat): 3322,
-
1
1
1
728, 1641, 1611, 1482, 1109, 1040, 1011, 798 cm ; H NMR
(400 MHz, DMSO-d ) δ (ppm): 10.15 (bs, 1H), 7.17 (t, J = 74.0
6
Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75
(d, J = 1.6 Hz, 1H), 6.72 (dd, J = 8.5, 2.4 Hz, 1H), 6.69 – 6.67 (m,
2H), 5.94 (d, 2.0 Hz, 2H), 4.93 (s, 1H), 4.93 (d, 15.4 Hz, 1H),
2
af’ (444 mg, 1.3 mmol, 53% yield). Pale yellow solid; R = 0.19
f
°
(acetone/hexanes; 35/65); m.p. 136-137 C; IR νmax (neat): 3222,
-
1
3
157, 3092, 1711, 1641, 1560, 1502, 1478, 1192, 1016, 831 cm ;
1
4.87 (d, 15.4 Hz, 1H); LC-MS (ESI): R = 7.3 mins, m/z 374.0
H NMR (400 MHz, DMSO-d ) δ (ppm): 9.84 (bs, 1H), 7.09 (d, J
t
6
(M+H), 396.0 (M+Na); HR-MS (ESI): m/z Calcd for
=
5
4
8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 6.54 (s, 1H), 6.51 (s, 1H),
.95 (s, 1H), 5.89 (s, 1H), 4.92 (d, J = 15.5 Hz, 1H), 4.85 (s, 1H),
.83 (d, J = 15.5 Hz, 1H), 3.69 (s, 3H); LC-MS (ESI): m/z 338.0
+
[
C H F NO +H] 374.0840, Found 374.0850 (+ 2.7 ppm).
19 13
2
5
6
-(difluoromethoxy)-9-(perfluorophenyl)-4,9-dihydrofuro[3,4-
(M+H).
b]quinolin-1(3H)-one (2cd’). Compound 2cd’ was prepared ac-
cordingly to the general procedure A for the one pot synthesis
using 3-(difluoromethoxy)benzamine 3c (159 mg, 1.0 mmol, 1.0
equiv.), pentafluorobenzaldehyde 4d’ (196 mg, 1.0 mmol, 1.0
equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-
pentanol (3.3 mL). The crude product was filtered and washed
with cold ethanol (3x 2.5 mL) to obtain compound 2cd’ (56 mg,
0
3
1
6
-hydroxy-9-(4-hydroxy-3-methoxyphenyl)-4,9-
dihydrofuro[3,4-b]quinolin-1(3H)-one (2bb’). Compound 2bb’
was prepared accordingly to the general procedure A for the one
pot synthesis using 3-hydroxy-4-methoxybenzaldehyde 4b’ (381
mg, 2.5 mmol, 1.0 equiv.) 3-aminophenol 3b (273 mg, 2.5 mmol,
1
.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in
.13 mmol, 13% yield). White solid; R = 0.29 (acetone/hexanes;
ethanol (10 mL). The product was filtered off the crude reaction
mixture and washed with cold ethanol (3x 2.5 mL) to obtain pure
compound 2bb’ (235 mg, 0.72 mmol, 29% yield). Off-white sol-
f
°
0/70); m.p. > 260 C; IR νmax (neat): 1726, 1644, 1495, 1190,
170, 1021, 991, 762 cm ; H NMR (400 MHz, DMSO-d ) δ
-
1 1
6
°
(ppm): 10.38 (bs, 1H), 7.23 (t, J = 73.7 Hz, 1H), 7.06 (d, J = 8.3,
H), 6.75 (dd, J = 8.3, 1.7 Hz, 1H), 6.71 (d, J = 2.0 Hz, 1H), 5.56
id; R = 0.24 (acetone/hexanes; 45/55); m.p. > 260 C; IR νmax
f
1
(neat): 3446, 3377, 3231, 1724, 1646, 1622, 1496, 1152, 1026,
1
3
-
1
1
(s, 1H), 4.96 (d, J = 16.1, 1H), 4.90 (d, J = 16.1, 1H); C NMR
100 MHz, DMSO-d ) δ (ppm): 171.4, 159.1, 150.7, 138.1 (2C),
1
018, 770 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 9.84
6
(
(
(
bs, 1H), 9.43 (s, 1H), 8.75 (s, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.79
d, J = 1.6 Hz, 1H), 6.62 (d, J = 8.1 Hz, 1H), 6.46 (dd, J = 8.1, 1.6
6

ACCEPTED MANUSCRIPT
1
9
31.9 (3C), 117.7, 116.2 (t, J = 258.6 Hz), 113.4 (3C), 106.3 (2C),
2.2, 65.4, 28.7; LC-MS (ESI): m/z 420.0 (M+H), 442.0 (M+Na);
(3x 2.5 mL) to obtain pure compound 2de’ (98 mg, 0.3 mmol,
30% yield). White solid; R = 0.29 (acetone/hexanes; 30/70); m.p.
f
°
>
1
260 C; IR νmax (neat): 3240, 1711, 1639, 1606, 1532, 1486,
6
1
-(difluoromethoxy)-9-phenyl-4,9-dihydrofuro[3,4-b]quinolin-
(3H)-one (2ch’). Compound 2ch’ was prepared accordingly to
-1
1
211, 1022, 920, 851 cm ; H NMR (400 MHz, DMSO-d ) δ
6
(ppm): 10.10 (bs, 1H), 7.22 (dd, J = 8.4, 5.6 Hz, 2H), 7.07 (t, J =
the general procedure B for the sequential one pot synthesis using
4
4
zamine 3c (159 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (110
mg, 1.1 mmol, 1.1 equiv.) in 2-pentanol (3.3 mL). The oily crude
product was taken in methanol (2.5 mL) and the precipitate was
filtered and washed with cold ethanol (3x 2.5 mL) to obtain pure
compound 2ch’ (12 mg, 0.4 mmol, 4% yield). Metallic white
crystals; R =0.35 (70/30 acetone/hexanes); m.p. > 260 C; IR
νmax (neat): 3231, 1723, 1636, 1617, 1495, 1118, 1012, 746, 696
cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.16 (bs, 1H),
7
5
8
2
1
1
1
.8 Hz, 2H), 6.95 (d, J = 8.0 Hz, 1H), 6.80 (dd, J = 10.1, 1.7 Hz,
-chloroaniline 3l (140 mg, 1.1 mmol, 1.1 equiv.), benzaldehyde
h’ (106 mg, 1.0 mmol, 1.0 equiv.), 3-(difluoromethoxy) ben-
H), 5.00 (s, 1H),4.97 (d, J = 15.8 Hz, 1H), 4.87 (d, J = 15.8 Hz,
13
H), 2.42 (s, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm):
6
72.1, 160.9 (d, J = 240.0 Hz,), 158.6, 143.1 (d, J = 3.0 Hz), ,
37.6, 136.7, 131.3, 129.5 (d, J = 8.0 Hz, 2C), 121.1, 120.8, 115.2
(d, J = 21.0 Hz, 2C), 113.0, 95.6, 65.3, 38.3, 14.6; LC-MS (ESI):
m/z 328.0 (M+H), 350.0 (M+Na).
°
f
9-(2,3-dimethoxyphenyl)-6-(methylthio)-4,9-dihydrofuro[3,4-
b]quinolin-1(3H)-one (2dg’). Compound 2dg’ was prepared
accordingly to the general procedure A for the one pot synthesis
using 3-methylthioaniline 3d (123 ꢀL, 1.0 mmol, 1.0 equiv.), 2,3-
dimethoxybenzaldehyde 4g’ (166 mg, 1.0 mmol, 1.0 equiv.), and
tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol (3.3
mL). The crude product was purified by recrystallization in etha-
nol (2.5 mL), then filtered and washed with cold ethanol (3 x 2.5
mL) to obtain pure compound 2dg’(66 mg, 0.2 mmol, 18% yield).
-
1
1
6
.18 (t, J = 74.0 Hz, 1H), 7.28-7.07 (m, 6H), 6.73-6.69 (m, 2H),
.01 (s, 1H), 5.00 (d, J = 15.8 Hz, 1H), 4.89 (d, J = 15.8 Hz, 1H);
LC-MS (ESI): m/z 368.0 (M+K).
6
-(methylthio)-9-(3,4,5-trimethoxyphenyl)-4,9-
dihydrofuro[3,4-b]quinolin-1(3H)-one (2da’). Compound 2da’
was prepared accordingly to the general procedure A for the one
pot synthesis using 3-methylthioaniline 3d (138 mg, 1.0 mmol,
White powder; R = 0.21 (acetone/hexanes; 30/70); m.p. > 260
f
°
C; IR νmax (neat): 1721, 1636, 1526, 1483, 1351, 1225, 1078,
1
.0 equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (196 mg, 1.0
-1
1
1
011, 740 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 9.98
6
mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0
equiv.) in 2-pentanol (3.3 mL). The crude product was recrystal-
lized in ethanol (2.5 mL), then filtered and washed with cold
ethanol (3x 2.5 mL) to obtain pure compound 2da’ (78 mg, 0.2
mmol, 20% yield). White crystals; R = 0.25 (acetone/hexanes;
3
1
(bs, 1H), 6.94 (t, J = 7.9 Hz, 1H), 6.87 – 6.81 (m, 2H), 6.75 (dd, J
=
8.2, 1.9 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H), 6.64 (dd, J = 7.7, 1.1
Hz, 1H), 5.25 (s, 1H), 4.96 (d, J = 15.4 Hz, 1H), 4.87 (d, J = 15.4
Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 2.40 (s, 3H); LC-MS (ESI):
m/z: 370.0 (M+H), 392.0 (M+Na).
f
°
5/65); m.p. > 260 C; IR νmax (neat): 3249, 2359, 2341, 1725,
635, 1483, 1128, 1008, 750 cm ; H NMR (400 MHz, DMSO-
-
1 1
5-(methylthio)-9-(3,4,5-trimethoxyphenyl)-4,9-
d6) δ (ppm): 10.00 (bs, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.81 (d, J =
dihydrofuro[3,4-b]quinolin-1(3H)-one (2ea’). Compound 2ea’
was prepared accordingly to the general procedure A for the one
pot synthesis using 3,4,5-trimethoxybenzaldehyde 4a’ (491 mg,
2.5 mmol, 1.0 equiv.), 2-methylthioaniline 3e (348 mg, 2.5 mmol,
1.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in
ethanol (10 mL). The crude product was purified by column
chromatography on silica gel using an isocratic solvent system of
acetone and dichloromethane (20:80) to obtain pure compound
8
4
2
1
1
.1 Hz, 1H), 6.77 (s, 1H), 6.49 (s, 2H), 5.02 (d, J = 15.7 Hz, 1H),
.91 (s, 1H), 4.87 (d, J = 15.7 Hz, 1H), 3.70 (s, 6H), 3.60 (s, 3H),
13
.42 (s, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm) 172.0,
6
58.7, 152.8 (2C), 142.6, 137.3, 136.5, 136.1, 131.1, 121.1,
20.7, 112.9, 104.9 (2C), 95.4, 65.1, 59.9, 55.9 (2C), 39.3, 14.6;
LC-MS (ESI): R = 6.5 mins, m/z 400.0 (M+1), 422.0 (M+Na);
t
+
HR-MS (ESI): m/z Calcd for [C H NO S+H] 400.1219, Found
21
21
5
4
00.1225 (+ 2.4 ppm).
2ea’ (771 mg, 1.93 mmol, 77% yield). Red solid; R = 0.29 (ace-
f
°
tone/hexanes; 45/55); m.p. > 260 C; IR νmax (neat): 2929, 1734,
6
-(methylthio)-9-(perfluorophenyl)-4,9-dihydrofuro[3,4-
-1
1
1
669, 1589, 1455, 1418, 1230, 1121, 1005, 770 cm ; H NMR
b]quinolin-1(3H)-one (2dd’). Compound 2dd’ was prepared
accordingly to the general procedure A for the one pot synthesis
using 3-methylthioaniline 3d (138 mg, 1.0 mmol, 1.0 equiv.),
pentafluorobenzaldehyde 4d’ (196 mg, 1.0 mmol, 1.0 equiv.) and
tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol (3.3
mL). The crude product was recrystallized in ethanol (2.5 mL),
then filtered and washed with cold ethanol (3x 2.5 mL) to obtain
pure compound 2dd’ (112 mg, 0.28 mmol, 28% yield). Off-white
solid; R = 0.30 (acetone/hexanes; 30/70); m.p. > 260 C; IR
νmax (neat): 3213, 1719, 1637, 1518, 1499, 1486, 1211, 1022,
(
400 MHz, DMSO-d ) δ (ppm): 7.09 (d, J = 2.1 Hz, 1H), 6.86 (dd,
6
J = 8.3, 2.1 Hz, 1H), 6.60 (d, J = 8.3 Hz, 1H) 6.59 (s, 2H), 4.86 (s,
H), 4.67 (s, 2H), 3.67 (s, 6H), 3.62 (s, 3H), 2.24 (s, 3H); LC-MS
ESI): m/z 418.1 (M+H O+H);
1
(
2
9-(4-hydroxy-3-methoxyphenyl)-5-(methylthio)-4,9-
dihydrofuro[3,4-b]quinolin-1(3H)-one (2eb’). Compound 2eb’
was prepared accordingly to the general procedure A for the one
pot synthesis using 3-hydroxy-4-methoxybenzaldehyde 4b’ (380
mg, 2.5 mmol, 1.0 equiv.), 2-methylthioaniline 3e (348 mg, 2.5
mmol, 1.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0
equiv.) in ethanol (10 mL). The crude product was purified by
column chromatography on silica gel using an isocratic solvent
system of acetone and dichloromethane (30:70) to obtain pure
°
f
-
1
1
9
1
1
1
92, 955, 916 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm):
6
0.26 (bs, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.82 (dd, J = 8.1, 1.7 Hz,
H), 6.77 (d, J = 1.7 Hz, 1H), 5.51 (s, 1H), 4.96 (d, J = 16.0 Hz,
H), 4.91 (d, J = 16.0 Hz, 1H), 2.44 (s, 3H); C NMR (100 MHz,
1
3
DMSO-d ) δ (ppm): 171.5, 159.3 (2C), 138.7 (2C), 137.2 (2C),
compound 2eb’ (733 mg, 2.1 mmol, 83% yield). Deep red solid;
R = 0.26 (acetone/hexanes; 45/55); m.p. > 260 C; IR νmax
f
6
°
1
30.6 (2C), 120.8 (2C), 117.3, 112.8 (2C), 91.7, 65.4, 28.8, 14.4;
LC-MS (ESI): m/z 422.0 (M+Na).
(neat): 2926, 1609, 1512, 1427, 1268, 1229, 1124, 1031, 779, 687
-
1
1
cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.75 (bs, 1H),
6
9
-(4-fluorophenyl)-6-(methylthio)-4,9-dihydrofuro[3,4-
7
3
3
.06 (d, J = 2.0 Hz, 1H), 6.85 – 6.81 (m, 2H), 6.68 – 6.56 (m,
H), 5.07 (bs, 1H), 4.82 (s, 1H), 4.64 (s, 2H), 3.65 (s, 3H), 2.23 (s,
H); LC-MS (ESI): m/z 356.2 (M+H).
b]quinolin-1(3H)-one (2de’). Compound 2de’ was prepared ac-
cordingly to the general procedure A for the one pot synthesis
using 3-methylthioaniline 3d (138 mg, 1.0 mmol, 1.0 equiv.), 4-
fluorobenzaldehyde 4e’ (124 mg, 1.0 mmol, 1.0 equiv.) and
tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol (3.3
mL). The crude product was filtered and washed with cold ethanol
11-(3,4,5-trimethoxyphenyl)-6,7,8,9-
tetrahydrobenzo[g]furo[3,4-b]quinolin-1(3H)-one (7fa’). Com-
pound 7fa’ was prepared accordingly to the general procedure B

ACCEPTED MANUSCRIPT
for the sequential one pot synthesis using 4-chloroaniline 3l (140
mg, 1.1 mmol, 1.1 equiv.), 5,6,7,8-tetrahydronaphthalen-2-amine
=16.1 Hz, 1H), 4.90 (s, 1H), 4.85 (d, J = 16.1 Hz, 1H), 3.38 (s,
2H), 2.63 (s, 2H), 1.65 (s, 4H). LC-MS (ESI): m/z 318.0 (M+H);
3
4
1
f (147 mg, 1.0 mmol, 1.0 equiv.), 3,4,5-trimethoxybenzaldehyde
a’ (196 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (110 mg,
.1 mmol, 1.1 equiv.) in 2-pentanol (1.7 mL). The crude product
8
-oxo-7-(3,4,5-trimethoxyphenyl)-7,8,10,11-
tetrahydrobenzo[h]furo[3,4-b]quinoline-5-carbonitrile (2ga’).
Compound 2ga’ was prepared accordingly to the general proce-
dure B for the sequential one pot synthesis using 4-chloroaniline
was taken in methanol (2.5 mL) and the precipitate was filtered
and washed with cold ethanol (3x 2.5 mL) to obtain pure com-
pound 7fa’ (25 mg, 0.06 mmol, 6% yield). White solid; R = 0.38
3
l (140mg, 1.1 mmol, 1.1 equiv.), 3,4,5-trimethoxybenzaldehyde
f
°
4a’ (196 mg, 1.0 mmol, 1.0 equiv.), 4-amino-1-naphthonitrile 3g
(168 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (110 mg, 1.1
mmol, 1.1 equiv.) in 2-pentanol (3.3 mL). The crude product was
recrystallized in ethanol (2.5 mL), then filtered and washed with
cold ethanol (3x 2.5 mL) to obtain pure compound 2ga’ (17 mg,
(ethyl acetate/hexanes; 1:1); m.p. > 260 C; IR νmax (neat): 2936,
-
1
1
768, 1599, 1579, 1445, 1412, 1121, 1039, 1029, 706, 687 cm ;
1
H NMR (400 MHz, DMSO-d ) δ (ppm): 7.89 (bs, 1H), 7.60 (s,
6
1
H), 6.77 (s, 2H), 5.46 (s, 2H), 3.79 (s, 3H), 3.77 (s, 6H), 3.03 (t,
J = 5.2 Hz, 2H), 2.90 (t, J = 5.3 Hz, 2H), 1.84 – 1.75 (m, 4H);
LC-MS (ESI): m/z 406.0 (M+H).
0
.04 mmol, 4% yield). Pale yellow solid; R = 0.29 (ace-
f
°
tone/hexanes; 40/60); m.p. >260 C; IR νmax (neat): 3367, 1741,
1676, 1595, 1456, 1331, 1118, 1029, 998, 749 cm ; H NMR
-
1
1
1
1-(3,4,5-trimethoxyphenyl)-4,6,7,8,9,11-
hexahydrobenzo[g]furo[3,4-b]quinolin-1(3H)-one (2fa’). To a
suspension of compound 7fa’ (21 mg, 0.05 mmol, 1.0 equiv.) in
glacial acetic acid (0.6 mL, 15.0 equiv.) was added sodium cya-
noborohydride (10 mg, 0.2 mmol, 3.0 equiv.) in one portion at
room temperature and the reaction mixture was stirred for 4 hours.
The reaction mixture was poured into an ice cold water (2 mL)
and the newly formed precipitate was filtered and finally washed
with cold ethanol (2x 1 mL) to obtain pure compound 2fa’ (12
(400 MHz, DMSO-d ) δ (ppm): 10.68 (bs, 1H), 8.40 – 8.35 (m,
6
1H), 8.14 – 8.01 (m, 3H), 7.95 (s, 1H), 7.81 (dd, J = 6.3, 3.3 Hz,
2H), 5.69 (s, 1H), 5.18 (d, J = 16.2 Hz, 1H), 5.13 (d, J = 16.2 Hz,
1H), 3.83 (s, 3H), 3.80 (s, 3H), 3.71 (s, 3H); LC-MS (ESI): m/z
451.0 (M+Na).
7
-(benzo[d][1,3]dioxol-5-yl)-8-oxo-7,8,10,11-
tetrahydrobenzo[h]furo[3,4-b]quinoline-5-carbonitrile (2gc’).
Compound 2gc’ was prepared accordingly to the general proce-
dure B for the sequential one pot synthesis using 4-chloroaniline
3l (140mg, 1.1 mmol, 1.1 equiv.), 1,3-benzodioxole-5-
carbaldehyde 4c’ (150 mg, 1.0 mmol, 1.0 equiv.), 4-amino-1-
naphthonitrile 3g (168 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid
mg, 0.03 mmol, 57% yield). White powder; R = 0.3 (hex-
f
o
anes/acetone 40 / 60); m.p. > 260 C; IR νmax (neat): 1734, 1658,
320, 1223, 1011, 750 cm ; H NMR (400 MHz, DMSO-d ) δ
-
1
1
1
6
(ppm): 9.85 (bs, 1H), 6.81 (s, 1H), 6.59 (s, 1H), 6.47 (s, 2H), 4.99
(d, J = 15.7 Hz, 1H), 4.85 (d, J = 15.8 Hz, 1H), 4.85 (s, 1H), 3.70
(d, J = 1.3 Hz, 6H), 3.60 (d, J = 1.5 Hz, 3H), 2.64 – 2.62 (m, 2H),
5
(110 mg, 1.1 mmol, 1.1 equiv.) in 2-pentanol (3.3 mL). The
crude product was recrystallized in ethanol (2.5 mL), then filtered
and washed with cold ethanol (3x 2.5 mL) to obtain pure com-
pound 2gc’ (50 mg, 0.13 mmol, 10% yield). Light brown solid;
2
.54 - 2.50 (m, 2H), 1.65-1.62 (m, 4H); LC-MS (ESI): R = 7.9
t
mins, m/z 408.0 (M+H); HR-MS (ESI): m/z Calcd for
+
[
C H NO +H] 408.1811, Found 408.1795 (- 1.5 ppm). Other
24 25 5
°
4e,4f
m.p. >260 C; IR νmax (neat): 2256, 1714, 1611, 1383, 1329,
spectral data match with the previous report from literature.
-
1 1
1
027, 920, cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.68
6
1
1-(perfluorophenyl)-4,6,7,8,9,11-hexahydrobenzo[g]furo[3,4-
(bs, 1H), 8.37 (dd, J = 6.4, 3.2 Hz, 1H), 8.03 (dd, J = 6.7, 3.1 Hz,
1H), 7.88 (s, 1H), 7.81 (dd, J = 4.5, 1.8 Hz, 2H), 6.88 (s, 1H),
6.82 (d, J = 8.0 Hz, 1H), 6.76 (dd, J = 8.0, 1.8 Hz, 1H), 5.95 (d, J
= 6.2 Hz, 2H), 5.16 (s, 1H), 5.14 (d, J = 15.5 Hz, 1H), 5.01 (d, J =
15.5 Hz, 1H); LC-MS (ESI; negative mode): m/z 381.0 (M-H).
b]quinolin-1(3H)-one (7fd’). Compound 2fd’ was prepared ac-
cordingly to the general procedure B for the sequential one pot
synthesis using 4-chloroaniline 3l (72 mg, 0.6 mmol, 1.1 equiv.),
5
,6,7,8-tetrahydro-2-naphthylamine 3f (75 mg, 0.5 mmol, 1.0
equiv.), pentafluorobenzaldehyde 4d’ (100 mg, 0.5 mmol, 1.0
equiv.) and tetronic acid 5 (56 mg, 0.6 mmol, 1.1 equiv.) in 2-
pentanol (1.7 mL). The crude product was filtered and washed
with cold ethanol (3 x 2.5 mL) to obtain pure compound 2fd’ (12
8
-oxo-7-(perfluorophenyl)-7,8,10,11-
tetrahydrobenzo[h]furo[3,4-b]quinoline-5-carbonitrile (2gd’).
Compound 2gd’ was prepared accordingly to the general proce-
dure B for the sequential one pot synthesis using 4-chloroaniline
3l (72 mg, 0.6 mmol, 1.1 equiv.), 4-amino-1-
naphthalenecarbonitrile 3g (86 mg, 0.5 mmol, 1.0 equiv.), pen-
tafluorobenzaldehyde 4d’ (100 mg, 0.5 mmol, 1.0 equiv.) and
tetronic acid 5 (56 mg, 0.6 mmol, 1.1 equiv.) in 2-pentanol (1.7
mL). The crude product was filtered and washed with cold ethanol
mg, 0.03 mmol, 5% yield). Off-white solid; R = 0.35 (ace-
f
°
tone/hexanes; 30/70); m.p. >260 C; IR νmax (neat): 1724, 1638,
467, 1021, 987, 946 cm ; H NMR (400 MHz, DMSO-d ) δ
-
1
1
1
6
(ppm): 10.12 (bs, 1H), 6.65 (s, 1H), 6.60 (s, 1H), 5.43 (s, 1H),
4
.91 (d, J = 15.8 Hz, 1H), 4.87 (d, J = 15.8 Hz, 1H), 2.61 - 2.65
1
3
(m, 2H), 2.50 – 2.53 (m, 2H), 1.62 – 1.67 (m, 4H); C NMR (100
(3 x 2.5 mL) to obtain pure compound 2gd’ (14 mg, 0.03 mmol,
MHz, DMSO-d ) δ (ppm): 171.7, 159.3, 137.0 (2C), 134.1, 132.2
6
6
>
6
% yield). White solid; R = 0.30 (acetone/hexanes; 30/70); m.p.
f
(2C), 130.1 (3C), 118.2, 116.1 (3C), 90.8, 65.3, 28.9 28.5, 28.1,
°
260 C; IR νmax (neat): 1592, 1444, 1364, 1205, 983, 764, 703,
85 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.90 (bs,
2
2.7, 22.6; LC-MS (ESI): m/z 408.0 (M+H), 430.0 (M+Na).
-
1
1
6
1
1
1-phenyl-4,6,7,8,9,11-hexahydrobenzo[g]furo[3,4-b]quinolin-
(3H)-one. Compound 2fh’ was prepared accordingly to the gen-
1H), 8.37 (d, J = 7.2 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.91 –
7.77 (m, 2H), 7.76 (s, 1H), 5.71 (s, 1H), 5.14 (d, J = 15.5 Hz, 1H),
13
eral procedure B for the sequential one pot synthesis using 4-
chloroaniline 3l (140mg, 1.1 mmol, 1.1 equiv.), 5,6,7,8-
tetrahydro-2-naphthylamine 3f (147 mg, 1.0 mmol, 1.0 equiv.),
benzaldehyde 4h’ (106 mg, 1.0 mmol, 1.0 equiv.) and tetronic
acid 5 (110 mg, 1.1 mmol, 1.1 equiv.) in 2-pentanol (3.3 mL). The
crude product was recrystallized in ethanol (2.5 mL), then filtered
and washed with cold ethanol (3x 2.5 mL) to obtain pure com-
pound 2fh’ (16 mg, 0.05 mmol, 5% yield). Light brown solid; R
=
1
4.87 (d, J = 15.5 Hz, 1H); C NMR (100 MHz, DMSO-d ) δ
6
(ppm):171.1, 158.9, 137.0 (2C), 135.3, 131.8, 129.6 (2C), 128.0
(2C), 124.9, 122.2 (2C), 121.9 (2C), 117.6, 115.4, 103.1 (2C),
94.8, 66.2, 29.5; LC-MS (ESI): m/z 429.1 (M+H).
7
-(3,4,5-trimethoxyphenyl)-7,11-dihydrobenzo[h]furo[3,4-
b]quinolin-8(10H)-one (2ha’). Compound 2ha’ was prepared
accordingly to the general procedure B for the one pot sequential
synthesis using 4-chloroaniline 3l (128 mg, 1.1 mmol, 1.1 equiv.),
4-amino-1-naphthalenecarbonitrile 3h (143 mg, 1.0 mmol, 1.0
equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (196 mg, 1.0 mmol,
f
°
0.41 (acetone/hexanes; 35/65); m.p. >260 C; IR νmax (neat):
-
1
1
754, 1593, 1487, 1429, 1143, 1040, 1028, 699 cm ; H NMR
(400 MHz, DMSO-d ) δ (ppm): 9.88 (bs, 1H), 7.25 (t, J = 7.6 Hz,
6
1
.0 equiv.) and tetronic acid 5 (100 mg, 1.1 mmol, 1.1 equiv.), in
2
H), 7.20 – 7.10 (m, 3H), 6.70 (s, 1H), 6.60 (s, 1H), 4.95 (d, J

ACCEPTED MANUSCRIPT
2
-pentanol (3.30 mL). The crude product was recrystallized in
ethanol (2.5 mL), then filtered and washed with cold ethanol (3x
.5 mL) to obtain pure compound 2ha’ (110 mg, 0.27 mmol, 27%
yield). White powder; IR νmax (neat): 1721, 1657, 1535, 1125,
122.3, 111.9, 104.9 (2C), 94.7, 65.0, 59.9, 55.8 (2C), 39.9, 32.0,
31.7, 25.2; LC-MS (ESI): m/z 394.0 (M+1), 416.0 (M+Na); HR-
+
2
MS (ESI): m/z Calcd for [C H NO +H] 394.1654, Found
23
23
5
394.1685 (+ 7.9 ppm).
-
1
1
1
021, 996, 763 cm ; R = 0.21 (hexanes/ethyl acetate 40 / 60); H
f
1
0-(benzo[d][1,3]dioxol-5-yl)-3,4,6,7,8,10-hexahydro-1H-
NMR (400 MHz, DMSO) δ (ppm): 10.23 (bs, 1H), 8.21 (d, J =
.6 Hz, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.66 – 7.52 (m, 2H), 7.50
d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 6.55 (s, 2H), 5.14 (s,
cyclopenta[g]furo[3,4-b]quinolin-1-one (2jc’). Compound 2jc’
was prepared accordingly to the general procedure B for the se-
quential one pot synthesis using 4-chloroaniline 3l (140mg, 1.1
mmol, 1.1 equiv.), 1,3-benzodioxole-5-carbaldehyde 4c’ (150 mg,
1.0 mmol, 1.0 equiv.), 5-aminoindan 3j (133 mg, 1.0 mmol, 1.0
equiv.) and tetronic acid 5 (110 mg, 1.1 mmol, 1.1 equiv.) in 2-
pentanol (3.3 mL). The crude product was then filtered and rinsed
with ethanol (3 x 2.5 mL) to obtain pure compound 2jc’ (93 mg,
8
(
1
6
H), 5.12 (d, J = 16.0 Hz, 1H), 4.99 (d, J = 16.0 Hz, 1H), 3.68 (s,
H), 3.59 (s, 3H). LC-MS (ESI): R = 7.7 mins, m/z 404.0
t
+
(M+H). HR-MS (ESI): m/z Calcd for [C H NO +H] 404.1498,
2
4
21
5
Found 404.1495 (- 0.5 ppm). Other spectral data match with the
previous report from literature.
4
e
7
8
-(perfluorophenyl)-7,11-dihydrobenzo[h]furo[3,4-b]quinolin-
(10H)-one (2hd’). Compound 2hd’ was prepared accordingly to
0.3 mmol, 27% yield). Pale yellow solid; R = 0.21 (ace-
f
°
tone/hexanes; 35/65); m.p. >260 C; IR νmax (neat): 3259, 3177,
-
the general procedure B for the sequential one pot synthesis using
-chloroaniline 3l (72 mg, 0.6 mmol, 1.1 equiv.), 4-amino-1-
3120, 1712, 1635, 1621, 1541, 1484, 1228, 1199, 1034, 1015 cm
1
1
4
; H NMR (400 MHz, DMSO-d ) δ (ppm): 9.89 (bs, 1H), 6.88 (s,
6
naphthalenecarbonitrile 3h (73 mg, 0.5 mmol, 1.0 equiv.), pen-
tafluorobenzaldehyde 4d’(100 mg, 0.5 mmol, 1.0 equiv.) and
tetronic acid 5 (56 mg, 0.6 mmol, 1.1 equiv.) in 2-pentanol (1.7
mL). The crude product was filtered and washed with cold ethanol
1H), 6.76 (s, 1H), 6.75 (d, J = 29.2 Hz, 2H), 6.65 (d, J = 8.9 Hz,
1H), 5.93 (d, J = 2.9 Hz, 2H), 4.90 (d, J = 15.6 Hz, 1H), 4.87 (s,
1H), 4.84 (d, J = 15.6 Hz, 1H), 2.76 (s, 2H), 2.68 (d, J = 7.2 Hz,
13
2H), 2.05 – 1.76 (m, 2H); C NMR (100 MHz, DMSO-d ) δ
6
(
3 x 2.5 mL) to obtain pure compound 2hd’ (21 mg, 0.05 mmol,
(ppm): 172.3, 158.6, 147.3, 145.6, 143.3, 141.7, 138.8, 134.4,
126.0, 122.6, 120.5, 112.0, 108.2 (2C), 108.0, 100.9, 94.9, 65.1,
1
3
9
1
7
0% yield). Light brown solid; Rf = 0.28 (acetone/hexanes;
°
0/70); m.p. >260 C; IR νmax (neat): 1729, 1644, 1499, 1107,
32.1, 31.7, 25.3; LC-MS (ESI): R = 8.9 mins, m/z 348.0 (M+H),
t
-
1
1
+
92, 951, 757 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm):
370.0 (M+Na); HR-MS (ESI): m/z Calcd for [C H NO +H]
6
21 17
4
0.47 (bs, 1H), 8.21 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H),
.64 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 7.4 Hz, 1H), 7.53 (d, J = 8.4
348.1236, Found 348.1244 (+ 2.3 ppm).
1
0-(perfluorophenyl)-3,4,6,7,8,10-hexahydro-1H-
Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.75 (s, 1H), 5.06 (d, J = 15.5
Hz, 1H), 5.01 (d, J = 15.5 Hz, 1H); C NMR (100 MHz, DMSO-
d6) δ (ppm): 171.6, 160.0, 133.0 (2C), 131.9, 128.4(3C), 127.4,
1
6
1
3
cyclopenta[g]furo[3,4-b]quinolin-1-one (2jd’). Compound 2jd’
was prepared accordingly to the general procedure B for the se-
quential one pot synthesis using 4-chloroaniline 3l (72 mg, 0.6
mmol, 1.1 equiv.), 5-aminoindan 3j (68 mg, 0.5 mmol, 1.0
equiv.), Pentafluorobenzaldehyde 4d’ (100 mg, 0.5 mmol, 1.0
equiv.) and tetronic acid 5 (56 mg, 0.6 mmol, 1.1 equiv.) in 2-
pentanol (1.7 mL). The crude product was filtered and washed
with cold ethanol (3 x 2.5 mL) to obtain pure compound 2jd’ (23
26.7,126.6 (2C), 123.5, 122.4 (2C), 120.9 (2C), 115.4, 92.6,
5.9, 30.1; LC-MS (ESI): m/z 426.0 (M+Na).
1
0-(3,4,5-trimethoxyphenyl)-3,6,7,8-tetrahydro-1H-
cyclopenta[g]furo[3,4-b]quinolin-1-one (7ja’). Compound 7ja’
was prepared accordingly to the general procedure B for the se-
quential one pot synthesis using 4-chloroaniline 3l (140 mg, 1.1
mmol, 1.1 equiv.), 5-aminoindane 3j (133 mg, 1.0mmol, 1.0
equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (196 mg, 1.0 mmol,
mg, 0.02 mmol, 12% yield). Off-White solid; R = 0.34 (ace-
f
°
tone/hexanes; 30/70); m.p. >260 C; IR νmax (neat): 1728, 1642,
-
1 1
1499, 1328, 1190, 1107, 991, 951, 776, 756 cm ; H NMR (400
1
2
.0 equiv.) and tetronic acid 5 (110 mg, 1.1 mmol, 1.1 equiv.) in
-pentanol (1.7 mL). The crude product was recrystallized in eth-
MHz, DMSO-d ) δ (ppm): 10.15 (bs, 1H), 6.82 (s, 1H), 6.79 (s,
1H), 5.50 (s, 1H), 4.89 (d, J = 15.6 Hz, 1H), 4.84 (d, J = 15.6 Hz,
6
anol (2.5 mL), then filtered and washed with cold ethanol (3x 2.5
mL) to obtain pure compound 2ja’ (47.1 mg, 0.1 mmol, 12%
1H), 2.80 - 2.70 (m, 2H),2.68 - 2.62 (m, 2H), 2.02 – 1.87 (m, 2H);
13
C NMR (100 MHz, DMSO-d ) δ (ppm): 171.7, 159.4, 144.5
6
yield). White solid; R = 0.3 (ethyl acetate/hexanes; 1:1); m.p.
(2C), 139.3 (2C), 135.0, 125.4 (3C), 118.6, 112.2 (3C), 90.8, 65.3,
32.1, 31.6, 29.4, 25.2; LC-MS (ESI): m/z 394.1 (M+H).
f
°
>
260 C; IR νmax (neat): 2960, 1764, 1579, 1440, 1455, 1248,
-
1 1
1
119, 1027, 1006, 718, 690 cm ; H NMR (400 MHz, DMSO-d )
6
1
0-phenyl-3,4,6,7,8,10-hexahydro-1H-cyclopenta[g]furo[3,4-
δ (ppm): 7.98 (s, 1H), 7.68 (s, 1H), 6.77 (s, 2H), 5.46 (s, 2H), 3.80
s, 3H), 3.77 (s, 6H), 3.12 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.3 Hz,
b]quinolin-1-one (2jh’). Compound 2jh’ was prepared accord-
ingly to the general procedure B for the sequential one pot syn-
thesis using 4-chloroaniline 3l (140 mg, 1.1 mmol, 1.1 equiv.),
benzaldehyde 4h’ (106 mg, 1.0 mmol, 1.0 equiv.), 5-aminoindan
3j (133 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (110 mg, 1.1
mmol, 1.1 equiv.) in 2-pentanol (3.3 mL). The crude product was
recrystallized in ethanol (2.5 mL), then filtered and washed with
cold ethanol (3x 2.5 mL) to obtain pure compound 2jh’ (30 mg,
(
2
H), 2.10 (p, J = 7.4 Hz, 2H); LC-MS (ESI): m/z 392.0 (M+H).
1
0-(3,4,5-trimethoxyphenyl)-3,4,6,7,8,10-hexahydro-1H-
cyclopenta[g]furo[3,4-b]quinolin-1-one (2ja’). To a suspension
of compound 7ja’ (25 mg, 0.1 mmol, 1.0 equiv.) in glacial acetic
acid (0.7 mL, 15.0 equiv.) was added with sodium cyanoborohy-
dride (11 mg, 0.2 mmol, 3.0 equiv.) in one portion at room tem-
perature and the reaction mixture was stirred for 4 hours. The
reaction mixture was poured into an ice cold water (2 mL) and the
newly formed precipitate was filtered and finally washed with
cold ethanol (2x 1 mL) to obtain pure compound 2ja’ (9 mg, 0.02
0.1 mmol, 10% yield). Off-white solid; R = 0.31 (ace-
f
°
tone/hexanes; 35/65); m.p. >260 C; IR νmax (neat): 3253, 3176,
3118, 1711, 1635, 1624, 1542, 1019, 701 cm ; H NMR (400
-
1
1
MHz, DMSO-d ) δ (ppm): 9.92 (bs, 1H), 7.64 – 7.43 (m, 3H),
6
mmol, 36% yield). White powder; R = 0.3 (hexanes/acetone 40 /
7.31 – 7.08 (m, 3H), 6.82 (d, J = 35.1 Hz, 1H), 5.49 (s, 1H), 4.96
(d, J = 15.6 Hz, 1H), 4.86 (d, J = 15.6 Hz, 1H), 3.14 – 2.96 (m,
2H), 2.78 – 2.63 (m, 2H), 2.20 – 1.88 (m, 2H); LC-MS (ESI): m/z
304.1 (M+H), 326.1 (M+Na).
f
°
6
1
0); m.p. >260 C; IR νmax (neat): 1742, 1657, 1479, 1324,
-
1 1
223, 1140, 1011, 748, 676 cm ; H NMR (400 MHz, DMSO-d )
6
δ (ppm): 9.87 (s, 1H), 6.97 (s, 1H), 6.78 (s, 1H), 6.49 (s, 2H), 5.00
(
(
2
1
d, J = 15.9 Hz, 1H), 4.90 (s, 1H), 4.86 (d, J = 15.9 Hz, 1H), 3.70
1
1-(3,4,5-trimethoxyphenyl)-8,11-dihydrofuro[3,4-
s, 6H), 3.60 (s, 3H), 2.77 (t, J = 6.8 Hz, 2H), 2.70 – 2.68 (m, 2H),
1
3
b][4,7]phenanthrolin-10(7H)-one (2ka’). Compound 2ka’ was
prepared accordingly to the general procedure B for the sequen-
.04 – 1.86 (m, 2H). C NMR (100 MHz, DMSO-d ) δ (ppm):
6
72.2, 158.7, 152.8 (2C), 143.2, 143.1 138.6, 136.0, 134.3, 125.9,

ACCEPTED MANUSCRIPT
1
2
tial one pot synthesis using 4-chloroaniline 3l (140mg, 1.1 mmol,
.1 equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (196 mg, 1.0
calculations application. A meaningful selected data set of these
properties is presented in the supporting information. All the cal-
culated physicochemical values for these compounds (cLogP <
1
mmol, 1.0 equiv.), 6-aminoquinoline 3k (144 mg, 1.0 mmol, 1.0
equiv.) and tetronic acid 5 (110 mg, 1.1mmol, 1.1 equiv.) in 2-
pentanol (3.3 mL). The crude product was recrystallized in etha-
nol (2.5 mL), then filtered and washed with cold ethanol (3x 2.5
mL) to obtain pure compound 2ka’ (168 mg, 0.4 mmol, 42%
2
2
3.7; 56 Å < TPSA <79 Å ; HBA+HBD<7) were in acceptable
range according to the literature.
20-23
Although the calculated
physicochemical values could not be directly correlated to the
biological activity and IC50 values (against leukemia), a trend can
be observed. For example, in the series of compounds 2bc’, 2lc’,
2dc’, 2cc’, 2hc’ and 2fc’, the lipophilicity increased drastically
(cLogP = 2.0, 2.2, 3.0, 3.1, 3.3 and 3.35) which also correspond to
an increase in cell permeation and in biological activity (corre-
sponding IC50 values of 883, 545, 37, 17, 11 and 50 nM). The
increase in lipophilicity resulting from the ether appendages at C7
(e.g. 2bc’ vs 2lc’ and 2cc’) may well favor a better cell permea-
tion without affecting the binding properties of the small-
molecules. The substitution change from a free-phenol at C7 to
various ethers and thioethers resulted in a large improvement of
cytotoxicity which could be related to the nature of the substitu-
ents (increased binding abilities for –OCHF and SCH ) and pos-
sibly to a diminished oxidative metabolism of degradation. There-
fore we could conclude that lead compounds such as 2ca’ and
cc’ may present an interesting balance between a relatively high
lipophilicity to facilitate cell permeation (cLogP ~ 3.0) while
presenting a relatively large TPSA (TPSA ~ 70 Å ) to maintain an
important binding affinity to the cellular target.
yield). Yellow solid; R = 0.19 (acetone/hexanes; 40/60); m.p.
f
°
>
1
260 C; IR νmax (neat): 3182, 1721, 1643, 1541, 1322, 1228,
205, 1113, 999, 819 cm ; H NMR (400 MHz, DMSO-d ) δ
-
1
1
6
(
ppm): 10.46 (bs, 1H), 8.68 (d, J = 4.0 Hz, 1H), 8.29 (d, J = 8.5
Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.39
dd, J = 8.6, 4.2 Hz, 1H), 6.49 (s, 2H), 5.67 (s, 1H), 5.00 (d, J =
(
1
5.8 Hz, 1H), 4.91 (d, J = 15.8 Hz, 1H), 3.60 (s, 6H), 3.54 (s, 3H);
1
3
C NMR (100 MHz, DMSO-d ) δ (ppm): 172.1, 157.3, 152.8
6
(2C), 148.0, 145.7, 141.4, 135.9, 135.2, 131.6, 130.0, 127.2,
1
21.9, 121.2, 114.8, 105.1 (2C), 97.0, 65.2, 59.9, 55.8 (2C), 36.7;
LC-MS (ESI): m/z 405.1 (M+H).
2
3
1
1-(perfluorophenyl)-8,11-dihydrofuro[3,4-
b][4,7]phenanthrolin-10(7H)-one (2kd’). Compound 2kd’ was
prepared accordingly to the general procedure B for the sequen-
tial one pot synthesis using 4-chloroaniline 3l (72 mg, 0.6 mmol,
2
1
.1 equiv.), 4-amino-1-naphthalenecarbonitrile 3k (74 mg, 0.5
2
mmol, 1.0 equiv.), pentafluorobenzaldehyde 4d’ (100 mg, 0.5
mmol, 1.0 equiv.) and tetronic acid 5 (56 mg, 0.6 mmol, 1.1
equiv.) in 2-pentanol (1.7 mL). The crude product was filtered and
washed with cold ethanol (3 x 2.5 mL) to obtain pure compound
2
kd’ (30 mg, 0.03 mmol, 15% yield). Off-White solid; R = 0.25
f
y = 2063.1159 nM+ (-637.3135 nM)*x
2bc'
°
2
(acetone/hexanes; 30/70); m.p. >260 C; IR νmax (neat): 1746,
(R = 0.89852)
-
1
1
800
1
500, 1209, 1021, 1007, 990, 832 cm ; H NMR (400 MHz,
DMSO-d ) δ (ppm): 10.73 (bs, 1H), 8.73 (d, J = 4.2 Hz, 1H), 7.96
6
2lc'
(
d, J = 9.1, 1H), 7.95 (d, J = 9.1, 1H), 7.52 - 7.50 (m, 2H), 6.14 (s,
H), 5.06 (d, J = 15.8 Hz, 1H), 5.00 (d, J = 15.8 Hz, 1H); LC-MS
ESI): m/z 405.0 (M+H).
1
(
4
00
9
-(benzo[d][1,3]dioxol-5-yl)-6-(methoxymethoxy)-4,9-
2dc'
dihydrofuro[3,4-b]quinolin-1(3H)-one (2lc’). To a flame dried
and argon purged 10 mL round-bottom flask, NaH (60% weight in
2hc' 2jc'
2cc'
0
mineral oil) (2 mg, 0.05 mmol, 1.5 equiv.) was suspended in DMF
°
(
200 µL, [0.15 M]) at 0 C. Compound 2bc’ (11 mg, 0.03 mmol,
1
.8
2.4
3.0
1
.0 equiv.) followed by chloromethylmethyl ether (3 mg, 0.033
cLogP
°
mmol, 1.1 equiv.) were then added at 0 C. The reaction mixture
was stirred under argon for an hour at room temperature and then
poured into cold water (1 mL), extracted with ethyl acetate (3 x 3
mL) and the combined organic extracts were dried over sodium
sulfate. Removal of solvents under reduced pressure afforded the
crude product which was further purified by column chromatog-
raphy on silica gel by using an isocratic solvent system (hex-
anes/ethyl acetate; 70:30) to obtain pure compound 2lc’ (3 mg,
Plot of IC50 results as function of clogP values for a series of ATP
molecules: 2bc’, 2lc’, 2dc’, 2cc’, 2hc’ and 2jc’.
Based on these physicochemical properties, the APT molecules
synthesized in this study demonstrated high solubility, a favorable
cell permeability and an important level of biological validation,
therefore these small-molecules should have the appropriate struc-
tural and electronic features (high TPSA and count of
HBA+HBD) to induce a high binding affinity to the biological
target. The degree of interaction or affinity of the APT molecules
and the biological target, leading to a potent activity on leukemia
THP-1 cells, can be related mostly to the stereoelectronic pattern
of substitution on the B-ring.
0
.01 mmol, 24% yield). White powder; Rf = 0.30 (hex-
o
anes/acetone 40 / 60); m.p. >260 C; IR νmax (neat): 3357, 1728,
1
-
1
1
621, 1610, 1485, 1109, 1035, 798 cm ; H NMR (400 MHz,
DMSO-d ) δ (ppm): 9.99 (bs, 1H), 6.95 (d, J = 8.2 Hz, 1H), 6.78
6
(d, J = 8.0 Hz, 1H), 6.72 (s, 1H), 6.65 (d, J = 7.9 Hz, 1H), 6.58 (d,
J = 7.8 Hz, 2H), 5.93 (d, J = 3.4 Hz, 2H), 5.20 – 5.04 (m, 2H),
4
3
.96 (d, J = 15.6 Hz, 1H), 4.87 (s, 1H), 4.84 (d, J = 15.6 Hz, 1H),
.35 (s, 3H); LC-MS (ESI): m/z 368.0 (M+H).
5
.3. Biological evaluation.
CellTiter-Glo® cell viability assays. Test compounds were solu-
bilized in 100% DMSO and added to polypropylene 384 well
plates (Greiner cat# 781280). 1,250 of THP-1, PSN-1, or HEK293
cells were plated in 384-well plates in 8 µl of serum-free media
5
.2. Calculated physicochemical properties.
Partition coefficient values (cLogP), hydrogen-bond acceptor and
donors sites at physiological pH (HBA and HBD) and the topo-
logical polar surface area (TPSA in Å ) were calculated for all
APT molecules from the library, using the Plexus discovery soft-
ware from the Plexus Suite 9.0 which is a web-based software
package that integrate the ChemAxon’s structure-based property
(RPMI-1640 for THP-1 and PSN-1, EMEM for HEK293). Test
2
compounds and 1 (pharmacological assay control) were prepared
as 10-point, 1:3 serial dilutions starting at 10 mM, then added to
P
the cells using the pin tool mounted on Biomek NX . Plates were

ACCEPTED MANUSCRIPT
incubated for 72 h at 37°C, 5% CO and 95% RH. After incuba-
R.; Shaik, T. B. Synthesis of
a
new 4-aza-2,3-
2
tion, 8 µL of CellTiter-Glo® (Promega cat#: G7570) were added
didehydropodophyllotoxin analogues as potent cytotoxic and antimi-
totic agents. Bioorg. Med. Chem. 2011, 19, 2349-2358. (d) Shi, F.;
Zeng, X.-N.; Zhang, G.; Ma, N.; Jiang, B.; Tu, S. Facile synthesis of
new 4-aza-podophyllotoxin analogs via microwave-assisted multi-
component reactions and evaluation of their cytotoxic activity.
Bioorg. Med. Chem. Lett. 2011, 21, 7119-7123. (e) Magedov, I. V.;
Frolova, L.; Manpadi, M.; Bhoga, U. D.; Tang, H.; Evdokimov, N.
M.; George, O.; Hadje, G. K.; Renner, S.; Getlic, M.; Kinnibrugh, T.
L.; Fernandes, M. A.; Van, S. S.; Steelant, W. F. A.; Shuster, C. B.;
Rogelj, S.; van, O. W. A. L.; Kornienko, A. Anticancer properties of
an important drug lead podophyllotoxin can be efficiently mimicked
by diverse heterocyclic scaffolds accessible via one-step synthesis. J.
Med. Chem. 2011, 54, 4234-4246. (f) Semenova, M. N.; Kiselyov, A.
S.; Tsyganov, D. V.; Konyushkin, L. D.; Firgang, S. I.; Semenov, R.
V.; Malyshev, O. R.; Raihstat, M. M.; Fuchs, F.; Stielow, A.; Lantow,
M.; Philchenkov, A. A.; Zavelevich, M. P.; Zefirov, N. S.; Kuznetsov,
S. A.; Semenov, V. V. Polyalkoxybenzenes from plants. 5. Parsley
seed extract in synthesis of azapodophyllotoxins featuring strong
tubulin destabilizing activity in the sea urchin embryo and cell culture
assays. J. Med. Chem. 2011, 54, 7138-7149. (g) Chernysheva, N. B.;
Tsyganov, D. V.; Philchenkov, A. A.; Zavelevich, M. P.; Kiselyov, A.
S.; Semenov, R. V.; Semenova, M. N.; Semenov, V. V. Synthesis and
comparative evaluation of 4-oxa- and 4-aza-podophyllotoxins as anti-
proliferative microtubule destabilizing agents. Bioorg. Med. Chem.
Lett. 2012, 22, 2590-2593. (h) Kamal, A.; Tamboli, J. R.; Nayak, V.
L.; Adil, S. F.; Vishnuvardhan, M. V. P. S.; Ramakrishna, S. Synthe-
sis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin
analogues as inhibitors of tubulin polymerization and apoptosis in-
ducers. Bioorg. Med. Chem. 2014, 22, 2714-2723.
to each well, and incubated for 15 min at room temperature. Lu-
minescence was recorded using a Biotek Synergy H4 multimode
microplate reader. Viability was expressed as a percentage rela-
tive to wells containing media only (0%) and wells containing
cells treated with 1% DMSO only (100%). Three parameters were
calculated on a per-plate basis: (a) the signal-to-background ratio
(S/B); (b) the coefficient for variation [CV; CV = (standard devia-
tion/mean) x 100)] for all compound test wells; and (c) the Z’-
26
factor.
The IC50 value of the pharmacological control (1, LC
Laboratories # E-4488) was also calculated to ascertain the assay
robustness. Each dose response curve was examined and curve
1
8
type was determined according to Inglese et al., . In cases where
a complete response was observed (viability < 20%, both asymp-
totes present) the IC50 value derived from GraphPad software was
used. In cases of partial response (viability 20-50%, both asymp-
totes present) the lowest concentration that induced > 50% viabil-
2
7
ity loss was assigned as EC50 value. In cases where the highest
efficacy observed was > 50% viability, IC50 value was assigned as
“
> highest concentration tested” (e.g., > 100 µM).
Trypan Blue cell viability assays. 10,000 of THP-1 or HEK293
cells were plated in 96-well plates in 100 µl of serum-free media
(RPMI-1640 for THP-1 and EMEM for HEK293). 10 µM of
Etoposide (1) and compound 2ha’ were added to the correspond-
ing wells of 96-well plates. Plates were incubated for 72 h at
3
7°C, 5% CO and 95% RH. After incubation, cells were harvest-
2
ed using standard tissue culture techniques, Trypan Blue was
added and cell counts and viability were analyzed using Cellome-
ter Auto T4 (Nexcelom Bioscience, Lawrence, MA) according to
the manufacturer’s instructions.
(
5) For a review on synthesis of APTs and biological evaluation,
see: Botes, M. G.; Pelly, S. C.; Blackie, M. A. L.; Kornienko, A.; van
Otterlo, W. A. L. Synthesis of 4-Azapodophyllotoxins with Anti-
cancer Activity by Multicomponent Reactions. Chem. Heterocycl.
Compd. 2014, 50, 119-138.
(
6) For selected reviews on MCR, see: (a) Dömling, A.; Ugi, I.
Multicomponent Reactions with Isocyanides. Angew. Chem., Int. Ed.
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Chem. 2003, 1133-1144. (c) Ramon, D. J.; Yus, M. Asymmetric mul-
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ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
.
HIGHLIGHTS
•
The modified Husson-3CR reported herein (multicomponent reaction) using a sacrificial aniline enabled
the rapid construction of a library of podophyllotoxin aza-analogues.
•
7 Compounds (2ca’, 2cc’, 2da’, 2fa’, 2ha’, 2ja’ and 2jc’) have been identified to have potent (low nano-
molar activity) and selective activity against leukemia while preserving a minimal toxicity against no-
malignant cells.
•
•
From this study, 2 novel lead small-molecules 2ca’and 2jc’ were identified to be more potent than the
reference drug etoposide (25-60 fold) against leukemia.
Our preliminary structure activity relationship studies demonstrated the importance of several pharmaco-
phore features on the azapodophyllotoxin B- and E-rings to enhance antitumoral activity.

ACCEPTED MANUSCRIPT
Multicomponent Assembly of 4-Aza-podophyllotoxins: A Fast Entry to Highly Selective and
Potent Anti-Leukemic Agents.
All Graphics
Scheme 1. MCR tactic to the 4-aza-podophyllotoxin chemotype

ACCEPTED MANUSCRIPT
a-c
d
Scheme 2. Synthesis and biological evaluation of a library of novel 4-aza-podophyllotoxins (APTs) 2xy’
a
12
Only novel analogues are presented above. Conditions. Method A: typical Husson-3CR with an equimolar ratio of
o
tetronic acid 5, aniline 3a-k and benzaldehyde 4a’-h’ under Ar was stirred at 120 C in 2-pentanol (1 mL) for 1 hour; Method
B using the sacrificial aniline 3l: aniline 3l (1.0 equiv.) and benzaldehyde 4a’-h’ (1.0 equiv.) was stirred under Ar at 120 C in
o
2
-pentanol (1 mL) for 2 hours, followed by addition of tetronic acid 5 (1.0 equiv.) for 10 mins and finally aniline 3a-k (1.0
b
equiv.), the resulting mixture was refluxed for an additional 30 mins. Yields refer to pure products 2xy’ isolated after
recrystallization of crude material in ethanol. Compound 2ja’ was isolated as over-oxidized quinolines and reduced in a
second step; overall yield for both steps is reported above. The reported IC50 or EC50 values are against the human THP-1
c
d
leukemia cell line.

ACCEPTED MANUSCRIPT
Table 1. Comparison of the Husson-3CR and modified-3CR
a
Figure 1. Anti-leukemic activity: Comparison of CellTiter Glo® and Trypan Blue viability assay using etoposide (1) and 2ha’.
a
Panel A: HEK293 in Trypan Blue assay; Panel B: THP-1 in Trypan Blue assay; Panel C: HEK293 in CellTiter Glo® assay;
Panel D: THP-1 in CellTiter Glo® assay.

ACCEPTED MANUSCRIPT
a,b,c
Table 2. SAR study
a
b
c
The reported IC50 or EC50 values are against: the human THP-1 leukemia cell line, the pancreas cancer PSN-1 cell line and the
non-malignant kidney HEK293 cell line.
a
Scheme 3. Modulations of the APTs pharmacophores
a
The reported IC values are against the human THP-1 leukemia cell line.
5
0

ACCEPTED MANUSCRIPT
a,b
Figure 2. Lead compounds having high potency and selectivity against THP-1 leukemia cells.
a
EC values are reported for HEK293 and PSN-1 cells. Panel A: selectivity chart for the 7 best APT
50
b
compounds of the library. Panel B: Compound comparison showing high selectivity and potency.
Figure 3. Best potencies against THP-1 leukemia cells for the APTs 2ca’, 2da’, 2fa’ and 2ha’

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Multicomponent Assembly of 4-Aza-podophyllotoxins: A Fast Entry
to Highly Selective and Potent Anti-Leukemic Agents
Nagalakshmi Jeedimalla, Madison Flint, Lyndsay Smith, Alberto Haces, Dmitriy Minond* and Stéphane P. Roche*
Table of Contents
I.
I. GENERAL INFORMATION............................................................................................................................2
A. Instrumentation and Methods ...........................................................................................................................2
B. Reagents and Solvents......................................................................................................................................2
II. Experimental procedures and compound characterization....................................................................................3
A. General Procedure A for the one pot synthesis of 4-aza-podophyllotoxins......................................................3
B. General Procedure B for the sequential one pot synthesis of 4-aza-podophyllotoxins.....................................3
1
13
III.
IV.
SELECTED H, C & LC-MS SPECTRA....................................................................................................17
BIOLOGICAL EVALUATION.....................................................................................................................47
1

ACCEPTED MANUSCRIPT
I. I. GENERAL INFORMATION
A. Instrumentation and Methods
Reactions were performed in flame-dried glassware under a positive pressure of argon. Yields refer to
chromatographically and spectroscopically pure compounds.
Analytical TLC was performed on 0.25 mm glass backed 60Å F-254 TLC plates (Silicycle, Inc.). The plates were
visualized by exposure to UV light (254 nm) and developed by a solution of cerium-ammonium-molybdate in
water/sulfuric acid and heat. Flash chromatography was performed using 200-400 mesh silica gels (Silicycle, Inc.).
1
Infrared spectra were recorded on a Nicolet IS5 FT-IR spectrophotometer. H NMR spectra were recorded on a
Varian Mercury400 (400 MHz) spectrometer and are reported in ppm using solvent as an internal standard (DMSO-
d6 at 2.50 ppm). NMR spectra were performed using standard parameter and data are reported as: (b = broad, s =
13
singlet, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constant(s) in Hz, integration). C NMR spectra
were recorded on Varian Mercury400 (100 MHz) spectrometer. Chemical shifts are reported in ppm, with solvent
resonance employed as the internal standard (DMSO-d at 39.5 ppm). Melting points were determined using
6
Digimelt digital melting point apparatus. The low resolution mass spectra were performed on Agilent 1200 series
HPLC system/6120 single quadrupole MSD (electrospray ionization; ESI) with dual detector (PDA and ELSD). A
purity of at least 95% was obtained for all the compounds by means of chromatography, crystallization, or
recrystallization. This level of purity was established by LC/MS on a Agilent 1200 series HPLC based on the ELSD
and UV chromatograms (λ= 360 nm) using a linear gradient, water + 0.1% formic acid and MeCN + 0.1% formic
acid, at 60:40 (0 min) to 0:100 (20 min) and a flow rate of 0.8 mL/min; Retention times are reported for the lead
compounds (R ). Accurate mass (High resolution HR-MS) was obtained from University of Florida using Agilent
t
6
210 TOF instrument.
B. Reagents and Solvents
All reagents used in the present paper were acquired from Alfa Aesar or Sigma Aldrich. 2-pentanol was purchased
from Sigma Aldrich.
2

ACCEPTED MANUSCRIPT
II. Experimental procedures and compound characterization
A. General Procedure A for the one pot synthesis of 4-aza-podophyllotoxins.
A round bottom flask was charged under argon with aniline 3 (1.0 equiv.), aldehyde 4 (1.0 equiv.), and tetronic acid
º
º
5
(1.0 equiv.) in 2-pentanol or ethanol [0.3 M]. Reaction was refluxed (at 125 C or 80 C respectively) for 1 hour,
then the solvent was evaporated under vacuum and the crude product purified by recrystallization in ethanol or by
flash chromatography on silica gel.
B. General Procedure B for the sequential one pot synthesis of 4-aza-podophyllotoxins.
A two neck round bottom flask under argon equipped with a condenser was charged with aldehyde 4 (1.0 equiv.)
and 4-chloroaniline 3b (1.1 equiv.) in 2-pentanol [0.3 M] and stirred at reflux for two hours. The tetronic acid 5 (1.1
equiv.) in 2-pentanol (minimum amount) was then added at reflux. After another 10 mins at reflux, the third
component aniline 3 (1.0 equiv.) was added neat. The reaction mixture was refluxed for an additional 30 mins. All
volatiles were evaporated under vacuum and the crude product purified by recrystallization in ethanol or by flash
chromatography on silica gel.
SI-1a-f
SI-1b
SI-1c
SI-5
SI-1b-d
SI-1d
SI-1a, b, d
SI-1f, SI-2
SI-1b, SI-3
SI-3
SI-1c
Compounds 2aa’,
2ac’,
2ae’,
2af’,
2ag’,
SI-5
2ah’,
2ba’,
2fa’,
2ha’, 2hc’,
SI-1f, SI-3
SI-1b, SI-4
SI-4
2
ia’,
2ic’,
2ih’, 2kc’, and 2kh’ were previously reported by others. Only biological activity
SI-6
1
13
was reported for Compound 2ja’ . Compounds 2bc’, 2ca’, 2dc’, 2dh’ and 2fc’ were reported by us with H,
C
SI-2
NMRs, IR, melting points and HR-MS.
[SI-1] a) Aillerie, A.; Talancé, V. L. d.; Moncomble, A.; Bousquet, T.; Pélinski, L. Org. Lett. 2014, 16, 2982-2985; b) Semenova,
M. N.; Kiselyov, A. S.; Tsyganov, D. V.; Konyushkin, L. D.; Firgang, S. I.; Semenov, R. V.; Malyshev, O. R.; Raihstat, M. M.;
Fuchs, F.; Stielow, A.; Lantow, M.; Philchenkov, A. A.; Zavelevich, M. P.; Zefirov, N. S.; Kuznetsov, S. A.; Semenov, V. V. J.
Med. Chem. 2011, 54, 7138-7149; c) Shi, C.; Wang, J.; Chen, H.; Shi, D. J. Comb. Chem. 2010, 12, 430-434; d) Frackenpohl, J.;
Adelt, I.; Antonicek, H.; Arnold, C.; Behrmann, P.; Blaha, N.; Böhmer, J.; Gutbrod, O.; Hanke, R.; Hohmann, S.; Houtdreve, M.
v.; Lösel, P.; Malsam, O.; Melchers, M.; Neufert, V.; Peschel, E.; Reckmann, U.; Schenke, T.; Thiesen, H.-P.; Velten, R.;
Vogelsang, K.; Weiss, H.-C. Bioorg. Med. Chem. 2009, 17, 4160-4184; e) Giorgi-Renault. S. Ann. Pharm. Fr. 2005, 63, 63-68; f)
Tratrat, C.; Giorgi-Renault, S.; Husson, H.-P. Org. Lett. 2002, 4, 3187-3189.
[SI-2]Jeedimalla, N.; Johns, J.; Roche, S. P. Tetrahedron Lett. 2013, 54, 5845-5848.
[SI-3]Magedov, I. V.; Frolova, L.; Manpadi, M.; Bhoga, U. d.; Tang, H.; Evdokimov, N. M.; George, O.; Hadje Georgiou, K.;
Renner, S.; Getlik, M. u.; Kinnibrugh, T. L.; Fernandes, M. A.; Van slambrouck, S.; Steelant, W. F. A.; Shuster, C. B.; Rogelj, S.;
van Otterlo, W. A. L.; Kornienko, A. J. Med. Chem. 2011, 54, 4234-4246.
[SI-4]Kozlov, N. G.; Bondarev, S. L.; Kadutskii, A. P.; Basalaeva, L. I.; Pashkovskii, F. S. Russ. J. Org. Chem. 2008, 44, 1031-
1
037.
[SI-5]Shi, F.; Zhou, D.; Tu, S.; Shao, Q.; Li, C.; Cao, L. J. heterocyc. Chem. 2008, 45, 1065-1070.
[SI-6]Hitotsuyanagi, Y.; Fukuyo, M.; Tsuda, K.; Kobayashi, M.; Ozeki, A.; Itokawa, H.; Takeya, K. Bioorg. Med. Chem. Lett.
2
000, 10, 315-317.
3

ACCEPTED MANUSCRIPT
a-c
Scheme SI-1.Synthesis of a library of 50 compounds: 4-aza-2,3didehydropodophyllotoxins (APT) 2
a
b
Note. Compounds presented in blue have been reported previously and compounds in black have ben synthesized for the first time in our library. Conditions.
o
Method A: typical Husson-3CR with an equimolar ratio of tetronic acid 5, aniline 3a-k and benzaldehyde 4a’-h’under N
2
was stirred at 120 C in 2-pentanol (1 mL)
o
for 1 hour; Method B using the sacrificial aniline 3l: aniline 3l (1.0 equiv.) and benzaldehyde 4a’-h’ (1.0 equiv.) was stirred under N
2
at 120 C in 2-pentanol (1 mL)
for 2 hours, followed by addition of tetronic acid 5 (1.0 equiv.) for 10 mins and finally aniline 3a-k (1.0 equiv.), the resulting mixture was refluxed for an additional
c
d
3
0 mins. Yields refer to pure products 2xy’ isolated after recrystallization of crude material in ethanol. Compounds 2fa’ and 2ja’ were isolated as over-oxidized
quinolines and reduced in a second step; overall yields for both steps are reported above.
4

ACCEPTED MANUSCRIPT
9
-(benzo[d][1,3]dioxol-5-yl)-6,9-dihydro-[1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (2ac’). Compound
2
ac’ was prepared accordingly to the general procedure A for the one pot synthesis using 1,3-
benzodioxole-5-carbaldehyde 4c’ (150 mg, 1.0 mmol, 1.0 equiv.), 3,4-
methylenedioxy)aniline 3a (137 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0
(
mmol, 1.0 equiv.) in 2-pentanol (3.3 mL). The crude product was filtered and washed with
cold ethanol (3x 2.5 mL) to obtain compound 2ac’ in a pure form (171 mg, 0.5 mmol, 49%
°
yield). Light brown solid; R = 0.29 (acetone/hexanes; 35/65); m.p. > 260 C; IR νmax (neat):
f
-
1
1
3
224, 3095, 1712, 1642, 1562, 1481, 1248, 1235, 1038, 1016, 933, 780 cm ; H NMR (400 MHz, DMSO-d ) δ
6
(
ppm): 9.87 (bs, 1H), 6.83 – 6.75 (m, 1H), 6.73 (s, 1H), 6.65 (dd, J = 7.7, 1.5 Hz, 1H), 6.59 (s, 1H), 6.51 (s, 1H),
.97 – 5.87 (m, 4H), 4.97 (d, J = 15.8 Hz, 1H), 4.83 (s, 1H), 4.83 (d, J = 15.8 Hz, 1H); LC-MS (ESI): m/z 374.0
M+Na);
SMILES: O=C(OC1)C2=C1NC3=CC(OCO4)=C4C=C3C2C5=CC(OCO6)=C6C=C5
5
(
9
-(perfluorophenyl)-6,9-dihydro-[1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (2ad’). Compound 2ad’ was
prepared accordingly to the general procedure A for the one pot synthesis using
pentafluorobenzaldehyde 4d’ (490 mg, 2.5 mmol, 1.0 equiv.), 3,4-(methylenedioxy)aniline 3a
(
(
343 mg, 2.5 mmol, 1.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in ethanol
10 mL). The crude product was filtered and washed with cold ethanol (3x 2.5 mL) to obtain
compound 2ad’ (99 mg, 0.3 mmol, 10% yield). White solid; R = 0.30 (acetone/hexanes;
f
°
3
9
5/65); m.p. >260 C; IR νmax (neat): 3230, 1727, 1650, 1498, 1481, 1190, 1040, 1020, 990,
-
1
1
42, 835, 761 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.12 (bs, 1H), 6.54 (s, 1H),
6
6
.51 (s, 1H), 5.96 (s, 1H), 5.93 (s, 1H), 5.43 (s, 1H), 4.90 (d, J = 16.0 Hz, 1H), 4.85 (d, J = 16.0 Hz, 1H); LC-MS
(
ESI): m/z 398.0 (M+H), 420.0 (M+Na);
SMILES: O=C(OC1)C2=C1NC3=CC(OCO4)=C4C=C3C2C5=C(F)C(F)=C(F)C(F)=C5F
9
-(4-methoxyphenyl)-6,9-dihydro-[1,3]dioxolo[4,5-g]furo[3,4-b]quinolin-8(5H)-one (2af’). Compound 2af’ was
prepared accordingly to the general procedure A for the one pot synthesis using 4-
methoxybenzaldehyde 4f’ (340 mg, 2.5 mmol, 1.0 equiv.), 3,4-(methylenedioxy)aniline 3a
(
(
343 mg, 2.5 mmol, 1.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in ethanol
10 mL). The crude product was filtered and sonicated in dichloromethane (4 mL) for 15
minutes, followed by centrifugation at 5,800 rpm for 10 minutes (x3) to obtain pure compound
af’ (444 mg, 1.3 mmol, 53% yield). Pale yellow solid; R = 0.19 (acetone/hexanes; 35/65);
2
f
°
-1 1
m.p. 136-137 C; IR νmax (neat): 3222, 3157, 3092, 1711, 1641, 1560, 1502, 1478, 1192, 1016, 831 cm ; H NMR
(
(
400 MHz, DMSO-d ) δ (ppm): 9.84 (bs, 1H), 7.09 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 6.54 (s, 1H), 6.51
6
s, 1H), 5.95 (s, 1H), 5.89 (s, 1H), 4.92 (d, J = 15.5 Hz, 1H), 4.85 (s, 1H), 4.83 (d, J = 15.5 Hz, 1H), 3.69 (s, 3H);
LC-MS (ESI): m/z 338.0 (M+H);
SMILES: O=C(OC1)C2=C1NC3=CC(OCO4)=C4C=C3C2C5=CC=C(OC)C=C5
5

ACCEPTED MANUSCRIPT
6
-hydroxy-9-(4-hydroxy-3-methoxyphenyl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2bb’). Compound 2bb’
was prepared accordingly to the general procedure A for the one pot synthesis using 3-
hydroxy-4-methoxybenzaldehyde 4b’ (381 mg, 2.5 mmol, 1.0 equiv.) 3-aminophenol 3b (273
mg, 2.5 mmol, 1.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in ethanol (10
mL). The product was filtered off the crude reaction mixture and washed with cold ethanol (3x
2
0
.5 mL) to obtain pure compound 2bb’ (235 mg, 0.72 mmol, 29% yield). Off-white solid; R =
f
°
.24 (acetone/hexanes; 45/55); m.p. > 260 C; IR νmax (neat): 3446, 3377, 3231, 1724, 1646,
-
1 1
1
622, 1496, 1152, 1026, 1018, 770 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 9.84 (bs, 1H), 9.43 (s, 1H), 8.75
6
(
s, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 1.6 Hz, 1H), 6.62 (d, J = 8.1 Hz, 1H), 6.46 (dd, J = 8.1, 1.6 Hz, 1H),
6
1
.33 (dd, J = 8.1, 1.6 Hz, 1H), 6.32 (d, J = 1.6 Hz, 1H), 4.92 (d, J = 15.6 Hz, 1H), 4.82 (d, J = 15.6 Hz, 1H), 4.76 (s,
H), 3.70 (s, 3H); LC-MS (ESI): m/z 326.1 (M+H);
SMILES: O=C(OC1)C2=C1NC3=CC(O)=CC=C3C2C4=CC=C(OC)C(O)=C4
6
-(difluoromethoxy)-9-(3,4,5-trimethoxyphenyl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one 2ca’. Compound
ca’ was prepared accordingly to the general procedure B for the one pot sequential
2
synthesis using 4-chloroaniline 3l (72 mg, 0.56 mmol, 1.1 equiv.), 3-difluoromethoxy
aniline 3c (81 mg, 0.51 mmol, 1.0 equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (100 mg, 0.51
mmol, 1.0 equiv.) and tetronic acid 5 (56 mg, 0.56 mmol, 1.1 equiv.), in 2-pentanol (1.7 mL
+
0.4 mL). The crude product was purified by column chromatography using isocratic
solvent system of 20% acetone in hexanes system to obtain pure compound 2ca’ (30 mg,
0
.06 mmol, 14% yield). Compound 2ca’ was also prepared accordingly to the general procedure A for the one pot
synthesis using the exact same quantities and was purified by column chromatography using isocratic solvent
system of 20% acetone in hexanes system to obtain pure compound 2ca’ (5 mg, 0.01 mmol, 2% yield). White
o
powder, R = 0.22 (Hexanes/acetone 65 / 35); m.p.>260 C; IR νmax (neat): 3246, 1727, 1644, 1548, 1492, 1174
f
-
1 1
cm ; H NMR (400 MHz, DMSO) δ (ppm):10.13 (s, 1H), 7.19 (s, 1H), 7.18 (t, J = 73.9 Hz, 1H), 6.73 (d, J = 8.1
13
Hz, 1H), 6.69 (s, 1H), 6.50 (s, 2H), 5.18 – 4.78 (m, 3H), 3.70 (s, 6H), 3.60 (s, 3H). C NMR (100 MHz, DMSO-d )
6
δ (ppm): 171.9, 158.5, 152.8 (2C), 150.0, 142.4, 137.3, 136.1, 132.1, 121.3, 116.3 (t, J = 278.6 Hz), 113.2, 106.2,
1
04.9 (2C), 95.7, 65.1, 59.9, 55.8 (2C), 30.7; LC-MS (ESI): R = 5.1 mins, m/z 420.0 (M+H); HR-MS (ESI): m/z
t
+
Calcd for [C H F NO +H] 420.1253, Found 420.1250 (- 0.7 ppm).
21
19
2
6
SMILES:COC1=C(OC)C(OC)=CC(C2C(C(OC3)=O)=C3NC4=C2C=CC(OC(F)F)=C4)=C1.
9
-(benzo[d][1,3]dioxol-5-yl)-6-(difluoromethoxy)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2cc’). Compound
cc’ was prepared accordingly to the general procedure B for the sequential one pot
2
synthesis using 4-chloroaniline 3l (140mg, 1.1 mmol, 1.1 equiv.), 1,3-benzodioxole-5-
carbaldehyde 4c’ (150 mg, 1.0 mmol, 1.0 equiv.), 3-(difluoromethoxy) benzamine 3c (159
mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (110 mg, 1.1 mmol, 1.1 equiv.) in 2-pentanol
(
3.3 mL). The oily crude product was taken in methanol (2.5 mL) and the precipitate was
filtered and washed with cold ethanol (3x 2.5 mL) to obtain pure compound 2cc’ (11 mg,
°
0
.03 mmol, 3% yield). Off-white crystals; R = 0.41 (acetone/hexanes; 40/60); m.p. > 260 C; IR νmax (neat): 3322,
f
6

ACCEPTED MANUSCRIPT
-
1 1
1
728, 1641, 1611, 1482, 1109, 1040, 1011, 798 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.15 (bs, 1H), 7.17
6
(
t, J = 74.0 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 1.6 Hz, 1H), 6.72 (dd, J = 8.5,
2
.4 Hz, 1H), 6.69 – 6.67 (m, 2H), 5.94 (d, 2.0 Hz, 2H), 4.93 (s, 1H), 4.93 (d, 15.4 Hz, 1H), 4.87 (d, 15.4 Hz, 1H);
+
LC-MS (ESI): R = 7.3 mins, m/z 374.0 (M+H), 396.0 (M+Na); HR-MS (ESI): m/z Calcd for [C H F NO +H]
t
19 13
2
5
3
74.0840, Found 374.0850 (+ 2.7 ppm).
SMILES: O=C(OC1)C2=C1NC(C=C(OC(F)F)C=C3)=C3C2C4=CC(OCO5)=C5C=C4
6
-(difluoromethoxy)-9-(perfluorophenyl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2cd’). Compound 2cd’
was prepared accordingly to the general procedure A for the one pot synthesis using 3-
(
difluoromethoxy) benzamine 3c (159 mg, 1.0 mmol, 1.0 equiv.), pentafluorobenzaldehyde
d’ (196 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-
4
pentanol (3.3 mL). The crude product was filtered and washed with cold ethanol (3x 2.5 mL)
to obtain compound 2cd’ (56 mg, 0.13 mmol, 13% yield). White solid; R = 0.29
f
°
(
acetone/hexanes; 30/70); m.p. > 260 C; IR νmax (neat): 1726, 1644, 1495, 1190, 1170,
-
1 1
1
8
1
021, 991, 762 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.38 (bs, 1H), 7.23 (t, J = 73.7 Hz, 1H), 7.06 (d, J =
6
.3, 1H), 6.75 (dd, J = 8.3, 1.7 Hz, 1H), 6.71 (d, J = 2.0 Hz, 1H), 5.56 (s, 1H), 4.96 (d, J = 16.1, 1H), 4.90 (d, J =
13
6.1, 1H); C NMR (100 MHz, DMSO-d ) δ (ppm): 171.4, 159.1, 150.7, 138.1 (2C), 131.9 (3C), 117.7, 116.2 (t, J
6
=
258.6 Hz), 113.4 (3C), 106.3 (2C), 92.2, 65.4, 28.7; LC-MS (ESI): m/z 420.0 (M+H), 442.0 (M+Na); SMILES:
FC(F)OC1=CC=C(C(C2=C(F)C(F)=C(F)C(F)=C2F)C(C(OC3)=O)=C3N4)C4=C1
6
-(difluoromethoxy)-9-phenyl-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2ch’). Compound 2ch’ was prepared
accordingly to the general procedure B for the sequential one pot synthesis using 4-
chloroaniline 3l (140 mg, 1.1 mmol, 1.1 equiv.), benzaldehyde 4h’ (106 mg, 1.0 mmol, 1.0
equiv.), 3-(difluoromethoxy) benzamine 3c (159 mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5
(
110 mg, 1.1 mmol, 1.1 equiv.) in 2-pentanol (3.3 mL). The oily crude product was taken in
methanol (2.5 mL) and the precipitate was filtered and washed with cold ethanol (3x 2.5 mL)
to obtain pure compound 2ch’ (12 mg, 0.4 mmol, 4% yield). Metallic white crystals; R =0.35
f
°
-1
(
70/30 acetone/hexanes); m.p. > 260 C; IR νmax (neat): 3231, 1723, 1636, 1617, 1495, 1118, 1012, 746, 696 cm ;
1
H NMR (400 MHz, DMSO-d ) δ (ppm): 10.16 (bs, 1H), 7.18 (t, J = 74.0 Hz, 1H), 7.28-7.07 (m, 6H), 6.73-6.69 (m,
6
2
H), 5.01 (s, 1H), 5.00 (d, J = 15.8 Hz, 1H), 4.89 (d, J = 15.8 Hz, 1H); LC-MS (ESI): m/z 368.0 (M+K); SMILES:
FC(F)OC(C=C1)=CC2=C1C(C(C(OC3)=O)=C3N2)C4=CC=CC=C4
6
-(methylthio)-9-(3,4,5-trimethoxyphenyl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2da’). Compound 2da’
was prepared accordingly to the general procedure A for the one pot synthesis using 3-
methylthioaniline 3d (138 mg, 1.0 mmol, 1.0 equiv.), 3,4,5-trimethoxybenzaldehyde 4a’ (196
mg, 1.0 mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol (3.3
mL). The crude product was recrystallized in ethanol (2.5 mL), then filtered and washed with
cold ethanol (3x 2.5 mL) to obtain pure compound 2da’ (78 mg, 0.2 mmol, 20% yield). White
7

ACCEPTED MANUSCRIPT
°
crystals; R = 0.25 (acetone/hexanes; 35/65); m.p. > 260 C; IR νmax (neat): 3249, 2359, 2341, 1725, 1635, 1483,
f
-
1 1
1
128, 1008, 750 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.00 (bs, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.81 (d, J
6
=
8.1 Hz, 1H), 6.77 (s, 1H), 6.49 (s, 2H), 5.02 (d, J = 15.7 Hz, 1H), 4.91 (s, 1H), 4.87 (d, J = 15.7 Hz, 1H), 3.70 (s,
1
3
6
1
H), 3.60 (s, 3H), 2.42 (s, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm) 172.0, 158.7, 152.8 (2C), 142.6, 137.3,
6
36.5, 136.1, 131.1, 121.1, 120.7, 112.9, 104.9 (2C), 95.4, 65.1, 59.9, 55.9 (2C), 39.3, 14.6; LC-MS (ESI): R = 6.5
t
+
mins, m/z 400.0 (M+1), 422.0 (M+Na); HR-MS (ESI): m/z Calcd for [C H NO S+H] 400.1219, Found 400.1225
2
1
21
5
(
+ 2.4 ppm).
SMILES: O=C(OC1)C2=C1NC3=CC(SC)=CC=C3C2C4=CC(OC)=C(OC)C(OC)=C4
6
-(methylthio)-9-(perfluorophenyl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2dd’). Compound 2dd’ was
prepared accordingly to the general procedure A for the one pot synthesis using 3-
methylthioaniline 3d (138 mg, 1.0 mmol, 1.0 equiv.), pentafluorobenzaldehyde 4d’ (196 mg,
1
.0 mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol (3.3
mL). The crude product was recrystallized in ethanol (2.5 mL), then filtered and washed with
cold ethanol (3x 2.5 mL) to obtain pure compound 2dd’ (112 mg, 0.28 mmol, 28% yield). Off-
°
white solid; R = 0.30 (acetone/hexanes; 30/70); m.p. > 260 C; IR νmax (neat): 3213, 1719,
f
-
1 1
1
6
1
637, 1518, 1499, 1486, 1211, 1022, 992, 955, 916 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 10.26 (bs, 1H),
6
.92 (d, J = 8.1 Hz, 1H), 6.82 (dd, J = 8.1, 1.7 Hz, 1H), 6.77 (d, J = 1.7 Hz, 1H), 5.51 (s, 1H), 4.96 (d, J = 16.0 Hz,
13
H), 4.91 (d, J = 16.0 Hz, 1H), 2.44 (s, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm): 171.5, 159.3 (2C), 138.7
6
(
(
2C), 137.2 (2C), 130.6 (2C), 120.8 (2C), 117.3, 112.8 (2C), 91.7, 65.4, 28.8, 14.4; LC-MS (ESI): m/z 422.0
M+Na). SMILES: CSC1=CC=C(C(C2=C(F)C(F)=C(F)C(F)=C2F)C(C(OC3)=O)=C3N4)C4=C1
9
-(4-fluorophenyl)-6-(methylthio)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2de’). Compound 2de’ was
prepared accordingly to the general procedure A for the one pot synthesis using 3-
methylthioaniline 3d (138 mg, 1.0 mmol, 1.0 equiv.), 4-fluorobenzaldehyde 4e’ (124 mg, 1.0
mmol, 1.0 equiv.) and tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol (3.3 mL).
The crude product was filtered and washed with cold ethanol (3x 2.5 mL) to obtain pure
compound 2de’ (98 mg, 0.3 mmol, 30% yield). White solid; R = 0.29 (acetone/hexanes; 30/70);
f
°
-1
m.p. > 260 C; IR νmax (neat): 3240, 1711, 1639, 1606, 1532, 1486, 1211, 1022, 920, 851 cm ;
1
H NMR (400 MHz, DMSO-d ) δ (ppm): 10.10 (bs, 1H), 7.22 (dd, J = 8.4, 5.6 Hz, 2H), 7.07 (t,
6
J = 8.8 Hz, 2H), 6.95 (d, J = 8.0 Hz, 1H), 6.80 (dd, J = 10.1, 1.7 Hz, 2H), 5.00 (s, 1H),4.97 (d, J = 15.8 Hz, 1H),
1
3
4
1
2
.87 (d, J = 15.8 Hz, 1H), 2.42 (s, 3H); C NMR (100 MHz, DMSO-d ) δ (ppm): 172.1, 160.9 (d, J = 240.0 Hz,),
6
58.6, 143.1 (d, J = 3.0 Hz), , 137.6, 136.7, 131.3, 129.5 (d, J = 8.0 Hz, 2C), 121.1, 120.8, 115.2 (d, J = 21.0 Hz,
C), 113.0, 95.6, 65.3, 38.3, 14.6; LC-MS (ESI): m/z 328.0 (M+H), 350.0 (M+Na); SMILES:
CSC1=CC=C(C(C2=CC=C(F)C=C2)C(C(OC3)=O)=C3N4)C4=C1
8

ACCEPTED MANUSCRIPT
9
-(2,3-dimethoxyphenyl)-6-(methylthio)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2dg’). Compound 2dg’ was
prepared accordingly to the general procedure A for the one pot synthesis using 3-
methylthioaniline 3d (123 μL, 1.0 mmol, 1.0 equiv.), 2,3-dimethoxybenzaldehyde 4g’ (166
mg, 1.0 mmol, 1.0 equiv.), and tetronic acid 5 (100 mg, 1.0 mmol, 1.0 equiv.) in 2-pentanol
(
3.3 mL). The crude product was purified by recrystallization in ethanol (2.5 mL), then filtered
and washed with cold ethanol (3 x 2.5 mL) to obtain pure compound 2dg’(66 mg, 0.2 mmol,
°
1
1
8% yield). White powder; R = 0.21 (acetone/hexanes; 30/70); m.p. > 260 C; IR νmax (neat): 1721, 1636, 1526,
f
-
1 1
483, 1351, 1225, 1078, 1011, 740 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 9.98 (bs, 1H), 6.94 (t, J = 7.9
6
Hz, 1H), 6.87 – 6.81 (m, 2H), 6.75 (dd, J = 8.2, 1.9 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H), 6.64 (dd, J = 7.7, 1.1 Hz, 1H),
.25 (s, 1H), 4.96 (d, J = 15.4 Hz, 1H), 4.87 (d, J = 15.4 Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 2.40 (s, 3H); LC-MS
ESI): m/z: 370.0 (M+H), 392.0 (M+Na);
SMILES: CSC1=CC=C(C(C2=C(OC)C(OC)=CC=C2)C(C(OC3)=O)=C3N4)C4=C1
5
(
5
-(methylthio)-9-(3,4,5-trimethoxyphenyl)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2ea’). Compound 2ea’
was prepared accordingly to the general procedure A for the one pot synthesis using 3,4,5-trimethoxybenzaldehyde
a’ (491 mg, 2.5 mmol, 1.0 equiv.), 2-methylthioaniline 3e (348 mg, 2.5 mmol, 1.0 equiv.) and
4
tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in ethanol (10 mL). The crude product was
purified by column chromatography on silica gel using an isocratic solvent system of acetone and
dichloromethane (20:80) to obtain pure compound 2ea’ (771 mg, 1.93 mmol, 77% yield). Red
°
solid; R = 0.29 (acetone/hexanes; 45/55); m.p. > 260 C; IR νmax (neat): 2929, 1734, 1669,
f
-
1 1
1
589, 1455, 1418, 1230, 1121, 1005, 770 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 7.09
6
(
d, J = 2.1 Hz, 1H), 6.86 (dd, J = 8.3, 2.1 Hz, 1H), 6.60 (d, J = 8.3 Hz, 1H) 6.59 (s, 2H), 4.86 (s, 1H), 4.67 (s, 2H),
.67 (s, 6H), 3.62 (s, 3H), 2.24 (s, 3H); LC-MS (ESI): m/z 418.1 (M+H O+H);
3
2
SMILES: O=C(OC1)C2=C1NC3=C(SC)C=CC=C3C2C4=CC(OC)=C(OC)C(OC)=C4
9
-(4-hydroxy-3-methoxyphenyl)-5-(methylthio)-4,9-dihydrofuro[3,4-b]quinolin-1(3H)-one (2eb’). Compound
eb’ was prepared accordingly to the general procedure A for the one pot synthesis using 3-
2
hydroxy-4-methoxybenzaldehyde 4b’ (380 mg, 2.5 mmol, 1.0 equiv.), 2-methylthioaniline 3e (348
mg, 2.5 mmol, 1.0 equiv.) and tetronic acid 5 (250 mg, 2.5 mmol, 1.0 equiv.) in ethanol (10 mL).
The crude product was purified by column chromatography on silica gel using an isocratic solvent
system of acetone and dichloromethane (30:70) to obtain pure compound 2eb’ (733 mg, 2.1 mmol,
°
8
3% yield). Deep red solid; R = 0.26 (acetone/hexanes; 45/55); m.p. > 260 C; IR νmax (neat):
f
-
1 1
2
1
3
926, 1609, 1512, 1427, 1268, 1229, 1124, 1031, 779, 687 cm ; H NMR (400 MHz, DMSO-d ) δ (ppm): 8.75 (bs,
6
H), 7.06 (d, J = 2.0 Hz, 1H), 6.85 – 6.81 (m, 2H), 6.68 – 6.56 (m, 3H), 5.07 (bs, 1H), 4.82 (s, 1H), 4.64 (s, 2H),
.65 (s, 3H), 2.23 (s, 3H); LC-MS (ESI): m/z 356.2 (M+H);
SMILES: O=C(OC1)C2=C1NC3=C(SC)C=CC=C3C2C4=CC=C(OC)C(O)=C4
9