Azobenzene derivatives show anti-cancer activity against pancreatic cancer cells only under nutrient starvation conditions via G0/G1 cell cycle arrest

Article abstract of DOI:10.1016/j.tet.2021.132077

Pancreatic cancer is one of the most aggressive cancers with a poor prognosis. Previous studies suggested that nutrient-deprived conditions may play a critical role in pancreatic cancer cell survival and resistance to chemotherapy. We describe a novel series of azobenzene derivatives including (E)-1-(4-methyl-3-((2-methyl-5-(naphthalen-1-yl)phenyl)diazenyl)phenyl)naphthalen-2-ol (9) with efficacy and selectivity in nutrient-deprived conditions. Although anticancer drug 5-fluorouracil (5-FU) was ineffective under nutrient-deprived conditions, five of our designed compounds, 9 and four other related compounds 11–14, showed anticancer activity with IC₅₀ values ranging from 1.5 to 9.6 μM. Interestingly, only 9 showed no cytotoxicity in normal conditions. This selectivity profile of 9 is clearly opposite to that of 5-FU. Furthermore, cell cycle analysis showed that, in contrast to S phase arrest induced by 5-FU, 9 caused G₀/G₁ phase arrest, which might block cancer cell growth by arresting them in quiescence. Therefore, it could be a novel and promising candidate for effective pancreatic cancer treatment under nutrient-deprived conditions.

Full text of DOI:10.1016/j.tet.2021.132077

Tetrahedron 85 (2021) 132077
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Azobenzene derivatives show anti-cancer activity against pancreatic
cancer cells only under nutrient starvation conditions via G0/G1 cell
cycle arrest
,
Kenta Shinzawa a, Daiki Kageta b, Robert J. Nash c, George W.J. Fleet d *, Tatsushi Imahori b,
,
**
Atsushi Kato a
a Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan
b Department of Industrial Chemistry, Tokyo University of Science, 1-3 Kagurazaka, Shinjyuku-ku, Tokyo, 162-8601, Japan
c Institute of Biological, Environmental and Rural Sciences / Phytoquest Limited, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, United Kingdom
d Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Pancreatic cancer is one of the most aggressive cancers with a poor prognosis. Previous studies suggested
that nutrient-deprived conditions may play a critical role in pancreatic cancer cell survival and resistance
to chemotherapy. We describe a novel series of azobenzene derivatives including (E)-1-(4-methyl-3-((2-
methyl-5-(naphthalen-1-yl)phenyl)diazenyl)phenyl)naphthalen-2-ol (9) with efficacy and selectivity in
nutrient-deprived conditions. Although anticancer drug 5-fluorouracil (5-FU) was ineffective under
nutrient-deprived conditions, five of our designed compounds, 9 and four other related compounds 11
Received 8 December 2020
Received in revised form
8 February 2021
Accepted 4 March 2021
Available online 9 March 2021
e14, showed anticancer activity with IC50 values ranging from 1.5 to 9.6 mM. Interestingly, only 9 showed
Keywords:
no cytotoxicity in normal conditions. This selectivity profile of 9 is clearly opposite to that of 5-FU.
Furthermore, cell cycle analysis showed that, in contrast to S phase arrest induced by 5-FU, 9 caused G0/
G1 phase arrest, which might block cancer cell growth by arresting them in quiescence. Therefore, it
could be a novel and promising candidate for effective pancreatic cancer treatment under nutrient-
deprived conditions.
Pancreatic cancer
Nutrient-deprived conditions
Selectivity
Azobenzene
Cell cycle analysis
G0/G1 phase arrest
© 2021 Elsevier Ltd. All rights reserved.
1. Introduction
To further elucidate the biological features of pancreatic cancer,
several studies have focused on metabolic changes and adaptations
Pancreatic cancer is one of the most aggressive cancers with
poor prognosis [1]. A recent study has reported that a 5-year sur-
vival rate of pancreatic cancer patients was approximately 9% [2].
Moreover, in the case of pancreatic cancer, anticancer agents have
only limited effects in clinical practice [3]. Also, a recent clinical trial
has shown that a median overall survival with gemcitabine (GEM),
which is widely used for chemotherapy of advanced pancreatic
cancer, was just 6.8 months [4]. Hence, the treatment of pancreatic
cancer is one of the most critical challenges, and a new anti-
pancreatic-cancer agent is sorely needed to improve treatment
outcomes.
under nutrient-deprived conditions. These studies have revealed
that pancreatic cancer displays the following unique features: (i)
some cells are exposed to nutrient-deprived conditions in the tu-
mor [5], (ii) the cells can survive under the nutrient-deprived
medium (NDM), which is inclusive of electrolytes and vitamins
but exclusive of glucose, amino acids, and serum [6,7], and (iii)
under such conditions, the cells exhibit resistance to anticancer
drugs like 5-fluorouracil (5-FU) and GEM [8]. Recent studies have
also shown that autophagy, which is an intracellular degradation
and recycling process induced by nutrient deprivation or cell stress,
is highly upregulated in pancreatic cancer [9] and may contribute to
survival under nutrient-deprived conditions [10,11]. These features
may be associated with poor prognoses [12]. It appears that
nutrient-deprived conditions may play a critical role in pancreatic
cancer cell survival and resistance to chemotherapy. This is a
difficult but important problem that needs to be addressed to
improve clinical outcomes. Thus, we focused on nutrient-deprived
* Corresponding author.
** Corresponding author.
E-mail addresses: george.fleet@sjc.ox.ac.uk (G.W.J. Fleet), kato@med.u-toyama.
ac.jp (A. Kato).
https://doi.org/10.1016/j.tet.2021.132077
0040-4020/© 2021 Elsevier Ltd. All rights reserved.

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
conditions as a potential target for chemotherapy.
presence of a catalytic amount of Pd(PPh3)4 formed the naphtha-
lene derivative 27 in quantitative yield. Removal of the methyl
ether in 27 by treatment with boron tribromide gave the naphthol
28 (70%). Reduction of the phosphine oxide group in 28 by tri-
chlorosilane gave the target phosphine 10 (24%) in an overall yield
of 13% from 25.
The amino azobenzene 6 was a divergent intermediate for the
efficient synthesis of a series of azobenzene derivatives 11e14
containing both pyridine and thiourea or urea moieties (Scheme 4).
The pyridine units were introduced by a palladium-catalysed
amination reaction; the thiourea or urea units were introduced
by the reactions of amines with isothiocyanate or isocyanate. The
Previous studies have demonstrated that some compounds,
including arctigenin [13] that has progressed to a clinical trial [14],
showed anticancer activity under nutrient-deprived conditions
[15e18]. To date, however, no drugs have been approved for use
against nutrient-deprived conditions. With this in mind, we
therefore aimed to investigate the effect of various synthesized
compounds under nutrient-deprived conditions. In addition to ef-
ficacy, selectivity is also evaluated by the use of normal culture
medium to avoid cytotoxic side effects in normal cells. Several
derivatives were then selected for further evaluation after initial
screening of the synthesized azobenzenes. Several previous studies
have shown an azobenzene-containing polymer is a carrier to
deliver anticancer agents into pancreatic cancer tissues [19,20].
Additionally, some azobenzene derivatives have been reported to
show photo-switching ability of the biological activities by photo-
irradiation based on photo-isomerizations [21,22]. However, anti-
pancreatic cancer activity of azobenzene derivatives themselves
has not been reported previously. This paper describes the syn-
thesis and preliminary evaluation of a novel series of azobenzene
derivatives (Fig. 1); the dimeric azonapthol (9) shows significant
anti-pancreatic cancer activity, with both efficacy and selectivity in
nutrient-deprived conditions.
azo
compound
6
was
coupled
with
the
iodo-
dimethylaminopyridine 29 in the presence of Pd2dba3 and 1,3-
bis(diphenylphosphino)propane to give the DMAP analogue 30
(47% yield). Subsequent treatment with the bis(trifluoromethyl)
phenyl isothiocyanate 31 gave the thiourea 11 (50%), or with the
corresponding isocyanate 32, afforded the urea 12 (42% yield).
Palladium-catalysed coupling of 6 with 2-iodopyridine 33
formed the pyridine analogue 34 (46%); subsequent reaction of 34
with the isothiocyanate 31 gave the corresponding thiourea 13
(57%). Similar coupling of 6 with the pyrrolidine 35 gave the amine
36 (42%); treatment of 36 with the isothiocyanate 31 afforded 14 in
40% yield.
2. Results and discussion
2.2. Evaluation of anti-cancer activity of azobenzene derivatives
2.1. Synthesis of azobenzene derivatives
The first aim of our study was to determine the efficacy of our
compounds under nutrient-deprived conditions. Initially, we
evaluated anticancer activity of nine azobenzene derivatives (1e9),
5-fluorouracil (5-FU), and gemcitabine (GEM) against the human
pancreatic cancer cell line PANC-1 [24] under nutrient-deprived
conditions by using nutrient-deprived medium (NDM), which is
exclusive of glucose, amino acids, and serum. Cell viability was
assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay [25]. Table 1 shows IC50 values for the
tested compounds.
A series of azobenzene compounds with acid and/or base
functionalities (1e14) were synthesized following the routes
described in Schemes 1e4. The symmetrical azo derivative 1, pre-
pared from coupling of the diazonium salt of 15 as previously
described [23] in 51% yield, was the divergent intermediate for the
synthesis of azocompounds 2e7 (Scheme 1). All the initial azo
compounds were isolated as mixtures of cis- and trans-isomers
which gave the pure trans-isomer when warmed at 50 ꢀC overnight.
As previously described [23], addition of phenylmagnesium
bromide and of 1-naphthyllithium to the ester 1 gave the tertiary
alcohols 2 and 3 in yields of 94% and 51% respectively. Reduction of
the esters in 1 by di-isobutylaluminum hydride afforded 4 (84%
yield); subsequent bromination of the primary alcohol 4 with tet-
rabromomethane and triphenyl phosphine provided the bromide 5
(74% yield). The amino azobenzene 6 was synthesized from 1 via
initial sodium hydroxide hydrolysis to the acid 16 (85%); treatment
of the acid 16 with diphenylphosphoryl azide in the presence of
benzyl alcohol resulted in a Curtius rearrangement to give the
carbamate protected amine 17 (69%). Basic solvolysis of the
carbamate 17 (92%) gave the amino azobenzene 6 in an overall yield
of 54% from 1. Reductive N-methylation of 6 by treatment with
formaldehyde and sodium borohydride gave the methylamino
azobenzene 7 in 42% yield.
When the bromide 20 was treated with the amino boronic acid
21 in the presence of Pd(PPh3)4, the biphenyl azo-amine 8 was
isolated in 89% yield. Similar Pd(PPh3)4-catalysed coupling of the
bromide 20 with 2-methoxy-1-naphthaleneboronic acid 22 gave
the methoxy naphthalene 23 (85% yield) which was demethylated
on treatment with boron tribromide to give the naphthols 9 (92%).
A similar strategy was successful in the synthesis of a diphe-
nylphosphine azobenzene 10 (Scheme 3). Condensation of the
iodoaniline 24 with the nitroso compound 19 [derived from 18]
gave the bromo iodoazo analogue 25 (73%). The iodide in 25 was
selectively replaced by treatment with a mixture of diphenyl-
phosphine and diphenylphosphine oxide in the presence of palla-
dium acetate to give the phosphine oxide 26 (77% yield). Coupling
of the bromide 26 with naphthalene boronic acid 22 in the
Reference anticancer drugs 5-FU and GEM showed no activity
even at 200
mM (Fig. 2A and B). Several studies have suggested
mechanisms for resistance to current anticancer drugs under
nutrient-deprived conditions [26]. One example is glucose-
regulated protein 78 (GRP78) which is one of the molecular chap-
erones and has been proposed as a possible factor contributing to
chemotherapy resistance [27]. It is not clear, however, which factors
responsible for this phenomenon. Our results and previous studies
highlight the issues of current anticancer drugs that lack activity
under nutrient-deprived conditions. In contrast to the results of 5-
FU and GEM, synthesized compound 9 showed anticancer activity
under nutrient-deprived conditions (IC50 ¼ 4.1 0.3
mM). In light of
this result, we further evaluated five additional azobenzene de-
rivatives (10e14). These results demonstrated that four related
compounds (11e14) showed anticancer activity with IC50 values
ranging from 1.5 to 9.6
mM under nutrient-deprived conditions.
Taken together, we identified five azobenzene derivatives (9, 11, 12,
13, and 14) that showed potent efficacy under nutrient-deprived
conditions, where the current anticancer drugs are not effective.
Our findings have also opened up possibilities of using azobenzene
derivatives for drug discovery in pancreatic cancer.
We next evaluated safety profiles of azobenzene derivatives to
minimize cytotoxicity under normal conditions. In clinical practice,
chemotherapy-related side effects, such as myelosuppression,
diarrhea, and neurotoxicity, are serious problems. These side effects
are caused by cytotoxic effects against normal cells [28e30]. Hence,
in addition to anticancer activity, minimizing cytotoxicity against
normal cells is required for safe and effective treatment. In the
2

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
Fig. 1. Structures of the azobenzene derivatives 1e14 and anti-cancer compounds 5-FU and GEM.
3

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
Scheme 1. Synthesis of azobenzenes 1e7.
Scheme 2. Pd-catalysed coupling in the synthesis of azobenzenes 8 and 9.
present study, we focused on nutrient-deprived conditions where
pancreatic cancer cells can survive, but not normal cells. Thus,
because normal cells cannot exist under such conditions, selective
targeting of nutrient-deprived conditions may contribute to
reducing toxicity against normal cells.
In light of these points, we examined whether their activities are
selective against nutrient-deprived conditions, by assessing their
cytotoxicity under normal conditions by using normal medium
(Dulbecco’s modified Eagle’s medium; DMEM). Unfortunately, most
of these compounds showed cytotoxicity under normal conditions
4

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
Scheme 3. Pd-catalysed coupling in the synthesis of a diphenylphosphine azobenzene 10.
Scheme 4. Amino azobenzene 6 as a divergent intermediate for pyridine azobenzenes 11e14.
(IC50 values ranging from 7.1 to 16.9
m
M) as well as anticancer ac-
In light of these results, we investigated the importance of the
naphthol unit in 9 and 10. Compound 9 has naphthol units on both
sides of the azobenzene core and shows anticancer activity only
under nutrient-deprived conditions. Compound 10 with only the
naphthol unit on one side does not show such activity both under
nutrient-deprived and normal conditions. These results suggested
that a combination of the naphthol unit may be important for se-
lective activity. In this study we elucidated that compounds 11e14
tivity under nutrient-deprived conditions, but interestingly, only 9
showed no cytotoxicity under normal conditions (Fig. 2C). We also
tested the toxicity of 9 against the human normal fibroblast cell line
GM05659 under normal conditions. In normal cells, 9 did not affect
the cell viability even at 20 mM (Fig. 3). These results indicated that
9 exhibits selective activity against nutrient-deprived conditions
with both potency and safety.
5

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
Table 1
its anticancer activity, and (ii) 9 caused G0/G1 phase arrest, which is
a different mode of action to that of S phase arrest induced by 5-FU
[34]. The anticancer activity results and cell cycle arrest may well
have a related mechanism. Although further detailed study is
needed, one possibility is that 9 might block cancer cell growth by
arresting cells in G0 phase, which is a state of cellular quiescence
[35].
IC50 values of test compounds (1e14, 5-fluorouracil, and gemcitabine) against PANC-
1 cells under normal conditions and nutrient-deprived conditions.
No.
IC50 [mM]
Normal conditions
72 h
Nutrient-deprived conditions
24 h
1
>80
>80
2
>80
>80
3
4
>80
>80
>80
>80
3. Conclusion
5
6
7
8
>80
>80
>80
>80
>80
>80
7.1 0.3
14.3 1.3
16.9 0.4
7.7 0.4
3.2 0.3
23.5 3.1
>80
>80
>80
>80
4.1 0.3
>80
1.5 0.1
3.6 0.5
5.4 0.8
9.6 0.1
>200
>200
In conclusion, five of the designed azobenzene showed signifi-
cant anticancer activity under nutrient-deprived conditions. These
findings provide unprecedented insight into the anticancer activity
of azobenzene derivatives. The symmetrical azodinapthol deriva-
tive 9 differed from the other four compounds in selectivity against
nutrient-deprived conditions. This selectivity of 9 is likely to be
important in the reduction of toxicity to normal cells under normal
conditions. Furthermore, 9 showed G0/G1 phase arrest, in contrast
to S phase arrest induced by 5-FU. The effect of 9 on cell cycle
progression was also selective against nutrient-deprived condi-
tions. Thus, the synthesized azobenzene derivative 9 could be a
novel and promising candidate for effective and selective pancre-
atic cancer treatment under nutrient-deprived conditions.
9
10
11
12
13
14
5-FU
GEM
Data are presented as mean SE (n ¼ 3). 5-FU; 5-fluorouracil, GEM; gemcitabine.
showed powerful anticancer effect under both normal conditions
and nutrient-deprived conditions. They have a combination of
DMAP (4-dimethilaminopyridine) and thiourea or related struc-
tures. In light of the activity profiles, these combinations may be
important for the powerful but non-selective activity of 11e14.
Further studies with different modification of the napthol moiety
may produce more selective agents.
4. Experimental section
4.1. Chemistry
4.1.1. General
To further characterize the anticancer activity of 9, we next
performed a cell cycle analysis by propidium iodide staining [31].
PANC-1 cells were treated with or without 9 under either normal or
nutrient-deprived conditions for 24 h, then stained with propidium
iodide and analyzed. Treatment with compound 9 significantly
In each synthesis, a mixture of both the cis- and trans-azo-
benzenes was initially formed; however when the mixture was
warmed at 50 ꢀC overnight, pure trans-azobenzene isomers were
isolated. The thermal isomerization of cis-to trans-azobenzenes
under these conditions is well-established [36]. NMR spectra were
recorded on JEOL JNM-EX 400 (400 MHz) spectrometer, Varian
INOVA 400 (400 MHz), BRUKER BioSpin-AVANCE DPX-400
(400 MHz), BRUKER BioSpin-AVANCE DPX-300 (300 MHz), and
BRUKER BioSpin-AVANCE 400 M (400 MHz). 13C NMR spectra were
recorded using broad band proton decoupling. Residual CHCl3 peak
or tetramethylsilane (TMS) were used as an internal reference for
1H NMR (CHCl3: 7.26 ppm, TMS: 0.00 ppm). For 13C NMR in CDCl3,
residual peak of CHCl3 was used as an internal reference
increased the percentage of cells in G0/G1 phase (47.5
p < 0.01 vs control cells) and decreased those in S phase
(18.7 0.6%, p < 0.01 vs control cells) under nutrient-deprived
conditions (Fig. 4A). In contrast, 9 did not affect the cell cycle un-
der normal conditions even at 100 M (Fig. 4B). There are possible
1.0%,
m
targets, including CDK4/6, PI3K/AKT, p21, that lead to cell cycle
arrest in G0/G1 phase. These factors might play a role in selective
activity under nutrient-deprived conditions. In light of this, further
research focused on these factors is needed to elucidate the
mechanisms underlying our findings [32,33].
(77.16 ppm). Chemical shifts are expressed in
d (ppm) values, and
coupling constants are expressed in hertz (Hz). The following ab-
breviations are used: s ¼ singlet, d ¼ doublet, m ¼ multiplet, br
s ¼ broad singlet, dt ¼ double-triplet, and dd ¼ double-doublet.
These results demonstrated two key points: (i) the effect of 9 on
the cell cycle was selective in nutrient-deprived conditions as was
Fig. 2. Anticancer activities of (A) gemcitabine, (B) 5-fluorouracil, and (C) 9 against PANC-1 cells under normal conditions and nutrient-deprived conditions. Results are expressed as
mean SEM (n ¼ 3).
6

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
Fig. 3. Toxicity of 9, 5-fluorouracil (5-FU), and gemcitabine (GEM) against GM05659 cells under normal conditions. GM05659 cells were cultured with or without compound for
72 h. Results are expressed as mean SEM (n ¼ 3).
Fig. 4. The cell cycle analysis. PANC-1 cells were cultured with or without 9 in (A) nutrient-deprived conditions or (B) normal conditions for 24 h. Statistical significance was
determined by using Welch’s t-test (**, p < 0.01 vs control cells). Each value represents the mean SEM (n ¼ 3).
Mass spectra and high-resolution mass spectra were recorded on a
JEOL JMS-700, Varian 910-MS FT-ICR. IR spectra were measured
with a PerkinElmer spectrum one S series FT-IR spectrometer and a
JASCO FT-IR 466ST. All reagents and solvents are commercial grade
and were used as supplied without further purification. Known
compounds were identified by 1H NMR and 13C NMR spectra.
sodium nitrite (7.04 g, 102 mmol) in water (600 mL) over 2 h
50 min at 0 ꢀC to form a solution of the corresponding diazonium
salt of 15 for use in the next step.
An aqueous solution of CuSO4$(H2O)5 (28.5 g, 114 mmol) in
water (100 mL) was treated with 30% aqueous ammonia (30 mL) at
0
ꢀC; the reaction mixture was allowed to warm to room temper-
ature. Then an aqueous solution of NH2OH$HCl (8.0 g, 115 mmol in
water (20 mL) was added until the deep blue color disappeared,
after which the solution of diazonium salt of 15 was added drop-
wise and the reaction mixture was stirred for 1 h. The resulting dark
orange solid was collected by filtration, washed with distilled water
4.1.2. Dimethyl 3,3’-(diazene-1,2-diyl)(E)-bis(4-methylbenzoate)1
[23]
Methyl 3-amino-4-methylbenzoate 15 (16.0 g, 96.9 mmol) in
7.4% aqueous HCl (200 mL) was added dropwise to a solution of
7

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
and dried under reduced pressure. The crude product was purified
by silica gel column chromatography (CH2Cl2 as eluent) and crys-
tallization (CH2Cl2/MeOH) to afford 1 as an orange solid (8.05 g,
24.7 mmol, 51%, mixture of trans- and cis-azobenzene isomer)
which was warmed at 50 ꢀC overnight to afford the pure trans-
azobenzene isomer 1.
filtered, and concentrated. The resulting solid was collected by
filtration, washed with dichloromethane and dried under reduced
pressure to give the mixture of trans- and cis-isomers 4 (0.31 g, 84%)
as an orange powder which was warmed at 50 ꢀC overnight to
afford the pure trans-azobenzene isomer 4.
1H NMR (400 MHz, DMSO)
d
(ppm): 2.67 (s, 6H), 4.52 (d,
J ¼ 5.8 Hz, 4H), 5.25e5.27 (t, J ¼ 5.7 Hz, 2H), 7.39 (s, 4H), 7.54 (s, 2H).
13C NMR (400 MHz, DMSO)
(ppm): 17.0, 62.4, 113.4, 129.2, 131.1,
1H NMR (400 MHz, CDCl3)
d (ppm): 2.82 (s, 6H), 3.94 (s, 6H), 7.44
(d, 2H, J ¼ 8.0 Hz), 8.05 (dd, 2H, J ¼ 7.9, 1.8 Hz), 8.23 (d, 2H,
d
J ¼ 1.7 Hz). 13C NMR (100 MHz, CDCl3)
d
(ppm): 18.0, 52.2, 117.2,
135.7, 141.1, 150.2. IR (solid, ATR): 3320, 2920, 1615, 1500, 1408, 895,
128.7, 131.4, 131.5, 143.4, 150.7, 166.8.
825, 764 cmꢁ1. MS(ESI) m/z: 271 [(M þ H)þ]. HRMS (ESI): Calcd. for
C
16H19N2Oþ2 : 271.1441 [(M þ H)þ], Found: 271.1442.
4.1.3. (E)-(Diazene-1,2-diylbis(4-methyl-3,1-phenylene))
bis(diphenylmethanol) 2 [23]
A 1.0 M THF solution of phenylmagnesium bromide (8.8 mL,
8.8 mmol) was added dropwise at 0 ꢀC under an argon atmosphere
to a solution of a mixture of cis- and trans-azobenzene isomers 1
(652 mg, 2.0 mmol) in THF (8.8 mL). The reaction mixture was then
stirred at 30 ꢀC for 26 h, quenched by the addition of saturated
aqueous solution of NH4Cl, and extracted with dichloromethane.
The organic extracts were dried (Na2SO4) The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography (CH2Cl2/hexane ¼ 2/1) to afford a mixture
of trans- and cis-azobenzene isomers 2 (1.08 g, 1.89 mmol, 94%)
which was warmed at 50 ꢀC overnight to afford the pure trans-
azobenzene isomer 2.
4.1.6. (E)-1,2-bis(5-(Bromomethyl)-2-methylphenyl)diazene 5
Triphenylphosphine (476 mg, 1.82 mmol) in THF (0.9 mL) was
added dropwise to a solution of a mixture of the trans- and cis-
azobenzene isomers 4 (196 mg, 0.73 mmol) and tetrabromo-
methane (602 mg, 1.82 mmol) in THF (7.0 mL) at room temperature.
The reaction mixture was stirred for 3 h, the solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography (hexane/ethyl acetate ¼ 5/1) to afford a
mixture of cis- and trans-isomers 5 (213.1 mg, 0.54 mmol, 74%) as a
solid which was warmed at 50 ꢀC overnight to afford the pure trans-
azobenzene isomer 5.
1H NMR (400 MHz, CDCl3)
d (ppm): 2.74 (s, 6H), 4.53 (s, 4H),
1H NMR (400 MHz, CDCl3)
2H), 7.28 (br s, 24H), 7.47 (br s, 2H). 13C NMR (100 MHz, CDCl3)
(ppm): 17.1, 81.9, 115.4, 127.3, 127.9, 128.0,130.2,130.9,137.1, 145.2,
d
(ppm): 2.53 (br s, 6H), 2.83 (br s,
7.33e7.35 (d, J ¼ 8.0 Hz, 2H), 7.41e7.43 (dd, J ¼ 2.0, 8.0 Hz, 2H), 7.62
(d, J ¼ 1.6 Hz, 2H). 13C NMR (400 MHz, C4D8O)
d (ppm): 16.6, 32.7,
d
116.2, 131.5, 131.7, 138.2, 151.0. IR (solid, ATR): 656, 744, 867, 1374,
1498, 1609, 2895, 2926 cmꢁ1. MS (ESI) m/z: 394 (Mþꢂ). HRMS (ESI):
Calcd. for C16H17N2Brþ2 [(M þ H)þ]: 394.9753, Found: 394.9751.
146.7, 150.2.
4.1.4. (E)-(Diazene-1,2-diylbis(4-methyl-3,1-phenylene))
bis(di(naphthalen-1-yl)methanol) 3 [23]
A solution of 1-naphthyllithium was prepared by addition of
4.1.7. (E)-3,30-Diazene-1,2-diyl)bis(4-methylaniline) 6
1.62
M
solution of nBuLi (6.17 mL, 10.0 mmol) to 1-
Aqueous sodium hydroxide (1 N, 240 mL) was added to a so-
lution of a mixture of the trans- and cis-azobenzene isomers 1
(4.00 g, 12.2 mmol) in THF (39.3 mL). The reaction mixture was
heated under reflux for 19.5 h, cooled to room temperature and
acidified by aqueous hydrochloric acid (3 N) to pH z 2 to afford the
corresponding acid 16 (3.12 g, 10.4 mmol, 85%) collected by filtra-
tion as an orange solid. A solution of the crude acid 16 in dry
toluene (229 mL) under argon atmosphere was treated sequentially
with triethylamine (5.37 mL, 38.5 mmol), benzyl alcohol (4.0 mL,
38.5 mmol), and diphenylphosphoryl azide (8.31 mL, 38.56 mmol).
The resulting reaction mixture was stirred at 80 ꢀC for 24 h, cooled
to room temperature, and the solvent removed under reduced
pressure. The solid residue was washed with dichloromethane and
MeOH to afford 17 (6.45 g, 12.6 mmol, 69%) as an orange solid.
The crude mixture of trans- and cis-azobenzene isomers 17
(376 mg, 0.74 mmol) in a saturated solution of KOH in ethanol was
heated at 100 ꢀC for 24 h. The reaction mixture was cooled to room
temperature, then neutralized by addition of saturated aqueous
ammonium chloride. The solvents were removed under reduced
pressure and the residue extracted with dichloromethane. The
combined organic layer was dried (Na2SO4). The solvent was
removed under reduced pressure and the residue was purified by
silica gel column chromatography (CH2Cl2/AcOEt ¼ 50/1) to afford a
mixture of trans- and cis-isomers 6 (163 mg, 0.68 mmol, 92%) as an
orange solid which was warmed at 50 ꢀC overnight to afford the
pure trans-azobenzene isomer 6.
bromonaphthalene (1.46 mL, 2.16 g, 9.91 mmol) in THF at ꢁ78 ꢀC
under an argon atmosphere and the mixture was stirred at ꢁ78 ꢀC
for an additional 30 min.
To the above solution of 1-naphthyllithium was added a mixture
of cis- and trans-azobenzene isomers 1 (652.3 mg, 2.0 mmol) in THF
(20 mL) dropwise at ꢁ78 ꢀC under argon. The reaction mixture was
allowed to warm to ꢁ10 ꢀC and stirred for 1 h and the reaction
quenched by addition of sat. aq. solution of NH4Cl and extracted
with dichloromethane. The organic extracts were dried (Na2SO4)
and the solvent removed under reduced pressure to give a residue
which was purified by silica gel column chromatography (hexane/
CH2Cl2 ¼ 1/1) to afford a mixture of trans- and cis-azobenzene
isomers 3 (795 mg, 1.0 mmol, 51%) which was warmed at 50 ꢀC
overnight to afford the pure trans-azobenzene isomer 3.
1H NMR (400 MHz, CDCl3)
d (ppm): 2.46 (s, 6H), 3.65 (s, 2H),
6.87 (d, J ¼ 6.8 Hz, 4H), 7.18e7.28 (m, 12H), 7.39 (d, J ¼ 7.8 Hz, 2H),
7.41 (d, J ¼ 6.8 Hz, 2H), 7.68 (br s, 2H), 7.80 (d, J ¼ 8.8 Hz, 4H), 7.86 (d,
J ¼ 7.8 Hz, 4H), 8.28 (d, J ¼ 7.8 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d
(ppm): 17.1, 85.2, 115.2, 124.3, 125.4, 125.6, 128.3, 128.6 (br m),
128.881, 129.3, 130.1, 130.9, 131.3, 135.1, 137.2, 141.9 (br m), 145.3,
150.4.
4.1.5. (E)-(Diazene-1,2-diylbis(4-methyl-3,1-phenylene))
dimethanol 4
A solution of di-isobutylaluminum hydride (1.0 M hexane,
6.8 mL, 6.8 mmol) was added to a mixture of cis- and trans-azo-
benzene isomers 1 (444 mg, 1.36 mmol) in dry toluene dropwise
at ꢁ78 ꢀC under an argon atmosphere. The reaction mixture was
allowed to warm to 0 ꢀC, stirred for 1.5 h, quenched by addition of
aqueous hydrochloric acid (1 N, 5.0 mL) and extracted with CH2Cl2-
THF (10:1); the combined organic extracts were dried (Na2SO4),
1H NMR (400 MHz, CDCl3)
d (ppm): 2.61 (s, 6H), 3.64 (brs, 4H,
NH2), 6.73 (dd, J ¼ 2.5, 8.1 Hz, 2H), 6.97 (d, J ¼ 2.5 Hz, 2H), 7.11 (d,
J ¼ 8.1 Hz, 2H). 13C NMR (400 MHz, CDCl3)
d (ppm): 16.7,101.9,118.3,
128.6, 131.8, 144.8, 151.5. IR (KBr, ATR): 720, 818, 874, 1157, 1292,
1499, 1635, 2362, 2729, 2920, 3024, 3214, 3312, 3403 cmꢁ1. HRMS
(EIþ): calcd. for C14H16Nþ4 (Mþꢂ), 240.1375, found, 240.1374.
8

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
4.1.8. (E)-3,3’-(Diazene-1,2-diyl)bis(N,4-dimethylaniline) 7
(d, J ¼ 1.5 Hz, 2H, AreH), 6.60 (d, J ¼ 8.1 Hz, 2H, AreH), 6.70 (t,
J ¼ 7.8 Hz, 2H, AreH), 6.81e6.83 (m, 2H, AreH), 7.07 (t, J ¼ 7.8 Hz,
2H, AreH), 7.14e7.20 (m, 2H, AreH), 7.31 (d, J ¼ 7.8 Hz, 2H, AreH).
A mixture of cis- and trans-azobenzene 6 was added to a solu-
tion of sodium methoxide [prepared by addition of lumps of so-
dium (139 mg, 6 mmol) to dry methanol (6.8 mL) in an eggplant
flask under argon at room temperature]. Then, paraformaldehyde
(45.7 mg) was added slowly to the reaction mixture which was
stirred at room temperature for 16 h. Sodium borohydride (39.8 mg,
1 mmol) was quickly added to the resulting orange suspension, and
a reflux condenser was attached. The mixture was heated under
reflux at 70 ꢀC for 6 h. Subsequent addition of aqueous potassium
hydroxide (1 N) precipitated a solid which was collected by filtra-
tion, washed with water and methanol, and dried under reduced
pressure. The residue was purified by silica gel column chroma-
tography (ethyl acetate/hexane ¼ 1/5) to a mixture of trans- and cis-
azobenzene isomers 7 (57 mg, 0.21 mmol, 42%) which on warming
at 50 ꢀC overnight, afforded the pure trans-isomer 7.
13C NMR (400 MHz, CDCl3)
d (ppm): 17.5, 115.6, 116.4, 118.7, 127.0,
128.6, 130.5, 131.5, 131.8, 137.1, 137.7, 143.6, 151.3. IR (solid, KBr):
754, 823, 1172, 1305, 1371, 1453, 1485, 1619, 2353, 1485, 1619, 2359,
2916, 3012, 3354, 3441 cmꢁ1. MS (ESI) m/z: 393 [(Mþ1)þ]. HRMS
(ESI) m/z: Calcd. for C26H25Nþ4 : 393.20707, Found: 393.20737.
4.1.10. (E)-1-(4-Methyl-3-((2-methyl-5-(naphthalen-1-yl)phenyl)
diazenyl)phenyl)naphthalen-2-ol 9
Argon was bubbled through a solution of Na2CO3 (123 mg) in
water (0.58 mL) for 10 min. Then, this solution was added to a
solution of 6 (a mixture of trans- and cis-azobenzene isomers)
(100 mg, 0.27 mmol), 2-methoxy-1-naphthaleneboronic acid 22
(136 mg, 0.68 mmol), and Pd(PPh3)4 (11.1 mg, 0.0096 mmol) in
degassed DME (1.2 mL) under an argon atmosphere. The reaction
mixture was refluxed for 24 h, and then the solvent was evaporated
under reduced pressure. Water was added to the residue, and the
reaction mixture extracted with dichloromethane; the extract was
dried (Na2SO4), and the solvent removed to give a residue which
was purified by silica gel column chromatography (ethyl acetate/
hexane ¼ 1/10) to provide 23 as a mixture of trans- and cis-azo-
benzene isomers (120 mg, 85%), an orange solid.
1H NMR (400 MHz, CDCl3)
d (ppm): 2.61 (s, 6H, CH3), 2.86 (s, 6H,
CH3), 6.68 (dd, J ¼ 2.6, 8.2 Hz, 2H, AreH), 6.90 (d, J ¼ 2.5 Hz, 2H,
AreH), 7.14 (d, J ¼ 8.2 Hz, 2H, AreH). 13C NMR (400 MHz, CDCl3)
d
(ppm): 16.6, 31.0, 99.0, 116.0, 127.3, 131.7, 148.0, 151.6. IR (KBr):
3447, 3295, 3077, 3040, 3012, 2978, 2911, 2801, 2366, 2344, 1609,
1516, 1450,1397, 1256, 1171, 1121, 860, 814 cmꢁ1. MS (ESI) m/z: 269
[(M þ H)þ]. HRMS (ESI) m/z: Calcd. for C16H21Nþ4 [(M þ H)þ]:
269.1766, Found: 269.1761.
To a solution of 23 (100 mg, 0.19 mmol) in CH2Cl2 (0.76 mL) was
added a 17% boron tribromide solution in dichloromethane
4.1.9. (E)-30,3000-(Diazene-1,2-diyl)bis(40-methyl-[1,10-biphenyl]-2-
amine) 8
(0.57 mL) at 0 ꢀC. The solution was gradually warmed to room
temperature and then stirred overnight. Then water and sat. aq.
NaHCO3 were added to quench the reaction. The reaction mixture
was extracted with dichloromethane, the combined organic layers
were dried (Na2SO4), filtered, and the solvent removed. The residue
was purified by silica gel column chromatography (hexane/
EtOAc ¼ 5/1) to afford 9 as a mixture of trans- and cis-azobenzene
isomers (86 mg, 92%) which on being warmed at 50 ꢀC overnight to
afforded the pure trans-azobenzene isomer 9.
A biphasic reaction mixture of Oxone® (13.25 g, 21.5 mmol) in
water (130 mL) was stirred with a solution of 5-bromo-2-
methylaniline 18 (2.0 g, 10.75 mmol) in dichloromethane (32 mL)
at room temperature for 3.5 h. The organic layer was then separated
and the aqueous layer was extracted with CH2Cl2. The combined
organic layers were washed with 1 N HCl, sat. aq. NaHCO3, water,
and brine. Then, the organic phase was dried over Na2SO4, filtered,
and evaporated under reduced pressure to give the nitroso com-
pound 19 which was used in the next step without further
purification.
5-Bromo-2-methylaniline 18 (2.2 g, 11.1 mmol) was added to the
solution of the crude 19 in acetic acid (50 mL) and the reaction
mixture stirred at room temperature for 25 h. The precipitate
formed was collected by filtration, washed with water and 1 N
AcOH (132 mL), and dried under reduced pressure to afford 5,50-
dibromo-2,20-dimethylazobenzene 20 (2.2 g, 53%) as an orange
solid.
1H NMR (400 MHz, CDCl3)
d (ppm): 2.76 (s, 6H), 5.17 (d,
J ¼ 2.3 Hz, 2H), 7.28 (d, J ¼ 8.9 Hz, 2H), 7.33e7.38 (m, 4H), 7.41e7.48
(m, 4H), 7.54 (d, J ¼ 7.7 Hz, 2H), 7.2 (d, J ¼ 1.5 Hz, 2H), 7.83 (d,
J ¼ 9.1 Hz, 2H). 13C NMR (400 MHz, CDCl3)
d (ppm): 17.6,117.4, 118.4,
120.4, 123.4, 124.6, 126.6, 128.1, 128.9, 129.6, 132.3, 132.7,
133.283 133.7, 138.7, 150.3, 151.6. IR (neat): 3536, 3364, 3057, 2359,
1938, 1622, 1598, 1496, 1465, 1434, 1390, 1334, 1271, 1227, 1214,
1196, 1146, 836, 816, 752, 743 cmꢁ1. MS (ESI) m/z: 495 [(M þ H)þ];
HRMS (ESI): Calcd. for C34H27N2Oþ2 : 495.2067, Found: 495.2064.
Argon was bubbled through a biphasic mixture of toluene
(26 mL), ethanol (2 mL), aqueous sodium carbonate (2 M, 1 mL) for
40 min in an ice bath. The mixture of trans- and cis-azobenzene
isomers 20 (200 mg, 0.54 mmol), (2-aminophenyl)boronic acid 21
(222 mg, 1.62 mmol), and Pd(PPh3)4 (37 mg, 6 mol%) were added to
another two-necked flask, dried, and placed under argon atmo-
sphere. The above Na2CO3 solution was added to the flask, and the
reaction mixture was heated under reflux at 100 ꢀC for 2 h. The
reaction mixture was then extracted with ethyl acetate; the solvent
was removed under reduced pressure to produce crude 8 which
was suspended in methanol and filtered. The filter was washed
with CH2Cl2 to provide red-black solid and orange filtrate. The
solvent of the filtrate was removed under reduced pressure. The
residue was passed through a short pad of silica gel prepared with
dichloromethane to obtain 8 as mixture of trans- and cis-azo-
benzene isomers (189 mg, 0.48 mmol, 89%) which when warmed at
50 ꢀC overnight afforded the pure trans-azobenzene isomer 8.
4.1.11. (E)-1-(3-((5-(Diphenylphosphaneyl)-2-methylphenyl)
diazenyl)-4-methylphenyl)naphthalen-2-ol 10
5-Bromo-2-methylaniline 18 (1.86 g, 10.0 mmol) was converted
to the corresponding nitroso compound 19 as described is section
4.1.9 above.
5-Iodo-2-methylaniline 24 (1.66 g, 7.1 mmol) was added to the
solution of the crude nitroso product 19 in acetic acid-EtOAc (2:1,
102 mL) at room temperature under argon. After stirring overnight,
a precipitate formed in the reaction mixture was collected by
filtration, washed with hexane, and dried under reduced pressure
to afford 5-bromo-50-iodo-2,20-dimethylazobenzene 25 (2.14 g,
73%) as an orange solid.
A solution of the iodoazo compound 25 (166 mg, 0.4 mmol),
diphenylphosphine (82
(64.6 mg, 0.32 mmol), palladium diacetate (9.06 mg, 0.04 mmol),
triethylamine (89 L, 0.64 mmol) and acetonitrile (7.2 mL) in a
mL, 0.48 mmol), diphenylphosphine oxide
m
1H NMR (400 MHz, CDCl3)
d
(ppm): 2.74 (s, 6H, CH3), 3.79 (s, 4H,
sealed test tube was stirred for 12.5 h at 80 ꢀC in an argon atmo-
sphere. The reaction mixture was evaporated to give crude product
26 which was purified by silica gel column chromatography (hex-
ane/EtOAc ¼ 1/2) to afford the azophosphine oxide 26 as a mixture
NH2), 6.78e6.86 (m, 4H, AreH), 7.14e7.20 (m, 4H, AreH), 7.40 (d,
J ¼ 7.8 Hz, 2H, AreH), 7.47 (dd, J ¼ 1.8, 7.8 Hz, 2H, AreH), 7.47 (d,
J ¼ 1.8 Hz, 2H, AreH). [cis] 2.44 (s, 6H, CH3), 3.19 (s, 4H, NH2), 6.32
9

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
of trans- and cis-azobenzene isomers (910 mg, 77%).
0.67 mmol) in dry CH2Cl2 (1.3 mL) at 0 ꢀC under an argon atmo-
sphere. The solution was gradually warmed to room temperature
and stirred for 5 h. The solvent of the reaction mixture was evap-
orated to give a residue which was washed with CHCl3 to provide 11
as a mixture of trans- and cis-azobenzene isomers (237 mg, 50%), a
yellow solid which was warmed at 50 ꢀC overnight to afford the
pure trans-azobenzene isomer 11.
A solution of Na2CO3 (123.4 mg) in water (0.58 mL) prepared as
in section 4.1.9 was added to a solution of the azo diphenylphos-
phine oxide 26 (1.47 g, 3.0 mmol), 2-methoxy-1-
naphthaleneboronic acid 22 (606 mg, 3 mmol), and Pd(PPh3)4
(124 mg, 0.108 mmol, 3.6 mol%) in degassed DME (1.2 mL) under an
argon atmosphere. The reaction mixture was heated under reflux
for 24 h, and then the solvent was removed under reduced pres-
sure. Water was added to the residue, and the reaction mixture
then extracted with dichloromethane, dried (Na2SO4), filtered, and
the solvent removed. The residue was purified by silica gel column
chromatography (ethyl acetate/hexane ¼ 1/2) to provide a pure
mixture of trans- and cis-azobenzene isomers 27 (1.85 g, quant.) as
an orange solid.
A boron tribromide solution in dichloromethane (1.0 M, 1.5 mL,
1.5 mmol) was added to the mixture of trans- and cis-azobenzenes
27 (566 mg,1.0 mmol) in dichloromethane (4.0 mL) at 0 ꢀC under an
argon atmosphere. The reaction mixture was gradually warmed to
room temperature, stirred overnight, quenched with water (4 mL)
and sat. aq. NaHCO3 and then extracted with dichloromethane. The
combined organic extracts were dried (Na2SO4), filtered, and
concentrated. The residue was purified by silica gel column chro-
matography (hexane/EtOAc ¼ 1/3) to afford 28 as a mixture of
trans- and cis-azobenzene isomers (388 mg, 70%).
1H NMR (400 MHz, CDCl3)
d (ppm): 2.64 (s, 3H), 2.71 (s, 3H), 2.96
(s, 6H), 6.07 (d, J ¼ 2.2 Hz, 1 H), 6.15 (dd, J ¼ 2.2, 6.1 Hz,1 H), 7.33 (m,
3 H), 7.43 (d, J ¼ 8.1 Hz, 1 H), 7.54 (d, J ¼ 2.3 Hz, 1H), 7.60 (d,
J ¼ 2.3 Hz, 1H), 7.68 (s, 1H), 7.85 (d, J ¼ 6.1 Hz, 1H), 8.02 (s, 1H). 13
C
NMR (400 MHz, DMSO) d (ppm): 16.3, 16.6, 91.0, 100.9, 104.1, 110.6,
116.8, 121.6, 121.9, 123.3, 124.6, 126.6, 129.1, 129.54, 129.9, 130.2,
130.5, 131.2, 131.5, 134.4, 137.3, 141.1, 141.9, 147.0, 150.2, 150.2, 155.4,
156.6, 179.9. IR (solid, ATR): 681, 884, 986, 1136, 1177, 1278, 1385,
1500, 1667, 2359, 2819, 2866, 2924, 3024, 3298, 3425 cmꢁ1. HRMS
(ESI): Calcd. for C30H28N7F6Sþ [(M þ H)þ]: 632.20256, Found:
632.20243.
4.1.13. (E)-1-(3,5-bisTtrifluoromethyl)phenyl)-3-(3-((5-((4-
(dimethylamino)pyridin-2-yl)amino)-2-methylphenyl)diazenyl)-4-
methylphenyl)urea 12
1-Isocyanato-3,5-bis(trifluoromethyl)benzene 32 (53.0
mL,
0.31 mmol) was added dropwise to a solution of the DMAP trans-
cis- isomers 30 (100 mg, 0.28 mmol) in dry CH2Cl2 (0.54 mL) at 0 ꢀC
under an argon atmosphere. The solution was gradually warmed to
room temperature and was stirred for 24 h. Then, the solvent of the
reaction mixture was evaporated, and the residue was purified by
A solution of the trans- and cis-azobenzene mixture 28 (276 mg,
0.5 mmol, 1.0 equiv.), diphenyl phosphate (10 mg, 0.04 mmol, 0.08
equiv.), and trichlorosilane (320
mL, 2 mmol, 4.0 equiv.) in 1,4-
dioxane (2 mL) in a sealed test tube under argon was stirred and
heated at 110 ꢀC for 48 h. Then, methanolic KOH (5.0 mL) was added
slowly at 0 ꢀC to the reaction mixture which was stirred vigorously
for 3 h at room temperature; water (3 mL) was added and the
mixture was extracted by ethyl acetate. The organic phase was
washed by aqueous hydrochloric acid (1 N, 5 mL) and sat. aq.
NaHCO3 (5 mL), dried (Na2SO4), filtered, and the solvent removed
under reduced pressure. The residue was purified by silica gel
column chromatography (hexane/CH2Cl2 ¼ 1/1) to afford 10 as a
mixture of trans- and cis-azobenzene isomers (66.25 mg, 24%) as an
orange solid which was warmed at 50 ꢀC overnight to produce the
pure trans-azobenzene isomer 10.
silica
gel
column
chromatography
(CHCl3/MeOH/
triethylamine ¼ 10/1/a few drops) to provide 12 as a mixture of
trans- and cis-azobenzene isomers (71.2 mg, 0.12 mmol, 42%) as a
yellow solid which was warmed at 50 ꢀC overnight to afford the
pure trans-azobenzene isomer 12.
1H NMR (400 MHz, CDCl3)
d (ppm): 2.43 (s, 3H), 2.45 (s, 3H), 3.01
(s, 6H), 5.98 (s, 1H), 6.20 (dd, J ¼ 2.17, 6.37 Hz, 1H), 7.06 (s, 2H), 7.12
(d, J ¼ 8.20 Hz, 1H), 7.30 (d, J ¼ 6.70 Hz, 1H), 7.45 (s, 2H), 7.81 (d,
J ¼ 6.30 Hz, 1H), 7.85 (s, 2H). 13C NMR (400 MHz, DMSO)
d (ppm):
16.5, 90.9, 101.0, 104.7, 105.7, 114.3, 118.0, 121.6, 121.9, 121.9, 124.7,
127.4, 129.1, 130.2, 130.5, 130.8, 131.2, 131.6, 131.6, 137.6, 140.8, 141.8,
146.4, 150.3, 150.4, 152.5, 155.5, 156.2. IR (solid, ATR): 893, 954, 987,
1011, 1064, 1128, 1176, 1281, 1386, 1448, 1472, 1499, 1566, 1604,
1643, 1714, 2324, 2351, 2872, 2925, 3273 cmꢁ1. HRMS (ESI): Calcd.
for C34H27N2Oþ2 [(M þ H)þ]: 616.22540, Found: 616.22509.
1H NMR (400 MHz, CDCl3)
7.28e7.35 (m, 3H), 7.40e7.58 (m,10H), 7.65e7.73 (m, 6H), 7.79e7.84
(m, 3H). 13C NMR (400 MHz, CDCl3)
(ppm): 17.4, 17.8, 117.5, 118.2,
d (ppm): 2.66 (s, 3H), 2.68 (s, 3H),
d
120.2, 120.3, 123.4, 124.5, 126.6, 128.1, 128.5, 128.6, 128.9, 129.6,
132.0, 132.1, 132.2, 132.68, 134.0, 150.3, 151.2. IR (neat): 3054, 1624,
1512,1496,1435,1345,1287,1174,1119,1106, 821, 748, 723, 705, 691,
546, 523 cmꢁ1. MS (ESI) m/z: 537.20 [(M þ H)þ]. HRMS (ESI): Calcd.
for C36H30N2OPþ [(M þ H)þ]: 537.20914, Found: 537.20903.
4.1.14. (E)-1-(3,5-bis(Trifluoromethyl)phenyl)-3-(4-methyl-3-((2-
methyl-5-(pyridin-2-ylamino)phenyl)diazenyl)phenyl)thiourea 13
A solution of 6 (200.0 mg, 0.83 mmol), 2-iodo-pyridine 33
(88 mL, 0.83 mmol), Pd2dba3 (73.2 mg, 0.08 mmol, 10 mol%), 1,3-
4.1.12. (E)-1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-((5-((4-
(dimethylamino)pyridin-2-yl)amino)-2-methylphenyl)diazenyl)-4-
methylphenyl)thiourea 11
bis(diphenylphosphino)propane (57.8 mg, 0.14 mmol), and so-
dium tert-butoxide (160 mg, 1.66 mmol) in degassed dry toluene
(6.6 mL) was heated at 105 ꢀC for 2 h under an argon atmosphere.
Then, the reaction mixture was cooled to room temperature and
filtered through a pad of celite which was rinsed with dichloro-
methane. The filtrate was washed with water and dried (Na2SO4),
and evaporated under reduced pressure to give a residue which
was purified by silica gel column chromatography (MeOH/
CHCl3 ¼ 1/20) to afford 34 (82.0 mg, 0.38 mmol, 46%).
A
solution of
6
(120 mg, 0.5 mmol), 2-iodo-4-
dimethylaminopyridine 29 (124.2 mg, 0.5 mmol), Pd2dba3
(49.8 mg, 0.05 mmol), 1,3-bis(diphenylphosphino)propane
(37.1 mg, 0.09 mmol) and sodium tert-butoxide (96.1 mg, 1.0 mmol)
in degassed toluene was heated at 105 ꢀC for 2 h under argon. The
reaction mixture was cooled to room temperature and was filtered
through a pad of celite which was washed with CH2Cl2. The filtrate
was washed with water, dried (Na2SO4), and evaporated under
reduced pressure to give a residue which was purified by silica gel
column chromatography (CHCl3/MeOH ¼ 20/1) to give DMAP azo
analogue 30 as a mixture of trans- and cis-azobenzene isomers
(83.7 mg, 47%) as a red solid.
3,5-bis(Trifluoromethyl)phenyl isothiocyanate 31 (232
mL,
1.27 mmol) was added dropwise to a solution of 34 as a mixture of
trans- and cis-azobenzene isomers (405 mg, 0.67 mmol) in dry
dichloromethane (1.3 mL) at 0 ꢀC under argon. The solution was
gradually warmed to room temperature and was stirred for 5 h.
Then, the solvent of the reaction mixture was evaporated, and the
residue was washed with CHCl3 to provide 13 as a mixture of trans-
and cis-azobenzene isomers (432 mg, 0.73 mmol, 57%) as a yellow
3,5-bis(Trifluoromethyl)phenyl isothiocyanate 31 (134
mL,
0.73 mmol) was added dropwise to a solution of 30 (240 mg,
10

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
solid which was warmed at 50 ꢀC overnight to afford the pure trans-
Pure Chemical, Osaka, Japan) supplemented with 15% BS and
azobenzene isomer.
penicillin-streptomycin (100 U/mL and 100 mg/mL, respectively) for
1H NMR (400 MHz, DMSO)
d
(ppm): 2.58 (s, 3H), 2.71 (s, 3H),
GM05659 cells. The cells were cultured at 37 ꢀC under 5% CO2. In
cases of experiments under nutrient-deprived conditions, the cells
were cultured in nutrient-deprived medium (NDM), which is in-
clusive of electrolytes and vitamins but exclusive of glucose, amino
acids, and serum. NDM was prepared as described previously [6].
6.75 (t, J ¼ 5.97 Hz, 1H), 6.83 (d, J ¼ 8.40 Hz, 1H), 7.31 (d, J ¼ 8.41 Hz,
1H), 7.44 (d, J ¼ 8.28 Hz, 1H), 7.57 (m, 2H), 7.72 (s, 1H), 7.81 (m, 2H),
8.03 (d, J ¼ 1.89 Hz, 1H), 8.15 (d, J ¼ 3.74 Hz, 1H), 8.25 (s, 2H), 9.15 (s,
1H), 10.31 (brs, 2H). 13C NMR (400 MHz, DMSO)
d (ppm): 16.8, 17.1,
104.9, 111.1, 111.3, 114.8, 117.3, 122.1, 122.4, 123.8, 125.1, 127.2, 130.3,
130.7, 131.8, 132.0, 135.0, 137.7, 137.9, 140.9, 142.4, 147.6, 150.7, 156.3,
180.4. IR (solid, ATR): 702, 890, 1126, 1278, 1385, 1530, 1602, 2322,
2351, 2372, 2972, 3224 cmꢁ1. HRMS (ESI): Calcd for C28H22N6F6Sþ
[(M þ H)þ]: 589.16036, Found: 589.16024.
4.2.2. Cell viability assay
Cell viability was assessed by MTT assay. All test samples were
dissolved in dimethyl sulfoxide (DMSO). Cells were seeded at
1.0 ꢃ 104 (PANC-1) or 5.0 ꢃ 103 (GM05659) cells/well in a 96-well
plate and precultured for 24 h. After preculture, the cells were
washed with phosphate-buffered saline (PBS) and exposed to serial
dilutions of test samples under either normal conditions (DMEM)
or nutrient-deprived conditions (NDM). The final concentration of
4.1.15. (E)-1-(3,5-bis(Trifluoromethyl)phenyl)-3-(4-methyl-3-((2-
methyl-5-((4-(pyrrolidin-1-yl)pyridin-2-yl)amino)phenyl)diazenyl)
phenyl)thiourea 14
A solution of 6 (500 mg, 2.08 mmol), 2-iodo-4-(pyrrolidin-1-yl)
pyridine 35 (571 mg, 2.08 mmol), Pd2dba3 (190 mg, 0.21 mmol),1,3-
bis(diphenylphosphino)propane (154 mg, 0.37 mmol), and sodium
tert-butoxide (400 mg, 4.16 mmol) in degassed dry toluene (27 mL)
was heated at 105 ꢀC for 15 h under an argon atmosphere. Then, the
reaction mixture was cooled to room temperature and filtered
through a pad of celite which was rinsed with dichloromethane.
The filtrate was washed with water and dried (Na2SO4) and the
solvent evaporated under reduced pressure to give a residue which
was purified by silica gel column chromatography (MeOH/
AcOEt ¼ 1/4) to afford the pyrrolidine azo analogue 36 (834 mg,
0.88 mmol, 42%).
DMSO was 0.1%. After 24 or 72 h of incubation, 20 mL of MTT so-
lution (2.0 mg/mL) was added to wells, and cells were incubated at
37 ꢀC for 4 h. Then, the culture medium was removed and crystals of
formazan were dissolved with DMSO (200 mL/well). Absorbance at
540 nm was measured by using the FilterMax F5 microplate reader
(Molecular Devices, San Jose, CA, USA). Results were expressed as
percentage of the values of control cells (mean SEM).
4.2.3. Cell cycle analysis by using flow cytometry
Cell cycle of PANC-1 cells was determined by flow cytometric
analysis after staining with propidium iodide (PI). PANC-1 cells
were seeded at 2.0 ꢃ 105 cells/well in a 6-well plate and precul-
tured for 24 h. The cells were washed with PBS and exposed to test
samples under either normal conditions (DMEM) or nutrient-
deprived conditions (NDM). After 24 h of incubation, the cells
were trypsinized and washed twice with PBS. The cells were fixed
in ice-cold 70% ethanol and placed overnight at ꢁ20 ꢀC. The fixed
cells were washed with PBS containing 2% BS, and then 0.25 mg/mL
RNAse (Nacalai Tesque Inc, Kyoto, Japan) was added and incubated
at 37 ꢀC for 15 min. The cells were stained with PI (FUJIFILM Wako
Pure Chemical, Osaka, Japan) solution at a final concentration of
3,5-bis(Trifluoromethyl)phenyl isothiocyanate 31 (73.1
mL,
0.39 mmol) was added dropwise to a solution of a mixture of trans-
and cis-azobenzene isomers of 36 (150 mg, 0.39 mmol) in dry
CH2Cl2 (3.9 mL) at 0 ꢀC under an argon atmosphere. The solution
was gradually warmed to room temperature and was stirred for 5 h.
Then, removal of the solvent gave a residue which was purified by
silica gel column chromatography (MeOH/CHCl3 ¼ 1/6) to provide
14 as a mixture of trans- and cis-azobenzene isomers (101 mg,
0.15 mmol, 40%) as an orange solid which was warmed at 50 ꢀC
overnight to afford the pure trans-azobenzene isomer 14.
50 mg/mL and finally analyzed by using the FACSCelesta cytometer
and FACSDiva software, ver. 9.0 (Becton Dickinson, Franklin Lakes,
NJ).
1H NMR (400 MHz, CDCl3)
d (ppm): 1.99 (m, 4H, CH2), 2.64 (s,
3H, CH3), 2.71 (s, 3H, CH3), 3.28 (m, 4H, CH2), 5.97 (d, J ¼ 2.1 Hz, 1H,
AreH), 6.04 (dd, J ¼ 2.1, 6.0 Hz, 1H, AreH), 7.08 (bs, 1H, NH),
7.28e7.36 (m, 3H, AreH), 7.44 (d, J ¼ 8.0 Hz, 1H, AreH), 7.55 (d,
J ¼ 2.2 Hz, 1H, AreH), 7.59 (d, J ¼ 2.2 Hz, 1H, AreH), 7.68 (s, 1H,
AreH), 7.86 (d, J ¼ 6.0 Hz, 1H, AreH), 8.02 (s, 2H, AreH). 13C NMR
Declaration of competing interest
None of the authors have any conflict of interest arising from
this work.
(400 MHz, DMSO) d (ppm): 16.3, 16.5, 46.6, 90.9, 101.3, 104.0, 110.6,
121.6,121.9,123.3,124.6,129.0,129.9,130.2,131.2,141.2,147.0,150.0,
152.6,156.4. IR (solid, ATR): 1179, 1209,1232, 1274,1330, 1354, 1387,
1484, 1506, 1529, 1557, 1607, 1641, 2325, 2351, 2373, 2840, 2874,
2974, 3088, 3296 cmꢁ1. HRMS (EI): Calcd. for C32H30N7F6Sþ
[(M þ H)þ]: 658.21821, Found: 658.21751.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2021.132077.
4.2. Biological assay
References
4.2.1. Cell culture
[1] J.D. Mizrahi, R. Surana, J.W. Valle, R.T. Shroff, Pancreatic cancer, Lancet 395
(2020) 2008e2020, https://doi.org/10.1016/S0140-6736(20)30974-0.
[2] P. Rawla, T. Sunkara, V. Gaduputi, Epidemiology of pancreatic cancer: global
trends, etiology and risk factors, World J. Oncol. 10 (2019) 10e27, https://
doi.org/10.14740/wjon1166.
[3] P. Sarantis, E. Koustas, A. Papadimitropoulou, A.G. Papavassiliou,
M.V. Karamouzis, Pancreatic ductal adenocarcinoma: treatment hurdles, tu-
mor microenvironment and immunotherapy, World J. Gastrointest. Oncol. 12
(2020) 173e181, https://doi.org/10.4251/wjgo.v12.i2.173.
[4] V. Vaccaro, I. Sperduti, M. Milella, FOLFIRINOX versus gemcitabine for meta-
static pancreatic cancer, N. Engl. J. Med. 365 (2011) 768e769, https://doi.org/
10.1056/NEJMc1107627.
[5] S. Li, H.X. Xu, C.T. Wu, W.Q. Wang, W. Jin, H.L. Gao, H. Li, S.R. Zhang, J.Z. Xu,
Z.H. Qi, Q.X. Ni, X.J. Yu, L. Liu, Angiogenesis in pancreatic cancer: current
research status and clinical implications, Angiogenesis 22 (2019) 15e36,
Human pancreatic cancer cell line PANC-1 was provided by Dr.
Yukiko Kurashima (Hokuriku University). Human normal fibroblast
cell line GM05659 was obtained from Coriell Institute for Medical
Research (Camden, NJ, USA). Cells were cultured in the following
media: Dulbecco’s modified Eagle’s medium (DMEM; FUJIFILM
Wako Pure Chemical, Osaka, Japan) supplemented with 10% heat-
inactivated bovine serum (BS; Thermo Fischer Scientific, MA,
USA), non-essential amino acids (FUJIFILM Wako Pure Chemical,
Osaka, Japan), and penicillin-streptomycin (100 U/mL and 100 mg/
mL, respectively; Thermo Fischer Scientific, MA, USA) for PANC-
1 cells; Eagle’s minimum essential medium (EMEM; FUJIFILM Wako
11

K. Shinzawa, D. Kageta, R.J. Nash et al.
Tetrahedron 85 (2021) 132077
https://doi.org/10.1007/s10456-018-9645-2.
echogenic polymersomes for drug delivery to solid tumors, Chem. Eur J. 24
(2018) 12490e12494, https://doi.org/10.1002/chem.201802229.
[21] W.A. Velema, W. Szymanski, B.L. Feringa, Photopharmacology: beyond proof
of principle, J. Am. Chem. Soc. 136 (2014) 2178e2191, https://doi.org/
10.1021/ja413063e.
[22] M.J. Fuchter, On the promise of photopharmacology using photoswitches: a
medicinal chemist’s perspective, J. Med. Chem. 63 (2020) 11436e11447,
https://doi.org/10.1021/acs.jmedchem.0c00629.
[23] T. Imahori, R. Yamaguchi, S. Kurihara, Azobenzene-tethered bis(trityl alcohol)
as a photoswitchable cooperative acid catalyst for Morita-Baylis-Hillman re-
actions, Chem. Eur J. 18 (2012) 10802e10807, https://doi.org/10.1002/
chem.201201383.
[6] K. Izuishi, K. Kato, T. Ogura, T. Kinoshita, H. Esumi, Remarkable tolerance of
tumor cells to nutrient deprivation: possible new biochemical target for
cancer therapy, Canc. Res. 60 (2000) 6201e6207.
[7] K. Kato, T. Ogura, A. Kishimoto, Y. Minegishi, N. Nakajima, M. Miyazaki,
H. Esumi, Critical roles of AMP-activated protein kinase in constitutive
tolerance of cancer cells to nutrient deprivation and tumor formation,
Oncogene 21 (2002) 6082e6090, https://doi.org/10.1038/sj.onc.1205737.
[8] J. Ueda, S. Athikomkulchai, R. Miyatake, I. Saiki, H. Esumi, S. Awale,
(þ)-Grandifloracin, an antiausterity agent, induces autophagic PANC-1
pancreatic cancer cell death, Drug Des. Dev. Ther. 8 (2014) 39e47, https://
doi.org/10.2147/DDDT.S52168.
[9] M. Piffoux, E. Eriau, P.A. Cassier, Autophagy as
a
therapeutic target in
[24] M. Lieber, J. Mazzetta, W. Nelson-Rees, M. Kaplan, G. Todaro, Establishment of
pancreatic cancer, Br. J. Canc. (2020) 1e12, https://doi.org/10.1038/s41416-
020-01039-5.
[10] S.E. Kim, H.J. Park, H.K. Jeong, M.J. Kim, M. Kim, O.N. Bae, S.H. Baek, Autophagy
sustains the survival of human pancreatic cancer PANC-1 cells under extreme
nutrient deprivation conditions, Biochem. Biophys. Res. Commun. 463 (2015)
205e210, https://doi.org/10.1016/j.bbrc.2015.05.022.
[11] A.M. Tawila, S. Sun, M.J. Kim, A.M. Omar, D.F. Dibwe, J. Ueda, N. Toyooka,
S. Awale, Highly potent antiausterity agents from callistemon citrinus and
their mechanism of action against the PANC-1 human pancreatic cancer cell
line, J. Nat. Prod. 83 (2020) 2221e2232, https://doi.org/10.1021/
acs.jnatprod.0c00330.
a continuous tumor-cell line (PANC-1) from a human carcinoma of the
exocrine pancreas, Int. J. Canc. 15 (1975) 741e747, https://doi.org/10.1002/
ijc.2910150505.
[25] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65
(1983) 55e63, https://doi.org/10.1016/0022-1759(83)90303-4.
[26] N. Yoshikawa, Y. Saito, H. Manabe, T. Nakaoka, R. Uchida, R. Furukawa,
T. Muramatsu, Y. Sugiyama, M. Kimura, H. Saito, Glucose depletion enhances
the stem cell phenotype and gemcitabine resistance of cholangiocarcinoma
organoids through AKT phosphorylation and reactive oxygen species, Cancers
11 (2019), https://doi.org/10.3390/cancers11121993.
[12] S. Fujii, S. Mitsunaga, M. Yamazaki, T. Hasebe, G. Ishii, M. Kojima, T. Kinoshita,
T. Ueno, H. Esumi, A. Ochiai, Autophagy is activated in pancreatic cancer cells
and correlates with poor patient outcome, Canc. Sci. 99 (2008) 1813e1819,
https://doi.org/10.1111/j.1349-7006.2008.00893.x.
[13] S. Awale, J. Lu, S.K. Kalauni, Y. Kurashima, Y. Tezuka, S. Kadota, H. Esumi,
Identification of arctigenin as an antitumor agent having the ability to elim-
inate the tolerance of cancer cells to nutrient starvation, Canc. Res. 66 (2006)
1751e1757, https://doi.org/10.1158/0008-5472.CAN-05-3143.
[14] M. Ikeda, A. Sato, N. Mochizuki, K. Toyosaki, C. Miyoshi, R. Fujioka,
S. Mitsunaga, I. Ohno, Y. Hashimoto, H. Takahashi, H. Hasegawa, S. Nomura,
R. Takahashi, S. Yomoda, K. Tsuchihara, S. Kishino, H. Esumi, Phase I trial of
GBS-01 for advanced pancreatic cancer refractory to gemcitabine, Canc. Sci.
107 (2016) 1818e1824, https://doi.org/10.1111/cas.13086.
[15] J. Lu, S. Kunimoto, Y. Yamazaki, M. Kaminishi, H. Esumi, D. Kigamicin, A novel
anticancer agent based on a new anti-austerity strategy targeting cancer cells’
tolerance to nutrient starvation, Canc. Sci. 95 (2004) 547e552, https://doi.org/
10.1111/j.1349-7006.2004.tb03247.x.
[27] B. Bhattacharya, M.F. Mohd Omar, R. Soong, The Warburg effect and drug
resistance, Br. J. Pharmacol. 173 (2016) 970e979, https://doi.org/10.1111/
bph.13422.
[28] J. Crawford, D.C. Dale, G.H. Lyman, Chemotherapy-induced neutropenia: risks,
consequences, and new directions for its management, Cancer 100 (2004)
228e237, https://doi.org/10.1002/cncr.11882.
[29] D. Schiff, P.Y. Wen, M.J. van den Bent, Neurological adverse effects caused by
cytotoxic and targeted therapies, Nat. Rev. Clin. Oncol. 6 (2009) 596e603,
https://doi.org/10.1038/nrclinonc.2009.128.
[30] C. Dumontet, M.A. Jordan, Microtubule-binding agents: a dynamic field of
cancer therapeutics, Nat. Rev. Drug Discov. 9 (2010) 790e803, https://doi.org/
10.1038/nrd3253.
[31] A. Krishan, Rapid flow cytofluorometric analysis of mammalian cell cycle by
propidium iodide staining, J. Cell Biol. 66 (1975) 188e193, https://doi.org/
10.1083/jcb.66.1.188.
[32] A. Recasens, L. Munoz, Targeting cancer cell dormancy, Trends Pharmacol. Sci.
40 (2019) 128e141, https://doi.org/10.1016/j.tips.2018.12.004.
[33] A. Kato, J. Koyama, K. Shinzawa, S. Imaeda, I. Adachi, R.J. Nash, G.W.J. Fleet,
[16] H. Esumi, J. Lu, Y. Kurashima, T. Hanaoka, Antitumor activity of pyrvinium
pamoate, 6-(dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)
ethenyl]-1-methyl-quinolinium pamoate salt, showing preferential cytotox-
icity during glucose starvation, Canc. Sci. 95 (2004) 685e690, https://doi.org/
10.1111/j.1349-7006.2004.tb03330.x.
M. Shintani, C. Takeuchi, F. Ishikawa, Ginnalin
B induces differentiation
markers and modulates the proliferation/differentiation balance via the
upregulation of NOTCH1 in human epidermal keratinocytes, Bioorg. Med.
Chem. 27 (2019) 2172e2180, https://doi.org/10.1016/j.bmc.2019.04.008.
[34] N. Yamane, M. Makino, N. Kaibara, S-phase accumulation precedes apoptosis
induced by preoperative treatment with 5-fluorouracil in human colorectal
carcinoma cells, Cancer 85 (1999) 309e317, https://doi.org/10.1002/(SICI)
1097-0142(19990115)85:2<309::AID-CNCR7>3.0.CO;2-X.
[35] J.A. Hensel, T.W. Flaig, D. Theodorescu, Clinical opportunities and challenges in
targeting tumour dormancy, Nat. Rev. Clin. Oncol. 10 (2013) 41e51, https://
doi.org/10.1038/nrclinonc.2012.207.
[36] While trans-azobenzene derivatives are generally thermally stable, cis-
azobenzene derivatives induce thermal isomerization to the trans-isomer.
Thus, warming the mixture of the cis- and trans-azobenzene isomers at 50
ꢀC provides the pure trans-azobenzene isomers through isomerization of the
cis-azobenzene isomer to the trans-isomer. For thermal isomerization of cis-
azobenene to trans-azobenzene, see H.M.D. Bandara, S.C. Burdette, Photo-
isomerization in different classes of azobenzene, Chem. Soc. Rev. 41 (2012)
1809e1825.
[17] A.M. Tawila, S. Sun, M.J. Kim, A.M. Omar, D.F. Dibwe, J. ya Ueda, N. Toyooka,
S. Awale, Chemical constituents of Callistemon citrinus from Egypt and their
antiausterity activity against PANC-1 human pancreatic cancer cell line, Bio-
org. Med. Chem. Lett 30 (2020) 127352, https://doi.org/10.1016/
j.bmcl.2020.127352.
€
[18] B.E. Alexander, S. Sun, M.J. Palframan, G. Kociok-Kohn, D.F. Dibwe,
S. Watanabe, L. Caggiano, S. Awale, S.E. Lewis, Sidechain diversification of
grandifloracin allows identification of analogues with enhanced anti-austerity
activity against human PANC-1 pancreatic cancer cells, ChemMedChem 15
(2020) 125e135, https://doi.org/10.1002/cmdc.201900549.
[19] P. Kulkarni, M.K. Haldar, P. Katti, C. Dawes, S. You, Y. Choi, S. Mallik, Hypoxia
responsive, tumor penetrating lipid nanoparticles for delivery of chemo-
therapeutics to pancreatic cancer cell spheroids, Bioconjugate Chem. 27
(2016) 1830e1838, https://doi.org/10.1021/acs.bioconjchem.6b00241.
[20] P. Kulkarni, M.K. Haldar, F. Karandish, M. Confeld, R. Hossain, P. Borowicz,
K. Gange, L. Xia, K. Sarkar, S. Mallik, Tissue-penetrating, hypoxia-responsive
12