Hypoxia-Activated Prodrugs of PERK Inhibitors

Article abstract of DOI:10.1002/asia.201801826

Tumour hypoxia plays an important role in tumour progression and resistance to therapy. Under hypoxia unfolded proteins accumulate in the endoplasmic reticulum (ER) and this stress is relieved through the protein kinase R-like ER kinase (PERK) signalling

Full text of DOI:10.1002/asia.201801826

CHEMISTRY
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Accepted Article
Title: Hypoxia Activated Prodrugs of PERK Inhibitors
Authors: Lydia P. Liew, Dean C. Singleton, Way W. Wong, Gary J.
Cheng, Stephen M.F. Jamieson, and Michael Hay
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10.1002/asia.201801826
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Hypoxia Activated Prodrugs of PERK Inhibitors
Lydia P. Liew [a,b], Dean C. Singleton [a,b], Way W. Wong [a], Gary J. Cheng [a], Stephen M.F. Jamieson
[a,b,c] and Michael P. Hay* [a,b]
[a]
Dr L.P. Liew (0000-0002-9322-2010), Dr D.C. Singleton (0000-0002-2044-8078), Ms W.W. Wong, Mr G.J. Cheng (0000-0002-2700-2415), Dr S.M.F.
Jamieson (0000-0002-5485-9211), Assoc. Prof. M.P. Hay *(0000-0002-7087-9087)
Auckland Cancer Society Research Centre,
School of Medical Sciences,
Faculty of Medical and Health Sciences,
University of Auckland,
Private Bag 92019,
Auckland, New Zealand
E-mail: m.hay@auckland.ac.nz
[b]
[c]
Dr L.P. Liew, Dr D.C. Singleton, Dr S.M.F. Jamieson, Assoc. Prof. M.P. Hay
Maurice Wilkins Centre for Molecular Biodiscovery
University of Auckland
Symonds St
Auckland 1010 New Zealand
Dr S.M.F. Jamieson,
Department of Pharmacology and Clinical Pharmacology,
School of Medical Sciences,
Faculty of Medical and Health Sciences,
University of Auckland,
Private Bag 92019,
Auckland, New Zealand
Supporting information for this article is given via a link at the end of the document.
Abstract: Tumour hypoxia plays an important role in tumour
progression and resistance to therapy. Under hypoxia unfolded
proteins accumulate in the endoplasmic reticulum (ER) and this stress
is relieved through the protein kinase R-like ER kinase (PERK)
signalling arm of the unfolded protein response (UPR). Targeting the
UPR through PERK kinase inhibitors provides tumour growth
inhibition, but also elicits on-mechanism normal tissue toxicity.
Hypoxia presents a target for tumour-selective drug delivery using
hypoxia-activated prodrugs. We designed and prepared hypoxia-
hypoxic conditions, unfolded proteins accumulate in the ER,
producing ER stress and activation of the unfolded protein
response (UPR).[1] Consequently, the UPR can be considered as
one of the key oxygen sensors, along with the hypoxia-inducible
factors (HIF), which mediate adaptation to hypoxia.[2]
The UPR mediates both pro-survival and pro-apoptotic
cellular responses. Accumulation of unfolded proteins activates
three different sensors located in the lumen of the ER, protein
kinase
R
(PKR)-like
endoplasmic
reticulum
kinase
activated prodrugs of modified PERK inhibitors using
a
2-
(PERK/EIF2AK3), inositol-requiring protein α
(IRE1α) and
nitromidazole bioreductive trigger. The new inhibitors retained PERK
kinase inhibitory activity and the corresponding prodrugs were
strongly deactivated. The prodrugs were able to undergo
fragmentation following radiolytic reduction, or bioreduction in
HCT116 cells, to release their effectors, albeit inefficiently. We
examined the effects of the prodrugs on PERK signalling in hypoxic
HCT116 cells. This study has identified a 2-substituted nitroimidazole
carbamate prodrug with potential to deliver PERK inhibitors in a
hypoxia selective manner.
activating transcription factor-6 (ATF6), which control
downstream signal transduction pathways to regulate ER
stress.[3] The UPR acts to lower protein synthesis and reduce
nascent protein translocation to the ER to manage protein load in
the short term. The UPR also upregulates expression of
chaperones involved in protein folding and degradative pathways
to provide longer-term relief from ER stress.[2,4] Following these
initial adaptive responses, the later stages of the UPR seek to
restore basal levels of protein synthesis.[5] If ER stress remains
unresolved this phase of the UPR produces excessive levels of
oxidative stress resulting in the induction of cell death.
In the context of cancer, the activated UPR supports the
growth and survival of tumour cells within a hostile environment.[6]
Tolerance to hypoxia-induced ER stress is mediated primarily
through the PERK pathway.[7] PERK activation occurs when the
chaperone GRP78 dissociates from the luminal domain of the
protein, allowing PERK to oligomerise and autophosphorylate.[8,9]
Subsequent phosphorylation of Ser51 on cytoplasmic eukaryotic
initiation factor 2α (eIF2α) promotes preferential translation of the
transcription factor ATF4. ATF4 induces the transcription of
chaperones, antioxidants and autophagy promoting genes.[10]
Inhibitors of PERK phosphorylation of eIF2α have been
developed.[11-13] For example, GSK2606414 (1) (IC50 = 0.4 nM) is
Introduction
The endoplasmic reticulum (ER) provides an appropriate
environment for the synthesis and maturation of proteins. Within
the ER, protein folding occurs co-translationally, as well as post-
translationally, through the introduction of disulphide bonds by the
protein disulphide isomerase (PDI).[1] PDI participates in a redox
relay with ER oxidases, requiring a terminal electron acceptor to
support disulphide bond formation. While the role of oxygen as
the terminal electron acceptor in cells is unclear, the post-
translational folding of protein is oxygen-dependent.[1] Under
1
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10.1002/asia.201801826
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a first-in-class PERK inhibitor and its analogue GSK2656157 (2)
(IC50 = 0.8 nM) was subsequently developed as a preclinical
candidate.[11,14] Crystal structures of PERK with 1 and 2 bound
show both inhibitors binding similarly (Figure 1).[11,12] The amino-
pyrimidine motif anchors the inhibitors to the hinge binding region,
while the central indoline core orientates the aryl acetamide to a
lipophilic back pocket in the ATP binding site.[11,12] The specificity
of these inhibitors for the PERK receptor is reliant on interactions
with this hydrophobic back pocket.[11] As potent PERK inhibitors,
both 1 and 2 were shown to inhibit tumour growth in pancreatic
and myeloma xenograft models.[11,14]
prodrugs (HAPs) are enzymatically reduced in hypoxic tissue to
release active drugs which may diffuse to adjacent tumour tissue
(a molecular “bystander effect”).[25-28] While early work focussed
on delivering DNA-reactive cytotoxins such as PR104,[29]
SN30000[30,31] and TH-302 (evofosfamide),[32] there is growing
interest in using HAPs to deliver molecularly targeted agents.[33-
35] We have explored various prodrug chemistries to deliver drugs
to hypoxic tumour cells[36-40] and, along with others,[41-44] have
identified 2-nitroimidazole methyl ethers and carbamates as
useful “triggers” for HAPs that fragment when reduced (Scheme
1).[45-47] One-electron reductases, such as cytochrome P450
oxidoreductase (POR),[48] reduce 2-nitroimidazoles to a nitro
radical anion which readily reacts with oxygen in a futile cycle. In
the absence of oxygen further reduction leads to the
hydroxylamine (and amine) which fragment to release a reactive
iminium cation and the corresponding phenol. In the case of
carbamates, a carbamic acid is released which readily fragments
to the amine. In this work we exemplify a HAP approach to deliver
PERK inhibitors to hypoxic cells.
NO2
N
NO2
N
One electron
reductases
CF3
Me
O
Me
O
N
N
F
NH2
NH2
N
N
N
O2
O2
N
N
Me
O
Effector
Effector
N
N
O
N
Me
N
Me
1
2
NHOH
Me
NHOH
N
N
-O Effector
Me
N
N
+
Figure 1. PERK inhibitors 1 (pink sticks) and 2 (green sticks)
adopt a similar orientation in the ATP binding site. The activation
loop (DFG motif) obscuring the binding site has been removed for
clarity. Image produced in PyMOL v 1.7.0.1 (pdb 4G31 and
4M7I).[11,12]
O
Effector
Scheme 1. Mechanism of bioreductive activation of 2-
nitroimidazole prodrugs.
While PERK is an attractive target for drug development, its well-
defined role in the function of specialised secretory cells raises
the spectre that UPR inhibition may be toxic to normal pancreas,
liver and immune cells.[15,16] Indeed, treatment of tumour-bearing
mice with 2 “caused damage to exocrine cells as well as
pancreatic β-cells. These effects were also observed in rats and
dogs treated with GSK2656157”,[14] thus precluding further
development. Nonetheless, the evidence that PERK acts as a
survival factor under hypoxia[7,17,18], and mediates metastasis[19,20]
provides a clear argument for the selective delivery of PERK
inhibitors to hypoxic areas of tumours. This would be expected to
maximise drug delivery to tumour cells that are dependent on
PERK signalling for survival, while minimizing normal tissue drug
exposure, and thus providing an improved therapeutic index.
Results and Discussion
Design and synthesis of PERK inhibitors and
prodrugs
The lipophilic pocket occupied by the 3-methylpyridyl group
(Figure 1) was identified as providing specificity for PERK and
presented an opportunity to design prodrugs where addition of
significant steric bulk would reduce PERK kinase activity. Our
starting point was to prepare a series of methoxy-substituted
inhibitors 3–5 based on the known inhibitor 4 (IC50 = 0.5 nM)
(Figure 2).[11] We designed a series of phenols 6a–8a as potential
PERK inhibitors (effectors), which could be released from the
corresponding 2-nitroimidazole methyl ether HAPs (6b–8b) after
bioreduction.
Hypoxia is
a
cardinal element of the tumour
microenvironment and plays a major role in tumour progression
and response to treatment.[21] Hypoxic cells also contribute to
resistance to therapy.[22-24] Nonetheless, hypoxia provides a
tumour-specific address for drug delivery.[25,26] Hypoxia-activated
R3
4
3
R2
O
O
O
N
NH2
N
NH2
N
NH2
2
R1
N
N
N
N
N
N
N
N
N
Me
Me
Me
R
= OMe
R = OMe
R
= OMe
3
5
8a
8b
11a
11b
11c
4
7a
7b
10a
10b
10c
R1 = OH
R3 = OH
R2 = OH
6a
6b
9a
9b
9c
R1 = O-prodrug
R1 = NH
R1 = NH2COO-prodrug
R3 = O-prodrug
R3 = NH
R2 = O-prodrug
R2 = NH
R2 = NH2COO-prodrug
R3 = NH2COO-prodrug
1 = NO
R
3 = NO
3
R
2 = NO
R
1
2
2
2
2
Figure 2. PERK inhibitors and prodrugs.
2
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10.1002/asia.201801826
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We also considered a series of anilines 9a–11a where the
prodrugs (9b–11b) are linked by a carbamate moiety.
respectively. Removal of the protecting group with TFA provided
anilines 10a and 11a in 73 and 76% over 3 steps, respectively.
The indoline coupling partner 12 was obtained in seven
steps from 4-chloropyrimidine and 5-bromoindoline using a
known, convergent synthetic route with minor modifications.[11]
The PERK inhibitors (3–5, 6a–8a) were prepared by coupling
indoline 12 with the appropriate phenylacetic acids using HATU
or EDCl in variable yields (Scheme 2). We observed that portion
wise addition of the acid or HATU to the reaction mixture resulted
in improved yields. Pre-treatment of phenylacetic acids with
HATU appears to form a stable intermediate species, which could
not be displaced by the indoline 12. This is consistent with the m/z
ion corresponding to an activated ester detected by LRMS when
the reaction was terminated.
O
O
N
NH2
N
NH2
NO2
NH2
N
N
N
N
a
N
N
Me
Me
9c
9a
O
N
NH2
R
O
N
N
c
R
HO
N
Me
R = 3-NHBoc
R = 3-NH
R = 4-NH2Boc
R = 3-NH
15
2
b
b
d
R = 3-NHBoc
R = 4-NH
13
10a
16
11a
2
d
R = 4-NH
R = 4-NHBoc
14
2
O
N
N
NH2
NH2
R
2HCl
NH
Scheme 3. Synthesis of aniline targets 9a–11a. Reaction
conditions. a) Zn dust, NH4Cl, DMF/acetone/water (3:3:2), 3 h,
72%; b) di-tert-butyl-dicarbonate, 1M NaOH, dioxane/water (2:1),
24 h, 90–91%; c) 12, HATU, DIPEA, DMF, overnight, 88–93% d)
TFA, DCM, 4–5 h, 86–96%.
a or b
N
N
N
N
N
Me
Me
R = OMe
R = OH
-
12
3
5
-
6a 8a
-
c R = NO
9c 11
2
With the effectors in hand, we turned our attention to the
synthesis of the prodrugs. The bioreductive trigger 2-
nitroimidazole-5-methanol (17) was prepared according to
literature procedure.[34] Alcohol 17 was converted to the chloride
18 with MsCl (Scheme 4). Alkylation of phenols 6a-8a gave the
ether prodrugs 6b–8b, respectively. In all examples, the reaction
did not proceed to completion, even with heating to 70 °C over 3
days in the case of 6b. A side-product in the crude reaction
mixture with a structure corresponding the formyl imidazole (See
Figure S1) was observed in the crude reaction mixtures in all
cases, which necessitated purification by preparative HPLC. The
alcohol 17 was activated as the 4-nitrophenylcarbonate 19,[46] and
reaction with amines 9a–11a gave carbamate prodrugs 9b-11b in
modest yields.
Scheme 2. Amide coupling. Reaction conditions. a) HATU,
DIPEA, DMF, overnight, 23–90%; b) EDCl, DIPEA, DMAP,
CH2Cl2, overnight, 41–80%.
We envisioned that the aniline targets 9a–11a would be
obtained by coupling the nitro-substituted phenylacetic acids to
indoline 12 followed by reduction. The nitro compounds 9c–11c
were prepared using HATU as a coupling agent (Scheme 2). We
were able to prepare 9a using zinc reduction in 72% yield, but met
with difficulties in reducing 10c. We attempted reduction of 10c
with SnCl2 and hydrogenation conditions (H2 at 40 psi, 10% Pd/C),
but the substrate 10c was poorly soluble in the solvents used. We
turned to a longer preparation, requiring an additional BOC-
protection step (Scheme 3). The phenylacetic acids 13 and 14
were coupled to 12 with HATU to give amides 15 and 16,
N
NO2
N
NH2
b
N
NMe
N
O
Me
Me
N
Cl
a
N
18
O
NO2
N
2-isomer
3-isomer
4-isomer
NO2
NMe
OH
6b
7b
8b
N
17
NO2
N
c
N
NH2
N
NMe
d
O
N
O
O
Me
Me
O
19
N
N
N
O
O
NO2
H
N
R = 2-isomer
R = 3-isomer
R = 4-isomer
9b
10b
11b
NO2
Scheme 4. Prodrug formation. Reaction conditions. (a) MsCl, DIPEA, THF, 30 min, 99%; (b) Phenol (6a–8a), KI, K2CO3, DMF, 28–
32%; (c) nitrophenyl chloroformate, DIPEA, THF, 16 h, 25%; (d) anilines (9a–11a), DIPEA, HOBt, 5 Å molecular sieves, THF, 72 h, 9–
58%.
myelin basic protein (10 µM) with 33P-ATP (10 µM). Ten point
titration curves were determined in duplicate and an IC50
calculated. (Reaction Biology Corp, Malvern, PA) (Table 1). The
new inhibitors were found to have retained substantial inhibitory
Inhibition of PERK activity
activity (IC50 1.2–60.7 nM) against PERK, while the prodrugs
demonstrated a considerable loss of potency. Inhibitors with a
The biochemical potency of the inhibitors and prodrugs against
PERK was determined for human EIF2AK3 phosphorylation of
3
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para-substituent were found to be less active (~10-fold) than their
corresponding ortho- or meta-substituted analogues, again
consistent with previous reports.[11] The ether prodrugs 6b–8b
demonstrated >100-fold deactivation as PERK inhibitors, while
the carbamate prodrugs 9b–11b demonstrated >1000-fold
deactivation compared to 9a–11a. The potency of the PERK
inhibitors was independent of the electronic or hydrogen bonding
nature of the substituents and was mostly influenced by the size
of the substituent, consistent with a steric interaction with the
pocket. Substitution at the 4-position was least favoured across
the series and this is consistent with previous findings.[11]
In all six examples, the prodrugs underwent fragmentation to
release the effector, albeit somewhat inefficiently. In the ether
series, the ortho-substituted compound 6b exhibited the greatest
loss of prodrug, followed by meta-and para- substituted
analogues, 7b and 8b, respectively. This order is reversed for the
carbamate series of prodrugs, with the para-substituted prodrug
11b showing the greatest fragmentation and followed by the
meta- and ortho-substituted prodrugs 10b and 9b, respectively.
The rates of reduction appear to be slower in the prodrugs
containing an ether linkage (38–58%) and in 9b, whereas the
carbamate prodrugs 10b and 11b demonstrated greater loss of
prodrug.
The 2-substituted carbamate prodrug 9b was able to
achieve the most effective prodrug to effector conversion, with a
Ggain to Gloss ratio of approximately 50%. All other examples
suggest that while the prodrugs are able to undergo fragmentation
to release its effector, the conversion to effector is inefficient and
will require further optimisation.
Table 1. Biochemical IC50 values for PERK inhibitors and prodrugs.
Compound
R Group
PERK/EIF2AK3
IC50 (nM)[a]
Selectivity
ratios
1 (GSK’414)
3-CF3
1.6
3
2-OMe
3-OMe
4-OMe
2-OH
1.4
Table 2. Rates of prodrug fragmentation
4
1.5
% Loss
of
prodrug
[a]
[b]
5
20.7
16.3
11.1
26.0
2080
1570
84000
1.2
Gloss
Ggain
#
Prodrug
(µM/Gy)
(µM/Gy)
6a
7a
8a
6b
7b
8b
9a
10a
11a
6b
2-O-
58
43
0.146
0.108
0.007
0.016
3-OH
7b
3-O-
4-OH
8b
4-O-
38
0.047
0.009
2-O-prodrug
3-O-prodrug
4-O-prodrug
2-NH2
128
9b
2-NHCO2-
3-NHCO2-
4-NHCO2-
27 ± 7[c]
84 ± 5
93 ± 1
0.07 ± 0.02
0.21 ± 0.01
0.233 ± 0.002
0.035 ± 0.003
0.056 ± 0.001
0.026 ± 0.0002
141
10b
11b
3231
3-NH2
3.2
[a] Gloss measures the loss of prodrug. [b] Ggain measures the effector being
released. [c] Values ± SEM.
4-NH2
60.7
2-NHCOO-
prodrug
9b
1619
1349
8694
1669
Target modulation of the UPR
3-NHCOO-
prodrug
10b
11b
27820
101335
We used HCT116 colorectal carcinoma cells to investigate the
ability of the PERK HAPs and their corresponding effectors to
modulate anoxia-induced PERK signalling. Four prodrug/effector
pairs were selected for this study, excluding the para-substituted
analogues 8a,b because of the poor aqueous stability of 8b (7%
over 24 h) and 11a,b because the low aqueous solubility of 11b
(Table S1). Three of the prodrugs (6b, 7b, 9b) and all four
effectors (6a, 7a, 9a, 10a) were stable, both in cell-free culture
medium incubated for 24 h at 37°C in oxic conditions, or in the
presence of HCT116 cells cultured for 24 h either in oxic
conditions (Figure S3). Loss of prodrug 10b was observed in both
the oxic and anoxic cell culture, but not in cell-free conditions,
suggesting cellular metabolism. Notably, anoxic prodrug
activation and release was observed with 6b, 7b, 9b and 10b,
generated effector concentrations of 0.27, 0.82, 0.27, and 1.2 μM,
respectively, in the extracellular culture medium after 24 h anoxic
exposure (Figure 3A). In contrast, none of the four prodrugs
generated effector species in oxic HCT116 cultures, confirming
the predicted oxygen-sensitive mechanism of prodrug activation.
We confirmed the hypoxia-selective activation and effector
4-NHCOO-
prodrug
9c
2-NO2
3-NO2
4-NO2
41.1
1.4
10c
11c
26.5
[a] IC50 determinations were performed by Reaction Biology Corp.
Prodrug fragmentation
Reduction of prodrugs and subsequent release of the
corresponding PERK inhibitors was measured following radiolytic
reduction (40 Gy) of anoxic solutions of prodrugs in formate buffer
with subsequent analysis by LC-MS.[37] Strongly reducing species
(aquated electron, e-(aq) and formate radical, CO2 ) are produced
by radiolysis which result in reduction of the nitroimidazole group.
·-
4
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10.1002/asia.201801826
Chemistry - An Asian Journal
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release for 7b using HCT116 POR-overexpressing cells (Figure
S5, 4h drug exposure).
DDIT3. All effectors suppressed anoxic PPP1R15A up-regulation
(to 0.69- to 1.4-fold) and appeared to modestly activate
expression under oxic conditions (by 1.3- to 2.5-fold). Of the
prodrugs, only 9b was effective in suppressing anoxic-induced
expression. 6b was inactive and 7b and 10b caused an
unexpected up-regulation of PPP1R15A expression in anoxic
conditions, possibly due to activation of apoptosis as evidenced
by an induction of PARP1 cleavage (data not shown).
Western immunoblotting was conducted with the same
prodrug/effector pairs. Both ATF4 and CHOP protein levels were
induced following treatment with anoxic ER stress, confirming
activation of the PERK-eIF2α-ATF4 branch of the UPR (Figure
3C). All effectors suppressed the up-regulation of CHOP
expression in response to anoxia, however, effects on ATF4 were
not observed. ATF4, and to a lesser extent CHOP, were also
induced in oxic cultures treated with each effector, and also with
prodrugs 6b and 7b, in agreement with the RT-qPCR analysis.
Two of the prodrugs (7b and 9b) suppressed anoxic CHOP up-
regulation, with minor effect on ATF4 and minimal effects in oxic
cultures with 9b showing marginal superiority. Prodrugs 6b and
10b were inactive. Overall, 9b appeared to be the best prodrug
demonstrating the greatest magnitude of PERK-eIF2α-ATF4
inhibition under anoxia with negligible effects on the pathway
under oxic conditions.
DDIT3 (CHOP) and PPP1R15A (GADD34) are well-
validated, downstream transcriptional targets of ATF4 and, thus,
act as sensitive biomarkers of PERK-eIF2α-ATF4 pathway
activity. DDIT3 transcript, assessed by RT-qPCR, was up-
regulated by 24.7- to 29.7-fold in HCT116 cells following 24 h
anoxic exposure, confirming anoxia-induced ER stress resulting
in PERK activation (Figure 3B). Of the PERK inhibitors tested, 7a,
9a and 10a all suppressed anoxic up-regulation of DDIT3 by 6.3-,
10.2- and 11.2-fold, respectively. 6a was inactive. Paradoxically,
the inhibitors all induced DDIT3 expression in normoxic
(unstressed) cultures by 4.3- to 10.7-fold, suggesting activation of
ATF4, at least with exposure to 10 μM compound. Both 7b and
9b demonstrated suppression of anoxic DDIT3 up-regulation to
17.4- and 6.0–fold, respectively, consistent with the observed
effector released in these conditions (Figure 3A). 6b and 10b did
not suppress anoxic DDIT3 up-regulation despite similar levels of
effector generated during anoxic exposure. Under oxic conditions
neither 7b nor 9b activated PERK signalling in keeping with the
lack of effector generated in oxic cultures.
PPP1R15A expression was modestly induced under anoxic
conditions. Suppression of anoxic PPP1R15A (GADD34)
expression was observed (2.9- to 5.1–fold) with all effectors and
with one prodrug (9b), in agreement with the effects observed
Figure 3. Evaluation of hypoxia-activated PERK inhibitor prodrugs and effectors in HCT116 cells. A. Concentrations of prodrugs and
effectors in HCT116 culture medium. HCT116 cultures were treated with 10 µM prodrug for 24 h in oxic or anoxic conditions (mean ±
SD, n = 1-2). B. Effect of 10 µM PERK prodrug/effector on DDIT3 and PPP1R15A transcript abundance in HCT116 cells cultured for
24 h in oxic or anoxic conditions (mean ± SEM, n = 2-3). C. Effect of 10 µM PERK prodrug/effector pairs on ATF4 and CHOP protein
abundance in HCT116 cells cultured for 24 h in oxic or anoxic conditions.
5
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Conclusions
I2. Column chromatography was carried out on silica gel (Merck
230–400 mesh). CH2Cl2 refers to dichloromethane, DIPEA refers
to
dimethylaminopyridine, DMF refers to dimethylformamide, DMSO
dimethyl sulfoxide, EDCI refers to 1-ethyl-3-
(dimethylaminopropyl)carbodiimide, EtOAc refers to ethyl acetate,
HATU refers to 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate, MeOH refers to
methanol, pet. ether refers to petroleum ether boiling fraction 40–
60 °C, THF refers to tetrahydrofuran, TFA refers to trifluoroacetic
acid.
diisopropylethylamine,
DMAP
refers
to
4-
We adapted a known class of PERK inhibitor and prepared new
analogues with phenol or aniline linkers and the corresponding
ether and carbamate hypoxia-activated prodrugs. The modified
inhibitors retained PERK inhibitory activity and the corresponding
prodrugs were substantial deactivated. We demonstrated the
prodrugs were able to undergo fragmentation following radiolytic
reduction to release their effectors, albeit inefficiently. The
prodrugs also underwent bioreduction in HCT116 cells under
anoxic conditions releasing effector, but not under oxic conditions,
demonstrating hypoxia-dependent fragmentation of the 2-
nitroimidazole trigger. However, the relatively high concentration
of both effectors and prodrugs did cause unexpected effects,
especially on ATF4 abundance and likely on cell survival. While
this class has been described as particularly selective for PERK,
analogues did engage with a variety of kinases at 10 µM.[11,12]
Subsequent work has also identified RIPK1 kinase as a target
kinase for compound 1.[49] Given these observations, it seems
plausible that the high concentrations of effectors and prodrugs
used in our cellular studies may have induced off-target effects.
Nonetheless, this study has identified the 2-substituted
nitroimidazole carbamate 9b as a prodrug with potential to deliver
PERK inhibitors in a hypoxia selective manner. Key steps to
further development is to increase aqueous solubility, enhance
the rates of fragmentation and gain a broader understanding of
the off-target effects of these compounds.
Amide coupling
Method A: HATU-mediated amide coupling
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(2-methoxyphenyl)ethan-1-one
(3).
2-
Methoxyphenylacetic acid (49 mg, 0.30 mmol) was added portion
wise to a stirred solution of amine 12 (100 mg, 0.30 mmol), HATU
(112 mg, 0.30 mmol) and DIPEA (207 µL, 1.18 mmol) in DMF (5
mL). The reaction mixture was stirred at 20 °C for 24 h. The
resulting mixture was diluted with EtOAc (50 mL), washed with
water (2 × 50 mL) and then brine (50 mL), dried and concentrated
in vacuo. The crude residue was purified by trituration with EtOAc
to give amide 3 (110 mg, 90 %) as an off-white solid: m.p. 185–
187°C; 1H NMR: δ=8.14 (s, 1H, H-2), 8.10 (d, J = 8.3 Hz, 1H, H-
7'), 7.30 (s, 1H, H-4'), 7.28–7.18 (m, 4H, H-6, H-6', H-4'', H-6''),
7.00 (d, J = 7.7 Hz, 1H, H-3''), 6.91 (td, J = 7.4, 1.0 Hz, 1H, H-5''),
6.06 (br s, 2H, NH2-4), 4.22 (t, J = 8.4 Hz, 2H, H2-2'), 3.77 (s, 5H,
CH2, OCH3-2''), 3.72 (s, 3H, NCH3-7), 3.23 ppm (t, J = 8.4 Hz, 2H,
H2-3'); 13C NMR: δ=168.9 (C=O), 157.2 (C-2''), 157.1 (C-4), 151.6
(C-2), 150.3 (C-7a), 141.9 (C-7'a), 132.8 (C-3'a), 130.8 (C-6''),
129.7 (C-5'), 128.1 (C-4''), 127.1 (C-6'), 124.9 (C-4'), 123.9 (C-6),
123.7 (C-1''), 120.2 (C-5''), 116.1 (C-7'), 115.0 (C-5), 110.7 (C-3''),
99.9 (C-4a), 55.4 (OCH3-2''), 47.9 (C-2'), 36.7 (CH2), 30.7 (NCH3-
7), 27.5 ppm (C-3'); MS (APCI): m/z (%): 414.2 (100) [M+H]+;
HRMS (ESI): m/z calcd for C24H24N5O2: 414.1925 [M+H]+; found:
414.1933; HPLC purity: 98.2 %.
Experimental Section
Chemistry Synthesis
General procedures. All final products were analysed by
reverse-phase HPLC, (ZORBAX Eclipse XDB C8 5 μm column,
4.6 × 150 mm; Agilent Technologies or an Altima C18 5μm
column, 3.2 × 150 mm) using an Agilent Technologies 1260
Infinity equipped with a diode-array detector. Mobile phases were
gradients of 80% acetonitrile/20% H2O (v/v) in 45 mM ammonium
formate at pH 3.5 and 0.7-1.2 mL/min. Final compound purity was
determined by monitoring at 330 ± 50 nM and was >95%, with the
exception of 8b (93.9%). Melting points were determined on an
Electrothermal 2300 Melting Point Apparatus. NMR spectra were
obtained on a Bruker Avance 400 spectrometer at 400 MHz for 1H
and 100 MHz for 13C spectra. Spectra were obtained in DMSO-d6
unless noted otherwise. Chemical shifts and coupling constants
were recorded in units of ppm and Hz, respectively. Low
resolution mass spectra were gathered by direct injection of
methanolic solutions into an Agilent 6120 mass spectrometer
using an atmospheric pressure chemical ionization (APCI) mode
with a fragmentor voltage of 50 V and a drying gas temperature
of 250 °C. High resolution mass spectra (HRMS) were measured
on an Agilent Technologies 6530 Accurate-Mass Quadrupole
Time of Flight (Q-TOF) LC / MS interfaced with an Agilent Jet
Stream Electrospray Ionization (ESI) source allowing positive or
negative ions detection. Organic solutions were dried over
Na2SO4 and solvents were evaporated under reduced pressure
on a rotary evaporator. Thin-layer chromatography was carried
out on aluminium-backed silica gel plates (Merck 60 F254) with
visualization of components by UV light (254 nm) or exposure to
Method B: EDCI-mediated amide coupling
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(3-methoxyphenyl)ethan-1-one (4). EDCI
(116 mg, 0.60 mmol) was added to a stirred solution of 3-
methoxyphenylacetic acid (100 mg, 0.60 mmol) and DIPEA (420
µL, 2.41 mmol) in CH2Cl2 (5 mL) and stirred for 5 min. DMAP (74
mg, 0.60 mmol) and 5-(indolin-4-yl)-7-methyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (12) (160 mg, 0.47 mmol), which have been
prepared according to literature procedure was added to the
mixture.[12] The reaction mixture was stirred at 20 °C for 24 and
diluted with CH2Cl2 (50 mL). The organic layer was washed with
water (50 mL) and with brine (50 mL), dried and concentrated to
give amide 4 (157 mg, 80 %) as a white solid: m.p. 230–233°C;
1H NMR: δ=8.14–8.12 (m, 2H, H-2, H-7'), 7.29 (s, 1H, H-4'), 7.27–
7.21 (m, 3H, H-6, H-6', H-5''), 6.88–6.87 (m, 2H, H-2'', H-6''), 6.83
(dd, J = 8.2, 2.4 Hz, 1H, H-4''), 6.05 (br s, 2H, NH2-4), 4.19 (t, J =
8.4 Hz, 2H, H2-2'), 3.83 (s, 2H, CH2), 3.75 (s, 3H, OCH3-3''), 3.72
(s, 3H, NCH3, 3.20 ppm (t, J = 8.4 Hz, 2H, H2-3'); 13C NMR:
δ=168.9 (C=O), 159.2 (C-3''), 157.1 (C-3''), 151.6 (C-2), 150.3 (C-
6
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10.1002/asia.201801826
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FULL PAPER
7a), 141.8 (C-7'a), 136.5 (C-1''), 132.8 (C-3'a), 129.8 (C-5'), 129.3
(C-5''), 127.1 (C-6'), 124.9 (C-4'), 124.0 (C-6), 121.6 (C-6''), 116.2
(C-7'), 115.2 (C-2''), 114.9 (C-5), 111.9 (C-4''), 99.8 (C-4a), 55.0
(OCH3-3''), 47.9 (C-2'), 42.1 (CH2), 30.7 (N-Me), 27.5 ppm (C-3');
MS (APCI): m/z (%): 414.2 (100) [M+H]+; HRMS (ESI): m/z calcd
for C24H24N5O2: 414.1925 [M+H]+; found: 414.1917; HPLC purity:
99.3 %. 1H NMR data was found to be consistent with literature.[11]
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(4-hydroxyphenyl)ethan-1-one
(8a).
Prepared using method A to obtain 8a (76 %) as an off-white solid:
1
m.p. 240–243°C; H NMR: δ=9.30 (br s, 1H, OH-4''), 8.14–8.12
(m, 2H, H-2, H-7'), 7.28 (s, 1H, H-4'), 7.23–7.20 (m, 2H, H-6, H-
6'), 7.09 (d, J = 8.4 Hz, 2H, H-3'', H-5''), 6.72 (ddd, J = 8.4, 2.8,
1.9 Hz, 2H, H-2'', H-6''), 6.04 (br s, 2H, NH2-4), 4.17 (t, J = 8.4 Hz,
2H, H2-2'), 3.72 (s, 5H, N-Me, CH2), 3.18 ppm (t, J = 8.4 Hz, 2H,
H2-3'); 13C NMR: δ=169.5 (C=O), 157.2 (C-4), 156.0 (C-4''), 151.6
(C-2), 150.3 (C-7a), 141.9 (C-7'a), 132.8 (C-3'a), 130.3 (C-2'', C-
6''), 129.7 (C-5'), 127.1 (C-6'), 125.0 (C-4'), 124.8 (C-1''), 124.0
(C-6), 116.2 (C-7'), 115.2 (C-3'', C-5''), 115.0 (C-5), 99.9 (C-4a),
47.9 (C-2'), 41.4 (CH2), 30.7 (N-Me), 27.5 ppm (C-3'); MS (APCI):
m/z (%): 400.2 (100) [M+H]+; HRMS (ESI): m/z calcd for
C23H22N5O2: 400.1768 [M+H]+; found: 400.1777; HPLC purity:
97.3 %.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(4-methoxyphenyl)ethan-1-one (5). Prepared
using method B. The resulting residue was diluted in EtOAc and
washed with 0.4M HCl to obtain 5 as the HCl salt (41 %) as a
1
white solid: m.p. 186–189°C; H NMR: δ=8.48 (s, 1H, H-2), 8.16
(d, J = 8.2 Hz, 1H, H-7'), 7.58 (s, 1H, H-6), 7.30 (s, 1H, H-4'), 7.25–
7.21 (m, 3H, H-6', H-2'', H-6''), 6.90 (ddd, J = 8.7, 2.9, 2.0 Hz, 2H,
H-3'', H-5''), 4.20 (t, J = 8.4 Hz, 2H, H2-2'), 3.83 (s, 3H, N-Me),
3.80 (s, 2H, CH2), 3.74 (s, 3H, OCH3-4), 3.20 ppm (t, J = 8.4 Hz,
2H, H2-3'), NH2 not observed; 13C NMR: δ=169.5 (C=O), 158.0 (C-
4''), 151.3 (C-4), 147.5 (C-7a), 142.7 (C-2), 142.6 (C-7'a), 133.1
(C-3'a), 130.4 (C-3'', C-5''), 127.3 (C-5'), 127.2 (C-6'), 127.1 (C-6),
126.8 (C-1''), 124.8 (C-4'), 117.9 (C-5), 116.4 (C-7'), 113.8 (C-3'',
C-5''), 98.4 (C-4a), 55.0 (OCH3-4''), 47.9 (C-2'), 41.3 (CH2), 31.5
(N-Me), 27.5 ppm (C-3'); MS (APCI): m/z (%): 414.2 (100) [M+H]+;
HRMS (ESI): m/z calcd for C24H24N5O2: 414.1925 [M+H]+; found:
414.1940; HPLC purity: 99.0 %.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(2-nitrophenyl)ethan-1-one (9c). Prepared
using method A to obtain 9c (quant.) as a yellow solid: m.p. 231–
1
233°C; H NMR: δ=8.14 (s, 1H, H-2), 8.11 (dd, 1H, J = 8.1, 1.2
Hz, 1H, H-3’’), 8.00 (d, J = 8.2 Hz, 1H, H-7’), 7.73 (td, J = 7.5, 1.2
Hz, 1H, H-5’’), 7.61–7.55 (m, 2H, H-4’’, H-6’’), 7.33 (s, 1H, H-4’),
7.24 (s, 1H, H-6), 7.20 (d, J = 8.2 Hz, 1H, H-6’), 6.06 (br s, 2H,
NH2-4), 4.33–4.29 (m, 4H, CH2, H2-2’), 3.72 (s, 3H, Me), 3.29 ppm
(t obscured, J = 8.8 Hz, 2H, H2-3’); 13C NMR: δ=167.8 (C=O),
157.6 (C-4), 152.1 (C-2), 150.8 (C-7a), 149.6 (C-2’’), 142.1 (C-
7’a), 134.2 (C-5’’), 134.2 (C-6’’), 133.2 (C-3’a), 131.3 (C-1’’), 130.4
(C-5’), 128.9 (C-4’’), 127.6 (C-6’), 125.4 (C-4’), 125.1 (C-3’’),
124.5 (C-6), 116.5 (C-7’), 115.4 (C-5), 100.3 (C-4a), 48.3 (C-2’),
40.9 (CH2), 31.2 (Me), 28.0 ppm (C-3’); MS (APCI): m/z (%): 429.2
(100) [M+H]+; HRMS (ESI): m/z calcd for C23H21N6O3: 429.1670
[M+H]+; found: 429.1690; HPLC purity: 99.4 %.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(2-hydroxyphenyl)ethan-1-one
(6a).
Prepared using method A and purified by column chromatography
(5% MeOH/EtOAc) to afford 6a (23 %) as an off-white white solid:
m.p. 252–255°C; 1H NMR: δ=9.47 (s, 1H, OH-2''), 8.15 (s, 1H, H-
2), 8.12 (d, J = 8.3 Hz, 1H, H-7'), 7.30 (s, 1H, H-4'), 7.26 (s, 1H,
H-6), 7.21 (dd, J = 8.3, 1.4 Hz, 1H, H-6'), 7.13–7.06 (m, 2H, H-4'',
H-6''), 6.82 (dd, J = 8.1, 0.8 Hz, 1H, H-3''), 6.76 (td, J = 7.4, 0.9
Hz, 1H, H-5''), 6.13 (br s, 2H, NH2), 4.22 (t, J = 8.4 Hz, 2H, H2-2'),
3.74–3.73 (m, 5H, CH2, Me), 3.21 ppm (t, J = 8.4 Hz, 2H, H2-3');
13C NMR: δ=169.2 (C=O), 156.8 (C-4), 155.3 (C-2''), 151.1 (C-2),
150.2 (C-7a), 142.0 (C-7'a), 132.8 (C-3'a), 130.8 (C-6''), 129.5 (C-
5'), 127.7 (C-4''), 127.1 (C-6'), 124.8 (C-4'), 124.1 (C-6), 121.8 (C-
1''), 118.8 (C-5''), 116.1 (C-7'), 115.1 (C-5), 114.9 (C-3''), 99.8 (C-
4a), 47.9 (C-2'), 36.7 (CH2), 30.7 (Me), 27.5 ppm (C-3'); MS
(APCI): m/z (%): 400.2 (100) [M+H]+; HRMS (ESI): m/z calcd for
C23H22N5O2: 400.1768 [M+H]+; found: 400.1783; HPLC purity:
98.0 %.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(3-nitrophenyl)ethan-1-one (10c). Prepared
using method A to obtain 10c (73 %) as a yellow solid: m.p. 246–
1
249°C; H NMR: δ=8.21 (t, J = 3.7 Hz, 1H, H-2''), 8.16–8.14 (m,
2H, H-2, H-4''), 8.11 (d, J = 8.3 Hz, 1H, H-7'), 7.77 (d, J = 7.8 Hz,
1H, H-6''), 7.65 (t, J = 7.8 Hz, 1H, H-5''), 7.32 (d, J = 1.0 Hz, 1H,
H-4’), 7.25 (s, 1H, H-6), 7.22 (d, J = 8.3 Hz, 1H, H-6'), 6.06 (br s,
2H, NH2-4), 4.27 (t, J = 8.4 Hz, 2H, H2-2'), 4.10 (s, 2H, CH2), 3.73
(s, 3H, N-Me), 3.25 ppm (t, J = 8.4 Hz, 2H, H2-3'); 13C NMR:
δ=168.3 (C=O), 157.1 (C-4), 151.6 (C-2), 150.3 (C-7a), 147.6 (C-
3''), 141.7 (C-7'a), 137.6 (C-1''), 136.9 (C-6''), 132.8 (C-3'a), 129.9
(C-5'), 129.5 (C-5''), 127.1 (C-6'), 124.9 (C-4'), 124.6 (C-2''), 124.0
(C-6), 121.5 (C-4''), 116.1 (C-7'), 114.9 (C-5), 99.8 (C-4a), 47.8
(C-2'), 40.9 (CH2), 30.7 (N-Me), 27.5 ppm (C-3'); MS (APCI): m/z
(%): 429.2 (100) [M+H]+; HRMS (ESI): m/z calcd for C23H21N6O3:
429.1670 [M+H]+; found: 429.1685; HPLC purity: 98.7 %.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(3-hydroxyphenyl)ethan-1-one
(7a).
Prepared using method A to obtain 7a (28 %) as an off-white solid:
1
m.p. 182–185°C; H NMR: δ=9.39 (br s, 1H, OH-3''), 8.14–8.13
(m, 2H, H-2, H-7'), 7.28 (s, 1H, H-4'), 7.23–7.21 (m, 2H, H-6, H-
6'), 7.14–7.10 (m, 1H, H-5''), 6.73–6.71 (m, 2H, H-2'', 6''), 6.66–
6.64 (m, 1H, H-4''), 6.04 (br s, 2H, NH2-4), 4.16 (t, J = 8.4 Hz, 2H,
CH2-2'), 3.76 (s, 2H, CH2), 3.72 (s, 3H, Me), 3.18 ppm (t, J = 8.4
Hz, 2H, CH2-3'); 13C NMR: δ=169.1 (C=O), 157.4 (C-4), 157.2 (C-
3''), 151.6 (C-2), 150.4 (C-7a), 141.9 (C-7'a), 136.2 (C-1''), 132.8
(C-3a), 129.8 (C-5'), 129.3 (C-5''), 127.1 (C-6'), 124.9 (C-4'), 124.0
(C-6), 120.0 (C-6''), 116.2 (C-7'), 116.1 (C-2''), 115.0 (C-5), 113.6
(C-4''), 99.9 (C-4a), 48.0 (C-2'), 42.4 (CH2), 30.7 (Me), 27.5 ppm
(C-3'); MS (APCI): m/z (%): 400.2 (100) [M+H]+; HRMS (ESI): m/z
calcd for C23H22N5O2: 400.1768 [M+H]+; found: 400.1760; HPLC
purity: 96.6 %.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(4-nitrophenyl)ethan-1-one (11c). Prepared
using method A to obtain 11c (80 %) as a yellow solid: m.p. 256–
1
258°C; H NMR: δ=8.22 (d, J = 8.7 Hz, 2H, H-3'', H-5''), 8.14 (s,
1H, H-2), 8.10 (d, J = 8.3 Hz, 1H, H-7'), 7.59 (d, J = 8.7 Hz, 2H,
H-2'', H-6''), 7.32 (br s, 1H, H-4'), 7.24 (s, 1H, H-6), 7.22 (d, J =
8.3 Hz, 1H, H-6'), 6.04 (br s, 2H, NH2-4), 4.25 (t, J = 8.4 Hz, 2H,
CH2-2'), 4.08 (s, 2H, CH2), 3.72 (s, 3H, Me), 3.24 ppm (t, J = 8.4
Hz, 2H, CH2-3'); 13C NMR: δ=168.5 (C=O), 157.6 (C-4), 152.1 (C-
2), 150.8 (C-7a), 146.8 (C-4''), 144.0 (C-1''), 142.2 (C-7'a), 133.3
7
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10.1002/asia.201801826
Chemistry - An Asian Journal
FULL PAPER
(C-3'a), 131.7 (C-2'', C-6''), 130.4 (C-5'), 127.6 (C-6'), 125.4 (C-
4'), 124.5 (C-6), 123.7 (C-3'', C-5''), 116.6 (C-7'), 115.4 (C-5),
100.3 (C-4a), 48.3 (C-2'), 42.0 (CH2), 31.2 (Me), 28.0 ppm (C-3');
MS (APCI): m/z (%): 429.2 (100) [M+H]+; HRMS (ESI): m/z calcd
for C23H21N6O3: 429.16697 [M+H]+; found: 429.16895; HPLC
purity 98.8 %.
1H), 3.60 (s, 2H), 1.51 (s, 9H); MS (APCI): m/z (%): 250.2 (100)
[M-H]-.
tert-Butyl
d]pyrimidin-5-yl)indolin-1-yl)-2-oxoethyl)phenyl)carbamate
(15). Prepared using method and purified by column
(3-(2-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-
A
chromatography (2–3% MeOH/EtOAc) to afford 15 (88 %) as an
1
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
off-white white solid: m.p. 208–211°C; H NMR: δ=9.32 (s, 1H),
yl)indolin-1-yl)-2-(2-aminophenyl)ethan-1-one (9a). To
suspension of nitrophenylacetamide 9c (190 mg, 0.44 mmol) in a
DMF/acetone/water mixture (3:3:2, 24 mL) was added Zn dust
(1.02 g, 15.5 mmol) and ammonium chloride (813 mg, 15.2 mmol). (s, 9H); MS (APCI): m/z (%): 499.2 (100) [M+H]+; HRMS (ESI):
a
8.14–8.12 (m, 2H), 7.45 (s, 1H), 7.32–7.29 (m, 2H), 7.24–7.18 (m,
3H), 6.89 (d, J = 7.3 Hz, 1H), 6.03 (br s, 2H), 4.19 (t, J = 8.4 Hz,
2H), 3.81 (s, 2H), 3.72 (s, 3H), 3.20 (t, J = 8.4 Hz, 2H), 1.47 ppm
The mixture was stirred at 20 °C for 3 h. The solids were removed
by filtration and rinsed with EtOAc (2 x 10 mL). The organic
fractions were combined and diluted with EtOAc (50 mL). The
organic layer was washed with sat. NaHCO3 (50 mL) and brine
(50 mL), dried (Na2SO4) and concentrated in vacuo. The resulting
residue was triturated with EtOAc to obtain the amine 9a (127 mg,
m/z calcd for C28H31N6O3: 499.2452 [M+H]+; found: 499.2468.
tert-Butyl (4-(2-(5-(4-amino-7-methyl-7H-pyrrolo[2,3-
d]pyrimidin-5-yl)indolin-1-yl)-2-oxoethyl)phenyl)carbamate
(16). Prepared using method A to obtain 16 (93 %) as a pale
brown solid: m.p. 239–242°C; 1H NMR: δ=9.29 (s, 1H), 8.13–8.11
(m, 2H), 7.41–7.39 (m, 2H), 7.29 (s, 1H), 7.24–7.16 (m, 4H), 6.02
(br s, 2H), 4.18 (t, J = 8.4 Hz, 2H), 3.77 (s, 2H), 3.72 (s, 3H), 3.19
(t, J = 8.4 Hz, 2H), 1.47 ppm (s, 9H); MS (APCI): m/z (%): 499.3
(100) [M+H]+; HRMS (ESI): m/z calcd for C28H31N6O3: 499.2452
[M+H]+; found: 499.2470.
1
72 %) as a white solid: m.p. 220–220°C; H NMR: δ=8.15–8.13
(m, 2H, H-2, H-7'), 7.29 (s, 1H, H-4'), 7.24–7.21 (m, 2H, H-6, H-
6'), 7.01–6.95 (m, 2H, H-4'', H-6''), 6.67 (dd, J = 7.9, 0.8 Hz, 1H,
H-3''), 6.53 (td, J = 7.4, 0.8 Hz, 1H, H-5''), 6.04 (br s, 2H, NH2-4),
5.05 (s, 2H, NH2-2''), 4.21 (t, J = 8.4 Hz, 2H, H2-2'), 3.72 (s, 3H,
Me), 3.67 (s, 2H, CH2), 3.20 ppm (t, J = 8.4 Hz, 2H, H2-3'); 13C
NMR: δ=169.1 (C=O), 157.1 (C-4), 151.6 (C-2), 150.3 (C-7a),
147.2 (C-2''), 141.9 (C-7'a), 132.8 (C-3'a), 130.3 (C-6''), 129.8 (C-
5'), 127.5 (C-4''), 127.1 (C-6'), 124.8 (C-4'), 123.9 (C-6), 118.4 (C-
1''), 116.2 (C-7', C-5''), 115.0 (C-3''), 114.9 (C-5), 99.8 (C-4a), 47.9
(C-2'), 39.0 (CH2), 30.7 (N-Me), 27.5 ppm (C-3'); MS (APCI): m/z
(%): 399.2 (100) [M+H]+; HRMS (ESI): m/z calcd for C23H23N6O:
399.1928 [M+H]+; found: 399.1923; HPLC purity: 99.1 %.
Boc-deprotection
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(3-aminophenyl)ethan-1-one (10a). TFA (0.5
mL) was added to a stirred solution of tert-butyl carbamate 15
(114 mg, 0.23 mmol) in CH2Cl2 (10 mL). The reaction was stirred
at 20 °C for 4 h and then quenched with sat. NaHCO3 (50 mL).
The reaction mixture was extracted with CH2Cl2 (50 mL), dried
and concentrated in vacuo. The resulting residue was triturated
with EtOAc to obtain the amine 10a (88 mg, 96 %) as an off-white
solid: m.p. 214–217°C; 1H NMR: δ=8.16–8.14 (m, 2H, H-2, H-7'),
7.28 (s, 1H, H-4'), 7.23–7.21 (m, 2H, H-6, H-6'), 6.96 (t, J = 7.7
Hz, 1H, H-5''), 6.51 (s, 1H, H-2''), 6.45–6.43 (m, 2H, H-4'', H-6''),
6.04 (br s, 2H, NH2-4), 5.04 (s, 2H, NH2-3''), 4.14 (t, J = 8.5 Hz,
2H, H2-2'), 3.72 (s, 3H, N-Me), 3.67 (s, 2H, CH2), 3.17 ppm (t, J =
8.5 Hz, 2H, H2-3'); 13C NMR: δ=169.2 (C=O), 157.1 (C-4), 151.6
(C-2), 150.3 (C-7a), 148.8 (C-3''), 141.9 (C-7'a), 135.3 (C-1''),
132.7 (C-3'a), 129.8 (C-5'), 128.9 (C-5''), 127.1 (C-6'), 124.8 (C-
4'), 123.9 (C-6), 116.6 (C-6''), 116.2 (C-7'), 114.9 (C-5), 114.3 (C-
2''), 112.3 (C-4''), 99.8 (C-4a), 47.9 (C-2'), 42.8 (CH2), 30.7 (N-
Me), 27.5 ppm (C-3'); MS (APCI): m/z (%): 399.2 (100) [M+H]+;
HRMS (ESI): m/z calcd for C23H23N6O: 399.1928 [M+H]+; found:
399.1942; HPLC purity: 97.7 %.
Boc-protected anilines
2-(3-((tert-Butoxycarbonyl)amino)phenyl)acetic acid (13). 1M
NaOH (4 mL) was added to a solution of 3-aminophenylacetic
acid (617 mg, 4.08 mmol) dissolved in 2:1 dioxane/water (12 mL)
and di-tert-butyl dicarbonate (980 mg, 4.49 mmol) was added.
The reaction mixture was stirred at 20 °C for 24 h, neutralised with
2N HCl and diluted with water (50 mL). EtOAc (2 × 50 mL) was
added and the organic layer was washed with brine (50 mL), dried
and concentrated to an orange gum. The crude residue was
purified by column chromatography, eluting with 25 % EtOAc/pet.
ether, to give acid 13 (928 mg, 90 %) as a white solid: m.p. 100–
102°C; 1H NMR (CDCl3): δ=7.35 (s, 1H), 7.25–7.22 (m, 2H), 6.97–
6.94 (m, 1H), 6.60 (br s, 1H), 3.62 (s, 2H), 1.51 (s, 9H); MS
(APCI): m/z (%): 250.2 (100) [M-H]-.
2-(4-((tert-Butoxycarbonyl)amino)phenyl)acetic acid (14).[50]
1M NaOH (5 mL) was added to a solution of 4-aminophenylacetic
acid (906 mg, 6.00 mmol) dissolved in 2:1 dioxane/water (15 mL)
and then di-tert-butyl dicarbonate (1.44 g, 6.59 mmol) was added.
The reaction mixture was stirred at 20 °C for 24 h, neutralised with
2N HCl and diluted with water (50 mL). EtOAc (2 × 50 mL) was
added and the organic layer was washed with brine (50 mL), dried
and concentrated to a white solid. The crude residue was
triturated with EtOAc and the carbamate 14 (1.37 g, 91 %) was
obtained as a white solid: m.p. 155–157°C; 1H NMR (CDCl3):
δ=7.32 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 6.48 (br s,
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(4-aminophenyl)ethan-1-one (11a). TFA (0.5
mL) was added to a stirred solution of tert-butyl carbamate 16
(120 mg, 0.24 mmol) in CH2Cl2 (10 mL). The reaction was stirred
at 20 °C for 5 h and quenched with sat. NaHCO3 (50 mL). The
reaction mixture was extracted with CH2Cl2 (40 mL), dried and
concentrated in vacuo. The resulting residue was triturated with
EtOAc to obtain the amine 11 (83 mg, 86 %) as an off-white solid:
1
m.p. 147–150°C; H NMR: δ=8.14–8.12 (m, 2H, H-2, H-7'), 7.27
(s, 1H, H-4'), 7.23–7.20 (m, 2H, H-6, H-6'), 6.95 (d, J = 8.3 Hz, 2H,
H-2'', H-6''), 6.52 (d, J = 8.3 Hz, 2H, H-3'', H-5''), 6.04 (br s, 2H,
NH2-4), 4.95 (s, 2H, NH2-4''), 4.15 (t, J = 8.4 Hz, 2H, H2-2'), 3.72
8
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10.1002/asia.201801826
Chemistry - An Asian Journal
FULL PAPER
(s, 3H, N-Me), 3.63 (s, 2H, CH2), 3.17 ppm (t, J = 8.4 Hz, 2H, H2-
3'); 13C NMR: δ=169.8 (C=O), 157.1 (C-4), 151.6 (C-2), 150.3 (C-
7a), 147.2 (C-4''), 142.0 (C-7'a), 132.7 (C-3'a), 129.7 (C-5', C-2'',
C-6''), 127.1 (C-6'), 124.8 (C-4'), 123.9 (C-6), 121.5 (C-1''), 116.2
(C-7'), 115.0 (C-5), 113.9 (C-3'', C-5''), 99.8 (C-4a), 47.9 (C-2'),
41.6 (CH2), 30.7 (N-Me), 27.5 ppm (C-3'); MS (APCI): m/z (%):
399.2 (100) [M+H]+; HRMS (ESI): m/z calcd for C23H23N6O:
399.1928 [M+H]+; found: 399.1942; HPLC purity: 98.5 %.
2H, H2-3'); 13C NMR: δ=169.0 (C=O), 157.2 (C-4), 155.5 (C-2''),
151.6 (C-2), 150.3 (C-7a), 145.9 (C-2'''), 141.6 (C-7'a), 133.6 (C-
5'''), 132.4 (C-3'a), 131.4 (C-6''), 129.8 (C-5'), 128.5 (C-4'''), 128.1
(C-4''), 127.0 (C-6'), 124.7 (C-4', C-1''), 124.0 (C-6), 121.1 (C-5''),
115.9 (C-7'), 115.0 (C-5), 111.9 (C-3''), 99.8 (C-4a), 59.1 (OCH2),
48.0 (C-2'), 37.0 (COCH2), 34.2 (NCH3-1'''), 30.7 (NCH3-7), 27.3
ppm (C-3'); MS (APCI): m/z (%): 539.2 (100) [M+H]+; HRMS
(ESI): m/z calcd for C28H27N8O4: 539.2150 [M+H]+; found:
539.2150; HPLC purity 98.7 % (0.32 % effector).
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(3-((1-methyl-2-nitro-1H-imidazol-5-
Preparation of prodrug triggers
yl)methoxy)phenyl)ethan-1-one (7b). Phenol 7a (71.5 mg, 0.18
mmol), chloride 18 (34.6 mg), K2CO3 (49.5 mg, 0.36 mmol) and
KI (32.7 mg, 0.20 mmol) were stirred in DMF (5 mL) at 20 °C
overnight (20 h). The resulting mixture was diluted with EtOAc (20
mL), washed with water (20 mL) and with brine (20 mL), dried and
concentrated. The crude residue was purified by preparative
HPLC [gradient (95–40–95 %) ammonium formate pH 3.45/90 %
CH3CN/H2O]. Solvent was removed and the residue was diluted
with EtOAc (20 mL), washed with sat. NaHCO3 (20 mL) and with
brine (20 mL), dried and concentrated. The ether 7b (30.4 mg,
5-(Chloromethyl)-1-methyl-2-nitro-1H-imidazole (18). MsCl
(149 µL, 1.93 mmol) was added to mixture of (1-methyl-2-nitro-
1H-imidazol-5-yl)methanol (17) (253 mg, 1.61 mmol) and DIPEA
(337 µL, 1.93 mmol) stirring in THF (5mL). The mixture was stirred
at 20 °C for 30 min and diluted with EtOAc (20 mL). The organic
layer was washed with 1M HCl (20 mL), dried and concentrated
in vacuo. The chloride 18 (280 mg, 99%) was obtained as a yellow
solid which was used without further purification: m.p. 82 °C
(decomp.) (lit. [34] 87–90°C, EtOAc); 1H NMR: δ=7.19 (s, 1H, H-4),
4.63 (s, 2H, CH2), 4.08 ppm (s, 3H, Me); MS (APCI): m/z (%):
176.1 (100) [M+H]+.
1
32 %) was obtained as a pale yellow solid: m.p. 128–131°C; H
NMR: δ=8.14–8.11 (m, 2H, H-2, H-7'), 7.35 (s, 1H, H-4'''), 7.31–
7.27 (m, 2H, H-4', H-5''), 7.24–7.21 (m, 2H, H-6, H-6'), 7.01–6.94
(m, 3H, H-2'', H-4'', H-6''), 6.04 (br s, 2H, NH2-4), 5.24 (s, 2H,
OCH2), 4.20 (t, J = 8.4 Hz, 2H, H2-2'), 3.96 (s, 3H, NCH3-1'''), 3.85
(s, 2H, COCH2), 3.72 (s, 3H, NCH3-7), 3.21 (t, J = 8.4 Hz, 2H, H2-
3'); 13C NMR: δ=168.8 (COCH2), 157.5 (C-3''), 157.1 (C-4), 151.6
(C-2), 150.3 (C-7a), 146.2 (C-2'''), 141.8 (C-7'a), 136.8 (C-1''),
133.6 (C-5'''), 132.8 (C-3''a), 129.8 (C-5'), 129.4 (C-4'), 128.5 (C-
4'''), 127.1 (C-6'), 124.9 (C-4'), 124.0 (C-6), 122.7 (C-6''), 116.3
(C-2''), 116.2 (C-7'), 114.9 (C-5), 113.0 (C-4''), 99.9 (C-4a), 59.1
(OCH2), 48.0 (C-2'), 42.0 (COCH2), 34.3 (NCH3-1'''), 30.7 (NCH3-
7), 27.5 ppm (C-3'); MS (APCI): m/z (%): 539.2 (100) [M+H]+;
HRMS (ESI): m/z calcd for C28H27N8O4: 539.2150 [M+H]+; found:
539.2168; HPLC purity: 97.6 % (0.27 % effector).
(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl
(4-nitrophenyl)
carbonate (19).[51] 4-Nitrophenylchloroformate (311 mg, 1.54
mmol) was added to a stirred suspension of alcohol 17 (220 mg,
1.40 mmol) and DIPEA (294 µL, 1.68 mmol) in THF (5 mL). The
reaction mixture was stirred at 20 °C for 16 h. The resulting
mixture was diluted with EtOAc (50 mL), washed with water (50
mL) and brine (50 mL), dried and concentrated in vacuo. The
crude residue was purified by column chromatography, eluting
with a gradient (60–70 %) of EtOAc/pet. ether to give the
carbonate 19 (113 mg, 25 %) as a white solid: m.p. 160–161°C
(lit.[45] m.p. 156.5–157.5 °C); 1H NMR: δ=8.35–8.31 (ddd, J = 9.2,
3.3, 2.2 Hz, 2H, H-3', H-5'), 7.61–7.57 (ddd, J = 9.2, 3.3, 2.2 Hz,
2H, H-2', H-6'), 7.36 (s, 1H, H-4), 5.46 (s, 2H, CH2), 3.98 ppm (s,
3H, N-Me); MS (APCI): m/z (%): 323.1 (100) [M+H]+.
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(4-((1-methyl-2-nitro-1H-imidazol-5-
yl)methoxy)phenyl)ethan-1-one (8b). Phenol 8a (99 mg, 0.25
mmol), chloride 18 (44 mg, 0.25 mmol), K2CO3 (69 mg, 0.50
mmol) and KI (41 mg, 0.25 mmol) were stirred in DMF (5 mL) at
20 °C overnight. A second equivalent of chloride 18 (44 mg, 0.25
mmol) was added to the mixture and was stirred at 20 °C for 43
h. The resulting mixture was diluted with EtOAc (50 mL), washed
with water (50 mL) and then brine (50 mL), dried and concentrated
in vacuo. The resulting crude residue was purified by column
chromatography, eluting in 4 % MeOH/CH2Cl2. Solvent was
removed and the mixture triturated with MeOH, filtered, and dried
to give the ether 8b (38 mg, 28 %) as an orange solid: m.p. 246–
249°C (decomp.); 1H NMR: δ=8.14–8.12 (m, 2H, H-2, H-7'), 7.34
(s, 1H, H-4'''), 7.30 (s, 1H, H-4'), 7.27–7.21 (m, 4H, H-6, H-6', H-
2'', H-6''), 7.05 (d, J = 8.68 Hz, 2H, H-3'', H-5''), 6.04 (br s, 2H,
NH2-4), 5.24 (s, 2H, OCH2), 4.20 (t, J = 8.5 Hz, 2H, H2-2'), 3.96 (s,
3H, NCH3-1'''), 3.81 (s, 2H, COCH2), 3.73 (s, 3H, NCH3-7), 3.21
ppm (t, J = 8.5 Hz, 2H, H2-3'); 13C NMR: δ=169.3 (C=O), 157.2
(C-4), 156.2 (C-4''), 151.6 (C-2), 150.3 (C-7a), 146.2 (C-2'''), 141.9
(C-7'a), 133.7 (C-1''), 132.8 (C-3'a), 130.6 (C-2'', C-6''), 129.8 (C-
5'), 128.5 (C-4'''), 128.1 (C-5'''), 127.1 (C-6'), 124.9 (C-4'), 124.0
(C-6), 116.2 (C-7'), 114.9 (C-5, C-3'', C-5''), 99.9 (C-4a), 59.2
Ether prodrug synthesis
1-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-
yl)indolin-1-yl)-2-(2-((1-methyl-2-nitro-1H-imidazol-5-
yl)methoxy)phenyl)ethan-1-one (6b). Phenol 6a (80 mg, 0.20
mmol), chloride 18 (39 mg, 0.22 mmol) and KI (37 mg, 0.22 mmol)
were suspended in DMF (5 mL). K2CO3 (55 mg, 0.40 mmol) was
added to the mixture, which was heated at 70 °C for 72 h. The
mixture was diluted with EtOAc (50 mL), washed with water (2 ×
50 mL), brine (50 mL), dried and concentrated in vacuo. The
crude residue was purified by preparative HPLC [gradient (95–
40–95 %) ammonium formate pH 3.45/90 % CH3CN/H2O].
Solvent was removed and the residue was diluted with EtOAc (50
mL), washed with sat. NaHCO3 (50 mL) and brine (50 mL), dried
and concentrated. The resulting residue was triturated with EtOAc
to obtain ether 6b (33 mg, 31 %) as a yellow solid: m.p. 228–
230 °C; 1H NMR: δ=8.14 (s, 1H, H-2), 8.03 (d, J = 8.3 Hz, 1H, H-
7'), 7.33–7.28 (m, 2H, H-4'', H-4'''), 7.26–7.17 (m, 5H, H-6, H-4',
H-6', H-3'', H-6''), 6.98 (td, J = 7.4, 0.8 Hz, 1H, H-5''), 6.10 (br s,
2H, NH2), 5.24 (s, 2H, OCH2), 4.11 (t, J = 8.6 Hz, 2H, H2-2'), 3.79
(s, 3H, NCH3-1'''), 3.73 (s, 5H, CH2, NCH3-7), 3.06 (t, J = 8.6 Hz,
9
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10.1002/asia.201801826
Chemistry - An Asian Journal
FULL PAPER
(OCH2), 47.9 (C-2'), 41.2 (COCH2), 34.3 (NCH3-1'''), 30.7 (NCH3-
7), 27.5 ppm (C-2'); HRMS (ESI): m/z calcd for C28H27N8O4:
539.2150 [M+H]+; found: 539.2175; HPLC purity: 93.9 % (0.03 %
effector).
(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl (4-(2-(5-(4-amino-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)indolin-1-yl)-2-
oxoethyl)phenyl)carbamate (11b). Prepared from 11a and 19
using the same procedure as for 9b. Purification by preparative
1
HPLC afforded 11b (9 %) as an orange solid: m.p. >300°C; H
NMR: δ=9.82 (s, 1H, NHCOO), 8.13–8.11 (m, 2H, H-2, H-7'), 7.41
(d, J = 8.24 Hz, 2H, H-3'', H-5''), 7.31–7.29 (m, 2H, H-4', H-4'''),
7.24–7.21 (m, 4H, H-6, H-6', H-2'', H-6''), 6.04 (br s, 2H, NH2-4),
5.28 (s, 2H, OCH2), 4.19 (t, J = 8.4 Hz, 2H, H2-2'), 3.97 (s, 3H,
NCH3-1'''), 3.79 (s, 2H, COCH2), 3.72 (s, 3H, NCH3-7), 3.20 ppm
(t, J = 8.4 Hz, 2H, H2-3'); 13C NMR: δ=169.2 (C=O), 157.1 (C-4),
152.7 (NHCOO), 151.6 (C-2), 150.3 (C-7a), 146.1 (C-2'''), 141.9
(C-7'a), 137.2 (C-4''), 133.4 (C-5'''), 132.8 (C-3'a), 129.8 (C-5', C-
2'', C-6''), 129.3 (C-1''), 128.8 (C-4'''), 127.1 (C-6'), 124.8 (C-4'),
124.0 (C-6), 118.3 (C-3'', C-5''), 116.2 (C-7'), 114.9 (C-5), 99.8 (C-
4a), 55.1 (OCH2), 47.9 (C-2'), 41.5 (COCH2), 34.2 (NCH3-1'''),
30.7 (NCH3-7), 27.5 ppm (C-3'); MS (APCI): m/z (%): 423.1 (100)
[M+H]+; HRMS (ESI): m/z calcd for C29H28N9O5: 582.2208 [M+H]+;
found: 582.2223; HPLC purity: 97.8 % (0.04 % effector).
Carbamate prodrug synthesis
(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl (2-(2-(5-(4-amino-7-
methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)indolin-1-yl)-2-
oxoethyl)phenyl)carbamate (9b). To a stirred solution of amine
9a (92 mg, 0.23 mmol) in THF (10 mL) was added DIPEA (80 µL,
0.46 mmol), HOBt (39 mg, 0.25 mmol) and 5 Å molecular sieves
(82 mg). 4-Nitrophenyl carbonate 19 (82 mg, 0.25 mmol) was
added and the mixture allowed to stir at 20 °C for 72 h. The
resulting mixture was diluted with EtOAc (50 mL), washed with
water (50 mL), brine (50 mL) and concentrated. The crude residue
was purified by preparative HPLC [gradient (95–40–95 %)
ammonium formate pH 3.45/90 % CH3CN/H2O]. Solvent was
removed and the residue diluted with EtOAc (20 mL), washed with
water (20 mL) and brine (20 mL) sequentially, dried and
concentrated in vacuo. The carbamate 9b (60 mg, 46 %) was
obtained as a pale orange solid: m.p. 172 (decomp.)°C; 1H NMR:
δ=9.26 (s, 1H, NHCOO), 8.28 (s, 1H, H-2), 8.08 (d, J = 8.2 Hz,
1H, H-7'), 7.52 (d, J = 7.3 Hz, 1H, H-3''), 7.39 (s, 1H, H-6), 7.31–
7.26 (m, 4H, H-4', H-4'', H-6'', H-4'''), 7.22 (dd, J = 8.2, 1.5 Hz, 1H,
H-6'), 7.14 (td, J = 7.5, 0.9 Hz, 1H, H-5''), 6.75 (br s, 2H, NH2),
5.26 (s, 2H, OCH2), 4.19 (t, J = 8.4 Hz, 2H, H2-2'), 3.91 (s, 3H,
NCH3-1'''), 3.89 (s, 2H, COCH2), 3.77 (s, 3H, NCH3-7), 3.21 ppm
(t, J = 8.4 Hz, 2H, H2-3'); 13C NMR: δ=168.9 (C=O), 163.0 (C-2''),
154.6 (C-4), 153.4 (NHCOO), 149.1 (C-7a), 147.8 (C-2), 146.0
(C-2'''), 142.0 (C-7'a), 136.7 (C-1''), 133.4 (C-5'''), 133.0 (C-3'a),
130.9 (C-6''), 128.9 (C-5'), 128.7 (C-4'''), 127.3 (C-4''), 127.1 (C-
6'), 125.3 (C-6), 124.9 (C-4'), 124.8 (C-5''), 124.2 (C-3''), 116.3 (C-
7'), 116.2 (C-5), 99.2 (C-4a), 55.3 (OCH2), 47.9 (C-2'), 38.5
(COCH2), 34.2 (NCH3-1'''), 31.1 (NCH3-7), 27.4 ppm (C-3'); MS
(APCI): m/z (%): 423.1 (100) [M+H]+; HRMS (ESI): m/z calcd for
C29H28N9O5: 582.2208 [M+H]+; found: 582.2231; HPLC purity:
99.6 % (0.03 % effector).
Radiolytic reduction
Solutions (10 µM) of the prodrugs in deoxygenated 0.1M sodium
formate/5 mM sodium phosphate buffer (pH 7.0), with a final
DMSO concentration of 0.1%, were prepared in a Bactron Pd/H2-
scrubbed anaerobic chamber (Sheldon Manufacturing Inc.
Cornelius, OR), transferred to HPLC vials and sealed. Solutions
were irradiated with 40 Gy (ca 2.5 Gy/min, Eldorado78 60Cobalt
source) and stored at -80 °C until analysis by LC-MS (See below).
The chromatograms of the irradiated samples were compared to
prodrug and effector samples without radiation (See Figure S2).
Cell culture
HCT116 colorectal carcinoma cells were cultured in alpha
minimum essential medium (αMEM, ThermoFisher Scientific,
Auckland, New Zealand) supplemented with 5% (v/v) fetal bovine
serum (FBS; Moregate Biotech, Hamilton, NZ). Cell line
authenticity was confirmed by short tandem repeat analysis and
cultures were demonstrated to be mycoplasma negative.
(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl(3-(2-(5-(4-amino-7-
methyl-7H-pyrrolo[2,3-d]
pyrimidin-5-yl)indolin-1-yl)-2-
oxoethyl)phenyl)carbamate (10b). Prepared from 10a and 19
using the same procedure as for 9b. Purification by preparative
HPLC afforded 10b (58 %) as an orange solid: m.p. 184°C
(decomp.); 1H NMR: δ=9.83 (s, 1H, NHCOO), 8.14–8.11 (m, 2H,
H-2, H-7'), 7.42 (s, 1H, H-2''), 7.36 (d, J = 8.0 Hz, 1H, H-4''), 7.30–
7.21 (m, 5H, H-6, H-4', H-6', H-5'', H-4'''), 6.95 (d, J = 7.5 Hz, 1H,
H-6''), 6.04 (s, 2H, NH2-4), 5.28 (s, 2H, OCH2), 4.19 (t, J = 8.4 Hz,
2H, H2-2'), 3.97 (s, 3H, NCH3-1'''), 3.83 (s, 2H, COCH2), 3.72 (s,
3H, NCH3-7), 3.20 ppm (t, J = 8.4 Hz, 2H, H2-3'); 13C NMR:
δ=168.9 (C=O), 157.1 (C-4), 152.7 (NHCO), 151.6 (C-2), 150.3
(C-7a), 146.1 (C-2'''), 141.8 (C-7'a), 138.8 (C-3''), 135.7 (C-1''),
133.3 (C-5'''), 132.8 (C-3'a), 129.8 (C-5'), 128.8 (C-5'', C-4'''),
127.1 (C-6'), 124.9 (C-4'), 124.0 (C-6, C-6''), 119.2 (C-2''), 116.6
(C-4''), 116.2 (C-7'), 114.9 (C-5), 99.8 (C-4a), 55.1 (OCH2), 47.9
(C-2'), 42.3 (COCH2), 34.2 (NCH3-1'''), 30.7 (NCH3-7), 27.5 ppm
(C-3'); MS (APCI): m/z (%): 423.1 (100) [M+H]+; HRMS (ESI): m/z
calcd for C29H28N9O5: 582.2208 [M+H]+; found: 582.2214; HPLC
purity: 99.8 % (0.01 % effector).
RT-qPCR
Cells (2 × 105) were seeded in 6-well plates (ThermoFisher
Scientific) in 1.5 mL αMEM + 5% FBS. An additional 1.5 mL of
αMEM + 5% FBS was added containing either compound to
achieve a final concentration of 10 μM, or an equivalent volume
of DMSO (final concentration of 0.1%). Samples were then
cultured in either 20%O2/5%CO2/N2 (oxic) or in 0%O2/5% CO2/N2
(anoxic) conditions (H45 HEPA Hypoxystation; Don Whitley
Scientific, West Yorkshire, United Kingdom). After 24 h incubation,
the culture medium was aspirated and RNA was extracted using
TRIzol reagent (ThermoFisher Scientific), quantified by NanoDrop
(ThermoFisher Scientific) and converted to cDNA using a High
Capacity cDNA Reverse Transcription Kit (ThermoFisher
Scientific). Quantitative PCR was carried out using SYBER Select
Master Mix (ThermoFisher Scientific) on a QuantStudio 6 Flex
Real-Time PCR instrument. Relative gene expression was
10
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10.1002/asia.201801826
Chemistry - An Asian Journal
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calculated using the comparative CT method (2-ΔΔCT method).
Primer sequences were as follows: DDIT3 (CHOP) Forward 5'-
Keywords: drug delivery, hypoxia, kinase inhibitor,
nitroimidazole, prodrug
GGAGCATCAGTCCCCCACTT,
TGTGGGATTGAGGGTCACATC;
Reverse
PPP1R15A
5'-
(GADD34)
Forward 5'-CCCAGAAACCCCTACTCATGATC, Reverse 5'-
GCCCAGACAGCCAGGAAAT;
CCAGTCAACAGGGGACATAAA,
HPRT1
Forward
Reverse
5'-
5'-
References:
CACAATCAAGACATTCTTTCCAGT.
[1]
M. Koritzinsky, F. Levitin, T. van den Beucken, R. A. Rumantir, N. J.
Harding, K. C. Chu, P. C. Boutros, I. Braakman, B. G. Wouters, J. Cell
Biol. 2013, 203, 615–627.
Western Immunoblotting
[2]
D. E. Feldman, V. Chauhan, A. C. Koong, Mol. Cancer Res. 2005, 3,
597–605.
Samples were prepared as above, except 1 × 106 cells were
seeded into 10 cm culture dishes (Corning NY) in 7.5 mL αMEM
+ 5% FBS and an additional 7.5 mL of αMEM + 5% FBS
containing compound/0.1% DMSO added. After 24 h oxic or
anoxic incubation, cell monolayers were washed twice using 10
mL of ice-cold PBS and then lysed in 0.5 mL RIPA buffer (50 mM
Tris-HCl pH 7.4, 1% IGEPAL® CA-630, 0.25% sodium
[3]
[4]
[5]
D. Ron, P. Walter, Nat. Rev. Mol. Cell Biol. 2007, 8, 519–529.
M. Wang, R. J. Kaufman, Nat. Rev. Cancer 2014, 14, 581–597.
J. Han, S. H. Back, J. Hur, Y.H. Lin, R. Gildersleeve, J. Shan, C. L. Yuan,
D. Krokowski, S. Wang, M. Hatzoglou, M. S. Kilberg, M. A. Sartor, R. J.
Kaufman, Nat. Cell Biol. 2013, 15, 481–490.
[6]
M. Bi, C. Naczki, M. Koritzinsky, D. Fels, J. Blais, N. Hu, H. Harding, I.
Novoa, M. Varia, J. Raleigh, D. Scheuner, R. J. Kaufman, J. Bell, D. Ron,
B. G. Wouters, C. Koumenis. EMBO J. 2005, 24, 3470–3481.
D. Cojocari, R. N. Vellanki, B. Sit, D. Uehling, M. Koritzinsky, B. G.
Wouters, Radiother. Oncol. 2013, 108, 541–547.
[7]
[8]
[9]
deoxycholate, 0.1% SDS, 150 mM NaCl,
1 mM EDTA)
supplemented with 1 × protease inhibitor (P8340; Sigma-Aldrich,
St Louis, MO). Protein lysates were separated on 4–12% Bis-Tris
gels (ThermoFisher Scientific). Proteins were then wet transferred
onto polyvinylidene fluoride membranes (Merck Millipore,
Burlington, MA), blocked for 1 h with 5% non-fat milk in TBS-T
and then incubated overnight at 4 °C with primary antibody diluted
1:1000 in blocking buffer. Detection of signal utilised HRP-
conjugated secondary antibodies (ThermoFisher Scientific),
chemiluminescent reagent (ThermoFisher Scientific) and imaging
using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA).
Primary antibodies were as follows; ATF4 (clone D4B8, Cell
Signaling Technology, Danvers, MA), CHOP (clone L63F7, Cell
Signaling Technology), β-actin (clone C4, Merck Millipore).
A. Bertolotti, Y. Zhang, L. M. Hendershot, H. P. Harding, D. Ron, Nat.
Cell. Biol. 2000, 2, 326–332.
M. Carrara, F. Prischi, P. R. Nowak, M. M. Ali, EMBO J. 2015, 34, 1589–
1600.
[10] H. P. Harding, Y. Zhang, H. Zeng, I. Novoa, P. D. Lu, M. Calfon, N. Sadri,
Y. Chi, B. Popko, R. Paules, D. F. Stojdl, J. C. Bell, T. Hettmann, J. M.
Leiden, D. Ron. Mol. Cell 2003, 11, 619–633.
[11] J. M. Axten, J. R. Medina, Y. Feng, A. Shu, S. P. Romeril, S. W. Grant,
W. H. H. Li, D. A. Heerding, E. Minthorn, T. Mencken, C. Atkins, Q. Liu,
S. Rabindran, R. Kumar, X. Hong, A. Goetz, T. Stanley, J. D. Taylor, S.
D. Sigethy, G. H. Tomberlin, A. M. Hassell, K. M. Kahler, L. M. Shewchuk,
R. T. Gampe, J. Med. Chem. 2012, 55, 7193–7207.
[12] J. M. Axten, S. P. Romeril, A. Shu, J. Ralph, J. R. Medina, Y. Feng, W.
H. H. Li, S. W. Grant, D. A. Heerding, E. Minthorn, T. Mencken, N. Gaul,
A. Goetz, T. Stanley, A. M. Hassell, R. T. Gampe, C. Atkins, R. Kumar,
ACS Med. Chem. Lett. 2013, 4, 964–968.
[13] A. L. Smith, K. L. Andrews, H. Beckmann, S. F. Bellon, P. J. Beltran, S.
Booker, H. Chen, Y.-A. Chung, N. D. D’Angelo, J. Dao, K. R.
Dellamaggiore, P. Jaeckel, R. Kendall, K. Labitzke, A. M. Long, S.
Materna-Reichelt, P. Mitchell, M. H. Norman, D. Powers, M. Rose, P. L.
Shaffer, M. M. Wu, J. R. Lipford, J. Med. Chem. 2015, 58, 1426–1441.
[14] C. Atkins, Q. Liu, E. Minthorn, S.-Y. Zhang, D. J. Figueroa, K. Moss, T.
B. Stanley, B. Sanders, A. Goetz, N. Gaul, A. E. Choudhry, H. Alsaid, B.
M. Jucker, J. M. Axten, R. Kumar, Cancer Res. 2013, 73, 1993–2002.
[15] H. P. Harding, A. F. Zyryanova, D. Ron. J. Biol. Chem. 2012, 287, 44338–
44344.
Mass spectrometry methods
Extracellular medium samples (from RT-qPCR plates) were
frozen at -80 °C after collection until analysis. Samples were
thawed on ice and vortexed vigorously. An aliquot (100 µL) of the
sample was added into 200 µL of ice cold methanol, spun at
13000 rpm, 4 °C for 2 minutes. The supernatant was collected
and analysed on an Agilent Technologies Single Quadrupole MS
6450b LC-MS interfaced with an Agilent Jet Stream Electrospray
Ionization (ESI) source allowing positive or negative ion detection.
Mobile phases were (A) 45 mM formate buffer, pH 4.5 and (B)
80 % acetonitrile + 0.01 % formic acid with a Zorbax, C18 3 x 150
mm, 5 µm column running at a flow rate of 0.5 mL/min with an
injection volume of 50 µL. Three point calibration curves (10, 3.33,
1.11 µM) were made fresh on the day of analysis.
[16] Q. Yu, B. Zhao, J. Gui, K. V. Katlinski, A. Brice, Y. Gao, C. Li, J. A.
Kushner, C. Koumenis, J. A. Diehl, S. Y. Fuchs, Proc. Natl. Acad. Sci. U.
S. A. 2015, 112, 15420–15425.
[17] J. D. Blais, C. L. Addison, R. Edge, T. Falls, H. Zhao, K. Wary, C.
Koumenis, H. P. Harding, D. Ron, M. Holcik, J. C. Bell, Mol. Cell. Biol.
2006, 26, 9517–9532.
[18] K. M. Rouschop, L. J. Dubois, T. G. Keulers, T. van den Beucken, P.
Lambin, J. Bussink, A. J. van der Kogel, M. Koritzinsky, B. G. Wouters,
Proc. Natl Acad. Sci. USA, 2013, 110, 4622-4627.
[19] H. Mujcic, A. Nagelkerke, K. M. Rouschop, S. Chung, N. Chaudary, P. N.
Span, B. Clarke, M. Milosevic, J. Sykes, R. P. Hill, M. Koritzinsky, B. G.
Wouters, Clin. Cancer Res. 2013, 19, 6126–6137.
Acknowledgements
The authors thank Dr Jack Flanagan for helpful discussions and
thank Dr Adrian Blaser, Ms Karin Tan, and Mr Sisira Kumara for
technical assistance. The authors would like to acknowledge the
support of the University of Auckland Faculty Research
Development Fund.
[20] Y. X. Feng, E. S. Sokol, C. A. Del Vecchio, S. Sanduja, J. H. Claessen,
T. A Proia, D. X. Jin, F. Reinhardt, H. L. Ploegh, Q. Wang, P. B. Gupta,
Cancer Discov. 2014, 4, 702–715.
[21] N. Dhani, A. Fyles, D. Hedley, M. Milosevic, Sem. Nuclear Med. 2015,
45, 110–121.
11
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10.1002/asia.201801826
Chemistry - An Asian Journal
FULL PAPER
[22] S. M. Evans, K. L. Du, A. A. Chalian, R. Mick, P. J. Zhang, S. M. Hahn,
H. Quon, R. Lustig, G. S. Weinstein, C. J. Koch, Int. J. Radiat. Oncol.
Biol. Phys. 2007, 69, 1024–1031.
[51] M. P. Hay, W. R. Wilson, W. A. Denny, Tetrahedron 2000, 56, 645–657.
[23] P. Vaupel, A. Mayer, Cancer Metastasis Rev. 2007, 26, 225–239.
[24] S. Chouaib, M.Z. Noman, K. Kosmatopoulos, M.A. Curran, Oncogene
2017, 36, 439–445.
[25] M. P. Hay, K. O. Hicks, J. Wang, in Tumor Microenvironment and Cellular
Stress (Eds.: C. Koumenis, E. Hammond, A. Giaccia), Springer New
York, New York, NY, 2014, pp. 111–145.
[26] W. R. Wilson, M. P. Hay, Nat. Rev. Cancer 2011, 11, 393–410.
[27] I. N. Mistry, M. Thomas, E. D. D. Calder, S. J. Conway, E. M. Hammond,
Int. J. Radiat. Oncol. Biol. Phys. 2017, 98, 1183–1196.
[28] R. M. Phillips, Cancer Chemother. Pharmacol. 2016, 77, 441-457.
[29] A. V. Patterson, D. M. Ferry, S. J. Edmunds, Y. Gu, R. S. Singleton, K.
Patel, S. M. Pullen, K. O. Hicks, S. P. Syddall, G. J. Atwell, S. Yang, W.
A. Denny, W. R. Wilson, Clin. Cancer Res. 2007, 13, 3922–3942.
[30] M. P. Hay, K. O. Hicks, W.R. Wilson, in Comprehensive Medicinal
Chemistry III, Vol. 8, Case Histories in Recent Drug Discovery (Eds.: D.
Rotella, S. Chackalamannil, S. Ward), Elsevier, Amsterdam, Netherlands,
2017, pp. 58–94.
[31] K. O. Hicks, B. G. Siim, J. K. Jaiswal, F. B. Pruijn, A. M. Fraser, R. Patel,
A. Hogg, H. D. S. Liyanage, M. J. Dorie, J. M. Brown, W. A. Denny, M.
P. Hay, W. R. Wilson, Clin. Cancer Res. 2010, 16, 4946–4956.
[32] J. D. Sun, Q. Liu, J. Wang, D. Ahluwalia, D. Ferraro, Y. Wang, J.-X. Duan,
W. S. Ammons, J. G. Curd, M. D. Matteucci, C. P. Hart, Clin. Cancer Res.
2012, 18, 758–770.
[33] G.-L. Lu, A. Ashoorzadeh, R. F. Anderson, A. V. Patterson, J. B. Smaill,
Tetrahedron 2013, 69, 9130–9138.
[34] L. J. O'Connor, C. Cazares-Körner, J. Saha, C. N. G. Evans, M. R. L.
Stratford, E. M. Hammond, S. J. Conway, Nat. Protoc. 2016, 11, 781–
794.
[35] R. Zhu, M.-C. Liu, M.-Z. Luo, P. G. Penketh, R. P. Baumann, K. Shyam,
A. C. Sartorelli, J. Med. Chem. 2011, 54, 7720–7728.
[36] M. P. Hay, G. J. Atwell, W. R. Wilson, S. M. Pullen, W. A. Denny, J. Med.
Chem. 2003, 46, 2456–2466.
[37] M. P. Hay, B. M. Sykes, W. A. Denny, C. J. O’Connor, J. Chem. Soc.,
Perkin Trans. 1 1999, 2759–2770.
[38] M. P. Hay, B. M. Sykes, W. A. Denny, W. R. Wilson, Bioorg. Med. Chem.
Lett. 1999, 9, 2237–2242.
[39] M. P. Hay, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. Lett. 1999,
9, 3417–3422.
[40] M. Tercel, A. E. Lee, A. Hogg, R. F. Anderson, H. H. Lee, B. G. Siim, W.
A. Denny, W. R. Wilson, J. Med. Chem. 2001, 44, 3511–3522.
[41] R. F. Borch, J. Liu, J. P. Schmidt, J. T. Marakovits, C. Joswig, J. J. Gipp,
R. T. Mulcahy, J. Med. Chem. 2000, 43, 2258–2265.
[42] J.-X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J. W. Evans, L. Lan, G.
Lorente, M. Banica, D. Jung, J. Wang, H. Ma, X. Li, Z. Yang, R. M.
Hoffman, W. S. Ammons, C. P. Hart, M. Matteucci, J. Med. Chem. 2008,
51, 2412–2420.
[43] S. A. Everett, M. A. Naylor, K. B. Patel, M. R. L. Stratford, P. Wardman,
Bioorg. Med. Chem. Lett. 1999, 9, 1267–1272.
[44] I. Parveen, D. P. Naughton, W. J. D. Whish, M. D. Threadgill, Bioorg.
Med. Chem. Lett. 1999, 9, 2031–2036.
[45] M. P. Hay, R. F. Anderson, D. M. Ferry, W. R. Wilson, W. A. Denny, J.
Med. Chem. 2003, 46, 5533–5545.
[46] M. P. Hay, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. 2005, 13,
4043-4055.
[47] L. J. O'Connor, C. Cazares-Korner, J. Saha, C. N. G. Evans, M. R. L.
Stratford, E. M. Hammond, S. J. Conway, Org. Chem. Front. 2015, 2,
1026–1029.
[48] F. W. Hunter, R. J. Young, Z. Shalev, R. N. Vellanki, J. Wang, Y. Gu, N.
Joshi, S. Sreebhavan, I. Weinreb, D. P. Goldstein, J. Moffat, T. Ketela,
K. R. Brown, M. Koritzinsky, B. Solomon, D. Rischin, W. R. Wilson, B. G.
Wouters, Cancer Res. 2015, 75, 4211–4223.
[49] D. Rojas-Rivera, T. Delvaeye, R. Roelandt, W. Nerinckx, K. Augustyns,
P. Vandenabeele, M. J. M. Bertrand, Cell Death Differentiat. 2017, 24,
1100–1110.
[50] B. Budke, J. H. Kalin, M. Pawlowski, A. S. Zelivianskaia, M. Wu, A. P.
Kozikowski, P. P. Connell, J. Med. Chem. 2013, 56, 254–263.
12
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Entry for the Table of Contents
NO2
N
HYPOXIA-
SELECTIVE
ACTIVATION
MeN
O
O
N
NH2
N
NH2
O
HN
NH2
N
N
N
N
O
N
N
Me
Prodrug
Me
Effector
Tumour hypoxia causes endoplasmic reticulum (ER) stress which is moderated through protein kinase R-like ER kinase (PERK)
mediated signaling. However, hypoxia also presents a tumour-selective target for prodrug delivery. We designed and prepared hypoxia-
activated prodrugs of PERK inhibitors and evaluated their activity as PERK inhibitors, their hypoxia-selective activation and their
modulation of PERK signalling in cells.
13
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