Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study

Article abstract of DOI:10.1016/j.bmcl.2019.03.015

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclis

Full text of DOI:10.1016/j.bmcl.2019.03.015

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1215–1219
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated
by tumour hypoxia: A chemical proof of concept study
Geraud N. Sansom , Nicholas S. Kirk , Christopher P. Guisec,d, Robert F. Andersonc,d,
a,b
a,b
c,d
c,d
a,b,⁎
Jeff B. Smaill , Adam V. Patterson , Michael J. Kelso
a
b
c
Molecular Horizons and School of Chemistry & Molecular Bioscience, University of Wollongong, NSW 2522, Australia
Illawarra Health & Medical Research Institute, Wollongong, NSW 2522, Australia
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
The Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as
prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was
shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase
inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell
testing and reduction potential measurements support optimisation of the compounds towards tumour-selective
mutual prodrugs.
Hypoxia
Mutual prodrug
Anticancer
Bioreductive activation
Sunitinib
Semaxinib
Floxuridine
4
-Aminoaniline mustard
Cytotoxic drugs have been the mainstay of many anticancer treat-
activated ‘mutual’ prodrug scaffolds,11 wherein hypoxia-dependent
bioactivation of the 2-nitrophenacetyl moiety and ensuing cyclisation
of the 2-aniline might simultaneously evolve an active cytotoxin (for-
merly incorporated within the prodrug as an amide or ester) along with
the kinase inhibitor semaxanib (SU5416) 3, a clinically evaluated pre-
ment regimens but their effectiveness is limited by low tumour cell
selectivity and, as a consequence, dose-limiting side effects. Tumour-
activated prodrugs that selectively deliver cytotoxins to the immediate
tumour microenvironment are sought as a means of improving the
pharmacodynamics of cytotoxic drugs and reducing their toxicity to-
1
2–14
decessor of sunitinib
as a functionalised version of Denny’s indolin-
1
–4
wards non-cancerous tissues.
Nitroaromatics have received intense
2-one waste fragment. Furthermore, we noted that a hydrogen bond
was invariably present between the pyrrole-NH and carbonyl oxygen
atoms in our 2-nitrophenylacetic amides and esters (Fig. 1(a)), sug-
gesting the scaffold may be pre-organised and activated for rapid in-
study as prodrugs that can undergo reductive metabolism (bioactiva-
tion) to hydroxylamine or amine derivatives within the hypoxic en-
5
–8
vironment of solid tumours.
The newly revealed amine nucleophiles
1
0
can then serve to trigger reactions that release the active cytotoxin. In
doline-2-one cyclisation following reduction to the aniline. Con-
ceptually, mutual prodrugs of this type could mitigate side effects
arising from either or both drugs while at the same time increasing
tumor concentrations of the actives and improving pharmacodynamics.
Two mutual prodrug prototypes were conceived to explore the
concept. Prodrug 1 incorporated Denny’s 4-aminoaniline mustard
linked to the 2-nitrophenylacetate scaffold via an amide (Fig. 1(b)). In
this compound, the amide would serve to withdraw electron density
from the mustard nitrogen, thereby reducing DNA reactivity of the
prodrug. The 4-aminoaniline mustard released into hypoxic tumors
following prodrug activation would be vastly more DNA-reactive and
cytotoxic due to increased electron density on the mustard nitrogen
1
994, the Denny group reported 2-nitrophenyl acetamides as hypoxia-
9
activated prodrug scaffolds, where spontaneous cyclisation of the
nascent 2-aminophenyl acetamide expelled an activated 4-aminoaniline
mustard cytotoxin and an indoline-2-one waste fragment. More re-
cently, we reported the synthesis and anti-angiogenic properties of 2-
nitrophenylacetate derivatives related to the drug sunitinib (Sutent®),
an indoline-2-one-based multi-kinase inhibitor used in the treatment of
highly vascularised renal cell carcinomas and gastrointestinal stromal
1
0
tumours (Fig. 1(a)).
9
When considered in the context of Denny and colleagues’ work, we
noted that our 2-nitrophenylacetates could potentially serve as tumor-
⁎
Corresponding author at: Molecular Horizons and School of Chemistry & Molecular Bioscience, University of Wollongong, NSW 2522, Australia.
E-mail address: mkelso@uow.edu.au (M.J. Kelso).
https://doi.org/10.1016/j.bmcl.2019.03.015
Received 9 December 2018; Received in revised form 7 March 2019; Accepted 10 March 2019
Available online 13 March 2019
0960-894X/ © 2019 Elsevier Ltd. All rights reserved.

G.N. Sansom, et al.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 1215–1219
Fig. 1. (a) Anti-angiogenic 2-nitrophenylacetate de-
1
0
rivatives related to sunitinib. (b) Prototype mutual
prodrugs 1 and 2 and their proposed mechanism of
bioreductive activation to selectively release the in-
doline-2-one kinase inhibitor semaxanib 3 and 4-
aminoaniline mustard or floxuridine cytotoxins
within hypoxic regions of tumors.
driving formation of DNA-alkylating aziridinium ions.9,15 Prodrug 2
linked the 2-nitrophenylacetate scaffold via an ester to the primary
alcohol of the anticancer drug floxuridine (Fig. 1(b)). As a nucleoside
antimetabolite requiring phosphorylation as part of its mechanism, the
drug should have reduced activity while tethered to the prodrug.
Within normoxic tissues, the nitro radical anion intermediate formed on
each prodrug following one-electron reduction of the nitro group by
human reductases is predicted to be back-scavenged by molecular
oxygen, regenerating the prodrug. Within hypoxic tissues, further re-
duction of this intermediate to the amine 6-electron reduction product
of each prodrug could occur, giving rise to hypoxia-selective trig-
precursor 8 in modest yield (21%) with no evidence of formation of the
1
E-isomer in the H NMR spectrum of the crude reaction mixture. De-
spite trialling many other pyrrole protecting groups, bases, solvents and
reaction conditions, the yield of 8 could not be improved. Performing
the Knoevenagel reaction with 7 and unprotected 5 gave none of the
desired bis-alcohol. Deprotection of 8 using TBAF buffered with acetic
acid in THF at 0 °C delivered the bis-alcohol 9 in 86% yield. Final
conversion to the target mustard 1 was accomplished in 80% yield over
2 steps by first converting 9 to the bis-mesylate (MsCl, triethylamine)
and then heating the freshly prepared crude with LiCl at 75 °C in DMF.
The synthesis of 2 commenced with bis-TBS protection of the 1° and
2° alcohols of commercially available floxuridine (Scheme 2). The re-
action proceeded well under standard conditions (TBDMSCl/imidazole)
to give 10 in excellent yield (97%). Similarly high yields were pre-
1
6
gering.
The synthesis of
1
commenced with the coupling of 2-ni-
trophenylacetic acid and p-phenylenediamine using HBTU/diisopro-
pylethylamine to give 4 in 80% yield (Scheme 1). N,N-dialkylation of 4
1
7
viously reported for the bis-TBS protection of thymidine. Treatment
of 10 with freshly distilled 2,4-dimethoxybenzoyl chloride, diisopro-
pylethylamine and DMAP gave the N-protected derivative 11 in 87%
yield. Selective silyl-deprotection of the less hindered 1° alcohol using
with 2-bromoethanol in the presence of K
2
CO afforded bis-alcohol 5 in
3
6
2% yield. Mono-alkylation was achieved within hours but prolonged
heating at 50 °C was required to obtain an acceptable yield of 5. Bis-
alcohol protection of using tert-butyldimethylsilyl chloride
TBDMSCl)/imidazole provided 6 in 86% yield. Reacting 6 with N-
1
8
5
aqueous trichloroacetic acid (4.2 M) in THF (1:4) delivered 12 in 50%
19
(
yield. Steglich esterification of 12 with 2-nitrophenylacetic acid
proceeded smoothly to give 13 (87%). Condensation of 13 with pyrrole
methylcarbamoyl pyrrole aldehyde 7 using our reported Knoevenagel
conditions10 (K
CO , 18-crown-6, THF, reflux) delivered the advanced
aldehyde 7 using our Knoevenagel conditions produced a mixture of
10
2
3
Scheme 1. Synthesis of mutual prodrug 1.
1216

G.N. Sansom, et al.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 1215–1219
Scheme 2. Synthesis of mutual prodrug 2.
cis and trans isomers in modest yield ((Z)-14 10%; (E)-14 11%). Ex-
ranging from 37 to 84%. N-hydroxy semaxanib 16, formed by cyclisa-
tion of the intermediate N-hydroxylamine 4-electron reduction product,
was also isolated from reactions in 11–51% yield. The combined yields
of indoline-2-ones 3 and 16 were invariably high (80–94%) suggesting
cyclisation within the scaffold is highly favoured. Downfield-shifting of
tensive efforts to improve the yield were unsuccessful. Removal of the
2
,4-dimethoxy benzoyl protecting group from (Z)-14 with ammonia in
methanol gave the penultimate precursor 15 (75%) and final depro-
tection of the TBS group with TBAF/acetic acid delivered prodrug 2
2
0
1
(
69%). Attempts to access the two prodrugs by an alternative route
the pyrrole-NH signals in the H NMR spectrum of 1 (δ 12.04 ppm) and
involving direct ester/amide coupling of cytotoxins to
a
2-ni-
2 (δ 11.66 ppm) (and all pyrrole-containing intermediates) confirmed
both cis stereochemistry for the compounds and the presence of an
intramolecular hydrogen bond between the pyrrole-NH and carbonyl
trophenylacetic acid derivative containing the pre-installed pyrrole
group (obtained from the allyl ester in Fig. 1(a)) were not successful,
but led to the discovery of novel bioactive 3-imino-2-(pyrrol-2-yl) isa-
1
0
oxygen atoms. This H-bond in 1 and 2 likely serves as a pseudo-acid
catalyst that promotes spontaneous cyclisation.
2
1
22
togens and pyrrolizin-3-ones.
Proof of concept reactions were performed with 1 and 2 to establish
whether chemical reduction of the arylnitro groups would trigger
spontaneous cyclisation of the nascent anilines to generate semaxanib 3
and, by inference, the respective 4-aminoaniline mustard and floxur-
idine cytotoxins. Three standard room temperature arylnitro reduction
Preliminary biological testing of 1 and 2 was carried out using
MDA-MB-468 triple-negative breast cancer cells and SW620 colon
cancer cells (Table 2). The prodrugs were assessed first for their degree
of deactivation relative to parent cytotoxin control compounds under
aerobic conditions. For prodrug 1, melphalan was selected as the
2
3
26
reactions were employed: Fe/CH
3
COOH in EtOH/H
2
O
O, FeCl
3
·6H
2
O/
clinically relevant nitrogen mustard control and for prodrug 2 the
2
4
25
Zn in DMF/H
2
O
and NaBH
4
/Pd-C in MeOH/H
2
(Table 1). Se-
comparator was Floxuridine. The degree of prodrug deactivation was
maxanib 3 was isolated from all reactions within 0.5–3 h in yields
determined using the deactivation ratio (DR = Aerobic IC50prodrug/
Table 1
Proof of concept chemical reductions with mutual prodrugs 1 and 2.
Prodrug
Reductant
Fe/CH COOH
Solvent
t (h)
Yield 3 (%)
Yield 16 (%)
Yield 3 + 16 (%)
1
3
EtOH/H
2
O
O
1
3
80
53
43
14
32
37
94
85
80
FeCl ·6H O/Zn
3
2
DMF/H
2
NaBH /Pd-C
4
MeOH/H
2
O
0.5
2
Fe/CH COOH
FeCl ·6H O/Zn
NaBH /Pd-C
3
EtOH/H
DMF/H
MeOH/H
2
O
1
84
40
37
11
51
45
95
91
82
3
2
2
O
3
4
2
O
0.5
1217

G.N. Sansom, et al.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 1215–1219
Table 2
Biological evaluation of mutual prodrugs.
Cell line
Drug
Aero IC50 (µM)
Anox IC50 (µM)
DRa
HCRb
MDA-MB-468
Melphalan
Floxuridine
1
1.00 ± 0.11
27.42 ± 12.52
8.31 ± 0.95
83.88 ± 40.93
105.2
0.81 ± 0.16
45.53 ± 6.10
7.38 ± 0.88
247.96 ± 80.56
1.304
–
1.2-fold
< 1-fold
1.1-fold
< 1-fold
81-fold
–
8.3-fold
2
3.1-fold
Tirapazamine
–
TH-302
4.403
0.0062
–
710-fold
SW620
Melphalan
Floxuridine
1
1.81 ± 0.25
0.96 ± 0.40
11.48 ± 6.57
18.70 ± 14.81
91.03
1.22 ± 0.10
1.48 ± 0.53
12.67 ± 4.93
13.96 ± 5.39
1.2
–
1.5-fold
< 1-fold
< 1-fold
1.3-fold
76-fold
–
6.3-fold
2
19.4-fold
Tirapazamine
TH-302
–
–
20.6
0.052
396-fold
Sensitivity of MDA-MB-468 and SW620 cells to hypoxia-activated mutual prodrugs 1 and 2 and their parent cytotoxins under aerobic (Aero) and anoxic (Anox)
2
7
conditions. Antiproliferative IC50 values are shown (mean ± std dev, n = 3). The activity of two positive control hypoxia-activated cytotoxins Tirapazamine and
2
8
a
TH-302 are included for comparison (n = 1). The deactivation ratio (DR) indicates the degree of deactivation of the prodrug relative to the effector under aerobic
b
conditions (i.e. DR = Aero prodrug IC50/Aero control cytotoxin IC50). The hypoxic cytotoxicity ratio (HCR) indicates whether cells are more sensitive to the
prodrugs or effectors under anoxic conditions (HCR = aerobic IC50/anoxic IC50).
Aerobic IC50control). For prodrug 1, 8.5-fold and 3.0-fold deactivation
was observed in the two cell-lines, while 3.0-fold and 6.9-fold deacti-
vation was seen with 2. It is proposed that for 1, the amide bond pulls
electron density away from the mustard nitrogen leading to decreased
DNA reactivity (and hence cytotoxicity) in the prodrug. In the case of 2,
Floxuridine is an antimetabolite and would be inactivated by the ester
linkage to the bulky 2-nitrophenylacetate moiety. The deactivation
observed with prodrug 2 relative to floxuridine suggests that the ester
bond remained intact under the assay conditions. The ability of the
prodrugs to undergo nitroaromatic bioreduction and cyclisation to re-
lease the respective cytotoxins was determined using the hypoxic cy-
totoxicity ratio (HCR = Aerobic IC50/Anoxic IC50). Under the assay
conditions used here, however, neither prodrug showed evidence for
activation under anoxia.
showed that electron withdrawing groups do not affect cyclisation rates
of 2-aminophenyl acetamides formed by reduction of 2-nitrophenyl
9
acetamide prodrugs. Based on this, a second generation of compounds
containing electron withdrawing groups on the nitroaryl ring are the
focus of efforts to optimise the mutual prodrugs for clinical use.
Funding sources
Funded in part by a Health Research Council of New Zealand grant
17/255 to Jeff Smaill and Adam Patterson and Australian National
Health and Medical Research Council (NHMRC) Project Grant
(APP1100432) to Michael Kelso.
Acknowledgment
To examine why 1 and 2 did not show enhanced cytotoxicity under
anoxia, one-electron reduction potentials (E[1]) for the arylnitro groups
in the prodrugs were measured using pulse radiolysis. This thermo-
dynamic parameter characterises the electron affinity of nitroaromatics
and hence their willingness to accept an electron from human oxidor-
eductases. Pulse radiolysis of 2 returned E[1] = −509 ± 8 mV, but
measurements for 1 were confounded by poor aqueous solubility.
Measurements were instead obtained using the bis-alcohol precursor 9
as a proxy. It was rationalised that the remoteness of the alcohols from
the arylnitro group would limit their influence on the reduction po-
tential. Pulse radiolysis of 9 returned E[1] = −550 ± 8 mV. Thus, we
conclude that the lack of bioreductive activation observed with the
prodrugs under anoxia arises from the low nitroaromatic one-electron
reduction potentials. It has been reported that nitroaromatic prodrugs
We thank the University of Wollongong (Wollongong, Australia)
and University of Auckland (Auckland, NZ) for supporting this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.03.015.
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