Facile synthesis of rapamycin-peptide conjugates as mTOR and Akt inhibitors

Article abstract of DOI:10.1039/d1ob00132a

A simple and straightforward process for the synthesis of rapamycin peptide conjugates in a regio and chemoselective manner was developed. The methodology comprises the tagging of chemoselective functionalities to rapamycin and peptides which enables the conjugation of free peptides, without protecting the functionality of the side chain amino acids, in high yield and purity. From this methodology, we successfully conjugate free peptides containing up to 15 amino acids. Rapamycin is also conjugated to the peptides known for inhibiting the kinase activity of Akt protein. These conjugates act as dual target inhibitors and inhibit the kinase activity of both mTOR and Akt. This journal is

Full text of DOI:10.1039/d1ob00132a

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Facile synthesis of rapamycin-peptide conjugates
as mTOR and Akt inhibitors†
Cite this: DOI: 10.1039/d1ob00132a
Shalini Singh,‡a Rafat Ali,‡a Javed Miyan,b Varsha Singh,b Sanjeev Meena,b
Mohammad Hasanain,b Smrati Bhadauria,b,c Dipak Datta,b,c Jayanta Sarkarb,c and
a,c
Wahajul Haq
*
A simple and straightforward process for the synthesis of rapamycin peptide conjugates in a regio and
chemoselective manner was developed. The methodology comprises the tagging of chemoselective
functionalities to rapamycin and peptides which enables the conjugation of free peptides, without pro-
tecting the functionality of the side chain amino acids, in high yield and purity. From this methodology,
we successfully conjugate free peptides containing up to 15 amino acids. Rapamycin is also conjugated
to the peptides known for inhibiting the kinase activity of Akt protein. These conjugates act as dual target
inhibitors and inhibit the kinase activity of both mTOR and Akt.
Received 23rd January 2021,
Accepted 6th April 2021
DOI: 10.1039/d1ob00132a
rsc.li/obc
Rapamycin (sirolimus), a 31-membered macrocyclic polyketide development.23–25 This was recognized during preclinical
produced by Streptomyces rapamycinicus, was first isolated in studies in which treatment with rapamycin or rapalogs
1975 and was initially used as antifungal agent.1–3 But later resulted in only minimal inhibition of tumor cell growth due
studies revealed its immunosuppressant,4 neuroprotective/ to the increase in Akt signaling and PI3K activity.26,27 To cope
neuroregenerative5–7 and anti-aging8 activities. The potential with this situation, several rapamycin or rapalog formulations
of rapamycin to act as an antiproliferative agent was first have been made in which rapamycin was combined or conju-
recognized by Dr Suren Shegal and was sent for anti-tumor gated with a PI3K inhibitor such as LY294002 or wortmannin,
activity screening against the standard 60 human tumor cell and the resulting combinations were shown to inhibit the Akt
lines of NCI.9,10 After that, a lot of studies on the antiprolifera- activation induced by rapamycin, resulting in the synergistic
tive activity of rapamycin were done.9,11–13
inhibition of tumor growth.28,29
Rapamycin is known as a potent inhibitor of the serine/
Therefore, a number of strategies have been developed for
threonine kinase mTOR (the mammalian target of rapamycin). the synthesis of new rapamycin derivatives, mainly involving
The inhibition of mTOR in turn inhibits cell progression from substitution at the hydroxyl group of the 42-and/or 31 posi-
the G1 to the S phase in most cells, and in some cells it tions of rapamycin.29–34 Recent efforts have been made to syn-
induces p53-independent apoptosis.14–16 mTOR forms two dis- thesize rapamycin peptide conjugates in which rapamycin is
tinct complexes, named as mTORC1 and mTORC2, as a result conjugated to peptides which are known for their antiprolifera-
of it binding with different regulatory proteins.17,18 Rapamycin tive activity. These rapamycin peptide conjugates were pre-
or rapamycin derivatives (rapalogs) are only partial inhibitors pared by the reaction of 42-O-(4-nitrophenoxycarbonyl) rapa-
of mTOR and inhibit only mTORC1 and not mTORC2, which mycin with a free amino group on the protected peptides
also plays an important role in cancer progression.19–22 The known for their CDK (cyclin dependent kinase) inhibitory
inhibition of only mTORC1 by rapamycin and not mTORC2 in activity (Scheme 1a).35 The resulting rapamycin peptide conju-
some tumors can stimulate PI3K/Akt signaling and antagonize gates have the capacity to target the two protein kinases and
its antitumor efficacy, leading to more aggressive tumor act as dual target inhibitors. These rapamycin peptide conju-
gates were found to be more efficient than rapamycin in inhi-
biting cell proliferation. In spite of the better bioactivity, this
aMedicinal and Process Chemistry Division, CSIR–Central Drug Research Institute,
Lucknow, Uttar Pradesh, 226031, India. E-mail: whaq001@gmail.com
bDivision of Cancer Biology, CSIR–Central Drug Research Institute, Lucknow,
Uttar Pradesh, 226031, India
cAcademy of Scientific and Innovative Research, New Delhi, 11000, India
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00132a
method for the synthesis of rapamycin peptide conjugates has
several drawbacks such as the use of only protected peptide
fragments, tedious purification, low yield etc. Herein, we devel-
oped a simple and efficient methodology for the synthesis of
rapamycin peptide conjugates. Subsequently, rapamycin was
conjugated to known Akt inhibitory peptides to overcome the
problem of the simultaneous activation of Akt by rapamycin.
‡These authors contributed equally.
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Scheme 1 Strategy for the synthesis of the rapamycin peptide conjugates.
Results and discussion
Rapamycin has a complex and delicate structure and the
reagents and chemical conditions used for the earlier syn-
thesis of rapamycin peptide conjugates restricts their further
development, in spite of the potential shown by the rapamycin
peptide conjugates. In view of this, we utilized hydrazone
chemistry in line with Solulink bioconjugation technology36
for the synthesis of rapamycin peptide conjugates, as shown in
Scheme 1b. The synthesis required appropriately functiona-
lized fragments, namely the aldehyde bearing component 42-
O-(4-formylphenylcarbonyl)-rapamycin (I) first and the hydrazi-
nonicotinyl (HyNic) modified peptide second.
The complex structure of rapamycin makes it difficult to
introduce aldehyde groups in a selective manner. For this, we
took 4-formyl benzoic acid, which was then regioselectively
attached to the 42-hydroxyl group of rapamycin by forming an
ester bond with the carboxylic group of 4-formyl benzoic acid
under mild conditions (Scheme 2). For esterification we used
different coupling reagents like HBTU and DCC with or without
using DMAP in its catalytic amount. We got the best result with
DCC/DMAP and the functionalization of rapamycin was suc-
cessfully carried out by the regioselective acylation of the
42-hydroxy group of rapamycin with 4-formyl benzoic acid to
form the corresponding 42-O-(4-formylphenylcarbonyl)-rapamy-
cin in good yield and purity. We next prepared the hydrazinoni-
cotinoyl modified peptide component by employing the solid
phase method of peptide synthesis using Wang resin as the
Scheme 2 Synthesis of 42-O-(4-formylphenylcarbonyl)-rapamycin.
Scheme 3 Synthesis of the hydrazinonicotinyl (HyNic) modified
solid support (Scheme 3). The Boc-hydrazinonicotinic acid 3 peptides.
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Scheme 4 Synthesis of rapamycin peptide conjugates.
used in the synthesis of the hydrazinonicotinyl modified pep- known for inhibiting the kinase activity of Akt. Akt over-
tides was prepared from 6-chloronicotinic acid 1 by following expression resulted in resistance to chemotherapy and Akt
the reported protocol.37 The peptide was finally deprotected and overexpression is also one of the major factors responsible for
cleaved by TFA/TIPS/acetone/water (92.5/2.5/2.5/2.5%) which resistance to cancer therapy by rapamycin or rapalogs.12,38 For
gave the isopropylidenyl-hydrazinonicotinoyl tagged peptides. the present study, we chose four Akt inhibitory peptides, Ala–
The addition of acetone in the peptide cleavage solution Val–Thr–Asp–His–Pro–Asp–Arg–Leu–Trp–Ala–Trp–Glu–Lys–Phe
resulted in the protection of the hydrazine group as hydrazone, (Akt-in),39 Arg–Pro–Arg–Nle–Tyr–Dap–Nle,40 Arg–Pro–Arg–Ala–
thus preventing hydrazide formation with trifluoroacetic acid.
Tyr–Dap–Nle and Arg–Pro–Arg–Nle–Tyr–Dap–Ala, to give the
Both the compounds have been synthesized separately, the rapamycin peptide conjugates 5, 6, 7 and 8, respectively. The
peptide rapamycin conjugates were prepared by mixing stoichio- peptide Akt-in is a well known potent inhibitor of the kinase
metric ratios of these components in a pH 5 sodium acetate activity of Akt, whereas the other peptides used for the conju-
buffer and ethanol mixture for a couple of hours (Scheme 4). gation were already found to be active in our in-house assays
Initially, tetrapeptide AVPI, which contains only amino acids as Akt inhibitors. It must be noted that all of these peptide
with no side chain functional groups, was conjugated to give conjugates were prepared in high yield and purity without pro-
conjugate 1. Then, more complicated peptides were selected, tecting the highly reactive side chain functionality like guani-
having amino acids with amino and hydroxy group functionality dino, carboxyl amino or phenolic groups, showing the poten-
in their side chains to give conjugates 2–4. The reaction pro- tial and versatility of the methodology.
gress was monitored by HPLC and, after completion, the reac-
The conjugates 5–8 were evaluated for their biological
tion mixture was subjected to purification by HPLC without activity for the inhibition of the kinase activity of mTOR and
additional workup to obtain the rapamycin peptide conjugates Akt proteins in vitro, as well as their ability to inhibit cell pro-
in almost quantitative yields. The progress of the formation of liferation in a cell-based assay. The mTOR kinase inhibition
the conjugates was easily identified as the bis-aryl hydrazone assay was performed using active recombinant mTOR protein
bond formed during the conjugation reaction is highly chromo- (Fig. 1a). mTOR phosphorylates the ribosomal protein S6
phoric and has a characteristic absorbance at 354 nm (Fig. S9†). kinase (S6K) at Thr389. It is clear from the western blots that
The conjugation chemistry used for the synthesis of the rapamy- the rapamycin peptide conjugates 7 and 8, like rapamycin,
cin conjugates is highly efficient and chemoselective, as demon- completely inhibited the kinase activity of recombinant mTOR
strated by the clean reaction despite the highly functional and at 10 nM concentration. However, conjugates 5 and 6 did not
complex nature of the peptides and rapamycin. One of the inhibit the kinase activity of the mTOR protein as much as
major advantages of this methodology is that the conjugation rapamycin. Different concentrations of conjugates 7 and 8
reaction was carried out using free peptides after the removal of were then evaluated for their inhibitory activity against the full
the side chain protecting groups of the hydrazinonicotinyl mTORC1 complex in the human breast cancer MCF-7 cell line
tagged peptides to obtain ready to use conjugates after purifi- (Fig. 1b). The conjugate 7 showed similar inhibition of
cation. The reaction conditions were extremely mild and did not mTORC1 to rapamycin, whereas conjugate 8 showed better
require any reagent for conjugation.
inhibition of mTORC1 as compared to rapamycin at 100 nM
Interestingly, after the conjugation of the peptides no concentration. It has been reported that incomplete mTOR
apparent loss in the biological activity of rapamycin in the con- inhibition by rapamycin (i.e. inhibiting only mTORC1 and not
jugates was observed in cell-based assays (Tables S1 and S2†). mTORC2) results in an increased level of Akt, which results in
This encouraged us to conjugate rapamycin to the peptides the development of an aggressive tumor like situation. It was
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Table 2 Percentage growth inhibition at 10 μM concentration in breast
cancer cell lines
S. no.
Compounds
MCF-7
MDA-MB-231
1
2
3
4
5
Rapamycin
Conjugate 5
Conjugate 6
Conjugate 7
Conjugate 8
42.42 4.3
42.94 4.7
32.69 1.5
51.1 8.8
15.73 0.57
No inhibition
No inhibition
10.46 2.1
57.19 8.43
No inhibition
inhibited the kinase activity of Akt and were comparable with
the standard Akt inhibitor A443654.
These conjugates were then evaluated for their antiproli-
ferative activity in two breast cancer cell lines (MCF-7 and
MDA-MB-231), which have different sensitivities towards rapa-
mycin, using the well established SRB (Sulforhodamine B)
assay.42 The MCF-7 cell line is sensitive to rapamycin and
MDA-MB-231 is almost rapamycin insensitive.43 Similarly to
rapamycin, no cytotoxicity of these conjugates was observed in
the MDA-MB-231 cell line, whereas these conjugates showed
cytotoxicity comparable to rapamycin in the MCF-7 cell line
and conjugate 8 was found to be most potent one (Table 2).
Fig. 1 The in vitro inhibition of the kinase activity of the recombinant
mTOR protein, mTOR complex 1 and mTOR complex 2 in the MCF-7
breast cancer cell line. Western blots showing the inhibition of the
kinase activity of; (a) recombinant mTOR protein; (b) mTOR complex 1 in
MCF-7 cells and (c) mTOR complex 2 in mCF-7 cells. Torin is used as a
standard inhibitor of the kinase activity of mTORC1 and mTORC2.
Conclusions
In conclusion, we have developed an efficient methodology for
the regioselective functionalization of rapamycin, which was
used for the preparation of rapamycin peptide conjugates in
excellent yield and purity under mild reaction conditions in a
chemoselective manner. One of the major advantages of this
process is that we could directly conjugate free peptides to rapa-
mycin without the need of protecting side chain functional
groups of the amino acids which are otherwise very difficult to
remove once the peptide is conjugated to rapamycin. We could
easily couple short and long peptides containing arginine,
lysine or serine amino acids to rapamycin, which is otherwise
difficult to conjugate, showing the versatility of the method-
ology. Furthermore, after making peptide conjugates of rapamy-
cin, no loss of the apparent activity of rapamycin was observed.
These conjugates may exhibit the ability to bind dual targets
depending upon the pharmacological properties of the other
ligand used for conjugation, for example conjugate 8 completely
inhibited the kinase activity of the recombinant mTOR protein
like rapamycin, and unlike rapamycin it significantly inhibited
the kinase activity of Akt as well. This will provide a new insight
into the area of research comprising the discovery of new rapa-
mycin conjugates with improved activity and selectivity and that
of target based new chemical entities.
therefore considered appropriate to evaluate the ability of the
novel conjugates for mTORC2 inhibition. mTORC2 phosphory-
lates the protein kinase Akt at Ser473 and is insensitive to
rapamycin. It is clear from Fig. 1c that conjugate 8, unlike
rapamycin, also significantly inhibits the kinase activity of
mTORC2 at 100 nM concentration, suggesting that conjugate 8
acts on dual targets which inhibit the kinase activities of
mTORC1 and mTORC2, resulting in less phosphorylation of
the S6K and Akt proteins, respectively. Quantitative analysis of
protein phosphorylation inhibition was performed by the den-
sitometric analysis of the respective western blots and the
results are given in Fig. S15.† A similar trend was observed
when we performed these experiments with isolated mTORC1
and mTORC2 complexes (Fig. S14†). To evaluate the potential
of these conjugates for inhibiting the kinase activity of the Akt
protein more precisely (through binding at the ATP binding
site), optimized non-radioactive in vitro ELISA based Akt
kinase assays were performed and the results are shown in
Table 1.41 The conjugates 6, 7 and 8 at 10 µM concentration
Table 1 Percentage inhibition of Akt kinase activity at 10 μM
concentration
S. no.
Compounds
% Inhibition SE
Experimental section
General information
1
2
3
4
5
A443654
85.44 4.1
Conjugate 5
Conjugate 6
Conjugate 7
Conjugate 8
No inhibition
80.20 8.89
80.53 7.4
The Fmoc protected amino acids and Wang resin
(100–200 mesh) used in the solid phase peptide synthesis were
62.97 14.68
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purchased from Novabiochem. Rapamycin was purchased from group of the assembled peptide was deprotected with 20%
APExBIO. Flame-dried or oven dried glassware was used to piperidine in DMF. To this resin a solution of 6-Boc-hydrazino-
carried out the reactions. All of the reactions were performed nicotinic acid (3 equiv.) and HBTU (3 equiv.) in DMF was
with magnetic stirring under nitrogen atmosphere using freshly added and the reaction mixture was stirred by purging nitro-
dried and distilled solvents, unless otherwise noted. Ready gen gas followed by the addition of DIPEA (3 equiv. to HBTU),
made TLC silica gel 60 F254 plates (Merck, Dermstadt, Germany) and the reaction mixture was stirred by purging nitrogen gas
were used for reaction monitoring. The TLC plates were either for 3 h under nitrogen. After the reaction was completed, the
developed under iodine vapors or visualised directly under UV resin was then washed with DMF (three times) and DCM
light at 254 nm. Silica gels of 100–200 and 230–400 mesh were (three times) and finally dried under vacuum. The peptide
used for column chromatography. High resolution mass spectra bound resin was then treated with
a
mixture of
were recorded using an Agilent 6520-Q-Tof MS/MS system. TFA : TIPS : acetone : water (92.5 : 2.5 : 2.5 : 2.5%) and the reac-
MALDI-MS spectra were recorded with an AB Sciex 4800 plus tion mixture was stirred for 3 h. The resin was removed by fil-
1
MALDI TOF–TOF Analyzer mass spectrometer. H NMR spectra tration and the filtrate was reduced to half by evaporation
were recorded on a Bruker Av III HD 400 MHz spectrometer under reduced pressure at 45 °C. The peptide was precipitated
operating at 400 MHz at 25 °C using 2–10 mM concentrations with diethyl ether. The precipitate obtained was filtered and
in appropriate solvents, using TMS as the internal standard or further washed three times with diethyl ether. The precipitate
the solvent signals as secondary standards, and the chemical obtained was dried under vacuum to give the crude peptide.
shifts (δ) are shown in ppm scales. Multiplicities of NMR Purification with RP-HPLC using a C-18 column gave the
signals are designated as s (singlet), d (doublet), t (triplet), q desired HyNic modified peptides.
(quartet), br (broad), td (triplet of a doublet), dt (doublet of a
triplet) and m (multiplet, for unresolved lines).
Synthesis of 42-O-(4-formylphenylcarbonyl) rapamycin (I)
To a solution of 4-carboxy benzaldehyde (40 mg, 0.2 mmol) in
DCM (8 mL) at 0 °C, DCC (41.28 mg, 0.2 mmol) was added.
Synthesis of 6-hydrazinonicotinic acid (2)
Hydrazine hydrate (5 mL) was added to 6-chloronicotinic acid
1 (980 mg, 6.22 mmol) and the reaction mixture was refluxed
at 100 °C for 4 h. The reaction mixture was concentrated to
dryness to give a solid residue. The residue was dissolved in a
minimum amount of water and acidified with hydrochloric
acid (35%) up to pH 5 to precipitate the HCl salt of 6-hydrazi-
nonicotinic acid. The precipitate was filtered off and washed
with ethanol to yield 800 mg (83.99%) of 6-hydrazinonicotinic
After 15 minutes, rapamycin (200 mg, 0.2 mmol) and DMAP
(4 mg, 0.03 mmol) was added to it at 0 °C. The reaction was
then stirred overnight at 25 °C. After that, DCU was removed
by filtration and washed with DCM. The organic layer was then
concentrated in vacuo, affording the crude product. The crude
residue was then purified on a silica gel column, eluting with
1% MeOH/DCM to give 100 mg (47.79%) of the title compound
as a light yellow solid. Characteristic 1H NMR peak (CDCl3,
400 MHz): δ/ppm = 10.11(s, CHO, 1H), 8.20 (d, J = 8.2, Ar–H,
2H) and 7.96 (d, J = 8.52, Ar–H, 2H), the detailed NMR spectra
is shown in Fig. S3;† MALDI-MS (M + Na)+ calculated for
C59H83NNaO15+ = 1068.56, found 1068.51.
1
acid. H NMR (DMSO-d6, 400 MHz): δ/ppm = 8.53 (d, J = 1.72,
1H, Ar–H), 8.27 (s, 1H, NH), 7.86 (dd, J = 2.28, 8.88, 1H, Ar–H),
6.86 (d, J = 8.8, 1H, Ar–H).
Synthesis of 6-Boc-hydrazinonicotinic acid (3)
Synthesis of the rapamycin peptide conjugates
To
a
solution of 6-hydrazinonicotinic acid (900 mg,
5.85 mmol) and triethylamine (0.7 mL, 5.85 mmol) in DMF
(6.3 mL), di-tert-butyl dicarbonate (1.34 mL, 5.85 mmol) was
added. The reaction mixture became homogeneous over 1 h
and stirring was continued for 16 h at room temperature. The
reaction mixture was concentrated to dryness under reduced
pressure to give a brown solid. The residue was dissolved in a
minimum amount of ethyl acetate and passed through silica
gel using ethyl acetate as the eluent to remove the coloured
impurities. The eluate was concentrated to dryness to give
1.3 g (87.75%) of 6-Boc-hydrazinonicotinic acid which was
To the solution of HyNic modified peptides (5–10 μmol) in
1–2 mL of sodium acetate buffer of pH 5.1 add solution of
equivalent amount of 42-O-(4-formylphenylcarbonyl) rapamy-
cin in 1–2 mL of ethanol. Reaction was stirred for 3 h at room
temperature (monitor by RP-HPLC). After that rapamycin
peptide conjugate was purified by RP-HPLC in C-4 column
using gradient solvent system of 0.1% TFA in DI water and
acetonitrile to give desired rapamycin peptide conjugate in
almost quantitative yield. The product is confirmed by mass
spectroscopy and the purity of peptide was checked with
analytical HPLC.
1
used for the next steps. H NMR (DMSO-d6, 400 MHz): δ/ppm
= 8.99 (s, 1H, NH), 8.89 (s, 1H, NH), 8.59 (d, J = 1.8, 1H, Ar–H),
7.97 (dd, J = 1.6, 10.88, 1H, Ar–H), 6.54 (d, J = 8.76, 1H, Ar–H),
+
1.43 (s, 9H); HRMS (ESI) calculated for C11H16N3O4
254.1135, found = 254.1134.
=
Author contributions
WH, SS and RA contributed to the designing, conceptualiz-
ation and synthesis. JM, VS and SB performed the mTOR
Synthesis of hydrazinonicotinyl (HyNic) modified peptides
The desired peptides were assembled on the Wang resin using related experiments. MH and JS performed the Akt kinase
standard Fmoc based solid phase peptide synthesis. The Fmoc assays. SM and DD performed the cell-based assays.
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18 D. D. Sarbassov, S. M. Ali, D. H. Kim, D. A. Guertin,
R. R. Latek, P. E. Bromage, H. Tempst and D. M. Sabatini,
Curr. Biol., 2004, 14, 1296–1302.
19 S. Wullschleger, R. Loewith and M. N. Hall, Cell, 2006, 124,
471–484.
Conflicts of interest
There are no conflicts to declare. An Indian Patent application
has been filed for part of this work with patent no. 341387.
20 E. Jacinto, V. Facchinetti, D. Liu, N. Soto, S. Wei, S. Y. Jung,
Q. Huang, J. Qin and B. Su, Cell, 2006, 127, 125–137.
21 M. A. Frias, C. C. Thoreen, J. D. Jaffe, W. Schroder,
T. Sculley, S. A. Carr and D. M. Sabatini, Curr. Biol., 2006,
16, 1865–1870.
Acknowledgements
R. A. and J. M. thank CSIR – New Delhi and S. S. thanks UGC –
New Delhi for the financial support. We also thank the SAIF
division, CSIR-CDRI, for the analytical facilities. CDRI 22 J. Martin, J. Masri, A. Bernath, R. N. Nishimura and
Communication No. 10235.
J. Gera, Biochem. Biophys. Res. Commun., 2008, 372, 578–
583.
23 A. Carracedo and P. P. Pandolfi, Oncogene, 2008, 27, 5527–
5541.
Notes and references
24 D. A. Guertin and D. M. Sabatini, Sci. Signaling, 2009, 2,
1 C. Vézina and A. Kudelski, J. Antibiot., 1975, 28, 721–726.
pe24.
2 S. N. Sehgal, H. Baker and C. Vézina, J. Antibiot., 1975, 28, 25 F. Meric-Bernstam and A. M. Gonzalez-Angulo, J. Clin.
727–732. Oncol., 2009, 27, 2278–2287.
3 K. Singh, S. Sun and C. Vézina, J. Antibiot., 1979, 32, 630– 26 S. Y. Sun, L. M. Rosenberg, X. Wang, Z. Zhou, P. Yue, H. Fu
645. and F. R. Khuri, Cancer Res., 2005, 65, 7052–7058.
4 R. R. Martel, J. Klicius and S. Galet, Can. J. Physiol. 27 Y. Shi, H. Yan, P. Frost, J. Gera and A. Lichtenstein, Mol.
Pharmacol., 1977, 55, 48–51. Cancer Ther., 2005, 4, 1533–1540.
5 C. Malagelada, Z. H. Jin, V. Jackson-Lewis, S. Przedborski 28 X. Wang, P. Yue, A. K. Young, H. Fu, F. R. Khuri and
and L. A. Greene, J. Neurosci., 2010, 30, 1166–1175. S. Y. Sun, Cancer Res., 2008, 68, 7409–7418.
6 T. Pan, S. Kondo, W. Zhu, W. Xie, J. Jankovic and W. Le, 29 S. Ayral-Kaloustian, J. Gu, J. Lucas, M. Cinque, C. Gaydos,
Neurobiol. Dis., 2008, 32, 16–25.
7 L. S. Tain, H. Mortiboys, R. N. Tao, E. Ziviani, O. Bandmann
and A. J. Whitworth, Nat. Neurosci., 2009, 12, 1129–1135.
A. Zask, I. Chaudhary, J. Wang, L. Di, M. Young,
M. Ruppen, T. S. Mansour, J. J. Gibbons and K. Yu, J. Med.
Chem., 2010, 53, 452–459.
8 D. E. Harrison, R. Strong, Z. D. Sharp, J. F. Nelson, 30 J. Gu, M. E. Ruppen and P. Cai, Org. Lett., 2005, 7, 3945–
C. M. Astle, K. Flurkey, N. L. Nadon, J. E. Wilkinson, 3948.
K. Frenkel, C. S. Carter, M. Pahor, M. A. Javors, 31 S. Rakhit, US Pat. no 4316885, 1982.
E. Fernandez and R. A. Miller, Nature, 2009, 460, 392–395.
9 K. Garber, J. Natl. Cancer Inst., 2001, 93, 1517–1519.
10 J. Douros and M. Suffness, Cancer Treat. Rev., 1981, 8, 63–
87.
11 D. P. Houchens, A. A. Ovejera, S. M. Riblet and D. E. Slagel,
Eur. J. Cancer Clin. Oncol., 1983, 19, 799–805.
32 C. E. Caufield, US Pat. no 5118677, 1992.
33 W. Kao, R. L. Vogel and J. H. Musser, US Pat. no 5118678,
1992.
34 A. A. Failli, C. E. Caufield and R. J. Steffan, US Pat. no
5130307, 1992.
35 S. K. Sharma, T. Woo and S. Naicker, US Pat. No US2007/
0129394A1, 2007.
12 T. F. Cloughesy, K. Yoshimoto, P. Nghiemphu, K. Brown,
J. Dang, S. Zhu, T. Hsueh, Y. Chen, W. Wang, D. Youngkin, 36 D. A. Schwartz, US Pat. No 20030013857A1, 2003.
L. Liau, N. Martin, D. Becker, M. Bergsneider, A. Lai, 37 M. J. Abrams, M. Juweid, C. I. TenKate, D. A. Schwartz,
R. Green, T. Oglesby, M. Koleto, J. Trent, S. Horvath,
P. S. Mischel, I. K. Mellinghoff and C. L. Sawyers, PLoS
Med., 2008, 5, 0139–0151.
M. M. Hauser, F. E. Gaul, A. J. Fuccello, R. H. Rubin,
H. W. Strauss and A. J. Fischman, J. Nucl. Med., 1990, 31,
2022–2028.
13 B. Seto, Clin. Transl. Med., 2012, 1, e29.
14 T. Seufferlein and E. Rozengurt, Cancer Res., 1996, 56,
3895–3897.
38 K. E. O’Reilly, F. Rojo, Q. B. She, D. Solit, G. B. Mills,
D. Smith, H. Lane, F. Hofmann, D. J. Hicklin, D. L. Ludwig,
J. Baselga and N. Rosen, Cancer Res., 2006, 66, 1500–1508.
15 H. Hosoi, M. B. Dilling, T. Shikata, L. N. Liu, L. Shu, 39 M. Hiromura, F. Okada, T. Obata, D. Auguin, T. Shibata,
R. A. Ashmun, G. S. Germain, R. T. Abraham and
P. J. Houghton, Cancer Res., 1999, 59, 886–894.
C. Roumestand and M. Noguchi, J. Biol. Chem., 2004, 279,
53407–53418.
16 S. Huang, L. Shu, M. B. Dilling, J. Easton, F. C. Harwood, 40 S. Singh, R. Shrivastava, G. Singh, R. Ali, R. Sankar
H. Ichijo and P. J. Houghton, Mol. Cell, 2003, 11, 1491–
1501.
Ampapathi, S. Bhadauria and W. Haq, ChemBioChem, 2017,
18, 1061–1065.
17 K. Hara, Y. Maruki, X. Long, K. I. Yoshino, N. Oshiro, 41 N. Rhodes, D. A. Heerding, D. R. Duckett, D. J. Eberwein,
S. Hidayat, C. Tokunaga, J. Avruch and K. Yonezawa, Cell,
2002, 110, 177–189.
V. B. Knick, T. J. Lansing, R. T. McConnell, T. M. Gilmer,
S. Y. Zhang, K. Robell, J. A. Kahana, R. S. Geske,
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2021

View Article Online
Organic & Biomolecular Chemistry
Paper
E. V. Kleymenova, A. E. Choudhry, Z. Lai, J. D. Leber, 42 V. Vichai and K. Kirtikara, Nat. Protoc., 2006, 1, 1112–1116.
E. A. Minthorn, S. L. Strum, E. R. Wood, P. S. Huang, 43 W. C. Noh, W. H. Mondesire, J. Peng, W. Jian, H. Zhang,
R. A. Copeland and R. Kumar, Cancer Res., 2008, 68,
2366–2374.
J. J. Dong, G. B. Mills, M. C. Hung and F. Meric-Bernstam,
Clin. Cancer Res., 2004, 10, 1013–1023.
This journal is © The Royal Society of Chemistry 2021
Org. Biomol. Chem.