Synthesis and structure-activity relationships of ring-opened 17-hydroxywortmannins: Potent phosphoinositide 3-kinase inhibitors with improved properties and anticancer efficacy

Article abstract of DOI:10.1021/jm7012858

The phosphoinositide 3-kinase (PI3K) signaling pathway is frequently up-regulated in human cancer and is a promising target for the treatment of cancer. Wortmannin and its analogues are potent inhibitors of PI3K but suffer from inherent defects such as instability, insolubility, and toxicity. Opening of the reactive furan ring of 17-hydroxywortmannin with amines gives compounds with improved properties such as greater stability and aqueous solubility and a larger therapeutic index. Ring-opened analogues such as compound 13 containing basic amine groups have significantly increased PI3K inhibitory potency and greater efficacy in nude mouse xenograft assays.

Full text of DOI:10.1021/jm7012858

J. Med. Chem. 2008, 51, 1319–1323
1319
Synthesis and Structure–Activity Relationships of Ring-Opened 17-Hydroxywortmannins:
Potent Phosphoinositide 3-Kinase Inhibitors with Improved Properties and Anticancer Efficacy
Arie Zask,*,† Joshua Kaplan,† Lourdes Toral-Barza,‡ Irwin Hollander,‡ Mairead Young,§ Mark Tischler,§ Christine Gaydos,‡
Michael Cinque,‡ Judy Lucas,‡ and Ker Yu‡
DiscoVery Medicinal Chemistry, Oncology Research, and Chemical Technologies, Wyeth Research, Pearl RiVer, New York 10965
ReceiVed October 11, 2007
The phosphoinositide 3-kinase (PI3K) signaling pathway is frequently up-regulated in human cancer and is
a promising target for the treatment of cancer. Wortmannin and its analogues are potent inhibitors of PI3K
but suffer from inherent defects such as instability, insolubility, and toxicity. Opening of the reactive furan
ring of 17-hydroxywortmannin with amines gives compounds with improved properties such as greater
stability and aqueous solubility and a larger therapeutic index. Ring-opened analogues such as compound
13 containing basic amine groups have significantly increased PI3K inhibitory potency and greater efficacy
in nude mouse xenograft assays.
The phosphoinositide 3-kinases (PI3K) signaling pathway is
one of the most frequently mutated in human cancer, leading
to amplification of signaling, and as such is a promising target
for small-molecule inhibition, with promising therapeutic op-
portunities for the treatment of cancer.1,2 The PI3Ks regulate
cellular metabolism and growth by phosphorylation of the
3-position of phosphatidylinositol to generate phosphatidyli-
nositol triphosphate that in turn couples the PI3Ks to down-
stream effectors.
The natural product wortmannin (1) and its analogue 17-
hydroxywortmannin (2, Figure 1) are potent, nonselective
inhibitors of PI3Ks that bind irreversibly to lysine-833 in the
ATP binding pocket of PI3K via opening of the electrophilic
furan ring at its C-20 position. The irreversible binding of
wortmannin to PI3K γ has been proven by X-ray crystal-
lography.3 Wortmannin is a potent cytotoxic agent against
human tumor cell lines in xenograft models in mice. The toxicity
of wortmannin, as seen in its low therapeutic index, as well as
its insolubility and aqueous instability, has hampered its
development into a useful anticancer agent.4,5 Recently, con-
jugates of 1 and 2 (e.g., 3, PWT-458) with greatly improved
properties were developed by the application of pegylation
technology (Figure 1).6,7
Another approach to improving the properties of wortmannin
(1) is through opening of the furan ring at C-20 with nucleo-
philes such as amines. Opening of the furan ring with dialky-
lamines was first reported in 1975 as a means of protecting the
ring under the basic conditions necessary for deacetylation of
the C-11 position.8 Subsequently, it was shown that ring-opened
wortmannin analogues retained PI3K inhibitory activity, al-
though with much reduced potency.9 Ring-opened wortmannin
analogue 4 (PX-866), the product of furan ring opening of
wortmannin with diallylamine,10 was recently reported to be
active in human tumor xenograft models in mice.11 Mechanisti-
cally, the PI3K inhibitory activity of ring-opened compounds
is consistent with the ability of the secondary amine to be
displaced by primary amines (e.g., lysine-833 in PI3K).9 We
Figure 1. Wortmannin (1) and analogues.
predicted that the greater stability of ring-opened compounds
would increase their half-lives under physiological conditions
where reactive amine groups are present. The reduced reactivity
of these analogues could lead to decreased toxicity as exempli-
fied by a higher therapeutic index. Described herein is the
synthesis and structure–activity relationships of ring-opened 17-
hydroxywortmannin analogues with greatly improved aqueous
solubility, in vivo stability, and therapeutic ratio relative to
wortmannin.
Synthesis
Treatment of 2 with primary or secondary amines in meth-
ylene chloride or ethyl acetate gave the ring-opened analogues
(Scheme 1). Similarly, 11-desacetyl 17-hydroxywortmannin8
was treated with amines in dichloromethane to give the
corresponding ring-opened analogues.
Results and Discussion
Preliminary libraries of ring-opened 17-hydroxywortmannin
analogues were prepared with structurally diverse amines
(Supporting Information Tables S1 and S2). This initial survey
of the structure–activity relationships revealed that aliphatic
monoamines gave analogues with PI3K IC50 values several
* To whom correspondence should be addressed. Phone: 845-602-2836.
Fax: 845-602-2836. E-mail: zaska@wyeth.com.
†
Discovery Medicinal Chemistry.
Oncology Research.
‡
§
Chemical Technologies.
10.1021/jm7012858 CCC: $40.75
2008 American Chemical Society
Published on Web 02/13/2008

1320 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Zask et al.
Scheme 1. Synthesis of Ring-Opened 17-Hydroxywortmannin
Ring-opened analogues were also found to be much more
stable in nude mouse plasma than 2 (Figure 3). Surprisingly,
the expected mechanism of degradation by amine displacement
at C-20 by plasma proteins did not take place to an appreciable
extent. Instead, deacetylation of the C-11 group by esterases
was the major metabolic pathway in plasma (e.g., 5 to 6 and
13 to 15). Addition of the esterase inhibitor potassium fluoride
suppressed the deacetylation reaction. Interestingly, analogue
8 with a bulky tert-butylethanolamine group was resistant to
enzymatic deacetylation and showed relatively little degradation
over 8 h. The loss of the 11-acetyl group led to ring-opened
analogues (6 and 15) that were much less potent PI3K inhibitors.
Analoguesa
The most potent inhibitor was the aliphatic diamine analogue
13 (IC50 ) 6.4 nM). This analogue had weak activity in a panel
of protein kinases but showed strong inhibition of PI3K isoforms
R, ꢀ, and δ (Table 3). Consistent with 2 and the ring-opened
analogues, 13 had diminished potency for the mammalian target
of rapamycin (mTOR) with a 426-fold selectivity for PI3K R.
a (a) HNR1R2, CH2Cl2 or EtOAc (R1 and R2 are as defined in the tables);
As with the wortmannins, the mode of action of these ring-
opened analogues is through irreversible inhibition of the
enzyme. Inhibition of the homologous enzyme mTOR, which
also contains the reactive lysine-833 residue, showed time
dependent inhibition consistent with irreversible binding (Figure
4). In contrast, the dual PI3K/mTOR inhibitor 2-(4-morpholi-
nyl)-8-phenyl-4H-1-benzopyran-4-one (19, LY294002) that
binds noncovalently showed no time dependence of inhibition
of mTOR. Inhibitor wash-out assays in U87MG glioma cells
also confirmed that these inhibitors irreversibly target PI3K.16
On the basis of its superior potency, 13 was further
investigated in biological systems. Compound 13 inhibited the
PI3K signaling cascade in the PTEN-negative U87MG glioma
cells. It inhibited phospho-AKT (S473) and phospho-S6K
(T389) phosphorylation at 30 ng/mL (Figure 5). Interestingly,
the aliphatic amine analogue 8 was only 3-fold less potent. The
enhanced cell activity of 8 is likely due to its superior stability
in plasma (Figure 3).
Compound 13 showed potent inhibition of tumor growth
(U87MG glioma) in the nude mouse xenograft model (Figure
6). The iv dosing was facilitated by the good aqueous solubility
of 13 (>10 mg/mL). A minimum efficacious dose of <1.25
mg/kg with twice weekly dosing and 2.5 mg/kg with once
weekly dosing was seen. Tumor suppression increased in a dose
dependent fashion with 10 mg/kg once weekly giving the
greatest inhibition of tumor growth, thus demonstrating a
therapeutic index of at least 4. In contrast, similar studies with
2 showed a therapeutic index of 2.6 As expected, the weaker
PI3K inhibitor 8 was less potent in the U87MG xenograft model,
requiring higher doses and/or more frequent dosing to achieve
efficacy, with a minimum efficacious dose of 15 mg/kg once
weekly, 7.5 mg/kg twice weekly, or 5 mg/kg 3× weekly (Fig-
ure 7).
The PK parameters of 13 were examined in the nude mouse
following a single bolus iv administration at 5 mg/kg. Com-
pound 13 exhibited moderate clearance (50 (mL/min)/kg), a low
volume of distribution (0.7 L/kg), and a short half-life (0.5 h).
A similar PK study conducted with 2 indicated that in contrast
to 13, levels of 2 are below the limits of detection upon iv
dosing. As expected from the in vitro stability studies in nude
mouse plasma, a proportionally high concentration of the 11-
deacetylated metabolite 15 was seen in vivo. At longer time
points (>1 h) the concentration of 15 surpassed that of 13.
(b) lysine-phenylalanine, pH 7.4.
orders of magnitude less potent than 2 (Table S1, Table 1), while
much more potent inhibitors were obtained by reacting 2 with
aliphatic diamines (Table S2, Table 2). The most potent
inhibitors were formed from acyclic diamines and inhibited PI3K
with IC50 values comparable to that of 2 (Table 2). To our
knowledge, these are the most potent ring-opened wortmannin
based inhibitors reported to date. Cyclic diamines typically gave
less potent PI3K inhibitors than the acyclic diamines (Table
S2). Although compound 4 is reported to have IC50 ) 0.5 nM
versus PI3K,10,11 in our assays its potency (IC50 ) 88 nM) is
much lower, similar to that of other aliphatic ring-opened
analogues in Table 1 and the literature.5 Polar groups such as
alcohols, esters, carboxylic acids, and nitriles had little effect
on the potency of aliphatic ring-opened analogues (Table S1).
Reaction of 2 with primary amines gave analogues (7, 10, 14)
that were orders of magnitude less potent than those synthesized
withthecorrespondingsecondaryamines(5,9,13,respectively).9,12
This decrease in potency is consistent with the reported inability
of primary amine analogues to revert to wortmannin13 or to have
their primary amine group displaced by amines.9 The inacces-
sibility of these mechanistic pathways would preclude irrevers-
ible inhibition of PI3K through reaction with lysine-833. Ring-
opened 17-hydroxywortmannin analogues, like 2, were much
weaker inhibitors of mTOR than PI3K. The X-ray structure of
11 shows the geometry of the olefin formed upon ring opening
with a secondary amine (methylbutylamine) as E (Figure 2).
This result confirms the olefin geometry of secondary amine
ring-opened wortmannins previously proposed, based on het-
eronuclear NMR coupling constants.9
Interestingly, inhibition of cell growth did not correlate
directly with the potency of PI3K inhibition of these analogues
(Tables 1 and 2). Ring-opened analogues of aliphatic monoam-
ines were often more potent inhibitors of cell growth than 2,
despite having much higher PI3K IC50 values (Table 1). One
explanation for this anomaly is the instability of 2 in culture
systems due to covalent binding via its furan ring to amine
groups in the serum proteins in the medium.12,14,15 In contrast
to 2, ring-opened analogues are resistant to reaction with amines,
thereby leading to longer exposure times of cells to these
compounds. For example, treatment of 5 and 2 with the
dipeptide lysine-phenylalanine at pH 7.4 led to compound 18
In summary, ring opening of 17-hydroxywortmannin (2) with
secondary amines leads to analogues with improved properties
such as aqueous solubility and stability toward amino acids and
(Scheme 1) 14 times more slowly from 5 than from 2 (T1/2
57 min vs 4 min, respectively).
)

17-Hydroxywortmannins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1321
Table 1. Alkyl- and Cycloalkylamino Ring-Opened 17-Hydroxywortmannin Analogues
a Average IC50 ( SEM (µM).
Table 2. Diaminoalkyl Ring-Opened 17-Hydroxywortmannin Analogues
a Average IC50 ( SEM (µM).
plasma. Aliphatic diamines lead to analogues with enhanced
PI3K inhibition potency relative to those with aliphatic monoam-
ines. In our hands, 13 is the most potent ring-opened wortmannin
based PI3K inhibitor reported to date with activity comparable
to that of 2. In human tumor xenograft assays in mice, 13 was
a potent inhibitor of tumor growth with an improved therapeutic
index over that of 2. These findings show that ring-opened 17-
hydroxywortmannin analogues have exciting potential as inhibi-
tors of the PI3K pathway and therefore as anticancer agents.
Experimental Section
Materials and Instruments. Wortmannin was obtained from
fermentation broths of the fungal culture ZIMV298 of the Wyeth
microbial collection. All solvents were HPLC grade, and all other

1322 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Zask et al.
Figure 4. Time dependent inhibition of mTOR by 13. Compounds 19 and
13 were assayed in the mTOR in vitro enzyme assay for various indicated
times. For each assay time, the IC50 value was determined and plotted.
Figure 2. X-ray structure of 11.
Figure 5. Inhibition of PI3K signaling in PTEN-negative U87MG cells.
U87MG cells were plated in six-well culture plates for 24 h and treated
with DMSO or the indicated doses of 13 or 8 in growth media for 6 h.
Protein lysates were prepared and immunoblotted for phospho-AKT,
phospho-S6K1, and phospho-ERK.
Figure 3. Stability of 8 and 13 in nude mouse plasma.
Table 3. IC50 Values (µM) of 13 and 8 in a Panel of Protein and Lipid
Kinases
kinase
13
8
PI3K R
PI3K ꢀ
PI3K γ
PI3K δ
AKT1
BTK
CDK4
EGFR
GSK3
IGFR
IKKꢀ
KDR
0.0064
0.013
0.008
0.011
1.110
>30
>30
>10
>50
>10
>10
>10
>10
>10
>30
>30
>10
>200
>10
>50
>10
>10
>10
>10
>10
>30
>30
>10
>200
Figure 6. Tumor efficacy of 13 in the xenograft glioma model. U87MG
glioma were grown in the nude mice as described.6 The tumors were
staged on day 0. Compound 13 was formulated in PBS and adminis-
trated intravenously (iv) at the indicated dosages and schedules for 2
weeks. Tumor growth was monitored twice a week. Tumor volume
was determined as described.6
LCK
LYN
MEK1
MK2
mTOR
PDK1
S6K1
Tpl2
2.92
28.6
(Echelon) and GST-GRP1 (expressed and purified from E. coli). The
reaction was carried out in 20 µL of reaction buffer (20 mM Hepes,
pH 7.5, 2 mM MgCl2, 0.05% CHAPS, and 0.01% ꢀME) containing
20 µM PIP2, 25 µM ATP with vehicle control or inhibitors. The assay
was incubated for 15–30 min at room temperature and stopped with
20 µL of stop/detection buffer (10 nM probe and 40 nM GST-GRP).
The plates were incubated for 1–2 h, and FP was measured in a Perkin-
Elmer Envision plate reader with TAMRA- FP filters. The mTOR
kinase assay was carried out as described previously.17 In vitro cell
growth assays18 and in vivo efficacy studies6 were carried out as
described previously.
(1E,4S,4aR,5R,6aS,7S)-1-{[[3-(Dimethylamino)propyl](methyl)
amino]methylene}-7,11-dihydroxy-4-(methoxymethyl)-4a,6a-di-
methyl-2,10-dioxo-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroinde-
no[4,5-h]isochromen-5-yl Acetate (13). To a slurry of 2 (15.3 g)
and EtOAc (107.7 mL) cooled in an ice bath was added N,N,N′-
>200
>30
>40
>200
>30
>40
chemicals were analytical reagents or equivalent. A BUCHI rotary
evaporation system (RE 260 and R 124) was from Buchi (Flawil,
Switzerland). 1H NMR spectra were recorded on a 400 MHz NMR
spectrophotometer using DMSO-d6 or CDCl3 neutralized with
alumina. Electrospray (ES) mass spectra were recorded on a
Micromass Platform spectrometer. High-resolution mass spectra
were recorded on a Finnigen MAT-90 spectrometer.
Biology. Inhibition of PI3K enzyme activity is measured in a
microtiter plate-based fluorescence polarization (FP) assay adapted
from Echelon’s K-1100 PI3K FP assay kit protocol with PI3K enzyme
purchased from Upstate Biotech, PIP2, and TAMRA-labeled detector

17-Hydroxywortmannins
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1323
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trimethyl-1,3-propanediamine (5.2 mL) dropwise over a period of
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1
88.9%). MS, m/z: 547.3 (M + H)+. H NMR (CDCl3) δ 0.75 (s,
3H), 1.43 (d, J ) 7.44, 1H), 1.46 (d, J ) 7.45, 1H), 1.54 (s, 3H),
1.50–1.78 (m, 5H), 2.07 (s, 3H), 2.18 (s, 7H), 2.23 (bd s, 2H),
2.54 (m, 3H), 2.71 (bd s, 2H), 3.11 (t, J ) 6.1, 1H), 3.21 (d, J )
2.1, 1H), 3.22 (s, 3H), 3.39 (bd s, 1H), 3.51 (bd s, 1H), 3.85 (t, J
) 5.6, 1H), 4.58 (bd s, 1H), 6.02 t, J ) 4.92, 1H), 8.10 (s, 1H). 1H
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Acknowledgment. The authors thank Drs. Tarek Mansour,
Semiramis Ayral-Kaloustian, and Jay Gibbons for support of
this work and useful discussions. Dr. Robert Mallon and Steven
Kim provided PI3K R enzyme for the assays. Dr. Ping Cai
prepared compounds S2, S27-S29. Dr. Madelene Antane
carried out the synthesis of 13 as described in the Experimental
Section. Dr. Jack Wang provided the data on the reactivity of
1 with lysine-phenylalanine, and Dr. Inder Chaudhary provided
the PK data of 13. Dr. Douglas Ho (Princeton University) solved
the X-ray structure of 11.
Supporting Information Available: CHN and HRMS data and
biological data for compounds prepared by parallel synthesis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JM7012858