Wortmannin-C20 conjugates generate wortmannin

Article abstract of DOI:10.1021/jm050699p

We report on C20-6-(N-methylamino)hexanoic conjugates of wortmannin featuring a tertiary enamine attached to the C20 that inhibit phosphoinositol-3-OH kinase (PI3K) by producing wortmannin (Wm) through an intramolecular attack. The generation of Wm by these conjugates permits the design of Wm based PI3K inhibitors that need not fit into the ATP pocket of PI3K, including Wm conjugates of BSA, IgG, or beads. Wm generating WmC20-N(Me)-hexanoate conjugates offer an approach to the design of targeted or slow release forms of Wm which may inhibit PI3K in tissues more selectively than the parent Wm, a compound which has desirable anti-inflammatory and anti-proliferative activities but which also has a variety of toxic effects.

Full text of DOI:10.1021/jm050699p

7
40
J. Med. Chem. 2006, 49, 740-747
Wortmannin-C20 Conjugates Generate Wortmannin
†
‡
†
‡
,†
Hushan Yuan, Ji Luo, Ralph Weissleder, Lewis Cantley, and Lee Josephson*
Center for Molecular Imaging Research, Massachusetts General Hospital and HarVard Medical School, 149 13th Street,
Charlestown, Massachusetts 02129, and Department of Systems Biology, HarVard Medical School, and DiVision of Signal Transduction,
Beth Israel Deaconess Medical Center, 77 AVenue Louis Pasteur, Boston, Massachusetts 02115
ReceiVed July 21, 2005
We report on C20-6-(N-methylamino)hexanoic conjugates of wortmannin featuring a tertiary enamine attached
to the C20 that inhibit phosphoinositol-3-OH kinase (PI3K) by producing wortmannin (Wm) through an
intramolecular attack. The generation of Wm by these conjugates permits the design of Wm based PI3K
inhibitors that need not fit into the ATP pocket of PI3K, including Wm conjugates of BSA, IgG, or beads.
Wm generating WmC20-N(Me)-hexanoate conjugates offer an approach to the design of targeted or slow
release forms of Wm which may inhibit PI3K in tissues more selectively than the parent Wm, a compound
which has desirable anti-inflammatory and anti-proliferative activities but which also has a variety of toxic
effects.
Introduction
improved fit for the C20 derivatives into the ATP site or because
the C20 substituents serve as superior leaving groups, enhancing
the rate of attack by the lysine residue and covalent modification
Wortmannin (Wm) is a steroid-like molecule that reacts
covalently with a lysine in the ATP binding site of the catalytic
32
subunit of phosphoinositol-3-OH kinase (PI3K).1,2 The central
of the enzyme as shown in Figure 1 (dotted arrow). However,
a second mechanism proposed here postulates that WmC20
derivatives generate Wm, which then binds PI3K (Figure 1, solid
arrow). Even though both mechanisms require detachment of
the substituents at C20, and covalent modification of Wm by
an amino group in the ATP site, the difference between them
has significant implications. The ability to generate Wm, or lack
thereof, might explain the relative potency of Wm based
compounds. In addition, the self-generation mechanism permits
the design of Wm based compounds as PI3K inhibitors that do
not need to fit into the ATP pocket of PI3K.
role played by PI3K in cell proliferation and immune regulation
suggests that PI3K inhibitors might be developed as anti-cancer
or anti-inflammtory drugs.3
-6
Wm and its derivatives have
long been investigated as anti-inflammatory agents (1970s-
5
,7-11
present)
and more recently for their anti-cancer activities
mid-1990s-present).12-15 Wm is growth inhibitory for tumor
(
1
6
17
15,18
cell lines from the pancreas, lung, and breast.
The activity
of PI3K, inclusive of all subtypes, is essential for normal
function in diverse areas of biology including platelet adhe-
19,20
21,22
sion,
insulin action,
growth factor activity (PDGF, VEGF
2
3
4,23,24
Our goals were (i) to demonstrate that a WmC20 derivative
could self-activate or generate Wm and (ii) to demonstrate that
WmC20 derivative maintained the property of self-activation
after conjugation to the amino groups of proteins or solid phases.
To accomplish these goals, we synthesized a C(20) 6-(N-
methylamino)-hexanoic derivative of Wm (2a), featuring a
tertiary enamine attached to the C20 (Figure 2). We also
synthesized a C(20) 6-amino-hexanoic derivative of Wm (2b),
featuring a secondary enamine attached to C20. Compound 2b,
a Wm derivative referred to as WmC20-NH-hexanoic acid, is
shown with the six membered hydrogen bond structure as
Her2/neu, EGF), cell proliferation,
and immune func-
tion.25-27
Wm is also used to inhibit PI3K and demonstrate the
role of the enzyme, and pathways it controls, in biological
phenomena.
The use of Wm as a pharmaceutical is hampered by its
toxicity which was described as hemorrhagic in the early
2
8-30
literature,
perhaps because of its ability to inhibit PI3K in
platelets.20 More recent studies have focused on Wm’s hepatic
and myelosuppressive toxicities.3
1,32
A second problem limiting
3
3
the use of Wm as a pharmaceutical is its instability. Thus,
despite considerable literature suggesting that Wm or Wm
derivatives have desirable therapeutic effects, development of
Wm based drugs has been hindered by their considerable toxicity
and instability. Attempts to overcome the drawbacks of Wm
1
3
proposed by Norman, which we confirmed by the presence
of a proton peak at 9.88 ppm (hydrogen bonded NH proton)
and by a shift in the position of other protons (Figure 6B). We
find that the self-activation of compound 2a, referred to as
WmC20-N(Me)-hexanoic acid, was maintained after conjugation
to proteins or solid phases. Our results suggest that self-
activating Wm conjugates might be designed to produce locally
high concentrations of Wm in vivo and offer a new approach
to the selective inhibition of PI3K.
have including synthesis of Wm derivatives at the C11 position
and C17 position.3
4,35
The chemistry and biology of Wm have
recently been reviewed.36
One class of efforts to develop Wm derivatives as PI3K based
pharmaceuticals has focused on the modification of Wm at the
C20 position by amines or thiol compounds.31,32 PX-866, formed
by reaction of Wm with diallylamine at C20, has an IC50 for
PI3K 12 times lower than the parent Wm and has shown
promise in animal tumor models. The superiority of WmC20
derivatives over Wm as PI3K inhibitors may be due to an
Materials and Methods
General Description. Wortmannin was a gift of the natural
products branch of the NCI. All reagents and solvents (Aldrich)
were of standard quality. Silica gel (open column chromatography)
was 60-20 mesh (J. T. Baker). BSA and mouse IgG were from
Sigma. Dynal M-270 amine magnetic beads were purchased from
Dynal Biotech. Pre-packed PD-10 columns were from Amersham
Biosciences. NMR was performed on Varian 400 MHz instrument
*
To whom correspondence should be addressed. Tel: (617) 726-6478.
Fax: (617) 726-5708. E-mail: ljosephson@partners.org.
†
‡
Massachusetts General Hospital and Harvard Medical School.
Harvard Medical School and Beth Israel Deaconess Medical Center.
1
0.1021/jm050699p CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/22/2005

Wortmannin-C20 Conjugates Generate Wortmannin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 741
Figure 1. Possible mechanisms of action of Wm C20 Derivatives. Wm binds to PI3K, with attack by lysine 833 of PI3K. For the general mechanism
of Wm action, see refs 1 and 2. We propose that Wm C20 derivatives generate Wm through an intermolecular attack, which then inhibits PI3K
solid arrows). Wm C20 derivatives could also bind to PI3K with attack by lysine as proposed (dotted arrows).3
1,32
(
Figure 2. Synthesis of WmC20 derivatives. Wm was modified by reaction with 6-N(Me)-hexanoic acid or 6-NH-hexanoic acid followed by
formation of the N-hydroxysuccinimide esters 3a or 3b. Compounds 3a or 3b are then attached to the amine groups of BSA, IgG, or amine
functionalized beads.
with CDCl
3
as solvent, while mass spectra were obtained on a
HPLC (Varian Prostar 210 with a variable wavelength PDA 330
detector) employed reverse phase C18 columns (VYDAC, Cat.#:
Micromass LCT instrument with time-of-flight ESI technique.

7
42 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
18TP1022 for synthesis; Varian, Cat. #: R0086200C5 for analysis)
Yuan et al.
2
NHS Ester, Cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-
trione, 5-(acetyloxy)-1-{[N-(5-carboxylpentyl)-N-methylamino]-
methylene}-4,4a,5,6,6a,8, 9,9a-octahydro-11-hydroxy-4-(meth-
oxymethyl)-4a,6a-dimethyl-, (4S,4aR,5R,6aS,9aR)- (9CI) (3a).
Compound 2a (15.8 mg, 0.0276 mmol) was mixed with N-
hydroxysuccinimide (38 mg, 0.33 mmol) and dicyclohexylcarboim-
ide (68.2 mg, 0.33 mmol) in anhydrous acetonitrile (1 mL). The
mixture was stirred at room temperature for 30 min. The solid
residue was obtained by filtration and washed with anhydrous
acetonitrile, and the solvent was removed by partial vacuum. The
residue was fractioned by silica gel chromatography with anhydrous
ethyl acetate:hexane (5:1-10:1). A light yellow solid was obtained
by precipitation with anhydrous hexane from trace amount of
with water (Millipore, containing 0.1% trifloroacetic acid) (buffer
A) and acetonitrile (containing 20% buffer A) (buffer B) as elution
buffer. System 1: buffer A/buffer B (80:20) isocratic for 5 min,
linear gradient to buffer A/buffer B (20:80) over 40 min, then the
gradient back to 80:20 in 5 min and isocratic for 5 min. Flow: 4.9
mL/min. λmax: 418 nm (used for purification of 2a). System 2:
buffer A/buffer B (80:20) isocratic for 5 min, linear gradient to
buffer A/buffer B (20:80) over 25 min and then isocratic for 5 min,
then the gradient back to 80:20 in 5 min and isocratic for 5 min.
Flow: 6.0 mL/ min. λmax: 408 nm (used for purification of 2b).
System 3: buffer A/buffer B (70:30) linear gradient to buffer
A/buffer B (20:80) over 15 min, then gradient back to 70:30 in 3
min and isocratic for another 5 min. Flow: 1.0 mL/min. λmax: 258
nm (Figure 3A,C). System 4: buffer A/buffer B (99:1) linear
gradient to buffer A/buffer B (40:60) over 46 min, then gradient
back to 90:1 in 5 min and isocratic for another 5 min. Flow: 1.0
mL/min. λmax: 250 nm (Figure 3B,D).
anhydrous ethyl acetate: 17 mg, 92%. MS: C34
H
42
N
2
O12, calcd
+
671.2816 (M + H ), found 671.2819.
NHS Ester, Cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-
trione, 5-(acetyloxy)-1-{[N-(5-carboxylpentyl)amino]methylene}-
,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-
a,6a-dimethyl-, (4S,4aR,5R,6aS,9aR)- (9CI) (3b). Compound 2b
11.2 mg, 0.02 mmol) was mixed with N-hydroxysuccinimide (27.6
mg, 0.24 mmol) and dicyclohexylcarboimide (49.5 mg, 0.24 mmol)
in anhydrous acetonitrile (1 mL) and handled as for 3a. The residue
was fractioned by silica gel chromatography with anhydrous ethyl
acetate:hexane 3:1. A light yellow solid was obtained by precipita-
tion with anhydrous hexane from trace amount of anhydrous ethyl
4
4
(
Synthesis of Wm C20 Derivatives and Conjugates. Relation-
ships between the compounds synthesized are shown in Figure 2.
6
-(N-Methylamino)-hexanoic acid was made according to litera-
37
ture.
Cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione,5-(acetyl-
oxy)-1-{[N-(5-carboxylpentyl)-N-methylamino]methylene}-4,4a,5,6,-
6
a,8, 9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-di-
methyl-, (4S,4aR,5R,6aS,9aR)- (9CI) (Referred To as WmC20-
N(Me)-hexanoic Acid 2a). Wortmannin (42.8 mg, 0.1 mmol), 6-(N-
methylamino)hexanoic acid hydrogen chloride (90 mg, 0.5 mmol),
and triethylamine (20 µL) were mixed in anhydrous DMSO (1 mL).
The mixture was stirred at room temperature until complete as
monitored by TLC (about 1.5 h). After dilution with 50%
acetonitrile in water (1:1) before the injection, the mixture was
purified by HPLC (system 1) and gave a yellow powder after
lyophilization. Analysis of 2a by HPLC (system 3) showed less
acetate: 12.4 mg, 94.6%. MS: C33
H
40
2
N O
12, calcd 657.2659 (M
+
+
H ), found 657.2669.
Conjugation of the WmC20 Derivatives 2a or 2b to Proteins
or Beads. Proteins were dissolved in PBS, pH 6.8 at 4-10 mg/
mL. The WmC20 NHS esters (3a or 3b) were dissolved in DMSO
at 40 mg/mL (60 mM), and 10-30 µL, depending on the loading
ratio, was added to 0.5 mL of protein. Reaction was for 1-1.5 h at
37 °C. Unreacted Wm derivative was removed with PD-10 columns.
Concentrations of proteins were determined spectrophotometrically,
subtracting the OD280 from the Wm derivative before determining
protein. The concentration of Wm was determined from its
than 3% of Wm: 39.5 mg, 68.9%. MS: C30
H
39NO10, calcd
74.2652 (M + H ), found 574.2648. H NMR (CDCl ): 0.85 (3H,
s, C18-CH ), 1.41-1.54 (2H, m, CH CH CH -), 1.58 (3H, s,
C19-CH ), 1.65-1.78 (4H, m, -CH CH CH -), 1.80-1.85 (1H,
dd, J ) 14.8 Hz, J ) 4.63 Hz, H-12), 2.06 (3H, s, CH CO), 2.09-
.20 (1H, m, H-16), 2.22-2.33 (1H, m, H-15), 2.37-2.43 (3H, m,
H-16, CH CH COOH), 2.54-2.61 (1H, m, H-12), 2.79 (3H, broad,
NCH ), 2.91-3.03 (2H, m, H-14, CH OCH ), 3.12-3.24 (2H, m,
H-15, CH OCH ), 3.25 (3H, s, CH O), 3.39-3.56 (2H, m, broad,
NHCH ), 4.05 (2H, broad, OH), 4.49 (1H, dd, J ) 2 Hz, J ) 7.6
Hz, H-1), 6.06 (1H, dd, J ) 4.8 Hz, J ) 7.2 Hz, H-11), 8.18
1H, s, H-20). The 13C NMR could not be obtained due to Wm
generation in the spectra recording.
+
1
5
3
3
2
2
2
-1
absorbance at 418 nm using an extinction coefficient of 7058 cm
3
2
2
2
-
1
M
we determined. Wm to protein molar ratios were as follows:
1
2
3
2
4a (2a/BSA) ) 4.9; 4b (2b/BSA) ) 8.7; 5a (2a/IgG) ) 23; 5b
(2b/IgG) ) 31. Beads (Dynal M-270, amine functionalized) in 0.6
2
2
9
3
2
3
mM PBS (2 × 10 beads/mL) were washed using a Dynal magnetic
2
3
3
separator and suspended in 0.5 mL of DMSO/PBS (50:50 v/v).
WmC20 NHS ester 3a or 3b (50 µL) dissolved in DMSO at 40
mg/mL (60 mM) was added to beads. After 1 h at 37 °C, unreacted
Wm derivatives were removed by magnetic separation of the beads.
2
1
2
1
2
(
Generation of Wm with WmC20 Derivatives and WmC20
Conjugates. Solutions of 2a and 2b (200 µL of about 1.5 mM in
Wm or Wm derivatives) were made by adding 30 mM WmC20
derivative in DMSO to PBS buffer (pH 6.8). All experiments were
in PBS, pH 6.8. Solutions were incubated (37 °C for the indicated
time) and analyzed by applying 10 µL plus 2 µL of internal standard
1 µg of methyl-4-hyrdroxybenzoate, MHB) to the HPLC. System
was used for 2a and Wm while system 4 was used for 2b.
Compounds 4a (1.8 mg) and 4b (1.3 mg) were dissolved in 0.4
Cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione,5-(acetyl-
oxy)-1-{[N-(5-carboxylpentyl)amino]methylene}-4,4a,5,6,6a,8,9,9a-
octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-, (4S,-
4
aR,5R,6aS,9aR)- (9CI) (Referred To as WmC20-NH-hexanoic
Acid 2b). Wm (42.8 mg, 0.1 mmol), 6-aminohexanoic acid (65.5
mg, 0.5 mmol), and triethylamine (20 µL) were mixed in anhydrous
DMSO (1 mL). The reaction was monitored as above, purified by
HPLC (system 2), and after lyophilization a yellow powder was
obtained. HPLC analysis showed a single peak to prove its high
(
3
mL of PBS buffer, incubated at 37 °C for the indicated time, and
applied to HPLC (system 3 for 4a, system 4 for 4b). Initial
concentrations used when modeling Wm generation (eq 1, Figure
purity: 42.5 mg, 76.0%. MS: C29H37NO10, calcd 560.2495 (M +
+
1
H ), found 560.2499. NMR (CDCl
3H, s, C18-CH ), 1.43-1.51 (2H, m, -CH
s, C19-CH ), 1.68-1.75 (4H, m, -CH CH
1H, dd, J ) 14.8 Hz, J ) 2.8 Hz, H-12), 2.05 (3H, s, CH
3
, ppm): H NMR (CDCl
CH CH -), 1.53 (3H,
CH -), 1.86-1.90
CO),
3
): 0.83
(
3
2
2
2
4
A) were determined by weight for 2a and 2b and spectrophoto-
3
2
2
2
-
1
-1
metrically (extinction coefficients: 2a, 7085 cm
2
M
at 418 nm;
at 408 nm) for 4a, 4b, 5a, and 5b.
Stability of Wm, WmC20 Derivatives, and WmC20 Conju-
(
2
1
2
3
-
1
-1
b, 5505 cm
M
.24-2.37 (3H, m, H-15, H-16), 2.42 (2H, t, J ) 7.4 Hz, CH
2
-
-
CH COOH), 2.54-2.63 (1H, m, H-12), 2.84-2.88 (1H, m, CH
2
3
OCH
CH OCH
6.5 Hz), 4.33-4.35 (1H, m, H-1), 4.84 (2H, broad, OH), 5.98-
.01 (1H, m, H-11), 8.55 (1H, d, J ) 13.9 Hz, H-20), 9.81-9.92
1H, m, NH). 13C NMR: 16.84, 21.14, 22.59, 24.29, 26.05, 26.43,
2
), 2.94-2.99 (1H, m, H-14), 3.14-3.26 (2H, m, H-15,
gates. To obtain the stability of Wm in the presence of BSA, Wm
(0.45 mg, 1.04 µmol) and BSA (1.8 mg) were incubated in PBS
buffer (400 µL) and acetonitrile (60 µL) at 37 °C for various times.
MHB was added as above followed by HPLC (system 3). To
determine the stability of Wm in the presence of N-acetyl lysine
or proline, Wm (about 1.5 mM) in PBS with 10 mM of N-acetyl
lysine or proline (pH 6.8, 400 µL) and acetonitrile (60ul) in a 1.5
mL tubes were incubated at 37 °C. At different times, a 10 µL
3
2
), 3.27 (3H, s, CH OCH ), 3.42 (2H, q, J ) 13.0 Hz, J
3
2
1
2
)
6
(
30.45, 33.62, 36.71, 38.59, 42.36, 42.41, 50.04, 59.42, 67.29, 73.33,
81.37, 88.43, 129.06, 137.09, 137.57, 151.13, 159.63, 166.40,
170.27, 178.01, 178.69, 218.29.

Wortmannin-C20 Conjugates Generate Wortmannin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 743
Figure 3. Formation of Wm from Wm C20 derivatives and conjugates. (A) Formation of Wm from 2a. The internal standard is MHB (methyl-
-hydroxybezoate). (B) Lack of formation of Wm from 2b. (C) Formation of wortmannin from 4a. (D) Lack of formation of Wm from 4b. HPLC
4
system 3 was used for A and C while HPLC system 4 was used for B and D.
solution was mixed with 2 µL internal standard solution in DMSO
min, the time scale was expanded on Figure 3B to clearly
visualize the presence of Wm, which was not detected. It can
be seen that 2a generated Wm while 2b did not, with a more
complete time course for Wm generation from 2a shown in
Figure 4A. We next examined if the formation of Wm would
occur if 2a was conjugated to BSA to yield 4a. After 3 h, 4a
generated Wm (Figure 3C), while Wm was not detected under
the same conditions with 4b (Figure 3D). The complete time
course of Wm formation from 4a is shown in Figure 4A. Figure
4A also shows the time course of Wm formation obtained when
2a was conjugated to IgG (5a) or beads (6a). Thus WmC20-
N(Me)-hexanoic acid (2a) and materials made with 2a (4a, 5a,
and 6a) generated Wm by our HPLC method. In contrast, Wm
formation could not be detected with WmC20-NH-hexanoic acid
(2b) or materials made from 2b (4b, 5b, 6b).
(2 µg MHB) and analyzed by HPLC (system 3).
For both Wm generation and Wm stability studies, the internal
standard (IS) ratio was calculated for each sample by dividing the
peak area of the samples by the area of internal standard (MHB).
To obtain the concentration of Wm generated (Figure 4A), standard
curves were obtained by using 1-5 µg of the appropriate WmC20
derivatives or conjugates and IS ratio.
PI3K activity was measured as described by Cantley and co-
workers,38 with minor modifications. The assay uses enzyme from
39
3
2
CHO-K1 cells and assesses lipid phosphorylation by P labeled
ATP after a 30 min incubation.
Results
We hypothesized that a Wm derivative with a tertiary enamine
at C20 would generate Wm, while a Wm derivative with a
secondary enamine at C20 would not, based on the work of
Norman discussed below. We therefore synthesized WmC20-
N(Me)hexanoic acid (2a) and conjugated it to the amino groups
of BSA, IgG, or amine functionalized beads to obtain the
WmC20-N(Me)hexanoic acid conjugates, 4a, 5a, or 6a. Similar
reactions were performed with WmC20-NH-hexanoic acid (2b)
to obtain 4b, 5b, and 6b. This strategy allowed WmC20-NH-
hexanoic acid based compounds to serve as negative controls
for their WmC20-N(Me)-hexanoic counterparts in experiments
where the generation of Wm was to be monitored.
We first examined the ability to generate Wm by diluting
our Wm derivatives and conjugates into PBS (pH 6.8), the buffer
used for all experiments, and analyzed the resulting samples
for the presence of Wm by RP-HPLC. Figure 3A-D shows
HPLC methods used to determine the formation of Wm in these
experiments. With increasing incubation time, WmC20-N(Me)-
hexanoic acid (2a) showed a progressive increase in Wm and
decrease in 2a (Figure 3A), which was not seen when a similar
experiment was performed with 2b (Figure 3B). Because 2b
and Wm had a difference in retention time of only about 1.0
A linear sequential model for the formation of Wm and
subsequent decay was used to generate the lines shown in Figure
4B. The model yields eq 1 in the case where the Wm
concentration as a function of time, [Wm]t, occurs from an initial
concentration of 2a, [2a]i.
[Wm] ) [2a] k [exp(-k t) - exp(-k t)]/(k - k ) (1)
t
i
1
1
2
2
1
A two parameter fit was employed to obtain k1 and k2 for
2a, 4a, and 5a with data provided in Table 1. The amount of
WmC20-N(Me)-hexanoic acid attached to beads (6a) could not
be determined either by weight or spectrophotometrically, so a
three parameter fit was employed to obtain values of k1, k2,
and [6a]i. The simple linear formation and decomposition model
(Figure 4B) satisfactorily described the time course of Wm
formation with WmC20-N(Me)-hexanoic acid (2a) and conju-
gates made with it (4a, 5a, and 6a).
As these studies were being conducted, Holleran33 reported
that Wm had a half-life of 8-13 min in culture media at 37
°C, a value that seemed inconsistent with the kinetics of Wm

7
44 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Yuan et al.
Figure 4. Time course of Wm generation from WmC20 N(Me)-hexanoic acid derivatives. (A) Time course of Wm generation and degradation by
HPLC. Data were analyzed according to the model shown in B which yields eq 1. Values of k and k are given in Table 1.
1
2
Table 1. Constants for Wm Generation from Wm C20 Derivatives (Fit
to Eq 1)
materials, with dramatically higher effects for the secondary
a
-1
amine of proline (kFOD )1.87 h ) compared to the single
-
1
initial concn of Wm
derivative (mM Wm)
primary amine of N-acetyl lysine (kFOD )0.30 h ). Addition
of 10 mM proline increased kFOD about 100-fold, yielding a
half-life of 22 min, which approached the half-lives of Wm in
compd
k1 (h-1)
0.051
0.045-0.056)
0.190
k2 (h-1
0.114
(0.095-0.132)
0.905
(0.707-1.103)
0.282
(0.166-0.398)
0.148
(0.076-0.220)
Concentrations of BSA and IgG were 0.065 mM and 0.011 mM,
)
2
4
5
6
a
0.94
(
33
RPM or MEM culture media. RPM and MEM are a mixture
a (2a-BSA) 0.35
a (2a-IgG) 0.35
a (2a-bead) 0.0750
of amino acids in the millimolar concentration range. The kFOD
(0.128-0.253)
was also measured as a function of the pH of the PBS buffer,
0.024
-
1
-1
(0.013-0.034)
with values of 0.018 h and 0.028 h obtained at pH 7.4 and
8.0, respectively. Thus Wm is quite stable in aqueous media
0.308
(0.0414-0.109)
(0.124-0.492)
(PBS), but its instability was greatly enhanced by biological
a
molecules such as proline, BSA, or N-acetyl lysine.
9
respectively. Concentration of beads was 3 × 10 beads/mL.
We also determined the kFOD for the WmC20 derivatives 2a
and 2b as shown in Figure 5B, with values given in Table 2.
disappearance shown in Figure 5. To resolve this issue, we
studied the disappearance of Wm in the presence of BSA,
N-acetyl-lysine, and proline as shown in Figure 5A. Lines were
obtained by fitting data to eq 2, which illustrates our treatment
of the data for Wm and 2a (initial concentration ) [2a]i or
-
1
-1
The kFODs for 2a and 2b were 0.080 h and 0.0053 h ,
-
1
respectively, compared to that of Wm which was 0.012 h .
However, the addition of 10 mM proline slightly decreased kFODs
for 2a and 2b, rather than dramatically increasing the kFOD as
was the case for Wm. A similar result was obtained for the
addition of 0.065 mM BSA, which had no effect on the kFOD of
[
Wm]i, concentration as a function of time ) [2a]t or [Wm]t),
with first order decay constants (kFOD) given in Table 2.
2
a and only slightly decreased that of 2b. Thus Wm modified
at C20 by the attachment of 6-N(Me)-hexanoic acid (2a) or
-NH-hexanoic acid (2b) was resistant to the increase in the
[
2a] ) [2a] exp(-kFODt)
(2)
t
i
6
-1
Wm was quite stable in PBS, with a kFOD of 0.012 h , which
corresponded to a half-life of 57.5 h. We repeated with
determination of kFOD in the presence of 0.065 mM BSA and
instability produced by the addition of 10 mM proline. It appears
that proline enhances the instability of Wm by reacting at the
C20 position, since this is the only difference between Wm and
2a or Wm and 2b. While the decay of 2a was not affected by
the addition of proline, a peak in addition to Wm was seen on
our HPLC chromatograms when 2a stability was measured. The
major peak with 2a decay was Wm, while the third peek had
the same retention time as Wm after reaction with proline (data
-
1
determined a kFOB of 0.18 h , or about 15-fold larger than the
kFOD in PBS. The addition of 10 mM proline or 10 mM N-acetyl
lysine also accelerated the decay of Wm (see Table 2 for values
of kFOD). Thus the first order decay constant for Wm was
increased by the addition of amine containing molecules or

Wortmannin-C20 Conjugates Generate Wortmannin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 745
Figure 6. Partial NMR spectra for 2a and 2b in CDCl
3
. (A) For 2a:
8
.26 ppm (s, H-C20 of Wm), 8.18 ppm (s, H-C20 of 2a), 6.16 ppm
(
m, H-C11 of Wm), 6.07 ppm (m, H-C11 of 2a). (B) For 2b: 9.86
ppm (m, C20NH of 2b), 8.55 ppm (d, J ) 13.9 Hz, H-C20 of 2b),
Figure 5. Stability of Wm or Wm C20 derivatives. (A) Stability of
Wm in PBS with the indicated compounds. (B) Stability of WmC20
derivative 2a in PBS (b), 10 mM proline (2), or 0.065 mM BSA (9).
Stability of WmC20 derivative 2b in PBS (O), 10 mM proline (4), or
5.99 ppm (m, H-C11 of 2b).
ppm (m, C20NH), 8.55 ppm (d, J ) 13.9 Hz, H-C20), and
5
.99 ppm (m, H-C11), respectively, and were independent of
0
.065 mM BSA (0). First order decay constants are given in Table 2.
time. The ability of 2a but not 2b to generate Wm occurs both
in PBS at pH 6.8 and in chloroform.
Table 2. First Order Decay (FOD) Constants of Wm and Wm C20
Amines
Finally, we sought to confirm our HPLC and NMR results
that WmC20 derivatives employing 6-N-methylaminohexanoic
acid linker (2a, 4a, 5a) would generate Wm, while WmC20
derivatives employing the 6-aminohexanoic acid linker (2b, 4b,
5b) could not do so, using a PI3K enzyme assay. We therefore
incubated all six compounds for 3 h at 37 °C in PBS to allow
significant Wm generation, the complete time course for which
is shown in Figure 4. PBS plus Wm C20 derivative was then
added to PI3K to assess its ability to inhibit PI3K activity. IC50s
for each compound are given in Table 3. Also shown are the
percentages of Wm released from the WmC20 adduct at the 3
h time point from Figure 4. WmC20 derivatives employing the
6-N-methylaminohexanoic acid linker (2a, 4a, 5a) inhibited
PI3K with IC50s that were 3- to 32-fold higher than Wm, while
the corresponding WmC20 derivatives employing the 6-N-
aminohexanoic acid linker (2b, 4b, 5b) did not inhibit PI3K at
kFOD (h-1)
(95% confidence intervals) half-life
compd
conditions
Wm
Wm
Wm
Wm
PBS
0.012 (0.0080-0.016)
0.18 (0.16-0.20)
1.87 (0.62-3.13)
0.30 (0.23-0.38)
0.080 (0.071-0.089)
0.065 (0.0062-0.068)
0.082 (0.075-0.088)
0.0053 (0.0040-0.0067)
0.0031 (0.0023-0.0038)
0.0028 (0.0021-0.0034)
4.02
57.8 h
3.85 h
22 min
2.31 h
8.7 h
10.7 h
8.5 h
131 h
223 h
248 h
10.3 min
8.3 min
PBS, 0.065 mM BSA
PBS, 10 mM proline
PBS, 10 mM NAcLys
PBS
PBS, 10 mM Proline
PBS, 0.065 mM BSA
PBS
2
2
2
2
2
2
a
a
a
b
b
b
PBS, 10 mM proline
PBS, 0.065 mM BSA
a
Wm
Wm
RPM
a
MEM
5.04
a
Values from ref 33.
not shown). This observation suggests that 2a produced Wm
which then reacted with proline.
1
000 nM or at IC50 at least 250-fold higher than Wm. Both
HPLC assays and a PI3K enzyme inhibition assay indicate that
Wm was produced from three WmC20 derivatives employing
the 6-N-methylaminohexanoic acid linker but not from those
employing the 6-aminohexanoic acid linker. The relative potency
of WmC20 derivatives employing the 6-(N-methylamino)-
hexanoic acid linker was similar as assayed by HPLC and as
inhibitors of Wm (4a > 2a > 5a).
We then examined the production of Wm from WmC20-
N(Me)-hexanoic acid (2a) and from WmC20-NH-hexanoic acid
(2b) in deuterated chloroform by NMR, a neutral organic solvent
at ambient temperature as shown in Figure 6. The 0 h spectra
of 2a (Figure 6A) exhibited a small peak at 8.26 ppm, due to
the C20 proton of Wm which was present at the initial time
point due to trace amounts of self-activation of 2a to yield Wm
during the purification and lyophlization. The peak at 8.26 ppm
increased with time, indicating a slow formation of Wm, while
a peak at 8.18 ppm, assigned to the C20 proton of 2a, decreased
with time. Thus 2a decreased 14% and Wm increased 16% over
a period of 50 h, which was calculated based on the ratio of
integration of these two peaks due to the conversion of 2a to
Wm. The spectra of 2b (Figure 6B) exhibited peaks at 9.86
Discussion
Wm derivatives were synthesized by reacting the C20 of Wm
with 6-N-methylaminohexanoic acid or 6-aminohexanoic acid
(2a, 2b), followed by the conjugation of 2a and 2b to amine
groups of proteins or beads. Wm compounds made with 6-(N-
methylamino)-hexanoic acid (2a, 4a, 5a, 6a) generated Wm

7
46 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Yuan et al.
Table 3. Inhibition of PI3K by Wm C20 Derivatives and Conjugate
bioassay: PI3K IC50 (nM)
IC50Wm/IC50
Wm generation (%)
(95% confidence intervals)
Wm-derivative
by HPLC (3 h, 37 °C)
Wm (1)
4.3 ( 1.3 (2.5-7.4)
36.7 (1.0 (34.5-39.0)
>1000
NA
NA
10.4
ND
16.5
ND
3.5
WmC20-N(Me)-hexanoic acid (2a)
WmC20-NH-hexanoic acid (2b)
WmC20-N(Me)-hexanoate-BSA (4a)
WmC20-NH-hexanoate-BSA (4b )
WmC20-N(Me)-hexanoate-IgG (5a)
WmC20-NH-hexanoate-IgG (5b)
0.117
<0.004
0.29
<0.004
0.033
<0.004
14.8 ( 1.1 (12.6-17.4)
>1000
129.9 (104.4 to 161.5)
>1000
ND
(
Figures 3 and 4) while the corresponding compounds made
57.5 h (in PBS) to 22 min (in PBS plus 10 mM proline). Wm
can be highly stable or highly unstable in an aqueous environ-
ment depending on the presence of biological molecules such
as amino acids or proteins.
with 6-aminohexanoic acid (2b, 4b, 5b, 6b) did not. The
production of Wm correlated with the inhibition of PI3K enzyme
activity; WmC20-N(Me)-hexanoic based compounds inhibited
PI3K while WmC20-NH-hexanoic based compounds failed to
Proposing that WmC20-N(Me)-hexanoic acid based com-
pounds are self-activating requires that they be inactive as
inhibitors of PI3K and converted to the active inhibitor (Wm).
However, because WmC20-N(Me)-hexanoic acid and com-
pounds made using it generate Wm under conditions of the PI3K
assay (phosphate buffer), the ability to demonstrate that they
are not inhibitors of PI3K is somewhat limited. The evidence
that WmC20-N(Me)-hexanoic acid based compounds fail to
inhibit PI3K until they generate Wm has been inferred from
different types of experiments: (i) WmC20-N(Me)-hexanoic
acid based compounds, but not WmC20-NH-hexanoic acid
based compounds, generated Wm with our HPLC assay (Figure
do so (Table 3). The pattern of inhibition of PI3K we obtained
was consistent with the observations of Norman.1
3,12
Norman
obtained an IC50 of 80 nM for a Wm reacted at C20 with the
secondary amine, diethylamine, compared to an IC50 of 36.7
nM we obtained for Wm reacted with the secondary amine,
6
-(N-methylamino)-hexanoic acid, see Table 3 (2a). Norman
obtained an IC50 of greater than 500 nM for Wm reacted at
C20 with the primary amine, ethylamine, which compared well
with the IC50 of greater than 1000 nM we obtained for Wm
reacted with the primary amine 6-aminohexanoic acid (2b). In
both studies the IC50 for Wm was 4.3 nM.
We propose that the WmC20-N(Me)-hexanoic acid derivative
2a), which cannot form the hydrogen bonded ring structure
Figure 2), inhibited PI3K by generating Wm through an
3
), (ii) WmC20-N(Me)-hexanoic acid, but not WmC20-NH-
hexanoic acid, generated Wm in chloroform evident by NMR,
iii) WmC20-N(Me)-hexanoic acid based compounds, but not
(
(
(
intramolecular attack of the C6 hydroxyl group (Figure 1, solid
arrow). The WmC20-NH-hexanoic acid derivative (2b) failed
to inhibit PI3K because it was stabilized by hydrogen bonded
ring, so that Wm formation was either nonexistent or too slow
to be detected. The control of the intramolecular attack by the
hydrogen bonded ring determined whether Wm was generated,
not only with the low molecular weight WmC20 N(Me)-
hexanoic acid (2a) or WmC20 NH-hexanoic (2b) derivatives
but also when these derivatives were covalently coupled to
amine groups, to yield the high molecular weight Wm conju-
gates (4a, b-6a, b). The intramolecular attack was due to a
combination of features of the WmC20 derivatives and conju-
gates including the enamine linkage of the nitrogen at C20, the
lack of the stabilizing hydrogen bond with carbonyl at C3, the
proximity of enol hydroxyl group at C6, and perhaps other
features of the Wm molecule. The intramolecular mechanism
for Wm generation was supported by the production of Wm
from 2a in chloroform (Figure 6), a nonaqueous solvent without
added acid or base. We know of no other case where a
pharmacologically active substance can be covalently conjugated
to a protein and regenerate itself under physiological conditions
through an enzyme independent, solvent independent, intra-
molecular attack.
WmC20-NH-hexanoic based compounds, inhibited PI3K. The
inhibition of PI3K through Wm generation is also supported
by the X-ray crystal structure of PI3K, which appears not to
allow WmC20 based compounds with very large modifications
at C20 (4a, 5a) to fit into the ATP binding pocket as Wm does.
2
Our studies have several implications for the use of Wm in
signal transduction research and for the development of low
molecular weight Wm derivatives and high molecular weight
Wm conjugates as pharmaceuticals. First, our observation that
Wm is stable in PBS but not in the presence of proline, together
with observations about its instability in tissue culture media,33
may result in new experimental designs or a reinterpretation of
older literature where Wm was used to evaluate the function of
the PI3 kinase pathway through its inhibition. Second, the
pharmacological potency of Wm based compounds may depend
on their ability to generate Wm and survive in biological media,
in addition to the usual parameters determining drug potency,
such as the ability of the synthesized compound to bind a
molecular target (PI3K) or the ability to undergo membrane
translocation. Wm is a highly unusual compound that is rapidly
degraded in biological media, whose derivatives vary widely
in their stability in aqueous media, and whose degradation is
influenced profoundly by common substances in biological
systems. Understanding these features of Wm, Wm derivatives,
or Wm conjugates may lead to new dosing schedules that
recognize these specific features. Third, the chemistry we have
described permits the attachment of the N-hydroxysuccinimide
ester of WmC20-N(Me)-hexanoic acid (3a) to the amines of
any amine bearing targeting biomolecule (antibody, peptide,
polysaccharide) or solid phase. This provides a general method
of designing self-activating WmC20 conjugates that maybe
useful as tissue or cell selective PI3K inhibitors. The develop-
ment of targeted PI3K inhibitors may be essential for the
development of successful PI3K inhibiting pharmaceuticals
The slow increase and decrease in Wm concentration when
WmC20 6-N(Me)-hexanoic based compounds were incubated
in PBS (Figure 4) requires considerable stability of Wm and
seemed to be inconsistent with the half-life of 8-13 min
reported for Wm in tissue culture media.33 We therefore
measured the stability of Wm as first order decay constants in
PBS and in PBS with amine bearing molecules added (Table
2). Wm was highly stable in PBS, but its stability was decreased
with the addition of BSA, N-acetyl-lysine, or proline. The kFOD
obtained with 10 mM proline of 1.87 h-1 was about half the
reported values for a similar constant for Wm stability in tissue
culture media.33 In our hands the half-life of Wm ranged from

Wortmannin-C20 Conjugates Generate Wortmannin
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 747
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Acknowledgment. This work was supported by NIH grants
to CMIR/R.W. (CA85240, HL078641, HL078641, EB004626)
and Beth Israel/L.C. (GM41890, CA89021).
Supporting Information Available: Information on purity of
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
(
(
(
(
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