Studies on the mechanism of phosphatidylinositol 3-kinase inhibition by wortmannin and related analogs

Article abstract of DOI:10.1021/jm950619p

Wortmannin, a fungal metabolite, was identified as a potent inhibitor (IC₅₀ = 4.2 nM) of phosphatidylinositol 3-kinase (PI 3-kinase). Due to the importance of PI 3-kinase in several intracellular signaling pathways, structure-activity studies on wortmannin analogs were performed in an effort to understand the structural requirements necessary for PI 3-kinase inhibition. Since wortmannin is an irreversible inhibitor of PI 3-kinase, it was postulated that covalent attachment at the electrophilic C-21 site was a possible mode of action for PI 3-kinase inhibition. We have prepared various wortmannin analogs which address the possibility of this mechanism. Of particular interest are compounds which affect the C-21 position of wortmannin either sterically or electronically. Our results support the conclusion that nucleophilic addition by the kinase onto the C-21 position of wortmannin is required for inhibition of PI 3-kinase by wortmannin analogs. Additionally, we have prepared several D-ring analogs of wortmannin, and their activities are reported herein. We conclude that the wortmannin D ring is an important recognition site since modifications have such a dramatic effect on inhibitor potency. Finally, the identification of 17β- hydroxywortmannin represents the first reported subnanomolar inhibitor of PI 3-kinase. These studies, along with in vivo antitumor experiments, suggest that the mechanism of PI 3-kinase inhibition correlates to the associated toxicity observed with wortmannin-based inhibitors of PI 3-kinase.

Full text of DOI:10.1021/jm950619p

1106
J . Med. Chem. 1996, 39, 1106-1111
Stu d ies on th e Mech a n ism of P h osp h a tid ylin ositol 3-Kin a se In h ibition by
Wor tm a n n in a n d Rela ted An a logs
Bryan H. Norman,* Chuan Shih, J ohn E. Toth, J ames E. Ray, J effrey A. Dodge, Doug W. J ohnson,
Pamela G. Rutherford, Richard M. Schultz, J ohn F. Worzalla, and Chris J . Vlahos
Eli Lilly and Company, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285
Received August 21, 1995X
Wortmannin, a fungal metabolite, was identified as a potent inhibitor (IC50 ) 4.2 nM) of
phosphatidylinositol 3-kinase (PI 3-kinase). Due to the importance of PI 3-kinase in several
intracellular signaling pathways, structure-activity studies on wortmannin analogs were
performed in an effort to understand the structural requirements necessary for PI 3-kinase
inhibition. Since wortmannin is an irreversible inhibitor of PI 3-kinase, it was postulated that
covalent attachment at the electrophilic C-21 site was a possible mode of action for PI 3-kinase
inhibition. We have prepared various wortmannin analogs which address the possibility of
this mechanism. Of particular interest are compounds which affect the C-21 position of
wortmannin either sterically or electronically. Our results support the conclusion that
nucleophilic addition by the kinase onto the C-21 position of wortmannin is required for
inhibition of PI 3-kinase by wortmannin analogs. Additionally, we have prepared several D-ring
analogs of wortmannin, and their activities are reported herein. We conclude that the
wortmannin D ring is an important recognition site since modifications have such a dramatic
effect on inhibitor potency. Finally, the identification of 17â-hydroxywortmannin represents
the first reported subnanomolar inhibitor of PI 3-kinase. These studies, along with in vivo
antitumor experiments, suggest that the mechanism of PI 3-kinase inhibition correlates to
the associated toxicity observed with wortmannin-based inhibitors of PI 3-kinase.
In tr od u ction
The signaling pathways associated with cell growth
and development are now recognized as potential new
targets for therapeutic intervention in various prolifera-
tive diseases, such as cancer.1 Research during the past
decade has resulted in a greater understanding of
oncogenes and their oncoproteins, giving us insight into
the factors responsible for cellular signal transduction,
influence the electrophilicity of that center, and observe
especially as it applies to cell growth. One enzyme
the effect on inhibitor potency. These studies, as well
which has been implicated in growth factor signal
as in vivo evaluation of wortmannin-based inhibitors of
PI 3-kinase, are presented here.
transduction is phosphatidylinositol 3-kinase (PI 3-ki-
nase).2 This enzyme exists as a heterodimer of a 110
kDa catalytic subunit and a 85 kDa regulatory subunit.3
PI 3-kinase, which selectively phosphorylates inositol
lipids at the 3′ position, is an important enzyme in a
number of cellular events which involve protein tyrosine
kinases.4 Recent studies show that PI 3-kinase is
regulated by p21ras via its association with the Ras
effector site in a GTP-dependent manner.5 Inhibitors
of PI 3-kinase would be useful tools for the study of PI
3-kinase function and are potential therapeutic agents
as well. Wortmannin (1) was recently identified as a
potent and selective inhibitor of PI 3-kinase.6 Structure-
activity relationship (SAR) studies were initiated in an
effort to better understand the structural requirements
necessary for PI 3-kinase inhibition.
Kinetic analysis of the PI 3-kinase inhibition by
wortmannin indicates that inhibition is irreversible.6 We
have postulated that the mechanism of PI 3-kinase
inhibition by wortmannin occurs via covalent attach-
ment of the kinase to the electrophilic C-21 position on
the furan ring (Figure 1).6 We felt that one strategy of
probing the mechanism would be to make changes in
the molecule (either steric or electronic), which may
F igu r e 1.
Resu lts
F u r a n Rin g Mod ifica tion s. The reaction of wort-
mannin with various amines and thiols has been shown
to result in nucleophilic addition to the C-21 position
followed by furan ring opening to give 2-4.7-9 Table 1
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1106$12.00/0 © 1996 American Chemical Society

PI 3-Kinase Inhibition by Wortmannin
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1107
Ta ble 1. PI 3-Kinase Activity of Wortmannin Nucleophilic
Addition Products
Sch em e 2
entry
R
PI 3-kinase IC50 (nM)
2
Et2N
NH2
MeNH
EtNH
n-PrNH
n-BuS
80
.500
.500
.500
.500
52
3a
3b
3c
3d
4
Sch em e 1
shows the compounds which we prepared and their
potency in PI 3-kinase inhibition assays.10 It is inter-
esting to note that 2 and 4 retain PI 3-kinase activity
(albeit dramatically reduced), whereas 3a -e show no
inhibition up to 500 nM. We determined that the
primary amine adducts 3 exist in a Z orientation with
relation to the lactone carbonyl, while the secondary
amine adduct 2 exists in the E configuration (Scheme
1).11 It is clear that the E configuration could more
closely mimic wortmannin in its ability to accept a
nucleophile, while covalent attachment of the kinase to
adducts such as 3 is precluded on steric grounds. In
fact, when 2 was treated with excess ethylamine,
efficient conversion to 3c was observed. However, when
3c was treated with diethylamine, no reaction was
observed. This observation is consistent with our belief
that nucleophilic addition to the inhibitor is required
for inhibition of PI 3-kinase in these analogs.
To further test our hypothesis, we felt that direct
substitution onto the C-21 position of wortmannin would
provide a useful derivative which could more accurately
test our steric hypothesis. When wortmannin was
treated with diazomethane, 21-methylwortmannin (6)
was produced in excellent yield (Scheme 2).9 The
structure of 6 was established by a single-crystal X-ray
structure (Figure 2). It is believed that this compound
is formed through an intermediate pyrrazoline, 5, which
undergoes loss of nitrogen to give the product. This
compound showed no inhibition of PI 3-kinase up to 500
nM. Additionally, when 6 was treated with diethyl-
amine, no reaction occurred. (It should be pointed out
F igu r e 2. Ortep drawing of 21-methylwortmannin (6).
that the ring-opening reaction of wortmannin with
diethylamine is instantaneous.) We feel that this is
convincing evidence for our belief that PI 3-kinase forms
a covalent attachment to wortmannin via the electro-
philic C-21 position.
We were interested in preparing 4,21-cyclopropyl-
wortmannin (7) via reaction of wortmannin with tri-
methylsulfoxoniumylide.9 Although none of the desired
product was observed, we did isolate an interesting ring
expansion product, 8, which could react with excess
ylide to give the cyclopropyl derivative 9 as a mixture
of diastereomers (Scheme 3).9 The initial product 8 was
a moderate inhibitor of PI 3-kinase (271 nM), while the
cyclopropyl derivative 9 did not inhibit up to 500 nM.
On the basis of our hypothesis, we were not surprised
that 9 did not inhibit PI 3-kinase. However, we had
expected 8 to be a more potent inhibitor. We can
suggest two possible explanations for this loss of activ-
ity. First, the pyran ring in 8 is no longer planar like
the furan ring in wortmannin. This slight perturbation
in the three-dimensional space of that region may
prevent efficient binding. An alternative explanation
invokes a thermodynamic comparison of the nucleophilic

1108 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Norman et al.
Sch em e 3
Sch em e 5
Sch em e 4
substituted inhibitors. We cannot draw many conclu-
sions from this data since it is such a small set of
compounds. However, this data suggests that the D
ring of wortmannin is an important recognition element
since even minor changes to the substitution pattern
precludes efficient binding to PI 3-kinase. This is in
contrast to our earlier studies on the C ring of wort-
mannin, which showed that changes in the C-ring
substitution pattern had only a slight effect on inhibitor
potency.6,13 We feel that it is unlikely that a nonselec-
tive alkylating agent would show such dramatic SAR
results so distant from the electrophilic site. The
changes to the D ring shown here would certainly have
a minimal effect on the electrophilicity of the C-21
position of wortmannin and its ability to form a covalent
complex with a nucleophilic species.
One D-ring modification gave a dramatic increase in
PI 3-kinase inhibitor potency. 17â-Hydroxywortmannin
(15), prepared from the reduction of wortmannin with
borane, showed a 10-fold increase in activity relative
to wortmannin and pushed the activity into the sub-
nanomolar range (Scheme 6).13 With an IC50 of 0.50 nM,
it is the most potent inhibitor of PI 3-kinase reported
to date.
addition products of 1 and 8. For our calculations,12 we
used dimethylamine as the nucleophile for the pre-
sumed reaction of the inhibitor with the kinase. Ad-
ditionally, we used a simplified inhibitor model (m od el
1 and m od el 8) which deletes many of the constants
between the two inhibitors, such as the C and D rings.
The ring-opening reaction of m od el 1 with dimethyl-
amine produces m od el 2. This reaction has a ∆Hf )
-27.1 kcal/mol and is irreversible under these condi-
tions. However, the reaction of m od el 8 with dimethyl-
amine produces m od el 10, which has a ∆Hf ) -9.0 kcal/
mol and is reversible (Scheme 4). This difference could
account for the dramatic difference in the activities of
1 vs 8.
D-Rin g Mod ifica tion s. Although it is remote from
the reactive furan ring, the D ring of wortmannin has
proven to be very sensitive to structural modifications
with relation to its PI 3-kinase activity. The enol
acetate 11, which showed about a 10-fold loss of activity
relative to wortmannin (IC50 ) 59 nM), was used as an
intermediate in the preparation of several 16-substi-
tuted wortmannin derivatives. All of these derivatives
(12-14) showed a sharp decrease in their ability to
inhibit PI 3-kinase (Scheme 5). Interestingly, the enol
acetate 11 was more active than any of the 16-
Cytotoxicity a n d An titu m or Activity of Wor t-
m a n n in . Wortmannin has been shown to inhibit PI
3-kinase in PDGF stimulated v-sis NIH 3T3 cells.6
Additionally, it has been reported that wortmannin
displays cytotoxicity in several human tumor cell lines,
with IC50’s in the 0.3-0.9 µg/mL range for GC3 colon

PI 3-Kinase Inhibition by Wortmannin
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1109
Sch em e 6
reported wortmannin analogs, 16-18.6 Included in
Table 3 is the in vitro PI 3-kinase activity of these
analogs.
Ta ble 2. In Vivo Evaluation of Wortmannin
tumor line
dose (mg/kg)a
inhibitionb
Ta ble 3. Antitumor Activity of Wortmannin Analogs in C3H
Mammary Model
6C3HED lymphosarcoma
0.75
1.50
0.75
1.50
0.75
1.50
0.75
1.50
1.25
2.50
0.75
1.50
0.38
0.75
0.75
1.50
0.75
1.50
0.75
1.50
0.38
0.75
0.75
1.50
0.75
1.50
0.75
1.50
0.75
1.50
0.75
1.50
0.75
1.50
-
toxic
-
PI 3-kinase
IC50 (nM)
dose
B-16 melanoma
BXPC-3 pancreatic
C-26 colon
compd
(mg/kg)a
inhibitionb
toxic
+
1
4.2
80
1.25
2.50
5.0
10.0
0.5
1.0
8.0
16
2.0
+
toxic
+
toxic
-
2
15
16
17
toxic
-
toxic
+
0.50
54
C3H mammary
CX-1 colon
toxic
+
toxic
-
toxic
-
toxic
toxic
toxic
-
17
GC3 colon
4.0
16
toxic
-
18
4600
HC1 colon
toxic
-
a,b
See Table 2 footnotes.
IGROV1 ovarian
Lewis lung
toxic
-
Discu ssion a n d Con clu sion s
toxic
-
One of the goals of our PI 3-kinase inhibitor program
was to further our understanding of the relationship
between PI 3-kinase activity and toxicity in our animal
models. The data in Table 3 show a relationship
between in vitro inhibitor potency and antitumor activ-
ity for the compounds which displayed a sufficient
therapeutic index. A relationship also exists between
in vitro potency and toxicity. The most potent PI
3-kinase inhibitors show toxicity at the lowest doses.
Additionally, the lactone ring-opened compound 18,
which was about 1000 times less potent than wortman-
nin in vitro, showed no activity and no toxicity in the
C3H mammary tumor model up to 16 mg/kg. Table 3
clearly shows that all of the potent inhibitors (1, 2, 16,
and 17) had a tolerable dose at one-half the toxic dose.
Compounds 1, 2, and 16 showed antitumor effects at
this dose. These data correlate well with the in vitro
potency. An additional interesting observation is the
fact that 21-methylwortmannin (6), which differs from
wortmannin only by the replacement of a hydrogen with
a methyl group, showed no toxicity in our C3H mam-
mary mouse lethality model up to 100 mg/kg (po, daily
×5), while wortmannin shows acute toxicity and death
at 3 mg/kg (po, daily ×4). Since 21-methylwortmannin
(6) does not inhibit PI 3-kinase (up to 500 nM) but is so
structurally similar to wortmannin, we feel that the
toxicity of wortmannin and related analogs is related
to the mechanism for PI 3-kinase inhibition. Addition-
ally, we have shown that this activity seems to be
associated with the ability of the inhibitor to bind
covalently to the kinase at the electrophilic C-21 posi-
tion.
LX-1 lung
toxic
-
M-5076 ovarian
MX-1 mammary
PACA-2 pancreatic
PANC-1 pancreatic
VRC5 colon
toxic
-
toxic
-
toxic
-
toxic
-
toxic
-
X-5563 plasma cell myeloma
toxic
a
Wortmannin was given po daily ×10 (except for the 6C3HED
b
lymphosarcoma tumor in which wortmannin was given po daily
×8). Antitumor activity (inhibition of tumor growth): -, <60%
inhibition; +, 60-79%; ++, 80-94%; +++, 95-100%; toxic, >25%
lethality.
carcinoma, IGROV1 ovarian carcinoma, and CCRF-
CEM leukemia.14 We have evaluated wortmannin in
16 different in vivo tumor models using daily po dosing
in an effort to find an appropriate model for antitumor
evaluation of wortmannin and related analogs. Our
results are shown in Table 2. The narrow therapeutic
index has made in vivo evaluation difficult for wort-
mannin and related analogs, but the C3H mammary
adenocarcinoma cell line was chosen to complete a more
thorough examination of issues such as dosage and
schedule dependency. Other routes (iv, sc, and ip) and
dosing schedules (q2d×5 and b.i.d. × 10) were investi-
gated in the C3H mammary tumor, but the antitumor
activity was not significantly different than seen for
daily ×10 po dosing.
In summary, we have evaluated wortmannin analogs
and studied the relationship between PI 3-kinase in-
hibitor potency and the ability of these analogs to react
with nucleophiles at the C-21 position. Our data
suggest that nucleophilic addition of the kinase to the
electrophilic C-21 position of wortmannin and related
We have evaluated some additional wortmannin
analogs in the C3H mammary antitumor model, and the
results are shown in Table 3. In addition to 2 and 15,
antitumor activity was evaluated for three previously

1110 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Norman et al.
144.2, 148.9, 158.6, 164.8, 169.5, 172.1, 216.2; IR (KBr) 1221,
analogs is required for inhibitor potency and antitumor
activity. Unfortunately, this mechanism appears to be
linked to the observed toxicity.
1582, 1653, 1680, 1740, 2920 cm-1; MS (FD) m/ e 443 (M+
+
1).
Wor tm a n n in P yr a n 8. Trimethylsulfoxonium iodide (321
mg, 1.46 mmol) was suspended in 4 mL of DMSO and stirred
at 25 °C as 58 mg (1.46 mmol) of sodium hydride (60% in
mineral oil) was added. The formation of the ylide was
complete in about 30 min, as evidenced by the cessation of
hydrogen evolution and the formation of a clear solution. To
this solution was added 500 mg (1.17 mmol) of wortmannin
in 2 mL of DMSO. The dark reaction mixture was stirred for
an additional 30 min at 25 °C and the reaction quenched by
pouring the mixture into 20 mL of water. The mixture was
extracted with ethyl acetate (2 × 30 mL), and the combined
organic extracts were washed once with brine. The resultant
orange solution was dried and concentrated in vacuo to give a
brown oil. A crude 1H NMR indicated that the reaction
mixture contained about 25% wortmannin, 50% pyran 8, and
25% cyclopropylpyran 9. Column chromatography on silica
gel using 50% ethyl acetate-hexane as the eluent provided
pure pyran 8, which was recrystallized using 50% ethyl
acetate-isooctane to give 182 mg (35%) of a white crystalline
solid (mp 130-131 °C): 1H NMR (300 MHz, CDCl3) δ 0.86 (s,
3H), 1.57 (s, 3H), 1.72 (dd, 1H, J ) 14.2, 5.7 Hz), 2.02 (m, 1H),
2.08 (s, 3H), 2.25 (m, 1H), 2.48 (dd, 1H, J ) 13.9, 7.6 Hz), 2.59
(m, 1H), 2.86-3.04 (m, 2H), 3.18 (dd, 1H, J ) 11.1, 6.4 Hz),
3.24 (s, 3H), 3.46 (dd, 1H, J ) 11.1, 2.2 Hz), 4.61 (dd, 1H, J )
6.3, 2.2 Hz), 4.74 (dd, 1H, J ) 16.1, 3.3 Hz), 5.14 (dd, 1H, J )
16.1, 5.5 Hz), 6.02 (m, 1H), 6.98 (dd, 1H, J ) 5.5, 3.3 Hz); IR
(CHCl3) 1236, 1653, 1743, 2936 cm-1; MS (FD) m/ e 442 (M+).
Anal. (C24H26O8) C, H.
Exp er im en ta l Section
Compounds 2 and 11-18 are not new and were prepared
according to the literature procedures outlined in ref 7.
Gen er a l P r oced u r e for th e Rea ction of Wor tm a n n in
w ith P r im a r y a n d Secon d a r y Am in es. Wortmannin (1.00
g, 2.33 mmol) was dissolved in 40 mL of methylene chloride
and stirred at 25 °C as 2.56 mmol of the amine was added
dropwise. The reaction mixture immediately turned dark
orange and was concentrated in vacuo. The resulting orange
amorphous solid was recrystallized from 50% ethyl acetate-
isooctane to give yellow-orange crystalline solids, whose
structures were assigned on the basis of their spectroscopic
and analytical data. This procedure was used for the prepara-
tion of diethylamino adduct 2, methylamino adduct 3b, ethyl-
amino adduct 3c, and n-propylamino adduct 3d . Each of these
compounds gave acceptable spectroscopic and analytical data
(1H NMR, IR, MS, elemental analysis). The spectroscopic data
was nearly identical with that of 3a in all of these cases.
Am in o Ad d u ct 3a . A solution of 25 mg (0.058 mmol) of
wortmannin, 9.0 mg (0.116 mmol) of ammonium acetate, and
16 mg (0.116 mmol) of anhydrous powdered potassium carbon-
ate in 2 mL of 50% THF-water was stirred at 25 °C for 30
min. The reaction mixture was poured into 10 mL of meth-
ylene chloride and the organic phase washed once with water,
dried over sodium sulfate, and concentrated in vacuo. The
resulting solid was recrystallized from 50% ethyl acetate-
isooctane to give 21 mg (81%) of a yellow solid (mp 140-143
°C): 1H NMR (300 MHz, CDCl3) δ 0.82 (s, 3H), 1.55 (s, 3H),
1.88 (dd, 1H, J ) 15.1, 3.3 Hz), 2.03 (s, 3H), 2.20-2.40 (m,
3H), 2.55 (m, 1H), 2.82-2.98 (m, 2H), 3.19 (m, 2H), 3.26 (s,
3H), 4.34 (dd, 1H, J ) 7.2, 1.2 Hz), 5.80 (bs, 1H), 5.99 (dd, 1H,
J ) 7.9, 3.2 Hz), 7.14 (s, 1H), 8.61 (dd, 1H, J ) 15.1, 8.3 Hz),
9.36 (m, 1H); IR (KBr) 1222, 1580, 1627, 1642, 1687, 1743,
3367 cm-1; MS (FAB) 446 (M+ + 1). Anal. (C23H27NO8) C, H,
N.
Wor tm a n n in Cyclop r op ylp yr a n 9. Using the above
procedure, 9 can be formed exclusively by simply using 2 equiv
of trimethylsulfoxoniumylide and sodium hydride. The white
crystalline product 9 (mp 122-125 °C) was isolted in 72% yield
as a 2:1 mixture of diastereomers (which were not separated).
Major isomer: 1H NMR (300 MHz, CDCl3) δ 0.82 (s, 3H), 2.70
(dd, 1H, J ) 9.1, 6.5 Hz), 2.79 (dd, 1H, J ) 9.1, 3.6 Hz), 2.05
(dd, 1H, J ) 6.5, 3.6 Hz), 2.10 (s, 3H), 2.22 (m, 2H), 2.51 (m,
2H), 2.78-2.97 (m, 2H), 3.00 (dd, 1H, J ) 8.6, 6.3 Hz), 3.33 (s,
3H), 3.48-3.75 (m, 3H), 4.53 (d, 1H, J ) 11.0 Hz), 4.59 (dd,
1H, J ) 7.8, 1.8 Hz), 5.97 (m, 1H); IR (CHCl3) 1146, 1653, 1744,
3019 cm-1; MS (FAB) m/ e 457 (M+ + 1). Anal. (C25H28O8),
C, H.
Bu ta n eth iol a d d u ct 4. To a solution of wortmannin (200
mg, 0.47 mmol) in methylene chloride (2 mL) were added
n-butanethiol (0.08 mL, 0.75 mmol) and 1 drop of triethyl-
amine. After the mixture had stirred overnight under nitro-
gen, another drop of triethylamine was added and the reaction
mixture stirred for an additional 1 h. The volatiles were
removed in vacuo, and the residue was chromatographed by
radial chromatography (silica gel, 1:1 EtOAc/hexanes) to give
186 mg (77%) of product as a bright yellow-orange solid (mp
88-90 °C): 1H NMR (300 MHz, CDCl3) δ 0.82 (s, 3H), 1.55 (s,
3H), 1.88 (dd, 1H, J ) 15.1, 3.3 Hz), 2.03 (s, 3H), 2.20-2.40
(m, 3H), 2.55 (m, 1H), 2.82-2.98 (m, 2H), 3.19 (m, 2H), 3.26
(s, 3H), 4.34 (dd, 1H, J ) 7.2, 1.2 Hz), 5.80 (bs, 1H), 5.99 (dd,
1H, J ) 7.9, 3.2 Hz), 7.14 (s, 1H), 8.61 (dd, 1H, J ) 15.1, 8.3
Hz), 9.36 (m, 1H); IR (CHCl3) 1197, 1317, 1421, 1625, 1743,
2976 cm-1; MS (FD) m/ e 518 (M+). Anal. (C27H34O8S) C, H.
In Vitr o Eva lu a tion of P I 3-Kin a se In h ibitor s. The PI
3-kinase inhibition assay has been described previously.6
In Vivo In h ibition of Tu m or Gr ow th . The antitumor
activity of wortmannin and its analogs was evaluated in a
series of solid murine16 and human xenograft17 tumor lines.
Tumors were carried by serial passage with subcutaneous
implants of tumor fragments via trochar in the axillary region.
Dosing was initiated 1 day after implant for the murine tumors
(6C3HED lymphosarcoma, B-16 melanoma, C-26 colon, C3H
mammary, Lewis lung, and X-5563 plasma cell myeloma)
except for the M-5076 ovarian for which dosing was initiated
5 days after implant. For the human xenograft tumors
(BXPC-3 pancreatic, CX-1 colon, GC3 colon, LX-1 lung, PACA-2
pancreatic, and PANC-1 pancreatic), dosing was initiated 7
days after the implant except for the HC1 colon and IGROV1
ovarian for which dosing was initiated 14 days after tumor
implant. Wortmannin and its analogs were dosed as a
suspension in 2.5% GAF emulphor EL-620 (Warren-Graham,
Cockeysville, MD) in 0.9% NaCl except for 2 which was dosed
in water. Tumor weights were calculated from measurements
of the width and length of tumors using electronic calipers
interfaced to a microcomputer using the following formula:18
21-Meth ylw or tm a n n in (6). Diazomethane (10 mmol) was
prepared according to the procedure of De Boer and Backer.15
The solution of diazomethane (about 30 mL) was transferred
to a fire-polished, 250 mL Ehrlenmeyer flask. To this solution
was added 500 mg (1.17 mmol) of wortmannin in 50 mL of
THF. The top of the flask was covered with parafilm, and the
solution was stirred at 25 °C and monitored by TLC. The
reaction required 3 days to go to completion, after which the
mixture was concentrated in vacuo and recrystallized from
50% ethyl acetate-isooctane to give 420 mg (81%) of a yellow
crystalline solid (mp 178-179 °C): 1H NMR (300 MHz, CDCl3)
δ 0.97 (s, 3H), 1.57 (m, 1H), 1.70 (s, 3H), 2.02 (m, 1H), 2.13 (s,
3H), 2.23 (m, 1H), 2.56-2.62 (m, 2H), 2.73 (s, 3H), 2.86 (ddd,
1H, J ) 12.6, 5.8, 2.8 Hz), 2.99 (dd, 1H, J ) 11.1 and 7.5 Hz),
3.16 (m, 1H), 3.21 (s, 3H), 3.43 (dd, 1H, J ) 11.1, 1.5 Hz), 4.71
(dd, 1H, J ) 7.4, 1.8 Hz), 6.13 (dt, 1H, J ) 7.7, 2.6 Hz); 13C
NMR (125 MHz, CDCl3) δ 13.9, 14.5, 21.0, 22.9, 26.5, 35.7,
36.2, 40.7, 44.1, 49.2, 59.4, 70.1, 72.9, 88.5, 109.3, 140.3, 142.8,
tumor weight in mg )
[tumor length in mm × (tumor width in mm)2] × 0.5
Tumor measurements were taken on the day after the last
dose for murine tumors and 5 days after the last dose for the
human xenograft tumors. Percentage inhibition of tumor
growth was calculated as:

PI 3-Kinase Inhibition by Wortmannin
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1111
the C and H). Less than 7 Hz is indicative of a Z orientation.
The coupling was measured using a gated 13C NMR spectrum
or a spectrum in which the proton was decoupled using low
power. The following coupling constants were observed. 2: J CH
) 4.5 Hz. 3c: J CH ) 9.8 Hz. These data confirm our assignments.
Additionally, an intramolecular hydrogen bond exists between
the lactone carbonyl (A ring) and the N-H hydrogen. This effect
results in a downfield shift in the N-H proton signal in the 1H
NMR and was observed for all of the primary amine adducts
(3a -d ). This effect has been previously addressed (see ref 9).
(12) Molecular modeling calculations were performed using the
program Spartan (version 3.1.1; Wavefunction, Inc., 18401 Von
Karmen, Suite 370, Irvine, CA 92715) running on an SGI IRIS
Indigo workstation. In vacuo heats of formation were obtained
with the semiemperical AM1 Hamiltonian; geometries were
optimized until the SCF energy converged such that the differ-
ence between two consecutive cycles was <1.0 × 10-5. It should
be pointed out that wortmannin reacts with dimethylamine to
give the product m od el 2, analogous to the reaction with
diethylamine. We have chosen to use dimethylamine to simplify
our thermodynamic calculations.
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No. 316.
percent inhibition of tumor growth )
100 × (1 - average tumor weight of treated group)
(average tumor weight of control group)
Groups consisted of 10 mice that were randomized from those
implanted. A group was considered toxic if lethality was
>25%.
Ack n ow led gm en t. We thank Laverne Boek, Patrick
Baker, Stephen Larson, and their co-workers for the
microbial production and chemical isolation of the
wortmannin used for this work. We thank Bill Matter
for the purification of the PI 3-kinase used in our in vitro
assay. We thank Donald Dudley, Deanna Marlow,
Tracey Self, Phyllis Seymour, and Karla Theobald for
the in vivo antitumor testing. We would like to thank
the Physical Chemistry Department at Eli Lilly and Co.
for help in the characterization of compounds. We
acknowledge Professor Garth Powis (University of
Arizona) and his research group for their contributions
to our collaborative PI 3-kinase program. Finally, we
would like to thank J ennifer Olkowski for her timely
solution of the X-ray crystal structure of 21-methyl-
wortmannin (6).
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