Structure - Activity relationship of Aza-steroids as PI-PLC inhibitors

Article abstract of DOI:10.1016/S0968-0896(00)00302-3

A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3β-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC₅₀ values of 7.4 and 7.5 μM, respectively. The 20α epimer, 8a2 (IC₅₀ = 0.64 μM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20β epimer, 8a1 (IC₅₀ = 32.2 μM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC₅₀ = 19.7 μM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC₅₀ = 7.4 μM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC₅₀ = 17.4 μM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors. However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI₅₀ value (MG-MID) of 5.75 μM for 54 tumors.

Full text of DOI:10.1016/S0968-0896(00)00302-3

Bioorganic & Medicinal Chemistry 9 (2001) 1073±1083
Structure±Activity Relationship of Aza-Steroids
as PI-PLC Inhibitors
a
a
a
b
b
Wenge Xie, Hairuo Peng, Deog-Il Kim, Mark Kunkel, Garth Powis
a,
and Leon H. Zalkow *
aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
bArizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-5024, USA
Received 16 August 2000; accepted 7 November 2000
AbstractÐA number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The
epimeric mixtures 22,25-diazacholesterol (8a) and 3b-hydroxy-22,25-diazacholestane (8b) were among the most active of these
inhibitors, with IC50 values of 7.4 and 7.5 mM, respectively. The 20a epimer, 8a2 (IC50=0.64 mM), whose stereochemistry at C-20
coincides with that of cholesterol, was found 50 times more potent than the 20b epimer, 8a1 (IC50=32.2 mM). In diaza-estrone
derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC =19.7 mM),
5
0
while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl
group (8a, IC50=7.4 mM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the
non-aromatic A-ring (26, IC =17.4 mM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent
5
0
3
PI-PLC inhibitors. However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of
activity. Epimeric mixture 8a showed selective growth inhibition e€ects in the NCI in vitro tumor cell screen with a mean GI50 value
(
MG-MID) of 5.75 mM for 54 tumors. # 2001 Published by Elsevier Science Ltd.
Introduction
kinase C. Together, the increase in intracellular free
2
+
Ca and the activation of protein kinase C result in a
series of profound cellular changes, such as DNA
synthesis, cell proliferation, and neuronal activity.
Phosphatidylinositol and its phosphorylated derivatives
represent less than 6±8% of the components of the
1
a
membrane of eukaryotic cells. Until recently, they had
been considered as relatively inert substances. However,
it has been recognized that inositol phospholipids are
crucially involved in the receptor-mediated activation
of signal transduction pathways initiated by action of
2
Abnormal functions of PIPLC have been linked to can-
cer and Alzheimer's disease. Increased PI-PLC activity
has been reported in a number of human tumors, espe-
3À6
cially in the more aggressive malignant tumors. The
growth inhibitory e€ect of tamoxifen on GH C cancer
1
hormones and growth factors. Phosphatidylinositol
4
1
7
phospholipase C (PI-PLC) is a key enzyme located at
the inner side of cell membrane and catalyzes hydrolysis
of a minor membrane phospholipid, phosphatidylinositol
cells has been linked to inhibition of PI-PLC. There-
fore, selective small molecule inhibitiors of over acti-
vated PI-PLC signaling pathways may provide potential
therapies for cancer.
(
4,5)-biphosphate (PI(4,5)P ), as a result of activation of
2
receptors at the cell surface. The resulting products are the
second messengers, inositol (1,4,5)-triphosphate
I(1,4,5)P ), which releases Ca from intracellular stores
We have recently described 6-aza-steroids as potent PI-
3
2
+
(
PLC inhibitors. The most active of these compounds,
3
2
+
to increase intracellular free Ca
diacylglycerol, which activates the Ca
lipid-dependent protein serine/threonine kinase, protein
concentration; and
3b-hydroxy-6-aza-cholestane (1, Fig. 1) showed an IC of
5
0
2
+
and phospho-
less than 1.8 mM against PI-PLC; it also showed sig-
ni®cant growth inhibition e€ects on HT-29 colon cancer
cells (IC =1.3 mM) and MCF-7 breast cancer cells
5
0
(
IC =1.3 mM). Structure±activity relationships revealed
5
0
that the free amino group at the 6-position played a
crucial role in PIPLC inhibition, and the presence of the
*Corresponding author. Tel.: +1-404-894-4002; fax: +1-404-894-7452;
e-mail: leon.zalkow@chemistry.gatech.edu
0968-0896/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0968-0896(00)00302-3

1074
W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
Figure 1.
Scheme 1. Reagents and conditions: (a) (i) RNH
re¯ux; (ii) 1.2 equiv NaBH , CH OH/THF, pH 6; K
O; or NH OAc, NaB(CN)H , CH OH/THF; (b) H , 5% Pd/C, AcOH.
2
, p-TsOH, toluene,
4
3
2
CO in CH OH/
3
3
H
2
4
3
3
2
hydrophobic cholesteryl side chain and the 3b-hydroxy
group are also critical for inhibitory activity.
was ®rst treated with primary amines and a catalytic
amount of p-TsOH in toluene under re¯ux overnight to
form the intermediate imines. Treatment of the resulting
Comparing the structure of compound 1 with that of
the well studied diaza steroidal PI-PLCg inhibitor,
U73122 (Fig. 1, IC =1.0±2.1 mM), lead us to reason
that other steroidal derivatives with amino groups on the
side chain and/or in the ring system might have improved
inhibitory potencies. Herein, we report a series of aza-
steroid derivatives of di€erent ring features and amino
group arrangements synthesized from readily available
starting materials, as PI-PLC inhibitors and potential
antitumor and anticancer agents.
imines with 1.2 equiv NaBH in methanol and THF
4
8
a€orded the corresponding 3b-hydroxy-20-aza-preg-
nene derivatives 2a±10a in 80±85% yield. The ratio of
20b/20a isomers was about 1.5±2.5:1, as determined by
5
0
1
H NMR spectroscopy. Pregnenolone acetate was trea-
ted with NH OAc in methanol/THF, and NaB(CN)H
4
3
at pH=6 to give compound 11a in 85% yield (20b : 20a
ꢀ 3:1). Hydrogenation of aza-steroids 2a±9a in glacial
acetic acid with 10% Pd/C at room temperature gave
the saturated 20-aza steroids 2b±9b in 80±90% yield.
As shown in Scheme 2, treatment of 8a with excess
NaBH4 (6.0 equiv) in methanol/THF, also a€orded
20% of the unnatural 17a epimers. The ratio of 17a,
20a-isomer/17a, 20b-isomer was about 1:1 as deter-
Chemistry
The syntheses9 of 3b-hydroxy-20-aza-pregnene deriva-
tives 2a±10a and 3b-hydroxy-20-aza-pregnane derivatives
1
2
b±10b are illustrated in Scheme 1. Pregnenolone acetate
mined by H NMR spectroscopy. Separation of the
Chart 1.

W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
1075
Scheme 3. Reagents and conditions: (a) (COCl)
2
/CH Cl
2
2
, pyridine;
(
(
b) NaN
e) 10% Pd/C/H
3
/acetone and H
, AcOH, then K
OH/THF, NaB(CN)H
3
2
O; (c) benzene, re¯ux; (d) 12 N HCl/acetone;
CO in methanol and H O; (f) N,N-
, pH 6.
2
2
3
2
Scheme 2. Reagents and conditions: (a) (i) N,N-dimethylethylenedia-
mine, p-TsOH, toluene, re¯ux; (ii) 6.0 equiv NaBH , CH OH/THF,
pH 6; K CO in CH OH/H O.
dimethylethylenediamine, CH
3
4
3
2
3
3
2
2
0,25-diaza-cholesterol diastereomers in mixture 8a was
performed by ¯ash column chromatography on silica gel
with ethyl acetate, petroleum ether and triethyl amine as
eluent to a€ord the four pure diastereomers. The sequence
by which these compounds being eluted were 8a1, 8a2,
and then the two 17a epimers, 8a3 and 8a4.
To establish the stereochemistry of the two epimers, 8a1
and 8a2, literature precedures were followed for com-
parison purpose. Pregnenolone acetate was treated with
10
N,N-dimethylethylenediamine and NaB(CN)H in metha-
3
nol/THF at pH 6, followed by treatment with K CO in
2
3
methanol and H O to a€ord 20,25-diaza-cholesterols
2
1
0
8
a1 and 8a2. It has been well documented that the
2
were reduced with NaB(CN)H . The H NMR spectrum
0b-epimer was the predominant product when 20-imines
1
3
showed a 4:1 ratio for the two isomers, indicating that
8
0% of the mixture should be the 20b-amine (8a1) and
0% of the product should be the 20a-amine (8a2). On
2
TLC, the major product, 20b-amine, was more mobile
than the minor product, 20a-amine.
Scheme 4. Reagents and conditions: (a) NaBH
b) MsCl, pyridine, CH Cl , 90%; (c) NaN
d) LiAlH , THF 63%; (e) (i) TsCl, pyridine, CH
4
, CH
, acetone, H
Cl , 18, 78%; (ii) phenyl
3
OH, THF, 96%;
(
(
2
2
3
2
O, 85%;
4
2
2
The assignment of the structures to the 17a, 20b (8a3) and
2 2
isocyanate, THF, 19, 95%; (3) benzyl chloroformate, THF, CH Cl ,
17a, 20a (8a4) isomers was not unequivocal. It was well
established that the side chain of cholesteryl derivatives
Et N, 20, 81%.
3
with 20b conformation oriented to the `left-handed' con-
formation. The less extended structure of 20b-amines
acetone and treated with NaN /water to give diazide 16 in
3
quantitative yield. Reduction of 16 with LiAlH in THF
4
1
1
8
8
8
a1 might account for the observed higher mobility of
a1 compared to 8a2. Therefore, the more mobile isomer
a3 in the two 17a epimers was assigned the 17a, 20b
a€orded 3a,20-diamine 17 (20b:20a ꢀ 3:1). Reaction of
diamine 17 with p-toluenesulfonyl chloride, phenyl iso-
cyanate, and benzyl chlorformate gave tosylate 18, urea
19, and carbamate 20, respectively.
stereochemistry.
The synthesis of 3b-hydroxy-6,20,25-triaza-cholestane 13
was carried out as shown in Scheme 3. Pregnenolone ace-
tate was converted to the intermediate 3b-hydroxy-6-aza-
pregnan-20-one (12) in six steps. Reaction of ketone 12
with N,N-dimethylethylenediamine and NaB(CN)H in
3
The syntheses of diaza-estrone derivatives 21 and 23 fol-
lowed the same procedure, and are illustrated in Scheme 5.
Estrone was allowed to react with (CH O) SO in ethanol
3
2
2
1
2
and NaOH in water to give 3-methoxy-estra-1,3,5(10)-
1
5
triene-20-one (22) in 57% yield. Treatment of estrone
or compound 22 with N,N-dimethylethylenediamine
and NaB(CN)H in CH OH/THF at pH 6 gave diaza-
CH OH/THF at pH 6 yielded 3b-hydroxy-6, 20,25-
3
triaza-cholestane 13 in 5% yield from pregnenolone
acetate. The ratio of the two isomers was 20b:20a=3:1.
3
3
estrone derivatives 21 or 23 in 85 and 80% yield,
respectively. In these reactions, only 17b-epimers were
found. Reaction of pregnenolone with TsCl in pyridine
gave a quantitative amount of ester 24. Re¯ux of com-
pound 24 in methanol a€orded pregnenolone methyl
The syntheses of 3,20-diaza-pregnene derivatives 17±20
are illustrated in Scheme 4. Reduction of pregnenolone
acetate in methanol/THF with NaBH yielded the cor-
4
1
3
16
responding diol 14 in 90% yield. Treatment of diol 14
with MsCl in pyridine and methylene chloride with cat.
DMAP a€orded the dimesylate 15, which was dissolved in
ether 25 in 80% yield. Reductive amination of 25, using
the same procedure described previously, gave 22,25-
diaza-cholesterol methyl ether 26 in 86% yield (Scheme 5).

1076
W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
compounds 4a/4b, 5a/5b, 6a/6b also showed reduced
inhibitory activities (IC =38.6±186.2 mM, Scheme 1).
50
It is interesting that when the terminal amino group was
masked by methyl groups, with two carbon atoms
between the two amino group, 8a and 8b, the most potent
PIPLC inhibition (IC =7.5, 7.4 mM, respectively) was
5
0
observed. However, when the number of carbon atoms
was increased to three as in 7a and 7b, the activity
dropped signi®cantly (Scheme 1). Without the terminal
amino group, (9a), 3b-hydroxy-20-aza-(N-butane)-
pregnane (9b), and 20-aza-cholesterol 10a were inactive.
3
b-Hydroxy-20-aza-pregn-5-ene (11a), also showed no
activity. It was observed that compounds (2a/2b, 5a/5b
and 8a/8b), with even numbers of carbon atoms
between the two amino groups in the side chain, gave
signi®cantly better inhibitory activities than those com-
pounds (4a/4b, 6a/6b, and 7a/7b) with odd numbers of
carbon atoms (Scheme 1). Better complementary
hydrogen bonding arrangements of compounds 2a/2b,
5a/5b and 8a/8b and their PIPLC bind site might
account for these di€erences in activities. Saturation of
the double bond at C-5 did not a€ect the inhibition
potencies of compounds with good PIPLC inhibitory
activity, such as 2a, 5a and 8a. However, removal of the
C-5 double bond greatly reduced inhibition activity of
those compounds (3a and 6a) with moderate PIPLC
inhibitory activity (Scheme 1). This may indicate
ampli®cation of unfavorable orientation by saturation
of the double bond.
It has been documented previously that the side-chain
stereochemistry at C-20 had dramatic e€ects on bio-
logical activity, with the natural 20a isomers exhibiting
1
7,18
Scheme 5. Reagents and conditions: (a) N,N-dimethylethylenedia-
more potent activities.
To di€erentiate PI-PLC
mine, CH
ethanol, NaOH in water, 57%; (c) TsCl, pyridine, CH
3
d) methanol, re¯ux, 95%; (e) (COCl) , DMSO, Et N, 85%.
3
OH/THF, NaB(CN)H
3
, pH 6, 80±85%; (b) (CH
3
O)
2 2
SO ,
inhibition activities, the two epimers, 20b,25-diaza-cho-
lesterol (8a1) and 20a,25-diaza-cholesterol (8a2) were
separated by ¯ash column chromatography on silica gel,
with ethyl acetate, petroleum ether and triethyl amine as
eluent; amine 8a1 was the more mobile component. As
shown in Scheme 2, the PIPLC inhibitory activities of
these two epimers were signi®cantly di€erent, with
compound 8a2 (IC =0.64 mM) being 50 times more
2
Cl
2
, 90%;
(
2
The synthesis of diaza-podocarpatriene derivative 29 is
illustrated in Scheme 5). Swern oxidation of 12-meth-
oxy-podocarpatrien-16-ol (27) gave the corresponding
aldehyde 28 in 85% yield. Reductive amination of 27
with N,N-dimethylethylenediamine using NaB(CN)H3
gave diaza-podocarpatriene derivative 29 in 80% yield.
5
0
active than 8a1 (IC =32.2 mM). This result is con-
50
sistent with the previous observation on the hypochol-
esterolemic activity of this series that with a natural 20a
stereocenter as that of cholesterol, epimer 8a2 has better
1
7
biological activity. Furthermore, when the stereo-
chemistry of C-17 was changed from the natural b as in
cholesterol to a as in 17a-20b,25-diaza-cholesterol (8a3)
and 17a-20a,25-diaza-cholesterol (8a4), the PIPLC
inhibitory activities were dramatically reduced. It seems
that departure from the natural stereochemistry of chol-
esterol leads to marked reduction in biological activity.
Results and Discussion
Structure±activity relationships
In the series of diaza-steroids with two free amino groups
a±6a and 2b±6b (Scheme 1), 3b-hydroxy-20-aza-(N-hex-
2
ylamine)-pregnene (2a, IC =26.0±30.0 mM) and 3b-
50
hydroxy-20-aza-(N-hexylamine)-pregnane (2b, IC =21.3±
50
2
8.7 mM) with six carbons between the two amino
We previously reported that 3b-hydroxy-6-aza-choles-
tane (1) was a potent PI-PLC inhibitor (IC =1.8 mM)
primarily due to the presence of the hydroxyl and the
groups gave the best PLC inhibition. Compounds with
longer side chains, as in 3b-hydroxy-20-aza-(N-octyla-
mine)-pregnene (3a, IC =78.8 mM) and 3b-hydroxy-20-
5
0
3
amino groups. 3b-Hydroxy-6,20,25-triaza-cholestane
5
0
aza-(N-octylamine)-pregnane (3b, IC =179.3 mM) with
(13) was synthesized in an attempt to combine the 6-aza
feature of compound 1 and 20,25-diaza feature of com-
pound 8a into one molecule. Unfortunately, the resulting
combination lead to acute loss of activity (Scheme 3).
50
eight carbon atoms between the two amino groups exhib-
ited much less PI-PLC inhibition. With ®ve, four and three
carbons between the two amino groups in the side chains,

W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
Table 1. NCI 60-human cell line screen of compound 8a
1077
This observation suggests that the hydrophobic side
chain in compound 1 contributes signi®cantly to its
binding anity to PIPLC. Compounds 1 and 8a2 might
not bind to the same site of the enzyme or they might
orient completely di€erently to ®nd the most favorable
hydrophobic and hydrophilic complements if they do
bind to the same site in the enzyme. Similarly, com-
pound 17, with simultaneous presence of amino groups
at C-3 and C-21, showed no PIPLC inhibitory activity.
Among the derivatives of diamine 17, including p-tosy-
late 18, urea 19, and carbamate 20, only carbamate 20
Cell lines
8a
GI50a
TGIb
LC50c
Leukemia
K-562
SR
4.17
0.003
11.5
7.94
>25.0
>25.0
Colon cancer
HT-29
SW-620
4.90
4.47
>25.0
12.5
>25.0
>25.0
Melanoma
LOX IMVI
MALME-3M
exhibited signi®cant PIPLC inhibition with an IC50
1.51
4.68
5.01
8.30
12.9
15.1
value of 4.1 mM (Scheme 4). Again, this compound
might bind to a completely di€erent site from those of
compounds 1 and 8a2.
Ovarian cancer
OVCAR-8
4.26
7.94
14.8
Prostate cancer
PC-3
DU-145
It was previously observed that removal of 3-methoxy
group in U73122 resulted in a pronounced loss of inhi-
bitory activity. To test the e€ects of a 3-methoxy group
in an ring A aromatic aromatic in the diaza series, diaza-
estrane derivative 21, diaza-3-methoxy-estrane deriva-
tive 23 and diaza-3-methoxy-aza-pregnane derivative 26
were synthesized. As shown in Scheme 5, with the
presence of aromatic A-ring, compound 23 containing
the 3-methoxy group and the diaza-side chain showed
an IC50 value of 19.7 mM, while compound 21, with a
4.68
3.09
8.51
6.92
15.1
15.8
9
Breast cancer
HS 578T
5.12
4.27
4.47
5.75
11.7
9.12
9.33
>25.0
19.4
>25.0
22.4
MDA-MB-435
MDA-N
d
MG-MID
15.1
aGI50 represents the compound concentration (mM) required to
achieve 50% inhibition of tumor cell growth.
b
TGI represents the compound concentration (mM) required to
achieve total growth inhibition of tumor cell.
3
-hydroxyl group, was not active. However, in the aza-
c
LC50 represents the compound concentration (mM) that is lethal to
the survival of 50% tumor cell.
pregnane PIPLC inhibitor series, with the 3-methoxy
group, 20, 25-diaza-cholesterol-methyl ester (26) was
less potent (IC =17.9 mM) than its 3-hydroxy coun-
d
MG-MID represents the calculated mean GI50, TGI and LC50 for all
panels.
5
0
terpart, 8a (IC =7.9 mM).
5
0
human non-small cell lung cancer and colon cancer.3,4
However, we cannot rule out cytotoxicity contributions
by inhibiting other crucial enzymes, such as 24,25-
reductase operating in the terminal stage of cholesterol
(+)-Podocarpic acid (29) is a representative member of
the podocarpene diterpenoids isolated from Podocarpus
1
9
capressina var. imbricata. Random screening of our in
house chemical library in the PIPLC in vitro assay
revealed 2-methoxy-podocarpatriene-16-ol (27) as a
moderate PIPLC inhibitor (IC =21.5 mM), whereas
2
0
synthesis.
5
0
(
+)-podocarpic acid (29, Chart 1) and 12-methoxy-
Conclusion
podocarpatrien-16-al (28) were not active. In hope of
discovery of potent non-steroidal PI-PLC inhibitors, we
synthesized diaza-podocarpatriene derivative 30.
Unfortunately, addition of diaza side chain as in 30 led
to loss of inhibition. Thus, the methylene hydroxyl
group in 27 must contribute greatly to its observed
activity.
A series of aza-steroids have been synthesized as
potent PI-PLC inhibitors. The most active compound
20a,25-diaza cholesterol 8a2 (IC =0.64 mM), whose
stereochemistry at C-20 coincides with that of choles-
terol, showed 50 times more potent PI-PLC inhibition
activity than that of the 20b epimer, 8a1 (IC =32.2
5
0
5
0
mM). The unnatural 17a-diaza isomers 8a3 and 8a4
were essentially inactive. These observations suggest
that retention of the natural stereochemistry of cholesterol
is important for the PIPLC inhibitory activity in this
series of aza-steroids. Simultaneous presence of the
active 6-aza and 22, 25-diaza characteristics of two lead
compounds (1 and 8a) in one molecule, 13, led to loss of
activity.
NCI 60-human tumor cell lines screen
The in vitro cytotoxicity of epimeric mixture 8a
(20a:20b=1:1) was evaluated at the National Cancer
Institute against a panel of 60 human tumor cell lines
representing nine di€erent cancer types. The GI , TGI
5
0
and GI50 values for some sensitive subpanels are shown
in Table 1. Mixture 8a showed a mean GI50 value (MG-
MID) of 5.75 mM for 54 tumors. It was more selective
against SR leukemia cancer cell line, HT-29 and SW-
It was also observed that the 3-methoxy group played a
key role in PI-PLC inhibition by diaza-estrone deriva-
tives. Replacing the 3-methoxy group on the aromatic
A-ring of compound 23 (IC =19.7 mM, Chart 1) with
6
20 colon cancer cell lines, and LOX IMVI melanoma
cancer cell lines. It also showed some selective inhibition
on MDA-MB-435 breast cancer cell, DU-145 and PC-3
prostate cancer. This is consistent with the observation
that PI-PLC activity is found to be increased in a num-
ber of human tumors, like human breast cancers,
5
0
a hydroxyl group, as in compound 21, led to loss of
activity. This is analogues to the previously reported
results for U73122 and its inactive 3-hydroxyl counter-
2
1
part.
However, in diaza-pregnane derivatives,

1078
W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
compounds with a 3-hydroxyl group (8a, IC =7.4 mM,
mL glacial acetic acid with 10% Pd on carbon as catalyst
at atmospheric pressure for 4 h. The solvent was then
removed under reduced pressure, and the residue obtained
was resuspended in water and the pH was adjusted to 8.
The solution was then extracted with chloroform and
the organic layer was washed with water. Removal of
solvent in vacuo a€orded the saturated 3b-hydroxy-20-
aza-pregnane analogues 2b±8b as white gums.
5
0
Chart 1) exhibited more potent PI-PLC inhibitory
activity than those with a 3-methoxy group in their non-
aromatic A-ring (26, IC =17.4 mM, Chart 1).
5
0
The side-chain-diaza inhibitors represented by 8a2
might bind to PIPLC in a di€erent orientation or even
to a di€erent site from 6-aza cholesterol (1). As shown
in Chart 1, Pregn-5-en-3, 20 diamino-bis(benzyloxy-
carbamate) 20 and 12-methoxy-podocarpatriene-16-ol
1
3ꢀ-Hydroxy-20-aza-(N-hexylamine)-pregn-5-ene (2a). H
NMR (CDCl , 300 MHz) d 0.66, 0.69 (1.2:1 (2s, 2CH ,
(27) represent two more unique leads for PIPLC inhibi-
tion. Di€erent from the phosphatidylinositol ether lipid
3
3
18-H)), 0.95, 1.07 (1:1.2 (2d, J=6.6 Hz, 2CH , 21-H)), 0.98
3
(s, CH , 19-H), 2.43 (m, 2H), 2.40, 2.51 (2m, each 1H),
3
2
2
analogues which likely inhibited PIPLC by mimicing
its natural substrate (PIP2), the mechanism by which
this series of aza-steroids inactive PIPLC is currently
unknown. Epimeric mixture 8a (20a:20b=1:1) showed
potent growth inhibition e€ects in the NCI in vitro
tumor cell screen with a mean GI50 value (MG-MID) of
2.66 (t, J=7.2 Hz, 2H), 3.49 (m, 1H), 5.32 (m, 1H); CIMS
+
+
m/z (relative intensity) 417 (50, M +1), 399 (25, M +
1-H O); HRMS m/z calcd for C H N O 417.3845
2
+
27 49
2
(M +1), found 417.3877.
1
5.75 mM for 54 tumors, indicating the potential of using
PIPLC as a molecular target for cancer drug discovery.
3ꢀ-Hydroxy-20-aza-(N-hexylamine)-pregnane 2b. H
NMR (CDCl , 300 MHz) d 0.81, 0.84 (1:1.2 (2s, 2CH ,
3
3
18-H)), 0.89 (s, CH , 19-H), 0.96, 1.17 (1.2:1 (2d, J=6.6
Hz, 2CH , 21-H)), 2.38 (m, 1H), 2.51 (m, 1H), 2.62 (t,
3
3
J=7.2 Hz, 2H), 3.58 (m, 1H); CIMS m/z (relative inten-
+
Experimental
+
sity) 419 (50, M +1), 401 (25, M +1-H O); HRMS m/z
2
+
Starting materials were purchased from Aldrich unless
otherwise indicated. Thin layer chromatography analy-
sis (TLC) was performed on aluminum sheets precoated
with 0.2 mm of silica gel containing 60F254 indicator.
Flash chromatography was run using 230±400 mesh
silica gel. Reverse phase high performance liquid chro-
calcd for C H N O 419.4003 (M +1), found 419.3973.
2
2
7
51
1
3ꢀ-Hydroxy-20-aza-(N-octylamine)-pregn-5-ene (3a). H
NMR (CDCl , 300 MHz) d 0.66, 0.69 (1:1 (2s, 2CH ,
3
3
18-H)), 0.94, 1.05 (1:1. (2d, J=6.0 Hz, 2CH , 21-H)),
0.98 (s, CH , 19-H), 2.64, 2.78 (2m, each 1H), 3.49 (m,
3
3
1
matography (HPLC) was run on a Phenomenex
1H), 5.32 (m, 1H); CIMS m/z (relative intensity) 445
+
+
LUNA 5m C18 semi-preparative column. TLC routinely
checked the homogeneity of all compounds on silica gel
plates, and by HPLC. Fourier transformed infrared
spectra were obtained on a Nicolet 520 FTIR spectro-
(50, M +1), 427 (25, M +1-H O); HRMS m/z calcd
2
+
for C H N O 445.4158 (M +1), found 445.4175.
2
9
53
2
3ꢀ-Hydroxy-20-aza-(N-octylamine)-pregnane (3b). 1H
NMR (CDCl , 300 MHz) d 0.81, 0.84 (1:1.2 (2s, 2CH ,
1
13
meter. H (300 or 400 MHz), C (75 or 100 MHz)
NMR and DEPT spectra were recorded on either a
Varian Gemini-300 or on a Varian XL-400 spectro-
meter. High-resolution mass spectrum (EI or FAB) were
recorded on a VG Analytical 70-SE mass spectrometer
equipped with a 11-250J data system. Melting points are
uncorrected. Elemental analyses were performed by
Atlantic Microlab, Norcross, GA.
3
3
18-H)), 0.89 (s, CH , 19-H), 0.96, 1.17 (1.2:1 (2d, J=6.6
Hz, 2CH , 21-H)), 2.38 (m, 1H), 2.51 (m, 1H), 2.62 (t,
3
3
J=7.2 Hz, 2H), 3.58 (m, 1H); CIMS m/z (relative
+
+
intensity) 447 (50, M +1), 429 (25, M +1-H O);
2
+
HRMS m/z calcd for C H N O 447.4316 (M +1),
2
9
55
2
found 447.4318.
3
ꢀ-Hydroxy-20-aza-(N-pentylamine)-pregn-5-ene (4a).
1
H NMR (CDCl , 300 MHz) d 0.66, 0.69 (1:1.3 (2s,
3
Syntheses of 3ꢀ-hydroxy-20-aza-pregnene analogues (2a±
8
2CH , 18-H)), 0.92, 1.03 (1.2:1 (2d, J=6.0 Hz, 2CH ,
21-H)), 0.98 (s, CH , 19-H), 2.24 (m, 2H), 2.40, 2.51 (2m,
3
3
a) and 3ꢀ-hydroxy-20-aza-pregnane analogues (2b±8b).
3
General procedure: To a solution of 1 g of pregneno-
lone acetate in 150 mL toluene was added 4 equiv amine
and cat. p-TsOH. The reaction mixture was re¯uxed
overnight with continuous extraction using a Dean-
Stack water separation trap. When the reaction is ®n-
ished, the solvent was removed under reduced pressure
and the resulted residue was dissolved in THF, then 1.2
each 1H), 2.62 (t, J=7.2 Hz, 2H), 3.51 (m, 1H), 5.31 (m,
+
1H); CIMS m/z (relative intensity) 403 (50, M +1),
+
26 47
385 (25, M +1-H O); HRMS m/z calcd for C H
2
+
N O 403.3690 (M +1), found 403.3694.
2
3ꢀ-Hydroxy-20-aza-(N-pentylamine)-pregnane (4b). 1
NMR (CDCl , 300 MHz) d 0.81, 0.84 (1:1.2 (2s, 2CH ,
H
3
3
equiv NaBH in 5 mL methanol was added dropwise.
4
18-H)), 0.89 (s, CH , 19-H), 0.96, 1.17 (1.2:1 (2d, J=6.6
Hz, 2CH , 21-H)), 2.38 (m, 1H), 2.51 (m, 1H), 2.62 (t,
3
The mixture was stirred at rt for 3 h. The solvent was
then removed completely in vacuo, and the residue
obtained was extracted with 10% HCl and ethyl acetate.
The solution was made alkaline with 10% NaOH solu-
tion, resulting in precipitates which was ®ltered, washed
well with water to a€ord the corresponding 20-aza-
pregnene analogues 2a±8a as white gums. Hydrogena-
tion of compounds 2a±8a (200 mg) was carried out in 30
3
J=7.2 Hz, 2H), 3.58 (m, 1H); CIMS m/z (relative
+
+
intensity) 405 (50, M +1), 387 (25, M +1-H O);
2
+
HRMS m/z calcd for C H N O 405.3846 (M +1),
2
6
49
2
found 405.3817.
1
3ꢀ-Hydroxy-20-aza-(N-butylamine)-pregn-5-ene (5a). H
NMR (CDCl , 300 MHz) d 0.65, 0.68 (1:1.2 (2s, 2CH ,
3
3

W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
1079
1
0
1
8-H)), 0.94, 1.05 (1.2:1 (2d, J=5.4 Hz, 2CH , 21-H)),
3
.98 (s, CH , 19-H), 2.20 (m, 2H), 2.40, 2.53 (2m, each
3
0.89 (s, CH , 19-H), 0.96, 1.17 (1.2:1 (2d, J=6.6 Hz,
3
2CH , 21-H)), 2.38 (m, 1H), 2.51 (m, 1H), 2.62 (t,
3
H), 2.66 (m, 2H), 3.49 (m, 1H), 5.31 (m, 1H); CIMS
+
J=7.2 Hz, 2H), 3.58 (m, 1H); CIMS m/z (relative
+
+
+
m/z (relative intensity) 389 (50, M +1), 371 (35, M +
-H O); HRMS m/z calcd for C H N O 389.3533
intensity) 391 (50, M +1), 373 (25, M +1-H O);
2
+
1
HRMS m/z calcd for C H N O 391.3690 (M +1),
2
2
+
25 45
2
25 47
(
M +1), found 389.3490.
found 391.3677.
ꢀ-Hydroxy-20-aza-(N-butylamine)-pregnane (5b). 1H
17ꢀ-20ꢀ,25-Diaza-cholesterol (8a1). H NMR (CDCl ,
3
1
3
NMR (CDCl , 300 MHz) d 0.81, 0.84 (1:1.2 (2s, 2CH ,
300 MHz) d 0.69 (s, CH , 18-H), 0.98 (s, CH , 19-H),
3
3
3
3
1
Hz, 2CH , 21-H)), 2.38 (m, 1H), 2.51 (m, 1H), 2.62 (t,
8-H)), 0.89 (s, CH , 19-H), 0.96, 1.17 (1.2:1 (2d, J=6.6
1.09 (d, J=6.6 Hz, CH , 21-H), 2.25 (s, 2CH ), 2.60 (m,
1H), 2.84 (m, 1H), 3.48 (m, 1H), 5.36 (m, 1H). CIMS
3
3 3
3
+
+
J=7.2 Hz, 2H), 3.58 (m, 1H); CIMS m/z (relative inten-
+
m/z (relative intensity) 389 (40, M +1), 371 (60, M +
1-H O), 283 (100). HRMS m/z calcd for C H N O
+
sity) 391 (50, M +1), 373 (25, M +1-H O); HRMS
2
2
25 45
2
+
+
m/z calcd for C H N O 391.3688 (M +1), found
2
389.3533 (M +1), found 389.3538. Anal. (C H N O+
25 44 2
5
47
2
3
91.3658.
1.5H O) C, H, N.
2
1
3
ꢀ-Hydroxy-20-aza-(N-propylamine)-pregn-5-ene (6a).
17ꢀ-20ꢁ,25-Diaza-cholesterol (8a2). H NMR (CDCl ,
3
1
H NMR (CDCl , 300 MHz) d 0.66, 0.68 (1:1.5 (2s,
300 MHz) d 0.65 (s, CH , 18-H), 0.98 (s, CH , 19-H),
3
3
3
2
CH , 19-H), 2.23 (m, 2H), 2.50, 2.65, 2.81 (3m, each
CH , 18-H)), 0.94 (d, J=6 Hz, CH , 21-H), 0.98 (s,
1.21 (d, J=6.6 Hz, CH , 21-H), 2.24 (s, 2CH ), 2.60
(2m, 2H), 2.84 (m, 1H), 3.48 (m, 1H), 5.33 (m, 1H).
3
3
3 3
3
+
1
intensity) 375 (50, M +1), 357 (25, M +1-H O);
H), 3.49 (m, 1H), 5.31 (m, 1H); CIMS m/z (relative
+
CIMS m/z (relative intensity) 389 (60, M +1), 371
+
+
(100, M +1-H O). HRMS m/z calcd for C H N O
2
2
2
25 45
+
+
HRMS m/z calcd for C H N O 375.3377 (M +1),
2
389.3533 (M +1), found 389.3534. Anal. (C H
25 44
2
4
43
found 375.3364.
N O+1.5H O) C, H, N.
2 2
1
1
3
ꢀ-Hydroxy-20-aza-(N-propylamine)-pregnane (6b). H
17ꢁ-20ꢀ,25-Diaza-cholesterol (8a3). H NMR (CDCl ,
3
NMR (CDCl , 300 MHz) d 0.81, 0.84 (1:1.2 (2s, 2CH ,
300 MHz) d 0.72 (s, CH , 18-H), 0.97(s, CH , 19-H),
3
3
3
3
1
Hz, 2CH , 21-H)), 2.38 (m, 1H), 2.51 (m, 1H), 2.62 (t,
8-H)), 0.89 (s, CH , 19-H), 0.96, 1.17 (1.2:1 (2d, J=6.6
0.99 (d, J=6.6 Hz, CH , 21-H), 2.60 (s, 2CH ), 2.79 (m,
3 3
1
3
3
2H), 3.00 (m, 1H), 3.46 (m, 1H), 5.36 (m, 1H).
NMR (CDCl , 100 MHz): 52.35, 52.18, 21.71, 20.17,
C
3
J=7.2 Hz, 2H), 3.58 (m, 1H); CIMS m/z (relative
+
3
+
intensity) 377 (50, M +1), 359 (25, M +1-H O);
2
19.36 (5 CH ); 64.82, 42.25, 42.16, 37.30, 33.74, 32.41,
3
31.63, 26.13, 22.58, 21.08 (10 CH ); 121.68, 71.72, 54.22,
2
+
HRMS m/z calcd for C H N O 377.3533 (M +1),
2
4
45
2
found 377.3490.
53.70, 52.93, 50.16, 32.21 (7 CH); 140.72, 43.22, 36.59 (3
+
C). CIMS m/z (relative intensity 389 (70, M +1), 371
+
0
3
ꢀ-Hydroxy-20-aza-(N-propanyl-N -dimethylamine)-
1
(60, M +1-H O), 330 (100); HRMS m/z calcd for
2
+
pregn-5-ene (7a). H NMR (CDCl , 300 MHz) d 0.66,
C H N O 388.3454 (M +1), found 388.3480. Anal.
25 44
3
2
0
J=6.6 Hz, 2CH , 21-H)), 0.98 (s, CH , 19-H), 2.23 (m,
.69 (1:1.5 (2s, 2CH , 18-H)), 0.85, 1.06 (1.5:1 (2d,
(C H N O+1.5H O) C, H, N.
25 44 2 2
3
3
3
1
2
H), 2.55, 2.59 (1.5:1 (2s, each 2CH )), 2.74 (m, 2H),
3
17ꢁ-20ꢁ,25-Diaza-cholesterol (8a4). H NMR (CDCl ,
3
3
sity) 403 (50, M +1), 385 (25, M +1-H O); HRMS
.49 (m, 1H), 5.32 (m, 1H); CIMS m/z (relative inten-
+
300 MHz) d 0.75 (s, CH , 18-H), 0.98 (s, CH , 19-H),
3
3
+
1.06 (d, J=6.6 Hz, CH , 21-H), 2.65 (s, 2CH ), 2.80 (m,
2H), 3.06 (2m, 2H), 3.46 (m, 1H), 5.33 (m, 1H). CIMS
2
3 3
+
m/z calcd for C H N O 403.3690 (M +1), found
2
6
47
2
+
+
4
03.3658.
m/z (relative intensity) 389 (40, M +1), 371 (65, M +
-H O). HRMS m/z calcd for C H N O 389.3533
1
2
+
25 45
2
0
3
ꢀ-Hydroxy-20-aza-(N-propanyl-N -dimethylamine)-
1
(M +1), found 389.3557. Anal. (C H N O+1.5H O)
25 44 2 2
pregnane (7b). H NMR (CDCl , 300 MHz) d 0.81,
C, H, N.
3
0
1
2
.84 (1:1.2 (2s, 2CH , 18-H)), 0.89 (s, CH , 19-H), 0.96,
.17 (1.2:1 (2d, J=6.6 Hz, 2CH , 21-H)), 2.38 (m, 1H),
.51 (m, 1H), 2.62 (t, J=7.2 Hz, 2H), 3.58 (m, 1H);
+
3 3
1
3ꢀ-Hydroxy-20-aza-(N-butane)-pregn-5-ene (9a). H
NMR (CDCl , 300 MHz) d 0.67, 0.70 (1.2:1 (2s, 2CH ,
3
3
3
CIMS m/z (relative intensity) 405 (50, M +1), 387 (25,
+
18-H)), 0.91 (t, J=7.2 Hz, CH ), 0.96, 1.07 (1:1.2 (2d,
J=6.6 Hz, 2CH , 21-H)), 0.98 (s, CH , 19-H), 2.59 (m,
3
M +1-H O); HRMS m/z calcd for C H N O
2
2
26 49
3
3
+
4
05.3847 (M +1), found 405.3804.
1H), 2.86 (m, 1H), 3.51 (m, 1H), 5.32 (m, 1H). CIMS
+
+
m/z (relative intensity) 374(85, M +1), 356 (100, M +
1-H O). HRMS m/z calcd for C H NO 374.3423
1
2
0,25-Diaza-cholesterol (8a). H NMR (CDCl , 300
3
2
+
25 44
MHz) d 0.63, 0.68 (1:1 (2s, 2CH , 18-H)), 0.96, 1.10 (1:1
(M + 1), found 374.3425.
3
(
2
2d J=6.6 Hz, 2CH , 21-H)), 0.98 (s, CH , 19-H), 2.22,
.28 (1:1 (2s, 2CH )). 2.60 (2m, 2H), 2.84 (m, 1H), 3.48
3 3
1
3ꢀ-Hydroxy-20-aza-(N-butane)-pregnane (9b). H NMR
(CDCl , 300 MHz) d 0.64, 0.66 (1:1 (2s, 2CH , 18-H)),
3
(
m, 1H), 5.32 (m, 1H); CIMS m/z (relative intensity) 389
+
3
3
+
(
m/z calcd for C H N O 388.3454, found 388.3480.
80, M +1), 371 (80, M +1-H O), 283(100); HRMS
0.73, 1.09 (1:1 (2d, J=6.6 Hz, 2CH , 21-H)), 0.77 (s,
CH , 19-H), 0.89 (t, J=8.0 Hz, CH ), 2.41 (m, 3H), 2.65
2
3
2
5
44
2
3
3
(m, 2H), 3.58 (m, 1H). CIMS m/z (relative intensity) 376
(100, M +1), 358 (40, M +1-H O). HRMS m/z calcd
for C H NO 376.3581 (M +1), found 376.3550.
25 45
ꢀ-Hydroxy-20,25-diaza-cholestane (8b). 1H NMR
CDCl , 300 MHz) d 0.81, 0.84 (1:1.2 (2s, 2CH , 18-H)),
+
+
3
(
2
+
3
3

1080
W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
1
3
(
0
ꢀ-Hydroxy-20-aza-cholestan-5-ene (10a). H NMR
(2m, 2H), 3.40 (m, 1H). CIMS m/z (relative intensity)
+
+
CDCl 300 MHz) d 0.67, 0.69 (1:1.2 (2s, 2CH , 18-H)),
392 (100, M +1), 374 (60, M +1-H O), 333 (40).
2
3
3
+
.87, 0.88 (1.2:1 (2d, J=6.6 Hz, 26, 27-CH )), 0.97, 1.08
HRMS m/z calcd for C H N O 392.3641 (M +1),
3
3
24 46
(
1.2:1 (2d, J=6.3 Hz, CH , 21-H)), 1.02 (s, CH , 19-H),
3
found 392.3634. Anal. (C H N O+1.5H O) C, H. N.
24 45 3 2
3
3
.49 (m, 1H), 5.32 (m, 1H). CIMS m/z (relative intensity)
+
+
3
calcd for C H NO 388.3580 (M +1), found 388.3574.
88 (85, M +1), 370 (100, M +1-H O). HRMS m/z
+
Pregn-5-en-3,20-diol (14). 3-Acetoxy-pregn-5-en-20-one
(500 mg, 1.39 mmol) was treated with LiAlH (106 mg,
2
2
6
46
4
ꢂ
excess amount of LiAlH was quenched with ethyl ace-
2
.79 mmol) in THF at 0 C and stirred for 1 h. The
1
3
ꢀ-Hydroxy-20-aza-pregn-5-ene (11a). H NMR (CDCl ,
3
4
3
CH , 19-H), 1.00 (d, J=6.6 Hz, CH , 21-H), 2.81 (m,
00 MHz) d 0.70, 0.72 (2.5:1 (2s, CH , 18-H)), 0.98 (s,
tate and the mixture was washed with 10% HCl. The
organic layer was dried over MgSO and concentrated
3
3
3
4
1
intensity) 318 (M +1, 40) for C H NO, 310 (100,
M +1-H O).
2
H), 3.50 (m, 1H), 5.32 (m, 1H). CIMS m/z (relative
+
to a€ord crude pregn-5-en-3,20-diol 14 as colorless solid
ꢂ
14
ꢂ
(425 mg, 96%): mp 234±237 C (lit. 236-238 C) which
was used without further puri®cation: FTIR (KBr)
2
1
36
+
À1
1
3
600±3200, 3000±2800, 1670, 1452, 1375, 1057 cm ; H
NMR (DMSO, 300 MHz) d 5.30 (d, J=3.9 Hz, 1H),
.65 (d, J=4.2 Hz, 1H), 4.15 (d, J=8.4 Hz, 1H), 3.50
m, 1H), 3.27 (m, 1H), 2.55.92 (m, 20H), 1.03 (d, J=6.3
Hz, 3H), 0.98 (s, 3H), 0.72 (s, 3H); CIMS m/z (relative
General procedures for reductive amination using NaB
CN)H3
4
(
(
Ketone precursor (200 mg) was dissolved in 10 mL
methanol, and 6 equiv N,N-dimethylethylene diamine
was added. pH of the solution was adjusted to ꢁ6 by
adding glacial acetic acid. Then 10 mL THF and 1.1
+
+
intensity) 374(85, M +1), 356 (100, M +1-H O);
2
+
HRMS m/z calcd for C H NO 374.3423 (M +1),
2
5
44
found 374.3425.
equiv NaBH CN in methanol was added, and the reac-
3
tion mixture was stirred under re¯ux overnight. After
evaporation of the solvent in vacuo, the resulted residue
was resuspended in 10 mL water, and the pH was adjus-
ted to ꢁ8. The solution was extracted with chloroform,
and the combined organic layer was washed with water
and dried over anhydrous Na SO . After evaporation,
Pregn-5-en-3,20-diamine (17). Crude 14 (400 mg, 1.26
mmol) was dissolved in CH Cl (3 mL) and pyridine (1
2
2
mL, 12.6 mmol), and mesyl chloride (490 mL, 6.33
mmol) was added to the solution at 0 C. After stirring
ꢂ
for 2 h, the mixture was diluted with water and extrac-
ted with methylene chloride. The combined organic
layer was dried over MgSO and concentrated to a€ord
2
4
the residue was washed with petroleum ether to give the
corresponding amine.
4
crude pregn-5-en-3,20-diol dimesylate 15 (568 mg):
FTIR (neat ®lm) 3068, 2944, 2898, 1608, 1536, 1487,
À1
1
3
5
ꢀ-Hydroxy-6,20,25-triaza-cholestane (13). 3b-Acetoxy-
-oxo-5,6-seco-pregnan-6-oic acid 1 g (2.5 mmol) was
1353, 1173, 938, 903, 758, 689 cm ; H NMR (CDCl ,
3
300 MHz) d 5.42 (m, 1H, ˆCH), 4.82 (m, 1H, CH-
+
allowed to react with oxalyl chloride in dichloro-
methane and cat. pyridine to give its acid chloride. The
solvent was evaporated and the residue was dissolved in
OMs), 4.52 (m, 1H, CH-OMs); FABMS m/z (relative
intensity) 475 (M +1, 25) for C H O S , 379 (100).
2
3
38
6 2
acetone. Then 10% NaN /water was added dropwise to
3
Crude 15 (550 mg, 1.16 mmol) was dissolved in DMF (5
mL) with excess amount of sodium azide (760 mg, 11.7
mmol) and heated to 80 C overnight. The mixture was
a€ord acyl azide. Formation of the intermediate acid
chloride and acyl azide was con®rmed by their charac-
ꢂ
À1
À1
teristic IR absorptions at 1801 cm and 2134 cm
respectively. Curtis rearrangement of the acyl azide was
,
diluted with water after cooling and extracted with ethyl
ether. The combined organic layer was dried over
MgSO and concentrated to a€ord crude pregn-5-en-
carried out in benzene at re¯ux to yield the isocyanate
À1
4
(n 2275 cm ), which, without further puri®cation, was
3,20-diazide 16 (321 mg): FTIR (neat ®lm) 2940, 2098
À1
treated with 12 N HCl in acetone at re¯ux and after
hydrogenation in acetic acid on 5% Pd/C gave the corre-
sponding aza-ketone 12. After being washed with petro-
leum ether, crude ketone 12 was allowed to undergo
reductive amination using NaB(CN)H . Following the
3
procedures described above, the crude amine product 13
in chloroform was further puri®ed by precipitation and
(strong), 1453, 1270, 1228 cm . Crude 16 (230 mg, 0.62
mmol) was reduced with LiAlH in THF to a€ord 17
4
(98 mg, 50%) or by hydrogenation with Pd/C in a 10:5:2
mixture of methanol/ethyl ether/acetic acid to a€ord 17
ꢂ
148 C); FTIR (neat ®lm) 3375 (strong), 2934, 1585, 1460,
13c
(123 mg, 63%) as yellowish solid: mp 147±149 C (lit.
ꢂ
À1 1
1387 cm ; H NMR (CDCl , 300 MHz,) d 5.33 (m, 1H),
3
®
ltration. Amine 13 was precipitated from its chloro-
3.12 (t, J=3 Hz, 1H, CHNH -3), 2.76 (m, 1H, CHNH -
20), 2.54 (d, J=15 Hz, 1H), 1.90.96 (m, 23H), 1.08 (d,
J=6.6 Hz, 3H), 0.97 (s, 3H) 0.65 (s, 3H); EIMS m/z
2
2
form solution by adding 2 M HCl. The solid obtained
from ®ltration was resuspended into water, and pH of
the solution was adjusted to ꢁ8. The solution was
extracted again with chloroform, and the combined
organic layer was washed with water, dried over anhy-
drous Na SO . Removal of the solvent gave the corre-
+
(relative intensity) 316 (M , 3), 301 (4), 273 (95), 44.0
+
(100); FABMS m/z (relative intensity) 317 (M +1, 100).
0
Bis(N -p-toluenesulfonyl)-pregn-5-en-3,20-diamine (18).
2
4
sponding 3b-hydroxy-6,20,25-triaza-chholestane 13 (60
mg, 5% from the acid) as a white gum: H NMR
To a solution of 17 (50 mg, 0.158 mmol) was added
p-toluenesulfonyl chloride (151 mg, 0.79 mmol) and
pyridine (128 mL, 1.58 mmol) at rt. After stirring for 2
days, the reaction mixture was treated with brine and
extracted with ethyl acetate three times. The combined
1
(
CDCl , 300 MHz) d 0.51, 0.53 (1:1.2, (2s, 2CH , 18-H)),
3
3
0
CH , 21-H)), 2.03 (s, 2CH ), 2.23 (m, 2H), 2.50, 2.80
.70 (s, CH , 19-H), 0.70, 0.80 (1.2:1, (2d, J=6.6 Hz,
3
3
3

W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
1081
organic layer was dried over MgSO , concentrated and
4
To the hot solution was added a solution of 3 equiv
NaOH in water and 6 equiv dimethyl sulfate. Then one
more equivalent of NaOH in water was added while
stirring under re¯ux. The reaction mixture was re¯uxed
for 3 h, then the solvent was removed under reduced
pressure. The resulted residue was resuspended in water
and extracted with ethyl acetate. The combined organic
layer was washed with brine and water, dried over anhy-
drous Na SO and concentrated to give a crude product
puri®ed by column chromatography (ethyl acetate/hex-
ane, 1/2) to a€ord 18 (77 mg, 78%) as colorless solid:
ꢂ
877, 1435, 1334, 1165, 1097 cm ; H NMR (CDCl ,
mp >300 C (decompose); FTIR (neat ®lm) 3283, 2938,
À1
1
2
3
300 MHz,) d 7.74 (dd, J=3.3 Hz, 8.1 Hz, 4H), 7.28 (d,
J=8.1 Hz, 4H), 5.17 (d, J=5.1 Hz, 1H), 4.74 (d, J=9.0
Hz, 1H), 4.46 (d, J=8.7 Hz, 1H), 3.52 (m, 1H), 3.23 (m,
1
1
H), 2.41 (s, 6H), 2.48.33 (m, 1H), 1.91.79 (m, 19H),
.01 (d, J=6.6 Hz, 3H), 0.89 (s, 3H), 0.59 (s, 3H);
2
4
containing 40% starting material. Recrystallization in
10±15% methanol/acetone solution to give pure methyl
+
FABMS m/z (relative intensity) 625 (M +1, 45), 471
(
ꢂ
ether (300 mg, 57%) as pellet: mp 160±162 C (lit.
15
19), 454 (76), 429 (25), 323 (53), 283 (100), 257 (24).
ꢂ
1
1
64±165 C): H NMR (CDCl , 300 MHz) d 7.21 (d,
3
0
19). A solution of 17 (52 mg, 0.164 mmol) and phenyl-
Bis(N -phenylaminocarbonyl)-pregn-5-en-3,20-diamine
(
J=8.7 Hz, 1H), 6.71 (dd, J=8.7, 3.0 Hz, 1H), 6.63 (d,
J=3.0 Hz, 1H), 3.76 (s, 3H), 2.87 (m, 2H), 0.89 (s, 3H);
+
+
isocyanate (38 mL, 0.345 mmol) in THF (3 mL) was
stirred for 3 days under nitrogen at rt. The reaction
mixture was concentrated and washed with ethyl ether
EIMS m/z (relative intensity) 284 (M , 100), 269 (M À
CH , 5), 256 (5), 199 (50); HRMS (EI) m/z calcd for
3
C H O 284.1776, found 284.1761.
1
9
24
2
ꢂ
to a€ord a white solid 19 (86 mg, 95%), mp 195±197 C;
FTIR (KBr) 3352 (strong) 3062, 2935, 1939, 1852, 1788,
Diaza derivative of 3-methoxy-estra-1,3,5(10)-trien-17-
one (23). Following the general procedures described
above for reductive amination using NaB(CN)H3,
3-methoxy-estra-1,3,5(10)-trien-17-one (22, 200 mg, 0.70
À1
1
654, 1601, 1542, 1444, 1313, 1232, 894, 754, 693 cm
;
1
H NMR (CD OD, 300 MHz,) d 7.25 (2s, 4H), 7.18 (t,
3
J=7.2 Hz, 4H), 6.87 (t, J=6.6 Hz, 2H), 5.34 (m, 1H),
3
2
.91 (m, 1H), 3.74 (m, 1H), 2.57 (m, 1H), 2.00±0.95 (m,
3H), 1.12 (d, J=6.6 Hz, 3H), 1.00 (s, 3H), 0.71 (s, 3H);
mmol) gave the corresponding diaza derivative 23 as
1
amorphous solid (200 mg, 80%): H NMR (CDCl , 300
3
+
FABMS m/z (relative intensity) 555 (M +1, 100), 462
(11), 419 (27), 323 (9), 283 (7); HRMS m/z calcd for
+
C H N O 555.3699 (M +1), found 555.3669.
MHz) d 7.15 (d, J=9.0 Hz, 1H), 6.64 (d, J=8.7, 2.7 Hz,
1H), 6.58 (d, J=2.7 Hz, 1H), 3.72 (s, 3H), 2.18 (s, 6H),
+
0.70 (s, 3H); EIMS m/z (relative intensity) 356 (M , 10),
+
3
5
47
4
2
3
41 (M À CH , 5), 298 (100), 58 (90); HRMS (EI) m/z
3
Pregn-5-en-3,20-diamino-bis(benzyloxycarbamate) (20).
To a solution of 17 (52 mg, 0.164 mmol) in CH Cl (3
calcd for C H N O 356.2828, found 356.2831.
23 36 2
2
2
mL) were added benzyl chloroformate (70 mL, 0.493
mmol) and triethylamine (120 mL, 0.86 mmol) under
nitrogen at rt. After stirring for 3 days, the mixture was
treated with brine and extracted with ethyl acetate. The
3ꢀ-Toluene-4-sulfonyloxy-pregn-5-en-20-one (24). To a
solution of pregnenolone (2 g, 6.3 mmol) in 20 mL pyri-
dine, 5 equiv toluene sulfonyl chloride was added with
cat. DMAP. After stirring at rt for 4 h, the reaction mix-
ture was poured into ice water resulting in white pre-
cipitates. The white powder obtained from ®ltration was
combined organic layer was dried over MgSO , con-
4
centrated and puri®ed by column chromatography
(THF/methylene chloride/hexane, 1/3/11) to a€ord 20
as colorless solid (78 mg, 81%): mp 64±67 C; FTIR
washed with water, dried in air to give quantitative
1
ꢂ
neat ®lm) 3431, 3332, 3067, 3032, 2941, 1699 (strong),
amount of product 24: H NMR (CDCl 300 MHz) d 7.78
3
(
(d, J=8.7 Hz), 7.30 (d, J=8.7 Hz), 5.28 (m, 1H), 4.31 (m,
1H), 2.42 (s, 3H), 2.09 (s, 3H), 0.94 (s, 3H), 0.58 (s, 3H).
1
6
5
530, 1504, 1456, 1335, 1264, 1241, 1221, 1072, 1031, 739,
À1
1
99 cm ; H NMR (CDCl , 300 MHz) d 7.33 (m, 10H),
3
.33 (m, 1H), 5.06 (m, 4H), 4.88 (d, J=Hz, 1H), 4.58 (d,
Pregnenolone methyl ether (25). 3b-Toluene-4-sulfonyl-
oxy-pregn-5-en-20-one (24, 500 mg, 1.06 mmol) was
dissolved in 50 mL methanol and stirred at rt for 2 h.
After evaporation of the solvent, the resulted residue
was resuspended in water and extracted with ethyl ace-
tate. The combined organic layer was washed with
saturated Na CO solution, brine and water, dried over
J=8.7 Hz, 1H), 3.92 (brs, 1H), 3.70 (m, 1H), 2.56 (d,
J=14.7 Hz, 1H), 1.94.07 (m, 19H), 1.16 (d, J=6.6 Hz,
3
H), 0.98 (s, 3H), 0.70 (s, 3H); FABMS m/z (relative
+
intensity) 585 (M +1, 100), 541 (12), 495 (11), 449 (17),
32 (11), 342 (13), 283 (18), 255 (8); HRMS m/z calcd for
+
4
C H N O 585.3692 (M +1), found 585.3724.
3
7
49
2
4
2
3
anhydrous Na SO . Removal of the solvent in vacuo
2
4
Diaza derivative of estrone (21). Following the general
procedures described above for reductive amination
using NaB(CN)H , estrone (500 mg, 1.85 mmol) gave the
3
gave a crude product, which was recrystalized in 10±
15% water/methanol solution to give pure methyl ether
ꢂ
16
ꢂ
(280 mg, 80%) as pellet: mp 121±123 C (lit. 123.5 C):
1
corresponding diaza derivative as amorphous solid (540
1
H NMR (CDCl , 300 MHz) d 5.33 (m, 1H), 3.33 (s,
3
mg, 85%): H NMR (CDCl , 300 MHz) d 7.11 (d, J=5.1
3H), 3.02 (m, 1H), 2.51 (m, 1H), 2.34 (m, 1H), 2.10 (s,
3H), 0.97 (s, 3H), 0.60 (s, 3H); EIMS m/z (relative
3
Hz, 1H), 6.60 (dd, J=5.1, 3.0 Hz, 1H), 6.69 (d, J=3.0 Hz,
+
+
+
1
0
3
H), 2.80 (m, 2H), 2.79 (m, 1H), 2.40 (m, 1H), 2.21 (s, 6H),
+
intensity) 330 (M , 50), 315 (M À CH , 10), 298 (M À
3
.70 (s, 3H); EIMS m/z (relative intensity) 342 (M , 20),
+
CH OH), 283 (50), 43 (100); HRMS (EI) m/z calcd for
3
27 (M - CH , 5), 284 (95), 58 (100); HRMS (EI) m/z
3
C H N O 330.2559, found 330.2554.
22 34 2
calcd for C H N O 342.2671, found 344.2668.
2
2
34
2
22,25-Diaza-cholesterol methyl ether (26). Following the
general procedures described above for reductive ami-
nation using NaB(CN)H , pregnenolone methyl ether
3
3
(
-Methoxy-estra-1,3,5(10)-trien-17-one (22). Estrone
1 g, 3.70 mmol) was dissolved in 20 mL ethanol by heating.

1082
W. Xie et al. / Bioorg. Med. Chem. 9 (2001) 1073±1083
3
3
2
5 (200 mg, 0.61 mmol) gave the corresponding diaza
1
[ H]-phosphatidylinositol-(4,5)-biphosphate ([ H]PIP )
2
3
derivative as amorphous solid (210 mg, 86%): H NMR
CDCl , 300 MHz) d 5.26 (m, 1H), 3.26 (s, 3H), 2.96 (m,
as the substrate. Unreacted [ H]PtdIns(4,5) biphosphate
was removed from its water soluble hydrolysis product
by acid coprecipitation with bovine serum albumin, to
(
3
1
H), 0.91 (s, 3H), 0.62, 0.60 (2s, 2:1, 3H, 18-H); EIMS
H), 2.13, 2.11 (2s, 1:2, 6H), 0.99, 0.89 (2d, 1:2, 3H, 21-
3
which [ H]PtdIns(4,5)biphosphate bind quantitatively.
+
+
m/z (relative intensity) 403 (M +1, 20), 402 (M , 10),
+
IC50 values were calculated from at least three indepen-
dent measurements liquid scintillation counting of the
supernatant. The variation from the mean value is 20%
or less. NA stands for IC50 value larger than 100 mg/mL.
3
HRMS (EI) m/z calcd for C H N O 402.3610, found
87 (M +1- CH , 5), 358 (10), 344 (100), 58 (90);
3
2
6
46
2
4
02.3611.
1
2-Methoxy-podocarpatrien-16-al (28). To a solution of
2-methoxy-podocarpatrien-16-ol (1 g, 3.6 mmol) in 5
Cytotoxicity assays
1
mL dichloromethane was added dropwise a cold solu-
The in vitro cytotoxicity assays were carried out at the
National Cancer Institute (NCI). Details of the assay
23
procedure have been reported previously.
tion of 2.2 equiv DMSO and 1.1 equiv oxalyl chloride in
ꢂ
5
mL dichloromethane at À60 C. After 5 min, 5 equiv
ꢂ
Et N was injected. After stirring at À60 C for 10 min,
3
the reaction mixture was poured into ice water and
extracted with dichloromethane. The combined organic
layer was washed with brine and water, dried over
anhydrous Na SO . After evaporation of the solvent,
Acknowledgements
We gratefully acknowledge support of this research by
the NCI/NIH (Grant 5U19 CA52995). We are grateful
to Dr. Leslie T. Gelbaum for his assistance in obtaining
high resolution NMR spectra, and Mr. David E. Bost-
wick and Ms. Sarah J. Shealy for their assistance in
obtaining high resolution mass spectra.
2
4
the residue obtained was recrystalized in acetone/
methanol to give the corresponding aldehyde as color-
ꢂ
17
less needles (850 mg, 85%): mp 134±136 C (lit. 135±
ꢂ
.95 (d, J=8.7 Hz, 1H), 6.76 (d, J=2.7 Hz, 1H), 6.70
1
1
6
37 C): H NMR (CDCl , 300 MHz) d 9.80 (s, 1H),
3
(
(
dd, J=8.7, 2.7 Hz, 1H), 3.78 (s, 3H), 2.80 (m, 2H), 2.20
m, 2H), 1.98 (m, 1H), 1.08 (s, 3H), 1.03 (s, 3H); EIMS
+
+
m/z (relative intensity) 272 (M , 100), 229 (M À
CH CO, 20), 147 (65); HRMS (EI) calcd for C H O
3
18 24
References
2
72.1776, found 272.1773.
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Compound 29
Following the general procedures described above for
reductive amination using NaB(CN)H , 12-methoxy-
podocarpatrien-16-al (28, 200 mg 0.73 mmol) gave the
corresponding diaza derivative 29 as amorphous solid,
2
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3
(
S. R.; Yue, X.; Bruzik, K. S.; Tsai, M. D. Biochemistry 1998,
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1
2
00 mg, 80% yield. H NMR (CDCl , 300 MHz) d 6.95
3
(
d, J=8.7 Hz, 1H), 6.76 (d, J=2.7 Hz, 1H), 6.69 (dd,
J=8.7, 2.7 Hz, 1H), 3.75 (s, 3H), 3.20 (m, 1H), 3.09 (m,
H), 2.43 (s, 6H), 1.17 (s, 3H), 1.13 (s, 3H); EIMS m/z
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4
. (a) Powis, G.; Seewald, M. J.; Gratas, C.; Melder, D.; Rie-
1
(
(
3
+
+
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4
50), 58 (100); HRMS (EI) m/z calcd for C H N O
2
2
36
2
44.2828, found 344.2818.
5
. (a) Yang, H.; Shan, F.; Herengiova, M.; Weber, G. Anti-
cancer Res. 1998, 12, 1399. (b) Arteaga, C. L.; Johnson, M. D.;
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PI-PLCꢂ in vitro inhibition assay
Inhibition of PI-PLC activity was measured as pre-
4
6. Robertson, J. B.; Hurd, S. D.; Koch, M. O.; Arteaga, C. L.
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viously described using bovine brain PI-PLCg and
Elemental analysis data for compounds 1, 8, 9, 13
Compound no.
8a1
+1.5H O
8a2
+1.5H O
8a3
+1.5H O
8a4+
1.5H O
13
+1.5 H O
2
2
2
2
2
Calcd
C
H
N
C
H
N
72.24
11.40
6.74
72.31
11.25
6.70
72.24
11.40
6.74
71.86
11.40
6.64
72.24
11.40
6.74
71.99
11.24
6.53
72.24
11.40
6.74
71.88
11.24
6.67
68.85
11.56
10.04
68.46
11.32
9.87
Found

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1
(
1
1
1
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1
1
4. Merck Index 12, 7908.
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1
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