Novel pyrrolo-quinoline derivatives as potent inhibitors for PI3-kinase related kinases

Article abstract of DOI:10.1016/S0968-0896(01)00260-7

Several pyrrolo-quinoline γ-lactones were found as novel inhibitors for two members of the PI3-kinase related kinase (PIKK) family. Ataxia-Telangiectasia-mutated (ATM) protein and the mammalian Target of Rapamycin (mTOR). Preliminary structure-activity relationship studies indicated that an electrophilic exocyclic double bond conjugated to the carbonyl group of the γ-lactone ring was crucial for the PIKK inhibitory potency. One of the best ATM inhibitors in this series, DK8G557, showed IC₅₀ values of 0.6 and 7.0 μM for ATM and mTOR, respectively. This compound exhibited potent and selective growth inhibition activities in the NCI 60 human tumor cell line screen with a GI₅₀ MG-MID value of 2.69 μM. The best mTOR inhibitor in this series, HP9912, exhibited IC₅₀ values of 0.5 and 6.5 μM for mTOR and ATM, respectively. These compounds suggest novel leads for the discovery of potent small molecule inibitors of PIKKs as potential anticancer drugs, with therapeutic activities as either single, or as sensitizing agents to conventional radio-, or chemo-therapeutic strategies. Copyright

Full text of DOI:10.1016/S0968-0896(01)00260-7

Bioorganic& Medicinal Chemistry 10 (2002) 167–174
Novel Pyrrolo-quinoline Derivatives as Potent Inhibitors
for PI3-Kinase Related Kinases
Hairuo Peng,a,* Doeg-Il Kim,a Jann N. Sarkaria,b Yong-Seo Cho,a
Robert T. Abrahamc and Leon H. Zalkowa
aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
bDepartment of Oncology, Mayo Clinic, Rochester, MN 55905, USA
cDepartment of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Received 25 May 2001; accepted 10 July 2001
Abstract—Several pyrrolo-quinoline g-lactones were found as novel inhibitors for two members of the PI3-kinase related kinase
(PIKK) family, Ataxia-Telangiectasia-mutated (ATM) protein and the mammalian Target of Rapamycin (mTOR). Preliminary
structure–activity relationship studies indicated that an electrophilic exocyclic double bond conjugated to the carbonyl group of the
g-lactone ring was crucial for the PIKK inhibitory potency. One of the best ATM inhibitors in this series, DK8G557, showed IC50
values of 0.6 and 7.0 mM for ATM and mTOR, respectively. This compound exhibited potent and selective growth inhibition
activities in the NCI 60 human tumor cell line screen with a GI50 MG-MID value of 2.69 mM. The best mTOR inhibitor in this
series, HP9912, exhibited IC50 values of 0.5 and 6.5 mM for mTOR and ATM, respectively. These compounds suggest novel leads
for the discovery of potent small molecule inhibitors of PIKKs as potential anticancer drugs, with therapeutic activities as either
single, or as sensitizing agents to conventional radio-, or chemo-therapeutic strategies. # 2001 Elsevier Science Ltd. All rights
reserved.
Introduction
exhibited by A-T cells, it is possible that small molecule
inhibitors of the ATM protein might sensitize tumor
cells to the cytotoxic effects of ionizing radiation or
DNA-damaging chemo-therapeutic agents.4 Recent
reports4ꢀ7 that wortmannin, an irreversible inhibitor of
the PIKK family members is a highly effective radio-
sensitizer, support this prediction.
PI3-kinase related kinases (PIKKs) are a novel family of
signaling proteins that share a common COOH-term-
inal region similar to that of phosphatidylinositol 3-
kinase (PI3-kinase).1 In mammalian cells, the PIKK
family comprises Ataxia-Telangiectasia-mutated (ATM)
protein, the mammalian target of rapamycin (mTOR),2
Ataxia-Telangiectasia-mutated and Rad3-related (ATR)
protein,3 and DNA-dependent protein kinase (DNA-
PK),4 which are different from PI3-kinase in the sense
that they do not possess lipid kinase activity, but are
serine/threonine protein kinases.
The mTOR protein is a critical enzyme in the transmis-
sion of mitogenic signals from cytokine receptors. It is
known that mTOR functions as a key component in
signaling pathways that regulate synthesis of proteins
required for cell-cycle progression in both lymphoid and
nonlympoid cells.2 Anticancer activity has been found
to be associated with two different types of mTOR
inhibitors: rapamycin and wortmannin. Therefore small
molecule mTOR inhibitors may have potential applica-
tions as anticancer agents.
The ATM protein functions in multiple signal-trans-
duction pathways that coordinate cell cycle arrest and
DNA repair in response to DNA damage. The gene that
encodes this protein is responsible for the human
genetic disorder Ataxia Telangiectasia (A-T), which is
characterized by cancer proneness and extreme radio-
sensitivity. In light of the radiosensitive phenotype
As shown in Figure 1, only a few PIKK inhibitors have
been reported. Wortmannin, a fungal metabolite, was
identified as
a
potent inhibitor of PI3-kinase
(IC50=4.2 nM).8 The mechanism by which wortmannin
inhibits PI3-kinase has been recently confirmed, in the
crystal structure of wortmannin bound PI3Kg,9 as irre-
versible modification of the primary amine, Lys-833, in
*Corresponding author at current address: Department of Chemistry,
Yale University, PO Box 208107, New Haven, CT 06520, USA. Tel.:
+1-203-432-5218; fax: +1-203-432-6144; e-mail: hairuo.peng@yale.
edu
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00260-7

168
H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
the active site. Wortmannin also showed differential
inhibitory activities for members of the PIKK family
(ATM, IC50=150 nM; mTOR, IC50=40 nM; DNA-PK,
IC50=16 nM; ATR, IC50=1.8 mM), which likely result
from inactivation of the PI3-kinase related catalytic
domains in these enzymes by a similar mechanism. The
radio-sensitizing effects of wortmannin correlated with
its ATM, DNA-PK inhibitory activities in intact A549
lung cancer cells.4 The competitive inhibitor LY294002
has been confirmed to occupy the ATP-binding site of
PI3-kinase9 and was reported to inhibit both PI3K
(IC50=1.4 mM)10 and DNA PK (Ki=6.0 mM).11 Dual
ligands that combined a LY294002-like kinase inhibitor
with geldanamycin, a high affinity binder of hsp90,
through different linkers, were reported as selective
inhibitors of the PI3K and DNA PK recently.12
search for direct inhibitors of the PIKK catalytic
domain was the observation that wortmannin, a potent
inhibitor of the catalytic activities of most members of
the PIKK family, possesses a near planar structure and
a highly electron-deficient center that is prone to react-
ing with nucleophiles through Michael addition15 (Fig.
2). In reviewing our in-house compound library, pyr-
rolo-quinoline g-lactone 13 (DK8G557), a by product
obtained in an earlier effort of synthesizing camptothe-
cin in this group,16 appears to provide a planar scaffold
with its pyrrolo-quinoline moiety and a Michael accep-
tor from the exocyclic double bond on the g-lactone
ring.
Chemistry
Scheme 1 illustrates the synthesis of compound 12 as
previously documented.16,17 Briefly, bis-g-lactone 7, a
racemic mixture, was synthesized in 4 steps using the
method of Piskov,18 which involved acid hydrolysis of
a-hydroxymethyl-b-carboethoxyparaconate (3) and
subsequent decarboxylation of the bis-g-lactone-a-car-
boxylicaicd 4. In the acid catalyzed hydrolysis of 3,
compounds 5 and 619 were obtained as by products.
Freidlander condensation of o-amino benzaldehyde (8)
and N-carbethoxy-3-pyrrolide (9) at 240 ꢁC gave com-
pound 10,20 which was hydrolyzed to 1,3-dihydro-2H-
pyrrolo(3, 4-b)quinoline (11).20 Coupling of 11 with bis-
g-lactone 7 in ethanol gave alcohol 12 as a racemic
mixture. However, delocalization of the amide bond,
and the bulky and asymmetrical pyrrolo-quinoline moi-
ety render chiral feature to the amide nitrogen, which
generally broadened and complicated the NMR signals.
Wortmannin and LY294002 have been invaluable tools
in the studying the biological roles of members of the
PIKK family in the intracellular signaling events. How-
ever, the significant toxicity and narrow therapeutic
indices associated with these two compounds have made
it difficult to develop them as drug candidates. There-
fore, there exists a need for the development of PIKK
inhibitors of distinct chemical identities that may pos-
sess more optimal in vitro and in vivo activities. We
report here the discovery a group of novel pyrrolo-
quinoline derivatives, as exemplified by DK8G557 and
HP9912 in Figure 1, as potent irreversible inhibitors for
PI3-kinase related kinases, specifically the ATM and
mTOR protein kinases.
Results and Discussion
As shown in Scheme 2, an improved procedure
employing treatment of alcohol 12 with triethyl amine
and oxalyl chloride/methacrylic acid (1:1) in chloroform
at 0 ꢁC for 1 h gave DK8G557 (13) as a racemic mixture
in 57% yield. The effect of the bulky amide bond in this
compound was evident from the split signals of one of
the exocyclic methylene protons, d 5.72 (d, J=2.1 Hz,
0.7H, H-30), 5.71 (d, J=2.7 Hz, 0.3H, H-30), indicating
existence of two rotamers. Conversion of 13 to 14 was
accomplished in 64% yield by stirring 13 with 1 equiv of
DMAP at rt for 1 day. Oxidation of compound 13 using
m-CPBA gave N-oxide derivative 15, while epoxide
derivative 16 was synthesized using urea/hydrogen per-
oxide complex.
There had been no structural information for the cata-
lyticdomain of any member of the PIKK family until
Williams and coworkers reported the crystal structures
of PI3-kinase recently.9,13,14 The starting point of our
As shown in Scheme 3, HP9912 (17) was synthesized by
treatment of compound 12 with the optically pure acid
chloride made from (S)-(+)-2-(4-chloro-phenyl)-2-
21
hydroxy-butyricaicd.
Formation of the ester was
1
confirmed by the extra H NMR signals at 4.42 ppm.
These signals result from protons on C-30, which was
located at d 3.97 (brs, 2H, H-30) in alcohol 12. However,
the hydroxyl group from the 2-(4-chloro-phenyl)-2-
hydroxy-butyricacid appeared to be eliminated during
the preparation of either the acid chloride or the ester.
1
In the H NMR spectrum of compound 17, extra peaks
at d 7.03 (q, J=7.2 Hz, H-300), d 1.53 (d, J=7.2 Hz, Me-
400) and d 1.58 (d, J=7.2 Hz, Me-400) may account for
the highly conjugated olefinic proton (H-300) and Me-400
Figure 1. Chemical structures of wortmannin, LY29004, DK8G557,
and HP9912.

H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
169
Figure 2. Proposed mechanism for irreversible inactivation of PI3 kinase by Wortmannin.
Scheme 1. Reagents and conditions: (a) Na/EtOH; I2/ether, rt, 3 h; (b) HCHO, Na/EtOH, 55 ꢁC, 2 h; (c) concd HCl, reflux, 3 h; (d) 185 ꢁC, nitrogen,
15 min; (e) TsOH/240 ꢁC, 5 min; (f) KOH/aq EtOH, overnight; (g) EtOH, reflux, 2 h.
Scheme 2. Reagents: (a) EtN3/(COCl)2–methacrylic acid (1:1), CHCl3, 0 ꢁC, 1 h; (b) DMAP/CHCl3, 1 day; (c) m-CPBA/CHCl3, rt, 2 h; (d) urea/
H2O2, trifluoroacetic anhydride, 40–50 ꢁC, 24 h.
(differentiated for Z or E configurations of the double
bonds, Z/E=1:1). Ester 18 was prepared in similar
condition using benzoyl chloride.
0.6 mM. The mechanism by which this compound inhi-
bits ATM appears to be similar to that employed by
wortmannin. The exocyclic double bond, like the one in
the furan ring of wortmannin, could well serve as an
excellent receptor for Michael addition of nucleophilic
groups. In addition to being conjugated to the lactone
carbonyl group, reaction of the double bond with a
nucleophile releases ring strain by conversion of the sp2
carbon on the five-membered ring into a sp3 center. The
activity of this moiety has been exemplified by the well-
known toxicities associated with the exocyclic double
bonds of sesquiterpenoid lactones. The irreversible
binding of DK8G557 (13) to ATM was confirmed by
the fact that extensive wash of the inhibited, protein A-
Sepharose beads-bound ATM against buffer did not
PIKK inhibitory activities
The inhibitory activities of the compounds described
above were evaluated in the immune complex kinase
assays for PI3-kinase related kinases as previously
described.4 Active compounds exhibited irreversible
binding to the PIKKs tested. The results are listed in
Table 1. Representative dose–response curve for
DK8G557 is shown in Figure 3. DK8G557 (13), which
contains an exocyclic double bond on the g-lactone ring,
inhibited the ATM protein with an IC50 value of

170
H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
recover the enzymatic activity. Compound 13 showed
significantly lower potency in inhibiting the other two
members of the PIKK family, mTOR (IC50=7.0 mM)
and DNA-PK (<50% inhibition at 10 mM).
center was changed to an epoxide, as in compound 16,
the inhibitory activity was significantly reduced, con-
firming the importance of the exocyclic double bond in
inhibiting these enzymes. One exception, however, was
found that benzoate derivative 18 exhibited moderate
inhibitory activity (IC50=10 mM) without the presence of
the exocyclic double bond. The activity might arise from
an effective fit in the active binding cleft, or it might
result from partial elimination of the benzoate, under
the assay condition, to give the active compound 13.
In contrast, alcohol 12 and the compound with an
endocyclic double bond, 14, were essentially inactive for
both ATM and mTOR. In addition, when the eletrophilic
Oxidation of the pyrrolo-quinoline moiety, as in com-
pound 15 (IC50=10 mM), led to reduced ATM inhibi-
tory activity. However, in the absence of the pyrrolo-
quinoline moiety, simple compounds with just the exo-
cyclic double bond-containing g-lactone also showed
medium (compound 5, IC50=6.0 mM) to excellent
(compound 6, IC50=0.25 mM) inhibitory activity against
ATM. The potency of compound 6 was similar to that
of wortmannin, which was used as a control to monitor
the assay. Surprisingly, simple lactones 5 and 6 exhibited
less than 50% inhibition of mTOR at 10 mM. The
excellent selectivity of 6 observed between the two
highly homologous members within the PIKK family was
unexpected, since simple electrophilc species like these
are usually predicted to exhibit non-specific inhibitions.
Scheme 3. Reagents: (a) (S)-(+)-2-(4-chloro-phenyl)-2-hydroxy-buty-
ric acid chloride, Et3N, DMAP, CHCl3; (b) benzoyl chloride, Et3N,
DMAP, CHCl3.
It appears that inhibition of mTOR requires more than
simply a Michael addition acceptor such as those exo-
cyclic double bonds in compounds 13 and 6. While the
pyrrolo-quinoline derivative 13 showed moderate
mTOR inhibitory activity, simple lactone 6 was inactive
at 10 mM for mTOR even though it showed excellent
ATM inhibition. It is likely that inhibition of mTOR
requires finer compound–enzyme interactions that could
be obtained from the pyrrolo-quinoline moiety. The
best mTOR inhibitor in this series is HP9912 (17), which
inhibited mTOR with an IC50 of approximately 0.5 mM.
This compound inhibited ATM with about 10 times less
potency (Table 1). The superior inhibitory activity of 17
toward mTOR may arise from the simultaneous pre-
sence of a Michael acceptor (the electrophilic double
bond activated by the adjacent carbonyl), the pyrrolo-
quinoline and the p-chloro phenyl moieties. The struc-
ture might be too bulky for the ATM active site to
produce excellent inhibition.
NCI 60-human tumor cell lines screen
Figure 3. Inhibition of ATM and mTOR by DK8G557. ATM and
mTOR were immunoprecipitaed from A549 cell extract and rat brain
extract, respectively. The immune complexes were treated with
DK8G557 for 30 min at rt, and kinase activity were assayed under
linear conditions. The kinase activities were normalized to those mea-
sured in the no-drugs controls
The growth inhibition activity of compounds 13, 15,
and 18 was evaluated at the National Cancer Institute
against a panel of 60 human tumor cell lines represent-
ing nine different cancer types. The GI50, TGI and LC50
values are shown in Table 2 for sensitive subpanels.
Table 1. PIKK inhibitory activities of compounds 5, 6, 12–18 and Wortmannin
Compound no.
5
6
1213
14
15
16
17
18
6.5
0.5
>10
Wortmannin4,22
ATM IC50 (mM)a
6.0
>10
ND
0.25
>10
ND
>10
ND
ND
0.6
7.0
>10
>10
>10
ND
10
>10
ND
>10
>10
ND
10
ND
ND
0.15
0.04
0.016
mTOR IC50 (mM)a
DNA PK IC50 (mM)a
aIC50 values were determined from at least two independent determinations.

H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
171
Table 2. Inhibition growth of in vitro human cancer cell lines by the pyrrolo-quinoline derived compounds
13
15
18
a
c
a
a
c
Disease type and cell lines
GI50
TGIb
LC50
GI50
GI50
TGIb
LC50
Leukemia
HL60TB
RPMI-8226
Non-Small Cell Lung Cancer
EKVX
NCI-H226
NCI-H522
Colon Cancer
HCT116
HT29
SW620
1.06
2.02
4.90
>25
21.6
>25
0.0036
7.37
5.31
2.24
14.1
9.50
>25
>25
3.69
0.38
0.35
7.39
0.71
0.83
14.8
1.32
2.00
>25
>25
4.62
11.7
0.95
1.05
>25
3.62
3.62
>25
9.51
10.1
1.06
4.01
0.50
4.05
7.58
1.64
10.1
14.3
7.42
>25
>25
3.88
9.58
1.51
8.34
>25
5.85
17.9
>25
18.9
9.07
CNS Cancer
SF-268
SF-539
3.58
0.56
8.15
1.70
18.5
8.62
>25
6.27
16.3
2.69
>25
7.09
>25
18.7
Melanoma
LOXIMVI
MALME-3M
M14
1.73
0.82
1.36
4.86
2.96
4.70
11.0
9.68
11.4
>25
9.11
>25
6.16
3.16
4.25
>25
>25
13.7
14.8
6.58
7.93
Ovarian Cancer
IGR-OV1
OVCAR3
OVCAR4
Renal Cancer
786-0
ACHN
CAKI-1
TK-10
UO-31
2.92
2.99
1.31
5.99
7.72
5.46
12.3
19.9
23.2
>25
>25
>25
7.22
6.73
3.69
13.9
>25
>25
>25
17.9
8.13
1.20
0.79
0.92
7.73
1.32
4.17
3.45
3.66
3.00
4.77
10.2
>25
3.77
3.11
3.36
4.21
4.50
7.47
6.63
7.40
9.17
14.8
14.2
16.3
20.0
9.28
10.6
8.77
11.5
15.9
>25
11.4
>25
14.3
>25
Prostate Cancer
PC-3
DU-145
3.45
3.12
6.81
6.89
13.4
15.2
>25
>25
13.5
8.35
>25
>25
>25
>25
Breast Cancer
MDA-MB-435
MDA-N
T-47D
MG-MID
1.00
2.74
1.18
2.69
4.27
5.84
6.40
5.49
14.1
12.5
>25
14.1
>25
>25
12.6
17.5
4.47
6.58
4.84
7.76
10.8
>25
13.9
15.8
>25
>25
>25
22.4
aGI50 represent the micromolar compound concentration required to achieve 50% inhibition of tumor cell growth.
bTGI represents the micromolar concentration required to achieve total growth inhibition of tumor cells.
cLC50 represent the micromolar concentration that is required to achieve 50% tumor cell killing.
As shown in Table 2, DK8G557 (13) exhibited potent
and selective growth inhibition activities, with a mean
GI50 value of 2.69 mM. The two most sensitive cell lines to
this compound are the NCI-H226 and NCI-H522 non-
small cell lung cancer cell lines, where high potency and
consistent selectivity in GI50 (0.38 and 0.35 mM, respec-
tively), TGI (0.71 and 0.83 mM, respectively) and LC50
(1.32 and 2.00 mM, respectively) were obtained. The HCT-
116 and SW-260 colon cancer cell lines also exhibited sen-
sitivity to this compound, with GI50s of 1.06 and 0.50mM,
respectively; the LC50 concentrations for these two tumor
cell lines were at about 10 mM. It is noteworthy that
DK8G557 showed selectivity in inhibiting growth of
several renal cancer cell lines (including ACHN, CAKI-1
and UO-31), and the selectivity was consistent through the
LC50s. Other sensitive subpanels included the SF539 CNS
cancer cell line (GI50=0.56 mM), the MALME-3M mela-
noma cell line (GI50=0.82 mM), and the MDA-MB-435
breast cancer cell line (GI50=1.00 mM).
oxide derivative 15 only showed limited growth inhibi-
tion effect in a few cancer cell lines (Table 2), and the
TGI and LC50 values are generally larger than 25 mM.
This may result from the presence of charges on the
molecule that could prevent it from crossing the plasma
membrane. Compounds 13 and 18 have been referred to
the Biological Evaluation Committee (BEC) at the
National Cancer Institute.
Conclusion
In conclusion, a novel group of pyrrolo-quinoline
derived g-lactones were found to be potent inhibitors
for two members of the PI3-kinase related kinase
family, ATM protein and mTOR. Initial structure–
activity relationships indicate that an electrophilic
exocyclic double bond conjugated to the carbonyl group
of the g-lactone ring, as in DK8G557 (Fig. 1), is
crucial for the ATM inhibitory activity. Furthermore,
simpler g-lactones (5, 6) also exhibited potent and
even more selective ATM inhibitory activities. The
inhibition appeared to be irreversible based on the
The benzoate derivative 18 showed sensitivity pattern
similar to that of DK8G557 (13), but the potencies for
different tumor cell lines are generally lower. The N-

172
H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
washout experiments. It is observed that compounds
6 and 13 showed inhibitory selectivity for ATM over
Synthesis of compound 13
mTOR, with
higher selectivity ratio as compared to wortmannin
(Table 1).
a
reversed selective preference and
To a suspension of 12 (26 mg, 0.096 mmol) and triethy-
lamine (500 mL) in CHCl3 (3 mL) was added a 1:1 mix-
ture (100 mL) of methacrylic acid and oxalic chloride at
0 ꢁC. After stirring for 1 h, the mixture was washed with
water and CHCl3. The combined organic layer was
dried, concentrated and purified by silica gel column
chromatography using methanol/ethyl acetate/chloro-
form (1:1:10) as mobile phase to give 13 as colorless
solid (13 mg, 57%), mp 202–205 ꢁC. FT-IR (KBr) 3271,
The significantly lower activity of lactones 5 and 6
toward mTOR suggest that in addition to a presence of
a Michael acceptor, inhibition of mTOR may require
more extended enzyme–inhibitor interaction than that
of ATM. The best of the mTOR inhibitors discovered is
pyrrolo-quinoline derived 2-(4-chloro-phenyl)-2-ene-
butyricacid ester HP9912 (Fig. 1, 0.5 mM). It showed
selectivity for mTOR over ATM, with the same selective
preference as that of wortmannin, but higher selectivity
ratio (Table 1). In the NCI 60 human tumor cell line
screen, DK8G557 (13) showed potent and selective
growth inhibition activities.
1
2928, 1777, 1646, 1449, 1412, 1266, 1121, 778 cmꢀ1; H
NMR (300 MHz, CDCl3) d 8.07 (m, 2H), 7.83 (d,
J=8.4 Hz, 1H), 7.74 (t, J=7.8 Hz, 1H), 7.58 (t,
J=7.8 Hz, 1H), 6.42 (d, J=3 Hz, 1H, H-30), 5.72 (d,
J=2.1 Hz, 0.7H, H-30), 5.71 (d, J=2.7 Hz, 0.3H, H-30),
5.11 (m, 4H, H-2, H-5), 4.72 (t, J=8.4 Hz, 1H, H-60),
4.59 (t, J=9.3 Hz, 1H, H-60), 4.30 (m, 1H, H-50); HRMS
(m/z) calcd for C17H14N2O3 294.1004, found 294.0994.
ꢂ
Anal. calcd for C17H14N2O3 0.25H2O: C, 68.33; H,
4.89; N, 9.37. Found: C, 68.30; H, 4.89; N, 9.20.
Experimental
Thin layer chromatography analysis (TLC) was per-
formed on aluminum sheets precoated with 0.2 mm of
silica gel containing 60F254 indicator. Spots were
detected with shortwave UV light or Ceric sulfate spray.
Flash chromatography was run using 230–400 mesh
silica gel. The homogeneity of all the compounds was
routinely checked by TLC on silica gel plates, and also
by HPLC. Melting points were measured on a Kofler
hot stage apparatus attached to a digital thermometer
and were uncorrected. Fourier transformed infrared
spectra were obtained on a Nicolet 520 FT-IR spectro-
meter. 1H (300 or 400 MHz), 13C (75 or 100 MHz)
NMR and DEPT spectra were recorded on either a
Varian Gemini-300 or on a Varian XL-400 spectro-
meter. Chemical shifts are reported relative to CDCl3 (d
7.24). High-resolution mass spectra (EI or FAB) were
recorded on a VG Analytical 70-SE mass spectrometer
equipped with a 11-250J data system. Elemental ana-
lyses were performed by Atlantic Microlab, Norcross,
GA, USA.
Synthesis of compound 14
A mixture of 13 (50 mg, 0.170 mmol) and DMAP
(23 mg, 0.187 mmol) in chloroform (3 mL) was stirred
for 1 day at room temperature. The mixture was directly
concentrated and purified by silica gel chromatography
using methanol/ethyl acetate/chloroform (1:1:15) as the
mobile phase to give 14 (32 mg, 64%) as colorless solid:
mp 235 ꢁC. FT-IR (KBr) 3044, 2929, 2873, 1857, 1778,
1749, 1651, 1600, 1505, 1435, 1410, 1378, 1295, 1095,
1031, 927, 778, 759 cmꢀ1; 1H NMR (300 MHz, CDCl3) d
8.05 (m, 2H), 7.84 (t, J=7.2 Hz, 1H), 7.74 (m, 1H), 7.57
(t, J=7.2 Hz, 1H), 5.11–5.00 (m, 4H, H-2, H-5), 4.94,
4.90 (s, 2H, H-60), 2.04 (t, J=2.1 Hz, 3H, H-30); EIMS m/
z (relative intensity) 294.0 (M+, 13), 169.1 (20), 97.1 (10),
83.1 (12), 69.1 (21), 57.1 (21), 44.0 (100); HRMS (m/z)
calcd for C17H14N2O3 294.1004, found 294.0994. Anal.
ꢂ
calcd for C17H14N2O3 0.25H2O: C, 68.33; H, 4.89; N,
9.37. Found: C, 68.41; H, 4.81; N, 9.24.
Synthesis of compound 15. To a solution of 13 (37 mg,
0.127 mmol) in chloroform (3 mL) was added 2 equiv of
m-CPBA(73 mg) in three portions. The reaction was
allowed to react at rt for 2 h, then the mixture was
quenched with satd NaHCO3 aqueous solution and
extracted with CHCl3. The combined organic layer was
dried over MgSO4, concentrated and purified by silica
gel chromatography using methanol/ethyl acetate/
chloroform (1:1:10) as mobile phase to give compound
15 (30 mg, 71%) as colorless solid, mp 190–192 ꢁC. IR
(KBr) 3087, 2987, 2922, 2864, 2246, 1765, 1666, 1650,
Syntheses of precursors 2–11 have been previously
documented.16,17,20
Synthesis of alcohol 12
To a solution of 1,3-dihydro-2H-pyrrolo[3, 4-b]quino-
line 11 (4.0 g, 23.5 mmol) in absolute ethanol (200 mL)
was added bis-g-lactone 7 (3.45 g, 24.3 mmol). The mix-
ture was refluxed in a nitrogen atmosphere for 2 h.
Concentration of the solution followed by titration with
chloroform precipitated alcohol 12 as white solid (4.7 g,
64%), mp 201–202 ꢁC. FT-IR (KBr) 3300, 3200, 1760,
1625 cmꢀ1; 1H NMR (300 MHz, CD3COCD3) d 8.09 (s,
1H), 7.82 (d, J=7.8 Hz, 1H), 7.74 (t, J=8.4 Hz, 1H),
7.57 (t, J=8.1 Hz, 1H), 5.07 (m, 4H, H-2, H-5), 4.72 (t,
J=8.7 Hz, 1H, H-60), 4.52 (t, J=8.4 Hz, 1H, H-60),
3.97(brs, 2H, H-30), 3.85(m, 1H, H-50), 3.05(m, 2H, H-20
and –OH); HRMS m/z calcd for C17H16N2O4 312.1110,
found 312.1091. Anal. calcd for C17H16N2O4: C, 45.38;
H, 5.13. Found: C, 45.22; H, 5.22.
1580, 1457, 1427, 1401, 1118, 737 cmꢀ1 1H NMR
;
(300 MHz, CDCl3) d 8.70 (d, J=9.3 Hz, 1H), 7.90 (d,
J=8.4 Hz, 1H), 7.79 (t, J=1.6 Hz, 1H), 7.69 (m, 2H),
6.43 (d, J=2.7 Hz, 1H), 5.74 (d, J=2.7 Hz, 0.7H), 5.70
(d, J=2.7 Hz, 0.3H), 5.33–5.06 (m, 4H), 4.73–4.68 (m,
1H), 4.59 (t, J=8.9 Hz, 1H), 4.32 (m, 1H); EIMS m/z
(relative intensity) 310.1 (M+, 28.7), 294.2 (36.4), 235.2
(9.6), 197.1 (10.8), 185.1 (10.1), 169.1 (100), 140.1 (15.1),
125.1 (32.4), 115.1 (12.5), 67.0 (15.1); HRMS (m/z)
calcd for C17H14N2O4 310.0954, found 310.0948. Anal.

H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
173
ꢂ
calcd for C17H14N2O4 0.29H2O: C, 64.71; H, 4.66; N,
8.88. Found: C, 64.71; H, 4.51; N, 8.80.
at 0 ꢁC. After stirring overnight at room temperature,
the mixture was poured into water and extracted with
chloroform. The combined organic layer was dried over
MgSO4 and concentrated at reduced pressure. The con-
centrate was purified by silica gel column chromato-
graphy using methanol/ethyl acetate/chloroform
(1:1:30) as mobile phase to afford compound 18 (12 mg,
41%) as colorless solid: mp 191–193 ꢁC. IR (KBr) 3056,
2929, 1788, 1712, 1636, 1452, 1280, 1117, 1039, 756,
Synthesis of compound 16. Trifluoroacetic anhydride
(60 mL, 0.425 mmol) was added to a mixture of
compound 13 (15 mg, 0.051 mmol), urea H2O2 complex
(130 mg,
1.38 mmol)
and
NaHCO3
(102 mg,
1.214 mmol) in chloroform (5 mL) at rt under Argon.
The reaction was allowed to react at 40–50 ꢁC for 24 h,
then the mixture was poured into water and washed
with chloroform. The combined organic layer was dried
over MgSO4 and concentrated under reduced pressure.
The concentrate was purified by silica gel chromato-
graphy using methanol/ethyl acetate/chloroform
(1:1:30) as mobile phase to afford compound 16 (2.2 mg,
14%) as a colorless solid: mp 171–174 ꢁC. IR (film)
2928, 2856, 1793, 1650, 1446, 1413, 1325, 1234, 1101,
1
713 cmꢀ1; H NMR (300 MHz, CD3COCD3) d 8.22 (s,
1H), 8.05–7.85 (m, 4H), 7.75 (t, J=7.6 Hz, 1H), 7.59 (m,
1H), 7.44 (m, 1H), 7.26 (t, J=7.8 Hz, 2H), 5.24 (s, 1H),
5.14 (d, J=1.8 Hz, 1H), 4.87 (s, 1H), 4.77 (s, 1H), 4.74–
4.52 (m, 4H), 4.24 (m, 1H), 3.72 (m, 1H); HRMS (FAB,
m/z) calcd 417.1450 for C24H21N2O5, observed
417.1447. Anal. calcd for C24H20N2O5: C, 69.22; H,
4.84; N, 6.73. Found: C, 69.24; H, 4.76; N, 6.56.
1019, 922, 726 cmꢀ1 1H NMR (300 MHz, CDCl3) d
;
8.06 (m, 2H), 7.84–7.81 (2s, 1H), 7.73 (tt, J=1.8 Hz, 7.2
Hz, 1H), 7.56 (2d, J=7.2 Hz, 1H), 5.16–4.90 (m, 5H),
4.73 (m, 1H), 4.09 (m, 1H), 3.23 (d, J=5.7 Hz, 1H),
3.07 (dd, J=1.8 Hz, 5.4 Hz, 1H); HRMS(FAB, m/z)
calcd for C17H15N2O4 (M++1) 311.1032, observed
311.1060.
PIKK immune complex kinase assays
The detailed procedure of ATM immune complex
kinase assays have been previously described.4 Briefly,
A549 cells were cultured and harvested during expo-
nential growth. The cells were washed twice with PBS
and then scraped on ice in lysis buffer (20 mM HEPES,
0.15 M NaCl, 1.5 mM MgCl2, and 1 mM EGTA, pH
7.4) containing 10 mg/mL aprotinin, 1 mM DTT, 5 mg/
mL leupeptin, 20 nM microcystin, 5 mg/mL pepstatin,
and 0.2% Tween 20. Lysates were cleared of insoluble
material by centrifugation, and equal amounts of lysate
protein (0.5 mg) were incubated with ATM-specific
antibody on ice for 2 h. The immune complexes were
precipitated with protein A-Sepharose beads, and the
immunoprecipitates were washed twice with the lysis
buffer, once with high-salt buffer (0.6 M NaCl, 0.1 M
Tris–HCl, pH 7.4) and once with kinase buffer (10 mM
HEPES, 50 mM NaCl, and 10 mM MgCl2, pH 7.4). The
resulting immunoprecipitates were incubated for 30 min
at room temperature with kinase buffer containing var-
ious concentrations of inhibitors. The kinase reaction
mixture was then added to yield final concentration of
10 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 10 mM
MnCl2, 1 mM DTT, 10 mM [g-32P]-ATP (specific activ-
ity, 50 Ci/mmol, ICN), and 25 ng/mL recombinant
PHAS-1 in a total volume of 40 mL. The reaction mix-
ture was incubated for 20 min at 30 ꢁC. The reactions
were terminated with equal volume of 30% acetic acid,
and duplicate aliquots were spotted on P81 phospho-
cellulose papers (Whatman). After four 5-min washes
with 1% phosphoric acid containing 10 mM sodium
PPi, the radioactivity retained in the paper was mea-
sured by liquid scintillation counting. All kinase reac-
tions were performed under linear reaction conditions.
The kinase activities for each PIKK were normalized to
that measured in the no-drug control. The IC50 values
were determined from two independent determinations,
each run in duplicate. The variation from the mean did
not exceed ꢃ30%.
Synthesis of ester 17
To a solution of (S)-(+)-2-(4-chloro-phenyl)-2-hydroxy-
butyricacid (100 mg, 0.5 mmol) in 10 mL CH 2Cl2 was
added DMF and then SOCl2 (43 mL, 0.6 mmol) drop-
wise at room temperature. After the reaction was com-
pleted, methylene chloride, excess SOCl2 and HCl were
removed under reduced pressure. The residue was dis-
solved in 5 mL CH2Cl2 and the distillation repeated to
give (S)-(+)-2-(4-chloro phenyl)-2-hydroxy-butyric acid
chloride, which was used for next reaction without fur-
ther purification.
To a suspension of 12 (100 mg, 0.32 mmol), triethyla-
mine (70 mL) and catalytic amount of DMAP in CHCl3
was added the above acid chloride via syringe as a con-
centrated CH2Cl2 solution. The reaction mixture was
stirred at 0 ꢁC for 2 h. Then the solution was poured
into ice water and extracted with CH2Cl2 twice. The
combined organic phases were dried over Na2SO4, and
concentrated and purified by silica gel column chroma-
tography to give ester 17 as a colorless gum: 1H NMR d
8.08 (s, 1H), 8.04 (d, J=10.2 Hz), 7.87 (d, J=8.4 Hz,
1H), 7.76 (t, J=8.1 Hz, 1H) 7.60 (t, J=8.4 Hz, 1H), 7.13
(m, 2H), 7.03 (q, J=7.2 Hz, 1H, H-300), 6.95 (m, 2H),
4.70–4.92 (m, 4H, H-2, H-5), 4.40–4.60 (m, 4H, H-30, H-
60), 3.69 (m, 1H, H-50), 3.28 (m, 1H, H-20), 1.58, 1.53 (d,
J=7.2 Hz, 3H, H-400, E and Z isomers); EIMS m/z
(relative intensity) 490 (M+, 10), 294 (62), 263 (11), 235
(25), 196 (54), 169 (100), 115 (77); HRMS (FAB, m/z)
calcd for C27H24ClN2O5 491.13737, found 491.13748.
ꢂ
Anal. calcd for C27H23ClN2O5 0.20H2O: C, 65.57; H,
4.77; N, 5.66. Found: C, 65.26; H, 4.72, N, 5.66.
Synthesis of compound 18. Benzoyl chloride (16 mL,
0.138 mmol) was added to a mixture of compound 12
(22 mg, 0.071 mmol), triethylamine (30 mL, 0.215 mmol)
and a catalytic amount of DMAP in chloroform (3 mL)
For measurements of DNA-PK activity, cells were lysed
as described above, with the exception that the lysates
were sonicated prior clearing. Cell extracts (0.75 mg
protein) were immunoprecipitated with anti-DNA-PK

174
H. Peng et al. / Bioorg. Med. Chem. 10 (2002) 167–174
antibodies. The immune complexes were washed twice
with lysis buffer and twice with kinase base buffer prior
to the kinase reaction. The kinase reaction conditions
were identical to those described above, except that the
reaction time was 15 min and the substrate used for
DNA-PK was a p53-derived peptide added to a final
concentration of 250 ng/mL in a total volume of 40 mL.
8. Powis, G.; Bonjoukian, R.; Berggren, M. M.; Gallegos, A.;
Abraham, R. T.; Ashendel, C. L.; Zalkow, L. H.; Matter,
W. F.; Dodge, J.; Grindey, G.; Vlahos, C. J. Cancer Res. 1994,
370, 2419.
9. Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.;
Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Molecular
Cell 2000, 6, 909.
10. Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F. J.
Biol. Chem. 1994, 269, 5241.
The mTOR immune complex assay was performed as
described previously.23 mTOR was immunoprecipitated
from rat brain extract with anti-mTOR antibodies. The
preincubation procedure and the kinase reactions were
performed using conditions that were identical those
used in the ATM assay.
11. Izzard, R. A.; Jackson, S. P.; Smith, G. C. M. Cancer Res.
1999, 59, 2581.
12. Chiosis, G.; Rosen, N.; Sepp-Lorenzino, L. Bioorg. Med.
Chem. Lett. 2001, 11, 909.
13. Walker, E. H.; Perisic, O.; Stephens, L.; Williams, R. L.
Nature 1999, 402, 313.
14. Pacold, M. E.; Suire, S.; Perisic, O.; Walker, E. H.; Haw-
kins, P. T.; Eccleston, J. F.; Williams, R. L. Cell 2000, 103, 931.
15. Norman, B. H.; Shih, C.; Toth, J. E.; Ray, J. E.;
Dodge, J. A.; Johnson, D. W.; Rutherford, P. G.; Schultz,
R. M.; Worzalla, J. F.; Vlahos, C. J. J. Med. Chem. 1996,
39, 1106.
16. Nabors, J. B. Studies in the synthesis of camptothecin.
Georgia Institute of Technology Doctoral Thesis, 1970.
17. French, K. A. Studies in the synthesis of camptothecin
and analogues. Georgia Institute of Technology Doctoral
Thesis, 1975.
Acknowledgements
We gratefully acknowledge support of this research by
the NCI /NIH (5U19-CA52995) and CA80829 (JNS).
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