Paullones, a series of cyclin-dependent kinase inhibitors: Synthesis, evaluation of CDK1/cyclin B inhibition, and in vitro antitumor activity

Article abstract of DOI:10.1021/jm9900570

The paullones represent a novel class of small molecule cyclin-dependent kinase (CDK) inhibitors. To investigate structure-activity relationships and to develop paullones with antitumor activity, derivatives of the lead structure kenpaullone (9-bromo-7,12

Full text of DOI:10.1021/jm9900570

J . Med. Chem. 1999, 42, 2909-2919
2909
P a u llon es, a Ser ies of Cyclin -Dep en d en t Kin a se In h ibitor s: Syn th esis,
Eva lu a tion of CDK1/Cyclin B In h ibition , a n d in Vitr o An titu m or Activity
Christiane Schultz,‡ Andreas Link,‡ Maryse Leost,§ Daniel W. Zaharevitz,† Rick Gussio,† Edward A. Sausville,†
Laurent Meijer,§ and Conrad Kunick*,‡
Institut fu¨r Pharmazie, Abteilung fu¨r Pharmazeutische Chemie, Universita¨t Hamburg, Bundesstrasse 45,
D-20146 Hamburg, Germany, Centre National de la Recherche Scientifique, Station Biologique, BP 74,
F-29682 Roscoff, France, and Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, Rockville, Maryland 20852
Received February 2, 1999
The paullones represent a novel class of small molecule cyclin-dependent kinase (CDK)
inhibitors. To investigate structure-activity relationships and to develop paullones with
antitumor activity, derivatives of the lead structure kenpaullone (9-bromo-7,12-dihydroindolo-
[3,2-d][1]benzazepin-6(5H)-one, 4a ) were synthesized. Paullones with different substituents
in the 2-, 3-, 4-, 9-, and 11-positions were prepared by a Fischer indole reaction starting from
1H-[1]benzazepine-2,5(3H,4H)-diones 5. Selective substitutions at either the lactam or the indole
nitrogen atom were accomplished by treating kenpaullone with alkyl halides in the presence
of sodium hydride/THF or potassium hydroxide/acetone, respectively. S-Methylation of the
kenpaullone-derived thiolactam 18 yielded the methylthioimidate 19, which gave the hydroxy-
amidine 20 upon reaction with hydroxylamine. The new paullones were tested both in a CDK1/
cyclin B inhibition assay and in the in vitro antitumor cell line-screening program of the
National Cancer Institute (NCI). With respect to the CDK1/cyclin B inhibition, electron-
withdrawing substituents in the 9-position as well as a 2,3-dimethoxy substitution on the
paullone basic scaffold turned out to be favorable. A 9-trifluoromethyl substituent was found
to be equivalent to the 9-bromo substituent of kenpaullone. Replacement of the 9-bromo
substituent of kenpaullone by a 9-cyano or 9-nitro group produced a substantial increase in
enzyme-inhibiting potency. Substitutions in other positions or the replacement of the lactam
moiety led to decreased CDK1 inhibition. Noteworthy in vitro antitumor activities (GI50 values
between 1 and 10 µM) were found with the 9-bromo-2,3-dimethoxy-7,12-dihydroindolo[3,2-d]-
[1]benzazepin-6(5H)-one (4t), its 9-trifluoromethyl analogue 4u , the 12-Boc-substituted paullone
15, and the methylthioimidate 19, respectively. The 9-nitro-7,12-dihydroindolo[3,2-d][1]-
benzazepin-6(5H)-one (4j, named alsterpaullone) showed a high CDK1/cyclin B inhibitory
activity (IC50 ) 0.035 µM) and exceeded the in vitro antitumor potency of the other paullones
by 1 order of magnitude (log GI50 mean graph midpoint ) -6.4 M).
In tr od u ction
The number of chemical agents that act selectively
as CDK inhibitors is limited; among them are a lactone
(butyrolactone I (1)), flavonoids (e.g., flavopiridol (2)),
and several purine derivatives (Chart 1). Butyrolactone
I (1) has shown antiproliferative activity for colon and
pancreatic carcinoma cell lines.10,11 Flavopiridol (2) is
the first CDK inhibitor that has entered clinical trials
as an anticancer agent.12,13 The class of purine-related
derivatives comprises the majority of known CDK
inhibitors, e.g., olomoucine,14 roscovitine (3),15,16 and the
purvalanols.17 X-ray structure determination of crystal-
lized inhibitor-enzyme complexes revealed that the
purine-derived compounds as well as flavopiridol bind
to the ATP-binding pocket of the CDKs.12,17-20
When flavopiridol was used in a COMPARE search21
performed at the National Cancer Institute (NCI) in a
database of compounds tested in the NCI in vitro cancer
cell line screening, the 9-bromo-7,12-dihydroindolo[3,2-
d][1]benzazepin-6(5H)-one (4a , kenpaullone)22 was iden-
tified as a potential CDK inhibitor.23 Inhibition experi-
ments revealed that 4a indeed is a potent inhibitor of
CDKs with selectivity for CDK1, CDK2, and CDK5. It
was shown, that kenpaullone acts as an ATP competi-
The cell division cycle is driven and regulated by a
variety of complex processes. Among the manifold
molecular entities that are involved in the surveillance
of the cell cycle, the cyclin-dependent kinases (CDKs)
play a central role. The CDKs are a group of serine
threonine kinases, which control the transmission be-
tween successive stages of the cell cycle.1 The activity
of the CDKs is regulated by multiple mechanisms,
including binding to cyclins, a diverse class of positive
regulatory CDK-binding proteins. The oscillating con-
centration of the cyclins during the cell cycle is the basis
for the stage-dependent activity of the CDKs. Binding
to CDK inhibitory proteins (CKIs) results in deactiva-
tion of CDKs. In various human tumors, deregulations
of CDK-related mechanisms have been found, e.g.,
overexpression of cyclins or deletion of genes encoding
for CKIs.2-5 Considering these observations, CDKs are
attractive targets for the development of antitumor
drugs.6-9
‡ Universita¨t Hamburg.
§ Centre National de la Recherche Scientifique.
† National Cancer Institute.
10.1021/jm9900570 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/30/1999

2910 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Schultz et al.
Ch a r t 1
Sch em e 1a
a
Reagents and conditions: (i) 1. HOAc, 70 °C, 2. concd H2SO4,
HOAc, 70 °C; (ii) CuCN, N-methyl-2-pyrrolidone, reflux; (iii) BBr3,
CH2Cl2, 20 °C.
hydrogen bond with a water molecule observed in the
crystal structure of CDK2 with ATP bound.24 It was
further postulated that a substituent with the proper
characteristics could enhance affinity by (a) increasing
the strength of the hydrogen bonds to Leu-83 by
increasing the resonant character of these bonds and
(b) increasing the strength of the interactions with the
hydrophobic side chains by rendering the ring systems
relatively charge deficient. Ab initio quantum mechan-
ical calculations, as well as molecular mechanics and
hydropathic analysis, were used to investigate possible
substituents. Results indicated that a 9-cyano substitu-
tion was likely to increase affinity. Details of the
theoretical investigations will be reported elsewhere.
Because of the remarkable improvement of the CDK1-
inhibiting activity experienced with 4i, the nitro ana-
logue 4j (named alsterpaullone) was prepared as an
additional example of a paullone with similar electronic
properties.
tive inhibitor of CDK1/cyclin B (IC50 ) 0.4 µM; apparent
Ki ) 2.5 µM).23 Although kenpaullone is equipotent to
flavopiridol with respect to its inhibiting potency for
CDK1/cyclin B, it exhibits only a modest antiprolifera-
tive activity in the in vitro cancer cell line screening (log
GI50 MG_MID ) -4.4 M), which is poor compared to
the in vitro antitumor potency of flavopiridol (log GI50
MG_MID ) -7.2 M).
A synthesis study was designed with a view to two
main objectives. First, it was intended to search for
paullone-related CDK-inhibitors with improved potency
and antitumor activity. Second, information on struc-
ture-activity relationships (SAR) within the paullone
series was needed for a further rational development.
Special interest was devoted to the identification of the
positions of the kenpaullone scaffold, which would
tolerate the attachment of substituents, and to the
question, which of these modifications would be detri-
mental for the CDK inhibitory activity.
Ch em istr y
Therefore, replacement of the 9-bromo substituent of
kenpaullone by other moieties was undertaken to
investigate the importance of this substitution position
for CDK inhibitory activity. Additional substituents
were introduced into the 2-, 3-, 4-, 10-, and 11-positions
with or without keeping the 9-bromo substituent in
place. The two nitrogen atoms of kenpaullone were
substituted alternatively with alkyl chains, and Boc
groups were introduced into the 5-, 7-, and 12-positions
of kenpaullone to explore the ability of the target to
accommodate bulky residues. To study the role of the
secondary lactam partial structure, it was replaced by
a thiolactam, a methylthioimidate, and a semicyclic
hydroxyamidine group, respectively.
Results of molecular modeling experiments have been
published, in which the energy-minimized structure of
kenpaullone was docked to the ATP binding site of the
CDK2 structure.23 Using this model as a guide, structure-
based considerations focused on the 9-position. It was
postulated that substituting a hydrogen bond acceptor
at this position could increase affinity by forming a
The preparation of the 7,12-dihydroindolo[3,2-d][1]-
benzazepin-6(5H)-ones 4a , 4c, 4d , 4k , and 4p by a
Fischer indole synthesis has been reported previously.22
Employing this method, the analogues 4c-h and 4j-w
were prepared by reacting 1H-[1]benzazepine-2,5(3H,4H)-
diones 5 with appropriate phenylhydrazines 6 in acetic
acid. The intermediate phenylhydrazones were not
isolated but were cyclized instantaneously by means of
concentrated sulfuric acid (Scheme 1). For the prepara-
tion of the 1H-[1]benzazepine-2,5(3H,4H)-diones 5 sev-
eral methods have been described.25-32 For the synthetic
sequences reported here, 5 were prepared by dealkoxy-
carbonylation of the corresponding 5-hydroxy-2-oxo-2,3-
dihydro-1H-[1]benzazepine-4-carboxylic acid esters.33
For example, the synthesis of the 9-methoxy-1H-[1]-
benzazepine-2,5(3H,4H)-dione (5a ), which was needed
as a starting material to prepare the 4-methoxy paullone
4w , is outlined in Scheme 2. The ethyl 2-amino-3-
methoxybenzoate (8) was obtained by esterification of
2-amino-3-methoxybenzoic acid (7) with ethanol in the
presence of gaseous hydrogen chloride. Acylation of 8

Synthesis and Properties of Paullones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2911
Sch em e 2a
Sch em e 3a
a
Reagents and conditions: (i) 2 equiv (Boc)2O, 2 equiv NaH,
THF, reflux; (ii) 3 equiv (Boc)2O, DMAP, CH2Cl2, 20 °C; (iii) TFA,
CH2Cl2, 20 °C; (iv) 3 equiv (Boc)2O, 3 equiv NaH, THF, reflux.
Sch em e 4a
a
Reagents and conditions: (i) HCl, EtOH, reflux; (ii) toluene,
pyridine, <10 °C; (iii) KH, toluene, DMF, N2, 80 °C; (iv) DMSO,
H2O, N2, 150 °C.
with ethyl succinyl chloride (9) led to the diester 10. A
Dieckmann cyclization of (10) catalyzed by potassium
hydride in a mixture of toluene and DMF furnished the
5-hydroxy-9-methoxy-2-oxo-2,3-dihydro-1H-[1]benzaze-
pine-4-carboxylic acid ethyl ester (11). Heating 11 in wet
DMSO yielded 5a , which was reacted with 4-bromo-
phenylhydrazine (6a ) to give the 9-bromo-4-methoxy-
7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one (4w ).
The 4-hydroxy derivative 4y of kenpaullone was ob-
tained by treating the methyl ether 4w with boron
tribromide in dichloromethane. Employing a similar
procedure, the hydroxy compound 4x was prepared by
ether cleavage from the corresponding methoxy precur-
sor 4t. The 9-cyano-substituted analogue 4i was pre-
pared by a Rosenmund-von Braun reaction upon
heating kenpaullone (4a ) with cuprous cyanide in
N-methyl-2-pyrrolidone (Scheme 1).
Employing varying reaction conditions and catalysts,
the Boc group was introduced into three different
positions of the kenpaullone molecule (Scheme 3). If
kenpaullone was refluxed with 2 equiv di-tert-butyl
dicarbonate in the presence of sodium hydride, the 5-
and 7-positions were substituted to yield compound 12.
Carrying out the reaction under identical conditions
with 3 equiv of di-tert-butyl dicarbonate led to the 3-fold
substituted derivative 14. The 5,12-disubstituted Boc
derivative 13 was formed upon reacting 4a with di-tert-
butyl dicarbonate and 4-(dimethylamino)pyridine in
dichloromethane. Treatment of 13 with 3 equiv of
a
Reagents and conditions: (i) NaH, THF, alkyl halide, reflux;
(ii) KOH, acetone, alkyl halide, 0-20 °C; (iii) LiAlH4, THF, reflux.
trifluoroacetic acid at ambient temperature resulted in
the selective cleavage of the Boc group in the 5-position,
furnishing the 12-substituted kenpaullone derivative 15.
A regioselective alkylation of the nitrogen atoms of
kenpaullone was achieved using two different basic
catalyst/solvent combinations (Scheme 4). On one hand,
deprotonation of kenpaullone with sodium hydride in
refluxing THF and the subsequent reaction with alkyl
halides generated the tertiary lactams 16a -d . On the
other hand, reaction of kenpaullone with alkyl halides
in the presence of potassium hydroxide in acetone led
to substitution of the indole nitrogen atom yielding the
12-substituted derivatives 17a -d .
For the reduction of the ester moiety of 17d , an excess
of lithium alumino hydride was employed to yield the
primary alcohol 17e (Scheme 4). The lactam moiety
remained unaffected under these conditions. A similar
observation on the resistance of a lactam group for
lithium alumino hydride reduction was reported for the
related class of 5H-pyrido[3,2-d][1]benzazepin-6(7H)-
ones.34

2912 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Schultz et al.
Ta ble 1. CDK1/Cyclin B Inhibition and in Vitro Antitumor Activity of the Paullones 4a -y
log GI50
IC50 CDK1/
compd
R1
R2
cyclin B (µM)a
HCT-116 (M)b,c
MG_MID (M)c,d
2 (flavopiridol)
0.3
0.65
0.4
1.3
7
0.6
1.6
0.9
2.0
-7.6e
-5.1/-5.1
-5.7/-5.7
-5.5
-7.2e
-4.8/-4.7
-4.4/-4.3
-4.8
3 (roscovitine)f
4a (kenpaullone)
4b
4c
4d
4e
4f
4g
4h
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9-Br
10-Br
H
9-Cl
9-F
9-OCH3
9-CH3
9-CF3
9-CN
9-NO2
11-Cl
11-Br
11-Me
11-Et
8,10-di-Cl
H
9-Br
9-CF3
H
9-Br
9-CF3
H
9-Br
9-Br
9-Br
-4.8
-4.5
-5.5
-4.1
-5.1
-4.6
-4.6
-4.6
>-4.0
-5.4
-4.0
0.4
-4.1
4i
0.024
0.035
1.4
1.3
3.0
3.8
2.5
3.3
0.3
0.24
4.3
0.2
0.28
430
250
3.0
-4.7
-4.1
4j (alsterpaullone)
4k
4l
4m
4n
4o
4p
4q
4r
-6.7/-7.4
>-4.0
NAg
-6.4/-6.5
-4.0
-4.3
-4.8
-4.1
>-4.0
>-4.0
>-4.0
>-4.0
-4.6
-5.8/-5.5
-5.7
>-4.0
-5.0
>-4.0
-4.3
-4.5
-4.2
-4.0
2-Br
2-Br
2-Br
2,3-di-OCH3
2,3-di-OCH3
2,3-di-OCH3
4-OCH3
4-OCH3
2,3-di-OH
4-OH
-4.0
-4.0
-4.0
4s
4t
-4.5
-5.2/-5.1
-5.2/-5.3
-4.1
4u
4v
4w
4x
4y
-4.6/-4.8
-4.0
-4.4
40
a
b
d
Tests were carried out in triplicate. Colon cancer cell line. c Results of two test runs are separated by a slash. MG_MID ) mean
graph midpoint. e Mean of six experiments. f Test results with racemic form. NA, not available.
g
Sch em e 5a
Ta ble 2. CDK1/Cyclin B Inhibition and in Vitro Antitumor
Activity of the Kenpaullone Derivatives 12-20
log GI50
IC50 CDK1/
compd
cyclin B (µM)a
HCT-116 (M)b,c
MG_MID (M)d,c
12
13
14
15
80
1000
150
70
20
470
35
6.4
6.2
23
>-4.0
NAe
-5.1/-4.4
-5.6
-4.7
>-4.0/-4.9
-4.7
-4.6/-4.8
-5.1/-5.1
-4.5
-4.0
-4.4
-4.4/-4.2
-5.3
16a
16b
16c
16d
17a
17b
17c
17d
17e
18
-4.5/-4.7
-4.6/-4.9
-4.5/-4.0
-4.7/-4.5
-4.5/-4.7
-4.3
60
>-4.0
>-4.0
-4.8
-4.2
1.4
3.0
2.3
43
-4.2
-4.7
NAe
ROf
19
20
-5.7/-5.8
-5.2
-5.5/-5.7
-5.0
1.0
a
b
Tests were carried out in triplicate. Colon cancer cell line.
c Results of two test runs are separated by a slash. MG_MID,
d
mean graph midpoint. e NA, not available. f RO, result omitted
because of irregular shape of the dose-response curve.
a
Reagents and conditions: (i) P2S5, NaHCO3, THF, N2, reflux;
assay, CDK1/cyclin B was harvested from starfish
oocytes and purified by affinity chromatography. CDK1/
cyclin B activity was determined by measurement of the
histone H1 phosphorylation catalyzed by the enzyme.
Test results are given in Tables 1 and 2, with Table 1
including results for the paullone derivatives 4a -y and
the results with flavopiridol and roscovitine for com-
parison. Table 2 contains the results for paullones
modified at the indole nitrogen atom or the lactam
moiety (compounds 12-20). The tests revealed that a
substitution in the 9-position of the 7,12-dihydroindolo-
[3,2-d][1]benzazepin-6(5H)-ones is favorable with re-
spect to the CDK1/cyclin B inhibitory activity. Thus, the
(ii) NaH, THF, ICH3, N2, reflux; (iii) H2NOH‚HCl, TEA, EtOH,
20 °C.
Treatment of kenpaullone with phosphorus pentasul-
fide led to the corresponding thiolactam 18, as was
reported previously for the parent compound 4c.35
S-Alkylation of 18 with iodomethane furnished the
methylthioimidate 19, which gave the hydroxyamidine
20 upon nucleophilic exchange of methanethiol for
hydroxylamine (Scheme 5).
Test Resu lts a n d Discu ssion
All new paullones were tested in a CDK1/cyclin B
kinase inhibition assay described previously.15 For this

Synthesis and Properties of Paullones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2913
9-bromo-substituted compounds 4a , 4q, 4t, and 4w
showed higher activity compared to their unsubstituted
counterparts 4c, 4p , 4s, and 4v, respectively. The shift
of the bromo substituent from the 9-position in ken-
paullone (4a ) to the 10-position (4b), 11-position (4l),
or 2-position (4p ) resulted in decreased activity. The
kinase selectivity has been investigated in detail with
4a and its 10-bromo analogue 4b, with results showing
a considerable change in the inhibition pattern caused
by the formal shift of the substituent.23 Thus 4b, which
shows decreased inhibitory potency for CDK1, CDK2,
and CDK5 (IC50 values > 1 µM, respectively), inhibits
some protein kinase C isozymes in submicromolar
concentrations. In contrast, kenpaullone (4a ) does not
inhibit protein kinase C isozymes in concentrations up
to 100 µM.
Diminished CDK inhibitory activity was also found
with analogues, in which the 9-bromo substituent of
kenpaullone was replaced for moieties with +M or +I
effects (4f and 4g, respectively). Replacement of the
9-bromo substituent by a 9-trifluoromethyl group led to
paullones with similar CDK1 inhibitory activity (e.g.,
pairs 4a /4h , 4q/4r , and 4t/4u ). The introduction of
substituents into position 4 was clearly detrimental, for
4v, 4w , and 4y were 2-3 orders of magnitude less active
than kenpaullone. A comparison of the derivatives 4s,
4t, and 4u with the analogues 4c, 4a , and 4h shows
that a substitution with two methoxy groups in the 2-
and 3-positions is favorable regarding the CDK1/cyclin
B inhibition. The replacement of the 9-bromo substitu-
ent of kenpaullone with hydrophilic, electron-withdraw-
ing -M substituents resulted in improved CDK inhibi-
tory potency. Thus, the 9-cyano paullone 4i (IC50 ) 0.024
µM) and the 9-nitro paullone 4j (now named alster-
paullone, IC50 ) 0.035 µM) exhibited a more than 10-
fold higher potency compared to kenpaullone, flavopir-
idol, and roscovitine, respectively. However, 4i and 4j
are less potent than the purvalanols (purvalanol A, IC50
) 0.004 µM; purvalanol B, IC50 ) 0.006 µM17). None of
the kenpaullone derivatives with substitution at either
the lactam or the indole nitrogen atoms were superior
to the lead structure with respect to the CDK1 inhibition
(derivatives 12-17e, Table 2). The introduction of bulky
Boc groups into the kenpaullone scaffold resulted in
dramatically decreased CDK1 inhibitory activity (ana-
logues 12-15). The thiolactam 18, the thioimidate 19,
and the hydroxyamidine 20 also were inferior to the lead
structure as CDK1 inhibitors.
For the calculation of the log MG_MID values, insensi-
tive cell lines are included with the highest test con-
centration.
In Tables 1 and 2, the antiproliferative activities of
the test compounds are given as log GI50 values both in
terms of results on the colon cancer cell line HCT-116
and the MG_MID parameter. The HCT-116 results were
selected, because this cell line was particularly sensitive
to the growth inhibitory activity of kenpaullone (4a ). A
similar selectivity for HCT-116 cells was observed with
several paullones (4b-e, 4h -j, 4t, 4u , 4w , see Table
1). The results of the 9-trifluoromethyl paullones 4h ,
4r , and 4u closely resembled the results for the corre-
sponding 9-bromo derivatives 4a , 4q, and 4t. This
observation is in accordance with the equivalence of the
9-bromo and 9-trifluoromethyl substituents with regard
to CDK1 inhibition. Both 2,3-dimethoxy-substituted
derivatives 4t and 4u exhibited interesting in vitro
antitumor potency with GI50 values in the low-micro-
molar range. Alsterpaullone (4j) was the only compound
from this study exhibiting GI50 activity in the submi-
cromolar concentration range (log MG_MID GI50
)
-6.40), representing a 100-fold improvement over ken-
paullone. In contrast, the 9-cyano paullone 4i was
devoid of noteworthy in vitro antitumor activity, al-
though it was more potent as CDK1 inhibitor compared
to alsterpaullone. The reason for the marked difference
between 4i and 4j with respect to antitumor activity is
not yet clear. Interestingly, all members of the 2-bromo-
substituted series of compounds (4p -r ) are further
examples exhibiting reasonable CDK1 inhibitory activ-
ity without showing in vitro antitumor potency. In
contrast, the 12-Boc-substituted paullone 15 as well as
the methylthioimidate 19, which both are weak CDK1
inhibitors only, gave promising results in the antitumor
cell line screening with GI50 concentrations between 1
and 10 µM. Apparently, the CDK1/cyclin B inhibition
is not likely to be the crucial biological property deter-
mining the antiproliferative activity of the latter deriva-
tives. Considering the structural similarity of the ATP
binding site in different kinases, it may be speculated
that the antitumor activity of the paullone derivatives
15 and 19 is due to the inhibition of other kinases
involved in cell division. For a further rational develop-
ment in the paullone series, inhibition studies on a
broad array of kinases with representative derivatives
would be helpful.
Con clu sion s
Furthermore, the new paullones were tested in the
NCIs in vitro anticancer drug discovery screen to
evaluate their antiproliferative activity. The ratio-
nale,36,37 applications,21,38-41 and experimental proce-
dures42-44 of this cell line-based screening program have
been described in detail. As a result of this screening,
the antitumor activity of a test compound is reported
for each out of 60 human tumor cell lines. For every
cell line, the following parameters are calculated: log
GI50 (GI50, molar concentration inhibiting 50% net cell
growth), log TGI (TGI, molar concentration for total
inhibition of net cell growth), and log LC50 (LC50, molar
concentration leading to 50% net cell death). Averaged
values, designated as mean graph midpoints (log
MG_MID), are calculated for each of the three param-
eters by averaging the log parameters of all cell lines.
The present structure-activity relationship study
revealed that electron-withdrawing substituents in the
9-position as well as a 2,3-dimethoxy substitution on the
paullone basic scaffold are favorable with respect to the
CDK1/cyclin B inhibitory activity. A 9-trifluoromethyl
substituent was found to be equivalent to a 9-bromo
substituent, whereas introduction of a 9-cyano or 9-nitro
group produced a substantial increase in enzyme-
inhibiting potency. Substitutions in the 4-, 5-, and 12-
positions as well as replacement of the lactam structure
for a thiolactam, a thioimidate, or a hydroxyamidine led
to decreased CDK1 inhibition. Evaluation of the new
paullones in a cancer cell line screening showed that
the antiproliferative activity is not necessarily paral-
leling the CDK inhibitory potency. Hence, CDK1 inhibi-

2914 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Schultz et al.
chloride (3764 mg, 25 mmol) (purchased from Aldrich) in
toluene (5 mL) was added dropwise to a solution of 2-amino-
4,5-dimethoxybenzoic acid methyl ester (purchased from Flu-
ka) (4224 mg, 20 mmol) and pyridine (2.3 mL) in toluene (30
mL) with stirring and cooling. The resulting suspension was
refluxed for 2 h. After the mixture cooled to room temperature,
water (2 mL) was added. The mixture was then poured into a
separating funnel, and a sufficient amount of dichloromethane
was added to ensure a plain separation of the organic and
aqueous layers. The organic layer was separated, washed
successively with 10% hydrochloric acid (10 mL) and 5%
aqueous sodium carbonate solution, dried over sodium sulfate,
and evaporated. The residue was crystallized from ethanol to
yield 87% colorless crystals: mp 115 °C; IR 3220 (NH), 1710,
1655 cm-1 (CdO); 1H NMR (400 MHz) 2.63-2.68 (m, 4H, CH2-
CH2), 3.61 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 7.39 (s, 1H), 8.13 (s, 1H), 10.81 (br
s, 1H, NH). Anal. (C15H19NO7) C, H, N.
tory paullones with a bromo substitution in the 2-posi-
tion (4p -r ) and the 9-cyano paullone 4i were devoid of
noteworthy in vitro antitumor activity. The 12-substi-
tuted paullone 15 and the methylthioimidate 19 exhib-
ited reasonable antitumor activity, although both com-
pounds turned out to be poor CDK1 inhibitors. The
9-nitro paullone (4j, alsterpaullone) exceeded the CDK1
inhibitory potency of both flavopiridol and roscovitine
and exhibited a remarkable in vitro antitumor activity.
Therefore, alsterpaullone was selected for preclinical
development in a NCI program. Future studies will be
directed to the design of new paullones incorporating a
combination of substituents with beneficial effects.
Furthermore, the biochemical mechanism underlying
the antitumor activity of paullones with poor CDK
inhibitory potency remains to be investigated.
7,8-Dim eth oxy-5-h ydr oxy-2-oxo-2,3-dih ydr o-1H-[1]ben z-
a zep in e-4-ca r boxylic Acid Meth yl Ester . A solution of 4,5-
dimethoxy-2-[(4-methoxy-1,4-dioxobutyl)amino]benzoic acid
methyl ester (650 mg, 2 mmol) and N,N-dimethylformamide
(1 mL) in toluene (12 mL) was added dropwise to a stirred
suspension of powdered potassium hydride (400 mg, 10 mmol)
in toluene (4 mL) with cooling and under nitrogen. [Be
ca u tiou s w h en h a n d lin g p ota ssiu m h yd r id e! Ca r efu lly
k eep a w a y w a ter or m oistu r e!] After the hydrogen evolu-
tion had ceased, the mixture was stirred for 3 h at 80 °C under
nitrogen. The mixture was cooled to room temperature, and
successively acetic acid (0.6 mL) and water (6 mL) were added
dropwise and with ca u tion under nitrogen. The mixture was
chilled in an ice bath and stirred for 15 min. The precipitate
was filtered off with suction, washed with water and hexanes
and crystallized from ethanol/toluene to yield 29% yellowish
crystals: mp 256 °C dec; IR 3180 (NH), 1665, 1640 cm-1 (Cd
Exp er im en ta l Section
Syn th etic Ch em istr y. Melting points were determined on
an electric variable heater (Gallenkamp) and are not corrected.
Elemental analyses were performed in the analytical depart-
ment of the Institut fu¨r Pharmazie, Universita¨t Hamburg.
Results obtained were within (0.4% unless indicated other-
wise. Infrared spectra were recorded using KBr pellets on a
Pye-Unicam SP 3-200 S, a Philips PU 9712, or a Perkin-Elmer
1660 FTIR spectrometer, respectively. Nuclear magnetic reso-
nance spectra were recorded on a Bruker AMX 400 instrument,
using dimethyl sulfoxide-d6 as solvent and tetramethylsilane
as internal standard. NMR signals are reported in ppm on a
δ scale. TLC analyses were carried out on fluorescent Polygram
Sil G/UV254 silica gel plates. Spots were visualized under 254-
nm UV illumination. The following compounds were prepared
according to literature methods: 1H-[1]benzazepine-2,5(3H,4H)-
dione,33 7-bromo-1H-[1]benzazepine-2,5(3H,4H)-dione,33 and
4a -d , 4k , 4p .22
Syn th esis of 7,12-Dih yd r oin d olo[3,2-d ][1]ben za zep in -
6(5H)-on es, Gen er a l P r oced u r e A. To a slurry of an
appropriate 1H-[1]benzazepine-2,5(3H,4H)-dione (1 mmol) in
glacial acetic acid (2 mL) was added a suspension of the
appropriate corresponding phenylhydrazine (1.5 mmol) [or the
appropriate substituted phenylhydrazine hydrochloride (1.5
mmol) and sodium acetate (123 mg, 1.5 mmol)] in glacial acetic
acid (5 mL) dropwise with stirring. After stirring at 70 °C for
1 h the mixture was cooled to room temperature. Concentrated
sulfuric acid (0.1 mL) was added, and stirring was continued
for 1 h at 70 °C. After cooling to room temperature, the mixture
was poured into a 5% aqueous sodium acetate solution (15 mL).
A precipitate was formed, which was filtered off with suction
and purified by crystallization from the given solvent.
Syn th esis of P h en ols by Clea va ge of Meth oxy Com -
p ou n d s, Gen er a l P r oced u r e B. Boron tribromide (1002 mg,
4 mmol) was added to a solution of the appropriate methoxy
compound (1 mmol) in dichloromethane (10 mL). The mixture
was stirred by means of a magnetic stirrer, and the reaction
was monitored by thin-layer chromatography (silica gel, eluent
acetone/toluene, 1:1). When the spot caused by the starting
methoxy compound was no longer detectable, water (10 mL)
was added and the mixture stirred for 1 h. A solid formed,
which was filtered off with suction, washed with water, and
crystallized for purification.
1
O); H NMR (400 MHz) 2.91 (s, 2H, CH2), 3.79 and 3.80 (2 s,
6 H, OCH3, overlapping signals), 3.82 (s, 3H, OCH3), 6.76 (s,
1H), 7.22 (s, 1H), 10.08 (s, 1H, NH), 12.54 (br s, 1H, OH). Anal.
(C14H15NO6) C, H, N.
7,8-Dim eth oxy-1H-[1]ben za zep in e-2,5(3H,4H)-d ion e. A
solution of 2,3-dihydro-7,8-dimethoxy-5-hydroxy-2-oxo-1H-[1]-
benzazepine-4-carboxylic acid methyl ester (293 mg, 1 mmol)
and water (0.5 mL) in dimethyl sulfoxide (10 mL) was stirred
at 150 °C under nitrogen. Portions of water (0.5 mL) were
added after 1 and 3 h of heating, respectively. After stirring
for an overall 4 h at 150 °C, the mixture was allowed to cool
to room temperature and then poured into water (20 mL). After
standing for 12 h at 4 °C, crystals formed, which were filtered
off and washed with water and hexanes. The material so
obtained was analytically pure and used without further
purification: yellowish crystals (90%), mp 234 °C (lit.45 mp 183
°C dec); IR 3230 (NH), 1660, 1640 cm-1 (CdO); 1H NMR (400
MHz) 2.60-2.85 (m, 4H, CH2-CH2), 3.76 (s, 3H, OCH3), 3.80
(s, 3H, OCH3), 6.79 (s, 1H), 7.32 (s, 1H), 9.86 (s, 1H, NH). Anal.
(C12H13NO4) C, H, N.
9-Flu or o-7,12-dih ydr oin dolo[3,2-d][1]ben zazepin -6(5H)-
on e (4e). Prepared according to general procedure A. Purifica-
tion by column chromatography (6-cm column of silica gel 60A,
100-200 mesh, eluent dichloromethane) yielded 52% cream-
colored powder: mp > 330 °C (shrinking starting at 180 °C);
1
IR 3220 (NH), 1635 cm-1 (CdO); H NMR (400 MHz) 3.50 (s,
2H, CH2), 7.00 (d “t“, 1H, 2.5/9.2/9.2 Hz), 7.23-7.31 (m, 2H),
7.35-7.44 (m, 2H), 7.48 (dd, 1H, 2.5/9.7 Hz), 7.73 (dd, 1H, 1.5/
7.6 Hz), 10.08 (s, 1H, lactam-NH), 11.67 (s, 1H, indole-NH).
Anal. (C16H11FN2O); C, H, N.
Syn th esis of 12-Su bstitu ted 9-Br om o-7,12-d ih yd r oin -
d olo[3,2-d ][1]ben za zep in -6(5H)-on es by Rea ction w ith
Alk yl Ha lid es, Gen er a l P r oced u r e C. Powdered potassium
hydroxide (56 mg, 1 mmol) was added with stirring and cooling
by an ice bath to a solution of 4a (327 mg, 1 mmol) in dry
acetone (120 mL). After the mixture stirred for 1 h at 0 °C,
the appropriate alkyl halide (10 mmol) was added and stirring
continued for 3 days at room temperature. After addition of
water (120 mL) a solid formed, which was filtered off and
crystallized from ethanol/toluene.
9-Meth oxy-7,12-d ih yd r oin d olo[3,2-d ][1]ben za zep in -6-
(5H)-on e (4f). Prepared according to general procedure A.
Purification by column chromatography (6-cm column of silica
gel 60A, 100-200 mesh, eluent dichloromethane) yielded 48%
cream-colored powder: mp > 330 °C (shrinking starting at 290
°C); IR 3200 (NH), 1640 cm-1 (CdO); 1H NMR (400 MHz) 3.49
(s, 2H, CH2), 3.80 (s, 3H, OCH3), 6.81 (dd, 1H, 2.0/8.6 Hz), 7.17
(d, 1H, 2.5 Hz), 7.22-7.28 (m, 2H), 7.30-7.38 (m, 2H), 7.72
4,5-Dim et h oxy-2-[(4-m et h oxy-1,4-d ioxob u t yl)a m in o]-
ben zoic Acid Meth yl Ester . A solution of methyl succinyl

Synthesis and Properties of Paullones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2915
(dd, 1H, 7.6 Hz), 10.04 (s, 1H, lactam-NH), 11.38 (s, 1H, indole-
NH). Anal. (C17H14N2O2) C, H, N.
5.9/1.1 Hz, 1H), 7.86 (dd, 6.2/1.1 Hz, 1H), 10.05 (s, 1H, NH),
11.25 (s, 1H, NH). Anal. (C18H16N2O) C, H, N.
8,10-Dich lor o-7,12-d ih yd r oin d olo[3,2-d ][1]ben za zep in -
6(5H)-on e (4o). Prepared according to general procedure A
to yield 55% colorless crystals: mp > 330 °C (ethanol/toluene);
9-Meth yl-7,12-dih ydr oin dolo[3,2-d][1]ben zazepin -6(5H)-
on e (4g). Prepared according to general procedure A to yield
59% cream-colored crystals: mp > 330 °C (ethanol); IR 3220
(NH), 1640 cm-1 (CdO); 1H NMR (400 MHz) 2.41 (s, 3H, CH3),
3.46 (s, 2H, CH2), 7.00 (dd, 1H, 1.0/8.1 Hz), 7.22-7.38 (m, 4H),
7.43 (s, 1H), 7.73 (d, 1H, 6.9 Hz), 10.05 (s, 1H, lactam-NH),
11.42 (s, 1H, indole-NH). Anal. (C17H14N2O) C, H, N.
9-(Tr iflu or om eth yl)-7,12-d ih yd r oin d olo[3,2-d ][1]ben z-
a zep in -6(5H)-on e (4h ). Prepared according to general pro-
cedure A to yield 33% cream-colored crystals: mp > 330 °C
(ethanol); IR 3200 (NH), 1650 cm-1 (CdO); 1H NMR (400 MHz)
3.61 (s, 2H, CH2), 7.27-7.32 (m, 2H), 7.40-7.47 (m, 2H), 7.62
(d, 1H, 8.6 Hz), 7.78 (dd, 1H, 1.5/7.6 Hz), 8.13 (s, 1H), 10.15
1
IR 3280 (NH), 1650 cm-1 (CdO); H NMR (400 MHz) 3.80 (s,
2H, CH2), 7.18 (d, 1H, 1.5 Hz), 7.27 (d, 1H, 6.6 Hz), 7.31 (t,
1H, 6.1 Hz), 7.42-7.44 (m, 1H), 7.45 (d, 1H, 1.2 Hz), 7.73-
7.75 (m, 1H), 10.19 (s, 1H, lactam-NH), 12.17 (s, 1H, indole-
NH). Anal. (C16H10Cl2N2O) C, H, Cl, N.
2,9-Dibr om o-7,12-d ih yd r oin d olo[3,2-d ][1]ben za zep in -
6(5H)-on e (4q). Prepared according to general procedure A
to yield 41% brown crystals: mp > 330 °C (ethanol/toluene);
IR 3200 (NH), 1640 cm-1 (CdO); 1H NMR (400 MHz) 3.55 (br
s, 2H, CH2), 7.20 (d, 1H, 8.6 Hz), 7.30 (dd, 1H, 1.5/8.6 Hz),
7.40 (d, 1H, 8.6 Hz), 7.56 (dd, 1H, 2.5/8.6 Hz), 7.92 (d, 1H, 2.6
Hz), 7.93 (d, 1H, 2.0 Hz), 10.20 (s, 1H, lactam-NH), 11.89 (s,
1H, indole-NH). Anal. (C16H10Br2N2O) C, H, Br, N.
(s, 1H, lactam-NH), 12.06 (s, 1H, indole-NH). Anal. (C17H11
FN2O) C, H, N.
-
9-Cyan o-7,12-dih ydr oin dolo[3,2-d][1]ben zazepin -6(5H)-
on e (4i). A mixture of 4a (327 mg, 1 mmol) and copper(I)
cyanide (180 mg, 2 mmol) in N-methyl-2-pyrrolidone (10 mL)
was refluxed for 2 h. After the mixture cooled to room
temperature, water (10 mL) was added. The precipitate was
filtered off with suction and washed with water. The precipi-
tate was then suspended in a mixture of water (10 mL) and
1,2-diaminoethane (25 mL). After stirring for 15 min, the solid
was filtered off with suction, washed twice with 10% aqueous
sodium cyanide solution, and crystallized twice from ethanol/
toluene to yield 74% colorless crystals: mp > 330 °C; IR 3350,
3180 (NH), 2200 (CN), 1670 cm-1 (CdO); 1H NMR (400 MHz)
3.59 (s, 2H, CH2), 7.27-7.32 (m, 2H), 7.43 (dt, 1H, 1.0/7.6 Hz),
7.51 (dd, 1H, 1.3/8.4 Hz), 7.59 (d, 1H, 8.1 Hz), 7.76 (dd, 1H,
1.0/7.6 Hz), 8.32 (s, 1H), 10.16 (s, 1H, lactam-NH), 12.19 (s,
1H, indole-NH). Anal. (C17H11N3O) C, H, N.
9-Nitr o-7,12-d ih yd r oin d olo[3,2-d ][1]ben za zep in -6(5H)-
on e (Alster p a u llon e, 4j). A mixture of 1H-[1]benzazepine-
2,5(3H,4H)-dione (175 mg, 1 mmol), 4-nitrophenylhydrazine
hydrochloride (284 mg, 1.5 mmol), and sodium acetate (123
mg, 1.5 mmol) in glacial acetic acid (10 mL) was stirred at 70
°C. After 1 h sulfuric acid (0.1 mL) was added. While stirring
was continued for 3 h at 70 °C, additional portions of
concentrated sulfuric acid (0.1 mL, respectively) were added
after each hour of stirring. The mixture was cooled to room
temperature and poured into 5% sodium acetate solution (20
mL). A precipitate was formed, which was filtered off with
suction, washed with water, and crystallized from ethanol/
toluene to yield 33% yellow crystals: mp > 330 °C; IR 3380
(NH), 1660 cm-1 (CdO); 1H NMR (400 MHz) 3.65 (s, 2H,
azepine-CH2), 7.29-7.34 (m, 2H), 7.43-7.47 (m, 1H), 7.60 (d,
9.2 Hz, 1H), 7.77-7.79 (m, 1H), 8.08 (dd, 8.6/2.0 Hz, 1H), 8.74
(d, 2.0 Hz, 1H), 10.22 (s, 1H, NH), 12.39 (s, 1H, NH). Anal.
(C16H11N3O3) C, H, N.
11-Br om o-7,12-d ih yd r oin d olo[3,2-d ][1]b en za zep in -6-
(5H)-on e (4l). Prepared according to general procedure A
yielding 58% yellow crystals: mp > 330 °C (ethanol); IR 3190
(NH), 1640 cm-1 (CdO); 1H NMR (400 MHz) 3.49 (s, 2H, CH2),
7.04 (“t”, 1H, 7.9 Hz), 7.22-7.31 (m, 2H), 7.40 (d, 2H, 7.6 Hz),
7.71 (d, 1H, 7.6 Hz), 7.91 (dd, 1H, 1.5/8.1 Hz), 10.10 (s, 1H,
lactam-NH), 11.58 (s, 1H, indole-NH). Anal. (C16H11BrN2O) H,
N; C: calcd, 58.74; found 58.18. Br: calcd, 24.42; found, 23.70.
11-Met h yl-7,12-d ih yd r oin d olo[3,2-d ][1]b en za zep in -6-
(5H)-on e (4m ). Prepared according to general procedure A
yielding 42% beige crystals: mp > 330 °C (ethanol); IR 3200
(NH), 1640 cm-1 (CdO); 1H NMR (400 MHz) 2.54 (s, 3H, CH3),
3.47 (s, 2H, azepine-CH2), 6.95-7.01 (m, 2H), 7.24-7.30 (m,
2H), 7.37 (dt, 7.6/1.5 Hz), 7.47 (dd, 6.9/1.3 Hz, 1H), 7.86 (dd,
7.6/1.5 Hz, 1H), 10.07 (s, 1H, NH), 11.26 (s, 1H, NH). Anal.
(C17H14N2O) H, N; C: calcd, 77.84; found, 77.21.
2-Br om o-9-(tr iflu or om eth yl)-7,12-d ih yd r oin d olo[3,2-d ]-
[1]ben za zep in -6(5H)-on e (4r ). Prepared according to general
procedure A, yielding 51% colorless crystals: mp > 330 °C
1
(ethanol/toluene); IR 3300 (NH), 1635 cm-1 (CdO); H NMR
(400 MHz) 3.64 (s, 2H, CH2), 7.23 (d, 1H, 9.2 Hz), 7.48 (dd,
1H, 1.0/8.7 Hz), 7.58-7.63 (m, 2H), 7.95 (d, 1H, 2.0 Hz), 8.15
(s, 1H), 10.25 (s, 1H, lactam-NH), 12.15 (s, 1H, indole-NH).
Anal. (C17H10BrF3N2O) C, H, Br, N.
2,3-Dim eth oxy-7,12-dih ydr oin dolo[3,2-d][1]ben zazepin -
6(5H)-on e (4s). Prepared according to general procedure A,
yielding 73% cream-colored crystals: mp > 330 °C (ethanol/
toluene); IR 3350, 3220 (NH), 1660 cm-1 (CdO); 1H NMR (400
MHz) 3.44 (s, 2H, CH2), 3.79 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
6.87 (s, 1H), 7.04-7.08 (m, 1H), 7.13-7.17 (m, 1H), 7.28 (s,
1H), 7.43 (d, 1H, 8.2 Hz), 7.62 (d, 1H, 7.6 Hz), 9.79 (s, 1H,
lactam-NH), 11.46 (s, 1H, indole-NH). Anal. (C18H16N2O3) C,
H, N.
9-Br om o-2,3-d im et h oxy-7,12-d ih yd r oin d olo[3,2-d ][1]-
ben za zep in -6(5H)-on e (4t). Prepared according to general
procedure A from 7,8-dimethoxy-1H-[1]benzazepine-2,5(3H,4H)-
dione, yielding 55% red-brown crystals: mp > 330 °C (ethanol/
toluene); IR 3340, 3210 (NH), 1660 cm-1 (CdO); 1H NMR (400
MHz) 3.45 (s, 2H, CH2), 3.80 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
6.87 (s, 1H), 7.23-7.27 (m, 2H), 7.39 (d, 1H, 8.6 Hz), 7.86 (d,
1H, 2.0 Hz), 9.83 (s, 1H, lactam-NH), 11.70 (s, 1H, indole-NH).
Anal. (C18H15BrN2O3) C, H, Br, N.
2,3-Dim eth oxy-9-(tr iflu or om eth yl)-7,12-d ih yd r oin d olo-
[3,2-d ][1]ben za zep in -6(5H)-on e (4u ). Prepared according to
general procedure A yielding 38% pale-yellow crystals: mp >
1
330 °C (ethanol); IR 3240 (NH), 1635 cm-1 (CdO); H NMR
(400 MHz) 3.55 (s, 2H, CH2), 3.81 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 6.90 (s, 1H), 7.31 (s, 1H), 7.43 (dd, 1H, 1.0/8.6 Hz),
7.61 (d, 1H, 8.6 Hz), 8.08 (s, 1H), 9.87 (s, 1H, lactam-NH), 11.96
(s, 1H, indole-NH). Anal. (C19H15F3N2O3) C, H, N.
4-Meth oxy-7,12-d ih yd r oin d olo[3,2-d ][1]ben za zep in -6-
(5H)-on e (4v). Prepared according to general procedure A
yielding 47% beige crystals: mp 285 °C (ethanol/toluene); IR
1
3380, 3250 (NH), 1640 cm-1 (CdO); H NMR (400 MHz) 3.48
(br s, 2H, CH2), 3.90 (s, 3H, OCH3), 7.05-7.10 (m, 2H), 7.15-
7.19 (m, 1H), 7.27 (t, 1H, 8.1 Hz), 7.35 (dd, 1H, 1.3/7.9 Hz),
7.44 (d, 1H, 7.6 Hz), 7.66 (d, 1H, 8.1 Hz), 8.77 (s, 1H, lactam-
NH), 11.58 (s, 1H, indole-NH). Anal. (C17H14N2O2) C, H, N.
9-Br om o-4-m eth oxy-7,12-d ih yd r oin d olo[3,2-d ][1]ben z-
a zep in -6(5H)-on e (4w ). Prepared according to general pro-
cedure A, yielding 67% brownish crystals: mp > 330 °C
1
(ethanol/toluene); IR 3300 (NH), 1650 cm-1 (CdO); H NMR
(400 MHz) 3.49 (s, 2H, CH2), 3.90 (s, 3H, OCH3), 7.12 (dd, 1H,
1.3/7.9 Hz), 7.27-7.35 (m, 3H), 7.40 (d, 1H, 8.6 Hz), 7.92 (d,
1H, 2.0 Hz), 8.84 (s, 1H, lactam-NH), 11.82 (s, 1H, indole-NH).
Anal. (C17H13BrN2O2) C, H, Br, N.
9-Br om o-2,3-d ih yd r oxy-7,12-d ih yd r oin d olo[3,2-d ][1]-
ben za zep in -6(5H)-on e (4x). Prepared according to general
procedure B from 4t, reaction time 1 h, yielding 30% colorless
crystals: mp > 330 °C (ethanol/toluene); IR 3400 (OH), 3260
(NH), 1610 cm-1 (CdO); 1H NMR (400 MHz) 3.39 (s, 2H, CH2),
6.68 (s, 1H), 7.06 (s, 1H), 7.20 (dd, 1H, 1.5/8.6 Hz), 7.33 (d,
11-Eth yl-7,12-dih ydr oin dolo[3,2-d][1]ben zazepin -6(5H)-
on e (4n ). Prepared according to general procedure A yielding
49% beige-yellow crystals: mp > 330 °C (ethyl acetate); IR
1
3250/3210 (NH), 1640 cm-1 (CdO); H NMR (400 MHz) 1.29
(t, 5.9 Hz, 3H, CH3), 2.96 (q, 6.0 Hz, 2H, CH2), 3.47 (s, 2H,
azepine-CH2), 6.99-7.03 (m, 2H), 7.25 (dd, 6.3/0.8 Hz, 1H),
7.28 (dt, 6.0/0.8 Hz, 1H), 7.37 (dt, 6.1/1.0 Hz, 1H), 7.47 (dd,

2916 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Schultz et al.
1H, 8.7 Hz), 7.79 (d, 1H, 1.5 Hz), 9.03 (s, 1H, OH), 9.52 (s, 1H,
OH), 9.69 (s, 1H, lactam-NH), 11.55 (s, 1H, indole-NH). Anal.
(C16H11BrN2O3) C, H, N; Br: calcd, 22.25; found, 21.75.
mL). Water (0.5 mL) was added, and the mixture was stirred
under nitrogen at 150 °C. After 1 and 3 h of stirring, portions
of water (0.5 mL, respectively) were added. After stirring for
an overall 4 h at 150 °C, the mixture was allowed to cool to
room temperature, and water (20 mL) was added. The mixture
was extracted three times with dichloromethane (portions of
20 mL). The combined organic layers were then washed with
water (20 mL), dried over sodium sulfate, and evaporated to
yield 87% beige crystals: mp 99 °C; IR 3220 (NH), 1645 cm-1
(CdO); 1H NMR (400 MHz) 2.65-2.94 (m, 4H, CH2-CH2), 3.87
(s, 3H, OCH3), 7.19 (t, 1H, 8.1 Hz), 7.29 (dd, 1H, 1.5/8.1 Hz),
7.32 (dd, 1H, 1.5/7.6 Hz), 8.73 (s, 1H, NH). Anal. (C11H11NO3)
C, H, N.
9-Br om o-4-h yd r oxy-7,12-d ih yd r oin d olo[3,2-d ][1]ben z-
a zep in -6(5H)-on e (4y). Prepared according to general pro-
cedure B from 4w (357 mg, 1 mmol), reaction time 24 h,
yielding 35% colorless crystals: mp > 330 °C (ethanol); IR
1
3420, 3180 (NH), 1615 cm-1 (CdO); H NMR (400 MHz) 3.49
(s, 2H, CH2), 6.94 (dd, 1H, 1.3/7.9 Hz), 7.12 (t, 1H, 7.9 Hz),
7.20 (dd, 1H, 1.3/7.9 Hz), 7.26 (dd, 1H, 2.0/8.7 Hz), 7.39 (d,
1H, 8.7 Hz), 7.90 (d, 1H, 2.0 Hz), 8.57 (s, 1H, lactam-NH), 10.23
(br s, 1H, OH), 11.74 (s, 1H, indole-NH). Anal. (C16H11BrN2O2)
C, H, Br, N.
9-Br om o-6-oxo-5,6,7,12-tetr ah ydr oin dolo[3,2-d][1]ben z-
a zep in e-5,7-d ica r boxylic Acid Di-ter t-bu tyl Ester (12). A
solution of 4a (327 mg, 1 mmol) in THF (40 mL) was refluxed
with sodium hydride (48 mg, 2 mmol, 60% suspension in white
oil) for 1 h. Di-tert-butyl dicarbonate (436 mg, 2 mmol) was
added, and refluxing was continued for 12 h. After cooling to
room temperature the mixture was poured on ice water (100
mL). A precipitate was formed, which consisted of a mixture
of unreacted starting material, a side product, and the desired
product. Repeated crystallization from ethanol afforded the
title compound as colorless crystals in 25% yield: mp > 330
°C (dec starting at 285 °C); IR 3260 (NH), 1755, 1680 cm-1
2-Am in o-3-m et h oxyb en zoic Acid E t h yl E st er (8). 2-
Amino-3-methoxybenzoic acid (167 mg, 1 mmol) (purchased
from Aldrich) was dissolved in dry ethanol (7 mL). Gaseous
hydrogen chloride was bubbled through the mixture, until 3 g
of hydrogen chloride was absorbed (requires approximately 20
min). The mixture was refluxed for 5 h and subsequently
evaporated to dryness. The residue was dissolved in water (20
mL), and the solution was neutralized by addition of 5%
sodium acetate solution. The neutral solution was extracted
five times with dichloromethane (each portion 10 mL). The
combined organic layers were washed with 5% aqueous sodium
carbonate solution (20 mL), dried by means of sodium sulfate,
and evaporated to dryness to yield 81% colorless crystals,
which were used without further purification: mp 40-41 °C,
IR 3480, 3360 (NH), 1680 cm-1 (CdO); 1H NMR (400 MHz)
1.30 (t, 3H, 7.1 Hz, CH2-CH3), 3.82 (s, 3H, OCH3), 4.26 (q,
2H, 7.1 Hz, CH2-CH3), 6.32 (br s, 2H, NH2), 6.52 (t, 1H, 8.1
1
(CdO); H NMR (400 MHz) 1.28 (br s, 9H, C(CH3)3), 1.45 (s,
9H, C(CH3)3), 6.82 (br s, 1H, azepine-CH), 7.32-7.37 (m, 2H),
7.44 (d, 1H, 8.6 Hz), 7.51-7.59 (m, 2H), 7.81 (dd, 1H, 1.5/7.6
Hz), 7.95 (d, 1H, 1.5 Hz), 12.15 (s, 1H, NH). Anal. (C26H27
BrN2O5) C, H, Br, N.
-
Hz), 6.98 (d, 1H, 7.6 Hz), 7.35 (d, 1H, 8.6 Hz). Anal. (C10H13
NO3) C, H, N.
-
9-Br om o-6-oxo-5,6,7,12-tetr ah ydr oin dolo[3,2-d][1]ben z-
a zep in e-5,12-d ica r boxylic Acid Di-ter t-bu tyl Ester (13).
A solution of 4a (327 mg, 1 mmol), (dimethylamino)pyridine
(DMAP) (122 mg, 1 mmol), and di-tert-butyl dicarbonate (655
mg, 3 mmol) in dry dichloromethane (50 mL) was stirred at
room temperature for 4 h. The mixture was then evaporated
to dryness, and the residue was crystallized from ethanol to
yield 85% colorless crystals: mp 196 °C; IR 1770, 1725, 1680
2-[(4-Eth oxy-1,4-d ioxobu tyl)a m in o]-3-m eth oxyben zo-
ic Acid Eth yl Ester (10). A solution of ethyl succinyl chloride
(9) (2633 mg, 16 mmol) (purchased from Aldrich) in toluene
(2 mL) was added dropwise to a stirred solution of 2-amino-
3-methoxybenzoic acid ethyl ester (2340 mg, 12 mmol) and
pyridine (1.4 mL) in toluene (5 mL) at 0-10 °C. After the
resulting suspension stirred for 1.5 h below 10 °C, water (2
mL) was added. The resulting organic layer was separated
using a separating funnel, washed with 10% hydrochloric acid
(2 mL) and 5% aqueous sodium carbonate solution (2 mL),
dried by means of sodium sulfate, and evaporated. After the
oily residue was heated with ethanol (2 mL), a crystalline solid
formed, which was crystallized from ethanol to yield 59%
colorless crystals: mp 59-60 °C; IR 3250 (NH), 1710, 1690,
1650 cm-1 (CdO); 1H NMR (400 MHz) 1.18 (t, 3H, 7.1 Hz,
CH2-CH3), 1.23 (t, 3H, 7.1 Hz, CH2-CH3), 2.50 (t, 2H, CH2-
CH2, overlapping the DMSO signal), 2.59 (t, 2H, 6.6 Hz, CH2-
CH2), 3.82 (s, 3H, OCH3), 4.04 (q, 2H, 7.1 Hz, CH2-CH3), 4.14
(q, 2H, 7.1 Hz, CH2-CH3), 7.20-7.25 (m, 3H), 9.49 (s, 1H, NH).
Anal. (C16H21NO6) C, H, N.
5-H yd r oxy-9-m et h oxy-2-oxo-2,3-d ih yd r o-1H -[1]b en z-
a zep in e-4-ca r boxylic Acid Eth yl Ester (11). 10 (323 mg,
1 mmol) was dissolved in a mixture of N,N-dimethylformamide
(0.44 mL) and toluene (2 mL). The solution was added
dropwise to a stirred suspension of powdered potassium
hydride (0.2 g, 5 mmol) in toluene (2 mL) under nitrogen with
cooling. [Be ca u tiou s w h en h a n d lin g p ota ssiu m h yd r id e!
Ca r efu lly k eep a w a y w a ter or m oistu r e!] After the hy-
drogen evolution had ceased, the mixture was stirred under
nitrogen for 1.5 h at 80 °C. Subsequently, the mixture was
neutralized by ca u tiou s addition of acetic acid (0.3 mL). Water
(3 mL) was then added dropwise, and the organic layer was
separated by means of a separating funnel. The organic layer
was evaporated, and the residue was crystallized from ethanol,
yielding 66% yellow crystals: mp 124 °C; IR 3160 (NH), 1650,
1625 cm-1 (CdO); 1H NMR (400 MHz) 1.31 (t, 3H, 7.1 Hz,
CH2-CH3), 2.91 (s, 2H, azepine-CH2), 3.87 (s, 3H, OCH3), 4.30
(q, 2H, 7.1 Hz, CH2-CH3), 7.22-7.28 (m, 2H), 7.37 (dd, 1H,
2.5/6.6 Hz), 9.23 (s, 1H, NH), 12.53 (br s, 1H, OH). Anal.
(C14H15NO5) C, H, N.
1
cm-1 (CdO); H NMR (400 MHz) 1.36 (s, 9H, C(CH3)3), 1.37
(s, 9H, C(CH3)3), 3.04 (d, 1H, 14.2 Hz, azepine-CH), 4.02 (d,
1H, 14.2 Hz, azepine-CH), 7.40-7.43 (m, 1H), 7.44-7.51 (m,
2H), 7.57 (dd, 1H, 2.0/9.1 Hz), 7.63 (dd, 1H, 1.8/7.4 Hz), 8.12-
8.14 (m, 2H). Anal. (C26H27BrN2O5) C, H, Br, N.
9-Br om o-6-oxo-5,6,7,12-tetr ah ydr oin dolo[3,2-d][1]ben z-
a zep in e-5,7,12-tr ica r boxylic Acid Tr i-ter t-bu tyl Ester
(14). A solution of 4a (327 mg, 1 mmol) in THF (40 mL) was
refluxed with sodium hydride (72 mg, 3 mmol, 60% suspension
in white oil) for 1 h. Di-tert-butyl dicarbonate (655 mg, 3 mmol)
was added, and refluxing was continued for 2 h. After the
mixture cooled to room temperature, water (100 mL) was
added. The mixture was extracted three times with dichlo-
romethane (20 mL each portion). The combined organic layers
were dried with sodium sulfate and evaporated to furnish a
residue, which was crystallized from ethanol to yield 48%
colorless crystals: mp 193 °C; IR 1760, 1715, 1660 cm-1 (Cd
1
O); H NMR (400 MHz) 1.30 (br s, 9H, C(CH3)3), 1.36 (s, 9H,
C(CH3)3), 1.47 (s, 9H, C(CH3)3), 6.87 (br s, 1H, azepine-CH),
7.35 (d, 1H, 8.1 Hz), 7.44-7.49 (m, 2H), 7.55-7.62 (m, 2H),
8.06-8.08 (m, 2H). Anal. (C31H35BrN2O7) C, H, Br, N.
9-Br om o-6-oxo-5,6,7,12-tetr ah ydr oin dolo[3,2-d][1]ben z-
a zep in e-12-ca r boxylic Acid ter t-Bu tyl Ester (15). Trifluo-
roacetic acid (0.25 mL, 3.25 mmol) was added dropwise to a
solution of 13 (527 mg, 1 mmol) in dry dichloromethane (20
mL). After the mixture stirred for 2 h at room temperature,
water (40 mL) was added and the mixture was neutralized by
addition of concentrated aqueous ammonia. The organic layer
was separated by means of a separating funnel, subsequently
dried with sodium sulfate, and evaporated to yield a solid,
which upon crystallization from ethanol furnished 60% color-
less crystals: mp > 330 °C; IR 3150 (NH), 1730, 1660 cm-1
1
(CdO); H NMR (400 MHz) 1.37 (s, 9H, C(CH3)3), 3.18 (br s,
1H, azepine-CH, overlapping the H2O signal), 3.66 (br s, 1H,
azepine-CH, overlapping the H2O signal), 7.23-7.27 (m, 2H),
7.38-7.43 (m, 1H), 7.49 (dd, 1H, 1.3/7.9 Hz), 7.54 (dd, 1H, 2.0/
9-Met h oxy-1H -[1]b en za zep in e-2,5(3H ,4H )-d ion e (5a ).
11 (277 mg, 1 mmol) was dissolved in dimethyl sulfoxide (10

Synthesis and Properties of Paullones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2917
1
9.2 Hz), 8.04 (d, 1H, 8.6 Hz), 8.08 (d, 1H, 1.5 Hz), 10.14 (s,
1H, NH). Anal. (C21H19BrN2O3) C, H, Br, N.
IR 3170 (NH), 1665 cm-1 (CdO); H NMR (400 MHz) 3.08-
3.99 (very broad signal, 2H, CH2, overlapping the H2O signal)
3.84 (s, 3H, CH3), 7.30-7.33 (m, 2H), 7.36 (dd, 1H, 1.6/6.9 Hz),
7.43-7.47 (m, 1H), 7.54 (d, 1H, 7.1 Hz), 7.74 (dd, 1H, 1.1/6.4
Hz), 7.95 (d, 1H, 1.5 Hz), 10.06 (s, 1H, NH). Anal. (C17H13
BrN2O) C, H, N.
9-Br om o-5-m et h yl-7,12-d ih yd r oin d olo[3,2-d ][1]b en z-
a zep in -6(5H)-on e (16a ). Sodium hydride (24 mg, 1 mmol,
60% suspension in white oil) was added to a solution of 4a
(327 mg, 1 mmol) in THF (25 mL). After the mixture refluxed
for 45 min, iodomethane (71 mg, 0.5 mmol) was added and
refluxing was continued. After 2 and 4 h portions of iodo-
methane (71 mg, 0.5 mmol, respectively) were added. After
refluxing for an overall 8 h, the mixture was poured into ice
water (50 mL). The precipitate which formed was filtered off
with suction, washed with water, and crystallized from ethanol
to yield 46% colorless crystals: mp 319 °C; IR 3250 (NH), 1630
cm-1 (CdO); 1H NMR (400 MHz) 3.25 (s, 3H, CH3), 3.00-3.95
(br s, 2H, CH2), 7.28 (dd, 1H, 2.0/8.7 Hz), 7.37-7.43 (m, 2H),
7.48-7.52 (m, 1H), 7.60 (dd, 1H, 1.0/8.1 Hz), 7.72 (dd, 1H, 1.3/
7.9 Hz), 7.92 (d, 1H, 1.5 Hz), 11.90 (s, 1H, indole-NH). Anal.
(C17H13BrN2O) C, H, Br, N.
9-Br om o-5-eth yl-7,12-d ih yd r oin d olo[3,2-d ][1]ben za ze-
p in -6(5H)-on e (16b). Sodium hydride (40 mg of a 60% sus-
pension in white oil, corresponding to 24 mg, 1 mmol, NaH)
was added to a solution of 4a (327 mg, 1 mmol) in THF (25
mL). After the mixture refluxed for 45 min, bromoethane (327
mg, 3 mmol) was added and refluxing was continued for 24 h.
The mixture was then cooled to room temperature and poured
into ice water (50 mL). A precipitate formed, which was filtered
off with suction, washed with water, and crystallized twice
from ethanol to yield 29% colorless crystals, mp 292 °C; IR
3260 (NH), 1630 cm-1 (CdO); 1H NMR (400 MHz) 0.89 (t, 6.9
Hz, 3H, CH3), 2.97 (br. s, 1H, azepine-CH), 3.74 and 3.93 (2
br s, together 3H, CH2 and azepine-CH, overlapping signals),
7.28 (dd, 8.6/1.5 Hz, 1H), 7.40-7.42 (m, 2H), 7.47-7.52 (m,
1H), 7.66 (d, 8.1 Hz, 1H), 7.73 (dd, 7.6/1.5 Hz, 1H), 7.91 (d, 1.5
Hz, 1H), 11.91 (s, 1H, NH). Anal. (C18H15BrN2O) C, H, Br, N.
5-Ben zyl-9-b r om o-7,12-d ih yd r oin d olo[3,2-d ][1]b en z-
a zep in -6(5H)-on e (16c). Sodium hydride (24 mg, 1 mmol,
60% suspension in white oil) was added to a solution of 4a
(327 mg, 1 mmol) in THF (25 mL). After the mixture refluxed
for 45 min, a solution of benzyl bromide (257 mg, 1.5 mmol)
in THF (5 mL) was added and refluxing was continued for 8
h. After the mixture cooled to room temperature, water (100
mL) was added and the mixture was extracted four times with
portions of dichloromethane (20 mL each). The combined
organic layers were dried with sodium sulfate and evaporated.
The residue was crystallized from ethanol to furnish 57%
yellowish crystals: mp 248 °C; IR 3300 (NH), 1640 cm-1 (Cd
O); 1H NMR (400 MHz) 3.20 (br s, 1H, azepine-CH, overlapping
the H2O signal), 4.01 (br s, 1H, azepine-CH), 5.08 (br s, 2H,
benzyl-CH2), 6.89-6.91 (m, 2H), 7.09-7.16 (m, 3H), 7.30 (dd,
1H, 1.5/8.7 Hz), 7.34 (d, 1H, 7.6 Hz), 7.39 (dd, 1H, 1.5/8.2 Hz),
7.43 (d, 1H, 8.6 Hz), 7.59 (d, 1H, 8.1 Hz), 7.67 (dd, 1H, 1.5/7.6
Hz), 7.96 (s, 1H), 11.93 (s, 1H, indole-NH). Anal. (C23H17BrN2O)
C, H, Br, N.
-
9-Br om o-12-eth yl-7,12-d ih ydr oin d olo[3,2-d ][1]ben za ze-
p in -6(5H)-on e (17b). Prepared according to general procedure
C employing iodoethane (1560 mg, 10 mmol) to furnish 24%
colorless crystals: mp 298 °C (ethanol/toluene); IR 3180 (NH),
1660 cm-1 (CdO); 1H NMR (400 MHz) 1.24 (t, 3H, 5.7 Hz,
CH2-CH3), 3.05 (br s, 1H, azepine-CH), 3.75 (br s, 1H, azepine-
CH), 4.33 (q, 2H, 5.5 Hz, CH2-CH3), 7.32-7.36 (m, 3H), 7.44-
7.47 (m, 1H), 7.58 (d, 1H, 7.0 Hz), 7.67-7.69 (m, 1H), 7.95 (d,
1H, 1.5 Hz), 10.03 (s, 1H, NH). Anal. (C18H15BrN2O); C, H, N;
Br: calcd, 22.49; found, 22.07.
9-Br om o-12-(2-p r op en yl)-7,12-d ih yd r oin d olo[3,2-d ][1]-
ben za zep in -6(5H)-on e (17c). Prepared according to general
procedure C employing allyl bromide (1210 mg, 10 mmol) to
furnish 34% colorless crystals: mp 318 °C dec (ethanol/
toluene); IR 3170 (NH), 1660 cm-1 (CdO); 1H NMR (400 MHz)
3.13 (br s, 1H, azepine-CH, overlapping the water signal), 3.78
(br s, 1H, azepine-CH), 4.80 (dd, 1H, 1.3/17.2 Hz), 4.89 (m,
2H, allyl-CH2), 5.17 (dd, 1H, 1.3/10.5 Hz), 6.07 (ddt, 1H, 4.3/
10.5/17.2 Hz), 7.28-7.32 (m, 2H), 7.35 (dd, 1H, 1.7/8.7 Hz),
7.42-7.46 (m, 2H), 7.62-7.64 (m, 1H), 7.98 (d, 1H, 1.6 Hz),
10.06 (s, 1H, NH). Anal. (C19H15BrN2O) C, H, Br, N.
(9-Br om o-6-oxo-5,6,7,12-tetr ah ydr oin dolo[3,2-d][1]ben z-
a zep in -12-yl)a cetic Acid Meth yl Ester (17d ). Prepared
according to general procedure C from bromoacetic acid ethyl
ester (1530 mg, 10 mmol), yielding 27% colorless crystals from
ethanol: mp 328 °C dec; IR 3220 (NH), 1745, 1665 cm-1 (Cd
1
O); H NMR (400 MHz) 3.20 (br s, 1H, azepine-CH), 3.79 (br
s, 1H, azepine-CH), 3.66 (s, 3H, OCH3), 5.16 (br s, 2H, CH2-
COOCH3), 7.27-7.32 (m, 2H), 7.36 (dd, 1H, 1.8/8.9 Hz), 7.43-
7.45 (m, 1H), 7.47-7.50 (m, 2H), 7.98 (d, 1H, 2.0 Hz), 10.08
(s, 1H, NH). Anal. (C19H15BrN2O3) C, H, Br, N.
9-Br om o-12-(2-h yd r oxyeth yl)-7,12-d ih yd r oin d olo[3,2-
d ][1]ben za zep in -6(5H)-on e (17e). A solution of 17d (399 mg,
1 mmol) in THF (80 mL) was added dropwise to a stirred
suspension of lithium alumino hydride (19 mg, 0.5 mmol).
After the addition was complete, the mixture was refluxed for
2 h. An additional portion of lithium alumino hydride was
added, and refluxing was continued for 1 h. After the mixture
cooled to room temperature, water was cautiously added until
the hydrogen evolution was finished. A precipitate of alumi-
num hydroxide was redissolved by dropwise addition of 25%
sulfuric acid. The solution was extracted twice with dichlo-
romethane (20 mL, each portion). The combined organic layers
were dried with sodium sulfate and evaporated. The residue
was crystallized from ethanol to yield 48% colorless crystals:
mp 267 °C; IR 3420, 3340 (OH), 3260 (NH), 1650 cm-1 (CdO);
1H NMR (400 MHz) 3.05 (br s, 1H, azepine-CH), 3.35 (br s,
1H, azepine-CH, overlapping the H2O signal), 3.68-3.75 (br
m, 2H, CH2-N), 4.32-4.35 (m, 2H, O-CH2), 5.02 (t, 1H, 5.3
Hz, OH), 7.29-7.35 (m, 3H), 7.42-7.46 (m, 1H), 7.59 (d, 1H,
8.6 Hz), 7.94 (d, 1H, 2.0 Hz), 7.97 (d, 1H, 7.5 Hz), 10.01 (s,
1H, NH). Anal. (C18H15BrN2O2) C, H, Br, N.
(9-Br om o-6-oxo-5,6,7,12-tetr ah ydr oin dolo[3,2-d][1]ben z-
a zep in -5-yl)a cetic Acid Meth yl Ester (16d ). A solution of
4a (327 mg, 1 mmol) in THF (35 mL) was refluxed with sodium
hydride (24 mg, 1 mmol, 60% suspension in white oil) for 1.5
h. Bromoacetic acid ethyl ester (153 mg, 1 mmol) was added,
and refluxing was continued for 5 h. After the mixture cooled
to room temperature, water (50 mL) was added. The mixture
was extracted three times with dichloromethane (20 mL,
respectively). The combined organic layers were dried with
sodium sulfate and evaporated to furnish a residue, which was
crystallized from ethanol to yield 46% colorless crystals: mp
9-Br om o-7,12-d ih yd r oin d olo[3,2-d ][1]b en za zep in e-6-
(5H)-th ion e (18). A solution of 4a (327 mg, 1 mmol) in THF
(30 mL) was stirred under nitrogen at 50 °C. Phosphorus
pentasulfide (250 mg, 1.12 mmol) and sodium hydrogen
carbonate (370 mg, 4.4 mmol) were added successively. After
refluxing for 3 h under nitrogen, the mixture was allowed to
cool to room temperature and then poured onto crushed ice
(50 g). The mixture was then stirred until the ice was molten,
and the precipitate which had formed was filtered off with
suction, washed with water, and crystallized from ethanol/
toluene yielding 67% pale-yellow crystals: mp > 330 °C; IR
3430, 3140 cm-1 (NH); 1H NMR (400 MHz) 3.91 (s, 2H, CH2),
7.30 (dd, 1H, 1.5/8.6 Hz), 7.39-7.45 (m, 4H), 7.79 (d, 1H, 7.1
Hz), 7.86 (d, 1H, 1.5 Hz), 11.92 (s, 1H, NH), 12.07(s, 1H, NH).
Anal. (C16H11BrN2S) C, H, Br, N, S.
1
240 °C; IR 3340 (NH), 1750, 1655 cm-1 (CdO); H NMR (400
MHz) 3.10 (br s, 1H, azepine-CH), 3.63 (s, 3H, COOCH3), 3.94
(br s, 1H, azepine-CH), 4.43 (br s, 2H, CH2COOCH3), 7.29 (dd,
1H, 1.8/8.4 Hz), 7.40-7.43 (m, 2H), 7.46-7.52 (m, 2H), 7.72-
7.74 (m, 1H), 7.93 (d, 1H, 1.5 Hz), 11.94 (s, 1H, NH). Anal.
(C19H15BrN2O3) C, H, Br, N.
9-Br om o-12-m eth yl-7,12-d ih yd r oin d olo[3,2-d ][1]ben z-
a zep in -6(5H)-on e (17a ). Prepared according to general pro-
cedure C employing iodomethane (1420 mg, 10 mmol) to
furnish 28% yellowish crystals: mp 313 °C (ethanol/toluene);

2918 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Schultz et al.
9-Br om o-6-(m eth ylth io)-7,12-d ih yd r oin d olo[3,2-d ][1]-
ben za zep in e (19). Sodium hydride (24 mg, 1 mmol, 60%
suspension in white oil) was added to a solution of 18 (343
mg, 1 mmol) in THF (20 mL). After refluxing for 1 h with
stirring under nitrogen, the mixture was cooled to room
temperature and a solution of iodomethane (170 mg, 1.2 mmol)
in THF (2 mL) was added. After further refluxing for 2 h, the
mixture was cooled to room temperature and poured into ice
water (150 mL). After stirring for 15 min, the precipitate was
filtered off with suction, washed with water, and crystallized
from ethanol to yield 44% colorless crystals: mp 199 °C; IR
3420 (NH), 1615 cm-1 (CdN); 1H NMR (400 MHz) 2.35 (s, 3H,
CH3), 3.51 (s, 2H, CH2), 7.26-7.32 (m, 2H), 7.36-7.43 (m, 3H),
7.80-7.82 (m, 1H), 7.97 (d, 1H, 1.5 Hz), 11.82 (s, 1H, NH).
Anal. (C17H13BrN2S) C, H, Br, N, S.
Refer en ces
(1) Morgan, D. Cyclin-dependent kinases: engines, clocks, and
microprocessors. Annu. Rev. Cell Dev. Biol. 1997, 13, 261-291.
(2) Draetta, G.; Pagano, M. Cell cycle control and cancer. Annu. Rep.
Med. Chem. 1996, 31, 241-248.
(3) Hartwell, L. H.; Kastan, M. B. Cell cycle control and cancer.
Science 1994, 266, 1821-1828.
(4) Hunter, T.; Pines, J . Cyclins and cancer. II: Cyclin D and CDK
inhibitors come of age. Cell 1994, 79, 573-582.
(5) Sherr, C. J . Cancer cell cycles. Science 1996, 274, 1672-1677.
(6) Meijer, L. Chemical inhibitors of cyclin-dependent kinases. In
Progress in Cell Cycle Research; Meijer, L., Guidet, S., Tung, H.
Y. L., Eds.; Plenum Press: New York, 1995; Vol. 1, pp 351-
363.
(7) Meijer, L. Chemical inhibitors of cyclin-dependent kinases.
Trends Cell Biol. 1996, 6, 393-397.
(8) Meijer, L.; Kim, S.-H. Chemical inhibitors of cyclin-dependent
kinases. Methods Enzymol. 1997, 283, 113-128.
9-Br om o-6-(h yd r oxya m in o)-7,12-d ih yd r oin d olo[3,2-d ]-
[1]ben za zep in e (20). Triethylamine (151 mg, 1.5 mmol) in
ethanol (20 mL) was added to a solution of 19 (357 mg, 1 mmol)
and hydroxylamine hydrochloride (104 mg, 1.5 mmol) in
ethanol (40 mL). After the mixture stirred for 8 h at 20 °C,
water (30 mL) was added. A precipitate formed, which was
filtered off with suction and crystallized from ethanol to yield
83% colorless crystals: mp 233 °C dec; IR 3350 cm-1 (NH),
(9) Coleman, K. G.; Lyssikatos, J . P.; Ynag, B. V. Chemical inhibitors
of cyclin-dependent kinases. Annu. Rep. Med. Chem. 1997, 32,
171-179.
(10) Yamamoto, H.; Monden, T.; Miyoshi, H.; Izawa, H.; Ikeda, K.;
Tsujie, M.; Ohnishi, T.; Sekimoto, M.; Tomita, N.; Monden, M.
Cdk2/cdc2 expression in colon carcinogenesis and effects of cdk2/
cdc2 inhibitor in colon cancer cells. Int. J . Oncol. 1998, 13, 233-
239.
(11) Wada, M.; Hosotani, R.; Lee, J .-U.; Doi, R.; Koshiba, T.; Fujimoto,
K.; Miyamoto, Y.; Tsuji, S.; Nakajima, S.; Okuyama, A.; Ima-
mura, M. An exogenous cdk inhibitor, butyrolactone-I, induces
apoptosis with increased Bax/Bcl-2 ratio in p53-mutated pan-
creatic cancer cells. Anticancer Res. 1998, 18, 2559-2566.
(12) Sedlacek, H. H.; Czech, J .; Naik, R.; Kaur, G.; Worland, P.;
Losiewicz, M.; Parker, B.; Carlson, B.; Smith, A.; Senderowicz,
A.; Sausville, E. Flavopiridol (L86-8275, NSC-649890), a new
kinase inhibitor for tumor therapy. Int. J . Oncol. 1996, 9, 1143-
1168.
(13) Senderowicz, A. M.; Headlee, D.; Stinson, S. F.; Lush, R. M.;
Kalil, N.; Villalba, L.; Hill, K.; Steinberg, S. M.; Figg, W. D.;
Tompkins, A.; Arbuck, S. G.; Sausville, E. A. Phase I trial of
continuous infusion flavopiridol, a novel cyclin-dependent kinase
inhibitor, in patients with refractory neoplasms. J . Clin. Oncol.
1998, 16, 2986-2999.
(14) Vesely, J .; Havlicek, L.; Strnad, M.; Blow, J . J .; Donella-Deana,
A.; Pinna, L.; Letham, D. S.; Kato, J .-Y.; Detivaud, L.; Leclerc,
S.; Meijer, L. Inhibition of cyclin-dependent kinases by purine
analogues. Eur. J . Biochem. 1994, 224, 771-786.
(15) Meijer, L.; Borgne, A.; Mulner, O.; Chong, J . P. J .; Blow, J . J .;
Inagaki, N.; Inagaki, M.; Delcros, J .-G.; Moulinoux, J .-P. Bio-
chemical and cellular effects of roscovitine, a potent and selective
inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.
Eur. J . Biochem. 1997, 243, 527-536.
(16) Havlicek, L.; Hanus, J .; Vesely, J .; Leclerc, S.; Meijer, L.; Shaw,
G.; Strnad, M. Cytokinin-derived cyclin-dependent kinase inhibi-
tors: Synthesis and cdc2 inhibitory activity of olomoucine and
related compounds. J . Med. Chem. 1997, 40, 408-412.
(17) Gray, N. S.; Wodicka, L.; Thunnissen, A.-M. W. H.; Norman, T.
C.; Kwon, S.; Espinoza, F. H.; Morgan, D. O.; Barnes, G.; Leclerc,
S.; Meijer, L.; Kim, S.-H.; Lockhart, D. J .; Schultz, P. G.
Exploiting chemical libraries, structure, and genomics in the
search of kinase inhibitors. Science 1998, 281, 533-538.
(18) De Azevedo, W. F.; Leclerc, S.; Meijer, L.; Havlicek, L.; Strnad,
M.; Kim, S.-H. Inhibition of cyclin-dependent kinases by purine
analogues. Crystal structure of human cdk2 complexed with
roscovitine. Eur. J . Biochem. 1997, 243, 518-526.
(19) Schulze-Gahmen, U.; Brandsen, J .; J ones, H. D.; Morgan, D. O.;
Meijer, L.; Vesely, J .; Kim, S.-H. Multiple modes of ligand
recognition: crystal structures of cyclin-dependent protein ki-
nase 2 in complex with ATP and two inhibitors, olomoucine and
isopentenyladenine. Proteins: Struct., Funct., Genet. 1995, 22,
378-391.
1
1635 cm-1 (CdN); H NMR (400 MHz) 3.48 (s, 2H, azepine-
CH2), 7.11-7.14 (m, 1H, 7.22-7.30 (m, 2H), 7.35-7.39 (m, 2H),
7.61-7.63 (m, 1H), 7.81 (d, 1.5 Hz, 1H), 8.34 (s, 1H, OH), 9.52
(s, 1H, NH), 11.65 (s, 1H, NH). Anal. (C16H12BrN3O) C, H, Br,
N.
Biologica l Eva lu a tion : p 34cd c2/Histon e H1 Kin a se As-
sa y. P34cdc2/cyclin B was purified from M phase oocytes of the
starfish Marthasterias glacialis by affinity chromatography on
p9CKhs1-Sepharose beads,15 from which it was eluted by free
p9CKhs1. The CDC2 assay mixture (final volume: 30 µL)
contained 0.5-1 µL of purified enzyme, 5 µL of histone H1 (5
mg/mL), 5 µL of [γ-32P]ATP (15 µM, 3000 Ci/mmol, 1 mCi/mL),
and 3 µL of the inhibitor (0.1-1000 µM), all in reaction buffer
C (60 mM â-glycerolphosphate, 15 mM p-nitrophenyl phos-
phate, 25 mM MOPS, pH 7.2, 5 mM EGTA, 15 mM MgCl2, 1
mM dithiothreitol, 1 mM sodium vanadate, 1 mM phenyl
phosphate, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/
mL soybean trypsin inhibitor, 100 µM benzamidine). For
determination of maximum phosphate incorporation, buffer C
was used instead of inhibitor. Nonspecific binding was deter-
mined in the absence of histone H1 in the reaction mixture
and subtracted from each volume. The assays were started
by addition of radioactive ATP, and after 10-min incubation
at 30 °C, 25-µL aliquots of the supernatant were spotted onto
2.5 × 3.0 pieces of Whatman P81 phosphocellulose paper. After
20 s, the filters were washed five times (for at least 5 min each
time) in 0.1% phosphoric acid. The wet filters were transferred
into 1 mL of ACS scintillation cocktail (Amersham), and after
mixing, 32P radioactivity was determined using a Packard Tri-
Carb counter. Control analyses were also performed with
appropriate dilutions of DMSO because the inhibitors were
dissolved in DMSO as 100 mM stock solutions. However, the
final DMSO concentration in the reaction mixture never
exceeded 1%. The kinase activity is expressed as pmol of
phosphate groups incorporated in histone H1 during a 10-min
incubation or in percent of the maximal kinase activity. Dose-
response curves were drawn for every compound tested and
used for calculating IC50s. All assays were carried out in
triplicate.
(20) Kim, S.-H. Structure-based inhibitor design for CDK2, a cell cycle
controlling protein kinase. Pure Appl. Chem. 1998, 70, 555-565.
(21) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,
D. A.; Rubinstein, L.; Plowman, J .; Boyd, M. R. Display and
analysis of patterns of differential activity of drugs against
human tumor cell lines: development of meangraph and COM-
PARE algorithm. J . Natl. Cancer Inst. 1989, 81, 1088-1092.
(22) Kunick, C. Synthese von 7,12-Dihydro-indolo[3,2-d][1]benzazepin-
6-(5H)-onen und 6, 11-Dihydro-thieno-[3′,2′:2,3]azepino[4,5-b]-
indol-5(4H)-on. Arch. Pharm. (Weinheim) 1992, 325, 297-299.
(23) Zaharevitz, D. W.; Gussio, R.; Leost, M.; Senderowicz, A.;
Lahusen, T.; Kunick, C.; Meijer, L.; Sausville, E. A. Discovery
and initial characterization of the paullones, a novel class of
small-molecule inhibitors of cyclin-dependent kinases. Cancer
Res., in press. A presentation of the molecular modeling results
and methods included in this paper is accessible via the Internet
from the following web site: Zaharevitz, D. W.; Gussio, R.; Leost,
Ack n ow led gm en t. We thank the fishermen of the
Station Biologique de Roscoff for collecting the starfish.
This research was supported by grants from the As-
sociation pour la Recherche sur le Cancer (ARC 9314)
(to L.M.) and the Conseil Re´gional de Bretagne (to L.M.)
and by a CNRS/NCI collaboration contract. Computer
time and software were provided, in part, by the
Advanced Biomedical Computing Center, Frederick,
MD.

Synthesis and Properties of Paullones
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2919
M.; Senderowicz, A.; Lahusen, T.; Kunick, C.; Meijer, L.; Saus-
ville, E. A. Model of kenpaullone (NSC-664704) fit in the ATP
site of CDK2. URL: http://dtp.nci.nih.gov/Docs/Branches/Itb/
tsddg/paullones/paullone.html.
(38) Boyd, M. R.; Paull, K. D.; Rubinstein, L. R. Data display and
analysis strategies for the NCI disease-oriented in vitro anti-
tumor drug screen. In Developments in Oncology. Twenty-Second
Annual Cancer Symposium 1990, Detroit, Michigan; Valeriote,
F. A., Corbett, T. H., Baker, L. H., Eds.; Kluwer Academic
Publishers: Boston, 1992; pp 11-34.
(39) Bates, S. E.; Fojo, A. T.; Weinstein, J . N.; Myers, T. G.; Alvarez,
M.; Paull, K. D.; Chabner, B. A. Molecular targets in the
National Cancer Institute drug screen. J . Cancer Res. Clin.
Oncol. 1995, 121, 495-500.
(40) Weinstein, J . N.; Myers, T. G.; O’Connor, P. M.; Friend, S. H.;
Fornace, A. J ., J r.; Kohn, K. W.; Fojo, T.; Bates, S. E.; Rubinstein,
L. V.; Anderson, N. L.; Buolamwini, J . K.; van Osdol, W. W.;
Monks, A. P.; Scudiero, D. A.; Sausville, E. A.; Zaharevitz, D.
W.; Bunow, B.; Viswanadhan, V. N.; J ohnson, G. S.; Wittes, R.
E.; Paull, K. D. An information-intensive approach to the
molecular pharmacology of cancer. Science 1997, 275, 343-349.
(41) Boyd, M. R.; Paull, K. D. Some practical considerations and
applications of the National Cancer Institute in vitro anticancer
drug discovery screen. Drug Dev. Res. 1995, 34, 91-109.
(42) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer drug screen-
ing. J . Natl. Cancer Inst. 1990, 82, 1107-1112.
(43) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.;
Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, A.; Boyd, M. R.
Comparison of in vitro anticancer drug-screening data generated
with a tetrazolium assay versus a protein assay against a diverse
panel of human tumor cell lines. J . Natl. Cancer Inst. 1990, 82,
1113-1118.
(24) Schulze-Gahmen, U.; De Bondt, H. L.; Kim, S.-H. High-resolution
crystal structures of human cyclin-dependent kinase 2 with and
without ATP: bound waters and natural ligand as guides for
inhibitor design. J . Med. Chem. 1996, 39, 4540-4546.
(25) Witte, J .; Boekelheide, V. Stereoselective syntheses of isoqui-
nuclidones. II. J . Org. Chem. 1972, 37, 2849-2853.
(26) Rivett, D. E.; Stewart, F. H. C. Formation of substituted
1-benzazepine-2,5-diones. Aust. J . Chem. 1978, 31, 439-443.
(27) Rees, A. H. Azatropolones. J . Chem. Soc. 1959, 3111-3116.
(28) Moriconi, E. J .; Maniscalco, I. A. π-Equivalent heterocyclic
congeners of tropone. Azatropones. J . Org. Chem. 1972, 37, 208-
215.
(29) J ones, G. Some basic and acidic derivatives of 2,5-dihydro-1H-
1-benzazepine as potential therapeutic agents. J . Chem. Soc.
1967, 1808-1813.
(30) J ames, D. M.; Rees, A. H. A chemical and pharmacological study
of some compounds derived from 3,4-xylidine. J . Med. Pharm.
Chem. 1962, 5, 1234-1239.
(31) Chen, W.-Y.; Gilman, N. W. Synthesis of 7-phenylpyrimido[5,4-
d][1]benzazepin-2-ones. J . Heterocycl. Chem. 1983, 20, 663-666.
(32) Rossi, S.; Micheli, M.; Giannotti, M.; Salvatori, A.; Peruzzi, G.
Benzocondensed heptaatomic heterocycles and their derivatives.
VII. Synthesis of benzazepines and benzazepinones analogues
of known anxiolytic drugs. Gazz. Chim. Ital. 1981, 11, 365-370.
(33) Kunick, C. Synthese [b]-kondensierter Azepindione durch De-
alkoxycarbonylierung. Arch. Pharm. (Weinheim) 1991, 324, 579-
581.
(44) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J .; Cronise, P.; Vaigro-Wolff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J .; Boyd, M. Feasibility
of a high-flux anticancer drug screen using a diverse panel of
cultured human tumor cell lines. J . Natl. Cancer Inst. 1991, 83,
757-766.
(45) Haq, M. A.; Raˆy, J . N.; Tuffail-Malkana, M. Quinoline Deriva-
tives. Part III. â-2-Amino-4:5-dimethoxy-benzoylpropionic acid
and its derivatives. J . Chem. Soc. 1934, 1326-1328.
(34) Kunick, C.; Link, A.; Hropot, M. Synthesis and in vivo testing
of a potential aquaretic agent. Pharmazie 1996, 51, 601-602.
(35) Kunick, C. Synthese und konformative Flexibilita¨t von 4,9-
Dihydroindolo[3,2-d][1,2,4]triazolo[4,3-a]benzazepinen. Liebigs
Ann. Chem. 1993, 1141-1143.
(36) Monks, A.; Scudiero, D. A.; J ohnson, G. S.; Paull, K. D.; Sausville,
E. A. The NCI anti-cancer drug screen: a smart screen to
identify effectors of novel targets. Anti-Cancer Drug Des. 1997,
12, 533-541.
(37) Robert, J . Anticancer screening with in vitro models. Drugs
Future 1997, 22, 739-746.
J M9900570