Synthesis and antitumor activity of 4-aminomethylthioxanthenone and 5- aminomethylbenzothiopyranoindazole derivatives

Article abstract of DOI:10.1021/jm9708083

Two new series of antitumor agents, 4-aminomethylthioxanthenones (9-50) and 5-amino-methylbenzothiopyranoindazoles (51-61), are described and compared. Nearly all members of both series display excellent in vivo activity versus murine pancreatic adenocarcinoma 03 (Panc03) although there is little to distinguish the two series from each other. In both series there is no discernible relationship between structure and in vivo efficacy. Selected analogues were evaluated in vitro; all were observed to have moderate to strong DNA binding via intercalation. However, varying degrees of in vitro P388 cytotoxicity and topoisomerase II inhibition were seen. In general, those molecules which exhibited strong topoisomerase II inhibition were significantly more cytotoxic than those which did not. In both series, those derivatives (45-50, 60, and 61) having a phenolic hydroxy substitution exhibited the most potent P388 cytotoxicity and topoisomerase II inhibition.

Full text of DOI:10.1021/jm9708083

J . Med. Chem. 1998, 41, 3645-3654
3645
Syn th esis a n d An titu m or Activity of 4-Am in om eth ylth ioxa n th en on e a n d
5-Am in om eth ylben zoth iop yr a n oin d a zole Der iva tives
Robert B. Perni,*,† Mark P. Wentland,*,† J ianhua I. Huang,† Ronald G. Powles,† Suzanne Aldous,†
Kristina M. Klingbeil,‡ A. Danielle Peverly,‡ Ronald G. Robinson,‡ Thomas H. Corbett,| J ulie L. J ones,|
Kenneth C. Mattes,§ J ames B. Rake,‡ and Susan A. Coughlin‡
Departments of Medicinal Chemistry and Oncopharmacology, Sanofi Winthrop Inc., 9 Great Valley Parkway,
Malvern, Pennsylvania 19355, Department of Internal Medicine, Division of Hematology and Oncology,
Wayne State University, Detroit, Michigan 48202, and Eastman Kodak Company, Rochester, New York 14650
Received December 1, 1997
Two new series of antitumor agents, 4-aminomethylthioxanthenones (6-50) and 5-amino-
methylbenzothiopyranoindazoles (51-61), are described and compared. Nearly all members
of both series display excellent in vivo activity versus murine pancreatic adenocarcinoma 03
(Panc03) although there is little to distinguish the two series from each other. In both series
there is no discernible relationship between structure and in vivo efficacy. Selected analogues
were evaluated in vitro; all were observed to have moderate to strong DNA binding via
intercalation. However, varying degrees of in vitro P388 cytotoxicity and topoisomerase II
inhibition were seen. In general, those molecules which exhibited strong topoisomerase II
inhibition were significantly more cytotoxic than those which did not. In both series, those
derivatives (48-50, 60, and 61) having a phenolic hydroxy substitution exhibited the most
potent P388 cytotoxicity and topoisomerase II inhibition.
In tr od u ction
series incorporating a pyrazolo ring fusion (51-61).5
This structural modification was commonly used to
study structure-activity relationships of similar anti-
tumor series such as the benzothiopyranoindazole CI
958 (2)6 and the pyrazoloacridine PD 115934 (3).7 In
addition to showing good activity in murine tumor
models, the pyrazoloacridines (e.g., 3) also display se-
lectivity against solid tumor cell lines in vitro similar
to SR 233377.8 We now present comparative in vitro
and in vivo antitumor data for the novel 5-aminometh-
ylbenzothiopyranoindazoles 51-61 and the structurally
related 4-aminomethylthioxanthenones 6-50.
Numerous reasons have been offered for the lack of
significant advances in the treatment of most solid
neoplasms over the last 30 years.1 Part of the difficulty
may lie in the historical preference for carrying out
cytotoxic screening regimens in vivo or in vitro with
leukemia cell lines. A screening program designed to
identify compounds which are selectively toxic to solid
tumor cell lines, as opposed to leukemias or normal cell
lines, has the effect of searching a completely different
region of structure space than conventional (i.e., leuke-
mia L1210) screening would have provided. Assuming
some correlation between in vitro and in vivo activity,
the desired result is that positive responses would be
obtained for compounds which are effective versus solid
tumors in vivo and which could have been missed in
earlier screens.2
Using this strategy to screen in-house compound
libraries, we recently reported3 the curative antisolid
tumor activity of a series of 4-aminomethylthioxan-
thenone derivatives, related to the known antitumor
agent hycanthone (1).4 One member of this series, SR
233377 (WIN 33377, 12), a compound that displayed
broad spectrum murine solid tumor activity, has com-
pleted phase I clinical trials. In this paper we describe,
in detail, the synthesis and antitumor properties of a
novel series of compounds structurally similar to SR
233377 (12)3 and also discuss a new structurally related
Ch em istr y
* Address correspondence to R. B. Perni, Vertex Pharmaceuticals
Inc., 130 Waverly St., Cambridge, MA 02139, or M. P. Wentland,
Department of Chemistry, Rensselaer Polytechnic Institute, Troy, NY
12180.
The syntheses of tricyclic derivatives 6-50 were
achieved via elaboration of the 4-position of the thiox-
anthenone nucleus as shown in Schemes 1 and 2. The
known aldehydes 49 and 634b were converted, via
Leuckart reaction, to N-methylformamide derivatives
5 and 64, respectively, using N-methylformamide/formic
† Department of Medicinal Chemistry.
‡ Department of Oncopharmacology.
| Department of Internal Medicine.
§ Eastman Kodak Co.
S0022-2623(97)00808-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/20/1998

3646 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Perni et al.
Sch em e 1a
Sch em e 3a
a
(a) R4′SO2Cl, Et3N, CH2Cl2; (b) NaH, THF; (c) n-PrI.
a
Sch em e 4a
(a) HCONCH3, HCOOH; (b) HCONH2, HCOOH; (c) HCON-
HEt, HCOOH; (d) HCONHC6H5, HCOOH; (e) 2 N HCl.
Sch em e 2a
a
(a) p-Toluenesulfonyl chloride, pyridine; (b) sodium saccharin,
tetra-n-butylammonium bromide.
chlorides under the same conditions as for sulfonamides
12-20. Likewise, phosphoramide 30 was produced
from the reaction of 8 and diethylphosphoryl chloride.
Two methyl carbamate derivatives, 29 and 46, were
synthesized from 8 and 65, respectively, by treatment
with methyl chloroformate. Phthalimide 31 was also
obtained cleanly from 8 and phthalic anhydride.
N-Methylsulfonamide analogues 21, 24, and 25 were
prepared in a fashion similar to sulfonamides (12-20)
from 6, while 22 was synthesized from 10 (Scheme 3).
Alternatively, deprotonation of 12 with NaH followed
by quenching with n-PrI gave 23.
Demethylation of the 7-methoxy group of 43, 45, and
46 to give the corresponding phenols 48-50 was effected
via standard conditions (Scheme 2). Treatment of 45
and 46 with BBr3 in CH2Cl2 at -78 °C followed by
warming to room temperature resulted in the formation
of 49 and 50, respectively. When 43 was allowed to
react under these same conditions, no identifiable
products were obtained. Phenol 48 was obtained by
heating 43 at 110 °C in 48% HBr for 5 h.
In an effort to incorporate both a sulfonamide and an
amide at the 4′-position of the thioxanthenone, the
saccharin derivative 32 was assembled by SN2 displace-
ment of tosylate from 66 with sodium saccharin and
catalytic tetra-N-butylammonium bromide. The un-
stable tosylate 66 was readily prepared from tosyl
chloride and hycanthone (1) in pyridine (Scheme 4).
Syntheses of analogues where the length of the
1-position side chain was varied was accomplished using
the same standard procedures as were used for making
a
(a) R4′Cl, Et3N, CH2Cl2; (b) 48% HBr; (c) BBr3, CH2Cl2; (d)
phthalic anhydride.
acid at 160 °C (Scheme 1). Substituting formamide,
N-ethylformamide, and N-phenylformamide for N-meth-
ylformamide under identical conditions afforded 7, 9,
and 62, respectively (from 4), and 44 (from 63). Sub-
sequent hydrolysis of 5, 7, 9, 62, 64, and 44 with hot 2
N aqueous HCl afforded high yields of 6, 8,3 10, 11, 43,
and 65, respectively.
Treatment of 8 with a variety of sulfonyl chlorides
afforded 12-20 (Scheme 2) in generally good yields.
Analogously, the 7-methoxy derivative 65 was reacted
with methanesulfonyl and benzenesulfonyl chlorides to
give 45 and 47, respectively. Amide derivatives 26-
28 were prepared from 8 and the corresponding acid

Synthesis and Activity of New Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3647
Sch em e 5a
In Vivo P r op er ties
All compounds underwent in vivo antitumor evalua-
tion versus subcutaneously implanted murine pancre-
atic adenocarcinoma 03 (Panc03)2 regardless of their in
vitro profile versus topoisomerase II, P388, or as inter-
calators. Tumor growth inhibition as expressed as a
%T/C value was measured at the maximum tolerated
dose (MTD) for each compound dosed intravenously;
these data and a more detailed description of these and
other terms are found in Table 1. Two other measures
of antitumor efficacy, long-term cures (LTC) and the
clonogenic log cell kill (LCK), are also presented in Table
1. Long-term cures require a minimum of 86 tumor free
days and an LCK > 4.5. Within both series of com-
pounds (thioxanthenones 6-50 and benzothiopyranoin-
dazoles 51-61) in vivo activity versus Panc03 was
generally high with 32 of 57 compounds showing
complete tumor growth inhibition (%T/C ) 0). Long-
term cures were obtained in 27 of those 32 cases. In
general, this activity is similar to that of adriamycin
(%T/C ) 0, with 1 LTC out of 5 test mice) and superior
to that of hycanthone (%T/C ) 4, no cures). The MTDs
ranged from 18 mg/kg for 61 to 2594 mg/kg for 19. The
MTD, defined as the LD10 or less, did not correlate with
LTC or LCK in vivo. With the exception of phenolic
derivatives 48-50, 60, and 61, all other compounds
tested required a higher total dose (i.e., less potent) than
adriamycin and mAMSA, as measured by the MTD.
Except for 53, the most potent compounds (48-50, 52-
55, 60, and 61) (MTDs < 100) were not curative
although significant antitumor activity was observed.
The log cell kill ranged from <0.5 to 8.8 (cures were
excluded from this calculation). In general, cures
represent >4.5 log kill in this tumor system. Com-
pounds which were curative represent an improvement
over hycanthone (1) and mAMSA which were not
curative in this model. Within the entire series (6-61)
the range of %T/C values, MTDs, the number of LTCs,
or LCKs did not correlate with structural modifications.
(a) HCOO-NH4+, HCONHCH3; (b) NaOH, aq MeOH.
a
the (diethylamino)ethylthioxanthenones.3 For example,
the reaction of 67 (Scheme 6) with (diethylamino)-
propylamine or (dimethylamino)propylamine followed
by elaboration of the 4-position according to the pub-
lished route3 afforded 34 and 38, respectively. This
standard methodology failed, however, when an attempt
was made to convert 7410 to 39 (Scheme 5). In this
instance, Leuckart conditions resulted in the loss of side
chain leaving only NHCH3 at the 1-position. To cir-
cumvent this presumed acid-catalyzed result, the Leuck-
art reaction was run under neutral conditions with
tetra-N-butylammonium formate and methylforma-
mide. The desired formamide was obtained along with
a small amount of the dealkylated product. Subsequent
hydrolysis of the intermediate formamide afforded 39
(Scheme 5).
Three derivatives (40-42) with a 7-bromo substitu-
tion were prepared from the 7-bromo analogue of 4 using
methodology4 previously described (Scheme 1) for mak-
ing the corresponding 7-H compounds 6, 8, and 12,
respectively.
The benzothiopyranoindazole ring system, which can
be considered a rigid variant of the parent 1-ami-
nothioxanthenone derivatives, was assembled as shown
in Scheme 6. Condensation of the 2-chlorothioxan-
thenones 6710 and 684b with N,N-diethylaminoethylhy-
drazine11 in pyridine at 100 °C afforded 69 and 71,
respectively. Formation of the desired regioisomer is
indicated by analogous chemical shifts of the ring proton
at C-2 of 4 and the C-3 proton of 70. Elaboration of the
5-position of 69 and 71 was initiated by introduction of
a formyl group by two different methods. Formylation
of 69 to 70 was carried out using CHCl2OCH3 and AlCl3
followed by aqueous workup,12 while reaction of 71 via
a standard Vilsmeier reaction (POCl3/DMF) gave 72.
From this point on, the syntheses paralleled the prepa-
ration of the thioxanthenone targets previously de-
scribed in Scheme 1. A Leuckart reaction effected the
transformation of aldehydes 70 to 52 and 72 to 56 using
formamide and formic acid (vide supra). Using identical
methodology as for the preparation of 5, 70 was con-
verted to 73 with N-methylformamide and formic acid.
The resulting formamide derivatives 52, 56, and 73
were hydrolyzed in high yield to amines 53, 57, and 51,
respectively, under basic conditions (NaOH, CH3OH/
H2O). Treatment of 53 with methanesulfonyl chloride
(Et3N, CH2Cl2) gave sulfonamide 54. Similarly, 58 was
obtained from 57 in good yield. Treatment of 53 and
57 with methyl chloroformate (Et3N, CH2Cl2) gave
carbamates 55 and 59, respectively, in good yield.
Analogous to the preparation of 49 and 50, demethyla-
tion of the 7-methoxy substituents of both 58 and 59
was effected in moderate yield with BBr3 in CH2Cl2 to
give 60 and 61, respectively.
Substitution of the 4′-position nitrogen of the thiox-
anthenone ring system (6-50, Table 1) with a variety
of functional groups resulted in a large percentage of
compounds (26 of 47) displaying high activity (%T/C <
10) with 22 compounds providing curative activity.
Particularly effective (%T/C ) 0) were sulfonamides 12-
14 and 21-25; however, not all sulfonamides (e.g., 15,
16, and 18) were active. While there is no discernible
trend in the activity of the sulfonamide derivatives,
phenylsulfonamides having a para substituent other
than H (e.g., 15-18 and 20) were generally the least
active. Formamides 7, 9, and 44 were also highly active.
Basic amines 6, 8, and 10 were curative while 11 was
inactive. The inactivity of the NHPh derivative 11 is
probably not related to steric bulk of the phenyl ring
since benzenesulfonamide derivative 14 was highly
active. The known tertiary amine 3313 was inactive.
While the acetamide 26 was curative, the trifluoroac-
etamido derivative 27 and benzamide 28 displayed only
moderate activity (%T/C ) 21 and 26, respectively). A
methyl carbamate analogue, 29, was also highly active
and provided an exceptionally high LCK of 8.8. Since
both methylsulfonamide 12 and acetamide 26 displayed
excellent activity, a saccharin derivative (32) which
incorporates both an amide and a sulfonamide moiety

3648 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Perni et al.
Sch em e 6a
a
(a) H2NNHCH2CH2NEt2, pyridine; (b) CHCl2OCH3, AlCl3, CH2Cl2; (c) POCl3, DMF; (d) HCONHCH3, HCOOH; (e) HCONH2, HCOOH;
(f) NaOH, CH3OH; (g) CH3SO2Cl, pyridine; (h) CH3OCOCl, Et3N, CH2Cl2; (i) BBr3, CH2Cl2.
was prepared. Unfortunately 32 displayed no antitu-
mor activity at its MTD of 740 mg/kg. A phthalimide
derivative, 31, showed marginal activity (%T/C ) 38).
The benefits of 7-methoxy and 7-hydroxy substitu-
tions were suggested by the reported activity of similarly
substituted hycanthone4b and ellipticine derivatives.14,15
While maintaining groups at the 4′-position that had
previously shown good activity (i.e., methylamino, for-
mamido, methylsulfonamido, and carbamoyl), incorpo-
ration of the 7-methoxy substituent provided compounds
43-47 which displayed uniformly high activity and
were very well-tolerated with rapid host recovery times.
Demethylation of 43, 45, and 46 gave phenols 48-50,
respectively, which retained significant activity; how-
ever, maximum tolerated doses dropped 4-7-fold, and
the agents were poorly tolerated with slow host recov-
ery. None of these phenols were curative. Replacement
of methoxy at the 7-position with bromine abolished
activity in 40-42.
Varying the (N,N-dialkylamino)alkylamino side chain
at the 1-position, while maintaining known active
substituents at the 4′-position, provided compounds 34-
39 that were generally less active in vivo than the 1-(2-
diethylamino)ethylamino derivatives (6-33). Only sul-
fonamide 37 was curative although 38 (propyl versus
ethyl side chain) also gave %T/C ) 0. N-Methylation
of the highly active derivative 6 provided 39 which was
inactive. This observation could be rationalized as
follows: The putative active conformation of these
agents is stabilized by an intramolecular H-bond be-
tween the N-H (donor) at position 1 and the carbonyl
group (acceptor) of the thioxanthenone ring. This
planar conformation locks the amine into conjugation
with the aromatic ring which in turn may affect the
reactivity of the 4-methylene group (e.g., enhance the
leaving group ability of the 4′-substituent in an SN2
alkylation of a biological nucleophile). The additional
methyl group of 39 obviously cannot form such an
H-bond; thus the planar conformation is relatively
destabilized resulting in loss of activity.
Incorporation of a pyrazolo ring into the thioxan-
thenone ring provided 51-61 (Table 1) which did not
significantly alter the in vivo properties of the series.
In this 5-aminomethylbenzothiopyranoindazole series,
all compounds displayed high levels of activity. In fact,
with the exception of two analogues, 53 and 61, %T/C
values were zero indicating complete growth inhibition.
For identical R7 and R4 substituents, changing from the
thioxanthenone ring system to the benzothiopyranoin-
dazole system did not appreciably change in vivo efficacy
in most cases. Pyrazolo analogues 51-55 of thioxan-
thenones 6-8, 12, and 29, respectively, shared the same
excellent in vivo efficacy; however, activity was not
curative. The benzothiopyranoindazole derivatives were

Synthesis and Activity of New Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3649
Ta ble 1. Physical and Antitumor Properties of 4-Aminomethylthioxanthenones and 5-Aminomethylbenzothiopyranoindazoles
murine antitumor
activity vs Panc03a
compd
R7
R4′
R1
mp, °C
formulab
%T/Cc MTDd LTCe LCKf
1
H
OH
NHCH2CH2NEt2
4
0
0
0
0
0
0
0
g
0
0
0
g
g
13
g
5
20
0
290 0/3
18 1/5
48 0/5
800 1/3
576 1/5
270 4/5
431 2/5
448 0/5
674
124 3/5
203 0/6
840 1/5
>1532
>1500
654 1/5
>1597
2594 1/5
1212 0/5
304 1/5
254 3/5
552 2/5
465 4/5
2403 2/5
540 3/5
1171 0/6
252 1/5
246 2/5
1298 0/5
2201 0/5
740
3.4
2.9
1.8
3.1
3.0
>4
1.1
1.8
adriamycin
mAMSA
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
NHCH3
NHCHO
NH2
N(Et)CHO
NHEt
NHC6H5
NHSO2CH3
NHSO2Et
NHSO2C6H5
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
237-239
154-155
270-272
75-77
C21H27N3OS‚2HCl‚1/2H2O
C21H25N3O2S
C20H25N3OS‚2HCl
9
C23H29N3O2S‚CH3SO3H‚1/2H2O
C22H29N3OS‚2HCl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
>160 dec
133-135
169-170
>135 dec
164-167
>58 dec
>57 dec
>109 dec
118-128
125-127
133-137
175-177
172-176
42-43
C26H29N3OS
C21H27N3O3S2
2.0
3.7
2.9
C22H29N3O3S2‚CH3SO3H
C26H29N3O3S2‚CH3SO3H
C27H31N3O3S2‚CH3SO3H‚3/2H2O
C26H28ClN3O3S2‚CH3SO3H‚3/2H2O
C26H27Cl2N3O3S2‚CH3SO3H‚1/2H2O
C26H28FN3O3S2
C26H28FN3O3S2
C27H31N3O4S2‚CH3SO3H
C22H29N3O3S2
C23H31N3O3S2
C24H33N3O3S2
C23H31N3O3S2‚CH3SO3H
C27H31N3O3S2‚CH3SO3H
C22H27N3O2S
C22H24N3F3O3S2
C27H29N3O2S
C22H27N3O3S
NHSO2-4-CH3-C6H4
NHSO2-4-Cl-C6H4
NHSO2-3,4-Cl-C6H3
NHSO2-4-F-C6H4
NHSO2-2-F-C6H4
h
2.6
h
NHSO2-4-CH3O-C6H4 NHCH2CH2NEt2
N(CH3)SO2CH3
N(Et)SO2CH3
N(nPr)SO2CH3
N(CH3)SO2Et
N(CH3)SO2C6H5
NHCOCH3
NHCOCF3
NHCOC6H5
NHCO2CH3
NHPO(OEt)2
2-phthalimidoyl
2-saccharinyl
NEt2
NHCH3
NHCH3
NHCH3
NHSO2CH3
NHSO2CH3
NHCH3
NHCH3
NH2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2CH2NEt2 222-224
NHCH2CH2NMe2 >177 dec
NHCH2CH2CH2NMe2 228-229
NHCH2CH2NMe2
NHCH2CH2CH2NMe2 >103 dec
N(CH3)CH2CH2NEt2 oil
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
NHCH2CH2NEt2
2.5
3.1
3.2
5.5
1.0
2.7
1.5
0.5
8.8
<0.5
h
0
0
0
6
159-161
171-174
182-183
152-154
161-163
129-131
108-110
212-214
>103 dec
0
21
26
0
36
38
g
C24H34N3O4PS
C28H27N3O3S‚CH3SO3H‚1/2H2O
C27H27N3O4S2‚CH3SO3H
205-20713 C24H33N3OS‚2HCl
g
1405
C22H29N3OS‚2HCl‚3/2H2O
C19H23N3OS‚2HCl‚5/4H2O
C20H25N3OS‚2HCl‚H2O
C19H23N3O3S2‚CH3SO3H
C20H25N3O3S2‚CH3SO3H‚1/2H2O
C22H29N3OS‚1/4H2O
C21H26BrN3OS
C20H24BrN3OS
C21H26BrN3O3S2
C22H29N3O2S
C22H27N3O3S
17
17
16
0
0
g
g
g
g
0
0
0
0
6
0
11
12
1060 0/5
608 0/5
952 0/5
160 1/5
256 0/5
1506
345
967
1281
880 1/5
763 5/5
248 1/5
240 5/5
382 0/3
60 0/5
47 0/5
34 0/5
1.0
1.6
1.8
3.0
5.1
>168 dec
oil
79-82
NHSO2CH3
134-139
55-56
OCH3 NHCH3
OCH3 NHCHO
5.1
>4.5
6.5
>4.5
h
4.6
3.7
3.0
95-99
OCH3 NHSO2CH3
OCH3 NHCO2CH3
OCH3 NHSO2C6H5
OH
OH
OH
>144 dec
139-140
134-137
167-169
>78 dec
149-157
C22H29N3O4S2
C23H29N3O4S
C27H31N3O4S2
NHCH3
NHSO2CH3
NHCO2CH3
C21H27N3O2S
C21H27N3O4S2‚3/4H2O
C22H27N3O4S
R9
R5′
R2
51
52
53
54
55
56
57
58
59
60
61
H
H
H
H
H
NHCH3
NHCHO
NH2
NHSO2CH3
NHCO2CH3
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
CH2CH2NEt2
189-193
160-161
C21H26N4S‚2HCl‚3/4H2O
0
0
326 0/5
81 0/5
72 0/5
72 0/5
78 0/5
231 3/5
240 3/4
208 3/5
170 4/4
31 0/5
18 0/5
3.0
2.4
h
C21H24N4OS
197-199.5 C20H24N4S‚HCl‚1/2H2O
25i
0
119-123
130-132
125-131
74-76
125-126
125-127
215-218
239-240
C21H26N4O2S2‚1/2H2O
C22H26N4O2S
2.3
2.5
3.6
>4.5
2.2
>4.5
2.0
h
0
OCH3 NHCHO
OCH3 NH2
OCH3 NHSO2CH3
OCH3 NHCO2CH3
OH
OH
C22H26N4O2S‚1/4H2O
C21H26N4OS
0
0i
0
C22H28N4O3S2
C23H28N4O3S
0i
0i
13i
NHSO2CH3
NHCO2CH3
C21H26N4O3S2‚1/4H2O
C22H26N4O3S
a
See ref 2 for the methods of tumor implantation, end point determination, and quantification. Mice were implanted bilaterally sc on
day 0 with 30-60-mg tumor fragments, and chemotherapy (iv) was started 3 days later. Animal use was approved by the Wayne State
b
University IACUC. Proton NMR, IR, and mass spectra were consistent with the assigned structures of all new compounds. Carbon,
hydrogen, and nitrogen elemental analyses were obtained for all new targets and most intermediates and were within (0.4% of the
theoretical values. c T/C value, tumor growth inhibition, where T is the median tumor burden in the treatment group × 100 at evaluation
d
and C is the median tumor burden in the control group at evaluation. MTD, maximum tolerated dose administered intravenously in
mg/kg. e LTC, long-term cures, the number of mice in a treatment group with no palpable tumor evident after a minimum of 86 days/total
number in treatment group. f LCK, log cell kill of tumor-bearing mice (cures excluded), a calculation based on tumor growth delay; cures
g
h
i
require >4.5 log kill. Not active, %T/C > 42. Not calculated. First dose administered 4 days after implantation.

3650 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Perni et al.
Ta ble 2. In Vitro Properties of 4-Aminomethylthioxanthones
and Benzothiopyranoindazoles
sponding unsubstituted derivatives 6, 7, 12, 29, and 51-
55. Hydroxy derivatives 48-50, 60, and 61 had similar
potency to each other and were more cytotoxic than
mAMSA and adriamycin. There is little or no correla-
tion between the in vitro P388 data and in vivo activity,
although there is some correlation between the P388
cytoxicity and the in vivo MTD. The most potent
compounds versus P388 were the hydroxy derivatives
48-50, 60, and 61 which also displayed the lowest
MTDs of all the compounds studied. The P388 cyto-
toxicity data for the benzothiopyranoindazole series
(51-56 and 59-61) did not correlate with the cytotox-
icity data for similarly substituted thioxanthenone
analogues (6-8, 12, 29, 44-46, 49, and 50, respec-
tively).
Recent molecular dynamics calculations suggest that
increased intercalative binding energy enhances anti-
tumor activity.18 As might be expected compounds with
basic amine substituents attached at the pendant 4′-
methylene carbon, 6, 8, 43, 51, and 53, are more potent
intercalators than their nonbasic counterparts probably
due to the additional site of electrostatic attraction
between the phosphate backbone of DNA and the
protonated nitrogen atom. For unknown reasons, the
7-OH substituent of 48 diminishes the intercalative
binding relative to the corresponding 7-OCH3 (43) and
7-H (6) derivatives. In general, benzothiopyranoinda-
zole analogues were more potent DNA intercalating
agents than their thioxanthenone counterparts. The
potency difference was as great as 47-fold as seen with
the pair 59 and 46.
cytotoxicitya topo IIb intercalationc
compd
R7
R4′
IC50, µM
EC50, µM
EC50, µM
1
H
OH
28
0.83
0.15
9.5
12
11
0.30
0.86
21
106
82
d
d
0.80
d
11
0.42
17
0.48
18
2.5
0.65
3.0
4.4
37
3.1
2.2
d
adriamycin
mAMSA
6
7
8
12
29
43
44
45
46
48
49
50
0.72
>270
510e
90e
H
H
H
H
H
NHCH3
NHCHO
NH2
NHSO2CH3
NHCO2CH3
3.0e
20
OCH3 NHCH3
>500
>480
>210
>220
10e
OCH3 NHCHO
OCH3 NHSO2CH3
OCH3 NHCO2CH3
OH
OH
OH
49
NHCH3
NHSO2CH3
NHCO2CH3
0.024
0.025
0.022
0.50
1.6
R9
R5′
51
52
53
54
55
56
57
58
59
60
61
H
H
H
H
H
NHCH3
NHCHO
NH2
NHSO2CH3
NHCO2CH3
16
0.33
5.8
0.15
0.5
55
13
30
45
0.0044
0.011
>220
>260
>250
>230
>240
>240
>260
>210
>220
0.55e
0.33e
0.18
1.5
0.12
1.8
2.6
1.9
0.13
1.6
0.79
2.4
OCH3 NHCHO
OCH3 NH2
OCH3 NHSO2CH3
OCH3 NHCO2CH3
OH
OH
As shown in Table 2, most analogues did not act as
topo II poisons in the assay system used. The only
nonphenolic compound that approached the potency of
mAMSA (IC50 ) 0.72 µM) was 12 having an IC50 value
of 3.0 µM. However, in both the benzothiopyranoinda-
zole and thioxanthenone series, the phenolic substitu-
tion had a profound effect on topo II inhibition. Potency
for phenolic compounds having the methanesulfonamido
(49 and 60) and methylcarbamido (50 and 61) groups
was very close to that of the standard, mAMSA. It is
unclear why the addition of the OH group is so benefi-
cial.
NHSO2CH3
NHCO2CH3
1.5
a
In vitro cytotoxicity was measured by quantifying clonogenic
survival in soft agar following a 1-h transient exposure of P388
mouse leukemia cells to drug. The IC50 is the concentration of drug
which reduced clonogenic survival by 50%. IC50 values were an
average of a minimum of two separate tests which were generally
b
within 2-3-fold agreement with one another. Topoisomerase II
inhibition: the quantification of topoisomerase II covalent complex
formation between 32P-end-labeled pBR322 DNA and extensively
purified HeLa cell topo II was determined by the method of Trask
and Miller (ref 19) as modified and fully described in ref 16. EC50
values were calculated to be the concentration of test compound
at which the amount of DNA precipitated was equivalent to 50%
of the maximum precipitated by mAMSA in a concomitant control
experiment. The EC50 for each test compound is based on the
average of at least two tests which were generally within 2-fold of
one another. However, reproducibility was not as great for strong
intercalators. c DNA intercalation: a known ethidium bromide
displacement assay was used to determine intercalation potency
(see ref 16). The EC50 value is the concentration of test agent that
causes a 50% reduction in the fluorescence of the calf thymus DNA/
Con clu sion s
Within these two series of new antitumor agents, the
only group that has a substantial impact on structure-
cytotoxicity function is the phenolic OH group (7-
position for the thioxanthenones or 9-position for the
benzothiopyranoindazoles); these compounds (48-50,
60, and 61) displayed greatly enhanced P388 cytotox-
icity, topo II inhibition potency, and DNA binding
relative to all other analogues. Otherwise, no relation-
ship between structure and topoisomerase II inhibition,
P388 cytotoxicity, or DNA binding was discernible.
Ring substituents (e.g., OCH3) and the 4-aminomethyl
appendage that contributed to the high in vivo activity
of the thioxanthenones (i.e., 6) also contributed to the
activity of the benzothiopyranoindazoles (e.g., 51).
Within both series, in vivo activity was observed in
analogues having varied physicochemical properties;
thus no clear relationship evolved between structure
and activity.
d
ethidium bromide complex. Not determined. e Bell-shaped dose-
response curve (see ref 20).
generally less potent than their corresponding thioxan-
thenone analogues.
In Vitr o P r op er ties
Selected compounds were evaluated in vitro for cy-
totoxicity against a P388 leukemia cell line,16 as inter-
calators in calf thymus DNA,17 and as inhibitors of
human topoisomerase II16 (Table 2). In vitro cytotoxic
potency spanned 4 orders of magnitude from 0.0044 µM
for 60 to 106 µM for 44. Activity of the methoxy
derivatives 43-46 and 56-59 was similar to that of 1,
but they were consistently less active than the corre-
In vivo efficacy did not correlate with in vitro P388
cytotoxicity. Of note is the fact that the 7-OCH3

Synthesis and Activity of New Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3651
Ta ble 3. In Vivo Activity (%T/C) of SR 233377 (12) versus
rected. Proton NMR spectra, obtained on a GE QE 300 NMR
spectrometer, and chemical ionization mass spectra, obtained
on a Nermag R 10-10 C spectrometer, were consistent with
the assigned structures. Proton NMR multiplicity data are
denoted by s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), and br (broad). Coupling constants are reported
in hertz. Combustion analyses (C, H, N) were performed by
Quantitative Technologies, Inc. (Whitehouse, NJ ) and were
within 0.4% of theoretical values. Reactions were performed
under an N2 atmosphere.
N-[[1-[[2-(Dieth yla m in o)eth yl]a m in o]-9-oxo-9H-th iox-
a n th en -4-yl]m eth yl]-N-m eth ylfor m a m id e (5). A solution
of 4 (35 g, 0.10 mol) in N-methylformamide (394 mL) and
formic acid (50 mL) was heated at 160 °C for 1 h, allowed to
cool to ambient temperature, and poured into water (2 L). The
aqueous solution was made basic with 35% aqueous NaOH;
the precipitate was collected and dried in vacuo. Recrystal-
lization from acetone afforded pure 5 (24.6 g, 62%): mp 127-
130 °C; 1H NMR (CDCl3) δ 10.40 (m, 1H), 8.50 and 8.18 (s
and s, 1H, amide rotamers), 7.62-7.36 (m, 4H), 7.31-7.20 (m,
1H), 6.60 (dd, 1H), 4.65 and 4.48 (s and s, 2H, amide rotamers),
3.35 (q, 2H), 3.80 (m, 5H), 2.64 (q, 4H), 1.09 (t, 6H). Anal.
(C22H27N3O2S) C, H, N.
N-[[1-[[2-(Dieth ylam in o)eth yl]am in o]-7-m eth oxy-9-oxo-
9H-th ioxa n th en -4-yl]m eth yl]for m a m id e (44) was prepared
in 69% yield from 63 via the same procedure as for 5 except
formamide was used instead of N-methylformamide. The
crude material was purified by flash chromatography (1%
isopropylamine in 1:1 CHCl3/hexane): mp 95-99 °C; 1H NMR
(CDCl3) δ 10.37 (br t, 1H), 8.28 (d, 1H), 7.97 (d, 1H), 7.37 (dd,
2H), 7.15 (dd, 1H), 6.54 (d, 1H), 4.56 and 4.45 (d and d, 2H,
amide rotamers), 3.91 (s, 6H), 3.32 (q, 2H), 2.82 (t, 2H), 2.64
(q, 4H), 1.09 (t, 6H); MS m/z 413 (M+). Anal. (C22H27N3O3S)
C, H, N.
N-[[1-[[2-(Dieth yla m in o)eth yl]a m in o]-9-oxo-9H-th iox-
a n th en -4-yl]m eth yl]m eth a n a m in e (6). A solution of 5 (9.34
g, 23 mmol) in 2 N aqueous HCl was refluxed for 3 h, allowed
to cool to ambient temperature, neutralized with 35% aqueous
NaOH, and chilled at 0 °C for 1 h. The resulting precipitate
was collected and dried in vacuo at 80 °C. The crude product
was passed through a pad of silica gel (isopropylamine/MeOH/
CHCl3-1:5:94). After concentration in vacuo the orange solid
was dissolved in MeOH, treated with 4 N HCl/Et2O, and
chilled at 0 °C. Ice cold i-PrOH was added and the precipitate
collected and dried in vacuo to yield 6 (12.81 g 58%) as the
dihydrochloride salt: mp 237-239 °C; 1H NMR (CD3OD) δ 8.31
(d, 1H), 7.55-7.30 (m, 4H), 6.76 (d, 1H), 4.14 (s, 2H), 4.73 (t,
2H), 3.27-3.05 (m, 6H), 2.58 (s, 3H), 1.26 (t, 6H). Anal.
(C21H27N3OS‚2HCl‚1/2H2O) C, H, N.
Murine Solid Tumorsa
Mam
16/C
Colon
38
Colon
26/A
Colon
51
Panc02
SR 233377 (12)
adriamycin
cytoxan
23
35
36
6
0b
0
c
29
7
8
14
21
c
0b
0
a
See refs 2 and 21 for the methods of tumor implantation, end
point determination, and quantification. %T/C value, tumor growth
inhibition, where T is the median tumor burden in the treatment
group × 100 at evaluation and C is the median tumor burden in
the control group at evaluation. Mice were implanted bilaterally
sc on day 0 with 30-60-mg tumor fragments, and chemotherapy
(iv) was started 3 days later. bCurative activity. c Not tested.
derivatives 43-46 in the thioxanthenone series and the
three 9-OCH3 analogues in the benzothiopyranoindazole
series (56-58) showed exceptional in vivo activity (%T/C
) 0 with long-term cures) despite being weakly potent
in the P388 cytotoxicity assay. It is likely that for the
phenolic derivatives 48-50, 60, and 61, their inherent
potent topoisomerase II inhibition properties and as-
sociated in vitro P388 cytotoxicity account for the
excellent in vivo activity (%T/C e 13) observed for these
analogues. However, these phenolic derivatives were
clearly less active and less well-tolerated than the
corresponding methoxy derivatives. For compounds
with moderate P388 cytotoxicity (e.g., 7-H derivatives
12 and 29 and 9-H analogues 52, 54, and 55), it is
unclear whether their inherent cytotoxicity or metabo-
lism of parent drug to a more cytotoxic species is
responsible for the observed highly efficacious in vivo
activity.
The methoxy derivatives 43-46 and 56-59 displayed
a very low level of P388 in vitro cytotoxicity and topo-
isomerase II inhibition. Nonetheless, they exhibited a
high level of antitumor activity in in vivo murine tumor
models. It is likely that this activity is a consequence
of metabolism to an active topo II inhibitor, presumed
to be the corresponding 7(or 9)-OH derivatives (e.g., 45
f 49 and 59 f 61). The consistently high levels of in
vivo activity associated with most of these compounds
render it difficult to elucidate a structure-activity
relationship particularly in the absence of pharmaco-
kinetic and tumor cell/normal cell metabolism data.
In conclusion, two related series of hycanthone de-
rivatives have been prepared and evaluated in vivo and
in vitro as antitumor agents. Both series were shown
to be efficacious and broadly active in vivo but displayed
varying in vitro properties. This work has led to the
clinical development of SR 233377 (12) based on its
breadth of in vivo activity and favorable toxicological
profile. The selection of SR 233377 was based not only
on its in vivo Panc03 and P388 in vitro cytotoxicity data
but also on a favorable comparison with adriamycin and
cytoxan across a range of in vivo solid tumor models.
As summarized in Table 3, SR 233377 was active
against the five murine solid tumors Panc 02, Mam 16/
C, Colon 38, Colon 26/A, and Colon 51. As previously
reported,21 SR 233377 showed no significant in vivo
activity against adriamycin-resistant or adriamycin-
sensitive P388.
[1-[[2-(Diet h yla m in o)et h yl]a m in o]-7-m et h oxy-9-oxo-
9H-th ioxa n th en -4-yl]m eth yla m in e (65). A solution of 44
(1.02 g, 2.5 mmol) in 35 mL of 2 N HCl was heated on a steam
bath for 1 h. The reaction mixture was cooled to room
temperature and made basic with 10% aqueous sodium
hydroxide, and the product was extracted into CHCl3. The
organic portion was washed with water and brine, dried over
Na2SO4, and concentrated in vacuo to give crude 65. Flash
chromatography (1:1 CHCl3/hexanes then 1% isopropylamine
in 1:1 CHCl3/hexane) afforded pure 65 (0.58 g, 60%): mp 75-
1
78 °C; H NMR (CDCl3) δ 10.28 (br t, 1H), 7.99 (d, 1H), 7.41
(t, 2H), 7.18 (dd, 1H), 6.59 (d, 1H), 3.95 (s, 2H), 3.92 (s, 3H),
3.36 (m, 2H), 2.82 (m, 2H), 2.64 (q, 4H), 1.09 (t, 6H); MS m/z
386 (MH+). Anal. (C21H27N3O2S) C, H, N.
N-[[1-[[2-(Dieth ylam in o)eth yl]am in o]-7-m eth oxy-9-oxo-
9H-th ioxa n th en -4-yl]m eth yl]m eth a n esu lfon a m id e (45).
A solution of 65 (5.60 g, 14.5 mmol) in dry pyridine (75 mL)
was cooled in an ice bath, and methanesulfonyl chloride (1.25
mL, 16 mmol) was added dropwise. The reaction mixture was
allowed to warm to ambient temperature over 2 h, poured into
ice water (800 mL), and extracted with CHCl3 (4 × 200 mL).
The combined extracts were washed with water (200 mL) and
brine (200 mL), dried over Na2SO4, and concentrated in vacuo
to afford crude 45. Purification by flash chromatography (1:1
CHCl3/hexane followed by 1% isopropylamine in 1:1 CHCl3/
Exp er im en ta l Section
Gen er a l. Melting points were determined on a Mel-Temp
melting point apparatus in open capillaries and are uncor-

3652 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Perni et al.
hexane then 1% isopropylamine in CHCl3) gave a solid which
was suspended in 1:1 EtOAc-hexane. The solid was collected
and dried in vacuo to afford (4.86 g, 78%): mp 144 °C dec; 1H
NMR (CDCl3) δ 10.42 (br t, 1H), 7.97 (d, 1H), 7.40 (dd, 2H),
7.18 (dd, 1H), 6.59 (d, 1H), 4.43 (d, 2H), 3.94 (s, 3H), 3.41 (br
q, 2H), 2.88 (m, 2H), 2.85 (s, 3H), 2.72 (q, 4H), 1.12 (t, 6H);
MS m/z 463 (M+). Anal. (C22H29N3O4S2) C, H, N.
(s, 2H), 3.89 (t, 2H), 3.40-3.17 (m, 6H), 2.85 (s, 3H), 1.40 (t,
6H). Anal. (C28H27N3O3S‚CH3SO3H‚1/2H2O) C, H, N.
N-[[1-[[2-(Dieth yla m in o)eth yl]a m in o]-9-oxo-9H-th iox-
a n th en -4-yl]m eth yl]-N-(1-p r op yl)su lfon a m id e (23). To a
mixture of NaH (0.20 g of 60% oil suspension washed with
pentane) in dry DMF (40 mL) was added 12 (2.00 g, 4.61
mmol), and the resulting mixture was heated to 50 °C for 2 h.
The reaction mixture was chilled in an ice bath for 15 min,
treated with N-propyl iodide (0.87 g, 5.12 mmol), and stirred
at room temperature overnight. Water was added (35 mL)
with rapid stirring. The resulting precipitate was washed with
additional water and dried in vacuo (50 °C) to give 23 (2.17 g,
99%): mp 142-143 °C; 1H NMR (CDCl3) δ 8.51 (d, 1H), 7.62-
7.38 (m, 4H), 6.65 (d, 1H), 4.46 (s, 2H), 3.38 (m, 2H), 3.15 (m,
2H), 2.89 (s, 3H), 2.80 (t, 2H), 2.63 (q, 4H), 1.70-1.45 (m, 2H),
1.05 (t, 6H), 0.83 (t, 3H). Anal. (C24H33N3O3S2) C, H, N.
Gen er a l P r oced u r e for Com p ou n d s 12-30, 46, a n d
47: Ca r ba m ic Acid , [[1-[[2-(Dieth yla m in o)eth yl]a m in o]-
9-oxo-9H-th ioxa n th eN-4-yl]m eth yl]-, Meth yl Ester (29).
A solution of 8 (prepared according to the same procedure as
for 65) (2.94 g, 8.3 mmol) in CH2Cl2 (50 mL) containing
triethylamine (5 mL) was cooled in an ice bath, treated with
methyl chloroformate (0.7 mL, 9.1 mmol, or the corresponding
chloroformate or acid chloride), and stirred for 2.5 h. The
solvent was removed in vacuo, and the residue was flash-
chromatographed (CHCl3 then 1% isopropylamine/CHCl3) to
N-[[1-[[2-(Dieth yla m in o)eth yl]a m in o]-9-oxo-9H-th iox-
a n th en -4-yl]m eth yl]sa cch a r in (32). A solution of hycan-
thone (1) (2.00 g, 5.6 mmol) in dry pyridine (50 mL) at 0 °C
was treated with p-toluenesulfonyl chloride (1.15 g, 6.0 mmol)
and stirred at ambient temperature for 90 min. The resulting
tosylate (66) was collected and dried under high vacuum. A
solution of 66 (2.04 g, 4.0 mmol) with sodium saccharin (1.94
g, 9.5 mmol) and tetra-n-butylammonium bromide (0.20 g) in
DMF (50 mL) was heated at 100 °C for 2 h. After cooling to
ambient temperature the solution was diluted with CHCl3 (500
mL) and filtered through a pad of silica gel. The product was
eluted with 2% isopropylamine/CHCl3 to give 32 (1.66 g, 80%)
as a yellow solid. Recrystallization from methanol containing
methanesulfonic acid afforded 32 as its methanesulfonate salt
1
afford 29 (2.36 g, 69%): mp 129-131 °C; H NMR (CDCl3) δ
10.32 (br t, 1H), 8.48 (dd, 1H), 7.50-7.28 (m, 4H), 6.56 (d, 1H),
4.43 (d, 2H), 3.69 (s, 3H), 3.33 (q, 2H), 2.79 (t, 2H), 2.63 (q,
4H); MS m/z 414 (MH+). Anal. (C22H27N3O3S) C, H, N.
N-[[1-[[2-(Dieth ylam in o)eth yl]am in o]-7-h ydr oxy-9-oxo-
9H-th ioxa n th en -4-yl]m eth yl]m eth a n a m in e (48). A solu-
tion of 43 (1.6 g, 4 mmol) in 48% HBr (10 mL) was heated to
110 °C for 5 h. After cooling, the reaction mixture was
neutralized with saturated NaHCO3 and extracted into CHCl3
(3 × 100 mL). The residual dark gum from the reaction vessel
was dissolved in MeOH and combined with the CHCl3 solution.
The solvents were removed in vacuo to afford 1.67 g of a dark-
orange solid. The crude product was purified by flash chro-
matography (isopropylamine/MeOH/CHCl3-1:1:98) followed by
a second silica gel column eluting with isopropylamine/MeOH/
CHCl3 (2:2:96) to afford 48 (0.56 g, 36%): mp 167-169 °C; 1H
NMR (CDCl3) δ 10.23 (br t, 1H), 7.64 (d, 1H), 7.30 (d, 1H),
7.19 (d, 1H), 6.93 (dd, 1H), 6.43 (d, 1H), 3.88 (s, 2H), 3.30 (q,
2H), 2.84 (t, 2H), 2.69 (q, 4H), 2.57 (s, 3H), 1.12 (t, 6H). Anal.
(C21H27N3O2S‚1/4H2O) C, H, N.
N-[[1-[[2-(Dieth ylam in o)eth yl]am in o]-7-h ydr oxy-9-oxo-
9H-th ioxa n th en -4-yl]m eth yl]m eth a n esu lfon a m id e (49).
To a solution of 45 (0.5 g, 1.1 mmol) in CH2Cl2 (45 mL) at -78
°C was added, dropwise, BBr3 (1.75 mL). The mixture was
warmed to room temperature, stirred overnight, and then
poured into ice-water (250 mL) containing 35% NaOH (8 mL).
The mixture was acidified with dilute HCl, neutralized with
solid Na2CO3, and extracted with EtOAc. The organic layer
was separated, washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by column
chromatography (5% MeOH/EtOAc) to afford 49 (0.28 g,
58%): mp 78 °C dec; 1H NMR (CDCl3) δ 10.35 (br t, 1H), 10.06
(br s, 1H), 7.86 (d, 1H), 7.55 (m, 2H), 7.23 (dd, 1H), 6.74 (d,
1H), 4.27 (d, 2H), 3.39 (s, 3H), 3.35 (m, 2H), 2.77 (t, 2H), 2.58
(m, 4H), 1.11 (t, 6H); MS m/z 449 (M+). Anal. (C21H27N3O4S2‚
3/4H2O) C, H, N.
1
(1.35 g); mp 103 °C dec; H NMR (CDCl3) δ 8.49 (d, 1H), 8.15
(m, 1H), 7.96-7.83 (m, 3H), 7.65-7.53 (m, 3H), 7.45 (m, 1H),
6.77 (d, 2H) 5.09 (s, 2H), 3.90 (t, 2H), 3.40-3.18 (m, 6H),
2.85 (s, 3H), 1.40 (t, 6H). Anal. (C27H27N3O4S2‚CH3SO3H)
C,H,N.
N-[[1-[[2-(Dieth yla m in o)eth yl]-1-m eth yla m in o]-9-oxo-
9H-th ioxa n th en -4-yl]m eth yl]m eth a n a m in e (39). A mix-
ture of 7410 (3.05 g, 8.3 mmol) and ammonium formate (3.00
g) in N-methylformamide (50 mL) was heated to 160 °C for 2
h, diluted with water (400 mL), and extracted with CHCl3 (5
× 100 mL). The combined extracts were flash chromato-
graphed on silica gel (CHCl3 then 2% isopropylamine/CHCl3).
The resulting orange gum was dissolved in MeOH (30 mL),
treated with 2 N NaOH (25 mL), and refluxed for 3 h. The
crude product was extracted with CHCl3 and flash chromato-
graphed (0.5% isopropylamine/1% MeOH/CHCl3). Final pu-
rification by MPLC (2% Et3N/CHCl3) furnished 39 (0.62 g,
19%) as an orange gum: 1H NMR (CDCl3) δ 8.21 (d, 1H), 7.51-
7.42 (m, 2H), 7.38-7.27 (m, 2H), 6.88 (dd, 1H), 3.85 (s, 2H),
3.35 (t, 2H), 2.85 (s, 3H), 2.67 (t, 2H), 2.46 (q, 4H), 2.44 (s,
3H) 0.92 (t, 6H). Anal. (C22H29N3OS‚1/4H2O) C, H, N.
N,N-Dieth yl-2H-(1)ben zoth iop yr a n o[4,3,2-cd ]in d a zole-
2-eth a n a m in e (69). A solution of 6710 (as a mixture with the
corresponding 3-Cl isomer; 6.20 g, 25.2 mmol), N,N-diethyl-
2-hydrazinoethanamine11 (4.20 g, 32.0 mmol), and pyridine (50
mL) was heated at reflux for 24 h, and the solvent was
removed in vacuo. The residue was dissolved in CHCl3 and
washed with water (3×). The organic layer was dried over
Na2SO4 and concentrated in vacuo, and the residue was
flushed through a pad of silica gel eluting with EtOAc/hexane
(10-40%) to give 69 (2.42 g, 30%). Recrystallization from
Ca r ba m ic Acid , [[1-[[2-(Dieth yla m in o)eth yl]a m in o]-7-
h yd r oxy-9-oxo-9H-th ioxa n th en -4-yl]m eth yl]-, Meth yl Es-
t er (50) was prepared from 46 in 52% yield via the same
procedure as for 49. The crude product was purified by flash
chromatography (isopropylamine/MeOH/CHCl3-1:1:98): mp
149-157 °C; 1H NMR (CDCl3) δ 10.33 (br t, 1H), 7.70 (d, 1H),
7.34 (d, 1H), 6.78 (dd, 1H), 6.46 (d, 1H) 4.37 (d, 2H), 3.73 (s,
3H), 3.39 (q, 2H), 2.90 (t, 2H), 2.75 (q, 4H), 1.16 (t, 6H). Anal.
(C22H27N3O4S) C, H, N.
1
hexanes gave the analytical sample: mp 49-50 °C; H NMR
(CDCl3) δ 8.02 (dd, 1H), 7.22-7.10 (m, 4H), 6.84 (d, 1H), 6.64
(d, 1H), 4.30 (t, 2H), 2.90 (t, 2H), 2.56 (q, 4H), 1.00 (t, 6H); MS
m/z 324 (MH+). Anal. (C19H21N3S) C, H, N.
N-[[1-[[2-(Dieth yla m in o)eth yl]a m in o]-9-oxo-9H-th iox-
a n th en -4-yl]m eth yl]p h th a lim id e (31). A mixture of 8 (1.67
g, 4.7 mmol) and phthalic anhydride (0.80 g, 5.4 mmol) in
toluene (200 mL) was refluxed with a Dean-Stark trap for 6
h. The resulting solution was filtered through a pad of silica
gel eluting with CHCl3 to remove an impurity followed by 1%
isopropylamine/CHCl3 to afford 1.92 g (84%) of 31 as an orange
solid. Recrystallization from i-PrOH/i-PrOAc/MeOH contain-
ing methanesulfonic acid afforded 31 as its methanesulfonate
2-[2-(Dieth ylam in o)eth yl]-2H-(1)ben zoth iopyr an o[4,3,2-
cd ]in d a zole-5-ca r boxa ld eh yd e (70). A suspension of alu-
minum chloride (1.76 g, 12.8 mmol) in CH2Cl2 (25 mL) was
stirred at room temperature for 15 min, and a solution of 69
(2.07 g, 6.38 mmol) in CH2Cl2 (15 mL) was added at 5 °C. The
resulting mixture was stirred for 10 min and cooled to 0 °C. A
solution of dichloromethyl methyl ether (1.52 g, 17.4 mmol)
in CH2Cl2 (10 mL) was added dropwise over a period of 15
min. The mixture was allowed to warm to ambient temper-
1
salt: mp 212-214 °C; H NMR (CDCl3) δ 8.46 (d, 1H), 7.90
(m, 2H), 7.75 (m, 2H), 7.60-7.38 (m, 4H), 6.74 (d, 1H), 4.96

Synthesis and Activity of New Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19 3653
ature, stirred overnight, and diluted with of 2 N HCl (10 mL)
and cool water (100 mL). The mixture was poured into CHCl3
(100 mL) and basified with 2 N NaOH solution to pH 8-9,
and the layers were separated. The aqueous layer was
extracted with CHCl3 (40 mL). The combined organic layers
were washed with water, dried over Na2SO4, and concentrated
in vacuo to give crude 70 (2.16 g, 96%) which could be used
without additional purification. The analytical sample was
obtained by silica gel chromatography (EtOAc) following by
recrystallization (1:2 EtOAc/hexane): mp 81-82 °C; 1H NMR
(CDCl3) δ 10.02 (s, 1H), 8.20-8.17 (m, 1H), 7.64 (d, 1H), 7.53
(m, 1H), 7.31 (m, 2H), 7.02 (d, 1H), 4.40 (t, 2H), 2.95 (t, 2H),
2.54 (q, 4H), 0.95 (t, 6H); MS m/z 352 (MH+). Anal. (C20H21N3-
OS) C, H, N.
N,N-Dieth yl-9-m eth oxy-2H-(1)ben zoth iop yr a n o[4,3,2-
cd ]in d a zole-2-eth a n a m in e (71) was prepared from 684b (as
a mixture with the corresponding 3-Cl isomer) and purified
via the same procedure as for 69 in 39% yield as a yellow oil:
1H NMR (CDCl3) δ 7.58 (d, 1H), 7.08 (2d, 2H), 6.80 (d, 1d),
6.76 (dd, 1H), 6.62 (d, 1H), 4.30 (t, 2H), 3.82 (s, 3H), 2.90 (t,
2H), 2.56 (q, 4H), 0.98 (t, 6H); MS m/z 354 (MH+). Anal.
(C20H23N3OS) C, H, N.
2-[2-(Dieth yla m in o)eth yl]-9-m eth oxy-2H-(1)ben zoth io-
p yr a n o[4,3,2-cd ]in d a zole-5-ca r boxa ld eh yd e (72). To a
stirred solution of 71 (1.60 g, 4.19 mmol) in DMF (45 mL) at
room temperature was added dropwise a solution of phospho-
rus oxychloride (1.6 mL) in DMF (5 mL). The reaction mixture
was heated at 95-100 °C for 18 h. Heat was removed, and
ice (50 g) was added. The mixture was stirred for 10 min and
basified with 2 N NaOH solution to pH 8-9. The basic
solution was extracted with CHCl3, and the extracts were
washed with water (3×), dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography (hexane, hexane/EtOAc-60:40, then EtOAc)
to give 72 as a yellow oil (1.06 g, 61%): 1H NMR (CDCl3) δ
10.04 (s, 1H), 7.72-7.62 (dd, 2H), 7.48 (d, 1H), 7.06-6.96 (m,
2H), 4.42 (t, 2H), 3.94 (s, 3H), 2.96 (t, 2H), 2.56 (q, 4H), 0.98
(t, 6H); MS m/z 382 (MH+). Anal. (C21H23N3O2S‚1/2H2O) C,
H, N.
4H), 6.87 (d, 1H), 4.34 (t, 2H), 3.79 (s, 2H), 2.93 (t, 2H), 2.54
(q, 4H), 0.99 (t, 6H); MS m/z 353 (MH+). Anal. (C20H24N4S‚
HCl‚1/2H2O) C, H, N.
2-[2-(Dieth yla m in o)eth yl]-9-m eth oxy-2H-(1)ben zoth io-
p yr a n o[4,3,2-cd ]in d a zole-5-m eth a n a m in e (57) was pre-
pared from 56 via the same procedure as for 53. Pure material
was obtained by flash chromatography (hexane/EtOAc-1:1,
followed by 100% EtOAc, followed by 100% CH2Cl2, and then
0.5-1% isopropylamine in CH2Cl2) in 90% yield: mp 74-76
°C; 1H NMR (CDCl3) δ 7.62 (d, 1H), 7.10 (d, 1H), 6.75 (m, 2H),
4.28 (t, 2H), 3.82 (s, 3H), 3.72 (s, 2H), 2.88 (t, 2H), 2,52 (q,
4H), 0.96 (t, 6H); MS m/z 383 (MH+). Anal. (C21H26N4OS) C,
H, N.
N-[[2-[2-(Dieth yla m in o)eth yl]-2H-(1)ben zoth iop yr a n o-
[4,3,2-cd ]in d a zol-5-yl]-m eth yl]m eth a n esu lfon a m id e (54).
To a solution of methanesulfonyl chloride (172 mg, 1.50 mmol)
in CH2Cl2 (30 mL) were added the free base of 53 (0.51 g, 1.28
mmol) and pyridine (0.4 mL) with stirring at 0 °C. After the
mixture was allowed to warm to ambient temperature over 5
h, CHCl3 (30 mL) and 2 N NaOH (5 mL) were added to the
mixture. The organic layers were separated, washed with
water, dried over Na2SO4, and concentrated in vacuo to give
the crude product. Purification by chromatography (0.5%
Et3N/CH2Cl2) gave 650 mg of gum. Crystallization from ether/
MeOH (90:10) gave 54 (460 mg, 84%): mp 119-123 °C; 1H
NMR (CDCl3) δ 8.06 (m, 1H), 7.19 (m, 4H), 6.90 (d, 1H), 4.48
(d, 1H), 4.38 (t, 2H), 4.26 (d, 2H), 2.92 (t, 2H), 2.84 (s, 3H),
2,58 (q, 4H), 0.98 (t, 6H); MS m/z 431 (MH+). Anal.
(C21H26N4O2S2‚1/2H2O) C, H, N.
N-[[2-[2-(Diet h yla m in o)et h yl]-9-m et h oxy-2H -(1)b en -
zot h iop yr a n o[4,3,2-cd ]in d a zol-5-yl]m e t h yl]m e t h a n e -
su lfon a m id e (58) was prepared from 57 via the same
procedure as for 54. Flash chromatography (hexane followed
by 2:3 hexane/EtOAc and then EtOAc) gave 58 as a gum (785
mg, 66%). Crystallization from ether and a few drops of
1
CH2Cl2 provided the analytical sample: mp 125-126 °C; H
NMR (CDCl3) δ 7.60 (d, 1H), 7.22 (d, 1H), 7.20 (d, 1H), 6.86
(m, 2H), 4.72 (t, 1H), 4.26 (d, 2H), 3.88 (s, 3H), 2.90 (t, 2H),
2.84 (s, 3H), 2.58 (q, 4H), 0.98 (t, 6H); MS m/z 461 (MH+). Anal.
(C22H28N4O3S2) C, H, N.
N-[[2-[2-(Dieth yla m in o)eth yl]-2H-(1)ben zoth iop yr a n o-
[4,3,2-cd ]in d a zol-5-yl]m eth yl]for m a m id e (52). A solution
of 70 (1.64 g, 4.67 mmol), formic acid (1.8 g, 39 mmol), and
formamide (30 mL) was heated at 140-150 °C for 5 h. The
reaction mixture was poured into 40 mL of ice-water and
basified with 2 N NaOH solution to pH 8-9. The basic
aqueous solution was extracted with CHCl3. The organic layer
was washed with water, dried, and concentrated to give the
crude product. The pure formamide was obtained by silica
gel chromatography, eluting with 0.5% isopropylamine in
Ca r ba m ic Acid , [[2-[2-(Dieth yla m in o)eth yl]-2H-(1)ben -
zoth iop yr a n o[4,3,2-cd ]in d a zol-5-yl]m eth yl]-, Meth yl Es-
ter (55). To a solution of the free base of 53 (974 mg, 2.45
mmol) in CH2Cl2 were added methyl chloroformate (0.23 mL)
and Et3N (0.8 mL) with stirring at 0 °C. The mixture was
stirred for 5 h and allowed to warm to room temperature. The
reaction mixture was partitioned between CHCl3/water and
basified to pH 10 by adding a few drops of 10% aqueous NaOH
solution. The organic layer was separated, washed with water,
dried over Na2SO4, and concentrated in vacuo to give crude
55. Purification by flash chromatography (0.5% isopropyl-
amine/EtOAc) gave pure 55 (0.62 g, 55%): mp 130-132 °C;
1H NMR (CDCl3) δ 8.04 (m, 1H), 7.19 (m, 4H), 6.86 (d, 1H),
4.98 (d, 1H), 4.30 (m, 4H), 3.66 (s, 2H), 2.90 (t, 2H), 2.56 (q,
4H), 0.98 (t, 6H); MS m/z 411 (MH+). Anal. (C22H26N4O2S)
C, H, N.
Ca r ba m ic Acid , [[2-[2-(Dieth yla m in o)eth yl]-2H-(1)-9-
m e t h oxyb e n zot h iop yr a n [4,3,2-cd ]in d a zol-5-yl]m e t h -
yl]-, Meth yl Ester (59) was prepared from 57 via the same
procedure as for 55. The crude product was flash chromato-
graphed (5% MeOH/EtOAc) to afford pure 59 (1.10 g, 63%):
mp 125-127 °C; 1H NMR (CDCl3) δ 7.60 (d, 1H), 7.22 (d, 2H),
7.85 (m, 2H), 4.89 (s, 3H), 3.70 (s, 3H), 4.35 (m, 4H), 2.95 (t,
2H), 2.59 (q, 4H), 0.98 (t, 6H). Anal. (C23H28N4O3S) C, H, N.
N-[[2-[2-(Diet h yla m in o)et h yl]-9-h yd r oxy-2H -(1)b en -
zot h iop yr a n o[4,3,2-cd ]in d a zol-5-yl]m e t h yl]m e t h a n e -
su lfon a m id e (60). A solution of 58 (1.02 g, 2.22 mmol) in
CH2Cl2 (50 mL) was cooled to -50 °C and treated dropwise
with BBr3 (15 mL of 1 M solution in CH2Cl2, 15 mmol) forming
a thick orange suspension. The mixture was allowed to warm
to -10 °C and stirred for an additional 2 h. The reaction
mixture was diluted with CH2Cl2 (100 mL) and the reaction
quenched with MeOH (10 mL). The resulting mixture was
poured into ice water, neutralized with aqueous NaOH, and
1
EtOAc to afford pure 52 (1.20 g, 67%): mp 160-161 °C; H
NMR (CDCl3) δ 8.26 (s, 1H), 8.18 (m, 1H), 7.24 (m, 3H), 7.15
(d, 1H), 6.83 (d, 1H), 4.42 (d, 2H), 4.36 (t, 2H), 2.92 (t, 2H),
2.56 (q, 4H), 0.95 (t, 6H); MS m/z 381 (MH+). Anal. (C21H24N4-
OS) C, H, N.
N-[[2-[2-(Diet h yla m in o)et h yl]-9-m et h oxy-2H -(1)b en -
zoth iopyr an o[4,3,2-cd]in dazol-5-yl]m eth yl]for m am ide (56)
was prepared in quantitative yield via the same procedure
used to synthesize 52 and could be used without additional
purification: mp 125-131 °C; 1H NMR (CDCl3) δ 8.08 (s, 1H),
7.42 (d, 1H), 7.10 (m, 2H), 6.75 (m, 2H), 4.28 (d, 2H), 4.22 (t,
2H), 3.80 (s, 3H), 2.82 (t, 2H), 2.48 (q, 4H), 0.92 (t, 6H); MS
m/z 412 (MH+). Anal. (C22H26N4O2S‚1/4H2O) C, H, N.
2-[2-(Dieth ylam in o)eth yl]-2H-(1)ben zoth iopyr an o[4,3,2-
cd ]in d a zole-5-m et h a n a m in e H yd r och lor id e H em ih y-
d r a te (53). To a solution of 52 (2.76 g, 7.25 mmol) in MeOH
(100 mL) was added 10% NaOH solution (50 mL), and the
mixture was heated at reflux for 5 h. The reaction mixture
was cooled to room temperature and extracted with CHCl3.
The organic layers were washed with water, dried over
Na2SO4, and concentrated in vacuo to give the crude product.
Purification by chromatography (2% Et3N/CHCl3) afforded
pure 53 as a viscous oil (1.78 g 62%). Recrystallization from
methanolic HCl/EtOAc (2:10) gave the analytical sample: mp
197-199.5 °C; 1H NMR (CDCl3) δ 8.01 (m, 1H), 7.10-7.30 (m,

3654 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 19
Perni et al.
Meeting of the American Association for Cancer Research,
Toronto, Canada, March 18-22, 1995; Abstr. 2289. (b) Wentland,
M. P.; Perni, R. B.; Huang, J . I.; Powles, R. G.; Aldous, S. A.;
Klingbeil, K. M.; Peverly, A. D.; Robinson, R. G.; Corbett, T. H.;
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extracted with 10% MeOH/CHCl3 (5×). The organic phase was
separated, washed with brine, dried over Na2SO4, and con-
centrated in vacuo to afford a yellow solid (0.87 g). Dry flash
chromatography (EtOAc followed by 1% MeOH/EtOAc and 1%
MeOH/0.2% IPA/EtOAc) provided 60 as a white solid (0.44 g,
45%): mp 215-218 °C dec; 1H NMR (DMSO-d6) δ 9.78 (s, 1H),
7.44 (t, J ) 6.0 Hz, 1H), 7.37 (d, J ) 2.6 Hz, 1H), 7.26 (d, J )
8.3 Hz, 2H), 7.16 (d, J ) 8.6 Hz, 1H), 6.74 (dd, J ) 2.7, 8.7 Hz,
1H), 4.33 (t, J ) 6.1 Hz, 2H), 4.02 (d, J ) 6.0 Hz, 1H), 2.86 (s,
3H), 2.82 (t, J ) 6.3 Hz, 2H), 2.44 (q, J ) 7.1 Hz, 4H), 0.80 (t,
J ) 7.1 Hz, 6H); MS m/z 447 (MH+). Anal. (C21H26N4O3S2‚
1/4H2O) C, H, N.
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2H -(1)b en zot h iop yr a n o[4,3,2-cd ]in d a zol-5-yl]m et h yl]-,
Meth yl Ester (61) was prepared from 59 via the same
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and 1.8% MeOH/0.2% IPA/EtOAc) to afford 61 as an off-white
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solid (0.42 g, 61%): mp 239-240 °C dec; H NMR (CDCl3) δ
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7.43 (d, J ) 2.9 Hz, 1H), 7.23 (m, 1H), 7.14 (d, J ) 8.6 Hz,
1H), 6.85 (d, J ) 8.6 Hz, 1H), 6.76 (dd, J ) 7.2, 8.5 Hz, 1H),
5.15 (br s, 1H), 4.25 (m, 2H), 3.65 (s, 3H), 2.86 (t, J ) 7.2 Hz,
2H), 2.54 (q, J ) 7.1 Hz, 4H), 0.98 (t, J ) 7.2 Hz, 6H); MS m/z
427 (MH+). Anal. (C22H26N4O3S) C, H, N.
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(21) Corbett, T.; Lowichik, N.; Pugh, S.; Polin, L.; Panchapor, C.;
White, K.; Knight, J .; Demchik, L.; J ones, J .; J ones, L.; Biernat,
L.; LoRusso, P.; Foster, B.; Heilbrun, L.; Rake, J .; Mattes, K.
C.; Perni, R.; Powles, R. G.; Hlavac, A. G.; Wentland, M. P.;
Coughlin, S. A.; Baker, L.; Valeriote, F. Antitumor activity of
N-[[1-[[2-(diethylamino)ethyl]amino]-9-oxo-9H-thioxanthen-4-yl]-
methyl]methanesulfonamide (WIN 33377) and analogues. Exp.
Opin. Invest. Drugs 1994, 3, 1281-1292.
Ack n ow led gm en t. The contributions of Chiab Pan-
chapor, Susan Pugh, Lisa Polin, Kathryn White, J ui-
wanna Knight, Lisa Demchik, and Lynne J ones for
generating in vivo tumor biology data at Wayne State
University are gratefully acknowledged. In vivo studies
at WSU were supported by CA 46560.
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