Design of cancer-specific antitumor agents based on aziridinylcyclopent[b]indoloquinones

Article abstract of DOI:10.1021/jm990466w

The merits of N-unsubstituted indoles and cyclopent[b]indoles as DNA- directed reductive alkylating agents are described. These systems represent a departure from N-substituted and pyrrolo[1,2-a]-fused systems such as the mitomycins and mitosenes. The cyclopent[b]indole-based aziridinylquinone system, when bearing an acetate leaving group with or without an N-acetyl group, was cytotoxic and displayed significant in vivo activity against syngeneic tumor implants. These analogues were superior to the others studied in terms of both high specificity for the activating enzyme DT-diaphorase and high percent DNA alkylation. Alkylation by a quinone methide intermediate as well as by the aziridinyl group could lead to cross-linking. The possible metabolites of the most active indole species were prepared and found to retain cytotoxicity, suggesting that in vivo activity could be sustained. The indole systems in the present study display selectivity for melanoma and, depending on the substituents present, selectivity for non-small-cell lung, colon, renal, and prostate cancers. The cancer specificities observed are believed to pertain to differential substrate specificities for DT- diaphorase.

Full text of DOI:10.1021/jm990466w

J . Med. Chem. 2000, 43, 457-466
457
Design of Ca n cer -Sp ecific An titu m or Agen ts Ba sed on
Azir id in ylcyclop en t[b]in d oloqu in on es
Chengguo Xing, Ping Wu, and Edward B. Skibo*
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604
Robert T. Dorr
Arizona Cancer Center, University of Arizona, Tucson, Arizona
Received September 13, 1999
The merits of N-unsubstituted indoles and cyclopent[b]indoles as DNA-directed reductive
alkylating agents are described. These systems represent a departure from N-substituted and
pyrrolo[1,2-a]-fused systems such as the mitomycins and mitosenes. The cyclopent[b]indole-
based aziridinylquinone system, when bearing an acetate leaving group with or without an
N-acetyl group, was cytotoxic and displayed significant in vivo activity against syngeneic tumor
implants. These analogues were superior to the others studied in terms of both high specificity
for the activating enzyme DT-diaphorase and high percent DNA alkylation. Alkylation by a
quinone methide intermediate as well as by the aziridinyl group could lead to cross-linking.
The possible metabolites of the most active indole species were prepared and found to retain
cytotoxicity, suggesting that in vivo activity could be sustained. The indole systems in the
present study display selectivity for melanoma and, depending on the substituents present,
selectivity for non-small-cell lung, colon, renal, and prostate cancers. The cancer specificities
observed are believed to pertain to differential substrate specificities for DT-diaphorase.
Ch a r t 1
In tr od u ction
Previous findings from this laboratory showed that
small molecule reductive alkylating agents called pyr-
rolobenzimidazoles or PBIs (Chart 1) show a high degree
of specific cytotoxicity for some cancers.1 This specificity
is very likely due to the presence of the two-electron
reducing enzyme DT-diaphorase, which reduces the PBI
in these cells.2,3 Reduction of the PBI to the correspond-
ing hydroquinone causes the aziridine nitrogen to
become more basic resulting in protonation and nucleo-
philic attack (alkylation). Similarly, indoloquinones such
as mitomycin C4 and EO95 (Chart 1) require two-
electron reductive activation by DT-diaphorase.
The present article discusses the design of selective
cytotoxic agents based on the aziridinylindoloquinones
shown in Chart 2. Although indoloquinone-based anti-
tumor agents are well-known, the compounds in Chart
2 possess one distinguishing feature: the N-unsubsti-
tuted indole system. Typically, the indoloquinone-based
antitumor agents (mitomycins and mitosenes) have an
alkyl substituent at this position. We were interested
in a possible hydrogen bond donor role in the DNA
major groove for the indole NH moiety. Previously, we
documented DNA major groove hydrogen-bonding in-
teractions with N-protonated reduced PBI antitumor
agents.6 Also, the quinone methide species derived from
the pyrrolo[1,2-a]-fused and the cyclopent[b]-fused sys-
tems, shown in the inset of Chart 2, was expected to
possess different stability and reactivity patterns. Fi-
nally, the differential DT-diaphorase substrate specific-
ity of these novel indole analogues could lead to cancer
specificity. Cyclopent[b]indole systems, 3c and its N-
acetyl analogue 14, possessed high in vivo activity and
high cancer specificity making them suitable compounds
for further development.
Resu lts a n d Discu ssion
Syn th esis. The preparation of the indole series 1 was
carried out as described in Scheme 1. The nitration and
reduction of the previously prepared indole 47 afforded
5, which was converted to quinone derivatives by
reduction/Fremy oxidation.8,9
The indole series 2, which have the methyl and
aziridinyl groups of 1 interchanged, were prepared by
starting with 7 as outlined in Scheme 2. Access to 7 was
possible employing the J app-Klingmann/Fischer indole
reactions starting with p-toluidine. This procedure is
analogous to a reported indole synthesis starting with
p-anisidine.10
* To whom correspondence should be addressed. Tel: 480-965-3581.
Fax: 480-965-2747. E-mail: ESkibo@ASU.EDU.
10.1021/jm990466w CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/19/2000

458 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Xing et al.
Ch a r t 2
Sch em e 2
Sch em e 3
Sch em e 1
borohydride and H2/Pd on carbon, respectively, followed
by Fremy oxidation to afford 12 and 13. The formation
of 12 is believed to arise from the following three-step
process: elimination of water from 11 resulting in a
quinone methide, addition of methanol solvent to this
species, and finally Fremy oxidation to the quinone
product. Cyclopent[b]indole 12 was converted to cyclo-
pent[b]indole 3d . The alcohol derivative 3b was then
converted to 3c, 3e, and 14.
The preparation of the cyclopentane-fused indole
series 3 was carried out as outlined in Scheme 3 starting
with 10. The preparation of 10 was possible by employ-
ing the Fischer indole reaction starting with 1,2-
cyclopentanedione11 and p-toluidine. The nitration of 10
afforded only the 8-nitro derivative, identifiable by an
AB quartet in the aromatic region with a J value ) 8.7
Hz. The carbonyl and nitro groups were reduced by

Cancer-Specific Antitumor Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 459
Ta ble 1. Log LC50 (concentration in mol/L causing 50%
lethality) Values and Histological Cancer Type for the Indole
Series 1a -ca
Ta ble 4. Results of Indoles 1c and 2c and Cyclopent[b]indole
3c in the B-16 Melanoma Model in C57/bl Mice (run at
University of Arizona Cancer Center)a
cancer
1a
1b
1c
dose
1c
2c
3c
leukemia
non-small-cell lung
colon
CNS
melanoma
ovarian
renal
prostate
breast
> -4
> -4
> -4
control
1 mg/kg
3 mg/kg
11.39
10.94
15.82
11.39
13.83
8.59
11.39
6.67
6.92
-4.29
-4.37
-4.48
> -4
> -4
-4.42
> -4
> -4
> -4
> -4
-4.32
> -4
-5.39
> -4
-4.53
-4.78
-4.56
-5.08
-4.83
-5.64
-4.46
-4.54
-5.01
-4.57
a
These compounds were studied at two dose levels: 1 and 3
mg/kg ip on days 1, 5, and 9 after tumor implantation into the
front flank muscle. Shown in the table are the values for the area
under the tumor growth curve (AUC) for indoles 1c and 2c and
cyclopent[b]indole 3c administered at 1 and 3 mg/kg doses. The
control was obtained with drug-free animals.
a
Each cancer type represents the arithmetic average of six to
eight different cancer cell lines. Melanomas are the most sensitive
cancer to this series of indoles, followed by prostate and colon for
the more active indole analogues 1b and 1c.
Ta ble 2. Log LC50 (concentration in mol/L causing 50%
lethality) Values and Histological Cancer Type for the Indole
Series 2a -ca
cancer
2a
2b
2c
leukemia
non-small-cell lung
colon
CNS
melanoma
ovarian
renal
prostate
breast
> -4
> -4
> -4
-4.3
-4.1
-4.1
-4.05
> -4
-4.15
> -4
> -4
> -4
> -4
-4.2
-4.05
-4.4
> -4
> -4
-4.15
> -4
> -4
-4.23
-4.2
-4.14
> -4
> -4
-4.05
a
Each cancer type represents the arithmetic average of six to
eight different cancer cell lines. The large log LC50 values in the
acetate-substituted indoles 2c and to a lesser extent 2b, compared
to 1c and 1b, respectively, in Table 1, reflect the substantial loss
of cytotoxicity observed when the aziridinyl group is moved from
the 5- to the 6-position.
F igu r e 1. Results of cyclopent[b]indole 14 in the B-16
melanoma model in C57/b1 mice (run at University of Arizona
Cancer Center). This compound was studied at three dose
levels: 2, 3, and 5 mg/kg ip on days 1, 5, and 9 after tumor
implantation into the front flank muscle. Shown in the plot
are the AUC values as a function of time for each dosage. The
control was obtained with drug-free animals.
Ta ble 3. Log LC50 (concentration in mol/L causing 50%
lethality) Values and Histological Cancer Type for the
Cyclopent[b]indole Series 3b-e and 14a
cancer
3e
3d
3c
3b
14
active indoles shown in Table 1, 1b and 1c, possess a
high specificity for lung, melanoma, and prostrate
cancers. Studies described in the following section reveal
that the position of the aziridinyl ring did not influence
either reduction by rat liver DT-diaphorase or the
percent alkylation of DNA.
leukemia
non-small-cell lung
colon
CNS
melanoma
ovarian
renal
prostate
breast
> -4
-4.06 -4.3 -4.06 > -4
-5.37
-4.2 -4.65 -4.22
-4.32 -4.85 -4.45
-4.17 -4.7 -4.54
-4.92 -4.99 -4.85
-4.42
-5.44
-5.13
-5.99
-4.77
-4.75
-5.06
-4.18
-4.78
-4.71
-4.5
-4.86 > -4
-4.4 -4.05
-5.34
-4.8
-4.9
-4.58 -4.72 -4.73
-4.46 -4.63 -4.5
-4.33 -4.53 -4.47
When the relatively inactive analogues 2 are func-
tionalized with the fused cyclopentane ring to afford
cyclopent[b]indoles 3, cytotoxic activity is substantially
restored despite the position of the aziridinyl group,
Table 3.
a
Each cancer type represents the average of six to eight
different cancer cell lines. The lower log LC50 values show the
increase of cytotoxicity with the addition of the fused cyclopentane
ring. Melanomas are again the most sensitive cancer to this series
of indoles followed by renal, colon, and non-small-cell lung cancers
for the more active cyclopent[b]indole analogues 3c, 3e, and 14.
Our studies with rat liver DT-diaphorase described
below revealed that the addition of a fused cyclopentane
ring to the indole system can increase the substrate
specificity for this enzyme greatly. Furthermore, this
fused ring increases the percent alkylation of DNA.
Consequently, the addition of the fused cyclopentane
ring enhances both cytotoxicity and in vivo activity. This
phenomenon is analogous to that seen when a fused
pyrrole ring is added to the benzimidazole system.14
In Vitr o Scr een in g Resu lts. The cytotoxicities of
the indole series 1-3 against a variety of cancer cell
lines are compared in Tables 1-3. These data were
derived from National Cancer Institute mean graph
data and are not direct reproductions thereof. For each
histologic cancer type, the average -log LC50 value was
determined from an NCI panel consisting of six to eight
human cancer cell lines.12,13
Comparison of the data in Tables 1 and 2 reveals the
importance of the position of the aziridinyl group: when
this group was moved from the 5- to the 6-position
cytotoxic activity was substantially decreased. The
importance of the position of the aziridinyl ring in
cytotoxicity was also apparent in the PBIs.14 The most
In Vivo Scr een in g Resu lts. Shown in Table 4 and
Figure 1 are B16 melanoma syngraft assays15 which
confirm the in vivo activity of the cyclo[b]indoles. Thus
cyclopent[b]indole 3c (1 and 3 mg/kg on days 1, 5, and
9 after subcutaneous implantation of 105 cells in the
front flank on day 0) was able to reduce the tumor mass
substantially. In contrast, indole 2c decreases tumor

460 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Xing et al.
Sch em e 4
F igu r e 2. Bar graph of the specificity (Vmax/KM) for rat liver
DT-diaphorase and the percent DNA base pair reductive
alkylation of 600-bp calf thymus DNA for the indole series 1-3.
Inspection of the data in Figure 2 reveals that the
position of the aziridinyl group influences the DT-
diaphorase reductase activity somewhat (compare 1a
with 2a ) but has little effect on the reductive alkylation
of DNA. In fact, for the indole series 1 and 2, neither
the position of the aziridinyl group nor the substituent
promote DNA alkylation. For the three indole series,
the acetate substituent substantially increases the
specificity for DT-diaphorase compared to the hydroxyl
substituent. Inspection of the components of the speci-
ficity (Vmax/KM) for the acetate-substituted indoles re-
veals that the acetate substituent influences the KM
much like the other substituents (10-14 × 10-4 M), but
also provides up to a 10-fold higher Vmax value (up to
263 × 10-9 M/s).
mass only at the 3 mg/kg dose, and 1c is inactive at
both concentrations.
Unexpectedly, the most cytotoxic compound of Table
3 (3e) was completely inactive in the B16 syngraft
assays. This finding suggests that oxidative metabolism
will deactivate the cyclopent[b]indole series 3. Similarly,
the PBIs are completely noncytotoxic (50% lethal con-
centration or LC50 > 10-4 M) upon oxidation to the
ketone at the 3-position, Scheme 4. However, metabo-
lism by acyl transfer to the indole NH group to afford
cyclopent[b]indole 14 will serve to enhance cytotoxicity.
Conversely, deacetylation reactions in the cyclopent[b]-
indole series will result in sustained activity.
The results enumerated above indicate that the
development of cancer-specific antitumor agents is still
possible, although the selection of active analogues is
still carried out in a random manner. This suggests that
a combinatorial synthesis/testing approach would be
required to identify the best compounds. For example,
our cytotoxicity studies show that 3c and 14 display in
vitro potency with high selectivity as well as in vivo
selectivity against melanoma, renal, and colon cancers.
DT-Dia p h or a se Su bstr a te Activity a n d P er cen t
DNA Red u ctive Alk yla tion . Although DT-diaphorase
is present in both normal and cancerous tissues,16 this
enzyme may be the key to developing selective anti-
tumor agents. Depending on the organ and species
source, the substrate and inhibition properties of the
enzyme can vary widely.17,18 The structure of the
enzyme even varies by ethnic origin, which may be a
factor in the success of cancer chemotherapy in some
patients.19 The differential cytotoxicity of the indole
series 1 and 3 discussed in the last section could
originate from differential DT-diaphorase specificity, as
well as differential concentrations of this enzyme.
In this section, we compare the Vmax/KM for reduction
of the indoles with purified rat liver DT-diaphorase as
well as the percent reductive alkylation of 600-bp calf
thymus DNA, Figure 2. The data in Figure 2 shows that
Addition of the fused cyclopentane ring increases both
the specificity for DT-diaphorase reduction as well as
the percent alkylation of DNA for all analogues. Com-
parison of the acetate derivatives 2c and 3c reveals a
large increase in substrate specificity for DT-diaphorase
as well as a large increase in the percent DNA alkyl-
ation. The Vmax rather than the KM is largely responsible
for this specificity difference. Addition of a fused ring,
either fused pyrrole or fused cyclopentane, appears to
be important for DT-diaphorase activity in some sys-
tems. Examples from this laboratory even show that two
fused pyrrolo rings are better than one, Chart 3. This
is not a general phenomenon since reductive alkylating
compounds such as DZQ20 and EO921 do not require
fused rings for activation by DT-diaphorase.
The importance of the fused ring in reductive alkyl-
ation is very likely related to quinone methide formation
upon elimination of acetate from the hydroquinone
species, Scheme 5. Shown in this scheme are the two
possible alkylation reactions of reduced 3c starting with
the nucleophile-mediated opening of the protonated
aziridinyl group followed by quinone methide formation
and nucleophile trapping of this species. The presence
of the fused ring would promote elimination because the
more substituted methide double bond is the product
(in contrast to the terminal quinone methide resulting
from reduced 1c and 2c). The indole shown in the inset
of Scheme 5 was prepared in this laboratory and found
to be noncytotoxic in the NCI’s 60-cancer cell line panel.
This result was surprising since this indole could form
a quinone methide species upon reduction and also was
substituted with a reactive aziridinyl ring. Apparently,
3c not only has the highest Vmax/KM (26.21 × 10-4 s-1
)
but also has the highest percent alkylation of DNA.
These observations provide a clear rationale for the
observed in vivo activity of 3c. Likewise the active
compound cyclpent[b]indole 14 is an excellent substrate
for DT-diaphorase and readily alkylates DNA.

Cancer-Specific Antitumor Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 461
Sch em e 5
Ch a r t 3
COMP ARE An a lysis. COMPARE was developed at
the NCI to compare the patterns of cytotoxicity in their
60-cell line cancer panel.12 Antitumor agents with
identical mechanisms of action possess identical or
nearly identical cytotoxicity patterns (correlation coef-
ficient > 0.8). For example anthracycline analogues
(doxorubicin, rubidazone, daunamycin) have a high
correlation (>0.9) with each other as do the DNA
alkylating agents (chlorambucil, thiotepa, triethylene-
melamine). The cytotoxicity (LC50) and growth inhibi-
tion (GI50) profiles of 3c were compared with those of
known antitumor agents in the NCI archives. We
observed a high correlation of the GI50 profile of 3c with
the concentration of cellular DT-diaphorase (correlation
coefficient ) 0.76). The cytotoxicity profile of 3c cor-
related well with that of mitomycin C (correlation
coefficient ) 0.7). These correlations are consistent with
the diaphorase-mediated activation and the possible
DNA cross-linking reaction of reduced 3c.
Con clu sion s
The above data show the merits of the cyclopent[b]-
indole ring system in the design of new DNA-directed
reductive alkylating agents. The combination of cyclo-
pent[b] fusion and the indole NH (cyclopent[b]indole 3c)
results in a system exhibiting both cytotoxicity and
antitumor activity. However, the addition of the N-
acetyl group (cyclopent[b]indole 14) enhances antitumor
activity greatly.
The role of the fused cyclopentane ring in cytotoxicity
is as yet unclear, and studies in this area are underway.
We postulate that decreased ring strain may play a role
in cyclopent[b]-fused quinone methide stability. The
cyclopent[b]indole system represents a departure from
the pyrrolo[1,2-a] fusion moieties typically found in the
mitomycins and mitosenes as well as the many synthetic
analogues thereof such as the PBI antitumor agents
developed in this laboratory.22-27
Compound series 1 and 3 show a high degree of cancer
selectivity with melanoma being the common target
cancer of both series. Similarly, the PBI compounds also
target this type of cancer, probably because of its high
DT-diaphorase concentrations. Agents in series 1 also
target colon and prostate cancers, while agents in series
3 target colon along with renal cancers. The mechanism
ring fusion and the aziridinyl substituent location must
be important components of cytotoxicity.
The role of ring fusion in cytotoxicity may be related
to ring strain. Models shown in Figure 3 reveal that the
quinone methide derived from cyclopent[b]indole is more
stable (based on a lower positive heat of formation) than
that derived from the pyrrolo[1,2-a]indole. The origin
of the difference in stability is the change in geometry
of the indole nitrogen when the quinone methide is
formed. While in the aromatic indole form, this nitrogen
has sp2 geometry but becomes tetrahedral when the
system becomes quinonoid. Strain results if this nitro-
gen is part of a five-membered ring. Thus the quinone
methide derived from the cyclopent[b]indole system may
form faster and have a low enough reactivity to trap
nucleophiles selectively.

462 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Xing et al.
F igu r e 3. Minimized structures (Austin model I, closed shell wave function) of the cyclopent[b]indole and pyrrolo[1,2-a]indole
quinone methides.
2,3,6-Tr im eth yl-5-m eth oxyin dole-4,7-dion e (6a) was pre-
pared from 5 by the following two-step procedure. A suspension
of 500 mg (1.71 mmol) of 5 in 20 mL of methanol, containing
250 mg of (5%) Pd on charcoal and a few drops of 2 N
hydrochloride, was shaken under 50 psi of H2 for 8 h. The
reaction mixture was filtered through Celite, and the filtered
cake was washed with methanol. The solvent was evaporated
off to afford the amine as a residue, which was used in the
next step without further purification.
The residue was dissolved in a solution of 1.5 g of Fremy
salt and 0.75 g of monobasic potassium phosphate in 150 mL
of water. The reaction was stirred at room temperature for 4
h and then extracted three times with 50 mL of chloroform.
The dried extracts (Na2SO4) were concentrated to an oil and
then chromatographed employing silica gel with chloroform
as the eluant. The product was recrystallized from chloroform/
hexane to afford pure 6a : 92 mg (24%) yield; mp 235 °C; TLC
(chloroform/methanol, 90:10) Rf ) 0.69; IR (KBr pellet) 3242,
2926, 2854, 1639, 1498, 1460, 1367, 1305, 1222, 1111, 999, 949,
763 cm-1; 1H NMR (CDCl3) δ 9.62 (1H, bs, indole proton), 4.00
(3H, s, 5-methoxyl) 2.25, 2.23 and 1.95 (9H, 3s, 2,3,6-tri-
methyl); MS (EI mode) m/z 219 (M+), 204 (M+ - CH3), 190,
176, 160, 148. Anal. (C12H13NO3) C, H, N.
of 3c cytotoxicity must closely resemble that of mito-
mycin C,22,28,29 based on the high COMPARE correla-
tion. These studies show that it is possible to design
cancer-specific agents based on small molecules.
Exp er im en ta l Section
All solutions and buffers for kinetic, DNA, and electrophore-
sis studies used doubly distilled water. All analytically pure
compounds were dried under high vacuum in a drying pistol
over refluxing toluene. Elemental analyses were run at
Atlantic Microlab, Inc., Norcross, GA. All TLCs were performed
on silica gel plates using a variety of solvents and a fluorescent
indicator for visualization. IR spectra were taken as thin films
1
and the strongest absorbances are reported. H NMR spectra
were obtained from a 300-MHz spectrometer. All chemical
shifts are reported relative to TMS.
The syntheses of new compounds are outlined below.
2-(H yd r oxym et h yl)-3,6-d im et h yl-5-m et h oxy-4-n it r o-
in d ole (5) was prepared from 4 by the following two-step
reaction. To a mixture consisting of 500 mg (2.02 mmol) of 4
and 15 mL of glacial acetic acid, cooled to 15 °C with a ice
bath, was added 0.8 mL of nitric acid (69-71%). The reaction
mixture was stirred for 15-20 min at room temperature and
then poured into 100 mL of ice water and stirred for 10 min.
The solids were filtered off and were washed with cold water.
The wet solid was dissolved in chloroform, dried over Na2SO4,
and concentrated to a residue, which was recrystallized from
chloroform/hexane to afford nitrated 4 as yellow needles: 423
mg (70%) yield; mp 100-102 °C; TLC (chloroform/methanol,
90:10) Rf ) 0.72; IR (KBr pellet) 3447, 3319, 2926, 1676, 1529,
1282, 1207, 1155, 1026, 869, 781 cm-1; 1H NMR (CDCl3) δ 8.76
(bs, 1H, indole), 7.29 (1H, s, H-7), 4.42 (2H, d, J ) 7.2 Hz,
methylene of ethyl), 3.87 (3H, s, 5-methoxy), 2.44 (6H, s, 3,6-
dimethyl), 1.42 (3H, t, J ) 7.2 Hz, methyl of ethyl); MS (EI
mode) m/z 292 (M+), 275 (M+ - OH), 263 (M+ - CH2CH3), 246,
229, 215. Anal. (C14H16N2O5) C, H, N.
To a solution of 200 mg of lithium aluminum hydride and 5
mL of dried THF, chilled to 0 °C, was added 200 mg (0.68
mmol) of nitrated 4 in 10 mL of THF. The reaction was stirred
at 0 °C for 15 min and then slowly combined with 5 mL of
ethyl acetate and stirred for 5 min more. The solid residue
was filtered off utilizing Celite and the filtrate was concen-
trated to dark red oil, which was dissolved in chloroform and
washed with water. The chloroform was dried over Na2SO4
and concentrated to a solid residue, which was recrystallized
from chloroform/hexane to afford 5a as a yellow powder: 148
mg (87%) yield; mp 152-153 °C; TLC (chloroform/methanol,
90:10) Rf ) 0.41; IR (KBr pellet) 3421, 3284, 2924, 1637, 1523,
1460, 1365, 1213, 1112, 1012, 866, 781 cm-1; 1H NMR (CDCl3)
δ 8.33 (1H, bs, indole), 7.23 (1H, s, H-7), 4.83 (2H, d, J ) 5.7
Hz, 2-methylene), 3.87 (3H, s, 5-methoxy), 2.42 and 2.09 (6H,
2s, 3,6-dimethyl), 1.73 (1H, t, J ) 5.7 Hz, hydroxy); MS (EI
mode) m/z 250 (M+ ), 249 (M+ - H), 235 (M+ - CH3) 233 (M+
- OH), 215, 202, 189, 174. Anal. (C12H14N2O4) C, H, N.
2-(Hyd r oxym eth yl)-3,6-d im eth yl-5-m eth oxyin d ole-4,7-
d ion e (6b) was prepared from 5b by the following two-step
procedure. A suspension of 100 mg (0.4 mmol) of 5b in 15 mL
of methanol, containing of 95 mg of 5% Pd on charcoal, was
shaken under 50 psi of H2 for 45 min. The reaction mixture
was filtered through Celite, and the filtered cake was washed
with methanol. The solvent was evaporated off to afford the
amine as a greenish oil, which was used without further
purification.
The amine was dissolved in 5 mL of acetone and then
combined with 5 mL of 0.055 M KH2PO4 buffer. This mixture
was combined with a solution of 800 mg of Fremy salt in 40
mL of 0.055 M KH2PO4 buffer and the reaction mixture was
stirred at room temperature for 45 min. The completed
reaction was extracted five times with 20-mL portions of
chloroform. The dried extracts (Na2SO4) were concentrated to
an oil and then chromatographed on silica gel with chloroform
as the eluant. The product was recrystallized from chloroform/
hexane to afford 6b as a yellow solid: 45 mg (47%) yield; mp
196-198 °C; TLC (chloroform/methanol, 90:10) Rf ) 0.43; IR
(KBr pellet) 3445, 3240, 2951, 1638, 1508, 1465, 1307, 1111,
949, 746; 1H NMR (CDCl3) δ 9.67 (1H, bs, indole proton), 4.72
(2H, d, J ) 5.4 Hz, 2 methylene), 4.02 (3H, s, 5-methoxy), 2.31
(1H, t, J ) 5.4 Hz, hydroxy), 2.26 and 1.96 (6H, 2s, 3.6-
dimethyl); MS (EI mode) m/z 235 (M+), 220 (M+ - CH3), 218
(M+ - OH), 202, 189, 174. Anal. (C12H13NO4) C, H, N.
5-Azir id in yl-2,3,6-tr im eth ylin d ole-4,7-d ion e (1a ). To a
solution of 42 mg (0.19 mmol) of 6a in 8 mL of methanol was
added 0.87 mL of ethylenimine. The reaction was stirred at
room temperature for 2.5 h and then the reaction mixture was
placed directly on a silica gel chromatography column employ-
ing chloroform as the eluant. The product was recrystallized

Cancer-Specific Antitumor Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 463
from chloroform/hexane to afford 1a as a red solid: 27 mg
(60%) yield; mp 251-253 °C; TLC (chloroform/methanol, 90:
10) Rf ) 0.63; IR (KBr pellet) 3213, 2922, 1664, 1626, 1587,
complete addition, the ice bath was removed and the reaction
stirred for 4 h at room temperature. The solution was vacuum-
dried and purified by a silica gel flash column using CHCl3 as
eluant. The nitrated derivative of 7 was recrystallized from
CHCl3 and hexane as a yellow solid: 43.3 mg (16%) yield; mp
1
1498, 1460, 1373, 1346, 1248, 1153, 1101, 960, 767 cm-1; H
NMR (DMSO-d6) δ 12.18 (1H, bs, indole proton), 2.19 (4H, s
ethylene) 2.09, 2.08 and 1.89 (9H, 3s, 2,3,6-trimethyl); MS (EI
mode) 230 (M+), 215 (M+ - CH3), 201, 185, 174. Anal.
(C13H14N2O2) C, H, N.
1
163-164 °C; TLC (CHCl3) Rf ) 0.28; H NMR (CDCl3) δ 8.87
(1H, bs, indole proton), 7.40 (1H, d, J ) 8.4 Hz, 6-proton) 7.18
(1H, d, J ) 8.4 Hz, 7-proton), 4.43 (2H, q, J ) 7.2 Mz,
methylene of ethyl), 2.47 and 2.40 (6H, s, 3,5-methyls) 1.43
(3H, t, J ) 7.2 Hz, methyl of ethyl); IR (KBr pellet) 3337, 2926,
1687, 1516, 1363, 1344, 1259, 1201, 1016, 775 cm-1, MS (EI
mode) 262 (M+), 245 (M+ - OH), 216, 199, 185, 169, Anal.
(C13H14N2O4) C, H, N.
5-Azir id in yl-2-(h yd r oxym et h yl)-3,6-d im et h ylin d ole-
4,7-d ion e (1b). To a solution of 15 mg (0.06 mmol) of 6b in 4
mL of methanol was added 0.35 mL (0.48 mmol) of ethylen-
imine. The reaction mixture was stirred at room temperature
for 2.5 h and then concentrated to a residue, which was
recrystallized from chloroform/hexane to afford 1b as a red
solid: 15 mg (93%) yield; mp 224-225 °C; TLC (chloroform/
methanol, 90:10) Rf ) 0.42; IR (KBr pellet) 3493, 3246, 2924,
The ester reduction step was the same as that employed
for the preparation of 6b: 92% yield; mp 120 °C; TLC
1
(chloroform/methanol, 90:10) Rf ) 0.30; H NMR (DMSO-d6
)
1685, 1618, 1502, 1377, 1350, 1253, 1151, 1020, 828, 752 cm-1
;
δ 11.38 (1H, bs, indole proton), 7.42 (1H, d, J ) 8.1 Hz, 6-H),
7.00 (1H, d, J ) 8.1 Hz, 7-H), 5.25 (1H, t, J ) 5.7 Hz, hydroxy
proton), 4.58 (2H, d, J ) 5.7 Hz, methylene), 2.29 (3H, s,
3-methyl) 1.98 (3H, s, 5-methyl); IR (KBr pellet) 3420, 3256,
2925, 1628, 1516, 1354, 1323, 1188, 999, 806 cm-1; MS (EI
mode) 220 (M+), 203 (M+ - OH), 185, 173, 156, 144, 130, 115.
Anal. (C11H12N2O3) C, H, N.
1H NMR (CDCl3) δ 9.39 (1H, bs, indole proton), 4.69 (2H, d, J
) 5.7 Hz, 2-methylene), 2.32 (4H, s, ethylene) 2.26 and 2.05
(6H, 2s, 3,6-dimethyl); MS (EI mode) m/z 246 (M+), 229 (M+
- OH), 213, 201, 190, 172. Anal. (C13H14N2O3) C, H, N.
2-(Acetoxym eth yl)-5-a zir id in yl-3,6-d im eth ylin d ole-4,7-
d ion e (1c). To a mixture of 10 mg of 1b (0.04 mmol) and 5
mg (0.04 mmol) of 4-(dimethylamino)pyridine (DMAP) in 5 mL
of chloroform was added 100 mg of acetic anhydride. The
reaction was stirred at room temperature for 25 min and then
added directly to a silica gel chromatography column employ-
ing chloroform as the eluant. The purified 1c was recrystallized
from chloroform/hexane: 11 mg (95%) yield; mp 210-212 °C;
TLC (chloroform/methanol, 90:10) Rf ) 0.68; 1H NMR (CDCl3)
δ 9.38 (1H, bs, indole proton), 5.03 (2H, s, 2-methylene), 2.32
(4H, s, ethylene), 2.31 (3H, s, 3-methyl) 2.08 and 2.05 (6H, 2s,
methyls); IR (KBr pellet) 3207, 2924, 1734, 1664, 1624, 1566,
2-(Hydr oxym eth yl)-3,5-dim eth ylin dole-4,7-dion e (9) was
prepared from 8 by the same procedure employed for the
preparation of 6b: 57% yield; mp 203-205 °C; TLC (chloroform/
1
methanol, 90:10) Rf ) 0.2; H NMR (DMSO-d6) δ 12.38 (1H,
bs, indole proton), 6.42 (1H, q, J ) 1 Hz, 6-proton), 5.02 (1H,
t, J ) 5.4 Hz, hydroxy proton), 4.38 (2H, d, J ) 5.4 Hz,
methylene), 2.20 (3H, s, 3-methyl), 1.94 (3H, d, J ) 1 Hz,
5-methyl); IR (KBr pellet) 3356, 3209, 2958, 1639, 1602, 1491,
1375, 1271, 1195, 1118, 993, 889, 781 cm-1; MS (EI mode) m/z
205 (M+), 188 (M+ - OH), 176, 160, 148, 131, 119. Anal.
(C11H11NO3) C, H, N.
1500, 1361, 1350, 1242, 1209, 1155, 1020, 962, 817 781 cm-1
;
MS (EI mode) m/z 288 (M+), 245 (M+ - CH3-CdO), 228, 213,
6-Azir id in yl-2-(h yd r oxym et h yl)-3,5-d im et h ylin d ole-
4,7-d ion e (2b). To a solution of 31.5 mg (0.15 mmol) of 9 in
10 mL of methanol was added 1 mL of ethylenimine and the
resulting mixture stirred at room temperature for 2 days. The
solution was concentrated to dryness and the solid residue was
purified by flash chromatography on silica gel using 1%
methanol in chloroform as the eluant. The red product fraction
was dried and recrystallized from chloroform and hexane: 8.1
mg (21%) yield; mp 227-231 °C; TLC (chloroform/methanol,
80:20) Rf ) 0.62; IR (KBr pellet) 3429, 3238, 2926, 1637, 1587,
201, 185. Anal. (C15H16N2O4) C, H, N.
Eth yl 3,5-Dim eth ylin d ole-2-ca r boxyla te (7). To a solu-
tion of 5.6 g (0.052 mol) of p-toluidine in 15 mL of concentrated
HCl and 25 mL of H2O was added dropwise a solution of 3.9
g (0.057 mol) of NaNO2 in 5 mL of H2O at -5 °C. After
complete addition, the mixture was stirred at 0 °C for 15 min
and brought to pH 3-4 by addition of 5 g of sodium acetate.
In a separate flask, a solution of 9 g (0.055 mol) of ethyl
R-ethylacetoacetate in 40 mL of EtOH was cooled to 0 °C and
combined with 3.5 g of KOH (0.064 mol) in 10 mL of H2O. To
this solution was added 70 g of ice followed by addition of the
diazonium salt prepared above. The mixture was then adjusted
to pH 5-6 and stirred at 0 °C for 15 h. The completed reaction
was extracted five times with 50-mL portions of CH2Cl2 and
the combined extracts were washed with brine and dried over
Na2SO4. Most of the solvent was removed under reduced
pressure, and the liquid residue was added dropwise to a
solution of 14.5% ethanolic HCl at reflux. After refluxing this
mixture for 2 h, the solvent was removed under reduced
pressure and the residue was combined with a mixture of 50
mL of water and 100 mL of CH2Cl2. The CH2Cl2 layer was
removed and the aqueous layer was extracted three times with
50-mL portions of CH2Cl2. The combined extracts were dried
over Na2SO4 and concentrated to a residue, which was applied
to a silica gel column prepared with CH2Cl2. Product fractions
were evaporated to afford a white solid: 5.74 g (51%) yield;
mp 131-133 °C; TLC (CHCl3) Rf ) 0.25; IR (KBr pellet) 3306,
2924, 2854, 1680, 1548, 1475, 1384, 1332, 1263, 798 cm-1; 1H
NMR (CDCl3) δ 8.56 (1H, bs, indole proton), 7.43 (1H, s,
4-proton), 7.26 (1H, d, J ) 8.4 Hz, 7-proton), 7.15 (1H, d, J )
8.4 Hz, 6-proton), 4.40 (2H, q, J ) 7.2 Hz, methylene) 2.59
and 2.46 (6H, 2s, 3,5- dimethyl), 1.42 (3H, t, J ) 7.2 Hz, methyl
of ethyl); MS (EI mode) m/z 217(M+), 188 (M+ - CH2CH3), 171,
142, 115. Anal. (C13H15NO2) C, H, N.
1506, 1378, 1336, 1271, 1155, 1058, 950, 827, 748 cm-1
NMR (DMSO-d6) δ 12.21 (1H, bs, indole proton), 4.96 (1H, t,
) 4.8 Hz, hydroxyl proton), 4.36 (2H, d, J ) 4.8 Hz,
;
1H
J
methylene), 2.20 (4H, s, ethylene), 2.17 & 1.90 (6H, 2s, 3,5-
dimethyl); MS (EI mode) m/z 246 (M+), 229 (M+ - OH) 213,
201, 190, 172. Anal. (C13H14N2O3) C, H, N.
2-(Acetoxym eth yl)-6-a zir id in yl-3,5-d im eth ylin d ole-4,7-
d ion e (2c). To a solution of 13 mg (0.052 mmol) of 2b and 12
mg of DMAP in 4 mL of dried methylene chloride was added
80 mg of acetic anhydride. The reaction mixture was stirred
for 1 min and then placed directly on a silica gel column with
chloroform/acetone (98:2) as the eluant. The product was
recrystallized from chloroform/hexane: 13 mg (89%) yield; mp
218-220 °C; TLC (chloroform/methanol, 90:10) Rf ) 0.78; IR
(KBr) 3464, 3240, 2926, 1751, 1637, 1571, 1334, 1251, 1334,
1251, 1057 cm-1; 1H NMR (CDCl3) δ 9.28 (1H, bs, indole NH),
5.04 (2H, s, methylene), 2.32, 2.09, 2.07 (9H, 3s, methyls), 2.27
(4H, s, aziridine); MS(EI mode) m/z 288 (M+), 245 (M+
CH3CO), 228, 213, 201, 185. Anal. (C15H16N2O4) C, H, N.
-
7-Meth yl-1,4-d ih yd r ocyclop en t[b]in d ol-3(2H)-on e (10).
To a solution of 290 mg (1.83 mmol) of p-tolylhydrazine
hydrochloride in 10 mL of ethanol was added a solution of 356
mg (3.62 mmol) of 1,2-cyclopentanedione in 10 mL of ethanol
and 10 mL of concentrated hydrochloric acid. This mixture was
refluxed at 110 °C for 3 h and then concentrated to a residue
under vacuum. This residue was purified by flash chromatog-
raphy on silica gel using 1% methanol in chloroform as the
eluant: 55 mg (16%) yield; mp 223 °C; TLC (chloroform/
methanol, 95:5) Rf ) 0.50; 1H NMR (CDCl3) δ 8.57 (1H, bs,
indole proton), 7.50 (1H, s, 8-proton), 7.35 & 7.24 (2H, AB
2-(Hyd r oxym eth yl)-3,5-d im eth yl-4-n itr oin d ole (8) was
prepared from 7 by the following two-step procedure. To a
solution of 219 mg (1.01 mmol) of 7 in 10 mL of acetic acid,
cooled in an ice/salt bath, was added dropwise a solution of
0.5 mL of nitric acid (69-71%) in 2 mL of acetic acid. After

464 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Xing et al.
quartet, J ) 8.1 Hz, 5 & 6 aromatic protons), 3.07 (2H, m,
1-methylene), 2.99 (2H, m, 2-methylene) 2.46 (3H, s, 5-methyl);
IR (KBr pellet) 3414, 3232, 2992, 1655, 1602, 1309, 1091, 802
1039, 952, 912 cm-1; MS (EI mode) m/z 217 (M+), 200 (M+
-
OH), 189 (M+ - CO), 174, 160, 146, 132. Anal. (C12H11NO3) C,
H, N.
cm-1; MS (EI mode) m/z 185 (M+), 170 (M+ - CH3), 157 (M+
-
6-Azir id in yl-1,4-d ih yd r o-3-m eth oxy-7-m eth yl-(2H)-cy-
clop en t[b]in d ole-5,8-d ion e (3d ) a n d 6-Azir id in yl-1,4-d i-
h yd r o-3-h yd r oxy-7-m eth yl-(2H)-cyclop en t[b]in d ole-5,8-
d ion e (3b). To a solution of 0.2 mmol of 12 or 13 in 20 mL of
methanol was added 0.5 mL of ethylenimine and the solution
was stirred at room temperature for 4 days. The solvent was
removed under vacuum and the residue was purified by flash
chromatography on silica gel using 5% acetone in chloroform
as the eluant, and either product was recrystallized from
chloroform/hexane as red prisms.
3b: 74% yield; mp 193-195 °C; TLC (chloroform/methanol,
90:10) Rf ) 0.25; 1H NMR (CDCl3) δ 9.24 (1H, bs, indole NH),
5.20 (1H, m, 3-methine proton). 3.51 (1H, d, J ) 4.5 Hz,
hydroxy proton), 2.91, 2.73 and 2.30 (4H, 3m, 1 & 2-methylene
protons) 2.29 (4H, s, aziridine), 2.07 (3H, s, 7-methyl); IR (KBr
pellet) 3448, 3160, 2960, 2926, 1655, 1325, 1041, 821, 748, 601
cm-1; MS (EI mode) m/z 258 (M+), 240 (M+ - H2O), 225 (M+
- H2O, CH3), 213, 184, 170, 158, 131. Anal. (C14H14N2O3‚H2O)
C, H, N.
3d : 81% yield; mp 186-188 °C; TLC (chloroform/methanol,
90:10) Rf ) 0.51; 1H NMR (CDCl3) δ 9.03 (1H, bs, indole NH),
4.79 (1H, m, 3-methine proton), 3.35 (3H, s, methoxy protons),
2.90, 2.76 and 2.42 (4H, 3m, 1 & 2-methylene protons), 2.29
(4H, s, aziridine), 2.07 (3H, s, 7-methyl protons); IR (KBr
pellet) 3277, 2924, 2859, 1655, 1643, 1591, 1340, 1097, 1026,
908, 825 cm-1; MS (EI mode) 272 (M+) 257 (M+ - CH3), 241
(M+ - CH3O), 225, 213, 200, 184, 172, 158, 132. Anal.
(C15H16N2O3) C, H, N.
3-Acet oxy-6-a zir id in yl-1,4-d ih yd r o-7-m et h yl-(2H )-cy-
clop en t[b]in d ole-5,8-d ion e (3c) a n d 3-Acetoxy-4-a cetyl-
6-azir idin yl-1,4-dih ydr o-7-m eth yl-(2H)-cyclopen t[b]in dole-
5,8-d ion e (14). To a solution of 21 mg (0.08 mmol) of 3b in 3
mL of methylene chloride with 22 mg of DMAP was added 32
mg (0.32 mmol) of acetic anhydride and the solution was
stirred at room temperature for 10 min. The reaction mixture
was then placed on a silica gel column employing methylene
chloride as the eluant.
CO), 128, 115. Anal. (C12H11NO) C, H, N.
1,4-Dih ydr o-3-h ydr oxy-7-m eth yl-8-n itr o-(2H)-cyclopen t-
[b]in d ole (11) was prepared from 10 by the following two-
step procedure. To a solution of 367 mg (1.98 mmol) of 10 in
20 mL of concentrated sulfuric acid was added dropwise over
15 min a solution of 199 mg (2.34 mmol) of sodium nitrate in
5 mL of concentrated sulfuric acid. The resulting solution was
stirred for another 15 min at 0 °C and then was poured over
200 g of crushed ice. The aqueous solution was extracted four
times with 100-mL portions of chloroform. The combined
extracts were washed with saturated sodium bicarbonate
solution, dried over sodium sulfate, and vacuum-dried. The
solid residue was recrystallized from chloroform/hexane to
afford the 8-nitro derivative of 10 as a yellow solid: 317 mg
(70%) yield; mp 268 °C dec; TLC (chloroform/methanol, 95:5)
Rf ) 0.62; 1H NMR (DMSO-d6) δ 12.29 (1H, s, indole proton),
7.66 & 7.57 (1H, AB quartet, J ) 8.7 Hz, 5 and 6 protons),
2.98 (m, 2H, 1-methylene), 2.87 (m, 2H, 2-methylene), 2.51 (3H,
s, 7-methyl); IR (KBr pellet) 3414, 3196, 2926, 1678, 1618,
1520, 1365, 1259, 1074, 835 cm-1; MS (EI mode) m/z 230 (M+),
213 (M+ - OH), 195, 183 (M+ - HNO2), 171, 154, 143. Anal.
(C12H10N2O3‚0.2H2O) C, H, N.
To a solution of 251 mg (1.09 mmol) of the product obtained
above in 40 mL of methanol was added a solution of 462 mg
(12.8 mmol) of sodium borohydride in 20 mL of methanol. This
solution was stirred at room temperature for 15 min followed
by the addition of 50 mL of water. The aqueous solution was
extracted four times with 50-mL portions of chloroform. The
extracts was dried over sodium sulfate and then vacuum-dried
to afford a solid residue, which was recrystallized from
chloroform/hexane: 213 mg (84%) yield; mp 142-144 °C; TLC
(chloroform/methanol, 90:10) Rf ) 0.50; 1H NMR (CDCl3) δ 8.42
(1H, bs, indole proton), 7.40 & 7.04 (2H, AB quartet, J ) 8.1
Hz, 5- and 6-protons), 5.36 (1H, m, 3-methine proton), 3.00 &
2.35 (4H, 2m, 1 & 2-methylenes protons), 2.59 (3H, s, 7-meth-
yl); IR (KBr pellet) 3508, 3237, 2970, 1631, 1500, 1354, 1074,
804 cm-1; MS (EI mode) m/z 232 (M+), 215 (M+ - OH), 185
(M+ - HNO2), 173, 156, 145. Anal. (C12H12N2O3) C, H, N.
1,4-Dih yd r o-3-m eth oxy-7-m eth yl-(2H)-cyclop en t[b]in -
d ole-5,8-d ion e (12) a n d 1,4-d ih yd r o-3-h yd r oxy-7-m eth yl-
(2H)-cyclop en t[b]in d ole-5,8-d ion e (13) were both prepared
from 11 by the following two-step procedure. A mixture of 101
mg (0.435 mmol) of 11 in 40 mL of methanol with 100 mg of
5% Pd on carbon was reduced under 50 psi H2 for 30 min. The
reaction mixture was filtered through Celite and the filtrate
concentrated to a residue, which was then dissolved in 5 mL
of acetone. To this solution was added a solution consisting of
390 mg (2.87 mmol) of monobasic potassium phosphate and
790 mg (2.94 mmol) of Fremy salt in 80 mL of water. The
resulting solution was stirred for 4.5 h and then extracted four
times with 50-mL portions of chloroform. The extracts were
dried (Na2SO4) and then concentrated to a residue. This
residue was purified by flash chromatography on silica gel
using 1% methanol in chloroform as the eluant and the
products were recrystallized from chloroform/hexane.
3c: 8.3 mg (34%) yield; mp 166-168 °C; TLC (chloroform/
methanol, 90:10) Rf ) 0.68; IR (KBr pellet) 3267, 3065, 2926,
2856, 1736, 1637, 1377, 1329, 1251, 1089, 601 cm-1; 1H NMR
(CDCl3) δ 9.26 (1H, bs, indole proton), 5.62 (1H, m, 3-methine);
2.96, 2.80 and 2.59 (4H, 3m, 1 & 2-methylene protons) 2.28
(4H, s, aziridine); 2.06 (6H, 2s, dimethyl); MS (EI mode) m/z
300 (M+), 257 (M+ - CH3CO), 240 (M+ - CH3COOH), 225,
213, 199, 184. Anal. (C16H16N2O4‚0.5H2O) C, H, N.
14: 7 mg (25%) yield; mp 135-137 °C; TLC (chloroform/
1
methanol, 90:10) Rf ) 0.75; H NMR (CDCl3) δ 6.23 (1H, m,
3-methine), 2.95, 2.84 (4H, 2m, 1 & 2 methylene), 2.73 (3H, s,
4-acetyl), 2.33 (4H, s, aziridine), 2.08 and 2.03 (6H, s, di-
methyl); IR (KBr pellet) 3211, 2924, 2854, 1741, 1655, 1591,
1373, 1336, 1251, 1224, 1030, 891 cm-1; MS (EI mode) 342
(M+), 300 (M+ - CH2CO), 283 (M+ - CH3COO), 258, 240, 225,
213, 189, 184. Anal. (C18H18N2O4) C, H, N.
6-Azir id in yl-1,4-d ih yd r o-7-m eth yl-(2H)-cyclop en t[b]in -
d ole-3,5,8-tr ion e (3e). To a solution of 10.2 mg (0.04 mmol)
of 3b in 2 mL of dried methylene chloride was added 60 mg of
pyridinium dichromate. The reaction mixture was stirred at
room temperature for 2 h and then flash chromatographed on
a silica gel column with chloroform as the eluant. The product
was recrystallized from chloroform/hexane: 2.7 mg (26%yield);
IR (KBr pellet) 3257, 3159, 2928, 1699, 1637, 1577, 1518, 1340,
12: 16 mg (16%) yield; mp 143-145 °C; TLC (chloroform/
1
methanol, 80:20) Rf ) 0.84; H NMR (CDCl3) δ 9.78 (1H, bs,
indole NH), 6.41(1H, q, J ) 1.5 Hz, 6-proton), 4.81 (1H, m,
3-methine proton), 3.36 (3H, s, methoxy), 2.92, 2.77 and 2.42
(4H, 3m, 1 & 2-methylene protons), 2.07 (3H, d, J ) 1.5 Hz,
7-methyl); IR (KBr pellet) 3425, 3246, 2943, 1655, 1587, 1465,
1165, 1100, 800, 669 cm-1; MS (EI mode) m/z 231 (M+), 216
(M+ - CH3), 200 (M+ - OCH3), 188, 174, 160, 132, 115. Anal.
(C13H13NO3) C, H, N.
1
1255, 1147, 1051, 985 cm-1; H NMR (CDCl3) δ 9.38 (1H, bs,
indole NH), 3.14 & 2.96 (4H, 2m, 1 & 2-methylenes), 2.33 (4H,
s, aziridine), 2.13 (3H, s, 7-methyl); MS (EI mode) m/z 256
(M+), 241 (M+ - CH3), 227, 213, 199, 174. Anal. (C14H12N2O3)
C, H, N.
13: 20.5 mg (22%) yield; mp 199-201 °C; TLC (chloroform/
1
methanol, 80:20) Rf ) 0.60; H NMR (CDCl3) δ 9.93 (1H, bs,
6-Azir id in yl-2,3-d ih yd r o-7-m et h yl-1H -p yr r olo[1,2-a ]-
ben zim id a zole-3,5,8-t r ion e (15). To a solution of 200 mg
(0.77 mmol) of the 3-hydroxy derivative30 in 60 mL of meth-
ylene chloride was added 1 g of pyridinium dichromate and
the reaction stirred for 15 h at room temperature. The solvent
indole NH), 6.38 (1H, q, J ) 1.5 Hz, 6-proton), 5.23 (1H, m,
3-methine proton), 2.92, 2.75 & 2.38 (4H, 3m, 1 & 2-methylene
protons), 2.06 (3H, d, J ) 1.5 Hz, 7-methyl); IR (KBr pellet)
3475, 3414, 3232, 2930, 1637, 1479, 1406, 1294, 1230, 1087,

Cancer-Specific Antitumor Agents
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 465
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the Pyrrolo[1,2- a]benzimidazole and Pyrrolo[1,2-a]indole Ring
Systems. J . Med. Chem. 1994, 37, 1625-1631.
was then evaporated and the residue chromatographed on a
silica gel column using chloroform and methanol (97:3). The
dark red product band was collected, concentrated to a residue,
and recrystallized from chloroform/hexane: 45 mg (23%) yield;
1H NMR (DMSO-d6) δ 4.42 and 3.2 (4H, 2t, ethylene bridge),
2.48 (4H, s, aziridinyl), 2.35 (3H, s, methyl). Anal. (C13H11N3O3)
C, H, N.
Alk yla tion of DNA by Red u ced In d oloqu in on es. To a
mixture of 1-2 mg of sonicated (600 bp) calf thymus DNA in
2.0 mL of 0.05 M pH 7.4 Tris buffer and 2 mg of Pd on carbon
was added a five-to-one base pair equivalent amount of the
indoloquinone dissolved in 0.5 mL of dimethyl sulfoxide
(DMSO). The resulting solution was degassed under argon for
30 min, after which the mixture was purged with H2 for 10
min. The solution was then purged with argon for 10 min and
placed in a 30 °C bath for 24 h. The reaction was opened to
the air, and the catalyst was removed with a Millex-PF 0.8-
µm syringe filter. The filtrate was adjusted to 0.3 M acetate
with a 3 M stock solution of pH 5.1 acetate and then diluted
with two volumes of ethanol. The mixture was chilled at -20
°C for 12 h and the DNA pellet collected by centrifuging at
12000g for 20 min. The pellet was redissolved in water and
then precipitated and centrifuged again. The resulting blue
or red pellet was suspended in ethanol, centrifuged, and dried.
The dried pellet was weighed and dissolved in 1 mL of double
distilled water resulting in a clear-colored solution with λmax
∼ 550 nm, ꢀ ∼ 750 M-1 cm-1 . This is the chromophore of the
aminoquinone resulting from nucleophile-mediated opening of
the aziridine ring. Model 2′-chloroethylaminoquinones for
extinction coefficient determination were prepared by treat-
ment of the indoloquinone with HCl.31
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R. The Organic Chemistry of the Pyrrolo[1,2-a]benzimidazole
Antitumor Agents. An Example of Rational Drug Design. Synlett
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DT-Dia p h or a se Red u ction Kin etics Stu d ies. Rat liver
DT-diaphorase was isolated as previously described.3,32 Kinetic
studies were carried out in 0.05 M pH 7.4 Tris‚HCl buffer,
under anaerobic conditions, employing Thunberg cuvettes. A
2 mM stock solution of each indoloquinone was prepared in
DMSO. To the top port was added the quinone stock and to
the bottom port were added DT-diaphorase and NADH in the
Tris buffer. The top and bottom ports were purged with argon
for 20 min and equilibrated to 30 °C. The ports were then
mixed, and the reaction was followed at 296 nm for 25 min in
order to obtain initial rates. The concentrations obtained after
mixing were: 0.3 mM NADH, 1-20 × 10-5 M quinone, and
14.5 nM (based on flavin) enzyme active sites. The value of
∆ꢀ was calculated from the initial and final absorbance values
for complete quinone reduction; usual value for ꢀ is 6000-8000
M-1 cm-1. The value of ∆ꢀ was used to calculate Vmax in M
s-1. The results were fitted to a Lineweaver-Burke plot from
which Vmax/Km values were calculated.
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F.; Spitz, M. R.; Wang, M.; Xu, X.; Lee, B. K.; Schwartz, B. S.;
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Ack n ow led gm en t. We thank the National Insti-
tutes of Health, National Science Foundation, and
Arizona Disease Control Research Commission for their
generous support.
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