Synthesis and biological evaluation of imidazoquinoxalinones, imidazole analogues of pyrroloiminoquinone marine natural products

Article abstract of DOI:10.1021/jm0700870

This report describes the synthesis and biological activity of imidazoquinoxalines, benzimidazole-based analogues of indole-based pyrroloiminoquinone marine natural products. Our analogues consist of series 1, which possesses the ethylene tether and exten

Full text of DOI:10.1021/jm0700870

J. Med. Chem. 2007, 50, 4561-4571
4561
Synthesis and Biological Evaluation of Imidazoquinoxalinones, Imidazole Analogues of
Pyrroloiminoquinone Marine Natural Products
Hung Hoang,† Daniel V. LaBarbera,†,‡ Kaleem A. Mohammed,‡ Chris M. Ireland,‡ and Edward B. Skibo*,†
Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604, and
Department of Medicinal Chemistry, UniVersity of Utah, Salt Lake City, Utah 84112
ReceiVed January 22, 2007
This report describes the synthesis and biological activity of imidazoquinoxalines, benzimidazole-based
analogues of indole-based pyrroloiminoquinone marine natural products. Our analogues consist of series 1,
which possesses the ethylene tether and extended amidine feature found in the pyrroloiminoquinone natural
products, and series 2, which also has the ethylene tether but with an electrostatically stabilized iminoquinone
rather than a resonance stabilized iminoquinone (i.e., extended amidine). The biological properties of series
1 analogues, bearing electron-rich side chain rings (indole and phenol), display cytostatic and cytotoxic
properties similar to that of the pyrroloiminoquinone natural products. In contrast, COMPARE analysis
suggests that analogues bearing benzyl and phenethyl side chains possess a different cytotoxicity mechanism.
Hollow fiber assays of analogs of 1 indicate promising antitumor activity and acceptable levels of toxicity.
One analogue of 2 is active only against breast cancer cell lines, but the cellular target is as yet unknown.
Chart 1
Introduction
The interest in biologically active pyrroloiminoquinone-based
alkaloid pigments from marine sponges, chiefly the Latrunculia
and Zyzzya species, began in the early 1990s1-3 and has
continued to the present day.4-9 Various cellular targets may
be responsible for the biological properties of these alkaloids,
including enzymes involved in DNA relaxation, topoisomerase
I and II, and signal transduction receptors. For example, both
catalytic inhibition and poisoning (stabilization of the cleavable
complex) of topoisomerase II was observed for makaluvamine
derivatives.10 The makaluvamine structure, shown in Chart 1,
possesses the pyrroloiminoquinone substructure with the viny-
logous amidine functional group. Vinylogous amidine resonance
is responsible for the red to blue pigmentation as well as the
iminoquinone hydrolytic stability.
At about the time the pyrroloiminoquinones were discovered,
our laboratory designed hydrolytically stable iminoquinones
based on the 6-acetamidopyrrolo[1,2-a]benzimidazoles (APBIa)
ring system, Chart 1.11-14 Iminoquinone stability arises from
electrostatic stabilization by the neighboring acetamido anion11
rather than by the vinylogous amidine resonance. It is note-
worthy that both the APBIs and the pyrroloiminoquinone natural
products inhibit topoisomerase II. Perhaps the similar ring sizes
(indole versus benzimidazole) as well as the presence of the
iminoquinone cation result in similar mechanisms of action. The
features of the pyrroloiminoquinone natural products (ethylene
tether and extended amidine) and the APBIs (benzimidazole
and zwitterionic iminoquinone) were combined to afford the
imidazoquinoxalinones 1 and 2 shown in Chart 2. It was
anticipated that the imidazoquinoxalines could possess new
biological and chemical properties: the presence of the purine-
like benzimidazole ring may affect signaling enzymes, the
ethylene tether may prevent the irreversible iminoquinone
hydrolysis observed with the imino-APBIs,11 and the zwitteri-
onic iminoquinones may be more bio-available than extended
amidines.
Recently, we reported the preparation of imidazoquinoxalines
1 and 2 without the amino substituents seen in the pyrroloimi-
noquinone natural products.15 These minimally substituted
imidazoquinoxalines displayed little activity in the NCI 60-cell-
line cancer panel.16 The present study describes the synthesis
of substituted analogues of 1 and 2 and the evaluation of their
biological properties. Two of the anticipated biological and
chemical properties were realized. Derivatives of 1 and 2 show
greater hydrolytic stability compared to the APBIs. Although
derivatives of 2 show little biological activity, select derivatives
* To whom correspondence should be addressed. Phone: 480-965-3581.
Fax: 480-965-2747. E-mail: eskibo@asu.edu.
† Arizona State University.
‡ University of Utah.
a Abbreviations: BAD, Bcl-2/Bcl-XL-associated death promotor; Bcl,
B cell lymphoma/leukemia; APBI, 6-acetamidopyrrolo[1,2-a]benzimida-
zoles; HBr, hydrobromic acid; HCl, hydrochloric acid; DMSO, dimethyl
sulfoxide; GI50, growth inhibition 50%; TGI, total growth inhibition; LC50,
lethal concentration 50%; akt/PKB, protein kinase B; RSK, ribosomal S6
kinase; IP, intraperitonial; SC, subcutaneaous; MALDI, matrix assisted laser
desorption/ionization; TLC, thin layer chromatography.
10.1021/jm0700870 CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/18/2007

4562 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Hoang et al.
Chart 2
Scheme 2
Scheme 1
Scheme 3
of 1 show inhibition of topoisomerase II-mediated relaxation
of supercoiled DNA and may promote apoptosis.
Results and Discussion
Synthesis. The preparation of compounds 1a,b started with
the functionalized benzene compound 3 that was previously
reported, Scheme 1.15 Catalytic reduction of the nitro groups
of 3 was followed by peracylation of the resulting amines with
either formic acid or methoxyacetic anhydride. Treatment of
the acylated product with 4 N HCl resulted in imidazo ring
closure and deacylation to afford intermediate 4. Oxidation of
4 with Fremy’s salt in pH 7 phosphate buffer11 afforded the
extended amidine products 1a,b.15 The purpose of preparing
1a,b was to determine the biological activity of imidazoqui-
noxalines devoid of substituents at the 8-amino center.
those previously reported for the preparation of simple imida-
zoquinoxalines.15
The preparation of substituted 8-amino derivatives 1c-g was
carried out by methanol displacement from the methoxy
derivative 10 using diverse alkylamine hydrochlorides, Scheme
2. Similarly, White et al. had prepared diverse makaluvamine
derivatives from methoxy pyrroloquinolines.17 The preparation
of 10 was carried out starting with the known compound 518 in
seven steps, Scheme 2. These synthetic steps are analogous to
The preparation of 2a began by nucleophilic aromatic
substitution of 4-chloro-3-nitrotoluene (11) with ethanolamine,
Scheme 3. Benzimidazole ring formation was carried out via a
Phillips cyclization reaction19 of the reduction product 12, using
formic acid in refluxing 4 N HCl, to give 13 in 82% yield. The
alcohol group of 13 was protected by acetylation with acetic
anhydride followed by nitration to obtain the key benzimidazole

Synthesis of Imidazoquinoxalinones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4563
Scheme 5
Scheme 4
intermediate 14, with an overall yield of 16%. Catalytic
reduction of 14 followed by amide formation with acetic
anhydride gave 15, which was selectively nitrated to afford the
7-nitro isomer 16.
Deprotection of 16 via acid-catalyzed trans-esterification gave
the alcohol as an HCl salt that was immediately used in the
next step, Scheme 4. Methanesulfonation of the alcohol
intermediate using diisopropyl ethylamine and methane sulfonyl
chloride gave the sulfonate ester 17. Catalytic reduction of 17
followed by stirring in refluxing methanol afforded the ring-
closed quinoxaline intermediate that was Fremy oxidized20 to
yield 2a.
Peracetylation of 12 with methoxyacetyl chloride in dry
dichloromethane, containing diisopropyl ethylamine, afforded
19 in 98% yield, Scheme 5. Nitration of 19 with fuming nitric
acid gave 20 in 55% yield, which was treated with 48% HBr.
The HBr treatment causes deacetylation, imidazo ring closure,
and O-demethylation, resulting in formation of benzimidazole
21 in 74% yield. Catalytic reduction of 21 with 5% palladium
on carbon in methanol afforded the aniline intermediate that
was peracetylated with acetic anhydride in acetic acid to give
22 in 70% yield from 21.
Table 1. In Vitro Assay Results of Imidazoquinoxalinones 1
mid-range Log
GI50 (range)
mid-range Log
TGI and range
mid-range Log
LC50 and range
cmpd
Selective nitration of 22 with a solution of 90% HNO3 and
H2SO4 gave the 7-nitro isomer 23 as the sole product. Alcohol
deacetylation via the acid-catalyzed trans-esterification of 23
with concentrated HCl in methanol followed by sulfonation with
methanesulfonyl chloride resulted in an unexpected product
containing the 2-chloromethyl substituent (24) instead of the
expected 2-sulfonoxymethyl substituent. The 2-sulfonoxymethyl
group likely formed, but rapidly reacted with chloride due to
the benzylic character of this center. The reduction and
cyclization of this intermediate proved futile due to hydro-
genolysis of the chloro during catalytic reduction to afford the
2-methyl substituent. To circumvent this problem, an acetate
group was substituted via an SN2 reaction with sodium acetate
in dimethyl sulfoxide (DMSO) to yield 25 as pale yellow
crystals in 75% yield. This substituent maintained the function
of a leaving group but was more robust than the chloro group
and was not susceptible to hydrogenolysis. Compound 2b was
obtained by the catalytic reduction of 25 followed by refluxing
in methanol to give the ring-closed quinoxaline intermediate,
which was Fremy oxidized20 in phosphate buffer (pH ) 7), with
an overall yield from 25 of 66% as yellow crystals.
1a
1c
1d
-5.34 (1.86)
-6.78 (2.62)
-5.98 (3.38)
-5.96 (3.28)
-6.72 (2.37)
-6.63 (2.34)
-5.60 (3.76)
-6.21 (3.02)
-6.29 (3.2)
-4.91 (1.52)
-5.97 (4.0)
-5.42 (3.78)
-5.37 (4.0)
-6.13 (2.57)
-6.06 (3.64)
-4.85 (3.47)
-5.55 (3.65)
-5.69 (3.52)
-4.45 (1.25)
-5.26 (4.0)
-4.89 (3.61)
-4.7 (3.42)
-5.35 (3.32)
-5.17 (3.3)
-4.39 (3.18)
-4.83 (3.02)
-5.18 (4.0)
1e
1f
1g
Cell Line Assays and COMPARE Analysis. The mean
cytostatic and cytotoxic parameters of 1 and 2, measured in a
60 human cancer cell line panel, are provided in Tables 1 and
2, respectively. The cytostatic parameters include GI50 and TGI,
which are the concentrations of drug required for 50% growth
inhibition and total growth inhibition, respectively. The cytotoxic
parameter is the LC50, which is the concentration required for
50% cell kill. These data were obtained under the In Vitro Cell
Line Screening Project at the National Cancer Institute.16,21,22
Also provided in Tables 1 and 2 are range values, which
represent the log of the maximum concentration difference

4564 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Table 2. In Vitro Assay Results of Imidazoquinoxalinones 2
Hoang et al.
COMPARE analysis16,25 provided insights into what might
be the molecular target of active analogues exhibiting high
histologic selectivity (compounds in bold font in Tables 1 and
2). The mean graphs of compounds with log range values of
>2 and cell line selectivity would have distinctive patterns
amenable to a meaningful COMPARE analysis. In contrast,
mean graphs with low log range values are essentially flat and
will correlate with other flat mean graphs, providing little
meaningful target information. The COMPARE analysis cor-
relates mean graph data with known molecular target levels in
cell lines and thereby generates hypotheses concerning the
agent’s mechanism of action. A molecular target is a protein or
enzyme that has been measured in the National Cancer
Institute’s panel of 60 human tumor cell lines. The levels of
over 1000 biologically relevant molecular targets have been
determined in these tumor cell lines from measurements of
mRNA and enzyme activity levels.26
mid-range GI50
(range)
mid-range TGI
and range
mid-range LC50
and range
cmpd
2a
2b
2c
-4.33 (0.8)
inactive
-4.28 (4.00)
inactive
inactive
-4.09(1.48)
inactive
inactive
inactive
X ) CH3
Chart 3
COMPARE correlations indicated that the cytostatic and
cytotoxic parameters of compounds 1 and 2 represent three
mechanistically distinct classes. The GI50, TGI, and LC50
parameters of compounds 1e and 1f correlate (0.5-0.6 correla-
tion coefficient) with the same molecular target: the ABC (ATP
binding cassette) family of membrane transporters.27 These
transporters are important in the transport of drugs into or out
of cells and could likely play a role in the transport of 1e and
1f into cancer cells. COMPARE correlations using 1e as a seed
against the natural products database revealed a good correlation
with makaluvamine H (Chart 3), giving correlation coefficients
of 0.748 (GI50) and 0.670 (TGI), <0.69 in LC50. The topoi-
somerase II poisoning exhibited by 1e is consistent with a
cytotoxic mechanism similar to that of the makaluvamines, see
the next section.
between the least-sensitive and the most-sensitive cell line. This
range parameter has been used to gain insights into the
selectivity of antitumor agents, because it provides a measure
of histological specificity.23
The criteria used for judging the activity of compounds
include: log mean values <-4 with log range values of >2,
and with high selectivity for 10 or more cell lines and selectivity
for one or more histological cancers. Compounds exhibiting the
highest activity according to these criteria are highlighted in
bold in Tables 1 and 2. Analogues of 1 bearing a two-carbon
tether and an electron-rich aromatic ring (1e and 1f) are the
most active, with histological selectivity exhibited toward
melanoma and colon cancer. Both of these compounds resemble
the natural products wakayin and makaluvamine, respectively.
Compound 1e resembles a wakayin-like synthetic makaluvamine
reported in the literature,24 Chart 3. Compounds 1c and 1g are
also potent compounds, but exhibit distinctly different histologi-
cal specificity than 1e or 1f, Figure 1. The N-unsubstituted
version 1a has little histologic specificity, while intermediate
10 in Scheme 2 is devoid of cytotoxic and cytostatic activity.
Thus, the presence of an aromatic substituent at the 8-position
is required for cytostatic/cytotoxic activity by 1. The presence
of a 2-methoxymethyl group in 1b resulted in cytotoxic activity
in two cell lines even though only an 8-amino is present, Figure
2. The importance of the 2-substituent on the cytostatic/cytotoxic
activity by 1 will require additional studies.
The only analogue of 2 with activity is 2c (X ) CH3, Chart
2), which exhibited cytostatic activity exclusively against breast
cancer cell lines.15 Observed log GI50 values in breast cancer
cell lines include: BT-549, <-8; MDA-MB-231/ATCC, -4.89;
MDA-MB-435, -4.72; and MDA-N, -4.73. These cell lines
are also prominent in the TGI panel. The data in Table 2 shows
that increasing the size of X beyond CH3, 2c, causes complete
loss of activity, while 2a (X ) H) possesses only modest
activity. These findings do not reflect the structure-activity
relationship of the APBIs, which showed a tolerance of bulky
leaving groups at the 3-position of the pyrrolobenzimidazole
ring.12
In contrast, COMPARE analysis of 1c revealed a dependence
on levels of the proteins BAD and cytochrome C without any
correlations with makaluvamine-type compounds. The molecular
target correlations suggest that 1c may be promoting apoptosis
by a BAD phosphorylation by either akt/PKB or p90RSK.
Compound 1g may promote apoptosis, with its cytostatic
properties (GI50) dependent on the levels ErbB proteins.28-30
At this point, we can only assert that compounds 1c,g have
different mean graphs than compounds 1e,f, suggesting different
mechanisms of cytotoxicity. The possible role of 1c,g in
promoting apoptosis will be addressed in another study.
Human Topoisomerase II Cleavage Assays. Makalu-
vamines are reported to be topoisomerase II poisons with
potencies similar to that of the clinically used drug etoposide.
To determine if 1e shares this feature, relaxation assays were
carried out with recombinant human topoisomerase II (p170)
in the presence of pRYG supercoiled DNA using etoposide as
a positive control. We assayed relaxation reactions on agarose
gels in the presence of ethidium bromide, Figure 3. The ethidium
bromide-containing gels readily detect the presence of linear
DNA, but will bearly resolve form I and form II DNAs (form
II traveled slightly faster than form I). If linear DNA is present,
then the agent acts as a topoisomerase II poison, that is, the
agent stabilizes the cleavable complex. Inspection of the agarose
gel shown in Figure 3 confirms that 1e, along with the etoposide
control, are topoisomerase II poisons. However, comparison of
the mean graph of 1e with that of etoposide revealed no
similarities. Etoposide displays potent cytostatic/cytotoxic activ-
ity (<10-6 M) against all histological cancer types.
Hollow Fiber Assays. To provide insights into drug toxicity
and antitumor efficacy, the National Cancer Institute determined
both acute toxicity of 1g and its activity in the hollow fiber

Synthesis of Imidazoquinoxalinones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4565
Figure 1. Bar graphs of the average -log GI50 values for 1f and 1c against histological cancer types. The different bar graph patterns are due to
the cytostatic mechanisms for 1f (resembles pyrroloiminoquinone natural products) and 1c (a promoter of apoptosis).
quinone natural products. The incorporation of various amino
side chains into 1 was readily carried out from the reaction of
intermediate 10 and diverse amines, Scheme 2. Currently, more
diverse libraries of series 1 are being prepared using this
methodology to optimize antitumor activity. In contrast, the
structural diversification of series 2 was made early in the
synthesis (Schemes 3-5), resulting in only a few analogues of
2. Nevertheless, we were able to evaluate the biological
properties of these novel compounds.
Series 1 structurally resembles pyrroloiminoquinone natural
products, specifically the makaluvamines. COMPARE analysis
verified that 1e,g possess similar mean graph profiles to those
of this class of natural products. Visual comparison of the
published mean graph of makaluvamine H34 with that of 1g
supports the COMPARE results. However, the mean graph data
indicate that 1e and 1g are less potent than makaluvanine H.
The major reason is likely the presence of the electron-deficient
benzimidazole ring (compared to the indole ring of the natural
products), resulting in a protonated amidine pKa of 8.2. Thus,
a substantial fraction of 1e,g will be neutral at physiological
pH, while cationic makaluvamines are likely the active form
given the activity of makaluvamine H. The presence of neutral
imidazoquinoxalines at physiological pH may be responsible
for the change in molecular target observed with some analogues
of series 1. Our COMPARE results suggest that Compound 1c
has a different mechanism of action than 1e,g based on a
different spectrum of cytostatic/cytotoxic activity. The phar-
macological basis for this difference will be the subject of
additional study.
Figure 2. Plots of percent cell viability/proliferation versus concentra-
tion of 1b against human colon tumor HCT-116 cells and human
epidermoid carcinoma A-431 cells. The IC50 for both tumor cell lines
is 3 µM.
assay. The maximum tolerated dose (MTD) for 1g is 12.5 mg/
kg (see Experimental Section for details). The hollow fiber
assays31,32 are carried out as previously described. The candidate
drug is injected intraperitoneal (IP) at 3.1 mg/kg/dose, according
to the schedule QD × 4, for 3 days. The score is broken down
into an IP and a subcutaneaous (SC) score that is based on the
percent of cancer left in the fiber (<50% ) 2 and >50% ) 0)
The highest total score (IP + SC) is 96. The typical antitumor
agent only has a hollow fiber score of ∼5, while the highest
score being 64 for the natural product cyclin-dependent kinase
inhibitor flavopiridol.33 A good SC score (g8) indicates that
the drug is able to get to the tumor site (subcutaneous) from a
distant site (intraperitoneal) of injection. Compound 1g had an
IP score of 4/48 and an SC score of 6/48. These results indicate
that compound 1g has minimal antitumor activity in this assay.
This find has prompted the synthesis of diverse analogues of
series 1 to optimize activity.
Experimental Section
Elemental analyses (CHN) were obtained for intermediates and
final products. Melting points and decomposition points were
determined with a Mel-Temp apparatus All TLCs were run with
VWR silica gel, 60 F254 plates, employing various solvents and a
fluorescent indicator for visualization. IR spectra were taken as KBr
pellets using a Nicolet MX-1 FTIR spectrophotometer; the strongest
IR absorbances are reported. 1H NMR spectra were obtained with
Conclusions. The synthesis of imidazoquinoxalines, benz-
imidazole-based analogues of the indole-based pyrroloimino-
quinone marine natural products, was successfully carried out
employing multistep syntheses. Series 1 possesses the ethylene
tether and extended amidine features found in pyrroloimino-

4566 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Hoang et al.
Figure 3. Agarose gel of topoisomerase IIa catalyzed relaxations of pRYG supercoiled DNA (form I) run in the absence of ethidium bromide. The
control lanes include relaxation reaction with pRYG supercoiled DNA in lane 1, pRYG supercoiled DNA only in lane 2, relaxation reactions
carried out with DMSO in lane 3, linear DNA standard in lane 4, relaxation reactions carried out in the presence of etoposide in lane 5, and
relaxation reactions carried out in the presence of 0.05 and 0.10 mM of 1e in lanes 6 and 7.
a 300 or 500 MHz spectrometer. All NMR chemical shifts (δ) are
reported relative to tetramethylsilane (TMS). Uncorrected melting
points were determined with an electrothermal Mel-Temp apparatus.
MS measurements were carried out with a high-resolution electron
impact mass spectrometer at the University of Nebraska, Center
for Mass Spectrometry or a Voyager DE STR MALDI-TOF mass
spectrometer.
Compounds in series 1 and 2 were screened in the National
Cancer Institute’s 60-cell-line screen.16,35 Mean graph results were
then analyzed with COMPARE online (http://itbwork.nci.nih.gov/
PublicServer/jsp/Form_Upload.jsp).16,25
3.37 (3 bs, 9H, methoxy), 2.18 (s, 3H, acetyl). Multiple amide
resonance structures cause the ethylene group resonances to become
very broad.
A solution consisting of 25 mg of the above product dissolved
in a minimal amount of 48% HBr was refluxed for 5 min, then
cooled, and concentrated to an oily liquid under vacuum. Adding
a small volume of ethanol and then ethyl acetate resulted in
crystallization of the trihydrobromide salt of 4b (15 mg, 56% yield).
This compound is somewhat unstable and was used immediately
in the Fremy oxidation step.
8-Amino-2-methyl-4,5-dihydroimidazo[1,5,4-de]quinoxalin-9-
one (1a). To a solution of 60 mg (0.14 mmol) of the trihydro-
bromide of 2 in 5 mL of 0.2 M pH 7.0 phosphate buffer was
added 150 mg of Fremy’s salt. The reaction mixture immediately
turned dark purple. The reaction mixture was placed on a
100 mL Baker Phenyl reverse phase column and eluted with
distilled H2O to remove salts. The product eluted as a purple band
with 0.5 M HCl. Concentration of the fractions containing
product and then precipitation of the residue from ethanol/ethyl
acetate afforded the dihydrochloride salt of 1a (15 mg, 41%
yield); mp > 233 °C dec; TLC (n-butanol, acetic acid, H2O [5:2:
3]) Rf ) 0.23; FTIR 3420, 3203, 3080, 2976, 1705, 1635, 1541,
1329 cm-1; 1H NMR (DMSO-d6) δ 11.49, 9.81, and 8.94 (3s, 3H,
NH protons), 8.21 (s, 1H, imidizole), 5.86 (s, 1H, 7-H), 4.4 and
3.9 (2t, J ) 7 Hz, 4H, ethylene bridge); MALDI m/z calcd for
C9H9N4O (M + 1), 189.07; found, 189.067. Anal. (C9H8N4O‚2HCl)
C, H, N.
8-Amino-5,6-dihydro-4H-imidazo[1,5,4-de]quinoxaline (4a).
A solution consisting of 200 mg (0.64 mmol) of 3 in 50 mL of
methanol was hydrogenated under 50 psi of H2 in the presence of
100 mg of 5% Pd on carbon at room temperature for 24 h. The
catalyst was filtered off using celite filter aid, and the filtrate
was concentrated in vacuo to afford a dark oil. To this oil was
added 3 mL of 88% formic acid, and the resulting mixture was
refluxed for 45 min. The solution was then concentrated in
vacuo to an oil, and 3 mL of 48% HBr was added followed by
heating at reflux for 40 min. The reaction was then concentrated
in vacuo to a solid, which was recrystallized from ethanol/ethyl
acetate as the trihydrobromide salt (150 mg, overall 56%
yield); mp > 270 °C dec; TLC (n-butanol, acetic acid, H2O [5:2:
3]) Rf ) 0.38; FTIR 3633, 3011, 2864, 2598, 2362, 1666, 1521,
1
1384, 1315, 1226, 1086, 815, 583 cm-1; H NMR (DMSO-d6) δ
9.37 (1s, 1H, imidazole), 6.84 and 6.51 (2d, J ) 1 Hz, 2H,
aromatic), 4.49 and 3.58 (2t, J ) 5 Hz, 4H, ethylene); MALDI m/z
175 (M + 1).
8-Amino-2-(methoxymethyl)-4,5-dihydroimidazo[1,5,4-de]
quinoxalin-9-one (1b). To a mixture of 50 mg (0.11 mmol)
of 4b in 5.0 mL of 0.2 M pH 7.4 phosphate buffer was added 120
mg of Fremy’s salt, and the resulting mixture was stirred
for 5 min. The reaction mixture was then placed on a 100 mL Baker
Phenyl reverse phase column and eluted with distilled H2O to
remove salts and excess Fremy’s salt. The product was then
eluted as a purple band with 1.0 N HCl. Concentration of the
product fractions and then precipitation of the residue from
ethanol/ethyl acetate to afford the dihydrochloride salt (23 mg, 68%
yield); mp > 220 °C dec; TLC (n-butanol, acetic acid, H2O [5:2:
3]) Rf ) 0.27; FTIR 3510, 3421, 3227, 3053, 3003, 2951, 2843,
1707, 1629, 1537, 1349, 1348, 1253, 1107, 1049, 952, 839, 738
8-Amino-5,6-dihydro-4H-2-(methoxymethyl)-imidazo[1,5,4-
de]quinoxaline (4b). Compound 4b was prepared by the following
three-step procedure. Reductive cyclization of 3 was carried out as
described under the preparation of 4a. To a solution of 100 mg of
the reductive cyclization product, dissolved in 5 mL of dry pyridine,
was added 200 mg of methoxyacetic anhydride. The reaction
mixture was stirred at room temperature for 3 h and then evaporated
in vacuo to an oil that was purified by column chromatography
using silica gel and acetone/ethyl acetate (1:1) as eluent. Recrys-
tallization of the product, 7-acetamido-1,4,5-tri(methoxyacetamido)-
1,2,3,4-tetrahydroquinoxaline, was carried out using chloroform/
hexane (40 mg, 45% yield); mp 165-170 °C; TLC (acetone/ethyl
acetate [1:1]) Rf ) 0.18; FTIR 3284, 3005, 2926, 2870, 1672, 1604,
cm-1 1H NMR (DMSO-d6) δ 5.58 (s, 1H, 7-H), 4.58 (s, 2H,
;
methylene), 4.37 and 3.98 (2t, J ) 7 Hz, 4H, ethylene bridge),
3.30 (s, 3H, methoxy); MALDI m/z calcd for C11H13N4O2
(M + 1), 233.104; found, 233.098. Anal. (C11H12N4O2‚2HCl) C,
H, N.
1
1529, 1467, 1425, 1267, 1118, 750, 700 cm-1; H NMR (CDCl3)
δ 8.9 (bs, 1H, amide NH), 8.0 and 7.6 (2s, 2H, aromatic), 4.40,
4.35 and 4.03 (3 very broad s, 6H, methylenes), 3.53, 3.47 and

Synthesis of Imidazoquinoxalinones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4567
4-(2-Hydroxyethylamino)-3,5-dinitroanisole (6). A mixture of
800 mg (3.5 mmol) of 5 and 2 mL of ethanolamine in 20 mL of
ethanol was heated until dissolution of solids and then stirred
without heating for 24 h. The product slowly crystallized from
solution as red needles upon chilling in a freezer (550 mg, 61%
yield); mp 101-102 °C; TLC (ethyl acetate) Rf ) 0.62; FTIR 3506,
814, 540, 451 cm-1; 1H NMR (CD3OD) δ 8.13 (1s, 1H, 2-H), 5.74
(s, 1H, 7-H), 5.52 and 4.09 (t, J ) 7 Hz, 4H, ethylene bridgehead),
3.84 and 3.60 (2t, J ) 5 Hz, 4H, ethylene side chain); MALDI m/z
calcd for C11H13N4O2 (M + 1), 233.104; found, 233.098. Anal.
(C11H13N4O2‚2HCl‚0.1H2O) C, H, N.
8-[2-(1H-Indol-3′-yl)-ethylamino]-4,5-dihydroimidazo[1,5,4-
de]quinoxalin-9-one (1e). Yield, 4.7 mg (22%); mp > 205 °C dec;
TLC (n-butanol, acetic acid, H2O [5:2:3]) Rf ) 0.48; IR 3450, 3080,
3024, 2931, 2831, 1631, 1535, 1350, 1236, 1182, 1099, 752,
3387, 1526, 1444, 1355, 1252, 1225, 1047, 894, 763, 697 cm-1
;
1H NMR (DMSO-d6) δ 8.1 (broad s, 1H, amino), 7.781 (s, 1H),
3.85 (s, 3H), 3.84 (q, J) 4.8 Hz, 2H), 3.12 (q, J ) 4.8 Hz, 2H),
1.60 (t, J ) 4.8 Hz, 1H, hydroxyl). Anal. (C9H11N3O6) C, H, N.
4-(2-Methanesulfonoxyethylamino)-3,5-dinitroanisole (7). To
a solution consisting of 6 (3 mmol) in 5 mL of pyridine was added
0.5 mL of methanesulfonyl chloride, and the resulting mixture
stirred for 1 h. The reaction mixture was combined with 100 mL
of methylene chloride and extracted 2× (100 mL) with H2O
followed by 1× (100 mL) with 1 N HCl. Compound 7 remained
in the organic layer after 1 N HCl extraction. The organic layer
was dried with Na2SO4, concentrated to a small volume, and diluted
with hexane, resulting in 7 as orange crystals. The product was
filtered off and dried without further purification (94% yield); mp
109-110 °C; TLC (ethyl acetate) Rf ) 0.64; IR 3302, 1666, 1535,
1350, 1283, 1062, 979, 526 cm-1; 1H NMR (DMSO-d6) δ 7.78 (s,
2H, aromatic), 4.36 and 3.37 (2t, 4H, J ) 4.8 Hz ethylene bridge),
3.85 (s, 3H, methoxy), 3.05 (s, 3H, methylsulfonyl). Anal.
(C10H13N3O8S) C, H, N.
702, 626 cm-1 1H NMR (CD3OD) δ 7.99 (s, 1H, 2-H), 7.47
;
(d, J ) 7 Hz, 1H, 7-indole), 7.35 (d, J ) 8 Hz, 1H, 4-indole),
7.04 (s, 1H, 2-indole) 6.97 (t, J ) 9 Hz, 1H, 5-indole), 6.89
(t, J ) 8 Hz 6-indole), 5.36 (s, 1H, 7-H), 4.36 and 3.91 (t, J ) 7
Hz, 2H, ethylene bridgehead), 3.68 and 3.11 (2t, J ) 7 Hz, 4H,
ethylene chain); MALDI m/z calcd for C19H18N5O2 (M + 1),
332.151; found, 332.148. Anal. (C19H17N5O‚3HCl‚0.4H2O) C,
H, N.
8-[2-(4′-Hydroxyphenyl)-ethylamino]-4,5-dihydroimidazo-
[1,5,4-de]quinoxalin-9-one (1f). Yield, 4.3 mg (23%); mp > 196 °C
dec; TLC (n-butanol, acetic acid, H2O [5:2:3]) Rf ) 0.32; IR 3441,
3225, 3117, 2935, 1639, 1562, 1516, 1352, 1234, 1105, 835, 648
1
cm-1; H NMR (CD3OD) δ 8.0 (1s, 1H, 2-H), 6.98 and 6.63 (2d,
4H, J ) 8 Hz, aromatic), 5.86 (s, 1H, 7-H), 4.38 and 3.97 (2t, J )
7 Hz, 4H, ethylene bridge), 3.55 and 2.84 (2t, J ) 7 Hz, 4H,
ethylene chain); MALDI m/z calcd for C17H17N4O2 (M + 1),
309.135; found, 309.142. Anal. (C19H17N5O‚2HCl) C, H, N.
8-Benzylamino-4,5-dihydroimidazo[1,5,4-de]quinoxalin-9-
one (1g). Yield, 5.8 mg (34%); mp > 240 °C dec; TLC (n-butanol,
acetic acid, H2O [5:2:3]) Rf ) 0.32; IR 3136, 3045, 1637, 1537,
8-Methoxy-4,5-dihydro-imidazo[1,5,4-de]quinoxalin-9-one (10).
A solution of 100 mg (0.30 mmol) of 7 in 50 mL of methanol was
hydrogenated under 50 psi of H2 in the presence of 80 mg of 5%
Pd on carbon for 24 h. The catalyst was filtered off using celite,
and the filtrate was concentrated in vacuo to afford 8 as a dark oil.
To this oil was added 3 mL of 88% formic acid, and the mixture
was refluxed for 1 h. After completion of reflux, the solution was
then concentrated in vacuo, 4 mL of 37% HCl was added, and the
mixture was refluxed for 2 h. The reaction was then concentrated
in vacuo to afford 9 as a dark oil. This oil was then dissolved in 10
mL of 0.2 M pH 7.0 phosphate buffer, to which was added 250
mg of Fremy’s salt. After a 5 min reaction time, the aqueous
solution was extracted 3× (15 mL) with chloroform. The organic
layer was dried with Na2SO4 and concentrated in vacuo, resulting
in an oil that was precipitated using ethyl acetate/hexanes to afford
10 (6.5 mg, 11% yield after three synthetic steps); mp > 120 °C
dec; TLC (10% MeOH in CHCl3) Rf ) 0.43; FTIR 3369, 3115,
1
1404, 1234, 1084, 700 cm-1; H NMR (CD3OD) δ 8.12 (1s, 1H,
2-H), 7.44 (mult, 5H, aromatic), 5.61 (s, 1H, 7-H), 4.49 (s, 2H,
methylene), 4.49 and 4.06 (2t, J ) 6 Hz, 4H, ethylene bridgehead);
MALDI m/z calcd for C16H15N4O (M + 1), 279.125; found,
279.126. Anal. (C16H15N4O‚2HCl) C, H, N.
2-(2-Hydroxyethylamino)-5-methylaniline (12). Compound 12
was prepared according to the following two-step process. A
solution of 4 mL (5.2 g, 0.03 mol) of 4-chloro-3-nitrotoluene (11)
in 4 mL (4.08 g, 0.07 mol) of ethanolamine was refluxed for 2 h.
The reaction was then cooled to room temperature and diluted with
dichloromethane and H2O. The organic phase was dried with
sodium sulfate and concentrated, giving a red oil. Triturating the
oil with hexane yielded 4-(2-hydroxyethylamino)-3-nitrotoluene,
a red solid. The solid was filtered, washed with hexanes, and dried
in the air. The crude product was recrystallized with DCM to give
red prisms (4.44 g, 75% yield); mp 80-81 °C; TLC (chloroform/
methanol [90:10]) Rf ) 0.44; IR (KBr pellet) 3445, 3346, 2957,
2926, 2872, 1637, 1568, 1523, 1433, 1406, 1356, 1311, 1219, 1178,
1151, 1039, 922, 812 cm-1; 1H NMR (CDCl3) δ 8.06 (s, 1H, N-4-
amino), 7.98 (s, 1H, C-2 aromatic proton), 7.29 (d, J ) 8.7 Hz,
1H, C-5 aromatic proton), 6.83 (dd, J ) 8.7 Hz, 1H, C-6 aromatic
proton), 3.95 and 3.53 (t, J ) 4.8 Hz, 4H, methylenes), 2.27 (3H,
s, C-1 methyl); MS (EI mode) m/z 196 (M+).
A solution of 8.1 g (142 mmol) of 4-(2-hydroxyethylamino)-3-
nitrotoluene in 100 mL of methanol was purged with nitrogen. To
this solution was added 2 g of 5% activated Pd on carbon. This
mixture was hydrogenated under 50 psi of H2 at room temperature
for 3 h. The catalyst was then filtered off, and the solids were
washed with DMF. The filtrates were combined and concentrated
in vacuo, resulting in a purple solid. This solid was recrystallized
with DCM to afford light purple crystals (6.3 g, 93% yield); mp
137-138 °C; TLC (ethyl acetate) R×c4 ) 0.55; IR (KBr pellet) 749,
868, 1053, 1155, 1269, 1462, 1521, 1620, 2939, 3198, 3356 cm
-1; 1H NMR (DMSO-d6) δ 6.36 (1H, s), 6.29 (2H, d), 4.62 (t, J )
5.7 Hz, 1H), 4.353 (br s, 2H), 4.11 (t, J ) 6 Hz, 1H), 3.57 (q, J )
6 Hz, 2H,), 3.02 (q, J ) 6 Hz, 2H), 2.06 (3H, s, methyl); MS (EI
mode) m/z 166 (M+). Anal. (C9H14N2O) C, H, N.
3043, 2939, 2850, 1649, 1539, 1246, 1087, 1028, 839, 661 cm-1
;
1H NMR (CDCl3) δ 7.69 (1s, 1H, imidazole), 6.09 (1s, 1H, 7-H),
4.33 and 4.21 (2t, J ) 6 Hz, 4H, ethylene), 3.86 (1s, 3H, methoxy);
MALDI m/z calcd for C10H10N3O2 (M + 1), 204.08; found, 204.075.
General Procedure for the Synthesis of 8-Substituted 4,5-
Dihydroimidazo[1,5,4-de]quinoxalin-9-ones (1c-g). To a solution
of 10 mg (0.005 mmol) of 10 in 15 mL of methanol was added 1.0
equiv of the amine hydrochloride. The reaction was allowed to stir
at room temperature for 24 h. The reaction mixture was then placed
on a 100 mL Baker Phenyl reverse phase column and eluded with
distilled H2O to remove salts and unreacted amine. The product
was eluted as a purple band with 25% methanol in 0.5 M HCl.
Concentration of the fractions containing product and then pre-
cipitation of the residue from ethanol/ethyl acetate afforded the
dihydrochloride salt of 1c-g. Physical properties are provided
below.
8-(2-Phenethylamino)-4,5-dihydroimidazo[1,5,4-de]quinoxa-
lin-9-one (1c). Yield, 6.0 mg (33%); mp > 218 °C dec; TLC (n-
butanol, acetic acid, H2O [5:2:3]) Rf ) 0.32; FTIR 3421, 3024,
2924, 2852, 1699, 1633, 1535, 1350, 1259, 1099, 752, 704 cm-1
;
1H NMR (CD3OD) δ 8.13 (1s, 1H, 2-H), 7.28 (mult, 5H, aromatic),
5.58 (s, 1H, 7-H), 4.51 and 4.07 (2t, J ) 6 Hz, 4H, ethylene bridge),
3.70 and 3.04 (2t, J ) 7 Hz, 4H, ethylene side chain); MALDI m/z
calcd for C17H17N4O (M + 1), 293.140; found, 293.137. Anal.
(C17H16N4O‚2HCl) C, H, N.
1-(2-Hydroxyethyl)-5-methylbenzimidazole (13). To a solution
consisting of 276 mg (6 mmol) of 96% formic acid and 20 mL of
4 N HCl was added 500 mg (3 mmol) of 12. The reaction mixture
was refluxed for 1 h, cooled to room temperature, and placed in an
ice bath. The cooled reaction was neutralized with ammonium
8-(2′-Hydroxyethylamino)-4,5-dihydroimidazo[1,5,4-de]qui-
noxalin-9-one (1d). Yield, 5.8 mg (34%); mp > 198 °C dec; TLC
(n-butanol, acetic acid, H2O [5:2:3]) Rf ) 0.12; IR 3431, 3213,
3047, 2937, 1699, 1639, 1562, 1541, 1450, 1356, 1126, 1066, 949,

4568 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Hoang et al.
hydroxide (pH ) 8). The resulting crystals were filtered and washed
with cold H2O and air-dried to afford 13 (435 mg, 82% yield); mp
135-136 °C; TLC (20% methanol in chloroform) R×c4 ) 0.4; IR
(KBr pellet) 790, 869, 1076, 1178, 1253, 1329, 1386, 1510, 1780,
nitrogen gas, the HCl salt (343 mg, 1.1 mmol) was suspended in
50 mL of dry dichloromethane. To this suspension was added of
574 µL of diisopropyl ethylamine and the mixture was stirred for
5 min followed by dropwise addition of 255 µL (3.3 mmol) of
methane sulfonyl chloride. The reaction was stirred for 2 h,
concentrated, and chromatographed on silica gel (5% methanol in
dichloromethane) to afford yellow crystals (250 mg, 64% yield);
mp 164-166 °C; TLC (20% methanol in chloroform) R×c4 ) 0.48;
IR (KBr pellet) 526, 800, 912, 1172, 1354, 1531, 1666, 3022, 3304,
1
2868, 3090, 3171, cm-1; H NMR (CDCl3) δ 7.71 (s, 1H), 7.18
(d, J ) 8 Hz, 1H), 7.11 (s, 1H), 7.03 (d, J ) 8 Hz, 1H), 4.26 (t, J
) 5 Hz, 2H), 4.04 (t, J ) 5 Hz, 2H), 2.35 (s, 3H, methyl). Anal.
(C10H12N2O) C, H, N.
1-(2-Acetoxyethyl)-5-methyl-6-nitrobenzimidazole (14). To a
solution consisting of 25 mL of acetic anhydride and 25 mL of
acetic acid was added 4 g (0.02 mol) of 13, and the resulting mixture
was refluxed for 1 h. The acetic acid was removed in vacuo leaving
an oily liquid (O-acetylated 13), which was pure by TLC and used
without further characterization.
1
cm-1; H NMR (CDCl3) δ 7.99 (s, 1H), 7.90 (s, 1H), 7.51 (br s,
1H), 4.52 (t, J ) 4.5 Hz, 2H, ethylene), 4.44 (t, J ) 4.5 Hz, 2H,
ethylene), 2.92 (s, 3H methyl), 2.41 (s, 3H, methyl), 2.23 (s, 3H,
methyl). Anal. (C13H16N4O6S) C, H, N.
7-Acetamido-8-methyl-4,5-dihydroimidazo[1,5,4-de]quinoxa-
lin-9-one (2a). A solution containing 100 mg of 17 in 25 mL of
methanol was purged with nitrogen. To this was added 200 mg of
5% Pd on carbon. This mixture was hydrogenated under 50 psi of
H2 at room temperature for 30 min. The catalyst was filtered off,
and the clear methanol solution was refluxed for 2 h. The methanol
was evaporated giving 60 mg of ring-closed product 18, which was
too unstable for complete characterization. The ring-closed product
was dissolved in 50 mL of phosphate buffer containing 300 mg of
Fremy’s salt. This mixture was stirred for 10 min followed by
extraction with dichloromethane. The organic extracts where dried
with Na2SO4 and concentrated to a yellow solid (20 mg, 29% yield);
mp > 185 °C dec; TLC (decomposed on TLC plate to form new
A 4:1 solution of 90% HNO3 and H2SO4 (20 mL) was cooled to
-5 °C with an ice-salt bath. To the cooled nitric acid solution was
added O-acetylated 13 in 10 mL of acetic acid dropwise over 1 h,
while keeping the temperature between -5 and 0 °C. After the
addition was complete, the reaction was warmed to 10 °C and stirred
for 1 h. The reaction was then warmed to room temperature,
followed by pouring over ice and a pH adjustment with sodium
bicarbonate (pH ∼ 5). This solution was extracted with dichlo-
romethane, and the extracts were dried with Na2SO4 and concen-
trated, giving an oily solid consisting of both nitro isomers. A small
portion of the desired isomer was isolated by column chromatog-
raphy with silica gel (20% acetone in ethyl acetate). The pure
crystals were then used to crystallize selectively the desired isomer
from ethanol to afford pale yellow crystals (1.5 g, 25% yield); mp
129-130 °C; TLC (20% methanol in chloroform) R×c4 ) 0.71;
FTIR (KBr pellet) 889, 1051, 1232, 1325, 1423, 1516, 1745, 2982,
1
product, thus, no Rf value was calculated); H NMR (CDCl3) δ,
7.65 (s, 1H), 7.45 (br s, 1H), 4.34 (t, J ) 7.2 Hz, 2H), 4.23 (t, J )
7.2 Hz, 2H), 2.24 (s, 3H, methyl), 2.05 (s, 3H, methyl); MS (EI
mode) m/z 244 (M+). Anal. (C12H12N4O2‚0.5H2O) C, H (the
nitrogen % deviated significantly from the calculated value).
Structure was verified using high-resolution MALDI calcd for
C12H12N4O2 (M + 2), 246.111; found, 246.113.
1
3109, cm-1; H NMR (CDCl3) δ 8.24 (s, 1H), 8.08 (s, 1H), 7.71
(s, 1H), 4.47 (m, 4H), 2.73 (s, 3H, methyl), 2.04 (s, 3H, methyl).
Anal. (C12H13N3O4) C, H, N.
6-Acetamido-1-(2-acetoxyethyl)-5-methylbenzimidazole (15).
A solution consisting of 100 mg (0.44 mmol) of 14 in 25 mL of
methanol was purged with nitrogen gas. To this solution was added
50 mg of 5% Pd on carbon. The mixture was hydrogenated under
50 psi of H2 at room temperature for 3 h. The catalyst was filtered
off and washed with methanol. The methanol solution was
concentrated, giving a clear residue that was dissolved in a solution
consisting of 2 mL of acetic acid and 2 mL of acetic anhydride,
and stirred for 24 h under a drying tube. The solution was
concentrated, giving a clear residue that was solidified with acetone
to afford 15 as white crystals (50 mg, 41% yield). When the reaction
was scaled up to 800 mg and the catalyst was washed with DMF,
the yield increased to 58%; mp 191-193 °C; TLC (20% methanol
in chloroform) R×c4 ) 0.5; IR (KBr pellet) 869, 1045, 1240, 1371,
1494, 1537, 1649, 1741, 3281, cm-1; 1H NMR (CDCl3) δ 8.14 (s,
1H), 7.85 (s, 1H), 7.59 (s, 1H), 7.10 (br s, 1H), 4.39 (s, 4H), 2.39
(s, 3H, methyl), 2.27 (s, 3H, methyl), 2.03 (s, 3H, methyl). Anal.
(C14H17N3O3‚0.7AcOH) C, H, N.
3-Methoxyacetamido-4-[N-methoxyacetyl-2-(2-methoxyace-
toxy)-ethylamino] Toluene (19). To an ice cold suspension of 1 g
(6 mmol) of 12 in 50 mL of dry dichloromethane was added 3.2
mL (183 mmol) of diisopropylethylamine with stirring. To this
mixture was added 1.7 mL (18 mmol) of methoxyacetyl chloride
dropwise. The reaction was allowed to warm to room temperature
while monitoring the progress of the reaction by TLC. At this time,
an additional 0.5 mL of methoxyacetyl chloride and 0.5 mL of
diisopropylethylamine were then added dropwise. When the reaction
was complete, 25 mL of H2O was added to quench the reaction.
The organic phase was separated and washed once with 25 mL of
H2O, followed by a washing with 25 mL of saturated NaHCO3,
and then with 25 mL of saturated brine solution. The organic phase
was then dried with Na2SO4 and concentrated, yielding a brown
oil that solidified upon triturating with hexanes, giving a cream-
colored solid (98% yield); mp 92-93 °C; TLC (1:1 acetone and
ethyl acetate) R×c4 ) 0.41; IR (KBr pellet) 1026, 1120, 1192, 1286,
1419, 1541, 1662, 1743, 2822, 2931, 3267 cm -1; 1H NMR (CDCl3)
δ 8.52 (s, 1H, amide), 8.16 (s, 1H), 7.09 (d, J ) 8 Hz, 1H), 6.98
(d, J ) 8 Hz, 1H), 4.36 (t, J ) 5.4 Hz, 2H), 4.01-3.59 (m, 8H)
3.45, 3.38, and 3.28 (3s, 9H, methoxy), 2.39 (s, 3H, methyl); MS
(HRE) m/z (M+) calcd for C18H26N2O7, 382.17; found, 382.173.
Anal. (C18H26N2O7) C, H, N.
6-Acetamido-1-(2-acetoxyethyl)-5-methyl-7-nitrobenzimida-
zole (16). To a 4:1 solution containing 90% HNO3 and H2SO4 (12
mL/3 mL) was added 550 mg (2 mmol) of 15 in small portions,
keeping the temperature between 0 and 5 °C. After the addition,
the reaction was stirred for 20 min at 0 °C, quenched with ice, and
the pH was adjusted to 8 with NaHCO3. The neutral solution was
then extracted with dichloromethane, dried with Na2SO4, and
concentrated, giving a yellow solid. The solid was recrystallized
with ethyl acetate and hexanes, yielding pale yellow crystals (400
mg, 63% yield); mp 186-187 °C; TLC (20% methanol in
chloroform) R×c4 ) 0.6; IR (KBr pellet) 1045, 1238, 1375, 1529,
3-Methoxyacetamido-4-[N-methoxyacetyl-2-(2-methoxyace-
toxy)ethylamino]-6-nitrotoluene (20). To 5 mL of 90% nitric acid,
cooled to 0 °C with an ice bath, was added 1 g (2.6 mmol) of 19
portionwise. During the addition, the temperature was kept below
10 °C, followed by stirring at 0 °C for 4 h. The completed reaction
was quenched with ice, and the product was extracted with
dichloromethane. The organic phase was dried with Na2SO4 and
concentrated to a yellow oil. The oil was crystallized with ethanol
to afford 20 as pale yellow crystals (614 mg, 55% yield); mp 126-
128 °C; TLC (1:1 acetone/ethyl acetate) R×c4 ) 0.43; IR (KBr pellet)
686, 754, 844, 954, 1028, 1122, 1188, 1344, 1508, 1670, 1747,
1
1660, 1737, 3074, 3286, cm-1; H NMR (CDCl3) δ 7.91, (s, 1H),
7.87 (s, 1H), 7.58 (br s, 1H), 4.44 (t, J ) 4 Hz, 2H, ethylene), 4.25
(t, J ) 4 Hz, 2H, ethylene) 2.41 (s, 3H methyl), 2.26 (s, 3H methyl),
1.96 (3H, s, methyl). Anal. (C14H16N4O5) C, H, N.
6-Acetamido-1-(2-methanesulfonoxyethyl)-5-methyl-7-nitroben-
zimidazole (17). A solution of methanol (50 mL) containing 350
mg (1.1 mmol) of 16 and 6 mL of concentrated HCl was stirred at
room temperature for 12 h. The reaction solution was evaporated
giving a yellow HCl salt of the O-deacetylated product. Under
1
2822, 2933, 3059 cm -1; H NMR (CDCl3) δ 876 (s, 1H, amide),
8.52 (s, 1H), 8.01 (s, 1H), 4.4 (m, J ) 2.1 Hz, 2H), 4.04-3.63 (m,
8H) 3.47 (s, 3H, methoxy), 3.37(s, 3H, methoxy), 3.27 (s, 3H,

Synthesis of Imidazoquinoxalinones
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4569
methoxy), 2.69 (s, 3H, methyl); MS (HRE) m/z (M+) calcd for
C18H25N3O9, 427.16; found, 427.1527. Anal. (C18H25N3O9) C,
H, N.
The reaction was stirred for 12 h at room temperature and
then concentrated in vacuo to afford a yellow oil that was
chromatographed on silica gel using ethyl acetate as the eluent.
The fractions containing product were concentrated, giving a yellow
residue that was crystallized with methanol to afford 24 as
yellow crystals (190 mg, 40% yield); mp > 180 °C dec; TLC
(10% methanol in chloroform) R×c4 ) 0.2; FTIR (KBr pellet)
1-(2-Hydroxyethyl)-2-(hydroxymethyl)-5-methyl-6-nitroben-
zimidazole (21). A solution of 3.04 g (7 mmol) of 20 in 50 mL of
48% HBr was refluxed at 130 °C for 22 min. The reaction was
then cooled in an ice bath immediately, and the pH was adjusted
to 8 with NaHCO3. The precipitated solids were filtered, washed
with H2O, and partially dried by vacuum filtration. The damp
crystals were recrystallized with ethanol yielding cream-colored
needles (1.32 g, 74% yield); mp 211-213 °C; TLC (1:1 acetone/
ethyl acetate) R×c4 ) 0.44; FTIR (KBr pellet) 879, 1074, 1332,
1431, 1516, 2908, 3171, 3344 cm-1; 1H NMR (DMSO-d6) δ 8.36
(s, 1H), 7.62 (s, 1H), 5.7 (t, J ) 6 Hz, 1H), 5.01 (t, J ) 5 Hz, 1H),
4.76 (d, J ) 5 Hz, 2H), 4.43 (t, J ) 5 Hz, 2H), 3.74 (q, J ) 5.7
Hz, 2H), 2.59 (t, 3H, CH3); MS (HRE) m/z (M+) calcd for
C11H13N3O4, 251.09; found, 251.089. Anal. (C11H13N3O4‚0.75H2O)
C, H, N.
925, 974, 1020, 1167, 1348, 1523, 1668, 3323, 3418, 3464 cm -1
;
1H NMR (DMSO-d6) δ 9.84 (s, 1H, amide), 7.90 (s, 1H), 5.13 (s,
2H, methylene), 4.55 (t, J ) 4 Hz, 2H), 4.396 (t, J ) 4 Hz,
2H), 3.01 (s, 3H, methyl), 2.31 (s, 3H, methyl), 2.04 (s, 3H, methyl);
MS (EI mode) m/z 404 (M+). Anal. (C14H17ClN4O6S) C,
H, N.
6-Acetamido-2-(acetoxymethyl)-1-(2-methansulfonoxyethyl)-
5-methyl-7-nitrobenzimidazole (25). To a solution of 122 mg (0.3
mmol) of 24 in 3 mL of DMSO was added 61 mg (0.732 mmol)
of sodium acetate dissolved in a minimum amount of methanol.
The reaction was stirred at room temperature for 5 h. After 5 h,
H2O was added to quench the reaction (exothermic), resulting in
the formation of pale yellow crystals. The crystals were filtered
off and dried to afford 25 (96 mg, 75% yield); mp > 215 °C dec;
TLC (10% methanol in chloroform) R×c4 ) 0.29; IR (KBr pellet)
810, 922, 1041, 1165, 1249, 1357, 1521, 1666, 1736, 3016, 3323
cm -1; 1H NMR (CDCl3) δ 7.85 (s, 1H), 7.36 (s, 1H), 5.40 (s, 2H,
methylene), 4.58 (t, J ) 5 Hz, 2H), 4.41 (t, J ) 5 Hz, 2H), 2.90 (s,
3H, methyl), 2.41 (s, 3H, methyl), 2.22 (s, 3H, methyl), 2.17 (s,
3H, methyl); MS (HRE) m/z 428.0995 (M+); Anal. (C16H20N4O8S)
C, H, N.
6-Acetamido-1-(2-acetoxyethyl)-2-(acetoxymethyl)-5-methyl-
benzimidazole (22). A solution of 2.18 g (8.6 mmol) of 21 in 110
mL of methanol was degassed with nitrogen. To this was added
492 mg of 5% Pd on carbon, and the mixture was hydrogenated
under 50 psi of H2 at room temperature for 3 h. The catalyst was
filtered off and washed with methanol. The methanol filtrate was
concentrated in vacuo leaving a white solid. The solid was dissolved
in a solution of 55 mL of acetic anhydride and 55 mL of acetic
acid and stirred at room temperature for 18 h. The completed
reaction was concentrated leaving a light purple solid that was taken
up in 25 mL of saturated NaHCO3 solution and 50 mL of
dichloromethane. The organic layer was dried with Na2SO4 and
concentrated to a white solid that was recrystallized with chloroform
and hexane to afford 22 as white crystals (2.11 g, 70% yield); mp
161-162 °C; TLC (10% methanol in chloroform) R×c4 ) 0.47; IR
7-Acetamido-2-acetoxymethyl-5, 6-dihydro-8-methyl-4H-imi-
dazo[1,5,4-de]quinoxaline (26). To a suspension of 96 mg (0.22
mmol) of 25 in 25 mL of methanol was added 256 mg of 5% Pd
on carbon. The mixture was hydrogenated under 50 psi of H2 for
30 min. The catalyst was filtered off and washed with methanol.
The methanol filtrate was heated at 60 °C for 1 h and concentrated,
giving a white powder that was recrystallized with methanol and
ethyl acetate to afford white crystals (52 mg, 78% yield); mp >
225 °C dec; TLC (20% methanol in chloroform) R×c4 ) 0.4; IR
(KBr pellet) 879, 1074, 1332, 1431, 1516, 2908, 3171, 3344 cm-1
;
1H NMR (CDCl3) δ 8.19 (s, 1H), 7.56 (s, 1H), 7.12 (s, 1H), 5.36
(s, 2H), 4.48 (t, J ) 6 Hz, 2H), 4.39 (t, J ) 6 Hz, 2H), 2.38 (s, 3H,
methyl), 2.25 (s, 3H, methyl) 2.15 (s, 3H, methyl), 2.02 (s, 3H,
methyl); MS (HRE) m/z (M+) calcd for C17H21N3O5, 347.15; found,
347.1479. Anal. (C17H21N3O5) C, H, N.
1
(KBr pellet) 1033, 1219, 1500, 1643, 1755, 3009, 3329 cm-1; H
NMR (DMSO-d6) δ 9.16 (s, 1H), 6.85 (s, 1H), 4.49 (s, 2H,
methylene) 4.45 (t, J ) 4.5 Hz, 2H), 3.57 (t, J ) 4.5 Hz, 2H), 2.28
(s, 3H, methyl), 2.21 (s, 3H, methyl), 2.05 (s, 3H, methyl); MS
(HRE) m/z (M+) calcd for C15H18N4O3, 302.1379; found, 302.138.
6-Acetamido-1-(2-acetoxyethyl)-2-(acetoxymethyl)-5-methyl-
7-nitrobenzimidazole (23). A solution of 12 mL of 90% nitric acid
and 3 mL of concentrated sulfuric acid was cooled to 0 °C on an
ice bath. To this mixture was added 525 mg (2 mmol) of 22 in
small portions while keeping the temperature below 5 °C. After
the addition was complete, the reaction was stirred at 0 °C for 75
min, followed by pouring over ice. The resulting mixture was
neutralized with sodium bicarbonate, and the product was extracted
with dichloromethane. The organic phase was dried with sodium
sulfate and concentrated to a yellow oil that crystallized with ethyl
acetate and hexane to afford light yellow crystals (462 mg, 78%
yield); mp 158-159 °C; TLC (10% methanol in chloroform) R×c4
) 0.45; IR (KBr pellet) 1049, 1240,1357, 1435, 1527, 1660, 1753,
7-Acetamido-2-acetoxymethyl-8-methyl-4,5-dihydroimidazo-
[1,5,4-de]quinoxalin-9-one (2b). To a solution consisting of 10
mL of phosphate buffer (pH ) 7) and 300 mg of Fremy’s salt was
added 40 mg (0.132 mmol) of 26 at room temperature. The reaction
was stirred for 10 min, followed by extraction with dichloromethane.
The organic layer was dried with sodium sulfate and concentrated,
giving a yellow solid. The solid was recrystallized with chloroform
and hexane to afford yellow crystals (22 mg, 56% yield); mp >
205 °C dec; TLC (20% methanol in chloroform) R×c4 ) 0.33; IR
(KBr pellet) 1035, 1238, 1373, 1535, 1662, 1743, 2987, 3161, cm-1
;
1
2982, 3292 cm -1; H NMR (CDCl3) δ 7.78 (s, 1H), 7.58 (s, 1H,
1H NMR (CDCl3) δ 7.54 (s, 1H), 5.21 (s, 2H, methylene), 4.32
(m, methylene, 2H), 4.21 (m, methylene, 2H), 2.32 (s, 3H, methyl),
2.11 (s, 3H, methyl), 2.04 (s, 3H, methyl); MS (HRE) m/z (M+)
calcd for C15H16N4O4, 316.1172; found, 316.1165. Anal. (C15H16N4O4‚
0.75H2O) C, H, N.
amide), 5.38 (s, 2H, methylene), 4.46 (t, J ) 5 Hz, 2H), 4.21 (t, J
) 5 Hz, 2H), 2.37 (s, 3H, methyl), 2.21 (s, 3H, methyl), 2.16 (s,
3H, methyl), 1.94 (s, 3H, methyl); MS (HRE) m/z (M+) calcd for
C17H20N4O7, 392.13; found, 392.1328. Anal. (C17H20N4O7)
C, H, N.
Cell Culture and MTT Assay. Cancer cell line HCT-116
(human colon tumor) was grown at 37 °C in 5% CO2 in McCoy’s
5a medium containing 10% fetal bovine serum, penicillin (50 IU/
mL), streptomycin (50 µg/mL), and 2 mM L-glutamine. Human
epidermoid carcinoma A-431 cells were maintained at 37 °C in
5% CO2 in Dulbecco’s Modified Eagle’s Medium containing 10%
fetal bovine serum, penicillin (50 IU/mL), streptomycin (50 µg/
mL), and 4 mM L-glutamine. Cells were plated at a density of
10 000 cells/well in a 96-well plate. A total of 24 hrs after plating,
they were exposed to medium containing compound at a concentra-
tion of 0.3 to 100 µM (HCT-116) and 0.1 to 100 µM (A-431) for
48 h at 37 °C. After a 48 h exposure of cells to compounds, 100
µL of fresh culture medium containing MTT at a final concentration
6-Acetamido-2-(chloromethyl)-1-(2-methansulfonoxyethyl)-5-
methyl-7-nitrobenzimidazole (24). To a solution of 500 mg (1.3
mmol) of 23 in 55 mL of methanol was added 7 mL of concentrated
HCl. The reaction was stirred at room temperature for 48 h. The
methanol was removed in vacuo leaving a yellow residue that was
crystallized from methanol and ethyl acetate to afford 408 mg of
yellow crystals.
This product was suspended in dry dichloromethane under a
nitrogen atmosphere. To this suspension was added 630 µL (36
mmol) of diisopropyl ethyl amine dropwise, and the solution was
stirred at room temperature for 5 min, followed by the drop-
wise addition of 557 µL (7.2 mmol) of methane sulfonyl chloride.

4570 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19
Hoang et al.
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of 0.3 mg/mL was added to each well and incubated for 3 h at
37 °C. The formazan crystals were solubilized in 100 µL of DMSO.
The absorbance of each well was measured by a microplate reader
(Multi-skan lab systems) at 570 nm. The percentage cytotoxicity
was calculated by comparison of the A570 reading from treated
versus control cells. Makaluvamine C was used as positive control
at a concentration of 10 µM.
Topoisomerase II Inhibition Assays. The topoisomerase II
relaxation reactions were carried out with 0.25 µg of pRYG
supercoiled DNA (form I) and 4 units of topoisomerase II in a
total volume of 20 µL 50 mM Tris buffer (pH 8.0) containing 120
mM of KCl, 10 mM of MgCl2, 0.5 mM of dithiothreitol, and 0.5
mM of ATP in microcentrifuge tubes. Varying amounts of
drugs were added to each reaction. The reactions were run at 37 °C
for 45 min. The reactions were then stopped by the addition of 2
µL of 10% SDS to each tube, and 1 µL of proteinase K (1 mg/mL)
was also added. The resulting mixtures were incubated
for 15 min at 37 °C. Each reaction mixture was extracted once
with 20 µL of chloroform/isoamyl alcohol (24:1) and then combined
with 2 µL of 10× loading dye (0.25% bromophenol blue and
50% aqueous glycerol). The resulting mixtures were then loaded
onto a 1% agarose gel in 1× TAE buffer and run at 2 V/cm for
5-7 h. The gels either contained or did not contain 0.5 µg/mL of
ethidium bromide. After running, gels without ethidium bromide
were soaked in 1× TAE buffer containing 0.5 µg/mL of ethidium
bromide for 30 min, followed by destaining in 1× TAE buffer for
20 min.
In Vitro Screening Procedures. Compounds from series 1 and
2 were tested against all cell lines included in the 60-cell-line panel,
as previously described.22
Acute Toxicity Assays. The drug was prepared at a concentration
consistent with administration of 400 mg/kg intraperitoneally in a
volume of 1 mL. Each mouse was treated with a single dose of
400, 200, and 100 mg/kg (1 mL, 0.5 mL, and 0.25 mL/mouse).
The body weight and strain of mouse used should be consistent
with that to be used in the subsequent hollow fiber assay. The mice
are held for a period of 14 days, observing for morbidity (body
weight loss) and mortality. If the mice do not survive for 14 days,
administer a 50% lower dose to a single mouse and continue this
process (50 mg/kg/25 mg/kg/12.5 mg/kg/etc.) until a tolerated
dosage is determined. Use this maximum tolerated dosage as the
basis for selecting doses for antitumor drug evaluations.
Acknowledgment. We thank the National Science Founda-
tion for their generous support.
(22) Boyd, M. R.; Paull, K. D. Some practical considerations and
applications of the National Cancer Institute in vitro anticancer drug
discovery screen. Drug DeV. Res. 1995, 34, 91-109.
(23) Xing, C.; Skibo, E. B.; Dorr, R. T. Aziridinyl quinone antitumor
agents based on indoles and cyclopent[b]indoles: Structure-activity
relationships for cytotoxicity and antitumor activity. J. Med. Chem.
2001, 44, 3545-3562.
Supporting Information Available: Elemental analyses of
reported compounds are included. This material is available free
of charge via the Internet at http://pubs.acs.org.
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