Dicationic dibenzofuran derivatives as anti-Pneumocystis carinii pneumonia agents: Synthesis, DNA binding affinity, and anti-P. carinii activity in an immunosuppressed rat model

Article abstract of DOI:10.1016/S0223-5234(99)80054-3

Previous work from our laboratory shows that compounds with two cations linked by a carbazole spacer were highly potent anti-P. carinii agents. A prodrug approach designed to increase oral activity of the dicationic carbazoles by converting amidine groups to amidoxime groups was unsuccessful. The ring nitrogen was implicated as playing a role in the lack of activity of carbazole amidoximes. The current study was designed to determine if replacement of the carbazole ring nitrogen by isosteric oxygen to form dibenzofurans would improve effectiveness of amidoxime prodrugs. Eight dibenzofuran dicationic derivatives were synthesized and evaluated for anti- P. carinii activity in an immunosuppressed rat model. Since DNA binding has been hypothesized to play a key role in antimicrobial activity of dicationic compounds, the compounds were examined for their binding affinity to calf thymus DNA and a poly-dA·poly-dT oligomer. While several of the compounds were more potent anti-P. carinii agents than pentamidine, the corresponding amidoximes were significantly less effective than the amidoxime of pentamidine. No direct quantitative correlation was determined between DNA binding affinity and anti-P. carinii activity, but all active compounds were strong DNA binding agents.

Full text of DOI:10.1016/S0223-5234(99)80054-3

Eur. J. Med. Chem. 34 (1999) 215−224
© Elsevier, Paris
215
Original article
Dicationic dibenzofuran derivatives as anti-Pneumocystis carinii pneumonia
agents: synthesis, DNA binding affinity, and anti-P. carinii activity
in an immunosuppressed rat model
Sihe Wanga, James Edwin Hallb,c, Farial A. Taniousd,
W. David Wilsond, Donald A. Patrickb, Donald R. McCurdyb,
Brenden C. Benderb, Richard R. Tidwella,b*
aDivision of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
bDepartment of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
cDepartment of Epidemiology, School of Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
dDepartment of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA
(Received 19 June 1998; accepted 3 September 1998)
Abstract – Previous work from our laboratory shows that compounds with two cations linked by a carbazole spacer were highly potent anti-
P. carinii agents. A prodrug approach designed to increase oral activity of the dicationic carbazoles by converting amidine groups to
amidoxime groups was unsuccessful. The ring nitrogen was implicated as playing a role in the lack of activity of carbazole amidoximes. The
current study was designed to determine if replacement of the carbazole ring nitrogen by isosteric oxygen to form dibenzofurans would
improve effectiveness of amidoxime prodrugs. Eight dibenzofuran dicationic derivatives were synthesized and evaluated for anti-P. carinii
activity in an immunosuppressed rat model. Since DNA binding has been hypothesized to play a key role in antimicrobial activity of dicationic
compounds, the compounds were examined for their binding affinity to calf thymus DNA and a poly-dA•poly-dT oligomer. While several of
the compounds were more potent anti-P. carinii agents than pentamidine, the corresponding amidoximes were significantly less effective than
the amidoxime of pentamidine. No direct quantitative correlation was determined between DNA binding affinity and anti-P. carinii activity,
but all active compounds were strong DNA binding agents. © Elsevier, Paris
Pneumocystis carinii pneumonia / dibenzofurans / amidines / amidoximes / DNA
1. Introduction
determined the potential effect of metabolism on antimi-
crobial activity and toxicity of pentamidine and related
compounds [11–15]; (2) provided evidence for potential
mechanisms of antimicrobial activity [7, 16–21] and
toxicity [22] of these molecules; (3) shown the com-
pounds to be active against a number of pathogenic
organisms [3, 5–9, 17, 20, 23–28]; (4) determined the
feasibility of utilizing amidoximes as prodrugs for dia-
midine compounds in order to increase bioavailabil-
ity [29, 30]. A major effort in our laboratory has been
directed toward the clinical development of a safe,
effective and orally available dicationic molecule for the
treatment of an important AIDS-related opportunistic
infection, Pneumocystis carinii pneumonia (PCP).
Dicationic molecules (pentamidine related) have been
shown to be active against a number of pathogenic
organisms including: Pneumocystis carinii [1–5], Plas-
modium falciparum [6], Giardia lamblia [7], Leishmania
mexicana amazonensis [6], Cryptosporidium parvum [8],
Toxoplasma gondii [9], Candida albicans [10], and Cryp-
tococcus neoformans [10]. Previous studies in our labo-
ratory directed toward the development of dicationic
molecules as potential antimicrobial agents have: (1)
*Correspondence and reprints
Abbreviations: HPLC: high-performance liquid chromatography;
DMF: N,N-dimethylformamide; DMSO: dimethylsulfoxide; TFA:
trifluoroacetic acid
While a large number of the dicationic molecules were
found to be active against P. carinii and have low toxicity

216
Figure 1. Bases corresponding to carbazole and dibenzofuran dications.
in an animal model of the disease, a major challenge has
been to develop an orally active drug. A promising
approach to obtaining orally active dicationic molecules
was the synthesis of prodrugs containing amidoximes in
place of amidine moieties. The amidoxime is less basic
than the parent amidine (pKa = 8–9 for amidoximes
compared to pKa = 12–13 for amidines) and has improved
oral uptake [29]. While the amidoxime is devoid of in
vitro antimicrobial activity, it is rapidly metabolized to
the corresponding active amidine [29–36]. Previous stud-
ies showed that this approach was highly effective for
pentamidine [29]. However, it was disappointing to find
that several of the more potent and less toxic dicationic
molecules were ineffective anti-PCP agents when given
orally as the amidoxime prodrug. An unexplained corre-
lation was observed in a series of prodrug experi-
ments [29, 30] when it was found that the amidoxime
prodrugs that were ineffective in treating PCP were those
having a nitrogen in the moiety linking the two dicationic
groups [29, 30]. The lack of activity of prodrugs with the
cations linked by a carbazole spacer (figure 1) was
particularly disappointing since the dicationic carbazoles
had proved to be highly potent antimicrobial agents with
low toxicity in animal models [4]. The current study was
designed to determine if replacement of the carbazole
nitrogen by oxygen to form a dibenzofuran would lead to
improved effectiveness of amidoxime prodrugs. The
current study contains the synthesis and anti-PCP testing
of a series of dicationic dibenzofurans (figure 1) and
selected amidoxime derivatives. Because DNA minor
groove binding is hypothesized to play a major role in the
antimicrobial activity of these molecules [7, 16–20], tests
were also performed to determine the binding affinity of
the molecules with calf thymus DNA and a poly-
dA•poly-dT oligomer.
2. Chemistry
Diamidine 1 was first made by Moffatt in 1950 [37],
while the other dications synthesized in this paper are
novel. The 2,8-disubstituted dibenzofurans (figure 2)
were synthesized from the commercially available diben-
zofuran 9. 2,8-Dibromodibenzofuran 10 [38], the major
product of bromination of dibenzofuran 9, underwent
nucleophilic reaction with copper(I) cyanide in refluxing
N,N-dimethylformamide to provide dicyanodibenzofuran
11 [39]. The diimidate intermediate 12 was prepared from
11 through Pinner reaction [24, 40–43] by reacting with
ethanol in hydrogen chloride saturated 1,4-dioxane at
room temperature for 9–14 days. The diimidate 12 was
isolated without being characterized after high conver-
sion was determined by HPLC and IR. The dried diimi-
date 12 reacted with excess ammonia, ethylenediamine,
isopropylamine, and hydroxylamine in anhydrous ethanol
to give the final compounds diamidine 1, diimidazoline 2,
di(isopropylamidine) 3, and diamidoxime 4 respectively.
All these dications were in hydrochloride salt forms after
final purification.
3,7-Disubstituted dibenzofuran derivatives (figure 3)
could not be synthesized effectively from the dibenzofu-
ran ring. Therefore, de novo furan ring preparation was
necessary to obtain the desired products. Ullman cou-
pling reaction [44] of 2,5-dibromonitrobenzene 13 with
copper powder in N,N-dimethylformamide at 120 °C
gave compound 14. Selective nucleophilic substitution of
14 by reacting with sodium methoxide between 0 °C and

217
Figure 2. Synthesis of 2,8-disubstituted dibenzofuran dications. Keys: (a) Br2/AcOH/reflux; (b) CuCN/DMF/reflux; (c)
EtOH/HCl/1,4-dioxane/room temperature; (d) Appropriate amine/EtOH.
room temperature provided the methoxy compound 15.
Since the aromatic nitro group is a better leaving group
than the aromatic bromo group, selective substitution
could be achieved [45, 46]. However, significant impuri-
ties were found in the reaction mixture, resulting from
different combinations of methoxy substitution. The key
condition for obtaining the desired product 15 was
temperature control (0 °C at the beginning of the reaction,
then increased to room temperature as the reaction
slowed). The nitro group of compound 15 was reduced to
the corresponding amine 16 using 5% ruthenium on
carbon as a catalyst in ethanol at 65–70 °C [47]. The
dibenzofuran ring was formed with minor modifications
of a reported procedure [48]. The amino group in com-
pound 16 was first diazotized in sodium nitrite and
aqueous sulfuric acid solution. The diazonium salt 17
obtained in situ was heated to reflux to give 3,7-
dibromodibenzofuran 18. The 3,7-dicyanodibenzofuran
19 was prepared from 18, by reacting with copper(I)
cyanide in refluxing N,N-dimethylformamide. Pinner re-
action [24, 40–43] of compound 19 with ethanol in HCl
saturated 1,4-dioxane at room temperature for 2 weeks
gave the intermediate 3,7-diimidatedibenzofuran 20,
which was isolated without characterization. 20 was
reacted with either excess ammonia, ethylenediamine,
isopropylamine, or hydroxylamine in ethanol solution to
give the corresponding diamidine 5, diimidazoline 6,
di(isopropylamidine) 7, or diamidoxime 8.
3. Results and discussion
3.1. Activity against P. carinii pneumonia
The activity of compounds 1–8 against PCP in the rat
model is shown in table I. Compounds 1–3 and 5–7 in
the initial screen were given once daily by tail vein
injection at a dose of 10 mmol/kg/d for 14 days, with
activities compared to pentamidine at approximately
one-half the effective i.v. dose of pentamidine, which is
22 mmol/kg/d. Two of the six dicationic compounds (3,7)
were found to be highly potent at the screening dose,
reducing the parasite load by over 99%. Both highly
active compounds contained isopropyl substituted dia-
midines, with the cationic moieties of 3 located para and
those of 7 meta to the furan oxygen atom. The 2,8-
diamidine 1 was found to be comparable in activity to
pentamidine at its screening dose and more active than
pentamidine when given at the same dose as pentamidine.
In contrast, the 3,7-diamidine 5 was found to be inactive.
The 2,8-diimidazoline 2 and 3,7-diimidazoline 6 were
both inactive at the screening dose. The observed toxici-
ties of compounds 1–3 and 5–7 were found to be less than
or equivalent to that of pentamidine at the doses used.
Compounds 4 and 8 were synthesized to test if diami-
doximes would be effective prodrugs of the dibenzofuran
diamidines. Both the diamidoximes were screened for

218
Figure 3. Synthesis of 3,7-disubstituted dibenzofuran dications. Key: (a) Cu/DMF/120 °C; (b) NaOMe/MeOH/DMF; (c)
NH2NH2•H2O/5% Ru/C/EtOH; (d) NaNO2/H2SO4/< 5 °C; (e) Reflux; (f) CuCN/DMF/reflux; (g) EtOH/HCl/1,4-dioxane; (h)
Appropriate amine/EtOH.
anti-P. carinii activity at 10 mmol/kg/d by tail vein injec-
tion and 33 mmol/kg/d by oral gavage for 14 days. The
diamidoximes 4 and 8 were compared for efficacy with
pentamidoxime, an effective prodrug. Not surprisingly,
3,7-diamidoxime 8 was not active since the corresponding
diamidine 5 was not active. Compound 4 had only
moderate activity against PCP, while the corresponding
diamidine 1 proved to be potent. Further studies of
diamidoxime metabolism are being performed using cul-
tured cell lines and cell-free enzyme preparations to
understand more about the metabolic mechanisms of
prodrug activation. Preliminary results showed that the
conversion of 2,8-dibenzofuran diamidoxime to diamidine
is less efficient than conversion of pentamidoxime to
pentamidine by whole cells. We hypothesize that either
transport or enzyme activity plays a key role in the
conversion of diamidoximes to diamidines.
zoles with the cations para to the ring nitrogen (3,6-
disubstituted carbazoles) [49]. As an example, 3,6-di(2-
imidazolinyl)carbazole bound in the minor groove via
hydrogen bonds from the two imidazoline groups result-
ing in an orientation with the ring nitrogen pointing away
from the DNA surface. The corresponding 2,7-
disubstituted compound was only able to establish two
hydrogen bonds with the surface of the minor groove by
flipping over and forming hydrogen bonds with the DNA
through one of the imidazoline groups and the ring
nitrogen. It was anticipated that the 3,7-dicationic benzo-
furans (cations meta to ring oxygen) would correspond
with the 2,7-disubstituted carbazoles in orientation rela-
tive to the DNA surface, and therefore, without the
hydrogen bond contributed by the ring nitrogen of the
carbazole, would be less potent DNA binders than the
corresponding 2,7-disubstituted carbazoles. Comparing
the DNA binding results in table I with previous results
from the carbazole study indicate only a modest reduction
in the ∆Tms of dibenzofurans compared to the carbazoles.
Furthermore, judging from the carbazole studies it was
anticipated that the 2,8-disubstituted dibenzofurans (two
hydrogen bond donors) would have highly increased
3.2. DNA binding
Previous studies with dicationic carbazoles indicated
that carbazoles with the cations substituted meta to the
ring heterocycle (2,7-substituted carbazole) bound to the
DNA minor groove in a different orientation from carba-

219
Table I. Anti-P. carinii pneumonia activity and DNA binding affinity of dibenzofuran dications.
d
Compound
R
Pneumocystis carinii
DNA binding ∆Tm (°C)
% Saline control ± standard Calf thymus
a
b
Dose
Toxicity
c
error
DNA poly-dA•poly-dT
Pentamidine
Pentamidoxime
–
–
–
–
22.0
22.0
33.0 *
++
+
0
0.80 ± 0.17
0.11 ± 0.02
13.76 ± 9.12
10.7
0.5
12.8 [51]
ND
10.0
22.0
+
++
7.15 ± 3.81
0.10 ± 0.02
1
2
2, 8
2, 8
14.5
15.0
10.0
10.0
10.0
+
97.36 ± 31.92
0.50 ± 0.27
14.9
3
2, 8
+
+
8.1
0.5
3.2
10.0
33.0 *
15.76 ± 14.60
45.06 ± 21.61
ND
4
5
6
2, 8
3, 7
3, 7
10.0
10.0
+
+
182.65 ± 38.32
219.93 ± 39.28
12.7
14.0
10.0
8.6
7
8
3, 7
3, 7
10.0
+
0.28 ± 0.27
12.7
0.9
1.8
0.5
10.0
33.0 *
+
0
165.58 ± 108.73
150.39 ± 54.05
a Dose units were µmol/kg/day. Each compound was administered to 8 rats once daily for 14 days. Doses marked with (*) were by oral gavage,
all others were i.v. via tail vein.
b Toxicity scores were subjective evalutions of overt toxicity in dexamethasone immunosuppressed rats. A score of 0 indicates no observable
deleterious effects from dosing whereas ++ indicates hypotension, ataxia, and dyspnea.
c Cysts/g lung scores for the saline and pentamidine control groups were pooled across experiments. Scores for each compound were reported
as % of the pooled saline score. Saline: 47.53 × 106 cysts/g lung (n = 45). Pentamidine: 0.38 × 106 cysts/g lung (n = 42).
d
Change in melting point determined on calf thymus DNA and a poly-dA•poly-dT oligomer.
DNA binding potency than the corresponding 3,7-
disubstituted dibenzofurans (one hydrogen bond donor).
As can be seen from table I, the 3,7-disubstituted
dibenzofurans were found to have ∆Tms in the same
range as the 2,8-disubstituted analogues. We are currently
studying the DNA interactions of 3,7-di(2-imida-
zolinyl)dibenzofuran utilizing UV-VIS absorbency, fluo-
rescence, kinetic measurement and circular dichroism in
conjunction with NMR spectroscopy in an attempt to
rationalize this unexpected finding.

220
Another interesting and unexpected result from the
acetonitrile was maintained at 3.75% from 0 to 0.5 min,
increased to 45% following a linear gradient in 19.5 min,
increased to 67.5% following a linear gradient over
5 min, then maintained at 67.5% for 7 min. HPLC meth-
ods 2, 3, and 4 employed a Dupont Zorbax SB-C8
column (3.5 µ, 3.0 mm × 15 cm) with a flow rate of
0.6 mL /min. In methods 2 and 3, the concentration of
acetonitrile was maintained at 3.75% from 0 to 0.5 min,
increased to 45% following a linear gradient in 13 min,
immediately increased to 67.5% following a linear gra-
dient over 3 min, then maintained at 67.5% for 4.5 min.
In method 4, the concentration of acetonitrile was main-
tained at 5% from 0 to 0.5 min, increased to 47%
following a linear gradient in 13 min, immediately in-
creased to 72.5% following a linear gradient over 3 min,
then maintained at 72.5% for 4.5 min. Electron impact
mass spectra were recorded on a VG 70-SE or a VG
70-SEQ Hybrid spectrometer. FAB mass spectra were
recorded on a VG 70-SEQ Hybrid spectrometer (Cesium
ion gun, 30 KV). Microanalyses were performed by
Atlantic Microlab, Norcross, GA., and all were within ±
0.4% of the theoretical values. Compounds 9 and 13 were
purchased from Aldrich Chemical Co., Milwaukee, WI.
DNA binding studies was the relatively low affinity for
poly-dA•poly-dT exhibited by two of the molecules
(Compounds 3 and 7). While these two molecules exhib-
ited good affinity for calf thymus DNA, they were found
to have low affinity for poly-dA•poly-dT. Both com-
pounds had isopropyl-substituted amidines as the cationic
group and were potent anti-P. carinii agents. The large
differential observed between binding of calf thymus
DNA and poly-dA•poly-dT for these molecules may
represent a different mode of binding as a result of GC
base pair interactions. Since the combination of strong
DNA affinity and low poly-dA•poly-dT appears to corre-
late with potent anti-P. carinii activity this would be an
exciting area for future study.
4. Biological studies
The activity of these compounds against P. carinii
pneumonia in the rat model was determined using a
standard procedure [5]. The binding affinities of the
compounds with calf thymus DNA and poly-dA•poly-dT
were measured by thermal melting experiments as re-
ported [49].
5.1. 2,8-Diamidinodibenzofuran dihydrochloride 1
5. Experimental protocol
Uncorrected melting points were measured on a Tho-
mas Hoover capillary melting point apparatus. IR spectra
were recorded in Nujol mulls or KBr pellets on a
A stirred suspension of 2,8-dicyanodibenzofuran (11,
2.07 g, 9.50 mmol) in anhydrous EtOH (3.5 mL,
60 mmol) and 1,4-dioxane (100 mL) was cooled in an
ice-salt bath and saturated with dry HCl gas at such a rate
that the reaction temperature was maintained below 5 °C.
The flask was then tightly sealed and the mixture was
maintained at room temperature for 2 weeks, until no or
only a small nitrile band (2230 cm–1) was detected by IR
analysis, and no starting material was detected by HPLC.
The reaction mixture was purged with dry N2 gas and
diluted with ether (100–200 mL). The crude diimidate
was filtered off and dried under N2, and then added
immediately to a solution of ethanolic ammonia (50 mL).
The resultant suspension was stirred at 50–60 °C over-
night in a tightly stoppered flask. The crude product was
filtered off and recrystallized several times from water-
–acetone to give a white powder (1.96 g, 63.3%): m.p. >
Perkin-Elmer 1320 spectrophotometer. 1H NMR and 13
C
NMR were recorded on Bruker AC 300, Varian XL 400,
and Bruker AMX 500 spectrometers. Chemical shifts
were expressed in parts per million downfield relative to
tetramethylsilane (TMS) or sodium 3-(trimethylsilyl)
propionate. Anhydrous ethanol was distilled over Mg
(with trace I2) immediately before use. Reaction products
were dried over P2O5 at room temperature, 40 °C or
77 °C at 0.2 mm Hg. Unless stated otherwise, reactions
were monitored by TLC on silica gel or by reverse phase
HPLC. HPLC chromatograms were recorded on a
Hewlett-Packard 1090 chromatograph using UV detec-
tion (230 nm). Chromatographic data were recorded and
analyzed with a Hewlett-Packard 3396 integrator. Mobile
phase consisted of mixtures of acetonitrile in water
containing 10 mM tetramethylammonium chloride,
10 mM sodium heptanesulfonate, and 2.2 mM phospho-
ric acid (Methods 1, and 2) or 10 mM potassium phos-
phate (Methods 3 and 4). HPLC method 1 employed a
Dupont Zorbax Rx-C8 column (5.0 µ, 4.6 mm × 25 cm)
with a flow rate of 1.5 mL /min. The concentration of
300 °C [Lit. [37] > 320 °C]; HPLC method 4 tR
=
1
9.48 min (98.1 area%); H NMR (300 MHz, DMSO-d6)
δ 9.64 (s, 4 H), 9.38 (s, 4 H), 8.79 (s, 2 H), 8.09 (s, 4 H);
13C NMR (75 MHz, DMSO-d6): δ 165.5, 158.7, 128.8,
123.9, 123.0, 122.3, 112.7; FAB-MS m/z 253 (MH+ of
free base). Anal. (C14H12N4O•2HCl•1.5H2O) C, H, N.

221
5.2. 2,8-Di(2-imidazolinyl)dibenzofuran dihydro-
chloride 2
124 mmol) in anhydrous ethanol (100 mL). The reaction
mixture was filtered through Celite 545 and the filtrate
was diluted with ether. The resultant precipitate was
filtered off and was recrystallized several times from 3–6
N HCl–acetone to give a white solid (0.55 g, 10%): m.p.
A stirred suspension of 2,8-dicyanodibenzofuran (11,
2.06 g, 9.46 mmol) in anhydrous EtOH (10.0 mL,
170 mmol) and 1,4-dioxane (300 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 3 weeks. A mixture of half of the diimidate
and ethylene diamine (22.74 g, 278.5 mmol) in anhy-
drous ethanol (80 mL) was refluxed under N2 for 5.5 h.
The reaction mixture was passed through decolorizing
carbon (1 cm thick), and the filtrate was evaporated. The
residue was further purified several times by recrystalli-
zation from 3 N HCl–acetone to give a white powder
(0.33 g, 17%): m.p. 299–302 °C (dec.); HPLC method 1
tR = 14.65 min (100 area%); 1H NMR (500 MHz,
DMSO-d6) δ 3.87 (s, 8 H), 7.96 (d, J = 8.6 Hz, 2 H), 8.14
(d, J = 8.6 Hz, 2 H), 8.77 (s, 2 H); FAB-MS m/z 305
(MH+ of free base). Anal. (C18H16N4O•2HCl•1.4H2O) C,
H, N.
1
300–305 °C; HPLC method 2 tR = 9.86 (100 area%); H
NMR (400 MHz, DMSO-d6) δ 13.22 (br s, 1 H), 11.39
(br s, 2 H), 9.13 (br s, 4 H), 8.69 (d, J = 1.7 Hz, 2 H), d
8.06 (d, J = 8.7 Hz, 2 H), d 7.99 (dd, J1 = 8.7 Hz, J2 = 1.7
Hz, 2 H); 13C NMR (75 MHz, DMSO-d6): δ 157.91,
157.82, 128.09, 123.04, 122.53, 121.52, 112.53; FAB-MS
m/z
285
(MH+
of
free
base).
Anal.
(C14H12N4O3•2HCl•0.85H2O) C, H, N.
5.5. 3,7-Diamidinodibenzofuran dihydrochloride 5
A stirred suspension of 3,7-dicyanodibenzofuran (19,
2.20 g, 10.1 mmol) in anhydrous EtOH (4.6 mL,
81 mmol) and 1,4-dioxane (100 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 2 weeks. A mixture of half of the diimidate
and ethanolic ammonia solution (80 mL) was stirred at
50–60 °C overnight. The crude product was filtered off,
and dilution of the filtrate with ether resulted in further
precipitation of product. The combined solids were re-
crystallized several times from 1 N HCl–acetone to give
an off-white powder (0.33 g, 18%): m.p. > 300 °C; HPLC
5.3. 2,8-Di(N-isopropylamidino)dibenzofuran dihydro-
chloride 3
A stirred suspension of 2,8-dicyanodibenzofuran (11,
2.00 g, 9.17 mmol) in anhydrous EtOH (4.3 mL,
73 mmol) and 1,4-dioxane (100 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 2 weeks. A mixture of the diimidate and
freshly distilled (KOH) isopropylamine (10 mL,
120 mmol) in ethanol (20 mL) was stirred at room
temperature for 4 days. The crude product was filtered off
and was recrystallized several times from water–acetone
to give a pale yellow powder (1.58 g, 42.0%): m.p.
279–282 °C; HPLC method 3 tR = 11.34 min (97.4
1
method 3 tR = 9.06 min (99.9 area%); H NMR (300
MHz, DMSO-d6) δ 9.70 (s, 4 H), 9.48 (s, 4 H), 9.56 (d,
J = 8.2 Hz, 2 H), 8.44 (s, 2 H), 8.00 (d, J = 8.2 Hz, 2 H);
FAB-MS m/z 253 (MH+). Anal. (C14H12N4O•2HCl) C, H,
N, Cl.
1
area%); H NMR (300 MHz, DMSO-d6) δ 9.86 (d, J =
5.6. 3,7-Di(2-imidazolinyl)dibenzofuran dihydro-
7.6 Hz, 2 H), 9.69 (s, 2 H), 9.33 (s, 2 H), 8.70 (d, J = 1.7
Hz, 2 H), 8.06 (d, J = 8.7, 2 H), d 7.98 (dd, J1 = 8.7 Hz,
J2 = 1.7 Hz, 2 H), 4.18 (m, 2 H), 1.33 (m, 12 H); FAB-MS
chloride 6
m/z
337
(MH+
of
free
base).
Anal.
A stirred suspension of 3,7-dicyanodibenzofuran (19,
2.20 g, 10.1 mmol) in anhydrous EtOH (4.6 mL,
81 mmol) and 1,4-dioxane (100 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 2 weeks. A mixture of half of the diimidate
and ethylene diamine (2.02 mL, 30.2 mmol) in ethanol
(80 mL) was refluxed overnight under nitrogen. The
crude product was filtered off, and recrystallized from 1
N HCl–acetone to give a white solid (0.55 g, 29%): m.p.
> 300 °C; HPLC method 3 tR = 9.61 min (95.3 area%);
1H NMR (300 MHz, TFA-d) δ 4.26 (s, 8 H), 7.86 (d, J =
8.2 Hz, 2 H), 8.17 (s, 2 H), 8.28 (d, J = 8.2 Hz, 2 H),
11.50 (s, 4 H); FAB-MS m/z 305 (MH+). Anal.
(C18H16N4O•2HCl•0.30H2O) C, H, N, Cl.
(C20H24N4O•2HCl•0.90H2O) C, H, N, Cl.
5.4. 2,8-Di(N-hydroxylamidino)dibenzofuran dihydro-
chloride 4
A stirred suspension of 2,8-dicyanodibenzofuran (11,
3.38 g, 15.5 mmol) in anhydrous EtOH (7.5 mL,
170 mmol) and 1,4-dioxane (110 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 2 weeks. The crude diimidate was stirred
overnight at 40 °C in a solution of hydroxylamine,
prepared from hydroxylamine hydrochloride (8.62 g,
124 mmol) and sodium ethoxide (21 wt.%, 46.3 mL,

222
5.7. 3,7-Di(isopropylamidino)dibenzofuran dihydro-
hot ethanol (100 mL) to give a white powder (6.97 g,
95.2%): m.p. 298–300 °C (dec.) (Lit. [37] 299 °C);
HPLC method 1 tR = 24.07 min (97.3 area%); H NMR
chloride 7
1
A stirred suspension of 3,7-dicyanodibenzofuran (19,
1.00 g, 4.58 mmol) in anhydrous EtOH (2.2 mL,
37 mmol) and 1,4-dioxane (80 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 2 weeks. A mixture of the diimidate and
freshly distilled (KOH) isopropylamine (3.1 mL,
37 mmol) in ethanol (20 mL) was stirred at room tem-
perature for 2 days. The crude product was filtered off,
and dilution of the filtrate with ether resulted in further
precipitation of product. The combined solids were re-
crystallized from water–acetone to give a pale yellow
solid (1.21 g, 64.8%): m.p. > 300 °C; HPLC method 3 tR
(400 MHz, DMSO-d6) δ 8.05 (d, J = 8.7 Hz, 2 H), 8.12
(dd, J1 = 8.7 Hz, J2 = 1.4 Hz, 2 H), 8.85 (d, J = 1.4 Hz,
2 H). Anal. (C14H6N2O•0.4H2O) C, H, N.
5.10. 4,4’-Dibromo-2,2’-dinitrobiphenyl 14
A suspension of 2,5-dibromonitrobenzene (13, 50.0 g,
178 mmol) and copper powder (100 mesh, 25.0 g,
391 mmol) in DMF (300 mL) was stirred at 137 °C (oil
bath) under N2 for 2 h. The mixture was poured into
toluene (1000 mL) and stirred for 4 h. Then the mixture
was passed through Celite 545. The filtrate was collected
and washed with water and dried over anhydrous sodium
sulfate. The solvent was evaporated, and the residue was
recrystallized from ethanol (650 mL) to give a pale
yellow solid (31.33 g, 87.57%): m.p. 143–146 °C (Lit.
150 °C [50] and 146–148 °C [44]); HPLC method 3 tR =
18.70 min (97.2%); NMR (300 MHz, CDCl3) δ 8.40 (d,
J = 2.0 Hz, 2 H), 7.85 (dd, J1 = 8.2 Hz, J2 = 2.0 Hz, 2 H),
7.18 (d, J = 8.2 Hz, 2 H).
1
= 10.86 min (95.9 area%); H NMR (300 MHz, DMSO-
d6) δ 9.54 (br s, 6 H), 8.51 (d, J = 8.1 Hz, 2 H), 8.27 (s,
2 H), 7.86 (dd, J1 = 8.1 Hz, J2 = 1.0 Hz, 2 H), 4.14 (m,
2 H), 1.32 (d, J = 6.3 Hz, 12 H); FAB-MS m/z 337 (MH+).
Anal. (C20H24N4O•2HCl•0.30H2O) C, H, N, Cl.
5.8. 3,7-Di(N-hydroxylamidino)dibenzofuran dihydro-
chloride 8
A stirred suspension of 3,7-dicyanodibenzofuran (19,
2.00 g, 9.17 mmol) in anhydrous EtOH (4.2 mL,
73 mmol) and 1,4-dioxane (100 mL) was saturated with
dry HCl gas as described above. The crude diimidate was
collected after 2 weeks. A mixture of the crude diimidate
in a solution of hydroxylamine, prepared from hydroxy-
lamine hydrochloride (5.095 g, 73.32 mmol) and sodium
ethoxide (21 wt.%, 27.4 mL, 73.3 mmol) in anhydrous
ethanol (100 mL) was stirred at 40–50 °C overnight. The
crude product was filtered off and recrystallized several
times from 1 N HCl–acetone to give a white solid (0.38 g,
12%): m.p. > 300 °C; HPLC method 3 tR = 8.83 min
(99.6 area%); 1H NMR (300 MHz, DMSO-d6) δ
11.3–11.6 (br s, 2 H), 8.8–9.6 (br s, 4 H), 8.51 (d, J = 8.4
Hz, 2 H), 8.29 (s, 2 H), 7.89 (d, J = 8.4 Hz, 2 H);
5.11. 2-Methoxy-2’-nitro-4,4’-dibromobiphenyl 15
A solution of sodium methoxide in methanol (22.7 mL,
99.4 mmol) was added dropwise to a solution of 4,4’-
dibromo-2,2’-dinitrobiphenyl (14, 33.28 g, 82.79 mmol)
in DMF (250 mL) cooled in an ice-bath. The reaction
mixture was stirred overnight at room temperature under
N2. The reaction mixture was then poured into ice-water
(700 mL). The precipitated solid was collected and re-
crystallized from acetonitrile (200 mL) and methanol
(100 mL) to give olive green crystals (18.88 g, 59.0%):
m.p. 127–129 °C; HPLC method 3 tR = 19.57 min (96.5
area%); 1H NMR (400 MHz, CDCl3) δ 3.69 (s, 3 H), 7.04
(d, J = 1.6 Hz, 1 H), 7.14 (d, J = 8.1 Hz, 1 H), 7.23 (dd,
J1 = 1.6 Hz, J2 = 7.3 Hz, 1 H), 7.26 (d, J = 7.3 Hz, 1 H),
7.76 (dd, J1 = 1.9 Hz, J2 = 8.1 Hz, 1 H), 8.09 (d, J = 1.9
Hz, 1 H); EI-MS m/z 385 (M+). Anal. (C13H9O3NBr2) C,
H, N.
FAB-MS
m/z
285
(MH+).
Anal.
(C14H12N4O3•2HCl•0.30H2O) C, H, N, Cl.
5.9. 2,8-Dicyanodibenzofuran 11
5.12. 2-Methoxy-2’-amino-4,4’-dibromobiphenyl 16
A mixture of 2,8-dibromodibenzofuran (10, 10.93 g,
33.53 mmol) and copper (I) cyanide (8.91 g, 102 mmol)
in DMF (80 mL) was refluxed under N2 for 9 h. The
reaction mixture was poured into ice-water (300 mL).
The precipitated solid was filtered off and stirred over-
night in a solution of ethylenediamine (50 mL) in water
(300 mL). The solid was filtered off, washed with water,
then stirred in 10% sodium cyanide solution (100 mL)
overnight. The solid was further purified by suspension in
A solution of hydrazine hydrate (19.4 mL, 400 mmol)
in ethanol (20 mL) was added dropwise into a suspension
of
2-methoxy-2’-nitro-4,4’-dibromobiphenyl
(15,
25.77 g, 66.59 mmol) and 5% Ru/C (2.69 g, 1.33 mmol)
in ethanol (250 mL) maintained at 65–70 °C. The reac-
tion mixture was then refluxed for 7 h. The hot reaction
mixture was passed through Celite 545 (1 cm thick), and
the filtrate was evaporated to give an off-white powder

223
(22.69 g, 95.46%): m.p. 94.5–97 °C; HPLC method 3 tR
References
1
= 18.57 min (97.9 area%); H NMR δ 3.40 (br s, 2 H),
3.80 (s, 3 H), 6.9–7.2 (m, 6 H); EI-MS m/z 355 (M+).
[1] Lane H.C., Laughon B.E., Falloon J., Kovacs J.S., Davey R.T.,
Polis M.A., Masur H., Annal. Intern. Med. 120 (1994) 945–955.
Anal. (C13H11ONBr2) C, H, N.
[2] Boykin D.W., Kumar A., Spychala J., Zhou M., Lombardy R.J.,
Wilson W.D., Dykstra C.C., Hall J.E., Jones S.K., Tidwell R.R., Laughton
C., Neidle S., J. Med. Chem. 38 (1995) 912–916.
5.13. 3,7-Dibromo-dibenzofuran 18
[3] Boykin D.W., Kumar A., Bajic M., Xiao G., Wilson W.D., Bender
B.C., McCurdy D.R., Hall J.E., Tidwell R.R., Eur. J. Med. Chem. (in press).
A solution of sodium nitrite (4.17 g, 60.5 mmol) in
water (55.7 mL) was added slowly to a suspension of
2-methoxy-2’-amino-4,4’-dibromobiphenyl (16, 21.60 g,
60.50 mmol) in H2SO4 (14.82 g, 151.3 mmol) and water
(52.7 mL), maintained below 2 °C. The mixture was
stirred for 2 h at 0 °C, and then an excess of urea was
added to destroy the unreacted nitrous acid. The reaction
mixture was stirred overnight at room temperature, then
stirred for 24 h at 70 °C. The solid formed was collected
and recrystallized from benzene (120 mL) and methanol
(100 mL) to give a beige solid (10.27g, 52.09%): m.p.
199–200.5 °C; HPLC method 3 tR = 20.99 min (100
[4] Patrick D.A., Boykin D.W., Wilson W.D., Tanious F.A., Spychala
J., Bender B.C., Hall J.E., Dykstra C.C., Ohemeng K.A., Tidwell R.R., Eur.
J. Med. Chem. 32 (1997) 781–793.
[5] Tidwell R.R., Bell C.A., Pneumocystis carinii Pneumonia (2nd
ed.), Marcel Dekker, New York, 1993.
[6] Bell C.A., Hall J.E., Kyle D.E., Grogl M., Ohemeng K.A., Allen
M.A., Tidwell R.R., Antimicrob. Agents Chemother. 34 (1990) 1381–1386.
[7] Bell C.A., Dykstra C.C., Naiman N.N., Cory M., Fairley T.A.,
Tidwell R.R., Antimicrob. Agents Chemother. 37 (1993) 2668–2673.
[8] Blagburn B.L., Sundermann C.A., Lindsay D.S., Hall J.E., Tidwell
R.R., Antimicrob. Agents Chemother. 35 (1991) 1520–1523.
[9] Lindsay D.S., Blagburn B.L., Hall J.E., Tidwell R.R., Antimicrob.
Agents Chemother. 35 (1991) 1914–1916.
1
[10] Del Poeta M.D., Schell W.A., Dykstra C.C., Jones S., Tidwell
R.R., Kumar A., Boykin D., Perfect J.R., Antimicrob. Agents Chemother.
42 (1998) 2503–2510.
area%); H NMR (300 MHz, CDCl3) δ 7.78 (d, J = 8.2
Hz, 2 H), 7.74 (d, J = 1.2 Hz, 2 H), 7.49 (dd, J1 = 8.2 Hz,
J2 = 1.2 Hz, 2 H); EI-Ms m/z 324 (M+). Anal.
(C12H6OBr2) C, H, N.
[11] Berger B.J., Hall J.E., Tidwell R.R., Pharmacol. Toxicol. 66 (1990)
234–236.
[12] Berger B.J., Lombardy R.J., Marbury G.D., Bell C.A., Dykstra
C.C., Hall J.E., Tidwell R.R., Antimicrob. Agents Chemother. 34 (1990)
1678–1684.
5.14. 3,7-Dicyano-dibenzofuran 19
[13] Berger B.J., Reddy V.V., Le S.T., Lombardy R.J., Hall J.E., Tidwell
R.R., J. Pharmacol. Exp. Therapeut. 256 (1991) 883–889.
A
suspension of 3,7-dibromo-dibenzofuran (18,
[14] Berger B.J., Naiman N.A., Hall J.E., Peggins J., Brewer T.G.,
Tidwell R.R., Antimicrob. Agents Chemother. 36 (1992) 1825–1831.
10.27 g, 31.51 mmol) and copper(I) cyanide (8.23 g,
94.5 mmol) in DMF (80 mL) was refluxed under N2 for
6 h. The reaction mixture was poured into ice-water
(300 mL). The precipitated solid was collected and stirred
for 5 h in a solution of ethylenediamine (50 mL) in water
(300 mL). The solid was filtered off, washed with water,
then stirred in 10% sodium cyanide solution (100 mL) for
4 h. The solid was further purified by suspension in hot
ethanol (100 mL) to give a pale yellow powder (6.69 g,
97.43%): m.p. 322–325 °C (dec.); HPLC method 3 tR =
16.56 min (96.6 area%); 1H NMR (300 MHz, DMSO-d6)
δ 8.51 (d, J = 8.3 Hz, 2 H), 8.48 (s, 2 H), 7.95 (dd, J1 =
8.3 Hz, J2 = 1.0 Hz, 2 H); EI-MS m/z 218 (M+). Anal.
(C14H6N2•0.23H2O) C, H, N.
[15] Berger B.J., Tidwell R.R., Clin. Pharmacokin. 22 (1992) 163–168.
[16] Dykstra C.C., Tidwell R.R., J. Protozool. 6 (1991) 78S–81S.
[17] Dykstra C.C., McClernon D.R., Elwell L.P., Tidwell R.R.,
Antimicrob. Agents Chemother. 38 (1994) 1890–1898.
[18] Cory M., Tidwell R.R., Fairley T.A., J. Med. Chem. 35 (1992)
431–438.
[19] Fairley T., Tidwell R.R., Donkor I., Naiman N., Ohemeng K.,
Lombardy R., Bentley J., Cory M., J. Med. Chem. 36 (1993) 1746–1753.
[20] Bell C.A., Cory M., Fairley T.A., Hall J.E., Tidwell R.R.,
Antimicrob. Agents Chemother. 35 (1991) 1099–1107.
[21] Hildebrandt E., Boykin D.W., Kumar A., Tidwell R.R., Dykstra
C.C., J. Euk. Microb. 45 (1998) 112–121.
[22] Wood D.H., Hall J.E., Rose B.G., Tidwell R.R., Eur. J. Pharm. 353
(1998) 97–103.
[23] Tidwell R.R., Jones S.K., Dykstra C.C., Gorton L., Hall J.E., J.
Protozool. 6 (1991) 148S–150S.
[24] Tidwell R.R., Jones S.K., Geratz J.D., Ohemeng K.A., Bell C.A.,
Berger B.J., Hall J.E., Ann. N. Y. Acad. Sci. 616 (1990) 421–441.
Acknowledgements
[25] Tidwell R.R., Jones S.K., Geratz J.D., Ohemeng K.A., Cory M.,
Hall J.E., J. Med. Chem. 33 (1990) 1252–1257.
[26] Tidwell R.R., Kilgore S.G., Ohemeng K.A., Geratz J.D., Hall J.E.,
J. Protozool. 36 (1989) 74S–76S.
This work was supported by NIH grant NIAID AI-
33363, and an award from Immtech International Inc.,
1890 Maple Ave., Evanston, IL 60201. We appreciate the
clerical expertise and assistance of Vicki J. Wingate.
[27] Tidwell R.R., Jones S.K., Naiman N.A., Berger L.C., Brake W.B.,
Dykstra C.C., Hall J.E., Antimicrob. Agents Chemother. 37 (1993)
1713–1716.

224
[28] Kumar A., Boykin D.W., Wilson W.D., Jones S.K., Bender B.K.,
Dykstra C.C., Hall J.E., Tidwell R.R., Eur. J. Med. Chem. 31 (1996)
767–773.
[40] Pinner A., Klein F., Chem. Ber. 11 (1878) 1475–1487.
[41] Pinner A., Chem. Ber. 16 (1883) 352–363.
[42] Pinner A., Chem. Ber. 16 (1883) 352–363.
[29] Hall J.E., Kerrigan J.E., Ramachandran K., Bender B.C., Stanko
J.P., Jones S.K., Tidwell R.R., Antimicrob. Agents Chemother. 42 (1998)
667–674.
[43] Chakrabarti A., Biswas G.K., Chakraborty D.P., Tetrahedron 45
(1989) 5059–5064.
[44] Yamato T., Hideshima C., Suehiro K., Tashiro M., Surya Prakash
G.K., Olah G.A., J. Org. Chem. 56 (1991) 6248–6250.
[30] Boykin D.W., Kumar A., Hall J.E., Bender B.C., Tidwell R.R.,
Biorg. Med. Chem. Lett. 6 (1996) 3017–3020.
[45] Beck J.R., Sobczak R.L., Suhr R.G., Yahner J.A., J. Org. Chem. 39
(1974) 1839–1841.
[31] Clement B., Jung F., Drug. Met. Disp. 22 (1994) 486–497.
[32] Clement B., German Patent DE 4321444A1, 1994.
[46] Effenberger F., Koch M., Streicher W., Chem. Ber. 124 (1991)
163–173.
[33] Clement B., Immel M., Terlinden R., Wingen F.J., Arch. Pharm.
(Weinheim) 325 (1992) 61–62.
[47] Miesel J.L., O’Doherty G.O.P., Owen J.M., In: Catalysis in
Organic Chemistry, Academic Press, New York, 1976.
[34] Clement B., Raether W., Arzneim. Forsch. 35 (1985) 1009–1014.
[35] Clement B., Zimmermann M., Xenobiotica 17 (1987) 659–667.
[36] Clement B., Xenobiotica 13 (1983) 467–473.
[48] Erdtman H., Haflid F., Stjernstrom N.E., Acta Chem. Scand. 15
(1961) 1761–1764.
[37] Moffatt J.S., J. Chem. Soc. (1951) 625–626.
[49] Tanious F.A., Ding D., Patrick D.A., Tidwell R.R., Wilson W.D.,
Biochemistry 36 (1997) 15315–15325.
[38] Turner W.R., Werbel L.M., J. Med. Chem. 28 (1985) 1728–1740.
[39] Friedman L., Schecter H., J. Org. Chem. 26 (1961) 2522–2524.
[50] Shaw F.R., Turner E.E., J. Chem. Soc. (1932) 285–297.