2,5-bis[4-(N-alkylamidino)phenyl]furans as anti-Pneumocystis carinii agents

Article abstract of DOI:10.1021/jm970570i

The syntheses of 12 new 2,5-bis[4-(N-alkylamidino)phenyl]furans are reported. The interaction of these dicationic furans with poly(dA-dT) and with the duplex oligomer d(CGCGAATTCGCG)₂ was determined by T(m) measurements, and the effectiveness o

Full text of DOI:10.1021/jm970570i

124
J . Med. Chem. 1998, 41, 124-129
2,5-Bis[4-(N-a lk yla m id in o)p h en yl]fu r a n s a s An ti-P n eu m ocystis ca r in ii Agen ts
David W. Boykin,*,† Arvind Kumar,† Ge Xiao,† W. David Wilson,† Brendan C. Bender,‡ Donald R. McCurdy,‡
J ames Edwin Hall,‡,§ and Richard R. Tidwell‡
Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University,
Atlanta, Georgia 30303-3083, and Department of Pathology, School of Medicine, and Department of Epidemiology,
School of Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Received August 25, 1997X
The syntheses of 12 new 2,5-bis[4-(N-alkylamidino)phenyl]furans are reported. The interaction
of these dicationic furans with poly(dA-dT) and with the duplex oligomer d(CGCGAATTCGCG)2
was determined by Tm measurements, and the effectiveness of these compounds against the
immunosuppressed rat model of Pneumocystis carinii was evaluated. At the screening dose of
10 µmol/kg, 9 of the 14 N-alkylamidino furans described here are more active than the parent
compound 1. Substitution of an alkyl group on the amidino nitrogen, except for in 9, 13, and
15, resulted in higher affinity for DNA than the parent compound as judged by the larger ∆Tm
values and suggests enhanced van der Waals interactions in the bis-amidine-DNA complex.
Five of the compounds, 3, 5, 7, 10, and 12, yield cyst counts of less than 0.1% of control when
administered at a dosage of 10 µmol/kg. Five compounds, 1, 6, 8, 10, and 12, show significant
activity at a dosage of approximately 1 µmol/kg; 12 is the most active derivative, and it is
approximately 100 times more effective than pentamidine in this animal model.
In tr od u ction
demonstrated that these compounds strongly bind to the
minor groove of DNA at AT-rich sites and weakly bind
by intercalation at GC sites of DNA.10 Similar dual
binding modes have been reported for the related
diamidines DAPI and berenil.12-14 The existence of an
intercalation component to the DNA binding of 1
continues to be debated.15,16 We recently published the
X-ray structures of the complexes between 2,5-bis[4-(N-
isopropylamindino)phenyl]furan (6) and 2,5-bis[4-(N-
cyclopropylamidino)phenyl]furan (7) and d(CGCGAAT-
TCGCG)2 and a preliminary report of their anti-P.
carinii activity.17 The X-ray structures for 1, 6, and 7
demonstrated the excellent fit of this class of compounds
in the minor groove at the AATT site.2,17,18 Moreover,
it has been shown that these types of furan amidines
also bind to RNA; however, the binding to RNA is
thought to be by intercalation.19 The intercalation
binding of 1 and its analogues to nucleic acids may be
a source of toxicity and/or drug loss for these com-
pounds. Based upon literature reports for related
minor-groove binders and our preliminary studies, it
was postulated that the mode of action of these furan
dications involves interference of the normal function
of the pathogen’s DNA-dependent enzymes, perhaps
topoisomerase II.2
A number of aromatic diamidines have been shown
to bind to the minor groove of DNA and to exhibit useful
antimicrobial activities.1-4 A number of hypotheses for
the mode of antimicrobial action of aryldiamidines have
been proposed;5 however, evidence is growing that these
compounds function by complex formation with DNA
and subsequent selective inhibition of DNA-dependent
microbial enzymes. Intervention in transcription con-
trol has been demonstrated and seems to be a plausible
mode of action for several structurally diverse minor-
groove binders.6 Several minor-groove binding com-
pounds have been shown to interfere with the function
of topoisomerase I and/or II.7 Studies with diamidino
bis-benzimidazoles show a direct correlation between
topoisomerase II inhibition and anti-giardial activity.8
Selective inhibition of topoisomerase II isolated from
Pneumocystis carinii relative to the mammalian enzyme
was noted for diamidino bis-benzimidazoles, although
a strong correlation between anti-Pneumocystis activity
and enzyme inhibition was not observed.9
Work from our laboratories has demonstrated the
antimicrobial and nucleic acid binding properties of
amidino and cyclic amidino 2,5-diarylfurans.2,10 2,5-Bis-
(4-amidinophenyl)furan (1) was recently reported to be
effective in vivo at submicromolar dosage against P.
carinii in the immunosuppressed rat model.2 Earlier,
1 was shown to be effective in vivo in both mouse and
simian models for Trypanosoma rhodesiense.1,11 An
X-ray structure of the complex between 1 and d(CGC-
GAATTCGCG)2 clearly shows that 1 fits well into the
DNA minor groove.2 Other biophysical studies have
The strong DNA affinity and effective antimicrobial
activity of 1 led us to consider other modifications of
this structure with the goal of improved efficacy and
reduced toxicity. From biophysical studies of other
minor-groove binding compounds20 and analysis of X-ray
crystallographic data for DNA complexes of 1, 6, and
7,17,18 we have concluded that groups which provide
increased van der Waals interactions with the walls of
the minor groove significantly enhance binding affinity.
A theoretical study of minor-groove binding molecules
reached the same conclusion.21 This report describes
the expansion of the series of bis-N-alkyl analogues of
† Georgia State University.
‡ Department of Pathology, UNC.
§ Department of Epidemiology, UNC.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
S0022-2623(97)00570-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/01/1998

Anti-Pneumocystis carinii Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 125
Sch em e 1a
a
(a) HCl, EtOH; (b) RNH2, EtOH.
Sch em e 2a
a
(a) NaOH, CH3OH, H2O; (b) SOCl2, C6H6; (c) RNH2, CH2Cl2; (d) SOCl2, CH2Cl2; (e) NH3, CH2Cl2.
1 with varying size of the alkyl groups, the determina-
tion of their DNA binding affinity, and the evaluation
of their effectiveness in the immunosuppressed rat
model for P. carinii pneumonia.
Most of the compounds, including the previously
reported parent compound 1, in Table 1 are more active
than pentamidine, with no overt toxicity at the screen-
ing dose, in the immunosuppressed rat model for P.
carinii pneumonia. Nine of the 14 N-alkylamidino
furans are more active at the screening dose than the
parent compound 1. Five of the compounds, 3, 5, 7, 10,
and 12, yield cyst counts of less than 0.1% of control
when administered at a dosage of approximately 10
µmol/kg. These five compounds are more active by
factors of 10-20 times that of 1. Five compounds, 1, 6,
8, 10, and 12, show significant activity at a dosage of
approximately 1 µmol/kg. The most active compound
of these five is 12 which is approximately 100 times
more effective than pentamidine in this animal model.
It appears for this series of furans that enhanced DNA
affinity indeed leads to improved anti-PCP activity. We
recently reported a similar relationship between cyto-
toxicity data for several of these compounds and DNA
binding affinity.22 All the compounds with ∆Tm values
of 14 °C or higher, 3, 6, 10, 12, and 14, show excellent
anti-P. carinii activity at the 10 µM dosage level.
However, at lower dosage all these compounds do not
show comparable activity; for example, note the results
for 12 and 14 which exhibit similar ∆Tm values, yet at
low dosages the anti-PCP activity of 14 is significantly
less than that of 12. Recently, Hilldebrandt et al.
demonstrated that several of the alkylamidino furans
listed in Table 1 inhibited an endo/exonuclease isolated
from P. carinii and thereby identified a new potential
mode of action for these types of compounds.23 Correla-
tions were noted between nuclease inhibition, DNA
binding affinity, and anti-P. carinii activity; when the
data for new compounds reported here are added to the
previously reported result, the correlation is retained
(not shown).23 Thus, it appears that the mode of anti-
P. carinii action of these dicationic furans may involve
inhibition of an endo/exonuclease.
Ch em istr y
The synthesis of all the 2,5-bis[4-(N-alkylamidino)-
phenyl]furans, except 2 and 3, was achieved in a
straightforward manner starting from 2,5-bis(4-cy-
anophenyl)furan and employing a classical Pinner-type
approach for conversion of the nitrile function into the
amidino one.1 The synthetic steps are outlined in
Scheme 1. Due to problems associated with obtaining
and using anhydrous methylamine and ethylamine,
preparation of 2 and 3 was achieved by the indirect
route outlined in Scheme 2.
Biologica l Resu lts
Table 1 contains the results from assessing the
affinity of the dications for poly(dA-dT) and d(CGC-
GAATTCGCG)2. It was necessary to employ the oligo-
mer in order to rank the relative DNA binding affinity
since several of the compounds (6, 7, 10-12) bind so
strongly to poly(dA-dT) that melting was not observed
within the temperature limits of the experiment. Sub-
stitution of an alkyl group on the amidino nitrogen,
except for 9, 13, and 15, resulted in higher affinity for
DNA than the parent compound (compare the ∆Tm
values for the oligomer in Table 1). Not only does the
minor groove accommodate rather bulky alkyl groups
at the termini of the dicatonic molecule (cf. 12, 14), but
these groups clearly enhance the binding affinity. These
results are consistent with enhanced van der Waals
interactions by the alkyl groups of greater surface area
with the walls of the DNA minor groove. It is notewor-
thy that the three compounds (9, 13, 15) which exhibit
significantly lower affinities for the oligomer than 1 are
ones in which it seems likely that entropic factors
contribute to the lower binding affinity. The fact that
the ∆Tm value for the 3-pentyl compound 13 is ap-
proximately one-half the value of that for the cyclopentyl
analogue 12 clearly illustrates this point.
In summary, the design strategy to increase minor-
groove DNA binding by enhancing the van der Waals
interactions with the wall of the groove by addition of
moderately bulky alkyl groups to the termini of the 2,5-

126 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Boykin et al.
Ta ble 1. Nucleic Acid Binding Results and in Vivo Activity of Dicationic Furans against P. Carinii
∆Tm
dosagec
(µmol/kg/day)
cysts/g of lungc
(% of control)
compd
saline
R
DNAa
25
oligomerb
toxicityc
100.0 ( 8.13d
1.46 ( 0.21
0.61 ( 0.27
5.65 ( 2.76
5.38 ( 2.61
21.97 ( 3.89
0.16 ( 0.13
85.37 ( 24.4
0.05 ( 0.02
0.60 ( 0.28
0.08 ( 0.04
107.83 ( 26.47
0.20 ( 0.18
8.41 ( 7.22
11.73 ( 4.68
169.0 ( 61.1
0.03 ( 0.01
129.0 ( 17.9
0.24 ( 0.14
3.25 ( 0.79
2.20 ( 1.35
0.05 ( 0.01
0.67 ( 0.25
2.38 ( 0.89
0.03 ( 0.02
0.08 ( 0.05
0.11 ( 0.02
93.5 ( 38.2
24.43 ( 14.7
0.02 ( 0.01
26.28 ( 5.51
43.9 ( 14.23
pentamidine
22.0
13.3
2.7
++
0
0
0
0
+
0
0
0
0
0
0
0
0
0
+
0
0
0
0
+
0
0
+
0
0
0
++
0
0
0
1
H
11.7
0.3
0.03
10.6
0.2
10.4
10.4
10.6
0.2
10.8
2.2
1.1
0.2
10.8
0.2
10.8
0.5
10.0
9.9
1.0
9.7
9.4
1.9
0.9
0.2
4.8
9.1
2
CH3
3
4
5
CH2CH3
CH2CH2OH
CH2CH2CH3
21.1
25.9
11.0
13.0
6
CH(CH3)2
>28
14.4
7
8
c-(CH2CH2CH2)
>28
12.4
13.3
CH2-c-(CH2CH2CH2)
26.8
9
10
CH2CH(CH3)2
c-(CH2CH2CH2CH2)
21.4
>28
10.0
14.8
11
12
CH2CH2CH(CH3)2
c-(CH2CH2CH2CH2CH2)
>28
>28
11.2
15.8
13
14
CH(CH2CH3)2
20.4
ppt
10.0
15.4
c-(C6H12
)
0.9
8.7
15
CH2-c-(C6H12
)
21.4
10.6
a
b
Increase in thermal melting of polyA‚polyT; see ref 19. Increase in thermal melting of the oligomer d(GCGCAATTGCGC)2; see ref
24. c Evaluation of iv dosage of the furan dications against P. carinii in rats as described in ref 4b. Mean cyst count for pooled controls;
d
saline (n ) 142) ) 63.77 ( 5.18 cysts/g of lung tissue; pentamidine (n ) 135) ) 0.93 ( 0.14 cysts/g of lung tissue.
1
394-396 °C; H NMR (DMSO-d6/D2O/70 °C) δ 8.42 (d, 4H, J
) 8 Hz) 8.34 (d, 4H, J ) 8 Hz), 7.67 (s, 2H); 13C NMR (DMSO-
d6/70 °C) δ 166.6, 152.5, 133.1, 130.1, 129.7, 123.2, 110.2; MS
m/e 308 (M+). Anal. (C18H12O5) C,H.
diarylfuran system appears to be successful. Further-
more, the improved DNA affinity has led to enhance-
ment of in vivo activity in the immunosuppressed rat
model for P. carinii pneumonia, and compound 12 has
emerged as a highly effective candidate for further
evaluation against P. carinii infections.
A suspension of 2,5-bis(4-carboxyphenyl)furan (3.08 g, 0.01
mol), 75 mL of dry benzene, thionyl chloride (7.08 g, 0.06 mol),
and 4 drops of DMF was allowed to reflux for 2.5 h. The
solvent was removed under vacuum, and the residue which
resulted was triturated with benzene; the benzene was re-
moved under vacuum and the residue triturated with dry
ether. The yellow solid was filtered and dried under vacuum
at 60 °C to yield 2.9 g (84%) of the diacid chloride: mp 358-
Exp er im en ta l Section
Melting points were recorded using a Thomas-Hoover (Uni-
Melt) capillary melting point apparatus and are uncorrected.
1H and 13C NMR spectra were recorded employing a Varian
GX400 spectrometer and chemical shifts (δ) are in ppm relative
to TMS unless otherwise noted. Mass spectra were recorded
on a VG Instruments 70-SE spectrometer (Georgia Institute
of Technology, Atlanta, GA). IR spectra were recorded using
a Michelson 100 (Bomem, Inc.) instrument. Elemental analy-
ses were obtained from Atlantic Microlab Inc. (Norcross, GA)
and are within (0.4 of the theoretical values. All chemicals
and solvents were purchased from Aldrich Chemical Co. or
Fisher Scientific. The synthesis of 6 and 7 was reported
previously.17 Tm values for compound-DNA complexes were
determined as previously described.2,3,19,24
2,5-Bis[4-(N-m eth yla m id in o)p h en yl]fu r a n (2). To a
slurry of 2,5-bis(4-cyanophenyl)furan25 (5.4 g, 0.02 mol) in 30
mL of methanol was added 100 mL of 30% NaOH, and the
mixture was heated at reflux for 40 h. The mixture was cooled,
100 mL of cold water was added, and the pH was adjusted to
a value of 2 with concentrated HCl. The solid was filtered,
washed with water and ether, and dried under vacuum at 85
°C to yield 4.65 g (75%) of 2,5-bis(4-carboxyphenyl)furan: mp
1
360 °C dec; H NMR (CDCl3) δ 8.17 (d, 4H, J ) 7.6 Hz), 7.86
(d, 4H, J ) 7.6 Hz), 7.02 (s, 2H); 13C NMR (CDCl3) δ 167.4,
153.1, 135.8, 132.0, 128.9, 123.8, 111.3; MS m/e 345 (M+). The
diacid chloride was used directly in the next step without
further characterization.
To a suspension of the diacid chloride (0.69 g, 0.002 mol) in
50 mL of dry CH2Cl2 was added methylamine (0.186 g, 0.004
mol), and the mixture was allowed to stir at room temperature
for 7 h. The solvent was removed under reduced pressure,
and water was added. The solid was filtered and washed with
10% HCl, 10% NaHCO3, and water. After drying the solid was
recrystallized from CHCl3-ether to give a white solid (0.53 g,
80%): mp 282-284 °C; 1H NMR (DMSO-d6) δ 8.29 (br s, 2H),
7.92 (d, 4H, J ) 8 Hz), 7.88 (d, 4H, J ) 8 Hz), 7.17 (s, 2H),
2.82 (br s, 6H); 13C NMR (DMSO-d6) δ 165.8, 152.4, 133.2,
131.8, 127.4, 123.0, 109.4; MS m/e 334 (M+). Anal. (C20H18
-
N2O3) C,H,N.
To a suspension of 2,5-bis[4-(N-methylcarbamoyl)phenyl]-
furan (0.835 g, 0.0025 mol) in 40 mL of dry CHCl3 and thionyl

Anti-Pneumocystis carinii Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 127
vacuo at 70 °C for 24 h to yield 0.43 g (88%): mp 190-192 °C
chloride (1.2 g, 0.01 mol) was added 4-5 drops of DMF, and
the mixture was allowed to reflux until the solution was clear
(ca. 4 h). The solution was filtered hot, and the filtrate was
distilled under reduced pressure to remove solvent. The
residue was dissolved in dry CH2Cl2, the solvent removed
under reduced pressure, and the solid triturated with dry
hexane. The solid was filtered (under nitrogen), and the yellow
solid was dried under vacuum at 40 °C to yield 0.88 g (81%) of
the bis-imidoyl chloride. The bis-imidoyl chloride was used
directly in the next step without further characterization.
1
dec; H NMR (DMSO-d6) δ 9.99 (s, 2H), 9.57 (s, 2H), 9.19 (s,
2H), 8.06 (d, 4H, J ) 7.93 Hz), 7.94 (d, 4H, J ) 8.55 Hz), 7.4
(s, 2H), 3.69 (t, 4H, J ) 4.9 Hz), 3.58 (t, 4H, J ) 4.9 Hz); 13C
NMR (DMSO-d6) δ 162.6, 152.4, 133.8, 129.1, 127.5, 123.6,
111.4, 58.4, 45.7. Anal. (C22H24N4O3‚2HCl‚1.25H2O) C,H,N.
2,5-Bis[4-(N-n -p r op yla m id in o)p h en yl]fu r a n (5). The
imidate ester was converted into the free base of 5 as described
above to yield (86%), after recrystallization from CHCl3-ether
(1:8), a pale yellow solid: mp 187-188 °C; IR (KBr) 3261, 3062,
2949, 2929, 2874, 2602, 1542, 1497, 1364, 1196, 1149, 1022,
847, 790, 750, 685; 1H NMR (DMSO-d6) δ 7.84 (s, 8H), 7.17 (s,
2H), 6.6-6.38 (vbr, 4H), 3.09 (t, 4H, J ) 6.9 Hz), 1.62 (tt, 4H,
J ) 6.9, 7.5 Hz), 0.96 (t, 6H, J ) 7.5 Hz); MS m/e 388 (M+).
A suspension of 0.88 g (0.002 mol) of the bis-imidoyl chloride
in 45 mL of dry CH2Cl2 was saturated with dry ammonia gas
at 0-5 °C. The mixture was stirred at room temperature for
24 h, and the solvent was removed under reduced pressure.
The residue was treated with ice-water, and the pH of the
slurry was adjusted to a value of 10 by adding 2 M NaOH.
The resulting solid was filtered, washed with water, and dried
under vacuum to yield 0.5 g (75%) of the free base of 2: mp
200-202 °C; 1H NMR (DMSO-d6) δ 7.81 (br s, 8H), 7.13 (s,
2H), 6.52-6.32 (br, 4H), 2.83 (s, 6H); MS m/e 332 (M+).
The free base was converted into the salt by taking up 0.4
g (0.0012 mol) in 6 mL of methanol saturated with HCl and
stirring for 1 h. After reducing the solvent volume under
reduced pressure, addition of dry ether caused precipitation
of the salt, which was filtered, washed with ether, and dried
under vacuum to yield 0.44 g (90%) of yellow solid: mp 330-
335 °C dec; 1H NMR (DMSO-d6/D2O/40 °C) δ 8.01 (d, 4H, J )
The free base of 5 was converted into the yellow dihydro-
chloride salt of 5 as described above in an 89% yield: mp 227-
230 °C dec; IR (KBr) 3399, 3228, 3042, 2878, 1670, 1614, 1501,
1375, 1292, 1127, 1027, 929, 849, 746 cm-1; 1H NMR (DMSO-
d6) δ 10.11 (br s, 2H), 9.65 (s, 2H), 9.3 (d, 2H), 8.05 (d, 4H, J
) 8.4 Hz), 7.95 (d, 4H, J ) 8.4 Hz), 7.4 (s, 2H), 3.46 (q, H, J )
6.3 Hz), 1.7 (dqt, 4H, J ) 7.2 Hz), 0.98 (t, 6H, J ) 7.2 Hz); 13
C
NMR (DMSO-d6) δ 161.8, 152.2, 133.5, 128.8, 127.2, 123.4,
111.0, 43.9, 20.6, 10.8. Anal. (C24H28N4O‚2HCl‚0.5H2O) C,H,N.
2,5-Bis[4-[N-(cyclop r op ylm eth yl)a m id in o]p h en yl]fu r -
a n (8). The imidate ester was converted into the free base of
8 as described above to yield (84%), after recrystallization from
1
CHCl3-ether (1:8), a pale yellow solid: mp 178-179 °C; H
8.4 Hz), 7.83 (d, 4H, J ) 8.4 Hz), 7.31 (s, 2H), 3.02 (s, 6H); 13
C
NMR (DMSO-d6) δ 7.83 (s, 8H), 7.14 (s, 2H), 6.34 (br, 4H),
3.09 (d, 4H, J ) 6.0 Hz), 1.13-1.05 (m, 2H), 0.47-0.41 (m,
4H), 0.28-0.21 (m, 4H); MS m/e 412 (M+).
NMR (DMSO-d6/D2O/40 °C) δ 163.2, 152.6, 134.1, 130.1, 128.9,
127.4, 124.1, 111.5, 29.6. Anal. (C20H20N4O‚2HCl) C,H,N.
2,5-Bis[4-(N-eth yla m id in o)p h en yl]fu r a n (3). Following
the procedure described above for 2, 2,5-bis[4-(N-ethylcarbam-
oyl)phenyl]furan was prepared in an 85% yield: mp 309-311
The free base of 8 was converted into the yellow dihydro-
chloride salt of 8 as described above in a 92% yield: mp 243-
245 °C; IR (KBr) 3390, 3245, 3197, 2865, 1673, 1614, 1503,
1
°C dec; H NMR (DMSO-d6/75 °C) δ 8.22 (br s, 2H), 7.92 (d,
1
1380, 1111, 929, 765, 746 cm-1; H NMR (DMSO-d6) δ 10.24
4H, J ) 8.4 Hz), 7.86 (d, 4H, J ) 8.4 Hz), 7.15 (s, 2H), 3.33 (q,
2H), 1.17 (t, 6H); 13C NMR (DMSO-d6/75 °C) δ 165.1, 152.3,
133.4, 131.7, 127.4, 122.8, 109.2, 33.7, 14.2; MS m/e 362 (M+).
Anal. (C22H22N2O3) C,H,N.
(br s, 2H), 9.65 (s, 2H), 9.3 (s, 2H), 8.06 (d, 4H, J ) 8.4 Hz),
7.97 (d, 4H, J ) 8.4 Hz), 7.41 (s, 2H), 3.42 (t, 4H, J ) 6.3 Hz),
1.26-1.06 (m, 2H), 0.58-0.51 (m, 4H), 0.44-0.38 (m, 4H); 13
C
NMR (DMSO-d6) δ 163.4, 153.3, 134.9, 129.7, 128.2, 124.9,
2,5-Bis[4-(N-ethylcarbamoyl)phenyl]furan was converted
into the corresponding bis-imidoyl chloride which was then
converted into the free base of 3 according to the procedure
described above for 2 to yield a white solid (80%): mp 213-
215 °C dec; 1H NMR (DMSO-d6) δ 7.81 (br s, 8H), 7.12 (s, 2H),
6.43-6.21 (br, 4H), 3.19 (q, 4H, J ) 7.6 Hz), 1.18 (t, 6H, J )
7.6 Hz); MS m/e 360 (M+).
The free base of 3 was converted into the yellow dihydro-
chloride salt of 3 as described above for 2 in an 88% yield:
mp 229-231 °C dec; 1H NMR (DMSO-d6/D2O) δ 7.96 (d, 4H, J
) 8.4 Hz), 7.79 (d, 4H, J ) 8.4 Hz), 7.26 (s, 2H), 3.42 (q, 4H,
J ) 7.2 Hz), 1.24 (t, 6H, J ) 7.2 Hz); 13C NMR (DMSO-d6/
D2O) δ 162.6, 152.8, 134.3, 129.3, 127.8, 124.3, 111.8, 38.2, 13.
2. Anal. (C22H24N4O‚2HCl‚0.75H2O) C,H,N.
2,5-Bis[4-[N-(2-h ydr oxyeth yl)am idin o]ph en yl]fu r an (4).
2,5-Bis(4-cyanophenyl)furan25 (2.79 g, 0.01 mol) was suspended
in 80 mL of ethanol (distilled from magnesium metal) and
stirred for ca. 1 week at room temperature to yield 3.93 g (90%)
of the corresponding imidate ester, which was used directly
without characterization. Dried (CaH2) and freshly distilled
aminoethanol (0.27 g, 0.0045 mol) was added to a suspension
of imidate ester hydrochloride (0.65 g, 0.0015 mol) in 10 mL
of ethanol, and the mixture was stirred under nitrogen at room
temperature for 12 h. The solvent was distilled under vacuum,
the residue was treated with ice-water, and the pale yellow
solution was made basic with 1 M NaOH to pH 9. The solid
was filtered, washed with water, dried, and crystallized from
CHCl3-ether (1:4) to yield a pale crystalline solid (0.43 g,
74%): mp >330 °C; 1H NMR (DMSO-d6/D2O) δ 7.92 (q, 8H, J
) 8.4 Hz), 7.20 (s, 2H), 4.13 (t, 4H, J ) 9.2 Hz), 3.97 (t, 4H, J
) 9.2); MS m/e 394 (M+).
112.3, 48.2, 9.8, 4.4. Anal. (C26H28N4O‚2HCl‚0.5H2O) C,H,N.
2,5-Bis[4-(N-isobu tyla m id in o)p h en yl]fu r a n (9). The
imidate ester was converted into the free base of 9 as described
above to yield (86%), after recrystallization from ethanol-ether
1
(1:5), a pale yellow solid: mp 178-179 °C; H NMR (DMSO-
d6/D2O) δ 7.84 (s, 8H), 6.65-6.31 (vbr, 4H), 2.94 (d, 4H, J )
7.7 Hz), 1.9 (sept, 2H, J ) 7.7, 6.1 Hz), 0.96 (d, 12H, J ) 6.1
Hz); 13C NMR (DMSO-d6/D2O) δ 156.1, 152.6, 136.0, 130.6,
127.1, 122.9, 109.1, 53.4, 28.9, 20.9; MS m/e 416 (M+).
The free base of 9 was converted into the yellow dihydro-
chloride salt of 9 as described above in a 94% yield: mp 293-
295 °C dec; IR (KBr) 3412, 3234, 3092, 2990, 1669, 1612, 1569,
1500, 1379, 1291, 1132, 1023, 929, 851, 792, 748 cm-1; 1H NMR
(DMSO-d6) δ 10.0 (br s, 2H), 9.58 (br s, 2H), 9.25 (br, 2H),
8.04 (d, 4H, J ) 8.6 Hz), 7.94 (d, 4H, J ) 8.6 Hz), 7.37 (s, 2H),
3.35 (d, 2H, J ) 7.7 Hz), 2.08 (sept, 2H, J ) 6.7 Hz), 1.0 (d,
12H, J ) 6.7 Hz); 13C NMR (DMSO-d6) δ 162.2, 152.3, 133.6,
128.9, 127.4, 123.5, 111.2, 49.4, 26.8, 19.7. Anal. (C26H32N4-
O‚2HCl‚0.5H2O) C,H,N.
2,5-Bis[4-(N-cyclobu tylam idin o)ph en yl]fu r an (10). The
imidate ester was converted into the free base of 10 as
described above to yield (76%), after recrystallization from
ethanol-ether (1:5), a pale yellow solid: mp 208-210 °C dec;
1H NMR (DMSO-d6/D2O) δ 7.82 (q, 8H, J ) 8.8 Hz), 7.13 (s,
2H), 4.08 (t, 2H, J ) 7.6 Hz), 2.40-2.31 (m, 4H), 2.0-1.88 (m,
4H), 1.77-1.64 (m, 4H); MS m/e 412 (M+).
The free base of 10 was converted into the yellow dihydro-
chloride salt of 10 as described above in a 90% yield: mp 293-
1
295 °C dec; H NMR (DMSO-d6) δ 10.2 (s, 2H), 10.18 (s, 2H),
9.6 (s, 2H), 9.1 (s, 2H), 8.04 (d, 4H, J ) 8.8 Hz), 7.94 (d, 4H, J
) 8.8 Hz), 7.38 (s, 2H), 4.44 (sext, 2H, J ) 7.2 Hz), 2.51-2.42
(m, 4H), 2.37-2.26 (m, 4H), 1.88-1.78 (m, 2H), 1.77-1.60 (m,
2H); 13C NMR (DMSO-d6) δ 160.7, 152.2, 133.6, 129.0, 127.0,
123.3, 110.0, 47.3, 28.4, 14.6. Anal. (C26H28N4O‚2HCl‚H2O)
C,H,N.
The free base (0.39 g, 0.001 mol) was dissolved in 5 mL of
dry ethanol, treated with 7 mL of saturated ethanolic HCl,
and heated at 40 °C for 2 h. The ethanol was removed under
vacuum and the residue triturated with dry ether; the yellow
crystalline solid was filtered, washed with ether, and dried in

128 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Boykin et al.
2,5-Bis[4-(N-isop en tyla m id in o)p h en yl]fu r a n (11). The
imidate ester was converted into the free base of 11 as
described above to yield (76%), after recrystallization from
ethanol-ether, a white solid: mp 185-186 °C; 1H NMR
(DMSO-d6/D2O) δ 7.85 (d, 4H, J ) 8.4 Hz), 7.81 (d, 4H, J )
8.4 Hz), 7.17 (s, 2H), 3.19 (t, 4H, J ) 7.5 Hz), 1.74 (sept, 2H,
J ) 6.6 Hz), 1.52 (q, 4H, J ) 6.6 Hz), 0.93 (d, 12H, J ) 6.6
Hz); MS m/e 444 (M+).
The free base of 11 was converted into the yellow dihydro-
chloride salt of 11 as described above in an 89% yield: mp
297-299 °C dec; IR (KBr) 3360, 3201, 2962, 2874, 1669, 1613,
1503, 1376, 1028, 847, 745 cm-1; 1H NMR (DMSO-d6) δ 10.1-
9.2 (vbr, 6H), 8.06 (d, 4H, J ) 8.3 Hz), 7.89 (d, 4H, J ) 8.3
Hz), 7.4 (s, 2H), 3.46 (t, 4H, J ) 7.3 Hz), 1.72 (sept, 2H, J )
6.35 Hz), 1.6 (dt, 4H, J ) 7.3, 6.35 Hz), 0.95 (d, 12H, J ) 6.35
Hz); 13C NMR (DMSO-d6) δ 161.9, 152.3, 133.7, 128.9, 127.5,
123.5, 111.2, 41.1, 35.8, 25.2, 22.1. Anal. (C28H36N4O‚2HCl)
C,H,N.
) 8.4 Hz), 7.88 (d, 4H, J ) 8.4 Hz), 7.35 (s, 2H), 3.24 (d, 4H,
J ) 6.9 Hz), 1.80-1.64 (m, 12H), 1.30-1.15 (m, 8H), 1.08-
0.93 (m, 4H); MS m/e 496 (M+).
The free base of 15 was converted into the yellow dihydro-
chloride salt of 15 as described above in an 88% yield: mp
245-247 °C dec; IR (KBr) 3418, 3236, 3061, 2921, 2852, 1668,
1614, 1501, 1448, 1289, 1025, 929, 847, 791, 747 cm-1; 1H NMR
(DMSO-d6) δ 10.0 (br, 2H), 9.61 (br, 2H), 9.24 (br, 2H), 8.06
(d, 4H, J ) 8.55 Hz), 7.93 (d, 4H, J ) 8.55 Hz), 7.4 (s, 2H),
3.35 (t, 4H, J ) 6.1 Hz), 1.81-1.65 (m, 12H), 1.26-1.14 (m,
6H), 1.09-1.0 (m, 4H); 13C NMR (DMSO-d6) δ 162.1, 152.3,
133.6, 128.9, 127.4, 123.5, 111.2, 48.2, 35.9, 29.8, 25.7, 25.1.
Anal. (C32H40N4O‚2HCl‚0.25H2O) C,H,N.
Ack n ow led gm en t. This work was supported by
NIH Grant NIAID AI-33363. An award by the Chemi-
cal Instrumental Program of NSF (CHE 8409599) and
the Georgia Research Alliance provided partial support
for acquisition of the NMR spectrometers used in this
work.
2,5-Bis[4-(N-cyclopen tylam idin o)ph en yl]fu r an (12). The
imidate ester was converted into the free base of 12 as
described above to yield (76%), after recrystallization from
1
ethanol-ether (1:6), a pale yellow solid: mp 200-201 °C; H
Refer en ces
NMR (DMSO-d6/D2O) δ 7.84 (s, 8H), 7.18 (s, 2H), 3.9 (br, 2H),
1.94-1.91 (br m, 4H), 1.88-1.7 (br m, 4H), 1.69-1.45 (br m,
12H); 13C NMR (DMSO-d6) δ 156.5, 152.5, 135.9, 130.7, 127.2,
122.9, 109.1, 55.0, 33.0, 24.0; MS m/e 440 (M+).
(1) Das, B. P.; Boykin, D. W. Synthesis and antiprotozoal activity
of 2,5-bis(4-guanylphenyl) furans. J . Med. Chem. 1977, 20, 531-
536.
(2) Boykin, D. W.; Kumar, A.; Spychala, J .; Zhou, M.; Lombardy,
R. J .; Wilson, W. D.; Dykstra, C. C.; J ones, S. K.; Hall, J . E.;
Tidwell, R. R.; Laughton, C.; Nunn, C. M.; Neidle, S. Dicationic
diarylfurans as anti-Pneumocystis carinii agents. J . Med. Chem.
1995, 36, 912-916.
(3) Kumar, A.; Boykin, D. W.; Wilson, W. D.; J ones, S. K.; Bender,
B. K.; Hall, J . E.; Tidwell, R. R. Anti-Pneumocystis carinii
pneumonia activity of dicationic 2,4-diarylpyrimidines. Eur J .
Med. Chem. 1996, 31, 767-773.
The free base of 12 was converted into the yellow dihydro-
chloride salt of 12 as described above in a 92% yield: mp 294-
296 °C dec; IR (KBr) 3400, 3045, 2939, 2880, 1667, 1609, 1502,
1358, 1291, 1189, 1024, 929, 845, 746 cm-1; 1H NMR (DMSO-
d6) δ 9.89 (s, 1H), 9.86 (s, 1H), 9.62 (s, 2H), 9.3 (s, 2H), 8.04
(d, 4H, J ) 8.3 Hz), 7.9 (d, 4H, J ) 8.3 Hz), 7.4 (s, 2H), 4.27
(br, 2H), 2.08-2.06 (m, 4H), 1.76-1.58 (m, 12H); 13C NMR
(DMSO-d6) δ 161.6, 152.2, 133.4, 128.9, 127.4, 123.2, 110.7,
54.1, 31.0, 23.3. Anal. (C28H32N4O‚2HCl‚H2O) C,H,N.
2,5-Bis[4-(N-3-p en tyla m id in o)p h en yl]fu r a n (13). The
imidate ester was converted into the free base of 13 as
described above to yield (76%), after recrystallization from
CHCl3-ether (1:3), a pale yellow solid: mp 155-156 °C; IR
(4) (a) Lombardy, R. J .; Tanious, F. A.; Ramachandran, K.; Tidwell,
R. R.; Wilson, W. D. Synthesis and DNA interactions of benz-
imidazole dications which have activity against opportunistic
infections. J . Med. Chem. 1996, 36, 912-916. (b) Tidwell, R. R.;
J ones, S. K.; Naiman, N. A.; Berger, L. C.; Brake, W. B.; Dykstra,
C. C.; Hall, J . E. Activity of cationically substituted bis-
benzimidazoles against experimental Pneumocystis carinii pneu-
monia. Antimicrob. Agents Chemother. 1993, 37, 1713-1716.
(5) Tidwell, R. R.; Bell, C. A. Pentamidine and related compounds
in treatment of Pneumocystis carinii Infection. Pneumocystis
carinii, Marcel Decker: New York, 1993; pp 561-583.
(6) (a) Henderson, D.; Hurley, L. H. Molecular Struggle for tran-
scription control. Nature Med. 1995, 1, 525-527. (b) Mote, J .,
J r.; Ghanounoi, P.; Reines, D. A minor-groove binding ligand
both potentiates and arrests transcription by RNA polymerase
II. J . Mol. Biol. 1994, 226, 725-737.
(7) (a) Beerman, T. A.; McHugh, M. M.; Sigmund, R.; Lown, J . W.;
Rao, K. E.; Bathini, Y. Effects of analogues of the DNA minor
groove binder Hoechst 33258 on topoisomerase I and II mediated
activities. Biochim. Biophys. Acta 1992, 1131, 52-61. (b) Chen,
A. Y.; Yu, C.; Gatto, B.; Liu, L. F. DNA Minor groove binding
ligands: A different class of mammalian DNA topoisomerase I
inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8131-8135.
(8) (a) Bell, C. A.; Dykstra, C. C.; Naiman, N. N.; Cory, M.; Fairley,
T. A.; Tidwell, R. R. Structure-activity studies of dicationic
substituted bis-benzimidazoles against Giardia lamblia: Cor-
relation of antigiardial activity with DNA binding affinity and
giardial topoisomerase II inhibition. Antimicrob. Agents Chemo-
ther. 1993, 37, 2668-2673. (b) Bell, C. A.; Cory, M.; Fairley, T.
A.; Hall, J . E.; Tidwell, R. R. Structure-activity relationships of
pentamidine analogues against Giardia lamblia and correlation
of anti-giardial activity with DNA binding affinity. Antimicrob.
Agents Chemother. 1991, 35, 1099-1107.
(9) Dykstra, C. C.; McClernon, D. R.; Elwell, L. P.; Tidwell, R. R.
Selective inhibition of topoisomerases from Pneumocystis carinii
versus topoisomerases from mammalian cells. Antimicrob. Agents
Chemother. 1994, 1890-1898.
(10) Tanious, F. A.; Spychala, J .; Kumar, A.; Greene, K.; Boykin, D.
W.; Wilson, W. D. Different binding mode in AT and GC
sequences for unfused-aromatic dications. J . Biomol. Struct. Dyn.
1994, 11, 1063-1083.
(11) Steck, E. A.; Kinnamon, K. K.; Davidson, D. E.; Duxbury, R. E.;
J ohnson, A. J .; Masters, R. E. Trypanosoma rhodesiense: Evalu-
ation of the antitrypanosomal action of 2,5-bis(4-guanylphen-
yl)furan Dihydrochloride. Exp. Parasitol. 1982, 53, 133-144.
(12) (a) Colson, P.; Houssier, C.; Bailly, C. Use of electric linear
dichroism and competition experiments with intercalating drugs
to investigate the mode of binding of Hoechst 33258, berenil and
DAPI to GC sequences. J . Biomol. Struct. Dyn. 1995, 13, 351-
(KBr) 3245, 3120, 2962, 1593, 1544, 1380, 1194, 848, 784 cm-1
;
1H NMR (DMSO-d6) δ 7.81 (q, 8H, J ) 8.4 Hz), 7.13 (s, 2H),
6.22 (br, 4H), 3.45 (br, 2H), 1.58-1.41 (m, 8H), 0.88 (t, 12H, J
) 7.2 Hz); 13C NMR (DMSO-d6) δ 157.3, 152.5, 136.7, 130.5,
1127.0, 122.8, 108.8, 55.0, 27.3, 10.5; MS m/e 444 (M+).
The free base of 13 was converted into the yellow dihydro-
chloride salt of 13 as described above in a 90% yield: mp >360
°C; IR (KBr) 3410, 3235, 3105, 1668, 1613, 1500, 1459, 1368,
1
1126, 1025 cm-1; H NMR (DMSO-d6/D2O) δ 7.93 (d, 4H, J )
8.5 Hz), 7.76 (d, 4H, J ) 8.5 Hz), 7.13 (s, 2H), 3.88-3.65 (m,
2H), 1.9-1.8 (m, 4H), 1.78-1.66 (m, 4H), 1.05 (t, 6H, J ) 7.3
Hz); 13C NMR (DMSO-d6/D2O) δ 165.1, 154.1, 136.2, 130.0,
129.0, 125.8, 112.9, 59.1, 27.8, 11.5. Anal. (C28H36N4O‚2HCl‚
1.5H2O) C,H,N.
2,5-Bis[4-(N-cycloh exylam idin o)ph en yl]fu r an (14). The
imidate ester was converted into the free base of 14 as
described above to yield (78%), after recrystallization from
ethanol-ether (1:6), a pale yellow solid: mp 270-275 °C dec;
1H NMR (DMSO-d6/D2O) δ 7.89 (d, 4H, J ) 8.4 Hz), 7.8 (d,
4H, J ) 8.4 Hz), 7.21 (s, 2H), 3.6 (br m, 2H), 1.9-1.57 (m,
5H), 1.41-1.14 (m, 5H); MS m/e 468.
The free base of 14 was converted into the yellow dihydro-
chloride salt of 14 as described above in a 91% yield: mp 298-
300 °C dec; IR (KBr) 3410, 3192, 3091, 2931, 2853, 1669, 1611,
1
1502, 1369, 1056, 791, 747 cm-1; H NMR (DMSO-d6) δ 9.65
(br s, 2H), 9.53 (br s, 2H), 9.28 (s, 2H), 8.07 (d, 4H, J ) 8.3
Hz), 7.85 (d, 4H, J ) 8.3 Hz), 7.43 (s, 2H), 3.75 (br m, 2H)
2.0-1.9 (m, 4H), 1.8-1.1 (m, 16H); 13C NMR (DMSO-d6) δ
161.1, 152.3, 133.6, 129.1, 127.7, 123.5, 111.2, 51.8, 30.9, 24.6,
24.1. Anal. (C30H36N4O‚2HCl‚0.5H2O) C,H,N.
2,5-Bis[4-[N-(cycloh exylm et h yl)a m id in o]p h en yl]fu r -
a n (15). The imidate ester was converted into the free base
of 15 as described above to yield (74%), after recrystallization
from ethanol-ether (1:4), a pale yellow solid: mp 280-282
1
°C dec; H NMR (DMSO-d6) δ 9.5-8.5 (br, 4H), 8.0 (d, 4H, J

Anti-Pneumocystis carinii Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 129
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J M970570I