Carbamate prodrugs of N-alkylfuramidines

Article abstract of DOI:10.1016/j.ejmech.2007.03.009

The synthesis and evaluation of 2,5-bis[4-(N-ethoxycarbonyl-N′-isopropyl)amidinophenyl]furan, 2,5-bis[4-(N-2,2,2-trichloroethoxycarbonyl-N′-isopropyl)amidinophenyl]furan and 2,5-bis[4-(N-cyclopentyl-N′-2,2,2-trichloroethoxycarbonyl)amidinophenyl]furan as

Full text of DOI:10.1016/j.ejmech.2007.03.009

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 174e177
http://www.elsevier.com/locate/ejmech
Short communication
Carbamate prodrugs of N-alkylfuramidines
Syed M. Rahmathullah a, Richard R. Tidwell b, Susan K. Jones b,
James E. Hall b,c, David W. Boykin a,
*
a Department of Chemistry, Georgia State University, University Plaza, Atlanta, GA 30303-3082, United States
b School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States
c School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States
Received 17 November 2006; received in revised form 12 March 2007; accepted 12 March 2007
Available online 3 April 2007
Abstract
The synthesis and evaluation of 2,5-bis[4-(N-ethoxycarbonyl-N0-isopropyl)amidinophenyl]furan, 2,5-bis[4-(N-2,2,2-trichloroethoxycarbonyl-
N0-isopropyl)amidinophenyl]furan and 2,5-bis[4-(N-cyclopentyl-N0-2,2,2-trichloroethoxycarbonyl)amidinophenyl]furan as prodrugs of bis-N-al-
kylamidines are reported. The results show that the bis-2,2,2-trichloroethyl carbamates function effectively in a rat model for Pneumocystis
pneumonia.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Prodrugs; Carbamates; N-Alkylfuramidines; Pneumocystis carinii
1. Introduction
antimicrobial agents [5]. In an effort to provide orally effective
diamidine antimicrobial agents we have explored various pro-
drug strategies including making bis-amidoximes, bis-O-alky-
lamidoximes and bis-carbamates of potent diamidine systems
[6,7]. Currently, pafuramidine, a methoxamidine of furami-
dine, is in two phase III clinical trials, one against early stage
human African trypanosomiasis and the other against human
Pneumocystis jiroveci (formerly Pneumocystis carinii) pneu-
monia [5b,8]. We have reported that several N-alkylfuramidine
compounds showed significantly improved activity compared
to furamidine against P. carinii when administered intrave-
nously in an immunosuppressed rat model [9]. Despite the
fact that there does not appear to be a carbamate specific mam-
malian enzyme [10], carbamates have been successfully used
as prodrugs for a number of different functional groups [11].
Presumably one or more of the plethora of mammalian ester-
ases is responsible for carbamate cleavage [12]. Despite a con-
siderable number of investigations focused on development of
prodrugs for unsubstituted amidines we are aware of only one
such study on bis-N-alkylamidines [13]. Thus, it is important
to explore approaches to provide oral bioavailability of the
highly active bis-N-alkylamidines. In view of our success by
using carbamates of furamidine as prodrugs [7] we decided
Amidines are quite basic functional groups and the pKs of
arylamidines range from 10 to 12 [1]. Consequently, drugs
containing amidine units are protonated at physiological pH
and typically exhibit low bioavailability. An extensive number
of monoamidines have been evaluated for their utility in
blocking various stages of the thrombin cascade and numerous
highly potent molecules have been reported [2]. In efforts to
markedly improve the oral bioavailability of this class of mol-
ecules, various prodrug approaches including conversion to
amidoximes, O-alkylamidoximes and carbamates have been
investigated [3]. A number of these prodrugs have been found
to significantly improve oral bioavailability relative to the
parent compounds and the amidoxime prodrugs sibrafiban and
ximelagatran have undergone clinical trials in an effort to pro-
vide orally effective thrombin inhibitors [4]. Since the discovery
of the potent antiprotozoan activity of pentamidine aryl diami-
dines, these compounds have been extensively studied as
* Corresponding author. Tel.: þ1 404 651 3798; fax: þ1 404 651 1416.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.009

S.M. Rahmathullah et al. / European Journal of Medicinal Chemistry 43 (2008) 174e177
175
to examine their effectiveness with N-alkylfuramidine ana-
logues. We report here an initial study of carbamates of two
active N-alkylfuramidines.
2-trichloroethyl carbamate of N-i-propyl furamidine 2b shows
excellent activity on iv dosing and quite good activity on oral
administration. These results and those reported earlier [7], sug-
gest that the trichloroethyl carbamate is either better absorbed
or metabolized (or both) than its ethyl carbamate analogue in
these bis-amidine systems. Only modest activity was found
on iv dosing of the 2,2,2-trichloroethyl carbamate of
N-c-pentyl furamidine 2c, however, on oral administration
good activity, approaching to that of 2b, was observed. Based
on these observations we conclude that carbamates of N-alkyla-
midines are suitable for prodrug approaches for these potent
and potentially quite useful molecules. No detectable overt tox-
icity has been noted for numerous other prodrugs of diamidines.
This initial study indicates that carbamates of N-alkylami-
dines can function effectively as prodrugs. Further studies
are required to optimize the carbamate structure for maximum
delivery of the active compounds.
2. Chemistry
Given the in vivo effectiveness of bis-2,2,2-trichloroethyl
furamidine carbamate we decided to explore the use of this
system with the highly active N-i-propyl and N-c-pentyl fura-
midine analogues. Earlier we reported the synthesis of a num-
ber of arylealkyl carbonates and their synthetic utility in the
preparation of carbamate prodrugs of 2,5-bis(4-amidinophe-
nyl)furan [7]. This method was applied to the synthesis of
the N-alkyl substituted amidines reported here (Scheme 1).
The reaction of the N-isopropyl amidine 1a with ethyl 4-nitro-
phenylcarbonate in DMF at room temperature gave 2a in 85%
yield and when a THF/CH3CN mixture was used the requisite
carbamate was obtained in a 90% yield. When 2b was pre-
pared by the reaction of 2,2,2-trichloroethyl chloroformate
with 1a only a 56% yield was obtained and the reaction of
1b with the same chloroformate failed to provide any isolat-
able carbamate (experimental not presented). However, when
1a was allowed to react with 4-nitrophenyl 2,2,2-trichloroe-
thylcarbonate in THF/CH3CN the carbamate 2b was obtained
in an 85% yield. The N-cyclopentyl-N0-2,2,2-trichloroethyl
carbamate 2c was synthesized in a 76% yield in a similar man-
ner using 4-nitrophenyl 2,2,2-trichloroethylcarbonate in THF/
CH3CN.
4. Experimental section
Melting points were recorded using a Thomas-Hoover
(Uni-Melt) capillary melting point apparatus and are uncor-
rected. TLC analysis was carried out on silica gel 60 F254
pre-coated aluminium sheets (0.20 mm layer thickness) (E.
Merck) and detected under UV light. IR spectra were recorded
using Perkin-Elmer Model 337 spectrometer. 1H and 13C
NMR spectra were recorded employing a Varian GX400 or
a Varian Unityplus 300 spectrometer and chemical shifts (d)
are in ppm relative to TMS as internal standard. Mass spectra
were recorded on a VG Analytical 70-SE spectrometer (Geor-
gia Institute of Technology, Atlanta, GA). IR spectra were re-
corded using a Perkin-Elmer 2000 instrument. Elemental
analyses were obtained from Atlantic Microlab Inc. (Norcross,
GA) and are within 0.4% of the theoretical values unless other-
wise mentioned. All chloroformates were purchased from
Aldrich Chemical Co. Other chemicals and solvents were
purchased from either Acros, Aldrich or Fischer Scientific.
Anti-P. carinii pneumonia screening was carried out ac-
cording to published methods [7,14]. Compounds were rou-
tinely tested orally at 33 mmol/kg/day and intravenously at
22 mmol/kg/day. Saline and pentamidine treated groups of
rats were included as negative and positive controls,
respectively.
3. Biology
Table 1 contains the results of evaluation of the carbamate
prodrugs of N-alkylfuramidines in an immunosuppressed rat
Pneumocystis model. The table also contains results for the con-
trol compounds pentamidine, furamidine, pafuramidine and the
bis-2,2,2-trichloroethyl carbamate of furamidine. As is well
known the parent diamidine, pentamidine and furamidine
show limited efficacy on oral administration and similar results
were observed with the N-i-propyl and N-c-pentyl furamidine
analogues 1a and 1b (Table 1). The ethyl carbamate of N-i-pro-
pyl furamidine 2a was not effective in the immunosuppressed
rat model (Table 1); similar results were observed for the ethyl
carbamate of furamidine [7]. In marked contrast, the 2,2,
HN
R1HN
RN
NR
O
NH
NHR1
O
(a)
R1HN
NHR1
1a R1 = i-Propyl
1b R1 = c-Pentyl
2a R1 = i-Propyl R = COOCH2CH3
2b R1 = i-Propyl R = COOCH2CCl3
2c R1 = c-Pentyl R = COOCH2CCl3
Reagents and conditions: (a) 4-nitrophenyl ethyl carbonate or 4-nitrophenyl 2,2,2-trichloroethyl carbonate, DMF or THF, MeCN, rt.
Scheme 1.

176
S.M. Rahmathullah et al. / European Journal of Medicinal Chemistry 43 (2008) 174e177
Table 1
In vivo evaluation of prodrugs and comparison with parent molecules versus P. carinii in a rat model (DB numbers will be deleted before submission)
RN
NR
O
R1HN
NHR1
Code
R
R1
Dosagea
(mmol/kg/day)
Cysts/g of lungb
(% of control)
Toxicityc
Saline
NA
NA
iv
po
100.0 ꢃ 12.72
100.00 ꢃ 35.16
2.75 ꢃ 1.27
0
0
Pentamidined
Furamidinee
Pafuramidinef
NA
NA
iv at 22.0
oral at 33.0
iv at 13.3
oral at 39.8
iv at 22.0
oral at 33.0
iv at 22.0
oral at 33.0
iv at 10.8
oral at 33.0
iv at 11.0
oral at 33.0
iv at 22.0
oral at 33.0
iv at 9.4
þþ
0
67.78 ꢃ 20.49
0.86 ꢃ 0.52
H
H
0
15.67 ꢃ 6.07
13.99 ꢃ 5.03
1.21 ꢃ 0.35
0
OMe
H
0
0
Furamidine trichloroethyl
carbamateg
1a
COOCH2CCl3
H
H
1.72 ꢃ 1.66
0
5.45 ꢃ 5.02
0
i-Propyl
i-Propyl
i-Propyl
c-Pentyl
c-Pentyl
0.26 ꢃ 0.23
0
78.52 ꢃ 24.52
82.21 ꢃ 43.74
51.92 ꢃ 19.53
0.02 ꢃ 0.01
0
2a
2b
1b
2c
a
COOCH2CH3
COOCH2CCl3
H
0
0
þþ
0
2.01 ꢃ 0.92
0.04 ꢃ 0.02
þ
0
oral at 33.0
iv at 22.0
oral at 33.0
125.30 ꢃ 39.98
39.84 ꢃ 2.81
3.70 ꢃ 2.22
COOCH2CCl3
0
0
Evaluation of iv dosage (by tail vein injection) of the compounds against P. carinii in rats as described in Refs. [7,13]; oral dosage by gavage.
Lower numbers of cysts/g reflect higher activity versus P. carinii. Values below 1% of control are within experimental error and can be considered a complete
b
cure of the infection in the immunosuppressed rats. Mean cyst count for pooled controls: saline (n ¼ 94) 50.63 ꢀ 106 cysts/g of lung tissue, pentamidine (n ¼ 89)
1.39 ꢀ 106 cysts/g of lung tissue for iv dosing and saline (n ¼ 11) 74.19 ꢀ 106 cysts/g of lung tissue, pentamidine (n ¼ 6) 50.28 ꢀ 106 cysts/g of lung tissue for po
dosing.
c
Toxicity of the compounds tested is evaluated according to a six level scale which ranges from 0 to þ4. The toxicity noted should not be considered definitive
and is essentially anecdotal. However, these values do allow for comparison of relative toxicities among tested compounds and the control drug pentamidine.
d
Ref. [13].
Ref. [9].
e
f
Ref. [6a].
Ref. [7].
g
4.1. 2,5-Bis[4-(N-ethoxycarbonyl-N0-isopropyl)-
amidinophenyl]furan (2a)
156.4, 155.0, 152.1, 148.1, 134.5, 91.1, 88.4, 39.0, 24.9; MS
m/z (FAB, thioglycerol) 533 (M þ 1), 492, 428, 402, 356, 330,
315, 288, 271. Anal. calcd. for C30H36N4O5: theory: C, 74.35;
H, 7.49; N, 11.56. Found: C, 74.21; H, 7.60; N, 11.30.
To a suspension of the N-isopropyl amidine (1a) [9] (0.4 g,
0.001 mol) in DMF (15 mL) at room temperature was added
ethyl 4-nitrophenylcarbonate (0.47 g, 0.0023 mol) and the mix-
ture was stirred over night. The solution was then cooled in an
ice bath, water (20 mL) was added and the resulting solid was
filtered, washed with water (3 ꢀ 40 mL), ether (2 ꢀ 30 mL)
and dried. The crude product was crystallized from CH2Cl2/
ether mixture to afford pure bis-carbamate 2a (0.46 g, 85%
yield) as a pale yellow solid: mp 186e188 ꢁC; IR (KBr)
3440e3140 (br, NH str), 3114 (m, CH-Ar str), 3080 (m, CH-
Ar str), 3050 (m, CH-Ar str), 2984 (s, CH-Alip str), 2935 (s,
4.2. 2,5-Bis[4-(N-2,2,2-trichloroethoxycarbonyl-N0-
isopropyl)amidinophenyl]furan (2b)
To a suspension of N-isopropyl amidine (1a) [9] (1.0 g,
0.0026 mol) and diisopropylethylamine (1 g, 0.0077 mol) in
THF/CH3CN mixture (20 mL, 1:1 v/v) at room temperature,
was added a solution of 4-nitrophenyl 2,2,2-trichloroethylcar-
bonate (1.76 g, 0.0056 mol) in THF (10 mL) and stirred over
night (16 h). The solution was concentrated on a rotary evapora-
tor at 30 ꢁC under reduced pressure and the residue was cooled
in an ice bath, triturated with anhydrous diethyl ether (20 mL),
filtered, washed with ether (3 ꢀ 20 mL), dried and crystallized
from CH2Cl2/ether mixture to obtain 2,2,2-trichloroethyl carba-
mate 2b (1.4 g, 90% yield) as a pale yellow solid: TLC (Rf) 0.68
(CHCl3/MeOH/NH4OH, 4:1:0.2, v/v/v); mp 156e158 ꢁC; IR
1
CH-Alip str), 1675 (s, C]O str) cmꢂ1. H NMR (DMSO-d6)
d 7.96 (s, 2H, NH), 7.88 (d, J ¼ 8.20 Hz, 4H, Ar-CH), 7.42 (d,
4H, J ¼ 8.1 Hz, Ar-CH), 7.22 (s, 2H, CH-furan), 4.05 (m, 2H,
CHMe2), 3.85 (q, 4H, OCH2Me, J ¼ 6.97 Hz), 1.20 (d, 6H,
J ¼ 6.7 Hz, CH3 of isopropyl), 0.99 (t, 6H, J ¼ 6.9 Hz,
OCH2CH3); 13C NMR (DMSO-d6) d 180.8, 178.3, 177.2,

S.M. Rahmathullah et al. / European Journal of Medicinal Chemistry 43 (2008) 174e177
177
(KBr) 3445e3140 (s, NH str), 3071 (m, CH-Ar str), 3050 (m,
CH-Ar str), 2978 (m, CH-Alip str), 2934 (m, CH-Ar str), 1669
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0.0017 mol) and diisopropylethylamine (0.52 g, 0.004 mol) in
a THF/CH3CN mixture (20 mL, 1:1 v/v) at room temperature,
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NH str), 3100 (m, CH-Ar str), 2960 (m, CH-Alip str), 2871
(m, CH-Alip str), 1674 (s, C]O str) cmꢂ1. 1H NMR (CDCl3)
d (tautomeric or geometric mixture) 10.21 (s, 2H, NH), 7.82
(d, J ¼ 7.93 Hz, Ar-CH), 7.75 (d, J ¼ 7.14 Hz, Ar-CH), 7.50
(d, J ¼ 7.93 Hz, Ar-CH), 6.87 (s, CH-furan), 6.86 (s, CH-furan),
5.45 (d, 2H, J ¼ 6.82 Hz, NH), 4.82 (s, CH2CCl3), 4.69 (s,
CH2CCl3), 4.50 (m, NHCH ), 3.97 (m, NHCH ), 2.16 (m, c-pen-
tyl), 1.96 (m, c-pentyl), 1.77 (m, c-pentyl), 1.60e1.76 (br m, c-
pentyl); 13C NMR (CDCl3) d (tautomeric mixture) 171.1, 165.2,
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124.2, 123.9, 109.3, 95.8, 75.8, 75.4, 57.3, 54.2, 34.7, 33.2,
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Acknowledgment
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This work was supported by NIH Grants AI-33363, AI-
42411 and AI-46365.