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T. b. gambiense infections, which has only recently been discovered,
further complicates control and eradication efforts.8,9
lipophilicity of the lead compounds, while maintaining an accept-
able level of biological activity and metabolic stability.
Polyamine biosynthesis gained recognition as a target for antit-
rypanosomal therapies upon the discovery by Bacchi et al. in 1980
that identified a-difluoromethylornithine (DFMO, eflornithine) as a
2. Results and discussion
curative agent against T. brucei infection in mice.10 Eflornithine is a
rationally designed mechanism-based suicide inhibitor of
ornithine decarboxylase (ODC), which catalyzes the first commit-
ted step in polyamine biosynthesis.3,11 Eflornithine has since been
registered (both on its own and as a NECT) for treatment of late
stage T. b. gambiense HAT confirming the polyamine pathway as
a very viable target for anti-HAT drug discovery.12,13
S-adenosylmethionine decarboxylase (AdoMetDC) is another
critical enzyme in the polyamine pathway required to generate
the aminopropyl group that is then transferred onto the ODC pro-
duct, putrescine, to make spermidine. Spermidine is essential in all
eukaryotic cells as a substrate for the hypusine modification of the
translation factor eIF5A.14 Trypanosomatid AdoMetDC is regulated
by a novel mechanism not found in mammalian cells.11 While
human AdoMetDC is a homodimer, T. brucei AdoMetDC requires
heterodimerization with an inactive paralog termed prozyme for
activity.15,16
Significant evidence that AdoMetDC will be a druggable target
in T. brucei has accumulated through the finding of inhibitors with
good antitrypanosomal activity.3 The most potent of these, 50-(((Z)-
4-amino-2-butenyl)methylamino)-50-deoxyadenosine (1, also
known as MDL 73811, or AbeAdo) was designed as a mecha-
nism-based suicide inhibitor of AdoMetDC.17 It inhibits AdoMetDC
from E. coli,17 T. brucei,18 rat,19 and human,20 acting through the
enzyme-activated transamination of the covalently bound pyru-
voyl prosthetic group.20 Potential for therapeutic use of AdoMetDC
inhibitors in general and 1 in particular was confirmed in a murine
model of the hemolymphatic stage of HAT using T. b. brucei and
clinical isolates of T. b. rhodesiense, where it showed acutely cyto-
static effect on the parasite.18,21 Despite the activity on the mam-
malian enzymes, selective toxicity against T. brucei was obtained,
and the low curative dosage in hemolymphatic model of HAT laid
a solid foundation for further lead development. Unfortunately, 1
was not efficacious in a mouse model of the CNS stage of HAT21
due to poor blood-brain barrier permeation.19,22,23
In attempt to improve on pharmacokinetic properties of 1, the
C8-methyl derivative Genz-644131 (2) was synthesized and
showed about 5-fold increased activity on T. brucei AdoMetDC
compared with 1.22 The improved potency on the enzyme trans-
lated to better T. brucei parasite growth inhibition.22 Overall, both
1 and 2 demonstrated favorable in vitro and in vivo stability pro-
files.22 However, neither of the compounds was able to achieve
good CNS exposure in mice, only showing 1.7% (1) and 7.3% (2)
brain-to-blood ratio.22 Not surprisingly, even though both com-
pounds resulted in sterile cure in mice infected with T. brucei with
a 7-day 50 mg/kg/day intraperitoneal dosage,22 neither compound
led to a cure of a mouse model of the CNS stage.21,24
2.1. Synthesis and evaluation of carbamate and amide analogs
We considered that decreasing the polarity of the basic buteny-
lamine side chain by conversion to an amide or carbamate would
improve overall permeability. Thus, treatment of 2 with the appro-
priate 4-nitrophenyl carbonate afforded carbamates 3a–c in good
yields (Scheme 1A), whereas several terminal amide analogs 5a–c
of MDL 73811 (1) were prepared via alkylation of amine 423 with
a series of 4-amidobuten-2-yl chlorides 7a–c, followed by acid-
mediated acetal deprotection (Scheme 1B). Given that Marasco
et al.25 had shown that both enzyme inhibition and antiparasitic
activity of 1 is retained after acetylation of the ribose hydroxyl
groups, we also prepared the bis-acetate derivatives 6a–c (Sche-
me 1B) hoping to further improve BBB penetration.
The T. brucei AdoMetDC enzyme inhibition, T. b. brucei cell
growth inhibition, monolayer permeability, and both murine and
human microsomal metabolic stability data are compiled in
Table 126 Unfortunately, none of the tested amide or carbamate
derivatives retained any meaningful activity in the AdoMetDC
enzyme assay, suggesting that the basic amine is essential for
activity within this series. This is in agreement with the estab-
lished mechanism of MDL 73811 (1) inhibition, which relies on
the primary amine for suicide transamination of the catalytic pyru-
voyl group.20 Similarity between relative IC50 values assessed with
and without pre-incubation in the case of the two compounds with
measurable activities, 5a–b, suggests that in the absence of the pri-
mary amine the mechanism of the enzyme inhibition is no longer
time-dependent. However, we were speculating that the amides or
carbamates might act as prodrugs, but unfortunately, the lack of
cellular antitrypanosomal activity indicated that no or insignificant
amounts of the active parent compounds 1 or 2 were liberated
within T. b. brucei parasites. This lack of cellular proteolytic amide
or carbamate hydrolysis therefore terminates meaningful pro-
spects for pro-drug strategies with these side-chain modifications.
With the exception of benzyl carbamate 3b, the microsomal stabil-
ity of carbamates 3a,c and amides 5a–c was excellent, indicating
that the short half-lives of the bis-acetate derivatives 6a–c could
be due to facile acetate hydrolysis. Most disappointingly however,
and contrary to our original hypothesis, these changes had no pos-
itive effect on CNS delivery as measured by the MDCKII-hMDR1
monolayer permeability assay and precluded any further interest
in pursuing these series.
2.2. Synthesis and evaluation of C50-amine and ribose ketal analogs
We next explored the effects of sterics, basicity, and lipophilic-
ity of the C50-amine substituent in both the MDL (8a, R1 = H) and
Genz (8b, R1 = Me) series (Scheme 2). The C50-amine modifications
(R2) were available through Fukuyama-Mitsunobu amination of 8a,
b to afford 9a–d in modest yields. N-Alkylation (?10a–d) was fol-
lowed by Boc deprotection to deliver 11a–d, or simultaneous Boc
and acetonide removal to afford 1 and 12b,c. Analog 12d was inac-
cessible under the later conditions due to extensive decomposition
of the starting material 10d.
Due to the promising activity of both MDL 73811 (1) and Genz-
644131 (2) (Fig. 1), we embarked upon a medicinal chemistry cam-
paign with the specific goal of improving the BBB penetration of
this class of compounds. We envisioned preparing a series of com-
pounds with structural modifications designed to increase the
The Boc- (10a–d) and acetonide-protected intermediates (11a–
d) were evaluated in addition to the final deprotected analogs 12b,
c in order to compile as many SAR data points as possible (Table 2).
As before, all of the analogs demonstrated severely impaired activ-
ity in the enzyme inhibition and cell growth assays, with the
exception perhaps of acetonide-protected MDL 73811 (11a), which
Fig. 1. Structure and activity of MDL 73811 (1) and Genz-644131 (2).