Design, synthesis, and evaluation of potential prodrugs of DFMO for reductive activation

Article abstract of DOI:10.1016/j.bmcl.2012.09.005

A series of potential DFMO prodrugs was designed through the incorporation of 4-nitrobenzyl ester or carbamate groups for potential activation by trypanosomal nitroreductase. It was found that only modification of N-amino group of DFMO by 4-nitro-2-fluorobenzyloxycarbonyl resulted in significant trypanocidal activity and could serve as a lead for further investigation.

Full text of DOI:10.1016/j.bmcl.2012.09.005

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6583–6586
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of potential prodrugs of DFMO
for reductive activation
Yanhui Yang a, Andrew Voak c, Shane R. Wilkinson c, Longqin Hu a,b,
⇑
a Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
b The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
c Queen Mary Pre-Clinical Drug Discovery Group, School of Biological & Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2012
Revised 31 August 2012
Accepted 4 September 2012
Available online 13 September 2012
A series of potential DFMO prodrugs was designed through the incorporation of 4-nitrobenzyl ester or
carbamate groups for potential activation by trypanosomal nitroreductase. It was found that only mod-
ification of N -amino group of DFMO by 4-nitro-2-fluorobenzyloxycarbonyl resulted in significant try-
e
panocidal activity and could serve as a lead for further investigation.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
a-Difluoromethylornithine
Nitroreductase
Prodrug design
Reductive activation
Trypanocidal
a
-Difluoromethylornithine (DFMO or Eflornithine) is an irre-
related to its high dose and cost of treatment, and the inconve-
nience of intravenous administration especially in understaffed
rural hospitals of disease endemic countries. Oral DFMO would
be logistically cheaper and much easier, especially for use in rural
settings. Even though the oral bioavailability of DFMO is not a
problem, its elimination from the blood is rapid so that high doses
are necessary to obtain the desired therapeutic effect.4 Therefore,
the development of DFMO prodrugs could be one approach to
overcome these drawbacks of DFMO.
Prodrug design is an important strategy that has proven to work
for many drugs by improving their undesirable physico-chemical
and biological properties.5–9 Nitroaromatic prodrug conjugates
have been developed that rely on specific activation by a desig-
nated E. coli type I nitroreductase, which can be delivered site-spe-
cifically to tumor cells and tissues using antibody-directed enzyme
prodrug therapy (ADEPT) or gene-directed enzyme prodrug
therapy (GDEPT).5,8,10,11 We have shown that trypanosomes also
express a type I nitroreductase that can activate nitrobenzyl
phosphoramide mustard and azirinindyl nitrobenzamidine pro-
drugs.12–15 Herein, we report the synthesis and evaluation of four
types of DFMO prodrugs that incorporate a nitroaromatic function-
ality designed to exploit the trypanosomal type I nitroreductase as
the activating enzyme to help potentially increase target specificity
and improve bioavailability.
versible inhibitor of ornithine decarboxylase (EC 4.1.1.17), an en-
zyme that catalyzes the first step in polyamine biosynthesis.
Inhibition of this enzyme activity results in depletion of putrescine
and spermidine, and consequently slows down cell proliferation.
Although originally developed in the 1970’s as a treatment for can-
cer, DFMO was shown to be ineffective in treating malignancies.
The lack of anti-tumor activity stemmed from the fast turnover
rate of ornithine decarboxylase displayed in mammalian cells
(t1/2 = 10–30 min) such that DFMO-inhibited enzyme is rapidly
cleared from the cell and replaced with newly synthesized active
protein.1 Subsequently, it was found that DFMO is highly effective
against human African trypanosomiasis (African sleeping sickness),
especially the West African form (Trypanosoma brucei gambiense),
due to the slow turn over (t1/2 = 18–19 h) of its ornithine
decarboxylase.2,3
Intravenous infusions of DFMO were approved in 1990 by US
Food and Drug Administration as a treatment for the meningo-
encephalic stage of the human African trypanosomiasis. The cur-
rent recommended regimen for all age groups is 400 mg/kg body
weight/day administered over 4 intravenous infusions daily for
14 days. Intravenous DFMO given either alone or in combination
with oral doses of nifurtimox has proven to be effective and safe
with 2-year cure rates of 97–100% against West African trypanoso-
miasis.3 The major drawback of intravenous DFMO is, however,
Design and synthesis
Examination of the structure of DFMO suggested that there are
three possible sites for the attachment of a promoiety. As shown in
⇑
Corresponding author. Tel.: +1 848 445 5291; fax: +1 732 445 6312.
E-mail address: LongHu@rutgers.edu (L. Hu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.005

6584
Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6583–6586
O
Biological evaluation
CHF2
NH2
Nε-carbamate
prodrugs
ester
HO
NH2
DFMO
prodrugs
All potential prodrugs were evaluated as substrates of T. brucei
nitroreductase (TbNTR) and screened for trypanocidal activity
against bloodstream form T. brucei using DFMO and nifurtimox as
the controls.12,13 As shown in Table 1, mono(4-nitrobenzyl)-DFMO
ester or carbamate conjugates (1a, 2a, 3a) without halogen
substitutions on the benzyl ring were not effective substrates of
TbNTR, giving substrate activity values similar to the background
DFMO control. The presence of two 4-nitrobenzylcarbamoyl
groups on the DFMO conjugate 4a did confer TbNTR substrate
activity. Addition of a 2-fluoro substitution to the phenyl ring in
the mono(4-nitrobenzyl)-DFMO ester or carbamate conjugates as
in 1b, 2b, and 3b resulted in an increased TbNTR activity while
conjugate 4b containing two nitrobenzyl carbamates both with
2-fluoro substituents had the highest activity of all. When tested
Nα-carbamate prodrugs
Figure 1. Structure of DFMO and sites for the attachment of a promoiety in the
design of potential prodrugs.
Figure 1, DFMO contains two amino groups and one carboxylic acid
group; each of these functional groups could be attached to a nitro-
benzyl promoiety to form either a carbamate or ester prodrug that
can potentially be activated by nitroreduction. We selected
4-nitrobenzyl and 4-nitro-2-fluorobenzyl as the promoieties since
both have been shown in our earlier efforts to be substrates of
trypanosomal nitroreducases.12,13
As shown in Scheme 1, the prodrugs were readily synthesized
through varying protection and deprotection strategies using
DFMO as the starting material. Boc protection of the primary
amines in DFMO followed by SN2 reaction of 4-nitrobenzyl tosyl-
ate with the protected DFMO carboxylate anion gave the diBoc
protected DFMO esters, which upon TFA deprotection afforded
the desired DFMO 4-nitrobenzyl ester prodrugs 1a and 1b. For
e
for their antitrypanosomal activity, only N -(4-nitro-2-fluoroben-
zyloxycarbonyl)-DFMO 2b out of the 8 DFMO conjugates synthe-
sized had significant inhibitory effect on parasite growth at
concentrations up to 100 lM with an IC50 of 40 lM (Table 1, entry
4). When 2b was tested against T. brucei induced to express ele-
vated levels of nitroreductase, we found that there was no
alteration in trypanocidal activity as compared to non-induced
controls, suggesting that T. brucei nitroreductase does not contrib-
ute towards the activation and trypanocidal activity of 2b; in con-
trast, cells expressing higher levels of nitroreductase were seven
fold more sensitive to the nitroaromatic prodrug nifurtimox than
controls. Precisely how 2b works in producing its trypanocidal
activity is still under investigation. As the T. brucei nitroreductase
is localized to the parasite’s single mitochondrion,15 2b may not
be able to access this sub-cellular compartment. Alternatively, if
2b can get into the mitochondrion, the product generated from
the nitroreductase-mediated reaction may not be able to gain ac-
cess into the cytoplasm, the site where the trypanosomal ornithine
decarboxylase is found. Why the other DFMO conjugates fail to af-
fect trypanosomal growth is also unclear. However, given that
DFMO is actively transported into the parasite by specific amino
acid permeases, then the modifications reported here may
e
the synthesis of N -(4-nitrobenzyloxycarbonyl)-DFMO prodrugs
2a and 2b, the N -primary amine was selectively protected
through copper(II) complexation during carbamoylation. For the
a
a
synthesis of N -(4-nitrobenzyloxycarbonyl)-DFMO prodrugs 3a
e
and 3b, the more basic N -amino group was first selectively
protected with Fmoc before carbamoylation. After reaction with
4-nitrobenzyloxycarbonyl imidazole, the Fmoc was removed
using 20% piperidine to the desired N -carbamate DFMO pro-
drugs 3a and 3b. The dicarbamate DFMO prodrugs 4a and 4b
were prepared from DFMO in the presence of excess 4-nitroben-
zyloxycarbonyl imidazole. The prodrugs were all purified to
homogeneity by preparative HPLC and structures confirmed by
NMR and MS. The overall yields for the synthesis of these pro-
drugs starting from DFMO were between 10% and 51% and were
not optimized.
a
O
1. (BOC)2O, DIEA/MeOH
2. 4-NO2BzlOTs, DIEA/THF, reflux
CHF2
O
NH2
3. 50% TFA/CH2Cl2
4. reversed phase HPLC
(11 - 25%)
NH2
O2N
HO
X
1a X = H
1b
X = F
O
O
O
X
1. CuSO4, aq.NaHCO3
2. 4-NO2BzlOCO-im, DMAP, DIEA/THF
CHF2
NH2
N
H
O
3. aq. HCl, pH 3
O
4. reversed phase HPLC
(34 - 36%)
NO2
2a X = H
CHF2
NH2
HO
NH2
2b
X = F
1. Fmoc-OSu, DIEA, THF
CHF2
2. 4-NO2BzlOCO-im, DMAP, DIEA/THF
DFMO
HO
HO
NH2
3. piperidine/NMP
HN
O
NO2
4. reversed phase HPLC
(10 - 32%)
O
3a
X = H
X
3b X = F
O
O
X
CHF2
1. 4-NO2BzlOCO-im, DMAP, DIEA/THF
N
H
O
NO2
2. reversed phase HPLC
(26 - 51%)
HN
O
NO2
O
4a
4b X = F
X = H
X
Scheme 1. Synthesis of DFMO prodrugs designed.

Y. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6583–6586
6585
Table 1
Biological activities of DFMO prodrugs as substrates of trypanosomal NTR and as inhibitors of trypanosomal growth
Entry Compounds Structure
TbNTR activitya (nmol NADH oxidized/min/mg) IC50
(l
M)b
wild-type T. brucei T. brucei with elevated NTR
O
O
CHF2
NH2
O
O
NH2
NH2
1
2
3
4
1a
1b
2a
2b
60
4
>100
—
O2N
CHF2
NH2
203 12
>100
—
O2N
HO
F
O
O
O
O
CHF2
NH2
N
O
O
33
2
8
>100
—
H
NO2
NO2
O
F
CHF2
NH2
HO
HO
N
H
260
39.5 0.9
39.9 2.0
CHF2
NH2
NH2
5
6
7
8
3a
3b
4a
4b
42 10
>100
>100
>100
>100
—
—
—
—
HN
O
NO2
NO2
O
O
CHF2
HO
HN
O
227
6
3
O
F
O
O
CHF2
HO
HO
N
H
O
NO2
140
HN
O
O
NO2
NO2
O
O
O
F
CHF2
N
H
O
NO2
HN
312 13
O
F
O
CHF2
NH2
9
DFMO
21
4
8.30 1.46
1.83 0.02
—
HO
NH2
N
N
O
O2N
10
Nifurtimox
423 45
0.26 0.01
S
O
O
a
Nitroreductase substrate activity expressed as the initial velocity (nmoles of NADH oxidized/min/mg) in the presence of purified His-tagged T. brucei nitroreductase
(TbNTR) by following the change in absorption at 340 nm.
b
The blood stream form T. brucei parasites were seeded at 1 Â 103 mLÀ1 in 200
lL growth medium containing different concentrations of test compounds. After incubation
at 37 °C for 3 days, 20 l
L alamarBlueÒ (Biosource UK Ltd) was added to each well and the plates incubated for a further 16 h. Cell viability was determined using fluorescence
at 530 nm/ex and 585 nm/em to establish the IC50 value for each test compound.
e
adversely affect prodrug uptake.16–18 Nevertheless, the weak anti-
trypanosomal activity of 2b could serve as a lead for further
investigation.
N -(4-nitro-2-fluorobenzyloxycarbonyl)-DFMO 2b. Because of the
increased urgency in finding effective treatments of trypanosomal
infections, 2b could potentially serve as
a lead for further
In summary, we attempted to incorporate 4-nitrobenzyl groups
into DFMO to form prodrugs for potential activation by trypanoso-
mal nitroreductase and found that only modification of N -amino
investigation.
e
Acknowledgments
group of DFMO by 4-nitro-2-fluorobenzyloxycarbonyl resulted in
significant but weak trypanocidal activity. Further assay against
T. brucei expressing elevated levels of nitroreductase did not show
any increased trypanocidal activity. Additional studies will be nec-
essary to understand the mechanism of the tryapnocidal activity of
We gratefully acknowledge the financial support of grant
SNJ-CCR 700–009 from the State of New Jersey Commission on
Cancer Research and grant RSG-03-004-01-CDD from American
Cancer Society.

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