Novel convenient synthesis of biologically active esters of hydroxylamine

Article abstract of DOI:10.1007/s00726-009-0410-0

Alkylation of ethyl N-hydroxyacetimidate with readily available methanesulfonates of functionally substituted alcohols and subsequent deprotection of aminooxy group is a novel and convenient method to prepare functionally substituted esters of hydroxylamine with high overall yield. This approach is a good alternative to wellknown reaction of N-hydroxyphthalimide with alcohols under the Mitsunobu conditions. The properties of ethoxyethylidene protection of aminooxy group on the contrary to that of N-alkoxyphthalimide group allow to perform a wide spectra of the transformations in the radical of N-protected hydroxylamine derivatives. This is essential for synthetic strategies consisting in the introduction of N-protected aminooxy group at one of the first steps of synthesis and subsequent transformations of the radical. The inhibitory effect of one of the newly synthesized compound, 1-guanidinooxy-3-aminopropane (GAPA), was compared with that of well-known inhibitors of ornithine decarboxylase namely, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA) on Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. GAPA, on the contrary with APA and DFMO, in micromolar concentrations, inhibited the growth of both amastigotes and promastigotes of sodium antimony gluconate-resistant forms of L. donovani. Springer-Verlag 2009.

Full text of DOI:10.1007/s00726-009-0410-0

Amino Acids (2010) 38:509–517
DOI 10.1007/s00726-009-0410-0
ORIGINAL ARTICLE
Novel convenient synthesis of biologically active esters
of hydroxylamine
•
Maxim A. Khomutov Swati Mandal Janne Weisell
•
•
•
•
Neiha Saxena Alina R. Simonian Jouko Vepsalainen
•
•
Rentala Madhubala Sergey N. Kochetkov
Received: 10 August 2009 / Accepted: 16 September 2009 / Published online: 8 December 2009
Ó Springer-Verlag 2009
Abstract Alkylation of ethyl N-hydroxyacetimidate with
readily available methanesulfonates of functionally substitu-
ted alcohols and subsequent deprotection of aminooxy
group is a novel and convenient method to prepare func-
tionally substituted esters of hydroxylamine with high
overall yield. This approach is a good alternative to well-
known reaction of N-hydroxyphthalimide with alcohols
under the Mitsunobu conditions. The properties of eth-
oxyethylidene protection of aminooxy group on the con-
trary to that of N-alkoxyphthalimide group allow to
perform a wide spectra of the transformations in the radical
of N-protected hydroxylamine derivatives. This is essential
for synthetic strategies consisting in the introduction of
N-protected aminooxy group at one of the first steps of
synthesis and subsequent transformations of the radical.
The inhibitory effect of one of the newly synthesized
compound, 1-guanidinooxy-3-aminopropane (GAPA), was
compared with that of well-known inhibitors of ornithine
decarboxylase namely, a-difluoromethylornithine (DFMO)
and 1-aminooxy-3-aminopropane (APA) on Leishmania
donovani, a protozoan parasite that causes visceral
leishmaniasis. GAPA, on the contrary with APA and
DFMO, in micromolar concentrations, inhibited the growth
of both amastigotes and promastigotes of sodium antimony
gluconate-resistant forms of L. donovani.
Keywords Hydroxylamines ꢀ Synthesis ꢀ
Enzyme inhibitors ꢀ Leishmaniasis ꢀ Polyamines
Abbreviations
AdoMet S-Adenosylmethionine
Agm
APA
DFMO
GAPA
Put
Agmatine (1-guanidino-4-aminobutane)
1-Aminooxy-3-aminopropane
a-Difluoromethylornithine
1-Guanidinooxy-3-aminopropane
Putrescine (1,4-diaminobutane)
Spermidine (1,8-diamino-4-azaoctane)
Sodium stibogluconate
Spd
SAG
Introduction
M. A. Khomutov, S. Mandal, and J. Weisell equally contributed to
this work.
During last decade, O-substituted hydroxylamines have
become increasingly important in enzymology and bioor-
ganic chemistry. One of the main reasons of this is well-
known spontaneous condensation of O-alkyl hydroxylamines
with aldehydes and ketones taking place in water solution
at physiological pH values and yielding stable oximes
with near quantitative yields. This was the reason: (i) to use
substrate-like O-substituted hydroxylamines as effective
inhibitors of carbonyl-dependent enzymes of amino acids
metabolism. The specificity and efficiency of the inhibition
is well interpreted in terms of structural similarity of the
oxime of the enzyme with the external aldimine—the first
M. A. Khomutov (&) ꢀ A. R. Simonian ꢀ S. N. Kochetkov
Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, Moscow, Russia
e-mail: hommaximus@mail.ru
S. Mandal ꢀ N. Saxena ꢀ R. Madhubala
School of Life Sciences, Jawaharlal Nehru University,
New Delhi, India
J. Weisell ꢀ J. Vepsalainen
Department of Biosciences, Biocenter Kuopio,
University of Kuopio, Kuopio, Finland
123

510
M. A. Khomutov et al.
intermediate of these types of enzymatic transformations
(Markovic-Housley et al. 1996; Capitani et al. 2003;
Liu et al. 2004). Hydroxylamine-containing analogs of
S-adenosylmethionine (AdoMet) and putrescine (1,4-diam-
inobutane) (Put) are nanmolar inhibitors of decarboxylases
of AdoMet and Orn (Wallace and Fraser 2004; Khomutov
2002) and in vitro showed antitrypanosomal (Guo et al.
1995), antimalarial (Das Gupta et al. 2005), and antile-
ishmanial activity (Heby et al. 2007). (ii) To use the
reaction of the oxime formation, which occurs with almost
complete functional group compatibility, as the key step of
a convenient chemoselective ligation strategy to prepare
different biologically important conjugates and semisyn-
thetic biopolymers using hydroxylamine-containing pep-
tides and oligonucleotides as building blocks (Peri et al.
1998; Chen et al. 2003; Salo et al. 1999). (iii) To use
hydroxylamine-containing resins for the isolation of
oligosaccharides (Shimaoka et al. 2007 and ref. within),
and even to prepare surfaces being coated with nanolayers
(Lee et al. 2004).
Here, we report that the alkylation of ethyl N-hydroxy-
acetimidate with methanesulfonates of functionally
substituted alcohols is an attractive alternative to both of
the above methods and results in required O-substituted
hydroxylamines in good or excellent yield. 1-Guanidi-
nooxy-3-aminopropane (GAPA), obtained by this method,
actively penetrated inside L. donovani and was very
effective toward sodium stibogluconate (SAG) resistant
forms of the parasite.
Materials and methods
Chemistry
Melting points were determined in open capillary tubes
with a ‘‘Manual Mel-Temp’’ apparatus (Barnstead Inter-
1
national) and are uncorrected. H and 13C NMR spectra
were recorded on Bruker Avance 500 DRX spectrometer
using tetramethylsilane (TMS) in CDCl3 or sodium tri-
methylsilylpropanesulfonate (TSP) in D2O as internal
standards. Chemical shifts are given in ppm, and J values
are given in Hz. TLC was carried out on precoated Kie-
selgel 60 F254 plates, elution systems are indicated in the
text. All reagents and solvents were used as received from
manufacturers unless otherwise specified. 1-Butyl meth-
anesulfonate (1a), 2-propyl methanesulfonate (1b), and
cyclohexyl methanesulfonate (1c) were prepared by known
method (Truce and Christensen 1968) and the rest of
methanesulfonates (1d–1g) were prepared following the
same method (Truce and Christensen 1968) and had the
following characteristics:
To solve the above and related applications, a lot of
different O-substituted hydroxylamines are required,
including compounds with different functional groups in
the alkyl substituent. Classic methods of the preparation
of the hydroxylamine esters start from corresponding
alkyl halogenides, which react with a proper N-protected
hydroxylamine derivative that after the removal of
N-protection group results in required O-substituted
hydroxylamines (Zeeh and Metzger 1971). However, a
family of functionally substituted alcohols is much
diverse, as compared with corresponding alkyl halides.
The only preparative method to synthesize functionally
substituted esters of hydroxylamines starting from alco-
hols is to use N-hydroxyphthalimide in Mitsunobu reac-
tion (Hughes 1992; Maillard et al. 2005). This allows
preparing different O-substituted hydroxylamines,
including nucleoside (Salo et al.1999), and carbohydrate
(Rodriguez et al. 1998) derivatives, and also hydroxyl-
amines bearing different reporter groups (Muddana
and Peterson 2004). Unfortunately, the properties of
N-alkoxyphthalimide group make it impossible to per-
form some of commonly used reactions of nucleophilic
substitution to introduce functional group(s) in the side-
chain of N-protected hydroxylamine ester. This makes
phthaloxime protection useless for many of synthetic
schemes, which involve introduction of N-hydroxyph-
thalimide at one of the first steps and subsequent ‘‘func-
tionalization of the radical.’’ Recently, an alkylation of
N-Boc-hydroxylamine with alkyl methanesulfonates of
alcohols was published. In the case of nonsubstituted
alcohols, the target hydroxylamines were prepared in
good or moderate yields (Albrecht et al. 2006).
(1d)—Yield 77%; mp 54–55°C (i-PrOH); Rf 0.41
1
(CHCl3–MeOH, 100:1). H NMR (CDCl3): d 7.35–7.26
(5H, m); 5.09 (2H, s); 5.00 (1H, s); 4.27 (2H, t, J 5.8);
3.35–3.30 (2H, m); 2.97 (3H, s); 1.98–1.91 (2H, m).
13C NMR (CDCl3): d 156.49, 136.45, 128.55, 128.19,
128.11, 67.26, 66.78, 37.36, 37.21, 29.52. Calculated, %:
C 50.16; H 5.96; N 4.87. C12H17NO7S. Found, %: C 50.09;
H 5.98; N 4.91.
(1e)—yield quant.; Rf 0.23 (CHCl3); 1H NMR (CDCl3):
d 4.23 (2H, t, J 6.5); 4.00 (2H, q, J 7.1); 3.88 (2H, t, J 6.5);
3.00 (3H, s); 1.92 (3H, s); 1.80–1.74 (2H, m); 1.67–1.61
(2H, m); 1.48–1.38 (4H, m); 1.27 (3H, t, J 7.1). 13C NMR
(CDCl3): d 162.19, 73.25, 70.04, 62.12, 37.39, 29.12,
28.69, 25.59, 25.34, 14.41, 13.62. Was used without
isolation to prepare (3e).
(1f)—Yield 72.5%; mp 82.5–83°C (EtOAc–hexane), Rf
0.60 (CHCl3–MeOH, 98:2). 1H NMR (CDCl3): d 4.37 (1H,
bs); 4.28 (2H, t, J 6.1); 3.81 (1H, bs); 3.01 (3H, s); 1.95–
1.72 (2H, m); 1.44 (9H, s); 1.20 (3H, d, J 6.7). 13C NMR
(CDCl3): d 155.38, 79.49, 67.29, 43.55, 37.34, 36.57,
123

O-Substituted hydroxylamines from alcohols
511
28.38, 21.38. Calculated, %: C 44.93; H 7.92; N 5.24.
C10H21NO5S. Found, %: C 44.98; H 7.98; N 5.17.
(1g)—Yield 91.1%; bp 125–126°C/0.7 mmHg. 1H NMR
(CDCl3): d 4.22 (2H, t, J 6.6); 3.53 (2H, t, J 6.5); 3.00 (3H, s);
1.81–1.75 (4H, m); 1.52–1.39 (4H, m). 13C NMR (CDCl3):
d 69.84, 44.82, 37.39, 32.31, 29.00, 26.27, 24.79. Found,
%: C 39.25; H 6.97. C7H15ClO3S. Calculated, %: C 39.16;
H 7.04.
J 7.2); 3.86 (4H, t, J 6.6); 1.91 (6H, s); 1.65–1.59 (4H, m);
1.39–1.36 (4H, m); 1.25 (6H, t, J 7.2). 13C NMR (CDCl3):
d 162.13, 73.54, 62.09, 28.09, 26.00, 14.41, 13.60. Calcu-
lated, %: C 58.31; H 9.79; N 9.71. C14H28N2O4. Found, %:
C 58.54; H 9.80; N 9.73.
(2f)—Reaction time 48 h; yield 68.6%; self-crystallized
1
oil, solid has mp 38°C. Rf 0.67 (CHCl3–MeOH, 98:2); H
NMR (CDCl3): d 4.63 (1H, s); 4.03–3.93 (4H, m); 3.78
(1H, bs); 1.94 (3H, s); 1.84–1.70 (2H, m); 1.44 (9H, s);
1.26 (3H, t, J 7.1); 1.17 (3H, d, J 6.6). 13C NMR (CDCl3):
d 162.36, 155.34, 78.88, 70.63, 62.20, 44.70, 35.73, 28.44,
21.18, 14.39, 13.73. Calculated, %: C 56.91; H 9.55; N
10.21. C13H26N2O4. Found, %: C 57.06; H 9.69; N 10.05.
(2g)—Reaction time 96 h; yield 82%; bp 72–73°C/
General method of ethyl N-hydroxyacetimidate
alkylation with alkyl methanesulfonates
To a stirred 2 M solution of sodium salt of ethyl
N-hydroxyacetimidate (ethyl N-hydroxyacetimidate from
‘‘Aldrich’’ was distilled before use, bp 62°C/12 mmHg) in
MeOH or i-PrOH, an equivalent of the corresponding alkyl
methanesulfonate was added slowly in small portions and
stirring was continued at 20°C until the neutral pH of the
reaction mixture. Precipitate was filtered off, filtrate was
evaporated to dryness in vacuo, and the residue was poured
into water and extracted with CHCl3 (Et2O was used in the
case of 2a and 2b). Combined organic extracts were
washed with 1 M NaHCO3, H2O, brine, dried (MgSO4),
and evaporated to dryness in vacuo, affording ethoxye-
thylidene derivatives (2a)–(2g):
1
0.7 mmHg; n2D0 1.4475. H NMR (CDCl3): 4.00 (2H, q,
J 7.2); 3.87 (2H, t, J 6.5); 3.52 (2H, t, J 6.9); 1.91 (3H, s);
1.81–1.74 (2H, m); 1.66–1.59 (2H, m); 1.50–1.34 (4H, m);
1.26 (3H, t, J 7.2).13C NMR (CDCl3): d 162.16, 73.35,
62.11, 45.03, 32.61, 28.76, 26.78, 25.45, 14.41, 13.61.
Calculated, %: C 54.17; H 9.09; N 6.32. C10H20ClNO2.
Found, %: C 54.28; H 9.11; N 6.18.
General procedure for the removal of ethoxyethylidene
protection of aminooxy group
(2a)—Reaction time 16 h; yield 78%; bp 91–91.5°C/
47 mmHg; n2D0 1.4178 (lit. (Nedospasov and Khomutov
1976): bp 64°C/14 mmHg, n1D6 1.4216). 1H NMR (CDCl3):
d 3.99 (2H, q, J 6.9); 3.86 (2H, t, J 6.5); 1.90 (3H, s); 1.62–
1.55 (2H, m); 1.41–1.31 (2H, m); 1.25 (3H, t, J 6.9); 0.91
(3H, t).
To the solution of 1 equiv. of ethyl N-alkoxyacetimidate’s
(2a)–(2e), and (2g) in i-PrOH was added 2.5 equiv. of 37%
aq. HCl and after 3–5 min at 20°C, the reaction mixture
was evaporated to dryness in vacuo. The residue was
co-evaporated twice with abs. i-PrOH and crystallized from
i-PrOH–Et2O that after drying in vacuo over P2O5/KOH
yielded compounds (3a)–(3e), and (3g):
(2b)—Reaction time 36 h; yield 65%; bp 74–74.5°C/
72 mmHg; n2D0 1.4096. 1H NMR (CDCl3): d 4.13–4.09 (1H,
m); 4.01 (2H, q, J 7.1); 1.90 (3H, s); 1.27 (3H, t, J 7.1);
1.12 (6H, d, J 6.2). 13C NMR (CDCl3): d 161.71, 74.45, 61.97,
21.51, 14.44, 13.66. Calculated, %: C 57.90; H 10.41; N 9.65.
C7H15NO2. Found, %: C 58.18; H 10.45; N 9.55.
(3a)—Yield 97%; mp 157°C (lit. Theilacker and Ebke
1956): mp 159–160°C).
(3b)—Yield 87%; mp 85–86°C (lit. Theilacker and
Ebke 1956): mp 84.8°C).
(3c)—Yield 86%; mp 184–185°C, dec. (lit. Albrecht et al.
2006): mp 210–220°C, subl. at 176–178°C; (lit. Theilacker
(2c)—Reaction time 7 days; yield 57.6%; bp 94–96°C/
1
1
12 mmHg; n2D0 1.4512. H NMR (CDCl3): d 4.01 (2H, q,
and Ebke 1956): mp 175°C. H NMR (D2O): d 4.09–4.00
J 7.1); 3.86–3.80 (1H, m); 1.93 (3H, s); 1.91–1.89 (2H, m);
1.72–1.68 (2H, m); 1.54–1.49 (1H, m); 1.41–1.21 (5H, m);
1.26 (3H, t, J 7.1). 13C NMR (CDCl3): d 161.89, 79.77,
61.99, 31.62, 25.98, 23.90, 14.46, 13.67. Calculated, %: C
64.83; H 10.34; N 7.56. C10H19NO2. Found, %: C 64.88;
H 10.44; N 7.62.
(1H, m), 1.98–1.89 (2H, m), 1.75–1.66 (2H, m), 1.53–1.45
(1H, m), 1.43–1.14 (5H, m). 13C NMR (D2O): d 86.13,
32.37, 27.25, 25.52. Calculated, %: C 47.53; H 9.31; N 9.24.
C6H14ClNO. Found, %: C 47.38; H 9.49; N 9.17.
(3d)—Yield 91%; mp 188–189°C (lit. Lee et al. 1995):
mp 191°C). 1H NMR (D2O): 7.46–7.42 (5H, m); 5.11
(2H, s); 4.11 (2H, t, J 6.1); 3.23 (2H, t, J 6.5); 1.89–1.84
(2H, m). 13C NMR (CDCl3): d 161.30, 139.34, 131.65,
131.24, 130.53, 75.66, 69.76, 39.67, 30.26.
(2d)—Reaction time 12 h; yield quant.; Rf 0.78 (CHCl3–
1
MeOH, 100:1); H NMR (CDCl3): d 7.37–7.27 (5H, m);
5.09 (3H, m); 3.99 (2H, q, J 7.2); 3.96 (2H, t, J 6.2); 3.35–
3.27 (2H, m); 1.90 (3H, s); 1.88–1.79 (2H, m); 1.25 (3H, t,
J 7.2). (2d) was used without isolation for the preparation
of (3d).
1
(3e)—Yield 93%; mp 227°C, dec. (MeOH–i-PrOH). H
NMR (D2O): d 4.02 (4H, t, J 6.4), 1.69–1.56 (4H, m),
1.41–1.30 (4H, m). 13C NMR (D2O): d 78.22, 29.42, 27.05.
Calculated, %: C 32.59; H; 8.20; N 12.67. C6H18Cl2N2O2.
Found, %: C 32.72; H 8.31; N 12.54.
(2e)—Reaction time 96 h; yield 70.5%; bp 121–122°C/
1
1 mmHg; n2D0 1.4480. H NMR (CDCl3): d 3.99 (4H, q,
123

512
M. A. Khomutov et al.
1
(3g)—Yield 87%; mp 127–128°C. 1H NMR (D2O):
(CHCl3–MeOH, 4:1). H NMR (CDCl3): d 9.00 (1H, s);
7.70 (1H, s); 7.34–7.26 (5H, m); 5.59 (1H, bs); 5.07 (2H,
s); 4.07 (2H, t, J 5.6); 3.31–-3.25 (2H, m); 1.87–1.80 (2H,
m); 1.46 (9H, s); 1.44 (9H, s). 13C NMR (CDCl3):
d 156.59, 151.85, 149.72, 140.85, 136.92, 128.33, 127.76,
83.26, 81.37, 71.13, 66.25, 37.93, 29.61, 28.09, 28.00.
d 4.05 (2H, t, J 6.5), 3.60 (2H, t, J 6.7), 1.79–1.71 (2H, m),
1.71–1.63 (2H, m), 1.48–1.34 (4H, m). 13C NMR (D2O):
d 78.24, 48.36, 34.33, 29.47, 28.35, 26.73. Calculated, %:
C 38.31; H; 8.04; N 7.45. C6H15Cl2NO. Found, %:
C 38.16; H 8.13; N 7.31.
1-Aminooxy-3-[N-(tert-butyloxycarbonyl)]
aminobutane (3f0)
3-Guanidinooxy-1-aminopropane dihydrobromide
(GAPA) (5)
To the solution of (2f) (0.85 g, 3.1 mmol) in MeOH (6 ml)
was added with stirring 0.5 M H2SO4 (4.65 ml) at 20°C
and after 5 min at 20°C the reaction mixture was cooled to
?4°C and pooled into cooled (?4°C) mixture of MeOH
(1 ml) and 25% NH4OH (1 ml). The resulted solution was
evaporated practically to dryness in vacuo, diluted with
2 M Na2CO3, extracted with CHCl3 (3 9 2 ml), washed
with H2O, brine and dried (MgSO4). The solvent was
removed in vacuo, the residue dried over P2O5 at 1 mmHg
that afforded 0.6 g (95%) of compound (3f00) as a vaxi oil,
which slowly solidified on standing. Rf 0.23 (CHCl3–
MeOH, 98:2); 1H NMR (CDCl3): d 5.40 (2H, s); 4.55 (1H,
bs); 3.79–3.69 (2H ? 1H, m); 1.76–1.65 (2H, m); 1.44
(9H, s); 1.15 (3H, d, J 6.6). 13C NMR (CDCl3): d 155.38,
79.02, 72.95, 44.24, 35.62, 28.45, 21.16. Calculated, %:
C 52.92; H 9.87; N 13.71. C9H20N2O3. Found, %: C 52.79;
H 9.91; N 13.62.
To a solution of (4) (0.49 g, 1.1 mmol) in glacial AcOH
(5 ml) a 32% HBr/AcOH (3 ml) was added and after 2 h at
20°C to the reaction mixture was added AcOH–Et2O, 1:1
(8 ml), the precipitated material was filtered off, washed
with Et2O and dried in vacuo over KOH. Recrystallisation
from abs. EtOH gave 0.26 g (70%) of the compound (5). Rf
0.25 (dioxane—25% NH4OH, 7:3); mp 172–173°C, dec.
1H NMR (D2O): d 4.06 (2H, t, J 6.2); 3.14 (2H, t, J 7.5);
2.15–2.00 (2H, m). 13C NMR (D2O): d 161.54, 77.07,
39.67, 28.08. Calculated, %: C 16.34, H 4.80, N 19.06.
C4H14Br2N4O. Found, %: C 16.48, H 4.77, N 19.20.
Biochemistry
Growth media and antibiotics were purchased from Sigma
(St. Louis, MO) and fetal bovine serum (FBS) from Gibco/
BRL (Life Technologies Scotland, United Kingdom).
1-Aminooxy-3-aminobutane dihydrochloride (3f00)
Parasite and culture condition
A solution of (2f) (90 mg, 0.3 mmol) in EtOH (2 ml) was
added 37% HCl (0.35 ml) and after 16 h at 20°C the
reaction mixture was evaporated to dryness in vacuo and
the residue was dried over P2O5/KOH in vacuo. Resulted
solid was treated with abs. EtOH, filtered off and dried over
P2O5 in vacuo to afford 45 mg (85%) of target (3f), mp
Promastigotes of Indian Leishmania donovani clone
GE1 (MHOM/IN/80/GE1F8R) (Bhattacharyya et al. 2001),
L. donovani strain MHOM/IN/80/AG83 and three untyped
clonal strains S-1, R-1, and R-2 were isolated from patients
with visceral leishmaniasis and were routinely cultured at
22°C in M199 medium with Hanks’ salts including 25 mM
HEPES buffer (Sigma, USA) supplemented with 10% heat
inactivated FBS and 0.13 mg/ml penicillin and strepto-
mycin. Clinical isolate S-1 was sensitive to sodium anti-
mony gluconate (SAG), whereas the three SAG-resistant
isolates were GE1-R, R-1, and R-2. The SAG-resistant
isolates were maintained in the absence of drug pressure in
vitro (Singh et al. 2007).
¨
157–159°C (lit. (Keinanen et al. 1994): mp 156–158°C); Rf
0.12 (dioxane—25% NH4OH, 9:1). 1H NMR (D2O): d
4.26–4.18 (2H, m); 3.57–3.53 (1H, m); 2.14–2.07 (2H, m);
1.35 (3H, d, J 6.5). 13C NMR (CDCl3): d 74.61, 48.41,
34.96, 20.43.
3-[N,N0-di-(tert-Butyloxycarbonyl)]guanidinooxy-1-
(benzyloxycarbonyl)aminopropane (4)
Drug susceptibility assay
A solution of (3d) (0.32 g, 1.4 mmol), N,N0-di-Boc-N00-
triflylguanidine (0.49 g, 1.26 mmol) and Et3N (0.2 ml,
1.4 mmol) in dry CHCl3 (6 ml) was incubated for 24 h at
37°C. Reaction mixture was diluted with CHCl3 (6 ml),
washed with 10% citric acid, H2O, 1 M NaHCO3, H2O,
brine and dried (MgSO4). The solvent was removed in
vacuo, the residue dried over P2O5 at 1 mmHg that affor-
ded 0.49 g (88%) of compound (4) as a vaxi oil. Rf 0.52
The effect of GAPA on the growth of the promastigotes
was determined in 96- wells microtiter plates. Briefly,
1 9 106 parasites in 0.2 ml of modified M199 medium
with 10% FBS were placed in each well and incubated with
various concentrations of the drug. Two wells in which
cells were permitted to grow in the absence of drug were
maintained in parallel as controls. After 72 h of incubation
123

O-Substituted hydroxylamines from alcohols
513
under normal growth condition, cell densities were deter-
mined by the Neubeur heamocytometer. The concentra-
tions of GAPA, which inhibited the growth of the cells by
50%, were determined. Two or more independent experi-
ments in triplicate were performed for the determination of
sensitivity to each drug.
nucleophilic substitution in N-alkoxy group and besides it
is resistant toward some oxidizing and reducing agents
(Khomutov et al. 1996 and ref. within), while phthaloxime
group, which is typically removed by hydrazine, or even
aliphatic amine treatment (Maillard et al. 2005), is not
stable even under standard conditions of preparation of
thiols from alkyl bromides (Bauer and Suresh 1963). The
main advantage of ethoxyethylidene protection group is
extremely mild conditions of its removal—normally
1 equiv. of strong acid and 1 equiv. of water within couple
of min at 20°C is enough to get required O-substituted
hydroxylamine with practically quantitative yield. The
removal conditions are so mild that it is possible to
deprotect aminooxy group in the presence of N-Boc-amino
group (Simonian et al. 2006). All above make ethyl
N-hydroxyacetimidate the best N-protected hydroxylamine
derivative, in the case synthetic strategy consists in the
introduction of the protected aminooxy group at one of the
first steps of synthesis and subsequent ‘‘functionalization’’
of the radical. However, the alkylation of ethyl N-hy-
droxyacetimidate with easily available methanesulfonates
of functionally substituted alcohols, which are more
available as compared with alkyl halogenides, remains
unknown.
DNA constructs and transfection
An episomal Leishmania expression vector (pGL-
aNEOaLUC) containing luciferase encoding DNA and
neomycin phosphotransferase selectable marker (Roy et al.
2000) was used in the present study. Twenty micrograms
of the construct was transfected into promastigotes of
L. donovani field isolates by electroporation in 2 mm gap
cuvettes, at 450 V, 500 lF (BTX Electro Cell Manipulator
600). Transfectants were selected for resistance to G418
(50 lg/ml) as described earlier (Papadopoulou et al. 1992).
Macrophage infection and intracellular amastigote drug
susceptibility assay
Stationary phase Leishmania promastigotes expressing the
luciferase gene (pGL-aNEOaLUC) were used to infect
J774A.1 macrophages. Macrophage cell line J774A.1
(American Type Culture Collection) was maintained at
37°C in RPMI-1640 medium (Sigma) containing 10% heat
inactivated FBS as described previously (Kapoor et al.
2001). Briefly, J774A.1 murine macrophages (1 9
105 cells/250 ll/per well) were infected with 1 9 106
promastigotes in M199 media with 10% FBS (Roy et al.
2000). After 3 h, the non-internalized parasites were
removed by washing and drug was added at different
concentrations. After 5 days of drug exposure, plates
containing adherent macrophages were washed and lucif-
erase activity was determined (Roy et al. 2000). The 50%
inhibitory concentration (IC50) was determined from the
graph representing different concentrations of drug plotted
against relative light units (RLU) produced by luciferase
expressing parasites.
To begin the study we compared the rates of the
alkylation of Na-salt of ethyl N-hydroxyacetimidate with
1-butyl- and 2-propyl methanesulfonates with that of
1-bromobutane. The reaction of 1-butyl methanesulfonate
(1a) with Na-salt of ethyl N-hydroxyacetimidate (2 M final
concentration of each) in MeOH was slightly exothermic
and practically completed within 5–6 h, while after 16 h
the pH of reaction mixture became neutral (Scheme 1).
2-Propyl methanesulfonate (1b) was less reactive under the
above conditions and after 12 h at 20°C, the reaction
mixture was still basic, while after 36 h the reaction
completed. The yields of pure (2a) and (2b) were only 78
and 65%, respectively. In the first case, it was decreased
due to enough laborious separation of (2a) from the traces
of nonreacted ethyl N-hydroxyacetamidate having close bp,
while (2b) turned to be enough volative like methyl and
ethyl esters of ethyl N-hydroxyacetamidate. The reaction
mixture containing 2 M of 1-bromobutane and 2 M of
Na-salt of ethyl N-hydroxyacetamidate in MeOH remained
alkaline even after 96 h at 20°C and heating was required
to get (2a) in a good yield. Hence, even 2-propyl meth-
anesulfonate turned to be more reactive toward ethyl
N-hydroxyacetamidate as compared with that of 1-bromo-
butane. As expected, cyclohexyl methanesulfonate (1c)
was much less reactive as compared with (1b). In this case,
the yield of (2c) was only 58% after 7 days at 20°C, but it
was much better than 36%, being obtained with N-Boc-
hydroxylamine and (1c) under the conditions outlined in
(Albrecht et al. 2006). One of the main advantages of
Results and discussion
Chemistry
Ethyl N-hydroxyacetimidate is not so widely used to pre-
pare O-substituted hydroxylamines as N-hydroxyphthali-
mide. However, ethyl N-hydroxyacetimidate easily and
with high yields adds to activated double bond according to
Michael and easily alkylated with alkyl halogenides
(Khomutov et al. 1996 and ref. within). Ethoxyethylidene
protection group is complementary to a wide set of
123

514
M. A. Khomutov et al.
CH3
N
i
ii
O
O
iii
.
HCl
NH2
R-OH
R-OMs
C2H5O
R
R'
(3a)-(3g)
(1a)-(1g)
(2a)-(2g)
R' = a: CH (CH ) CH -; b: (CH ) CH-;
R = a: CH (CH ) CH -; b: (CH ) CH-;
3
2 2
2
3 2
3
2 2
2
3 2
c: Cyclohexyl; d: CbzNHCH CH CH -;
c: Cyclohexyl; d: CbzNHCH CH CH -;
2
2
2
2
2
2
e: HCl*H NO(CH ) CH -;
e: C H OC(CH )=NO(CH ) CH -;
2
2 5
2
2
5
3
2 5
2
f': BocNHCH(CH )CH CH -;
f: BocNHCH(CH )CH CH -;
3
2
2
3
2
2
f'': HCl*H NCH(CH )CH CH -;
g: Cl(CH ) CH -
2
3
2
2
2 5
2
g: Cl(CH ) CH -
2 5
2
Scheme 1 Synthesis of O-substituted hydroxylamines by the alkyl-
ation of ethyl N-hydroxyacetimidate with alkyl methanesulfonates
and subsequent deprotection of aminooxy group. i—MsCl/C6H6/
Et3N; ii—C2H5OC(CH3) = NONa/i-PrOH, or MeOH/20°C; iii—
HCl/H2O for 3a-3e, 3f00, and 3g; 0.75 mol. equiv. H2SO4/H2O/MeOH
for 3f0
ethoxyethylidene protection group is the possibility to
remove it under mild conditions with practically quantita-
tive yield. This made possible to obtain the required (3a)–
(3c) with the overall yields of 76, 57, and 50%, as calcu-
lated for ethyl N-hydroxyacetimidate, that is much better as
compared with the overall yields of 64, 40, and 20%, for
the same (3a)–(3c) being prepared from N-Boc-hydroxyl-
amine and mesylates (1a)–(1c) under earlier published
protocol (Albrecht et al. 2006).
H
H
N
i
ii
Cbz
NH2
HCl
Cbz
N
*
N
O
N
H
O
Boc
H
(4)
(3d)
N
Boc
H
N
NH2
2HBr
*
H2N
O
NH
GAPA (5)
Scheme 2 Synthesis of 1-guanidinooxy-3-aminopropane (GAPA).
i—Tf-Boc2-Guanidine/Et3N/CH2Cl2; ii—HBr/AcOH
First functionally substituted alcohols of this study were
6-chloro-1-hexanol and 6-(1-ethyxyethylideneamino-oxy)-
1-hexanol, the last was prepared by alkylation of Na-salt of
ethyl N-hydroxyacetimidate with 6-chloro-1-hexanol in
boiling alcohol. Corresponding mesylates (1 g) and (1e),
the last was used without isolation, smoothly alkylated
Na-salt of ethyl N-hydroxyacetimidate in MeOH and
required ethoxyethylidene derivatives (2g) and (2e) were
isolated by distillation with the yields of 82 and 71%,
respectively (Scheme 1). Mild acidic hydrolysis resulted in
dihydrochloride (3g) and hydrochloride (3e) with the yields
of 93 and 87%, respectively. The conditions of the removal
of the ethoxyethylidene protection of the aminooxy group
are so that, that it turned possible to deprotect aminooxy
group of (2f) selectively using slight excess of H2SO4 in
water–MeOH mixture and (3f0) was finally obtained as a
base with 95% yield. Simutaneous removal of ethoxye-
thylidene and N-Boc protections gave dihydrochloride
(3f00) in 85% yield (Scheme 1).
afforded target (3d) with the yield of 91% (two steps). Thus,
obtained 1-aminooxy-3-[N-(benzyloxycarbonyl)amino]pro-
pane was reacted (Scheme 2) with an equivalent of N,N0-di-
Boc-N00-triflylguanidine at 37°C for 24 h, that gave protected
GAPA (4) with practically quantitative yield. Subsequent
removal of N-Boc- and N-Cbz-groups with HBr/AcOH
afforded target GAPA (5) with the yield of 61% (two steps).
Above data clearly proves that the alkylation of ethyl
N-hydroxyacetimidate with alkyl methanesulfonates is a
convenient approach to prepare functionally substituted
esters of hydroxylamine with excellent or high yields.
Biochemistry
A protozoan parasite L. donovani is the causative agent of
visceral leishmaniasis. The pentavalent antimonials SAG
(Fig. 1) and meglumine antimonate, the first line of drugs
for visceral and cutaneous leishmaniasis and resistance to
these drug is one of the major problem in the field. This
increase in resistance has led to the need for more selective
and efficacious drugs. As polyamine biosynthetic pathway
is essential for growth and survival of L. donovani, inhi-
bition of this pathway is an important leishmanicidal
strategy (Heby et al. 2007). a-Difluoromethylornithine
(DFMO), an irreversible inhibitor of ODC, is used to treat
late stage of African sleeping sickness caused by the pro-
tozoan parasite Trypanosoma brucei gambiense (Burri and
Brun 2003), and it is effective against other genera of
protozoan parasites including Plasmodium species (Bitonti
et al. 1989), Giardia (Gillin et al. 1984), and Leishmania
N-(Benzyloxycarbonyl)-3-amino-1-propanol was selec-
ted as another example of functionally substituted alcohol
(Scheme 1). Corresponding methanesulfonate (1d) was
prepared with an excellent yield and the alkylation was
performed by adding small portions of solid (1d) to a
stirred 2 M solution of Na-salt of ethyl N-hydroxyacetim-
idate in i-PrOH at 20°C. The reaction practically completed
after 4–5 h, while after 12 h starting (1d) could not be
detected by TLC. Intermediate ethoxyethylidene derivative
(2d) was without isolation treated with 2–3 equiv. of 37%
aq. HCl for 3–5 min at 20°C, that after recrystallisation
123

O-Substituted hydroxylamines from alcohols
515
Fig. 1 Structures of
1-guanidinooxy-3-
aminopropane (GAPA),
1-aminooxy-3-aminopropane
(APA), and sodium
NH
O
HOCH2-(CHOH)2-CHO
OCH-(CHOH)2-CH2OH
-
O
NH2
NH2
GAPA
APA
Sb
H2N
N
-
HOC-COO
2Na+
H
OOC-CHO
O
H2N
Sodium antimony gluconate (SAG)
stibogluconate (SAG)
Table 1 Comparative analysis of the effect of sodium antimony gluconate (SAG), APA, and GAPA on the SAG-sensitive and SAG-resistant
clinical isolates
Isolates
IC50 (lM)
Promastigotes
SAGa
Intracellular amastigotes
SAGa
APAb
GAPAc
APAb
GAPAd
WT
S-1
R-1
R-2
GE1
a
57 5.0
42 8.0
19 0.7
[400
36 7.0
10 4.2
15.0 6.0
5
2.0
9
9
1.0
0.7
47 5.6 (1)
128 3.3 (2.7)
68 4.2 (1.4)
164 5.1 (3.4)
13.0 1.5 (1)
62.0 3.4 (5)
23.0 1.4 (1.8)
[95.0 ([ 7.6)
10 2.5
[200
[200
[200
8
1.4
78 1.4
1.4
18 2.0
22 3.0
46 2.8
[400
8
3
0.7
Data for IC50 values for SAG are adopted from (Singh et al. 2007)
Data for IC50 values for APA are adopted from (Singh et al. 2007)
IC50s were determined in promastigotes after 72 h of drug addition
b
c
d
IC50s were determined from the graph representing concentration of drug plotted against RLU produced by luciferase expressing parasites. The
stationary phase L. donovani isolates transfected with pGL-aNeo a Luc were used to infect J774A.1 macrophages as described in ‘‘Materials and
methods’’ section. After 5 days of drug exposure, luciferase activity was determined. IC50 are given as means standard deviations of at least
three independent determinations. Fold change in the IC50 compared to S-1 is given in parenthesis
(Kaur et al. 1986). 1-Aminooxy-3-aminopropane (APA)
(Fig. 1) being another very effective inhibitor of ODC
(KI toward recombinant L. donovani ODC is 1 nM (Singh
et al. 2007) inhibits the growth of amastigotes and prom-
astigotes of wild type L. donovani at 5 and 45 lM,
respectively (Singh et al. 2007). However, both DFMO and
APA passively penetrate inside cells and parasites.
Recently, we made an attempt to transform APA into
actively transported form, converting terminal H2NO-
group into H2NC(=NH)NHO-group (Singh et al. 2008).
Thus, designed GAPA (Fig. 1) is a structural analog of
Agm, but not a Put as parent APA is. Terminal guanidi-
nooxy group of GAPA has the pKa 6.71 that makes GAPA
to be enough protonated at physiological pH in order to
mimic charge distribution of Put. GAPA turned to be not
very effective inhibitor of ODC, having KI of only 60 lM
toward recombinant L. donovani ODC (Singh et al. 2008).
However, in vitro GAPA inhibited the growth of wild type
L. donovani amastigotes and promastigotes at 9 and
36 lM, respectively (Singh et al. 2008), i.e., was as
effective as APA. The relationship between KI of ODC and
activity toward amastigotes may be considered as an
indication on the active transport of GAPA inside the
parasite.
L. donovani and the ability of exogenous Put and Spd to
reverse the growth of the parasite clearly indicate that the
cellular target of GAPA is closely related to metabolism of
polyamines (Singh et al. 2008).
Effect of GAPA on promastigotes and amastigotes
of antimony susceptible/resistant clinical isolates
In the present study the efficacy of GAPA was analyzed on
the promastigotes of field isolates from SAG-responsive
and SAG-unresponsive patients. The IC50 values of the
promastigotes of wild type L. donovani and SAG-sensitive
isolate S-1 to GAPA were 36 7.0 and 10 4.2 lM,
respectively (Table 1). The promastigotes of SAG-unre-
sponsive field isolates R-1, R-2, and GE1 had IC50 values
of 8 1.4, 78 1.4, and 8 1.4 lM, respectively, with
GAPA (Table 1). The sensitivity of these clinical isolates
to GAPA was also tested in intracellular amastigotes
using amastigote-macrophage model. GAPA was shown to
effectively inhibit growth of amastigotes of wild type and
S-1 isolate coming from SAG-responsive patients with an
IC50 value of 9 1.0 and 9 0.7 lM, respectively
(Table 1). The amastigotes of the SAG-resistant isolates
R-1, R-2, and GE1 had IC50s of 18 2.0, 22 3.0, and
Cellular effects of GAPA, i.e., the decrease of Put
and spermidine (1,8-diamino-4-azaoctane) (Spd) levels in
3 0.7 lM, respectively, when treated with GAPA
(Table 1). Amastigotes of GE1 strain, which was most
123

516
M. A. Khomutov et al.
Bhattacharyya A, Roy M, Mandal A, Duttagupta S (2001) Effect of
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resistant to SAG, was most sensitive to GAPA when
compared to all the other isolates.
Comparison has also been made between the effect of
GAPA on these SAG-sensitive and SAG-resistant isolates
and the effect of APA. As depicted in Table 1, the con-
centration of APA as high as 200 lM did not inhibit
SAG-resistant amastigotes, R-1, R-2, and GE1, while
among promastigotes only R-2 was sensitive to APA.
ODC activity as well as Put and Spd levels were
reported to be higher in the SAG-resistant isolates com-
pared to the sensitive isolates (Singh et al. 2007). To
show whether GAPA promotes its effect on SAG-sensi-
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concentrations, Put and Spd contents were measured.
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promastigotes of SAG-sensitive and SAG-resistant iso-
lates showed that GAPA (40 lM) inhibited polyamine
levels in both cases. Inhibition of Put was *1.3-, *2.7-,
*2.4-fold, respectively, in R1, R2, and GE1 compared to
the corresponding untreated control values (data not
shown). However, no significant change was observed in
the Spd levels in the resistant isolates when compared to
the corresponding untreated control values (data not
shown).
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Hence, both in wild type and SAG-resistant forms of
L. donovani the cellular target(s) of GAPA are the system
of polyamine metabolism and polyamine-responsible cel-
lular processes. However, the mechanism of GAPA’s
action cannot be described only in terms of intracellular
transformation of the inhibitor into APA, which might take
place in macrophages possessing agmatinase-like activity
(Sastre et al. 1998), because APA is not active against
SAG-resistant forms of L. donovani. It is more likely that
the mechanism has a complex nature and includes the
inhibition of ODC with GAPA ‘‘as such’’; the decrease of
Put level might also contribute into growth inhibition
effect; and finally GAPA itself might target some important
metabolic pathway, being different from the conversion of
ornithine into Put.
¨
¨
¨ ¨
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Acknowledgments This work was supported by the Russian
Foundation for Basic Research (project nos. 09-04-01272, 08-04-
91317, and 08-04-91777), the program Molecular and Cell Biology of
the Presidium of the Russian Academy of Sciences, grant from the
Academy of Finland (project no. 124185), grant from the Council of
Scientific and Industrial Research (CSIR), Government of India, and
joint Indo-Russian DST-RFBR grant (project no. RUSP-883).
Lee DC, Chang BJ, Yu L et al (2004) Polymer cushions function-
alized with lipid molecules. Langmuir 20:11297–11300
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Toney MD (2004) Crystal structures of unbound and amin-
ooxyacetate-bound Escherichia coli c-aminobutyrate amino-
transferase. Biochemistry 43:10896–10905
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