Synthesis of L-indospicine

Article abstract of DOI:10.1081/SCC-120004860

Preparation of L-indospicine in eight steps by a reaction sequence starting with L-lysine.

Full text of DOI:10.1081/SCC-120004860

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SYNTHESIS OF L-INDOSPICINE
Aimee K. Bence a & Peter A. Crooks a
a Department of Pharmaceutical Sciences , College of Pharmacy , University of Kentucky ,
Lexington, KY, 40536-0082, U.S.A.
Published online: 16 Aug 2006.
To cite this article: Aimee K. Bence & Peter A. Crooks (2002) SYNTHESIS OF L-INDOSPICINE, Synthetic Communications:
An International Journal for Rapid Communication of Synthetic Organic Chemistry, 32:13, 2075-2082, DOI: 10.1081/
SCC-120004860
To link to this article: http://dx.doi.org/10.1081/SCC-120004860
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SYNTHETIC COMMUNICATIONS, 32(13), 2075–2082 (2002)
SYNTHESIS OF L-INDOSPICINE
*
Aimee K. Bence and Peter A. Crooks
Department of Pharmaceutical Sciences,
College of Pharmacy, University of Kentucky,
Lexington, KY 40536-0082, USA
ABSTRACT
Preparation of L-indospicine in eight steps by a reaction
sequence starting with L-lysine.
Key Words:
analog
L-indospicine; Non-protein amino acid; Arginine
L-Indospicine [2(S)-2,7-diamino-7-iminohepatanoic acid] (1) is the ami-
dino analog of L-arginine (2), and is found in the seed and leaf of several
tropical legumes in the genus Indigofera, including the creeping indigo plant
(Indigofera spicta Forsk), and Indigofera linnaei[1] (Figure 1). Several studies
have investigated the biological effects of L-indospicine. Exposure to L-indos-
picine resulted in the inhibition of protein synthesis and subsequent inhibition
of DNA synthesis in several different cell systems.[2–5] L-Indospicine also
inhibited the in vitro activity of rat liver arginase, and may be an inhibitor
of both the constitutive form of nitric oxide synthase (NOS) in vascular
endothelium, and the inducible form of NOS in macrophages.[6] Due to the
structural similarity between L-arginine and L-indospicine, L-indospicine
appears to have the capacity to be charged to arginyl-tRNA and to be incor-
porated into nascent protein chains in place of L-arginine.[7] L-Canavanine,
*Corresponding author. E-mail: pcrooks@uky.edu
2075
Copyright & 2002 by Marcel Dekker, Inc.
www.dekker.com

2076
BENCE AND CROOKS
Figure 1.
another L-arginine analog, is currently in development as an anticancer
agent;[8] it is postulated that the antineoplastic properties of L-canavanine
result from its capacity to be charged to arginyl-tRNA and incorporated
into protein in place of L-arginine. Studies indicate that L-indospicine may
be charged to arginyl-tRNA and incorporated into protein in place of
L-arginine more efficiently than L-canavanine.[9] Consequently, the anticancer
properties of L-indospicine are currently under active investigation.[10]
Despite the interesting and promising biological activity of
L-indospicine, research on the compound is limited because of the difficulty in
obtaining sufficient quantities of the pure L-enantiomer by synthetic methods.
Currently, the only detailed literature reports on the total synthesis of indos-
picine are for the synthesis of DL-indospicine;[11,12] L-indospicine can be sub-
sequently separated through acylation and enzymatic resolution.[11] The
overall yield of DL-indospicine obtained by this method was only 2%.
Although the yields of the acylation and enzymatic resolution steps were
not reported, it is likely that the overall yield of the reaction was less than
1%.[11] In 1996, a short note describing a chirally pure synthesis of
L-indospicine and the biological effects of L-indospicine on NOS was pub-
lished by Feldman and Chi.[13] Unfortunately, this synthesis cannot be readily
repeated, since the authors do not provide full experimental details for the
synthesis, or report the spectral data for structure confirmation.
To our knowledge, this is the first report to fully describe a total syn-
thesis of L-indospicine. This synthesis, drawing on the work of Culvenor and
coworkers[12] and Baldwin and colleagues,[14] was developed to facilitate the
characterization of the biological effects of this interesting L-arginine analog.
The synthesis of L-indospicine was achieved from readily available L-lysine
in eight steps via L-aminoadipic acid (Scheme 1). It was necessary to protect
only the ꢀ-amino moiety of L-lysine. Initially, the e-amino was masked by
formation of the benzylidene imine (4), and the ꢀ-amino group was pro-
tected with the benzyloxycarbonyl group. The ꢀ-imine was then selectively
hydrolyzed to yield (5). In a key transformation, the e-amino group of (5)
was converted to a hydroxyl group to form Cbz-protected L-ꢀ-aminoadipic
acid (6). The carboxylic acid moiety was then protected as an ethyl ester (7),
and the e-hydroxyl function was tosylated (8). After displacement of the

SYNTHESIS OF L-INDOSPICINE
2077
Scheme 1. a) 2M LiOH, 4ꢀC, benzaldehyde; b) (i) EtOH/1M NaOH, benzyl chloro-
formate, À20ꢀC; (ii) HCl, 50ꢀC; c) 4M NaOH, 60ꢀC, pH 9.5, sodium nitroprusside;
d) EtOH, H2SO4, reflux; e) (i) p-toluenesulfonyl chloride/pyridine, 4ꢀC, (ii) HCl;
f) EtOH/KCN/H2O, 90ꢀC; g) (i) CH2Cl2, EtOH/HCl (g), (ii) MeOH, NH3 (g),
0ꢀC; h) 6M HCl, rt.
tosyl group with cyanide ion to afford (9), the Pinner synthesis was utilized
to form the amidino analog (10). Finally, 10 was deprotected by hydrolysis
in aqueous hydrochloric acid to yield L-indospicine (1) in 6% overall yield.
EXPERIMENTAL
1H and 13C NMR spectra were determined using a Varian 300 MHz
NMR. All spectra were referenced, and chemical shifts determined using
tetramethylsilane (TMS) as the internal standard. Melting points were deter-
mined on a Fisher–Johns visual melting point apparatus, and are uncorrected.
Infrared spectra (IR) were obtained using a Bio-Rad Excalibur infrared
spectrophotometer. MALDI (matrix-assisted laser desorption ionization)
experiments were carried out on a Kratos KOMPACT SEQ. The mass

2078
BENCE AND CROOKS
spectroscopy (MS) experiments were performed at the University of
Kentucky Mass Spectroscopy Laboratory, Lexington, KY. Amino acid
analysis was done on a Hewlet-Packard 1100 amino acid analyzer fitted
with a Pickering PCX 5200 column. Elemental analyses were conducted
by Atlantic Microlabs (Norcross, GA) using automated combustion tube
analysis. Optical rotation measurements were determined on a Perkin Elmer
241 polarimeter.
2(S)-2-Amino-6-(benzylideneamino)hexanoic Acid (4)
L-Lysine monohydrochloride (3) (0.3 mol) was dissolved in 2 M LiOH
(0.3 mol) and cooled to 4ꢀC. Benzaldehyde (0.31 mol) was slowly added, and
the cooled solution was carefully shaken. The reaction mix was allowed to
stir for two hours at 4ꢀC. After the addition of 250mL of cold ethanol, the
solution was filtered. The resulting white precipitate was suspended in 60mL
of cold ethanol and filtered again. The product was dried in vacuo to yield
0.19 mol (63.3%) of (4); m.p. 187–89ꢀC, lit. (14). Found: 185–186ꢀ; 1H NMR
(300 MHz, D2O) d 3–1.5m (2H, ꢁ-CH2), 1.6–1.8 m (4H, ꢂ, d-CH2), 3.0t (2H,
e-CH2), 3.5m (1H, ꢀ-CH), 3.6m (1H, CH–Ar), 7.6–7.8m (5H, Ar) ppm.
13C NMR (D2O) d 20.7 (ꢁ-CH2) 25.8 (d-CH2), 31.0( ꢂ-CH2), 38.2 (e-CH2),
54.1 (ꢀ-CH), 74.2 (CH–Ar), 127.2 (Ar), 127.9 (Ar), 130.3 (Ar), 133.9 (Ar),
179 (CO) ppm.
2(S)-2-(Benzyloxycarbonylamino)-6-aminohexanoic Acid (5)
N-e-Benzylidene L-lysine (4) (0.316 mol) was dissolved in 600 mL of an
ethanol–1 M NaOH solution (1 : 1). The solution was cooled to ꢁ20ꢀC. Over
the period of one hour, precooled benzyl chloroformate (0.316 mol) was
added alternatively in portions with 1 M NaOH in ethanol (2.5 : 1). The
temperature was allowed to rise to ꢁ5ꢀC, and 50mL of precooled HCl
was added. After stirring for five minutes, the reaction mixture was
warmed to 50ꢀC for five minutes and then concentrated under vacuum.
The pH of the solution was adjusted to 6.2 and the solution was allowed
to stand for 14 h at 4ꢀC. The brown precipitate was separated, and the
remaining solution evaporated. The resulting white precipitate was recrys-
tallized from water to yield 0.275 mol (86.7%) of the product; TLC (silica)
Rf ¼ 0.15 (95% ethanol); 1H NMR (300 MHz, D2O) d 1.4–1.5m (2H,
ꢁ-CH2), 1.6–1.9m (4H, ꢂ, d-CH2), 3.0t (2H, e-CH2), 4.0m (1H, ꢀ-CH), 5.1s
(2H, CH2–Ar), 7.5m (5H, Ar) ppm. 13C NMR (D2O) d 21.8 (ꢁ-CH2), 26.0
(d-CH2), 30.9 (ꢂ-CH2), 39.0( e-CH2), 54.1 (ꢀ-CH), 66.6 (CH2–Ar), 127.4
(Ar), 128.0(Ar), 128.5 (Ar), 136.3 (Ar), 157.6 (NCO), 179.0(CO) ppm.

SYNTHESIS OF L-INDOSPICINE
2079
2(S)-2-(Benzyloxycarbonylamino)-6-hydroxyhexanoic Acid (6)
Compound (5) (25 mmol) was dissolved in 50mL of water, the sol-
ution warmed to 60ꢀC, and 4 M NaOH was added to adjust the pH to 9.5.
Sodium nitroprusside dihydrate (31 mmol) was slowly added with stirring.
The reaction was stirred at 60ꢀC for 6.5 h with periodic additions of NaOH
to maintain the pH at 9.5. The mixture was filtered through celite, and the
filtrate acidified to pH 1 and extracted with ethyl acetate (3 Â 100 mL). The
combined organic extracts were washed with brine, dried over anhydrous
MgSO4, and evaporated, to yield 13 mmol of the crude oil (52.2%). MS:
(m/z) ¼ 304 (M þ Kþ); (m/z) ¼ 320(M þ Naþ); 1H NMR (300 MHz, CDCl3)
d 1.4–1.9m (6H, ꢁ,ꢂ,d-CH2), 3.6t (2H, e-CH2), 4.4m (1H, ꢀ-CH), 5.1s (2H,
CH2–Ar), 5.6d (1H, NH), 6.2–6.5s (1H, OH), 7.4m (5H, Ar) ppm. 13C NMR
(CDCl3) d 22.1 (ꢁ-CH2), 32 (ꢂ-CH2), 32.5 (d-CH2), 53.8 (ꢀ-CH), 61.5
(e-CH2), 67.0(CH 2–Ar), 128.1 (Ar), 128.2 (Ar), 128.5 (Ar), 136.3 (Ar),
155.9 (NCO), 179.5 (CO) ppm.
Ethyl 2(S)-2-(Benzyloxycarbonylamino)-6-hydroxyhexanoate (7)
Crude (6) (11.3 mmol) was dissolved in 70mL of absolute ethanol.
Sulfuric acid (1.4 mL) was added and the solution heated under reflux for
5 h. The mixture was neutralized with 2 M NaOH and the ethanol evaporated
on a rotary evaporator. The resulting oil was taken up into water and
extracted with ethyl acetate (3 Â 50mL). After silica gel column chromatog-
raphy (100 : 1, chloroform : methanol), 7.9 mmol of the product was obtained
(73%). TLC (silica): Rf ¼ 0.21 (80 : 1, chloroform : methanol); 1H NMR
(300 MHz, CDCl3) d 1.2t (3H, CH3), 1.35–1.45m (2H, ꢁ-CH2), 1.5–1.9m
(4H, ꢂ, d-CH2), 2.1s (1H, OH), 3.6t (2H, e-CH2), 4.2q (2H, CH2–O), 4.35m
(1H, ꢀ-CH), 5.1s (2H, CH2–Ar), 5.5d (1H, NH), 7.3m (5H, Ar) ppm.
13C NMR (CDCl3) d 14.2 (CH3), 21.5 (ꢁ-CH2), 32 (ꢂ-CH2), 32.4 (d-CH2),
53.9 (ꢀ-CH), 61.4 (CH2–O), 62.3 (e-CH2), 67.0(CH –Ar), 128.1 (Ar), 128.2
2
(Ar), 128.5 (Ar), 136.3 (Ar), 156 (NCO), 172.6 (CO) ppm.
Ethyl 2(S)-2-(Benzyloxycarbonylamino)-6-tosylhexanoate (8)
A solution of (7) (34.3 mmol) in 25 mL of dry pyridine was cooled to
ꢁ25ꢀC. The temperature was kept below 0ꢀC and p-toluenesulfonyl chloride
(69 mmol) was slowly added. The mixture was stirred for six hours at 4ꢀC
and then acidified to pH 1 with aqueous HCl. The mixture was extracted
with chloroform, dried over anhydrous MgSO4, and purified by silica gel

2080
BENCE AND CROOKS
column chromatography (80: 1 chloroform : methanol). 26.4 mmol (77%
yield) of the product was obtained; MS: (m/z) ¼ 502 (M þ Kþ); (m/z) ¼
486 (M þ Naþ); TLC (silica) Rf ¼ 0.18 (200 : 1, chloroform : methanol); IR
1
(CHCl3): 3431, 3020, 1718, 1509 cmꢁ1; H NMR (300 MHz, CDCl3) d 1.2t
(3H, CH3), 1.3 (2H, ꢁ-CH2), 1.5–1.8m (4H, ꢂ, d-CH2), 2.4s (3H, CH3–Ar),
3.9t (2H, e-CH2), 4.1q (2H, CH2–O), 4.2m (1H, ꢀ-CH), 5.1s (2H, CH2–Ar),
5.2d (1H, NH), 7.3m (5H, Ar), 7.7m (5H, Ar) ppm. 13C NMR (CDCl3) d
14.1 (CH3), 21.1 (ꢁ-CH2), 21.6 (Ar–CH3), 28.3 (d-CH2), 32.0( ꢂ-CH2), 53.5
(ꢀ-CH), 61.5 (CH2–O), 67.0(CH 2–Ar), 69.9 (e-CH2), 128.7 (Ar), 128.2 (Ar),
129.8 (Ar), 133 (Ar), 136.2 (Ar), 144.7 (Ar), 155.8 (NCO), 172.1 (CO) ppm.
Ethyl 2(S)-2-(Benzyloxycarbonylamino)-6-cyanohexanoate (9)
26.5 mmol of (8) was dissolved in 10mL of ethanol. A solution of
KCN (78.5 mmol) in 10mL of water was slowly added. The mixture was
kept at 90ꢀC for two hours, the ethanol was evaporated, and the resulting
solution was acidified with aqueous HCl. The mixture was extracted with
diethyl ether (3 Â 20mL) and the combined organic extracts were washed
with water, brine, and dried over anhydrous sodium sulfate. After evapora-
tion and purification by silica gel chromatography (chloroform : methanol
gradient 100 : 1 to 1 : 1), 7.4 mmol (28%) of the ester product (9) was isolated
along with 16 mmol (60%) of the free acid, which could be subsequently
reesterified with ethanol to afford a second crop of (9) (total yield: 88%);
MS: (m/z) ¼ 318 (M þ Kþ); (m/z) ¼ 357 (M þ Naþ); TLC (silica) Rf ¼ 0.21
1
(100 : 1, chloroform : methanol); H NMR (300 MHz, CDCl3) d 1.3t (3H,
CH3), 1.4–2.0m (6H, ꢁ, ꢂ, d-CH2), 2.3t (2H, e-CH2), 4.2q (2H, CH2–O),
4.3m (1H, ꢀ-CH), 5.1s (2H, CH2–Ar), 5.35d (1H, NH), 7.3m (5H, Ar)
ppm. 13C NMR (CDCl3) d 14.1 (CH3), 17.0( e-CH2), 24.2 (ꢁ-CH2), 24.8
(ꢂ-CH2), 32 (d-CH2), 53.4 (ꢀ-CH), 61.7 (CH2–O), 67.0(CH –Ar), 119.3
2
(CN), 128.1 (Ar), 128.2 (Ar), 128.5 (Ar), 136.1 (Ar), 155.8 (NCO), 172.6
(CO) ppm.
Ethyl 2(S)-2-(Benzyloxycarbonylamino)-
7-diamino-7-iminoheptanoate (10)
Compound(9)(1.4 mmol)wasdissolvedin10mLdrymethylenechloride
under nitrogen. Anhydrous ethanol (1.5 mL) was added and dry HCl gas was
bubbled through the solution until it was saturated. The reaction was stirred
at room temperature overnight. The following morning, more dry HCl gas
was bubbled through the reaction mixture and the solution was allowed to stir

SYNTHESIS OF L-INDOSPICINE
2081
at room temperature for an additional five hours. At this time, the solvent was
evaporated and anhydrous methanol was added and evaporated. This pro-
cedure was repeated an additional two times. The resulting residue was dis-
solved in anhydrous methanol under nitrogen and the solution cooled to 0ꢀC.
Ammonia gas was bubbled through the solution until it was saturated. The
reaction was stirred at 0ꢀC for six hours and allowed to stand at 4ꢀC over-
night. TLC (silica) analysis (25 : 1, chloroform : methanol) indicated the reac-
tion had gone to completion. The reaction mixture was evaporated and the
residue dissolved in hot distilled water, decolorized with charcoal, filtered and
evaporated. The resulting product was purified via column chromatography
(7 : 1, chloroform : methanol þ1% acetic acid) to afford 0.67 mmol (48%)
of (10). A small, but significant amount of methyl 2(S)-2,7-diamino-7-imino-
heptanoate (the transesterification product) was recovered during the column
chromatographic separation, which was subsequently hydrolyzed to L-indos-
picine (1). MS: (m/z) ¼ 336 (M þ 1); TLC (silica) Rf ¼ 0.15 (7 : 1, chloro-
1
form : methanol þ1% glacial acetic acid; H NMR (300 MHz, CD3OD) d
1.3t (3H, CH3), 1.4–1.8m (6H, ꢁ,ꢂ,d-CH2), 2.4t (2H, e-CH2), 4.2q
(2H, CH2–O), 4.2m (1H, ꢀ-CH), 5.1s (2H, CH2–Ar), 7.3m (5H, Ar) ppm.
13C NMR (CD3OD) d 14.7 (CH3), 26.3 (ꢁ-CH2), 27.7 (d-CH2), 32.2 (ꢂ-CH2),
33.4 (e-CH2), 55.3 (ꢀ-CH), 62.5 (CH2–O), 67.8 (CH2–Ar), 128.8 (Ar), 129.0
(Ar), 129.5 (Ar), 138.1 (Ar), 158.6 (NCO), 172.6 (C¼N), 174.3 ppm.
2(S)-2,7-Diamino-7-iminoheptanoic Acid Dihydrochloride
(L-Indospicine) (1)
Compound (10) (0.2 mmol) was dissolved in 5 mL of 6M HCl and
allowed to stir at room temperature for 72 h. The solvent was evaporated,
the residue was taken up in methanol, and the methanol evaporated. This
procedure was repeated two more times. Aqueous methanol was used to
crystallize 0.146 mmol (73%) of the extremely hygroscopic product. MS (m/z):
22
174.09 (M þ 1); optical rotation ð½ꢀŠD Þ lit. þ18 ꢂ 1 (c. 1 in 5N HCl)
[172]; Found: þ18.5 ꢂ 0.6ꢀ (c. 0.25 in 5N HCl); amino acid analysis Rf
¼ 60.6 min (Pickering PCX 5200 amino acid analysis column,
T ¼ 40ꢀꢂ 20ꢀ, flow rate ¼ 0.30 mL/min, with gradient pH elution); Anal.
Calc. for C7H17Cl2N3O2 ꢃ ð3=4ÞH2O ꢃ ð1=4Þ CH3OH: C, 33.48; H, 7.51; N,
15.28. Found: C, 33.41; H, 7.06; N, 14.90 (Note: Natural L-Indospicine
hydrochloride has been shown to crystallize with 0.75 molecule of water
1
per L-indospicine molecule[15]). H NMR (300 MHz, CD3OD) d 1.8m (2H,
ꢁ-CH2), 1.9m (2H, d-CH2), 2.1m (ꢂ-CH2), 2.6m (2H, e-CH2), 4.1m (1H,
ꢀ-CH), 8.5s and 9.0s (CNH2(NH)) ppm. 13C NMR (CD3OD) d 25.5

2082
BENCE AND CROOKS
(ꢁ-CH2), 27.8 (ꢂ-CH2), 31.1 (d-CH2), 33.3 (e-CH2), 53.8 (ꢀ-CH), 171.6
(C¼O), 172.8 (C¼N) ppm.
ACKNOWLEDGMENTS
The authors would like to acknowledge the University of Kentucky
Graduate School and the American Foundation for Pharmaceutical
Education for their generous fellowship support of AKB.
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