Synthesis of a new diaminodithiol bifunctional chelator for radiolabeling biomolecules with indium(III)

Article abstract of DOI:10.1016/S0040-4020(00)00424-5

The synthesis of a new bifunctional ligand 1-(p- carboxybenzyl)-N,N'-bis-[1,1-dimethyl-1-(p- methoxybenzylthio)ethyl]ethylenediamine-N,N'-diacetic acid, di-t- butyl ester (1, nbi6ss) is described. It consists of a carboxybenzyl group substituted on a carb

Full text of DOI:10.1016/S0040-4020(00)00424-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5093±5103
Synthesis of a New Diaminodithiol Bifunctional Chelator for
Radiolabeling Biomolecules with Indium(III)
b,³
Yizhen Sun,a,² Arthur E. Martella, and Michael J. Welch
*
aDepartment of Chemistry, Texas A&M University, College Station, TX 77842-3012, USA
bThe Edward Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
Received 21 March 2000; accepted 19 May 2000
AbstractÐThe synthesis of a new bifunctional ligand 1-(p-carboxybenzyl)-N,N0-bis-[1,1-dimethyl-1-(p-methoxybenzylthio)ethyl]-
ethylenediamine-N,N0-diacetic acid, di-t-butyl ester (1, nbi6ss) is described. It consists of a carboxybenzyl group substituted on a carbon
atom of the ethylenediamine moiety of a hexadentate ligand, which has been found to have a very high af®nity for In(III). The gem-
dimethylthiol groups and the carboxylic acid groups of the ligand were protected by groups that can be removed under mild conditions after
conjugation to a peptide or protein. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
are perfectly suitable for diagnostic applications, many of
the In-111-radiopharmaceuticals are nevertheless limited by
a high liver uptake. One of the reasons is probably due to the
large formation constant of In(III)-transferrin (log K1ˆ
18.74)12 and the high plasma concentration of this protein
(0.25 g/100 mL) thermodynamically favor the in vivo
exchange of many In(III) complexes with transferrin.
Meares et al. in 19741 synthesized the ®rst bifunctional
chelator (BFC). In this ligand a linkage of L-1 (see Chart
1) is used to combine a chelating agent, EDTA (ethylene-
dinitrilotetraacetic acid) to protein by amidation through an
a-bromoacetamido group (in BrCH2CONH±C6H4±CH2±
group), which was formed from catalytic hydrogenation of
the nitro group and then acylation with bromoacetyl
chloride. This linkage was further modi®ed by Meares
et al. (L-2,2 L-33). Since then, L-3 is the most widely used
intermediate for the design and synthesis of BFC. Altman et
al. (1984) synthesized L-44 as the linkage for the BFC of
EDTA. Here an aliphatic carboxylic acid is used for conju-
gating to protein. Gansow et al. (1986) designed L-55 for the
BFC of DTPA (diethylenetrinitrilopentaacetic acid). The
Meares' group also successfully prepared the 1,4,7,10-tetra-
azacyclododecane (cyclen) and 1,4,7-triazacyclononane
(tacn) BFC linkage with the aromatic amine for conjugation
(L-66 and L-77). Parker's group prepared the L-8,8 L-98 with
aliphatic amines for conjugation. Other compounds are of
interest for the design of BFC appeared in the recent
literatures are: L-10,9 with a thioester group for conjugation;
L-11,10 with a protected ethyleniminyl group to form the
ethylenediamine moiety, and L-12,11 use a trans-1,2-di-
aminocyclohexyl group as linkage.
The design and synthesis of new chelating agents for
effective coordination of In(III) has long been an important
objective of this research group. Not long ago, DTPA was
considered to be the best candidate for the design of bifunc-
tional chelators for In(III) (see Table 1). Since 1995, several
ligands containing aminoethanethiol donor groups were
found to have very high af®nity for In(III).14±16 Examples
are the two EDTA analogues: 6ss, N,N0-bis(2,2-dimethyl-2-
mercaptoethyl)ethylenediamine-N,N0-diacetic acid (2, Chart2,
pMˆ30.9)14 and EDDASS, N,N0-bis(2-mercaptoethyl)ethyl-
enediamine-N,N0-diacetic acid (3, Chart 2, pMˆ30.4).15
A
logical approach to the use of these complexes is to prepare
their BFCs. The challenge for the design and synthesis of
bifunctional chelators containing thiol groups are: thiol groups
are not as stable as carboxylic acid or amines (in EDTA,
DTPA or polyazamacrocycles); and the a-bromoacetamido
group (BrCH2CONH±C6H4±CH2±) which is widely used
for the amidation reaction to bond proteins, also reacts with
the thiol groups of the ligand.17 Based on the structure of 6ss, a
bifunctional ligand: 1-(4-carboxymethoxybenzyl)-N,N0-bis-
[(2-mercapto-2,2-dimethyl)ethyl]-1,2-ethylenediamine-N,N0-
diacetic acid (4, bi6ss Chart 2) was designed and synthesized
in this laboratory.18 In this ligand a carboxymethoxy group
was used for conjugation. However, it was found that the
labeled complexes to be dif®cult to conjugate to proteins.
One reason may be due to the fact that carboxymethoxy
Indium-111 is a radioisotope whose physical characteristics
Keywords: In(III) complexes; bifunctional ligand; diaminodithiol.
* Corresponding author. Tel.: 11-979-845-2011; fax: 11-979-845-4719;
e-mail: martell@mail.chem.tamu.edu
²
sun@mail.chem.tamu.edu
welchm@mir.wustl.edu.
³
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00424-5

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Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
Chart 1. Some intermediates for the preparation of BFC.

Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
5095
Table 1. Formation constants (log KML) and pM (pM is 2log [M] of In(III)
at pHˆ7.4 with 100% excess of the ligand) of In(III) (Ref. 13)
Results and Discussion
1-(p-Carboxybenzyl)-ethylenediamine (7, see Scheme 1) is
a simple and very useful intermediate for the synthesis of
bifunctional ligands for covalently bonded metal ions to
protein. However this intermediate has not been described
in the literature. Kidwai20 published `A New Route for
the Synthesis of Substituted Ethylenediamines', including
1-benzylethylenediamine, by catalytic hydrogenation of the
azalactones. (See Scheme 2). Following his idea, a para-
substituted azalactone was prepared and hydrogenated.
All that could be obtained is the glycinamide and the
2-(p-carboxybenzyl)glycine. It was concluded that it is
impossible to reduce the amide by catalytic hydrogenation
under such mild conditions as Kidwai described in his
paper.20 The reaction probably requires very high pressure
and temperature. However, it is well known that cyano
groups can be reduced to amines by hydrogenation with
Raney Nickel under low pressure and room temperature.
Thus, 2-benzamidoacetonitrile (9) was used instead of
N-benzoylglycine ethyl ester, to prepare the ethylene-
diamine derivative: compound 7.
Ligand
log KML
pM
EDTA
DTPA
DOTA
NOTA
HBED
6ss
24.9
29.0
23.9
26.2
27.9
39.8
22.1
24.9
17.8
21.6
17.9
30.9
30.4
18.3
EDDASS
Transferrina
37.0
18.74, 16.86
a Ref. 12.
group may be involved in a dissociation mechanism before
the ligand can be bonded to the amino groups of a protein.
Rogers et al.19 published a paper on two bifunctional poly-
azamacrocycles (see Chart 2, CPTA, 5 and PCBA, 6), in
which benzoic acid groups were used for conjugation. Here
we design and synthesize a bifunctional ligand of 6ss with the
chelating donor groups protected and with a benzoic acid
group as the linkage for conjugation. After conjugation, the
protecting groups, tert-butyl and 4-methoxybenzyl, can be
removed under mild conditions.
For the preparation of the new bifunctional ligand, the
strategy similar to that used for the previous bifunctional
Chart 2.

5096
Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
Scheme 1.
ligand, bi6ss: 1-(4-carboxymethoxybenzyl)-N,N0-bis[(2-
mercapto-2,2-dimethyl)ethyl]-1,2-ethylenediamine-N,N0-
diacetic acid18 was employed (see Scheme 3). The ®rst four
steps of this route run smoothly (see Experimental).
However, in the last step: the opening of the disul®de
bond by Na/NH3 liq., the aromatic ring of the benzyl group
is also hydrogenated by Birch reduction.21 Although aerial
oxidation of the cyclohexadiene may allow reformation of
the aryl ring, the thiol groups will also be oxidized. The
gem-dimethyl disul®de bond is much more stable than the
unsubstituted disul®de. Several other reducing reagents (Na
metal, NaBH4, and LiBH4) were tried; none of them gave a
clean product. At this time, another route, Scheme 4, was
designed.
base formation before chlorination with N-chlorosuccini-
mide. The remaining steps are simple (see Scheme 7), and
the overall yield is above 70%.
This new BFC (nbi6ss, 1) is designed as a pre-conjugation
type bifunctional ligand. After conjugation, the protecting
groups (t-butyl and methoxybenzyl) will be removed.22 This
strategy may work when conjugating small peptides or non-
biological macromolecules. For conjugating bio-macra-
molecules, during the removal of these protecting groups,
acid treatments may compromise the biological activity of
biomolecules irreversibly. This new BFC (nbi6ss, 1) may
also be used as the precursor of a precomplexation type
BFC, i.e. to remove the t-butyl and methoxybenzyl protect-
ing groups and prepare its In-111 complexes, then conjugate
to proteins.
For the preparation of multidentate ligands containing gem-
dimethyl thiol groups, compound 10: 2-(p-methoxybenzyl-
thio)-2-methylpropanal is a very useful intermediate, since
the condition for the deprotection of the methoxybenzyl
group is much milder than that of the benzyl group.22 Koji
Yoneda et al. 199323 published a three step route for the
synthesis of this aldehyde (see Scheme 5). The starting
material is not commercially available. Jones and Elmaleh24
(1990) reported a one-pot synthesis of the benzyl-protected
aldehyde, 2-benzylthio-2-methylpropanal (Scheme 6).
However, due to the fact that the aldehyde group also can
be attacked, the yield is low. The Jones and Elmalehs' route
was modi®ed by protecting the aldehyde group by Schiff
Experimental
Materials and methods
Aminoacetonitrile disulfate, benzoyl chloride, methyl
4-formylbenzoate, iso-butylaldehyde, sec-butylamine,
N-chlorosuccinimide,
p-methoxybenzylthiol,
t-butyl
bromoacetate, sodium cyanoborohydride and bis-trimethyl-
silyl acetamide (BSA), were obtained from Aldrich
Chemical Co. and were used as supplied.

Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
5097
Scheme 2.
The proton and carbon-13 NMR were recorded on a Varian
XL-200 spectrometer operating at 200 MHz, and the chemi-
cal shifts are reported in ppm relative to tetramethylsilane.
The mass spectra were obtained with a VG analytical 70S
high resolution double focusing magnetic sector spectro-
meter with an attached VG analytical 11/250J data system.
Fast atom bombardment (FAB) technique was used for
ionization. The C, H, N analyses were performed by
Galbraith Laboratories, Inc., Knoxville, TN. The melting
point was determined with a Fisher-Johns melting point
apparatus and was uncorrected.
®nished, the reaction mixture was stirred at room tempera-
ture for 20 h. The crude product was taken out by ®ltration
and washed with 3£100 mL of benzene and 3£100 mL of
ethyl ether and was vacuum dried over P2O5 for 16 h. To
49 g of this crude product 150 mL of methanol was added
and heated to re¯ux for 20 min. The hot suspension was
®ltered and cooled at 08C for 16 h. A large amount of crys-
talline material was separated. The product was collected by
®ltration and washed with dry ethanol and vacuum drying;
44 g of crystalline product was obtained, yield 80%.
25
1
Mpˆ146±1488C (lit. 1408C). H NMR (in CDCl3): 7.80
(d, 2H, arom.); 7.51 (m, 3H, arom.); 6.6 (b,H, CONH); 4.40
(d, 2H, ±CH2±).
Synthetic procedures
2-(p-Carbomethoxybenzylidine)-2-benzamidoacetonitrile
(9). A 500 mL three-necked ¯ask was equipped with a
mechanical stirrer and HClg inlet and outlet tubes. A solu-
tion of 4.26 g (0.026 mol) methyl 4-formylbenzoate in
250 mL of dry ethyl ether and 4 g (0.025 mol) of N-benz-
amidoacetonitrile (8) was added to the ¯ask. The reaction
mixture was cooled to about 2108C by a NaCl±ice-water
bath. Dry HCl gas was bubbled through the reaction mixture
for about 30 min. The mixture was then stirred at 258C for
another half hour. It was ®ltered with a sintered glass funnel
and washed with 2£100 mL of benzene. The wet residue
was suspended in about 50 mL of benzene. This suspension
was evaporated under reduced pressure to nearly dryness.
The route for the synthesis of 1-(p-carboxybenzyl)-ethyl-
enediamine dihydrochloride (7) and 1-(p-carbethoxy-
benzyl)-ethylenediamine dihydrochloride (70) involving
intermediates 8 and 9 is shown in Scheme 1
2-Benzamidoacetonitrile (8). The procedure of Li, Gang
et al.25 was followed and modi®ed. A solution of 60 g
(0.39 mol) of aminoacetonitrile disulfate in 150 mL water
was placed in a 1 L three-necked ¯ask in a 5±68C bath with
mechanical stirring. A solution of 60 g (0.43 mol) of
benzoyl chloride in 200 mL of benzene was added
dropwise. At the same time anhydrous Na2CO3, 82 g
(0.78 mol) was added portionwise. After the addition was

5098
Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
Scheme 3.
More benzene was added and the solid material was sepa-
rated by ®ltration and washed with benzene and ethyl ether.
It was air-dried and 6.1 g of yellow solid was obtained. This
yellow solid was suspended in 50 mL 5% Na2CO3 in
aqueous solution and was stirred in an ice-water bath for
2 h. The insoluble material was separated by ®ltration and
washed with 3£50 mL cold 2% Na2CO3 solution and
5£60 mL cold water, until the washings became neutral.
This solid was vacuum dried over P2O5 at room temperature
for 2 days; 4.8 g crude product was obtained. It was
dissolved in 70 mL boiling toluene. After hot ®ltration,
the clear ®ltrate was allowed to stand at 08C, 2.9 g white
pure product was obtained, yield 30%. 1H NMR (in CDCl3):
8.03 (d, 2H, arom.); 7.79 (d, 2H, arom.); 7.55±7.47 (m, 5H,
arom.); 7.85 (b, H, CONH); 6.84 (s, H, CHvC±); 3.88 (s,
3H, CH3OOC±). FAB. MS: [M1H1]ˆ307. Anal. Calcd
for: C18H14N2O3: C, 70.36; H, 4.56; N, 9.12. Found: C,
70.00; H, 4.64; N, 9.36.
with water, 90% ethanol and absolute ethanol, 3±4 g, were
added and the reaction mixture was hydrogenated at 50±
55 psi and room temperature for 20 h. The reaction mixture
was degassed (remove H2 and NH3 by aspiration), and then
®ltered through decolorized charcoal. The insoluble mate-
rial was washed with 6£50 mL of absolute ethanol. The
®ltrate and washings were combined and acidi®ed with
6 M HCl to pH,1. The NH4Cl that was separated was
removed by ®ltration. The ®ltrate was concentrated under
reduced pressure until a yellow residue was obtained. To
this solid, 140 mL of conc. HCl was added and heated in a
1208C bath for 48 h. After cooling, it was ®ltered and the
clear ®ltrate was evaporated under reduced pressure to
nearly dryness. Then it was further dried with 250 mL of
benzene by azeotropic distillation, and 4.5 g of yellow
powder was obtained. This is the crude product of 1-(p-
carboxybenzyl)-ethylenediamine dihydrochloride (7). 1H
NMR (in D2O, pDˆ1.5, t-BuOH as internal standard,
1.29 ppm): 7.88 (d. 2H, arom.); 7.33 (d, 2H, arom.); 3.83
(quint. H, ±CH±); 3.25±2.94 (m, 4H, ±CH2CHCH2).13C
NMR (in D2O, pDˆ1.5, t-BuOH as internal standard,
31.1 ppm): 169.3 (±CvO), 138.9, 129.7, 128.8, 128.5
(arom.); 49.51 (±CH±); 39.81 and 35.30 (±CH2CHCH2±).
FAB. MS.: [M1H1]ˆ195.
1-(p-Carbethoxybenzyl)-ethylenediamine dihydrochloride
(70) Compound 9, 5 g (16 mmol) was added to a 500 mL
Parr glass bottle containing 55 mL of absolute ethanol satu-
rated with NH3 gas in an ice-water bath. Raney Nickel
(CAUTION! dry solid is ¯ammable), which was prewashed

Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
5099
Scheme 4.
The 4.5 g yellow powder of the above crude product was
esteri®ed with 600 mL of absolute ethanol and HClg. The
product was recrystallized with 10±15 mL of absolute
ethanol, and 1.98 g of the ethyl ester (70) of compound 7
was obtained, yield 41%. 1H NMR (in D2O, pDˆ1.5,
t-BuOH as internal standard, 1.29 ppm): 7.88 (d. 2H,
arom.); 7.33 (d, 2H, arom.); 4.24 (quat. 2H, ±CH2CH3);
3.82 (quint. H, ±CH±); 3.25±2.96 (m, 4H, CH2CHCH2±);
1.22 (t, 3H, ±CH3). 13C NMR (in D2O, pDˆ1.5, t-BuOH as
internal standard, 31.1 ppm): 167.9 (±CvO), 139.0, 129.8,
129.0, 128.8 (arom.); 61.68 (±CH2CH3); 49.66 (±CH±);
39.96 and 35.45 (±CH2CHCH2±); 12.71 (±CH3). FAB.
MS: [M1H1]ˆ223. Anal. Calcd for: C12H18N2O2´
2HCl.NH4Cl´3/4H2O: C, 39.78; H, 7.04; N, 11.60. Found:
C, 39.77; H, 6.70; N, 11.40.
The route used for the synthesis of nbi6ss (1) involving
intermediates 11 and 12 is shown in Scheme 4.
1-(p-Carbethoxybenzyl)-N,N0-[1,1-dimethyl-1-(p-methoxy-
benzylthio) ethyl]ethylenediamine (11). A sodium ethano-
late solution was prepared by dissolving 0.14 g (6 mmol) of
Scheme 5. Koji Yoneda et al., 1993.

5100
Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
removed, a solution of 1.2 g of 50% NaOH in 15 mL of
saturated NaCl solution was added; 3£30 mL of ethyl
ether were used to extract the product. The combined
ether solutions were dried with anhydrous MgSO4 for
16 h. After the solvents and drying agent were removed,
1.7 g pale yellow oil was obtained. It was puri®ed by ¯ash
chromatography; 0.76 g pure product was obtained, yield
1
40%. H NMR (in CDCl3): 7.95 (d. 2H, arom.); 7.25±7.16
(m, 6H, arom.); 6.81±6.77 (d, 4H, arom.); 4.35 (quat. 2H,
CH2CH3); 3.75 (s, 6H, CH3O±); 3.63±3.56 (two singlet, 4H,
CH2S±); 2.8±2.2 (m. 9H, ±CH2CHCH2, ±CH2C(CH3)2);
1.9 (b, 2H, ±NH±); 1.4±1.3 (m, 15H, ±CH3). 13C NMR
(in CDCl3): 166.2 (±CvO of benzoate), 158.1, 144.5,
130.1, 129.5, 129.2, 128.0 (arom.);113.5 (C-3 of methoxy-
benzyl); 60.4(±CH2CH3); 59.1 and 58.9(±CH±); 56.8, 54.8,
52.7, 46.6, (±CH2CHCH2±, NH±CH2±, CH3O±); 39.3
(±SCH2±); 31.7 (±C(CH3)2); 26.9 (±C(CH3)2); 13.9
(±CH2CH3). FAB. MS: [M1H1]ˆ639. Anal. Calcd
for:C36H50N2O4S2: C, 67.71; H, 8.14; N, 4.39. Found: C,
67.60; H, 8.04; N, 4.48.
Scheme 6. Jones and Elmaleh, 1990.
sodium into 4.5 mL of absolute ethanol. This solution was
added to a suspension of 0.89 g (3 mmol) of compound 70 in
3 mL of absolute ethanol; then a solution of 1.49 g (6 mmol)
of compound 10 (see the procedures bellow) in 10 mL of
CCl4 was added. This reaction mixture was stirred at room
temperature for 10 min. The solvents were removed by
evaporation under reduced pressure. To the residue 10 mL
of CCl4 and 3±4 g of anhydrous MgSO4 were added and the
mixture was stirred at room temperature for another 20 h.
The insoluble material was removed by ®ltration and the
solvents were removed by evaporation under reduced pres-
sure. To the residue 12 mL of methanol was added; 0.35 g of
NaBH3CN was added portionwise, while the pH of the solu-
tion was maintained at about 4. The reaction was stirred at
room temperature for 1.5 h. After the methanol was
1-( p-Carbethoxybenzyl)-N,N0-[1,1-dimethyl-1-( p-meth-
oxybenzylthio)ethyl]ethylenediamine-N,N0-diacetic acid,
di-t-butyl ester (12). Compound 11, 0.64 g (1 mmol),
t-butyl bromoacetate, 3.9 g (20 mmol) and 2,6-lutidine,
0.24 g (2 mmol) were mixed and stirred at room tempera-
ture for 24 h. The excess reactants were removed by distil-
lation under reduced pressure. The crude product was dried
at 408C (0.1 mm Hg) for 6 h, and 0.9 g of pale yellow oil
was obtained. It was puri®ed by ¯ash chromatography, the
product was eluted by hexane/ethyl acetateˆ95:5; and
1
0.14 g pure product was obtained, yield 16%. H NMR (in
CDCl3): 7.90 (d, 2H, arom.); 7.26±7.19 (m, 6H, arom.);
6.82±6.79 (d, 4H, arom.); 4.33 (quat. 2H, CH2CH3); 3.77
(s, 6H, CH3O±); 3.63±3.58 (two singlet, 4H, CH2S±); 3.5±
3.0 (m, 9H, ±CH2CO±, ±CH2CHCH2±); 2.8±2.3 (m, 4H,
CH2C(CH3)2); 1.5±1.1 (m, 33H, CH2CH3, ±C(CH3)2 and
±C(CH3)3). 13C NMR (in CDCl3): 171.8 and 171.1 (CvO
of acetate) 166.4 (±CvO of benzoate), 158.1, 146.1, 130.0,
129.7, 129.2, 127.8 (arom.); 113.6 (C-3 of methoxybenzyl);
80.6 and 80.4 (±CH2± of the two acetate); 60.4 (CH2CH3);
58.0 and 57.3 (±CH±); 66.7, 65.5, 63.2, 54.9, 53.4, 47.7,
(±CH2CHCH2±, NH±CH2±, CH3O±); 36.4 (±SCH2±);
31.7 (C(CH3)2); 27.8 (±CH3 of t-butyl); 26.9(±C(CH3)2);
14.0 (CH2CH3). FAB. MS.: [M1H1]ˆ867. Anal. Calcd
for: C48H70N2O8S2´1.5H2O: C, 64.50; H, 8.17; N, 3.14.
Found: C, 64.66; H, 8.15; N, 3.01.
1-( p-Carboxybenzyl)-N,N0-[1,1-dimethyl-1-( p-methoxy-
benzylthio) ethyl]ethylenediamine-N,N0-diacetic acid,
di-t-butyl ester (1). Compound 2, 0.135 g (0.156 mmol)
was dissolved in 6.8 g of KOH±H2O±1, 2-dimethoxyethane
(0.12 g KOH (87%), 3.8 g H2O and 5.6 g of 1,2-dimethoxy-
ethane) solution, and was heated to re¯ux under Ar for 1 h.
After the solution was cooled to rt the solvents were
removed by evaporation under reduced pressure. To the
residue 10 mL of water was added and 2.5 mL HCl was
used to acidify the potassium salt. Dichloromethane was
used to extract the product. After it was worked up and
dried, 0.12 g of pure product (pale yellow oil) was obtained,
yield 80%. 1H NMR (in CDCl3): 7.90(d. 2H, arom.); 7.26±
7.19 (m, 6H, arom.); 6.82±6.79 (d, 4H, arom.); 3.75 (s, 6H,
CH3O±); 3.63±3.58 (two singlet, 4H, CH2S±); 3.5±2.5 (m,
Scheme 7.

Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
5101
13H, ±CH2CO±, ±CH2CHCH2±, ±CH2C(CH3)2); 1.5±1.1
(m, 30H, ±C(CH3)3 and ±CH2C(CH3)2). 13C NMR (in
CDCl3): 171.9 and 171.2 (CvO of acetate); 158.2
(±CvO of benzoic acid), 147.2, 129.8, 127.8 126.7
(arom.); 114.0 (C-3 of methoxybenzyl); 80.7 and 80.6
(±CH2± of the two acetate); 58.0 and 57.3 (±CH±); 66.6,
65.4, 63.4, 54.9, 53.6, 47.7, (±CH2CHCH2±, NH±CH2±,
CH3O±); 36.5 (±SCH2±); 31.8 (±C(CH3)2); 27.9
(±C(CH3)3); 26.8 (±C(CH3)2); FAB. MS: [M1H1]ˆ839.
Anal. Calcd For C46H66N2O8S.22.5H2O: C, 62.51; H, 8.04;
N, 3.17. Found: C, 62.77; H, 7.62; N, 3.18.
dried with anhydrous MgSO4. After the solvent and drying
agent were removed, 5.1 g of colorless oil was obtained,
which was crystallized after cooling to about 2208C. H
NMR (in CDCl3): 9.12 (s, H, CHvO); 7.18 (d, 2H,
arom.); 6.82 (d. 2H, arom.); 3.78 (s, 3H, methoxy-); 3.47
(s, 2H, ±CH2S±); 1.38 (s, 6H, two methyl groups). 13C
NMR (in CDCl3): 193.7 (±CHvO); 158.8, 130.5, 128.6,
113,9 (arom.); 55.22 (methoxy-), 42.76 (±C± (CH3)2±);
32.81 (±CH2S±); 21.08 (the two methyl groups).
1
The synthetic procedures of the new compounds in Scheme
3 are shown as follows:
The route used for the synthesis of 2-(4-methoxybenzyl-
thio)-2-methylpropanal (10) involving intermediates 13
and 14 is shown in Scheme 7.
6-(p-Carbethoxybenzyl)-3,3,10,10-tetramethyl-1,2-dithia-
5,8-diaza-cyclodeca-4,8-diene (15) Compound 70, 1.77 g
(6 mmol), was placed in a 50 mL two-necked ¯ask, which
was connected with a trap and vacuum distillation equip-
ment. The whole system was ¯ushed with Ar for three times
before BSA, 15 mL (60 mmol) were added. The reaction
mixture was heated to 508C bath for 1 h. Then vacuum at
30±40 mm Hg for 30 min was used to remove the excess
BSA. A solution of 2,20-dithio-bis(2-methylpropanal),26
1.26 g (6.12 mmol) in 8 mL of benzene was added, and
the reaction mixture was heated to 70±808C for 3 h. The
solvents and by-products were removed by distillation under
reduced pressure. About 40 mL of benzene and 40 mL of
water were added to extract the product and deprotect the
trimethylsilyl groups. The organic phase was ®ltered and
dried with anhydrous MgSO4 for 16 h. The crude product
was loaded on 45 g of silica gel 60 and eluted with benzene,
CH2Cl2 and CH2Cl2±MeOH (v/vˆ98/2); 1.63 g of pure
N-(2-Chloro-isobutylidene)-sec-butylamine
(13).
A
mixture of 7.2 g (0.1 mol) of iso-butylaldehyde and 7.3 g
(0.1 mol) of sec-butylamine was stirred at room temperature
for 5 min. To the reaction mixture 150 mL of carbon tetra-
chloride and anhydrous MgSO4 were added; this suspension
was stirred at room temperature for another 2 h. The
insoluble material was removed by ®ltration. To the ®ltrate
14.7 g (0.11 mol) of N-chlorosuccinimide was added
portionwise while the temperature was maintained at rt by
means of a water bath. After it was stirred thoroughly for
3 h, the reaction mixture was ®ltered and washed with a
small amount of CCl4. The solvent was then removed in
vacuo and the turbid colorless oil was distilled under
reduced pressure; 11.8 g pure product was obtained,
1
bpˆ63± 648C/30 mmHg. H NMR (in CDCl3): 7.65 (s, H,
1
CHvN±); 3.03 (quint. H, N±CH±); 1.69 (s, 6H,
(CH3)2CCl±); 1.50 (quat. 2H, ±CH2±), 1.13 and 1.15 (d,
3H, CH±CH3) 0.78 (t. 3H, CH2CH3). 13C NMR (in
CDCl3): 162.5(±CHvN±); 67.76 (±CCl±), 66.52 (vN±
CH±); 29.87 (±CH2CH3); 29.08 ((CH3)2CCl); 21.53
(CH±CH3); 10.43 (CH2CH3).
product was obtained, yield 68%. H NMR (in CDCl3):
7.95 and 7.92 (d, 2H, 3,5-arom.) 7.25 and 7.23(d, 2H, 2,6-
arom.); 6.90 and 6.78 (two s, 2H, ±CHvN±); 4.37±4.35
(quadri. 2H, ±CH2CH3); 4.12±4.07 (m, 1H, 1 of ethylene);
3.4 and 3.1±2.8 (m, 4H, ±CH2± of benzyl and ethylene);
1.43±1.22 (m, 15H, methyl). 13C NMR (in CDCl3): 167.9
and 166.0 (±CHvN±); 160.4 (±COO); 144.4 (4-benzene);
129.3±129.2 (2,3,5,6-benzene); 128.4 (1-benzene); 73.27 (1
of ethylene); 66.52 (2 of ethylene); 60.76 (±CH2CH3); 52.7
(±CH2± of benzyl); 39.98 (±C(CH3)3); 24.4±21.1 (methyl).
FAB.MS: [M1H]ˆ393.
N-[2-( p-methoxybenzylthio)-iso-butylidene]-sec-butyl-
amine (14). A sodium methanolate solution was prepared
by dissolving 0.8 g (0.033 mol) of Na in 17 mL of methanol.
To this solution, 12 g (0.035 mol) of 4-methoxybenzylthiol
was added dropwise. After the addition was complete, 5.3 g
(0.033 mol) of compound 13 was added slowly. The reac-
tion mixture was heated to 858C for 30 min. The cooled
mixture was poured into 70 mL of water and extracted
with dichloromethane. The organic extractions were
combined and dried with anhydrous MgSO4 for 16 h.
After the solvent and drying agent were removed, 10 g of
colorless oil was obtained. It was crystallized after cooling
to about -208C. 1H NMR (in CDCl3): 7.48(s, H, ±CHvN±);
7.16 (d, 2H, arom.); 6.80 (d, 2H, arom.); 3.74 (d, 3H,
methoxy); 3.58 (s, 2H, ±CH2-S±); 3.04 (hexad. H,
N±CH±); 1.50 (quint. 2H, CH2CH3); 1.41 (s, 6H,
CH3)2CS±).
6-(p-Carbethoxybenzyl)-3,3,10,10-tetramethyl-1,2-dithia-
5.8-diaza-cyclodecane (16). A 1.2 M HCl±CH3OH solu-
tion (conc. HCl/CH3OHˆ1:9, v/v) was added to a solution
of 1.6 g (4.1 mmol) of compound 15 in 25 mL of methanol
until pH was about 5. Sodium cyanoborohydride
(NaBH3CN), 0.51 g (8.2 mmol) was added portionwise
within 2 h. The pH of the reaction mixture was maintained
at 5.0±5.3 with 1.2 M HCl±CH3OH. More acid was added
until pH was 1.8. About 5 mL of water was added and the
methanol was removed by distillation under reduced pres-
sure. To the resulting mixture 16 mL of water were added,
and 7.5 M of NH3±H2O was added until pH is about 9.5.
The product was extracted with 32 mL of CH2Cl2, which
was then ®ltered and dried, with anhydrous MgSO4 for 2
days. After working up, 1.6 g of yellow oil was obtained. It
was puri®ed by ¯ash chromatography; 0.99 g of pure
product was obtained from CH2Cl2/MeOHˆ98:2, 9:1 and
2-(p-Methoxybenzylthio)-2-methylpropanal (10).
A
warm solution of 3.78 g (0.03 mol) of oxalic acid
(C2H2O.42H2O) was added to a solution of 5.6 g of
compound 14 in 20 mL of dichloromethane with vigorous
stirring at rt. for 1 h. The organic phase was separated and
the aqueous phase was extracted with 2£15 mL of dichloro-
methane. The combined dichloromethane solutions were
1
CH2Cl2/MeOH/conc.NH3±H2Oˆ100:15:2, yield 62%. H
NMR (in CDCl3): 7.97 (m, 2H, 3,5-benzene); 7.26 (m,
2H, 2,6-benzene); 4.37 (quadri, 2H, ±CH2CH3); 3.2±2.2

5102
Y. Sun et al. / Tetrahedron 56 (2000) 5093±5103
(m, 9H, ±NH±CH2, ±NH±CH and CH2± of benzyl); 2.1±
1.8 (b, 2H, ±NH±); 1.4±1.1 (m, 15H, methyl). 13C NMR (in
CDCl3): 160.2 (±COO); 144.6 (4 of benzene); 129.5, 128.9
and 128.7 (1,2,3,5,6 of benzene); 61.6 (±CH2CH3); 60.6±
50.4 (±NH±CH2, ±NH±CH); 40.9 (±CH2± of benzyl);
29.3±23.7 (±C(CH3)2, ±C(CH3)3); 14.1 (±CH2CH3). FAB
MS: [M1H]ˆ397. Anal. Calcd for C20H32N2O2S2´1/2H2O:
C, 59.26; H, 8.15; N, 6.91. Found: C, 59.47; H, 8.33; N,
6.86.
±N±CH2C±, ±CH2± of benzyl); 32.3, 31.7, (±C(CH3)2);
30.8, 29.0 27.7, 27.0 (±C(CH3)2); FAB. MS:
[M1H1]ˆ484. Anal. Calcd for C22H32N2O6S2. 1.5.H2O: C,
51.66; H, 6.85; N, 5.48. Found: C, 51.37; H, 6.34; N, 5.20.
Preliminary experiment about conjugation
About 0.11 g (0.13 mmol) of compound 1 and 11.6 mg
(0.13 mmol) of dl-alanine in of CH2Cl2 (0.5 mL) were
treated with a solution of 26.8 mg (0.13 mmol) of DCC
(N,N0-dicyclohexylcarbodiimide) in 0.5 mL of CH2Cl2.
After 15 min., the conjugated precipitate was ®ltered and
washed with small amount of ethyl acetate. The 13C NMR
of this product shows a benzamide peak at 162 ppm.
6-(p-Carbethoxybenzyl)-3,3,10,10-tetramethyl-1,2-dithia-
5,8-diaza-cyclodecane-N,N0-diacetic acid, di-tert-butyl)
ester (17). A mixture of 0.24 g (0.61 mmol) of compound
16, 0.72 g (3.7 mmol) of t-butyl bromoacetate; 0.25 g
(1.8 mmol) K2CO3 and 0.1 g (0.61 mmol) of KI was
prepared and was stirred at rt for 19 h. It was ®ltered and
washed with CH2Cl2. The ®ltrate and washings were
combined and the solvents and excess BrCH2COO±t-Bu
were removed by distillation under reduced pressure.
After vacuum drying at 1 mmHg/508C for 6 h, 0.45 g of
pale yellow oil was obtained. It was puri®ed with silica
gel 60 and was eluted by benzene and benzene±ethyl
Acknowledgements
This research was supported by the US Public Health
Service, National Cancer Institute, CA-42925 (A. E. M.
and M. J. W.). National Science Foundation Grant
CHE-8705697 supported the purchase of the mass
spectrometers.
1
ether; 0.3 g of pure product was obtained, yield 79%. H
NMR (in CDCl3): 7.97 (d, 2H, 3,5-benzene); 7.26 (d, 2H,
2,6-benzene); 4.36 (quadri, 2H, ±CH2CH3); 4.33.8 and 3.4±
2.8 (m, 9H, ±N±CH2C±, ±N±CH and CH2± of benzyl);
2.2±2.5 (m, 4H, ±CH2±COOt-Bu); 1.5±1.1 (m, 33H,
methyl). 13C NMR (in CDCl3): 171.5 and 170.7 (±CvO
of the ester); 166.6 (CvO± of the benzoate); 145.6 (4 of
benzene); 129.5, 129.4 and 128.3 (1,2,3, 5,6 of benzene);
80.0 (±CH2± of the acetate); 60.7, 58.8, 56.0, 53.7, 50.1
(±CH2CH3, ±N±CH2CH±, ±N±CH2C±, ±CH2± of benzyl);
38.3 (±C(CH3)3); 33.2 and 32.3 (±C(CH3)2); 28.1
(±C(CH3)3); 27.0 and 25.3 (±C(CH3)2); 14.3 (±CH2CH3).
FAB. MS: [M1H1]ˆ625. Anal. Calcd for C32H52N2O6S2´1/
2H2O: C, 60.06; H, 8.37; N, 4.42. Found: C, 60.23; H, 8.36;
N, 4.26.
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