Structure-immunosuppressive activity relationships of new analogues of 15-deoxyspergualin. 1. Structural modifications of the hydroxyglycine moiety

Article abstract of DOI:10.1021/jm980431g

A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent currently commercialized in Japan, was synthesized and tested in a graft-versus-host disease (GVHD) model in mice. Using the general concept of bioisosteric replacement, variations of the hydroxyglycine central 'C' region were made in order to determine its optimum structure in terms of in vivo immunosuppressive activity. By this way, the malonic derivative 13a was discovered as the first example of a new series of potent immunosuppressive agents encompassing a retro-amide bond linked to the hexyl- guanidino moiety. Structure-activity relationships of this series were studied by synthesizing compounds 13g-i and 13k-s. Variation of the 'right- amide' of 13a led to the urea 19a and the carbamates 23 and 27a which proved to be equally active as DSG in our GVHD model. Finally 27a was found to be the most potent derivative, being slightly more active than DSG in a heart allotransplantation model in rats. Due to the absence of chiral center in its structure and to its improved chemical stability compared to DSG, 27a was selected as a candidate for clinical evaluation.

Full text of DOI:10.1021/jm980431g

J . Med. Chem. 1999, 42, 277-290
277
Str u ctu r e-Im m u n osu p p r essive Activity Rela tion sh ip s of New An a logu es of
15-Deoxysp er gu a lin . 1. Str u ctu r a l Mod ifica tion s of th e Hyd r oxyglycin e Moiety
Luc Lebreton, J ocelyne Annat, Philippe Derrepas,† Patrick Dutartre, and Patrice Renaut*
Laboratoires Fournier S.A., Axe Immunologie, 50 Rue de Dijon, 21121 Daix, France
Received J uly 24, 1998
A series of new analogues of 15-deoxyspergualin (DSG), an immunosuppressive agent currently
commercialized in J apan, was synthesized and tested in a graft-versus-host disease (GVHD)
model in mice. Using the general concept of bioisosteric replacement, variations of the
hydroxyglycine central “C” region were made in order to determine its optimum structure in
terms of in vivo immunosuppressive activity. By this way, the malonic derivative 13a was
discovered as the first example of a new series of potent immunosuppressive agents
encompassing a retro-amide bond linked to the hexyl-guanidino moiety. Structure-activity
relationships of this series were studied by synthesizing compounds 13g-i and 13k -s. Variation
of the “right-amide” of 13a led to the urea 19a and the carbamates 23 and 27a which proved
to be equally active as DSG in our GVHD model. Finally 27a was found to be the most potent
derivative, being slightly more active than DSG in a heart allotransplantation model in rats.
Due to the absence of chiral center in its structure and to its improved chemical stability
compared to DSG, 27a was selected as a candidate for clinical evaluation.
In tr od u ction
In vivo experiments clearly showed that (-)DSG is the
only immunosuppressive enantiomer.7 However, most
of the biological, pharmacological, and clinical data have
been obtained with the more readily available (+,-)-
DSG whose first total synthesis was patented in 1982.13
Since its discovery, DSG has been widely studied and
demonstrated efficacy first as an anticancer agent,14
then in animal transplantation either in allografts15 or
xenografts,16 and more recently in graft-versus-host
disease (GVHD) animal models.17 DSG has also been
widely investigated18 in autoimmune diseases. The high
potential of DSG to reverse renal graft rejection was
first reported in 1990.19 This drug was therefore launched
in J apan (Spanidin) in this indication in 1994.19
In the last three decades, major advances have been
achieved in the development of immunosuppressive
drugs which have made possible organ transplantation
and treatment of autoimmune diseases. Azathioprine1
was first introduced to control graft rejection and is still
used in combination with other immunosuppressive
agents. However, its myelotoxicity and other side effects
limit its use. The discovery of cyclosporin A2 allowed
major improvements in transplantation. It induced
dramatic increase of organ allograft survival which
made possible liver and cardiac transplants. More
recently, FK-5063 has been introduced as a more potent
immunosuppressant although mechanistically related
to cyclosporin A. These two immunosuppressive com-
pounds block T-cell activation via the inhibition of
interleukin-2 expression.4 Recently, mycophenolate
mofetil5 which inhibits the purine biosynthesis has been
approved for the prevention of kidney graft rejection.
However, although more specific than azathioprine,
these drugs do show nephro-, neuro-, and myelotoxicities
as well as other side effects.6 Therefore, there is a need
for new less toxic compounds and with new mechanisms
of action, which could lead to a more specific immuno-
suppression.
Among the different compounds recently developed
in this area (-)15-deoxyspergualin ((-)DSG)7 appeared
to be attractive considering its biological profile and its
new mechanism of action.8 (-)DSG is a derivative of (-)-
spergualin,9 a fermentation product isolated from Bacil-
lus laterosporus by Umezawa’s group. It was first
obtained by dehydroxylation of (-)spergualin in 1982.10
Its first total synthesis was published in 1987,11 and
despite its low overall yield,12 this process afforded (-)-
DSG and (+)DSG which could be separately evaluated.
However DSG suffers from three main drawbacks
which limit its use. The first one is related to its low
chemical stability in aqueous solution where hydrolysis
of the hydroxyglycine part leads to 7-guanidinoheptan-
amide and glyoxyloylspermidine. This cleavage is a
problem for large scale synthesis, purification, and
standard galenic development. The second problem is
due to the presence of the inactive (+)DSG isomer
present in the racemic mixture. This compound is not
reported to be immunosuppressive but can contribute
to the acute toxicity.7 Until now, industrial synthesis
of the active (-)DSG form or another equivalent achiral
compound was not available. The last limitation is
related to a very low oral bioavailability (less than 5%20),
preventing oral administration.
We present here the first part of our program aimed
at the discovery of new DSG analogues (Chart 1), devoid
of the above-mentioned drawbacks. For this the DSG
structure has been divided into four regions. The
purpose of the present study was to find the optimal
“C” region which could alleviate problems associated
with chirality and hydrolytic sensitivity brought by the
hydroxyglycine moiety. Umezawa and co-workers have
† Deceased.
10.1021/jm980431g CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/08/1999

278 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Lebreton et al.
Ch a r t 1
molecule using concepts encountered in peptide medici-
nal chemistry such as retro-inverso approach and other
bioisosteric replacements for the amide bonds. We also
replaced the hydroxymethylene moiety by various link-
ers (Chart 1).
Ch em istr y
The wide range of structural modifications planned
forced us to use rather diverse synthetic schemes.
Moreover, the choice of these schemes was dictated by
availability of certain starting materials.
The malonamide-related compounds 13a -t and 15
were made by the route illustrated in Schemes 1, 2, and
3 from key intermediates 4a -t, 9, and 10. Compounds
4a -t were prepared in three steps from N,N′-bis(tert-
butoxycarbonyl)-S-methylisothiourea 1 which was ob-
tained according to the procedure described by Bergeron
et al.22 (Scheme 1). Condensation of 1 with hexanedi-
amine led to the intermediate 2 which was coupled to
commercially available monoester malonic acid deriva-
tives using standard coupling techniques. Saponification
of the ester groups led to compounds 4a -t. We used a
shorter procedure (Scheme 2) than the one described
by Bergeron et al.23 to obtain 9. Alkylation of propane
diamine by n-chloroalkanol in refluxing butanol led to
compounds 7a -c with good yields. The amines were
then protected by Boc groups to give 8a -c. Conversion
of alkanol 8a to its methanesulfonate derivative followed
by nucleophilic displacement with ammonia or meth-
ylamine yielded the desired spermidine derivatives 9
and 10. Target compounds 13a -t and 15 were obtained
by the coupling of key intermediates 4a -t with 9 or 4a
with 10 using standard coupling techniques followed by
removal of Boc protecting groups of the intermediates
12a -t or 14 with trifluoroacetic acid (TFA) in CH2Cl2
(Scheme 3).
Sch em e 1a
a
Reagents: (a) hexanediamine, THF; (b) HOOCACOOEt, CH2Cl2,
DCC, HOBT; (c) 1 M NaOH, DME (v/v); (d) amino-6-hexanol, THF;
(e) Et3N, CH2Cl2, -20 °C, methanesulfonyl chloride; (f) DME,
MeNH2 (40%); (g) PDC, CH2Cl2.
already explored the structure-activity relationship
(SAR) of this part of the molecule, synthesizing numer-
ous analogues where the hydroxyglycine has been
replaced by various R-, â-, and γ-amino acids.21 O-
methylhydroxyglycine, L-serine, and glycine were the
only tolerated R-amino acids. γ-Aminobutyric and γ-ami-
no R- or â-hydroxybutyric acids were also tolerated, but
none of these derivatives were as active as DSG.
â-Amino acids and all other R-amino acids led to inactive
compounds. This stringent structural requirement for
this “C” region is remarkable. However, all these
analogues retained the two amide bonds with the same
orientation as in the parent DSG. We thus decided to
extend the investigation of this crucial part of the
The synthetic sequences utilized for the preparation
of derivatives 19a -c, 21a -b, 23, 35, and 42 are
outlined in the Scheme 4. The key intermediates 17a -c
were obtained by coupling 2 with N-benzyloxycarbon-
ylglycine, N-benzyloxycarbonyl-N-methyl-glycine, or
N-benzyloxycarbonyl-â-alanine using standard coupling
techniques followed by cleavage of the Cbz protecting
group with Pd/C under a hydrogen atmosphere. The
synthesis of urea derivatives (19a -c) was best achieved
using a strategy involving condensation of 17a -c with
bis(4-nitrophenyl)carbonate followed by the action of 9
to give the N-Boc protected compounds 18a -c. Final
Sch em e 2a
a
Reagents: (a) KI, K2CO3, n-BuOH reflux; (b) (Boc)2O, dioxane, H2O; (c) Et3N, CH2Cl2, -20 °C, methanesulfonyl chloride; (d) EtOH,
NH4OH (v/v); (e) EtOH, MeNH2; (f) PDC, DMF.

SAR of New Analogues of 15-Deoxyspergualin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 279
Sch em e 3a
chloroformate led to the intermediate 32. Saponification
of 32 with NaOH in DME followed by coupling with 9,
according to the standard techniques, and deprotection
afforded 35. Compound 42 was prepared via the key
intermediate 40 which was obtained by the coupling of
2 with thioglycolic acid. Compound 40 was further
converted to the thiocarbamate 42 by reaction of the
nonisolated nitrophenyl thiocarbonate with 9 and depro-
tection with the standard acidic procedure.
The synthetic route leading to the DSG analogues
27a -d , where the carbamate function is reversed
compared to 23, is outlined in Scheme 5. Acylation of
spermidine derivatives 9 or 10 (Scheme 2) with phenyl
carbonates derived from methyl hydroxyacetate or hy-
droxypropionate afforded, after saponification, 25a -c.
These acids were converted to the final products 27a -d
by coupling with 2 or 6 (Scheme 1) followed by removal
of protecting groups.
The compounds 31, 39, 46, 50, 55, and 60 were made
starting from 9 using specific routes illustrated in
Scheme 6 and which are explained below. The inter-
mediate 10-[(phenylmethoxy)carbonylamino]decanoic acid
was coupled with 9 using standard coupling technique
to give 28. Removal of the Cbz group by hydrogenolysis,
condensation with 1, and final deprotection yielded the
desired compound 31. Carbobenzyloxyglycine was coupled
to 9, and the Cbz group of the coupling product 36 was
then cleaved. The obtained intermediate was then
a
Reagents: (a) DCC, HOBT, CH2Cl2; (b) TFA, CH2Cl2 (v/v).
deprotection was then performed to provide the desired
DSG analogues 19a -c. In the same way, we adopted a
similar strategy for the synthesis of the carbamate 23.
To the nonisolated intermediate nitrophenyl carbonate,
generated by reaction of 4-nitrophenyl chloroformate
with 8a , was added 17a to afford 22, which was further
deprotected to yield the desired carbamate derivative
23. Oxidation of alkanol 8b-c by pyridinium dichro-
mate in DMF provided the carboxylic derivatives 11a -b
(Scheme 2). Compounds 21a -b were obtained via the
coupling of 17a with intermediates 11a -b to yield 20a -
b. Cleavage of the Boc protecting groups was then
carried out by the standard method, i.e., TFA in CH2-
Cl2 to give the target compounds 21a -b.
Addition of 2 to the methyl [(phenoxycarbonyl)oxy]
acetate generated from methyl glycolate and phenyl
Sch em e 4a
a
Reagents: (a) PhCH2OCONR(CH2)nCO2H with n ) 1, 2 and R ) H, CH3, isobutylchloroformate, THF, -20 °C, NMM; (b) Pd/C 10%,
EtOH, H2; (c) bis-(4-nitrophenyl)carbonate, THF, 9; (d) CH2Cl2, TFA (v/v); (e) 11a or 11b, isobutylchloroformate, THF, -30 °C, NMM; (f)
8a , Et3N, THF, 4-nitrophenyl chloroformate; (g) methyl[(phenoxycarbonyl)oxy]acetate, toluene, 40 °C; (h) 1 M NaOH, DME (v/v); (i) DCC,
HOBT, CH2Cl2, 9; (j) thioglycolic acid, carbonyldiimidazole, CH2Cl2; (k) 4-nitrophenyl chloroformate, Et3N, THF then 9.
Sch em e 5a
a
Reagents: (a) PhOCO2(CH2)nCO2Me with n ) 1, 2, toluene, reflux; (b) 1 M NaOH, DME (v/v); (c) DCC, HOBT, CH2Cl2, 2 or 6; (d)
CH2Cl2, TFA (v/v).

280 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Lebreton et al.
Sch em e 6a
a
Reagents: (a) CbzHN(CH2)9CO2H, CHCl3, DCC, HOBT; (b) Pd/C 10%, EtOH, H2; (c) 1, THF; (d) CH2Cl2, TFA (v/v); (e) carbobenzyl-
oxyglycine, CHCl3, DCC, HOBT; (f) 4-nitrophenyl chloroformate, THF, Et3N, 2; (g) methyl 2-(chlorosulfonyl)acetate, CH2Cl2, -20 °C,
Et3N; (h) 1 M NaOH, DME (v/v); (i) carbonyldiimidazole, CH2Cl2, Et3N, 2; (j) ethyl hydrogen malonate, CHCl3, DCC, HOBT; (k) CH2Cl2,
DCC, HOBT, 6; (l) methylacrylate, MeOH reflux; (m) (Boc)2O, Et3N, THF; (n) isobutyl chloroformate, THF, -30 °C, NMM, 2; (o)
PhOCO2(CH2)2NHCbz, Et3N, toluene; (p) EtOH, 56, AcOH then NaBH3CN.
Sch em e 7a
a
Reagents: (a) 6-bromohexanoic acid, CHCl3, DCC, HOBT; (b) EtOH, reflux, KCN; (c) 1 M NaOH, DME (v/v); (d) H2, Ni Raney, EtOH,
1 M NaOH; (e) EtOH, NaHCO3, benzylchloroformate; (f) Et3N, THF, DPPA then 8a , toluene, reflux; (g) H2, Pd/C 5%, EtOH; (h) THF, 1;
(i) CH2Cl2, TFA (v/v).
reacted with 4-nitrophenylchloroformate to give a reac-
tive carbamate derivative which in the presence of 2 at
room temperature provided the desired protected com-
pound. Final Boc protecting groups cleavage was then
performed to give 39.
Methyl methyl 2-(chlorosulfonyl)acetate reacted with
9 in dichloromethane to afford the key intermediate 43.
Then usual sequence involving saponification, amide
bond coupling of 2 using carbonyldiimidazole, and final
deprotection was performed to obtain 46.
The reactions carried out to obtain 50 are similar to
those used for 46; however, they include coupling 6
instead of 2 and using the DCC, HOBT procedure.
Michael addition of 9 to methylacrylate in refluxing
methanol afforded the key intermediate 51 in which the
amine was further protected by a Boc group. Saponifica-
tion, coupling with 2 using the mixed anhydride method,
and final deprotection allowed us to obtain the desired
compound 55. N-carbobenzyloxy-2-aminoethanol re-
acted with phenyl chloroformate in toluene to afford the
reactive carbonate which in the presence of 9 led to the
intermediate 57. Removal of Cbz group of 57 by hydro-
genolysis afforded the corresponding amine which was
converted to the final compound 60 by a reductive
amination reaction with 56 (see Scheme 1) in the
presence of NaBH3CN followed by cleavage of Boc
protecting groups.
The synthetic sequence leading to 68 with a reversed
amide bond compared to 23 is outlined in Scheme 7.
N-(6-bromohexanoyl)-glycine ethyl ester was obtained
by coupling bromohexanoic acid with glycine ethyl ester.
Nucleophilic substitution of the bromine by KCN fol-
lowed by saponification of the ester allowed the produc-
tion of the acid 62. Hydrogenation of the nitrile group
carried out with Raney nickel in the presence of sodium
hydroxide afforded the amine which was protected by
a Cbz group to give 64. The crucial conversion of 64 to
66 was carried out as follows: reaction of 64 with
diphenylphosphorazide gave the corresponding acyl
azide derivative which upon Curtius rearrangement in
toluene at reflux led to the corresponding isocyanate
which was further reacted with 8a (Scheme 2) to give
66 after cleavage of the Cbz protecting group. Reaction
of the primary amine of 66 with 1 (Scheme 1) in THF
and further acidic cleavage of the Boc protecting groups
afforded the desired diacylaminal compound 68.

SAR of New Analogues of 15-Deoxyspergualin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 281
Ta ble 1. Physicochemical and Biological Data of 15-DSG Analogues with X and Y Variations
GVHD mean survival
HRMS
(days) (M ( SD)a
compd
X
Y
scheme
calcd
found
3 mg/kgb
1 mg/kgb
21a
13a
31
35
39
55
46
19a
21b
27a
23
68
42
27b
50
15
19c
27c
60
NHCO
NHCO
CH2CH2
NHCOO
NHCONH
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
NHCO
NMeCO
NMeCO
NHCO
NHCO
NHCO
NHCH2
NHCO
CONH
CONH
CONH
CONH
CH2NH
SO2NH
NHCONH
NHCOCH2
OCONH
NHCOO
NHCOO
SCONH
OCONH
CONH
CONMe
NMeCONH
OCONMe
OCONH
4
3
6
4
6
6
6
4
4
5
4
7
4
5
6
3
4
5
6
372.3087
372.3087
357.3342
388.3036
387.3196
358.3294
408.2757
387.3196
386.3243
388.3036
388.3036
388.3036
404.2808
402.3193
386.3243
386.3243
401.3352
402.3193
374.3243
372.3089
372.3086
357.3339
388.3038
387.3200
358.3294
408.2755
387.3173
386.3243
388.3037
388.3035
388.3034
404.2805
402.3193
386.3247
386.3246
401.3352
402.3192
374.3245
22 ( 6
52 ( 20
16 ( 1
13 ( 1
24 ( 7
13 ( 4
15 ( 1
48 ( 12
18 ( 4
58 ( 6
48 ( 16
58 ( 5
17 ( 2
14 ( 1
14 ( 1
12 ( 2
17 ( 4
15 ( 2
34 ( 9
53 ( 13
42 ( 21
46 ( 10
55 ( 4
32 ( 3
a
The mean survival of control is 15 ( 2 days and, respectively, 38 ( 12 and 57 ( 7 days for animals treated with DSG at 1 mg/kg and
b
3 mg/kg. All compounds were administered daily by i.p. route.
Resu lts a n d Discu ssion
of activity as that of DSG. However, NH cannot be
replaced by a methylene (21b). It is worth noting that
compound 23 was the first example of a very active
analogue of DSG not encompassing the full spermidine
moiety. The carbamate is clearly a very suitable function
in this position whatever the orientation of the “left
amide” (see activity of 68). Interestingly the correspond-
ing thiocarbamate (42) was inactive, possibly due to its
poor stability at physiological pH. N-Methylation of
either the amides 27b, 50, and 15, the urea 19c, or the
carbamate 27c was absolutely detrimental. Finally,
reduction of the “left amide” of 27a gave rise to the
slightly active compound 60.
Biological and analytical data are shown in Tables 1,
2, and 3. Immunosuppressive activity was assayed in
vivo in a mouse GVHD model which was adapted from
the literature.24 Briefly, GVHD is induced at day 0,
treatments are then given from day 1 to day 10 (day 6
omitted), and survival is followed until sacrifice at day
60. The mean survival of the controls is 15 ( 2 days. In
such conditions, DSG administered by i.p. route in-
creases survival to 38 ( 12 days at 1 mg/kg/day and to
57 ( 7 days at 3 mg/kg/day. All compounds were then
studied by the same route first at 3 mg/kg/day and then
at 1 mg/kg/day if they demonstrated a significant
activity. The first step of our investigation dealt with
the modification of the two amide bonds. Our goal was
to get more stable compounds that were easier to
synthesize and devoid of chirality. Thus, we decided to
make glycine instead of hydroxyglycine analogues of
DSG. We first synthesized 21a (Table 1) where the two
amide bonds are reversed compared to DSG. This
modification which destroys the spermidine integrity
completely abolishes the immunosuppressive activity.
Table 2 shows results obtained with analogues of 13a ,
19a , and 27a where the nature and length of the central
linker have been modified. Obviously a single bond (13e)
or a methylene (13a ) were the only tolerated linkers. It
is interesting to notice that a CH2CH2 linker leads to a
totally inactive compound (13c) when its replacement
with a CH2O (27a ) affords one of the most active
derivatives of this series (Table 1).
In Umezawa’s derivatives, the presence of OH or, to
a lesser extent, of OCH3 in the “C” region increases the
potency compared to hydrogen. We investigated (Table
3) the effect of such groups or related ones such as F,
NH2, or NHAc on compound 13a . We also introduced
lipophilic aliphatic and aromatic substituents. Finally
we made some disubstituted derivatives. In sharp
contrast to the DSG series, introduction of OH (13s) or
OCH3 (13i) does not really increase activity. However,
only OH, OCH3, or F (13t) groups are tolerated in
contrast to NH2 (13p ), NHAc (13o), or OCH2Ph (13r )
which, though susceptible to sharing a hydrogen bond,
are detrimental for activity.
However 13a , where only the “left amide” was re-
versed compared to DSG, was active at 3 and 1 mg/kg
i.p. Other replacements of the “left amide” such as a
two-methylene unit (31), a carbamate (35), or an urea
(39) gave inactive compounds. Similarly, bioisosteric
replacement of the “right amide” with a reduced amide
(55) or a sulfonamide (46) was detrimental to activity.
By contrast, 19a , which can be seen as an analogue of
inactive compound 21a where the nitrogen of spermi-
dine has been reintroduced giving a urea, was active at
3 mg/kg i.p.. Further modification of the urea of 19a
was successful and afforded potent and less toxic
analogues.25 Either NH can be favorably replaced by an
O atom leading to 27a and 23 which were very active
at 3 and 1 mg/kg i.p. and which were in the same range
Disubstitution with OCH3 (13q) and CH2OH (13l) is
also totally ineffective. Finally, as it is the case for DSG
series, alkyl or aryl substituents led to inactive com-
pounds 13g, 13h , 13k , 13m , and 13n .

282 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Lebreton et al.
Ta ble 2. Physicochemical and Biological Data of 15-DSG Analogues with A Modifications
GVHD mean survival
(days) (M ( SD)a
HRMS
compd
A
scheme
calcd
found
3 mg/kgb
1 mg/kgb
13b
13c
13d
13f
13j
13e
19b
27d
CHdCH (Z)
CH2CH2
CH2CH2CH2
CHdCH (E)
CH2CH (OH)
3
3
3
3
3
3
4
5
384.3087
386.3243
400.3400
384.3087
402.3193
358.2930
401.3352
402.3193
384.3084
386.3246
400.3403
384.3086
402.3184
358.2932
401.3350
402.3196
17 ( 3
16 ( 4
15 ( 1
17 ( 8
15 ( 1
34 ( 8
15 ( 3
13 ( 1
53 ( 13
CH2CH2NH
CH2CH2O
a,b
See footnotes in Table 1.
Ta ble 3. Physicochemical and Biological Data of Malonic Derivatives with R1 and R2 Substituents
GVHD mean survival
(days) (M ( SD)a
HRMS
compd
R1
R2
calcd
found
3 mg/kgb
1 mg/kgb
13s
13i
13t
OH
OCH3
F
H
H
H
H
H
H
388.3036
402.3193
390.2993
387.3196
429.3302
478.3506
432.3298
432.3298
386.3243
400.3400
448.3400
462.3556
400.3400
388.3030
402.3193
390.2996
387.3196
429.3299
478.3501
432.3298
432.3296
386.3243
400.3399
448.3403
462.3553
400.3407
53 ( 13
55 ( 12
60 ( 0
16 ( 1
16 ( 1
14 ( 2
12 ( 3
17 ( 2
16 ( 2
19 ( 3
19 ( 4
16 ( 2
15 ( 1
12 ( 1
27 ( 3
18 ( 11
13p
13o
13r
13q
13l
13h
13m
13k
13g
13n
NH2
NHAc
OCH2C6H5
OCH3
CH2OH
CH3
CH2CH3
C6H5
CH2C6H5
CH3
OCH3
CH2OH
H
H
H
H
CH3
a,b
See footnotes in Table 1.
Ta ble 4. Prevention of Graft Rejection after Heart Allotransplantation in the Dark Agouti to Lewis Rat Combination
dosea
(mg/kg)
mean survival
(day) (M ( SD)
statistical
analysisb
compd
survival (day)
6, 6, 6, 7, 7, 7, 7
13, 20, 20, 26, 42, 46, >100
17, 27, 27, 45, 63, 99, >100
5, 7, 8, 10, 10, 13
11, 11, 11, 11, 11, 11, 15
6, 6, 12, 12, 13, 15, 17
15, 20, 24, 25, 28, 37, 79
control
DSG
27a
13a
13e
0
6
6
6
6
6
6
7 ( 1
38 ( 30
54 ( 34
9 ( 3
12 ( 2
12 ( 4
33 ( 22
S
S
NS
S
S
19a
13s
S
a
b
All compounds were administered daily by i.p. route for 10 days starting the day after surgery. Manntel and Haenszel test according
to SAS (Cary, NC).
Selection of the best immunosuppressor compounds
obtained in this study was then made using a more
demanding model. Some of the more active compounds
were then tested in a model for acute organ rejection
after heart allotransplantation in the rat. Genetic
combination was selected to be completely histoincom-
patible, and the recipient was considered to be a high
responder in which control of rejection would be espe-
cially difficult.26,27 DSG induces a 38 ( 30 days survival
of the graft in these experimental conditions (10 days,
6 mg/kg, i.p.) whereas rejection is observed at 7 ( 1 days
in controls (Table 4). Despite its good activity in the
mouse GVHD test, 13a was unable to control graft
rejection in rats at 6 mg/kg. Compounds 13e and 19a
were only slightly active in this model, showing that it
is a good discrimination step. However 13s, which can
be seen as a retro-analogue of DSG, induced a 4- to
5-fold graft survival increase compared to the control.
Finally, 27a proved to be the best tested compound of
the series, being slightly more active than DSG. This
compound was thus selected as a candidate for further
preclinical investigation. In particular, it proved to be
far more stable than DSG as assessed by its chemical
stability studies. Figure 1 shows the kinetics of degra-
dation of DSG and 27a in aqueous solution at different
pH at 37 °C. DSG chemical stability is clearly pH
dependent. Its half-life is around 15 days at pH 1, 2 days
at pH 7, and a few hours at pH 10. As anticipated, the
stability of 27a is much better: at acidic or neutral pH,
27a degradation is less than 10% after 50 days. However
at pH 10, its half-life is around 1 day. This high
improvement in chemical stability makes the handling

SAR of New Analogues of 15-Deoxyspergualin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 283
Exp er im en ta l Section
1
Ch em istr y. H Nuclear magnetic resonance spectra were
obtained on a Bruker 300 at 300 MHz. The chemical shifts
are expressed in δ values (part per million) relative to
tetramethylsilane as internal standard. Significant 1H NMR
data are reported in the following order: multiplicity (s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet), number of
protons.
The identity of final compounds was verified by exact mass
spectral determinations performed by the University of Rennes
I (Centre Re´gional de Mesures Physiques de l’Ouest). High-
resolution mass spectra (HRMS) and low resolution mass
spectra (LRMS) were performed using a ZABSpec TOF from
V. G. Analytical (L-SIMS ionization mode, Cs+, mNBA).
Thin-layer chromatography was performed on silica gel
60F254 plates or RP-18F254S (Merck). Merck silica gel 60 (230-
400 mesh) was used for flash column chromatography and
lichroprep RP-18 Merck (5-20 µm) for medium-pressure liquid
chromatography.
DSG was prepared following Umezawa’s procedure.14
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
13a -t a n d 15. Compounds of the general structure 13a -t
(Scheme 3) were synthesized from carboxylic acid derivatives
4a -t, and 4-aminobutyl[3-[(1,1-dimethylethoxy)carbonylami-
no]propyl]carbamic acid, 1,1-dimethyl-ethyl ester 9 by the
representative procedure illustrated for analogue 13a .
Carboxylic acid derivatives 4a -t were obtained in three
steps (Scheme 1) from N,N′-bis(tert-butoxycarbonyl)-S-methoxy
isothiourea 1 which was prepared according to the procedure
described by R. J . Bergeron and J . S. MacManis.23
The compounds 9 and 10 were prepared in three steps as
follows (Scheme 2).
F igu r e 1. The chemical stability of DSG and 27a in aqueous
buffers at pH 1 (0), pH 7 (]), and pH 10 (O) at 37 °C.
4-[(3-Am in op r op yl)a m in o]bu ta n ol (7a ). 4-Chloro-1-bu-
tanol (2.2 g, 20.0 mmol) was added into a solution of 1,3-
diaminopropane (3.0 g, 40.0 mmol), potassium iodide (0.33 g,
2.0 mmol), and potassium carbonate (1.4 g, 10.0 mmol) in
butanol (50 mL). The mixture was stirred at reflux for 24 h.
Then the mixture was slowly cooled to room temperature,
filtered, and concentrated under vacuum to afford 7a as a
colorless oil which was used without further purification (2.8
g, 86% yield). 1H NMR (DMSO-d6) δ: 1.4-1.5 (m, 6H), 2.4-
2.6 (m, 6H), 3.4-3.5 (m, 2H).
of 27a much easier in aqueous medium. In particular,
purification, formulation, and dosing can be carried out
without any problem of degradation. This practical
aspect of this freely water soluble compound intended
to be administered by injection is crucial for its develop-
ment.
(4-Hyd r oxybu tyl)[3-(1,1-d im eth yleth oxy)ca r bon yla m i-
n o]p r op yl]ca r ba m ic Acid , 1,1-Dim eth yleth yl Ester (8a ).
This compound was obtained from 4-[(3-aminopropyl)amino]-
butan-1-ol (2 g, 13.67 mmol) according to the procedure
described by F. Veznik et al.31 as a colorless oil (4.45 g, 94%
yield).
(4-Am in obu tyl)[3-[(1,1-d im eth yleth oxy)ca r bon yla m i-
n o]p r op yl]ca r ba m ic Acid , 1,1-Dim eth yleth yl Ester (9).
To a stirred solution of 8a (1 g, 2.9 mmol) and triethylamine
(0.7 g, 3.76 mmol) in CH2Cl2 (10 mL) cooled to -20 °C was
added dropwise a solution of methanesulfonyl chloride (0.43
g, 3.76 mmol) in CH2Cl2 (5 mL). The stirring was continued
at -20 °C for 10 min, and then the reaction mixture was
allowed to warm to room temperature. An additional portion
of CH2Cl2 was added, and the solution was washed with
saturated K2CO3 solution. The layers were separated, and the
aqueous phase was extracted with CH2Cl2. The organic layers
were combined, dried over MgSO4, filtered, and concentrated
under vacuum.
The obtained crude product was taken up in ethanol (20 mL)
and ammonium hydroxide (20 mL). The reaction mixture was
then stirred for 48 h at room temperature. The solvents were
removed under vacuum, and the oily residue was purified by
flash chromatography on silica gel (EtOAc then EtOAc/EtOH/
NH4OH 6/3/0.1) to afford 9 as a clear oil (0.46 g, 46%). 1H NMR
(CDCl3) δ: 1.4-1.6 (m, 24H), 2.7 (t, 2H), 3.1-3.25 (m, 6H),
4.8 (s, 1H), 5.3 (s, 1H).
[4-(Meth yla m in o)bu tyl] [3-[(1,1-d im eth yleth oxy)ca r -
bon yl a m in o]p r op yl]ca r ba m ic Acid , 1,1-Dim eth yleth yl
Ester (10). Starting with 8a , the same procedure described
for the synthesis of 9 but using methylamine as nucleophilic
agent was applied to 10 obtained in 72% yield. 1H NMR
Con clu sion
In the present study we have synthesized a series of
new DSG analogues in order to investigate the struc-
tural requirement for the so-called “C” region of the
molecule in term of immunosuppressive activity. It
proved to be rather stringent. However, we have shown
that the left amide bond can be present in both direc-
tions but other functions such as urea, carbamate, and
a two methylene unit are not compatible. N-Methylation
of this amide is also precluded. Nevertheless the right
amide bond can be favorably replaced by a carbamate
in both directions and less successfully with an urea.
N-Methylation of these functionalities led to inactive
compounds. The central linker should be either a single
bond or a methylene which can also be monosubstituted
by a methoxy or hydroxyl group or by a fluorine atom.
This study allowed us to select some DSG analogues
which have the same range of potency as DSG without
containing chirality. Moreover, their chemical stability
was strongly improved in comparison to DSG, which
makes their industrial synthesis much more accessible.
Due to its optimum biological profile in terms of immu-
nosuppressive properties in the heart allograft model28,29
and in a GVHD model after bone marrow transplanta-
tion30 and also in terms of safety pharmacology and
toxicology, 27a was selected as a drug candidate and is
currently undergoing clinical evaluation.

284 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Lebreton et al.
(CDCl3) δ: 1.4-1.8 (m, 24H), 2.7 (s, 3H), 2.9-3.3 (m, 8H), 4.8-
5.4 (br s, 1H), 8.1-9.5 (br s, 1H).
solvents were then removed under reduced pressure. The
obtained residue was taken up in 150 mL of distilled water
and then lyophilized. The residue was purified by MPLC
(medium-pressure liquid chromatography) on a RP18 silica
(type grafted, particle size 5 to 20 µm) using a water/CH3CN/
TFA mixture (7/2/1, v/v/v) as eluent to afford 13a (0.43 g, 66%
5-[N-[3[(1,1-d im eth yleth oxy)ca r bon yla m in o]-p r op yl]-
N-[(d im et h ylet h oxy)ca r b on yl]-a m in o]-p en t a n oic Acid
(11a ). Pyridinium dichromate (77.5 g, 206.0 mmol) was added
in small portions to a solution of 8b (21.2 g, 58.88 mmol) in
DMF (100 mL). The reaction medium was stirred for 6 h at
room temperature, then diluted with H2O (700 mL) and
extracted with Et2O (3 × 150 mL). The organic phase was
washed with a CuSO4 solution, dried, concentrated, and
purified by flash chromatography on silica gel (EtOAc/hexane
5/5 then EtOAc) to afford 11a (20.0 g, 91% yield) as a yellow
oil. 1H NMR (CDCl3) δ: 1.4-1.7 (m, 24H), 2.3 (t, 2H), 3.0-3.3
(m, 6H), 4.7 (br s, 1H), 5.3 (br s, 1H).
[(6-Am in oh exyl)car bon im idoyl]bis-car bam ic Acid, Bis-
(1,1-d im eth yleth yl) Ester (2). Hexane-1,6-diamine (17.23 g,
0.148 mol) was added at room temperature to a stirred solution
of N,N′-bis (tert-butoxycarbonyl)-S-methylisothiourea (43.0 g,
0.148 mol) in THF (300 mL). The reaction medium was stirred
for 16 h. After evaporation of the solvent, the obtained residue
was purified by flash chromatography on silica gel (CHCl3/
EtOH 3/1 then EtOAc/MeOH/32% aqueous ammonia mixture
6/3/0.1) to afford 2 (19.7 g, 37%) as a yellow oil. 1H NMR
(CDCl3) δ: 1.25-1.60 (m, 28H), 2.7 (t, 2H), 3.5 (q, 2H), 8.3 (t,
1H), 11.5 (s, 1H).
3-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-12-oxo-2,4,-
11-tr ia za -tetr a d ec-2-en ed ioic Acid , 1-[1,1-Dim eth yleth yl
Ester ]14-eth yl Ester (3a ). To a stirred solution of ethyl
hydrogen malonate (3.7 g, 28.0 mmol) in CH2Cl2 (50 mL) were
added at 0 °C 1,3-dicyclohexyl carbodiimide (DCC) (5.8 g, 28.0
mmol) and 1-hydroxybenzotriazole hydrate (HOBT) (0.4 g, 3
mmol). The mixture was allowed to stand for half an hour at
0 °C. Then 2 (5 g, 14 mmol) in solution in CH2Cl2 (30 mL) was
added dropwise at 0 °C. After the addition was complete, the
mixture was allowed to warm gradually to room temperature
overnight. The solvent was evaporated off under reduced
pressure, and the oily residue was purified by flash chroma-
tography on silica gel (EtOAc/methylcyclohexane 1/1) to afford
3a (6.27 g, 95% yield) as a viscous oil. 1H NMR (CDCl3) δ: 1.2-
1.7 (m, 29H), 3.2-3.3 (m, 4H), 3.4 (q, 2H), 4.2 (q, 2H), 7.2 (br
s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
3-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-12-oxo-2,4,-
11-tr ia za -tetr a d ec-2-en ed ioic Acid , 1-[1,1-Dim eth yleth yl
Ester ] (4a ). 3a (1.97 g, 4.19 mmol) was dissolved in a mixture
of 1 N NaOH (20 mL) and DME (20 mL). The reaction mixture
was stirred for 1 h at room temperature, concentrated to a
third of its volume, and then acidified to pH 2 with 1 N HCl.
Extraction of the water layer with 2 × 50 mL of CHCl3,
followed by evaporation of the organic phases under reduced
pressure, afforded a residue which was purified by flash
chromatography on silica gel (EtOAc/EtOH 1/9) to give 4a (1.29
g, 70%). 1H NMR (CDCl3) δ: 1.2-1.7 (m, 24H), 3.1-3.4 (m,
6H), 7.7 (br s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
1
yield) in the form of a translucent white amorphous solid. H
NMR (DMSO-d6) δ: 1.2-1.6 (m, 12H), 1.9 (m, 2H), 2.9-3.1
(m, 14H), 7.2 (br s, 4H), (t, 1H), 8 (m, 5H), 8.7 (br s, 2H). 13C
NMR (DMSO-d6) δ: 22.5, 23.4, 25.4, 25.6, 25.7, 28.0, 28.5, 35.8,
37.6, 38.2, 40.3, 43.0, 43.5, 46.1, 156.6, 166.9 (2C).
3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-20-[(1,1-d i-
m et h ylet h oxy)ca r b on yl]-15-m et h yl-12,14-d ioxo-2,4,11,-
15,20,24-h exa a za p en t a cos-2-en ed ioic Acid , Bis(1,1-d i-
m eth yleth yl) Ester (14). 14 (1,15 g, 65%) was obtained in
the form of an oil according to the method described for 12a
starting from 4a (1 g, 2.25 mmol) and 10 (0.86 g, 2.4 mmol).
1H NMR (CDCl3) δ: 1.3-1.7 (m, 50H), 3.1-3.3 (m, 15H), 3.4
(q, 2H), 4.8 and 5.3 (br s, 1H), 6.8 (br s, 1H), 8.3 (t, 1H), 11.5
(s, 1H).
N-[4-[(3-Am in op r op yl)a m in o]bu tyl]-N-[6[(a m in oim in o-
m eth yl)a m in o]h exyl]-N-m eth yl-p r op a n ed ia m id e, Tr is-
(tr iflu or oa ceta te) (15). 15 (0.72 g, 72%) was obtained as an
oil according to the method described for 13a starting from
14 (1 g, 1.27 mmol). 1H NMR (DMSO-d6) δ: 1.2-1.6 (m, 12H),
1.8-2.0 (m, 2H), 2.8-3.1 (m, 13H), 3.2-3.4 (m, 4H),7.8-8.0
(m, 3H), 8.0-8.1 (m, 1H), 8.5-8.8 (m, 4H). 13C NMR (DMSO-
d6) δ: 23.5, 24.1, 24.5, 26.2, 26.3, 26.4, 28.1, 28.9, 35.2, 36.5,
37.3, 40.3, 41.9, 45.2, 48.2, 158.5, 168.6, 168.8.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
27a -d . Compounds of the general structure 27a -d (Scheme
5) were synthesized from carboxylic acid derivatives 25a -d
and 2 or 6 by the representative procedure illustrated for 27a .
Carboxylic acid derivatives 25a -d were obtained in two steps
from 9 or 10. 6 was prepared in three steps as follows (Scheme
1).
P r ep a r a tion of Com p ou n d 6. [(6-Hyd r oxyh exyl)ca r -
bon im idoyl]bisca r ba m ic Acid , Bis(1,1-dim eth yleth yl) Es-
ter (5). Amino-6 hexanol (4.24 g, 35.0 mmol) was added at
room temperature, with stirring, to a solution of N,N′-bis (tert-
butoxycarbonyl)-S-methylisothiourea (9,54 g, 32.8 mmol) in
THF (100 mL). The reaction medium was stirred for 4 days.
After evaporation of the solvent, the obtained residue was
purified by flash chromatography on silica gel (methylcyclo-
hexane/EtOAc 7.5/2.5) to afford 5 (9.05 g, 77% yield). 1H NMR
(CDCl3) δ: 1.2-1.7 (m, 26H), 3.35-3.5 (m, 2H), 3.6-3.7 (m,
2H), 8.2-8.4 (br s, 1H), 11.5 (br s, 1H).
[(6-Am in om e t h ylh e xyl)ca r b on im id oyl]b isca r b a m ic
Acid , Bis(1,1-d im eth yleth yl) Ester (6). To a stirred solution
of 5 (2 g, 5.57 mmol) and triethylamine (1.4 g, 13.8 mmol) in
CH2Cl2 (20 mL) cooled to - 20 °C was added dropwise a
solution of methanesulfonyl chloride (0.8 g, 7 mmol) in CH2-
Cl2 (5 mL). The stirring was continued at - 20 °C for 15 min,
and the mixture was then allowed to warm to room temper-
ature. An additional portion of CH2Cl2 was added, and the
solution was washed with a saturated K2CO3 solution. The
layers were separated, and the aqueous phase was extracted
with CH2Cl2. The organic layers were combined, dried over
MgSO4, filtered, and concentrated under vacuum. The obtained
crude product was taken up in DME (5 mL) and methylamine
in aqueous solution (40%) (6.2 mL, 80 mmol). The reaction
mixture was then stirred for 18 h. The solvents were removed
under vacuum, and the oily residue was taken up in EtOAc
(100 mL) and washed with a brine solution. The organic layer
was dried over MgSO4, filtered, and concentrated under
vacuum. The obtained oily residue was purified by flash
chromatography on silica gel (EtOAc/EtOH/NH4OH 6/3/0.1) to
3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-20-[(1,1-d i-
m eth yleth oxy)car bon yl]-12,14-dioxo-2,4,11,15,20,24-h exa-
a za p en ta cosen ed ioic Acid , Bis(1,1-d im eth yleth yl) Ester
(12a ). To a stirred solution of 4a (1.25 g, 2.8 mmol) in CH2Cl2
(20 mL) were added at 0 °C DCC (0.57 g, 2.8 mmol) and HOBT
(0.07 g, 0.5 mmol). The mixture was allowed to stand for 0.5
h at 0 °C. Then 9 (1 g, 2.9 mmol) in solution in CH2Cl2 (10
mL) was added dropwise at 0 °C. After the addition was
complete, the mixture was allowed to warm gradually to room
temperature and left to stand one night. The solvent was
evaporated off under reduced pressure, and the obtained
residue was purified by flash chromatography on silica gel
(EtOAc/methylcyclohexane 1/1 then EtOAc/MeOH 9/1) to
afford 12a (1.6 g, 73%) as a viscous oil. 1H NMR (CDCl3) δ:
1.3-1.7 (m, 50H), 3.1-3.3 (m, 12H), 3.4 (q, 2H), 4.8 and 5.3
(br s, 1H), 6.8 and 7.15 (br s, 2H), 8.3 (t, 1H), 11.5 (s, 1H).
1
afford 6 as a yellow oil (1.7 g, 83% yield). H NMR (CDCl3) δ:
1.3-1.7 (m, 26H), 2.50 (s, 3H); 2.68 (t, 2H), 3.40 (t, 2H), 8.3 (t,
1H), 11.5 (s, 1H).
N-[4-[[3-(Am in o)p r op yl]a m in o]bu tyl]-N′-[6-[(a m in oim -
in om eth yl)a m in o]h exyl]p r op a n ed ia m id e, Tr is(tr iflu o-
r oa ceta te) (13a ). 12a (0.7 g, 0.9 mmol) was dissolved in 10
mL of TFA and 10 mL of anhydrous CH2Cl2. The reaction
mixture was stirred for 24 h at room temperature, and the
P r epar ation of Com pou n d 27a. 6-[(1,1 Dim eth yleth oxy)-
ca r b on yl]-13-oxa -12-oxo-2,6,11-t r ia za p en t a d eca n ed ioic
Acid , 1-(1,1-Dim eth yleth yl) Ester 15-Meth yl Ester (24a ).
A solution of 9 (7.4 g, 21.4 mmol) in toluene (20 mL) was added

SAR of New Analogues of 15-Deoxyspergualin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 285
to a stirred solution of methyl [(phenoxycarbonyl) oxy] acetate
(4.5 g, 21.4 mmol) in toluene (100 mL). The reaction medium
was refluxed for 15 h. The solvent was evaporated off under
reduced pressure, and the obtained residue was purified by
flash chromatography on silica gel (methylcyclohexane/EtOAc
7/3 and then EtOAc) to afford 24a (8.4 g, 85%) as an oil. 1H
NMR (CDCl3) δ: 1.45-1.65 (m, 24H), 3.05-3.25 (m, 8H), 3.8
(s, 3H), 4.7 (s, 2H), 4.9 and 5.3 (br s, 1H).
13 mmol) in anhydrous THF (50 mL) was added dropwise.
Stirring was continued for 24 h at room temperature, and the
solvent was evaporated off under reduced pressure. The
obtained residue was purified by flash chromatography on
silica gel (EtOAc) to afford 18a (6.01 g, 64% yield) as an oil.
1H NMR (CDCl3) δ: 1.3-1.7 (m, 50H), 3.1-3.35 (m, 12H), 3.8
(d, 2H), 4.8 and 5.8 (br s, 3H), 6.9 (t, 1H), 8.3 (t, 1H); 11.5 (s,
1H).
6[(1,1-Dim eth yleth oxy)ca r bon yl]-13-oxa -12-oxo-2,6,11-
tr ia za p en ta d eca n ed ioic Acid , 1-(1,1-Dim eth yleth yl Es-
ter ) (25a ). This compound was obtained (6.7 g, 81% yield) as
an oil by following a procedure analogous to preparation of
4a and starting from 24a (8.45 g, 18.3 mmol). 1H NMR (CDCl3)
δ: 1.3-1.8 (m, 24H), 3.10-3.25 (m, 8H), 4.7 (s, 2H), 5.0 (t, 1H),
6.8 (s, 1H).
3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-21-[(1,1-d i-
m eth yleth oxy)ca r bon yl]-14-oxa -12,15-d ioxo 2,4,11,16,21,-
25-h exa a za h exa cos-2-en ed ioic Acid , Bis(1,1-d im eth yl-
eth yl) Ester (26a ). This compound was obtained (3.85 g, 73%
yield) in the form of an oil by following a procedure analogous
to preparation of 12a and starting from 25a (3.0 g, 6.7 mmol)
and 2 (2.4 g, 6.7 mmol). 1H NMR (CDCl3) δ: 1.35-1.65 (m,
50H), 3.10-3.30 (m, 12H), 4.55 (s, 2H), 5.2-5.5 (br s, 1H), 6.4
(br s, 1H).
4-[3-(Am in op r op yl)a m in o]bu t ylca r b a m ic Acid , 2-[[6-
[(am in oim in om eth yl)am in o]-h exyl]am in o]-2-oxoeth yl Es-
ter , Tr is(tr iflu or oa ceta te) (27a ). This compound was ob-
tained as a translucent white amorphous solid (2.61 g, 73%
yield) according to the procedure described for 13a and starting
from 26a (3.85 g, 4.9 mmol). 1H NMR (DMSO-d6) δ: 1.25-1.55
(m, 12H), 1.9 (m, 2H), 2.80-3.10 (m, 12H), 4.35 (s, 2H), 5.5-
8.6 (m, 12H). 13C NMR (D2O) δ: 23.6, 24.5, 26.2, 26.3, 26.7,
28.6, 29.0, 37.3, 39.8, 40.6, 41.9, 45.2, 48.2, 63.7, 157.5, 158.2,
171.4.
N-[4-[[3-(Am in o)p r op yl]a m in o]bu tyl]-N′-[[[[6-[(a m in o-
im in om e t h yl)a m in o]h e xyl]a m in o]ca r b on yl]m e t h yl]-
u r ea , Tr is(tr iflu or oa ceta te) (19a ). This compound was
obtained as an oil (4.75 g, 86% yield) according to the procedure
described for 13a and starting from 18a (6 g, 7.3 mmol). 1H
NMR (DMSO-d6) δ: 1.2-1.65 (m, 12H), 1.9 (m, 2H), 2.9-3.15
(m, 12H), 3.6 (d, 2H), 6.1 (t, 1H), 6.3 (t, 1H), 6.8-9 (m, 11H).
13C NMR (DMSO-d6) δ: 22.8, 23.7, 25.6, 25.8, 27.0, 28.3, 28.9,
36.1, 38.3, 38.5, 40.5, 42.7, 43.7, 46.5, 156.7, 157.9, 169.6.
P r epar ation of Com pou n d 21a. 3-[(1,1-Dim eth yleth oxy)-
ca r bon yla m in o]-20-[(1,1-d im eth yleth oxy)ca r bon yl]-12,-
15-dioxo-2,4,11,14,20,24-h exaaza pen tacos-2-en edioic Acid,
Bis(1,1-d im eth yleth yl) Ester (20a ). A solution of isobutyl
chloroformate (0.97 g, 7.1 mmol) in THF (5 mL) was added
dropwise to a solution cooled to -30 °C of 11a (2.7 g, 7.1 mmol)
and N-methylmorpholine (0.72 g, 7.1 mmol) in THF (40 mL).
The reaction medium was stirred for 0.5 h and a solution of
17a (2.9 g, 7.1 mmol) in THF (30 mL) was added. Stirring was
maintained for 2 h at - 30 °C and then for 24 h at room
temperature. After filtration of the reaction mixture and
evaporation of the filtrate under vacuum, the residue obtained
was purified by flash chromatography on silica gel (EtOAc) to
afford 20a as an oil (1.9 g, 34% yield). 1H NMR (CDCl3) δ: 1.1-
1.6 (m, 50H), 2.1-2.2 (t, 2H), 2.8-2.9 (q, 2H), 3.0-3.1 (m, 6H),
3.2-3.3 (q, 2H), 3.6 (d, 2H), 6.7-6.9 (bs, 1 H), 7.7 (t, 1H), 8.0
(t, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
5-[(3-Am in op r op yl)a m in o]-N-[2-[6-[(a m in oim in om eth -
yl)am in o]h exylam in o]-2-oxoeth yl]pen tan am ide (21a). This
compound was obtained as an oil (0.7 g, 90%) according to the
procedure described for 13a and starting from 20a (0.84 g, 1.1
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
19a -c, 21a -b, a n d 23. Compounds of the general structure
19a -c, 21a -b, and 23 were synthesized according to the
Scheme 4 by the representative procedures illustrated for
analogues 19a , 21a , and 23.
1
mmol). H NMR (DMSO-d6) δ: 1.2-1.7 (m, 12H), 1.8-1.9 (m,
2H), 2.2 (t, 2H), 2.8-3.2 (m, 10H), 3.6 (d, 2H), 7.7 (t, 1H), 7.8-
8.0 (m, 4H), 8.1 (t, 1H), 8.5-8.7 (m, 3H). 13C NMR (D2O) δ:
22.8, 24.6, 25.8, 26.2, 26.3, 28.6, 29.0, 35.4, 37.4, 40.0, 41.9,
43.4, 45.2, 48.2, 157.5, 172.0, 177.4.
P r epar ation of Com pou n d 19a. 3-[[(1,1-Dim eth yleth ox-
y)ca r bon yl]a m in o]-12-oxo-2,4,11,14-tetr a a za p en ta d ec-2-
en ed ioic Acid , 1-(1,1-Dim eth yleth yl) Ester 15-P h en yl-
m eth yl Ester (16a ). A solution of isobutyl chloroformate (1.6
g, 14.0 mmol) in THF (5 mL) was added dropwise to a solution
cooled to -30 °C of carbobenzyloxyglycine (3.0 g, 14.0 mmol)
and N-methylmorpholine (2.8 g, 28 mmol) in THF (50 mL).
The reaction medium was stirred for 0.5 h and a solution of 2
(5.4 g, 14 mmol) in THF (20 mL) was added. Stirring was
maintained for 2 h at -30 °C and then for 24 h at room
temperature. After filtration of the reaction medium and
evaporation of the filtrate under vacuum, the residue obtained
was purified by flash chromatography on silica gel (EtOAc/
methylcyclohexane 1/1) to afford 16a as an oil (7.16 g, 91%
P r epar ation of Com pou n d 23. 3-[[(1,1-Dim eth yleth oxy)-
ca r bon yl]a m in o]-21-[(1,1-d im eth yleth oxy)ca r bon yl]-1,2,-
15-d ioxo-16-oxa -2,4,11,14,21,25-h exa a za h exa cos-2-en e-
d ioic Acid , Bis(1,1-d im eth yleth yl) Ester (22). To a stirred
solution of 8a (1.0 g, 2.89 mmol) and triethylamine (0.45 g,
4.5 mmol) in THF (20 mL) was added a solution of 4-nitro-
phenyl chloroformate (0.58 g, 2.89 mmol) in THF (5 mL). The
reaction mixture was stirred for 15 h at room temperature,
and a solution of 17a (1.2 g, 2.89 mmol) in THF (10 mL) was
then added. The reaction mixture was heated to 40 °C and
stirred for 5 h. After concentration of the reaction mixture
under reduced pressure, the residue was purified by MPLC
on silica gel (methylcyclohexane/EtOAc 7/3 and then EtOAc)
1
yield). H NMR (CDCl3) δ: 1.3-1.7 (m, 26H), 3.2 (q, 2H), 3.4
(q, 2H), 3.8 (d, 2H), 5.15 (s, 2H), 5.5 (br s, 1H), 6.0 (br s, 1H),
7.3 (s, 5H).
1
to afford 22 (1.0 g, 44% yield) as a transparent oil. H NMR
13-Am in o-3-[[(1,1-d im et h ylet h oxy)ca r b on yl]a m in o]-
12-oxo-2,4,11-tr ia za tr id ec-2-en oic Acid , 1,1-Dim eth yleth -
yl Ester (17a ). A mixture of 16a (7.1 g, 13 mmol) and 10%
palladium on carbon (0.7 g) in ethanol (120 mL) was stirred
at room temperature and under a hydrogen atmosphere for 2
h at atmospheric pressure. The catalyst was then filtered off,
and the organic phase was evaporated to give 17a (5.3 g, 98%
yield) in the form of an oily residue which was used without
further purification for the preparation of 18a . 1H NMR
(CDCl3) δ: 1.3-1.6 (m, 28H), 3.25-3.45 (m, 6H), 7.3 (s, 1H),
8.3 (t, 1H), 11.5 (s, 1H).
3-[[(1,1-Dim et h ylet h oxy)ca r b on yl]a m in o]-21-[(1,1-d i-
m et h ylet h oxy)ca r b on yl]-12,15-d ioxo-2,4,11,14,16,21,25-
h ep ta a za h exa cos-2-en ed ioic Acid , Bis(1,1-d im eth yleth -
yl) Ester (18a ). Bis(4-nitrophenyl)carbonate (4.3 g, 13 mmol)
was added in small portions to a solution of 17a (5.3 g, 12
mmol) in anhydrous THF (50 mL). The reaction medium was
stirred for 1 h at room temperature, and a solution of 9 (4.5 g,
(CDCl3) δ: 1.3-1.9 (m, 50H), 3.05-3.55 (m, 10H), 3.85 (d, 2H),
4.10 (t, 2H), 4.7-5.3 (br s, 1H), 5.4-5.6 (br s, 1H), 6.0-6.2 (br
s, 1H), 8.3-8.5 (br s, 1H), 11.5 (s, 1H).
[4-[(3-Am in op r op yl)a m in o]bu toxyca r bon yla m in o]-N-
[6-[(a m in oim in om et h yl)a m in o]h exyl]a cet a m id e, Tr is-
(tr iflu or oa ceta te) (23). This compound was obtained in the
form of a translucent white amorphous solid (0.78 g, 84% yield)
according to the procedure described for 13a and starting from
22 (1.0 g, 1.25 mmol). 1H NMR (DMSO-d6) δ: 1.2-1.55 (m,
8H), 1.6-1.75 (m, 4H), 1.8-1.95 (m, 2H), 2.8-3.1 (m, 10H),
3.55 (d, 2H), 3.95 (t, 2H), 6.7-7.4 (m, 4H), 7.5 (t, 1H), 7.75-
8.0 (m, 4H), 8.5-8.65 (m, 3H). 13C NMR (D2O) δ: 23.0, 24.5,
26.1, 26.2, 26.3, 28.5, 28.9, 31.0, 37.3, 39.9, 41.8, 45.2, 48.1,
65.7, 158.0, 159.0, 178.5.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 31. 31
was synthesized from 10-[(phenylmethoxy)carbonyl amino]-
decanoic acid and 9 in four steps as follows:

286 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Lebreton et al.
18-[(1,1-Dim et h ylet h oxy)ca r b on yl]-12-oxo-2,13,18,22-
tetr a a za tr icosa n ed ioic Acid , 23-(1,1-Dim eth yleth yl) Es-
ter 1-P h en ylm eth yl Ester (28). To a stirred solution of 10-
[(phenylmethoxy)carbonylamino]decanoic acid (0.92 g, 2.92
mmol) in CHCl3 (30 mL) were added at 0 °C DCC (1.05 g, 5.1
mmol) and HOBT (69 mg, 0.51 mmol). The mixture was
allowed to stand for half an hour at 0 °C. Then 9 (1.2 g, 3.47
mmol) in solution in CHCl3 (20 mL) was added dropwise at 0
°C. After the addition was complete, the mixture was allowed
to warm gradually to room temperature overnight. The solvent
was evaporated off under reduced pressure and the oily residue
was purified by flash chromatography on silica gel (hexane/
3-[(1,1-Dim e t h yle t h oxy)ca r b on yla m in o]-21-[(1,1-d i-
m eth yleth oxy)ca r bon yl]-13-oxa -12,15-d ioxo-2,4,11,16,21,-
25-h exa a za cos-2-en ed ioic Acid , Bis(1,1-d im eth yleth yl)
Ester (34). To a stirred solution of 33 (3.0 g, 6.52 mmol) in
CH2Cl2 (60 mL) were added at 0 °C DCC (2.7 g, 13.1 mmol)
and HOBT (0.176 g, 1.31 mmol). This mixture was allowed to
stand for 0.5 h at 0 °C. Then 9 (2.25 g, 6.52 mmol) in solution
in CH2Cl2 (30 mL) was added dropwise at 0 °C. After the
addition was complete, the mixture was allowed to warm
gradually to room temperature and left to stand 2 days. The
solvent was evaporated off under reduced pressure and the
obtained residue was purified by flash chromatography on
silica gel (methylcyclohexane/EtOAc 3/7) to afford 34 (4.5 g,
1
EtOAc 1/1 then EtOAc) as a viscous oil. H NMR (CDCl3) δ:
1.2-1.7 (m, 38H), 2.1 (t, 2H), 3.0-3.3 (m, 10H), 4.7-4.9 (br s,
1H), 5.1 (s, 2H), 5.2 (br s, 1H), 5.7 (br s, 1H), 7.3-7.4 (m, 5H).
1
87.8% yield) as a viscous oil. H NMR (CDCl3) δ: 1.3-1.8 (m,
50H), 3.1-3.5 (m, 12H), 4.5 (s, 2H), 4.8-5.0 (br s, 1H), 5.2-
5.4 (br s, 1H), 6.2-6.6 (br s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
10-Am in o-N-[4-[(1,1-d im eth yleth oxyca r bon yl)-[3-[(1,1-
dim eth yleth oxycar bon yl)am in o]pr opyl]am in o]bu tyl]dec-
a n a m id e (29). A mixture of 28 (1.1 g, 1.69 mmol) and 10%
palladium on carbon (0.1 g) in ethanol (20 mL) was stirred at
room temperature and under a hydrogen atmosphere for 4 h
at atmospheric pressure. The catalyst was then filtered off,
and the organic phase was evaporated to give an oily residue
which was purified by flash chromatography on silica gel
(EtOAc then EtOAc/MeOH/NH4OH/6/4/0.1) to afford 29 (0.78
g, 90% yield). 1H NMR (CDCl3) δ: 1.2-1.7 (m, 38H), 2.1 (t,
2H), 2.3-2.5 (br s, 2H), 2.6-2.8 (br s, 2H), 3.0-3.3 (m, 8H),
5.1 (br s, 1H), 5.8 (br s, 1H).
[6-(Am in oim in om et h yla m in o)h exyl]ca r b a m ic Acid ,
2-[[4-[(3-Am in opr opyl)am in o]-bu tyl]am in o]-2-oxoeth yl Es-
ter , Tr is(tr iflu or oa ceta te) (35). This compound was ob-
tained in the form of a translucent white amorphous solid (4.0
g, 84.7% yield) according to the procedure described for 13a
1
and starting from 34 (4.5 g, 5.72 mmol). H NMR (DMSO-d6)
δ: 1.2-1.6 (m, 12H), 1.8-2.0 (m, 2H), 2.3-3.2 (m, 12H), 4.3
(s, 2H), 7.2 (t, 1H),7.6 (t, 1H), 7.8-8.0 (m, 4H), 8.5-8.7 (br s,
3H). 13C NMR (D2O) δ: 23.6, 24.5, 26.2, 26.3, 26.4, 28.5, 29.4,
37.3, 38.9, 41.8, 45.2, 48.1, 63.7, 157.9, 158.2, 172.0.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 39. 39
was synthesized from 9 and carbobenzyloxyglycine in four
steps as follows.
3-[(1,1-Dim e t h yle t h oxy)ca r b on yla m in o]-20-[(1,1-d i-
m et h ylet h oxy)ca r b on yl]-14-oxo-2,4,15,20,24-p en t a a za -
p en ta cos-2-en d ioic Acid , Bis(1,1-d im eth yleth yl) Ester
(30). 29 (0.78 g, 1.52 mmol) was added at room temperature
to a stirred solution of N,N′-bis(tert-butoxycarbonyl)-S-meth-
ylisothiourea 1 (1.76 g, 6.0 mmol) in THF (20 mL). The reaction
mixture was stirred at room temperature for 3 days. After
evaporation of the solvent, the obtained residue was purified
by flash chromatography on silica gel (hexane/EtOAc 1/1 then
EtOAc) to afford 30 (0.67 g, 59% yield) as a colorless oil. 1H
NMR (CDCl3) δ: 1.2-1.7 (m, 56H), 2.1 (t, 2H), 2.3-3.3 (m,
8H), 3.4 (q, 2H), 5.1 (br s, 1H), 5.7 (br s, 1H), 8.3 (t, 1H), 11.5
(s, 1H).
10-[(1,1-Dim eth yleth oxy)ca r bon yl]-4-oxo-2,5,10,14-tet-
r a a za p en ta d eca n ed ioic Acid , 1-P h en ylm eth yl 15-(1,1-
Dim eth yleth yl) Ester (36). To a stirred solution of carboben-
zyloxyglycine (7.06 g, 34.0 mmol) in CHCl3 (100 mL) were
added DCC (7.0 g, 34 mmol) and HOBT (0.95 g, 7 mmol). The
mixture was allowed to stand for half an hour at 0 °C. Then 9
(9.32 g, 27 mmol) in solution in CHCl3 (100 mL) was added
dropwise at 0 °C. After the addition was complete, the mixture
was allowed to warm gradually to room temperature overnight.
The solvent was evaporated off under reduced pressure, and
the oily residue was purified by flash chromatography on silica
gel (methylcyclohexane/EtOAc 1/1 then EtOAc) to afford 36
10-(Am in oim in om eth yla m in o)-N-[4-[(3-a m in op r op yl)-
am in o]bu tyl]decan am ide, Tr is(tr iflu or oacetate) (31). This
compound was obtained as a white amorphous solid (0.46 g,
75.4% yield) according to the procedure described for 13a and
1
(12.5 g, 86% yield) as a white solid. H NMR (CDCl3) δ: 1.4-
1.8 (m, 24H), 3.0-3.4 (m, 8H), 3.8 (d, 2H), 4.8-5.0 (br s, 1H),
5.1 (s, 2H),5.3-5.7 (br s, 1H), 6.1-6.6 (br s, 1H), 7.3-7.4 (m,
5H).
1
starting from 30 (0.67 g, 0.88 mmol). H NMR (DMSO-d6) δ:
[3-[[(1,1-Dim eth yleth oxy)ca r bon yl][4-[2-a m in o-1-oxo-
eth yl)a m in o]bu tyl]a m in o]p r op yl]ca r ba m ic Acid , (1,1-
Dim eth yleth yl) Ester (37). A mixture of 36 (12.5 g, 23.32
mmol) and 10% palladium on carbon (0.88 g) in ethanol (200
mL) was stirred at room temperature under a hydrogen
atmosphere for 5 h at atmospheric pressure. The catalyst was
then filtered off, and the organic phase was evaporated to give
37 (9.3 g, quantitative yield) in the form of an oily residue
which was used without further purification for the prepara-
1.2-1.35 (m, 10H), 1.4-1.6 (m, 8H), 1.8-1.9 (m, 2H), 2.0 (t,
2H), 2.8-3.1 (m, 10H),7.6 (t, 1H), 7.8-8.0 (m, 4H), 8.5-8.7
(br s, 3H). 13C NMR (D2O) δ: 23.8, 24.5, 26.2, 26.4, 26.5, 28.6,
28.9, 29.0, 29.1, 29.2, 36.6, 37.3, 39.2, 42.0, 45.2, 48.2.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 35. 35
was synthesized from 9 and 3-[(1,1-dimethylethoxy)carbonyl-
amino]-13-oxa-12-oxo-2,4,11-triazapentadec-2-enedioic acid,
1-(1,1-dimethylethyl) ester, 33 which was obtained in two steps
as follows.
1
tion of 38. H NMR (CDCl3) δ: 1.4-1.8 (m, 24H), 1.9-2.0 (br
3-[(1,1-Dim eth yleth oxy)ca r bon yla m in o]-13-oxa -12-oxo-
2,4,11-tr ia za p en ta d ec-2-en d ioic Acid , 1-(1,1-Dim eth yl-
eth yl) Ester 15-Meth yl Ester (32). A solution of 2 (18.78 g,
47.6 mmol) in toluene (700 mL) was added to a stirred solution
of methyl [(phenoxycarbonyl)oxy] acetate (10.0 g, 47.6 mmol)
in toluene (100 mL). The stirred reaction mixture was heated
at 40 °C for 5 h. The solvent was evaporated off under reduced
pressure, and the obtained residue was purified by flash
chromatography on silica gel (hexane/EtOAc 8/2) to afford 32
s, 2H), 3.0-3.4 (m, 10H), 4.5 (br s, 1H), 5.3-5.4 (br s, 1H).
3-[(1,1-Dim e t h yle t h oxy)ca r b on yla m in o]-21-[(1,1-d i-
m et h ylet h oxy)ca r b on yl]-12,15-d ioxo-2,4,11,13,16,21,25-
h ep ta a za h exa cos-2-en ed ioic Acid , Bis(1,1-d im eth yleth -
yl) Ester (38). 4-Nitrophenyl chloroformate (0.5 g, 2.5 mmol)
was added in small portions to a solution of 37 (1.0 g, 2.5 mmol)
and triethylamine (2.5 mmol) in anhydrous THF (50 mL). The
reaction medium was stirred for 1 h at room temperature, and
then a solution of 2 (0.9 g, 2.5 mmol) in anhydrous THF (20
mL) was added dropwise. Stirring was continued overnight
at room temperature, and the solvent was evaporated off under
reduced pressure. The obtained residue was purified by flash
chromatography on silica gel (EtOAc, MeOH 9.5/0.5) to afford
1
(11.8 g, 52% yield). H NMR (CDCl3) δ: 1.3-1.6 (m, 26H), 3.2
(q, 2H), 3.4 (q, 2H), 3.8 (s, 3H), 4.6 (s, 2H), 4.8-5.0 (br s, 1H),
8.3 (t, 1H), 11.5 (s, 1H).
3-[(1,1-Dim eth yleth oxy)ca r bon yla m in o]-13-oxa -12-oxo-
2,4,11-tr ia za p en ta d ec-2-en ed ioic Acid , 1-(1,1-Dim eth yl-
eth yl) Ester (33). This compound was obtained (8.9 g, 78.5%
yield) as an oil by following a procedure analogous to prepara-
1
38 (1.1 g, 56% yield). H NMR (CDCl3) δ: 1.2-1.7 (m, 50H),
2.8-3.1 (m, 12H), 3.2 (q, 2H), 5.9 (t, 1H), 6.1 (t, 1H), 6.7-6.8
(br s, 1H), 7.8 (t, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
1
tion of 4a and starting from 32 (11.7 g, 24.7 mmol). H NMR
N-[6-(Am in oim in om eth ylam in o)h exyl]-N′-[2-[[4-[(3-am i-
n op r op yl)a m in o]bu tyl]a m in o]-2-oxoeth yl]u r ea , Tr is(tr i-
flu or oa ceta te) (39). This compound was obtained in the form
(CDCl3) δ: 1.3-1.7 (m, 26H), 3.2-3.4 (m, 4H), 4.5 (s, 2H), 4.9
(t, 1H), 8.4 (s, 1H), 11.0-11.5 (br s, 1H).

SAR of New Analogues of 15-Deoxyspergualin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 287
of an oil (0.58 g, 53% yield) according to the procedure
described for 13a and starting from 38 (1.2 g, 1.5 mmol). H
mmol) in CH2Cl2 (30 mL) was added carbonyldiimidazole (1.2
g, 6.5 mmol) in small portions and at room temperature. The
reaction mixture was stirred for 3 h. Then 2 (2.28 g, 5.8 mmol)
and triethylamine (0.9 mL, 6.0 mmol) in solution in CH2Cl2
(20 mL) were added dropwise at room temperature. After the
addition was complete, stirring was continued overnight. The
solvent was evaporated off under reduced pressure, and the
obtained residue was purified by flash chromatography on
silica gel (EtOAc/cyclohexane 1/1 then 4/1) to give 45 (2.17 g,
46% yield). 1H NMR (CDCl3) δ: 1.3-1.7 (m, 50H), 3.0-3.5 (m,
12H), 3.9 (s, 2H), 4.7-5.0 (br s, 1H), 5.1-5.3 (br s, 1H), 6.4-
6.6 (br s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
2-[[4-[(3-Am in op r op yl)a m in o]bu tyl]a m in osu lfon yl]-N-
[6-a m in oim in om eth yla m in o)h exyl]-a ceta m id e, Tr is(tr i-
flu or oa ceta te) (46). This compound was obtained as an oil
(1.47 g, 73% yield) according to the procedure described for
13a and starting from 45 (2.17 g, 2.7 mmol). 1H NMR (DMSO-
d6) δ: 1.2-1.7 (m, 12H), 1.9-2.0 (m, 2H), 2.9-3.2 (m, 12H),
3.9 (s, 2H), 7.2 (t, 1H), 7.7 (t, 1H), 7.8-8.0 (m, 4H), 8.2 (t, 1H),
8.6-8.7 (br s, 2H). 13C NMR (D2O) δ: 23.2, 24.3, 26.0, 26.1,
27.0, 28.3, 28.5, 37.2, 40.8, 41.7, 42.9, 45.0, 47.8, 58.1, 157.4,
173.3.
1
NMR (D2O) δ: 1.2-1.7 (m, 12H), 1.9-2.1 (m, 2H), 3.0-3.2 (m,
14H). 13C NMR (D2O) δ: 23.8, 24.6, 26.3, 26.4, 26.5, 28.7, 29.7,
37.6, 38.3, 40.3, 41.9, 44.1, 45.1, 48.1, 157.8, 161.5, 174.3.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 42. 42
was synthesized from 2 and thioglycolic acid in three steps as
follows.
[6[(2-Mer capto-1-oxoeth yl)am in o]h exyl]car bon im idoyl]-
bisca r ba m ic Acid , Bis(1,1-d im eth yleth yl) Ester (40). Car-
bonyldiimidazole (1.4 g, 8.4 mmol) was added in small portions
to a solution of thioglycolic acid (0.55 mL, 7.7 mmol) in CH2-
Cl2 (30 mL). The reaction medium was stirred overnight at
room temperature, and then a solution of 2 (2.5 g, 7 mmol) in
CH2Cl2 (20 mL) was added dropwise. Stirring was continued
for 5 h at room temperature, and the solvent was evaporated
off under reduced pressure. The obtained residue was purified
by flash chromatography on silica gel (EtOAc/cyclohexane 1/1)
to afford 40 as an oil (1.5 g, 55% yield). 1H NMR (CDCl3) δ:
1.3-1.7 (m, 26H), 1.9 (t, 1H), 3.2-3.4 (m, 4H), 3.5-3.6 (q, 2H),
6.6-6.8 (br s, 1H),8.3 (t, 1H), 11.5 (s, 1H).
3-[(1,1-Dim e t h yle t h oxy)ca r b on yla m in o]-21-[(1,1-d i-
m et h ylet h oxy)ca r b on yl]-12,15-d ioxo-14-t h ia -2,4,11,16,-
21,25-h exa a za h exa cos-2-en ed ioic Acid , Bis(1,1-d im eth -
yleth yl) Ester (41). To a stirred solution of 40 (1.37 g, 3.5
mmol) and triethylamine (0.7 g, 7.0 mmol) in THF (25 mL)
was added a solution of 4-nitrophenyl chloroformate (0.7 g,
3.5 mmol) in THF (5 mL). The reaction mixture was stirred
for 1 h at room temperature, and a solution of 9 (1.2 g, 3.5
mmol) in THF (10 mL) was then added. Stirring was continued
for one night at room temperature. After concentration of the
reaction medium under reduced pressure, the residue was
purified by flash chromatography on silica gel (EtOAc/cyclo-
hexane 6/4) to afford 41 (2.0 g, 71% yield) as a transparent
P r oced u r e for th e P r ep a r a tion of Com p ou n d 50. 50
was synthesized from 6 and 1-(1,1-dimethylethyl)-6-[(1,1-
dimethylethoxy)carbonyl]-12-oxo-2,6,11,triazatetradecane dio-
ate 48 obtained in two steps as follows.
6-[(1,1-Dim eth yleth oxy)ca r bon yl]-12-oxo-2,6,11-tr ia za -
t et r a d eca n ed ioic Acid , 1-(1,1-Dim et h ylet h yl)-14-et h yl
Ester (47). To a stirred solution of ethyl hydrogen malonate
(1.60 g, 12.0 mmol) in CHCl3 (50 mL) were added at 0 °C DCC
(2.50 g, 12.0 mmol) and HOBT (0.13 g, 1 mmol). The mixture
was allowed to stand for half an hour at 0 °C. Then 5 (3.12 g,
9.0 mmol) in solution in CHCl3 (20 mL) was added dropwise
at 0 °C. After the addition was complete, the mixture was
allowed to warm gradually to room temperature overnight. The
solvent was evaporated off under reduced pressure, and the
oily residue was purified by flash chromatography on silica
gel (EtOAc/hexane 1/1 then EtOAc) to afford 47 (2.67 g, 69%)
1
oil. H NMR (CDCl3) δ: 1.3-1.8 (m, 50H), 3.1-3.4 (m, 12H),
3.5 (s, 2H), 4.7-5.3 (br s, 1H), 6.7-6.8 (br s, 1H), 7.0-7.2 (br
s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
[4-[(3-Am in op r op yl)a m in o]bu tyl]ca r ba m oth ioic Acid ,
S-[2-[[6-(Am in oim in om eth yla m in o)h exyl]a m in o]-2-oxo-
eth yl Ester , Tr is(tr iflu or oa ceta te) (42). This compound was
obtained in the form of a translucent white amorphous solid
(1.45 g, 77% yield) according to the procedure described for
13a and starting from 41 (2 g, 2.5 mmol). 1H NMR (DMSO-
d6) δ: 1.2-1.7 (m, 12H), 1.8-2.0 (m, 2H), 2.9-3.2 (m, 12H),
3.5 (s, 2H), 7.5 (t, 1H), 7.8-8.0 (m, 5H), 8.3 (t, 1H), 8.5-8.6
(br s, 2H). 13C NMR (D2O) δ: 23.7, 24.5, 26.1, 26.3, 26.5, 28.5,
28.8, 33.8, 37.3, 40.1, 41.1, 41.8, 45.2, 48.1, 157.5, 169.1, 171.8.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 46. 46
was synthesized from 9 and methyl 2-(chlorosulfonyl)acetate
in four steps as follows.
1
as a viscous oil. H NMR (CDCl3) δ: 1.25 (t, 3H), 1.4-1.7 (m,
24H), 3.10-3.35 (m, 10H), 4.2 (q, 2H).
6-[(1,1-Dim eth yleth oxy)ca r bon yl]-12-oxo-2,6,11-tr ia za -
d eca n ed ioic Acid , 1-(1,1-Dim eth yleth yl) Ester (48). This
compound was obtained (0.75 g, 84% yield) as an oil by
following a procedure analogous to preparation of 4a and
starting from 47 (0.95 g, 2.0 mmol). 1H NMR (DMSO-d6) δ:
1.40 (s, 18H), 1.55 (m, 6H), 2.9 (m, 4H), 3.15 (m, 6H).
3-[(1,1-Dim eth yleth oxyca r bon yl)a m in o]-11-m eth yl-20-
[1,1-d im et h ylet h oxyca r b on yl)-12,14-d ioxo-2,4,11,15,20,-
24-h exa a za p en ta cos-2-en ed ioic Acid , Bis(1,1-d im eth yl-
eth yl) Ester (49). To a stirred solution of 48 (1.2 g, 2.7 mmol)
in CH2Cl2 (50 mL) were added at 0 °C DCC (1.2 g, 5.4 mmol)
and HOBT (0.2 g, 1.5 mmol). The mixture was allowed to stand
0.5 h at 0 °C. Then 6 (1.0 g, 2.7 mmol) in solution in CH2Cl2
(30 mL) was added dropwise at 0 °C. After the addition was
complete, the mixture was allowed to warm gradually to room
temperature and left to stand one night. The solvent was
evaporated off under reduced pressure, and the obtained
residue was purified by flash chromatography on silica gel
(EtOAc/EtOH 95/5) to afford 49 (1.8 g, 83% yield) as a viscous
9-[(1,1-Dim eth yleth oxy)ca r bon yl]-3-th ia -3,3-d ioxo-4,9,-
13-tr ia za tetr a d eca n ed ioic Acid , 14-(1,1-Dim eth yleth yl)
1-Meth yl Ester (43). To a stirred solution of 9 (6.2 g, 18 mmol)
and triethylamine (2.8 mL, 20 mmol) in CH2Cl2 (40 mL) cooled
to -20 °C was added dropwise a solution of methyl 2-(chloro-
sulfonyl)acetate in CH2Cl2 (20 mL). The stirring was continued
at -20 °C for 10 min, and the mixture was then allowed to
warm to room temperature for 4 h. The solvent was evaporated
off under reduced pressure, and the oily residue was purified
by flash chromatography on silica gel (cyclohexane/EtOAc 1/1)
1
oil. H NMR (CDCl3) δ: 1.3-1.7 (m, 50H), 2.9-3.3 (m, 15H),
1
3.4 (q, 2H), 4.8 and 5.3 (br s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
N-Meth yl-N-[6-(a m in oim in om eth yla m in o)h exyl]-N′-[4-
[(3 a m in op r op yl)a m in o]bu tyl]-m a lon od ia m id e (50). This
compound was obtained in the form of a translucent white
amorphous solid (1.44 g, 85% yield) according to the procedure
to afford 43 (3.54 g, 41% yield). H NMR (CDCl3) δ: 1.4-1.7
(m, 24H), 3.0-3.3 (m, 8H), 3.8 (s, 3H), 4.0 (s, 2H), 4.6-5.3 (br
s, 2H).
9-[(1,1-Dim eth yleth oxy)ca r bon yl]-3-th ia -3,3-d ioxo-4,9,-
13-tr ia za tetr a d eca n ed ioic Acid , 14-(1,1-Dim eth yleth yl)
Ester (44). This compound was obtained (2.9 g, 85% yield) as
a yellow oil by following a procedure analogous to preparation
of 4a and starting from 43 (3.5 g, 7.3 mmol). 1H NMR (CDCl3)
δ: 1.4-1.8 (m, 24H), 3.0-3.3 (m, 8H), 4.0 (s, 2H), 4.7-5.4 (br
s, 2H).
3-[(1,1-Dim e t h yle t h oxy)ca r b on yla m in o]-20-[(1,1-d i-
m et h ylet h oxy)ca r b on yl]-14-t h ia -14,14-d ioxo-2,4,11,15,-
20,24-h exa a za p en ta cos-2-en ed ioic Acid , Bis(1,1-d im eth -
yleth yl) Ester (45). To a stirred solution of 44 (2.7 g, 5.8
1
described for 13a and starting from 49 (1.8 g, 2.3 mmol). H
NMR (DMSO-d6) δ: 1.2-1.35 (m, 4H), 1.4-1.7 (m, 8H), 1.5-
2.0 (m, 2H), 2.8-3.0 (m, 9H), 3.05-3.15 (m, 4H), 3.2-3.3 (m,
4H), 7.5-7.65 (br s, 1H), 7.75-7.9 (br s, 3H), 8.0-8.1 (br s,
1H), 8.4-8.6 (br s, 3H). 13C NMR (D2O) δ: 23.7, 24.5, 26.3,
26.4, 26.9, 28.0, 28.5, 36.6, 37.3, 39.5, 41.8, 45.2, 48.1, 48.7,
51.5, 157.5, 170.0, 170.5.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 55. 55
was synthesized from 2 and 3-[[4-[[(1,1-dimethylethoxy)car-

288 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Lebreton et al.
bonyl][3-[[(1,1-dimethylethoxy)carbonyl]amino]propyl]amino]-
butyl] [(1,1-dimethylethoxy)carbonyl]amino]propanoic acid 53
which was obtained in three steps as follows.
for 1 day at room temperature, then filtered, and washed with
CH2Cl2 (3 × 25 mL). The filtrates were evaporated off under
reduced pressure, and the obtained residue was purified by
flash chromatography on silica gel (EtOAc/methylcyclohexane
2.5/7.5) to afford 56 (0.42 g, 30%) as a colorless oil. H NMR
(CDCl3) δ: 1.3-1.8 (m, 24H), 2.43 (t, 2H), 3.42 (t, 2H), 8.3 (t,
1H), 9.77 (t, 1H), 11.5 (s, 1H).
3-[[4-[[(1,1-Dim eth yleth oxy)ca r bon yl][3-[[(1,1-d im eth -
ylet h oxy)ca r b on yl]a m in o]p r op yl]a m in o]b u t yl]a m in o]
p r op a n oic Acid , Meth yl Ester (51). Methylacrylate (0.5 g,
5.8 mmol) in solution in methanol (30 mL) was added with
stirring to a solution of 9 (2.0 g, 5.8 mmol) in methanol (20
mL). The reaction was refluxed for 3 h. The solvent was
evaporated off under reduced pressure, and the obtained
residue was purified by flash chromatography on silica gel
1
12-[1,1-Dim et h ylet h oxyca r b on yl]-5-oxa -6-oxo-2,7,12,-
16-tetr a a za h ep ta d eca n ed ioic Acid , 1-(P h en ylm eth yl)-17-
(1,1-Dim eth yleth yl) Ester (57). To a stirred solution of
carbamic acid (2-hydroxyethyl)-phenylmethyl ester (2.0 g,
10.25 mmol) and triethylamine (2.0 g, 20.5 mmol) in anhydrous
toluene (100 mL) was added dropwise at room temperature a
solution of phenylchloroformate (2.8 g, 10.25 mmol) in toluene
(20 mL). The reaction mixture was stirred for 2 h at room
temperature, and a solution of 9 (3.53 g, 10.25 mmol) in toluene
(100 mL) was then added. The reaction mixture was heated
to 60 °C and stirred for 16 h. After evaporation of the solvent,
the obtained residue was purified by flash chromatography
on silica gel (methylcyclohexane/EtOAc 8/2) to afford 57 (3.3
g, 57%). 1H NMR (CDCl3) δ: 1.4-1.8 (m, 24H), 3.0-3.3 (m,
8H), 3.4-3.5 (m, 2H), 4.1-4.2 (m, 2H), 4.7-5.0 (br s, 2H), 5.10
(s, 2H), 5.15-5.4 (br s, 1H), 7.3-7.4 (m, 5H).
N-[4[(2-Am in oet h oxy)ca r b on yla m in o]b u t yl]-1,3-p r o-
p a n e-bisca r b a m ic Acid , Bis(1,1-d im et h ylet h yl) E st er
(58). A mixture of 57 (3.3 g, 5.8 mmol) and 10% palladium on
carbon (0.4 g) in ethanol (70 mL) was stirred at room
temperature under a hydrogen atmosphere for 5 h at atmo-
spheric pressure. The catalyst was then filtered off and the
organic phase was evaporated to give 58 (2.45 g, 97% yield)
in the form of an oily residue which was used without further
purification for the preparation of 59. 1H NMR (CDCl3) δ: 1.4-
1.8 (m, 26H), 2.9 (t, 2H), 3.0-3.4 (m, 8H), 4.08 (t, 2H), 4.6-
5.4 (br s, 2H).
3-[(1,1-Dim eth yleth oxyca r bon yla m in o]-21-[1,1-d im eth -
yleth oxyca r bon yl)-14-oxa -15-oxo-2,4,11,16,21,25-h exa a za -
h exa cos-2-en ed ioic Acid , Bis(1,1-d im eth yleth yl) Ester
(59). To a stirred solution of 56 (0.43 g, 1.2 mmol) and 59 (0.52
g, 1.2 mmol) in EtOH (40 mL) acidified to pH 6 with AcOH
was added in small portions NaBH3CN (0.15 g, 2.5 mmol). The
reaction medium was stirred for 1 day. After concentration of
the reaction medium under reduced pressure, the residue was
taken up in CH2Cl2-H2O (100 mL), and the aqueous phase
was extracted with CH2Cl2. The combined organic layers were
dried, concentrated, and purified by flash chromatography on
silica gel (EtOAc then EtOAc/EtOH/NH4OH 8/2/0.02) to afford
59 (0.45 g, 48% yield) as a yellow oil. 1H NMR (CDCl3) δ: 1.2-
2.2 (m, 50H), 2.7 (t, 2H), 2.8-3.0 (m, 2H), 3.05-3.35 (m, 8H),
3.5 (q, 2H), 4.2 (t, 2H), 4.8 (br s, 1H), 5.0 (br s, 1H), 5.3 (br s,
1H), 8.3 (t, 1H), 11.5 (s, 1H).
1
(EtOAc then EtOAc/EtOH 6/4) to afford 51 (0.98 g, 39%). H
NMR (CDCl3) δ: 1.4-1.9 (m, 25H), 2.9-3.3 (m, 12H), 3.72 (s,
3H), 4.7-5.4 (br s, 1H).
3-[[4-[[(1,1-Dim eth yleth oxy)ca r bon yl][3-[[(1,1-d im eth -
ylet h oxy)ca r b on yl]a m in o]p r op yl]a m in o]b u t yl][(1,1-d i-
m eth yleth oxy)ca r bon yl]a m in o]p r op a n oic Acid , Meth yl
Ester (52). To a stirred solution of 51 (0.86 g, 2 mmol) and
triethylamine (0.14 g, 1.4 mmol) in THF (30 mL) was added a
solution of diterbutyl dicarbonate (0.48 g, 2 mmol) in THF (20
mL). The reaction mixture was stirred for 2 h at room
temperature. Then the solvent was evaporated off under
reduced pressure, and the obtained residue was purified by
flash chromatography on silica gel (EtOAc/cyclohexane 5/5) to
1
afford 52 (0.9 g, 85%). H NMR (CDCl3) δ: 1.4-1.8 (m, 33H),
2.58 (t, 2H), 3.3-3.3 (m, 8H), 3.45 (t, 2H), 3.68 (s, 3H), 4.7-
5.4 (br s, 1H).
3-[[4-[[(1,1-Dim eth yleth oxy)ca r bon yl][3-[[(1,1-d im eth -
ylet h oxy)ca r b on yl]a m in o]p r op yl]a m in o]b u t yl][(1,1-d i-
m eth yleth oxy)ca r bon yl]a m in o]p r op a n oic Acid (53). This
compound was obtained (0.74 g, 90% yield) as a yellow oil by
following a procedure analogous to preparation of 4a and
1
starting from 52 (0.34 g, 1.6 mmol). H NMR (CDCl3) δ: 1.4-
1.8 (m, 33H), 2.61 (t, 2H), 3.0-3.3 (m, 8H), 3.5 (t, 2H), 4.7-
5.4 (br s, 1H).
3-[(1,1-Dim eth yleth oxy)ca r bon yla m in o]-15,20-bis[(1,1-
dim eth yleth oxy)car bon yl]-12-oxo-2,4,11,15,20,24-h exaaza-
p en ta cos-2-en ed ioic Acid , Bis(1,1-d im eth yleth yl) Ester
(54). A solution of isobutylchloroformate (0.165 mL, 1.3 mmol)
in THF (5 mL) was added dropwise to a solution, cooled to -
30 °C, of 53 (0.66 g, 1.3 mmol) and N-methylmorpholine (0.28
mL, 2.6 mmol) in THF (30 mL). The reaction medium was
stirred for 0.5 h, and a solution of 2 (0.46 g, 1.3 mmol) in THF
(20 mL) was added dropwise. Stirring was maintained for 2 h
at -30 °C and then overnight at room temperature. After
filtration of the reaction medium and evaporation of the filtrate
in vacuo, the residue obtained was purified by flash chroma-
tography on silica gel (EtOAc/cyclohexane 8/2) to afford 54 (0.9
g, 66% yield). 1H NMR (CDCl3) δ: 1.3-1.8 (m, 59H), 2.43 (t,
2H), 3.0-3.3 (m, 10H), 3.4 (t, 2H), 3.46 (t, 2H), 4.7-5.4 (br s,
1H), 6.2-6.4 (br s, 1H), 8.3 (t, 1H), 11.5 (s, 1H).
3-[[4-[(3-Am in op r op yl)a m in o]bu tyl]a m in o]-N-[6-(a m i-
n oim in om eth yla m in o)h exyl]-p r op a n a m id e, Tetr a k is(tr i-
flu or oa ceta te) (55). This compound was obtained in the form
of an oil (0.6 g, 74% yield) according to the procedure described
for 7a and starting from 54 (0.85 g, 1 mmol). 1H NMR (DMSO-
d6) δ: 1.2-1.5 (m, 8H), 1.55-1.7 (m, 4H), 1.8-2.0 (m, 2H), 2.5
(t, 2H), 2.8-3.2 (m, 14H), 7.67 (t, 1H), 7.8-8.0 (br s, 3H), 8.12
(t, 1H), 8.5-8.65 (br s, 2H), 8.7-8.85 (br s, 2H). 13C NMR (D2O)
δ: 23.4, 23.5, 24.5, 26.2, 26.4, 28.5, 28.8, 31.8, 37.3, 40.2, 41.9,
44.3, 45.3, 47.6, 47.8, 157.4, 170.3.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 60. This
compound was synthesized starting from [(6-oxohexyl)car-
boimidoyl]biscarbamic acid, bis(1,1-dimethylethyl) ester, 56
and N-[4[(2 aminoethoxy)carbonylamino]butyl]-1,3-propane-
biscarbamic acid, bis(1,1-dimethyl ethyl ester, 58 obtained,
respectively, in one step from [(6-aminomethylhexyl)carboim-
idoyl]biscarbamic acid, bis(1,1-dimethylethyl) ester, 6 and in
two steps from carbamic acid, (2-hydroxyethyl)-phenylmethyl
ester as follows.
[(6-Oxoh exyl)ca r boim id oyl]bisca r ba m ic Acid , Bis(1,1-
d im eth yleth yl) Ester (56). Pyridinium dichromate (2.6 g, 7.0
mmol) was added in small portions to a solution of 5 (1.4 g,
3.9 mmol) in CH2Cl2 (50 mL). The reaction medium was stirred
[4-[(3-Am in op r op yl)a m in o]bu tyl]ca r ba m ic Acid , 2[[6-
(Am in oim in om eth yla m in o)h exyl]a m in o]eth yl Ester (60).
This compound was obtained in the form of a translucent white
amorphous solid (0.4 g, 44% yield) according to the procedure
1
described for 13a and starting from 59 (0.85 g, 1.1 mmol). H
NMR (DMSO-d6) δ: 1.2-1.4 (m, 4H), 1.45-1.7 (m, 8H), 1.8-
2.0 (m, 2H), 2.8-3.3 (m, 14H), 4.17 (t, 2H), 7.29 (t, 1H), 7.71
(t, 1H), 7.8-8.0 (br s, 3H), 8.5-8.8 (br s, 4H). 13C NMR (D2O)
δ: 23.7, 24.5, 26.0, 26.1, 26.7, 28.4, 37.3, 40.6, 41.8, 45.2, 47.3,
48.0, 48.2, 48.5, 61.1, 158.4, 170.5.
P r oced u r e for th e P r ep a r a tion of Com p ou n d 68. This
compound was synthesized with N,N′-bis(tert-butoxycarbonyl)-
S-methylisothiourea 1 and 22-amino-6[(1,1-dimethylethoxy)-
carbonyl]-12,16-dioxo-11-oxa-2,6,13,15-tetraaza-docosanoic acid,
(1,1-dimethylethyl) ester 66 obtained in six steps from N-(6-
bromohexanoyl)glycine, ethyl ester and 8a .
N-(6-Cya n oh exa n oyl)glycin e, Eth yl Ester (61). Pow-
dered potassium cyanide (6.5 g, 100 mmol) was added in small
portions to a solution of N-(6-bromohexanoyl)glycine, ethyl
ester (23.73 g, 84.7 mmol) in EtOH (200 mL). The reaction
medium was refluxed for 15 h. After concentration of the
mixture under reduced pressure, the residue was taken up
with CH2Cl2, and the organic phase was washed with a brine
solution. The organic phase was dried and concentrated under

SAR of New Analogues of 15-Deoxyspergualin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 289
1
reduced pressure to give 61 (19.0 g, 99%) which was used
as a colorless oil. H NMR (CDCl3) δ: 1.0-1.8 (m, 50H), 2.16
1
without purification. H NMR (CDCl3) δ: 1.29 (t, 3H), 1.45-
(t, 2H), 3.05-3.3 (m, 6H), 3.4 (q, 2H), 4.07 (t, 2H), 4.55 (t, 2H),
5.2-5.3 (br s, 1H), 5.7-5.8 (br s, 1H), 6.45-6.55 (br s, 1H),
8.3 (t, 1H), 11.5 (s, 1H).
1.55 (m, 2H), 1.6-1.8 (m, 4H), 1.27 (t, 2H), 2.36 (t, 2H), 4.03
(d, 2H), 4.22 (q, 2H), 5.35-6.05 (br s, 1H).
7-[(Am in oim in om eth yl)a m in o]-N-[[4-[(3-a m in op r op yl)-
am in o]bu toxy]car bon ylam in om eth yl]h eptan am ide, Tr is-
(tr iflu or oa ceta te) (68). This compound was obtained (0.050
g, 54% yield) in the form of a translucent white amorphous
solid according to the procedure described for 13a and starting
from 67 (0.1 g, 0.127 mmol). 1H NMR (DMSO-d6) δ: 1.2-1.35
(m, 4H), 1.4-1.7 (m, 8H), 1.8-1.95 (m, 2H), 2.06 (t, 2H), 2.8-
3.1 (m, 8H), 3.96 (t, 2H), 4.32 (t, 2H), 6.85-7.4 (br s, 3H), 7.55-
7.7 (m, 2H), 7.8-8.0 (m, 4H), 8.33 (t, 1H), 8.55-8.7 (m, 2H).
Biology. Experimental graft-versus-host disease was in-
duced in cyclophosphamide immunosuppressed B6D2F1 mice24
by intravenous injection of 2 × 108 viable spleen cells from B6
origin. Generally, untreated control animals died between days
14 and 21 post-GVHD induction. Compounds were dissolved
in aqueous solution and administered from days 1 to 10 (day
6 omitted) by intraperitoneal route. In such experimental
conditions, control vehicles were unable to modify survival.
Survival was followed until day 60 post-cell injection.
Heterotopic abdominal heart transplant was done according
to the method of Ono and Lindsey.32 End-to-side anastomoses
of the aorta and pulmonary artery of the donor heart (Dark
Agouti) to the recipient (Lewis rat) abdominal aorta and vena
cava were performed. Graft function was assessed by daily
palpation until day 100, and rejection was defined by the
cessation of palpable contractions. Treatment was done as
previously described including day 6. All results were analyzed
by the Manntel & Haenszel procedure for the nonparametric
Log Rank test, validated by the Statistical Analysis System
(SAS, Cary, NC).
N-(6-Cya n oh exa n oyl)glycin e (62). This compound was
obtained (10.2 g, 61% yield) as a yellow oil by following a
procedure analogous to preparation of 4a and starting from
61 (19.0 g, 84.0 mmol). 1H NMR (CDCl3) δ: 1.45-1.55 (m, 2H),
1.6-1.8 (m, 4H), 2.3 (t, 2H), 2.36 (t, 2H), 4.08 (d, 2H), 6.15-
6.25 (br s, 1H).
N-(7-Am in oh ep ta n oyl)glycin e, Sod iu m Sa lt (63). A
mixture of N-(6-cyanohexanoyl)glycine (8.2 g, 41.4 mmol) and
Raney nickel (800 mg) in ethanol (100 mL) and 1 M NaOH
solution (60 mL) was stirred at room temperature under a
hydrogen atmosphere and under a pressure of 3.5. 105 Pa for
8 h. Then 1 M HCl (20 mL) was added, and the mixture was
concentrated under reduced pressure to give 63 (10.2 g) as a
white pasty solid which contains the expected salt and sodium
chloride and which can be used without further purification
in the next step. 1H NMR (DMSO-d6) δ: 1.15-1.6 (m, 8H),
2.08 (t, 2H), 2.56 (t, 2H), 3.32 (d, 2H), 7.1-7.25 (br s, 1H).
N-[7-[(P h en ylm eth oxyca r bon yl)a m in o]h ep ta n oyl]gly-
cin e (64). To a stirred solution of 63 (2 g, 9.9 mmol) in EtOH
(50 mL) and water (50 mL) were added sodium carbonate (1.0
g, 10 mmol) and then dropwise benzylchloroformate (2.3 g, 19.8
mmol). After stirring overnight at room temperature, the
reaction medium was brought to pH 1 with 1 M hydrochloric
acid and then extracted with CH2Cl2 (3 × 100 mL). The organic
layers were combined, dried, and concentrated under reduced
pressure, and the obtained crude product was purified by flash
chromatography on silica gel (EtOAc then EtOAc/EtOH/NH4-
OH 6/3/0.5) to afford 64 (1.3 g, 40% yield) as a white pasty
solid. 1H NMR (DMSO-d6) δ: 1.2-1.6 (m, 8H), 2.09 (t, 2H),
2.98 (td, 2H), 3.64 (d, 2H), 5.01 (s, 2H), 7.25 (t, 1H), 7.3-7.4
(m, 5H), 7.94 (t, 1H).
19-[(1,1-Dim eth yleth oxy)ca r bon yl]-9,13-d ioxo-14-oxa -
2,10,12,19,23-p en ta za -tetr a cosa n ed ioic Acid , 1-(P h en yl-
m eth yl) 24-(1,1-Dim eth yleth yl) Ester (65). To a stirred
solution of 64 (0.5 g, 1.49 mmol) and triethylamine (0.18 g,
1.8 mmol) in THF (25 mL) was added dropwise at 0 °C a
solution of diphenylphosphoryl azide (0.46 g, 1.67 mmol). The
reaction mixture was stirred at room temperature for 45 min.
After concentration of the mixture under reduced pressure,
the residue was taken up with toluene (20 mL). To this solution
were added 8a (1.04 g, 3.0 mmol) and triethylamine (0.18 g,
1.8 mmol). The solution medium was stirred at reflux for 24
h. After evaporation of the solvent, the obtained residue was
purified by flash chromatography on silica gel (methylcyclo-
hexane/EtOAc 1/1 to 1/9) to afford 65 (0.4 g, 40% yield). 1H
NMR (CDCl3) δ: 1.2-1.7 (m, 32H), 2.15 (t, 2H), 3.05-3.3 (m,
8H), 4.05 (t, 2H), 4.5 (t, 2H), 4.7-4.9 (br s, 1H), 5.08 (s, 2H),
5.7-5.9 (br s, 1H), 6.4-6.6 (br s, 1H), 7.3-7.4 (m, 6H).
22-Am in o-6-[(1,1-dim eth yleth oxy)car bon yl]-12,16-dioxo-
11-oxa -2,6,13,15-tetr a a za -d ocosa n oic Acid , (1,1-Dim eth -
yleth yl) Ester (66). A mixture of 65 (0.33 g, 0.48 mmol) and
5% palladium on carbon (0.1 g) in ethanol (15 mL) was stirred
at room temperature and under a hydrogen atmosphere for 5
h at atmospheric pressure. The catalyst was then filtered off,
and the organic phase was evaporated to give 66 (0.26 g,
quantitative yield) in the form of an oily residue which was
used without further purification for the preparation of 67.
1H NMR (CDCl3) δ: 1.2-1.75 (m, 32H), 2.2 (t, 2H), 2.8 (t, 2H),
2.85-3.3 (m, 8H), 4.07 (t, 2H), 4.53 (t, 2H), 4.7-5.4 (br s, 1H),
5.7-6.1 (br s, 1H), 6.4-7.1 (br s, 1H).
3-[[(1,1-Dim eth yleth oxyl)ca r bon yl]a m in o]-21-[(1,1-d i-
m eth yleth oxy)ca r bon yl]-11,15-d ioxo-16-oxa -2,4,12,14,21,-
25-h exa a za h exa cos-2-en ed ioic Acid , Bis(1,1-d im eth yl-
eth yl) Ester (67). 66 (0.26 g, 0.48 mmol) was added at room
temperature to a stirred solution of N,N′-bis(tert-butoxycar-
bonyl)-S-methylisothiourea 1 (0.28 g, 0.95 mmol) in THF (20
mL). The reaction medium was stirred at room temperature
for 4 days. After evaporation of the solvent, the obtained
residue was purified by flash chromatography on silica gel
(methylcyclohexane/EtOAc 4/6) to afford 67 (0.10 g, 27% yield)
Ack n ow led gm en t. The authors thank Philippe
Peralba, Vale´rie Lepais, Anne-Marie Dhennequin, Gilles
Martin-Gousset, Florence Chirade, Agne`s Martinien,
Marina Tripier, and Herve´ Duclos for their skillfull
experimental work.
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