Synthesis and activity of dipeptides, linked to targeting ligands, as specific NK cell enhancers

Article abstract of DOI:10.1021/jm970734v

Water soluble analogues of the lipophilic immunostimulant, octadecyl D- alanyl-L-glutamine, BCH-527, were synthesized and evaluated for the ability to stimulate natural killer (NK) cells. One of these compounds in which the octadecyl chain of BCH-527 was

Full text of DOI:10.1021/jm970734v

J . Med. Chem. 1998, 41, 1909-1926
1909
Syn th esis a n d Activity of Dip ep tid es, Lin k ed to Ta r getin g Liga n d s, a s Sp ecific
NK Cell En h a n cer s
Shaun D. Abbott, Lyne Gagnon,† Mouna Lagraoui, Salam Kadhim, Giorgio Attardo, Boulos Zacharie, and
Christopher L. Penney*
Department of Medicinal Chemistry, BioChem The´rapeutique Inc., 275 Boulevard Armand-Frappier,
Laval, Que´bec, H7V 4A7, Canada
Received October 28, 1997
Water soluble analogues of the lipophilic immunostimulant, octadecyl D-alanyl-L-glutamine,
BCH-527, were synthesized and evaluated for the ability to stimulate natural killer (NK) cells.
One of these compounds in which the octadecyl chain of BCH-527 was replaced with a shorter
chain alcohol, 6-(D-alanyl-L-glutaminylamino)hexan-1-ol, 9, displayed an in vitro stimulation
of NK cells comparable to that of interleukin 2 (IL 2). However, when the hydroxyl of 9 was
linked to L-fucose to yield 1-â-[6-(D-alanyl-L-glutaminylamino)hex-1-yl]-L-fucopyranose (BCH-
2537, 1), the observed stimulation of NK cells was greater than that observed with IL 2. Further
evaluation of these compounds revealed that the improved in vitro activity of BCH-2537 was
more pronounced in vivo. That is, while both compounds significantly increased splenic NK
cells, only BCH-2537 significantly increased the activity of these cells in vivo. In terms of a
structure-activity relationship, NK cell activity was sensitive to minor structural modifications.
It was influenced by conservative substitutions within the dipeptide, the length of the
hydrocarbon chain, and the functionality at the end of the chain. No other compound enhanced
NK cell activity to the extent exhibited by BCH-2537, although a few were equipotent to 9.
In tr od u ction
dipeptide. NK cells possess receptors for monosaccha-
rides (e.g., lectins such as NKR-P1)5 which may permit
intracellular delivery of the immunostimulant signal.
Indeed, it has been reported that L-fucose enhances
human NK cell activity.6
Over the years, octadecyl L-tyrosine has been de-
scribed in the literature as an inert, insoluble carrier
of immunological activation signals provided by vaccine
antigens, allergens, etc.1 More recently, we reported
that an immunomodulatory “signal” could be incorpo-
rated into the insoluble carrier, as exemplified by
octadecyl D-alanyl-L-glutamine; BCH-527.2 The dipep-
tide signal sequence was developed from the well-
known, but toxic, immunomodulator muramyl dipeptide
(MDP; N-acetylmuramyl-L-alanyl-D-isoglutamine).3 BCH-
527 induces a significant activation of NK cells and
macrophages in vivo, which is reflected by an antiviral
activity against murine cytomegalovirus. However, the
insolubility of BCH-527 limits its utility as a therapeutic
agent. It was therefore desired to develop an analogue
of BCH-527 with reasonable aqueous solubility and
selectivity toward NK cell or macrophage populations.
With regard to solubility, it was recognized that
obvious modifications to the BCH-527 structure, such
as shortening the hydrocarbon chain, could solve the
problem. However, for a range of chain length, deter-
gency and accompanying toxicity can become an issue.
Removal of the ester chain would yield a dipeptide that
is expected to be cleared rapidly. Interestingly, lower
alkyl esters of dipeptides have been reported to be
inhibitory toward cytolytic immune cells.4 Therefore,
an approach was taken which was expected to introduce
polarity into the molecule, prevent rapid degradation
and clearance of the molecule, and target the dipeptide
signal to NK cells and/or macrophages. That is, intro-
duction of L-fucose at the carboxyl terminus of the
The importance of NK cells is illustrated by their role
in immunosurveillance, and much work is underway to
elucidate their mechanism of action.7 They act as a first
line of defense by the recognition and cytolysis of, for
example, metastatic tumors or virus-infected cells. A
number of immunostimulants, primarily interferon
inducers, stimulate NK cells along with other immune
cell subsets. Well-known examples include tilorone, CL
246738, 7-thia-8-oxoguanosine, loxoribine, bropirimine,
and imiquimod. Nevertheless, few compounds have
been reported which specifically increase NK cell activ-
ity and/or populations. Only recently have examples
appeared in the literature. These include agelasphin-
11, an R-galactosylceramide isolated from sponge,8 and
eisenin, a tripeptide isolated from algae.9 Yet, com-
pounds which specifically activate NK cells are expected
to be less toxic than immunostimulants, such as inter-
feron inducers, which stimulate multiple immune cell
subsets. The advantages of a selective immune cell
stimulant have recently been discussed with respect to
organ transplantation.10 In this paper, we report on the
immunological activity of synthetic dipeptide com-
pounds which selectively activate NK cells.
Ch em istr y
The synthesis of 1-â-[6-(D-alanyl-L-glutaminylamino)-
hex-1-yl]-L-fucopyranose (BCH-2537, compound 1), is
shown in Scheme 1. Thus a suitably N-protected
glutamate ester was used as a glutamine precursor. This
was necessary since the carboxamide of glutamine led
† Present address: Biophage Inc., 6100 Royalmount, Montre´al,
Que´bec H4P 2R2, Canada
S0022-2623(97)00734-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/28/1998

1910 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
Sch em e 1: Synthesis of
1-â-[6-(D-Alanyl-L-glutaminylamino)hex-1-yl]fucopyranose,
BCH-2537, 1
lowed by treatment with methanolic ammonia) to give
compound 1.
This strategy was followed for most other members
of the series, using appropriately protected starting
materials. However, the synthesis of certain analogues
dictated the use of alternative synthetic routes. These
analogues were prepared by standard procedures, which
are described in the Experimental Section. Thus,
analogues with no linker, 15, 16, and 19, and the
thioanalogue 10 were prepared from the amino- and
thioglycosides, respectively.11,12 Interestingly, in the
case of the glucose analogue 7, the Ko¨nigs-Knorr
methodology gave unacceptable yields. This compound
was therefore synthesized using the trichloroacetimi-
date coupling procedure.13
In Vitr o An a lysis: SAR Stu d ies
Activation of murine NK cells is routinely measured
in vitro by determination of the ability to lyze YAC-1
target cells in a conventional chromium release assay.
Therefore, splenocytes were incubated for 4 h with
51Cr-labeled YAC-1 tumor cells and the released chro-
mium determined in the lysate. With this assay,
compound 1 (BCH-2537) was shown to have a direct
stimulatory effect on NK cells which was consistently
greater than the positive control; interleukin 2 (IL 2).
Additionally, the desfucose analogue, compound 9,
consistently displayed a stimulatory effect on NK cells
which was equivalent to IL 2. The assays were per-
formed seven times (n ) 7), and the results are
presented in Table 1. On the basis of these data, BCH-
2537 was selected as a lead upon which to define a
structure-activity relationship for dipeptide-linker-
monosaccharide. As shown in Tables 1-4, this was
undertaken by systematic modification of the monosac-
charide, linker, and amino acid portions of the molecule.
(i) Va r ia tion of th e Mon osa cch a r id e. The in vitro
data for modification of the monosaccharide portion of
the molecule is shown in Table 1, entries 1-9. The data
indicates a preference for L-fucose. Substitution by
D-glucose, 7, results in complete loss of activity. Moder-
ate activity is observed with the D-mannose derivative
8. This suggests that it is not the physiochemical
properties of the sugar, such as increased polarity,
which are important. That is, there is a specific
monosaccharide-cell surface receptor interaction. Omis-
sion of the sugar to give alcohol 9 yielded a compound
with activity which was surpassed only by 1. Hence,
the incorrect sugar configuration functions as an an-
tagonist. Even substitution of L-fucose for the thioana-
logue 10 resulted in significantly weaker stimulation,
although the potency was similar. Simple substitution
of the hydroxyl function of 9 with chlorine, 11, or the
carboxylate, 12, resulted in the loss of all activity.
However, activity was seen with an amino replacement,
13, but the analogue was not very potent. Capping of
alcohol 9 with an O-acetyl group, 14, also resulted in
the complete loss of activity.
to intermediates with poor solubility and gave coupling
yields which were not satisfactory. Although the ben-
zyloxycarbonyl-protected intermediate was used in the
synthesis of 1, during analogue preparation it was found
that tert-butoxycarbonyl protection strategy gave supe-
rior yields in the deprotection/coupling sequence. Use
of a glutamate ester allowed facile conversion by ami-
nolysis with ammonia to yield the glutamine side chain,
and use of the acetyl-protected sugar allowed deprotec-
tion of this portion of the molecule in the same step.
Initial syntheses used the methyl ester. However, it
was found that the benzyl ester gave shorter reaction
times without a decrease in product yield. For the
synthesis of 1, 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihyd-
roquinoline (EEDQ) coupling of the glutamic acid de-
rivative 2 with unprotected 6-aminohexan-1-ol pro-
ceeded selectively, in high yield, to give the alcohol 3.
Glycosidic coupling with the protected bromofucose 4,
prepared quantitatively from the peracetate, was found
to occur readily under traditional Ko¨nigs-Knorr condi-
tions. This afforded the glycopeptide 5 exclusively as
the â-anomer. N-Deprotection and carbodiimide cou-
pling gave the fully protected intermediate 6, which was
smoothly deprotected in two steps (hydrogenolysis fol-
(ii) Va r ia tion of th e Lin k er . It was next decided
to vary the length and type of the linker between the
D-alanyl-L-glutamine dipeptide and the monosaccharide,
as shown in Table 1, entries 10-18. Omission of the
linker from 1 to give compound 15 resulted in a loss of
approximately half of the activity, but little change in

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1911
Ta ble 1. In Vitro Natural Killer Cell Activity of D-Alanyl-L-glutaminyl-Linker-Sugars and Analogues Relative to Interleukin 2
D-Ala-L-Gln-linker-R
entry
1
linker
NH(CH2)6O
R
compd
activitya
concentrationb
L-fucose
BCH-2537
+++++
10-12-10-7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
NH(CH2)6O
NH(CH2)6O
NH(CH2)6O
NH(CH2)6S
NH(CH2)6
NH(CH2)5
NH(CH2)6
D-glucose
7
8
9
0
D-mannose(2-O-Ac)c
++++
++++
++
0
0
++++
0
+++
0
0
0
++
+
10-4
H
10-12-10-4
10-12-10-8
L-fucose
Cl
CO2H
NH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
10-4
L-Ala-D-Gln- NH(CH2)6OAc
NH
NH
OH
â-L-fucose
R-L-fucose
10-12-10-4
D-Ala-L-glutarimide
NH
â-D-mannose
10-12-10-5
10-8-10-4
10-12-10-8
10-10-10-4
NH(CH2)12
NH(CH2)12
NH(CH2)12
O
O
O
L-fucose
D-mannose(2-O-Ac)c
H
++
++
0
NH-1,4-C6H4(CH2)2O
L-fucose
a
All compounds were tested in the concentration range 10-4-10-12 M. Reported concentrations represent the peak or the range of
peak activity. Each active compound was tested two to five times. Inactive compounds were tested once. Peak activities are reported,
relative to positive IL 2 control. Therefore, with IL 2 control as 100% stimulation, then 0 ) 0-20% of IL 2; + ) 21-40% of IL 2; ++ )
b
41-60% of IL 2; +++ ) 61-80% of IL 2; ++++ ) 81-100% of IL 2; +++++ ) 101-120% of IL 2. Quoted over the range of activity
observed. c The 2-O-acetyl function and other protecting groups could not be removed under an array of conditions without degradation
of the product.
Ta ble 2. In Vitro Natural Killer Cell Activity of
Dipeptide-Aminohex-1-ylfucopyranoses Relative to
Interleukin 2
significant loss of activity, regardless of the sugar
present. Introduction of an aromatic ring into the linker
of 1, to give compound 23, also abolished activity.
X-Y-NH(CH2)6O-L-fucose
compd activitya concentrationb
Taken together, these data suggest that a flexible
hexamethylene chain provides an optimum spatial
arrangement between the dipeptide and monosaccha-
ride.
entry
X
Y
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
D-Ala
L-Ala
D-Val
L-Gln
L-Gln
L-Gln
1
24
25
26
27
28
29
30
31
32
33
34
40
41
42
43
44
45
46
+++++
+++
++
+++
+
0
0
0
0
10-12-10-7
10-12-10-5
10-12-10-4
10-12-10-4
10-8-10-4
(iii) Va r ia tion of th e Dip ep tid e. Numerous ana-
logues of BCH-2537 were synthesized which contained
variations of the dipeptide signal, as shown in Table 2.
Activity is significantly affected by variations in the
D-alanyl-L-glutamine sequence. For example, conserva-
tive substitution of D-alanine with other R-amino acids
gave analogues with moderate activity, compounds 24-
26. Note that this includes the L-alanine diastereomer
24. Interestingly, even with the removal of the first
amino acid to give 27, weak activity remains. However,
acetylation of alanine, compounds 28 and 29, or replace-
ment of alanine with an acetyl group, compound 30,
abolished activity. Activity is also lost when alanine is
replaced with â-alanine, compound 31. These data
illustrate the requirement for an appropriately placed
primary amino function or positive charge.
L-amino-Ala L-Gln
L-Gln
Ac-D-Ala
Ac-L-Ala
Ac
L-Gln
L-Gln
L-Gln
L-Gln
D-Gln
L-Glu
â-Ala
L-Ala
D-Ala
0
++
++
0
0
0
0
0
0
0
10-12-10-4
L-amino-Ala L-Glu
L-pyro-Glu
10-7
Me
L-pyro-Glu
Gly
L-Orn
L-Asn
L-Pro
D-Ala
D-Ala
D-Ala
L-Ala
L-Ile
L-Pro
All compounds were tested in the concentration range 10-4
-
a
10-12 M. Reported concentrations represent the peak or the range
of peak activity. Each active compound was tested two to five
times. Inactive compounds were tested once. Peak activities are
reported, relative to positive IL 2 control. Therefore, with IL 2
control as 100% stimulation, then 0 ) 0-20% of IL 2; + ) 21-
40% of IL 2; ++ ) 41-60% of IL 2; +++ ) 61-80% of IL 2; ++++
Very little change is tolerated at the L-glutamine
position. In contrast to D-alanine, the enantiomeric
D-glutamine resulted in an inactive compound 32.
Replacement with L-glutamate resulted in compounds
33 and 34 which displayed moderate activity. All other
changes, compounds 40-46, afforded inactive ana-
logues. For example, compound 44, which contained the
glutamine homologue asparagine, was devoid of activity.
L-Alanyl-L-proline and L-isoleucyl-L-proline analogues,
compounds 45 and 46, were examined because of the
structural similarity to alanylglutamine and its occur-
rence at the N-terminus of cytokine immunostimu-
lants.14
b
) 81-100% of IL 2; +++++ ) 101-120% of IL 2. Quoted over
the range of activity observed.
potency. However, a change in the stereochemistry of
the glycosidic linkage to yield the R-L-fucose analogue
16 resulted in complete loss of activity. A similar
abrogation of activity was observed with the dipeptide
17 (no linker, no sugar) or the carboximide of 17,
compound 18. The D-mannose equivalent of 15, com-
pound 19, displayed a loss of activity relative to its
parent 8 and relative to 15. Extension of the linker to
a 12-carbon chain, compounds 20, 21, and 22, led to a
Analogues were also examined in which the dipeptide
was varied and the fucose portion was replaced with

1912 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
Ta ble 3. In Vitro Natural Killer Cell Activity of Signal-Aminohex-1-yl-Sugar Relative to Interleukin 2
X-Y-NH(CH2)6O-R
entry
X-Y
R
compd
activitya
concentrationb
1
2
3
4
5
6
7
8
9
10
11
12
13
D-Ala-L-Gln
D-Ala-L-Gln
L-Ala-L-Gln
Ac-D-Ala-L-Gln
L-Gln
L-pyro-Glu
cyclo(D-Ala-L-Glu)
L-Ala-L-Gln
L-Ala-D-Gln
L-Gln
L-fucose
D-mannose(2-O-Ac)
D-mannose(2-O-Ac)
D-mannose(2-O-Ac)
D-mannose(2-O-Ac)
D-mannose(2-O-Ac)
D-mannose(2-O-Ac)
H
H
H
H
H
H
1
7
+++++
++++
++++
+++
++++
+++
0
10-12-10-7
10-4
47
48
49
50
51
52
53
54
55
56
57
10-10
10-7-10-4
10-5
10-12-10-7
++
10-12-10-4
10-12-10-8
++
0
L-pyro-Glu
L-Ala-L-Pro
L-Ile-L-Pro
+++
0
+
10-12-10-5
10-12-10-6
a
All compounds were tested in the concentration range of 10-4-10-12 M. Reported concentrations represent the peak or the range of
peak activity. Each active compound was tested two to five times. Inactive compounds were tested once. Peak activities are reported,
relative to positive IL 2 control. Therefore, with IL 2 control as 100% stimulation, then 0 ) 0-20% of IL 2; + ) 21-40% of IL 2; ++ )
b
41-60% of IL 2; +++ ) 61-80% of IL 2; ++++ ) 81-100% of IL 2; +++++ ) 101-120% of IL 2. Quoted over the range of activity
observed.
Ta ble 4. In Vitro Natural Killer Cell Activity of Other Fucose
Analogues of BCH-2537, 1, Relative to Interleukin 2
greater variation of dipeptide signal than seen with the
L-fucose series.
(iv) Oth er An a logu es. Table 4 shows the results
of further investigation of the activity of analogues of
BCH-2537, 1. These compounds represent an extension
of the general dipeptide-linker-monosaccharide struc-
ture. First, analogues were synthesized which con-
tained either two dipeptide signal sequences, 35, or two
fucose monosaccharides, 36. The goal was to exploit the
known clustering of cell surface receptors as a primary
event in cell-cell contact and cell signaling processes.
Indeed, individual protein-carbohydrate (receptor-
ligand) interactions are generally weak. To enhance the
strength of cell surface binding, protein-saccharide
interactions often occur via multivalent presentation in
order to achieve the necessary avidity.15 Therefore,
while these initial results were disappointing, the length
of the linker or distance between the bivalent groups
may not be appropriate for a synergistic binding inter-
action.
Additionally, the hybrid molecule 37 was synthesized
in which the L-fucose sugar is linked to the purine
portion of the cytotoxic T-lymphocyte (CTL) activator
6-(N,N-dimethylamino)purin-9-yl pentoxycarbonyl-D-
arginine; BCH-1393,16 38. This expanded the specific
CTL activity of BCH-1393 to include a significant NK
cell stimulation. In view of the recently described
commonality of CTL and NK cells and their collective
role in immune surveillance,17 compound 37 exhibits an
interesting in vitro immune profile.
Finally, the activity of BCH-2537, 1, was compared
to the well-described NK cell activator 7-allyl-8-oxogua-
a
All compounds were tested in the concentration range 10-4
-
nosine, 39 (loxoribine).18 The lack of activity of loxor-
ibine in our direct-stimulation (4 h incubation) assay is
consistent with the indirect activation of NK cells via
the induction of interferon. Demonstration of the activ-
ity of loxoribine in the chromium release assay requires
a 14 h incubation of the NK cells with YAC-1 target
cells.
10-12 M. Reported concentrations represent the peak or the range
of peak activity. Each active compound was tested two to five
times. Inactive compounds were tested once. Peak activities are
reported, relative to positive IL 2 control. Therefore, with IL 2
control as 100% stimulation, then 0 ) 0-20% of IL 2; + ) 21-
40% of IL 2; ++ ) 41-60% of IL 2; +++ ) 61-80% of IL 2; ++++
b
) 81-100% of IL 2; +++++ ) 101-120% of IL 2. Quoted over
the range of activity observed.
(v) In Vitr o Im m u n e P r ofile of 1. The in vitro
activity of BCH-2537, 1, was both potent and selective
for NK cells. The latter was evidenced by a lack of
activity in B- and T-cell mitogenic proliferation assays,
macrophage phagocytosis and respiratory burst, and the
D-mannose or removed to yield the corresponding alco-
hol. This is presented in Table 3, compounds 47-57.
In all cases the analogue exhibited less activity than 1.
However, some activity was maintained through a

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1913
Ta ble 5. Summary of the in Vitro Immunological Analysis of
BCH-2537, 1
Ta ble 6. Summary of Immunophenotyping Data for Mice
Administered Four Doses of BCH-2537, 1
assaya
cell sourceb
activityc
cell subsets
0 mg/kg
25 mg/kg
50 mg/kg
NK cell cytolytic activity
(YAC-1 target)
NK cell cytolytic activity
(K 562 target)
phagocytosis
respiratory burst
murine splenocytes +++++
Blood Immunophenotyping BCH-2537, 1a
CD4+CD45+
NK+
mean
STD
P value
mean
STD
21.3
2.8
32.5
5.3
0.02
6.70
0.2
30.9
4.8
0.002
5.90
0.6
human PBML
++++
murine whole blood NE
murine whole blood NE
5.40
0.5
phagocytosis
respiratory burst
cytotoxic T lymphocyte activity
mixed lymphocyte reaction
human PBML
human PBML
murine splenocytes
murine splenocytes NE
NE
NE
+
P value
mean
STD
0.015
3.95
2.2
0.2
1.45
0.3
CD4+CD11b+
0.75
0.4
P value
0.048
0.032
T cell proliferation (PHA, ConA) murine splenocytes NE
B cell proliferation (PWM, LPS) murine splenocytes NE
Spleen Immunophenotyping BCH-2537, 1
CD4+CD45+
mean
STD
22.8
1.4
26.2
1.0
26.5
1.6
0.008
2.33
0.2
a
Experimental protocol for murine NK cell assay is described
P value
mean
STD
0.02
2.13
0.1
in the Experimental Section, while the protocol for human NK
cell assay is given in ref 22. Protocol for measurement of mac-
rophage/monocyte phagocytosis and respiratory burst is given in
ref 23. Protocol for CTL/MLR assay is given in ref 16. Protocol for
mitogen-induced lymphocyte (T and B cell) assay is given in ref
NK+CD3+
1.90
0.2
P value
0.039
0.021
a
Groups of four C57BL/6 mice were injected ip for four
consecutive days with BCH-2537. No effect was observed on NK
cells when mice were given 5 mg/kg BCH-2537.
b
22. PBML ) peripheral blood mononuclear leukocytes: obtained
from heparinized blood by density gradient centrifugation on
Ficoll-Hypaque. Splenocytes: obtained as described in the Ex-
perimental Section. c NE ) no effect. For NK cell and CTL assays,
activity is reported relative to IL 2 control. Therefore, with IL 2
control as 100% stimulation, then 0 ) 0-20% of IL 2; + ) 21-
40% of IL 2; ++ ) 41-60% of IL 2; +++ ) 61-80% of IL 2; ++++
) 81-100% of IL 2; +++++ ) 101-120% of IL 2.
Ta ble 7. Summary of Immunophenotyping Data for Mice
Administered Four Doses of Either BCH-2537, 1, or the
Desfucose Analogue 9
cell subsets
0 mg/kg
5 mg/kg
25 mg/kg
Blood Immunophenotyping BCH-2537, 1a
CD4+CD11b+
CD11b+
mean
STD
P value
mean
STD
5.70
2.6
9.00
1.7
0.049
38.6
3.8
7.20
2.0
0.46
20.6
4.6
mixed lymphocyte reaction. However, compound 1
weakly enhances cytotoxic T-cells. A summary of the
in vitro analysis of BCH-2537, 1, is presented in Table
5.
24.7
8.6
P value
0.043
0.26
In Vivo Activity of BCH-2537, 1
Spleen Immunophenotyping BCH-2537, 1
CD4+
mean
STD
P value
mean
STD
P value
mean
STD
P value
mean
STD
24.3
2.5
29.6
2.5
0.02
3.88
0.6
0.006
3.64
0.8
0.027
4.30
1.0
0.007
5.56
0.8
29.5
3.7
0.15
3.90
0.7
0.041
2.82
0.2
0.146
4.34
0.9
In view of the significant in vitro NK cell activity of
BCH-2537, 1, this compound was selected for in vivo
evaluation. Also, because of the similar activity of the
desfucose analogue 9, and its relatively easier synthesis,
it was also selected for a more limited in vivo evaluation.
It was possible that the small difference in activity
between BCH-2537 and 9 would become insignificant
upon in vivo analysis. Therefore, two immunopheno-
typing experiments were undertaken with normal im-
mune status mice. The first experiment was performed
in the presence or absence of BCH-2537 to determine
the increase or decrease in immune cell subset popula-
tions, relative to each other. Mice were injected intra-
peritoneally for four consecutive days with 5, 25, or 50
mg/kg of BCH-2537. At the end of the experiment, blood
and spleens were collected for determination by flow
cytometry of the effects of BCH-2537 on the immune
cell subsets within these tissues. The second experi-
ment was performed in the presence or absence of 9 or
BCH-2537 in order to undertake a direct comparison of
the two compounds. Mice were injected intraperito-
neally for four consecutive days with 5 or 25 mg/kg of 9
or BCH-2537. Again, blood and spleens were collected
at the end of the experiment for analysis by flow
cytometry. A summary of the results is presented in
Tables 6 and 7.
NK+
2.13
0.7
NK+CD3+
CD4+CD11b+
CD11b+
2.30
0.3
2.14
0.6
P value
mean
STD
0.006
7.08
1.2
3.78
0.2
P value
0.008
0.015
Blood Immunophenotyping, compound 9
no significant effect
Spleen Immunophenotyping, compound 9
CD4+
mean
STD
P value
mean
STD
P value
mean
STD
P value
mean
STD
24.3
2.5
32.1
3.6
0.003
26.4
3.6
0.023
2.76
0.1
0.080
3.04
1.0
0.094
3.00
1.1
36.0
3.9
0.005
31.6
4.5
0.001
3.08
0.5
0.008
2.96
0.3
CD8+
19.3
3.1
NK+
2.13
0.7
NK+CD3+
CD4+CD11b+
2.30
0.3
P value
mean
STD
0.004
3.04
0.6
2.14
0.6
The first experiment demonstrated that BCH-2537
induces a significant increase in NK cells. In blood, 25
mg/kg of BCH-2537 resulted in a significant increase
in NK cells (24%, P e 0.015). Significant increases also
occur in T-helper cells (CD4+CD45+, 45%, P e 0.02)
and monocytes (CD11b+, 93% to 427%, P e 0.048). In
P value
0.073
0.013
a
Groups of five C57BL/6 mice were injected ip for four consecu-
tive days with BCH-2537, 1, or compound 9.
spleen, a significant increase in NK cells (12% to 23%,
P e 0.039) is observed at 25 and 50 mg/kg of BCH-2537.

1914 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
Discu ssion
In this study, the determination of the in vitro NK
cell activity of more than 50 water soluble analogues of
the lipophilic immunomodulator octadecyl D-alanyl-L-
glutamine (BCH-527) was undertaken. On the basis of
these results, a lead molecule, 1-â-[6-(D-alanyl-L-glutami-
nylamino)hex-1-yl]-L-fucopyranose (BCH-2537, 1), was
selected for in vivo immunological evaluation. A struc-
ture-activity relationship centered about this dipep-
tide-linker-monosaccharide was approximated as fol-
lows:
(1) Activity is dependent upon the type and stereo-
chemistry of the monosaccharide attached to the linker
portion of the dipeptide. However, the presence of a
monosaccharide is not mandatory for activity, as il-
lustrated by the desfucose analogue 9. Indeed, the
incorrect monosaccharide can function as an antagonist.
This is illustrated by the loss of activity with replace-
ment of fucose with glucose; compound 7. Generally,
the lack of any monosaccharide abrogates activity, as
illustrated by compounds 11, 12, and 14.
(2) Activity is dependent upon the distance and
relative flexibility or spatial freedom between the dipep-
tide and monosaccharide. No separation between the
dipeptide and monosaccharide results in reduced activ-
ity relative to a six-carbon spacer (compounds 15-19).
Similarily, too large a separation also results in reduced
activity (compounds 20-22). Compound 23 illustrates
the importance of a highly flexible linker. Additionally,
compound 23 suggests that the hexamethylene linker
adopts a bent configuration, which could perhaps be
replaced with a shorter chain.
(3) Activity is dependent upon the sequence and
stereochemistry of the dipeptide. D-Alanyl-L-glutamine,
as represented by BCH-2537, is the most active com-
pound. Conservative substitution of D-alanine resulted
in loss of activity, as illustrated by compounds 24-26.
However, the N-terminal amino function must be
present, 28 and 29, to presumably provide an ap-
propriately positioned positive charge. Very little change
is tolerated for L-glutamine (compounds 32-34 and 40-
46).
F igu r e 1. Dose-response profile of the functional ex vivo NK
cell activity of BCH-2537, 1.
This is accompanied by a significant increase in T-helper
cells (13% to 14%, P e 0.02).
The second experiment with BCH-2537 confirmed a
significant increase in NK cells. However, the immune
cell subset profile is different. For example, there is no
significant increase in NK cells in the blood. Nonethe-
less, in the spleen, significant increases in NK cells (60%
to 83%, P e 0.041) occur which are of greater magnitude
than in the first experiment. Further, these increases
occur at two doses of BCH-2537; 5 and 25 mg/kg. Again,
the increase in NK cells is accompanied by increases in
T-helper cells (22%, P e 0.02) and monocytes (47% to
103%, P e 0.015). Additionally, the second experiment
revealed that 9 also induces a significant increase in
NK cells, but to a lesser extent than BCH-2537. In
blood, 9 did not afford a significant increase in any
immune cell subset. In spleen, however, a significant
increase in NK cells (28% to 45%, P e 0.008) is observed,
but the magnitude of the increase is less than with
BCH-2537 and it is seen only at 25 mg/kg 9. As with
BCH-2537, significant increases occur in T-helper cells
(32% to 48%, P e 0.005) and monocytes (42%, P e
0.013). Unlike BCH-2537, a significant increase also
occurs in cytotoxic T-lymphocytes (37% to 64%, P e
0.023).
To fully understand the immunological effect of BCH-
2537 on NK cells, an important component of the
immunophenotyping experiments was to assess any
changes in the functional activity of NK cells along with
changes in their relative numbers. That is, to determine
the ability of BCH-2537 to stimulate NK cells. There-
fore, upon sacrifice of the mice at the end of the
experiment, a portion of the splenic cell suspension was
assayed for lytic activity by the same chromium release
assay used for the in vitro determination of NK cell
activity. The results from the first experiment are
shown in Figure 1. NK cell activity is enhanced at all
three doses of BCH-2537. Further, this increase in
functional NK cell activity is significant at 5 mg/kg (P
e 0.002) and 25 mg/kg (P e 0.022) BCH-2537. The
second experiment confirmed this significant (P e 0.010)
augmentation of NK cell activity at 5 and 25 mg/kg
BCH-2537. Interestingly, the increase in NK cell activ-
ity induced by 5 and 25 mg/kg 9 is not significant. Thus,
there is a significant difference in the in vivo stimulation
of NK cell activity induced by BCH-2537 and the
desfucose analogue 9.
The model structure which emerges from the above
is one in which the N-terminal amino function, the
glutamine carboxamide, and the linker (terminal) oxy-
gen atom participate in attractive (e.g., hydrogen bond)
interactions with receptor protein(s). Once established,
these interactions may be reinforced by binding of the
appropriate portion of the fucose sugar and the alanine
methyl group. Alternatively, the alternating (D-alanine)
stereochemistry may facilitate bending of the dipeptide.
Oligopeptides composed of D-alanyl-L-glutamine have
been reported to have a propensity for a bend.19 Evi-
dence for the importance of the linker oxygen atom is
provided by comparison of the activity of the thio
analogue 10 which retains moderate activity. Addition-
ally, a specific (hydrogen bond) interaction is implied
by the superior activity of compound 9 when compared
to compounds 11-14 which also do not possess a
monosaccharide. In summary, while the structure-
activity data is consistent with a drug-receptor interac-
tion, and BCH-2537 was designed to bind to a monosac-
charide receptor, it is not known with which NK cell
receptor(s) BCH-2537 and analogues interact.

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1915
Ta ble 8. Summary of the Antimetastatic Effect of BCH-2537,
1, in the Lungs of Mice
The potent and selective in vitro NK cell activity of
BCH-2537, 1, justified selection for in vivo immunologi-
cal evaluation. Indeed, it is interesting that a low
molecular weight synthetic (MW ) 463) could mimic the
activity of interleukin 2 (MW ≈ 15 000). Immunophe-
notyping experiments with BCH-2537 revealed a sig-
nificant increase in spleen NK cells. The magnitude of
this increase, up to 83% relative to control, was greater
in the second experiment. However, a significant
increase in blood NK cells was observed in the first
experiment. While these results may appear somewhat
variable, this reflects the variability between experi-
ments with two groups of mice. Further, the consistent
increase in spleen NK cells suggests a more sustained
response indicative of a greater therapeutic potential.
In support of the latter was the concomitant increase
in NK cell functional activity. This increase in NK cells
induced by BCH-2537 was significant at 5 and 25 mg/
kg concentrations of drug in both experiments. On the
other hand, the desfucose analogue 9 induced a smaller
increase in NK cells relative to BCH-2537. Unlike
BCH-2537, this increase was only significant at one dose
in the same (second) phenotyping experiment. Ad-
ditionally, the stimulation of NK cells induced by
compound 9 was not significant. Finally, significant
increases in other immune cell subsets were also
observed with BCH-2537 and the desfucose analogue 9
in spite of the selective in vitro activity. This likely
reflects cellular communication processes arising from
the direct stimulation of NK cells. For example, in-
creases in monocyte/macrophage (CD11b+) populations
may result from increased γ interferon produced by
activated NK cells.
number of lung colonies (SEM)a
BCH-2537, 1
exp 1 (n ) 8)
exp 2 (n ) 4)
0
142 ( 5
109 ( 10
(P < 0.02)
nd
101 ( 3
86 ( 11
25 mg/kg
50 mg/kg
60 ( 20
a
C57BL/6 mice were injected iv on day 0 with 2.5 × 105 B16
melanoma cells. Mice were injected ip with BCH-2537 on days -2,
-1, 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. All mice were
sacrificed on day 24. Lungs were fixed and counted for the number
of lung tumor colonies.
Exp er im en ta l Section
Ch em istr y. Melting points were taken on a Fisher-J ohns
melting point apparatus and are uncorrected. Merck silica gel
(Kieselgel 60) was used for TLC and flash column chromatog-
raphy (230-400 mesh). NMR spectra were recorded on a
Bruker DRX-400 or a Varian VXR-300 spectrometer. Chemi-
cal shifts (δ) are expressed in ppm, relative to the solvent as
internal standard unless otherwise stated. Mass spectra were
recorded on a Kratos MS-50 TA instrument. Analytical HPLC
results were obtained using a Waters instrument with either
a Hamilton PRP-1, 4.1 × 150 mm (“Hamilton”) or YMC C-4 (5
µm) 120 Å, 4.6 × 250 mm (“YMC”) reverse phase column.
Liquid phase: A ) MeOH; B ) MeCN; C ) 10 mM NH4OAc;
D ) 10 mM NH4HCO3; E ) 0.01% aqueous TFA, with
gradients as described, flow rate of 1 mL min-1 (0.5 mL min-1
for TFA system), and using a UV detector at an absorption of
210 nm. Combustion analyses were carried out by Chemisar
Laboratories (Guelph, ON). All amino acids and dipeptide 17
were obtained from Bachem Bioscience Inc. (King of Prussia,
PA), and all other reagents were obtained from Aldrich
Chemical Co. (Milwaukee, WI), Fluka (Ronkonkoma, NY), or
VWR Inc. (Montre´al, PQ). All nonaqueous reactions were
carried out under an inert atmosphere.
Gen er a l P r oced u r e A: Cou p lin g of Am in o Alcoh ols
w ith Am in o Acid s. A solution of the suitably protected
amino acid, the unprotected amino alcohol (1.2 equiv) and
2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline (EEDQ; 1.2
equiv) in CHCl3 was stirred at ambient temperature overnight.
The solvent was evaporated in vacuo, and the crude product
was purified by flash silica gel chromatography.
Gen er a l P r oced u r e B: Am in o Acid Cou p lin g P r oce-
d u r e. A solution of the free amine (or amine salt and 1 equiv
of triethylamine) and the free acid (1.2 equiv) in dry CH2Cl2
was treated with anhydrous 1-hydroxybenzotriazole (HOBT;
1.2 equiv), and the mixture was cooled to 0 °C. The reaction
was then treated with either 1,3-dicyclohexylcarbodiimide
(DCC; 1.4 equiv) or 1-(3-(dimethylamino)propyl)-3-ethylcar-
bodiimide hydrochloride (EDC‚HCl; 1.4 equiv) and stirred from
0 °C to ambient temperature overnight. The reaction was
filtered through Celite, and the residue was washed with CH2-
Cl2. Combined filtrate and washings were evaporated in vacuo
to give the crude product, which was purified by flash silica
gel chromatography.
Gen er a l P r oced u r e C: Cou p lin g of Alcoh ols w ith
Br om o Su ga r s. The free hydroxy compound and silver(I)
carbonate (1.1 equiv) were dried in the dark at ambient
temperature in vacuo for 24 h prior to reaction. Freshly
powdered, oven-dried calcium sulfate was placed in an oven-
dried flask, which was then cooled to ambient temperature
under an argon atmosphere. Solutions of the dried hydroxy
compound and bromo sugar (1.1 equiv) in dry CH2Cl2 were
introduced. This mixture was treated with the dried silver(I)
carbonate and iodine (0.3 equiv). The reaction was wrapped
in aluminum foil to exclude light and stirred at ambient
temperature overnight. The reaction was filtered through
Celite, and the residue was washed with CH2Cl2. Combined
filtrate and washings were evaporated in vacuo to give the
crude product, which was purified by flash silica gel chroma-
tography.
In conclusion, BCH-2537, 1, induces a significant
stimulation and expansion of NK cells. Further, on the
basis of this study, it appears to be well-tolerated and
devoid of any apparent toxicity. Irritation was not
observed at the site of injection, thereby indicating a
lack of an inflammatory response. BCH-2537 is very
soluble and stable in aqueous solution.20 Therefore,
BCH-2537 possesses the properties desirable for an
injectable therapeutic. However, the question remains
whether the effect of BCH-2537 on NK cells results in
a significant therapeutic response. In an effort to
address this question, a preliminary evaluation was
undertaken as to the ability of BCH-2537 to inhibit the
metastasis of murine B16 melanoma cells. In this
model, NK cell stimulants (interferon inducers) such as
loxoribine, 39, inhibit metastasis in the lung.21 BCH-
2537 does reduce the number of lung colonies when
administered by intraperitoneal injection, as shown in
Table 8. Although the activity is moderate, this experi-
ment does not represent an optimized protocol (e.g., dose
schedule, route of administration). Also, most immu-
nostimulants, such as interferon inducers, offer multiple
effector mechanisms (e.g., NK, B cell, and macrophage)
which result in a relatively more robust response.
Therefore, specific stimulants such as BCH-2537 may
offer more potential as a nontoxic adjunct in prophy-
lactic or therapeutic regimens. Additionally, such com-
pounds may prove valuable as tools for the delineation
of the precise role of NK cells in host defense mecha-
nisms.

1916 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
Gen er a l P r oced u r e D: Dep r otection of th e BOC-
P r otected Am in o Gr ou p . A solution of the BOC-protected
compound in Et2O and EtOH (1:1) was cooled to 0 °C and was
treated with toluene-4-sulfonic acid, monohydrate. Once dis-
solution was complete, the solvents were evaporated in vacuo,
and the residue was heated at 40 °C in vacuo for 2-3 h to
give the free amine, toluene-4-sulfonate salt.
Gen er a l P r oced u r e E: Dep r otection of Z-P r otected
Am in o Gr ou p . A solution of the Z-protected compound in
denatured EtOH, or MeOH, under N2, was treated with
palladium on activated carbon (10% w/w, of 10% w/w Pd/C).
The mixture was thoroughly degassed and then stirred at room
temperature, under 1 atm of H2 overnight. The mixture was
diluted with MeOH and filtered through Celite. The residue
was washed with MeOH, and combined filtrate and washings
were evaporated in vacuo to give the free amine.
Gen er a l P r oced u r e F : Am m on olysis. A solution of the
protected compound in MeOH was cooled to 0 °C. Anhydrous
ammonia gas was then bubbled through the solution for 10
min, and the flask was sealed. The reaction was stirred at
room temperature overnight, and solvent was evaporated in
vacuo. The residue was purified by flash chromatography to
give the desired compound.
6-[[N-(Ben zyloxyca r bon yl)-5-O-m eth yl-(S)-glu ta m yl]-
a m in o]h exa n -1-ol, 3. Z-L-glutamic acid, 5-methyl ester 2
(5.34 g, 18.1 mmol) and 6-aminohexanol were coupled accord-
ing to general procedure A, to give, after purification, 3 as a
white solid (6.18 g, 87%): 1H NMR (CDCl3, 300 MHz) 1.30-
1.44 (4H, m, hexyl H-3 and H-4), 1.45-1.60 (4H, m, hexyl H-2
and H-5), 1.64 (1H, br s, OH), 1.87-1.96 and 2.05-2.10 (2H,
2 × m, Glu H-3), 2.36 (1H, A of ABX2, J 17.0, 6.5 Hz, 1H of
Glu H-4), 2.51 (1H, B of ABX2, J 17.0, 7.0 Hz, 1H of Glu H-4),
3.23 (2H, dt, J 6.5, 6.5 Hz, hexyl H-6) 3.61 (2H, t, J 6.5 Hz,
hexyl H-1), 3.65 (3H, s, OMe), 4.14-4.22 (1H, m, Glu H-2),
5.08 (2H, s, PhCH2), 5.64 (1H, d, J 8.0 Hz, ZNH), 6.27-6.34
(1H, m, NHCH2), 7.32 (5H, s, Ph).
according to general method E to give the intermediate amine,
which was coupled with Z-D-alanine (1.34 g, 6.0 mmol) accord-
ing to general method B (using DCC). This gave, after
purification, 6 as a white solid (2.52 g, 68%): 1H NMR (CDCl3,
300 MHz) 1.19 (3H, d, J 6.5 Hz, fucose H-6), 1.22-1.38 (4H,
m, hexyl H-3 and H-4), 1.35 (3H, d, J 7.0 Hz, Ala H-3), 1.40-
1.58 (4H, m, hexyl H-2 and H-5), 1.85-2.20 (2H, m, Glu H-3),
1.95 (3H, s, OAc), 2.01 (3H, s, OAc), 2.14 (3H, s, OAc), 2.28-
2.58 (2H, m, Glu H-4), 3.17 (2H, dt, J 6.5, 6.5 Hz, hexyl H-6),
3.40 (1H, A of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 3.65 (3H,
s, OMe), 3.76 (1H, qd, J 6.5, 1.0 Hz, fucose H-5), 3.85 (1H, B
of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 4.10-4.20 and 4.34-
4.42 (2H, 2 × m, Ala H-2 and Glu H-2), 4.38 (1H, d, J 8.0 Hz,
fucose H-1), 4.98 (1H, dd, J 10.0, 3.5 Hz, fucose H-3), 5.05-
5.16 (3H, m, CH2Ph + fucose H-2), 5.19 (1H, dd, J 3.5, 1.0 Hz,
fucose H-4), 5.39 (1H, d, J 6.5 Hz, ZNH), 6.60-6.67 (1H, m,
NHCH2), 7.06 (1H, d, J 7.0 Hz, Glu NH), 7.27-7.34 (5H, m,
Ph).
1-â-[6-((R)-Ala n yl-(S)-glu ta m in yla m in o)h ex-1-yl]-L-fu -
cop yr a n ose, BCH-2537, 1. Compound 6 (177 mg, 0.17 mmol)
was N-deprotected according to general method E to give the
intermediate free amine as a colorless oil. This material was
converted to BCH-2537, 1, by ammonolysis according to
general method F to give, after purification by flash silica gel
chromatography (gradient elution of 10-20% concentrated
aqueous ammonium hydroxide in propan-2-ol), compound 1
as a white solid (43 mg, 56%): mp 180-183 °C (softens 173
°C); 1H NMR (CD3OD + D2O, 300 MHz) 1.25 (3H, d, J 6.5 Hz,
fucose H-6), 1.27 (3H, d, J 7.0 Hz, Ala H-3), 1.30-1.42 (4H,
m, hexyl H-3 and H-4), 1.46-1.54 (2H, m, hexyl H-5), 1.55-
1.66 (2H, m, hexyl H-2), 1.87-2.00 and 2.02-2.12 (2H, 2 ×
m, Gln H-3), 2.28-2.33 (2H, m, Gln H-4), 3.11-3.25 (2H, m,
hexyl H-6), 3.44 (1H, dd, J 10.0, 7.5 Hz, fucose H-2), 3.48 (1H,
q, J 7.0 Hz, Ala H-2), 3.51 (1H, dd, J 10.0, 3.5 Hz, fucose H-3),
3.54 (1H, A of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 3.64 (1H,
dd, J 3.5, 1.0 Hz, fucose H-4), 3.65 (1H, qd, J 6.5, 1.0 Hz, fucose
H-5), 3.83 (1H, B of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1),
4.22 (1H, d, J 7.5 Hz, fucose H-1), 4.28 (1H, dd, J 9.0, 5.0 Hz,
Gln H-2); 13C NMR (CD3OD, 75.5 MHz) 16.78 (fucose C-6),
21.31 (Ala C-3), 26.73, 27.69, 29.23, 30.32, 30.69 and 32.57
(Gln C-3 and C-4 and hexyl C-2, C-3, C-4, and C-5), 40.36
(hexyl H-6), 51.51 and 54.22 (Ala C-2 and Gln C-2), 70.58
(hexyl H-1), 71.84, 72.34, 73.07 and 75.20 (fucose C-2, C-3, C-4,
and C-5), 104.82 (fucose C-1), 173.56 (Gln C-5), 177.67 and
178.41 (Ala C-1 and Gln C-1); HRMS (FAB) m/z 463.27832,
calculated for MH+ (C20H39N4O8+) 463.27679; HPLC, system
A (Hamilton; 0-50% (60 min) B in C, pH 8.45), 17.4 min, 99%;
system B (YMC; 0-35% (30 min) A in E), 18.4 min. Anal.
(C20H38N4O8‚0.5H2O) C, H, N.
2,3,4,6-Tetr a -O-a cetyl-â-D-glu cop yr a n ose. A solution of
2,3,4,6-tetra-O-acetyl-R-D-glucopyranosyl bromide (97%; 1.00
g, 2.43 mmol) in acetone (10 mL) was treated with silver(I)
carbonate (704 mg, 2.55 mmol) and water (200 µL, 11.1 mmol).
The mixture was stirred in the dark at ambient temperature
for 16 h. The mixture was filtered through Celite and the
residue washed with CH2Cl2. Combined filtrate and washings
were evaporated in vacuo to give the title compound as a white
solid (820 mg, 97%): 1H NMR (CDCl3, 300 MHz) 1.96 (3H, s,
OAc), 1.97 (3H, s, OAc), 2.02 (3H, s, OAc), 2.04 (3H, s, OAc),
3.71 (1H, ddd, J 10.0, 4.5, 2.5 Hz, H-5), 4.09 (1H, A of ABX, J
12.5, 2.5 Hz, 1H of H-6), 4.18 (1H, D2O exchangeable, d, J 8.0
Hz, OH), 4.19 (1H, B of ABX, J 12.5, 4.5 Hz, 1H of H-6), 4.70
(1H, dd, J 8.0, 8.0 Hz [collapses to d, J 8.0 Hz on addition of
D2O], H-1), 4.85 (1H, dd, J 9.5, 8.0 Hz, H-2), 5.03 (1H, dd, J
10.0, 9.5 Hz, H-4), 5.19 (1H, dd, J 9.5, 9.5 Hz, H-3).
2,3,4-Tr i-O-a cetyl-r-L-fu cop yr a n osyl Br om id e, 4.
A
solution of R-L-fucose tetraacetate (5.00 g, 15.0 mmol) in HBr/
AcOH (45% w/v HBr; 350 mL) was stirred at room tempera-
ture, in a sealed flask (septum only), for 5 h. Acids were then
removed in vacuo, and the residue was dissolved in CH2Cl2
(200 mL). This solution was treated with water (100 mL). This
well-stirred mixture was treated with saturated aqueous
NaHCO3 solution until the pH of the aqueous phase reached
7. The layers were separated, the aqueous phase was ex-
tracted with CH2Cl2 (100 mL), and the combined organic
extracts were dried (Na2SO4), filtered, and evaporated in vacuo
to give 4 as a pale yellow oil (100%): TLC Rf ) 0.80 (SiO2; 3:2
hexane/EtOAc); 1H NMR (CDCl3, 300 MHz) 1.22 (3H, d, J 6.5
Hz, H-6), 2.02 (3H, s, OAc), 2.12 (3H, s, OAc), 2.18 (3H, s, OAc),
4.41 (1H, qd, J 6.5, 1.0 Hz, H-5), 5.03 (1H, dd, J 10.5, 4.0 Hz,
H-2), 5.37 (1H, dd, J 3.0, 1.0 Hz, H-4), 5.42 (1H, dd, J 10.5,
3.0 Hz, H-3), 6.70 (1H, d, J 4.0 Hz, H-1).
1-â-[6-[[N-(Benzyloxycarbonyl)-5-O-methyl-(S)-glutamyl]-
a m in o]h ex-1-yl]-2,3,4-t r i-O-a cet yl-â-L-fu cop yr a n ose, 5.
Compounds 3 (4.32 g, 11.0 mmol) and 4 (12.0 mmol) were
reacted according to general procedure B to give, after
purification, 5 as a colorless foam (3.24 g, 44%): 1H NMR
(CDCl3, 300 MHz) 1.18 (3H, d, J 6.5 Hz, fucose H-6), 1.22-
1.38 (4H, m, hexyl H-3 and H-4), 1.41-1.62 (4H, m, hexyl H-2
and H-5), 1.85-1.96 (1H, m, 1H of Glu H-3), 1.86 (3H, s, OAc),
2.01 (3H, s, OAc), 2.04-2.17 (1H, m, 1H of Glu H-3), 2.14 (3H,
s, OAc), 2.30-2.41 and 2.44-2.55 (2H, 2 × m, Glu H-4), 3.20
(2H, dt, J 7.0, 6.5 Hz, hexyl H-6), 3.41 (1H, A of ABX2, J 8.5,
6.5 Hz, 1H of hexyl H-1), 3.64 (3H, s, OMe), 3.76 (1H, qd, J
6.5, 1.0 Hz, fucose H-5), 3.86 (1H, B of ABX2, J 9.5, 6.0 Hz,
1H of hexyl H-1), 4.12-4.21 (1H, m, Glu H-2), 4.38 (1H, d, J
8.0 Hz, fucose H-1), 4.97 (1H, dd, J 10.5, 3.5 Hz, fucose H-3),
5.07 (2H, s, PhCH2), 5.14 (1H, dd, J 10.5, 8.0 Hz, fucose H-2),
5.20 (1H, dd, J 3.5, 1.0 Hz, fucose H-4), 5.63 (1H, d, J 8.0 Hz,
ZNH), 6.20-6.27 (1H, m, NHCH2), 7.28-7.35 (5H, m, Ph).
O-(2,3,4,6-Tet r a -O-a cet yl-r-D-glu cop yr a n osyl)t r ich lo-
r oa cetim id a te. A solution of 2,3,4,6-tetra-O-acetyl-â-D-glu-
copyranose (820 mg, 2.35 mmol) and trichloroacetonitrile (725
µL, 7.1 mmol), in dry CH2Cl2 (10 mL) was treated with oven-
dried K2CO3 (553 mg, 4.00 mmol), and the reaction was stirred
at ambient temperature for 64 h. The mixture was filtered
through Celite and the residue washed with CH2Cl2. Com-
bined filtrate and washings were evaporated in vacuo to give
a pale brown foam, which was purified by flash silica gel
1-â-[6-[[N-(Ben zyloxyca r bon yl)-(R)-a la n yl-5-O-m eth yl-
(S)-glu ta m yl]a m in o]h exyl]-2,3,4-tr i-O-a cetyl-L-fu cop yr a -
n ose, 6. Compound 5 (3.33 g, 5.0 mmol) was deprotected

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1917
1
chromatography (100:1, gradient elution, 30-60% EtOAc in
hexane) to give the title compound as a white foam (978 mg,
84%): 1H NMR (CDCl3, 300 MHz) 1.99 (3H, s, OAc), 2.01 (3H,
s, OAc), 2.02 (3H, s, OAc), 2.05 (3H, s, OAc), 4.10 (1H, A of
ABX, J 12.0, 2.0 Hz, 1H of H-6), 4.18 (1H, ddd, J 10.0, 4.0, 2.0
Hz, H-5), 4.25 (1H, B of ABX, J 12.0, 4.0 Hz, 1H of H-6), 5.10
(1H, dd, J 10.0, 3.5 Hz, H-2), 5.15 (1H, dd, J 10.0, 10.0 Hz,
H-3), 5.54 (1H, dd, J 10.0, 10.0 Hz, H-4), 6.53 (1H, d, J 3.5
Hz, H-1), 8.67 (1H, s, NH).
(SiO2; 8:2 IPA concentrated aqueous NH3); mp 82-86 °C; H
NMR (D2O, 300 MHz, MeCN ) 2.05 ppm) 1.29 (3H, d, J 7.0
Hz, Ala H-3), 1.29-1.40 (4H, m, hexyl H-3 and H-4), 1.45-
1.53 (2H, m, hexyl H-5), 1.54-1.65 (2H, m, hexyl H-2), 1.90-
2.17 (2H, m, Gln H-3), 2.35 (2H, t, J 7.5 Hz, Gln H-4), 3.12-
3.25 (2H, m, hexyl H-6), 3.23 (1H, dd, J 9.0, 8.0 Hz, glucose
H-2), 3.35 (1H, dd, J 9.0, 9.0 Hz, glucose H-4), 3.45 (1H, ddd,
J 9.0, 6.0, 2.0 Hz, glucose H-5), 3.47 (1H, dd, J 9.0, 9.0 Hz,
glucose H-3), 3.59 (1H, q, J 7.0 Hz, Ala H-2), 3.65 (1H, A of
ABX2, J 10.0, 7.5 Hz, 1H of hexyl H-1), 3.70 (1H, A of ABX, J
12.0, 6.0 Hz, 1H of glucose H-6), 3.90 (1H, B of ABX2, J 10.0,
6.5 Hz, 1H of hexyl H-1), 3.90 (1H, B of ABX, J 12.0, 2.0 Hz,
1H of glucose H-6), 4.24 (1H, dd, J 8.5, 5.5 Hz, Gln H-2), 4.43
(1H, d, J 8.0 Hz, glucose H-1); 13C NMR (D2O, 75.5 MHz, MeCN
) 1.30 ppm) 19.93 (Ala C-3), 25.10, 26.13, 27.40, 28.56, 29.08
and 31.62 (Gln C-3 and C-4, + hexyl C-2 to C-5), 39.72 (hexyl
C-6), 50.24 and 53.88 (Ala C-2 and Gln C-2), 61.21 and 70.94
(hexyl C-1 and glucose C-6), 70.10, 73.59, 76.26 and 76.34
(glucose C-2, C-3, C-4, and C-5), 102.61 (glucose C-1), 173.50
1-â-[6-[[N-Ben zyloxyca r b on yl-5-O-m et h yl-(S)-glu t a -
m yl]a m in o]h ex-1-yl]-2,3,4,6-tetr a -O-a cetyl-â-D-glu cop yr a -
n ose. A solution of compound 3 (434 mg, 1.10 mmol) and
O-(2,3,4,6-tetra-O-acetyl-â-D-glucopyranosyl)trichloroacetim-
idate (493 mg, 1.00 mmol) in dry CH2Cl2 (40 mL) at -15 °C
was treated with BF3.Et2O (25 µL, 0.20 mmol), and the
reaction was stirred at -15 °C for 90 min. The reaction was
quenched by addition of NaHCO3 (500 mg) followed by addition
of saturated aqueous NaHCO3 (25 mL). The layers were
separated, and the aqueous phase was further extracted with
CH2Cl2 (25 mL). Combined organic extracts were dried (Na2-
SO4), filtered, and evaporated in vacuo. The crude material
was purified by flash silica gel chromatography (100:1, gradi-
ent elution, 50-100% EtOAc in hexane) to give the title
compound as a colorless oil (222 mg, 31%): 1H NMR (CDCl3,
300 MHz) 1.23-1.38 (4H, m, hexyl H-3 and H-4), 1.41-1.51
(2H, m, hexyl H-5), 1.51-1.61 (2H, m, hexyl H-2), 1.85-2.17
(2H, m, Glu H-3), 1.98 (3H, s, OAc), 2.00 (3H, s, OAc), 2.02
(3H, s, OAc), 2.06 (3H, s, OAc), 2.32-2.41 and 2.45-2.56 (2H,
2 × m, Glu H-4), 3.21 (2H, dt, J 6.5, 6.5 Hz, hexyl H-6), 3.44
(1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.62-3.69
(1H, m, glucose H-5), 3.65 (3H, s, OMe), 3.83 (1H, B of ABX2,
J 9.5, 6.0 Hz, 1H of hexyl H-1), 4.11-4.20 (1H, m, Glu H-2),
4.12 (1H, A of ABX, J 12.5, 2.5 Hz, 1H of glucose H-6), 4.24
(1H, B of ABX, J 12.5, 4.5 Hz, 1H of glucose H-6), 4.46 (1H, d,
J 8.0 Hz, glucose H-1), 4.96 (1H, dd, J 9.5, 8.0 Hz, glucose
H-2), 5.06 (1H, dd, J 10.0, 9.5 Hz, glucose H-4), 5.08 (2H, s,
PhCH2), 5.18 (1H, dd, J 9.5, 9.5 Hz, glucose H-3), 5.60 (1H, d,
J 7.0 Hz, ZNH), 6.20-6.28 (1H, m, NHCH2), 7.32 (5H, s, Ph).
1-â-[6-[[N-(Ben zyloxyca r bon yl)-(R)-a la n yl-5-O-m eth yl-
(S)-glu t a m yl]a m in o]h exyl]-2,3,4,6-t et r a -O-a cet yl-D-glu -
cop yr a n ose. 1-â-[6-[[N-(Benzyloxycarbonyl)-5-O-methyl-(S)-
glu t a m yl]a m in o]h ex-1-yl]-2,3,4,6-t et r a -O-a cet yl-â-D -
glucopyranose (220 mg, 0.30 mmol) was deprotected according
to general method E to give the intermediate amine. This was
coupled with Z-D-alanine (90 mg, 0.33 mmol) according to
general method B (using EDC‚HCl). This gave, after purifica-
tion, the title compound as a white solid (118 mg, 54%). 1H
NMR (CDCl3, 300 MHz) 1.21-1.32 (4H, m, hexyl H-3 and H-4),
1.32 (3H, d, J 7.0 Hz, Ala H-3), 1.37-1.46 (2H, m, hexyl H-5),
1.46-1.55 (2H, m, hexyl H-2), 1.87-2.16 (2H, m, Glu H-3),
1.95 (3H, s, OAc), 1.97 (3H, s, OAc), 1.98 (3H, s, OAc), 2.03
(3H, s, OAc), 2.24-2.48 (2H, m, Glu H-4), 3.09-3.19 (2H, m,
hexyl H-6), 3.40 (1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl
H-1), 3.62 (3H, s, OMe), 3.64 (1H, ddd, J 9.5, 4.5, 2.5 Hz,
glucose H-5), 3.79 (1H, B of ABX2, J 9.5, 6.5 Hz, 1H of hexyl
H-1), 4.09 (1H, A of ABX, J 12.0, 2.5 Hz, 1H of glucose H-6),
4.17 (1H, qd, J 7.0, 7.0 Hz, Ala H-2), 4.21 (1H, B of ABX, J
12.0, 4.5 Hz, 1H of glucose H-6), 4.38 (1H, dt, J 7.5, 7.5, 5.0
Hz, Glu H-2), 4.44 (1H, d, J 8.0 Hz, glucose H-1), 4.92 (1H,
dd, J 9.5, 8.0 Hz, glucose H-2), 5.01 and 5.08 (2H, AB q, J
12.0 Hz, PhCH2), 5.03 (1H, dd, J 9.5, 9.5 Hz, glucose H-4),
5.16 (1H, dd, J 9.5, 9.5 Hz, glucose H-3), 5.63 (1H, d, J 7.0 Hz,
ZNH), 6.73-6.80 (1H, m, NHCH2), 7.19 (1H, d, J 7.5 Hz, Glu
NH), 7.28 (5H, s, Ph).
(Gln C-5), 178.14 and 178.20 (Ala C-1 and Gln C-1); HRMS
(FAB) m/z 479.270 80, calculated for MH+ (C20H39N4O9
)
+
479.271 70; HPLC, system A (Hamilton; 0-50% (60 min) B in
C, pH 7.7), 14.4 min, 93%; system B (YMC; 0-35% (30 min) A
in E), 12.4 min. Anal. (C20H38N4O9‚2H2O) C, H, N.
2,3,4,6-Tetr a -O-a cetyl-r-D-m a n n op yr a n osyl Br om id e. A
solution of R-D-mannose pentaacetate (1.09 g, 2.8 mmol) in
HBr/AcOH (45% w/v HBr; 80 mL) was stirred at room
temperature, in a sealed flask (septum only), for 4 h. Acids
were then removed in vacuo, the residue was dissolved in CH2-
Cl2 (200 mL), and this solution was treated with water (100
mL). This well-stirred mixture was treated with saturated
aqueous NaHCO3 solution until the pH of the aqueous phase
reached 7. The layers were separated, the aqueous phase was
extracted with CH2Cl2 (100 mL), and combined organic ex-
tracts were dried (Na2SO4), filtered, and evaporated in vacuo
to give the title compound as a yellow oil (100%): TLC Rf )
0.50 (SiO2; 3:2 hexane/EtOAc); 1H NMR (CDCl3, 300 MHz) 1.97
(3H, s, OAc), 2.03 (3H, s, OAc), 2.06 (3H, s, OAc), 2.13 (3H, s,
OAc), 4.09 (1H, A of ABX, J 12.5, 2.0 Hz, 1H of H-6), 4.18 (1H,
ddd, J 10.0, 5.0, 2.0 Hz, H-5), 4.29 (1H, B of ABX, J 12.5, 5.0
Hz, 1H of H-6), 5.33 (1H, dd, J 10.0, 10.0 Hz, H-4), 5.41 (1H,
dd, J 3.5, 1.5 Hz, H-2), 5.67 (1H, dd, J 10.0, 3.5 Hz, H-3), 6.26
(1H, d, J 1.5 Hz, H-1).
1-â-[6-[[N-Ben zyloxyca r b on yl-5-O-m et h yl-(S)-glu t a -
m yl]a m in o]h ex-1-yl]-2,3,4,6-tetr a -O-a cetyl-â-D-m a n n op y-
r a n ose. Compound 3 (1.00 g, 2.50 mmol) and 2,3,4,6-tetra-
O-acetyl-R-D-mannopyranosyl bromide (12 mmol) were reacted
according to general procedure B to give, after purification,
the title compound as a colorless oil (1.50 g, 74%): 1H NMR
(CDCl3, 300 MHz) 1.22-1.64 (8H, m, hexyl H-2 to H-5), 1.73
(3H, s, OAc), 1.88-2.18 (2H, m, Glu H-3), 2.05 (3H, s, OAc),
2.06 (3H, s, OAc), 2.11 (3H, s, OAc), 2.32-2.57 (2H, m, Glu
H-4), 3.16-3.29 (2H, m, hexyl H-6), 3.46 (2H, t, J 6.5 Hz, hexyl
H-1), 3.64-3.72 (1H, m, mannose H-5), 3.66 (3H, s, OMe),
4.10-4.27 (3H, m, Glu H-2 and mannose H-6), 4.59 (1H, dd, J
4.0, 2.0 Hz, mannose H-2), 5.09 (2H, s, PhCH2), 5.14 (1H, dd,
J 10.0, 4.0 Hz, mannose H-3), 5.29 (1H, dd, J 10.0, 9.5 Hz,
mannose H-4), 5.47 (1H, d, J 2.0 Hz, mannose H-1), 5.67 (1H,
d, J 7.5 Hz, ZNH), 6.22-6.32 (1H, m, NHCH2), 7.30-7.39 (5H,
m, Ph).
1-â-[6-[[N-(Ben zyloxyca r bon yl)-(R)-a la n yl-5-O-m eth yl-
(S)-glu ta m yl]a m in o]h exyl]-2,3,4,6-tetr a -O-a cetyl-D-m a n -
n op yr a n ose. 1-â-[6-[[N-(Benzyloxycarbonyl)-5-O-methyl-(S)-
glu t a m yl]a m in o]h ex-1-yl]-2,3,4,6-t et r a -O-a cet yl-â-D -
mannopyranose (362 mg, 0.50 mmol) was deprotected according
to general method E to give the intermediate amine, which
was coupled with Z-D-alanine (134 mg, 0.60 mmol) according
to general method B (using DCC). This gave, after purifica-
tion, the title compound as colorless crystals (290 mg, 73%):
1H NMR (CDCl3, 300 MHz) 1.20-1.33 (4H, m, hexyl H-3 and
H-4), 1.32 (3H, d, J 7.0 Hz, Ala H-3), 1.37-1.52 (4H, m, hexyl
H-2 and H-5), 1.68 (3H, s, OAc), 1.82-2.15 (2H, m, Glu H-3),
2.01 (3H, s, OAc), 2.02 (3H, s, OAc), 2.07 (3H, s, OAc), 2.25-
2.50 (2H, 2, Glu H-4), 3.14 (2H, dt, J 6.5, 6.5 Hz, hexyl H-6),
1-â-[6-((R)-Ala n yl-(S)-glu ta m in yla m in o)h ex-1-yl]-D-glu -
cop yr a n ose, 7. 1-â-[6-[[N-(Benzyloxycarbonyl)-(R)-alanyl-5-
O-methyl-(S)-glutamyl]amino]hexyl]-2,3,4,6-tetra-O-acetyl-D-
glucopyranose (118 mg, 0.15 mmol) was N-deprotected according
to general method E to give the intermediate free amine as a
colorless oil. This material was converted to 7 by ammonolysis
according to general method F to give, after purification by
flash silica gel chromatography (gradient elution of 10-20%
concentrated aqueous ammonium hydroxide in propan-2-ol),
compound 7 as a white solid (23 mg, 32%): TLC, Rf ) 0.25

1918 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
3.37-3.45 (1H, m, hexyl H-1), 3.60-3.77 (1H, m, mannose
H-5), 3.62 (3H, s, OMe), 4.04-4.22 (3H, m, Glu H-2 and
mannose H-6), 4.34-4.42 (1H, m, Ala H-2), 4.54 (1H, dd, J
4.0, 2.5 Hz, mannose H-2), 5.00 and 5.08 (2H, AB q, J 12.5
Hz, PhCH2), 5.11 (1H, dd, J 10.0, 4.0 Hz, mannose H-3), 5.25
(1H, dd, J 10.0, 9.5 Hz, mannose H-4), 5.42 (1H, d, J 2.5 Hz,
mannose H-1), 5.60 (1H, d, J 7.0 Hz, ZNH), 6.70-6.77 (1H,
m, NHCH2), 7.19 (1H, d, J 7.5 Hz, Glu NH), 7.25-7.32 (5H,
m, Ph).
2.13 (2H, 2 × m, Gln H-3), 2.24-2.32 (2H, m, Gln H-4), 3.18
(2H, t, J 7.0 Hz, hexyl H-6), 3.46 (1H, q, J 7.0 Hz, Ala H-2),
3.53 (2H, t, J 6.5 Hz, hexyl H-1), 4.29 (1H, dd, J 9.0, 5.0 Hz,
Gln H-2); 13C NMR (CD3OD, 75.5 MHz) 21.17 (Ala C-3), 26.59,
27.74, 29.21, 30.33, 32.55, and 33.52 (Gln C-3 and C-4 and
hexyl C-2, C-3, C-4, and C-5), 40.39 (hexyl H-6), 51.46 and
54.25 (Ala C-2 and Gln C-2), 62.84 (hexyl H-1), 173.58 (Gln
C-5), 177.66 and 178.16 (Ala C-1 and Glu C-1); HRMS (FAB)
m/z 317.217 17, calculated for MH+ (C14H29N4O4+) 317.218 87;
HPLC, system A (Hamilton; 0-35% (40 min) B in C, pH 6.00),
18.3 min; system B (YMC; 0-25% (30 min) A in E), 12.5 min.
Anal. (C14H28N4O4‚H2O) C, H, N; C: calcd. 50.28; found 50.71;
N: calcd. 16.75; found 16.28.
2,3,4-Tr i-O-a cetyl-1-th io-â-L-fu cop yr a n osid e. A solution
of 2-S-(2,3,4-tri-O-acetyl-â-L-fucopyranosyl)-2-pseudothiourea,
hydrobromide12 (1.00 g, 2.33 mmol) in methanol (10 mL) was
treated with a solution of K2CO3 (483 mg, 3.49 mmol) in water
(100 mL). The mixture was stirred at ambient temperature,
under a nitrogen atmosphere for 90 min. The solution was
extracted with dichloromethane (2 × 50 mL), and the combined
extracts were dried (Na2SO4), filtered, and evaporated in vacuo
to give the title compound as a white foam (702 mg, 98%): 1H
NMR (CDCl3, 300 MHz) 1.20 (3H, d, J 6.5 Hz, H-6), 1.96 (3H,
s, OAc), 2.06 (3H, s, OAc), 2.16 (3H, s, OAc), 2.45-2.90 (1H,
br s, SH), 3.81 (1H, qd, J 6.5, 1.0 Hz, H-5), 4.47 (1H, d, J 9.5
Hz, H-1), 4.98 (1H, dd, J 10.0, 3.5 Hz, H-3), 5.13 (1H, dd, J
10.0, 9.5 Hz, H-2), 5.25 (1H, dd, J 3.5, 1.0 Hz, H-4).
2-O-Acetyl-1-â-[6-((R)-a la n yl-(S)-glu ta m in yla m in o)h ex-
1-yl]-D-m a n n op yr a n ose, 8. 1-â-[6-[[N-(Benzyloxycarbonyl)-
(R)-alanyl-5-O-methyl-(S)-glutamyl]amino]hexyl]-2,3,4,6-tetra-
O-acetyl-D-mannopyranose (147 mg, 0.18 mmol) was
N-deprotected according to general method E to give the
intermediate free amine as a colorless oil. This material was
converted to BCH-2549, 8, by ammonolysis according to
general method F to give, after purification by flash silica gel
chromatography (gradient elution of 10-20% concentrated
aqueous ammonium hydroxide in propan-2-ol), compound 8
as a white solid (33 mg, 36%): mp 79.5-82 °C; 1H NMR (CD3-
OD, 300 MHz) 1.27 (3H, d, J 7.0 Hz, Ala H-3), 1.30-1.44 (4H,
m, hexyl H-3 and H-4), 1.45-1.58 (4H, m, hexyl H-2 and H-5),
1.62 (3H, s, OAc), 1.85-1.98 and 2.01-2.14 (2H, 2 × m, Gln
H-3), 2.28 (2H, dd, J 7.5, 7.0 Hz, Gln H-4), 3.18 (2H, t, J 7.0
Hz, hexyl H-6), 3.22 (1H, ddd, J 9.5, 6.0, 2.5 Hz, mannose H-5),
3.45 (1H, q, J 7.0 Hz, Ala H-2), 3.49-3.56 (2H, m, hexyl H-1),
3.55 (1H, dd, J 9.5, 9.5 Hz, mannose H-4), 3.65 (1H, A of ABX,
J 12.0, 6.0 Hz, 1H of mannose H-6), 3.69 (1H, dd, J 9.5, 4.0
Hz, mannose H-3), 3.84 (1H, B of ABX, J 12.0, 2.5 Hz, 1H of
mannose H-6), 4.30 (1H, dd, J 9.0, 5.0 Hz, Gln H-2), 4.43 (1H,
dd, J 4.0, 2.5 Hz, mannose H-2), 5.43 (1H, d, J 2.5 Hz, H-1);
13C NMR (CD3OD, 75.5 MHz) 21.30 (Ala C-3), 25.83 (MeCO),
26.83, 27.56, 29.21, 30.26, 30.49, and 32.55 (Gln C-3 and C-4
and hexyl C-2, C-3, C-4, and C-5), 40.37 (hexyl H-6), 51.49 and
54.23 (Ala C-2 and Gln C-2), 62.83 (hexyl C-1), 68.54, 73.30,
76.92 and 81.18 (mannose C-2, C-3, C-4, and C-5), 99.07
(mannose C-1), 124.84 (mannose C-6), 173.58 (Gln C-5), 177.67
6-[[N-(ter t-Bu toxyca r bon yl)-5-O-ben zyl-(S)-glu ta m yl]-
a m in o]-1-iod oh exa n e. A solution of triphenylphosphine (629
mg, 2.40 mmol) and iodine (609 mg, 2.40 mmol) in dry
tetrahydrofuran (40 mL) was stirred at ambient temperature
for 10 min. The mixture was then treated with compound 3
(873 mg, 2.00 mmol) and imidazole (163 mg, 2.40 mmol) and
was stirred at ambient temperature for 20 h. Solvent was
evaporated in vacuo to give the crude product, which was
purified by flash silica gel chromatography (gradient elution,
20-100% EtOAc in hexane) to give the title compound as a
pale yellow solid (275 mg, 25%), together with unreacted 3 (576
mg, 66%): 1H NMR (CDCl3, 300 MHz) 1.22-1.50 (6H, m, hexyl
and 178.42 [2C] (MeCO, Ala C-1 and Glu C-1); HRMS (FAB)
+
m/z 543.264 310, calculated for MNa+ (C22H40N4NaO10
)
t
543.264 214; HPLC, system A (Hamilton; 0-40% (50 min) B
in C, pH 8.5), 17.9 min (solvated NH2) and 23.5 min (NH2
H-bonded to sugar ring [confirmed by NOESY spectroscopy]),
98% total; system B (YMC; 0-50% (30 min) A in E), 9.8 and
13.7 min. Anal. (C22H40N4O10‚H2O) C, H, N.
H-2, H-3 and H-4), 1.39 (9H, s, Bu), 1.76 (2H, tt, J 7.0, 7.0
Hz, hexyl H-5), 1.90 (1H, A of ABX2Y, J 14.5, 7.5, 7.0 Hz, 1H
of Glu H-3), 2.09 (1H, B of ABX2Y, J 14.5, 7.5, 6.5 Hz, 1H of
Glu H-3), 2.40 (1H, A of ABX2, J 17.0, 7.5 Hz, 1H of Glu H-4),
2.50 (1H, B of ABX2, J 17.0, 7.5 Hz, 1H of Glu H-4), 3.13 (2H,
t, J 7.0 Hz, CH2I), 3.18 (1H, dt, J 7.0, 7.0 Hz, hexyl H-6), 4.06-
4.16 (1H, m, Glu H-2), 5.09 (2H, s, CH2Ph), 5.38 (1H, d, J 7.5
Hz, BOCNH), 6.38-6.44 (1H, m, NHCH2), 7.31 (5H, s, Ph).
N-(Ben zyloxycar bon yl)-(R)-alan yl-1-N-(6-acetoxyh exyl)-
5-O-m et h yl-(S)-glu t a m a m id e. 1-N-(6-Acetoxyhexyl)-2-N-
(benzyloxycarbonyl)-5-O-methyl-(S)-glutamamide (isolated as
a biproduct during the formation of compound 5; 437 mg, 1.00
mmol) was deprotected according to general method E to give
the intermediate free amine as a colorless oil (315 mg, quant.),
which was coupled with Z-D-alanine (274 mg, 1.20 mmol)
according to general method B (using DCC). This gave, after
purification, the title compound as a white solid (251 mg,
49%): 1H NMR (CDCl3, 300 MHz) 1.22-1.38 (4H, m, hexyl
H-3 and H-4), 1.33 (3H, d, J 7.0 Hz, Ala H-3), 1.39-1.65 (4H,
m, hexyl H-2 and H-5), 1.85-2.17 (2H, m, Glu H-3), 1.99 (3H,
s, OAc), 2.26-2.50 (2H, m, Glu H-4), 3.16 (2H, dt, J 6.5, 6.5
Hz, hexyl H-6), 3.62 (3H, s, OMe), 3.99 (2H, t, J 6.5 Hz, hexyl
H-1), 4.13-4.24 (1H, m, Ala H-2), 4.35-4.45 (1H, m, Glu H-2),
5.01 and 5.08 (2H, AB q, J 12.0 Hz, PhCH2), 5.66 (1H, d, J 7.0
Hz, ZNH), 6.75-6.84 (1H, m, NHCH2), 7.20-7.35 (6H, m, Ph
and Glu NH).
6-[[N-(ter t-Bu toxyca r bon yl)-5-O-ben zyl-(S)-glu ta m yl]-
a m in o]h ex-1-yl 2,3,4-Tr i-O-a cetyl-1-th io-â-L-fu cop yr a n o-
sid e. A solution of 2,3,4-tri-O-acetyl-1-thio-â-L-fucopyranoside
(185 mg, 0.60 mmol) and 6-[[N-tert-butoxycarbonyl-5-O-benzyl-
(S)-glutamyl]amino]-1-iodohexane (275 mg, 0.50 mmol) in dry
CH2Cl2 (5 mL) was treated with triethylamine (85 µL, 0.61
mmol). The mixture was stirred at ambient temperature for
41 h. Solvent was evaporated in vacuo to give the crude
product, which was purified by flash silica gel chromatography
(gradient elution, 50-70% EtOAc in hexane) to give the title
compound as a colorless oil (285 mg, 78%): 1H NMR (CDCl3,
300 MHz) 1.14 (3H, d, J 6.5 Hz, fucose H-6), 1.21-1.45 (6H,
t
m, hexyl H-2, H-3 and H-4), 1.35 (9H, s, Bu), 1.47-1.57 (2H,
m, hexyl H-5), 1.78-2.15 (2H, m, Glu H-3), 1.91 (3H, s, OAc),
1.99 (3H, s, OAc), 2.10 (3H, s, OAc), 2.30-2.50 (2H, m, Glu
H-4), 2.58 (1H, A of ABX2, J 12.0, 7.0 Hz, 1H of hexyl H-1),
2.64 (1H, B of ABX2, J 12.0, 6.5 Hz, 1H of hexyl H-1), 3.14
(1H, dt, J 7.0, 7.0 Hz, hexyl H-6), 3.76 (1H, q*, J 6.5 Hz, fucose
H-5), 4.03-4.12 (1H, m, Glu H-2), 4.38 (1H, d, J 10.0 Hz, fucose
H-2), 4.98 (1H, dd, J 10.0, 3.5 Hz, fucose H-3), 5.05 (2H, s,
CH2Ph), 5.14 (1H, dd, J 10.0, 10.0 Hz, fucose H-2), 5.20 (1H,
d*, J 3.5 Hz, fucose H-4), 5.36 (1H, d, J 8.0 Hz, BOCNH), 6.34-
6.41 (1H, m, NHCH2), 7.28 (5H, s, Ph), *fucose J 4-5 unresolved.
6-((R)-Ala n yl-(S)-glu t a m in yla m in o)h exa n -1-ol, 9. N-
(Benzyloxycarbonyl)-(R)-alanyl-1-N-(6-acetoxyhexyl)-5-O-methyl-
(S)-glutamamide (200 mg, 0.39 mmol) was N-deprotected
according to general method E to give the intermediate free
amine as a colorless oil. This material was converted to
compound 9 by ammonolysis according to general method F
to give, after purification by flash silica gel chromatography
(isocratic elution with 5% concentrated aqueous ammonium
hydroxide in propan-2-ol), compound 9 as a white solid (71 mg,
1-â-[[6-((R)-Ala n yl-(S)-glu ta m in yla m in o)h ex-1-yl]th io]-
1-d eoxy-L-fu cop yr a n ose, 10. Using general methods D, B,
D and F, 6-[[N-tert-butoxycarbonyl-5-O-benzyl-(S)-glutamyl]-
amino]hex-1-yl 2,3,4-tri-O-acetyl-1-thio-â-L-fucopyranoside was
1
70%): mp 98-101 °C; H NMR (CD3OD, 300 MHz) 1.28 (3H,
d, J 7.0 Hz, Ala H-3), 1.30-1.43 (4H, m, hexyl H-3 and H-4),
1.45-1.58 (4H, m, hexyl H-2 and H-5), 1.84-1.97 and 2.01-

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1919
converted in four steps to compound 10 in a manner similar
to that seen for the previous analogues. Compound 10 was
obtained as a white solid (97 mg, 52% over four steps): mp
122-125 °C; 1H NMR (CD3OD + D2O, 300 MHz) 1.24 (3H, d,
J 6.5 Hz, fucose H-6), 1.28 (3H, d, J 7.0 Hz, Ala H-3), 1.30-
1.44 (4H, m, hexyl H-3 and H-4), 1.45-1.55 (2H, m, hexyl H-2),
1.56-1.67 (2H, m, hexyl H-5), 1.87-1.98 and 2.01-2.12 (2H,
2 × m, Gln H-3), 2.30 (2H, t, J 7.5 Hz, Gln H-4), 2.64 (1H, A
of ABX2, J 12.5, 7.0 Hz, 1H of hexyl H-1), 2.72 (1H, B of ABX2,
J 12.5, 7.0 Hz, 1H of hexyl H-1), 3.18 (2H, t, J 7.0 Hz, hexyl
H-6), 3.45-3.53 (3H, m, Ala H-2 and fucose H-2 and H-3), 3.67
(1H, qd, J 6.5, 1.0 Hz, fucose H-5), 3.69 (1H, dd, J 3.0, 1.0 Hz,
fucose H-4), 4.29 (1H, dd, J 9.0, 5.0 Hz, Gln H-2), 4.30 (1H, d,
J 9.5 Hz, fucose H-1); 13C NMR: δC (CD3OD + D2O, 75.5 MHz)
17.03 (fucose C-6), 21.16 (Ala C-3), 27.20, 28.96, 29.22, 29.87,
30.70, 30.93 and 32.53 (Gln C-3 and C-4, + hexyl C-1 to C-5),
40.38 (hexyl H-6), 51.30 and 54.31 (Ala C-2 and Gln C-2),
70.93, 73.00, 75.96 and 76.02 (fucose C-2, C-3, C-4, and C-5),
87.27 (fucose C-1), 173.72 (Gln C-5), 178.07 and 178.56 (Ala
C-1 and Gln C-1); MS (high resolution; FAB) 479.251 90,
calculated for [MH+] (C20H39N4O7S+) 479.253 94. HPLC,
system A (Hamilton; 0-50% (60 min) B in C, pH 7.7), 18.8
min, 100%; system B (YMC; 0-50% (25 min) A in E), 18.4 min.
Anal. (C20H39N4O7S‚2H2O) C, H, N.
2,3,4-Tr i-O-a cetyl-â-L-fu cop yr a n osyl Azid e. A solution
of compound 4 (1.06 g, 3.00 mmol) in dry MeCN (3 mL) was
treated with powdered NaN3 (293 mg, 4.51 mmol), and the
mixture was heated at reflux for 90 min and then stirred at
ambient temperature for 17 h. The mixture was diluted with
CH2Cl2 and filtered through Celite, and the residue was
washed with CH2Cl2. Combined filtrates and washings were
evaporated in vacuo, and the residue was purified by flash
silica gel chromatography (100:1, gradient elution, zero to 30%
EtOAc in CH2Cl2) to give the title compound as white crystals
(635 mg, 67%); IR (film) γmax 1220 s (C-O), 1750 s (CdO), 2121
s (N3); 1H NMR (CDCl3, 300 MHz) 1.23 (3H, d, J 6.5 Hz, H-6),
1.96 (3H, s, OAc), 2.06 (3H, s, OAc), 2.17 (3H, s, OAc), 3.88
(1H, qd, J 6.5, 1.0 Hz, H-5), 4.56 (1H, d, J 8.5 Hz, H-1), 5.00
(1H, dd, J 10.5, 3.5 Hz, H-3), 5.12 (1H, dd, J 10.5, 8.5 Hz, H-2),
5.24 (1H, dd, J 3.5, 1.0 Hz, H-4).
2,3,4-Tr i-O-a cetyl-â-L-fu cop yr a n osyla m in e. A solution
of 2,3,4-tri-O-acetyl-â-L-fucopyranosyl azide (315 mg, 1.00
mmol) in EtOAc (2 mL) was treated with palladium on carbon
(10% w/w, of 10% w/w Pd/C; 30 mg). The mixture was
thoroughly degassed and then stirred at room temperature
under 1 atm of H2 for 16 h. The mixture was diluted with
EtOAc and filtered through Celite. The residue was washed
with EtOAc, and combined filtrates and washings were
evaporated in vacuo to give the title compound as a white foam
(292 mg, 100%): 1H NMR (CDCl3, 300 MHz) 1.14 (3H, d, J
6.5 Hz, H-6), 1.95 (3H, s, OAc), 2.04 (3H, s, OAc), 2.14 (3H, s,
OAc), 3.76 (1H, qd, J 6.5, 1.0 Hz, H-5), 4.09 (1H, d, J 8.0 Hz,
H-1), 4.96 (1H, dd, J 10.5, 8.0 Hz, H-2), 5.02 (1H, dd, J 10.5,
3.0 Hz, H-3), 5.21 (1H, dd, J 3.0, 1.0 Hz, H-4). NH2 signal too
broad to be observed.
N-(Ben zyloxyca r bon yl)-(R)-a la n yl-(S)-glu ta m in e. A so-
lution of (R)-alanyl-(S)-glutamine (2.17 g, 10.0 mmol) in 2.0
M aqueous sodium hydroxide (5 mL, 10.0 mmol) at 0 °C was
treated alternatively with four portions each of benzyl chlo-
roformate (1.65 mL, 11 mmol total) and 2.0 M aqueous sodium
hydroxide (6.5 mL, 11 mmol total). The reaction was then
allowed to warm to ambient temperature and was stirred at
ambient temperature for 75 min. The resultant slurry was
diluted with water, brought to pH 12 with 1 M aqueous sodium
hydroxide, and washed with EtOAc (2 × 50 mL). The aqueous
phase was then brought to pH 1 with 5% aqueous hydrochloric
acid and extracted with EtOAc (3 × 100 mL). Combined
extracts were dried (Na2SO4), filtered, and evaporated in vacuo
to give the title compound as a white solid (1.71 g, 49%): 1H
NMR (DMSO-d6, 300 MHz) 1.20 (3H, d, J 7.0 Hz, Ala H-3),
1.68-1.80 and 1.88-1.99 (2H, 2 × m, Gln, H-3), 2.08 (2H, t, J
8.0 Hz, Gln H-4), 4.08 (1H, dq, J 7.0, 7.0 Hz, Ala H-2), 4.16
(1H, ddd, J 8.0, 8.0, 5.0 Hz, Gln H-2), 4.08 and 5.03 (2H, AB
q, J 12.5 Hz, PhCH2), 6.75 (1H, br s, 1H of CONH2), 7.26 (1H,
br s, 1H of CONH2), 7.30-7.38 (6H, m, Ph and Ala NH), 8.11
(1H, d, J 8.0 Hz, Gln NH), 12.60 (1H, br s, CO2H).
1-â-[[N -(Be n zyloxyca r b on yl)-(R )-a la n yl-(S )-glu t a m -
in yl]a m in o]-1-d eoxy-2,3,4-t r i-O-a cet yl-L-fu cop yr a n ose.
2,3,4-Tri-O-acetyl-â-L-fucopyranosylamine (292 mg, 1.00 mmol)
and N-(benzyloxycarbonyl)-(R)-alanyl-(S)-glutamine (421 mg,
1.20 mmol) were coupled according to general method B (using
DCC), except that sufficient DMF was added to allow dissolu-
tion. The title compound was obtained as a white solid (208
mg, 33%): 1H NMR (CDCl3, 300 MHz) 1.14 (3H, d, J 6.5 Hz,
fucose H-6), 1.37 (3H, d, J 7.0 Hz, Ala H-3), 1.80-2.10 (2H,
m, Gln H-3), 1.85 (3H, s, OAc), 2.01 (3H, s, OAc), 2.02 (3H, s,
OAc), 2.18-2.40 (2H, m, Gln H-4), 3.91 (1H, q*, J 6.5 Hz,
fucose H-5), 4.27 (1H, qd, J 7.0, 7.0 Hz, Ala H-2), 4.30-4.37
(1H, m, Gln H-2), 5.08-5.20 (5H, m, PhCH2 + fucose H-1, H-2
and H-3), 5.23-5.26 (1H, m, fucose H-4), 5.62 (1H, d, J 7.0
Hz, Ala NH), 5.80 and 5.86 (2H, 2 × br s, CONH2), 7.29-7.40
(5H, m, Ph), 7.60-7.65 (2H, m, 2 × NH), *fucose J 4-5
unresolved.
1-â-[(R)-Alan yl-(S)-glu tam in ylam in o]-1-deoxy-L-fu copy-
r a n ose Aceta te Sa lt, 15. 1-â-[[N-(Benzyloxycarbonyl)-(R)-
alanyl-(S)-glutaminyl]amino]-1-deoxy-2,3,4-tri-O-acetyl-L-fu-
copyranose (205 mg, 0.33 mmol) was deprotected by
ammonolysis according to general method F, followed by
N-deprotection according to general method E (using H2O/1,4-
dioxane as solvent) to give, after purification by reverse phase
HPLC (Hamilton PRP-1, isocratic C, pH 8.5), compound 15
as a very hygroscopic white solid (86 mg, 55%): 1H NMR (D2O,
300 MHz, MeOH ) 3.35 ppm) 1.23 (3H, d, J 6.5 Hz, fucose
H-6), 1.53 (3H, d, J 7.0 Hz, Ala H-3),1.91 (6H, s, AcO- and
complexed AcOH), 1.97-2.22.(2H, m, Gln H-3), 2.39 (2H, t, J
7.5 Hz, Gln H-4), 3.60 (1H, dd, J 9.5, 9.0 Hz, fucose H-2), 3.70
(1H, dd, J 9.5, 3.0 Hz, fucose H-3), 3.80 (1H, d*, J 3.0 Hz,
fucose H-4), 3.88 (1H, q*, J 6.5 Hz, fucose H-5), 4.08 (1H, q, J
7.0 Hz, Ala H-2), 4.41 (1H, dd, J 8.5, 5.5 Hz, Gln H-2), 4.91
(1H, d, J 9.0 Hz, fucose H-1), *fucose J 4-5 unresolved; 13C NMR
(D2O, 75.5 MHz, MeCN ) 1.30 ppm) 15.98 (fucose C-6), 17.49
(Ala C-3), 23.71 (2 × MeCO2H), 27.40 (Gln C-3), 31.43 (Gln
C-4), 49.61 and 53.73 (Ala C-2 and Gln C-2), 69.40, 71.76,
73.20, and 73.92 (fucose C-2, C-3, C-4, and C-5), 80.15 (fucose
C-1), 172.60 (2 × MeCO2H), 178.04 (Gln C-5), 181.84 and
177.86 (Ala C-1 and Gln C-1); HRMS (FAB) 385.168 10,
calculated for [M - 2AcOH + Na+] (C14H26N4NaO7+) 385.169 92.
HPLC, system A (Hamilton; isocratic C, pH 8.5), 4.3 min, 99%;
system B (YMC; isocratic E), 4.1 min. Anal. (C14H26N4O7.CH3-
CO2H‚5H2O) C, H, N.
Other analogues were prepared by similar procedures:
6-((R)-Alan yl-(S)-glu tam in ylam in o)-1-ch lor oh exan e, 11:
1
mp 158-161 °C (softens 135-140 °C); H NMR (CD3OD, 300
MHz) 1.27 (3H, d, J 7.0 Hz, Ala H-3), 1.31-1.57 (6H, m, hexyl
H-2, H-3 and H-4), 1.76 (2H, tt, J 6.5, 6.5 Hz, hexyl H-5), 1.91
(1H, A of ABXY2, J AB 14.0, J AX 9.0, J AY 7.5 Hz, 1H of Gln H-3),
2.08 (1H, B of ABXY2, J AB 14.0, J BY 7.5, J BX 5.0 Hz, 1H of Gln
H-3), 2.28 (2H, t, J 7.5 Hz, Gln H-4), 3.18 (2H, t, J 7.0 Hz,
hexyl H-6), 3.45 (1H, q, J 7.0 Hz, Ala H-2), 3.55 (2H, t, J 6.5
Hz, hexyl H-1), 4.30 (1H, dd, J 9.0, 5.0 Hz, Gln H-2); 13C NMR
(CD3OD, 75.5 MHz) 21.21 (Ala C-3), 27.16, 27.57, 29.18 and
30.19 (hexyl C-2 to C-5), 32.54 and 33.67 (Gln C-3 and C-4),
40.30 (hexyl C-6), 45.65 (hexyl C-1), 51.45 and 54.24 (Ala C-2
and Gln C-2), 173.59 (Gln C-5), 177.63 and 178.19 (Ala C-1
and Gln C-1); HRMS (FAB) 335.183 90, calculated for
M[35Cl]H+ (C14H28N4O335Cl+) 335.185,00; HPLC (Hamilton;
40-80% (50 min) A in C, pH 7.85), 14.9 min, 93%.
6-((R)-Ala n yl-(S)-glu ta m in yla m in o)h exa n oic a cid , h y-
1
d r och lor id e sa lt, 12: mp 127-129 °C; H NMR (DMSO-d6,
300 MHz) 1.20-1.50 (6H, m, hexyl H-3, H-4 and H-5), 1.36
(3H, d, J 7.0 Hz, Ala H-3), 1.68-1.78 and 1.85-1.95 (2H, 2 ×
m, Gln H-3), 2.08 (2H, t, J 7.0 Hz, hexyl H-5), 2.18 (2H, t, J
7.0 Hz, Gln H-4), 2.96-3.06 (2H, m, hexyl H-6), 3.83-3.88 (1H,
m, Ala H-2), 4.18-4.28 (1H, m, Gln H-2), 6.76 and 7.31 (2H,
2 × br s, CONH2), 8.09 (1H, t, J 7.0 Hz, NHCH2), 8.20 (3H, br
s, NH3+) 8.66-8.63 (1H, m, Gln NH); 13C NMR (DMSO-d6, 75.5
MHz) 17.65 (Ala C-3), 24.56, 26.28, 28.49, and 29.12 (hexyl
C-2 to C-5), 31.66 and 33.98 (Gln C-3 and C-4), 40.23 (hexyl

1920 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
C-6), 48.61 and 52.82 (Ala C-2 and Gln C-2), 169.72, 170.81,
173.76 and 174.80 (hexyl C-1, Ala C-1, Gln C-1 and C-5);
mannose H-2), 4.42 (1H, dd, J 9.0, 5.0 Hz, Gln H-2), 5.24 (1H,
s*, mannose H-1), *mannose J H1-H2 unresolved; 13C NMR (D2O,
75.5 MHz, MeCN ) 1.30 ppm) 20.04 (Ala C-3), 27.18 (Gln C-3),
31.55 (Gln C-4), 50.22 and 52.37 (Ala C-2 and Gln C-2), 61.29
(mannose C-6), 66.83, 70.48, 73.68, 78.25, and 78.29 (mannose
C-1, C-2, C-3, C-4, and C-5), 174.13 (CONH2), 178.26 and
178.60 (2 × CONH); HRMS (FAB) 379.181 10, calculated for
[MH+] (C14H27N4O8+) 389.182 89; HPLC (Partisil 10 SCX, 4.6
× 150 mm; 0-40% (50 min) 7 mM potassium phosphate, pH
7.0 in 250 mM potassium phosphate, pH 7.0), 12.0, 94%.
1-â-[12-((R)-Ala n yl-(S)-glu ta m in yla m in o)d od ec-1-yl]-L-
fu cop yr a n ose, 20: mp 140-143 °C; 1H NMR (CD3OD + D2O,
300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.25-1.40 (19H,
m, Ala H-3 and dodecyl H-3 to H-10), 1.41-1.52 (2H, m,
dodecyl H-11), 1.53-1.64 (2H, m, dodecyl H-2), 1.85-2.12 (2H,
m, Gln H-3), 2.30 (2H, t, J 7.5 Hz, Gln H-4), 3.09-3.23 (2H,
m, dodecyl H-12), 3.44 (1H, dd, J 10.0, 7.5 Hz, fucose H-2),
3.49 (1H, q, J 7.0 Hz, Ala H-2), 3.52 (1H, dd, J 10.0, 3.0 Hz,
fucose H-3), 3.54 (1H, A of ABX2, J 9.5, 7.0 Hz, dodecyl H-1),
3.64 (1H, dd, J 3.0, 1.0 Hz, fucose H-4), 3.66 (1H, qd, J 6.5,
1.0 Hz, fucose H-5), 3.82 (1H, B of ABX2, J 9.5, 7.0 Hz, dodecyl
H-1), 4.22 (1H, d, J 7.5 Hz, fucose H-1), 4.28 (1H, dd, J 9.0,
5.0 Hz, Gln H-2); 13C NMR (CD3OD + D2O, 75.5 MHz) 16.66
(fucose C-6), 21.10 (Ala C-3), 26.66, 27.61, 28.85, 29.92, 30.04,
30.19, 30.33 [4 × CH2 unresolved], 30.41 and 32.48 (Gln C-3
and C-4, + dodecyl C-2 to C-11), 40.47 (NHCH2), 51.24 and
54.29 (Ala C-2 and Gln C-2), 71.23 (dodecyl C-1), 71.83, 71.95,
72.76, and 74.69 (fucose C-2, C-3, C-4, and C-5), 104.37 (fucose
C-1), 173.72 (Gln C-5), 178.17 and 178.63 (Ala C-1 and Gln
C-1); HRMS (FAB) 547.373 50, calculated for [MH+]
(C26H51N4O8+) 547.370 67; HPLC (Hamilton; 10-50% (50 min)
B in C, pH 8.5), 24.4 min, 100%.
2-O-Acet yl-1-â-[12-((R)-a la n yl-(S)-glu t a m in yla m in o)-
d od ec-1-yl]-D-m a n n op yr a n ose, 21: mp 81-85 °C; 1H NMR
(CD3OD, 300 MHz) 1.27-1.38 (16H, m, dodecyl H-3 to H-10),
1.45-1.57 (4H, m, dodecyl H-2 and H-11), 1.49 (3H, d, J 7.0
Hz, Ala H-3), 1.63 (3H, s, OAc), 1.87-1.99 and 2.01-2.12 (2H,
2 × m, Gln H-3), 2.27-2.32 (2H, m, Gln H-4), 3.17 (2H, t, J
7.0 Hz, dodecyl H-12), 3.22 (1H, ddd, J 9.5, 6.0, 2.5 Hz,
mannose H-5), 3.51 (1H, A of ABX2, J 9.0, 6.5 Hz, 1H of dodecyl
H-1), 3.55 (1H, B of ABX2, J 9.0, 6.5 Hz, 1H of dodecyl H-1),
3.55 (sic) (1H, dd, J 9.5, 9.5 Hz, mannose H-4), 3.65 (1H, A of
ABX, J 12.0, 6.0 Hz, 1H of mannose H-6), 3.68 (1H, dd, J 9.5,
4.0 Hz, mannose H-3), 3.84 (1H, B of ABX, J 12.0, 2.5 Hz, 1H
of mannose H-6), 3.90 (1H, q, J 7.0 Hz, Ala H-2), 4.31 (1H, dd,
J 9.0, 5.0 Hz, Gln H-2), 4.42 (1H, dd, J 4.0, 2.5 Hz, mannose
H-2), 5.42 (1H, d, J 2.5 Hz, mannose H-1); 13C NMR (CD3-
OD, 75.5 MHz) 19.97 (Ala C-3), 25.86 (MeCO), 27.24, 27.98,
29.08, 30.35, 30.40 [2C], 30.44, 30.65 [4C] and 32.42 (Gln C-3
and C-4, and dodecyl C-2 to C-11), 40.55 (dodecyl C-12), 50.45
and 54.48 (Ala C-2 and Gln C-2), 62.81 (dodecyl C-1), 68.55,
73.31, 76.93, and 81.20 (mannose C-2, C-3, C-4, and C-5), 99.09
(mannose C-1), 124.85 (mannose C-6), 171.71 [2C], 173.33 and
177.48 (4 × CdO); HRMS (FAB) 605.378 50, calculated for
[MH+] (C28H53N4O10+) 605.376 16; HPLC (Hamilton; 10-50%
(50 min) B in C, pH 8.5, 25.6 min.
12-((R)-Ala n yl-(S)-glu ta m in yla m in o)d od eca n -1-ol, 22:
mp 164-167 °C; 1H NMR (CD3OD, 300 MHz) 1.27-1.40 (16H,
m, dodecyl H-3 to H-10), 1.44-1.56 (4H, m, dodecyl H-2 and
H-11), 1.52 (3H, d, J 7.0 Hz, Ala H-3), 1.87-2.13 (2H, m, Gln
H-3), 2.30 (2H, t, J 7.5 Hz, Gln H-4), 3.12-3.21 (2H, m, dodecyl
H-12), 3.53 (2H, t, J 6.5 Hz, dodecyl H-1), 3.98 (1H, q, J 7.0
Hz, Ala H-2), 4.32 (1H, dd, J 9.0, 5.0 Hz, Gln H-2); 13C NMR
(CD3OD, 75.5 MHz) 17.62 (Ala C-3), 26.95, 28.00, 29.06, 30.36,
30.43, 30.61, 30.67, 30.71 [2C], 30.74, 32.45, and 33.66 (Gln
C-3 and C-4, and dodecyl C-2 to C-11), 40.56 (dodecyl C-12),
50.36 and 54.55 (Ala C-2 and Gln C-2), 63.00 (dodecyl C-1),
HRMS (FAB) 331.196 80, calculated for [M
-
Cl-]+
(C14H27N4O5+), HPLC (YMC; 0-25% (30 min) A in C, pH 6.00),
10.6 min, 92%.
6-((R)-Alan yl-(S)-glu tam in ylam in o)-1-am in oh exan e, 13:
1
mp 111-114 °C; H NMR (CD3OD, 300 MHz) 1.33 (3H, d, J
7.0 Hz, Ala H-3), 1.35-1.47 (4H, m, hexyl H-2 and H-4), 1.52-
1.59 (2H, hexyl H-5), 1.61-1.70 (2H, m, hexyl H-2), 1.89-2.00
and 2.06-2.14 (2H, 2 × m, Gln H-3), 2.30 (2H, t, J 7.5 Hz,
Gln H-4), 2.90 (2H, t, J 7.5 Hz, hexyl H-1), 3.18 (1H, A of ABX2,
J 13.5, 6.5 Hz, 1H of hexyl H-6), 3.23 (1H, B of ABX2, J 13.5,
7.0 Hz, 1H of hexyl H-6), 3.66 (1H, q, J 7.0 Hz, Ala H-2), 4.28
(1H, dd, J 9.0, 5.0 Hz, Gln H-2); 13C NMR (CD3OD, 75.5 MHz)
20.26 (Ala C-3), 26.70, 26.91, 28.52, 28.72, 29.07, and 29.72
(Gln C-3 and C-4 and hexyl C-2 to C-5), 32.71 (hexyl C-1), 40.04
(hexyl C-6), 51.16 and 54.70 (Ala C-2 and Gln C-2), 174.04 (Gln
C-5), 176.73 and 177.96 (Ala C-1 and Gln C-1); HRMS (FAB)
316.232 60, calculated for [MH+] (C14H30N5O3+) 316.234 86;
HPLC (Hamilton; 0-20% (40 min) A in C, pH 7.85), 14.9 min,
92%.
6-((S)-Alan yl-(R)-glu tam in ylam in o)h ex-1-yl acetate, 14:
mp 100-104 °C (softens ∼80 °C); 1H NMR (CD3OD, 400 MHz)
1.29 (3H, d, J 7.0 Hz, Ala H-3), 1.32-1.40 (4H, m, hexyl H-3
and H-4), 1.48-1.56 (2H, m, hexyl H-5), 1.60-1.67 (2H, m,
hexyl H-2), 1.87-1.96 (1H, m, 1H of Gln H-3), 2.01 (3H, s,
OAc), 2.03-2.11 (1H, m, 1H of Gln H-3), 2.28 (2H, t, J 8.0 Hz,
Gln H-4), 3.18 (2H, t, J 7.0 Hz, hexyl H-6), 3.48 (1H, q, J 7.0
Hz, Ala H-2), 4.05 (2H, t, J 6.5 Hz, hexyl H-1), 4.29 (1H, dd,
J 9.0, 5.0 Hz, Gln H-2); 13C NMR (CD3OD, 75.5 MHz) 21.17
(Ala C-3), 26.62 and 27.48 (hexyl C-3 and C-4), 27.80 (MeCO),
29.17, 29.57 and 30.20 (Gln C-3 and hexyl C-2 and C-5), 32.52
(Gln C-4), 40.29 (hexyl C-6), 51.47 and 54.27 (Ala C-2 and Gln
C-2), 65.62 (hexyl C-1), 173.29 and 173.85 (Gln C-5 and MeCO),
177.92 and 178.53 (Ala C-1 and Gln C-1); HRMS (FAB)
359.230 80, calculated for [MH+] (C16H31N4O5+) 359.229 43;
HPLC (Hamilton; 0-50% (60 min) A in C, pH 7.9), 59.2 min,
92%.
r-L-F u cop yr a n osyl [(R)-a la n yl-(S)-glu ta m in yl]a m in e,
a ceta te sa lt, 16: 1H NMR (D2O, 300 MHz, MeOH ) 3.35 ppm)
1.18 (3H, d, J 6.5 Hz, fucose H-6), 1.53 (3H, d, J 7.0 Hz, Ala
H-3), 1.91 (3H, s, AcO-), 1.96-2.20 (2H, m, Gln H-3), 2.39 (2H,
t, J 7.5 Hz, Gln H-4), 3.81 (1H, d*, J 3.5 Hz, fucose H-4), 3.88
(1H, dd, J 10.5, 3.5 Hz, fucose H-3), 3.89 (1H, q*, J 6.5 Hz,
fucose H-5), 4.03 (1H, dd, J 10.5, 5.5 Hz, fucose H-2), 4.08 (1H,
q, J 7.0 Hz, Ala H-2), 4.53 (1H, dd, J 9.0, 5.0 Hz, Gln H-2),
5.59 (1H, d, J 5.5 Hz, fucose H-1), *fucose J H4-H5 unresolved;
13C NMR (D2O, 75.5 MHz, MeCN ) 1.30 ppm) 16.05 and 17.42
(fucose C-6 and Ala C-3), 23.71 (MeCO2-), 27.67 (Gln C-3),
31.29 (Gln C-4), 49.59 and 53.36 (Ala C-2 and Gln C-2), 66.36,
68.25, 69.92 and 71.83 (fucose C-2, C-3, C-4, and C-5), 77.35
(fucose C-1), 172.32 (MeCO2-), 174.92 (Gln C-5), 178.01 and
181.88 (Ala C-1 and Gln C-1); HRMS (FAB) 385.168 50,
calculated for [M - AcOH + Na+] (C14H26N4NaO7+) 385.169 92;
HPLC (Hamilton; isocratic C, pH 8.5), 3.1 min, 98%.
2-((R)-Ala n yla m in o)-(S)-glu ta r im id e, 18: mp 245-248 °C
(darkens >180 °C to brown); 1H NMR (D2O, 300 MHz, MeCN
) 2.05 ppm) 1.43 (3H, d, J 7.0 Hz, Ala H-3), 2.05-2.24 (2H,
m, imide H-3), 2.35 (2H, t, J 7.0 Hz, imide H-4), 4.20-4.26
(1H, m, imide H-2), 4.22 (1H, q, J 7.0 Hz, Ala H-2); 13C NMR
(D2O, 75.5 MHz, MeCN ) 1.30 ppm) 18.72 (Ala C-3), 28.95
(imide C-3), 30.35 (imide C-4), 50.49 (Ala C-2), 54.51 (imide
C-2), 170.24 and 172.15 (Ala C-1 and imide C-5), 178.66 (imide
C-1); HPLC (YMC; 0-10% (20 min) B in C, pH 6.0), 20.5 min,
95%.
â-O-Man n opyr an osyl [(R)-alan yl-(S)-glu tam in yl]am in e,
19: mp 138-142 °C dec; 1H NMR (D2O, 300 MHz, Me2CHOH
) 1.18 ppm) 1.31 (3H, J 7.0 Hz, Ala H-3), 1.96-2.08 and 2.12-
2.22 (2H, 2 × m, Gln H-3), 2.42 (2H, t, J 7.5 Hz, Gln H-4),
3.48 (1H, ddd, J 9.5, 6.0, 2.0 Hz, mannose H-5), 3.57-3.65 (1H,
m, Ala H-2), 3.61 (1H, dd, J 9.5, 9.5 Hz, mannose H-4), 3.73
(1H, A of ABX, J 12.0, 6.0 Hz, 1H of mannose H-6), 3.73 (sic)
(1H, dd, J 9.5, 3.0 Hz, mannose H-3), 3.92 (1H, B of ABX, J
12.0, 6.0 Hz, 1H of mannose H-6), 3.98 (1H, d*, J 3.0 Hz,
171.02 (Gln C-5), 173.29 and 177.46 (Ala C-1 and Gln C-1);
+
HRMS (FAB) 401.314 20, calculated for [MH+] (C20H41N4O4
)
401.312 77; HPLC (YMC; 10-50% (50 min) B in C, pH 6.0),
33.1 min.
1-â-[2-[4-((R)-Alan yl-(S)-glu tam in ylam in o)ph en yl]eth yl]-
L-fu cop yr a n ose, 23: mp 107-110 °C (softens 103 °C); 1H
NMR (CD3OD + D2O, 400 MHz) 1.28 (3H, d, J 6.5 Hz, fucose

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1921
H-6), 1.33 (3H, d, J 7.0 Hz, Ala H-3), 1.97-2.06 and 2.12-
2.21 (2H, 2 × m, Gln H-3), 2.36 (2H, t, J 7.5 Hz, Gln H-4),
2.90 (2H, t, J 7.0 Hz, ethyl H-2), 3.45 (1H, dd, J 9.5, 7.0 Hz,
fucose H-2), 3.48 (1H, dd, J 9.5, 3.0 Hz, fucose H-3), 3.52 (1H,
q, J 7.0 Hz, Ala H-2), 3.61 (1H, d*, J 3.0 Hz, fucose H-4), 3.63
(1H, q*, J 6.5 Hz, fucose H-5), 3.72 (1H, A of ABX2, J 9.5, 7.0
Hz, 1H of ethyl H-1), 4.01 (1H, B of ABX2, J 9.5, 7.5 Hz, 1H of
ethyl H-1), 4.23 (1H, d, J 7.5 Hz, fucose H-1), 4.47 (1H, dd, J
9.0, 5.0 Hz, Gln H-2), 7.22 (2H, d, J 8.5 Hz, phenylene H-2
and H-6), 7.46 (2H, d, J 8.5 Hz, phenylene H-3 and H-5),
*fucose J H4-H5 unresolved; 13C NMR (CD3OD + D2O, 75.5 MHz)
16.61 (fucose C-6), 11.62 (Gln C-3), 32.32 (Ala C-3), 32.41 (Gln
C-4), 36.34 (ethyl C-2), 51.26 and 54.75(Ala C-2 and Gln C-2),
71.70 (ethyl C-1), 71.92, 72.05, 72.81, and 74.78 (fucose C-2,
C-3, C-4, and C-5), 104.56 (fucose C-1), 121.98 (phenylene C-2
and C-5), 130.57 (phenylene C-2 and C-6), 136.67 and 137.16
(phenylene C-1 and C-4), 172.41 (Gln C-5), 178.27 and 178.86
(Ala C-1 and Gln C-1); HRMS (FAB) 483.244 40, calculated
for [MH+] (C22H35N4O8+) 483.245 48; HPLC (Hamilton; 10-
60% (60 min) A in C, pH 7.85), 25.6 min, 99%.
3.44 (1H, dd, J 10.0, 7.5 Hz, fucose H-2), 3.53 (1H, dd, J 10.0,
3.5 Hz, fucose H-3), 3.56 (1H, A of ABX2, J 9.5, 7.0 Hz, 1H of
hexyl H-1), 3.63-3.69 (1H, m, amino-Ala H-2), 3.66 (1H, d*, J
3.5 Hz, fucose H-4), 3.67 (1H, q*, J 6.5 Hz, fucose H-5), 3.84
(1H, B of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 4.24 (1H, d, J
7.5 Hz, fucose H-1), 4.32 (1H, dd, J 9.0, 5.0 Hz, Gln H-2),
*fucose J H4-H5 unresolved; 13C NMR (CD3OD + D2O, 75.5 MHz)
16.71 (fucose C-6), 24.11 (MeCO2-), 26.47, 27.51, 29.06, 29.97,
30.42, and 33.10 (Gln C-3 and C-4, + hexyl C-2 to C-5), 40.42
(hexyl C-6), 44.42 (amino-Ala C-3), 54.01 and 54.30 (amino-
Ala C-2 and Gln C-2), 70.92 (hexyl C-1), 71.86, 72.11, 72.86,
and 74.86 (fucose C-2, C-3, C-4, and C-5), 104.51 (fucose C-1),
174.90, 175.03, 176.39, and 180.53 (MeCO2-, amino-Ala C-1,
Gln C-1 and Gln C-5); HRMS (FAB) 478.285 80, calculated for
[M - AcOH + H+] (C20H40N5O8+) 478.287 69; HPLC (YMC;
0-40% (50 min) B in C, pH 7.0), 29.3 min, 99%.
1-â-[6-((S )-Glu t a m in yla m in o)h e x-1-yl]-L -fu cop yr a -
n ose a ceta te sa lt, 27: 1H NMR (CD3OD + D2O, 300 MHz)
1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.30-1.45 (4H, m, hexyl
H-3 and H-4), 1.47-1.67 (4H, m, hexyl H-2 and H-5), 1.81 (3H,
s, AcO-), 1.94-2.11 (2H, m, Gln H-3), 2.36 (2H, t, J 7.5 Hz,
Gln H-4), 3.14-3.27 (3H, m, hexyl H-6 and Gln H-2), 3.44 (1H,
dd, J 9.5, 7.0 Hz, fucose H-2), 3.50 (1H, dd, J 9.5, 3.0 Hz, fucose
H-3), 3.54 (1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1),
3.63 (1H, dd, J 3.0, 1.0 Hz, fucose H-4), 3.64 (1H, qd, J 6.5,
1.0 Hz, fucose H-5), 3.84 (1H, B of ABX2, J 9.5, 7.0 Hz, 1H of
hexyl H-1), 4.20 (1H, d, J 7.5 Hz, fucose H-1); 13C NMR (CD3-
OD + D2O, 75.5 MHz) 16.73 (fucose C-6), 23.76 (MeCO2-),
26.48, 27.53, 29.91, 29.97, 30.42, 31.90, and 32.58 (Gln C-3
and C-4, + hexyl C-2 to C-5), 40.50 (hexyl C-6), 58.25 (Gln
C-2), 70.91 (hexyl C-1), 71.87, 72.12, 72.87, and 74.88 (fucose
C-2, C-3, C-4, and C-5), 104.53 (fucose C-1), 174.73 (Gln C-5),
177.60 (Gln C-1), 180.12 (MeCO2-); HRMS (FAB) 392.239 00,
calculated for [M - AcOH + H+] (C17H34N3O7+) 392.239 69;
HPLC (Hamilton; 0-40% (60 min) B in C, pH 8.5), 21.4 min,
98%.
1-â-[6-((S)-Ala n yl-(S)-glu ta m in yla m in o)h ex-1-yl]-L-fu -
1
cop yr a n ose, 24: mp 188-190 °C; H NMR (CD3OD + D2O,
300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.27 (3H, d, J 7.0
Hz, Ala H-3), 1.30-1.42 (4H, m, hexyl H-3 and H-4), 1.44-
1.54 (2H, m, hexyl H-5), 1.55-1.65 (2H, m, hexyl H-2), 1.87-
2.11 (2H, m, Gln H-3), 2.31 (2H, t, J 7.5 Hz, Gln H-4), 3.15
(1H, A of ABX2, J 13.5, 7.0 Hz, 1H of hexyl H-6), 3.21 (1H, B
of ABX2, J 13.5, 7.0 Hz, 1H of hexyl H-6), 3.44 (1H, dd, J 10.0,
7.5 Hz, fucose H-2), 3.48 (1H, q, J 7.0 Hz, Ala H-2), 3.52 (1H,
dd, J 10.0, 3.5 Hz, fucose H-3), 3.55 (1H, A of ABX2, J 9.5, 7.0
Hz, 1H of hexyl H-1), 3.65 (1H, dd, J 3.5, 1.0 Hz, fucose H-4),
3.66 (1H, qd, J 6.5, 1.0 Hz, fucose H-5), 3.83 (1H, B of ABX2,
J 9.5, 7.0 Hz, 1H of hexyl H-1), 4.23 (1H, d, J 7.5 Hz, fucose
H-1), 4.29 (1H, dd, J 9.0, 5.5 Hz, Gln H-2); 13C NMR (CD3OD,
75.5 MHz) 16.62 (fucose C-6), 21.23 (Ala C-3), 26.18, 27.23,
28.79, 29.70, 30.19, and 32.38 (Gln C-3 and C-4 and hexyl C-2,
C-3, C-4, and C-5), 40.28 (hexyl C-6), 51.22 and 54.16 (Ala C-2
and Gln C-2), 71.09 (hexyl C-1), 71.82 71.89, 72.70, and 74.59
(fucose C-2, C-3, C-4, and C-5), 104.27 (fucose C-1), 173.66 (Gln
C-5), 178.24 and 178.80 (Ala C-1 and Glu C-1); HRMS (FAB)
m/z 463.275 00, calculated for MH+ (C20H39N4O8+) 463.276 79;
HPLC (Hamilton; 0-40% (50 min) B in C, pH 8.5), 17.6 min,
100%.
1-â-[6-([N-Acetyl-(R)-a la n yl-(S)-glu ta m in yl]a m in o)h ex-
1-yl]-L-fu cop yr a n ose, 28: mp 171-174 °C; 1H NMR (CD3-
OD + D2O, 300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.28-
1.42 (4H, m, hexyl H-3 and H-4), 1.36 (3H, d, J 7.0 Hz, Ala
H-3), 1.44-1.53 (2H, m, hexyl H-5), 1.54-1.64 (2H, m, hexyl
H-2), 1.84-2.00 and 2.08-2.19 (2H, 2 × m, Gln H-3), 2.00 (3H,
s, NAc), 2.29 (2H, t, J 7.0 Hz, Gln H-4), 3.14 (1H, A of ABX2,
J 13.5, 7.0 Hz, 1H of hexyl H-6), 3.21 (1H, B of ABX2, J 13.5,
7.0 Hz, 1H of hexyl H-6), 3.44 (1H, dd, J 9.5, 7.0 Hz, fucose
H-2), 3.50 (1H, dd, J 9.5, 3.5 Hz, fucose H-3), 3.53 (1H, A of
ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.62 (1H, dd, J 3.5, 1.0
Hz, fucose H-4), 3.64 (1H, qd, J 6.5, 1.0 Hz, fucose H-5), 3.83
(1H, B of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 4.21 (1H, d, J
7.0 Hz, fucose H-1), 4.24 (1H, q, J 7.0 Hz, Ala H-2), 4.25 (1H,
dd, J 9.5, 4.5 Hz, Gln H-2); 13C NMR (CD3OD, 75.5 MHz) 16.68
and 17.52 (fucose C-6 and Ala C-3), 22.56 (NCOMe), 26.39,
27.36, 28.34, 39.87, 30.36, and 32.55 (Gln C-3 and C-4 and
hexyl C-2, C-3, C-4, and C-5), 40.40 (hexyl C-6), 51.14 and
54.50 (Ala C-2 and Gln C-2), 70.99 (CH2O), 71.84, 72.03, 72.82,
and 74.77 (fucose C-2, C-3, C-4, and C-5), 104.43 (fucose C-1),
173.58 and 174.21 (Gln C-5 and NCOMe), 175.88 and 178.17
(Ala C-1 and Gln C-1); HRMS (FAB) m/z 505.290 20, calculated
for MH+ (C22H41N4O9+) 505.287 35; HPLC, system A (Hamil-
ton; 0-25% (50 min) B in C, pH 8.5), 24.6 min, 100%.
1-â-[6-([N-Acetyl-(S)-a la n yl-(S)-glu ta m in yl]a m in o)h ex-
1-yl]-L-fu cop yr a n ose, 29: no mp observed <250 °C (darkens
1-â-[6-((R)-Va lyl-(S)-glu t a m in yla m in o)h ex-1-yl]-L-fu -
cop yr a n ose, 25: mp 68-71 °C; 1H NMR (CD3OD + D2O, 300
MHz) 0.91 (3H, d, J 7.0 Hz, Val H-4), 0.94 (3H, d, J 7.0 Hz,
Val H-4′), 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.30-1.42 (4H,
m, hexyl H-3 and H-4), 1.46-1.52 (2H, m, hexyl H-5), 1.55-
1.66 (2H, m, hexyl H-2), 1.88-1.99 (2H, m, Val H-3 and 1H of
Gln H-3), 2.03-2.12 (1H, m, 1H of Gln H-3), 2.31 (2H, t, J 7.5
Hz, Gln H-4), 3.13 (1H, d, J 6.0 Hz, Val H-2), 3.18 (2H, t, J
7.0 Hz, hexyl H-6), 3.44 (1H, dd, J 9.5, 7.0 Hz, fucose H-2),
3.50 (1H, dd, J 9.5, 3.0 Hz, fucose H-3), 3.54 (1H, A of ABX2,
J 9.5, 6.5 Hz, 1H of hexyl H-1), 3. 63 (1H, dd, J 3.0, 1.0 Hz,
fucose H-4), 3.65 (1H, qd, J 6.5, 1.0 Hz, fucose H-5), 3.83 (1H,
B of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 4.21 (1H, d, J 7.0
Hz, fucose H-1), 4.30 (1H, dd, J 9.0, 5.0 Hz, Gln H-2); 13C NMR
(CD3OD + D2O, 75.5 MHz) 16.71 (fucose C-6), 18.02 (Val C-4),
19.82 (Val C-4′), 26.43, 27.45, 28.91, 29.95, 30.41, and 32.58
(Gln C-3 and C-4, + hexyl C-2 to C-5), 33.26 (Val C-3), 40.35
(hexyl C-6), 54.29 and 61.66 (Val C-2 and Gln C-2), 70.90 (hexyl
C-1), 71.82, 72.06, 72.84, and 74.84 (fucose C-2, C-3, C-4, and
C-5), 104.49 (fucose C-1), 173.65 (Gln C-5), 177.35 and 177.95
(Val C-1 and Gln C-1); HRMS (FAB) 491.307 20, calculated
for [MH+] (C22H43N4O8+) 491.308 07; HPLC (Hamilton; 0-50%
(60 min) B in C, pH 7.7), 20.8 min, 94%.
1
to brown above 165 °C); H NMR (CD3OD + D2O, 300 MHz)
1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.30-1.41 (4H, m, hexyl
H-3 and H-4), 1.36 (3H, d, J 7.0 Hz, Ala H-3), 1.48-1.55 (2H,
m, hexyl H-5), 1.57-1.68 (2H, m, hexyl H-2), 1.86-2.00 and
2.07-2.23 (2H, 2 × m, Gln H-3), 2.01 (3H, s, NAc), 2.24-2.30
(2H, m, Gln H-4), 3.12-3.18 (2H, m, hexyl H-6), 3.44 (1H, dd,
J 10.0, 7.5 Hz, fucose H-2), 3.51 (1H, dd, J 10.0, 3.0 Hz, fucose
H-3), 3.55 (1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1),
3.64 (1H, dd, J 3.0, 1.0 Hz, fucose H-4), 3.66 (1H, qd, J 6.5,
1.0 Hz, fucose H-5), 3.83 (1H, B of ABX2, J 9.5, 6.5 Hz, 1H of
hexyl H-1), 4.22 (1H, d, J 7.5 Hz, fucose H-1), 4.24 (1H, q, J
7.0 Hz, Ala H-2), 4.26 (1H, dd, J 9.5, 4.5 Hz, Gln H-2); 13C
NMR (CD3OD + D2O, 100.6 MHz, protonated carbons only
1-â-[6-([(S)-3-Am in oa la n yl-(S)-glu ta m in yl]a m in o)h ex-
1-yl]-L-fu cop yr a n ose, m on oa ceta te sa lt, 26: 1H NMR (CD3-
OD + D2O, 300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.29-
1.41 (4H, m, hexyl H-3 and H-4), 1.43-1.54 (2H, m, hexyl H-5),
1.55-1.67 (2H, m, hexyl H-2), 1.89 (3H, s, AcO-), 1.91-2.03
and 2.05-2.20 (2H, 2 × m, Gln H-3), 2.32 (2H, t, J 7.0 Hz,
Gln H-4), 3.01 (1H, A of ABX, J 13.0, 7.5 Hz, 1H of amino-Ala
H-3), 3.12-3.21 (3H, m, hexyl H-6 and 1H of amino-Ala H-3),

1922 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
from HMQC assignment) 16.40 (fucose C-6), 17.21 (Ala C-3),
22.18 (NCOMe), 25.66 and 27.41 (hexyl C-3 and C-4), 29.22
(hexyl C-5), 29.81 (hexyl C-2), 30.08 (Gln C-3), 32.72 (Gln C-4),
40.12 (hexyl C-6), 50.80 and 53.51 (Ala C-2 and Gln C-2), 70.72
(hexyl C-1), 71.48 (fucose C-2), 71.51 (fucose C-5), 72.42 (fucose
C-4), 74.42 (fucose C-3), 103.93 (fucose C-1); HRMS (FAB) m/z
505.289 70, calculated for MH+ (C22H41N4O9+) 505.287 35;
HPLC, system A (Hamilton; 0-25% (50 min) B in C, pH 8.5),
26.2 min, 100%.
72.67 and 74.51 (fucose C-2, C-3, C-4, and C-5), 104.26 (fucose
C-1), 174.00 (Gln C-5), 178.50 and 178.95 (Ala C-1 and Gln
C-1); HRMS (FAB) 463.279 30, calculated for [MH+]
(C20H39N4O8+) 463.276 79; HPLC (Hamilton; 10-50% (40 min)
A in C, pH 7.85), 21.5 min, 96%.
1-â-[6-((R)-Alan yl-(S)-glu tam ylam in o)h ex-1-yl]-L-fu copy-
1
r a n ose, 33: mp 126-129 °C (softens 117 °C); H NMR (D2O
+ CD3OD, 300 MHz) 1.24 (3H, d, J 6.5 Hz, fucose H-6), 1.29-
1.40 (4H, m, hexyl H-3 and H-4), 1.47-1.58 (2H, m, hexyl H-5),
1.55 (3H, d, J 7.0 Hz, Ala H-3), 1.59-1.65 (2H, m, hexyl H-2),
1.85-2.09 (2H, m, Glu H-3), 2.24 (2H, t, J 7.0 Hz, Glu H-4),
3.15-3.22 (2H, m, hexyl H-6), 3.43 (1H, dd, J 10.0, 8.0 Hz,
fucose H-2), 3.57 (1H, dd, J 10.0, 3.0 Hz, fucose H-3), 3.57-
3.63 (1H, m, 1H of hexyl H-1), 3.68 (1H, d* J 3.0 Hz, fucose
H-4), 3.72 (1H, q*, J 6.5 Hz, fucose H-5), 3.86 (1H, B of ABX2,
J 9.5, 6.5 Hz, 1H of hexyl H-1), 4.04 (1H, q, J 7.0 Hz, Ala H-2),
4.23 (1H, dd, J 9.0, 5.0 Hz, Glu H-2), 4.29 (1H, d, J 7.5 Hz,
fucose H-1), *fucose J H4-H5 unresolved; 13C NMR (D2O, 75.5
MHz, MeCN ) 1.30 ppm) 19.79 (fucose C-6), 21.20 (Ala C-3),
29.01, 30.11, 32.26, 32.52 and 33.01 (Glu C-3 + hexyl C-2 to
C-5), 37.89 (Glu C-4), 43.70 (hexyl C-6), 53.54 and 58.47 (Ala
C-2 and Glu C-2), 74.85 (hexyl C-1), 74.90, 75.23, 75.74, and
77.36 (fucose C-2, C-3, C-4, and C-5), 107.02 (fucose C-1),
175.80 and 177.63 (Ala C-1 and Glu C-1), 185.56 (Glu C-5);
HRMS (FAB) 486.244 80, calculated for [MNa+] (C20H37N3-
NaO9+) 486.24274; HPLC (Hamilton; 0-50% (60 min) B in C,
pH 8.45), 16.3 min.
1-â-[6-([N-Acet yl-(RS)-glu t a m in yl]a m in o)h ex-1-yl]-L-
1
fu cop yr a n ose, 30: mp 71-74 °C; H NMR (CD3OD + D2O,
300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.29-1.40 (4H,
m, hexyl H-3 and H-4), 1.44-1.53 (2H, m, hexyl H-5), 1.55-
1.66 (2H, m, hexyl H-2), 1.83-2.14 (2H, m, Gln H-3), 2.01 (3H,
s, AcN), 2.25-2.32 (2H, m, Gln H-4), 3.12-3.20 (2H, m, hexyl
H-6), 3.44 (1H, dd, J 9.5, 7.5 Hz, fucose H-2), 3.50 (1H, dd, J
9.5, 3.0 Hz, fucose H-3), 3.54 (1H, A of ABX2, J 9.5, 6.5 Hz,
1H of hexyl H-1), 3.63 (1H, dd, J 3.0, 1.0 Hz, fucose H-4), 3.65
(1H, qd, J 6.5, 1.0 Hz, fucose H-5), 3.83 (1H, B of ABX2, J 9.5,
7.0 Hz, 1H of hexyl H-1), 4.21 (1H, d, J 7.5 Hz, fucose H-1),
4.27 (∼0.5H, dd, J 9.0, 5.0 Hz, (S)-Gln H-2), 4.28 (∼0.5H, dd,
J 9.0, 5.0 Hz, (R)-Gln H-2); 13C NMR (CD3OD + D2O, 75.5
MHz) 16.68 (fucose C-6), 22.60 (MeCO), 26.35, 27.38, 27.45,
28.89, 29.02, 29.87, 30.34, 32.51, 33.24 (Gln C-3 and C-4, +
linker C-2 to C-5 for (R)-Gln and (S)-Gln diastereomers), 40.33
and 40.41 (hexyl C-6 for (R)-Gln and (S)-Gln diastereomers),
54.03 and 54.44 ((R)-Gln C-2 and (S)-Gln C-2), 71.01 (hexyl
C-1), 71.85, 72.02, 72.81 and 74.77 (fucose C-2, C-3, C-4, and
C-5), 104.43 (fucose C-1), 173.81, 174.04, 174.12, 175.10,
176.80, and 178.17 (MeCO, Gln C-1, and Gln C-5 for (R)-Gln
and (S)-Gln diastereomers); HRMS (FAB) 434.248 40, calcu-
lated for [MH+] (C19H36N3O8+) 434.250 24; HPLC (Hamilton;
0-50% (60 min) B in C, pH 7.7), 18.8 min, 100% (diastereo-
mers not resolved).
1-â-[6-([(S)-3-Am in oa la n yl-(S)-glu ta m yl]a m in o)h ex-1-
yl]-L-fu cop yr a n ose, 34: mp 175-180 °C (decomp; darkens
1
> 150 °C); H NMR (D2O, 300 MHz, MeOH ) 3.35 ppm) 1.25
(3H, d, J 6.5 Hz, fucose H-6), 1.30-1.43 (4H, m, hexyl H-3
and H-4), 1.48-1.56 (2H, m, hexyl H-5), 1.56-1.67 (2H, m,
hexyl H-2), 1.91-2.11 (2H, m, Gln H-3), 2.29 (2H, t, J 7.5 Hz,
Glu H-4), 3.16 (1H, A of ABX, J 13.0, 7.5 Hz, 1H of amino-Ala
H-3), 3.17 (1H, A of ABX2, J 9.0, 7.0 Hz, 1H of hexyl H-6),
3.25 (1H, B of ABX2, J 9.0, 7.0 Hz, 1H of hexyl H-6), 3.32 (1H,
B of ABX, J 13.0, 5.5 Hz, 1H of amino-Ala H-3), 3.45 (1H, dd,
J 10.0, 8.0 Hz, fucose H-2), 3.63 (1H, dd, J 10.0, 3.5 Hz, fucose
H-3), 3.65 (1H, A of ABX2, J 10.0, 7.0 Hz, 1H of hexyl H-1),
3.74 (1H, d*, J 3.5 Hz, fucose H-4), 3.78 (1H, q*, J 6.5 Hz,
fucose H-5), 3.85 (1H, dd, J 7.5, 5.5 Hz, amino-Ala H-2), 3.88
(1H, B of ABX2, J 10.0, 7.0 Hz, 1H of hexyl H-1), 4.26 (1H, dd,
J 8.5, 5.5 Hz, Glu H-2), 4.36 (1H, d, J 8.0 Hz, fucose H-1),
*fucose J H4-H5 unresolved; 13C NMR (D2O, 75.5 MHz, MeOH
) 49.0 ppm) 15.54 (fucose C-6), 24.76, 25.86, 27.91, 28.27,
28.80, and 33.61 (Gln C-3 and C-4, + hexyl C-2 to C-5), 39.46
and 40.03 (amino-Ala C-3 and hexyl C-6), 52.02 and 54.28
(amino-Ala C-2 and Glu C-2), 70.63 (hexyl C-1), 70.66, 71.00,
71.51, 73.13 (fucose C-2, C-3, C-4, and C-5), 102.79 (fucose C-1),
173.09 and 173.39 (amino-Ala C-1 and Glu C-1), 181.38 (Glu
C-5); HRMS (FAB) 479.270 30, calculated for [MH+]
(C20H39N4O9+) 479.271 70; HPLC (YMC; 0-20% (40 min) B in
C, pH 7.0), 26.5 min, 99%.
1-â-[6-(â-Alan yl-(S)-glu tam in ylam in o)h ex-1-yl]-L-fu copy-
r a n ose, 31: mp 121-124 °C; 1H NMR (CD3OD + D2O, 300
MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.29-1.41 (4H, m,
hexyl H-3 and H-4), 1.45-1.53 (2H, m, hexyl H-5), 1.54-1.66
(2H, m, hexyl H-2), 1.87-2.12 (2H, m, Gln H-3), 2.33 (2H, t, J
7.5 Hz, Gln H-4), 2.71 (2H, t, J 6.5 Hz, â-Ala H-2), 3.10-3.24
(4H, m, â-Ala H-3 and hexyl H-6), 3.44 (1H, dd, J 10.0, 7.5
Hz, fucose H-2), 3.54 (1H, dd, J 10.0, 3.5 Hz, fucose H-3), 3.56
(1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.66 (1H, d*,
J 3.5 Hz, fucose H-4), 3.68 (1H, q*, J 6.5 Hz, fucose H-5), 3.83
(1H, B of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 4.25 (1H, d, J
7.5 Hz, fucose H-1), 4.27 (1H, dd, J 9.0, 5.5 Hz, Gln H-2),
*fucose J H4-H5 unresolved; 13C NMR (CD3OD + D2O, 75.5 MHz)
16.76 (fucose C-6), 26.52, 27.51, 28.95, 30.03, 30.50 and 32.67
(Gln C-3 and C-4, + hexyl C-2 to C-5), 33.95 (â-Ala C-2), 37.29
(â-Ala C-3), 40.36 (hexyl C-6), 54.64 (Gln C-2), 70.81 (hexyl
C-1), 71.85, 72.17, 72.92, and 74.95 (fucose C-2, C-3, C-4, and
C-5), 104.57 (fucose C-1), 173.05 and 173.89 (â-Ala C-1 and
Gln C-5), 178.09 (Gln C-1); HRMS (FAB) 463.278 80, calcu-
lated for [MH+] (C20H39N4O8+) 463.276 79; HPLC (Hamilton;
0-50% (50 min) B in C, pH 8.4), 14.9 min, 96%.
1-â-[6-((S)-Ala n yl-(R)-glu ta m in yla m in o)h ex-1-yl]-L-fu -
cop yr a n ose, 32: mp 188-190 °C (darkens slowly >150 °C);
1H NMR (CD3OD + D2O, 300 MHz) 1.24 (3H, d, J 6.5 Hz,
fucose H-6), 1.26 (3H, d, J 7.0 Hz, Ala H-3), 1.29-1.40 (4H,
m, hexyl H-3 and H-4), 1.51 (2H, tt, J 6.5, 6.5 Hz, hexyl H-5),
1.60 (2H, tt, J 7.0, 6.5 Hz, hexyl H-2), 1.89-2.13 (2H, m, Gln
H-3), 2.33 (2H, t, J 7.5 Hz, Gln H-4), 3.16 (1H, A of ABX2, J
13.0, 6.5 Hz, 1H of hexyl H-6), 3.21 (1H, B of ABX2, J 13.0,
7.0 Hz, 1H of hexyl H-6), 3.43 (1H, dd, J 10.0, 8.0 Hz, fucose
H-2), 3.51 (1H, q, J 7.0 Hz, Ala H-2), 3.57 (1H, dd, J 10.0, 3.5
Hz, fucose H-3), 3.60 (1H, A of ABX2, J 9.5, 7.0 Hz, 1H of hexyl
H-1), 3.69 (1H, d*, J 3.5 Hz, fucose H-4), 3.73 (1H, q*, J 6.5
Hz, fucose H-5), 3.85 (1H, B of ABX2, J 9.5, 7.0 Hz, 1H of hexyl
H-1), 4.24 (1H, dd, J 9.0, 5.5 Hz, Gln H-2), 4.30 (1H, d, J 8.0
Hz, fucose H-1), *fucose J H4-H5 unresolved; 13C NMR (CD3OD
+ D2O, 75.5 MHz) 16.54 (fucose C-6), 20.97 (Ala C-3), 26.02
and 27.09 (hexyl C-3 and C-4), 28.57 (Gln C-3), 29.54 and 30.04
(hexyl C-2 and C-5), 32.35 (Gln C-4), 40.29 (hexyl C-6), 51.11
and 54.28 (Ala C-2 and Gln C-2), 71.20 (hexyl C-1), 71.84 [2C],
1-â-[6-([2,6-N,N′-Di((R)-alan yl-(S)-glu tam in yl)-(S)-lysyl]-
a m in o)h ex-1-yl]-L-fu cop yr a n ose, 35: mp 107-110 °C; 1H
NMR (CD3OD + D2O, 300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose
H-6), 1.29 (6H, d, J 7.0 Hz, 2 × Ala H-3), 1.31-1.43 (6H, m,
Lys H-4 and hexyl H-3 and H-4), 1.45-1.55 (4H, m, Lys H-5
and hexyl H-5), 1.60 (2H, tt, J 6.5, 6.5 Hz, hexyl H-2), 1.64-
1.83 (2H, m, Lys H-3), 1.86-2.15 (4H, m, 2 × Gln H-3), 2.30
(2H, t, J 7.5 Hz, Gln H-4), 2.32 (2H, t, J 7.5 Hz, Gln H-4),
3.09-3.25 (2H, m, hexyl H-6), 3.19 (2H, t, J 7.0 Hz, Lys H-6),
3.45 (1H, dd, J 10.0, 7.5 Hz, fucose H-2), 3.47-3.56 (4H, m, 2
× Ala H-2, fucose H-3 and 1H of hexyl H-1), 3.62 (1H, d*, J
3.0 Hz, fucose H-4), 3.64 (1H, q*, J 6.5 Hz, fucose H-5), 3.83
(1H, B of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 4.20 (1H, d, J
7.5 Hz, fucose H-1), 4.25 (1H, dd, J 9.0, 5.5 Hz, Gln H-2, Gln′
H-2 or Lys H-2), 4.28 (1H, dd, J 9.0, 5.0 Hz, Gln H-2, Gln′ H-2
or Lys H-2), 4.32 (1H, dd, J 8.0, 6.0 Hz, Gln H-2, Gln′ H-2 or
Lys H-2), *fucose J H4-H5 unresolved; 13C NMR (CD3OD + D2O,
75.5 MHz) 16.69 (fucose C-6), 21.21 (2 × Ala C-3), 23.97 (Lys
C-4), 26.51 and 27.51 (hexyl C-3 and C-4), 28.78 and 28.94
(Lys C-5 and 2 × Gln C-3), 29.57, 30.03, and 32.46 (Lys C-3
and hexyl C-2 and C-5), 32.46 (2 × Gln C-4), 40.03 and 40.29

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1923
(Lys C-6 and hexyl C-6), 51.38, 54.40, and 54.90 (Lys C-2, 2 ×
Ala C-2 and 2 × Gln C-2), 70.90 (hexyl C-1), 71.89, 72.22, 73.01,
and 75.01 (fucose C-2, C-3, C-4, and C-5), 104.71 (fucose C-1),
174.05 and 174.37 (2 × Gln C-5), 178.22, 178.91 and 179.10
(Lys C-1, 2 × Ala C-1 and 2 × Gln C-1); HRMS (FAB)
790.464 400, calculated for [MH+] (C34H64N9O12+) 790.467 444;
HPLC (Hamilton; 0-40% (50 min) B in C, pH 8.35), 22.8 min,
95%.
C-6), 26.71, 26.84, 27.64, 30.23, 30.52, and 30.64 (pyro-Glu C-3
and C-4, + hexyl C-2 to C-5), 40.36 (hexyl C-6), 58.24 (pyro-
Glu C-2), 70.53 (hexyl C-1), 71.80, 72.29, 73.00, and 75.14
(fucose C-2, C-3, C-4, and C-5), 104.77 (fucose C-1), 174.75 and
181.51 (pyro-Glu C-1 and C-5); HRMS (FAB) 375.215 90,
calculated for [MH+] (C17H31N2O7+) 375.213 13; HPLC (Hamil-
ton; 0-40% (50 min) B in C, pH 8.5), 18.1 min, 100%.
1-â-[6-((N-Meth yl-(S)-p yr oglu ta m yl)a m in o)h ex-1-yl]-L-
fu cop yr a n ose, 41: mp 143-145 °C; 1H NMR (D2O, 300 MHz,
MeCN ) 2.05 ppm) 1.24 (3H, d, J 6.5 Hz, fucose H-6), 1.30-
1.41 (4H, m, hexyl H-3 and H-4), 1.50-1.56 (2H, m, hexyl H-5),
1.58-1.63 (2H, m, hexyl H-2), 1.90-2.01 (1H, m, 1H of pyro-
Glu H-3), 2.32-2.58 (3H, m, pyro-Glu H-4 and 1H of pyro-
Glu H-3), 2.75 (3H, s, NMe), 3.24 (2H, t, J 7.0 Hz, hexyl H-6),
3.44 (1H, dd, J 9.5, 8.0 Hz, fucose H-2), 3.61 (1H, dd, J 10.0,
3.5 Hz, fucose H-3), 3.63 (1H, A of ABX2, J 10.0, 7.0 Hz, 1H of
hexyl H-1), 3.72 (1H, d*, J 3.5 Hz, fucose H-4), 3.76 (1H, q*, J
6.5 Hz, fucose H-5), 3.87 (1H, B of ABX2, J 10.0, 7.0 Hz, 1H of
hexyl H-1), 4.23 (1H, dd, J 9.0, 5.0 Hz, pyro-Glu H-2), 4.34
(1H, d, J 8.0 Hz, fucose H-1), *fucose J H4-H5 unresolved; 13C
NMR (CD3OD + D2O, 75.5 MHz) 16.75 (fucose C-6), 23.94,
26.51, 27.55, 30.06, 30.50, and 30.71 (pyro-Glu C-3 and C-4 +
hexyl C-2 to C-5), 29.03 (NMe), 40.41 (hexyl C-6), 64.56 (pyro-
Glu C-2), 70.80 (hexyl C-1), 71.85, 72.16, 72.90, and 74.96
(fucose C-2, C-3, C-4, and C-5), 104.60 (fucose C-1), 173.62 and
178.79 (pyro-Glu C-1 and C-5); HRMS (FAB) 389.229 50,
calculated for [MH+] (C18H33N2O7+) 389.228 79; HPLC (Hamil-
ton; 10-60% (60 min) A in C, pH 7.85), 28.2 min, 96%.
1-â-[6-([(R)-Ala n yl-(S)-glu ta m in yl-5-N-[6-(â-L-fu cop yr a -
n osyl)oxyh exyl]-(S)-glu t a m in yl]-a m in o)h ex-1-yl]-L-fu -
1
cop yr a n ose, 36: mp 125-128 °C; H NMR (CD3OD + D2O,
300 MHz) 1.25 (6H, d, J 6.5 Hz, 2 × fucose H-6), 1.28-1.42
(8H, m, 2 × [hexyl H-3 and H-4]), 1.45-1.55 (4H, m, 2 × hexyl
H-5), 1.49 (3H, d, J 7.0 Hz, Ala H-3), 1.61 (4H, tt, J 7.0, 7.0
Hz, 2 × hexyl H-2), 1.90-2.20 (4H, m, 2 × Gln H-3), 2.22-
2.38 (4H, m, 2 × Gln H-4), 3.10-3.23 (2H, m, hexyl H-6), 3.15
(2H, t, J 7.0 Hz, hexyl H-6), 3.45 (2H, dd, J 10.0, 7.5 Hz, 2 ×
fucose H-2), 3.52 (2H, dd, J 10.0, 3.0 Hz, 2 × fucose H-3), 3.51-
3.59 (2H, m, 1H of each hexyl H-1’s), 3.64 (2H, d*, J 3.0 Hz, 2
× fucose H-4), 3.66 (2H, q*, J 6.5 Hz, 2 × fucose H-5), 3.84
(2H, B of ABX2, J 9.5, 7.0 Hz, 1H of each hexyl H-1’s), 4.01
(1H, q, J 7.0 Hz, Ala H-2), 4.23 (2H, d, J 7.5 Hz, 2 × fucose
H-1), 4.23-4.28 (1H, m, Gln H-2), 4.30 (1H, dd, J 9.0, 5.0 Hz,
Gln H-2), *fucose J H4-H5 unresolved; 13C NMR (CD3OD + D2O,
75.5 MHz) 16.68 (2C, 2 × fucose C-6), 18.15 (Ala C-3), 26.39,
26.45, 27.40, and 27.50 (2 × [hexyl C-3 and C-4]), 28.42 and
28.97 (2 × Gln C-3), 29.89, 29.95, and 30.38 [2C] (2 × [hexyl
C-2 and C-5]), 32.49 and 33.31 (2 × Gln C-4), 40.35 and 40.43
(2 × hexyl C-6), 50.50 (Ala C-2), 54.71 and 54.92 (2 × Gln C-2),
70.97 (2C, 2 × hexyl C-1), 71.90 [2C], 72.14 [2C], 72.89, 72.93
and 74.91 [2C] (2 × [fucose C-2, C-3, C-4, and C-5]), 104.61
(2C, 2 × fucose C-1), 173.41, 173.87, 174.05, 175.42 and 178.22
(Ala C-1, 2 × Gln C-1 and 2 × Gln C-5); HRMS (FAB)
837.478 400, calculated for [MH+] (C37H69N6O15+) 837.482 0 91;
HPLC (Hamilton; 0-50% (60 min) B in C, pH 8.5), 27.3 min,
95%.
1-â-[6-((R)-Ala n ylglycyla m in o)h e x-1-yl]-L-fu cop yr a -
1
n ose, 42: mixture of anomers (R:â ) 1:4). mp 52-54 °C; H
NMR (CD3OD + D2O, 300 MHz) â-anomer 1.25 (3H, d, J 6.5
Hz, fucose H-6), 1.28 (3H, d, J 7.0 Hz, Ala H-3), 1.31-1.42
(4H, m, hexyl H-3 and H-4), 1.47-1.54 (2H, m, hexyl H-5),
1.56-1.66 (2H, m, hexyl H-2), 3.19 (2H, t, J 7.0 Hz, hexyl H-6),
3.44 (1H, dd, J 9.5, 7.5 Hz, fucose H-2), 3.48 (1H, q, J 7.0 Hz,
Ala H-2), 3.49 (1H, dd, J 9.5, 3.0 Hz, fucose H-3), 3.53 (1H, A
of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.62 (1H, dd, J 3.0,
1.0 Hz, fucose H-4), 3.63 (1H, qd, J 6.5, 1.0 Hz, fucose H-5),
3.80 (1H, A of AB q, J 16.5 Hz, 1H of Gly H-2), 3.83 (1H, B of
ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.88 (1H, B of AB q, J
16.5 Hz, 1H of Gly H-2), 4.20 (1H, d, J 7.5 Hz, fucose H-1).
For R-anomer (where different) 1.20 (d, J 6.5 Hz, fucose H-6),
4.75 (d, J 2.5 Hz, fucose H-1); 13C NMR (CD3OD + D2O, 75.5
MHz) â-anomer 16.66 (fucose C-6), 21.06 (Ala C-3), 26.30,
27.31, 29.84, and 30.26 (hexyl C-2 to C-5), 40.35 (hexyl C-6),
43.35 (Gly C-2), 51.33 (Ala C-2), 71.04 (hexyl C-1), 71.83, 71.95,
72.75, and 74.68 (fucose C-2, C-3, C-4, and C-5), 104.35 (fucose
C-1), 171.63 and 179.29 (Ala C-1 and Gly C-1). For R-anomer
(where different) 16.54 (fucose C-6), 26.61 and 30.13 (hexyl
C-2 and C-3), 67.53, 69.54, 71.26, 73.24 (fucose C-2, C-3, C-4,
and C-5), 69.27 (hexyl C-1), 99.98 (fucose C-1); HRMS (FAB)
392.238 50, calculated for [MH+] (C17H34N3O7+) 392.239 69;
HPLC (Hamilton; 0-50% (60 min) B in D, pH 7.7), 17.7 min,
98%.
1-â-[6-[6-(Dim et h yla m in o)p u r in -9-yl]h exyl]-L-fu cop y-
r a n ose, 37:16 mixture of anomers (R:â ) 0.15:0.85); mp 141-
145 °C; νmax 214.2 and 276.0 nm (ꢀ1/ꢀ2 ) 1.1); 1H NMR (CD3OD
+ D2O, 300 MHz, MeOH ) 3.35 ppm) â-anomer 1.23 (3H, d, J
6.5 Hz, fucose H-6), 1.26-1.35 (2H, m, hexyl H-4), 1.36-1.47
(2h, m, hexyl H-3), 1.53-1.61 (2H, m, hexyl H-2), 1.86 (2H, tt,
J 7.0, 7.0 Hz, hexyl H-5), 3.37-3.53 (9H, m, fucose H-2 and
H-3, 1H of hexyl H-1, and NMe2), 3.61 (1H, d*, J 3.5 Hz, fucose
H-4), 3.61 (1H, q*, J 6.5 Hz, fucose H-5), 3.80 (1H, B of ABX2,
J 10.0, 6.5 Hz, 1H of hexyl H-1), 4.17 (1H, d, J 7.5 Hz, fucose
H-1), 4.21 (2H, t, J 7.0 Hz, linker H-6), 8.04 (1H, s, purine
H-8), 8.19 (1H, s, purine H-2), *fucose J H4-H5 unresolved. For
R-anomer (where different) 1.16 (d, J 6.5 Hz, fucose H-6), 4.76
(d, J 2.0 Hz, fucose H-1); 13C NMR (CD3OD + D2O, 75.5 MHz)
â-anomer 16.72 (fucose C-6), 26.29, 27.12, 30.32, and 30.73
(hexyl C-2 to C-5), 39.41 (NMe2), 44.82 (hexyl C-6), 70.71 (hexyl
C-1), 71.84, 72.12, 72.88, and 74.91 (fucose C-2, C-3, C-4, and
C-5), 104.56 (fucose C-1), 120.61 (purine C-5), 141.36 (purine
C-8), 150.90 (purine C-4), 152.76 (purine C-2), 155.88 (purine
C-6). For R-anomer (where different) 16.58 (fucose C-6), 26.60,
27.18, 30.19, and 30.52 (hexyl C-2 and C-5), 67.50, 69.74, 71.46,
and 73.39 (fucose C-2, C-3, C-4, and C-5), 69.08 (hexyl C-1),
100.19 (fucose C-1); HRMS (FAB) 410.239 50, calculated for
[MH+] (C19H32N5O5+) 410.240 36; HPLC (Hamilton; 50-80%
(40 min) A in D, pH 7.85), 23.9 min, 99%.
1-â-[6-((R)-Alan yl-(S)-or n ith ylam in o)h ex-1-yl]-L-fu copy-
r a n ose, 43: mp 82-86 °C; 1H NMR (CD3OD + D2O, 300 MHz)
1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.27 (3H, d, J 6.5 Hz, Ala
H-3), 1.31-1.41 (4H, m, hexyl H-3 and H-4), 1.47-1.55 (2H,
m, hexyl H-5), 1.56-1.75 (5H, m, hexyl H-2, Orn H-4 and 1H
of Orn H-3), 1.77-1.89 (1H, m, 1H of Orn H-3), 2.76 (2H, t, J
7.0 Hz, Orn H-5), 3.18 (2H, t, J 6.5 Hz, hexyl H-6), 3.44 (1H,
dd, J 10.0, 7.0 Hz, fucose H-2), 3.49 (1H, q, J 6.5 Hz, Ala H-2),
3.49 (1H, dd, J 10.0, 3.5 Hz, fucose H-3), 3.53 (1H, A of ABX2,
J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.62 (1H, d*, J 3.0 Hz, fucose
H-4), 3.63 (1H, q*, J 6.5 Hz, fucose H-5), 3.83 (1H, B of ABX2,
J 9.5, 6.5 Hz, 1H of hexyl H-1), 4.20 (1H, d, J 7.0 Hz, fucose
H-1), 4.27 (1H, dd, J 8.5, 5.5 Hz, Orn H-2), *fucose J H4-H5
unresolved; 13C NMR (CD3OD + D2O, 75.5 MHz) 15.94 (fucose
C-6), 20.59 (Ala C-3), 26.22, 26.25, 28.69, 29.15, and 29.21 (Orn
C-3 and C-4, + hexyl C-2 to C-5 [one peak unresolved]), 39.76
and 39.95. (hexyl C-6 and Orn C-5), 50.39 and 54.22 (Ala C-2
and Orn C-2), 71.05 (hexyl C-1), 71.09, 71.42, 71.94, and 73.60
(fucose C-2, C-3, C-4, and C-5), 103.31 (fucose C-1), 174.29 and
1-â-[6-((S)-P yr oglu t a m yla m in o)h ex-1-yl]-L-fu cop yr a -
1
n ose, 40: mp 91-92 °C (softens 81 °C); H NMR (CD3OD +
D2O, 300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.30-1.46
(4H, m, hexyl H-3 and H-4), 1.47-1.56 (2H, m, hexyl H-5),
1.56-1.67 (2H, m, hexyl H-2), 1.97-2.08 (1H, m, 1H of pyro-
Glu H-3), 2.28-2.53 (3H, m, pyro-Glu H-4 and 1H of pyro-
Glu H-3), 3.13-3.27 (2H, m, hexyl H-6), 3.44 (1H, dd, J 10.0,
7.5 Hz, fucose H-2), 3.52 (1H, dd, J 10.0, 3.0 Hz, fucose H-3),
3.55 (1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1), 3.64 (1H,
dd, J 3.0, 1.0 Hz, fucose H-4), 3.66 (1H, qd, J 6.5, 1.0 Hz, fucose
H-5), 3.84 (1H, B of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1),
4.20 (1H, dd, J 9.5, 5.5 Hz, pyro-Glu H-2), 4.22 (1H, d, J 7.5
Hz, fucose H-1); 13C NMR (CD3OD, 75.5 MHz) 16.77 (fucose

1924 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
179.53 (Ala C-1 and Orn C-1); HRMS (FAB) 449.296 70,
calculated for [MH+] (C20H41N4O7+) 449.297 52.
2-O-Acetyl-1-â-[6-((S)-a la n yl-(S)-glu ta m in yla m in o)h ex-
1-yl]-D-m a n n op yr a n ose, 47: 1H NMR (CD3OD, 300 MHz)
1.27 (3H, d, J 7.0 Hz, Ala H-3), 1.30-1.42 (4H, m, hexyl H-3
and H-4), 1.45-1.57 (4H, m, hexyl H-2 and H-5), 1.62 (3H, s,
OAc), 1.84-1.98 and 2.02-2.11 (2H, 2 × m, Gln H-3), 2.26-
2.31 (2H, m, Gln H-4), 3.15-3.20 (2H, m, hexyl H-6), 3.22 (1H,
ddd, J 9.5, 6.0, 2.5 Hz, mannose H-5), 3.45 (1H, q, J 7.0 Hz,
Ala H-2), 3.50-3.57 (2H, m, hexyl H-1), 3.55 (1H, dd, J 9.5,
9.5 Hz, mannose H-4), 3.65 (1H, A of ABX, J 12.0, 6.0 Hz, 1H
of mannose H-6), 3.70 (1H, dd, J 9.5, 4.0 Hz, mannose H-3),
3.84 (1H, B of ABX, J 12.0, 2.5 Hz, 1H of mannose H-6), 4.32
(1H, dd, J 9.0, 5.5 Hz, Gln H-2), 4.43 (1H, dd, J 4.0, 2.5 Hz,
mannose H-2), 5.43 (1H, d, J 2.5 Hz, H-1); 13C NMR (CD3OD,
75.5 MHz) 21.38 (Ala C-3), 25.83 (MeCO), 26.83, 27.56, 29.32,
30.26, 30.50, and 32.50 (Gln C-3 and C-4 and hexyl C-2, C-3,
C-4, and C-5), 40.35 (hexyl H-6), 51.55 and 54.10 (Ala C-2 and
Gln C-2), 62.83 (hexyl C-1), 68.55, 73.30, 76.93, and 81.18
(mannose C-2, C-3, C-4, and C-5), 99.08 (mannose C-1), 124.84
(mannose C-6), 173.51, 173.59, 177.27, and 178.30 (MeCO, Ala
C-1, Gln C-1 and Gln C-5); HRMS (FAB) m/z 521.284 00,
calculated for MH+, (C22H41N4O10+) 521.282 29; HPLC (Hamil-
ton; 10-50% (50 min) A in C, pH 8.5), 23.7 min, 100%.
1-â-[6-((R)-Ala n yl-(S)-a sp a r a gin yla m in o)h ex-1-yl]-L-fu -
1
cop yr a n ose, 44: mp 100-103 °C; H NMR (CD3OD + D2O,
300 MHz) 1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.28 (3H, d, J 7.0
Hz, Ala H-3), 1.30-1.43 (4H, m, hexyl H-3 and H-4), 1.49 (2H,
tt, J 7.0, 7.0 Hz, hexyl H-5), 1.61 (2H, tt, J 7.0, 7.0 Hz, hexyl
H-2), 2.56-2.74 (2H, m, Asn H-3), 3.11-3.19 (2H, m, hexyl
H-6), 3.44-3.54 (1H, m, Ala H-2), 3.45 (1H, dd, J 10.0, 7.0
Hz, fucose H-2), 3.49 (1H, dd, J 10.0, 3.0 Hz, fucose H-3), 3.53
(1H, A of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 3.62 (1H, d*,
J 3.0 Hz, fucose H-4), 3.64 (1H, q*, J 6.5 Hz, fucose H-5), 3.83
(1H, B of ABX2, J 9.5, 7.0 Hz, 1H of hexyl H-1), 4.20 (1H, d, J
7.0 Hz, fucose H-1), 4.71 (1H, dd, J 8.0, 5.5 Hz, Asn H-2),
*fucose J H4-H5 unresolved; 13C NMR (CD3OD + D2O, 75.5 MHz)
16.57 (fucose C-6), 21.03 (Ala C-3), 26.21 and 27.28 (hexyl C-3
and C-4), 29.76 and 30.20 (hexyl C-2 and C-5), 36.56 (Asn C-3),
40.40 (hexyl C-6), 51.20 and 51.41 (Ala C-2 and Asn C-2), 71.10
(hexyl C-1), 71.83, 71.96, 72.75, and 74.69 (fucose C-2, C-3,
C-4, and C-5), 104.42 (fucose C-1), 172.32 (Asn C-4), 176.16
and 178.68 (Ala C-1 and Asn C-1); HRMS (FAB) 449.262 50,
calculated for [MH+] (C19H37N4O8+) 449.261 14; HPLC (Hamil-
ton; 0-50% (60 min) A in C, pH 7.9), 38.2 min, 94%.
2-O-Ac e t y l-1-â-[6-(N -a c e t y l-(R )-Ala n yl-(S )-g lu t a m -
in yla m in o)h ex-1-yl]-D-m a n n op yr a n ose, 48: 1H NMR (CD3-
OD, 300 MHz) 1.26-1.42 (4H, m, hexyl H-3 and H-4), 1.34
(3H, d, J 7.0 Hz, Ala H-3), 1.44-1.57 (4H, m, hexyl H-2 and
H-5), 1.62 (3H, s, OAc), 1.84-1.97 and 2.09-2.20 (2H, 2 × m,
Gln H-3), 1.98 (3H, s, NAc), 2.23-2.31 (2H, m, Gln H-4), 3.15
(1H, A of ABX2, J 11.5, 7.0 Hz, 1H of hexyl H-6), 3.20 (1H, B
of ABX2, J 11.5, 7.0 Hz, 1H of hexyl H-6), 3.22 (1H, ddd, J 9.5,
6.0, 2.5 Hz, mannose H-5), 3.47-3.56 (2H, m, hexyl H-1), 3.55
(1H, dd, J 9.5, 9.5 Hz, mannose H-4), 3.65 (1H, A of ABX, J
12.0, 6.0 Hz, 1H of mannose H-6), 3.70 (1H, dd, J 9.5, 4.0 Hz,
mannose H-3), 3.84 (1H, B of ABX, J 12.0, 2.5 Hz, 1H of
mannose H-6), 4.22 (1H, q, J 7.0 Hz, Ala H-2), 4.26 (1H, dd, J
9.5, 4.5 Hz, Gln H-2), 4.43 (1H, dd, J 4.0, 2.5 Hz, mannose
H-2), 5.43 (1H, d, J 2.5 Hz, H-1); 13C NMR (CD3OD, 75.5 MHz)
17.47 (NCOMe), 22.45 (Ala C-3), 25.84 (OCOMe), 26.84, 27.50,
28.49, 30.19, 30.47, and 32.65 (Gln C-3 and C-4 and hexyl C-2,
C-3, C-4, and C-5), 40.41 (hexyl H-6), 51.16 and 54.50 (Ala C-2
and Gln C-2), 62.80 (hexyl C-1), 68.52, 73.26, 76.88, and 81.15
(mannose C-2, C-3, C-4, and C-5), 99.04 (mannose C-1), 124.80
(mannose C-6), 173.40, 173.53, 175.59, and 177.75 (2 × MeCO,
Ala C-1, Gln C-1 and Gln C-5); HRMS (FAB) m/z 585.2767 00,
calculated for MNa+ (C24H42N4NaO11+) 585.274 778; HPLC
(Hamilton; 0-40% (50 min) B in C, pH 8.5), 18.7 min, 98%.
1-â-[6-((S)-Ala n yl-(S)-p r olyla m in o)h ex-1-yl]-L-fu cop y-
r a n ose, 45: mp 66-70 °C; 1H NMR (CD3OD + D2O, 300 MHz)
1.25 (3H, d, J 6.5 Hz, fucose H-6), 1.27 (3H, d, J 7.0 Hz, Ala
H-3), 1.31-1.44 (4H, m, hexyl H-3 and H-4), 1.50 (2H, tt, J
7.0, 7.0 Hz, hexyl H-5), 1.62 (2H, tt, J 7.0, 7.0 Hz, hexyl H-2),
1.85-1.93 and 1.94-2.01 (2H, 2 × m, Pro H-4), 2.02-2.11 (1H,
m, 1H of Pro H-3), 2.20 (1H, A of ABX2Y, J 12.0, 7.5, 7.5 Hz,
1H of Pro H-3), 3.12 (1H, A of ABX2, J 13.5, 7.0 Hz, 1H of
hexyl H-6), 3.21 (1H, B of ABX2, J 13.5, 7.0 Hz, 1H of hexyl
H-6), 3.45 (1H, dd, J 9.5, 7.5 Hz, fucose H-2), 3.50 (1H, dd, J
9.5, 3.0 Hz, fucose H-3), 3.54 (1H, A of ABX2, J 9.5, 6.5 Hz,
1H of hexyl H-1), 3.59-3.65 (1H, m, Ala H-2), 3.63 (1H, d*, J
3.0 Hz, fucose H-4), 3.64 (1H, q*, J 6.5 Hz, fucose H-5), 3.66-
3.77 (2H, m, Pro H-5), 3.83 (1H, B of ABX2, J 9.5, 7.0 Hz, 1H
of hexyl H-1), 4.20 (1H, d, J 7.5 Hz, fucose H-1), 4.36 (1H, dd,
J 8.5, 5.0 Hz, Pro H-2), *fucose J H4-H5 unresolved; 13C NMR
(CD3OD + D2O, 75.5 MHz) 16.66 (fucose C-6), 19.94 (Ala C-3),
25.80 and 26.40 (hexyl C-3 and C-4), 27.40 (Pro C-4), 29.97,
30.39, and 30.62 (Pro C-3 and hexyl C-2 and C-5), 40.24 (hexyl
C-6), 48.39 (Pro C-5), 61.41 and 61.73 (Ala C-2 and Pro C-2),
70.97 (hexyl C-1), 71.87, 72.10, 72.90, and 74.89 (fucose C-2,
C-3, C-4, and C-5), 104.61 (fucose C-1), 174.73 and 176.34 (Ala
C-1 and Pro C-1)); HRMS (FAB) 432.269 20, calculated for
[MH+] (C20H38N3O7+) 432.270 97; HPLC (Hamilton; 0-40% (50
min) B in D, pH 7.8), 24.2 min, 99%.
1-â-[6-((S)-Isoleu cyl-(S)-pr olylam in o)h ex-1-yl]-L-fu copy-
r a n ose, 46: mp 58-61 °C; 1H NMR (CD3OD + D2O, 300 MHz)
0.91 (3H, t, J 7.5 Hz, Ile H-5), 0.97 (3H, d, J 6.5 Hz, Ile H-4′),
1.08-1.21 (1H, m, 1H of Ile H-4), 1.25 (3H, d, J 6.5 Hz, fucose
H-6), 1.31-1.44 (4H, m, hexyl H-3 and H-4), 1.50 (2H, tt, J
7.0, 7.0 Hz, hexyl H-5), 1.57-1.71 (4H, m, hexyl H-2, Ile H-3
and 1H of Ile H-4), 1.86-2.00 (2H, m, Pro H-4), 2.02-2.11 and
2.12-2.23 (2H, 2 × m, Pro H-3), 3.13 (1H, A of ABX2, J 13.5,
7.0 Hz, 1H of hexyl H-6), 3.20 (1H, B of ABX2, J 13.5, 7.0 Hz,
1H of hexyl H-6), 3.43-3.49 (3H, m, Ile H-2 and fucose H-2
and H-3), 3.53 (1H, A of ABX2, J 9.5, 6.5 Hz, 1H of hexyl H-1),
3.59-3.67 (1H, m, 1H of Pro H-5), 3.61 (1H, d*, J 2.5 Hz, fucose
H-4), 3.64 (1H, q*, J 6.5 Hz, fucose H-5), 3.71 (1H, B of ABX2,
J 9.5, 7.0 Hz, 1H of Pro H-5), 3.83 (1H, B of ABX2, J 9.5, 7.0
Hz, 1H of hexyl H-1), 4.19 (1H, d, J 6.5 Hz, fucose H-1), 4.38
(1H, dd, J 8.0, 5.0 Hz, Pro H-2), *fucose J H4-H5 unresolved;
13C NMR (CD3OD + D2O, 75.5 MHz) 11.61 (Ile C-5), 16.04 (Ile
C-4′), 16.69 (fucose C-6), 24.67 (Ile C-4), 25.92, 26.55 and 27.53
(Pro C-4 and hexyl C-3 and C-4), 30.15 and 30.52 [2C] (Pro
C-3 and hexyl C-2 and C-5), 39.71 (Ile C-3), 40.27 (hexyl C-6),
48.88 (Pro C-5), 58.20 and 61.76 (Ile C-2 and Pro C-2), 70.87
(hexyl C-1), 71.87, 72.20, 72.96, and 75.03 (fucose C-2, C-3,
C-4, and C-5), 104.73 (fucose C-1), 174.55 and 175.54 (Ile C-1
and Pro C-1); HRMS (FAB) 474.316 50, calculated for [MH+]
(C23H44N3O7+) 474.317 93; HPLC (Hamilton; 5-35% (40 min)
B in D, pH 7.8), 25.4 min, 99%.
2-O-Acetyl-1-â-[6-(S)-glu tam in ylam in o)h ex-1-yl]-D-m an -
n op yr a n ose, a ceta te sa lt, 49: 1H NMR (CD3OD + D2O, 300
MHz) 1.28-1.42 (4H, m, hexyl H-3 and H-4), 1.46-1.58 (4H,
m, hexyl H-2 and H-5), 1.64 (3H, s, OAc), 1.91 (3H, s, AcO-),
1.97-2.14 (2H, m, Gln H-3), 2.37 (2H, t, J 7.5 Hz, Gln H-4),
3.13-3.31 (4H, m, hexyl H-6, mannose H-5 and Gln H-2),
3.50-3.58 (2H, m, hexyl H-1), 3.58 (1H, dd, J 9.5, 9.5 Hz,
mannose H-4), 3.67 (1H, A of ABX, J 12.0, 6.0 Hz, 1H of
mannose H-6), 3.76 (1H, dd, J 9.5, 4.0 Hz, mannose H-3), 3.84
(1H, B of ABX, J 12.0, 2.5 Hz, 1H of mannose H-6), 4.46 (1H,
dd, J 4.0, 2.5 Hz, mannose H-2), 5.46 (1H, d, J 2.5 Hz, H-1);
-
13C NMR (CD3OD, 75.5 MHz) 25.05 and 25.49 (MeCO2 and
OCOMe), 26.54, 27.35, 29.21, 29.85, 30.17, and 31.77 (Gln C-3
and C-4 and hexyl C-2, C-3, C-4, and C-5), 40.52 (hexyl H-6),
58.19 (Gln C-2), 62.38 (hexyl C-1), 68.20, 72.66, 76.53, and
80.83 (mannose C-2, C-3, C-4, and C-5), 98.81 (mannose C-1),
124.68 (mannose C-6), 174.61, 174.94, 177.54, and 180.35 (2
× MeCO, Gln C-1 and Gln C-5); HRMS (FAB) m/z 450.244 00,
calculated for MH+ (C19H36N3O9+) 450.245 15; HPLC (Hamil-
ton; 0-40% (50 min) B in C, pH 8.5), 22.6 min, 97%.
2-O-Acet yl-1-â-[6-(S)-p yr oglu t a m yla m in o)h ex-1-yl]-D-
1
m a n n op yr a n ose, 50: mp 53-55 °C; H NMR (CD3OD, 300
MHz) 1.30-1.43 (4H, m, hexyl H-3 and H-4), 1.47-1.59 (4H,
m, hexyl H-2 and H-5), 1.62 (3H, s, OAc), 1.97-2.08 (1H, m,
1H of pyro-Glu H-3), 2.23-2.51 (3H, m, pyro-Glu H-4 and 1H
of pyro-Glu H-3), 3.20 (2H, t, J 7.0 Hz, hexyl H-6), 3.21 (1H,
ddd, J 9.5, 6.0, 2.5 Hz, mannose H-5), 3.50-3.57 (2H, m, hexyl
H-1), 3.55 (1H, dd, J 9.5, 9.5 Hz, mannose H-4), 3.65 (1H, A of

Dipeptides as Specific NK Cell Enhancers
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1925
ABX, J 12.0, 6.0 Hz, 1H of mannose H-6), 3.69 (1H, dd, J 9.5,
4.0 Hz, mannose H-3), 3.84 (1H, B of ABX, J 12.0, 2.5 Hz, 1H
of mannose H-6), 4.14 (1H, dd, J 8.5, 5.0 Hz, pyro-Glu H-2),
4.42 (1H, dd, J 4.0, 2.5 Hz, mannose H-2), 5.43 (1H, d, J 2.5
Hz, mannose H-1); 13C NMR (CD3OD, 75.5 MHz) 25.86
(OCOMe), 26.86, 26.88, 27.51, 30.27, 30.46, and 30.53 (pyro-
Glu C-3 and C-4 + hexyl C-2 to C-5), 40.38 (hexyl H-6), 58.28
(pyro-Glu C-2), 62.78 (hexyl C-1), 68.52, 73.30, 76.92, and 81.20
(mannose C-2, C-3, C-4, and C-5), 99.07 (mannose C-1), 124.85
(mannose C-6), 174.80 and 181.56 (MeCO and pyro-Glu C-1
and C-5); HRMS (FAB) m/z 455.201 70, calculated for MNa+
(C19H32N2NaO9+) 455.200 56; HPLC (Hamilton; 0-40% (50
min) B in C, pH 8.5), 19.5 min, 100%.
C-3 and C-4, and hexyl C-2 to C-5), 40.26 (hexyl C-6), 55.61
(Gln H-2), 62.82 (hexyl C-1), 175.15 (Gln C-5), 178.11 (Gln C-1);
HRMS (FAB) 246.181 80, calculated for [MH+] (C11H24N3O3
)
+
246.181 76; HPLC (Hamilton; 10-50% (50 min) A in C, pH
8.5), 9.9 min, 100%.
6-((S)-P yr oglu ta m in yla m in o)h exa n -1-ol, 55: mp 170.5-
171.5 °C; 1H NMR (CD3OD, 300 MHz) 1.31-1.43 (4H, m, hexyl
H-3 and H-4), 1.46-1.58 (4H, m, hexyl H-2 and H-5), 1.97-
2.08 (1H, m, 1H of pyro-Glu H-3), 2.23-2.51 (3H, m, pyro Glu
H-4 and 1H of pyro-Glu H-3), 3.20 (2H, t, J 7.0 Hz, hexyl H-6),
3.54 (2H, t, J 6.5 Hz, hexyl H-1), 4.14 (1H, dd, J 8.5, 5.0 Hz,
pyro-Glu H-2); 13C NMR (CD3OD + D2O, 75.5 MHz) 26.48,
26.79, 27.63, 30.18, 30.50, and 33.33 (pyro-Glu C-3 and C-4,
and hexyl C-2 to C-5), 40.43 (hexyl C-6), 58.26 (pyro-Glu H-2),
2-O-a cetyl-1-â-[6-([3R, 8S]-1,4-d ia za -3-m eth yl-2,5-d ioxo-
cyclooct a n -8-yl]-ca r b on yla m in o]h e xyl-D-m a n n op yr a -
n ose, 51: mp 136-139 °C; 1H NMR (CD3OD, 300 MHz) 1.30-
1.42 (4H, m, hexyl H-3 and H-4), 1.41 (3H, d, J 7.0 Hz,
cyclooctyl C(3)Me), 1.43-1.57 (4H, m, hexyl H-2 and H-5), 1.62
(3H, s, OAc), 2.04-2.13 (2H, m, cyclooctyl H-7), 2.22-2.33 (2H,
m, cyclooctyl H-6), 3.15 (2H, t, J 7.5 Hz, hexyl H-6), 3.18-
3.25 (1H, m, mannose H-5), 3.51 (1H, A of ABX2, J 9.0, 6.0
Hz, 1H of hexyl H-1), 3.55 (1H, B of ABX2, J 9.0, 6.5 Hz, 1H
of hexyl H-1), 3.55 (sic) (1H, dd, J 9.5, 9.5 Hz, mannose H-4),
3.65 (1H, A of ABX, J 12.0, 6.0 Hz, 1H of mannose H-6), 3.69
(1H, dd, J 9.5, 4.0 Hz, mannose H-3), 3.84 (1H, B of ABX, J
12.0, 2.5 Hz, 1H of mannose H-6), 4.01 (1H, td, J 5.5, 1.0 Hz,
cyclooctyl H-8), 4.06 (1H, qd, J 7.0, 1.0 Hz, cyclooctyl H-3),
4.43 (1H, dd, J 4.0, 2.5 Hz, mannose H-2), 5.42 (1H, d, J 2.5
Hz, mannose H-1); 13C NMR (CD3OD, 75.5 MHz) 19.21
(cyclooctyl C(3)Me), 25.85 (MeCO), 26.87, 27.60, 30.30, 30.42,
30.49, and 32.16 (cyclooctyl C-6 and C-7 and hexyl C-2, C-3,
C-4, and C-5), 40.42 (hexyl H-6), 51.42 and 55.78 (cyclooctyl
C-3 and C-8), 62.84 (hexyl C-1), 68.57, 73.34, 76.95, and 81.22
(mannose C-2, C-3, C-4, and C-5), 99.11 (mannose C-1), 124.29
(mannose C-6), 174.23, 174.58, 178.87, and 178.95 (MeCO,
CONHCH2, cyclooctyl C-2 and C-5); HRMS (FAB) m/z
526.240 500, calculated for MNa+ (C22H37N3NaO10+) 526.237 665;
HPLC (Hamilton; 0-40% (50 min) B in C, pH 8.5), 19.5 min,
89%.
62.80 (hexyl C-1), 174.87 and 181.88 (2 × CONH); HRMS
(FAB) 229.154 00, calculated for [MH+] (C11H21N2O3
)
+
229.155 21; HPLC (Hamilton; 10-40% (40 min) A in C, pH
6.0), 15.7 min, 100%.
6-((S)-Ala n yl-(S)-p r olyla m in o)h exa n -1-ol, 56: H NMR
1
(CD3OD, 400 MHz) 1.27 (3H, d, J 7.0 Hz, Ala H-3), 1.32-1.44
(4H, m, hexyl H-3 and H-4), 1.47-1.59 (4H, m, hexyl H-2 and
H-5), 1.86-2.01 (2H, m, Pro H-4), 2.02-2.22 (2H, m, Pro H-3),
3.10-3.26 (2H, m, hexyl H-6), 3.54 (2H, t, J 6.5 Hz, hexyl H-1),
3.60-3.75 (2H, m, Pro H-5), 4.37 (1H, dd, J 8.0, 4.5 Hz, Pro
H-2); 13C NMR (CD3OD, 75.5 MHz) 20.30 (Ala C-3), 25.89,
26.52, 27.63, 30.31, 30.65, and 33.51 (Pro C-3 and C-4, + hexyl
C-2 to C-5), 40.24 (hexyl C-6), 48.26 (Pro C-5), 61.42 and 61.67
(Ala C-2 and Pro C-2), 62.84 (hexyl C-1), 174.55 and 176.48
(Ala C-1 and Pro C-1); HRMS (FAB) 286.214 30, calculated
for [MH+] (C14H28N3O3+) 286.213 07; HPLC (Hamilton; 0-40%
(50 min) B in D, pH 7.8), 21.9 min, 97%.
6-((S)-Isoleu cyl-(S)-pr olylam in o)h exan -1-ol, 57: 1H NMR
(CD3OD, 300 MHz) 0.93 (3H, t, J 7.5 Hz, Ile H-5), 1.00 (3H, d,
J 7.0 Hz, Ile H-4′), 1.10-1.21 (1H, m, 1H of Ile H-4), 1.32-
1.40 (4H, m, hexyl H-3 and H-4), 1.48-1.57 (4H, m, hexyl H-2
and H-5), 1.58-1.66 (1H, m, 1H of Ile H-4), 1.67-1.75 (1H,
m, Ile H-3), 1.85-1.99 (2H, m, Pro H-4), 2.02-2.12 and 2.13-
2.20 (2H, 2 × m, Pro H-3), 3.14 (1H, A of ABX2, J 13.5, 7.0
Hz, 1H of hexyl H-6), 3.19 (1H, B of ABX2, J 13.5, 7.0 Hz, 1H
of hexyl H-6), 3.52 (1H, d, J 6.0 Hz, Ile H-2), 3.54 (2H, t, J 6.5
Hz, hexyl H-1), 3.62 (1H, A of ABX2, J 10.0, 6.5 Hz, 1H of Pro
H-5), 3.71 (1H, B of ABX2, J 10.0, 6.5 Hz, 1H of Pro H-5), 4.38
(1H, dd, J 8.0, 3.0 Hz, Pro H-2); 13C NMR (CD3OD, 75.5 MHz)
11.61 (Ile C-5), 16.04 (Ile C-4′), 24.78 (Ile C-4), 25.98 and 26.57
(hexyl C-3 and C-4), 27.68 (Pro C-4), 30.38 and 30.59 (hexyl
C-2 and C-5), 33.52 (Pro C-3), 40.02 and 40.29 (hexyl C-6 and
Ile C-3), 48.80 (Pro C-4), 58.30 (Ile C-2), 61.71 (Pro C-2), 62.89
6-((S)-Ala n yl-(S)-glu ta m in yla m in o)h exa n -1-ol, 52: mp
1
104-106 °C; H NMR (CD3OD, 300 MHz) 1.28 (3H, d, J 7.0
Hz, Ala H-3), 1.30-1.42 (4H, m, hexyl H-3 and H-4), 1.45-
1.59 (4H, m, hexyl H-2 and H-5), 1.85-1.97 and 2.01-2.12 (2H,
2 × m, Gln H-3), 2.25-2.32 (2H, m, Gln H-4), 3.18 (2H, t, J
7.0 Hz, hexyl H-6), 3.46 (1H, q, J 7.0 Hz, Ala H-2), 3.53 (2H,
t, J 6.5 Hz, hexyl H-1), 4.31 (1H, dd, J 9.0, 5.0 Hz, Gln H-2);
13C NMR (CD3OD, 75.5 MHz) 21.26 (Ala C-3), 26.59, 27.74,
29.31, 30.33, 32.50, and 33.53 (Gln C-3 and C-4 and hexyl C-2,
C-3, C-4, and C-5), 40.36 (hexyl H-6), 51.50 and 54.06 (Ala C-2
and Gln C-2), 62.84 (hexyl H-1), 173.51 (Gln C-5), 177.70 and
177.97 (Ala C-1 and Glu C-1); HRMS (FAB) m/z 317.217 90,
calculated for MH+ (C14H29N4O4+) 317.218 87; HPLC (Hamil-
ton; 0-40% (50 min) B in C, pH 8.5), 14.9 min, 96%.
(hexyl C-1), 174.51 and 175.70 (Ile C-1 and Pro C-1); HRMS
+
(FAB) 328.258 80, calculated for [MH+] (C17H34N3O3
)
328.260 01; HPLC (Hamilton; 30-70% (50 min) A in D, pH
7.9), 31.7 min, 99%.
Biology. NK cell a ctivity Assa y: Splenocyte cell suspen-
sions were prepared by homogenization of spleens from 6-8-
week old female C57BL/6 (H-2b) in tissue culture medium
[RPMI 1640 (Gibco, Burlington, Canada) supplemented with
10% fetal calf serum (Hyclone, Logan, USA) and 2 mM
L-glutamine]. The homogenate was centrifuged on a density
gradient (Lympholyte M, Cedarlane, Hornby, Canada) (800g,
20 min), and the mononuclear leukocytes were collected,
washed three times in phosphate buffer saline (PBS), and
resuspended in RPMI for evaluation of cell viability by the use
of trypan blue. The assay was performed as follows:
NK cell activity was assayed by incubation of 5 × 105
splenocytes for 4 h with 5 × 103 sodium chromate labeled
(51CrO4; Amersham, Oakville, Canada) NK-sensitive YAC-1
cells. After the incubation, chromium in the lysate was
quantified and the percent specific release or lysis calculated
using the expression
6-((S)-Ala n yl-(R)-glu ta m in yla m in o)h exa n -1-ol, 53: mp
84-87 °C; 1H NMR (CD3OD, 300 MHz) 1.31 (3H, d, J 7.0 Hz,
Ala H-3), 1.34-1.40 (4H, m, hexyl H-3 and H-4), 1.48-1.54
(4H, m, hexyl H-2 and H-5), 1.87-1.96 and 2.03-2.12 (2H, 2
× m, Gln H-3), 2.28 (2H, t, J 8.0 Hz, Gln H-4), 3.18 (2H, t, J
7.0 Hz, hexyl H-6), 3.51-3.57 (1H, m, Ala H-2), 3.53 (2H, t, J
6.5 Hz, hexyl H-1), 4.30 (1H, dd, J 9.0, 5.0 Hz, Gln H-2); 13C
NMR (CD3OD, 75.5 MHz) 20.76 (Ala C-3), 25.54 and 27.69
(hexyl C-3 and C-4), 29.13, 30.29, 32.50, and 33.48 (Gln C-3
and C-4 and hexyl C-2 and C-5), 40.38 (hexyl C-6), 51.34 and
54.30 (Ala C-2 and Gln C-2), 62.86 (hexyl C-1), 173.79 (Gln
C-5), 177.68 and 177.87 (Ala C-1 and Gln C-1); HRMS (FAB)
m/z 317.217 50, calculated for MH+ (C14H29N4O4+) 317.218 87;
HPLC (Hamilton; 0-50% (60 min) A in D, pH 7.9), 29.8 min,
99%.
6-((S)-Glu ta m in yla m in o)h exa n -1-ol, 54: mp 89-92 °C;
1H NMR (CD3OD, 300 MHz) 1.30-1.43 (4H, m, hexyl H-3 and
H-4), 1.46-1.58 (4H, m, hexyl H-2 and H-5), 1.73-1.83 and
1.85-2.01 (2H, 2 × m, Gln H-3), 2.27 (2H, dd, J 8.0, 7.5 Hz,
Gln H-4), 3.13-3.24 (2H, m, hexyl H-6), 3.27-3.35 (1H, m,
Gln H-2), 3.54 (2H, t, J 6.5 Hz, hexyl H-1); 13C NMR (CD3OD,
75.5 MHz) 26.60, 27.79, 30.37, 32.39, 32.65, and 33.51 (Gln
(ER - SR)
(TR - SR)
% specific lysis )
× 100
where ER ) experimental release of 51Cr, ET ) total release
of 51Cr, and SR ) spontaneous release of 51Cr.

1926 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Abbott et al.
(5) Yokoyama, W. M. Natural Killer Cell Receptors. Curr. Opin.
Immunol. 1995, 7, 110-120.
Due to variability between individual experiments, data is
compared to the positive control; human interleukin 2 (IL 2,
15 ng/mL, R and D Systems, Minneapolis, MN) and presented
in a semiquantitative (+) format. Therefore, the percent
specific lysis induced by IL 2 in each experiment is taken as
100% or the maximum stimulation. As such, 0-20% activa-
tion of NK cells relative to IL 2 is assigned 0; 21-40%, +; 41-
60%, ++; 61-80%, +++; 81-100%, ++++; and 100% and
more, +++++.
Im m u n op h en otyp in g Assa y. Female, 6-8-week old,
C57BL/6 mice were injected intraperitoneally for four consecu-
tive days with BCH-2537 at different concentrations. Mice
were sacrificed on day five by cardiac puncture. Gross
pathological observations were recorded at the end of the
experiment. Blood and spleens were collected and the cell
suspension was prepared and lysed in ACK buffer (155 mM
NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH 7.3) for 5 min.
The cells were washed three times in phosphate buffered
saline, pH 7.4 (PBS), and resuspended in tissue culture
medium. The cells were then incubated for 45 min on ice with
fluorescein (FITC) or phycoerythrin (PE) conjugated anti-cell
surface marker monoclonal antibodies according to the manu-
facturer’s (Gibco/BRL, Cedarlane, Boehringer Mannheim)
recommendation. The cells were then washed in PBS, fixed
with 1% paraformaldehyde, and analyzed with a Coulter XL
flow cytometer. Analysis of the cell subsets was undertaken
by determination of standard cells surface markers which were
as follows; CD3 (T-cells), TCR (T-cell receptor), CD4 (T helper),
CD8 (T cytotoxic/suppressor), CD45 (tyrosine phosphatase;
activation marker), CD11b (macrophage), NK (NK cells), and
Ly5 (B-cells).
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S. C.; Burr, L. E.; Chourmouzis, E.; Come, J .; Goodman, J . H.;
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mL. No degradation was observed over a 24 h period.
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An titu m or Exp er im en t. Female 6-8-week old C57BL/6
mice were injected intravenously on day zero with 2.5 × 105
B16F10 melanoma cells from DCTDC Tumor Repository, NCI,
Frederick, MD (source of cell culture, Dr. I. J . Fidler). Animals
were then injected intraperitoneally with BCH-2537 according
to the treatment schedule described in Table 8.
Sta tistica l An a lysis. The immunophenotyping data are
presented as ( standard error. The means are compared using
an unpaired student’s t test. The differences are considered
significant when P e 0.05.
Ack n ow led gm en t. We gratefully acknowledge the
excellent technical assistance of Sylvie Taillon. We
appreciate the thoughtful reading of the manuscript by
Dr. T. Bowlin.
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