Synthesis and activity of 6-substituted purine linker amino acid immunostimulants

Article abstract of DOI:10.1021/jm960844m

A series of 6-substituted purinyl alkoxycarbonyl amino acids were synthesized and evaluated for their ability to stimulate cytotoxic T lymphocytes (CTLs) and the mixed lymphocyte reaction (MLR). A few of these compounds, in particular [[5-[6-(N,V-dimethylamino)purin-9- yl]pentoxy]carbonyl]D-arginine (BCH-1393, 4a), displayed an in vitro stimulation of CTLs comparable to interleukin 2 (IL 2). BCH-1393 increased the CTL response between 10^-9 M and 10^-5 M. Further, this potent in vitroactivity was reflected as a significant increase in CTL cell number in vivo. However, immunophenotyping of some of the other equipotent compounds did not reveal a parallel relative increase in CTLs in vivo. It was difficult to formulate a rigorous structure-activity relationship based on in vitro CTL activity. Nevertheless, the activity was dependent upon the nature of the 6- substituent on the purine, the type and stereochemistry of the amino acid, and the distance and spatial freedom between the purine and amino acid as defined by the length and rigidity of the linker. These compounds were generally nontoxic, as exemplified by BCH-1393. BCH-1393 is a promising immunostimulant which may be targeted for those disease states which require an increased CTL or TH1 type response.

Full text of DOI:10.1021/jm960844m

J . Med. Chem. 1997, 40, 2883-2894
2883
Syn th esis a n d Activity of 6-Su bstitu ted P u r in e Lin k er Am in o Acid
Im m u n ostim u la n ts†
Boulos Zacharie, Lyne Gagnon, Giorgio Attardo, Timothy P. Connolly, Yves St-Denis, and
Christopher L. Penney*
Department of Medicinal Chemistry, BioChem Therapeutic Inc., 275 Armand-Frappier Boulevard, Laval, Quebec, H7V 4A7
Received December 12, 1996X
A series of 6-substituted purinyl alkoxycarbonyl amino acids were synthesized and evaluated
for their ability to stimulate cytotoxic T lymphocytes (CTLs) and the mixed lymphocyte reaction
(MLR). A few of these compounds, in particular [[5-[6-(N,N-dimethylamino)purin-9-yl]pentoxy]-
carbonyl]D-arginine (BCH-1393, 4a ), displayed an in vitro stimulation of CTLs comparable to
interleukin 2 (IL 2). BCH-1393 increased the CTL response between 10-9 M and 10-5 M.
Further, this potent in vitro activity was reflected as a significant increase in CTL cell number
in vivo. However, immunophenotyping of some of the other equipotent compounds did not
reveal a parallel relative increase in CTLs in vivo. It was difficult to formulate a rigorous
structure-activity relationship based on in vitro CTL activity. Nevertheless, the activity was
dependent upon the nature of the 6-substituent on the purine, the type and stereochemistry of
the amino acid, and the distance and spatial freedom between the purine and amino acid as
defined by the length and rigidity of the linker. These compounds were generally nontoxic, as
exemplified by BCH-1393. BCH-1393 is a promising immunostimulant which may be targeted
for those disease states which require an increased CTL or TH1 type response.
In tr od u ction
isoprinosine, has weak immunostimulant activity in
vivo and is licensed in many countries as an antiviral
agent.5 As such, isoprinosine has served as an impor-
tant standard for the hypoxanthine class of thymomi-
metic immunostimulants. Indeed, numerous hypoxan-
thine derivatives have been described such as NPT
15392, NPT 16416, and more recently methyl inosine
monophosphate (MIMP).6 Although these compounds
are nontoxic, they are generally not very active, espe-
cially as regards the ability to stimulate CTLs.
Pharmaceutically induced stimulation of the immune
system offers an important approach to the control of
disease. In addition to the toxicity associated with the
use of chemotherapeutics, there is increasing concern
regarding the development of resistance to antibacterial,
antiviral, and antitumor agents. Yet, research into this
important area of immunology has lagged behind the
more traditional pharmaceutical sector. Few well de-
fined synthetic immunostimulants have been approved
for use in North America. One example is the substi-
tuted thiazole, levamisole, which was approved for
Duke’s class C colorectal cancer in combination with
5-fluorouracil. Levamisole has the ability to act prima-
rily on the T-cell lineage in a manner similar to thymic
hormones and so is classified as a “thymomimetic”
immunostimulant.1 More recently, another T-cell stimu-
lant, tucaresol, has been described2 and is now in clinical
trials.
Among the classes of molecules which can nonspe-
cifically stimulate the immune system, nucleosides have
displayed promising activity. In particular, a group of
substituted guanosines, exemplified by loxoribine, cause
significant B and NK-cell stimulation.3 This is prima-
rily attributed to the ability of these compounds to
induce interferon. However, another group of nucleo-
sides with important thymomimetic properties comprise
analogues of inosine or the base portion, hypoxanthine.
The immunological importance of inosine is demon-
strated by the severe immunodeficiency which occurs
in the congenital absence of adenosine deaminase.4
While inosine may be expected to function as an
immunostimulant in vivo, the high activity of purine
nucleoside phosphorylase in lymphoid cells rapidly
metabolizes it. A slow-release formulation of inosine,
In an effort to enhance the immunostimulant activity
of hypoxanthine derivatives, a compound was synthe-
sized which contained hypoxanthine and the amino acid
L-arginine connected by a pentamethylene linker. L-
Arginine was selected because it plays a role in immune
activation.7 The biology of this immunostimulant, hy-
poxanthine pentoxycarbonyl L-arginine (ST 789, 1b),7
has been thoroughly described, but the data reported
does not indicate that it is significantly superior.
Interestingly, a number of analogues have been reported
in the patent and scientific literature in which the
L-arginine portion of ST 789 has been replaced with
small peptides. For example, introduction of the bomb-
esin carboxy terminal dipeptide, leucyl methionine, gave
a compound which was reported to modulate the pro-
liferation of hematopoietic cells.8 However, little has
been stated as regards replacement of hypoxanthine
with other purine moieties. One report9 cites work with
other naturally occurring bases (adenine, guanine), but
no details were provided regarding immunological activ-
ity. In summary, the literature suggests that thymo-
mimetic activity arises from the presence of hypoxan-
thine, and any improved or altered immunological
activity requires the presence of L-arginine or naturally
occurring peptide sequences covalently linked to hypo-
xanthine.
† Part of this work was presented at the 87th Annual Meeting of
the American Association for Cancer Research, Washington D.C., April
20-24, 1996.
In this paper, we report on the immunological activity
of compounds which possess a structural motif resem-
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0022-2623(96)00844-8 CCC: $14.00 © 1997 American Chemical Society

2884 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Zacharie et al.
Sch em e 1a
dioxolane methanol 22b followed by selective hydrolysis
of the silylated acetonide 22c with trifluoroacetic acid
(TFA) afforded the monoprotected triol 22d . Treatment
of this compound or thiol 23c with (benzoyloxy)acetal-
dehyde gave benzoate 22e and 23d , respectively. Al-
kaline hydrolysis gave the corresponding monoprotected
alcohol 22f or 23e. The Mitsunobu approach was also
successfully applied to amino alcohols. In this case, the
amino function of compounds 16d and 32a was pro-
tected as the carbobenzyloxy (Cbz) derivative, and the
remaining diol of 16e was monoprotected as a TBDPS
ether using the procedure described above.
The Mitsunobu reaction proceeded in moderate yield
(50-60%) and is general for different diols and amino
alcohols. In addition, the desired N-9 adduct was
formed in high yield relative to the N-7 adduct. How-
ever, this procedure is limited by the difficult removal
of the triphenylphosphine oxide byproduct, which makes
this approach awkward for large scale synthesis. There-
fore, it was decided to explore a more practical approach
for the preparation of chloropurine II. Recently, we
demonstrated that coupling between 4-substituted 1H-
pyrazolo[3,4-d]pyrimidine bases and protected or un-
protected iodoalkanols proceeded efficiently and in
reasonable yield.11 We therefore investigated the reac-
tion between 6-chloropurine and iodoalkanols. These
compounds were prepared by treatment of the corre-
sponding mesylate with sodium iodide in refluxing
acetone.11 Other routes were also examined for the
preparation of iodoalkanols. For example, treatment of
protected diol with triphenylphosphine, imidazole, and
iodine12 gave the expected iodoalkanol in good yield.
Specific linkers of four or five carbon atoms length were
prepared by reaction of tetrahydrofuran or tetrahydro-
pyran with iodotrimethylsilane (TMSI) to give ring
opening of the cyclic ether and the corresponding silyl
protected iodoalkanol.13a-c This latter approach is
practical and amenable to scale-up. Therefore, prepara-
tion of purine derivative II was achieved in a single step
by preparation of iodoalkanol in situ, followed by reac-
tion with chloropurine and cesium carbonate (Scheme
3). Similar results were obtained with protected io-
doalkylamine. For example, treatment of protected
iodoamine 32c with 6-chloropurine and cesium carbon-
ate in DMF at 0 °C afforded 32d in high yield.
a
X ) acyclic or cyclic alkyl, or aryl group, with or without
heteroatoms; Z ) amino acid, peptide, or second alkylpurine unit.
For BCH 1393, X ) (CH2)3, Z ) D-ARG.
bling ST 789: arginine covalently attached to a purine
via a pentamethylene linker. However, these com-
pounds are significantly different from ST 789. The
purine is not hypoxanthine or another naturally occur-
ring base, and the amino acid is the unnatural D-
enantiomer. Further, immunological activity does not
require the presence of an amino acid, and the natural
L-enantiomer can be antagonistic.
Ch em istr y
Our first approach to the synthesis of substituted
purine compounds followed that described for ST 789
and analogues.9 That is, the purine ring was built from
5-amino-4,6-dichloropyrimidine while incorporating the
linker portion at the same time. However, this route
was long and the yield of product poor. Therefore, our
goal was to develop a simplified synthesis starting from
commercially available substituted purine. An ap-
proach was selected in which the linker was attached
to 6-chloropurine I by means of a Mitsunobu reaction
with monoprotected diol (Scheme 1).
The diols were protected by reaction of tert-butylchlo-
rodiphenylsilane (TBDPSCl) with acyclic or cyclic alkyl
or aralkyl diols, in the presence of base. These precur-
sor diols are commercially available or conveniently
prepared according to Scheme 2. For example, pro-
tected butynol 17e was treated with n-butyllithium and
methyl chloroformate to give ester 17f which was
reduced to the corresponding alcohol 17g. We also
developed a simple procedure to prepare ester 21e.
Ozonolysis of norbornylene in methanol10 gave aldehyde
21c which was reduced with sodium borohydride to
alcohol 21d . The latter was protected and converted
to the monoprotected alcohol 21f by reduction of ester
21e with DIBAL in dichloromethane. The preparation
of other cyclic diols, 1,3-dioxolane- and 1,3-oxathiolane-
2,5-dimethanol, was also undertaken. Protection of 1,3-
Removal of the silyl group of purine II was ac-
complished by treatment with fluoride, followed by
neutralization with dilute HCl or acetic acid, to give the
corresponding alcohol. These compounds were then
linked to amino acids, peptides, or a second purine
derivative by a carbamate or ester function. The latter
was prepared in good yield by activation of the acid with
carbodiimide, followed by treatment with the alcohol.
The carbamate group was introduced by carbonylation
of alcohol III followed by treatment of the resulting
chloroformate with different amines (Scheme 1).
A
number of procedures were examined for the prepara-
tion of the carbamates. These included 1,1′-carbonyl-
diimidazole and triphosgene, of which neither gave
satisfactory results. However, phosgene in toluene gave
the desired purine derivatives IV. In the case of amino
acids, the carboxylic function was converted in situ to
the sodium salt, thus requiring no protection. The free
amine of these salts was then treated with the chloro-
formate in aprotic solvents such as THF or DMF. The
reactions proceeded slowly and product yield was low.

6-Substituted Purine Linker Amino Acid Immunostimulants
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2885
Sch em e 2a
a
Key: (a) Benzyl chloroformate, K2CO3, CH3CN; (b) imidazole, TBDPSCl, CH2Cl2; (c) n-BuLi, methyl chloroformate, hexane, -78 °C;
(d) DIBAL-H, CH2Cl2, -78 °C; (e) Ozone, CH2Cl2, MeOH, -78 °C; (f) Ac2O, Et3N, CH2Cl2, 0 °C to rt; (g) NaBH4, MeOH, 0 °C; (h) CF3COOH,
THF/H2O; (i) BzOCH2CHO, PPTS, toluene; (j) MeONa/MeOH; (k) benzyl chloroformate, aq. Na2CO3; (l) MsCl, TEA, CH2Cl2; (m) NaI,
acetone; (n) 6-chloropurine, Cs2CO3, DMF.
Sch em e 3
This was due presumably to the lower solubility of the
anion in those solvents. Homogeneous reaction condi-
tions were examined in an attempt to improve product
yields. In a mixture of THF:H2O (1:1), the amine reacts
efficiently with the chloroformate to give compound IV
in high yield. This procedure is applicable to all purine
derivatives and has been employed to supply gram
quantities of BCH-1393. It offers the advantage of a
practical and general route for the preparation of a
variety of analogues.
In order to prepare N,N-dimethylpurine derivatives
III and IV, two approaches were considered (Scheme

2886 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Zacharie et al.
Ta ble 1. In Vitro Cytotoxic T-Lymphocyte Activity of
6-Substituted Purine Pentoxycarbonyl Arginine Relative to
Interleukin 2
1). The first route was the deprotection of the silyl
group of II followed by introduction of the carbamate
function to give IV. The second approach was the
conversion of the 6-chloropurine of II to N,N-dimethyl
group, followed by removal of the protecting group, to
afford alcohol III. The latter was converted to the final
product IV as described above. Both approaches gave
high yield of N,N-dimethyl derivative IV and were
appropriate for large-scale synthesis.
In Vitr o An a lysis: SAR Stu d ies
RG
RG
The two commonly used in vitro models for T-cell
mediated responses are the cytotoxic T lymphocyte
(CTL) reaction and the mixed lymphocyte reaction
(MLR). A unidirectional CTL/MLR assay was under-
taken by incubation of murine splenocytes, along with
irradiated allogeneic splenocytes, for five days in the
presence or absence of candidate immunostimulant.
Early in the program, a sample of ST 789, N-[(hypo-
xanthin-9-ylpentoxy)carbonyl]-L-arginine (1b), was syn-
thesized, along with the D-enantiomer (1a ), for evalu-
ation of in vitro CTL activity. The adenine (6-amino-
purine) analogues, with L- and D-arginine, were also
made, along with the (monomethylamino)- and (di-
methylamino)purine equivalents. Measurement of the
CTL activity induced by each of these eight compounds
revealed some interesting points, as shown in Table 1.
ST 789, which was predicated upon the importance of
L-arginine, possesses the same CTL activity as the
D-enantiomer. However, amongst the L-arginine com-
pounds, the adenine analogue 2b is the most active.
Indeed, in the case of the monomethylamino and di-
methylamino analogues 3b and 4b, L-arginine appears
to function as an antagonist. The most active compound
in the group is the (dimethylamino)purine analogue
BCH-1393 (4a ) which contains D-arginine. Clearly, the
substituent at the 6-position of the purine base, along
with the stereochemistry of arginine, determines the
extent of CTL activity. The base does not have to be
restricted to hypoxanthine. Other 6-position substitu-
ents were therefore examined, as shown in Table 1.
None yielded a compound with greater CTL activity
than BCH-1393. However, the hydrazino analogues 5a
and 5b possess good activity. Whereas the activity of
BCH-1393 is sensitive to arginine stereochemistry, the
activity of the hydrazino compounds is hardly influenced
by stereochemistry.
On the basis of the above results, compound 4a (BCH-
1393) was selected as a lead upon which to further
define a structure-activity relationship for 6-(N,N-
dimethylamino)purine-linker-amino acid. As shown
in Table 2, analogues were synthesized in which the
linker length and type were varied while maintaining
6-(dimethylamino)purine and arginine components. The
BCH-1393 analogue with a shorter (tetramethylene)
linker, 13a , retains potent CTL activity. However, the
potency is reduced with a longer (hexamethylene) linker,
as seen with 12a . The corresponding analogues with
L-arginine, 12b and 13b, demonstrate a consistent
trend. Regardless of the length of the linker, four to
six carbons, there is no CTL activity when L-arginine
is linked to 6-(N,N-dimethylamino)purine. Therefore,
a series of BCH-1393 analogues were synthesized with
five-atom linkers wherein the middle carbon is replaced
with a heteroatom. Replacement with oxygen, 14a , or
nitrogen, 16a , is well tolerated but activity declines with
introduction of a larger sulfur atom, 15a . CTL activity
is abrogated upon introduction of a more rigid (π
electron) five-carbon alkyne, 17a . Interestingly, no CTL
activity is observed when these five-atom linkers are
used to connect L-arginine with 6-(N,N-dimethylamino)-
purine, unless nitrogen is present in the linker, as seen
in 16b. Both 16a and 16b display good CTL activity.
It was next decided to introduce rigidity into the
linker to examine if a defined geometry or spatial
arrangement could improve the CTL activity of BCH-
1393. Replacement of three of the five carbons of the
linker in BCH-1393 with rigid five-membered rings, 21
to 23, abolished CTL activity (Table 3). However,
replacement with a six-membered cyclohexyl ring, 19
and 20, preserved some weak activity especially if the
purine base and arginine amino acid were in a trans-
relationship. Nonetheless, these initial results were not
encouraging, and so work was not pursued with L-
arginine analogues or other constrained linkers.
Attention was next given to the role of the amino acid
arginine. Therefore, analogues of BCH-1393 were made
in which D-arginine was replaced with glycine, 24;

6-Substituted Purine Linker Amino Acid Immunostimulants
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2887
Ta ble 2. In Vitro Cytotoxic T-Lymphocyte Activity of
Analogues of the Lead Compound 4a , BCH-1393, with Different
Length/Type Linkers
Ta ble 4. In Vitro Cytotoxic T-Lymphocyte Activity of
Analogues of the Lead Compound 4a , BCH-1393, with Different
Amino Acids/Peptides
a
b
CTL Activityb
(*) ) D-ARG
(*) ) L-ARG
Compounda
X
a
b
4
-CH2CH2CH2-
-CH2CH2CH2CH2-
-CH2CH2-
+ + + + (10-7 M)
+ + (10-7 M)
0
0
0
0
0
12
13
14
15
16
17
+ + (10-9 M)
-CH2OCH2-
+ + + (10-7 M)
+ + (10-7 M)
-CH2SCH2-
-CH2NHCH2-
-CtCCH2-
+ + + (10-9 M) + + + (10-9 M)
0
0
a,b
See Table 1 footnotes.
Ta ble 3. In Vitro Cytotoxic T-Lymphocyte Activity of
Analogues of the Lead Compound 4a , BCH-1393, with Different
Rigid Linkers
D
a
D-ornithine, 25; and D-citrulline, 26. Replacement of the
guanidino function of arginine with an amino, 25, or
urea function, 26, severely impaired CTL activity (Table
4). Complete removal of the D-amino acid side chain is
better tolerated, as is observed with the glycine com-
pound, 24. However, among these compounds, the best
CTL activity is seen when â-alanine was esterified to
the linker, compound 27. Although 27 is not as active
as BCH-1393, it possesses CTL activity which is similar
to the desarginine analogue of BCH-1393, compound 28.
Interestingly, the desarginine analogue of 1b (not shown
in Table 4) displayed no CTL activity. It was noted in
the introduction that small peptides have been linked
to hypoxanthine to provide corresponding analogues of
ST 789. Therefore, two analogues of BCH-1393, 29 and
30, were made wherein arginine was replaced with the
tripeptides diprotin A and B. These tripeptides inhibit
the enzyme dipeptidyl peptidase IV (CD 26).14 This
enzyme is present on the cell surface of T-lymphocytes.
However, 29 and 30 do not activate CTLs. Finally,
three analogues of BCH-1393, 31-33, were made
wherein arginine was replaced with a second unit of
6-(N,N-dimethylamino)purine. Essentially, these com-
pounds are dimers of desarginine BCH-1393, 28. How-
ever, one of these dimers, 31, has improved CTL
activity, compared to 28. Compound 32 has the same
activity, while 33, which is extended by a glycine linker

2888 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Zacharie et al.
F igu r e 2. Comparison of the in vitro cytotoxic T-lymphocyte
activity of BCH-1393 with tucaresol, MIMP, and FPA. Con-
centration is expressed as µM.
F igu r e 1. Dose response profile of the in vitro cytotoxic
T-lymphocyte activity of BCH-1393 in comparison to interleu-
kin-2.
Ta ble 5. Summary of Immunophenotyping Data for Mice
Administered One Dose of BCH-1393
0
25
50
100
cell subsets
mg/kg
mg/kg
mg/kg
mg/kg
connecting the two units, has no CTL activity. None-
theless, these dimers are not as active as BCH-1393.
All of the compounds assayed for CTL activity were
also examined for their ability to enhance MLR. The
majority of compounds had no effect, while a few
displayed a modest enhancement of the MLR; ap-
proximately 1.5 times at 10-7 M. The latter included
BCH-1393, analogues of BCH-1393 where the linear
linker contained a heteroatom, and dimers of desargi-
nine BCH-1393.
Blood Immunophenotyping BCH-1393a
CD4-CD8-
mean
STD
P value
mean
STD
P value
mean
STD
65.9
11.1
70.7
3.97
0.187
21.9
3.00
0.289
6.30
74.7
4.92
0.072
19.8
3.89
0.274
5.10
82.1
8.59
0.002
14.0
6.87
0.026
3.53
1.59
0.020
CD4+
CD8+
20.9
1.76
8.10
3.54
0.54
0.131
1.14
0.047
P value
Spleen Immunophenotyping BCH-1393
CD8+CD45+
mean
STD
P value
7.81
1.06
7.46
0.75
0.300
8.92
2.36
0.185
9.13
1.24
0.035
In Vitr o P r ofile of BCH-1393 (4a )
Since the structure-activity study did not reveal any
compounds with activity superior to BCH-1393, with
regard to the ability to stimulate CTLs, it was decided
to examine the reproducibility of this stimulation. This
is necessary in view of the variable nature of cell based
proliferation assays, variation in cell populations be-
tween experiments, and sensitivity to environmental
influences. Nonetheless, it was observed that as long
as the IL 2 positive control was able to stimulate a
significant (maximal) increase in specific cell lysis, BCH-
1393 displayed a robust CTL stimulation. The dose-
response profile for BCH-1393, as a mean of 13 experi-
ments, is shown in Figure 1. The stimulation of CTLs
achieved with 10-7 M BCH-1393 is comparable to IL 2.
It was also decided to compare the CTL stimulation
induced by BCH-1393 with the aromatic aldehyde
tucaresol, MIMP, and formylphenoxyacetic acid. T-cell
stimulation was reported to be a general property of
aromatic aldehydes,2 and the latter compound is a
metabolite of the tuberculosis drug aconiazide.15 As can
be seen in Figure 2, only BCH-1393 at 10-7 M induces
a significant stimulation of CTLs (P < 0.001, n ) 5)
relative to the negative control.
a
Groups of seven C57BL/6 mice were injected ip for one day
with BCH-1393.
of BCH-1393, to determine the increase or decrease of
immune cell subset populations, relative to each other.
Mice were injected intraperitoneally for one, four, or
nine consecutive days with BCH-1393 at different
concentrations. At the end of the experiment, blood and
spleens were collected for determination by flow cytom-
etry of the effect of BCH-1393 on the immune cell
subsets within these tissues. The results of the analysis
are presented in Tables 5-7.
In blood, one dose of the highest concentration of
BCH-1393, 100 mg/kg, significantly increases the rela-
tive percentage of non-B- non-T-cells (30%). While this
subset was not identified, the probable candidate is a
neutrophil population, based on relative occurrence.
However, four doses (50 mg/kg) of BCH-1393 shifted the
cell subset distribution so that significant (P e 0.041)
increases (23% to 70%) occur in the CTL (CD8+
CD45+), NK (CD3(), and monocytes (CD11b+) popula-
tions. Nine doses (50 mg/kg) of BCH-1393 significantly
(P e 0.004) increases (26%) T-helper (CD4+ CD45+)
cells. In spleen, one dose of the highest concentration
of BCH-1393 induces a significant (P e 0.035) increase
(17%) in CTLs. This correlates with the observed
increases in the blood upon administration of multiple
doses of BCH-1393. Although not apparent after four
doses, it is noteworthy that after nine doses of BCH-
1393, the significant (P e 0.035) increase (44%) in CTLs
persists. There is no indication of paradoxical effects16
or immunoparalysis. Gross pathological examination
of the mice at the end of the experiment revealed no
abnormalities, including splenomegaly.
In addition to stimulation of CTL activity and a
modest effect on the mixed lymphocyte reaction, BCH-
1393 displays weak T-cell proliferative activity (data not
shown) in the presence of concanavalin A mitogen.
However, BCH-1393 alone does not stimulate NK and
B-cell populations (data not shown).
In Vivo P r ofile of BCH-1393 (4a )
In view of the significant in vitro CTL activity of BCH-
1393, it was of interest to determine if this activity
would translate into an in vivo effect. Therefore,
immunophenotyping experiments were undertaken with
normal immune status mice, in the presence or absence
Although none of the analogues of BCH-1393 possess

6-Substituted Purine Linker Amino Acid Immunostimulants
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2889
Ta ble 6. Summary of Immunophenotyping Data for Mice
Administered Four Doses of BCH-1393
compound and a smaller sample size (n ) 4; data not
shown). Interestingly, the results obtained with nine
doses of BCH-1393 paralleled those observed with four
doses of desarginine BCH-1393 (28). That is, in blood
there is a significant increase in T-helper cells, while
in spleen there is a significant increase in CTLs.
However, a significant decrease in blood monocytes was
observed which was not seen at all with BCH-1393.
Throughout these studies, it was observed that BCH-
1393 and most of the analogues were well tolerated and
devoid of any apparent toxicity. As such, a preliminary
chronic toxicity study was undertaken in rats whereby
they were administered BCH-1393 by intravenous
injection for five consecutive days. Lethality occurs at
1 g/kg, but BCH-1393 is well tolerated at doses up to
500 mg/kg.
0
25
50
cell subsets
mg/kg
mg/kg
mg/kg
Blood Immunophenotyping BCH-1393a
CD8+CD45+
mean
STD
P value
mean
STD
P Value
mean
STD
P value
mean
STD
8.25
1.59
11.7
5.54
0.108
5.90
1.39
0.500
5.84
2.08
0.021
13.7
2.68
10.1
2.16
0.041
8.14
1.35
0.005
3.25
0.57
0.289
9.68
3.59
0.404
NK+CD3-
6.01
0.98
NK+CD3+
3.43
0.76
CD4-CD11b+
9.60
2.79
P value
0.015
Spleen Immunophenotyping BCH-1393
TCR
Ly5
mean
STD
P value
mean
STD
38.9
3.83
39.8
7.61
0.421
54.1
7.30
0.367
45.1
7.34
0.035
50.4
6.72
0.034
Discu ssion
In this study, the in vitro CTL activity of more than
50 6-substituted purine-linker amino acid hybrids and
related structures was determined and compared to
known T-cell stimulants. On the basis of these results,
a lead molecule, BCH-1393 (4a ), was selected for in vivo
immunological evaluation. While it was difficult to
formulate a rigorous structure-activity relationship as
pertains to CTL activity, a few trends emerged which
are as follows:
55.5
3.44
P value
a
Groups of ten C57BL/6 mice were injected ip for four consecu-
tive days with BCH-1393.
Ta ble 7. Summary of Immunophenotyping Data for Mice
Administered Nine Doses of BCH-1393
0
25
50
100
cell subsets
mg/kg
mg/kg
mg/kg
mg/kg
(1) Activity is dependent upon the nature of the
substituent at the 6-position of the purine ring. While
no substituent, compound 8a , results in minimal activ-
ity, the 6-chloro compounds, 6a and 6b, are inactive.
Indeed, the nature of the 6-substituent appears to
determine if arginine will improve, maintain, or abolish
immunological activity.
Blood Immunophenotyping BCH-1393a
CD4+CD45+
mean
STD
P value
17.5
3.26
18.5
2.01
0.347
22.1
2.01
0.004
20.6
8.85
0.228
Spleen Immunophenotyping BCH-1393
CD8+CD45+
mean
STD
P value
6.90
1.36
8.39
1.91
0.061
9.06
2.82
0.066
9.96
2.28
0.035
(2) Activity is dependent upon the type and stereo-
chemistry of the amino acid covalently linked to the
purine base. D-Arginine is the most effective amino
acid, as exemplified by compounds 4a , 5a , 13a , 14a , and
16a . L-Arginine can be as effective as the respective
D-enantiomer, for example compounds 1b, 2b, 5b and
16b, but more often it abrogates CTL activity. Other
amino acid substituents such as glycine or isosteric
citrulline result in reduced activity and the absence of
an amino acid can result in the same or better relative
activity. This is illustrated by comparison of compounds
24, 25, and 26 with 28 or desarginine BCH-1393.
(3) Activity is dependent upon the distance and
relative degrees of freedom between the purine and
amino acid as defined by the length and rigidity of the
linker. A distance of four to five (carbon) atoms appears
to represent an optimum, as illustrated by compounds
4a and 13a . Introduction of rigidity, for example
compounds 18-23, significantly diminishes activity
relative to the acyclic linker in 4a or BCH-1393.
Activity was not improved, relative to BCH-1393, by
linking two 6-N,N-dimethylaminopurine units together
as shown by compounds 31, 32, and 33. It is interesting
to observe that activity decreases as the distance
between the two purine units increases which suggests
a dimeric binding site or receptor. This somewhat
parallels the relationship noted above between activity
and distance between 6-N,N-dimethylaminopurine and
D-arginine. However, this may be coincidence and the
possibility cannot be ignored that 31 is, for example, a
prodrug form of 28 or desarginine BCH-1393.
a
Groups of seven C57BL/6 mice were injected ip for nine
consecutive days with BCH-1393.
superior in vitro CTL activity, six of them (5a , 5b, 13a ,
14a , 16a , and 16b) exhibited similar activity. There-
fore, two of these compounds, 5a and 16a , were selected
for preliminary immunophenotyping to determine if
their in vivo activity was similar to BCH-1393. This
work was undertaken with four consecutive doses of
compound and a smaller sample size (n ) 4; data not
shown). Nonetheless, the results clearly demonstrate
that unlike BCH-1393, in vitro CTL activity does not
translate in vivo into a corresponding increase in CTLs.
In blood, 5a induces a significant increase in non-T-cells
(CD4- CD8-) relative to all other immune cell popula-
tions. No significant increases are observed in immune
cell subsets in the spleen. In blood, 16a also induces a
significant increase in non-T-cells (CD4- CD8-) relative
to all other immune cell populations except monocytes.
This significant increase in non-T-cells is also seen in
the spleen, along with a significant increase in mac-
rophages.
In view of the unique ability of BCH-1393 to increase
T-cell populations after four and nine doses, it was of
interest to determine if the desarginine analogue 28
could induce a similar effect. Compound 28 displays
good in vitro CTL activity, although less than BCH-
1393. However, it is reasonable to expect that 28
represents a metabolite of BCH-1393, and so the ques-
tion of in vivo activity becomes more important. There-
fore, another preliminary immunophenotyping experi-
ment was undertaken with four consecutive doses of
The impetus for the design of BCH-1393 (4a ) came
from the compound ST 789; hypoxanthine pentoxycar-

2890 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Zacharie et al.
bonyl L-arginine (1b). Therefore, a sample of ST 789
was made early in the program and evaluated for in
vitro CTL activity. The literature7,17 indicates that ST
789 is able to enhance NK-cell and phagocytic popula-
tions and production of IL 6. However, there is no
report of CTL activation or induction of IL 2. Therefore,
the observation of in vitro CTL activity in our assay was
unexpected. This was not pursued further since ST 789
is not under development as a T-cell stimulant. Instead,
the CTL activity of BCH-1393 was compared with the
two well documented T-cell stimulants, Tucaresol and
MIMP.6 While the results in Figure 2 indicate that only
BCH-1393 has significant CTL activity, both Tucaresol
and MIMP did display greater activity than the control.
Furthermore, this is murine data which does not neces-
sarily reflect the results that would be obtained with
human CTLs. Nonetheless, these results, along with
the extensive in vitro analysis of BCH-1393, as exempli-
fied in Figure 1, justified an in vivo evaluation of BCH-
1393.
of CD26 is therefore uncertain with regard to the
mechanism by which BCH-1393 stimulates CTLs. How-
ever, an important role for BCH-1393 which merits
further investigation may be the potential to influence
or alter T-cell apoptosis, in particular the survival of
CTLs. Recently, it was reported that tumor necrosis
factor (TNF) mediates the death of most CD8+ T-cells
(CTLs) while Fas ligand (FasL) mediates the death of
most CD4+ T-cells (T-helper).21 Further, CD26 influ-
ences the expression of the receptor for FasL, CD95
(Fas/Apo-1).19 The effect of BCH-1393 on the in vivo
expansion of T-helper cells could be accounted for by
its effect on the activity of CD26. More importantly,
BCH-1393 may inhibit the production of TNF and
thereby permit an in vivo expansion of CTLs. This
inhibition may in turn result from interaction with
adenosine deaminase (adenosine sparing effect) or the
adenosine receptor. Adenosine has been reported to
inhibit the production of TNF.22 With regard to the
structure of BCH-1393, interaction with adenosine
deaminase appears more likely and would account for
the expansion of both T-cell populations. Regardless of
the mechanism by which BCH-1393 stimulates CTLs
and subsequently expands T-cell populations, the effect
on CTLs offers significant potential for the treatment
of those diseases, cancer, viral and parasitic infections,
where CTLs play an important role.
Immunophenotyping experiments with BCH-1393
revealed a significant increase in the spleen and blood
CTL populations. However, this increase was ac-
companied by significant increases in other immune cell
subsets, and BCH-1393 appeared less specific in vivo
relative to the in vitro profile. Nonetheless, it is difficult
to delineate which cell populations are enhanced directly
by BCH-1393 as opposed to indirect activation by means
of cellular communication. One dose of BCH-1393 did
not result in the immediate increase in CTLs in the
blood. However, the increase in non-B- non-T-cells
(neutrophils?) followed by T-cell, NK, and monocyte
populations with repeated doses of BCH-1393 suggests
a potential immunorestorative function. On the other
hand, the persistent increase in CTLs after nine con-
secutive doses of BCH-1393 without immunoparalysis
or tolerance suggests a potential role in chronic (e.g.:
cancer, virus) treatment regimens. Indeed, recent ef-
forts to enhance antitumor CTLs by means of thera-
peutic vaccination with a peptide representing a specific
CTL epitope resulted in tolerance and subsequent
increased tumor growth.18
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), with solvent systems (A) MeOH, (B)
CH3COONH4, 0.01M pH 6, (C) 20% EtOAc/toluene, (D) 5%
EtOAc/hexane, (E) 10% EtOAc/hexane, (F) 20% EtOAc/hexane,
(G) 25% EtOAc/hexane, (H) 30% EtOAc/hexane, (I) 50% EtOAc/
hexane, (J ) EtOAc, (K) 10% MeOH/EtOAc, (L) 15% MeOH/
EtOAc, (M) 20% MeOH/EtOAc, (N) 30% MeOH/EtOAc, (O)
40% MeOH/EtOAc, (P) 80% MeOH/EtOAc, (Q) BuOH/AcOH/
H2O, 5:1:2, (R) 10% NH4OH/MeOH, (S) CH3CN, 0.01% TFA,
(T) H2O, 0.01% TFA pH 2.9. NMR spectra were recorded on
a Bruker DRX-400 or a Varian VXR-300 spectrometer. Chemi-
cal shifts (δ) are expressed in ppm. Mass spectra were
recorded on a Kratos MS-50 TA instrument. Analytical HPLC
results were obtained using a Waters instrument with a YMC
C-4 (5 µm) 120 Å reverse phase column with a gradient of 20%
to 50% (A)/(B), flow rate of 1 mL/min, or with a YMC ODS-A
(5 µm) 120 Å reverse phase column with a graident of 0% to
25% (S)/(T), flow rate of 0.5 mL/min, and using a UV detector
at an absorption of 256 nm. Tucaresol and MIMP were
synthesized according to literature procedures.23,24 All non-
commercial 6-substituted purines were prepared by standard
procedures.25 All amino acids 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. (Montreal, Canada).
All nonaqueous reactions were carried out under an inert
atmosphere. Standard workup consisted of pouring the reac-
tion solution into the indicated solvent system (1:1 mixture)
followed by extraction with CH2Cl2 (2 ×). The organic phase
was dried over MgSO4, solids were filtered, and the solvent
was removed in vacuo.
The specificity of BCH-1393 observed in vitro for
stimulation of CTLs and the corresponding lack of
stimulation of T-cell proliferation is unusual and does
not adhere to a strict classification of BCH-1393 as a
thymomimetic drug. It appears unlikely that BCH-1393
shares a mechanistic pathway of immunological activa-
tion in common with hypoxanthine-containing thymo-
mimetics. For example, unlike BCH-1393, MIMP in-
creased the mitogenic responses to concanavalin A and
phytohemagglutin.6 Indeed, whereas hypoxanthine com-
pounds may interact with an inosine receptor,4 BCH-
1393 may interact with an adenosine receptor. Perhaps
more likely is a transient inhibition by BCH-1393 of
adenosine deaminase or purine nucleoside phosphory-
lase, as has also been suggested for hypoxanthine
thymomimetics.4 Adenosine deaminase, present on the
cell surface of T-lymphocytes, is ligated to CD26.19 In
preliminary work,20 in addition to inducing a significant
stimulation of human CTLs, BCH-1393 was shown to
increase the specific enzyme activity of CD26 in IL
2/PHA-stimulated human blood T-cells (PBMLs). How-
ever, CD26 is preferentially restricted to T-helper
(memory) populations.19 This is contrary to the stimu-
lation of CTLs induced by BCH-1393. The importance
Gen er a l P r oced u r e for th e Syn th esis of Mon op r o-
tected Diols. Compounds 4d , 12d , 13d , 14d , 15d , 16f, 18d ,
20b, and 21b were prepared from commercially available diols.
Compounds 17g, 21f, 22f, 23e and 32b were prepared as
outlined in Scheme 2 (synthetic details are available as
Supporting Information). All commercial diols were protected
using the same procedure as described for 16f; however,
sodium hydride was substituted for imidazole in the protection
of diols 4d , 12d , 13d , 20b, and 21b.
N-(Ca r boben zyloxy)d ieth a n ola m in e (16e). To a solu-
tion of 16d (2.0 g, 19.0 mmol) in acetonitrile (19 mL) were

6-Substituted Purine Linker Amino Acid Immunostimulants
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2891
added successively powdered K2CO3 (5.3 g, 38 mmol) and
benzyl chloroformate (2.9 mL, 20.0 mmol). The white suspen-
sion was stirred vigorously for 3 h and treated by standard
workup (H2O/CH2Cl2) to give 16e, as a clear oil that was used
without purification or characterization.
tions were dried in vacuo, and the product was taken up in
minimal methanol. Trace amounts of silica were removed by
either of two methods: (1) the solution was cooled (4 °C) for
2-4 h and then centrifuged (375 g, 10 min), and the super-
natant was removed, or (2) the solution was passed through a
micron filter The solution was then added dropwise to vigor-
ously stirred ether (500 mL). The product was collected on
sintered glass, washed with ether, and dried in vacuo to give
1a (0.85 g, 2.01 mmol, 48%) as a white solid: mp 182 °C; TLC
(Q) Rf 0.18; 1H NMR (DMSO-d6, 300 MHz) δ 9.28 (1H, br,
COOH), 8.10 (1H, s, purine), 8.04 (1H, s, purine), 7.8-7.2 (4H,
br, guanidine), 6.38 (1H, d, NH), 4.32 (1H, br, OH), 4.13 (2H,
t, NCH2), 3.87 (2H, t, CH2O), 3.61 (1H, m, CRH), 3.04 (2H, br,
CδH2), 1.8-1.1 (10H, m, CâH2, CγH2, (CH2)3); HRMS m/ z calcd
for C17H27N8O5 (MH+) 423.2104, found 423.2124; HPLC 99%.
Nr-[[5-(6-Hyd r oxyp u r in -9-yl)p en toxy]ca r bon yl]-L-a r gi-
n in e (1b, ST 789):9 yield 42%; mp 184 °C; TLC (A) Rf 0.20;
spectral properties were identical with 1a ; HPLC 98%.
Nr-[[5-(6-Am in op u r in -9-yl)p en toxy]ca r bon yl]-D-a r gin -
in e (2a ): yield 52%; mp 150 °C; TLC (A) Rf 0.20; 1H NMR
(DMSO-d6, 300 MHz) δ 9.40(1H, br, COOH), 8.14 (1H, s,
purine), 8.13 (1H, s, purine), 8.0-7.0 (6H, m, guanidine, NH2),
6.36 (1H, br, NH), 4.13 (2H, t, NCH2), 3.87 (2H, t, CH2O), 3.65
(1H, m, CRH), 3.03 (2H, b, CδH2), 1.9-1.2 (10H, m, CâH2, CγH2,
(CH2)3); HRMS m/ z calcd for C17H28N9O4 (MH+) 422.2264,
found 422.2242; HPLC 97%.
N-(Ca r b ob en zyloxy)d iet h a n ola m in e
ter t-Bu t yld i-
p h en ylsilyl Eth er (16f). To a solution of 16e (1.29 g, 19.0
mmol) in dry CH2Cl2 (190 mL) at 0 °C were added imidazole
(1.29 g, 19.0 mmol) and tert-butylchlorodiphenylsilane (5 mL,
19.0 mmol). The solution was stirred at 0 °C for 45 min and
rt for 90 min. It was then treated by standard workup
(saturated NH4Cl/CH2Cl2). The crude product was purified by
silica gel column chromatography, eluting with 20% ethyl
acetate/toluene to give 16f (2.09 g, 4.37 mmol, 23%) as a clear
oil: 1H NMR (CDCl3, 400 MHz): δ 7.65-7.63 (4H, m, aro-
matic), 7.46-7.22 (11H, m, aromatic), 5.16 (2H, AB quartet,
CH2Ph), 5.06 (2H, AB quartet, CH2Ph), 3.86-3.74 (4H, m,
CH2), 3.55-3.45 (4H, m, CH2), 1.59 (1H, br s, OH), 1.05 (9H,
s, t-Bu).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Alk yl-
p u r in es (Sch em e 1). Purine derivatives IV were prepared
by Mitsunobu coupling of 6-chloropurine and monoprotected
diols. The sequence is illustrated for 6c.
5-(6-Ch lor op u r in -9-yl)-1-[(ter t-bu tyld ip h en ylsilyl)oxy]-
p en ta n e (II). To a stirred solution of triphenylphosphine (4.7
g., 17.9 mmol) and 6-chloropurine (2.3 g., 15.1 mmol) in THF
(100 mL) was added diethyl azodicarboxylate (2.8 mL, 17.9
mmol). After 10 min, 4d (4.7 g., 13.7 mmol) dissolved in THF
(20 mL) was added dropwise to the reaction, which was then
stirred at ambient temperature overnight. The solvent was
removed in vacuo, and the crude product was purified by silica
gel column chromatography eluting with 30% ethyl acetate/
hexane to give II (3.7 g, 7.6 mmol, 55%) as a colorless oil: TLC
(K) Rf 0.30; 1H NMR (CDCl3, 300 MHz) δ 8.74 (1H, s, purine);
8.07 (1H, s, purine); 7.62 (5H, m, aromatic); 7.39 (5H, m,
aromatic); 4.27 (2H, t, NCH2); 3.65 (2H, t, CH2O); 2.0-1.3 (6H,
m, (CH2)3); 1.05 (9H, s, t-Bu).
5-(6-Ch lor op u r in -9-yl)p en ta n ol (6c). To a stirred solu-
tion of II (2.3 g, 4.4 mmol) in THF (40 mL) was added
tetrabutylammonium fluoride (5.3 mL, 5.1 mmol), and the
reaction was stirred overnight. To the solution was added
glacial acetic acid (0.3 mL, 5.1 mmol) and solvent removed in
vacuo. The crude product was purified by silica gel column
chromatography, eluting with 10% methanol/ethyl acetate. The
solvent was removed in vacuo to give 6c (1.0 g., 4.2 mmol, 95%)
as a white solid: mp 84 °C; TLC (K) Rf 0.20; 1H NMR (CDCl3,
300 MHz) δ 8.75 (1H, s, purine); 8.13 (1H, s, purine); 4.32 (2H,
t, NCH2); 3.65 (2H, t, CH2O); 2.1-1.3 (7H, m, (CH2)3, OH).
Alt er n a t e P r oced u r e for t h e P r ep a r a t ion of Alk yl-
p u r in es (Sch em e 2). Compound 32d was prepared by
coupling of 6-chloropurine and alkyl iodide.
5-(6-Ch lor op u r in -9-yl)-N -(ca r b ob e n zyloxy)p e n t a n -
a m in e (32d ). To a solution of 6-chloropurine (1.55 g, 10.0
mmol) and Cs2CO3 in DMF (45 mL) was added 32c (4.18 g,
12.0 mmol). The reaction was stirred for 16 h. The solvent
was evaporated, and the crude product was purified by silica
gel column chromatography, eluting with 80% ethyl acetate/
hexane to give 32d (2.53 g, 6.77 mmol, 67.7%) as a pale yellow
oil: TLC (J ) Rf 0.41; 1H NMR (CDCl3, 400 MHz) δ 8.08 (1H, s,
purine), 7.27 (5H, m, aromatic), 5.15 (1H, br s, NH), 5.03 (2H,
s, PhCH2), 4.22 (2H, t, purine-NCH2), 3.18-3.10 (2H, m,
NCH2), 1.93-1.83 (2H, p, CH2), 1.57-1.47 (2H, p, CH2), 1.35-
1.25 (2H, p, CH2).
Gen er a l P r oced u r e for Cou p lin g of Alcoh ol w ith
Am in o Acid s or P ep tid es. To a stirred solution of 5-(6-
hydroxypurin-9-yl)pentanol, 1c (1.0 g, 4.2 mmol) in THF (75
mL) was added toluenic phosgene (4.4 mL, 8.8 mmol), and the
reaction was stirred for 6-8 h. Formation of the chloroformate
intermediate was monitored by TLC (A). The solvent and
unreacted phosgene were removed in vacuo, and the residue
Nr-[[5-(6-Am in op u r in -9-yl)p en toxy]ca r bon yl]-L-a r gin -
in e (2b): yield 49%; mp 153-155 °C; TLC (A) Rf 0.22; spectral
properties were identical with 2a ; HRMS m/ z (MH+) found
422.2259; HPLC 100%.
Nr-[[5-[6-(N-Meth ylam in o)pu r in -9-yl]pen toxy]car bon yl]-
1
D-a r gin in e (3a ): yield 61%; mp 130 °C; TLC (A) Rf 0.20; H
NMR (DMSO-d6, 300 MHz) δ 9.30(1H, br, COOH), 8.21 (1H,
s, purine), 8.13 (1H, s, purine), 7.8-7.2 (4H, br, guanidine),
6.29 (1H, d, NH), 4.13 (2H, t, NCH2), 3.86 (2H, t, CH2O), 3.61
(1H, m, CRH), 3.17 (1H, m, CH3NH), 3.02 (3H, br, NHCH3),
2.97 (2H, br, CδH2), 1.9-1.2 (10H, m, CâH2, CγH2, (CH2)3);
HRMS m/ z calcd for C18H30N9O4 (MH+) 436.2421, found
436.2400; HPLC 97%.
Nr-[[5-[6-(N-Meth ylam in o)pu r in -9-yl]pen toxy]car bon yl]-
L-a r gin in e (3b): yield 48%; mp 130 °C; TLC (A) Rf 0.20;
spectral properties were identical with 3a ; HRMS m/ z (MH+)
found 436.2430; HPLC 98%.
Nr-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]-D-a r gin in e (4a , BCH 1393): yield 43%; mp 123-125
°C; TLC (Q) Rf 0.23; 1H NMR (DMSO-d6, 300 MHz) δ 9.40 (1H,
br, COOH), 8.20 (1H, s, purine), 8.15 (1H, s, purine), 8.0-7.3
(4H, br, guanidine), 6.33 (1H, d, NH), 4.13 (2H, t, NCH2), 3.86
(2H, t, CH2O), 3.63 (1H, m, CRH), 3.36 (6H, br s, N(CH3)2),
3.02 (2H, br, CδH2), 1.8-1.2 (10H, m, CâH2, CγH2, (CH2)3);
HRMS m/ z calcd for C19H32N9O4 (MH+) 450.2577, found
450.2578; HPLC 99%.
Nr-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]-L-a r gin in e (4b): yield 47%; mp 123-125 °C; TLC (Q)
Rf 0.25; spectral properties were identical with 3a ; HRMS m/ z
(MH+) found 450.2575; HPLC 98%.
Nr-[[5-(6-H yd r a zin op u r in -9-yl)p en t oxy]ca r b on yl]-D-
a r gin in e (5a ): yield 65%; mp 134 °C; TLC (A) Rf 0.27; 1H NMR
(DMSO-d6, 300 MHz) δ 9.20 (1H, br, COOH), 8.23 (1H, s,
purine), 8.14 (1H, s, purine), 8.0-7.3 (4H, br, guanidine), 6.60
(2H, br, NH2), 6.43 (1H, d, NH), 4.14 (2H, t, NCH2), 3.87 (2H,
t, CH2O), 3.64 (1H, m, CRH), 3.04 (2H, br, CδH2), 1.8-1.3 (10H,
m, CâH2, CγH2, (CH2)3); HRMS m/ z calcd for C17H29N10O4
(MH+) 437.2373, found 437.2350; HPLC 99%.
Nr-[[5-(6-Hydr azin opu r in -9-yl)pen toxy]car bon yl]-L-ar gi-
n in e (5b): yield 68%; mp 134 °C; TLC (A) Rf 0.26; spectral
properties were identical with 5a ; LRMS m/ z (MH+) found
437; HPLC 98%.
Nr-[[5-(6-Ch lor op u r in -9-yl)p en toxy]ca r bon yl]-D-a r gin -
in e (6a ): yield 41%; mp 145-148 °C; TLC (Q) Rf 0.26; 1H NMR
(DMSO-d6, 300 MHz) δ 9.40 (1H, br, COOH), 8.77 (1H, s,
purine), 8.73 (1H, s, purine), 8.1-7.2 (4H, br, guanidine), 6.35
(1H, d, NH), 4.29 (2H, t, NCH2), 3.87 (2H, t, CH2O), 3.64 (1H,
m, CRH), 3.02 (2H, br, CδH2), 1.9-1.2 (10H, m, CâH2, CγH2,
(CH2)3); LRMS m/ z (MH+) found 441; HPLC 100%.
Nr-[[5-(6-Ch lor op u r in -9-yl)p en toxy]ca r bon yl]-L-a r gin -
in e (6b): yield 54%; mp 143-146 °C; TLC (Q) Rf 0.28; spectral
was taken up in THF (75 mL). In
a separate beaker,
D-arginine (0.94 g, 5.4 mmol) was dissolved in minimal water,
and added to the chloroformate suspension. After 10 min
NaHCO3 (1.06 g,12.6 mmol) was added, and the reaction was
stirred overnight. The solvent was removed in vacuo, and the
residue was purified by silica gel column chromatography,
eluting with 80% MeOH/EtOAc. The combined product frac-

2892 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Zacharie et al.
properties were identical with 6a ; LRMS m/ z (MH+) found
441; HPLC 94%.
Nr-[[4-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]bu toxy]ca r -
bon yl]-L-a r gin in e (13b): yield 75%; mp 139-142 °C; TLC (P)
Rf 0.20; spectral properties were identical with 13a ; LRMS m/ z
(MH+) found 436.2421; HPLC 100%.
Nr-[[5-(6-Mer captopu r in -9-yl)pen toxy]car bon yl]-D-ar gi-
1
n in e (7a ): yield 61%; mp 200 °C; TLC (A); Rf 0.50; H NMR
(DMSO-d6, 300 MHz) δ 9.15 (1H, b, COOH), 8.29 (1H, s,
purine), 8.18 (1H, s, purine), 7.5-7.3 (4H, br, guanidine), 6.39
(1H, d, NH), 4.13 (2H, t, NCH2), 3.87 (2H, t, CH2O), 3.65 (1H,
m, CRH), 3.04 (2H, br, CδH2), 1.90-1.23 (10H, m, CâH2, CγH2,
(CH2)3); HRMS m/ z calcd for C17H27N8O4S (MH+) 439.1876,
found 439.1858; HPLC 100%.
Nr-[[[[6-(N,N-Dim eth ylam in o)pu r in -9-yl]eth oxy]eth oxy]-
ca r bon yl]-D-a r gin in e (14a ): yield 33%, mp 140-145 °C; TLC
(A) Rf 0.35; 1H NMR (DMSO-d6, 300 MHz) δ 8.25 (1H, s,
purine), 8.14 (1H, s, purine), 6.5 (1H, br, NH), 4.37 (2H, t,
NCH2), 4.03 (2H, m, CH2), 3.81 (2H, m, CH2), 3.72 (1H, m,
CRH), 3.60 (2H, m, CH2), 3.72 (6H, m, N(CH3)2), 3.05 (2H, m,
CδH2), 1.78-1.39 (4H, m, CâH2, CγH2); HRMS m/ z calcd for
C18H30N9O5 (MH+) 452.2370, found 452.2400; HPLC 98%.
Nr-[[[[6-(N,N-Dim eth ylam in o)pu r in -9-yl]eth oxy]eth oxy]-
ca r bon yl]-L-a r gin in e (14b): yield 41%; mp 130-135 °C; TLC
(A) Rf 0.35; spectral properties were identical with 14a ; HRMS
m/ z (MH+) found 452.2361; HPLC 100%.
Nr-[[5-(6-Mer captopu r in -9-yl)pen toxy]car bon yl]-L-ar gi-
n in e (7b): yield 60%; mp 200 °C; TLC (A); Rf 0.50; spectral
properties were identical with 7a ; LRMS m/ z (MH+) found
439; HPLC 100%.
Nr-[[5-(P u r in -9-yl)p en toxy]ca r bon yl]-D-a r gin in e (8a ):
yield 68%; hygroscopic solid; TLC (A); Rf 0.23; 1H NMR
(DMSO-d6, 300 MHz) δ 9.10 (1H, s, purine), 8.93 (1H, s,
purine), 8.64 (1H, s, purine), 7.8-7.2 (4H, br, guanidine), 6.25
(1H, br s, NH), 4.28 (2H, t, NCH2), 3.87 (2H, t, CH2O), 3.58
(1H, m, CRH), 3.09 (2H, m, CδH2), 1.20-1.90 (10H, m, CâH2,
CγH2, (CH2)3); HRMS m/ z calcd for C17H27N8O4 (MH+) 407.2155,
found 407.2179; HPLC 99%.
Nr-[[[[[6-(N,N-Dim eth yla m in o)p u r in -9-yl]eth yl]th io]et-
h oxy]ca r bon yl]-D-a r gin in e (15a ): yield 26%; mp 150-155
°C; TLC (Q) Rf 0.38; 1H NMR (DMSO-d6) δ 8.26 (1H, s, purine),
8.22 (1H, s, purine), 6.61 (1H, br, NH), 4.39 (2H, t, NCH2),
4.09 (2H, t, CH2O), 3.72 (1H, m, CRH), 3.48 (8H, m, N(CH3)2,
CH2), 3.06 (2H, t, CH2), 2.78 (2H, t, CδH2), 1.75-1.40 (4H, m,
CâH2, CγH2); HRMS m/ z calcd for C18H30N9O4S (MH+) 468.2141,
found 468.2114; HPLC 99%.
Nr-[[5-(P u r in -9-yl)p en toxy]ca r bon yl]-L-a r gin in e (8b):
yield 64%; hygroscopic solid; TLC (A); Rf 0.23; spectral proper-
ties were identical with 8a ; HRMS m/ z (MH+) found 407.2180;
HPLC 92%.
Nr-[[[[[6-(N,N-Dim eth yla m in o)p u r in -9-yl]eth yl]th io]et-
h oxy]ca r bon yl]-L-a r gin in e (15b): yield 57%; mp 145-150
°C; TLC (A) Rf 0.35; spectral properties were identical with
15a ; HRMS m/ z (MH+) found 468.2129; HPLC 99%.
Nr-[[[[[6-(N,N-Dim eth ylam in o)pu r in -9-yl]eth yl]am in o]-
eth oxy]ca r bon yl]-D-a r gin in e (16a ): yield 56%; mp 160-165
°C; TLC (A) Rf 0.10; 1H NMR (CDCl3, 300 MHz) δ 9.36 (m,
1H), 8.19 (1H, s, purine), 8.11 (1H, s, purine), 6.38 (1H, d, NH),
4.17 (2H, t, CH2), 3.91-3.87 (2H, m, CH2), 3.65 (1H, m, CRH),
3.43 (6H, br s, N(CH3)2), 3.03-3.01 (2H, m, CδH2), 2.90 (2H, t,
CH2), 2.68 (2H, t, CH2), 1.64-1.44 (4H, m, CâH2, CγH2); HRMS
m/ z calcd for C18H31N10O4 (MH+) 451.2530, found 451.2519;
HPLC 96%.
Nr-[[5-[6-(N-Cyclopr opylam in o)pu r in -9-yl]pen toxy]car -
bon yl]-D-a r gin in e (9a ): yield 58%; mp 151 °C; TLC (A); Rf
0.34; 1H NMR (DMSO-d6, 300 MHz) δ 8.22 (1H, s, purine),
8.14 (1H, s, purine), 8.0-7.0(4H, br s, guanidine), 6.28 (1H, d,
NH), 4.13 (2H, t, NCH2), 3.87 (2H, m, CH2O), 3.62 (1H, m,
CRH), 3.02 (2H, m, CδH2), 1.8-1.2 (11H, m CâH2, CγH2, (CH2)3),
CH), 0.69 (2H, m, CH2), 0.67 (2H, m, CH2); HRMS m/ z calcd
for C20H32N9O4 (MH+) 462.2577, found 462.2588; HPLC 100%.
Nr-[[5-[6-(N-Cyclopr opylam in o)pu r in -9-yl]pen toxy]car -
bon yl]-L-a r gin in e (9b): yield 55%; mp 144-146 °C; TLC (A);
Rf 0.35; spectral properties were identical with 9a ; HRMS m/ z
(MH+) found 462.2578; HPLC 99%.
Nr-[[[[[6-(N,N-Dim eth ylam in o)pu r in -9-yl]eth yl]am in o]-
eth oxy]ca r bon yl]-L-a r gin in e (16b): yield 54%; mp 125-130
°C; TLC (R) Rf 0.30; spectral properties were identical with
16a ; HRMS m/ z (MH+) found 451.2551; HPLC 100%.
Nr-[[[[6-(N,N-Dim eth yla m in o)p u r in -9-yl]-3-p en tyn yl]-
oxy]ca r bon yl]-D-a r gin in e (17a ): yield 30%; mp 130-135 °C;
Nr-[[5-[6-(N-Azetid in yl)p u r in -9-yl]p en toxy]ca r bon yl]-
D-a r gin in e (10a ): yield 74%; mp 190-192 °C; TLC (A); Rf 0.25;
1H NMR (CD3OD, 300 MHz) δ 7.96 (1H, s, purine), 7.89 (1H,
s, purine), 4.27 (4H, m, 2 × CH2-N-azetidine), 4.02 (2H, t,
NCH2), 3.79 (3H, m, CH2O and CRH), 2.99 (2H, m, CH2-
azetidine), 2.32 (2H, m, CH2), 1.71-1.17 (10H, m, CâH2, CγH2,
(CH2)3); HRMS m/ z calcd for C20H32N9O4 (MH+) 462.2577,
found 462.2593; HPLC 100%.
1
TLC (Q) Rf 0.32; H NMR (DMSO-d6) δ 8.27 (1H, s, purine),
8.26 (1H, s, purine), 6.55 (1H, m, NH), 5.05 (2H, br, CH2N),
4.02 (2H, m, CH2O), 3.69 (1H, m, CRH), 3.60-3.35 (8H, m,
N(CH3)2, CH2), 3.06 (2H, m, CδH2), 1.70-1.40 (4H, m, CâH2,
CγH2); HRMS m/ z calcd for C19H27N9O4 (MH+) 446.2264, found
446.2280; HPLC 100%.
Nr-[[5-[6-(N-Azetid in yl)p u r in -9-yl]p en toxy]ca r bon yl]-
L-a r gin in e (10b): yield 60%; mp 187 °C; TLC (A); Rf 0.27;
spectral properties were identical with 10a ; LRMS m/ z (MH+),
found 462; HPLC 100%.
Nr-[[[[6-(N,N-Dim eth yla m in o)p u r in -9-yl]-3-p en tyn yl]-
oxy]ca r bon yl]-L-a r gin in e (17b): yield 17%; mp 130-135 °C;
TLC (A) Rf 0.30; spectral properties were identical with 17a ;
HRMS m/ z (MH+) found 446.2263; HPLC 100%.
Nr-[[5-[6-Meth yla zir id in -N-yl)p u r in -9-yl]p en toxy]ca r -
bon yl]-D-a r gin in e (11a ): yield 64%; mp 200 °C; TLC (A); Rf
0.40; 1H NMR (CD3OD, 300 MHz) δ 8.29 (1H, s, purine), 8.14
(1H, s, purine), 4.10 (2H, t, NCH2), 3.79 (3H, m, CH2O, CRH),
2.97 (2H, m, CδH2), 2.62 (1H, m, CH-aziridine), 2.45 (2H, d,
CH2-aziridine), 1.76-1.2 (13H, m, CâH2, CγH2, (CH2)3), CH3);
HRMS m/ z calcd for C20H32N9O4 (MH+) 462.2604, found
462.2602; HPLC 95%.
Nr-[[[m -[[6-(N,N-Dim et h yla m in o)p u r in -9-yl]m et h yl]-
ben zyl]oxy]ca r bon yl]-D-a r gin in e (18): yield 25%; mp 165-
1
170 °C; TLC (A) Rf 0.30; H NMR (DMSO-d6) δ 8.31 (1H, s,
purine), 8.26 (1H, s, purine), 7.36-7.19 (4H, m, aromatic), 6.67
(1H, br, NH), 5.41 (2H, s, CH2N), 4.98 (2H, s, CH2O), 3.73 (1H,
m, CRH), 3.41 (6H, br s, N(CH3)2), 3.07 (2H, m, CδH2), 1.69-
1.50 (4H, m, CâH2, CγH2); HRMS m/ z calcd for C22H30N9O4
(MH+) 484.2421, found 484.2410; HPLC 100%.
Nr-[[6-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]h exoxy]ca r -
bon yl]-D-a r gin in e (12a ): yield 60%; mp 123 °C; TLC(A); Rf
0.48; 1H NMR (CD3OD, 300 MHz) δ 7.98 (1H, s, purine), 7.81
(1H, s, purine), 3.98 (2H, t, NCH2), 3.78 (3H, m, CH2O and
CRH), 3.27 (6H, br s, N(CH3)2), 2.96 (2H, m, CδH2), 1.78-1.10
(12H, m, CâH2, CγH2, (CH2)4); HRMS m/ z calcd for C20H34N9O4
(MH+) 464.2734, found 464.2749; HPLC 100%.
tr a n s-Nr-[[[4-[[(6-(N,N-Dim eth yla m in o)p u r in -9-yl]m e-
th yl]cycloh exyl]m eth oxy]ca r bon yl]-D-a r gin in e (19): yield
25%; mp 164-166 °C; TLC (A) Rf 0.35, 1H NMR (DMSO-d6,
400 MHz) δ 9.4 (1H, br s, COOH), 8.19 (1H, s, purine), 8.11
(1H, s, purine), 8.0-7.2 (4H, br, guanidine), 6.28 (1H, d, NH),
3.98 (2H, d, NCH2), 3.69 (2H, d, CH2O), 3.61 (1H, m, CRH),
3.43 (6H, br s, N(CH3)2), 3.00 (2H, br, CδH2), 1.9-0.8 (14H, m,
CâH2, CγH2, 2 × CH, 4 × CH2); HRMS m/ z calcd for
C22H36N9O4 (MH+) 490.2890, found 490.2899; HPLC 100%
cis-Nr-[[[4-[[(6-(N,N-Dim eth yla m in o)p u r in -9-yl]m eth -
yl]cycloh exyl]m eth oxy]ca r bon yl]-D-a r gin in e (20): yield
Nr-[[6-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]h exoxy]ca r -
bon yl-L-a r gin in e (12b): yield 61%; mp 131 °C; TLC (A); Rf
0.47; spectral properties were identical with 12a ; HRMS m/ z
(MH+) found 464.2754; HPLC 100%.
Nr-[[4-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]bu toxy]ca r -
bon yl]-D-a r gin in e (13a ): yield 86%; mp 140-142 °C; TLC (P)
Rf 0.20; 1H NMR (DMSO-d6, 300 MHz) δ 8.20 (1H, s, purine),
8.16 (1H, s, purine), 8.1-7.3 (4H, br, guanidine), 6.40 (1H, d,
NH), 4.16 (2H, t, NCH2), 3.91 (2H, t, CH2O), 3.65 (1H, m, CRH),
3.40 (6H, br s, N(CH3)2), 3.02 (2H, m, CδH2), 1.9-1.3 (8H, m,
CâH2, CγH2, (CH2)2); HRMS m/ z calcd for C18H30N9O4 (MH+)
436.2421, found 436.2392; HPLC 99%.
1
27%; mp 168-170 °C; TLC (A) Rf 0.35; H NMR (DMSO-d6,
400 MHz) δ 9.28 (1H, br s, COOH), 8.19 (1H, s, purine), 8.13
(1H, s, purine), 8.0-7.2 (4H, br, guanidine), 6.34 (1H, d, NH),
4.10 (2H, d, NCH2), 3.85 (2H, d, CH2O), 3.65 (1H, m, CRH),
3.44 (6H, br s, N(CH3)2), 3.02 (2H, m, CδH2), 2.09 (1H, m, CH),

6-Substituted Purine Linker Amino Acid Immunostimulants
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2893
1.8-1.2 (14H, m, CâH2, CγH2, 2 × CH, 4 × CH2); HRMS m/ z
calcd for C22H36N9O4 (MH+) 490.2890, found 490.2885; HPLC
100%.
N-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]-L-va lyl-L-p r olyl-L-leu cin e (30): yield 11%; mp 168 °C;
1
TLC (O) Rf 0.40; H NMR (CD3OD, 400 MHz) δ 8.22 (1H, s,
purine); 8.05 (1H, s, purine), 4.60 (1H, t, CRH), 4.3-3.6 (8H,
m, NCH2, CδH2, CH2O, 2 × CRH), 3.51 (6H, br s, N(CH3)2),
2.2-1.2 (14H, m, (CH2)3, 2 × CâH2, CâH, CγH2, CγH), 1.0-0.8
(12H, m, 2 × CâCH3, 2 × CγCH3); LRMS m/ z (MH+) found
603.5; HPLC 97%.
(1S,3R)- a n d (1R,3S)-1-[[(6-(N,N-Dim eth yla m in o)p u r in -
9-yl]m eth yl]-3-[[(D-a r gin in -Nr-ylca r bon yl)oxy]m eth yl]cy-
clop en ta n e (21): yield 33%; mp 140-155 °C; TLC (A) Rf 0.32;
1H NMR (CDCl3, 400 MHz) δ 8.19 (1H, s, purine), 8.15 (1H, s,
purine), 6.32 (1H, br, NH), 4.09 (2H, d, NCH2), 3.81 (2H, d,
CH2O), 3.65 (1H, m, CRH), 3.39 (6H, m, N(CH3)2), 3.02 (2H,
m, CδH2), 2.45 (1H, m), 2.10 (1H, m), 1.72-1.30 (9H, m), 0.95
(1H, m). HRMS m/ z calcd for C21H34N9O4 (MH+) 476.2738,
found 476.2734; HPLC 100%.
(2S,4S)-2-[[(6-(N,N-Dim eth yla m in o)p u r in -9-yl]m eth yl]-
4-[[(D-a r gin in -Nr-ylca r b on yl)oxy]m et h yl]-1,3-d ioxola n e
(22): yield 41%; mp 155-160 °C; TLC (A) Rf 0.30; 1H NMR
(DMSO-d6) δ 8.43 (1H, s, purine), 8.11 (1H, s, purine), 6.6 (1H,
br, NH), 5.28 (1H, m, H-2-dioxolane), 4.39 (2H, m, NCH2), 4.26
(1H, m, H-4-dioxolane), 3.97-3.81 (3H, m, CRH, CH2O), 3.71
(2H, m, H-5-dioxolane), 3.39 (6H, br s, N(CH3)2), 3.07 (2H, m,
CδH2), 1.70-1.45 (4H, m, CâH2, CγH2); HRMS m/ z calcd for
C19H30N9O6 (MH+) 480.2319, found 480.2306; HPLC 99%.
(2S,4S)- a n d (2R,4R)-2-[[(6-(N,N-Dim eth yla m in o)p u r in -
9-yl]m et h yl]-4-[[(D-a r gin in -Nr-ylca r b on yl)oxy]m et h yl]-
1,3-oxa th iola n e (23): yield 30%; mp 160-165 °C; TLC (Q)
Rf 0.32; 1H NMR (CDCl3, 300 MHz) δ 8.49 (1H, s, purine), 8.47
(1H, s, purine), 6.60 (1H, br, NH), 6.22 (1H, m, H-2-oxathi-
olane), 4.26-4.03 (3H, m), 3.63-3.00 (11H, m), 2.78-2.69 (2H,
m, H-5-oxathiolane), 1.53-1.40 (4H, m, CâH2, CγH2); HRMS
m/ z calcd for C19H30N9O5S (MH+) 496.2091, found 496.2100;
HPLC 97%.
6-[6-(N,N-Dim et h yla m in o)p u r in -9-yl]h exa n oic Acid
5-[[6-(N,N-d im eth yla m in o)p u r in -9-yl]p en tyl Ester (31):
yield 73%; mp 70-72 °C; TLC (L) Rf 0.42; 1H NMR (CDCl3
300 MHz) δ 8.33 (2H, s, purine), 7.70 (2H, s, purine), 4.17 (4H,
m, 2 × NCH2), 4.02 (2H, m, CH2O), 3.53 (12H, br s 2 ×
N(CH3)2), 2.25 (2H, t, COCH2), 1.92 (4H, m, 2 × CH2), 1.64
(4H, m, 2 × CH2), 1.37 (4H, m, 2 × CH2); HRMS m/ z calcd for
C25H37N10O2 (MH+) 509.3101, found 509.3072; HPLC 99%.
N-[5-[6-(N,N-Dim eth ylam in o)pu r in -9-yl]pen tyl]car bam -
ic Acid 5-[6-(N,N-d im eth yla m in o)p u r in -9-yl]p en tyl Ester
(32): yield 64%; oil; TLC (M) Rf 0.40; 1H NMR (CDCl3 300
MHz) δ 8.34 (2H, s, purine), 7.71 (2H, s, purine), 4.93 (1H, br
s, NH), 4.18 (4H, 2t, 2 × NCH2), 4.02 (2H, t, CH2O), 3.53 (12H,
br s, 2 × N(CH3)2); 3.15 (2H, t, CH2) 1.96-1.2 (12H, 3m, 3 ×
CH2), HRMS m/ z calcd for C25H38N11O2 (MH+) 524.3210, found
524.3233; HPLC 99%.
[[[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]-p en toxy]ca r -
bon yl]a m in o]a cetic Acid 5-[6-(N,N-d im eth yla m in o)p u -
r in -9-yl]p en tyl Ester (33): yield 60%; oil; TLC (M) Rf 0.4;
1H NMR (CDCl3 300 MHz) δ 8.33 (2H, s, purine), 7.71 (2H, s,
purine), 5.42 (1H, t, NH), 4.19-3.90 (10H, m, 2 × NCH2, 2 ×
CH2O, CH2NH), 3.52 (12H, br s, 2 × N(CH3)2), 1.91 (4H, m, 2
× CH2), 1.70 (4H, m, 2 × CH2), 1.40 (4H, m, 2 × CH2); HRMS
m/ z calcd for C27H41N11O4 (MH+) 582.3265, found 582.3280;
HPLC 99%.
Biology. Cytotoxic T-Lym p h ocyte/Mixed Lym p h ocyte
Rea ction (CTL/MLR) Assa y: Splenocyte cell suspensions
were prepared by homogenization of spleens from 6- to 8-week-
old female C57BL/6 (H-2b) and DBA/2 (H-2d) mice in tissue
culture medium [RPMI 1640 (Gibco, Burlington, Canada)
supplemented with 10% fetal calf serum (Hyclone, Logan, UT)
and 2 µM L-glutamine]. The homogenate was centrifuged on
a density gradient (Lympholyte M, Cedarlane, Hornby, Canada)
(800 g, 20 min), and the mononuclear leukocytes were collected
and washed three times in phosphate buffered saline, pH )
7.4 (PBS) and resuspended in RPMI for evaluation of cell
viability by the use of trypan blue. DBA/2 spleen cells were
irradiated (3000 rads) for the unidirectional MLR and to prime
CTLs. The assay was performed as follows: Splenocytes (107
cells from C57BL/6 and DBA/2 mice) were incubated in a 5%
CO2 humidified incubator at 37 °C for 120 h and collected, and
an aliquot was pulsed with 0.1 µCi of [3H]thymidine (Amer-
sham, Oakville, Canada). After 6 hours these cells were
collected by filtration, and newly synthesized DNA was
measured by determination of the uptake of tritiated thymi-
dine.
N-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
1
bon yl]glycin e (24): yield 14%; mp 93 °C; TLC (O) Rf 0.; H
NMR (DMSO-d6, 300 MHz) δ 8.20 (s, 1H, purine), 8.16 (s, 1H,
purine), 6.05 (1H, d, NH), 4.14 (2H, t, NCH2), 3.86 (2H, t,
CH2O), 3.33 (6H, br s, N(CH3)2), 3.18 (2H, d, CRH2), 1.80 (2H,
m, CH2), 1.55 (2H, m, CH2), 1.26 (2H, m, CH2); HRMS m/ z
calcd for C15H23N6O4 (MH+) 351.1780, found 351.1768; HPLC
100%.
Nr-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]-D-or n ith in e (25): yield 19%; mp 189-190 °C; TLC (A)
Rf 0.20; 1H NMR (DMSO-d6, 400 MHz) δ 8.20 (1H, s, purine),
8.12 (1H, s, purine), 6.21 (1H, d, NH), 4.10 (2H, t, NCH2), 3.87
(2H, t, CH2O), 3.59 (1H, m, CRH), 3.4 (8H, br, N(CH3)2, NH2),
2.70 (2H, m, CδH2), 1.9-1.2 (10H, m, (CH2)3, CâH2, CγH2);
HRMS m/ z calcd for C18H30N7O4 (MH+) 408.2359, found
408.2379; HPLC 100%.
N-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]-D-citr u lin e (26): yield 30%; mp 178-181 °C; TLC (O)
1
Rf 0.2; H NMR (DMSO-d6, 400 MHz) δ 8.19 (1H, s, purine),
8.15 (1H, s, purine), 6.21 (1H, d, NH), 6.11 (1H, s, NCO-NH),
5.42 (2H, s, NH2), 4.14 (2H, t, NCH2), 3.86 (2H, m, CH2O),
3.56 (1H, m, CRH), 3.35 (6H, br s, N(CH3)2), 2.87 (2H, m, CδH2),
1.9-1.2 (10H, m, (CH2)3, CγH2, CâH2); HRMS m/ z calcd for
C19H31N8O5 (MH+) 451.2417, found 451.2437; HPLC 100%.
2-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]eth yla m in e (27): yield 87%; hygroscopic solid; TLC (A)
Cells were simultaneously assayed for cell-mediated cytoly-
sis by incubation of 5 × 105 cells from the culture for 4 h with
sodium chromate labeled (51CrO4; Amersham, Oakville, Canada)
P815 mastocytoma cells (5 × 103). After the incubation,
chromium in the lysate was quantified and the percent specific
release or lysis calculated as follows:
1
Rf 0.3; H NMR (DMSO-d6, 300 MHz) δ 8.43 (1H, s, purine),
8.40 (1H, s, purine), 8.04 (3H, br s, NH3), 4.24 (2H, t, NCH2),
4.02 (2H, t, CH2O), 3.69 (6H, br s, N(CH3)2), 2.99 (2H, m, CH2),
2.67 (2H, m, CH2), 1.83 (2H, m, CH2), 1.62 (2H, m, CH2), 1.28
(2H, m, CH2); LRMS m/ z (MH+) found 321; HPLC 100%.
5-[6-(N,N-Dim eth ylam in o)pu r in -9-yl]pen tan ol (28): yield
80%; mp 57-59 °C; TLC (N) Rf 0.48; 1H NMR (CDCl3, 300
MHz) δ 8.35 (1H, s, purine), 7.71 (1H, s, purine), 4.19 (2H, t,
NCH2), 3.64 (2H, t, CH2O), 3.54 (6H, br s, N(CH3)2), 1.92 (2H,
m, CH2), 1.62 (2H, m, CH2), 1.44 (2H, m, CH2); HRMS m/ z
calcd for C12H20N5O (MH+) 250.1668, found 250.1682; HPLC
100%.
percent specific 51Cr release ) (ER - SR)/(TR - SR) ×
100%; where ER ) experimental release, TR )
total release, and SR ) spontaneous release
Due to variability between individual experiments, data is
compared to the positive control (human IL 2, 15 ng/mL, R&D
Systems, Minneapolis, MN) and presented in a semiquanti-
tative (+) format. Therefore, the percent specific lysis induced
by IL 2 in each experiment is taken as 100% or the maximum
stimulation. As such, 0 to 20% activation of CTLs relative to
IL 2 is assigned 0; 20 to 40%, +; 40 to 60%, ++; 60 to 80%,
+++; and 80 to 100%, ++++.
Im m u n op h en otyp in g Assa y. Female, 6- to 8-week-old,
C57BL/6 mice were injected intraperitoneally for one, four, or
nine consecutive days with BCH-1393, dissolved in physiologi-
cal saline, at different concentrations. Mice given one and four
N-[[5-[6-(N,N-Dim eth yla m in o)p u r in -9-yl]p en toxy]ca r -
bon yl]-L-isoleu cyl-L-p r olyl-L-isoleu cin e (29): yield 12%; mp
93 °C; TLC (O) Rf 0.35; 1H NMR (CD3OD, 400 MHz) δ 8.21
(1H, s, purine), 8.03 (1H, s, purine), 4.62 (1H, t, CRH), 4.3-
3.6 (8H, m, NCH2, CδH2, CH2O, 2 × CRH), 3.50 (6H, br s,
N(CH3)2), 2.1-1.0 (16H, m, (CH2)3, CâH2, 2 × CâH, 3 y CγH2),
0.95 (6H, d, 2 × CâCH3), 0.87 (6H, t, 2 × CγCH3); HRMS m/ z
calcd for C30H49N8O6 (MH+) 617.3775, found 617.3783; HPLC
100%.

2894 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Zacharie et al.
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doses of BCH-1393 were sacrificed on day five by cardiac
puncture, while mice given nine doses were sacrificed in the
same manner on day ten. Gross pathological observations
were recorded at the end of the experiment. Blood and spleens
were collected and cell suspension prepared and lysed in ACK
buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH
) 7.3) for five minutes. 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 cell surface marker monoclonal antibodies accord-
ing to the manufacturer’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 cell 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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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 < 0.05.
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was examined in vitro in enriched human T-cells. Expression of
CD26 was not affected by BCH-1393, as ascertained by immu-
nophenotyping. However, a modulation of the enzyme activity
in excess of 100% was observed at 10-7 M to 10-5 M BCH-1393
on IL 2/PHA stimulated cells. Further details of this work, as
well as the stimulation of human CTLs by BCH-1393, will be
published elsewhere.
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Ack n ow led gm en t. We gratefully acknowledge the
excellent technical assistance of Sylvie Taillon. The
participation of J . Duchaine, J . Heibein, D. J utras, and
M. Lagraoui in the immunophenotyping experiments is
appreciated. The preparation of gram quantities of
BCH-1393 for in vivo studies by Dr. T. Mansour and
his group is also acknowledged, as is the preliminary
toxicity study by Dr. R. Martel and his group. We
appreciate the thoughtful reading of this manuscript by
Dr. T. Bowlin and Dr. S. Abbott, and a useful discussion
with Dr. J . W. Hadden. We also thank Ms. Sandy
MacLellan for the preparation of this document.
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