Synthesis and evaluation of analogues of 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine as inhibitors of tumor cell growth, trypanosomal growth, and HIV-1 infectivity

Article abstract of DOI:10.1021/jm0201621

A well-defined series of 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine analogues was designed and synthesized in order to further ascertain the optimal structural requirements for S-adenosylmethionine decarboxylase inhibition and potentially t

Full text of DOI:10.1021/jm0201621

5112
J . Med. Chem. 2002, 45, 5112-5122
Syn th esis a n d Eva lu a tion of An a logu es of
5′-([(Z)-4-Am in o-2-bu ten yl]m eth yla m in o)-5′-d eoxya d en osin e a s In h ibitor s of
Tu m or Cell Gr ow th , Tr yp a n osom a l Gr ow th , a n d HIV-1 In fectivity†
Canio J . Marasco, J r.,‡,§ Debora L. Kramer,‡ J ohn Miller,‡ Carl W. Porter,‡ Cyrus J . Bacchi,| Donna Rattendi,|
Louis Kucera, Nathan Iyer, Ralph Bernacki,‡ Paula Pera,‡ and J anice R. Sufrin*,‡
Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute,
Buffalo, New York 14263, Haskins Laboratories and Department of Biology, Pace University, New York, New York 10038, and
Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Received April 17, 2002
A well-defined series of 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine analogues
was designed and synthesized in order to further ascertain the optimal structural requirements
for S-adenosylmethionine decarboxylase inhibition and potentially to augment and perhaps
separate their antiproliferative and antitrypanosomal activities. Most structural modifications
had a deleterious affect on both the antitrypanosomal and antineoplastic activity of 5′-([(Z)-
4-amino-2-butenyl]methylamino)-5′-deoxyadenosine. However, di-O-acetylation of the parent
compound produced a potential prodrug that caused markedly pronounced inhibition of
trypanosomal and neoplastic cell growth and viability. Moreover, the acetylated derivative of
5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine did inhibit HIV-1 growth and
infectivity, whereas the parent compound did not.
In tr od u ction
S-Adenosylmethionine decarboxylase (AdoMetDC) is
a key enzyme in the regulation and synthesis of
polyamines.1 The decarboxylation of S-adenosylme-
thionine (AdoMet) by AdoMetDC represents a potential
metabolic switching point between cellular methylation
reactions and polyamine biosynthesis.2 AdoMet can
donate a methyl group by acting as a cofactor in a
variety of cellular reactions (e.g., DNA methyltrans-
ferase).3,4 Furthermore, if AdoMet is decarboxylated by
F igu r e 1. Structure of AbeAdo (MDL73811).
AdoMetDC, it can donate a propylamine moiety in the
synthesis of the polyamines, spermidine, and spermine.2
Since polyamines play a critical role in both the origin
and progression of neoplastic phenotypes and in the life
cycle of trypanosomes, inhibition of AdoMetDC by a
variety of drugs has led to the development of agents
possessing both antineoplastic and antiparasitic activ-
ity.2,3 Of interest to our research group is a prototypical
irreversible inhibitor of AdoMetDC, 5′-([(Z)-4-amino-2-
butenyl]methylamino)-5′-deoxyadenosine (AbeAdo), orig-
inally synthesized by chemists at Merrell Dow Research
Institute where it was designated MDL73811 (Figure
1).5 As illustrated in Figure 2, it has been hypothesized
that binding of AbeAdo to AdoMetDC results in the
formation of a Schiff base between the butenylamine
functionality of AbeAdo and a pyruvate prosthetic group
within the enzyme’s active site. Subsequent enzyme-
mediated abstraction of a proton R to the Schiff base
causes the formation of a conjugated imine that is highly
susceptible to nucleophilic attack by other residues
within the enzyme.2,5 Despite AbeAdo’s high affinity and
pronounced inhibition of AdoMetDC in cell-free as-
says2,5,6 and its salient in vitro activity against a variety
of cancer cell lines, it possessed only moderate growth
inhibitory activity when tested in vivo.2,7,8 Conversely,
AbeAdo has shown good efficacy, in vivo, against a
variety of trypanosomal strains such as Trypanosoma
brucei brucei and Trypanosoma brucei rhodesiense.2,9,10
The greater efficacy of AbeAdo as an inhibitor of
trypanosomal growth, as opposed to mammalian cells,
has been attributed to the active uptake of AbeAdo by
trypanosomes via a purine transport system not found
in mammalian cells.2,11
† A preliminary report of these studies was presented at the 86th
Annual Meeting of the American Association for Cancer Research,
Toronto, Ontario, Canada, March 1995. An abstract was published in
Proc. Am. Assoc. Cancer Res. 1995, 36, 301.
The promising antitrypanosomal and antineoplastic
activity of AbeAdo clearly warranted further investiga-
tion. Therefore, a well-defined series of AbeAdo ana-
logues was designed and synthesized in order to further
ascertain the optimal structural requirements for
AdoMetDC inhibition and potentially to augment and
perhaps separate their antiproliferative and antitrypa-
nosomal activities.
* To whom correspondence should be addressed. Phone: 716-845-
4582. Fax: 716-845-8857. Email: J anice.sufrin@roswellpark.org.
‡ Roswell Park Cancer Institute.
§ Present address: Department of Mathematics and Natural Sci-
ences, D’Youville College, Buffalo, New York 14213.
| Pace University.
Wake Forest University School of Medicine.
10.1021/jm0201621 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/15/2002

S-Adenosylmethionine Decarboxylase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5113
F igu r e 2. Proposed mechanism of AbeAdo. This figure was adapted from the following: Casara, P.; Marchal, P.; Wagner, J .;
Danzin, C. 5′-{[(Z)-4-Amino-2-butenyl]methylamino}-5′-deoxyadenosine: A Potent Enzyme-Activated Irreversible Inhibitor of
S-Adenosyl-L-methionine Decarboxylase from Escherichia coli. J . Am. Chem. Soc 1989, 111, 9111-9113.
Sch em e 1. Synthesis of Acetylated Analogues of AbeAdo
Ch em istr y
analogue. Intermediate 7 was O-acetylated and then
treated with trifluoroacetic acid for selective removal
of the tert-butyloxycarbonyl protecting groups to give
10, the di-O-acetylated analogue of AbeAdo.
Syntheses of 9, the 2′-deoxy derivative of AbeAdo and
of 10, the di-O-acetylated derivative of AbeAdo, are
shown in Scheme 1. The 5′-hydroxyl group of 2′-
deoxyadenosine or adenosine was chlorinated with
thionyl chloride according to Robbins et al.12 The
respective halogenated intermediates 2 and 3 were then
aminated with anhydrous methylamine to give the
corresponding methylamino analogues 4 and 5, respec-
tively. Compounds 4 and 5 were N-alkylated with cis-
tert-butoxycarbonyl-4-chloro-2-butenyl-1-amine to yield
6 and 7.5 Intermediate 6, after treatment with trifluo-
roacetic acid, gave 9, the desired 2′-deoxy AbeAdo
As illustrated in Scheme 2, the synthesis of the 2′,3′-
seco-adenosine analogues 14 and 16 was accomplished
by an oxidative-reductive cleavage of 5′-deoxy-5′-chlo-
roadenosine to give 5′-deoxy-5′-chloro-2′,3′-seco-adenos-
ine, 11,13 which was aminated and N-alkylated as
previously described5 to give 13. Deprotection of 13
yielded analogue 14 directly. When intermediate 13 was
O-acetylated and then selectively deprotected, analogue
16 was obtained.

5114 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Marasco et al.
Sch em e 2. Synthesis of 2′,3′-Seco Analogues of
Evaluation of the biological data presented in Table
1 offered serveral key insights into the structure-
activity relationships (SAR) of these analogues. Unsur-
prisingly, absence of the butenyl moiety resulted in a
loss of activity, as seen with AbeAdo versus analogue
4. This result supports the previously proposed mech-
anism of action of AbeAdo, whereby AbeAdo binds to
the active site of AdoMetDC and forms a Schiff base
with a pyruvate prosthetic group. The enzyme then ab-
stracts a proton from the substrate to give a conjugated
imine.5 The lack of activity displayed by the phenyl
derivatives of AbeAdo, 24, and 25 is consistent with this
proposed mechanism. The phenyl substituents of 24 and
25, which can be viewed as isosteric replacements for
the cis-butenyl arm of AbeAdo, can potentially form a
Schiff base, but not a conjugated imine (which requires
proton abstraction) within the enzyme’s active site.
AbeAdoa
Several analogues of AbeAdo, 9, 14, and 36, all
containing modifications of the ribose ring, were syn-
thesized. A previous observation that 2′-deoxyadenosine
is a substrate of 5′-methylthioadenosine phosphorylase
in trypanosomes but not in human tumor cells14
prompted us to synthesize 9, the 2′-deoxy derivative of
AbeAdo, as a possible means to increase its selectivity
toward trypanosomes. In fact, the in vitro antitrypano-
somal activity of 9 was ∼40 times lower than that of
AbeAdo as seen in the standard EATRO 110 strain of
T. b. brucei (Table 1). This suggests that the 2′-hydroxyl
group of AbeAdo plays an active role in the inhibition
of this enzyme. The uptake of 2′-deoxyAbeAdo (not
determined) may also be a contributing factor to the
reduced activity of 9 compared to AbeAdo. Moreover,
when the inhibitory potencies of 9 and AbeAdo toward
mammalian AdoMet DC were compared, an even greater
differential in activity was observed; compound 9 was
>250 times less active than AbeAdo (Table 2). This
result indicates that the 2′-hydroxyl group of AbeAdo
makes a substantial contribution to inhibition of the
mammalian enzyme. The 2′, 3′-seco derivative of AbeAdo,
14, was synthesized in order to increase the conforma-
tional freedom of the ribose ring, yet maintain a
structure very similar to that of a ribose moiety. As seen
with the 2′-deoxy derivative 9, the acyclic analogue 14
displayed significantly less activity than the parent
compound. Similarly, the antitrypanosomal activity of
compound 36, whose hydroxyethoxymethyl functionality
can serve as a ribose ring isostere (e.g., acyclovir), was
substantially decreased compared to AbeAdo (Table 1).
The findings that compounds 9, 14, and 36 display
consistently lowered biological activities compared to
AbeAdo indicate that the ribose ring of AbeAdo is highly
sensitive to structural modifications.
a
(a) NaIO4/NaBH3; (b) methylamine; (c) cis-tert-butoxycarbonyl-
4-chloro-2-butenyl-1-amine; (d) acetic anhydride/DMAP; (e) trif-
luoroacetic acid.
Synthesis of the phenyl derivatives of AbeAdo, 24 and
25, is shown in Scheme 3. The 2′,3′-isopropylidene
derivative of adenosine was chlorinated and aminated
to give analogues 18 and 19, respectively. Intermediate
19 was then N-alkylated with either R-bromo-p-tolu-
enitrile or R-bromo-o-toluenitrile to yield 20 and 21,
respectively. The nitrile groups of 20 and 21 were
reduced with LiAlH4, and the isopropylidene groups
were removed under acidic conditions to give the desired
analogues 24 and 25. The 2-aminoadenosine derivative
of AbeAdo, 30, was synthesized in an analogous manner
(Scheme 4).
The synthesis of 36, a ribose isostere of AbeAdo, is
illustrated in Scheme 5. Adenine was N-alkylated with
acetylethoxymethyl bromide to give 31. The acetyl group
was saponified, and the corresponding alcohol was
chlorinated as previously indicated to yield 33. Amina-
tion with methylamine, followed by alkylation with the
butenyl arm moiety and final deprotection, was done
as previously described to yield the expected final
analogue 36.
Biologica l Resu lts a n d Discu ssion
AbeAdo analogue 30, which contains 2,6-diaminopu-
rine in place of adenine, was synthesized to determine
what effects, if any, modifications to the purine sub-
stituent might have on the antitrypanosomal activity
of AbeAdo. Disappointingly, this modification caused at
least an order of magnitude decrease in activity.
In Vitr o An titr yp a n osom a l Activity. The in vitro
antitrypanosomal activities of AbeAdo and its related
analogues were determined in the LAB 110 EATRO
isolate of Trypanosoma brucei brucei and three clinical
isolates of Trypanosoma brucei rhodesiense: KETRI 243
(melarsoprol- and diamidine-resistant), KETRI 269, and
KETRI 243 As 10-3 (highly arsenical resistant). The
data are shown in Table 1. In this assay, compound 10
displayed the most pronounced inhibition of trypano-
somal growth. The remaining analogues, with the
exception of 4, 24, and 25, were also growth inhibitory.
Finally, to potentially improve the cellular uptake of
these AbeAdo-derived structures, the O-acetylated de-
rivatives of certain analogues were prepared. Acetyla-
tion of 2′,3′-seco-AbeAdo, 14, gave 16, whose in vitro
antitrypanosomal activity showed an increase, on aver-
age, of 6-fold in the EATRO and 243 strains, compared

S-Adenosylmethionine Decarboxylase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5115
Sch em e 3. Synthesis of Benzyl Derivatives of AbeAdoa
a
(a) Thionyl chloride; (b) methylamine; (c) R-bromo-p-tolunitrile or R-bromo-o-tolunitrile; (d) LiAlH4; (e) H2SO4.
Sch em e 4. Synthesis of 2-Amino Derivative of AbeAdoa
In Vivo An titr yp a n osom a l Activity. AbeAdo and
its acetylated derivative, 10, were further tested in mice
against several trypanosomal infections (Table 3). When
mice infected with T. b. brucei LAB 110 EATRO were
treated with 10, this AbeAdo derivative was curative
in several dose regimens. The activity of 10 paralleled
that of the parent compound, AbeAdo, when dosed in
two equal injections (b.i.d). Compound 10 was also
active in the same strain when administered by con-
tinuous infusion using 7 day pumps. The di-O-acetylated
derivative, 10, was protective but not curative against
T. b. rhodesiense (KETRI 243), a strain that is resistant
to the arsenical, melarsoprol (Arsobal), and the diami-
dine pentamidine. This strain was more sensitive to the
parent compound. AbeAdo was curative for four out of
five mice receiving 100 mg kg-1 day-1 in 7 day pump
dosing. In contrast, compound 10, at the highest dose
administered, increased life span by ∼68% (Table 4).
Since compound 10 was ∼4 times more active in vitro
a
(a) Thionyl chloride; (b) methylamine; (c) cis-tert-butoxycar-
bonyl-4-chloro-2-butenyl-1-amine; (d) H2SO4.
to that of its unacetylated parent compound, 14 (Table
1). Similarly, 10, the di-O-acetylated derivative of
AbeAdo, was 10 times more active in vitro than AbeAdo
in the EATRO and 269 strains (Table 1).

5116 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Marasco et al.
Sch em e 5. Synthesis of Ethoxymethyl Ribose Analogue of AbeAdoa
a
(a) Stir at room temp; (b) NaH; (c) sodium methoxide; (d) thionyl chloride; (e) methylamine; (f) cis-tert-butoxycarbonyl-4-chloro-2-
butenyl-1-amine; (g) H2SO4.
Ta ble 1. In Vitro Antitrypanosomal Activity of AbeAdo
Derivatives (IC50, µM)a
Ta ble 3. In Vivo Activity of AbeAdo and Its Acetylated
Derivative, 10, against T. b. brucei LAB 110 EATRO
trypanosomal strain
daily dose
(mg/kg)
treatment MSDa
time (days) (days) (no. cured)/total
compound
control
compound 110b
243c
269c 243 As 10-3c
3
0/5
AbeAdo
0.1
0.04
0.22
d
74
0.02
d
3.8
d
d
0.1
d
30
0.054
d
2.9
d
d
4
44% @ 100 µM 26% @ 100 µM
AbeAdo
10 ip (b.i.d.)
25 ip (b.i.d.)
50 ip (b.i.d.)
3
3
3
12
>34
>34
4/5
5/5
5/5
9
4.2
2.9
10
14
16
24
25
30
36
0.014
16
0.014
15
10
10 ip (b.i.d.)
25 ip (b.i.d.)
50 ip (b.i.d.)
25 (pump)
50 (pump)
100 (pump)
3
3
3
7
7
7
17
33
>34
>34
>34
26
4/5
4/5
5/5
5/5
5/5
4/5
1.8
4.6
21% @ 100 µM 23% @ 100 µM
14
4.5
10
42% @ 100 µM
3.8
6.6
19.0
11
2.0
0.96
a
IC50 is the concentration required to inhibit the growth of
a
MSD is the mean survival in days.
trypanosomes by 50%. EATRO strain. c KETRI strain. Not
b
d
determined.
Ta ble 4. In Vivo Activity of AbeAdo and Its Acetylated
Derivative, 10, against T. b. rhodesiense KETRI 243
Ta ble 2. In Vitro Inhibition of AdoMetDC from L1210 Murine
Leukemia Cells
daily dose
(mg/kg)
treatment MSDa
time (days) (days) (no. cured)/total
compound
compound
ID50 (µM)a
control
AbeAdo
8.8
31
31.3
32
0/5
2/5
2/5
4/5
AbeAdo
9
10
14
24
25
36
0.078
25 (pump)
50 (pump)
100 (pump)
7
7
7
20
0.82
27% inhibition at 100 µM
21% inhibition at 100 µM
41% inhibition at 100 µM
>100
control
10
13.4
15.6
18.0
21.2
0/5
0/5
0/5
0/5
10 (pump)
25 (pump)
50 (pump)
7
7
7
a
ID50 value is the concentration required to inhibit the assay
a
MSD is the mean survival in days.
of AdoMetDC activity by 50%.
Since KETRI 243 isolate consists of a heterogeneous
population with respect to drug resistance, derivative
10 was also examined for activity against four cloned
subpopulations of KETRI 243. This agent, administered
at 100 mg kg-1 day-1 (7 day pump), was 40-100%
curative against clones selected for resistance to dia-
midines but only 20% curative against an arsenical-
resistant clone (K243 As-10-3). Compound 10 was also
against KETRI 243 than AbeAdo (Table 1). The finding
that 10 is less active than AbeAdo against KETRI 243
in vivo at the same doses (25 and 50 mg kg-1 day-1 in
7 day pumps) was unexpected. The dose-response
relationship observed at the low concentrations tested
suggested that at higher doses 10 might produce cures
against KETRI 243. No further in vivo experiments
were done to substantiate this prediction.

S-Adenosylmethionine Decarboxylase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5117
Ta ble 5. In Vitro Antineoplastic Activity of AbeAdo Derivatives in Human and Murine Tumor Cell Lines
b
cell linea/IC50 (µM)
compound
A121
A549
HT-29
MCF7
MCF7-Adr
L1210
AbeAdo
4
9
10
14
16
24
25
36
>100
>100
d
(3.0-100)c
>100
>100
>100
d
>100
>100
d
51 ( 8.7
63 ( 6.1
>100
>100
d
200
>100
d
>100
78 ( 6.1
>100
>100
d
>600
500
20
>600
d
>600
>600
d
d
(1-30) ( 3.4c
41 ( 1.9
>100
>100
d
(3-10) ( 2.8c
>100
(3-30) ( 3.2c
112 ( 3.9
>100
>100
d
>100
>100
d
>100
>100
>100
>100
>100
a
Cell lines are A121 (human ovarian carcinoma), A549 (human non-small-cell lung carcinoma), HT-29 (human colon adenocarcinoma),
MCF7 (human breast adenocarcinoma), MCF7-Adr (adriamycin-resistant human breast adenocarcinoma), and L1210 (murine leukemia).
b
Concentration of drug necessary to inhibit cell growth by 50% after 72 h; see Experimental Section. c A plateau in cell growth was
observed over a range of drug concentrations. This profile is typical of inhibitors of polyamine biosynthesis, since the cells are slowly
d
depleted of polyamine pools. Not determined.
Ta ble 6. In Vitro Anti-HIV Activity of AbeAdo and Its
curative against two other T. b. rhodesiense strains:
Acetylated Derivative, 10
KETRI 2002 and KETRI 2538. KETRI 2002 infected
differential
selectivity
(TC50/EC50)
mice were cured by a daily dose of 100 mg/kg adminis-
tered in a 7 day pump, while KETRI 2538 infected mice
responded to ip dosing and 3 day pump regimens. In
an earlier study, the parent compound AbeAdo had
equivalent activity against both strains.
compound
EC50 (µM)a
TC50 (µM)b
AbeAdo
10
0.93
0.05
<6.25
12.0
∼6.7
240
a
EC50 is the concentration required to inhibit plaque formation
by 50%. TC50 is the concentration that causes morphological
In Vitr o An tin eop la stic Activity. The in vitro
antineoplastic activity of AbeAdo and its related ana-
logues was determined against a variety of human
tumor cell lines (A121 ovarian carcinoma, A549 non-
small-cell lung carcinoma, HT-29 colon adenocarcinoma,
MCF7-S breast adenocarcinoma, and MCF7-Adr, adria-
mycin-resistant breast carcinoma) and is shown in Table
5. In general, both AbeAdo and its analogues were
devoid of significant antineoplastic activity against all
the tumor cell lines assayed. However, as seen previ-
ously, acetylation of AbeAdo caused a significant in-
crease (10-fold) in antineoplastic activity as evidenced
by analogue 10 versus AbeAdo. As previously stated,
the greater efficacy of AbeAdo as an inhibitor of trypa-
nosomal growth, as opposed to mammalian cells, may
be due to the active uptake of AbeAdo into trypanosomes
via a purine transport system not found in mammalian
cells.11 Consequently, the enhanced antineoplastic activ-
ity of analogue 10 may be due to an increase in the
passive uptake and activation of this analogue in
mammalian cells.
In Vitr o En zym e In h ibitor y Activity. To deter-
mine whether the acetylated analogues 10 and 16 could
serve as substrates of the target enzyme, AdoMetDC,
or were likely acting as prodrugs of AbeAdo, they were
tested for their ability to inhibit AdoMetDC in vitro. The
results are shown in Table 2. Although there is no direct
correlation between the inhibition of AdoMetDC in vitro
and antitrypanosomal activity, for the most part, inhibi-
tors of this enzyme are also inhibitors of trypanosomal
growth. Interestingly, the acetylated derivative of
AbeAdo, 10, is a substrate/inhibitor of AdoMetDC and
may not be acting solely as a prodrug.
b
changes in noninfected CEM-SS cells.
results, the antiviral activity of AbeAdo and compound
10 was determined by use of a syncytial plaque assay
in which HIV-1 infected CEM-SS cells, a syncytial clone,
were treated with various concentrations of the sensitive
compounds. The results, shown in Table 6, are ex-
pressed as EC50 and TC50 values. The EC50 value is a
measure of the concentration required to inhibit plaque
formation by 50% (EC ) effective concentration). The
TC50 value is a measure of the concentration that causes
morphological changes in noninfected CEM-SS cells (TC
) toxic concentration). The TC50 value is predictive of
cell cytotoxicity and inhibition of cell growth. Dif-
ferential selectivity, defined as TC50/EC50, is used to
identify potential anti-HIV compounds. An analogue
with a differential selectivity greater than 100 is
considered to be an effective inhibitor of HIV infectivity,
as opposed to a nonspecific inhibitor of CEM-SS cell
growth. The differential selectivity of AbeAdo (∼6.7)
suggests that its inhibition of HIV infectivity is due to
nonspecific cytotoxicity in CEM-SS cells. In contrast, the
differential selectivity of compound 10 is 240. Thus,
compound 10 effectively inhibits HIV infectivity in
CEM-SS cells at a concentration 240-fold less than that
required to induce cytotoxicity in uninfected CEM-SS
cells. The difference in activity between these two
analogues will have to be further investigated.
Con clu sion s
The majority of structural modifications made to
AbeAdo had a deleterious affect on both the antitrypa-
nosomal and antineoplastic activity of this compound.
However, di-O-acetylation of the parent compound
produced 10, a prodrug compound with markedly pro-
nounced inhibition of trypanosomal and neoplastic cell
growth and viability.
New structural inhibitors of AdoMet decarboxylase
continue to be designed and synthesized as potential
antitrypanosomal and antineoplastic agents.26,27 Our
In Vitr o An tivir a l Activity. Polyamine pools, es-
pecially at the levels of putrescine, are elevated in the
lymphocytes of patients with overt AIDS.15 However,
in another study, White et al.16 did not observe elevated
polyamine pools in HIV-1 infected CEM cells. Further-
more, AdoMetDC inhibitors such as MHZPA (5′-deoxy-
5′-[(3-hydrazinopropyl)methylamino]-5′-adenosine) did
not inhibit HIV-1 infectivity. Undaunted by these

5118 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Marasco et al.
2.5 × 105 trypanosomes from a rat infection. Compounds were
dissolved in water. Treatment was begun 24 h after infection
and consisted of twice per day (b.i.d.) intraperitoneal injections
(9 a.m. and 4 p.m.) for 3 days. Alternatively, 7 day Alza
miniosmotic pumps were loaded and implanted aseptically
according to the manufacturer’s instructions. Mice were
monitored daily for viability, and tail vein blood smears were
taken weekly and checked for trypanosomes. Animals surviv-
ing >30 days beyond the deaths of untreated control mice with
no evidence of blood parasitemia were considered cured.
Deter m in a tion of An tivir a l Activity. This assay was
conducted as previously reported.25 Individual wells of a 96-
well plate were coated with 50 µL of poly-L-lysine (50 µg/mL,
MW ) 90 000), incubated for 30 min at room temperature and
washed 3 times with 0.01 M phosphate-buffered 0.85% (w/v)
sodium chloride (PBS). Aliquots of CEM-SS cells in log-phase
growth were washed 3 times with PBS, suspended at a density
of 50 000 CEM-SS cells per 50 µL of RPMI-1640 medium
without serum, transferred to the precoated wells, and incu-
finding that compound 10 (but not its parent compound
AbeAdo) inhibits HIV-1 growth and infectivity identifies
a new target for possible therapeutic intervention by
these nucleoside inhibitors of AdoMet decarboxylase.
Exp er im en ta l Section
Hu m a n Tu m or Cell Lin es. The A121 ovarian carcinoma
cell line was obtained from Dr. Kent Crickard, SUNY Buffalo.17
Other human tumor cell lines including A549 non-small-cell
lung carcinoma, HT-29 colon adenocarcinoma, MCF7-S breast
adenocarcinoma. and MCF7-R adriamycin-resistant breast
adenocarcinoma were purchased from the American Type
Culture Collection. All cell lines are propagated as monolayers
in RPMI-1640 containing 5% FCS, 5% NuSerum IV, 20 mM
HEPES, and 2 mM L-glutamine at 37 °C in a 5% CO2
humidified atmosphere. The doubling times for the cell lines
range between 20 and 28 h.
Gr ow th In h ibition Assa y in 96-Well Micr otiter P la tes.
Assessment of cell growth inhibition was determined according
to the methods of Skehan et al.18 Briefly, cells were plated
between 400 and 1200 cells/well in 96-well plates and incu-
bated at 37 °C 15-18 h prior to drug addition to allow
attachment of cells. Compounds tested were solubilized in
100% DMSO and further diluted in RPMI-1640 containing 10
mM HEPES to a maximal final DMSO concentration of
e0.25%, which is not growth inhibitory. Each cell line was
treated with 10 concentrations of compound (5 log range). After
a 72 h incubation, 100 µL of ice-cold 50% TCA was added to
each well and incubated for 1 h at 4 °C. Plates were then
washed 5 times with tap water to remove TCA, low-molecular-
weight metabolites, and serum proteins. A 50 µL aliquot of
0.4% sulforhodamine B (SRB), an anionic protein stain, was
added to each well. At cell densities ranging from very sparse
to supraconfluent, SRB staining changes linearly with in-
creases or decreases in number of cells and protein concentra-
tions. These staining characteristics provide an accurate
assessment of cell growth.18 Following a 5 min incubation at
room temperature, plates were rinsed 5 times with 0.1% acetic
acid and were air-dried. Bound dye was solubilized with 10
mM Tris base (pH 10.5) for 5 min on a gyratory shaker. Optical
density was measured at 570 nm.
Da ta An a lysis. Data were fit with the Sigmoid-Emax
concentration-effect model19 with nonlinear regression, weight-
ed by the reciprocal of the square of the predicted response.
The fitting software was developed at RPCI with MicroSoft
FORTRAN and uses the Marquardt20 algorithm as adapted
by Nash21 for the nonlinear regression. The concentration of
drug that inhibited growth by 50% (IC50 ) was determined.
Tr yp a n osom e Str a in s. The T. b. brucei LAB 110 EATRO
isolate was obtained from W. Trager of Rockefeller Univer-
sity.22 Clinical isolates of T. brucei rhodeiense (KETRI 243 and
KETRI 269) were obtained from A. R. Njogu of the Kenya
Trypanosomiasis Research Institute (KETRI). The KETRI 243
isolate is resistant to diamidines and melarsoprol; KETRI 243
As 10-3 is a clone of KETRI 243, which is highly resistant to
melarsoprol.23 These strains were adapted to grow under
axenic conditions in the bloodstream form in HMI-18 medium
with hypoxanthine at 1 µM and 20% horse serum instead of
synthetic serum.24
bated for 30 min at 37 °C in
a humidified atmosphere
containing 5% CO2. The cell monolayer was then inoculated
with 50 µL of HIV-1 (60-90 plaque forming units) diluted in
growth medium. Compounds were formulated in growth
medium for testing. After 120 min, the cell monolayer was
overlaid with 100 µL of growth medium with or without test
compounds and was incubated as before at 37 °C. Serial 2-fold
dilutions of test compounds (0.00015-20 µM) were tested.
After 3 days, each well received a second 100 µL overlay of
growth medium with or without compound, and incubation
was continued for an additional 2 days. On day 5, the syncytial
plaques were counted and the concentration required to inhibit
50% of plaque formation (EC50) was determined.
Syn th etic Meth od s. 1H NMR spectra were recorded on
either a Bruker 400 MHz or Varian EM 390 spectrometer.
Chemical shifts are reported in parts per million (δ) relative
to tetramethylsilane. Column chromatography was performed
using silica gel 60 (230-400 mesh). TLC was done using EM
Industries aluminum sheets (precoated with silica gel 60 F254),
and preparative TLC was done using Analtech uniplates (silica
gel GF, 20 cm × 20 cm, and 1000 µm thick). The purity of all
final compounds was determined by HPLC (McPherson FL75,
C18 column, mobile phase CH3CN/10 mM ammonium phos-
phate, pH 4.4) and verifed by elemental analysis (Robertson
Microlit Laboratories, Inc. of Madison, NJ ).
cis-ter t-Bu toxycar bon yl-4-ch lor o-2-bu ten yl-1-am in e (1).
This intermediate was prepared according to literature pro-
cedures with analogous yields.5
5′-Deoxy-5′-ch lor oa d en osin e (2). This intermediate was
prepared according to the procedure of Robbins et al.12
5′-Deoxy-5′-ch lor o-2′-d eoxya d en osin e (3). This interme-
diate was prepared according to the procedure of Robbins et
al.12
5′-Deoxy-5′-m eth yla m in oa d en osin e (4). 5′-Deoxy-5′-chlo-
roadenosine (2) (5.224 g, 18.3 mmol) was chilled to -70 °C in
a metal bomb, and 45 mL of condensed anhydrous methyl-
amine was added. The bomb was sealed, heated to 55 °C for
72 h, and then cooled to -70 °C. The bomb was unsealed and
allowed to come slowly to room temperature. After the excess
methylamine had boiled off, the resulting thick oil was taken
up in H2O (50 mL) and allowed to stand for 18 h at room
temperature. The mixture was filtered to remove unreacted
2, and the filtrate was concentrated in vacuo. The thick oil
was then extracted with EtOAc/H2O (11:1) until TLC (CH2-
Cl2/MeOH, 5:1) indicated only product was present. The thick
oil was dried under vacuum to give 4 (2.489 g, 49%) as a
hygroscopic solid. 1H NMR (DMSO-d6): δ 2.50 (s, 3H, CH3-
NH), 3.30 (m, 2H, 5′-CH2), 4.25 (m, 2H, 2′- and 3′-CH), 4.75
(m, 1H, 4′-CH), 5.65 (br m, 2H, OH), 5.90 (m, 1H, 1′-CH), 7.25
(s, 2H, NH2), 8.15 (s, 1H, Ad-H8), and 8.40 (s, 1H, Ad-H2).
Anal. (C11H16N6O3‚1.25H2O) C, H, N.
Deter m in a tion of in Vitr o An titr yp a n osom a l Activity.
Drug studies were done in duplicate 24-well plates (1 mL per
well), with final inhibitor concentrations of 0.1, 1, 10, 25, and
100 µM. After 48 h, the number of parasites was determined
in a Z1 Coulter counter and the approximate range of activity
was determined. The 50% inhibitory concentrations (IC50
values) were then determined from additional studies with
closely spaced inhibitor concentrations. Inhibitors with <50%
inhibition at 100 µM were not studied further. Most analogues
were dissolved in water, and several were dissolved in 100%
dimethyl sulfoxide. Dilutions were made with HMI-18 medium
so that dimethyl sulfoxide concentrations never exceeded 0.3%,
a noninhibitory concentration.
5′-Deoxy-5′-m eth yla m in o-2′-d eoxya d en osin e (5). This
intermediate was prepared from compound 3 (0.186 g, 0.7
mmol) and 25 mL of anhydrous methylamine (96 h) in a
manner analogous to that of 4. The crude product was purified
In Vivo Activity vs Tr yp a n osom e Mou se Mod el In fec-
tion s. Mice (20 g of female Swiss-Webster) were infected with

S-Adenosylmethionine Decarboxylase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5119
on preparative silica gel plates developed in CH2Cl2/MeOH (4:
1). The appropriate bands were scraped and strirred with 100
mL of CH2Cl2/MeOH (7:3), the silica gel was filtered, and the
filtrate was concentrated in vacuo to give 0.119 g (65%) of
product.
CH2), 3.50-3.65 (m, 4H, CH2NHCHdCHCH2), 4.15-4.45 (m,
2H, 3′- and 4′-CH), 5.85 (m, 2H, CHdCH), 6.40 (m, 1H, 1′-
CH), 7.3 (s, 2H, NH2), 8.15 (s, 1H, Ad-H8), and 8.30 (s, 1H,
Ad-H2). MS: (M + H) 334.5, (M + H + 1.2TFA) 467.9.
cis-5′-Deoxy-5′-(4-a m in o-2-bu ten yl)m eth yla m in o-2′,3′-
d i-O-a cetyla d en osin e (10). A solution of 8 (0.107 g, 0.2
mmol) in trifluoroacetic acid/CH2Cl2 (1:1) was allowed to stand
at room temperature for 90 min. The reaction mixture was
concentrated in vacuo and coevaporated 3× with CH2Cl2. The
resulting oil was taken up in 5 mL of CH2Cl2, 1 mL of MeOH,
and 50 mL of ether and placed at -20 °C for 18 h. The solution
was decanted, and the precipitate was tritrated 2× with ether.
The white solid was dried under vacuum to give 0.010 g (66%)
cis-5′-Deoxy-5′-(4-ter t-bu toxycar bon ylam in o-3-bu ten yl)-
m eth yla m in oa d en osin e (6). A solution of 4 (0.228 g, 0.81
mmol), 1 (0.182 g, 0.88 mmol), K2CO3 (0.113 g, 0.82 mmol),
and NaI (0.123 g, 0.82 mmol) in DMF (10 mL) was heated to
85 °C for 72 h. The reaction mixture was cooled to room
temperature, diluted with H2O (50 mL), and extracted with
CH2Cl2 until TLC (CH2Cl2/MeOH, 4:1) indicated the absence
of product in the aqueous layer. The CH2Cl2 extracts were
pooled, dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by silica gel chromatography
(continuous gradient of CH2Cl2/MeOH, 4:1, and 2% triethyl-
amine) and the appropriate fractions were pooled, treated with
charcoal, filtered through Celite, and concentrated in vacuo
to give 0.248 g (69%) of product as a white solid. 1H NMR
(CDCl3): δ 1.45 [s, 9H, C(CH3)3], 2.50 (s, 3H, CH3NH), 3.15
(m, 2H, 5′-CH2), 3.75 (m, 2H, CH2NHCH), 4.25 (m, 4H, 2′- and
3′-CH, t-BOC-NH-CH2), 4.75 (m, 1H, 4′-CH), 5.65 (br m, 2H,
OH), 5.50 (m, 2H, CHdCH), 5.95 (m, 1H, 1′-CH), 6.6-6.9 (m,
3H, NH2, NH), 8.05 (s, 1H, Ad-H8), and 8.20 (s, 1H, Ad-H2).
1
of product. H NMR (CD3OD): δ 2.00 [d, 6H, (COCH3)2], 2.80
(s, 3H, CH3NH), 3.30-3.95 (m, 6H, 5′-CH2-N-CH2-CHdCH-
CH2-NH3), 4.30 (m, 2H, 2′- and 3′-CH), 4.95 (m, 1H, 4′-CH),
5.95 (m, 2H, CHdCH), 6.25 (m, 1H, 1′-CH), 8.25 (m, 2H, Ad-
H8 and Ad-H2). Anal. (C19H28N7O5‚21/2CF3COOH‚CH3OH) C,
H, N, F.
5′-Deoxy-5′-ch lor o-2′,3′-seco-a d en osin e (11). This inter-
mediate was prepared as previously reported from 2 (1.30 g,
4.6 mmol) to yield 1.14 g of product as a waxy solid (86%).12
1H NMR (DMSO-d6): δ 3.40-3.90 (m, 7H, 2′,3′-CH2, 4′-CH,
5′-CH2), 5.10 (br m, 2H, HO), 5.90 (t, 1H, 1′-CH), 7.20 (m, 2H,
NH2), and 8.20-8.40 (m, 2H, Ad-H2 and H8).
cis-5′-Deoxy-5′-(4-ter t-bu toxycar bon ylam in o-3-bu ten yl)-
m eth yla m in o-2′-d eoxya d en osin e (7). This intermediate
was prepared in an analogous manner to that of 6. Compound
5 (0.113 g, 0.43 mmol), 1 (0.111 g, 0.54 mmol), K2CO3 (0.071
g, 0.51 mmol), and NaI (0.077 g, 0.51 mmol) in DMF (15 mL)
were heated to 85 °C for 72 h. The crude product was applied
to preparative silica gel plates and was developed with CH2-
Cl2/MeOH (3:1) and the appropriate bands were scraped,
stirred with CH2Cl2/MeOH (7:3), filtered, and concentrated in
5′-Deoxy-5′-m eth yla m in o-2′,3′-seco-a d en osin e (12). This
intermediate was prepared from 11 (0.348 g, 1.2 mmol) and
anhydrous methylamine (8 mL) in a manner analogous to that
of 4. The crude product was purified on preparative silica gel
plates developed in CH2Cl2/MeOH (4:1). The appropriate bands
were scraped and stirred with 100 mL of CH2Cl2/MeOH (7:3).
The silica gel was filtered, and the filtrate was concentrated
1
in vacuo to give 0.105 g (31%) of product. H NMR (CD3OD-
1
vacuo to give 0.099 g (53%) of product. H NMR (CD3OD): δ
d6): δ 2.30 (s, 3H, CH3NH), 2.80 (m, 2H, 5′-CH2), 3.75-4.15
(m, 5H, 2′,3′-CH2, 4′-CH), 6.10 (t, 1H, 1′-CH), and 8.20-8.40
(m, 2H, Ad-H2 and H8).
1.45 [s, 9H, C(CH3)3], 2.30 (overlapping m, 5H, 2′-CH2, CH3-
NH), 2.75 (m, 2H, 5′-CH2), 3.20 (m, 2H, CH2NHCH), 3.60 (m,
2H, t-BOC-NH-CH2), 4.1-4.4 (m, 2H, 3′- and 4′-CH), 5.50
(m, 2H, CHdCH), 6.35 (m, 1H, 1′-CH), 8.20 (s, 1H, Ad-H8),
and 8.30 (s, 1H, Ad-H2).
cis-5′-Deoxy-5′-(4-ter t-bu toxycar bon ylam in o-3-bu ten yl)-
m eth yla m in o-2′,3′-seco-a d en osin e (13). This intermediate
was prepared from 12 (0.105 g, 0.37 mmol), 1 (0.091 g, 0.44
mmol), K2CO3 (0.057 g, 0.41 mmol), and NaI (0.061 g, 0.41
mmol) in DMF (15 mL), and the mixture was heated to 85 °C
for 72 h in a manner analogous to that of 6. The crude product
was applied to preparative silica gel plates and was developed
with CH2Cl2/MeOH (2.5:1) and the appropriate bands were
scraped, stirred with CH2Cl2/MeOH (7:3), filtered, and con-
cis-5′-Deoxy-5′-(4-ter t-bu toxycar bon ylam in o-3-bu ten yl)-
m eth yla m in o-2′,3′-d i-O-a cetyla d en osin e (8). A solution of
6 (0.248 g, 0.55 mmol), acetic anhydride (125 µL, 1.32 mmol),
triethylamine (208 µL, 1.45 mmol), and 4-(dimethylamino)-
pyridine (5 mg, 0.04 mmol) in CH3CN (10 mL) was stirred at
room temperature for 3 h. MeOH (5 mL) was added to the
reaction mixture, and the solution was stirred an additional
30 min. The reaction mixture was concentrated in vacuo, and
the resulting oil was taken up in CH2Cl2 (50 mL), washed
once× with saturated NaHCO3 (50 mL) and once with H2O
(50 mL), dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was applied to two preparative silica gel
plates and was developed in CH2Cl2/MeOH (4:1). The ap-
propriate bands were scraped, stirred with CH2Cl2/MeOH (7:
3), and filtered, and the filtrate was concentrated in vacuo to
yield 0.211 g (72%) of product as a white solid. 1H NMR
(CDCl3): δ 1.45 [s, 9H, C(CH3)3], 2.10 [d, 6H, (COCH3)2], 2.25
(s, 3H, CH3NH), 2.80 (m, 2H, 5′-CH2), 3.10 (m, 2H, CH2NHCH),
3.80 (m, 2H, t-BOC-NH-CH2), 4.30 (m, 2H, 2′- and 3′-CH),
4.95 (m, 1H, 4′-CH), 5.50 (m, 2H, CHdCH), 5.90-6.10 (m, 4H,
1′-CH, NH2, NH), 8.00 (s, 1H, Ad-H8), and 8.30 (s, 1H, Ad-
H2).
cis-5′-De oxy-5′-(4-a m in o-2-b u t e n yl)m e t h yla m in o-2′-
d eoxya d en osin e (9). This intermediate was prepared from
7 (0.118 g, 0.28 mmol) and trifluoroacetic acid/CH2Cl2 (1:1, 8
mL) in a manner analogous to that of 10. The reaction mixture
was concentrated in vacuo and azeotroped 3× with CH2Cl2.
The resulting orange oil was taken up in MeOH and treated
with charcoal, and the solution was filtered (0.2 µm) and
concentrated in vacuo. The pale-yellow oil was precipitated 4×
from MeOH and ether, and the product was taken up in
MeOH, decolorized with charcoal, filtered (0.2 µm) into a tared
vial, and dried to constant weight to give 78 mg (60%) of
product as a very hygroscopic solid. 1H NMR (DMSO-d6): δ
2.30 (m, 2H, 2′-CH2), 2.55 (s, 3H, CH3NH), 3.35 (m, 2H, 5′-
1
centrated in vacuo to give 0.048 g (29%) of product. H NMR
(DMSO-d6): δ 1.30 [s, 9H, C(CH3)3], 2.50-2.70 (s, 5H, CH3-
NH, 5′-CH2), 3.30-3.90 (m, 9H, 2′ , 3′-CH2, 4′-CH, CH2CHd
CHCH2), 5.10-5.40 (m, 4H, CHdCH, OH), 5.90 (t, 1H, 1′-CH),
7.20 (s, 2H, NH2), and 8.10-8.30 (m, 2H, Ad-H2 and H8).
cis-5′-Deoxy-5′-(4-a m in o-2-bu ten yl)m eth yla m in o-2′,3′-
seco-a d en osin e (14). This intermediate was prepared from
13 (0.048 g, 0.10 mmol) in trifluoroacetic acid/CH2Cl2 (1:1, 2
mL) in a manner analogous to that of 9 to afford 0.056 g (88%)
of product as a pale-yellow hygroscopic solid. 1H NMR (DMSO-
d6): δ 2.50-2.60 (m, 5H, CH3NH, 5′-CH2), 3.60-4.10 (m, 9H,
2′,3′-CH2, 4′-CH, CH2CHdCHCH2), 5.35 (br m, 2H, OH), 5.85
(m, 2H, CHdCH), 6.00 (t, 1H, 1′-CH), 7.50 (s, 2H, NH2), 8.15
(m, 2H, NH2), and 8.20-8.50 (m, 2H, Ad-H2 and H8). Anal.
(C15H24N7O3‚1.2CF3COOH‚0.5CH3OH‚1H2O) C, H, N, F.
cis-5′-Deoxy-5′-(4-ter t-bu toxycar bon ylam in o-3-bu ten yl)-
m eth yla m in o-2′,3′-d i-O-a cetyl-seco-a d en osin e (15). This
intermediate was prepared from 13 (0.07 g, 0.16 mmol), acetic
anhydride (360 µL, 0.38 mmol), triethylamine (545 µL, 0.38
mmol), and (dimethylamino)pyridine (0.002 g, 0.08 mmol) in
CH3CN (20 mL) in a manner analogous to that of 8. The crude
product was applied to preparative silica gel plates and was
developed in CH2Cl2/MeOH (6:1). The appropriate bands were
scraped, stirred with CH2Cl2/MeOH (7:3), and concentrated in
vacuo to give 0.068 g (79%) of product. 1H NMR (CDCl3): δ
1.80-2.30 (m, 9H, CH3NH, 2′,3-CH3CO), 2.80 (m, 2H, 5′-CH2),
3.40-4.50 (m, 9H, 2′,3′-CH2, 4′-CH, CH2CHdCHCH2), 5.50 (m,
2H, NH2), 6.00 (m, 2H, CHdCH), 6.20 (t, 1H, 1′-CH), and 8.10-
8.30 (m, 2H, Ad-H2 and H8).

5120 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Marasco et al.
cis-5′-Deoxy-5′-(4-a m in o-2-bu ten yl)m eth yla m in o-2′,3′-
d i-O-a cetyl-seco-a d en osin e (16). This congener was pre-
pared from 15 (0.068 g, 0.13 mmol) in trifluoroacetic acid/
CH2Cl2 (1:1, 6 mL) in a manner analogous to that of 10 to
afford 0.070 g (76%) of product as a hygroscopic solid. 1H NMR
(DMSO-d6): δ 1.90-2.10 (m, 6H, 2′,3-CH3CO), 2.50 (s, 3H,
CH3NH), 3.15 (m, 2H, 5′-CH2), 3.50 (br m, 8H, CH2CHd
CHCH2, 2′,3′-CH2), 4.70 (m, 1H, 4′-CH), 5.60-5.80 (m, 2H,
CHdCH), 6.20 (t, 1H, 1′-CH), 7.50 (br s, 2H, NH2), 8.10 (br s,
2H, NH2), and 8.20-8.40 (m, 2H, Ad-H2 and H8). Anal.
(C19H30N7O5‚2.5CF3COOH‚0.75H2O) C, H, N, F.
decomposed by the slow addition of H2O. The reaction mixture
was then diluted with H2O (100 mL) and allowed to stand at
room temperature overnight. The solution was filtered to
remove inorganic salts, and the filtrate was concentrated in
vacuo. The resulting thick oil was taken up in a minimum
amount of MeOH to further remove inorganic salts and was
filtered, and the filtrate was concentrated in vacuo. The crude
product was applied to preparative silica gel plates and was
developed with CH2Cl2/MeOH/Et3N (20:5:0.5). The appropriate
bands were scraped, stirred with CH2Cl2/MeOH (7:3) and 2%
Et3N (v/v), filtered, and concentrated in vacuo to give 0.146 g
(52%) of a white, foamy solid. 1H NMR (CD3OD): δ 1.4-1.6
[2s, 6H, C(CH3)3], 2.25 (s, 3H, CH3NH), 2.50 (m, 2H, H2NCH2),
3.35 (m, 2H, 5′-CH2), 3.85 [m, 2H, CH2-N(CH3)CH], 4.30 (m,
1H, 4′-CH), 4.90-5.30 (m, 2H, 2′- and 3′-CH), 6.10 (m, 2H,
1′-CH), 7.10 (s, 4H, aromatic Hs of phenyl), 7.90 (s, 1H, Ad-
H8), and 8.10 (s, 1H, Ad-H2).
5′-Deoxy-5′-ch lor o-2′,3′-isop r op ylid en ea d en osin e (18).
This intermediate was prepared according to the procedure of
Robbins et al.11
5′-Deoxy-5′-m et h yla m in o-2′,3′-isop r op ylid en ea d en o-
sin e (19). This intermediate was prepared from 17 (0.569 g,
1.75 mmol) and anhydrous methylamine (15 mL) in a manner
analogous to that of 3. The crude product was purified on
preparative silica gel plates developed in CH2Cl2/MeOH (7:1).
The appropriate bands were scraped and stirred with 100 mL
of CH2Cl2/MeOH (7:3), the silica gel was filtered, and the
filtrate was concentrated in vacuo to give 0.392 g (63%) of
product. 1H NMR (CDCl3/CD3OD): δ 1.4-1.6 [2s, 6H, C(CH3)3],
2.50 (s, 3H, CH3NH), 3.30 (m, 2H, 5′-CH2),), 4.50 (m, 1H, 4′-
CH), 5.10-5.50 (m, 2H, 2′- and 3′-CH), 6.10 (m, 2H, 1′-CH),
8.00 (s, 1H, Ad-H8), and 8.30 (s, 1H, Ad-H2).
5′-Deoxy-5′-(o-a m in om eth ylp h en yl)m eth yla m in o-2′,3′-
isop r op ylid en ea d en osin e (23). This intermediate was pre-
pared from 21 (0.481 g, 1.0 mmol) and LiAlH4 (0.128 g, 3.0
mmol) in a manner analogous to that of 22. The crude product
was applied to a silica gel column packed in CH2Cl2/MeOH
(16:1 + 5% Et3N v/v) and eluted with a continuous gradient
to 2:1 + 5% Et3N v/v. The appropriate fractions were pooled,
concentrated in vacuo, and dried under vacuum to give 0.133
1
g (28%) of product as a foamy white solid. H NMR (CD3OD):
δ 1.4-1.6 [2s, 6H, C(CH3)3], 2.25 (s, 3H, CH3NH), 2.55 (m, 2H,
H2NCH2), 3.50 (m, 2H, 5′-CH2), 3.75 [m, 2H, CH2-N(CH3)-
CH], 4.30 (m, 1H, 4′-CH), 4.90-5.30 (m, 2H, 2′- and 3′-CH),
6.10 (m, 2H, 1′-CH), 7.10 (s, 4H, aromatic Hs of phenyl), 7.90
(s, 1H, Ad-H8), and 8.10 (s, 2H, Ad-H2).
5′-Deoxy-5′-(p -t olu en it r ile)m et h yla m in o-2′,3′-isop r o-
p ylid en ea d en osin e (20). This intermediate was prepared in
a manner analogous to that of 6. Compound 19 (0.392 g, 1.1
mmol), R-bromo-p-toluenitrile (0.237 g, 1.2 mmol), K2CO3
(0.166 g, 1.2 mmol), and NaI (0.181 g, 1.2 mmol) were placed
in CH3CN (25 mL) and refluxed for 18 h. The reaction mixture
was cooled to room temperature, diluted with 100 mL of
EtOAc, and washed with brine, and the organic layer was dried
over MgSO4, filtered, and concentrated in vacuo. The crude
product was applied to preparative silica gel plates and was
developed with CH2Cl2/MeOH (7:1). The appropriate bands
were scraped, stirred with CH2Cl2/MeOH (7:3), filtered, and
concentrated in vacuo to give 0.281 g (54%) of a clear waxy
5′-Deoxy-5′-(p -a m in om et h ylp h en yl)m et h yla m in oa d -
en osin e (24). Compound 22 (0.141 g, 0.3 mmol) was taken
up in 1.0 N H2SO4 (5 mL) and was allowed to stand at room
temperature for 18 h. The solution was diluted with 200 mL
of EtOH and placed at -20° C for 18 h. The solution was
decanted, and the precipitate was redissolved in a minimum
amount of H2O, diluted with 200 mL of EtOH, and placed at
-20° C for 18 h. The solution was decanted, and the solid was
dissolved in MeOH, transferred to a tared vial, and dried under
vacuum to constant weight to give 0.133 g (65%) of product as
1
solid. H NMR (CDCl3): δ 1.4-1.6 [2s, 6H, C(CH3)3], 2.30 (s,
3H, CH3NH), 3.50 (m, 2H, 5′-CH2),), 4.50 (m, 1H, 4′-CH), 5.00-
5.50 (m, 2H, 2′- and 3′-CH), 5.85 (m, 2H, CH2-toluenitrile), 6.10
(m, 2H, 1′-CH), 7.40 (m, 4H, aromatic Hs of toluenitrile), 8.00
(s, 1H, Ad-H8), and 8.30 (s, 1H, Ad-H2).
1
a white hygroscopic powder. H NMR (DMSO-d6): δ 2.25 (s,
3H, CH3NH), 2.50 (m, 2H, H2NCH2), 3.45 (m, 2H, 5′-CH2), 3.85
[m, 2H, CH2-N(CH3)CH], 4.20-4.50 (m, 3H, 2′-, 3′-, 4′-CH),
5.40 (m, 1H, OH), 5.80 (m, 1H, OH), 6.10 (m, 1H, 1′-CH), 6.30
(bs, 2H, NH2), 7.10-7.30 (m, 6H, aromatic Hs of phenyl, NH2),
7.90 (s, 1H, Ad-H8), and 8.10 (s, 1H, Ad-H2). Anal. (C19H26N7O3‚
2.3H2SO4‚1.5CH3OH‚1H2O) C, H, N, S.
5′-Deoxy-5′-(o-t olu en it r ile)m et h yla m in o-2′,3′-isop r o-
p ylid en ea d en osin e (21). This intermediate was prepared in
a manner analogous to that of 20. Compound 19 (0.472 g, 1.3
mmol), R-bromo-o-toluenitrile (0.317 g, 1.6 mmol), K2CO3
(0.224 g, 1.6 mmol), and NaI (0.241 g, 1.6 mmol) were placed
in CH3CN (25 mL) and refluxed for 18 h. The reaction mixture
was cooled to room temperature, diluted with 100 mL of
EtOAc, and washed with brine, and the organic layer was dried
over MgSO4, filtered, and concentrated in vacuo. The crude
product was applied to a silica gel column packed in EtOAc
and eluted isocratically with EtOAc/MeOH (25:1). The ap-
propriate fractions were pooled, concentrated in vacuo, and
dried under vacuum to give 0.481 g (77%) of product. 1H NMR
(CDCl3) δ 1.4-1.6 [2s, 6H, C(CH3)3], 2.30 (s, 3H, CH3NH), 3.65
(m, 2H, 5′-CH2), 4.45 (m, 1H, 4′-CH), 5.00-5.50 (m, 2H, 2′-
and 3′-CH), 6.10 (m, 2H, CH2-toluenitrile), 6.20 (m, 2H, 1′-
CH), 7.40 (m, 4H, aromatic Hs of toluenitrile), 7.90 (s, 1H, Ad-
H8), and 8.25 (s, 1H, Ad-H2).
5′-Deoxy-5′-(p-a m in om eth ylp h en yl)m eth yla m in o-2′,3′-
isop r op ylid en ea d en osin e (22). A solution of 20 (0.281 g,
0.59 mmol) in anhydrous THF (10 mL) was added dropwise
to a solution of LiAlH4 (0.035 g, 0.88 mmol) in anhydrous THF
(20 mL) under an inert atmosphere. The solution was stirred
at room temperature for 2 h, at which time TLC indicated the
presence of starting material (∼40%). An additional 0.88 mmol
of hydride was added to the reaction mixture. After an
additional 2 h, TLC still indicated the presence of starting
material (∼20%). Another 0.88 mmol of hydride was added to
the reaction mixture. After an additional hour, TLC indicated
the absence of starting material. Unreacted hydride was
5′-Deoxy-5′-(o-a m in om et h ylp h en yl)m et h yla m in oa d -
en osin e (25). This intermediate was prepared from 23 (0.113
g, 0.24 mmol) in 1 N H2SO4 (5 mL) in a manner analogous to
that of 23. The product was isolated as previously detailed to
yield 0.102 g (69%) of product as a white hygroscopic powder.
1H NMR (DMSO-d6): δ 2.30 (s, 3H, CH3NH), 2.50 (m, 2H, H2-
NCH2), 3.50 (m, 2H, 5′-CH2), 3.80 [m, 2H, CH2-N(CH3)CH],
4.25-4.45 (m, 3H, 2′-, 3′-, 4′-CH), 5.60 (m, 1H, OH), 5.80 (m,
1H, OH), 6.10 (m, 1H, 1′-CH), 6.45 (bs, 2H, NH2), 7.10 (s, 4H,
aromatic Hs of phenyl), 7.40 (bs, 2H, NH2), 7.90 (s, 1H, Ad-
H8), and 8.10 (s, 1H, Ad-H2). Anal. (C19H26N7O3‚1.9H2SO4‚CH3-
OH) C, H, N, S.
2′,3′-Isop r op ylid en e-2-a m in oa d en osin e (26). To a solu-
tion of 2-aminoadenosine (0.793 g, 2.8 mmol) in acetone (340
mL) and 2,2-dimethoxypropane (1 mL) was added 70% per-
chloric acid (1.3 mL). The solution was stirred at room
temperature for 1 h and was neutralized by the addition of
pyridine. The solution was concentrated in vacuo, and the
resulting oil was taken up in 15 mL of MeOH and allowed to
stand for 18 h at room temperature. The solution was filtered,
and the filtrate was concentrated in vacuo. The obtained crude
product was purified on preparative silica gel plates developed
in CH2Cl2/MeOH (8:1). The appropriate bands were scraped
and stirred with 100 mL of CH2Cl2/MeOH (7:3), the silica gel
was filtered, and the filtrate was concentrated in vacuo to give
0.873 g (97%) of product as a foamy solid. 1H NMR (DMSO-

S-Adenosylmethionine Decarboxylase Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5121
d6): δ 1.3-1.5 [2s, 6H, C(CH3)3], 2.50 (m, 1H, OH), 3.55 (m,
2H, 5′-CH2), 4.20 (m, 1H, 4′-CH), 5.00-5.30 (m, 2H, 2′- and
3′-CH), 5.95 (m, 1H, 1′-CH), 7.50 (m, 2H, NH2), 8.20 (s, 1H,
8-CH), and 8.45 (m, 2H, NH2).
5′-Deoxy-5′-ch lor o-2′,3′-isop r op ylid en e-2-a m in oa d en o-
sin e (27). This intermediate was prepared according to the
procedure of Robbins et al.11 1H NMR (CDCl3): δ 1.3-1.5 [2s,
6H, C(CH3)3], 3.80 (m, 1H, 4′-CH), 4.40 (m, 2H, 5′-CH2), 4.95
(m, 2H, NH2), 5.10-5.40 (m, 2H, 2′- and 3′-CH), 5.95 (m, 1H,
1′-CH), 6.10 (2, 2H, NH2), and 7.55 (s, 1H, 8-CH).
up into MeOH, absorbed onto silica gel, and then applied to
the top of a silica gel column packed in CH2Cl2. The column
was eluted with 150 mL portions of CH2Cl2/MeOH (from 100:0
to 95:5) and then isocratically at 94:6 until all desired product
eluted from the column. The appropriate fractions were pooled,
concentrated in vacuo, and dried under vacuum to give 0.948
g (85%) of product. 1H NMR (DMSO-d6): δ 3.25 (m, 2H,
ClCH2CH2O), 3.65 (m, 2H, ClCH2CH2O), 5.60 (s, 2H, OCH2-
Ad), 8.20 (s, 1H, Ad-H8), and 8.30 (s, 1H, Ad-H2).
9-(Meth yla m in oeth ylm eth oxy)a d en in e (34). This inter-
mediate was prepared from 33 (0.109 g, 0.36 mmol) in
anhydrous methylamine (20 mL) in a manner analogous to
that of 4 (60 °C for 72 h). The excess methylamine was
evaporated, the crude product was then tritrated 3× with CH2-
Cl2, and the resulting oil was dried under vacuum to give 1.116
5′-Deoxy-5′-m et h yla m in o-2′,3′-isop r op ylid en e-2-a m i-
n oa d en osin e (28). This intermediate was prepared from 27
(0.109 g, 0.36 mmol) and anhydrous methylamine (20 mL,
reaction time 96 h) in a manner analogous to that of 4. The
crude product was purified on preparative silica gel plates
developed in CH2Cl2/MeOH (5:1). Appropriate bands were
scraped and stirred with 100 mL of CH2Cl2/MeOH (7:3), the
silica gel was filtered, and the filtrate was concentrated in
vacuo to give 0.134 g of product. 1H NMR (CD3OD): δ 1.3-
1.5 [2s, 6H, C(CH3)3], 2.55 (s, 3H, CH3NH), 3.10 (m, 2H, 5′-
CH2), 3.60 (m, 1H, 4′-CH), 5.00-5.30 (m, 2H, 2′- and 3′-CH),
6.00 (m, 1H, 1′-CH), and 7.80 (s, 1H, 8-CH).
cis-5′-Deoxy-5′-(4-ter t-bu toxycar bon ylam in o-3-bu ten yl)-
m eth ylam in o-2′,3′-isopr opyliden e-2-am in oaden osin e (29).
This intermediate, prepared from 28 (0.134 g, 0.40 mmol), 1
(0.093 g, 0.46 mmol), K2CO3 (0.069 g, 0.46 mmol), and NaI
(0.074 g, 0.50 mmol) in CH3CN (25 mL), was refluxed for 18 h
in a manner analogous to that of 6. The crude product was
applied to preparative silica gel plates and was developed with
CH2Cl2/MeOH (1:1) and the appropriate bands were scraped,
stirred with CH2Cl2/MeOH (7:3), filtered, and concentrated in
vacuo to give 0.058 g (29%) of product. 1H NMR (CDCl3): δ
1.45 [s, 12H, C(CH3)3, C-CH3), 1.51 (s, 3H, C-CH3), 2.40 (s,
3H, CH3NH), 3.15 (m, 2H, 5′-CH2), 3.75 (m, 2H, CH2NHCH),
4.30 (m, 4H, 2′- and 3′-CH, t-BOC-NH-CH2), 4.85 (m, 1H,
4′-CH), 5.50 (m, 2H, CHdCH), 5.95 (m, 1H, 1′-CH), and 7.65
(s, 1H, Ad-H8).
cis-5′-Deoxy-5′-(4-am in o-2-bu ten yl)m eth ylam in o-2-am i-
n oa d en osin e (30). This intermediate was prepared from 29
(0.0.58 g, 0.12 mmol) in 1 N H2SO4 (5 mL) in a manner
analogous to that of 24. The product was isolated as previously
detailed to yield 0.053 g (69%) of product as a white hygro-
scopic powder. NMR (DMSO-d6): δ 2.40 (s, 3H, CH3NH), 3.15
(m, 2H, 5′-CH2), 3.45-3.70 (m, 4H, CH2NHCHdCHCH2),
4.20-4.40 (m, 2H, 2′- and 3′-CH), 4.55 (m, 1H, 4′-CH), 5.70
(m, 2H, CHdCH), 6.0 (m, 1H, 1′-CH), 6.80 (bs, 2H, NH2), 7.25
(bs, 2H, NH2), and 7.65 (s, 1H, Ad-H8). Anal. (C15H25N8O3‚2.3H2-
SO4‚1.5CH3CH2OH) C, H, N, S.
9-(Acetoxyeth ylm eth oxy)a d en in e (31). This intermedi-
ate was synthesized according to the procedure of Robbins et
al.12 1H NMR (CDCl3): δ 2.00 (s, 3H, CH3), 3.85 (m, 2H,
AcOCH2CH2O), 4.30 (m, 2H, AcOCH2CH2O), 5.70 (s, 2H,
OCH2-Ad), 8.20 (s, 1H, Ad-H8), and 8.30 (s, 1H, Ad-H2).
9-(Hyd r oxyeth ylm eth oxy)a d en in e (32). Compound 27
was suspended in MeOH (20 mL). To this solution was added
1 M sodium methoxide (1.7 mL), and the solution was allowed
to stir at room temperature for 2 h. Several grams of silica
gel were added to the reaction mixture, and the solution was
then concentrated in vacuo. The crude product, once absorbed
onto silica gel, was placed on top of a silica gel column packed
in CH2Cl2 and eluted with 150 mL portions of CH2Cl2/MeOH
(from 100:0 to 95:5) and then isocratically at 94:6 until all
desired product eluted from the column. The appropriate
fractions were pooled, concentrated in vacuo, and dried under
vacuum to give 0.991 g (99%) of product as a white powder.
1H NMR (DMSO-d6): δ 3.35-3.50 (m, 4H, AcOCH2CH2O), 4.65
(m, 1H, HO), 5.60 (s, 2H, OCH2-Ad), 8.20 (s, 1H, Ad-H8), and
8.30 (s, 1H, Ad-H2).
1
g (92%) of a white hygroscopic foam. H NMR (DMSO-d6): δ
2.55 (s, 3H, CH3NH), 3.15 (m, 2H, HNCH2CH2O), 3.85 (m, 2H,
HNCH2CH2O), 5.65 (s, 2H, OCH2-Ad), 8.20 (s, 1H, Ad-H8), and
8.30 (s, 1H, Ad-H2).
cis-(4-ter t-Bu toxycar bon ylam in o-3-bu ten yl)m eth ylam i-
n o(eth ylm eth oxy)a d en in e (35). This intermediate was pre-
pared from 34 (0.522 g, 2.5 mmol), 1 (0.758 g, 3.8 mmol), K2CO3
(0.494 g, 3.6 mmol), and NaI (0.531 g, 3.5 mmol) in DMF (20
mL) heated to 85 °C for 48 h in a manner analogous to that of
6. The reaction mixture was concentrated in vacuo. The crude
product was applied to a silica gel column packed in CH2Cl2
and eluted with 150 mL portions of CH2Cl2/MeOH (from 100:0
to 91:9) and then isocratically at 90:10 until all desired product
eluted from the column. The appropriate fractions were pooled,
concentrated in vacuo, and dried under vacuum to give 0.183
1
g (19%) of product as a waxy solid. H NMR (CDCl3): δ 1.45
[s, 12H, C(CH3)3], 2.45 (s, 3H, CH3NH), 2.95 (m, 2H, NCH2CH2O),
3.45 (m, 2H, t-BOC-NHCH2), 3.80 (m, 4H, CHdCHCH2NCH2-
CH2O), 5.65 (m, 4H, CHdCH, OCH2-Ad), 8.15 (s, 1H, Ad-H8),
8.30 (s, 1H, Ad-H2).
cis-(4-Am in o-2-bu ten yl)m eth yla m in o(eth ylm eth oxy)-
a d en in e (36). This intermediate was prepared from 35 (0.162
g, 0.40 mmol) in 1 N H2SO4 (2 mL) in a manner analogous to
that of 24. The reaction mixture was diluted with EtOH (100
mL), and a minimum amount of H2O/MeOH was added to
achieve a homogeneous mixture. The solution was further
diluted with 50 mL of ether and placed at -20 °C for 18 h.
The solution was decanted, and the precipitate was redissolved
in H2O (2 mL), EtOH (150 mL), and ether (100 mL) and was
placed at -20 °C for 18 h. The solution was decanted, and the
precipitate was dissolved in H2O, filtered (0.2 µm), concen-
trated in vacuo, azeotroped 4× with EtOH/MeOH, and dried
under vacuum at 45 °C for 18 h to give 0.138 g (64%) of product
as a hygroscopic solid. 1H NMR (DMSO-d6): δ 2.45 (s, 3H, CH3-
NH), 2.95 (m, 2H, NCH2CH2O), 3.60-3.75 (m, 6H, H2NCH2-
CHdCHCH2NCH2CH2O), 5.75 (m, 4H, CHdCH, OCH2-Ad),
7.20 (bs, 2H, NH2), 8.15 (s, 1H, Ad-H8), 8.30 (s, 1H, Ad-H2).
Anal. (C13H21N7O‚H2SO4‚CH3OH‚H2O) C, H, N, S.
Ack n ow led gm en t. These studies were supported in
part by Grant 920082 (J .R.S.) and Grant 950594 (C.J .B.)
from the United Nations Development Programme/
World Bank World Health Organization Special Pro-
gramme for Research and Training in Tropical Diseases.
We gratefully acknowledge the efforts of Dr. J ames
Alderfer, Director of the NMR facility at Roswell Park
Cancer Institute and support by the Roswell Park
Cancer Institute Core Grant (Grant CA16056). The
authors also thank Paula Diegelman for her assistance
in determining the purity of our final analogues via
HPLC, and Marc Vargas for assistance with the trypa-
nosome model infections.
9-(Ch lor oeth ylm eth oxy)a d en in e (33). CCl4 (664 µL, 7.0
mmol) was added dropwise to a solution of 32 (0.972 g, 4.65
mmol) and triphenylphosphine (2.444 g, 9.30 mmol) in anhy-
drous pyridine (80 mL). The reaction mixture was stirred
overnight at room temperature under an inert atmosphere of
argon. The reaction mixture was concentrated in vacuo, taken
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