New insights into the design of inhibitors of human S-adenosylmethionine decarboxylase: studies of adenine C8 substitution in structural analogues of S-adenosylmethionine

1388

J. Med. Chem. 2009, 52, 1388–1407

New Insights into the Design of Inhibitors of Human S-Adenosylmethionine Decarboxylase:

Studies of Adenine C⁸Substitution in Structural Analogues of S-Adenosylmethionine^†

Diane E. McCloskey,^§Shridhar Bale,^‡John A. Secrist, III,^|Anita Tiwari,^|Thomas H. Moss, III,^|Jacob Valiyaveettil,^|

,#

Wesley H. Brooks, Wayne C. Guida, Anthony E. Pegg,^§and Steven E. Ealick*^,‡

Department of Chemistry and Chemical Biology, Cornell UniVersity, Ithaca, New York 14853-1301, Departments of Cellular and Molecular

Physiology and of Pharmacology, Milton S. Hershey Medical Center, PennsylVania State UniVersity College of Medicine, Hershey,

PennsylVania 17033, Southern Research Institute, Birmingham, Alabama 35225-5305, Drug DiscoVery Program, H. Lee Mofﬁtt Cancer Center

& Research Institute, Tampa, Florida 33612, Departments of Chemistry and Oncologic Sciences, UniVersity of South Florida,

Tampa, Florida 33620

ReceiVed September 9, 2008

S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway

and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme

with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual

syn conformation when bound to the enzyme. To determine whether compounds that favor the syn

conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate

analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their

ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in

more potent inhibitors, yet most other 8-substitutions provided no beneﬁt over the parent compound. To

understand these results, we used computational modeling and X-ray crystallography to study C⁸-substituted

adenine analogues bound in the active site.

Introduction

polyamine pathway is a therapeutic strategy for treatment of

various types of cancers. AdoMetDC catalyzes the conversion

of S-adenosylmethionine (AdoMet) to decarboxylated S-adeno-

sylmethionine (dcAdoMet), which then donates the aminopropyl

group to putrescine or spermidine to form spermidine and

spermine, respectively. AdoMetDC is at a key branch point in

the pathway and its action commits AdoMet to polyamine

biosynthesis and removes it from the pool available for methyl

transfer to a variety of substrates.

S-Adenosylmethionine decarboxylase (AdoMetDC^a) is a

pyruvoyl dependent decarboxylase and a critical enzyme in the

polyamine biosynthetic pathway, which is found in mammals,

Protista, and many other species.^1-4The polyamines putrescine,

spermidine, and spermine are essential for cell growth and play

important roles in cell proliferation and differentiation.^5-7

Polyamines have been found to be elevated in various types of

cancer including non-small-cell lung cancer, prostate cancer,

melanoma, and pancreatic cancer.^8,9Polyamine levels in cells

depend on the polyamine biosynthetic and catabolic pathways

as well as on import and export of polyamines across the cellular

membrane. Altering regulation of the key enzymes in the

Attempts to regulate polyamine levels have resulted in the

development of inhibitors that target the biosynthetic enzymes

ornithine decarboxylase (ODC),¹⁰AdoMetDC, and the catabolic

enzyme spermidine/spermine N¹-acetyltransferase (SSAT).¹¹The

best-known inhibitor of ODC is R-diﬂuoromethylornithine

(DFMO), which irreversibly inactivates the enzyme. The success

of DFMO in cancer therapy has been limited as the cells

compensate for the decreased synthesis of polyamines through

increased cellular uptake of polyamines.¹²DFMO is currently

being investigated as a chemopreventive agent against carcino-

genesis.^13-17The development of drugs to inhibit AdoMetDC

(Figure 1A) started with the competitive inhibitor methylglyoxal

bis(guanylhydrazone) 1 (MGBG), which is similar to spermidine

in structure.¹⁸Use of 1 caused extreme toxicity in humans, and

many analogues of 1 were developed in attempts to decrease

the toxicity. One such AdoMetDC inhibitor that resulted was

4-amidinoindan-1-one-2′-amidinohydrazone 2 (CGP48664A¹⁹),

which progressed into clinical trials as a cancer chemothera-

peutic agent.¹⁹Alternatively, inhibitors such as 5′-deoxy-5′-[(3-

hydrazinopropyl)methylamino]adenosine 3(MHZPA), 5′-deoxy-

5′-[(3-hydrazinoethyl)methylamino)adenosine 4 (MHZEA),

and 5′-[(2-aminooxyethyl)methylamino]-5′-deoxyadenosine

5 (MAOEA), which are structural analogues of the natural

substrate, were developed (Figure 1B). These compounds

inactivate AdoMetDC by forming a Schiff base to the active

site pyruvoyl group.²⁰Another known nucleoside inhibitor

of AdoMetDC is 5′-[[(Z)-4-amino-2-butenyl]methylamino]-

†

The Protein Data Bank codes for the complexes are under the following

accession numbers: wild type AdoMetDC with 12a (3DZ5), 14e (3DZ6),

17d (3DZ4), 17f (3DZ7) and 21c (3DZ2), and F223A mutant AdoMetDC

with MeAdoMet (3DZ3).

* To whom correspondence should be addressed. Phone: (607) 255-7961.

Fax: (607) 255-1227. E-mail: see3@cornell.edu.

‡

Department of Chemistry and Chemical Biology, Cornell University.

Departments of Cellular and Molecular Physiology and of Pharmacol-

§

ogy, Milton S. Hershey Medical Center, Pennsylvania State University

College of Medicine.

|

Southern Research Institute.

Drug Discovery Program, H. Lee Mofﬁtt Cancer Center & Research

Institute.

#

Departments of Chemistry and Oncologic Sciences, University of South

Florida.

^aAbbreviations: AdoMetDC, S-adenosylmethionine decarboxylase; Ado-

Met, S-adenosylmethionine; ODC, ornithine decarboxylase; SSAT, sper-

midine/spermine N¹-acetyltransferase; DFMO, R-diﬂuoromethylornithine;

MGBG 1, methylglyoxal bis(guanylhydrazone); MHZPA 3, 5′-deoxy-5′-

[(3-hydrazinopropyl)methylamino]adenosine; MAOEA 5, 5′-[(2-amino-

oxyethyl)methylamino]-5′-deoxyadenosine; MHZEA 4, 5′-deoxy-5′-[(3-

hydrazinoethyl)methylamino)adenosine; MeAdoMet, methyl ester of S-

adenosylmethionine; hAdoMetDC, human S-adenosylmethionine decar-

boxylase; DMF, dimethylformamide; DTT, dithiothreitol; Tris, tris(hydroxy-

methyl)aminomethane; PEG, poly(ethylene glycol); CCD, charge-coupled

device; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid;

DIEA, diisopropylethylamine.

10.1021/jm801126a CCC: $40.75

2009 American Chemical Society

Published on Web 02/11/2009

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1389

The syntheses of some of the 8-unsubstituted compounds date

back to our earlier work,²⁶and these compounds dictated our

initial synthetic approaches. We began by assuming that we

needed to block the 2′- and 3′-hydroxyl groups, which we did

with an isopropylidene group. Later, we discovered that it was

possible to conduct the chemistry without blocking these two

hydroxyl groups and that the new schemes were superior to

those that utilized a blocking-deblocking sequence. In situations

where we had already prepared a target compound utilizing a

blocked precursor, we did not go back and resynthesize the

compound without using a blocking group, and the schemes

below reﬂect that fact. Scheme 1 presents the precursor

nucleoside series 8 and 9 that we have used along with their

syntheses.

Target compounds with an aminooxyalkylamino side chain

at C-5′ were prepared using two different routes, as shown in

Scheme 2. In our original sequence, which utilized a 2′,3′-O-

isopropylidene group for protection, we generated the hydroxy-

alkylamino precursor 15 by displacement of a tosyl group with

the requisite amine. Using N-hydroxyphthalimide, triphenylphos-

phine, and DEAD,²⁷the aminooxy precursor 16 was produced

and then converted to the desired target 5 under acidic

conditions. Later, we found that it was more effective to ﬁrst

generate the aminooxy precursors ethyl N-(2-bromoethoxy)

ethanimidate²⁸and ethyl N-(N-4-bromobutoxy)ethanimidate²⁹

(Scheme 2), which could be appended to C-5′ by halide

displacement with a 5′-methylamino-5′-deoxynucleoside to

produce product series 11 and 13. Initially, we carried out this

displacement with an isopropylidene protecting group on the

nucleoside but subsequently determined that the reaction works

as well or better without the protecting group. By the above

means targets 12a-c and 14a-f were prepared.

All of the amides and hydrazides were made by similar

procedures, as shown in Scheme 3. The 5′-methylamino-5′-

deoxynucleosides were treated with the appropriate ω-chlo-

roester, followed by treatment with either ammonia or hydrazine.

If an isopropylidene group was involved, then it was removed

with an acidic deprotection step. In this manner targets 17d-f,

j-m, with two different linker lengths and various 8-substitu-

ents, were prepared.

Targets with an aminoalkylamino side chain at C-5′ were

mainly prepared utilizing the displacement of a C-5′ leaving

group with the unsymmetrical amine (Scheme 4). For example,

treatment of 8a with 3-methylaminoethylamine produced a

mixture of 18f and 19d, which were separated to afford pure

18f, our desired target. In the case where this procedure involved

a starting material with an isopropylidene group, treatment with

acid produced the desired ﬁnal product. In early work, com-

pounds 21c,d were prepared by treatment of a 5′-methylamino-

5′-deoxynucleoside with 3-bromopropylphthalimide followed by

two deprotection steps.

Building on the aminoalkylamino side chain, reaction of 18e

with 1-carboxamidinopyrazole³⁰produced the guanidine target

22a. In a related sequence, the target amidoxime 22c was

prepared by treating 8j with 3-(methylamino)propionitrile to

produce the nitrile 22b,³¹which was treated with hydroxylamine

hydrochloride under basic conditions.

Figure 1. Previously described inhibitors of hAdoMetDC.

5′-deoxyadenosine. This butenyl analogue was designed as

an enzyme-activated irreversible inhibitor,²¹but subsequent

experiments showed that it acted via transamination of the

pyruvate prosthetic group.²⁰

The crystal structure of AdoMetDC and its S68A and H243A

mutants were solved to aid understanding of the mechanisms

of decarboxylation and autoprocessing.^22-24The crystal struc-

tures of AdoMetDC with inhibitors like 5, 3, and the methyl

ester of S-adenosylmethionine (MeAdoMet) have been solved

previously.²⁵These structures show that the adenine base of

the inhibitors assumes an unusual syn conformation within the

active site. The preference for the unusual conformation has

led us to develop new structural analogues of AdoMet with

modiﬁcations on the adenine base and to investigate, through

biochemical analysis, computational modeling and analysis of

crystal structures, whether these compounds would be more

potent inhibitors of AdoMetDC than the unsubstituted parent

compounds. Substitution at the 8-position of adenine is expected

to result in ligands that favor the syn conformation in solution,

and it was hoped that this would increase their ability to inhibit

AdoMetDC. We now describe the synthesis of several series

of structural analogues of AdoMet with 8-substituted adenine

and present AdoMetDC inhibition data. We report the crystal

structures of the AdoMetDC F223A mutant complexed with

MeAdoMet and the wild-type protein complexed with several

8-substituted inhibitors.

Results

Chemical Synthesis. Our synthetic efforts relating to AdoMet-

DC date back many years, when we prepared an early series of

related compounds that included 3 and 5.²⁶In our current

research, we have prepared a series of compounds with various

8-substituents on an adenosine template having a chain extension

at C-5′. These compounds fall into four broad categories with

respect to the various substituents at C-5′, and synthetic schemes

will be organized based upon these categories. For comparison

purposes, we have included available compounds with an 8-H

within the four categories. End groups of the C-5′ substituent

such as an aminooxyalkyl will bind covalently and to a large

extent irreversibly to the pyruvoyl group within the active site

of the enzyme, while groups ending in an amide will not even

bind reversibly covalently to the pyruvoyl group. Amino end

groups will bind covalently, but entirely reversibly, while a

hydrazide group binds with some reversibility. In addition to

these compounds, we have prepared several compounds without

a chain extension at C-5′, i.e., compounds that do not reach the

vicinity of the pyruvate group within the binding site.

The 5′-dimethylamino and 5′-dimethylsulfonio compounds

23a,b and 25a-d were prepared by routine methods (Scheme

5). The dimethylamino group was introduced by displacement

of a 5′-chlorine on 8a or 8g³²with dimethylamine. The

5′-methylthio compounds 24a,b were treated with methyl

bromide to produce 25a and 25c. Ion exchange was utilized to

prepare the chloride salts 25b and 25d. 8-Methyl-5′-methylthio

1390 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

Scheme 1^a

^a(a) (CH₃)₄Sn or (CH₃CH₂)₄Sn, HMDS/dioxane, NMP, (Ph₃P)₄Pd, 110 °C; (b) CH₃NH₂, MeOH, 110 °C; (c) C₆H₅B(OH)₂, K₂CO₃, (Ph₃P)₄Pd, 1,2-DME-

H₂O (2:1), 90 °C; (d) SOCl₂, CH₃CN/pyridine, 0 °C-RT, NH₄OH, RT; (e) MsCl, pyridine, 0 °C; (f) 33% CH₃NH₂, EtOH, RT (9e,f,g) or 90 °C (9a-d); (g)

NaOMe/MeOH, RT.

nucleoside 24a was prepared by displacement of the 5′-chlorine

in 8a with sodium thiomethoxide.

in the AdoMetDC active site was performed using the mixed

Monte Carlo/low mode conformational search method within

the MacroModel program.^34-36The conformational search

started with MeAdoMet in either the anti or syn conformation,

and in each case the ﬁve lowest energy structures from the

search exhibited a syn conformation for the adenine nucleoside.

A superposition of the modeled structure with the crystal

structure (Figure 2) indicates that the results of the conforma-

tional search match well with those observed crystallographi-

cally. Conformational searches were also done for AdoMet, 5′-

deoxy-5′-(dimethylsulfonio)adenosine (MMTA), 3, and 5 binding

to AdoMetDC, and each yielded a syn conformation for the

glycosidic bond (data not shown). The ribose makes key

hydrogen bonds to Glu247 and the adenine base stacks between

Phe7 and Phe223 and also makes hydrogen bonds to the

backbone amide and C-terminal carboxyl group of Glu67. These

interactions together with π-π stacking of the adenine base

with Phe223 and with Phe7 constrain the glycosidic bond to

the syn conformation.

Modeling of MeAdoMet in the Active Site of AdoMetDC.

The crystal structures of AdoMetDC complexed with MeAdo-

Met or the inhibitors 3 and 5 have shown that the ligand binds

with the adenine base in the unusual syn conformation.²⁵The

active site residues of AdoMetDC with MeAdoMet bound are

shown in Figure 2. However, NMR data, coupled with molecular

modeling studies, suggest that in solution AdoMet assumes an

anti conformation as an energy minimum.³³A survey of crystal

structures in which AdoMet is bound showed that the substrate

assumes a range of glycosidic torsion angles but that the anti

conformation is preferred.³³To explain the conformational

preferences and the related energetics of ligand binding to

AdoMetDC, the modeling of MeAdoMet in the active site of

AdoMetDC was done. Because MeAdoMet is tethered to the

active site of AdoMetDC through covalent bonding to the

pyruvoyl group, docking involving positional and orientational

sampling was not performed. Instead, a conformational search

to locate the populated low energy conformations of MeAdoMet

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1391

Scheme 2^a

^a(a) CH₃(OEt)CdNO(CH₂)₂Br (ref 28), DMF, DIEA, 50 °C; (b) 1 N H₂SO₄, RT; (c) CH₃(OEt)CdNO(CH₂)₄Br (ref 29), DMF, DIEA, 50 °C; (d)

2-(methylaminoethanol), RT; (e) N-hydroxyphthalimide, PPh₃, DEAD, THF (ref 40), RT; (f) 1 N H₂SO₄, 60 °C.

Scheme 3^a

^a(a) Cl(CH₂)_nCO₂Et (n ) 1 or 2), DMF, DIEA, 60 °C; (b) NH₃/MeOH, RT; (c) 1 N H₂SO₄, RT; (d) NH₂NH₂, H₂O, EtOH, reﬂux.

Virtual Mutations in the Active Site of AdoMetDC. Virtual

mutations were made to study the effect of various residues on

the conformation of the bound nucleoside. Conformational

searching with MacroModel employing the AdoMetDC F223A

and F7A single amino acid mutants, with MeAdoMet in the

active site, resulted in a mixture of syn and anti conformations

in the low energy ensemble. With each of the mutations, the

global minimum was an anti conformation of the adenine base,

closely followed by a syn conformation with an energy

difference of ∼2 kJ/mol. The global minimum energy confor-

mation of the ligand bound in the anti conformation in the

F223A mutant exhibits major changes compared to the second

lowest energy conformer which adopts the syn conformation.

In the F223A binding site, the ribose of the global minimum

energy structure is displaced and makes hydrogen bonds to

Glu247 and Cys226 instead of to Glu247 alone (Figure 3A).

This change causes the ligand to twist back upon itself, the

sulfonium stacks over the adenine base, and the adenine base

makes three hydrogen bonds to Ser66. In the F7A binding

site, the ligand assumes a similar conformation as with the

F223A mutant. The Phe223 side chain undergoes a torsional

change to accommodate the conformational change of the

ligand and also stacks with the adenine base (Figure 3B).

The presence of the anti conformation in low energy

structures of the ligand in the enzyme active site where virtual

mutations have been made suggests the importance of the

phenyl groups in maintaining the syn conformation of the

ligand within the wild-type enzyme binding site. However,

since we observed a syn conformation of the nucleoside as

the second lowest energy structure in our conformational

search on the F223A mutant and since the relative energy of

that structure compared to the global minimum (∆E ) 2.5

kJ/mol) is well within the error limit of our calculations, we

were prompted to obtain the crystal structure of the F223A

mutant complexed with MeAdoMet.

Structure of F223A Mutant Complexed with MeAdoMet.

The structure of the F223A mutant is similar to that of the wild

type protein.²²The human AdoMetDC (hAdoMetDC) protomer

has a four layer RꢀꢀR fold in which two ꢀ-sheets are

sandwiched between two layers of R-helices. The secondary

1392 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

Scheme 4^a

^a(a) CH₃NH(CH₂)_nNH₂(n ) 1 or 2), RT; (b) 1 N H₂SO₄, RT; (c) 3-bromopropylphthalimide, DMF, DIEA, 60 °C; (d) NH₂NH₂, H₂O, reﬂux; (e) 1H-

pyrazole-1-carboxamidine·HCl (ref 30), DMF, DIEA, RT; (f) 3-(methylamino)propionitrile, RT (ref 31); (g) NH₂OH·HCl, MeOH, DMF, KOH, RT.

Scheme 5^a

^a(a) (CH₃)₂NH, 2 M solution in MeOH, 90 °C; (b) CH₃SNa, DMF, RT; (c) CH₃Br, Et₂O, HCO₂H, HOAc, RT; (d) IRA-400 (Cl^-) ion exchange resin.

structural elements are related by a pseudo 2-fold axis, sug-

gesting that the protomer resulted from gene duplication. The

proenzyme consists of 334 amino acid residues, and the enzyme

undergoes autoprocessing to give the R and the ꢀ subunits.²²

The autoprocessing reaction yields the active enzyme with the

pyruvoyl cofactor. The pyruvoyl group is located at the end of

the N-terminal ꢀ-sheet and the active site involves residues from

both of the ꢀ-sheets. The binding site of putrescine, which

activates both the autoprocessing and decarboxylation reactions

of hAdoMetDC, is located well away from the ligand binding

site within the wild-type enzyme. Experimental conditions for

the puriﬁcation of the enzyme included putrescine at sufﬁcient

concentration to ensure high occupancy of the putrescine binding

site. The loops between the residues 1-4, 21-27, 165-173,

288-299, and 329-334 are disordered in the crystal struc-

tures.

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1393

Figure 2. Comparison in stereoview of the crystal structure of hAdoMetDC complexed with MeAdoMet to that of a structure derived from

the modeling of the complex. The crystal structure has all atoms colored gray. For the model, the active site pyruvoyl group is shown in

magenta and MeAdoMet carbon atoms are shown in green. MeAdoMet makes a Schiff base to the pyruvoyl group. The ribose makes two

hydrogen bonds to Glu247 (shown as red dashed lines). The adenine base stacks between Phe223 and Phe7 in the unusual syn conformation.

The hydrogen bonds between the adenine base and the backbone of Glu67 stabilize the syn conformation. The modeling result agrees well

with the experimentally determined crystal structure.

Figure 3. Comparison in stereoview of modeling of hAdoMetDC F223A and hAdoMetDC F7A mutants, each complexed with MeAdoMet,

with the crystal structure of the F223A mutant with MeAdoMet bound. Global minimum of modeling of MeAdoMet in the active site of the

F223A mutant superposed with the crystal structure (A) and the F7A mutant (B) of hAdoMetDC (see Experimental Section for details). The

crystal structure has all atoms colored gray. The pyruvoyl group is shown in magenta and the ligand carbon atoms are shown in green for

the models. Hydrogen bonds are shown as dashed lines. The adenine base attains an anti conformation in the models. The ribose makes one

hydrogen bond to Glu247 and the other to the backbone carbonyl of Cys226. The adenine base makes three hydrogen bonds to Ser66. In the

F7A model (B), the Phe223 residue changes its conformation to stack with the adenine base of MeAdoMet in the anti conformation.

1394 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

Table 1. Inhibition of hAdoMetDC^a

The crystal structure of hAdoMetDC F223A complexed with

MeAdoMet was solved using molecular replacement. The

difference F_o- F_cdensity shows that MeAdoMet is covalently

bound to the enzyme, and the nucleoside adopts a clear syn

conformation. As expected, the composite omit map density

shows no density for the Phe223 side chain. The ribose makes

two hydrogen bonds to Glu247, which anchor the ligand, and

the base is held in syn conformation by stacking interactions

with Phe7 and hydrogen bonds between the adenine and Glu67.

One molecule of putrescine per monomer is present in the

expected putrescine binding site. A superposition of the F223A/

MeAdoMet structure and the wild type structure with MeAdoM-

et shows that there is no appreciable change in the position or

conformation of the ligand (Figure 3A). The loops disordered

in the mutant are also disordered in the wild type protein.

compd

IC₅₀

12a

12b

12c

14a

14b

14c

14d

14e

14f

5

17d

17e

17f

17j

17k

17l

7 nM

86 nM

<5% inhibition at 100 µM

49 nM

<5% inhibition at 100 µM

11 nM

5 nM

15 nM

18 nM

55 nM

400 nM

4 µM

<5% inhibition at 100 µM

7 µM

170 nM

1.5 µM

Biochemical Analysis of Potential Inhibitors of hAdoMetDC.

The demonstrated importance of the syn conformation of the

adenine base of the AdoMet substrate for binding in the active

site of the enzyme led us to explore whether this could be

exploited in designing better hAdoMetDC inhibitors. It is known

that 8-substitution on adenine rings causes the nucleotide to

favor a syn conformation in solution.^26,27,37,38It was thought

that structural analogues of AdoMet that preferred the syn

conformation in solution would lead to improved hAdoMetDC

inhibition. Modeling of the active site had indicated that there

was sufﬁcient room to accommodate even rather large substit-

uents at C⁸of adenine. Several series of AdoMet structural

analogues were synthesized with substituents ranging from a

methyl group to a phenyl group at the 8-position of adenine.

Each of these compounds was then assayed for its ability to

inhibit hAdoMetDC and IC₅₀values for the inhibition were

determined (Table 1).

17m

18a

18b

18d

18e

18f

19a

19b

19c

19d

21c

21d

22a

22c

23a

23b

25b

25d

31 µM

440 µM

<5% inhibition at 100 µM

500 µM

<5% inhibition at 100 µM

88 µM

<5% inhibition at 100 µM

70 µM

420 µM

<5% inhibition at 100 µM

157 µM

600 nM

9 µM

3 µM

15 µM

^aEach of the potential inhibitors was assayed for the ability to inhibit

hAdoMetDC. At least ﬁve concentrations of each compound were used

and IC₅₀values were calculated from curve ﬁts to plots of inhibitor

concentration versus % inhibition of hAdoMetDC.

The inhibitors tested fall into four groups as described in

the “Chemical Synthesis” section. One group (12a-c, 14a-f,

5) has an aminooxyalkyl side chain at C-5′, which can form

a Schiff base with the pyruvate of AdoMetDC^20,39-41

Compounds of this group were potent inhibitors, with a

4-aminooxybutyl group being slightly superior to a 2-ami-

resulted in slightly increased potency compared to no substituent

but was not as effective as the 8-methyl substituent (compare

14c to 14d and 14f). Larger 8-substitutions did not improve

the effectiveness. An 8-phenyl addition to compounds 5, 14f,

and 18d abolished the inhibitory activity. Smaller additions such

as 8-ethyl (compare 14d and 14e, 17d and 17e, and 21c and

21d) or 8-methylamino (compare 12a and 12b and 21c and 18a)

were tolerated but were worse than 8-methyl.

Crystal Structures of hAdoMetDC Complexes. The crystal

structure of native hAdoMetDC with 12a was solved using

molecular replacement (Figure 4A). As noted above, 12a is

structurally similar to the previously studied inhibitor 5 except

that it has a methyl substitution at the 8-position on the adenine

base. The electron density indicates that the amino terminus of

12a forms a Schiff base with the pyruvoyl group of the enzyme.

The adenine base of 12a adopts a syn conformation in the crystal

structure as expected. There is one molecule of putrescine bound

in the putrescine binding site.

The crystal structure of native hAdoMetDC with 14e was

solved using molecular replacement (Figure 4B). Compound

14e is similar to 5 except for an ethyl substituent on the

8-position of the adenine base and two extra carbon atoms

between the tertiary nitrogen (near ribose) and the terminal

nitrogen. The presence of a three-carbon linker between the

ribose and the amino terminus makes this ligand interesting to

study. The electron density maps show no density for Schiff

base formation between the pyruvoyl group and the amino

terminus of the ligand. There is no density for the terminal three

atoms of the ligand, but there is good density for the rest of the

ligand. The position of the last three atoms was obtained by

nooxyethyl addition.

A second group of compounds

(17d,e,f,j,k,l,m) had an amide or a hydrazide side chain at

C-5′, and a third group of inhibitors (18a,b,d,e,f; 19a,b,c,d;

21c,d) had an aminoalkylamino side chain at C-5′. Also

related to the third group by the synthetic method are 22a

and 22c, which, respectively, have a guanidine and an

amidoxime at the end of the C-5′ side chain. The compounds

of groups 2 and 3 were less potent (particularly those with

the aminoalkylamino, guanidine, or amidoxime side chain)

but are more likely to be stable under in vivo conditions.

The ﬁnal group of compounds consisted of 5′-dimethylamino

(23a,b) or 5′-dimethylsulfonio (25b,d) compounds. Com-

pound 25d has been previously reported to be an AdoMetDC

inhibitor with a K_iin the µM range.³²As shown in Table 1,

the replacement of sulfur by nitrogen slightly improves the

AdoMetDC inhibition.

Within each of these groups, there was a consistent improve-

ment of inhibitory activity when an 8-methyl substituent was

added to the adenine ring. The reduction in the IC₅₀value varied

from 3.4-fold for compound 14d to 15-17-fold for compounds

23a and 17d. There was an 8-fold increase in potency when an

adenine 8-methyl substituent was added to compound 5, forming

compound 12a. This is consistent with the concept that the

8-methyl substitution on adenine biases the corresponding

nucleoside toward the syn conformation and that this is the form

that is bound at the active site. An adenine 8-hydroxy substituent

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1395

Figure 4. Complexes of hAdoMetDC with inhibitors having 8-substitutions. The carbon atoms of the inhibitor are shown in green and the pyruvoyl

group is shown in magenta. Water molecules are shown as red spheres and hydrogen bonds are shown as dashed lines. (A) Complex with 12a. The

ligand makes a Schiff base linkage to the active site pyruvoyl group. (B) Complex with 14e. There is no evidence from the electron density for the

formation of a Schiff base, and there is no density for the terminal three atoms of the ligand. The position of the terminal three atoms is determined

by modeling. (C) Complex with 17d. The carboxamide terminus of the ligand makes hydrogen bonds to Leu65 and Ser229. (D) Complex with 17f.

The carboxamide terminus of the ligand makes water mediated hydrogen bonds to Glu11 and Gly9. (E) Complex with 21c. The amino terminus

of the ligand makes water mediated hydrogen bonds to Glu11 and Gly9. The inhibitors 17d, 17f, and 21c do not make a Schiff base to the enzyme

and are hence competitive inhibitors. The adenine base of all the inhibitors attains a syn conformation.

modeling them to an energetically favorable conformation using

molecular modeling. The density around the pyruvoyl group

ﬁts it well and does not show any evidence of formation of a

Schiff base. The ribose makes the critical hydrogen bonds to

Glu247 and anchors the ligand. The nucleoside is held in the

syn conformation and is stabilized by π-π stacking. The density

of the ethyl substituent on the base is well deﬁned indicating

that the substituent is not disordered.

stituents; the ﬁrst two have carboxamide end groups at the 5′-

tail, while the third ligand has an amino group in this position.

All three ligands showed clear electron density and all three

ligands bound in the syn conformation.

Discussion

The active site of AdoMetDC contains a bound pyruvoyl

cofactor. The interactions of various ligands at the active site

were elucidated previously from the crystal structures obtained

from complexes of the enzyme with the inhibitors 3 (PDB 1I79),

5 (PDB 1I72), MeAdoMet (PDB 1I7B), 1 (PDB 1I7C), and 2

The crystal structures of hAdoMetDC with 17d (Figure 4C),

17f (Figure 4D), and 21c (Figure 4E) were also determined by

molecular replacement. The three ligands have 8-methyl sub-

1396 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

(PDB 1I7M).²⁵The crystal structure of MeAdoMet covalently

bound to the enzyme most closely approximates the substrate

AdoMet in the active site. The crystal structure shows key

interactions of MeAdoMet with the enzyme including: (1)

hydrogen bonding of the ribose oxygens with Glu247, (2) π-π

stacking interactions of the adenine ring with Phe223 and Phe7,

(3) hydrogen bonding of the 6-amino substituent of the adenine

ring with Glu67, the C-terminal residue of the ꢀ-chain, and (4)

hydrogen bonding of N-1 of the adenine ring with the backbone

amide group of Glu67 (Figure 2A). Similar interactions are also

present in the structures of 3 and 5 complexed with hAdoMet-

DC. The glycosidic angle for the adenine base in the structures

ranges from 128° to 139°, which demonstrates a preference for

the syn conformation of the nucleoside when bound to the active

site. Crystal structures of 1 and 2 with the enzyme show that

they stack between the two phenyl rings and make hydrogen

bonds to Glu247.

be within the error limit of our molecular mechanics based

calculations. The energy difference between the syn and anti

conformation of both structures is low, and based on the X-ray

structure of hAdoMetDC F223A with bound MeAdoMet, the

enzyme binds the ligands in the syn conformation, suggesting

that π-π interactions with Phe7 are sufﬁcient to maintain the

syn conformation. Thus, although the modeling studies were

incapable of accurately predicting that the syn conformation of

the nucleoside would be maintained in the F223A mutant, it

was possible to infer from these studies that the binding afﬁnity

of the nucleoside for the enzyme would be diminished.

Our attempts to exploit the requirement by AdoMetDC for a

ligand with a syn conformation were successful, as demonstrated

by the 8- to 18-fold improvement in inhibition when a methyl

group is attached to C-8. However, the larger substituents that

we tested provided no beneﬁt over the unsubstituted parent

compounds. In fact, the 8-phenyl substituent rendered the

compounds much less potent than the unsubstituted analogue.

Modeling studies of the active site had indicated that there

should be sufﬁcient space to accommodate the larger groups

with the adenine in the syn conformation. A more detailed look

at the area occupied by adenine C-8 substituents has indicated

that this area is near the solvent interface. On the basis of our

biochemical results, although large 8-substituents were structur-

ally compatible with the active site, the penalty of incompletely

burying a large hydrophobic group within a hydrophobic cavity

is apparently greater than the gain from favoring the syn

conformation. We are now exploring the effect of more

hydrophilic C-8 adenine substituents that should be more

compatible with proximity to the solvent. Such substituents may

be useful in maintaining the inhibitory potency associated with

the syn structure while still allowing species speciﬁc binding.

Structures of many of the AdoMet analogues bound to

AdoMetDC have shown that they inhibit the enzyme through

Schiff base formation with the pyruvoyl group of the enzyme.

The linker length between the tertiary ammonium/sulfur and

the terminal nitrogen of those inhibitors is typically 3-4 atoms,

which makes the formation of a Schiff base geometrically and

sterically feasible. Compound 14e has a linker length of ﬁve

atoms. The electron density map for the complex of 14e shows

a break in the density after the pyruvoyl group, suggesting that

there is no Schiff base formation. There is good density for the

ligand except at the three terminal atoms, which are disordered

and have no density. The positions of the last three atoms were

ﬁxed in an energetically favorable conformation using computer

modeling. The ﬁve atoms of the linker region appear to cause

a sterically unfavorable orientation for formation of the Schiff

base. The ligand is still held rigidly in the active site by hydrogen

bonds to Glu247 and the π-π stacking interactions with Phe7

and Phe223, and thus little movement is allowed to accom-

modate Schiff base formation for the longer linker region. Even

though compound 14e is not covalently attached to the pyruvoyl

group, its potency is better than 5 and nearly as good as

compound 12a.

The molecular modeling of MeAdoMet in the active site

of hAdoMetDC was performed by using mixed Monte Carlo/

low mode conformational searching as described above. The

glycosidic torsional angle was free to rotate during the

conformational search, which would allow a wide range of

rotamers that are compatible with the steric constraints of

the active site before energy minimization. The low energy

structures show that the adenine-derived nucleosides prefer

the syn conformation in the active site of hAdoMetDC.

Markham et al. have studied the conformational preferences

of AdoMet in solution and in vacuo.³³These studies based

on ¹H NMR and calculations based on NMR constraints have

shown that AdoMet prefers an anti conformation in solution

and a syn conformation in vacuo. In solution, the energy

difference between the anti and the corresponding syn

conformation, which includes steric, electrostatic, and the

solvation contributions, is around -34 kJ/mol. However,

these calculations were based on molecular mechanics

without polarization effects and it is likely that the energy

difference is much less negative. Our crystal structures and

modeling results show that hAdoMetDC binds ligands in the

syn conformation and that the energy difference is overcome

by hydrogen bonding and π-π interactions with Phe7 and

Phe233. Typical π-π interactions of parallel geometry

account for a stabilization of 8-12 kJ/mol,⁴²suggesting that

other factors may be involved.

The roles of Phe223 and Phe7 in AdoMetDC were studied

previously through crystal structures and kinetic experiments.²⁵

Kinetic data from reaction of hAdoMetDC F223A and F7A

mutants with the substrate AdoMet have shown that there is a

45-fold reduction of efﬁciency (k_cat/k_m) for the F7A mutant and

a 1400-fold decrease with the F223A mutant. In addition, 1 and

2 show a signiﬁcant increase in the IC₅₀values for both the

F7A and F233A mutants when compared to the wild-type

enzyme, with decrease in binding greater for F223A than for

F7A. Therefore, we chose to investigate the structural and

conformational properties of MeAdoMet in the active site of

the F223A mutant.

Conclusion

Our conformational searches with virtual mutations were done

to understand the roles of Phe223 and Phe7 in stabilizing the

syn conformation. In contrast to calculations done with the wild

type enzyme structure, in which only the syn conformation was

observed for the ensemble of lowest energy structures, the global

minimum from both the mutations has the base in an anti

conformation and the next higher energy structure has the base

in a syn conformation. The difference in the energy between

these conformations is about 2 kJ/mol, which we estimate to

Previous structural studies showed that AdoMet binds to the

active site of hAdoMetDC in the syn conformation, suggesting

that adenosine analogues favoring the syn conformation in

solution might be more potent inhibitors than corresponding

analogues favoring the lower energy anti conformation. 8-Sub-

stituted nucleoside analogues favor the syn conformation

because of unfavorable interactions in the anti conformation

between a bulky 8-substituent and ribose. We used computer

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1397

Table 2. Data Collection Statistics for hAdoMetDC Complexes

F223A + MeAdoMet

12a

14e

17d

17f

21c

wavelength

space group (Å)

a (Å)

0.9795

C2

95.98

1.5418

C2

96.78

0.9795

C2

94.43

0.9790

C2

99.82

0.9792

C2

99.65

0.9771

C2

100.08

50.75

b (Å)

44.25

44.46

50.04

50.95

50.75

c (Å)

70.83

70.55

70.41

68.98

68.90

69.04

ꢀ

104.52

2.62

104.17

2.43

105.34

1.83

105.52

1.84

105.34

1.91

105.56

1.86

resolution (Å)

total/unique reﬂections

redundancy

% complete

I/σ

23532/8160

2.9(2.6)^a

92.9(91.2)

13.3(2.0)

7.7(45.2)

1.90

26010/10403

2.5 (1.9)

93.6(86.8)

10.9(2.9)

9.0(33.8)

1.92

83134/26894

3.1(3.1)

95.6(95.5)

13.5(2.7)

7.2(54.8)

2.09

89749/28243

3.2(2.6)

97.6(94.1)

17.4(8.0)

6.0(14.0)

2.21

97188/25449

3.8(2.6)

98.8(91.0)

16.6(3.9)

7.6(25.0)

2.19

77769/27505

2.8(2.5)

98.7(96.8)

14.2(2.2)

7.1(39.1)

2.21

b

R_sym

Matthews no.

solvent content (%)

34.1

34.8

39.7

43.2

42.9

43.2

^aValues in parenthesis are for the highest resolution shell. ^bR_sym) ∑∑_i|I_i- I |/∑ I , where I is the mean intensity of the N reﬂections with intensities

I_iand common indices h,k,l.

separated by centrifugation at 12000g. Talon metal afﬁnity resin

was equilibrated with the wash buffer, and then the lysate and the

resin were gently spun together for 1.5 h. The resin was loaded

onto a column and washed with a volume of wash buffer equivalent

to 15-20 times the column volume. Next, the column was washed

in the same manner with wash buffer containing 25 mM imidazole.

The protein was then eluted with buffer containing 100-200 mM

imidazole. The eluted protein solution was concentrated to around

10 mL and passed through a Sephadex G-75 column pre-

equilibrated with 10 mM N-(2-hydroxyethyl)piperazine-N′-2-

ethanesulfonic acid (HEPES), pH 7.5, 2.5 mM putrescine, 5 mM

dithiothreitol (DTT), 0.1 mM ethylenediaminetetraacetic acid,

0.02% Brij-35, and 300 mM NaCl. The buffer was run through the

column, and the fractions containing the protein were identiﬁed by

UV absorbance at 280 nm. The protein was concentrated to ∼10

mg/mL and stored at -80 °C. The puriﬁcation of the F223A mutant

was similar to that of the native enzyme.

Structure Determination. The protein was thawed on ice and

buffer exchanged to 10 mM HEPES, pH 7.5, 200 mM NaCl and 1

mM DTT using Bio-Rad buffer exchange chromatography columns

(Bio-Rad Laboratories, Hercules, CA 94547). The wild type protein

was incubated with a 4-6 M excess of inhibitor for 24 h prior to

crystallization. The F223A mutant was diluted to ∼6 mg/mL and

incubated with a 4-6 M excess of MeAdoMet for 24 h prior to

crystallization. Crystals of both the native and the mutant complexes

were grown using the hanging drop method at 22 °C in 13-16%

PEG 8000, 100 mM Tris, pH 8.0-9.0, and 10 mM DTT. Crystals

appeared overnight and were stable for 1-2 weeks.

simulations to predict 8-substituted compounds that might bind

to hAdoMetDC and synthesized and assayed the most promising

candidates. We also determined crystal structures of several

compounds bound to hAdoMetDC to validate the predictions;

the structures conﬁrmed that the 8-substituted analogues bound

in the syn conformation and retained the previously identiﬁed

features of AdoMet binding, namely, purine stacking between

Phe7 and Phe223, and hydrogen bonding between the ribose

hydroxyl groups and Glu247. A group of adenosine analogues

was generated by varying the size and nature of the both the

8-substituent and the 5′-modiﬁcation. In general, 8-substituted

analogues bound with a potency of 8- to 18-fold higher

compared to the corresponding compound with a hydrogen atom

at the 8-position; however, 8-substituents larger than methyl

often showed lower potency than the corresponding 8-H

compound. The observation results from excessive solvent

exposure for large 8-substituents. Computer modeling and X-ray

crystallography were also used to understand the preference for

the syn conformation. Modeling studies suggested an important

role for the two active site phenylalanine residues in addition

to Glu247; however, the crystal structure of the F223A mutant

hAdoMetDC showed that AdoMet still binds in the syn

conformation, suggesting that other factors that favor the syn

conformation remain to be identiﬁed.

Experimental Section

The data for the 12a complex were collected at a home source

with a Rigaku R-AxisIV⁺⁺image plate detector using Cu KR

radiation from a Rigaku RU-300 rotating anode generator. The data

for the 14e complex were collected at NE-CAT beamline 8-BM at

the Advanced Photon Source (APS) using a ADSC Q315 detector.

Data for the 17f complex were collected at NE-CAT beamline 24-

ID-C using an ADSC Q315 detector. The data for the AdoMetDC

F223A mutant with MeAdoMet and the complexes with 17d and

21c were collected at the F2, A1, and A1 stations of CHESS,

respectively, using an ADSC Q210 detector. The diffraction quality

of the crystals strongly depended on cryoprotection conditions. The

crystals were sequentially transferred to a solution containing the

well solution with 2, 5, 8, 15, and 18% glycerol with 1-2 min

equilibration between each step. The data for all of the complexes

were indexed, integrated, and scaled using the HKL2000⁴³program

suite. The data collection statistics are summarized in Table 2.

The structures of all of the complexes were determined by

molecular replacement using the structure of native AdoMetDC

with MeAdoMet bound (PDB 1I7B) as the search model, and the

CNS program suite.⁴⁴The model building was done using the

program O⁴⁵or Coot.⁴⁶The conformations of the ligand molecules

were determined using difference F_o- F_cand composite omit maps.

The parameters and the topology ﬁles for the ligands were generated

using the HIC-Up server.⁴⁷The difference maps also showed

Protein Production. For crystallography of wild type and F223A

mutant hAdoMetDC, plasmids in the pQE30 vector in E. coli were

produced as described previously.²⁵This construct replaces the

N-terminal methionine with MRGS(H)₆GS- for puriﬁcation by

immobilized metal afﬁnity chromatography. A different plasmid

also based on the pQE30 vector was used for the production of

protein for the hAdoMetDC enzyme assays. In this plasmid, the

(H)₆tag was located at the carboxyl end replacing the terminal

-QQQQQS. The position of the (H)₆tag did not alter the activity

of the puriﬁed enzyme.

The wild type hAdoMetDC was puriﬁed based on the protocol

described by Ekstrom et al.²²The plasmid encoding the enzyme is

in the pQE30 vector and was transformed into JM109 strain E.

coli cells. The cells were grown as an overnight culture in LB media

at 37 °C and then introduced into larger cell cultures with both of

the cultures containing 100 mg/mL ampicillin. The cells were grown

until they reached an OD₆₀₀of 0.6 and then were induced with 100

mg/L isopropyl ꢀ-D-thiogalactopyranoside (IPTG). The cells were

allowed to grow overnight at 15 °C and were then harvested by

centrifugation, washed using a wash buffer that contained 20 mM

Na₂HPO₄, pH 7.0, 500 mM NaCl, 2.5 mM putrescine, 0.02% Brij-

35 and 10 mM imidazole, and stored at -80 °C. The frozen cell

pellet was thawed, suspended in the wash buffer, and lysed using

a French press at 1500 psi. The cellular debris and the lysate were

1398 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

Table 3. Reﬁnement Statistics for AdoMetDC Complexes

F223A + MeAdoMet

12a

14e

17d

17f

21c

resolution (Å)

R factor^a

2.62

0.203

0.280

2.43

0.199

0.247

1.83

0.208

0.231

1.84

0.204

0.237

1.91

0.197

0.208

1.86

0.200

0.232

R-free^b

no. of non-H atoms

protein

ligand

2473

28

2419

25

2381

28

2489

26

2454

25

2470

25

water

79

73

137

222

212

217

B factors

protein (Å²)

ligand (Å²)

putrescine (Å²)

41.3

63.4

32.4

31.5

42.1

27.9

29.6

32.3

40.0

26.8

26.0

22.4

28.2

43.9

24.7

32.4

39.9

29.8

rms deviations

bonds (Å)

angles (deg)

dihedrals (deg)

0.010

1.4

24.9

0.011

1.4

25.2

0.007

1.3

25.3

0.006

1.3

25.3

0.012

1.4

25.8

0.008

1.3

25.2

Ramachandran plot

most favored region (%)

additional favored region (%)

generously allowed region (%)

disallowed region (%)

84.2

14.7

0.8

89.3

9.5

0.8

91.4

7.8

0.4

91.8

7.8

0.4

92.1

7.5

0.4

92.5

7.5

0.0

0.4

0.0

over a subset of reﬂections that were excluded from all stages of reﬁnement.

density for a molecule of putrescine bound in all of the structures.

The reﬁnement statistics of the complexes are given in Table 3.

Molecular Modeling. Determination of the conformational

preference of ligands in the active site of AdoMetDC was carried

out with Macromodel version 7.2,³⁶available from Schro¨dinger,

LLC. To make the computational studies tractable, the protein was

truncated to a shell of atoms that included any residue that contained

an atom within 20.0 Å of MeAdoMet located in the active site of

AdoMetDC (from PDB 1I7B) and was used as the starting model

for conformational searching/energy minimization. Removal of

water molecules from this “docking shell” was followed by

appropriate hydrogen treatment using Schro¨dinger’s protein prepa-

ration utility that aids in the generation of appropriate ionic states

and histidine tautomers for active site amino acids and minimizes

the protein’s potential energy gradient through a series of con-

strained energy minimizations. For the conformational searches,

the appropriate ligand was added to the active site and, where

appropriate, the covalent bond between the amino terminus of the

ligand and the pyruvoyl group was formed.

The resulting structures were subjected to 50,000 mixed Monte

Carlo MCMM/low mode conformational search steps,^34,35allowing

residues within a 5 Å shell surrounding the active site to freely

move during each Monte Carlo/low mode step and subsequent

energy minimization step of the search. All other protein atoms

were constrained to their starting position. Residues His5, Glu67,

Cys226, and Glu247 were also constrained to their starting position.

The energy minimization step was considered to have converged

when the energy gradient was less than 0.05 kJ/mol. The AMBER*

force ﬁeld,^48,49with a distance dependent dielectric “constant”

further attenuated by a factor of 4, was employed for the

calculations, and the energy minimizations relied upon the TNCG

minimization technique.⁵⁰The global minimum and low energy

ensemble of structures within 15 kJ/mol of the global minimum

(after convergence) were further reﬁned by energy minimization

until a gradient less than 0.01 kJ/mol was obtained with just the

ligand allowed to move during this subsequent energy minimization

procedure. All protein atoms during this process were constrained

to their starting position. The jobs were run with the nucleoside

starting in both the syn and anti conformations for completeness.

The AMBER* parameters for the sulfonium ion were adapted from

the work done by Markham et al.³³

above. Because the position of the rest of the ligand and the protein

was determined to high accuracy by ﬁtting to the electron density

determined by X-ray diffraction, all of the protein and the ligand

atoms except the last three non-hydrogen atoms and their attached

hydrogens were ﬁxed during the conformational search. Torsional

rotation was allowed around the last two bonds of the C-5′ extension

during the conformational search. A visual survey of the ﬁve lowest

energy structures, which spanned an energy range of 6.5 kJ/mol,

showed that they were similar and the global minimum of the search

was utilized to obtain the coordinates of the disordered terminal

atoms of 14e.

AdoMetDC Activity and Inhibition. AdoMetDC was assayed

by measuring the release of ¹⁴CO₂from S-adenosyl-L-[carboxy-

¹⁴C]methionine (Amersham Pharmacia Biotech, ∼60 mCi/mmol).⁵¹

Assay of 30 ng of C-terminal his-tagged AdoMetDC under these

conditions results in ∼7000 cpm with a background of 30 and an

activity of ∼1.5 pmol/min/ng protein. For determination of the

abilities of compounds to inhibit AdoMetDC, the enzyme activity

was determined in the presence of no inhibitor and at least ﬁve

concentrations of each potential inhibitor. The enzyme concentation

was 1 nM. The IC₅₀values were determined from curve ﬁtting to

plots of the inhibitor concentration versus the % inhibition of

AdoMetDC.

Target Synthesis. TLC analysis was performed on Analtech

precoated (250 µm) silica gel GF plates. Melting points were

determined on a Mel-Temp apparatus and are uncorrected. Puriﬁca-

tions by ﬂash chromatography were carried out on Merck silica

gel (230-400 mesh). Evaporations were performed with a rotary

evaporator, higher boiling solvents (dimethylformamide (DMF),

pyridine) were removed in vacuo (<1 mm, bath to 35 °C). Products

were dried in vacuo (<1 mm) at 22-25 °C over P₂O₅. The mass

spectral data were obtained with a Varian-MAT 311A mass

spectrometer in the fast atom bombardment (FAB) mode or with a

Bruker BIOTOF II by electrospray ionization (ESI). ¹H NMR

spectra were recorded on a Nicolet NT-300 NB spectrometer

operating at 300.635 MHz. Chemical shifts in CDCl₃and Me₂SO-

d₆are expressed in parts per million downﬁeld from tetramethyl-

silane (TMS) and in D₂O chemical shifts are expressed in parts

per million downﬁeld from sodium 3-(trimethylsilyl)propionate-

2,2,3.3-d₄(TMSP). Chemical shifts (δ) listed for multiplets were

measured from the approximate centers, and relative integrals of

peak areas agreed with those expected for the assigned structures.

UV absorption spectra were determined on a Perkin-Elmer Lambda

The modeling of the terminal three atoms of 14e was done using

conformational searching with Macromodel version 7.2 as described

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1399

19 spectrometer by dissolving each compound in MeOH or EtOH,

and diluting 10-fold with 0.1 N HCl, pH 7 buffer, or 0.1 N NaOH.

Numbers in parentheses are extinction coefﬁcients (ε × 10^-3).

Microanalyses were performed by Atlantic Microlab, Inc. (Atlanta,

GA) or the Spectroscopic and Analytical Department of Southern

Research Institute. Analytical results indicated by element symbols

were within (0.4% of the theoretical values, and where solvents

J_2′,3′) 5.5 Hz, J_2′-2′OH) 6.3 Hz), 4.16 (bs, 1H, H-3′), 3.96-4.0

(m, 1H, H-4′), 2.64-2.77 (bm, 2H, 5′-CH₂), 2.53 (s, 3H, 8-CH₃),

2.29 (s, 3H, 5′NH-CH₃).

5′-Deoxy-5′-methylamino-8-ethyladenosine (9b). The procedure

was the same as reported above for 9a using 8b (1.00 g, 3.18 mmol)

and 33% methylamine/ethanol solution (30 mL). After column

chromatography (elution with 5:1:0.3 chloroform:methanol:

NH₄OH), a yellow glassy solid was obtained: 498 mg (50%). MS

1

are indicated in the formula, their presence was conﬁrmed by H

1

m/z 309 (M + H)⁺. H NMR (DMSO-d₆) δ 8.08 (bs, 1H, H-2),

NMR.

7.13 (bs, 2H, 6-NH₂), 5.70 (d, 1H, H-1′, J_1′,2′) 6.6 Hz), 5.30 (d,

1H, 2′-OH, J_2′-2′OH) 6.4 Hz), 5.16 (d, 1H, 3′-OH, J_3′-3′OH) 4.5

5′-Chloro-5′-deoxy-8-methyladenosine (8a). To a stirred sus-

pension of 7a³⁸(892 mg, 3.17 mmol) in anhydrous pyridine (501

mg, 0.51 mL, 6.33 mmol) and CH₃CN (2.5 mL) cooled in an ice

bath was slowly added SOCl₂(1.88 g, 1.15 mL, 15.80 mmol).

Stirring was continued at 0-5 °C for 3-4 h, with subsequent

warming to ambient temperature overnight. The resulting suspension

was concentrated in vacuo. To this reaction mixture was added

methanol (20 mL), water (2 mL), and NH₄OH (4 mL), followed

by stirring for 0.5 h at room temperature. The reaction mixture

was concentrated to dryness. The compound was dissolved in

MeOH, silica gel (3 g) was added and then solvent was removed.

The mixture on silica gel was poured onto a column ﬁlled with

silica gel and eluted with chloroform:methanol (7:1) to yield 661

Hz), 5.08 (ddd, 1H, H-2′, J_1′,2′) 6.6 Hz, J_2′,3′) 5.4 Hz, J_2′-2′OH

)

6.4 Hz), 4.14-4.18 (m, 1H, H-3′), 3.96-4.0 (m, 1H, H-4′), 2.88

(q, 2H, 8-CH₂CH₃), 2.64-2.78 (m, 2H, 5′-CH₂), 2.29 (s, 3H, 5′NH-

CH₃), 1.30 (t, 3H, 8CH₂CH₃).

5′-Deoxy-5′,8-bis(methylamino)adenosine (9c). Compound 9c

was prepared by the same procedure as described for the preparation

of 9a using 8c (1.00 g, 3.17 mmol) and 33% methylamine/ethanol

solution (30 mL). After column chromatography (elution with 7:1:

0.4 chloroform:methanol:NH₄OH), a yellow glassy solid was

obtained: 505 mg (51%). MS m/z 310 (M + H)⁺. ¹H NMR (DMSO-

d₆) δ 7.89 (bs and q, 2H, H-2 and 8CH₃-NH), 6.44 (bs, 2H, 6-NH₂),

1

mg (70%). MS m/z 300 (M + H)⁺. H NMR (DMSO-d₆) δ 8.09

5.84 (d, 1H, H-1′, J_1′,2′) 7.0 Hz), 5.22 (d, 1H, 2′-OH, J_2′-2′OH

)

6.4 Hz), 5.12 (d, 1H, 3′-OH, J_3′-3′OH) 4.0 Hz), 4.66 (ddd, 1H,

H-2′, J_1′,2′) 7.0 Hz, J_2′,3′) 5.4 Hz, J_2′-2′OH) 6.4 Hz), 4.14 (bm,

1H, H-3′), 3.94-4.0 (bm, 1H, H-4′), 2.90 (d, 3H, 8NH-CH₃, J )

4.4 Hz), 2.77-2.83 (m, 1H, 5′-CH₂), 2.57-2.62 (m, 1H, 5′-CH₂),

2.35 (s, 3H, 5′NH-CH₃).

(bs, 1H, H-2), 7.15 (bs, 2H, 6-NH₂), 5.81 (d, 1H, H-1′, J_1′,2′) 5.7

Hz), 5.49 (d, 1H, 2′-OH, J_2′-2′OH) 6.1 Hz), 5.45 (d, 1H, 3′-OH,

J_3′-3′OH) 5.3 Hz), 5.13 (ddd, 1H, H-2′, J_1′,2′) 5.7 Hz, J_2′,3′) 4.8

Hz, J_2′-2′OH) 6.1 Hz), 4.31 (ddd, 1H, H-3′, J_2′,3′) 4.8 Hz, J_3′,4′

)

4.0 Hz, J_3′-3′OH) 5.3 Hz), 4.03-4.10 (bm, 1H, H-4′), 3.93-3.99

(m, 1H, 5′-CH₂), 3.82-3.88 (m, 1H, 5′-CH₂), 2.55 (s, 3H, 8-CH₃).

5′-Chloro-5′-deoxy-8-ethyladenosine (8b). The procedure was

the same as reported above for 8a using 7b³⁸(1.28 g, 4.33 mmol),

pyridine (685 mg, 0.70 mL, 8.65 mmol), CH₃CN (10 mL), and

SOCl₂(2.57 g, 1.58 mL, 21.60 mmol): yield 498 mg (37%). MS

5′-Deoxy-5′-methylamino-8-phenyladenosine (9d). Compound 9d

was prepared by the same procedure as described for the preparation

of 9a using 8d (2.00 g, 5.52 mmol) and 33% methylamine/ethanol

solution (40 mL). After column chromatography (elution with 4:1:

0.2 chloroform:methanol:NH₄OH), a yellow glassy solid was

obtained: 963 mg (49%). MS m/z 357 (M + H)⁺. ¹H NMR (DMSO-

d₆) δ 8.19 (s, 1H, H-2), 7.72-7.76 (m, 2H, 8-phenyl o-H’s),

7.58-7.61 (m, 3H, 8-phenyl m- and p-H’s), 7.40 (bs, 2H, 6-NH₂),

1

m/z 314 (M + H)⁺. H NMR (DMSO-d₆) δ 8.09 (bs, 1H, H-2),

7.14 (bs, 2H, 6-NH₂), 5.80 (d, 1H, H-1′, J_1′,2′) 5.7 Hz), 5.48 (d,

1H, 2′-OH, J_2′-2′OH) 6.1 Hz), 5.45 (d, 1H, 3′-OH, J_3′-3′OH) 5.4

5.70 (d, 1H, H-1′, J_1′,2′) 6.4 Hz), 5.38 (d, 1H, 2′-OH, J_2′-2′OH

)

Hz), 5.20 (ddd, 1H, H-2′, J_1′,2′) 5.7 Hz, J_2′,3′) 4.6 Hz, J_2′-2′OH

)

6.2 Hz), 5.29 (ddd, 1H, H-2′, J_1′,2′) 6.4 Hz, J_2′,3′) 5.1 Hz, J_2′-2′OH

) 6.2 Hz), 5.14 (bs, 1H, 3′-OH), 4.19 (bs, 1H, H-3′), 3.95-4.0

(m, 1H, H-4′), 2.82 (d, 2H, 5′-CH_2,J ) 5.2 Hz), 2.34 (s, 3H, 5′NH-

CH₃).

6.1 Hz), 4.30-4.37 (bm, 1H, H-3′), 4.03-4.10 (bm, 1H, H-4′),

3.94-4.0 (m, 1H, 5′-CH₂), 3.83-3.89 (m, 1H, 5′-CH₂), 2.89 (q,

2H, 8-CH₂CH₃), 1.31 (t, 3H, 8-CH₂CH₃).

5′-Chloro-5′-deoxy-8-(methylamino)adenosine (8c). Compound

8c was prepared by the same procedure as described for the

preparation of 8a using 7c^39,41(2.9 g, 9.78 mmol), pyridine (1.54

g, 1.57 mL, 19.46 mmol), CH₃CN (5 mL), and SOCl₂(5.82 g, 3.56

mL, 48.91 mmol): yield 1.95 g (63%). MS m/z 315 (M + H)⁺. ¹H

NMR (DMSO-d₆) δ 7.91 (bs, 1H, H-2),), 6.78 (q, 1H, 8CH₃-NH),

6.50 (bs, 2H, 6-NH₂), 5.70 (d, 1H, H-1′, J_1′,2′) 5.0 Hz), 5.41 (d,

1H, 2′-OH, J_2′-2′OH) 5.6 Hz), 5.32 (d, 1H, 3′-OH, J_3′-3′OH) 5.3

5′-Deoxy-5′-[[2-[[(1-ethoxyethylidene)amino]oxy]ethyl]methyl-

amino]-8-methyladenosine (11a). A mixture of compound 9a (416

mg, 1.41 mmol), ethyl N-(2-bromoethoxy)ethanimidate²⁸(350 mg,

1.66 mmol), and diisopropylethylamine (DIEA) (11 mg, 0.014 mL,

0.085 mmol) in DMF (5 mL) was heated at 50 °C overnight under

nitrogen. The reaction mixture was concentrated to dryness. The

resulting syrup was puriﬁed by column chromatography (elution

with 7:1:0.1 chloroform:methanol:NH₄OH) to yield 50 mg (8%)

Hz), 5.18 (ddd, 1H, H-2′, J_1′,2′) 5.0 Hz, J_2′,3′) 5.4 Hz, J_2′-2′OH

)

1

of a yellow glassy sticky solid. MS m/z 424 (M + H)⁺. H NMR

5.6 Hz), 4.33 (ddd, 1H, H-3′, J_2′,3′) 5.4 Hz, J_3′,4′) 4.4 Hz, J_3′-3′OH

) 5.3 Hz),), 3.91-4.02 (bm, 2H, H-4′, 5′-CH₂), 3.76-3.82 (m,

1H, 5′-CH₂), 2.88 (d, 3H, 8NH-CH₃, J ) 4.5 Hz).

(DMSO-d₆) δ 8.07 (s, 1H, H-2), 7.11 (bs, 2H, 6-NH₂), 5.73 (d,

1H, H-1′, J_1′,2′) 5.5 Hz), 4.31 (d, 1H, 2′-OH, J_2′-2′OH) 6.3 Hz),

5.15 (d, 1H, 3′-OH, J_3′-3′OH) 5.5 Hz), 5.04 (ddd, 1H, H-2′, J_1′,2′

) 5.5 Hz, J_2′,3′) 5.1 Hz, J_2′-2′OH) 6.3 Hz), 4.13 (ddd, 1H, H-3′,

J_2′,3′) 5.1 Hz, J_3′,4′) 4.7 Hz, J_3′-3′OH) 5.5 Hz), 3.92 (q, 2H,

CH₂CH₃), 3.88 (t, 2H, NO-CH₂), 2.72-2.78 (m, 1H, 5′-CH₂),

2.56-2.62 (m, 4H, 5′-CH_2,H-4′_,N(CH₃)-CH₂), 2.53 (s, 3H, 8-CH₃),

2.21 (s, 3H, N-CH₃), 1.83 (s, 3H, C-CH₃), 1.19 (t, 3H, OCH₂CH₃).

5′-Deoxy-5′-[[2-[[(1-ethoxyethylidene)amino]oxy]ethyl]methyl-

amino]-8-(methylamino)adenosine (11b). Compound 11b was

prepared by the same procedure as reported for 11a using 9c (500

mg, 1.61 mmol), ethyl N-(2-bromoethoxy)ethanimidate²⁸(407 mg,

1.93 mmol), DIEA (104 mg, 0.14 mL, 0.80 mmol), and DMF (5

mL). After column chromatography (elution with 7:1:0.3 chloroform:

methanol:NH₄OH), a yellow glassy sticky solid was obtained: yield

5′-Chloro-5′-deoxy-8-phenyladenosine (8d). The procedure

described for 8a was used to prepare 8d from 7d⁴⁰(4.5 g, 13.10

mmol), pyridine (2.07 g, 2.12 mL, 26.2 mmol), CH₃CN (6 mL),

and SOCl₂(7.79 g, 4.78 mL, 65.47 mmol): yield 2.21 g (47%).

MS m/z 362 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.20 (s, 1H, H-2),

7.71-7.76 (m, 2H, 8-phenyl o-H’s), 7.59-7.64 (m, 3H, 8-phenyl

m- and p-H’s), 7.40 (bs, 2H, 6-NH₂), 5.75 (d, 1H, H-1′, J_1′,2′) 6.0

Hz), 5.52 (d, 1H, 2′-OH, J_2′-2′OH) 5.6 Hz), 5.39-5.44 (m, 2H,

H-2′, 3′-OH), 4.33 (bs, 1H, H-3′), 3.98-4.06 (bm, 2H, H-4′, 5′-

CH₂), 3.88-3.94 (m, 2H, 5′-CH₂).

5′-Deoxy-5′-methylamino-8-methyladenosine (9a). A mixture of

8a (660 mg, 2.20 mmol) in 33% methylamine/ethanol solution (30

mL) in a steel bomb was heated for two days at 90 °C. The reaction

mixture was concentrated to dryness and puriﬁed by column

chromatography (elution with 4:1:0.3 chloroform:methanol:NH₄OH

to yield 294 mg (45%). MS m/z 295 (M + H)⁺. ¹H NMR (DMSO-

d₆) δ 8.08 (bs, 1H, H-2), 7.14 (bs, 2H, 6-NH₂), 5.72 (d, 1H, H-1′,

J_1′,2′) 6.5 Hz), 5.30 (d, 1H, 2′-OH, J_2′-2′OH) 6.3 Hz), 5.17 (bd,

1H, 3′-OH, J_3′-3′OH) 3.5 Hz), 5.01 (ddd, 1H, H-2′, J_1′,2′) 6.5 Hz,

1

209 mg (30%). MS: m/z 439 (M + H)⁺. H NMR (DMSO-d₆) δ

7.89 (s, 1H, H-2), 6.85 (q, 1H, 8CH₃-NH), 6.47 (bs, 2H, 6-NH₂),

5.69 (d, 1H, H-1′, J_1′,2′) 5.0 Hz), 5.27 (d, 1H, 2′-OH, J_2′-2′OH

)

5.5 Hz), 5.06 (d, 1H, 3′-OH, J_3′-3′OH) 5.0 Hz), 4.88 (ddd, 1H,

H-2′, J_1′,2′) 5.0 Hz, J_2′,3′) 5.8 Hz, J_2′-2′OH) 5.5 Hz), 4.17 (ddd,

1H, H-3′, J_2′,3′) 5.8 Hz, J_3′,4′) 4.7 Hz, J_3′-3′OH) 5.0 Hz),

1400 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

3.86-3.95 (m, 3H, H-4′, NO-CH₂), 3.92 (q, 2H, CH₂CH₃), 2.88

(d, 3H, 8NH-CH_3,J ) 4.6 Hz), 2.71-2.77 (m, 1H, 5′-CH₂),

2.56-2.67 (m, 3H, 5′-CH_2,N(CH₃)-CH₂), 2.24 (s, 3H, N-CH₃),

1.83 (s, 3H, C-CH₃), 1.19 (t, 3H, OCH₂CH₃).

1H, H-2), 6.87 (q, 1H, 8CH₃-NH), 6.46 (bs, 2H, 6-NH₂), 5.69 (d,

1H, H-1′, J_1′,2′) 4.8 Hz), 5.25 (d, 1H, 2′-OH, J_2′-2′OH) 5.6 Hz),

5.06 (d, 1H, 3′-OH, J_3′-3′OH) 5.4 Hz), 4.91 (ddd, 1H, H-2′, J_1′,2′

) 4.8 Hz, J_2′,3′) 5.4 Hz, J_2′-2′OH) 5.6 Hz), 4.16 (ddd, 1H, H-3′,

J_2′,3′) 5.4 Hz, J_3′,4′) 4.9 Hz, J_3′-3′OH) 5.4 Hz), 3.85-3.94 (m,

1H, H-4′), 3.92 (q, 2H, OCH₂CH₃), 3.80 (t, 2H, NO-CH₂), 2.88 (d,

3H, 8NH-CH_3,J ) 4.6 Hz), 2.65-2.74 (m, 1H, 5′-CH₂), 2.46-2.58

(m, 1H, 5′-CH₂), 2.34 (t, 2H, N(CH₃)-CH₂), 2.17 (s, 3H, N-CH₃),

1.83 (s, 3H, C-CH₃), 1.37-1.61 (bm, 4H, NOCH₂-CH₂CH₂), 1.19

(t, 3H, OCH₂CH₃).

5′-Deoxy-5′-[[2-[[(1-ethoxyethylidene)amino]oxy]ethyl]methyl-

amino]-8-phenyladenosine (11c). The procedure described for 11a

was used to prepare 11c from 9d (400 mg, 1.12 mmol), ethyl N-(2-

bromoethoxy)ethanimidate²⁸(283 mg, 1.34 mmol), and DIEA (72

mg, 0.10 mL, 0.55 mmol). After column chromatography (elution

with 7:1 chloroform:methanol), a yellow glassy sticky solid was

1

obtained: yield 105 mg (20%). MS: m/z 486 (M + H)⁺. H NMR

5′-Deoxy-5′-[[4-[[(1-ethoxyethylidene)amino]oxy]butyl]methyl-

amino]-8-phenyladenosine (13b). The same procedure as described

for 6a was used to prepare 13b from 9d (450 mg, 1.26 mmol),

ethyl N-(4-bromobutoxy)ethanimidate²⁹(360 mg, 1.51 mmol), and

DIEA (81 mg, 0.10 mL, 0.62 mmol). After column chromatography

(elution with 7:1 chloroform:methanol), a yellow glassy sticky solid

(DMSO-d₆) δ 8.17 (s, 1H, H-2), 7.72-7.78 (m, 2H, 8-phenyl

o-H’s), 7.58-7.63 (m, 3H, 8-phenyl m- and p-H’s), 7.36 (bs, 2H,

6-NH₂), 5.67 (d, 1H, H-1′, J_1′,2′) 5.2 Hz), 5.29-5.34 (m, 2H, 2′-

OH, H-2′), 5.13 (d, 1H, 3′-OH, J_3′-3′OH) 5.3 Hz), 4.14-4.18 (m,

1H, H-3′), 3.88-3.97 (m, 3H, H-4′, NO-CH₂), 3.92 (q, 2H,

CH₂CH₃), 2.78-2.84 (m, 1H, 5′-CH₂), 2.60-2.70 (m, 3H, 5′-CH_2,

N(CH₃)-CH₂), 2.24 (bs, 3H, N-CH₃), 1.83 (s, 3H, C-CH₃), 1.19 (t,

3H, OCH₂CH₃).

1

was obtained: yield 312 mg (48%). MS: m/z 514 (M + H)⁺. H

NMR (DMSO-d₆) δ 8.17 (s, 1H, H-2), 7.71-7.78 (m, 2H, 8-phenyl

o-H’s), 7.58-7.64 (m, 3H, 8-phenyl m- and p-H’s), 7.36 (bs, 2H,

6-NH₂), 5.67 (d, 1H, H-1′, J_1′,2′) 5.7 Hz), 5.32 (bs, 1H, 2′-OH),

5.31 (t, 1H, H-2′, J_1′,2′) 5.7 Hz, J_2′,3′) 5.4 Hz), 5.11 (d, 1H, 3′-

OH, J_3′-3′OH) 4.8 Hz), 4.16 (bddd, 1H, H-3′, J_2′,3′) 5.4 Hz, J_3′,4′

) 4.0 Hz), 3.92-3.97 (m, 1H, H-4′), 3.91 (q, 2H, OCH₂CH₃), 3.79

(t, 2H, NO-CH₂), 2.72-2.80 (m, 1H, 5′-CH₂), 2.54-2.59 (m, 1H,

5′-CH₂), 2.34 (bt, 2H, N(CH₃)-CH₂), 2.17 (bs, 3H, N-CH₃), 1.83

(s, 3H, C-CH₃), 1.39-1.60 (bm, 4H, NOCH₂-CH₂CH₂), 1.18 (t,

3H, OCH₂CH₃).

5′-[(2-Aminooxyethyl)methylamino]-5′-deoxy-8-methyladenos-

ine sulfate (2.2:1 salt) (12a). Compound 11a (50 mg, 0.11 mmol)

was dissolved in 2 mL of 2 N H₂SO₄and stirred for two days at

room temperature. The reaction mixture was neutralized with

NaHCO₃and lyophilized. The compound was extracted with EtOH

(2 × 10 mL) and concentrated to dryness. The residue was puriﬁed

by column chromatography (silica gel 230-400 mesh, elution with

7:1:0.3 chloroform:methanol:NH₄OH). The desired fractions were

combined, concentrated, and dried in vacuo. The product was

dissolved in 3 mL of EtOH and 2 N H₂SO₄was added dropwise.

The resulting sulfate salt that precipitated out was ﬁltered and then

washed with EtOH. This product, which was hygroscopic in nature,

was dissolved in water (2 mL) and lyophilized to give a white solid:

yield 20 mg (29%). MS: m/z 354 (M + H)⁺. ¹H NMR (DMSO-d₆)

δ 8.25 (s, 1H, H-2), 7.80 (bs, 2H, O-NH₂), 5.87 (d, 1H, H-1′, J_1′,2′

) 5.7 Hz), 4.88 (t, 1H, H-2′, J_2′,3′) 5.2 Hz), 4.35-4.40 (bm, 1H,

H-4′), 4.23 (t, 1H, H-3′, J_2′,3′) 3.2 Hz), 4.10 (t, 2H, NH₂O-CH₂),

3.50-3.57 (m, 1H, 5′-CH₂), 3.65-3.72 (m, 1H, 5′-CH₂), 3.45 (bm,

2H, N(CH₃)-CH₂), 2.85 (s, 3H, N-CH₃), 2.58 (s, 3H, 8-CH₃). UV

5′-Deoxy-5′-[[4-[[(1-ethoxyethylidene)amino]oxy]butyl]methyl-

amino]-8-oxoadenosine (13c). The procedure described for 11a was

used to prepare 13c from 9i⁵²(500 mg, 1.68 mmol), ethyl N-(4-

bromobutoxy)ethanimidate²⁹(481 mg, 2.01 mmol), DIEA (109 mg,

0.14 mL, 0.84 mmol), and DMF (5 mL). After column chroma-

tography (elution with 4:1:0.2 chloroform:methanol:NH₄OH), a

yellow glassy sticky solid was obtained: yield 200 mg (26%). MS:

m/z 454 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 10.34 (bs, 1H, 8-OH),

8.02 (s, 1H, H-2), 6.49 (bs, 2H, 6-NH₂), 5.62 (d, 1H, H-1′, J_1′,2′

)

5.0 Hz), 4.99 (bs, 1H, 3′-OH), 5.19 (bs, 1H, 2′-OH), 4.90 (t, 1H,

H-2′, J_2′,3′) 5.4 Hz), 4.16-4.24 (bm, 1H, H-3′), 3.83-3.89 (m,

1H, H-4′), 3.92 (q, 2H, OCH₂CH₃), 3.77 (t, 2H, NO-CH₂),

2.62-3.68 (m, 1H, 5′-CH₂), 2.40-2.46 (m, 1H, 5′-CH₂), 2.30 (t,

2H, N(CH₃)-CH₂), 2.13 (s, 3H, N-CH₃), 1.84 (s, 3H, C-CH₃),

1.35-1.60 (bm, 4H, NOCH₂-CH₂CH₂), 1.21 (t, 3H, OCH₂CH₃).

5′-Deoxy-2′,3′-isopropylidene-5′-[[4-[[(1-ethoxyethylidene)ami-

no]oxy]butyl]methylamino]-8-methyladenosine (13d). Compound

13d was prepared by the same procedure as reported for 11a using

9e (1.00 g, 3.11 mmol), MsCl (392 mg, 0.26 mL, 3.42 mmol),

methylamine (25 mL), ethyl N-(4-bromobutoxy)ethanimidate (853

mg, 3.58 mmol), DIEA (200 mg, 0.27 mL, 1.54 mmol), and DMF

(8 mL). After column chromatography (95:5 chloroform:methanol),

a glassy solid was obtained: yield 176 mg (12%). MS: m/z 492 (M

λ

_max, nm, pH 1, 274 (ε 15200), pH 7, 276 (ε 15500), pH 13, 277

(ε 15900). Anal. (C₁₄H₂₃N₇O₄·2.2H₂SO₄·0.1C₂H₅OH·0.5H₂O) C,

H, N.

5′-[(2-Aminooxyethyl)methylamino]-5′-deoxy-8-(methylamino)ad-

enosine sulfate (2.1:1 salt) (12b). The procedure described for 12a

was used to prepare 12b from 11b (200 mg, 0.45 mmol): yield

1

125 mg (46%). MS: m/z 369 (M + H)⁺. H NMR (DMSO-d₆) δ

8.16 (s, 1H, H-2), 7.50-7.65 (bm, 2H, O-NH₂), 5.83 (d, 1H, H-1′,

J_1′,2′) 5.3 Hz), 6.56 (bs, 2H, 6-NH₂), 4.96 (t, 1H, H-2′, J_2′,3′) 4.8

Hz), 4.28-4.35 (bm, 1H, H-4′), 4.25 (t, 1H, H-3′, J_3′,4′) 4.1 Hz),

3.96 (t, 2H, NH₂O-CH₂), 3.59-3.66 (m, 1H, 5′-CH₂), 3.49-3.57

(m, 1H, 5′-CH₂), 3.36-3.40 (bm, 2H, N(CH₃)-CH₂), 2.94 (s, 3H,

8NH-CH₃), 2.81 (s, 3H, N-CH₃). UV λ_max, nm, pH 1, 274 (ε 14300),

pH 7, 276.7 (ε 17100), pH 13, 276.1 (ε 17500). Anal.

(C₁₄H₂₄N₈O₄·2.1H₂SO₄·0.3C₂H₅OH·0.2H₂O) C, H, N, S.

5′-[(2-Aminooxyethyl)methylamino]-5′-deoxy-8-phenyladenos-

ine sulfate (2:1 salt) (12c). Compound 12c was prepared by the

same procedure as described for the preparation of 12a using 11c

(99 mg, 0.20 mmol): yield 57 mg (42%). MS: m/z 416 (M + H)⁺.

¹H NMR (D₂O) δ 8.37 (s, 1H, H-2), 7.73-7.76 (m, 2H, 8-phenyl

o-H’s), 7.60-7.70 (m, 3H, 8-phenyl m- and p-H’s), 6.02 (d, 1H,

H-1′, J_1′,2′) 5.7 Hz), 5.25 (t, 1H, H-2′, J_2′,3′) 4.9 Hz), 4.46-4.54

(bm, 2H, H-3′, 4′), 4.03 (t, 2H, NH₂O-CH₂), 3.87-4.0 (m, 1H,

5′-CH₂), 3.61-3.67 (m, 1H, 5′-CH₂), 3.50-3.55 (m, 2H, N(CH₃)-

CH₂), 3.0 (s, 3H, N-CH₃). UV λ_max, nm, pH 1, 275 (ε 21600), pH

7, 275 (ε 17100), pH 13, 274.4 (ε 16800). Anal. (C₁₉H₂₅N₇O₄

·2.0H₂SO₄·3H₂O) C, H, N, S.

1

+ H)⁺. H NMR (CDCl₃) δ 8.27 (s, 1H, H-2), 5.99 (d, 1H, H-1′,

J_1′,2′) 1.8 Hz), 5.75 (dd, 1H, H-2′, J_1′,2′) 1.8 Hz, J_2′,3′) 6.4 Hz),

5.39 (bs, 2H, 6-NH₂), 5.08 (dd, 1H, H-3′, J_2′,3′) 6.4 Hz, J_3′,4′

)

3.5 Hz), 4.27-4.34 (m, 1H, H-4′), 4.0 (q, 2H, OCH₂CH₃), 3.84 (t,

2H, NO-CH₂), 2.64 (s, 3H, 8-CH₃), 2.55-2.61 (m, 1H, 5′-CH₂),

2.45-2.55 (m, 1H, 5′-CH₂), 2.29-2.34 (m, 2H, N(CH₃)-CH₂), 2.21

(s, 3H, N-CH₃), 1.91 (s, 3H, C-CH₃), 1.61 and 1.40 (2s, 6H,

C(CH₃)₂), 1.51-1.60 (m, 2H, NOCH₂-CH₂), 1.37-1.45 (m, 2H,

N(CH₃)CH₂-CH₂), 1.27 (t, 3H, OCH₂CH₃).

5′-Deoxy-2′,3′-isopropylidene-5′-[[4-[[(1-ethoxyethylidene)ami-

no]oxy]butyl]methylamino]-8-ethyladenosine (13e). The procedure

described for 11a was used to prepare 13e from 9f (1.00 g, 2.98

mmol), MsCl (375 mg, 0.25 mL, 3.27 mmol), methylamine (25

mL), ethyl N-(4-bromobutoxy)ethanimidate (852 mg, 3.57 mmol),

DIEA (192 mg, 0.25 mL, 1.48 mmol), and DMF (10 mL). After

column chromatography (95:5 chloroform:methanol), a glassy solid

5′-Deoxy-5′-[[4-[[(1-ethoxyethylidene)amino]oxy]butyl]methyl-

amino]-8-(methylamino)adenosine (13a). Compound 13a was pre-

pared by the same procedure as reported for 11a using 9c (1.00 g,

3.23 mmol), ethyl N-(4-bromobutoxy)ethanimidate²⁹(924 mg, 3.87

mmol), and DIEA (209 mg, 0.28 mL, 1.6 mmol): yield 635 mg

1

was obtained: yield 159 mg (11%). MS: m/z 506 (M + H)⁺. H

NMR (CDCl₃) δ 8.27 (s, 1H, H-2), 5.99 (d, 1H, H-1′, J_1′,2′) 2.0

Hz), 5.73 (dd, 1H, H-2′, J_1′,2′) 2.0 Hz, J_2′,3′) 6.4 Hz), 5.40 (bs,

2H, 6-NH₂), 5. 09 (dd, 1H, H-3′, J_2′,3′) 6.4 Hz, J_3′,4′) 3.6 Hz),

4.26-4.33 (m, 1H, H-4′), 4.0 (q, 2H, O CH₂CH₃), 3.84 (t, 2H,

1

(42%). MS: m/z 467 (M + H)⁺. H NMR (DMSO-d₆) δ 7.89 (s,

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1401

NO-CH₂), 2.91-2.99 (m, 2H, CH₂of 8-Et), 2.59-2.65 (m, 1H,

5′-CH₂), 2.46-2.53 (m, 1H, 5′-CH₂), 2.30-2.35 (m, 2H, N(CH₃)-

CH₂), 2.21 (s, 3H, N-CH₃), 1.91 (s, 3H, C-CH₃), 1.61 and 1.40

(2s, 6H, C(CH₃)₂), 1.51-1.59 (m, 2H, NOCH₂-CH₂), 1.49-1.38

(m, 2H, N(CH₃)CH₂-CH₂), 1.43 (s, 3H, CH₃of 8-Et), 1.27 (t, 3H,

OCH₂CH₃).

5′-[(4-Aminooxybutyl)methylamino]-5′-deoxy-8-methyladenos-

ine sulfate (1.9:1 salt) (14d). Compound 13d (149 mg, 0.30 mmol)

was dissolved in 2.5 mL of 1 N H₂SO₄and stirred for 12 days at

room temperature. After a work up identical with that used for 7a,

the residue was puriﬁed by column chromatography (silica gel

230-400 mesh, elution with 4:1:0.2 chloroform:methanol:NH₄OH).

The product was dissolved in 8 mL of EtOH and 2 N H₂SO₄was

added dropwise. The sulfate salt that precipitated out was ﬁltered

and washed with EtOH. This product was dissolved in water (2

mL) and lyophilized to give a white solid: yield 59 mg (33%). MS:

5′-Deoxy-2′,3′-isopropylidene-5′-[[4-[[(1-ethoxyethylidene)ami-

no]oxy]butyl]methylamino]-adenosine (13f). Compound 13f was

prepared by the same procedure as reported for 11a using 9g³²

(1.00 g, 3.25 mmol), MsCl (447 mg, 0.30 mL, 3.90 mmol),

methylamine (25 mL), ethyl N-(4-bromobutoxy)ethanimidate (511

mg, 2.15 mmol), DIEA (125 mg, 0.17 mL, 0.96 mmol), and DMF

(5 mL). After column chromatography (95:5 chloroform:methanol),

a pale-yellow syrup was obtained: yield 839 mg (86%). MS: m/z

478 (M + H)⁺. ¹H NMR (CDCl₃) δ 8.36 (s, 1H, H-2), 7.96 (s, 1H,

H-8), 6.07 (d, 1H, H-1′, J_1′,2′) 2.2 Hz), 5.60 (bs, 2H, 6-NH₂),

5.49 (dd, 1H, H-2′, J_1′,2′) 2.2 Hz, J_2′,3′) 6.4 Hz), 4.95 (dd, 1H,

H-3′, J_2′,3′) 6.4 Hz, J_3′,4′) 3.4 Hz), 4.36-4.40 (m, 1H, H-4′), 4.0

(q, 2H, OCH₂CH₃), 3.86 (t, 2H, NO-CH₂), 2.61 (dd, 1H, 5′-CH₂),

2.55 (dd, 1H, 5′-CH₂), 2.38 (bt, 2H, N(CH₃)-CH₂), 2.26 (s, 3H,

N-CH₃), 1.91 (s, 3H, C-CH₃), 1.61 and 1.40 (2s, 6H, C(CH₃)₂),

1.55-1.61 (m, 2H, NOCH₂-CH₂), 1.44-1.52 (m, 2H, N(CH₃)CH₂-

CH₂), 1.27 (t, 3H, OCH₂CH₃).

1

m/z 382 (M + H)⁺. H NMR (D₂O) δ 8.39 (s, 1H, H-2), 6.08 (d,

1H, H-1′, J_1′,2′) 5.5 Hz), 5.0-5.19 (bm, 1H, H-2′), 4.51-4.58

(bm, 2H, H-3′, 4′), 4.08 (t, 2H, NH₂O-CH₂), 3.73-4.0 (m, 1H,

5′-CH₂), 3.44-3.69 (m, 1H, 5′-CH₂), 3.16-3.36 (bm, 2H, N(CH₃)-

CH₂), 2.92 (bs, 3H, N-CH₃), 2.70 (s, 3H, 8-CH₃), 1.68-1.88 (bm,

4H, NH₂OCH₂-CH₂CH₂). UV λ_max, nm, pH 1, 258.2 (ε 15400),

pH 7, 259.7 (ε 15500), pH 13, 260.9 (ε 15900). Anal.

(C₁₆H₂₇N₇O₄·1.9H₂SO₄·0.4C₂H₅OH) C, H, N.

5′-[(4-Aminooxybutyl)methylamino]-5′-deoxy-8-ethyladenos-

ine sulfate (1.9:1 salt) (14e). The procedure was the same as reported

above for 14d using 13e (155 mg, 0.30 mmol): yield 55 mg (30%).

MS: m/z 396 (M + H)⁺. ¹H NMR (D₂O) δ8.39 (s, 1H, H-2), 6.09

(d, 1H, H-1′, J_1′,2′) 5.6 Hz), 5.07-5.23 (bm, 1H, H-2′), 4.50-4.60

(bm, 2H, H-3′, 4′), 4.06 (t, 2H, NH₂O-CH₂), 3.82-3.96 (m, 1H,

5′-CH₂), 3.45-3.69 (m, 1H, 5′-CH₂), 3.27 (bs, 2H, N(CH₃)-CH₂),

3.0-3.10 (m, 2H, 8CH₂CH₃), 2.91 (bs, 3H, N-CH₃), 1.68-1.86

5′-[(4-Aminooxybutyl)methylamino]-5′-deoxy-8-(methylamino)ad-

enosine sulfate (0.4:1 salt) (14a). The same procedure used to

prepare 12a was used to prepare 14a using 13a (600 mg, 1.28

mmol) and 2 N H₂SO₄(10 mL): yield 514 mg (87%). MS: m/z

(bm, 4H, NH₂OCH₂-CH₂CH₂), 1.39 (t, 3H, 8CH₂CH₃). UV λ_max

,

1

510 (M + H)⁺. H NMR (DMSO-d₆) δ 7.91 (s, 1H, H-2), 6.88

nm, pH 1, 259.1 (ε 16400), pH 7, 260 (ε 15700), pH 13, 260.2 (ε

15900). Anal. (C₁₇H₂₉N₇O₄·1.9H₂SO₄·0.2C₂H₅OH) C, H, N.

5′-[(4-Aminooxybutyl)methylamino]-5′-deoxyadenosine sulfate

(2:1 salt) (14f). The procedure was the same as reported above for

14d using 13f (750 mg, 1.5 mmol): yield 457 mg (48%). MS: m/z

368 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.43 (s, 1H, H-8), 8.23 (s,

1H, H-2), 7.58 (bs, 2H, 6-NH₂), 6.03 (d, 1H, H-1′, J_1′,2′) 5.4 Hz),

4.75 (t, 1H, H-2′, J_1′,2′) 5.4 Hz, J_2′,3′) 4.8 Hz), 4.32-4.40 (bm,

1H, H-4′), 4.23 (t, 1H, H-3′, J_3′,4′) 3.8 Hz), 3.93 (t, 2H, NH₂O-

CH₂), 3.68 (dd, 1H, 5′-CH₂), 3.49 (bdd, 1H, 5′-CH₂), 3.13 (bt, 2H,

N(CH₃)-CH₂), 2.80 (s, 3H, N-CH₃), 1.50-1.78 (bm, 4H,

NH₂OCH₂-CH₂CH₂). Anal. (C₁₅H₂₅N₇O₄·2.0H₂SO₄·0.3C₂H₅OH·

1.5H₂O) C, H, N, S.

(bq, 1H, 8CH₃-NH), 6.50 (bs, 2H, 6-NH₂), 5.72 (d, 1H, H-1′, J_1′,2′

) 5.1 Hz), 5.08-5.39 (bm, 2H, 2′, 3′-OH), 4.0-5.02 (m, 1H, H-2′),

4.19 (t, 1H, H-3′), 3.94-4.06 (bm, 1H, H-4′), 3.45 (bt, 2H, NH₂O-

CH₂), 3.36-3.54 (m, 2H, 5′-CH₂), 2.89 (d, 3H, 8NH-CH₃),

2.78-2.95 (bm, 2H, NCH₃-CH₂), 2.04 (bs, 3H, N-CH₃), 1.38-1.52

(bm, 4H, NH₂OCH₂-CH₂CH₂). UV λ_max, nm, pH 1, 274.8 (ε

14300), pH 7, 276 (ε 16700), pH 13, 277 (ε 17400). Anal.

(C₁₆H₂₈N₈O₄·0.4H₂SO₄·0.2C₂H₅OH·0.9H₂O) C, H, N, S.

5′-[(4-Aminooxybutyl)methylamino]-5′-deoxy-8-phenyladenos-

ine sulfate (1.75:1 salt) (14b). Compound 14b was prepared by the

same procedure as described for the preparation of 12a using 13b

(305 mg, 0.59 mmol) and 2 N H₂SO₄(4 mL): yield 252 mg (64%).

MS: m/z 444 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.18 (s, 1H, H-2),

7.20-7.78 (m, 2H, 8-phenyl o-H’s), 7.59-7.61 (m, 3H, 8-phenyl

m- and p-H’s), 7.37 (bs, 2H, 6-NH₂), 5.85 (s, 2H, O-NH₂), 5.68 (d,

1H, H-1′, J_1′,2′) 5.7 Hz), 5.31 (t, 1H, H-2′, J_2′,3′) 5.5 Hz), 5.13

(bd, 1H, 3′-OH), 4.18 (t, 1H, H-3′, J_3′,4′) 3.9 Hz), 3.94-3.99 (bm,

1H, H-4′), 3.49 (t, 2H, NH₂O-CH₂), 2.73-2.79 (m, 1H, 5′-CH₂),

2.54-2.62 (m, 1H, 5′-CH₂), 2.27-2.36 (bm, 2H, N(CH₃)-CH₂),

2.16 (bs, 3H, N-CH₃), 1.33-1.53 (bm, 4H, NH₂OCH₂-CH₂CH₂).

¹H NMR (D₂O) δ 8.36 (s, 1H, H-2), 7.72-7.78 (m, 2H, 8-phenyl

o-H’s), 7.63-7.71 (m, 3H, 8-phenyl m- and p-H’s), 6.02 (d, 1H,

H-1′, J_1′,2′) 5.8 Hz), 5.28-5.41 (bm, 1H, H-2′), 4.43-4.53 (bm,

2H, H-3′, 4′), 3.92-4.03 (m, 1H, 5′-CH₂), 3.90 (t, 2H, NH₂O-CH₂),

3.49-3.59 (m, 1H, 5′-CH₂), 3.22- 3.32 (bm, 2H, N(CH₃)-CH₂),

2.92 (bs, 3H, N-CH₃), 1.61-1.83 (bm, 4H, NH₂OCH₂-CH₂CH₂).

UV λ_max, nm, pH 1, 275 (ε 21400), pH 7, 274.5 (ε 16900), pH 13,

274.8 (ε 16,700). Anal. (C₂₁H₂₉N₇O₄· 1.75H₂SO₄· 0.05C₂H₅OH ·

2.4H₂O) C, H, N, S.

5′-Deoxy-2′,3′-O-isopropylidene-5′-[(2-hydroxyethyl)methylami-

no]adenosine (15). Compound 8i⁵²(8.20 g, 17.79 mmol) was

dissolved in 2-(methylamino)ethanol (54 mL, 673 mmol) and stirred

at room temperature for 41 h. The solvent was evaporated to give

a yellow residue. The residue was dissolved in 100 mL of

chloroform and washed with NaHCO₃(3 × 50 mL). The organic

layer was dried over Na₂SO₄and concentrated to dryness to give

yellow foam. The residue was puriﬁed by column chromatography

(silica gel 230-400 mesh, elution with 9:1:0.1 chloroform:

methanol:NH₄OH) to yield 2.55 g (39%). MS: m/z 365 (M + H)⁺.

¹H NMR (DMSO-d₆) δ 8.34 (s, 1H, H-2), 8.18 (s, 1H, H-8), 7.33

(bs, 2H, 6-NH₂), 6.13 (d, 1H, H-1′, J_1′,2′) 2.5 Hz), 5.48 (dd, 1H,

H-2′, J_2′,3′) 6.3 Hz), 4.96 (dd, 1H, H-3′, J_3′,4′) 3.0 Hz), 4.33 (t,

1H, OH), 4.24 (dt, 1H, H-4′), 3.44 (t, 2H, OH-CH₂), 2.64 (dd, 1H,

5′-CH₂), 2.35-2.49 (m, 3H, 5′-CH₂, N(CH₃)-CH₂), 2.18 (s, 3H,

N-CH₃), 1.54 and 1.33 (2s, 6H, C(CH₃)₂).

5′-Deoxy-2′,3′-O-isopropylidene-5′-[(2-phthalimidooxyethyl)meth-

ylamino]-adenosine (16). To a solution of compound 15 (989 mg,

2.714 mmol), N-hydroxyphthalimide (1.107 g, 6.786 mmol) and

P(Ph)₃(1.780 g, 6.787 mmol) in 50 mL of anhydrous THF was

added DEAD (1.07 mL, 6.8 mmol) in THF (10 mL) under nitrogen

over a period of 3 min at room temperature. After 5 min, 2% of

sodium carbonate (75 mL) was added to the reaction mixture

followed by dichloromethane (100 mL). The organic layer was

washed with 2% Na₂CO₃(75 mL) and then with saturated NaCl (2

× 75 mL). The organic layer was dried over Na₂SO₄and

concentrated to dryness to give a foam. The residue was puriﬁed

by column chromatography and eluted from the column with 1:3

dichloromethane:acetone to yield 842 mg (61%). MS: m/z 510 (M

+ H)⁺)⁺. ¹H NMR (CDCl₃) δ 8.36 (s, 1H, H-2), 8.07 (s, 1H, H-8),

7.79-7.83 (m, 2H, phthalimido aromatic H’s), 7.63-7.72 (m, 2H,

5′-[(4-Aminooxybutyl)methylamino]-5′-deoxy-8-oxoadenosine sul-

fate (1.9:1 salt) (14c). The procedure was the same as reported above

for 12a using 13c (190 mg, 0.41 mmol) and 2 N H₂SO₄(3 mL).

The compound was puriﬁed by column chromatography (elution

with 4:1:0.5 chloroform:methanol:NH₄OH): yield 208 mg (82%).

1

MS: m/z 384 (M + H)⁺. H NMR (DMSO-d₆) δ 10.45 (bs, 1H,

8-OH), 8.05 (s, 1H, H-2), 6.58 (bs, 2H, 6-NH₂), 5.77 (d, 1H, H-1′,

J_1′,2′) 5.0 Hz), 5.37-5.71 (bm, 2H, O-NH₂), 4.83 (t, 1H, H-2′,

J_2′,3′) 4.2 Hz), 4.18-4.29 (m, 2H, H-3′, H-4′), 3.88 (t, 2H, NH₂O-

CH₂), 3.34-3.54 (m, 2H, 5′-CH₂), 3.06 (bt, 2H, N(CH₃)-CH₂), 2.73

(s, 3H, N-CH₃), 1.46-1.74 (bm, 4H, NH₂OCH₂-CH₂CH₂). UV

λ

_max, nm, pH 1, 263.3 (ε 12200), pH 7, 268.9 (ε 13600), pH 13,

279.9 (ε 15,600). Anal. (C₁₅H₂₅N₇O₅·1.9H₂SO₄·0.1C₂H₅OH·2H₂O)

C, H, N, S.

1402 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

phthalimido aromatic H’s), 6.12 (d, 1H, H-1′, J_1′,2′) 2.2 Hz), 5.87

(bs, 2H, 6-NH₂), 5.49 (dd, 1H, H-2′, J_1′,2′) 2.2 Hz, J_2′,3′) 6.4

Hz), 5.05 (dd, 1H, H-3′, J_2′,3′) 6.4 Hz, J_3′,4′) 3.3 Hz), 4.40-4.45

(m, 1H, H-4′), 4.29 (t, 2H, NO-CH₂), 2.89 (t, 2H, NOCH₂-CH₂),

2.83-2.89 (m, 1H, 5′-CH₂), 2.72-2.79 (m, 1H, 5′-CH₂), 2.40 (s,

3H, N-CH₃), 1.62 and 1.40 (2s, 6H, C(CH₃)₂).

15900), pH 13, 260.1 (ε 16200). Anal. (C₁₆H₂₄N₆O₅·0.5CHCl₃·

0.3CH₃OH) C, H, N.

5′-[(2-Carboxamidoethyl)methylamino]-5′-deoxy-8-methylade-

nosine sulfate (1.5:1 salt) (17d). Compound 17a (89 mg, 0.22 mmol)

was dissolved in 5 mL of methanolic ammonia and stirred for ﬁve

days at room temperature. The reaction mixture was concentrated

to dryness and puriﬁed by column chromatography (4:1:0.2

chloroform:methanol:NH₄OH). The product was dissolved in 8 mL

of EtOH and 2 N H₂SO₄was added dropwise. The salt that

precipitated out was ﬁltered and washed with EtOH. This product,

which was hygroscopic in nature, was dissolved in water (2 mL)

and lyophilized to give a white solid: yield 65 mg (55%). MS: m/z

5′-[(2-Aminooxyethyl)methylamino]-5′-deoxyadenosine sulfate

(1:1 salt) (5). A solution of 16 (373 mg, 0.73 mmol) in 1 N H₂SO₄

(5 mL) was heated at 60 °C for 3 h. The reaction mixture was

neutralized with NaHCO₃and lyophilized. The compound was

extracted with EtOH (2 × 20 mL) and concentrated to dryness.

The residue was puriﬁed by column chromatography, eluting with

77:20:3 chloroform:methanol:NH₄OH). The product was dissolved

in 10 mL of EtOH and 1 N H₂SO₄was added dropwise with cooling

to precipitate the salt, which was ﬁltered and washed with EtOH

1

366 (M + H)⁺. H NMR (D₂O) δ 8.43 (s, 1H, H-2), 6.09 (d, 1H,

H-1′, J_1′,2′) 5.9 Hz), 5.0-5.30 (bm, 1H, H-2′), 4.61-4.70 (bm,

1H, H-4′), 4.51-4.54 (bm, 1H, H-3′), 3.30-3.89 (bm, 5H, N-CH₃

and N(CH₃)-CH₂), 2.96 (bs, 2H, 5′-CH₂), 2.77 (bs, 2H, NH₂CO-

CH₂), 2.70 (s, 3H, 8-CH₃). UV λ_max, nm, pH 1, 258.4 (ε 14900),

pH 7, 260.1 (ε 14900), pH 13, 260.1 (ε 15,300). Anal.

(C₁₅H₂₃N₇O₄·1.5H₂SO₄·0.8H₂O) C, H, N, S.

1

and dried in vacuo: yield 100 mg. MS: m/z 340 (M + H)⁺. H

NMR (DMSO-d₆) δ 8.42 (s, 1H, H-8), 8.25 (s, 1H, H-2), 7.68 (bs,

2H, 6-NH₂), 5.99 (d, 1H, H-1′, J_1′,2′) 5.3 Hz), 5.67 (t, 1H, H-2′,

J_2′,3′) 4.6 Hz), 4.33-4.39 (bm, 1H, H-4′), 4.22 (t, 1H, H-3′, J_3′,4′

) 4.7 Hz), 4.06 (bt, 2H, NH₂O-CH₂), 3.63-3.71 (dd, 1H, 5′-CH₂),

3.52-3.59 (bdd, 1H, 5′-CH₂), 3.43 (bm, 2H, N(CH₃)-CH₂), 2.84

(s, 3H, N-CH₃). UV λ_max, nm, pH 1, 258.2 (ε 14,300), pH 7, 259

(ε 14,600), pH 13, 259 (ε 15,500). Anal. (C₁₃H₂₁N₇O₄· 1.0

H₂SO₄·0.5C₂H₅OH·1.0H₂O) C, H, N.

5′-[(2-Carboxamidoethyl)methyamino]-5′-deoxy-8-ethyladenos-

ine sulfate (1.1:1 salt) (17e). The procedure was the same as reported

above for 17d using 17b (149 mg, 0.36 mmol) and methanolic

ammonia (5 mL): yield 94 mg (51%). MS: m/z 380 (M + H)⁺. ¹H

NMR (DMSO-d₆) δ 8.08 (s, 1H, H-2), 7.31 (bs, 1H, CO-NH₂),

7.10 (bs, 2H, 6-NH₂), 6.71 (bs, 1H, CO-NH₂), 5.72 (d, 1H, H-1′,

J_1′2′) 5.4 Hz), 5.31 (d, 1H, OH-2′, J_2′-2′OH) 6.2 Hz), 5.16 (d,

1H, OH-3′, J_3′-3′OH) 5.5 Hz), 5.09 (ddd, 1H, H-2′, J_1′,2′) 5.4 Hz,

J_2′,3′) 5.7 Hz, J_2′-2′OH) 6.2 Hz), 4.17 (ddd, 1H, H-3′, J_2′,3′) 5.7

Hz, J_3′,4′) 4.3 Hz, J_3′-3′OH) 5.5 Hz), 3.92-3.99 (m, 1H, H-4′),

2.87 (q, 2H, 8-CH₂CH₃), 2.69-2.75 (m, 1H, 5′-CH₂), 2.52-2.60

(m, 3H, CO-CH_2,5′-CH₂), 2.18 (bs, 2H, N(CH₃)-CH₂), 2.16 (s,

3H, N-CH₃), 1.30 (t, 3H, CH₃of 8-Et). ¹H NMR (D₂O) δ 8.38 (s,

1H, H-2), 6.09 (d, 1H, H-1′, J_1′,2′) 6.2 Hz), 5.33 (bs, 1H, H-2′),

4.56-4.62 (m, 1H, H-4′), 4.51-4.54 (m, 1H, H-3′), 3.87-3.96 (bm,

2H, NH₂CO-CH₂), 3.56 (s, 3H, N-CH₃), 2.98-3.80 (bm, 2H,

8-CH₂CH₃), 2.96 (bs, 2H, 5′-CH₂), 2.72-2.82 (m, 2H, N(CH₃)-

CH₂), 1.39 (s, 3H, CH₃of 8-Et). UV λ_max, nm, pH 1, 259.4 (ε

15200), pH 7, 260.8 (ε 15100), pH 13, 260.6 (ε 15500). Anal.

(C₁₆H₂₅N₇O₄·1.1H₂SO₄·1.05H₂O) C, H, N, S.

5′-[(2-Carboethoxyethyl)methylamino]-5′-deoxy-8-methyl-

adenosine (17a). A mixture of 9a (500 mg, 1.69 mmol), ethyl

3-chloropropionate (270 mg, 1.97 mmol), DIEA (109 mg, 0.14 mL,

0.84 mmol), and DMF (5 mL) was heated at 60 °C for two days.

Starting material remained but because the solution was getting

darker, heating was stopped. The reaction mixture was concentrated

to dryness. The product was puriﬁed by column chromatography

(7:1:0.1 chloroform:methanol:NH₄OH) to give a sticky solid: yield

1

210 mg (31%). MS m/z 395 (M + H)⁺. H NMR (DMSO-d₆) δ

8.08 (s, 1H, H-2), 7.11 (bs, 2H, 6-NH₂), 5.74 (d, 1H, H-1′, J_1′2′

)

5.6 Hz), 5.33 (bd, 1H, OH-2′), 5.16 (bd, 1H, OH-3′), 5.12 (bdd,

1H, H-2′, J_1′,2′) 5.6 Hz, J_2′,3′) 5.5 Hz), 4.21 (bdd, 1H, H-3′, J_2′,3′

) 5.5 Hz, J_3′,4′) 4.3 Hz), 4.01 (q, 2H, OCH₂CH₃), 3.91-4.00 (m,

1H, H-4′), 2.70-2.77 (m, 1H, 5′-CH₂), 2.54-2.66 (m, 3H, 5′-CH_2,

CO-CH₂), 2.53 (s, 3H, 8-CH₃), 2.38 (t, 2H, N(CH₃)-CH₂), 2.16 (bs,

3H, N-CH₃), 1.15 (t, 3H, OCH₂CH₃).

5′-[(Carboxamidomethyl)methylamino]-5′-deoxy-8-methylade-

nosine sulfate (1.45:1 salt) (17f). The same procedure used to prepare

17d was used to prepare 17f using 17c (200 mg, 0.52 mmol) and

methanolic ammonia (5 mL): yield 105 mg (39%). MS: m/z 352

5′-[(2-Carboethoxyethyl)methylamino]-5′-deoxy-8-ethyladenos-

ine (17b). Compound 17b was prepared by the same procedure as

described for the preparation of 17a using 9b (260 mg, 0.84 mmol),

ethyl 3-chloropropionate (138 mg, 1.0 mmol), DIEA (53 mg, 0.07

mL, 0.41 mmol), and DMF (4 mL). After column chromatography

(elution with 7:1:0.1 chloroform:methanol:NH₄OH), a glassy sticky

solid was obtained: yield 153 mg (44%). MS: m/z 409 (M + H)⁺.

¹H NMR (DMSO-d₆) δ 8.08 (s, 1H, H-2), 7.10 (bs, 2H, 6-NH₂),

1

(M + H)⁺. H NMR (DMSO-d₆) δ 8.08 (s, 1H, H-2), 7.11 (bs,

2H, CO-NH₂), 7.07 (bs, 2H, 6-NH₂), 5.74 (d, 1H, H-1′, J_1′2′) 5.2

Hz), 5.34 (d, 1H, OH-2′, J_2′-2′OH) 5.9 Hz), 5.21 (d, 1H, OH-3′,

J_3′-3′OH) 5.7 Hz), 4.97 (ddd, 1H, H-2′, J_1′,2′) 5.2 Hz, J_2′,3′) 5.5

Hz, J_2′-2′OH) 5.9 Hz), 4.21 (ddd, 1H, H-3′, J_2′,3′) 5.5 Hz, J_3′,4′

)

5.2 Hz, J_3′-3′OH) 5.7 Hz), 3.94-4.01 (m, 1H, H-4′), 2.94 (d, 1H,

N(CH₃)-CH₂, J ) 15.7 Hz), 2.88 (d, 1H, N(CH₃)-CH₂, J ) 15.7

Hz), 2.75-2.80 (m, 1H, 5′-CH₂), 2.63-2.70 (m, 1H, 5′-CH₂), 2.53

5.71 (d, 1H, H-1′, J_1′2′) 5.5 Hz), 5.32 (bd, 1H, OH-2′, J_2′-2′OH

)

5.0 Hz), 5.16 (bd, 1H, OH-3′, J_3′-3′OH) 5.1 Hz), 5.12 (ddd, 1H,

H-2′, J_1′,2′) 5.5 Hz, J_2′,3′) 5.7 Hz, J_2′-2′OH) 5.0 Hz), 4.14 (ddd,

1H, H-3′, J_2′,3′) 5.7 Hz, J_3′,4′) 4.1 Hz, J_3′-3′OH) 5.1 Hz), 4.01

(q, 2H, OCH₂CH₃), 3.91-3.98 (m, 1H, H-4′), 2.87 (q, 2H, CH₂of

8-Et), 2.71-2.79 (m, 1H, 5′-CH₂), 2.51-2.65 (m, 3H, 5′-CH_2,CO-

CH₂), 2.38 (t, 2H, N(CH₃)-CH₂), 2.16 (bs, 3H, N-CH₃), 1.30 (t,

3H, CH₃of 8-Et), 1.15 (t, 3H, OCH₂CH₃).

1

(s, 3H, 8-CH₃), 2.23 (s, 3H, N-CH₃). H NMR (D₂O) δ 8.40 (s,

1H, H-2), 6.08 (d, 1H, H-1′, J_1′,2′) 4.8 Hz), 5.01 (t, 1H, H-2′, J_2′,3′

) 5.2 Hz), 4.60 (t, 1H, H-3′, J_3′,4′) 4.9 Hz), 4.50-4.59 (m, 1H,

H-4′), 4.03-4.17 (m, 2H, NH₂CO-CH₂), 3.82-3.92 (m, 1H, 5′-

CH₂), 3.68-3.76 (m, 1H, 5′-CH₂), 3.03 (s, 3H, N-CH₃), 2.70 (s,

3H, 8-CH₃). UV λ_max, nm, pH 1, 259 (ε 15900), pH 7, 259.7 (ε

16100), pH 13, 260.2 (ε 16100). Anal. (C₁₄H₂₁N₇O₄· 1.45-

H₂SO₄·0.2C₂H₅OH·1.3H₂O) C, H, N, S.

5′-[(Carboethoxymethyl)methyamino]-5′-deoxy-8-methyladenos-

ine (17c). Compound 17c was prepared by the same procedure as

described for the preparation of 17a using 9a (415 mg, 1.41 mmol),

ethyl chloroacetate (207 mg, 0.18 mL, 1.68 mmol), DIEA (91 mg,

0.12 mL, 0.70 mmol), and DMF (5 mL): yield 204 mg (38%). MS:

m/z 381 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.06 (s, 1H, H-2), 7.11

(bs, 2H, 6-NH₂), 5.73 (d, 1H, H-1′, J_1′2′) 5.4 Hz), 5.33 (bd, 1H,

OH-2′, J_2′-2′OH) 4.7 Hz), 5.19 (bd, 1H, OH-3′, J_3′-3′OH) 4.9 Hz),

5.03 (ddd, 1H, H-2′, J_1′,2′) 5.4 Hz, J_2′,3′) 5.7 Hz, J_2′-2′OH) 4.7

5′-[(2-Carboethoxyethyl)methylamino]-5′-deoxyadenosine (17g).

The general procedure previously described for 17a was used to

prepare 17g using 9h⁵³(400 mg, 1.42 mmol), ethyl 3-chloropro-

pionate (214 mg, 1.56 mmol), DIEA (92 mg, 0.12 mL, 0.71 mmol),

and DMF (5 mL). The reaction mixture was heated at 60 °C for

two days. The product was puriﬁed by column chromatography

(7:1 chloroform:methanol) to give a solid: yield 102 mg (19%).

MS m/z 381 (M + H)⁺.

Hz), 4.17 (ddd, 1H, H-3′, J_2′,3′) 5.7 Hz, J_3′,4′) 4.4 Hz, J_3′-3′OH

)

4.9 Hz), 4.02 (q, 2H, OCH₂CH₃), 3.92-3.99 (m, 1H, H-4′), 3.27

(bs, 2H, N(CH₃)-CH₂), 2.83-2.90 (m, 1H, 5′-CH₂), 2.70-2.79 (m,

1H, 5′-CH₂), 2.53 (s, 3H, 8-CH₃), 2.31 (s, 3H, N-CH₃), 1.13 (t,

3H, OCH₂CH₃). UV λ_max, nm, pH 1, 258.9 (ε 16100), pH 7, 260 (ε

5′-Deoxy-5′-[(2-carboethoxyethyl)methylamino]-2′,3′-O-isopro-

pylideneadenosine (17h). To compound 9g (380 mg, 1.18 mmol)

in anhydrous CH₃CN (5 mL) was added ethyl 3-chloropropionate

(195 mg, 0.18 mL, 1.42 mmol) and diisopropylethylamine (153

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1403

mg, 0.2 mL, 1.18 mmol) and the reaction mixture was stirred at

75-80 °C for 72 h under nitrogen. The reaction mixture was

evaporated to dryness, and the residue was puriﬁed by column

chromatography (silica gel 230-400 mesh, elution with 9:1

methylene chloride:methanol) to yield 260 mg (52%), MS: m/z 421

(M + H)⁺.

5′-[(2-Carboxamidoethyl)methylamino]-5′-deoxy-2′,3′-O-isopro-

pylideneadenosine (17i). A mixture of 17h (80 mg, 0.19 mmol) in

saturated methanolic ammonia (15 mL) was stirred for 12 days at

room temperature. The resulting solution was concentrated to

dryness, and the residue was puriﬁed by column chromatography

(elution with 9:1:0.1 methylene chloride:methanol:NH₄OH) to yield

40 mg (55%). MS: m/z 392 (M + H)⁺.

) 5.6 Hz), 3.94-3.99 (m, 1H, H-4′), 3.01 (d, 1H, CO-CHa Hb, J

) 15.2 Hz), 2.95 (d, 1H, CO-CHa Hb, J ) 15.2 Hz), 2.78 (dd, 1H,

5′-CH₂), 2.66 (dd, 1H, 5′-CH₂), 2.53 (s, 3H, 8-CH₃), 2.22 (s, 3H,

N-CH₃). UV λ_max, nm, pH 1, 258.9 (ε 15300), pH 7, 259.3 (ε

15600), pH 13, 260.1 (ε 16100). Anal. (C₁₄H₂₂N₈O₄· 0.2

CH₃OH·0.4H₂O) C, H, N.

5′-[(3-Aminopropyl)methylamino]-5′-deoxy-8-(methylamino)ad-

enosine sulfate (2.4:1 salt) (18a) and 5′-deoxy-8-(methylamino)-5′-

(3-methylaminopropylamino)adenosine sulfate (2.4:1 salt) (19a).

A solution of 8c (175 mg, 0.55 mmol) in 2 mL of N-methyl-1,3-

propanediamine was stirred for four days at ambient temperature.

The mixture was poured into diethyl ether (20 mL). Decantation

of the ether layer left an oil, which was puriﬁed by column

chromatography. The column was eluted with 4:1:0.5 chloroform:

methanol:NH₄OH to afford two isomers. These separated isomers

were dissolved in 6 mL of EtOH and 5 mL of EtOH respectively,

and 2 N H₂SO₄was added dropwise. The sulfate salts that

precipitated out were ﬁltered and washed with EtOH. These salts,

which were hygroscopic in nature, were dissolved in water (2 mL)

and lyophilized to give white solids: 18a: yield 50 mg (15%). MS:

m/z 367 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 7.91 (s, 1H, H-2), 6.90

(bs, 1H, 8CH₃-NH), 6.49 (bs, 2H, 6-NH₂), 5.70 (d, 1H, H-1′, J_1′,2′

) 4.8 Hz), 4.92 (t, 1H, H-2′, J_2′,3′) 5.5 Hz), 4.17 (t, 1H, H-3′,

J_3′,4′) 5.2 Hz), 3.89-3.96 (bm, 1H, H-4′), 2.89 (s, 3H, 8NH-CH₃),

2.53-2.70 (m, 4H, NH₂-CH_2,5′-CH₂), 2.38 (t, 2H, N(CH₃)-CH₂),

2.17 (s, 3H, N-CH₃), 1.48-1.60 (m, 2H, NH₂CH₂-CH₂). ¹H NMR

(D₂O) δ 8.28 (s, 1H, H-2), 5.87 (d, 1H, H-1′, J_1′,2′) 5.1 Hz), 5.18

(t, 1H, H-2′, J_2′,3′) 5.1 Hz), 4.54 (t, 1H, H-3′, J_3′,4′) 5.1 Hz),

4.46-4.51 (m, 1H, H-4′), 3.01-3.82 (bm, 6H, 5′-CH_2,NH₂-CH_2,

N(CH₃)-CH₂), 3.05 (s, 3H, 8NH-CH₃), 2.94 (s, 3H, N-CH₃),

2.05-2.18 (bm, 2H, NH₂CH₂-CH₂). UV λ_max, nm, pH 1, 275.5 (ε

14500), pH 7, 275 (ε 17200), pH 13, 277 (ε 17500). Anal.

(C₁₅H₂₆N₈O₃·2.4H₂SO₄·0.2C₂H₅OH) C, H, N. 19a: yield 29 mg

5′-[(2-Carboxamidoethyl)methylamino]-5′-deoxyadenosine sul-

fate (1.9:1 salt) (17j). Compound 17i (20 mg) was dissolved in 1 N

H₂SO₄(2 mL) and the solution was stirred at room temperature

for 36 h. The reaction mixture was concentrated to 0.5 mL, absolute

ethanol (3 mL) was added to the solution to produce a slight

turbidity, and the mixture was chilled at 0 °C. The solid was ﬁltered,

washed with ethanol, and dried under high vacuum: yield 15 mg

1

(51%). MS: m/z 352 (M + H)⁺. H NMR (DMSO-d₆) δ 8.64 (s,

1H, H-8), 8.43 (s, 1H, H-2), 7.59 (bs, 1H, NH₂), 7.17 (bs, 1H,

NH₂), 6.02 (bdd, 1H, H-1′, J_1′,2′) 3.4 Hz), 4.65 (t, 1H, H-2′, J_2′,3′

) 6.4 Hz), 4.17-4.21 (bm, 1H, H-3′), 3.34-3.42 (bm, 1H, H-4′),

3.52-3.67 (bm, 4H, 5′-CH₂, N(CH₃)-CH₂), 2.80 (bs, 3H, N-CH₃),

2.54-2.60 (bm, 2H, CO-CH₂). UV λ_max, nm, pH 1, 256.3 (ε 15200),

pH 7, 258.5 (ε 15400), pH 13, 259.6 (ε 16100). Anal.

(C₁₄H₂₁N₇O₄·1.9H₂SO₄·1.6H₂O) C, H, N, S.

5′-Deoxy-5′-[(2-hydrazinocarbonylethyl)methylamino]-8-methyl-

adenosine sulfate (2:1 salt) (17k). Compound 17a (115 mg, 0.29

mmol) was dissolved in 10 mL of anhydrous ethanol, and hydrazine

monohydrate (73 mg, 0.07 mL, 1.46 mmol) was added to the

solution. The reaction mixture was heated to reﬂux overnight.

Hydrazine monohydrate (0.07 mL) was added again, and heating

was continued overnight. The resulting solution was evaporated to

dryness. The crude product was puriﬁed by column chromatography

(4:1:0.5 chloroform:methanol:NH₄OH), affording a sticky solid. The

product was dissolved in 8 mL of EtOH, and 2 N H₂SO₄was added

dropwise. The salt that was precipitated out was ﬁltered and washed

with EtOH. This salt, which was hygroscopic in nature, was

dissolved in water (2 mL) and lyophilized to give a white solid:

1

(7%). MS: m/z 367 (M + H)⁺. H NMR (DMSO-d₆) δ 7.96 (s,

1H, H-2), 7.0 (bs, 1H, 8CH₃-NH), 6.57 (bs, 2H, 6-NH₂), 5.80 (d,

1H, H-1′, J_1′,2′) 5.5 Hz), 4.97 (t, 1H, H-2′, J_2′,3′) 5.5 Hz), 5.4

(bs, 2H, 2′, 3′-OH’s), 4.33 (t, 1H, H-3′, J_3′,4′) 3.7 Hz), 4.17-4.24

(bm, 1H, H-4′), 3.20-3.50 (m, 2H, 5′-CH₂), 3.05-3.85 (m, 4H,

NH-CH₂), 2.88 (s, 3H, 8NH-CH₃), 2.49 (s, 3H, NH-CH₃), 1.85-2.0

(bm, 2H, NHCH₂-CH₂). ¹H NMR (D₂O) δ 8.27 (s, 1H, H-2), 5.86

(d, 1H, H-1′, J_1′,2′) 5.2 Hz), 5.16 (t, 1H, H-2′, J_2′,3′) 5.5 Hz),

4.55 (t, 1H, H-3′, J_3′,4′) 4.5 Hz), 4.36-4.44 (m, 1H, H-4′),

3.47-3.69 (m, 2H, 5′-CH₂), 3.22 (t, 2H, NH-CH₂), 3.10 (t, 2H,

CH₃NH-CH₂), 3.05 (s, 3H, 8NH-CH₃), 2.71 (s, 3H, NH-CH₃),

2.04-2.18 (bm, 2H, NHCH₂-CH₂). UV λ_max, nm, pH 1, 274.1 (ε

14300), pH 7, 276 (ε 17100), pH 13, 277.1 (ε 18700). Anal.

(C₁₅H₂₆N₈O₃·2.4H₂SO₄·0.2C₂H₅OH) C, H, N.

1

yield 71 mg (65%). MS: m/z 381 (M + H)⁺. H NMR (D₂O) δ

8.44 (s, 1H, H-2), 6.09 (d, 1H, H-1′, J_1′,2′) 5.6 Hz), 5.10 (dd, 1H,

H-2′, J_2′,3′) 4.5 Hz), 4.53-4.62 (bm, 2H, H-3′, H-4′), 3.86-3.94

(m, 1H, 5′-CH₂), 3.52-3.66 (m, 3H, 5′-CH₂, N(CH₃)-CH₂), 2.95

(s, 3H, N-CH₃), 2.86 (t, 2H, NHCO-CH₂), 2.70 (s, 3H, 8-CH₃).

UV λ_max, nm, pH 1, 258.4 (ε 15000), pH 7, 259.3 (ε 15200), pH

13, 260.4 (ε 15700). Anal. (C₁₅H₂₄N₈O₄·2.0 H₂SO₄·2.7H₂O) C,

H, N, S.

5′-[(3-Aminopropyl)methylamino]-5′-deoxy-8-phenyladenos-

ine sulfate (2.2:1 salt) (18b) and 5′-deoxy-5′-(3-methylaminopro-

pylamino)-8-phenyladenosine sulfate (1.7:1 salt) (19b). The same

procedure as described above for 18a was used to prepare 18b and

19b from 8d (200 mg, 0.55 mmol), and N-methyl-1,3-propanedi-

amine (3 mL) except in this case, after one day of stirring at room

temperature, the reaction mixture was heated at 65 °C for two days.

The column was eluted with 4:1:0.2 chloroform:methanol:NH₄OH.

After the same work up, two isomers were obtained. 18b: yield

5′-Deoxy-5′-[(2-hydrazinocarbonylethyl)methylamino]-adenos-

ine sulfate (2:1 salt) (17l). The procedure was the same as reported

above for 17k using 17g (95 mg, 0.25 mmol) and hydrazine

monohydrate (63 mg, 0.06 mL, 1.25 mmol): yield 39 mg (43%).

MS: m/z 367 (M + H)⁺. ¹H NMR (D₂O) δ 8.47 (s, 1H, H-8), 8.45

(s, 1H, H-2), 6.17 (d, 1H, H-1′, J_1′,2′) 4.7 Hz), 4.90 (dd, 1H, H-2′,

J_2′,3′) 5.2 Hz), 4.56-4.60 (bm, 1H, H-4′), 4.48 (dd, 1H, H-3′,

J_3′,4′) 5.0 Hz), 3.82 (dd, 1H, 5′-CH₂), 3.66 (dd, 1H, 5′-CH₂), 3.59

(bt, 2H, N(CH₃)-CH₂), 2.97 (s, 3H, N-CH₃), 2.86 (t, 2H, NHCO-

CH₂). UV λ_max, nm, pH 1, 256.8 (ε 16600), pH 7, 258.5 (ε 16800),

pH 13, 259.5 (ε 17,600). Anal. (C₁₄H₂₂N₈O₄·2.0 H₂SO₄·2.0H₂O)

C, H, N.

1

124 mg (34%). MS: m/z 414 (M + H)⁺. H NMR (DMSO-d₆) δ

8.38 (s, 1H, H-2), 7.61-7.84 (m, 7H, 8-phenyl, 6-NH₂), 5.88 (d,

1H, H-1′, J_1′,2′) 6.3 Hz), 5.17-5.30 (bm, 1H, H-2′), 4.34-4.38

(bm, 1H, H-3′), 4.25-4.31 (bm, 1H, H-4′), 3.10-3.85 (bm, 4H,

N(CH₃)-CH_2,5′-CH₂), 2.80-2.94 (m, 2H, NH₂-CH₂), 2.78 (s, 3H,

N-CH₃), 1.81-2.0 (bm, 2H, NH₂CH₂-CH₂). UV λ_max, nm, pH 1,

274.2 (ε 20500), pH 7, 275.8 (ε 16300), pH 13, 274.5 (ε 16400).

Anal. (C₂₀H₂₇N₇O₃·2.2H₂SO₄·0.1C₂H₅OH·2.5H₂O) C, H, N, S.

19b: yield 151 mg (42%). MS: m/z 414 (M + H)⁺. ¹H NMR

(DMSO-d₆) δ 8.23 (s, 1H, H-2), 7.0-7.74 (m, 2H, 8-phenyl o-H’s),

7.60-7.65 (m, 3H, 8-phenyl m- and p-H’s), 7.46 (bs, 2H, 6-NH₂),

5.80 (d, 1H, H-1′, J_1′,2′) 6.2 Hz), 5.25 (t, 1H, H-2′, J_2′,3′) 4.9

Hz), 5.5-6.0 (m, 2H, NH’s), 4.29-4.35 (bm, 1H, H-3′), 4.12-4.20

(m, 1H, H-4′), 3.40-3.47 (m, 1H, 5′-CH₂), 3.19-3.25 (m, 1H, 5′-

CH₂), 2.94 (m, 4H, NH-CH₂, CH₃NH-CH₂), 2.54 (s, 3H, NH-CH₃),

5′-Deoxy-5′-[(hydrazinocarbonylmethyl)methylamino]-8-methyl-

adenosine (17m). The same procedure used to prepare 17k was

used to prepare 17m using 17c (167 mg, 0.44 mmol) and hydrazine

monohydrate (109 mg, 0.11 mL, 2.18 mmol). After the column it

yielded a white solid: yield 154 mg (96%). MS: m/z 367 (M +

H)⁺. ¹H NMR (DMSO-d₆) δ 8.75 (bs, 1H, NH), 8.09 (s, 1H, H-2),

7.10 (bs, 2H, NH-NH₂), 5.74 (d, 1H, H-1′, J_1′,2′) 5.2 Hz), 5.31 (d,

1H, OH-2′, J_2′-2′OH) 6.0 Hz), 5.18 (d, 1H, OH-3′, J_3′-3′OH) 5.6

Hz), 5.0 (ddd, 1H, H-2′, J_1′,2′) 5.2 Hz, J_2′,3′) 5.7 Hz, J_2′-2′OH

)

6.0 Hz), 4.19 (ddd, 1H, H-3′, J_2′,3′) 5.7 Hz, J_3′,4′) 4.9 Hz, J_3′-3′OH

1404 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

1.80-1.95 (bm, 2H, NHCH₂-CH₂). UV λ_max, nm, pH 1, 275.5 (ε

20600), pH 7, 274.9 (ε 16200), pH 13, 274.9 (ε 16,300). Anal.

(C₂₀H₂₇N₇O₃·1.7H₂SO₄·0.05C₂H₅OH·3.3H₂O) C, H, N, S.

5′-[(3-Aminopropyl)methylamino]-5′-deoxy-2′,3′-O-isopropylide-

neadenosine (18c). A mixture of 8h⁴²(1.0 g, 2.60 mmol) and

N-methyl-1,3-propanediamine (1.35 mL, 13.0 mmol) was stirred

overnight under an argon atmosphere. The reaction mixture was

concentrated to dryness, and the crude product was puriﬁed by

column chromatography using chloroform:methanol:NH₄OH (6:1:

0.1) as eluent to give a semisolid. This material was dissolved in

3 mL of water and lyophilized: yield 361 mg (37%). MS: m/z 378

(M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.33 (s, 1H, H-8), 8.17 (s, 1H,

H-2), 7.33 (bs, 2H, 6-NH₂), 6.13 (d, 1H, H-1′, J_1′,2′) 2.3 Hz),

5.49 (dd, 1H, H-2′, J_2′,3′) 4.0 Hz), 4.94 (dd, 1H, H-3′, J_3′,4′) 2.9

Hz), 4.21-4.27 (m, 1H, H-4′), 2.52-2.58 (m, 2H, NH₂-CH₂),

2.26-2.38 (m, 4H, 5′-CH₂, N(CH₃)-CH₂), 2.12 (s, 3H, N-CH₃),

1.53 and 1.33 (2s, 6H, C(CH₃)₂), 1.37-1.47 (m, 2H,

NH₂CH₂-CH₂).

4.17 (dd, 1H, H-3′, J_3′,4′) 3.1 Hz), 3.94-3.99 (m, 1H, H-4′), 2.80

(dd, 1H, 5′-CH₂), 2.74 (dd, 1H, 5′-CH₂), 2.54-2.63 (bm, 4H_,

NHCH₃-CH₂, NH-CH₂), 2.54 (s, 3H, 8-CH₃), 2.24 (s, 3H, NH-

CH₃). UV λ_max, nm, pH 1, 258.4 (ε 15400), pH 7, 259.5 (ε 15400),

pH 13, 260 (ε 15900). Anal. (C₁₄H₂₃N₇O₃·0.4CH₃OH·0.7H₂O) C,

H, N.

5′-Deoxy-2′,3′-O-isopropylidene-5′-[(3-phthalimidopropyl)methyl-

amino]-8-methyladenosine (20a). To a cold solution of compound

7e (500 mg, 1.5 mmol) in anhydrous pyridine (2 mL) was added

methanesulfonyl chloride (196 mg, 0.13 mL, 1.7 mmol) and the

solution was stirred for 2 h at 0 °C. The reaction mixture was

concentrated to dryness to afford crude 8e. Methylamine (33%

solution in EtOH, 12 mL) was added to this crude mixture, and

the solution was stirred for three days at room temperature. The

reaction mixture was evaporated to dryness. The resulting crude

9e was dissolved in anhydrous DMF (3 mL), DIEA (0.07 mL),

and N-(3-bromopropyl)phthalimide (502 mg, 1.87 mmol) were

added, and the reaction mixture was heated overnight at 60 °C.

The solution was evaporated to dryness, and the residue was

dissolved in CHCl₃(10 mL), washed with water, dried over Na₂SO₄,

and concentrated to dryness. The resulting syrup was puriﬁed by

column chromatography. The column was eluted with 97:3 chlo-

roform:methanol to yield 108 mg (13%). MS m/z 522 (M + H)⁺.

¹H NMR (CDCl₃) δ 8.26 (s, 1H, H-2), 7.82-7.86 (m, 2H,

phthalimido aromatic H’s), 7.69-7.73 (m, 2H, phthalimido aromatic

5′-[(3-Aminopropyl)methylamino]-5′-deoxyadenosine sulfate (2:1

salt) (18d). The procedure described for 17j was used to prepare

18d from 18c (200 mg, 0.53 mmol): yield 121 mg (41%). MS: m/z

1

338 (M + H)⁺. H NMR (DMSO-d₆) δ 8.47 (bs, 1H, H-8), 8.29

(bs, 1H, H-2), 7.0-10.0 (broad peaks, NH₂s + H₂SO₄), 5.97 (d,

1H, H-1′, J_1′,2′) 4.7 Hz), 5.70 (bs, 1H, 2′-OH), 5.56 (bs, 1H, 3′-

OH), 4.72 (bt, 1H, H-2′), 4.30 (bm, 1H, H-4′), 4.21 (bt, 1H, H-3′),

3.30-3.70 (bm, 2H, 5′-CH₂), 3.08 (bs, 2H, N(CH₃)-CH₂), 2.83 (bm,

2H, NH₂-CH₂), 2.70 (bs, 3H, N-CH₃), 1.84-1.92 (m, 2H,

NH₂CH₂-CH₂). UV λ_max, nm, pH 1, 257(ε 14700), pH 7, 259.2 (ε

15000), pH 13, 260 (ε 15300). Anal. (C₁₄H₂₃N₇O₃· 2.0H₂SO₄·

0.25C₂H₅OH·0.7H₂O) C, H, N.

H’s), 5.98 (d, 1H, H-1′, J_1′,2′) 1.8 Hz), 5.76 (dd, 1H, H-2′, J_1′,2′

)

1.8 Hz, J_2′,3′) 6.5 Hz), 5.38 (bs, 2H, 6-NH₂), 5.10 (dd, 1H, H-3′,

J_2′,3′) 6.5 Hz, J_3′,4′) 3.5 Hz), 4.26-4.32 (m, 1H, H-4′), 3.60-3.76

(m, 2H, N-CH₂), 2.64 (s, 3H, 8-CH₃), 2.58-2.63 (m, 1H, 5′-CH₂),

241-2.48 (m, 1H, 5′-CH₂), 2.38 (t, 2H, N(CH₃)-CH₂), 2.21 (s, 3H,

N-CH₃), 1.68-1.80 (m, 2H, NCH₂-CH₂), 1.61 and 1.40 (2s, 6H,

C(CH₃)₂).

5′-[(2-Aminoethyl)methylamino]-5′-deoxyadenosine (18e) and

5′-deoxy-5′-(2-methylaminoethylamino)adenosine (19c). A mixture

of 8g³²(1.0 g, 3.5 mmol) and N-methylethylenediamine (8 mL)

was stirred at room temperature for 12 days. The reaction mixture

was poured into diethyl ether (50 mL). The ether layer was decanted

and the resulting syrup was puriﬁed by column chromatography

(silica gel 230-400 mesh, elution with 4:1:0.5 chloroform:

methanol:NH₄OH) to give the two isomers. 18e: yield 576 mg

5′-Deoxy-2′,3′-O-isopropylidene-5′-[(3-phthalimidopropyl)methyl-

amino]-8-ethyladenosine (20b). The same procedure as described

for 20a was used to prepare 20b from 7f (600 mg, 1.78 mmol),

MsCl (225 mg,0.15 mL, 1.96 mmol), methylamine (12 mL), and

N-(3-bromopropyl)phthalimide (553 mg, 2.06 mmol): yield 81 mg

(8.5%). MS: m/z 536 (M + H)⁺. ¹H NMR (CDCl₃) δ 8.26 (s, 1H,

H-2), 7.81-7.86 (m, 2H, phthalimido aromatic H’s), 7.67-7.72

(m, 2H, phthalimido aromatic H’s), 5.98 (d, 1H, H-1′, J_1′,2′) 2.0

Hz), 5.75 (dd, 1H, H-2′, J_1′,2′) 2.0 Hz, J_2′,3′) 6.5 Hz), 5.37 (bs,

2H, 6-NH₂), 5.12 (dd, 1H, H-3′, J_2′,3′) 6.5 Hz, J_3′,4′) 3.5 Hz),

4.25-4.33 (m, 1H, H-4′), 3.61-3.75 (m, 2H, N-CH₂), 2.94 (q, 2H,

CH₂of 8-Et), 2.62-2.68 (m, 1H, 5′-CH₂), 243-2.49 (m, 1H, 5′-

CH₂), 2.38 (bt, 2H, N(CH₃)-CH₂), 2.22 (s, 3H, N-CH₃), 1.70-1.80

(m, 2H, NCH₂-CH₂), 1.60 and 1.40 (2s, 6H, C(CH₃)₂), 1.42 (t,

3H, CH₃of 8-Et).

1

(51%). MS: m/z 324 (M + H)⁺. H NMR (DMSO-d₆) δ 8.35 (s,

1H, H-8), 8.15 (s, 1H, H-2), 7.27 (bs, 2H, 6-NH₂), 5.85 (d, 1H,

H-1′, J_1′,2′) 5.3 Hz), 4.64 (dd, 1H, H-2′, J_2′,3′) 5.3 Hz), 4.11 (dd,

1H, H-3′, J_3′,4′) 4.3 Hz), 3.95-4.0 (m, 1H, H-4′), 2.90-3.60 (bs,

4H, 2′, 3′-OHs + NHs), 2.70 (dd, 1H, 5′-CH₂), 2.50-2.60 (bm,

3H, 5′-CH_2,N(CH₃)-CH₂), 2.36 (t, 2H, NH₂-CH₂), 2.19 (s, 3H,

N-CH₃). UV λ_max, nm, pH 1, 256.9 (ε 14100), pH 7, 259.5 (ε

14700), pH 13, 259.2 (ε 15300). Anal. (C₁₃H₂₁N₇O₃·

0.25CHCl₃·0.5H₂O) C, H, N. 19c: yield 323 mg (29%). MS: m/z

324 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.35 (s, 1H, H-8), 8.14 (s,

1H, H-2), 7.28 (bs, 2H, 6-NH₂), 5.84 (d, 1H, H-1′, J_1′,2′) 5.9 Hz),

4.69 (dd, 1H, H-2′, J_2′,3′) 5.3 Hz), 4.12 (dd, 1H, H-3′, J_3′,4′) 3.5

Hz), 3.94-3.99 (m, 1H, H-4′), 2.90-3.60 (bs, 4H, 2′, 3′-OHs +

NHs), 2.80 (dd, 1H, 5′-CH₂), 2.73 (dd, 1H, 5′-CH₂), 2.50-2.62

5′-Deoxy-2′,3′-O-isopropylidene-5′-[(3-aminopropyl)methylamino]-

8-methyladenosine (21a). To a boiling solution of 20a (100 mg,

0.19 mmol) in 3 mL of ethanol was added hydrazine monohydrate

(50 mg, 0.048 mL, 0.99 mmol) and the solution was heated to reﬂux

for 1 h. The reaction mixture was cooled down to room temperature,

and the solid was ﬁltered and washed with ethanol. The ﬁltrate

was evaporated to dryness. This crude product was puriﬁed by

column chromatography using chloroform:methanol:NH₄OH (7:1:

(bm, 4H_,NHCH₃-CH₂, NH-CH₂), 2.25 (s, 3H, NH-CH₃). UV λ_max

,

nm, pH 1, 256.4 (ε 13900), pH 7, 259 (ε 13900), pH 13, 259.8 (ε

14000). Anal. (C₁₃H₂₁N₇O₃·0.05CH₃OH·0.1H₂O) C, H, N.

5′-[(2-Aminoethyl)methylamino]-5′-deoxy-8-methyladenosine (18f)

and 5′-deoxy-8-methyl-5′-(2-methylaminoethylamino)adenosine (19d).

The procedure described above for 18e/19c was used to prepare

18f/19d from 8a (300 mg, 1.0 mmol) and N-methylethylenediamine

1

0.2) for elution: yield 69 mg (92%). MS: m/z 392 (M + H)⁺. H

NMR (DMSO-d₆) δ 8.12 (s, 1H, H-2), 7.18 (bs, 2H, 6-NH₂), 6.06

(d, 1H, H-1′, J_1′,2′) 1.9 Hz), 5.79 (dd, 1H, H-2′, J_1′,2′) 1.9 Hz,

J_2′,3′) 6.3 Hz), 5.0 (dd, 1H, H-3′, J_2′,3′) 6.3 Hz, J_3′,4′) 3.1 Hz),

4.15-4.21 (m, 1H, H-4′), 2.56 (s, 3H, 8-CH₃), 2.40-2.50 (m, 2H,

N(CH₃)-CH₂), 2.13-2.31 (bm, 4H, NH₂-CH_2,5′-CH₂), 2.07 (s,

3H, N-CH₃), 1.53 and 1.33 (2s, 6H, C(CH₃)₂), 1.22-1.32 (m, 2H,

NH₂CH₂-CH₂).

1

(3 mL). 18f: yield ( 69 mg (18.3%). MS: m/z 338 (M + H)⁺. H

NMR (DMSO-d₆) δ 8.08 (s, 1H, H-2), 7.11 (bs, 2H, 6-NH₂), 5.74

(d, 1H, H-1′, J_1′,2′) 5.4 Hz), 5.31 (bs, 1H, 2′-OH), 5.04 (dd, 1H,

H-2′, J_2′,3′) 5.6 Hz), 4.17 (dd, 1H, H-3′, J_3′,4′) 4.5 Hz), 4.08 (bs,

1H, 3′-OH), 3.93-3.98 (m, 1H, H-4′), 2.70 (dd, 1H, 5′-CH₂),

2.53-2.57 (bm, 3H, 5′-CH_2,N(CH₃)-CH₂), 2.53 (s, 3H, 8-CH₃),

2.30-2.35 (m, 2H, NH₂-CH₂), 2.16 (s, 3H, N-CH₃). UV λ_max, nm,

pH 1, 258.5 (ε 15600), pH 7, 259.1 (ε 16000), pH 13, 260 (ε 16200).

Anal. (C₁₄H₂₃N₇O₃·0.5CH₃OH·0.3H₂O) C, H, N. 19d: yield ( 58

mg (15.4%). MS: m/z 338 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.07

(s, 1H, H-2), 7.12 (bs, 2H, 6-NH₂), 5.72 (d, 1H, H-1′, J_1′,2′) 6.4

Hz), 5.27 (bs, 1H, 2′-OH), 5.02 (dd, 1H, H-2′, J_2′,3′) 5.4 Hz),

5′-Deoxy-2′,3′-O-isopropylidene-5′-[(3-aminopropyl)methylamino]-

8-ethyladenosine (21b). Compound 21b was prepared by the same

procedure as reported for 21a using 20b (76 mg, 0.14 mmol) and

hydrazine monohydrate (38 mg, 0.036 mL, 0.76 mmol): yield 47

1

mg (82%). MS: m/z 406 (M + H)⁺. H NMR (DMSO-d₆) δ 8.12

(s, 1H, H-2), 7.16 (bs, 2H, 6-NH₂), 6.03 (d, 1H, H-1′, J_1′,2′) 2.0

Hz), 5.76 (dd, 1H, H-2′, J_1′,2′) 2.0 Hz, J_2′,3′) 6.4 Hz), 5.01 (dd,

1H, H-3′, J_2′,3′) 6.4 Hz, J_3′,4′) 3.0 Hz), 4.15-4.22 (m, 1H, H-4′),

Adenine C⁸-Substituted AdoMetDC Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1405

2.87-2.94 (bm, 2H, 5′-CH₂), 2.38-2.53 (m, 2H, CH₂of 8-Et),

2.13-2.34 (m, 4H, N(CH₃)-CH₂, NH₂-CH₂), 2.08 (s, 3H, N-CH₃),

1.53 and 1.33 (2s, 6H, C(CH₃)₂), 1.22-1.39 (m, 2H,

NH₂CH₂-CH₂), 1.31 (t, 3H, CH₃of 8-Et).

was extracted with EtOAc (2 × 40 mL) and washed with brine

solution (20 mL). The organic layer was dried over Na₂SO₄, ﬁltered,

washed with EtOAc, and concentrated to dryness. The product was

puriﬁed by column chromatography. The column was eluted with

4:1:0.5 chloroform:methanol:NH₄OH to yield 165 mg (32%). MS:

5′-[(3-Aminopropyl)methylamino]-5′-deoxy-8-methyladenos-

ine sulfate (2:1 salt) (21c). Compound 21a (66 mg, 0.168 mmol)

was dissolved in 2 mL of 1 N H₂SO₄and stirred overnight. To this

solution was added ethanol (10 mL), causing a ﬁne solid to separate.

The solvent was decanted, the compound was dissolved in water

(1 mL), and 15 mL of ethanol was added. The resulting solid was

dissolved in water (2 mL) and lyophilized to give a white solid:

yield 66 mg (67%). MS: m/z 352 (M + H)⁺. ¹H NMR (DMSO-d₆)

δ 8.16 (s, 1H, H-2), 7.74 (bs, 2H, CH₂-NH₂), 7.24 (bs, 2H, 6-NH₂),

5.84 (d, 1H, H-1′, J_1′,2′) 6.0 Hz), 5.62 (bs, 2H, 2′, 3′-OH’s), 4.98

(t, 1H, H-2′, J_2′,3′) 4.4 Hz), 4.29-4.36 (bm, 1H, H-4′), 4.23 (t,

1H, H-3′), 3.40-3.68 (bm, 2H, 5′-CH₂), 2.99-3.19 (bm, 2H,

N(CH₃)-CH₂), 2.83 (t, 2H, NH₂-CH₂), 2.70 (bs, 3H, N-CH₃), 2.55

(s, 3H, 8-CH₃), 1.80-1.93 (m, 2H, NH₂CH₂-CH₂). UV λ_max, nm,

pH 1, 258.7 (ε 14900), pH 7, 259.5 (ε 15100), pH 13, 260.7 (ε

15300). Anal. (C₁₅H₂₅N₇O₃·2.0H₂SO₄·2.5H₂O) C, H, N, S.

5′-[(3-Aminopropyl)methylamino]-5′-deoxy-8-ethyladenosine sul-

fate (2.5:1 salt) (21d). The procedure described for 21c was used

to prepare 21d from 21b (44 mg, 0.108 mmol). In this case after

the addition of EtOH a ﬁne solid came out, which was collected

by centrifugation. It was then dissolved in water (2 mL) and

lyophilized: yield 30 mg (69%). MS: m/z 366 (M + H)⁺. ¹H NMR

(DMSO-d₆) δ; 8.15 (s, 1H, H-2), 7.68 (bs, 2H, CH₂-NH₂), 7.21

(bs, 2H, 6-NH₂), 5.82 (d, 1H, H-1′, J_1′,2′) 6.0 Hz), 5.61 (bs, 2H,

2′, 3′-OH’s), 5.04 (t, 1H, H-2′), 4.19-4.27 (bm, 2H, H-3′, 4′),

3.22-3.43 (bm, 4H, 5′-CH₂, N(CH₃)-CH₂), 2.90 (q, 2H, 8CH₂CH₃),

2.81 (t, 2H, NH₂-CH₂), 2.50 (bs, 3H, N-CH₃), 1.73-1.88 (bm,

2H, NH₂CH₂-CH₂), 1.32 (t, 3H, 8CH₂CH₃). UV λ_max, nm, pH 1,

259.3 (ε 15100), pH 7, 260.5 (ε 15100), pH 13, 260.3 (ε 15100).

Anal. (C₁₆H₂₇N₇O₃·2.5H₂SO₄·2.5H₂O) C, H, N.

5′-Deoxy 5′-[(2-guanidinoethyl)methylamino]adenosine (22a). To

a stirred solution of 18e (218 mg, 0.67 mmol) and 1H-pyrazole-

1-carboxamidine hydrochloride³⁰(196 mg, 1.34 mmol) in anhy-

drous DMF (5 mL) was added DIEA (479 mg, 0.65 mL, 3.7 mmol)

under nitrogen at 5 °C. Stirring was continued at room temperature

overnight. The reaction mixture was concentrated to dryness, and

the product was puriﬁed by column chromatography (silica gel

230-400 mesh, elution with 4:1:0.3 chloroform:methanol:NH₄OH)

to afford 219 mg (89%). MS: m/z 366 (M + H)⁺. ¹H NMR (DMSO-

d₆) δ 8.34 (s, 1H, H-8), 8.15 (s, 1H, H-2), 7.48, 7.37, 7.28 (bs,

NHs), 5.87 (d, 1H, H-1′, J_1′,2′) 5.2 Hz), 5.50 (d, 1H, 2′-OH, J_2′-2′OH

) 5.7 Hz), 5.28 (d, 1H, 3′-OH, J_3′-3′OH) 4.5 Hz), 4.65 (ddd, 1H,

H-2′, J_1′,2′) 5.2 Hz, J_2′,3′) 5.1 Hz, J_2′-2′OH) 5.7 Hz), 4.12 (ddd,

1H, H-3′, J_2′,3′) 5.1 Hz, J_3′,4′) 4.6 Hz, J_3′-3′OH) 4.5 Hz), 4.0-4.10

(bm, 1H, H-4′), 3.30-3.50 (bm, 2H, 5′-CH₂), 3.12-3.28 (bm, 2H,

NH-CH₂), 2.55-2.65 (bm, 2H_,N(CH₃)-CH₂), 2.25 (bs, 3H, N-CH₃).

UV λ_max, nm, pH 1, 256.3 (ε 10900), pH 7, 259 (ε 11300), pH 13,

259 (ε 11000). Anal. (C₁₄H₂₃N₉O₃·0.05CHCl₃·3.5H₂O) C, H, N.

5′-[(2-Cyanoethyl)methylamino]-5′-deoxyadenosine (22b). A so-

lution of 8j⁵⁴(1.0 g, 2.37 mmol) in 10 mL of 3-(methylamino)

propionitrile was stirred at room temperature for ﬁve days. The

reaction mixture was poured into diethyl ether (50 mL). The ether

layer was decanted, and the resulting syrup was puriﬁed by column

chromatography (silica gel, elution with 7:1:0.1 chloroform:

methanol:NH₄OH) to yield 685 mg (87%). MS: m/z 324 (M + H)⁺.

¹H NMR (DMSO-d₆) δ 8.34 (s, 1H, H-8), 8.15 (s, 1H, H-2), 7.29

(bs, 2H, 6-NH₂), 5.87 (d, 1H, H-1′, J_1′,2′) 5.4 Hz), 5.46 (bd, 1H,

2′-OH), 5.22 (bd, 1H, 3′-OH), 4.65 (m, 1H, H-2′), 4.13 (m, 1H,

H-3′), 3.97-4.03 (m, 1H, H-4′), 2.78 (dd, 1H, 5′-CH₂), 2.58-2.68

(bm, 5H, 5′-CH₂, NC-CH₂CH₂), 2.24 (s, 3H, N-CH₃).

1

m/z 367 (M + H)⁺. H NMR (DMSO-d₆) δ 8.70 (bs, 1H, NOH),

8.33 (s, 1H, H-8), 8.15 (s, 1H, H-2), 7.27 (bs, 2H, 6-NH₂), 5.86 (d,

1H, H-1′, J_1′,2′) 5.3 Hz), 5.43 (d, 1H, 2′-OH, J_2′-2′OH) 6.0 Hz),

5.35 (bs, 2H, C-NH₂), 5.20 (d, 1H, 3′-OH, J_3′-3′OH) 5.2 Hz), 4.63

(ddd, 1H, H-2′, J_1′,2′) 5.3 Hz, J_2′,3′) 4.9 Hz, J_2′-2′OH) 6.0 Hz),

4.10 (ddd, 1H, H-3′, J_2′,3′) 4.9 Hz, J_3′,4′) 3.5 Hz, J_3′-3′OH) 5.2

Hz), 3.96-4.0 (m, 1H, H-4′), 2.64-2.75 (bm, 2H, 5′-CH₂), 2.56

(t, 2H_,N(CH₃)-CH₂), 2.20 (s, 3H, N-CH₃), 2.09 (t, 2H_,C-CH₂).

Anal. (C₁₄H₂₂N₈O₄·1.2C₂H₅OH·0.2CH₃OH) C, H, N.

5′-Deoxy-5′-(N,N-dimethylamino)-8-methyladenosine (23a). A

mixture of 8a (150 mg, 0.50 mmol) and a 2 M solution of

dimethylamine in methanol (10 mL) in a steel bomb was heated

for two days at 90 °C. The reaction mixture was concentrated to

dryness and puriﬁed by column chromatography (elution with 4:1:

0.15 chloroform:methanol:NH₄OH) to afford 38 mg (25%). MS

m/z 309 (M + H)⁺. ¹H NMR (DMSO-d₆) δ 8.08 (s, 1H, H-2), 7.11

(bs, 2H, 6-NH₂), 5.74 (d, 1H, H-1′, J_1′,2′) 5.5 Hz), 5.31 (d, 1H,

OH-2′, J_2′-2′OH) 5.9 Hz), 5.18 (d, 1H, OH-3′, J_3′-3′OH) 5.4 Hz),

5.03 (ddd, 1H, H-2′, J_1′,2′) 5.5 Hz, J_2′,3′) 4.6 Hz, J_2′-2′OH) 5.9

Hz), 4.15 (ddd, 1H, H-3′, J_2′,3′) 4.6 Hz, J_3′,4′) 5.5 Hz, J_3′-3′OH

)

5.4 Hz), 3.91-3.96 (m, 1H, H-4′), 2.55-2.61 (m, 1H, 5′-CH₂),

2.53 (s, 3H, 8-CH₃), 2.42-2.48 (m, 1H, 5′-CH₂), 2.14 (bs, 6H,

N-(CH₃)₂). UV λ_max, nm, pH 1, 258.5 (ε 15300), pH 7, 259.3 (ε

15300), pH 13, 260.1 (ε 15500). Anal. (C₁₃H₂₀N₆O₃·

0.35CHCl₃·0.5C₂H₅OH) C, H, N.

5′-Deoxy-5′-(N,N-dimethylamino)adenosine (23b). Compound

23b was prepared by the same procedure as described for the

preparation of 23a using 8g³²(500 mg, 1.75 mmol) and a 2 M

solution of dimethylamine in methanol (20 mL): yield 238 mg

1

(46%). MS m/z 295 (M + H)⁺. H NMR (DMSO-d₆) δ 8.33 (s,

1H, H-8), 8.15 (s, 1H, H-2), 7.29 (bs, 2H, 6-NH₂), 5.86 (d, 1H,

H-1′, J_1′,2′) 5.4 Hz), 5.45 (d, 1H, OH-2′, J_2′-2′OH) 5.9 Hz), 5.22

(bd, 1H, OH-3′, J_3′-3′OH) 3.9 Hz), 4.65 (ddd, 1H, H-2′, J_1′,2′

)

5.4 Hz, J_2′,3′) 5.5 Hz, J_2′-2′OH) 5.9 Hz), 4.10 (ddd, 1H, H-3′,

J_2′,3′) 5.5 Hz, J_3′,4′) 4.5 Hz, J_3′-3′OH) 3.9 Hz), 3.94-4.0 (m,

1H, H-4′), 2.62 (dd, 1H, 5′-CH₂), 2.48 (dd, 1H, 5′-CH₂), 2.18 (bs,

6H, N-(CH₃)₂). UV λ_max, nm, pH 1, 256.3 (ε 15100), pH 7, 259.2

(ε 15500), pH 13, 259.7 (ε 15600). Anal. (C₁₂H₁₈N₆O₃·0.35CH₃OH)

C, H, N.

5′-Deoxy-5′-methylthio-8-methyladenosine (24a). A solution of

8a (200 mg, 0.66 mmol) and sodium thiomethoxide (47 mg, 0.67

mmol) in 2 mL of anhydrous DMF was stirred for two days at

room temperature and then concentrated to dryness. The crude

product was puriﬁed by column chromatography using chloroform:

methanol (7:1) as eluent to yield 102 mg (49%). MS m/z 312 (M

1

+ H)⁺. H NMR (DMSO-d₆) δ 8.09 (s, 1H, H-2), 7.12 (bs, 2H,

6-NH₂), 5.77 (d, 1H, H-1′, J_1′,2′) 5.7 Hz), 5.38 (bd, 1H, OH-2′,

J_2′-2′OH) 4.4 Hz), 5.31 (d, 1H, OH-3′, J_3′-3′OH) 4.2 Hz), 5.15

(bddd, 1H, H-2′, J_1′,2′) 5.7 Hz, J_2′,3′) 5.7 Hz, J_2′-2′OH) 4.4 Hz),

4.20 (ddd, 1H, H-3′, J_2′,3′) 5.7 Hz, J_3′,4′) 3.6 Hz, J_3′-3′OH) 4.2

Hz), 3.97-4.05 (m, 1H, H-4′), 2.74-2.92 (m, 2H, 5′-CH₂), 2.54

(s, 3H, 8-CH₃), 2.03 (s, 3H, S-CH₃).

5′-Deoxy-5′-dimethylsulfonio-8-methyladenosine bromide (25a).

Compound 24a (78 mg, 0.25 mmol) in a 2:1 mixture (4 mL) of

formic and acetic acid was treated with a 2 M solution of

bromomethane in diethyl ether (5 mL) and stirred for six days in

darkness at room temperature. Solvents were removed in vacuo,

and a solution of the residue in water (10 mL) was extracted with

(3 × 10 mL) ether. The aqueous layer was concentrated to dryness.

The resulting product was dissolved in MeOH (10 mL), ﬁltered,

and treated with diethyl ether to precipitate out the salt. The salt

was ﬁltered, washed with ether, and dried in vacuo to give white

5′-Deoxy-5′-[(2-hydroxyamidinoethyl)methylamino]adenosine

(22c). To a solution of 22b (470 mg, 1.4 mmol) in 20 mL of

anhydrous MeOH and 4 mL of anhydrous DMF under nitrogen

was added hydroxylamine hydrochloride (258 mg, 3.7 mmol) and

potassium hydroxide (206 mg, 3.7 mmol) and the resulting

suspension was stirred at room temperature for two days. The

reaction mixture was concentrated to dryness, and the crude product

1

solid: yield 79 mg (78%). MS m/z 326 (M)⁺. H NMR (D₂O) δ

8.24 (s, 1H, H-2), 6.03 (d, 1H, H-1′, J_1′,2′) 5.4 Hz), 5.29 (t, 1H,

H-2′, J_2′,3′) 5.7 Hz), 4.80 (t, 1H, H-3′, J_3′,4′) 4.8 Hz), 4.52-4.60

1406 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

McCloskey et al.

boxylase (EC 4.1.1.17) by substrate and product analogues. J. Am.

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(m, 1H, H-4′), 4.11-4.20 (m, 1H, 5′-CH₂), 3.81-3.90 (m, 1H, 5′-

CH₂), 2.92 and 2.89 (2s, 6H, S-(CH₃)₂), 2.67 (s, 3H, 8-CH₃).

5′-Deoxy-5′-dimethylsulfonio-8-methyladenosine chloride (25b).

Ion exchange resin (IRA-400, Cl^-form) was washed repeatedly

with water and loaded into the column. The column was left

overnight and washed again repeatedly with water. The bromide

salt 25a (50 mg) was dissolved in water (1 mL) and put on the

column. The column was eluted with water very slowly in the dark.

The desired fractions were combined and lyophilized to yield 30

mg (68%). MS m/z 326 (M)⁺. ¹H NMR (D₂O) δ 8.24 (s, 1H, H-2),

6.03 (d, 1H, H-1′, J_1′,2′) 5.4 Hz), 5.29 (t, 1H, H-2′, J_2′,3′) 5.6

Hz), 4.80 (t, 1H, H-3′, J_3′,4′) 4.9 Hz), 4.52-4.60 (m, 1H, H-4′),

3.82-4.11 (m, 1H, 5′-CH₂), 3.75-3.83 (m, 1H, 5′-CH₂), 2.95 and

2.92 (2s, 6H, S-(CH₃)₂), 2.69 (s, 3H, 8-CH₃). UV λ_max, nm, pH 1,

258.8 (ε 15000), pH 7, 259.1 (ε 14700), pH 13, 263.5 (ε 13000).

Anal. (C₁₃H₂₀ClN₅O₃S·2H₂O) C, H, N.

5′-Deoxy-5′-dimethylsulfonioadenosine bromide (25c). The

procedure described for 25a was used to prepare 25c from 24b

(58 mg, 0.19 mmol): yield 49 mg (65%). MS m/z 312 (M)⁺.

5′-Deoxy-5′-dimethylsulfonioadenosine chloride (25d). The pro-

cedure used was identical to that for 25b but starting with 25c.

The desired fractions were combined together and lyophilized to

1

yield 30 mg (44%). MS m/z 312 (M)⁺. H NMR (D₂O) δ 8.29 (s,

1H, H-8), 8.27 (s, 1H, H-2), 6.12 (d, 1H, H-1′, J_1′,2′) 4.4 Hz),

4.99 (t, 1H, H-2′, J_2′,3′) 5.1 Hz), 4.55-4.62 (m, 2H, H-3′, H-4′),

3.84-4.0 (m, 2H, 5′-CH₂), 2.95 and 2.93 (2s, 6H, S-(CH₃)₂). UV

λ

_max, nm, pH 1, 256.4 (ε 14300), pH 7, 259.6 (ε 14400), pH 13,

266 (ε 12300). Anal. (C₁₂H₁₈ClN₅O₃S·1.5H₂O·0.1C₂H₅OH) C, H,

N, S.

Acknowledgment. This work was supported by grant PO1

CA-94000 from the National Cancer Institute. S.E.E. is indebted

to the W. M. Keck Foundation and the Lucille P. Markey

Charitable Trust. We thank the staffs of NE-CAT beamlines

8-BM and 24-ID-C at the Advanced Photon Source (NIH grant

RR-15301) and the F2 beam line of the Cornell High Energy

Synchrotron Source (NIH grant RR-01646) for providing beam

time. We thank Leslie Kinsland and Stephanie Brannon for

assistance in the preparation of this manuscript and thank Rachel

Robbins for her assistance in performing some of the confor-

mational searches reported in this manuscript.

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Supporting Information Available: Elemental analyses used

to determine the degree of purity for all target compounds. This

material is available free of charge via the Internet at http://

pubs.acs.org.

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JM801126A

Article Doi

New insights into the design of inhibitors of human S-adenosylmethionine decarboxylase: studies of adenine C8 substitution in structural analogues of S-adenosylmethionine

DOI: 10.1021/jm801126a

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