Transition state analogue inhibitors of human methylthioadenosine phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine nucleosidase incorporating acyclic ribooxacarbenium ion mimics

Article abstract of DOI:10.1016/j.bmc.2012.07.006

Several acyclic hydroxy-methylthio-amines with 3-5 carbon atoms were prepared and coupled via a methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and Escherichia coli and Neisseria meningitidis MTANs and gave K_i values as low as 0.23 nM. These results were compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-5-methylthio-d-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-yl)methyl]3-hydroxy-4- methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly weaker than 4. Cleavage of the 2,3 carbon-carbon bond in MT-Immucillin-A (3) gave an acyclic product (79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain secondary amine resulted in a 250-fold loss of activity against the same enzyme [(±)-65 vs (±)-68]. The inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case were found to be more potent than their cyclic counterparts.

Full text of DOI:10.1016/j.bmc.2012.07.006

Bioorganic & Medicinal Chemistry 20 (2012) 5181–5187
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Transition state analogue inhibitors of human methylthioadenosine
phosphorylase and bacterial methylthioadenosine/S-adenosylhomocysteine
nucleosidase incorporating acyclic ribooxacarbenium ion mimics
Keith Clinch a, , Gary B. Evans a, Richard F. G. Fröhlich a,à, Shivali A. Gulab a, Jemy A. Gutierrez b,
,
⇑
a
Jennifer M. Mason a, Vern L. Schramm b, Peter C. Tyler a, , Anthony D. Woolhouse
⇑
a Carbohydrate Chemistry, Industrial Research Limited, PO Box 31310, Lower Hutt 5040, New Zealand
b Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several acyclic hydroxy-methylthio-amines with 3–5 carbon atoms were prepared and coupled via a
methylene link to 9-deazaadenine. The products were tested for inhibition against human MTAP and
Escherichia coli and Neisseria meningitidis MTANs and gave Ki values as low as 0.23 nM. These results were
compared to those obtained with 1st and 2nd generation inhibitors (1S)-1-(9-deazaadenin-9-yl)-1,4-
Received 30 May 2012
Revised 28 June 2012
Accepted 4 July 2012
Available online 14 July 2012
dideoxy-1,4-imino-5-methylthio-D-ribitol (MT-Immucillin-A, 3) and (3R,4S)-1-[9-deazaadenin-9-
yl)methyl]3-hydroxy-4-methylthiomethylpyrrolidine (MT-DADMe-Immucillin-A, 4). The best inhibitors
were found to exhibit binding affinities of approximately 2- to 4-fold those of 3 but were significantly
weaker than 4. Cleavage of the 2,3 carbon–carbon bond in MT-Immucillin-A (3) gave an acyclic product
(79) with a 21,500 fold loss of activity against E. coli MTAN. In another case, N-methylation of a side chain
secondary amine resulted in a 250-fold loss of activity against the same enzyme [( )-65 vs ( )-68]. The
inhibition results were also contrasted with those acyclic derivatives previously prepared as inhibitors
for a related enzyme, purine nucleoside phosphorylase (PNP), where some inhibitors in the latter case
were found to be more potent than their cyclic counterparts.
Keywords:
Human MTAP
Bacterial MTANs
Ribooxacarbenium ion mimics
Inhibitors
Acyclic hydroxy-methylthio-amines
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
erating MTA as a by-product of the reaction.23 Efficient inhibitors
of MTAN will lead to elevated concentrations of MTA which may
The human methylthioadenosine phosphorylase (MTAP)1,2 and
bacterial methylthioadenosine/S-adenosylhomocysteine nucleosi-
dase (MTAN)3,4 enzymes are targets for drug design. MTAP is a target
for cancer therapy through its importance in polyamine biosynthe-
sis and S-adenosylmethionine (SAM) metabolism.5–10 Efficient
inhibitors of MTAP are expected to lead to accumulation of methyl-
thioadenosine (MTA) and consequent inhibition of spermidine and
spermine synthases while MTA itself has antiproliferative proper-
ties.11–17 In addition, the metabolism of SAM is likely to be prevented
along with the disruption of SAM-dependent methylation. We have
developed potent inhibitors of MTAP that show strong anti-cancer
effects18,19 thereby providing validation for MTAP as a cancer target.
MTAN is involved in bacterial polyamine biosynthesis and quo-
rum sensing.20–22 Certain gram-negative organisms utilize species-
specific acyl-homoserine lactones as quorum sensing molecules.
These are synthesized from N-acylated S-adenosylmethionine gen-
induce feedback inhibition of acyl-homoserine lactone synthesis.
S-Ribosylhomocysteine, the product of MTAN-catalysed hydrolysis
of S-adenosylhomocysteine, is catabolized by S-ribosylhomocy-
steinase (LuxS) into L-homocysteine and (4S)-4,5-dihydroxy-2,3-
pentanedione.24 The latter forms the basis of the bacterial quorum
sensing signal AI-2.21,25,26 The LuxS enzyme has been identified in
more than 55 gram-negative and gram-positive bacteria and the
evidence suggests that the autoinducing signal AI-2 is common
to many bacterial species. Thus, without MTAN activity there can
be no synthesis of AI-2. In consequence, inhibition of MTAN would
be expected to inhibit polyamine biosynthesis, block salvage path-
ways for methionine and adenine as well as blocking the synthesis
of two types of quorum sensing autoinducer molecules.
We have been engaged for a number of years in the design and
synthesis of transition state analogue inhibitors of a number of N-
ribosyltransferase enzymes, including those mentioned above. The
use of measured kinetic isotope effects has enabled the identifica-
tion of the nature of the transition states of many of these
enzymes.27–29 With this knowledge, inhibitor design and synthesis
has provided exceedingly potent inhibitors of human and Plasmo-
dium falciparum purine nucleoside phosphorylases (PNPs),30–33 hu-
man MTAP,34–37 protozoan nucleoside hydrolases38,39 and bacterial
⇑
Corresponding authors. Fax: +64 4 931 3055.
E-mail addresses: k.clinch@irl.cri.nz (K. Clinch), p.tyler@irl.cri.nz (P.C. Tyler).
Present address: Pfizer Worldwide Research and Development, 620 Memorial
Drive, Cambridge, MA 02139, USA.
à
Present address: piCHEM Forschungs- und Entwicklungs GmbH, Kahngasse 25, A
8045 Graz, Austria.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.006

5182
K. Clinch et al. / Bioorg. Med. Chem. 20 (2012) 5181–5187
O
N
NH
R
MeS
O
N
MeS
2
b
b
Boc
N
+
-
H
N
H
N
H
N
NH Cl
NHBoc
3
NH
HO
N
NH
HO
MeS
H
N
H
N
O
N
TBDMSO
HO
N
11 I, II R = OH
12 I, II R = SMe
14 I, II, III
13 III
a
HO
OH
HO
HO
HO
OH
c
I
=
(HO) MeS
3
2
1
2
O
N
NH
O
N
1
R
2
R
H
N
H
N
H
II
=
(HO) MeS
MeS
N
N
NH
HO
N
NH
N
MeS
H
N
MeS
H
H
N
N
X
N
N
III =
and enantiomer
N
HO
OH
HO
15 I, II, III
16 I, II, III R = BOM, R = NH
HO
HO
6 X = H
7 X = NH
1
2
5
4
R
= BOM, R = Cl
2
d
e
1
2
2
1
2
17 I, II, III R = H, R = NH
2
Figure 1. Structures of known transition state inhibitors of various N-
ribosyltransferases.
Scheme 2. Reagents and conditions: (a) (i) MsCl, Et3N, CH2Cl2, 0 °C ? rt, 30 min, (ii)
NaSMe, DMF, rt, 1 h, 75% (I), 91% (II); (b) aq. HCl (37%), MeOH, 0 °C ? rt, 1 h, 100%
(I, II and III); (c) 10, 2-picoline-borane complex (for I and II) or NaCNBH3 (for III),
MeOH, Et3N (for I and II) or NaHCO3 (for III), rt, 16 h, 67% (I), 78% (II), 71% (III); (d)
7 M NH3–MeOH, 135 °C, sealed tube, 24–30 h, 74% (I), 86% (II), 59% (III); (e)
NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt 1 h, 84% (I), 79% (II), 76% (III).
MTANs.36,40–47 Immucillin-H (1, Forodesine) and DADMe-Immucil-
lin-H (2, Ulodesine) are first and second generation inhibitors of
the PNPs, and are in clinical development for the treatment of T-
cell proliferative disorders and gout (Fig. 1). MT-Immucillin-A (3)
and MT-DADMe-Immucillin-A (4) are corresponding first and sec-
ond generation inhibitors of MTAP and bacterial MTANs and the
second generation inhibitor 4 is in preclinical development for
oncology indications.18,19 Recently, we reported on third genera-
tion inhibitors of PNP with acyclic aza-sugar mimics, some of
which showed surprising activity. For example, DATMe-Immucil-
lin-H (5) and SerMe-Immucillin-H (6) had exceptional activity with
the achiral serinol derivative 7 being the most potent PNP inhibitor
yet discovered (Ki⁄ = 2.1 pM).48
Therefore we wished to investigate whether replacement of the
hydroxymethyl and 9-deazahypoxanthine or 9-deazaguanine
units, found in acyclic PNP inhibitors, with methylthio and 9-dea-
zaadenine ones, respectively, would lead to potent inhibitors of the
related MTAP and MTAN enzymes. In our previous work on the
preparation of acyclic PNP inhibitors, including compounds 5–7,
we found reductive amination/alkylation and Mannich reactions
useful in their construction.48 In both cases several primary and
secondary amino alcohols were prepared and coupled through
their amino functions, via a methylene link, to the 9-position of
either deazahypoxanthine or deazaguanine. In this paper we de-
scribe the synthesis of several hydroxy-methylthio-substituted
primary and secondary amines and their couplings to aldehyde
10 or 9-deazaadenine,49 substrates for the reductive amination/
alkylation (Schemes 1–8) and Mannich reactions (Schemes 9–11),
respectively. In addition, the direct conversion of MT-Immucillin-
A (3) into an acyclic derivative is also described (Scheme 12).
changed derivative with N,N-dimethylformamide (DMF)
(Scheme 1). The structure of bromide 9 was characterized spectro-
51
scopically and further confirmed by X-ray crystallography.
Serinol-linked 9-deazahypoxanthine or 9-deazaguanine deriva-
tives 6 and 7 (Fig. 1) have been shown to be low pM inhibitors of
human PNP48 and so we prepared the analogous methylthio-seri-
nol-based 9-deazaadenine derivative 17 III to see if this would also
be the case with human MTAP and bacterial MTANs. Treatment of
racemic methylthio-serinol derivative 13 III52 with hydrochloric
acid gave amine salt 14 III. This was reductively alkylated with
aldehyde 10 using sodium cyanoborohydride,53 after adjusting
the pH of the mixture to between 6 and 7 with sodium hydrogen
carbonate, to give the benzyloxymethyl (BOM) protected chloride
15 III (Scheme 2). Displacement of the chloride in 15 III with
ammonia was effected in a sealed tube at 135 °C to give 16 III. Fi-
nally, the BOM protecting group was efficiently removed hydro-
genolytically using hydrazine hydrate and palladium black in
methanolic ammonia solution to produce 17 III.
Encouraged by the enzyme inhibition results (Table 1) obtained
for 17 III, we next prepared the individual enantiomers 17 I and 17
II. Readily obtainable (S)-alcohol 11 I, prepared as for its enantio-
mer,54 was converted through its mesylate into methylthio com-
pound 12 I and then into (R)-amine salt 14 I on treatment with
hydrochloric acid (Scheme 2). Similarly, (R)-alcohol 11 II54 was
transformed into (S)-amino hydrochloride 14 II.
Conversions of 14 I and 14 II into enantiomeric products 17 I
and 17 II, respectively, were achieved as for the racemate above ex-
cept 2-picoline-borane complex55 was used as a less toxic alterna-
tive to sodium cyanoborohydride, and triethylamine was used as a
more convenient base for adjusting the pH. Treatment of amines
2. Chemistry
2.1. Synthesis using the reductive amination/alkylation reaction
14 I and 14 II with (S)-a-methoxy-a-trifluoromethylphenylacetyl
The key aldehyde 10 was synthesized by brominating chloro-
deazapurine 850 followed by trapping of the lithium-bromine-ex-
chloride [(S)-MTPACl]56 gave Mosher amides with d.e.’s of ꢀ94%.
Cl
BOM
N
N
N
R
8 R = H
9 R = Br
10 R = CHO
a
b
Scheme 1. Reagents and conditions: (a) NBS, 0 °C ? rt, 1 h, 71%; (b) (i) n-BuLi, anisole-Et2O (1:3), À78 °C, 3 min, (ii) DMF,À78 ? À40 °C, (iii) H2O, À40 °C ? rt, 57%.

K. Clinch et al. / Bioorg. Med. Chem. 20 (2012) 5181–5187
5183
Table 1
O
-
PhCO
Inhibitory constants (Ki) for acyclic compounds against human MTAP and bacterial
MTANs
2
OH
HO
+
O
NH
H
N
3
NH
a
c
2
O
Compound
Ki human
Ki E. coli
Ki N. meningitidis
MTAPa (nM)
MTANa (nM)
MTANa (nM)
O
1
2
O
O
R O
OR
3
4
1.0 0.5b,c
0.08 0.02d
0.002 .0002d
1.7 0.2
0.23 0.2
1.1 0.4
0.36 0.04
0.8 0.1
3.9 0.5
0.36e
29
SMe
1
2
27 R , R = C(Me)
0.09 0.01c
0.14e
b
1
2
26
28 R = R = H
d
17 III
17 I
17 II
( )-25
33
12
1
0.80 .06
2
NDf
1
NH
R
4.4 0.2
2
R
34
10
3
1
NDf
H
N
N
N
N
HO
1.2 0.1
0.9 0.1
HO
HO
H
H
NH
2
5.2 0.4
87 11
e
N
N
N
N
ent-33
40
6
1
87
8
10
2
10
1
HO
SMe
ent-40
47
54
( )-65
( )-68
( )-72
( )-78
79
34 18
34 24
602 83
2.1 0.5
5.8 0.8
1.8 0.4
HO
SMe
HO
SMe
30
4
36
5
2
1
1
2
31 R = BOM, R = Cl
ent-33
40
9
5
2
4
5
f
g
1
2
32 R = BOM, R = NH
2
34
12
6
2
1
2
33
R
= H, R = NH
2
2300 600
401 37
202 35
61
105 30
368 153
130 20
132
85
47
9
6
4
Scheme 4. Reagents and conditions: (a) (i) Amberlyst A26 (OHÀ) resin, MeOH; (ii)
triphosgene, CH2Cl2, Et3N, rt, 90 min, 92%; (b) AcCl, MeOH, rt, 5 h, 93%; (c) (i) TsCl,
pyridine, 0 °C ? rt, 16 h; (ii) NaSMe, DMF, rt, 3 h, 44%; (d) KOH, i-PrOH, 80 °C, 4 h,
94%; (e) 10, AcCl, MeOH, NaCNBH3, rt, 3 h, 52%; (f) 7 M NH3–MeOH, 135 °C, sealed
tube, 24 h, 60%; (g) NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt 1 h, 74%.
1720 850
a
b
c
d
e
f
for compounds 3 and 4 these are Ki* values.
Ref 34.
Ref 35.
Ref 44.
Ref 40.
ND – not determined.
The free amine present in salt 26, prepared as for its enantiomer61
and liberated from the benzoic acid with basic ion exchange resin,
was converted to the oxazolidinone 27 with triphosgene, then
deacetalized under acid-catalysed conditions to give diol 28. Tosy-
lation of the primary hydroxyl then displacement with sodium
thiomethoxide in DMF led unexpectedly to the rearranged oxazo-
The (S)-enantiomer 17 II structurally resembles 3 with a CHOH
group removed from the latter’s pyrrolidine ring. Similarly, the (S)-
enantiomer present in ( )-25 resembles the structure resulting
from the formal removal of a methylene at the 2-position of the
pyrrolidine ring in 4. We therefore synthesized ( )-25 as shown
(Scheme 3). Acetonide alcohol 1857 was methylthiolated via its
mesylate, then the isopropylidene protecting group was removed
by acid-catalysed transacetalization, and the resulting diol selec-
tively mono-silylated58 to give alcohol ( )-20. Displacement of
the mesylate derivative of ( )-20 with azide followed by hydroge-
nation furnished amine ( )-22 which was de-silylated then reduc-
tively alkylated with aldehyde 10 to afford ( )-23. Transformations
( )-23 ? ( )-24 ? ( )-25 were carried out as described for the con-
versions of 15 I to III ? 17 I to III above.
DATMe-Immucillin-H (5) has been identified, amongst its enan-
tiomer and diastereomers, as a powerful PNP inhibitor (Fig. 1).48
Human MTAP and PNP share similar active sites and overall struc-
tural homology59 and this, together with a crystal structure of 5 in
the active site of human PNP,60 suggested that the methylthio 9-
deazadenine analogue 33 was preferred as a target for MTAP/
MTANs inhibition, rather than the structure in which the alterna-
tive hydroxymethyl was substituted by methylthio (Scheme 4).
lidinone 29. X-ray crystallography62 of 29 with molybdenum K
a
radiation confirmed both the relative and absolute stereochemis-
try. Base-catalysed hydrolysis of the oxazolidinone ring in 29 led
to amine 30 which was reductively alkylated with aldehyde 10
to give 31. The standard conversion of 31 ? 32 ? 33 proceeded
uneventfully as shown in the Scheme. The enantiomer (ent-33) of
33 was also prepared from ent-2661 in the same way as described
for 33 (Scheme 4).
Two further diastereomers (40 and its enantiomer ent-40) of 33
were also prepared (Scheme 5). Azido triol 3463 was converted into
methylthio derivative 36 through regioselective formation of an
intermediate 5-membered ring stannylene acetal. The structure
of 36 was confirmed by 1H–1H DQF-COSY NMR. After reduction
of the azido group in 36, the resulting amine 37 was reductively
2
1
R
N
2
R
N
N
HO
HO
HO
H
N
N
NH
3
2
d
c
2
HO
34
HO
HO
38
R
SMe
SMe
1
R
N
R
1
1
R = OH
35 R = OMs
R
= BOM, R = Cl
2
a
b
e
f
N
N
37
39 R = BOM, R = NH
2
MeS
R
36
MeS
R = MeS
1
2
40 R = H, R = NH
2
R
b
e
N
H
O
NH
2
O
H
N
TBDMSO
HO
N
HO
H
N
1
1
2
18
R = OH
19 R = SMe
20
23
( )- R = OH
(
(
(
)-
R
= BOM, R = Cl
2
N
a
c
d
f
g
( )-21 R = N
( )-22 R = NH
)-24 R = BOM, R = NH
3
2
1
2
2
)-25 R = H, R = NH
2
HO
SMe
ent -40
Scheme 3. Reagents and conditions: (a) (i) MsCl, Et3N, 0 °C ? rt, 30 min, (ii)
NaSMe, DMF, rt, 16 h, 76%; (b) (i) AcCl, MeOH, rt, 1 h, (ii), NaH, TBDMSCl, rt, 2 h,
75%; (c) (i) MsCl, Et3N, 0 °C ? rt, 30 min, (ii) NaN3, DMF, 80 °C, 3 h, 80%; (d)
NH2NH2ÁH2O, Pd black, MeOH, rt 1 h, 82%; (e) (i) aq HCl (37%), MeOH, rt, 1 h, (ii) 10,
NaCNBH3, NaHCO3, MeOH, (iii) 7 M NH3–MeOH, 135 °C, sealed tube, 24 h, 20%; (f)
NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt 1 h, 54%.
Scheme 5. Reagents and conditions: (a) (i) Bu2SnO, toluene, reflux, 30 min, (ii)
MsCl, rt, 16 h; (b) NaSMe, DMF, rt, 2 h, 37% over 2 steps; (c) LAH, THF, 0 °C ? rt, 1 h,
65%; (d) 10, NaCNBH3, HCl, MeOH, 61%; (e) 6 M NH3–EtOH, 130 °C, sealed tube,
48 h, 73%; (g) NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt 1 h, 61%.

5184
K. Clinch et al. / Bioorg. Med. Chem. 20 (2012) 5181–5187
alkylated with aldehyde 10 to give 38 then further transformed
into product by the standard end sequence 38 ? 39 ? 40. The
enantiomer (ent-40) of 40 was made in the same way starting from
ent-34.63
chains (Scheme 8). The 1,3-dioxepine acetal 5566 underwent a
1,3-dipolar cycloaddition with N-benzyl hydroxylamine and form-
aldehyde to afford ( )-56 which after reductive cleavage of the
1,2-isoxazolidine ring, then amino protection as a benzyl carbamate
(Cbz), gave ( )-57. Acid-catalysed rearrangement of the acetonide
moiety in ( )-57 was accomplished in acetone and gave a separable
mixture of the 1,3-dioxane ( )-58 and 1,3-dioxolane ( )-59. Stan-
dard transformation of the hydroxyl in ( )-59 into a methylthio
substituent followed by base-catalysed hydrolysis of the carbamate
moiety gave amine ( )-61. Reductive amination of aldehyde 10
with ( )-61, using sodium triacetoxyborohydride67 as a less toxic
alternative to sodium cyanoborohydride, gave the expected
chloro-deazapurine ( )-62. At this stage we tried a different end se-
quence that avoided the use of sealed pressure vessels. Treatment
of chloride ( )-62 with sodium azide gave ( )-63 which underwent
a Staudinger reaction to produce protected amine ( )-64. The aceto-
nide and BOM protecting groups were then removed by acid-cata-
lysed hydrolysis and hydrogenolysis, respectively, to furnish ( )-65.
In order to explore steric congestion effects on the side chain
adjacent to the amino group, the site that when protonated mimics
the ribosyl cation in the transition state, we prepared compound
47—formally equivalent to adding a hydroxymethyl group to com-
pounds 17 I–III. In similar fashion to the methods presented above,
the tris(hydroxymethyl)aminomethane acetonide 4164 was con-
verted into the N-donor 44 and coupled to aldehyde 10 to provide
the chloro-deazapurine derivative 45 (Scheme 6). The usual trans-
formation of the deazapurine Cl ? NH2 (45 ? 46) then BOM re-
moval furnished 47.
On the other hand, removal of a hydroxymethyl group from 33
would lead to compound 54 containing just one hydroxyl on a 3-
carbon acyclic framework. The synthesis of 54 was accomplished
through conversion of N-benzyl amine 4848 to the known tert-bu-
tyl carbamate (Boc) protected amino alcohol 4965 followed by suc-
cessive treatment with tosyl chloride then sodium thiomethoxide
to give oxazolidinone 51 in addition to the expected product 50
(Scheme 7). After removal of the N-Boc group in 50 with hydro-
chloric acid, the resulting amine salt was reductively alkylated
with aldehyde 10 and the deazapurine ring modified as shown in
the Scheme to give 54.
2.2. Synthesis using the Mannich reaction
N-Methylation of the secondary amine products above would
provide derivatives with an altered affinity towards protonation
whilst at the same time removing an N–H bond which may be
important for enzyme inhibitor interaction. We therefore prepared
a small subset of such compounds. Carbamate ( )-60 was N-meth-
ylated under anhydrous, basic conditions then the Cbz group was
removed by basic solvolysis to give N-methyl amine ( )-66
(Scheme 9). This latter compound underwent a Mannich reaction
with formaldehyde and 9-deazaadenine49 to afford ( )-67, then
the acetal function was hydrolysed with hydrochloric acid to give,
after neutralization, ( )-68, the N-methyl analogue of ( )-65 as the
free base form.
An isomer [( )-72] of ( )-68 was also prepared by Mannich
reaction except in this case the amine precursor ( )-70 was ob-
tained by direct reduction of the N-Cbz group in ( )-69 with lith-
ium aluminium hydride (Scheme 10).
By way of further example we also prepared ( )-78, equivalent
to the formal removal of a methylene from the hydroxymethyl side
chain found in ( )-72 (Scheme 11). It was conveniently prepared
from racemic diethyl tartrate-derived amino alcohol ( )-7348 using
similar chemistry to that used in the previous two schemes. In this
case N-methylation was accomplished by reduction of the N-Boc
function in ( )-75 with lithium aluminium hydride.
The compounds described so far were constructed with either
3- or 4-carbon atoms in their acyclic side chains. We expanded
the scope of the study by preparing products with 5-carbon side
R2
R1
N
N
HO
MeS
MeS
HO
H
N
NH3+Cl-
d
c
NHBoc
N
O
HO
O
HO
44
HO
a
b
45 R1 = BOM, R2 = Cl
41 R = OH
e
f
46 R1 = BOM, R2 = NH2
42 R = OMs
43
R = SMe
47
R
1 = H, R2 = NH2
Scheme 6. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2, 0 °C ? rt, 90 min; (b)
NaSMe, DMF, rt, 15 h; (c) aq HCl (37%), MeOH, rt, 65% over 3 steps; (d) 10, NaCNBH3,
MeOH, rt, 15 h; (e) 7 M NH3–MeOH, 135 °C, sealed tube, 20 h, 78% over 2 steps; (f)
NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt 1 h, 56%.
2.3. Synthesis from a cyclic precursor
2
1
R
N
R
N
N
MT-Immucillin-A (3), a first generation MTAP/MTAN inhibitor,
is ideally set up for conversion to an acyclic system through oxida-
tive cleavage of the vicinal diol. In a one-pot reaction 3 was selec-
tively oxidized with periodate and the resulting dialdehyde
reduced with sodium borohydride to afford 79 (Scheme 12).
NHBn
O
NHBoc
R
H
N
c
a
O
HO
HO
SMe
1
2
48
49 R = OH
50 R = SMe
+
52 R = BOM, R = Cl
d
e
b
1
2
53 R = BOM, R = NH
2
1
2
3. Biological evaluation
54
R
= H, R = NH
2
SMe
The inhibition of the phosphorolysis or hydrolysis of MTA cata-
lysed by human MTAP or bacterial MTANs, respectively, was eval-
uated with 3rd generation acyclic inhibitors and the results
compared with 1st and 2nd generation inhibitors, 3 and 4 (Table 1).
Compounds 17 I–III, ( )-25 and 33 were the most potent inhibitors
with the (R)-enantiomer (17 I) having Ki’s of 4.4 (Km/Ki = 1200) and
0.23 (Km/Ki = 1870) nM against human MTAP and E. coli MTAN,
respectively. Although 17 I was not tested against Neisseria menin-
gitidis MTAN it is likely that this compound would also be a strong
inhibitor of this enzyme given that the racemate (17 III) was the
O
NH
O
51
Scheme 7. Reagents and conditions: (a) (i) H2, Pd/C, EtOH, rt, 16 h, (ii) 6 M HCl,
100 °C, 30 min, (iii) (Boc)2O, Et3N, MeOH, rt, 2 h, 66%; (b) (i) TsCl, pyridine, 0 °C ? rt,
42 h, (ii) NaSMe, DMF, rt, 2 h, 22% (50), 16% (51); (c) (i) aq HCl (37%), MeOH, rt,
5 min, (ii) 10, NaCNBH3, MeOH, rt, 60 h, 44%; (d) 7 M NH3-MeOH, 135 °C, sealed
tube, 24 h, 72%; (e) NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt, 40 min, 76%.

K. Clinch et al. / Bioorg. Med. Chem. 20 (2012) 5181–5187
5185
Bn
NHCbz
OH
N
O
NHCbz
OH
b
a
c
O
O
O
O
O
O
O
O
55
( )-56
( )-57
( )-58
+
MeS
MeS
HO
NH
O
NHCbz
NHCbz
2
d
e
O
O
O
O
O
( )-61
( )-60
( )-59
f
2
NH
1
R
2
R
H
N
N
N
N
MeS
HO
MeS
H
N
H
N
N
i
+
N
H
O
1
-
CF CO
3
2
OH
O
( )-65
2
( )-62 R = BOM, R = Cl
g
h
1
2
( )-63 R = BOM, R = N
3
1
2
64
( )-
R
= BOM, R = NH
2
Scheme 8. One enantiomer series present in the racemic mixtures is drawn to illustrate relative stereochemistry. Reagents and conditions: (a) BnNHOHÁHCl, aq CH2O, NaOAc,
EtOH, reflux, 6 h, 43%; (b) (i) H2, Pd/C. EtOH, rt, 2 days, (ii) BnOCOCl, Et3N, 0 °C ? rt, 30 min, 63%; (c) camphor sulfonic acid, Me2CO, rt, 40 min, ( )-58 (52%), ( )-59 (44%); (d)
(i) MsCl, i-Pr2NEt, CH2Cl2, rt 15 min, (ii) NaSMe, DMF, rt, 1 h, 83%; (e) KOH, i-PrOH, reflux, 2 h, 83%; (f) 10, Na(OAc)3BH, 1,2-dichloroethane, rt, 40 min, 64%; (g) NaN3, DMF,
90 °C, 1 h, 100%; (h) PMe3, THF, aq NH3, rt, 1.5 h, 91%; (i) TFA–H2O, rt, 10 min, then NH2NH2ÁH2O, Pd black, 7 M NH3–MeOH, rt 1 h, 100%.
NH2
N
NH2
N
H
N
H
N
MeS
c
MeS
N
N
d
NHR2
R1
N
RO
RO
a
b
N
O
NHMe
O
60
( )-
Me
Me
O
R1O
OR2
( )-67 R1, R2 = C(Me)2
68
SMe
O
(
( )-73 R1 = OH, R2 = H
( )-77 R = C(Me)2
( )-78 R = H
e
a
)-66
( )-74 R1 = OH, R2 = Boc
( )-
R
1 = R2 = H
b
c
1
75
( )-
R
= SMe, R2 = Boc
Scheme 9. One enantiomer series present in the racemic mixtures is drawn to
illustrate relative stereochemistry. Reagents and conditions: (a) (i) NaH, THF, MeI,
rt, 30 min, (ii) KOH, i-PrOH, reflux, 5 h, 77%; (b) 9-deazaadenine, CH2O, 1,4-dioxane,
H2O, 90 °C, 1 h, (c) aq HCl (37%)- MeOH, rt, 61% over 2 steps.
( )-76 R1 = SMe, R2 = Me
Scheme 11. One enantiomer series present in the racemic mixtures is drawn to
illustrate relative stereochemistry. Reagents and conditions: (a) (Boc)2O, MeOH, rt,
1 h, 85%; (b) (i) MsCl, Et3N, CH2Cl2, rt, 30 min, (ii) NaSMe, DMF, rt 2 h, 60%; (c) LAH,
THF, reflux, 1 h, 74%; (d) 9-deazaadenine, CH2O, 1,4-dioxane, H2O, 90 °C, 1 h; (e) aq
HCl (37%), MeOH, rt, 63% over 2 steps.
NH2
H
N
N
As shown in the schemes, compounds 17 I and ( )-25 can be
drawn such that they resemble cyclic compounds 3 and 4, respec-
tively, with one carbon atom removed. Compound 17 I had binding
affinities in the order of 3- and 4-fold that of 3 aginst E. coli MTAN
and human MTAP, respectively. The racemate 17 III was about half
as potent against N. meningitidis MTAN indicating 17 I could have a
similar potency to 3 against the latter enzyme. Thus it appeared
that compounds 17, despite the larger number of degrees of free-
dom, were able to adopt conformations similar to that taken by 3
in the enzymes’ active sites, resulting in comparable enzyme inhi-
bition. Compared with 4 however, 17 I and ( )-25 showed approx-
imately 110- and 180-fold weaker binding affinities, respectively,
against E. coli MTAN and approximately 50- and 110-fold weaker
binding affinities, respectively, against human MTAP. In contrast
17 III and ( )-25, had smaller 6- and 9-fold differences, respec-
tively, with N. meningitidis MTAN. The large differences seen with
the first two enzymes were attributed to compound 4 being a
better match for the late dissociative transition state found with
R1O
N
NHR
SMe
N
a
c
O
( )-58
Me
R2O
SMe
O
( )-69 R = Cbz
( )- R = Me
( )-71 R1, R2 = C(Me)2
( )-72 R1 = R2 = H
b
d
70
Scheme 10. One enantiomer series present in the racemic mixtures is drawn to
illustrate relative stereochemistry. Reagents and conditions: (a) (i) MsCl, i-Pr2NEt,
CH2Cl2, rt 15 min, (ii) NaSMe, DMF, rt, 1 h, 86%; (b) LAH, THF, rt, 18 h, 57%; (c) 9-
deazaadenine, CH2O, 1,4-dioxane, H2O, 85 °C, 15 min, 68%; (d) aq HCl (37%), MeOH,
rt, 79%.
strongest inhibitor tested with a Ki of 0.80 nM (Km/Ki = 1750). Race-
mic compound ( )-25 was found to be just 1.5 times weaker an
inhibitor than 17 I against E. coli MTAN suggesting one of the enan-
tiomers present in ( )-25 could be of similar potency to 17 I.

5186
K. Clinch et al. / Bioorg. Med. Chem. 20 (2012) 5181–5187
NH
therefore we have prepared a series of acyclic compounds to see
2
H
N
if improved activity and simpler structures could be found as
was the case with the related enzyme PNP.48 Compounds 17 I–
III, ( )-25 and 33 were identified as the best inhibitors with 17 I
being around 3- and 4-fold as active as compound 3 against
E. coli MTAN and human MTAP, respectively, but significantly
weaker than 4 against the same enzymes. (S)-enantiomer 17 II,
the product of the formal removal of a CHOH group from the pyr-
rolidine ring of 3, was found to be a weaker inhibitor than its
(R)-enantiomer 17 I. Racemate ( )-25 containing the enantiomer
present in the product of formal removal of the 2-methylene from
the pyrrolidine in 4 was the second most potent inhibitor found
against E. coli MTAN but it was not determined which enantiomer
had the best activity. It was also found that simple bond cleavage
of a cyclic inhibitor (3 ? 79) or N-methylation of a secondary
amine to a tertiary one [( )-65 ? ( )-68] led to dramatic losses
in activity. In our previous work on the related PNP enzyme, acyclic
compounds were found that were comparable or better inhibitors
than the best cyclic forms and it was further shown that unfavour-
able binding entropies present in these acyclic derivatives were
offset by favourable enthalpies of binding.68 In the present study
such thermodynamic properties remain to be determined but it
is clear that with MTAP/MTANs the acyclic inhibitors described
here have not achieved the same level of potency as seen with
our previous PNP study, particularly when compared to 4. The best
compounds were nevertheless novel and potent inhibitors of
human MTAP and E. coli and N. meningitidis MTANs.
N
a
MeS
H
N
3
N
HO
OH
79
Scheme 12. Reagents and conditions: (a) (i) NaIO4, H2O, rt, 1 h, (ii) NaBH4, rt,
15 min, 47%.
human MTAP37 and E. coli MTAN44 than for the early transition
state of N. meningitidis MTAN.40
Compound 54 contains a side chain one carbon atom shorter
than one of the enantiomers found in ( )-25 and had significantly
reduced inhibitory activity, particularly against E. coli MTAN, expe-
riencing a drop in binding affinity in the region of 110-fold. Alter-
natively, compound 54 can be viewed as the product produced by
the removal of a hydroxymethyl group from 33. In contrast, com-
pound 47 which had an extra hydroxymethyl group compared to
compounds 17 I–III still retained moderate activity.
Compounds 33, 40 and their respective enantiomers ent-33 and
ent-40 are the structures that would be formed from the formal
cleavage of the 1,2-bond in the pyrrolidine ring of 3 with subse-
quent transposition of the terminal CH2SMe and CH2OH groups.
Compound 33 was the strongest inhibitor of this group having
approximately 7- to 17-fold greater affinity over the other com-
pounds for human MTAP and approximately 3- to 13-fold in-
creased affinity for the bacterial MTANs. Again compound 33
showed comparable efficacy to compound 3 being within about 2
to 10 fold of its activity against all three enzymes. Compound 33
was also of a similar potency as 17 I was against human MTAP
and as 17 III was against N. meningitidis MTAN, respectively, but
was about 4-fold less active than 17 I was against E. coli MTAN.
By comparison, oxidative cleavage of the vicinal diol bond in 3
without transposition of the CH2SMe and CH2OH units gave 79,
and resulted in a severe loss of inhibitory activity against E. coli
MTAN together with more moderate losses suffered against the
other two enzymes.
Acknowledgements
This work was supported by research grant C08X0701 from the
New Zealand Foundation for Research, Science & Technology and
by research grants GM41916 and CA135405 from the National
Institutes for Health. We are grateful to Drs Herbert Wong and Yin-
rong Lu for NMR and MS measurements, respectively.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.006.
A dramatic 250-fold loss of binding affinity occured in E. coli
MTAN when ( )-65, a moderate inhibitor against all three en-
zymes, was N-methylated to ( )-68, making it the weakest inhibi-
tor against the former enzyme with a Ki of 2300 nM (Km/Ki = 19). In
contrast, inhibition of N. meningitidis MTAN suffered only an
approximate 12-fold loss in activity by this change but inhibition
improved about 3-fold against human MTAP. This loss of activity
against E. coli MTAN may be due to removal of a critical N–H bond.
A similar case was also observed in the related enzyme PNP where
N-methylation of DATMe-Immucillin-H (5) and SerMe-Immucillin-
H (6) also resulted in a significant loss of activity.48
The isomeric N-methyl compounds ( )-68 and ( )-72 differ only
by having their CH2SMe and CH2OH groups transposed. The latter
compound showed a gain in binding affinity in the order of 6-fold
with E. coli MTAN but a reduction of approximately 2- and 9-fold
for N. meningitidis MTAN and human MTAP, respectively. On the
other hand compound ( )-78 which differs from ( )-72 by one car-
bon atom, resulting in the transformation of a primary alcohol to a
secondary one, produced around a 2-fold increases in potency for
the bacterial MTANs but an approximate 4-fold loss in activity with
human MTAP.
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