Tight binding enantiomers of pre-clinical drug candidates

Article abstract of DOI:10.1016/j.bmc.2015.07.059

MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the enantiomers of MTDIH and MTDIA, compounds 1 and 2, respectively, were prepared and their enzyme binding properties studied. Despite binding less tightly to their target enzymes than their enantiomers compounds 1 and 2 are nanomolar inhibitors.

Full text of DOI:10.1016/j.bmc.2015.07.059

Bioorganic & Medicinal Chemistry 23 (2015) 5326–5333
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tight binding enantiomers of pre-clinical drug candidates
Gary B. Evans a, , Scott A. Cameron a,b, Andreas Luxenburger a, Rong Guan b, Javier Suarez b,
⇑
Keisha Thomas b, Vern L. Schramm b, Peter C. Tyler a
a Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Rd, Lower Hutt 5010, New Zealand
b Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., 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:
MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase
and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be
a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a
variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent
inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered
enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa. Since the enantiomers
of some pharmaceuticals have revealed surprising biological activities, the enantiomers of MTDIH and
MTDIA, compounds 1 and 2, respectively, were prepared and their enzyme binding properties studied.
Despite binding less tightly to their target enzymes than their enantiomers compounds 1 and 2 are
nanomolar inhibitors.
Received 8 June 2015
Revised 26 July 2015
Accepted 27 July 2015
Available online 30 July 2015
Keywords:
Transition state analogue
Enzyme
Cancer
Enantiomer
Drug
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
described enzyme methylthioinosine phosphorylase (MTIP) has
also been published and the potential of inhibitors of MTIP as
Transition state analogues,1 of enzyme catalysed reactions, are
often potent inhibitors of the target enzyme.2–4 Both the enzyme,
and more often than not, the inhibitors are chiral. With regards
antibiotics discussed.23
Another immucillin with considerable potential as a drug candi-
date is (3R,4S)-50-methylthio-DADMe-ImmA (MTDIA, ent-2)24
(Fig. 1) and hence we are interested in the synthesis of its enan-
tiomer. In particular, where whole-body methylthioadenosine
phosphorylase (MTAP) is blocked by MTDIA,24 dramatic effects
to
a-amino acids and sugars this chirality is often manifested in
the form of L- and D-modifications, respectively. Surprisingly,
given the chirality of the enzyme target, and biological targets in
general, the D form of the
a
-amino acids and the L-form of the sug-
are observed in mouse xenografts of a variety of human
ars often retain or offer improved biological activity when com-
pared with their enantiomers.5–10 Coupled with this improved
biological activity, enantiomers may exhibit useful pharmacologi-
cal activities in terms of improved solubility, bioavailability, stabil-
ity, and reduced toxicity in vivo as well as extending the patent life
of existing drugs.11 In particular the study of the biological activity
of a variety of L-nucleosides has often revealed surprising activ-
ity8,9 and our laboratories have demonstrated the biological activ-
ity of the L-nucleoside analogues12,13 of the immucillins.14
Immucillins, based on transition state analogue design of purine
nucleoside phosphorylase (PNP), have been described.14 Syntheses
of the enantiomers of the two clinical leads, Forodesine15–18 and
Ulodesine,19–21 have been reported together with their biological
activity (Fig. 1). (3R,4S)–MT-DADMe-ImmH (MTDIH, ent-1) is a
related compound and its activity as a PNP inhibitor has also been
described.22 Recently the activity of MTDIH against the newly
tumours.25,26 Also, MTDIA is a femtomolar inhibitor of the dual-
substrate enzyme methylthioadenosine/S-adenosylhomocysteine
nucleosidase (MTAN)27 from Escherichia coli and MTDIA and its
analogues may act as antibiotics that do not induce resistance.28,29
Therefore, given the biological activity of MTDIH and MTDIA,
and the surprising activity of the enantiomers of Forodesine and
Ulodesine, we investigated the synthesis and enzyme inhibitory
activity of their enantiomers, (3S,4R)-MTDIH
1 and (3S,4R)-
MTDIA 2 (Fig. 2). In the case of 2 we also investigated its binding
to EcMTAN both in terms of the thermodynamics of binding and
through the crystal structure of EcMTAN with 2 in the active site.
2. Results and discussion
2.1. Chemistry
Previously, Clinch et al. described the large scale synthesis of
(3R,4R)-hydroxymethylpyrrolidin-3-ol from diethyl maleate via
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmc.2015.07.059
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
5327
methanol, was doped with 1% aqueous ammonia (v:v). Boc protec-
tion of amine 9 could be achieved in situ but we preferred a step-
wise process to afford carbamate 10 in excellent overall yield for
the two steps. Methanesulfonation of compound 10 was carried
out at ꢁ60 °C and although the reaction was not entirely regiose-
lective, the over-reacted side-product could be carried through to
the next step along with the desired product 11, and the resulting
impurity removed later by chromatography. Dibutyltin oxide can
also be used to achieve regioselective mesylation, however due
to the inherent toxicity of organotin compounds we were not pre-
pared to use this method. Treatment of the crude product from the
mesylation step with sodium thiomethoxide at room temperature
afforded the Boc protected 5-methylthiopyrrolidine 12 in moder-
ate yield for the two steps. Removal of the Boc protecting group
was achieved using concentrated HCl in methanol to afford the
desired amine hydrochloride 13, the right-hand side of the two tar-
get molecules, in quantitative yield.
OH
OH
H
N
H
N
N
N
HO
HO
H
N
N
N
N
HO OH
OH
Forodesine
Ulodesine
NH2
N
OH
H
N
H
N
N
5
MeS
MeS
1
N
N
4
N
N
3
2
OH
(3R,4S) - MTDIA ent-
OH
2
(3R,4S) - MTDIH ent-1
Figure 1.
Synthesis of the 9-deazapurine component of the target mole-
cule 1, 9-deazahypoxanthine, has been well described and it was
prepared using a method reported previously.31,32
The synthesis of 9-deazaadenine is less well described33–35 and
although our preferred route has been previously outlined36 the
synthetic details of this robust and scalable process have not been
previously revealed (Scheme 2).
OH
N
NH2
H
N
H
N
N
N
SMe
SMe
Ethyl(ethoxymethylene)cyano acetate (14) was treated with
aminoacetonitrile to afford enamine 15 in excellent yield. The
enamine nitrogen of compound 15 was protected using
methylchloroformate, again in excellent yield, to afford 16 and this
was then cyclised using DBU to provide pyrrole 17 in good yield.
Reaction of pyrrole 17 with formamidine acetate in ethanol gave
ester 18 which was efficiently decarboxylated to afford 9-deaza-
adenine as a crystalline solid in 25% overall yield for the 5 steps
without chromatography.
N
N
N
OH
OH
1
2
(3S,4R) - MTDIA
(3S,4R) - MTDIH
Figure 2.
isoxazolidine 3 (Scheme 1).30 The key step in the synthesis involves
an enantioselective enzymatic resolution of compound 4, using the
lipase Novozyme 435, to afford the carboxylic acid (3S,4R)-5 and
the ethyl ester (3R,4S)-6. Compound (3S,4R)-5 has been converted
to ent-7 and then to (3R,4R)-hydroxymethylpyrrolidin-3-ol ent-9.
The other product from the enantioselective resolution process,
compound (3R,4S)-6 can be used to synthesise the desired enan-
tiomers of MTDIA and MTDIH. Therefore reduction of 6 was
achieved with borane, generated in situ from BF3ꢀOEt2 and
NaBH4, to afford diol 7 in good yield which could be readily
separated from a minor contaminant, the ethyl ether 8 (8%).
Hydrogenolysis of the N-benzyl protecting group was readily
achieved using Pearlman’s catalyst where the reaction solvent,
Target compounds 1 and 2 were synthesized in a similar fashion
to that previously described for their enantiomers24 utilizing
Mannich chemistry.37 The amine hydrochloride 13 was dissolved
in a 1,4-dioxane:water mixture which was buffered with sodium
acetate. Formaldehyde was added followed by either 9-deazahy-
poxanthine or 9-deazaadenine and the resulting mixtures heated
to 95 °C to afford the target compounds 1 and 2 in good to moder-
ate yields, respectively, (Scheme 3).
2.2. Inhibition studies
The inhibition of human and Plasmodium falciparum PNP,
Pseudomonas aeruginosa MTIP, human MTAP and Escherichia coli
EtO2C
EtO2C
EtO2C
HO2C
CO2Et
CO2Et
ref 30
ref 30
a
NBn
O
NBn
O
BnN
O
+
NBn
O
CO2Et
OH
OH
OH
diethyl maleate
3
4
( ) -
5
6
( ) -
b
SMe
R
OH
R
g
e
c
HCl.N
BocN
RN
BnN
OH
OH
OH
OH
13
11
12
9
10
7
8
100%
R = OMs
R = SMe
R = H
82%
89%
R = OH
R = OEt
53%
8%
f
d
38% 2 steps
R = Boc
Scheme 1. (a) Novozyme 435, H2O, acetone, pH 7.5, 27 °C, reflux; (b) BF3ꢀOEt2, NaBH4, THF, 0 °C ? room temperature; (c) Pd(OH)2, H2, conc NH4OH, MeOH, room
temperature; (d) Boc2O, MeOH, room temperature; (e) MsCl, Hunig’s base, CH2Cl2, ꢁ60 °C; (f) NaSMe, DMF, room temperature; (g) MeOH; conc HCl, room temperature.

5328
G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
MeO
O
H
OEt
CN
N
CN
N
CN
a
b
EtO2C
EtO2C
CN
EtO2C
CN
16 95%
14
15 98%
c
NH2
N
H
N
NH2
N
H
N
H
N
CN
e
d
N
EtO2C
NH2
N
EtO2C
9-deazaadenine 65%
18 46%
17 88%
Scheme 2. (a) Aminoacetonitrile bisulfate, NaOAc, MeOH, room temperature; (b) Methyl chloroformate, Et3N, CH2Cl2, room temperature; (c) CH2Cl2, DBU, MeOH, room
temperature; (d) Formamidine acetate, EtOH, reflux; (e) KOH, H2O, reflux.
R
H
N
N
SMe
R
N
H
N
a
SMe
HCl.HN
N
+
N
N
OH
OH
69 %
48 %
1
R = OH
13
9-deazahypoxanthine R = OH
9-deazaadenine R = NH2
2
R = NH2
Scheme 3. (a) aq Formaldehyde, NaOAc, 1,4-dioxane, H2O, 95 °C.
Table 1
Summary of Ki values (nM)
driven by large enthalpic values: ꢁ15.2 kcal molꢁ1 at the first active
site; and ꢁ13.5 kcal molꢁ1 at the second active site of the MTAN
homodimer.38 These large enthalpy contributions were accompa-
nied by smaller entropic binding contributions of ꢁ2.5 kcal molꢁ1
at the first active site and 3.4 kcal molꢁ1 at the second active site.38
When 2 binds, the enthalpic binding contribution is reduced to ꢁ8.1
and ꢁ5.6 kcal molꢁ1 at the first and second sites, respectively. The
entropic binding contribution is smaller but favorable at ꢁ3.6 and
ꢁ4.4 kcal molꢁ1 at the first and second binding sites generated in
the case of 2 is likely due to the release of bound water molecules
from the active site. Overall, the thermodynamic signature suggests
different modes of binding between the enantiomers of MTDIA and
E. coli MTAN, consistent with the crystal structures.
Enzyme
Ki
Ki⁄
Ki
Ki⁄
1
2
Ent-1
Hs PNP
Pf PNP
Pa MTIP
110 14
298 37
8.6 0.2
84 17
93
0.3 0.01
0.07 0.01
4
11
4
0.9 0.1
0.8 0.1
0.34 0.02
Ent-2
Hs MTAP
Ec MTAN
750 10
4.5 0.5
0.53 0.05
0.048 0.003
0.078 0.008
0.002 0.0002
MTAN was evaluated with enantiopure immucillins 1, ent-1, 2 and
ent-2 (Table 1). While the inhibition constants of compounds 1 and
2 indicate binding, that is, considerably weaker for their respective
target enzymes, than their enantiomers, somewhat surprisingly,
they remain high to low-range nanomolar inhibitors for all
enzymes.
The L-enantiomer compound 1 bound to human PNP some
1200-fold less tightly to the enzyme than (3R,4S)-MTDIH (ent-1).
Despite this difference, 1 remains a nanomolar inhibitor of human
PNP and in some drug design programs, could be considered a
potent inhibitor. Against MTIP, compound 1 was a low nanomolar
inhibitor with slow-onset tight binding characteristics and was
only 2–3 times less potent than its enantiomer. Alternatively, com-
pound 2 exhibited no slow-onset tight binding inhibition against
either MTAP or MTAN and showed some 3–4 orders of magnitude
weaker binding to these enzymes than its enantiomer ent-2.
2.4. Crystal structure of EcMTANꢀ(3S,4R)-MTDIA 2
Consistent with all MTAN structures deposited thus far with
DADMe-Immucillin-based inhibitors bound, the purine-like por-
tion of 2 is hydrogen bonded to Ile152 and Asp197. Likewise, the
50-alkylthio group in both enantiomers have similar conformations,
and do not make any strong interactions. In contrast, the aza-sugar
portion of the molecule must adopt a very different orientation in 2
compared to ent-2 (Fig. 3). This is illustrated by the torsion angle
C9-C10-N10-C10, which changes almost 110° from 46.6° to
ꢁ60.3°. The aza-sugar in both enantiomers (in the bound state) is
in an N10-endo conformation. The latter inhibitor ent-2 is a
transition state analogue, which matches the stereochemistry of
the adenosine-derived substrates, whereas the former inhibitor 2
has the opposite stereochemistry at C30 and C40. The hydrogen
bonding contacts to the aza-sugar are somewhat different between
the enantiomers. Thus far, all deposited structures with the
(3R,4S)-alkylthio-DADMe-Immucillin A based inhibitors have a
2.3. Thermodynamics of binding for (3S,4R)-MTDIA 2
The energetics of binding of compound 2 to E. coli MTAN is differ-
ent from that of E. coli MTAN with ent-2. With ent-2, binding was

G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
5329
Figure 3. EcMTAN bound to (3S,4R)-MTDIA (left) and (3R,4S)-MTDIA (right). The key residues are shown as sticks. The nearby residues are shown as lines.
water molecule (and sometimes two) distal to the aza-sugar in the
active site cavity: the ‘nucleophilic’ water, which would act to
hydrolyze the native substrate(s); and a supporting water mole-
cule if space permits. Neither of these water molecules are present
in the 2-bound EcMTAN structure. In the (3R,4S)-inhibitor-bound
structures, N10 is always hydrogen bonded to the nucleophilic
water, which in turn is hydrogen bonded to both Glu12 and
Glu174. Additionally, the C30-hydroxyl group appears to make a
single hydrogen bond to Glu174. In contrast, in the (3S,4R)-in-
hibitor-bound structure N10 faces away from Glu12 and instead,
hydrogen bonds to the neighboring Ser76. The C30-hydroxyl group
now has a bifurcated hydrogen bond to Glu174. The capacity of
EcMTAN to bind 2 appears to be positively influenced by Ser76,
which provides a key hydrogen bonding interaction. In MTANs
from several other organisms, such as Clostridium difficile,
Mycobacterium tuberculosis and Helicobacter pylori, the analogous
residue is a valine and so the binding affinity to 2 might be
expected to be much worse. Comparison of the ent-2 and 2-bound
EcMTAN structures shows the active site residues are positioned
very similarly (Fig. 3), with residues closer than 5 Å to the inhibi-
tors having an RMSD of 0.70 Å (167 atoms in alignment). As
expected, the alignment for the entire structure is also very similar,
having an RMSD of 0.91 Å (3344 atoms in alignment). In both
alignments, no atoms were excluded due to poor fit.
performed on glass or aluminium sheets coated with 60 F254 silica
gel. Organic compounds were visualized under uv light or use of a
dip of ammonium molybdate (5 wt %) and cerium(IV) sulfate 4 H2O
(0.2 wt %) in aq H2SO4 (2 M), one of I2 (0.2%) and KI (7%) in H2SO4
(1 M), or 0.1% ninhydrin in EtOH. Chromatography (flash column)
was performed on silica gel (40–63
system with continuous gradient facility. Optical rotations
were recorded at a path length of 10 cm and are in units of 10ꢁ1
lm) or on an automated
-
deg cm2 gꢁ1; concentrations are in g/100 ml. 1H NMR spectra were
measured in CDCl3, CD3OD, DMSO d6 (internal Me4Si, d 0) or D2O
(HOD, d 4.79), and 13C NMR spectra in CDCl3 (centre line, d 77.0),
CD3OD (centre line, d 49.0), DMSO d6 (centre line, d 39.5) or D2O
(no internal reference or internal CH3CN, d 1.47 where stated).
Assignments of 1H and 13C resonances were based on 2D (1H–1H
DQF-COSY, 1H–13C HSQC, HMBC) and DEPT experiments. Positive
electrospray mass spectra were recorded on a Q-TOF Tandem
Mass Spectrometer. Microanalyses were performed by the
Campbell Microanalytical Department, University of Otago,
Dunedin, New Zealand.
4.1.1. (3S,4S)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol (7)
To a suspension of lactam 6 (9.90 g, 37.6 mmol) and sodium
borohydride (5.69 g, 150 mmol) in dry THF (190 ml) was added
boron trifluoride diethyl etherate (23.2 ml, 188 mmol) dropwise
at 0 °C and the resulting reaction mixture was stirred for 3 days
at rt. The reaction was then quenched carefully with methanol
under ice cooling and the solvent was concentrated in vacuo. 6 M
hydrochloric acid (60 ml) was added and after being stirred for
10 min the mixture was again concentrated in vacuo.
Subsequently, a 15% aqueous sodium hydroxide solution was
added (ca. 60 ml) to the resulting residue until the pH reached
14. After concentrating the mixture again the residue was sus-
pended in chloroform, stirred for 1 h at room temperature and fil-
tered through CeliteÒ. The crude product was then purified by
3. Conclusion
Enantiomers of two compounds that show promise as drug can-
didates have been prepared and assayed against a series of target
enzymes. While not as potent as their enantiomers, 1 and 2 are still
nanomolar inhibitors and their biological activity may warrant fur-
ther investigation. The thermodynamic data suggests different
modes of binding by 2 and ent-2 which are borne out by the struc-
tural data. A structural analysis of 2 bound to E. coli MTAN shows
that in order to accommodate the opposite stereochemistry the
water molecules normally found in the active site are absent and
new hydrogen bond connection is revealed to Ser76.
silica
gel
chromatography
(eluent
methanol/chloroform
1:9 ? 1:4) to yield 4.10 g (53%) of the title compound 7 as a color-
less oil. In addition, 726 mg (8%) of the corresponding ethyl ether 8
were isolated as a by-product.
4. Experimental protocols
4.1. Synthesis
4.1.2. (3S,4S)-1-Benzyl-4-(hydroxymethyl)pyrrolidin-3-ol (7)
½
a 2D2
ꢂ
= ꢁ33.1 (c 1.095, MeOH); 1H NMR (500 MHz, CD3OD) d
7.34–7.28 (m, 4H), 7.26–7.21 (m, 1H), 3.99 (dt, J = 4.1 Hz, 6.3 Hz,
1H); 3.64 (d, J = 12.7 Hz, 1H), 3.62 (dd, J = 5.9, 10.7 Hz, 1H), 3.55
(d, J = 12.7 Hz, 1H), 3.50 (dd, J = 7.7, 10.7 Hz, 1H), 2.89 (dd, J = 8.1,
9.5 Hz, 1H), 2.72 (dd, J = 6.3, 10.1 Hz, 1H), 2.56 (dd, J = 4.1,
10.1 Hz, 1H), 2.33 (dd, J = 6.6, 9.7 Hz, 1H), 2.21–2.13 (m, 1H); 13C
NMR (125 MHz, CD3OD) d 139.36, 130.28, 129.36; 128.33, 74.17,
Air sensitive reactions were performed under argon. Organic
solutions were dried over anhydrous MgSO4 and the solvents were
evaporated under reduced pressure. Anhydrous and chromatogra-
phy solvents were obtained commercially and used without any
further purification. Thin layer chromatography (TLC) was

5330
G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
64.25, 63.10, 61.52, 51.19; HRMS (ESI) m/z calcd for C12H17NO2H+
208.1332, obsd 208.1343.
148.0796, obsd 148.0803. ½a D20
= ꢁ34.5 (c = 1, MeOH) equal and
ꢂ
opposite to that of the (3R,4S)-enantiomer.41
4.1.3. (3S,4S)-1-Benzyl-4-(ethoxymethyl)pyrrolidin-3-ol (8)
4.1.6. (E)-Ethyl 2-cyano-3-(cyanomethylamino)acrylate (15)
Yellow oil; ½a 2D0
ꢂ
= ꢁ24 (c = 1.21, MeOH). 1H NMR (500 MHz,
A suspension of aminoacetonitrile bisulfate (14) (73 g,
CD3OD)
d
7.34–7.28 (m, 4H), 7.26–7.22 (m, 1H), 3.98 (dt,
474 mmol) and sodium acetate (117 g, 1421 mmol) in methanol
was stirred at rt for 20 min. Ethyl(ethoxymethylene) cyano acetate
(80 g, 474 mmol) was then added in one portion with vigorous stir-
ring and the mixture left for 1 h. The mixture was evaporated to
dryness and the solid suspended in a satd aq NaHCO3 solution
(1.0 L) which was then extracted with EtOAc (2 ꢃ 400 ml). The
organic layers were combined and washed with water (250 ml),
dried (MgSO4), filtered and the solvent evaporated in vacuo to
afford compound 15 (83.4 g, 98% yield) as a yellow solid (charac-
terised as a 60:40 isomeric mixture, H0 = minor isomer protons).
Mp 95 °C. 1H NMR (500 MHz, d6-DMSO) d 9.27 (brs, 1H), 9.00
(brs, 1H), 8.16 (s, 1H), 7.86 (brd, J = 11.3 Hz, 1H), 4.53 (s, 2H),
4.44 (s, 2H), 4.17 (q, J = 7.2 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 1.23
(t, J = 7.2 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, d6-
DMSO) d 165.7, 164.3, 160.1, 159.9, 118.2, 116.9, 116.9, 115.7,
72.7, 71.9, 60.1, 36.8, 36.1, 14.3, 14.0. HRMS (ESI) m/z calcd for
C8H9N3O2Na 202.0592, obsd 202.0590.
J = 4.1 Hz, 6.3 Hz, 1H); 3.63 (d, J = 12.7 Hz, 1H), 3.54 (d,
J = 12.6 Hz, 1H), 3.51–3.46 (m, 3H), 3.38 (dd, J = 7.7, 9.3 Hz, 1H),
2.88 (dd, J = 7.9, 9.2 Hz, 1H), 2.88 (dd, J = 7.9, 9.2 Hz, 1H), 2.70
(dd, J = 6.3, 10.1 Hz, 1H), 2.29 (dd, J = 6.7, 9.4 Hz, 1H), 2.27–2.20
(m, 1H), 1.16 (t, J = 7 Hz, 3H); 13C NMR (125 MHz, CD3OD) d
139.38, 130.29, 129.35, 128.33, 74.37, 72.86, 67.48, 62.94, 61.51,
57.56; HRMS (ESI) m/z calcd for C14H21NO2H+ 236.1645, obsd
236.1644.
4.1.4. (3S,4S)-N-tert-Butoxycarbonyl-3-hydroxy-4-hydroxyme-
thylpyrrolidine (10)
A
suspension
of
(3S,4S)-N-benzyl-3-hydroxy-4-hydrox-
ymethylpyrrolidine (7) (2.7 g, 13.03 mmol, Pearlmans catalyst
(200 mg, 1.879 mmol, aqueous ammonia (0.2 ml, 28%, 13 mmol,
and methanol (20 ml were stirred under and atmosphere of hydro-
gen gas (0.026 g, 13.03 mmol) for 14 h. The resulting mixture was
filtered through CeliteÒand concentrate in vacuo to afford a crude
syrup. The crude product was purified by flash chromatography
(eluent 1:1 1,4-dioxane:conc NH4OH) to afford (3S,4S)-3-hy-
droxy-4-hydroxymethylpyrrolidine (9) (1.34 g, 88%). The 1H and
13C NMR were in agreement with the data for the (3R,4R)-enan-
tiomer reported by Filichev et al.39 Boc anhydride (4.99 g,
23 mmol) was added to a solution of 10 (1.34 g, 11.4 mmol) in
methanol (20 ml) and the resulting stirred for 1 h. The solution
was concentrated in vacuo and the crude residue purified by chro-
matography (eluent 10 ? 20% MeOH:CHCl3) to afford 10 (2.20 g,
89% yield) as a colourless syrup. The 1H and 13C NMR were in
agreement with the data for the (3R,4R)-enantiomer reported by
4.1.7. (E)-Ethyl 2-cyano-3-((cyanomethyl)(methoxycarbonyl)-
amino)acrylate (16)
Compound 15 (76 g, 424 mmol) was suspended in CH2Cl2
(1.0 L) and to this mixture was added methyl chloroformate
(45.9 ml, 594 mmol). Triethylamine (83 ml, 594 mmol) was then
added dropwise at such a rate as to ensure the reaction tempera-
ture is maintained below 30 °C. After the addition was complete
the mixture was stirred for a further 30 min at which point the
reaction was complete. The organic layer was then washed with
water (500 ml) and satd aq NaHCO3 ensuring the pH of the aque-
ous layer is basic. The organic layer was dried (MgSO4), filtered
and concentrated in vacuo to afford 16 (96 g, 95%) as a dark red
syrup. 1H NMR (500 MHz, d6-DMSO) d 8.44 (1H, s), 5.07 (2H, s),
4.27 (q, J = 7.1 Hz, 2H), 3.94 (3H, s), 1.27 (t, J = 7.1 Hz, 3H). 13C
NMR (125 MHz, d6-DMSO) d 162.6, 151.9, 149.4, 115.3, 114.0,
83.6, 62.1, 56.0, 34.2, 14.0. HRMS (ESI) m/z calcd for
Evans et al.40
½
a 2D0
ꢂ
= ꢁ15.2 (c = 1, MeOH) equal and opposite to that
reported for the (3R,4R)-enantiomer.40
4.1.5. (3S,4R)-3-Hydroxy-4-(methylthiomethyl)pyrrolidine (13)
Hunig’s base (3.53 ml, 20.25 mmol) was added to a solution of
(3S,4S)-10 (2.2 g, 10.13 mmol) in dichloromethane (35 ml), and
the resulting mixture cooled to ꢁ60 °C. Methanesulfonyl chloride
(0.784 ml, 10.1 mmol) was added dropwise and the internal tem-
perature maintained at ꢁ60 °C. After 30 min the reaction was
judged to be complete and was diluted with chloroform and
washed with water, 10% HCl and satd NaHCO3, dried and concen-
trated in vacuo. The crude mesylate 11 (2.99 g), was committed
to the next synthetic step without purification or characterisation.
Sodium thiomethoxide (1.42 g, 20.2 mmol) was added to a solu-
tion of mesylate 11 (2.99 g, 10.1 mmol) in dimethylformamide
(40 ml) and the resulting suspension left to stir for 14 h. The mix-
ture was then diluted with toluene and washed with water, brine,
dried (MgSO4), and concentrated in vacuo. The crude residue was
purified by chromatography (eluent 20% ? 60% ? 100% EA) to
afford compound 12, presumably (3S,4R)-1-tert-butoxycarbonyl-
3-hydroxy-4-(methylthiomethyl)pyrrolidine (0.96 g) as a syrup
which was committed to the next step without characterisation.
Concentrated HCl (2 ml, xs) was added to a solution of compound
12 (0.96 g, 3.9 mmol in methanol (2 ml) and the solution concen-
trated in vacuo. The resulting residue was dissolved in additional
conc HCl (2 ml) and concentrated in vacuo to afford the title com-
pound 13 (0.71 g, 38% yield for 3 steps) as a low melting crystalline
solid. 1H NMR (500 MHz, D2O) d 4.41 (brs, 1H), 3.68 (dd, J = 12,
6.5 Hz, 1H), 3.51 (dd, J = 12.6, 5.1 Hz, 1H), 3.29 (brd, J = 12.6 Hz,
1H), 3.22 (dd, J = 12.3, 5.1 Hz, 1H), 2.71 (q, J = 16.9, 10.5 Hz, 1H),
2.55 (m, 2H), 2.14 (s, 3H). 13C NMR (500 MHz, D2O) d 73.5, 51.5,
48.6, 45.2, 34.3, 14.9. HRMS (ESI) m/z calcd for C6H13NOSH+
C10H11N3O4Na 260.0647, obsd 260.0652.
4.1.8. Ethyl 4-amino-5-cyano-1H-pyrrole-3-carboxylate (17)
Compound 16 (109 g, 460 mmol) was dissolved in CH2Cl2 (1.0 L,
460 mmol) and DBU (34.6 ml, 230 mmol) added dropwise. A small
exotherm occured (19 ? 28 °C) on addition and the reaction was
complete after 30 min. 10% aq HCl (250 ml) was added to the
organic layer and the two layers stirred together for 10 min. The
two layers were partitioned and the organic layer washed with
an additional quantity of 10% aq HCl (250 ml), satd aq NaHCO3,
and water. The organic layer (MgSO4) was dried, filtered and con-
centrated in vacuo to afford 17 (107 g, 98%) as a brown solid (com-
pound characterised in ꢄ90% purity). Mp 205 °C. 1H NMR
(500 MHz, d6-DMSO) d 11.8 (brs, 1H), 7.35 (s, 1H), 5.65 (s, 2H),
4.20 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz,
d6-DMSO) d 164.0, 146.0, 127.4, 114.8, 102.3, 84.4, 59.1, 14.3.
HRMS (ESI) m/z calcd for C8H9N3O2Na 202.0592, obsd 202.0591.
4.1.9. Ethyl 4-amino-5H-pyrrolo[3,2-d]pyrimidine-7-carboxylate
(18)
A mixture of compound 17 (107 g, 451 mmol) and formamidine
acetate (70.4 g, 677 mmol) in ethanol (1.0 L, 451 mmol) was
heated at reflux overnight. The mixture was cooled to 0 °C and fil-
tered, washing the solid with cold ethanol (1.0 L) to afford ethyl 4-
amino-5-cyano-1H-pyrrole-3-carboxylate
(42.4 g,
206 mmol,
45.6% yield) as a gun-metal grey solid. Mp >300 °C. 1H NMR
(500 MHz, d6-DMSO) d 8.22 (s, 1H), 8.17(s, 1H), 4.25 (q, J = 7.0 Hz,

G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
5331
2H), 1.30 (t, J = 7.0 Hz, 3H).35 13C NMR (125 MHz, d6-DMSO) d
163.0, 152.0, 150.9, 145.0, 134.0, 114.5, 107.0, 58.9, 14.5.
described.23 The recombinant E. coli MTAN was expressed in BL-21
DE3 Star (Invitrogen), IPTG inducible E. coli cells and purified using
Ni-NTA chromatography. All five enzymes contain N-terminal His-
6-tags, but are catalytic equivalents of the native proteins.
4.1.10. 9-Deazaadenine35
Potassium hydroxide (34.1 g, 517 mmol) was added to a suspen-
sion of ethyl 4-amino-5-cyano-1H-pyrrole-3-carboxylate (42.62 g,
207 mmol) in water (500 ml) and refluxed for 24 h. The mixture
was cooled to rt concentrated in vacuo and resuspended in water
(100 ml). The resulting suspension was filtered to afford a brown
solid which was washed with water (50 ml) and ether (100 ml),
and then air-dried to afford 9-deazaadenine (18 g, 65%) as a tan
solid. Mp >300 °C. 1H NMR (500 MHz, d6-DMSO) d 8.12 (s, 1H),
7.51 (brd, J = 2.1 Hz, 1H), 6.36 (brd, J = 2.1 Hz, 1H). 13C NMR
(125 MHz, d6-DMSO) d 150.4, 150.3, 147.2, 127.8, 113.6, 101.2.
4.2.2. Inhibition assays
Enzyme inhibition assays were carried out using xanthine oxi-
dase as the coupling enzyme and monitored for 60–120 min. For
the PNP’s and MTIP, hypoxanthine formed by the phosphorolysis
of inosine and MTI, respectively, is converted to uric acid by xan-
thine oxidase (Sigma) and monitored spectromerically at 293 nm
(e
293 = 12.9 mMꢁ1 cmꢁ1).44 For the PNP’s, the assay was initiated
by the addition of either 0.5 nM (human) and 10 nM (P. falciparum)
enzyme into a 1 ml reaction mixture containing 1 mM inosine,
50 mM potassium phosphate (pH = 7.5), with xanthine oxidase
added to a final concentration of 60 milliunits. For P. aureginosa
MTIP, the assay was initiated by the addition of 1 nM enzyme into
a 1 ml reaction mixture containing 2 mM MTI, 100 mM HEPES
(pH = 7.4), 100 mM potassium phosphate, 5 mM dithiothreitol
and 0.05 units of xanthine oxidase.
4.1.11. (3S,4R)-1-[(9-Deazahypoxanthin-9-yl)methyl]-3-hydroxy-
4-(methylthiomethyl)pyrrolidine (1)
Aqueous formaldehyde (0.245 ml, 1.6 mmol, 37% bw) was
added dropwise to a mixture of amine hydrochloride 13 (200 mg,
1.1 mmol) and sodium acetate (179 mg, 2.2 mmol) in water
(2 ml) and 1,4-dioxane (1 ml) mixture. After 5 min 9-deazahypox-
anthine (0.177 g, 1.3 mmol) was added and the resulting suspen-
sion heated to 95 °C for 14 h. The crude reaction mixture was
absorbed onto silica gel and the resulting residue purified by flash
For both MTAP and MTAN reactions, the adenine formed by
phosphorolysis of MTA, is converted to 2,8-hydroxyladenine by
xanthine oxidase and monitored spectrophotometrically at 305
nm (e
305 = 15.5 mMꢁ1 cmꢁ1).45 For human MTAP, the reaction
chromatography
MeOH:CHCl3 ? 20% ? 30%) to afford the title compound
on
silica
gel
(15%
7 N
NH3
in
1
was initiated by the addition of 0.8 nM enzyme into a 1 ml reaction
mixture containing 1 mM methylthioadenosine (MTA), 50 mM
HEPES (pH = 7.4), 100 mM potassium phosphate (pH = 7.4), 1 mM
dithiothreitol and 1 unit of xanthine oxidase. For E. coli MTAN,
the reaction was initiated by the addition of 1 nM enzyme into a
1 ml reaction mixture containing 1 mM MTA, 100 mM HEPES (pH
7.8) and 1 unit of xanthine oxidase. Controls without inhibitor
and without enzyme were included in all experiments. Ki values
were obtained using the follow equation for competitive
inhibition:
(220 mg, 69% yield) as a white solid. Mp 239–240 °C. 1H NMR
(500 MHz, d6-DMSO) d 7.79 (s, 1H), 7.28 (s, 1H), 4.84 (brs, 1H),
3.79 (bs, 1H), 3.62 (dd, J = 23.4, 13.3 Hz, 2H), 2.78 (dd, J = 9.2,
7.8 Hz, 1H), 2.72 (dd, J = 9.6, 6.6 Hz, 1H), 2.62 (dd, J = 12.7, 5.9 Hz,
1H), 2.40 (m, 2H), 2.24 (dd, J = 9.3, 6.2 Hz, 1H), 2.02 (s, 3H). 13C
NMR (125 MHz, d6-DMSO) d 153.7, 143.4, 141.2, 126.9, 117.5,
113.3, 74.9, 61.5, 57.3, 47.7, 46.7, 36.8, 14.9. HRMS (ESI) m/z calcd
for C13H18N4O2SH+ 295.1229, obsd 295.1226. ½a 2D0
= ꢁ45.9 (c = 1,
ꢂ
MeOH) equal and opposite to that of the (3R,4S)-enantiomer.42
Found: C, 53.08; H, 6.30; N, 19.08; S, 10.79. C13H18N4O2S requires:
C, 53.04; H, 6.16; N, 19.03; S, 10.89.
v00
v0
Km þ ½Sꢂ
¼
Km½Iꢂ
Km þ ½Sꢂ þ
Ki
where v00 and v0 are initial rates in the presence and absence of
inhibitor, respectively; Km and [S] are the Michaelis constant and
concentration of either inosine (for both PNP’s), MTI (for MTIP)
and MTA (for MTAP and MTAN), respectively, and Ki and [I] are
the inhibition constant and inhibitor concentration, respectively. If
[I] is less than 10 times the enzyme concentration, the following
correction is applied:
4.1.12. (3S,4R)-1-[(9-Deazaadenin-9-yl)methyl]-3-hydroxy-4-
(methylthiomethyl)pyrrolidine (2)
Aqueous formaldehyde (0.257 ml, 1.72 mmol, 37% bw) was
added to a mixture of amine hydrochloride 13 (210 mg, 1.14 mmol)
and sodium acetate (188 mg, 2.29 mmol) in water (2 ml) and 1,4-
dioxane (1 ml). After 5 min 9-deazaadenine (0.184 g, 1.372 mmol)
was added and the resulting suspension warmed to 95 °C for 3 h.
The crude reaction mixture was absorbed onto silica gel and the
resulting residue purified by flash chromatography on silica gel
(15% 7 N NH3 in MeOH:CHCl3 ? 20% ? 30%) to afford the title com-
pound 2 (160 mg, 48%) as a white solid. Mp decomposed >180 °C.
1H NMR (500 MHz, d4-MeOH) 8.15 (s, 1H), 7.49 (s, 1H), 3.96 (dt,
J = 6.3, 4.1 Hz, 1H), 3.83 (dd, J = 13.4, 9.8 Hz, 2H), 3.05 (dd, J = 9.7,
7.9 Hz, 1H), 2.85 (dd, J = 10.2, 6.4 Hz, 1H), 2.70–2.65 (m, 2H), 2.47
(dd, J = 12.7, 8.9 Hz, 1H), 2.38 (dd, J = 9.8, 7.1 Hz, 1H), 2.21 (m,
1H), 2.06 (s, 3H). 13C NMR (125 MHz, d4-MeOH) 152.1, 151.0,
147.0, 130.1, 115.2, 112.5, 76.8, 62.3, 58.9, 48.9, 48.2, 38.1, 15.6.
ꢀ
ꢁ
v00
I0 ¼ I ꢁ
Et
1 ꢁ v0
where I0 is the effective inhibitor concentration to be used in the
equation for competitive inhibition and Et is the final enzyme con-
centration. In some instances, a second slower rate phase is
observed in progress curves of tight binding inhibitors. These steady
state rates are taken instead (v0s
/vs) and used in the inhibition equa-
tion above to determine the equilibrium inhibition constant K*i,
which reflects the slow onset inhibition of the enzyme–inhibitor
complex.
½
a 2D0
ꢂ
= ꢁ41.6 (c = 1.09, MeOH) equal and opposite to that of the
(3R,4S)-enantiomer.43 HRMS (ESI) m/z calcd for C13H19N5OSH+
294.1389, obsd 294.1385. Found: C, 51.91; H, 6.73; N, 23.04; S,
10.10. C13H19N5OSꢀ½H2O requires: C, 51.63; H, 6.67; N, 23.16; S,
10.60.
4.2.3. ITC experimental details
Titrations were performed at 25 °C in a VP-ITC (MicroCal).
EcMTAN was dialyzed against charcoal overnight to remove any
bound adenine. Further dialysis followed in 100 mM phosphate
buffer, pH 7.8. The dialysate was filtered and used to wash the cell
4.2. Biology
and syringe, and to dissolve the ligand. Using 8
ligand, (3S,4R)-MTDIA (225 M) was titrated into the cell contain-
ing EcMTAN (25 M of the single subunit) to post-saturation. Past
saturation, only heats of dilution were observed. The data was fit in
lL injections, the
l
4.2.1. Protein preparation
The recombinant human and P. falciparum PNPs, P. aeruginosa
MTIP and human MTAP were expressed and purified as previously
l

5332
G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
Origin7 (MicroCal) using a standard equation for the fitting of two
independent binding sites. Ka was constrained to the externally
determined Ki reported within.
1–230) were sufficiently ordered enough to be built into the model,
however none of the residues in the cleavage/HIS6-tag could be
modeled. The structure was refined to Rcryst 17.3% and Rfree
19.3%. Analysis of the structures in Coot revealed good stereo-
chemistry with one residue (SER155) falling into the disallowed
regions of the Ramachandran plot. The refinement statistics are
reported in Table 2.
4.2.4. Co-crystallization of E. coli MTAN with (3S,4R)-MTDIA
EcMTAN was expressed and purified as described previously.38
The enzyme, with C-terminal TEV-cleavage site and HIS6 tag intact,
was concentrated to 10 mg/ml and incubated with 5 equiv of inhi-
bitor. The crystallization experiments was performed at 22 °C
using the sitting drop vapor diffusion technique. Rod-shaped crys-
Acknowledgments
This work was supported by funds from the U.S. NIH GM041916
(VLS) and the New Zealand Ministry for Business, Innovation, and
Employment. Use of the Advanced Photon Source, an Office of
Science User Facility operated for the U.S. Department of Energy
(DOE) Office of Science by Argonne National Laboratory, was sup-
ported by the U.S. DOE under Contract No. DE-AC02-06CH11357.
Use of the Lilly Research Laboratories Collaborative Access Team
(LRL-CAT) beamline at Sector 31 of the Advanced Photon Source
was provided by Eli Lilly Company, which operates the facility.
tals (25 ꢃ 35 ꢃ 230
lm) of inhibitor-bound EcMTAN were obtained
over 4 days using 0.1 M BIS-TRIS pH 7.0 and 2.4 M sodium
malonate.
4.2.5. Data collection and processing
The inhibitor-bound E. coli nucleosidase crystal was flash cooled
in liquid nitrogen, without auxiliary cryoprotectant. Diffraction
data for this crystal was collected with 0.9793 Å wavelength radi-
ation at the LRL-CAT beamline (Argonne National Laboratory) on a
Rayonix 225 HE CCD detector to 1.75 Å resolution. Diffraction
intensities were integrated and scaled with XDS.46 The diffraction
data statistics are summarized in Table 2.
References and notes
1. Muller, P. Pure Appl. Chem. 1994, 66, 1077.
2. Schramm, V. L. J. Biol. Chem. 2007, 282, 28297.
3. Singh, V.; Schramm, V. L. J. Am. Chem. Soc. 2006, 128, 14691.
4. Taylor Ringia, E. A.; Schramm, V. L. Curr. Top. Med. Chem. 2005, 5, 1237.
5. Simons, C. In Nucleoside Mimetics: Their Chemistry and Biological Properties; CRC
Press, 2000; Vol. 3,
6. Schneller, S. W.; Seley, K. L.; Hegde, V. R.; Rajappan, V. P.; Chu, C. Recent
Advances in Nucleosides: Chemistry and Chemotherapy: Chemistry and
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2002, 2, 1065.
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11. Darrow, J. J. Stan. Tech. L. Rev. 2007, 2.
12. Rinaldo-Matthis, A.; Murkin, A. S.; Ramagopal, U. A.; Clinch, K.; Mee, S. P.;
Evans, G. B.; Tyler, P. C.; Furneaux, R. H.; Almo, S. C.; Schramm, V. L. J. Am. Chem.
Soc. 2008, 130, 842.
4.2.6. Structure determination
The structure was determined by molecular replacement in
Molrep,47 using the previously published structure of
EcMTANꢀ(3R,4S)-MTDIA (PDB code 1Y6Q) as a search model. The
refinement of the initial solution and subsequent refinements were
carried out using a restrained refinement in Refmac,48 using all
data between 25.0 Å and 1.75 Å. Manual model rebuilding was car-
ried out using Coot.49 Weighted difference Fourier maps revealed
ordered water molecules and strong unambiguous positive
Fo ꢁ Fc difference electron density corresponding to the inhibitor.
The inhibitor model was generated using JLigand.50 Water mole-
cules with proper hydrogen-bonding coordination and electron
densities greater than 1 RMSD and 3 RSMD in maps calculated with
2Fobs ꢁ Fcalc and Fobs ꢁ Fcalc coefficients, respectively, were included
in the model. All but two of the Ec-associated residues (residues
13. Clinch, K.; Evans, G. B.; Fleet, G. W.; Furneaux, R. H.; Johnson, S. W.; Lenz, D. H.;
Mee, S. P.; Rands, P. R.; Schramm, V. L.; Taylor Ringia, E. A.; Tyler, P. C. Org.
Biomol. Chem. 2006, 4, 1131.
14. Schramm, V. L. Annu. Rev. Biochem 2011, 80, 703.
15. Al-Kali, A.; Gandhi, V.; Ayoubi, M.; Keating, M.; Ravandi, F. Future Oncol. 2010,
6, 1211.
16. Dummer, R.; Duvic, M.; Scarisbrick, J.; Olsen, E.; Rozati, S.; Eggmann, N.;
Goldinger, S.; Hutchinson, K.; Geskin, L.; Illidge, T. Ann. Oncol. 2014, 25,
1807.
Table 2
Data collection and refinement statistics for EcMTANꢀ(3S,4R)-MTDIA
Data collection
Space group
No. of mol. in asym. unit
C2221
1
17. Balakrishnan, K.; Ravandi, F.; Bantia, S.; Franklin, A.; Gandhi, V. Clin. Lymphoma
Myeloma Leuk. 2013, 13, 458.
Cell dimensions
a, b, c (Å)
72.010, 91.840, 69.530
50.0–1.75 (1.85–1.75)
38054 (7212)
0.074 (0.629)
14.0 (2.9)
18. Ogura, M.; Tsukasaki, K.; Nagai, H.; Uchida, T.; Oyama, T.; Suzuki, T.; Taguchi, J.;
Maruyama, D.; Hotta, T.; Tobinai, K. Cancer Sci. 2012, 103, 1290.
19. Bantia, S.; Parker, C.; Upshaw, R.; Cunningham, A.; Kotian, P.; Kilpatrick, J. M.;
Morris, P.; Chand, P.; Babu, Y. S. Int. Immunopharmacol. 2010, 10, 784.
20. Edwards, N. L.; So, A. Rheum. Dis. Clin. North Am. 2014, 40, 375.
21. Diaz-Torne, C.; Perez-Herrero, N.; Perez-Ruiz, F. Curr. Opin. Rheumatol. 2015,
27.
22. Murkin, A. S.; Clinch, K.; Mason, J. M.; Tyler, P. C.; Schramm, V. L. Bioorg. Med.
Chem. Lett. 2008, 18, 5900.
23. Guan, R.; Ho, M. C.; Almo, S. C.; Schramm, V. L. Biochemistry 2011, 50, 1247.
24. Evans, G. B.; Furneaux, R. H.; Lenz, D. H.; Painter, G. F.; Schramm, V. L.; Singh,
V.; Tyler, P. C. J. Med. Chem. 2005, 48, 4679.
Resolutiona (Å)
No. of unique reflectionsa
a
Rmerge
I
I/r a
Completenessa,b (%)
84.1 (98.9)
Refinement
Resolution (Å)
Rcryst
25.0–1.75
0.173
0.193
Rfree
R.m.s deviations
Bond lengths (Å)
Bond angles (°)
25. Basu, I.; Cordovano, G.; Das, I.; Belbin, T. J.; Guha, C.; Schramm, V. L. J. Biol.
Chem. 2007, 282, 21477.
0.008
1.29
26. Basu, I.; Locker, J.; Cassera, M. B.; Belbin, T. J.; Merino, E. F.; Dong, X.; Hemeon,
I.; Evans, G. B.; Guha, C.; Schramm, V. L. J. Biol. Chem. 2011, 286, 4902.
27. Singh, V.; Evans, G. B.; Lenz, D. H.; Mason, J. M.; Clinch, K.; Mee, S.; Painter, G.
F.; Tyler, P. C.; Furneaux, R. H.; Lee, J. E.; Howell, P. L.; Schramm, V. L. J. Biol.
Chem. 2005, 280, 18265.
28. Gutierrez, J. A.; Luo, M.; Singh, V.; Li, L.; Brown, R. L.; Norris, G. E.; Evans, G. B.;
Furneaux, R. H.; Tyler, P. C.; Painter, G. F. ACS Chem. Biol. 2007, 2, 725.
29. Gutierrez, J. A.; Crowder, T.; Rinaldo-Matthis, A.; Ho, M. C.; Almo, S. C.;
Schramm, V. L. Nat. Chem. Biol. 2009, 5, 251.
No. of atoms (average B-factor)
Protein
Inhibitor
Waters
Other
PDB entry
1699 (23.5)
20 (24.4)
132 (31.7)
5 (29.5)
4YML
a
Numbers in parentheses indicate values for the highest resolution shell.
Several diffraction zones were excluded due to ice rings, resulting in a low total
completeness.
b
30. Clinch, K.; Evans, G. B.; Furneaux, R. H.; Lenz, D. H.; Mason, J. M.; Mee, S. P. H.;
Tyler, P. C.; Wilcox, S. J. Org. Biomol. Chem. 2007, 5, 2800.

G. B. Evans et al. / Bioorg. Med. Chem. 23 (2015) 5326–5333
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Jr. Org. Proc. Res. Dev. 2009, 13, 928.
32. Furneaux, R. H.; Tyler, P. C. J. Org. Chem. 1999, 64, 8411.
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Gorzalka, S.; Knospe, M.; Schiedel, A. C.; Cristalli, G.; Müller, C. E. J. Med.
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34. Goeminne, A.; Berg, M.; McNaughton, M.; Bal, G.; Surpateanu, G.; Van der
Veken, P.; De Prol, S.; Versées, W.; Steyaert, J.; Haemers, A. Bioorg. Med. Chem.
2008, 16, 6752.
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36. Compain, P.; Martin, O. R. Iminosugars: From Synthesis to Therapeutic
Applications; John Wiley & Sons, 2007.
41. (3R,4S)-13 ½a D20
ꢂ
= +34.3 (c = 1, MeOH).
= +47.9 (c = 1, MeOH).
42. (3R,4S)-20 ½a D20
ꢂ
43. ½ ꢂ = +40.2 (c = 1.05, MeOH).
a 2D0
44. Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L.
Biochemistry 1998, 37, 8615.
45. Kung, P.-P.; Zehnder, L. R.; Meng, J. J.; Kupchinsky, S. W.; Skalitzky, D. J.;
Johnson, M. C.; Maegley, K. A.; Ekker, A.; Kuhn, L. A.; Rose, P. W. Bioorg. Med.
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46. Kabsch, W. Acta Crystallogr., Sect. D 2010, D66, 125.
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