Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

Article abstract of DOI:10.1038/s41429-019-0199-3

We have found cyclophane-type adenosine derivatives having p-quinone amide moieties (1 and 2) as weak inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase (CpIMPDH) from the Hokkaido University Chemical Library via the luciferase-based high-throughput screening. To obtain more potent inhibitors, we synthesized four new derivatives free from cyclophane rings (3–6). The N-H derivatives 3 and 5 showed more potent activities (24.4 and 11.1 μM, respectively) in the presence of dithiothreitol (DTT), whereas the N-methyl derivative 4 indicated more potent activity (2.1 μM) without DTT. Conformational analysis of compounds 3 and 4 suggested that N-H amide 3 binds to IMP-binding site in the DTT mediated manner.

Full text of DOI:10.1038/s41429-019-0199-3

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0199-3
SPECIAL FEATURE: ARTICLE
Novel adenosine-derived inhibitors of Cryptosporidium parvum
inosine 5′-monophosphate dehydrogenase
2
1
Kengo Shigetomi1 Albertus Eka Yudistira Sarwono1 Satoshi Ichikawa
Makoto Ubukata
●
●
●
Received: 28 March 2019 / Revised: 22 May 2019 / Accepted: 27 May 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
We have found cyclophane-type adenosine derivatives having p-quinone amide moieties (1 and 2) as weak inhibitors of
Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase (CpIMPDH) from the Hokkaido University Chemical
Library via the luciferase-based high-throughput screening. To obtain more potent inhibitors, we synthesized four new
derivatives free from cyclophane rings (3–6). The N-H derivatives 3 and 5 showed more potent activities (24.4 and 11.1 μM,
respectively) in the presence of dithiothreitol (DTT), whereas the N-methyl derivative 4 indicated more potent activity
(2.1 μM) without DTT. Conformational analysis of compounds 3 and 4 suggested that N-H amide 3 binds to IMP-binding
site in the DTT mediated manner.
Introduction
inhibitors from the Pharmakon repositioning library of off-
patent US Food and Drug Administration approved com-
pounds (Microsouce Discovery Systems, Inc., CT). The
screening of 1600 compounds resulted in the discovery of
three irreversible inhibitors: disulfiram, bronopol, and
ebselen. In the efficacy experiment against cryptosporidiosis
in severe combined immunodeficiency mouse, a decrease in
the number of oocyst shed was observed on the oral
administration of disulfiram and bronopol [4].
Cryptosporidium parvum is a waterborne pathogen, which
causes watery or mucoid diarrhea and abdominal pain in
many mammal species, including human. The infection
is usually self-limiting in immunocompetent patients.
Unfortunately, the infections could be severe in immuno-
compromised patients, such as children, elderly person, or
AIDS patients [1–3].
For the prevention of such infections, we have focused
on the inhibition of protozoan inosine 5′-monophosphate
dehydrogenase (IMPDH) because these microbes rely
solely on the IMPDH-mediated guanosine nucleotides
biosynthesis. In previous work, we established a luciferase-
based high-throughput screening system to identify IMPDH
Next, we focused on the chemical library from the
Faculty of Pharmacy, Hokkaido University (Hokkaido
University Chemical Library). The identified cyclophane-
type compounds 1 and 2 as hit compounds in the first
screening, showed relatively weak activities. Therefore,
four novel derivatives of 1 and 2 were synthesized and three
compounds 3–5 showed potent inhibitory activities. In this
article, we report synthesis and biological activity of these
novel adenosine-derived CpIMPDH inhibitors.
This article is dedicated to the 88th anniversary of the birth of Dr.
Kiyoshi Isono.
Results and discussion
* Kengo Shigetomi
sgtm@for.agr.hokudai.ac.jp
High-throughput screening of CpIMPDH inhibitors
from a chemical library
* Makoto Ubukata
m-ub@for.agr.hokudai.ac.jp
1
For the discovery of new protozoan IMPDH inhibitors,
a high-throughput screening was performed by using
Hokkaido University Chemical Library, which comprises
1600 compounds derived from the Faculty of Pharmacy,
Graduate School of Agriculture, Hokkaido University, Kita-9,
Nishi-9, Kita-ku, Sapporo 060-8589, Japan
2
Faculty of Pharmaceutical Science, Hokkaido University,
Sapporo 060-0812, Japan

K. Shigetomi et al.
Fig. 1 Structures of hit compounds in high-throughput screening of CpIMPDH and designed derivatives
Scheme 1 Synthesis of adenosine unit 12
Hokkaido University. The screening system providing high
accuracy (Z′-factor value of 0.7) [4] has been previously
established by the authors based on the detection of NADH
consumed by luciferase. In addition, the assay was designed
so as to sift out NAD+ mimics as hits by adding the excess
amount of NAD+ (1.6 mM), because the cofactor ubiqui-
tously functions in diverse biological systems. After
screening, the activities of 1260 were successfully evaluated
from 1600 compounds without mechanical errors, and 44
compounds showed comparable activities to mycophenolic
acid, a known IMPDH inhibitor. These hits were next
subjected to reproducibility test with triplicating assays. The
reproducibility was affirmed only in 19 compounds out of
44. Interestingly, 15 of 19 compounds were adenosine
derivatives as typified by cyclophane-type 1 and 2 (Fig. 1),
which had been originally developed targeting heat shock
protein 90 [5, 6]. The ribonucleoside substructures are fre-
quently conserved in IMPDH inhibitors mimicking both
IMP and NAD+ [7]. Thus, these compounds could be
regarded as the promising lead compounds to develop novel
IMPDH inhibitors. On the other hand, the cyclophane
skeleton is rarely seen in IMPDH inhibitors, except for
naturally occurring halicyclamine A [8–13]. Therefore, we
next designed new adenosine derivatives 3–6 of which
p-quinones were liberated from cyclophane and undertook
syntheses of these compounds.
Synthesis of compounds 3–6
Nucleoside unit 12 was first synthesized as shown in
Schemes 1. 2′,3′-Hydroxy groups and 5′-hydroxy group
of adenosine 7 were protected with isopropylidene and
TBS groups, respectively to give 9 [14, 15]. After pro-
tecting 6-amino group with trityl group, TBS group was
effectively deprotected by tetrabutylammonium fluoride
to give 5′-alcohol 11. Dess–Martin oxidation of 11
afforded 5′-aldehyde 12, which was subjected to
Horner–Wadsworth–Emmons (HWE) reaction without
further purification.
Phosphonate units, 18 and 21 were synthesized as shown
in Scheme 2. Methoxyhydroquinone 13 was protected with
MOM group followed by nitration [16] with ammonium
nitrate in the presence of trifluoroacetic anhydride to give
desired 15 and deprotected 16, which was converted to 15
by protection with MOM group. Nitro group of 15 was
converted to amine to give 17, which was converted to N-H

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase
Scheme 2 Synthesis of phosphonate units 18 and 21
phosphonate 18 by coupling with diethylphosphonoacetic
acid in 95% in two steps [17]. To synthesize N-methyl
phosphonate 21, compound 15 was converted to tri-
fluoroacetoamide 19 by catalytic hydrogenation followed
by protection with trifluoroacetyl group. Methylation,
deprotection of trifluoroacetyl group, and acylation with
bromoacetyl chloride afforded 20. Arbuzov reaction of 20
with triethylphosphite gave desired N-methyl phosphonate
21 in 84% yield from 15.
HWE reaction of aldehyde 12 with N-H phosphonate 18
gave desired compound 22 under the neutral conditions
using zinc triflate, triethylamine, and N,N,N′,N″-tetra-
methylethylenediamine developed by Schauer et al. [18].
Similarly, compound 23 was synthesized from 12 and N-
methyl phosphonate 21. Compounds 22 and 23 were converted
to 5′-substituted adenosine 3 and 4, respectively. Hydrogena-
tion of compound 3 with Pd/C gave saturated amide 5.
Although the same treatment of 4 gave compound 6, the
activity was not tested due to the limited amount (Scheme 3).
showed remarkable activities (IC50: 0.7–1.3 μM) compared
with hit compounds (>50 μM) as we expected. Next, we
determined IC50 values on fluorescence-based low-
throughput assay (Ex 340 nm/Em 465 nm) to eliminate the
possible effect by luciferase in the activity. As shown in
Table 2, compounds 3 and 5 showed inferior activities to
those in luciferase-based assay (IC50: 209.8 and >500.0 μM,
respectively), whereas 4 retained same activity. The result
would reflect partial inhibitions by 3 and 5 against lucifer-
ase activity. Computational conformation analyses pre-
dicted that N-H amide 3 is in an equilibrium between trans-
amide (M3–007 in Fig. 2a) and cis-amide (M3–078) at the
approximate ratio 96:4, while N-methyl amide 4 is uni-
formly present as cis-amide in any possible conformer
(M4–015, −003, −009, −011, and −071 in Fig. 2b). It can
be speculated that cis conformation of 4 enables selective
inhibition against CpIMPDH.
In the assay of enzymes that require cysteine as a cata-
lytic residue, reducing agents such as dithiothreitol (DTT)
or glutathione are commonly added to protect the residue
from inactivators. Also in IMPDH, the cysteine residue
positioned near IMP-binding site (e.g. Cys331 in human
type II IMPDH) is known to play a critical role in the
catalysis. Thus, we next evaluated the activities of 3–5 in
the presence of 1.0 mM DTT. These inhibitors showed
the IC50 values at 11.1–76.8 μM against CpIMPDH in DTT-
supplemented conditions, and the values were similar
against human type II IMPDH (Table 2). CpIMPDH is
Evaluation of IMPDH inhibitory activities
The inhibitory activities of hit compounds 1 and 2, synthetic
compounds 3–5 against recombinant CpIMPDH were
evaluated. The apparent IC50 values were determined using
essentially the same conditions as the luciferase-based high-
throughput assay, except that different concentrations were
used (Table 1). Interestingly, synthetic compounds 3–5

K. Shigetomi et al.
MeO
OMOM
NHTr
N
OMOM
N
O
N
N
N
MeO
12, Zn(OTf)2, Et3N,
TMEDA, THF
MOMO
O
O
R
N
P(OEt)2
O
MOMO
R
O
O
18 R = H
21 R = Me
22 R = H, 67%
23 R = Me, 56%
MeO
O
N
MeO
O
O
N
NH2
NH2
O
O
N
N
N
N
1) 80% aq. TFA
2) O2, Pd/C, MeOH
H2, Pd/C, MeOH
then O2
O
R
N
R
N
N
N
O
O
3 (R = H) 73%
4 (R = Me) 77%
5 (R = H) 73%
6 (R = Me) 29%
HO
OH
HO
OH
Scheme 3 Synthesis of adenosine derivatives 3–6
Table 1 Apparent IC50 values of hit compounds 1 and 2, synthetic
compounds 3–5 measured by luciferase-based assay
between Cp- and hIMPDH II may be due to the less con-
tribution of adenine ring recognition. The addition of
reducing agents often attenuates the activity due to the
competition or the direct contact with inhibitors [20], and
the same phenomena were observed in bronopol, ebselen,
and disulfiram, previously discovered Cys-targetting inhi-
bitors [4] (Table 2). Among the synthetic compounds, only
N-methyl derivative 4 behaves according to these precedent
examples (76.8 μM with DTT), which in turn suggests that
cysteine residue is involved in this inhibition, such as via
Michael addition to quinone moiety. In contrast, it would be
noteworthy that compounds 3 and 5 showed superior
activities in the DTT-supplemented conditions. This pro-
nounced difference would stem from the conformational
preference of each compound. The “stretched” conforma-
tion in compounds 3 and 5 conjure a binding model seen in
Compounds
IC50 (μM) against CpIMPDH
Without DTT
1
2
3
4
5
>50
>50
1.1
0.7
1.3
Table 2 IC50 values of previously discovered Cys-targetting
compounds and synthetic compounds 3–5 measured by luciferase-
free low-throughput assay
Compounds
IC50 (μM) against
CpIMPDH (0.15 μg/mL)
IC50 (μM) against
hIMPDH
C2-mycophenolic adenine dinucleotide (C2-MAD),
a
(0.75 μg/mL)
ligand mimicking NAD+ [21]. Given that 3 and 5 bind in
the same manner as C2-MAD, the improved activity may be
explained by crosslinking via DTT between Cys and
p-quinone moieties, because they are to be positioned away
by 5–10 Å. Meanwhile, these compounds can possibly
adopt a different binding manner from C2-MAD consider-
ing the screening system set to eliminate NAD+ mimics and
the above-discussed similarity in IC50 values with hIMPDH.
Obviously, this is true also for compound 4. Although
further investigations, such as X-ray crystallography or
detailed docking simulation are required, each N-H and
N-methyl amide was proved to have clearly different
mode of binding against CpIMPDH. This variance
may provide the optional application of these compounds,
such as combined or selective use depending on different
physiological conditions.
With DTT Without DTT
With DTT
Disulfiram [4] >500.0
Ebselen [4] 148.7
Bronopol [4] 291.0
1.4
>500.0
>500.0
>500.0
30.3
1.1
1.3
3
4
5
24.4
76.8
11.1
209.8
2.1
43.0
>500.0
11.3
known to have a dramatic difference in NAD+ binding site
from that of hIMPDH II. Especially the residues related to
the binding of adenine ring, such as His253, Phe282, Thr45,
and Gln469, are not conserved in CpIMPDH, which dis-
criminates the specificity to NAD+ versus nicotinamide
hypoxanthine dinucleotide [19]. The similar IC50 values

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase
Fig. 2 Predicted stable
a)
conformers of a compound 3
and b compound 4. The
numbers in parentheses are
relative energies to the most
stable conformers M3–007
and M4–015
M3-007
M3-078
(+7.99 kJ/mol)
b)
M4-015
M4-003
M4-009
(+0.56 kJ/mol)
(+3.74 kJ/mol)
M4-011
M4-071
(+4.43 kJ/mol)
(+4.56 kJ/mol)
Conclusion
Silica Gel 60 (0.040–0.063 mm), respectively. All NMR
spectra were measured with JNM-ECA-500, JNM-AL-400,
or JNM-ECX-400P (JEOL, Tokyo, Japan). Chemical shifts
are defined using tetramethylsilane as the internal standard,
except for the measurement with DMSO-d6 in which sol-
By the use of high-throughput screening system,
cyclophane-type adenosine derivatives furnishing p-qui-
none moieties (1 and 2) were discovered as weak inhibitors
of CpIMPDH from the Hokkaido University Chemical
Library. To ameliorate the activities, we designed and
synthesized four new derivatives of which p-quinones were
liberated from cyclophane (3–6). All tested compounds
(3–5) showed more potent activities at the absence of DTT
than the hit compounds (1 and 2) in luciferase-based high-
throughput assay. Interestingly, the supplement of DTT in
fluorescence-based low-throughput assay differentiated the
activity of N-methyl amide 4 from those of N-H amides 3
and 5, which would stem from the conformational differ-
ence between cis- and trans-amides. These insights raised a
significance of impact by reducing agents in the IMPDH
assay and will be helpful for the development of novel
IMPDH inhibitors.
1
vent peaks (2.49 ppm for H and 39.7 ppm for 13C) were
used. Coupling constants (J) are given in Hz. Mass spectra
were acquired with ESI techniques using JMS-HX 110,
JMS-700TZ, or JMS-T100LP (JEOL, Tokyo, Japan).
2′,3′-O-Isoprppylideneadenosine (8)
Adenosine (20 g, 75 mmol) was dissolved in 800 mL of
acetone–DMF (3:1, v/v) and to the mixture was added 14 g
of TsOH monohydrate (75 mmol). After 4 h stirring, 19 mL
of 2,2-dimethoxypropane (150 mmol) was added and the
reaction was allowed to stir for another 6 h. The mixture
was partially evaporated and partitioned between EtOAc
(200 mL) and sat. Na2CO3 aq. The water layer was washed
with CHCl3 and CH2Cl2. Combined organic layer was then
washed with brine (100 mL) and dried over anhydrous
Na2SO4. After the removal of solvent in vacuo, 2′,3′-O-
isoprppylideneadenosine (8) was recovered by trituration
with EtOAc–hexane as colorless crystals (8.0 g, 35%). 1H
NMR (400 MHz, DMSO-d6) δ 8.32 (1H, s, H, H-2), 8.14
(1H, s, H-8), 7.34 (2H, s, H-6), 6.10 (1H, d, J = 2.8 Hz,
H-1′), 5.33 (1H, dd, J = 6.4, 3.2 Hz, H-2′), 4.95 (1H, dd,
J = 6.2, 2.5 Hz, H-3′), 4.20 (1H, m, H-4′), 3.52 (2H, m,
H-5′), 1.53 (3H, s, Me), 1.31 (3H, s, Me).
Experimental procedures
Chemistry
Organic solvents were purified by normal methods. Unless
otherwise stated, all commercially available chemicals of
the highest purity were used without further purification.
Thin layer and column chromatography were performed
with Merck Silica Gel 60 (0.063–0.020 mm) and Merck

K. Shigetomi et al.
5′-O-tert-Butyldimethylsilyl-2′,3′-O-
7.98 (1H, s, H-2), 7.79 (1H, s, H-8), 7.30 (15H, m, Tr), 7.05
(1H, s, H-6), 5.83 (1H, d, J = 5.0 Hz, H-1′), 5.17 (1H, t,
J = 5.7 Hz, H-2′), 5.08 (1H, d, J = 6.0 Hz, H-3′), 4.52 (1H,
s, H-4′), 3.93 (1H, dt, J = 12.8, 1.8 Hz, H-5′a), 3.75 (1H, dt,
J = 12.8, 1.8Hz, H-5′b), 1.64 (3H, s, Me), 1.36 (3H, s, Me).
isopropylideneadenosine (9)
Imidazole (5.4 g, 79 mmol) and 7 were dissolved in 50 mL
of DMF and tert-butyldimethylchlorosilane (6.0 g, 40
mmol) was added to the solution at 0 °C under argon
atmosphere. The reaction was quenched with MeOH in 1 h
and was partitioned between EtOAc (300 mL) and sat.
Na2CO3 aq. (250 mL). The organic layer was washed with
water (150 mL × 3) and brine (100 mL), dried over anhy-
drous Na2SO4 and evaporated in vacuo. The product 9 was
recovered as colorless crystals by filtration, and also from
the filtrate after evaporation and trituration with hexane
(E)-N-(3-Methyl-1,4-bismethoxymethylphenyl)-3-
{(3aR,4R,6R,6aR)-6-(6-triphenylmethylamino-9H-purin-9-yl)-
2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}prop-2-
enamide (22)
Alcohol 4 (550 mg, 1.0 mmol) was dissolved in CH2Cl2 (10
mL) and excess amount (>4.0 eq.) of Dess–Martin period-
inane was added at 0 °C under argon atmosphere. After
complication of the reaction, sat. Na2S2O3 aq. and sat.
Na2CO3 aq. were added. To the mixture was added 20 mL
of water and extracted with EtOAc (30 mL). The organic
layer was washed with sat. Na2CO3 aq. (20 mL) and brine
(20 mL), dried over anhydrous Na2SO4 and evaporated in
vacuo. Resulting crude product (330 mg) was used in next
step without further purification.
1
(14 g, quant.). H NMR (400 MHz, DMSO-d6) δ 8.39 (lH,
s, H-2), 8.05 (1 H, s, H-8), 6.17 (1 H, d, J = 2.8 Hz, H-1′),
5.48 (2 H, br s, H-6), 5.27 (lH, dd, J = 6.4, 2.3 Hz, H-2′),
4.94 (1 H, dd, J = 6.4, 2.3 Hz, H-3′), 4.43 (1 H, dd, J = 6.9,
4.1 Hz, H-4′), 3.88 (1 H, dd, J = 11.4, 4.1 Hz, H-5′a), 3.76
(1 H, dd, J = 11.4, 4.1 Hz, H-5′b), l.64 (3 H, s, Me), 1.41 (3
H, s, Me), 0.84 (9 H, s, t-Bu), 0.02 and 0.01 (6 H, s, Me).
5′-O-tert-Butyldimethylsilyl-2′,3′-O-isopropylide-6-N-
The aldehyde 18, Zn(OTf)2 (440 mg, 1.2 mmol), triethy-
lamine (560 μL, 4.0 mmol), and TMEDA (180 μL, 1.2 mmol)
were added to 3.0 mL of THF. A solution of 11 in THF (12
mL) was added to the mixture, which was allowed to stir for
15 under argon atmosphere. The mixture was evaporated and
partitioned between EtOAc (40 mL) and water (40 mL). The
organic layer was washed with 20 mL of water, 0.1 M HCl
aq., sat. Na2CO3 aq., and brine. The EtOAc layer was dried
over anhydrous Na2SO4 and evaporated in vacuo. Subsequent
silica gel column chromatography eluting with EtOAc:hex-
triphenylmethyladenosine (10)
To a solution of 9 (10 g, 24 mmol) and triethylamine (7.9
mL, 57 mmol) in 100 mL of CH2Cl2, 7.8 g of trityl chloride
(28 mmol) was added at 0 °C. The reaction was allowed to
stir at room temperature then 50 °C under argon atmo-
sphere. After complication of the reaction, sat. Na2CO3 aq.
was added and resulting white precipitate was filtered off.
The filtrate was evaporated and dissolved in 200 mL of
EtOAc. The organic layer was washed with 200 mL of
water, 0.1 M HCl aq., sat. Na2CO3 aq. and brine. The
EtOAc layer was dried over anhydrous Na2SO4 and eva-
porated in vacuo. The crude product was purified with silica
gel column chromatography eluting with EtOAc:hexane =
1:10–3:1 then MeOH:CHCl3 = 1:4 to give 11 g of 10 (71%)
and 2.8 g of starting material 9 (20%). 1H NMR (400 MHz,
CDCl3) δ 8.06 (1H, s, H-2), 7.97 (1H, s, H-8), 7.30 (15H,
m, Tr), 6.92 (1H, s, H-6), 6.12 (1H, d, J = 2.7 Hz, H-1′),
5.28 (1H, dd, J = 6.2, 2.7 Hz, H-2′), 4.93 (1H, dd, J = 6.4,
4.1 Hz, H-4′), 3.84 (1H, dd, J = 11.5, 4.1 Hz, H-5′a), 3.75
(1H, dd, J = 11.5, 4.1 Hz, H-5′b), 1.61 (3H, s, Me), 1.38
(3H, s, Me), 0.83 (9H, s, t-Bu), 0.00 and –0.02(6H, s, Me).
1
ane = 2:3–2:1 gave 14 as brown foam (420 mg, 67%). H
NMR (400 MHz, CDCl3) δ 8.05 (1H, s, purinyl H-2), 7.85
(1H, s, purinyl H-8), 7.58 (1H, s, phenyl), 7.30 (15H, m, Tr),
6.99 (1H, dd, J = 15.6, 5.0 Hz, olefinic H-3), 6.95 (1H, s,
purinyl H-6), 6.77 (1H, s, phenyl), 6.13 (1H, dd, J = 15.6,
1.4 Hz, olefinic H-2), 6.10 (1H, d, J = 6.1 Hz, H-6), 5.49 (1H,
dd, J = 6.1, 2.5 Hz, H-6a), 5.18 (2H, s, OCH2OCH3), 5.05
(3H, m, H-3a and OCH2OCH3), 4.83 (1H, t, J = 3.4 Hz, H-4),
3.83 (3H, s, OMe), 3.53 (3H, s, OCH2OCH3), 3.41 (3H, s,
OCH2OCH3), 1.63 (3H, s, Me), 1.38 (3H, s, Me); LR-ESI-
MS m/z = 814.33 [M]+.
(E)-N-methyl-N-(3-Methyl-1,4-bismethoxymethylphenyl)-3-
{(3aR,4R,6R,6aR)-6-(6-triphenylmethylamino-9H-purin-9-yl)-
2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}prop-2-
enamide (23)
2′,3′-O-Isopropylidene-6-N-triphenylmethyladenosine (11)
To a solution of 10 in THF (150 mL) was added 17 mL of
tetra-n-butylammonium bromide 1 M solution at 0 °C under
argon atmosphere. After 40 min, the mixture was evapo-
rated and directly subjected to a silica gel column chro-
matography (EtOAc:hexane = 1:1–5:1) to isolate 11 as a
Following the same procedure as synthesis of 22, N-methyl
product 23 was prepared (1.7 g, 57%) from the reaction
between crude 12 and 1.6 g of 21 (3.6 mmol).
1H NMR (400 MHz, CDCl3, mixture of rotamers) δ
8.05 (1H, s, purinyl H-2), 8.03 (1H, s, purinyl H-2), 7.73
1
yellow foam (9.1 g, quant.). H NMR (400 MHz, CDCl3) δ

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase
(1H, s, purinyl H-8), 7.71 (1H, s, purinyl H-8), 7.30
(30H, m, Tr), 6.90 (6H, m, purinyl H-6, olefinic H-3 and
phenyl), 6.79 (1H, s, phenyl), 6.78 (1H, s, phenyl), 6.02
(4H, m, H-6 and olefinic H-2), 5.33 (1H, dd, J = 6.4,
2.8Hz, H-6a), 5.26 (1H, dd, J = 6.4, 2.8 Hz, H-6a), 5.12
(8H, m, OCH2OCH3), 4.88 (2H, m, H-3a), 4.59 (2H, m,
H-4), 3.87 (6H, s, OMe), 3.49 (3H, s, OCH2OCH3), 3.46
(3H, s, OCH2OCH3), 3.39 (3H, s, OCH2OCH3), 3.35
(3H, s, OCH2OCH3), 3.22 (3H, s, NMe), 3.21 (3H, s,
NMe), 1.56 (3H, s, Me), 1.55 (3H, s, Me), 1.32 (6H, s,
Me); ESI-MS m/z = 828 .35 [M]+.
(1H, s, quinonyl), 5.85 (1H, d, J = 5.6 Hz, H-6), 4.63 (1H, t,
J = 5.0 Hz, H-6a), 4.05 (1H, t, J = 5.0 Hz, H-3a), 3.87 (1H,
m, H-4), 3.79 (3H, s, OMe), 2.63 (2H, t, J = 7.5 Hz, olefinic
H-2), 1.93 (2H, m, olefinic H-3); 13C NMR (125 MHz,
DMSO-d6) δ 182.7, 182.2, 173.8, 159.2, 156.1, 152.7,
149.4, 139.8, 139.8, 119.2, 111.6, 105.1, 87.4, 83.0, 73 .1,
73.0, 56.6, 32.9, 28.4, 22.1; ESI-MS m/z = 467.13 [M +
Na]+.
(E)-N-Methyl-N-(4-methoxy-p-benzoquinonyl)-3-
{(3aR,4R,6R,6aR)-6-(6-triphenylmethylamino-9H-purin-9-yl)-
2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}prop-2-
enamide (4)
(E)-N-(4-Methoxy-p-benzoquinonyl)-3-{(3aR,4R,6R,6aR)-6-(6-
triphenylmethylamino-9H-purin-9-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}prop-2-
enamide (3)
Following the same procedure as synthesis of 3, N-methyl
product 4 was prepared in 77% yield. Recrystallization was
1
performed with the combination of MeOH and EtOAc. H
A precursor 22 was dissolved in 80% trifluoroacetic acid
aqueous solution. After stirring for 25 min, dilution with
EtOH and evaporation was repeated several times. The
concentrate was dissolved in 15 mL of MeOH and Pd/C (Pd
10%) (31 mg) was added to the solution. The suspension
was allowed to stir at O2 atmosphere. After 25 min, Pd/C
was removed by celite filtration and solvent was removed
in vacuo. Subsequent recrystallization from MeOH gave
82 mg of 3 as yellow crystals (73%).
NMR (500 MHz, DMSO-d6) δ 8.35 (1H, s, purinyl H-2),
8.20 (1H, s, purinyl H-8), 7.75 (2H, br s, purinyl H-6), 6.83
(1H, dd, J = 14.9, 6.3 Hz, olefinic H-3), 6.78 (1H, s, qui-
nonyl), 6.16 (1H, dd, J = 14.9, 1.5 Hz, olefinic H-2), 6.12
(1H, s, quinonyl), 5.92 (1H, d, J = 5.2 Hz, H-6), 4.64 (1H, t,
J = 5.2 Hz, H-6a), 4.46 (1H, t, J = 6.3 Hz, H-4), 4.15 (1H, t,
J = 5.2 Hz, H-3a), 3.77 (3H, s, OMe), 3.12 (3H, s, NMe);
13C NMR (100 MHz, DMSO-d6) δ 182.9, 181.5, 165.3,
159.1, 153.9, 150.0, 149.0, 147.2, 141.6, 140.9, 127.1,
123.5, 119.1, 106.7, 87.8, 83.0, 73.7, 72.8, 56.8, 36.4; ESI-
MS m/z = 479.13 [M + Na]+.
1H NMR (400 MHz, DMSO-d6) δ 8.49 (1H, s, purinyl H-
2), 8.26 (1H, s, purinyl H-8), 7.80 (2H, br s, purinyl H-6),
7.42 (1H, s, quinonyl), 7.01 (1H, dd, J = 16.0, 5.5 Hz,
olefinic H-3), 6.82 (1H, dd, J = 16.0, 1.5 Hz, olefinic H-2),
6.15 (1H, s, quinonyl), 5.99 (1H, d, J = 5.0 Hz, H-6), 4.62
(1H, t, J = 5.0 Hz, H-6a), 4.53 (1 H, t, J = 5.5 Hz, H-4),
4.22 (1 H, t, J = 5.0 Hz, H-3a), 3.79 (3H, s, OMe); 13C
NMR (125 MHz, DMSO-d6) δ 183.3, 182.8, 165.7, 159.9,
158.9, 153.5, 149.4, 144.1, 142.0, 140.7, 124.8, 119.6,
113.0, 105.8, 88.6, 83.5, 74.2, 73 .7, 57.3; ESI-MS m/z =
442.12 [M]+.
Computational conformation analysis
All calculations were performed using the Spartan’16
(Wavefunction Inc., Irvine, CA, USA) in gas conditions.
The structures of compounds 3 and 4 were built and the
conformer distributions were first analyzed by MMFF.
For the given conformers of 3 and 4, IDs were assigned as
“M3-NNN” or “M4-NNN”, respectively, where the
numbers after prefixes are the order of relative energies
after molecular mechanics calculation. The conformers
within relative energies of 40 kJ/mol were geometrically
optimized by Hartree–Fock/3–21G, and energies of
resulting conformers within 40 kJ/mol were evaluated
using density functional (DF)/ωB97 X-D/6–31G* calcu-
lation. The conformers with >15 kJ/mol relative energies
were discarded and geometry optimization was again
performed using DF/ωB97 X-D/6–31G*. This series of
DF calculations returned M3–007 and M4–015 as the
lowest-energy conformers together with the conformers
within 10 kJ/mol relative energies (one conformer for
M3–007 and four conformers for M4–015; see Fig. 2).
Finally, energies and Boltzmann distribution at 297 K of
these conformers were calculated using DF/ωB97 X-V/
6–311 + G (2df, 2p).
(E)-N-(4-Methoxy-p-benzoquinonyl)-3-{(3aR,4R,6R,6aR)-
6-(6-triphenylmethylamino-9H-purin-9-yl)-2,2-
dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl}propanamide (5)
Amide 3 (15 mg, 27 μmol) was dissolved in 6.0 mL of
MeOH and to the solution was added 10 mg of Pd/C (Pd
10%). The reaction was allowed to stir under H2 atmosphere
for 8 h followed by O2 atmosphere for another 70 min. Pd/C
was removed by celite filtration and solvent was evaporated
in vacuo. Recrystallization with the mixed solvent of
MeOH, EtOAc, and hexane gave propanamide 5 (8.1 mg,
54%) as yellow crystals.
1H NMR (400 MHz, DMSO-d6) δ 8.45 (1H, d, J = 3.6
Hz, purinyl H-2), 8.24 (1H, d, J = 3.2 Hz, purinyl H-8),
7.86 (2H, br s, purinyl H-6), 7.24 (1H, s, quinonyl), 6.09

K. Shigetomi et al.
Biology
Determination of IC50 values of hit and synthetic
compounds
Expression and purification of recombinant CpIMPDH and
human IMPDH II
Standard IMPDH assay solution containing 50 mM Tris-
HCl pH 8.0, 100 mM KCl, 3 mM EDTA, 0.1 mg/mL BSA,
1.0 mM DTT, and appropriate amounts of inhibitors with a
total volume of 270 μL. Substrate concentrations in the
solution were 500 μM NAD+ and 250 μM IMP for
CpIMPDH. Reaction was started by the addition of 30 μL
enzyme solution (CpIMPDH: 0.15 μg/mL; hIMPDH:
0.75 μg/mL). The activity of the enzyme was measured by
monitoring NADH production in either absorbance at
340 nm or fluorescence emissions at 465 nm. IC50 values
were calculated by plotting the recorded NADH yield
against log of compound concentration in GraFit ver. 7
(Erithacus Software, UK). All screening assays in this study
was conducted with 0.5% DMSO as vehicle and library
compound concentration of 10 μM unless stated otherwise.
The PCR product coding CpIMPDH amplified with the
primer sets 5′-TTTTGGATCCTCAAACATGGGTAC
A-3′ and 5′-TTTTGAATTCCTATTTACT-ATAATT-3′
was cloned into pCR2.1-TOPO vector (Invitrogen Japan
KK, Tokyo, Japan). The target gene was digested by
BamHI and EcoRI, and inserted into a pGEX-6P-2 plas-
mid (GE Healthcare Bio-Science Corp., UK). The plasmid
was then transformed to ECOS E. coli BL21 (DE3) (Wako
Pure Chemical Ind., Ltd., Japan). The cells were grown
overnight at 30 °C in 50 mL 2× YT broth containing 100
μg/mL ampicillin. Afterwards, the broth was subcultured
to 700 ml of medium containing a final concentration of
1.0 mM isopropyl-1-thio-β-galactopyranoside (Sigma-
Aldrich Japan, Tokyo, Japan) and 100 μg/mL ampicillin.
After 5 h incubation at 25 °C, cells were harvested by
centrifugation, washed with PBS solution, and stored
frozen at –80 °C until usage.
Acknowledgements The preliminary screening study was partly sup-
ported by Platform Project for Supporting Drug Discovery and Life
Science Research (Basis for Supporting Innovative Drug Discovery
and Life Science Research (BINDS)) from AMED under Grant
Number 18am0101093j0002, Hokkaido University, Global Facility
Center (GFC), Pharma Science Open Unit (PSOU), funded by MEXT
under “Support Program for Implementation of New Equipment
Sharing System” and Takeda Science Foundation. We sincerely thank
the Institute for Fermentation, Osaka (IFO) Japan for the financial
support. This work was supported in part by Grant-in-Aid for Chal-
lenging Exploratory Research (JSPS No. 25660051).
hIMPDH II plasmid was a generous gift from Prof.
Lizbeth Hedstrom, Brandeis University, USA. The plasmid
was transformed in the same manner as CpIMPDH.
High-throughput screening of IMPDH inhibitors
The HTS assay was established using the NAD(P)H-Glo
Assay Kit (Promega, Madison, WI, USA). Briefly, 10 μL
of substrate solution containing 50 mM Tris-HCl pH 8.0,
200 mM KCl, 0.1 mg/mL BSA, 1.6 mM β-NAD+
(Oriental Yeast Co., Tokyo, Japan), 100 μM IMP (Sigma-
Aldrich Japan, Tokyo, Japan), and assay kit was added to
white 384-well plates. Mycophenolic acid (APAC Phar-
maceutical LLC, Hangzhou, China), a known IMPDH
inhibitor, or the chemical library from the Faculty of
Pharmacy, Hokkaido University was also added to the
wells in concentration of 10 μM. Reaction was started by
addition of 10 μL enzyme solution containing 50 mM
Tris-HCl pH 8.0, 0.1 mg/mL BSA, and CpIMPDH.
Reaction was carried out at 30 °C for 30 min in dark.
Solution handlings was carried out using Mosquito LCP
(TTP LabTech Ltd., Melbourn, UK) and Multidrop
Combi (Thermo Scientific, Waltham, MA, USA). The
production of NADH was monitored by the measurement
of luminescence (RLU) using either VERITAS Microplate
Luminometer (Turner Biosystems, Sunnyvale, CA, USA)
or EnSpire Multimode Reader (Perkin-Elmer, Waltham,
MA, USA). The experiments were performed without
repetition in first screening, and in triplicate in other
assays.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
1. DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB,
Jakubowski W. The infectivity of Cryptosporidium parvum in
healthy volunteers. N Engl J Med. 1995;332:855–9.
2. Hunter PR, Nichols G. Epidemiology and clinical features of
Cryptosporidium infection in immunocompromised patients. Clin
Microbiol Rev. 2002;15:145–54.
3. Fayer R. Cryptosporidium. A wter-borne zoonotic parasite. Vet
Parasitol. 2004;126:37–56.
4. Sarwono AEY, Mitsuhashi S, Kabir MHB, Shigetomi K, Okada
T, Ohsaka F, Otsuguro S, Maenaka K, Igarashi M, Kato K,
Ubukata M. Repurposing existing drugs: identification of irre-
versible IMPDH inhibitors by high-throughput screening. J
Enzym Inhib Med Chem. 2019;34:171–8.
5. Muranaka K, Ichikawa S, Matsuda A. Development of the car-
boxamide protecting group, 4-(tert-butyldimethylsiloxy)-2-meth-
oxybenzyl. J Org Chem. 2011;76:93.

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase
6. Muranaka K, Ichikawa S, Matsuda A. Design and synthesis of
3′,5′-ansa-adenosines as potential Hsp90 inhibitors. Tetrahedron
Lett. 2009;50:5102–6.
14. Townsend AP, Roth S, Williams HE, Stylianou E, Thomas NR.
New S-adenosyl-L-methionine analogues: synthesis and reactivity
studies. Org Lett. 2009;11:2976–9.
7. Sintchak MD, Nimmersgern E. The structure of inosine 5′-
monophosphate dehydrogenase and the design of novel inhibitors.
Immunopharmacol. 2000;47:163–84.
8. Jaspars M, Pasupathy V, Crews P. A tetracyclic diamine alkaloid,
halicyclamine A, from the marine sponge Haliclona sp. J Org
Chem. 1994;59:3253–5.
15. Enomoto K, Nagasaki T, Yamauchi A, Onoda J, Sakai K, Yoshida
T, Maekawa K, Kinoshita Y, Nishino I, Kikuoka S, Fukunaga T,
Kawamoto K, Numata Y, Takemoto H, Nagata K. Development
of high-throughput spermidine synthase activity assay using
homogeneous time-resolved fluorescence. Anal Biochem. 2006;
351:229–40.
9. Felczak K, Vince R, Pankiewicz KW. NAD-based inhibitors with
anticancer potential. Bioorg Med Chem Lett. 2014;24:332–6.
10. Franchetti P, Cappellacci L, Pasqualini M, Petrelli R, Jayapraka-
san V, Jayaram HN, Boyd DB, Jain MD, Grifantini M. Synthesis,
conformational analysis, and biological activity of new analogues
of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP
dehydrogenase inhibitors. Bioorg Med Chem. 2005;13:2045–53.
11. Chen L, Wilson DJ, Xu Y, Aldrich CC, Felczak K, Sham YY,
Pankiewicz KW. Triazole-linked inhibitors of inosine monopho-
sphate dehydrogenase from human and Mycobacterium tubercu-
losis. J Med Chem. 2010;53:4768–78.
12. Franchetti P, Cappellacci L, Perlini P, Jayaram HN, Butler A,
Schneider BP, Collart FR, Huberman E, Grifantini M. Isosteric
analogues of nicotinamide adenine dinucleotide derived from
furanfurin, thiophenfurin, and selenophenfurin as mammalian
inosine monophosphate dehydrogenase (type I and II) inhibitors. J
Med Chem. 1998;41:1702–7.
16. Andrus MB, Hicken EJ, Meredith EL, Simmons BL, Cannon JF.
Selective synthesis of the para-quinone region of geldanamycin.
Org Lett. 2003;5:3859–62.
17. Clevenger RC, Blagg BS. Design, synthesis, and evaluation of a
radicicol and geldanamycin chimera, radamide. Org Lett. 2004;
6:4459–62.
18. Schauer DJ, Helquist P. Mild zinc-promoted Horner–
Wadsworth–Emmons reactions of diprotic phosphonate reagents.
Synthesis. 2006;21:3654–60.
19. Umejiego NN, Li C, Riera T, Hedstrom L, Striepen B. Cryptos-
poridium parvum IMP dehydrogenase: identification of func-
tional, structural, and dynamic properties that can be exploited for
drug design. J Biol Chem. 2004;279:40320–7.
20. Lee H, Torres J, Truong L, Chaudhuri R, Mittal A, Johnson ME.
Reducing agents affect inhibitory activities of compounds: results
from multiple drug targets. Anal Biochem. 2012;423:46–53.
21. Risal D, Strickler MD, Goldstein BM. Crystal structure of human
inosine monophosphate dehydrogenase type II complexed with
the MPA/NAD analog C2-MAD. PDB ID: 1NF7. https://doi.org/
10.2210/pdb1NF7/pdb. Released 27 Jan 2004.
13. Digits JA, Hedstrom L. Drug selectivity is determined by coupling
across the NAD+ site of IMP dehydrogenase. Biochemistry. 2000;
39:1771–7.