Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c01835

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5′-(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

Full text of DOI:10.1021/acs.jmedchem.0c01835

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Article
Discovery of Potent and Selective Methylenephosphonic Acid CD73
Inhibitors
Ehesan U. Sharif,* Jaroslaw Kalisiak, Kenneth V. Lawson, Dillon H. Miles, Eric Newcomb,
Erick A. Lindsey, Brandon R. Rosen, Laurent P. P. Debien, Ada Chen, Xiaoning Zhao,
̈
Stephen W. Young, Nigel P. Walker, Norbert Strater, Emma R. Scaletti, Lixia Jin, Guifen Xu,
Manmohan R. Leleti, and Jay P. Powers
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ABSTRACT: Solid tumors are often associated with high levels of
extracellular ATP. Ectonucleotidases catalyze the sequential
hydrolysis of ATP to adenosine, which potently suppresses T-
cell and NK-cell functions via the adenosine receptors (A2a and
A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP
to adenosine. Thus, increased CD73 enzymatic activity in the
tumor microenvironment is a potential mechanism for tumor
immune evasion and has been associated with poor prognosis in
the clinic. CD73 inhibition is anticipated to restore immune
function by skirting this major mechanism of adenosine generation.
We have developed a series of potent and selective methyl-
enephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5′-
(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure−activity
relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73,
exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.
clinical use. Therefore, the development of new CD73
inhibitors continues to be vitally important.
INTRODUCTION
■
5′-Ribonucleotide phosphohydrolase, also known as cluster of
differentiation 73 (CD73), is a Zn-binding GPI-anchored
extracellular protein that functions as a noncovalent homo-
dimer with two structural domains connected by a small hinge
region.1 CD73 has high affinity for adenosine monophosphate
(AMP) and catalyzes the conversion of AMP to adenosine.2
Numerous studies have demonstrated that CD73 is upregu-
lated by hypoxia within the tumor microenvironment.3 High
concentrations of CD73 within the tumor microenvironment
contribute substantially to elevated levels of adenosine, which
results in immune suppression by activating the adenosine
receptors (A2a and A2b) on infiltrating lymphocytes.
RESULTS AND DISCUSSIONS
■
We started our investigation based on the known CD73
inhibitor AMPCP. The crystal structure of AMPCP bound to
̈
human CD73 (hCD73) was disclosed by Strater et al. in 2012
(PDB: 4H2I).9 Our initial lead scaffold was designed by
investigation of key binding interactions of this known
inhibitor with hCD73, i.e., coordination of the phosphono-
methyl-phosphonic acid with di-zinc, hydrogen-bonding
interaction between syn-diol of ribose with Asp-506 and Asn-
390 and the hydrophobic π−π stacking between adenine and
Phe-500 and Phe-417 shown in Scheme 1.
CD73 represents a novel therapeutic target for cancer
immunology, and several efforts have been undertaken to
leverage the role it plays in the development of anticancer
therapeutics.4 For example, anti-CD73 antibody therapy has
been shown to inhibit tumor growth and metastasis in animal
Early structure−activity relationship (SAR) studies on
APMCP were reported by Muller et al., where substitution
̈
at the C6 and C8 positions and the methylene bridging the two
models.5 Mu
̈
ller and co-workers have reported derivatives of α-
Received: October 20, 2020
Published: January 5, 2021
β-methylene-ADP (AMPCP), one of the earlier metabolically
stable, potent, and selective CD73 inhibitors known.6 Similarly,
in industrial settings, several efforts have been directed toward
the discovery of CD73 inhibitors in recent years.7,8 Despite
these efforts, no CD73 inhibitors are currently approved for
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resulted in successful replacement of the bisphosphonic acid 1
with methylenephosphonic acid 1c. Additionally, methylene-
phosphonic acid analogs offered us the prospect for developing
prodrugs to improve oral bioavailability because contrary to
earlier experience with bisphosphonic acids, successful
development of prodrugs for monophosphonic acids have
been reported.11
Scheme 1. Key Interactions of AMPCP with hCD73
To improve the potency of the methylenephosphonic acid
analog 1c, we carried out a systematic SAR analysis of the
adenine core, which began with modification at the C6
position (Table 2).12 A wide variety of benzylamines and
phenethylamines were tolerated at the C6 position. Sub-
stitution of the phenyl ring in the phenethylamine moiety with
electron-donating or electron-withdrawing groups did not
significantly affect the potency (2a−2c). Similar observations
were made with substitution of the benzylamine (1c vs 2f). In
contrast, the stereochemistry at the benzylic position appeared
to be important for the observed potency. For example, (S)-
(−)-α-methylbenzylamine as a C6 substituent (2d) exhibited
higher potency than its enantiomer (2e) (IC50 = 65 nM for 2d
vs IC50 = 156 nM for 2e). Heteroaromatic amine substituents
like 2-picolylamine 2h, N-methylpyrazole-4-methylamine 2i,
and 4-methyltriazole-3-methylamine 2j showed decreased
potency. C6-amine substituents containing an aliphatic group
(2k) or carbocyclic groups (2l−2o) were also investigated.
The most potent analog 2n was obtained when cyclopentyl-
amine was introduced at the C6 position (IC50 = 26 nM).
Compounds containing a cyclic secondary amine at the C6
position, such as pyrrolidine (2p) or morpholine (2r), were
found to be less potent. Similarly, C6 amines that contain a
heterocyclic substituent as in the case of (R)-3-amino-
tetrahydrofuran (2q) did not improve the potency (Table 2).
To better understand the binding interactions of methyl-
enephosphonic acid analog 2n with hCD73, we obtained an X-
ray cocrystal structure of 2n bound to hCD73 (PDB: 6YE1).
The X-ray structure revealed that the methylene phosphonate
interacts extensively with the di-zinc, as was observed in the
case of AMPCP. Similarly, other polar interactions of the
ribose moiety and hydrophobic interactions of the adenine
base are also maintained (Figure 1D). From the overlaid
cocrystal structures of AMPCP and 2n, it can be seen that the
terminal phosphonates superimpose well but the ribose and
the adenine rings of 2n are shifted 1−1.5 Å relative to AMPCP
(Figure 1A,B). This shift of the adenine base toward the di-
zinc center indicates that when 2n is bound to CD73, the N-
and C-terminal domains of CD73 move closer together to
accommodate the shorter phosphate linker. Further study of
the X-ray cocrystal structure revealed a narrow hydrophobic
pocket around the C2 chlorine atom (Figure 1C). This
presented us with an opportunity to evaluate C2 modifications
to improve the binding affinity and boost the potency.
phosphonic acid units of AMPCP were investigated.6 Their
study found that substitution of the C6 amine with a benzyl
group led to analog PSB-12379 (Ki = 2.2 nM) with
substantially improved potency relative to AMPCP (Ki =
88.4 nM). While the results were encouraging, their reported
SAR was limited to the aforementioned modifications.
During the course of our early investigations, in a parallel
program also targeting CD73 using bisphosphonic acids, it was
discovered that introduction of a chlorine atom at the C2
position of PSB-12379 led to a 14-fold improvement in
potency (IC50 = 0.4 nM for 1, Scheme 2).7a−c Similar
beneficial effects of C2 chlorine substitution have been
reported subsequentially by Mu
̈
ller et al.10 Our early discovery
led us to focus our SAR on 1. In general, bisphosphonates are
poorly absorbed from the gastrointestinal tract; thus, they
suffer from poor oral bioavailability. To alleviate the potential
liability arising from bisphosphonic acid, we embarked on this
simultaneous SAR study by replacing the bisphosphonic acid
moiety of 1.
Our initial effort to replace the bisphosphonic acid moiety in
1 with malonic acid led to a significant drop in potency (1a,
IC50 = 1 μM), presumably due to the loss of interaction with
di-zinc (Table 1). When this malonic acid was replaced with
phosphonoacetic acid 1b, an improvement in potency was
observed (IC50 = 110 nM). However, the ability of the
phosphonic acid moiety to engage in the binding interaction
with di-zinc was realized to a greater extent when the steric
congestion around it was removed. Thus, replacing the
phosphonoacetic acid with a methylenephosphonic acid (1c)
led to adequate interaction with di-zinc and further improve-
ment in potency (IC50 = 58 nM). The position of the
phosphonic acid also played a key role in the observed
potency. For example, if the phosphonic acid moiety was
extended by an ethylene (1d) or a propylene (1e) linker,
complete loss of potency was observed. This SAR interrogation
Replacing the C2 chlorine with a phenyl group (3a) led to a
decrease in potency, presumably due to the conformational
Scheme 2. SAR Study of AMPCP-Based CD73 Inhibitors
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Table 1. Replacement of Bisphosphonic Acid Residue
Table 2. Modification of C6 Position
constraints induced by the biaryl system, which might not be
accommodated in the nucleotide binding site of the enzyme.
As expected, this effect was particularly severe for the o-tolyl
strain imposed by the biaryl rings could be relieved by a
methylene-ether bridge (3c and 3d). In the case of 3d, a boost
in potency was observed when the C2 chlorine was replaced
with a phenoxy methyl ether (IC50 = 13 nM). Unsurprisingly,
when the C2 chlorine was replaced with pyrrolidine-3-methyl
group (3b), which showed a > 130-fold loss in potency (IC50
=
3.5 μM for 3b vs IC50 = 26 nM for 2n). The conformational
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Figure 1. (A) Superposition of AMPCP (PDB: 4H2I) in gray with 2n (PDB: 6YE1) in yellow. (B) Lateral view of superposition of AMPCP and
2n. (C) X-ray structure of 2n bound to CD73, represented by a water-accessible surface in gray for selected residues within 10 Å of the ligand,
reveals a narrow hydrophobic pocket around the C2 chlorine. (D) Molecular interactions of 2n in the cocrystal structure with hCD73.
Table 3. Modification of C2 Position
Figure 2. (A) X-ray structure of 4a cocrystallized with hCD73 (PDB: 6YE2) showing molecular interactions. (B) Overlay of X-ray crystals of 2n
(PDB: 6YE1) in green and 4a in yellow.
ether, complete loss of activity was observed (3e, IC50 > 10
μM, Table 3) presumably because of the negative interaction
between the polar pyrrolidine and the hydrophobic pocket
around the C2 position. Taking into account the potency,
synthetic tractability, and physiochemical properties of the C2-
modified analogs, we concluded that the C2-chlorine
substituent (2n) was optimal.
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Next, we focused on the modification of the adenine base by
changing its electron distribution to modulate hydrophobic
interactions arising from the π−π stacking of Phe-500 and Phe-
417 (Figure 2D). When adenine base 2n (IC50 = 26 nM) was
replaced with pyrazolopyrimidine 4a, a 10-fold improvement in
potency (IC50 = 2.6 nM) was observed (Table 3). Similarly,
introduction of pyrazolopyridine (4b) led to a fivefold
improvement in potency (IC50 = 4.5 nM). However, replacing
the adenine base with imidazolopyridine (4c) did not improve
potency (IC50 = 33 nM) (Table 4).
Table 5. Potency and Selectivity of 4a
potency
selectivity
target
IC50 (nM)
target
CD39
A2aR
hNTPDase-2
hNTPDase-3
hNTPDase-8
IC50 (nM)
hCD73-CHO
hCD73 (soluble)
mCD73-CHO
mCD73 (soluble)
SKOV-3
2.6
0.86
13
3.0
0.55
>10 000
>10 000
>10 000
>10 000
>10 000
mCD73 (IC50 = 13 nM), and the human ovary cancer cell line
SKOV-3 (IC50 = 0.55 nM). It exhibits excellent selectivity
(IC50 > 10 000 nM) against CD39, A2aR, and NTPDase2, 3,
and 8. Furthermore, compound 4a was also found to be stable
when cultured with human, dog, rat, or mouse hepatocytes
(see the SI) and exhibited low clearance in rats (CL = 0.13 L/
h/kg).
Table 4. Modification of the Purine Base
During our earlier investigation of C6 substituents on the
adenine series (vide supra), we found that introduction of α-
(S)-methylbenzylamine at the C6 position (2d) led to
decreased clearance, albeit with some loss in potency
compared to the corresponding cyclopentylamine (2n)
(Table 6). Based on this observation, we decided to re-
evaluate C6 substitutions on the most potent analogs, 4a in the
pyrazolopyrimidine series and 4b in the pyrazolopyridine
series, to further decrease clearance in rats. The observed trend
in potency and clearance of the pyrazolopyrimidine series was
similar to that of the adenine series. When cyclopentylamine in
4a was replaced with α-(S)-methylbenzylamine (5a), it
resulted in an 11-fold loss in potency but a 39-fold decrease
in systemic clearance. To improve potency while maintaining
low clearance, fluoro-substituted α-(S)-methylbenzylamines
were explored. The loss in potency due to introduction of α-
(S)-methylbenzylamine was substantially reclaimed with 2-
fluoro- and 4-fluoro-α-(S)-methylbenzylamines 5b (IC50 = 7.6
nM) and 5d (IC50 = 12 nM), respectively. Contrary to this, 3-
fluoro-α-(S)-methylbenzylamine (5c) had a deleterious effect
on potency (IC50 = 39 nM). A similar trend in potencies was
also reflected in the pyrazolopyridine series when the C6
cyclopentylamine of 4b was replaced with the above-
mentioned fluoro-benzyl amines (5e−5h). The positive effect
of fluorine incorporation on rat pharmacokinetic (PK) was
demonstrated with 5d in the pyrazolopyrimidine series and
with 5h in the pyrozolopyridine series. Both 5d and 5h
exhibited remarkably low clearance and improved half-life (CL
= 0.020 and 0.018 L/h/kg; T1/2 = 4.5 and 4.0 h, respectively).
Chemistry. The bisphosphonic acid replacement analogs
(1a−e) were synthesized according to Scheme 3a. Introduc-
tion of the malonate and phosphonoacetate at the C5′
hydroxyl group of commercially available 2,6-dichloro-9-[2,3-
O-(1-methylethylidene)-β-D-ribofuranosyl]-9H-purine 6 was
carried out via rhodium catalysis using dimethyl 2-diazomal-
onate and ethyl 2-diazo-2-(diethoxyphosphoryl)acetate in
refluxing toluene to obtain 7 and 8, respectively.7d The
benzylamine was then introduced at the C6 position via a SNAr
reaction to obtain 9 and 10, respectively. Hydrolysis of the
ethyl phosphate and removal of the acetonide were carried out
with TMS-Br and a successive aqueous quench. Saponification
of dimethylmalonate 11 and phosphonoethylacetate 12 with
LiOH gave 1a and 1b, respectively. Monophosphonic acid
derivatives (1c−e) were synthesized using a four-step sequence
(steps e−h) shown in Scheme 3a. The first step in the
synthesis involves a SNAr reaction between commercially
It is worth noting that heterocyclic core modifications 4a
and 4b, which incorporate a nitrogen atom at the 8-position,
both led to a significant boost in potency. To further
understand how these modifications influence the protein-
binding interactions that improve potency, we obtained an X-
ray cocrystal of 4a with hCD73 (PDB: 6YE2). Figure 2A
shows key ligand interactions. In comparison to the adenine
base in 2n, the position of the pyrazolopyrimidine base in 4a
shifted 0.8 Å away from the N-terminal domain of CD73. This
shift can be visualized clearly from the crystal structure overlay
of 2n with 4a (Figure 2B). These two compounds differ only
in the position of the nitrogen atom at the 7- and 8-positions,
and the close alignment of the protein atoms of both CD73
domains indicates an almost identical relative domain
orientation. Repositioning of the nitrogen atom affects electron
distribution of the nucleobase, which presumably influences
the hydrophobic stacking interactions of 4a, resulting in the
observed shift. The shifting of the pyrazolopyrimidine away
from the phosphonate residue also indicates that the C2
chlorine is being extended deeper into the narrow hydrophobic
pocket compared to 2n. The potentially stronger interaction
established due to this conformational orientation might have
contributed toward the improvement in potency.
Compound 4a was characterized further against related
targets and evaluated for off-target selectivity (Table 5). It was
found to exhibit excellent potency against soluble hCD73
(IC50 = 0.86 nM) and soluble mouse CD73 (mCD73; IC50
=
3.0 nM), CHO cells overexpressing hCD73 (IC50 = 2.6 nM) or
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Table 6. Further Improvements in Rat PK
a
b
IC50 measured in hCD73-CHO cells. PK experiments were performed on male SD rats at IV dose of 0.25 or 0.5 mg/kg using 31.6%
dimethylacetamide (DMA) + 36.8% ethanol + 31.6% poly(ethylene glycol) (PEG) as the vehicle; N/D, not determined.
a
Scheme 3a. Replacement of Bisphosphonic Acid
a
Reagents and conditions: (a) Rh2(OAc)4, dimethyl 2-diazomalonate for 7 or ethyl 2-diazo-2-(diethoxyphosphoryl)acetate for 8, toluene, reflux,
yield = 60% for 7 and 64% for 8; (b) benzylamine, Et3N, EtOH, room temperature (rt), yield = quantitative; (c) TMS-Br, CH3CN, rt and then
H2O, yield = 38% for 11 and 23% for 12; (d) LiOH, tetrahydrofuran (THF)/H2O, rt, yield = 89% for 1a, 15% for 1b; (e) benzylamine, Et3N,
EtOH, rt and then 7 N NH3 in MeOH, yield = 55%; (f) 2,2-dimethoxypropane, p-TSA, acetone, rt; (g) diethyl p-toluenesulfonyloxy
methylphosphonate for 1c or diethyl 2-bromoethylphosphonate for 1d or diethyl 2-bromopropylphosphonate for 1e, NaH, DMF, 0 °C−rt; (h)
TMS-Br, CH3CN, rt and then H2O, yield = 20% for 1c, 6% for 1d, 23% for 1e (three steps).
available 2,6-dichloropurine riboside triacetate 13 and benzyl-
amine in ethanol in the presence of triethylamine. The crude
reaction mixture was then subjected to global acetate
deprotection using methanolic ammonia to obtain 14. Next,
the 2,3-syn-diol was protected as acetonide 15. At this stage,
alkyl phosphonates were introduced at the C5′ hydroxyl of the
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a
Scheme 3b. Synthesis of C2-Modified Analogs
a
Reagents and conditions: (a) two steps: (1) phenylboronic acid or tolylboronic acid, Pd(PPh3)4, Na3CO3, THF/H2O, reflux; (2) TMS-Br,
CH3CN, rt and then H2O, yields = 20% for 3a and 10% for 3b; (b) cinnamylboronic acid, Pd(PPh3)4, Na3CO3, THF/H2O, reflux; (c)
K2OsO4·2H2O, NaIO4, 2,6-lutidine, THF, H2O, rt; (d) NaBH(OAc)3, CH2Cl2, rt, yield = 60% (three steps); (e) SOCl2, pyridine, CH3CN, 0 °C,
yield = 40%; (f) two steps: (1) K2CO3, MeOH or phenol, rt−40 °C; (2) TMS-Br, CH3CN, rt and then H2O, yields = 13% for 3c and 37% for 3d;
(g) N-Boc-3-pyrrolidinol, NaH, DMF; (h) TMS-Br, CH3CN, rt and then H2O, yield = 15% (two steps).
ribose unit using diethyl p-toluenesulfonyloxy methylphosph-
onate, diethyl 2-bromoethylphosphonate, and diethyl 2-
bromopropylphosphonate in the presence of NaH in N,N-
dimethylformamide (DMF) to obtain phosphonates 16, 17,
and 18, respectively. As before, hydrolysis of ethylphospho-
nates and concomitant acetonide removal were achieved with
TBS-Br and a successive aqueous quench to yield 1c−e. The
above-mentioned four-step sequence (Scheme 3a, steps e−h)
was also used for the synthesis of all C6-modified analogs (2a−
2r) shown in Table 2. Similar synthesis of 5′-methylphosph-
onate nucleosides has been reported previously.13
Variation of the C2 position was carried out according to
Scheme 3b. Following Steps e−g in Scheme 3a, commercially
available 2,6-dichloropurine riboside triacetate 13 was
converted to 19, which was used for various C2 modifications.
Introduction of a phenyl or o-tolyl substituent (3a and 3b,
respectively) was achieved via Suzuki coupling of 19 with the
corresponding arylboronic acids followed by hydrolysis of the
ethylphosphonate using TMS-Br and a successive aqueous
quench. For the synthesis of the methylene-ether substituents,
intermediate 19 was first coupled with cinnamylboronic acid to
obtain 20. Oxidative cleavage of 20 furnished aldehyde 21,
which was further reduced to carbinol 22.14 The carbinol 22
was then treated with thionyl chloride to obtain 23, which was
used for the synthesis of various methylene-ether analogs.
Methoxymethyl ether 3c and phenoxymethylether 3d were
synthesized via SN2 displacement of the chlorine atom of 23 by
methanol and phenol, respectively, in the presence of
potassium carbonate followed by hydrolysis of the phospho-
nate ester. For the synthesis of compound 3e, commercially
available N-Boc-3-pyrrolidinol was used in the presence of
sodium hydride in N,N-dimethylformamide. Removal of the
Boc group and the hydrolysis of the ethylphosphonate were
carried out in one-pot using TMS-Br.
Synthesis of analogs with modifications to the central core
was carried out according to Scheme 3c. N-Glycosylation of
commercially available central cores pyrazolopyrmidine 25 and
pyrazolopyridine 26 with ribose tetraacetate were carried out
using bis(trimethylsilyl)amine (HMDS) and trimethylsilyl
trifluoromethanesulfonate (TMSOTf) to obtain 27 and 28,
respectively. Similar to the synthesis of 1c, cyclopentylamine
was introduced at the C6 position of pyrazolopyrimidine 27 at
room temperature in methanol in the presence of triethyl-
amine. Successive removal of the acetate groups with
methanolic ammonia gave compounds 29. Syn-diol of 29 was
then protected as acetonide 30. The methylphosphate moiety
was then introduced at C5′ hydroxyl to obtain 31. Finally,
global deprotection of 31 gave 4a. This four-step sequence
(steps b−e) was also used for the synthesis of analogs 5a−d.
An alternative approach was adopted for the synthesis of the
pyrazolopyridine-derived analog 4b, in which cyclopentyl-
amine was introduced at the C6 position at the end of the
sequence. The glycosylated product 28 was first converted to
acetonide 32, followed by introduction of the phosphate
residue at C5′ to obtain 33. Contrary to the pyrazolopyr-
imidine 27, installation of C6 amines on pyrazolopyridine 33
required elevated temperature (refluxing ethanol). As before,
hydrolysis of the ethylphosphonate and removal of acetonide
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a
Scheme 3c. Synthesis of Analogs with Modified Purine Base
a
Reagents and conditions: (a) HMDS, (NH4)2SO4, reflux and then β-D-ribofuranose 1,2,3,5-tetraacetate, TMSOTf, CH3CN, rt, yield = 25% for 27
and 32% for 28; (b) cyclopentylamine, Et3N, MeOH, rt and then 7 N NH3 in MeOH; (c) 2,2-dimethoxypropane, p-TSA, acetone, rt; (d)
NaHMDS, diethyl p-toluenesulfonyloxy methylphosphonate THF, 0 °C−rt; yield = 73% (three steps); (e) TMS-Br, CH3CN, rt and then MeOH/
HCl, yield = 93%; (f) two steps: (1) 7 N NH3 in MeOH, rt; (2) 2,2-dimethoxypropane, p-TSA, acetone, rt, yield = 80%; (g) diethyl p-
toluenesulfonyloxy methylphosphonate, LiHMDS, THF, −78 °C−rt, yield = 59%; (h) two steps: (1) cyclopentylamine, Et3N, EtOH, reflux; (2)
TMS-Br, CH3CN, rt and then H2O, yield = 48%; (i) BSA, CH3CN, reflux then β-D-ribofuranose 1,2,3,5-tetraacetate, TMSOTf, CH3CN, reflux; (j)
cyclopentylamine, dioxane, reflux, yield = 40%, two steps; (k) 2,2-dimethoxypropane, p-TSA, acetone, rt, yield = 78%; (l) NaHMDS, diethyl p-
toluenesulfonyloxy methylphosphonate THF, 0 °C−rt; yield = 62%; (m) TMS-Br, CH3CN, rt and then H2O, yield = 15%.
were carried out in one-pot using TMS-Br to obtain analogs
4b. Analogs 5e−h were synthesized in a similar fashion to 4b.
Glycosylation of imidazolopyridine 34 was achieved using
N,O-bis(trimethylsilyl)acetamide (BSA) and TMSOTf. Similar
to pyrazolopyridine core 33, installation of the C6 cyclo-
pentylamine on imidazolopyridine 35 was achieved at elevated
temperature using refluxing dioxane to obtain 36. Analogous to
the synthesis of 4a, compound 36 was then subjected to steps
k−m to furnish 4c.
CONCLUSIONS
■
A series of methylenephosphonic acid analogs were synthe-
sized in a systematic SAR study of CD73 inhibitors. Our
structure-based design relied on information from the X-ray
cocrystal structures of hCD73 with the known inhibitor
AMPCP as well as our early lead methylenephosphonic acid
2n. We have demonstrated that the bisphosphonic acid moiety
can be successfully replaced with a methylenephosphonic acid
on nucleoside-derived CD73 inhibitors. With additional
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for 4 h or until LCMS analysis showed complete cleavage of the
acetonide protecting group. The reaction mixture was evaporated and
purified by reverse-phase HPLC (C18 column, 0−30% gradient of
acetonitrile and water with 0.1% TFA) to give the product 11 as a
white solid in 38% yield (210 mg) over two steps: ESI MS [M + H]+
for C22H24ClN5O8, calcd 522.1, found 522.2.
Compound 11 (170 mg, 0.27 mmol) was dissolved in 4 mL of a
1:1 mixture of THF and H2O. To this mixture was added LiOH·H2O
(46 mg, 1.1 mmol) and was stirred overnight at rt and then
neutralized with AcOH. The solvent was evaporated from the reaction
mixture, and the crude material was purified by reverse-phase HPLC
(C18 column, 0−30% gradient of acetonitrile and water with 0.1%
TFA) to give the product 1a as a white solid in 89% yield (145 mg)
over two steps: 1H NMR (400 MHz, DMSO-d6) δ 8.92 (t, J = 6.2 Hz,
1H), 8.57 (s, 1H), 7.40−7.17 (m, 5H), 5.87 (d, J = 6.3 Hz, 1H), 4.65
(t, J = 5.5 Hz, 1H), 4.59 (d, J = 9.4 Hz, 2H), 4.17 (dd, J = 4.8, 2.7 Hz,
1H), 4.10 (q, J = 3.0 Hz, 1H), 3.76 (dd, J = 10.4, 3.1 Hz, 1H), 3.65
(dd, J = 10.6, 3.3 Hz, 1H). ESI MS [M + H]+ for C20H20ClN5O8,
calcd 494.1, found 494.1.
2-{[(2R,3S,4R,5R)-5-[6-(Benzylamino)-2-chloro-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}-2-phosphonoacetic Acid (1b).
Compound 1b was synthesized in a similar fashion to example 1a as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.93 (dt, J = 12.6, 6.4
Hz, 1H), 8.61 (d, J = 7.2 Hz, 1H), 7.43−7.17 (m, 5H), 5.86 (d, J =
6.5 Hz, 1H), 4.74−4.53 (m, 2H), 4.19 (dd, J = 18.5, 4.2 Hz, 2H), 4.08
(q, J = 3.2 Hz, 1H), 3.85−3.60 (m, 2H). ESI MS [M + H]+ for
C19H21ClN5O9P, calcd 530.1, found 530.1.
({[(2R,3S,4R,5R)-5-[6-(Benzylamino)-2-chloro-9H-purin-9-yl]-3,4-
dihydroxyoxolan-2-yl]methoxy}methyl) Phosphonic Acid (1c). A
mixture of 2,6-dichloro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine
6 (29 g, 65 mmol), benzylamine (28.4 mL, 260 mmol), and
triethylamine (9 mL, 165 mmol) in EtOH (200 mL) was stirred at rt
for overnight. Then, 7 M NH3 in MeOH (50 mL) was added to the
above reaction and was stirred at rt for 24 h. The solvent was
evaporated, and the mixture was reconstituted in 400 mL of EtOAc
and 300 mL of H2O. Layers were separated, and the aqueous layer
was extracted with EtOAc (2 × 300 mL). The combined organic layer
was dried over Na2SO4, concentrated, and purified by silica gel
chromatography give 14 as a white solid in 55% yield (14 g). ESI MS
[M + H]+ for C17H19ClN5O4, calcd 392.1, found 392.1.
Compound 14 (5.6 g, 14.3 mmol) was dissolved in acetone (100
mL) and 2,2-dimethoxypropane (30 mL). To this mixture was added
p-TsOH·H2O (3.4 g, 17.9 mmol). The reaction mixture was stirred at
rt overnight, diluted with brine (100 mL), and carefully quenched
with saturated NaHCO3 (200 mL). After extraction with EtOAc (2 ×
200 mL), combined organics were dried over MgSO4 and evaporated
to give the crude product 15 (6.1 g), which was used in the next step
without purification. ESI MS [M + H]+ for C20H22ClN5O4, calcd
432.1, found 432.1.
A sample of the crude product 15 (431.5 mg, 1 mmol) was
dissolved in anhydrous DMF (5 mL) and cooled to 0 °C; then, 60%
NaH (60 mg, 1.5 mmol) was added and the reaction mixture was
stirred at 0 °C for 1 h. Diethyl p-toluenesulfonyloxy methylphosph-
onate (386 mg, 1.2 mmol, 1.2 equiv) was added, and the reaction was
slowly warmed up to rt and stirred overnight. H2O (20 mL) was
added to dilute the reaction mixture and extracted with MTBE (2 ×
10 mL). The combined organics were dried over MgSO4, filtered, and
evaporated to give crude product 16, which was used in the next step
without purification. ESI MS [M + H]+ for C25H33ClN5O7P, calcd
582.2, found 582.1.
All of the crude product 16 was dissolved in anhydrous CH3CN (5
mL), TMS-Br (0.5 mL) was added, and the reaction was stirred at rt
overnight. It was quenched with H2O (1 mL) and stirred at rt for 4 h
or until LCMS analysis showed complete cleavage of the acetonide
protecting group. The reaction mixture was evaporated and purified
by reverse-phase HPLC (C18 column, 0−30% gradient of acetonitrile
and water with 0.1% TFA) to give the product 1c as a white solid in
20% yield (123 mg) over three steps: %: 1H NMR (400 MHz,
DMSO-d6) δ 8.93 (t, J = 6.2 Hz, 1H), 8.44 (s, 1H), 7.37−7.22 (m,
5H), 5.85 (d, J = 6.0 Hz, 1H), 4.68−4.61 (m, 2H), 4.54 (t, J = 5.5 Hz,
modifications of the adenine base, improved potency and
selectivity can be achieved. Our structure-guided approach led
to systematic investigation of the C2 position to take advantage
of a hydrophobic pocket and nucleobase modification to
influence π−π stacking to boost potency. Similarly, we
demonstrated that introduction of 4-fluoro-α-(S)-methylben-
zylamine at C6 led to a remarkable improvement in rat PK
properties. In combination with the results from our additional
SAR studies on CD73 inhibitors in a related program,7c
currently in human clinical trials, this study describes progress
toward understanding the molecular requirements for nucleo-
tide-like small-molecule CD73 inhibitors and the discovery of
several promising candidates against this promising therapeutic
target.
EXPERIMENTAL SECTION
■
General Chemistry. All reactions were performed using a Teflon-
coated magnetic stir bar at the indicated temperature and were
conducted under an inert atmosphere when stated. All chemicals were
used as received. Reactions were monitored by TLC (silica gel 60
with fluorescence F254, visualized with a short-wave/long-wave UV
lamp) and/or liquid chromatography−mass spectrometry (LCMS,
Agilent 1100 series LCMS with UV detection at 254 nm using a
binary solvent system [0.1% trifluoroacetic acid (TFA) in MeCN/
0.1% TFA in H2O] using either of the following columns: Agilent
Eclipse Plus C18 [3.5 μm, 4.6 mm i.d. × 100 mm] or Aeris Widepore
C4 [3.6 μm, 2.1 mm i.d. × 50 mm]). Flash chromatography was
conducted on silica gel using an automated system (CombiFlash RF+
manufactured by Teledyne ISCO), with detection wavelengths of 254
and 280 nm. Reverse-phase preparative high-performance liquid
chromatography (HPLC) was conducted on an Agilent 1260 Infinity
series HPLC. Samples were eluted using a binary solvent system
(0.1% TFA in MeCN/0.1% TFA in H2O) with gradient elution on a
Gemini C18 110 Å column (21.2 mm i.d. × 250 mm) with detection
at 254 nm. Final compounds obtained through preparative HPLC
were concentrated through lyophilization. All reported yields are
isolated yields. All assayed compounds were purified to ≥95% purity
as determined by LCMS (Agilent 1100 series LCMS with UV
detection at 254 nm using a binary solvent system [0.1% TFA in
MeCN/0.1% TFA in H2O] using either of the following columns:
Agilent Eclipse Plus C18 column [3.5 μm, 4.6 mm i.d. × 100 mm] or
1
Aeris Widepore C4 column [3.6 μm, 2.1 mm i.d. × 50 mm]). H
NMR spectra were recorded on a Varian 400 MHz NMR
spectrometer equipped with an Oxford AS400 magnet. Chemical
shifts (δ) are reported as parts per million (ppm) relative to residual
undeuterated solvent as an internal reference. The abbreviations s, br.
s, d, t, q, dd, dt, ddd, and m stand for singlet, broad singlet, doublet,
triplet, quartet, doublet of doublets, doublet of triplets, doublet of
doublet of doublets, and multiplet, respectively.
2-{[(2R,3S,4R,5R)-5-[6-(Benzylamino)-2-chloro-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}propanedioic Acid (1a). A
mixture of 2,6-dichloro-9-[2,3-O-(1-methylethylidene)-β-D-ribofura-
nosyl]-9H-purine 6 (1.05 g, 2.9 mmol), dimethyl 2-diazomalonate
(648 mg, 4.1 mmol), and Rh2(OAc)4 (128 mg, 0.29 mmol) in toluene
(50 mL) was stirred at 100 °C overnight. After cooling to rt, the
solvent was evaporated from the reaction mixture and the crude
material was purified by column chromatography to obtain 7 as a
yellow oil in 60% yield (850 mg). ESI MS [M + H]+ for
C18H20Cl2N4O0, calcd 491.1, found 490.8.
A mixture of 7 (420 mg, 0.86 mmol), benzylamine (98 μL, 0.9
mmol), and triethylamine (125 μL, 0.9 mmol) in EtOH (5 mL) was
stirred at rt overnight. The solvent was evaporated from the reaction
mixture, and the crude product 9 was used in the next step without
purification. ESI MS [M + H]+ for C25H29ClN5O8, calcd 562.2, found
562.2.
The crude product 9 was dissolved in anhydrous CH3CN (10 mL).
TMS-Br (1.0 mL) was added, and the reaction was stirred at rt
overnight. Then, it was quenched with H2O (2 mL) and stirred at rt
853
https://dx.doi.org/10.1021/acs.jmedchem.0c01835
J. Med. Chem. 2021, 64, 845−860

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1H), 4.15−4.09 (m, 1H), 4.05 (q, J = 3.8 Hz, 1H), 3.77−3.65 (m,
2H), 3.61 (d, J = 8.9 Hz, 2H). ESI MS [M + H]+ for C18H22ClN5O7P,
calcd 486.1, found 486.2.
8.9 Hz, 2H). ESI MS [M + H]+ for C18H21Cl2N5O7P, calcd 520.1,
found 520.1.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(2-chlorophenyl)methyl]-
(methyl)amino}-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]-
methoxy}methyl)phosphonic Acid (2g). Compound 2g was
(2-{[(2R,3S,4R,5R)-5-[6-(Benzylamino)-2-chloro-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}ethyl)phosphonic Acid (1d).
Compound 1d was synthesized in a similar fashion to example 1c
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 29.8
Hz, 1H), 7.31 (m, 6H), 5.87 (d, J = 5.8 Hz, 1H), 5.71−5.41 (m, 1H),
4.97 (d, J = 12.7 Hz, 1H), 4.65−3.25 (m, 7H), 2.13−1.84 (m, 2H).
ESI MS [M + H]+ for C19H23ClN5O7P, calcd 500.1, found 500.1.
(3-{[(2R,3S,4R,5R)-5-[6-(Benzylamino)-2-chloro-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}propyl)phophonic Acid (1e).
Compound 1e was synthesized in a similar fashion to example 1c
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 32.3
Hz, 1H), 7.34−7.05 (m, 5H), 5.80 (d, J = 6.1 Hz, 1H), 5.48 (q, J =
16.6 Hz, 1H), 5.02−4.71 (m, 1H), 4.55−4.36 (m, 1H), 4.18−3.93
(m, 2H), 3.88 (q, J = 3.8 Hz, 1H), 3.70−3.22 (m, 3H), 1.88−1.39 (m,
4H). ESI MS [M + H]+ for C20H25ClN5O7P, calcd 514.1, found
514.1.
({[(2R,3S,4R,5R)-5-{2-Chloro-6-[(2-phenylethyl)amino]-9H-purin-
9-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid
(2a). Compound 2a was synthesized in a similar fashion to example
1c as a white solid: 1H NMR (400 MHz, DMSO-d6) δ 8.50−8.33 (m,
2H), 7.33−7.17 (m, 5H), 5.84 (d, J = 6.1 Hz, 1H), 4.54 (t, J = 5.5 Hz,
1H), 4.12 (dd, J = 4.9, 3.2 Hz, 1H), 4.05 (q, J = 3.7 Hz, 1H), 3.79−
3.57 (m, 6H), 2.92 (t, J = 7.4 Hz, 2H). ESI MS [M + H]+ for
C19H24ClN5O7P, calcd 500.1, found 500.2.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[2-(2-methoxyphenyl)ethyl]-
amino}-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]metoxy}methyl)-
phosphonic Acid (2b). Compound 2b was synthesized in a similar
fashion to example 1c as a white solid: 1H NMR (400 MHz, DMSO-
d6) δ 8.46−8.33 (m, 2H), 7.22−7.11 (m, 2H), 6.96 (d, J = 8.2 Hz,
1H), 6.84 (t, J = 7.4 Hz, 1H), 5.84 (d, J = 6.1 Hz, 1H), 4.57−4.51 (m,
1H), 4.12 (dd, J = 4.9, 3.2 Hz, 1H), 4.05 (q, J = 3.7 Hz, 1H), 3.82−
3.56 (m, 9H), 2.89 (t, J = 7.3 Hz, 2H). ESI MS [M + H]+ for
C20H26ClN5O8P, calcd 530.1, found 530.2.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[2-(2-fluorophenyl)ethyl]amino}-
9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (2c). Compound 2c was synthesized in a similar
fashion to example 1c as a white solid: 1H NMR (400 MHz, DMSO-
d6) δ 8.54−8.39 (m, 2H), 7.33−7.07 (m, 5H), 5.84 (d, J = 6.1 Hz,
1H), 4.57−4.52 (m, 1H), 4.12 (dd, J = 4.9, 3.2 Hz, 1H), 4.05 (q, J =
3.8 Hz, 1H), 3.77−3.57 (m, 6H), 2.96 (t, J = 7.2 Hz, 2H). ESI MS [M
+ H]+ for C19H23ClFN5O7P, calcd 518.1, found 518.2.
1
synthesized in a similar fashion to example 1c as a white solid: H
NMR (400 MHz, DMSO-d6) δ 8.56−8.33 (m, 1H), 7.54−7.48 (m,
1H), 7.36−7.27 (m, 2H), 7.23−7.08 (m, 1H), 5.88 (d, J = 6.1 Hz,
1H), 5.63 (s, 1H), 5.00 (s, 1H), 4.54 (s, 1H), 4.15−4.01 (m, 2H),
3.78−3.55 (m, 5H), 3.17 (s, 2H). ESI MS [M + H]+ for
C19H23Cl2N5O7P, calcd 534.1, found 534.1
({[(2R,3S,4R,5R)-5-{2-Chloro-6-[(pyridin-2-ylmethyl)amino]-9H-
purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic
Acid (2h). Compound 2h was synthesized in a similar fashion to
1
example 1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 9.05
(t, J = 6.2 Hz, 1H), 8.82−8.78 (m, 1H), 8.72−8.68 (m, 1H), 8.49 (s,
1H), 8.26 (d, J = 8.1 Hz, 1H), 7.83−7.76 (m, 1H), 5.86 (d, J = 6.1
Hz, 1H), 4.78 (d, J = 6.0 Hz, 2H), 4.54 (dd, J = 6.1, 4.9 Hz, 1H), 4.12
(dd, J = 4.9, 3.2 Hz, 1H), 4.05 (q, J = 3.7 Hz, 1H), 3.77−3.66 (m,
2H), 3.61 (dd, J = 8.9, 1.5 Hz, 2H). ESI MS [M + H]+ for
C17H21ClN6O7P, calcd 487.1, found 487.1.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(1-methyl-1H-pyrazol-4-yl)-
methyl]amino}-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}-
methyl)phosphonic Acid (2i). Compound 2i was synthesized in a
1
similar fashion to example 1c as a white solid: H NMR (400 MHz,
DMSO-d6) δ 8.72 (t, J = 6.1 Hz, 1H), 8.43 (s, 1H), 7.58 (s, 1H), 7.38
(s, 1H), 5.84 (d, J = 6.0 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 4.48−4.43
(m, 2H), 4.12 (dd, J = 4.9, 3.3 Hz, 1H), 4.05 (q, J = 3.7 Hz, 1H), 3.76
(s, 3H), 3.75−3.65 (m, 2H), 3.62 (d, J = 8.9 Hz, 2H). ESI MS [M +
H]+ for C16H22ClN7O7P, calcd 490.1, found 490.2.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(4-methyl-4H-1,2,4-triazol-3-yl)-
methyl]amino}-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}-
methyl)phosphonic Acid (2j). Compound 2j was synthesized in a
1
similar fashion to example 1c as a white solid: H NMR (400 MHz,
DMSO-d6) δ 9.12−8.99 (m, 2H), 8.51 (s, 1H), 5.86 (d, J = 6.1 Hz,
1H), 4.87 (s, 2H), 4.54 (t, J = 5.5 Hz, 1H), 4.12 (dd, J = 4.9, 3.2 Hz,
1H), 4.06 (q, J = 3.7 Hz, 1H), 3.86 (s, 3H), 3.77−3.65 (m, 2H), 3.61
(dd, J = 8.9, 1.6 Hz, 2H). ESI MS [M + H]+ for C15H21ClN8O7P,
calcd 491.1, found 491.2.
({[(2R,3S,4R,5R)-5-{2-Chloro-6-[(propan-2-yl)amino]-9H-purin-9-
yl}-3,4-dihydroxyoxolan-2-yl]methoxy}methyl) Phosphonic Acid
(2k). Compound 2k was synthesized in a similar fashion to example
1
1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H),
8.21−8.09 (m, 1H), 5.79 (d, J = 7.1 Hz, 1H), 4.23 (dd, J = 4.8, 1.9
Hz, 1H), 4.02 (s, 1H), 3.66−3.50 (m, 3H), 3.42−3.19 (m, 3H), 1.21
(dd, J = 6.5, 2.1 Hz, 6H). ESI MS [M + H]+ for C14H22ClN5O7P,
calcd 438.1, found 438.1.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(1S)-1-phenylethyl]amino}-9H-
purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic
Acid (2d). Compound 2d was synthesized in a similar fashion to
1
example 1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 8.89
({[(2R,3S,4R,5R)-5-[2-Chloro-6-(cyclopropylamino)-9H-purin-9-
yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid
(2l). Compound 2l was synthesized in a similar fashion to example
(d, J = 8.4 Hz, 1H), 8.45 (s, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.31 (t, J =
7.6 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 5.84 (d, J = 6.0 Hz, 1H), 5.41
(p, J = 7.6 Hz, 1H), 4.51 (t, J = 5.4 Hz, 1H), 4.15−4.08 (m, 1H),
4.07−4.01 (m, 1H), 3.78−3.65 (m, 2H), 3.61 (d, J = 9.0 Hz, 2H),
1.54 (d, J = 7.0 Hz, 3H). ESI MS [M + H]+ for C19H24ClN5O7P, calcd
500.1, found 500.2.
1
1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H),
8.42 (s, 1H), 5.85 (d, J = 6.0 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 4.12
(dd, J = 4.9, 3.2 Hz, 1H), 4.05 (q, J = 3.7 Hz, 1H), 3.77−3.65 (m,
2H), 3.61 (dd, J = 8.9, 1.4 Hz, 2H), 2.97 (s, 1H), 0.84−0.56 (m, 4H).
ESI MS [M + H]+ for C14H20ClN5O7P, calcd 436.1, found 436.1.
({[(2R,3S,4R,5R)-5-[2-Chloro-6-(cyclobutylamino)-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid (2m).
Compound 2m was synthesized in a similar fashion to example 1c as a
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(1R)-1-phenylethyl]amino}-9H-
purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic
Acid (2e). Compound 2e was synthesized in a similar fashion to
1
example 1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 8.89
1
white solid: H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 7.7 Hz,
(d, J = 8.4 Hz, 1H), 8.46 (s, 1H), 7.44 (d, J = 7.4 Hz, 2H), 7.31 (t, J =
7.6 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 5.84 (d, J = 6.1 Hz, 1H), 5.41
(p, J = 7.6 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 4.15−4.07 (m, 1H),
4.07−4.01 (m, 1H), 3.77−3.65 (m, 2H), 3.61 (dd, J = 8.9, 1.8 Hz,
2H), 1.54 (d, J = 6.9 Hz, 3H). ESI MS [M + H]+ for C19H24ClN5O7P,
calcd 500.1, found 500.2.
1H), 8.43 (s, 1H), 5.84 (d, J = 5.9 Hz, 1H), 4.61 (q, J = 8.1 Hz, 1H),
4.56−4.50 (m, 1H), 4.15−4.09 (m, 1H), 4.05 (q, J = 3.7 Hz, 1H),
3.78−3.65 (m, 2H), 3.61 (d, J = 8.8 Hz, 2H), 2.35−2.00 (m, 4H),
1.74−1.61 (m, 2H). ESI MS [M + H]+ for C15H22ClN5O7P, calcd
450.1, found 450.1.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(2-chlorophenyl)methyl]amino}-
9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (2f). Compound 2f was synthesized in a similar
fashion to example 1c as a white solid: 1H NMR (400 MHz, DMSO-
d6) δ 8.97−8.90 (m, 1H), 8.49 (s, 1H), 7.50−7.43 (m, 1H), 7.33−
7.25 (m, 3H), 5.87 (d, J = 6.0 Hz, 1H), 4.75−4.67 (m, 2H), 4.56 (t, J
= 5.5 Hz, 1H), 4.16−4.02 (m, 2H), 3.78−3.65 (m, 2H), 3.62 (d, J =
({[(2R,3S,4R,5R)-5-[2-Chloro-6-(cyclopentylamino)-9H-purin-9-
yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid
(2n). Compound 2n was synthesized in a similar fashion to example
1
1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H),
8.39−8.26 (m, 1H), 5.84 (d, J = 5.9 Hz, 1H), 4.53 (t, J = 5.5 Hz, 1H),
4.48−4.35 (m, 1H), 4.12 (dd, J = 4.9, 3.3 Hz, 1H), 4.05 (q, J = 3.8
Hz, 1H), 3.79−3.65 (m, 2H), 3.62 (d, J = 8.9 Hz, 2H), 2.05−1.85 (m,
854
https://dx.doi.org/10.1021/acs.jmedchem.0c01835
J. Med. Chem. 2021, 64, 845−860

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Article
2H), 1.78−1.44 (m, 6H). ESI MS [M + H]+ for C16H24ClN5O7P,
calcd 464.1, found 464.2.
obtain the crude product. ESI MS [M + H]+ for C29H40N5O7P, calcd
602.3, found 602.4.
({[(2R,3S,4R,5R)-5-{2-Chloro-6-[(cyclopentylmethyl)amino]-9H-
purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic
Acid (2o). Compound 2o was synthesized in a similar fashion to
The crude product obtained above was dissolved in anhydrous
CH3CN (2.5 mL), TMS-Br (0.25 mL) was added, and the reaction
was stirred at rt overnight. It was quenched with H2O (0.25 mL) and
stirred at rt for 4 h or until LCMS analysis showed complete cleavage
of the acetonide protecting group. The reaction mixture was
evaporated and purified by reverse-phase HPLC (C18 column, 0−
30% gradient of acetonitrile and water with 0.1% TFA) to give the
1
example 1c as a white solid: H NMR (400 MHz, DMSO-d6) δ 8.45
(t, J = 2.4 Hz, 1H), 8.41 (s, 1H), 5.84 (d, J = 6.0 Hz, 1H), 4.61−4.47
(m, 1H), 4.12 (t, J = 4.1 Hz, 1H), 4.04 (d, J = 3.7 Hz, 1H), 3.85−3.51
(m, 5H), 3.36 (m, 1H), 2.38−2.13 (m, 1H), 1.77−1.39 (m, 6H),
1.34−1.12 (m, 2H). ESI MS [M + H] for C17H25ClN5O7P, calcd
478.1, found 478.2.
({[(2R,3S,4R,5R)-5-[2-Chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid (2p).
Compound 2p was synthesized in a similar fashion to example 1c as a
white solid: 1H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 5.86 (d,
J = 6.1 Hz, 1H), 4.53 (dd, J = 6.2, 4.9 Hz, 1H), 4.11 (dd, J = 4.9, 3.2
Hz, 1H), 4.09−4.00 (m, 3H), 3.76−3.65 (m, 2H), 3.65−3.56 (m,
4H), 2.05−1.86 (m, 4H). ESI MS [M + H]+ for C15H22ClN5O7P,
calcd 450.1, found 450.2.
({[(2R,3S,4R,5R)-5-(2-Chloro-6-{[(3R)-oxolan-3-yl]amino}-9H-
purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic
Acid (2q). Compound 2q was synthesized in a similar fashion to
example 1c as a white solid: 1H NMR (400 MHz, DMSO-d6) δ 8.63−
8.51 (m, 1H), 8.45 (s, 1H), 5.86 (d, J = 6.0 Hz, 1H), 4.63 (s, 1H),
4.57−4.51 (m, 1H), 4.12 (dd, J = 4.9, 3.3 Hz, 1H), 4.05 (q, J = 3.7
Hz, 1H), 3.96−3.83 (m, 2H), 3.79−3.66 (m, 3H), 3.62 (d, J = 8.9 Hz,
3H), 2.29−1.83 (m, 2H). ESI MS [M + H]+ for C15H22ClN5O8P,
calcd 466.1, found 466.1.
1
product 3a as a white solid in 20% yield (46 mg). H NMR (400
MHz, DMSO-d6) δ 8.44−8.37 (m, 3H), 7.55−7.41 (m, 4H), 6.03 (d,
J = 6.0 Hz, 1H), 4.73 (t, J = 5.6 Hz, 1H), 4.27−4.17 (m, 1H), 4.08
(app. q, J = 4.0 Hz, 1H), 3.89−3.74 (m, 1H), 3.70 (dd, J = 10.6, 4.8
Hz, 1H), 3.61 (d, J = 8.8 Hz, 2H), 3.52 (dd, J = 8.5, 3.6 Hz, 1H),
2.10−1.98 (m, 3H), 1.80−1.54 (m, 5H). ESI MS [M − H]− for
C22H28N5O7P, calcd 504.2, found 504.3.
({[(2R,3S,4R,5R)-5-[6-(Cyclopentylamino)-2-(2-methyl phenyl)-
9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (3b). Compound 3b was synthesized in a similar
fashion to example 3a as a white solid: 1H NMR (400 MHz, DMSO-
d6) δ 8.49 (s, 1H), 7.55−7.09 (m, 4H), 5.97 (d, J = 5.8 Hz, 1H), 4.64
(t, J = 5.4 Hz, 1H), 4.32−4.08 (m, 1H), 4.05 (app. q, J = 3.9 Hz, 1H),
3.75 (dd, J = 10.7, 3.6 Hz, 1H), 3.68 (dd, J = 10.7, 4.6 Hz, 1H), 3.60
(dd, J = 8.9, 1.9 Hz, 2H), 1.97 (br. s, 3H), 1.80−1.45 (m, 8H). ESI
MS [M − H]− for C23H30N5O7P, calcd 518.2, found 518.2.
({[(2R,3S,4R,5R)-5-[6-(Cyclopentylamino)-2-(methoxymethyl)-
9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (3c). In a round-bottom flask, compound 19 (1.5 g,
2.68 mmol), phenylvinylboronic acid (595 mg, 4.02 mmol), and
sodium carbonate (845 mg, 8.04 mmol) were suspended in 3:1 THF/
H2O (15 mL). This mixture was degassed by N2 sparge for 10 min.
Subsequently, Pd(PPh3)4 (310 mg, 0.27 mmol) was added, and the
resulting mixture was degassed for an additional 5 min and then
heated to reflux overnight. After cooling to rt, the reaction was diluted
with EtOAc and washed with water and brine. The organics were
dried over MgSO4, filtered, and concentrated under reduced pressure
to obtain the crude product 20 (1.89 g), which was used directly in
the following step without purification. ESI MS [M + H]+ for
C31H42N5O7P, calcd 628.3, found 628.4.
({[(2R,3S,4R,5R)-5-[2-Chloro-6-(morpholin-4-yl)-9H-purin-9-yl]-
3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid (2r).
Compound 2r was synthesized in a similar fashion to example 1c as
1
a white solid: H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 5.82
(d, J = 7.5 Hz, 1H), 5.14 (dd, J = 7.5, 4.7 Hz, 1H), 4.27−4.21 (m,
1H), 4.05−4.00 (m, 1H), 3.78−3.66 (m, 4H), 3.61−3.48 (m, 2H),
3.35 (dd, J = 12.1, 7.6 Hz, 1H), 3.27−3.15 (m, 1H). ESI MS [M +
H]+ for C15H22ClN5O8P, calcd 466.1, found 466.1.
({[(2R,3S,4R,5R)-5-[6-(Cyclopentylamino)-2-phenyl-9H-purin-9-
yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)phosphonic Acid
(3a).
A mixture of 2,6-dichloro-9-(2,3,5-tri-O-acetyl-β-D-
ribofuranosyl)purine 13 (13.5 g, 30 mmol), cyclopentylamine (3.2
mL, 33 mmol), and triethylamine (4.6 mL, 33 mmol) in MeOH (60
mL) was stirred at rt overnight. Then, 7 M NH3 in MeOH (20 mL)
was added to the above reaction and stirred at rt for 1 day. The
reaction mixture was evaporated, the crude product was redissolved in
acetone (100 mL), and 2,2-dimethoxypropane (40 mL) and p-TsOH·
H2O (7.1 g, 37.5 mmol) were added. The reaction mixture was stirred
at rt overnight, diluted with brine (100 mL), and carefully quenched
with saturated NaHCO3 (200 mL). After extraction with EtOAc (2 ×
200 mL), combined organics were dried over MgSO4, filtered, and
evaporated to give crude acetonide (12.2 g), which was used in the
next step without purification. A sample of the crude product (410
mg, 1 mmol) was dissolved in anhydrous DMF (5 mL) and cooled to
0 °C; then, 60% NaH (60 mg, 1.5 mmol) was added and the reaction
mixture was stirred at 0 °C for 1 h. Diethyl p-toluenesulfonyloxy
methylphosphonate (386 mg, 1.2 mmol, 1.2 equiv) was added, and
the reaction was slowly warmed up to rt and stirred overnight. It was
diluted with H2O (20 mL) and extracted with MTBE (2 × 10 mL);
combined organics were dried over MgSO4, filtered, and evaporated
to give the crude product 19, which was used in the next step without
purification. ESI MS [M + H]+ for C23H36ClN5O7P, calcd 560.2,
found 560.1.
In a glass vial, compound 19 (250 mg, 0.45 mmol), phenylboronic
acid (82 mg, 0.67 mmol), and sodium carbonate (142 mg, 3.75
mmol) were suspended in 3:1 THF/H2O (3 mL). This mixture was
degassed by N2 sparge for 10 min. Subsequently, Pd(PPh3)4 (52 mg,
0.045 mmol) was added, and the resulting mixture was degassed for
an additional 5 min and then sealed and heated to 80 °C overnight.
After cooling to room temperature, the reaction was diluted with
EtOAc and washed with water and brine. The organics were dried
over MgSO4, filtered, and concentrated under reduced pressure to
To a suspension of the crude product 20 (1.62 g), sodium
periodate (3.31 g, 15.48 mmol), and 2,6-lutidine (601 μL, 5.16
mmol) in 2:1 THF/H2O (28 mL) was added potassium osmate
dihydrate (24 mg, 0.065 mmol). The resulting thick suspension was
stirred overnight at room temperature and then partitioned between
EtOAc and water. The organics were washed sequentially with water
and brine, dried over MgSO4, and concentrated under reduced
pressure to obtain the crude product 7, which was used directly in the
following step without purification. ESI MS [M + H]+ for
C24H36N5O8P, calcd 554.2, found 554.3.
To a solution of the crude compound 21 (471 mg) in
dichloroethane (5.7 mL) was added sodium triacetoxyborohydride
(216 mg, 1.02 mmol) in a single portion. The reaction was stirred at
room temperature overnight and then partitioned between EtOAc
and water. The organics were washed with brine, dried over MgSO4,
and concentrated under reduced pressure. The title compound 22
(285 mg, 60% for three steps) was obtained following column
chromatography (SiO2, 0−15% gradient of MeOH and CH2Cl2). ESI
MS [M + H]+ for C24H38N5O8P, calcd 556.3, found 556.3.
To a solution of compound 22 (285 mg, 0.51 mmol) in acetonitrile
(5 mL) at 0 °C were added pyridine (125 μL, 1.54 mmol) followed
by thionyl chloride (56 μL, 0.77 mmol). The reaction was kept at 0
°C for 1 h. Subsequently, saturated NaHCO3 (aq) was added slowly
until gas evolution ceased. The reaction was diluted with EtOAc and
washed with sat. NaHCO3, water, and brine. The organics were dried
over MgSO4 and concentrated under reduced pressure. The title
compound 23 (115 mg, 40%) was obtained following column
chromatography (SiO2, 0−10% gradient of MeOH and CH2Cl2). ESI
MS [M + H]+ for C24H37ClN5O7P, calcd 574.2, found 574.3.
To a flask charged with compound 23 (115 mg, 0.20 mmol) in
methanol (5 ml) was added potassium carbonate (138 mg, 1.0
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
mmol). The resulting suspension was stirred overnight at room
temperature, diluted with EtOAc, and washed with water and brine.
The organics were dried over MgSO4 and concentrated under
reduced pressure. The crude mixture (35 mg) so obtained comprised
the desired product as well as an inconsequential mixture of methyl-
phosphonates resulting from transesterification. ESI MS [M + H]+ for
C25H40N5O8P, calcd 570.3, found 570.3.
The crude product obtained above (35 mg) was dissolved in
anhydrous CH3CN (2.5 mL), TMS-Br (0.25 mL) was added, and the
reaction was stirred at rt for 1.5 h. It was quenched with H2O (0.25
mL) and stirred at rt for 1 h or until LCMS analysis showed complete
cleavage of the acetonide protecting group. The reaction mixture was
evaporated and purified by reverse-phase HPLC (C18 column, 0−
30% gradient of acetonitrile and water with 0.1% TFA) to give the
product 3c as a white solid in 13% yield (12 mg), two steps: 1H NMR
(400 MHz, DMSO-d6) δ 8.48 (br. s, 1H), 5.94 (d, J = 6.2 Hz, 1H),
4.61 (t, J = 5.6 Hz, 1H), 4.42 (s, 3H), 4.15 (dd, J = 4.9, 3.1 Hz, 1H),
4.05 (t, J = 3.8 Hz, 1H), 3.82−3.65 (m, 2H), 3.61 (d, J = 8.9 Hz, 2H),
3.39 (d, J = 1.3 Hz, 3H), 1.97 (br. s, 2H), 1.82−1.65 (m, 2H), 1.58
(br. s, 4H). ESI MS [M − H]− for C18H28N5O8P, calcd 472.2, found
472.3.
({[(2R,3S,4R,5R)-5-[6-(Cyclopentylamino)-2-(phenoxy methyl)-
9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (3d). Compound 3d was synthesized in a similar
fashion to example 3c as a white solid: 1H NMR (400 MHz, DMSO-
d6) δ 8.33 (s, 1H), 7.30−7.12 (m, 3H), 6.93 (d, J = 7.8 Hz, 2H), 6.84
(t, J = 7.3 Hz, 1H), 5.84 (d, J = 5.9 Hz, 1H), 4.99 (s, 3H), 4.53 (dd, J
= 6.1, 5.0 Hz, 1H), 4.07 (dd, J = 5.0, 3.4 Hz, 1H), 3.95 (q, J = 3.9 Hz,
1H), 3.65 (dd, J = 10.7, 3.7 Hz, 1H), 3.59−3.49 (m, 3H), 1.81 (s,
3H), 1.63 (br. s, 3H), 1.46 (br. s, 5H). ESI MS [M + H]+ for
C23H30N5O8P, calcd 536.2, found 536.3.
({[(2R,3S,4R,5R)-5-[6-(Cyclopentylamino)-2-[(pyrrolidin-3-yloxy)-
methyl]-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}-
methyl)phosphonic Acid (3e). To a vial containing 1-boc-3-
hydroxypyrrolidine (107 mg, 0.57 mmol) in DMF (1.5 mL) was
added sodium hydride (60 wt % oil dispersion, 23 mg, 0.57 mmol).
After 2 min, compound 23 (65 mg, 0.11 mmol) in DMF (500 μL)
was added and the reaction was stirred overnight at rt. The reaction
was quenched with water, diluted with EtOAc, and washed with water
and brine. The organics were dried over MgSO4 and concentrated
under reduced pressure. The crude product 24 was used directly in
the following step without further purification. ESI MS [M + H]+ for
C33H53N6O10P, calcd 724.4, found 725.4.
saturated NaHCO3 (1 L) and extracted with EtOAc (3 × 500 mL).
Combined organics were dried over MgSO4, filtered, and evaporated
to give a dark-brown oil that was purified by column chromatography
(SiO2, hexane/EtOAc, 100−40%) to give 27 (yellow oil, 28.5 g, 25%).
ESI MS [M + Na]+ for C16H16Cl2N4NaO7, calcd 469.0, found 469.0.
Compound 27 (22 g, 49.3 mmol) was dissolved in MeOH (100
mL) and cooled to 0 °C. Cyclopentylamine (5.1 g, 51.8 mmol) and
triethylamine (7.2 mL, 51.8 mmol) were added, and the reaction
mixture was stirred at 0 °C for 15 min and then at rt for 4 h. Then, 7
M NH3 in MeOH (60 mL) was added and the reaction was stirred at
rt for 1 day. The reaction mixture was evaporated, and the crude
product 29 was used in the next step without purification. ESI MS [M
+ H]+ for C15H21ClN5O4, calcd 370.1, found 370.2.
The crude product 29 (10.5 g, 28.4 mmol) was dissolved in
acetone (50 mL), and 2,2-dimethoxypropane (50 mL) and p-TsOH·
H2O (6.7 g, 35.5 mmol) were added. The reaction mixture was stirred
at rt overnight, diluted with brine (100 mL), and carefully quenched
with saturated NaHCO3 (100 mL). After extraction with EtOAc (2 ×
100 mL), combined organics were dried over MgSO4, filtered, and
evaporated to give the crude product 30 (6.2 g), which was used in
the next step without purification. ESI MS [M + H]+ for
C18H25ClN5O4, calcd 410.2, found 410.2.
The crude compound 30 (3.2 g, 7.8 mmol) in anhydrous THF (50
mL) was cooled to 0 °C, and 1 M NaHMDS in THF (12.4 mL, 12.4
mmol) was added dropwise. The reaction mixture was stirred at 0 °C
for 1 h; then, (diethoxyphosphoryl)methyl trifluoromethanesulfonate
(3.4 g, 12.4 mmol) was added. The cooling bath was removed, and
the reaction was stirred at rt overnight. It was quenched with a
saturated solution of NH4Cl (50 mL) and diluted with MTBE (100
mL). The organic layer was separated, dried over MgSO4, filtered, and
evaporated. The crude product was purified by column chromatog-
raphy (SiO2, Hex → Hex:EtOAc, 2:8) to give compound 31 as a
white foamy solid (3.2 g, 73%). ESI MS [M + H]+ for
C23H36ClN5O7P, calcd 560.2, found 560.3.
Product 31 (3.2 g, 5.7 mmol) was dissolved in anhydrous CH3CN
(30 mL), TMS-Br (1.6 mL) was added, and the reaction was stirred at
rt overnight. The solvent was evaporated to obtain a white foamy
solid. To a portion of the white solid (250 mg) were added 4 mL of
MeOH and 2 mL of 1 N HCl. The reaction mixture was stirred for 8
h at rt, after which the solvent was evaporated and purified by reverse-
phase HPLC (C18 column, 0−30% gradient of acetonitrile and water
with 0.1% TFA) to give the product 4a as a white solid in 93% yield
(267 mg): 1H NMR (400 MHz, DMSO-d6) 8.71 (d, J = 7.2 Hz, 1H),
8.25 (s, 1H), 6.00 (d, J = 3.9 Hz, 1H), 4.51−4.37 (m, 2H), 4.19 (t, J =
5.2 Hz, 1H), 4.07−3.98 (m, 1H), 3.71 (dd, J = 10.7, 4.0 Hz, 1H),
3.59−3.48 (m, 3H), 2.06−1.93 (m, 3H), 1.79−1.47 (m, 5H). ESI MS
[M + H]+ for C16H24ClN5O7P, calcd 464.1, found 464.2.
All of the crude product 24 was dissolved in anhydrous CH3CN
(3.0 mL), TMS-Br (0.3 mL) was added, and the reaction was stirred
at rt for 1.5 h. It was quenched with H2O (0.3 mL) and stirred at rt
for 4 h or until LCMS analysis showed complete cleavage of the
acetonide and BOC protecting groups. The reaction mixture was
evaporated and purified by reverse-phase HPLC (C18 column, 0−
30% gradient of acetonitrile and water with 0.1% TFA) to give the
product 3e as a white solid in 15% yield (9 mg, two steps): 1H NMR
(400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.82 (s, 1H), 5.91 (d, J = 6.1
Hz, 1H), 4.62 (t, J = 5.6 Hz, 1H), 4.54−4.41 (m, 4H), 4.16 (t, J = 4.1
Hz, 1H), 4.04 (q, J = 4.0 Hz, 1H), 3.78−3.67 (m, 2H), 3.61 (d, J =
8.8 Hz, 2H), 3.40−3.13 (m, 5H), 2.20−2.09 (m, 1H), 2.04−1.88 (m,
4H), 1.66−1.47 (m, 4H). ESI MS [M − H]− for C21H33N6O8P, calcd
527.2, found 527.4.
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-phenylethyl]amino}-1H-
pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]-
methoxy}methyl)phosphonic Acid (5a). Compound 5a was
1
synthesized in a similar fashion to example 4a as a white solid. H
NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 7.55−7.09 (m, 4H),
5.97 (d, J = 5.8 Hz, 1H), 4.64 (t, J = 5.4 Hz, 1H), 4.32−4.08 (m, 1H),
4.05 (app. q, J = 3.9 Hz, 1H), 3.75 (dd, J = 10.7, 3.6 Hz, 1H), 3.68
(dd, J = 10.7, 4.6 Hz, 1H), 3.60 (dd, J = 8.9, 1.9 Hz, 2H), 1.97 (br. s,
3H), 1.80−1.45 (m, 8H). ESI MS [M − H]− for C19H23ClN5O7P,
calcd 498.1, found 498.3.
({[(2R,3S,4R,5R)-5-[6-Chloro-4-(cyclopentylamino)-1H-pyrazolo-
[3,4-d]pyrimidin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (4a). The mixture of 4,6-dichloro-1H-pyrazolo[3,4-
d]pyrimidine 25 (47.7 g, 252.4 mmol) and (NH4)2SO4 (300 mg) in
HMDS (200 mL) was stirred under reflux for 5 h and then
concentrated under a vacuum to give a dark-brown oil that was
immediately used in the next step. The crude TMS-protected
heterocycle was dissolved in anhydrous CH3CN (500 mL), and
1,2,3,5-tetraacetate-β-D-ribofuranose (88.3 g, 277.6 mmol) was added.
The reaction mixture was stirred at rt until all starting materials
dissolved, and then, TfO-TMS (68.3 mL, 378.6 mmol) was added
dropwise. It was stirred at rt overnight, concentrated under a vacuum
to ∼50% of the original volume, and then carefully quenched with
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]-
amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-
yl]methoxy}methyl)phosphonic Acid (5b). Compound 5b was
1
synthesized in a similar fashion to example 4a as a white solid. H
NMR (400 MHz, DMSO-d6) δ 9.21 (t, J = 6.6 Hz, 1H), 8.32 (dt, J =
4.7, 1.0 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.36−7.23 (m, 1H), 7.22−
7.12 (m, 2H), 6.03−5.92 (m, 1H), 5.58 (q, J = 7.0 Hz, 1H), 4.44 (t, J
= 4.5 Hz, 1H), 4.16 (d, J = 5.6 Hz, 1H), 4.05−3.93 (m, 1H), 3.69
(dd, J = 10.8, 4.0 Hz, 1H), 3.52 (t, J = 8.2 Hz, 2H), 1.53 (d, J = 6.9
Hz, 3H). ESI MS [M + H]+ for C19H22ClFN5O7P, calcd 518.1, found
518.1.
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-(3-fluorophenyl)ethyl]-
amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-
856
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
yl]methoxy}methyl)phosphonic Acid (5c). Compound 5c was
synthesized in a similar fashion to example 4a as a white solid. H
protecting group. The reaction mixture was evaporated and purified
by reverse-phase HPLC (C18 column, 0−30% gradient of acetonitrile
and water with 0.1% TFA) to give the product 4b as a white solid
(200 mg; 48%): 1H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.65
(d, J = 6.7 Hz, 1H), 6.22 (s, 1H), 6.06 (d, J = 3.9 Hz, 1H), 4.47 (t, J =
4.5 Hz, 1H), 4.17 (t, J = 5.2 Hz, 1H), 4.02−3.92 (m, 2H), 3.69 (dd, J
= 10.9, 3.9 Hz, 1H), 3.50 (d, J = 8.6 Hz, 3H), 2.06−1.88 (m, 2H),
1.75−1.47 (m, 6H). ESI MS [M + H]+ for C17H25ClN4O7P, calcd
463.1, found 463.2.
1
NMR (400 MHz, DMSO-d6) δ 9.16 (d, J = 7.8 Hz, 1H), 8.35−8.21
(m, 1H), 7.50−7.28 (m, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.07 (t, J = 8.7
Hz, 1H), 5.99 (d, J = 4.1 Hz, 1H), 5.41 (t, J = 7.2 Hz, 1H), 4.44 (t, J =
4.4 Hz, 1H), 4.17 (t, J = 5.1 Hz, 1H), 3.99 (s, 1H), 3.69 (dd, J = 10.7,
3.9 Hz, 1H), 3.51 (d, J = 8.7 Hz, 2H), 1.53 (d, J = 6.8 Hz, 3H). ESI
MS [M + H]+ for C19H22ClFN5O7P, calcd 518.1, found 518.1.
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]-
amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-
yl]methoxy}methyl)phosphonic Acid (5d). Compound 5d was
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-phenylethyl]amino}-1H-
pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}-
methyl)phosphonic Acid (5e). Compound 5e was synthesized in a
1
synthesized in a similar fashion to example 4a as a white solid. H
1
NMR (400 MHz, DMSO-d6) δ 9.14 (d, J = 7.8 Hz, 1H), 8.29 (s, 1H),
7.42 (t, J = 6.8 Hz, 2H), 7.22−7.05 (m, 2H), 5.98 (d, J = 4.1 Hz, 1H),
5.48−5.31 (m, 1H), 4.44 (t, J = 4.6 Hz, 1H), 4.17 (t, J = 5.1 Hz, 1H),
3.99 (d, J = 5.9 Hz, 1H), 3.69 (dd, J = 10.9, 4.0 Hz, 1H), 3.51 (d, J =
8.6 Hz, 2H), 1.57−1.48 (m, 3H). ESI MS [M + H]+ for
C19H22ClFN5O7P, calcd 518.1, found 518.1
similar fashion to example 4b as a white solid. H NMR (400 MHz,
DMSO-d6) δ 8.37 (s, 1H), 8.19 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 8.1
Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.24−7.18 (m, 1H), 6.04 (d, J = 4.1
Hz, 1H), 6.02−5.92 (m, 1H), 4.85 (s, 1H), 4.46 (t, J = 4.6 Hz, 1H),
4.17 (t, J = 5.4 Hz, 1H), 3.96 (t, J = 5.5 Hz, 1H), 3.67 (td, J = 13.5,
12.1, 7.3 Hz, 1H), 3.51 (d, J = 8.3 Hz, 2H), 1.52 (d, J = 6.7 Hz, 3H).
ESI MS [M + H]+ for C20H24ClN4O7P, calcd 499.9, found 499.8.
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]-
amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]-
methoxy}methyl)phosphonic Acid (5f). Compound 5f was synthe-
({[(2R,3S,4R,5R)-5-[6-Chloro-4-(cyclopentylamino)-1H-pyrazolo-
[3,4-b]pyridin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (4b). A mixture of 4,6-dichloro-1H-pyrazolo[3,4-
b]pyridine 26 (9.5 g, 50.5 mmol) and (NH4)2SO4 (100 mg) in
HMDS (50 mL) was stirred under reflux for 5 h and then
concentrated under a vacuum to give a dark-brown oil that was
immediately used in the next step. The crude TMS-protected
heterocycle was dissolved in anhydrous CH3CN (100 mL), and
1,2,3,5-tetraacetate-β-D-ribofuranose (17.6 g, 55.5 mmol) was added.
The reaction mixture was stirred at rt until all starting materials
dissolved; then, TfO-TMS (13.7 mL, 75.7 mmol) was added
dropwise. It was stirred at rt overnight, concentrated under a vacuum
to ∼50% of the original volume, and then carefully quenched with
saturated NaHCO3 (300 mL) and extracted with EtOAc (3 × 100
mL). Combined organics were dried over MgSO4, filtered, and
evaporated to give a dark-brown oil that was purified by column
chromatography (SiO2, hexane/EtOAc, 100−40%) to give 28 (yellow
oil, 7.2 g, 32%). ESI MS [M + Na]+ for C17H17Cl2N3NaO7, calcd
468.0, found 468.0.
Compound 28 (3.1 g, 6.9 mmol) was dissolved in MeOH (10 mL)
and cooled to 0 °C. Then, 7 M NH3 in MeOH (5 mL) was added and
the reaction was stirred at rt for 1 day. The reaction mixture was
evaporated, the crude material was redissolved in acetone (10 mL),
and 2,2-dimethoxypropane (10 mL) and p-TsOH·H2O (1.63 g, 8.6
mmol) were added. The reaction mixture was stirred at rt overnight,
diluted with brine (20 mL), and carefully quenched with saturated
NaHCO3 (30 mL). After extraction with EtOAc (2 × 50 mL),
combined organics were dried over MgSO4, filtered, and evaporated.
The crude material was purified by column chromatography (SiO2,
hexane/EtOAc, 1:1) to give 32 (white solid, 2.0 g, 80%, overall). ESI
MS [M + H]+ for C14H16Cl2N3O4, calcd 360.1, found 360.1.
A solution of compound 32 (2.7 g, 7.5 mmol) in anhydrous THF
(20 mL) was cooled to 0 °C, and 1 M LiHMDS in THF (9.0 mL, 9.0
mmol) was added dropwise. The reaction mixture was stirred at −78
°C for 1 h; then, (diethoxyphosphoryl)methyl trifluoromethanesulfo-
nate (2.4 g, 9.0 mmol) was added. The cooling bath was removed,
and the reaction was stirred at rt overnight. It was quenched with a
saturated solution of NH4Cl (6.0 mL) and diluted with MTBE (20
mL). The organic layer was separated, dried over MgSO4, filtered, and
evaporated. The crude product was purified by column chromatog-
raphy (SiO2, Hex → Hex:EtOAc, 2:8) to give compound 33 as a
white foamy solid (2.0 g, 59%). ESI MS [M + H]+ for
C19H27Cl2N3O7P, calcd 510.1, found 510.0.
1
sized in a similar fashion to example 4b as a white solid. H NMR
(400 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.23 (d, J = 7.1 Hz, 1H),
7.44−7.34 (m, 1H), 7.34−7.26 (m, 1H), 7.26−7.09 (m, 2H), 6.05 (d,
J = 4.0 Hz, 1H), 6.00−5.73 (m, 1H), 5.04 (s, 1H), 4.53−4.40 (m,
1H), 4.17 (t, J = 5.2 Hz, 1H), 3.98 (dt, J = 6.4, 4.5 Hz, 1H), 3.69 (dd,
J = 10.8, 4.0 Hz, 1H), 3.51 (t, J = 7.4 Hz, 3H), 1.56 (d, J = 6.7 Hz,
3H). ESI MS [M + H]+ for C20H23ClFN4O7P, calcd 517.1, found
517.1.
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-(3-fluorophenyl)ethyl]-
amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]-
methoxy}methyl)phosphonic Acid (5g). Compound 5g was
1
synthesized in a similar fashion to example 4b as a white solid. H
NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.18 (d, J = 7.3 Hz, 1H),
7.42−7.31 (m, 1H), 7.31−7.18 (m, 2H), 7.10−6.99 (m, 1H), 6.04 (d,
J = 4.0 Hz, 1H), 4.91 (s, 1H), 4.51−4.41 (m, 1H), 4.17 (t, J = 5.3 Hz,
1H), 4.04−3.90 (m, 1H), 3.69 (dd, J = 10.8, 4.0 Hz, 1H), 3.51 (d, J =
8.3 Hz, 3H), 1.57−1.46 (m, 3H). ESI MS [M + H]+ for
C20H23ClFN4O7P, calcd 517.1, found 517.1.
({[(2R,3S,4R,5R)-5-(6-Chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]-
amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]-
methoxy}methyl)phosphonic Acid (5h). Compound 5h was
1
synthesized in a similar fashion to example 4b as a white solid. H
NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.17 (d, J = 7.2 Hz, 1H),
7.43 (ddd, J = 8.8, 5.5, 1.5 Hz, 2H), 7.14 (td, J = 8.8, 1.4 Hz, 2H),
6.04 (d, J = 4.1 Hz, 2H), 4.89 (s, 1H), 4.52−4.40 (m, 1H), 4.17 (t, J =
5.2 Hz, 1H), 3.97 (dt, J = 6.3, 4.4 Hz, 1H), 3.68 (dd, J = 10.8, 4.0 Hz,
1H), 3.60−3.38 (m, 3H), 1.50 (d, J = 6.5 Hz, 3H). ESI MS [M + H]+
for C20H23ClFN4O7P, calcd 517.1, found 517.1.
({[(2R,3S,4R,5R)-5-[5-Chloro-7-(cyclopentylamino)-3H-imidazo-
[4,5-b]pyridin-3-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}methyl)-
phosphonic Acid (4c). To a solution of 5,7-dichloroimidazo[4,5-
b]pyridine 34 (376 mg, 2 mmol) in MeCN (14 mL) at rt was added
N,O-bis(trimethylsilyl)acetamide (0.523 mL, 2.14 mmol) dropwise,
and the reaction mixture was heated to 85 °C for 1 h. The mixture
was cooled to rt, and a solution of β-D-ribofuranose 1,2,3,5-
tetraacetate (726 mg, 2.28 mmol) in MeCN (7 mL) and trimethylsilyl
trifluoromethanesulfonate (0.471 mL, 2.60 mmol) were added
sequentially dropwise. The reaction mixture was heated to 85 °C
for 4 h. After cooling to rt, aqueous saturated sodium bicarbonate (50
mL) was added, subsequently extracted three times with EtOAc (100
mL), dried over sodium sulfate, and concentrated. The crude material
35 thus obtained was redissolved in dioxane (2 mL), and
cyclopentylamine (0.987 mL, 10 mmol) was added. The mixture
was heated to 100 °C for 16 h. The reaction mixture was then directly
loaded onto silica gel and purified by silica gel chromatography (0−
10% MeOH in DCM) to afford the desired product 36 as a brown
solid (298 mg, 40%, two steps). ESI MS [M + H]+ for C16H22ClN4O4,
calcd 369.1, found 369.1.
In a glass vial, compound 33 (400 mg, 0.78 mmol) was dissolved in
EtOH (1.0 mL). To this solution were added cyclopentylamine (115
μL, 1.17 mmol) and triethylamine (163 μL, 1.17 mmol). The reaction
mixture was heated to reflux in the sealed vial for 16 h. Volatiles were
removed under reduced pressure, and the crude mass was redissolved
in anhydrous CH3CN (5.0 mL), followed by addition of TMS-Br (0.5
mL). The reaction mixture was stirred at rt overnight before
quenching with H2O (1 mL) and stirring for an additional 4 h at rt
or until LCMS analysis showed complete cleavage of the acetonide
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A solution of the compound 36 (298 mg; 0.808 mmol) and p-
toluenesulfonic acid monohydrate (154 mg, 0.808 mmol) in 2,2-
dimethoxypropane (1.6 mL) and acetone (1.6 mL) was stirred at rt
for 20 h. Triethylamine (0.5 mL) was added, the solvent was
removed, and the residue was purified by silica gel chromatography
(50−100% EtOAc in hexanes) to afford the desired product 37 as a
white solid (258 mg; 78%). ESI MS [M + H]+ for C19H26ClN4O4,
calcd 409.2, found 409.2.
Compound 37 (126 mg, 0.308 mmol) in anhydrous THF (1.5 mL)
was cooled to 0 °C, and 1 M NaHMDS in THF (0.43 mL, 0.43
mmol) was added dropwise. The reaction mixture was stirred at 0 °C
for 1 h; then, (diethoxyphosphoryl)methyl trifluoromethanesulfonate
(139 mg, 0.47 mmol) was added. The cooling bath was removed, and
the reaction was stirred at rt overnight. It was quenched with a
saturated solution of NH4Cl (1.0 mL) and diluted with MTBE (100
mL). The organic layer was separated, dried over MgSO4, filtered, and
evaporated. The crude product was purified by column chromatog-
raphy to obtain 38 as a white solid (107 mg; 62%). ESI MS [M + H]+
for C24H37ClN4O7P, calcd 559.9, found 559.9.
μL of the PiColorLock Gold colorimetric assay reagent (Cat. No. 30
300 30, Thermo Fisher). The inorganic phosphate reaction product
was assessed as a function of color developed by reaction with the
PiColorLock Gold reagent and quantitated using an Envision 2102
Multilabel Reader fitted with a 620 nm filter. The CD73 enzymatic
activity was evaluated as a correlate of phosphate product levels.
Percentage maximum activity in each test well was calculated based
on DMSO (maximum activity) and wells not containing cells
(baseline activity). The IC50 values of the test compounds were
determined from the compound’s dose−response curve fitted using a
standard four-parameter fit equation.
CD39 CHO Cellular Assay. CD39 stable cell lines were generated
by Lake Pharma (Belmont, CA) using the same protocol as the
generation of CD73 stable cells. The CD39 CHO cellular assay was
carried out using the same protocol as the CD73 CHO cellular assay
but with modification to cell number, substrate concentration, and
compound preincubation time. Final assay conditions comprised 500
cells per well in 2% DMSO and 15 μM ATP substrate with 4 h of
compound preincubation.
Compound 38 (107 mg, 0.19 mmol) was dissolved in anhydrous
CH3CN (0.46 mL), TMS-Br (0.25 mL) was added, and the reaction
was stirred at rt overnight. It was quenched with H2O (1 mL) and
stirred at rt for 4 h or until LCMS analysis showed complete cleavage
of the acetonide protecting group. The reaction mixture was
evaporated and purified by reverse-phase HPLC (C18 column, 0−
30% gradient of acetonitrile and water with 0.1% TFA) to give the
Preparation of Chinese Hamster Ovary (CHO) K1 Cells
Transiently Transfected with NTPDase2, NTPDase3, or
NTPDase8. One day prior to the assay, 5 ×106 CHO-K1 cells
(ATCC, #CCL-61) were seeded in 11 mL of cell media (Hams F-12,
(Fisher, 11765047), supplemented with 10% FBS (Gibco, Cat. No.
A3160502), and 1% GlutaMax (Invitrogen, Cat. No. 35050-061)) in
T75 flasks (Corning, 430641U). Then, 10 μL of 100 ng/μL
NTPDase2, NTPDase3, or NTPDase8 plasmid DNA was added to
2 mL of serum-free Opti-MEM media (Fisher, 31985062). The
diluted plasmid was combined with 2 mL of serum-free Opti-MEM
media containing 100 μL of the Lipofectamine LTX Reagent (Fisher,
#15338030). The final DNA/LTX mixture was incubated for 30 min
at ∼25 °C and added to the T75 flask containing CHO-K1 cells. The
cells were incubated at 37 °C and 5% CO2 overnight. The following
day, media were removed by gentle aspiration and cells were washed
with 10 mL of phosphate-buffered saline (PBS, Gibco, 14190250)
twice. The cells were harvested by adding 2 mL of Accutase (Fisher,
#00-4555-56) to the flask. The flask was then incubated at 37 °C until
the cells detached from the flask when lightly tapped by hand. Next, 8
mL of PBS was added to the flask to dislodge cells and then the cells
were transferred to a 15 mL conical centrifuge tube (Falcon, 339650)
and centrifuged at 225g for 5 min. PBS was removed by aspiration,
and the cells were suspended in 5 mL of an assay buffer consisting of
20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2,
4.2 mM NaHCO3, and 0.1% glucose. Cell density was determined
using ViaCell XR (Becman Coulter). The cells were then diluted to
0.25 × 106 cells/mL.
NTPDase2, NTPDase3, or NTPDase8 Cellular Assays. A 16-
point, 1:3 master serial dilution of the test compound was prepared in
100% DMSO to span a final concentration range of 10−0 μM. Then,
18 μL of an assay buffer was added to each well of a 96-well
polypropylene plate (Corning, Cat no. 3365) followed by addition of
2 μL of the test compound at 50-fold final concentration. Then, 40 μL
of NTPDase2, 3, or 8 cells in an assay buffer was added to each well
and the plate was incubated for a further 1 h at 37 °C prior to
addition of 40 μL of 62.5 μM (NTPDase2), 40 μL of 37.5 μM
(NTPDase3), 40 μL of 75 μM (NTPdase8), and adenosine-5′-
triphosphate monohydrate (ATP) (Sigma, Cat. No. A26209-5G) in
the assay buffer. Final assay conditions comprised 10 000 cells per
well in 2% DMSO and 25/15/30 μM ATP substrate. After a further
50 min of incubation at 37 °C and 5% CO2, the plate was centrifuged
at 225g for 5 min. Then, 80 μL of the supernatant was transferred to a
96-well Spectra Plate (Perkin Elmer, Cat. No. 6005640), the wells of
which had been preloaded with 20 μL of PiColorLock Gold
colorimetric assay reagent (Thermo Fisher, Cat. No. 30 300 30).
The inorganic phosphate reaction product was quantitated as a
function of color developed by reaction with PiColorLock Gold
reagent and determined using an Envision 2102 Multilabel Reader
(Perkin Elmer) fitted with a 620 nm filter. NTPDase2, 3, or 8 activity
was assessed as a correlate of phosphate product levels. Percentage
maximum activity in each of the compound test wells was calculated
1
product 4c as a white solid (14 mg; 15%). H NMR (400 MHz,
DMSO-d6) δ 8.47 (s, 1H), 7.12 (d, J = 7.4 Hz, 1H), 6.41 (s, 1H), 5.90
(d, J = 5.8 Hz, 1H), 4.68−4.45 (m, 1H), 4.16−4.10 (m, 1H), 4.03 (q,
J = 3.9 Hz, 1H), 3.78−3.65 (m, 2H), 3.61 (d, J = 8.9 Hz, 2H), 2.05−
1.89 (m, 2H), 1.77−1.63 (m, 2H), 1.63−1.47 (m, 4H). ESI MS [M −
H]− for C17H23ClN4O7P, calcd 461.8, found 461.2.
Generation and Expansion of the Human CD73 Stable CHO
Cell Line. Stable cell lines were generated by Lake Pharma (Belmont,
CA) using a standard protocol to transform CHO cells with a
pcDNA3.1(+) vector carrying the human NT5E (CD73) gene.
Antibiotic selection was performed in CD OptiCHO cell media
containing 5 μg/mL Puromycin and 200 μg/mL Hygromycin B.
Pools of surviving CHO-CD73 were collected and frozen in 7.5%
dimethyl sulfoxide (DMSO) in CD OptiCHO cell media.
Cryopreserved cells were defrosted in a water bath at 37 °C by
agitating the vial until the cells were completely thawed. Cells were
then transferred to a 15 mL Falcon tube prior to centrifuging at 225g
for 5 min to pellet the cells. The cell pellet was resuspended in a fresh
warm CD OptiCHO Growth Medium supplemented with 2 mM
Glutamax and transferred to a T175 flask. After two days and on
reaching ∼80% confluence (∼20 million cells/flask), cells were split
1:3 into three fresh T175 flasks. After a further three days, cells were
transferred to 15 mL Falcon tubes and centrifuged at 250g for 5 min
to pellet. Cells were resuspended at a density of 3 million cells/mL in
CellBanker2 cryopreservation media and aliquoted into cryogenic
vials. Cell aliquots were stored at −80 °C until needed.
CD73 CHO Cellular Assay. On the day of the experiment, one
vial of CHO-CD73 cells was thawed and cells were resuspended in 10
mL of assay buffer consisting of 20 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES), pH 7.4, 137 mM NaCl, 5.4
mM KCl, 1.3 mM CaCl2, 4.2 mM NaHCO3, and 0.1% glucose. A 16-
point, 1:3 master serial dilution of the test compound was prepared in
100% DMSO to span a final concentration range of 100−0 μM or
0.1−0 μM. Twenty microliters of assay buffer was added to each well
of a 96-well round-bottomed polypropylene plate followed by
addition of 10 or 1 μL of the test compound at 10-fold or 100-fold
final concentration. Fifty or thirty microliters of cells was added to
each well, and the plate was incubated for a further 60 min at 37 °C
prior to addition of 20 μL of 125 μM AMP or 49 μL of 51 μM AMP
in an assay buffer. Final assay conditions comprised 1500 cells per
well in 10 or 2% DMSO and 25 μM AMP substrate. After a further 60
min of incubation at 37 °C and 5% CO2, the plate was centrifuged at
225g for 5 min. Eighty microliters of supernatant was transferred to a
96-well Spectra Plate, the wells of which had been preloaded with 20
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based on DMSO (maximum activity) and no cell control wells
(baseline activity). The IC50 values of the test compounds were
determined using Prism software (Graphpad Software Inc., version
7.02, La Jolla, CA 92037), and the compound’s dose−response curve
was fitted using a standard four-parameter fit equation.
Dillon H. Miles − Arcus Biosciences, Inc., Hayward, California
94545, United States
Eric Newcomb − Arcus Biosciences, Inc., Hayward, California
94545, United States
Erick A. Lindsey − Arcus Biosciences, Inc., Hayward,
California 94545, United States
Brandon R. Rosen − Arcus Biosciences, Inc., Hayward,
California 94545, United States; orcid.org/0000-0002-
0790-6433
Laurent P. P. Debien − Arcus Biosciences, Inc., Hayward,
California 94545, United States
Ada Chen − Arcus Biosciences, Inc., Hayward, California
94545, United States
Xiaoning Zhao − Arcus Biosciences, Inc., Hayward, California
94545, United States
Stephen W. Young − Arcus Biosciences, Inc., Hayward,
California 94545, United States
Nigel P. Walker − Arcus Biosciences, Inc., Hayward,
California 94545, United States
A2aR Assay. The compound inhibition constant (IC50) was
determined as a function of cellular cAMP levels in NECA-stimulated
CHO cells stably transfected with adenosine 2Aa receptor. Clonal
CHO cells stably transfected with ADORA2A and induced with
tetracycline were seeded into 384-well OptiPlates at a density of
between 2000 cells per well and preincubated with 20-point, 1:2 serial
dilution of the compound to span a final concentration range of 10−0
μM. A 30 nM fixed concentration of NECA was prepared and added
to the wells to generate a full serial dilution of NECA against each of
the five compound concentrations. Cells were incubated for a further
30 min at 37 °C. The reaction was quenched by addition of 5 μL of
Ulight-anti-cAMP diluted 1:150 with a conjugate and lysis buffer. Five
microliters of Eu-cAMP tracer diluted 1:50 dilution in the conjugate
and lysis buffer was then added to each well, and the plate was
incubated for an additional 60 min at 37 °C. Cellular cAMP levels
were then quantitated as a function of the fluorescence resonance
energy transfer (FRET) signal measured using an Envision 2102
Multilabel Reader (Perkin Elmer) fitted with 615 nm excitation and
665 nm emission filters. The IC50 potency was performed using Prism
software (Graphpad Software Inc., version 7.02, La Jolla, CA 92037)
to calculate the compound IC50.
Pharmacokinetic Studies. All animal procedures were con-
ducted under protocols approved by the Arcus Institutional Animal
Care and Use Committee (IACUC). Male rats, weighing between
0.27 and 0.30 kg, were purchased from Charles River Laboratories
(Hollister, CA) and were acclimated before use. Test articles were
dissolved in 31.6% DMA, 36.8% ethanol, and 31.6% PEG and
administered intravenously via the femoral vein at 0.25 or 0.5 mg/kg.
Blood was sampled via the jugular vein at predose, 5, 15, and 30 min,
1, 2, 4, 6, 8, and 24 h post dose. Blood samples were placed in
polypropylene tubes containing K2-EDTA as the anticoagulant and
centrifuged at 8000 rpm and 4 °C for 7 min to obtain plasma, which
were stored at −80 °C until analysis. Plasma samples were prepared
by protein precipitation and analyzed by LCMS/MS (Shimadzu
Nexera X2 UHPLC and API4000 mass spectrometer). Pharmacoki-
netic analysis was performed using Phoenix WinNonlin v6.4.
̈
Norbert Strater − Institute of Bioanalytical Chemistry, Center
for Biotechnology and Biomedicine, Leipzig University, D-
04103 Leipzig, Germany; orcid.org/0000-0002-2001-
0500
Emma R. Scaletti − Institute of Bioanalytical Chemistry,
Center for Biotechnology and Biomedicine, Leipzig University,
D-04103 Leipzig, Germany
Lixia Jin − Arcus Biosciences, Inc., Hayward, California 94545,
United States
Guifen Xu − Arcus Biosciences, Inc., Hayward, California
94545, United States
Manmohan R. Leleti − Arcus Biosciences, Inc., Hayward,
California 94545, United States
Jay P. Powers − Arcus Biosciences, Inc., Hayward, California
94545, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01835
Author Contributions
All authors have given approval to the final version of the
manuscript.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01835.
■
sı
Notes
The authors declare no competing financial interest.
Molecular formula strings (CSV)
Hepatocyte stability assay, CYP inhibition assay, and
crystallographic methods (PDF)
ABBREVIATIONS
■
SAR, structure−activity relationship; GPI, glycosylphosphati-
dylinositol; hCD73, human cluster of differentiation 73; CYP,
cytochrome P450; PDB, protein data bank; hNTPDases,
human nucleoside triphosphate diphosphohydrolases; POM,
pivaloyloxymethyl; POC, isopropyloxycarbonyloxymethyl;
HDP, hexadecyloxypropyl; DMA, dimethylacetamide; PEG,
poly(ethylene glycol); DMF, N,N-dimethylformamide; THF,
tetrahydrofuran; pTsOH, para-toluenesulfonic acid; HMDS,
bis(trimethylsilyl)amine; BSA, N,O-bis(trimethylsilyl)-
acetamide; HPLC, high-performance liquid chromatography;
LCMS, liquid chromatography−mass spectrometry; ESI,
electrospray ionization; rt, room temperature; NMR, nuclear
magnetic resonance; TLC, thin-layer chromatography
Accession Codes
Atomic coordinates have been deposited in the Protein Data
Bank (PDB: 6YE1 for 2n and PDB: 6YE2 for 4a). Structures
will be released upon article publication.
AUTHOR INFORMATION
Corresponding Author
■
Ehesan U. Sharif − Arcus Biosciences, Inc., Hayward,
California 94545, United States; orcid.org/0000-0002-
2605-6745; Email: esharif@arcusbio.com
Authors
Jaroslaw Kalisiak − Arcus Biosciences, Inc., Hayward,
California 94545, United States
Kenneth V. Lawson − Arcus Biosciences, Inc., Hayward,
California 94545, United States; orcid.org/0000-0001-
5094-6337
REFERENCES
■
̈
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