Identification of Novel Carbocyclic Pyrimidine Cyclic Dinucleotide STING Agonists for Antitumor Immunotherapy Using Systemic Intravenous Route

Article abstract of DOI:10.1021/acs.jmedchem.1c00374

Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.

Full text of DOI:10.1021/acs.jmedchem.1c00374

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Article
Identification of Novel Carbocyclic Pyrimidine Cyclic Dinucleotide
STING Agonists for Antitumor Immunotherapy Using Systemic
Intravenous Route
Stepan Vyskocil,* David Cardin, Jeffrey Ciavarri, Joe Conlon, Courtney Cullis, Dylan England,
Rachel Gershman, Kenneth Gigstad, Krista Gipson, Alexandra Gould, Paul Greenspan, Robert Griffin,
Nanda Gulavita, Sean Harrison, Zhigen Hu, Yongbo Hu, Akito Hata, Jian Huang, Shih-Chung Huang,
Dave Janowick, Matthew Jones, Vihren Kolev, Steven P. Langston, Hong Myung Lee, Gang Li,
David Lok, Liting Ma, Doanh Mai, Jenna Malley, Atsushi Matsuda, Hirotake Mizutani, Miho Mizutani,
Nina Molchanova, Elise Nunes, Sandeep Pusalkar, Christelle Renou, Scott Rowland, Yosuke Sato,
Michael Shaw, Luhua Shen, Zhan Shi, Robert Skene, Francois Soucy, Steve Stroud, He Xu, Tianlin Xu,
Adnan O. Abu-Yousif, and Ji Zhang
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ABSTRACT: Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon
production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation
in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to
activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route.
Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with
optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an
acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic
T-cells, which ultimately provides robust antitumor efficacy.
adaptive immune responses against tumors.3 This pathway is
INTRODUCTION
■
activated in the presence of cytosolic DNA detected by the
sensor cyclic GMP−AMP synthase (cGAS), which generates
cyclic GMP−AMP (cGAMP) (1, Figure 1), which sub-
sequently directly binds and activates STING.4
Novel immunotherapy approaches, such as the PD-1 and
CTLA-4 immune checkpoint inhibitors, have been trans-
forming the treatment of cancer. Despite this progress,
immunotherapies are ineffective in many patients because the
tumor microenvironment fails to support recruitment of
immune cells, including CD8+ T-cells, to abolish the tumor.1
Conversion of such poorly immune-infiltrated tumors, or so-
called “cold” tumors, to immune-infiltrated “hot” tumors
represents an exciting avenue for improving the treatment of
cancer.2 Spontaneous activation of the Stimulator of
Interferon Genes (STING) pathway within tumor-resident
dendritic cells leads to type I IFN production and elicits
Received: March 1, 2021
Published: May 17, 2021
© 2021 The Authors. Published by
American Chemical Society
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Figure 1. Structures of reference CDN STING agonists.
Figure 2. Key interactions between cGAMP and STING from X-ray structure (PDB code 4KSY).18 Carbon is shown in white (1), cyan, or gold
(STING); nitrogen in blue; and oxygen in red. Water molecules are shown as red spheres. Hydrogen bonds are indicated by gray dashed lines,
with the length of the cylinder indicating a relative strength of interaction.
Figure 3. Docking model of a rationally designed carbocyclic pyrimidine CDN showing preferred (R,R)-diphosphorothioate configuration.
Crystal structure 4KSY was used as the starting point for molecular modeling. Carbon is shown in white (15a), cyan, or gold (STING);
nitrogen in blue; oxygen in red; and sulfur in yellow. Water molecules are shown as red spheres. Hydrogen bonds are indicated by dashed lines,
with the length of the cylinder indicating a relative strength of interaction.
Lack of cellular penetration and rapid degradation by
plasma phosphatases and hydrolases prevents in vivo utility of
cGAMP.5 A number of synthetic cyclic dinucleotide (CDN)
STING agonists with improved ADME properties are
currently being investigated in clinical trials.6 ADU-S100/
MIW815 (2, Figure 1),7 featuring diphosphorothioate groups,
was the first one to enter the clinic, followed by others with
undisclosed structures including MK-1454, E7766,8 SB-
11285, BMS-986301, and IMSA-101. The majority of clinical
CDN STING agonists are administered by intratumoral
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a b c
, ,
Table 1. Structure−Activity Relationship of Diphosphate CDN Analogues with a 3′,5′-Carbocyclic Pyrimidine Function
a
b
Competitive TR-FRET STING binding assay. Reporter assay in HEK293T cells transiently transfected with STING variant, with or without the
c
permeabilization agent digitonin. Reporter assay in THP-1 cells endogenously expressing STING HAQ variant. n.d., not determined.
injection.9 While the intratumoral route delivers the agent
directly to the tumor site, it has disadvantages including
limited eligibility of patient populations,10 the risk of local
overdosing with an ablative effect on immune cells,11 and
significant leakage from tumors due to the physicochemical
properties of CDNs.11 To address these concerns and provide
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a differentiated treatment approach, our team initiated a
search for CDN STING agonists for systemic iv admin-
istration. We hypothesized that structural distinction from
cGAMP may reduce the likelihood of plasma degradation and
focused on additional ADME improvements, particularly
maximizing passive permeability to enable access to tumors.
We were encouraged in our search by recent reports on non-
CDN STING agonists, where short-duration drug exposures
proved sufficient for robust antitumor activities.12
agonist activity on STING. Profiles of prepared compounds
were compared to natural cGAMP ligand 1 and clinical
ADU-S100 2.
Initial SAR Exploration. It was anticipated, based on
early reports,9 that the incorporation of phosphorothioates at
both phosphate locations would be advantageous for potency,
stability, and permeability. Due to the synthetic complexity of
the diphosphorothioate scaffold, and with a need for
diastereomeric separation of up to four phosphorus stereo-
isomers, our initial SAR exploration focused on the more
tractable diphosphate scaffold. At the beginning of our search,
we set out to replace the adenosine moiety in cGAMP.
Unexpectedly and to our satisfaction, the N-linked pyrimidine
analogue 3 demonstrated encouraging STING binding
activity with EC50 = 1.31 μM, only a two-fold weaker than
ADU-S100 (2; Table 1). STING agonism was confirmed by
cellular testing with EC50 = 0.68 μM in the permeabilized
reporter assay. The weak cellular activity under standard
(nonpermeabilized) conditions and a high permeability ratio
(25-fold) was consistent with expectations for a poorly
permeable diphosphate scaffold. Addition of a 6-amino group
to the pyrimidine scaffold 4 resulted in markedly weaker
binding activity (EC50 = 11.6 μM), while the triazine
Carbocyclic nucleotides are well established as nucleotide
mimetics13 with potentially improved chemical stability and
resistance from cleavage by phosphatases and hydrolases.14
Carbocyclic systems may also offer expanded options for
nucleobase modifications, including an N-linked pyrimidine
moiety, which would be incompatible with a furanose ring
due to the unstable hemiaminal ether linkage.15,16 At the
outset of this work, examples of carbocyclic CDN analogues
were rare,17 leading to uncertainty as to the ability to replace
CDN ribose rings with cyclopentane. A thorough computa-
tional analysis of the crystal structure of cGAMP18 suggested
that ribose can be replaced with cyclopentane because the
oxygen atom is not making any specific interactions with the
protein (Figure 2). It also revealed that the binding
contributions from the imidazole portion of adenine ring
are not significant and that pyrimidine ring should be capable
of maintaining key π−π interaction with Tyr167. In contrast,
N7 of the guanosine makes a strong hydrogen bond to
Arg238, and the interaction between phosphate and guanine
C8 stabilizes the active conformation of cGAMP. These
findings suggested that the adenosine moiety in cGAMP
could be possibly replaced with heteroatom-linked carbocyclic
pyrimidine, as shown in the STING docking model of
rationally designed carbocyclic pyrimidine CDN (Figure 3).
We therefore set about to explore carbocyclic pyrimidine
opportunities within CDN scaffolds, establish SAR, and seek
to identify analogues most suitable for the systemic iv route.
Herein, we report the structure-guided discovery of novel
carbocyclic pyrimidine cyclic dinucleotide 15a with properties
suitable for systemic iv administration.
analogue 6 demonstrated improvement in binding (EC50
=
0.25 μM). Bicyclic compound 5 was prepared with
anticipation of enhancing potency through the interaction
of pyrazolo-triazine N6 and Arg238, but this modification was
unsuccessful. Replacement of the N-linker between cyclo-
pentane and pyrimidine with oxygen in 7 led to improved
binding activity compared to that of 3, resulting in superior
cellular potency under standard conditions and the lowest
permeability ratio of 9.3 from analogues prepared thus far.
The critical contribution of both pyrimidine nitrogens was
demonstrated by significantly inferior potency of pyridine,
pyrazine, and thiazoline 8−10. Finally, in agreement with
modeling predictions, pyrimidine replacement of the
guanosine portion of the CDN led to a complete loss in
potency (11−13; Figure 4). Importantly, all tested analogues
RESULTS AND DISCUSSION
■
Biological Methods and Assays. All synthesized
analogues were assessed for STING binding using com-
petitive time-resolved fluorescence energy-transfer (TR-
FRET) assay with a cGAMP-derived tracer.19 The ability to
activate the STING pathway was determined using reporter
assays in human R232 STING-transfected HEK293T cells
and human THP-1 cells that endogenously express the
STING HAQ variant. A permeabilized HEK293T cell assay
(hSTING R232) under digitonin treatment was used to
determine STING agonist EC50 values without permeability
restrictions, and the ratio of nonpermeabilized/permeabilized
EC50 values was used as a quantitative measure of
permeability potential. Mouse STING activation was
determined in an mSTING-transfected HEK293T cell assay
and in a RAW-ISG mouse monocyte line that endogenously
expresses mSTING. Serum stability was used to quantify
compound degradation by plasma enzymes and prioritize
analogues with superior stability potential. Selected com-
pounds were tested in a broader panel of mouse and human
STING-transfected HEK293T cell lines, including human
STING variants21 HAQ, AQ, and R232H. STING KO THP-
1 reporter cell line was used to confirm the dependence of
Figure 4. Inactive analogues with the 2′,5′-carbocyclic pyrimidine
function - STING binding EC50 > 25 μM.
demonstrated good serum stability (Table 1), significantly
superior to the natural cGAMP ligand. Given the matched
guanosine and diphosphate moieties, this stability improve-
ment is clearly due to the non-natural carbocyclic pyrimidine
portion of the molecules.
SAR of Diphosphorothioate Analogues with En-
hanced Passive Permeability. Having completed the SAR
survey using diphosphate CDN scaffolds, we next targeted
diphosphorothioate analogues for enhanced cellular activity
and superior ADME properties. Scaffolds 3 and 7 bearing
pyrimidine groups were selected due to preferred cellular
activities, and all synthetically accessible diphosphorothioate
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Table 2. Structures and Key Biological and ADME Properties of N- and O-Linked Pyrimidine STING Agonists
2 (ADU-S100)
14a
15a
g
g
in vitro STING activities
af
EC50 (μM)
EC50 (μM)
Emax
EC50 (μM)
Emax
,
STING Binding EC50
0.70
0.85
15.0
17.6
17.0
13.0
>25
7.2
15.3
>25
7.16
7.38
0.085 ( 0.03)
0.27 ( 0.15)
3.52 ( 0.96)
13.0
1.41
0.54
>25
1.09 ( 0.21)
n.d.
>25
0.70 ( 0.15)
0.50 ( 0.01)
0.102 ( 0.05)
0.28 ( 0.15)
0.94 ( 0.31)
3.3
1.01 ( 0.36)
0.39 ( 0.12)
>25
0.83 ( 0.18)
1.27 ( 0.31)
>25
0.28 ( 0.09)
0.76 ( 0.27)
b
hSTING R232 Cell EC50 w digitonin
124.8%
114.2%
130.8%
146.7%
b
hSTING R232 Cell EC50
permeability ratio
c
b
hSTING HAQ Cell EC50
218%
152.1%
n.d.
107.7%
117%
n.d.
288.9%
222.4%
n.d.
112.1%
122.7%
n.d.
b
hSTING AQ Cell EC50
b
hSTING R232H Cell EC50
THP-1 Cell EC50
d
d
THP-1 R232 STING Cell EC50
d
THP-1 STING KO Cell EC50
d
mouse STING Cell EC50
97.1%
120.5%
110.1%
119.7%
e
mouse RAW-ISG Cell EC50
ADME properties
Clint Eh rat; human
0.5; 0.3
93; >289
n.d.
0.4; 0.2
>289; >289
7.59
1.52
0.15
6.82 3.35
4.36 5.31
1.4 1.6
<0.16; <0.19
>289; >289
1.33
0.11
0.39
3.21 0.20
0.419 0.055
0.27 0.01
serum stability t1/2 (min)rat, human
mouse PK (1 mg/kg iv)
rat PK (1 mg/kg iv)
Clp (L/h/kg)
Vss (L/kg)
t1/2 (h)
Clp (L/h/kg)
Vss (L/kg)
t1/2 (h)
n.d.
a
b
Competitive TR-FRET STING binding assay. Reporter assay in HEK293T cells transiently transfected with STING variant, with or without the
c
permeabilization agent digitonin. The ratio of cellular EC50 values under standard/digitonin-permeabilized conditions is used as the quantitative
measure of cellular permeability. Reporter assay in THP-1 cells endogenously expressing STING HAQ variant, STING knockout, or STING R232
knock-in. Reporter assay in the RAW-ISG line stably expressing mouse STING. STING binding TR-FRET EC50 (μM) values of other isolated
isomers were 3.1 (14b), 1.91 (15b), and 52.97 (15c). Maximum activation response in reporter assays relative to ADU-S100 (2) control at 25
d
e
f
g
μM.
isomers were characterized for STING activity. Two isolated
N-linked pyrimidine isomers 14a and 14b had binding EC50
values of 0.085 and 3.1 μM, respectively (Table 2). In
comparison to 3, the more potent isomer 14a showed
significantly stronger standard (nonpermeabilized) cellular
potency and attenuated permeability ratio. O-Linked
pyrimidines 15a−c demonstrated similar patterns, with
diphosphorothioate 15a displaying the strongest potency
(0.102 μM) and a permeability ratio of 3.3 (Table 2). X-ray
analysis of 14a and 15a in STING complex (Figure 5)
revealed (RP,RP)-phosphorothioate configuration, consistent
with the preferred stereochemistry of reported phosphor-
othioate cGAMP analogues9 and absolute configuration of
ADU-S100.7,20
Characterization of Prioritized Pyrimidine Ana-
logues. Characterization of 14a and 15a in the extended
cellular panel confirmed the activity in major human
variants,21 except for R232H (Table 2). As the side chain
of His is shorter than that of Arg, we speculated that this
inferiority could be due to the inability of His to maintain the
key interaction between Arg232 and the corresponding
phosphorothioate function. Both compounds displayed
mouse cross-reactivity, and STING dependence was con-
firmed by THP-1 STING −/− line, where both compounds
were inactive. We next evaluated ADME properties of both
analogues. Good serum stability with no signs of degradation
during 120 min of treatment and low predicted microsomal
clearance was seen in both cases, setting the stage for in vivo
PK evaluation. Rat and mouse PK with systemic iv dosing,
however, revealed mostly high clearance and short terminal
half-lives. As nucleotide analogues are frequently directly
eliminated via renal clearance,22 we analyzed pooled 0−24 h
urine samples for the presence of unchanged parent
molecules. In both cases, <5% of the dose was recovered in
urine, suggesting that renal clearance of unchanged nucleotide
analogues does not play a significant role in compound
elimination. Further analysis of samples from animals treated
with an iv dose of 15a revealed that 42% of unchanged
parent recovered from feces, suggesting a biliary excretion of
parent mechanism. This hypothesis was confirmed by PK
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Figure 5. X-ray structures of 14a and 15a. Carbon is shown in white (14a, 15a), cyan, or gold (STING); nitrogen in blue; oxygen in red; and
sulfur in yellow. Water molecules are shown as red spheres. Hydrogen bonds are indicated by dashed lines.
analysis in bile duct-cannulated animals, where 84% of the
15a dose was recovered in bile. As overall in vitro and in vivo
profiles of O-linked pyrimidine 15a were superior to those of
N-linked analogue 14a, subsequent SAR exploration was
carried out on the O-linked pyrimidine scaffold.
(Figure 6A). Dose-dependent responses of 15a at 3.3 and 10
μM were clearly superior to that of 2 at 10 μM, in line with
the ∼9-fold shift seen in the THP-1 reporter cell assay
(Table 2). To further evaluate compound effects on primary
human cells, human CD14+ monocyte-derived dendritic cells
(Mo-DCs)23 were treated with 2 and 15a. In line with THP-
1 assay observations, 15a induced superior dose-dependent
expression of activation marker CD86 on Mo-DCs compared
to 2 (Figure 6B).
In Vivo PD and Efficacy Characterization of Lead 15a.
Activation of STING results in a subsequent type I interferon
response, which activates interferon signaling in tumor-
resident antigen-presenting cells, leading to T-cell dependent
antitumor immunity. Analysis of the mouse PK of 15a at 1
mg/kg dose revealed a 3.9 μM drug concentration 5 min
after iv administration.24 As this systemic exposure was at or
above cellular concentrations required for activity (Table 2
and Figure 6) and the PK profile was not significantly inferior
to diABZI STING agonist with robust antitumor activity,12a
we chose 1 mg/kg iv dose for initial PD evaluation. Murine
colon cancer CT-26 mice were treated with a single dose of
15a, leading to a significant increase in type I IFN and
proinflammatory markers, such as IFNα, MIP-1β, IL-18
(Figure 7A), and additional 12 cytokines (data not shown).
The antitumor activity was confirmed in the efficacy study
using 1 and 3 mg/kg iv doses of 15a with intermittent days
0, 3, and 6 schedule in the CT-26 model. Compound
treatment led to significant tumor growth inhibition as
evidenced by tumor growth analysis. The 1 mg/kg treatment
mostly led to tumor stasis with 1/8 tumor-free animals, while
SAR Expansion of O-Linked Carbocyclic Pyrimidine
Scaffold. SAR of the guanosine modification is summarized
in Table 3, with data shown for the most potent
phosphorothioate stereoisomers. Except for azaguanosine
16a, the remaining analogues 17a and 18a displayed inferior
potency. We next turned our attention to the substitution of
ribose and cyclopentane rings (Table 3). Both deoxyribose
19a and fluorodeoxyribose 20a analogues showed comparable
TR-FRET activity to 15a. Interestingly, even large sub-
stituents such as trifluoroethyl in 21a did not attenuate the
potency. Introduction of fluorine to cyclopentane ring in 22a
demonstrated an ∼3-fold STING binding activity improve-
ment over 15a. While 20a and 22a had marginal improve-
ments in STING binding or cellular activities compared to
15a, the permeability ratio was not decreased, and the
potency benefit was offset by more challenging access to
building blocks. We therefore selected 15a for further
pharmacology evaluation.
In Vitro Mechanistic Characterization of Lead 15a. To
gain additional confidence in STING pathway activation, the
human monocyte cell line THP-1 was incubated with the
prioritized analogue 15a and responses were compared to
vehicle control and clinical benchmark 2. As anticipated,
compound treatments led to activation of STING pathway
markers pSTING, pTBK1, and pIRF3 at the protein level
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Table 3. Impact of Guanosine or Ribose/Cyclopentane Modifications on SAR of O-Linked Pyrimidine STING Agonists
7/8 animals in the 3 mg/kg group remained tumor-free at
the end of the study (Figure 7B,C). Both dosing levels were
well tolerated with 3.5 and 11.2% maximum body weight
losses followed by full recovery (Figure 7D). All animals with
complete tumor regression were subsequently rechallenged
with CT-26 cells. In mice with complete response (n = 4),
the absence of tumor growth was suggestive of long-term
antitumor immunity, contrasting to the normal tumor growth
rate in age-matched control animals (Figure 7E). CD8 T-cell
dependence of antitumor immunity was confirmed by co-
administration of anti-CD8α depletion antibody at the
rechallenge, resulting in the restoration of tumor growth
(Figure 7E).
Chemistry. Synthesis of Diphosphate Analogues.
Several synthetic routes were developed to access diphos-
phate analogues 3−13 to allow SAR exploration of adenosine
and guanosine replacements with carbocyclic pyrimidines.
Routes utilized individual monomer building blocks bearing
H-phosphonate and phosphoroamidite groups installed to
facilitate their coupling to form noncyclic dimers, which were
further transformed into target cyclic dinucleotides.
Scheme 1 shows coupling transformation of cyclopentane
pyrimidine 5′-hydroxy-3′-H-phosphonate i with 2′-guanosine
phosphoroamidite ii using ethylthiotetrazole and subsequent
oxidation with t-butyl hydrogen peroxide, resulting in the
formation of a 2′,5′-phosphate connection.25 Removal of the
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remaining 5′-DMTr protecting group with aqueous acetic
acid26 provided dimeric phosphate ester iii. Subsequent
treatment of iii with 2-chloro-5,5-dimethyl-1,2,3-dioxaphos-
phorinane 2-oxide followed by oxidation with iodine afforded
protected cyclic diphosphate iv.27 Removal of protecting
groups from the diphosphate iv with methylamine followed
by hydrogen fluoride triethylamine resulted in the fully
deprotected cyclic diphosphate compound vi (analogues 4−
9).
An alternative method for the formation of the cyclic
diphosphate scaffold vi is shown in Scheme 2. Cyclopentane
pyrimidine 3′-phosphoroamidite vii was treated with 5′-
hydroxy-2′-guanosine H-phosphonate viii in a similar manner
as shown in Scheme 1 to create intermediate ix with a 3′,5′-
phosphate connection. Subsequent transformations using the
same conditions shown in Scheme 1 afforded cyclic
diphosphate compound vi (analogues 3 and 10).
Figure 6. STING activation in cellular systems by 2 and 15a. (A)
Expression of STING pathway markers in human monocyte cell line
THP-1 following a 2 h treatment. (B) Expression of activation
marker CD86 on human monocyte-derived dendritic cells following
a 24 h treatment.
Scheme 3 shows the synthesis of guanosine-site carbocyclic
pyrimidine CDNs 11−13. Starting from cyclopentane 2′-
phosphoroamidite xii and 3′-adenosine H-phosphonate xiii
Figure 7. PD and efficacy profile of 15a in CT-26 murine tumor-bearing mice. (A) Response of type I IFN and proinflammatory cytokines in
the plasma at 6 h after single treatment of 15a (1 mg/kg, iv). (B) In vivo efficacy study for 15a using 1 or 3 mg/kg iv treatments on days 0, 3,
and 6. (C) Tumor growth curves of individual animals in control (left) and 15a treatment groups (middle and right). Complete regression
(CR) rates on day 51 are shown. (D) Body weight change (BWC) during in vivo efficacy study. (E) Rechallenge study with CT-26 tumor
̈
cellscomparison of tumor growth between naive animals and complete responders from 15a treatment arms with or without anti-CD8α
depletion antibody.
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a
Scheme 1. Representative Synthetic Route for Cyclic Diphosphate Analogues 4−9
a
Reagents and conditions: (a) (i) ethylthiotetrazole, MeCN; (ii) t-BuOOH; (iii) AcOH, H2O; (b) (i) 2-chloro-5,5-dimethyl-1,2,3-
dioxaphosphorinane 2-oxide, pyridine; (ii) H2O, I2; (c) MeNH2, EtOH; and (d) 3HF-Et3N, pyridine, Et3N, 50 °C.
and utilizing transformations shown in Scheme 1, the final
cyclic dinucleotide analogues 11−13 were prepared.28
center, after the cyclization step, or after the deprotection
with methylamine.
Another method for the preparation of cyclic diphosphor-
othioates is shown in Scheme 6. It begins from 3′-
cyclopentane H-phosphonate xxxi and phosphoroimidite
xxxii bearing a 3′-5′-cyclic silyl protecting group, which is
subsequently selectively deprotected at the 5′-position to
afford intermediate xxxiv. The presence of the bulky silyl
protecting group led to the preferential formation of the
desired (R)-phosphorothioate center in xxxv.32 Transforma-
tions to the intermediate xxxvi and separation of stereo-
isomers to afford analogues 16 and 18 were carried out
similarly as in Scheme 4.
Synthesis of Cyclic Diphosphorothioate Analogues.
Scheme 4 depicts the synthesis of cyclic diphosphorothioate
analogues. Following the formation of phosphite ester
between cyclopentane 3′-H-phosphonate xix and phosphor-
oamidite xx, oxidation with a sulfur transfer reagent29
followed by DMTr deprotection leads to the formation of
protected phosphorothioate intermediates xxi. Both stereo-
isomers are formed at approximately a 1:1 ratio, and their
separation using reverse-phase chromatography is possible in
some cases (see the Experimental Methods and Supporting
Methods section). The formation of 3′-5′-connection is
carried out using a similar two-step process, forming
phosphite ester followed by oxidation29 to mixture of
phosphorothioate isomers.30 Separation of the stereoisomers
is either accomplished at this point or at intermediates xxiii
after treatment with methylamine. 3′-Silyl group (if needed)
is then removed to give analogues 14, 15, and 19−22.31
An alternative method for the formation of cyclic
diphosphorothioate analogues is shown in Scheme 5. Initial
3′-5′-phosphorothioate connection is built from 2′-inosine H-
phosphonate xxv and 3′-cyclopentane phosphoramidite xxvi
and then follows similar steps as in Scheme 4 to afford the
final analogues 17. Isolation of individual stereoisomers is
either accomplished after the formation of the first stereo-
CONCLUSIONS
■
In summary, we have identified novel carbocyclic pyrimidine
STING agonists with suitable properties for systemic
intravenous administration. As the majority of current clinical
STING CDN agonists are administered using the intra-
tumoral route, systemic dosing represents an important
option for patients who cannot benefit from localized
tumor administration. Characterization of the prioritized
analogue 15a demonstrated binding, cellular activity, and
permeability significantly superior to natural cGAMP and the
clinical benchmark ADU-S100. While rapid biliary excretion
was the main driver of high in vivo clearance, compound
optimization minimized other elimination pathways, including
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a
Scheme 2. Representative Synthetic Route for Cyclic Diphosphate Analogues 3 and 10
a
Reagents and conditions: (a) (i) ethylthiotetrazole, MeCN, 1 h; (ii) t-BuOOH; (iii) AcOH, H2O; (b) (i) 2-chloro-5,5-dimethyl-1,2,3-
dioxaphosphorinane 2-oxide, pyridine; (ii) H2O, I2; (c) MeNH2, EtOH; and (d) 3HF-Et3N, pyridine, Et3N, 50 °C.
a
Scheme 3. Representative Synthetic Route for Cyclic Diphosphate Analogues 11−13
a
Reagents and conditions: (a) (i) ethylthiotetrazole, MeCN; (ii) t-BuOOH; (b) TFA, H2O, 0 °C; (c) (i) 2-chloro-5,5-dimethyl-1,2,3-
dioxaphosphorinane 2-oxide, pyridine; (ii) H2O, I2; (d) MeNH2, EtOH; and (e) 3HF-Et3N, pyridine, MeOH, 50 °C.
plasma degradation and microsomal clearance. In vivo testing
demonstrated that the intravenous PK profile was sufficient
to drive robust type I IFN response and T-cell dependent
long-term antitumor immunity at well-tolerated doses in
mouse models. Additional characterization of 15a to
understand its suitability for patient administration is
underway and will be reported in due course.
EXPERIMENTAL METHODS
■
Permeability Ratio. The ratio of cellular EC50 values under
standard (nonpermeabilized)/permeabilized conditions was used as
a quantitative measure of permeability potential. Permeabilization of
cells was achieved using treatment with digitonin. High permeability
ratio indicates low cellular penetration, while favorable permeability
profile is indicated by a low ratio. It is worth noting that factors
beyond passive permeability contribute to cellular transport,
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a
Scheme 4. Representative Synthetic Route for Diphosphorothioate Analogues 14, 15, and 19−22
a
Reagents and conditions: (a) (i) ethylthiotetrazole, MeCN; (ii) ((dimethylamino methylidene)amino)-3H-1,2,4-dithiazoline-3-thione; (iii)
AcOH, H2O; (b) (i) 2-chloro-5,5-dimethyl-1,2,3-dioxaphosphorinane 2-oxide, pyridine; or diphenyl chlorophosphite, −35 °C; (ii) H2O, 3H-1,2-
benzodithiol-3-one 1,1-dioxide or 3H-1,2-benzodithiol-3-one; (c) MeNH2, EtOH; and (d) 3HF-Et3N, pyridine, Et3N, 50 °C (if necessary).
including active cellular transporters aiding intracellular import of
cyclic dinucleotides.33
from dry ACN (3 × 10 mL), dissolved in ACN (1.06 mL), and
added to the reaction mixture under an atmosphere of argon. The
reaction mixture was allowed to stir at rt for 50 min. tert-Butyl
hydroperoxide (5.5 M in nonane, 0.35 mL, 1.91 mmol) was added,
and the reaction mixture was allowed to stir at rt for 45 min. The
reaction mixture was quenched by the addition of sodium thiosulfate
(353 mg, 2.23 mmol) in water (0.353 mL). The solvents were
evaporated and then placed under vacuum for 2 min. The residue
was dissolved in a mixture of acetic acid (2.53 mL, 44.2 mmol) and
water (0.63 mL). The reaction mixture was allowed to stir at rt for 1
h and then concentrated from toluene and concentrated. The
residue was concentrated again with toluene and then purified by
silica gel chromatography (0−50% MeOH in DCM) to give I-7
[(1S,2R,4R)-2-[[[(2R,3R,4R,5R)-4-[tert-butyl(dimethyl)silyl]oxy-5-
(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-
yl]tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-
4-pyrimidin-4-yloxy-cyclopentoxy]phosphinic acid (372 mg, 68%).
LC−MS (FA): m/z = 857.3 (M + H).
Step 2: intermediate I-7 (370 mg, 0.432 mmol) was concentrated
from dry ACN (3 × 15 mL) and then placed under vacuum for 15
min. The residue was dissolved in pyridine (8.64 mL) under an
argon atmosphere. 2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane
2-oxide (291 mg, 1.51 mmol) was then added, and the reaction
mixture was allowed to stir at rt for 30 min. Water was added
(0.272 mL) followed by iodine (143 mg, 0.561 mmol). The mixture
was allowed to stir at rt under argon for 8 min. Sodium thiosulfate
(91.5 mg, 0.561 mmol) in water (0.5 mL) was added. The reaction
mixture was allowed to stir at rt for 10 min. Dry toluene was added
(15 mL) and concentrated. The residue was concentrated from
Chemistry. Nuclear magnetic resonance (NMR) spectra were
recorded in the solvent reported on a 400 MHz Bruker spectrometer
using residual solvent peaks as the reference. Compound purity was
determined by analysis of the diode array UV trace of a liquid
chromatography−mass spectrometry (LC−MS) spectrum using the
following procedure: compounds were dissolved in dimethyl
sulfoxide (DMSO), methanol, or acetonitrile, and the solutions
were analyzed using a Hewlett Packard HP1100 or Agilent 1100
Series LC system connected to a Micromass mass spectrometer
using reverse-phase C18 columns. One of two gradients was used to
elute the compounds: either a formic acid (FA) gradient
(acetonitrile containing 0−100% of 0.1% formic acid in water) or
an ammonium acetate (AA) gradient (acetonitrile containing 0−
100% of 10 mM ammonium acetate in water). All compounds were
determined to be >95% pure unless otherwise noted.
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2,10-dioxido-14-(pyrimidin-4-yloxy)decahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (7). Step 1: a mixture of I-4 [(1S,2R,4R)-2-(hydroxymethyl)-4-
pyrimidin-4-yloxy-cyclopentoxy]phosphinic acid (175 mg, 0.638
mmol) and N-[9-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-
methoxy]methyl]-4-[tert-butyl(dimethyl)silyl]oxy-3-[2-cyanoethoxy-
(diisopropylamino)phosphanyl]oxy-tetrahydrofuran-2-yl]-6-oxo-1H-
purin-2-yl]-2-methyl-propanamide (743 mg, 0.766 mmol) was
concentrated from dry ACN (3 × 15 mL). The residue was then
suspended in ACN (2.34 mL) under argon. In a separate flask, 5-
(ethylthio)-1H-tetrazole (249 mg, 1.91 mmol) was concentrated
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a
Scheme 5. Synthetic Route for Diphosphorothioate 17
a
Reagents and conditions: (a) (i) ethylthiotetrazole, MeCN; (ii) ((dimethylamino methylidene)amino)-3H-1,2,4-dithiazoline-3-thione; (iii)
AcOH, H2O; (b) (i) 2-chloro-5,5-dimethyl-1,2,3-dioxaphosphorinane 2-oxide, pyridine; (ii) H2O, 3H-1,2-benzodithiol-3-one 1,1-dioxide; (c)
MeNH2, EtOH; and (d) 3HF-Et3N, pyridine, Et3N, 50 °C.
a
Scheme 6. Representative Synthetic Route for Diphosphorothioate Analogues 16 and 18
a
Reagents and conditions: (a) (i) ethylthiotetrazole, MeCN; (ii) ((dimethylamino methylidene)amino)-3H-1,2,4-dithiazoline-3-thione; (b) TFA,
H2O, 0 °C, 2 h; (c) (i) 2-chloro-5,5-dimethyl-1,2,3-dioxaphosphorinane 2-oxide, pyridine; (ii) H2O, 3H-1,2-benzodithiol-3-one 1,1-dioxide; (d)
MeNH2, EtOH; and (e) HF-Et3N, pyridine, Et3N, 50 °C.
toluene (2 × 15 mL). The crude mixture was purified by silica gel
chromatography (0−50% MeOH in DCM) to provide I-8 N-{9-
[(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-
10-(2-cyanoethoxy)-2-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-
decahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-
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2-yl}-2-methylpropanamide (366 mg, 99%). LC−MS (FA): m/z =
855.3 (M + H).
(D2O) δ -0.73 (s, 1P), -0.77 (s, 1P). HRMS m/z [M + H]+ calcd
for C21H26N10O11P2 657.1331, observed 657.1336.
Step 3: intermediate I-8 (366 mg, 0.428 mmol) was taken up in
methylamine (33% in EtOH, 12.8 mL, 103 mmol), and the mixture
was allowed to stir at rt for 90 min. The reaction mixture was
concentrated. The residue was then dry-loaded on silica gel and
purified by silica gel chromatography (0−80% MeOH in DCM) to
provide 2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl-
(dimethyl)silyl]oxy}-2,10-dihydroxy-2,10-dioxido-14-(pyrimidin-4-
yloxy)decahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one
I-9 (159 mg, 51%) as a white solid. LC−MS (FA): m/z = 732.3 (M
+ H).
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2,10-dioxido-14-(1,3,5-triazin-2-ylamino)decahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
1
one (6). LC−MS (FA): m/z = 618.0 (M + H). H NMR (D2O) δ
8.48 (s, 1H), 8.36 (s, 1H), 8.02 (s, 1H), 6.00 (d, J = 8.4 Hz, 1H),
5.33 (dt, J = 4.2, 8.5 Hz, 1H), 4.77−4.73 (m, 1H), 4.62 (d, J = 4.2
Hz, 1H), 4.43−4.35 (m, 2H), 4.22−4.08 (m, 2H), 3.98−3.91 (m,
1H), 3.81−3.73 (m, 1H), 3.14 (q, J = 7.3 Hz, 12H), 2.44−2.34 (m,
2H), 2.30−2.22 (m, 1H), 2.18−2.09 (m, 1H), 1.33−1.26 (m, 1H),
1.22 (t, J = 7.3 Hz, 18H). 31P NMR (D2O) δ −0.50 (s, 1P), −0.78
(s, 1P). HRMS m/z [M + H]+ calcd for C19H25N9O11P2 618.1222,
observed 618.1236.
Step 4: to a suspension of I-9 (159 mg, 0.217 mmol) in pyridine
(1.09 mL) was added triethylamine trihydrofluoride (0.175 mL, 1.09
mmol) and then TEA (2.72 mL, 19.3 mmol). The reaction mixture
was sealed in a propylene tube and vigorously stirred at 50 °C for 4
h. The reaction mixture was cooled to rt. The reaction mixture was
diluted with water (6.4 mL) and then CaCl2 (0.339 M in water, 6.4
mL, 2.17 mmol) was added. The cloudy white mixture was allowed
to stir at rt for 30 min. The suspension was filtered through celite
and the celite was washed with water (5 × 5 mL). The clear
aqueous filtrate was concentrated to a solid residue. No HF was
observed by 19F NMR. The crude residue was suspended in ACN
(25 mL) and then adsorbed on celite. The crude compound was
purified by reverse-phase flash column chromatography (10% ACN
in aqueous triethylammonium acetate (10 mM)) to provide
compound 7 2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-tri-
hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)decahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one
(70 mg, 39%) as an N,N-diethylethanamine salt. LC−MS (FA): m/z
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2 , 1 0 - d i o x i d o - 1 4 - ( p y r i d i n - 2 - y l o x y ) d e c a h y d r o - 5 , 8 -
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
1
one (8). LC−MS (AA): m/z = 617.2 (M + H). H NMR (MeOD)
δ 8.03 (s, 1H), 7.98 (dd, J = 5.0, 1.1 Hz, 1H), 7.53 (ddd, J = 8.7,
7.2, 1.9 Hz, 1H), 6.79 (dd, J = 6.4, 5.5 Hz, 1H), 6.61 (d, J = 8.3 Hz,
1H), 5.94 (d, J = 8.3 Hz, 1H), 5.28−5.40 (m, 2H), 4.85 (quin, J =
4.3 Hz, 1H), 4.60 (d, J = 4.0 Hz, 1H), 4.17−4.23 (m, 2H), 4.00−
4.10 (m, 1H), 3.92−4.00 (m, 1H), 3.64−3.78 (m, 1H), 3.03 (d, J =
7.4 Hz, 12H), 2.24−2.45 (m, 4H), 1.37−1.49 (m, 1H), 1.16 (t, J =
7.3 Hz, 18H); 31P NMR (MeOD) δ 0.42 (s, 1P), 0.56 (s, 1P).
HRMS m/z [M+H]+ calcd for C21H26N6O12P2 617.1157, observed
617.1170.
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2 , 1 0 - d i o x i d o - 1 4 - ( p y r a z i n - 2 - y l o x y ) d e c a h y d r o - 5 , 8 -
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
1
1
= 618.1 (M + H). H NMR (D2O) δ 8.63 (s, 1H), 8.40 (d, J = 6.0
one (9). LC−MS (AA): m/z = 618.2 (M + H). H NMR (MeOD)
Hz, 1H), 8.04 (s, 1H), 6.78 (dd, J = 6.1, 1.1 Hz, 1H), 5.99 (d, J =
8.4 Hz, 1H), 5.31−5.43 (m, 2H), 4.77−4.87 (m, 1H), 4.62 (d, J =
4.2 Hz, 1H), 4.38 (q, J = 2.3 Hz, 1H), 4.14−4.19 (m, 2H), 3.95 (dt,
J = 10.6, 3.4 Hz, 1H), 3.82 (dt, J = 10.6, 6.5 Hz, 1H), 3.15 (q, J =
7.3 Hz, 12H), 2.33−2.56 (m, 3H), 2.20−2.31 (m, 1H), 1.54−1.68
(m, 1H), 1.23 (t, J = 7.3 Hz, 18H). 31P NMR (D2O) δ 0.57 (s, 1P),
0.65 (s, 1P). HRMS m/z [M + H]+ calcd for C20H25N7O12P2
618.1109, observed 618.112.
δ 8.02 (dd, J = 2.8, 1.3 Hz, 1H), 7.99−8.01 (m, 2H), 7.95 (d, J =
2.8 Hz, 1H), 5.93 (d, J = 8.4 Hz, 1H), 5.37−5.46 (m, 1H), 5.33
(ddd, J = 8.8, 5.4, 4.5 Hz, 1H), 4.84 (quin, J = 5.8 Hz, 1H), 4.58
(d, J = 4.2 Hz, 1H), 4.11−4.21 (m, 2H), 4.03 (ddd, J = 11.2, 8.1,
2.2 Hz, 1H), 3.96 (ddd, J = 10.5, 5.9, 3.1 Hz, 1H), 3.71 (ddd, J =
10.8, 8.4 Hz, 1H), 3.02 (q, J = 7.3 Hz, 15H), 2.33−2.46 (m, 3H),
2.28 (dt, J = 14.4, 5.5 Hz, 1H), 1.44 (ddd, J = 17.6, 10.4, 4.8 Hz,
1H), 1.15 (t, J = 7.3 Hz, 23H); 31P NMR (MeOD) δ 0.42 (s, 1P),
0.58 (s, 1P). HRMS m/z [M+H]+ calcd for C20H25N7O12P2
618.1109, observed 618.1116.
General Procedure for the Synthesis of Compounds 4−6, 8,
and 9. The compounds listed below were prepared according to the
procedures used for the synthesis of compound 7, using H-
phosphonate intermediates I-1, I-2, I-3, I-5, and I-6, respectively, in
step 1. Compounds were isolated as N,N-diethylethanamine salts.
2-Amino-9-{(5R,7R,8R,12aR,14R,15aS,16R)-14-[(6-aminopyrimi-
din-4-yl)amino]-2,10,16-trihydroxy-2,10-dioxidodecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl}-1,9-dihydro-6H-purin-6-
one (4). The benzoyl group in this compound was removed by
treatment with methylamine in step 3. LC−MS (AA): m/z = 632.2
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2,10-dioxido-14-(pyrimidin-4-ylamino)decahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (3). Step 1: a mixture of I-10 (1S,2R,4R)-2-{[bis(4-
methoxyphenyl)(phenyl)methoxy]methyl}-4-(pyrimidin-4-ylamino)
cyclopentyl 2-cyanoethyl diisopropylphosphoramidoite (630 mg,
0.885 mmol) and I-12 (2R,3R,4R,5R)-4-{[tert-butyl(dimethyl)silyl]-
oxy}-5-(hydroxymethyl)-2-[2-(isobutyrylamino)-6-oxo-1,6-dihydro-
9H-purin-9-yl]tetrahydrofuran-3-yl hydrogen phosphonate (360 mg,
0.677 mmol) was dissolved in dry acetonitrile and concentrated to
dryness (3 × 25 mL). The residue was then dissolved in ACN (2.63
mL) under an atmosphere of nitrogen. In a separate flask, 5-
(ethylthio)-1H-tetrazole (265 mg, 2.03 mmol) was dissolved in dry
acetonitrile and concentrated to dryness (3 × 10 mL), taken up in
ACN (1.20 mL), and added to the reaction mixture. The reaction
mixture was allowed to stir at rt for 1 h. tert-Butyl hydroperoxide
(5.50 M in nonane, 0.370 mL, 2.03 mmol) was added, and the
reaction mixture was allowed to stir at rt for 45 min. The reaction
mixture was quenched by the addition of sodium thiosulfate (375
mg, 2.37 mmol) in water (0.398 mL, 22.1 mmol), concentrated, and
dried on vacuum for 2 min. The residue was dissolved in a mixture
of acetic acid (2.85 mL, 49.8 mmol) and water (0.713 mL, 39.6
mmol), sonicated for 2 min, and allowed to stir at rt for 1 h. The
reaction mixture was diluted with toluene, concentrated, and the
crude compound was purified by silica gel chromatography (0−70%
MeOH in DCM) to provide I-13 (2R,3R,4R,5R)-4-{[tert-butyl-
1
(M + H). H NMR (D2O) δ 7.99 (s, 1H), 7.96 (s, 1H), 5.97 (d, J
= 8.4 Hz, 1H), 5.56 (s, 1H), 5.39 (td, J = 8.6, 4.2 Hz, 1H), 4.72−
4.80 (m, 1H), 4.60 (d, J = 4.2 Hz, 1H), 4.38 (q, J = 2.6 Hz, 1H),
4.03−4.22 (m, 3H), 3.94 (dt, J = 10.4, 2.6 Hz, 1H), 3.74−3.83 (m,
1H), 3.15 (q, J = 7.3 Hz, 12H), 2.33−2.47 (m, 2H), 2.12−2.21 (m,
2H), 1.28−1.36 (m, 1H), 1.23 (t, J = 7.3 Hz, 18H); 31P NMR
(D2O) δ 0.77 (s, 1P), 0.83 (s, 1P). HRMS m/z [M + H]+ calcd for
C20H27N9O11P2 632.1378, observed 632.1387.
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2,10-dioxido-14-(pyrazolo [1,5-a][1,3,5]triazin-4-ylamino)-
decahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10] pentaoxadi-
phosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (5).
LC−MS (AA): m/z = 657.2 (M + H). 1H NMR (D2O) δ 8.07
(s, 1H), 8.04 (s, 1H), 7.90 (d, J = 2.2 Hz, 1H), 6.37 (d, J = 2.2 Hz,
1H), 5.99 (d, J = 8.4 Hz, 1H), 5.43 (dt, J = 4.2, 7.8 Hz, 1H), 4.91−
4.83 (m, 1H), 4.69 (d, J = 4.2 Hz, 1H), 4.63−4.55 (m, 1H), 4.43−
4.40 (m, 1H), 4.25−4.15 (m, 2H), 4.13−4.07 (m, 1H), 3.97−3.89
(m, 1H), 3.16 (q, J = 7.3 Hz, 12H), 2.60−2.39 (m, 3H), 2.35−2.26
(m, 1H), 1.62−1.53 (m, 1H), 1.25 (t, J = 7.3 Hz, 18H); 31P NMR
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(dimethyl)silyl]oxy}-5-({[(S)-(2-cyanoethoxy){[(1S,2R,4R)-2-(hy-
droxymethyl)-4-(pyrimidin-4-ylamino)cyclopentyl]oxy}phosphoryl]-
oxy}methyl)-2-[2-(isobutyrylamino)-6-oxo-1,6-dihydro-9H-purin-9-
yl]tetrahydrofuran-3-yl hydrogen phosphonate (320 mg, 55%) as a
mixture of diastereomers. LC−MS (AA): m/z = 856.4 (M + H).
Step 2: intermediate I-13 (300 mg, 0.351 mmol) was dissolved in
dry acetonitrile and concentrated to dryness (3 × 10 mL) and then
dried under vacuum for 15 min. The residue was dissolved in
pyridine (7.99 mL, 98.7 mmol) under an argon atmosphere. Then,
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (236 mg,
1.23 mmol) was added, and the reaction mixture was allowed to
stir at rt for 45 min. Water (0.221 mL, 12.3 mmol) and iodine (116
mg, 0.469 mmol) were added, and stirring was continued for 8 min.
Sodium thiosulfate (74.3 mg, 0.456 mmol) in water (0.5 mL) was
added. The reaction mixture was allowed to stir at rt for 15 min.
Toluene (15 mL) was added, and the reaction mixture was
concentrated. The residue was concentrated from toluene (15 mL)
and dried on vacuum for 15 min. The crude compound was purified
by silica gel chromatography (0−100% MeOH in DCM) to provide
I-14 N-{9-[(5R,7R,8R,12aR,14R,15aS,16R)-16-{[tert-butyl-
(dimethyl)silyl]oxy}-2-(2-cyanoethoxy)-10-hydroxy-2,10-dioxido-14-
(pyrimidin-4-ylamino)decahydro-5,8-methanocyclopenta[l]-
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-di-
hydro-1H-purin-2-yl}-2-methylpropanamide (170 mg, 57%) as a
mixture of diastereomers. LC−MS (AA): m/z = 854.3 (M + H).
Step 3: intermediate I-14 (170 mg, 0.200 mmol) was taken up in
methylamine (33% in EtOH, 5.87 mL, 47.1 mmol), and the reaction
mixture was allowed to stir at rt for 90 min. The reaction mixture
was concentrated and dried on vacuum for 10 min. The crude
compound was purified by silica gel chromatography (0−100%
MeOH in DCM) to provide I-15 2-amino-9-[(5R,7R,8R,12aR,14-
R,15aS,16R)-16-{[tert-butyl(dimethyl)silyl]oxy}-2,10-dihydroxy-2,10-
dioxido-14-(pyrimidin-4-ylamino)decahydro-5,8-methanocyclopenta-
[l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihy-
dro-6H-purin-6-one (135 mg, 93%). LC−MS (FA): m/z = 731.3
(M + H).
7.96 (s, 1H), 7.88 (s, 1H), 5.90 (d, J = 8.4 Hz, 1H), 5.29−5.19 (m,
2H), 4.74−4.68 (m, 1H), 4.53 (d, J = 4.1 Hz, 1H), 4.30−4.26 (m,
1H), 4.09−4.05 (m, 2H), 3.89−3.83 (m, 1H), 3.77−3.69 (m, 1H),
3.04 (q, J = 7.3 Hz, 12H), 2.47−2.34 (m, 2H), 2.34−2.25 (m, 1H),
2.22−2.13 (m, 1H), 1.57−1.50 (m, 1H), 1.12 (t, J = 7.3 Hz, 18H).
31P NMR (D2O) δ -0.56 (s, 1P), -0.65 (s, 1P). HRMS m/z [M +
H]+ calcd for C18H23N7O12P2S 624.0673, observed 624.0699.
(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-Amino-9H-purin-9-yl)-
7-(pyrimidin-4-yloxy)octahydro-4H,12H-5,8-methanofuro[3,2-d]
[1,3,7,9,2,8]tetraoxadiphosphacyclotetradecine-2,10,15,16-tetrol
2,10-dioxide (12). Step 1: a mixture of I-17 3-[[(6aR,8R,9S,9aR)-
2,2,4,4-tetraisopropyl-8-pyrimidin-4-yloxy-6,6a,7,8,9,9a-
hexahydrocyclopenta[f ][1,3,5,2,4]trioxadisilocin-9-yl]oxy-(diisopro-
pylamino) phosphanyl]oxypropanenitrile (745.6 mg, 1.114 mmol)
and I-19 [(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl-
(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl] oxy-
phosphinic acid (490 mg, 0.892 mmol) was dissolved in dry
acetonitrile and concentrated to dryness (3 × 25 mL). The residue
was then dissolved in a mixture of ACN (2.80 mL) and THF (0.80
mL) under an atmosphere of nitrogen. In a separate flask, 5-
(ethylthio)-1H-tetrazole (365 mg, 2.80 mmol) was dissolved in dry
acetonitrile and concentrated to dryness (3 × 10 mL), taken up in
ACN (1.60 mL), and added to the reaction mixture. The reaction
mixture was allowed to stir at rt for 1.5 h. tert-Butyl hydroperoxide
(5.50 M in nonane, 0.49 mL, 2.70 mmol) was added, and the
reaction mixture was allowed to stir at rt for 60 min. The reaction
mixture was quenched by the addition of sodium thiosulfate
pentahydrate (780 mg, 3.14 mmol) in water (1.50 mL, 83 mmol).
The reaction mixture was concentrated and dried on vacuum for 2
min. The crude compound was purified by silica gel chromatography
(0−80% MeOH in DCM) to provide I-20 [(2R,3R,4R,5R)-2-
[[[(6aR,8R,9S,9aR)-2,2,4,4-tetraisopropyl-8-pyrimidin-4-yloxy-
6,6a,7,8,9,9a-hexahydrocyclopenta[f][1,3,5,2,4]trioxadisilocin-9-yl]-
oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-5-(6-benzamidopurin-9-
yl)-4-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-3-yl]-
oxyphosphinic acid (880 mg, 87%) as a mixture of diastereomers.
LC−MS (AA): m/z = 1133 (M + H).
Step 2: intermediate I-20 (880 mg, 0.776 mmol) was taken up in
THF (8.4 mL) and water (2.1 mL) and cooled to 0 °C. TFA (2.1
mL, 28.0 mmol) was added dropwise, and the reaction mixture was
allowed to stir at 0 °C for 2 h. Sodium bicarbonate (3.54 g, 41.9
mmol) was added portionwise, followed by water and EtOAc. The
reaction mixture was allowed to warm to rt and extracted into
EtOAc. The combined organic phases were washed with brine, dried
with Na2SO4, and concentrated. The crude compound was purified
by silica gel chromatography (0−20% MeOH in DCM) to provide
I-21 [(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl-
(dimethyl)silyl]oxy-2-[[2-cyanoethoxy-[(1S,2R,3R,5R)-2-[[hydroxy-
(diisopropyl)silyl]oxy-diisopropyl-silyl]oxy-3-(hydroxymethyl)-5-pyri-
midin-4-yloxy-cyclopentoxy]phosphoryl] oxymethyl]tetrahydrofuran-
Step 4: to a suspension of I-15 (131 mg, 0.179 mmol) in pyridine
(0.896 mL, 11.1 mmol) were added triethylamine trihydrofluoride
(0.149 mL, 0.896 mmol) and then TEA (2.14 mL, 15.2 mmol). The
reaction mixture was sealed in a propylene tube and allowed to stir
at 50 °C for 5 h. The reaction mixture was cooled to rt, diluted with
water (3.21 mL), and then CaCl2 (207 mg, 2.17 mmol) in water
(3.21 mL) was added. The cloudy white mixture was allowed to stir
at rt for 60 min, filtered through celite, and the celite plug was
washed with water (5 × 5 mL). The clear aqueous filtrate was
concentrated to a solid residue. No HF was observed by 19F NMR.
The crude residue was suspended in ACN (25 mL), adsorbed onto
celite, and purified by reverse-phase flash column chromatography
(10−100% ACN in aqueous triethylammonium acetate (10 mM))
to provide compound 3 2-amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-
2,10,16-trihydroxy-2,10-dioxido-14-(pyrimidin-4-ylamino)decahydro-
5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one
(73 mg, 50%) as the N,N-diethylethanamine salt. LC−MS (AA): m/
3-yl]oxyphosphinic acid (602 mg, 67%) as
a mixture of
diastereomers. LC−MS (FA): m/z = 1151 (M + H).
Step 3: intermediate I-21 (602 mg, 0.523 mmol) was dissolved in
dry acetonitrile and concentrated to dryness (3 × 25 mL) and then
dried under vacuum for 15 min. The residue was dissolved in
pyridine (10.6 mL) under an argon atmosphere, and then 2-chloro-
5,5-dimethyl-1,2,3-dioxaphosphorinane 2-oxide (560 mg, 2.91
mmol) was added and the reaction mixture was allowed to stir at
rt for 30 min. This reaction mixture was cooled to 0 °C, and then
water (0.40 mL, 22 mmol) and iodine (175 mg, 0.668 mmol) were
added and stirring was continued for 10 min. Sodium thiosulfate
pentahydrate (185 mg, 0.745 mmol) in water (0.5 mL) was added.
The reaction mixture was allowed to stir at rt for 15 min. Toluene
(15 mL) was added, and the reaction mixture was concentrated.
The residue was concentrated from toluene (15 mL) and dried on
vacuum for 15 min. The crude compound was purified by silica gel
chromatography (0−10% MeOH in DCM) to provide I-22 (425
mg, 71%) as a mixture of diastereomers. LC−MS (AA): m/z =
1149.5 (M + H).
1
z = 617.1 (M + H). H NMR (D2O) δ 8.50 (br s, 1H), 8.02 (s,
1H), 7.95 (br s, 1H), 6.59 (br s, 1H), 5.99 (d, J = 8.3 Hz, 1H), 5.37
(td, J = 8.7, 4.2 Hz, 1H), 4.73−4.80 (m, 1H), 4.61 (d, J = 4.2 Hz,
1H), 4.47 (br s, 1H), 4.39 (q, J = 2.2 Hz, 1H), 4.11−4.23 (m, 2H),
3.95 (dt, J = 10.6, 2.7 Hz, 1H), 3.75−3.84 (m, 1H), 3.16 (q, J = 7.3
Hz, 8H), 2.35−2.48 (m, 2H), 2.23 (br d, J = 6.2 Hz, 2H), 1.37 (s,
1H), 1.24 (t, J = 7.3 Hz, 12H); 31P NMR (D2O) δ 0.72 (s, 1P),
0.79 (s, 1P). HRMS m/z [M + H]+ calcd for C20H26N8O11P2
617.1269, observed 617.1286.
2-Amino-9-[(5R,7R,8R,12aR,14R,15aS,16R)-2,10,16-trihydroxy-
2,10-dioxido-14-(1,2,5-thiadiazol-3-yloxy)decahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (10). The compound was prepared according to the procedure
used for the synthesis of compound 3 using intermediate I-11 in
1
step 1. LC−MS (AA): m/z = 624.0 (M + H). H NMR (D2O) δ
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Step 4: intermediate I-22 (420 mg, 0.365 mmol) was taken up in
methylamine (33% in EtOH, 13.6 mL, 109 mmol), and the reaction
mixture was allowed to stir at rt for 3 h. The reaction mixture was
concentrated and dried on vacuum for 10 min. The crude
compound was purified by reverse-phase flash column chromatog-
raphy (0−70% ACN in aqueous ammonium bicarbonate (5 mM))
to provide I-23 (70 mg, 19%). LC−MS (FA): m/z = 992.3 (M +
H).
methoxy]methyl]-4-[tert-butyl(dimethyl)silyl]oxy-3-[2-cyanoethoxy-
(diisopropylamino)phosphanyl]oxy-tetrahydrofuran-2-yl]-6-oxo-1H-
purin-2-yl]-2-methyl-propanamide (2.24 g, 2.31 mmol) was
dissolved in ACN and concentrated to dryness (3 × 25 mL). The
residue was then dissolved in ACN (6.51 mL) under argon. In a
separate flask, 5-(ethylthio)-1H-tetrazole (694 mg, 5.33 mmol) was
dissolved in ACN and concentrated (3 × 10 mL), dissolved in ACN
(3 mL), and added to the reaction mixture. The resulting reaction
mixture was allowed to stir at rt for 40 min. ((Dimethylamino-
methylidene)amino)-3H-1,2,4-dithiazoline-3-thione (437 mg, 2.13
mmol) was added as a solid, and the reaction mixture was allowed
to stir at rt for 45 min. The solvent was evaporated, and then the
residue was dried under vacuum for 10 min. The residue was
dissolved in a mixture of acetic acid (7.0 mL) and water (1.8 mL),
sonicated briefly, and then allowed to stir at rt for 1 h. The reaction
mixture was diluted with dry toluene and evaporated to dryness (3
× 15 mL) to give a yellow oil. The crude mixture was purified by
silica gel chromatography (0−50% MeOH in DCM) to provide I-28
(971 mg, 63%) as a mixture of two diastereomers. LC−MS (FA):
m/z = 873.3 (M + H).
Step 2: intermediate I-28 (969 mg, 1.11 mmol) was dissolved in
dry acetonitrile and concentrated to dryness (3 × 10 mL), dried
under vacuum for 10 min, and then dissolved in pyridine (22 mL)
under a nitrogen atmosphere. 2-Chloro-5,5-dimethyl-1,3,2-dioxa-
phosphorinane 2-oxide (717 mg, 3.89 mmol) was added. The
reaction mixture was allowed to stir at rt for 45 min. Water was
added (0.70 mL, 38.9 mmol) followed by 3H-1,2-benzodithiol-3-one
1,1-dioxide (269 mg, 1.33 mmol), and the reaction mixture was
allowed to stir at rt under nitrogen for 60 min. An additional
portion of 3H-1,2-benzodithiol-3-one 1,1-dioxide (67.0 mg, 0.331
mmol) was added, and stirring was continued for 10 min. Toluene
was added, the mixture was concentrated, and then concentrated
from toluene (3 × 25 mL) to provide a mixture of four
diastereomers, two major and two minor. The crude mixture was
adsorbed onto silica gel and purified by silica gel chromatography
(0−50% MeOH in DCM). I-29a−c eluted as a mixture (520 mg,
53%), and some clean I-29b was cleanly obtained (230 mg, 23%).
LC−MS (FA): m/z = 887.3 (M + H).
Step 5: to a suspension of I-23 (70 mg, 0.071 mmol) in pyridine
(0.70 mL) were added triethylamine trihydrofluoride (0.081 g, 0.809
mmol) and then TEA (2.14 mL, 15.2 mmol). The reaction mixture
was sealed in a propylene tube and allowed to stir at 50 °C for 16 h.
The reaction mixture was cooled to rt. To the reaction mixture,
CaCl2 (170 mg, 1.47 mmol) in water (3.21 mL) was added. The
cloudy white mixture was allowed to stir at rt for 60 min. Then, the
suspension was filtered through celite and the celite was washed
with water (5 × 5 mL). The clear aqueous filtrate was concentrated
to a solid residue. The crude residue was suspended in ACN (25
mL) and adsorbed onto celite. The crude mixture was purified by
reverse-phase flash column chromatography (0−15% ACN in
aqueous triethylammonium acetate (10 mM)) to provide compound
12 (42 mg, 73%) as an N,N-diethylethanamine salt. LC−MS (AA):
1
m/z = 618.2 (M + H). H NMR (D2O) δ 1.19 (t, J = 7.34 Hz,
18H) 1.44−1.57 (m, 1H) 2.19−2.32 (m, 1H) 2.56−2.70 (m, 1H)
3.11 (q, J = 7.32 Hz, 12H) 3.78 (m, 1H) 4.11 (m, 2H) 4.16−4.23
(m, 1H) 4.23−4.34 (m, 1H) 4.36−4.57 (m, 1H) 4.73 (m, 1H)
4.81−4.86 (m, 1H) 4.98 (m, 1H) 5.35−5.48 (m, 1H) 6.04−6.17
(m, 1H) 6.66 (d, J = 6.02 Hz, 1H) 8.18 (s, 1H) 8.28 (d, J = 5.90
Hz, 1H) 8.39 (s, 1H) 8.56 (s, 1H); 31P NMR (D2O) δ 0.90 (s, 1P),
0.59 (s, 1P). HRMS m/z [M + H]+ calcd for C20H25N7O12P2
618.1109, observed 618.1123.
General Procedure for the Synthesis of Compounds 11 and 13.
The compounds were prepared according to the procedure used for
the synthesis of compound 12 and intermediates I-16 and I-18 in
step 1.
(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-Amino-9H-purin-9-yl)-
7-(6-(methylamino)pyrimidin-4-yloxy)octahydro-4H,12H-5,8-
m e t h a n o f u r o [ 3 , 2 - d ] [ 1 , 3 , 7 , 9 , 2 , 8 ] -
tetraoxadiphosphacyclotetradecine-2,10,15,16-tetrol 2,10-dioxide
(11). The 6-chloro group of pyrimidine in starting material I-16 was
converted to methylamino group in step 4. LC−MS (AA): m/z =
Step 3: the mixture of I-29a−c (473 mg, 0.533 mmol) was
dissolved in methylamine (33% in EtOH, 16 mL, 129 mmol), and
the reaction mixture was allowed to stir under an atmosphere of
nitrogen at rt for 90 min. The reaction mixture was concentrated
and dried on vacuum for 10 min. The LC−MS of this crude mixture
shows three peaks with the desired product mass (one major peak).
The crude mixture was purified by reverse-phase flash column
chromatography (10−100% ACN in aqueous ammonium acetate
(10 mM)) to give I-30a (27 mg, 7%), I-30b (55 mg, 11%), and I-
30c (131 mg, 32%). LC−MS (AA): m/z = 764.2 (M + H).
Step 4: intermediate I-30c (131 mg, 0.172 mmol) was taken up
in pyridine (0.86 mL) to give a suspension. Triethylamine
trihydrofluoride (0.14 mL, 0.86 mmol) was added, followed by
TEA (2.1 mL). The reaction mixture was sealed in a propylene tube
and allowed to stir at 50 °C overnight. The reaction mixture was
diluted with water (3.21 mL), and then a solution of CaCl2 in water
(0.556 M in water, 3.2 mL, 1.72 mmol) was added. The cloudy
white mixture was allowed to stir at rt for 60 min. The suspension
was filtered through celite, and the celite was washed with water (5
× 5 mL). The clear aqueous filtrate was concentrated to a solid
residue. No HF was observed by 19F NMR. The crude compound
was adsorbed onto celite and purified by reverse-phase flash column
chromatography (10% ACN in aqueous triethylammonium acetate
(10 mM)) to provide clean compound 15a as the N,N-
diethylethanamine salt (78 mg, 53%). LC−MS (AA) m/z = 650.1
1
647.0 (M + H). H NMR (CD3OD) δ 1.29 (t, J = 7.34 Hz, 18H)
1.58 (m, 1H) 2.24 (m, 1H) 2.57 (m, 1H) 2.86 (s, 3H) 3.18 (d, J =
7.34 Hz, 12H) 3.83 (m, 1H) 4.05−4.24 (m, 3H) 4.31−4.45 (m,
2H) 4.62−4.70 (m, 1H) 5.09−5.16 (m, 2H) 5.41 (q, J = 8.64 Hz,
1H) 5.85 (s, 1H) 6.08 (d, J = 5.50 Hz, 1H) 8.10 (s, 1H) 8.23 (s,
1H) 8.38 (s, 1H). 31P NMR (CD3OD) δ -1.13 (s, 1P), -0.07 (s,
1P). HRMS m/z [M + H]+ calcd for C21H28N8O12P2 647.1375,
observed 647.1393.
(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-Amino-9H-purin-9-yl)-
7-(pyrimidin-4-ylamino)octahydro-4H,12H-5,8-methanofuro[3,2-
d] [1,3,7,9,2,8]tetraoxadiphosphacyclotetradecine-2,10,15,16-te-
trol 2,10-dioxide (13). TBS and DMTr were used as the protecting
groups for 3′,5′-hydroxyls in I-18 replacing 3′5′-OSi(iPr)2OSi-
(iPr)2O disiloxane group. Acetic acid/water was used to deprotect
1
DMTr in step 2. LC−MS (AA): m/z = 617.1 (M + H). H NMR
(D2O) δ 1.27 (t, J = 7.34 Hz, 18H) 1.44−1.56 (m, 1H) 2.30 (m,
1H) 2.53 (m, 1H) 3.19 (q, J = 7.34 Hz, 12H) 3.85 (m, 1H) 4.06−
4.19 (m, 2H) 4.22 (d, J = 4.03 Hz, 1H) 4.28−4.35 (m, 1H) 4.37−
4.46 (m, 1H) 4.47−4.63 (m, 2H) 4.86−4.95 (m, 2H) 6.16 (d, J =
3.55 Hz, 1H) 6.66 (br d, J = 5.87 Hz, 1H) 8.03 (br d, J = 6.48 Hz,
1H) 8.26 (s, 1H) 8.41 (s, 1H) 8.42 (s, 1H). 31P NMR (D2O) δ
−0.21 (s, 1P), -0.49 (s, 1P). HRMS m/z [M + H]+ calcd for
C20H26N8O11P2 617.1269, observed 617.1267.
1
(M + H). H NMR (D2O) δ 1.28 (t, J = 7.34 Hz, 18H) 1.62−1.77
2-Amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-
2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (15a). Step 1: a mixture of I-4 [(1S,2R,4R)-2-(hydroxymethyl)-
4-pyrimidin-4-yloxy-cyclopentoxy] phosphinic acid (487 mg, 1.78
mmol) and N-[9-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-
(m, 1H) 2.26−2.42 (m, 1H) 2.43−2.64 (m, 3H) 3.20 (q, J = 7.34
Hz, 12H) 3.82−3.97 (m, 1H) 4.03-4.05 (m, 1H) 4.14 (br dd, J =
11.68, 2.75 Hz, 1H) 4.31−4.43 (m, 1H) 4.48 (br s, 1H) 4.76−4.80
(m, 1H) 4.96−5.10 (m, 1H) 5.44 (m, 1H) 5.51 (td, J = 8.96, 3.97
Hz, 1H) 6.03 (d, J = 8.44 Hz, 1H) 6.80 (d, J = 5.99 Hz, 1H) 8.07
(s, 1H) 8.44 (d, J = 5.99 Hz, 1H) 8.68 (s, 1H). 31P NMR (D2O) δ
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Article
54.12 (s, 1P), 52.59 (s, 1P). HRMS m/z [M+H]+ calcd for
C20H26N7O10P2S2 650.0652, observed 649.0672.
Compounds 15b and 15c, the phosphorothioate diastereomers of
compound 15a, were prepared following step 4 used for the
synthesis of compound 15a, substituting the starting material I-29c
with I-29a and I-29b, respectively.
General procedure for the synthesis of 19a−c, 20a,b, 21a,b, and
22a,b. Individual analogues and their phosphorothioate diaster-
eomers were prepared according to the procedures used for the
synthesis of compound 15a−c substituting the starting phosphoa-
midite ii with (2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)-
methoxy)methyl)-2-(2-(((E)-(dimethylamino)methylene)amino)-6-
oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl)
diisopropylphosphoramidite, I-26, I-27, and I-25, respectively, in
step 1. Compounds were isolated as N,N-diethylethanamine salts.
2-Amino-9-[(2R,5S,7R,8R,10S,12aR,14R,15aS)-2,10-dioxido-14-
(pyrimidin-4-yloxy)-2, 10-disulfanyldecahydro-5, 8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5S,7R,8R,10S,12aR,14R,15aS)-2,10-dioxido-
14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2R,5S,7R,8R,10R,12aR,14R,15aS)-2,10-dioxi-
do-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5S,7R,8R,10R,12aR,14R,15aS)-2,10-dioxido-
14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (19a−c). Isomer 19a. LC−MS (AA): m/z = 634.1 (M + H);
1H NMR (D2O) δ 8.64 (br s, 1H), 8.40 (br d, J = 6.0 Hz, 1H),
7.97 (br s, 1H), 6.82 (br d, J = 5.7 Hz, 1H), 5.84−5.91 (m, 1H),
5.34−5.45 (m, 2H), 4.85−4.97 (m, 1H), 4.53−4.64 (m, 1H), 4.29−
4.42 (m, 1H), 3.96−4.10 (m, 2H), 3.83-3.95 (m, 1H), 3.15 (q, J =
7.1 Hz, 12H), 2.65 (br d, J = 4.4 Hz, 1H), 2.25−2.59 (m, 5H), 1.69
(br s, 1H), 1.23 (t, J = 7.2 Hz, 18H); 31P NMR (D2O) δ 54.89 (s,
2-Amino-9-[(2R,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hydroxy-
2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hy-
droxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahy-
d r o - 5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hy-
droxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahy-
d r o - 5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (15b,c). Isomer 15b (N,N-diethylethanamine salt): LC−MS
1
(AA) m/z = 650.1 (M + H). H NMR (D2O) δ 8.64 (s, 1H), 8.47
(s, 1H), 8.40 (d, J = 6.1 Hz, 1H), 6.88 (d, J = 6.1 Hz, 1H), 6.02 (d,
J = 8.4 Hz, 1H), 5.32−5.46 (m, 2H), 4.96−5.06 (m, 1H), 4.60 (d, J
= 4.2 Hz, 1H), 4.40 (d, J = 2.0 Hz, 1H), 4.02−4.22 (m, 3H), 3.85
(dt, J = 11.1, 3.1 Hz, 1H), 3.15 (q, J = 7.3 Hz, 8H), 2.40−2.57 (m,
4H), 1.51−1.63 (m, 1H), 1.23 (t, J = 7.3 Hz, 12H); 31P NMR
(D2O) δ 58.98 (s, 1P), 53.96 (s, 1P).
Isomer 15c (N,N-diethylethanamine salt): LC−MS (AA) m/z =
1
650.0 (M + H). H NMR (D2O) δ 8.64 (s, 1H), 8.40 (d, J =6.0 Hz,
1H), 8.10 (s, 1H), 6.86 (d, J = 6.0 Hz, 1H), 5.99 (d, J = 8.4 Hz,
1H), 5.44 (spt, J = 4.4 Hz, 2H), 5.02−5.10 (m, 1H), 4.54 (d, J =
4.3 Hz, 1H), 4.41 (d, J = 2.3 Hz, 1H), 4.31 (ddd, J = 12.2, 10.1, 2.2
Hz, 1H), 3.98−4.13 (m, 2H), 3.83 (dt, J = 10.5, 2.9 Hz, 1H), 3.15
(q, J = 7.3 Hz, 12H), 2.37−2.55 (m, 4H), 1.54−1.66 (m, 1H), 1.23
(t, J = 7.2 Hz, 18H); 31P NMR (D2O) δ 58.60 (s, 1P), 54.10 (s,
1P).
1P), 53.29 (s, 1P). HRMS m/z [M
+
H]+ calcd for
C20H25N7O9P2S2 634.0703, observed 634.0718. Isomer 19b. LC−
1
MS (AA): m/z = 634.1 (M + H); H NMR (D2O) δ 8.64 (s, 1H),
8.40 (d, J = 6.0 Hz, 1H), 8.07 (s, 1H), 6.87 (d, J = 6.0 Hz, 1H),
5.86 (d, J = 6.5 Hz, 1H), 5.37−5.48 (m, 2H), 4.96−5.06 (m, 1H),
4.56−4.64 (m, 1H), 4.25 (td, J = 11.3, 2.1 Hz, 1H), 3.97−4.08 (m,
2H), 3.85−3.94 (m, 1H), 3.15 (q, J = 7.3 Hz, 12H), 2.35−2.67 (m,
6H), 1.55−1.72 (m, 1H), 1.23 (t, J = 7.3 Hz, 18H); 31P NMR
(D2O) δ 57.15 (s, 1P), 54.61 (s, 1P). Isomer 19c. LC−MS (AA):
General Procedure for the Synthesis of Compounds 14a,b.
Phosphorothioate diastereomers were prepared according to the
procedures used for the synthesis of compound 15a−c, replacing H-
phosphonate intermediate I-4 with I-24 in step 1.
2-Amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-
2,10-dioxido-14-(pyrimidin-4-ylamino)-2,10-disulfanyldecahydro-
5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
1
m/z = 634.2 (M + H); H NMR (D2O) δ 8.64 (s, 1H), 8.44 (s,
1H), 8.40 (br d, J = 5.7 Hz, 1H), 6.87 (d, J = 6.1 Hz, 1H), 5.86 (d,
J = 7.2 Hz, 1H), 5.34−5.47 (m, 2H), 4.96−5.07 (m, 1H), 4.52−
4.62 (m, 1H), 4.06 (br d, J = 9.0 Hz, 3H), 3.84−3.95 (m, 1H), 3.15
(q, J = 7.2 Hz, 12H), 2.41−2.65 (m, 6H), 1.52−1.68 (m, 1H), 1.23
(t, J = 7.3 Hz, 18H); 31P NMR (D2O) δ 57.75 (s, 1P), 53.77 (s,
1P).
1
one (14a). LC−MS (FA): m/z = 649.1 (M + H). H NMR (D2O)
δ 8.50 (s, 1H), 8.02 (s, 1H), 7.94−8.01 (m, 1H), 6.55 (br s, 1H),
6.02 (d, J = 8.4 Hz, 1H), 5.55 (ddd, J = 10.5, 8.4, 4.2 Hz, 1H), 4.98
(quin, J = 7.0 Hz, 1H), 4.68 (d, J = 4.0 Hz, 1H), 4.43−4.52 (m,
2H), 4.40 (ddd, J = 12.0, 7.9, 2.9 Hz, 1H), 4.13 (ddd, J = 11.9, 3.2,
1.3 Hz, 1H), 4.00 (ddd, J = 10.1, 3.7, 2.1 Hz, 1H), 3.85−3.94 (m,
1H), 3.20 (q, J = 7.3 Hz, 10H), 2.40−2.54 (m, 2H), 2.23−2.35 (m,
2H), 1.44 (s, 1H), 1.28 (t, J = 7.3 Hz, 15H). 31P NMR (D2O) δ
51.83 (s, 1P), 50.55 (s, 1P). HRMS m/z [M + H]+ calcd for
C20H26N8O9P2S2 649.0812, observed 649.0802.
2-Amino-9-[(2S,5R,7R,8S,10S,12aR,14R,15aS,16R)-16-fluoro-
2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2R,5R,7R,8S,10R,12aR,14R,15aS,16R)-16-fluo-
ro-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-
5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2R,5R,7R,8S,10S,12aR,14R,15aS,16R)-16-fluo-
ro-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-
5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5R,7R,8S,10R,12aR,14R,15aS,16R)-16-fluo-
ro-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-
5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
2-Amino-9-[(2R,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hydroxy-
2,10-dioxido-14-(pyrimidin-4-ylamino)-2,10-disulfanyldecahydro-
5 , 8 - m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hy-
droxy-2,10-dioxido-14-(pyrimidin-4-ylamino)-2,10-disulfanyldeca-
hy d r o -5 , 8 -m et h a no c y c l o p en t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hy-
droxy-2,10-dioxido-14-(pyrimidin-4-ylamino)-2,10-disulfanyldeca-
hy d r o -5 , 8 -m et h a no c y c l o p en t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
1
one (20a,b). Isomer 20a. LC−MS (FA): m/z = 652.1 (M + H); H
1
NMR (D2O) δ 8.52 (s, 1H), 8.28 (d, J = 6.0 Hz, 1H), 7.88 (s, 1H),
6.65 (d, J = 5.9 Hz, 1H), 5.91 (d, J = 8.5 Hz, 1H), 5.20−5.34 (m,
2H), 4.86 (quin, J = 6.8 Hz, 1H), 4.28 (ddd, J = 11.2, 8.7, 2.3 Hz,
1H), 3.96 (br d, J = 12.3 Hz, 1H), 3.85 (br s, 1H), 3.69−3.80 (m,
1H), 3.02 (q, J = 7.3 Hz, 12H), 2.24−2.47 (m, 4H), 2.10−2.22 (m,
1H), 1.42-1.60 (m, 1H), 1.10 (t, J = 7.3 Hz, 18H), 31P NMR
(D2O) δ 54.08 (s, 1P), 53.20 (s, 1P). HRMS m/z [M+H]+ calcd
for C20H24FN7O9P2S2 652.0609, observed 652.0622. Isomer 20b.
one (14b). LC−MS (FA): m/z = 649.2 (M + H). H NMR (D2O)
δ 8.43 (s, 1H), 8.00 (s, 1H), 7.91 (br d, J = 5.9 Hz, 1H), 6.57 (br
d, J = 6.1 Hz, 1H), 5.98 (d, J = 8.4 Hz, 1H), 5.54 (ddd, J = 12.1,
8.2, 3.9 Hz, 1H), 4.97−5.09 (m, 1H), 4.57 (d, J = 4.2 Hz, 1H), 4.43
(br q, J = 1.2 Hz, 1H), 4.30−4.41 (m, 2H), 4.09 (dt, J = 11.6, 2.5
Hz, 1H), 3.85−3.98 (m, 2H), 3.16 (q, J = 7.3 Hz, 14H), 2.39−2.54
(m, 2H), 2.24 (br s, 2H), 1.34 (br d, J = 3.7 Hz, 1H), 1.23 (t, J =
7.3 Hz, 21H); 31P NMR (D2O) δ 56.48 (s, 1P), 53.93 (s, 1P).
6917
https://doi.org/10.1021/acs.jmedchem.1c00374
J. Med. Chem. 2021, 64, 6902−6923

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
LC−MS (FA): m/z = 652.1 (M + H); H NMR (D2O) δ 8.54 (s,
1H), 8.29 (d, J = 6.0 Hz, 1H), 7.97 (s, 1H), 6.75 (d, J = 5.9 Hz,
1H), 5.95 (d, J = 8.5 Hz, 1H), 5.27−5.38 (m, 2H), 4.87−5.01 (m,
1H), 4.28 (t, J = 12.1 Hz, 1H), 3.86−4.06 (m, 2H), 3.75 (m, 1H),
3.05 (q, J = 7.3 Hz, 12H), 2.25−2.48 (m, 5H), 1.42−1.55 (m, 1H),
1.13 (t, J = 7.3 Hz, 18H), 31P NMR (D2O) δ 58.59 (s, 1P), 54.01
(s, 1P).
9-[(2S,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-Hydroxy-2,10-dioxi-
do-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 9-[(2S,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-Hydroxy-2,10-
dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 9-[(2R,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-Hydroxy-2,10-
dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-Hydroxy-2,10-
dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one (17a−d). Step 1: a mixture of I-31 (2R,3R,4R,5R)-4-((tert-
butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2-(6-oxo-1,6-dihydro-9H-
purin-9-yl)tetrahydrofuran-3-yl hydrogen phosphonate (873 mg,
1.60 mmol) and I-32 (1S,2R,4R)-2-((bis(4-methoxyphenyl)-
(phenyl)methoxy)methyl)-4-(pyrimidin-4-yloxy)cyclopentyl (2-cya-
noethyl) diisopropylphosphoramidite (1.67 g, 2.34 mmol) was
concentrated from dry ACN (3 × 10 mL), dried on vacuum for 1 h,
and then suspended in ACN (6.59 mL) and THF (1.00 mL) under
an atmosphere of argon. Separately, 5(ethylthio)1H-tetrazole (703
mg, 5.40 mmol) was concentrated from ACN (3 × 10 mL),
dissolved in ACN (3.00 mL), and then added to the reaction
mixture. The reaction mixture was allowed to stir at rt for 30 min.
((Dimethylaminomethylidene)amino)3H-1,2,4-dithiazoline-3-thione
(442 mg, 2.15 mmol) was added to the reaction mixture, and
stirring was continued for 45 min at rt. The reaction mixture was
concentrated and dried on vacuum for 10 min. The residue was
taken up in acetic acid (7.13 mL) and water (1.78 mL). The
reaction mixture was sonicated for 2 min and then allowed to stir at
rt for 1 h. The reaction mixture was concentrated from toluene (2 ×
10 mL) and adsorbed onto celite. The crude compound was purified
by silica gel chromatography (0−60% MeOH in DCM) to provide
I-33 (2R,3R,4R,5R)-5-((((((1S,2R,4R)-2-((bis(4-methoxyphenyl)-
(phenyl)methoxy)methyl)-4-(pyrimidin-4-yloxy)cyclopentyl)oxy)(2-
cyanoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-
butyldimethylsilyl)oxy)-2-(6-oxo-1,6-dihydro-9H-purin-9-yl)-
tetrahydrofuran-3-yl hydrogen phosphonate (1.17 g, 76%) as a
mixture of two diastereomers that was taken on without further
purification. LC−MS (FA): m/z = 788.3 (M + H).
Step 2: intermediate I-33 (1.17 g, 1.48 mmol) was dissolved in
dry pyridine and concentrated to dryness (3 × 10 mL), dried under
vacuum for 10 min, and then dissolved in pyridine (25.3 mL) under
an atmosphere of argon. 2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphor-
inane 2-oxide (815 mg, 4.42 mmol) was added, and the reaction
mixture was allowed to stir at rt for 1.5 h. An additional portion of
2-chloro-5,5-dimethyl-1,2,3-dioxaphosphorinane 2-oxide (100 mg,
0.542 mmol) was added, and stirring was continued at rt for 15
min. Water was added (0.796 mL), followed by 3H-1,2-
benzodithiol-3-one 1,1-dioxide (306 mg, 1.51 mmol). The reaction
mixture was allowed to stir at rt for 1 h. An additional portion of
3H-1,2-benzodithiol-3-one 1,1-dioxide (77.0 mg, 0.380 mmol) was
added, and stirring was continued at rt for 20 min. The reaction
mixture was concentrated, concentrated from toluene (2 × 10 mL),
and adsorbed onto celite. The crude compound was purified by
silica gel chromatography (0−50% MeOH in DCM). I-34 (a,b)
eluted as a mixture (640 mg, 46%), followed by a mixture of I-34
(c,d) (500 mg, 32%). LC−MS (FA): m/z = 802.2 (M + H).
Step 3: the mixture of I-34 (a,b) (640 mg, 0.798 mmol) was
dissolved in methylamine (33% in EtOH, 29.0 mL, 233 mmol), and
the reaction mixture was allowed to stir at rt for 2 h. The reaction
mixture was concentrated and adsorbed onto celite. The crude
compound was purified by reverse-phase flash column chromatog-
raphy (10−100% ACN in aqueous ammonium acetate (10 mM)) to
provide I-35a (92 mg, 20%) and I-35b (32 mg, 7%). LC−MS (FA):
m/z = 749.2 (M + H).
2-Amino-9-[(2S,5R,7R,8R,10S,12aR,14R,15aS,16R)-2,10-dioxido-
14-(pyrimidin-4-yloxy)-2,10-disulfanyl-16-(2,2,2-trifluoroethoxy)-
decahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one or 2-amino-9-[(2R,5R,7R,8R,10S,12aR,14R,15aS,16R)-2,10-di-
oxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyl-16-(2,2,2-
trifluoroethoxy)decahydro-5,8-methanocyclopenta[l]-
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihy-
dro-6H-purin-6-one or 2-amino-9-[(2S,5R,7R,8R,10R,12aR,14-
R,15aS,16R)-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyl-
16-(2,2,2-trifluoroethoxy)decahydro-5,8-methanocyclopenta[l]-
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihy-
dro-6H-purin-6-one or 2-amino-9-[(2R,5R,7R,8R,10R,12aR,14-
R,15aS,16R)-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyl-
16-(2,2,2-trifluoroethoxy)decahydro-5,8-methanocyclopenta[l]-
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihy-
dro-6H-purin-6-one (21a,b). Isomer 21a. LC−MS (AA): m/z =
1
732.1 (M + H); H NMR (D2O) δ 8.69 (s, 1H), 8.46 (d, J = 5.9
Hz, 1H), 8.07 (s, 1H), 6.87 (dd, J = 1.0, 6.1 Hz, 1H), 6.04 (d, J =
8.6 Hz, 1H), 5.61−5.51 (m, 1H), 5.46 (td, J = 3.0, 6.1 Hz, 1H),
5.05−4.95 (m, 1H), 4.66 (d, J = 4.3 Hz, 1H), 4.63−4.58 (m, 1H),
4.55−4.25 (m, 3H), 4.13 (ddd, J = 1.7, 4.3, 12.2 Hz, 1H), 4.06−
3.98 (m, 1H), 3.90 (td, J = 7.1, 10.4 Hz, 1H), 3.20 (q, J = 7.3 Hz,
12H), 2.61−2.46 (m, 3H), 2.43−2.34 (m, 1H), 1.70−1.59 (m, 1H),
1.28 (t, J = 7.3 Hz, 18H). 31P NMR (D2O) δ 54.18 (s, 1P), 53.39
(s, 1P). HRMS m/z [M+H]+ calcd for C22H26F3N7O10P2S2
732.0683, observed 732.0693. Isomer 21b. LC−MS (AA): m/z =
1
732.1 (M + H); H NMR (D2O) δ 8.69 (s, 1H), 8.45 (d, J = 6.0
Hz, 1H), 8.15 (s, 1H), 6.93 (dd, J = 1.1, 6.1 Hz, 1H), 6.05 (d, J =
8.7 Hz, 1H), 5.59−5.47 (m, 2H), 5.14−5.07 (m, 1H), 4.63−4.57
(m, 1H), 4.54 (d, J = 4.4 Hz, 1H), 4.52−4.23 (m, 3H), 4.18−4.08
(m, 2H), 3.89 (td, J = 2.9, 10.5 Hz, 1H), 3.20 (q, J = 7.3 Hz, 12H),
2.60−2.46 (m, 4H), 1.69−1.59 (m, 1H), 1.28 (t, J = 7.3 Hz, 18H).
31P NMR (D2O) δ 59.19 (s, 1P), 54.12 (s, 1P).
2-Amino-9-[(2S,5R,7R,8R,10S,12aR,14R,15S,15aR,16R)-15-fluoro-
16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanylde-
cahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2S,5R,7R,8R,10R,12aR,14R,15S,15aR,16R)-15-
fluoro-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disul-
fanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2R,5R,7R,8R,10S,12aR,14R,15S,15aR,16R)-15-
fluoro-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disul-
fanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
one, or 2-Amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15S,15aR,16R)-15-
fluoro-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disul-
fanyldecahydro-5,8-methanocyclopenta[l][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-
1
one (22a,b). Isomer 22a. LC−MS (FA): m/z = 668.1 (M + H); H
NMR (D2O) δ 8.73 (s, 1H), 8.48 (d, J = 6.0 Hz, 1H), 8.03 (s, 1H),
6.79 (d, J = 6.0 Hz, 1H), 6.01 (d, J = 8.4 Hz, 1H), 5.55−5.29 (m,
3H), 4.96−4.83 (m, 1H), 4.75−4.73 (m, 1H), 4.51−4.44 (m, 2H),
4.17−4.12 (m, 1H), 4.07−3.98 (m, 2H), 3.20 (q, J = 7.3 Hz, 12H),
2.67−2.56 (m, 2H), 1.77−1.68 (m, 1H), 1.28 (t, J = 7.3 Hz, 18H);
31P NMR (D2O) δ 54.57 (s, 1P), 52.26 (s, 1P). HRMS m/z [M
+H]+ calcd for C20H24FN7O10P2S2 668.0558, observed 668.0566.
Isomer 22b. LC−MS (FA): m/z = 668.1 (M + H); 1H NMR
(D2O) δ 8.73 (s, 1H), 8.46 (d, J = 5.9 Hz, 1H), 8.09 (s, 1H), 6.87
(dd, J = 1.0, 6.0 Hz, 1H), 6.03 (d, J = 8.3 Hz, 1H), 5.56−5.29 (m,
3H), 5.01−4.90 (m, 1H), 4.63 (d, J = 4.3 Hz, 1H), 4.51−4.43 (m,
2H), 4.18−4.13 (m, 1H), 4.08−3.94 (m, 2H), 3.20 (q, J = 7.3 Hz,
12H), 2.68−2.58 (m, 2H), 1.72−1.62 (m, 1H), 1.28 (t, J = 7.3 Hz,
18H); 31P NMR (D2O) δ 57.04 (s, 1P), 54.39 (s, 1P).
6918
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Article
The mixture of I-34 (c,d) (320 mg, 0.399 mmol) was dissolved
in methylamine (33% in EtOH, 19.9 mL, 160 mmol), and the
reaction mixture was allowed to stir at rt for 2 h. The reaction
mixture was concentrated and adsorbed onto celite. The crude
compound was purified by reverse-phase flash column chromatog-
raphy (10−100% ACN in aqueous ammonium acetate (10 mM)) to
provide I-35c (74 mg, 24%) LC−MS (FA): m/z = 749.1 (M + H)
and I-35d (82 mg, 26%). LC−MS (FA): m/z = 749.1 (M + H).
Step 4: to a suspension of I-35b (137 mg, 0.183 mmol) in
pyridine (0.915 mL) and TEA (2.29 mL) in a propylene tube was
added triethylamine trihydrofluoride (0.152 mL, 0.915 mmol). The
tube was capped and sealed, and the reaction mixture was allowed
to stir at 50 °C for 8 h and then at rt overnight. The reaction
mixture was diluted with water (3.43 mL), a solution of CaCl2 (212
mg, 1.83 mmol) in water (3.43 mL) was added, and the cloudy
white reaction mixture was sonicated for 2 min and allowed to stir
at rt for 1 h. The reaction mixture was filtered through celite, the
celite plug was washed with water (5 × 3 mL), and the filtrate was
concentrated to give an off-white solid. No HF was observed by 19F
NMR. The residue was concentrated from toluene (3 × 5 mL),
adsorbed onto celite, and the crude mixture was purified by reverse-
phase flash column chromatography (0−20% ACN in aqueous
triethylammonium acetate (10 mM)) to provide compound 17a (99
mg, 61%) as an N,N-diethylethanamine salt. LC−MS (AA): m/z =
dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
methanocyclopenta[l][1,3,6,9,11,2,10] pentaoxadiphosphacyclote-
tradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one or 2-
Amino-7-[(2R,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hydroxy-2,10-
dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
methanocyclopenta[l][1,3,6,9,11,2,10] pentaoxadiphosphacyclote-
tradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one or 2-
Amino-7-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-
dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
methanocyclopenta[l][1,3,6,9,11,2,10] pentaoxadiphosphacyclote-
tradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(18a,b). Step 1: a mixture of I-4 [(1S,2R,4R)-2-(hydroxymethyl)-4-
pyrimidin-4-yloxy-cyclopentoxy]phosphinic acid (598 mg, 2.06
mmol) and I-36 (1.83 g, 2.30 mmol) was dissolved in dry
acetonitrile and concentrated to dryness (3 × 10 mL). The residue
was then dissolved in ACN (7.52 mL) under an atmosphere of
argon. In a separate flask, 5-(ethylthio)-1H-tetrazole (801 mg, 6.15
mmol) was dissolved in dry acetonitrile and concentrated to dryness
(3 × 6 mL), and the residue was dissolved in ACN (3.42 mL) and
added to the reaction mixture under an atmosphere of argon. The
reaction mixture was allowed to stir at rt for 1 h. ((Dimethylamino-
methylidene)amino)-3H-1,2,4-dithiazoline-3-thione (505 mg, 2.46
mmol) was added to the reaction mixture, and stirring was
continued for 45 min at rt. The reaction mixture was concentrated
and dried on vacuum for 10 min. The crude compound was purified
by silica gel chromatography (0−80% MeOH in DCM) to provide
I-38 (206 mg, 10%) as a mixture of diastereomers. LC−MS (FA):
m/z = 1000.4 (M + H).
Step 2: I-38 (204 mg, 0.20 mmol) was taken up in THF (2.3
mL) and water (0.57 mL) and cooled to 0 °C. TFA (0.57 mL, 7.50
mmol) was added dropwise, and the reaction mixture was allowed
to stir at 0 °C for 2 h. Sodium bicarbonate (930 mg, 11.0 mmol)
was added portion-wise, followed by water and EtOAc. The reaction
mixture was allowed to warm to rt and extracted into EtOAc. The
combined organic phases were washed with brine, dried with
Na2SO4, and concentrated. The crude compound was purified by
silica gel chromatography (0−60% MeOH in DCM) to provide I-39
(155 mg, 75%) as a mixture of diastereomers. LC−MS (FA): m/z =
1018.4 (M + H).
Step 3: I-39 (155 mg, 0.15 mmol) was concentrated from dry
pyridine (3 × 5 mL), dried under vacuum for 10 min, and dissolved
in pyridine (3.03 mL) under an atmosphere of argon. 2-Chloro-5,5-
dimethyl-1,2,3-dioxaphosphorinane 2-oxide (97.7 mg, 0.53 mmol)
was added, and the reaction mixture was allowed to stir at rt for 45
min. Water (0.10 mL) was added followed by 3H-1,2-benzodithiol-
3-one 1,1-dioxide (37.7 mg, 0.18 mmol), and then stirring was
continued at rt for 1 h. An additional portion of 3H-1,2-
benzodithiol-3-one 1,1-dioxide (36.3 mg, 0.18 mmol) was added,
and stirring was continued for 10 min. The reaction mixture was
concentrated and concentrated from toluene. The crude compound
was purified by silica gel chromatography (0−40% MeOH in DCM)
to provide slightly impure I-40 (204 mg) as a mixture of
diastereomers. LC−MS (FA): m/z = 1032.3 (M + H).
Step 4: I-40 (216 mg, 0.21 mmol) was dissolved in methylamine
(33% in EtOH, 10.4 mL, 83.7 mmol), and the reaction mixture was
allowed to stir under an atmosphere of nitrogen at rt overnight. The
reaction mixture was concentrated and dried on vacuum for 10 min.
The crude compound was purified by reverse-phase flash column
chromatography (10−100% ACN in aqueous ammonium acetate
(10 mM)) to provide I-41a (67 mg, 34%); LC−MS (AA): m/z =
909.4 (M + H) and I-41b (22 mg, 11%); LC−MS (AA): m/z =
909.4 (M + H).
1
635.2 (M + H). H NMR (D2O) δ: 8.68 (s, 1H), 8.56 (s, 1H), 8.44
(d, J = 6.0 Hz, 1H), 8.22 (s, 1H), 6.88 (dd, J = 6.1, 1.0 Hz, 1H),
6.29 (d, J = 8.3 Hz, 1H), 5.44−5.51 (m, 1H), 5.33 (ddd, J = 10.0,
8.4, 4.2 Hz, 1H), 4.87−4.94 (m, 2H), 4.51−4.54 (m, J = 2.4 Hz,
1H), 4.40 (ddd, J = 12.0, 10.0, 1.7 Hz, 1H), 4.02−4.15 (m, 2H),
3.83 (dt, J = 10.5, 7.9 Hz, 1H), 3.20 (q, J = 7.3 Hz, 12H), 2.47−
2.57 (m, 3H), 2.36−2.47 (m, 1H), 1.53−1.64 (m, 1H), 1.28 (t, J =
7.3 Hz, 18H). 31P NMR (D2O) δ 54.31 (s, 1P), 53.13 (s, 1P).
HRMS m/z [M+H]+ calcd for C20H24N6O10P2S2 635.0543, observed
635.0555.
The three phosphorothioate diastereomers of compound 17a
were prepared following step 4 used for the synthesis of compound
15a, substituting the starting material I-35b with I-35a, I-35c, and I-
35d, respectively.
1
Isomer 17b. LC−MS (AA): m/z = 635.0 (M + H). H NMR
(D2O) δ 8.86 (s, 1H), 8.68−8.67 (m, 1H), 8.44 (d, J = 6.0 Hz,
1H), 8.22 (s, 1H), 6.86 (d, J = 6.0 Hz, 1H), 6.29 (d, J = 8.3 Hz,
1H), 5.48−5.42 (m, 1H), 5.34 (ddd, J = 4.2, 8.3, 9.9 Hz, 1H),
4.91−4.85 (m, 1H), 4.83 (d, J = 4.0 Hz, 2H), 4.53−4.51 (m, 1H),
4.30−4.16 (m, 2H), 4.12−4.06 (m, 1H), 3.91−3.83 (m, 1H), 3.20
(q, J = 7.3 Hz, 12H), 2.63−2.46 (m, 3H), 2.40−2.31 (m, 1H), 1.27
(t, J = 7.3 Hz, 18H); 31P NMR (D2O) δ 54.09 (s, 1P), 52.79 (s,
1P).
Isomer 17c. LC−MS (AA): m/z = 635.0 (M + H). 1H NMR
(D2O) δ 8.90 (s, 1H), 8.62 (s, 1H), 8.39 (d, J = 6.1 Hz, 1H), 8.16
(s, 1H), 6.87 (dd, J = 6.0, 1.0 Hz, 1H), 6.23 (d, J = 8.3 Hz, 1H),
5.41 (m, 1H), 5.32 (ddd, J = 12.7, 8.3, 4.3 Hz, 1H), 4.95−5.05 (m,
1H), 4.59 (d, J = 4.3 Hz, 1H), 4.43 (d, J = 1.8 Hz, 1H), 4.20 (ddd,
J = 11.9, 7.5, 1.3 Hz, 1H), 4.00-4.13 (m, 2H), 3.82 (dt, J = 10.5, 3.1
Hz, 1H), 3.13 (q, J = 7.3 Hz, 12H), 2.39−2.56 (m, 4H), 1.55 (qd, J
= 9.4, 3.2 Hz, 1H), 1.21 (t, J = 7.3 Hz, 18H); 31P NMR (D2O) δ
59.23 (s, 1P), 54.26 (s, 1P).
1
Isomer 17d. LC−MS (AA): m/z = 635.2 (M + H). H NMR
(D2O) δ 8.62 (s, 1H), 8.57 (s, 1H), 8.39 (d, J = 6.1 Hz, 1H), 8.16
(s, 1H), 6.88 (dd, J = 6.0, 1.1 Hz, 1H), 6.23 (d, J = 8.3 Hz, 1H),
5.40−5.47 (m, 1H), 5.25 (ddd, J = 12.8, 8.3, 4.4 Hz, 1H), 4.96−
5.05 (m, 1H), 4.55 (d, J = 4.4 Hz, 1H), 4.45 (d, J = 2.2 Hz, 1H),
4.26−4.36 (m, 1H), 3.96−4.10 (m, 2H), 3.80 (dt, J = 10.6, 3.4 Hz,
1H), 3.13 (q, J = 7.3 Hz, 12H), 2.39−2.53 (m, 4H), 1.48−1.60 (m,
1H), 1.21 (t, J = 7.3 Hz, 18H); 31P NMR (D2O) δ 59.17 (s, 1P),
54.35 (s, 1P).
Step 5: I-41b (58.0 mg, 0.0638 mmol) was suspended in pyridine
(0.32 mL) and TEA (0.80 mL) in a propylene tube. Triethylamine
trihydrofluoride (0.053 mL, 0.32 mmol) was added, and the tube
was sealed and the reaction mixture was allowed to stir at 50 °C
overnight. The reaction mixture was cooled to rt, and water (1.20
mL) was added followed by the dropwise addition of a solution of
calcium chloride (148 mg, 1.28 mmol) in water (1.20 mL). The
reaction mixture was allowed to stir at rt for 30 min, then filtered
through celite, and the celite was rinsed with water (5 × 3 mL).
2-Amino-7-[(2S,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hydroxy-
2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
methanocyclopenta[l][1,3,6,9,11,2,10] pentaoxadiphosphacyclote-
tradecin-7-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one or 2-
Amino-7-[(2S,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-
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Article
The filtrate was concentrated to give a white solid. No HF was
observed by 19F NMR. The crude mixture was purified by reverse-
phase flash column chromatography (0−15% ACN in aqueous
triethylammonium acetate (10 mM)) to provide compound 18a (27
mg, 50%) as an N,N-diethylethanamine salt. LC−MS (AA): m/z =
(q, J = 7.3 Hz, 14H), 2.54−2.41 (m, 3H), 2.40−2.30 (m, 1H),
1.68−1.58 (m, 1H), 1.21 (t, J = 7.3 Hz, 20H). 31P NMR (D2O) δ
57.34 (s, 1P), 53.77 (s, 1P). Isomer 16d. LC−MS (AA): m/z =
1
651.1 (M + H); H NMR (MeOD) δ 8.51 (s, 1H), 8.21 (d, J = 5.9
Hz, 1H), 6.68 (d, J = 5.9 Hz, 1H), 6.07 (d, J = 8.2 Hz, 1H), 5.91−
5.79 (m, 1H), 5.43−5.35 (m, 1H), 4.84−4.70 (m, 2H), 4.17−4.10
(m, 1H), 3.96−3.85 (m, 1H), 3.78−3.70 (m, 1H), 3.68−3.60 (m,
1H), 2.97 (q, J = 7.6 Hz, 8H), 2.60−2.49 (m, 1H), 2.39−2.24 (m,
4H), 1.35−1.23 (m, 1H), 1.10 (t, J = 7.3 Hz, 11H). 31P NMR
(MeOD) δ 59.66 (br s, 1P), 54.32 (br s, 1P).
1
648.9 (M + H); H NMR (D2O) δ 8.69 (s, 1H), 8.44 (d, J = 6.0
Hz, 1H), 7.26 (d, J = 3.8 Hz, 1H), 6.85 (d, J = 6.1 Hz, 1H), 6.56
(d, J = 3.7 Hz, 1H), 6.19 (d, J = 8.4 Hz, 1H), 5.51−5.43 (m, 1H),
5.28 (dt, J = 4.2, 8.9 Hz, 1H), 4.99 (quin, J = 6.3 Hz, 1H), 4.58−
4.57 (m, 1H), 4.42 (d, J = 2.1 Hz, 1H), 4.39−4.31 (m, 1H), 4.12−
4.02 (m, 2H), 3.92−3.82 (m, 1H), 3.19 (q, J = 7.3 Hz, 12H), 2.60−
2.47 (m, 3H), 2.46−2.36 (m, 1H), 1.68−1.57 (m, 1H), 1.27 (t, J =
7.3 Hz, 18H); 31P NMR (D2O) δ 54.13 (s, 1P), 52.70 (s, 1P).
HRMS m/z [M+H]+ calcd for C21H26N6O10P2S2 649.0700, observed
649.0714.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00374.
■
sı
Phosphorothioate diastereomer 18b was prepared according to
step 5 using starting material I-41a. LC−MS (AA): m/z = 648.9 (M
Molecular formula strings (CSV)
1
+ H); H NMR (D2O) δ 8.69 (s, 1H), 8.46 (d, J = 6.0 Hz, 1H),
Docking model of 15a shown in Figure 3 (PDB)
In vitro assay procedures, protocols and PK tables for
in vivo evaluation of 14a and 15a, synthetic
experimental details, molecular modeling methods,
crystallography protocols, and HPLC chromatograms
(PDF)
7.31 (d, J = 3.8 Hz, 1H), 6.94 (dd, J = 1.0, 6.0 Hz, 1H), 6.55 (d, J
= 3.7 Hz, 1H), 6.20 (d, J = 8.6 Hz, 1H), 5.53−5.47 (m, 1H), 5.33−
5.25 (m, 1H), 5.13−5.06 (m, 1H), 4.59 (d, J = 4.4 Hz, 1H), 4.40
(br s, 1H), 4.36−4.27 (m, 1H), 4.14−4.01 (m, 2H), 3.93−3.86 (m,
1H), 3.20 (q, J = 7.3 Hz, 12H), 2.58−2.48 (m, 4H), 1.67−1.57 (m,
1H), 1.28 (t, J = 7.3 Hz, 18H); 31P NMR (D2O) δ 58.48 (s, 1P),
54.33 (s, 1P).
General Procedure for the Synthesis of 16a−d. Compounds
were prepared according to the procedures used for the synthesis of
compound 18, substituting the starting material phosphoramidate I-
36 with I-37 in step 1. Compounds were isolated as N,N-
diethylethanamine salts.
Accession Codes
PDB depositions are available for STING-bound agonist
compounds 14a (7KVX) and 15a (7KW1). The authors will
release the atomic coordinates and experimental data upon
article publication.
5-Amino-3-[(2S,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hydroxy-
2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]-
t r i a z o l o [ 4 , 5 - d ] p y r i m i d i n - 7 - o n e o r 5 - A m i n o - 3 -
[(2S,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-
14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]-
t r i a z o l o [ 4 , 5 - d ] p y r i m i d i n - 7 - o n e o r 5 - A m i n o - 3 -
[(2R,5R,7R,8R,10S,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-
14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]-
t r i a z o l o [ 4 , 5 - d ] p y r i m i d i n - 7 - o n e o r 5 - A m i n o - 3 -
[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-
14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-
m e t h a n o c y c l o p e n t a [ l ] [ 1 , 3 , 6 , 9 , 1 1 , 2 , 1 0 ] -
pentaoxadiphosphacyclotetradecin-7-yl]-3,6-dihydro-7H-[1,2,3]-
triazolo[4,5-d]pyrimidin-7-one (16a−d). Isomer 16a. LC−MS
(AA): m/z = 651.1 (M + H); 1H NMR (D2O) δ 8.52 (s, 1H),
8.29 (d, J = 6.5 Hz, 1H), 6.69 (dd, J = 1.1, 6.0 Hz, 1H), 6.15 (d, J
= 8.6 Hz, 1H), 5.70 (ddd, J = 4.1, 8.5, 9.8 Hz, 1H), 5.29−5.23 (m,
1H), 5.05 (quin, J = 7.1 Hz, 1H), 4.56 (br s, 1H), 4.36−4.34 (m,
1H), 4.29−4.22 (m, 1H), 3.97−3.92 (m, 1H), 3.86−3.81 (m, 1H),
3.79−3.73 (m, 1H), 3.03 (q, J = 7.3 Hz, 12H), 2.46−2.38 (m, 1H),
2.38−2.26 (m, 2H), 2.19−2.10 (m, 1H), 1.62−1.54 (m, 1H), 1.10
(t, J = 7.3 Hz, 18H). 31P NMR (D2O) δ 53.86 (s, 1P), 52.23 (s,
1P). HRMS m/z [M+H]+ calcd for C19H24N8O10P2S2 651.0605,
observed 651.0615. Isomer 16b. LC−MS (AA): m/z = 651.2 (M +
AUTHOR INFORMATION
Corresponding Author
■
#Stepan Vyskocil − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States; orcid.org/0000-
0001-8178-7113; Email: Vyskocil_stepan@yahoo.com
Authors
David Cardin − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
∇Jeffrey Ciavarri − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Joe Conlon − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Courtney Cullis − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Dylan England − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Rachel Gershman − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Kenneth Gigstad − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Krista Gipson − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Alexandra Gould − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
1
H); H NMR (MeOD) δ 8.60 (s, 1H), 8.30 (d, J = 5.6 Hz, 1H),
6.74 (d, J = 5.7 Hz, 1H), 6.16 (d, J = 8.3 Hz, 1H), 5.98−5.87 (m,
1H), 5.52−5.41 (m, 1H), 4.98−4.87 (m, 2H), 4.27−4.18 (m, 1H),
4.13−4.04 (m, 1H), 3.91−3.81 (m, 1H), 3.80−3.68 (m, 1H), 3.02
(q, J = 7.2 Hz, 10H), 2.54−2.30 (m, 5H), 1.52−1.40 (m, 1H), 1.17
(t, J = 7.5 Hz, 15H). 31P NMR (MeOD) δ 54.50 (s, 1P), 53.72 (s,
1
1P). Isomer 16c. LC−MS (AA): m/z = 651.1 (M + H); H NMR
(D2O) δ 8.62 (s, 1H), 8.37 (d, J = 6.0 Hz, 1H), 6.83 (dd, J = 1.0,
6.0 Hz, 1H), 6.26 (d, J = 8.5 Hz, 1H), 5.77 (ddd, J = 4.1, 8.3, 12.4
Hz, 1H), 5.43−5.37 (m, 1H), 5.15−5.08 (m, 1H), 4.56 (d, J = 4.3
Hz, 1H), 4.47−4.43 (m, 1H), 4.35 (ddd, J = 3.2, 8.4, 11.8 Hz, 1H),
4.14−4.08 (m, 1H), 4.01−3.93 (m, 1H), 3.85−3.79 (m, 1H), 3.13
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Article
Paul Greenspan − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Robert Griffin − DMPK, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
Hirotake Mizutani − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Miho Mizutani − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Nanda Gulavita − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Sean Harrison − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Nina Molchanova − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Elise Nunes − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Zhigen Hu − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Yongbo Hu − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Sandeep Pusalkar − DMPK, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
Christelle Renou − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Akito Hata − Global Biologics, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
Jian Huang − Global Biologics, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
Scott Rowland − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Yosuke Sato − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Shih-Chung Huang − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Dave Janowick − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Michael Shaw − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Luhua Shen − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Matthew Jones − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Vihren Kolev − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Zhan Shi − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Robert Skene − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, San Diego,
California 92121, United States
Steven P. Langston − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States; orcid.org/0000-
0001-5840-0005
Hong Myung Lee − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Gang Li − Drug Discovery Sciences, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
David Lok − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Liting Ma − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Doanh Mai − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Jenna Malley − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Atsushi Matsuda − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Francois Soucy − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Steve Stroud − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
He Xu − Drug Discovery Sciences, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
Tianlin Xu − Drug Discovery Sciences, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Adnan O. Abu-Yousif − Immuno-oncology Biology, Takeda
Pharmaceuticals International Company, Cambridge,
Massachusetts 02139, United States
Ji Zhang − Drug Discovery Sciences, Takeda Pharmaceuticals
International Company, Cambridge, Massachusetts 02139,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00374
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript. Y.S. and H.X. contributed equally to the
preparation of the manuscript.
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J. Med. Chem. 2021, 64, 6902−6923

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Article
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Notes
The authors declare no competing financial interest.
#Present Address: Stepan Vyskocil Consulting, Ke Stadionu
627, Benesov, 256 01, Czech Republic.
∇Present Address: Mitobridge Inc. (an Astellas company),
1030 Massachusetts Avenue, Cambridge, Massachusetts
02138, United States.
ACKNOWLEDGMENTS
■
The authors thank Masanori Okaniwa, Michiyo Mochizuki,
and Yusuke Tominari for sharing their experience with CDN
chemistry, Saurabh Menon for input on biological assays, and
Pedro Serrano for biophysical and structural biology
consultations.
ABBREVIATIONS
■
BDC, bile duct cannulated; BWC, body weight change;
CDN, cyclic dinucleotide; IFN, type 1 interferon; PD,
pharmacodynamic; STING, Stimulator of Interferon Genes
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