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

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Identiﬁcation of Novel Carbocyclic Pyrimidine Cyclic Dinucleotide

STING Agonists for Antitumor Immunotherapy Using Systemic

Intravenous Route

Stepan Vyskocil,* David Cardin, Je ﬀ rey Ciavarri, Joe Conlon, Courtney Cullis, Dylan England,

Rachel Gershman, Kenneth Gigstad, Krista Gipson, Alexandra Gould, Paul Greenspan, Robert Gri ﬃ n,

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.³This 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.²Spontaneous 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

American Chemical Society

https://doi.org/10.1021/acs.jmedchem.1c00374

J. Med. Chem. 2021, 64, 6902−6923

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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).¹⁸Carbon 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.⁵A number of synthetic cyclic dinucleotide (CDN)

STING agonists with improved ADME properties are

currently being investigated in clinical trials.⁶ADU-S100/

MIW815 (2, Figure 1),⁷featuring diphosphorothioate groups,

was the ﬁrst one to enter the clinic, followed by others with

undisclosed structures including MK-1454, E7766,⁸SB-

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.⁹While the intratumoral route delivers the agent

directly to the tumor site, it has disadvantages including

limited eligibility of patient populations,¹⁰the risk of local

overdosing with an ablative eﬀect on immune cells,¹¹and

signiﬁcant leakage from tumors due to the physicochemical

properties of CDNs.¹¹To 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,⁹that 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

mimetics¹³with 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.^15,16At the

outset of this work, examples of carbocyclic CDN analogues

were rare,¹⁷leading to uncertainty as to the ability to replace

CDN ribose rings with cyclopentane. A thorough computa-

tional analysis of the crystal structure of cGAMP¹⁸suggested

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.¹⁹The 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 EC₅₀values without permeability

restrictions, and the ratio of nonpermeabilized/permeabilized

EC₅₀values 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 variants²¹HAQ, 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

in vitro STING activities

af

EC₅₀(μM)

E_max

EC₅₀(μM)

E_max

,

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 EC₅₀w 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

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 t_1/2(min)rat, human

mouse PK (1 mg/kg iv)

rat PK (1 mg/kg iv)

Clp (L/h/kg)

V_ss(L/kg)

t_1/2(h)

Clp (L/h/kg)

V_ss(L/kg)

t_1/2(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 (R_P,R_P)-phosphorothioate conﬁguration, consistent

with the preferred stereochemistry of reported phosphor-

othioate cGAMP analogues⁹and 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,²¹except 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,²²we 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)²³were 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.²⁴As 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.²⁵Removal of the

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remaining 5′-DMTr protecting group with aqueous acetic

acid²⁶provided 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.²⁷Removal 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.³²Transforma-

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 oxidation²⁹to mixture of

phosphorothioate isomers.³⁰Separation 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 EC₅₀values 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 C₂₁H₂₆N₁₀O₁₁P₂657.1331, 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 C₁₉H₂₅N₉O₁₁P₂618.1222,

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 C₂₁H₂₆N₆O₁₂P₂617.1157, 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

= 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

C₂₀H₂₇N₉O₁₁P₂632.1378, 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 CaCl₂in 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 C₂₁H₂₈N₈O₁₂P₂647.1375,

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

C₂₀H₂₆N₈O₁₁P₂617.1269, 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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54.12 (s, 1P), 52.59 (s, 1P). HRMS m/z [M+H]+ calcd for

C₂₀H₂₆N₇O₁₀P₂S₂650.0652, 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

C₂₀H₂₅N₇O₉P₂S₂634.0703, 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

C₂₀H₂₆N₈O₉P₂S₂649.0812, 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 C₂₀H₂₄FN₇O₉P₂S₂652.0609, 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 C₂₀H₂₄FN₇O₁₀P₂S₂668.0558, 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).

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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 C₂₀H₂₄N₆O₁₀P₂S₂635.0543, 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 C₂₁H₂₆N₆O₁₀P₂S₂649.0700, 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 C₁₉H₂₄N₈O₁₀P₂S₂651.0605,

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

6920

https://doi.org/10.1021/acs.jmedchem.1c00374

J. Med. Chem. 2021, 64, 6902−6923

Journal of Medicinal Chemistry

pubs.acs.org/jmc

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.

6921

https://doi.org/10.1021/acs.jmedchem.1c00374

J. Med. Chem. 2021, 64, 6902−6923

Journal of Medicinal Chemistry

pubs.acs.org/jmc

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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Article Doi

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

DOI: 10.1021/acs.jmedchem.1c00374

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