Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING)

Article abstract of DOI:10.1021/acs.jmedchem.6b01300

We describe novel STING-activating cyclic dinucleotides whose constituent nucleosides are adenosine and inosine and that vary by ribose substitution, internucleotide linkage position, and phosphate modification. In mammalian cells in vitro, some of these

Full text of DOI:10.1021/acs.jmedchem.6b01300

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Article
Design, synthesis, and biological evaluation of novel
cyclic adenosine-inosine monophosphate (cAIMP) analogs
that activate stimulator of interferon genes (STING)
Thierry Lioux, Marc-Antoine Mauny, Alain Lamoureux, Nicolas Bascoul, Mathieu Hays, Fabienne Vernejoul,
Anne-Sophie Baudru, Cédric Boularan, Justine Lopes-Vicente, Gregory Qushair, and Gérard Tiraby
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01300 • Publication Date (Web): 26 Oct 2016
Downloaded from http://pubs.acs.org on October 26, 2016
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Design, synthesis, and biological evaluation of novel
cyclic adenosineꢀinosine monophosphate (cAIMP)
analogs that activate stimulator of interferon genes
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(STING)
Thierry Lioux*, Marc-Antoine Mauny, Alain Lamoureux, Nicolas Bascoul, Mathieu Hays,
Fabienne Vernejoul, Anne-Sophie Baudru, Cédric Boularan, Justine Lopes-Vicente, Gregory
Qushair and Gérard Tiraby
InvivoGen, 5 rue Jean Rodier, 31400 Toulouse, France
ABSTRACT
We describe novel STINGꢀactivating cyclic dinucleotides whose constituent nucleosides are
adenosine and inosine, and that vary by ribose substitution, internucleotide linkage position and
phosphate modification. In mammalian cells in vitro, some of these cAIMP analogs induce
greater STINGꢀdependent IRF and NFꢀκB pathway signaling than do the reference agonists for
murine (DMXAA) or human (2’,3’ꢀcGAMP) STING. In human blood ex vivo, they induce type I
interferons (IFNs) and proꢀinflammatory cytokines: for the former, 3’,3’ꢀcAIMP (9; EC : 6.4
5
0
µM), and analogs 52ꢀ56 (EC : 0.4 µM to 4.7 µM), which contain one or two 2’ꢀfluoroꢀ2’ꢀ
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deoxyriboses and/or bisꢀphosphorothioate linkages, are more potent than 2’,3’ꢀcGAMP (EC :
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19.6 µM). Interestingly, 9 induces type I IFNs more strongly than do its linkage isomers 2’,3’ꢀ
cAIMP (10), 3’,2’ꢀcAIMP (23) and 2’,2’ꢀcAIMP (27). Lastly, some of the cAIMP analogs are
more resistant than 2’,3’ꢀcGAMP to enzymatic cleavage in vitro. We hope to exploit our findings
to develop STINGꢀtargeted immunotherapies.
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INTRODUCTION
Stimulator of interferon genes (STING) is an adaptor protein that is paramount in innate
immunity, especially in orchestrating the body’s responses to cytoplasmic DNA, whether
pathogen DNA (e.g. viral or microbial), self DNA (e.g. mitochondrial or junk) or tumor DNA. It
is widely expressed in immune cells (e.g. dendritic cells, macrophages and B cells) and
endothelial cells, among others. Activation of STING leads to production of type I interferons
such as IFNꢀα and IFNꢀβ, via the interferon regulatory factor (IRF) pathway; and to production
of proꢀinflammatory cytokines such as tumorꢀnecrosis factor alpha (TNFꢀα) and interleukin 1
beta (ILꢀ1β), via the nuclear factor kappaꢀlightꢀchainꢀenhancer of activated B cells (NFꢀκB)
pathway. Human STING has four known natural agonists, which correspond to the four known
naturally occurring cyclic dinucleotides (CDNs; Figure 1). Three of these CDNs (cꢀdiGMP, cꢀ
1
diAMP and cGAMP) are released by microbes into host cells during infection . However, the
fourth, 2’,3’ꢀcGAMP, is produced by the DNA sensor cyclic GMPꢀAMP synthase (cGAS) in
mammalian cells after it detects self or foreign DNA in the cytoplasm (see, for example:
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Ablasser et al. and Zhang et al. ). Microbial CDNs contain a (3’,5’)(3’,5’) phosphodiester
linkage (denoted as “3’,3’”), whereas the mammalian CDN contains a (2’,5’)(3’,5’) linkage
(denoted as “2’,3’”).
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Figure 1. The known natural agonists of STING are the four known natural CDNs. Clockwise
from top left: the microbial (3’,3’) CDNs cGAMP, cꢀdiGMP and cꢀdiAMP, and the mammalian
CDN 2’,3’ꢀcGAMP.
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Since its discovery in 2008 , STING has been widely imputed in infectious diseases, cancer
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and autoꢀimmunity . Accordingly, it is attracting increasing attention as a potential
immunotherapy target. Synthetic STING agonists that exhibit favorable drugꢀlike properties are
currently being developed as adjuvants, antiꢀtumor agents and other immunotherapy agents. In
fact, a CDN STING agonist has very recently entered Phase I clinical trials for monotherapy of
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Herein we report the design, synthesis, and biological screening of novel CDNs that activate
STING signaling. In the context of immunotherapy drug development, we sought to develop
CDNs that could offer certain advantages over natural CDNs in terms of STINGꢀdependent
cytokine induction potency and selectivity, and drugability. Unlike natural CDNs, whose
constituent nucleosides are guanosine and/or adenosine, the CDNs we describe here contain one
adenosine nucleoside and one inosine nucleoside. Beginning with the parent molecule 3’,3’ꢀ
cAIMP (9), we designed, synthesized and biologically tested eleven cAIMP analogs that vary by
sugar (ribose, 2’ꢀdeoxyribose or 2’ꢀfluoroꢀ2’ꢀdeoxyribose) and by internucleotide linkage
position (2’,2’; 2’,3’; 3’,3’; or 3’,2’) and phosphate modification (bisꢀphosphodiester or bisꢀ
phosphorothioate). We screened all eleven analogs for STINGꢀdependent activities in vitro in
two mammalian cell lines: RAW 264.7 murine macrophages (for induction of the IRF pathway)
and THPꢀ1 human leukemic monocytes (for induction of the IRF and NFꢀκB pathways). We also
screened these analogs for ex vivo production of type I IFNs and proꢀinflammatory cytokines in
human blood from healthy donors. Lastly, we explored the resistance of the cAIMP analogs to
cleavage in vitro by two enzymes: nuclease P1 (NP1) and snakeꢀvenom phosphodiesterase
(SVPD). We summarize our findings and discuss our preliminary structureꢀactivity relationship
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(SAR) observations on these cAIMP analogs, highlighting those features that appear to enhance
STINGꢀpathway activity and/or resistance to enzymatic cleavage relative to 2’,3’ꢀcGAMP.
RESULTS
Design and synthesis of cAIMP analogs
We began our study by reflecting on the fact that all four known natural CDNs are based on
the purine bases guanosine and/or adenosine. We decided to explore how using another purine
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nucleoside, inosine, in CDNs might influence STINGꢀpathway agonism. Thus, we designed,
synthesized and screened eleven cAIMP analogs, whose structures are summarized in Figure 2
and Table 1. Unless otherwise specified, the cAIMP analogs of the present study were
synthesized using methods described, or adapted from those described, in the following
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references: Hyodo et al. , Gaffney et al. , Rammler et al. , Shanahan et al. , Zhou et al. and
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Ora et al. .
Figure 2. General structure of the cAIMP analogs of the present study.
Table 1. Structures of the cAIMP analogs of the present study
Compound
Linkage
3’,3’
X1
X2
Z
O
O
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S
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OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
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2’,3’
3’,2’
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2’,2’
3’,3’
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3’,3’
3’,3’
3’,3’
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3’,3’
H
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We started by preparing 9, its linkage isomer 10 and its bisꢀphosphorothioate analog 3’,3’ꢀ
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cAIM(PS) (13) (Scheme 1). Synthesis began with the commercially available compounds N ꢀ
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Bzꢀ2’ꢀOꢀTBDMSꢀ5’ꢀOꢀDMTr adenosine phosphoramidite (1) or N ꢀBzꢀ3’ꢀOꢀTBDMSꢀ5’ꢀOꢀ
DMTr adenosine phosphoramidite (3). Phosphoramidites 1 and 3 were converted to their
corresponding Hꢀphosphonates and subsequently detritylated with dichloroacetic acid to give the
3
'ꢀHꢀphosphonate 2 and the 2'ꢀHꢀphosphonate 4, respectively. Then, 3’ꢀOꢀTBDMSꢀ5’ꢀOꢀDMTr
inosine phosphoramidite was coupled with the 5’ꢀOꢀfree alcohol of 2 or 4, using an ACN
solution of Activator 42® (0.1 M) as promoter in the presence of 3 Å molecular sieves (MS 3 Å).
The resulting phosphite products were oxidized with either 5.5 M tertꢀbutyl hydroperoxide
(tBuOOH) in decane, or sulfurized phenylacetyl disulfide (PADS). The 5’ꢀOꢀDMTr group in
each compound was removed by treatment with a 20% solution of dichloroacetic acid in
dichloromethane to give the linear dimers 5 and 11 (from 2), and 6 (from 4). The dimers were
then cyclized in a highꢀdilution mixture of 5,5ꢀdimethylꢀ2ꢀoxoꢀ2ꢀchloroꢀ1,3,2ꢀdioxaphosphinane
(
DMOCP) in dry pyridine, and then treated with either iodine and water (for bisꢀphosphodiester)
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or elemental sulfur (for bisꢀphosphorothioate diester) to provide the corresponding fully
protected CDNs 7, 8 and 12. Finally, these CDNs were deprotected by treatment with 33%
methylamine in EtOH at 50 °C to remove the benzoyl (Bz) and cyanoethyl protecting groups,
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and then treated with Et N·3HF in pyridine, and triethylamine, to remove the tert-
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butyldimethylsilyl (TBDMS) protecting groups, to give the target compounds 9, 10 and 13.
a
Scheme 1. Preparation of the cAIMP analogs 9, 10 and 13
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Reagents and conditions: (a) H O, pyridinium trifluoroacetate (Pyr·TFA), dry acetonitrile
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(ACN), RT; (b) tertbutyl amine (tBuNH ); (c) dichloroacetic acid (DCA), H O, dichloromethane
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CH Cl ); (d) 5’ꢀOꢀDMTrꢀ2’ꢀOꢀTBDMSꢀinosine phosphoramidite, Activator 42®, ACN, 3 Å
2 2
molecular sieves (MS); (e) 5.5 M tertꢀbutyl hydroperoxide (tBuOOH) in decane for Z = O, or
phenylacetyl disulfide (PADS) for Z = S; (f) DCA, H O, CH Cl ; (g) 5,5ꢀdimethylꢀ2ꢀoxoꢀ2ꢀ
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chloroꢀ1,3,2ꢀdioxaphosphinane (DMOCP), pyridine (Pyr); (h) iodine (I ), H O for Z = O or S
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for Z = S; (i) methylamine (MeNH ) 33% in ethanol (EtOH), RT or 50 °C; and (j) triethylamine
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Et N), pyr. Et N·3HF.
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To synthesize the next two analogs (Scheme 2), the cAIMP linkage isomers (3’,2’)cAIMP (23)
and (2’,2’)cAIMP (27), we began by simultaneously protecting the hydroxy groups in the 3’ and
5’ positions of commercially available inosine via treatment with 1,3ꢀdichloroꢀ1,1,3,3ꢀ
13, 14
tetraisopropyldisiloxane (TIPSCl)
. The resulting protected nucleoside 14 was treated with
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,4ꢀdihydroꢀ2Hꢀpyran and pyridinium p-toluenesulfonate (PPTS) in dry CH Cl to give the
2 2
corresponding fully protected inosine intermediate 15, which was subsequently treated with
tetrabutylammonium fluoride (TBAF) on silica gel in tetrahydrofuran (THF) at room
temperature to afford 2’ꢀOꢀTHPꢀinosine (16). After sequential protection of the 5’ position with
DMTr and the 3’ position with TBDMS, the 5’ position of the resulting intermediate was
deprotected in acidic media to afford compound 19. Coupling of 19 with phosphoramidites 1 or 3
gave the dimers 21 or 25, respectively. These dimers were then converted into their
corresponding 5ꢀHꢀphosphonates and deprotected in acid media to remove the THP groups.
Finally, the resulting intermediates were cyclized and deprotected as described for preparation of
9
, to give 23 and 27.
a
Scheme 2. Preparation of the cAIMP analogs 23 and 27
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Reagents and conditions: (a) TIPSCl Pyr; (b) 3,4ꢀdihydroꢀ2Hꢀpyran, PPTS, dry CH Cl ; (c)
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TBAF on silica gel, THF; (d) DMTrꢀCl, Pyr/CH Cl (4:1); (e) TBDMSꢀCl, imidazole, Pyr; (f)
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ZnBr (0.5 M) iPrOH/CH Cl (1:1); (g) adenosine 3’ꢀ or 2’ꢀOꢀphosphoramidite, Activator 42®,
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ACN, 3 Å MS; (h) 5.5M tBuOOH in decane; (i) diphenylphosphite, pyr. TEAA 0.1 M; (j) DCA
0% in CH Cl ; (k) DMOCP, Pyr; (l) I , H O; (m) 33% MeNH in EtOH, RT or 50 °C; and (n)
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triethylamine (Et N), pyr. Et N·3HF.
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Finally, we synthesized (Scheme 3) cꢀ(dAMPꢀdIMP) (51), cꢀ(2’FdAMPꢀ2’FdIMP) (52), cꢀ
2’FdAMP(S)ꢀ2’FdIMP(S)] (53), cꢀ(dAMPꢀ2’FdIMP) (54), cꢀ(2’FdAMPꢀdIMP) (55) and cꢀ
2’FdAM(PS)ꢀdIM(PS)] (56), all of which, relative to cAIMP, differ by the 2’ꢀsubstitution at
[
[
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either or both sugars, and by the internucleotide linkage position and phosphate substitution.
Thus, inosine phosphoramidites 28 and 29 were each condensed with the 5’ꢀOꢀfree nucleoside
allyl phosphotriesters 30ꢀ32, using the standard procedure, to afford the corresponding linear
dimers 33ꢀ38. Subsequently, the allyl group in each dimer was removed by treatment of sodium
iodide in acetone (for compound 33), or tetrakis(triphenylphosphine)palladium(0) and Nꢀ
methylaniline in THF (for compounds 34ꢀ38). The resulting products were cyclized with
DMOCP in dry pyridine. Finally, the cyclic compounds 45ꢀ50 were fully deprotected by
successive treatment with 33% methylamine in ethanol to afford the desired analogs 51ꢀ56. Due
to their high polarity, these compounds had to be purified using reverseꢀphase HPLC.
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a
Scheme 3. Preparation of cAIMP analogs 51ꢀ56
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a
Reagents and conditions: (a) allyl alcohol, Activator 42®, ACN, 3 Å MS; (b) 5.5 M tBuOOH)
in decane for Z = O or PADS for Z = S; (c) DCA, H O, CH Cl ; (d) 5’ꢀOꢀDMTrꢀ2’ꢀX ꢀinosine
2
2
2
2
phosphoramidite, Activator 42®, ACN, 3 Å MS; (e) NaI, acetone, reflux, 2 h; (f)
0
1
2
3
4
5
6
7
8
9
0
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9
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3
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5
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7
8
9
0
tetrakis(triphenylphosphine)palladium(0), Nꢀmethylaniline, THF; (g) 1ꢀ(mesityleneꢀ2ꢀsulfonyl)ꢀ
3ꢀnitroꢀ1,2,4ꢀtriazole (MSNT), pyridine; (h) 33% MeNH in EtOH.
2
Biological Evaluation.
In vitro screening of cAIMP analogs in a murine macrophage cell line
We first screened the cAIMP analogs for IRF pathway induction in a murine immune cell line
that expresses STING, and then compared their activities to that of the reference agonists for
1
5,16
murine (DMXAA
) and human (2’,3’ꢀcGAMP) STING. We used the murine macrophage
TM
reporter cell line RAWꢀLucia ISG, which reports on IRF pathway activation as an indirect
measure of type I IFN induction. Each cAIMP analog induced IRF activation to distinct levels
(
Table 2). Interestingly, compared to DMXAA (EC : 35.2 µM) and 2’,3’ꢀcGAMP (EC : 18.8
50 50
µM), most of the cAIMP analogs gave a superior IRF response, with 53 (EC : 0.6 µM) and 56
50
(EC : 2.2 µM) exhibiting strikingly higher activity. We next screened the analogs in the
5
0
TM
corresponding STING knockout cell line, RAWꢀLucia ISGꢀKOꢀSTING. As expected, none of
them exhibited any IRF activity in the knockout cells (data not shown). Together, these findings
indicate that in a murine immune cell line, the cAIMP analogs trigger STINGꢀdependent IRF
pathway induction and that their activities differ to that of DMXAA and 2’,3’ꢀcGAMP.
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Table 2. Biological activities of the cAIMP analogs of the present study, and of DMXAA and
a,b
2’,3’ꢀcGAMP
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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50
51
52
53
54
55
56
57
58
59
60
TM
TM
Assay
RAWꢀLucia ISG
THP1ꢀDual
Blood
Readout
IRF
IRF
NFꢀκB
Type I IFNs
a
b
a
b
a
b
c
Compound
9
EC50 (µM)
Emax (%)
EC50 (µM)
5.1 ± 1.4
15.1 ± 0.8
12.5 ± 0.3
17.9 ± 1.2
1.6 ± 1.3
10 ± 4.1
1.1 ± 0.4
0.3 ± 0.2
1.4 ± 0.4
2.3 ± 0.9
1.9 ± 1.7
n.t.
Emax (%)
134 ± 13
126 ± 2
141 ± 1
115 ± 2
94 ± 7
133 ± 5
79 ± 4
81 ± 15
91 ± 1
81 ± 3
87 ± 24
n.t.
EC50 (µM)
15.9 ± 4.1
60.6 ± 2.6
Emax (%)
97 ± 21
116 ± 1
100 ± 9
EC50 (µM) Emax (%)
8.4 ± 5.7
107.0 ± 24
85.0 ± 2
85 ± 9
6.4 ± 2.3
11.2 ± 3.8
15.5 ± 4.9
20.5 ± 5.5
10.6 ± 3.9
9.9 ± 2.5
0.7 ± 0.1
0.4 ± 0.1
3.6 ± 1.1
2.8 ± 0.9
4.7 ± 2.1
n.t
96 ± 8
101 ± 13
104 ± 13
100 ± 17
104 ± 9
108 ± 12
101 ± 4
103 ± 3
98 ± 8
1
0
15.9 ± 12.2
6.1 ± 4.8
17.4 ± 12.3
4.0 ± 2.7
11.6 ± 4.3
5.1 ± 0.1
0.6 ± 0.4
9.1 ± 7.1
5.0 ± 2.6
2.2 ± 0.5
35.2 ± 14.1
18.8 ± 9.7
23
54.3 ± 12.1
110.9 ± 10.2
7.8 ± 1.3
2
1
5
5
5
7
3
1
2
3
95 ± 1
162 ± 15
100 ± 24
109 ± 12
99 ± 23
94 ± 26
93 ± 8
115 ± 30
91 ± 1
62.3 ± 18.7
15.4 ± 3.2
1.6 ± 0.3
90 ± 14
104 ± 5
99 ± 7
54
55
25.5 ± 0.5
32.3 ± 10.2
9.9 ± 0.2
93 ± 36
83 ± 1
105 ± 18
106 ± 25
n.t.
103 ± 4
103 ± 5
n.t.
5
6
DMXAA
68 ± 40
100
n.t
2
',3'ꢀcGAMP
7.2 ± 3.2
100
39.1 ± 22.6
100
19.6 ± 6.7
100
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6
a. Each value is the mean ± SD from three independent experiments. b. Emax values are reported
as a mean percentage ± SD relative to the corresponding 2’,3’cGAMP response from three
independent experiments. c. Each value is the mean ± SD from three independent experiments on
a total of fifteen healthy donors.
0
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0
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9
0
In vitro screening of cAIMP analogs in a human monocyte cell line
We next evaluated the ability of the cAIMP analogs to induce both the IRF and the NFꢀκB
pathways in a human immune cell line that expresses STING, and then compared their activities
to that of 2’,3’ꢀcGAMP (in this case we did not test DMXAA, as it does not bind to human
1
6
TM
STING ). We employed the THPꢀ1 human monocyte reporter cell line THP1ꢀDual , which
uses a dualꢀreporter system to report on IRF activation as an indirect measure of type I IFN
induction, and NFꢀκB activation as an indirect measure of proꢀinflammatory cytokine induction.
Each cAIMP analog activated both pathways to varying degrees (Table 2). In terms of IRF
activity relative to that of 2’,3’ꢀcGAMP (EC : 7.2 µM), six of the cAIMP analogs (13 and 52ꢀ
5
0
56) exhibited greater than threefold activity, whereas the remaining five showed roughly similar
activity. Alternatively, for NFꢀκΒ pathway activation, 13, 53 and 56 were also more active (>
threefold) than 2’,3’ꢀcGAMP (EC : 39.1 µM), with 53 showing nearly 20ꢀfold greater activity;
50
in contrast, the remaining analogs had similar or lower activity than 2’,3’ꢀcGAMP (in all cases,
EC : ca. 15.4 µM to 110.9 µM). Together, these findings indicate that in a human immune cell
50
line, the cAIMP analogs induce the IRF and the NFꢀκB pathways and that some of them are
more active than 2’,3’ꢀcGAMP at activating either or both pathways.
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Page 15 of 52
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9
Ex vivo screening of cAIMP analogs in human blood
Having demonstrated that the cAIMP analogs trigger STING signaling in vitro, we then
assessed their cytokineꢀinduction activities in human blood ex vivo. To this end, we tested blood
samples collected from healthy donors (three experiments; five donors per experiment) by
incubating them with either a cAIMP analog or 2’,3’ꢀcGAMP at a range of CDN concentrations,
and then evaluating the induction of type I IFNs (IFNꢀα and IFNꢀβ), type III interferons (ILꢀ28
a/b), interleukin 1 (ILꢀ1) and interleukin 6 (ILꢀ6). Figure 3A shows doseꢀresponse curves for
these activities for 9, as a representative example. However, the only activity for which we were
able to calculate EC values was type I IFN induction (see Table 2 and Figure 3B), as the other
10
11
12
13
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19
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50
51
52
53
54
55
56
57
58
59
60
5
0
activities were characterized by a lack of saturating responses, even at the highest test dose. In
terms of inducing type I IFNs, every one of the cAIMP analogs showed either significantly
greater (up to ~ 45ꢀfold; p < 0.05), or similar, activity relative to 2’,3’ꢀcGAMP (EC : 19.6 µM ±
50
6.7 µM) (Table 2 and Figure 3B). The most active analogs were 53 (EC : 0.4 µM ± 0.1 µM; p =
5
0
0
.003) and 52 (EC : 0.7 µM ± 0.1 µM; p = 0.002), which also gave the strongest ILꢀ1
50
production (see Figure 3C). In contrast, 9, whose EC for type I IFN induction activity (6.4 µM
50
± 2.3 µM; p = 0.046) was three times lower than that of 2’,3’ꢀcGAMP, was highly selective for
this activity over induction of ILꢀ1 (see Figure 3C). Together, the above findings indicate that in
whole blood from healthy human donors, the cAIMP analogs induce type I IFNs and other
cytokines to varying degrees and that they exhibit distinct cytokine induction activities to that of
2
’,3’ꢀcGAMP. Importantly, some of these analogs preferentially induce type I IFNs over ILꢀ1,
whereas others give a more balanced response.
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0
1
2
3
4
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6
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8
9
0
Figure 3. Ex vivo cytokine induction in human blood. (A) Mean cytokine induction for
stimulation of blood with 9. Doseꢀresponse curves for production of type I IFNs, ILꢀ6, ILꢀ28 or
ILꢀ1 were calculated using GraphPad Prism software. (B) Plot of EC values for induction of
50
type I IFNs for the indicated compounds (points: values for individual donors; bars: mean values
± SEM acquired from three independent experiments). Significance (p 0.05) was determined
<
by a twoꢀtailed, unpaired tꢀtest relative to 2’,3’ꢀcGAMP. (C) Type I IFN induction vs. ILꢀ1
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Page 17 of 52
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9
induction at a CDN concentration of 10 ꢁg/mL. Each value represents the mean from a total of
fifteen donors (in three experiments), expressed as fold induction (normalized to the mean value
for unstimulated cells).
10
11
12
13
14
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17
18
19
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23
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27
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50
51
52
53
54
55
56
57
58
59
60
Enzymatic stability
As previously mentioned, we are endeavoring to design and synthesize STINGꢀactivating
CDNs that can offer advantages over natural CDN STING agonists, not only in terms of activity,
but also as concerns drugability. Considering that CDNs can be degraded by circulating and
cytoplasmic enzymes such as nucleases and phosphodiesterases (see, for example: Shanahan et
17
al. ), which can ultimately influence their PK, we subjected our cAIMP analogs and 2’,3’ꢀ
cGAMP to a preliminary in vitro enzyme cleavage assay. To this end, we incubated each analog
for up to 2 h with either nuclease P1 (NP1) or snake venom phosphodiesterase (SVPD), both of
1
8, 19
which cleave CDNs at their phosphodiester linkages
. To measure the resistance of each CDN
to each enzyme, we monitored the reactions by HPLC. In terms of resistance to NP1, some of the
cAIMP analogs were more resilient than 2’,3’ꢀcGAMP (83% remaining): in fact, 13, 52 and 53
did not show any signs of degradation (Figure 4, top). The remaining analogs showed a similar
level of resistance to that of 2’,3’ꢀcGAMP, except for 9, 23 and 51, which had been totally, or
near totally, degraded within 2 hours, exhibiting halfꢀlives (T ) of 23.1 minutes (9), 7.1 minutes
1
/2
(
23) and 5.7 minutes (51). Regarding resistance to SVPD, all of the cAIMP analogs exhibited
either markedly greater (13, 51, 52, 53, 55 and 56), or similar, stability relative to 2’,3’ꢀcGAMP.
Interestingly, even after 2 hours incubation, 53 and 56 had not undergone any degradation
(
Figure 4). These results reveal that some of the cAIMP analogs are more stable than 2’,3’ꢀ
cGAMP to cleavage by NP1 and/or SVPD.
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9
0
Figure 4. In vitro enzymatic cleavage resistance assays. The cAIMP analogs and 2’,3’ꢀcGAMP
were each incubated for 2 h with either (A) nuclease P1 (NP1) or (B) snake venom
phosphodiesterase (SVPD). The amount of remaining CDN was determined by HPLC (% peak
area).
DISCUSSION
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Page 19 of 52
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9
We provide here a preliminary SAR analysis for the cAIMP analogs. Based on the ensemble of
biological results, we compare these analogs to the parent compound, 9, based on each structural
parameter explored. We also consider the activities of all the analogs relative to those of 2’,3’ꢀ
cGAMP.
10
11
12
13
14
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60
Based on literature precedent describing substantial differences in the STINGꢀbinding activity
3
, 20, 21
and modes of cGAMP linkage isomers
, we sought to evaluate the role of the
internucleotide linkage position in the four linkage isomers of cAIMP (9, 10, 23 and 27).
Intriguingly, whereas 2’,3’ꢀcGAMP has been described to bind to STING approximately 300
3
times more strongly than does 3’,3’ꢀcGAMP , our screening results suggest that 10 and 9 induce
STING signaling to similar levels. We did observe that 9 was generally more active in vitro and
gave slightly stronger induction of type I IFNs in blood. Extending this analysis to the remaining
cAIMP linkage isomers 23 and 27, we noted that all four linkage isomers gave comparable
results for induction of ILꢀ1 in blood but differed in their ability to induce type I IFNs.
Specifically, 9 was the strongest inducer of type I IFNs, especially relative to 27. Thus, in terms
of internucleotide linkage geometry, it appears that AI CDNs do not exhibit the same SAR as the
corresponding (naturally occurring) cGAMP linkage isomers. Accordingly, for the future design
of CDN STING agonists, the choice of internucleotide linkage should account for the two
constituent bases, and vice versa.
In terms of enzymatic degradation, 10 and 2’,3’ꢀcGAMP were similarly more resistant than 9
to NP1, but all three CDNs were susceptible to hydrolysis by SVPD, which suggests that the
linkage position influences stability in an enzymeꢀspecific fashion. In line with these findings,
2
2
23
Danilchanka and Mekalanos , and Gao et al. , have previously reflected on the possible
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9
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existence, in eukaryotic cells, of linkageꢀspecific phosphodiesterases that could distinguish
between self (i.e. 2’,3’ꢀcGAMP) and foreign (i.e. 3’,3’) CDNs.
Considering the ribose modifications, we first observed that 9 and 51 generally had similar
activity in vitro, although the former gave mildly higher induction of type I IFNs in blood (based
on EC ). In terms of enzymatic stability, although both CDNs had been totally degraded by NP1
0
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2
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0
1
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0
1
2
3
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7
8
9
0
50
within 2 hours, 51 did show a fourfold shorter halfꢀlife. Interestingly, Li et al. reported that 2’,3’ꢀ
cGAMP and its monoꢀ2’ꢀdeoxyribose analog 2’,3’ꢀcdGAMP underwent similar levels of
degradation (T1/2
=
ca.
1
hour for both compounds) by ectoꢀnucleotide
24
pyrophosphatase/phosphodiesterase 1 (ENPP1) ; however, the authors did not test the
corresponding bisꢀ2’ꢀdeoxyribose analog. Nevertheless, whereas 9 had also been degraded by
SVPD within 2 hours, 51 was partially resistant, having undergone less than 40% degradation.
Thus, the bisꢀ2’ꢀdeoxyribose substitution appears to increase or decrease the resistance of
cAIMP to enzymatic cleavage on an enzymeꢀspecific basis.
We observed that the cAIMP analogs containing at least one 2’ꢀfluoroꢀ2’ꢀdeoxyribose sugar
(52ꢀ56) were typically among the most active compounds in all assays. Specifically, in blood the
bisꢀ2’ꢀfluoroꢀ2’ꢀdeoxyribose analogs 52 and 53 were markedly more active for induction of type
I IFNs and of ILꢀ1 than were the monoꢀ2’ꢀfluoroꢀ2’ꢀdeoxyribose compounds 54ꢀ56, which in
turn were more active than all the remaining (i.e. nonꢀfluoro) analogs for induction of type I
IFNs. Likewise, 52 and 53 were also among the most resistant analogs to enzymatic degradation
and were more resistant than 54 and 55. Interestingly, 54 and 55, which differ only by the
location of the 2’ꢀfluoro group (2’ꢀFdI vs. 2’ꢀFdA, respectively), exhibited similar activities in
nearly all of the assays, which suggests that this moiety enhances STINGꢀpathway signaling and
enzymatic stability relative to 9 irrespective of which nucleoside is modified. Regardless, we
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Page 21 of 52
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9
cannot yet conclude whether the apparently superior activity of these fluoroꢀcAIMP analogs
relative to both 9 and 2’,3’ꢀcGAMP is due to superior enzymatic stability, stronger STING
binding, greater cellular uptake (e.g. owing to the lipophilicity of the 2’ꢀF atoms relative to the
corresponding 2’ꢀOH groups in 9), or a combination of these or other factors known to be
10
11
12
13
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2
5
conferred by fluorine atoms .
In oligonucleotide chemistry, replacement of phosphodiester linkages with phosphorothioate
linkages is a wellꢀknown strategy for lengthening the halfꢀlife of drugs in vivo, as it confers
partial or total resistance to enzymatic cleavage (for a review on the PK of phosphorothioate
26
oligonucleotides, see, for example: Srinivasan et al. ). Furthermore, phosphorothioate analogs
of oligonucleotides have been reported to exhibit distinct activities relative to their parent
(phosphodiester) compounds. In fact, phosphorothioate CDNs have been described to be more
24
stable to enzymatic resistance than their corresponding phosphodiester CDNs in vitro ; to
2
7
induce more STINGꢀdependent production of type I IFNs in vitro in monocytes , lung
2
4
24
fibroblasts and modified HEK293 cells ; and to provide greater efficacy as STING agonists
27
for immunotherapy of cancer in mouse tumor models in vivo . Consistent with these and other
reports on the relative activity of phosphorothioate CDNs, in our study all three phosphorothioate
analogs (13, 53 and 56) were more active than their corresponding phosphodiester analogs (9, 52
and 55, respectively) for cytokine induction in vitro. The cytokine induction activity of CDNs
depends partly on their stability to enzymatic cleavage. For example, McWhirter et al. reported
that cꢀdiGMP induced IFNꢀβ in bone marrowꢀderived macrophages, but the product resulting
2
8
from hydrolysis of cꢀdiGMP by SVPD was inactive . Similarly, Li et al. attributed the superior
in vitro cytokine induction capacity of 2’,3’ꢀcGAM(PS) relative to 2’,3’ꢀcGAMP exclusively to
2
the greater resistance of the former to phosphodiesterase digestion, rather than to superior
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STING binding, as the two compounds exhibited similar affinities in a STINGꢀbinding assay .
Consistent with this premise, we also observed that our phosphorothioate analogs were among
the most stable to enzymatic degradation. However, in the present work we did not directly
measure the STINGꢀbinding affinity of the cAIMP analogs and thus, cannot speculate on this
parameter. Regardless of whether the superior in vitro activity of the phosphorothioates relative
to the phosphodiesters was indeed due to the greater enzymatic stability of the former, we cannot
yet explain why this advantage did not consistently translate to better cytokine induction activity
in blood. For example, concerning type I IFN induction, although 53 was slightly more active
than 52, 13 and 56 were actually slightly less active than 9 and 55, respectively.
0
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0
A crucial factor to consider when assessing the activity of phosphorothioate CDNs is
stereochemical purity: since each of the two internucleotide linkages in a CDN can exist as either
of two diastereomers (Rp or Sp), each CDN can theoretically exist as four possible stereoisomers
(
[Rp, Rp]; [Rp, Sp]; [Sp, Sp]; or [Sp, Rp]). Interestingly, Corrales et al. reported that for
induction of type I IFNs in THP1 cells by bisꢀphosphorothioate CDNs such as 2’,3’ꢀcdiAM(PS)2,
27
each Rp,Rp stereoisomer was more active than its corresponding Rp,Sp stereoisomer. In our
study, we screened 13, 53 and 56 as mixtures of their constituent stereoisomers.
The results for cytokine induction in blood demonstrated that the cAIMP analogs vary in their
relative abilities to drive STINGꢀdependent induction of type I interferons and of ILꢀ1. For drug
development, such differences might ultimately represent a therapeutic advantage according to
the desired activity: for example, for selective induction of type I IFNs (as with 9 and 10), or for
a more balanced response between type I IFNs and ILꢀ1 (as with 52ꢀ54).
Not only did we observe that type I IFN induction activity in blood differed with each CDN,
but we also observed that it varied widely by donor. Interestingly, this variability was the
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smallest for the two most active CDNs (52 and 53). There is some precedent for such
heterogeneity in assays of STINGꢀactivating compounds. Fu et al. stimulated PBMCs from
human donors with different CDN STING agonists, and found that production of IFNꢀβ and
10
11
12
13
14
15
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2
9
TNFꢀα varied not only by CDN, but also by each donor’s STING (Tmem173) genotype .
Among the four genotypes in their study, they observed the weakest cytokine induction for the
30
heterozygous variant R232H/R71HꢀG230AꢀR293Q . Given their findings, and the variability
that we observed in the present study, one can reason that the therapeutic utility of a STINGꢀ
modulating drug might ultimately have to be ascertained on a patientꢀbyꢀpatient basis: for
example, by using a bloodꢀbased companion diagnostic test.
CONCLUSIONS
We have reported the design, synthesis and biological screening of a set of novel STINGꢀ
activating cAIMP analogs. Compared to the reference agonists for murine (DMXAA) and human
(
2’,3’ꢀcGAMP) STING, some of these cAIMP analogs trigger greater induction of the IRF
and/or NFꢀκB pathways in vitro in human and murine immune cell lines. Similarly, these cAIMP
analogs induce cytokine production ex vivo in human blood from healthy donors, with some of
them, such as 9, showing selective induction of type I IFNs over ILꢀ1. In contrast to literature
reports that 2’,3’ꢀcGAMP is a dramatically stronger STING agonist than is its linkage isomer
3
’,3’ꢀcGAMP, we did not observe any marked difference in activity between 10 and 9 in most of
our assays, although in human blood, 9 did demonstrate slightly stronger induction of type I
IFNs relative to all three of its linkage isomers. Provisional SAR analysis suggests that the
combination of adenosine and inosine as constituent nucleosides, the 3’,3’ internucleotide
linkage geometry, the presence of one or two 2’ꢀfluoroꢀ2’ꢀdeoxyribose sugars and, in some
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cases, a bisꢀphosphorothioate linkage, each seems to enhance STING activation potency relative
to 2’,3’ꢀcGAMP. However, the advantages of each of these structural features are not necessarily
additive. Moreover, the 2’ꢀdeoxyribose and 2’ꢀfluoroꢀ2’ꢀdeoxyribose sugars, and the bisꢀ
phosphorothioate linkage, also seem to confer resistance to cleavage by certain
phosphodiesterases. It would be interesting to ascertain how these same features influence the
PK/PD of such compounds in vivo. Accordingly, we are currently evaluating these cAIMP
analogs in healthy animals and in animal models of disease, with the aim of identifying viable
preclinical candidates for STINGꢀbased immunotherapy of cancer, infectious diseases and other
disorders.
0
1
2
3
4
5
6
7
8
9
0
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0
EXPERIMENTAL SELECTION
Chemistry Materials, General Procedures, and Methods. Anhydrous solvents were
purchased from SigmaꢀAldrich and all reagents for nucleoside and nucleotide synthesis were
purchased from SigmaꢀAldrich, TCI, Carbosynth or ChemGenes. Unless noted otherwise, all
commercially obtained solvents and reagents were used directly. Moistureꢀ or airꢀsensitive
reactions were run under argon atmosphere in ovenꢀdried (120 °C) glassware and in the presence
of activated 3Å molecular sieves. Amidite coupling reactions and cyclizations were done in
anhydrous acetonitrile or pyridine under dry argon or nitrogen. The starting materials for all
reactions in dry pyridine were dried by concentration (three times) from pyridine. Preparative
silicaꢀgel flash chromatography was done on Fluka 60 Å highꢀpurity grade or Merck Grade 9385
silica, using gradients of methanol in dichloromethane. LC/ESꢀMS was performed on an Agilent
1290 Infinity UHPLC system coupled to an Agilent 1260 Infinity Diode Array Detector (DAD)
and an Agilent 6130 Quadrupole mass spectrometer equipped with an electrospray ionization
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source (ESI) and controlled by Chemstation software. The LC system was equipped with an
Aquity CSH C18 column (50 × 2.1 mm 1.7 µm) using gradients of 10 mM aq. triethylammonium
sodium bicarbonate and acetonitrile at a flowꢀrate of 300 µl/min. The UV detection wavelength
was 254 nm. The mass spectrometer was run in positive and negative ESI modes. Purity for all
final compounds was confirmed to be greater than 95% by LCꢀMS. Preparative HPLC was done
on a Waters preparative 150Q HPLC system with monitoring at 254 nm on a SunFire Prep C18
column (5 µm OBD; 30 x 150 mm) using gradients of 10 mM aq. triethylammonium bicarbonate
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
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27
28
29
30
31
32
33
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36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
31
and acetonitrile at a flowꢀrate of 100 mL/min. HꢀNMR and PꢀNMR spectra were acquired on a
Bruker Fourier 300 at room temperature and are reported in ppm. Molecular sieves (3 Å;
Aldrich) were employed after drying the commercially supplied product at 250 °C for 12 h under
vacuum. Exact mass characterization was done on a Waters ESIꢀQTOF operating in negativeꢀion
acquisition mode. The compounds were dissolved in a 1:1 (v/v) mixture of water (0.1% formic
acid) and acetonitrile. Flow Injection Analysis (FIA) was performed at a rate of 0.15 mL/min.
6
2
’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(Hꢀphosphonate)ꢀN ꢀ(Bz)adenosine
(2).
The
adenosine
phosphoramidite 1 (11.7 g, 14.73 mmol) was dissolved in a solution of ACN (50 mL). To the
solution were added water (0.53 mL, 29.46 mmol) and pyridine·TFA (3.41 g, 17.67 mmol), and
the resulting mixture was stirred for 15 min at RT. Then, tertꢀbutylamine was added, the solution
was stirred for 15 min, and the solvents were removed in vacuo. The residue was treated with a
3% solution of DCA in CH Cl (100 mL) and water (10 equiv.) for 15 min. The reaction was
2
2
quenched with MeOH and pyridine. The solvents were removed in vacuo, and the residue was
purified by silicaꢀgel column chromatography, using CH Cl /EtOH/MeOH (80/10/10) as eluent,
2
2
+
ꢀ
to give 2 (6.8 g; 84% yield). LCꢀMS: Rt = 4.41 min, m/z = 550 [M+H] , m/z = 548 [MꢀH] .
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
4
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5
5
6
6
3
’ꢀOꢀ(TBDMS)ꢀ2’ꢀOꢀ(Hꢀphosphonate)ꢀN ꢀ(Bz)adenosine
(4).
The
adenosine
phosphoramidite 3 was subjected to a similar procedure to that described for preparation of 2, to
+
ꢀ
give 4 (1.10 g; 94% yield). LCꢀMS: Rt = 4.47 min, m/z = 550 [M+H] , m/z = 548 [MꢀH] .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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9
0
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6
7
8
9
0
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2
3
4
5
6
7
8
9
0
[
2’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(CE)phosphotriesterꢀinosine]ꢀ(3’,5’)ꢀ[2’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(Hꢀ
6
phosphonate)ꢀN ꢀ(Bz)adenosine] (5). To a solution of compound 2 (2.18 g, 3.97 mmol) in a
®
solution of Activator 42 (0.1 M in ACN) (79.4 mL, 7.94 mmol) in the presence of molecular
sieves (3 Å) was added, in one portion, the commercial available inosine phosphoramidite (9.21
g, 10.4 mmol). The mixture was stirred for 30 min at RT. Then, a 5.5 M solution of tertꢀbutyl
hydroperoxide in decane (1.4 mL, 7.7 mmol) was added to the mixture, which was stirred for 40
min. The solution was filtered, and the molecular sieves were washed with CH Cl . The filtrate
2
2
was concentrated in vacuo. The residue was treated with 3% DCA in CH Cl (65 mL) in the
2
2
presence of water (10 equiv.) for 15 min. The reaction was quenched with MeOH and pyridine.
The solvents were removed in vacuo, and the residue was purified by silicaꢀgel column
chromatography, using CH Cl /MeOH (5% to 20%) as eluent to give 5 (3.27 g; 78% yield). LCꢀ
2
2
+
ꢀ
MS: Rt = 5.22 min, m/z = 1048 [M+H] , m/z = 1046 [MꢀH] .
[2’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(CE)phosphotriesterꢀinosine]ꢀ(3’,5’)ꢀ[3’ꢀOꢀ(TBDMS)ꢀ2’ꢀOꢀ(Hꢀ
6
phosphonate)ꢀN ꢀ(Bz)adenosine] (6). Compound 4 was reacted with inosine phosphoramidite
using a similar procedure to that described for preparation of 5, to give 6 (410 mg; 44% yield).
+
ꢀ
Rt = 4.70 min, m/z = 1048 [M+H] , m/z = 1046 [MꢀH] .
3’,3’)Cyclicꢀ[2’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(phosphodiester)ꢀN ꢀ(Bz)adenosine]ꢀ[2’ꢀOꢀ(TBDMS)ꢀ
’ꢀOꢀ(CE)phosphotriesterꢀinosine] (7). Compound 5 (3.27 g, 3.12 mmol) was coevaporated
6
(
3
with dry pyridine three times. The residue was suspended in dry pyridine (67 mL), and to the
resulting solution was added 5,5ꢀdimethylꢀ2ꢀoxoꢀ2ꢀchloroꢀ1,3,2ꢀdioxaphosphinane (DMOCP)
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9
(
1.99 g, 10.8 mmol). The resulting mixture was stirred at RT for 3 h. Then, iodine (0.99 g, 3.92
mmol) and water (30 equiv.) were added to the mixture. After 10 min, aq. NaHSO (0.15%) (670
3
mL) was added until complete discoloration was observed, and then aq. NaHCO was added.
3
10
11
12
13
14
15
16
17
18
19
20
21
22
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The aqueous layer was extracted three times with a 1:1 (v/v) mixture of EtOAc/Et O (800 mL).
2
The organic layers were pooled, dried over MgSO , and filtered. The solvent was removed in
4
vacuo to give 7 (3.01 g, 92% yield). This crude intermediate was used in the next step without
+
ꢀ
any further purification. LCꢀMS: Rt = 5.41 min, m/z = 1046 [M+H] , m/z = 1044 [MꢀH] .
6
(2’,3’)cyclicꢀ[3’ꢀOꢀ(TBDMS)ꢀ2’ꢀOꢀ(phosphodiester)ꢀN ꢀ(Bz)adenosine]ꢀ[2’ꢀOꢀ(TBDMS)ꢀ
3’ꢀOꢀ(phosphodiester)ꢀinosine] (8). Compound 6 was subjected to a similar procedure to that
described for preparation of 7, to give 8 (364 mg; 89% yield). LCꢀMS: Rt = 5.66 min, m/z =
+
ꢀ
1046 [M+H] , m/z = 1044 [MꢀH] .
cAIMP (9). Compound 7 (4.43 g, 4.24 mmol) was treated with a solution of methylamine 33%
in EtOH (190 mL), and the resulting mixture was stirred at 50 °C for 4 h. The reaction mixture
was concentrated, and the resulting residue was dried in vacuo. The dried material was dissolved
in dry pyridine (14 mL) and treated with Et N·3HF (14.0 mL, 85.89 mmol). The mixture was
3
stirred at 50 °C for 3 h. Then, the pH of the solution was adjusted to 6 with 1M TEAA (75 mL).
The solution was concentrated in vacuo (20 mL), and then treated with cooled acetone (210 mL)
to provide a solid precipitate. The crude product was filtered, and then purified by preparative
HPLC using a C Sunfire column (30 x 150 mm, 5 µm) with aq. ammonium formate/ACN as
1
8
eluent. The fractions containing the desired compound were pooled, and the resulting solution
was concentrated and lyophilized. The ammonium salt was exchanged on sodium Amberlite
column to give the sodium salt of 9 (0.87 g; 32% yield). LCꢀMS: Rt = 2.72 min, m/z = 660
+
ꢀ 1
[
M+H] , m/z = 658 [MꢀH] . H NMR (D O, 300 MHz) δ (ppm) 8.34 (s, 1H), 8.27 (s, 1H), 8.10
2
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9
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
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31
(
s, 1H), 7.86 (s, 1H), 5.94 (s, 2H), 5.06ꢀ4.80 (m, 4H), 4.42 (m, 4H), 4.03 (m, 2H). P NMR
ꢀ
(
D O, 202 MHz) δ (ppm) ꢀ0.88 and ꢀ1.95. Exact MS (ESIꢀTOF for C H N O P [MꢀH]
2
20 22
9
13 2
calculated 658.0812; observed mass: 658.0801.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2’,3’)cAIMP (10). Compound 8 was subjected to a similar procedure to that described for
+
preparation of 9, to give 10 (50 mg; 19% yield). LCꢀMS: Rt = 1.53 min, m/z = 660 [M+H] , m/z
ꢀ
1
=
658 [MꢀH] . H NMR (D O, 300 MHz) δ (ppm) 8.50 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 8.09 (s,
2
1
H), 6.24 (d, 1H), 6.08 (s, 1H), 5.20 (m, 1H), 4.83ꢀ4.73 (m, 2H), 4.58 (d, 1H), 4.40 (m, 2H), 4.26
3
1
(m, 2H), 4.04 (m, 2H). P NMR (D O, 202 MHz) δ (ppm) ꢀ1.03 and ꢀ1.93. Exact MS (ESIꢀTOF
2
ꢀ
for C H N O P [MꢀH] calculated 658.0812; observed mass: 658.0810.
2
0
22
9
13 2
[
2’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(CE)phosphorothioateꢀtriesterꢀinosine]ꢀ(3’,5’)ꢀ[2’ꢀOꢀ(TBDMS)ꢀ3’ꢀ
6
Oꢀ(Hꢀphosphonate)ꢀN ꢀ(Bz)adenosine] (11). To a solution of 2 (2.78 g, 5.06 mmol) in a
®
solution of Activator 42 (0.1 M in ACN) (101 mL, 10.1 mmol) in the presence of molecular
sieves (3 Å) was added, in one portion, inosine phosphoramidite (7.22 g, 8.1 mmol). The mixture
was stirred for 30 min at RT. Then, a solution of phenylacetyl disulfide (PADS) (3.1 g, 10.0
mmol) in dry pyridine (50 mL) was added, and the mixture was stirred at RT for 45 min. The
resulting solution was filtered, and the molecular sieves were washed with CH Cl . The filtrate
2
2
was concentrated in vacuo and coevaporated with ACN three times. The residue was treated with
% DCA in CH Cl (65 mL), in the presence of water (10 equiv.), for 15 min. The reaction was
3
2
2
quenched with MeOH and pyridine. The solvents were removed in vacuo, and the residue was
purified by silicaꢀgel column chromatography, using CH Cl /MeOH (5% to 20%) as eluent, to
2
2
+
give 11 (4.94 g; 91% yield). LCꢀMS: Rt = 5.42 and 5.52 min, m/z = 1064 [M+H] , m/z = 1062
ꢀ
[
MꢀH] .
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(
3’,3’)Cyclicꢀ[2’ꢀOꢀ(TBDMS)ꢀ3’ꢀOꢀ(phosphorothioateꢀdiester)ꢀN ꢀ(Bz)adenosine]ꢀ[2’ꢀOꢀ
(
TBDMS)ꢀ3’ꢀOꢀ(CE)phosphorothioateꢀtriesterꢀinosine] (12). Compound 11 (4.94 g, 4.65
mmol) was coevaporated with dry pyridine three times. The residue was suspended in dry
pyridine (286 mL), and to the solution was added DMOCP (2.4 g, 13.0 mmol). The resulting
mixture was stirred at RT for 2 h. Then, elemental sulfur (0.51 g, 15.9 mmol) was added, and the
mixture was stirred at RT for 45 min. Then, the mixture was concentrated in vacuo, coevaporated
with toluene three times, precipitated in ACN to remove the excess sulfur. The solvents were
removed in vacuo to give 12 (3.18 g; 64% yield). This crude intermediate was used in the next
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
step without any further purification. LCꢀMS: Rt = 5.61 and 5.71 min, m/z = 1078 [M+H] , m/z
ꢀ
=
1076 [MꢀH] .
cꢀAIM(PS) (13). Compound 12 (1.25 g, 1.16 mmol) was treated with 33% methylamine in
2
EtOH (85 mL), and the resulting mixture was stirred at 50 °C for 3 h. The mixture was
concentrated, and the resulting residue was dried in vacuo. The solid was dissolved in dry
pyridine (8 mL), and the solution was heated to 50 °C, and then treated simultaneously with Et N
3
(
7.2 mL, 51.66 mmol) and Et N·3HF (4.2 mL, 25.77 mmol). The mixture was stirred for 5 h.
3
The resulting solution was cooled to RT and treated with a large volume of acetone (100 mL) to
yield a precipitate, which was then filtered, and washed with cold acetone. The crude product
purified by preparative HPLC, using a C18 Sunfire column (30 x 150 mm, 5 µm) with TEAB
(aq.)/ACN as eluent. The fractions containing the desired compound were pooled, and the
solution was concentrated and lyophilized. The triethylammonium salt was exchanged on a
sodium Amberlite column to provide the sodium salt of 13 (0.287 g; 40% yield). LCꢀMS: Rt =
+
ꢀ 1
3
.45 min, m/z = 692 [M+H] , m/z = 690 [MꢀH] . H NMR (D O, 300 MHz) δ (ppm) 8.54 (s,
2
1
H), 8.37 (s, 1H), 8.22 (s, 1H), 7.97 (s, 1H), 6.65 (dd, 1H), 6.15 (dd, 1H), 4.60ꢀ4.50 (m, 4H),
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