Clicking 3′-azidothymidine into novel potent inhibitors of human immunodeficiency virus

Article abstract of DOI:10.1021/jm401232v

3′-Azidothymidine (AZT) was the first approved antiviral for the treatment of human immunodeficiency virus (HIV). Reported efforts in clicking the 3′-azido group of AZT have not yielded 1,2,3-triazoles active against HIV or any other viruses. We report herein the first AZT-derived 1,2,3-triazoles with submicromolar potencies against HIV-1. The observed antiviral activities from the cytopathic effect (CPE) based assay were confirmed through a single replication cycle assay. Structure-activity-relationship (SAR) studies revealed two structural features key to antiviral activity: a bulky aromatic ring and the 1,5-substitution pattern on the triazole. Biochemical analysis of the corresponding triphosphates showed lower ATP-mediated nucleotide excision efficiency compared to AZT, which along with molecular modeling suggests a mechanism of preferred translocation of triazoles into the P-site of HIV reverse transcriptase (RT). This mechanism is corroborated with the observed reduction of fold resistance of the triazole analogue to an AZT-resistant HIV variant (9-fold compared to 56-fold with AZT).

Full text of DOI:10.1021/jm401232v

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Article
Clicking 3’-azidothymidine into novel potent
inhibitors of human immunodeficiency virus
Venkata Ramana Sirivolu, Sanjeev Kumar V. Verneka, Tatiana Ilina,
Nataliya S Myshakina, Michael A Parniak, and Zhengqiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401232v • Publication Date (Web): 08 Oct 2013
Downloaded from http://pubs.acs.org on October 9, 2013
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Clicking 3’-azidothymidine into novel potent inhibitors of human
immunodeficiency virus
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a§
a§
b
Venkata Ramana Sirivolu Sanjeev Kumar V. Vernekar Tatiana Ilina , Nataliya S.
,
,
b
b
a
Myshakina , Michael A. Parniak and Zhengqiang Wang*
a
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455
Department of Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
b
§
These authors contributed equally
Key Words
3
'ꢀ[5ꢀArylꢀ(1,2,3ꢀtriazolꢀ1ꢀyl)]ꢀ3'ꢀdeoxythymidine, HIV, AZT, click chemistry
Abstract
3
’ꢀAzidothymidine (AZT) was the first approved antiviral for the treatment of human
immunodeficiency virus (HIV). Reported efforts in clicking the 3’ꢀazido group of AZT have not
yielded 1,2,3ꢀtriazoles active against HIV or any other viruses. We report herein the first AZTꢀ
derived 1,2,3ꢀtriazoles with subꢀmicromolar potencies against HIVꢀ1. The observed antiviral
activities from the cytopathic effect (CPE) based assay were confirmed through a single
replication cycle assay. Structureꢀactivityꢀrelationship (SAR) studies revealed two structural
features key to antiviral activity: a bulky aromatic ring and the 1,5ꢀsubstitution pattern on the
triazole. Biochemical analysis of the corresponding triphosphates showed lower ATPꢀmediated
nucleotide excision efficiency compared to AZT, which along with molecular modeling,
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suggests a mechanism of preferred translocation of triazoles into the Pꢀsite of HIV reverse
transcriptase (RT). This mechanism is corroborated with the observed reduction of fold
resistance of the triazole analogue to an AZTꢀresistant HIV variant (9ꢀfold compared to 56ꢀfold
with AZT).
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Introduction
Nucleoside RT inhibitors (NRTIs) constitute the cornerstone of chemotherapies against HIV
1
infection. Typically these inhibitors lack the 3’ꢀOH and act as obligate chain terminators. Such a
mechanism of inhibition requires successive phosphorylation by cellular kinases, competitive
active site binding against endogenous deoxynucleoside triphosphates (dNTPs) and effective
incorporation by RT. Although NRTIs generate a relatively large genetic barrier for viruses to
select resistant strains, the common pathway of intracellular phosphorylation, particularly the
2
shared cellular kinases, could easily lead to intraꢀclass drugꢀdrug interactions. Among numerous
nucleoside antivirals, AZT was the first approved for the treatment and prophylaxis of HIV /
3
,4
AIDS. Long term clinical use of AZT is unfortunately associated with significant side effects,
including myopathy, cardiomyopathy and anemia, presumably due to the sensitivity of γꢀDNA
5
6
polymerase in some cell mitochondria, and / or the depletion of thymidine triphosphate as AZT
can be both a substrate and inhibitor of human thymidine kinases (hTKs).
7
Mechanistically the unique 3’ꢀazido group of AZT can contribute critically to HIV RT binding.
Chemically modifying the 3’ꢀazido group may yield novel classes of nucleoside inhibitors with a
distinct binding mode and toxicity profile. One convenient channel for the azido modification
would be through click chemistry to form 1,2,3ꢀtriazoles which can occupy a much bigger
binding space with the three consecutive nitrogen atoms taking a profoundly different geometry.
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The potential of this particular transformation to rapidly provide new opportunities for antiviral
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discovery has been recognized and pursued by many in the field.
Unfortunately, none of the
resulting 1,2,3ꢀtriazole analogues were found to inhibit HIV or any other DNA or RNA viruses
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even at very high concentrations (100—500 ꢀM). Habich et al and Hirota et al reported that
the AZTꢀderived 1,2,3ꢀtriazoles exhibited no appreciable activity in HIVꢀ1 infected CEMꢀV and
MTꢀ2V cells, nor did they inhibit syncytium formation in infected human peripheral blood
monocytes. Herdewijn’s group showed that replacing the 3’ꢀazido group of AZT with other fiveꢀ
1
2
13
membered heterocyclic rings eliminated antiviral activity. More recently, Zhou et al found
that even with a more elaborated side chain, AZTꢀderived 1,2,3ꢀtriazoles showed no inhibitory
activity against any of these viruses: parainfluenza type 3, reo type 1, Sindbis, Coxsackie B4,
Punta Toro, vesicular stomatitis, respiratory syncytial, herpes simplex, vaccinia, cowpox, and
HIV. Notably, AZTꢀderived 1,2,3ꢀtriazoles reported to date typically feature an alkyl substituent
at the Cꢀ4 site of the triazole (Figure 1, 2). Their lack of antiviral activity is likely due to
inefficient cellular activation as 1,2,3ꢀtriazole nucleosides are poor substrates for the cytosolic
1
4
human thymidine kinase 1 (hTKꢀ1) when compared to AZT. Intriguingly, when the 4ꢀalkyl
group of the triazole is replaced with a substituted phenyl ring, the resulting analogues (1d–1e)
15
showed significantly improved affinity for hTKꢀ2 (Figure 1). This observation led us to
hypothesize that a bulky aryl substituent on the triazole scaffold may provide crucial binding for
the target enzyme, thus fundamentally change the antiviral profile. Based on this hypothesis, we
have synthesized both 4ꢀaryl (scaffold 3) and 5ꢀaryl (scaffold 4) 1,2,3ꢀtriazoles (Figure 1) and
have identified a few analogues with confirmed antiviral activity in the subꢀmicromolar range.
To the best of our knowledge, this represents the first successful attempt of clicking AZT into
1,2,3ꢀtriazoles with potent antiviral activity.
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Figure 1. Design of 1,2,3ꢀtriazole HIV inhibitors. (a) AZTꢀderived 1,2,3ꢀtriazoles as inhibitors
of hTKꢀ2. The C4 aromatic substituent yields significantly improved inhibitory activity (1d–1e);
(b) introducing a bulky group at C4 or C5 may turn the inactive 1,2,3ꢀtriazoles 2 into potent HIV
inhibitor scaffolds 3–4.
Results and Discussion
Chemistry. Click chemistry provides a powerful tool in chemical biology and drug discovery as
16
it allows efficient and clean creation of compounds under extremely mild conditions. The
Huisgen thermal cycloaddition between an organic azide and an alkyne typically yields a mixture
1
7,18
of two regioisomeric 1,2,3ꢀtriazoles.
This reaction was rendered clickable through two
dramatic Sharpless modifications: the copper(I)ꢀcatalyzed azide–alkyne cycloaddition
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(
CuAAC) which results in the exclusive formation of 1,4ꢀdisubstituted 1,2,3ꢀtriazoles; and the
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ruthenium(II)ꢀcatalyzed variant (RuAAC) that specifically generates 1,5ꢀdisubstituted 1,2,3ꢀ
triazoles. The CuAAC click chemistry has gained particular significance and popularity from its
2
1
22,23
24
wide range of applications in drug discovery, bioconjugation
and material science. By
contrast, the RuAAC click chemistry remains rather underexplored due to its relatively low
applicability and efficiency. As mentioned earlier, reported efforts on clicking AZT have
primarily focused on the CuAAC with alkyl alkynes and none of the resulting 1,4ꢀdisubstituted
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,2,3ꢀtriazoles showed appreciable activity in antiviral assays. A close examination on their
inhibition of hTKꢀ2 revealed that a bulkier substituent, preferably a large aromatic ring, may
1
5
provide key interactions for target binding. Toward this end, we were prompted to explore both
the CuAAC and RuAAC click reactions to gain access to both 1,4 and 1,5 regioisomers. The
reactions were carried out with a broad range of aromatic and aliphatic alkynes as shown in
schemes 1 and 2. The propargyl aryl ethers (7a–f) were readily prepared via the alkylation of
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phenols with propargyl bromide and K CO in good yields (scheme 1). The aromatic and
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aliphatic alkynes were either commercially available or prepared from aldehydes via the
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SeyferthꢀGilbert homologation with the Bestmann reagent (14) (scheme 2).
a
Scheme 1. Synthesis of 1,2,3ꢀtriazoles 8 and 9
a
O
N
O
N
Ar OH
Ar
b (for 8)
c (for 9)
O
Br
HN
HN
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AZT
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MeO
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HO
+
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N1
O
a
d
b
c
f
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N
O
N
Ar
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MeO
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a
Reagents and conditions: a) propargyl bromide, K CO , DMF, rt, 12–15 h, 60–76%; b) sodium
2
3
ascorbate, CuSO ·5H O, THF/H O (3:1), rt, 12 h, 54–96%; c) Cp*RuCl(PPh ) , THF, 60 ºC, 1–2
4
2
2
3 2
d, 24–48%.
The two versions of the Huisgen–Sharpless cycloaddition proceeded with considerably different
efficiencies: the CuAAC with alkynes (7a–f) (scheme 1) and (16a–m) (scheme 2) typically
completed within 12 hours under ambient temperature and produced 1,4ꢀdisubstituted 1,2,3ꢀ
triazoles (8a–f) and (17a–m) in good yields, whereas the RuAAC variant with alkynes (7a–f)
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scheme 1) and (16a–m) (scheme 2) required longer reaction time and elevated temperature for
completion and generated 1,5ꢀdisubstituted 1,2,3ꢀtriazoles (9a–f) and (18a–m) in only moderate
yields. The isolation of 1,4ꢀ and 1,5ꢀdisubstituted triazoles from reaction mixture proved to be
rather straightforward by flash column chromatography. However, caution has to be taken for
analogues with a similar Rf to AZT to avoid any AZT contamination which will likely cause
false positive in antiviral assays. In our case, the purity of final triazoles was confirmed by NMR
and HPLC analysis. The formation of 1,4ꢀ and 1,5ꢀdisubstituted triazoles were clearly evident
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from 1H NMR that the 2’ꢀCH protons were split into two distinct peaks around 2–3 ppm, which
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was not observed with AZT.
a
Scheme 2. Synthesis of 1,2,3ꢀtriazoles 17 and 18
a
Reagents and conditions: a) KI, acetone/CH CN, rt–50 ºC, 12 h, 72%; b) NaN , acetone/H O, 0
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ºC–rt, 14 h, 94%; c) NaH, THF/benzene, then 13, 0 ºC–rt, 12h, 65%; d) K CO , MeOH, rt, 12 h,
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5–90%; e) sodium ascorbate, CuSO ·5H O, THF/H O (3:1), rt, 12 h, 54–96%; f)
4 2 2
Cp*RuCl(PPh ) , THF, 60 ºC, 1–2 d, 24–48%.
3
2
The preparation of triphosphates for selected nucleoside triazoles is necessitated by the need of
biochemical studies. In this event the nucleoside triazoles (18a, 18e–18f) were directly converted
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to the corresponding triphosphates (21a, 21e–21f) using the oneꢀpot procedure by Eckstein
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(
Scheme 3). The triazole nucleotides were purified by reversedꢀphase HPLC using the C18
reverse phase column and ion exchange chromatography. The triphosphates were obtained in
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moderate yields (33–43%) and were characterized by HRMS, H and P NMR.
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a
Scheme 3. Synthesis of triazole nucleoside triphosphates 21
a
Reagents and conditions: a) 2ꢀChloroꢀ4Hꢀ1,3,2ꢀbenzodioxaphosphorinꢀ4ꢀone, pyridine/dioxane
(
1:3), rt, 15 min; b) (Bu HN) H P O , Bu N, DMF, rt, 15 min; c) I , pyridine/H O, NH (aq), rt, 1
3 2 2 2 7 3 2 2 3
h, Naꢀion exchange, 33ꢀ43%.
Antiviral Screening. To test the hypothesized beneficial effects of the bulky aromatic group, all
synthesized 1,2,3ꢀtriazole compounds were screened for antiviral activity. The primary antiviral
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screening was conducted with a wellꢀestablished colorimetric cytoprotection assay
based on
viral CPE in CMEꢀSS cells. This assay measures cell viability in relation to CPE and requires
multiple rounds of viral replication. Initial screening was done at a single concentration (10 ꢀM)
and cell viability determined through the addition of (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyl
tetrazolium bromide (MTT), a colorless compound that is reduced only by metabolically viable
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cells to produce a purple compound. We first tested the 1,2,3ꢀtriazole series where the C4 / C5
bulky substituent is separated from the triazole ring with a methylene ether linkage (scaffolds 8–
9
). As shown in Table 1, analogues of this series all have good viability (85–100%) under 10
M. The most striking SAR trend is the observation that most 1,5ꢀsubstituted 1,2,3ꢀtriazoles (9a,
c–9e) showed significant antiviral activity (42–88% CPE reduction) at 10 ꢀM whereas the 1,4ꢀ
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regioꢀisomers remained inactive (Table 1), strongly suggesting that the substitution pattern may
be crucial to conferring antiviral activity. The relatively flat SAR in regard to the size and
positioning of the aromatic ring for 1,5 analogues may simply reflect the deleterious effect of the
linkage. Nevertheless, this level of antiviral activity is in stark contrast with all reported AZTꢀ
derived 1,2,3ꢀtriazoles where no appreciable activity was observed at concentrations up to 500
ꢀM.
Table 1. Single concentration screening of scaffolds 8 and 9 in the cytoprotection assay against
HIVꢀ1 in CEMꢀSS cells.
Inhibition % @ 10
Compound
R
Viability % @ 10 ꢀM
ꢀM
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9
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a
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Next we tested analogues with the bulky substituent immediately connected to the triazole ring
scaffolds 17–18). Again these triazole analogues typically do not show cytotoxicity at 10 ꢀM
(
(Table 2). As for antiviral activity, 1,5ꢀsubstituted analogues in general remained superior to
their 1,4 regioisomers, though many 1,4 analogues (17b–17f, Table 2) also exhibited discernible
antiviral activities (33–44%) at 10 ꢀM, implying that effective bulkiness without a linkage
greatly benefits target binding. This bulky group effect is further illustrated within the 1,5ꢀseries,
where a small cycloalkyl group (18k) confers little inhibitory activity, while large aromatic rings
(18a, 18c–18f, 18h–18j) consistently support substantial antiviral activity. Smaller aromatic
rings, the phenyl ring (17h, 18h) or substituted phenyl rings (1d, 1e, 18m, 18n), do not appear to
provide sufficient target binding, hence the observed less potent antiviral activities. In the
meantime, the positioning of the aromatic ring also seems to considerably impact the antiviral
activity as evidenced by the much higher potency of the α positioned naphthyl substituent (18a)
when compared to the β positioned naphthyl analogues (18d–18e). Furthermore, the highly
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privileged biphenyl substituent appears to benefit the target binding of both 1,4 (17j) and 1,5
(18j) regioisomers. This beneficial effect for the 1,4 series, however, is drastically reduced when
the biphenyl is separated by an O atom (17i vs 17j). Finally, the screening assay initially
identified 18l as a promising hit (100% inhibition @ 10 ꢀM). However, this activity was not
confirmed upon testing in the single replication cycle assay, in which 18l did not produce
significant antiviral activity at concentrations up to 10 ꢀM. Retesting in the screening assay
yielded a much reduced percentage inhibition for 18l, corroborating the single replication assay
result.
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Single concentration screening of scaffolds 17 and 18 in the cytoprotection assay
against HIVꢀ1 in CEMꢀSS cells.
Inhibition % @ 10
Compound
R
Viability % @ 10 ꢀM
ꢀM
1
1
7a
8a
1
100
100
100
82
83
35
12
44
74
33
37
35
17b
18b
1
7c
8c
100
94
1
1
7d
8d
98
1
100
95
1
7e
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8e
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100
100
17f
18f
100
11
0
100
100
97
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1
7g
8g
1
1
7h
8h
24
44
0
100
100
100
97
1
17i
18i
67
91
63
1
1
7j
8j
88
1
80
1
7k
8k
100
100
100
1
17
0
1
7l
a
a
1
8l
100 (43 )
100 (88 )
1d
0
23
6
100
18m
100
1
e
100
1
8n
31
100
a
Retest results
Dose-Response Antiviral Potency. The antiviral potential of these novel 1,2,3ꢀtriazoles were
further assessed in doseꢀresponse fashion using the same cytoprotection (CPE reduction) assay
with three selected 1,5 analogues (9d, 18a, 18f) and AZT as control. Gratifyingly, all three
compounds showed exceptional antiviral activity with low cytotoxicity (Table 3). Of particular
significance are analogues 18a and 18f each inhibiting HIVꢀ1 IIIB with submicromolar potency
and a very large therapeutic window. The other analogue tested (9d) was also active at low
micromolar concentrations. The EC of 1.9 ꢀM represents a nearly 30ꢀfold drop in antiviral
50
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1
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potency when compared to 18a, strongly suggesting that the bulkiness provided by the aromatic
substituent is crucial to target binding and that its separation from the triazole ring through a
linkage tremendously compromises the antiviral potency.
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
Table 3. Doseꢀresponse antiviral testing of selected compounds in two distinct assays.
CPE Reduction Assay
Single Replication
Compound
a
a
b
c
Cycle Assay EC50
(ꢀ
M)
EC50
(ꢀM)
CC50
(ꢀM)
TI
AZT
0.0053
1.9
>1.0
70
>48
37
910
210
ꢀꢀ
0.14
> 5.0
1.0
9
d
18a
0.067
0.10
61
18f
21
> 5.0
7.2
d
17d
17f
ND
ND
ND
ND
ND
ND
ꢀꢀ
4.7
18e
ꢀꢀ
4.1
a
b
c
Concentration inhibiting virus replication by 50%. Concentration resulting in 50% cell death.
d
Therapeutic index, defined by CC /EC . Not determined.
5
0
50
To verify the observed doseꢀresponse antiviral potency, we have also tested these compounds in
a distinct single replication cycle antiviral assay. This assay quantitatively measures HIV
infection in indicator cells (P4R5) through the expression of a Tatꢀdependent reporter (βꢀ
galactosidase) and offers the advantage of quickly determining the infectivity / inhibition with
only one replication cycle by colorimetric analysis after incubating with a βꢀgalactosidase
substrate. Through this single replication cycle assay the exceptional potency of 18a was
confirmed as it inhibited WT HIVꢀ1 in dose response fashion with an EC of 1.0 ꢀM (Table 3).
5
0
While this potency is lower than the submicromolar activity (0.067 ꢀM) observed in the CPE
based assay, it may reflect largely the intrinsic difference between these two assays, as AZT also
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showed considerably different potencies in the two assay methods. Dose response inhibition was
also observed with the other two selected analogues (9d and 18f) in low micromolar range
(
curves not shown), though definite EC values were not determined. Significantly, this assay
50
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also identified three additional triazole analogues (17d, 17f and 18e) with low micromolar
activities (EC50 = 4.1–7.2 ꢀM) against WT HIVꢀ1, further establishing the antiviral potential of
these nucleoside triazoles. A few other hits (9c, 9e, 18i, 18j, 18l and 18n) from the screening
assay were also tested in the single replication cycle assay, and none showed significant antiviral
activity at concentrations up to 10 ꢀM (data not shown).
We then further characterized the antiviral profile of the most active analogue 18a using AZTꢀ
resistant and NNRTIꢀresistant HIV strains. When tested against HIV possessing mutations
associated with AZT resistance (D67N/K70R/T215F/K219Q), 18a showed an EC of 9.1 ꢀM
50
which amounts to a resistance of 9 fold (Table 4). However, the degree of HIV resistance
imparted by these mutations to 18a was substantially less than that to AZT (56 fold). By contrast,
testing of 18a against the NNRTIꢀresistant HIV (L100I/K103N) yielded better potency than
against WT HIV (Table 4). Interestingly, the same NNRTIr HIV did not show enhanced
susceptibility to AZT (Table 4).
Table 4. EC (ꢀM) values for AZT and AZTꢀtriazole derivative 18a against wild type (WT),
50
AZTꢀresistant (AZTr), and NNRTIꢀresistant (NNRTIr) HIVs.
a
Compound
WT HIV
0.14
AZTr HIV
NNRTIr HIV
0.12 (1)
b
AZT
7.9 (56)
1
8a
1.0
9.1 (9)
0.6 (0.6)
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a
NNRTIr HIV possesses L100I/K103N mutations in RT that confer highꢀlevel resistance to most
b
NNRTIs; Fold resistance relative to WT HIV.
3
1,32
33,34
Viral hypersusceptibility of NRTIꢀresistant HIVs to NNRTIs
and NRTIs
is well known.
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
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8
9
0
1
2
3
4
5
6
7
8
9
0
However, hypersusceptibility of NNRTIꢀresistant HIVs to other classes of antivirals is rarely
reported. That mutations associated with NNRTI resistance confer hypersusceptibility of HIV to
our AZTꢀtriazoles is particularly intriguing. Expanded antiviral profiling to include most of our
active analogues resulted in 2ꢀ to 5ꢀfold enhanced potencies against NNRTIꢀresistant HIV as
compared to WT virus (Table 5). These results substantiate the hypersusceptibility of NNRTIꢀ
resistant HIV to our AZTꢀtriazole analogues, though the mechanism involved is presently
unclear.
Table 5. Hypersusceptibility of NNRTIr HIV to AZTꢀtriazole NRTIs
Compound
EC WT HIV (
ꢀM)
EC NNRTIr HIV (
ꢀM)
ratio NNRTIr/WT
5
0
50
9
d
>5.0
7.2
4.7
1.0
4.1
2.4
2.1
1.0
0.6
1.8
< 0.5
0.3
17d
17f
0.2
18a
0.6
18e
0.4
AZT-triazole Inhibition of HIV-1 RT-directed DNA Synthesis. AZTꢀ5’ꢀtriphosphate is readily
used as a substrate by HIV RT, and once incorporated into the viral DNA, acts as a chain
terminator to prevent further DNA chain elongation. The antiꢀHIV activity of the AZTꢀtriazole
analogues suggested that they might function in a similar manner. To evaluate this, we carried
out biochemical studies with the triphosphate forms (21a and 21f) of the two most potent
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inhibitors (18a and 18f). The triphosphate derivatives 21a and 21f inhibited HIV RT DNA
polymerase activity in vitro with IC50 values (inhibitory concentration required for 50%
inhibition of the enzyme activity) of 3.7 and 11.8 ꢀM, respectively. We then focused on 21a for
detailed biochemical mechanism of action studies. This analogue was a substrate for HIV RT
and was incorporated into DNA albeit with reduced efficiency compared to TTP or AZTꢀTP
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(data not shown), consistent with the reduced antiviral potency of 18a compared to AZT (Table
4). HIV RT bound a nucleic acid template/primer terminated with either AZT or 21a with similar
affinity (K values of 5.4 and 10.9 nM, respectively), indicating that reduced dissociation of 21aꢀ
D
terminated template/primer was unlikely to contribute to the observed inhibitory activity.
Figure 2. (a) ATPꢀmediated excision of chainꢀterminating nucleotides by AZTr RT. Excision
reactions were carried out as described in Materials and Methods. Assays were quenched at
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different times of reaction and monitored by gel electrophoresis. (b) Rate of nucleotide excision
2
+
for AZTꢀterminated (●) and 21aꢀterminated (○) primers. (c). Fe ꢀ mediated siteꢀspecific
footprinting assay. Reactions were carried out as described in Materials and Methods. The next
0
1
2
3
4
5
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9
0
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0
2+
incoming nucleotide (TTP) was added in increasing concentrations prior to the Fe ꢀmediated
cleavage. (d) Graphical representation of data shown in panel (c).
Pyrophosphorolytic Removal of Incorporated 21a. HIV resistance to AZT arises from RTꢀ
3
5,36
catalyzed phosphorolytic removal of the chainꢀterminating AZT.
The partially reduced
sensitivity to 18a seen with HIV containing mutations associated with AZT resistance (Table 4)
suggested that, like AZT, incorporated AZTꢀtriazole analogues might also be susceptible to
phosphorolytic excision, although with lesser efficiency than AZT. We therefore investigated the
efficiency of in vitro ATPꢀmediated excision catalyzed by AZTꢀresistant (AZTr) HIV RT for the
investigated 21a compound compare to the AZT. As seen in figure 2 (a & b), the rate of
ꢀ1
nucleotide excision of terminal 21a (0.0126 min ) was substantially slower than that of terminal
ꢀ1
AZT (0.024 min ), consistent with the reduced level of resistance to 18a shown by AZTꢀ
resistant HIV (Table 4).
2
+
Fe -directed Site-specific Footprinting Analysis of 21a-terminated Template/Primers. The
efficiency of phosphorolytic removal of chainꢀterminating nucleotides for the primer 3’ꢀterminus
37ꢀ40
depends on the translocation state of the RTꢀprimer/template complex.
During active DNA
synthesis, the primer 3’ꢀterminal nucleotide resides in the Pꢀsite (primer site) which allows
binding and positioning of the incoming complementary nucleotideꢀtriphosphate for
incorporation. Immediately following this incorporation, the new primer 3’ꢀterminal nucleotide
occupies the Nꢀsite (nucleotide site). To enable further nucleotide incorporation, the primer
terminus must translocate to the Pꢀsite again. Thus, the Nꢀ and Pꢀsites correspond to preꢀ
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translocation and postꢀtranslocation states, respectively. Phosphorolytic excision of the primer
3
9,40
3’ꢀterminal nucleotide can occur only when this terminal nucleotide is in the Nꢀsite.
The
relative occupancy of Nꢀ and Pꢀsites by any given 3’ꢀterminal nucleotide (translocation
equilibrium) will therefore directly impact on the efficiency of phosphorolytic removal of that
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3
7ꢀ39
terminal nucleotide.
The degree of Nꢀ and Pꢀsite occupancy can be assessed by the technique
2
+
37
2+
of Fe ꢀmediated siteꢀspecific footprinting in which Fe bound in the RT RNase H active site
under appropriate conditions generates hydroxyl radicals that cleave the template nucleic acid
strand at a position directly correlated with the position of the primer terminus in the RT
polymerase active site. This technique showed that AZTꢀterminated primers preferentially
3
7,39
occupy the Nꢀsite in AZTrꢀRT
terminal AZT.
, thereby enabling facile phosphorolytic excision of the
We used this footprinting approach to compare AZTꢀ and 21aꢀterminated template/primer
positioning in RT (Figure 2 c & d). The latter showed much more facile Nꢀ to Pꢀsite translocation
than did AZTꢀterminated template/primers. The increased translocation of 21aꢀterminated
primers correlates well with the reduced rate of ATPꢀmediated phosphorolysis of primer 3’ꢀ
terminal 21a (Figure 2b), and is consistent with the reduced degree of resistance conferred to the
parent nucleoside 18a by AZTꢀresistance mutations (Table 4).
Computational Modeling. Potential interactions of chainꢀterminating 18a were assessed by
computational modeling approaches. Two different models were constructed: (i) 18a was
inserted in place of primer 3’ꢀterminal AZT in the Nꢀsite of the RT/nucleic acid complex (PDB
4
1
3
KLG ), and (ii) 18a was inserted in place of primer 3’ꢀterminal AZT in the Pꢀsite of the
4
1
RT/nucleic acid complex (PDB 3KLH ). In constructing these models, care was taken to ensure
that the furanose ring puckering and base glycosidic angle of the 18a conformer corresponded
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exactly to that of the terminal AZT in the crystal structures (Figure 3). Geometrical constraints
applied on the furanose ring puckering and on the spatial orientation of the thymine ring during
final geometry optimization resulted in an 18a conformer with the naphthalene ring almost
coplanar to the thymine ring (Figure 3).
0
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3
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. AZTMP and 18a conformational descriptors: (A) furanose ring puckering and
glycosidic dihedral angle; (B) Minimized conformer of 18a with furanose ring conformation and
glycosidic angle similar to the conformation of AZTMP in the crystal structures.
The triazole and naphthalene ring substituents at C3’ of the furanose ring were allowed free
rotation during structural geometry optimization, leading to the generation of a series of rotamers
(Figure 4A). The global minimum of χ’ (C2’ꢀC3’ꢀNꢀN torsional angle) corresponds to that
obtained from geometry optimization (χ’= ꢀ126°). Nonetheless, we chose several rotamers with
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χ’ values between ꢀ146° and ꢀ66° for superimposition over the primer 3’ꢀterminal AZTMP in the
Nꢀ and Pꢀcomplexes of HIVꢀ1 RT.
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Figure 4. 18a modeled in place of primer 3’ꢀterminal AZTMP in the Nꢀ and Pꢀsites of HIV RT.
(A) Conformational flexibility of the furanose C3’ triazoleꢀnaphthalene ring substituents of 18a;
(B) Primer 3’ꢀterminal 18a in the Nꢀsite may sterically conflict with Gln151 and Arg72, but this
conflict can be resolved by rotation of the triazoleꢀnaphthalene substituent; (C) No steric
conflicts noted for primer 3’ꢀterminal 18a in the Pꢀsite; (D) The triazoleꢀnaphthalene substituent
of primer 3’ꢀterminal 18a in the Pꢀsite partially occupies the Nꢀsite.
The optimized structure ((χ’= ꢀ126°) of primer 3’ꢀterminal 18a in the Nꢀsite complex suggests
potential steric clashes of the furanose C3’ triazoleꢀnaphthalene substituent with the side chains
of RT residues Gln151 and Arg72. However, the rotational flexibility of the furanose substituent
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allows the naphthalene ring to rotate in a manner that minimizes this steric conflict. For example,
the 18a rotamer with χ’=ꢀ66° is readily accommodated without steric issues (Figure 4B).
Interestingly, in this structure, the naphthalene ring “shields” the phosphodiester bond of the
primer 3’ꢀterminal 18a. This might interfere with phosphorolytic excision of chainꢀterminating
18a, consistent with the reduced rate of phosphorolysis noted in our biochemical studies (Figures
0
1
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0
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0
2A & B).
In contrast to the Nꢀsite complex, the Pꢀsite complex with primer 3’ꢀterminal 18a has no steric
issues, and in this complex the triazoleꢀnaphthalene substituent of 18a can adopt a more
energetically favorable torsional angle (χ’ = ꢀ126°) (Figure 4C). This more energetically
favorable conformation may account in part for the relatively facile translocation of 18aꢀ
terminated template/primers (Figures 2C & D). In this structure, the naphthalene ring is coplanar
to the thymine ring of 18a. Thus, the Pꢀsite complex of primer 3’ꢀterminal 18a mimics two layers
of bases occupying both Pꢀ and Nꢀsites, with one (the thymine base of 18a) being in the Pꢀsite
and the other (the naphthalene ring) residing in the Nꢀsite, potentially blocking binding of the
next incoming nucleotide.
Conclusions
Reported efforts in clicking AZT into 1,2,3ꢀtriazoles all failed to achieve any antiviral activities.
By introducing a bulky aromatic group at the C5 position of the 1,2,3ꢀtriazole, we have
identified the first AZTꢀderived 1,2,3ꢀtriazoles with low to subꢀmicromolar potencies against
HIVꢀ1. The observed antiviral activities from CPE based cytoprotection assay were confirmed
through a single replication cycle assay with βꢀgal as the reporter. Mutations associated with
AZT resistance also conferred some degree of HIV resistance to the AZTꢀderived triazoles,
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whereas mutations associated with nonnucleoside RT inhibitor resistance resulted in viral
hypersusceptibility to the compounds. Biochemical analysis of the corresponding triphosphates
showed lower ATPꢀmediated nucleotide excision efficiency than AZT, presumably due to the
preferable translocation into the Pꢀsite of HIV RT. This finding corroborates the reduced fold
resistance (9 for 18a versus 56 for AZT) to AZTꢀresistant HIV variant. Molecular modeling
simulations provided insights into the binding mode and possible residue contacts in Nꢀ and Pꢀ
complexes of HIVꢀ1 RT, and showed that in the Nꢀcomplex triazole ring substituents shield the
3’ꢀterminal phosphodiester bond from pyrophosphorolysis, consistent with the biochemical
observations. AZTꢀderived 1,2,3ꢀtriazoles may provide interesting nucleoside candidates for the
future drug development.
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Experimental
Chemistry
General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Dry solvents (THF, Et O, CH Cl and DMF) were dispensed under argon from an
2
2
2
anhydrous solvent system with two packed columns of neutral alumina or molecular sieves.
Flash chromatography was performed on a Teledyne Combiflash RFꢀ200 with RediSep columns
(silica) and indicated mobile phase. All reactions were performed under inert atmosphere of
1
13
ultraꢀpure argon with ovenꢀdried glassware. H and C NMR spectra were recorded on a Varian
600 MHz spectrometer. Mass data were acquired on an Agilent TOF II TOS/MS spectrometer
capable of ESI and APCI ion sources. Analysis of sample purity was performed on a Varian
Prepstar SDꢀ1 HPLC system with a Phenomenex Gemini, 5 micron C18 column (250 mm x 4.6
mm). HPLC conditions: solvent A = H O, solvent B = MeCN; flow rate = 1.0 mL/min;
2
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compounds were eluted with a gradient of 5% MeCN/H O to 100% MeCN/H O for 25 min.
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Purity was determined by total absorbance at 254 nm. All tested compounds have a purity ≥
6%.
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General procedure 1 for the synthesis of 3’-Deoxy-3’-(4-substituted-1H-1,2,3-triazol-1-
yl)thymidine derivatives via CuACC. To the mixture of AZT (0.375 mmol, 1.0 equiv.) and
alkyne (0.375 mmol, 1.0 equiv.) in 4.0 mL of THF/H O (3:1) was added freshly prepared 1 M
2
solution of sodium ascorbate (0.1 equiv.) in water, followed by the addition of freshly prepared
1 M solution of CuSO • 5H O (0.06 equiv.) in water. The heterogeneous reaction mixture was
4
2
stirred at room temperature for 12 h and monitored by TLC and MS. After the completion, the
reaction was evaporated to dryness. The crude product was purified by column chromatography,
eluted with 4ꢀ10% MeOH in DCM, yielded the desired 1,4ꢀtriazole.
General procedure 2 for the synthesis of 3’-Deoxy-3’-(5-substituted-1H-1,2,3-triazol-1-
yl)thymidine derivatives via RuACC. To the mixture of AZT (0.5 mmol, 1.0 equiv.) and
alkyne (0.75 mmol, 1.5 equiv.) in 4.0 mL of dry THF was added catalytic amount of
Cp*RuCl(PPh ) (0.05 equiv.) and stirred at 60 ºC for 1ꢀ2 days. The reaction was monitored by
3
2
TLC and MS. The reaction mixture was evaporated to dryness and the crude product was
purified by column chromatography, eluted with 4ꢀ10% MeOH in DCM, yielded the desired 1,5ꢀ
triazole.
General Procedure 3 for the Synthesis of deoxynucleoside triphosphates. The
deoxynucleoside triazole (0.119 mmol, 1.0 equiv.) and tributylammonium pyrophosphate (0.238
mmol, 2.0 equiv.) were dried under high vacuum for 1 h at ambient temperature in separate
round bottom flasks. Throughout the entire experiment, the reaction was maintained under an
argon atmosphere. The deoxynucleoside triazole was dissolved in anhydrous pyridine (0.1 mL)
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and anhydrous dioxane (0.3 mL), a solution of 2ꢀchloroꢀ4Hꢀ1,3,2ꢀ benzodioxaphosphorinꢀ4ꢀone
(0.143 mmol, 1.2 equiv.) in anhydrous dioxane (0.2 mL) was added and stirred at room
temperature for 15 min. To the mixture a solution of tributylammonium pyrophosphate (0.213
mmol, 2.0 equiv.) in anhydrous DMF (0.2 mL) was added, which was followed by quick
addition of tributylamine (0.596 mmol, 5.0 equiv.) and stirred for 15 min at room temperature. A
solution of iodine (1% solution in pyridine/water, 9:1) was then added dropwise till a permanent
brown color of iodine was persisted and stirred for 20 min. The excess of iodine was quenched
by adding 5% aqueous solution of Na S O . The reaction mixture was evaporated to dryness
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under vacuum and dissolved in 25% ammonia solution and stirred for 1 h at room temperature.
The reaction was monitored by TLC (isopropanol/aq.NH /H O = 5:3:2) and MS. The reaction
3
2
mixture was concentrated under reduced pressure and crude product was purified by reverseꢀ
phase preparative HPLC [eluted with a linear gradient of 5% to 40% CH CN in buffer triethyl
3
ammonium bicarbonate solution (TEAB, 0.1 M, pH=8.0) over 30 min, on a reverseꢀphase
preparative Varian Dynamax Microsorb 100ꢀ8 C18 column (250 mm x 44.1 mm, 10 ꢁm) total
absorbance at 254 nm at a flow rate of 20.0 mL/min]. The TEAB buffer solution was evaporated
by lyophilization afforded the desired 5'ꢀtriphosphates triethylammonium salt, which was
exchanged into sodium salt by performing Naꢀion exchange column chromatography to yield 5'ꢀ
triphosphates sodium salt as a white solid. The synthesized nucleoside 5'ꢀtriphosphates were
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confirmed by HꢀNMR, PꢀNMR, and HRꢀMS analyses.
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-(phenoxymethyl)-1H-1,2,3-triazol-1yl)tetrahydro
furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8a). The reaction of AZT (130 mg, 0.486
mmol) with alkyne (64.2 mg, 0.486 mmol) yielded compound 8a (0.18 g, 93%) as a white
1
powder. mp 165–167 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.32 (s, 1H, 3ꢀNH), 8.40 (s, 1H),
6
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.78 (s, 1H), 7.27 (t, J = 6.2 Hz, 2H), 7.01 (d, J = 6.1 Hz, 2H), 6.93 (t, J = 6.2 Hz, 1H), 6.39 (t, J
=
6.6 Hz, 1H), 5.34ꢀ5.36 (m, 1H), 5.25 (t, J = 4.8 Hz, 1H, 5’ꢀOH), 5.11 (s, 2H), 4.19ꢀ4.20 (m,
1
3
1
H), 3.58ꢀ3.68 (m, 2H), 2.62ꢀ2.72 (m, 2H), 1.77 (s, 3H); C NMR (150 MHz, DMSOꢀd ) δ
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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164.1, 158.5, 150.9, 143.4, 136.7, 129.9, 124.7, 121.3, 115.1, 110.1, 84.9, 84.3, 61.4, 61.1, 59.7,
+
37.6, 12.7; HRMSꢀESI(+) m/z calcd for C H N O 400.1621 [M+H] , found 400.1641.
1
9
22
5
5
1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)tetrahydro
furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9a). The reaction of AZT (135 mg, 0.505
mmol) with alkyne (100 mg, 0.758 mmol) yielded compound 9a (93 mg, 48%) as a white solid.
1
mp 182–184 ºC; H NMR (600 MHz, CD OD) δ 7.89 (s, 1H), 7.83 (s, 1H) 7.29 (t, J = 7.2 Hz,
3
,
2H), 6.96ꢀ7.02 (m, 3H), 6.06 (t, J = 6.0 Hz, 1H), 5.40ꢀ5.42 (m, 1H), 5.28 (s, 2H) 4.44ꢀ4.45 (m,
,
1
2
1
H), 3.87 (dd, J = 3.4 Hz, J = 12.6 Hz, 1H), 3.75 (dd, J = 3.0 Hz, J = 12.6 Hz, 1H), 2.68ꢀ2.88 (m,
13
H), 1.84 (s, 3H, CH ); C NMR (150 MHz, CD OD)
δ 166.3, 158.9, 152.2, 138.1, 135.1,
3
3
34.9, 130.7, 122.9, 115.8, 111.6, 87.1, 86.6, 62.4, 59.7, 58.8, 39.4, 12.4; HRMSꢀESI(+) m/z
+
calcd for C H N O 400.1621 [M+H] , found 400.1647.
1
9
22
5
5
Methyl-2-((tert-butoxycarbonyl)amino)-3-(4-((1-((2S,3S,5R)-2-(hydroxymethyl)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)propanoate (8b). The reaction of AZT (300 mg, 1.12 mmol) with alkyne
1
(410 mg, 1.12 mmol) yielded compound 8b (0.58 g, 86%) as a white solid. mp 103–105 ºC; H
NMR (600 MHz, DMSOꢀd6)
δ 11.34 (s, 1H), 8.40 (s, 1H), 7.79 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H,
NH), 7.13 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 6.40 (t, J = 6.5 Hz, 1H), 5.35ꢀ5.36 (m,
1H), 5.25 (m, 1H, OH), 5.08 (s, 2H), 4.18ꢀ4.20 (m, 1H), 4.05ꢀ4.09 (m, 1H), 3.61ꢀ3.69 (m, 1H),
13
3.58 (s, 3H, OMe), 2.62ꢀ2.90 (m, 3H), 2.05 (s, 2H), 1.78 (s, 3H), 1.29 (s, 9H); C NMR (150
MHz, DMSOꢀd ) δ 173.1, 164.1, 157.2, 155.8, 150.8, 143.4, 136.7, 130.5, 130.3, 124.6, 114.8,
6
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10.1, 84.8, 84.3, 78.7, 61.4, 61.2, 59.7, 55.9, 52.2, 37.6, 36.1, 31.1, 28.5, 12.7; HRMSꢀESI(+)
+
m/z calcd for C H N O 601.2622 [M+H] , found 601.2620.
28 37
6
9
Methyl-2-((tert-butoxycarbonyl)amino)-3-(4-((1-((2S,3S,5R)-2-(hydroxymethyl)-5-(5-
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11
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methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-triazol-5-
yl)methoxy)phenyl)propanoate (9b). The reaction of AZT (267 mg, 1.0 mmol) with alkyne
1
(
499 mg, 1.5 mmol) yielded compound 9b (18 mg, 30%) as a yellow solid. H NMR (600 MHz,
DMSOꢀd ) δ 11.34 (s, 1H, 3ꢀNH), 7.86 (s, 1H), 7.80 (s, 1H), 7.27 (br, 1H), 7.15 (d, J = 8.40 Hz,
6
1H), 6.94 (d, J = 7.90 Hz, 1H), 6.53 (t, J = 7.2 Hz, 1H), 5.32 (t, J = 5.4 Hz, 1H, 5’ꢀOH), 5.25ꢀ
5.28 (m, 3H), 4.23ꢀ4.25 (m, 1H), 4.05ꢀ4.07 (s, 1H), 3.62ꢀ3.68 (m, 2H), 3.56 (s, 3H, OMe), 2.73ꢀ
1
3
2
.90 (m, 2H), 2.57ꢀ2.55 (m, 2H), 1.75 (s, 3H), 1.29 (s, 9H, Boc); C NMR (150 MHz, DMSOꢀ
d )
6
δ
178.6, 173.1, 164.1, 156.4, 155.8, 150.9, 145.2, 136.4, 134.6, 133.5, 130.9, 130.6, 114.9,
111.8, 110.1, 85.4, 78.7, 78.7, 61.8, 57.8, 52.2, 46.9, 37.9, 31.1, 28.5, 12.7; HRMSꢀESI(+) m/z
+
calcd for C H N O 601.2622 [M+H] , found 601.2632.
2
8
37
6
9
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((naphthalen-1-yloxy)methyl)-1H-1,2,3-triazol-1-
yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8c). The reaction of AZT
(100 mg, 0.375 mmol) with alkyne (68 mg, 0.375 mmol) yielded compound 8c (156 mg, 93%) as
1
a white solid. H NMR (600 MHz, DMSOꢀd ) δ 11.33 (s, 1H, 3ꢀNH), 8.52 (s, 1H), 8.12 (d, J =
6
6
6
1
.0 Hz, 1H), 7.85 (d, J = 6.2 Hz, 1H), 7.81 (s, 1H), 7.40ꢀ7.51 (m, 4H), 7.17 (d, J = 6.2 Hz, 1H),
.43 (m, 1H), 5.39ꢀ5.41 (m, 1H), 5.34 (s, 2H), 5.25 (t, J = 4.8 Hz, 1H, 5’ꢀOH), 4.23ꢀ4.25 (m,
13
H), 3.62ꢀ3.71 (m, 2H), 2.61ꢀ2.76 (m, 2H), 1.79 (s, 3H); C NMR (150 MHz, DMSOꢀd ) δ
6
164.1, 153.9, 150.9, 143.6, 136.7, 134.5, 127.9, 126.9, 126.6, 125.8, 125.3, 124.6, 122.0, 120.8,
110.1, 106.2, 84.9, 84.3, 62.2, 61.2, 59.7, 37.6, 12.7; HRMSꢀESI(+) m/z calcd for C H N O
23 24 5 5
+
4
50.1777 [M+H] , found 450.1733.
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1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-((naphthalen-1-yloxy)methyl)-1H-1,2,3-triazol-1-
yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9c). The reaction of AZT
(150 mg, 0.56 mmol) with alkyne (150 mg, 0.84 mmol) yielded compound 9c (68 mg, 27%) as a
0
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2
3
4
5
6
7
8
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2
3
4
5
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7
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yellow solid. mp 112–115 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.31 (s, 1H, 3ꢀNH), 8.06 (d, J
6
= 6.2 Hz, 1H), 8.00 (s, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.76 (s, 1H), 7.43ꢀ7.52 (m, 4H), 7.16 (d, J =
6
.4 Hz, 1H), 6.55 (t, J = 6.0 Hz, 1H), 5.53 (d, J = 12.0 Hz, 1H), 5.47 (d, J = 12.0 Hz, 1H), 5.36ꢀ
.38 (m, 1H), 5.27 (t, J = 4.8 Hz, 1H), 4.32ꢀ4.34 (m, 1H), 3.59ꢀ3.65 (m, 2H), 2.57ꢀ 2.67 (m, 2H),
5
1
3
1.70 (s, 3H); C NMR (150 MHz, DMSOꢀd ) δ 164.1, 153.3, 150.9, 136.4, 134.7, 133.5, 127.9,
6
1
1
1
27.1, 126.4, 126.1, 125.1, 121.7, 121.3, 111.9, 110.1, 106.3, 85.3, 84.9, 61.7, 58.8, 58.4, 37.9,
+
2.7; HRMSꢀESI(+) m/z calcd for C H N O 450.1777 [M+H] , found 450.1807.
23 24
5
5
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-
yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8d). The reaction of AZT
(100 mg, 0.375 mmol) with alkyne (65 mg, 0.375 mmol) yielded compound 8d (141 mg, 84%)
1
as a white solid. mp 233–234 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.34 (s, 1H, 3ꢀNH), 8.46 (s,
6
1
H), 7.81 (s, 1H), 7.80ꢀ7.82 (m, 3H), 7.50 (s, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz,
1H), 7.17 (dd, J = 3.0 Hz, J = 9.6 Hz, 1H), 6.41 (t, J = 7.2 Hz, 1H), 5.36ꢀ5.39 (m, 1H), 5.27 (t, J
5.4 Hz, 1H), 5.25 (s, 2H), 4.21ꢀ4.23 (m, 1H), 3.59ꢀ3.69 (m, 2H), 2.61ꢀ2.75 (m, 2H), 1.78 (s,
=
1
3
3
H); C NMR (150 MHz, DMSOꢀd ) δ 164.1, 156.3, 150.9, 143.3, 136.7, 134.6, 129.8, 129.1,
6
127.9, 126.9, 124.8, 124.2, 119.1, 110.1, 107.5, 84.8, 84.3, 61.6, 61.2, 59.7, 53.3, 37.6, 12.7;
+
HRMSꢀESI(+) m/z calcd for C H N O 450.1777 [M+H] , found 450.1786.
2
3
24
5
5
1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)
tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9d). The reaction of AZT (100
mg, 0.375 mmol) with alkyne (102 mg, 0.56 mmol) yielded compound 9d (91 mg, 36%) as a
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white solid. mp 170–172 ºC; H NMR (600 MHz, CD OD) δ 7.90 (s, 1H) 7.86 (s, 1H) 7.76ꢀ7.79
3
,
,
(
m, 3H), 7.41ꢀ7.45 (m, 2H), 7.34 (t, J = 6.2 Hz, 1H), 7.16 (dd, J = 2.1 Hz, J = 12.0 Hz, 1H), 6.60
(
t, J = 12.2 Hz, 1H), 5.43ꢀ5.46 (m, 1H), 5.41 (s, 2H) 4.47ꢀ4.49 (m, 1H), 3.87 (dd, J = 3.6 Hz, J =
,
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
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58
59
60
1
2.0 Hz, 1H), 3.76 (dd, J = 3.0 Hz, J = 12.0 Hz, 1H), 2.85ꢀ2.89 (m, 1H), 2.69ꢀ2.72 (m, 1H), 1.78
1
3
(s, 3H, CH ); C NMR (150 MHz, CD OD) δ 164.9, 155.4, 150.8, 136.7, 134.5, 133.6, 129.5,
3
3
1
29.4, 127.2, 126.5, 126.2, 123.8, 117.8, 110.2, 107.2, 85.8, 85.2, 61.2, 58.3, 57.5, 38.2, 11.1;
+
HRMSꢀESI(+) m/z calcd for C H N O 450.1777 [M+H] , found 450.1804.
2
3
24
5
5
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-(((6-methoxynaphthalen-2-yl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8e). The reaction
of AZT (100 mg, 0.375 mmol) with alkyne (79 mg, 0.375 mmol) yielded compound 8e (160 mg,
1
89%) as a white solid. mp 220–221 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.32 (s, 1H, 3ꢀNH),
6
8
.44 (s, 1H), 7.79 (s, 1H), 7.70ꢀ7.72 (m, 2H), 7.43 (s, 1H), 7.25 (s, 1H), 7.11ꢀ7.15 (m, 2H), 6.41
(
(
t, J = 6.6 Hz, 1H), 5.36ꢀ5.38 (m, 1H), 5.25 (t, J = 5.1 Hz, 1H, 5’ꢀOH), 5.21 (s, 2H), 4.21ꢀ4.23
1
3
m, 1H), 3.81 (s, 3H, OMe), 3.61ꢀ3.69 (m, 2H), 2.63ꢀ2.73 (m, 2H), 1.78 (s, 3H); C NMR (150
MHz, DMSOꢀd ) δ 164.1, 156.2, 154.8, 150.9, 143.4, 136.7, 130.0, 129.7, 128.7, 128.6, 124.7
6
119.3, 111.8, 110.1, 107.9, 106.6, 84.8, 84.3, 61.6, 61.2, 59.7, 55.5, 37.6, 12.7; HRMSꢀESI(+)
+
m/z calcd for C H N O 480.1883 [M+H] , found 480.1874.
2
4
26
5
6
1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-(((6-methoxynaphthalen-2-yl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9e). The reaction
of AZT (150 mg, 0.56 mmol) with alkyne (178 mg, 0.83 mmol) yielded compound 9e (80 mg,
1
3
7
7
0%) as a white solid. mp 228–230 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.33 (s, 1H, 3ꢀNH),
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.99 (s, 1H), 7.80 (s, 1H), 7.72ꢀ7.74 (m, 2H), 7.43 (d, J = 6.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H),
.12ꢀ7.17 (m, 2H), 6.54 (t, J = 6.8 Hz, 1H), 5.39 (s, 2H), 5.30ꢀ5.38 (m, 2H, Hꢀ3’, 5’ꢀOH ), 4.28ꢀ
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Journal of Medicinal Chemistry
Page 28 of 55
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.29 (m, 1H), 3.81 (s, 3H, OMe), 3.63ꢀ3.70 (m, 2H), 2.61ꢀ 2.64 (m, 2H), 1.74 (s, 3H); C NMR
(
150 MHz, DMSOꢀd ) δ 164.1, 156.4, 154.1, 150.9, 136.5, 134.7, 133.5, 130.3, 129.5, 128.8,
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1
28.6, 119.4, 119.0, 110.1, 108.3, 106.6, 85.4, 85.0, 61.9, 59.0, 58.0, 55.5, 37.9, 12.8; HRMSꢀ
0
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0
+
ESI(+) m/z calcd for C H N O 480.1883 [M+H] , found 480.1899.
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4
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5
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1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-(((7-methoxynaphthalen-2-yl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione 8f. The reaction of
AZT (100 mg, 0.375 mmol) with alkyne (79 mg, 0.375 mmol) yielded compound 8f (154 mg,
1
86%) as a white solid. mp 237–240 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.33 (s, 1H, 3ꢀNH),
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8
.45 (s, 1H), 7.79 (s, 1H), 7.71ꢀ7.72 (m, 2H), 7.39 (s, 1H), 7.20 (s, 1H), 6.96ꢀ7.00 (m, 2H), 6.41
(
t, J = 6.6 Hz, 1H), 5.37ꢀ5.39 (m, 1H), 5.26 (t, J = 5.4 Hz, 1H, 5’ꢀOH), 5.23 (s, 2H), 4.21ꢀ4.22
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3
(m, 1H), 3.84 (s, 3H, OMe), 3.58ꢀ3.68 (m, 2H), 2.62ꢀ2.74 (m, 2H), 1.79 (s, 3H); C NMR (150
MHz, DMSOꢀd ) δ 164.1, 158.2, 156.9, 150.8, 143.4, 136.7, 136.1, 129.6, 129.4, 124.8, 124.3,
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1
16.4, 116.3, 110.1, 107.0, 105.8, 84.8, 84.3, 61.5, 61.2, 59.7, 55.5, 37.6, 12.7; HRMSꢀESI(+)
+
m/z calcd for C H N O 480.1883 [M+H] , found 480.1874.
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4
26
5
6
1
-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-(((7-methoxynaphthalen-2-yl)oxy)methyl)-1H-1,2,3-
triazol-1-yl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9f). The reaction
of AZT (150 mg, 0.56 mmol) with alkyne (177 mg, 0.84 mmol) yielded compound 9f (91 mg,
1
3
4%) as a white solid. mp 249ꢀ251 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.33 (s, 1H, 3ꢀNH),
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7.96 (s, 1H), 7.80 (s, 1H), 7.72ꢀ7.74 (m, 2H), 7.39 (s, 1H), 7.22 (s, 1H), 6.98ꢀ7.03 (m, 2H), 6.55
(
t, J = 6.6 Hz, 1H), 5.41 (s, 2H), 5.30ꢀ5.33 (m, 2H), 4.28ꢀ4.29 (m, 1H), 3.84 (s, 3H, OMe), 3.66ꢀ
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3
3
.70 (m, 2H), 2.63ꢀ2.65 (m, 2H), 1.74 (s, 3H); C NMR (150 MHz, DMSOꢀd ) δ 164.1, 158.3,
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1
56.2, 150.9, 136.4, 135.9, 134.7, 133.5, 129.7, 129.5, 124.6, 116.7, 116.0, 110.1, 107.4, 105.8,
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Page 29 of 55
Journal of Medicinal Chemistry
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5.4, 85.0, 78.5, 61.9, 59.0, 55.5, 37.9, 12.7; HRMSꢀESI(+) m/z calcd for C H N O 480.1883
2
4
26
5
6
+
[
M+H] , found 480.1894.
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)tetrahydro
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furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dion (8g). The reaction of AZT (130 mg, 0.486
mmol) with alkyne (28 mg, 0.486 mmol) yielded compound 8g (150 mg, 96%) as a white solid.
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mp 184ꢀ185 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.26 (s, 1H) 8.07 (s, 1H), 7.73 (s, 1H) 6.32
6
,
,
(t, J = 6.2 Hz, 1H), 5.26ꢀ5.28 (m, 1H), 5.17ꢀ5.25 (m, 2H), 4.44 (s, 2H), 4.11ꢀ4.13 (m, 1H), 3.61
(dd, J = 3.8 Hz, J = 12.2 Hz, 1H), 3.52 (dd, J = 4.2 Hz, J = 12.2 Hz, 1H), 2.53ꢀ2.65 (m, 2H), 1.73
13
(
s, 3H); C NMR (150 MHz, DMSOꢀd )
δ
164.1, 150.9, 148.7, 136.6, 122.7, 110.0, 84.9, 84.3,
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+
61.1, 59.4, 55.4, 37.6, 12.7; HRMSꢀESI(+) m/z calcd for C H N O 324.1308 [M+H] , found
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3
18
5
5
324.1302.
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)tetrahydro
furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (9g). The reaction of AZT (135 mg, 0.505
mmol) with alkyne (42 mg, 0.758 mmol) yielded compound 9g (69 mg, 45%) as a white solid.
1
mp 177–178 ºC; H NMR (600 MHz, CD OD) δ 7.92 (s, 1H), 7.65 (s, 1H) 6.61 (t, J = 6.0 Hz,
3
,
1H), 5.43ꢀ5.42 (m, 1H), 4.74 (s, 2H), 4.38ꢀ4.37 (m, 1H), 3.87 (dd, J = 3.0 Hz, J = 12.6 Hz, 1H),
13
3
.78 (dd, J = 3.6 Hz, J = 12.6 Hz, 1H), 2.68ꢀ2.92 (m, 2H), 1.89 (s, 3H); C NMR (150 MHz,
CD OD)
δ
164.9, 150.9, 137.4, 136.8, 132.1, 110.2, 85.7, 85.4, 61.1, 58.1, 51.5, 37.8, 11.0;
3
+
HRMSꢀESI(+) m/z calcd for C H N O 324.1308 [M+H] , found 324.1306.
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3
18
5
5
1-((2R,4S,5S)-5-(Hydroxymethyl)-4-(4-(naphthalen-1-yl)-1H-1,2,3-triazol-1-yl)tetrahydro
furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (17a). The reaction of AZT (100 mg, 0.374
mmol) with alkyne (60 mg, 0.375 mmol) yielded compound 17a (139 mg, 89%) as a white solid.
1
mp 238–240 ºC; H NMR (600 MHz, DMSOꢀd ) δ 11.34 (s, 1H, 3ꢀNH), 8.90 (s, 1H), 8.39 (s,
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