Synthesis and anti-HIV activities of glutamate and peptide conjugates of nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1021/jm201551m

Mono-, di-, and trinucleoside conjugates of glutamate or peptide scaffolds containing nucleoside reverse transcriptase inhibitors were synthesized. Among dinucleoside glutamate ester derivatives, N-myristoylated derivatives showed significantly higher anti-HIV activity than the corresponding N-acetylated conjugates against cell-free virus. Myristoyl-Glu(3TC)-FLT (46, EC₅₀ = 0.3-0.6 μM) and myristoyl-Glu(FTC)-FLT (47, EC₅₀ = 0.1-0.4 μM) derivatives were the most active glutamate-dinucleoside conjugates. A trinucleoside glutamate derivative containing AZT, FLT, and 3TC (34, EC ₅₀ = 0.9-1.4 μM) exhibited higher anti-HIV activity than AZT and 3TC against cell-free virus. Compound 34 also exhibited higher anti-HIV activity against multidrug (IC₅₀ = 5.9 nM) and NNRTI (IC₅₀ = 12.9 nM) resistant viruses than parent nucleosides. The physical mixture containing FLT-succinate, AZT, 3TC, and glutamic acid exhibited 115-fold less activity against cell associated virus (EC₅₀ = 91.9 μM) when compared to 34 (EC₅₀ = 0.8 μM). Other conjugates showed less or comparable potency to that of the corresponding physical mixtures.

Full text of DOI:10.1021/jm201551m

Article
pubs.acs.org/jmc
Synthesis and Anti-HIV Activities of Glutamate and Peptide
Conjugates of Nucleoside Reverse Transcriptase Inhibitors
Hitesh K. Agarwal,† Bhupender S. Chhikara,† Megrose Quiterio,† Gustavo F. Doncel,*,‡
and Keykavous Parang*,†
†Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 41 Lower College Road,
Kingston, Rhode Island 02881, United States
‡CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 601 Colley Avenue, Norfolk, Virginia,
23507, United States
S
* Supporting Information
ABSTRACT: Mono-, di-, and trinucleoside conjugates of glutamate or peptide scaffolds containing nucleoside reverse
transcriptase inhibitors were synthesized. Among dinucleoside glutamate ester derivatives, N-myristoylated derivatives showed
significantly higher anti-HIV activity than the corresponding N-acetylated conjugates against cell-free virus. Myristoyl-Glu(3TC)-
FLT (46, EC50 = 0.3−0.6 μM) and myristoyl-Glu(FTC)-FLT (47, EC50 = 0.1−0.4 μM) derivatives were the most active
glutamate−dinucleoside conjugates. A trinucleoside glutamate derivative containing AZT, FLT, and 3TC (34, EC50 = 0.9−1.4
μM) exhibited higher anti-HIV activity than AZT and 3TC against cell-free virus. Compound 34 also exhibited higher anti-HIV
activity against multidrug (IC50 = 5.9 nM) and NNRTI (IC50 = 12.9 nM) resistant viruses than parent nucleosides. The physical
mixture containing FLT−succinate, AZT, 3TC, and glutamic acid exhibited 115-fold less activity against cell associated virus
(EC50 = 91.9 μM) when compared to 34 (EC50 = 0.8 μM). Other conjugates showed less or comparable potency to that of the
corresponding physical mixtures.
generation of steric hindrance at amino acid 184 residue have
been proposed as reasons for development of resistance.2,3
Similar to HIV, mutation at Met552 with Val and Ile (M552 V/
I) results in development of 3TC resistant HBV strains.5
Furthermore, nucleoside analogues are polar and have limited
cellular uptake. Thus, designing new lipophilic conjugates
containing different NRTIs on a multivalent scaffold may have
major advantages such as broad-spectrum activity against drug-
resistant HIV, dose simplicity, and simultaneous delivery of
several NRTIs to the HIV-infected cells.
Diversity in the structure and physicochemical properties of
peptides allow their applications as scaffolds and for targeted
drug delivery. Peptides with different chain lengths have been
used as spacers to deliver active drugs through lysosomal
hydrolysis.6−8 Peptides have also been used as linkers to deliver
INTRODUCTION
■
The introduction of highly active antiretroviral therapy
(HAART) in the mid-1990s resulted in a significant decrease
of morbidity and mortality in the human immunodeficiency
virus-1 (HIV-1)-infected patient population. A combination of
nucleoside reverse transcriptase inhibitors (NRTIs) is used in
HAART to reduce the patient’s viral load. Each nucleoside
analogue, however, has different cellular uptake rates and
pharmacokinetics and poor patient compliance during long-
term use of combination therapy and emergence of drug
resistance compromise the sustained efficacy of HAART.
NRTIs succumb to newly developed resistant virus. For
example, (−)-2′,3′-dideoxy-3′-thiacytidine (3TC) is a nucleo-
side analogue commonly used in the treatment of both HIV-1
and hepatitis B virus (HBV).1 Although 3TC has potent activity
against wild type HIV, a single point mutation at methionine
184 residue (M184) results in 3TC-resistant mutant virus
(M184 V/I).2−4 Cytidine deamination in 3TC and the
Received: November 16, 2011
Published: February 21, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of Fmoc-Glu(FLT)-OH (5), Fmoc-Glu(AZT)-OH (6), and Fmoc-Ser(OMy)-OH (8) as Building Blocks
a
Reagents and conditions: (a) Fmoc-Glu(OH)OtBu, HBTU, DIPEA, DMF; (b) TFA/H2O (95:5 v/v); (c) myristoyl chloride, DIPEA, DMF.
Scheme 2. Solid-Phase Synthesis of Dipeptide My-Glu(FLT)-Lys(My)-OH (10) and Tripeptide−Nucleoside Conjugates Ac-
a
Glu(FLT)-βAla-Lys(My)-OH (13) and Ac-Glu(AZT)-βAla-Lys(My)-OH (14)
a
Reagents and conditions: (a) (1) piperidine/DMF (20%), (2) HBTU, 5, NMM, (3) piperidine/DMF (20%), (4) TFA/DCM (5:95 v/v), (5)
myristic anhydride, DIPEA, DMF, (6) TFA/H2O/anisole (95:2.5:2.5 v/v/v); (b) (1) piperidine/DMF (20%), (2) HBTU, Fmoc-βAla-OH, NMM,
(3) piperidine/DMF (20%), (4) HBTU, 5 or 6, NMM, (5) piperidine/DMF (20%); (c) (1) DIPEA, acetic anhydride, (2) TFA/DCM (5:95 v/v),
(3) myristic anhydride, DIPEA, DMF, (4) TFA/H2O/anisole (95:2.5:2.5 v/v/v).
drugs at desired sites where they undergo site-specific
enzymatic hydrolysis releasing the bioactive compounds.
Chau et al. reported a specific peptide sequence of matrix
metalloproteinase, an enzyme overexpressed in cancer cells for
delivering anticancer drugs, such as methotrexate, directly to
the cancer cells.9−11
Peptide esters have also been shown to improve the
bioavailability of active drugs. For example, peptide prodrugs
of lopinavir showed higher oral bioavailability than lopinavir.12
Peptide conjugates of 5-aminolevulinic acid showed improved
pharmacological response compared to that of the parent
analogue as a result of better cellular uptake.13
Furthermore, peptide derivatives have been shown to
produce direct pharmacological activity against some targeted
enzymes. Peptide-based drugs, ramipril, enalapril, and captopril,
were developed as angiotensin converting enzyme inhibitors.14
Enfuvirtide is an approved peptide-based anti-HIV drug which
blocks gp41 and prevent HIV-1 cell entry.15 HIV protease
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inhibitors, such as lopinavir and saquinavir, are also peptide-
based drugs.12,16
Scheme 3. Synthesis of Tetrapeptide−Nucleoside
a
Conjugates of FLT and AZT 16 and 17
Herein, we report the synthesis and anti-HIV evaluation of
mono-, di-, and trinucleoside conjugates of NRTIs substituted
on a glutamate or a multivalent peptide scaffold containing a
glutamic acid. N-Myristoylated and N-acetylated peptide-
mononucleoside conjugates contained one myristoyl group
attached to the side chains of glutamic acid and were
synthesized by using Fmoc solid-phase peptide synthesis. The
mononucleoside conjugates were used as control for com-
parative studies with di- and trinucleosides. N-Myristoylated
and N-acetylated dinucleoside peptide ester derivatives
included different nucleosides attached on carboxylic acid
groups and were synthesized by using multistep solution phase
methods. A glutamic acid conjugate with three different
nucleosides was synthesized as a trinucleoside ester conjugate.
The nucleosides included 3′-azido-2′,3′-deoxythymidine (AZT),
3′-fluoro-2′,3′-deoxythymidine (FLT), 2′,3′-dideoxy-3′-thiacyti-
dine (3TC), and (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine
(FTC).
Nucleoside−glutamate scaffold conjugates were designed
with the expectation that attachment of more than one
nucleoside analogue to the scaffold would generate a
substituted multivalent conjugate capable of delivering different
nucleosides to the HIV-target cells. Myristic acid was attached
to the scaffolds to improve the conjugate’s lipophilicity and
cellular uptake. Several fatty acyl esters of nucleoside analogues
have been previously synthesized to improve their lipophilicity
and cellular uptake,17−19 as shown with fatty acyl esters of 2′,3′-
didehydro-2′,3′-dideoxythymidine (stavudine, d4T).17 It is
expected that once the conjugate enters the cells, it will be
hydrolyzed by esterase and/or peptidases, releasing the parent
nucleoside analogues. Simultaneous intracellular release of
different nucleosides would help to increase the barrier to viral
resistance. Combined multidrug conjugates could also bear the
benefits of synergistic anti-HIV effects, broader-spectrum
activity, simpler dosing, and improved pharmacokinetic proper-
ties.
a
Reagents and conditions: (a) (1) piperidine/DMF (20%), (2)
HBTU, 5 or 6, NMM, (3) piperidine/DMF (20%), (4) HBTU, Fmoc-
βAla-OH, NMM, (5) piperidine/DMF (20%), (6) HBTU, 8, NMM,
(7) piperidine/DMF (20%), (8) DIPEA, acetic anhydride, (9) TFA/
anisole/water (95:2.5:2.5 v/v/v).
at room temperature. Lysine side chain protecting group 4-
methyltrityl (Mtt) was removed by adding TFA/DCM (5:95 v/
v) to the resin NH2-Glu(FLT)-Lys(Mtt)-Wang resin. The free
amino groups were myristoylated by using myristic anhydride.
The peptide was cleaved from the resin by using TFA/H2O/
anisole (95:2.5:2.5 v/v/v) to yield dimyristoylated FLT−
dipeptide conjugate 10 (Scheme 2).
For the synthesis of tripeptide-nucleoside conjugates 13 and
14 containing a myristoylated lysine group, the peptide was
assembled on Fmoc-Lys(Mtt)-Wang resin by Fmoc solid-phase
peptide synthesis strategy at room temperature using Fmoc-
protected amino acids [Fmoc-β-Ala-OH and Fmoc-Glu-
(nucleoside)-OH (5 or 6)], followed by acetylation. After
acidic removal of Mtt group, the amino group of lysine side
chain was myristoylated using myristic anhydride in the
presence of DIPEA as a base to afford mysristoylated tripeptide
derivatives Ac-Glu(FLT)-βAla-Lys(My)-OH (13) and Ac-
Glu(AZT)-βAla-Lys(My)-OH (14) (Scheme 2).
The synthesis of tetrapeptide−nucleoside conjugates con-
taining a myristoylated serine residue is depicted in Scheme 3.
Deprotection of Fmoc-Gly-Wang resin (15) in the presence of
piperidine, coupling reactions with building blocks, 5 or 6,
Fmoc-β-Ala-OH, and 8, respectively, in the presence of HBTU,
followed by cleavage in the presence of TFA/anisole/water
(95:2.5:2.5 v/v/v), afforded tetrapeptide−nucleoside conju-
gates Ac-Ser(My)-βAla-Glu(FLT)Gly-OH (16) and Ac-Ser-
(My)-βAla-Glu(AZT)Gly-OH (17) (Scheme 3).
Synthesis of Dinucleoside and Trinucleoside Glutamate
Ester Derivatives Functionalized with Acetyl or Myristoyl
Moiety. The synthesis of glutamate esters containing two
nucleosides (AZT-FLT, FLT-3TC, AZT-3TC) with acetyl or
myristic acid (Scheme 4) and glutamate esters containing three
nucleosides (AZT-FLT-3TC) (Scheme 5) was accomplished by
solution phase synthesis.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of Myristoylated Mononucleoside
Dipeptide, Tripeptide, and Tetrapeptide Conjugates. Dipep-
tide (10), tripeptide (13, 14), and tetrapeptide (16, 17)
nucleoside conjugates containing one nucleoside and at least
one myristoyl moiety attached in the peptide side chain were
synthesized in three steps: (i) Fmoc-protected building block
synthesis (Scheme 1), (ii) peptide assembly on the resin, and
(iii) deprotection followed by acylation of the free amino
groups (Schemes 2 and 3).
Fmoc-Glu(nucleoside)-OH building blocks (5 or 6, nucleo-
side = FLT or AZT) were synthesized by the reaction of Fmoc-
Glu(OH)-tBu with the corresponding nucleoside (AZT or
FLT) in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) and N,N-
diisopropylethylamine (DIPEA), followed by the deprotection
of tBu group with TFA (Scheme 1). Fmoc-Ser(OMy)OH (8),
a fatty acyl-substituted building block, was synthesized by the
reaction of Fmoc-Ser(OH)-OH (7) with myristoyl chloride in
the presence of DIPEA (Scheme 1).
Dipeptide-nucleoside conjugate My-Glu(FLT)-Lys(My)-OH
(10) was synthesized by assembling Fmoc-protected building
block, Fmoc-Glu(FLT)-OH (5), on Fmoc-Lys(Mtt)-Wang
resin 9 by using Fmoc solid-phase peptide synthesis strategy
Dinucleoside−glutamate conjugates were synthesized by the
reaction of an appropriate building block, such as 5 or 6
containing a free α-carboxylic acid, with other nucleosides, such
as dimethoxytrityl (DMTr)-protected 3TC (DMTr-3TC) or
AZT, in the presence of HBTU and DIPEA to afford 18, 22, or
23. The synthesis of DMTr-3TC has been previously reported
by us.20 In the next step, Fmoc deprotection was accomplished
in the presence of piperidine and n-octanethiol to afford 19, 24,
or 25. n-Octanethiol was used in excess as a scavenger because
the conjugates were not stable in the presence of piperidine or
other bases like DMAP. The use of piperidine in ratios more
than 0.1 equiv or increasing cleavage reaction time resulted in
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from t-butyl protected glutamic acid. Glu(OtBu)-OH (40) was
reacted with myristic anhydride in the presence of DIPEA to
generate My-Glu(OtBu)-OH (41). Conjugation of 41 with
FLT in the presence of HBTU, DIPEA, and HOBt afforded
My-Glu(OtBu)-OFLT (42). HOBt was used to protect the
racemization of glutamic acid. Deprotection of tBu in 42 was
accomplished in the presence of TFA/DCM (95:5 v/v) to yield
My-Glu(OH)-OFLT (43) (Scheme 7).
Scheme 4. Synthesis of Glutamic Acid Esters of
Dinucleosides (AZT and FLT) (20 and 21)
a
Reaction of DMTr protected 3TC and FTC (35 and 39)
with 43 in the presence of DIPEA, HBTU, and HOBt, followed
by cleavage of DMTr protecting group with acetic acid/TFA
(98:2 v/v) mixture, afforded myristoylated dinucleoside−
glutamate conjugates, 46 and 47 conjugates, in 58−64% yield
(Scheme 7).
Anti-HIV Activity Evaluation. The anti-HIV activity and
cytotoxicity of glutamate and peptide conjugates of nucleosides
were primarily evaluated as previously reported17,21−23 using
X4 (IIIB) and R5 (BaL) HIV-1 and P4R5 cells expressing CD4
and HIV-1 coreceptors, stably transfected with β-galactosidase
reporter gene under the control of HIV-1 LTR. The impact of
compounds on cell viability was assessed using an MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, re-
duction of tetrazolium salt) assay. Compound antiviral activity
was evaluated using a single-round infection assay.24 Tables
1−3 illustrate the anti-HIV-1 activity of selected compounds
and their corresponding physical mixtures against cell-free lab-
adapted virus. In general, minimal cytotoxicity was observed
(EC50 > 100 μM) for all conjugates. A selected conjugate was
further evaluated against wild-type and mutant HIV-1 clinical
isolates in a multiround infection assay.
Anti-HIV Activities of Myristoylated Mononucleoside
Dipeptide, Tripeptide, and Tetrapeptide Conjugates. The
mononucleoside−peptide conjugates were used as control for
comparative studies with di- and trinucleosides. As shown in
Table 1, dipeptide−nucleoside (10), tripeptide−nucleoside
(13), and tetrapeptide−nucleoside (16, 17) conjugates
containing one nucleoside and one myristoyl moiety displayed
EC50 values ranging from 6.9 to 86.4 μM against cell-free virus
and were not active against cell associated virus (EC50 > 100
μM; data not shown). Myristoylated peptide−AZT conjugate
17 (EC50 = 63.1−86.4 μM) was approximately 6 times less
active than AZT (EC50 = 10.8−14.2 μM). Dimyristoylated
peptide−FLT conjugate 10 (EC50 = 26.8−29.1 μM) was 34- to
73-fold less active than FLT (EC50 = 0.4−0.8 μM).
Monomyristoylated peptide−FLT conjugates 13 and 16
(EC50 = 6.9−11.1 μM) were nearly 14−17 times less active
than FLT. Reduced anti-HIV activity could be due to low
cellular uptake of compounds containing peptide backbones or
limited intracellular hydrolysis to parent nucleosides under
assay conditions. The attachment of myristic acid did not
improve the anti-HIV profile of the nucleosides, suggesting that
increasing the lipophilicity of the mononucleoside−peptide
conjugates was not an effective approach to enhancing the anti-
HIV activity of the NRTIs.
a
Reagents and conditions: (a) AZT, HBTU, DIPEA, DMF; (b)
piperidine, n-octanethiol; (c) [RCO]2O, DIPEA, DMF.
the loss of the nucleoside from side chain of glutamate at δ-
carboxylic acid. After Fmoc deprotection, free N-terminal was
reacted with myristic anhydride or acetic anhydride in the
presence of DIPEA to afford 20, 21, 26, 28, 30, and 32. The
deprotection of DMTr group in 26, 28, 30, and 32 was
accomplished in the presence of acetic acid. This strategy
afforded the dinucleoside glutamate esters containing two
different nucleosides with acetyl or myristic group My-AZT-
FLT (20), Ac-AZT-FLT (21) (Scheme 4), My-FLT-3TC (27),
Ac-FLT-3TC (29), My-AZT-3TC (31), and Ac-AZT-3TC
(33) (Scheme 5). Myristoyl and acetyl capping at N-terminal
was carried out to provide peptides with high and low
lipophilicity, respectively.
A trinucleoside−glutamate conjugate containing three differ-
ent nucleosides (FLT, 3TC, AZT) was synthesized (34,
Scheme 5) from DMTr protected 3TC-AZT conjugate 25. In
compound 34, 3TC, AZT, and FLT were attached to the C-
terminal, the side chain carboxylate of glutamate, and the N-
terminal through a linker, respectively. A glutamate conjugate
containing two different nucleosides, 3TC and AZT, at α and δ-
carboxylic acids (NH2-Glu(AZT)-3TC-DMTr) (25) was
reacted first with FLT−succinate in the presence of DIPEA,
followed by DMTr cleavage with acetic acid to afford a
trinucleoside−glutamate conjugate 34 (Scheme 5).
DMTr-protected FTC (39) was synthesized according to the
previously reported synthesis for DMTr-protected 3TC (35).20
FTC was reacted with tert-butyldimethylsilyl chloride and
imidazole in dry DMF to yield 5′-protected TBDMs-FTC
derivative 37. Subsequent reaction with DMTr-Cl in the
presence of pyridine afforded 4-amino DMTr-protected
derivative 38. Removal of 5′-TBDMS group in the presence
of tetrabutylammonium fluoride afforded DMTr-protected
FTC (39) (Scheme 6).
Anti-HIV Activities of Dinucleoside and Trinucleoside
Glutamate Ester Derivatives Functionalized with Acetyl or
Myristoyl Moiety. Anti-HIV activity of glutamate esters of two
and three different nucleosides was evaluated and compared
with that of mononucleoside−peptide conjugates, the corre-
sponding physical mixtures of nucleosides with or without
myristic acid, and with glutamate ester 43 containing one
nucleoside (FLT) (Tables 2 and 3). The data showed improved
anti-HIV activity against cell-free virus for dinucleoside and
Myristoylated dinucleoside−glutamate conjugates, My-FLT-
3TC (46) and My-FLT-FTC (47), were synthesized starting
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a
Scheme 5. Synthesis of Glutamic Acid Esters of Dinucleosides and Trinucleosides (AZT, FLT, and 3TC) (26−34)
a
Reagents and conditions: (a) 3TC-DMTr, HBTU, DIPEA, DMF; (b) piperidine, octanethiol; (c) (1) [R2CO]2O, DIPEA, DMF, (2) acetic acid;
(d) (1) FLT-succinate, HBTU, DIPEA, DMF, (2) acetic acid.
trinucleoside compounds (EC50 = 0.1−17.0 μM, Table 2)
versus that of mononucleoside−peptide conjugates (EC50
several of lipophilic fatty acyl derivatives of d4T,17 AZT,18 and
FLT,19 when compared to those of the parent nucleosides.
Fatty acid nucleoside derivatives possess dramatically different
physicochemical properties from the parent nucleoside
compounds. The compounds are lipophilic and are expected
to enhance drug penetration through membranes.
Among all glutamate esters with two nucleosides, compound
47, containing conjugated FLT and FTC, was the most potent
anti-HIV agent (EC50 = 0.1−0.4 μM) and was superior to the
individual parent nucleosides FLT and FTC with EC50 values of
0.4−2.0 μM. The anti-HIV activity of the remaining glutamate
esters containing two nucleosides was higher or comparable to
that of 3TC and AZT but less than that of FLT.
=
6.9−86.4 μM, Table 1) against cell-free virus. Furthermore,
conjugates containing myristic acid showed superior anti-HIV
activity compared to the corresponding compounds without the
fatty acid, possibly due to improved lipophilicity and cellular
uptake. For example, glutamate ester of FLT and 3TC with
myristic acid (27 and 46) (EC50 = 0.3−2.7 μM) exhibited a 5-
to 41-fold higher anti-HIV activity against cell-free virus than
the corresponding conjugate without myristic acid (29) (EC50
= 12.3−17.0 μM). Similarly, improved anti-HIV activity of
lipophilic N-myristoylated conjugates 20 and 31 was observed
versus the corresponding N-acetylated derivatives 21 and 33,
respectively. These data suggest the potential contribution of
higher lipophilicity in improving cellular uptake and generating
compounds with better anti-HIV profile. These data are in
consistence with our previously reported data exhibiting
significantly higher anti-HIV profile and/or cellular uptake of
A myristoylated glutamate ester of FLT (43, EC50 = 6.3−7.7
μM) was also evaluated for antiviral activity. Addition of AZT
as a second nucleoside (20, EC50 = 2.4−3.6 μM) improved the
activity of the conjugate by 2.1- to 2.6-fold (versus that of 43).
Addition of 3TC as the second nucleoside (27, EC50 = 2.0−2.7
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a
Position of nucleoside on glutamic acid also played an
important role in antiviral activity as observed in compounds 27
versus 46 with glutamate esters of FLT and 3TC. Because FLT
is more potent than 3TC, the substitution of FLT in these
derivatives can be considered more crucial for anti-HIV activity.
Compound 46 (EC50 = 0.3−0.6 μM) with FLT substituted at
the C-terminal of glutamic acid displayed 3- to 9-fold higher
antiviral activity than 27 (EC50 = 2.0−2.7 μM) with FLT
substituted at the side chain of glutamate ester, possibly due to
higher intracellular release of FLT from 46 than from 27.
A glutamate derivative with three different nucleosides (34,
FLT-AZT-3TC) showed higher anti-HIV activity (EC50 = 0.9−
1.4 μM) against cell-free virus than the conjugates containing
two of the corresponding nucleosides [21 (FLT-AZT), 29
(FLT-3TC), and 33 (AZT-3TC)] (Table 2). Compound 34
showed nearly 10−12- and 8−36-fold higher anti-HIV activity
than AZT and 3TC, respectively, but slightly less activity than
FLT. Although compound 34 exhibited less anti-HIV activity
than FLT against cell-free virus, the conjugate showed
significantly higher anti-HIV activity (EC50 = 0.8 μM) against
cell associated virus when compared with FLT, AZT, 3TC, and
other glutamate esters with two nucleosides (data not shown).
These data suggest that the incorporation of the third
nucleoside using a glutamate as a scaffold could generate
conjugates with high anti-HIV activity against both cell-free and
cell-associated virus.
Scheme 6. Synthesis of FTC-DMTr (39)
a
Reagents and conditions: (a) TBDMS-Cl, imidazole, DMF; (b)
DMTr-Cl, pyridine; (c) TBAF.
μM) enhanced anti-HIV activity approximately 3-fold when
compared to that of 43. Compound 47 with FTC as the second
nucleoside exhibited significantly (EC50 = 0.1−0.4 μM) higher
anti-HIV activity by 16- to 77-fold. Improved antiviral activity
suggests synergistic or additive contribution of the second
nucleoside to the overall anti-HIV activity. Because AZT and
3TC (EC50 = 10.8−32.7 μM) have lower activity against HIV
compared to FTC (EC50 = 0.8−2.0 μM), their cooperative
effect in 20 and 27 was less than that observed in 47.
Physical mixtures were prepared by mixing glutamic acid with
the nucleosides with or without myristic acid in similar
equimolar ratios to that of the corresponding conjugates.
Table 3 depicts the anti-HIV activity of synthesized glutamate−
a
Scheme 7. Synthesis of Glutamic Acid Esters of Nucleosides (FLT, FTC, and 3TC) (46 and 47)
a
Reagents and conditions: (a) (C13H27CO)2O, DIPEA, DMF; (b) FLT, DIPEA, HBTU, HOBt, DMF; (c) TFA/DCM (95:5 v/v); (d) DIPEA,
HBTU, HOBt, DMF; (e) acetic acid/TFA (98:2 v/v).
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Table 1. Anti-HIV1 Activity of Peptide−Nucleoside Conjugates
antiviral ativity
a
b
c
d
compd
chemical name
cytotoxicity EC50 (μM)
X4 EC50 (μM)
R5 EC50 (μM)
FLT
AZT
10
3′-fluoro-2′,3′-dideoxythymidine
3′-azido-2′,3′-dideoxythymidine
>400
>374
>108
>121
>119
>116
>1000
>25
0.8
10.8
26.8
10.6
11.1
63.1
>1000
0.4
14.2
29.1
6.9
myristoyl-Glu(FLT)-Lys(Myristoyl)-OH
Ac-Glu(FLT)-β-Ala-Lys(myristoyl)-OH
Ac-Ser(myristoyl)-β-Ala-Glu(FLT)-Gly-OH
Ac-Ser(myristoyl)-β-Ala-Glu(AZT)-Gly-OH
dimethyl sulfoxide
13
16
6.9
17
86.4
>1000
0.4
DMSO
e
positive control
dextran sulfate (50 KDa)
0.02
a
b
c
d
Cytotoxicity assay (MTS). 50% Effective concentration. Single-round infection assay (lymphocytotropic strain). Single-round infection assay
e
(monocytotropic strain). Assay control.
Table 2. Anti-HIV Activity of Nucleoside−Glutamate Derivatives with or without Myristoyl Moiety
antiviral activity
a
b
c
d
compd
chemical name
cytotoxicity EC50 (μM)
(X4) EC50 (μM)
(R5) EC50 (μM)
FLT
AZT
3TC
FTC
20
3′-fluoro-2′,3′-dideoxythymidine
3′-azido-2′,3′-dideoxythymidine
(−)-2′,3′-dideoxy-3′-thiacytidine
(−)-5-fluoro-2′,3′-dideoxy-3′-thiacytidine
mysristoyl-Glu(FLT)-AZT
acetyl-Glu(FLT)-AZT
>400
>374
>436
>404
>120
>151
>126
>160
>122
>154
>107
>51
0.8
10.8
32.7
2.0
0.4
14.2
11.3
0.8
2.4
3.6
21
13.5
2.0
13.1
2.7
27
mysristoyl-Glu(FLT)-3TC
acetyl-Glu(FLT)-3TC
29
17.0
6.0
12.3
6.5
31
myristoyl-Glu(AZT)-3TC
acetyl-Glu(AZT)-3TC
33
12.0
0.9
13.4
1.4
34
FLT-succinate-Glu(AZT)-3TC
myristoyl-Glu(OH)-FLT
myristoyl-Glu(3TC)-FLT
myristoyl-Glu(FTC)-FLT
dimethyl sulfoxide
43
6.3
7.7
46
>38
0.6
0.3
47
>37
0.4
0.1
DMSO
>1000
>25
>1000
>1000
0.4
e
positive control
dextran sulfate (50 KDa)
0.02
a
b
c
d
Cytotoxicity assay (MTS). 50% Effective concentration. Single-round infection assay (lymphocytotropic strain). Single-round infection assay
e
(monocytotropic strain). Assay control.
nucleoside conjugates when compared with the corresponding
physical mixtures in μM. For example, the activity of 34 was
compared to the corresponding physical mixtures either with or
without myristic acid (56 and 55, Table 3). The physical
mixture containing FLT, AZT, 3TC, and glutamic acid (55)
showed comparable activity to conjugate 34 against cell-free
virus. However, compound 55 (EC50 = 30.5 μM) exhibited 38-
fold lower activity than 34 (EC50 = 0.8 μM) against cell-
associated virus. Replacing FLT in 55 with FLT−succinate in a
physical mixture (54) (corresponding to conjugate 34) resulted
in reduced anti-HIV activity by 2-fold against X4 cell free virus
and 115-fold against cell associated virus (EC50 = 91.9 μM)
when compared to 34 (EC50 = 0.8 μM). This reduction in the
activity might be due to slow hydrolysis of FLT−succinate to
FLT, the molecule responsible for anti-HIV activity.
In general, the physical mixtures of nucleosides and glutamic
acid with myristic acid showed higher or comparable potency to
that of other corresponding conjugates, possibly because of the
presence of completely free nucleosides in the mixtures. For
example, the physical mixture of FLT, AZT, glutamic acid, and
myristic acid in equimolar ratio (48) exhibited 8−18-fold
higher active than its corresponding conjugate 20 against cell-
free virus. The physical mixture of FLT, 3TC, glutamic acid,
and myristic acid in equimolar ratio (50) showed comparable
activity with corresponding conjugate 46, but was 5−14-fold
more potent than conjugate 27. These data were expected
because the physical mixtures contain free nucleosides that can
be immediately converted to active triphosphosphates after
cellular uptake. On the other hand, the corresponding lipophilic
conjugates need to undergo intracellular hydrolysis to parent
free nucleosides. The rate of regeneration of the nucleoside
from the conjugate is dependent upon the rate of hydrolysis of
the conjugate by esterase. Although the conjugates displayed
comparable or lower anti-HIV activity than the corresponding
physical mixture, it is expected that the lipophilic compounds
may have a prolonged effect possibly due to the sustained
intracellular hydrolysis to parent nucleosides.
Furthermore, physical mixtures containing myristic acid
generally exhibited 2−4-fold higher anti-HIV activity (see 49
vs 48, 51 vs 50, and 55 vs 56) than that of compounds without
myristic acid. Addition of myristic acid in equivalent ratio in
physical mixture 56 improved the activity against cell-free virus
by 3−6-fold in comparison with 34 and the physical mixture
55. These data suggest that the presence of myristic acid plays
an important role in improving the anti-HIV activity of the
physical mixtures. These data are consistent with previously
reported results indicating that medium chain fatty acids
enhance the permeability and cellular uptake of other
compounds and can be used as absorption enhancers.25
These data were also consistent with those obtained for the
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Table 3. Anti-HIV Activity of Nucleoside−Glutamate Derivatives with or without Myristoyl Moiety and the Corresponding
Physical Mixtures
antiviral activity
a
b
c
d
compd
composition
myristoyl-Glu(FLT)-AZT
cytotoxicity EC50 (μM)
(X4) EC50 (μM)
(R5) EC50 (μM)
20
48
21
49
27
46
50
29
51
31
52
33
53
34
54
55
56
>120
>113
>151
>152
>126
>38
2.4
0.3
3.6
0.2
FLT + AZT + glutamic acid + myristic acid
acetyl-Glu(FLT)-AZT
13.5
1.5
13.1
1.1
FLT + AZT + glutamic acid
myristoyl-Glu(FLT)-3TC
2.0
2.7
myristoyl-Glu(3TC)-FLT
0.6
0.3
FLT + 3TC + glutamic acid + myristic acid
acetyl-Glu(FLT)-3TC
>118
>160
>161
>122
>115
>154
>155
>107
>101
113
0.4
0.2
17.0
1.0
12.3
1.3
FLT + 3TC + glutamic acid
myristoyl-Glu(AZT)-3TC
6.0
6.5
AZT + 3TC + glutamic acid + myristic acid
acetyl-Glu(AZT)-3TC
2.2
1.0
12.0
2.6
13.4
1.4
AZT + 3TC + glutamic acid
FLT-succinate-Glu(AZT)-3TC
FLT-succinate + AZT + 3TC + glutamic acid
FLT + AZT + 3TC + glutamic acid
FLT + AZT + 3TC + glutamic acid + myristic acid
dimethyl sulfoxide
0.9
1.4
1.8
1.6
0.9
0.6
>90
0.3
0.2
DMSO
>1000
>25
>1000
>1000
0.4
e
positive control
dextran sulfate (50 KDa)
0.02
a
b
c
d
Cytotoxicity assay (MTS). 50% Effective concentration. Single-round infection assay (lymphocytotropic strain). Single-round infection assay
e
(monocytotropic strain). Assay control.
conjugates containing two nucleosides and myristic acid (20,
27, 29, and 31) as described above. These results indicate that
the addition of myristic acid to 34 may further improve the
anti-HIV activity against cell-free virus.
Table 4. Anti-HIV Evaluation against Wild-Type Clinical
Isolates and Resistant Viruses
a
compd
virus
clade/resistance IC50 (nM) IC90 (nM)
3TC
94US3393IN
98USMSC5016
A-17 MDR
B-wild type
C-wild type
B-NNRTI
B-MDR
87.3
21.8
305.6
305.6
Anti-HIV Activities of Glutamate−Trinucleoside Conjugate
34 (FLT-AZT-3TC) Against Clinical Isolates and Multidrug
Resistant HIV. Because glutamate−trinucleoside conjugate 34
was found to be consistently effective against both X4 and R5
lab-adapted viruses, it was further evaluated against four clinical
isolates of HIV-1 (two wild type isolates from clades B and C,
and two resistant mutants) (Table 4). The anti-HIV activity of
34 was compared with that of its precursors, AZT, FLT, and
3TC. Conjugate 34 showed high antiviral activity against the
multidrug resistant virus 4755-5 (IC50 = 5.9 nM) and its activity
was 2219, 411, and 18 times better than that of 3TC (IC50
>13097 nM), AZT (IC50 = 2425 nM), and FLT (IC50 = 107.3
nM), respectively. Compound 34 also exhibited higher antiviral
activity (IC50 = 12.9 nM) against virus A17 bearing NNRTI-
resistant mutations when compared with parent nucleosides
3TC (IC50 = 92.1 nM), FLT (IC50 = 26.0 nM), and AZT (IC50
= 21.7 nM). However, the antiviral activity of 34 against the
wild-type isolates from B and C subtypes was weaker than FLT.
These results indicate that conjugate 34 can be used against
MDR and NNRTI-resistant strains, improving the anti-HIV
profile of the parent nucleosides.
92.1
282.9
4755-5 MDR
>13097
>13097
FLT
AZT
34
94US3393IN
98USMSC5016
A-17 MDR
B-wild type
C-wild type
B-NNRTI
B-MDR
2.0
2.0
20.4
12.3
87.2
1065
26.0
107.3
4755-5 MDR
94US3393IN
98USMSC5016
A-17 MDR
B-wild type
C-wild type
B-NNRTI
B-MDR
2.4
1.95
21.7
2425
21.3
34.2
152.3
10657
4755-5 MDR
94US3393IN
98USMSC5016
A-17 MDR
B-wild type
C-wild type
B-NNRTI
B-MDR
26.7
22.7
12.9
5.9
160.6
84.5
28.3
46.0
4755-5 MDR
a
Multiround infection assay using HIV-1 clinical isolates (wild type
and NNRTI and multidrug (MDR) resistant mutants) and peripheral
blood mononuclear cells.
nucleosides. Addition of two myristic acids was not beneficial
either, and the antiviral activity was reduced drastically.
CONCLUSIONS
■
Dinucleoside and trinucleoside conjugates of multivalent
scaffolds (e.g., glutamic acid and peptides) were synthesized
with the goal of improving anti-HIV activity by simultaneously
On the other hand, the glutamate esters containing two
nucleosides exhibited higher anti-HIV activity than conjugates
substituted with one nucleoside only and other peptide
analogues. The presence of a myristic acid in the glutamate
conjugates and their corresponding physical mixtures improved
anti-HIV activity. Myristoylated glutamate conjugates 46 (EC50
= 0.3−0.6 μM, containing FLT and 3TC) and 47 (EC50 = 0.1−
̈
delivering different nucleosides to infected or HIV-naive target
cells. Improved activity may be associated with increased
potency, broader antiviral spectrum, and higher barrier to drug
resistance. Peptide conjugates with one nucleoside and myristic
acid did not improve the anti-HIV activity of their parent
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TFA was removed at reduced pressure, and the residue was purified
with HPLC using a C18 column and water/acetonitrile as solvents as
described above to yield 5 and 6.
0.4 μM, containing FLT and FTC) were the most active
glutamate conjugates.
Among all the tested compounds, a trinucleoside glutamate
derivative containing AZT, FLT, and 3TC (34, EC50 = 0.9−1.4
μM) exhibited higher anti-HIV activity than AZT and 3TC
against cell-free virus. Conjugate 34 was also more active
against multidrug resistant and NNRTI-resistant HIV clinical
isolates when compared to 3TC, FLT, and AZT. Furthermore,
compound 34 was more potent against cell-associated virus
when compared with the corresponding physical mixtures.
Compounds 46, 47, and 34 showed better antiviral profiles
than their parent nucleosides, suggesting that multinucleoside
conjugation on a glutamate scaffold may be considered for the
development of new anti-HIV drugs for therapeutic and
preventive purposes.
Fmoc-Glu(3′-fluoro-2′,3′-dideoxythymidine-5′-yl)-OH (5).
1
Overall yield (5.0 g, 70%). H NMR (400 MHz, CD3OD, δ ppm):
7.78 (d, J = 7.4 Hz, 2H, Fmoc Ar-H), 7.60−7.69 (m, 2H, Fmoc Ar-H),
7.35−7.42 (m, 3H, H-6, Fmoc Ar-H), 7.31 (dt, J = 7.4 and 3.3 Hz, 2H,
Fmoc Ar-H), 6.23 (dd, J = 6.4 and 9.3 Hz, 1H, H-1′), 5.27 (dd, J = 5.9
and 53.3 Hz, 1H, H-3′), 4.34−4.51 (m, 5H, H-4′, H-5″, Glu CH(α),
and Fmoc NHCOOCH2), 4.28 (dd, J = 5.9 and 9.1 Hz, 1H, H-5′),
4.22 (t, J = 6.7 Hz, 1H, Fmoc NHCOOCH2CH), 2.10−2.58 (m, 6H,
Glu CH2CH2COO (β and γ methylene), H-2″, and H-2′), 1.87 (s, 3H,
CH3). HR-MS (ESI-TOF) (m/z): C30H30FN3O9, calcd 595.1966;
found 596.1989 [M + H]+, 1191.4610 [2 M + H]+.
Fmoc-Glu(3′-azido-2′,3′-dideoxythymidine-5′-yl)-OH (6).
1
Overall yield (5.2 g, 70%). H NMR (400 MHz, CD3OD, δ ppm):
7.78 (d, J = 7.4 Hz, 2H, Fmoc Ar-H), 7.35−7.70 (m, 2H, Fmoc Ar-H),
7.44 (s, 1H, H-6), 7.37 (t, J = 7.4 Hz, 2H, Fmoc Ar-H), 7.30 (t, J = 7.4
Hz, 2H, Fmoc Ar-H), 6.10 (t, J = 6.4 Hz, 1H, H-1′), 4.15−4.45 (m,
7H, H-3′, H-5′, H-5″, Glu-α-CH, Fmoc NHCOOCH2CH), 4.05 (dd, J
= 4.8 and 9.0 Hz, 1H, H-4′), 2.52 (t, J = 7.2 Hz, 2H, Glu-CH2COO (β-
methylene), 2.32−2.48 (m, 2H, Glu-CH2CH2COO (γ-methylene)),
2.14−2.30 (m, 1H, H-2′), 1.90−2.05 (m, 1H, H-2″), 1.85 (s, 3H, 5-
CH3). 13C NMR (CD3OD, 100 MHz, δ ppm): 175.27 (COOH),
173.97 (COOAZT), 162.39 (C-4 CO), 152.25 (C-2 CO), 145.40,
142.73 (Fmoc Ar-C), 137.85 (C-6), 128.95, 128.33, 126.42, 121.08
(Fmoc Ar-C), 112.02 (C-5), 86.70 (C-1′), 83.176 (C-4′), 68.16
(CH2OCONH), 64.88 (C-5′), 62.15 (Glu CH(α), 54.57 (C-3′), 37.88
(C-2′), 31.47 (Glu γ-CH2), 27.99 (Glu β-CH2), 12.73 (5-CH3). HR-
MS (ESI-TOF) (m/z): C30H30N6O9, calcd 618.2074; found 619.2564
[M + H]+.
Fmoc-Ser(O-myristoyl)-OH (8). Compound 7 (5 g, 15.3 mmol)
and DIPEA (10 mL, 75 mmol) were dissolved in DMF (20 mL).
Myristoyl chloride (10 g, 23 mmol) was added to the solution. The
reaction mixture was stirred for overnight. The solvent was removed at
reduced pressure, and the residue was purified using silica gel column
chromatography using dichloromethane (DCM)/methanol (0−5%) as
eluents. The compound was eluted at 5% methanol in DCM to yield 8
(5.3 g, 63%).
EXPERIMENTAL SECTION
■
Materials and Methods. PS3 automated peptide synthesizer
(Rainin Instrument Co., Oakland, CA) was used to synthesize
peptides. In general, all peptides were synthesized by the solid-phase
synthesis strategy employing N-(9-fluorenyl)methoxycarbonyl
(Fmoc)-based chemistry and Fmoc-L-amino acid building blocks.
HBTU and N-methylmorpholine (NMM) in N,N-dimethylformamide
(DMF) were used as coupling and activating reagents, respectively.
Wang resin, Fmoc-amino acid Wang resins, coupling reagents, Fmoc-
protected amino acids (Fmoc-Glu-OtBu, Fmoc-Ser-OH, Fmoc-
Lys(Mtt)-OH, Fmoc-β-Ala-OH, Fmoc-Gly-OH), and HBTU were
purchased from Novabiochem. 3TC (Lamivudine), AZT (Zidovu-
dine), FTC (Emtricitabine), and FLT (Alovudine) were purchased
from Euro Asia Tran Continental (Bombay, India). DIPEA and all the
other reagents including solvents were purchased from Fisher
Scientific.
The chemical structures of final products were characterized by
nuclear magnetic resonance spectrometry (1H NMR and 13C NMR)
determined on a Bruker NMR spectrometer (400 MHz). Chemical
shifts are reported in parts per millions (ppm). The chemical
structures of final products were confirmed by a high-resolution PE
Biosystems Mariner API time-of-flight electrospray mass spectrometer
or Biosystems QStar Elite time-of-flight electrospray mass spectrom-
eter. The purity of final products (>95%) was confirmed by analytical
HPLC. The analytical HPLC was performed on a Hitachi analytical
HPLC system using a C18 Shimadzu Premier 3 μm column (150 cm
× 4.6 mm) using two different systems at a flow rate of 1 mL/min with
detection at 265 nm.
Synthesis of Myristoylated Mononucleoside Dipeptide,
Tripeptide, and Tetrapeptide Conjugates. Glutamate ester
conjugates of AZT, FLT, and myristic acid were synthesized
employing a PS3 automated peptide synthesizer and Fmoc solid-
phase peptide synthesis using Fmoc-L-amino acid building blocks. The
glutamate−nucleoside conjugates were assembled on Wang resin solid
support at room temperature. The building blocks, 5, 6, and 8, were
synthesized from Fmoc-Glu(OH)-OtBu and Fmoc-Ser-OH, respec-
tively, as described below:
Fmoc-Glu(3′-fluoro-2′,3′-dideoxythymidine-5′-yl)-OH (5)
and Fmoc-Glu(3′-azido-2′,3′-dideoxythymidine-5′-yl)-OH (6).
Fmoc-Glu(OH)OtBu (5 g, 11.8 mmol), 1 or 2, 14.1 mmol), and
HBTU (6.7 g, 17.6 mmol) were dissolved in DMF (25 mL). DIPEA
(5 mL, 38 mmol) was added to the solution, and the reaction mixture
was stirred at room temperature overnight. The solvent was removed
under reduced pressure to yield crude Fmoc-Glu(3′-fluoro-2′,3′-
dideoxythymidine-5′-yl)OtBu (3) or Fmoc-Glu(3′-azido-2′,3′-dideox-
ythymidine-5′-yl)OtBu (4).
1H NMR (400 MHz, CDCl3, δ ppm): 7.70 (d, J = 7.4 Hz, 2H, Fmoc
Ar-H), 7.53 (d, J = 6.9 Hz, 2H, Fmoc Ar-H), 7.34 (t, J = 7.4 Hz, 2H,
Fmoc Ar-H), 7.25 (t, J = 7.4 Hz, 2H, Fmoc Ar-H), 5.50 (d, J = 8.0 Hz,
serine CH(α)), 4.65−4.71 (m, 1H, serine CH″ (β)), 4.45 (dd, J = 3.9
and 11.4 Hz, 1H, serine CH′ (β)), 4.32−4.39 (m, 2H, Fmoc
NHCOOCH2), 4.17 (t, J = 6.9 Hz, 1H, Fmoc NHCOOCH2CH), 2.26
(t, J = 7.5 Hz, 2H, CH2COO), 1.54 (t, J = 6.3 Hz, 2H, CH2CH2COO),
1.13−1.25 (br m, 20H, methylene protons), 0.80 (t, J = 6.6 Hz, 3H,
CH3). HR-MS (ESI-TOF) (m/z): C32H43NO6, calcd 537.3090; found
538.2673 [M + H]+, 1075.5442 [2 M + H]+.
N-Myristoyl-Glu(3′-fluoro-2′,3′-dideoxythymidine-5′-yl)-Lys-
(myristoyl)OH [My-Glu(FLT)-Lys(My)-OH (10)]. Resin 9 (300 mg,
0.45 mmol/g) was swelled in DMF for 30 min. Compound 5 (320 mg,
0.54 mmol), HBTU (200 mg, 0.54 mmol), and NMM (0.54 mmol)
were added to the swelled resin suspension in DMF. The mixture was
shaken overnight at room temperature. The resin was filtered and
washed two times with DMF (10 mL). Fmoc deprotection was carried
out using piperidine in DMF (20%, 10 mL). The resin was washed
with DMF (3 × 10 mL). To the resin was added TFA/DCM mixture
(5:95 v/v, 10 mL) to remove methyltrityl protecting group (Mtt) at
lysine side chain. The mixture was shaken for 1 h at room temperature.
The resin was washed with DCM (3 × 10 mL) and DMF (10 mL) and
swelled in DMF (10 mL). Myristic anhydride (100 mg, 1.08 mmol)
and DIPEA (2 mL, 15 mmol) were added to the swelled resin. The
mixture was shaken for 2 h at room temperature. The resin was
washed with DMF (2 × 10 mL). A mixture of TFA/anisole/water
(95:2.5:2.5 v/v/v, 10 mL) was added to the resin, and the mixture was
shaken for 1 h. After filtration, the solution was concentrated and dried
under reduced pressure. The crude peptide conjugates were purified
with reversed phase HPLC using a C18 column and water/acetonitrile
3: HR-MS (ESI-TOF) (m/z): C34H38FN3O9, calcd 651.2592; found
652.1312 [M + H]+; 1303.2873 [2 M + H]+. 4: HR-MS (ESI-TOF)
(m/z): C34H38N6O9, calcd 674.2700; found 697.0440 [M + Na]+.
Trifluoroacetic acid/water (TFA/water, 95:5 v/v, 20 mL) was
added to the reaction mixture containing 3 or 4. The reaction mixture
was stirred for 1 h to remove t-butyl protecting group at C-terminal.
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1
as solvents as described above and were lyophilized to yield 10 (10 mg,
8.0%).
16: Overall yield (20 mg, 8%). H NMR (400 MHz, CD3OD, δ
ppm): 7.48 (s, 1H, FLT H-6), 6.28 (dd, J = 5.6 and 8.8 Hz, 1H, FLT
H-1′), 5.29 (dd, J = 53.5 and 4.4 Hz, 1H, FLT H-3′), 4.58−4.66 [m,
1H, Ser COCHNH (CH-α)], 4.22−4.50 (m, 5H, FLT H-4′, FLT H-
5″, and Glu COCHNH (CH-α), Ser CH2O (CH-β)], 3.98 (dd, J = 3.9
and 17.8 Hz, 1H, FLT H-5′), 3.72−3.92 [m, 2H, Gly COCH2NH
(CH-α)], 3.38−3.58 (m, 2H, β-Ala CH2NH), 2.24−2.64 (m, 8H, FLT
H-2′, FLT H-2″, β-Ala CH2COO, Glu CH2CH2COO), 2.16 (t, J = 7.2
Hz, 3H, myristate CH2COO), 1.94−2.08 (m, 3H, Ac CH3), 1.88 (s,
3H, FLT 5-CH3), 1.52−1.66 (m, 2H, myristate CH2CH2COO), 1.22−
1.38 (br m, 20H, methylene protons), 0.90 (t, J = 6.3 Hz, 3H,
myristate CH3). HR-MS (ESI-TOF) (m/z): C39H61FN6O13, calcd
840.4281; found 841.3842 [M + H]+.
HR-MS (ESI-TOF) (m/z): C49H84FN5O10, calcd 921.6202; found
922.9989 [M + H]+, 1845.9558 [2 M + H]+.
N-Acetyl-Glu(3′-fluoro-2′,3′-dideoxythymidine-5′-yl)-β-Ala-
Lys(myristoyl)-OH [Ac-Glu(FLT)-βAla-Lys(My)-OH (13)] and N-
Acetyl-Glu(3′-azido-2′,3′-dideoxythymidine-5′-yl)-βAla-Lys-
(myristoyl)-OH [Ac-Glu(AZT)-βAla-Lys(My)-OH (14)]. The pep-
tide was assembled on Fmoc-Lys(Mtt)-Wang resin (600 mg, 0.45
mmol/g) by Fmoc solid-phase peptide synthesis strategy at room
temperature using Fmoc protected amino acids [Fmoc-β-Ala-OH (C +
1) and Fmoc-Glu(nucleoside)-OH (C + 2) (5 or 6, 1.08 mmol)].
HBTU (1.08 mmol) and NMM (1.08 mmol) in DMF were used as
coupling and activating reagents, respectively. Fmoc deprotection at
each step was carried out using piperidine in DMF (20%). NH2-
Glu(FLT)-βAla-Lys(Mtt)-Wang resin (11) or NH2-Glu(AZT)-βAla-
Lys(Mtt)-Wang resin (12) was transferred to the reaction vessel and
swelled in DMF (2 mL) for 30 min. Acetic anhydride (2 mL) and
DIPEA (2 mL, 15 mmol) were added to the mixture. The reaction was
shaken at room temperature for 30 min to cap the N-terminal with
acetyl group. N-Acetylated resin was washed with DMF (2 × 10 mL).
TFA/DCM (5%, 10 mL) to the resin. The mixture was shaken for 1 h
at room temperature. The resin was washed with DCM (3 × 10 mL)
and DMF (10 mL) and swelled in DMF (10 mL). Free amino group at
lysine side chain was further myristoylated by adding myristic
anhydride (100 mg, 1.08 mmol) and DIPEA (2 mL, 15 mmol) to
the swelled resin. The mixture was shaken for 2 h at room
temperature. The resin was washed with DMF (3 × 10 mL). A
mixture of TFA/anisole/water (95:2.5:2.5 v/v/v, 10 mL) was added to
the resin, and the mixture was shaken for 1 h. After filtration, the
solution was concentrated and dried under reduced pressure. The
crude peptide conjugates were purified with reversed phase HPLC
using a C18 column and water/acetonitrile as solvents as described
above and were lyophilized to yield 13 and 14.
1
17: Overall yield (20 mg, 8%). H NMR (400 MHz, CD3OD, δ
ppm): 7.48 (s, 1H, H-6 AZT), 6.14 (t, J = 6.6 Hz, 1H, AZT H-1′),
4.20−4.70 [m, 6H, AZT H-5′, AZT H-5″, AZT H-3′, Ser COCHNH
(CH-α), Ser CH2O (CH-β), Glu COCHNH, (CH-α)], 4.07 (dd, J =
4.9 and 8.9 Hz, 1H, AZT H-4′), 3.84−4.05 [m, 2H, Gly COCH2NH
(CH-α)], 3.44 (dd, J = 6.6 and 12.5 Hz, 2H, β-Ala CH2NH), 2.10−
2.66 (m, 10H, FLT H-2′, FLT H-2″, β-Ala CH2COO, Glu
CH2CH2COO, myristate CH2COO), 1.92−2.09 (m, 3H, acetyl
CH3), 1.89 (s, 3H, 5-CH3-AZT), 1.52−1.72 (m, 2H, myristate-
CH2CH2COO), 1.24−1.50 (br m, 20H, methylene protons), 0.90 (t, J
= 6.5 Hz, 3H, myristate CH3). HR-MS (ESI-TOF) (m/z):
C39H61N9O13, calcd 863.4389; found 864.9022 [M + H]+,
1729.8398 [2 M + H]+.
Synthesis of Dinucleoside and Trinucleoside Glutamate
Ester Derivatives Functionalized with Acetyl or Myristoyl
Moiety. Glutamate−nucleoside conjugates containing more than one
nucleoside with or without myristoyl group were synthesized by using
solution phase synthesis.
Fmoc-Glu(OFLT)-OAZT (18). Compound 5 (500 mg, 0.84 mmol),
AZT (269 mg, 1 mmol), and HBTU (650 mg 1.7 mmol) were
dissolved in DMF (10 mL). DIPEA (5 mL, 37 mmol) was added to
the solution, and the reaction mixture was stirred overnight. The
solvent was removed, and the residue was dried under reduced
pressure. The residue was purified with reversed phase HPLC using a
C18 column and water/acetonitrile as solvents as described above and
was lyophilized to yield 18 (620 mg, 85%).
1
13: Overall yield (20 mg, 8%). H NMR (400 MHz, CD3OD, δ
ppm): 7.48 (s, 1H, FLT H-6), 6.28 (dd, J = 5.7 and 8.8 Hz, 1H, FLT
H-1′), 5.29 (dd, J = 53.6 and 4.8 Hz, 1H, FLT H-3′), 4.20−4.70 (m,
6H, FLT H-4′, FLT H-5′, FLT H-5″, Lys COCHNH (CH-α), Glu
COCHNH (CH-α)], 3.40−3.50 (m, 2H, β-Ala CH2NH), 3.17 (t, J =
6.7 Hz, 2H, Lys CH2-NHCO), 2.20−2.60 (m, 6H, FLT H-2′, FLT H-
2″, β-Ala CH2COO, Glu CH2CH2COO), 2.17 (t, J = 7.6 Hz, 3H,
myristate CH2COO), 1.94−2.05 (m, 3H, Ac CH3), 1.88 (s, 3H, FLT
5-CH3), 1.20−1.70 (m, 20H, myristate methylene and Lys methylene
protons), 0.90 (t, J = 6.6 Hz, 3H, myristate CH3). HR-MS (ESI-TOF)
(m/z): C40H65FN6O11, calcd 824.4695; found 825.6640 [M + H]+,
1651.2282 [2 M + H]+.
1H NMR (400 MHz, CDCl3, δ ppm): 9.24 (s, 1H, FLT NH), 9.09
(s, 1H, AZT NH), 7.75 (d, J = 7.2 Hz, 1H, Fmoc Ar-H), 7.57 (d, J =
3.6 Hz, 1H, Fmoc Ar-H), 7.38 (t, J = 7.2 Hz, Fmoc Ar-H), 7.29 (t, J =
7.2 Hz, 2H, Fmoc Ar-H), 7.15 (s, 1H, FLT H-6), 7.07 (s, 1H, AZT H-
6), 6.14 (t, J = 8.1 Hz, 1H, FLT H-1′), 5.80 (t, J = 6.9 Hz, 1H, AZT H-
1′), 5.17 (dd, J = 2.0 and 55.6 Hz, 1H, FLT H-3′), 4.23−4.60 (m, 6H,
AZT H-5′, AZT H-5″, AZT H-3′, FLT H-4′, FLT H-5′, and FLT H-5″),
4.19 (t, J = 6.7 Hz, 1H, Glu HN-CH-COO CH(α)), 3.95−4.05 (m,
1H, AZT H-4′), 2.16−2.69 (m, 8H, AZT H-2′, AZT H-2″, FLT H-2′,
FLT H-2″, and Glu CH2CH2COO), 1.88 (br s, 6H, FLT 5-CH3 and
AZT 5-CH3). 13C NMR (CDCl3, 100 MHz, δ ppm): 172.3, 171.4
(FLT COO, AZT COO), 163.85, 163.72 (FLT C-4 CO, AZT C-4
CO), 156.23 (Fmoc OCONH), 150.10, 149.98 (AZT C-2 CO
and FLT C-2 CO), 143.74, 143.55, 141.31 (Fmoc Ar-C), 137.23,
135.88 (AZT C-6, FLT C-6), 127.88, 127.10, 125.05, 125.00, 120.08,
(Fmoc Ar-C), 111.37, 111.28 (AZT C-5, FLT C-5), 93.23 (J = 177.8
Hz, FLT C-3′), 87.82 (FLT C-1′), 86.53 (AZT C-1′), 82.21 (J = 26.3
Hz, FLT C-4′), 81.72 (AZT C-4′), 68.00 (Fmoc CH2-OCONH), 63.82
(FLT C-5′), 60.29 (AZT C-5′), 53.48, 53.31 (AZT C-3′, CH(α)),
47.04 (Fmoc CH-CH2-OCONH), 37.58 (J = 20.1 Hz, FLT C-2′),
36.94 (AZT C-2′), 29.72 (Glu γ-CH2), 25.60 (Glu β-CH2), 12.57,
12.43 (AZT 5-CH3, FLT 5-CH3). HR-MS (ESI-TOF) (m/z):
C40H41FN8O12, calcd 844.2828; found 845.0241 [M + H]+.
14: Overall yield (20 mg, 8%). HR-MS (ESI-TOF) (m/z):
C40H65N9O11, calcd 847.4804; found 848.9452 [M + H]+.
N-Acetyl-Ser(myristoyl)-β-Ala-Glu(3′-fluoro-2′,3′-dideoxy-
thymidine-5′-yl)-Gly-OH [Ac-Ser(My)-βAla-Glu(FLT)Gly-OH
(16)] and N-Acetyl-Ser(myristoyl)-β-Ala-Glu(3′-azido-2′,3′-di-
deoxythymidine-5′-yl)-Gly-OH [Ac-Ser(My)-βAla-Glu(AZT)Gly-
OH (17)]. The peptides were assembled on resin 15 (600 mg, 0.45
mmol/g) by Fmoc solid-phase peptide synthesis strategy using Fmoc
protected amino acids (5 or 6) (C + 1), Fmoc-β-Ala-OH (C + 2), and
8 (C + 3), each 1.08 mmol]. HBTU (1.08 mmol) and NMM (1.08
mmol) in DMF were used as coupling and activating reagents,
respectively. Fmoc deprotection at each step was carried out using
piperidine in DMF (20%). NH2-Ser(My)-βAla-Glu(FLT)Gly-Wang
resin or NH2-Ser(My)-βAla-Glu(AZT)-Gly-Wang resin was trans-
ferred to the reaction vessel and swelled in DMF (2 mL) for 30 min.
Acetic anhydride (2 mL) and DIPEA (2 mL, 15 mmol) were added to
the mixture. The reaction was shaken at room temperature for 30 min
to cap N-terminal amino group with acetyl group. N-Acetylated resins
were washed with DMF (2 × 10 mL). A mixture of TFA/anisole/
water (95:2.5:2.5 v/v/v, 10 mL) was added to the resins. The mixtures
were shaken for 1 h to cleave the peptides. After filtration, the solution
was concentrated and dried under reduced pressure. The crude
peptide conjugates were purified with reversed phase HPLC using a
C18 column and water/acetonitrile as solvents as described above and
were lyophilized to yield 16 and 17.
NH2-Glu(OFLT)-OAZT (19). Compound 18 (610 mg, 0.72 mmol)
was dissolved in THF (10 mL). Piperidine (7.18 μL, 0.072 mmol) and
1-octanethiol (7.3 mmol, 10 mM solution in THF, 0.73 mL) were
added to the reaction mixture. The mixture was stirred at room
temperature for 1 h. The solvent was removed, and the residue was
dried under reduced pressure. The residue purified with reversed
phase HPLC using a C18 column and water/acetonitrile as solvents as
described above and lyophilized to yield 19 (225 mg, 50%).
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1H NMR (400 MHz, CD3OD, δ ppm): 7.44 (s, 1H, FLT H-6), 7.38
(s, 1H, AZT H-6), 6.22 (dd, J = 8.1 Hz, 1H, FLT H-1′), 6.02 (t, J = 6.9
Hz, 1H, AZT H-1′), 5.23 (dd, J = 2.0 and 55.6 Hz, 1H, FLT H-3′),
4.20−4.55 (m, 6H, AZT H-5′, AZT H-5″, AZT H-3′, FLT H-4′, FLT
H-5′, and FLT H-5″), 4.15 (t, J = 6.7 Hz, 1H, Glu HN-CH-COO
CH(α)), 3.98−4.04 (m, 1H, AZT H-4′), 2.16−2.69 (m, 8H, AZT H-
2′, AZT H-2″, FLT H-2′, FLT H-2″, CH2CH2COO), 1.87 (br s, 6H,
FLT 5-CH3, AZT 5-CH3). 13C NMR (CD3OD, 100 MHz, δ ppm):
173.29, 170.17 (FLT COO, AZT COO), 166.40, 163.72 (FLT C-4
CO, AZT C-4 CO), 152.33, 152.19 (AZT C-2 CO, FLT C-2
CO), 139.13, 137.89 (AZT C-6, FLT C-6), 114.03, 112.08 (AZT C-
5, FLT C-5), 95.07 (J = 176.7 Hz, FLT C-3′), 88.12 (FLT C-1′), 87.34
(AZT C-1′), 83.86 (J = 27.3 Hz, FLT C-4′), 82.72 (AZT C-4′), 66.90
(FLT C-5′), 61.99 (AZT C-5′), 53.23, 52.28 (AZT C-3′, CH(α)),
38.26 (J = 21.2 Hz, FLT C-2′), 37.10 (AZT C-2′), 30.14 (Glu γ-CH2),
26.63 (Glu β-CH2), 12.73, 12.53 (AZT 5-CH3, FLT 5-CH3). HR-MS
(ESI-TOF) (m/z): C25H31FN8O10, calcd 622.2147; found 622.9532
[M + H]+, 1244.9406 [2 M + H]+.
N-Myristoyl-Glu(OFLT)-OAZT (20). Compound 19 (75 mg, 0.12
mmol) and myristic anhydride (100 mg, 0.24 mmol) were dissolved in
DMF (10 mL). DIPEA (5 mL, 37 mmol) was added to the solution.
The mixture was stirred for 2 h at room temperature. The solvent was
removed under reduced pressure. The residue was purified with
reversed phase HPLC using a C18 column and water/acetonitrile as
solvents as described above and was lyophilized to yield 20 (40 mg,
40%).
C-1′), 82.42 (J = 26.0 Hz, FLT C-4′), 81.55 (AZT C-4′), 64.31 (FLT
C-5′), 60.94 (AZT C-5′), 51.75 (AZT C-3′, CH(α)), 36.96 (J = 20.6
Hz, FLT C-2′), 36.14 (AZT C-2′), 29.39 (Glu γ-CH2), 26.04 (Glu β-
CH2), 20.88 (acetyl CH3), 12.73, 12.53 (AZT 5-CH3, FLT 5-CH3).
HR-MS (ESI-TOF) (m/z): C27H33FN8O11, calcd 664.2253; found
664.9298 [M + H]+, 1328.8401 [2 M + H]+.
Fmoc-Glu(OFLT)-O(3TC(DMTr)) (22). (−)-N4-(4,4′-Dimethoxy-
trityl)-2′,3′-dideoxy-3′-thiacytidine (DMTr-3TC) was synthesized by
using a previously reported method.20 Compound 5 (500 mg, 0.84
mmol), DMTr-3TC (535 mg, 1 mmol), and HBTU (650 mg 1.7
mmol) were dissolved in DMF (10 mL). DIPEA (5 mL, 37 mmol)
was added to the solution, and the reaction mixture was stirred
overnight at room temperature. The solvent was removed, and the
residue was dried under reduced pressure. The residue was purified
with reversed phase HPLC using a C18 column and water/acetonitrile
as solvents as described above and was lyophilized to yield 22 (820
mg, 75%).
1H NMR (400 MHz, CDCl3, δ ppm): 8.99 (s, 1H, FLT NH), 7.74
(d, J = 7.5 Hz, 1H, Fmoc Ar-H), 7.55 (d, J = 7.6 Hz, 1H, 3TC H-6),
7.38 (t, J = 7.5 Hz, Fmoc Ar-H), 7.06−7.33 (m, 13H, DMTr Ar-H,
Fmoc Ar-H, and FLT H-6), 6.76−6.87 (m, 4H, DMTr Ar-H protons),
6.34 (m, 1H, 3TC H-1′), 6.14−6.22 (m, 1H, FLT H-1′), 5.64 (d, J =
7.6 Hz, 1H, 3TC H-5), 5.05−5.27 (m, 2H, FLT H-3′ and 3TC H-4′),
4.24−4.45 (m, 8H, 3TC H-5′, 3TC H-5″, Fmoc NHCOOCH2CH,
FLT H-4′, FLT H-5′, FLT H-5″), 4.17 (t, J = 6.7 Hz, 1H, Glu HN-CH-
COO CH(α)), 3.75 and 3.78 (two s, 6H, DMTr CH3O), 3.46 (dd, J =
11.8 and 5.3 Hz, 1H, 3TC H-2″), 2.97 (dd, J = 11.8 and 5.3 Hz, 1H,
3TC H-2′), 1.97−2.67 (m, 6H, FLT H-2′, FLT H-2″, and Glu
CH2CH2COO), 1.86 (s, 3H, 5-CH3). 13C NMR (CDCl3, 100 MHz, δ
ppm): 172.00 (FLT COO), 171.25 (3TC COO), 165.06 (3TC C-4),
163.50 (FLT C-4 CO), 158.69 (DMTr Ar-C-OCH3), 156.00 (3TC
C-2 CO), 150.09 (FLT C-2 CO), 144.24 (3TC C-6), 135.38
(FLT C-6), 129.91, 128.52, 128.37, 127.82, 127.53, 127.11, 125.00,
120.08, 113.60 (Fmoc Ar-C and DMTr Ar-C), 111.41 (FLT C-5),
95.17 (3TC C-5), 93.20 (J = 178.3 Hz, FLT C-3′), 87.64 (3TC C-1′),
86.01 (FLT C-1′), 83.16 (3TC C-4′), 82.16 (J = 26.9 Hz, FLT C-4′),
81.69 (DMTr Ph3C-NH), 70.34 (Fmoc CH2-OCONH), 67.15 (FLT
C-5′), 65.76 (3TC C-5′), 55.28 (DMTr OCH3), 53.40 (CH(α)), 47.04
(Fmoc CH-CH2-OCONH), 38.16 (FLT C-2′), 37.36 (3TC C-2′),
29.86, (Glu γ-CH2), 27.16 (Glu β-CH2), 12.64 (FLT 5-CH3). HR-MS
(ESI-TOF) (m/z): C59H57FN6O13S, calcd 1108.3688; found
1109.4804 [M + H]+, 1131.4395 [M + Na]+, 1147.4739 [M + K]+,
2218.5704 [2 M + H]+.
Fmoc-Glu(OAZT)-O(3TC(DMTr)) (23). Compound 6 (520 mg,
0.84 mmol), DMTr-3TC (535 mg, 1 mmol), and HBTU (650 mg 1.7
mmol) were dissolved DMF (10 mL). DIPEA (5 mL, 37 mmol) was
added to the solution, and the reaction mixture was stirred overnight at
room temperature. The solvent was removed, and the residue was
dried under reduced pressure. The residue was purified with reversed
phase HPLC using a C18 column and water/acetonitrile as solvents as
described above and was lyophilized to yield 23 (840 mg, 87%).
HR-MS (ESI-TOF) (m/z): C59H57N9O13S, calcd 1131.3797; found
1132.3485 [M + H]+, 2265.1887 [2 M + H]+.
NH2-Glu(OFLT)-O(3TC(DMTr)) (24). Compound 22 (800 mg,
0.72 mmol) was dissolved in THF (10 mL). Piperidine (7.2 μL, 0.072
mmol) and 1-octanethiol (7.3 mmol, 10 mM solution in THF, 0.73
mL) were added to the reaction mixture. The mixture was stirred for 1
h at room temperature. The solvent was removed, and the residue was
dried under reduced pressure. The residue was purified with reversed
phase HPLC using a C18 column and water/acetonitrile as solvents as
described above and lyophilized to yield 24 (300 mg, 50%).
1H NMR (400 MHz, CD3OD, δ ppm): 7.45, 7.44 (two s, 2H, AZT
H-6, FLT H-6), 6.23 (dd, J = 8.9 and 5.6 Hz, 1H, FLT H-1′), 6.09 (t, J
= 6.7 Hz, 1H, AZT H-1′), 5.24 (dd, J = 5.0 and 53.6 Hz, 1H, FLT H-
3′), 4.32−4.48 (m, 6H, AZT H-5′, AZT H-5″, AZT H-3′, FLT H-4′,
FLT H-5′, and FLT H-5″), 4.22 (dd, J = 3.8 and 11.4 Hz, 1H, Glu HN-
CH-COO CH(α)), 4.06 (dd, J = 4.8 and 8.6 Hz, 1H, AZT H-4′),
2.15−2.60 (m, 10H, AZT H-2′, AZT H-2″, FLT H-2′, FLT H-2″,
myristate CH2COO, Glu CH2CH2COO), 1.87 (s, 6H, FLT 5-CH3,
AZT 5-CH3), 1.58 (t, J = 6.6 Hz, 2H, CH2CH2COO), 1.23−1.33 (br
m, 20H, methylene protons), 0.87 (t, J = 6.7 Hz, 3H, 5-CH3). 13C
NMR (CDCl3, 100 MHz, δ ppm): 173.78, 172.37, 171.56 (FLT COO,
AZT COO, CONH), 164.01 (FLT C-4 CO and AZT C-4 CO),
150.27 (AZT C-2 CO, FLT C-2 CO), 137.14, 135.77 (AZT C-6,
FLT C-6), 111.37 (AZT C-5, FLT C-5), 93.27 (J = 179.4 Hz, FLT C-
3′), 87.56 (FLT C-1′), 86.32 (AZT C-1′), 82.19 (J = 26.6 Hz, FLT C-
4′), 81.68 (AZT C-4′), 63.83 (FLT C-5′), 60.33 (AZT C-5′), 51.48,
51.35 (AZT C-3′, CH(α)), 37.59 (J = 20.8 Hz, FLT C-2′), 36.96 (AZT
C-2′), 36.39 (CH2COO), 34.14, (Glu γ-CH2), 31.93 (Glu β-CH2),
30.04, 29.90, 29.83, 29.66, 29.60, 29.51, 29.46, 29.36, 29.31, 29.16,
27.27 25.59, 24.97, 22.70 (methylene carbons), 14.15 (My-CH3)
12.73, 12.53 (AZT 5-CH3, FLT 5-CH3). HR-MS (ESI-TOF) (m/z):
C39H57FN8O11, calcd 832.4131; found 832.8583 [M]+, 1665.8057 [2
M + H]+.
N-Acetyl-Glu(OFLT)-OAZT (21). Compound 19 (75 mg, 0.12
mmol) was dissolved in DMF (10 mL). DIPEA (5 mL, 37 mmol) and
acetic anhydride (2 mL, 20 mmol) were added to the solution. The
reaction mixture was stirred for 2 h at room temperature. The solvent
was removed under reduced pressure, and the residue was purified
with reversed phase HPLC using a C18 column and water/acetonitrile
as solvents as described above and was lyophilized to yield 21 (30 mg,
35%).
1H NMR (400 MHz, CD3OD, δ ppm): 7.45, 7.43 (two s, 2H, AZT
H-6, FLT H-6), 6.23 (dd, J = 8.8 and 5.6 Hz, 1H, FLT H-1′), 6.09 (t, J
= 6.6 Hz, 1H, AZT H-1′), 5.24 (dd, J = 5.0 and 53.6 Hz, 1H, FLT H-
3′), 4.33−4.47 (m, 6H, AZT H-5′, AZT H-5″, AZT H-3′, FLT H-4′,
FLT H-5′, FLT H-5″), 4.22 (dd, J = 3.2 and 11.0 Hz, 1H, Glu HN-CH-
COO CH(α)), 4.07 (dd, J = 4.6 and 8.8 Hz, 1H, AZT H-4′), 2.11−
2.61 (m, 8H, AZT H-2′, AZT H-2″, FLT H-2′, FLT H-2″, and Glu
CH2CH2COO), 1.96 (s, 3H, acetyl CH3), 1.87 (s, 6H, FLT 5-CH3,
AZT 5-CH3). 13C NMR (CD3OD, 100 MHz, δ ppm): 172.17, 172.10,
171.40 (FLT COO, AZT COO, CONH), 164.93, 164.82 (FLT C-4
CO, AZT C-4 CO), 150.93, 150.73 (AZT C-2 CO, FLT C-2
CO), 136.57, 136.07 (AZT C-6, FLT C-6), 110.55 (AZT C-5, FLT
C-5), 93.69 (J = 176.6 Hz, FLT C-3′), 85.80 (FLT C-1′), 85.51 (AZT
1H NMR (400 MHz, CDCl3, δ ppm): 7.44 (d, J = 7.8 Hz, 1H, 3TC
H-6), 7.25−7.33 (m, 4H, DMTr Ar-H), 7.18−7.21 (m, 2H, DMTr Ar-
H and FLT H-6), 7.12 (d, J = 8.8 Hz, 4H, DMTr Ar-H), 6.84 (d, J =
8.8 Hz, 4H, DMTr Ar-H), 6.20 (dd, J = 5.7 and 8.6 Hz, 1H, FLT H-
1′), 6.13 (t, J = 5.2 Hz, 1H, 3TC H-1′), 5.33 (dd, J = 4.0 and 6.0 Hz,
1H, 3TC H-4′), 5.10−5.31 (m, 2H, FLT H-3′ and 3TC H-5), 4.28−
4.45 (m, 4H, 3TC H-5′, 3TC H-5″, FLT H-5′, and FLT H-5″), 4.18−
4.26 (m, 1H, FLT H-4′), 4.07 (t, J = 6.6 Hz, 1H, HN-CH(CH2)-COO
(CH(α)), 3.77 (s, 6H, DMTr-CH3O), 3.41 (dd, J = 11.9 and 5.2 Hz,
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1H, 3TC H-2″), 3.05 (dd, J = 11.9 and 5.2 Hz, 1H, 3TC H-2′), 2.10−
2.66 (m, 6H, FLT H-2′, FLT H-2″, and Glu CH2CH2COO), 1.85 (s,
3H, 5-CH3). 13C NMR (CDCl3, 100 MHz, δ ppm): 172.56 (FLT
COO), 169.13 (3TC COO), 165.55 (3TC C-4, FLT C-4 CO),
159.68 (DMTr Ar-C-OCH3), 159.16 (3TC C-2 CO), 151.51 (FLT
C-2 CO), 145.56 (3TC C-6), 136.96 (FLT C-6), 130.77, 130.72,
129.30, 129.09 128.86, 128.41, 127.35, 114.33, 113.70 (DMTr Ar-C),
111.90 (FLT C-5), 96.74 (3TC C-5), 94.11 (J = 177.9 Hz, FLT C-3′),
88.58 (3TC C-1′), 86.72 (FLT C-1′), 82.92 (J = 26.6 Hz, FLT C-4′),
82.78 (3TC C-4′), 71.72 (DMTr Ph3C-NH), 67.15 (FLT C-5′), 64.53
(3TC C-5′), 55.66 (DMTr OCH3), 52.36 (CH(α)), 37.85 (J = 21.1
Hz, FLT C-2′), 37.27 (3TC C-2′), 29.70, (Glu γ-CH2), 25.80 (Glu β-
CH2), 12.73 (FLT 5-CH3). HR-MS (ESI-TOF) (m/z):
C44H47FN6O11S, calcd 886.3008; found 887.2456 [M + H]+,
1774.5611 [2 M + H]+.
NH2-Glu(OAZT)-O(3TC(DMTr)) (25). Compound 23 (815 mg,
0.72 mmol) was dissolved in THF (10 mL). Piperidine (7.2 μL, 0.072
mmol) and 1-octanethiol (7.3 mmol, 10 mM solution in THF, 0.73
mL) were added to the reaction mixture. The mixture was stirred for
1 h at room temperature. The solvent was removed under reduced
pressure, and the residue was purified with HPLC using a C18 column
and water/acetonitrile as solvents as described above to yield 25 (325
mg, 50%).
CH2CH2COOH), 1.20−1.35 (br m, 20H, methylene protons), 0.85 (t,
J = 6.6 Hz, 3H, CH3). 13C NMR (CDCl3, 100 MHz, δ ppm): 175.92,
172.97 (FLT COO, 3TC COO), 172.13 (CONH), 165.61 (3TC C-4),
160.80 (FLT C-4 CO), 151.57 (3TC C-2 CO), 147.96 (FLT C-2
CO), 145.21 (3TC C-6), 136.97 (FLT C-6), 111.34 (FLT C-5),
97.92 (3TC C-5), 93.27 (J = 179.4 Hz, FLT C-3′), 87.99 (3TC C-1′),
86.32 (FLT C-1′), 82.19 (J = 26.6 Hz, FLT C-4′), 81.68 (3TC C-4′),
64.96 (FLT C-5′), 64.14 (3TC C-5′), 54.23 (CH(α)), 37.59 (J = 20.8
Hz, FLT C-2′), 36.96 (3TC C-2′), 36.40 (CH2COO), 31.93, 26.34,
30.34, 30.21, 30.09, 29.91, 29.76, 29.69, 26.80, 23.16 (methylene
carbons, Glu γ-CH2, Glu β-CH2), 13.88 (myristate CH3), 12.10 (FLT
5-CH3). HR-MS (ESI-TOF) (m/z): C37H55FN6O10S, calcd 794.3684;
found 795.1608 [M + H]+, 1590.3674 [2 M + H]+.
N-Acetyl-Glu(OFLT)-O(3TC(DMTr)) (28). Compound 24 (100
mg, 0.12 mmol) and acetic anhydride (2 mL, 20 mmol) were dissolved
in DMF (10 mL). DIPEA (5 mL, 37 mmol) was added to the solution.
The reaction mixture was stirred for 2 h at room temperature. The
solvent was removed under reduced pressure, and the residue was
purified with HPLC using a C18 column and water/acetonitrile as
solvents as described above to yield 28.
HR-MS (ESI-TOF) (m/z): C46H49FN6O12S, calcd 928.3113; found
929.1423 [M + H]+.
N-Acetyl-Glu(OFLT)-O3TC (29). Compound 28 was dissolved in
acetic acid (80% in water, 10 mL) and was heated at 80 °C for 30 min
to remove DMTr protection. Acetic acid was removed under reduced
pressure, and the residue was purified with reversed phase HPLC using
a C18 column and water/acetonitrile as solvents as described above and
was lyophilized to yield 29 (30 mg, 40%).
1H NMR (400 MHz, CDCl3, δ ppm): 8.04 (d, J = 7.8 Hz, 1H, 3TC
H-6), 7.02−7.47 (m, 10H, DMTr Ar-H and AZT H-6), 6.82 (d, J = 8.8
Hz, 4H, DMTr Ar-H), 6.18−6.35 (m, 1H, 3TC H-1′), 6.15 (d, J = 7.8
Hz, 1H, 3TC H-5), 6.07 (t, J = 6.5 Hz, 1H, AZT H-1′), 5.30−5.55 (m,
1H, 3TC H-4′), 4.66 (dd, J = 6.4 and 12.1 Hz, 1H, 3TC H-5′), 4.57
(dd, J = 3.2 and 12.1 Hz, 1H, 3TC H-5″), 4.26−4.46 (m, 3H, AZT H-
3′, AZT H-5′, and AZT H-5″), 4.21 (t, J = 6.6 Hz, 1H, Glu HN-CH-
COO CH(α)), 3.93−4.07 (m, 1H, AZT H-4′), 3.75 (s, 6H, DMTr
CH3O), 3.63−3.70 (m, 1H, 3TC H-2″), 3.75 (dd, J = 12.3 and 5.5 Hz,
1H, 3TC H-2′), 2.67 (t, J = 7.0 Hz, 2H, CH2COO), 2.07−2.56 (m,
4H, AZT H-2′, AZT H-2″, Glu CHCH2CH2COO), 1.85 (s, 3H, AZT
5-CH3). 13C NMR (CDCl3, 100 MHz, δ ppm): 173.20 (AZT COO),
169.95 (3TC COO), 166.25 (3TC C-4, AZT C-4 CO), 161.54
(DMTr Ar-C-OCH3), 160.03 (3TC, C-2 CO), 148.70 (AZT C-2
CO), 145.48 (3TC C-6), 138.34 (AZT C-6), 137.37, 131.25,
129.32, 128.7, 127.69, 114.00 (DMTr Ar-C), 111.86 (AZT C-5), 95.32
(3TC C-5), 88.88 (AZT C-1′), 87.15 (3TC C-1′), 84.02 (AZT C-4′),
82.93 (3TC C-4′), 67.30 (AZT C-5′), 65.16 (3TC C-5′), 62.06 (DMTr
OCH3), 55.69 (AZT C-3′), 53.03 (CH(α)), 37.48 (AZT C-2′), 37.38
(3TC C-2′), 30.15 (Glu γ-CH2), 26.53 (Glu β-CH2), 12.54 (AZT 5-
CH3). HR-MS (ESI-TOF) (m/z): C44H47N9O11S, calcd 909.3116;
found 910.4154 [M + H]+, 1821.5154 [2 M + H]+.
1H NMR (400 MHz, CD3OD, δ ppm): 8.13 (d, J = 7.8 Hz, 1H,
3TC H-6), 7.46 (s, 1H, FLT H-6), 6.28 (dd, J = 3.7 and 5.4 Hz, 1H,
FLT H-1′), 6.23 (dd, J = 5.7 and 8.8 Hz, 1H, 3TC H-1′), 6.16 (d, J =
7.8 Hz, 1H, 3TC H-5), 5.44 (dd, J = 3.1 and 4.9 Hz, 1H, 3TC H-4′),
5.25 (dd, J = 5.2 and 53.7 Hz, 1H, FLT H-3′), 4.60 (dd, J = 4.9 and
12.4 Hz, 1H, 3TC H-5″), 4.32−4.55 (m, 4H, 3TC H-5′, FLT H-5′,
FLT H-5″, FLT H-4′), 4.26 (dd, J = 3.3 and 11.4 Hz, 1H, Glu HN-CH-
COO CH(α)), 3.71 (dd, J = 5.7 and 12.6 Hz, 1H, 3TC H-2″), 3.58
(dd, J = 5.7 and 12.6 Hz, 1H, 3TC H-2′), 2.11−2.63 (m, 6H, FLT H-
2′, FLT H-2″, and Glu CH2CH2COO), 1.96 (s, 3H, acetyl CH3), 1.86
(s, 3H, FLT 5-CH3). 13C NMR (CD3OD, 100 MHz, δ ppm): 173.71,
173.65 (FLT COO, 3TC COO), 172.86 (CONH), 161.46 (3TC C-4),
161.21 (3TC C-2 CO), 152.33 (FLT C-2 CO), 146.00 (3TC C-
6), 137.69 (FLT C-6), 112.06 (FLT C-5), 95.23 (J = 176.4 Hz, FLT
C-3′), 95.14 (3TC C-5), 88.73 (3TC C-1′), 87.13 (FLT C-1′), 85.55
(3TC C-4′), 84.03 (J = 26.0 Hz, FLT C-4′), 65.69 (FLT C-5′), 64.99
(3TC C-5′), 53.03 (CH(α)), 38.64 (FLT C-2′), 38.42 (3TC C-2′),
31.04 (Glu γ-CH2), 27.55 (Glu β-CH2), 22.48 (acetyl CH3), 12.78
(FLT 5-CH3). HR-MS (ESI-TOF) (m/z): C25H31FN6O10S, calcd
626.1806; found 627.6073 [M + H]+, 1254.9359 [2 M + H]+.
N-Myristoyl-Glu(AZT)-3TC-DMTr (30). Compound 25 (100 mg,
0.12 mmol) and myristic anhydride (100 mg, 0.24 mmol) were
dissolved in DMF (10 mL). DIPEA (5 mL, 37 mmol) was added to
the solution. The mixture was stirred for 2 h at room temperature. The
solvent was removed under reduced pressure, and the residue was
purified with HPLC using a C18 column and water/acetonitrile as
solvents as described above to yield 30.
N-Myristoyl-Glu(OFLT)-O(3TC(DMTr)) (26). Compound 24 (100
mg, 0.12 mmol) and myristic anhydride (100 mg, 0.24 mmol) were
dissolved in DMF (10 mL). DIPEA (5 mL, 37 mmol) was added to
the solution. The mixture was stirred for 2 h at room temperature. The
solvent was removed under reduced pressure, and the residue was
purified with HPLC using a C18 column and water/acetonitrile as
solvents as described above to yield 26.
HR-MS (ESI-TOF) (m/z): C58H73FN6O12S, calcd 1096.4991;
found 1097.2874 [M + H]+, 2193.8167 [2 M + H]+.
N-Myristoyl-Glu(OFLT)-O3TC (27). Compound 26 was dissolved
in acetic acid (80% in water, 10 mL) and was heated at 80 °C for 30
min to remove DMTr protection. Acetic acid was removed under
reduced pressure, and the residue was purified with reversed phase
HPLC using a C18 column and water/acetonitrile as solvents as
described above and was lyophilized to yield 27 (40 mg, 45%).
1H NMR (400 MHz, CD3OD, δ ppm): 8.13 (d, J = 7.8 Hz, 1H,
3TC H-6), 7.45 (s, 1H, FLT H-6), 6.28 (dd, J = 3.6 and 5.4 Hz, 1H,
FLT H-1′), 6.22 (dd, J = 5.7 and 8.8 Hz, 1H, 3TC H-1′), 6.17 (d, J =
7.8 Hz, 1H, 3TC H-5), 5.43 (dd, J = 3.2 and 4.9 Hz, 1H, 3TC H-4′),
5.25 (dd, J = 5.2 and 53.7 Hz, 1H, FLT H-3′), 4.59 (dd, J = 4.9 and
12.4 Hz, 1H, 3TC H-5″), 4.31−4.55 (m, 4H, 3TC H-5′, FLT H-5′,
FLT H-5″, FLT H-4′), 4.27 (dd, J = 3.0 and 10.8 Hz, 1H, Glu HN-CH-
COO CH(α)), 3.54−3.65 (m, 2H, 3TC H-2′ and H-2″), 2.11−2.65
(m, 8H, FLT H-2′, FLT H-2″, myristate CH2COO, and Glu
CH2CH2COO), 1.86 (s, 3H, 5-CH3), 1.58 (t, J = 6.8 Hz, 2H,
HR-MS (ESI-TOF) (m/z): C58H73N9O12S, calcd 1119.5099; found
1120.3216 [M + H]+.
N-Myristoyl-Glu(OAZT)-O3TC (31). Acetic acid (80% in water,
10 mL) was added to compound 30. The mixture was heated at 80 °C
for 30 min to remove DMTr protection. Acetic acid was removed
under reduced pressure, and the residue was purified with reversed
phase HPLC using a C18 column and water/acetonitrile as solvents as
described above to yield 30 (35 mg, 50%). 1H NMR (400 MHz,
CDCl3, δ ppm): 7.91 (d, J = 7.8 Hz, 1H, 3TC H-6), 7.17 (s, 1H, AZT
H-6), 6.20 (dd, J = 3.7 and 5.2 Hz, 1H, 3TC H-1′), 6.14 (d, J = 7.8 Hz,
1H, 3TC H-5), 5.94 (t, J = 6.5 Hz, 1H, AZT H-1′), 5.31 (dd, J = 4.3
and 3.6 Hz, 1H, 3TC H-4′), 4.32−4.58 (m, 4H, 3TC H-5′, 3TC H-5″,
AZT H-3′), 4.21−4.29 (m, 3H, AZT H-5′, AZT H-5″, Glu HN-CH-
COO (CH(α)), 3.94 (dd, J = 3.7 and 5.1 Hz, 1H, AZT H-4′), 3.47
(dd, J = 5.2 and 12.6 Hz, 1H, 3TC H-2″), 3.13 (dd, J = 3.7 and 12.6
2683
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Hz, 1H, 3TC H-2′), 2.07−2.51 (m, 8H, AZT H-2′, AZT H-2″,
myristate CH2COO, and Glu CH2CH2COO), 1.81 (s, 3H, AZT 5-
CH3), 1.40−1.58 (m, 2H, CH2CH2COOH), 1.04−1.27 (br m, 20H,
methylene protons), 0.78 (t, J = 7.0 Hz, 3H, CH3). 13C NMR (CDCl3,
100 MHz, δ ppm): 175.92, 172.79 (AZT COO, 3TC COO), 171.70
(CONH), 164.82 (3TC C-4), 160.74 (AZT C-4 CO), 150.67 (3TC
C-2 CO), 148.15 (AZT C-2 CO), 143.44 (3TC C-6), 136.83
(AZT C-6), 111.32 (AZT C-5), 94.90 (3TC C-5), 87.07 (AZT C-1′),
86.45 (3TC C-1′), 84.35 (AZT C-4′), 81.86 (3TC C-4′), 64.38 (AZT
C-5′), 63.75 (3TC C-5′), 60.80 (AZT C-3′), 51.54 (CH(α)), 38.22
(AZT C-2′), 37.05 (CH2COO), 36.24 (3TC C-2′), 32.11, 30.11, 29.99,
29.83, 29.55, 29.47, 29.40, 25.80 (methylene carbons), 26.77 (Glu γ-
CH2), 22.85 (Glu β-CH2), 14.19 (myristate CH3), 12.40 (AZT 5-
CH3). HR-MS (ESI-TOF) (m/z): C37H55N9O10S, calcd 817.3793;
found 818.2535 [M + H]+, 1636.0917 [2 M + H]+.
N-Acetyl-Glu(OAZT)-O3TC-DMTr (32). To compound 25 (110
mg, 0.12 mmol) in DMF (10 mL) was added DIPEA (5 mL, 37
mmol) and acetic anhydride (2 mL, 20 mmol). The reaction mixture
was stirred for 2 h at room temperature. The solvent was removed
under reduced pressure, and the residue was purified with HPLC using
a C18 column and water/acetonitrile as solvents as described above to
yield 32.
CH3). 13C NMR (CD3CN, 100 MHz, δ ppm): 174.50, 173.95, 172.95
(3TC COO, FLT COO, AZT COO), 166.38 (3TC C-4), 161.62
(AZT C-4 CO and FLT C-4 CO), 152.38, 152.27, 148.86 (AZT
C-2, FLT C-2, 3TC C-2 CO), 145.83, 138.25, 137.50 (FLT C-6,
AZT C-6, 3TC C-6), 112.22, 112.04 (FLT C-5, AZT C-5), 95.43 (J =
176.5 Hz, FLT C-3′), 96.42 (3TC C-5), 88.78 (3TC C-1′), 86.85
(FLT C-1′), 86.81 (AZT C-1′), 86.81 (3TC C-4′), 84.11 (J = 26.0 Hz,
FLT C-4′), 83.16 (AZT C-4′), 65.93, 65.05, 64.99 (3TC C-5′, FLT C-
5′, AZT C-5′), 62.20 (AZT C-3′), 52.64 (CH(α)), 38.79 (3TC C-2′),
38.48 (J = 19.0 Hz, FLT C-2′), 37.76 (AZT C-2′), 31.86, 31.06
(succinate two CH2 groups), 30.24 (Glu γ-CH2), 27.71 (Glu β-CH2),
12.89, 12.75 (AZT 5-CH3 and FLT 5-CH3). HR-MS (ESI-TOF) (m/
z): C37H44FN11O15S, calcd 933.2723; found 934.2864 [M + H]+,
1867.6791 [2 M + H]+.
(−)-5′-O-(t-Butyldimethylsilyl)-5-fluoro-2′,3′-dideoxy-3′-
thiacytidine (5′-TBDMS FTC, 37). FTC (500 mg, 2.18 mmol), tert-
butyldimethylsilyl chloride (1 g, 6.54 mmol), and imidazole (440 mg,
6.54 mmol) were dissolved in dry DMF (10 mL), and the reaction
mixture was stirred for 18 h at room temperature. The solvent was
concentrated under reduced pressure, and the residue was purified
with silica gel column chromatography using dichloromethane and
methanol (0−5%) as eluents to yield 37 (700 mg, 90%).
1H NMR (400 MHz, CDCl3, δ ppm): 8.50−9.20 (br s, 2H, NH2),
8.17 (d, J = 6.4 Hz, 1H, H-6), 6.25 (dd, J = 5.2 and 2.4 Hz, 1H, H-1′),
5.17−5.21 (t, J = 2.4 Hz, 1H, H-4′), 4.15 (dd, J = 11.8 and 2.4 Hz, 1H,
H-5″), 3.90 (dd, J = 11.8 and 2.4 Hz, 1H, H-5′), 3.47 (dd, J = 12.4 and
5.2 Hz, 1H, H-2″), 3.14 (dd, J = 12.4 and 2.4 Hz, 1H, H-2′), 0.90 (s,
9H, (CH3)3C), 0.10 (s, 6H, CH3Si). 13C NMR (CDCl3, 100 MHz, δ
ppm): 158.24 (J = 14.5 Hz, C-4), 153.73 (C-2 CO), 136.32 (J =
240.7 Hz, C-5), 126.04 (J = 32.7 Hz, C-6), 88.22 (C-1′), 87.30 (C-4′),
63.66 (C-5′), 39.11 (C-2′), 25.85, 18.56 (CH3)3C-Si), −5.47, −5.49
(CH3-Si). HR-MS (ESI-TOF) (m/z): C14H25FN3O3SSi, calcd
361.1292; found 362.4160 [M + H]+, 723.8098 [2 M + H]+.
(−)-5′-O-(t-Butyldimethylsilyl)-N4-(4,4′-dimethoxytrityl)-5-
fluoro-2′,3′-dideoxy-3′-thiacytidine (5′-TBDMS-N4-DMTr-FTC,
38). Compound 37 (600 mg, 1.75 mmol) was dissolved in dry
pyridine (10 mL). A solution of DMTr-Cl (700 mg, 1.2 equiv) in 10
mL of pyridine was added to the reaction mixture dropwise at 0 °C.
The reaction mixture was stirred for 30 min. The temperature was
raised to room temperature, and stirring was continued for overnight.
The reaction mixture was neutralized with saturated sodium
bicarbonate solution (100 mL) and was extracted with dichloro-
methane (3 × 100 mL). The organic layer was separated and
concentrated under reduced pressure. The residue was purified with
silica gel column chromatography using dichloromethane and
methanol (0−1%) as eluents to yield 38 (1.0 g, 86%).
HR-MS (ESI-TOF) (m/z): C46H49N9O12S, calcd 951.3221; found
952.2062 [M + H]+, 1903.9995 [2 M + H]+.
N-Acetyl-Glu(OAZT)-O3TC (33). Acetic acid (80% in water, 10
mL) was added to compound 32. The reaction mixture was heated at
80 °C to remove DMTr protection. Acetic acid was removed under
reduced pressure, and the residue was purified with reversed phase
HPLC using a C18 column and water/acetonitrile as solvents as
described above to yield 33 (35 mg, 55%).
1H NMR (400 MHz, CD3OD, δ ppm): 8.02 (d, J = 7.9 Hz, 1H,
3TC H-6), 7.33 (s, 1H, FLT H-6), 6.20−6.30 (m, 1H, 3TC H-1′),
6.03−6.16 (m, 2H, AZT H-1′, 3TC H-5), 5.42 (t, J = 3.7 Hz, 1H, 3TC
H-4′), 4.43−4.62 (m, 3H, 3TC H-5″, 3TC H-5′, AZT H-3′), 4.25−4.35
(m, 3H, AZT H-5′, AZT H-5″, Glu HN-CH-COO (CH(α)), 4.02 (dd,
J = 4.5 and 9.1 Hz, 1H, AZT H-4′), 3.57 (dd, J = 5.5 and 12.4 Hz, 1H,
3TC H-2″), 3.24 (dd, J = 3.4 and 12.4 Hz, 1H, 3TC H-2′), 2.11−2.53
(m, 6H, AZT H-2′, AZT H-2″, Glu CH2CH2COO), 1.96 (s, 3H, acetyl
CH3), 1.87 (s, 3H, 5-CH3). 13C NMR (CD3OD, 100 MHz, δ ppm):
173.22, 172.50 (AZT COO, 3TC COO), 172.24 (CONH), 165.41
(3TC C-4), 161.32 (AZT C-4 CO), 151.49 (3TC C-2 CO),
148.53 (AZT C-2 CO), 144.96 (3TC C-6), 137.32 (AZT C-6),
111.56 (AZT C-5), 95.03 (3TC C-5), 87.92 (AZT C-1′), 86.03 (3TC
C-1′), 84.92 (AZT C-4′), 82.40 (3TC C-4′), 65.19 (AZT C-5′), 64.42
(3TC C-5′), 61.36 (AZT C-3′), 52.36 (CH(α)), 38.29 (AZT C-2′),
37.36 (3TC C-2′), 30.65 (Glu γ-CH2), 27.06 (Glu β-CH2), 22.60
(acetyl CH3), 12.60 (AZT 5-CH3). HR-MS (ESI-TOF) (m/z):
C25H31N9O10S, calcd 649.1915; found 650.1956 [M + H]+, 692.1986
[M + Na]+.
1H NMR (400 MHz, CDCl3, δ ppm): 8.73−9.40 (br s, 1H, NH),
8.49 (d, J = 6.0, 1H, H-6), 7.27 (d, J = 3.6 Hz, 5H, DMTr protons),
7.17 (d, J = 8.7 Hz, 4H, DMTr protons), 6.83 (d, J = 8.7 Hz, 4H,
DMTr protons), 6.26−6.29 (br s, 1H, H-1′), 5.23−5.25 (br s, 1H, H-
4′), 4.25 (dd, J = 10.2 and 1.8 Hz, 1H, H-5″), 3.94 (d, J = 10.2 Hz, 1H,
H-5′), 3.80 (s, 6H, DMTr-OCH3), 3.53 (dd, J = 12.2 Hz, J = 4.6 Hz,
1H, H-2″), 3.24 (d, J = 12.2 Hz, 1H, H-2′), 0.94 (s, 9H, (CH3)3C),
0.15 (s, 6H, CH3Si). 13C NMR (CDCl3, 100 MHz, δ ppm): 158.63
(C-4), 155.85 (C-2 CO), 151.47, 147.33 (DMTr-C), 139.46 (C-5),
129.14, 127.86, 127.77 (DMTr-C), 127.09 (C-6), 113.17 (DMTr-C),
89.14 (C-1′), 87.26 (C-4′), 81.44 (DMTr-C-NH), 63.22 (C-5′), 55.26
(DMTr-OCH3), 39.59 (C-2′), 25.86 (CH3-C), 18.66 ((CH3)3C-Si),
−5.46, −5.50 (CH3-Si). HR-MS (ESI-TOF) (m/z): C35H43FN3O5SSi,
calcd 663.2598; found 663.9596 [M + H]+, 765.0250 [M + TEA]+.
(−)-N4-(4,4′-Dimethoxytrityl)-5-fluoro-2′,3′-dideoxy-3′-thia-
cytidine (DMTr-FTC, 39). Compound 38 (1 g, 1.55 mmol) was
dissolved in 1 M solution of tetrabutylammonium fluoride (4.5 mL,
1M, 3 equiv) and stirred for 3 h. The reaction mixture was
concentrated under reduced pressure, and the residue was purified
with silica gel column chromatography using dichloromethane (2%
triethylamine) and methanol (2%) as eluents to yield 39 (750 mg,
90%).
FLT-Succ-Glu(OAZT)-O3TC (34). Compound 25 (110 mg, 0.12
mmol), DIPEA (5 mL, 37 mmol), and FLT−succinate (100 mg, 0.30
mmol) were dissolved in DMF (10 mL). The reaction mixture was
stirred for 2 h at room temperature. The solvent was removed under
reduced pressure. Acetic acid (80% in water, 10 mL) was added to the
residue. The reaction mixture was heated at 80 °C to remove DMTr
protection. Acetic acid was removed under reduced pressure, and the
residue was purified with HPLC using a C18 column and water/
acetonitrile as a solvent as described above to yield 34 (55 mg, 50%).
1H NMR (400 MHz, CD3CN, δ ppm): δ 10.7 (s, 1H, NH), 9.59 (s,
2H, NH), 7.97 (d, J = 7.6 Hz, 1H, 3TC H-6), 7.77 (s, 1H, NH), 7.36
(s, 1H, FLT H-6), 7.29 (s, 1H, AZT H-6), 6.96 (d, 1H, NH), 6.17−
6.26 (m, 2H, 3TC H-1′ and FLT H-1′), 6.14 (d, J = 7.6 Hz, 1H, 3TC
H-5), 6.06 (t, J = 6.3 Hz, 1H, AZT H-1′), 5.40 (t, J = 3.6 Hz, 1H, 3TC
H-4′), 5.24 (dd, J = 4.8 and 53.7 Hz, 1H, FLT H-3′), 4.16−4.59 (m,
9H, 3TC H-5′, 3TC H-5″, FLT H-5′, FLT H-5″, FLT H-4′, AZT H-5′,
AZT H-5″, AZT H-3′, Glu HN-CH-COO (CH(α)), 4.00 (dd, J = 6.6
Hz, 1H, AZT H-4′), 3.56 (dd, J = 11.9 and 5.2 Hz, 1H, 3TC H-2″),
3.24 (dd, J = 11.9 and 5.2 Hz, 1H, 3TC H-2′), 2.05−2.66 (m, 12H,
succinate OOCCH2CH2CON, FLT H-2′, FLT H-2″, AZT H-2′, AZT
H-2″, and Glu CH2CH2COO), 1.83 (s, 6H, FLT 5-CH3 and AZT 5-
1H NMR (400 MHz, CDCl3, δ ppm): 7.83−7.96 (m, 1H, H-6),
7.10−7.35 (m, 9H, DMTr protons), 6.80 (s, 4H, DMTr protons),
2684
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6.42−6.46 (br s, 1H, OH), 6.15−6.19 (br s, 1H, H-1′), 5.14−5.18 (br
s, 1H, H-4′), 4.06−4.20 (m, 1H, H-5″), 3.93 (d, J = 12.5 Hz, 1H, H-5′),
3.78 (s, 6H, DMTr-OCH3), 3.34−3.50 (m, 1H, H-2″), 2.98−3.15 (m,
1H, H-2′). HR-MS (ESI-TOF) (m/z): C29H29FN3O5S, calcd
549.1734; found 550.5078 [M + H]+, 651.6923 [M+TEA]+,
1122.0138 [2 M + Na]+.
N-Myristoyl-Glu(OtBu)-OH (41). DIPEA (390 mg, 3 mmol) was
added to the mixture of gamma-tert-butoxy-glutamic acid (40, 203 mg,
1 mmol) and myristic anhydride (525 mg, 1.2 mmol) in dry DMF (10
mL) at room temperature. The reaction mixture was stirred for 4 h.
After completion of the reaction, the solvent was removed under
reduced pressure. Water (40 mL) was added, and the mixture was
extracted with dichloromethane (3 × 10 mL). The organic layer was
dried over MgSO4 (anhydrous) and concentrated under reduced
pressure. The crude residue was purified on silica gel column
chromatography eluted with DCM and methanol (0−10%) to give 41
(375 mg, 91%).
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude products were carried forward for the next
reaction.
44: HR-MS (ESI-TOF) (m/z): C58H73FN6O12S, calcd 1096.4990;
found 1097.2520 [M + 1]+.
45: HR-MS (ESI-TOF) (m/z): C58H72F2N6O12S, calcd 1114.4897;
found 1115.2175 [M + 1]+.
General Method for the Synthesis of N-Myristoyl-Glu-
(O3TC)-OFLT (46) and N-Myristoyl-Glu(OFTC)-OFLT (47).
Compounds 44 and 45 were treated with a mixture of acetic acid/
TFA (98:2 v/v, 20 mL). The reaction mixture was stirred for 1 h at
room temperature. After completion of the reaction, acetic acid/TFA
was removed under reduced pressure. The crude products were
dissolved in DCM and purified on a silica gel column eluted with
DCM and methanol (0−10%) to afford 46 and 47, respectively.
1
46: Overall yield 174 mg, 64%. H NMR (CDCl3, 500 MHz, δ
ppm): 8.49 (s, 1H, FLT-NH), 7.99 (s, 1H, 3TC-C6), 7.18 (s, 1H, FLT
H-6), 6.67−6.62 (m, 1H, FLT H-1′), 6.18 (s, 1H, 3TC H-1′), 6.08−6.0
(br m, 1H, 3TC H-4′), 5.31 (s, 1H, FLT H-4′), 5.20 (d, J = 57.7 Hz,
1H, FLT H-3′), 4.58−4.28 (m, 5H, 3TC H-5′, FLT H-5′ and Glu α
−CH-COOFLT), 3.49 (br m, 1H, 3TC H-2′), 3.33 (br s, 1H, 3TC H-
2″), 2.58−2.31 (br m, 3H, FLT H-2′, Glu γ-CH2), 2.19−1.16 (br m,
2H, Glu β −CH2CH2-COOFTC), 1.94 (br s, 1H, FLT H-2″), 1.83 (s,
3H, FLT 5-CH3), 1.53 (t, J = 7.2 Hz, 2H, −CH2-CONH−), 1.22 (br s,
22H, methylene protons −(CH2)11), 0.85 (t, J = 8 Hz, 3H, −CH3).
13C NMR (CDCl3, 100 MHz, δ ppm): 174.96, 172.43 (COO3TC,
COOFLT), 171.59 (CONH), 164.75, 163.65 (FLT C-4), 160.45
(3TC C-4), 150.66 (FLT C-2), 147.47 (3TC C-2), 143.51 (3TC C-6),
136.77 (FLT C-6), 114.45 (3TC C-5), 111.56 (FLT C-5), 94.73 (FLT
C-3′), 87.28 (3TC C-1′), 86.72 (FLT C-1′), 84.94 (3TC C-4′), 82.30
(d, J = 25.0 Hz, FLT C-4′), 64.29 (FLT C-5′), 63.80 (3TC C-5′), 51.81
(Glu α NH-CH−), 37.23 (3TC C-2′), 36.32 (−CH2−CONH−),
32.06 (FLT C-2′), 29.84, 29.82, 29.80, 29.66, 29.50, 29.40, 26.94,
25.82, 22.83 (methylene carbons, Glu β-CH2 and Glu γ-CH2), 14.27
(CH3), 12.43 (FLT 5-CH3). HR-MS (ESI-TOF) (m/z):
C37H55FN6O10S, calcd 794.3680; found 795.1980 [M + 1]+.
1H NMR (CDCl3, 400 MHz, δ ppm): 7.98 (s, 1H, COOH), 6.51
(d, J = 8.0 Hz, 1H, NH), 4.52 (q, J = 4.0 Hz, 1H, −CHCOOH), 2.33−
2.16 (m, 4H, −CH2CH2COOC−), 1.58 (t, J = 4.0 Hz,
−CH2CONH−), 1.39 (s, 9H, −C(CH3)3), 1.20 (s, 22H,
−(CH2)11), 0.83 (t, J = 8.0 Hz, 3H, −CH3). 13C NMR (CDCl3, 100
MHz, δ ppm): 174.01 (COOH), 172.75 (COOC), 163.06 (CONH),
80.89 (COOC−), 51.97 (NHCH−), 36.80 (−CH2COOC), 36.67
(−CH2CONH−), 32.05, 31.78, 29.81, 29.78, 29.62, 29.48, 29.39,
28.16, 27.39, 25.73, 22.82 ((CH3)3, methylene −CH2−), 14.26 (CH3).
HR-MS (ESI-TOF) (m/z) (negative mode): C23H43NO5, calcd
413.3140; found 412.280 [M − 1]−.
N-Myristoyl-Glu(OtBu)-OFLT (42). DIPEA (390 mg, 3 mmol)
was added to the mixture of 41 (413 mg, 1 mmol), FLT (244 mg, 1
mmol), and HBTU (1.13 g, 3 mmol) in dry DMF (30 mL). The
mixture was stirred at room temperature for 6 h. After completion of
reaction, the solvent was removed under reduced pressure. Water (40
mL) was added and extracted with dichloromethane (3 × 20 mL). The
organic layer was dried over Na2SO4 (anhydrous) and concentrated
under reduced pressure. The crude residue was purified on silica gel
column chromatography eluted with dichloromethane and methanol
(0−10%) to give 42 (581 mg, 91%).
1
47: Overall yield 161 mg, 58%. H NMR (CDCl3 + CD3OD, 500
MHz, δ ppm): 8.08 (d, J = 8.1 Hz, 1H, FTC H-6), 7.25 (s, 1H, FLT
H-6), 6.75 (dd, J = 15.4 Hz, J = 7.7 Hz, 1H, FLT H-1′), 6.24 (br s, 1 H,
FTC H-1′), 6.21−6.09 (m, 1H, FTC H-4′), 5.34 (t, J = 2.9 Hz, 1 H,
FLT H-4′), 5.28−5.13 (m, 1H, FLT H-3′), 4.65−4.36 (m, 5H, FTC H-
5′, FLT H-5′ and Glu α-CH-COOFLT), 3.55 (dd, J = 12 Hz, J = 5.2
Hz, 1H, FTC H-2′), 3.26−3.22 (m, 1H, FTC H-2″), 2.35−2.41 (br m,
6H, FLT H-2′, Glu γ-CH2, myristate CH2CH2COO), 2.10−1.96 (m,
2H, Glu β −CH2CH2COOFTC), 1.86 (s, 3H, FLT 5-CH3), 1.57 (t, J
= 7.2 Hz, 2H, −CH2CONH−), 1.22 (s, 20H, methylene protons
−(CH2)10), 0.85 (t, J = 8.0 Hz, 3H, −CH3). 13C NMR (CDCl3, 100
MHz, δ ppm): 174.38, 172.41 (COOFTC, COOFLT), 171.79
(CONH), 164.22 (FLT C-4), 156.46 (FTC C-4), 150.56 (FLT C-
2), 150.49 (FTC C-2), 136.36 (FLT C-6), 136.08 (FTC C-6), 126.85
(d, J = 33.0 Hz, FTC C-5), 111.66 (FLT C-5), 93.12 (d, J = 179.65
Hz, FLT C3′), 87.37 (FTC C-1′), 86.95 (FLT C-1′), 86.13 (FTC C-
4′), 82.18 (d, J = 27.3 Hz, FLT C-4′), 64.21 (FLT C-5′), 63.39 (FTC
C-5′), 51.59 (Glu NHCH−), 39.06 (FTC C2′), 36.41
(−CH2CONH−), 32.03 (FLT C-2′), 29.94, 29.80, 29.76, 29.63,
29.47, 29.40, 25.71, 22.80 (methylene −CH2−), 27.06 (Glu β −
CH2CCOOFTC), 14.23 (−CH3), 12.49 (FLT 5-CH3). HR-MS (ESI-
TOF) (m/z): C37H54F2N6O10S, calcd 812.3590; found 813.1642 [M +
1]+.
Anti-HIV Assays. The anti-HIV activity of the compounds was
evaluated according to the previously reported procedure17,21−23 in
single-round (MAGI) infection assays using X4 (IIIB) and R5 (BaL)
HIV-1 and P4R5 cells expressing CD4 and coreceptors. In summary,
P4R5MAGI cells were cultured at a density of 1.2 × 104 cells/well in a
96-well plate approximately 18 h prior to infection. Cells were
incubated for 2 h at 37 °C with purified, cell-free HIV-1 laboratory
strains IIIB or BaL (Advanced Biotechnologies, Inc., Columbia, MD)
in the absence or presence of each agent. After 2 h, cells were washed,
cultured for an additional 46 h, and subsequently assayed for HIV-1
infection using the Galacto-Star β-Galactosidase Reporter Gene Assay
HR-MS (ESI-TOF) (m/z): C33H54FN3O8, calcd 639.3895; found
640.2220 [M + 1]+.
N-Myristoyl-Glu(OH)-OFLT (43). Compound 42 (500 mg, 0.86
mmol) was treated with TFA/DCM (95%, 20 mL). The reaction
mixture was stirred for 2 h at room temperature. After completion of
the reaction, TFA/DCM was removed under reduced pressure. The
crude product was dissolved in DCM and purified on silica gel column
eluted with DCM and methanol (0−10%) to give 43 (355 mg, 78%).
1H NMR (CDCl3, 500 MHz, δ ppm): 10.37 (s, 1H, COOH), 8.05
(br s, 1H, CONH), 7.41 (s, 1H, FLT C-6), 6.46−6.28 (m, 1 H, FLT
H-1′), 5.22 (d, J = 53.4 Hz, 1H, FLT H-3′), 4.65−4.25 (m, 4H, FLT
H-4′, FLT H-5′, −CHCOOH), 2.84−2.56 (m, 1H, FLT H-2′), 2.49−
2.40 (m, 3H, Glu γ CH2-COO and FLT H-2″), 2.27−2.16 (m, 2H, Glu
β −CH2-CH2-COO), 1.86 (s, 3H, FLT 5-CH3), 1.63−1.56 (m,
−CH2CONH−), 1.24 (br s, 22H, −(CH2)11), 0.87 (t, J = 8.0 Hz, 3H,
−CH3). 13C NMR (CDCl3, 125 MHz, δ ppm): 177.29 (COOH),
174.66 (COOC), 171.65 (CONH), 165.84 (FLT C-4), 149.81 (FLT
C-2), 136.87 (FLT C-6), 111.22 (FLT C-5), 93.95 (d, J = 177.5 Hz,
FLT C-3′), 85.98 (FLT C-1′), 82.32 (FLT C-4′), 82.10, 64.86 (FLT C-
5′), 51.66 (NHCH−), 38.21, 36.28 (FLT C-2′), 31.90 (Glu γ-CH2),
29.85, 29.67, 29.63, 29.60, 29.46, 29.33, 29.28, 29.21, 27.53, 25.59,
22.67 (methylene −CH2− and Glu β-CH2), 14.10 (CH3), 12.20 (FLT
C5 CH3). HR-MS (ESI-TOF) (m/z): C29H46FN3O8, calcd 583.32;
found 584.186 [M + 1]+.
General Method for the Synthesis of N-Myristoyl-Glu(O3TC-
DMTr)-OFLT (44) and N-Myristoyl-Glu(OFTC-DMTr)-OFLT (45).
DIPEA (260 mg, 2 mmol) was added to the mixture of 3 (200 mg,
0.34 mmol), 3TC-DMTr/FTC-DMTr (0.34 mmol), and HBTU (758
mg, 2 mmol) in dry DMF (40 mL) at room temperature. The mixture
was stirred for 6 h. After completion of the reaction, the solvent was
removed under reduced pressure. Water (40 mL) was added, and the
mixture was extracted with DCM (3 × 20 mL). The organic layer was
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Journal of Medicinal Chemistry
Article
System for Mammalian Cells (Applied Biosystems, Bedford, MA).
Dextran sulfate, a polyanionic HIV entry inhibitor,27,28 was used as
assay control. Reductions in infection were calculated as a percentage
relative to the level of infection in the absence of agents, and 50%
inhibitory concentrations (EC50) were derived from regression
analysis. Each compound concentration was tested in triplicate wells.
Cell toxicity was evaluated using the same experimental design but
without the addition of virus. The impact of compounds on cell
viability was assessed using an MTT (reduction of tetrazolium salts)
assay (Invitrogen, Carlsbad, CA).
ABBREVIATIONS USED
■
AZT, 3′-azido-2′,3′-dideoxythymidine; d4T, 2′,3′-didehydro-
2′,3′-dideoxythymidine; FLT, 3′-fluoro-2′,3′-dideoxythymidine;
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylfor-
mamide; Fmoc, fluorenylmethyloxycarbonyl; FTC, (−)-5-
fluoro-2′,3′-dideoxy-3′-thiacytidine; HAART, highly active anti-
retroviral therapy; HBTU, 1,1,3,3-tetramethyluronium hexa-
fluorophosphate; HBV, hepatitis B virus; HOBt, hydroxybenzo-
triazole; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; NMM, N-methylmorpholine; Mtt, 4-
methyltrityl; NRTI, nucleoside reverse transcriptase inhibitors;
3TC, (−)-2′,3′-dideoxy-3′-thiacytidine; TFA, trifluoroacetic acid
For the assessment of compounds against wild-type (WT; R5;
clones = 94US3393IN [B subtype] and 98USMSC5016 [C subtype])
and drug resistant (clones = 4755−5 and A17) HIV-1 clinical isolates,
PHA-P stimulated cells from at least two normal donors were pooled,
diluted in fresh media, and plated in the interior wells of a 96-well
round-bottom microplate. Pooling PBMCs from more than one donor
is used to minimize the variability observed between individual donors,
which results from quantitative and qualitative differences in HIV
infection and overall response to the PHA and IL-2 of primary
lymphocyte populations. Each plate contains virus/cell control wells
(cells + virus), experimental wells (drug + cells + virus), and
compound control wells (drug + media, no cells, necessary for MTS
monitoring of cytotoxicity). Test drug dilutions were prepared in
microtiter tubes, and each concentration was placed in appropriate
wells. Following addition of the drug dilutions to the PBMCs, a
predetermined dilution of virus stock was then placed in each test well
(final MOI ≅ 0.1). Because HIV-1 is not cytopathic to PBMCs, the
same assay plate can be used for both antiviral efficacy and cytotoxicity
measurements. Compounds were incubated with virus and cells in a
96-well format for 6 h. The cells were then washed by removing 75%
of the medium (150 μL) and replacing with 150 μL of fresh (no drug)
medium. The plates were then centrifuged (∼200g) for 10 min, after
which 150 μL of medium was removed and an additional 150 μL of
fresh medium was added to each well and further incubated for 6 days
or until peak reverse transcriptase (RT) activity was detected. A
microtiter plate-based RT reaction was utilized.26 Incorporated
radioactivity (counts per minute, CPM) was quantified using standard
liquid scintillation techniques. Compound IC50 (50%, inhibition of
virus replication) was calculated using statistical software and
regression analysis.
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ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC purification method, characterization of compounds,
analytical HPLC methods, 1H NMR, 13C NMR, and/or
analytical HPLC of compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For K.P.: phone, +1-401-874-4471; fax, +1-401-874-5787; E-
mail, kparang@uri.edu. For G.F.D.: phone, +1-757-446-8908;
fax, +1-757-446-8998; E-mail, DoncelGF@evms.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this subproject (MSA-03-367) was provided by
CONRAD, Eastern Virginia Medical School, under a
Cooperative Agreement (GPO-A-8-00-08-00005-00) with the
United States Agency for International Development (USAID).
The views expressed by the authors do not necessarily reflect
the views of USAID or CONRAD.
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