Synthesis and Evaluation of Anti-HIV Activity of Mono- andDi-Substituted Phosphonamidate Conjugates of TenofovirAaminat Qureshi 1, Louise A. Ouattara 2, Naglaa Salem El-Sayed 3,4, Amita Verma 3,5 , Gustavo F. Doncel 2,Muhammad Iqbal Choudhary 1,6,7,8,*, Hina Siddiqui 1,* and Keykavous Parang 3,*
1 H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,University of Karachi, Karachi 75270, Pakistan; [email protected]
2 CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School,Norfolk, VA 23507, USA; [email protected] (L.A.O.); [email protected] (G.F.D.)
3 Center for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences,Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus,Irvine, CA 92618, USA; [email protected] (N.S.E.-S.); [email protected] (A.V.)
4 Cellulose and Paper Department, National Research Center, Dokki, Cairo 12622, Egypt5 Bioorganic and Medicinal Chemistry Research Laboratory, Department of Pharmaceutical Sciences,
Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj 211007, India6 Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi 75270, Pakistan7 Department of Biochemistry, King Abdul Aziz University, Jeddah 21452, Saudi Arabia8 Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Komplek Campus C,
Abstract: The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphory-lation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to thepresence of ionizable negatively-charged groups. To circumvent these limitations, several prodrugapproaches have been proposed. Herein, we hypothesized that the conjugation or combination ofthe lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIVactivity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated withthe amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty esterhydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized.The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. Inaddition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIVactivity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV (1) was usedas a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, thedisubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate (69), pentanolyl esteralanine phosphonamidate with phenol oleate (62), and butanolyl ester alanine phosphonamidatewith naphthol oleate (87) ester conjugates of TFV were more potent than parent drug TFV with 79.0%,76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates oftenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF andTFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL andshowed comparable activity to TAF (97–99% inhibition) at 10–100 ng/mL but was more potent thanTAF when compared at molar concentration.
Acquired immunodeficiency syndrome (AIDS) caused by the human immune defi-ciency virus (HIV) is still a major global health challenge [1]. According to the Joint UnitedNations Programmes on HIV/AIDS statistics, in the year 2021, about 28.2 million peoplewere accessing antiretroviral therapy. Moreover, millions of people have died from thedisease. Despite the success of highly active antiretroviral therapies, the rapid emergenceof drug-resistant mutants has sharply limited the clinical applications of existing anti-HIVdrugs, requiring an active pipeline of new antiretrovirals [2].
The US Food and Drug Administration (FDA) approved several drugs to treat HIVinfection [3]. An important limitation of antiviral drugs as therapeutic agents is, in manycases, their low oral bioavailability (less than 20%) and poor transport into cells, which inthe case of nucleotide-based drugs is attributed to their ionizable groups [4].
A prodrug is a compound that undergoes a transformation within the body beforeeliciting its therapeutic action. The prodrug approach is extensively used to increase drugbioavailability, as well as drug targeting after oral administration [5]. This strategy is basedon chemically modifying an active substance by attaching pro-moieties, which ideallyovercome the biochemical and physical barriers associated with the parent compound.Limited oral bioavailability is usually attributed to poor membrane permeability, lowaqueous solubility (in the gastrointestinal fluids), or extensive first-pass metabolism [6].
Several prodrug strategies have been applied to circumvent this problem in antiviraldrugs (Figure 1). Tenofovir (1) (TFV) is a nucleotide analog of deoxyadenosine monophos-phate. Sofosbuvir (2) is a masked phosphorylated nucleotide drug that has cell permeabilityand oral bioavailability against the hepatitis C virus [7]. Valacyclovir (VACV) (3) (Figure 1),the valine ester prodrug of acyclovir (ACV) targeting intestinal oligopeptide transporter1 (PepT1), has proven to be a safe and effective drug [8–11]. Valganciclovir (4), an acyclicguanosine analog, was first used intravenously to treat CMV infection in AIDS patients. Tocircumvent the inconvenience and risks associated with frequent ganciclovir intravenousadministration and its low bioavailability, an oral formulation was further developed [12].Famciclovir (5) (Figure 1) is a prodrug of penciclovir containing acetyl diester and 6-deoxy promoieties. The prodrug is efficiently converted to the parent drug via enzymaticdeacetylation and oxidation after oral administration [13]. Fosamprenavir (6) (Figure 1) is aprodrug of amprenavir and has the advantage of increased water solubility and improvedoral bioavailability. This allows for a reduction in the daily dose [14].
Adefovir is an acyclic analog of deoxyadenosine. It displays low oral bioavailabilitycompared to other acyclic nucleoside phosphonate analogs due to the limited intestinalpermeability of the anionic phosphonate moiety. Hence, various prodrugs of adefovir,such as adefovir dipivoxil (7) (Figure 1) were designed to mask the charged phosphonategroups and improve the oral absorption of adefovir [15,16]. Other pronucleotides includeβ-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine nucleotide prodrug (PSI-7977) that has beeninvestigated for the treatment of hepatitis C Virus [17]. Remdesivir is also among the firstexamples of a phosphoramidate prodrug aimed at delivering a nucleoside monophosphateinto lung cells to efficiently generate the nucleoside triphosphate inhibitor of viral RNApolymerases [18].
Interestingly, many water-soluble compounds have been shown to move well acrosscell membranes, utilizing specialized carrier-mediated transport mechanisms [19]. Thesemembrane transporters play a key role in determining the exposure of the cells or organ-isms to a variety of solutes, including nutrients and cellular byproducts, as well as drugmolecules. Efforts have been made to improve drug bioavailability by conjugating differentpro-moieties, targeting various active transportation systems present in the small intestine.
Molecules 2022, 27, 4447 3 of 33Molecules 2022, 27, x FOR PEER REVIEW 3 of 34
POH
OHOO
N
NN
N
NH2
Tenofovir (TFV)(1)
HN
N N
N
O
O
NH2O
O
NH2
Valacyclovir(3)
HN
N N
N
O
O
NH2O
O
NH2
Valgancyclovir
OH
(4)
HN
N N
N O
O
O
O
O
NH2
Famciclovir(5)
O N SO
ONH2P
OHHO
O
OO
O
Fosamprenavir
HN
(6)
Adefovir dipivoxil
(7)
PO
O
N
NN
N
NH2
O O
O
O
O
O
Tenofovir disoproxil Fumerate (TDF)
(8)
PO
OOO
N
NN
N
NH2
OO O
O
O
O
Tenofovir alafenamide (TAF)
(9)
NH
OO
PO O
O
N
NN
N
NH2
Figure 1. Chemical Structures of Antiviral Prodrugs.
Tenofovir (1) (TFV) (Figure 1) has activity against HIV-1, HIV-2, and hepatitis B vi-ruses (HBV) [20]. Tenofovir disoproxil fumarate (8) (TDF, Viread®) (Figure 1) is an ester prodrug of TFV that is hydrolyzed to TFV intracellularly, and phosphorylated to the ac-tive metabolite, TFV diphosphate. TDF is used in combination with other antiviral medi-cations, such as 2’,3’-dideoxy-5-fluoro-3’-thiacytidine (emtricitabine, FTC). Resistance to TDF is conferred by the reverse transcriptase (RT) K65R and/or K70E mutations. Tenofo-vir alafenamide fumarate (TAF) (9) (Figure 1) is another prodrug of tenofovir. TAF has higher antiviral activity and distribution in the lymphatic system with fewer side effects, such as impaired kidney function [21–24]. All prodrugs are safe and effective and are used as part of combination therapy or for prevention [25].
With the constant emergence of HIV mutants of clinical relevance and the need to reduce the number of ARVs for chronic treatment [26], it is logical to develop new long-acting and more potent nucleoside conjugates that display broad-spectrum activity against drug-resistant HIV. We previously demonstrated that several fatty acids, such as 2-methoxydodecanoic acid, 4-oxatetradecanoic acid, and 12-thioethyldodecanoic acid, re-duced HIV-1 replication in acutely infected T-lymphocytes [27]. For example, 12-thioe-thyldodecanoic acid was moderately active (EC50 = 9.4 μM) against HIV-infected T4 lym-phocytes. Protein N-myristoylation in HIV-1 is catalyzed by NMT, which is inhibited by myristic acid derivatives. Myristoylated proteins include PR160gag-pol, Pr55gag, p17gag, and p27nef proteins of HIV-1 [28]. Furthermore, fatty acyl derivatives of 3-fluoro-2′,3′-dideoxythymidine (FLT), 3′-azido-2′,3′-dideoxythymidine (AZT) [29], 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) [30], FTC [31], and 3TC [32] exhibited a significantly higher cellular uptake and anti-HIV profile against wild-type cell-free, cell-associated, and re-sistant viruses when compared with the corresponding parent nucleosides. The fatty acids were found to also have modest anti-HBV activity [33]. For example, myristic acid conju-gate of FTC (IC90 = 15.7–16.1 nM) exhibited 6.6- and 35.2 times higher activity than FTC (IC90 = 103–567 nM) against multidrug-resistant viruses B-NNRTI and B-K65R, indicating
O
OHN
PO
O
OO
HO F
NHN O
O
Sofosbuvir(2)
Figure 1. Chemical Structures of Antiviral Prodrugs.
Tenofovir (1) (TFV) (Figure 1) has activity against HIV-1, HIV-2, and hepatitis Bviruses (HBV) [20]. Tenofovir disoproxil fumarate (8) (TDF, Viread®) (Figure 1) is an esterprodrug of TFV that is hydrolyzed to TFV intracellularly, and phosphorylated to the activemetabolite, TFV diphosphate. TDF is used in combination with other antiviral medications,such as 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (emtricitabine, FTC). Resistance to TDF isconferred by the reverse transcriptase (RT) K65R and/or K70E mutations. Tenofoviralafenamide fumarate (TAF) (9) (Figure 1) is another prodrug of tenofovir. TAF has higherantiviral activity and distribution in the lymphatic system with fewer side effects, such asimpaired kidney function [21–24]. All prodrugs are safe and effective and are used as partof combination therapy or for prevention [25].
With the constant emergence of HIV mutants of clinical relevance and the needto reduce the number of ARVs for chronic treatment [26], it is logical to develop newlong-acting and more potent nucleoside conjugates that display broad-spectrum activityagainst drug-resistant HIV. We previously demonstrated that several fatty acids, suchas 2-methoxydodecanoic acid, 4-oxatetradecanoic acid, and 12-thioethyldodecanoic acid,reduced HIV-1 replication in acutely infected T-lymphocytes [27]. For example,12-thioethyldodecanoic acid was moderately active (EC50 = 9.4 µM) against HIV-infectedT4 lymphocytes. Protein N-myristoylation in HIV-1 is catalyzed by NMT, which is inhib-ited by myristic acid derivatives. Myristoylated proteins include PR160gag-pol, Pr55gag,p17gag, and p27nef proteins of HIV-1 [28]. Furthermore, fatty acyl derivatives of 3-fluoro-2′,3′-dideoxythymidine (FLT), 3′-azido-2′,3′-dideoxythymidine (AZT) [29], 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) [30], FTC [31], and 3TC [32] exhibited a significantly highercellular uptake and anti-HIV profile against wild-type cell-free, cell-associated, and resistantviruses when compared with the corresponding parent nucleosides. The fatty acids werefound to also have modest anti-HBV activity [33]. For example, myristic acid conjugate ofFTC (IC90 = 15.7–16.1 nM) exhibited 6.6- and 35.2 times higher activity thanFTC (IC90 = 103–567 nM) against multidrug-resistant viruses B-NNRTI and B-K65R, in-
Molecules 2022, 27, 4447 4 of 33
dicating that FTC conjugation with myristic acid generates a more potent analog with abetter resistance profile than its parent compound. The fatty acyl conjugation changes theuptake, activity profile, and mechanism of activity, presumably by interfering with theposttranslational myristoylation of proteins in the HIV life cycle. Intracellular hydrolysis tothe parent nucleoside is one of the factors that contribute to overall anti-HIV activity.
Based on these reports, we hypothesized that the modification of the nucleotide-basedTFV conjugated with different amino acids and fatty acids would improve its lipophilicity,thereby leading to improved anti-HIV activity. Thus, several mono- and disubstitutedclasses of fatty ester conjugates of TFV were synthesized and evaluated for HIV inhibition.The design of compounds was based on generating diversity in the phosphonate groupby conjugating different amino acids, alcohol, phenols, naphthols, and fatty acids. Thesynthesis of compounds consists of L-alanine monosubstituted phosphonamidate deriva-tives (16–20), L-leucine monosubstituted phosphonamidate derivatives (26–28), L-alaninedisubstituted esters (32–34, and 36), phosphonadiamidate (35, 37, and 38), phosphonami-date ester derivatives (39–46), L-alanine 2-pentanolyl ester substituted phosphonamidateesters (52–54), and diester derivatives of phenolated and naphtholated TFV (56–63), L-alanine 2-heptanolyl substituted phosphonamidate ester (68), diester derivatives of naph-tholated TFV (69), L-alanine 1-butanolyl substituted phosphonamidate ester (74), anddiester derivatives of phenolated TFV (75–77), L-valine 1-butanolyl substituted phospho-namidate ester (84), diester derivatives of phenolated and naphtholated TFV (84–87), andglycine1-butanolyl phosphonamidate diester derivatives of phenolated TFV (94) (Figure 2).Alternatively, tetradecanoyl and 12-azidododecanoyl amide conjugates of TAF (95 and 96)(Figure 3) were synthesized for comparative studies with TAF and TFV conjugates. Allcompounds containing the stereocenter at phosphorus were evaluated as racemic mixtures.It is worth emphasizing that this work was a preliminary study to screen and identifylead compounds and templates and develop the structure–activity relationship. Furtheroptimization, biostability, and in vivo work are planned in future studies and are beyondthe scope of this manuscript.
Molecules 2022, 27, 4447 5 of 33Molecules 2022, 27, x FOR PEER REVIEW 5 of 34
POH
OHOO
N
NN
N
NH2
1
N
NN
N
OP
N
O
H
O
OR
NH2
OH
16-20
N
NN
N
OP
N
O
H
O
OR
NH2
OH
26-28
32-34
N
N
NN
OP
N
O
H
O
O
NH2
NH O
RO
N
N
NN
OP
N
O
H
O
O
NH2
NH NH2
36
68
N
NN
N
OP
N
O
H
O
O
NH2
O
69
O R
O
(16) R = -CH(CH
3)CH2CH3
(17) R = -CH(CH
3)CH2CH2CH3
(18) R = -CH(CH
3CH2)2
(19) R = CH3CH(CH=CH2)
(20) R = -C(CH3)3
(26) R = -CH
2CH(CH3)CH2CH3
(27) R = -CH2CH=CH(CH3)CH2CH2CH=CH(CH3)2
(28) R =
(32) R = -CH
2CH2CH2CH3
(33) R = -CH(CH
3)CH2CH2CH3
(34) R = -CH
2CH2CH2CH2(OCH3)
R = CH3(CH2)7-CH=CH-(CH2)7
-
Z
O
O
OOH
N
NN
N
OP
N
O
H
O
O
NH2
O
OH
E E
N
N
NN
OPN
O
H
O
O
NH2
O
O R
O
39-41, 46
(39) R = (CH3(CH2)16-,
(40) R = CH3(CH2)14-, (41) R = CH3(CH2)7-CH=CH-(CH2)7-, (46) R
=N3(CH2)10
-Z
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
42-45
Z
(42) R =N
3(CH2)10- (43) R = CH3(CH2)14-,
(44) R = (CH3(CH2)16-,
(45) R = CH3(CH2)7-CH=CH-(CH2)7-
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
N
N
NN
OPN
O
H
O
O
NH2
O
OH
52
(56) R = CH3(CH2)7-CH=CH-(CH2)7-
(57) R = N3(CH2)10-, Z
Fatty acids
N
N
NN
OPN
O
H
O
O
NH2
O
OH
53
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
(58) R = CH3(CH2)13CH2-,
(59) R = CH3(CH2)7-CH=CH-(CH2)7-
(60) R = N3(CH2)10-, Z
Fatty acids
N
N
NN
OPN
O
H
O
O
NH2
O
54
N
NN
N
OP
N
O
H
O
O
NH2
O
OH
O O
R
(61) R = CH3(CH2)15CH2-,
(62) R = CH3(CH2)7-CH=CH-(CH2)7-
(63) R = N3(CH2)10-, Z
Fatty acidsFatty acid
Fatty acids
N
NN
N
OP
N
O
H
O
O
NH2
O
74 (a)N
N
NN
OPN
O
H
O
O
NH2
O O R
O
(75) R = CH3(CH2)14-,
(76) R = N3(CH2)10-,
HO
N
NN
N
OP
N
O
H
O
O
NH2
O
74 (b)
OH
(77) R = CH3(CH2)7-CH=CH-(CH2)7-
Z
N
NN
N
OP
N
O
H
O
O
NH2
O
O
R
O
Fatty acid
f
N
NN
N
OP
N
O
H
O
O
NH2
O
N
N
NN
OPN
O
H
O
O
NH2
O
OH
83(a)
O
(84) R = CH3(CH2)7-CH=CH-(CH2)7-
Z
O
R
Fatty acid
N
N
NN
OPN
O
H
O
O
NH2
O
OHN
N
NN
OPN
O
H
O
O
NH2
O
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
(85) R = CH3(CH2)7-CH=CH-(CH2)7-
(86) R = N3(CH2)10-,
(87) R = CH3(CH2)7-CH=CH-(CH2)7-
Z
Z
83(b)
83(c)
OH
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
Fatty acid
Fatty acid
94
N
NN
N
OP
N
O
H
O
O
NH2
O
OH
N
NN
N
OP
N
O
H
O
O
NH2
O
O
O7 7
Figure 2. Chemical Structures of Synthesized TFV Conjugates.
95 96
N
NN
N
OP
N
O
H
O
O
HN
O
O12 N
NN
N
OP
N
O
H
O
O
HN
O
O
N311
Figure 3. Chemical Structures of Synthesized TAF Conjugates.
Figure 2. Chemical Structures of Synthesized TFV Conjugates.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 34
POH
OHOO
N
NN
N
NH2
1
N
NN
N
OP
N
O
H
O
OR
NH2
OH
16-20
N
NN
N
OP
N
O
H
O
OR
NH2
OH
26-28
32-34
N
N
NN
OP
N
O
H
O
O
NH2
NH O
RO
N
N
NN
OP
N
O
H
O
O
NH2
NH NH2
36
68
N
NN
N
OP
N
O
H
O
O
NH2
O
69
O R
O
(16) R = -CH(CH
3)CH2CH3
(17) R = -CH(CH
3)CH2CH2CH3
(18) R = -CH(CH
3CH2)2
(19) R = CH3CH(CH=CH2)
(20) R = -C(CH3)3
(26) R = -CH
2CH(CH3)CH2CH3
(27) R = -CH2CH=CH(CH3)CH2CH2CH=CH(CH3)2
(28) R =
(32) R = -CH
2CH2CH2CH3
(33) R = -CH(CH
3)CH2CH2CH3
(34) R = -CH
2CH2CH2CH2(OCH3)
R = CH3(CH2)7-CH=CH-(CH2)7
-
Z
O
O
OOH
N
NN
N
OP
N
O
H
O
O
NH2
O
OH
E E
N
N
NN
OPN
O
H
O
O
NH2
O
O R
O
39-41, 46
(39) R = (CH3(CH2)16-,
(40) R = CH3(CH2)14-, (41) R = CH3(CH2)7-CH=CH-(CH2)7-, (46) R
=N3(CH2)10
-Z
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
42-45
Z
(42) R =N
3(CH2)10- (43) R = CH3(CH2)14-,
(44) R = (CH3(CH2)16-,
(45) R = CH3(CH2)7-CH=CH-(CH2)7-
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
N
N
NN
OPN
O
H
O
O
NH2
O
OH
52
(56) R = CH3(CH2)7-CH=CH-(CH2)7-
(57) R = N3(CH2)10-, Z
Fatty acids
N
N
NN
OPN
O
H
O
O
NH2
O
OH
53
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
(58) R = CH3(CH2)13CH2-,
(59) R = CH3(CH2)7-CH=CH-(CH2)7-
(60) R = N3(CH2)10-, Z
Fatty acids
N
N
NN
OPN
O
H
O
O
NH2
O
54
N
NN
N
OP
N
O
H
O
O
NH2
O
OH
O O
R
(61) R = CH3(CH2)15CH2-,
(62) R = CH3(CH2)7-CH=CH-(CH2)7-
(63) R = N3(CH2)10-, Z
Fatty acidsFatty acid
Fatty acids
N
NN
N
OP
N
O
H
O
O
NH2
O
74 (a)N
N
NN
OPN
O
H
O
O
NH2
O O R
O
(75) R = CH3(CH2)14-,
(76) R = N3(CH2)10-,
HO
N
NN
N
OP
N
O
H
O
O
NH2
O
74 (b)
OH
(77) R = CH3(CH2)7-CH=CH-(CH2)7-
Z
N
NN
N
OP
N
O
H
O
O
NH2
O
O
R
O
Fatty acid
f
N
NN
N
OP
N
O
H
O
O
NH2
O
N
N
NN
OPN
O
H
O
O
NH2
O
OH
83(a)
O
(84) R = CH3(CH2)7-CH=CH-(CH2)7-
Z
O
R
Fatty acid
N
N
NN
OPN
O
H
O
O
NH2
O
OHN
N
NN
OPN
O
H
O
O
NH2
O
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
(85) R = CH3(CH2)7-CH=CH-(CH2)7-
(86) R = N3(CH2)10-,
(87) R = CH3(CH2)7-CH=CH-(CH2)7-
Z
Z
83(b)
83(c)
OH
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
Fatty acid
Fatty acid
94
N
NN
N
OP
N
O
H
O
O
NH2
O
OH
N
NN
N
OP
N
O
H
O
O
NH2
O
O
O7 7
Figure 2. Chemical Structures of Synthesized TFV Conjugates.
95 96
N
NN
N
OP
N
O
H
O
O
HN
O
O12 N
NN
N
OP
N
O
H
O
O
HN
O
O
N311
Figure 3. Chemical Structures of Synthesized TAF Conjugates. Figure 3. Chemical Structures of Synthesized TAF Conjugates.
Molecules 2022, 27, 4447 6 of 33
2. Results and Discussion2.1. Chemistry
The synthesis of monosubstituted phosphonamidate conjugates of TFV is depictedin Schemes 1 and 2 in order to evaluate their anti-HIV activities. The synthesis wasaccomplished by the protection of the NH2 group of amino acid L-alanine 10 or L-leucine21 with di-tert-butyl dicarbonate 11 in the presence of NaHCO3 and H2O/THF (1:1 v/v)as a solvent under reflux until the starting materials were consumed. N-Boc protectedamino acids 12 and 22 were further esterified with alcohols 13 and 23, respectively, inthe presence of thionyl chloride and chloroform (used as a solvent) to form the protectedN-Boc protected amino esters 14 and 24. The protected amino esters 14 and 24 weredeprotected with trifluoracetic acid in the presence of methanol to form amino esters 15and 25, respectively. Further reaction of 15 and 25 with TFV (1) in the presence of thionylchloride in chloroform and triethylamine (TEA) as a base generated compounds 16–20and 26–28, respectively (Schemes 1 and 2). Compounds 16–20 and 26–28 did not containmasked phosphates and were synthesized as a control for comparative studies.
Molecules 2022, 27, x FOR PEER REVIEW 6 of 34
2. Results and Discussion 2.1. Chemistry
The synthesis of monosubstituted phosphonamidate conjugates of TFV is depicted in Schemes 1 and 2 in order to evaluate their anti-HIV activities. The synthesis was accom-plished by the protection of the NH2 group of amino acid L-alanine 10 or L-leucine 21 with di-tert-butyl dicarbonate 11 in the presence of NaHCO3 and H2O/THF (1:1 v/v) as a solvent under reflux until the starting materials were consumed. N-Boc protected amino acids 12 and 22 were further esterified with alcohols 13 and 23, respectively, in the presence of thionyl chloride and chloroform (used as a solvent) to form the protected N-Boc protected amino esters 14 and 24. The protected amino esters 14 and 24 were deprotected with tri-fluoracetic acid in the presence of methanol to form amino esters 15 and 25, respectively. Further reaction of 15 and 25 with TFV (1) in the presence of thionyl chloride in chloroform and triethylamine (TEA) as a base generated compounds 16–20 and 26–28, respectively (Schemes 1 and 2). Compounds 16–20 and 26–28 did not contain masked phosphates and were synthesized as a control for comparative studies.
H2NO
OHO
O
O
O
OO
O
NH O
OH
O
O
NH O
OR H2N
O
OR
N
NN
N
OP
N
O
H
O
OR
NH2
OH
10 1112
14a-e15a-e
16-20
Reagents and conditions: (a) H2O/THF (1:1)/NaHCO3, 3-4 h, 70%, reflux 60 °C; (b) Chloroform/SOCl2, 3 h, reflux 70 °C, 60-70%; (c) TFA/MeOH, 1 h, reflux 60 °C, 60-70%; (d) TFV (1), Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-80%.
(a)
(b)
(c) (d)
ROH (13a-e)
(16) R = -CH(CH
3)CH2CH3
(17) R = -CH(CH
3)CH2CH2CH3
(18) R = -CH(CH
3CH2)2
(19) R = CH3CH(CH=CH2)
(20) R = -C(CH3)3
(a) R = -CH(CH
3)CH2CH3
(b) R = -CH(CH
3)CH2CH2CH3
(c) R = -CH(CH
3CH2)2
(d) R = CH3CH(CH=CH2)
(e) R = -C(CH3)3
Scheme 1. Synthesis of L-alanine monosubstituted phosphonamidate derivatives of TFV. Scheme 1. Synthesis of L-alanine monosubstituted phosphonamidate derivatives of TFV.
Molecules 2022, 27, 4447 7 of 33Molecules 2022, 27, x FOR PEER REVIEW 7 of 34
NH2
O
OHO
O
O
O
O O
O
NH O
OH
O
O
NH O
OR
NH2
O
OR
N
NN
N
OP
N
O
H
O
OR
NH2
OH
21 11 22
24a-c25a-c
Reagents and conditions: (a) H2O/THF (1:1)/NaHCO3, 3-4 h, 70%, reflux 60 °C; (b) Chloroform/SOCl2, 3 h, reflux 70 °C, 60-70%; (c) TFA / MeOH, 1 h, reflux 60 °C, 60-70% (d) TFV (1), Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-80%
.
(a) ROH (23a-c)
(c)
(b)
(d)
26-28(26) R =
-CH2CH(CH3)CH2CH3
(27) R = -CH2CH=C(CH3)CH2CH2CH=C(CH3)2
(28) R =
O
O
OOH
(a) R = -CH
2CH(CH3)CH2CH3
(b) R = -CH2CH=C(CH3)CH2CH2CH=C(CH3)2
(c) R =
O
O
OOH
E
E
Scheme 2. Synthesis of L-leucine monosubstituted phosphonamidate derivatives of TFV.
Figure 1 shows the chemical structures of phosphondiamidate and phosphonamidite ester derivatives of TFV. Phosphondiamidate derivatives of TFV (32–34) were synthe-sized, as depicted in Scheme 3. The NH2 group of L-alanine 10 was protected with BOC (11) in the presence of NaHCO3 and H2O/THF (1:1 v/v), as described above, to afford N-Boc-protected amino acid 12 that was further esterified with different alcohols (1-butanol, 2-pentanol, and 4-methoxy-1-butanol, 29a–c) in the presence of thionyl chloride and chlo-roform (used as a solvent) to obtain the protected amino esters 30a–c. The protected amino ester was deprotected with trifluoracetic acid in the presence of methanol to form amino esters 31a–c. Further reaction of 31a–c with compound 20 in the presence of thionyl chlo-ride afforded compounds 32–34.
Phosphondiamidate 36 and phosphonamidate esters 35 and 37 were synthesized by the reaction of compound 20 with phenol, ethane diamine, and naphthol in the presence of thionyl chloride and chloroform.
A library of phosphonamidate ester derivatives of TFV was prepared via the reaction of compound 20 with substituted phenols (1,3-dihydroxybenzene and 1,4-dihydroxyben-zene). The reaction was performed in chloroform (used as a solvent) in the presence of thionyl chloride. The precipitates were formed, which were filtered to afford the interme-diate product 38 that was further substituted with different fatty acids (stearic acid, pal-mitic acid, oleic acid, and 11-azidoundecanoic acid) in the presence of thionyl chloride and chloroform (used as a solvent) to afford fatty ester derivatives of amino esters of TFV (39–46, Scheme 3). The chemical structures of all synthetic compounds were elucidated by different spectroscopic techniques, such as ESI-MS, HR-ESI-MS, 1H NMR, UV, I.R., and/or 13C NMR, COSY, NOESY, and HMBC.
Scheme 2. Synthesis of L-leucine monosubstituted phosphonamidate derivatives of TFV.
Figure 1 shows the chemical structures of phosphondiamidate and phosphonamiditeester derivatives of TFV. Phosphondiamidate derivatives of TFV (32–34) were synthesized,as depicted in Scheme 3. The NH2 group of L-alanine 10 was protected with BOC (11) in thepresence of NaHCO3 and H2O/THF (1:1 v/v), as described above, to afford N-Boc-protectedamino acid 12 that was further esterified with different alcohols (1-butanol, 2-pentanol,and 4-methoxy-1-butanol, 29a–c) in the presence of thionyl chloride and chloroform (usedas a solvent) to obtain the protected amino esters 30a–c. The protected amino ester wasdeprotected with trifluoracetic acid in the presence of methanol to form amino esters 31a–c.Further reaction of 31a–c with compound 20 in the presence of thionyl chloride affordedcompounds 32–34.
Phosphondiamidate 36 and phosphonamidate esters 35 and 37 were synthesized bythe reaction of compound 20 with phenol, ethane diamine, and naphthol in the presence ofthionyl chloride and chloroform.
A library of phosphonamidate ester derivatives of TFV was prepared via the reaction ofcompound 20 with substituted phenols (1,3-dihydroxybenzene and 1,4-dihydroxybenzene).The reaction was performed in chloroform (used as a solvent) in the presence of thionylchloride. The precipitates were formed, which were filtered to afford the intermediateproduct 38 that was further substituted with different fatty acids (stearic acid, palmiticacid, oleic acid, and 11-azidoundecanoic acid) in the presence of thionyl chloride andchloroform (used as a solvent) to afford fatty ester derivatives of amino esters of TFV(39–46, Scheme 3). The chemical structures of all synthetic compounds were elucidated bydifferent spectroscopic techniques, such as ESI-MS, HR-ESI-MS, 1H NMR, UV, I.R., and/or13C NMR, COSY, NOESY, and HMBC.
Molecules 2022, 27, 4447 8 of 33Molecules 2022, 27, x FOR PEER REVIEW 8 of 34
O
O
NH O
OH O
O
NH O
OR
NH2
O
OR
1230a-c 31a-c
32-34
Reagents and conditions: (a) H2O/THF (1:1 v/v)/NaHCO3, 3-4 h, 70%, reflux 60 °C (b) Chloroform/SOCl2,3 h, reflux 70 °C, 60-70% (c) TFA/MeOH, 1 h, reflux 60 °C,
60-70% (d) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-80%, (e) Chloroform/SOCl2, 1 h, reflux 60 °C, 60-70%, (f) DMF/SOCl2, 1 h, reflux 60 °C, 40-50%, (g) Chloroform/SOCl2, 1 h, reflux 60 °C, 60-70%, (h) Chloroform/SOCl2, 1 h, reflux 60 °C, 60-70%, (i) Chloroform/SOCl2, 1 h, reflux 60 °C, 60-70%, (j) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-85%, (k) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-85%,.
(b)(c) (d)
(a) R = -CH
2CH2CH2CH3
(b) R = -CH(CH
3)CH2CH2CH3
(c) R = -CH
2CH2CH2CH2(OCH3)
N
N
NN
OPN
O
H
O
O
NH2
OH
N
N
NN
OP
N
O
H
O
O
NH2
NH O
RO
(20)
N
N
NN
OP
N
O
H
O
O
NH2
NH NH2
H2N NH2
N
N
NN
OP
N
O
H
O
O
NH2
O
OH
OH
N
N
NN
OP
N
O
H
O
O
NH2
O
(h)
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
42-45
(Stearic acid/Palmitic acid/Oleic acid/ or 11-Azidoundecanoic acid)
N
N
NN
OPN
O
H
O
O
NH2
O
38 b
(k)
36
35
37
(f)
(e)
(g)
ROH (29a-c)
(32) R = -CH
2CH2CH2CH3
(33) R = -CH(CH
3)CH2CH2CH3
(34) R = -CH
2CH2CH2CH2(OCH3)
Z
N
N
NN
OPN
O
H
O
O
NH2
OH
(20)
OH
OH
N
N
NN
OPN
O
H
O
O
NH2
O
38 a
OH
OH
HO
(i)
N
N
NN
OPN
O
H
O
O
NH2
O
O R
O
OH
39-41, 46(39) R = (CH3(CH2)16
-, (40) R = CH3(CH2)14-,
(41) R = CH3(CH2)7-CH=CH-(CH2)7-, (46) R =N
3(CH2)10-
Z
(j)
(42) R =N
3(CH2)10- (43) R = CH3(CH2)14-,
(44) R = (CH3(CH2)16-,
(45) R = CH3(CH2)7-CH=CH-(CH2)7-
(Stearic acid/Palmitic acid/Oleic acid/ or 11-Azidoundecanoic acid)
Scheme 3. Synthesis of L-alanine substituted phosphondiamidate and phosphonamidate ester de-rivatives of TFV.
The synthesis of phosphonamidate diester derivatives of phenolated TFV was ac-complished, as shown in Schemes 4 and 5. L-Alanine N-Boc protected amino acid (12) was esterified with different alcohols, 2-pentanol (47) and 3-heptanol (64), in the presence of thionyl chloride and chloroform (as a solvent) to form the protected amino esters 48 and 65, respectively. N-Boc was deprotected with trifluoroacetic acid in the presence of meth-anol to form amino esters 49 and 66, which were further reacted with TFV (1) in the pres-ence of thionyl chloride to form intermediate compounds 50 and 67. Compounds 50 and
Scheme 3. Synthesis of L-alanine substituted phosphondiamidate and phosphonamidate esterderivatives of TFV.
The synthesis of phosphonamidate diester derivatives of phenolated TFV was accom-plished, as shown in Schemes 4 and 5. L-Alanine N-Boc protected amino acid (12) wasesterified with different alcohols, 2-pentanol (47) and 3-heptanol (64), in the presence ofthionyl chloride and chloroform (as a solvent) to form the protected amino esters 48 and 65,respectively. N-Boc was deprotected with trifluoroacetic acid in the presence of methanolto form amino esters 49 and 66, which were further reacted with TFV (1) in the presence
Molecules 2022, 27, 4447 9 of 33
of thionyl chloride to form intermediate compounds 50 and 67. Compounds 50 and 67were then treated with different phenols (1,3-dihydroxybenzene, 1,4-dihydroxybenzene,and 1,4-dihydroxynaphthalene). The reactions were performed in chloroform to synthesizealcohol-substituted intermediates 52–54, and 68, which were further reacted with differentfatty acids (stearic acid, palmitic acid, oleic acid, and 11-azidoundecanoic acid) to formphosphonamidate ester derivatives of phenolated TFV 56–63, and 69 in the presence ofthionyl chloride and chloroform (Schemes 4 and 5). The chemical structures of all syn-thetic compounds were confirmed by different spectroscopic techniques such as ESI-MS,HR-ESI-MS, 1H NMR, 13C NMR, UV, and the I.R. Terminal azido group was incorpo-rated into a number of compounds, such as 42 and 63, since we previously reported that12-azidodecanoic acid has modest anti-HIV activities.
Molecules 2022, 27, x FOR PEER REVIEW 9 of 34
67 were then treated with different phenols (1,3-dihydroxybenzene, 1,4-dihydroxyben-zene, and 1,4-dihydroxynaphthalene). The reactions were performed in chloroform to synthesize alcohol-substituted intermediates 52–54, and 68, which were further reacted with different fatty acids (stearic acid, palmitic acid, oleic acid, and 11-azidoundecanoic acid) to form phosphonamidate ester derivatives of phenolated TFV 56–63, and 69 in the presence of thionyl chloride and chloroform (Schemes 4 and 5). The chemical structures of all synthetic compounds were confirmed by different spectroscopic techniques such as ESI-MS, HR-ESI-MS, 1H NMR, 13C NMR, UV, and the I.R. Terminal azido group was in-corporated into a number of compounds, such as 42 and 63, since we previously reported that 12-azidodecanoic acid has modest anti-HIV activities.
O
O
NH O
OH
12
a b cO
O
NH O
O
48
H2NO
O
49
(a) 1,4-Dihydroxynaphthalene
d
N
NN
N
OP
N
O
H
O
O
NH2
OH
50
Reagents and conditions: (a) 2-Pentanol (47), Chloroform/SOCl2, 3 h, reflux 70 °C, 60-70%; (b) TFA/MeOH, 1 h, reflux 60 °C, 60-70%; (c) TFV (1), Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-80%; (d) Chloroform/SOCl2, 1 h, reflux, 60 °C, 60-70%; (e) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-85%, (f) Chloroform/SOCl2, 1 h, reflux, 60 °C, 60-70%; (g) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-85%.(h) Chloroform/SOCl2, 1 h, reflux, 60 °C, 60-70%; (i) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-85%.
51
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
N
N
NN
OPN
O
H
O
O
NH2
O
OH
(b) 1,4-Dihydroxybenzene or
51
(c) 1,3-Dihydroxybenzene51
f
h
N
N
NN
OPN
O
H
O
O
NH2
O
OH
N
N
NN
OPN
O
H
O
O
NH2
O
52
53
54
N
NN
N
OP
N
O
H
O
O
NH2
O
O R
O
N
NN
N
OP
N
O
H
O
O
NH2
O
OH O O
R
(58) R = CH3(CH2)13CH2-,
(59) R = CH3(CH2)7-CH=CH-(CH2)7-
(60) R = N3(CH2)10-,
(56) R = CH3(CH2)7-CH=CH-(CH2)7-
(57) R = N3(CH2)10-, Z
(61) R = CH3(CH2)15CH2-,
(62) R = CH3(CH2)7-CH=CH-(CH2)7-
(63) R = N3(CH2)10-, Z
Z
Fatty acids
Fatty acids
Fatty acids
e
g
i
Scheme 4. Synthesis of L-alanine 2-pentanolyl ester substituted phosphonamidate diester deriva-tives of phenolated and naphtholated TFV.
Scheme 4. Synthesis of L-alanine 2-pentanolyl ester substituted phosphonamidate diester derivativesof phenolated and naphtholated TFV.
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Molecules 2022, 27, x FOR PEER REVIEW 10 of 34
OH
1,4-Dihydroxynaphthalene
d
O
O
NH O
OH
N
NN
N
OP
N
O
H
O
O
NH2
OH
12
67
Reagents and conditions: (a) 2-Heptanol (64), Chloroform/SOCl2, 3 h, reflux 70 °C, 60-70%; (b) TFA/MeOH, 1 h, reflux 60 °C, 60-70%; (c) TFV (1), Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-80%; (e) Chloroform/SOCl2, 1 h, reflux 60 °C, 60-70%,; (f) Chloroform/SOCl2, 7-8 h, reflux 65 °C, 60-85%.
a b
c
O
O
NH O
O
65
H2NO
O
66
N
NN
N
OP
N
O
H
O
O
NH2
O
e
N
N
NN
OPN
O
H
O
O
NH2
O
5168
69
Oleic acid
OH
OH
O
O7 7
Scheme 5. Synthesis of L-alanine 2-heptanolyl substituted phosphonamidate diester derivatives of naphtholated TFV 69 containing 2-heptanol.
Similarly, the synthesis of additional phenolated and naphtholated phosphonamidate diester derivatives of L-alanine, L-valine, and glycine (Schemes 6–8) was accomplished by the protection of the NH2 group of amino acids 10 (L-alanine), 78 (L-valine), and 88 (glycine) with BOC (11) in the presence of NaHCO3, and H2O/THF (1:1 v/v) (as a solvent) under reflux until the starting material was consumed. The protected amino acids 12, 79, and 89 were further esterified with 1-butanol in the presence of thionyl chloride and chloroform (as a solvent) to form the protected amino esters 71, 80, and 90, respectively. The protected amino esters were deprotected with trifluoracetic acid in the presence of methanol to form amino esters 72, 81, and 91, which were further treated with TFV (1) in the presence of thionyl chloride to form intermediate compounds 73, 82, and 92, respectively. The intermediate compounds were re-acted with different alcohols (1,3-dihydroxybenzene, 1,4-dihydroxybenzene, and 1,4-dihy-droxynaphthalene) to synthesize compounds 74, 83, and 93. Phenol and naphthol-substituted intermediates were further reacted with different fatty acids (palmitic acid, oleic acid, and 11-azidoundecanoic acid) to form fatty ester derivatives (75–77, 84–87, and 94) in the presence of
Similarly, the synthesis of additional phenolated and naphtholated phosphonamidatediester derivatives of L-alanine, L-valine, and glycine (Schemes 6–8) was accomplishedby the protection of the NH2 group of amino acids 10 (L-alanine), 78 (L-valine), and 88(glycine) with BOC (11) in the presence of NaHCO3, and H2O/THF (1:1 v/v) (as a solvent)under reflux until the starting material was consumed. The protected amino acids 12,79, and 89 were further esterified with 1-butanol in the presence of thionyl chloride andchloroform (as a solvent) to form the protected amino esters 71, 80, and 90, respectively.The protected amino esters were deprotected with trifluoracetic acid in the presence ofmethanol to form amino esters 72, 81, and 91, which were further treated with TFV (1) in thepresence of thionyl chloride to form intermediate compounds 73, 82, and 92, respectively.The intermediate compounds were reacted with different alcohols (1,3-dihydroxybenzene,1,4-dihydroxybenzene, and 1,4-dihydroxynaphthalene) to synthesize compounds 74, 83,and 93. Phenol and naphthol-substituted intermediates were further reacted with differentfatty acids (palmitic acid, oleic acid, and 11-azidoundecanoic acid) to form fatty esterderivatives (75–77, 84–87, and 94) in the presence of thionyl chloride and chloroform. Thechemical structures of all synthetic compounds were confirmed by different spectroscopictechniques, such as ESI-MS, HR-ESI-MS, 1H NMR, 13C NMR, UV, and IR.
Molecules 2022, 27, 4447 11 of 33
Molecules 2022, 27, x FOR PEER REVIEW 11 of 34
thionyl chloride and chloroform. The chemical structures of all synthetic compounds were
confirmed by different spectroscopic techniques, such as ESI-MS, HR-ESI-MS, 1H NMR, 13C
NMR, UV, and IR.
Scheme 6. Synthesis of L-alanine 1-butanolyl substituted phosphonamidate diester derivatives of
Scheme 7. Synthesis of L-valine 1-butanolyl substituted phosphonamidate diester derivatives of
phenolated and naphtholated TFV. Scheme 7. Synthesis of L-valine 1-butanolyl substituted phosphonamidate diester derivatives ofphenolated and naphtholated TFV.
Molecules 2022, 27, 4447 12 of 33Molecules 2022, 27, x FOR PEER REVIEW 13 of 34
Scheme 8. Synthesis of glycine1-butanolyl phosphonamidate diester derivatives of phenolated and
naphtholated TFV.
Finally, the synthesis of fatty acyl amino substituted TAF conjugates was conducted
through the reaction of TAF (9) with myristoyl chloride in the presence of N,N-diisopro-
pylethylamine (DIPEA) in dimethylformamide (DMF) at 70 °C to afford myristoyl conju-
gate 95. Alternatively, 9 was reacted with 12-azidododecanoic acid in the presence of 1-
hydroxy-7-benzotriazole (HOAt) and DIPEA to yield 12-azidododecanoyl TAF conjugate
96 (Scheme 9). The anti-HIV activities of the conjugates were compared with the physical
mixture of myristic acid and TAF (50:50 mole/mole, 97) and TAF (9).
Scheme 9. Synthesis of fatty acyl amide substituted derivatives of TAF (95 and 96).
Scheme 8. Synthesis of glycine1-butanolyl phosphonamidate diester derivatives of phenolated andnaphtholated TFV.
Finally, the synthesis of fatty acyl amino substituted TAF conjugates was conductedthrough the reaction of TAF (9) with myristoyl chloride in the presence of N,N-diisopropylethylamine (DIPEA) in dimethylformamide (DMF) at 70 ◦C to afford myris-toyl conjugate 95. Alternatively, 9 was reacted with 12-azidododecanoic acid in the presenceof 1-hydroxy-7-benzotriazole (HOAt) and DIPEA to yield 12-azidododecanoyl TAF con-jugate 96 (Scheme 9). The anti-HIV activities of the conjugates were compared with thephysical mixture of myristic acid and TAF (50:50 mole/mole, 97) and TAF (9).
Molecules 2022, 27, x FOR PEER REVIEW 13 of 34
Scheme 8. Synthesis of glycine1-butanolyl phosphonamidate diester derivatives of phenolated and
naphtholated TFV.
Finally, the synthesis of fatty acyl amino substituted TAF conjugates was conducted
through the reaction of TAF (9) with myristoyl chloride in the presence of N,N-diisopro-
pylethylamine (DIPEA) in dimethylformamide (DMF) at 70 °C to afford myristoyl conju-
gate 95. Alternatively, 9 was reacted with 12-azidododecanoic acid in the presence of 1-
hydroxy-7-benzotriazole (HOAt) and DIPEA to yield 12-azidododecanoyl TAF conjugate
96 (Scheme 9). The anti-HIV activities of the conjugates were compared with the physical
mixture of myristic acid and TAF (50:50 mole/mole, 97) and TAF (9).
Scheme 9. Synthesis of fatty acyl amide substituted derivatives of TAF (95 and 96). Scheme 9. Synthesis of fatty acyl amide substituted derivatives of TAF (95 and 96).
Molecules 2022, 27, 4447 13 of 33
2.2. Biological Activities
Selected compounds were evaluated for their cytotoxicity on TZM-bl cells and werefound to be non-toxic to the cells at 100 ng/mL, except for in compound 87, which showedcytotoxicity similar to the positive control (nonoxynol-9) (Figure 4). TAF conjugates 95 and96 and the physical mixture 97 were found to show no significant toxicity at concentrationsof 1–100 ng/mL (Figure 5) in TZM-bl cells.
Molecules 2022, 27, x FOR PEER REVIEW 14 of 34
2.2. Biological Activities
Selected compounds were evaluated for their cytotoxicity on TZM-bl cells and were
found to be non-toxic to the cells at 100 ng/mL, except for in compound 87, which showed
cytotoxicity similar to the positive control (nonoxynol-9) (Figure 4). TAF conjugates 95
and 96 and the physical mixture 97 were found to show no significant toxicity at concen-
trations of 1–100 ng/mL (Figure 5) in TZM-bl cells.
Figure 4. TZM-bl cells were exposed to TFV conjugates for 48 h. TZM-bl cells were plated in 96-well
plates and exposed the following day to 100 ng/mL of compounds, except if specified otherwise in
the graph. The experiments were repeated twice with triplicate wells plated per concentration tested
in each experiment. The cells were also exposed to nonoxynol-9 (N9) as a positive control of cyto-
toxicity. After 48 h exposure, the viability of the cells was measured by MTS assay.
Figure 5. TZM-bl cells were exposed to TAF, TAF conjugates (95 and 96) and the physical mixture
of TAF and myristic acid for 48 h. TZM-bl cells were plated in 96-well plates and exposed the fol-
lowing day to 1–100 ng/mL of compounds. The experiments were repeated twice with triplicate
wells plated per concentration tested in each experiment. The cells were also exposed to nonoxynol-
9 (N9) as a positive control of cytotoxicity. After 48 h exposure, the viability of the cells was meas-
ured by MTS assay.
Selected compounds were then screened for their efficacy against HIV infection in a
single round infection assay using TZM-bl cells at 100 ng/mL (50 ng/mL for 87) (Figure 6).
The median Relative Luminescence Unit (RLU) adjusted per assay was calculated and
Figure 4. TZM-bl cells were exposed to TFV conjugates for 48 h. TZM-bl cells were plated in 96-wellplates and exposed the following day to 100 ng/mL of compounds, except if specified otherwisein the graph. The experiments were repeated twice with triplicate wells plated per concentrationtested in each experiment. The cells were also exposed to nonoxynol-9 (N9) as a positive control ofcytotoxicity. After 48 h exposure, the viability of the cells was measured by MTS assay.
Molecules 2022, 27, x FOR PEER REVIEW 14 of 34
2.2. Biological Activities
Selected compounds were evaluated for their cytotoxicity on TZM-bl cells and were
found to be non-toxic to the cells at 100 ng/mL, except for in compound 87, which showed
cytotoxicity similar to the positive control (nonoxynol-9) (Figure 4). TAF conjugates 95
and 96 and the physical mixture 97 were found to show no significant toxicity at concen-
trations of 1–100 ng/mL (Figure 5) in TZM-bl cells.
Figure 4. TZM-bl cells were exposed to TFV conjugates for 48 h. TZM-bl cells were plated in 96-well
plates and exposed the following day to 100 ng/mL of compounds, except if specified otherwise in
the graph. The experiments were repeated twice with triplicate wells plated per concentration tested
in each experiment. The cells were also exposed to nonoxynol-9 (N9) as a positive control of cyto-
toxicity. After 48 h exposure, the viability of the cells was measured by MTS assay.
Figure 5. TZM-bl cells were exposed to TAF, TAF conjugates (95 and 96) and the physical mixture
of TAF and myristic acid for 48 h. TZM-bl cells were plated in 96-well plates and exposed the fol-
lowing day to 1–100 ng/mL of compounds. The experiments were repeated twice with triplicate
wells plated per concentration tested in each experiment. The cells were also exposed to nonoxynol-
9 (N9) as a positive control of cytotoxicity. After 48 h exposure, the viability of the cells was meas-
ured by MTS assay.
Selected compounds were then screened for their efficacy against HIV infection in a
single round infection assay using TZM-bl cells at 100 ng/mL (50 ng/mL for 87) (Figure 6).
The median Relative Luminescence Unit (RLU) adjusted per assay was calculated and
Figure 5. TZM-bl cells were exposed to TAF, TAF conjugates (95 and 96) and the physical mixture ofTAF and myristic acid for 48 h. TZM-bl cells were plated in 96-well plates and exposed the followingday to 1–100 ng/mL of compounds. The experiments were repeated twice with triplicate wells platedper concentration tested in each experiment. The cells were also exposed to nonoxynol-9 (N9) asa positive control of cytotoxicity. After 48 h exposure, the viability of the cells was measured byMTS assay.
Selected compounds were then screened for their efficacy against HIV infection in asingle round infection assay using TZM-bl cells at 100 ng/mL (50 ng/mL for 87) (Figure 6).The median Relative Luminescence Unit (RLU) adjusted per assay was calculated andplotted. The experiments were repeated three or four times, and each experiment included
Molecules 2022, 27, 4447 14 of 33
triplicates per condition. The objective was to determine the relative anti-HIV activities ofthe conjugates in comparison to the parent molecule, TFV (1).
Molecules 2022, 27, x FOR PEER REVIEW 15 of 34
plotted. The experiments were repeated three or four times, and each experiment included
triplicates per condition. The objective was to determine the relative anti-HIV activities of
the conjugates in comparison to the parent molecule, TFV (1).
Figure 6. Anti-HIV activity of TFV and TFV analogs in TZMbl cells. The cells were plated in 96-well
plates and exposed the following day to 100 ng/mL of compounds except for compound 87 (50
ng/mL) in presence of HIVBAL for 48 h. The experiments were repeated 3 or 4 times with triplicate
wells per condition in each experiment.
L-Alanine (16–20), and L-leucine (26–28) monosubstituted phosphonamidite deriva-
dian = 35%), suggesting that monosubstitution is not an effective strategy for improving
the HIV inhibition (Table S1, Supplementary Materials).
Among the disubstituted conjugates, i.e., tert-butyl alanine substituted phosphondi-
amidates (32–34), compound 32 (15%) showed less activity to TFV. Other conjugates, 33
and 34, showed significantly less inhibitory activity (5.0–11.7%), suggesting that having
two phosphonamidates does not confer improved HIV inhibitory activity as compared to
TFV (1), presumably due to the limited hydrolysis of the amidate linkage. tert-Butyl ala-
nine disubstituted phosphonamidate ester derivatives (35 and 37–46) exhibited 0–25.8.%
inhibition, with compounds 39 and 40 showing the highest inhibition. These data suggest
that no dramatic improvement of inhibitory activity was shown by tert-butyl alanine
phosphonamidate ester or phosphondiamidate derivatives. Less activity to TFV (1) was
shown by compounds 42 (10.2%) and 76 (18.5%), both containing the same long fatty ester
11-azidoundecanoyl chain at the meta position. The only difference between these two
compounds was the presence of tert-butyl alanine phosphonamidate in compound 42, and
n-butanolyl alanine phosphonamidate in compound 76. Comparable activity was ob-
served in compound 61 (39.7%) with a long fatty ester chain at the meta position of phe-
nolate ring, and 2-pentanolyl alanine phosphonamidate. However, less activity was ob-
served with compound 32 (15%) with butyl alanine and tert-butyl alanine phosophdi-
amidate. These data indicate that the size and nature of the substituents contribute to the
anti-HIV activity, possibly due to an alteration in the rate of uptake and differential release
profile of the compounds.
Selected 2-pentanolyl alanine phosphonamidate ester conjugates of TFV (52–63)
showed more diverse anti-HIV activities depending on the substituents. Meta and para-
substituted 11-azidoundecanoyl phenolate conjugates 60 (42.5%) and 63 (58%) exhibited
higher inhibitory activity than TFV. The only difference between compounds 60 and 63
Figure 6. Anti-HIV activity of TFV and TFV analogs in TZMbl cells. The cells were plated in 96-well plates and exposed the following day to 100 ng/mL of compounds except for compound 87(50 ng/mL) in presence of HIVBAL for 48 h. The experiments were repeated 3 or 4 times withtriplicate wells per condition in each experiment. Median adjusted relative luminescence units (RLU)with interquartile range are displayed on the graph for each compound tested. Each data point isrepresented by a black or gray symbol with different shapes according to compound. HIV only valuesare represented by gray circles.
L-Alanine (16–20), and L-leucine (26–28) monosubstituted phosphonamidite deriva-tives exhibited significantly lower anti-HIV activity (0–17% inhibition) vs. TFV (1)(median = 35%), suggesting that monosubstitution is not an effective strategy for improvingthe HIV inhibition (Table S1, Supplementary Materials).
Among the disubstituted conjugates, i.e., tert-butyl alanine substituted phosphondi-amidates (32–34), compound 32 (15%) showed less activity to TFV. Other conjugates, 33and 34, showed significantly less inhibitory activity (5.0–11.7%), suggesting that havingtwo phosphonamidates does not confer improved HIV inhibitory activity as comparedto TFV (1), presumably due to the limited hydrolysis of the amidate linkage. tert-Butylalanine disubstituted phosphonamidate ester derivatives (35 and 37–46) exhibited 0–25.8.%inhibition, with compounds 39 and 40 showing the highest inhibition. These data sug-gest that no dramatic improvement of inhibitory activity was shown by tert-butyl alaninephosphonamidate ester or phosphondiamidate derivatives. Less activity to TFV (1) wasshown by compounds 42 (10.2%) and 76 (18.5%), both containing the same long fatty ester11-azidoundecanoyl chain at the meta position. The only difference between these twocompounds was the presence of tert-butyl alanine phosphonamidate in compound 42, andn-butanolyl alanine phosphonamidate in compound 76. Comparable activity was observedin compound 61 (39.7%) with a long fatty ester chain at the meta position of phenolatering, and 2-pentanolyl alanine phosphonamidate. However, less activity was observedwith compound 32 (15%) with butyl alanine and tert-butyl alanine phosophdiamidate.These data indicate that the size and nature of the substituents contribute to the anti-HIVactivity, possibly due to an alteration in the rate of uptake and differential release profile ofthe compounds.
Selected 2-pentanolyl alanine phosphonamidate ester conjugates of TFV (52–63) showedmore diverse anti-HIV activities depending on the substituents. Meta and para-substituted11-azidoundecanoyl phenolate conjugates 60 (42.5%) and 63 (58%) exhibited higher in-
Molecules 2022, 27, 4447 15 of 33
hibitory activity than TFV. The only difference between compounds 60 and 63 was thepresence of the 11-azidoundecanoyl long chain group chain at para position rather than meta.Both compounds have 2-pentanolyl alanine phosphonamidite. Meta oleic acid phenolateconjugate 62 (76.5%) was 2.2-fold more potent than TFV, while the same fatty acid onnaphthol conjugate 56 (40.6%) showed slightly higher activity, although 57 (4.6%) was lesseffective. The absence of fatty acyl esters on naphthol or phenol in compounds 52–54 (noinhibition to 35.1%) or the presence of another type of fatty acid in compound 61 (39.7%)impaired or did not significantly change the antiviral activity. Compound 59 with oleicacid on the para position of phenolate demonstrated a complete loss of activity whencompared with meta-substituted oleic phenolate conjugate 62 (76.5%). These data suggestthat the nature and position of the fatty acid contribute significantly to the anti-HIV activity,presumably due to changes in cellular uptake and release.
Changing the 2-pentanolyl in compound 56 (40.6%) to 3-heptanolyl in compound69 (79%), significantly enhanced the anti-HIV activity by 2.3-fold, making it more potentthan TFV (1). This suggested that the nature of ester substitution on the alanine is criticalfor generating compounds with higher anti-HIV activity. The corresponding butanolyl-substituted naphtholated conjugate 77 showed no activity. The importance of alkyl estersubstitution was also obvious when comparing compounds 84 (24.3%) and 62 (76.5%), withthe only difference being the presence of butyl instead of a pentyl phenolated ester. Ingeneral, 2-butanolyl alanine conjugates were less active than the corresponding 2-pentanylalanine conjugates, as observed for compounds 75 (22.1%), 61 (39.7%), 76 (18.5%) and 60(42.5%), 77 (no inhibition), and 56 (40.6%).
2-Butanolyl ester valine naphtholated conjugate 87 (71.5%) was more potent thanthe corresponding butanoyl ester alanine naphtholated conjugate 77 (no inhibition%), butless active than 3-heptanoyl ester alanine naphtholated conjugate 69 (79%), suggestingthat the selection of the amino acid, substituted ester on the amino acid and the fatty acidester on the naphtholated conjugate are critical for optimal activity. Compound 87 with along oleic acid ester chain at the para position of the naphthol ring and butyl-substitutedvaline attached similarly to phosphonamidite inhibited the HIV infection by 71.5% at100 ng/mL. 2-Butyl glycine conjugate 94 (no inhibition) was not potent as compared to thecorresponding isoleucine conjugate 85 (11.4%).
Among all the selected compounds, compounds 62, 69, and 87 (Figure 6 and Table S1,Supplementary Materials), demonstrated higher HIV inhibition than TFV (1). Compound69 significantly inhibited HIV infection by 79.0% at 100 ng/mL. The compound contains along hydrocarbon chain of oleic acid with a double bond at para position of the naphtholring and 3-heptanoyl-substituted alanine on the phosphonamidate (Figure 7). A slightdecrease in activity was observed in compound 62 (76.5% inhibition) when the oleate esterwas positioned at meta position of the phenolate ring, and 2-pentanolyl alanine was attachedas phosphonamidate (Figure 7). The compound was still more potent than the parent drugTFV (1). Compounds 56 (40.6%), 60 (42.5%), 63 (58.0%), and 93 (58.0%) (Figure 7) alsoexhibited slightly higher activity than TFV.
Molecules 2022, 27, 4447 16 of 33Molecules 2022, 27, x FOR PEER REVIEW 17 of 34
Figure 7. Chemical structures of selected screened compounds against HIV with higher activity than
TFV.
For the TAF conjugates 95, 96, and 97 the inhibitory activity was more than 99% at
100 ng/mL and was comparable to TAF (Figure 8 and Table S2, Supplementary Materials).
Indeed, compound 95 exhibited 99.6% inhibition at a lower molar concentration of 0.145
µM (100 ng/mL) vs. 0.210 µM (100 ng/mL) for TAF (9). Thus, lower concentrations (10, 1,
0.1, 0.01 and 0.001 ng/mL) were examined. Among these two compounds (95 and 96) and
the physical mixture (97), tetradecanoyl conjugate of TAF (95) was found to be signifi-
cantly more potent. Tetradecanoyl conjugate 95 showed 98.4%, 70.9%, and 33% inhibition
vs. the corresponding physical mixture of myristic acid and TAF, which showed 91.7%,
60.4%, and 27.9% inhibition at concentrations of 10, 1, and 0.1 ng/mL, respectively. Fur-
thermore, compound 95 showed a comparable 98.4% inhibition at a lower molar concen-
tration of 0.0145 µM (10 ng/mL) vs. 0.021 µM (10 ng/mL) for TAF. These data indicate the
importance of conjugation in improving anti-HIV activity. Compound 95 generated com-
parable anti-HIV activity (~99% inhibition) to TAF at lower molar concentrations, suggest-
ing higher potency for the fatty acyl conjugated TAF. We previously observed that fatty
acyl conjugates of FTC demonstrated higher potency against the resistant virus when
compared with the parent FTC [26]. Further investigations are required to determine
whether the fatty acyl conjugation of TAF can enhance the long-acting anti-HIV activity
and potency against the TFV-resistant virus in a similar way. Further optimization, bio-
stability, and in vivo characterization are needed to determine the biological relevance
and added value of these conjugates.
Figure 7. Chemical structures of selected screened compounds against HIV with higher activitythan TFV.
For the TAF conjugates 95, 96, and 97 the inhibitory activity was more than 99% at100 ng/mL and was comparable to TAF (Figure 8 and Table S2, Supplementary Materials).Indeed, compound 95 exhibited 99.6% inhibition at a lower molar concentration of 0.145 µM(100 ng/mL) vs. 0.210 µM (100 ng/mL) for TAF (9). Thus, lower concentrations (10, 1, 0.1,0.01 and 0.001 ng/mL) were examined. Among these two compounds (95 and 96) and thephysical mixture (97), tetradecanoyl conjugate of TAF (95) was found to be significantlymore potent. Tetradecanoyl conjugate 95 showed 98.4%, 70.9%, and 33% inhibition vs. thecorresponding physical mixture of myristic acid and TAF, which showed 91.7%, 60.4%,and 27.9% inhibition at concentrations of 10, 1, and 0.1 ng/mL, respectively. Furthermore,compound 95 showed a comparable 98.4% inhibition at a lower molar concentration of0.0145 µM (10 ng/mL) vs. 0.021 µM (10 ng/mL) for TAF. These data indicate the importanceof conjugation in improving anti-HIV activity. Compound 95 generated comparable anti-HIV activity (~99% inhibition) to TAF at lower molar concentrations, suggesting higherpotency for the fatty acyl conjugated TAF. We previously observed that fatty acyl conjugatesof FTC demonstrated higher potency against the resistant virus when compared with theparent FTC [26]. Further investigations are required to determine whether the fatty acylconjugation of TAF can enhance the long-acting anti-HIV activity and potency againstthe TFV-resistant virus in a similar way. Further optimization, biostability, and in vivocharacterization are needed to determine the biological relevance and added value ofthese conjugates.
Molecules 2022, 27, 4447 17 of 33Molecules 2022, 27, x FOR PEER REVIEW 18 of 34
Figure 8. Anti-HIV activity of TAF conjugates in TZMbl cells (concentrations listed below are in
ng/mL). The median adjusted Relative Luminescence units (RLU) for each concentration per exper-
iment are shown in Figure 8 (Experiments were repeated 2 or 4 times, with 3 replicates per concen-
tration in each experiment). The median percentage of HIV inhibition in cells exposed to different
concentrations of TAF or TAF conjugates in presence of HIV per experiment is shown in Table S2.
3. Materials and Methods
3.1. General
The experimental part defines different methods and technical characteristics of the
present work, which include the synthesis of fatty ester conjugates and different purifica-
tion methods. Characterization of the synthesized compounds was achieved through var-
ious spectroscopic techniques, such as 1H NMR, 13C NMR, NOESY, COSY, HMBC, HSQC,
IR, UV, and mass spectrometry. The synthesized analogs were also evaluated for their
anti-HIV activity.
All chemicals were of analytical grade and were directly used without any purifica-
tion. TFV (1) was purchased from Supelco by Sigma-Aldrich (St. Louis, USA), Pennsylva-
nia, United States. L-Leucine and L-alanine, sodium iodide, palmitic acid, 1-butanol, 2-
methane, acetone, acetonitrile, methanol, hexane, ethyl acetate, and sodium azide were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Tert-Butyloxycarbonyl (BOC)
and triethylamine (TEA) were acquired from E. Merck (Darmstadt, Germany). Thionyl
chloride and oxalyl chloride were purchased from Tokyo Chemical Industry (Tokyo, Ja-
pan). Ethanol, trifluoroacetic acid, stearic acid, and oleic acid were acquired from E Merck,
(Darmstadt, Germany). 11-Bromodecanoic acid was purchased from ICN Biomedicals Inc.
(Costa Mesa, CA 92626 United States). 2,4-Pentanediol and 2-pentanol were purchased
from Merck (Kenilworth, NJ, United States). Dimethyl sulfoxide (DMSO) was purchased
from Fisher Scientific (Schwerte, Germany). All reagents were of analytical grade and
used directly without purification.
Thin-layer chromatography (TLC) was performed on pre-coated silica gel GF-254
aluminum plates (Kieselgel 60, 254 mm thick, E. Merck, Darmstadt, Germany). Spots on
Figure 8. Anti-HIV activity of TAF conjugates in TZMbl cells (concentrations listed below are inng/mL). Median adjusted Relative Luminescence units (RLU) with interquartile range for eachconcentration per experiment are shown in Figure 8 (Experiments were repeated 2 or 4 times, with3 replicates per concentration in each experiment). Each data point is represented by a black or graysymbol with different shapes according to compound. HIV only values are represented by graycircles. The median percentages of HIV inhibition in cells exposed to different concentrations of TAFor TAF conjugates in the presence of HIV are shown in Table S2.
3. Materials and Methods3.1. General
The experimental part defines different methods and technical characteristics of thepresent work, which include the synthesis of fatty ester conjugates and different purificationmethods. Characterization of the synthesized compounds was achieved through variousspectroscopic techniques, such as 1H NMR, 13C NMR, NOESY, COSY, HMBC, HSQC,IR, UV, and mass spectrometry. The synthesized analogs were also evaluated for theiranti-HIV activity.
All chemicals were of analytical grade and were directly used without any purifica-tion. TFV (1) was purchased from Supelco by Sigma-Aldrich (St. Louis, USA), Pennsyl-vania, United States. L-Leucine and L-alanine, sodium iodide, palmitic acid, 1-butanol, 2-methyl-1-butanol, orcinol 2-methoxyethanol, 3-pentanol, phenol, resorcinol, hydroquinone,1,4-dihydroxynaphthalene, N,N-dimethylformamide, tetrahydrofuran, dichloromethane,acetone, acetonitrile, methanol, hexane, ethyl acetate, and sodium azide were purchasedfrom Sigma-Aldrich Co. (St. Louis, MO, USA). Tert-Butyloxycarbonyl (BOC) and triethy-lamine (TEA) were acquired from E. Merck (Darmstadt, Germany). Thionyl chloride andoxalyl chloride were purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethanol,trifluoroacetic acid, stearic acid, and oleic acid were acquired from E Merck, (Darmstadt,Germany). 11-Bromodecanoic acid was purchased from ICN Biomedicals Inc. (Costa Mesa,CA 92626 United States). 2,4-Pentanediol and 2-pentanol were purchased from Merck(Kenilworth, NJ, United States). Dimethyl sulfoxide (DMSO) was purchased from Fisher
Molecules 2022, 27, 4447 18 of 33
Scientific (Schwerte, Germany). All reagents were of analytical grade and used directlywithout purification.
Thin-layer chromatography (TLC) was performed on pre-coated silica gel GF-254aluminum plates (Kieselgel 60, 254 mm thick, E. Merck, Darmstadt, Germany). Spots onTLC plates were visualized under ultraviolet light at 254 and 366 nm. Melting points wererecorded on an electronic melting point apparatus (SMP3), Sigma-Aldrich Co. (St. Louis,MO, USA). Electron impact mass spectra (EI-MS) were recorded on a Finnigan MAT-311A(Bremen, Germany) mass spectrometer (MASPEC Data System). 1H NMR and 13C NMRspectra were recorded in deuterated DMSO or CD3OD with Avance Bruker AM-300, AMX-400, and AMX-500 MHz equipment (Zurich, Switzerland). The chemical shifts (δ) wereshown on a ppm scale, and coupling constants or J values were expressed in Hz relativeto internal standard tetramethyl silane SiMe4. IR spectra (KBr discs) were run on a FTS3000 MX, Bio-RAD Merlin (Excalibur Model) spectrophotometer. Reagents and solventswere obtained from Sigma-Aldrich (St. Louis, USA) and Merck (Germany).
3.2. Chemistry
General Procedure for the Synthesis of Amino Ester Conjugates of TFV (16–28).Amino acids (L-alanine/L-leucine) (1 mmol) were protected with tert-butyloxycarbonyl(1 mmol) in the presence of sodium bicarbonate (NaHCO3) (1 mol). The reaction mixturewas refluxed for 3–4 h at 60 ◦C in H2O/THF (1:1) (10 mL) as a solvent. The progress ofthe reaction was monitored periodically with TLC. The precipitates were formed, filtered,washed with water, and dried under vacuum at 40 ◦C. The yields of the compounds werein the range of (60–70%).
The protected amino acid (1 mmol) was reacted with different alcohols (1 mmol) in thepresence of thionyl chloride (SOCl2) (1 mmol). The compounds were mixed and refluxedfor 3 h at 70 ◦C in chloroform to form the protected amino ester. The progress of the reactionwas monitored by thin-layer chromatography. After the reaction was completed, waterwas added to afford precipitates. These precipitates were filtered, washed with water, anddried under vacuum (yield 60–70%). The protected amino ester (1 mmol) was deprotectedwith trifluoracetic acid (TFA) (1 mmol) in the presence of methanol (as a solvent), and thereaction mixture was stirred at 60 ◦C for 1 h. The advancement in reaction was monitoredfrom time to time with TLC. An oily product was formed. The purification of compoundswas accomplished by solvent extraction DCM: H2O (1:1).
The amino ester (1 mmol) and TFV (1 mmol) were added to chloroform (10 mL) inthe presence of thionyl chloride SOCl2 (1 mmol) as a catalyst. The reaction mixture wasrefluxed for 7–8 h at 65 ◦C. The reaction progress was periodically monitored using thin-layer chromatography. The final product was purified by using column chromatographyand solvent extraction DCM: H2O (1:1 v/v). The compound was dried under a vacuum(60–80%). All compounds were characterized with 1H NMR, 13C NMR, NOESY, COSY,HMBC, HSQC, IR, UV, and mass spectrometry.
General Procedure for the Synthesis of Phosphonadiamidate Diester Derivativesof TFV (32–46). L-Alanine (1 mmol) was protected with di-tert-butyl dicarbonate (1 mmol)and sodium bicarbonate (NaHCO3) (1 mmol). The reaction mixture was refluxed for 3–4 hat 60 ◦C in H2O/THF (1:1 v/v) (10 mL), and used as a solvent. The reaction progress wasmonitored with TLC. The yield of the compounds was in the range of 60–70%.
The protected amino acid (1 mmol) reacted with different alcohols (1 mmol) in thepresence of thionyl chloride (SOCl2) (1 mmol) and refluxed for 3 h at 70 ◦C in chloroformto form the protected amino ester. After the reaction was completed, water was addedto afford the precipitates. These precipitates were filtered, washed with water, and driedunder vacuum (yield 60–70%). The protected amino esters (1 mmol) were deprotectedwith trifluoracetic acid (TFA) (1 mmol) in the presence of methanol (as a solvent), and thereaction mixture was stirred at 60 ◦C for 1 h. An oily product was formed. The purificationof compounds was achieved by solvent extraction with DCM: H2O (1:1 v/v).
Molecules 2022, 27, 4447 19 of 33
The amino ester (1 mmol) and intermediate compound 26 (1 mmol) were added inchloroform (10 mL) in the presence of thionyl chloride SOCl2 (1 mmol) which was used asa catalyst. The reaction mixture was refluxed for 7–8 h at 65 ◦C. The final products 32–34were purified using column chromatography and solvent extraction DCM:H2O (1:1 v/v),and dried under vacuum (60–80%).
Intermediate compound 26 (1 mmol), triethylamine (TEA) (0.05 mL), and thionyl chlo-ride (SOCl2) (1 mmol) were taken along with chloroform (10 mL); then, the correspondingalcohol (1 mmol) was added to the reaction flask, followed by further stirring for 1 h at60 ◦C. The completion of the reaction was monitored with TLC, and the products wereextracted with dichloromethane through solvent extraction with water to form compounds35, 37, and 38. Pure product 38 (1 mmol) was refluxed with various fatty acids (1 mmol)for 7–8 h at 65 ◦C. This reaction resulted in good yields of compounds 36–46 (60–85%).Intermediate compound 26 (1 mmol) was reacted with ethane diamine (1 mmol) in thepresence of DMF as a solvent and thionyl chloride. The reaction mixture was refluxedfor 1 h at 60 ◦C (40–50% yield). All compounds were characterized using 1H NMR, IR,UV, and mass spectrometry. The representative compounds 29, 32, 39, 58, and 65 werefurther evaluated with 13C NMR. Compounds 28 and 69 were also evaluated using 31PNMR. NOESY, COSY, HMBC, and HSQC were used for compounds 28 and 58.
General Procedure for the Synthesis of Phosphonadiamidate Diester Derivativesof Phenolated TFV (52–94). In the reaction flask containing the intermediate compounds50, 67, 73, 82, and 92 (1 mmol), a few drops of triethylamine (TEA) (0.05 mL) and thionylchloride (SOCl2) (1 mmol) were added along with chloroform (10 mL); then, the corre-sponding alcohol (1 mmol) was added, followed by further stirring for 1 h at 60 ◦C. Theprogress of the reaction was monitored with TLC, and the products were extracted withdichloromethane through solvent extraction with water. Pure intermediate products wereobtained after washing with hexane. The intermediate products (1 mmol) were refluxedwith various fatty acids (1 mmol) for 7–8 h at 65 ◦C. This reaction furnished good yields ofthe compounds (60–85%). All compounds were characterized using 1H NMR, IR, UV, andmass spectrometry.
13C NMR broad-band decoupled spectrum (DMSO-d6) showed a total of twenty-threecarbon signals, including two methyl, three methylene, eight methine, and eight quaternarycarbons. Quaternary C-15, C-17, and C-24 of the ester group were the most downfieldsignals that appeared at δC 167.0, 166.2, and 165.0 ppm, respectively. Methine C-3 and C-8resonated at δC 145.7 and δC 140.6 ppm, respectively, and were located between the twonitrogen groups. C-1 appeared downfield at δC 144.8 ppm, due to being directly attachedto the amino group. Another quaternary C-5 appeared at δC 135.1 ppm. Quaternary C-18and C-23 resonated at δC 134.1 and δC 131.3 ppm, respectively, because of the proximity ofcarbonyl groups. Another quaternary C-16 appeared at δC 130.3 ppm. Methine C-19, C-20,C-21, and C-22 resonated at δC 129.5, 128.9, 127.7, and 127.3, respectively. Quaternary C-6appeared at δC 123.7 ppm. Methine C-11, C-14, and C-26 appeared at δC 69.2, 50.4, and 36.6ppm, respectively. Methylene C-13, C-10, and C-25 appeared at δC 66.3, 59.3, and 45.3 ppm,respectively. Methyl C-12, C-27, and C-28 appeared at δC 18.0, and 14.0 ppm, respectively.
The structure of the coupling part in the compound was elucidated by 2D NMR.Proton–proton coupling connectivity was found in the HH-COSY spectrum due to H-10,H-11, H12, H-14, H-19, H-20, H-25, H-26, H-27, and H-28. Carbon proton long-rangecoupling connectivity was observed in the HMBC spectrum. The phase-sensitive NOESYspectrum of the compound showed a strong correlation between H-8, and H-10. The stereorelationship between H-11, and H-12 was also deduced between the two protons. Otherclear connectivities from methyl protons H-27, and H-28 to H-26 were also observed. Thearomatic protons H-19, and H-22 showed coupling with H-20, and H-21.
13C-NMR broad-band decoupled spectrum (DMSO-d6) exhibited a total of eighteencarbon signals, including three methyl, four methylene, five methine, and five quaternarycarbons. Quaternary carbons C-15, C-2′, C-5, and C-6 of ester and amine groups were themost downfield signals, appearing at δC 169.0, δC 166.2, δC 164.8, and δC 149.7, respectively.Methine carbon C-3 resonated at δC 144.9, and C-8 at δC 141.0, being present between thetwo nitrogen groups. C-1 appeared downfield at δC 127.1 due to being directly attached tothe -NH2 group. Quaternary carbon C-16 appeared at δC 94.3. Methine carbons C-11, C-14,and C-1′ appeared at δC 89.7, δC 50.4, and δC 59.3. Methylene carbons C-10, C-13, and C-3′
appeared at δC 66.3, δC 71.3, and δC 69.2. Other methylene carbons C-4′ and C-5′ appearedat δC 36.6. Methyl carbons C-12, C-20, and C-6′ appeared at δC 20.8, and δC 18.0. Othermethyl carbons, including C-17, C-18, and C-19 appeared at δC 14.0.
13C-NMR broad-band decoupled spectrum (DMSO-d6) of the compound displayed atotal of twenty-six carbon signals, including three methyl, four methylene, ten methine, andnine quaternary carbons. Quaternary carbons C-15 and C-11′ of the ester group were themost downfield signals at δC 167.0, and δC 166.2, respectively. C-1 appeared downfield atδC 156.8 due to being directly attached to the -NH2 group. Another quaternary carbon C-5appeared at δC 145.7. Methine C-3 resonated at δC 145.2, and C-8 at δC 144.6, being presentbetween the two nitrogen groups. Quaternary carbons C-4′, and C-1′ directly attached tocarbonyl groups resonating at δC 142.0, and δC 130.8, respectively. Quaternary carbons C-6appeared at δC 130.5. Other quaternary carbons C-5′ and C-6′ appeared at δC 128.9, andδC 127.7, respectively. Methine carbons C-2′, and C-3′resonated at δC 102.4, and δC 102.1,respectively. Quaternary carbons C-16 appeared at δC 69.0. Methine carbons C-11, and C-14appeared at δC 68.6, and δC 50.4, respectively. Methylene carbons C-13 and C-12′ appearedat δC 66.4, and δC 36.6, respectively. Methylene carbons C-13′-C-27′ resonated at δC 29.0.Methyl carbons C-17, C-18, and C-19 appeared at δC 28.0, while methyl carbons C-28′, C-12,and C-20 resonated at δC 18.0, and δC 14.0.
13C NMR broad-band decoupled spectrum (DMSO-d6) showed a total of twenty-three carbon signals, including two methyl, five methylene, nine methine, and sevenquaternary carbons. Quaternary C-7′, C-15 of ester, and the amine group were the mostdownfield signals that appeared at δC 167.0 ppm and δC 166.2 ppm, respectively. MethineC-3 resonated at δC 145.7 ppm and C-8 at δC 135.7 ppm, being present between the twonitrogen groups. C-1 appeared downfield at δC 159.1 ppm due to being directly attached tothe -NH2 group. Another quaternary C-5 appeared at δC 130.9 ppm. Methine C-6′ and C-2′
appeared at δC 129.3 ppm and δC 128.9 ppm. Another methine C-3′ and C-5′ appeared atδC 127.7 ppm and δC 127.3 ppm. Quaternary C-1′ and C-4′ resonated at δC 119.3 ppm andδC 112.3 ppm directly attached to the carbonyl group. Another quaternary C-6 appeared atδC 117.0 ppm. Methine C-11, C-14, and C-16 appeared at δC 69.2 ppm, 55.2, and 50.4 ppm.Methylene C-13, C-10, and C-9′ appeared at δC 66.3 ppm, δC 59.3 ppm, and δC 36.6 ppm.Other methylene C-17 and C-18 resonated at δC 34.6 ppm. Methylene C-10′-C-22′ wasobserved at δC 29.0 ppm. Methyl C-12, C-19, C-20, C-21, and C-23′ appeared at δC 18.0 ppmand δC 14.0 ppm.
The structure of the coupling part in the compound was elucidated by 2D NMR.Proton–proton coupling connectivity occurred due to H-8′, H-9′, H-10, H-11, H12, H-14,H-20, H-12′, H-25, H-26, H-27, and H-28 being found in the HH-COSY spectrum andcarbon–proton long-range coupling connectivity in the HMBC spectrum. The phase-sensitive NOESY spectrum of the compound showed a strong correlation between H-8 andH-10. The stereo relationship between H-11 and H-12 was also deduced between the twoprotons. Other clear connectivities were observed from the methyl protons H-27 and H-28to H-26. The aromatic protons H-9′ and H-8′ also showed coupling with H-7′ and H-10.
13C-NMR broad-band decoupled spectrum (DMSO-d6) displayed a total of twenty-sixcarbon signals, including three methyl, six methylene, ten methine, and seven quaternarycarbons. Quaternary carbons C-7′, C-15, and C-6 of the ester and amine groups werethe most downfield signals that appeared at δC 167.0, δC 166.4, and δC 166.2, respectively.Methine carbons C-3 resonated at δC 145.7, and C-8 at δC 131.5, being present between thetwo nitrogen groups. C-1 appeared downfield at δC 144.9 due to being directly attachedto the -NH2 group. Another quaternary carbon C-5 appeared at δC 145.3. The quaternarycarbons C-3′ and C-6′ resonated at δC 141.0 which directly attached to the carbonyl group.Methine carbons C-15′, and C-16′ appeared at δC 131.0, and δC 130.9. Methine carbons C-1′,and C-5′ appeared at δC 128.9 and δC 128.7, respectively. C-4′, and C-2′ appeared at δC127.3, and δC 127.1. Methine carbons C-11, C-14, and C-15 appeared at δC 69.3, and δC 50.4.Methylene carbons C-13, C-10, C-16, and C-8′ appeared at δC 66.3, δC 59.3, and δC 36.6.Other methylene carbons C-17-C-18 resonated at δC 36.6. Methylene carbons C-9′-C-14′,and C-17′-C-23′ showed at δC 29.0, and δC 27.0. Methyl carbons C-12, C-21, and C-22appeared at δC 20.8, and δC 18.0, respectively. Other methyl carbons, C-24′ and C-19 wereobtained at δC 14.0.
Isopropyl(phenoxy((((S)-1-(6-tetradecanamido-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)-L-alaninate (95). Compound 95 was obtained by the reaction of (9, 9.3 mg,2 mmol) with myristoyl chloride (1.5 mmol) using DIPEA in 5 mL of (DMF/DCM 1:1) for24 h. The compound was purified by silica gel column chromatography. Yield (5.23 mg,55.7%, colorless solid). 1H NMR (400 MHz, CDCl3): δH 8.89-8.73 (1H, br-s, NHCO), 8.70(1H, s, H-3 of purine ring), 8.24 (1H, s, H-8 of purine ring), 7.23 (2H, t, J3′-2′ , J5′-6′ = 8.0 Hz,H-3′and H-5′ of phenoxy ring), 7.11 (1H, t, J4′-3′ , J4′-5′ = 7.2 and 7.6 Hz, H-4′ of phenoxyring), 7.00 (2H, d, J2′-3′ , J6′-5′ = 8.8 Hz, H-2′ and H-6′ of phenoxy ring), 5.03-4.94 (1H, m, CHof propan-2-yl moiety), 4.44 (1H, dd, J =2.8, 14 Hz, CH2 of propan-2-yl moiety), 4.18 (1H,dd, J = 8.0 and 7.6 Hz, CH2 of propan-2-yl moiety), 3.99 (2H, m, NH of alanine moiety andCH of propyloxy group), 3.67 (2H, m, CH2 of methane group), 2.83 (2H, t, J =7.2 Hz, α-CH2of the myristoyl moiety), 1.67-1.57 (2 H, m, β-CH2 of myristoyl moiety), 1.29- 1.22 (32 H, m,CH3 of propyloxy, CH3 of alanine moiety and 2 CH3 groups of the isopropyl moiety, and 10CH2 of the myristoyl moiety), 0.87 (3H, t J′ = 6.8 Hz, CH3 of myristoyl moiety); 13C NMR(101 MHz, CDCl3): δ 174.39, 173.12, 172.86, 152.53, 151.43, 149.99, 149.06, 143.77, 129.74,125.01, 120.24, 69.26, 65.36, 63.81, 51.46, 49.85, 48.60, 37.93, 34.13, 31.93, 29.65, 29.52, 29.44,29.37, 29.27, 29.16, 24.92, 22.70, 21.73, 21.58, 16.47, 14.14.; ESI-MS m/z (rel. int %): Calcd.Formula [C35H55N6O6P]: 686.4, Found: 687.4 [M+H]+.
Isopropyl(((((S)-1-(6-(12-azidododecanamido)-9H-purin-9-yl)propan-2-yl)oxy)methyl)(phenoxy)phosphoryl)-L-alaninate (96). Compound 96 was obtained by the reaction ofTAF (9, 9.3 mg, 2 mmol) with 12-azidododecanoic acid (1.5 mmol) using HOAt as an acti-vating agent and DIPEA as a base in 5 mL of DMF/DCM (1:1 v/v) for 24 h. The compoundwas purified by silica gel column chromatography. Yield (4.5 mg, 55.7%, colorless solid).
The physical mixture of TAF with myristic acid (97). The physical mixture wasprepared by mixing 1mmole of TAF (9) with 1 mmole of myristic acid. The mixture
Molecules 2022, 27, 4447 31 of 33
was dissolved in 6 mL of THF:MeOH (1:2 v/v), and then the solvents were evaporatedcompletely and dried under a vacuum overnight.
3.3. Cytotoxicity and Anti-HIV Assays
Compounds were dissolved in DMSO typically at a concentration of 1–4 mg/mL.Certain compounds were dissolved at 30 mg/mL in DMSO, and the vial was rocked at RTfor complete dissolution. The compounds were then stored at −20 ◦C. TZM-bl cells wereplated (104 cells per well in 96-well plate), and 100 ng/mL (or 50 ng/mL for compound87, 0.1–100 ng/mL for 95 and 96) of the compound was applied to the cells the followingday with or without HIV in triplicates. For toxicity testing, 100 µL of medium with orwithout compounds was added to each well for 48 h. The surfactant nonoxynol-9 (N9)was used as a cytotoxic positive control. The media was removed and replaced with20 µL of CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA),and 100 µL of cDMEM media for 3–4 h. Absorbance was read at 490 nm. An HIV-1BALstrain, generously obtained from Dr. Susana Asin’s lab (originally from NIH AIDS repos-itory) was amplified in our lab using Interleukin-2 stimulated human peripheral bloodmononuclear cells and also tittered in our lab in TZMbl cells (ATCC). The TCID50s werecalculated according to the Kaerber formula [34]. For antiviral activity (inhibition) testing,we used the Bright-Glo Luciferase Assay System (Promega, USA) following the manufac-turer’s instructions. Briefly, 100 µL of medium +/− TFV compounds containing HIV-1BaL(5× 103 TCID50) was added to each well. After 48 h, the cells were lysed with 100 µL of GloLysis buffer. Lysate 50 µL was transferred into a 96-well black microtiter plate, after which50 µL of Bright-Glo assay reagent was added, and the luminescence was measured andexpressed in relative luminescence units (RLU). The average percentage of infection of theHIV-1BaL growth in three wells exposed to TFV compounds was calculated, and comparedto the control (cells exposed to growth medium and HIV; no compounds) 100% infectionwas detected. The % HIV inhibition is 100 minus % HIV infection.
4. Conclusions
Several classes of amino esters conjugates of TFV (1) were synthesized. Three com-pounds 62, 69, and 87 showed higher potency than TFV (1), while others showed compa-rable or lower activity. Compound 69 significantly inhibited HIV infection by 79.0% at100 ng/mL, and was about 2.2-fold more active than TFV. Compound 69 contains a longhydrocarbon chain of oleic acid with a double bond at para position of the naphthol ringand 3-heptyl-substituted alanine on the phosphonamidate. Comparable or slightly loweranti-HIV activity was obtained for compounds 62 and 87 with similar structural features.Due to its chemical modification and inferred physicochemical properties, we speculatethat this compound would be better suited than TFV as a long-acting formulation. In thisstudy, the data revealed that the nature of the amino acid on phosphonamidate, the size ofthe alkyl ester moiety on the amino acid, and the presence of a specific fatty acid on thephenolate or naphtholate ester within the nucleotide-based compound are key featuresunderpinning their performance. Furthermore, fatty acyl amide conjugation generatedcompounds with comparable activity to TAF. Thus, this strategy may be used to improvethe anti-HIV activity of TFV. This work represents a preliminary study to show the proofof concept. However, more research is needed based on the identified templates for theoptimization and development of long-acting TFV-based anti-HIV agents. The most potentcompounds will be further evaluated in vivo to determine their potential application aslong-acting antiretrovirals.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/molecules27144447/s1. Supplementary data to this article can be found online to reportdetailed structure elucidation of representative compounds 28 and 58, and 1H NMR, UV, IR and massspectrometry spectra of all other synthesized compounds. Figure S1. COSY, NOESY, and HMBCcorrelation of compound 28; Figure S2. COSY, NOESY, and HMBC correlation of compound 58.13C NMR spectra were provided for 28, 32, 39, 58, and 85, and 31P NMR for 28 and 69. UPLC of
representative compounds 28, 53, 58, 60, 61, 63, 69, and 87. Table S1. Percentage of HIV inhibitionin TZM-bl cells co-exposed to TFV or TFV derivatives and HIVBAL. Table S2. Percentage of HIVinhibition in TZM-bl cells co exposed to TAF (9) and TAF conjugates (95, 96) and the physical mixtureof TAF with myristic acid (97) and HIVBAL.
Author Contributions: K.P., H.S. and M.I.C. planned and designed the experiments; A.Q., A.V. andN.S.E.-S. performed the chemistry; L.A.O. and G.F.D. conducted the antiviral and cytotoxicity assays;K.P. and H.S. contributed reagents/materials/analysis tools; A.Q., K.P., M.I.C. and H.S. wrote themanuscript. L.A.O. and G.F.D. provided edits. All authors have read and agreed to the publishedversion of the manuscript.
Funding: The biological activities reported in this study was supported from funds from ChapmanUniversity School of Pharmacy (KP) and Director’s Funds from Eastern Virginia Medical School(LAO, and GFD).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors also acknowledge the support of the core facility at the ChapmanUniversity School of Pharmacy (USA). We also acknowledge the financial support of the SearleCompany (Pakistan).
Conflicts of Interest: The authors declare no conflict of interest.
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