Amidate Prodrugs of Cyclic 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine with Potent Anti-Herpesvirus Activity

Article abstract of DOI:10.1021/acsmedchemlett.8b00079

A series of amidate prodrugs of cyclic 9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (cHPMPA) featuring different amino acid motifs were synthesized. All phosphonamidates derived from (S)-cHPMPA displayed a broad spectrum activity against herpesviruses

Full text of DOI:10.1021/acsmedchemlett.8b00079

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Amidate Prodrugs of Cyclic 9-(S)-[3-Hydroxy-2-
(phosphonomethoxy)propyl]adenine with Potent Anti-Herpesvirus Activity.
Min Luo, Elisabetta Groaz, Steven De Jonghe, Robert Snoeck, Graciela Andrei, and Piet Herdewijn
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00079 • Publication Date (Web): 16 Mar 2018
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Amidate
Prodrugs
of
Cyclic
9-(S)-[3-Hydroxy-2-
(phosphonomethoxy)propyl]adenine with Potent Anti-Herpesvirus
Activity
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Min Luo,† Elisabetta Groaz,† Steven De Jonghe,§ Robert Snoeck,§ Graciela Andrei,§ and Piet
Herdewijn*,†
† Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000 Leuven, Belgium
§ Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Herestraat 49 bus 1043, 3000
Leuven, Belgium
KEYWORDS: acyclic nucleoside phosphonates, phosphonamidate prodrugs, DNA viruses, antiviral activity, metabolic sta-
bility
ABSTRACT: A series of amidate prodrugs of cyclic 9ꢀ[3ꢀhydroxyꢀ2ꢀ(phosphonomethoxy)propyl]adenine (cHPMPA) featuring
different amino acid motifs were synthesized. All phosphonamidates derived from (S)ꢀcHPMPA displayed a broad spectrum activiꢀ
ty against herpesviruses with EC50 values in the low nanomolar range. A phosphonobisamidate prodrug of (S)ꢀHPMPA also exhibꢀ
ited a remarkably potent antiviral activity. In addition, the leucine ester prodrug of (S)ꢀcHPMPA and phosphonobisamidate valine
ester prodrug of (S)ꢀHPMPA proved stable in human plasma. These data warrant further development of cHPMPA prodrugs, espeꢀ
cially against human cytomegalovirus (HCMV), for which there is a high need for treatment in transplant recipients.
Human herpesviruses are doubleꢀstranded DNA viruses beꢀ
longing to the Herpesviridae family, which includes nine huꢀ
man viruses causing a wide range of diseases. In particular, the
Alphaherpesvirinae subfamily comprises herpes simplex virus
types 1 and 2 (HSVꢀ1 and HSVꢀ2) and varicellaꢀzoster virus
(VZV), while human cytomegalovirus (HCMV) and human
herpes viruses 6A, 6B, and 7 (HHVꢀ6A, HHVꢀ6B, and HHVꢀ7)
are classified as Betaherpesvirinae. EpsteinꢀBarr virus and
Kaposi’s sarcomaꢀassociated herpesvirus, considered as oncoꢀ
genic viruses, belong to the Gammaherpesvirinae.1, 2
analogues are metabolically stable and do not depend on viral
enzymes for activation, but undergo only two phosphorylation
steps by cellular kinases.
In order to reduce the recognized toxic side effects associatꢀ
ed with the use of HPMPꢀtype nucleosides, their correspondꢀ
ing cyclic forms (S)ꢀcHPMPC and (S)ꢀcHPMPA (Figure 1)
were synthesized and shown to retain the remarkable antiviral
potency of the parent compounds, while allowing for imꢀ
proved selectivity indexes.10, 11
All drugs currently marketed for the treatment of herpesviꢀ
rus infections, such as acyclovir, ganciclovir, penciclovir, and
brivudine, target viral DNA polymerases.3ꢀ5 For several of
these compounds, some prodrugs also received marketing
approval (valacyclovir, valganciclovir and famciclovir). The
antiviral activity of these “classical” nucleosides depends upon
their intracellular metabolism within virusꢀinfected cells to be
sequentially converted to the monoꢀ, diꢀ, and triphosphates.
The latest are the pharmacologically active species, as they
can be incorporated into a growing viral DNA strand by viral
DNA polymerases, resulting in chain termination or fraudulent
Figure 1. Structures of (S)ꢀHPMP and (S)ꢀcHPMP nucleoside
analogues active against DNA viruses.
However, all these HPMP derivatives invariably suffer from
a low oral bioavailability, due to the presence of negatively
charged group(s) at physiological pH that restrict their ability
to penetrate the lipidꢀrich cell membrane. Therefore, the deꢀ
velopment of orally bioavailable, less toxic prodrug forms of
cHPMPs is highly desirable. Up to now, no prodrugs of (S)ꢀ
HPMPA have been approved for clinical use, while the deriꢀ
vatization of cHPMPA has purely focused on the synthesis of
phosphoester prodrugs.12, 13
DNA.
Cidofovir
[(S)ꢀ1ꢀ(3ꢀhydroxyꢀ2ꢀ
phosphonylmethoxypropyl)cytosine, (S)ꢀHPMPC)] is an acyꢀ
clic nucleoside phosphonate analogue (Figure 1), which is
essentially a monophosphate mimic, officially approved as
injectable medication for the treatment of cytomegalovirus
(CMV) retinitis in AIDS patients.6 Its adenine counterpart,
namely (S)ꢀHPMPA (Figure 1), was also described to exhibit a
similarly potent inhibitory activity against a broad spectrum of
DNA viruses,7 while showing greater toxicity in vitro.8, 9 Owꢀ
ing to the presence of the phosphonate moiety, such nucleotide
One of the most promising prodrug strategies that has been
developed over the past few years for nucleoside phosphates
and phosphonates are aryloxy monoamidates.14 This approach
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TMSBr, 2,6ꢀlutidine, CH3CN, 0 °C to rt, 12 h, 70%; (f) (i) Lꢀ
has not been applied to HPMPꢀbased compounds, owing to the
potential chemical instability of the resulting phenoxyphosꢀ
phonoamidate with concomitant formation of a cyclic phosꢀ
phonate analogue. Thus, we decided to exploit this reactivity
in order to synthesize a series of phosphonoꢀamidate prodrugs
of cHPMPA bearing different amino acid motifs.
aspartic acid amyl ester HCl salt, PhOH, 2,2'ꢀdithiodipyridine,
PPh3, Et3N, Pyr, 60 °C, 12 h; (g) TCA (6% in DCM), rt, 2 h, 37ꢀ
42% over 2 steps.
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Additionally, guided by the preliminary data obtained for
8a/b (Table 1) and in order to determine the influence of the
amino acid side chain on the antiviral activity, a restricted
library of (S)ꢀcHPMPA phosphonoamidate prodrugs 10-16
was prepared by introducing a range of structural variations.
To this aim, phosphonoacid 6a was coupled with a variety of
Lꢀamino acid (phenylalanine, methionine, alanine, glutamic
acid, valine, leucine, and isoleucine) esters yielding the deꢀ
sired compounds in moderate yields over two steps (Scheme
2).
Given the wellꢀestablished beneficial effect of a Lꢀaspartic
acid amyl ester moiety instead of the classical Lꢀalanine ester
on the antiviral activity of different phosphonamidate proꢀ
drugs,15ꢀ17 our study started with the synthesis of the Lꢀ
aspartate prodrugs of (R)ꢀ and (S)ꢀcHPMPA. Both enantiomers
of MMTꢀprotected HPMPA 6a/b could be readily obtained
from commercially available (R)ꢀ(+)ꢀ and (S)ꢀ(‒)ꢀglycidol
2a/b, as illustrated in Scheme 1. Interestingly, when 2a/b were
subjected to a tritylation reaction in the presence of pyridine as
base, ring opened compounds 3a/b were formed as the sole
products instead of the anticipated MMTꢀglycidols.18 The folꢀ
lowing alkylation of 3ꢀchloroꢀ1ꢀOꢀmonomethoxytritylꢀ1,2ꢀ
propanediols 3a/b with adenine in presence of 0.9 equivalents
of sodium hydride afforded moderate yields (40ꢀ50%) of comꢀ
pounds 4a/b in a regioselective and stereospecific manner.
Subsequently, the phosphonate group was introduced using
diethyl tosyloxymethylphosphonate and NaH, furnishing 5a/b
in 35ꢀ60% yield. Standard hydrolysis of the diester group with
TMSBr in dry acetonitrile was carried out overnight to form
6a/b in good yield. The free phosphonic acid moiety of 6a/b
was then converted into the corresponding key intermediate
aryloxyphosphonamidates 7a/b by treatment with Lꢀaspartic
acid diamyl ester (Aspꢀdiamyl)17 and phenol using 2,2'ꢀ
dithiodipyridine and triphenylphosphine as activating agents.19
Under acid conditions, the phenoxy group served as a good
leaving group, efficiently generating cyclic (S)/(R)ꢀ
phosphonamidates 8a/b.
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Scheme 2. Synthesis of (S)-cHPMPA Phosphonamidates
10-16a
Scheme 1. Synthesis of (S)- and (R)-cHPMPA L-Asp-
aReagents and conditions: (a) (i) Lꢀamino acid (di)amyl ester
HCl salts 9a-g, PhOH, 2,2'ꢀdithiodipyridine, PPh3, Et3N, Pyr,
60 °C, 12 h; (ii) TCA (6% in DCM), rt, 2 h, 28ꢀ35% over 2 steps.
diamyl-phosphonamidates 8a and 8ba
Phosphonobisamidate 17 was also prepared using a slightly
modified protocol from the parent nucleoside phosphonate 6a
and featured Lꢀvaline as amino acid motifs (Scheme 3).
Scheme 3. Synthesis of (S)-cHPMPA Phosphonobisamidate
17a
aReagents and conditions: (a) (i) Lꢀvaline amyl ester HCl salt
9e, 2,2'ꢀdithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h; (ii) TCA (6%
in DCM), rt, 2 h, 32% over 2 steps.
Compounds 8a/b and 10-16 were evaluated for their antiviꢀ
ral activity against HSVꢀ1, HSVꢀ2, VZV [strains (TK+) Oka
and thymidine kinase deficient (TK−) 07ꢀ1], and HCMV
(strains ADꢀ169 and Davis) in human embryonic lung (HEL)
cells (Table 1). In parallel, the potential toxic effects were
assessed on the same cell line. The (S)ꢀenantiomer of
cHPMPA diamyl aspartate phosphonoamidate prodrug 8a
aReagents and conditions: (a) MMTCl, Pyr, 50 °C, 20 h, 80%;
(b) NaH, DMF, 110 °C,
6 h, 40ꢀ50%; (c) diethyl toꢀ
syloxymethylphosphonate, NaH, THF, 0 °C to rt, 5 h, 35ꢀ40%; (d)
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lized in liver cells. The microsomal stability of prodrug 15 was
showed good activity against HSV, VZV, and HCMV with
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EC50 values in the 0.00073ꢀ0.0032, 0.0014ꢀ0.0015, and 0.009ꢀ
0.027 µM ranges, respectively. On the other hand, the (R)ꢀ
counterpart 8b displayed 24ꢀ to 180ꢀfold decreased potency.
Notably, all other tested analogues 10-16 displayed very poꢀ
tent antiꢀHSV, VZV, and HCMV activity. Especially, (S)ꢀPheꢀ
cHPMPA (10), (S)ꢀGluꢀcHPMPA (13), (S)ꢀValꢀcHPMPA (14),
(S)ꢀLeuꢀcHPMPA (15), and (S)ꢀIleꢀcHPMPA (16) emerged as
the most potent compounds against HSV with EC50 values
consistently in the 0.0012ꢀ0.0042 µM range. Good selectivity
indexes (SI: CC50/EC50 > 4400) were obtained, in particular
for (S)ꢀPheꢀcHPMPA (10) and (S)ꢀIleꢀcHPMPA (16). Comꢀ
pounds 10-16 proved also very potent against all VZV and
HCMV strains with EC50 values in the 0.00045ꢀ0.0043 and
0.0038ꢀ0.064 µM range, respectively. Among these comꢀ
pounds, (S)ꢀIleꢀcHPMPA (16) emerged as the most selective
one with SI’s of 20000 and 1800 in the antiꢀVZV and antiꢀ
HCMV assays, respectively.
slightly better with a t1/2 value of 16 min, indicating that the
structural variation of the amino acid moiety offers the possiꢀ
bility to improve the microsomal stability.
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When compared to cidofovir, all compounds displayed a
dramatically improved antiviral activity. This can be explained
by an improved cellular permeability, due to an increased lipꢀ
ophilicity, as evidenced by the clogP values. Importantly,
HPMPA prodrugs proved more potent than the reference drugs,
i.e., acyclovir (HSV and VZV) and ganciclovir (HCMV). Furꢀ
thermore, the newly synthesized compounds were equally
active against TK+ and TKꢀ HSV and VZV strains.
The main drawback of these amidate prodrugs of (S)ꢀ
cHPMPA is that diastereomeric mixtures are formed due to
the chirality of the phosphorus atom. To avoid the tedious
separation of both diastereomers, the symmetrical phosꢀ
phonodiamidate prodrug 17 was also evaluated. This comꢀ
pound was endowed with potent antiꢀHSV and VZV activity,
displaying EC50 values in the range of 0.0009ꢀ0.0015 µM,
along with decreased CC50 values. With regard to the antiꢀ
HCMV activity, compound 17 was less potent than the (S)ꢀ
cHPMPA prodrugs, with EC50 values in the range of 0.072ꢀ
0.10 µM.
The amidate prodrugs of (S)ꢀcHPMPA showed promising in
vitro antiviral activity against HSV, VZV, and HCMV. Howꢀ
ever, to achieve antiviral efficacy in vivo, such prodrugs must
be stable during the absorption and distribution processes,
since their partial or full hydrolysis before reaching the target
organs may reduce their antiviral effect. To exert such effect,
the phosphonoamidate prodrugs must penetrate the target cells
efficiently, and undergo intracellular metabolism, yielding
HPMPA, which is further phosphorylated to the pharmacologꢀ
ically active diphosphophosphonate analogue. The intracelluꢀ
lar activation pathway of ProTides has been extensively studꢀ
ied.20ꢀ23 McGuigan et al. proposed an activation route for
phosphorodiamidates.24 It has been reported that cHPMPC is
intracellularly converted to cidofovir by a cyclic CMP phosꢀ
phodiesterase.25
The in vitro stability of three representative prodrugs (8a,
15 and 17) in human plasma and human liver microsomes was
investigated (Table 2). (S)ꢀLeuꢀcHPMPA (15) was found to be
stable in human plasma with a t1/2 value of almost 1.5 h,
whereas the halfꢀlife of prodrug 8a (bearing an aspartic acid as
amino acid motif) was only 22 min. The phosphonobisamidate
prodrug of HPMPA (17) displayed high stability in human
plasma (t1/2 > 2 h). On the other hand, very short halfꢀlives of
prodrugs 8a and 17 (t1/2 = 4 min) were observed in human
liver microsomes, suggesting that they are quickly metaboꢀ
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Table 2. Metabolic Stability of Prodrugs 8a, 15, and 17 in
Human Plasma and Human Liver Microsomes
lung; TK, thymidine kinase; Phe, phenylalanine; Glu, glutamic
acid; Val, valine; Leu, leucine; Ile, isoleucine
8
9
Compound Human plasma
Human liver microsomes
t
1/2 (min)a
22
t1/2 (min)a
REFERENCES
8a
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17
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aResults are the mean of two independent experiments.
In summary, the synthesis of amidate prodrugs of cHPMPA
was performed using an aryloxyphosphonamidate as key inꢀ
termediate. In addition, one phosphonobisamidate prodrug of
(S)ꢀHPMPA was synthesized. All prodrugs based on (S)ꢀ
HPMPA exhibited potent antiviral activity against HSV, VZV,
and HCMV with EC50 values in the low nanomolar range,
whereas a prodrug derived from (R)ꢀHPMPA resulted much
less active. Three representative prodrugs were tested for metꢀ
abolic stability in the presence of human plasma and human
liver microsomes. (S)ꢀLeuꢀcHPMPA 15 and the phosphonoꢀ
bisamidate prodrug of HPMPA 17 were found to be stable in
human plasma with t1/2 values exceeding 1 h. On the other
hand, all prodrugs underwent fast metabolism in the presence
of human liver microsomes. Further chemistry could focus on
structural variations of the amino acid side chain and ester
moieties in order to further optimize the prodrug structure in
function of antiviral activity and metabolic stability in plasma
and microsomes.
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ASSOCIATED CONTENT
Supporting Information
(8) Andrei, G.; Snoeck, R.; Schols, D.; Goubau, P.;
Desmyter, J.; De Clercq, E. Comparative activity of selected
antiviral compounds against clinical isolates of human
cytomegalovirus. Eur. J. Clin. Microbiol. Infect. Dis. 1991,
10, 1026ꢀ1033.
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental details and characterization data for the reported
compounds, NMR spectra, and biological assays (PDF)
(9) De Clercq, E. Therapeutic potential of HPMPC as an
antiviral drug. Rev. Med. Virol. 1993, 3, 85ꢀ96.
AUTHOR INFORMATION
Corresponding Author
(10) Snoeck, R.; Schols, D.; Andrei, G.; Neyts, J.; De
Clercq, E. Antiviral activity of antiꢀcytomegalovirus agents
(HPMPC, HPMPA) assessed by a flow cytometric method and
DNA hybridization technique. Antiviral Res. 1991, 16, 1ꢀ9.
* Phone: +32 16 32 26 57. Eꢀmail:
Piet.Herdewijn@kuleuven.be.
Notes
(11) Bischofberger, N.; Hitchcock, M.; Chen, M. S.;
Barkhimer, D. B.; Cundy, K. C.; Kent, K. M.; Lacy, S. A.;
Lee, W. A.; Li, Z.ꢀH.; Mendel, D. B. 1ꢀ[((S)ꢀ2ꢀhydroxyꢀ2ꢀoxoꢀ
1, 4, 2ꢀdioxaphosphorinanꢀ5ꢀyl) methyl] cytosine, an
intracellular
phosphonylmethoxypropyl)
The authors declare no competing financial interest.
ACKNOWLEDGMENT
prodrug
for
cytosine
(S)ꢀ1ꢀ(3ꢀhydroxyꢀ2ꢀ
with improved
M.L. acknowledges the China Scholarship Council (CSC) for
funding and FWO for financial support. The authors are grateful
to Ellen De Waegenaere for excellent technical assistance.
therapeutic index in vivo. Antimicrob. Agents Chemother.
1994, 38, 2387ꢀ2391.
(12) Peterson, L. W.; SalaꢀRabanal, M.; Krylov, I. S.; Serpi,
M.; Kashemirov, B. A.; McKenna, C. E. Serine side chainꢀ
linked peptidomimetic conjugates of cyclic HPMPC and
HPMPA: Synthesis and interaction with hPEPT1. Mol. Pharm.
2010, 7, 2349ꢀ2361.
ABBREVIATIONS
HSV, herpes simplex virus; VZV, varicella zoster virus;
HCMV, human cytomegalovirus; HHV, human herpesvirus;
cHPMPC, cyclic cidofovir; TAF, tenofovir alafenamide; TDF,
tenofovir
disoproxil;
HPMPA,
1ꢀ(3ꢀhydroxyꢀ2ꢀ
(13) Zakharova, V. M.; Serpi, M.; Krylov, I. S.; Peterson, L.
W.; Breitenbach, J. M.; Borysko, K. Z.; Drach, J. C.; Collins,
M.; Hilfinger, J. M.; Kashemirov, B. A. Tyrosineꢀbased 1ꢀ(S)ꢀ
[3ꢀhydroxyꢀ2ꢀ(phosphonomethoxy) propyl] cytosine andꢀ
phosphonylmethoxypropyl)adenine; HPMPC, 1ꢀ(3ꢀhydroxyꢀ2ꢀ
phosphonylmethoxypropyl)cytosine; TMSBr, bromotrimeꢀ
thylsilane; NaH, sodium hydride; HEL, human embryonic
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Figure 1. Structures of (S)-HPMP and (S)-cHPMP nucleoside analogues active against DNA viruses.
52x17mm (300 x 300 DPI)
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Scheme 1. Synthesis of (S)- and (R)-cHPMPA L-Asp-diamyl-phosphonamidates 8a and 8ba
189x217mm (300 x 300 DPI)
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Scheme 2. Synthesis of (S)-cHPMPA Phosphonamidates 10-16a
126x104mm (300 x 300 DPI)
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Scheme 3. Synthesis of (S)-cHPMPA Phosphonobisami-date 17a
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