Amidate Prodrugs of O-2-Alkylated Pyrimidine Acyclic Nucleosides Display Potent Anti-Herpesvirus Activity

Article abstract of DOI:10.1021/acsmedchemlett.0c00090

Three series of amidate prodrugs of O-2-alkylated acyclic nucleosides of the 3-fluoro-2-(phosphonomethoxy)propyl (FPMP), cyclic 3-hydroxy-2-(phosphonomethoxypropyl) (cHPMP), and 2-(phosphonomethoxypropyl) (PMP)-type featuring cytosine and 5-fluorocytosine

Full text of DOI:10.1021/acsmedchemlett.0c00090

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Letter
Amidate Prodrugs of O‑2-Alkylated Pyrimidine Acyclic Nucleosides
Display Potent Anti-Herpesvirus Activity
Min Luo, Elisabetta Groaz, Robert Snoeck, Graciela Andrei, and Piet Herdewijn*
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ABSTRACT: Three series of amidate prodrugs of O-2-alkylated acyclic nucleosides of the 3-fluoro-2-(phosphonomethoxy)propyl
(FPMP), cyclic 3-hydroxy-2-(phosphonomethoxypropyl) (cHPMP), and 2-(phosphonomethoxypropyl) (PMP)-type featuring
cytosine and 5-fluorocytosine as nucleobases were readily synthesized. Both the aspartic acid ester and valine ester prodrugs of (R)-
O-2-alkylated FPMPC exhibited potent anti-HCMV and VZV activity in the micromolar range. In addition, the valine ester prodrugs
of 5-fluorocytosine (R)-O-2-alkylated FPMP and (R)-O-2-alkylated cHPMPC showed inhibitory activity at molar concentrations
against these viruses.
KEYWORDS: Acyclic nucleoside phosphonates, prodrugs, human cytomegalovirus, varicella zoster virus, antiviral activity
C i d o f o v i r ( H P M P C , ( S ) - 1 - ( 3 - h y d r o x y - 2 -
phosphonylmethoxypropyl)cytosine) is currently being used
for the treatment of human cytomegalovirus (HCMV) retinitis
in acquired immune deficiency syndrome (AIDS) patients,
while adefovir (PMEA, 9-(2-phosphonylmethoxyethyl)-
adenine) was approved as antihepatitis B virus (HBV) agent.
Upon derivatization of the active drug tenofovir (PMPA, (R)-
9-(2-phosphonylmethoxypropyl)adenine), two nucleoside
phosphonate prodrugs, i.e., tenofovir disoproxil and alafena-
mide, have been developed and licensed for the treatment of
HBV as well as human immunodeficiency virus (HIV)
infections.
cyclic nucleoside phosphonates (ANPs) have gained
Aincreasing attention in nucleos(t)ide research due to a
number of significant advantages, including potent antiviral
activity, easy and economical chemical synthesis, and potential
as genetic materials.1−4 Extensive investigation of their
synthesis and therapeutic properties has led to the marketing
approval of three ANPs as antiviral agents (Figure 1).5,6
The generation of structurally modified ANPs remains an
attractive platform for the development of new bioactive
compounds (Figure 1).7 Among these, the cyclic forms of
HPMP-type nucleosides such as (S)-cHPMPA and (S)-
cHPMPC, which were prepared aiming to reduce the toxic
side effects of the parent ANPs, retained a remarkable antiviral
Received: February 20, 2020
Accepted: June 19, 2020
Figure 1. Chemical structures of cidofovir (HPMPC), adefovir
dipivoxil, tenofovir alafenamide, (S)-cHPMPA, (S)-cHPMPC,
FPMPA, and FPMPC.
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potency against DNA viruses.8,9 On the other hand, the
replacement of the hydroxyl group of HPMC derivatives with a
fluorine atom led to 3-fluoro-2-(phosphonomethoxy)propyl
(FPMP) nucleoside analogues that lacked anti-DNA virus
activity, while being moderately active inhibitors of HIV and
HBV.10−12 Similarly, the PMP series characterized by the
presence of a methyl group at the acyclic side chain, as
exemplified by tenofovir, showed potent activity against
retroviruses but were completely devoid of anti-DNA virus
activity.12
Scheme 1. Synthesis of (S)/(R)-Phosphonamidates 10a,
10b, 11a, 11b, 12a, and 12b
a
It should be noted that the majority of reported nucleoside
phosphonate analogues belong to the N-nucleoside series, i.e.,
where a heterocyclic base (purine or pyrimidine) is linked to
an acyclic chain through a nitrogen atom (Figure 1). On the
other hand, O-alkylated nucleosides, especially O-alkylated
cytosine derivatives, were not widely investigated and never
commercialized as either anticancer or antiviral agents. The
best studied class of O-alkylated acyclic phosphonate nucleo-
sides comprises substituted 4-amino-6-hydroxypyrimidine
derivatives as nucleobase moiety (Figure 2).13,14 For instance,
a
Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/
MeOH, 45 °C, 15 h, 48−50% over 2 steps; (c) 1 M HCl, CH3CN/
H2O 12 h, 30−31% over 2 steps; (d) TMSBr, 2,6-lutidine, CH3CN,
rt, 12 h, 65−70%; (e) L-Aspartic acid amyl diester HCl salt or L-valine
amyl ester HCl salt, PhOH, 2,2′-dithiodipyridine, PPh3, Et3N, Pyr, 60
°C, 12 h, 28−54%.
Figure 2. 4-Amino-6-hydroxypyrimidine (A) and selected examples of
6-O-alkylated acyclic nucleosides (B and C).
successfully deprotected by treatment with 1 M HCl at room
temperature to afford the desired products 7a and 7b. All
phosphonate esters underwent hydrolysis in the presence of
TMSBr to give compounds 8a, 8b, 9a, and 9b in good yields.
Phosphonic acids 8a, 8b, 9a, and 9b were then converted to
their aryloxyphosphonamidates 10a, 10b, 11a, 11b, 12a, and
12b upon reacting with either L-aspartic acid amyl diester HCl
salt or L-valine amyl ester HCl salt and phenol using 2,2′-
dithiodipyridine and triphenylphosphine as activating agents.
All compounds were isolated as diastereoisomeric mixtures
(ratios in the 1/1−1/1.5 range).
Subsequent efforts were directed toward the synthesis of
phosphonamidates 22a, 22b, 23a, and 23b, as illustrated in
Scheme 2. In this case, compounds 13a and 13b were prepared
according to the same method used for the synthesis of diethyl
alcohols (Scheme S-1, Supporting Information).23 Under
Mitsunobu conditions (Ph3P, DIAD), the condensation
reaction between 13a and 13b and either nucleobase 1 or 2
occurred smoothly to afford nucleoside phosphonates 14a, 14b
and 15a, 15b, respectively. After deprotection of either the
acetyl group or triphenylphosphine adduct, standard TMSBr-
promoted hydrolysis of the phosphonate diesters afforded
compounds 18a, 18b, 19a, and 19b in 70−75% yields. The key
aryloxyphosphonamidate intermediates 20a, 20b, 21a, and 21b
were then obtained as described above. Lewis-acid assisted
removal of the benzyl moiety using boron trichloride and
sequential treatment with triethylamine in DCM led to a
concomitant cyclization, affording the desired cyclic L-valine
amyl ester containing (S)/(R)-phosphonamidates 22a, 22b,
23a, and 23b as diastereoisomeric mixtures (ratios in the 1/
1.1−1/1.2 range) in 25−30% yields.
5-methyl derivative B (Figure 2) showed excellent antiviral
activity against HIV and Moloney murine sarcoma virus in
vitro (EC50 = 0.00016−0.00043 μmol/mL). In addition, 5-
halogen-substituted derivatives C (Figure 2) were endowed
with pronounced antiretroviral activity with EC50 values in the
0.0023−0.0110 μmol/mL range.
Among the various available strategies for nucleotide
prodrug design,15 amidate prodrugs have been shown on
many occasions to lead to superior antiviral activities by
masking the negatively charged phosphate or phosphonate
group, which commonly limits the ability of the parent
compounds to penetrate the lipid-rich cell membrane,
consequently contributing to higher levels of active cellular
metabolites.16−19 In particular, we previously demonstrated
that the use of L-aspartic acid diamyl ester18 and L-valine amyl
ester20 moieties afforded analogues with an enhanced antiviral
efficacy.
Herein, we report the discovery of a new family of O-
alkylated acyclic nucleoside phosphonates and their amidate
prodrugs. For this specific class of molecules, various aliphatic
pseudosugar side chains (i.e., FPMP, cHPMP, and PMP) were
linked to the 2-O position of a pyrimidine base rather than the
4-O-position.
As shown in Scheme 1, N4-acetylcytosine 1 was successfully
condensed under Mitsunobu conditions (Ph3P, DIAD) with 3a
and 3b to provide compounds 4a and 4b. The initial
enantiomeric fluorinated acyclic phosphonate ester synthons
3a and 3b were synthesized according to a previously reported
method.18,21 Subsequent removal of the N4-acetyl protecting
group using methanolic ammonia furnished compounds 6a
and 6b in 48 and 50% yield, respectively. On the other hand,
the alkylation of 5-fluorocytosine 2 with 3a and 3b generated
the corresponding triphenylphosphine adducts 5a and 5b in
agreement with a previous report,22 which could be
Lastly, phosphonamidates 31a, 31b, 32a, and 32b were
easily accessible from nucleobases 1 and 2 and compounds
24a24 and 24b (Scheme S-2, Supporting Information), as
illustrated in Scheme 3. Compounds 27a, 27b, 28a, and 28b
B
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phosphonates against HCMV (strains AD-169 and Davis) and
VZV [strains (TK+) Oka and thymidine kinase deficient
(TK−) 07-1] in human embryonic lung (HEL) cells, along
with the assessment of their toxic effects on the same cell line
(Table 1). Ganciclovir, cidofovir, acyclovir, and brivudin were
also included in this study as reference drugs.
Scheme 2. Synthesis of (S)/(R)-Phosphonamidates 22a,
22b, 23a, and 23b
a
As discussed earlier, N-alkylated FPMP nucleoside deriva-
tives bearing canonical and modified nucleobases showed no
or very weak antiviral activity against HCMV and VZV at
subtoxic concentrations.18,25 However, we previously estab-
lished that the use of a diamyl aspartate phenoxyamidate group
as phosphonate prodrug moiety was beneficial for enhancing
the antiviral activity of FPMP nucleosides and particularly
enlarging their spectrum of activity against herpesviruses.18 In
particular, amyl aspartate phosphonamidate prodrugs of purine
containing analogues exhibited submicromolar anti-VZV
potency, while (S)-aspartate-FPMPC displayed an EC50 value
of 0.76 μM against HCMV.
Interestingly, for the O-2-alkylated counterparts, (R)-O-2-
alkylated FPMPC 8b showed moderate antiviral activity
against both HCMV and VZV with EC50 values in the 0.80−
2.36 and 3.79−25.3 μM range, respectively, while its (S)-
enantiomer 8a was found to be inactive.
a
Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/
MeOH, 45 °C, 15 h, 42−53% over 2 steps; (c) 1 M HCl, CH3CN/
H2O, 12 h, 31−46% over 2 steps; (d) TMSBr, 2,6-lutidine, CH3CN,
rt, 12 h, 70−75%; (e) L-Valine amyl ester HCl salt, PhOH, 2,2′-
dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h; (f) (i) BCl3, DCM,
−78 to 0 °C, 2 h; (ii) Et3N, DCM, rt, 1 h, 25−30% over 2 steps.
Further derivatization of (R)-O-2-alkylated FPMPC 8b
afforded amidate prodrugs 10b and 11b, which exhibited
potent antiviral activity against different strains of HCMV and
VZV (including a thymidine kinase mutant virus) with EC50
values ranging between 0.094 and 0.54 μM and concomitantly
low cytotoxicity and cytostatic effects. These compounds
displayed interesting selectivity indices (ratio CC50/EC50), i.e.,
96−551 (10b) and 220−818 (11b). In contrast, the
corresponding (S)-enantiomers 10a and 11a showed minimal
antiviral activity against HCMV and VZV with EC50 values in
the range of 7.61 to >100 μM. The L-valine amyl ester prodrug
of (R)-O-2-alkylated FPMP carrying 5-fluorocytosine (12b)
was found to be moderately active against both HCMV strains
with EC50 values in the 2.94−11.2 μM range, while no activity
was observed for its (S)-counterpart 12a. It is worth
mentioning that the lack of anti-herpesvirus activity was
associated with the opposite configuration at the pseudosugar
chiral center for the N- and O-alkylated series of prodrug
analogues [(R) and (S), respectively]. Moreover, when
compared to (R)-O-2-alkylated FPMPC 8b, the corresponding
prodrugs 10b and 11b displayed a dramatically improved
cellular permeability due to increased lipophilicity, as
evidenced by their clogP values (Table 1).
Scheme 3. Synthesis of (S)/(R)-Phosphonamidates 31a,
31b, 32a, and 32b
a
In previous studies, N-alkylated (S)-cHPMPC and (S)-
cHPMPA (Figure 1) were synthesized and displayed good
antiviral potency against DNA viruses.26,27 It was also
demonstrated that their corresponding amidate prodrugs
showed excellent antiviral potency against DNA viruses.20
However, the alkylation of cytosine or 5-fluorocytosine at the
2-O-position as in the case of compounds 22a, 22b, 23a, and
23b generally resulted in loss of antiviral activity, except for the
prodrug of (R)-O-2-cHPMPC (22b) that proved to be
moderately active against both VZV strain mutants expressing
either a functional or deficient thymidine kinase with EC50
values of 14.0 and 4.76 μM, respectively.
a
Reagents and conditions: (a) Ph3P, DIAD, THF, rt, 12 h; (b) NH3/
MeOH, 45 °C, 15 h, 35% over 2 steps; (c) 1 M HCl, CH3CN/H2O,
12 h, 30−35% over 2 steps; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 12
h, 65−70%; (e) L-Valine amyl ester HCl salt, PhOH, 2,2′-
dithiodipyridine, PPh3, Et3N, Pyr, 60 °C, 12 h, 40−45%.
were obtained under the established Mitsunobu conditions as
diastereoisomeric mixtures (ratios in the 1/1−1/1.3 range),
followed by hydrolysis of either the acetyl moiety or
triphenylphosphine adduct. Cleavage of the phosphonate
ester groups furnished the corresponding phosphonic acids,
which were converted to the desired prodrugs.
With regard to the O-2-alkylated nucleoside phosphonates
and prodrug analogues of the PMP-type, i.e., compounds 29a,
29b, 30a, 30b, 31a, 31b, 32a, and 32b, no inhibition of
HCMV and VZV replication was observed.
Next, we proceeded with the antiviral activity evaluation of
all synthesized phosphonamidate prodrugs and their parent
C
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Table 1. Antiviral Activity and Cytotoxicity of Acyclic Nucleoside Phosphonates and Phosphonamidates against HCMV and
VZV in HEL Cells
a
Antiviral activity EC50 (μM)
Cytotoxicity (μM)
Cell morphology
Cell growth
b
c
HCMV
Compound AD-169 strain Davis strain
>100
VZV
TK+ VZV strain (OKA) TK− VZV strain (07-1)
(MCC)
(CC50)
d
HEL
HEL
clogP
8a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>394
>317
>444
>300
>100
>100
>100
51.84
−0.70
−0.70
6.28
6.28
5.25
5.25
5.34
5.34
4.00
4.00
4.09
4.09
5.38
5.38
5.47
5.47
8b
10a
10b
11a
2.36 0.24
62.8 4.20
0.54 0.37
>100
0.80
48.2 7.60
0.094
3.79 0.30
38.2 7.04
0.36 0.16
38.6
25.3 9.75
24.7 12.9
0.098 0.003
35.9
e
7.61
ND
11b
12a
0.26 0.14
>100
0.097 0.004
>100
0.36 0.035
>100
0.18 0.014
>100
79.3
e
ND
12b
22a
11.2 3.64
>100
4.70 2.76
>100
6.73 3.64
>100
2.94 1.76
>100
>100
e
ND
22b
23a
14.0 1.88
>100
4.76 1.07
>100
53.6 25.1
>100
35.7 13.5
>100
>100
e
ND
e
23b
>100
>100
>100
>100
ND
e
31a
>100
>100
>100
>100
ND
e
31b
>100
>100
>100
>100
ND
e
32a
>100
>100
>100
>100
ND
e
32b
>100
>100
>100
>100
ND
e
e
Ganciclovir
Cidofovir
Acyclovir
Brivudin
13.0 3.40
1.74 0.77
4.44 2.23
0.94 0.86
ND
ND
>394
>317
>444
>300
e
e
ND
ND
−2.39
e
e
ND
ND
7.06 3.56
0.089 0.058
42.7 13.7
0.18
e
e
ND
ND
a
b
Effective concentration required to reduce virus-induced cytopathicity (HCMV) or plaque formation (VZV) by 50%. Minimum concentration
c
required to cause a microscopically detectable alteration of cell morphology. Cytotoxic concentration required to reduce cell viability by 50%.
d
e
cLogP values were calculated using ChemBioDraw Ultra version 14.0 from CambridgeSoft. Not determined.
In addition, the ability of all synthesized amidate prodrugs to
Furthermore, prodrugs of (R)-O-2-alkylated FPMP containing
5-fluorocytosine and (R)-O-2-cHPMPC showed a moderate
ability to inhibit HCMV replication or VZV plaque formation.
These results can help guide the development of novel
potential drug candidates against DNA viruses.
inhibit the replication of HBV was also assessed in the human
hepatoblastoma cell line HepG2.2.15, which is a widely used
cell line containing two copies of the HBV wild-type strain
ayw1 genome and constitutively produces high levels of
HBV.28 Real-time qPCR (TaqMan) was used to measure the
extracellular HBV DNA copy number associated with virions
released from HepG2 2.2.15 cells. A tetrazolium dye uptake
assay was employed to measure cell viability and calculate the
CC50 values. Only compounds 10b, 11b, and 12b proved
moderately active against HBV with EC50 values in the range of
2.10−8.71 μM (Table S1).
The amidate prodrugs of (R)-O-2-alkylated FPMPC, i.e.,
compounds 10b and 11b, showed promising antiviral activity
against HCMV and VZV in vitro. To achieve this efficacy,
prodrugs 10b and 11b must efficiently penetrate the target
cells and undergo further intracellular metabolism. Previously,
we suggested that deoxythreosyl and 3-fluoro-2-
(phosphonomethoxy)propyl aryloxyphosphonamidate pro-
drugs containing a L-aspartate acid diester could be hydrolyzed
to afford the nucleoside phosphonates in the cell, which then
formed the pharmacologically active diphosphates of phos-
phonates.18,19 Therefore, these two prodrugs can be expected
to generate intracellularly phosphonate 8b, which then
undergoes activation to its diphosphate species.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00090.
■
sı
Experimental details and characterization data for the
reported compounds, NMR spectra, and biological
assays (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Piet Herdewijn − Medicinal Chemistry, Rega Institute for
Medical Research, KU Leuven, 3000 Leuven, Belgium;
orcid.org/0000-0003-3589-8503; Phone: +32 16 32 26
57; Email: Piet.Herdewijn@kuleuven.be
Authors
Min Luo − Medicinal Chemistry, Rega Institute for Medical
Research, KU Leuven, 3000 Leuven, Belgium
Elisabetta Groaz − Medicinal Chemistry, Rega Institute for
Medical Research, KU Leuven, 3000 Leuven, Belgium
Robert Snoeck − Laboratory of Virology and Chemotherapy,
Rega Institute for Medical Research, KU Leuven, 3000 Leuven,
Belgium
Graciela Andrei − Laboratory of Virology and Chemotherapy,
Rega Institute for Medical Research, KU Leuven, 3000 Leuven,
Belgium
In summary, a number of amidate prodrugs of O-2-alkylated
acyclic nucleosides bearing cytosine or 5-fluorocytosine as
nucleobase in combination with three structurally diverse
aliphatic side chains were synthesized. Such modification
proved to be especially successful within the FPMP series, as
demonstrated by the potent activity exhibited by the prodrugs
of (R)-O-2-alkylated FPMPC against both HCMV and VZV.
D
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Letter
(12) Balzarini, J.; Holy, A.; Jindrich, J.; Naesens, L.; Snoeck, R.;
Schols, D.; De Clercq, E. Differential antiherpesvirus and anti-
retrovirus effects of the (S) and (R) enantiomers of acyclic nucleoside
phosphonates: potent and selective in vitro and in vivo antiretrovirus
activities of (R)-9-(2-phosphonomethoxypropyl)-2, 6-diaminopurine.
Antimicrob. Agents Chemother. 1993, 37, 332−338.
(13) Holy, A.; Votruba, I.; Masojídkova, M.; Andrei, G.; Snoeck, R.;
Naesens, L.; De Clercq, E.; Balzarini, J. 6-[2-(Phosphonomethoxy)
alkoxy] pyrimidines with antiviral activity. J. Med. Chem. 2002, 45,
1918−1929.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00090
Notes
The authors declare no competing financial interest.
́
́
ACKNOWLEDGMENTS
■
We wish to thank Raj Kalkeri and Roger G. Ptak at the
Southern Research Institute for testing against HBV. Testing
was conducted using federal funds from the Division of
Microbiology and Infectious Diseases (DMID), NIAID/NIH
under contract IV19VB12839 entitled “Anti-HBV screening of
(R)-O-2-alkylated cytosine Asp-prodrug analogues”. In addi-
tion, we are grateful to Ellen De Waegenaere, Brecht Dirix, and
Luc Baudemprez for excellent technical assistance.
́
́
́
(14) Hockova, D.; Holy, A.; Masojídkova, M.; Andrei, G.; Snoeck,
R.; De Clercq, E.; Balzarini, J. 5-Substituted-2, 4-diamino-6-[2-
(phosphonomethoxy) ethoxy] pyrimidines acyclic nucleoside phos-
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ABBREVIATIONS
(16) Mcguigan, C.; Murziani, P.; Slusarczyk, M.; Gonczy, B.; Vande
Voorde, J.; Liekens, S.; Balzarini, J. Phosphoramidate protides of the
anticancer agent fudr successfully deliver the preformed bioactive
monophosphate in cells and confer advantage over the parent
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(17) Mehellou, Y.; Balzarini, J.; Guigan, M. Aryloxy phosphor-
amidate triesters: a technology for delivering monophosphorylated
nucleosides and sugars into cells. ChemMedChem 2009, 4, 1779−
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(18) Luo, M.; Groaz, E.; Andrei, G.; Snoeck, R.; Kalkeri, R.; Ptak, R.
G.; Hartman, T.; Buckheit, R. W., Jr; Schols, D.; De Jonghe, S.;
Herdewijn, P. Expanding the antiviral spectrum of 3-fluoro-2-
(phosphonomethoxy) propyl acyclic nucleoside phosphonates: diamyl
aspartate amidate prodrugs. J. Med. Chem. 2017, 60, 6220−6238.
(19) Liu, C.; Dumbre, S. G.; Pannecouque, C.; Huang, C.; Ptak, R.
G.; Murray, M. G.; De Jonghe, S.; Herdewijn, P. Amidate prodrugs of
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hbv replication. J. Med. Chem. 2016, 59, 9513−9531.
(20) Luo, M.; Groaz, E.; De Jonghe, S.; Snoeck, R.; Andrei, G.;
Herdewijn, P. Amidate prodrugs of cyclic 9-(S)-[3-hydroxy-2-
(phosphonomethoxy) propyl] adenine with potent anti-herpesvirus
activity. ACS Med. Chem. Lett. 2018, 9, 381−385.
■
ANPs, acyclic nucleoside phosphonates; HPMPC, (S)-1-(3-
hydroxy-2-phosphonylmethoxypropyl)cytosine); PMEA, 9-(2-
phosphonylmethoxyethyl)adenine; PMPA, (R)-9-(2-
phosphonylmethoxypropyl)adenine; FPMP, 3-fluoro-2-
(phosphonomethoxy)propyl; HIV, human immunodeficiency
virus; HCMV, human cytomegalovirus; VZV, varicella zoster
virus; cHPMPC, cyclic cidofovir; TMSBr, bromotrimethylsi-
lane; HEL, human embryonic lung; TK, thymidine kinase; Val,
valine
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