Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

Article abstract of DOI:10.1002/cmdc.201500183

Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC₅₀=0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough. Whooping cough combatted: With the aim to establish a new strategy against pertussis, C2-modified adefovir analogues in their bisamidate prodrug form were found to efficiently inhibit adenylate cyclase toxin (ACT) from Bordetella pertussis. The compounds show favorable plasma stability, effective distribution to target tissues, and good selectivity for ACT over human adenylate cyclase isoforms.

Full text of DOI:10.1002/cmdc.201500183

DOI: 10.1002/cmdc.201500183
Full Papers
Bisamidate Prodrugs of 2-Substituted 9-[2-
(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as
Selective Inhibitors of Adenylate Cyclase Toxin from
Bordetella pertussis
[a]
[a]
[a]
[a]
ˇ
ˇ
Michal Cesnek, Petr Jansa, MarkØta Smídkovµ,* Helena Mertlíkovµ-Kaiserovµ,
[a]
[d]
[b, c]
[b]
[d]
ˇ
´
ˇ
Martin Dracínsky, Tarsis F. Brust, Petr Pµvek,
Frantisek Trejtnar, Val J. Watts, and
Zlatko Janeba*[a]
Novel small-molecule agents to treat Bordetella pertussis infec-
tions are highly desirable, as pertussis (whooping cough) re-
mains a serious health threat worldwide. In this study, a series
of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethy-
l]adenine (PMEA, adefovir), in their isopropyl ester bis(l-phenyl-
alanine) prodrug form, were designed and synthesized as
potent inhibitors of adenylate cyclase toxin (ACT) isolated from
B. pertussis. The series consists of PMEA analogues bearing
either a linear or branched aliphatic chain or a heteroatom at
the C2 position of the purine moiety. Compounds with a small
C2 substituent showed high potency against ACT without cyto-
toxic effects as well as good selectivity over human adenylate
cyclase isoforms AC1, AC2, and AC5. The most potent ACT in-
hibitor was found to be the bisamidate prodrug of the 2-fluoro
PMEA derivative (IC50 =0.145 mm). Although the bisamidate
prodrugs reported herein exhibit overall lower activity than
the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their
toxicity and plasma stability profiles are superior. Furthermore,
the bisamidate prodrug was shown to be more stable in
plasma than in macrophage homogenate, indicating that the
free phosphonate can be effectively distributed to target tis-
sues, such as the lungs. Thus, ACT inhibitors based on acyclic
nucleoside phosphonates may represent a new strategy to
treat whooping cough.
Introduction
Pertussis (whooping cough) is a highly contagious respiratory
disease caused by a strictly human pathogen, Bordetella pertus-
sis.[1] Despite high levels of vaccination, pertussis remains a seri-
ous worldwide health threat, especially for infants.[2] As B. per-
tussis is generally sensitive to antibiotics, this approach has
been the primary recourse for therapy.[3] However, antibiotics
are ineffective against toxemia and antibiotic-resistant B. per-
tussis strains.[4] Moreover, the protection imparted by pertussis
vaccinations has been reported to wane after 4–12 years.[5] As
a result, whooping cough still annually affects some 16 million
people worldwide, and about 195000 children died from this
disease in 2008.[6] Thus, the need for new effective therapies to
combat pertussis remains.
B. pertussis is transmitted from infected to susceptible indi-
viduals through aerosols from a cough or sneeze. The coloniza-
tion of the upper respiratory tract begins by adherence of bac-
teria to the ciliated epithelial cells in the nasopharynx and tra-
chea.[7] Afterward, a host defense response is neutralized by
the secretion of several types of toxins.[7,8] Among these, the
adenylate cyclase toxin (ACT or CyaA) is considered an impor-
tant virulence factor for B. pertussis infection.[9] A study by
Weiss et al.[10] was the first demonstration that an ACT- and he-
molysin-deficient double mutant B. pertussis strain was nonvir-
ulent in infant mouse models. This provided direct evidence
that ACT may be essential for lethal infection in mice.
ˇ
ˇ
[a] Dr. M. Cesnek, Dr. P. Jansa, Dr. M. Smídkovµ, Dr. H. Mertlíkovµ-Kaiserovµ,
ˇ
´
Dr. M. Dracínsky, Dr. Z. Janeba
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
v.v.i. Flemingovo nµm. 2, 16610 Prague 6 (Czech Republic)
E-mail: janeba@uochb.cas.cz
[b] Prof. P. Pµvek, Dr. F. Trejtnar
Charles University in Prague, Faculty of Pharmacy in Hradec KrµlovØ
HeyrovskØho 1203, 50005 Hradec KrµlovØ (Czech Republic)
The entry of ACT into phagocyte cytosol is a two-step pro-
cess:[11] The toxin first binds the integrin CD11b/CD18 and is in-
serted into the phagocyte membrane to mediate calcium ion
influx into cells.[12] This promotes relocation of the toxin–recep-
tor complex into specific lipid microdomains (rafts) within the
cell membrane; the increased cholesterol concentrations in
rafts and their particular organization then support transloca-
tion of the adenylate cyclase domain from ACT directly into cy-
toplasm.[11] Increased intracellular calcium concentrations lead
to a calmodulin-mediated activation of ACT, which converts cy-
[c] Prof. P. Pµvek
Institute of Molecular and Translational Medicine
Faculty of Medicine and Dentistry, Palacky University Olomouc
ˇ
Hnevotínskµ 5, 77515 Olomouc (Czech Republic)
[d] T. F. Brust, Prof. V. J. Watts
Department of Medicinal Chemistry and Molecular Pharmacology
College of Pharmacy, Purdue University
575 Stadium Mall Drive, West Lafayette, IN, 47907 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500183.
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tosolic ATP into the key signaling molecule cAMP.[9] This pro-
cess completely subverts the cellular signaling pathways by es-
tablishing non-physiological intracellular cAMP levels. In vitro,
ACT has been shown to impair a number of important meta-
bolic functions of human immune effector cells,[13] facilitating
an effective invasion and colonization of bacteria in the host
organism. Moreover, such disruption of the immune response
can clearly make the host organism susceptible to secondary
infections.
Acyclic nucleoside phosphonates (ANPs)[14] belong to a rec-
ognized class of highly effective nucleotide analogues with
a broad spectrum of antiviral activity.[15] ANPs also exert
a myriad of other biological processes including cytostatic,[16]
antibacterial,[17] antiparasitic,[18] and immunomodulatory activi-
ties.[19] Furthermore, adefovir diphosphate (PMEApp, 1,
Figure 1), the active cellular metabolite of adefovir dipivoxil
(bis(POM)PMEA, 2, Figure 1) approved for the treatment of
that it has the potential for use at concentrations that effec-
tively neutralize the bacterial toxins without pronounced inter-
ference with host ACs.
Analogously to other ANPs, PMEA prodrugs provide tempo-
rary protection of the polar phosphonate moiety, allowing
them to enter cells, where they undergo metabolic transforma-
tion into free PMEA and subsequent phosphorylation to active
PMEApp (1), thus inhibiting adenylate cyclase activity. The
drawbacks of adefovir dipivoxil (2) are its rapid hydrolysis to
PMEA by plasma esterases[22] and its dose-dependent cytotox-
icity.[23] Therefore, a bisamidate series of PMEA prodrugs was
recently studied by our research group as potential inhibitors
of B. pertussis ACT. Of those, compounds 3 and 4 (Figure 1)
were selected as the most promising prodrugs owing to their
attractive in vitro activity, bioavailability, and synthetic accessi-
bility.[24] Based on in vivo pharmacokinetic tests, the isopropyl
ester bis(l-phenylalanine) moiety (present in 4, Figure 1) was
selected for synthesis of the cell-permeable prodrugs of the
new series of ACT inhibitors.
Based on the ACT crystal structure in the presence of
PMEApp,[20] we proposed possible structural modifications of
the PMEA molecule, especially at the C2 or C8 positions of the
purine moiety. Because none of the 8-substituted PMEA ana-
logues[25] exhibited promising activity,[26] we focused on the 2-
substituted derivatives of PMEA. Our in silico predictions sug-
gested that substitution at the C2 position of PMEApp (1)
could be well tolerated by the ACT active site, as demonstrated
with 2-ethyl- and 2-butyl-substituted PMEApp, for example
(Figure 2). The two alkyl substituents are nicely accommodated
in the hydrophobic pocket of the ACT active site, with possible
attractive interactions between the lipophilic substituents and
the uncharged portion of the ACT protein (Figure 2).
The aim of this work was the synthesis of a series of 2-sub-
stituted PMEA prodrugs 5 (Figure 3) and evaluation of their
ability to selectively inhibit B. pertussis ACT. Furthermore, the
pharmacological and biochemical properties of the lead struc-
ture were investigated in vitro and in vivo and were compared
with those of the previous generation of compounds, repre-
sented by compound 4 (Figure 1).
Figure 1. Structures of adefovir diphosphate (1), adefovir dipivoxil (2), and
PMEA bisamidate prodrugs 3 and 4.
chronic hepatitis B virus infection, was shown to effectively in-
hibit both B. pertussis adenylate cyclase toxin[20] and Bacillus
anthracis edema factor (EF).[21] PMEApp (1) also exhibited lower
affinity for mammalian adenylate cyclases (ACs),[21] suggesting
Figure 2. Docking of A) 2-ethyl-PMEApp and B) 2-butyl-PMEApp with B. pertussis ACT. Images were generated in Discovery Studio 4.0 using the implemented
protocols. The protein surface is colored according to interpolated charge (red: negative, blue: positive, grey: uncharged).
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(Scheme 1, Table 1 entries 10 and 11) were also prepared by
Method C using the corresponding commercially available al-
kylzinc bromide reagents in 82 and 69% yields, respectively.
Because the preliminary docking studies (Figure 2) suggest-
ed that there may be sufficient space in the ACT binding
pocket for even bulkier C2 substituents, we decided to explore
the spatial tolerance of ACT and to prepare PMEA analogues
bearing tert-butyl and adamantyl substituents at the C2 posi-
tion. However, the reaction of tertiary zinc bromide reagents
(1-adamantylzinc bromide or tert-butylzinc bromide) with com-
pound 8 using Method C (Pd(dppf)2Cl2·CH2Cl2, THF) failed, and
2-tetrahydrofuranyl derivative 9i was formed as the sole reac-
tion product in both cases. Compound 9i (Scheme 1, Table 1
entry 9) was isolated in 78% yield in the case of reaction with
1-adamantylzinc bromide; 9i was not isolated from the reac-
tion with tert-butylzinc bromide. Product 9i probably results
Figure 3. General structure of the target 2-substituted PMEA derivatives 5 in
their bisamidate prodrug form for subsequent cell-based assays.
Results
Chemistry
Because nonaqueous diazotation–dediazonation reactions on
purine moieties have been thoroughly studied and optimize-
d,[27a,b]
2-amino-6-chloropurine
derivative 6 (Scheme 1)[28] was
selected for subsequent modifi-
cations of the purine moiety at
the C2 position. Compound 6
was converted by diazotation–
dediazonation[27a,b] into 6-chloro-
2-iodo intermediate 7 in 77%
yield. Key 6-amino-2-iodopurine
intermediate 8 was then pre-
pared by ammonolysis of com-
pound
7
in
86%
yield
(Scheme 1).
The coupling reaction of inter-
mediate 8 with trimethylalumi-
num and Pd(PPh3)4 in THF af-
forded 6-amino-2-methyl deriva-
tive 9a as its mono-isopropyl
ester in 65% yield (Scheme 1,
Scheme 1. Synthesis of 2-substituted PMEA analogues and their prodrugs 5 (see Table 1 for R1 groups). Reagents
and conditions: a) I2, CH2I2, CuI, THF, isoamylnitrite, 808C, 30 min; b) NH3/EtOH, RT, 24 h; c) see Table 1 for methods
and yields; d) TMSBr, pyridine, RT, 12 h, then l-phenylalanine isopropyl ester hydrochloride, Et3N, 2,2’-dipyridyldi-
sulfide, PPh3, pyridine, 708C, 24–72 h; e) CuSCN, DMF, 1208C.
Table 1. Synthesis of 2-substituted PMEA derivatives 9 and isopropyl ester bis(l-phe-
Table 1 entry 1). Derivatives 9b, 9d, and 9h
(Scheme 1, Table 1 entries 2, 4, and 8) were prepared
in high yields (78–93%) from compound 8 and the
corresponding alkylmagnesium bromides by a trans-
metalation reaction using zinc bromide (Method A).
The other 2-alkylpurine derivatives were synthe-
sized by Negishi coupling of the corresponding alkyl-
zinc bromides with intermediate 8. Derivatives 9c
and 9e (Scheme 1, Table 1 entries 3 and 5) bearing
linear alkyl groups were prepared in high yields (70
and 97%, respectively) by the standard cross-cou-
pling procedure with Pd(PPh3)4 in DMF (Method B).[29]
2-sec-Butyl derivative 9 f (Scheme 1) was obtained
under analogous reaction conditions (Method B) in
poor yield (31%), as the reaction also afforded 2-n-
butyl derivative 9e (17%) and the 2-dehalogenated
product (PMEA diester) as the major product (45%).
Modification of the reaction conditions (Method C),
that is, replacement of solvent (DMF for THF) and
catalyst (Pd(PPh3)4 for Pd(dppf)2Cl2), yielded 2-sec-
butyl derivative 9 f in 77% yield (Scheme 1, Table 1
entry 6). Subsequently, compounds 9j and 9k
nylalanine) prodrugs 5.
Entry
Compd
R1
Yield [%]
Compd
Yield [%]
1
2
9a
9b
Me[a]
65
93
70
86
97
77
49
78
78
82
69
59
NA[h]
NA[h]
NA[h]
NA[h]
NA[h]
5a
5b
5c
5d
5e
5 f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
70
55
57
69
65
53
54
51
29
70
66
41
46
45
61
48
55
Et[b]
3
9c
Pr[c]
4
9d
iPr[b]
5
9e
Bu[c]
6
9 f
sBu[d]
7
9g
iBu[e]
8
9h
pentan-3-yl[b]
9
9i
tetrahydrofuran-2-yl[f]
10
11
12
13
14
15
16
17
9j
9k
cyclopentyl[d]
bicyclo[2.2.1]heptan-2-yl[d]
9l[31]
9m[28]
9n[28]
9o[28]
9p[28]
8
2-(cyclopropylamino)[g]
NH2
F
Cl
OH
I
Reaction conditions: [a] Me3Al, Pd(PPh3)4, THF, 708C. [b] Method A (alkMgBr, ZnBr2,
Pd(dppf)2Cl2·CH2Cl2, THF). [c] Method B (alkZnBr, Pd(PPh3)4, DMF). [d] Method C
(alkZnBr, Pd(dppf)2Cl2·CH2Cl2, THF). [e] tBuZnBr, BHT, Pd(dppf)Cl2·CH2Cl2, NMP, 808C.
[f] 1-AdamantylZnBr, Pd(dppf)Cl2·CH2Cl2, À408C, then RT. [g] Cyclopropylamine, CuI,
(iPr)2NEt, NMP. [h] Not applicable.
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from the solvent trapping the adamantyl or tert-butyl radical
formed during the reaction.[30] Thus, 2,6-di-tert-butyl-4-methyl-
phenol (BHT) was used as a radical scavenger in the subse-
quent experiments, and an increase in BHT concentration ac-
tually caused a decrease in yields of 9i in both cases. Ultimate-
ly, the reactions of intermediate 8 with 1-adamantylzinc bro-
mide and tert-butylzinc bromide were carried out with excess
BTH and in N-methylpyrrolidone to yield the corresponding 2-
dehalogenated product (PMEA diester, not isolated) and iso-
merized 2-isobutyl derivative 9g (49% yield; Scheme 1, Table 1
entry 7), respectively.
Table 2. ACT inhibition and cytotoxic effects of bis(POM)PMEA (2), PMEA
bisamidate prodrug 4, and 2-substituted PMEA bisamidate prodrugs 5.
Compd
IC50 [mm][a]
Viability [%][b]
2
4
0.006Æ0.001
0.16Æ0.01
1.14Æ0.34
5.43Æ0.10
59
93
94
5a
5b
5c
5d
5e
5 f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
105
ND
ND
93
ND
ND
ND
102
ND
ND
87
160
83
142
182
81
>10
>10
1.56Æ0.35
>10
>10
>10
All of the above-mentioned PMEA derivatives 9a–9k contain
a lipophilic group attached to the C2 position of the purine
moiety by a CÀC bond. To extend the structure–activity rela-
tionship (SAR) study of this group of compounds, a series of
PMEA derivatives bearing various heteroatoms at the C2 posi-
tion were prepared according to previously published proce-
dures: namely, 2-(cyclopropylamino) derivative 9l (59% yield)
by copper-catalyzed nucleophilic aromatic substitution with cy-
clopropylamine (Scheme 1, Table 1 entry 12);[31] 2-amino, 2-
fluoro, and 2-chloro derivatives 9m–9o (Scheme 1, Table 1 en-
tries 13–15) by alkylation of the corresponding 6-amino-2-sub-
stituted purines;[28] and 2-hydroxy derivative 9p (Scheme 1,
Table 1 entry 16) by treatment of 2,6-diaminopurine analogue
9m with isoamylnitrite in 80% acetic acid.[28]
5.46Æ0.60
>10
>10
2.68Æ0.75
0.97Æ0.13
0.14Æ0.04
0.54Æ0.06
10.93Æ1.05
1.24Æ0.42
4.38Æ0.88
101
[a] Data are the meanÆSD of at least three independent experiments.
[b] Data are the percent cell viability at a fixed prodrug concentration
(10 mm) versus untreated control; ND: not determined.
All prepared 2-substituted PMEA analogues 8 and 9a–9p
were converted into their corresponding isopropyl ester bis(l-
phenylalanine) prodrugs 5a–5q (Scheme 1, Table 1) by stan-
dard methodologies.[32] Furthermore, 2-thiocyano derivative 5r
(Scheme 1) was prepared in 21% yield directly from 2-iodo
PMEA bisamidate prodrug 5q by the reaction using CuSCN in
DMF.
the murine J774A.1 cell line, but also in human tumor (CCRF-
CEM, HL-60, HeLa-S3, HepG2) and normal (HUVEC-2, NHDF-Ad)
cells. From the compounds tested, only 5m caused higher cy-
totoxic effects than 4 in all cell lines. This effect was similar to
that of adefovir dipivoxil (2, Table 3). The rest of the com-
pounds of series 5 showed a marked improvement in cytotoxic
properties, and, unlike compound 4, no cytotoxicity toward
normal human cell lines was observed (Table 3). Compound 5n
showed no cytotoxicity for either cell line (Table 3).
Biological activity
Inhibition of ACT
Selectivity for ACT over mAC
Bisamidate prodrugs 5a–r were tested for their ability to inhib-
it ACT activity in J774A.1 macrophage cells (Table 2). Murine
macrophage cells J774A.1 were incubated with vari-
ous concentrations of bisamidate prodrugs 5a–r and
The ability of 2-substituted PMEA prodrugs 5 to inhibit host
mammalian adenylate cyclases (mACs) was also examined
Table 3. Long-term cytotoxic effects of bis(POM)PMEA (2), PMEA bisamidate prodrugs
4, and 2-substituted PMEA bisamidate prodrugs 5 in tumor and normal cells.
subsequently exposed to B. pertussis ACT. The cells
were lysed, and the cAMP content was determined.
Compounds that showed activity in the low micro-
molar and sub-micromolar range (Table 2) were also
assessed for their effects on the viability of J774A.1
cells under conditions identical to those of the cAMP
assay, in order to exclude cytotoxic effects during the
experiment. In contrast to bis(POM)PMEA (2), no sig-
nificant cytotoxic effects were observed if the cells
were treated with bisamidate prodrugs 5 at a concen-
tration of 10 mm for 5 h (Table 2).
Compd
Viability [%][a]
CCRF-CEM HL-60 HepG2 HeLa-S3 J774A.1 HUVEC-2 NHDF-Ad
2
4
10
19
114
100
107
31
87
73
9
74
11
14
84
101
111
84
90
25
11
92
44
65
92
100
93
106
92
99
41
100
101
133
18
40
83
96
89
42
92
91
12
74
92
94
34
68
93
92
75
77
65
93
29
96
72
91
38
72
91
98
92
89
95
86
33
107
99
98
32
65
93
102
93
90
96
87
29
95
5n
5a
5b
5e
5i
5l
5m
5o
5p
5q
115
91
117
95
91
101
Long-term cytotoxicity
[a] Data are the percent cell viability at a fixed prodrug concentration (10 mm) versus
untreated control.
The three-day cytotoxic effect of prodrugs 2, 4, and 5
at a concentration of 10 mm was tested not only in
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Table 4. Mammalian AC1, AC2, and AC5 inhibition by bisamidate pro-
drugs 5.
% mAC inhibition[a]
Compd
AC1
AC2
AC5
3 mm A23187
100 nm PMA
300 nm FSK
5a
5b
5c
5d
5e
5 f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
180Æ21
163Æ34
178Æ31
168Æ12
118 Æ12
122Æ15
156Æ23
94Æ10
189Æ15
119 Æ12[b]
39Æ4
147Æ15
161Æ16
164Æ23
159Æ30
172Æ34
146Æ10
149Æ26
149Æ27
193Æ48
90Æ12[b]
31Æ9
97Æ8
98Æ14
110 Æ16
103Æ10
121Æ23
114 Æ18
101Æ5
97Æ11
118 Æ18
96Æ10[b]
84Æ19
106Æ7
101Æ5
75Æ6
Figure 4. Susceptibility of prodrug 5n (100 mm) to enzymatic hydrolysis in
macrophage homogenate. PMEA-F, free PMEA-F retention time: 7.9 min; in-
termediate products IP1, IP2, and IP3 respective retention times: 8.2, 8.5,
and 10.2 min; 5n: parent prodrug.
141Æ26
161Æ21
ND
166Æ24
130Æ26
151Æ27
196Æ27
119 Æ23
253Æ78
336Æ83
À2Æ4
spective retention times of 8.2, 8.5, and 10.2 min (Figure 4).
The free phosphonate PMEA-F (retention time 7.9 min) was
also observed; interestingly, this was further partially metabo-
lized to PMEA (Figure 4). Thus, the bisamidate prodrug 5n was
shown to be more stable to enzymatic degradation than bis(-
POM)PMEA (2), which was completely degraded to free PMEA
within one hour (data not shown).
189Æ29
125Æ15
204Æ28
23Æ5
110 Æ13
97Æ6
116 Æ8
192Æ58
83Æ10
64Æ1
W300
SKF83566
loratadine
95Æ9
102Æ25
84Æ5
[a] Test compound concentration: 30 mm; data are the meanÆSD of
three independent experiments; ND: not determined. [b] Tested at
18 mm; data listed are the percentage of the mAC response treated with
stimulant only.
Pharmacokinetic study in rats
The results of a pilot experiment in a rat model revealed that
prodrug 5n is more stable in plasma (Figure 5A) than in mac-
rophage homogenate (Figure 4). Compared with macrophage
homogenate, only the IP3 intermediate was detected in
plasma, not free phosphonate (Figure 5A). Moreover, prodrug
5n and/or its metabolites were distributed to the organs (liver,
kidney, lung) within 2 h. The IP3 intermediate was detected in
these tissues predominantly (Figure 5B). These findings indi-
cate that significant amounts of free phosphonate could be
distributed to the target tissue.
(Table 4). These assays were carried out with HEK293 cells
stably expressing mAC1, mAC2, or mAC5. Each of these mACs
represents one of the three major mAC families. Each com-
pound was tested on three separate occasions at both 30 and
3 mm, with one exception: compound 5j was tested at 18 and
3 mm. Specific activation of the mAC overexpressed in the cells
was accomplished as previously described.[33] Briefly, the calci-
um ionophore A23187 was employed to stimulate AC1, the
protein kinase C (PKC) activator phorbol 12-myristate 13-ace-
tate (PMA) to stimulate AC2, and forskolin (FSK) to stimulate
AC5. None of the compounds significantly inhibited any mAC
at 3 mm (data not shown). Most of the compounds did not in-
hibit the mACs, with the exception of prodrugs 5k and 5n. In
fact, several compounds potentiated the selectively stimulated
cAMP response at AC1 and AC2, but not AC5.
Discussion
2-Substituted PMEA analogues 5, in their isopropyl ester bis(l-
phenylalanine) prodrug form, were efficiently synthesized, and
their ACT inhibitory properties were studied in vitro in J774A.1
macrophages. Results of this SAR study demonstrate that small
substituents at the C2 position of PMEA are well tolerated and
that this trend is consistent in both 2-alkylpurine and 2-halo-
Hydrolysis of 5n in macrophage
homogenate
Hydrolysis of isopropyl ester
bis(l-phenylalanine) prodrug 5n
into free phosphonate in macro-
phage homogenate (i.e., in the
target tissue) was next evaluat-
ed. During the course of the ex-
periment (18 h), hydrolysis of 5n
led to the appearance of the
partially hydrolyzed intermedi-
Figure 5. Susceptibility of prodrug 5n to enzymatic hydrolysis in A) plasma and B) specific organs. PMEA-F; IP3 in-
ates IP1, IP2, and IP3 with re- termediate (monoester/monoamidate) retention time: 10.2 min; 5n: parent prodrug.
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purine analogues. 2-Methylpurine derivative 5a (IC50 =1.14 mm)
is the most active compound in the aliphatic series, as is the 2-
fluoropurine derivative 5n (IC50 =145 nm) in the halogenated
series. Among the aliphatic compounds, the inhibitory proper-
ties decrease in the following order: Me 5a > Et 5b > Pr 5c ~
iPr 5d ~ sBu 5 f ~ iBu 5g, whereas in the 2-halogenated series,
the order is: F 5n > Cl 5o > I 5q. Interestingly, the 2-butyl de-
rivative 5e retained some inhibitory activity (IC50 =1.56 mm),
suggesting that the AC binding pocket accommodates even
longer linear lipophilic substituents, providing some space for
further investigation in this direction. Moreover, unlike bis(-
POM)PMEA (2), prodrugs of the series 5 maintain low cytotox-
icity.
Thus, the stability of 5n far exceeds that of bis(POM)PMEA (2).
Although prodrug 5n did not exceed the potency of bis(-
POM)PMEA (2; IC50 =6 nm) for ACT inhibition, an increased in
vivo bioavailability of its active metabolite is expected. More-
over, the in vivo study in rats confirmed that the prodrug 5n
and/or its metabolites are distributed to different tissues, in-
cluding lungs.
Compared with compound 4 (IC50 =158 nm), 5n (IC50 =
145 nm) demonstrates similar ACT inhibitory activity. Likewise,
there is no significant difference in stability in rat plasma be-
tween 4 and 5n. However, modification at the C2 position of
5n caused a decrease in the rate of release of the free phos-
phonate in macrophage homogenate. This may be expected
to result in steady tissue concentrations of the active compo-
nent.
The prepared compounds 5 were also tested in cell-based
assays for mammalian ACs, namely mAC1, mAC2, and mAC5.
Most of the 2-substituted PMEA analogues (with the exception
of 5k) did not inhibit mACs at the concentrations tested, indi-
cating these compounds are selective inhibitors of the bacteri-
al AC. The observed selectivity for ACT over mAC suggests that
2-substituted PMEA derivatives 5, or related compounds, may
be used therapeutically at concentrations that effectively neu-
tralize bacterial toxins without deleterious side effects through
interference with host ACs.
The high level of adefovir (PMEA) in kidneys is connected
with its nephrotoxicity, which has been shown to be mediated
by inhibition of mtDNA replication associated with damage to
mitochondrial function.[36] Although the compounds of series 5
did not decrease the viability of J774A.1 cells during the first
5 h, they were evaluated for their possible long-term cytotoxic
effects on several cancer and normal cell lines. Relative to the
previous PMEA series,[24] the substitution at C2 resulted in a de-
crease in cytotoxicity toward all cancer lines. Moreover, unlike
4, the compounds of series 5 were not cytotoxic toward
normal cell lines (HUVEC-2, NHDF-Ad). The only exception is
compound 5m with a 2,6-diaminopurine moiety. The introduc-
tion of other substituents at the C2 position appears to
weaken the effect on mtDNA replication. Thus, presence of
a fluorine atom at the C2 position of PMEA together with the
isopropyl ester bis(l-phenylalanine) prodrug moiety substan-
tially improved the cytotoxic profile. In summary, compound
5n is a lead structure of series 5, with sufficient plasma stabili-
ty and capacity to deliver the active component to lung tissue
without the side effects connected with PMEA analogues.
As the main target of B. pertussis infection are lung phago-
cytes, the most important property of an ideal prodrug for po-
tential oral administration of an ACT inhibitor seems to be its
plasma stability as well as its ability to be cleaved in the target
tissue. Recently, monoester monoamidate and bisamidate pro-
drugs of PMEA have been shown to exhibit good stability, with
t
1/2 >1 h in human plasma, but undergo rapid hydrolysis in
lymphoid cells and cellular extracts.[16a,34] This pharmacokinetic
profile is in contrast with that of adefovir dipivoxil (bis(POM)P-
MEA), which displays limited stability in both rat and human
plasma (t1/2 <10 min) and no selectivity for intracellular activa-
tion over systemic hydrolysis.[35]
Based on our previous study,[24] bisamidate PMEA prodrugs
3 (ethyl ester bis(l-alanine)) and 4 (isopropyl ester bis(l-phenyl-
alanine)) (Figure 1) were chosen for pilot pharmacokinetic
studies in rats because they displayed optimal stability profiles
in macrophage homogenate. Both 3 and 4 exhibited rapid hy-
drolysis in macrophage homogenate, but 4 manifested higher
stability in rat plasma (Figures S1–S4, Supporting Information).
Thus, for the present study, the prodrug moiety of 4 (i.e., iso-
propyl ester bis(l-phenylalanine)) was chosen for the novel
series of 2-substituted PMEA derivatives 5. Because the stability
of prodrugs can be affected not only by the prodrug moiety,
but also by substitution on the purine moiety of PMEA, the
stability of the most potent prodrug of series 5, 2-fluoro PMEA
derivative 5n, was evaluated.
The hydrolysis of 5n in macrophage homogenate is rapid,
as only some 10% of 5n remains uncleaved after 1 h, and
~30% of free 2-fluoro PMEA is present (Figure 4). Interestingly,
free dehalogenated PMEA also appeared in 2 h. Evidently, 2-
fluoro PMEA is slowly metabolized by cellular enzymes to
PMEA. The pilot pharmacokinetic study in rats showed that
a large portion of the parent prodrug 5n remained unchanged
in rat plasma over the course of a 2 h experiment (Figure 5).
Conclusions
A series of 18 PMEA analogues substituted at the C2 position
of the purine moiety and in their isopropyl ester bis(l-phenyla-
lanine) prodrug (compounds 5) form were synthesized and
studied as potent inhibitors of B. pertussis ACT. The SAR study
showed that a small substituent at C2 is well tolerated, for
which 2-fluoro derivative 5n was found to be the most potent
inhibitor of the entire series (IC50: 145 nm). The bisamidate pro-
drug 5n was shown to be more stable in rat plasma than in
macrophage homogenate, and 5n, and/or its metabolites,
were efficiently distributed to organs, including lungs. The
tested compounds demonstrated promising selectivity for ACT
over mammalian ACs and no cytotoxicity. As ACT is an signifi-
cant invasive toxin secreted by B. pertussis, its successful neu-
tralization could prevent toxemia in the host. Thus, potent in-
hibitors of ACT activity based on acyclic nucleoside phospho-
nates could be used for the prophylaxis or even treatment of
whooping cough (and possibly of anthrax), especially in com-
bination with the appropriate antibacterial agent.
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moved in vacuo, and the residue was purified by column chroma-
tography.
Experimental Section
Unless otherwise stated, solvents were evaporated at 408C/2 kPa,
and the compounds were dried over P2O5 at 2 kPa. Solvents were
dried by standard procedures. Tetrahydrofuran (THF) was freshly
distilled from sodium/benzophenone under Ar. N,N-Dimethylforma-
mide (DMF) and acetonitrile were distilled from P2O5 and stored
over molecular sieves (4 A). TLC was performed on plates of Kiesel-
gel 60 F254 (Merck). Mass spectra were measured on an LCQ classic
spectrometer using electrospray ionization (ESI). NMR spectra were
recorded on Bruker Avance 500 (1H at 500 MHz; 13C at 125.8 MHz)
and Bruker Avance 400 (1H at 400 MHz; 13C at 100.6 MHz) spec-
trometers, with TMS as internal standard or referenced to the resid-
ual solvent signal. Reagents were obtained from commercial sour-
ces (Sigma–Aldrich). Starting compounds were prepared according
to published procedures.[32] Preparative HPLC purifications were
performed on columns packed with 10 mm C18 reversed-phase
resin (Phenomenex Gemini 10 mm 21”250 mm) on a Waters
Delta 600 chromatograph system, in ~200 mg batches of mixtures
using a gradient of MeOH/H2O as eluent. Flash chromatography on
normal phase and deionization on reversed phase was performed
on a Reveleris Flash Chromatography System. Deionization was
performed on a Redisep Rf Gold C18 Teledyne ISCO column. All
tested ANPs were characterized by 1H NMR, 13C NMR, and mass
spectrometry. The purity of the compounds was determined by
combustion elemental analysis (C, H, N). Compound 6 was pre-
pared according to a published procedure.[28]
6-Chloro-2-iodo-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}-
9H-purine (7). CH2I2 (55 mL, 682 mmol) was added to a mixture of
6
(18.3 g, 21.3 mmol), I2 (16.5 g, 65 mmol), and CuI (13.2 g,
69 mmol) in dry THF (330 mL) under Ar at room temperature. Isoa-
mylnitrite (57 mL, 424 mmol) was subsequently added dropwise to
the reaction mixture. The resulting slurry was placed on a preheat-
ed oil bath (808C) and held at reflux for 30 min. The reaction mix-
ture was cooled to room temperature, and the solid was filtered
off. The filtrate was evaporated in vacuo, dissolved in EtOAc
(300 mL), and filtered once again. The filtrate was washed with sa-
turated aqueous Na2S2O3 (2”100 mL), brine (100 mL), and dried
over Na2SO4. Volatiles were removed in vacuo, and the residue was
purified by flash chromatography (MeOH/CHCl3 3:97) to give 18 g
(77%) of 7 as a pale-yellow solid. ESIMS m/z (%): 503 (35) [M+H]+;
1H NMR (400 MHz, CDCl3): d=1.26 (d, J=6.2 Hz, 6H, CH3), 1.29 (d,
J=6.2 Hz, 6H, CH3), 3.72 (d, J=8.2 Hz, 2H, P-CH2), 3.91–3.96 (m,
2H, O-CH2), 4.47–4.44 (m, 2H, N-CH2), 4.68 (m, 2H, CH-iPr),
8.21 ppm (s, 1H, H-8); 13C NMR (100 MHz, CDCl3): d=23.97 (2C, d,
J
P,C =4.6 Hz, CH3), 23.98 (2C, d, JP,C =3.6 Hz, CH3), 44.08 (C1’), 66.10
(d, JP,C =168.0 Hz, P-C), 70.52 (d, JP,C =9.7 Hz, C2’), 71.26 (2C, d, JP,C
=
6.7 Hz, POC), 116.31 (C2), 131.42 (C5), 146.25 (C8), 150.29 (C6),
152.53 ppm (C4); HRMS (ESI+): m/z [M+H]+ calcd for
C14H21ClN4O4P: 503.0112, found: 503.0110.
6-Amino-2-iodo-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}-
9H-purine (8). A mixture of compound 7 (15 g, 30 mmol) and
EtOH/NH3 (250 mL) was stirred at room temperature for 24 h. The
solvent was removed, and the residue was purified by flash chro-
matography (MeOH/CHCl3, 2:98) to give 12 g (83%) of compound
General cross-coupling reaction procedures
1
8. ESIMS m/z (%): 484 (45) [M+H]+; H NMR (400 MHz, CDCl3): d=
Method A: A solution of ZnBr2 (1m in THF; 7 mL, 7 mmol) was
added dropwise to the appropriate Grignard reagent (6 mmol) in
10 mL THF at À788C under Ar. The reaction mixture was stirred at
À788C for 15 min, allowed to warm to room temperature (30 min),
and stirred at 508C for 30 min. The resulting mixture was cooled to
room temperature. The solution of compound 8 (0.48 g, 1 mmol)
and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in THF (15 mL) under Ar was added
dropwise to the reaction mixture at room temperature. Saturated
aqueous NH4Cl (10 mL) was added to the reaction mixture, and it
was stirred for 15 min. The reaction was poured into EtOAc
(150 mL). The organic layer was washed with a saturated EDTA so-
lution (30 mL), brine (30 mL), and then dried over Na2SO4. Volatiles
were removed in vacuo, and the residue was purified by flash chro-
matography.
1.27 (d, J=6.2 Hz, 6H, CH3), 1.30 (d, J=6.2 Hz, 6H, CH3), 3.73 (d,
J=8.3 Hz, 2H, P-CH2), 3.90 (t, 2H, O-CH2), 4.35 (t, 2H, N-CH2), 4.63–
4.76 (m, 2H, CH-iPr), 7.92 ppm (s, 1H, H-8); 13C NMR (100 MHz,
CDCl3): d=23.98 (2C, d, JP,C =4.5 Hz, CH3), 24.02 (2C, d, JP,C =3.8 Hz,
CH3), 43.61 (C1’), 66.04 (d, JP,C =168.0 Hz, P-C), 70.97 (d, JP,C
=
10.4 Hz, C2’), 71.24 (2C, d, JP,C =6.7 Hz, POC), 118.61 (C5), 119.65
(C2), 141.20 (C8), 150.21 (C4), 154.99 ppm (C6); HRMS (ESI+): m/z
[M+H]+ calcd for C14H24IN5O4P: 484.0611, found 405.0614.
6-Amino-2-methyl-9-{2-[(isopropoxyphosphoryl)methoxy]ethyl}-
9H-purine (9a). Compound 8 (0.48 g, 1 mmol) and Pd(PPh3)4
(0.1 g) were purged with Ar and dissolved in THF (20 mL). A solu-
tion of Al(CH3)3 in toluene (2m, 1 mL, 2 mmol) was added dropwise
to the above solution. The reaction mixture was heated at 708C for
3 h, cooled and poured into saturated aqueous NaH2PO4 (20 mL).
The precipitated solid was removed by filtration, the filtrate was
evaporated in vacuo, and the residue was purified by flash chroma-
tography (CHCl3/MeOH/H2O, 5:4:1) to give 9a (0.21 g, 65%). ESIMS
Method B: Compound 8 (0.48 g, 1 mmol) and Pd(PPh3)4 (0.06 g)
were dissolved in DMF (5 mL) under Ar. A solution of alkylZnBr
(0.5m in THF; 10 mL, 5 mmol) was added dropwise to the reaction
mixture, and it was stirred at room temperature for 2 h. Saturated
NH4Cl solution (10 mL) was added to the reaction mixture, and it
was stirred for 15 min and then poured into EtOAc (150 mL). The
organic layer was washed with a saturated solution of EDTA
(30 mL), brine (30 mL), and then dried over Na2SO4. Volatiles were
removed in vacuo, and the residue was purified by column chro-
matography.
1
m/z (%): 330 (100) [M+H]+; H NMR (400 MHz, [D6]DMSO): d=0.96
(d, J=4.5 Hz, 6H, CH3), 2.37 (s, 3H, H-1’’), 3.41 (d, J=8.7 Hz, 2H, P-
CH2), 3.77 (t, J=5.0 Hz 2H, O-CH2), 4.12–4.21 (m, 1H, CH-iPr), 4.27
(t, J=5.0 Hz, 2H, N-CH2), 8.09 ppm (s, 1H, H-8); 13C NMR (100 MHz,
[D6]DMSO): d=24.18 (2C, d, JP,C =3.7 Hz, CH3), 25.12 (C1’’), 42.47
(C1’), 67.41 (d, JP,C =147.4 Hz, P-C), 70.14 (d, JP,C =11.2 Hz, C2’), 66.57
(C, d, JP,C =5.8 Hz, POC), 116.55 (C5), 140.83 (C8), 150.06 (C4), 155.18
(C6), 160.44 ppm (C2); HRMS (ESI+): m/z [M+H]+ calcd for
C12H21N5O4P: 330.1326, found: 330.1326.
Method C: A solution of alkylZnBr (0.5m in THF; 10 mL, 5 mmol)
was added dropwise to a solution of compound 8 (0.48 g, 1 mmol)
and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in THF (15 mL) under Ar. Saturated
NH4Cl solution (10 mL) was added to the reaction mixture and
stirred for 15 min. The reaction was poured into EtOAc (150 mL).
The organic layer was washed with a saturated solution of EDTA
(30 mL), brine (30 mL), and dried over Na2SO4. Volatiles were re-
6-Amino-2-ethyl-9-{2-[(diisopropoxyphosphoryl)methoxy]ethyl}-
9H-purine (9b). Prepared from compound 8 by Method A (reac-
tion time 4 h), yield 0.70 g (93%). ESIMS m/z (%): 386 (100) [M+
1
H]+; H NMR (400 MHz, CDCl3): d=1.24 (d, J=6.2 Hz, 6H, CH3), 1.27
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(d, J=6.2 Hz, 6H, CH3), 1.31 (t, J=7.6 Hz, 3H, H-2’’), 2.78 (q, J=
7.6 Hz, 2H, H-1’’), 3.71 (d, J=8.3 Hz, 2H, P-CH2), 3.92 (t, J=5.1 Hz,
2H, O-CH2), 4.36 (t, J=5.1 Hz, 2H, N-CH2), 4.61–4.72 (m, 2H, CH-
iPr), 6.04 (brs, 2H, NH2), 7.90 ppm (s, 1H, H-8); 13C NMR (100 MHz,
CDCl3): d=13.05 (C2’’), 23.89 (2C, d, JP,C =6.9 Hz, CH3), 23.97 (2C, d,
2H, NH2), 7.92 ppm (s, 1H, H-8); 13C NMR (100 MHz, CDCl3): d=
12.09 (C4’’), 19.68 (C1’’), 23.96 (2C, d, JP,C =6.6 Hz, CH3), 24.00 (2C, d,
J
J
P,C =6.0 Hz, CH3), 29.24 (C3’’), 43.27 (C1’), 44.32 (C2’’), 66.06 (d,
P,C =167.8 Hz, P-C), 71.15 (d, JP,C =10.6 Hz, C2’), 71.15 (C, d, JP,C
=
6.6 Hz, POC), 117.43 (C5), 141.37 (C8), 150.63 (C4), 154.59 (C6),
168.82 ppm (C2); HRMS (ESI+): m/z [M+H]+ calcd for C18H33N5O4P:
414.2265, found: 414.2264.
J
P,C =6.9 Hz, CH3), 32.51 (C1’’), 43.13 (C1’), 66.00 (d, JP,C =167.8 Hz, P-
C), 71.24 (d, JP,C =10.5 Hz, C2’), 71.09 (C, d, JP,C =6.7 Hz, POC), 117.55
(C5), 140.93 (C8), 150.69 (C4), 155.20 (C6), 166.73 ppm (C2); HRMS
(ESI+): m/z [M+H]+ calcd for C16H29N5O4P: 386.1952, found:
386.1950.
6-Amino-2-isobutyl-9-{2-[(disopropoxyphosphoryl)methoxy]eth-
yl}-9H-purine (9g). A solution of tert-butylzinc bromide in THF
(0.5m, 10 mL, 5 mmol) was added to the mixture of 8 (0.48 g,
1.0 mmol), 2,6-di-tert-butyl-4-methylphenol (BHT) (0.22 g, 1 mmol),
and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in N-methylpyrrolidone (50 mL)
under Ar at 808C. The reaction mixture was stirred at 808C for 4 h
and cooled to room temperature. Saturated NH4Cl solution
(100 mL) was added to the reaction mixture and stirred for 15 min.
The reaction mixture was poured into EtOAc (150 mL). The organic
layer was washed with saturated solution of EDTA (30 mL), brine
(30 mL), and dried over Na2SO4. Volatiles were removed in vacuo,
and the residue was purified by flash chromatography (MeOH/
CHCl3, 3:97) to give 0.21 g (49%) of 9g as a pale-yellow solid.
6-Amino-2-propyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-
9H-purine (9c). Prepared from compound 8 by Method B (reaction
time 2 h), yield 0.27 g (70%). ESIMS m/z (%): 400 (100) [M+H]+;
1H NMR (400 MHz, CDCl3): d=0.98 (t, J=7.4 Hz, 3H, H-3’’), 1.26 (d,
J=6.2 Hz, 6H, CH3), 1.29 (d, J=6.2 Hz, 6H, CH3), 1.75–1.87 (m, 2H,
H-2’’), 2.77 (t, J=7.7 Hz, 2H, H-1’’), 3.72 (d, J=8.3 Hz, 2H, P-CH2),
3.93 (t, J=4.9 Hz, 2H, O-CH2), 4.37 (t, J=4.9 Hz, 2H, N-CH2), 4.66–
4.72 (m, 2H, CH-iPr), 5.95 (brs, 2H, NH2), 7.90 ppm (s, 1H, H-8);
13C NMR (100 MHz, CDCl3): d=13.93 (C3’’), 22.31 (C2’’), 23.94 (2C, d,
J
P,C =6.9 Hz, CH3), 23.99 (2C, d, JP,C =5.7 Hz, CH3), 41.06 (C1’’), 43.26
1
ESIMS m/z (%): 414 (100) [M+H]+; H NMR (400 MHz, CDCl3): d=
(C1’), 66.06 (d, JP,C =167.8 Hz, P-C), 71.20 (d, JP,C =10.4 Hz, C2’),
71.14 (C, d, JP,C =6.8 Hz, POC), 117.49 (C5), 141.27 (C8), 150.62 (C4),
154.71 (C6), 165.14 ppm (C2); HRMS (ESI+): m/z [M+H]+ calcd for
C17H31N5O4P: 400.2108, found: 400.2107.
0.95 (d, J=6.6 Hz, 6H, CH3-iBu), 1.25 (d, J=6.2 Hz, 6H, CH3), 1.29
(d, J=6.2 Hz, 6H, CH3), 2.22–2.29 (m, 1H, CH-1’’), 2.67 (d, J=7.3 Hz,
2H, C1’’), 3.72 (d, J=8.3 Hz, 2H, P-CH2), 3.93 (t, J=4.9 Hz, 2H, N-
CH2), 4.37 (t, J=4.9 Hz, 2H, O-CH2), 4.64–4.74 (m, 2H, CH-iPr), 5.97
(s, 2H, NH2), 7.90 ppm (s, 1H, H-8); 13C NMR (100 MHz, CDCl3): d=
6-Amino-2-isopropyl-9-{2-[(disopropoxyphosphoryl)methox-
y]ethyl}-9H-purine (9d). Prepared from compound 8 by Method A
(reaction time 2 h), yield 0.34 g (86%). ESIMS m/z (%): 400 (100)
[M+H]+; 1H NMR (400 MHz, CDCl3): d=1.25 (d, J=6.2 Hz, 6H, H-
2’’), 1.29 (d, J=6.2 Hz, 6H, CH3), 1.30 (t, J=6.9 Hz, 6H, CH3), 3.04
(td, J=13.7, J=6.8 Hz, 1H, H-1’’), 3.72 (d, J=8.3 Hz, 2H, P-CH2),
3.94 (t, J=5.0 Hz, 2H, O-CH2), 4.37 (t, J=4.9 Hz, 2H, N-CH2), 4.66–
4.71 (m, 2H, CH-iPr), 5.90 (brs, 2H, NH2), 7.88 ppm (s, 1H, H-8);
13C NMR (100 MHz, CDCl3): d=21.93 (C2’’), 23.94 (2C, d, JP,C =6.9 Hz,
CH3), 23.99 (2C, d, JP,C =6.1 Hz, CH3), 37.23 (C1’’), 43.18 (C1’), 66.03
22.36 (2CCH3), 23.95 (2C, d, JP,C =6.5 Hz, CH3), 24.00 (2C, d, JP,C
5.8 Hz, CH3), 28.52 (C2’’), 43.31 (C1’), 47.64 (C1’’), 66.08 (d, JP,C
167.8 Hz, P-C), 71.15 (2C, d, JP,C =6.9 Hz, POC), 71.18 (d, JP,C
=
=
=
10.1 Hz, C2’), 117.43 (C5), 141.38 (C8), 150.54 (C4), 154.50 (C6),
164.33 ppm (C2); HRMS (ESI+): m/z [M+Na]+ calcd for
C18H32N5O4PNa: 436.2084, found: 436.2082.
6-Amino-2-(pentan-3-yl)-9-{2-[(disopropoxyphosphoryl)methoxy]-
ethyl}-9H-purine (9h). Prepared from 8 by Method A, (reaction
time 4.5 h), yield 0.33 g (78%). ESIMS m/z (%): 428 (100) [M+H]+;
1H NMR (400 MHz, CDCl3): d=0.81 (t, J=7.4 Hz, 6H, H-3’’), 1.26 (d,
J=6.2 Hz, 6H, CH3), 1.29 (d, J=6.2 Hz, 6H, CH3), 1.61–1.87 (m, 4H,
H-2’’-CH2), 2.55–1.65 (m, 1H, H-1’’), 3.72 (d, J=8.3 Hz, 2H, P-CH2),
3.94 (t, J=5.0 Hz, 2H, O-CH2), 4.38 (t, J=5.0 Hz, 2H, N-CH2), 4.65–
4.74 (m, 2H, CH-iPr), 5.96 (brs, 2H, NH2), 7.90 ppm (s, 1H, H-8);
(d, JP,C =167.8 Hz, P-C), 71.19 (d, JP,C =10.5 Hz, C2’), 71.13 (C, d, JP,C
=
6.8 Hz, POC), 117.56 (C5), 141.16 (C8), 150.65 (C4), 154.82 (C6),
169.56 ppm (C2); HRMS (ESI+): m/z [M+H]+ calcd for C17H31N5O4P:
400.2108, found: 400.2107.
6-Amino-2-butyl-9-{2-[(disopropoxyphosphoryl)methoxy]ethyl}-
9H-purine (9e). Prepared from compound 8 by Method B (reaction
time 2 h), yield 0.40 g (97%). ESIMS m/z (%): 414 (100) [M+H]+;
1H NMR (400 MHz, CDCl3): d=0.92 (t, J=7.4 Hz, 3H, 4’-CH3), 1.24
(d, J=6.2 Hz, 6H, CH3), 1.28 (d, J=6.2 Hz, 6H, CH3), 1.33–1.45 (m,
2H, H-3’’), 1.69–1.81 (m, 2H, H-2’’), 2.75 (t, J=7.9 Hz, 2H, H-1’’),
3.71 (d, J=8.3 Hz, 2H, P-CH2), 3.91 (t, J=4.9 Hz, 2H, O-CH2), 4.36 (t,
J=4.9 Hz, 2H, N-CH2), 4.61–4.74 (m, 2H, CH-iPr), 6.01 (brs, 2H,
NH2), 7.87 ppm (s, 1H, H-8); 13C NMR (100 MHz, CDCl3): d=13.92
(C4’’), 23.96 (2C, d, JP,C =6.0 Hz, CH3), 23.92 ppm (2C, d, JP,C =6.7 Hz,
CH3): d=22.58 (C3’’), 31.18 (C2’’), 38.90 (C1’’), 43.21 (C1’), 66.02 (d,
13C NMR (100 MHz, CDCl3): d=12.09 (C3’’-CH3), 23.96 (2C, d, JP,C
=
6.9 Hz, CH3), 24.00 (2C, d, JP,C =6.2 Hz, CH3), 27.50 (C2’’-CH2), 43.30
(C1’), 51.79 (C1’’), 66.08 (d, JP,C =167.9 Hz, P-C), 71.15 (C, d, JP,C =6.4
Hz, POC), 71.16 (d, JP,C =10.9 Hz, C2’), 117.52 (C5), 141.34 (C8),
150.59 (C4), 154.48 (C6), 168.83 ppm (C2); HRMS (ESI+): m/z [M+
H]+ calcd for C19H35N5O4P: 428.2421, found: 428.2419.
6-Amino-2-(tetrahydrofuran-2-yl)-9-{2-[(disopropoxyphosphoryl)-
methoxy]ethyl}-9H-purine (9i). A solution of 1-adamantylzinc bro-
mide in THF (0.5m, 10 mL, 5 mmol) was added to the mixture of 8
(0.48 g, 1.0 mmol) and Pd(dppf)Cl2·CH2Cl2 (0.04 g) in THF under Ar
at À408C. The reaction mixture was allowed to warm to room tem-
perature and stirred for 2 h at room temperature. Saturated NH4Cl
solution (100 mL) was added to the reaction mixture and stirred
for 15 min. The reaction was poured into EtOAc (150 mL) and the
organic layer was washed with saturated solution of EDTA (30 mL),
brine (30 mL), and dried over Na2SO4. Volatiles were removed in
vacuo and the residue was purified by flash chromatography
(MeOH/CHCl3 6:94) to give 0.33 g (78%) of 9i as a white solid.
J
P,C =167.8 Hz, P-C), 71.19 (d, JP,C =10.5 Hz, C2’), 71.13 (C, d, JP,C =
6.7 Hz, POC), 117.38 (C5), 141.09 (C8), 150.56 (C4), 154.89 (C6),
165.51 ppm (C2); HRMS (ESI+): m/z [M+H]+ calcd for C18H33N5O4P:
414.2265, found: 414.2263.
6-Amino-2-(sec-butyl)-9-{2-[(disopropoxyphosphoryl)methox-
y]ethyl}-9H-purine (9 f). Prepared from compound 8 by Method C
(reaction time 1.5 h), yield 0.32 g (77%). ESIMS m/z (%): 414 (100)
[M+H]+; 1H NMR (400 MHz, CDCl3): d=0.86 (t, J=7.4 Hz, 3H, H-
4’’), 1.26 (d, J=6.2 Hz, 6H, CH3), 1.29 (d, J=6.2 Hz, 6H, CH3), 1.28
(d, 3H, H-1’’), 1.60 and 1.86 2x (m, 1H, H-3’’), 2.77–2.85 (m, 1H, H-
2’’), 3.72 (d, J=8.3 Hz, 2H, P-CH2), 3.94 (t, J=5.0 Hz, 2H, O-CH2),
4.38 (t, J=5.0 Hz, 2H, N-CH2), 4.68–4.72 (m, 2H, CH-iPr), 5.93 (brs,
1
ESIMS m/z (%): 450 (100) [M+Na+]; H NMR (400 MHz, CDCl3): d=
1.28 (d, J=6.2 Hz, 3H, CH3), 1.28 (d, J=6.2 Hz, 3H, CH3), 1.31 (d,
J=6.2 Hz, 6H, CH3), 1.98–2.07 (m, 2H, H-4’’), 2.14–2.35 (m, 2H, H-
3’’), 3.74 (d, J=8.4 Hz, 2H, P-CH2), 3.94 (t, J=5.0 Hz, 2H, O-CH2),
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3.97–4.24 (m, 1H, H-5’’), 4.43 (m, 2H, N-CH2), 4.67–4.76 (m, 2H, CH-
iPr), 4.95 (dd, 1H, J=7.2 Hz, J=6.4 Hz, H-3’’), 6.64 (brs, 2H, NH2),
8.21 ppm (s, 1H, H-8); 13C NMR (100 MHz, CDCl3): d=23.95–24.03
stirred at room temperature overnight. After evaporation of the
volatiles and co-distillation with dry pyridine (20 mL) in vacuo
(without any contact with air), the flask was purged with Ar and
amino acid ester hydrochloride (4 mmol, dried in vacuo at 308C
and 0.1 mbar for 1 day), dry triethylamine (2 mL) and dry pyridine
(8 mL) were added, and the mixture was heated at 708C to obtain
a homogenous solution. Then, a solution of 2,2’-dipyridyldisulfide
(6 mmol) and triphenylphosphine (6 mmol) in dry pyridine (10 mL)
under Ar was added. The resulting mixture was heated at 708C.
After cooling, the solvent was removed in vacuo, and the residue
was purified by flash chromatography (2% MeOH in a mixture of
hexane/EtOAc, 60:40) to remove impurities, followed by hexane/
EtOAc 60:40 and gradient of MeOH (2–30%) to obtain the desired
product. Subsequently, polar impurities were removed by reversed-
phase chromatography on C18 silica gel (aqueous MeOH 1–100%).
The products were lyophilized from dioxane.
(m, CH3iPr), 25.68 (C4’’), 32.16 (C3’’), 43.68 (C1’), 66.04 (d, JP,C
=
168.2 Hz, P-C), 69.39 (C5’’), 70.86 (d, JP,C =10.4 Hz, C2’), 71.35 (2C, d,
J
P,C =6.6 Hz, POC), 80.97 (C2’’), 116.73 (C5), 142.66 (C8), 150.27 (C4),
154.35 (C6), 164.30 ppm (C2); HRMS (ESI+): m/z [M+H]+ calcd for
C18H31N5O5P: 428.2057, found: 428.2057.
6-Amino-2-cyclopentyl-9-{2-[(disopropoxyphosphoryl)methox-
y]ethyl}-9H-purine (9j). Prepared from 8 by Method C (reaction
time 1.5 h), yield 0.35 g (82%). ESIMS m/z (%): 426 (100) [M+H]+;
1H NMR (400 MHz, CDCl3): d=1.26 (d, J=6.2 Hz, 6H, CH3), 1.30 (d,
J=6.2 Hz, 6H, CH3), 1.60–1.72 (m, 2H, CH2), 1.80 (m, 2H, (m, 2H,
CH2), 1.86–1.95 (m, 2H, CH2), 1.98–2.08 (m, 2H, CH2), 3.18 (pent, J=
8.3 Hz, 1H, H-1’), 3.73 (d, J=8.3 Hz, 2H, P-CH2), 3.94 (t, J=5.0 Hz,
2H, N-CH2), 4.39 (t, J=5.0 Hz, 2H, O-CH2), 4.66–4.74 (m, 2H, CH-
iPr), 6.26 (s, 2H, NH2), 8.05 ppm (s, 1H, H-8); 13C NMR (100 MHz,
CDCl3): d=23.94 (2C, d, JP,C =5.6 Hz, CH3), 24.01 (2C, d, JP,C =4.9 Hz,
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
methyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5a). Start-
ing compound 9a (0.11 g, 0.40 mmol), Method D, reaction time
48 h, yield 0.18 g (70%) of 5a. ESIMS m/z (%): 688 (100) [M+Na]+;
1H NMR (500 MHz, [D6]DMSO): d=1.01 (d, J=6.3 Hz, 3H, CH3), 1.06
(d, J=6.3 Hz, 3H, CH3), 1.11 (d, J=6.3 Hz, 3H, CH3), 1.16 (d, J=
6.3 Hz, 3H, CH3), 2.71–2.90 (m, 4H, CH2-Bn), 3.24 (dd, J=8.1 Hz, J=
13.3 Hz, 1H, Pb-CH2), 3.31 (dd, J=7.8 Hz, J=13.3 Hz, 1H, Pa-CH2),
3.68 (t, J=5.3 Hz, 2H, O-CH2), 3.84–3.98 (m, 2H, NH-CH), 4.11 (dd,
J=10.6 Hz, J=12.1 Hz, 1H, NH), 4.21 (t, J=5.3 Hz, 2H, N-CH2), 4.42
(dd, J=10.8 Hz, J=12.0 Hz, 1H, NH), 4.78–4.82 (sept, J=6.3 Hz,
CHiPr), 7.04 (bs, 2H, NH2), 7.09–7.26 (m, 10H, H-arom), 8.01 ppm (s,
1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=21.45, 21.52, 21.58 and
21.65 (CH3-iPr), 25.65 (C1’’), 40.17 (CH2-Bn), 42.45 (C1’), 54.02 and
54.21 (NH-CH), 67.51 (d, JP,C =135.3 Hz, P-C), 67.97 and 68.10 (2C,
POC), 70.47 (d, JP,C =11.3 Hz, C2’), 116.90 (C5), 126.57 and 126.63
(C4’’ arom), 128.20 and 128.24 (C3’’ arom), 129.62 (C2’’ arom),
137.18 and 137.26 (C1’’ arom), 140.74 (C8), 150.45 (C4), 155.75 (C6),
161.11 (C2), 172.30–172.44 ppm (m, COO); Anal. calcd for
C33H44N7O6P·5/4H2O: C (57.59), H (6.81), N (14.15), P (4.50), found: C
(57.94), H (6.73), N (13.70), P (4.30).
CH3), 32.87 (C2’’, C3’’), 43.45 (C1’), 48.19 (C1’’), 66.07 (d, JP,C
=
167.9 Hz, P-C), 70.95 (d, JP,C =10.4 Hz, C2’), 71.25 (2C, d, JP,C =6.7 Hz,
POC), 116.64 (C5), 141.76 (C8), 150.57 (C4), 154.40 (C6), 168.67 ppm
(C2); HRMS (ESI+): m/z [M+H]+ calcd for C19H33N5O4P: 426.2265,
found: 426.2263.
6-Amino-2-(bicyclo[2.2.1]heptan-2-yl)-9-{2-[(disopropoxyphos-
phoryl)methoxy]ethyl}-9H-purine (9k). Prepared from 8 by Meth-
od C (reaction time 1.5 h), yield 0.31 g (69%). ESIMS m/z (%): 452
(100) [M+H]+; 1H NMR (400 MHz, CDCl3): d=1.09 (dm, J=9.2 Hz,
1H, H-7b’’-CH2), 1.25 (d, J=6.2 Hz, 6H, CH3), 1.29 (d, J=6.2 Hz, 6H,
CH3), 1.23 (m, 1H, endo H-5’’), 1.32 (m, 1H, endo H-6’’), 1.46–1.55
(m, 3H, endo H-3’’, exo H-5’’, exo H-6’’), 1.72 (dm, J=9.2 Hz, 1H, H-
7a’’-CH2), 2.15–2.18 (m, 1H, exo H-3’’), 2.28 (m, 1H, H-4’’), 2.83–2.87
(m, 1H, H-1’’), 3.72 (d, J=8.3 Hz, 2H, P-CH2), 3.94 (t, J=5.0 Hz, 2H,
O-CH2), 4.34 (t, J=5.0 Hz, 2H, N-CH2), 4.60–4.76 (m, CHiPr), 5.84
(brs, 2H, NH2), 7.87 ppm (s, 1H, H-8); 13C NMR (125 MHz,
[D6]DMSO): d=23.91 (2C, d, JP,C =6.9 Hz, CH3), 23.97 (2C, d, JP,C
5.7 Hz, CH3), 29.10 (C5’’), 30.16 (C6’’), 35.97 and 36.18 (C3’’, C7’’),
36.58 (C4’’), 42.87 (C1’), 42.95 (C1’’), 50.09 (C2’’), 66.06 (d, JP,C
=
=
167.8 Hz, P-C), 71.13 (d, JP,C =9.9 Hz, C2’), 71.17 (2C, d, JP,C =7.2 Hz,
POC), 117.34 (C5), 141.16 (C8), 150.49 (C4), 154.52 (C6), 168.15 ppm
(C2); HRMS (ESI+): m/z [M+H]+ calcd for C21H35N5O4P: 452.2421,
found: 452.2419.
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
ethyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5b). Starting
compound 9b (0.50 g, 1.3 mmol), Method D, reaction time 72 h,
yield 0.49 g (55%) of 5b. ESIMS m/z (%): 680 (100) [M+H]+;
1H NMR (500 MHz, CDCl3): 1.27 (d, J=6.3 Hz, 3H, CH3), 1.32 (d, J=
6.3 Hz, 6H, CH3), 1.37 (d, J=6.3 Hz, 3H, CH3), 1.46 (t, J=7.6 Hz, 3H,
H-2’’), 2.86–3.20 (m, 8H, CH2-Bn, P-CH2, H-1’’), 3.33–3.45 (m, 2H, O-
CH2), 3.80–3.83 (m, 2H, NH-CH), 4.10–4.18 (m, 1H, NH), 4.27–4.35
(m, 1H, NH), 4.39 (t, J=4.9 Hz, 2H, N-CH2), 5.03–5.15 (m, 2H,
CHiPr), 5.79 (bs, 2H, NH2), 7.18–7.38 (m, 10H, H-arom), 7.91 ppm (s,
1H, H-8); 13C NMR (125 MHz, CDCl3): d=13.28 (C2’’), 21.62, 2x 21.72
and 21.80 (CH3-iPr), 32.58 (C1’’), 40.67 (CH2-Bn), 43.10 (C1’), 53.65
and 54.02 (NH-CH), 68.04 (d, JP,C =135.9 Hz, P-C), 69.97 and 69.17
(2C, POC), 71.12 (d, JP,C =12.1 Hz, C2’), 117.66 (C5), 126.86 and
126.89 (C4’’ arom), 128.33 and 128.40 (C3’’ arom), 129.60 and
129.62 (C2’’ arom), 136.30 and 136.56 (C1’’ arom), 141.02 (C8),
150.75 (C4), 155.12 (C6), 166.80 (C2), 172.37–172.61 ppm (m, COO);
Anal. calcd for C34H46N7O6P·1/2H2O: C (59.29), H (6.88), N (14.24), P
(4.50), found: C (59.68), H (7.08), N (14.07), P (4.44).
6-Amino-2-cyclopropylamino-9-{2-[(disopropoxyphosphoryl)me-
thoxy]ethyl}-9H-purine (9l). Prepared from 8 (0.60 g, 1.24 mmol)
according to lit.[31] yield 0.30 g (59%). ESIMS m/z (%): 413 (100)
[M+H]+; 1H NMR (400 MHz, CDCl3): d=0.57–0.46 (m, 2H) and
0.82–0.69 (m, 2H, CH2-cypr), 1.26 (d, J=6.2 Hz, 6H, CH3), 1.29 (d,
J=6.2 Hz, 6H, CH3), 2.79–2.72 (m, 1H, CH-1’’), 3.73 (d, J=8.4 Hz,
2H, P-CH2), 3.92 (t, J=5.0 Hz, 2H, N-CH2), 4.24 (t, J=5.0 Hz, 2H, O-
CH2), 4.67–4.71 (m, 2H, CH-iPr), 5.24 (s, 1H, NH), 5.73 (s, 2H, NH2),
7.64 ppm (s, 1H, H-8); 13C NMR (100 MHz, CDCl3): d=7.32 (2Ccypr),
23.95 (2C, d, JP,C =7.9 Hz, CH3), 23.99 (2C, d, JP,C =7.2 Hz, CH3), 24.19
(C1’’), 42.92 (C1’), 66.03 (d, JP,C =167.8 Hz, P-C), 71.17 (d, JP,C
=
10.7 Hz, C2’), 71.13 (2C, d, JP,C =6.8 Hz, POC), 113.85 (C5), 138.83
(C8), 152.11 (C4), 155.05 (C6), 159.70 ppm (C2); HRMS (ESI+): m/z
[M+H]+ calcd for C17H30N6O4P: 413.2061, found: 413.2062.
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(propyl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5c). Start-
ing compound 9c (0.26 g, 0.67 mmol), Method D, reaction time
24 h, yield 0.26 g (57%) of 5c. ESIMS m/z (%): 694 (100) [M+H]+;
1H NMR (500 MHz, [D6]DMSO): d=0.91 (t, J=7.4 Hz, 3H, H-3’’-CH3),
1.01 (d, J=6.2 Hz, 3H, CH3), 1.07 (d, J=6.2 Hz, 3H, CH3), 1.11 (d, J=
General procedure for preparation of the bisamidate
prodrugs 5
Method D: Diester 9 (1 mmol) was dissolved in dry pyridine
(10 mL), and TMSBr (1 mL) was added. The reaction mixture was
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6.2 Hz, 3H, CH3), 1.16 (d, J=6.2 Hz, 3H, CH3), 1.73 (m, 2H, H-2’’),
2.62 (m, 2H, H-1’’), 2.72–2.90 (m, 4H, CH2-Bn), 3.24 (dd, J=8.2 Hz,
J=13.3 Hz, 1H, Pb-CH2), 3.31 (dd, J=7.8 Hz, J=13.3 Hz, 1H, Pb-
CH2), 3.69 (t, J=5.3 Hz, 2H, O-CH2), 3.84–3.98 (m, 2H, NH-CH), 4.11
(dd, J=10.6 Hz, J=11.9 Hz, 1H, NH), 4.21 (t, J=5.3 Hz, 2H, N-CH2),
4.43 (m, 1H, NH), 4.78–4.82 (sept, J=6.3 Hz, CH-iPr), 7.01 (brs, 2H,
NH2), 7.09–7.26 (m, 10H, H-arom), 8.01 ppm (s, 1H, H-8); 13C NMR
(125 MHz, [D6]DMSO): d=14.05 (C3’’), 21.45, 21.52, 21.58 and 21.65
(CH3-iPr), 21.92 (C2’’), 40.18 (CH2-Bn), 40.89 (C1’’), 42.47 (C1’), 54.02
and 54.21 (NH-CH), 67.49 (d, JP,C =134.8 Hz, P-C), 67.97 and 68.10
(2C, POC), 70.39 (d, JP,C =11.1 Hz, C2’), 117.08 (C5), 126.56 and
126.63 (C4’’ arom), 128.19 and 128.24 (C3’’ arom), 129.63 (C2’’
arom), 137.19 and 137.26 (C1’’ arom), 140.80 (C8), 150.41 (C4),
155.83 (C6), 164.42 (C2), 172.31–172.44 ppm (m, COO); Anal. calcd
for C35H48N7O6P·H2O: C (59.06), H (7.06), N (13.77), P (4.35), found: C
(58.89), H (7.06), N (13.40), P (4.36).
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(sec-butyl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5 f).
Starting compound 9 f (0.63 g, 1.5 mmol), Method D, reaction time
24 h, yield 0.57 g (53%) of 5 f. ESIMS m/z (%): 730 (100) [M+Na]+;
1H NMR (500 MHz, [D6]DMSO): d=0.79 (t, J=7.4 Hz, 3H, H-4’’), 1.01
(d, J=6.2 Hz, 3H, CH3), 1.07 (d, J=6.2 Hz, 3H, CH3), 1.11 (d, J=
6.2 Hz, 3H, CH3), 1.16 (d, J=6.2 Hz, 3H, CH3), 1.20 (d, J=6.9 Hz, 3H,
H-1’’), 1.52–1.79 (m, 1H, H-3’’), 2.69 (m, 1H, H-2’’), 2.79 (m, 4H, CH2-
Bn), 3.23 (dd, J=8.1 Hz, J=13.2 Hz, 1H, Pb-CH2), 3.31 (m, 1H, Pa-
CH2), 3.69 (t, J=5.4 Hz, 2H, O-CH2), 3.84–3.98 (m, 2H, NH-CH), 4.11
(dd, J=10.6 Hz, J=11.9 Hz, 1H, NH), 4.21 (t, J=5.3 Hz, 2H, N-CH2),
4.43 (dd, J=10.7 Hz, J=11.9 Hz, 1H, NH), 4.78–4.82 (m, CHiPr), 6.98
(brs, 2H, NH2), 7.08–7.26 (m, 10H, H-arom), 8.01 ppm (s, 1H, H-8);
13C NMR (125 MHz, [D6]DMSO): d=12.31 (C4’’-CH3), 20.12 (C1’’),
21.49 (2C, d, JP,C =8.5 Hz, CH3), 21.62 (2C, d, JP,C =8.5 Hz, CH3), 29.08
(C3’’), 40.16 (CH2-Bn), 42.44 (C1’), 44.13 (C2’’), 54.02 and 54.21 (NH-
CH), 67.47 (d, JP,C =135.3 Hz, P-C), 67.97 and 68.10 (2C, POC), 70.30
(d, JP,C =11.1 Hz, C2’), 117.19 (C5), 126.54 and 126.62 (C4’’ arom),
128.19 and 128.23 (C3’’ arom), 129.62 (C2’’ arom), 137.19 and
137.26 (C1’’ arom), 140.82 (C8), 150.36 (C4), 155.91 (C6), 168.10
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
isopropyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5d).
Starting compound 9d (0.30 g, 0.75 mmol), Method D, reaction
time 48 h, yield 0.36 g (69%) of 5d. ESIMS m/z (%): 716 (100) [M+
Na]+; 1H NMR (500 MHz, [D6]DMSO): d=1.01 (d, J=6.3 Hz, 3H,
CH3), 1.06 (d, J=6.3 Hz, 3H, CH3), 1.11 (d, J=6.3 Hz, 3H, CH3), 1.15
(d, J=6.3 Hz, 3H, CH3), 1.23 (d, J=6.9 Hz, 6H, CH3), 2.72–2.90 (m,
4H, CH2-Bn), 2.93 (sept, J=6.9 Hz, 1H, CH), 3.25 (dd, J=8.2 Hz, J=
13.3 Hz, 1H, Pb-CH2), 3.32 (dd, J=7.8 Hz, J=13.3 Hz, 1H, Pa-CH2),
3.70 (t, J=5.4 Hz, 2H, O-CH2), 3.84–3.98 (m, 2H, NH-CH), 4.10 (dd,
J=10.6 Hz, J=12.0 Hz, 1H, NH), 4.22 (t, J=5.3 Hz, 2H, N-CH2), 4.42
(dd, J=10.7 Hz, J=11.9 Hz, 1H, NH), 4.77–4.82 (sept, J=6.3 Hz,
CHiPr), 6.99 (bs, 2H, NH2), 7.09–7.26 (m, 10H, H-arom), 8.01 ppm (s,
1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=21.46, 21.52, 21.58 and
21.65 (CH3-iPr), 22.22 (2xC2’’), 36.96 (C1’’), 40.18 (CH2-Bn), 42.43
(C1’), 54.03 and 54.21 (NH-CH), 67.48 (d, JP,C =135.2 Hz, P-C), 67.97
and 68.11 (2C, POC), 70.32 (d, JP,C =11.1 Hz, C2’), 117.20 (C5), 126.56
and 126.63 (C4’’ arom), 128.19 and 128.24 (C3’’ arom), 129.63 (C2’’
arom), 137.19 and 137.26 (C1’’ arom), 140.85 (C8), 150.36 (C4),
155.91 (C6), 168.73 (C2), 172.31–172.44 ppm (m, COO); Anal. calcd
for C35H48N7O6P·1/2H2O: C (59.82), H (7.03), N (13.85), P (4.41),
found: C (59.43), H (7.07), N (13.41), P (4.11).
(C2),
172.30–172.43 ppm
(m,
COO);
Anal.
calcd
for
C36H50N7O6P·H2O: C (59.57), H (7.22), N (13.51), P (4.27), found: C
(59.76), H (7.17), N (13.21), P (4.16).
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(isobutyl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5g).
Starting compound 9g (0.20 g, 0.48 mmol), Method D, reaction
time 24 h, yield 0.18 g (54%) of 5g. ESIMS m/z (%): 730 (100) [M+
Na]+; 1H NMR (500 MHz, [D6]DMSO): d=0.89 (t, J=6.7 Hz, 6H, H-
3’’), 1.01 (d, J=6.3 Hz, 3H, CH3), 1.07 (d, J=6.3 Hz, 3H, CH3), 1.10
(d, J=6.3 Hz, 3H, CH3), 1.16 (d, J=6.3 Hz, 3H, CH3), 2.19 (m, 1H, H-
2’’), 2.52 (d, J=7.2 Hz, 2H, H-1’’), 2.72–2.79 (m, 4H, CH2-Bn), 3.22
(dd, J=8.2 Hz, J=13.3 Hz, 1H, Pb-CH2), 3.30 (dd, J=7.8 Hz, J=
13.3 Hz, 1H, Pa-CH2), 3.68 (t, J=5.3 Hz, 2H, O-CH2), 3.84–3.98 (m,
2H, NH-CH), 4.11 (dd, J=10.6 Hz, J=12.0 Hz, 1H, NH), 4.21 (t, J=
5.3 Hz, 2H, N-CH2), 4.43 (dd, J=10.8 Hz, J=12.0 Hz, 1H, NH), 4.77–
4.82 (sept, J=6.3 Hz, CHiPr), 7.04 (bs, 2H, NH2), 7.08–7.26 (m, 10H,
H-arom), 8.02 ppm (s, 1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=
21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 22.64 (C3’’), 28.14 (C2’’),
40.16 (CH2-Bn), 42.50 (C1’), 47.94 (C1’’), 54.02 and 54.21 (NH-CH),
67.49 (d, JP,C =135.2 Hz, P-C), 67.97 and 68.11 (2C, POC), 70.36 (d,
J
P,C =11.0 Hz, C2’), 117.03 (C5), 126.55 and 126.63 (C4’’ arom),
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
butyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5e). Starting
compound 9e (0.36 g, 0.87 mmol), Method D, reaction time 24 h,
yield 0.40 g (65%) of 5e. ESIMS m/z (%): 730 (100) [M+Na]+;
1H NMR (500 MHz, [D6]DMSO): d=0.89 (t, J=7.4 Hz, 3H, H-4’’-CH3),
1.01 (d, J=6.2 Hz, 3H, CH3), 1.07 (d, J=6.2 Hz, 3H, CH3), 1.11 (d, J=
6.2 Hz, 3H, CH3), 1.16 (d, J=6.2 Hz, 3H, CH3), 1.33 (m, 2H, H-3’’),
1.69 (m, 2H, H-2’’), 2.64 (m, 2H, H-1’’), 2.72–2.89 (m, 4H, CH2-Bn),
3.23 (dd, J=8.0 Hz, J=13.3 Hz, 1H, Pb-CH2), 3.31 (dd, J=7.8 Hz,
J=13.3 Hz, 1H, Pb-CH2), 3.69 (t, J=5.3 Hz, 2H, O-CH2), 3.85–3.97
(m, 2H, NH-CH), 4.10 (dd, J=10.6 Hz, J=12.1 Hz, 1H, NH), 4.21 (t,
J=5.3 Hz, 2H, N-CH2), 4.42 (dd, J=10.8 Hz, J=12.0 Hz, 1H, NH),
4.77 and 4.82 (sept, J=6.3 Hz, 2H, CHiPr), 7.01 (bs, 2H, NH2), 7.09–
7.26 (m, 10H, H-arom), 8.01 ppm (s, 1H, H-8); 13C NMR (125 MHz,
[D6]DMSO): d=14.06 (C4’’), 21.45, 21.52, 21.57 and 21.64 (CH3-iPr),
22.20 (C3’’), 30.89 (C2’’), 38.62 (C1’’), 40.16 (CH2-Bn), 42.46 (C1’),
54.02 and 54.20 (NH-CH), 67.48 (d, JP,C =135.1 Hz, P-C), 67.96 and
68.09 (2C, POC), 70.37 (d, JP,C =11.2 Hz, C2’), 117.05 (C5), 126.55 and
126.62 (C4’’ arom), 128.19 and 128.23 (C3’’ arom), 129.62 (C2’’
arom), 137.18 and 137.26 (C1’’ arom), 140.78 (C8), 150.41 (C4),
155.84 (C6), 164.62 (C2), 172.30–172.43 ppm (m, COO); Anal. calcd
for C36H50N7O6P·2/3H2O: C (60.07), H (7.15), N (13.62), P (4.30),
found: C (60.10), H (6.95), N (13.46), P (4.07).
128.19 and 128.24 (C3’’ arom), 129.63 (C2’’ arom), 137.19 and
137.26 (C1’’ arom), 140.88 (C8), 150.34 (C4), 155.67 (C6), 163.78
(C2),
C36H50N7O6P·H2O: C (59.57), H (7.22), N (13.41), P (4.27), found: C
(59.55), H (7.25), N (12.93), P (3.99).
172.30–172.43 ppm
(m,
COO);
Anal.
calcd
for
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(pentan-3-yl)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5h).
Starting compound 9h (0.31 g, 0.72 mmol), Method D, reaction
time 72 h, yield 0.27 g (51%) of 5h. ESIMS m/z (%): 722 (100) [M+
H]+; H NMR (500 MHz, [D6]DMSO): d=0.75 (t, J=7.4 Hz, 6H, H-3’’),
1
1.01 (d, J=6.3 Hz, 3H, CH3), 1.07 (d, J=6.3 Hz, 3H, CH3), 1.11 (d, J=
6.3 Hz, 3H, CH3), 1.16 (d, J=6.3 Hz, 3H, CH3), 1.58 and 1.73 (m, 4H,
H-2’’), 2.47 (m, 1H, H-1’’), 2.72–2.90 (m, 4H, CH2-Bn), 3.22 (dd, J=
8.2 Hz, J=13.2 Hz, 1H, Pb-CH2), 3.30 (dd, J=7.7 Hz, J=13.2 Hz, 1H,
Pa-CH2), 3.68 (t, J=5.3 Hz, 2H, O-CH2), 3.84–3.98 (m, 2H, NH-CH),
4.11 (dd, J=10.6 Hz, J=12.0 Hz, 1H, NH), 4.21 (t, J=5.3 Hz, 2H, N-
CH2), 4.44 (dd, J=10.7 Hz, J=12.0 Hz, 1H, NH), 4.78–4.82 (sept, J=
6.3 Hz, CHiPr), 6.98 (bs, 2H, NH2), 7.08–7.26 (m, 10H, H-arom),
8.01 ppm (s, 1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=12.33
(C3’’), 21.46, 21.53, 21.58 and 21.65 (CH3-iPr), 27.45 (C2’’), 40.15
(CH2-Bn), 42.47 (C1’), 51.66 (C1’’), 54.03 and 54.22 (NH-CH), 67.47
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Full Papers
(d, JP,C =135.0 Hz, P-C), 67.97 and 68.11 (2C, POC), 70.28 (d, JP,C
=
2H, P-CH2), 3.70 (t, J=5.3 Hz, 2H, O-CH2), 3.84–3.97 (m, 2H, NH-
CH), 4.09 (m, 1H, NH), 4.21 (t, J=5.3 Hz, 2H, N-CH2), 4.42 (m, 1H,
NH), 4.77–4.82 (sept, J=6.3 Hz, CHiPr), 6.98 (brs, 2H, NH2), 7.08–
7.26 (m, 10H, H-arom), 8.00 ppm (s, 1H, H-8); 13C NMR (125 MHz,
[D6]DMSO): d=21.45, 21.52, 21.58 and 21.64 (CH3-iPr), 29.01 (C5’’),
29.85 (C6’’), 35.56 and 35.64 (C3’’, C7’’), 36.17 (C4’’), 40.16 (CH2-Bn),
42.46 (C1’), 42.95 (C1’’), 49.95 (C2’’), 54.02 and 54.19 (NH-CH), 67.47
11.2 Hz, C2’), 117.17 (C5), 126.54 and 126.62 (C4’’ arom), 128.18 and
128.23 (C3’’ arom), 129.63 (C2’’ arom), 137.20 and 137.26 (C1’’
arom), 140.80 (C8), 150.34 (C4), 155.86 (C6), 167.15 (C2), 172.31–
172.44 ppm (m, COO); Anal. calcd for C37H52N7O6P·1/2H2O: C
(60.81), H (7.31), N (13.42), P (4.24), found: C (60.68), H (7.18), N
(13.20), P (4.08).
(d, JP,C =135.1 Hz, P-C), 67.96 and 68.10 (2C, POC), 70.29 (d, JP,C
=
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(tetrahydrofuran-2-yl)-9H-purin-9-yl)ethoxy)methyl)phosphonic
acid (5i). Starting compound 9i (0.34 g, 0.78 mmol), Method D, re-
action time 48 h, yield 0.16 g (29%) of 5i. ESIMS m/z (%): 744 (100)
11.2 Hz, C2’), 116.99 (C5), 126.54 and 126.62 (C4’’ arom), 128.18 and
128.23 (C3’’ arom), 129.62 (C2’’ arom), 137.18 and 137.26 (C1’’
arom), 140.84 (C8), 150.23 (C4), 155.67 (C6), 167.35 (C2), 172.29–
172.42 ppm (m, COO); Anal. calcd for C39H52N7O6P·4/3H2O: C
(60.84), H (7.16), N (12.74), P (4.02), found: C (61.22), H (7.04), N
(12.37), P (3.86).
1
[M+Na]+; H NMR (500 MHz, [D6]DMSO): d=1.00 (d, J=6.3 Hz, 3H,
CH3), 1.06 (d, J=6.3 Hz, 3H, CH3), 1.10 (d, J=6.3 Hz, 3H, CH3), 1.15
(d, J=6.3 Hz, 3H, CH3), 1.87 (m, 1H, H-4b’’), 2.00–2.10 (m, 2H, H-
3b’’, H-4a’’), 2.15 (m, 1H, H-3a’’), 2.71–2.90 (m, 4H, CH2-Bn), 3.22–
3.32 (m, 2H, P-CH2), 3.70 (t, J=5.3 Hz, 2H, O-CH2), 3.77–4.02 (m,
4H, NH-CH, C5’’), 4.12 (m, 1H, NH), 4.23 (t, J=5.3 Hz, 2H, N-CH2),
4.43 (m, 1H, NH), 4.74–4.84 (m, 3H, C2’’, CH-iPr), 7.09–7.26 (m,
10H, H-arom), 8.07 ppm (s, 1H, H-8); 13C NMR (125 MHz,
[D6]DMSO): d=21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 25.79 and
31.56 (C3’’, C4’’), 40.17 (CH2-Bn), 42.53 (C1’), 54.02 and 54.22 (NH-
CH), 67.46 (d, JP,C =135.3 Hz, P-C), 67.97 and 68.10 (2C, POC), 68.22
(C5’’), 70.34 (d, JP,C =11.2 Hz, C2’), 81.62 (C2’’), 117.75 (C5), 126.56
and 126.63 (C4’’ arom), 128.20 and 128.24 (C3’’ arom), 129.62 (C2’’
arom), 137.19 and 137.26 (C1’’ arom), 141.35 (C8), 150.11 (C4),
156.00 (C6), 164.34 (C2), 172.31–172.44 ppm (m, COO); Anal. calcd
for C36H48N7O7P·4/3H2O: C (57.98), H (6.85), N (13.15), P (4.15),
found: C (58.13), H (6.84), N (12.68), P (3.89).
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(cyclopropylamino)-9H-purin-9-yl)ethoxy)methyl)phosphonic
acid (5l). Starting compound 9l (0.30 g, 0.72 mmol), Method D, re-
action time 24 h, yield 0.21 g (41%) of 5l. ESIMS m/z (%): 707 (100)
1
[M+H]+; H NMR (500 MHz, [D6]DMSO): d=0.42 (m, 2H, H-2’’-CH2),
0.58 (m, 2H, H-2’’-CH2), 1.01 (d, J=6.3 Hz, 3H, CH3), 1.07 (d, J=
6.3 Hz, 3H, CH3), 1.11 (d, J=6.3 Hz, 3H, CH3), 1.16 (d, J=6.3 Hz, 3H,
CH3), 2.70–2.90 (m, 5H, CH2-Bn, H-1’’), 3.24–3.31 (m, 2H, P-CH2),
3.67 (t, J=5.4 Hz, 2H, O-CH2), 3.85–3.98 (m, 2H, NH-CH), 4.07 (t, J=
5.4 Hz, 2H, N-CH2), 4.10 (dd, J=10.6 Hz, J=12.1 Hz, 1H, NH), 4.41
(dd, J=10.8 Hz, J=11.9 Hz, 1H, NH), 4.78–4.82 (sept, J=6.3 Hz, CH-
iPr), 6.41 (bd, J=3.9 Hz, 2-NH), 6.60 (brs, 2H, NH2), 7.09–7.27 (m,
10H, H-arom), 7.68 ppm (s, 1H, H-8); 13C NMR (125 MHz,
[D6]DMSO): d=6.67 (C2’’), 21.46, 21.53, 21.59 and 21.65 (CH3-iPr),
24.40 (C1’’), 40.17 (CH2-Bn), 42.10 (C1’), 54.04 and 54.18 (NH-CH),
67.54 (d, JP,C =135.0 Hz, P-C), 67.99 and 68.12 (2C, POC), 70.38 (d,
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
cyclopentyl-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5j).
Starting compound 9j (0.35 g, 0.81 mmol), Method D, reaction
time 24 h, yield 0.41 g (70%) of 5j. ESIMS m/z (%): 721 (100) [M+
JP,C =11.0 Hz, C2’), 113.41 (C5), 126.58 and 126.64 (C4’’ arom),
128.22 and 128.25 (C3’’ arom), 129.52 and 129.63 (C2’’ arom),
137.18 and 137.27 (C1’’ arom), 137.90 (C8), 151.76 (C4), 156.02 (C6),
160.47 (C2), 172.31–172.45 ppm (m, COO); Anal. calcd for
C35H47N8O6P·H2O: C (58.00), H (6.86), N (15.40), P (4.27), found: C
(58.10), H (6.80), N (14.78), P (4.13).
1
H]+; H NMR (500 MHz, [D6]DMSO): d=1.01 (d, J=6.3 Hz, 3H, CH3),
1.06 (d, J=6.3 Hz, 3H, CH3), 1.11 (d, J=6.3 Hz, 3H, CH3), 1.15 (d, J=
6.3 Hz, 3H, CH3), 1.59 and 1.74 2x (m, 2H, H-3’’), 1.84–1.95 (m, 4H,
H-2’’), 2.72–2.89 (m, 4H, CH2-Bn), 3.09 (p, J=8.3 Hz, 1H, H-1’’), 3.25
(dd, J=8.1 Hz, J=13.3 Hz, 1H, Pb-CH2), 3.31 (dd, J=7.7 Hz, J=
13.4 Hz, 1H, Pa-CH2), 3.70 (t, J=5.4 Hz, 2H, O-CH2), 3.85–3.97 (m,
2H, NH-CH), 4.10 (dd, J=10.6 Hz, J=12.1 Hz, 1H, NH), 4.21 (t, J=
5.4 Hz, 2H, N-CH2), 4.42 (dd, J=10.7 Hz, J=12.0 Hz, 1H, NH), 4.77
and 4.82 (sept, J=6.3 Hz, 2H, CH-iPr), 7.08–7.26 (m, 10H, H-arom),
8.01 ppm (s, 1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=21.44,
21.51, 21.57 and 21.64 (CH3-iPr), 25.66 (C3’’), 32.60 (C2’’), 40.16
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(2,6-diamino-
9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5m). Starting
compound 9m[32] (0.48 g, 1.3 mmol), Method D, reaction time 72 h,
yield 0.40 g (46%) of 5m. ESIMS m/z (%): 689 (100) [M+Na]+;
1H NMR (500 MHz, CDCl3): d=1.26 (d, J=6.3 Hz, 3H, CH3), 1.30 (d,
J=6.3 Hz, 3H, CH3), 1.32 (d, J=6.3 Hz, 3H, CH3), 1.36 (d, J=6.3 Hz,
3H, CH3), 2.89–3.18 (m, 4H, CH2-Bn), 3.26–3.33 (m, 1H, Pb-CH2),
3.46 (dd, J=13.1 Hz, 8.0 Hz, 1H, Pa-CH2), 3.52 (m, 2H, P-CH2), 3.52
(m, 2H, O-CH2), 3.67–3.79 (m, 2H, NH-CH), 4.15–4.23 (m, 1H, NH),
4.20 (t, J=4.91 Hz, 2H, N-CH2), 4.26–4.36 (m, 1H, NH), 5.04–5.14
(m, 2H, CH-iPr), 5.23 (bs, 2H, NH2), 5.91 (bs, 2H, NH2), 7.20–7.38 (m,
10H, H-arom), 7.64 ppm (s, 1H, H-8); 13C NMR (125 MHz, CDCl3):
d=21.61, 2”21.70 and 21.79 (CH3-iPr), 40.64 (CH2-Bn), 42.81 (C1’),
53.82 and 54.05 (NH-CH), 67.97 (d, JP,C =135.9 Hz, P-C), 69.07 and
69.12 (2C, POC), 71.12 (d, JP,C =12.3 Hz, C2’), 114.19 (C5), 126.81 and
126.84 (C4’’ arom), 128.31 and 128.36 (C3’’ arom), 129.60 and
129.64 (C2’’ arom), 136.43 and 136.59 (C1’’ arom), 138.58 (C8),
151.99 (C4), 155.98 and 160.03 (C2, C6), 172.72–172.74 ppm (m,
COO); Anal. calcd for C32H43N8O6P: C (57.65), H (6.50), N (16.81), P
(4.65), found: C (57.31), H (6.80), N (16.58), P (4.54).
(CH2-Bn), 42.43 (C1’), 54.02 and 54.19 (NH-CH), 67.47 (d, JP,C
=
135.0 Hz, P-C), 67.96 and 68.09 (2C, POC), 70.31 (d, JP,C =11.0 Hz,
C2’), 117.17 (C5), 126.54 and 126.62 (C4’’ arom), 128.18 and 128.23
(C3’’ arom), 129.61 (C2’’ arom), 137.18 and 137.25 (C1’’ arom),
140.82 (C8), 150.32 (C4), 155.79 (C6), 167.53 (C2), 172.30–
172.42 ppm (m, COO); Anal. calcd for C37H50N7O6P·H2O: C (60.23), H
(7.10), N (13.29), P (4.20), found: C (60.26), H (7.10), N (12.91), P
(4.00).
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(bicyclo[2.2.1]heptan-2-yl)-9H-purin-9-yl)ethoxy)methyl)phos-
phonic acid (5k). Starting compound 9k (0.26 g, 0.57 mmol),
Method D, reaction time 48 h, yield 0.28 g (66%) of 5k. ESIMS m/z
(%): 746 (100) [M+H]+; 1H NMR (500 MHz, [D6]DMSO): d=1.03
(dm, J=9.2 Hz, 1H, H-7b’’-CH2), 1.01 (d, J=6.2 Hz, 3H, CH3), 1.06
(d, J=6.2 Hz, 3H, CH3), 1.10 (d, J=6.2 Hz, 3H, CH3), 1.15 (d, J=
6.2 Hz, 3H, CH3), 1.23 (m, 1H, endo H-5’’), 1.32 (m, 1H, endo H-6’’),
1.46–1.55 (m, 3H, endo H-3’’, exo H-5’’, exo H-6’’), 1.70 (dm, J=
9.2 Hz, 1H, H-7a’’-CH2), 2.20 (m, 1H, exo H-3’’), 2.28 (m, 1H, H-4’’),
2.44 (m, 1H, H-1’’), 2.71–2.88 (m, 5H, H-2’’, CH2-Bn), 3.21–3.32 (m,
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
fluoro-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5n). Start-
ing compound 9n[28] (0.44 g, 1.2 mmol), Method D, reaction time
72 h, yield 0.35 g (45%) of 5n. ESIMS m/z (%): 692 (100) [M+Na]+;
1H NMR (500 MHz, [D6]DMSO): d=1.00 (d, J=6.3 Hz, 3H, CH3), 1.05
(d, J=6.3 Hz, 3H, CH3), 1.10 (d, J=6.3 Hz, 3H, CH3), 1.15 (d, J=
ChemMedChem 2015, 10, 1351 – 1364
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Full Papers
6.3 Hz, 3H, CH3), 2.73–2.87 (m, 4H, CH2-Bn), 3.23 (dd, J=8.1 Hz, J=
13.3 Hz, 1H, Pb-CH2), 3.30 (dd, J=7.8 Hz, J=13.3 Hz, 1H, Pa-CH2),
3.68 (t, J=5.3 Hz, 2H, O-CH2), 3.86–3.93 (m, 2H, NH-CH), 4.14 (dd,
J=10.6 Hz, J=12.2 Hz, 1H, NH), 4.18 (t, J=5.3 Hz, 2H, N-CH2), 4.45
(dd, J=10.8 Hz, J=12.0 Hz, 1H, NH), 4.76–4.81 (sept, J=6.3 Hz, 2H,
CH-iPr), 7.09–7.27 (m, 10H, H-arom), 7.75 (bs, 2H, NH2), 8.08 ppm
(s, 1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=21.48, 21.55, 21.61
and 21.68 (CH3-iPr), 40.10 (CH2-Bn), 42.87 (C1’), 54.06 and 54.24
(NH-CH), 67.48 (d, JP,C =135.5 Hz, P-C), 68.01 and 68.14 (2C, POC),
70.24 (d, JP,C =11.0 Hz, C2’), 117.13 (d, JCÀF =4.0 Hz, C5), 126.62 and
126.68 (C4’’ arom), 128.24 and 128.29 (C3’’ arom), 129.66 (C2’’
arom), 137.22 and 137.30 (C1’’ arom), 141.78 (d, JCÀF =2.2 Hz, C8),
151.03 (d, JCÀF =20.3 Hz, C4), 157.75 (d, JCÀF =21.3 Hz, C6), 158.87
(d, JCÀF =203.2 Hz, C2), 172.37–172.48 ppm (m, COO); HRMS (ESI+):
m/z [M+Na]+ calcd for C32H41FN7O6P: 692.2732, found: 692.2730.
CH3), 21.60 and 21.66 (2C, CH3), 42.86 (C1’), 54.01 and 54.22 (NH-
CH), 67.47 (d, JP,C =135.0 Hz, P-C), 67.97 and 68.10 (2C, POC), 70.17
(d, JP,C =11.0 Hz, C2’), 118.68 (C5), 120.82 (C2), 126.58 and 126.64
(C4’’ arom), 128.21 and 128.25 (C3’’ arom), 129.62 (C2’’ arom),
137.19 and 137.26 (C1’’ arom), 141.31 (C8), 150.13 (C4), 156.02 (C6),
172.31–172.43 ppm (m, COO); Anal. calcd for C32H41IN7O6P·1/2H2O:
C (48.86), H (5.38), N (12.46), P (3.94), found: C (48.99), H (5.42), N
(12.02), P (3.86).
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
(thiocyanato)-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5r).
A
solution of 5q (0.28 g, 0.36 mmol) and CuSCN (0.13 g,
1.08 mmol) in DMF (10 mL) was heated at 1208C for 8 h. The sol-
vent was removed in vacuo, the residue was co-distilled with tolu-
ene and dissolved in EtOAc (20 mL). The solid was filtered off. The
filtrate was washed with a solution that contains saturated EDTA
and brine, 1:1 (3”30 mL). The organic layer was collected, evapo-
rated, and the residue was purified by flash chromatography
(MeOH/CHCl3, 3:97) to give 0.07 g (21%) of 5r. ESIMS m/z (%): 731
(100) [M+Na]+; 1H NMR (500 MHz, [D6]DMSO): d=0.99–1.16 (m,
12H, CH3), 2.70–2.86 (m, 4H, CH2-Bn), 3.25 (dd, J=8.0 Hz, J=
13.2 Hz, 1H, Pb-CH2), 3.31 (dd, J=7.9 Hz, J=13.3 Hz, 1H, Pa-CH2),
3.71 (m, 2H, O-CH2), 3.78–3.99 (m, 2H, NH-CH), 4.12 (dd, J=
10.6 Hz, J=12.1 Hz, 1H, NH), 4.21 (m, 2H, N-CH2), 4.43 (m, 1H, NH),
4.73–4.84 (m, 2H, CHiPr), 7.08–7.27 (m, 10H, H-arom), 7.83 (bs, 2H,
NH2), 8.14 ppm (s, 1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=
21.45, 21.52, 21.58 and 21.65 (CH3-iPr), 40.00 (CH2-Bn), 43.01 (C1’),
54.01 and 54.20 (NH-CH), 67.45 (m, P-C), 67.96 and 68.09 (2C, POC),
70.01 (d, JP,C =11.3 Hz, C2’), 109.19 (CN), 117.86 (C5), 126.58 and
126.63 (C4’’ arom), 128.20 and 128.24 (C3’’ arom), 129.62 (C2’’
arom), 137.17 and 137.27 (C1’’ arom), 141.76 (C8), 150.24 (C4),
154.04 and 156.47 (C2 and C6), 172.30–172.43 ppm (m, COO);
HRMS (ESI+): m/z [M+H]+ calcd for C33H42N8O6PS: 709.2680,
found: 709.2679.
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
chloro-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5o). Start-
ing compound 9o[28] (0.50 g, 1.3 mmol), Method D, reaction time
72 h, yield 0.53 g (61%) of 5o. ESIMS m/z (%): 708 (100) [M+Na]+;
1H NMR (500 MHz, CDCl3): d=1.27 (d, J=6.3 Hz, 3H, CH3), 1.32 (d,
J=6.3 Hz, 3H, CH3), 1.33 (d, J=6.3 Hz, 3H, CH3), 1.38 (d, J=6.3 Hz,
3H, CH3), 2.90–3.26 (m, 6H, CH2-Bn, NH-CH), 3.34–3.45 (m, 2H, P-
CH2), 3.79 (t, J=4.9 Hz, 2H, O-CH2), 4.09–4.17 (m, 1H, NH), 4.17–
4.27 (m, 1H, NH), 4.36 (m, 2H, N-CH2), 5.06–5.22 (m, 2H, CH-iPr),
6.72 (bs, 2H, NH2), 7.19–7.41 (m, 10H, H-arom), 7.94 ppm (s, 1H, H-
8); 13C NMR (125 MHz, CDCl3): d=21.61, 21.70, 21.72 and 21.80
(CH3-iPr), 40.59 (CH2-Bn), 43.42 (C1’), 53.57 and 54.03 (NH-CH),
68.07 (d, JP,C =136.0 Hz, P-C), 69.23 and 69.32 (2C, POC), 71.00 (d,
J
P,C =12.3 Hz, C2’), 118.15 (C5), 126.90 (C4’’ arom), 128.34 and
128.40 (C3’’ arom), 129.59 and 129.61 (C2’’ arom), 136.23 and
136.55 (C1’’ arom), 141.80 (C8), 150.92 (C4), 154.01 and 156.28 (C2,
C6), 172.44–172.70 ppm (m, COO); Anal. calcd for C32H41ClN7O6P·1/
2H2O: C (55.29), H (6.09), Cl (5.10), N (14.10), P (4.46), found: C
(55.59), H (5.88), Cl (4.82), N (13.96), P (4.49).
Bis(l-phenylalanine isobutyl ester) prodrug of ((2-(6-amino-2-
oxo-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5p). Starting
compound 9p[28] (0.30 g, 1.03 mmol), Method D, reaction time
72 h, yield 0.35 g (48%) of 5p. ESIMS m/z (%): 696 (100) [M+H]+;
1H NMR (500 MHz, [D6]DMSO): [808C] d=0.82–0.85 (m, 12H, CH3),
1.74–1.86 (m, 2H, CHiPr), 2.81–2.94 (m, 4H, CH2-Bn), 3.38 (d, J=
7.8 Hz, 2H, P-CH2), 3.68 (t, J=5.4 Hz, 2H, O-CH2), 3.75–3.79 (m, 4H,
COO-CH2), 3.90–4.20 (m, 6H, NH, NH-CH, N-CH2), 7.08–7.27 (m,
12H, H-arom, NH2), 7.65 ppm (s, 1H, H-8); 13C NMR (125 MHz,
[D6]DMSO): [808C] d=18.54 and 18.58 (CH3-iPr), 26.96 and 26.97
Biological assays
ACT inhibitory activity. J774A.1 cells were seeded in a 96-well plate
at 5”104 cells per well and left to attach overnight. Prior to the ex-
periment, cells were washed with HBSS (135 mm NaCl, 5.9 mm KCl,
1.5 mm CaCl2, 1.2 mm MgCl2, 25 mm glucose, 10 mm HEPES
[pH 7.4]) and pre-incubated with compounds at concentrations of
0.001–30 mm for 5 h. Cells were then exposed to ACT (0.4 mg mLÀ1
)
from B. pertussis for 30 min. Finally, the cAMP content was deter-
mined by using the CatchPointcAMP immunoassay kit (Molecular
Devices, Wokingham, UK). After the addition of lysis buffer (50 mL
per well) provided by the manufacturer, the cellular content was
extracted by shaking the plate at 250 rpm for 10 min. The plate
was then centrifuged to remove cell debris, the supernatant was
replaced to the assay plate, and immunoassays were carried out
according to the manufacturer’s instructions. Fluorescence signal
was acquired using an Infinite M1000 plate reader (Tecan Systems
Inc., San Jose, CA, USA).
(CH-iPr), 41.91 (C1’), 53.81 and 53.88 (NH-CH), 67.57 (d, JP,C
=
135.4 Hz, P-C), 70.08–70.20 (m, C2’, COOCH2), 126.18 (C4’’ arom),
127.86 (C3’’ arom), 129.08 (C2’’ arom), 136.89 and 136.98 (C1’’
arom), 139.25 (C8), 152.07 (C2), 156.00 (C4), 172.38–172.50 ppm (m,
COO); HRMS (ESI+): m/z [M+H]+ calcd for C34H47N7O67P: 696.3269,
found: 696.3270.
Bis(l-phenylalanine isopropyl ester) prodrug of ((2-(6-amino-2-
iodo-9H-purin-9-yl)ethoxy)methyl)phosphonic acid (5q). Starting
compound 8 (1.00 g, 2.1 mmol), Method D, reaction time 24 h,
yield 0.88 g (55%) of 5q. ESIMS m/z (%): 800 (100) [M+Na]+;
1H NMR (500 MHz, [D6]DMSO): d=1.04 (d, J=6.3 Hz, 6H, CH3), 1.14
(d, J=6.3 Hz, 6H, CH3), 2.71–2.90 (m, 4H, CH2-Bn), 3.23 (dd, J=
8.1 Hz, J=13.3 Hz, 1H, Pb-CH2), 3.30 (dd, J=7.7 Hz, J=13.3 Hz, 1H,
Pa-CH2), 3.67 (t, J=5.3 Hz, 2H, O-CH2), 3.84–3.97 (m, 2H, NH-CH),
4.12 (dd, J=10.6 Hz, J=12.1 Hz, 1H, NH), 4.19 (m, 2H, N-CH2), 4.43
(dd, J=10.7 Hz, J=12.0 Hz, 1H, NH), 4.77–4.82 (sept, J=6.3 Hz CH-
iPr), 7.09–7.27 (m, 10H, H-arom), 7.60 (brs, 2H, NH2), 8.03 ppm (s,
1H, H-8); 13C NMR (125 MHz, [D6]DMSO): d=21.46 and 21.53 (2C,
Effect on the viability of J774A.1 cells. J774A.1 cells were plated
onto white 96-well assay plates at 5”104 cells per well and allowed
to attach overnight. Cells were then washed with HBSS and treated
with compounds at 10 mm for 5 h. Cell viability was then assessed
with a Cell Titer-Glo Luminescent Cell Viability assay (Promega,
Madison, WI, USA) according to the manufacturer’s instructions.
Measurement of luminescence signal was performed by use of
a GENiosmicro plate reader (Tecan Systems). Data were expressed
as percent of control, represented by untreated cells.
ChemMedChem 2015, 10, 1351 – 1364
www.chemmedchem.org
1362
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
In vitro long-term viability tests. The sensitivity of the cells to com-
pound 5n was assessed with XTT cell proliferation kit II (Roche Di-
agnostics GmbH, Mannheim, Germany) according to manufactur-
er’s instructions. The following cell lines were used: murine macro-
phage cell line J774A.1; two normal human cell lines, HUVEC-2 and
NHDF-Ad; and four human cancer cell lines, CCRF-CEM, HL-60,
HeLa-S3, HepG2. Cells were seeded in a 96-well plate at a density
of 13500 cells per well and left to rest overnight. After 24 h 5n
was added at a concentration of 10 mm to the culture media and
incubated for 72 h before addition of the XTT dye. The absorbance
at l 495 nm was measured after 1 h incubation with the dye.
were housed under a 12 h light/dark cycle at 228C. They were
fasted for 16 h before experiments, with free access to water.
Plasma levels of tested compounds and their metabolites were an-
alyzed after i.v. administration of parent compounds into vena sa-
phena. Animals were anesthetized with pentobarbital (55 mgkgÀ1
i.p.). A silastic cannula was inserted into the carotid artery and
a blank sample of blood was taken. Tested compounds were then
administered as bolus dose into the saphenous vein (5n and 4,
10 mgkgÀ1, or 25 mgkgÀ1 for 3 dissolved in DMSO). Blood samples
(0.4 mL) were collected from the carotid artery into heparinized
tubes at 5, 10, 20, 45, 60, and 120 min post-administration. Be-
tween each blood sample, the cannula was filled with heparinized
0.9% saline. After the experiments, liver, kidney, and lung were
sampled and weighed. The blood samples were centrifuged and
the plasma separated. All samples were stored at À708C until anal-
ysis. The organs were cut into pieces and homogenized using
a Dounce tissue grinder in 50 mm Tris·HCl buffer (pH 7.4) contain-
ing 1 mm dithiothreitol, 5 mm MgCl2, and protease inhibitor cock-
tail. Finally, all blood and organ samples were deproteinized by
methanol precipitation and analyzed by HPLC, which was per-
formed as described above for hydrolysis in macrophage homoge-
nate. Research involving animals was performed in accordance
with the respective Czech laws concerning animal protection. The
experimental protocol was approved by the Ethical Committee of
the Pharmaceutical Faculty, Charles University in Prague, Czech Re-
public as well as by the Ministry of Education, Youth and Sport,
Czech Republic.
Preparation of crude macrophage extracts. J774A.1 cells (2”109)
were washed with PBS and suspended in 1.5 mL Tris·HCl buffer
(50 mm, pH 7.4) containing 1 mm dithiothreitol, 5 mm MgCl2, and
protease inhibitor cocktail. The cells were then homogenized four
times for 30 s at speed 4.0 msÀ1 in a FastPrep24 instrument. The
homogenate was centrifuged at 30000”g for 30 min. The superna-
tant was incubated with streptomycin sulfate for 1 h to remove nu-
cleic acids and centrifuged at 30000”g for 30 min. The resulting
crude cell extract was desalted on PD-10 columns (GE Healthcare,
Piscataway, NJ, USA), divided into aliquots, and stored at À808C.
The protein content was determined with a QuantiPro BCA assay
kit (Sigma–Aldrich) according to the manufacturer’s instructions.
Assays with mACs. HEK293 cells stably expressing AC1, AC2, or AC5
were cultured and frozen as previously described.[33,37] Cells were
thawed and plated in white-bottom 384-well plates (PerkinElmer,
Shelton, CT, USA). Inhibitor compounds were added to cells using
a Multipette-mounted 384-well pintool and incubated at room
temperature for 30 min. Then, the specific mAC stimulator (3 mm
A23187 for AC1, 100 nm PMA for AC2, and 300 nm forskolin for
AC5) in 500 mm 3-isobutyl-1-methyxanthine was added to the cells.
Cells were incubated at room temperature for 1 h, and cAMP accu-
mulation was measured using Cisbio’s dynamic 2 kit (Cisbio Bioas-
says, Bedford, MA, USA) according to the manufacturer’s instruc-
tions.
Acknowledgements
The study was supported by the Ministry of Interior of the Czech
Republic (VG20102015046) and Gilead Sciences (Foster City, CA,
USA).
Keywords: adenylate cyclase toxin · bisamidates · Bordetella
pertussis · nucleosides · phosphonates · PMEA · prodrugs
Hydrolysis of bis(POM)PMEA (2) and bisamidate prodrugs 3, 4, and
5n in macrophage homogenate. A total of 70 mL of cellular extract
(5.5 mgmLÀ1 of total protein) was mixed with 30 mL reaction buffer
(1 mm dithiothreitol, 5 mm MgCl2, 0.4 mm EDTA, 20 mm Tris·HCl
[pH 7.5]) and incubated for various time intervals (2–18 h) at 378C
in the presence of the corresponding prodrug (100 mm). Simultane-
ously, prodrugs were incubated in reaction buffer only serving as
negative (no enzyme) controls. Finally, samples were deproteinized
by methanol precipitation and analyzed by ion-pairing reversed-
phase high-pressure liquid chromatography using a Supelco Dis-
covery C8 HPLC column (150”4.6 mm [i.d.: 5 mm]) under a gradient
of 50!100 mm CH3COONH4 (pH 4.7) and acetonitrile. The flow
rate was 0.9 mLminÀ1. The absorption spectra of eluted com-
pounds were recorded by a photodiode array detector (PDA) at
l 260 nm. The retention times under the conditions used were as
follows: 5n, 18.7 min; bis(POM)PMEA, 14.7 min; MIC-768, 16.6 min;
MIC-747, 11.4 min; PMEA-F, 7.9 min; PMEA, 6.7 min. The concentra-
tions of PMEA and intact prodrugs in the samples were calculated
from the peak areas by using calibration curves generated from
intact prodrug standards. The concentrations of the PMEA-based
intermediate metabolites were calculated using the intact prodrug
calibration curve. Blank samples were obtained from untreated
macrophage homogenate. No interfering peaks were observed,
and thus selectivity was ensured.
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ChemMedChem 2015, 10, 1351 – 1364
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