Synthesis and anti-HSV activity of tricyclic penciclovir and hydroxybutylguanine derivatives

Article abstract of DOI:10.1016/j.bmc.2019.02.005

A series of tricyclic penciclovir (PCV) and hydroxybutylguanine (HBG) derivatives have been prepared with enhanced lipophilicity following an efficient synthetic route. All the novel tricyclic derivatives were evaluated for inhibitory activity against her

Full text of DOI:10.1016/j.bmc.2019.02.005

Accepted Manuscript
Synthesis and anti-HSV activity of tricyclic penciclovir and hydroxybutylgua-
nine derivatives
Anber F. Mohammed, Graciela Andrei, Alaa M. Hayallah, Samia G. Abdel-
Moty, Robert Snoeck, Claire Simons
PII:
S0968-0896(18)32087-X
https://doi.org/10.1016/j.bmc.2019.02.005
BMC 14741
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 December 2018
18 January 2019
1 February 2019
Please cite this article as: Mohammed, A.F., Andrei, G., Hayallah, A.M., Abdel-Moty, S.G., Snoeck, R., Simons,
C., Synthesis and anti-HSV activity of tricyclic penciclovir and hydroxybutylguanine derivatives, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.005
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Synthesis and anti-HSV activity of tricyclic penciclovir and
hydroxybutylguanine derivatives
a,b
c
b,d
Anber F. Mohammed, Graciela Andrei, Alaa M. Hayallah, Samia G. Abdel-
b
c
a*
Moty, Robert Snoeck, and Claire Simons
a
School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII
Avenue, Cardiff CF10 3NB, UK;
b
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut,
Egypt
c
Rega Institute for Medical Research, KU Leuven, Herestraat 49 box 1030, Leuven3000, Belgium
dPresent address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Deraya University,
El-Minia, Egypt
*
Address for correspondence: Claire Simons, School of Pharmacy and
Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII
Avenue, Cardiff CF10 3NB, Wales, UK. Tel: 02920-876307. E-mail:
simonsc@cardiff.ac.uk
1

Abstract
A series of tricyclic penciclovir (PCV) and hydroxybutylguanine (HBG) derivatives
have been prepared with enhanced lipophilicity following an efficient synthetic route.
All the novel tricyclic derivatives were evaluated for inhibitory activity against herpes
simplex virus 1 and 2 (HSV-1, HSV-2) and thymidine kinase deficient (ACV
resistant) HSV-1. The tricyclic HBG derivatives were devoid of inhibitory activity
however several of the tricyclic PCV derivatives showed promising antiviral activity,
in particular 9g (R=4-MeO-C H )displayed good inhibitory activity (HSV-1 EC 1.5
6
4
50
-

M, HSV-2 EC 0.8 M) and retained inhibitory activity in HSV-1 TK cells (EC
5
0
50
0
.8 M). Computational docking experiments supported the biological data observed
and this preliminary study provides useful data for further development of tricyclic
acyclic nucleoside derivatives with improved lipophilicity with retention of activity in
HSV-1 TK deficient strains. Also, the new tricyclic derivatives were evaluated against
a broad range of other DNA and RNA viruses, but were found to be inactive at
subtoxic concentrations. In addition, weak to moderate cytostatic effect was observed
for the new compounds.
Keywords
Tricyclic penciclovir (PCV) derivatives; tricyclic hydroxybutylguanine (HBG)
derivatives; herpes simplex virus (HSV); herpes simplex encephalitis (HSE);
molecular modelling.
2

1
.Introduction
The herpes simplex virus (HSV), like all herpes viruses, establishes lifelong latency
following a primary infection. HSV-associated diseases can range from mild orolabial
1
ulcers (cold sores) to severe encephalitis. Herpes simplex viruses are categorised into
two types: HSV-1, which is mainly transmitted through oral-oral contact resulting
1
mainly in cold sores; and HSV-2, which is mainly sexually transmitted resulting in
2
the most common infection of genital herpes. Globally two thirds of the population
under the age of 50 are estimated to be infected with HSV-1.3
Herpes simplex encephalitis (HSE) is the most common cause of sporadic fatal viral
4
encephalitis, accounting for almost 20% of all cases of encephalitis. HSE presents as
an acute focal necrotising infection of the brain, which, if untreated, has a 70%
5
mortality rate. The first line treatment of HSE is aciclovir (ACV), administered
intravenously for 14-21 days, however even when treated, HSE is associated with a
3
0% mortality rate and permanent neurological damage.6-8 Limitations of ACV
9
10
therapy for HSE are the development of viral resistance and low CNS uptake.
Parent drug Tricyclic derivative
Figure 1. Parent acyclic nucleosides and tricyclic derivatives
3

Tricyclic ACV and ganciclovir (GCV) derivatives (Figure 1) were shown to be
effective inhibitors of HSV-1 thymidine kinase (TK) and are of interest as they
maintain the inhibitory activity against the enzyme and, as they are more lipophilic
than the current medications, may be valuable agents for the treatment of CNS
infections.11-13 Moreover, these tricyclic compounds were found to exhibit strong
intrinsic fluorescent properties, which allows for sensitive concentration monitoring
and optimal therapeutic dosing.11-13 Penciclovir (PCV) and hydroxybutylguanine
(HBG) (Figure 1) are the carbocyclic analogues of GCV and ACV, respectively.The
spectrum of activity of PCV against human herpesviruses was found to be similar to
that of ACV, however in certain circumstances PCV was more active than ACV
1
4
owing to the higher stability of PCV-TP in infected cells relative to ACV-TP. Also,
at concentrations up to 100 µg/mL, it was confirmed that PCV, like ACV, was highly
1
4
selective and subsequently did not exhibit any toxicity in cell culture. Similarly,
HBG is reported to have higher affinity for HSV-1 TK than ACV in addition to
relatively comparable selectivity for inhibition of viral DNA synthesis and good
safety profile in cell culture.15
Application of the tricyclic scaffold modification to PCV and HBG was of interest to
determine whether antiviral activity was maintained and, importantly, modification of
the nucleobase moiety with an additional ring may result in beneficial
physicochemical properties (enhanced lipophilicity, fluorescence) and /or enhanced
enzyme binding interaction. A library of tricyclic PCV and HBG derivatives (Figure
2
) was designed and synthesised to evaluate antiviral activity and study structure-
activity relationships, supported by computational docking analysis.
4

Figure 2. Library for the designed tricyclic PCV and HBG nucleosides
2
. Results and discussion
2
.1 Chemistry
PCV (6) and HBG (7) were prepared as previously described by reaction of 2-amino-
-chloropurine (1) with 5-(2-bromoethyl)-2-phenyl-1,3-dioxane (2)16 or 4-
6
1
7
bromobutylacetate (3) with subsequent acid hydrolysis of the protected derivatives
4) and (5) (Scheme 1).
The new tricyclic analogues of PCV and HBG, 3-[4-hydroxy-3-
(
(
hydroxymethyl)butyl]-6-(aryl)-3,5 dihydro-9H-imidazo[1,2-a]purin-9-one (9) and 3-
4-hydroxybutyl)-6-(aryl)-3,5-dihydro-9H-imidazo[1,2-a]purin-9-one (10) were
(
obtained upon reaction of the sodium salt of PCV (6) and HBG (7), generated in situ
on treatment with NaH, and the appropriate bromoketone (8) in 38-79 % yield (Table
1
). The new compounds were purified by flash column chromatography using a
gradient eluting mixture of CH Cl -MeOH followed by recrystallisation (Scheme 1).
2
2
5

The procedure used for the preparation of the new derivatives mostly followed a
previously reported method for the preparation of analogously modified guanosine
and other tricyclic derivatives.12
Scheme 1. Reagents and conditions: (i) K CO , 18-crown-6, DMF, r.t. 72 h for synthesis of 4,
2
3
o
9
4 %, 16 h for synthesis of 5, 81% (ii) (a) 2M aqueous HCl, 90 C, 3h (b) 10% aqueous
o
o
NaOH, 4 C 24 h, 77 % (iii) (a) 2M aqueous HCl, THF, 75 C overnight (b) 10% aqueous
o
NaOH, 4 C 24 h, 81% (iv) NaH, DMF, r.t. 6 h, 38-79%.
The tricyclic derivatives showed some sensitivity towards light and air, so were
protected by wrapping with foil and rapid storage in amber coloured vials. The 6-
thien-2-yl group was highly unstable to light and air and in the case of the PCV
6

derivative decomposed rapidly during collection. Conversely, its HBG congener
10h) was stable enough to be collected safely.
(
Table 1. Yields and mp data for final tricyclic PCV and HBG derivatives 9 and 10
No.
R
Yield
m.p.
( C)
No.
R
Yield
m.p.
( C)
o
o
(%)
(%)
9
9
9
9
9
9
9
a
b
c
d
e
f
68
212-214 10a
>300 10b
61
248-250
288-290
280-282
260-261
292-293
252-254
272-274
65
62
61
55
38
79
62
62
67
71
61
69
258-260 10c
112-114 10d
>300
>300
10e
10f
g
202-203 10g
9
9
h
i
77
68
276-278 10h
64
246-247
259-260
9
j
75
276-278
2
.2 Biological evaluation
2
.2.1 Antiviral activity
The tricyclic compounds (9 and 10) were screened as inhibitors of HSV using herpes
simplex virus-1 (KOS strain), herpes simplex virus-2 (G strain), thymidine kinase
–
r
deficient (aciclovir resistant) herpes simplex virus-1 (HSV-1 TK KOS ACV strain) in
7

human embryonic lung (HEL) cells. None of the tricyclic HBG derivatives (10)
showed any inhibitory activity (EC >100 M) or cytotoxicity (>100 M) against
5
0
HSV-1 or HSV-2. However, some of the tricyclic PCV derivatives (9) exhibited anti-
herpetic activity (Table 2).
Table 2. Inhibitory activity of tricyclic PCV derivatives (9) against HSV and
cytotoxicity data
a
Antiviral activity EC (M)
Cytotoxicity (M)
5
0
-
Compound HSV-1 (KOS) HSV-2 (G)
HSV-1 TK
KOS ACV
0.2
Cell Morphology
r
b
(MCC)
PCV (6)
0.8
>100
8.9
0.2
>100
28
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
i
>100
12
>100
>100
20
>100
>100
31.7
2.3
>100
>100
>100
>100
0.8
>100
>100
>100
7.6
1.5
0.8
76.5
44.7
>100
>20
40.9
>100
j
HBG (7)
GCV
CDV
ACV
BVDU
1.5
0.07
2.0
0.9
0.04
2.1
0.4
2
0.9
>250
10
0.8
5
100
14
>100
>250
>250
>250
>250
a
Required to reduce virus-induced cytopathogenicity by 50 %.
Required to cause a microscopically detectable alteration of normal cell morphology.
b
Aciclovir (ACV), Brivudin (BVDU), Cidofovir (CDV), Ganciclovir (GCV)
Of the newly synthesised tricyclic PCV derivatives, compounds 9a, 9c, 9dand 9j were
inactive as antiviral agents. Compounds 9h and 9i showed anti-HSV-1 activity only at
a relatively high concentration (EC = 76.5 and 44.7 µM, respectively), compounds
5
0
9
b and 9e showed moderate anti-HSV-1 activity (EC50 = 8.9 and 20.00 µM/mL,
respectively), whereas 9g and 9f were the most active and were inhibitory to HSV-1
replication with EC = 1.5 and 7.6 µM, respectively. Similar to anti-HSV-1 results,
5
0
8

among the synthesised tricyclic PCV compounds, compounds 9g and 9f were the
most active against the HSV-2 strain and showed marked inhibition (EC = 0.8 and
5
0
2
.3µM, respectively). Also, compounds 9b, 9e and 9i showed moderate activity
against HSV-2 strain (EC = 28 - 40.9 µM), whereas the remaining compounds did
5
0
not show any activity against the HSV-2 strain. Two compounds, 9b and 9g, retained
-
activity against the TK ACV resistant strain of HSV-1. Compound 9g showed potent
-
inhibitory activity against HSV-1 TK (EC = 0.8 µM), whereas compound 9b was
5
0
-
moderately active against HSV-1 TK (EC = 12 µM). The data suggest that in the
5
0
-
case of the resistant strain TK , compounds 9f and 9g are independent of viral TK
catalysed phosphorylation for antiviral activity and/or may use a different metabolic
activation pathway and/or display a unique mechanism of antiviral action by the un-
metabolised nucleoside analogue.
Table 3. Inhibitory activity of tricyclic PCV derivatives (9) against VZV and HCMV
and cytotoxicity data
a
Antiviral activity EC (M)
Cytotoxicity
5
0
Compound
(M)
VZV TK+
Oka strain
3.5
VZV TK-
07-1 strain
33.4
HCMV
AD-169 strain
>100
HCMV
Davis strain
>100
Cell Morphology
b
(MCC)
PCV (6)
>100
>100
>100
>100
>100
100
>100
>100
>100
100
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
i
>100
>100
>100
63.14
49.5
>100
>100
>100
>100
100
≥74.5
100
>100
>100
>100
>100
>100
>100
63.1
>100
>100
>100
>100
>100
>100
>100
76.5
>100
>100
>100
9.6
66.87
>100
>20
>100
>100
>100
>100
j
62.8
>100
>100
HBG (7)
GCV
CDV
ACV
BVDU
76.5
ND
ND
5.0
0.037
>100
ND
ND
59.2
7.9
>100
6.5
0.72
ND
>100
4.6
0.87
ND
>100
>250
>250
>250
>250
ND
ND
9

aRequired to reduce virus-induced cytopathogenicity (HCMV) or plaque formation (VZV) by
0 %.
5
b
Required to cause a microscopically detectable alteration of normal cell morphology.
Aciclovir (ACV), Brivudin (BVDU), Cidofovir (CDV), Ganciclovir (GCV).
ND: not determined.
In addition to HSV all the tricyclic derivatives (9 and 10) were screening against a
broad range of DNA and RNA viruses including vaccinia virus, adenovirus-2, human
coronavirus (229E), human cytomegalovirus (HCMV) (AD-169 strain and Davis
+
-
strain), varicella-zoster virus (TK VZV OKA strain and TK VZV 07-1 strain),
vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytial virus,
parainfluenza-3 virus, reovirus-1, Sindbis virus, Punta Toro virus, yellow fever virus,
influenza A virus (H N and H N subtypes) and influenza B virus. Of all the
1
1
3
2
compounds, only a few displayed any inhibitory activity < 100 M against VZV and
HCMV (Table 3): 9e (R = CF -C H -) showed weak inhibitory activity against
3
6
4
cytomegalovirus AD-169 strain and Davis strain (EC50 = 63.1 µM and 76.5 µM
respectively) compared with GCV as the reference compound (EC = 6.5 µM and 4.6
5
0
µM respectively) and the TK+ VZV Oka strain (EC = 49.5 µM); 9f (R = HO-C H -)
5
0
6
4
+
showed inhibitory activity against varicella-zoster virus TK VZV OKA strain and
-
TK VZV 07-1 strain (EC = 9.6 µM and ≥74.5 µM respectively) compared with
5
0
ACV as the reference compound (EC = 5.0 µM and 59.2 µM respectively); 9d and
5
0
9
j only displayed minimal activity against the VZV TK+ strain Oka (EC = 63 µM).
50
It should be noted that in contrast to HSV, HBG (7) proved inactive against HCMV
and poorly inhibited the VZV TK+ strain (EC = 76.5 µM). PCV was not inhibitory
5
0
to HCMV but inhibited VZV with EC values comparable with those of ACV, i.e.
5
0
3
.5 and 33.4 µM, respectively for TK+ and TK- VZV strains (Table 3).
2
.2.2 Cytostatic activity
1
0

The new compounds were screened for their potential cytostatic activity against eight
human cancer cell lines. The 50% cytostatic inhibitory concentration (IC ) causing a
5
0
5
0% decrease in cell proliferation was determined against retina (non-cancerous)
hTERT RPE-1, pancreatic adenocarcinoma Capan-1, chronic myeloid leukaemia
Hap1, lung carcinoma NCI-H460, acute lymphoblastic leukaemia DND-41, acute
myeloid leukaemia HL-60, chronic myeloid leukaemia K-562 and non-Hodgkin
lymphoma Z-138 (Table 4).
Table 4: Cytostatic activity results of compounds 9 and 10
a
IC (M)
5
0
hTERT
RPE-1
Capan-
1
NCI-
DND-
41
HL-
60
K-
Hap1
Z-138
No.
H460
562
PCV
>100
78.6
>100
>100
>100
51.5
93.0
58.3
82.8
84.8
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
9
a
9
b
9
c
9
d
>100 >100 >100
17.2 37.8 12.5
9
e
56.5
55.3 >100 76.1
9
f
>100
>100
>100
>100
16.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.0
>100 >100 >100
>100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
9
g
9
h
>100 >100
95.4
>100 >100
44.0 47.2
>100 >100
>100 >100
9
9
i
j
78.8
46.4
72.7
69.1
56.1
64.4
78.9
36.1
98.9
92.8
90.4
63.3 >100 >100
HBG
0a
0b
0c
0d
0e
0f
0g
0h
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
>100 >100 >100 >100
1
1
>100
55.5
1
>100 >100
>100 >100
1
1
63.8
52.5
1
>100 >100
>100 >100
1
1
>100 >100 >100
Docetaxel
Etoposide
Stauroporine
0.95
0.15
0.66
1.19
0.04
3.55 11.50
0.89
1.35
1.63
0.06
21.5
1.94 3.00 3.00
0.03 0.54 1.06
9.24 23.10 21.50
0.23
0.25
a
5
0% Inhibitory concentration or compound concentration required to inhibit cell proliferation by 50%.
hTERT RPE-1 retina (non cancerous); Capan-1 pancreatic adenocarcinoma; Hap1 chronic myeloid
leukemia; NCI-H460 lung carcinoma; DND-41 acute lymphoblastic leukemia; HL-60 acute myeloid
leukemia; K-562 chronic myeloid leukemia; Z-138 non-Hodgkin lymphoma
1
1

Among the tricyclic PCV derivatives compounds 9e(R = CF -C H -) and 9j(R =
3
6
4
C H -O-C H -) showed moderate cytostatic activity against at least six cell lines in
6
5
6
4
the range of 12.5 - 76.1 µM and 16.9 - 90.4 µM respectively. The tricyclic HBG
derivatives (10) were generally inactive or displayed very weak cytostatic activity. Of
the tricyclic HBG derivatives the trifluoromethyl aryl derivative of HBG 10e (R =
CF -C H -) was the most effective against the range of cancer cell lines. Proliferation
3
6
4
of the pancreatic adenocarcinoma Capan-1 cell line was slightly inhibited by most of
the new tricyclic compounds as well as the parent compounds PCV and HBG (Table
4
).
2
.3 Molecular Modelling
Molecular modelling of the compounds was performed using Molecular Operating
1
8
Environment (MOE) software and the crystal structure of HSV-1 thymidine kinase
1
9
co-crystallised with PCV (PDB 1KI3) and 9-(4-hydroxybutyl)-N2-phenylguanine
2
0
(
HBPG) (PDB 1QHI) .
The crystal structures of HSV-1 TK do not include an ATP molecule and a sulphate
ion is observed at the supposed position of the β-phosphate of ATP.19,20 Therefore, the
distance between OH of the acyclic chain and SO was measured to give an idea
4
about the proper orientation of the ligand within the active site and the possibility for
the compound acyclic OH to be phosphorylated by the enzyme. Results indicated that
PCV analogues 9a-j display distances in the range of 2.73-6.82 Å compared with
PCV (4.61 Å). The HSV-1 TK pocket is predominately lipophilic with several regions
of hydrophilicity. PCV and HBPG probe the space of the enzyme active site in a
manner that maintains both the base moiety (guanine) and the acyclic sugar hydroxyl
groups in a good geometry.
1
2

A
B
Figure 3. 2D
Ligplots
illustrating
the
amino acids in
HSV-1 TK active
site involved in
binding
interactions with
(A) PCV and (B)
HBPG
Overall docking results revealed some important interactions for the binding of the
reference ligands PCV and HBPG within the active site (Figure 3). The base moiety is
lying in a location that stacks with Tyr172 and makes hydrophobic interactions with
the amino acid residues; Tyr172, Met128, His58. Also, H-bond interactions are
observed between the carbonyl group as well as the top ring nitrogens with Arg176
1
3

and Gln125. The acyclic sugar moiety forms two important H-bond interactions with
amino acid residues Arg163and Glu83 (Figure 3).
Figure 4. Docking of representative tricyclic PCV derivative 9f (cyan) in the HSV-1 TK
active site compared with PCV (magenta).
The tricyclic PCV compounds lead to interactions with additional amino acids and,
amongst the new compounds, the observed interactions of the most active compounds
9
f and 9g (R= 4-HO-C H , and 4-MeO-C H ) closely mimic the interactions observed
6 4 6 4
with PCV binding. However, rather than Gln125, the 4-HO-C H and 4-MeO-C H
4
6
4
6
form a H-bond interaction with Ala168 or water-bridged H-bonds with Ala168 and
Gln125 (Figure 4). The moderately active fluoro substituted analogues, 9b and 9e,
showed almost similar binding modes, although the small fluoro substituent was
better docked within the base pocket with the tricyclic moiety forming π-π interaction
with Tyr172.
1
4

As the tricyclic purine moiety is longer than the guanine base of PCV, the orientation
of the 6-arylgroup has an impact on the geometry of the acyclic hydroxyl groups and
hence the stability of the formed complex for further phosphorylation. PCV analogues
9
h, 9i and 9j with relatively more bulky ether moieties (4-EtO-C H , 3-OCH O-4-
6 4 2
C H and 4-C H O-C H ) formed weak interactions within the acyclic sugar binding
6
3
6
5
6
4
pocket. In addition a steric clash was observed in case of compound 9j.The steric
effect might consequently diminish or abolish their complex stability.
A
B
Figure 5: Docking of HBG analogues 10a-h showing two different orientations adopted by
the compounds on binding within the HSV-1 TK active site.
Analysis of the docked HBG analogues 10a-h compared with HBPG suggests that the
ligand likely competes between two different binding orientations within the active
site. In one orientation the tricyclic base moiety stacks with Tyr172 with the carbonyl
group forming a H-bond from Gln125, while the acyclic hydroxyl group is situated
5
.73-7.13 Å from the sulphate group (HPBG 5.66 Å) and donates a H-bond to Glu83
and/or accepts water bridged H-bonds from Arg163, Glu83, Arg222 and Lys62. Also,
Arg176, in some derivatives, donates a H-bond to N-1 (Figure 5A). In the other
orientation the tricyclic structure is lying in a location that allows a stacking
1
5

interaction with His58 and the carbonyl group is being upturned to accept a H-bond
from Arg163 and /or water-bridged H-bonds from Arg163, Glu83, Arg222 and
Lys62.In contrast, the same acyclic hydroxyl group in the second ligand orientation is
1
2.75 Å away from the sulfate compared with the former orientation where it accepts
a H-bond from Arg176 (Figure 5B). In both orientations no contribution in ligand
binding was observed for the 6-aryl group resulting in more flexibility and weaker
binding of the whole compound within the active site.
3
. Conclusion
Comparing antiviral activity to compound structure provides an insight into which
functional groups may be beneficial towards HSV-1 inhibitory activity.
Figure 6: Summary for SAR of the new tricyclic PCV compounds
The SAR study suggested that a di-hydroxyl acyclic sugar moiety was essential for
the activity. For the PCV derivatives, activity was only retained in those that have a
hydrogen bond forming group on the tricyclic ring, however the size of the hydrogen
bond forming group is limited with larger groups inducing a steric effect that exhibits
an apparent impact on activity, which was supported by molecular docking (Figure 6).
1
6

The new tricyclic analogues of PCV and HBG are the carbocyclic analogues of the
previously reported tricyclic analogues of GCV and ACV11,12 (Figure 1). For both the
,
tricyclic ACV and GCV derivatives, inhibitory activity against HSV-1 and HSV-2
–
were generally comparable, however this activity was not retained in HSV-1 TK KOS
r
ACV strain. Replacing oxygen in the acyclic chain of the tricyclic ACV analogues
by carbon in the new tricyclic HBG derivatives abolished their activity, highlighting
the preference of O rather than CH in the acyclic chain of the tricyclic guanine
2
derivative. In the case of the tricyclic PCV derivatives the activity generally decreased
noticeably however activity was retained only in the tricyclic PCV derivatives with
R=6-(4-MeO-C H ) and R=6-(4-HO-C H ) and R=6-(4-F-C H ) groups as discussed
6
4
6
4
6
4
in the present study. Importantly, for two of the tricyclic PCV derivatives with R=6-
4-MeO-C H ) and R=6-(4-F-C H ) groups activity was retained in the ACV resistant
(
6
4
6
4
–
HSV-1 TK KOS strain, which was not observed for the tricyclic GCV
compounds11,12
.
This preliminary study provides useful data for further development of tricyclic
acyclic nucleoside derivatives with improved lipophilicity with retention of activity in
TK deficient strains.
4
. Experimental
4
.1 General Experimental
All reagents and solvents were of general purpose or analytical grade and purchased from
1
13
19
Sigma-Aldrich Ltd, Fisher Scientific, Fluka and Acros. H, C and F NMR spectra
were recorded with a Bruker Avance DPX500 spectrometer operating at 500, 125 and
4
70 MHz respectively, with Me Si as internal standard. Elemental analysis was
4
performed (for compounds 10e and 10g) by MEDAC Ltd (Chobham, Surrey, UK).
1
7

High resolution mass spectra (HRMS) were determined (for compounds 9a-j, 10a-d,
0f, and 10h) at the EPSRC National Mass Spectrometry Facility at Swansea
1
University and Medac Ltd (Chobham, Surrey, UK), using ESI (Electrospray
Ionisation). Flash column chromatography was performed with silica gel 60 (230-400
mesh) (Merck) and TLC was carried out on precoated silica plates (kiesel gel 60 F ,
2
54
BDH). Compounds were visualised by illumination under UV light (254 nm). Melting
points were determined on an electrothermal instrument and are uncorrected. All
solvents were dried prior to use and stored over 4 Å molecular sieves, under nitrogen.
All the compounds were ≥ 95% pure.
1
6
The following compounds were prepared as previously described: PCV (6), HBG
1
7
21-25
(
7), and the bromoketones (8).
4
4
1
0
.2 Chemistry
.2.1. General method for the synthesis of tricyclic PCV and HBG derivatives 9 and
0.To a suspension of penciclovir (6) or hydroxybutylguanine (7) (0.2 g, 0.8 mmol or
.9 mmol, respectively) in dry dimethylformamide (16 mL), sodium hydride (60%
suspension in mineral oil, 1.2 eq.) was added and the reaction stirred at room
temperature (21 C) for 1.5 h. Bromoketone (8) (1.3 eq.) was then added and the
̊
reaction stirred for 6 h. Aqueous ammonia (25% solution 5 mL) was added to quench
the reaction, which was concentrated under reduced pressure and the residual oil
purified by flash column chromatography using gradient elution (CH Cl -MeOH).
2
2
The obtained products were recrystallised from a mixture of CH Cl and MeOH.
2
2
4
.2.1.1.
imidazo[1,2-a]purin-9-one(9a). Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-
-(phenyl)ethan-1-one (8a) (0.21 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 95:5 to 90:10 v/v to give the product as
3-(4-Hydroxy-3-(hydroxymethyl)butyl)-6-phenyl-3,5-dihydro-9H-
1
2
2
1
8

o
a white solid. Yield: 0.19 g (68%), m.p. 212-214 C, TLC (CH Cl -MeOH 95:5 v/v)
2
2
1
Rf 0.26. H-NMR (DMSO-d ): δ 1.51 (septet, 1H, J=6.0 Hz, H-3'), 1.82 (q, 2H, J=7.0
6
Hz, H-2'), 3.40 and 3.47 (two quint, 4H, J=5.4Hz, H-4'), 4.17 (t, 2H, J= 7.5Hz, H-1'),
4
.48 (t, 2H, J= 5.3 Hz, 2 x OH), 7.39 (t, 1H, J=7.0 Hz, H-4''), 7.49 (t, 2H, J= 7.7 Hz,
H-3" and H-5"), 7.91 (d, 2H, J= 7.4 Hz, H-2'' and H-6''), 7.95 (s, 1H, H-2), 8.21 (s,
1
3
1
4
H, H- 7), 13.07 (br.s, 1H, H-5). C-NMR (DMSO-d ): δ 29.1 (C-2'), 41.3 (C-3'),
6
2.0 (C-1'), 61.8 (C-4'), 103.6 (C-7), 115.9 (C-9a), 125.4 (C-4''), 128.4 (C-6), 129.2
(C-2" and C-6"), 129.4 (C-1''), 129.5 (C-3'' and C-5''), 139.0 (C-2), 146.9 (C-4a),
1
50.6 (C-3a), 151.7 (C-9). [ESI-HRMS] calculated for C H N O : 354.1561
1
8
20
5
3
+
+
[
M+H] . Found: 354.1562 [M+H] .
4
.2.1.2. 6-(4-Fluorophenyl)-3-(4-hydroxy-3-(hydroxymethyl)butyl)-3,5-dihydro-9H-
imidazo[1,2-a]purin-9-one (9b).Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-
-(4-fluorophenyl)ethanone (8b) (0.23 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 98:2 to 91:9 v/v to give the product as a
1
2
2
o
white solid. Yield: 0.19 g (65%), m.p. > 300 C, TLC (CH Cl -MeOH 9:1 v/v) Rf
2
2
1
0
.49. H-NMR (DMSO-d ): δ 1.50 (septet, 1H, J=6.0 Hz, H-3'), 1.81 (q, 2H, J=7.0
6
Hz, H-2'), 3.38 and 3.46 (two quint, 4H, J=5.4 Hz, H-4'), 4.17 (t, 2H, J= 7.5 Hz, H-
'), 4.48 (t, 2H, J=5.25 Hz, 2 x OH), 7.34 (t, 2H, J = 8.8 Hz, H-3" and H-5"), 7.97
1
HF
(
s, 1H, H-2), 7.98 (dd, 2H, J ,J = 6.0, 8.5 Hz, H-2'' and H-6''), 8.21 (s, 1H, H-7),
HH HF
1
3
1
6
1
1
3.10 (br.s, 1H, H-5). C-NMR(DMSO-d ): δ 29.1 (C-2'), 41.3 (C-3'), 42.0 (C-1'),
6
1.8 (C-4'), 103.6 (C-7), 116.1 (C-9a), 116.6 (d, 2C, JCF =22 Hz, C-3'' and C-5''),
25.1 (C-1''), 127.7 (d, 2C, JCF = 8.0 Hz, C-2'' and C-6"), 128.5 (C-6), 139.6 (C-2),
1
9
46.8 (C-4a), 150.6 (C-3a), 151.8 (C-9), 162.6 (d, 1C, JCF = 245 Hz, C-4''). F-
NMR(DMSO-d ): δ-112.40. [ESI-HRMS] calculated for C H N O F: 372.1472
6
18 19
5
3
+
+
[
M+H] . Found: 372.1483 [M+H] .
1
9

4
.2.1.3. 6-(4-Chlorophenyl)-3-(4-hydroxy-3-(hydroxymethyl)butyl)-3,5-dihydro-9H-
imidazo[1,2-a]purin-9-one, (9c).Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-
-(4-chlorophenyl)ethanone (8c) (0.24 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 95:5 to 90:10 v/v to give the product as
1
2
2
o
a white solid. Yield: 0.19 g (62%), m.p. 258-260 C, TLC (CH Cl -MeOH 95:5 v/v)
2
2
1
Rf 0.29. H-NMR (DMSO-d ): δ 1.50 (septet, 1H, J=6.0 Hz, H-3'), 1.81 (q, 2H, J=7.0
6
Hz, H-2'), 3.38 and 3.46 (two quint, 4H, J=5.5 Hz, H-4'), 4.17 (t, 2H, J= 7.5 Hz, H-
1
1
'), 4.48 (t, 2H, J= 5.2 Hz, 2 x OH), 7.56 (d, 2H, J=8.6 Hz, H-2" and H-6"), 7.94 (s,
H, H-2), 7.95 (d, 2H, J=8.6 Hz, H-3" and H-5"), 8.29 (s, 1H, H-7), 13.13 (br.s, 1H,
1
3
H-5). C-NMR (DMSO-d ): δ 29.1 (C-2'), 41.3 (C-3'), 42.0 (C-1'), 61.8 (C-4'), 104.3
6
(C-7), 116.0 (C-9a), 127.2 (C-2" and C-6"), 127.5 (C-1"), 128.4 (C-6), 129.6 (C-3"
and C-5"), 133.7 (C-4"), 139.5 (C-2), 146.9 (C-4a), 150.7 (C-3a), 151.8 (C-9). [ESI-
+
+
HRMS] calculated for C H N O Cl: 388.1176 [M+H] . Found: 388.1187 [M+H] .
1
8
19
5
3
4
.2.1.4.
imidazo[1,2-a]purin-9-one (9d).Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-
-(p-tolyl)ethanone (8d) (0.22 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 100:0 to 85:15 v/v to give the product as
3-(4-Hydroxy-3-(hydroxymethyl)butyl)-6-(p-tolyl)-3,5-dihydro-9H-
1
2
2
o
a white solid. Yield: 0.18 g (61%), m.p. 112-114 C, TLC (CH Cl -MeOH 95:5 v/v)
2
2
1
Rf 0.47. H-NMR (DMSO-d ): δ 1.50 (septet, 1H, J=6.0 Hz, H-3'), 1.82 (q, 2H, J=7.0
6
Hz, H-2'), 2.34 (s, 3H, CH3), 3.39 and 3.46 (two quint, 4H, J=5.4 Hz, H-4'), 4.17 (t,
2
H, J= 7.5 Hz, H-1'), 4.48 (t, 2H, J= 5.3 Hz, 2 x OH), 7.28 (d, 2H, J=8.0 Hz, H-3"
and H-5"), 7.79 (d, 2H, J=8.0 Hz, H-2" and H-6"), 7.95 (s, 1H, H-2), 8.13 (s, 1H, H-
1
3
7
3
), 13.01 (br.s, 1H, H-5). C-NMR (DMSO-d ): δ 21.3 (CH ), 29.1 (C-2'), 41.3 (C-
6
3
'), 44.0 (C-1'), 61.8 (C-4'), 103.0 (C-7), 116.0 (C-9a), 125.4 (C-3" and C-5"), 125.7
(C-1"), 129.5 (C-6), 130.0 (C-2" and C-6"), 138.8 (C-4"), 139.5 (C-2), 146.8 (C-4a),
2
0

1
3
4
50.6 (C-3a), 151.8 (C-9). [ESI-HRMS] calculated for C H N O Na:
19 21 5 3
+
+
90.1537[M+Na] . Found: 390.1533[M+Na] .
.2.1.5. 3-(4-Hydroxy-3-(hydroxymethyl)butyl)-6-(4-(trifluoromethyl)phenyl)-3,5-
dihydro-9H-imidazo[1,2-a]purin-9-one (9e). Prepared from PCV (0.2 g, 0.8 mmol)
and 2-bromo-1-(4-(trifluoromethyl)phenyl)ethanone (8e) (0.28 g, 1.04 mmol) and
purified by flash column chromatography eluting with CH Cl -MeOH 95:5 to 92:8
2
2
o
v/v to give the product as a white solid. Yield: 0.18 g (55%), m.p. > 300 C, TLC
1
(
CH Cl -MeOH 95:5 v/v) Rf 0.43. H-NMR (DMSO-d ): δ 1.51 (septet, 1H, J= 6.0
2
2
6
Hz, H-3'), 1.82 (q, 2H, J= 7.0 Hz, H-2'), 3.39 and 3.46 (two quint, 4H, J =5.4 Hz, H-
4
2
'), 4.18 (t, 2H, J =7.0 Hz, H-1'), 4.45 (br s, 2H, 2 x OH), 7.85 (d, 2H, J = 7.8 Hz, H-
" and H-6"), 7.98 (s, 1H, H-2), 8.15 (d, 2H, J =7.7 Hz, H-3" and H-5"), 8.42 (s, 1H,
1
3
H-7), 13.24 (br.s, 1H, H-5). C-NMR (DMSO-d ): δ 29.2 (C-2'), 42.0 (C-3'), 42.3 (C-
6
1
2
'), 61.8 (C-4'), 105.7 (C-7), 116.1 (C-9a), 124.5 (d, 1C, J = 272 Hz, CF3), 126.1(C-
CF
'' and C-6''), 126.4 (d, 2C, J = 3.8 Hz, C-3'' and C-5''), 128.0 (C-6), 128.5 (q, 1C,
CF
JCF = 31.8 Hz, C-4''), 132.6 (C-1''), 139.7 (C-2), 147.0 (C-4a), 150.8 (C-3a), 151.7 (C-
1
9
9
4
4
).
22.1440 [M+H] , Found: 422.1430 [M+H] .
.2.1.6. 3-(4-Hydroxy-3-(hydroxymethyl)butyl)-6-(4-hydroxyphenyl)-3,5-dihydro-9H-
F-NMR(DMSO-d ): δ-61.11.[ESI-HRMS] calculated for C H N O F :
6 19 19 5 3 3
+
+
imidazo[1,2-a]purin-9-one (9f).Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-1-
4-hydroxyphenyl)ethanone (8f) (0.22 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 95:5 to 90:10 v/v to give the product as
(
2
2
o
a pale yellow solid. Yield: 0.11 g (38%), m.p. > 300 C, TLC (CH Cl -MeOH 9:1
2
2
1
v/v) Rf 0.25. H-NMR (DMSO-d ): δ 1.51 (septet, 1H, J= 6.0 Hz, H-3'), 1.81 (q, 2H,
6
J= 7.0 Hz, H-2'), 3.38 and 3.45 (two quint, 4H, J =5.4 Hz, H-4'), 4.17 (t, 2H, J=7.0
Hz, H-1'), 4.47 (br s, 2H, 2 x OH), 6.86 (d, 2H, J =8.7 Hz, H-3" and H-5"), 7.72 (d,
2
1

2
H, J =8.7 Hz, H-2" and H-6"), 7.95 (br s, 2H, H-2 and H-7) 9.87 (br s, 1H, phenolic
1
3
OH), 12.89 (br.s, 1H, H-5). C-NMR (DMSO-d ): δ 29.1 (C-2'), 41.3 (C-3'), 42.0 (C-
6
1
'), 61.8 (C-4'), 101.4 (C-7), 116.0 (C-9a), 116.3 (C-3'' and C-5''), 119.2 (C-1''), 127.1
(C-2'' and C-6''), 129.9 (C-6), 139.6 (C-2), 146.6 (C-4a), 150.5 (C-3a) 151.8 (C-9),
+
1
3
4
58.6 (C- 4''). [ESI-HRMS] calculated for C H N O : 368.1359 [M-H] . Found:
1
8
18
5
4
68.1349 [M-H]+.
.2.1.7. 3-(4-Hydroxy-3-(hydroxymethyl)butyl)-6-(4-methoxyphenyl)-3,5-dihydro-9H-
imidazo[1,2-a]purin-9-one (9g). Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-
-(4-methoxyphenyl)ethanone (8g) (0.24 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 97.5:2.5 to 87:13 v/v to give the product
1
2
2
o
as a white solid. Yield: 0.24 g (79%), m.p. 202-203 C, TLC (CH Cl -MeOH 9:1 v/v)
2
2
1
Rf 0.45. H-NMR (DMSO-d ): δ 1.51 (septet, 1H, J=6.0 Hz, H-3'), 1.81 (q, 2H, J=7.0
6
Hz, H-2'), 3.39 and 3.46 (two quint, 4H, J=5.5 Hz, H-4'), 3.81 (s, 3H, OCH ), 4.17 (t,
3
2
H, J=7.5 Hz, H-1'), 4.47 (t, 2H, J= 5.2 Hz, 2 x OH), 7.04 (d, 2H, J=8.0 Hz, H-3" and
H-5"), 7.85 (d, 2H, J=8.0 Hz, H-2" and H-6"), 7.95 (s, 1H, H-2), 8.07 (s, 1H, H-7),
1
3
1
5
1
1
2.97 (br.s, 1H, H-5). C-NMR (DMSO-d ): δ 29.1 (C-2'), 41.3 (C-3'), 42.0 (C-1'),
6
5.8 (OCH ), 61.8 (C-4'), 102.2 (C-7), 115.0 (C-3" and C-5"), 116.1 (C-9a), 120.9 (C-
3
"), 127.0 (C-2" and C-6"), 129.4 (C-6), 139.5 (C-2), 146.7 (C-4a), 150.5 (C-3a),
51.8 (C-9), 160.1 (C-4"). [ESI-HRMS] calculated for C H N O : 384.1666
1
9
22
5
4
+
+
[
M+H] . Found: 384.1666 [M+H] .
4
.2.1.8. 6-(4-Ethoxyphenyl)-3-(4-hydroxy-3-(hydroxymethyl)butyl)-3,5-dihydro-9H-
imidazo[1,2-a]purin-9-one (9h). Prepared from PCV (0.2 g, 0.8 mmol) and 2-bromo-
-(4-ethoxyphenyl)ethanone (8h) (0.25 g, 1.04 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 95:5 to 91:9 v/v to give the product as a
1
2
2
o
pale yellow solid. Yield: 0.24 g (77%), m.p. 276-278 C, TLC (CH Cl -MeOH 9:1
2
2
2
2

1
v/v) Rf 0.5. H-NMR (DMSO-d ): δ 1.35 (t, 3H, J= 7.0 Hz, CH ), 1.51 (septet, 1H,
6
3
J=6.0 Hz, H-3'), 1.82 (q, 2H, J=7.0 Hz, H-2'), 3.39 and 3.46 (two quint, 4H, J=5.5 Hz,
H-4'), 4.08 (q, 2H, J= 7.0 Hz, OCH ), 4.17 (t, 2H, J=7.0 Hz, H-1'), 4.44 (br s, 2H, 2 x
2
OH), 7.02 (d, 2H, J= 8.3 Hz, H-3" and H-5"), 7.83 (d, 2H, J= 8.3 Hz, H-2" and H-6"),
1
3
7
1
7
.94 (s, 1H, H-2), 8.05 (s, 1H, H-7), 12.93 (br.s, 1H, H-5). C-NMR (DMSO-d ): δ
6
5.1 (CH ), 29.2 (C-2'), 41.4 (C-3'), 42.0 (C-1'), 61.8 (C-4'), 63.7 (OCH ), 102.1 (C-
3
2
), 115.4 (C-3" and C-5"), 116.1 (C-9a), 120.8 (C-1"), 127.0 (C-2" and C-6"), 129.4
(C-6), 140.0 (C-2), 146.7 (C-4a), 150.5 (C-3a), 151.8 (C-9), 159.4 (C-4"). [ESI-
+
+
HRMS] calculated for C H N O : 396.1672 [M-H] , Found: 396.1668 [M-H] .
2
0
22
5
4
4
.2.1.9.
6-(Benzo[d][1,3]dioxol-5-yl)-3-(4-hydroxy-3-(hydroxymethyl)butyl)-3,5-
dihydro-9H-imidazo[1,2-a]purin-9-one (9i).Prepared from PCV (0.2 g, 0.8 mmol)
and 1-(benzo[d][1,3]dioxol-5-yl)-2-bromoethanone (8i) (0.25 g, 1.04 mmol) and
purified by flash column chromatography eluting with CH Cl -MeOH-acetone
2
2
8
2
0:10:10 v/v/v to give the product as a pale yellow solid. Yield: 0.21 g (68%), m.p.
o
1
59-260 C, TLC (CH Cl -MeOH 9:1 v/v) Rf 026. H-NMR (DMSO-d ): δ 1.51
2
2
6
(septet, 1H, J=6.0 Hz, H-3'), 1.82 (q, 2H, J=7.0 Hz, H-2'), 3.38 and 3.46 (two quint,
4
H, J=5.5 Hz, H-4'), 4.17 (t, 2H, J=7.5 Hz, H-1') 4.45 (t, 2H, J= 5.2 Hz, 2 x OH), 6.09
(s, 2H, H-2''), 7.03 (d, 1H, J= 8.1 Hz, H-7''), 7.41 (dd, 1H, J= 1.8, 8.1 Hz, H-6''), 7.53
(d, 1H, J= 1.7 Hz, H-4''), 7.94 (s, 1H, H-2), 8.11 (s, 1H, H-7), 12.92 (br.s, 1H, H-5).
1
3
C-NMR (DMSO-d ): δ 29.2 (C-2'), 41.4 (C-3'), 42.0 (C-1'), 61.8 (C-4'), 101.9 (C-
6
2
1
1
3
4
''), 102.8 (C-7), 106.0 (C-4''), 109.2 (C-7''), 116.1 (C-9a), 119.5 (C- 6''), 122.4 (C-5''),
29.3 (C-6), 139.6 (C-2), 146.7 (C-4a), 148.2 (C-7a''), 148.5 (C-3a''), 150.6 (C-3a)
+
51.8 (C-9). [ESI-HRMS] calculated for C H N O : 398.1459 [M+H] . Found:
1
9
20
5
5
98.1460 [M+H]+.
.2.1.10. 3-(4-Hydroxy-3-(hydroxymethyl)butyl)-6-(4-phenoxyphenyl)-3,5-dihydro-
2
3

9
H-imidazo[1,2-a]purin-9-one (9j).Prepared from PCV (0.2 g, 0.8 mmol) and 2-
bromo-1-(4-phenoxyphenyl)ethanone (8j) (0.3 g, 1.04 mmol) and purified by flash
column chromatography eluting with CH Cl -MeOH 100:0 to 92:8 v/v to give the
2
2
o
product as a white solid. Yield: 0.26 g (75%), m.p. 278-280 C, TLC (CH Cl -MeOH
2
2
1
9
2
7
:1 v/v) Rf 0.53. H-NMR (DMSO-d ): δ 1.52 (septet, 1H, J=6.0 Hz, H-3'), 1.82 (q,
6
H, J=7.0 Hz, H-2'), 3.38 and 3.46 (two quint, 4H, J=5.5 Hz, H-4'), 4.18 (t, 2H, J=
.5 Hz, H-1'), 4.44 (br s, 2H, 2 x OH), 7.09 (d, 4H, J= 8.1 Hz, H-3'', H-5'', H-2''' and
H-6'''), 7.20 (t, 1H, J= 7.4 Hz, H-4'''), 7.44 (t, 2H, J=8.0 Hz, H-3''' and H-5'''), 7.93 (d,
H, J= 8.8 Hz, H- 2" and H-6"), 7.98 (s, 1H, H-2), 8.14 (s, 1H, H-7), 13.04 (br.s, 1H,
2
1
3
H-5). C-NMR (DMSO-d ): δ 29.1 (C-2'), 41.4 (C-3'), 42.0 (C-1'), 61.8 (C-4'), 103.1
6
(C-7), 115.9 (C-9a), 119.2 and 119.6 (C-3", C-5", C-2''' and C-6'''), 123.6 (C-1"),
1
1
24.5 (C-4'''), 127.4 and 130.7 (C-2", C-6", C-3''' and C-5'''), 129.0 (C-6), 139.5 (C-2),
46.8 (C-4a), 150.6 (C-3a), 151.7 (C-9), 156.5 and 157.7 (C- 4" and C-1'''). [ESI-
+
+
HRMS] calculated for C H N O :444.1672 [M-H] , Found: 444.1675 [M-H] .
2
4
22
5
4
4
.2.1.11.
one (10a). Prepared from HBG (0.2 g, 0.9 mmol) and 2-bromo-1-(phenyl)ethan-1-one
8a) (0.23 g, 1.17 mmol) and purified by flash column chromatography eluting with
CH Cl -MeOH 95:5 to 93:7 v/v to give the product as a white solid. Yield: 0.18 g
3-(4-Hydroxybutyl)-6-phenyl-3,5-dihydro-9H-imidazo[1,2-a]purin-9-
(
2
2
o
1
(
61%), m.p. 248-250 C, TLC (CH Cl -MeOH 95:5 v/v) Rf 0.32. H-NMR (DMSO-
2 2
d6): δ 1.43 (quint., 2H, J=7.0 Hz, H-3'), 1.85 (quint, 2H, J= 7.0 Hz, H- 2'), 3.42 (q,
H, J= 5.5 Hz, H-4'), 4.11 (t, 2H, J=7.2 Hz, H-1'), 4.49 (t, 1H, J= 4.8 Hz, OH), 7.39
t, 1H, J= 7.4 Hz, H-4"), 7.48 (t, 2H, J= 7.7 Hz, H-3" and H-5"), 7.91 (d, 2H, J= 7.6
2
(
1
3
Hz, H-2'' and H-6''), 7.95 (s, 1H, H-2), 8.22 (s, 1H, H-7), 13.07 (br.s, 1H, H-5). C-
NMR (DMSO-d ): δ 26.7 (C-2'), 30.0 (C-3'), 43.5 (C-1'), 60.6 (C-4'), 103.6 (C-7),
6
1
16.1 (C-9a), 125.4 (C-4''), 128.5 (C-6), 129.2 (C-2'' and C-6''), 129.4 (C-1''), 129.5
2
4

(C-3'' and C-5''), 139.7 (C-2), 146.9 (C-4a), 150.9 (C-3a), 151.8 (C-9). [ESI-HRMS]
+
+
calculated for C H N O : 322.1309 [M+H] . Found: 322.1302 [M+H] .
1
7
28
5
2
4
.2.1.12.
6-(4-Fluorophenyl)-3-(4-hydroxybutyl)-3,5-dihydro-9H-imidazo[1,2-
a]purin-9-one (10b). Prepared from HBG (0.2 g, 0.9 mmol) and 2-bromo-1-(4-
fluorophenyl)ethanone (8b) (0.25 g, 1.17 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 100:0 to 94:6 v/v to give the product as
2
2
o
a white solid. Yield: 0.19 g (62%), m.p. 288-290 C, TLC (CH Cl -MeOH 95:5 v/v)
2
2
1
Rf 0.31. H-NMR (DMSO-d ): δ 1.43 (quint, 2H, J=7.5 Hz, H-3'), 1.85 (quint, 2H, J=
6
7
.5 Hz, H-2'), 3.42 (q, 2H, J=5.4 Hz, H-4'), 4.11 (t, 2H, J=7.0 Hz, H-1'), 4.49 (t, 1H,
J= 5.0 Hz, OH), 7.34 (t, 2H, J = 8.9 Hz, H-3'' and H-5''), 7.96 (dd, 2H, J = 5.0, 9.0
HF
HF
Hz, H-2" and H-6"), 7.96 (s, 1H, H-2), 8.22 (d, 1H, J= 2.0 Hz, H-7), 13.09 (br.s, 1H,
1
3
H-5). C-NMR (DMSO-d ): δ 26.7 (C-2'), 30.0 (C-3'), 43.5 (C-1'), 60.6 (C-4'), 103.6
6
(
C-7), 116.1 (C-9a), 116.6 (d, 2C, J = 22.6 Hz, C-3'' and C-5''), 125.1 (d, 1C, J= 3.8
CF
Hz, C-1''), 127.7 (d, 2C, J= 7.5 Hz, C-2'' and C-6''), 128.5 (C-6), 139.7 (C- 2), 146.8
C-4a), 150.7 (C-3a), 151.8 (C-9), 162.6 (d, 1C, J= 246.5 Hz, C-4''). 19F-
(
NMR(DMSO-d ): δ-112.40.[ESI-HRMS] calculated for C H N O F: 342.1366
6
17 17
5
2
+
+
[
M+H] . Found: 342.1373 [M+H] .
4
.2.1.13.
6-(4-Chlorophenyl)-3-(4-hydroxybutyl)-3,5-dihydro-9H-imidazo[1,2-
a]purin-9-one (10c). Prepared from HBG (0.2 g, 0.9 mmol) and 2-bromo-1-(4-
chlorophenyl)ethanone (8c) (0.27 g, 1.17 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 95:5 to 92:8 v/v to give the product as a
2
2
o
white solid. Yield: 0.2 g (62%), m.p. 280-282 C, TLC (CH Cl -MeOH 95:5 v/v) Rf
2
2
1
0
7
.36. H-NMR (DMSO-d ): δ 1.43 (quint, 2H, J = 7.0 Hz, H-3'), 1.85 (quint, 2H, J=
6
.5 Hz; H-2'), 3.42 (q, 2H, J=5.5 Hz, H-4'), 4.10 (t, 2H, J=7.0 Hz, H-1'), 4.48 (t, 1H,
J= 5.0 Hz, OH), 7.53 (d, 2H, J= 8.4 Hz, H-2" and H-6"), 7.92 (d, 2H, J= 8.4 Hz, H-3''
2
5

1
3
and H-5''), 7.94 (s, 1H, H-2), 8.27 (s, 1H, H-7), 14.00 (br.s, 1H, H-5). C-NMR
(
DMSO-d ): δ 26.7 (C-2'), 30.0 (C-3'), 43.5 (C-1'), 60.6 (C-4'), 104.3 (C-7), 116.1 (C-
6
9
4
a), 127.1 (C-2'' and 6''), 127.4 (C-1''), 128.3 (C-6), 129.5 (C-3'' and C-5''), 133.6 (C-
''), 139.7 (C-2), 146.9 (C-4a), 150.7 (C-3a), 151.7 (C-9).[ESI-HRMS] calculated for
+
+
C H N O Cl: 358.1071 [M+H] . Found: 358.1075 [M+H] .
1
7
17
5
2
4
.2.1.14. 3-(4-Hydroxybutyl)-6-(p-tolyl)-3,5-dihydro-9H-imidazo[1,2-a]purin-9-one
(10d). Prepared from HBG (0.2 g, 0.9 mmol) and 2-bromo-1-(p-tolyl)ethanone (8d)
(0.25 g, 1.17 mmol) and purified by flash column chromatography eluting with
CH Cl -MeOH 100:0 to 94:6 v/v to give the product as a white solid. Yield: 0.2 g
2
2
o
1
(
67%), m.p. 260-261 C, TLC (CH Cl -MeOH 95:5 v/v) Rf 0.63. H-NMR (DMSO-
2 2
d6): δ 1.43 (quint, 2H, J=7.0 Hz, H-3'), 1.85 (quint, 2H, J= 7.5 Hz, H-2'), 2.34 (s, 3H,
CH ), 3.42 (q, 2H, J=5.5 Hz, H-4'), 4.11 (t, 2H, J=7.2 Hz, H-1'), 4.49 (t, 1H, J= 5 Hz,
3
OH), 7.28 (d, 2H, J= 8.0 Hz, H-3'' and H-5''), 7.79 (d, 2H, J= 8.2 Hz, H-2" and H-6"),
1
3
7
2
9
4
.95 (s, 1H, H-2), 8.14 (s, 1H, H-7), 13.01 (br.s, 1H, H- 5). C-NMR (DMSO-d ): δ
6
1.3 (CH ), 26.7 (C-2'), 30.0 (C-3'), 43.5 (C-1'), 60.6 (C-4'), 103.0 (C-7), 116.0 (C-
3
a), 125.4 (C-3'' and 5''), 125.6 (C-1''), 129.5 (C- 6), 130.1 (C-2'' and C-6''), 138.8 (C-
''), 139.6 (C-2), 146.8 (C-4a), 150.6 (C- 3a), 151.8 (C-9). [ESI-HRMS] calculated for
+
+
C H N O : 338.1617 [M+H] . Found: 338.1626 [M+H] .
1
8
20
5
2
4
.2.1.15.
3-(4-Hydroxybutyl)-6-(4-(trifluoromethyl)phenyl)-3,5-dihydro-9H-
imidazo[1,2-a]purin-9-one (10e). Prepared from HBG (0.2 g, 0.9 mmol) and 2-
bromo-1-(4-(trifluoromethyl)phenyl)ethanone (8e) (0.3 g, 1.17 mmol) and purified
by flash column chromatography eluting with CH Cl -MeOH 97.5:2.5 to 92:8 v/v to
2
2
o
give the product as a white solid. Yield: 0.25 g (71%), m.p. 292-293 C, TLC
1
(
CH Cl -MeOH 95:5 v/v) Rf 0.58. H-NMR (DMSO-d ): δ 1.44 (quint, 2H, J=7.0 Hz,
2
2
6
H-3'), 1.86 (quint, 2H, J= 7.5 Hz, H-2'), 3.43 (q, 2H, J=5.5 Hz, H-4'), 4.12 (t, 2H,
2
6

J=7.0 Hz, H-1'), 4.45 (t, 1H, J= 5.0 Hz, OH), 7.84 (d, 2H, J= 8.0 Hz, H-2'' and H-6''),
.96 (s, 1H, H-2), 8.14 (d, 2H, J= 8.0 Hz, H-3" and H-5"), 8.42 (s, 1H, H-7), 13.23
7
1
3
(
br.s, 1H, H-5). C-NMR (DMSO-d ): δ 26.7 (C-2'), 30.0 (C-3'), 43.5 (C-1'), 60.6 (C-
6
4
6
'), 105.7 (C-7), 116.1 (C-9a), 124.5 (d, 1C, J= 272.0 Hz, CF ), 126.0 (C-2'' and C-
3
''), 126.4 (d, 2C, J= 3.6 Hz, C-3'' and C-5''), 128.0 (C-6), 128.5 (q, 1C, J = 32.0 Hz,
CF
C-4''), 132.6 (C-1''), 139.8 (C-2), 147.1 (C-4a), 150.8 (C-3a), 151.7 (C-9). 19F-
NMR(DMSO-d ): δ -61.14. Anal. Calcd for C H F N O 0.5H O (400.1309): C,
6
18 16
3
5
2
2
5
4.00; H, 2.38; N, 17.49. Found: C, 53.56; H, 3.84; N, 17.33.
.2.1.16. 3-(4-Hydroxybutyl)-6-(4-methoxyphenyl)-3,5-dihydro-9H-imidazo[1,2-
4
a]purin-9-one (10f). Prepared from HBG (0.2 g, 0.9 mmol) and 2-bromo-1-(4-
methoxyphenyl)ethanone (8g) (0.27 g, 1.17 mmol) and purified by flash column
chromatography eluting with CH Cl -MeOH 95:5 to 93:7 v/v to give the product as a
2
2
o
white solid. Yield: 0.19 g (61%), m.p. 252-254 C, TLC (CH Cl -MeOH 95:5 v/v) Rf
2
2
1
0
7
.23. H-NMR (DMSO-d ): δ 1.43 (quint, 2H, J=7.0 Hz, H-3'), 1.85 (quint, 2H, J=
6
.5 Hz; H-2'), 3.42 (q, 2H, J=5.4 Hz, H-4'), 3.81 (s, 3H, OCH ) 4.11 (t, 2H, J=7.0 Hz,
3
H-1'), 4.48 (t, 1H, J= 5.0 Hz, OH), 7.04 (d, 2H, J= 8.8 Hz, H-3" and 5''), 7.84 (d, 2H,
J= 8.8 Hz, H-2" and H-6"), 7.94 (s, 1H, H-2), 8.08 (s, 1H, H-7), 12.97 (br.s, 1H, H-5).
1
3
C-NMR (DMSO-d ): δ 26.7 (C-2'), 30.1 (C-3'), 43.5 (C-1'), 55.7 (OCH ), 60.6 (C-
6
3
4
'), 102.2 (C-7), 115.0 (C-3'' and C-5''), 116.0 (C-9a), 120.9 (C-1''), 126.9 (C-2'' and
C-6''), 129.4 (C-6), 139.6 (C-2), 146.7 (C-4a), 150.5 (C-3a), 151.8 (C-9), 160.1 (C-
+
4
'').ꢀ[ESI-HRMS] calculated for C H N O : 354.1566 [M+H] . Found: 354.1563
1
8
20
5
3
[
M+H]+.
4
.2.1.17.
6-(Benzo[d][1,3]dioxol-5-yl)-3-(4-hydroxybutyl)-3,5-dihydro-9H-
imidazo[1,2-a]purin-9-one (10g). Prepared from HBG (0.2 g, 0.9 mmol) and 1-
benzo[d][1,3]dioxol-5-yl)-2-bromoethanone (8i) (0.28 g, 1.17 mmol) and purified by
(
2
7

flash column chromatography eluting with CH Cl -MeOH 97.5:2.5 to 92:8 v/v to give
2
2
o
the product as a pale yellow solid. Yield: 0.23 g (69%), m.p. 272-274 C, TLC
1
(
CH Cl -MeOH 95:5 v/v) Rf 0.38. H-NMR (DMSO-d ): δ 1.43 (quint, 2H, J=7.5 Hz,
2
2
6
H-3'), 1.85 (quint, 2H, J= 7.5 Hz; H-2'), 3.42 (q, 2H, J=5.5 Hz, H-4'), 4.11 (t, 2H, J=7
Hz, H-1'), 4.45 (t, 1H, J= 5 Hz, OH), 6.09 (s, 1H, H-2''), 7.03 (d, 1H, J= 8.2 Hz, H-
7
''), 7.41 (d, 1H, J= 8.1 Hz, H-6"), 7.53 (s, 1H, H-4''), 7.93 (s, 1H, H-2), 8.12 (s, 1H,
1
3
H-7), 12.92 (br.s, 1H, H- 5). C-NMR (DMSO-d ): δ 26.7 (C-2'), 30.1 (C-3'), 43.5
6
(C-1'), 60.7 (C- 4'), 101.9 (C-2''), 102.8 (C-7), 106.0 (C-4''), 109.2 (C-7''), 116.0 (C-
9
1
a), 119.4 (C-6''), 122.5 (C-5''), 129.4 (C-6), 139.9 (C-2), 146.7 (C-4a), 148.2 (C-7a''),
48.5 (C-3a''), 150.6 (C-3a), 151.8 (C-9). Anal. Calcd for C H N O 0.3H O
1
8
17
5
4
2
(372.5312): C, 58.00; H, 4.76; N, 18.79. Found: C, 57.60; H, 4.37; N, 18.56.
4
.2.1.18.
3-(4-Hydroxybutyl)-6-(thiophen-2-yl)-3,5-dihydro-9H-imidazo[1,2-
a]purin-9-one (10h).Prepared from HBG (0.2 g, 0.9 mmol) and 2-bromo-1-(thiophen-
-yl)ethanone (8k) (0.24 g, 1.17 mmol) and purified by flash column chromatography
eluting with CH Cl -MeOH 95:5 to 92:8 v/v to give the product as a pale yellow
2
2
2
o
solid. Yield: 0.19 g (64%), m.p. 246-247 C, TLC (CH Cl -MeOH 9:1 v/v) Rf 0.67.
2
2
1
H-NMR (DMSO-d ): δ 1.43 (quint., 2H, J=7.0 Hz, H-3'), 1.85 (quint, 2H, J= 7.5 Hz;
6
H-2'), 3.42 (q, 2H, J=5.5 Hz, H-4'), 4.11 (t, 2H, J=7.0 Hz, H-1'), 4.45 (t, 1H, J= 5.0
Hz, OH), 7.18 (t, 1H, J= 4.2 Hz, H-4''), 7.61 (d, 1H, J= 3.3 Hz, H-3''), 7.66 (d, 1H, J=
1
3
4
.9 Hz, H-5''), 7.91 (s, 1H, H-2), 7.95 (s, 1H, H-7), 13.14 (br.s, 1H, H-5). C-NMR
(
DMSO-d ): δ 26.6 (C-2'), 30.0 (C-3'), 43.5 (C-1'), 60.6 (C-4'), 102.7 (C-7), 116.1 (C-
6
9
1
a), 124.6 (C-6), 126.6 (C-3''), 127.5 (C-4''), 128.6 (C-5''), 139.7 (C-2), 146.5 (C-4a),
50.6 (C-3a), 151.7 (C-9). [ESI-HRMS] calculated for C H N O S: 330.1019
1
5
16
5
2
+
+
[
M+H] . Found: 330.1021 [M+H] .
4
.3. Antiviral and Cytotoxicity assays
2
8

The antiviral assays were based on inhibition of virus-induced cytopathicity or plaque
formation in HEL [herpes simplex virus 1 (HSV-1) (KOS), HSV-2 (G), vaccínia
virus, vesicular stomatitis virus, cytomegalovirus (HCMV), varicella-zoster virus
(VZV), adenovirus-2, and human corona virus (299E)], Vero (para-influenza-3,
reovirus-1, Sindbisvirus, and Coxsackie B4), HeLa (vesicular stomatitis virus,
Coxsackie virus B4, and respiratorysyncytial virus), or MDCK [influenza A (H1N1;
H3N2) and influenza B] cell cultures. Confluent cell cultures (or nearly confluent for
MDCK cells) in microtiter 96-well plates were inoculated with 100 CCID of virus (1
5
0
CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque-
forming units (PFU). After 1–2h virus adsorption period, residual virus was removed,
and the cell cultures were incubated in the presence of varying concentrations (200,
4
0, 8, 1.6, 0.32 M) of the test compounds. Viral cytopathicity was recorded as soon
as it reached completion in the control virus-infected cell cultures that were not
treated with the test compounds. Antiviral activity was expressed as the EC (50%
5
0
effective concentration) or compound concentration required to reduce virus-induced
cytopathogenicity or viral plaque formation by 50%. The MCC (minimal cytotoxic
concentration) values were determined as the compound concentration required to
afford a microscopically visible alteration of cell morphology. The new compounds
were screened for their potential cytostatic activity against eight human cancer cell
lines. The 50% cytostatic inhibitory concentration (IC ) causing a 50% decrease in
5
0
cell proliferationrelative to the number of cells in the untreated controls was
determined against retina (non cancerous) hTERT RPE-1, pancreatic
adenocarcinomaCapan-1, chronic myeloid leukaemia Hap1, lung carcinoma NCI-
H460, acute lymphoblastic leukaemia DND-41, acute myeloid leukaemia HL-60,
chronic myeloid leukaemia K-562 and non-Hodgkin lymphoma Z-138.
2
9