Efficient synthesis of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) and 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG)

Article abstract of DOI:10.1016/j.jfluchem.2017.08.007

A new, high radiochemical yield synthesis of [¹⁸F]FHBG and [¹⁸F]FHPG, the most popular imaging agents currently in use for monitoring gene therapy using positron emission tomography (PET), is reported in this work. Protection of sens

Full text of DOI:10.1016/j.jfluchem.2017.08.007

Accepted Manuscript
Title: Efficient synthesis of
1
8
18
9
-(4-[ F]fluoro-3-hydroxymethylbutyl)guanine ([ F]FHBG)
1
8
and 9-[(3-[ F]fluoro-1-hydroxy-2-propoxy)methyl]guanine
1
8
([ F]FHPG)
Authors: Jie Liu, Jorge R. Barrio, Nagichettiar Satyamurthy
PII:
S0022-1139(17)30299-3
DOI:
Reference:
http://dx.doi.org/10.1016/j.jfluchem.2017.08.007
FLUOR 9033
To appear in:
FLUOR
Received date:
Revised date:
Accepted date:
6-7-2017
11-8-2017
13-8-2017
Please cite this article as: Jie Liu, Jorge R.Barrio, Nagichettiar Satyamurthy, Efficient
synthesis of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) and 9-[(3-
[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG), Journal of Fluorine
Chemistryhttp://dx.doi.org/10.1016/j.jfluchem.2017.08.007
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1
8
18
Efficient synthesis of 9-(4-[ F]fluoro-3-hydroxymethylbutyl)guanine ([ F]FHBG)
1
8
18
and 9-[(3-[ F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ F]FHPG)
Jie Liu, Jorge R. Barrio, Nagichettiar Satyamurthy*
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine
at UCLA, University of California, Los Angeles, California 90095-6948, USA
*
Author for correspondence (E-mail: nsaty@mednet.ucla.edu)
Graphical abstract
C(CH3)3
O
N
O
N
N
N
N
N
N
HN
O
(CH ) C
3 3
O
N
H N
2
1
8
(
1) K F/Kryptofix
2) H+
MMTr
MMTrO
(
X
HO
X
18F
TsO
1
8
X = CH2
X = O
X = CH2 ([ FFHBG)
18
X = O ([ F]FHPG)
1

Highlights
6
18


New O -carbamoyal protected precursors for [ F]FHBG synthesized.
6
2
18
Novel O -tert-butyl-N -monomethoxytrityl precursors for [ F]FHBG and
18
[
F]FHPGsynthesized.


These precursors were fully characterized by NMR and MS.
18
These precursors gave excellent radiochemical yields for [ F]FHBG and
18
[
F]FHPG.
Abstract
1
8
18
A new, high radiochemical yield synthesis of [ F]FHBG and [ F]FHPG, the most popular
imaging agents currently in use for monitoring gene therapy using positron emission
tomography (PET), is reported in this work. Protection of sensitive sites in the precursors
18
18
generally utilized for the preparation of [ F]FHBG and [ F]FHPG using the nucleophilic
18F-fluorination reaction was found to be critical for good radiochemical yields, reliability
6
and reproducibility of the synthetic process. As an initial approach, protection at O -oxygen
in the guanine moiety of the currently used monomethoxytrityl-protected penciclovir
tosylate derivative 9 with carbamoyl groups was carried out. Subsequently, full protection
6
2
of both O -oxygen and N -nitrogen in the monomethoxytrityl-protected penciclovir and
ganciclovir tosylate analogs 9 and 10 were achieved by their reaction with di-tert-butyl
6
2
dicarbonate, which resulted in O -tert-butyl-N -Boc-monomethoxytrityl-protected
6
2
penciclovir tosylate 18 and O -tert-butyl-N -Boc-monomethoxytrityl-protected
ganciclovir tosylate 19, respectively. The newly synthesized carbamoyl- and the Boc-
protected precursors were first reacted with non-radioactive KF complexed with Kryptofix
2
22 to isolate the fluorinated products. Acid hydrolysis of the purified fluorinated
intermediates provided the nucleosides FHBG and FHPG. Full characterization of the new
2

precursors as well as the products obtained by fluorination and hydrolysis reactions were
carried out by one- and two-dimensional NMR spectroscopy and high resolution mass
spectrometry. Single crystal X-ray crystallographic analysis of a model ganciclovir analog
2
1
2 confirmed the structural characterization of the new Boc-protected tosylate precursors
8 and 19 by NMR spectroscopy. The carbamoyl- and the Boc-protected precursors were
further subjected to radiofluorination followed by acid hydrolysis reactions to furnish
[18
F]FHBG and [ F]FHPG reliably and reproducibly in excellent radiochemical yields (>
18
6
5%), much higher than those previously achieved.
1
8
18
18
Keywords: [ F]FHBG; [ F]FHPG; Penciclovir; Ganciclovir; F-fluorination.
1
. Introduction
Acyclic guanosine nucleosides 9-[4-hydroxy-3-(hydroxymethyl)butyl] guanine
[Pencicolvir (1)] and 9-[ (1,3-dihydroxy-2-propoxy)methyl]guanine [Ganciclovir (2)]
(Figure 1) are potent antiviral drugs useful for the treatment of herpes simplex virus (HSV)
[1, 2]. The antiviral activity of these drugs stems from the broad substrate specificity of the
phosphorylating viral thymidine kinase (TK) enzyme as compared with the restrictive
specificity of the mammalian TK enzyme. The established antiviral activity of guanine
based nucleoside analogues has spurred renewed interest in the development of a new class
of derivatives in order to further study the effects of structural modifications on their
9
antiviral activity and toxicity [3]. These efforts led to the preparation of two N - fluorinated
chain substituted guanine derivatives, namely 9-(4-fluoro-3-hydroxymethylbutyl)guanine
(FHBG, 3) and 9-[(3-fluoro-1-hydroxy-2-propoxy)methyl]guanine (FHPG, 4) (Fig. 1).
3

The fluorinated analogs 3 and 4 displayed slightly lower antiviral activity [3] and altered
cellular transport mechanism [4], but maintained good substrate activity for the HSV type
-1 thymidine kinase (HSV-1 TK) enzyme [5,6]. These fluorinated substrates are
specifically monophosphorylated by HSV-1 TK enzyme in cells that express the gene
HSV-1-tk (this gene expresses the enzyme HSV-1 TK in high levels) [5,6]. The
monophosphorylated nucleosides are subsequently converted to their corresponding di-
and triphosphates by the endogenous cellular kinases. When these nucleoside triphosphates
are incorporated into the host DNA, chain termination and cell death occurs. Thus, specific
cells (e.g., malignant cells) can be selectively targeted and destroyed by delivering HSV-
1
-tk gene into them followed by treatment with these antiviral nucleosides. Similarly, this
process of combination of a reporter gene (e.g., HSV-1-tk) and its complimentary reporter
1
8
18
18
probe labeled with a positron emitter [e.g., [ F]FHBG (4) or [ F]FHPG (6), Figure 1; F-
isotope half-life = 109.74 min] forms the basis of in vivo imaging of endogenous gene
expression in cancer and several other diseases by positron emission tomography (PET)
[
6]. As a prelude to mapping the thymidine kinase gene using PET, we previously
developed a regiospecific fluorination (at carbon-8) method for purines with elemental
fluorine [7] and extended that technique to the preparation of the corresponding 8-
[18
F]fluoro-analogs as reporter probes [8]. Among all the PET reporter probes, [ F]FHBG
18
18
(
4) [9-12], and to a lesser extent [ F]FHPG (6) [13-15], have convincingly become the
most useful purine based gene imaging agents for HSV1-tk enzyme and its mutated
counterparts HSV1-sr39tk and HSV1-A167Ysr39tk [6,16]. Investigations have also
1
8
18
demonstrated that [ F]FHBG is more preferred over [ F]FHPG for imaging gene
4

expression in tumors because of its higher accumulation in transduced cells [6,11]. The
18
importance and usefulness of [ F]FHBG as a PET imaging agent for monitoring gene
expression in cancer gene therapy in humans is further underscored by its recent US Food
and Drug Administration (FDA) approval as an investigational new drug (IND) [17].
18
18
The synthesis of [ F]FHPG [18-23] and [ F]FHBG [19,20,22-31] have been well
described in the literature. However, many of these methods provide low radiochemical
yields and/or suffer from reproducibility related issues. In the present investigation, we
have developed a strategy using several new precursor compounds with specific design for
1
8
18
the synthesis of both [ F]FHBG and [ F]FHPG. This approach has provided reliable and
1
8
18
significantly higher radiochemical yields for [ F]FHBG and [ F]FHPG than all the
previously reported methods. Described herein are the details on the synthesis of these
novel precursors, their spectroscopic characterization as well as their reactions with
[18
F]fluoride ion to yield pure [ F]FHBG and [ F]FHPG suitable for human utilization.
18
18
2
. Results and Discussion
.1. Probable causes for the low radiochemical yields of [ F]FHBG and [ F]FHPG in
18
18
2
the previous synthetic approaches
18
The enticing property of [ F]FHBG as a gene imaging agent in humans has elicited
1
8
considerable interest in its synthesis. As the demand for [ F]FHBG is steadily increasing,
there is a compelling need for its production with high synthesis yields. To date, the various
18
18
published radiosyntheses of [ F]FHBG [19,20,22-31] and [ F]FHPG [18-23] have all
used tosylate precursors 9 and 10 (Scheme 1) in which the hydroxyl group in the side chain
5

and the exo-NH
2
group of the guanine moiety are protected by monomethoxytrityl (MMTr)
groups.
1
8
18
After nucleophilic F-fluorination of the tosylate precursors with [ F]fluoride ion, the
18
18
MMTr groups are deprotected by acid hydrolysis to provide [ F]FHBG and [ F]FHPG,
which in all cases provide modest radiochemical yields. More importantly, the
radiochemical yields obtained for these tracers frequently fluctuate and are erratic for
unreported reasons [20,26]. Great efforts have been made by several research groups in
the past fifteen years to improve the reliability and reproducibility of the radiosynthesis of
[18
F]FHBG and [ F]FHPG by changing the various reaction conditions, such as reaction
18
solvent, temperature or purification procedure. For instance, conducting the reaction in
acetonitrile [18,19,24], mixture of acetonitrile and DMF [26] or DMSO [23,30], or heating
o
the reaction mixture at 90 C [19] or higher [22,24,27,28,30], or heating by microwaves
[
26] has achieved varying levels of success with the radiochemical yields ranging between
0 and 34% (decay corrected). One-pot synthesis [19] (radiochemical yield:10-15%) or an
optimized solid phase extraction method for purification [20,27] (radiochemical yield:15-
0%) decreased the overall synthesis time to < 60 min [26]. but did not enhance the
1
3
radiochemical yield. Similarly, the decay corrected radiochemical yields obtained for
[18
F]FHBG with fully automated [25,28-31] modules ranged between 10 and 35% while
utilization of a robotic module [23] provided a radiochemical yield of 31%.
18
The instability of the hemiaminal structure in the side chain of [ F]FHPG under strong
acidic deprotection condition (1M HCl) was suspected to be one of the reasons for the low
6

radiochemical yield. Accordingly, use of 1M acetic acid instead of 1M hydrochloric acid
18
for the deprotection step was found to be beneficial, at least for the synthesis of [ F]FHPG
1
8
[
22]. However, in the case of [ F]FHBG which lacks the hemiaminal structure in the side
1
8
chain, unlike [ F]FHPG, little improvement in radiochemical yield was attained by using
M acetic acid for the hydrolytic deprotection step. Use of alternative leaving groups such
as mesylate, tresylate or brosylate in place of the tosylate in precursors 9 and 10 (Scheme
) (Barrio et al, unpublished data from our laboratories) also provided radiochemical yields
1
1
18
in the range of 8-35% for [ F]FHBG, as previously realized with other methods. In spite
of all these efforts from various laboratories, achievement of satisfactory reliability,
1
8
reproducibility and good radiochemical yields for the synthesis of [ F]FHBG has remained
18
elusive. Only limited enhancement in the efficiency of the synthesis of [ F]FHBG, over
the original experimental conditions that provided radiochemical yields of 8-22% [24], has
been demonstrated so far.
1
8
From the standpoint of the nucleophilic F-fluorination method used for the preparation
1
8
18
of the nucleosides [ F]FHBG and [ F]FHPG (Scheme 1), it is important to consider the
1
2
active hydrogens present on N - and N -nitrogens in their precursors 9 and 10, respectively.
These two active hydrogens could potentially be sensitive for attack by a base (i.e., K CO )
2
3
18
that is commonly present in the F-fluorination reaction medium. For example, the lactam
1
N -nitrogen in guanosine derivatives is known to be deprotonated under basic conditions
[
32]. Interestingly, it has been hypothesized that during the 18F-fluorination of the
1
precursors 9 and 10 (Scheme 1) a base-catalyzed abstraction of the N -hydrogen of the
lactam, followed by an intramolecular cyclization with concomitant elimination of the
tosylate took place and was the cause for the decrease in the radiochemical yield of
7

products 4 and 6 [22]. Since fluoride ion is a Lewis base and a strong hydrogen bond
acceptor, the anhydrous reaction conditions generally required for the nucleophilic
fluorination reactions unfortunately also enhances the basicity of the fluoride ion [33]. The
1
8
precise role of [ F]fluoride ion as a base and an abstractor of active hydrogens, however,
is difficult to discern under the radiofluorination reaction conditions. We thus presumed
1
2
the presence of the active hydrogens on N - and N -nitrogens in the tosylate precursors 9
and 10 as likely impediments for an efficient radiofluorination reaction. Accordingly, in
this work we have developed and report synthetic schemes for the preparation of
appropriately protected precursors devoid of active hydrogens for the preparation of
[18
F]FHBG and [ F]FHPG.
18
2
.2. Optimization of the preparation of the monomethoxytrityl analogs 7 and 8
The monomethoxytrityl (MMTr) is a very useful protecting group for amines and
alcohols [34]. The MMTr group is quite stable under basic conditions typically used for
nucleophilic fluorination reactions and is easily removed by dilute mineral acids.
18
18
Accordingly, it has found use in the synthesis of [ F]FHBG and [ F]FHPG (Scheme 1)
wherein a selective protection of one of the hydroxyl groups on the side chain on N9-
nitrogen along with the primary amino group at 2-position of the guanine moiety in
penciclovir (1) and ganciclovir (2) has been achieved to provide the derivatives 7 and 8
(
Scheme 1) [3,18,24]. The original literature procedure utilized 4-dimethylaminopyridine
DMAP) as catalyst in dimethylformamide and triethylamine as a base for the
(
monomethoxytritylation reaction [3]. Subsequently, conditions for this reaction have been
modified by Zheng and co-workers and a good discussion on the product formation has
8

been reported [21,27]. For slightly better yield, decrease of by-product formation and ease
of product purification, we further modified the reaction conditions for this protection step
adopting the method reported by Ogilvie et al [35]. In our syntheses, triethylamine was
first added to a concentrated solution of the nucleosides 1 and 2 in DMSO followed by
monomethoxytrityl chloride. Under this condition, the formation of side products was
diminished and pure products 7 and 8 were easily obtained by flash column
chromatography. The MMTr protected derivatives 7 and 8 were previously characterized
1
by H NMR spectroscopy [21, 27]. However, the presence of six phenyl rings in these
1
13
products relatively confounds simple interpretation of H and C NMR signals. We have
therefore extensively utilized two-dimensional COSY, HMQC and HMBC NMR
1
3
techniques to alleviate this predicament to some extent and were able to assign the C
signals for the anisole part of the MMTr groups. For example, in the HMBC spectrum of
2
13
the analog 7, the N -H proton NMR signal (ppmcorrelated with the C signals of
the anisyl carbon C-1′ (136.87 ppm) and two phenyl C-1 carbons (144.74 and 144.91 ppm)
in the N-MMTr group on the guanine moiety (Fig. 2).
̋
This pattern clearly distinguished the corresponding NMR signal arising from the O-MMTr
group on the side chain. Further, the characteristic doublets due to H-3′/H-5′ protons in the
anisole rings of both MMTr groups at 6.89 ppm (CH
CH OPh-C-N) were shifted up-field from other aromatic protons (ppm due
to the electron donating nature of the 4′-OCH and exhibited correlations with the 4-
methoxyphenyl C-1′ C signals at 135.41 ppm and 136.87 ppm, respectively. The proton
3
OPh-C-O) and  6.75 ppm
(
3
3
1
3
singlets for the CH
3
O groups in both MMTr moieties at 3.73 ppm (O-MMTr), 3.64
9

ppm (N-MMTr) correlated with the C-4′ carbon NMR signals at 158.10 ppm (CH
3
OPh-C-
O), 157.62 ppm (CH OPh-C-N). The HMBC spectrum (Fig. 2) was also helpful in the
3
1
3
assignment of C signals in the guanine part of the molecule 7. For instance, the C-5 carbon
1
signal at 116.97 ppm showed correlation to the singlets of N -hydrogen (ppmand
H-8 (ppm, while C-4 carbon (149.46 ppm), as expected, correlated with only H-8.
Surprisingly, H-8 displayed a relatively weak correlation with C-6 carbon (156.56 ppm),
likely due to the four-bond separation between them. Curiously though the C-2 carbon
1
2
(
150.36 ppm) did not exhibit any correlation with N -H or N -H proton signals. The
assignments of these NMR chemical shifts are all consistent with the data previously
reported in the literature [36].
Standard tosylation reaction of the two monomethoxytrityl protected products 7 and 8
with p-toluenesulfonyl chloride in pyridine gave the precursors 9 and 10 in yields similar
to a recently published work [37].
6
2
.3. O -Carbamoyl protected penciclovir anaogs
A variety of protecting groups for the lactam moiety in guanine nucleosides (on N1-
6
nitrogen or O -oxygen) are known [34]. Examples among them are methoxymethyl
(MOM) [38], 2,2,2-trichloro-tert-butyloxycarbonyl (TCBOC) [39] groups that protect the
1
N - nitrogen (lactam tautomer) while 4-nitrophenylethyl (NPE)[40], diphenylcarbamoyl
6
(
DPC) [41,42] and dimethylcarbamoyl (DMC) [41,43] groups protect O -oxygen (lactim
tautomer). We selected the carbamoyl groups DPC and DMC in an initial attempt to block
1
the adverse effect, during the nucleophilic fluorination reaction, of N -H in the tosylate
6
precursor 9. Both these protecting groups are easily incorporated on the O -oxygen of
1
0

guanine nucleosides in excellent yields (> 80%) and also are efficiently deprotected by
bases [41-43]. Thus, the penciclovir tosylate analog 9 reacted with ease with diphenyl- and
dimethylcarbamoyl chloride to yield the corresponding carbamates 11 and 12 (Scheme 2).
The structures of the analogs 11 and 12 were confirmed by 1-D and 2-D NMR
1
spectroscopic and mass spectrometric analyses. In the H NMR spectrum, the down field
1
2
lactam N -H signal of precursor 9 disappeared while the N -H signals of 11 and 12
1
3
remained around 7.8 ppm. In C NMR spectrum, the carbonyl signal of the carbamoyl
groups in 11 and 12 appeared at 150.32 and 151.76 ppm, respectively. In the HBMC
spectrum of the compound 12, correlation between the N-methyl proton singlets ( 2.90
1
3
and 3.01 ppm) of the carbamoyl group and the C resonance for the carbonyl group
151.76 ppm) was also observed.
(
2
.4. Nucleophilic fluorination of the carbamoyal precursors 11 and 12
6
The O -diphenylcarbamoyal precursor 11 was subjected to nucleophilic fluorination
o
with KF/Kryptofix in DMSO at 145 C (Scheme 3).
After workup, the crude reaction mixture was separated by silica column flash
chromatography to yield two distinct fractions. In fraction one, the fluorinated product 13
and the elimination product 14 (Scheme 3) were obtained as an inseparable mixture in 42%
yield. Surprisingly, NMR spectroscopic and mass spectrometric analyses indicated the
carbamoyl group was found to be cleaved off in all the products. The deprotection of the
carbamoyl group is generally achieved with concentrated ammonia [41-43]. To our
1
1

knowledge, this is the first observation of the deprotection of the carbamoyl moiety by
fluoride ion. The NMR spectroscopic analysis indicated the ratio of the fluorinated to the
elimination product in the inseparable mixture was 8:2. In the electrospray ionization (ESI)
mass spectrum, the molecular ion signal intensity of the elimination product 14 was about
3
0% of that of the desired fluorinated product 13. The vinyl proton signal in the product
4 appeared as two broad singlets at 4.64 ppm and 5.24 ppm, each integrating for one
1
1
3
proton. The C NMR signal for the vinyl-CH
2
carbon appeared at 111.0 ppm. As expected,
the presence of fluorine in the product 13 caused down field shifts for the proton (4.22
ppm, 1JH,F = 47.5 Hz) and C (83.77 ppm, JC,F = 166.5 Hz) signals for the methylene
13
1
chain carrying it compared with the corresponding signals in the tosylate precursor 11 or
19
the hydroxyl derivative 7. The F NMR chemical shift for this product was observed at
−225.4 ppm consistent for an aliphatic fluorinated compound.
The second fraction from the silica column chromatography was obtained in 22 % yield.
The NMR spectroscopic analysis of this fraction showed that it was also a mixture of two
products in a ratio of 3:1 and resisted all attempts at separation. Hence, the unseparated
mixture was analyzed as such. The mass spectrometry data of this mixture revealed a
molecular weight of exactly 20 Daltons less than that of the fluorinated product 13,
suggesting a loss of HF from it. The 2-D NMR spectroscopic analysis of the major
3
component of this mixture suggested the possibility of a side chain CH
2
-N -cyclized
structure 15 for it (Scheme 3). Thus, in the HMBC spectrum clear correlations of the
3
protons on the N -CH
2
group (4.66 ppm) with C-2 (141.27 ppm) and C-4 (144.42 ppm)
carbons were observed, strongly implying a cyclization of the side chain with the N3-
nitrogen. Further, the C-2, C-4 and C-5 NMR signals in the compound 15 were shifted up
1
2

field when compared with the corresponding carbons in the analogs 13 and 14 due to the
well-known γ shielding effect [44] arising from the cyclic carbon chain attached to N3-
nitrogen. Accordingly, the C-2, C-4 and C-5 chemical shifts in the cyclic derivative 15
1
3
occur at 141.27, 144.42 and 114.05 ppm, respectively while the corresponding C signals
in the fluoro analog 13 appear at 150.93, 149.95 and 117.44, respectively. Support for the
cyclic structure for compound 15 is also partly derived from the report of Holmes and
Robins [45] who have previously synthesized an analogues cyclonucleoside from a 5′-
tosylate derivative of guanosine. More recently, the formation of such a cyclonucleoside,
as a side product, has been observed during the nucleophilic fluorination reaction of the
tosylate 9 [22]. Since the formation of the cyclic product 15 from the precursor 11 would
1
first require an availability of N -hydrogen [22], this side reaction must have happened
after the cleavage of carbamoyl protecting group which would provide the necessary N1-
hydrogen. The cycloguanine derivatives in both these reports [22, 45] were shown to have
2
an amidine tautomeric moiety wherein the exocylic N -nitrogen carrying a hydrogen and
1
N existing as a tertiary nitrogen. In contrast, the HMBC spectrum indicated the presence
1
of an imidine tautomeric structure in 15 with N -nitrogen carrying the hydrogen instead of
2
1
the N -nitrogen. Correspondingly, N -hydrogen (ppmdisplayed correlations with
both C-5 (114.05 ppm) and C-6 (155.41 ppm) carbons in the HMBC spectrum.
1
In the H NMR spectrum for the minor component in this mixture, chemical shifts
1
presumed to be due to H-8, N -H of guanine moiety as well as signals resembling to those
of the methylene protons in compound 15 were observed. It could be speculated that this
2
3
product might have a side chain cyclized structure involving N -nitrogen instead of N -
1
3

nitrogen [45]. However, no further attempts were made to characterize this minor
component.
6
The fluorination reaction of O -dimethylcarbamoyal precursor 12 (Scheme 4) provided
a mixture of the fluorinated product 13 and the elimination product 14 in a total yield of
6
1%.
Similar to the diphenylcarbamoyal precursor (Scheme 3), the dimethylcarbamoyal group
was also deprotected during the fluorination reaction. Interestingly, however, the product
mixture was totally devoid of the cycloguanine derivative 15. The lack of formation of the
derivative 15 suggests a higher stability of the precursor 12 under the reaction conditions
than that of the diphenylcarbamoyl analog 11 since the formation of the cyclic product
1
would first require the presence of N -hydrogen [22], creation of which is possible only by
the cleavage of carbamoyl protecting group.
Both the diphenylcarbamoyl precursor 11 and dimethylcarbamoyl precursor 12 were
18
used in the radiosynthesis of [ F]FHBG (4) (Scheme 5).
Decay corrected radiochemical yields of 38% and 43%, respectively were obtained for
[18
F]FHBG with the precursors 11 and 12 with a total synthesis time of ~110 min. Relevant
details are summarized in Table 1 below and full synthesis details are provided in the
Experimental section.
1
4

These radiochemical yields are still more consistent and higher than those obtained with
the commonly used precursor 9 [19,20,22-24]. The side products (i.e., 14 and 15) formed
during the fluorination reaction did not affect the final chemical purity of the product
[18
F]FHBG. They were readily removed by semi-preparative HPLC purification after acid
hydrolysis (see Experimental). Further discussion on the radiochemistry conditions is
provided later in this section.
2
.5. The need for fully protected tosylate precursors
1
Substitution of the hydrogen on the N -nitrogen with a methyl group in the tosylate
precursors 9 and 10 was first strategized to suppress the formation of the cyclonucleoside
1
5 during the radiofluorination reactions and thereby improve the final radiochemical
1
yields [22,46]. The results of the radiofluorination reactions of such N -blocked analogs of
1
8
1
9
and 10 were rather confounding. The F-fluorination followed by acid hydrolysis of N -
methyl substituted FHBG tosylate precursor surely provided a better radiochemical yield
1
18
18
of 19% for N -methyl-[ F]FHBG vs. 10% obtained for [ F]FHBG with the tosylate
1
derivative 9 (Scheme 1) attesting to the beneficial effect of N -nitrogen blockage.
1
Unfortunately, analogous radiofluorination of the N -methyl substituted FHPG tosylate
1
18
gave a lower radiochemical yield of 12% for N -methyl-[ F]FHBG while the
corresponding non-methylated precursor 10 provided a higher radiochemical yield of
1
8
1
1
5% for [ F]FHPG. Thus, the effect of blockage of N -nitrogen with a methyl group in
guanosine analogs seems rather unpredictable from the standpoint of improving the
radiochemical yields. Besides, no viable reactions are known to de-methylate the N1-
1
5

1
8
1
18
methyl-[ F]FHBG or N -methyl-[ F]FHPG to provide the desired radiolabeled
nucleosides.
6
The nucleophilic fluorination reaction results with the O -carbamates (Schemes 3-5), in
1
contrast to the N -methyl blocking studies [22,46] are quite encouraging. These results
6
further indicate that protection of O -oxygen in the lactim tautomer is preferable to the
1
protection of the N -nitrogen in the lactam tautomer of the tosylate derivatives 9 and 10.
1
8
6
The radiochemical yields for the F-nucleosides obtained with the O -carbamate
precursors 11 and 12 (Scheme 5) in this investigation are higher than those obtained with
1
the free lactam tosylate precursors 9 and 10 (Scheme 1) [18,24]. The N -methyl group
1
protection studies discussed above, however, clearly implies that N -hydrogen abstraction
under the nucleophilic fluorination conditions [22,46] might not be the only reason for the
1
8
18
poor radiochemical yields for preparation of [ F]FHBG and [ F]FHPG from the tosylates
2
9
and 10. It is quite likely that the hydrogen on the exocyclic N -nitrogen of the guanine
moiety in these tosylate precursors 9 and 10 may also be subjected to abstraction by base
during the nucleophilic fluorination reaction in detriment to the radiochemical yields. We
6
2
therefore envisioned that protection of the O -oxygen (lactim tautomer) as well as the N -
nitrogen of the tosylate precursors 9 and 10 might be a prudent plan for more efficient and
1
8
reproducible F- labeling of these guanine derivatives.
2
The judicious approach of selectively protecting the N -nitrogen and only one of the
hydroxyl groups in the side chain of penciclovir (1) and ganciclovir (2) with
monomethoxytrityl groups [3] strategically permits the synthesis of the tosylate precursors
9
and 10 (Scheme 1) [18,24]. After careful considerations, we elected to maintain this core
6
structure and embarked on to protect the O -oxygen (lactim tautomer) and the exocyclic
1
6

2
N -nitrogen in the tosylates 9 and 10 with appropriate groups. An alternative to the
carbamoyl group was also considered because of its labile nature during the nucleophilic
fluorination reaction condition (Schemes 3 and 4). A quite promising protecting group in
this regard is tert-butyloxycarbonyl (Boc) which can be used to block both the exocyclic
2
6
N -nitrogen and the O -oxygen in the lactim moiety [47,48]. An attractive feature of the
Boc protection is the ease with which it can be deprotected by acid hydrolysis. Thus, we
endeavored the preparation of new derivatives of tosylates 9 and 10 with Boc protection
2
on the lactim moiety as well as on the N -nitrogen.
6
2
2
.6. Boc protection of O -oxygen and N -nitrogen in the tosylate precursors 9 and 10
For the Boc protection, a solution of the tosylates 9 and 10 in dichloromethane were
reacted, under reflux, with ~10 M excess of di-tert-butyl dicarbonate (Boc anhydride) in
the presence of DMAP and triethylamine (Scheme 6) [47]. The reaction mixture underwent
a quick color change from yellow to orange-red accompanied by a vigorous evolution of a
gas, presumably carbon dioxide.
The bright red color of the reaction mixture soon faded back to yellow. The product was
2
6
conveniently purified by flash column chromatography. Surprisingly, the expected N , O-
di-Boc products 16 or 17 were not obtained after the column chromatographic purification.
2
6
Instead, N -Boc-O -tert-butyl ethers 18 and 19 resulted in 52% and 72% yields,
respectively, as carefully confirmed by spectroscopic analyses.
In the high resolution mass spectrum, the presence of both M+1 and M+23 (M+Na) ion
signals corresponding to the products 18 and 19, and not the di-Boc analogs 16 and 17,
1
7

were observed. One- and two-dimensional (COSY, HMBC and HMQC) NMR
spectroscopic analyses also indicated the products obtained in the Boc protection reaction
2
6
13
were indeed N -Boc-O -tert-butyl ethers 18 and 19. For example, in the 150 MHz C
NMR (600 MHz NMR spectrometer) spectrum of the derivative 18, only a single carbonyl
signal at 153.33 ppm was observed corresponding to the presence of only one Boc group
1
3
in the molecule. The C NMR carbonyl carbon signal in the Boc group usually appears in
1
3
the region of 148 -158 ppm. The four C NMR signals (apart from the carbonyl resonance
at 153.33 ppm) of 151.92 ppm (C-4), 152.41ppm (C-2), 157.32 ppm (C-4' of CH OPh-C-
N) and 158.18 ppm (C-4' of CH OPh-C-O) in this region were all assigned to other carbons
3
3
1
3
in the compound 18 based on the HMBC spectrum. Only one C carbonyl signal at ~153
ppm was observed even at a higher resolution and greater sensitive NMR frequency of 200
MHz for carbon-13 (800 MHz NMR spectrometer) indicating the presence of only one Boc
1
13
group in the compound 18. Overall, many correlations discerned between H and C NMR
signals in the Boc protected analogs 18 and 19 mirrored those observed in the products 7-
1
2
1
1
2. Incidentally, the H NMR signals for the protons in the N -MMTr group in compounds
8 and 19 were slightly broadened due to overlapping of closely spaced resonances
2
suggesting the presence of rotamers due to the N -Boc group.
Reaction of Boc anhydride with alcohols to form tert-butyl ethers in the presence of
Lewis acid catalysts is known [49]. In the reaction of the Boc anhydride with the tosylates
6
9
and 10 (Scheme 6), an acyl transfer from the Boc anhydride to O -oxygen [50] likely
leads to the formation of the intermediates 16 and 17. Spontaneous elimination of a
6
molecule of CO
2
from these intermediates forms the O -tert-butyl ethers 18 and 19.
1
Conversely, the reaction could also proceed by first forming N -Boc derivative by an acyl
1
8

6
transfer from the O -Boc analog [50], which upon elimination of CO
2
followed by an
6
internal transpositionof tert-butyl groupcould lead to the O -tert-butyl ethers. In any event,
6
analogous formation of O -tert-butyl ether has recently been well documented during the
Boc protection reaction of closely related guanosine nucleoside analogs [51-54].
The tosylate derivatives 18 and 19, after nucleophilic fluorination reaction, would be
subjected to acid hydrolysis for deprotection of the monomethoxytrityl, Boc and tert-butyl
groups. To test the ease with which these protecting groups could be hydrolyzed, a model
guanine nucleoside containing all these groups was prepared (Scheme 7).
Monomethoxytritylation of ganciclovir (2) with six equivalents of MMTrCl gave the
analog 20 in 76% yield which upon reaction with Boc anhydride provided the product 21.
The product 21 was fully characterized by NMR spectroscopy and mass spectrometry. As
1
2
expected, the signals in the H NMR spectrum for N -MMTr group were slightly broadened
due to rotamers while sharp proton signals were observed for the other two MMTr groups
1
3
on the oxygen in the side chain. The C NMR spectrum of the derivative 21 showed the
presence a single Boc carbonyl group. The high resolution mass spectrum was consistent
with the structure 21.
o
Deprotection of the Boc-protected derivative 21 with 1M HCl at 115 C for 1 h gave
ganciclovir (2) while the same hydrolysis for 3 min led to a partially deprotected product
2
2 containing a Boc group (Scheme 7). The structure of the Boc analog 22 was consistent
with high resolution mass spectrometric and NMR spectroscopic data. Furthermore,
1
9

compound 22 was crystallized from methanol/water and analyzed by single crystal X-ray
crystallography (Fig. 3).
As discussed earlier, the NMR spectroscopic and mass spectrometric data clearly showed
the presence of only one Boc and one tert-butyl group in the tosylates 18 and 19. The above
X-ray structure (Fig. 3) now confirms the Boc group in the derivatives 18 and 19 must also
2
6
be on the N -position and the tert-butyl group on the O -oxygen.
2
.7. Nucleophilic fluorination of fully protected tosylate precursors 18 and 19
Nucleophilic fluorination reaction of the fully protected precursor 18 was carried out
o
with potassium fluoride in the presence of Kryptofix 222 in DMSO at 145 C (Scheme 8).
The NMR spectroscopic analysis of the reaction mixture, after work up (see Experimental),
2
6
indicated the N -Boc and O -tert-butyl ether moieties were stable under the fluorination
reaction condition unlike the carbamoyl groups, as discussed earlier. Normal phase semi-
preparative HPLC was used to cleanly separate the closely eluting fluorinated product 23
(53.9% yield) from the elimination side product 24 (29.6% yield). Interestingly, the HPLC
analysis did not reveal the presence of the cycloguanine derivative 15, attesting to the
suitability of tert-butyl group for the protection of the lactam in the precursor 18 to prevent
the formation of this undesired side product during nucleophilic fluorination reaction. The
1
fluorinated product 23 and the elimination side product 24 were fully characterized by H,
13C and 19
F NMR spectroscopy and mass spectrometry. For decisive NMR signal
assignments, HMBC and HMQC spectra were extensively utilized. As representative
examples, Fig. 4 and 5 illustrate our efforts in this regard for the NMR analysis of the
fluorinated analog 23.
2
0

In the HMBC spectrum, many of the correlations previously observed for the
monomethoxytrityl protected analog 7 (Fig. 2) in the aromatic region were also discerned
for the fluorinated analog 23 (Fig. 4). In the guanine moiety in 23, the H-8 singlet at 
ppm correlated with C-4 (152.00 ppm) and C-5 (119.63 ppm) carbon signals. As expected,
both C-2 and C-6 carbons having no two- or three-bond separated hydrogens showed no
9
correlations in the HMBC spectrum. However, protons on the side chain on the N -nitrogen
displayed rich HMBC correlations with carbons on the ring as well as on the aliphatic
chain. For instance, the multiplet signal for H-10 at ppmcorrelated with C-4 and
C-8 (143.72 ppm) carbon resonances. The proton NMR signal for the methylene group (H-
1
3) carrying the fluorine appeared as a doublet of doublets (J=47.4 and 4.6 Hz) at
ppm andalsodisplayed two and three bond correlations with C-12 (37.16 ppm)
and C-11 (27.71 ppm) and C-14 (61.73 ppm) carbons, respectively. Similarly, H-11 (1.53
ppm) correlated with C-10 (41.21 ppm), C-12, C-13 (83.33 ppm) and C-14 carbons. The
1
H NMR multiplet signal for H-14 (ppmfurther manifested correlations with
the resonances of carbons 11, 12 and 13. These HMBC correlations thus permitted
definitive assignment of the chemical shifts for various carbons in the derivative 23. The
1
3
HMQC spectrum (Fig. 5) was also valuable for the assignment of C NMR signals of
carbons carrying a hydrogen.
1
For example, the H NMR doublet of doublets at ppm for H-13 showed correlation
1
3
to a C signal at 83.33 ppm which could undoubtedly be ascribed to the C-13 carbon in
2
1

13
the CH
2
F group. The correlation of the H-10 proton signal at ppm with the C
resonance of 41.21 ppm allowed the assignment of the C-10 carbon. Similarly, the
1
3
correlation of the H-12 methine proton multiplet at 1.84 ppm with a C signal at 37.16
ppm was surely attributed to the C-12 carbon on the side chain. The one bond correlation
of the proton multiplet at 3.02-3.06 with the carbon signal of 61.73 ppm led to the
assignment of C-14 carbon. In the guanine part of the molecule 23, the lone H-8 proton at
 ppmcleanlycorrelated with C-8 carbon resonance at 143.72 ppm establishing their
one bond connectivity.
Overall, the HMBC and HMQC spectra complemented each other in the unambiguous
assignment of the NMR chemical shifts in all the products analyzed.
o
As described in the Experimental section, acid hydrolysis (at 115 C) of the HPLC
2
purified fluorinated analog 23 for a short duration expectedly provided the N -Boc
protected derivative 25 in 93.6% yield while conducting the reaction for a longer period
led to full deprotection resulting in the formation of FHBG (3) in 75% yield (Scheme 9).
The crude fluorination reaction mixture containing the products 23 and 24 was also
1
8
subjected to acid hydrolysis to mimic the conditions of F-labeling process as described
below. Semi-preparative HPLC purification of this reaction mixture provided pure FHBG
(3) along with the deprotected elimination side product 26.
The nucleophilic fluorination of the fully protected ganciclovir precursor 19 also
provided only the fluorinated derivative 27 and the elimination product 28 (Scheme 10).
2
2

However, semi-preparative HPLC could not fully separate these two products. The HPLC
purification provided a mixture containing the fluorinated product 27 (56.3% yield) along
1
with the elimination product 28 as judged by H NMR spectroscopy and mass
spectrometry. The NMR and mass spectral characterization of the products 27 and 28 was
performed only as a mixture.
The partial deprotection (see Experimental) of the mixture of derivatives 27 and 28 with
o
2
1
M HCl at 115 C for short duration followed by HPLC purification provided the N -Boc
protected product 29 in 64.6% yield. The partially hydrolyzed product 29 could
subsequently be fully deprotected to give pure FHPG (5) in 74.4% yield by further
hydrolysis with 4M HOAc (Scheme 11).
1
8
18
2
.8. Preparation of [ F]FHBG and [ F]FHPG from the carbamoyl- (11, 12) and Boc-
protected (18, 19) tosylate precursors
The nucleophilic fluorination reaction of the carbamoyl- (11 and 12) and Boc-protected
18
tosylate precursors (18 and 19) with [ F]fluoride ion complexed with Kryptofix 222
followed by acid hydrolysis and semi-preparative HPLC purification provided chemically
1
8
18
and radiochemically pure [ F]FHBG (4) and [ F]FHPG (6) (Schemes 5 and 12) in
decicurie amounts.
The radiochemical yields obtained for these experiments are provided in Table 1. The
radiofluorination reaction of the tosylate precursors 9 and 10 (Scheme 1) were generally
2
3

o
conducted in acetonitrile medium at 90-120 C [18-21,24,27]. Alternatively, DMSO has
1
8
o
also been used as a solvent for this F-fluorination reaction at 100-135 C [23,30]. Initially,
we conducted a few selected radiofluorination reactions of the precursor 18 in acetonitrile
o
o
18
medium in a sealed reaction vessel at 150 -165 C and obtained [ F]FHBG in 55%
radiochemical yield (Table 1). However, when the same reaction was conducted in DMSO
o
at 150 C, a significant improvement in the radiochemical yield of 68% was realized (Table
1
8
1
). Thus, all further nucleophilic F-fluorination reactions with the carbamoyl- and the
Boc-protected precursors were conducted in DMSO medium. Protection of the lactam
moiety in the tosylate 9 with diphenylcarbamoyl group (precursor 11) was found to be
1
8
beneficial and provided a radiochemical yield of ~38 % for [ F]FHBG (Table 1), which is
higher than those of all the previously reported methods [19,20,22-24,26,27]. Interestingly,
the radiochemical yield was further improved to ~43 % when the dimethylcarbamoyl group
(precursor 12) was used to protect the lactam in the tosylate 9. This strongly suggests the
dimethylcarbamoyl group is more stable than the diphenylcarbamoyl moiety under the
fluorination reaction condition. It is, however, quite gratifying that protection of both O6-
2
oxygen and N -nitrogen in the tosylates 9 and 10, as realized with the derivatives 18 and
1
8
1
9, had the most significant effect on the radiofluorination yields for [ F]FHBG and
[18
F]FHPG. The radiochemical yield obtained for [ F]FHBG with the fully protected
18
precursor 18 (68%) is substantially higher than that attained even with the derivative 12
18
(
43%) (Table 1). Similarly, the 65% radiochemical yield accomplished for [ F]FHPG with
the precursor 19 (Table 1) is the highest achieved to date for this molecular imaging probe
1
8
18
[
18-23]. Thus, starting typically with 500-550 mCi of [ F]fluoride ion, [ F]FHBG or
[18
F]FHPG can be reproducibly obtained in > 150 mCi yields (end of synthesis) and ready
2
4

for human administration in a total synthesis time of 110 min. In addition, an extremely
attractive feature of the Boc- protected tosylate precursors 18 and 19 is their stability. No
ill effects on the radiochemical yields were observed for these precursors stored for more
o
than a year in a freezer at – 10 C.
3
. Conclusions
Several novel precursors for the high yield synthesis of the very valuable gene imaging
18
18
PET imaging probes [ F]FHBG (4) and [ F]FHPG (6) have been developed. Previously
utilized precursors 9 and 10 for the preparation of these tracers seem to suffer from
undesired side reactions during the radiofluorination process, diminishing the
radiochemical yields, as well as the reliability and reproducibility of the procedure. The
new precursor strategies developed in this investigation is capable of mitigating these
6
shortcomings. Protection of the O -oxygen in the precursor 9 with diphenyl- or
dimethylcarbamoyl group (11, 12) decidedly improved the radiochemical yield for
[18
F]FHBG. More impressively, when the O -oxygen and the N -nitrogen in the precursors
and 10 were fully protected with tert-butyl and Boc groups, respectively (18, 19)
6
2
9
18
excellent radiochemical yields (> 60%) were reliably obtained for [ F]FHBG and
[18
F]FHPG, in decicurie amounts ready for human administration. The radiochemical
yields achieved with these fully protected precursors are the highest reported to date.
2
5

4
. Experimental
4
.1. General
Penciclovir (1) was synthesized as previously reported [55] or purchased from Eurasia
Trans Continental (Mumbai, India). Ganciclovir (2) was obtained by neutralization of an
aqueous solution of Cytovene-IV (Roche Laboratories Inc, New Jersey, USA) with 1M
HCl to yield a white powder which was filtered, washed with water and methanol and then
dried over phosphorous pentoxide in a vacuum desiccator. All reagent chemicals and
solvents were purchased from Aldrich. All solvents prior to use were distilled and dried
under standard conditions. Flash column chromatography was carried out with 0.063-0.200
mm silica gel.
One and two dimensional [gradient selected heteronuclear multiple quantum coherence
1
(
HMQC) and gradient selected heteronuclear multiple bond coherence (HMBC)] H and
13C NMR spectra were acquired on Bruker Avance 300, DRX 500 or Avance 600 NMR
spectrometer with a 5 mm broadband probe. The 800 MHz NMR spectra were recorded on
1
9
a Bruker Avance III HD 800 spectrometer equipped with an inverse cryoprobe. The F
NMR was acquired on a Bruker Avance 300 MHz or 400 MHz spectrometer with a 5 mm
QNP probe. The chemical shifts () are expressed in parts per million (ppm) downfield
1
13
from internal tetramethylsilane (TMS) for H and C and external fluorotrichloromethane
Freon 11) for 19F NMR. Atmospheric pressure chemical ionization (APCI) and
(
electrospray ionization (ESI) high resolution mass (HRMS) spectra were obtained on an
Agilent LC TOF spectrometer at the analytical chemistry instrumentation facility of the
University of California, Riverside. Single crystal X-ray crystallography was performed on
a Bruker Smart 1000 CCD diffractometer.
2
6

4
4
.2. Preparation of precursor compounds
2
.2.1. N -(p-Anisyldiphenylmethyl)-9-[3-(p-anisyldiphenylmethoxymethyl)-4-
hydroxybutyl] guanine (7)
To a suspension of penciclovir (1) (251 mg, 0.99 mmol) in DMSO (1.5 mL) was first
added triethylamine (0.84 mL, 6 mmol) and then p-anisyldiphenylmethyl chloride (862
mg, 2.8 mmol). The reaction mixture was stirred at room temperature overnight. This
resulted in a thick brown mixture to which brine (30 mL) was added. The solid that
separated was collected on Celite 521 (Aldrich) by filtration. The solid was washed with
water (30 mL) and then stirred with CH
dried over anhydrous Na SO . Evaporation of the solvent at reduced pressure gave the
crude product as a yellow foam (1.32 g). The crude product was purified by flash
2
Cl
2
(30 mL). The CH
2
Cl
2
solution was filtered and
2
4
chromatography on silica gel (40 g), eluted with CH
2
Cl
2
/MeOH gradient (0-10% MeOH),
1
to give the product 7 (453 mg, 56.8% yield) as a beige solid. H NMR (600 MHz, DMSO-
d
2
2
4
2
6
):  1.20 (m, 2H, CH
.80 (dd, J = 9.2 and 5.3 Hz, 1H, CH
H, NCH ), 3.64 (s, 3H, OCH of N-MMTr), 3.73 (s, 3H, OCH
.9 Hz, 1H, OH), 6.75 (d, J = 8.9 Hz, 2H, H-3'/H-5' of CH OPh-C-N), 6.89 (d, J = 9.0 Hz,
H, H-3'/H-5' of CH OPh-C-O), 7.07-7.33 (m, 24H, Ar-H), 7.44 (s, 1H, H-8), 10.48 (s,
H, N -H); C NMR (150 MHz, DMSO-d ):  28.50 (CH ), 35.30 (CH), 41.27 (NCH ),
4.92 (OCH of N-MMTr), 55.02 (OCH of O-MMTr), 61.14 (CH OMMTr), 63.19
OH), 69.60 (N-tC), 85.48 (O-tC), 112.77 (C3'/C5' of CH OPh-C-N), 113.11 (C3'/C5'
of CH OPh-C-O), 116.97 (C5), 126.32, 126.73, 127.37, 127.42, 127.75, 127.89, 128.34,
28.43 (Ph-C), 129.78 (C2'/C6' of CH OPh-C-N), 129.87 (C2'/C6' of CH OPh-C-O),
2
), 1.40 (m, 1H, CH), 2.71 (dd, J = 9.2 and 6.9 Hz, 1H, CH
OMMTr), 3.14 and 3.29 (m, 2H, CH OH), 3.39 (m,
of O-MMTr), 4.36 (t, J =
2
OMMTr),
2
2
2
3
3
3
3
1
13
1
5
6
2
2
3
3
2
(CH
2
3
3
1
3
3
2
7

1
35.41 (C1' of CH
Ph-C-O), 144.74 and 144.91 (C1” of Ph-C-N), 149.46 (C4), 150.36 (C2), 156.56 (C6),
57.62 (C4' of CH OPh-C-N), 158.10 (C4' of CH OPh-C-O); HRMS Calcd for
(M+H): 798.3655; Found: 798.3670.
3
OPh-C-O), 136.87 (C1' of CH OPh-C-N), 137.41 (C8), 144.58 (C1” of
3
1
3
3
C
50
H
48
N
5
O
5
2
4
.2.2. N -(p-Anisyldiphenylmethyl)-9-[(1-(p-anisyldiphenylmethoxy)-3-hydroxy-2-
propoxy)methyl]guanine (8)
Ganciclovir (2) (510 mg, 2 mmol) was dissolved in DMSO (3 mL) and triethylamine
(1.67 mL, 12 mmol) was added to it followed by monomethoxytrityl chloride (1.85 g, 6
mmol). After stirring the reaction mixture at room temperature overnight brine (40 mL)
was added to it. A solid that precipitated was collected on Celite 521 (Aldrich) by filtration.
The filter cake was washed with water (30 mL) and stirred with CH
CH Cl solution was filtered, dried over anhydrous Na SO and evaporated at reduced
pressure to give the crude product. The crude product was purified by flash
2
Cl
2
(50 mL). The
2
2
2
4
chromatography on silica gel (80 g), eluted with CH
2
Cl
2
/MeOH gradient (0-5% MeOH),
1
to give the product 8 as a beige solid (950 mg, 59.4% yield). H NMR (600 MHz, DMSO-
d
6
):  2.59 (d, J = 9.1 Hz, 1H, CH
CH OMMTr), 2.91 and 3.06 (m, 2H, CH
MMTr), 3.76 (s, 3H, OCH of O-MMTr), 4.49 (t, J = 5.3 Hz, 1H, OH), 4.95 and 5.06 (d, J
11.2 Hz, 2H, NCH O), 6.73 (d, J = 8.9 Hz, 2H, H-3'/H-5' of CH
.9 Hz, 2H, H-3'/H-5' of CH
.80 (s, 1H, H-8), 10.73 (s, 1H, N -H); C NMR (150 MHz, DMSO-d
of O-MMTr), 60.88 (CH OMMTr), 63.28 (CH
2
OMMTr), 2.93 (dd, J = 9.1 and 6.6 Hz, 1H,
2
2
OH), 3.41 (m, 1H, CH), 3.62 (s, 3H, OCH of N-
3
3
=
2
3
OPh-C-N), 6.89 (d, J =
2
8
7
3
OPh-C-O), 7.03-7.31 (m, 24H, Ar-H), 7.71 (s, 1H, N -H),
1
13
6
):  54.88 (OCH
3
of N-MMTr), 54.98 (OCH
3
2
2
OH), 69.65 (tC
2
8

of N-MMTr), 70.62 (NCH
CH OPh-C-N), 113.05 (C3'/C5' of CH
27.47, 127.50, 127.65, 127.67, 127.87, 127.97, 128.39, 128.44 (Ph-C), 130.21 (C2'/C6'
of CH OPh-C-N and CH OPh-C-O), 135.20 (C1' of CH OPh-C-O), 136.68 (C1' of
CH OPh-C-N), 137.88 (C8), 144.16 and 144.41 (C1” of Ph-C-O), 144.78 and 144.83 (C1”
of Ph-C-N), 150.97 (C2), 156.70 (C6), 157.62 (C4' of CH OPh-C-N), 158.08 (C4' of
: 799.3370; Found: 800.3448 (M+H).
2
), 78.90 (CH), 85.04 (tC of O-MMTr), 112.79 (C3'/C5' of
3
3
OPh-C-O), 116.68 (C5), 126.33, 126.61, 126.34,
1
3
3
3
3
3
CH
3
OPh-C-O); HRMS Calcd for C49
H
46
N
5
O
6
2
4
.2.3. N -(p-Anisyldiphenylmethyl)-9-[3-(p-anisyldiphenylmethoxymethyl)-4-
tosyloxybutyl]guanine (9)
To a solution of the penciclovir derivative 7 (254 mg, 0.32 mmol) in pyridine (4 mL) at
0o C, under argon, was added p-toluenesulfonyl chloride (182 mg, 0.96 mmol). The
o
solution was stirred at 0 C for 30 min and then at room temperature overnight. The dark
yellow reaction mixture was quenched with ice-water and the solution was evaporated in a
rotary evaporator. The residual pyridine from the reaction mixture was removed by co-
evaporation with toluene under reduced pressure. The oily residue of the crude product was
purified by flash chromatography on silica gel (40 g) with CH
2
Cl
2
/MeOH gradient (0-4%
1
MeOH) eluent to give the product 9 (167 mg, 55.1% yield) as a white solid. H NMR (600
MHz, DMSO-d
6
):  1.15 (m, 2H, CH
dd, J = 9.6 and 7.7 Hz, 1H, CH OMMTr), 2.74 (dd, J = 9.6 and 4.5 Hz, 1H, CH
), 3.64 (s, 3H, OCH of N-MMTr), 3.74 (s, 3H, OCH
OTs), 3.84 (dd, J = 9.7 and 5.2 Hz, 1H, CH OTs), 6.75 (d, J =
.7 Hz, 2H, H-3'/H-5' of CH OPh-C-N), 6.88 (d, J = 8.8 Hz, 2H, H-3'/H-5' of CH OPh-C-
2
), 1.46 (m, 1H, CH), 2.38 (s, 3H, Ar-CH
3
of Ts), 2.64
OMMTr),
of O-
(
2
2
3
.29 (t, J = 7.1 Hz, 2H, NCH
2
3
3
MMTr), 3.73 (m, 1H, CH
2
2
8
3
3
2
9