The 4-N-acyl and 4-N-alkyl gemcitabine analogues with silicon-fluoride-acceptor: Application to 18F-Radiolabeling

Article abstract of DOI:10.1016/j.ejmech.2018.02.017

The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of 4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabin

Full text of DOI:10.1016/j.ejmech.2018.02.017

Accepted Manuscript
The 4-N-acyl and 4-N-alkyl gemcitabine analogues with silicon-fluoride-acceptor:
18
Application to F-Radiolabeling
Cesar Gonzalez, Andersson Sanchez, Jeffrey Collins, Ksenia Lisova, Jason T. Lee,
R. Michael van Dam, M. Alejandro Barbieri, Cheppail Ramachandran, Stanislaw F.
Wnuk
PII:
S0223-5234(18)30139-9
10.1016/j.ejmech.2018.02.017
EJMECH 10194
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 October 2017
Revised Date: 24 January 2018
Accepted Date: 6 February 2018
Please cite this article as: C. Gonzalez, A. Sanchez, J. Collins, K. Lisova, J.T. Lee, R. Michael van
Dam, M. Alejandro Barbieri, C. Ramachandran, S.F. Wnuk, The 4-N-acyl and 4-N-alkyl gemcitabine
18
analogues with silicon-fluoride-acceptor: Application to F-Radiolabeling, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.017.
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ACCEPTED MANUSCRIPT
Graphical Abstract

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The 4-N-Acyl and 4-N-Alkyl Gemcitabine Analogues with Silicon-Fluoride-Acceptor:
Application to 18F-Radiolabeling
Cesar Gonzalez,† Andersson Sanchez,† Jeffrey Collins,‡ Ksenia Lisova,‡ Jason T. Lee,‡ R.
Michael van Dam,‡ M. Alejandro Barbieri,§ Cheppail Ramachandran∥ and Stanislaw F. Wnuk*,†
†Department of Chemistry and Biochemistry, Florida International University, Miami, Florida
33199, United States
‡Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology,
Los Angeles, California, United States
§Department of Biological Sciences, Florida International University, Miami, Florida 33199,
United States
∥Nicklaus Children’s Hospital, Miami, Florida, United States
wnuk@fiu.edu
Abstract
The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling
conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of
4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabine with a terminal
azido group. Click reaction with silane building blocks afforded 4-N-alkanoyl or 4-N-alkyl
gemcitabine analogues suitable for fluorination. RP-HPLC analysis indicated better chemical
stability of 4-N-alkyl gemcitabine analogues versus 4-N-alkanoyl analogues in acidic aqueous
conditions. The 4-N-alkanoyl gemcitabine analogues showed potent cytostatic activity against
L1210 cell line, but cytotoxicity of the 4-N-alkylgemcitabine analogues was low. However, 4-N-
alkanoyl and 4-N-alkyl analogues had comparable antiproliferative activities in the HEK293
cells. The 4-N-alkyl analogue with a terminal azide group was shown to be localized inside
HEK293 cells by fluorescence microscopy after labelling with Fluor 488-alkyne. The [18F]4-N-
alkyl or alkanoyl silane gemcitabine analogues were successfully synthesized using microscale
and conventional silane-labeling radiochemical protocols. Preliminary positron-emission
tomography (PET) imaging in mice showed the biodistribution of [18F]4-N-alkyl to have initial
concentration in the liver, kidneys and GI tract followed by increasing signal in the bone.

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1. Introduction
Gemcitabine (2′,2′-difluoro-2′-deoxycytidine, dFdC), is a chemotherapeutic nucleoside
analogue used as first line therapy in pancreatic and lung cancers.[1] Gemcitabine, like many
other nucleosides, enters cells via the human equilibrative nucleoside transport protein 1
(hENT1) and is subsequently activated via phosphorylation by deoxycytidine kinase (dCK) and
other kinases to its triphosphate form.[2, 3] The triphosphate is a substrate for DNA synthesis
inhibiting DNA polymerase by chain termination during replication and repair processes,
triggering apoptosis.[4]
Gemcitabine, even if beneficial for the treatment of a variety of tumors, can have its efficacy
diminished by increased toxicity to normal cells and rapid intracellular deamination into inactive
2′,2′-difluorouridine (dFdU) by cytidine deaminase (CDA).[5-7] In order to tackle these issues,
various prodrug strategies have been developed through hydrolysable lipophilic acyl
modifications on the exocyclic 4-N-amine or 5′-hydroxyl group of the nucleoside,[8-14] and
such approaches were recently reviewed.[15] These modifications facilitate the uptake by cells
slowing also the release of gemcitabine through hydrolysis, which increases its bioavailability
while also providing resistance to enzymatic deamination by CDA. Gemcitabine analogues have
been also designed and synthesized as theranostic delivery systems with cellular specificity and
imaging capabilities.[16-20] Increased bioavailability of gemcitabine 4-N-alkyl analogues have
been scarcely explored and thus their synthesis and mode of activity need further investigation.
Recently, Pulido et al. showed that 4-N-alkyl analogues had modest cytostatic activity with
neither measurable deamination nor conversion to gemcitabine observed.[21, 22] This increased
stability of the 4-N-alkyl analogues within the cell, compared to 4-N-alkanoyl counterparts,
provides an opportunity for their development as novel derivatives of gemcitabine and to study
their distribution via PET.

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Although 18F-radiolabeling has progressed tremendously in recent years, the incorporation of
the fluorine-18 label into biomolecules normally involves a large number of steps and
burdensome labeling procedures.[23-28] This is definitely the case in the synthesis of 18F-
labelled nucleoside analogues, which usually involves multiple protection and deprotection steps,
and therefore can lead to an overall increased reaction time and decreased radiochemical yield.
Since practical syntheses of gemcitabine analogues are based on incorporation of a geminal
difluoro unit in their early synthetic stages,[29, 30] preparation of 2'-[18F]dFdC is not feasible
due to short half-life of the 18F isotope.[31] Recently, the gemcitabine analogue 1-(2-deoxy-2-
[18F]fluoro-β-D-arabinofuranosyl)cytosine ([18F]FAC) was developed enabling noninvasive
prediction of tumor responses to gemcitabine and is utilized as a probe for PET imaging of dCK
activity.[32-34] Although FAC generally follows the known ex vivo biodistribution of
gemcitabine, it is missing a geminal difluoromethylene unit at C2' which is reported to be critical
for anticancer properties of dFdC and its inhibitory activity of ribonucleotide reductases.[35]
Therefore, we have undertaken efforts to investigate dFdC derivatives with the geminal difluoro
unit already incorporated and having a silane moiety that would allow convenient 18F labelling.
The use of compounds bearing Si-F bond in radiochemistry has been explored since
1958,[36] with in vivo studies reported as early as the 1970s.[37] The Si-F bond was considered
as an alternative to C-F bonds due to its increased bond strength (565 kJ/mol for Si–F versus 485
kJ/mol for C–F).[38] However, early on, it was discovered that even with this increased stability,
the Si-F bond is highly susceptible to hydrolysis in physiological conditions.[38] This tendency
can be prevented by the use of bulky substituents on Si-atom since they have been shown to
shield the Si-F bond from hydrolysis while also increasing lipophilicity. This makes these
silicon-fluoride acceptors potential 18F-labeled tracers for PET imaging.[39-43] Herein, we

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report synthesis of clickable 4-N-alkanoyl and 4-N-alkyl gemcitabine analogues with silicon-
fluoride acceptors, high-yield 18F-radiolabeling via a conventional and micro-droplet approach,
in vitro anti-cancer and cell localization evaluation, and preliminary in vivo biodistribution
studies with PET imaging.
2. Results and Discussion
2.1. Chemistry
2.1.1 Synthesis of silane precursors
The strategy for the synthesis of gemcitabine analogues bearing silicon fluoride acceptors
attached to an exo-amino group of cytosine ring involve: (a) synthesis of gemcitabine analogues
having terminal azido or alkyne group at an alkyl chain attached to 4-amino group, and (b)
copper(I) catalyzed click reaction with the corresponding silane reagent having terminal alkyne
or azido group. Thus, condensation of gemcitabine 1 with 11-azidoundecanoic acid S4 under
peptide coupling conditions [(N-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC)/1-
hydroxybenzotriazole (HOBt)/ N,N-Diisopropylethylamine (DIPEA)] in DMF at 65°C afforded
4-N-(11-azidoundecanoyl)gemcitabine 2 (70%; Scheme 1). The 11-azidoundecanoic acid S4 was
prepared by esterification of the commercially available 11-bromoundecanoic acid and
subsequent azidation (NaN3/DMF) followed by saponification with the overall 81% yield
(Scheme S1, see Supporting Information Section). Condensation of 1 with 5-hexynoic acid under
similar conditions gave 4-N-(hexynoyl)gemcitabine 3 in lower yield contaminated with mono
and/or di sugar 5-hexynoate esters. However, transient protection[44] of 1 with trimethylsilyl
group followed by condensation with 5-hexynoic acid in the presence of EDC provided 3 (63%).

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Scheme 1. Synthesis of 4-N-alkanoyl gemcitabine analogues with silicon-fluoride acceptors.a
a Reagents and conditions: (a) (i) TMSCl/Pyr/MeCN, 3 h, (ii) CH
≡C(CH2)3COOH/EDC/MeCN/60 °C;
(iii) EtOH, 45°C, 4 h; (b) Sodium ascorbate/Cu2SO4/t-BuOH/water (3:1), 6 h; (c)
HOBt/DIPEA/EDC/DMF/65 °C/overnight; (d) KF/18-crown-6/AcOH/MeCN/80 oC/25 min. Synthesis
and chemical characterization of substrates S4, S9 and S10 are described in SI Section.
Copper-catalyzed click reaction of alkyne 3 with azido building block S9 [4-
(azidomethyl)phenyldiisopropylsilane] in the presence of sodium ascorbate and copper(I) sulfate
gave silicon-fluoride acceptor 4 (92%). Alternatively azide 2 coupled with alkyne building block
S10 [diisopropyl(4-((prop-2-yn-1-yloxy)methyl)phenyl)silane] gave 5 (87%). The two
bifunctional silicon building blocks S9 and S10 were prepared from 4-
(diisopropylsilyl)benzylalcohol S8 as depicted in Scheme S3 (see SI section).

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The synthesis of the 4-N-alkyl gemcitabine analogues with silicon-fluoride acceptor are
based on displacement of a 4-N-tosylamine group from 7 with freshly prepared 7-
azidohepylamine S7. Thus, transient protection of gemcitabine 1 with trimethylsilyl group and
subsequent treatment with TsCl followed by deprotection with methanolic ammonia afforded 4-
N-tosylgemcitabine[21] 7 (90%, Scheme 2). Treatment of 7 with 7-azidohepylamine S7 effected
displacement of the p-toluenesulfonamido group from the C4 position of the cytosine ring to give
4-N-(7-azidoheptanyl) gemcitabine 8 (82%). The 7-azidoheptylamine S7 was prepared from 1,7-
dibromoheptane by treatment with 2 eq. of NaN3, followed by selective Staudinger reduction of
one of the azido group in intermediary 1,7-diazidoheptane S6 with triphenylphosphine in 83%
overall yield (Scheme S2, see SI section). Click reaction of azido 8 with alkyne building block
S10 gave silane 9 (90%).
Scheme 2. Synthesis of 4-N-alkyl gemcitabine analogues with silicon-fluoride acceptors.a

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a Reagents and conditions: (a) (i) TMSCl/Pyr, (ii) TsCl, (iii) MeOH/NH3; (b) TEA/1,4-dioxane/65 oC; (c)
Sodium ascorbate/Cu2SO4/t-BuOH/H2O (3:1)/6 h; (d) KF/18-crown-6/AcOH/MeCN/80 oC/25 min.
2.1.2. Fluorination and stability of Si-F products
Treatment of 4-N-acyl silanes 4 or 5 with KF in the presence of 18-crown-6 in MeCN at 80
ºC for 20 min followed by quick cooling, filtration, and silica column chromatography (5 → 10%
MeOH/CHCl3) gave the respective fluorinated products 11 (65%) or 6 (63%, Scheme 1).
Analogous fluorination of 4-N-alkyl silanes 9 afforded (fluoro)diisopropylsilane 10 (62%,
Scheme 2). Besides desired silyl fluorides 6, 10 and 11 the corresponding silanols resulting from
the hydrolysis from Si-OH were also isolated during purification on column (~20-25%). The
structure of the silanols were confirmed by the absence of hydrogen from of Si-H bond (e.g., in 5
1
at 3.92 ppm) and lack of fluorine signal (e.g., in 6 at -188.86 ppm) in H or 19F NMR and
additionally defined by HRMS.
Stability of 4-N-alkanoyl 5 and 4-N-alkyl 9 substrates as well as their fluorinated products 6
and 10 were examined employing RP-HPLC with isocratic mobile phase of CH3CN/water
containing 0.1% of TFA which is compatible with the purification protocols for the [18F]-labeled
products (vide infra). The 4-N-alkanoyl 5 were found to be prone to hydrolysis of the amide
bond. For example, gemcitabine (10-15%, 30 min) was detected after 5 was dissolved in 35%
CH3CN/0.1% TFA (Figure 1). The RP-HPLC of the fluoro product 6 showed both hydrolysis of
the acyl chain to gemcitabine (15%, 30 min) and Si-F bond to the corresponding silanol (20%, 30
min; Figure S1, see SI section). HPLC after 2 h showed larger amounts of silanol (30%) and
gemcitabine (20%; Figure S2).

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Figure 1. Stability of 4-N-alkanoyl 5 (chromatogram A: 1, chromatogram B: 5 after 30 min) in
35 %CH3CN /0.1% TFA
On the other hand, the 4-N-alkyl substrate 9 was found to be stable with only very minor
formation of byproduct peak(s) (e.g., gemcitabine) observed after long exposure (8 h) to 35%
CH3CN/0.1% TFA (Figure S3, see SI). The fluorinated product 10 in the 25% CH3CN/0.1%
TFA in water hydrolyzes to silanol (25%, 1 h, 55%, 3 h; Figure S4). However, hydrolysis of 10
in TFA-free system (25% CH3CN/water) occurred to a lesser extent (20%, 1 h, 30%, 3 h; Figure
S5). These studies show that 4-N-alkyl analogues are more stable under acidic conditions than 4-
N-acyl counterparts, indicating their advantage in developing them as PET imaging agents for
gemcitabine prodrugs.
2.1.3. Radiosynthesis of [18F]Fluoro-silane probes
The conventional one-pot syntheses of 4-N-alkanoyl [18F]6 and 4-N-alkyl [18F]10 were
performed on the ELIXYS FLEX/CHEM radiosynthesizer employing silane-labeling
protocols.[42] Thus, by adding silane precursor 9 in DMSO with 1% v/v AcOH to the previously

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dried [18F]KF/K222 complex and reacting at 100°C for 25 min, followed by HPLC purification, 4-
N-alkyl [18F]10 was produced with 6.6 ± 3.2 % (n = 5) decay-corrected isolated radiochemical
yield and >99% radiochemical purity (Scheme 3, Table 1, Figure 2; see SI for more detailed
protocols, Figure S6). Analogously, the 18F-fluorination of precursor 5 gave 4-N-alkanoyl [18F]6
in ~0.5% (n = 1) decay-corrected crude radiochemical yield. Optimization of the protocol for the
synthesis of [18F]6 was not performed.
Scheme 3. Radiosynthesis of [18F] 4-N-alkanoyl and alkyl gemcitabine analogues with silicon-
fluoride acceptors.
Table 1. 18F radiosynthetic yields of 4-N-modified gemcitabine analogues 6 and 10.
Entry
Analogue
Conventional
radiosynthesisa
Average decay-corrected
isolated RCY (%)
0.5
Microscale
radiosynthesisb
Decay-corrected
crude RCY (%)
10
1
2
6
10
6.6 ± 3.2 (n=5)
24.4 ± 4.1 (n=5)
a 2-3 mg scale reactions
b 0.2 mg scale reactions

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Figure 2. Analytical HPLC chromatogram of purified 4-N-alkyl [18F]10 (produced at
conventional scale) (chromatogram A: UV detector, chromatogram B: gamma detector).
The [18F] labeled 6 and 10 were synthesized more efficiently using microscale approach on
simple microfluidic chips (Figure S7 in SI).[45, 46] By adding silane precursor 5 or 9 in DMSO
with 1% v/v AcOH to the previously dried [18F]KF/K222 residue on one chip, covering with a
second chip, and heating at 100 °C for 20 min, a decay-corrected crude radiochemical yield (i.e.
without purification) for 4-N-alkanoyl [18F]6 was 10% (n = 1) and for 4-N-alkyl [18F]10 was 24.4
± 4.1 (n = 5) (Figure S8, see SI for more detailed protocol). Additionally, 4-N-alkyl [18F]10 is
radiochemically stable at ambient temperature for over 4 hours after formulation in saline with
10% v/v EtOH, with no significant radiolysis or other degradation observed (Figure S9, see SI).
2.2. Biological evaluation and imaging
2.2.1. Cytostatic activity
The antiproliferation capabilities of the 4-N-alkanoyl (2, 3, 5 and 6) and 4-N-alkyl (8 and 9)
gemcitabine analogues were assessed (72 h) in L1210 mouse lymphocytic leukemia cells (Table

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2). The 4-N-alkanoyl analogues 2 and 5 demonstrated potent antiproliferative activities with IC50
= 8.0 M and IC50 = 7.5 M, respectively. The reported IC50 value of gemcitabine for an
identical incubation time (72 h) is 23.7 M.[47] Derivatives 2 and 5 have also comparatively
µ
µ
µ
low IC75 values indicating superior antiproliferative effects. However, 4-N-alkylgemcitabine
derivatives 8 and 9 showed less antiproliferative activities having IC values of >200 µM.
Table 2. In vitro cytostatic activity of 4-N-modified gemcitabine analogues in L1210
lymphocytic leukemia cell line.
IC50 (µM)a
8.0 ±0.4
65.3±0.6
7.5±0.4
IC75 (µM)a
14.9± 2.4
88.3±1.1
13.3±1.1
78.3±5.6
Compounds
2
3
5
6
8
9
40.0±1.9
>200
>200
>200
>200
a Values are the mean of three independent experiments and reported as mean ± SD.
Additionally, we tested proliferation of human embryonic kidney HEK293 cells (48h) with
4-N-alkanoyl 2 and 5 and 4-N-alkyl analogues 8 and 9. These analogues were found to have
different levels of inhibition in a dose dependent manner and blocked cell proliferation (Table 3).
These results show comparable antiproliferative activities for 4-N-alkanoyl and 4-N-alkyl
analogues. This might be due to the fact that HEK293 cells have higher CDA activity than that
reported for many other cells and organs;[48, 49] and that CDA expression/activity is known to
affect anticancer therapy.[50],[51] Also, the low cytotoxicity of or high resistance to cytidine
analogues such as gemcitabine have been associated with high levels of CDA expression in
cancer cells.[52, 53] Conversely, the fact that 4-N-alkyl cytidine analogues (including 4-N-alkyl
analogues of gemcitabine) are not substrates of CDA[22, 54] can contribute to their cytotoxicity

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in HEK293 cells since their metabolism to inactive uracil metabolites is either very slow or
prohibited.
Table 3. In vitro inhibitory activity of gemcitabine and 4-N-modified gemcitabine analogues (at
50 and 100 µM concentrations) in human embryonic kidney HEK293 cell line.
Compounds % inhibition % inhibition
(50 µM)a
38±4%
(100 µM)a
45±6%
1
2
79±10%
89±15%
42±5%
45±9%
50±9%
63±6%
61±11%
78±15%
5
8
9
a Values are the mean of three independent experiments and reported as mean ± SD.
Even though 4-N-alkyl analogues 8 and 9 showed lower cytotoxic activities in the cells
tested, we decided to study cellular uptake of 8 with HEK293 cells since the lipophilic[55] 4-N-
alkyl derivatives of gemcitabine show a stability toward fluorination protocols compatible with
18F fluorination. We were interested to investigate whether nucleoside 8 would be internalized
and/or localized in the nucleus of cells and incorporated into DNA, or they are trapped in an
extranuclear compartment.
Figure 3. Incorporation of 8 in HEK293 cells after 24 h, followed by fixation and addition of
Fluor 488-Alkyne and copper (I): A) Nuclear staining with DAPI, B) Cell staining with Fluor 488-

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Alkyne. C) Merged A & B microscopy images. Inset: Staining of DNA during cell division. Scale
bars: 10 µm.
To determine whether 4-N-alkyl analogue 8 was localized inside cells, we carried out in vitro
fluorescence studies with HEK293 cells. The cells were incubated with 100 µM of 8 for 24 h,
fixed and labelled with Fluor 488-alkyne in presence of copper (I). Fluor 488-labeled cells
(Figure 3, B; seen in green) was evidence that 4-N-alkyl 8 was internalized by the cells.[56]
Also, strong nuclear staining was evident by its co-localization with DAPI (Figure 3, A; seen in
blue), as well as on the DNA during cell division. As expected, when cells were incubated
without 8 no fluorescent green signal was observed (data not shown).
2.2.2. In Vivo Imaging of [18F]4-N-alkyl gemcitabine radioligand 10
To determine the distribution and uptake of [18F]10, preliminary in vivo, static and dynamic
PET imaging studies were performed in wild-type mice. For static PET imaging, a WT C57BL/6
mouse was injected with [18F]10 via tail vein and imaged for 10 min after 1 h uptake. For
dynamic PET imaging, a 1 h PET acquisition was started concurrently at the beginning of a 10
sec infusion via a tail vein catheter of [18F]10 and histogrammed into a series of images (Figures
S10 and S11 in SI). Both static and dynamic PET imaging show the tracer first in the liver,
kidneys, and GI tract (also the gallbladder in the static scan), but significant bone signal is
evident after 15-20 min post-injection (Figure 4 and 5, see SI for more detailed protocol). One
cause of bone signal is in vivo defluorination of the PET tracer, releasing [18F]fluoride, which is
strongly incorporated into hydroxyapatite crystals of bone. More detailed analysis of tracer
metabolites in vivo is needed to determine whether defluorination is occurring or whether bone
signal is actually uptake in bone marrow.

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Figure 4. (Left) Dynamic biodistribution of [18F]10 in WT C57BL/6 mouse. (Right) Maximum intensity
projection at 1 h post-injection (left: coronal; right: sagittal).
Figure 5. (Left) Biodistribution of [18F]10 from 10 min static scan in WT C57BL/6 mouse at 1 h post-
injection. (Right) Maximum intensity projection of 10 min static scan at 1 h post-injection (left: coronal;
right: sagittal).
It appears that [18F]4-N-alkyl gemcitabine radioligand 10 undergoes renal (high kidney,
bladder) and possibly hepatobiliary (high gut and liver) clearances, with possible defluorination.
It is noteworthy that biodistribution of the 4-N-alkyl radioligand does not reflect biodistribution
of [14C]gemcitabine.[5]

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3. Conclusion
We have demonstrated that copper(I) catalyzed click reaction of the 4-N-alkanoyl or alkyl
gemcitabine analogues having terminal azide or alkyne group on the alkyl chain with the silane
reagents having terminal alkyne or azido group provided 4-N-alkanoyl and alkyl gemcitabine
analogues with silicon-fluoride acceptors. RP-HPLC analysis showed that the 4-N-alkyl
analogues were more stable in aqueous acidic conditions, indicating their advantage in
developing them as PET imaging tracers. The silane 4-N-alkyl precursor 9 was successfully
fluorinated to [18F]10 using conventional and microscale radiosynthetic methods in high
radiochemical purity and stability of up to 4 h. Preliminary static and dynamic PET studies in
wild-type mice have shown the initial biodistribution of [18F]4-N-alkyl tracer 10 in the liver,
kidneys and GI tract followed by increasing signal in the bone. The 4-N-alkanoylgemcitabine
analogues showed more potent antiproliferative activity in L1210 cells compared to the 4-N-
alkylgemcitabine derivatives. However, 4-N-alkanoyl and 4-N-alkyl analogues had comparable
antiproliferative activities in the HEK293 cells, which have high levels of CDA expression. The
4-N-alkyl gemcitabine derivatives were shown to be localized inside HEK293 cells by
fluorescence microscopy after click labelling with Fluor 488-alkyne. The chemical and
enzymatic stabilities of 4-N-alkylcytidine analogues and their compatibility with 18F-labeling
provide a potential avenue for developing new PET imaging tracers for gemcitabine derivatives.
Future work would involve optimizing the structure to improve the in vivo stability and the
biodistribution of these analogs.
4. Experimental Section
1
The H (400 MHz), 13C (100 MHz), or 19F (376 MHz) NMR spectra were recorded at
ambient temperature in solutions of CDCl3 or MeOH-d4 as noted. The reactions were followed

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by TLC with Merck Kieselgel 60-F254 sheets and products were detected with a 254 nm light or
with Hanessian’s stain. Column chromatography was performed using Merck Kieselgel 60 (230-
400 mesh). Reagent grade chemicals were used and solvents were dried by reflux distillation
over CaH2 under nitrogen gas, unless otherwise specified, and reactions carried out under N2
atmosphere. The carboxylic acid and amine derivatives used for the coupling with gemcitabine
were commercially available or synthesized [11-azidoundecanoic acid (S4) and 7-Azido-1-
aminoheptane (S7)] as described in Supporting Information. The purity of some of the
synthesized compounds was determined by HPLC on Phenomenex Gemini RP-C18 with
isocratic at various mobile phases (CH3CN/H2O) and flow rates.
4-N-(11-Azidoundecanoyl)-2
L, 26 mg, 0.2 mmol), 1-hydroxybenzotriazole (27 mg, 0.2 mmol), S4 (46 mg, 0.2 mmol),
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (45 L, 31 mg, 0.2 mmol) were
′-deoxy-2′,2′-difluorocytidine (2). N,N-Diisopropylethylamine
(35
µ
µ
sequentially added to a stirred solution of gemcitabine hydrochloride (1; 50 mg, 0.17 mmol) in
DMF (4 mL) at ambient temperature under N2 atmosphere The reaction mixture was then
gradually heated to 65 °C (oil-bath) and was kept stirring overnight. The crude mixture was
evaporated and the residue column chromatographed (0
mg, 70%): UV (CH3OH) max 299 nm ( 6500),
→
10% MeOH/CHCl3) to give 2 (56
12 900); 1H NMR (CD3OD)
δ
λ
ε
λ
min 250 nm (
ε
1.24−1.47 (m, 12H, 6 × CH2), 1.51−1.75 (m, 4H, 2 × CH2), 2.45 (t, J = 7.4 Hz, 2H, CH2), 3.27 (t,
J = 6.9 Hz, 2H, CH2), 3.81 (dd, J = 2.8, 12.4 Hz, 1H, H5 ), 3.89−4.05 (m, 2H, H5′′, H4 ), 4.30
), 7.50 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J =
25.92, 27.80, 29.90, 30.11, 30.20, 30.33, 30.41, 30.50, 38.17,
), 82.92 (d, J = 8.6 Hz, C4 ), 86.42 (dd, J =
26.6, 37.6 Hz, C1), 98.25 (C5), 120.87 (t, J = 259.9 Hz, C2 ), 145.96 (C6), 157.68 (C2), 164.83
′
′
(td, J = 8.5, 12.2 Hz, 1H, H3′), 6.17−6.35 (m, 1H, H1′
7.6 Hz, 1H, H6); 13C NMR
δ
52.47, 60.30 (C5
′), 70.27 (dd, J = 22.2, 23.6 Hz, C3
′
′
′

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17
(C4), 176.00 (CO); 19F NMR
Hz, 1F). HRMS (ESI+) m/z calcd for C20H30F2N6NaO5 [M+Na]+ 495.2146; found 495.2141.
δ -120.05 (d of m, J = 239.7 Hz, 1F), -119.10 (d of m, J = 240.1
4-N-(5-Hexynoyl)-2′-deoxy-2′,2′-difluorocytidine (3). Trimethylsilyl chloride (250 µL, 2
mmol) was added to a solution of 1 (200 mg, 0.7 mmol) in MeCN (2 mL) and pyridine (3 mL) at
0 °C. The mixture was stirred for 4 h from 0 °C to room temperature. A solution of commercially
available 5-hexynoic acid (230
µL, 2.1 mmol) in MeCN (2 mL), previously activated by EDC
(50 L, 1 mmol), was added to the reaction mixture, which was heated at 60°C for 18 h. After
µ
the solution was cooled down to room temperature, ethanol (5 mL) was added and the mixture
was heated at 45 °C for 4 h. After evaporation under vacuum, the resulting residue was column
1
chromatographed (80
(CD3OD)
→
100% EtOAc/hexane) to give 3[44] (157.6 mg, 63%): H NMR
δ
1.84−1.86 (m, 2H, CH2), 2.26−2.28 (m, 3H, CH2, CH), 2.60 (t, J = 7.3 Hz, 2H, CH2),
3.82−3.84 (m, 1H, H5′), 3.89−4.07 (m, 2H, H5′′, H4
′
), 4.31 (dd, J = 12.1, 20.6 Hz, 1H, H3
′),
6.24−6.26 (m, 1H, H1
′
), 7.49 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J = 7.6 Hz, 1H, H6); 13C NMR
δ
18.42, 24.96, 33.47, 60.50 (C5
′
), 68.14 (CH), 70.20 (C) 70.34 (t, J = 23.1 Hz, C3
′
), 82.35 (d, J =
), 142.51
-120.14 (d of m, J = 244.4 Hz, 1F), -
8.6 Hz, C4 ), 86.1 (dd, J = 26.6, 38.3 Hz, C1
′
′), 96.30 (C5
′), 123.91 (t, J = 259.3 Hz, C2
′
19
(C6), 157.78 (C2), 167.74 (C4), 175.97 (CO); F NMR
δ
119.23 (d of m, J = 243.6 Hz, 1F).
4-N-(7-Azidoheptanyl)-2′-deoxy-2′,2′-difluorocytidine (8). Freshly prepared 7-Azido-1-
aminoheptane (S7; 112.5 mg, 0.72 mmol) was added to a suspension of 7[21] (100 mg, 0.24
mmol) in 1,4-dioxane (5 mL) and TEA ( 0.100 mL, 63 mg, 0.72 mmol) and the mixture was left
stirring at 65°C. After 24 h, volatiles were evaporated and the resulting residue was column
chromatographed (5% MeOH/CHCl3) to give 8 (238 mg, 82%): UV (CH3OH)
λ max 267 nm (ε
8200),
λ
min 227 nm (
ε
7400); 1H NMR (CD3OD)
δ 1.31−1.41 (m, 6H, 3 × CH2), 1.51−1.70 (m,

ACCEPTED MANUSCRIPT
18
4H, 2 × CH2), 3.25 (t, J = 6.9 Hz, 2H, CH2), 3.45 (t, J = 7.0 Hz, 2H, CH2) 3.77−3.83 (m, 1H,
H5
′
), 3.91−3.99 (m, 2H, H5′′, H4
′
), 4.25 (dt, J = 10.5, 20.8 Hz, 1H, H3
′
), 5.85 (d, J = 7.6 Hz, 1H,
27.75, 27.86, 29.84,
), 82.22 (d, J = 8.6 Hz, C4 ),
), 97.32 (C5), 124.02 (t, J = 259.2 Hz, C2 ), 140.80 (C6), 158.29
-119.86 (d of m, J = 246.2 Hz, 1F), -119.89 (d of m, J = 240.2 Hz,
H5), 6.23−6.25 (m, 1H, H1
′
) , 7.71 (d, J = 7.6 Hz, 1H, H6); 13C NMR
δ
29.88, 29.92, 41.65, 52.45, 60.56 (C5
′
), 70.66 (t, J = 23.1 Hz, C3
′
′
86.26 (dd, J = 26.6, 38.3 Hz, C1
′
′
(C2), 165.38 (C4); 19F NMR
δ
1F). HRMS (ESI+) m/z calcd for C16H25F2N6O4 [M+H]+ 403.1902; found 403.1913.
General procedure for click reactions
Sodium ascorbate (0.02 mmol), copper sulfate (0.02 mmol), nucleoside (0.1 mmol) and
silicon-fluoride acceptor (0.1 mmol) were suspended in a mixture of tert-butanol/water (3:1
(v/v), 3 mL). The reaction mixture was left at room temperature for 1-6 hours. The reaction
mixture was extracted with CHCl3 (10 mL). The organic layer was washed with sat NH4Cl (10
mL), brine (10 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. The
crude product was purified by chromatography (CHCl3/MeOH 95:5) to afford the desired
triazole adducts:
4-N-[4-(1-(4-(Diisopropylsilyl)benzyl)-1H-1,2,3-triazol-4-yl)butanoyl]-2
′-deoxy-
2′,2′difluorocytidine (4). Treatment of 3 (35 mg, 0.1 mmol) with S9 (24.7 mg, 0.1 mmol), as
described in general procedure, gave 4 (52.6 mg, 87%). UV (CH3OH)
λ max 299 nm (ε 6700),
1
248 nm (
ε
13000); H NMR (CD3CN)
δ
0.96 (d, J = 7.3 Hz, 6H, iPr), 1.03 (d, J = 7.3 Hz, 6H,
iPr), 1.18−1.20 (m, 4H, 2 × CH2), 2.46 (t, J = 7.3 Hz, 2H, CH2). 2.71 (t, J = 7.4 Hz, 2H, CH2),
3.74−3.76 (m, 1H, H5 ), 3.89−3.95 (m, 3H, H5′′, H4 , Si-H), 4.24−4.26 (m, 1H, H3 ), 5.50 (s,
2H, CH2), 6.19 (t, J = 7.5 Hz, 1H, H1
′
′
′
′), 7.28 (d, J = 8.0 Hz, 2H, Ar), 7.35 (d, J = 7.6 Hz, 1H,
13
H5), 7.51 (d, J = 8.0 Hz, 2H, Ar), 7.56 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H, H6); C NMR
δ 11.18,

ACCEPTED MANUSCRIPT
19
), 69.68 (CH), 70.34 (t, J = 23.1 Hz, C3 ),
), 122.39 (t, J = 259.3
18.65, 18.83, 24.96, 25.17, 37.04, 54.09, 59.93 (C5
′
′
81.82 (d, J = 8.6 Hz, C4′), 85.70 (dd, J = 26.6, 38.3 Hz, C1′), 97.03 (C5′
Hz, C2′), 128.07, 134.96, 136.86, 138.11, 145.43 (C6), 148.04, 155.77 (C2), 163.85 (C4), 174.28
(CO); 19F NMR
δ
-120.10 (d of m, J = 233.4 Hz, 1F), -119.20 (d of m, J = 240.9 Hz, 1F). HRMS
(ESI+) m/z calcd for C28H38F2N6NaO5Si [M+Na]+ 627.2538; found 627.2522.
4-N-[11-(4-((4-(Diisopropylsilyl)benzyloxy)methyl)-1H-1,2,3-triazol-1-yl)undecanoyl]-2′-
deoxy-2′,2′difluorocytidine (5). Treatment of 2 (30 mg, 0.06 mmol) with S10 (15.6 mg, 0.06
mmol), as described in general procedure, gave 5 (40.5 mg, 92%). UV (CH3OH)
λ max 299 nm
1
(ε
6500), 250 nm (
ε
12900),
λ
min 278 nm (
ε
3800); H NMR (CD3CN)
δ
0.96 (d, J = 7.3 Hz,
6H, iPr), 1.05 (d, J = 7.3 Hz, 6H, iPr), 1.24−1.47 (m, 14H, 7 × CH2), 1.51−1.75 (m, 4H, 2 ×
CH2), 2.38 (t, J = 7.3 Hz, 2H, CH2), 3.81 (dd, J = 2.8, 12.4 Hz, 1H, H5 ), 3.89−3.96 (m, 3H, H5′′
H4 , Si-H), 4.30 (td, J = 8.5, 12.2 Hz, 1H, H3 ), 4.33 (t, J = 7.1 Hz, 2H), 4.55 (s, 2H, CH2), 4.61
(s, 2H, CH2), 6.19 (t, J = 7.5 Hz, 1H, H1
′
,
′
′
′), 7.33−7.35 (m, 3H, H5, Ar), 7.51 (d, J = 8.0 Hz, 2H,
13
Ar), 7.74 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H, H6); C NMR
δ
25.92, 27.80, 29.90, 30.11, 30.20,
), 82.92 (d, J =
30.33, 30.41, 30.50, 38.17, 52.47, 60.30 (C5
′
), 70.27 (dd, J = 22.2, 23.6 Hz, C3
′
8.6 Hz, C4
′), 86.42 (dd, J = 26.6, 37.6 Hz, C1), 98.25 (C5), 120.87 (t, J = 259.9 Hz, C2′), 145.96
19
(C6), 157.68 (C2), 164.83 (C4), 176.00 (CO); F NMR
δ -120.09 (d of m, J = 239.2 Hz, 1F), -
119.13 (d of m, J = 240.2 Hz, 1F). HRMS (ESI+) m/z calcd for C36H54F2N6NaO6Si [M+Na]+
755.3738; found 755.3731.
4-N-[7-(4-((4-(Diisopropylsilyl)benzyloxy)methyl)-1H-1,2,3-triazol-1-yl)heptanyl]-2
′-
deoxy-2 ,2 difluorocytidine (9) Treatment of 8 (40 mg, 0.1 mmol) with S10 (26.0 mg,
′
′
0.1mmol), as described in general procedure, gave 9 (59.6 mg, 90%). UV (CH3OH)
λ max 267
nm (
ε
8200),
λ
min 228 nm (
ε
7500); 1H NMR (acetone-d6)
δ 0.99 (d, J = 7.3 Hz, 6H, iPr), 1.05

ACCEPTED MANUSCRIPT
20
(d, J = 7.3 Hz, 6H, iPr), 1.31−1.41 (m, 10H, 5 × CH2), 1.50−1.52 (m, 2H, CH2), 1.90-1.93 (m,
2H, CH2), 3.41−3.43 (m, 2H, CH2), 3.80−3.82 (m, 1H, H5 ), 3.91−4.02 (m, 3H, H5′′, H4 , Si-H),
4.42−4.44 (m, 3H, H3 , CH2), 4.60 (s, 2H, CH2), 4.65 (s, 2H, CH2), 5.82 (d, J = 7.6 Hz, 1H,
H5), 6.23−6.25 (m, 1H, H1 ) , 7.38 (d, J = 8.0 Hz, 2H, Ar), 7.48 (d, J = 8.0 Hz, 2H, Ar), 7.71 (d,
J = 7.6 Hz, 1H, H6), 7.99 (s, 1H, CH); C NMR
40.97, 50.50, 60.31 (C5 ), 64.42, 70.26 (t, J = 23.1 Hz, C3
′
′
′
′
13
δ
11.26, 18.76, 18.96, 27.01, 27.34, 30.93,
), 72.37, 81.75 (d, J = 8.6 Hz, C4 ),
′), 95.97 (C5′), 124.04 (t, J = 259.3 Hz, C2′), 127.88, 133.36,
′
′
′
84.92 (dd, J = 26.6, 38.3 Hz, C1
140.78, 135.56, 136.34, 138.84, 140.78 (C6), 141.02, 155.92 (C2), 164.77 (C4); 19F NMR
δ
-
119.86 (d of m, J = 246.2 Hz, 1F), -119.89 (d of m, J = 240.2 Hz, 1F). HRMS (ESI+) m/z calcd
for C32H48F2N6NaO5Si [M+Na]+ 685.3322; found 685.3324.
General Procedure for Fluorination Reactions
Solid KF (4.7 mg, 0.08 mmol, 4 eq.) was added to a stirred solution of 5 (15 mg, 0.02 mmol,)
and 18-Crown-6 ether (21 mg, 0.08 mmol, 4 eq.) in CH3CN (3 mL) in a round bottom flask
under N2 atmosphere. To this mixture acetic acid (2 µL, 0.02 mmol. 1 eq.) was then added and
the resulting reaction mixture was stirred at 80 ºC for 25 min. The reaction mixture was then left
to cool (~5 min.) and filtered to remove the left over 18-crown ether and KF. The filtrate was
washed with CH3CN (2 mL) and the combined mother liquors were then concentrated under
reduced pressure to give crude 6. The resulting residue was chromatographed (MeOH/CHCl3
10:90) to give pure 9 (9.5 mg, 63%).
4-N-[11-(4-((4-(Fluorodiisopropylsilyl)benzyloxy)methyl)-1H-1,2,3-triazol-1-
yl)undecanoyl]-2′-deoxy-2′,2′difluorocytidine (6). Treatment of 5 (15 mg, 0.02 mmol) with KF
(4.7 mg, 0.08 mmol), as described in general procedure, gave 6 (9.5 mg, 63%): UV (CH3OH)
λ
max 299 nm (
ε
6500), 250 nm (
ε
12900),
λ
min 278 nm (
ε
3800); 1H NMR (CD3OD)
δ 0.99 (d,

ACCEPTED MANUSCRIPT
21
J = 7.3 Hz, 6H, iPr), 1.07 (d, J = 7.3 Hz, 6H, iPr), 1.24−1.47 (m, 14H, 7 × CH2), 1.78−1.80 (m,
2H, CH2), 1.90-1.92 (m, 2H, CH2), 2.44 (t, J = 7.4 Hz, 2H, CH2), 3.81 (dd, J = 2.8, 12.4 Hz, 1H,
H5′), 3.89−3.99 (m, 2H, H5′′, H4′), 4.29 (t, J = 10.4 Hz, 1H, H3′), 4.40 (t, J = 7.0 Hz, 2H), 4.60
(s, 2H, CH2), 4.66 (s, 2H, CH2), 6.26 (t, J = 7.5 Hz, 1H, H1
′), 7.41 (d, J = 7.8 Hz, 2H, Ar), 7.49
(d, J = 7.6 Hz, H5), 7.54 (d, J = 8.1 Hz, 2H, Ar), 7.99 (s, 1H), 8.34 (d, J = 7.6 Hz, 1H, H6); 13C
NMR
δ
13.22, 25.92, 27.80, 29.90, 30.11, 30.20, 30.33, 30.41, 30.50, 38.17, 52.47, 60.30 (C5
′),
70.27 (dd, J = 22.2, 23.6 Hz, C3
′
), 82.92 (d, J = 8.6 Hz, C4 ), 86.42 (dd, J = 26.6, 37.6 Hz, C1),
′
98.25 (C5), 120.87 (t, J = 259.9 Hz, C2
′), 128.07, 135.53, 135.56, 136.34, 138.84, 145.93 (C6),
148.76, 145.93 (C6), 157.66 (C2), 164.78 (C4), 175.25 (CO); 19F NMR
δ
- 188.86 (s, 1F), -
120.09 (d of m, J = 239.2 Hz, 1F), -119.13 (d of m, J = 240.2 Hz, 1F). HRMS (ESI+) m/z calcd
for C36H54F3N6O6Si [M+H]+ 751.3821; found 751.3770.
4-N-[7-(4-((4-(Fluorodiisopropylsilyl)benzyloxy)methyl)-1H-1,2,3-triazol-1-yl)heptanyl]-
2′-deoxy-2′,2′difluorocytidine (10). Treatment of 9 (15 mg, 0.023 mmol) with KF (5.4 mg, 0.09
mmol), as described in general procedure, gave 10 (9 mg, 62 %): UV (CH3OH)
λ max 267 nm (ε
8200),
λ
min 228 nm (
ε
7500); 1H NMR (CD3OD)
δ 0.99 (d, J = 7.3 Hz, 6H, iPr), 1.05 (d, J = 7.3
Hz, 6H, iPr), 1.31−1.41 (m, 10H, 5 × CH2), 1.50−1.52 (m, 2H, CH2), 1.90−1.92 (m, 2H, CH2),
3.38−3.40 (m, 2H, CH2), 3.80−3.82 (m, 1H, H5 ), 3.91−4.02 (m, 3H, H5′′, H4 , Si-H), 4.24−4.26
(m, 1H, H3 ), 4.41 (t, J = 6.9 Hz, 2H, CH2), 4.60 (s, 2H, CH2), 4.65 (s, 2H, CH2), 5.82 (d, J =
′
′
′
7.6 Hz, 1H, H5), 6.21−6.23 (m, 1H, H1
′) , 7.41 (d, J = 8.0 Hz, 2H, Ar), 7.52 (d, J = 8.0 Hz, 2H,
Ar), 7.71 (d, J = 7.6 Hz, 1H, H6), 8.01 (s, 1H, CH); 13C NMR
δ
11.26, 18.76, 18.96, 27.01,
), 72.37, 81.75 (d, J =
), 124.04 (t, J = 259.3 Hz, C2 ),
127.88, 133.36, 140.78, 135.56, 136.34, 138.84, 140.78 (C6), 141.02, 155.92 (C2), 164.77 (C4).
27.34, 30.93, 40.97, 50.50, 60.31 (C5
′), 64.42, 70.26 (t, J = 23.1 Hz, C3
′
8.6 Hz, C4 ), 84.92 (dd, J = 26.6, 38.3 Hz, C1
′
′
), 95.97 (C5
′
′

ACCEPTED MANUSCRIPT
22
19F NMR
1F). HRMS (ESI+) m/z calcd for C32H47F3N6NaO5Si [M+Na]+ 703.3227; found 703.3234.
δ - 188.82 (s, 1F), -119.75 (d of m, J = 244.1 Hz, 1F), -119.98 (d of m, J = 239.1 Hz,
4-N-[4-(1-(4-(Fluorodiisopropylsilyl)benzyl)-1H-1,2,3-triazol-4-yl)butanoyl]-2
′-deoxy-
2′,2′difluorocytidine (11). Treatment of 4 (15 mg, 0.025 mmol) with KF (5.9 mg, 0.1 mmol), as
described in general procedure, gave 11 (10 mg, 65%): UV (CH3OH)
λ max 299 nm (ε 6700),
1
248 nm (
ε
13000); H NMR (CD3OD)
δ
0.99 (d, J = 7.3 Hz, 6H, iPr), 1.06 (d, J = 7.3 Hz, 6H,
iPr), 1.18−1.20 (m, 2H, CH2), 1.91−1.93 (m, 2H, CH2), 2.50 (t, J = 7.3 Hz, 2H, CH2). 2.78 (t, J =
7.4 Hz, 2H, CH2), 3.82−3.84 (m, 1H, H5 ), 3.93−4.05 (m, 2H, H5′′, H4 ), 4.29−4.31 (m, 1H,
H3 ), 5.59 (s, 2H, CH2), 6.21 (t, J = 7.5 Hz, 1H, H1 ), 7.35 (d, J = 7.9 Hz, 2H, Ar), 7.47 (d, J =
7.6 Hz, 1H, H5), 7.57 (d, J = 8.1 Hz, 2H, Ar), 7.81 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H, H6); C
NMR 13.22, 13.35, 16.82, 17.01, 25.46, 25.53, 37.18, 54.70, 60.30 (C5 ), 70.01 (CH), 70.34 (t,
J = 23.1 Hz, C3 ), 81.72 (d, J = 8.6 Hz, C4 ), 82.92 (dd, J = 26.6, 38.3 Hz, C1 ), 98.27 (C5 ),
′
′
′
′
13
δ
′
′
′
′
′
123.93 (t, J = 259.3 Hz, C2
′), 128.39, 134.45, 135.53, 135.56, 136.34, 138.84, 145.93 (C6),
148.76, 157.66 (C2), 164.78 (C4), 175.25 (CO); 19F NMR
δ
- 188.89 (s, 1F), -120.10 (d of m, J =
233.4 Hz, 1F), -119.20 (d of m, J = 240.9 Hz, 1F). HRMS (ESI+) m/z calcd for
C28H37F3N6NaO5Si [M+Na]+ 645.2445; found 645.2392.
Radiosynthesis of [18F]Fluoro-silane probes
Unless otherwise stated, reagents and solvents were commercially available and used without
further purification. Purification of [18F]10 was performed by semi-preparative HPLC using a
WellChrom K-501 HPLC pump and reversed-phase Gemini-NX (5 µm, 10 x 250 nm,
Phenomenex) column. Analytical HPLC was performed to confirm identity and radiochemical
purity of the compound in a Knauer Smartline HPLC system with C18 reversed-phase Luna
column (5 µm, 10 x 250 nm, Phenomenex). Radio-TLC was performed on precut silica plates

ACCEPTED MANUSCRIPT
23
(Baker-flex®, J.T.Baker). The radiochemical purity (RCP) was determined by using both radio-
TLC and radio-HPLC radiation-detector chromatograms. For more detailed information, see
supporting information.
[18F]4-N-alkyl (10). Microscale radiochemical synthesis. The microdroplet synthesis was
performed using two Teflon-coated glass chips as shown in Figure S6 (see supporting
information). The first was placed (Teflon-coated side up) on the heater. [18F]fluoride in
[18O]H2O (10
containing K222 (0.23 µg) and K2CO3 (0.07 µg) and deposited in the center of the chip.
Additional CH3CN (10 L) was added to aid in azeotropic drying, and the chip was heated at
105°C until the droplet on chip shrank to a small volume (~1 L). Next, 0.2 mg precursor 9 in 30
µL; ~150 MBq [4 mCi]) was mixed with 12µL of a 70:30 v/v CH3CN/H2O solution
µ
µ
µL of DMSO with 1% v/v AcOH was added to the dried [18F]KF/K222 residue, and the reaction
droplet was covered with the second glass chip (Teflon-coated side down). Tape affixed to the
edges of the top chip resulted in a gap of ~150
at 100°C for 20 min. Crude product was extracted from chip by adding 20
MeOH:H2O solution and then collecting the diluted mixture with a pipette. This process was
repeated 2x for each substrate (~80 L total volume). Without purification, the microscale
µ
m between the substrates. The chip was heated
µL of 1:1 v/v
µ
synthesis took ~45 min. Decay-corrected crude radiochemical yield for 4-N-alkyl [18F]10 was
24.4 ± 4.1% (n=5).
Note: See supporting information for more detailed protocol and conventional synthesis.
Preclinical PET Imaging
Animal studies were approved by the UCLA Animal Research Committee and were carried
out according to the guidelines of the Division of Laboratory Animal Medicine at UCLA.

ACCEPTED MANUSCRIPT
24
PET/CT was performed on the Genisys 8 PET/CT (Sofie Biosciences, USA). The Genisys 8
PET/CT is an integrated scanner with a PET subsection that consists of 8 detectors with BGO
scintillator array arranged in a box geometry and a back section consisting of a rotating CT
gantry. For static PET scans, a WT C57BL/6 mouse was injected with approximately 75 µCi
[18F]10 via tail vein. After 60 min of conscious uptake, mice were anesthetized with 1.5%
isoflurane and placed in a dedicated imaging chamber. MicroPET images were acquired for 600
sec with an energy window of 150-650 keV, reconstructed using maximum-likelihood
expectation maximization as recommended by the vendor. All images were corrected for CT-
based photon attenuation, detector normalization and radioisotope decay (scatter correction was
not applied) and converted to units of percent injected dose per gram (%ID/g). For dynamic PET
scans, a WT C57BL/6 mouse was anesthetized with 2% isoflurane, placed in a dedicated
imaging chamber with heating, and catheterized. Dynamic microPET imaging was started
concurrently at the beginning of a 10 sec infusion via the catheter with approximately 75 µCi of
[18F]10. Data was acquired in listmode for 3600 sec with an energy window of 150-650 keV and
histogrammed into a frame sequence of 4 x 15 sec, 8 x 30 sec, 5 x 60 sec, 4 x 300 sec, 3 x 600
sec. Images were reconstructed using maximum-likelihood expectation maximization as
recommended by the vendor. All images were corrected for photon attenuation, detector
normalization and radioisotope decay (scatter correction was not applied) and converted to units
of percent injected dose per gram (%ID/g). All PET acquisitions were immediately followed by
CT acquisition. The CT section consists of a gantry and flywheel that uses a 50 kVp, 200 uA x-
ray source and flat-panel detector. The CT acquires images in a continuous-rotation mode and
with standard CT acquisition time of 50 s. Standard scans are acquired with 720 projections at 55
ms per projection, and reconstructed using a Feldkamp algorithm.

ACCEPTED MANUSCRIPT
25
Cytostatic activity assays
Murine leukemia L1210 cells were obtained from American Type Culture Collection
(ATCC, Manassas, VA). The gemcitabine analogues tested (Table 2) were added to murine
leukemia L1210 cell cultures in 96-well microtiter plates. After 72 h of incubation at 37 °C, the
percentage surviving cells were assayed according to method described in the Cell Proliferaiton
kit I (MTT) (Roche Biochemicals, Indianapolis, IN). The 50% and 75% inhibitory
concentrations (IC50 and IC75) were defined as the compound concentration required to inhibit
cell proliferation by 50% and 75%, respectively.
Human HEK293 cells were obtained from ATCC (Manassas, VA). The gemcitabine
analogues tested (2, 5, 8 and 9; Table 3) were added (50µM and 100 µM) to human HEK293 cell
cultures in 12-well plates. After 48 h of incubation at 37 °C, the number of living cells was
determined by MTT assays using Coulter counter.
Fluorescence studies with HEK293 cells
HEK293 cells were seeded in plates containing glass coverslips and incubated overnight.
After incubation, fresh media solutions containing 8 (100 µM) was added and then incubated for
various durations (6, 12 and 24 hr). Cells were fixed in paraformaldehyde (3.7%) for 15 min at
room temperature, quenched, and washed with PBS as essentially described by Neef et al.[57]
Cells on coverslips were incubated upside-down with 50
µL of freshly prepared staining mix
(10
µ
M Fluor alkyne, 1 mM CuSO4, and 10 mM sodium ascorbate in PBS) for 1 h at room
temperature in the dark. Cells were then washed with 0.1% Triton X-100 and PBS, respectively.
Cells were then stained with DAPI for 15 min at room temperature in the dark. After incubation,
cells were washing with PBS and coverslips were mounted and viewed on an Olympus FV1200
Laser confocal microscope.

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Supplementary Material. Detailed synthesis and characterization data for 11-azidoundecanoic
acid (S4), 7-azido-1-aminoheptane (S7), 4-(azidomethyl)phenyldiiso-propylsilane (S9) and
diisopropyl(4-((prop-2-yn-1-yloxy)methyl)phenyl)silane (S10) as well as experimental
radiochemical and biological procedures, representative HPLC chromatograms, and stability and
metabolite analysis protocols can be found at https://.
Corresponding Author Information. *Telephone 305-348-6195; e-mail wnuk@fiu.edu.
Abbreviations used. dFdC, 2',2'-difluoro-2'-deoxycytidine (Gemcitabine); hENT1, human
equilibrative nucleoside transport protein 1; dCK, deoxycytidine kinase; dFdU, 2',2'-
difluorouridine; CDA, cytidine deaminase; [18F]FAC, 1-(2-deoxy-2-[18F]fluoro-
β-D-
arabinofuranosyl)cytosine; EDC, N-dimethylaminopropyl)-N'-ethyl-carbodiimide; HOBt, 1-
hydroxybenzotriazole; DIPEA, N,N-Diisopropylethylamine; TEA, triethylamine.
Acknowledgment. A.S. is supported by MBRS RISE program (NIGMS; R25 GM061347). This
work was supported in part by the National Institutes of Health (R21 AG049918, P30
CA016042, T32 EB002101), and the UCLA Foundation from a donation made by Ralph and
Marjorie Crump for the Crump Institute for Molecular imaging. The authors thank Dr. Roger
Slavik and the staff of the UCLA Biomedical Cyclotron Facility for generously providing
[18F]fluoride for these studies, and Dr. Tove Olafsen and Charles Zamilpa of the Crump
Institute’s Preclinical Imaging Technology Center for assistance with PET/CT imaging.
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