Probing the effects of pyrimidine functional group switches on acyclic fleximer analogues for antiviral activity

Article abstract of DOI:10.3390/molecules24173184

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as “fleximers” have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.

Full text of DOI:10.3390/molecules24173184

molecules
Article
Probing the Effects of Pyrimidine Functional Group
Switches on Acyclic Fleximer Analogues for
Antiviral Activity
Mary K. Yates 1, Payel Chatterjee 2, Mike Flint 2 , Yafet Arefeayne 1, Damjan Makuc 3,
Janez Plavec 3 , Christina F. Spiropoulou 2 and Katherine L. Seley-Radtke 1,
*
1
Department of Chemistry and Biochemistry, University of Maryland, Baltimore County,
Baltimore, MD 21250, USA
Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA
Slovenian NMR Center, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
Correspondence: kseley@umbc.edu; Tel.: +410-45508684
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Luigi A. Agrofoglio
Received: 2 August 2019; Accepted: 31 August 2019; Published: 2 September 2019
Abstract: Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have
been used for decades as potent antiviral therapeutics. However, one of the major limitations of
nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside
analogues known as “fleximers” have garnered attention over the years due to their ability to survey
different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral
resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous
viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV),
and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these
interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the
effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity,
and conformation. The results of those studies are presented herein.
Keywords: nucleoside; SAR; filovirus; flavivirus; fleximers
1. Introduction
Nucleoside analogues have long served as the cornerstone for antiviral therapeutics due to
their ability to inhibit viral DNA or RNA replication [1,2]. By altering different components of the
nucleoside, such as the nucleobase, sugar moiety, and phosphate group, medicinal chemists can
develop novel analogues for their use in various therapeutics [1,2]. Over the past decade, research in
the Seley-Radtke lab has focused on the development of various types of flexible nucleoside analogues,
called “fleximers”, that have demonstrated the ability to overcome point mutations within the binding
site of biologically significant enzymes (Figure 1) [3–20].
Fleximers feature a purine ring that is split into its imidazole and pyrimidine components, but
remain connected by a single carbon–carbon bond between the C5 of the imidazole and the C6 of the
pyrimidine in distal fleximers, and between the C4 of the imidazole and the C5 of the pyrimidine in
proximal fleximers [3,4,7] This strategic design endows fleximers with an inherent flexibility that (1)
retains hydrogen bond motifs that are necessary for enzyme recognition, (2) adapts to flexible binding
site environments, and (3) allows access to different amino acids in enzyme binding sites that were
previously unattainable by the more rigid parent nucleoside [6,8,21].
Molecules 2019, 24, 3184; doi:10.3390/molecules24173184
www.mdpi.com/journal/molecules

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Figure 1. Structure of guanosine compared to the corresponding proximal and distal fleximer analogues.
Most of the recent studies within the Seley-Radtke lab have focused on the development of
fleximer analogues for use in antiviral and anticancer therapeutics [11 17 20]. In particular, fleximer
,
,
analogues based on the structure of the Food and Drug Administration (FDA)-approved drug Acyclovir
have demonstrated great promise, with compound 1 demonstrating low micromolar activity against
coronaviruses [17], filoviruses [20], and most recently, flaviviruses (Figure 2, unpublished results).
Figure 2. Parent fleximer analogues demonstrating broad spectrum antiviral activity [3–20].
In parallel, a secondary series featuring a methoxy at the 2-position on the pyrimidine was
developed in order to better analyze the effect of hydrogen bonding motifs on antiviral activity. This
series was also tested against the aforementioned viruses; however, these analogues were not more
active than compound 1. Furthermore, the addition of an acetate protecting group (1-Ac) led to a
decrease in antiviral activity, but also a large decrease in cytotoxicity (unpublished results). In order to
explore the effects of different hydrogen bond donor and acceptor groups on the fleximer pyrimidine
ring, as well as the effect of various acetate protecting groups on antiviral activity, an SAR study was
pursued. Herein, we report the synthesis and antiviral evaluation of series 1–5 (Figure 3).

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Figure 3. Target analogues for the SAR study.
2. Results
Chemistry
Synthesis of these analogues began with modifying the acyclic sugar moiety
different acyl protecting groups (Scheme 1). The synthesis of and
our group [17 20], and all intermediates were obtained in a similar fashion, with the addition of the
6
by the addition of
6
7 was previously published by
,
appropriate anhydride followed by triethylamine and diisopropylamine (DIPEA) in anh. CH2Cl2 to
give 8–10 as clear to pale yellow oils in high yields (78–99%).
Scheme 1. Reagents and conditions: a. Acetic anhydride (for
7
), propionic anhydride (for
8),
isobutyric anhydride (for
CH2Cl2, rt, 3h.
9), or trimethylacetic anhydride (for 10), triethylamine (TEA), DIPEA, anh.
As the synthesis of 1, 1-Ac, 2, and 2-Ac have already been reported [17,20], the remaining analogues
in these series were pursued first. Using the appropriate stannyl pyrimidine under Stile conditions, 1-
EtAc, 1-iPrAc, 1-tBuAc, 2-EtAc, 2-iPrAc, and 2-tBuAc were all realized in good yields (Scheme 2).

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Scheme 2. Reagents and conditions: a. 4-methoxy-5-(tributylstannyl)pyrimidin-2-amine (for
series 1) or 2,4-dimethoxy-5-(tributylstannyl)pyrimidine (for series 2), Pd(PPh3)4, CuI, CsF, anh.
dimethylformamide (DMF), 65 ◦C, 3 h.
After the successful completion of series
1 and 2, attention turned toward the synthesis
of the 4-methoxypyrimidine series (Scheme 3). Synthesis began with commercially available
3
4-chloro-5-iodopyrimidine (11), where the addition of sodium methoxide (30% by weight in CH3OH)
gave 5-iodo-4-methoxypyrimidine 12 in good yields. Following purification, the stannyl pyrimidine 13
was realized with bistributyltin, the tris(dibenzylideneacetone)dipalladium catalyst, and mild heating.
Scheme 3. Reagents and conditions: a. NaOMe (30% by weight in CH3OH), CH3OH, rt, 6h; b. (SnBu3)2,
Pd2dba3
•
CHCl3, anhydrous (anh.) DMF, 65 ◦C, 2.5 h; c.
6 (for 3), 7 (for 3-Ac), 8 (for 3-EtAc), 9 (for
3-iPrAc), or 10 (for 3-tBuAc), Pd(PPh3)4, CuI, CsF, anh. DMF, 65 ◦C, 3 h.
Coupling of the various sugars with the stannyl pyrimidine 12 was then accomplished using Stille
palladium catalyzed cross-coupling methods to give analogues 3, 3-Ac, 3-EtAc, 3-iPrAc, and 3-tBuAc
in good yields (30–45%).
Once the 4-methoxypyrimidine series
3
was successfully synthesized, the 2-aminopyrimidine
started with commercially
series was pursued. Similar to the previous series, synthesis of series
4
5
available 2-amino-5-iodopyrimidine (14) and bis(tributyltin) to give the stannyl pyrimidine 15 in good
yields (86%, Scheme 4). Following purification of the stannyl pyrimidine, Stille palladium catalyzed
cross-coupling methods were employed to realize compounds
compared to the previous series (>50% for all analogues compared to an average of 15% for series 1).
Finally, the 4-aminopyrimidine series was originally pursued using similar methods, starting
with commercially available 4-aminopyrimidine (16, Scheme 5).
4
through 4-tBuAc in high yields
5

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Scheme 4. Reagents and conditions: a. (SnBu3)2, Pd2dba3
•
CHCl3, anh. DMF, 65 ◦C, 2.5 h; b.
6 (for 4), 7
(for 4-Ac), (for 4-EtAc),
8
9
(for 4-iPrAc), or 10 (for 4-tBuAc), Pd(PPh3)4, CuI, CsF, anh. DMF, 65 ◦C, 3 h.
◦
Scheme 5. Reagents and conditions: a. N-iodosuccinimide (NIS), AcOH, 85 C, 3 h; b. (SnBu3)2,
Pd2dba3 6 (for 5), 7 (for 5-Ac), or 8 (for 5-EtAc), CsF, CuI, PPh3,
CHCl3, DMF, 65 ◦C, 2.5 h; c.
•
Pd2dba3•CHCl3, DMF, 65 ◦C, 3–5 h.
Using N-iodosuccinimide in glacial acetic acid, 4-amino-5-iodopyrimidine 17 was realized,
followed by the addition of bis(tributyltin) to give the stannyl coupling partner 18. Subsequent
coupling with
6
,
7
, and
8
gave very low yields (<10%) and gave rise to purifications issues, as it was
to 5-EtAc were also fairly
difficult to isolate the pure product without residual tin impurities. Products
5
soluble in water; thus, using a 1-M potassium fluoride (KF) wash to convert the soluble tin impurity to
the insoluble SnBu3F was unsuccessful. Furthermore, the addition of the acyl protecting group had no
effect on tin impurity removal, thus the deprotection of 5-Ac to give
5 with methanolic ammonia was
ineffective as well. As such, a protecting group for the 4-aminopyrimidine was envisioned in attempts
to make purification more facile and increase the overall yields.
First, protection of the 4-amino group was tried using two equivalents of di-tert-butyl dicarbonate
to give a di-boc protected amine, which could easily be deprotected after coupling to the sugar using
mild acidic conditions (Scheme 6).
Protection of the 5-iodo intermediate 17 using di-tert-butyl dicarbonate was successful in producing
the di-boc compound 19; however, subsequent attempts at installing the stannyl component (20) were
unsuccessful. Furthermore, attempts at installing the boronic ester (21) for use in more mild Suzuki
coupling conditions were also unsuccessful. Final attempts at employing the di-boc protecting group
tried protecting the stannyl pyrimidine 18; however, this method also proved ineffective. As such,
attention turned toward using an in situ protection of the amine group followed by Negishi conditions
for the 5-iPrAc and 5-tBuAc compounds, as these conditions had previously been successful for other
fleximers developed in our lab (Scheme 7) [22].

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Scheme 6. Reagents and conditions: a. Boc2O, dimethylaminopyridine (DMAP), dichloromethane
(DCM), rt, 2h; b. (SnBu3)2, Pd2dba3
CHCl3, DMF, 65 ◦C, 3 h; c. bis(pinacolato)diboron, KOAc,
PdCl2(dppf) CHCl3, DMF, 120 ◦C.
•
Scheme 7. Reagents and conditions: a. EtMgBr, trimethylsilyl chloride (TMSCl), tetrahydrofuran
(THF),
78 ◦C; b. EtMgBr, ZnCl2, THF, 78◦C – rt, 2 h; c. (for 5-iPrAc), or 10 (for 5-tBuAc), Pd(PPh3)4,
CuI, THF, rt, 18 h.
−
−
9
Compound 17 was first protected in situ with chlorotrimethylsilane to give intermediate 23
which was subsequently complexed with ZnCl2 to give intermediate 24. Dropwise addition of the
zinc complex to either
or 10 gave 5-iPrAc and 5-tBuAc, respectively. While the coupling itself went
,
9
well, later purification of these analogues proved difficult. However, the addition of 10% NH4OH to
CH3OH in the chromatography gradient greatly decreased streaking and allowed for purification of
these products (yields ~48%).
3. Discussion
As the different series were completed, they were sent for testing to collaborators at the Centers for
Disease Control (CDC) for testing against EBOV. Briefly, a recombinant reporter virus, EBOV-Makona
expressing ZsGreen (rEBOV/ZsG), was used to infect compound-treated Huh7 cells at a multiplicity of
infection (MOI) of 0.3. Three days later, ZsGreen fluorescence was measured, and EC50 values were
determined. Concurrently, the compound effects on cell viability were determined by measuring the
cellular ATP content of compound-treated, mock-infected cells [20,23,24]. The results of this study are
summarized in Table 1.

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Table 1. Antiviral activity of series 1–5 against Ebola virus (EBOV) in Huh7 cells.
1
2
3
4
5
R Group
aEC50 (µM)
EC50 (µM)
EC50 (µM)
EC50 (µM)
EC50 (µM)
H
Ac
EtAc
iPrAc
tBuAc
1.49 ± 0.98
23.44 ± 20.28
16.34 ± 11.46
>100
78.88 ± 17.14
35.36 ± 20.37
51.87 ± 19.58
38.29 ± 28.15
51.40 ± 10.01
>100
>100
>100
>100
>100
52.89 ± 28.25
62.18 ± 18.99
>100
7.21 ± 1.85
>100
31.96 ± 9.12
53.99 ± 23.74
34.42 ± 20.01
>100
81.34 ±10.15
72.29 ± 18.74
aEC50: effective concentration showing 50% inhibition of virus-expressed ZsGreen fluorescence.; all compounds
were nontoxic up to 100 µM in uninfected cells.
None of the analogues tested demonstrated any cytotoxicity up to 100
µM, and while compound
1
remained the most active against EBOV, interesting trends emerged regarding the acyl protecting
groups as well as the moieties on the pyridmine ring.
When the parent compounds of each series were tested, it was found that changing the hydrogen
bond-donating amino group at the 2-poistion (compound
group (compound ) decreased activity (1.49 M compared to 23.44
when there is a hydrogen atom at the 2-position (compound ), a decrease in activity compared to
compound was also observed (1.49 M compared to 78.88 M, p = 0.0061). Compounds and
which feature only an amino group in the 2-position and 4-position, respectively, did not display any
activity against EBOV. Interestingly, activity could be recovered for series and 5, as well as improved
for series 3, by altering the acyl protecting group. For instance, the addition of the acetate protecting
group with 3-Ac greatly increased activity compared to the parent analogue (35.36 M compared
to 78.88 M, p = 0.0162). However, the addition of the acyl protecting group seemed to affect each
series differently, as compound 1-Ac was not active against EBOV. In contrast, the parent compound
was very active, but 4-Ac was active against EBOV (EC50 = 52.89 M), whereas the parent compound
was not active. For series 2, the activity of 2-Ac appeared to increase, but did not reach statistical
significance (p = 0.7922).
For series and , there was a loss in activity with the ethyl acyl (EtAc) analogues compared to
1) to a hydrogen bond-accepting methoxy
2
µ
µM, p = 0.0082). Furthermore,
3
1
µ
µ
4
5,
4
3
µ
µ
1
µ
4
2
4
analogues with only an acetate group, such as 2-Ac compared to 2-EtAc as well as 4-Ac compared to
4-EtAc. However, activity returned with the isopropyl acyl (iPrAc) protected compounds. Finally,
the tert-butyl acyl (tBuAc) protected analogues were variable in activity across the series, though
for the most part, these analogues were not significantly different in activity from the acetate (Ac).
For example, 3-Ac demonstrated activity to 35.36
(p = 0.1709), and 4-Ac demonstrated activity to 52.89
The adenosine series
results, along with the potent activity observed for compound
µ
M, 3-tBuAc demonstrated activity to 51.40
µM
µ
M compared to 4-tBuAc at 34.42
µM
(p = 0.1949).
5
were not active except for the tBuAc, which demonstrated weak activity. These
1
, which also bears a 4-methoxy group,
suggests that the 4-methoxy group is an important determinant for antiviral activity.
The difference in activity between the Ac, EtAc, and iPrAc is an interesting trend, as the EtAc
analogues were consistently the least active analogues. In order to analyze if this trend was due to
a change in lipophilicity or due to branching, the logP values were determined for each analogue
(Table 2).
LogP values are a common measure of lipophilicity, and the greater the value, the more lipophilic
the compound. Not surprisingly, all the parent analogues (R group = H) were the least lipophilic of the
series, with the amino series 4 and 5 demonstrating the lowest lipophillicity. On average, the difference
between the parent analogues and the acetate analogues (R = Ac) was not substantial; however,
the ethyl acyl analogues (R = EtAc) were substantially more lipophilic than the acetate analogues (for
example,
−
0.14 compared to 0.52 for series 5). Not surprisingly, the increased branching also led to an
increase in lipophilicity, as the tBuAc analogues were the most lipophilic of their series.

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Table 2. Lipophilicity (logP) values of series 1–5.
Series (logP)
R Group
1
2
3
4
5
H
Ac
EtAc
iPrAc
tBuAc
0.02
0.25
0.9
1.47
2.18
0.7
0.31
0.54
1.2
1.76
2.47
−0.57
−0.34
0.31
0.88
1.59
−0.36
−0.14
0.52
1.09
1.79
0.93
1.58
2.15
2.85
While increased lipophillicity is typically associated with a better drug candidate, there is a
limit where increased lipophillicity affects potency. For instance, the isopropyl acyl analogue 2-iPrAc
demonstrated an EC50 of 31.96
of 81.34 10.15
an EC50 of 38.29
a logP of 2.47. This suggests that compounds with logP values over 2.8 are too lipophilic, and thus
are less active against EBOV. Interestingly, compound 4-iPrAc is less active than the more branched
4-tBuAc and is less lipophilic (logP = 0.88 vs 1.59), suggesting that compound with logP values greater
than 1 may be more active. All together, these results suggest that lipophillicity and not branching
affects antiviral activity.
±
9.12 µ
M against EBOV and a logP of 2.15, whereas 2-tBuAc has an EC50
±
µ
M and a logP of 2.85. Furthermore, the isopropyl acyl analogue 3-iPrAc demonstrates
±
28.15
µ
M and a logP of 1.76, whereas 3-tBuAc has an EC50 of 51.40 ± 10.01 µM and
Previous studies with the Seley-Radtke lab found that in solution, ribose fleximers preferred the
typical anti-conformation found with nucleosides [21]. As such, studies were pursued in order to
1
determine if the acyclic fleximer analogues also preferred anti-conformation in solution. H-NMR
spectra were recorded on 300-MHz NMR spectrometer at 3-mM concentration in 1% dimethylsulfoxide
(DMSO)-d6/D2O.
Using well-resolved NMR signals of H6, H8, and H11 aromatic protons (Figure 4A), an assessment
of the conformational preferences along the C11–C10–C5–C6 torsion angle was possible through the
use of 1D Nuclear Overhauser Effect Spectroscopy (NOESY) experiments.
A
B
C
1
Figure 4.
(
A
) H-NMR spectrum of
1
in 1% DMSO/D2O at 25 ◦C; 1D NOESY Nuclear Magnetic
in 1% DMSO/D2O at 25 ◦C; selective inversion of (
) H11 and ( ) H6
Resonance (NMR) spectra of
proton; mixing time 1.5 s.
1
B
C

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Selective inversion of the H11 proton resulted in strong NOE enhancement of 4.3% at H1’, whereas
other NOE enhancements were relatively weak (Figure 4B). The lack of strong NOE enhancements
between H11 and H6 suggests that anti-orientation along the C11–C10–C5–C6 torsion angle is the
predominant conformation of compound
1 in solution. Additionally, inversion of the H6 proton
showed no NOE enhancements (Figure 4C), which confirms that H6 and H11 are spatially apart,
overall supporting anti-conformation.
These studies also found that compounds 1-Ac, 2, and 2-Ac also favored anti-conformation along
the C11–C10–C5–C6 torsion angle. Interestingly, selective inversion of the H11 proton of compound
5
resulted in relatively strong NOE enhancement of 4.6% at H1’, as well as moderate NOE enhancement of
2.8% at H6 (Figure 5B). These results suggest that syn orientation along the C11–C10–C5–C6 torsion angle
is predominant. This conformation was also determined to be prominent in the acetate prodrug 5-Ac.
A
B
Figure 5.
1% DMSO/D2O at 25 ◦C; selective inversion of H11; mixing time 1.5 s.
(A 5 B) 1D NOESY NMR spectra of 5 in
) 1H-NMR spectrum of in 1% DMSO/D2O at 25 ◦C. (
These results are particularly interesting due to the lack of antiviral activity seen with series
5
against EBOV. The difference in the conformations between compounds 1, 2, and may offer a reason
5
for the drastic differences in antiviral activity observed. The differences in orientation are particularly
highlighted in Figure 6.
A
B
Figure 6. (A) Anti-conformation of compound 1 compared to (B) syn conformation of compound 5.
While compounds 1, 1-Ac, 2, 2-Ac, 5, and 5-Ac have been analyzed in these studies, it will be
interesting to see if the 4-methoxy series
compared to series or 5. Due to the antiviral activity found with series
these analogues also are predominately found in the anti-conformation.
3
or the 2-amino series
4
also demonstrate different orientations
1
3
and , it is expected that
4

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4. Materials and Methods
4.1. Chemistry General Information
All reactions were performed using oven-dried glassware under a nitrogen atmosphere with
magnetic stirring. Reagents were purchased from Sigma-Aldrich (Rockville, MD, USA), Alfa Aesar
(Haverhill, MA, USA), and Combiblocks (San Diego, CA, USA). Solvents were either purchased
as anhydrous or were dried using the MBRAUN solvent purification system (MB-SPS) (MBRAUN,
Stratham, NH, USA). Reactions were monitored by thin layer chromatography (TLC) using EMD silica
gel 60 F254 coated glass-backed TLC plates (Milliporesigma, Rockville, MD, USA) and visualized with
a UV lamp and/or KMnO4 stain (Fisher Scientific, Pittsburgh, PA, USA). Column chromatography
was performed on a CombiFlash®Rf automated chromatography using REdiSep Rf silica (Teledyne
Isco, Lincoln, NE, USA). High-Pressure Liquid Chromatography (HPLC) purification was performed
on an LC-8A Shimadzu preparation liquid chromatograph (Shimadzu, Columbia, MD, USA) using a
Zorbax SB C18 column 9.4
1 L lyophilizer. H-NMR spectra was recorded on a JEOL Eclipse ECX (400 MHz) (JEOL USA, Inc.,
Peabody, MA, USA) or a Bruker Avance III HD (500 MHz) spectrometer (Bruker BioSpin Corporation,
×
250 mm, 5 µm. Samples were lyophilized utilizing a Labconco Freezone
1
Billerica, MA, USA) Chemical shifts are reported in parts per million (δ ppm) from tetramethylsilane
with the solvent resonance as an internal standard. Data for the 1H-NMR are reported as follows:
br = broad, s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet.
13C-NMR were recorded on a JOEL Eclipse ECX (126 MHz) or a Bruker Advance III HD (101 MHz)
spectrometer with complete proton decoupling. Data for 13C-NMR are reported in terms of chemical
shifts (δ ppm) with the solvent resonance as the internal standard. Mass spectrometry was performed
on a Bruker AmaZon X quadrupole ion trap mass spectrometer using electrospray ionization (ESI)
(Bruker BioSpin Corporation, Billerica, MA, USA). The purity of all compounds tested in biological
assays were verified by elemental analyses performed by Atlantic Microlabs (Atlantic Microlabs,
Norcross, GA, USA). LogP values were calculated using the ChemDraw program by PerkinElmer
Informatics (PerkinElmer Informatics, Cambridge, MA, USA).
4.2. Synthesis
General procedure for the acyl-protected sugars 8–10. To a solution of 6 (1.5 g, 5.96 mmol) in anh.
CH2Cl2 (100 mL) under a nitrogen balloon was added the corresponding anhydride (2 equivalents),
triethylamine (2.50 mL, 17.85 mmol), and DMAP (0.18 g, 1.49 mmol) with stirring at room temperature.
The reaction stirred for 3 hours at room temperature; then, the solvent was removed in vacuo.
Purification by flash column chromatography on silica gave the products in good yields.
Characterization of 2-((4-iodo-1H-imidazol-1-yl)methoxy)ethyl propionate (8). The anhydride used was
propionic anhydride (1.52 mL, 11.90 mmol). The crude mixture was purified by flash column
chromatography on silica (0–100% EtOAc in hexanes) to give the product as a clear oil (1.78 g, 99%);
Rf = 0.42 (1:1 EtOAc: Hex); 1H-NMR (500 MHz, CDCl3)
δ
7.41 (s, 1H), 7.05 (s, 1H), 5.19 (s, 2H), 4.01–4.03
(t, J = 4.7 Hz, 2H), 3.47–3.49 (t, J = 4.6 Hz, 2H), 2.14–2.19 (m, 2H), 0.93–0.96 (t, J = 7.6 Hz, 3H); 13C-NMR
(126 MHz, CDCl3)
δ 174.43, 139.03, 124.30, 83.12, 76.41, 66.72, 62.80, 27.58, 9.13; MS (ESI+) m/z calcd for
C9H13IN2O3 [M + H]+ 325.00 found: 325.01.
Characterization of 2-((4-iodo-1H-imidazol-1-yl)methoxy)ethyl isobutyrate (9). The anhydride used was
isobutyric anhydride (1.98 mL, 11.90 mmol). The crude mixture was purified by flash column
chromatography on silica (0–100% EtOAc in hexanes) to give the product as a clear oil (2.0 g, 99%);
Rf = 0.53 (1:1 EtOAc: Hex;1H-NMR (500 MHz, CDCl3)
δ
7.26 (s, 1H), 6.89 (s, 1H), 5.01 (s, 2H), 3.77–3.78
(m, 2H), 3.27–3.28 (m, 2H), 2.14–2.15 (m, 1H), 0.72–0.78 (d, J = 4.4 Hz, 6H); 13C-NMR (101 MHz, CDCl3)
176.45, 139.17, 124.53, 82.33, 76.12, 66.70, 62.46, 33.47, 18.79. MS (ESI+) m/z calcd for C10H15IN2O3 [M
+ Na]+ 361.15 found: 361.03.
δ

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Characterization of 2-((4-iodo-1H-imidazol-1-yl)methoxy)ethyl pivalate (10). The anhydride used was
trimethylacetic anhydride (2.40 mL, 11.90 mmol). The crude mixture was purified by flash column
chromatography on silica (0–100% EtOAc in hexanes) to give the product as a clear oil (1.55 g, 78%);
Rf = 0.53 (1:1 EtOAc: Hex; 1H NMR (400 MHz, CDCl3)
δ 7.44 (s, 1H), 7.07 (s, 1H), 5.22 (s, 2H), 4.06–4.13
(t, J = 4.1 Hz, 2H), 3.48–3.52 (t, J = 4.1 Hz, 2H), 1.07–1.15(s, 9H); 13C NMR (125 MHz, CDCl3)
δ 177.42,
139.53, 130.08, 124.92, 76.02, 66.76, 62.70, 38.35, 28.88. MS (ESI+) m/z calcd for C11H17IN2O3 [M + Na]+
375.17 found: 375.05.
General procedure for Stille cross coupling. The sugar (1 equivalent) and stannyl reagent (1.2 equivalents)
were dried in a three-neck flash in vacuo for two hours, and then were suspended in anh. DMF
(100 mL) under direct nitrogen bubbling. CsF (2 equivalents) and CuI (0.4 equivalents) were added,
and the reaction was stirred for 10 min. Tetrakis (0.2 equivalents) was then added, and the reaction
was heated to 65 ◦C and stirred with direct nitrogen bubbling for 2.5 h. After completion, the reaction
was cooled, and the solvent was removed in vacuo. The crude mixture was then purified three to four
times by flash column chromatography on silica to give the pure products.
2-((4-(2-Amino-4-methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl propionate (1-EtAc). The sugar
8
(0.75 g, 2.31 mmol) and 4-methoxy-5-(tributylstannyl)pyrimidin-2-amine (1.15 g, 2.77 mmol) were used.
The product was purified three times by flash column chromatography on silica (0–25% CH3OH in
CH2Cl2) to give the product as a white solid (0.041 g, 9%); 1H NMR (500 MHz, CD3OD)
δ
8.83 (s, 1H),
7.62 (s, 1H), 7.40 (s, 1H), 5.32 (s, 2H), 4.20–4.21 (t, J = 4.7 Hz, 2H), 4.01 (s, 3H), 3.62–3.64 (t, J = 4.7 Hz,
2H), 2.30–2.35 (m, 2H), 1.09–1.12 (t, J = 7.6 Hz, 3H); 13C NMR (126 MHz, CDCl3)
174.30, 166.23, 161.08,
δ
154.83, 136.85, 135.41, 117.00, 105.92, 76.46, 66.58, 62.70, 53.66, 27.35, 9.00; Elemental analysis calcd for
C14H19N5O4 + 0.25 CH4OH + 0.15 H2O: C, 51.55; H, 6.16; N, 21.09; Found: C, 51.58; H, 6.12, N, 21.06.
2-((4-(2-Amino-4-methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl isobutyrate (1-iPrAc). The sugar
9
(0.80 g, 2.37 mmol) and 4-methoxy-5-(tributylstannyl)pyrimidin-2-amine (1.18 g, 2.84 mmol) were used.
The product was purified three times by flash column chromatography on silica (0–25% CH3OH in
CH2Cl2) to give the product as a pale yellow oil (0.09 g, 12%); 1H NMR (500 MHz, CDCl3)
δ
8.80 (s, 1H),
7.60 (s, 1H), 7.39 (s, 1H), 5.31 (s, 2H), 4.17–4.20 (t, J = 5.6 Hz, 2H), 3.98 (s, 3H), 3.59–3.61 (t, J = 5.3 Hz,
2H), 2.48–2.55 (m, 1H), 1.10–1.12 (d, J = 9.2 Hz, 6H); 13C NMR (126 MHz, CDCl3)
176.86, 166.42,
δ
161.37, 155.24, 137.35, 135.49, 117.03, 106.04, 76.41, 66.58, 62.66, 53.58, 33.78, 18.96; Elemental analysis
calcd for C15H21N5O4 + 0.65 H2O: C, 51.91; H, 6.48; N, 20.18; Found: C, 51.60; H, 6.09; N, 20.18.
2-((4-(2-Amino-4-methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl pivalate (1-tBuAc). The sugar 10
(0.75 g, 2.13 mmol) and 4-methoxy-5-(tributylstannyl)pyrimidin-2-amine (1.06 g, 2.56 mmol) were used.
The product was purified three times by flash column chromatography on silica (0–20% CH3OH in
CH2Cl2) to give the product as a clear oil (0.08 g, 11%); 1H NMR (500 MHz, CDCl3)
δ
8.78 (s, 1H), 7.67
(s, 1H), 7.41 (s, 1H), 5.36 (s, 2H), 4.23–4.25 (t, J = 4.0 Hz, 2H), 4.11 (s, 3H), 3.65–3.67 (t, J = 4.0 Hz, 2H),
1.19–1.27 (s, 9H); 13C NMR (126 MHz, CDCl3)
176.48, 166.35, 153.71, 137.37, 134.27, 117.33, 104.72,
δ
76.09, 66.45, 62.77, 52.73, 38.31, 26.05; Elemental analysis calcd for C16H23N5O4 + 0.1 CH4OH + 0.95
H2O: C, 52.31; H, 6.90; N, 18.94; Found: C, 52.41; H, 6.74; N, 18.78.
2-((4-(2,4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl propionate (2-EtAc). The sugar
8
(0.75 g, 2.31 mmol) and 2,4-dimethoxy-5-(tributylstannyl)pyrimidine (1.19 g, 2.78 mmol) were used.
The product was purified three times by flash column chromatography on silica (50–100% EtOAc in
hexanes) to give the product as a pale yellow oil (0.25 g, 36%); 1H NMR (500 MHz, CDCl3)
δ
8.82 (s,
1H), 7.91 (s, 1H), 7.65 (s, 1H), 5.47 (s, 2H), 4.19–4.21 (t, J = 3.5 Hz, 2H), 4.14 (s, 3H), 4.02 (s, 3H), 3.71–3.72
(t, J = 3.6 Hz, 2H), 2.28–2.33 (m, 2H), 1.05–1.08 (t, J = 7.6 Hz, 3H); 13C NMR (126 MHz, CD3OD)
174.44, 166.98, 163.58, 154.80, 138.21, 132.99, 118.74, 108.50, 76.34, 66.67, 62.73, 54.08, 53.49, 26.74, 8.01;
δ

Molecules 2019, 24, 3184
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Elemental analysis calcd for C15H20N4O5 + 0.05 H2O + 0.2 CH2Cl2: C, 51.54; H, 5.83; N, 15.82; Found:
C, 51.67; H, 5.64; N, 15.61.
2-((4-(2,4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl isobutyrate (2-iPrAc). The sugar
9
(0.25 g, 0.74 mmol) and 2,4-dimethoxy-5-(tributylstannyl)pyrimidine (0.38 g, 0.89 mmol) were used.
The product was purified twice times by flash column chromatography on silica (25–100% EtOAc in
hexanes) to give the product as a light orange oil (0.06 g, 24%); 1H NMR (400 MHz, CDCl3)
δ
8.96 (s,
1H), 7.61 (s, 1H), 7.43 (s, 1H), 5.32 (s, 2H), 4.14–4.19 (t, J = 3.4 Hz, 2H), 4.03 (s, 3H), 3.95 (s, 3H), 3.55–3.57
(t, J = 3.4 Hz, 2H), 2.45–2.47 (m, 1H), 1.05–1.09 (d, J = 6.9 Hz, 6H); 13C NMR (101 MHz, CDCl3)
δ
176.95, 166.89, 163.73, 155.65, 137.12, 134.63, 117.99, 109.21, 76.91, 66.71, 62.61, 54.86, 54.12, 33.84, 18.95;
Elemental analysis calcd for C16H22N4O5: C, 54.85; H, 6.33; N, 15.99; Found: C, 54.56; H, 6.50; N, 15.81.
2-((4-(2,4-Dimethoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl pivalate (2-tBuAc). The sugar 10
(0.30 g, 0.85 mmol) and 2,4-dimethoxy-5-(tributylstannyl)pyrimidine (0.44 g, 1.02 mmol) were used.
The product was purified twice by flash column chromatography on silica (50–100% EtOAc in hexanes)
to give the product as a light orange oil (0.16 g, 51%); 1H NMR (400 MHz, CDCl3)
δ
9.05 (s, 1H), 7.95 (s,
1H), 7.53 (s, 1H), 5.42 (s, 2H), 4.22–4.23 (t, J = 4.7 Hz, 2H), 4.12 (s, 3H), 4.04 (s, 3H), 3.65–3.68 (t, J = 4.6 Hz,
2H), 1.18–1.20 (s, 9H); 13C NMR (101 MHz, CDCl3)
178.40, 167.03, 163.64, 154.57, 137.83, 133.21, 118.62,
δ
108.77, 76.15, 66.55, 62.74, 54.04, 53.44, 38.30, 26.06; Elemental analysis calcd for C17H24N4O5 + 0.35
CH3OH + 0.05 H2O: C, 55.35; H, 6.83; N, 14.88; Found: C, 55.36; H, 6.75; N, 14.83.
5-Iodo-4-methoxypyrimidine (12). To a solution of 4-chloro-5-iodopyrimidine (0.5 g, 2.08 mmol) in anh.
CH3OH (20 mL) under nitrogen was added NaOMe (30% by weight in CH3OH, 0.53 mL, 2.88 mmol)
at room temperature. Then, the reaction was stirred at room temperature under nitrogen for 6 hours.
Upon completion, the solvent was removed in vacuo to give a yellow solid. The crude mixture was
purified by flash column chromatography on silica (0–50% EtOAc in hexanes) to give the product as a
white solid (0.45 g, 92%); Rf = 0.86 (1:4 EtOAc: Hex); 1H NMR (500 MHz, CDCl3)
δ
8.79 (s, 1H), 8.72 (s,
1H), 4.10 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ 167.1, 163.9, 157.4, 80.1, 54.8; MS (ESI+) m/z calcd for
C5H5IN2O [M + H]+ 236.95, found: 236.56.
4-Methoxy-5-(tributylstannyl)pyrimidine (13). To a solution of 12 (0.89 g, 3.79 mmol) in anh. DMF (50 mL)
under direct nitrogen bubble was added bis(tributyltin) (2.29 mL, 4.55 mmol) and Pd2dba3 CHCl3
•
(0.39 g, 0.38 mmol). Then, the reaction was stirred at 65 ◦C for 2.5 h. After completion, the reaction was
cooled to room temperature, and the solvent was removed in vacuo to give a black oil. The crude mixture
was resuspended in EtOAc (100 mL) and filtered over a celite pad. The filtrated was concentrated in
vacuo to give a brown oil. Then, the crude product was purified by flash column chromatography on
silica (0–70% EtOAc in hexanes) to give the pure product as a pale yellow oil (1.40 g, 93%); Rf = 0.64
(1:1 EtOAc: Hex); 1H NMR (500 MHz, CDCl3)
1.03–1.06 (m, 6H), 0.81–0.89 (m, 6H), 0.58–0.62 (m, 9H); 13C NMR (126 MHz, CDCl3)
128.7, 53.1, 28.9, 27.3, 13.6, 9.6; MS (ESI+) m/z calcd for C17H32N2OSn [M + H]+ 401.15, found: 401.18.
δ
8.46 (s, 1H), 8.11 (s, 1H), 3.65 (s, 3H), 1.20–1.31 (m, 6H),
188.5, 163.3, 158.5,
δ
2-((4-(4-Methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethan-1-ol (3). The sugar 6 (0.35 g, 1.31 mmol)
and stannyl pyrimidine 13 (0.63 g, 1.57 mmol) were used. The product was purified three times by
flash column chromatography on silica (0–25% CH3OH in CH2Cl2) to give the product as a shiny white
solid (0.135 g, 41%); 1H NMR (500 MHz, CD3OD)
δ
9.05 (s, 1H), 8.62 (s, 1H), 7.97 (s, 1H), 7.83 (s, 1H),
5.51 (s, 2H), 4.16 (s, 3H), 3.67–3.69 (t, J = 5.0 Hz, 2H), 3.57–3.59 (t, J = 4.4 Hz, 2H); 13C NMR (126 MHz,
CD3OD) 164.99, 154.93, 151.84, 138.32, 132.41, 120.82, 115.24, 76.54, 70.09, 60.51, 53.41; Elemental
analysis calcd for C11H14N4O3: C, 52.79; H, 5.64; N, 22.39. Found: C, 52.62, H, 5.62, N, 22.17.
δ
2-((4-(4-Methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl acetate (3-Ac). The sugar
7 (0.35 g,
1.13 mmol) and stannyl pyrimidine 13 (0.54 g, 1.35 mmol) were used. The product was purified three

Molecules 2019, 24, 3184
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times by flash column chromatography on silica (0–20% CH3OH in CH2Cl2) to give the product as a
pale yellow-white solid (0.093 g, 30%); 1H NMR (500 MHz, CDCl3)
9.05 (s, 1H), 8.65 (s, 1H), 7.65 (s,
1H), 7.60 (s, 1H), 5.33 (s, 2H), 4.13–4.15 (t, J = 4.8 Hz, 2H), 4.06 (s, 3H), 3.58–3.60 (t, J = 4.6 Hz, 2H), 1.98
(s, 3H); 13C NMR (126 MHz, CDCl3)
170.89, 164.37, 155.43, 153.56, 137.72, 134.37, 121.32, 115.17, 76.55,
δ
δ
66.90, 62.88, 54.44, 21.43; Elemental analysis calcd for C13H16N4O4: C, 53.42; H, 5.52; N, 19.17. Found:
C, 53.14, H, 5.56, N, 18.88.
2-((4-(4-Methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl propionate (3-EtAc). The sugar 8 (0.35 g,
1.08 mmol) and stannyl pyrimidine 13 (0.52 g, 1.30 mmol) were used. The product was purified three
times by flash column chromatography on silica (0–10% CH3OH in CH2Cl2) to give the product as a pale
yellow oil (0.15 g, 45%); 1H NMR (400 MHz, CDCl3)
δ
9.19 (s, 1H), 8.60 (s, 1H), 7.63 (s, 1H), 7.57 (s, 1H), 5.31
(s, 2H), 4.13–4.14 (t, J = 5.0 Hz, 2H), 4.05 (s, 3H), 3.57–3.59 (t, J = 4.6 Hz, 2H), 2.22–2.26 (m, 2H), 1.01–1.05 (t,
J = 7.8 Hz, 3H); 13C NMR (126 MHz, CDCl3)
174.27, 164.74, 155.58, 153.57, 137.50, 134.17, 119.98, 115.11,
δ
76.62, 66.79, 62.66, 54.05, 27.38, 9.03; Elemental analysis calcd for C14H18N4O4 + 0.1% CH3OH + 0.5%
H2O: C, 53.17; H, 6.14; N, 17.59. Found: C, 52.99, H, 5.95, N, 17.40.
2-((4-(4-Methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl isobutyrate (3-iPrAc). The sugar
9 (0.35 g,
1.04 mmol) and stannyl pyrimidine 13 (0.50 g, 1.24 mmol) were used. The product was purified
three times by flash column chromatography on silica (first purification 40–100% EtOAc in hexanes,
subsequent purifications 0–5% CH3OH in CH2Cl2) to give the product as a pale yellow oil (0.12 g,
1
37%); H NMR (400 MHz, CDCl3)
δ 9.24 (s, 1H), 8.63 (s, 1H), 7.63 (s, 1H), 7.57 (s, 1H), 5.31 (s, 2H),
4.12–4.14 (t, J = 9.6 Hz, 2H), 4.04 (s, 3H), 3.57–3.58 (t, J = 5.0 Hz, 2H), 2.44–2.47 (m, 1H), 1.04–1.06 (d,
J = 6.9 Hz, 6H); 13C NMR (126 MHz, CDCl3)
δ
177.42, 165.12, 155.43, 153.66, 137.54, 134.37, 120.02,
115.26, 76.59, 66.34, 62.43, 54.04, 34.19, 19.99; Elemental analysis calcd for C15H20N4O4 + 0.3% H2O: C,
55.31; H, 6.37; N, 17.20. Found: C, 55.29, H, 6.33, N, 17.09.
2-((4-(4-Methoxypyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl pivalate (3-tBuAc). The sugar 10 (0.38 g,
1.06 mmol) and stannyl pyrimidine 13 (0.51 g, 1.28 mmol) were used. The product was purified
three times by flash column chromatography on silica (first purification 25–100% EtOAc in hexanes,
subsequent purifications 0–5–10% CH3OH in CH2Cl2) to give the product as a clear oil (0.042 g,
12%); 1H NMR (500 MHz, CD3OD)
4.19–4.21 (m, 2H), 4.17 (s, 3H), 3.71–3.73 (m, 2H), 1.15 (s, 9H); 13C NMR (126 MHz, CD3OD)
δ
9.07 (s, 1H), 8.65 (s, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 5.50 (s, 2H),
178.41,
δ
165.02, 155.01, 151.91, 138.36, 132.60, 120.77, 115.23, 76.21, 66.61, 62.73, 53.43, 38.30, 26.04; Elemental
analysis calcd for C16H22N4O4 + 0.35 CH3OH + 0.1 H2O: C, 56.53; H, 6.85; N, 16.13; Found: C, 56.52;
H, 6.83; N, 16.09.
5-(Tributylstannyl)pyrimidin-2-amine (15). To a solution of 2-amino-5-iodopyrimidine (0.50 g, 2.26 mmol)
in anh. DMF (100 mL) under direct nitrogen bubbling was added bis(tributyltin) (◦1.37 mL, 2.71 mmol)
and Pd2dba3 CHCl3 (0.23 g, 0.23 mmol). Then, the reaction was heated to 65 C with stirring for
•
3 h under direct nitrogen bubbling. After completion, the solvent was removed in vacuo to give a
black thick oil. The crude mixture was resuspended in EtOAc and filtered over a celite pad; then,
the filtrate was concentrated in vacuo to give a dark liquid. Purification was performed using flash
column chromatography on silica (0–100% EtOAc in hexanes) to give the pure product as a yellow
oil (0.75 g, 86%); Rf = 0.76 (1:1 EtOAc: Hex); 1H NMR (500 MHz, CDCl3)
1.45–1.51 (m, 6H), 1.39–1.44 (m, 6H), 1.21–1.29 (m, 6H), 0.83–1.06 (m, 9H); 13C NMR (126 MHz, CDCl3)
164.32, 163.16, 119.22, 28.84, 27.44, 13.57, 8.09; MS (ESI+) m/z calcd for C16H31N3Sn [M + H]+ 386.15,
δ 8.17 (s, 2H), 6.08 (br, 2H),
δ
found: 386.19.
2-((4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethan-1-ol (4). The sugar 6 (0.44 g, 1.63 mmol)
and stannyl pyrimidine 15 (0.75 g, 1.95 mmol) were used. The product was purified four times by
flash column chromatography on silica (0–30% CH3OH in CH2Cl2) to give the product as a pale pink

Molecules 2019, 24, 3184
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solid (0.19 g, 50%); 1H NMR (500 MHz, CD3OD)
δ
8.64 (s, 2H), 7.91 (s, 1H), 7.62 (s, 1H), 5.48 (s, 2H),
3.67–3.69 (t, J = 4.9 Hz, 2H), 3.56–3.58 (t, J = 4.6 Hz, 2H); 13C NMR (126 MHz, CD3OD)
δ 163.2, 154.8,
138.5, 137.0, 117.6, 114.4, 77.4, 70.9, 60.2; Elemental analysis calcd for C10H13N5O2: C, 51.06; H, 5.57; N,
29.77. Found: C, 51.01, H, 5.47, N, 29.83.
2-((4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl acetate (4-Ac). The sugar
7 (0.48 g,
1.54 mmol) and stannyl pyrimidine 15 (0.71 g, 1.86 mmol) were used. The product was purified three
times by flash column chromatography on silica (0–30% CH3OH in CH2Cl2) to give the product as a
white solid (0.10 g, 24%); 1H NMR (400 MHz, CD3OD)
δ
8.71 (s, 2H), 7.70 (s, 1H), 7.29 (s, 1H), 5.38
(s, 2H), 4.22–4.24 (t, J = 4.6 Hz, 2H), 3.66–3.68 (t, J = 4.6 Hz, 2H), 2.02 (s, 3H); 13C NMR (126 MHz,
CD3OD) 171.5, 162.6, 154.6, 138.9, 136.8,117.2, 114.3, 75.6, 67.0, 63.1, 19.2; Elemental analysis calcd for
C12H15N5O3 + 0.1 CH3OH + 0.05 CH2Cl2: C, 51.25; H, 5.49; N, 24.60. Found: C, 51.21, H, 5.47, N, 24.54.
δ
2-((4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl propionate (4-EtAc). The sugar
8 (0.30 g,
0.93 mmol) and stannyl pyrimidine 15 (0.43 g, 1.11 mmol) were used. The product was purified four
times by flash column chromatography on silica (0–20% CH3OH in CH2Cl2) to give the product as a
white solid (0.09 g, 26%); 1H NMR (400 MHz, CD3OD)
δ
8.60 (s, 2H), 7.87 (s, 1H), 7.57 (s, 1H), 5.42 (s,
2H), 4.15–4.17 (t, J = 4.6 Hz, 2H), 3.67–3.69 (t, J = 4.6 Hz, 2H), 2.24–2.28 (m, 2H), 1.02–1.06 (t, J = 7.8 Hz,
3H); 13C NMR (101 MHz, CD3OD)
174.58, 162.29, 154.81, 138.47, 136.81, 117.60, 114.45, 76.38, 66.75,
δ
62.79, 26.78, 8.03; Elemental analysis calcd for C13H17N5O3 + 0.05 CH3OH + 0.95 H2O: C, 50.56 H, 6.21;
N, 22.59. Found: C, 50.45, H, 6.07, N, 22.49.
2-((4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl isobutyrate (4-iPrAc). The sugar
9 (0.45 g,
1.33 mmol) and stannyl pyrimidine 15 (0.61 g, 1.60 mmol) were used. The product was purified four
times by flash column chromatography on silica (0–20% CH3OH in CH2Cl2) to give the product as a
pink solid (0.07 g, 17%); 1H NMR (400 MHz, CD3OD)
δ
8.59 (s, 2H), 7.87 (s, 1H), 7.56 (s, 1H), 5.42 (s,
2H), 4.15–4.17 (t, J = 4.6 Hz, 2H), 3.67–3.69 (t, J = 4.6 Hz, 2H), 2.46–2.50 (m, 1H), 1.06–1.08 (d, J = 6.8 Hz,
6H); 13C NMR (101 MHz, CD3OD)
177.42, 162.32, 155.05, 138.65, 136.98, 117.69, 114.61, 76.41, 66.72,
δ
62.80, 33.55, 18.45; Elemental analysis calcd for C14H19N5O3: C, 55.07 H, 6.27; N, 22.94. Found: C,
55.02, H, 6.13, N, 22.73.
2-((4-(2-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl pivalate (4-tBuAc). The sugar 10 (0.30 g,
0.85 mmol) and stannyl pyrimidine 15 (0.39 g, 1.02 mmol) were used. The product was purified twice
by flash column chromatography on silica (0–20% CH3OH in CH2Cl2) to give the product as a white
solid (0.06 g, 20%); 1H NMR (400 MHz, CDCl3)
δ
8.63 (s, 2H), 7.63 (s, 1H), 7.22 (s, 1H), 5.48 (br, 2H), 5.30
(s, 2H), 4.15–4.16 (t, J = 3.7 Hz, 2H), 3.58–3.60 (t, J = 3.7 Hz, 2H), 1.09–1.11 (s, 9H); 13C NMR (101 MHz,
CDCl3) 178.40, 162.17, 155.12, 138.17, 137.85, 118.49, 113.33, 76.58, 66.82, 62.64, 38.78, 27.17; Elemental
δ
analysis calcd for C15H21N5O3: C, 56.41; H, 6.63; N, 21.93. Found: C, 56.11, H, 6.47, N, 21.65.
5-Iodopyrimidin-4-amine (17).14 To a solution of 4-aminopyrimidine (0.80 g, 8.5 mmol) in glacial AcOH
(20 mL) under nitrogen was added N-iodosuccinimide (2.30 g, 10.20 mmol) with stirring. The reaction
refluxed at 80 ◦C for two hours. Upon completion, the reaction was cooled to room temperature, and
the solvent was removed in vacuo. The resulting yellow solid was resuspended in EtOAc, quenched
with NaHCO3 (50 mL), washed with Na2S2O3 (50 mL), and the organic layer was dried with MgSO4
and gravity filtered to give a yellow solid. Then, the crude product was purified by flash column
chromatography on silica (0–10% CH3OH in CH2Cl2) to give the pure product as a pale yellow solid
(1.5 g, 80%); Rf = 0.48 (1:1 EtOAc: Hex); Spectral data is in agreement with literature values.14
5-(tributylstannyl)pyrimidin-4-amine (18).14 To a solution of 17 (0.75 g, 3.39 mmol) in anh. DMF (50 mL)
under direct bubbling nitrogen was added bis(tributyl)tin (2.05 mL, 4.07 mmol) and Pd2dba3•CHCl3

Molecules 2019, 24, 3184
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(0.35 g, 0.34 mmol) with stirring. Then, the reaction was heated to 65 ◦C for 2.5 h. Upon completion, the
reaction was cooled to room temperature and the solvent was removed in vacuo. Then, the resulting
oil was resuspended in EtOAc, filtered over a celite pad, and the filtrate was concentrated in vacuo to
give a yellow oil. Then, the crude product was purified by flash column chromatography on silica
(10–60% EtOAc in hexanes) to give the pure product as a pale yellow solid (0.80 g, 61%); Rf = 0.65 (1:1
EtOAc: Hex); Spectral data is in agreement with literature values.14
2-((4-(4-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethan-1-ol (5). The sugar 6 (0.25 g, 0.93 mmol)
and stannyl pyrimidine 18 (0.43 g, 1.12 mmol) were used. The product was purified five times by flash
column chromatography on silica (0–15% CH3OH in CH2Cl2) to give the product as a pale pink solid
(0.03 g, 11%); 1H NMR (500 MHz, CD3OD)
δ
8.44 (s, 1H), 8.29 (s, 1H), 7.93 (s, 1H), 7.78 (s, 1H), 5.50 (s,
2H), 3.67–3.69 (m, 2H), 3.56–3.58 (m, 2H); 13C NMR (126 MHz, CD3OD)
δ
160.25, 155.37, 150.03, 137.43,
136.56, 116.33, 110.63, 76.53, 70.11, 60.50. Elemental analysis calcd for C10H13N5O2 + 0.05 CH2Cl2: C,
50.40; H, 5.41; N, 29.24; Found: C, 50.55; H, 5.47; N, 29.10.
2-((4-(4-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl acetate (5-Ac). The sugar
7 (0.27 g,
0.87 mmol) and stannyl pyrimidine 18 (0.40 g, 1.04 mmol) were used. The product was purified five
times by flash column chromatography on silica (0–15% CH3OH in CH2Cl2) to give the product as a
pink solid (0.03 g, 11%); 1H NMR (500 MHz, DMSO-d6)
δ
8.52 (s, 1H), 8.26 (s, 1H), 8.00 (s, 1H), 7.91
(s, 1H), 5.45 (s, 2H), 4.10–4.12 (t, J = 4.7 Hz, 2H), 3.65–3.67 (t, J = 4.7 Hz, 2H), 2.01 (s, 3H); 13C NMR
(125 MHz, CDCl3) 171.13, 159.92, 156.76, 151.23, 138.36, 133.76, 114.90, 109.90, 76.58, 66.57, 62.71,
δ
20.42; Elemental analysis calcd for C12H15N5O3 + 0.55 CH2Cl2: C, 46.52; H, 5.01; N, 21.62; Found: C,
46.42; H, 4.87; N, 21.60.
2-((4-(4-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl propionate (5-EtAc). The sugar
8 (0.27 g,
0.84 mmol) and stannyl pyrimidine 18 (0.39 g, 1.01 mmol) were used. The product was purified four
times by flash column chromatography on silica (0–10% CH3OH in CH2Cl2) to give the product as a
clear oil (0.015 g, 6.1%); 1H NMR (500 MHz, CD3OD)
δ
8.45 (s, 1H), 8.29 (s,1H), 7.93 (s, 1H), 7.79 (s, 1H),
5.49 (s, 2H), 4.19–4.21 (m, 2H), 3.72–3.74 (m, 2H), 2.29–2.34 (q, J = 7.6 Hz, 2H), 1.06–1.09 (t, J = 7.6 Hz,
3H); 13C NMR (126 MHz, CD3OD)
174.53, 160.25, 155.41, 150.04, 137.43, 136.69, 116.28, 110.56, 76.31,
δ
66.73, 62.71, 26.70, 7.93; Elemental analysis cald for C13H17N5O3 + 0.1 CH3OH + 0.4 H2O: C, 52.12; H,
6.08; N, 23.21; Found: C, 52.26; H, 5.82; N, 23.03.
2-((4-(4-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl isobutyrate (5-iPrAc). A solution of 17 (0.2 g,
◦
0.91 mmol) in anh. THF (20 mL) was cooled to
−
78 C in an acetone/CO2(s) bath under nitrogen.
EtMgBr (0.33 mL, 1.0 mmol) was added dropwise and the reaction was stirred for two minutes,
followed by the addition of TMSCl (0.14 mL, 1.09 mmol), after which the reaction was stirred for 5 min.
Another addition of EtMgBr (0.33 mL, 1.0 mmol) was added dropwise, and the reaction was stirred
for 2 min; then, another addition of TMSCl (0.14 mL, 1.09 mmol) was added, and the reaction was
stirred for 5 min. A final addition of EtMgBr (0.33 mL, 1.0 mmol) was added, and the reaction stirred
for 10 min; then, ZnCl2 (1.81 mL, 1.81 mmol) was added dropwise. The solution was stirred at
for 10 min, and then warmed to room temperature and stirred for 2 h. The organozinc mixture was
then added dropwise to a solution of (0.34 g, 1.0 mmol), Pd(PPh3)4 (0.11 g, 0.09 mmol), and CuI
−
78 ◦C
9
(0.01 g, 0.05 mmol) in anh. THF (10 mL). Then, the reaction stirred under nitrogen for 18 hours at room
temperature. Upon completion, the reaction was quenched with saturated ethylenediaminetetraacetic
acid (EDTA) (10 mL), after which it was concentrated in vacuo, extracted three times with CH2Cl2,
washed with brine, and the organics were dried with MgSO4. After gravity filtration, the solvent was
removed in vacuo to give a dark oil. Then, the crude mixture was purified twice via flash column
chromatography on silica (0–10% CH3OH (10% NH4OH) in DCM) to give the product as a pink oil
(0.16 g, 48%); 1H NMR (500 MHz, CD3OD)
2H), 4.20–4.21 (m, 2H), 3.72–3.74 (m, 2H), 2.52–2.53 (m, 1H), 1.11–1.13 (d, J = 7.0 Hz, 6H); 13C NMR
δ 8.45 (s, 1H), 8.29 (s, 1H), 7.94 (s, 1H), 7.80 (s, 1H), 5.50 (s,

Molecules 2019, 24, 3184
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(126 MHz, (CD3)3CO)
δ
176.26, 160.13, 156.41, 151.08, 137.78, 137.24, 115.69, 109.97, 76.43, 66.84, 62.68,
33.54, 18.42; Elemental analysis calcd for C14H19N5O3 + 0.75 H2O: C, 52.74; H, 6.48; N, 21.96; Found: C,
52.55; H, 6.21; N, 21.82.
2-((4-(4-Aminopyrimidin-5-yl)-1H-imidazol-1-yl)methoxy)ethyl pivalate (5-tBuAc). A solution of 17 (0.2 g,
◦
0.90 mmol) in anh. THF (15 mL) was cooled to
−
78 C in an acetone/CO2(s) bath under nitrogen.
EtMgBr (0.33 mL, 1.0 mmol) was added dropwise, and the reaction was stirred for two minutes,
followed by the addition of TMSCl (0.14 mL, 1.09 mmol) and the reaction was stirred for another 5 min.
Another addition of EtMgBr (0.33 mL, 1.0 mmol) was added dropwise, after which the reaction stirred
for 2 min; then, another addition of TMSCl (0.14 mL, 1.09 mmol) was added, and the reaction was
stirred for 5 min. A final addition of EtMgBr (0.33 mL, 1.0 mmol) was added, and the reaction stirred
for 10 min; then, ZnCl2 (1.81 mL, 1.81 mmol) was added dropwise. The solution was stirred at
78 ◦C
−
for 10 min, and then warmed to room temperature and stirred for 2 h. Then, the organozinc mixture
was added dropwise to a solution of 10 (0.35 g, 1.0 mmol), Pd(PPh3)4 (0.01 g, 0.09 mmol), and CuI
(0.01 g, 0.04 mmol) in anh. THF (10 mL). Then, the reaction was stirred under nitrogen for 18 hours at
room temperature. Upon completion, the reaction was quenched with saturated EDTA (10 mL), after
which it was concentrated in vacuo, extracted three times with CH2Cl2, washed with brine, and the
organics were dried with MgSO4. After gravity filtration, the solvent was removed in vacuo to give a
dark oil. Then, the crude mixture was purified twice via flash column chromatography on silica (0–10%
CH3OH (10% NH4OH) in DCM) to give the product as a light pink oil (0.03, 9.4%); 1H NMR (500 MHz,
CD3OD)
δ
8.54 (s, 1H), 8.30 (s, 1H), 7.98 (s, 1H), 7.87 (s, 1H), 5.58 (s, 2H), 4.19–4.21 (m, 2H), 3.72–3.78
179.09, 159.91, 156.79, 151.26, 138.42, 136.21, 114.81,
(m, 2H), 1.16 (s, 9H); 13C NMR (126 MHz, CDCl3)
δ
109.90, 76.66, 66.93, 62.52, 38.74, 27.71; Elemental analysis calcd for C14H19N5O3 + 0.05 CH3OH + 0.8
H2O: C, 54.01; H, 6.53; N, 20.50.
4.3. Biological Assays
All work with infectious virus was conducted in a Biological Safety Level 4 (BSL-4) laboratory
at the Centers for Disease Control and Prevention. Laboratorians adhered to international practices
appropriate for this biosafety level.
Huh7 cells were seeded at 4
×
103 cells/well (384-well plates) in FluoroBrite Dulbecco’s modified
Eagle’s medium (ThermoFisher, Waltham, MA, USA), supplemented with 10% (v/v) fetal calf serum
(Hyclone, GE Healthcare, Chicago, IL, USA) and 1x penicillin and streptomycin (ThermoFisher).
The following day, compounds were added using an Echo 550 acoustic liquid handler (Labcyte,
San Jose, CA, USA), and the plates were transferred to a BSL-4 laboratory. Cells were infected with
EBOV-ZsGreen reporter virus [23,24] 2–3 h after the addition of compounds. ZsGreen fluorescence
was determined 72 h post-infection using a Synergy H1MD plate reader (BioTek, Winooski, VT, USA).
Cell viability was determined at the same time on compound-treated, mock-infected cells using
CellTiter-Glo (Promega, Madison, WI). GraphPad Prism (GraphPad Software, La Jolla, CA, USA) was
used to fit a four-parameter equation to semilog plots of the concentration–response data and to derive
the concentration of compound that inhibited 50% of the virus-induced cell death (EC50).
For statistical analysis, EC50 values from repeat experiments were compared using the
Mann–Whitney test in GraphPad Prism. Statistical significance was assumed with a p value < 0.05.
4.4. NMR Conformational Analysis
1H NMR spectra were recorded on an Agilent Technologies DD2 300 NMR spectrometer at
the Slovenian NMR center at 297.8 MHz. Spectra were acquired at 3-mM concentration in 1%
DMSO-d6/D2O. Gradient-based 1D NOESY pulse sequence was used for the selective inversion and
assessment of conformational features of fleximers based on observed NOE enhancements.

Molecules 2019, 24, 3184
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5. Conclusions
In this study, an SAR was pursued in order to increase antiviral activity against EBOV and decrease
cytotoxicity. While none of the analogues analyzed were cytotoxic up to 100 M, none of the analogues
were more active against EBOV than compound . Regardless, it was determined that the 4-methoxy
µ
1
series were overall more active than the 2-amino series, suggesting that the 4-methoxy moiety may be
more important for activity. Furthermore, compounds with logP values greater than 1 but less than 2.4
offer the necessary lipophilicity for activity. It was also found that compounds within series
1 and 2
adopt the typical anti-conformation, whereas compounds within series adopt a syn conformation,
5
thus offering another possible explanation for the differences in antiviral activity. With these initial
results in hand, the synthesis of additional analogues, especially those with a methoxy group at the 4
position will be considered, in order to continue to try and further explore antiviral activity. The results
of those studies will be reported elsewhere as they become available.
Author Contributions: Conceptualization, M.K.Y. and K.L.S.-R.; synthesis, M.K.Y. and Y.A.; antiviral analysis, P.C.
and M.F.; NMR studies, D.M. and J.P.; writing—original draft preparation, M.K.Y.; writing—review and editing,
M.K.Y., J.P., M.F., C.F.S. and K.L.S.-R.
Funding: This work was funded by the National Institutes of Health – T 32 GM066706 (KLSR and MKY) and R21
AI135252 (KLSR). The authors acknowledge the CERIC-ERIC Consortium for the access to experimental facilities.
Acknowledgments: The findings and conclusions in this report are those of the authors and do not necessarily
represent the official position of the Centers for Disease Control and Prevention.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).