Validating the 1,2-Difluoro Motif As a Hybrid Bioisostere of CF3and et Using Matrix Metalloproteinases As Structural Probes

Article abstract of DOI:10.1021/acs.jmedchem.0c00648

Matrix metalloproteinases (MMPs) are involved in a spectrum of physiological processes, rendering them attractive targets for small-molecule drug discovery. Strategies to achieve selective inhibition continue to be intensively pursued, facilitated by advances in structural biology. Herein, we harness MMPs 2, 8, 9, and 13 to validate the vicinal difluoro motif as a hybrid bioisostere of CF3 and Et (BITE) in a series of modified barbiturate inhibitors. Crystallographic analyses of representative structures reveal conformations of the vicinal difluoro motif that manifest stabilizing hyperconjugative interactions consistent with the stereoelectronic gauche effect. Detailed docking studies of a potent difluorinated probe with MMP-9 are also disclosed and indicate that the structural basis of inhibition is a consequence of the anisotropic nature of the motif. Significant selectivity of MMP 13 versus MMP-2 can be achieved by subtle chain contraction in a BITE-modified inhibitor.

Full text of DOI:10.1021/acs.jmedchem.0c00648

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Article
Validating the 1,2-Difluoro (BITE) Motif as a Hybrid Bioisostere of
CF3 and Et using Matrix Metalloproteases as Structural Probes
Nathalie Erdeljac, Christian Thiehoff, Ravindra Jumde, Constantin G. Daniliuc,
Sandra Hoeppner, Andreas Faust, Anna K.H Hirsch, and Ryan Gilmour
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00648 • Publication Date (Web): 07 May 2020
Downloaded from pubs.acs.org on May 7, 2020
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Journal of Medicinal Chemistry
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Validating the 1,2-Difluoro (BITE) Motif as a Hybrid Bioisostere of
CF3 and Et using Matrix Metalloproteases as Structural Probes
Nathalie Erdeljac,a Christian Thiehoff,a Ravindra P. Jumde,b Constantin G. Daniliuc,a Sandra Höppner,c
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Andreas Faust,c Anna K. H. Hirsch*b,d and Ryan Gilmour*a
a Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, 48149 Münster, Germany
b Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helm-
holtz Centre for Infection Research (HZI), University Campus E8.1, 66123 Saarbrücken, Germany
c
European Institute for Molecular Imaging, Westfälische Wilhelms-Universität Münster, Waldeyerstraße 15, 48149 Müns-
ter, Germany
d Department of Pharmacy, Saarland University, 66123 Saarbrücken, Germany
ABSTRACT: Matrix metalloproteases (MMPs) are involved in a spectrum of physiological processes, rendering them attractive
targets for small molecule drug discovery. Strategies to achieve selective inhibition continue to be intensively pursued, facilitated
by advances in structural biology. Herein, we harness MMPs 2, 8, 9 and 13 to validate the vicinal difluoro motif (BITE) as a hybrid
bioisostere of CF3 and Et in a series of modified barbiturate inhibitors. Crystallographic analyses of representative structures reveal
conformations of the vicinal difluoro-motif that manifest stabilizing hyperconjugative interactions consistent with the stereoelec-
tronic gauche effect. Detailed docking studies of a potent difluorinated probe with MMP-9 are also disclosed and indicate that the
structural basis of inhibition is a consequence of the anisotropic nature of the motif. Significant selectivity of MMP 13 versus
MMP-2 can be achieved by subtle chain contraction in a BITE-modified inhibitor.
KEYWORDS: bioisostere; conformational analysis; molecular docking; fluorine; MMP-Inhibition
INTRODUCTION
difluoroethyl group,11 and explore its effect in differentiating
between closely similar MMPs.
Fluorination patterns are pervasive in synthetic bio-active small
molecules,1 but are rarely encountered in the natural product
repertoire.2 The value of these motifs in drug discovery stems
from a unique juxtaposition of steric subtlety, electronegativity
and stability of the C-F bond.3 Consequently, bioisosterism strate-
gies with fluorine continue to be intensively pursued in pharma-
ceutical design to optimize physicochemical parameters.4 This
prominence continues to provide a powerful impetus to develop
more efficient routes to validated structural motifs, and to facili-
tate exploration of new areas of chemical space.5 The emergence
of increasingly complex multiply fluorinated arrays will require
continued innovation to understanding and harness their biologi-
cal potential.6 Conventional bioisosterism strategies involve the
substitution of H or OH groups, to enable respective partial charge
inversion or strategic deletion of a hydrogen bond whilst retaining
electronic character. In contrast, the venerable trifluoromethyl
group has extended bioisosterism into achiral 3D space and is
predicated on a steric equivalency with the isopropyl group.4a,d,7
However, consideration of the van der Waals volumes of the
trifluoromethyl group with various alkyl substituents suggests that
a comparison with the ethyl group is more appropriate.1a,8 This is
further supported by the findings of Zanda and co-workers who
have employed matrix metalloproteases (MMPs) as steric probes
to evaluate relative binding in the lipophilic S1´ cavity of the
enzyme (Figure 1).9 By assessing the inhibitory potency of barbi-
turate derivatives, the following trend was established: Et ≈ CF3 <
iPr. In view of the clinical significance of MMPs, and challenges
associated with selective inhibition,10 this sensitive platform
provided an opportunity to simultaneously validate a chiral hybrid
bioisostere of CF3 and CF3 Et (BITE) based on the 1,2-
Figure 1. Generic 5-alkyl-5-benzyl substituted barbiturate and
representation of the S1´ and S2´ binding pockets in MMPs.
Confidence in this BITE group as a hybrid bioisostere stemmed
from a calculation of its van der Waals volume (51.0 Å3) using a
literature method (Figure 2, top).12 Although this indicates a series
i
Et ≈ CF3 < BITE < Pr, there is conformity with the shape of the
Et motif (anisotropic), since the steric and dipolar properties of
the BITE group are directionally dependent as opposed to the CF3
group (isotropic). It is also pertinent to note that the BITE unit
preferentially adopts a syn-clinal orientation to satisfy the stereoe-
lectronic gauche effect,3,13 and is characterized by a temperature
dependent dipole moment (dμ/dT<0).14 The introduction of a
chiral C(sp3)-F center remote to the Zn-binding site and the stere-
ochemical consequences of the enol tautomers are notable fea-
tures that must be considered when placing biological activity on
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Page 2 of 15
a structural footing (Figure 2, bottom). To date, the favorable
the trifluoromethyl derivative Y was constructed through radical
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characteristics of the BITE group have found application in me-
dicinal chemistry due to advances in contemporary catalysis.15
Contemporaneous reports from this laboratory16 and from Jacob-
sen and co-workers17 have described an I(I)/I(III) platform to
facilitate the vicinal difluorination of alkenes via in situ genera-
tion of ArIF2 species.18
hydrotrifluoromethylation of hept-6-en-1-yl benzoate (see ESI).20
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Scheme 1. Preparation of targeted barbiturates 4a-e and 5a-e. a)
NaH, benzyl bromide or 4-methoxybenzyl chloride, DMF, 0 °C to
rt (up to 61% yield); b) NaH, C8H17Br, C9H19Br, X, Y or Z, DMF,
0 °C to rt (up to 81% yield); c) urea, BuOK, DMSO, rt (up to
96% yield).
t
Figure 2. Top: Incorporating the BITE group at the R1 position
via I(I)/I(III) catalysis, shape and van der Waals volumes of the
functional groups analyzed in this study. Bottom: Stereochemical
considerations in the difluorinated scaffolds.
The direct catalytic, vicinal difluorination of terminal alkenes via
an I(I)/I(III) manifold enabled the synthesis of the key difluoro-
ethyl motif in X.16a Finally, cyclization of malonate derivatives
2a-e and 3a-e with urea and tBuOK furnished the desired barbitu-
rates 4a-e and 5a-e. To explore the influence of chain length, two
shorter (octyl) BITE-containing barbiturates (4f and 5f) were
prepared as reference compounds, following the same synthetic
route as for 4e and 5e (please see the ESI for full details). Gratify-
ingly, single crystals suitable for X-ray diffraction analysis were
obtained for barbiturates 5c, 5d and 5f (Figure 3). In addition to
providing unequivocal proof of structure, the postulated stereoe-
lectronic gauche effect is clearly visible in derivative 5f
(σC−H→σ∗C−F; torsion angle ΦFCCO= -66.29°). Interestingly, in
1,2-difluoroethane, the gauche / anti conformers are partitioned
by ca. 1 kcal•mol-1 which provides the structural basis for the
curious temperature dependent dipole moment.14
Herein, the ability of a vicinal difluoro motif to act as a chiral
hybrid of Et and CF3 is established in an MMP assay using MMPs
2, 8, 9 and 13. The findings are complemented by a docking study
using MMP-9 that considers the importance of keto-enol tauto-
mers, and the configuration of the quaternary and C(sp3)-F cen-
ters (Figure 2).
RESULTS AND DISCUSSION
Initially, a series of test probes were conceived that varied at the
termini to allow comparison of the BITE group with the CF3 and
Et analogues in vitro. To that end, a simple three-step synthesis
route provided access to the target compounds 4a-e and 5a-e
(Scheme 1). Sequential alkylation of diethyl malonate efficiently
installed the aryl and alkyl fragments required. Preparation of
electrophile
Z required treatment of benzyl-protected 6-
bromohexanol with iPrMgCl in the presence of Li2CuCl4,19 whilst
Figure 3. X-Ray crystallographic analyses of barbiturates 5c, 5d and 5f. The fluorine gauche effect is visible in 5f (ΦFCCO= -66.29°).
CCDC 1995218 (5c), 1995219 (5d), and 1995220 (5f) contain the supplementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
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MMP-inhibition: To evaluate the BITE group as a hybrid bi-
With regards to MMPs 2 and 8, the BITE-modified barbiturates
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8
oisostere of Et and CF3, inhibition of various MMPs via a fluoro-
genic in vitro assay20 was performed with the compounds de-
scribed in Scheme 1. In addition to MMPs 2 and 9, which were
used in the original Zanda study,9 the investigation was extended
to include MMPs 8 and 13 (Table 1). In general, effective inhibi-
tion across the four MMPs was observed with the exception of the
iPr derivative 4c (R2= H) analog, whose IC50 value could not be
determined due to solubility issues.
4e and 5e, show an inhibitory potency that is between that of the
corresponding Et (4b and 5b) and CF3 (4d and 5d) systems (Fig-
ure 4A, R2 = H). In both compound series, the BITE-modified
compounds outperform the trifluoromethyl species but are less
active than the hydrocarbon-scaffolds. In addition, the inhibition
pattern of the two series (R2 = H and OMe) is almost identical
towards MMP-13, in which the S1’ cavity is known to be more
structurally distinguished in comparison to other MMPs due to an
additional side pocket.21 In this case, the BITE-group containing
barbiturates 4e and 5e exhibit inhibitory potencies similar to those
of their Et group equivalents, whereas the activities of the CF3
bearing compounds are much lower. This observation highlights
the anisotropic similarities that relate the Et and BITE groups, in
contrast to the isotropic trifluoromethyl group. This is a particu-
larly striking example of the sensitivity of value of this enzymatic
steric volume analysis. Intriguingly, the contracted BITE scaffold
4f also proved to be a highly efficient inhibitor with values that
out-competed the C9 (4e) scaffolds, with the exception of MMP-
9. Again, the analogy to the hydrocarbon scaffold is evident from
the comparison to the Me (C8, 4a) derivatives (pink).
Upon closer inspection of the MMP-inhibition activities of the R2
= OMe test series (Figure 4B), similarities between the CF3 and
iPr derivatives were observed, particularly for MMPs 2 and 9.
This is in contrast with Zanda´s observations.9 Again, the hybrid
Et/CF3 bioisosterism analogy of the BITE group is further appar-
ent from a comparison of MMPs 2 and 8, where intermediate IC50
values were noted for the C8 chains. The high inhibitory potencies
of the one carbon contracted systems (Me) reflects the lower
steric bulk of the substituent and is unsurprising. Interestingly, the
shorter BITE-bearing reference compounds, which are less steri-
cally demanding than their longer nonyl equivalents, generally
show more encouraging inhibitory activities with the exception of
MMPs 2 and 8, where R2 = OMe. Repeated attempts to separate
rac-5e failed and it was not possible to investigate the effect of
the constituent enantiomers on MMP-9 inhibition. However,
given the encouraging experimental data, the structural basis of
inhibition was investigated by a molecular docking study to estab-
lish potential interactions in the S1’ cavity (Figures 5-7).
Particularly illuminating was the direct comparison of the Et (dark
gray) and CF3 (light gray) containing analogs with the BITE-
containing inhibitors (dark blue, C9 chains). These data reveal a
similar activity pattern across the two compound groups (R2 = H
and OMe, Figures 4A and B, respectively).
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Table 1. Inhibition against MMPs 2, 8, 9 and 13.
IC50 (nM)a
Compound
MMP-2
288 ± 38
210 ± 26
n.d.
MMP-8
157 ± 3
96 ± 28
n.d.
MMP-9
22 ± 2
5 ± 0.5
n.d.
MMP-13
214 ± 43
180 ± 6
n.d.
4a
4b
4c
4d
4e
4fb
5a
5b
5c
567 ± 58
320 ± 14
253 ±79
42 ± 17
94 ± 23
207 ± 74
209 ± 19
139 ± 49
411± 124
229 ± 35
180 ± 22
125 ± 10
73 ± 10
84 ± 22
281 ± 74
187 ± 33
110 ± 27
165 ± 59
52 ± 6
60 ± 4
123 ± 16
3 ± 1
753 ± 81
163 ± 59
127 ± 53
94 ± 16
83 ± 7
93 ± 1
60 ± 5
62 ± 14
26 ± 4
47 ± 16
225 ± 19
177 ± 35
64 ± 2
5d
5e
5fb
17 ± 7
a The IC50 values are the mean ±SD of three experiments. n.d. =
not determined due to solubility issues. b Contracted carbon chain.
R2 = H
B
R2 = OMe
A
800
700
600
500
400
300
200
100
0
500
400
300
200
100
0
MMP-2
Et
MMP-8
MMP-9
MMP-13
MMP-2
MMP-8
MMP-9
MMP-13
R1 =
R1 =
BITE (CC4C9e99)
CF33
Me
BITE (CC88)
4f
Et
BITE (CC59e9)
CF333
iPr
Me
BITE (CC88)
5f
a
Figure 4. Inhibition of various MMPs. (A) Compounds 4a-f. (B) Compounds 5a-f. IC50 values corresponding the mean of three experi-
ments.
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Docking Studies of compound 5e (BITE) in MMP-9: Initially,
These docking analyses indicate that the configuration of the ring
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the enantiomers of compound 5e (5e (R) and 5e (S)) were docked
into the active site of MMP9 (PDB code: 6ESM, Figure 5).22
Immediately obvious from the analysis was that the binding
modes were closely similar suggesting that inhibition is grounded
in the anisotropic shape of the BITE group and not determined by
the configuration of the C(sp3)-F center. A comparison of 5e (R)
and 5e (S) also reveals that the 4-methoxybenzyl groups are ac-
commodated in the narrow, lipophilic pocket of the enzyme and
that the barbiturate rings chelate to the zinc cation as expected.
The aliphatic chains with BITE groups are positioned in the large
exposed part of the enzyme with no clear interactions with the
surrounding amino acid residues.
plays a crucial role in regulating the orientation of these com-
pounds in the active site of MMP-9, irrespective of the configura-
tion of the distant C(sp3)-F center. The enol-tautomers (5e (R,S)
and 5e (R,R) have the aliphatic chain with the BITE group located
in the narrow, lipophilic part of the pocket. In contrast, the S-
configured systems 5e (S,S) and 5e (S,R) have this moiety posi-
tioned towards the exposed part of the pocket (compare Figures 6
and Figure 7).
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Figure 7. Docked top-ranked poses of compound 5e (S,S) and 5e
(S,R) in the active site of MMP-9 (PDB code: 6ESM).
CONCLUSION
Figure 5. Docked top-ranked poses of compound 5e (R) and 5e
(S) in the active site of MMP-9. (PDB code: 6ESM).
Motivated by the increasing importance of C(sp3)-F containing
patterns in contemporary medicinal chemistry, an enzymatic
validation of the BITE group as a hybrid bioisostere of CF3 and Et
is disclosed. Harnessing MMPs 2, 8, 9 and 13 as Zanda-type steric
probes, a series of barbiturates were modified at a common site to
allow for comparative analyses. In both test sets, the IC50 values
of the BITE-modified inhibitors intersect those of the correspond-
ing Et and CF3 derivatives. In the case of MMP-9, the BITE de-
rivative (when R2 = H) behaved similarly to the CF3 derivative,
but its inclusion generally led to improved IC50 values against the
other MMPs in the screen. The sensitivity of the screen is evident
from Figure 4B, where selectivity for MMP-13 over MMP-2
results from the contraction of the pendant chain by a single car-
bon atom. Molecular docking studies have revealed the im-
portance of the configuration at the quaternary center and that the
positive influence of the BITE group is likely a consequence of its
anisotropic nature.
To investigate the effect of the relative stereochemistry of the
quaternary center (barbiturate ring) and C(sp3)-F center on bind-
ing mode, the four enol-tautomers of 5e (please see Figure 2,
bottom) were docked in the active site of MMP-9 (Figures 6 and
7).
EXPERIMENTAL SECTION
General Information. Commercially available chemicals were
purchased as reagent grade and were used as received. Teflon®
reaction vessels and caps for 1,2-difluorination reactions were
prepared by the mechanical workshop at the Westfälische Wil-
helms Universität Münster. Thin layer chromatography (TLC)
was performed on pre-coated silica gel 60 F254 aluminium plates
from Merck and were visualized under UV-light and/or stained
with a solution of KMnO4. Solvents for extractions and flash
chromatography were of technical grade and distilled prior to use.
Dry solvents were dried via a Grubbs purification system. Flukas
silica gel (230-400 mesh) was used for purification by flash
Figure 6. Docked top-ranked poses of compound 5e (R,S) and 5e
(R,R) in the active site of MMP-9 (PDB code: 6ESM).
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Journal of Medicinal Chemistry
chromatography. NMR measurements were performed by the
1454 (w), 1362 (w), 1306 (w), 1258 (w), 1241 (w), 1202 (w),
1
2
3
4
5
6
7
8
NMR service of the Organic Chemistry institute (WWU, Münster)
on a Bruker AV300, AV400 or an Agilet DD2 600 at indicated
temperature. Chemical shifts (δ, ppm) are referenced to residual
solvent signals and the coupling constants (J) are given in Hz. The
resonance multiplicities are reported as: s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet). Additional 1D and 2D
NMR experiments (e.g., 1H{19F}, 19F{1H}, DEPT, COSY, HMBC
and HSQC) were used to assign the resonances of previously
unknown compounds. High resolution mass spectra (HR-ESI)
were measured by the MS service of the Organic Chemistry de-
partment (WWU, Münster). Melting points were determined in
oPentane capillaries on a Büchi B-540 apparatus. IR spectra were
recorded on a Perkin Elmer 100 FT-IR (ATR) spectrometer. The
adsorption bands are given in wavenumbers (cm-1) and intensities
are abbreviated and described as: w (weak), m (medium) or s
(strong). Purity of target compounds were determined to on an
Agilent Infinity 1260 HPLC system; DAD detector; column:
Chiralcel OJ-H; flow rate: 1.00 mL/min; injection volume: 5.0
µL; detection: λ = 210 nm; eluent (n-hexane / isopropanol): 4a-f
and 5a-d, 5f = 80 / 20 and 5e = 90 / 10. The purity of all target
compounds (4a-f and 5a-f) was determined as ≥95% by HPLC
(see the ESI).
General Procedure 1 (GP1): alkylation of the benzylated
diethyl malonates. A flame-dried Schlenk flask was charged with
the specified malonate and DMF (0.1 – 0.2 M) was added under
argon followed by NaH (60% dispersion in mineral oil, 1.2 eq.) at
0 °C. After stirring at 0 °C for 15 – 30 min, the specified alkyl
chain was added and the mixture was stirred at room temperature
for the stated time. The reaction was quenched by the addition of
an aqueous saturated solution of NH4Cl and diluted with H2O.
The mixture was extracted with Et2O (3x) and the combined
organic layers were washed with brine, dried over MgSO4 or
Na2SO4 and concentrated under reduced pressure.
1100 (m), 1028 (w), 906 (w), 814 (w), 733 (m), 696 (m). Analyti-
cal data in agreement with literature.26
(((8-Methylnonyl)oxy)methyl)benzene (S2, ESI). Prepared
according to a modified literature procedure.27 S1 (960 mg,
3.54 mmol, 1.00 eq.) was dissolved in dry THF (30 ml) under
argon. The solution was then cooled to -10 °C and a solution of
Li2CuCl4 (46.0 mg, 0.21 mmol, 0.06 eq.) [prepared from
LiCl•H2O (17.8 mg, 0.42 mmol, 0.12 eq.) and CuCl2 (28.2 mg,
0.21 mmol, 0.06 eq.)]28 in THF (1 ml) was added followed by
i
dropwise addition of PrMgCl (2.0 M in THF, 825 µl, 1.65 mmol,
9
5.00 eq.). The solution was stirred at -10 °C for 4 h and then
stirred at room temperature for 43 h. The reaction was quenched
by the addition of an aqueous saturated solution of NH4Cl (40 ml)
and extracted with Et2O (3x). The combined organic layers were
dried over MgSO4 and the solvent was removed under reduced
pressure. Purification by column chromatography on silica gel (n-
pentane/Et2O 150:1) afforded the desired product as a colourless
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1
liquid (784 mg, 89%). Rf = 0.45 (n-pentane/Et2O 80:1). H NMR
(500 MHz, CD2Cl2): δ 7.36 – 7.32 (m, 4H, H-C12/13), 7.30 – 7.25
3
(m, 1H, H-C14), 4.47 (s, 2H, H-C10), 3.46 (t, JHH = 6.6 Hz, 2H,
3
H-C1), 1.63 – 1.57 (m, 2H, H-C2), 1.53 (dh, JHH = 13.3, 6.7 Hz,
1H, H-C8), 1.39 – 1.25 (m, 8H, H-C3/4/5/6), 1.20 – 1.14 (m, 2H,
3
H-C7), 0.87 (d, JHH = 6.6 Hz, 6H, H-C9). 13C{1H} NMR
(126 MHz, CD2Cl2): δ 139.7 (C11), 128.8 (C13), 128.1 (C12),
127.9 (C14), 73.3 (C10), 71.2 (C1), 39.6 (C7), 30.5 (C2), 30.4
(C6), 30.1 (C4), 28.6 (C8), 27.9 (C5), 26.8 (C3), 23.0 (C9). ESI-
MS: (m/z) required: [(C17H28O)Na]+ = 271.2032; (m/z) found:
[(C17H28O)Na]+ = 271.2019. FT-IR (휈̃= cm-1): 3065 (w), 3033
(w), 2925 (m), 2854 (m), 1496 (w), 1455 (w), 1384 (w), 1365 (w),
1308 (w), 1261 (w), 1203 (w), 1102 (m), 1028 (w), 906 (w), 804
(w), 723 (m), 696 (m). Analytical data in agreement with litera-
ture.29
8-Methylnonan-1-ol (S3, ESI). S2 (500 mg, 2.0 mmol, 1.0 eq.)
was dissolved in Et2O (20 ml) and Pd/C (50 mg, 10 wt%) was
added. The atmosphere was then changed to H2 and the mixture
was stirred at room temperature for 27 h. The reaction mixture
was filtered through a plug of Celite and the filtrate was concen-
trated under reduced pressure. Purification by column chromatog-
raphy on silica gel (n-pentane/Et2O 5:1) afforded the desired
product as a colourless liquid (293 mg, 92%). Rf = 0.19 (n-
General Procedure 2 (GP2): synthesis of barbiturates. The
corresponding malonate (1.0 eq.) was dissolved in DMSO (0.5 M)
and urea (5.8 eq.) was added followed by a solution of KOtBu in
DMSO (1 M, 2.1 eq.). The reaction mixture was then stirred at
room temperature. After full consumption of starting material as
indicated by TLC (CyH/EtOAc 10:1), the reaction was diluted
with EtOAc and was then washed with 1 M HCl. The aqueous
layer was extracted with EtOAc (3x) and the combined organic
layers were washed with H2O and brine, dried over MgSO4 and
concentrated under reduced pressure.
pentane/Et2O 5:1). 1H NMR (400 MHz, CD2Cl2): δ 3.58 (t, 3JHH
=
6.7 Hz, 2H, H-C1), 1.57 – 1.46 (m, 3H, H-C2/8), 1.39 (br, 1H,
OH), 1.36 – 1.23 (m, 8H, H-C3/4/5/6), 1.20 – 1.12 (m, 2H, H-C7),
3
0.86 (d, JHH = 6.6 Hz, 6H, H-C9). 13C{1H} NMR (101 MHz,
Synthetic sequence for the preparation of substrate Z:
(((6-Bromohexyl)oxy)methyl)benzene (S1, ESI). Prepared
according to a modified literature procedure.9 6-Bromo-1-hexanol
(723 µl, 5.05 mmol, 1.0 eq.) was dissolved in dry THF (8 ml)
under argon. The solution was then cooled to 0 °C and NaH (60%
dispersed in mineral oil, 264 mg, 6.60 mmol, 1.2 eq.) was added
followed by benzyl bromide (720 µl, 6.05 mmol, 1.1 eq.). After
stirring at room temperature overnight, the reaction mixture was
poured into H2O at 0 °C and extracted with EtOAc (3x). The
combined organic layers were dried over MgSO4 and the solvent
was removed under reduced pressure. Purification by column
chromatography on silica gel (CyH/EtOAc 150:1 → 50:1) afford-
ed the desired product as a colourless liquid (960 mg, 64%). Rf =
CD2Cl2): δ 63.4 (C1), 39.6 (C7), 33.5 (C2), 30.5 (C6), 30.1 (C4),
28.5 (C8), 27.9 (C5), 26.3 (C3), 23.0 (C9). ESI-MS: (m/z)
required:
[(C10H22O)Na]+ =
181.1563;
(m/z)
found:
[(C10H22O)Na]+ = 181.1539. FT-IR (휈̃= cm-1): 3317 (w), 2925
(m), 2855 (w), 1466 (w), 1384 (w), 1366 (w), 1057 (w), 723 (w).
Analytical data in agreement with literature.30
8-Methylnonyl 4-methylbenzenesulfonate (Z). Prepared ac-
cording to
a
modified literature procedure.31 S3 (200 mg,
1.26 mmol, 1.00 eq.) was dissolved in CH2Cl2 (5 ml) and DMAP
(3 mg, 0.03 mmol, 0.02 eq.) and Et3N (230 µl, 1.64 mmol,
1.30 eq.) were added. TsCl (241 mg, 1.26 mmol, 1.00 eq.) was
added at 0 °C. After stirring for 90 min at 0 °C, the ice-bath was
removed and stirring was continued for 21 h at room temperature.
The mixture was concentrated under reduced pressure. Purifica-
tion by column chromatography on silica gel (CyH/EtOAc 8:1)
afforded the desired product as a yellow liquid (307 mg, 78%).
1
0.35 (CyH/EtOAc 50:1). H NMR (400 MHz, CDCl3): δ 7.38 –
7.33 (m, 4H, H-C9/10), 7.32 – 7.27 (m, 1H, H-C11), 4.51 (s, 2H,
3
3
H-C7), 3.48 (t, JHH = 6.5 Hz, 2H, H-C1), 3.41 (t, JHH = 6.8 Hz,
3
2H, H-C6), 1.87 (dq, JHH = 7.8, 6.7 Hz, 2H, H-C5), 1.69 – 1.57
(m, 2H, H-C2), 1.51 – 1.36 (m, 4H, H-C3/4). 13C{1H} NMR
(101 MHz, CDCl3): δ 138.7 (C8), 128.5 (C10), 127.8 (C9), 127.7
(C11), 73.0 (C7), 70.3 (C1), 34.0 (C6), 32.9 (C5), 29.7 (C2), 28.1
(C4), 25.5 (C3). ESI-MS: (m/z) required: [(C13H19OBr)Na]+ =
293.0529; (m/z) found: [(C13H19OBr)Na]+ = 293.0511. FT-IR
(휈̃= cm-1): 3064 (w), 3031 (w), 2934 (w), 2856 w), 1496 (w),
1
Rf = 0.66 (CyH/EtOAc 4:1). H NMR (400 MHz, CDCl3):  7.79
(m, 2H, H-C11), 7.35 (m, 2H, H-C12), 4.01 (t, 3JHH = 6.5 Hz, 2H,
H-C1), 2.45 (s, 3H, H-C14), 1.68 – 1.58 (m, 2H, H-C2), 1.49
(hept, 3JHH = 6.5 Hz, 1H, H-C8), 1.34 – 1.25 (m, 2H, H-C3), 1.25
– 1.16 (m, 6H, H-C4/5/6), 1.11 (q, 3JHH = 6.4 Hz, 2H, H-C7), 0.85
3
(d, JHH = 6.6 Hz, H-C9). 13C{1H} NMR (101 MHz, CDCl3): 
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Journal of Medicinal Chemistry
Page 6 of 15
144.8 (C13), 133.4 (C10), 130.0 (C12), 128.0 (C11), 70.9 (C1),
stirred at room temperature overnight. The mixture was filtered
1
2
3
4
5
6
7
8
39.1 (C7), 29.8 (C5), 29.1 (C4), 29.0 (C2), 28.1 (C8), 27.4 (C6),
25.5 (C3), 22.8 (C9), 21.8 (C14). ESI-MS: (m/z) required:
[(C17H28O3S)Na]+ = 335.1651; (m/z) found: [(C17H28O3S)Na]+ =
335.1650. FT-IR (휈̃= cm-1): 2926 (w), 2856 (w), 1599 (w), 1467
(w), 1360 (m), 1307 (w), 1188 (m), 1175 (s), 1098 (w), 1020 (w),
955 (m), 925 (m), 813 (m), 763 (w), 723 (w), 662 (s).
through a plug of Celite and the residue was concentrated under
reduced pressure. Purification by column chromatography on
silica gel (n-pentane/Et2O 4:1) afforded the desired product as a
colourless oil (28 mg, 76%). Rf = 0.37 (CyH/EtOAc 2:1). 1H
3
NMR (600 MHz, CDCl3): δ 3.64 (t, JHH = 6.6 Hz, 2H, H-C8),
2.11 – 2.01 (m, 2H, H-C2), 1.62 – 1.52 (m, 4H, H-C3/C7), 1.45
(s, 1H, OH), 1.41 – 1.32 (m, 6H, H-C4/5/6). 13C{1H} NMR (151
Synthetic sequence for the preparation of substrate Y:
1
MHz, CDCl3): δ 127.4 (q, JFC = 276.3 Hz, C1), 63.1 (C8), 33.9
Hept-6-en-1-yl benzoate (S4, ESI). Prepared according to a
modified literature procedure.32 K2CO3 (415 mg, 3.0 mmol,
2.0 eq.) was flame-dried in a Schlenk flask. Then benzoic acid
(183 mg, 1.5 mmol, 1.0 eq.), dry DMF (3 ml) and 7-bromo-1-
heptene (230 µl, 1.5 mmol, 1.0 eq.) were added under argon. The
resulting mixture was heated to 70 °C for 8 h. The solvent was
removed under reduced pressure and the residue was diluted with
H2O (10 ml) followed by extraction with CH2Cl2 (3 x 50 ml). The
combined organic layers were washed with an aqueous solution of
LiCl (5%), dried over MgSO4 and concentrated under reduced
pressure. Purification by column chromatography on silica gel
(CyH/EtOAc 20:1) afforded the desired product as a colourless oil
(305 mg, 93%). Rf = 0.46 (CyH/EtOAc 10:1). 1H NMR (400
MHz, CDCl3): δ 8.07 – 8.01 (m, 2H, H-C10), 7.58 – 7.53 (m, 1H,
H-C12), 7.47 – 7.41 (m, 2H, H-C11), 5.81 (ddt, 3JHH = 16.9, 10.2,
2
(q, JFC = 28.3 Hz, C2), 32.8 (C7), 29.1 (C5), 28.8 (C4), 25.7
(C6), 21.9 (q, 3JFC = 2.9 Hz, C3). 19F NMR (564 MHz, CDCl3): δ
9
3
-66.5 (t, JFH = 11.0 Hz, 3F, F-C1). ESI-MS: (m/z) required:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[(C8H15OF3)Na]+ = 207.0967; (m/z) found: [(C8H15OF3)Na]+ =
207.0966. FT-IR (휈̃= cm-1): 3340 (w), 2934 (w), 2860 (w), 1463
(w), 1441 (w), 1388 (w), 1336 (w), 1317 (w), 1252 (m), 1212 (w),
1174 (w), 1135 (m), 1049 (m), 924 (w), 836 (w), 727 (w), 691
(w), 654 (w). Analytical data in agreement with literature.9
8,8,8-Trifluorooctyl 4-methylbenzenesulfonate (Y). Prepared
according to a modified literature procedure.31 S6 (26 mg,
0.14 mmol, 1.00 eq.) was dissolved in CH2Cl2 (2 ml) and DMAP
(1 mg, 0.01 mmol, 0.05 eq.) and Et3N (26 µl, 0.18 mmol,
1.30 eq.) were added. After addition of TsCl (27 mg, 0.14 mmol,
1.00 eq.) at 0 °C, the ice-bath was removed and stirring was con-
tinued at room temperature overnight. The mixture was diluted
with CH2Cl2, washed with brine, dried over MgSO4 and filtered
through a plug of silica gel. Concentration of the filtrate under
reduced pressure afforded the desired product as a colourless
3
2
6.7 Hz, 1H, H-C2), 5.01 (ddt, JHH = 17.2 Hz, JHH = 2.1 Hz,
4JHH = 1.6 Hz, 1H, HZ-C1), 4.95 (ddt, JHH = 10.2 Hz, JHH
=
3
2
2.1 Hz, JHH = 1.2 Hz, 1H, HE-C1), 4.32 (t, JHH = 6.6 Hz, 2H, H-
C7), 2.13 – 2.04 (m, 2H, H-C3), 1.84 – 1.72 (m, 2H, H-C6), 1.52
– 1.41 (m, 4H, H-C4/5). 13C{1H} NMR (101 MHz, CDCl3): δ
166.8 (C8), 138.9 (C2), 133.0 (C12), 130.6 (C9), 129.7 (C10),
128.5 (C11), 114.7 (C1), 65.2 (C7), 33.8 (C3), 28.7 (C6), 28.7
(C4), 25.7 (C5). ESI-MS: (m/z) required: [(C14H18O2)Na]+ =
241.1199; (m/z) found: [(C14H18O2)Na]+ = 241.1213. FT-IR (휈̃=
cm-1): 3074 (w), 2932 (w), 2858 (w), 1717 (s), 1641 (w), 1602
(w), 1585 (w), 1492 (w), 1452 (w), 1387 (w), 1314 (w), 1269 (s),
1176 (w), 1111 (m), 1070 (m), 1027 (m), 994 (w), 953 (w), 910
(m), 850 (w), 806 (w), 708 (s), 687 (m), 675 (s).
4
3
1
liquid (45 mg, 95%). Rf = 0.26 (CyH/EtOAc 6:1). H NMR (600
MHz, CDCl3): δ 7.79 (m, 2H, H-C10), 7.34 (m, 2H, H-C11), 4.02
(t, 3JHH = 6.4 Hz, 2H, H-C8), 2.45 (s, 3H, H-C13), 2.07 – 1.98 (m,
2H, H-C2), 1.68 – 1.61 (m, 2H, H-C7), 1.54 – 1.47 (m, 2H, H-
C3), 1.35 – 1.28 (m, 4H, H-C4/6), 1.28 – 1.22 (m, 2H, H-C5).
13C{1H} NMR (151 MHz, CDCl3): δ 144.8 (C12), 133.4 (C9),
1
129.9 (C11), 128.0 (C10), 127.3 (q, JFC = 276.3 Hz, C1), 70.6
2
(C8), 33.8 (q, JFC = 28.4 Hz, C2), 28.9 (C7), 28.6 (C5), 28.6
(C4), 25.3 (C6), 21.9 (q, 3JFC = 2.8 Hz, C3), 21.8 (C13). 19F NMR
3
(564 MHz, CDCl3): δ -66.4 (t, JFH = 11.0 Hz, 3F, F-C1). ESI-
8,8,8-Trifluorooctyl benzoate (S5, ESI). Prepared according
to a modified literature procedure.33 K2CO3 (170 mg, 1.23 mmol,
1.6 eq.) was flame-dried in a Schlenk flask. Then Togni reagent II
(531 mg, 1.01 mmol, 1.3 eq.) and a solution of S4 (170 mg,
0.78 mmol, 1.0 eq.) in dry DMF (10 ml) were added under argon.
The resulting suspension was stirred at 80 °C overnight. After
cooling to room temperature, the reaction mixture was diluted
with EtOAc and washed with brine. The organic layer was dried
over MgSO4 and concentrated under reduced pressure. Purifica-
tion by preparative thin layer chromatography (CyH/EtOAc 30:1
→ 20:1) afforded the desired product as a colourless oil (109 mg,
48%). Rf = 0.38 (CyH/EtOAc 20:1). 1H NMR (600 MHz,
CDCl3): δ 8.06 – 8.03 (m, 2H, H-C11), 7.58 – 7.54 (m, 1H, H-
MS: (m/z) required: [(C15H21O3SF3)Na]+ = 361.1056; (m/z) found:
[(C15H21O3SF3)Na]+ = 361.1074. FT-IR (휈̃= cm-1): 2931 (w),
2860 (w), 1722 (w), 1599 (w), 1495 (w), 1463 (w), 1387 (w),
1360 (m), 1308 (w), 1253 (m), 1212 (w), 1189 (m), 1175 (s),
1136 (m), 1097 (m), 1048 (m), 1020 (w), 960 (m), 925 (m), 814
(m), 770 (m), 740 (w), 725 (w), 688 (w), 662 (m).
Synthetic sequence for the preparation of substrate X:
8-Bromo-1,2-difluorooctane (S7, ESI). Prepared according to
a modified literature procedure.16a A Teflon® vessel was charged
with 8-bromooct-1-ene (76 mg, 0.40 mmol, 1.0 eq.), p-
iodotoluene (17 mg, 0.08 mmol, 0.2 eq.), DCE (1 ml), amine:HF
1:5 (1 ml) and Selectfluor (213 mg, 0.60 mmol, 1.5 eq.). The
reaction vessel was then sealed with a Teflon® screw cap. After
stirring at room temperature for 40 h, the mixture was poured into
an aqueous saturated solution of NaHCO3 (15 ml). Once the gas
evolution has ceased the aqueous layer was extracted with CH2Cl2
(3 x 10 ml). The combined organic layers were dried over Na2SO4
and concentrated under reduced pressure. Purification by column
chromatography on silica gel (n-pentane/Et2O 20:1, dry load)
afforded the desired product as a colourless liquid (66 mg, 72%).
Rf = 0.42 (CyH/EtOAc 15:1). 1H NMR (600 MHz, CDCl3): δ
4.67 (dddddd, 2JFH = 49.2 Hz, 3JFH = 20.7 Hz, 3JHH = 11.2, 5.3, 4.2,
3
C13), 7.47 – 7.42 (m, 2H, H-C12), 4.32 (t, JHH = 6.6 Hz, 2H, H-
C8), 2.11 – 2.02 (m, 2H, H-C2), 1.78 (dq, 3JHH = 8.0, 6.7 Hz, 2H,
H-C7), 1.61 – 1.53 (m, 2H, H-C3), 1.50 – 1.43 (m, 2H, H-C6),
1.43 – 1.37 (m, 4H, H-C4/C5). 13C{1H} NMR (151 MHz,
CDCl3): δ 166.8 (C9), 133.0 (C13), 130.6 (C10), 129.7 (C11),
1
128.5 (C12), 127.4 (q, JFC = 276.3 Hz, C1), 65.1 (C8), 33.8 (q,
2JFC = 28.3 Hz, C2), 29.0 (C5), 28.8 (C7), 28.7 (C4), 26.0 (C6),
21.9 (q, 3JFC = 2.9 Hz, C3). 19F NMR (564 MHz, CDCl3): δ -66.5
3
(t, JFH
= 11.0 Hz, 3F, F-C1). ESI-MS: (m/z) required:
[(C15H19O2F3)Na]+ = 311.1229; (m/z) found: [(C15H19O2F3)Na]+ =
311.1233. FT-IR (휈̃= cm-1): 2940 (w), 2861 (w), 1717 (m), 1603
(w), 1585 (w), 1492 (w), 1468 (w), 1452 (w), 1388 (w), 1336 (w),
1315 (w), 1271 (s), 1252 (m), 1221 (w), 1177 (m), 1136 (m),
1111 (m), 1098 (m), 1070 (m), 1050 (m), 1027 (m), 936 (w), 915
(w), 836 (w), 807 (w), 709 (s), 688 (w), 675 (w), 654 (w).
2
3
2.9 Hz, 1H, H-C2), 4.49 (dddd, JFH = 47.3 Hz, JFH = 25.2 Hz,
2JHH = 10.8 Hz, JHH = 2.6 Hz, 1H, H-C1), 4.45 (dddd, JFH
47.7 Hz, JFH = 23.7 Hz, JHH = 10.8 Hz, JHH = 5.5 Hz, 1H, H-
=
3
2
3
2
3
3
C1), 3.41 (t, JHH = 6.8 Hz, 2H, H-C8), 1.90 – 1.83 (m, 2H, H-
3
2
C7), 1.72 (ddddd, JFH = 15.6 Hz, JHH = 13.7 Hz, 3JHH = 9.6, 8.5,
3
2
4.9 Hz, 1H, H-C3), 1.59 (ddddd, JFH = 29.4 Hz, JHH = 13.9 Hz,
8,8,8-Trifluorooctan-1-ol (S6, ESI). To a solution of S5
(107 mg, 0.37 mmol, 1.0 eq.) in MeOH (2 ml) Na2CO3 (64 mg,
0.60 mmol, 3.0 eq.) was added and the resulting mixture was
3JHH = 10.1, 5.4, 4.4 Hz, 1H, H-C3), 1.55 – 1.32 (m, 6H, H-
C4/5/6). 13C{1H} NMR (151 MHz, CDCl3): δ 91.9 (dd, JFC
=
1
ACS Paragon Plus Environment

Page 7 of 15
Journal of Medicinal Chemistry
2
1
2
172.6 Hz, JFC = 19.2 Hz, C2), 84.2 (dd, JFC = 173.6 Hz, JFC
=
1333 (w), 1271 (m), 1224 (m), 1146 (m), 1113 (w), 1095 (w),
1058 (w), 1031 (m), 915 (w), 858 (w), 790 (w), 751 (m), 699 (m).
Analytical data in agreement with literature.35
2
1
2
3
4
5
6
7
8
23.1 Hz, C1), 33.9 (C8), 32.7 (C7), 30.1 (dd, JFC = 20.9 Hz,
3JFC = 6.4 Hz, C3), 28.6 (C5), 28.1 (C6), 24.8 (d, JFC = 4.6 Hz,
3
C4). 19F NMR (564 MHz, CDCl3): δ -189.2 (dddddd, JFH
=
Diethyl 2-(4-methoxybenzyl)malonate (1b). Prepared accord-
ing to a modified literature procedure.34 A flame-dried Schlenk
flask was charged with NaH (60% dispersed in mineral oil,
295 mg, 7.40 mmol, 1.0 eq.) and DMF (25 mL) under argon and
diethyl malonate (1.12 mL, 7.37 mmol, 1.0 eq.) was added drop-
wise at 0 °C. After stirring for at 0 °C 20 min, 4-methoxybenzyl
chloride (1.00 mL, 7.37 mmol, 1.0 eq.) was added dropwise and
the resulting mixture was stirred at room temperature overnight.
The reaction was quenched with an aqueous saturated solution of
NH4Cl, diluted with H2O and extracted with Et2O (3x). The com-
bined organic layers were washed with brine, dried over MgSO4
and concentrated under reduced pressure. Purification by column
chromatography on silica gel (n-pentane/Et2O 15:1) afforded the
desired product as a colourless oil (1.02 g, 49%). Rf = 0.23 (n-
2
3
3
49.2 Hz, JFH = 29.4, 25.2, 23.7, 15.6 Hz, JFF = 13.5 Hz, 1F, F-
2
3
3
C2), -230.1 (tdd, JFH = 47.5 Hz, JFH = 20.6 Hz, JFF = 13.5 Hz,
1F, F-C1). ESI-MS: (m/z) required: [(C8H15F2Br)]+ = 228.0; (m/z)
found: [(C8H15F2Br)]+ = 228.0. FT-IR (휈̃= cm-1): 2936 (w), 2861
(w), 1456 (w), 1433 (w), 1355 (w), 1259 (w), 1233 (w), 1148 (w),
1038 (w), 920 (w), 864 (w), 727 (w).
9-Bromo-1,2-difluorononane (X). Prepared according to a
modified literature procedure.16a A Teflon® vessel was charged
with 9-bromonon-1-ene (103 mg, 0.50 mmol, 1.0 eq.), p-
iodotoluene (22 mg, 0.10 mmol, 0.2 eq.), DCE (1.25 ml),
amine:HF 1:5 (1.25 ml) and Selectfluor (266 mg, 0.75 mmol,
1.5 eq.). The reaction vessel was then sealed with a Teflon®
screw cap. After stirring at room temperature for 26 h, the mixture
was poured into an aqueous saturated solution of NaHCO3
(15 ml). Once the gas evolution has ceased the aqueous layer was
extracted with CH2Cl2 (3 x 10 ml). The combined organic layers
were dried over Na2SO4 and concentrated under reduced pressure.
Purification by column chromatography on silica gel
(n-pentane/Et2O 20:1) afforded the desired product as a colourless
liquid (80 mg, 66%). Rf = 0.49 (CyH/EtOAc 14:1). 1H NMR (599
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
pentane/Et2O 9:1). H NMR (400 MHz, CDCl3):  7.12 (m, 2H,
H-C5), 6.81 (m, 2H, H-C6), 4.15 (m, 4H, H-C9), 3.77 (s, 3H, H-
C8), 3.59 (t, 3JHH = 7.8 Hz, 1H, H-C2), 3.15 (d, 3JHH = 7.8 Hz, 2H,
3
H-C3), 1.21 (t, JHH = 7.1 Hz, 6H, H-C10). 13C{1H} NMR
(101 MHz, CDCl3):  169.1 (C1), 158.5 (C7), 130.0 (C4), 130.0
(C5), 114.0 (C6), 61.6 (C9), 55.4 (C8), 54.3 (C2), 34.0 (C3), 14.2
(C10). ESI-MS: (m/z) required: [(C15H20O5)Na]+ = 303.1213;
(m/z) found: [(C15H20O5)Na]+ = 303.1203. FT-IR (휈̃= cm-1):
2983 (w), 2937 (w), 2838 (w), 1729 (s), 1613 (w), 1585 (w), 1513
(m), 1465 (w), 1444 (w), 1391 (w), 1369 (w), 1337 (w), 1300 (m),
1277 (m), 1246 (s), 1177 (m), 1147 (s), 1110 (m), 1096 (w), 1062
(w), 1031 (s), 858 (w), 822 (m), 753 (w), 719 (w), 686 (w). Ana-
lytical data in agreement with literature.36
2
3
MHz, CD2Cl2): δ 4.66 (dddddd, JFH = 49.6 Hz, JFH = 21.1 Hz,
3JHH = 8.3, 5.6, 4.7, 2.2 Hz, 1H, H-C2), 4.49 (dddd, JFH
=
2
3
2
3
47.5 Hz, JFH = 26.6 Hz, JHH = 10.9 Hz, JHH = 2.2 Hz, 1H, H-
2
3
2
C1), 4.44 (dddd, JFH = 47.5 Hz, JFH = 24.1 Hz, JHH = 10.9 Hz,
3JHH = 5.7 Hz, 1H, H-C1), 3.42 (t, 3JHH = 6.8 Hz, 2H, H-C9), 1.89
3
2
– 1.82 (m, 2H, H-C8), 1.69 (ddddd, JFH = 15.8 Hz, JHH
=
=
3
3
13.9 Hz, JHH = 9.7, 8.4, 5.1 Hz, 1H, H-C3), 1.56 (ddddd, JFH
Diethyl 2-benzyl-2-octylmalonate (2a). The compound was
synthesized from malonate 1a (250 mg, 1.0 mmol, 1.0 eq.) and 1-
bromooctane (190 µl, 1.1 mmol, 1.1 eq) following GP1 (reaction
time: 21 h). Purification by column chromatography on silica gel
(n-pentane/Et2O 60:1 → 30:1) afforded the desired product as a
colourless oil (238 mg, 66%). Rf = 0.28 (n-pentane/Et2O 30:1). 1H
NMR (400 MHz, CDCl3):  7.28 – 7.18 (m, 3H, H-C6/7), 7.09 –
7.05 (m, 2H, H-C5), 4.33 – 3.99 (m, 4H, H-C16), 3.23 (s, 2H, H-
C3), 1.79 – 1.72 (m, 2H, H-C8), 1.31 – 1.25 (m, 12H, H-
28.8 Hz, 2JHH = 13.9 Hz, 3JHH = 10.0, 5.6, 4.6 Hz, 1H, H-C3), 1.50
– 1.31 (m, 8H, H-C4/5/6/7). 13C{1H} NMR (151 MHz, CD2Cl2): δ
1
2
1
92.6 (dd, JFC = 171.7 Hz, JFC = 18.8 Hz, C2), 84.9 (dd, JFC
=
2
172.7 Hz, JFC = 22.3 Hz, C1), 34.7 (C9), 33.4 (C8), 30.4 (dd,
2JFC = 20.7 Hz, JFC = 6.7 Hz, C3), 29.7 (C5), 29.1 (C6), 28.6
3
(C7), 25.2 (d, 3JFC = 4.7 Hz, C4). 19F NMR (564 MHz, CD2Cl2): δ
2
3
-187.1 (dddddd, JFH = 49.6 Hz, JFH = 28.8, 26.6, 24.1, 15.8 Hz,
3JFF = 13.3 Hz, 1F, F-C2), -228.3 (tdd, JFH = 47.5 Hz, JFH
=
2
3
3
3
21.1 Hz, JFF = 13.2 Hz, 1F, F-C1). ESI-MS: (m/z) required:
[(C9H17F2Br)Na]+ = 265.0374; (m/z) found: [(C9H17F2Br)Na]+ =
265.0383. FT-IR (휈̃= cm-1): 2934 (w), 2858 (w), 1456 (w), 1439
(w), 1392 (w), 1355 (w), 1251 (w), 1227 (w), 1146 (w), 1029 (w),
920 (w), 851 (w), 820 (w), 725 (w), 667 (w).
C9/10/11/12/13/14), 1.24 (t, JHH = 7.1 Hz, 6H, H-C17), 0.88 (t,
3JHH = 6.7 Hz, 3H, H-C15). 13C{1H} NMR (101 MHz, CDCl3): 
171.6 (C1), 136.5 (C4), 130.0 (C5), 128.4 (C6), 127.0 (C7), 61.3
(C16), 59.0 (C2), 38.0 (C3), 32.0 (C9), 31.8 (C8), 29.9 (C11),
[29.5, 29.3] (C12/13), 24.2 (C10), 22.8 (C14), 14.3 (C15), 14.2
(C17). ESI-MS: (m/z) required: [(C22H34O4)Na]+ = 385.2349;
(m/z) found: [(C22H34O4)Na]+ = 385.2339. FT-IR (휈̃= cm-1):
3032 (w), 2939 (w), 2862 (w), 1729 (m), 1605 (w), 1497 (w),
1455 (w), 1390 (w), 1367 (w), 1262 (w), 1182 (m), 1095 (m),
1031 (m), 919 (w), 862 (w), 784 (w), 743 (w), 701 (w). Analytical
data in agreement with literature.9
Diethyl 2-benzylmalonate (1a). Prepared according to a modi-
fied literature procedure.34 A flame-dried flask was charged with
NaH (60% dispersed in mineral oil, 240 mg, 6.0 mmol, 1.2 eq.)
and DMF (25 ml) under argon and diethyl malonate (915 µl,
6.0 mmol, 1.2 eq.) was added dropwise at 0 °C. After stirring at
0 °C for 45 min, benzyl bromide (595 µl, 5.0 mmol, 1.0 eq.) was
carefully added and the reaction was stirred at room temperature
for 20 h. The mixture was diluted with H2O (30 ml) and then
extracted with Et2O (3 x 50 ml). The combined organic layers
were washed with brine (2x), dried over MgSO4 and concentrated
under reduced pressure. Purification by column chromatography
on silica gel (n-pentane/Et2O 1:0 → 19:1 → 9:1) afforded the
desired product as a colourless liquid (760 mg, 61%). Rf = 0.32
Diethyl 2-(4-methoxybenzyl)-2-octylmalonate (3a). The
compound was synthesized from malonate 1b (200 mg,
0.71 mmol, 1.0 eq.) and 1-bromooctane (135 µl, 0.78 mmol,
1.1 eq.) following GP1 (reaction time: 22 h). Purification by
column chromatography on silica gel (CyH/EtOAc 19:1) afforded
the desired product as a slightly yellow oil (219 mg, 78%). Rf =
0.29 (CyH/EtOAc 19:1). 1H NMR (400 MHz, CDCl3):  6.98 (m,
2H, H-C5), 6.78 (m, 2H, H-C6), 4.17 (m, 4H, H-C17), 3.77 (s,
1
(n-pentane/Et2O 9:1). H NMR (400 MHz, CDCl3):  7.30 -7.25
(m, 2H, H-C6), 7.24 – 7.18 (m, 3H, H-C5/7), 4.16 (q, JHH
3
=
3H, H-C8), 3.17 (s, 2H, H-C3), 1.79 – 1.71 (m, 2H, H-C9), 1.32 –
3
3
7.1 Hz, 2H, H-C8), 4.15 (q, JHH = 7.1 Hz, 2H, H-C8), 3.64 (t,
1.21 (m, 18H, H-C10/11/12/13/14/15, H-C18), 0.88 (t, JHH
=
3JHH = 7.9 Hz, 1H, H-C2), 3.22 (d, 3JHH = 7.9 Hz, 2H, H-C3), 1.20
6.9 Hz, 3H, H-C16). 13C{1H} NMR (101 MHz, CDCl3):  171.6
(C1), 158.6 (C7), 131.0 (C5), 128.4 (C4), 113.8 (C6), 61.2 (C17),
59.0 (C2), 55.3 (C8), 37.2 (C3), 31.7 (C9), [32.0, 29.9, 29.5, 29.3,
24.2, 22.8](C10/11/12/13/14/15), 14.3 (C16), 14.2 (C18). ESI-
MS: (m/z) required: [(C23H36O5)Na]+ = 415.2484; (m/z) found:
[(C23H36O5)Na]+ = 415.2460. FT-IR (휈̃= cm-1): 2956 (w), 2926
(t, JHH = 7.1 Hz, 6H, H-C9). 13C{1H} NMR (101 MHz, CDCl3):
3
 169.0 (C1), 138.0 (C4), 129.0 (C5), 128.6 (C6), 126.8 (C7),
61.6 (C8), 54.0 (C2), 34.8 (C3), 14.1 (C9). ESI-MS: (m/z)
required:
[(C14H18O4)Na]+ =
273.1097;
(m/z)
found:
[(C14H18O4)Na]+ = 273.1101. FT-IR (휈̃= cm-1): 2983 (w), 2939
(w), 1729 (s), 1605 (w), 1496 (w), 1455 (w), 1391 (w), 1369 (w),
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Journal of Medicinal Chemistry
Page 8 of 15
(w), 2856 (w), 1731 (m), 1613 (w), 1584 (w), 1513 (m), 1465 (w),
1299 (w), 1259 (w), 1179 (m), 1132 (w), 1094 (w), 1033 (m), 916
(w), 863 (w), 779 (w), 742 (w), 700 (m).
1
2
3
4
5
6
7
8
1445 (w), 1367 (w), 1301 (w), 1248 (m), 1223 (m), 1205 (m),
1176 (m), 1115 (m), 1096 (w), 1034 (m), 848 (w), 822 (w), 723
(w), 683 (w). Analytical data in agreement with literature.9
Diethyl
2-(4-methoxybenzyl)-2-(8-methylnonyl)malonate
(3c). The compound was synthesized from malonate 1b (117 mg,
0.42 mmol, 1.00 eq.) and Z (130 mg, 0.42 mmol, 1.00 eq.) follow-
ing GP1 (reaction time: overnight). Purification by column chro-
matography on silica gel (n-pentane/Et2O 30:1) afforded the
desired product as a colourless oil (52 mg, 30%). Rf = 0.30
(CyH/EtOAc 3:1). 1H NMR (400 MHz, CDCl3) δ 6.98 (m, 2H, H-
C5), 6.78 (m, 2H, H-C6), 4.17 (m, 4H, H-C18), 3.77 (s, 3H, H-
C8), 3.17 (s, 2H, H-C3), 1.79 – 1.71 (m, 2H, H-C9), 1.51 (hept,
Diethyl 2-benzyl-2-nonylmalonate (2b). The compound was
synthesized from malonate 1a (60 mg, 0.24 mmol, 1.00 eq.) and
1-bromononane (55 µl, 0.30 mmol, 1.20 eq.) following GP1
(reaction time: overnight). Purification by column chromatog-
raphy on silica gel (n-pentane/Et2O 30:1) afforded the desired
product as a colourless liquid (65 mg, 72%). Rf = 0.24 (n-
1
pentane/Et2O 20:1). H NMR (600 MHz, CD2Cl2): δ 7.28 – 7.24
9
(m, 2H, H-C6), 7.24 – 7.21 (m, 1H, H-C7), 7.11 – 7.05 (m, 2H,
H-C5), 4.20 – 4.11 (m, 4H, H-C17), 3.20 (s, 2H, H-C3), 1.74 –
3JHH
C10/11/12/13/14/19), 1.18 – 1.11 (m, 2H, H-C15), 0.86 (d, 3JHH
= 6.7 Hz, 1H, H-C16), 1.29 – 1.21 (m, 16H, H-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
=
1.69 (m, 2H, H-C8), 1.34
–
1.25 (m, 14H, H-
6.7 Hz, 6H, H-C17). 13C{1H} NMR (101 MHz, CDCl3) δ 171.6
(C1), 158.6 (C7), 130.9 (C5), 128.4 (C4), 113.8 (C6), 61.2 (C18),
59.0 (C2), 55.3 (C8), 39.2 (C15), 37.2 (C3), 31.7 (C9), [29.9,
29.9, 29.5, 27.5, 24.2](C10/11/12/13/14), 28.1 (C16), 22.8 (C17),
14.2 (C19). ESI-MS: (m/z) required: [(C25H40O5)Na]+ = 443.2768;
(m/z) found: [(C25H40O5)Na]+ = 443.2751. FT-IR (휈̃= cm-1): 2984
(w), 2926 (w), 2855 (w), 1732 (m), 1613 (w), 1584 (w), 1513 (m),
1465 (w), 1445 (w), 1385 (w), 1366 (w), 1301 (w), 1248 (m),
1218 (m), 1176 (m), 1131 (w), 1114 (w), 1096 (w), 1035 (m), 865
(w), 845 (w), 822 (w), 723 (w), 681 (w).
3
C9/10/11/12/13/14/15), 1.23 (t, JHH = 7.1 Hz, 6H, H-C18), 0.89
3
(t, JHH = 7.1 Hz, 3H, H-C16). 13C{1H} NMR (151 MHz,
CD2Cl2): δ 171.8 (C1), 137.2 (C4), 130.5 (C5), 128.7 (C6), 127.4
(C7), 61.7 (C17), 59.3 (C2), 38.3 (C3), 32.5 (C15), 32.1 (C8),
30.3 (C9), [30.1, 29.92, 29.86] (C11/12/13), 24.6 (C10), 23.3
(C14), 14.5 (C16), 14.4 (C18). ESI-MS: (m/z) required:
[(C23H36O4)Na]+ = 399. 2505; (m/z) found: [(C23H36O4)Na]+ =
399.2501. FT-IR (휈̃= cm-1): 3031 (w), 2957 (w), 2925 (m), 2855
(w), 2359 (w), 1732 (s), 1605 (w), 1497 (w), 1455 (w), 1367 (w),
1298 (w), 1258 (m), 1180 (m), 11267 (m), 1095 (w), 1084 (m),
1032 (m), 915 (w), 863 (w), 781 (w), 741 (w), 700 (m).
Diethyl 2-(4-methoxybenzyl)-2-nonylmalonate (3b). The
compound was synthesized from malonate 1b (200 mg,
0.71 mmol, 1.00 eq) and 1-bromononane (163 µl, 0.78 mmol,
1.10 eq.) following GP1 (reaction time: overnight). Purification
by column chromatography on silica gel (CyH/EtOAc 19:1) af-
forded the desired product as a colourless oil (212 mg, 73%). Rf =
0.23 (CyH/EtOAc 19:1). 1H NMR (400 MHz, CDCl3): δ 6.98 (m,
2H, H-C5), 6.78 (m, 2H, H-C6), 4.17 (m, 4H, H-C18), 3.77 (s,
Diethyl 2-benzyl-2-(8,8,8-trifluorooctyl)malonate (2d). The
compound was synthesized from malonate 1a (49 mg, 0.20 mmol,
1.5 eq.) and Y (44 mg, 0.13 mmol, 1.00 eq.) following GP1 (reac-
tion time: 30 h. As TLC (CyH/EtOAc 4:1) indicated incomplete
consumption of Y, the mixture was heated to 60 °C for 24 h).
Purification by column chromatography on silica gel (n-
pentane/Et2O 50:1) afforded the desired product as a white solid
(12 mg, 22%). Rf = 0.29 (CyH/EtOAc 15:1). M.p. 42.3-43.0 °C.
1H NMR (600 MHz, CDCl3):  7.25 – 7.23 (m, 2H, H-C6), 7.23 –
7.20 (m, 1H, H-C7), 7.08 – 7.05 (m, 2H, H-C5), 4.22 – 4.13 (m,
4H, H-C16), 3.23 (s, 2H, H-C3), 2.10 – 2.00 (m, 2H, H-C14),
3H, H-C8), 3.17 (s, 2H, H-C3), 1.80 – 1.70 (m, 2H, H-C9), 1.31 –
1.22 (m, 20H, H-C10/11/12/13/14/15/16/19), 0.88 (t, JHH
3
3
=
1.76 (dd, JHH = 10.8, 5.1 Hz, 2H, H-C8), 1.56 – 1.50 (m, 2H, H-
6.8 Hz, 3H, H-C17). 13C{1H} NMR (101 MHz, CDCl3): δ 171.6
(C1), 158.6 (C7), 131.0 (C5), 128.4 (C4), 113.8 (C6), 61.2 (C18),
59.0 (C2), 55.3 (C8), 37.2 (C3), 31.7 (C9), [32.0, 29.9, 29.6, 29.5,
29.4, 24.2, 22.8](C10/11/12/13/14/15/16), 14.3 (C17), 14.2 (C19).
ESI-MS: (m/z) required: [(C24H38O5)Na]+ = 429.2611; (m/z)
found: [(C24H38O5)Na]+ = 429.2611. FT-IR (휈̃= cm-1): 2956 (w),
2925 (w), 2855 (w), 1731 (m), 1613 (w), 1584 (w), 1514 (m),
1465 (w), 1445 (w), 1367 (w), 1301 (w), 1248 (m), 1217 (m),
1177 (m), 1096 (w), 1034 (m), 864 (w), 845 (w), 822 (w), 796
(w), 722 (w), 682 (w).
C13), 1.37 – 1.25 (m, 8H, H-C9/10/11/12), 1.24 (t, 3JHH = 7.1 Hz,
6H, H-C17). 13C{1H} NMR (151 MHz, CDCl3):  171.5 (C1),
1
136.4 (C4), 130.0 (C5), 128.4 (C6), 127.4 (q, JFC = 276.2 Hz,
C15), 127.0 (C7), 61.3 (C16), 58.9 (C2), 38.2 (C3), 33.8 (q, 2JFC
=
28.4 Hz, C14), 31.8 (C8), 29.6 (C9), 29.0 (C10/11), 28.7 (C12),
24.1 (C10/11), 21.9 (q, 3JFC = 2.9 Hz, C13), 14.2 (C17). 19F NMR
3
(564 MHz, CDCl3): δ -66.4 (t, JFH = 11.0 Hz, 3F, F-C15). ESI-
MS: (m/z) required: [(C22H31O4F3)Na]+ = 439.2067; (m/z) found:
[(C22H31O4F3)Na]+ = 439.2072. FT-IR (휈̃= cm-1): 2980 (w), 2956
(w), 2938 (w), 2864 (w), 1746 (w), 1723 (s), 1497 (w), 1451 (w),
1392 (w), 1368 (w), 1306 (w), 1285 (m), 1275 (m), 1264 (m),
1253 (m), 1228 (m), 1211 (m), 1187 (m), 1172 (m), 1160 (m),
1135 (s), 1094 (m), 1031 (m), 982 (w), 961 (w), 923 (w), 861 (w),
826 (w), 795 (w), 776 (w), 746 (m), 726 (w), 703 (m), 655 (w).
Analytical data in agreement with literature.9
Diethyl 2-benzyl-2-(8-methylnonyl)malonate (2c). The com-
pound was synthesized from malonate 1a (113 mg, 0.45 mmol,
1.5 eq.) and Z (94 mg, 0.30 mmol, 1.00 eq.) following GP1 (reac-
tion time: 30 h. As TLC (CyH/EtOAc 4:1) indicated incomplete
consumption of Z, the mixture was heated to 60 °C for 24 h).
Purification by column chromatography on silica gel
(n-pentane/Et2O 60:1 → 30:1) afforded the desired product as a
colourless liquid (46 mg, 40%). Rf = 0.23 (n-pentane /Et2O 30:1).
1H NMR (600 MHz, CDCl3):  7.26-7.23 (m, 2H, C-H6), 7.22-
7.19 (m, 1H, H-C7), 7.09-7.06 (m, 2H, H-C5), 4.22 – 4.13 (m,
4H, H-C17), 3.23 (s, 2H, H-C3), 1.76 (dd, JHH = 10.7, 5.1 Hz, 2H,
H-C8), 1.51 (dq, JHH = 13.3, 6.7 Hz, 1H, H-C15), 1.30 – 1.24 (m,
10H, H-C9/10/11/12/13), 1.24 (t, JHH = 7.1 Hz, 6H, H-C18), 1.17-
1.12 (m, 2H, H-C14), 0.86 (d, JHH = 6.6 Hz, 6H, H-C16). 13C{1H}
NMR (151 MHz, CDCl3):  171.5 (C1), 136.5 (C4), 130.0 (C5),
128.3 (C6), 127.0 (C7), 61.2 (C17), 59.0 (C2), 39.2 (C14), 38.1
(C3), 31.8 (C8), [29.91, 29.88, 29.5, 27.5, 24.2](C9/10/11/12/13),
28.1 (C15), 22.8 (C16), 14.2 (C18). ESI-MS: (m/z) required:
[(C24H38O4)Na]+ = 413.2662; (m/z) found: [(C24H38O4)Na]+ =
413.2662. FT-IR (휈̃= cm-1): 2955 (w), 2926 (w), 2855 (w), 1732
(m), 1605 (w), 1496 (w), 1465 (w), 1455 (w), 1385 (w), 1366 (w),
Diethyl
2-(4-methoxybenzyl)-2-(8,8,8-
trifluorooctyl)malonate (3d). The compound was synthesized
from malonate 1b (108 mg, 0.39 mmol, 1.00 eq.) and Y (132 mg,
0.39 mmol, 1.00 eq.) following GP1 (reaction time: 24 h). Purifi-
cation by column chromatography on silica gel (n-pentane/Et2O
20:1) afforded the desired product as a colourless oil (140 mg,
81%). Rf = 0.81 (n-pentane/Et2O 5:1). 1H NMR (600 MHz,
Methanol-d4): δ 7.03 (m, 2H, H-C5), 6.84 (m, 2H, H-C6), 4.19
(m, 4H, H-C17), 3.79 (s, 3H, H-C8), 3.18 (s, 2H, H-C3), 2.16 (m,
2H, H-C15), 1.78 – 1.73 (m, 2H, H-C9), 1.61 – 1.55 (m, 2H, H-
3
C14), 1.45 – 1.30 (m, 8H, H-C10/11/12/13), 1.27 (t, JHH
=
7.1 Hz, 6H, H-C18). 13C{1H} NMR (151 MHz, Methanol-d4): δ
172.8 (C1), 160.2 (C7), 132.0 (C5), 128.9 (q, JFC = 275.4 Hz,
1
C16), 114.7 (C6), 62.3 (C17), 60.2 (C2), 55.6 (C8), 38.2 (C3),
2
34.4 (q, JFC = 28.2 Hz, C15), 32.8 (C9), 30.6 (C11), 30.0 (C12),
3
29.6 (C13), 25.0 (C10), 23.0 (q, JFC = 3.0 Hz, C14), 14.4 (C18).
19F NMR (564 MHz, Methanol-d4): δ -68.0 (t, 3JFH = 11.2 Hz, 3F,
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Journal of Medicinal Chemistry
F-C16). ESI-MS: (m/z) required: [(C23H33O5F3)Na]+ = 439.2067;
Diethyl 2-benzyl-2-(8,9-difluorononyl)malonate (2e). The
(m/z) found: [(C23H33O5F3)Na]+ = 439.2072. FT-IR (휈̃= cm-1):
2939 (w), 1730 (m), 1613 (w), 1514 (m), 1465 (w), 1389 (w),
1367 (w), 1248 (m), 1177 (m), 1134 (m), 1034 (m), 845 (w), 737
(w). Analytical data in agreement with literature.9
1
2
3
4
5
6
7
8
compound was synthesized from malonate 1a (113 mg,
0.45 mmol, 1.5 eq.) and X (73 mg, 0.30 mmol, 1.0 eq.) following
GP1 (reaction time: 48 h). Purification by column chromatog-
raphy on silica gel (n-pentane/Et2O 25:1 → 15:1) afforded the
desired product as a colourless liquid (72 mg, 58%). Rf = 0.23
Diethyl 2-benzyl-2-(7,8-difluorooctyl)malonate (2f). The
compound was synthesized from malonate 1a (69 mg,
0.275 mmol, 1.1 eq.) and S7 (57 mg, 0.25 mmol, 1.0 eq) follow-
ing GP1 (reaction time: 17 h). Purification by column chromatog-
raphy on silica gel (CyH/EtOAc 20:1) afforded the desired prod-
uct as a colourless oil (76 mg, 69%). Rf = 0.15 (CyH /EtOAc
15:1). 1H NMR (599 MHz, CDCl3):  7.26 – 7.23 (m, 2H, H-C6),
7.23 – 7.19 (m, 1H, H-C7), 7.08 – 7.05 (m, 2H, H-C5), 4.66
1
(CyH /EtOAc 10:1). H NMR (600 MHz, CDCl3):  7.26-7.23
(m, 2H, H-C6), 7.23-7.19 (m, 1H, H-C7), 7.08-7.05 (m, 2H, H-
2
3
3
C5), 4.65 (dddddd, JFH = 49.2 Hz, JFH = 20.8 JHH = 8.3, 5.4,
3
3
4.5, 2.5 Hz, 1H, H-C15), 4.48 (ddd, JFH = 25.4 Hz, JHH = 10.8,
2.5 Hz, 1H, H-C16), 4.43 (dd, JHH = 10.8, 5.5 Hz, 1H, H-C15),
3
9
4.22-4.13 (m, 4H, H-C17), 3.23 (s, 2H, H-C3), 1.78-1.74 (m, 2H,
H-C8), 1.74-1.65 (m, 1H, H-C14), 1.62-1.51 (m, 1H, H-C14),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
2
3
3
1.47 (dtt, JHH = 10.4, 8.1, 4.9 Hz, 1H, H-C13), 1.41 – 1.35 (m,
(dddddd, JFH = 49.2 Hz, JFH = 20.6 Hz, JHH = 8.2, 5.5, 4.6,
3
2
3
1H, H-C13), 1.35 – 1.26 (m, 8H, H-C9/10/11/12), 1.24 (t, JHH
=
2.5 Hz, 1H, H-C14), 4.49 (dddd, JFH = 47.3 Hz, JFH = 25.3 Hz,
2JHH = 10.8 Hz, JHH = 2.5 Hz, 1H, H-C15), 4.44 (dddd, JFH
=
3
2
7.1 Hz, 6H, H-C18). 13C{1H} NMR (151 MHz, CDCl3):  171.5
3
2
3
47.8 Hz, JFH = 23.7 Hz, JHH = 10.8 Hz, JHH = 5.5 Hz, 1H, H-
C15), 4.23 – 4.13 (m, 4H, H-C16), 3.23 (s, 2H, H-C3), 1.78 – 1.74
(m, 2H, H-C8), 1.74 – 1.65 (m, 1H, H-C13), 1.62 – 1.51 (m, 1H,
H-C13), 1.50 – 1.43 (m, 1H, H-C12), 1.42 – 1.26 (m, 7H, H-
(C1), 136.5 (C4), 130.0 (C5), 128.4 (C6), 127.0 (C7), 91.9 (dd,
1JFC = 172.5 Hz, JFC = 19.0 Hz, C15), 84.3 (dd, JFC = 173.6 Hz,
2
1
2JFC = 22.9 Hz, C16), 61.3 (C17), 59.0 (C2), 38.1 (C3), 31.8 (C8),
2
3
30.1 (dd, JFC = 20.7 Hz, JFC = 6.4 Hz, C14), 29.7 (C10), [29.3,
C9/10/11/12), 1.24 (t, JHH = 7.2 Hz, H-C17). 13C{1H} NMR
3
3
29.2](C11/12), 24.8 (d, JFC = 4.6 Hz, C13), 24.2 (C9), 14.2
3
(C18). 19F NMR (564 MHz, CDCl3):  -188.93 (dm, JFF
=
(151 MHz, CDCl3):  171.5 (C1), 136.4 (C4), 130.0 (C5), 128.4
(C6), 127.0 (C7), 91.9 (dd, 1JFC = 172.5 Hz, 2JFC = 19.2 Hz, C14),
13.5 Hz, 1F, F-C15), -229.91 (tdd, 2JFH = 47.5 Hz, 3JFH = 20.6 Hz,
3JFF
13.6 Hz, 1F, F-C16). ESI-MS: (m/z) required:
1
2
84.3 (dd, JFC = 173.6 Hz, JFC = 23.0 Hz, C15), 61.3 (C16), 58.9
=
2
3
[(C23H34O4F2)Na]+ = 435.2317; (m/z) found: [(C23H34O4F2)Na]+ =
435.2323. FT-IR (휈̃= cm-1): 2937 (w), 2858 (w), 1729 (m), 1605
(w), 1497 (w), 1455 (w), 1390 (w), 1367 (w), 1299 (w), 1259 (w),
1181 (m), 1117 (w), 1096 (w), 1081 (w), 1030 (m), 918 (w), 861
(w), 780 (w), 743 (w), 701 (m), 671 (w).
(C2), 38.2 (C3), 31.8 (C8), 30.1 (dd, JFC = 20.7 Hz, JFC
=
3
6.4 Hz, C13), 29.7 (C10), 29.2 (C11), 24.8 (d, JFC = 4.6 Hz,
C12), 24.1 (C9), 14.2 (C17). 19F NMR (564 MHz, CDCl3):  -
2
3
189.0 (dddddd, JFH = 49.2 Hz, JFH = 29.3, 25.3, 23.7, 15.9 Hz,
3JFF = 13.6 Hz, 1F, F-C14), -229.9 (tdd, JFH = 47.5 Hz, JFH
=
2
3
3
20.6 Hz, JFF = 13.6 Hz, 1F, F-C15). ESI-MS: (m/z) required:
[(C22H32O4F2)Na]+ = 421.2161; (m/z) found: [(C22H32O4F2)Na]+ =
421.2162. FT-IR (휈̃= cm-1): 3032 (w), 2939 (w), 2862 (w), 1729
(m), 1605 (w), 1497 (w), 1455 (w), 1390 (w), 1367 (w), 1262 (w),
1182 (m), 1095 (m), 1031 (m), 919 (w), 862 (w), 784 (w), 743
(w), 701 (m).
Diethyl 2-(8,9-difluorononyl)-2-(4-methoxybenzyl)malonate
(3e). The compound was synthesized from malonate 1b (115 mg,
0.41 mmol, 1.0 eq.) and X (100 mg, 0.41 mmol, 1.0 eq.) following
GP1 (reaction time: overnight). Purification by column chroma-
tography on silica gel (n-pentane/Et2O 10:1) afforded the desired
product as a colourless oil (76 mg, 42%). Rf = 0.19 (n-
1
pentane/Et2O 5:1). H NMR (600 MHz, CDCl3): δ 6.98 (m, 2H,
Diethyl 2-(7,8-difluorooctyl)-2-(4-methoxybenzyl)malonate
(3f). The compound was synthesized from malonate 1b (81 mg,
0.29 mmol, 1.0 eq.) and S7 (60 mg, 0.26 mmol, 1.0 eq following
GP1 (reaction time: overnight). Purification by column chroma-
tography on silica gel (CyH/EtOAc 7:1 followed by n-
pentane/Et2O 10:1) afforded the desired product as a colourless oil
(70 mg, 63%). Rf = 0.16 (n-pentane/Et2O 9:1). 1H NMR
(600 MHz, CDCl3): δ 6.98 (m, 2H, H-C5), 6.78 (m, 2H, H-C6),
4.66 (dddddd, 2JFH = 49.2 Hz, 3JFH = 20.6 Hz, 3JHH = 8.3, 5.4, 4.5,
2
3
H-C5), 6.78 (m, 2H, H-C6), 4.66 (dddddd, JFH = 49.2 Hz, JFH
=
3
20.7 Hz, JHH = 8.3, 5.5, 4.6, 2.5 Hz, 1H, H-C16), 4.49 (dddd,
2JFH = 47.3 Hz, 3JFH = 25.4 Hz, 2JHH = 10.8 Hz, 3JHH = 2.5 Hz, 1H,
2
3
2
H-C17), 4.44 (dddd, JFH = 47.8 Hz, JFH = 23.7 Hz, JHH
=
10.8 Hz, 3JHH = 5.5 Hz, 1H, H-C17) 4.17 (m, 4H, H-C18), 3.77 (s,
3H, H-C8), 3.17 (s, 2H, H-C3), 1.74 (m, 2H, H-C9), 1.72 – 1.65
(m, 1H, H-C15), 1.62 – 1.51 (m, 1H, H-C15), 1.50 – 1.43 (m, 1H,
H-C14), 1.42 – 1.22 (m, 15H, H-C10/11/12/13, H-C14, H-C19).
13C{1H} NMR (151 MHz, CDCl3): δ 171.6 (C1), 158.7 (C7),
3
2.5 Hz, 1H, H-C15), 4.49 (dddd, 2JFH = 47.3 Hz, JFH = 25.3 Hz,
1
3
2
2JHH = 10.8 Hz, JHH = 2.5 Hz, 1H, H-C16), 4.44 (dddd, JFH
=
130.9 (C5), 128.4 (C4), 113.8 (C6), 91.9 (dd, JFC = 172.5 Hz,
1
2
2JFC = 19.1 Hz, C16), 84.3 (dd, JFC = 173.6 Hz, JFC = 23.0 Hz,
3
2
3
47.8 Hz, JFH = 23.7 Hz, JHH = 10.8 Hz, JHH = 5.5 Hz, 1H, H-
C16), 4.17 (m, 4H, H-C17), 3.77 (s, 3H, H-C8), 3.17 (s, 2H, H-
C3), 1.74 (m, 2H, H-C9), 1.72 – 1.65 (m, 1H, H-C14), 1.62 – 1.44
(m, 2H, H-C13, H-C14), 1.42 – 1.26 (m, 7H, H-C13, H-
C17), 61.2 (C18), 59.0 (C2), 55.3 (C8), 37.3 (C3), 31.8 (C9), 30.2
2
3
(dd, JFC = 20.7 Hz, JFC = 6.4 Hz, C15), 29.8 (C11), [29.3,
3
29.2](C12/13), 24.8 (d, JFC = 4.6 Hz, C14), 24.2 (C10), 14.2
(C19). 19F NMR (564 MHz, CDCl3): δ -188.9 (dddddd, JFH
=
2
C10/11/12), 1.24 (t, JHH = 7.1 Hz, 6H, H-C18). 13C{1H} NMR
3
3
3
49.2 Hz, JFH = 29.1, 25.4, 23.7, 15.7 Hz, JFF = 13.6 Hz, 1F, F-
(151 MHz, CDCl3): δ 171.6 (C1), 158.7 (C7), 130.9 (C5), 128.3
2
3
3
(C4), 113.8 (C6), 91.9 (dd, 1JFC = 172.5 Hz, 2JFC = 19.2 Hz, C15),
C16), -229.9 (tdd, JFH = 47.5 Hz, JFH = 20.6 Hz, JFF = 13.6 Hz,
1F, F-C17). ESI-MS: (m/z) required: [(C24H36O5F2)Na]+ =
465.2429; (m/z) found: [(C24H36O5F2)Na]+ = 465.2429. FT-IR
(휈̃= cm-1): 2936 (w), 2858 (w), 1729 (m), 1613 (w), 1583 (w),
1513 (m), 1464 (w), 1446 (w), 1389 (w), 1367 (w), 1301 (w),
1248 (m), 1215 (m), 1178 (m), 115 (m), 1096 (w), 1032 (m), 919
(w), 846 (w), 822 (w), 797 (w), 724 (w), 670 (w).
1
2
84.3 (dd, JFC = 173.6 Hz, JFC = 23.0 Hz, C16), 61.2 (C17), 59.0
(C2), 55.3 (C8), 37.3 (C3), 31.8 (C9), 30.1 (dd, 2JFC = 20.8, 3JFC
=
3
6.3 Hz, C14), 29.7 (C12), 29.2 (C11), 24.8 (d, JFC = 4.5 Hz,
C13), 24.1 (C10), 14.2 (C18). 19F NMR (564 MHz, CDCl3): δ -
2
3
189.0 (dddddd, JFH = 49.2 Hz, JFH = 29.1, 25.3, 23.7, 15.7 Hz,
3JFF = 13.6 Hz, 1F, F-C15), -229.9 (tdd, JFH = 47.5 Hz, JFH
=
2
3
3
20.6 Hz, JFF = 13.6 Hz, 1F, F-C16). ESI-MS: (m/z) required:
[(C23H34O5F2)Na]+ = 451.2272; (m/z) found: [(23H34O5F2)Na]+ =
451.2269. FT-IR (휈̃= cm-1): 2939 (w), 2860 (w), 1728 (m), 1612
(w), 1584 (w), 1513 (m), 1465 (w), 1446 (w), 1390 (w), 1367 (w),
1301 (w), 1248 (m), 1221 (m), 1178 (m), 1114 (m), 1095 (m),
1033 (m), 922 (w), 864 (w), 845 (m), 822 (w), 749 (w), 726 (w),
702 (w), 675 (w).
5-Benzyl-5-octylpyrimidine-2,4,6(1H,3H,5H)-trione
(4a).
This compound was synthesized from 2a (188 mg, 0.52 mmol)
following GP2 (reaction time: 2 h). Purification by column chro-
matography on silica gel (CyH/EtOAc 3:1) afforded the desired
product as a white solid (129 mg, 75%). HPLC: tr = 5.1 min,
purity = 99%. Rf = 0.34 (CyH/EtOAc 4:1). M.p. 148.2 –
1
148.6 °C. H NMR (400 MHz, CDCl3):  8.42 (s, 2H, NH), 7.24
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
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– 7.18 (m, 3H, H-C6/7), 7.11 – 7.06 (m, 2H, H-C5), 3.27 (s, 2H,
H-C3), 2.18 2.10 (m, 2H, H-C8) 1.26 (m, 12H, H-
13C{1H} NMR (101 MHz, CDCl3): δ 172.3 (C1), 159.2 (C7),
1
2
3
4
5
6
7
8
–
148.1 (C18), 130.6 (C5), 126.5 (C4), 114.3 (C6), 59.2 (C2), 55.3
(C8), 44.5 (C3), 39.0 (C9), [32.0, 29.6, 29.6, 29.4, 29.3, 25.4,
22.8] (C10/11/12/13/14/15/16), 14.2 (C17). ESI-MS: (m/z)
required: [(C21H30N2O4)Na]+ = 397.2098; (m/z) found:
[(C21H30N2O4)Na]+ = 397.2098. FT-IR (휈̃= cm-1): 3237 (w),
2967 (w), 2922 (m), 1853 (w), 1754 (m), 1690 (s), 1612 (w), 1582
(w), 1513 (m), 1447 (m), 1401 (w), 1380 (m), 150 (m), 1301 (m),
1275 (m), 1248 (m), 1178 (m), 1112 (w), 1028 (m), 806 (m), 779
(m), 746 (m), 723 (w), 666(w). Analytical data in agreement with
literature.9
C9/10/11/12/13/14), 0.87 (t, JHH= 6.9 Hz, 3H, H-C15). 13C{1H}
NMR (101 MHz, CDCl3):  172.3 (C1), 148.3 (C16), 134.6 (C4),
129.5 (C5), 128.9 (C6), 128.0 (C7), 59.0 (C2), 45.1 (C3), 39.2
(C8), [31.9, 29.6, 29.28, 29.26, 25.4, 22.7](C9/10/11/12/13/14),
14.2 (C15). ESI-MS: (m/z) required: [(C19H26N2O3)Na]+ =
353.1836; (m/z) found: [(C19H26N2O3)Na]+ = 353.1834. FT-IR
(휈̃= cm-1): 3221 (m), 3121 (m), 3030 (w), 2959 (w), 2925 (m),
2854 (m), 1760 (s), 1743 (m), 1721 (m), 1698 (s), 1583 (w), 1537
(w), 1491 (w), 1429 (s), 1378 (m), 1355 (s), 1336 (m), 1300 (m),
1291 (m), 1274 (m), 1234 (w), 1210 (m), 1073 (w), 1029 (m), 926
(w), 833 (s), 792 (m), 753 (m), 735 (m), 702 (s), 665(w). Analyti-
cal data in agreement with literature.9
3
9
5-Benzyl-5-(8-methylnonyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (4c). This compound was synthesized 2c (18 mg,
0.04 mmol) following GP2 (reaction time: 4 h). Purification by
column chromatography on silica gel (CyH/EtOAc 3:1) afforded
the desired product as a white solid (8 mg, 50%). HPLC: tr = 4.5
min, purity = 95%. Rf = 0.21 (CyH /EtOAc 4:1). M.p. 157.7 –
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(4-Methoxybenzyl)-5-octylpyrimidine-2,4,6(1H,3H,5H)-
trione (5a). This compound was synthesized from 3a (150 mg,
0.38 mmol) following GP2 (reaction time: 2 h). Purification by
column chromatography on silica gel (CyH/EtOAc 3:1) afforded
the desired product as a white solid (57 mg, 41%). HPLC: tr = 6.2
min, purity = 99%. Rf = 0.32 (CyH/EtOAc 2:1). M.p.: 133 °C. 1H
NMR (400 MHz, CDCl3): δ 8.10 (s, 2H, HN), 6.96 – 6.91 (m, 2H,
H-C5), 6.71 – 6.66 (m, 2H, H-C6), 3.69 (s, 3H, H-C8), 3.15 (s,
2H, H-C3), 2.09 – 2.01 (m, 2H, H-C9), 1.25 – 1.10 (m, 12H, H-
C10/11/12/13/14/15), 0.80 (t, 3JHH = 6.8 Hz, 3H, H-C16). 13C{1H}
NMR (101 MHz, CDCl3): δ 172.3 (C1), 159.2 (C7), 148.1 (C17),
130.6 (C5), 126.5 (C4), 114.3 (C6), 59.2 (C2), 55.3 (C8), 44.5
(C3), 39.0 (C9), [31.9, 29.6, 29.3, 29.3, 25.4,
22.7](C10/11/12/13/14/15), 14.2 (C16). ESI-MS: (m/z) required:
1
159.0 °C. H NMR (500 MHz, CDCl3):  7.67 (s, 2H, NH), 7.25
– 7.22 (m, 3H, H-C6/7), 7.11 – 7.07 (m, 2H, H-C5), 3.26 (s, 2H,
H-C3), 2.18 – 2.10 (m, 2H, H-C8), 1.50 (hept, 3JHH = 6.8 Hz, 1H,
H-C15), 1.33 – 1.11 (m, 12H, H-C9/10/11/12/13/14), 0.86 (d,
3JHH = 6.6 Hz, 6H, H-C16). 13C{1H} NMR (126 MHz, CDCl3): 
171.8 (C1), 147.4 (C17), 134.5 (C4), 129.5 (C5), 129.0 (C6),
128.0 (C7), 59.0 (C2), 45.2 (C3), 39.2 (C8), 39.1 (C14), [29.8,
29.6, 29.4] (C10/11/12), 28.1 (C15), 27.4 (C13), 25.4 (C9), 22.8
(C16). ESI-MS: (m/z) required: [(C21H30N2O3)Na]+ = 381.2149;
(m/z) found: [(C21H30N2O3)Na]+ = 381.2154. FT-IR (휈̃= cm-1):
3225 (m), 3121 (w), 2924 (m), 2854 (w), 1759 (m), 1698 (s),
1492 (w), 1451 (m), 1430 (s), 1380 (m), 1354 (s), 1341 (m), 1302
(m), 1265 (w), 1215 (w), 1194 (w), 1077 (w), 1029 (w), 831 (s),
776 (w), 753 (w), 734 (m), 702 (s), 666 (w). Analytical data in
agreement with literature.9
[(C20H28N2O4)Na]+ =
383.1937;
(m/z)
found:
[(C20H28N2O4)Na]+ = 383.1947. FT-IR (휈̃= cm-1): 3233 (m),
3119 (w), 2959 (w), 2924 (m), 2854 (w), 1755 (m), 1690 (s), 1612
(w), 1582 (w), 1413 (m), 1422 (m), 1403 (m), 1380 (m), 1353
(m), 1301 (m), 1248 (m), 1211 (m), 1194 (m), 1178 (m), 1111
(w), 1028 (m), 953 (w), 938 (w), 809 (m), 779 (m), 747 (m), 666
(w). Analytical data in agreement with literature.9
5-(4-Methoxybenzyl)-5-(8-methylnonyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (5c). This compound was synthesized
from 3c (32 mg, 0.08 mmol) following GP2 (reaction time: 3 h).
Purification by column chromatography on silica gel (CyH/EtOAc
4:1) afforded the desired product as a white solid (22 mg, 71%).
HPLC: tr = 5.5 min, purity = 97%. Rf = 0.12 (CyH/EtOAc 4:1).
5-Benzyl-5-nonylpyrimidine-2,4,6(1H,3H,5H)-trione (4b).
The compound was synthesized from 2b (34 mg, 0.09 mmol)
following GP2 (reaction time: 3.5 h). Purification by column
chromatography on silica gel (CyH/EtOAc 4:1) afforded the
desired product as a white solid (12 mg, 39%). HPLC: tr = 4.7
min, purity = 98%. Rf = 0.12 (CyH/EtOAc 4:1). M.p.: 145 °C. 1H
NMR (400 MHz, CDCl3): δ 8.13 (br, 2H, NH), 7.25 – 7.19 (m,
3H, H-C6/7)), 7.11 – 7.05 (m, 2H, C5), 3.27 (s, 2H, C3), 2.17 –
1
M.p.: 141 °C. H NMR (400 MHz, CDCl3): δ 8.39 (s, 2H, HN),
7.00 (m, 2H, H-C5), 6.74 (m, 2H, H-C6), 3.75 (s, 3H, H-C8), 3.22
(s, 2H, H-C3), 2.11 (m, 2H, H-C9), 1.50 (hept, 3JHH = 6.6 Hz, 1H,
H-C16), 1.32 – 1.09 (m, 12H, H-C10/11/12/13/14/15), 0.85 (d,
3JHH = 6.6 Hz, 6H, H-C17). 13C{1H} NMR (101 MHz, CDCl3): δ
172.4 (C1), 159.2 (C7), 148.3 (C18), 130.6 (C5), 126.5 (C4),
114.2 (C6), 59.2 (C2), 55.3 (C8), 44.4 (C3), 39.1 (C9), 39.1
(C15), [29.9, 29.6, 29.4](C11/12/13), 28.1 (C16), 27.4 (C14), 25.4
(C10), 22.8 (C17). ESI-MS: (m/z) required: [(C22H32N2O4)Na]+ =
411.2254; (m/z) found: [(C22H32N2O4)Na]+ = 411.2252. FT-IR
(휈̃= cm-1): 3229 (m), 2958 (w), 2921 (m), 2853 (w), 1755 (m),
1691 (s), 1611 (w), 1582 (w), 1514 (m), 1450 (m), 1403 (m),
1381 (m), 1355 (m), 1338 (m), 1304 (m), 1279 (m), 1247 (s),
1216 (m), 1191 (w), 1178 (m), 1112 (w), 1027 (m), 807 (m), 779
(s), 745 (m), 724 (m), 667 (m). Analytical data in agreement with
literature.9
2.11 (m, 2H, H-C8), 1.35
–
1.16 (m, 14H, H-
C9/10/11/12/13/14/15), 0.87 (t, JHH = 6.8 Hz, 3H, H-C16). 13C
NMR (101 MHz, CDCl3): δ 172.1 (C1), 148.0 (C17), 134.6 (C4),
129.5 (C5), 128.9 (C6), 128.1 (C7), 59.0 (C2), 45.2 (C3), 39.2
(C8), 32.0 (C15), 29.6 (C9), [29.5, 29.4, 29.3] (C11/12/13), 25.4
(C10), 22.8 (C14), 14.2 (C16). ESI-MS: (m/z) required:
3
[(C20H28N2O3)Na]+ =
367.1992;
(m/z)
found:
[(C20H28N2O3)Na]+ = 367.1988. FT-IR (휈̃= cm-1): 3217 (m),
3108 (w), 2958 (w), 2921 (m), 2852 (m), 1752 (m), 1698 (s),
1605 (w), 1545 (w), 1496 (w), 1447 (m), 1427 (m), 1408 (m),
1380 (m), 1354 (m), 1343 (m), 1301 (m), 1207 (w), 1194 (w),
1073 (w), 1032 (w), 967 (w), 917 (w), 858 (m), 846 (m), 799 (m),
778 (m), 753 (m), 700 (s), 670 (m). Analytical data in agreement
with literature.9
5-(4-Methoxybenzyl)-5-nonylpyrimidine-2,4,6(1H,3H,5H)-
trione (5b). This compound was synthesized from 3b (28 mg,
0.07 mmol) following GP2 (reaction time: 3 h). Purification by
column chromatography on silica gel (CyH/EtOAc 3:1) afforded
the desired product as a white solid (25 mg, 96%). HPLC: tr = 5.9
min, purity = 98%. Rf = 0.23 (CyH/EtOAc 2:1). M.p.: 111 °C. 1H
NMR (400 MHz, CDCl3): δ 8.21 (s, 2H, HN), 7.00 (m, 2H, H-
C5), 6.75 (m, 2H, H-C6), 3.76 (s, 3H, H-C8), 3.21 (s, 2H, H-C3),
5-Benzyl-5-(8,8,8-trifluorooctyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (4d). This compound was synthesized
from 2d (11 mg, 0.03 mmol) following GP2 (reaction time: 4 h).
Purification by column chromatography on silica gel (CyH/EtOAc
3:1) afforded the desired product as a white solid (6.0 mg, 60%).
HPLC: tr = 6.9 min, purity = 96%. Rf = 0.13(CyH /EtOAc 4:1).
M.p.: 126.2 – 127.3 °C. 1H NMR (599 MHz, CDCl3):  7.74 (br,
2H, NH), 7.25 – 7.22 (m, 3H, H-C6/7), 7.10 – 7.06 (m, 2H, H-
C5), 3.26 (s, 2H, H-C3), 2.17 – 2.12 (m, 2H, H-C8), 2.09 – 2.01
(m, 2H, H-C14), 1.55 – 1.50 (m, 2H, C-13), 1.36 – 1.27 (m, 6H,
H-C10/11/12), 1.25 – 1.18 (m, 2H, H-C9). 13C{1H} NMR
(151 MHz, CDCl3):  171.8 (C1), 147.5 (C16), 134.4 (C4), 129.5
2.11 (m, 2H, H-C9), 1.33
C10/11/12/13/14/15/16), 0.87 (t, JHH = 6.8 Hz, 3H, H-C17).
–
1.14 (m, 14H, H-
3
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(C5), 129.0 (C6), 128.1 (C7), 127.4 (q, JFC = 276.4 Hz, C15),
Purification by column chromatography on silica gel (CyH/EtOAc
2:1) afforded the desired product as a white solid (40 mg, 91%).
HPLC: tr = 31.3 min, purity = 95%. Rf = 0.12 (CyH/EtOAc 2:1).
M.p.: 94 °C. 1H NMR (600 MHz, CDCl3): δ 8.43 – 8.32 (m, 2H,
HN), 7.00 (m, 2H, H-C5), 6.75 (m, 2H, H-C6), 4.65 (dddddd,
2JFH = 49.1 Hz, 3JFH = 20.8 Hz, 3JHH = 8.3, 5.4, 4.5, 2.5 Hz, 1H, H-
2
1
2
3
4
5
6
7
8
59.0 (C2), 45.4 (C3), 38.9 (C8), 33.8 (q, JFC = 28.3 Hz, C14),
29.3 (C10), 28.9 (C11), 28.6 (C12), 25.2 (C9), 21.9 (q, JFC
2.9 Hz, C13). 19F NMR (564 MHz, CDCl3):  -66.4 (t, JFH
3
=
=
3
11.0 Hz,
3F,
F-C15).
ESI-MS:
407.1553;
(m/z)
(m/z)
required:
found:
[(C19H23N2O3F3)Na]+ =
2
C15), 4.48 (dddd, JFH = 47.3 Hz, 3JFH = 26.1 Hz, 2JHH = 10.8 Hz,
[(C19H23N2O3F3)Na]+ = 407.15549. FT-IR (휈̃= cm-1): 3223 (m),
3120 (w), 2935 (w), 2857 (w), 1759 (m), 1699 (s), 1583 (w), 1539
(w), 1492 (w), 1432 (m), 1380 (m), 1356 (m), 1341 (m), 1303
(m), 1253 (s), 1197 (m), 1180 (m), 1137 (m), 1103 (m), 1078 (m),
1054 (m), 1029 (m), 976 (w), 834 (m), 792 (m), 733 (m), 701 (s),
654 (m). Analytical data in agreement with literature.9
2
3
3JHH = 2.5 Hz, 1H, H-C16), 4.44 (dddd, JFH = 47.8 Hz, JFH
=
2
3
23.9 Hz, JHH = 10.8 Hz, JHH = 5.5 Hz, 1H, H-C16), 3.75 (s, 3H,
H-C8), 3.21 (s, 2H, H-C3), 2.12 (m, 2H, H-C9), 1.74 – 1.64 (m,
1H, H-C14), 1.61 – 1.49 (m, 1H, H-C14), 1.49 – 1.41 (m, 1H, H-
C13), 1.40 – 1.27 (m, 5H, H-C13, H-C11/12), 1.25 – 1.17 (m, 2H,
H-C10). 13C{1H} NMR (151 MHz, CDCl3): δ 172.4 (C1), 159.2
(C7), 148.3 (C17), 130.6 (C5), 126.4 (C4), 114.3 (C6), 91.8 (dd,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
34
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49
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59
60
5-(4-Methoxybenzyl)-5-(8,8,8-trifluorooctyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (5d). This compound was synthesized
from 3d (60 mg, 0.13 mmol) following GP2 (reaction time: 4 h).
Purification by column chromatography on silica gel (CyH/EtOAc
3:1) afforded a white solid. The solid was recrystallized from
CH2Cl2 and CyH, which afforded the desired product as colour-
less needles (27 mg, 48%). HPLC: tr = 8.5 min, purity = 95%.
1JFC = 172.5 Hz, JFC = 19.3 Hz, C15), 84.2 (dd, JFC = 173.6 Hz,
2
1
2JFC = 23.1 Hz, C16), 59.1 (C2), 55.3 (C8), 44.6 (C3), 38.7 (C9),
2
3
30.0 (dd, JFC = 20.8 Hz, JFC = 6.4 Hz, C14), 29.3 (C11), 28.9
(C12), 25.2 (C10), 24.7 (d, JFC = 4.6 Hz, C13). 19F NMR
3
(564 MHz, CDCl3): δ -189.0 (dddddd, 2JFH = 49.1 Hz, 3JFH = 29.2,
3
1
26.1, 23.9, 15.6 Hz, JFF = 13.5 Hz, 1F, F-C15), -229.9 (tdd,
Rf = 0.26 (CyH/EtOAc 2:1). M.p.: 110 °C. H NMR (600 MHz,
3
3
2JFH = 47.5 Hz, JFH = 20.6 Hz, JFF = 13.5 Hz, 1F, F-C16). ESI-
MS: (m/z) required: [(C20H26N2O4F2)Na]+ = 419.1758; (m/z)
found: [(C20H26N2O4F2)Na]+ = 419.1748. FT-IR (휈̃= cm-1): 3240
(w), 2932 (w), 2858 (w), 1755 (m), 1692 (m), 1612 (w), 1583 (w),
1513 (m), 1446 (m), 1381 (m), 1352 (m), 1300 (m), 1247 (m),
1179 (m), 1111 (w), 1069 (w), 1027 (m), 923 (w), 838 (m), 778
(m), 746 (m), 665 (w).
CDCl3): δ 8.19 – 8.14 (m, 2H, HN), 7.00 (m, 2H, H-C5), 6.75 (m,
2H, H-C6), 3.75 (s, 3H, H-C8), 3.21 (s, 2H, H-C3), 2.12 (m, 2H,
H-C9), 2.05 (m, 2H, H-C15), 1.56 – 1.50 (m, 2H, H-C14), 1.36 –
1.26 (m, 6H, H-C11/12/13), 1.24 – 1.17 (m, 2H, H-C10). 13C{1H}
NMR (151 MHz, CDCl3): δ 172.2 (C1), 159.3 (C7), 148.1 (C17),
1
130.6 (C5), 127.3 (q, JFC = 275.8 Hz, C16), 126.4 (C4), 114.3
(C6), 59.1 (C2), 55.3 (C8), 44.6 (C3), 38.7 (C9), 33.8 (q, JFC
2
=
5-Benzyl-5-(8,9-difluorononyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (4e). This compound was synthesized from 2e (70 mg,
0.17 mmol) following GP2 (reaction time: 4.5 h). Purification by
column chromatography on silica gel (CyH/EtOAc 3:1) afforded
the desired product as a white solid (35 mg, 55%). HPLC: tr =
21.1 min, purity = 96%. Rf = 0.27 (CyH/EtOAc 3:1). M.p.: 163.6
28.4 Hz, C15), 29.3 (C11), 28.8 (C12), 28.6 (C13), 25.3 (C10),
3
21.9 (q, JFC = 2.8 Hz, C14). 19F NMR (564 MHz, CDCl3): δ -
3
66.4 (t, JFH = 11.0 Hz, 3F, F-C16). ESI-MS: (m/z) required:
[(C20H25N2O4F3)Na]+ =
437.1664;
(m/z)
found:
[(C20H25N2O4F3)Na]+ = 437.1648. FT-IR (휈̃= cm-1): 3233 (w),
2931 (w), 2855 (w), 1755 (m), 1693 (m), 1611 (w), 1513 (m),
1450 (m), 1381 (m), 1338 (m), 1304 (m), 1247 (s), 1192 (m),
1177 (m), 1136 (s), 1052 (m), 1026 (m), 974 (m), 836 (m), 806
(m), 779 (m), 743 (m), 656 (m). Analytical data in agreement with
literature.9
1
– 164.2 °C. H NMR (600 MHz, CDCl3):  8.58 (br, 2H, H-N),
7.23 – 7.19 (m, 3H, H-C6/7), 7.09 – 7.07 (m, 2H, H-C5), 4.65
2
2
(dm, JFH = 49.2 Hz, 1H, H-C15), 4.48 (dddd, JFH = 47.3 Hz,
3JFH = 25.4 Hz, JHH = 10.8 Hz, JHH = 2.4 Hz, 1H, H-C16), 4.44
(dddd, JFH = 47.7 Hz, JFH = 23.6 Hz JHH = 10.8 Hz, JHH
2
3
2
3
2
3
=
5-Benzyl-5-(7,8-difluorooctyl)pyrimidine-2,4,6(1H,3H,5H)-
trione (4f). This compound was synthesized from 2f (54 mg,
0.135 mmol) following GP2 (reaction time: 2 h). Purification by
column chromatography on silica gel (CyH/EtOAc 3:1) afforded
the desired product as a white solid (27 mg, 55%). HPLC: tr =
27.4 min, purity = 99%. Rf = 0.21 (CyH /EtOAc 3:1). M.p.: 130.0
5.5 Hz, 1H, H-C16), 3.27 (s, 2H, H-C3), 2.20 – 2.08 (m, 2H, H-
3
2
3
C8), 1.69 (dddt, JFH = 18.2 Hz, JHH = 9.8, JHH = 8.5, 4.8 Hz,
1H, H-C14), 1.62 – 1.50 (m, 1H, H-C14), 1.50 – 1.43 (m, 1H, H-
3
C13), 1.38 (ddq, JHH = 8.2, 5.5, 2.7 Hz, 1H, H-C13), 1.35 – 1.25
(m, 6H, H-C10/11/12), 1.25 – 1.18 (m, 2H, H-C9). 13C{1H} NMR
(151 MHz, CDCl3):  172.4 (C1), 148.5 (C18), 134.5 (C4), 129.5
1
– 130.5 °C. H NMR (600 MHz, CDCl3):  8.45 (s, 2H, NH),
1
2
(C5), 128.9 (C6), 128.0 (C7), 91.9 (dd, JFC = 172.5 Hz, JFC
19.1 Hz, C15), 84.2 (dd, JFC = 173.5 Hz, JFC = 23.0 Hz C16),
58.9 (C2), 45.2 (C3), 39.0 (C8), 30.1 (dd, JFC = 20.7 Hz, JFC
=
7.25 – 7.20 (m, 3H, H-C6/7), 7.10 – 7.06 (m, 2H, H-C5), 4.73 –
4.57 (m, 1H, H-C14), 4.57 – 4.35 (m, 2H, H-C15), 3.27 (s, 2H, H-
C3), 2.19 – 2.10 (m, 2H, H-C8), 1.76 – 1.64 (m, 1H, H-C13), 1.62
– 1.50 (m, 1H, H-C13), 1.46 (ttd, 3JHH = 9.7, 7.7, 6.9, 5.1 Hz, 1H,
H-C12), 1.41 – 1.29 (m, 5H, H-C9/11/12), 1.27 – 1.19 (m, 2H, H-
C10). 13C{1H} NMR (151 MHz, CDCl3):  172.3 (C1), 148.3
(C16), 134.5 (C4), 129.5 (C5), 128.9 (C6), 128.0 (C7), 91.8 (dd,
1
2
2
3
=
6.4 Hz, C14), 29.4 (C10), 29.2 (C12), 29.0 (C11), 25.2 (C9), 24.8
(d, 3JFC = 6.4 Hz, C13). 19F NMR (564 MHz, CDCl3):  - 188.8 –
189.1 (m, 1F, F-C15), -229.9 (tdd, 2JFH = 47.5 Hz, 3JFH = 20.7 Hz,
3JFF
[(C20H26N2O3F2)Na]+ =
=
13.5 Hz, 1F, F-C16). ESI-MS: (m/z) required:
403.1804; (m/z) found:
2
1
1JFC = 172.6 Hz, JFC = 19.2 Hz, C14), 84.2 (dd, JFC = 173.6 Hz,
2JFC = 23.0 Hz, C15), 58.9 (C2), 45.2 (C3), 38.9 (C8), 30.0 (dd,
[(C20H26N2O3F2)Na]+ = 403.1804. FT-IR (휈̃= cm-1): 3216 (w),
3105 (w), 2932 (w), 2855 (w), 1749 (m), 1696 (s), 1497 (w), 1431
(s), 1410 (w), 1380 (m), 1354 (s), 1302 (m), 1203 (w), 1124 (w),
1091 (w), 1072 (w), 1035 (w), 1010 (w), 983 (w), 918 (w), 855
(m), 794 (m), 778 (w), 731 (m), 700 (s), 674 (w).
2JFC = 20.8 Hz, JFC = 6.4 Hz, C13), 29.3 (C9), 28.9 (C11), 25.2
3
(C10), 24.7 (d, 3JFC = 4.4 Hz, C12). 19F NMR (564 MHz, CDCl3):
2
 -188.9 – -189.2 (m, 1F, F-C14), -229.9 (tdd, JFH = 47.5 Hz,
3
3JFH = 20.6 Hz, JFF = 13.5 Hz, 1F, F-C15). ESI-MS: (m/z)
required: [(C19H24N2O3F2)Na]+ = 389.1647; (m/z) found:
[(C19H24N2O3F2)Na]+ = 389.1653. FT-IR (휈̃= cm-1): 3226 (m),
3118 (w), 2933 (w), 2857 (w), 1759 (m), 1696 (s), 1584 (w), 1536
(w), 1492 (w), 1452 (m), 1428 (m), 1380 (m), 1355 (m), 1341
(m), 1295 (m), 1208 (w), 1120 (w), 1081 (w), 1028 (m), 1008
(m), 926 (w), 831 (m), 753 (m), 734 (s), 702 (s), 664 (m).
5-(7,8-Difluorooctyl)-5-(4-methoxybenzyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (5f). This compound was synthesized
from 3f (48 mg, 0.11 mmol) following GP2 (reaction time: 2.5 h).
5-(8,9-Difluorononyl)-5-(4-methoxybenzyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (5e). This compound was synthesized
from 3e (74 mg, 0.17 mmol) following GP2 (reaction time: 4 h).
Purification by twofold column chromatography on silica gel
(CyH/EtOAc 2:1, followed by CyH/EtOAc 2.2:1) afforded the
desired product as a white solid (47 mg, 68%). HPLC: tr = 67.5
min (enantiomer 1), 87.7 min (enantiomer 2), purity = 97%. Rf =
0.28 (CyH/EtOAc 2:1). M.p.: 119 °C. 1H NMR (600 MHz,
CDCl3): δ 8.43 (s, 2H, HN), 7.00 (m, 2H, H-C5), 6.74 (m, 2H, H-
C6), 4.65 (dddddd, 2JFH = 49.2 Hz, 3JFH = 20.7 Hz, 3JHH = 8.3, 5.4,
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2
3
4.6, 2.5 Hz, 1H, H-C16), 4.48 (dddd, JFH = 47.3 Hz, JFH
=
comparison of poses from different substrates and to generate the
2
3
1
2
3
4
5
6
7
8
25.6 Hz, JHH = 10.8 Hz, JHH = 2.5 Hz, 1H, H-C17), 4.44 (dddd,
figures.25
2JFH = 47.8 Hz, JFH = 23.8 Hz, JHH = 10.8 Hz, JHH =5.5 Hz, H-
C17), 3.75 (s, 3H, H-C8), 3.21 (s, 2H, H-C3), 2.11 (m, 2H, H-C9),
1.74 – 1.64 (m, 1H, H-C15), 1.61 – 1.50 (m, 1H, H-C15), 1.50 –
1.42 (m, 1H, H-C14), 1.40 – 1.26 (m, 7H, H-C14, H-C11/12/13),
1.24 – 1.17 (m, 2H, H-C10). 13C{1H} NMR (151 MHz, CDCl3): δ
172.5 (C1), 159.2 (C7), 148.4 (C18), 130.6 (C5), 126.5 (C4),
3
2
3
ASSOCIATED CONTENT
Supporting information, including experimental and analytical
data, is provided as a PDF file.
AUTHOR INFORMATION
Corresponding Authors
1
2
114.3 (C6), 91.9 (dd, JFC = 172.4 Hz, JFC = 19.2 Hz, C16), 84.3
(dd, JFC = 173.5 Hz, JFC = 23.0 Hz, C17), 59.1 (C2), 55.3 (C8),
44.5 (C3), 38.8 (C9), 30.1 (dd, JFC = 20.7 Hz, JFC = 6.5 Hz,
C15), 29.4 (C11), 29.2 (C13), 29.0 (C12), 25.3 (C10), 24.8 (d,
3JFC = 4.5 Hz, C14). 19F NMR (564 MHz, CDCl3): δ -188.9
1
2
2
3
9
anna.hirsch@helmholtz-hips.de
ryan.gilmour@uni-muenster.de
10
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49
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2
3
3
Author Contributions
(ddddd, JFH = 49.2 Hz, JFH = 29.2, 25.6, 23.8, 15.7 Hz, JFF
=
2
3
13.5 Hz, 1F, F-C16), -229.9 (tdd, JFH = 47.5 Hz, JFH = 20.7 Hz,
3JFF
13.5 Hz, 1F, F-C17). ESI-MS: (m/z) required:
[(C21H28N2O4F2)Na]+ =
433.1909; (m/z) found:
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
=
[(C21H28N2O4F2)Na]+ = 433.1910. FT-IR (휈̃= cm-1): 3233 (w),
3120 (w), 2930(w), 2856 (w), 1756 (m), 1691 (s), 1612 (w), 1582
(w), 1514 (m), 1444 (m), 1435 (m), 1381 (m), 1353 (m), 1339
(m), 1302 (m), 1247 (s), 1206 (m), 1178 (m), 1113 (w), 1077 (w),
1028 (m), 982 (w), 957 (w), 918 (w), 839 (m), 808 (m), 779 (m),
745 (m), 725 (m), 710 (w), 667 (w).
Funding Sources
We gratefully acknowledge generous financial support from the
WWU Münster, the German Research Foundation, DFG (“Cells
in Motion, Cluster of Excellence” CiMIC, and SFB 858) and the
Helmholtz Association’s Initiative and Networking Fund. The
European Commission is acknowledged for an Intra-European
Marie Skłodowska-Curie actions fellowship under Horizon-2020
(796089–NovInDXS, R. P. J.).
METHODS
MMP Assays: As previously described,20 the synthetic fluorogen-
ic substrate (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-
ACKNOWLEDGMENT
We thank the analytical departments of the Institute for Organic
Chemistry at the WWU Münster for technical support.
(3-(2,4-dinitrophenyl)-l-2,3-diamino-propionyl)-Ala-Arg-NH
2
(R&D Systems, Minneapolis, USA) was used to measure MMP-2,
MMP-8, MMP-9 and MMP-13 activities. The inhibition of human
active MMPs by the barbituric acid derivatives were measured by
preincubating MMP-2, MMP-8, MMP-9, MMP-13 (each at 2 nM)
and test compounds at varying concentrations (10 pM–1 mM) in
ABBREVIATIONS
BITE, bioisostere of the trifluoromethyl and ethyl groups; Et,
ethyl; CF3, trifluoromethyl; HF, hydrogen fluoride; r.t., room
temperature.
Tris·HCl (50 mM), pH 7.5, containing NaCl (0.2 M), CaCl (5
2
mM), ZnSO (20 mM) and 0.05 % Brij 35 at 37 °C for 30 min.
4
An aliquot of substrate (10 mL of a 50 mM solution) was then
added to 90 mL of the preincubated MMP/inhibitor mixture, and
the fluorescence was determined by following the product release
over time at 37 °C. The fluorescence changes were monitored
using a Fusion Universal Microplate Analyzer (Packard Biosci-
ence, Massachusetts, USA) with excitation and emission wave-
lengths set to 330 and 390 nm, respectively. Reaction rates were
measured from the initial 10 min of the reaction profile where
product release was linear with time and plotted as a function of
inhibitor concentration. From the resulting inhibition curves, the
REFERENCES
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D. Biosynthesis of an Organofluorine Molecule. Nature
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Crystal Structure and Mechanism of a Bacterial Fluorinat-
ing Enzyme. Nature 2004, 427, 561−565. (e) Zhu, X.; Rob-
inson, D. A.; McEwan, A. R.; O’Hagan, D.; Naismith, J. H.
Mechanism of Enzymatic Fluorination in Streptomyces
cattleya. J. Am. Chem. Soc. 2007, 129, 14597−14604; (f)
Aldemir, H.; Kolhepp, S. V.; Gulder, T.; Gulder, T. A. M.
Structure of a Putative Fluorinated Natural Product from
Streptomyces sp. TC1. J. Nat. Prod. 2014, 77, 2331−2334.
IC value for each inhibitor was calculated by nonlinear regres-
50
sion analysis, performed using the Grace 5.1.8 software (Linux).
Docking Studies: The X-ray crystal structure of MMP9 in com-
plex with inhibitor BE4 (pdb code: 6ESM) was used for our dock-
ing studies.23 The binding site of MMP9 was defined to include
residues within 7.0 Å radius around the bound reference ligand
BZ9 in the LeadIT program. The binding site includes 35 amino
acid residues and a zinc ion. The enantiomers and enol-tautomers
of compound 5e were used as ligands for docking using the FlexX
docking module in the LeadIT suite v2.3.2.24 Top-30 (FlexX)-
scored solutions were retained, and subsequently post-scored with
the HYDE module in LeadIT v2.3.2. After careful visualization to
exclude poses with significant inter- or intramolecular clash terms
or unfavorable conformations, the resulting solutions were subse-
quently ranked according to their binding energies. The top-
ranked pose from each compound was chosen and used for the
comparison. The program SeeSAR 9.2 was used to visualize the
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(3) O´Hagan, D. Understanding Organofluorine Chemistry: An
(difluoromethoxy)-1,1,1-Trifluoroethane. J. Phys. Chem.
1992, 96, 5521-5526.
Introduction to the C-F Bond. Chem. Soc. Rev. 2008, 37,
308-319.
1
2
3
4
5
6
7
8
(15) For examples, see (a) Erdeljac, N.; Kehr, K.; Ahlqvist, M.;
Knerr, L.; Gilmour, R. Exploring Physicochemical Space
via a Bioisostere of the Trifluoromethyl and Ethyl Groups
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