Synthesis and Biological Evaluation of Imidazole-Bearing α-Phosphonocarboxylates as Inhibitors of Rab Geranylgeranyl Transferase (RGGT)

Article abstract of DOI:10.1002/cmdc.201700791

Rab geranylgeranyl transferase (RGGT) is an interesting therapeutic target, as it ensures proper functioning of Rab GTPases, a class of enzymes responsible for the regulation of vesicle trafficking. Relying on our previous studies, we synthesized a set of new α-phosphonocarboxylic acids as potential RGGT inhibitors, with emphasis on the elaboration of imidazole-containing analogues. We identified two compounds with activity similar to that of previously reported RGGT inhibitors, showing structural similarity to imidazo[1,2-a]pyridine-containing analogues in terms of their substitution pattern. Interestingly, analogues of the N-series, derived from another phosphonocarboxylate RGGT inhibitor, 2-fluoro-3-(1H-imidazol-1-yl)-2-phosphonopropanoic acid, turned out to be inactive in our model, indicating that an additional substituent localized at positions C2 or C4 of the imidazole ring, may adversely affect the potency against the targeted enzyme.

Full text of DOI:10.1002/cmdc.201700791

DOI: 10.1002/cmdc.201700791
Full Papers
Synthesis and Biological Evaluation of Imidazole-Bearing
a-Phosphonocarboxylates as Inhibitors of Rab
Geranylgeranyl Transferase (RGGT)
[
a]
[b]
[b]
Łukasz Joachimiak, Aleksandra Marchwicka, Edyta Gendaszewska-Darmach, and
[a]
Katarzyna M. Bła z˙ ewska*
In memory of Prof. Henryk Krawczyk
Rab geranylgeranyl transferase (RGGT) is an interesting thera-
peutic target, as it ensures proper functioning of Rab GTPases,
a class of enzymes responsible for the regulation of vesicle
trafficking. Relying on our previous studies, we synthesized a
set of new a-phosphonocarboxylic acids as potential RGGT in-
hibitors, with emphasis on the elaboration of imidazole-con-
taining analogues. We identified two compounds with activity
similar to that of previously reported RGGT inhibitors, showing
structural similarity to imidazo[1,2-a]pyridine-containing ana-
logues in terms of their substitution pattern. Interestingly, ana-
logues of the N-series, derived from another phosphonocar-
boxylate RGGT inhibitor, 2-fluoro-3-(1H-imidazol-1-yl)-2-phos-
phonopropanoic acid, turned out to be inactive in our model,
indicating that an additional substituent localized at positions
C2 or C4 of the imidazole ring, may adversely affect the poten-
cy against the targeted enzyme.
Introduction
Rab geranylgeranyl transferase (RGGT, Rab GGTase, GGT-II) is re-
sponsible for post-translational prenylation of Rab GTPases, en-
zymes from the Ras superfamily of small GTP-binding proteins,
which are regulators of vesicle trafficking and their secretion
into the extracellular matrix. Most Rab GTPases are modified
by RGGT by formation of thioether bonds between two C-ter-
minal cysteines and lipophilic geranylgeranyl chains (Figure 1).
The abnormal activity of RGGT and some Rab proteins is asso-
ciated with a number of diseases, including neurodegenerative
[1–3]
and infectious disorders as well as several types of cancer.
Figure 1. The isoprenoid biosynthesis pathway.
[
a] Ł. Joachimiak, Dr. K. M. Bła z˙ ewska
Faculty of Chemistry, Lodz University of Technology, Institute of Organic
Chemistry, Z
˙
eromskiego Str. 116, 90-924 Łꢀd z´ (Poland)
E-mail: katarzyna.blazewska@p.lodz.pl
The effect of disrupting geranylgeranylation is also associat-
ed with inhibition of upstream enzymes of the isoprenoid bio-
synthetic pathway, such as farnesyl pyrophosphate synthase
(FPPS), whereas alternative prenylation of the oncogenic K-Ras
GTPase by geranylgeranyl transferases might be responsible
for the low cytotoxicity of farnesyl transferase (FT) inhibitors
[
b] A. Marchwicka, Dr. E. Gendaszewska-Darmach
Faculty of Biotechnology and Food Sciences, Lodz University of Technology,
Institute of Technical Biochemistry, Stefanowskiego Str. 4/10, 90-924 Łꢀdz´
(Poland)
[4,5]
and their failure in clinical trials.
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201700791.
Numerous RGGT inhibitors have been developed in recent
years, with possible application in the therapies of diverse dis-
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[
6–10]
eases.
a-Phosphonocarboxylates (PCs) constitute one class
of RGGT inhibitors, with the most active analogues containing
imidazole in their structure, either in the N-substituted form
[
11]
(
Figure 1: compound F-ZolPC) or as built into the imida-
zo[1,2-a]pyridine ring (Figure 1: compound 3-IPEHPC and its
[
12–14]
analogues).
The N-substituted imidazole ring seems to be
advantageous for inhibitory activity against RGGT, as it is also
present in the most potent RGGT inhibitors reported to date,
[
8]
bearing a tetrahydrobenzodiazepine scaffold.
In our previous studies we evaluated the influence of a sub-
stituent’s type and localization in imidazo[1,2-a]pyridine on ac-
[
14]
tivity toward RGGT. We showed that the introduction of a
substituent of different size, geometry, and electronegativity at
position C6 of this heterocycle results in increased inhibitor ac-
tivity, whereas substitution at any other location abolishes the
potency of such analogues.
Our current efforts are focused on determining the struc-
ture–activity relationship for analogues of 2-fluoro-3-(1H-imida-
zol-1-yl)-2-phosphonopropanoic acid (F-ZolPC). We studied
both the influence of the point of attachment of a-phospho-
nopropionic acid to the imidazole ring (either via N- or C-sub-
stitution), as well as the effect of the substituent’s localization
in the imidazole ring on the compound’s potency toward
RGGT (Figure 2). For clarity, we discuss these two classes, N-
and C-substituted analogues, separately.
Figure 2. Structures of the studied compounds; *1d was obtained as a mix-
ture of 4- and 5-methyl-substituted regioisomers (ratio 1.0:0.3).
subjected the Michael adduct 5 immediately to fluorination,
using sodium hydride and the electrophilic fluorinating agent,
N-fluorobenzenesulfonimide (NFSI). This new one-pot proce-
dure led to triesters 6a–f with significantly improved yields
(76–87%) and purity. The use of Selectfluor as a fluorinating
agent led to lower yields due to formation of side products,
such as double Michael adducts mentioned above. Both
desoxy-5a–f as well as fluoro analogues 6a–f were subjected
to hydrolysis in concentrated hydrochloric acid to afford target
products with 11–91% yields.
Results and Discussion
Synthesis
We first synthesized six new analogues of the N-series, 1a–1 f,
derived from F-ZolPC, modified with a methyl or phenyl group,
or bromine atom at either the 2- or 4(5)-position of the imida-
[
15]
zole ring (Figure 2a). Target compounds 1a–f were prepared
Analogues of the C-series, 1g–l, in which the phosphonocar-
boxylate group is connected to the imidazole ring via carbon,
constituted the second class of studied compounds. For their
synthesis we applied a method previously developed by us for
according to a modified procedure previously developed by
[
11]
us. It involved aza-Michael addition of commercially available
substituted imidazoles 4 to the ethyl 2-(diethoxyphosphoryl)-
acrylate 3 (Scheme 1). Thus obtained triesters 5 were relatively
unstable and spontaneously underwent subsequent reaction
with an acceptor, forming so-called “double Michael addition”
[14]
imidazo[1,2-a]pyridine analogues,
which is based on the
condensation of appropriate aldehydes with triethyl phospho-
noacetate (Scheme 2). We used N-methylated (7h and 7j) as
well as nitrogen-unsubstituted aldehydes (7g, 7i, 7k, 7l) with
[
16,17]
products (Scheme S1).
To circumvent this side reaction, we
Scheme 1. Reagents and conditions: a) THF, RT, 30 min; b) 1. NaH, THF, ꢀ108C, 40 min, 2. NFSI, THF, ꢀ608C, 20 min, <ꢀ208C, 1 h; c) 12m HCl, reflux, 4 h.
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Scheme 2. Reagents and conditions: a) Ph
7
4
3
CCl, TEA, RT, (DMF, 48 h for 7g; CH
h,j,k; THF for 7l) ꢀ208C!RT, 24 h; c) for 9g–i: NaBH , NiCl ·6H O, MeOH, ꢀ208C, 15 min, for 9h,j,k,l: H
8 h; e) 1. NaH, THF, ꢀ10–58C, 50 min, 2. NFSI, THF, ꢀ608C, 20 min, <ꢀ208C, 1 h; f) 12m HCl, reflux, 4 h.
2
Cl
2
, 24 h for 7i); b) triethyl phosphonoacetate, TiCl
4
, TEA, (CH
2 2
Cl for 8g, 8i and
4
2
2
2
, Pd/C, MeOH, RT, 5 h; d) Ph
3
CCl, TEA, DMF, 608C,
or without the additional C-attached methyl substituent. Some
N-unsubstituted aldehydes have limited solubility in the reac-
tion medium, CH Cl , and therefore they were either tritylated
the imidazole ring, could lead to a mixture of regioisomers,
with preference for the formation of the less sterically hindered
4-substituted analogue. Such was the case in the reaction of
ethyl 2-(diethoxyphosphoryl)acrylate 3 with 4(5)-methylimida-
zole 4d (ratio of regioisomers 1:0.3). We used this product mix-
ture for confirmation of the structure of the major regioisomer
by NMR analysis (COSY, HMQC, DEPT135, HMBC, NOESY). In the
case of bromo- and phenyl-substituted imidazoles, only one re-
gioisomer was formed. Our analysis was supported by previ-
ously reported structure determinations of t- and p-regioisom-
2
2
prior to condensation (e.g., compounds 8g and 8i were ob-
[
18–20]
tained in 61 and 99% yields, respectively)
or the conden-
sation was carried out in THF (7l). Other aldehydes were used
[
21]
without prior tritylation.
The condensation of aldehydes with triethyl phosphonoace-
tate led to vinyl analogues 9, obtained as a mixture of E/Z iso-
mers for compounds 9i–l (predominantly the E isomer) or as
the single Z isomer (compounds 9g,h), with yields ranging
from 30 to 78%. In the next step compounds 9 were subjected
[23]
ers of histidinoalanines.
Because the formation of particular regioisomer(s) was de-
termined in the aza-Michael addition, for NMR structural analy-
sis we could choose the analogues among ester (6d) and/or
acids (2d/1d), depending on the satisfactory separation of sig-
[
22]
to hydrogenolysis on Pd-C or NaBH /NiCl ·H O, and thus ob-
4
2
2
tained triesters 10 (49–91%) were fluorinated in the a-position,
using NFSI along with NaH. Triesters 10k,l, bearing two unsub-
stituted nitrogen atoms in the imidazole ring, required trityla-
tion prior to fluorination. Otherwise only traces of products
were detected, whereas tritylated analogues were fluorinated
with 51–53% yields. The other fluorinated products 12 were
obtained with good yields (65–87%). Thus obtained desoxy-
1
13
nals in H and C NMR spectra. The most convenient model
[24]
turned out to be the mixture of esters 6d (Figure 3). We
used the HMBC technique to assign the position of the methyl
group in the imidazole ring, thanks to correlation of the corre-
sponding quaternary aromatic carbon atom with protons in
the b position. While in the minor regioisomer, C3 (128.3 ppm)
shows strong correlation with protons in the b position (Fig-
ure 3a, arrow a), the analogous correlation for the correspond-
10g–l and fluorinated esters 12g–l were subjected to acidic
hydrolysis in concentrated hydrochloric acid, which simultane-
ously cleaved the trityl group, if present. Final acids were ob-
tained with 41–94% yields.
13
ing C signal in the major regioisomer, C6 (138.7 ppm), is not
observed (Figure 3a, arrow h; see spectra in Figure S3). This
observation indicates that the methyl group in the major
isomer is connected to C6 (or position 4 according to the
proper numbering of the imidazole ring). That assignment is
supported by the fact that only in the case of the major
NMR analysis
Synthesis of analogues by the aza-Michael reaction (so-called
N-series), bearing substituents at the 4- and/or 5-positions of
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Cytotoxicity assays with HeLa cells
Inhibition of prenyltransferases or enzymes in the mevalonate
[25–27]
pathway may be associated with induction of cell death.
Therefore, we investigated the antiproliferative activity of the
newly synthesized phosphonocarboxylate analogues on
human cervical carcinoma HeLa cells. Taking into account that
activities of tested compounds could be attenuated or modu-
[28]
lated by serum proteins, cell viability was assessed during
serum deprivation as well as in the presence of FBS (Table 1).
All analogues 1a–f, in which the a-phosphonopropionic
acid moiety is linked through N-substitution with the imidazole
ring, did not influence the number of viable cells up to the
maximum concentration tested (2 mm), except for compound
1
d, which showed negligible inhibition in fasting medium (IC50
1.88 mm). C-substituted analogues 1g,h and 1k did not show
any effect on HeLa cell viability either, whereas 1l indicated in-
significant cytotoxic effect only in serum-free medium (IC50
1.01 mm). However, analogues 1i,j demonstrated cytotoxic
effect under both medium conditions, with IC50 values of 566
and 546 mm in fasting medium and 1313 and 1277 mm in FBS-
containing medium, respectively. Under serum-free conditions,
they showed stronger effects than the reference compound F-
[11]
ZolPC (decrease in cell viability: IC =850 mm) Relative to re-
50
[14]
Figure 3. Key correlations observed in the a) HMBC and b) NOESY spectra ac-
quired for the mixture of regioisomers 6d. For details see Figure S3–S4, Sup-
porting Information.
cently described analogues, these newly synthesized com-
pounds showed slightly weaker antiproliferative activity, which
was especially conspicuous in complete medium, but less pro-
nounced in fasting medium.
isomer, two signals from aromatic tertiary carbon atoms (C5:
137.5 ppm and C8: 116.6 ppm) correlate with protons in the
b position (Figure 3a, arrows f and e), whereas for the minor
isomer only one tertiary carbon (C1, 137.8 ppm) shows such a
correlation (arrow b). Additional confirmation comes from
NOESY experiments (Figure 3b and Figure S2). In the minor re-
gioisomer, the methyl group (2.19 ppm) and one aromatic
proton (H-1, 7.44 ppm) interact with protons in the b position
Effect on prenylation of Rab11A and Rap1A/Rap1B
The novel PCs were screened for their ability to inhibit the ac-
tivity of RGGT and GGT-1 in intact HeLa cells by detection of
un- and misprenylated forms of Rab11A and Rap1A/Rap1B,
which are modified with geranylgeranyl groups by RGGT and
GGT-1, respectively. Inhibition of Rab11A prenylation alone is
indicative of a specific RGGT inhibitor, whereas inhibition of
prenylation of both types of GTPases at similar concentrations
might be indicative of either unselective inhibition of both
prenyl transferases (RGGT and GGT-1), or of inhibition of meval-
onate pathway enzymes, such as GGPPS or FPPS, which play
key roles in providing substrates for prenylation (GGPP and
FPP, respectively).
(
Figure 3b, arrows b and a, respectively). In the case of the
major regioisomer such correlation of the methyl group
2.16 ppm) was not observed, but instead both aromatic pro-
(
tons (H-5, 7.35 ppm and H-8, 6.64 ppm) interacted with pro-
tons in the b position (arrows d and e).
Based on comparative analysis of the correlations observed
in the 2D spectra for bromo- and phenyl-substituted esters 6
and acids 1–2, which showed the same patterns as those ob-
served for the major regioisomer 6d’, we determined that
those analogues bear a substituent at the 4-position.
The inhibitory activity of the set of newly synthesized com-
pounds 1a–1l was initially tested at 50 mm under serum-free
conditions, and Rab11A enrichment in the cytosol fraction was
[14]
examined by western blot analysis (Table 1).
Interestingly,
only two analogues of the C-series, compounds 1i and 1j,
were found to be active at the tested concentration (Figure 4).
They contain the a-phosphonopropionic residue connected to
the imidazole ring through C5, whereas new analogues bear-
ing such a linkage at the C2 or N1 positions were inactive. No-
tably, the active compounds 1i and 1j also had observable cy-
totoxic effects. None of the analogues of the N-series, 1a–f,
derived from the known RGGT inhibitor, F-ZolPC, showed sig-
nificant potency against RGGT, which demonstrates that addi-
Biological studies
All synthesized fluorine-containing compounds
1
were
screened for their biological activity, while desoxy analogues 2
were excluded from such tests as potentially less potent, as
[
13,14]
was indicated in our previous studies.
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Table 1. Effects of synthesized PC analogues on HeLa cell viability in serum-supplemented (SS, 10% FBS) and serum-free (SF) medium, and inhibition of
Rab11A prenylation.
1
2
3
[a]
[b]
Compd
Structure
R/R /R /R
HeLa IC50 [mm]
Rab11A
SS
SF
1
1
1
a
b
c
Me
Ph
Br
NE
NE
NE
NE
NE
NE
ꢀ
ꢀ
ꢀ
1
2
R =Me; R =H
1
d
NE
1880
ꢀ
1
2
R =H; R =Me
1
2
1
1
e
f
R =Ph, R =H
NE
NE
NE
NE
ꢀ
1
2
R =Br, R =H
ꢀ
1
1
g
h
H
Me
NE
NE
NE
NE
ꢀ
ꢀ
[
c]
1
2
3
1
1
1
1
1
1
i
j
R , R , R =H
1313
1277
NE
728
NE
566
546
1701
297
NE
+
+
ꢀ
+
ꢀ
ꢀ
[
c]
[
[
c,d]
c,d]
1
2
3
j-E1
j-E2
k
R =Me; R , R =H
1
2
3
R , R =H; R =Me
1
3
2
l
R , R =H; R =Me
NE
1014
[
a] HeLa cells were treated with test compounds for 72 h and then viable cell number was determined. Data were calculated from the means of eight test
concentrations from at least three independent experiments. NE stands for lack of response (“no effect”) in cells treated with inhibitor up to 1 mm.
b] HeLa cells were treated for 48 h with compounds at 50 mm, then lysed and separated into cytosolic and membrane-rich fractions and western blotted
[
for Rab11A and b-actin in cytosolic fractions. Data are from at least three independent experiments, and indicate for which compounds higher band inten-
sity relative to control (untreated cells) was observed. [c] Compounds in bold were evaluated to inhibit Rab11A and Rap1A/Rap1B prenylation across a
wide concentration range. [d] The enantiomers of 1j are distinguished as E1 and E2, based on their retention times during chiral HPLC separation. The
enantiomer with the shorter retention time is labeled as E1, and the one with the longer retention time, as E2.
dose (LED) inhibition of Rab11A and Rap1A/Rap1B prenylation
(
Table 2, Figure 5). Compound 1i, bearing no additional sub-
stituent in the imidazole ring, showed inhibitory activity
against Rab11A prenylation at 25 mm, whereas analogue 1j,
which has an additional methyl group at the N1 position of
the imidazole ring, was found to be more active, as shown by
a decrease in the LED value to 10 mm. Similar to previous find-
Figure 4. Effect of F-ZolPC analogues (1a–l) on Rab11A prenylation in HeLa
cells. Cells were treated for 48 h with PCs (50 mm) in serum-free medium.
Cells were then lysed and fractionated into cytosolic and membrane-rich
fractions. Cytosolic fractions containing unprenylated proteins were separat-
ed by electrophoresis and western blotted for Rab11A and b-actin.
[12,14]
ings,
only one enantiomer of 1j, compound 1j-E2, had the
desired biological activity to inhibit Rab11A prenylation as
tional substitution at the C2 or C4 positions adversely affects
the activity of this class of compounds.
Table 2. Effects of selected PC analogues on the inhibition of Rab11A
Because 1d and 1l did not affect Rab11A prenylation, their
effect on HeLa cell viability could be associated with a different
mechanism of action. However, it cannot be excluded that in
the case of the methyl-substituted analogue 1d, which had
been obtained and tested as a mixture of 4- and 5-regioisom-
ers, such activity stems from the presence of ꢁ23% of the 5-
substituted analogue (ꢁ12% of the more active stereoisomer),
which seems to posses an advantageous substitution pattern
that corresponds to 3-IPEHPC and 1j.
and Rap1A/Rap1B prenylation in HeLa cells.
[
a]
Compound
LED [mm]
[b]
Rab11A
Rap1A/Rap1B
1
1
1
i
j
25
10
NE
10
NE
NE
NE
NE
j-E1
1j-E2
[
a] LED=lowest effective dose; NE=LED is not included at a concentra-
tion up to 50 mm. [b] The reference compound 3-(6-bromoimidazo[1,2-
a]pyridin-3-yl)-2-fluoro-2-phosphonopropanoic acid was evaluated in par-
allel, showing the same activity as reported previously.
Two selected analogues—1i and 1j—were subjected to a
full-panel five-dose assay to determine the lowest effective
[
14]
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Figure 5. Effect of 1i, 1j, and their enantiomers (1j-E1 and 1j-E2) on Rab11A and Rap1A/Rap1B prenylation in HeLa cells. Cells were treated for 48 h with the
indicated concentrations of PCs (mm) and 10 mm lovastatin (Lov) acting as a positive control in serum-free medium. Cells were then lysed and fractionated
into cytosolic and membrane-rich fractions. Cytosolic fractions containing unprenylated proteins were separated by electrophoresis and western blotted for
Rab11A, Rap1A/Rap1B, and b-actin.
measured by LED (Table 2, Figure 5). Stronger effect of 1j-E2
was also observed comparing the cytotoxic activity of enantio-
mers (Table 1, no effect and 728 mm in full medium and
ized solely at positions C5 and/or N1. The potentially different
interaction mechanism between analogues bearing the 2-
phosphonopropionic acid moiety at either the N1 or C5 posi-
tions cannot be excluded. Studies on the synthesis (including
asymmetric variant) and evaluation of analogues substituted at
those two positions are in progress.
1701 mm and 297 mm in serum-free medium for 1j-E1 and 1j-
E2, respectively). Simultaneously, the selected compounds did
not affect Rap1A/Rap1B prenylation at the concentrations
tested (LED>250 mm, Figure S2).
Because the connection between imidazole and the a-phos-
phonopropionic acid’s residue through the C5 atom is a pref-
erable structural pattern for activity against RGGT, the influence
of additional methyl substituents at other positions of the imi-
dazole ring was analyzed. While addition of the methyl sub-
stituent at N1 slightly increased activity (as with compound
Experimental Section
All reagents were purchased from commercial sources and were
used as obtained, unless specified otherwise. Thin-layer chroma-
tography was performed with silica gel 60 with the F254 indicator
on alumina plates. Precursors of final compounds were purified
using by liquid chromatography (Gilson PLC 2250) coupled with a
mass spectrometer (Advion expression) and 40–63 mm silica gel as
stationary phase. Chromatography was performed with the report-
ed solvent system with gradient elution. Preparative HPLC for pu-
rification of compounds 2c and 2 f was performed using a Gilson
Prep equipped with a UV/Vis-156 detector (237 nm) and semi-
preparative column Kromasil 100-5-C18 (5 mm, 10ꢁ250 mm). Sepa-
ration of enantiomers of compound 1j was performed on a Chiral-
pak QN-AX column (Chiral Technologies Europe; 0.46 cmꢁ15 cm),
1
j), analogues with the methyl group at the C2 or C4 positions
showed no potency toward RGGT at the concentrations tested.
Interestingly, all compounds bearing a substituent at the C2
position (1a–c, 1g,h, 1l), regardless of the position connecting
the a-phosphonopropionic acid with imidazole, did not show
[
29]
any potency, possibly due to steric bulk. The same applies to
compounds bearing a substituent at position C4 of the imida-
zole ring (1d–f, 1k). These compounds bear structural similari-
ty to 2-substituted imidazo[1,2-a]pyridine analogues, which
[12]
according to a previously described method. The column was
[
14]
were found to be inactive toward RGGT.
ꢀ1
eluted isocratically (1 mLmin ) with 0.7m TEAAc containing 75%
Our data indicate that 1i,j are selective micromolar RGGT in-
hibitors with similar or slightly weaker activity than the refer-
ence compound F-ZolPC (LED for inhibition of Rab11A:
MeOH at pH 5.8. Confirmation of the optical purity of enantiomers
was evaluated based on HPLC analysis using a Chiralpak QN-AX
column with a more sensitive detection method provided by a
Gilson prepELS II detector (with temperatures set at 658C and
[
11]
10 mm) and a-fluorinated analogues of 3-IPEHPC (LED for in-
5
08C for drift tube and spray chamber, respectively). The enantio-
hibition of Rab11A: 10 or 25 mm, depending on the substitu-
mer with the shorter retention time, 3.6 min, was termed 1j-E1,
whereas that with the longer retention time, 4.6 min, was termed
[
14]
ent’s character).
1
j-E2. Optical rotations of enantiomers 1j-E1 and 1j-E2 were mea-
20
sured on a PerkinElmer 241 polarimeter, and ½aꢂ values are given
Conclusions
D
ꢀ
1
ꢀ1
in deg·cmg ·dm ; concentration c=1 for 1 g in 100 mL.
We elaborated convenient protocols for N- and C-functionaliza-
tion of the imidazole ring with a 2-phosphonopropionic acid
group. Among the new compounds, we identified two phos-
phonocarboxylates (1i and 1j) that show micromolar potency
as inhibitors of Rab11A prenylation, and which have the ca-
pacity to decrease HeLa cell viability. Both compounds struc-
turally resemble the preferable substitution pattern found in
other phosphonocarboxylate-derived RGGT inhibitors, confirm-
ing the structure–activity relationship disclosed recently for an-
alogues bearing an imidazo[1,2-a]pyridine ring. Simultaneously,
we found that the small imidazole ring constitutes the core
scaffold necessary for the activity of phosphonocarboxylates
against RGGT, and preferable points of modifications are local-
1
NMR spectra were measured at 250.13 or 700 MHz for H NMR,
6
13
31
2.90 or 170 MHz for C NMR, and 283 or 101.30 MHz for P NMR
on Bruker Avance DPX 250 and Bruker Avance II Plus 700 spec-
trometers. Chemical shifts (d) are reported in parts per million
1
(ppm) relative to: internal residual CHCl in CDCl (d=7.26 H NMR)
3
3
13
or CDCl signal in C NMR (d=77.16); internal residual HDO in D O
3 2
d=4.79 H NMR) or external 85% H PO (d=0 ppm P NMR).
and C NMR spectra were proton-decoupled. Assignment of the
signals in H and C NMR was supported by two-dimensional ex-
periments (COSY, HMQC, HMBC, DEPT135). For compounds ob-
tained as mixtures of regioisomers (5d, 6d, 1d, 2d) or isomers E
and Z (9g–l), when two complementary signals overlap, the total
integration of those two signals is given in the NMR description
(e.g., in case of a mixture of isomers in a ratio of 1.0:0.6, total inte-
1
31
31
(
P
3
4
13
1
13
&
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Full Papers
gration of overlapping signals from complementary CH2 group
equals 1.0ꢁ2+0.6ꢁ2=3.2). All final compounds were obtained as
white solids, decomposing before melting point was reached.
Compounds used in biological testing possess a purity of no less
than 95%.
the reaction mixture was stirred for 24 h at room temperature.
Water (50 mL) was the added, and the solution was adjusted to
pH 9 with a saturated Na CO solution. The product was extracted
2
3
with Et O (5ꢁ100 mL). Combined organic phases were dried over
2
MgSO and concentrated. Products were purified by column chro-
4
matography using a CH Cl /acetone eluent system, except for com-
2
2
PrestoBlue Cell Viability Reagent, Mem-PER Plus Membrane Protein
Extraction Kit, and all reagents for cell culture were purchased
from Life Technologies (Carlsbad, CA, USA). Mem-PERꢂ Plus Mem-
brane Protein Extraction Kit was used for cell lysis and separation
of cytosolic and membrane-rich fractions. Bradford Protein Assay
and Clarity Western ECL Substrate were obtained from Bio-Rad
pounds 9j–l, for which a CHCl /MeOH system was used. For prod-
3
ucts containing triethyl phosphonoacetate after unsuccessful purifi-
cation, the approximate yields were calculated for the quantity of
31
1
the product determined based on P and H NMR data.
(
Hercules, CA, USA). Protease inhibitor cocktail and lovastatin were
purchased from Sigma (St. Louis, MO, USA). Primary antibodies
against Rab11A and Rap1A/Rap1B were obtained from Abcam
General procedure for reduction of the double bond in com-
pounds 9g and 9i under NaBH /NiCl conditions on the exam-
(
Cambridge, UK), and primary antibodies against b-actin along
4
2
with secondary HRP-linked antibodies were purchased from Cell
Signaling Technology (Beverly, MA, USA).
ple of the synthesis of compound 10i
To a solution of compound 9i (1.07 g, 1.96 mmol) in MeOH (10 mL)
NiCl ·6H O (0.575 g, 2.42 mmol, 1.2 equiv) was added. The solution
2
2
was cooled to ꢀ208C in a CO /acetone bath, and NaBH (0.229 g,
2
4
General procedure for the synthesis of compounds 5a–f on
the example of compound 5b
6
.06 mmol, 3 equiv) was carefully added so as to maintain temper-
ature below ꢀ108C. The mixture was then stirred for 10 min. Then
Reaction performed in dry flaks purged with Ar. To ethyl 2-(dieth-
oxyphosphoryl)acrylate 3 (300 mg, 1.27 mmol) in THF (6 mL), imi-
dazole 4b (198 mg, 1.37 mmol, 1.1 equiv) at room temperature
was added, and the resulting solution was stirred for 30 min. The
obtained adduct was directly subjected to fluorination or, after
evaporation of THF, to hydrolysis.
the cooling bath was removed, and when the temperature reached
108C, saturated NH
extracted with CH Cl
anhydrous Mg SO and concentrated. The product was purified by
flash liquid chromatography using CH Cl /MeOH system as eluent.
Cl (20 mL) was added. The aqueous layer was
4
(4ꢁ50 mL). The organic layer was dried over
2
2
2
4
2
2
General procedure for the synthesis of compounds 6a–f on
the example of compound 6b
General procedure for reduction of the double bond in com-
pounds 9h,j,k,l upon hydrogenolysis on the example of the
synthesis of compound 10j
Aza-Michael adduct 5b (obtained from 300 mg of 3 and 198 mg of
4
b) in THF (6 mL) was added under argon to a cooled (ꢀ108C)
Reaction was carried out in a single-neck flask equipped with two-
way stopcock, which enabled degassing the system (vacuum–
argon–vacuum, three times) prior to hydrogen supply. In a single-
neck flask compound 9j (0.9 g, 2.84 mmol) was placed in MeOH
suspension of NaH (60 mg, 1.52 mmol, 1.2 equiv, 60% suspension
in oil) in THF (9 mL) within 3 min. It was stirred for 40 min at <58C
and then cooled to ꢀ608C followed by the addition of NFSI
(
480 mg, 1.52 mmol, 1.2 equiv) in THF (6 mL) within 3 min. It was
(
20 mL). The flask was then equipped with a two-way stopcock.
The system was degassed (see above). 10% Pd/C (110 mg, 40 mg/
mmol substrate, 3.64 mol%) was carefully added, and the system
stirred for 20 min at ꢀ608C and then for 60 min at <ꢀ208C. The
reaction was quenched by the addition of a saturated NH Cl solu-
4
1
tion (3 mL) and H O (3 mL). After addition of CHCl3 (20 mL) the
mixture was agitated, and the organic and aqueous phases were
separated. The aqueous phase was additionally extracted with
CHCl (2ꢁ20 mL). The combined organic phases were dried over
MgSO and concentrated. The thus obtained oil was purified by
2
was again degassed. Finally, the system was supplied with hydro-
gen atmosphere in the same manner as for argon (three times),
and this suspension was stirred for 5 h at room temperature. The
catalyst was then filtered off through a thin layer of Celite 500, and
the filtrate was evaporated to dryness. The product was purified
3
4
column chromatography with gradient elution using CH Cl /MeOH
system as eluent.
2
2
by flash liquid chromatography using CHCl /MeOH system as
3
eluent.
General procedure of the Knoevenagel reaction for the syn-
thesis of compounds 9g–l on the example of compound 9i
General tritylation procedure for the synthesis of compounds
11k–l on the example of compound 11l
In a dry and argon-purged double-neck flask equipped with ther-
mometer and septum, triethyl phosphonoacetate was placed
In a dry single-neck flask compound 10l was placed (280 mg,
(
1.09 g, 4.87 mmol) in 20 mL of CH Cl . The solution was cooled to
2 2
0
.88 mmol), and TEA (178 mg, 1.76 mmol, 2 equiv) dissolved in
ꢀ
208C in a CO /acetone bath, and neat TiCl (0.65 mL, 5.84 mmol,
.2 equiv, 110 mL/1 mmol TiCl4) and TEA (1.9 mL, 13.6 mmol,
2
4
DMF (4 mL) was added via syringe. After 10 min of stirring at RT,
trityl chloride was added (270 mg, 0.97 mmol, 1.1 equiv), and the
mixture was stirred for a further 48 h at 608C. The mixture was
then transferred to a separatory funnel in 40 mL EtOAc. It was
washed with brine (15 mL), a saturated solution of Na CO (15 mL)
1
2
.8 equiv) were then added. After 15 min, a solution of aldehyde
1
8
i (1.65 g, 4.87 mmol, 1 equiv) in CH Cl (10 mL) was added, and
2
2
1
2
3
For the synthesis of compounds 9k and 9l, corresponding aldehydes were
and H O (15 mL), dried over MgSO4 and concentrated using a
added without prior dissolution, and therefore the initial triethyl phosphonoa-
2
cetate solution was prepared by using 35 mL CH
tively.
2
Cl
2
and 40 mL THF, respec-
rotary evaporator. The crude product was purified by column chro-
matography using CHCl /MeOH system as eluent.
3
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These are not the final page numbers! ÞÞ

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1
3
2
3
General procedure for the synthesis of compounds 12g–l on
the example of compound 12i
H NMR (283 MHz, D
2
O): d=4.54 (ddd, JHH =15.0 Hz, JFH =7.6 Hz,
3
2
JPH =4.9 Hz, CH H C(F)P, 1H), 4.88 (bdd, JFH =35.8 Hz, JHH
5.0 Hz, CH H C(F)P, 1H), 7.02 (d, JHH =1.6, Hz CHar(IM-4), 1H),
a b
.30 ppm (bs, CHar(IM-5), 2H); C NMR (283 MHz, D O): d=51.48 (dd,
2
=
a
b
3
1
7
Compound 10i (400 mg, 0.732 mmol) was added under argon at-
mosphere to a cooled (ꢀ108C) suspension of NaH (35 mg,
13
2
2
1
JFH =19.8, JPH =8.2 Hz, CH C(F)P, 1C), 98.58 (dd, JFC =194.3 Hz,
2
0
1
.878 mmol, 1.2 equiv, 60% suspension in oil) in THF (6 mL,
.5 mL/100 mg of substrate) within 4 min. It was stirred for 50 min
1
JPC =134.4 Hz, CH C(F)P, 1C), 121.31 (s, C
, 1C), 123.66 (s,
2
ar(IM-2)
2
CHar(IM-5), 1C), 128.49 (s, CHar(IM-4), 1C), 174.60 ppm (d, JFC =22.0,
CO H, 1C).
at ꢀ10 to +58C and then cooled to ꢀ608C followed by the addi-
2
tion of NFSI (276 mg, 0.878 mmol, 1.2 equiv) in THF (4 mL) within
2
-Fluoro-3-(4-methyl-1H-imidazol-1-yl)-2-phosphonopropanoic
4
ꢀ
min. It was stirred for 20 min at ꢀ608C and for 60 min at <
1
acid (R ) and 2-fluoro-3-(5-methyl-1H-imidazol-1-yl)-2-phospho-
nopropanoic acid (R ) (1d): Yield: 63%. Obtained from 0.54 mmol
208C. The reaction was quenched by the addition of saturated
2
NH Cl solution (4 mL) and H O (4 mL). After the addition of CHCl
4
2
3
1
2
31
(182 mg) of 6b. Mixture of regioisomers: R /R =1.0:0.3. P NMR
(
20 mL) the mixture was agitated and then the organic and aque-
1
(283 MHz, D O, pH 8): d=8.08–8.64 ppm (2d overlapping, JPF
ous phases were separated. The aqueous phase was additionally
2
1
2
1
ꢁ
65.0 Hz, R and R , 1.3P); H NMR (700 MHz, D O): d=2.27 (bs,
2
extracted with CHCl (2ꢁ20 mL). Combined organic phases were
3
1
2
CH IM, R , 3H), 2.34 (bs, CH IM, R , 0.9H), 4.50–4.57 (m, CH H C(F)P,
dried over MgSO and concentrated. Thus obtained oil was purified
3
3
a
b
4
1
2
1
2
R and R , 1.3H), 4.87–5.98 (m, CH H C(F)P, R and R , 1.3H), 7.03 (s,
CHar(IM-4), R , 0.3H), 7.07 (s, CH
8
by column chromatography using CH Cl /MeOH system as eluent.
a
b
1
2
2
2
1
, R , 1H), 8.18 (s, CH
, R , 0.3H); C NMR (283 MHz, D O): d=8.52
, R , 1H),
ar(IM-5)
13
ar(IM-2)
2
.25 ppm (s, CH
ar(IM-2)
2
2
1
(bs, CH IM, R , 0.3C) 10.27 (s, CH IM, R , 1C), 49.60–49.81 (m,
General procedure for the synthesis of compounds 1a–f and
a–f on the example of 1b
3
3
2
2
2
CH C(F)P, R , 0.3C), 52.65 (dd, J =20.1 Hz, J =7.7 Hz, CH C(F)P,
R , 1C), 98.38 (dd, J =193.6 Hz, J =133.6 Hz, CH C(F)P, R , 1C),
97.71–99.55 (mutliplet overlapping with dd at 98.38, CH C(F)P, R ,
0.3C), 118.50 (bs, CH
131.42 (s, C
CHar(IM-2), R , 1C), 135.92 (bs, CH
CO H, R and R , 1.3C); Elemental analysis: C H FN O P(H O)1.95,
calcd: C 29.27, H 4.88, N 9.75, found: C 29.42, H 4.72, N 9.54.
2
FC
PC
2
1
2
1
1
1
FC
PC
2
2
In a single-neck flask compound 6b (190 mg, 0.477 mmol) was
placed and 36% HCl (5 mL, ꢁ1 mL/0.1 mmol) was added. The mix-
ture was held a reflux for 5 h. Excess HCl was evaporated, and the
residue was co-evaporated with EtOH (3ꢁ1 mL). EtOH (1 mL) was
then added, and the resulting precipitate was filtered off and
rinsed with 0.5 mL ice-cold EtOH. It was then dried under vacuum,
dissolved in water (3 mL) and lyophilized. Compounds 1c, 1e and
2
2
1
, R , 0.3C), 118.72 (s, CH
, R , 0.3C), 132.37 (bs, C
, R , 1C),
ar(IM-5)
, R , 1C), 135.66 (s,
ar(IM-4)
2
1
ar(IM-5)
ar(IM-4)
1
2
, R , 0.3C), 174.19 ppm (m,
ar(IM-2)
1
2
2
7
10
2
5
2
2
-Fluoro-3-(4-phenyl-1H-imidazol-1-yl)-2-phosphonopropanoic
1
f, which were insoluble in water, were instead lyophilized from
acid (1e): Yield: 45%. Obtained from 0.41 mmol (165 mg) of 6d.
suspension, followed by trituration of thus obtained powder. Com-
pounds 2c and 2 f required purification by preparative HPLC using
3
1
3
1
1
P NMR (283 MHz, D O, pH 9): d=8.08 ppm (d, J =65.9 Hz);
2
PF
3
2
H NMR (700 MHz, D O): d=4.52 (ddd, J =15.0 Hz, J =8.7 Hz,
JPH =4.6 Hz, CH H C(F)P, 1H), 4.93 (bdd, JFH =36.1 Hz, JHH
2
HH
FH
H O as eluent followed by lyophilization. Remaining compounds 2
2
3
2
=
a
b
were only precipitated from EtOH.
1
5.0 Hz, CH H C(F)P, 1H), 7.36–7.39 (m, C H -, 1H), 7.49–7.52 (m,
a b 6 5
2
-Fluoro-3-(2-methyl-1H-imidazol-1-yl)-2-phosphonopropanoic
C H -, 2H), 7.58 (bs, CH
7.79 ppm (dd, JHH =8.4 Hz, JHH =1.2 Hz, C H -, 2H); C NMR
6 5
5), 1H), 7.76 (bs, CHar(IM-2 or 5), 1H),
6
5
ar(IM-2 or
3
4
13
acid (1a): Yield: 64%. Obtained from 0.55 mmol (184 mg) of 6a.
3
1
1
2
2
P NMR (283 MHz, D O, pH 8): d=8.67 ppm (d, J =65.9 Hz);
(176 MHz, D O): d=51.86 (dd, J =20.5 Hz, J =6.8 Hz, CH C(F)P,
2 FH PH 2
2
PF
1
2
1
1
H NMR (700 MHz, D O): d=2.54 (s, CH IM, 3H), 4.50 (ddd, J
=
=
1C), 99.16 (dd, J =193.4 Hz, J =133.2 Hz, CH C(F)P, 1C), 117.88
FC PC 2
2
3
HH
3
3
3
1
5.1 Hz, J =7.8 Hz, J =4.7 Hz, CH H C(F)P, 1H), 4.87 (bdd, J
(s, CHar(IM-2 or 5), 1C), 124.82 (s, C H -, 2C), 127.41 (s, C H -, 1C), 129.27
6 5 6 5
(s, C H -, 2C), 133.54 (s, C H -, 1C), 139.84 (s, CHar(IM-2 or 5), 1C), 139.96
6 5 6 5
(s, Car(IM-4), 1C), 175.06 ppm (bd, J =20.8 Hz, CO H, 1C).
FC 2
FH
PH
a
b
FH
2
3
3
5.1 Hz, J =15.1 Hz, CH H C(F)P, 1H), 7.06 (d, J =1.8 Hz, CHar(IM-
HH
a
b
HH
13
2
4),
1H), 7.21 ppm (bs, CHar(IM-5), 1H); C NMR (176 MHz, D O): d=
2
2
2
11.40 (s, CH IM, 1C), 50.87 (dd, J =20.0 Hz, J =8.0 Hz, CH C(F)P,
3
FH
PH
2
1
1
3-(4-Bromo-1H-imidazol-1-yl)-2-fluoro-2-phosphonopropanoic
acid (1 f): Yield: 76%. Obtained from 0.38 mmol (152 mg) of 6 f.
1
C), 98.98 (dd, J =194.2 Hz, J =133.7 Hz, CH C(F)P, 1C), 121.74
FC
PC
2
(
s, CHar(IM-4), 1C), 121.97 (s, CHar(IM-5), 1C), 146.62 (s, Car(IM-2), 1C),
31
1
2
P NMR (283 MHz, D
2
O, pH 8): d=8.43 ppm (d,
JPF =66.5 Hz);
1
74.76 ppm (d, JFC =21.3 Hz, CO H, 1C); Elemental analysis:
2
1
2
3
H NMR (700 MHz, D O): d=4.46 (ddd, J =15.0 Hz, J =8.8 Hz,
2
HH
FH
C H FN O P(H O) , calcd: C 30.92, H 4.52, N 10.30, found: C 31.04,
H 4.39, N 10.10.
7
10
2
5
2
1.1
3
3
2
JPH =4.4 Hz, CH H C(F)P, 1H), 4.93 (bdd, JFH =35.7 Hz, JHH
=
a
b
1
5.0 Hz, CH H C(F)P, 1H), 7.20, 7.61 ppm (2bs, CH 5), 2H);
ar(IM-2 and
a
b
13
2
-Fluoro-3-(2-phenyl-1H-imidazol-1-yl)-2-phosphonopropanoic
C NMR (176 MHz, D O, residua signal of EtOH as a reference): d=
2
2
2
1
acid (1b): Yield: 69%. Obtained from 0.48 mmol (190 mg) of 6c.
52.65 (dd, J =20.2, J =7.8 Hz, CH C(F)P, 1C), 99.46 (dd, J =
FH PH 2 FC
193.2 Hz, JPC =133.2 Hz, CH C(F)P, 1C), 113.06 (s, C
3
1
3
1
1
1
P NMR (283 MHz, D O, pH 3): d=8.68 ppm (d, J =67.8 Hz);
, 1C),
ar(IM-4)
2
FC
2
PF
3
2
2
H NMR (700 MHz, D O): d=4.65 (ddd, J =15.0 Hz, J =7.9 Hz,
JPH =4.9 Hz, CH H C(F)P, 1H), 4.97 (ddd,
121.15, 139.58 (2 s, CHar(IM-2 and 5), 2C), 175.30 ppm (bd, J =21.3,
2
HH
FH
3
2
J
=34.3 Hz, JHH
=
CO H, 1C); Elemental analysis: C H BrFN O P, calcd: C 22.73, H 2.23,
a
b
FH
3
2
6
7
2
5
3
1
5.0 Hz, J =1.5 Hz, CH H C(F)P, 1H), 7.28 (d, J =1.8 Hz, CHar(IM-
N 8.84, found: C 22.66, H 2.33, N 8.75.
PH
a
b
HH
4),
1H), 7.47 (bs, CHar(IM-5), 1H), 7.63–7.66 (m, C H -, 3H), 7.70–
6 5
13
7
.73 ppm (m, C H -, 2H); C NMR (176 MHz, D O): d=50.80 (dd,
6 5 2
2
2
1
JFH =19.7, JPH =8.4 Hz, CH C(F)P, 1C), 98.24 (dd, JFC =194.6 Hz,
2
1
5
General procedure for the synthesis of compounds 1g–l and
2g–l on the example of compound 1i
JPC =134.9 Hz, CH C(F)P, 1C), 122.37 (d, J =2.9 Hz, CHar(IM-5), 1C),
2
PC
1
1
1
23.74 (s, CHar(IM-4), 1C), 127.00 (s, C H -, 1C), 129.01(s, C H -, 2C),
6 5 6 5
29.62 (s, C H -, 2C), 130.46 (s, C H -, 1C), 147.99 (s, Car(IM-2), 1C),
6
5
6
5
2
In a single-neck flask compound 12i (320 mg, 0.567 mmol) was
placed and 36% HCl (5.5 mL, ꢁ1ml/0.1 mmol) was added. The mix-
ture was held at reflux for 3 h. Excess HCl was evaporated, and the
74.00 ppm (bd, J =21.1 Hz, CO H, 1C).
FC
2
3
-(2-Bromo-1H-imidazol-1-yl)-2-fluoro-2-phosphonopropanoic
acid (1c): Yield: 41%. Obtained from 0.38 mmol (153 mg) of 6e.
residue was transferred to a separatory funnel in 5 mL of H O and
2
3
1
1
P NMR (283 MHz, D O, pH 8): d=8.85 ppm (d, JPF =66.1 Hz);
5 mL of CH Cl . The organic phase was discarded, and the aqueous
2
2
2
&
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3
3
3
phase was additionally washed with CH Cl (3ꢁ20 mL). Water was
15.6 Hz, J =8.1 Hz, J =8.1 Hz, CH H C(F)P, 1H), 3.62 (ddd, J
=
2
2
FH
PH
3
a
b
FH
2
evaporated, and the residue was co-evaporated with EtOH (2ꢁ
mL). Then 1 mL of EtOH was added, and the resulting precipitate
38.6 Hz,
J
HH =15.6 Hz,
J
PH =2.9 Hz, CH
O) d=8.71 (s, CH
PC =3.4 Hz, CH C(F)P, 1C), 98.47 (dd,
191.2 Hz, JPC =140.3 Hz, CH C(F)P, 1C), 124.02 (bdd, JPC =13.3 Hz,
a
H
b
C(F)P, 1H), 8.29 ppm (s,
13
1
CHar(IM-2), 1H); C NMR (176 MHz, D
2
3
IM, 1C), 29.46
3
3
1
was filtered off and rinsed with 0.5 mL EtOH. The precipitate was
(dd,
J
FC =21.4 Hz,
J
2
J =
FC
1
3
dissolved in H O (2 mL) and lyophilized. When non-tritylated sub-
2
2
3
strates were used (10h, j, k, l and 12h or 12j), CH Cl washing was
JFC =2.4 Hz, Car[IM-4(5)], 1C), 127.50 (s, Me-Car[IM-5(4)], 1C), 131.54 (s,
CHar(IM-2), 1C), 176.03 ppm (dd, J =19.9 Hz, J =2.4 Hz, CO H, 1C).
2
2
2
2
omitted.
PC
FC
2
2
-Fluoro-3-(2-methyl-1H-imidazol-4(5)-yl)-2 phosphonopropano-
2
-Fluoro-3-(1H-imidazol-2-yl)-2-phosphonopropanoic acid (1g):
31
ic acid (1l): Yield, 74%. Obtained from 0.31 mmol (180 mg) of 12l.
Yield: 50%. Obtained from 0.41 mmol (229 mg) of 12g. P NMR
31
1
1
1
P NMR (283 MHz, D O, pH 8): d=10.56 ppm (d, J =69.9 Hz);
(
283 MHz, D O, pH 8): d=10.16 ppm (d, J =68.9 Hz); H NMR
2
PF
2
PF
1
2
2
3
3
H NMR (700 MHz, D O): d=2.53 (s, CH IM, 3H), 3.26 (dt, JHH
=
=
(
700 MHz, D O): d=3.49 (ddd, JHH =15.5 Hz, JFH =10.5 Hz, JPH
=
2
3
3
2
3
3
3
2
1
3
5.7 Hz, J =7.7 Hz, J =7.7 Hz, CH H C(F)P, 1H), 3.58 (ddd, J
9.8 Hz, JHH =15.7 Hz, JPH =2.6 Hz CH H C(F)P, 1H), 6.99 ppm (s,
7
.3 Hz, CH H C(F)P, 1H), 3.77 (ddd, JFH =36.6 Hz, JHH =15.5 Hz,
FH
PH
3
a
b
FH
a
b
2
3
J
=4.1 Hz, CH H C(F)P, 1H), 7.17 ppm (s, CH 5), 2H);
ar(IM-4 and
a
b
PH
a
b
13
1
3
2
CHar[IM-4(5)], 1H); C NMR (176 MHz, D O): d=10.92 (s, CH -IM, 1C),
C NMR (176 MHz, D O): d=32.92 (d, J =21.4 Hz, CH C(F)P, 1C),
2
3
2
FH
2
2
2
1
1
1
3
1
1
1
0.59 (dd, J =21.3, J =3.3 Hz, CH C(F)P, 1C), 99.10 (dd, J =
90.9 Hz,
28.63 (d, JPC =14.1 Hz, Car[IM-5(4)], 1C), 143.84 (s, Car(IM-2), 1C),
76.19 ppm (dd, J =20.7 Hz, J =2.6 Hz, CO H, 1C).
9
7.77 (dd, JFC =192.2 Hz,
J
PC
3
=138.1 Hz, CH C(F)P, 1C), 120.27 (s,
FC PC 2 FC
2
1
J
=139.2 Hz, CH C(F)P, 1C), 116.52 (s, CHar[IM-4(5)], 1C),
CH
, 2C), 144.16 (d, J =14.2 Hz, Car(IM-2), 1C), 175.91 ppm
PC
3
2
ar(IM-4 and 5)
2
PC
2
(
dd, JFC =20.6 Hz, JPC =2.8 Hz CO H, 1C); Elemental analysis:
2
2
2
C H FN O P(H O)1.15, calcd: C 27.84, H 4.01, N 10.82, found: C 27.86,
FC
PC
2
6
8
2
5
2
H 3.88, N 10.67.
2
-Fluoro-3-(1-methyl-1H-imidazol-2-yl)-2-phosphonopropanoic
Biological studies
acid (1h): Yield: 80%. Obtained from 0.54 mmol (180 mg) of 12h.
3
1
3
1
1
HeLa cell culture: The cervical epithelial carcinoma HeLa cell line
was purchased from the American Type Cell Collection (ATCC).
Cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) contain-
P NMR (283 MHz, D O, pH 8): d=10.13 ppm (d, J =70.6 Hz);
2
PF
3
2
H NMR (700 MHz, D O): d=3.34 (dt, JHH =15.5 Hz, JFH =7.8 Hz,
JPH =7.8 Hz, CH H C(F)P, 1H), 3.64 (ddd, JFH =37.7 Hz, JHH
2
3
2
=
a
b
3
1
5.5 Hz, J =2.7 Hz, CH H C(F)P, 1H), 3.64 (s, CH IM, 3H), 6.92 (d,
PH a b 3
ꢀ1
ꢀ1
3
3
ing 100 IUmL penicillin and 100 mgmL streptomycin. Cells were
incubated at 378C in a humidified atmosphere of 95% air and 5%
J
=1.6 Hz, CHar(IM-4), 1H), 7.01 ppm (d, J =1.6 Hz, CHar(IM-5), 1H);
HH
HH
1
3
2
C NMR (700 MHz, D O): d=30.66 (d, J =21.9 Hz, CH C(F)P, 1C),
2
FC
2
1
1
CO . For biological studies, PCs were dissolved in phosphate-buf-
3
4.37 (bs, CH IM, 1C), 97.19 (dd, JFC =193.7 Hz, JPC =137.7 Hz,
2
3
fered saline (PBS) at stock concentrations of 10 mm at approxi-
mately pH 7. Compounds were stored at 48C or ꢀ208C (for longer
storage) prior to use.
CH C(F)P, 1C), 119.21 (s, CH
, 1C), 123.01 (s, CH
, 1C), 143.60
2
3
ar(IM-4)
ar(IM-5)
2
2
(
d, J =13.0 Hz, Car(IM-2), 1C), 175.36 ppm (dd, J =20.1 Hz, J =
PC FC PC
1
.9 Hz, CO H, 1C).
2
Determination of cytotoxicity: HeLa cells were seeded into 96-
well cell culture plates at a density of 4ꢁ10 cells per well in
2
-Fluoro-3-(1H-imidazol-4(5)-yl)-2-phosphonopropanoic acid (1i):
3
31
Yield: 70%. Obtained from 0.57 mmol (320 mg) of 12i. P NMR
1
1
100 mL of complete culture medium. On the following day, cells
were washed with PBS, and 100 mL fresh serum-containing or
serum-free (fasting) medium was added. Subsequently HeLa cells
were treated with PCs at eight concentrations; 72 h later PrestoBl-
ue Cell Viability Reagent was applied. Following 50 min incubation
(
283 MHz, D O, pH 8): d=10.39 ppm (d, J =71.1 Hz); H NMR
2 PF
2
3
3
(
700 MHz, D O): d=3.27 (dt, JHH =15.5 Hz, JFH =7.6 Hz, JPH
=
2
3
2
7
.6 Hz, CH H C(F)P, 1H), 3.59 (ddd, JFH =41.8 Hz, JHH =15.5 Hz,
a b
3
JPH =2.2 Hz, CH H C(F)P, 1H), 6.91 (s, CH , 1H), 7.70 ppm (s,
ar[IM-4(5)]
a
3
b
1
2
CHar(IM-2), 1H); C NMR (176 MHz, D O): d=30.41 (dd, JFC =21.5,
2
2
1
1
time at 378C and 5% CO , cell viability was determined by measur-
JPC =3.3 Hz, CH C(F)P, 1C), 98.47 (dd, J =191.6 Hz, J =142.0 Hz,
2
2
FC
PC
3
ing the fluorescence signal (l /l =530/590 nm) on a Synergy 2
CH C(F)P, 1C), 117.43 (s, CH
Car[IM-4(5)], 1C), 133.26 (s, CHar(IM-2), 1C), 175.58 ppm (bd, J =21.2 Hz,
CO H, 1C); Elemental analysis: C H FN O P(H O) , calcd: C 27.94, H
, 1C), 129.20 (bd, J =14.2 Hz,
Ex Em
2
ar[(IM-4(5)]
PC
2
microplate reader (BioTek, VT, USA). The obtained fluorescence
magnitudes were used to calculate cell viability expressed as a per-
centage of untreated control cell viability. The data, expressed as
the mean of at least four independent experiments, were used to
calculate IC50 values.
FC
2
6
8
2
5
2
1.1
3
.99, N 10.86, found: C 28.02, H 4.00, N 10.69.
2
-Fluoro-3-(1-methyl-1H-imidazol-5-yl)-2-phosphonopropanoic
acid (1j): Yield: 78%. Obtained from 0.46 mmol (156 mg) of 12j.
2
D
0
20
D
Assessment of inhibition of Rab11 and Rap1A/Rap1B prenyla-
1
j-E1: [a] =ꢀ18.1 (c=0.5, H O); 1j-E2: [a] =23.5 (c=0.5, H O);
2
2
3
1
3
1
1
tion: HeLa cells were seeded into six-well cell culture plates at a
P NMR (283 MHz, D O, pH 8): d=10.87 ppm (d, J =71.0 Hz);
2
PF
3
5
2
density of 4ꢁ10 cells per well in 3 mL of complete medium. On
H NMR (700 MHz, D O): d=3.33 (dt, JHH =16.0 Hz, JFH =6.9 Hz,
JPH =6.9 Hz, CH H C(F)P, 1H), 3.63 (ddd, JFH =41.0 Hz, JHH
2
3
2
the following day, 1.5 mL of fresh serum-free medium was supple-
mented with PCs as well as lovastatin. After 48 h of incubation, cell
monolayers were rinsed with PBS and detached using trypsin–
EDTA solution. The cytosolic and membrane-rich fractions, contain-
ing protease inhibitor cocktail, were isolated from cell pellets using
Mem-PER Plus Membrane Protein Extraction Kit according to the
manufacturers’ instructions. The protein concentration in both frac-
tions was determined by Bradford Protein Assay. Equal amounts of
protein (20 mg) from cytosolic fractions were resolved by 12% SDS-
PAGE and transferred onto 0.2 mm nitrocellulose membranes. Mem-
branes were probed with b-actin, Rab11A, or Rap1A/Rap1B anti-
bodies and detected using the appropriate horseradish peroxidase
(HRP)-conjugated secondary antibody, followed by electrochemilu-
minescence (ECL) assay. Visualization of the chemiluminescent pro-
=
a
b
3
1
6.0 Hz, J =1.9 Hz, CH H C(F)P, 1H), 3.74 (s, CH IM, 3H), 6.99 (s,
PH a b 3
13
CHar(IM-4), 1H), 7.91 ppm (s, CHar(IM-2), 1H); C NMR (176 MHz, D O):
d=28.16 (bd, J =20.9 Hz, CH C(F)P, 1C), 32.20 (bs, CH IM, 1C),
2
2
FC
2
3
1
1
9
9.60 (dd, J =190.9 Hz, J =139.3 Hz, CH C(F)P, 1C), 123.10 (bs,
FC
PC
3
2
CHar(IM-4), 1C), 129.72 (bd, JPC =14.0 Hz, C
CHar(IM-2), 1C), 176.35 ppm (dd, JFC =22.3 Hz, JPC =3.9 Hz, CO H,
, 1C), 137.07 (s,
ar(IM-5)
2
2
2
1
C); Elemental analysis: C H FN O P(H O)0.35, calcd: C 32.53, H 4.17,
7 10 2 5 2
N 10.84, found: C 32.64, H 4.09, N 10.64.
2
-Fluoro-3-(4(5)-methyl-1H-imidazol-5(4)-yl)-2-phosphonopropa-
noic acid (1k): Yield: 41%. Obtained from 0.38 mmol (217 mg) of
3
1
1
1
2k. P NMR (283 MHz, D O, pH 8): d=9.98 ppm (d, J =71.2 Hz);
2 PF
1
2
H NMR (700 MHz, D O) d=2.27 (s, CH IM, 3H), 3.27 (dt, JHH
=
2
3
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www.chemmedchem.org
9
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
tein bands was performed with a ChemiDoc MP Imaging System
Bio-Rad).
[12] C. E. McKenna, B. A. Kashemirov, K. M. Bła z˙ ewska, I. Mallard-Favier, C. A.
Stewart, J. Rojas, M. W. Lundy, F. H. Ebetino, R. A. Baron, J. E. Dunford,
M. L. Kirsten, M. C. Seabra, J. L. Bala, M. S. Marma, M. J. Rogers, F. P.
Coxon, J. Med. Chem. 2010, 53, 3454–3464.
(
[
[
[
13] K. M. Bła z˙ ewska, F. Ni, R. Haiges, B. A. Kashemirov, F. P. Coxon, C. A.
Stewart, R. Baron, M. J. Rogers, M. C. Seabra, F. H. Ebetino, C. E. McKen-
na, Eur. J. Med. Chem. 2011, 46, 4820–4826.
14] A. Ka z´ mierczak, D. Kusy, S. P. Niinivehmas, J. Gmach, Ł. Joachimiak, O. T.
Pentikꢃinen, E. Gendaszewska-Darmach, K. M. Bła z˙ ewska, J. Med. Chem.
Abbreviations
RGGT (Rab GGTase, GGT-2): Rab geranylgeranyl transferase; FPPS:
farnesyl pyrophosphate synthase; GGPPS: geranylgeranyl pyro-
phosphate synthase; FT: farnesyl transferase; GGT-1: geranylgeranyl
transferase 1; FPP: farnesyl pyrophosphate; GPP: geranyl pyrophos-
phate; GGPP: geranylgeranyl pyrophosphate; 3-IPEHPC: 3-(3-pyrid-
yl)-2-hydroxy-2-phosphonopropanoic acid; PC: phosphonocarboxy-
late; F-ZolPC: 2-fluoro-3-(1H-imidazol-1-yl)-2-phosphonopropanoic
acid; NFSI: N-fluorobenzenesulfonimide; IM: imidazole ring.
2
017, 60, 8781–8800.
15] The introduced modification represents substituents with diverse elec-
tronegativity, size and shape, and allows determination of these factors’
[14]
influence on the activity toward RGGT. The selection of such substitu-
ents stems from our previous studies, in which we have shown that an-
alogues containing an imidazo[1,2-a]pyridine ring can be modified with
diverse substituents and retain activity toward RGGT, as long as the po-
sition of modification is at C6.
Acknowledgements
[16] J. Gmach, Ł. Joachimiak, K. M. Bła z˙ ewska, Synthesis 2016, 48, 2681–
704.
17] M. N. Feofanov, M. V. Anokhin, A. D. Averin, I. P. Beletskaya, Synthesis
017, 49, 5045–5058.
2
[
This work was supported financially by the National Science
Centre, Poland (2014/14/E/ST5/00491). We thank Dr. Marek
Domin (Boston College) for HRMS data of esters 6 and 12.
2
[18] T. Fujimoto, M. Tobisu, N. Konishi, M. Kawamura, N. Tada, T. Takagi, K.
Kubo, Bioorg. Med. Chem. 2009, 17, 7993–8002.
[
[
[
19] S. Hack, B. Woerlein, G. Hoefner, J. Pabel, K. T. Wanner, Eur. J. Med.
Chem. 2011, 46, 1483–1498.
20] J. Ohkanda, J. W. Lockman, M. A. Kothare, Y. Qian, M. A. Blaskovich, S. M.
Sebti, A. D. Hamilton, J. Med. Chem. 2002, 45, 177–188.
21] Tritylation of aldehyde 7k led to an equimolar mixture of two re-
gioisomers (67%). Upon subsequent Knovenagel condensation, a mix-
ture of four isomers was formed (ratio of 1.3:0.6:0.6:0.7), which required
tedious chromatography purification. Therefore, such approach was dis-
continued as inefficient.
Conflict of interest
The authors declare no conflict of interest.
Keywords: inhibitors
·
Michael
addition
·
phosphonocarboxylates · prenylation · Rab geranylgeranyl
transferase
[22] The second option was used only for tritylated analogues, 9g and 9i.
[
[
23] C. M. Taylor, S. T. De Silva, J. Org. Chem. 2011, 76, 5703–5708.
24] For clarity of presentation and discussion, in Figure 3 we have labeled
the atoms in the imidazole ring of the minor and major isomers of 6d
with consecutive numbers, which do not correspond to the proper
numbering according to nomenclature rules.
[1] E. E. Kelly, C. P. Horgan, B. Goud, M. W. McCaffrey, Biochem. Soc. Trans.
2012, 40, 1337–1347.
[
[
2] A. Guichard, V. Nizet, E. Bier, Nat. Rev. Microbiol. 2014, 12, 624–634.
3] W. Zhao, M. Jamshidiha, T. Lanyon-Hogg, C. Recchi, E. Cota, E. W. Tate,
Curr. Top. Med. Chem. 2017, 17, 16–29.
[
25] F. P. Coxon, M. H. Helfrich, B. Larijani, M. Muzylak, J. E. Dunford, D. Mar-
shall, A. D. McKinnon, S. A. Nesbitt, M. A. Horton, M. C. Seabra, F. H. Ebe-
tino, M. J. Rogers, J. Biol. Chem. 2001, 276, 48213–48222.
[4] J. D. Ochocki, M. D. Distefano, MedChemComm 2013, 4, 476–492.
[5] S. S. Agabiti, Y. Liang, A. J. Wiemer, Mol. Membr. Biol. 2016, 33, 1–11.
[6] M. Watanabe, H. D. G. Fiji, L. Guo, L. Chan, S. S. Kinderman, D. J. Slamon,
O. Kwon, F. Tamanoi, J. Biol. Chem. 2008, 283, 9571–9579.
[
[
[
[
26] A. Dudakovic, A. J. Wiemer, K. M. Lamb, L. A. Vonnahme, S. E. Dietz, R. J.
Hohl, J. Pharmacol. Exp. Ther. 2008, 324, 1028–1036.
27] A. J. Wiemer, R. J. Hohl, D. F. Wiemer, Anti-Cancer Agents Med. Chem.
2
009, 9, 526–542.
28] R. S. Ehrick, M. Capaccio, D. A. Puleo, L. G. Bachas, Bioconjugate Chem.
008, 19, 315–321.
[
[
[
7] K.-T. Tan, E. Guiu-Rozas, R. S. Bon, Z. Guo, C. Delon, S. Wetzel, S. Arndt,
K. Alexandrov, H. Waldmann, R. S. Goody, Y.-W. Wu, W. Blankenfeldt, J.
Med. Chem. 2009, 52, 8025–8037.
8] E. A. Stigter, Z. Guo, R. S. Bon, Y.-W. Wu, A. Choidas, A. Wolf, S. Menning-
er, H. Waldmann, W. Blankenfeldt, R. S. Goody, J. Med. Chem. 2012, 55,
2
29] Position C2 in imidazole corresponds to position C9 in the imidazo[1,2-
a]pyridine ring, while the methyl substituent at C2 in imidazole may re-
semble C8 in imidazo[1,2-a]pyridine. Therefore, it might be surprising
that 2-substituted analogues (1a–c, 1g,h, 1l) do not show activity
against RGGT. However, we hypothesize that such a difference may
stem from different geometry of the corresponding atoms (e.g., planar
8330–8340.
9] R. S. Bon, Z. Guo, E. A. Stigter, S. Wetzel, S. Menninger, A. Wolf, A. Choi-
das, K. Alexandrov, W. Blankenfeldt, R. S. Goody, H. Waldmann, Angew.
Chem. Int. Ed. 2011, 50, 4957–4961; Angew. Chem. 2011, 123, 5059–
2
3
sp of C8 in imidazo[1,2-a]pyridine vs. the tetrahedral sp methyl group
5063.
on imidazole).
[
[
10] C. Deraeve, Z. Guo, R. S. Bon, W. Blankenfeldt, R. DiLucrezia, A. Wolf, S.
Menninger, E. A. Stigter, S. Wetzel, A. Choidas, K. Alexandrov, H. Wald-
mann, R. S. Goody, Y.-W. Wu, J. Am. Chem. Soc. 2012, 134, 7384–7391.
11] F. P. Coxon, Ł. Joachimiak, A. K. Najumudeen, G. Breen, J. Gmach, C.
Oetken-Lindholm, R. Way, J. E. Dunford, D. Abankwa, K. M. Bła z˙ ewska,
Eur. J. Med. Chem. 2014, 84, 77–89.
Manuscript received: December 15, 2017
Revised manuscript received: February 6, 2018
Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
&
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10
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
Vesicle traffic light: Novel phosphono-
carboxylates bearing imidazole in their
structure were synthesized and evaluat-
ed for inhibitory activity against Rab
geranylgeranyl transferase (RGGT). The
preferred positions for modifications in
the heterocyclic ring, ensuring micro-
molar potency toward RGGT, harmonize
with bicyclic analogues containing the
imidazo[1,2-a]pyridine core.
Ł. Joachimiak, A. Marchwicka,
E. Gendaszewska-Darmach,
K. M. Bła z˙ ewska*
&& – &&
Synthesis and Biological Evaluation of
Imidazole-Bearing a-
Phosphonocarboxylates as Inhibitors
of Rab Geranylgeranyl Transferase
(RGGT)
ChemMedChem 2018, 13, 1 – 11
www.chemmedchem.org
11
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ