Chirality-Driven Mode of Binding of α-Aminophosphonic Acid-Based Allosteric Inhibitors of the Human Farnesyl Pyrophosphate Synthase (hFPPS)

Article abstract of DOI:10.1021/acs.jmedchem.9b01104

Thienopyrimidine-based allosteric inhibitors of the human farnesyl pyrophosphate synthase (hFPPS), characterized by a chiral α-aminophosphonic acid moiety, were synthesized as enantiomerically enriched pairs, and their binding mode was investigated by X-ray crystallography. A general consensus in the binding orientation of all (R)- and (S)-enantiomers was revealed. This finding is a prerequisite for establishing a reliable structure-activity relationship (SAR) model.

Full text of DOI:10.1021/acs.jmedchem.9b01104

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Article
Chirality-Driven Mode of Binding of #-Aminophosphonic Acid-Based
Allosteric Inhibitors of the Human Farnesyl Pyrophosphate Synthase (hFPPS)
Yuting Feng, Jaeok Park, Shi-Guang Li, Rebecca Boutin, Peter
Viereck, Matthew Schilling, Albert M. Berghuis, and Youla S. Tsantrizos
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01104 • Publication Date (Web): 02 Oct 2019
Downloaded from pubs.acs.org on October 5, 2019
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Chirality-Driven Mode of Binding of α-Aminophosphonic Acid-Based Allosteric In-
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hibitors of the Human Farnesyl Pyrophosphate Synthase (hFPPS)
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Yuting Feng,†,⊥ Jaeok Park,†,‡,⊥ Shi-Guang Li,† Rebecca Boutin,† Peter Viereck,†,∇ Matthew A. Schil-
ling,‡,∇ Albert M. Berghuis,*‡ Youla S. Tsantrizos*†,‡
†Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8,
Canada
‡Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC,
H3G 0B1, Canada
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ABSTRACT:
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Thienopyrimidine-based allosteric inhibitors of the human farnesyl pyrophosphate synthase (hFPPS),
characterized by a chiral α-aminophosphonic acid moiety, were synthesized as enantiomerically en-
riched pairs and their binding mode was investigated by X-ray crystallography. A general consensus in
the binding orientation of all (R)- and (S)-enantiomers was revealed. This finding is a prerequisite for
establishing a reliable structure-activity relationship (SAR) model.
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INTRODUCTION
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Human farnesyl pyrophosphate synthase (hFPPS) is the gatekeeper of mammalian isoprenoid biosyn-
thesis and a validated therapeutic target for bone-related diseases.1 The catalytic product of hFPPS is the
C-15 isoprenoid farnesyl pyrophosphate (FPP), which is formed from the successive condensation of two
C-5 isopentenyl pyrophosphate (IPP) units with dimethyl allyl pyrophosphate (DMAPP). FPP is a key
precursor to many essential metabolites, including geranylgeranyl pyrophosphate (GGPP). Blocking post-
translational farnesylation or geranylgeranylation of small GTP-binding proteins (GTPases), particularly
members of the RAS superfamily that are known drivers of oncogenesis, has been proposed as a potential
novel mechanism for treating cancer.2 However, bona fide and clinically validated antitumor agents that
selectively target hFPPS have not yet been identified. Currently, nitrogen-containing bisphosphonates (N-
BPs; e.g. zoledronic acid 1) are the only approved drugs that selectively target hFPPS. These inhibitors
bind to the allylic sub-pocket of the active site and exhibit low nanomolar intrinsic potency (e.g. the IC50
value of the most potent known inhibitor 1 is ~2-4 nM).3 It has been assumed that the highly charged
nature of the bisphosphonate pharmacophore of N-BPs, which exists as the trianion under physiological
conditions, limits their cell membrane permeability, systemic circulation and exposure to non-skeletal
tissues. Nonetheless, clinical data obtained from patients treated with 1, in addition to standard chemo-
therapy, suggest that this compound may have some therapeutic value in the treatment of cancers, such
as multiple myeloma4 and breast cancer.5 Consequently, the identification of more “drug-like” (in the
classical sense) inhibitors of hFPPS has attracted significant attention in recent years.
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The discovery of a catalytically relevant, allosteric pocket near the IPP binding sub-pocket of the active
site6 has fueled interest in the discovery of non-bisphosphonate inhibitors that can bind to this pocket and
may exhibit superior biopharmaceutical properties than those of N-BPs. This allosteric pocket was found
to play a critical role in the feed-back mechanism of the enzyme, which is Nature’s way of controlling the
intracellular concentrations of FPP.7 To date, many non-bisphosphonate, allosteric inhibitors of hFPPS
have been reported; some examples are shown in Figure 1.8
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Figure 1. Examples of active site (1) and allosteric inhibitors (2-7) of hFPPS.
In the course of our own investigations, we discovered a new chemotype of allosteric inhibitors, the
C6-substituted thienopyrimidine-based monophosphonate (C6-ThP-MP) analogs 6 (Fig. 1). These analogs
are characterized by a chiral α-aminophosphonic acid moiety, which was previously shown to adopt two
different binding orientations.9 A general consensus in the binding mode of all analogs within a structural
class of inhibitors is a prerequisite for establishing a reliable structure-activity relationship (SAR) model.
A predictive SAR model is the hallmark of medicinal chemistry and drug discovery. Therefore, we em-
barked on an investigation into the role of the α-aminophosphonic acid in controlling the binding orien-
tation of this class of inhibitors. In this report, we describe the synthesis of several enantiomeric pairs of
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6 and demonstrate that their chirality controls the binding orientation and interactions of these inhibitors
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with the hFPPS allosteric pocket.
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RESULTS AND DISCUSSION
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The asymmetric synthesis of α-aminophosphonic acids is of considerable interest in drug discovery.10
These bioisosteres of amino acids are useful building blocks in the design of potential therapeutic agents,
including HCV protease inhibitors.11 A number of methodologies for their asymmetric synthesis have
been previously reported. Recent examples include metal-catalyzed asymmetric hydrogenation of α,β-
dehydroaminophosphonates,12 α-iminophosphonates,13 and α-hydrazono phosphates,14 asymmetric hy-
drophosphonylation of imines,15 [3+2] cycloaddition of dehydroaminophosphonates and N-tosylhydra-
zones16 and other enantioselective C-C bond forming reactions that can lead to the formation of chiral
quaternary Cα centers, as well as the asymmetric three component Kabachnik-Fields condensations.17
In this study, we adopted the asymmetric 1,4-addition of aryltrifluoroborates to dehydroaminophospho-
nate catalyzed by a Rh-Difluorphos complex for the preparation of the (R)-α-aminophosphonates with a
benzylic side chain as previously reported by Darses (Scheme 1; Path A).18 Preparation of various (R)-
acetamido phosphonate esters 10 was initiated with the preparation of diethyl (1-acetamidovinyl)phos-
phonate 8, following the protocol previously reported.18 The subsequent 1,4-addition of various aryltri-
fluoroborates or boronic acids (9) to 8 proceeded to give the desired products (R)-10 in 70-75% isolated
yields and 85-90% enantiomeric excess (Scheme 1; Path A). Since the (R)-Difluorphos ligand is not com-
mercially available, we decided to screen several other commonly used phosphorus-based ligands (e.g.
BINAP, SEGPHOS and many others) for the preparation of intermediates (S)-10. However, all of the
ligands tested led to significantly lower enantiomeric purity of the (S)-10 products. For example, only
55% ee was observed with both (S)-BINAP and (S)-SEGPHOS in the synthesis of analog (S)-10a. Re-
cently, a site-selective and enantioselective modification of an amino acid moiety in the natural product
thiostrepton was reported by Key and Miller.19 In this transformation, 1,4-addition of an arylboronic acids
to an olefinic moiety was catalyzed by [Rh(nbd)2]BF4 in the presence of (S,S)-Chiraphos as the ligand.
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We adopted this method and successfully synthesized various (S)-10 building blocks, in good yield
(~70%) and enantiomeric purity (70-80% ee; Scheme 1, Path B).
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Unfortunately, neither of the above methods (i.e. Paths A or B) were successful in the preparation of
analogs 10 with an alkyl side chain (e.g. cyclohexyl derivative 10c; Scheme 1, Path C). Such analogs were
prepared by asymmetric phosphorylation of the N-acylimine intermediate 11 catalyzed by diethyl zinc in
the presence of Trost’s (S,S)- or (R,R)- ProPhenol ligands,20 as previously reported by Wang.21 Although
both the (R)- and (S)-10c were obtained in high enantiomeric purity (~95% ee), the isolated yields were
low (~30%). Isomerization of imine 11 to the corresponding enamide 12 was observed as the main side
product and the likely reason for the low yields. This isomerization has been previously observed with
other similar systems22 and suggests that this methodology (i.e. Scheme 1, Path C) would likely be even
more problematic for the preparation of benzylic analogs, such as 10a and 10b.
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The subsequent removal of the N-acyl or N-benzoyl protecting groups of all intermediates 10 was
achieved under mild conditions using a 3-step protocol, which involves the amide-to-carbamate transfor-
mation originally reported by Burk and Allen.23 The Boc group was cleaved under standard acidic condi-
tions to liberate the free amine intermediates 13 (Scheme 1). Nucleophilic addition of amines 13 to the 4-
fluoro-6-(p-tolyl)thieno[2,3-d]pyrimidine scaffold 14a,b via SNAr, followed by deprotection of the phos-
phonate ethyl esters, as previously reported,9 gave the final C6-ThP-MP inhibitors (R)- and (S)-6d to 6h
(Fig. 1).
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Scheme 1. Asymmetric synthesis of α-aminophosphonic acid hFPPS allosteric inhibitors. Conditions: (a)
[Rh(C2H4)2Cl]2 (1.5 mol%), (S)-Difluorphos (3.3 mol%), NaHCO3 (2 eq.), iPrOH, 90°C, 18 h (70-80%
yield); (b) Rh(nbd)2BF4, (7.5 mol%), (S,S)-Chiraphos (8 mol%), Na2CO3 (1 eq.), MeOH (8 eq.), diox-
ane/H2O, 70°C, 20 h (60-70% yield); (c) i. Boc2O, DMAP (cat.), THF, 80°C, 4h, ii. LiOH.H2O, THF/H2O
(2:1), RT, 20 h iii. HCl (4M in dioxane), DCM, RT, 3 h (50%); (d) Et3N, DMSO, 100°C, 2 h (50-80%);
(e) TMSBr/MeOH, RT, 24 h (40-80% yiels); (f) HPO(OEt)2, (R,R)- or (S,S)-ProPhenol/Et2Zn (10 mol%)
toluene, 0oC, 0.5 h (30% yield); (g) [Rh(cod)2]BF4 (3 mol%), (S)-BINAP (6 mol%), guaiacol (1 eq.),
toluene, 110oC, 16 h (50% yield).
To probe the binding contributions of the phosphonate group, we also synthesized the α-amino acid
derivative of our most potent inhibitor 6g. The synthesis of the amino acid (S)-17 was achieved starting
with 1,4-addition of aryltrifluoroborate 9d to commercially available methyl 2-acetamidoacrylate
(Scheme 1), followed by deprotection of both the amine and the carboxylic acid moieties using the same
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protocol as for the conversion of the α-aminophosphonates 10 to 13. For the synthesis of (R)-17, the best
enantiomeric purity was achieved using the rhodium-catalyzed 1,4-addition of 9d in the presence of (S)-
BINAP and guaiacol, as previously reported by Darses.24
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Previously, in an effort to expedite optimization of our initial hit compound 6a and establish SAR, a
library of C6-ThP-MP analogs was prepared in racemic mixtures (Fig. 1).9 In this approach, a presumption
was made that the potency observed with the racemic compounds would be mainly due to one enantiomer.
In addition, it was assumed that the potent (or more potent) enantiomer was more likely to bind and co-
crystallize with the enzyme. However, when enantiomerically enriched inhibitors (R)- and (S)-6 were
prepared, to our surprise, only a 2-3-fold difference in potency was observed, suggesting that more than
one binding mode was possible for this class of compounds. This assumption was confirmed by the co-
crystal structures of several analogs, including those of inhibitors 6a, 6b and 6g, which bound to the
hFPPS allosteric pocket in two distinct orientations (Fig. 2; co-crystallization were carried out with the
racemic mixtures of the inhibitors).9 Interestingly, the aromatic 6-tolyl thienopyrimidine core was the only
part of these molecules that adopted consistent binding interactions with the protein, engaging in an edge-
to-face π-stacking interaction with the side chain of Phe239 and a sandwich of π-stacking interactions
with the side chains of Phe206 and Asn59. In contrast, the entire molecule 6g was flipped by approxi-
mately 180o as compared to 6a and 6b and consequently, the α-aminophosphonate moiety of each com-
pound adopted a completely different orientation (Fig. 2). For example, in analog 6a, the phosphonate
moiety was bound facing the active site and close to the guanidinium ion of Arg60 (from here on referred
to as the “up orientation”; Fig. 2a), whereas in analog 6g, the phosphonate was bound nearly in the oppo-
site direction and towards the solvent-exposed surface of the cavity (from here on referred to as the “down
orientation”; Fig. 2c).
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Figure 2. Previously determined co-crystal structures of C6-ThP-MP inhibitors 6 (racemic samples of
inhibitors 6b and 6g were used):9 (a) 6a, (b) 6b and (c) 6g bound in the allosteric pocket of hFPPS. For
clarity, protein residues are only labeled in panel (a).
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In this study, we re-investigated the molecular interactions between hFPPS and several pairs of (R)-
and (S)-enantiomers of the C6-ThP-MP inhibitors (6) by X-ray crystallography. A general consensus in
the binding orientation for all (S)-6/enzyme complexes (Fig. 3a, c, e) and a different consensus in binding
for all the (R)-6/enzyme complexes (Fig. 3b, d, f) were observed. Regardless of the presence of a substit-
uent on the C-6 tolyl moiety attached to the thienopyrimidine core and/or a substituent on the Cα benzyl
moiety of the α-aminophosphonate moiety (i.e. R1), all of the inhibitors with the (S)-stereochemistry were
found to bind with the phosphonate in the “up-orientation” (Fig. 3a, c, e), whereas all the (R)-enantiomers
bound with the phosphonate in the “down-orientation” (Fig. 3b, d, f). It should be noted that all crystals
were obtained under slightly basic conditions (i.e. pH ~7.5-8.5) to assure (in as much as it is possible)
that the phosphonate moiety of the enzyme-bound inhibitors were fully deprotonated; reported pKas of
α-aminophosphonates are in the range of 2.3 (1st pKa) to 6.0 (2nd pKa).31 It is noteworthy that inhibitor
(R)-6b, which has a larger aromatic substituent directly attached to the Cα carbon, displays a different
binding orientation (Fig. 2b). As we previously reported,9 this is likely due to anticipated steric clashes
between the dihydrobenzofuran moiety and residues of the αH and αJ helices of the protein, which prevent
this compound from binding in the “down orientation”. Such unfavorable interactions may also induce an
unusual microenvironment protonation of the D243 carboxylate side chain, which is found to bind close
to the phosphonate moiety of the inhibitor. However, since inhibitors with an aromatic moiety directly
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attached to the Cα carbon are less potent that the benzylic analogs, we did not explore these analogs any
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further.
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Figure 3. Co-crystal structures of inhibitor/ hFPPS complexes. Inhibitors: (a) (S)-6d (PDB code: 6N7Z),
(b) (R)-6d (PDB code: 6N7Y), (c) (S)-6f (PDB code: 6OAG), (d) (R)-6f (PDB code: 6OAH), (e) (S)-6g
(PDB code: 6N82), and (f) (R)-6g (PDB code: 6N83). The red spheres represent water molecules.
We further examined the effects of the 3-chloro substituent on the C-6 tolyl moiety, which was previ-
ously found to improve the in vitro potency of these compounds. The co-crystal structures of inhibitors
(R)-6f and (R)-6g (Fig. 3d and f, respectively), which bind in the “down orientation”, revealed that the 3-
chloro moiety contributed to the binding affinity of these inhibitors by making the entire tolyl side chain
occupy the allosteric pocket more tightly (Fig. 4b, c vs a). Additionally, the binding of the chlorine sub-
stituent induced some movement to the scaffold, which led to either a direct (Fig. 3d) or a water-mediated
H-bond between the exocyclic C-4 nitrogen of the thienopyrimidine core and the carbonyl oxygen of
Lys347 (Fig. 3f). In contrast, with inhibitors (S)-6f and (S)-6g (Fig. 3c and e, respectively), which bind in
the “up orientation”, the 3-chloro moiety pushed the scaffold upwards by ~1Å, causing the loss of several
water-mediated interactions between the phosphonate moiety and the protein (e.g. to Asn59 and Arg60;
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Fig. 3a). These water-mediated interactions were previously observed with several analogs, including our
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initial hit, compound 6a (Fig. 2a), and presumed to contribute to the binding affinity of these inhibitors.
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Figure 4. Sideview of (R)-enantiomeric C6-ThP-MP inhibitor binding in space-filling representation. (a)
(R)-6d, (b) (R)-6f, and (c) (R)-6g.
Substitution on the Cα benzyl (i.e. Fig. 1; inhibitor 6, R1) of the α-aminophosphonate also played a
significant role in the inhibitor/enzyme complex formation. For example, the 3-fluorobenzyl substituent
in analogs 6e and 6g introduced additional binding interactions with the protein. The fluorine atom in (S)-
6g formed water-mediated H-bonds with the carbonyl of Phe239 and Gln242 (Fig. 3e), whereas in the
(R)-enantiomer of 6g, the fluorobenzyl moiety packed more tightly against the protein surface engaging
in stronger Van der Waals interactions, and the benzyl ring made a stacking interaction with the side chain
of Phe239 (Fig. 3f). Additionally, the fluorine atom appeared to form a direct non-classical H-bond with
the NH amide of Gln242. These interactions pushed the thienopyrimidine scaffold significantly towards
the active site, while burying the 3-chlorotolyl group deeper into the allosteric pocket (Fig. 4c) and con-
tributed to the formation of the water-mediated H-bond between the exocyclic C-4 NH of the thienopy-
rimidine and the carbonyl oxygen of Lys347, mentioned above (Fig. 3f). Although the position of the
phosphonate group was also shifted, the interaction with the water molecule bound to Asn59 was retained
(Fig. 3f). Interestingly, all hFPPS inhibitors having the (R)-absolute stereochemistry and a 3-chloro sub-
stituent on the C-6 tolyl moiety were found to be more potent than their corresponding (S)-enantiomers
in our in vitro hFPPS inhibition assay (Table 1). These results are consistent with the more extensive
network of inhibitor/protein interactions observed by X-ray crystallography.
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In our previous studies, we compared our initial hit, the α-aminophosphonate 6a with the correspond-
ing carboxylic acid derivative 7a and found the latter to be significantly less potent (IC50 values of 4.5
µM and >20 µM for compounds 6a and 7a, respectively).8a Although the carboxylic acid analog (R)-7b
was found to be considerably more potent (IC50 value of 1 µM) than 7a, the (S)-7b was essentially inactive
at the highest concentration test of 10 µM (Table 1). Unfortunately, we were not able to obtain a co-crystal
structures of (R)-7b bound to hFPPS. In the absence of direct structural evidence, we cautiously rationalize
that binding of (R)-7b should correspond to that of the phosphonate derivatives (S)-6g (i.e. binding in the
“up orientation”), and since the phosphonate moiety of this analog [i.e. (S)-6g] does not form any inter-
actions with the protein, its replacement with a carboxylic acid should have negligible impact on potency.
This hypothesis is consistent with the almost identical potency observed for the carboxylic acid analog
(R)-7b and the corresponding phosphonate derivative (S)-6g (Table 1; IC50 values of 1.1 vs. 1.0 µM,
respectively). By analogy, inhibitor (S)-7b would be expected to bind in the “down orientation” [analo-
gous to (R)-6g] and the smaller electron density and size of the carboxylate moiety could lead to loss of
the water-mediated H-bond that was previously observed between the phosphonate moiety and Asn59
(Fig. 3f), leading to significant loss of potency. Currently, we cannot exclude the possibility that without
the phosphonate group engaging in this interaction, the fluorobenzyl moiety may not be able to position
itself correctly for optimal binding, leading to further loss in binding affinity [i.e. as seen with (R)-6g].
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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17
18
19
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Enzyme inhibition (IC50) of representative allosteric hFPPS inhibitors
Com-
pound
2a
hFPPS IC50
(µM)
1.3α
5.0
R2
%ee
-
H
-
-
6a
(S)-6d
(R)-6d
(S)-6e
(R)-6e
(S)-6f
(R)-6f
(S)-6g
(R)-6g
(S)-6h
2.8
7.7
1.2
2.8
3.7
1.5
H
H
H
H
Cl
Cl
Cl
Cl
Cl
71β
90β
86β
80β
71β
92β
87β
78β
91β
1.1
0.54α
8.1
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(R)-6h
(S)-7b
(R)-7b
3.0
>10
1.0
Cl
Cl
Cl
99β
83
80
1
2
3
IC50 values were determined with 10 min pre-incubation of the enzyme with each inhibitor; the values
4
5
6
shown are average of n ≥3 determinations with standard deviation of ±5-10%. Inhibitor 2a was used as
7
8
9
α
the positive control in each assay. Assays were run in parallel, average values of n=6 determinations.
βThe enantiomeric purity (% ee) of analogs 6 was estimated from the chiral HPLC data of their precursor
10
11
12
13
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17
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diethyl esters 15.
CONCLUSION
In summary, we synthesized a cluster of enantiomerically enriched pairs of Cα-substituted aminophos-
phonate-based allosteric inhibitors 6 of the human FPPS and demonstrated that the binding mode of these
compounds is driven primarily (if not exclusively) by their chirality. A general consensus in the binding
mode of all (S)-enantiomers (Fig. 3a, c, e) and all (R)-enantiomers (Fig. 3b, d, f) was observed. These
results can guide our future efforts in establishing a reliable SAR model. Independent optimization of the
(R)- and (S)-enantiomers, expected to lead to divergent SAR, could also lead to more potent and selective
allosteric inhibitors of hFPPS that can be used in vivo to confirm hFPPS as a validated biological target
for oncology.
EXPERIMENTAL SECTION
General Information. All reactions were carried out under an atmosphere of dry argon unless otherwise
specified. Completion of all reactions was monitored by TLC and/or LC-MS. Flash column chromatog-
raphy was performed on silica gel (Sigma Aldrich, 60 Å, 230-400 mesh, 40-63 μm) as the stationary
phase. Thin Layer Chromatography (TLC) was performed on alumina plates pre-coated with silica gel
(MilliporeSigma, 60 Å, F254), and visualized by UV fluorescence when applicable (λmax = 254 nm) and/or
by staining with Seebach’s Magic Stain (acidic aqueous solution of polymolybdic acid and cerium (IV)
1
sulfate), ninhydrin, or basic aqueous KMnO4 solution. All compounds were fully characterized by H,
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13C, 19F, 31P NMR and HRMS. 1H NMR were recorded at 400 MHz or 500 MHz and coupling constants
(J) are reported to ± 0.5 Hz. 13C{1H} NMR were recorded at 125 MHz and 31P{1H} NMR were recorded
at 202 MHz, unless otherwise indicated. Chemical shifts (δ) are reported in ppm relative to the internal
deuterated solvent. Enantiomeric purity of chiral compounds was determined by chiral HPLC using an
Agilent 1100 series instrument and the column type and solvent system indicated. Enantiomeric purity (%
ee) was determined at two stages: intermediates 10 and 15 for the α-aminophosphonate analogs, as well
as 16 and final compound 7b for the carboxylic acid inhibitor. The absolute stereochemistry of the major
enantiomer in all new α-aminophosphonates and α-amino acids were assigned by analogy with previously
reported compounds in the literature. These assignments were further confirmed from the co-crystal struc-
tures of inhibitors (R)- and (S)-6d, (R)- and (S)-6f and (R)- and (S)-6g. The homogeneity of all final in-
hibitors, compounds (R)- and (S)-6d-h and 7b were confirmed to >95% by 1H-NMR and C18 reversed
phase HPLC, using an Atlantis T3 OBD, 5 µm, 4.6 mm x 100 mm column and a linear gradient of
H2O:MeCN from 95:5 to 5:95 in 13 min then 100% MeCN for 2 min at a flow rate of 1 mL/min (all
solvents contained 0.1% formic acid). LRMS were obtained on a Waters LC-MS instrument with ESI±
modes. HRMS were obtained on a TOF instrument with ESI± modes, and the quoted masses are accurate
to ±5 ppm. The names of the molecules that appear in the following pages were generated using Chem-
BioDraw Ultra 12.0.
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General procedure for the preparation of final inhibitors 6. Synthesis of the thienopyrimidine scaf-
folds 14, the SNAr reaction between 13 and 14, as well as the final deprotection of the phosphonate esters
15 to obtain the phosphonic acid inhibitors 6 were previously reported.9
(R)- or (S)-(2-phenyl-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid [(R)-
or (S)-6d]. Both compounds were isolated as off-white solids, (R)-6d, 3 mg, 37% yield; (S)-6d, 15 mg,
90% yield (yields indicated are for the last deprotection step). 1H NMR (500 MHz, D2O): δ 7.88 (s, 1H),
7.75 (s, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.13 (t, J = 7.4
Hz, 2H), 7.06 (t, J = 7.3 Hz, 1H), 4.59 (ddd, J = 15.2, 12.4, 3.0 Hz, 3H), 3.46 (d, J = 13.8 Hz, 1H), 2.92
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(td, J = 13.1, 6.5 Hz, 1H), 2.37 (s, 3H); 31P{1H}NMR (203 MHz, D2O): δ 15.84; 13C{1H}NMR (126 MHz,
D2O): δ 162.6, 156.7, 152.10, 140.1, 139.9 (d, J = 13.5 Hz), 138.7, 129.7, 129.6, 129.4, 128.1, 126.2,
125.3, 118.1, 112.9, 52.4 (d, J = 138.2 Hz), 38.1, 20.2. HRMS-ESI (m/z) [M-H+]- calcd for C21H19N3O3PS:
424.0890, found 424.0878.
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6
7
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12
13
14
15
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(R)- or (S)-(2-(3-fluorophenyl)-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic
acid [(R)- or (S)-6e]. Both compounds were isolated as off-white solids, (R)-6e, 6 mg, 40% yield; (S)-6e,
5 mg, 45% yield (yields indicated are for the last deprotection step). 1H NMR (500 MHz, D2O): δ 7.71 (s,
1H), 7.61 (s, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 6.90 (dd, J = 16.5, 9.5 Hz, 3H), 6.60
(t, J = 8.4 Hz, 1H), 4.38 (ddd, J = 15.6, 12.1, 3.1 Hz, 1H), 3.27 (d, J = 13.6 Hz, 1H), 2.73 (td, J = 12.9,
6.5 Hz, 1H), 2.23 (s, 3H); 19F{1H}NMR (471 MHz, D2O): δ -115.07; 31P{1H}NMR (203 MHz, D2O): δ
15.62; 13C{1H}NMR (126 MHz, D2O): δ 163.3, 162.1 (d, J = 187.3 Hz), 156.8, 152.2, 142.6 (d, J = 21.2
Hz), 140.3, 139.0, 129.8, 129.6 (d, J = 8.4 Hz), 129.5, 125.6, 125.5, 118.2, 116.2 (d, J = 21.0 Hz), 113.1,
112.7 (d, J = 20.6 Hz), 52.3 (d, J = 136.6 Hz), 38.0, 20.3. HRMS-ESI (m/z) [M+H]+ calcd for
C21H20FN3O3PS: 444.0942, found 444.0949.
(R)-
or
(S)-(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-phe-
nylethyl)phosphonic acid [(R)- or (S)-6f]. Both compounds were isolated as off-white solids, (R)-6f, 18
mg, 90% yield; (S)-6f, 8 mg, 88% yield (yields indicated are for the last deprotection step). 1H NMR (500
MHz, D2O): δ 7.84 (s, 1H), 7.65 (s, 1H), 7.50 (s, 1H), 7.34 (d, J = 7.5 Hz, 2H), 7.25 (d, J = 7.9 Hz, 1H),
7.12 (t, J = 7.5 Hz, 2H), 7.03 (t, J = 7.3 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.58 (t, J = 13.7 Hz, 1H), 3.44
(d, J = 13.8 Hz, 1H), 2.90 (td, J = 12.7, 6.1 Hz, 1H), 2.18 (s, 3H); 31P{1H}NMR (203 MHz, D2O): δ 15.79.
13C{1H}NMR (126 MHz, D2O): δ 162.9, 156.7, 152.5, 139.8 (d, J = 7.1 Hz), 138.4, 135.9, 134.0, 131.6,
131.0, 129.4, 128.1, 126.1, 125.3, 123.5, 117.9, 113.8, 52.3 (d, J = 143.0 Hz), 38.0, 18.8. HRMS-ESI
(m/z) [M-H+]- calcd for C21H18ClN3O3PS: 458.0501, found 458.0489.
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(R)-
or
(S)-(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(3-fluoro-
1
2
3
phenyl)ethyl) phosphonic acid [(R)- or (S)-6g]. Both compounds were isolated as off-white solids, (R)-
4
5
6
1
6g, 10 mg, 41% yield; (S)-6g, 5 mg, 50% yield (yields indicated are for the last deprotection step). H
7
8
9
NMR (400 MHz, D2O): δ 7.88 (s, 1H), 7.79 (d, J = 1.5 Hz, 2H), 7.58 (dd, J = 8.0, 1.8 Hz, 1H), 7.37 (d, J
= 8.0 Hz, 1H), 7.10 – 7.00 (m, 3H), 6.75 (t, J = 9.0 Hz, 1H), 4.53 (ddd, J = 15.6, 12.3, 3.0 Hz, 1H), 3.42
(dt, J = 13.3, 3.0 Hz, 1H), 2.88 (td, J = 13.3, 6.6 Hz, 1H), 2.41 (s, 3H); 31P{1H}NMR (162 MHz, D2O): δ
15.31; 19F{1H}NMR (377 MHz, D2O) δ -115.57;
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13C{1H}NMR (126 MHz, D2O): δ 163.0, 162.3 (d, J = 242.4 Hz), 156.9 (d, J = 6.6 Hz), 152.5, 142.6 (dd,
J = 13.6, 7.2 Hz), 138.4, 136.3, 134.2, 131.9, 131.2, 129.5 (d, J = 8.5 Hz), 125.6, 125.6 (d, J = 2.4 Hz),
123.8, 117.9, 116.2 (d, J = 20.8 Hz), 114.1, 112.7 (d, J = 21.0 Hz), 52.2 (d, J = 138.4 Hz), 38.0, 18.8.
HRMS-ESI (m/z) [M-H+]- calcd for C21H17ClFN3O3PS: 476.0406, found 476.0387.
(R)- or (S)-(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-cyclohexylethyl)
phosphonic acid [(R)- or (S)-6h]. Both compounds were isolated as off-white solids, (R)-6h, 3 mg, 50%
yield; (S)-6h, 5 mg, 35% yield (yields indicated are for the last deprotection step). 1H NMR (500 MHz,
D2O): δ 8.21 (s, 1H), 7.59 (s, 1H), 7.47 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 4.43 (t,
J = 13.7 Hz, 1H), 2.16 (s, 3H), 2.01 (d, J = 12.2 Hz, 1H), 1.81 (t, J = 12.4 Hz, 1H), 1.72 – 1.55 (m, 5H),
1.31 (d, J = 11.8 Hz, 1H), 1.22 – 1.01 (m, 4H), 0.92 (q, J = 11.8 Hz, 1H); 31P{1H}NMR (202 MHz, D2O):
δ 17.32; 13C{1H}NMR (201 MHz, D2O): δ 163.1, 153.2, 138.2, 136.3, 134.3, 132.0, 131.3, 125.7, 123.9,
114.2, 48.5, 47.8, 39.5, 34.2, 32.1, 26.2, 25.9, 18.9. HRMS-ESI (m/z) [M-H+]- calcd for C21H25ClN3O3PS:
464.0970, found 464.0982.
((R)-
or
(S)-2-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-3-(3-fluoro-
phenyl)propanoic acid [(R)- or (S)-7b]. The SNAr reaction between α-amino acid 17 and 14b was con-
ducted based on previously reported procedure with minor modifications.8a,9 To a stirred solution of 14b
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(28 mg, 0.1 mmol, 1 eq.) in 1.6 ml dioxane, HCl salt of 17 (33 mg, 0.15 mmol, 1.5 eq) in dioxane:H2O
mixture (1:1, 1.6 mL, pH = ~8-9 adjusted with 2.5 eq. Na2CO3) was added. The mixture was stirred at
100°C for 30 min and then acidified using 1 M HCl (10 mL). EtOAc (10 mL x 2) was used to extract
organic compounds from aqueous phase. The crude was concentrated under reduced pressure. First
purification was done by flash chromatography on silica gel using a solvent gradiant from 20-50% EtOAc
in hexane followed by 0-10% MeOH in Et2O. The concentrated polar fractions were repurified using
semi-preparative HPLC on a C18 reversed phase column (Atlantis T3 OBD Prep Column, 100Å, 5 µm,
19 mm x 50 mm) to give both enantiomers as white solids: (R)-7b (6 mg) in 20% yield, 80% ee; (S)-7b
(15 mg) 32% yield, 83% ee. 1H NMR (400 MHz, DMSO-d6): δ 8.29 (s, 1H), 8.15 (s, 1H), 8.01 (d, J = 7.8
Hz, 1H), 7.74 (d, J = 1.9 Hz, 1H), 7.55 (dd, J = 7.9, 1.9 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.26 (td, J =
8.0, 6.3 Hz, 1H), 7.16 – 7.09 (m, 2H), 7.00 – 6.92 (m, 1H), 4.86 (q, J = 8.8, 7.5 Hz, 1H), 3.13 (dd, J =
13.9, 9.3 Hz, 2H), 2.37 (s, 3H); 19F{1H}NMR (471 MHz, DMSO-d6): δ -114.18; 13C{1H}NMR (101 MHz,
DMSO-d6): δ 172.5, 164.9, 161.9 (d, J = 242.3 Hz), 156.2, 154.0, 142.2 (d, J = 6.9 Hz), 136.1, 135.6,
134.1, 132.8, 132.0, 129.6 (d, J = 8.3 Hz), 125.4, 125.3 (d, J = 2.5 Hz), 124.3, 117.4, 116.0 (d, J = 10.8
Hz), 115.7, 112.6 (d, J = 20.8 Hz), 55.8, 36.9, 19.3. HRMS-ESI (m/z) [M+H]+ calcd for C22H17ClFN3O2S:
442.0787, found 442.0775. Chiral HPLC (UV abs. 254 nm), Lux® Cellulose-3 (250 mm x 4.6 mm), 60:40
MeCN:H2O (0.1% formic acid), 1 ml/min: (R)-enantiomer tR = 6.43 min, (S)-enantiomer tR = 6.81 min.
1
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Diethyl (1-acetamidovinyl)phosphonate (8). The synthesis of compound 8 was achieved in two steps as
previously reported.18, 25
Step 1: Triethyl phosphite (9.2 mL, 54 mmol) was slowly added to acetyl chloride (3.8 mL, 60 mmol) at
0°C under Ar. The mixture was stirred for 18 h at RT and concentrated under reduced pressure to remove
excessive starting material and by-product. Diethyl acetylphosphonate was isolated as a colorless oil (9.25
g, 95% yield). 1H NMR (500 MHz, CDCl3): δ 4.23 (p, J = 7.2 Hz, 4H), 2.48 (d, J = 5.1 Hz, 3H), 1.38 (t,
J = 7.1 Hz, 6H); 31P{1H}NMR (203 MHz, CDCl3): δ -2.89. NMR data consistent with those previously
reported.
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Step 2: In a 250 mL three-neck round-bottom flask connected to Dean-Stark apparatus with a condenser,
cyclohexane (90 mL) was added to acetamide (4.35 g, 73.5 mmol, 1.5 eq.), pTsOH monohydrate (0.932
g, 4.9 mmol, 0.1 eq.) and p-methoxyphenol (6.1 mg, 0.049 mmol, 0.1 mol%) under Ar. The mixture was
refluxed at 102°C and diethyl acetylphosphonate (9.29 g, 49 mmol) was added dropwise over 60 min with
a syringe pump. After refluxing for another 2 h, the mixture was cooled to RT and concentrated. Water
(100 mL) and EtOAc (50 mL) were added to the crude; the two phases were separated and the aqueous
phase was further extracted with EtOAc (50 mL x 2). The combined organic phase was dried over anhy-
drous MgSO4 and concentrated. The crude pale yellow oil was purified by flash chromatography using
20-85% EtOAc in hexane (desired product eluted at 60-70% EtOAc) to yield an off-white solid (1.6 g,
15% yield). 1H NMR (500 MHz, CDCl3): δ 7.18 (s, 1H), 6.66 (d, J = 41.6 Hz, 1H), 5.50 (d, J = 19.4 Hz,
1H), 4.18 – 4.07 (m, 4H), 2.10 (s, 3H), 1.35 (t, J = 7.1 Hz, 6H); 31P{1H}NMR (203 MHz, CDCl3): δ 12.63;
13C{1H}NMR (126 MHz, CDCl3): δ 169.5, 131.4 (d, J = 198.8 Hz), 113.3 (d, J = 9.2 Hz), 63.2 (d, J = 5.5
Hz), 24.8, 16.3 (d, J = 6.4 Hz). NMR data consistent with those previously reported. LRMS (ESI) m/z:
222.4 [M+H]+.
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Potassium 3-Fluorophenyl trifluoroborate salt (9d). The synthesis of trifluoroborate salt 9d was
achieved using standard literature procedures.26
Step 1: Anhydrous THF (15 mL) was added to an oven-dried 100 mL round bottom flask under Ar, cooled
to -78°C and n-BuLi (6 mL, 2.5M in hexane, 20 mmol, 1.5 eq.) was added slowly. 3-bromofluorobenzene
(1.1 mL, 10 mmol, 1 eq.) was added dropwise and the mixture was stirred for 30 min at -78°C. Triisopro-
pyl borate (4.6 mL, 20 mmol, 2 eq.) was added dropwise. Stirring was continued at -78 °C for an additional
20 min, and then the mixture was warmed up and stirred at RT for 30 min. HCl (1M, 15 mL) was subse-
quently added to hydrolyze the borate and the mixture was stirred for 20 min. The aqueous phase was
extracted with anhydrous Et2O (30 mL x 2). The combined organic phase was dried over anhydrous
MgSO4 and concentrated. The crude solid was washed with hexane to remove any by-products and 3-
fluorophenylboronic acid was isolated as a white solid (1 g, 65% yield), which was used as such in the
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1
following step. H NMR (500 MHz, CDCl3): δ 8.00 (d, J = 7.3 Hz, 1H), 7.88 (dt, J = 9.0, 4.6 Hz, 1H),
7.52 (m, 1H), 7.31 (m, 1H). 4.79 (s, boronic acid); 19F{1H}NMR (471 MHz, CDCl3): δ -113.53.
Step 2: 3-Fluorophenylboronic acid (0.62 g, 4.5 mmol, 1 eq.) was dissolved in MeOH (1.5 mL) and an
excess of saturated KHF2 aqueous solution (0.88 g in 2.5 mL MilliQ water, ~4.5M, 11.3 mmol) was added
slowly under vigorous stirring. After 30 min, the solvent was removed and the mixture was taken up in
small quantity of MeCN and filtered to remove KHF2. The filtrate was concentrated to obtain a white
solid (0.89 g, quantitative yield), which was used directly in next step. 1H NMR (400 MHz, DMSO-d6):
δ 7.11 (t, J = 5.0 Hz, 2H), 6.96 (dd, J = 10.2, 2.5 Hz, 1H), 6.83 – 6.74 (m, 1H); 19F{1H}NMR (377 MHz,
DMSO-d6): δ -116.38; 13C{1H}NMR (101 MHz, DMSO-d6): δ 128.5 (d, J = 6.5 Hz), 127.5, 117.4 (d, J
= 16.0 Hz), 111.8 (d, J = 20.8 Hz).
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7
8
9
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Diethyl (R)-(1-acetamido-2-phenylethyl)phosphonate [(R)-10a]. The synthesis of (R)-10a was based
on the procedure provided below for the preparation of (R)-10b with one modification; due to the poor
solubility of potassium phenyl trifluoroborate in iPrOH, phenyl boronic acid was used instead. The desired
product (R)-10a was isolated as a yellow oil (275 mg, 95% yield, 93% ee). 1H NMR (400 MHz, CDCl3):
δ 7.34 – 7.25 (m, 4H), 7.24 (d, J = 6.8 Hz, 1H), 6.15 (d, J = 10.0 Hz, 1H), 4.79 (dtd, J = 16.3, 10.2, 4.9
Hz, 1H), 4.12 (m, 4H), 3.24 (ddd, J = 14.0, 8.7, 4.9 Hz, 1H), 2.93 (dt, J = 14.4, 10.5 Hz, 1H), 1.90 (d, J =
1.1 Hz, 3H), 1.31 (t x 2, J = 7.1 Hz, 6H); 31P{1H}NMR (162 MHz, CDCl3): δ 24.21; 13C{1H}NMR (126
MHz, CDCl3): δ 169.6 (d, J = 5.2 Hz), 136.7 (d, J = 12.8 Hz), 129.2, 128.5, 126.9, 63.0 (d, J = 6.9 Hz),
62.6 (d, J = 6.8 Hz), 46.8, 45.5, 35.8 (d, J = 3.3 Hz), 23.0, 16.5 (d x 2, J = 6.2 Hz). LRMS (ESI) m/z:
300.6 [M+H]+. Chiral HPLC (UV abs. 220 nm) condition: Chiralcel OD (250 mm x 4.6 mm), 95:5 hexane:
iPrOH, 0.8 ml/min; tR = 16.4 min (major), 18.6 min (minor).
Diethyl (S)-(1-acetamido-2-phenylethyl)phosphonate [(S)-10a]. Synthesis of (S)-10a was achieved us-
ing the procedure described below for the preparation of (S)-10b. The product was isolated as a yellow
oil (115 mg) in 65% yield, 70% ee. 1H/31P/13C NMR and LC-MS data were identical to those of the (R)-
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isomer. Chiral HPLC (UV abs. 220 nm) condition: Chiralcel OD (250 mm x 4.6 mm), 95:5 hexane: iPrOH,
0.8 ml/min; tR = 16.7 min (minor), 18.4 min (major).
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Diethyl (R)-(1-acetamido-2-(3-fluorophenyl)ethyl)phosphonate [(R)-10b]. Diethyl (1-acetamido-vi-
nyl) phosphonate 8 (335 mg, 1.5 mmol, 1 eq.), potassium 3-fluorophenyl trifluoroborate 9d (576 mg, 2.85
mmol, 1.9 eq.), NaHCO3 (252 mg, 3 mmol, 2 eq.), di-μ-chloro-tetraethylene dirhodium(I) (8.8 mg, 0.023
mmol, 1.5 mol%) and (S)-difluorphos (33.8 mg, 0.05 mmol, 3.3 mol%) were dissolved in anhydrous,
degassed iPrOH (4 mL) under Ar in a microwave tube (Biotage) and the mixture was further degassed for
10 min with Ar. The resulting orange suspension was stirred at 90°C for 20 h. After cooling to RT, satu-
rated aqueous NH4Cl solution (10 mL) was added into the mixture. The aqueous phase was extracted with
DCM (10 mL x 3); the combined organic phase was washed with brine and dried with anhydrous MgSO4.
The solvent was evaporated to yield crude product as brown viscous oil, which was purified by flash
column chromatography on silica gel (from 1:1 EtOAc : hexane to 9:1 EtOAc : MeOH) to give the desired
product as a yellow oil (330 mg, 70% yield, 90% ee). 1H NMR (500 MHz, CDCl3): δ 7.25 (m, 1H), 7.00
(d, J = 7.7 Hz, 1H), 6.92 (m, 2H), 5.65 (d, J = 10.1 Hz, NH), 4.75 (dd, J = 16.8, 4.9 Hz, 1H), 4.13 (m,
4H), 3.20 (ddd, J = 14.4, 9.5, 4.9 Hz, 1H), 2.88 (dt, J = 14.6, 10.2 Hz, 1H), 1.91 (d, J = 0.8 Hz, 3H), 1.31
(t x 2, J = 7.1 Hz, 6H); 19F{1H}NMR (471 MHz, CDCl3): δ -113.32; 31P{1H}NMR (203 MHz, CDCl3): δ
23.80; 13C{1H}NMR (126 MHz, CDCl3): δ 169.4 (d, J = 5.0 Hz), 139.2 (d, J = 5.4 Hz), 130.0 (d, J = 8.3
Hz), 124.9, 116.4 (d, J = 21.4 Hz), 114.0 (d, J = 20.8 Hz), 63.0 (d, J = 7.1 Hz), 62.8 (d, J = 6.7 Hz), 46.6,
45.49, 35.7, 23.2, 16.5 (d x 2, J = 5.5 Hz). LRMS (ESI) m/z: 318.3 [M+H]+. Chiral HPLC (UV abs. 220
nm) condition: Chiralcel OD (250 mm x 4.6 mm), 95:5 hexane: iPrOH, 1 ml/min; tR = 12.3 min (major),
15.1 min (minor)
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Diethyl (S)-(1-acetamido-2-(3-fluorophenyl)ethyl)phosphonate [(S)-10b].19 Diethyl (1-acetamido-vi-
nyl) phosphonate 8 (223 mg, 1 mmol, 1 eq.), 3-fluorophenyl boronic acid (280 mg, 2 mmol, 2 eq.) and
Na2CO3 (106 mg, 1 mmol, 1 eq.) were mixed in dry and degassed dioxane (3 mL) under Ar in a microwave
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Journal of Medicinal Chemistry
tube (Biotage). Bis(norbornadiene)rhodium(I) tetrafluoroborate (26 mg, 0.07 mmol, 7 mol%) and (S,S)-
Chiraphos (32 mg, 0.075 mmol, 7.5 mol%) were premixed with degassed dioxane/water (0.9/0.45 mL)
and injected into the microwave tube. The mixture was then further degassed for 10 min with Ar, and
MeOH (0.32 mL, 8 eq.) was added dropwise. The orange suspension was heated to 70°C for 20 h (moni-
tored by TLC). After cooling to RT, solvent was removed under reduced pressure. The residue was re-
dissolved in DCM, washed with water and brine, and dried over anhydrous MgSO4 to give the crude
product as a brown viscous oil, which was purified by flash column chromatography on silica gel (using
a solvent gradient from 1:1 EtOAc:hexane to 9:1 EtOAc:MeOH). Compound (S)-10b was isolated as a
yellow oil (0.23 g) 70% yield in 89% ee. 1H/19F/31P/13C NMR and LC-MS data were identical to those of
the (R)-enantiomer. Chiralcel OD (250 mm x 4.6 mm), 95:5 hexane: iPrOH, 1 ml/min; tR = 12.7 min
(minor), 14.9 min (major)
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Diethyl (S)- or (R)-(1-benzamido-2-cyclohexylethyl)phosphonate [(S)- or (R)-10c]. 21 Diethylzinc
(0.15 mL, 1.3 M in toluene, 0.2 mmol) was added slowly to a stirred solution of (R,R) or (S,S)-ProPhenol
(ligand L1; 64 mg, 0.1 mmol) in toluene (1.85 mL) under Ar. The mixture was stirred at RT for 0.5 h to
generate zinc catalyst (0̚ .05 M). The resulting catalyst solution (1 mL, 0.05 mmol) was added in one
portion to freshly made 11 (120 mg, 0.5 mmol, ca. 80% purity) and diethyl phosphite (0.05 mL, 0.6 mmol,
1.2 eq.) in toluene (4 mL) at 0°C under Ar. After stirring for 30 min, the reaction was quenched with
saturated aqueous NH4Cl solution. After extraction with anhydrous Et2O (20 mL x 2), the combined or-
ganic phase was washed with brine, dried over anhydrous MgSO4 and concentrated under reduced pres-
sure. The crude mixture was purified by flash column chromatography on silica gel using 20%-70%
EtOAc in hexane to obtain (R)-10c or (S)-10c as a white solids (50 mg, 30% yield for both enantiomers)
with >95% ee (after recrystallization using DCM-pentane at -20°C) and side product 12 (due to isomeri-
zation of starting material 11). 1H NMR (400 MHz, CDCl3): δ 7.81 – 7.74 (m, 2H), 7.57 – 7.49 (m, 1H),
7.49 – 7.41 (m, 2H), 6.10 (d, J = 10.1 Hz, 1H), 4.86 – 4.71 (m, 1H), 4.24 – 4.03 (m, 4H), 1.94 (d, J = 12.9
Hz, 1H), 1.84 – 1.67 (m, 1H), 1.66 (m, 6H), 1.44 – 1.35 (m, 1H), 1.35 (s, 1H), 1.35 – 1.24 (m, 6H), 1.17
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(m, 2H), 1.09 – 0.94 (m, 1H), 0.89 (t, J = 11.9 Hz, 1H); 31P {1H} NMR (202 MHz, CDCl3): δ 25.55; 13C
{1H} NMR (126 MHz, CDCl3): δ 167.0, 134.3, 131.9, 128.9, 127.1, 62.8 (dd, J = 19.2, 6.8 Hz), 42.9,
37.4, 34.1 (d, J = 12.7 Hz), 26.6, 26.1, 16.6 (d x 2, J = 5.8 Hz). LRMS (ESI) m/z: 368.3 [M+H]+. HPLC
(UV abs. 220 nm) condition: Chiralcel OD (250 mm x 4.6 mm), 98:2 hexane: iPrOH, 0.8 ml/min; (R)-
enantiomer tR = 10.3 min (major), 12.9 min (minor); (S)-enantiomer tR = 9.9 min (minor), 12.0 min (ma-
jor).
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(E)-N-(2-cyclohexylethylidene)benzamide (11). The synthesis of 11 was achieved in three steps using
the procedure recently reported by Lupton27 with minor modifications.
Step 1: Pyridinium chlorochromate (3.23 g, 15 mmol, 1.5 eq.) was added in one portion to cyclohexyleth-
anol (1.4 mL, 10 mmol) in DCM (50 mL) and the mixture was stirred at RT for 2 h. Silica gel (3.75 g)
and anhydrous diethyl ether (50 mL) were subsequently added and stirring was continued for an additional
1 h. The mixture was concentrated and the solid residue was loaded directly onto a silica gel column and
eluted using 0%-25% EtOAc in hexane to yield the 2-cyclohexylacetaldehyde as a colorless oil (0.83 g,
63% yield). 1H NMR (500 MHz, CDCl3): δ 9.75 (t, J = 2.4 Hz, 1H), 2.28 (dd, J = 6.8, 2.4 Hz, 2H), 1.88
13
(m, 1H), 1.71 (m, 5H), 1.29 (m, 2H), 1.17 (m, 1H), 1.00 (m, 2H); C{1H}NMR (126 MHz, CDCl3): δ
203.2, 51.5, 33.4, 32.8, 26.2.
Step 2: Benzenesulfinic acid sodium salt (2.73 g, 15 mmol, 1.5 eq) and benzamide (1.82 g, 15 mmol, 1.5
eq) were mixed with acetonitrile (100 mL) under Ar. After purging Ar for 15 min, 2-cyclohexylacetalde-
hyde (1.41 g, 10 mmol) was added to the slurry in one portion. The suspension was cooled to 0°C and
chlorotrimethylsilane (2.68 mL, 20 mmol, 2 eq) was added slowly. The reaction was warmed up to RT
and stirred for 16 h. Water (80 mL) was added to the suspension and the mixture was stirred for 30 min.
N-(2-cyclohexyl-1-tosylethyl)benzamide precipitated as fine white solid and collected by filtration. The
1
product was used in the next step without further purification (3.0 g, 80% yield). H NMR (500 MHz,
CDCl3): δ 7.79 – 7.72 (m, 2H), 7.61 – 7.55 (m, 2H), 7.55 – 7.50 (m, 1H), 7.46 – 7.38 (m, 2H), 7.25 – 7.23
(m, 1H), 6.35 (d, J = 10.5 Hz, 1H), 5.52 (ddd, J = 11.7, 10.5, 3.1 Hz, 1H), 2.39 (s, 3H), 2.19 (ddd, J =
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14.2, 9.8, 3.1 Hz, 1H), 1.89 – 1.78 (m, 2H), 1.69 (d, J = 11.0 Hz, 3H), 1.62 (d, J = 6.9 Hz, 1H), 1.41 (dddt,
J = 14.4, 10.8, 7.2, 3.7 Hz, 1H), 1.29 – 1.00 (m, 4H), 0.95 (tt, J = 12.0, 5.9 Hz, 1H); 13C{1H}NMR (101
MHz, CDCl3): δ 166.4, 145.3, 133.8, 133.2, 132.4, 129.9, 129.2, 128.9, 127.1, 67.6, 34.2, 34.0, 33.9, 32.0,
26.4, 26.3, 26.0, 21.9. LRMS (ESI) m/z: 408.3 [M+Na]+.
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Step 3: Cs2CO3 (2.45 g,7.5 mmol) and Na2SO4 (1.06 g, 7.5 mmol) were flame-dried under high vacuum,
cooled under Ar and mixed with anhydrous DCM (20 mL). N-(2-cyclohexyl-1-tosylethyl)benzamide (577
mg, 1.5 mmol) was added as DCM solution (5 mL). The reaction mixture was stirred vigorously (>1000
rpm) at RT for 4 h. Pentane (40 mL) was added to the mixture and the solid was filtered. The solution
was concentrated under reduced pressure at 20°C to obtain 11 as colorless oil, which was used directly in
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the next step without purification. H NMR (400 MHz, CDCl3): δ 8.12 (t, J = 5.0 Hz, 1H), 8.06 – 7.98
(m, 2H), 7.61 – 7.54 (m, 1H), 7.46 (dd, J = 8.4, 7.0 Hz, 2H), 2.35 (dd, J = 6.5, 5.0 Hz, 2H), 1.86 – 1.59
13
(m, 7H), 1.36 – 1.13 (m, 3H), 1.11 – 0.98 (m, 2H); C{1H}NMR (101 MHz, CDCl3): δ 180.9, 169.7,
133.5, 132.9, 130.0, 128.6, 77.5, 77.2, 76.8, 44.1, 35.3, 33.4, 26.3, 26.2. LRMS (ESI) m/z: 230.2 [M+H]+.
(E)-N-(2-cyclohexylvinyl)benzamide (12). Compound 12 was isolated as a white solid as major by-
product in the preparation of 10c. 1H NMR (500 MHz, CDCl3): δ 7.92 – 7.72 (m, 2H), 7.61 (d, J = 9.8
Hz, 1H), 7.58 – 7.47 (m, 1H), 7.44 – 7.38 (m, 2H), 6.94 (ddd, J = 14.4, 10.5, 1.3 Hz, 1H), 5.27 (dd, J =
14.4, 7.1 Hz, 1H), 2.04 (qdd, J = 8.1, 4.2, 1.2 Hz, 1H), 1.79 – 1.72 (m, 5H), 1.66 (dddt, J = 12.9, 5.1, 3.4,
1.6 Hz, 2H), 1.28 (tt, J = 12.7, 3.2 Hz, 2H), 1.23 – 1.16 (m, 1H), 1.16 – 1.08 (m, 2H).
General procedure for the synthesis of α-aminophosphonate diethyl esters 13. A typical procedure
for the N-deacetylation of intermediate 10 (regardless stereochemistry) at 1 mmol scale was achieved in
three steps according to literature procedure.23
Step 1: Intermediate 10 in anhydrous THF (4.5 mL) was transferred to a 10 mL microwave tube loaded
with Boc2O (437 mg, 2 mmol, 2 eq.) and DMAP (24 mg, 0.2 mmol, 0.2 eq.) under Ar. The tube was
sealed and the reaction mixture was stirred for 4 h at 80°C. After cooling to RT, the solvent was removed
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under reduced pressure and the crude product was re-dissolved in DCM. The organic phase was washed
with saturated aqueous NH4Cl and NaHCO3 solutions, dried over anhydrous Na2SO4 and concentrated to
give crude as orange to brown oil.
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Step 2: The crude from Step 1 was diluted in THF-water mixture (2:1, in total 6 mL) and LiOH·H2O (84
mg, 2 mmol, 2 eq.) was added. The suspension was stirred for 20-24 h at RT. The crude was diluted with
anhydrous Et2O (10 mL) and washed with 1 M HCl (4 mL) followed by brine. The organic phase was
dried over anhydrous Na2SO4 and concentrated to give orange oil.
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Step 3: The crude from Step 2 was diluted in anhydrous DCM (8 mL) under Ar. HCl (4 M in Dioxane, 5
mL, 20 eq.) was added dropwise to the solution. The mixture was stirred at RT for 2-4 h and monitored
by TLC. After completion, the solvent was removed under reduced pressure and slowly taken up in 5%
MeOH in DCM (10 mL). The solvent was removed again after stirring for 5 min and the residue was re-
dissolved in DCM. The organic phase was washed with 1 M NaOH (25 mL) followed by brine, and dried
over anhydrous Na2SO4. The solution was concentrated to give light orange oil. The crude product was
purified by flash column chromatography on silica gel (from 1:1 EtOAc : hexane to 1.5:8.5 MeOH :
EtOAc to yield light yellow oil (50-80% isolated yield over 3 steps).
Diethyl (R)- or (S)-(1-amino-2-phenylethyl)phosphonate [(R)- or (S)-13a]. Light yellow oils (130-140
mg, ~50% yield). 1H NMR (500 MHz, CDCl3): δ 7.33 – 7.29 (m, 2H), 7.25 – 7.21 (m, 3H), 4.17 (dqd, J
= 8.0, 7.0, 4.3 Hz, 4H), 3.31 – 3.18 (m, 2H), 2.66 (ddd, J = 13.9, 10.8, 9.1 Hz, 1H), 1.34 (t x 2, J = 7.1
Hz, 6H); 31P{1H} NMR (203 MHz, CDCl3): δ 28.01; 13C{1H}NMR (126 MHz, CDCl3): δ 138.1 (d, J =
16.0 Hz), 129.4, 128.7, 126.97, 62.4 (t, J = 7.5 Hz), 50.5 (d, J = 154.2 Hz), 38.0, 16.7 (d, J = 5.6 Hz).
LRMS (ESI) m/z: 258.2 [M+H]+.
Diethyl (R)- or (S)- (1-amino-2-(3-fluorophenyl)ethyl)phosphonate [(R)- or (S)-13b]. Light yellow
oils (~130 mg, ~50% yield). 1H NMR (500 MHz, CDCl3): δ 7.29 (dd, J = 7.9, 6.1 Hz, 1H), 7.02 (d, J =
7.7 Hz, 1H), 6.95 (ddd, J = 10.7, 9.0, 2.3 Hz, 2H), 4.18 (qd, J = 7.0, 3.0 Hz, 4H), 3.26 (dd, J = 10.7, 3.3
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Journal of Medicinal Chemistry
Hz, 1H), 3.20 (dd, J = 13.9, 3.5 Hz, 1H), 2.68 (dd, J = 13.9, 10.9 Hz, 1H), 1.35 (t x 2, J = 7.1 Hz, 6H);
19F{1H}NMR (471 MHz, CDCl3): δ -113.21; 31P{1H}NMR (203 MHz, CDCl3): δ 27.56. 13C{1H}NMR
(126 MHz, CDCl3): δ 163.1 (d, J = 245.8 Hz), 140.7 (d, J = 7.3 Hz), 130.1 (d, J = 8.4 Hz), 125.1 (d, J =
2.8 Hz), 116.3 (d, J = 21.1 Hz), 113.8 (d, J = 21.1 Hz), 62.6 (d, J = 7.0 Hz), 62.5 (d, J = 7.0 Hz), 50.3 (d,
J = 153.6 Hz), 37.8, 16.7 (d, J = 5.6 Hz). LRMS (ESI) m/z: 276.3 [M+H]+.
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Diethyl (R)- or (S)-(1-amino-2-cyclohexylethyl)phosphonate [(R)- or (S)-13c]. Light yellow oils (~30
1
mg, ~80% yield). H NMR (400 MHz, CDCl3): δ 4.27 – 4.05 (m, 4H), 3.07 (td, J = 11.0, 3.2 Hz, 1H),
1.83 – 1.60 (m, 7H), 1.48 – 1.37 (m, 1H), 1.34 (t x 2, J = 7.1 Hz, 6H), 1.31 – 1.08 (m, 3H), 1.05 – 0.93
(m, 1H), 0.91 – 0.77 (m, 1H); 31P{1H}NMR (162 MHz, CDCl3): δ 29.98; 13C{1H}NMR (126 MHz,
CDCl3): δ 62.2 (dd, J = 10.5, 7.3 Hz), 46.6, 45.5, 38.6, 34.4, 33.6 (d, J = 13.1 Hz), 32.0, 26.7, 26.5, 26.2,
16.7 (d, J = 5.5 Hz). LRMS (ESI) m/z: 264.3 [M+H]+.
4-Fluoro-6-(p-tolyl)thieno[2,3-d]pyrimidine
(14a)
and
6-(3-chloro-4-methylphenyl)-4-
fluorothieno[2,3-d]pyrimidine (14b). The preparation and characterization of 14a and 14b were previ-
ously reported.9
General procedure for the synthesis of aminophosphonate esters 15d-h. The SNAr reaction between
α-aminophosphonates 13 and scaffolds 14 was previously reported.9
Diethyl (R)- or (S)-(2-phenyl-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonate
[(R)- or (S)- 15d]. Both enantiomers were isolated as off-white solids; (R)-15d in 47% yield (9 mg) and
90% ee and (S)-15d in 67% yield (21 mg) and 71% ee. 1H NMR (400 MHz, CDCl3): δ 8.37 (s, 1H), 7.53
(dd, J = 5.6, 2.7 Hz, 3H), 7.34 – 7.27 (m, 2H), 7.24 – 7.16 (m, 4H), 7.16 – 7.10 (m, 1H), 6.50 (s, 1H),
5.55 – 5.43 (m, 1H), 4.26 – 3.93 (m, 4H), 3.38 (ddd, J = 13.9, 8.6, 5.0 Hz, 1H), 3.29 – 3.14 (m, 1H), 2.38
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(s, 3H), 1.31 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0 Hz, 3H); P{1H}NMR (162 MHz, CDCl3): δ 24.18;
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13C{1H}NMR (126 MHz, CDCl3): δ 166.1, 156.2 (d, J = 5.0 Hz), 153.4, 141.2, 138.8, 136.9 (d, J = 12.7
Hz), 131.0, 129.8 (d, J = 2.2 Hz), 129.32 (d, J = 3.5 Hz), 128.5, 126.9, 126.4, 126.3, 118.2, 112.5, 63.5
(d, J = 6.9 Hz), 62.5 (d, J = 7.3 Hz), 47.3 (d, J = 156.2 Hz), 36.2 (d, J = 4.1 Hz), 21.4, 16.5 (d x 2, J = 5.8
Hz). LRMS (ESI) m/z: 482.4 [M+H]+. Chiral HPLC (UV abs. 254 nm) condition: Chiralcel OD (250 mm
x 4.6 mm), 90:10 hexane: iPrOH, 1 ml/min; (R)-enantiomer tR = 7.0 min (minor), 15.0 min (major), (S)-
enantiomer tR = 6.9 min (major), 15.2 min (minor).
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Diethyl
(R)-
or
(S)-(2-(3-fluorophenyl)-1-((6-(p-tolyl)thieno
[2,
3-d]pyrimidin-4-
yl)amino)ethyl)phosphonate [(R)- or (S)-15e]. Both enantiomers were isolated as off-white solids; (R)-
15e in 72% yield (21 mg) and 80% ee; (S)-15e in 50% yield (15 mg) and 86% ee. 1H NMR (400 MHz,
CDCl3): δ 8.41 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.31 (s, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.16 (td, J = 8.1,
6.1 Hz, 1H), 7.08 – 7.01 (m, 2H), 6.85 (ddd, J = 10.6, 8.1, 2.6 Hz, 1H), 5.58 (d, J = 9.9 Hz, 1H), 5.44
(dtd, J = 16.4, 9.7, 5.0 Hz, 1H), 4.23 – 3.95 (m, 4H), 3.37 (ddd, J = 14.7, 9.9, 5.0 Hz, 1H), 3.13 (dt, J =
19
14.6, 10.3 Hz, 1H), 2.40 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H), 1.17 (t, J = 7.1 Hz, 3H); F{1H}NMR (377
MHz, CDCl3): δ -113.33; 31P{1H}NMR (162 MHz, CDCl3): δ 23.87; 13C{1H}NMR (126 MHz, CDCl3):
δ 166.1, 162.7 (d, J = 245.7 Hz), 156.1 (d, J = 5.0 Hz), 153.2, 141.1, 139.5 (dd, J = 13.5, 7.4 Hz ), 138.7,
130.9, 129.8 (d, J = 8.5 Hz), 129.7, 126.2, 124.8 (d, J = 2.8 Hz), 118.1, 116.3 (d, J = 21.5 Hz), 113.7 (d,
J = 21.0 Hz), 112.6, 63.0 (dd, J = 143.4, 7.1 Hz), 47.0 (d, J = 156.6 Hz), 35.7 (d, J = 4.4 Hz), 21.3, 16.3
(d x 2, J = 5.9 Hz). LRMS (ESI) m/z: 500.4 [M+H]+. Chiral HPLC (UV abs. 254 nm) condition: Chiralcel
OD (250 mm x 4.6 mm), 90:10 hexane: iPrOH, 1 ml/min: (R)-enantiomer tR = 5.8 min (minor), 21.4 min
(major), (S)-enantiomer tR = 7.0 min (major), 24.3 min (minor).
Diethyl (R)- or (S)-(1-((6-(3-chloro-4-methylphenyl)thieno[2, 3-d]pyrimidin-4-yl)amino)-2-phe-
nylethyl) phosphonate [(R)- or (S)-15f]. Both enantiomers were isolated as off-white solids; (R)-15f in
76% yield (26 mg) and 92% ee; (S)-15f in 44% yield (11 mg) and 71% ee. 1H NMR (500 MHz, CDCl3):
δ 8.37 (s, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.61 (s, 1H), 7.41 (dd, J = 7.9, 2.0 Hz, 1H), 7.31 (d, J = 7.0 Hz,
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2H), 7.24 (d, J = 7.8 Hz, 1H), 7.19 (t, J = 7.5 Hz, 2H), 7.13 (m, 1H), 5.50 (dtd, J = 14.9, 9.9, 4.8 Hz, 1H),
4.27 – 3.91 (m, 2H), 3.39 (ddd, J = 13.5, 8.3, 4.8 Hz, 1H), 3.25 (dt, J = 14.4, 10.6 Hz, 1H), 2.40 (s, 3H),
1.33 (t, J = 7.1 Hz, 3H), 1.10 (t, J = 7.0 Hz, 3H); 31P{1H}NMR (203 MHz, CDCl3): δ 24.16; 13C{1H}NMR
(126 MHz, CDCl3): δ 166.1, 156.2 (d, J = 4.9 Hz), 153.4, 141.2, 138.8, 136.9 (d, J = 12.7 Hz), 131.0,
129.8 (d, J = 2.2 Hz), 129.3 (d, J = 3.5 Hz), 128.5, 126.9, 126.4, 126.3, 118.2, 112.5, 63.5 (d, J = 6.9 Hz),
62.5 (d, J = 7.3 Hz), 47.3 (d, J = 156.2 Hz), 36.2 (d, J = 4.1 Hz), 21.4, 16.5 (d x 2, J = 5.8 Hz). LRMS
(ESI) m/z: 517.3 [M+H]+. Chiral HPLC (UV abs. 254 nm), Chiralcel OD (250 mm x 4.6 mm), 90:10
hexane: iPrOH, 1 ml/min: (R)-enantiomer tR = 5.5 min (minor), 12.3 min (major); (S)-enantiomer tR =
5.5 min (major), 12.2 min (minor).
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Diethyl (R)- or (S)-(1-((6-(3-chloro-4-methylphenyl)thieno[2, 3-d]pyrimidin-4-yl)amino)-2-(3-fluor-
ophenyl)ethyl) phosphonate [(R)- or (S)-15g]. Both enantiomers were isolated as off-white solids; (R)-
15g in 44% yield (25 mg) and 78% ee; (S)-15g in 26% yield (14 mg) and 87% ee. 1H NMR (400 MHz,
CDCl3): δ 8.41 (s, 1H), 7.65 (d, J = 1.8 Hz, 1H), 7.43 (dd, J = 7.9, 1.8 Hz, 1H), 7.35 (s, 1H), 7.28 (d, J =
8.3 Hz, 1H), 7.21 – 7.13 (m, 1H), 7.04 (t, J = 9.3 Hz, 2H), 6.85 (td, J = 8.6, 1.9 Hz, 1H), 5.64 (d, 1H),
5.44 (dtd, J = 19.6, 9.7, 4.9 Hz, 1H), 4.28 – 3.98 (m, 4H), 3.38 (ddd, J = 14.3, 9.6, 4.6 Hz, 1H), 3.13 (dt,
J = 14.3, 10.3 Hz, 1H), 2.41 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H), 1.17 (t, J = 7.0 Hz, 3H); 19F{1H}NMR (471
MHz, CDCl3): δ -113.29; 31P{1H}NMR (162 MHz, CDCl3): δ 23.79;
13C{1H}NMR (126 MHz, CDCl3): δ 166.4, 162.8 (d, J = 245.8 Hz), 156.4 (d, J = 4.7 Hz), 153.7, 139.6
(dd, J = 13.7, 7.4 Hz), 139.3, 136.5, 135.1, 133.2, 131.6, 129.9 (d, J = 8.2 Hz), 126.6, 124.9 (d, J = 2.8
Hz), 124.8, 118.1, 116.4 (d, J = 21.4 Hz), 113.8, 113.8 (d, J = 21.0 Hz), 63.8 (d, J = 6.9 Hz), 62.6 (d, J =
7.3 Hz), 47.1 (d, J = 156.6 Hz), 35.8, 20.0, 16.6 (d, J = 6.0 Hz), 16.3 (d, J = 6.0 Hz). LRMS (ESI) m/z:
534.4 [M+H]+. Chiral HPLC (UV abs. 254 nm): Chiralcel OD (250 mm x 4.6 mm), 90:10 hexane: iPrOH,
1 ml/min: (R)-enantiomer tR = 5.4 min (minor), 20.8 min (major); (S)-enantiomer tR = 5.4 min (major),
21.5 min (minor).
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Diethyl (R)- or (S)-(1-((6-(3-chloro-4-methylphenyl)thieno [2,3-d]pyrimidin-4-yl)amino)-2-cyclo-
hexylethyl) phosphonate [(R)- or (S)-15h]. Both enantiomers were isolated as off-white solids; (R)-15h
in 60% yield (15 mg) and 99% ee; (S)-15h in 52% yield (13 mg) and 91% ee. 1H NMR (500 MHz, CDCl3):
δ 8.45 (s, 1H), 7.69 (d, J = 4.3 Hz, 1H), 7.64 (d, J = 1.9 Hz, 1H), 7.42 (dd, J = 7.8, 1.9 Hz, 1H), 7.24 (d,
J = 7.9 Hz, 1H), 6.58 (s, 1H), 5.34 – 5.22 (m, 1H), 4.28 – 4.07 (m, 3H), 2.39 (s, 4H), 1.91 (tt, J = 16.4,
7.6 Hz, 4H), 1.82 – 1.71 (m, 1H), 1.63 – 1.57 (m, 1H), 1.41 (s, 1H), 1.37 – 1.22 (m, 4H), 1.16 - 0.95 (m,
7H), 0.95 – 0.83 (m, 1H); 31P{1H}NMR (203 MHz, CDCl3): δ 25.55; 13C{1H}NMR (126 MHz, CDCl3):
δ 166.4, 156.6 (d, J = 3.8 Hz), 154.0, 139.2, 136.5, 135.2, 133.1, 131.6, 126.6, 124.7, 118.0, 113.9, 63.3
(d, J = 7.0 Hz), 62.5 (d, J = 7.2 Hz), 43.8 (d, J = 155.7 Hz), 37.5, 34.2, 33.9 (d, J = 13.1 Hz), 32.2, 26.6,
26.3, 26.0, 20.0, 16.5 (d x 2, J = 5.9 Hz). Chiral HPLC (UV abs. 254 nm), Chiralcel OD (250 mm x 4.6
mm), 90:10 hexane: iPrOH, 1 ml/min: (R)-enantiomer tR = 5.3 min (minor), 7.2 min (major); (S)-enanti-
omer tR = 4.9 min (major), 6.5 min (minor).
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Methyl (R)-2-acetamido-3-(3-fluorophenyl)propanoate [(R)-16].24 Methyl-2-acetamidoacrylate (1
mmol, 146 mg), potassium 3-fluorophenyl trifluoroborate 9d (364 mg, 1.8 mmol, 1.8 eq.), bis(1,5-cy-
clooctadiene)rhodium(I) tetrafluoroborate (12.2 mg, 3 mol%) and (S)-BINAP (37 mg, 6 mol%) were
mixed in degassed, anhydrous toluene (3 mL) under Ar. Freshly distilled guaiacol (74 µL, 0.66 mmol, 1.2
eq.) was added and the mixture was stirred at 110°C for 16 h (monitored by TLC). After cooling to RT,
the mixture was diluted with anhydrous Et2O (10 mL) and directly dry-loaded into a silica gel column.
The crude was purified using 10%-75% EtOAc in hexane to obtain white solid (118 mg, 50% yield, 85%
ee). 1H/19F/13C NMR and LC-MS data were identical to those of the (S)-isomer. Chiral HPLC (UV abs.
220 nm), Chiralcel OD (250 mm x 4.6 mm), 90:10 hexane: iPrOH, 1 ml/min: (S)-enantiomer tR = 10.3
min (minor), (R)-enantiomer tR = 13.4 min (major).
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Methyl (S)-2-acetamido-3-(3-fluorophenyl)propanoate [(S)-16]. Synthesis of (S)-16 was achieved us-
ing the procedure described for preparing (R)-10b. The crude product was purified by flash column chro-
matography on silica gel using a solvent gradient from 10-70% EtOAc in hexane to obtain the desired
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product as an off-white solid (125 mg, 67% yield, 81% ee). H NMR (400 MHz, CDCl3): δ 7.29 – 7.21
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(m, 1H), 6.94 (tdd, J = 8.5, 2.7, 1.0 Hz, 1H), 6.87 (dt, J = 7.6, 1.3 Hz, 1H), 6.80 (ddd, J = 9.7, 2.5, 1.6 Hz,
1H), 5.96 (d, J = 7.7 Hz, 1H), 4.88 (dt, J = 7.7, 5.7 Hz, 1H), 3.74 (s, 3H), 3.12 (qd, J = 13.9, 5.7 Hz, 2H),
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2.00 (s, 3H); F{1H}NMR (377 MHz, CDCl3): δ -112.99; C{1H}NMR (126 MHz, CDCl3): δ 172.0,
169.7, 162.9 (d, J = 246.3 Hz), 138.5 (d, J = 7.3 Hz), 130.2 (d, J = 8.4 Hz), 125.1 (d, J = 2.9 Hz), 116.3
(d, J = 21.1 Hz), 114.2 (d, J = 20.9 Hz), 53.1, 52.6, 37.7 (d, J = 1.8 Hz), 23.29. LRMS (ESI) m/z: 240.2
[M+H]+. Chiral HPLC (UV abs. 220 nm): (R)-enantiomer tR = 11.4 min (major), (S)-enantiomer tR = 14.8
min (minor).
(R)- or (S)-2-Amino-3-(3-fluorophenyl)propanoic acid [(R)- or (S)-17]. The synthesis of 17 was
achieved using the same procedure as for 13, however, in Step 2, during LiOH hydrolysis, both the N-
deacetylation and the ester hydrolysis take place to give the desired amino acid HCl salts as white solids;
(R)-17 in 70% yield (41 mg) and (S)-17 in 74% yield (86 mg). The enantiomeric purity of amino acids 17
was not determined at this stage, but assumed to be very similar to the final inhibitors (R)- and (S)-7b,
which were analyzed by chiral HPLC and found to have 80% ee and 83% ee for (R) and (S), respectively.
1H NMR (400 MHz, D2O): δ 7.43 (td, J = 7.9, 6.1 Hz, 1H), 7.22 – 7.06 (m, 3H), 4.30 (dd, J = 7.5, 5.8 Hz,
1H), 3.37 (dd, J = 14.7, 5.6 Hz, 1H), 3.23 (dd, J = 14.6, 7.7 Hz, 1H); 19F{1H}NMR (377 MHz, D2O): δ -
113.13; 13C{1H}NMR (126 MHz, D2O): δ 171.7, 162.8 (d, J = 244.1 Hz), 136.7 (d, J = 7.5 Hz), 130.9 (d,
J = 8.5 Hz), 125.2 (d, J = 2.8 Hz), 116.1 (d, J = 21.9 Hz), 114.7 (d, J = 21.1 Hz), 54.3, 35.4 (d, J = 1.8
Hz). LRMS (ESI) m/z: 184.2 [M+H]+.
Expression and purification of hFPPS
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