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Pharmacophore Mapping of Thienopyrimidine-Based Monophosphonate (ThP-MP) Inhibitors of the Human Farnesyl Pyrophosphate Synthase Jaeok Park, Chun Yuen Leung, Alexios N. Matralis, Cyrus M. Lacbay, Michail Tsakos, Guillermo Fernandez De Troconiz, Albert M. Berghuis, and Youla S. Tsantrizos J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01888 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Pharmacophore mapping of thienopyrimidine-based monophosphonate (ThP-MP) 5 7
6
inhibitors of the human farnesyl pyrophosphate synthase 8 9 10 12
1
Jaeok Park,†# Chun Yuen Leung,‡# Alexios N. Matralis,‡ Cyrus M. Lacbay,‡ Michail Tsakos, ‡ 14
13
Guillermo Fernandez De Troconiz,‡ Albert M. Berghuis,†‡ Youla S. Tsantrizos*‡†‡ 15 16 17 18 20
19 ‡Department
2
21
of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC,
Canada H3A 0B8 24
23
†
25
Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal,
27
26
QC, Canada H3G 0B1 29
28
‡
30
Groupe de Recherche Axé sur la Structure des Protéines, McGill University, 3649 Promenade Sir
32
31
William Osler, Montreal, QC, Canada 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 #
50
J. Park and C. Y. Leung contributed equally to the work described in this manuscript
51 52 53 54 5 56 57 58 59
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ABSTRACT 5
4
The human farnesyl pyrophosphate synthase (hFPPS), a key regulatory enzyme in the 6 7
mevalonate pathway, catalyzes the biosynthesis of the C-15 isoprenoid farnesyl pyrophosphate 8 10
9
(FPP). FPP plays a crucial role in the post-translational prenylation of small GTPases that 12
1
perform a plethora of cellular functions. Although hFPPS is a well-established therapeutic target 13 15
14
for lytic bone diseases, the currently available bisphosphonate drugs exhibit poor cellular uptake 17
16
and distribution into non-skeletal tissues. Recent drug discovery efforts have focused primarily 19
18
on allosteric inhibition of hFPPS and the discovery of non-bisphosphonate drugs for potentially 20 2
21
treating non-skeletal diseases. Hit-to-lead optimization of a new series of thienopyrimidine24
23
based monosphosphonates (ThP-MPs) led to the identification of analogs with nanomolar 25 26
potency in inhibiting hFPPS. Their interactions with the allosteric pocket of the enzyme were 27 29
28
characterized by crystallography and the results provide further insight into the pharmacophore 31
30
requirements for allosteric inhibition. 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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INTRODUCTION 4 5 6 7
Human farnesyl pyrophosphate synthase (hFPPS) is the gate-keeper for the biosynthesis 8 10
9
of all mammalian isoprenoids (Fig. 1). The catalytic product of hFPPS, farnesyl pyrophosphate 12
1
(FPP), is the precursor of many human metabolites, including geranylgeranyl pyrophosphate 13 15
14
(GGPP). FPP and GGPP are essential for the post-translational prenylation of all small GTPase 17
16
proteins (Fig. 1),1 which play a crucial role in a plethora of cellular biological functions, 19
18
including cell signaling, proliferation, and synaptic plasticity.2 20 21
Although hFPPS has been a therapeutic target of interest for over 30 years,3 the highly 24
23
2
charged nature of its active site cavity has prevented the development of active site inhibitors 25 26
with good drug-like molecules (in the classical sense). Currently, nitrogen-containing 27 29
28
bisphosphonates (N-BPs) are the only clinically validated drugs that target hFPPS (e.g. 1, 2). N31
30
BPs bind avidly to bone, inhibiting osteoclasts,4 but suffer from poor oral bioavailability, rapid 32 3
clearance from the systemic circulation, and almost negligible distribution to non-skeletal 34 36
35
tissues.5 Consequently, these drugs do not allow clear validation of hFPPS as a therapeutic target 38
37
for treating non-skeletal diseases, such as cancer. Nonetheless, biochemical studies have shown 39 41
40
that inhibition of hFPPS can directly downregulate the activity of mutated H-Ras, K-Ras and N43
42
Ras proteins that are major drivers in oncogenesis.6 We have also previously shown that active 4 45
site inhibitors of hFPPS induce apoptosis and inhibit phosphorylation of ERK in multiple 46 48
47
myeloma cells (ERK is a signaling protein downstream from Ras).7 Additionally, clinical 50
49
evidence suggests that inhibition of hFPPS with the most potent of the N-BP drug, zoledronic 51 52
acid (1), improves survival of cancer patients via mechanism(s) unrelated to the skeletal effects 53 5
54
of this drug.8 For example, a randomized clinical trial (with >1,500 patients) has shown that 57
56
treatment with standard care chemotherapy plus zoledronic acid (1) increases the progression58 59
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free survival of multiple myeloma patients by a statistically significant period as compared to 5
4
clodronate.8 Although clodronate is also a bisphosphonate compound, it is not a potent inhibitor 6 8
7
of hFFPS. 10
9
Inhibition of hFPPS impacts both the down-stream and the up-stream events in the 12
1
mevalonate pathway, inducing cell apoptosis9 and activation of the human innate immune 13 15
14
response.10 Inhibition of hFPPS also blocks the biosynthesis of GGPP, an indirect consequence 17
16
of intracellular depletion of the FPP substrate required by the human geranylgeranyl 18 19
pyrophosphate synthase (hGGPPS;Fig. 1). Not surprisingly, treatment with zoledronic acid (1) 20 2
21
was found to cause intracellular decrease in Rap1A geranylgeranylation and activation, 24
23
providing an additional (or alternative) mechanism that could account for the in vivo 25 26
antimyeloma effects observed with this drug.8,11 Intracellular depletion of GGPP is also expected 27 29
28
to have a significant effect on the prenylation cascade from GGPP → RhoA-cdc42, which leads 31
30
to activation of GSK3-β and upregulation of phosphorylated tau (P-Tau) protein in the brain. P32 34
3
Tau accumulation is associated with the physiology of tauopathies, such as frontotemporal 36
35
dementia, neuronal damage and the progression of the Alzheimer’s-associated tangle formation 38
37
in neurons.12,17 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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Figure 1: Biosynthesis of FPP and GGPP and their role in protein prenylation; key enzymes are highlighted in blue 38
37 39 40 41
The presumed potential value of hFPPS as a therapeutic target for non-skeletal diseases has 42 4
43
fueled efforts towards the identification of non-bisphosphonates inhibitors of this target. Jahnke and 46
45
co-workers at Novartis were the first to employ fragment-based screening by NMR and X-ray 47 49
48
crystallography to identify a catalytically relevant allosteric pocket, near the IPP substrate binding 51
50
site of the hFPPS active site.13 Recently, we explored the biological role of this allosteric pocket 53
52
and showed that FPP product can bind to this allosteric pocket, locking the enzyme in an inactive 54 56
5
conformation and consequently, providing a feed-back mechanism for controlling the intracellular 57 58 59
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levels of isoprenoid biosynthesis in vivo.14 To date, numerous structurally diverse non5
4
bisphosphonate inhibitors have been reported that bind to the hFPPS allosteric pocket with high 6 7
affinity; these include analogs 3, 4 and 5.13,15 In vitro potencies in the low nanomolar range were 10
9
8
observed with some of these compounds (e.g. 4) using an hFPPS inhibition assay based on 12
1
LC/MS/MS.13,15 Efforts by other research groups have also contributed to this field,16 although 13 15
14
some of the compounds reported do not actually bind inside the hFPPS allosteric pocket.16a 17
16
In the course of our own investigations, we reported the discovery of thienopyrimidine-based 18 19
bisphosphonates (ThP-BPs; e.g. compound 6) that exhibit a promiscuous binding mode and interact 20 2
21
with both the active site and the allosteric pocket of hFPPS under physiologically relevant 24
23
conditions.17 Structural re-modeling of this class of compounds allowed us to probe the 25 27
26
pharmacophore elements that discriminate between binding in the allosteric pocket of hFPPS as 29
28
opposed to the allylic sub-pocket of its active site cavity.18 Truncation of one phosphonate moiety 31
30
led to the identification of a new chemotype of hFPPS inhibitors (e.g. compound 9) that binds 32 34
3
exclusively in the allosteric pocket and engage in similar interactions with the protein as those 36
35
observed with inhibitors 3-5.13,15 In this report, we describe our efforts towards the optimization of 37 38
our initial hit and the development of a structure-activity relationship (SAR) model that could guide 39 41
40
further the optimization of thienopyrimidine-based inhibitors. Herein, we describe the identification 43
42
of analogs with nanomolar potency and characterization of the protein-inhibitor interactions of 4 45
structurally diverse analogs with hFPPS. These studies provide further insight into the essential 46 48
47
minimal pharmacophore that is required for allosteric inhibition of hFPPS and point towards new 50
49
directions in the design of non-phosphonate thienopyrimidine-based allosteric inhibitors targeting 51 53
52
this enzyme. 54 5 56 57 58 59
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CHEMISTRY 30
29
Potency optimization and structure-activity relationship studies of the thienopyrimidine 31 3
32
monophosphonate inhibitors of hFPPS were initiated with the synthesis of compound libraries 35
34
focusing mainly on (a) the linker connecting the lipophilic thienopyrimidine core to the charged 36 37
phosphate moiety and (b) the substitutions at R4 and R6 of the scaffold. Representative examples 38 40
39
from these libraries are shown in Figures 2 and 3 (analogs 7 to 48). It should be noted that although 42
41
all analogs with a Cα substituent at R4 were initially synthesized (as part of the parallel library 4
43
synthesis) in racemic form, the role of the Cα substitution was also evaluated with enantiomerically 45 47
46
enriched compounds, such as the (R)- and (S)-13, which were synthesized in 98% and 92% ee, 49
48
respectively. In addition to the compound libraries, and in order to better understand the binding 50 51
contributions of the presumed essential negatively charged moiety,13,15,16b,17 we also synthesized 52 54
53
analogs 49 and 50 as individual compounds. For brevity, the experimental procedures for the 5 56 57 58 59
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preparation of (R)- and (S)-13 analogs and compounds 49 and 50 are summarized in the Supporting 5
4
Information. 6 7 8
The synthesis of ThP-BP analogs was initiated from thieno[2,3-d]pyrimidin-4(3H)-ones 9 1
10
(51a or 51b), prepared as previously reported (Scheme 1).17,19,20,21,22 In general, the R6 moieties 13
12
were installed via Pd-catalyzed Suzuki cross-coupling reactions with the C-6 bromide, as 14 15
previously reported.17,19,20 Installation of the R4 moiety (in library synthesis mode) turned out 16 18
17
to be more challenging. We attempted to directly couple the thienopyrimidinones 51 with 20
19
various α-aminophosphonate esters using the phosphonium-mediated SNAr protocol reported by 21 2
Wan and co-workers;23 however, in our hands this approach was unsuccessful using our 25
24
23
thienopyrimidine scaffold. In contrast, SNAr displacement of the C-4 chloride of intermediates 26 27
54 or 55 was possible, but the conversion was often slow, requiring high temperatures and 28 30
29
leading to modest or poor yields, particularly with sterically hindered amines having an electron 32
31
withdrawing groups at Cα. We also contemplated the conversion of the starting 3 34
thienopyrimidinones (51 or 53) directly to the corresponding 4-fluoropyrimidine, in order to 35 37
36
improve the outcome of the subsequent SNAr reaction. In the past, Ritter and co-workers reported 39
38
the conversion of quinazolin-4(3H)-one to the 4-fluoroquinazoline using 1,3-bis(2,640 42
41
diisopropylphenyl)-2,2-difluoro-2,3-dihydro-1H-imidazole as the fluorinating agent in 34% 4
43
yield.24 However, in addition to the modest yields, the cost of this reagent ($375.00 CAD/10 mL 45 46
of 0.1M solution), discouraged us from using this methodology. The alternative approach of 47 49
48
preparing intermediate 56 via the triflate (instead of the chloride 55) and then fluorinating under 51
50
very mild reaction conditions, as reported by Buchwald and co-workers25 was also considered. 52 53
Ultimately, given the high thermal and chemical stability of the thienopyrimidine scaffold, we 54 56
5
chose to prepare the fluoride intermediate 56, by treating 55 with KF in DMSO, as reported by 57 58 59
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Shestopalov, Gakh and co-workers;26 this approach proved to be the most efficient and atom5
4
economical path to the fluoride 56 and in most cases, provided excellent yields (≥85%). The 6 7
subsequent SNAr displacement of the C-4 fluoride with various α-aminophosphonate esters in 10
9
8
DMSO under basic conditions gave the diethyl esters of compounds 7-48 in moderate to 1 12
excellent isolated yields (45-90%). Finally, the diethyl monosphosphonate esters of compounds 13 15
14
7-48 were first treated with TMSBr and the trimethylsilyl esters generated were then cleaved 17
16
with methanol to give the final allosteric inhibitors, thienopyrimidine-based phosphonic acids 718 19
48 (Fig. 2 and 3). 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43
Scheme 1: Parallel synthesis of a permutation library of hFPPS allosteric inhibitors; fragments 45
4
f1-f32 were used for R1 and R6 (Fig. 4). Reagents and conditions: (a) Br2, AcOH, 80 oC, 1.5 h 47
(98%); (b) 5 mol% Pd(PPh3)4, aryl boronic acid or boronate ester, dioxane:H2O (1:1), 110 oC, 49
48
46
12 h (60-80%); (c) POCl3, 95 oC, 5 h (50%); (d) KF.2H2O, DMSO, 120 oC, MW, 15 min (85%); 50
(e) diethyl α-aminophosphonate ester 60, Et3N, DMSO, 100 oC, 3 h (47-87%); (f) TMSBr then 52
51
MeOH, 50% to quantitative. 53 54 5 56 57 58 59
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The synthesis of racemic diethyl α-aminophosphonate esters with general structure 60 5
4
(Scheme 2) was previously achieved using the Kabachnick-Fields three-component condensation 6 7
of aldehydes, amines and diethyl phosphite (Scheme 2I).27,28 In our hands, this method was 10
9
8
reasonably successful (40-90% yield) with simple aryl aldehydes (e.g. benzaldehyde-type 12
1
reagents), particularly in the presence of catalytic amounts of zirconium(IV),29 but led to low yields 13 15
14
and numerous side products with more complex and benzylic-type of aldehydes. Given these results 17
16
and the limited availability of commercial aldehydes, the direct Cα alkylation of diethyl 18 19
(aminomethyl)phosphonate appeared more desirable and was explored (Scheme 2-II). We 20 2
21
examined in parallel the deprotonation at Cα of imine 5930 and dibenzylaminomethyl phosphonate 24
23
5831 and subsequent alkylation with an alkyl halide. The latter approach was found to be generally 25 27
26
more reliable in providing higher yields of the desired products with many structurally diverse 29
28
halides (26-95%). The synthesis of the β-aminophosphonate building blocks 6132 and 6233, leading 31
30
to compounds 14-16 (Fig. 2) was achieved as previously described (with only minor modifications) 32 34
3
and summarized briefly in Scheme 2-III and -IV, respectively (experimental details are provided in 36
35
the Supporting Information). 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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Scheme 2: Synthesis of diethyl α-aminophosphonate esters; R1 selected from fragments f1-f32 30
(Fig. 1). Reagents and conditions: (a) ZrOCl2.H2O, neat, 80 oC (40-90%); (b) H2, Pd/C, MeOH; 31
29
(c) THF, 50 oC, 24h (63%); (d) LDA, R1Br, THF, -78 oC to RT, 2 h (25-95%); (e) i. 3
32
CH3PO(OEt)2, n-BuLi, THF, -78 oC to RT, ii. NaBH4, AcOH, RT (60%); (f) allyl bromide, n35
BuLi, THF, -78 oC to reflux (75%); (g) i. 4-methyl-N-morpholine oxide, OsO4, dioxane:H2O, RT, 37
36
34
ii. NaIO4, RT (92%); (h) H2O2, SeO2, THF, reflux (70%); (i) EtOH, H2SO4, reflux (89%); (j) 39
38
NH4OH, RT (97%; (k) HCO2H, CH3CN:H2O, RT (83%). 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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Figure 2: Permutation library of hFPPS allosteric inhibitors; the structure of all the fragments 42
used (i.e. f1 to f32) are shown in Fig. 4. 43
41
4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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Figure 3: Permutation library of hFPPS allosteric inhibitors; the structure of all the fragments 26
25
used (i.e. f1 to f32) are shown in Fig. 4. 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54
Figure 4: Structures of fragments f1 to f32 used in the synthesis of the compound libraries (Fig. 2 56
5
and 3) 57 58 59
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The synthesis of (2-aminoacetyl)phosphoramidic acid inhibitors, such as compounds 29 and 5
4
30 (Fig. 2) is outlined in Scheme 3. The amides of L-phenylalanine34 and a-phenylglycine35 were 6 8
7
synthesized according to literature procedures, and their reaction with the thienopyrimidine scaffold 10
9
was achieved via SNAr to give the primary amides 63 and 65. These intermediates were 1 12
subsequently treated with tetrabenzyl pyrophosphate in the presence of t-BuOLi to afford the 13 15
14
respective phosphoroamidates. The benzyl protecting group could be removed with TMSBr fairly 17
16
fast (2h compared to 3-4 days for removal of the ethyl groups) and its use eliminated concerns over 18 19
any potential hydrolysis of the P-N bond. As indicated earlier, analogs (R)- and (S)-13, 49 and 50, 20 2
21
were not synthesized as part of the libraries (Fig. 2 and 3); the synthesis of these compounds is 24
23
summarized in the Supporting Information. 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39
Scheme 3: Synthesis of (2-aminoacetyl)phosphoramidic acid analogs (Fig 2). Reagents and 41
40
conditions: (a) NH4OH, EDC, HOBt, DMF/CH2Cl2, 0oC to RT, 60-70%; (b) TFA, CH2Cl2, RT, 43
quant.; (c) intermediate 55a, DIPEA, dioxane, 100oC, 75-85%; (d) [PO(OBn)2]2O, t-BuOLi, THF, 45
4
42
Ar, RT, 45-52%; (e) i. TMSBr, CH2Cl2, 0oC to RT then ii. MeOH, RT, 64-95%. 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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RESULTS AND DISCUSSION 20 21
Dysregulation of the mevalonate pathway and upregulation of isoprenoid biosynthesis has 2 24
23
been 26
25
implicated
in
numerous
human
diseases,
including
metabolic
disorders
(e.g.
hypercholesterolemia and osteoporosis), cancers that are mostly prevalent in aging populations (e.g. 28
27
multiple myeloma, breast and prostate cancers),36 and neurodegeneration.37,38 Consequently, the 29 31
30
discovery of therapeutic agents that can selectively block the catalytic function of key enzymes in 3
32
this pathway are promising areas of research. Amongst the biological targets of interest is the human 34 35
FPPS, the gate-keeper of isoprenoid biosynthesis and consequently, prenylation/activation of all 36 38
37
small GTPases. Until recently, the only known inhibitors of this enzyme were the bisphosphonate 40
39
active site inhibitors, such as the N-BP drugs zoledronic acid (1)39 and risedronic acid (2).40 41 42
However, the discovery of a catalytically relevant allosteric pocket,13 located near the active site of 43 45
4
the enzyme, opened a new area of drug discovery leading towards the identification of non47
46
bisphosphonate inhibitors, with potentially superior drug-like properties and consequently, 48 50
49
therapeutic value as compared to the N-BPs. 52
51
A number of non-bisphosphonate allosteric inhibitors of hFFPs (e.g. compounds 3-5), with 53 54
good potency in vitro, have been reported and their binding interactions with the allosteric pocket 5 57
56
have been characterized by crystallography;13,15 unfortunately, none of these compounds have 58 59
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shown promising cell-based potency. In our own search for new structural classes of hFPPS 5
4
allosteric inhibitors, we identified thienopyrimidine-based bisphosphonates (ThP-BPs; e.g. 6 8
7
compound 6) that exhibit a highly unusual binding mode (for bisphosphonate-type compounds) in 10
9
that they can bind to both the active site and the allosteric pocket of hFPPS under physiological 1 12
conditions. We investigated this dual binding mode by DSF, NMR and ITC and finally confirmed 13 15
14
our results by co-crystallizing compound 6 with hFPPS in the presence and absence of Mg2+ ions.17 17
16
As expected, in the presence of Mg2+ ions, compound 6 was driven to bind in the allylic subpocket 19
18
of the active site (refered to as the hFPPS/6/Pi(act); PDB code: 4JVJ). In contrast, co-crystallization 20 2
21
of 6 in the absence of Mg2+ ions revealed binding exclusively in the allosteric pocket of the enzyme 24
23
(refered to as the hFPPS/6/Pi(allo) complex; PDB code: 4LPG; Fig. 5a). Removal of one phosphonate 25 27
26
moiety led to the corresponding monophosphonate analog 9, which was shown to selectively bind 29
28
in the allosteric pocket of hFPPS (Fig 5b; PDB code: 4LPH). 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 46
45
Figure 5: Co-crystal structures of hFPPS and ligand complexes. (a) Structure of the 48 49
hFPPS/6/Pi(allo) complex (PDB ID: 4LPG). Polar and stacking interactions are indicated with 50
dashes. An inorganic phosphate ion is bound in the IPP subpocket of hFPPS; its interactions 52
51
47
with the protein are omitted for simplicity. The side chain of K57 could not be fully modeled 53
due to disorder. (b) Structure of the hFPPS/9/Pi complex (PDB ID: 4LPH). Represented residues 5
54
are the same as in panel (a). 56 57 58 59
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4
Our crystallographic data also confirmed that the interactions between 9 and the protein surface 6 7
within the allosteric pocket were very similar to those previously observed in the hFPPS/6/Pi(allo) 8 10
9
complex (Fig 5a). In both cases, the predominant inhibitor/protein interactions involve π-stacking 12
1
of the 6-phenylthieno[2,3-d]pyrimidin-4-amine core with F206 and F239; in fact, the p-tolyl 13 14
substituent of 9 was buried in the allosteric pocket, engaging in a “sandwich” of π-stacking 15 17
16
interactions with the side chains of N59 and F206. At the time, we presumed that the low potency 19
18
of inhibitor 9 was mainly due to the fact that its phosphonate moiety formed only water-mediated 20 2
21
interaction with the guanidinium side chain of R60, whereas the carboxylic acid moieties of the 24
23
more potent inhibitors 3 and 4 engages in direct interactions with the side chains of K57 and 26
25
R60.13,15 To gain more insight into the importance of each key interaction between the ThP-MP 27 29
28
inhibitors and the allosteric pocket, we generated permutation libraries of structurally diverse 31
30
analogs, starting from the “naked” thienopyrimidine monophosphonate core (i.e. analog 7 ) and the 3
32
building blocks shown in Figure 4 as the variable substituents at the Cα/Cβ of R4 and at R6 (Fig. 2 34 36
35
and 3). 38
37
Initial biological screening of all compounds was carried out using a previously described 39 40
in vitro M2 hFPPS inhibition assay at a fixed concentration of 10 μM.19 A full dose-response 41 43
42
inhibition curve (IC50) was determined only with compounds exhibiting more than 80% inhibition 45
4
at 10 μM (IC50 average values for assays run in triplicate or more are shown in Table 1). For the 46 48
47
purpose of our investigations, the IC50 values shown in Table 1, were fairly consistent with the SAR 50
49
suggested by the preliminary screening data shown in Figures 6, 7 and 8. 51 52
It is noteworthy that compound 7 was virtually inactive in our in vitro assay at 53 5
54
concentrations up to 10 μM and only slightly active even at 100 μM (Table 1; Fig. 6). However, a 57
56
sharp increase in potency was observed when the R6 of 7 was substituted with a phenyl or a p-tolyl 58 59
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moiety (i.e. analogs 7 vs 8 vs 9; Fig. 6a), confirming the importance of the π-stacking interactions 5
4
between this group and residues F206 and N59 observed previously (Fig. 5). Having achieved a 6 8
7
reasonable potency in inhibiting hFPPS, we turned our attention to probing the impact of the 10
9
phosphonate pharmacophore. Initially, we replaced the α-aminophosphonate moiety of 9 with the 1 12
corresponding ethylphosphonic acid and glycine amino acid (i.e. analogs 10 and 26, respectively; 13 15
14
Fig. 2) and observed a strong correlation between loss in potency and decrease in the acidity of the 17
16
negatively-charged pharmacophore (i.e. analogs 9 vs 10 or 26; Fig. 6).17 Similarly, capping of one 18 19
hydroxyl group of the phosphonate as the ethyl ester led to a significant loss of potency (i.e. analog 20 2
21
9 vs 18; Fig 6), once again, suggesting that a phosphonate dianion may be essential for binding. In 24
23
addition, since interactions between the negatively charged pharmacophore of 9 and the 25 27
26
guanidinium side chain of R60 were previously shown to be water mediated (Fig. 5), we presumed 29
28
that introducing modifications to the linker that would allow similar binding of the thienopyrimidine 31
30
core and direct interactions of the phosphonate with R60 would lead to improvement in the potency 32 34
3
of our inhibitors. Several modifications were attempted, including linker extension (i.e. analog 9 vs 36
35
11; Fig 6b), partial rigidification of the extended linker via substitution at both the α- and β-carbons 37 38
of a (2-aminoethyl)phosphonic acid moiety [e.g. analog 12 vs 14 (Fig. 6c) and 17 vs 15 or 16 (Fig. 39 41
40
6d)] and replacement of the α-aminophosphonate moiety with a (2-aminoacetyl)phosphoramidic 43
42
acid (e.g. analog 29 and 30; Fig. 6e). Contrary to our expectations, these extended linkers 4 45
connecting the thienopyrimidine core with the phosphonate moiety led to compounds that were 46 48
47
either equipotent or slightly less potent than the derivatives having an α-aminophosphonate moiety 50
49
at R4. For example, the difference in potency between the α-amino-(2-phenylethyl)phosphonic acid 51 52
derivative 13 (IC50 = 1.4 μM) and the corresponding (2-aminoacetyl)phosphoramidic acid analog 53 5
54
29 (IC50 = 3.7 μM) was minimal. 56 57 58 59
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Since the enzyme-bound conformation of inhibitor 9 (Fig. 5b) placed the phosphonate moiety 5
4
folded over the C-5 position of the thienopyrimidine core, we then assumed that connecting the 6 8
7
phosphonate moiety to the C-5 would allow direct interaction with R60. Based on this assumption 10
9
and preliminary in silico docking studies, we designed analogs such as 49 or 50 that were expected 1 12
to produce the desired outcome. Unfortunately, both of these compounds were found to be 13 14
significantly less active than anticipated (IC50 values of ≥100μM; Table 1), in spite of the fact that 17
16
15
the phosphonate moiety indeed interacts directly with the guanidinium ion of R60 at least for analog 18 19
49 (confirmed with the co-crystal structure of hFPPS/49; Fig. 9a). A likely reason is that the 20 2
21
methylene linker between the C-5 of the scaffold and the phosphonate clashed with the protein 24
23
surface and consequently, the aromatic core is pushed out of its preferred binding mode for strong 25 26
π-staking interactions. Additionally, unfavorable torsional stress was observed in the bound 27 29
28
compound 49 (i.e. loss of the co-planarity between the two aromatic rings; Fig. 9a). Given these 31
30
observations, it is reasonable to assume that the same effects are (at least in part) responsible for 32 34
3
the poor potency of the cyclic phostone 50. 36
35
We then turned our attention to the SAR at the Cα position of the R4 α-aminophosphonic acid 37 38
moiety. Initially, we observed some minor improvements in potency when a phenyl or benzyl 39 41
40
moiety was attached to the Cα as compared to the unsubstituted hit 9. For example, the racemic 43
42
benzyl-substituted derivative 13 was approximately 4-fold more potent than the unsubstituted 4 45
compound 9, whereas the racemic phenyl-substituted analog 19 was only 2-fold more potent than 46 48
47
the corresponding unsubstituted derivative 17 (Table 1). Analogs such as 17, having an additional 50
49
meta-halo substituent on the R6 p-tolyl group, appeared to be slightly more potent than the initial 51 52
hit 9 (Table 1). These preliminary results suggested that further exploration at both the Cα position 53 5
54
of the C-4 α-aminophosphonate moiety and at R6 was justified. 56 57 58 59
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1 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 3
32
Figure 6: Screening data for select compounds from the 1st library of hFPPS allosteric inhibitors at a fixed concentration of 10 μM (average of % inhibition from triplicate test runs); preliminary SAR at R4 and R6 of the thienopyrimidine core (fragment A to I are as shown in Fig. 2) 37
36
35
34
38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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Table 1: Inhibition data for key compounds 4 5
hFPPS 6 8
7
hFPPS
Compound
IC50 (μM)
Compound
IC50 (μM)
15
14
13
12
1
10
3α
0.85
28
2.6
7
>100
29
3.7
9
5.0
33
3.0
12
4.8
36
2.8
13
1.4
37
1.6
17
3.3
38
1.1
19
1.6
42α
0.86
20
2.3
43
1.5
21
8.8
44
1.0
22
1.6
47α
0.87
24
8.4
48
4.6
25
4.6
49
~100
27
10
9
17
16
24
23
2
21
20
19
18
26
25
3
32
31
30
29
28
27
35
34
38
37
36
40
39
Average IC50 values from three determinations; αaverage IC50 values from six determinations; (standard deviation ≤10%). 42
41 43 4 45 47
46
A library of compounds with variables substituents at R6 of the thienopyrimidine scaffold 49
48
having a phenyl or benzyl substituent at the Cα position of R4 (i.e. R4 = D or E; Fig. 2) was 50 52
51
synthesized, examples are shown in Figure 7 (R6 = f1-f21). In general, R6 phenyl derivatives with 54
53
a large meta or para moiety (e.g. OCH3, CF3, N(CH3)2) were unfavorable (data not shown). In 5 56
contrast, when the p-tolyl moiety (i.e. R6 = f2) was further substituted with a 3-fluoro or 3-chloro 57 58 59
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(i.e. f9 and f10, respectively) or 2-methoxy-5-fluoro/chloro (i.e. f20 and 21, respectively) a modest 5
4
improvement in potency was observed. Confirming our previous observations (i.e. Fig. 6), a very 6 8
7
modest improvement in potency was usually observed when a phenyl or benzyl substituent was 10
9
introduced at the Cα position of the R4 moiety (i.e. scaffolds II and III vs I; Fig. 7). For example, 1 12
the IC50 values of the most potent compounds confirmed a ~2-4-fold improvement in potency when 13 14
the Cα position of R4 was substituted with a benzyl group (e.g. IC50 values of analogs 9 and 13 are 17
16
15
5.0 µM and 1.4 µM, respectively; Table 1). Since all analogs were initially synthesized as racemic 18 19
mixtures, these results seemed promising for further optimization; initially, we presumed that only 20 2
21
one enantiomer was responsible for all the activity observed. Analogs (R)- and (S)-13 were 24
23
synthesized in 98% and 92% ee, respectively, and their IC50 values determined to be 4 μM and 1 25 26
μM, respectively; this small difference in potency between the two enantiomers hinted to a 27 29
28
potentially inconsistent binding mode for these molecules. Nonetheless, a small library of racemic 31
30
compounds with structural diversity at the Cα of the α-aminophosphonic acid was investigated in 32 34
3
combination with fragment f2, f10, f20 and f21 at the R6 position (i.e. sub-scaffold IV; Fig. 8); 36
35
representative examples from this library are shown in Figure 8. In general, both aromatic and alkyl 37 38
groups were well tolerated (e.g. analogs 13 with R1=f22 was found to be equipotent to analog 37 39 41
40
with R1=f24 (Fig. 8; Table 1), suggesting some van der Waals interactions between the R1 moiety 43
42
and the protein. Significant loss in potency was observed when R1 was a basic moiety, such as 4 45
pyridinyl derivatives (e.g. R1 = f29, f30 or f31) or a disubstituted alkyl derivatives (e.g. f32; Fig. 46 48
47
8). From this cluster of compounds, the most potent hFPPS inhibitors identified were those having 50
49
a combinations of fragments f23 at the R1 position and either f10 or f20 at R6 (i.e. analogs 42 and 51 53
52
47, respectively; Fig. 8; Table 1). The IC50 values of compounds 42 (IC50 = 0.86 µM) and 47 (IC50 54 5 56 57 58 59
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= 0.87 µM) were found to be in the same potency range as allosteric inhibitor 3 (IC50 = 0.85 µM), 5
4
when tested in parallel in our in vitro M2 hFPPS inhibition assay (Table 1).19 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 34
3
Figure 7: Screening inhibition data for select compounds from the 2nd library of hFPPS allosteric inhibitors at a fixed concentration of 10 μM (average of % inhibition from triplicate test runs); the R6 position of the (aminomethyl)- phosphonic acid (I), (amino(phenyl)methyl)phosphonic acid (II) and an (1-amino-2-phenylethyl)phosphonic acid (III) analogs were substituted with fragments f1 to f21 (Fig. 4). 40
39
38
37
36
35
41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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18
Figure 8: Screening inhibition data for select compounds from the 3rd library of hFPPS allosteric inhibitors at a fixed concentration of 10 μM (average of % inhibition from triplicate test runs); xaxis and z-axis indicate R1 and R6 fragments, respectively, from Fig. 4. 23
2
21
20
24 25 27
26
Mindful of the conformational plasticity of this enzyme, which permits the binding of many 29
28
structurally diverse compounds at its allosteric pocket (e.g. 3, 4, 5 and 9), several representative 30 31
molecules, including analogs 29, 33, 38, 42 and 49, were co-crystallized with hFPPS in order to 32 34
3
identify a consensus binding mode. All compounds were found to bind at the allosteric site of 36
35
hFPPS. However, three compounds (analogs 29, 38, and 42) were found to bind in an “upside 37 38
down” orientation with respect to the binding of our initial hit 9, in which the sulfur atom of the 39 41
40
thienopyrimidine scaffold is facing the active site cavity instead of the C-terminal helix (Fig. 9c,d,e 43
42
vs Fig. 5b). In addition to the sandwich π-stacking interactions of the thienopyrimidine core, the 4 46
45
exocyclic nitrogen of the analogs 29 and 38 formed a H-bond with the carbonyl oxygen of K347 48
47
(Fig. 9c,d). The substituents (2-aminoacetyl)phosphoramidate and α-aminophosphonate also made 49 50
an electrostatic interaction with K347 and a water-mediated H-bond with N59 (Fig. 9c,d). 51 53
52
Compound 42 did not form any of these additional interactions; however, its fluorobenzyl moiety 5
54
at R1 (i.e. the Cα of R4) made a new stacking interaction with F239, tucked tightly in the small 56 57 58 59
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depression formed by F239, Q242, D243, and I348 (Fig. 9e; SI Fig. S3). It is interesting that 5
4
compound 33, unlike the three analogs above, assumed the orientation observed with the original 6 8
7
hit 9 (Fig. 9b vs Fig. 5). Comparison with the other structures indicates that the dihydrobenzofuran 10
9
derivative 33, with a bulkier and less flexible R1 moiety, likely causes steric clashes with residues 12
1
of αH and αJ in the upside down binding mode (Fig. 9b; SI Fig. S3). 13 15
14
Interestingly, when we superimposed all of our co-crystal structures, we found that the 17
16
thienopyrimidine cores of the inhibitors generally occupy the same 3D space within the allosteric 19
18
binding site, regardless of the binding orientation of each molecule (i.e. “up” or “down”). This 20 2
21
finding highlights the importance of the π-stacking interactions provided by the scaffold for the 24
23
binding of these compounds. Notably, the thienopyrimidine of compound 42 aligns slightly off 25 27
26
from those of the rest of the inhibitors (Fig. 9f) and makes the closest contact with F239 of the 29
28
protein (Fig. 9e). Since 42 differs from 38 only by a single chloride atom, its distinct binding pose 31
30
is attributed to this substitution. A close examination reveals that the m-chloro tolyl group fits more 32 34
3
tightly in the allosteric pocket and in a slightly tilted angle that pushes the thienopyrimidine core 36
35
towards the active site cavity (Fig. 9g). This binding pose not only allows stronger interaction 38
37
between the thienopyrimidine core and F239 but also enables the aforementioned burial of the Cα 39 41
40
fluorobenzyl moiety in the protein. The monophosphonate of 42 in this binding pose is too far from 43
42
K347 to interact with this residue. 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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38
Figure 9. Binding poses of ThP-MP inhibitors at the allosteric site of hFPPS. Inhibitors 49, 33, 29, 38, and 42 are shown in (a), (b), (c), (d), and (e), respectively. The red and green spheres represent water molecules and a chloride ion, respectively. (f) Superposition of the new ThP-MPs and the initial hit, compound 9 (cyan). The thienopyrimidine of 42 is marked with a black arrow. (g) Binding of the R6 tolyl groups of 38 (left panel) and 42 (right). The inhibitors are shown as spacefilling models, and the protein surfaces as meshes. 46
45
4
43
42
41
40
47 48 49 50 51 53
52
Based on all previous co-crystal structures of inhibitors bound to the hFPPS allosteric 5
54
pocket,13,15,17 we expected the polar interactions between the monophosphonate group and the side 56 57 58 59
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chain(s) of N59 and/or R60 to be crucial for inhibitor/enzyme molecular recognition. Consequently, 5
4
the upside down binding mode of analogs 29, 38 and 42 was surprising, particularly since the 6 7
binding orientation of the ThP-MPs (i.e. “up” or “down”) does not seem to significantly affect their 10
9
8
potency (e.g. IC50 values of analog 33 and 42 are 3.0 and 0.86 μM, respectively, Table 1). More 1 12
importantly, our results indicate that, given the right combination of substituent around the 13 15
14
thienopyrimidine core, the phosphonate-mediated interactions are entirely dispensable; the 17
16
monophosphonate of compound 42 when bound to hFPPS is exposed to bulk solvent without 18 19
making any contact with the protein (Fig. 9e; SI Fig. S3e). The loss in the binding energy seems to 20 2
21
be fully compensated by the non-polar interactions provided by the m-chloro substituent on the R6 24
23
p-tolyl group and the fluorobenzyl substituent at the Cα of the R4 in compound 42, which shows 25 27
26
the greatest potency amongst all of our ThP-MP compounds and is equipotent to the allosteric 29
28
inhibitor 3 (when tested in parallel in our M2 in vitro assay; Table 1). In contrast, compound 49 is 31
30
approximately 100-fold less potent than 42 (Table 1), in spite of the fact that its phosphonate moiety 32 34
3
makes direct interactions with both N59 and R60 (Fig. 9a). Therefore, analogs such as 42 may 36
35
suggest a new direction towards the identification of potent hFPPS allosteric inhibitors that do not 37 38
require a highly charged moiety. 39 40 41 42 43 4 45
CONCLUSIONS: 46 48
47
Over the last few years, there has been an on-going debate in the literature as to whether N50
49
BP inhibitors of hFPPS possess antitumor therapeutic properties, in addition to their undisputed 51 53
52
skeletal benefits. This debate has been fueled by somewhat controversial (or unclear) clinical data 5
54
from multiple myeloma8 and breast cancer41 patients treated with standard chemotherapy plus 56 57 58 59
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zoledronic acid (1). However, given the extremely poor soft-tissue distribution and low systemic 5
4
exposure of N-BP drugs, it is unlikely that a better understanding of the role of hFPPS in 6 8
7
oncogenesis can be gained from in vivo studies using these compounds. Herein, we disclose the 10
9
synthesis of compound libraries used to conduct hit-to-lead optimization of a new series of 1 12
thienopyrimidine-based allosteric inhibitors of hFPPS. These studies led to the identification of 13 15
14
hFPPS allosteric inhibitors with nanomolar potency, such as compounds 42 and 47. X-ray 17
16
crystallography was used to identify the molecular recognition elements dictating the 18 19
inhibitor/protein interactions. These studies strongly suggest that the 6-tolylthienopyrimidine 20 2
21
scaffold drives the binding by providing a number of consistent π-staking interactions with residues 24
23
F239, F206 and N59. Interestingly, in the co-crystal structure of our most potent inhibitor, analog 25 27
26
42, the phosphonate moiety was not involved in any interactions with the protein. This observation 29
28
may appear to contradict our previous SAR data, which indicated that the phosphonate 31
30
pharmacophore was essential for binding. However, it suggests that it may be possible to design 32 34
3
allosteric inhibitors of hFPPS that binding in a different orientation from that of the original 36
35
thienopyrimidine hits (i.e. analogs 6 and 9; Fig. 5), yet maintain the same strong π-stacking 37 38
interactions observed with residues F239, F206 and N59 (in addition to other interactions), but 39 41
40
lacking the highly charged phosphonate moiety. Such investigations are currently in progress and 43
42
if successful, they may provide the molecular tools needed to clearly validate (or disqualify) hFPPS 4 45
as a therapeutic target for non-skeletal diseases, such as cancer. 46 47 48 49 50 51 52 53 54 5 56 57 58 59
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EXPERIMENTAL 5
4
General Procedures for Characterization of Compounds: 6 8
7
The phosphonate ester precursor of inhibitors 7 - 48 were purified by normal phase flash column 10
9
chromatography using a CombiFlash instrument on silica gel and a solvent gradient from 5% 1 12
EtOAc in hexanes to 100% EtOAc and then to 20% MeOH in EtOAc, unless otherwise indicated. 13 14
Only phosphonate esters with homogeneity ≥95% were processed further though the hydrolysis 17
16
15
step to the final ThP-MP phosphonic acids. The purity and homogeneity of all final inhibitors to 19
18
≥95% was confirmed by NMR (1H, and 20
31
P) and reversed-phase HPLC. HPLC Analysis was
2
21
performed using a Waters ALLIANCE® instrument (e2695 with 2489 UV detector and 3100 mass 24
23
spectrometer). Key compounds and intermediates were fully characterized by 1H, 13C and 31P NMR, 25 26
and HRMS. Chemical shifts () are reported in ppm relative to the internal deuterated solvent (1H, 29
28
27
13
C) or external H3PO4 ( 0.00 31P), unless indicated otherwise. The NMR spectra of all final
30 32
31
inhibitors were acquired either in DMSO-d6 or in D2O (with ~2%-5% ND4OD or as the 34
3
corresponding di-sodium salt). The high-resolution MS spectra were recorded using electrospray 35 36
ionization (ESI+/-) and Fourier transform ion cyclotron resonance mass analyzer (FTMS). 37 39
38
Method (homogeneity analysis using a Waters Atlantis T3 C18 5 µm column): 41
40
Solvent A: H2O, 0.1% formic acid 42 43
Solvent B: CH3CN, 0.1% formic acid 4 46
45
Mobile phase: linear gradient from 95%A and 5%B to 5%A and 95%B in 13 min, then 2 min at 48
47
100% B 49 50
Flow rate: 1 mL/min 51 52 53 54 5 56 57 58 59
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General synthetic protocols: 5
4
Synthesis of analogs of 54, through chlorination of the thienopyrimidinone derivatives 52 followed 6 8
7
by Suzuki cross-coupling reactions and deprotection of the monophosphonate esters to the 10
9
phosphonic acids 7-48, was carried out using the general protocols previously described.17 12
1
Intermediates 61,33a 6233b, 51b and 52b42 were synthesized as previously reported with slight 13 15
14
modifications; details are provided in the Supporting Information. The synthesis and 17
16
characterization of inhibitors 9, 10, 11, and 26 was previously reported.17 Details of the synthesis 18 19
of 58, key boronic acid building blocks, analogs 55, inhibitors 49, 50, and (S)-13 are provided in 20 2
21
the Supporting Information. 23 24 25 27
26
General procedures for the preparation of the final inhibitors: 29
28
Step 1 - SNAr reactions: In a pressure vessel, the 4-chlorothienopyrimidine (54 or 55) or 431
30
fluorothienopyrimidine (56) was dissolved in DMSO or dioxane, the amine reagents 60, 61 or 62 32 34
3
(1.5 eq) and Et3N or DIPEA (3 - 5 eq) were added, and the pressure vessel was sealed. The reaction 36
35
mixture was stirred at 100°C for 3 − 24 h; completion of the reaction was monitored by LC-MS. 37 38
The reaction mixture was cooled to RT and diluted with EtOAc. The organic layer was washed with 39 41
40
an aqueous solution of saturated NaHCO3, water, brine, and dried over anhydrous MgSO4. The 43
42
crude product was purified by silica-gel column chromatography to afford intermediates 57 (if 4 45
starting from 54) or the diethyl esters of the final compounds 7-48 (if starting from 56) in yield 46 48
47
ranging from 45% to 90%. 50
49
Step 2: Suzuki cross-coupling with intermediate 57 to obtain the diethyl esters of the final 51 53
52
compounds 7-48 (yields ranging from 60-80%) and final deprotection were carried out as 5
54
previously reported (yields 50% to quantitative).17,19,20 56 57 58 59
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1 2 3 5
4
((Thieno[2,3-d]pyrimidin-4-ylamino)methyl)phosphonic acid (7): Isolated as a yellow solid. 6 7 1
10
9
8
H NMR (500 MHz, D2O) δ 8.15 (s, 1H), 7.38 (d, J = 6.1 Hz, 1H), 7.32 (d, J = 6.1 Hz, 1H),
3.35 (d, J = 13.3, 2H). 13C NMR (126 MHz, D2O) δ 163.7, 157.34, 157.27, 123.3, 118.7, 12
1
116.9, 40.2 (d, J = 136 Hz). 31P NMR (202 MHz, D2O): δ 13.6. HRMS (ESI-) m/z 243.9951 13 15
14
calculated for C7HN3O3PS; found m/z 243.9952 [M - H]16 17 18 19 21
20
(((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic acid (8):Isolated as a white 23
2
solid. 1H NMR (500 MHz, D2O) δ 8.00 (s, 1H), 7.55 (s, 1H), 7.48 (d, J = 7.1 Hz, 2H), 7.27 (t, J = 25
24
7.5 Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H), 3.31 (d, J=13.3, 2H). 13C NMR (126 MHz, D2O) δ 162.9, 26 28
27
156.7, 156.6, 152.8, 139.8, 132.5, 129.0, 128.5, 125.5, 118.2, 114.0, 40.3 (d, J = 136 Hz). 31P NMR 30
29
(202 MHz, D2O): δ 12.87. HRMS (ESI-) m/z 320.0264 calculated for C13H11N3O3PS; found m/z 32
31
320.0267 [M - H]3 34 35 36 37 38
(Phenyl((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic acid (12): Isolated 39 41
40
43
42
as a white solid. 1H NMR (400 MHz, D2O) δ 7.72 (s, 1H), 7.58 (s, 1H), 7.48 (d, J = 7.7 Hz, 2H), 7.32 (t, J = 7.5Hz, 1H), 7.20 (t, J = 7.3Hz, 1H), 7.04 (d, J = 7.5Hz, 2H), 6.57 (d, J = 7.5Hz, 45
4
2H), 4.98 (d, J = 20 Hz, 1H), 1.92 (s, 3H). 13C NMR (126 MHz, D2O) δ 161.7, 154.8, 151.0, 46 48
47
139.8, 139.1, 137.4, 128.3, 128.0, 127.0, 126.7, 125.4, 123.9, 117.3, 112.1, 54.4 (d, J = 129 Hz), 50
49
19.0. 51
31
P NMR (81 MHz, D2O) δ 13.82. HRMS (ESI-) m/z 410.0734 calculated for
52
C20H17N3O3PS; found m/z 410.0725 [M - H]53 54 5 56 57 58 59
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(2-phenyl-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid (13): Isolated 5
4
as a pale beige solid. 1H NMR (500 MHz, D2O) δ 7.73 (s, 1H), 7.58 (s, 1H), 7.33 – 7.25 (m, 4H), 6 8
7
7.06 (t, J = 7.4 Hz, 2H), 6.96 (t, J = 7.2 Hz, 1H), 6.91 (d, J = 7.7 Hz, 2H), 4.49 (t, J = 12.6 Hz, 10
9
1H), 3.36 (d, J = 13.9 Hz, 1H), 2.82 (td, J = 13.2, 6.3 Hz, 1H), 2.17 (s, 3H). 31P NMR (203 MHz, 12
1
D2O) δ 15.9. HRMS (ESI-) m/z 424.0890 calculated for C19H19N3O3PS; found m/z 424.0892 [M 13 15
14
H]16 17 18 19 21
20
(2-Phenyl-2-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid (14): Isolated 23
2
as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 2.06 (m, 1H), 2.36 (s, 3H), 2.39-2.45 (m, 1H), 24 25
5.69-5.75 (m, 1H), 7.21 (t, 1H, J = 7.3 Hz), 7.30-7.34 (m, 4H), 7.45 (d, 2H, J = 7.5 Hz), 7.59 (d, 26 28
27
2H, J = 8.0 Hz), 8.03 (s, 1H), 8.27 (s, 1H), 8.30 (d, 1H, J = 7.7 Hz). 13C NMR (125 MHz, DMSO30
29
d6): 21.2, 35.7 (d), 50.1, 115.1, 118.1, 126.7, 127.2, 128.8, 130.4, 130.9, 138.7, 144.5, 144.7, 154.1, 32
31
156.0, 165.0. 31P NMR (202 MHz, DMSO-d6): δ 21.6. HRMS (ESI-) m/z 424.0890 calculated for 3 35
34
C21H19N3O3PS, found 424.0897 [M-H-]-. 36 37 38 40
39
(2-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-phenylethyl)phosphonic 42
41
acid (15): Isolated as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 2.10 (ddd, 1H, J1 = 19.3 Hz, 43 45
4
J2 = 15.2 Hz, J3 = 4.4 Hz), 2.38 (s, 3H), 2.40-2.45 (m, 1H), 5.69-5.76 (m, 1H), 7.21 (t, 1H, J = 7.3 47
46
Hz), 7.32 (t, 2H, J = 7.7 Hz), 7.45 (d, 2H, J = 7.4 Hz), 7.49 (d, 1H, J = 8.0 Hz), 7.54 (dd, 1H, J1 = 49
48
7.9 Hz, J2 = 1.8 Hz), 7.72 (d, 1H, J = 1.8 Hz), 8.13 (s, 1H), 8.28 (s, 1H), 8.34 (d, 1H, J = 7.8 Hz). 50 51 13
52
C NMR (125 MHz, DMSO-d6): 19.8, 35.7 (d), 50.1, 116.6, 117.9, 124.8, 125.7, 126.7, 127.3,
54
53
128.8, 132.6, 133.2, 134.6, 136.2, 136.6, 144.5, 144.6, 154.4, 156.1, 165.3. 5
31
P NMR (202 MHz,
56 57 58 59
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DMSO-d6): δ 21.7. HRMS (ESI-) m/z 458.0501 calculated for C21H18ClN3O3PS, found 458.0506 5
4
[M-H-]-. 6 7 8 9 10 12
1
(2-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-1-phenylethyl)phosphonic 14
13
acid (16): Isolated as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 2.80 (s, 3H), 4.02 (ddd, 1H, 15 16
J1 = 22.2 Hz, J2 = 10.4 Hz, J3 = 4.9 Hz), 4.49-4.61 (m, 2H), 7.60 (t, 1H, J = 6.9 Hz), 7.69 (t, 1H, J 17 19
18
= 7.6 Hz), 7.75 (t, 2H, J = 7.9 Hz), 7.88-7.92 (m, 2H), 8.07 (s, 1H), 8.31 (s, 1H), 8.33-8.34 (m, 1H), 21
20
8.81 (s, 1H). 2
13
C NMR (125 MHz, DMSO-d6): 23.8, 39.7 (d), 54.0, 120.5, 121.9, 128.8, 129.7,
23
130.7, 131.2, 132.7, 136.6, 137.1, 138.6, 140.2, 140.6, 148.4, 148.6, 158.4, 160.1, 169.2. 31P NMR 24 26
25
(202 MHz, DMSO-d6): δ 21.1. HRMS (ESI-) m/z 458.0501 calculated for C21H18ClN3O3PS, found 28
27
458.0479 [M-H-]-. 29 30 31 3
32
6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic acid (17): 34 35
Isolated as a pale beige solid. 1H NMR (400 MHz, D2O) δ 8.05 (s, 1H), 7.42 (s, 1H), 7.28 (s, 38
37
36
1H), 7.23 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 3.44 (d, J = 13.2 Hz, 2H), 2.19 (s, 3H). 40
39
C NMR (126 MHz, D2O) δ 162.6, 156.4, 152.5, 138.2, 136.0, 134.0, 131.5, 131.0, 125.0, 123.5,
13
41 42
118.0, 114.2, 40.3 (d, J = 134 Hz), 19.0. 31P NMR (202 MHz, D2O): δ 13.67. HRMS (ESI-) m/z 45
4
43
368.0031 calculated for C14H12ClN3O3PS; found m/z 368.0017 [M - H]46 47 48 49 50 52
51
Synthesis of inhibitor 18: 54
53
Ethyl hydrogen (((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonate (18). To a 5 56
dry round bottom flask, diethyl (((6-(p-tolyl)thieno[2,3-d]pyrimidin-457 58 59
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yl)amino)methyl)phosphonate (40.0 mg, 0.10 mmol) was dissolved in EtOH (5.0 mL) and the 5
4
solution was cooled to 0oC. NaOH pellets (28.6 mg, 0.72 mmol, 7 eq) were added and the 6 8
7
resulting mixture was stirred at RT for 11 days. Solvent was then removed in vacuo and the 10
9
resulting residue was dissolved in deionized H2O (2.0 mL) and acidified using 1N HCl (1.0 1 12
mL). The white precipitate formed was collected via filtration and purified by trituration. The 13 14
product was isolated as a white solid (29.4 mg, 79%). 1H NMR (500 MHz, D2O) δ 7.86 (s, 17
16
15
1H), 6.99 (s, 1H), 6.95 (d, J = 8.0 Hz, 2H), 6.77 (d, J = 8.0 Hz, 2H), 3.89 (m, 2H), 3.47 (d, J = 18 19
12.7 Hz, 2H), 2.11 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H). HRMS (ESI-) m/z 362.0734 calculated for 20 2
21
C16H17N3O3PS; found m/z 362.0739 [M - H]23 24 25 26 28
27
(((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)(phenyl)methyl)phosphonic 30
29
acid (19): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 7.77 (s, 1H), 7.49 (d, J = 7.7 31 32
Hz, 3H), 7.35 (t, J = 7.2 Hz, 2H), 7.25, -7.22 (m, 1H), 7.05 (s, 1H), 6.79 (s, 1H), 6.27 (d, J = 7.2 3 35
34
Hz, 1H), 5.00 (d, J = 19.7 Hz, 1H), 1.75 (s, 3H). 37
36
C NMR (126 MHz, D2O) δ 162.8, 155.8,
13
152.2, 140.9, 138.4, 135.6, 131.2, 128.1, 127.8, 126.6, 124.8, 123.2, 118.1, 114.0, 110.0, 55.5 38 39
(d, J = 134 Hz), 18.6. 31P NMR (81 MHz, D2O) δ 13.84. HRMS (ESI-) m/z 444.0344 calculated 40 42
41
for C20H16ClN3O3PS; found m/z 444.0357 [M - H]43 4 45 46 47
(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-248 50
49
phenylethyl)phosphonic acid (20): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 7.75 52
51
(s, 1H), 7.52 (s, 1H), 7.28 (d, J = 9.0 Hz, 2H), 7.26 (s, 1H), 7.04 (t, J = 7.5 Hz, 3H), 6.94 (t, J = 53 5
54
7.3 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 4.50 (t, J = 12.8 Hz, 1H), 3.36 (d, J = 14.1 Hz, 1H), 2.82 56 57 58 59
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(td, J = 13.4, 6.3 Hz, 1H), 1.99 (s, 3H). 31P NMR (202 MHz, D2O) δ 15.83. HRMS (ESI-) m/z 5
4
458.0501 calculated for C21H18ClN3O3PS; found m/z 458.0488 [M - H]6 7 8 9 10 12
1
(((6-(5-Chloro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic 14
13
acid (21): Isolated as a light beige solid. 1H NMR (500 MHz, D2O) δ 8.20 (s, 1H), 7.67 (s, 1H), 16
15
7.40 (s, 1H), 6.81 (s, 1H), 4.03 (s, 3H), 3.63 (d, J = 13.3 Hz, 2H), 2.28 (s, 3H). 31P NMR (203 MHz, 17 19
18
D2O) δ 13.78. 13C NMR (125 MHz, D2O) δ 162.8, 156.3 (d, JC-P = 8.9 Hz), 153.6, 152.2, 136.8, 21
20
134.2, 126.6, 125.4, 119.9, 116.7, 115.5, 114.2, 55.9, 40.7 (d, JC-P = 135.8 Hz), 19.5. HRMS (ESI+) 23
2
m/z 443.9921 calculated for C15H14ClN3Na2O4PS; found m/z 443.9930 [M + 2 Na]+. 24 25 26 27 28 30
29
(1-((6-(5-Fluoro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-232
31
phenylethyl)phosphonic acid (22): Isolated as a light yellow solid. 1H NMR (500 MHz, DMSO3 35
34
d6) δ 8.33 (s, 1H), 8.28-8.20 (m, 2H), 7.46 (d, J = 10.5 Hz, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.1837
36
7.12 (m, 3H), 7.07 (t, J = 7.3 Hz, 1H), 5.09-4.95 (m, 1H), 3.93 (s, 3H), 3.30-3.22 (m, 1H), 3.0739
38
3.01 (m, 1H), 2.29 (d, J = 1.0 Hz, 3H). 31P NMR (203 MHz, DMSO-d6) δ 19.42. 13C NMR (125 40 42
41
MHz, DMSO-d6) δ 156.0, 154.1, 152.1, 151.3, 138.5 (d, J = 15.8 Hz), 132.9, 128.8, 128.1, 126.1, 4
43
125.6 (d, J = 18.5 Hz), 120.2 (d, J = 7.5 Hz), 117.1, 115.6, 112.8 (d, J = 25.6 Hz), 56.6, 49.5 (d, 45 46
JC-P = 151.3 Hz), 34.9, 14.4. HRMS (ESI+) m/z 518.0686 calculated for C22H20FN3Na2O4PS; 49
48
47
found m/z 518.0692 [M + 2 Na]+. 50 51 52 54
53
(((6-(5-fluoro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-45 56
yl)amino)(phenyl)methyl)phosphonic acid (23): Isolated as a white solid. 1H NMR (500 MHz, 57 58 59
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D2O) δ 7.94 (s, 1H), 7.74 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.21 (t, J = 5
4
7.2 Hz, 1H), 6.92 (d, J = 10.0 Hz, 1H), 6.28 (d, J = 5.0 Hz, 1H), 5.09 (d, J = 19.9 Hz, 1H), 3.67 6 7
(s, 3H), 1.68 (s, 3H). 31P NMR (203 MHz, D2O) δ 13.77. HRMS (ESI+) m/z 504.0530 calculated 10
9
8
for C21H18FN3Na2O4PS; found m/z 504.0546 [M + 2 Na]+ 1 12 13 14 16
15
(((6-(5-chloro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-418
17
yl)amino)(phenyl)methyl)phosphonic acid (24): Isolated as a white solid. 1H NMR (500 MHz, 19 20
D2O) δ 7.98 (s, 1H), 7.71 (s, 1H), 7.51 (d, J = 7.6 Hz, 2H), 7.30 (t, J = 7.0 Hz, 1H), 7.21 (t, J = 23
2
21
7.0 Hz, 1H), 7.08 (s, 1H), 6.22 (s, 1H), 5.12 (d, J = 19.6 Hz, 1H), 3.70 (s, 3H), 1.60 (s, 3H). 31P 25
24
NMR (203 MHz, D2O) δ 13.92. HRMS (ESI-) m/z 474.0450 calculated for C21H18ClN3O4PS; 26 28
27
found m/z 474.0448 [M - H]29 30 31 32 34
3
(1-((6-(5-Chloro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-236
35
phenylethyl)phosphonic acid (25): Isolated as a light yellow solid. 1H NMR (500 MHz, DMSO-d6) 39
38
37
δ 8.34 (s, 1H), 8.28-8.20 (m, 2H), 7.73 (s, 1H), 7.28 (d, J = 7.3 Hz, 2H), 7.23 (s, 1H), 7.16 (t, J = 41
40
7.6 Hz, 2H), 7.07 (t, J = 7.3 Hz, 1H), 5.07-5.00 (m, 1H), 3.95 (s, 3H), 3.30-3.21 (m, 1H), 3.09-2.98 42 43
(m, 1H), 2.38 (s, 3H). 31P NMR (203 MHz, DMSO-d6) δ 19.49. 13C NMR (125 MHz, DMSO-d6) δ 46
45
4
156.1, 153.8, 152.4, 138.6 (d, JC-P = 15.9 Hz), 136.9, 132.4, 128.7, 128.1, 126.6, 126.1, 125.3, 48
47
120.9, 117.0, 116.0, 115.5, 56.4, 49.4 (d, JC-P = 151.3 Hz), 34.9, 19.8. HRMS (ESI+) m/z 534.0391 49 50
calculated for C22H20ClN3Na2O4PS; found m/z 534.0415 [M + 2 Na]+. 52
51 53 54 5 56 57 58 59
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(((5-methyl-6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic acid (27): 5
4
Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 8.04 (s, 1H), 7.07 (dd, J = 18.8, 7.8 Hz, 6 7
4H), 3.42 (d, J = 13.1 Hz, 2H), 2.42 (s, 3H), 2.22 (s, 3H). 31P NMR (203 MHz, D2O) δ 14.25. 10
9
8
HRMS (ESI-) m/z 348.0577 calculated for C15H15N3O3PS; found m/z 348.0576 [M - H]1 12 13 14 15 16
(1-((5-methyl-6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-phenylethyl)phosphonic acid 17 19
18
(28): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 7.78 (s, 1H), 7.26 (d, J = 7.4 Hz, 21
20
2H), 7.13 (d, J = 7.8 Hz, 2H), 7.08 – 6.99 (m, 4H), 6.91 (t, J = 7.2 Hz, 1H), 4.65 – 4.50 (m, 2 23
1H), 3.36 (d, J = 13.8 Hz, 1H), 2.89 – 2.76 (m, 1H), 2.57 (s, 3H), 2.23 (s, 3H). 24
31
P NMR (202
26
25
MHz, D2O) δ 15.59. HRMS (ESI-) m/z 438.1047 calculated for C22H11N3O3PS; found m/z 28
27
438.1056 [M - H]29 30 31 3
32
Synthesis of 29 and 30: 34 36
35
To a solution of 63 or 65 (0.26 mmol) in dry THF (20 mL), was added t-BuOLi (0.51 mmol) in 37 38
small portions and the resulting red solution was stirred under Ar and at RT for 3 h. Then, 39 41
40
tetrabenzyl pyrophosphate (0.39 mmol) was added in small portions over 5 min and the resulting 43
42
mixture was stirred under Ar for 2 h. The solvent was evaporated under vacuum and the residue 4 46
45
was purified by flash column chromatography eluted with hexane:EtOAc (1:1) affording the 48
47
phosphonate esters (yield of 45%-52%). Deprotection of the phosphonate esters using TMSBr (5.0 50
49
eq) was done as previously described. 17,19,20 However, for these scaffolds the reaction was finished 51 53
52
in 2h (yield of 64%-95%). 54 5 56 57 58 59
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(S)-(3-phenyl-2-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)propanoyl)phosphoramidic acid 5
4
(29): Isolated as a white solid. 1H NMR (500 MHz, DMSO-d6): δ 2.36 (s, 3H), 2.97 (t, 1H, J = 11.8 6 8
7
Hz), 3.23 (d, 2H, J = 11.9 Hz), 5.11 (br_s, 1H), 7.13 (t, 1H, J = 7.3 Hz), 7.23 (t, 2H, J = 7.6 Hz), 10
9
7.33 (d, 2H, J = 8.0 Hz), 7.46 (d, 2H, J = 7.5 Hz), 7.58 (d, 2H, J = 8.0 Hz), 8.06 (d, 1H, J = 8.2 Hz), 12
1
8.09 (s, 1H), 8.24 (s, 1H), 9.52 (br_s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 21.2, 37.5, 56.6, 13 15
14
115.1, 118.0, 126.0, 126.7, 128.5, 129.6, 130.4, 130.8, 138.68, 138.74, 139.0, 153.9, 156.7, 165.2, 17
16
173.9. 19
18
31
P NMR (202 MHz, DMSO-d6): δ -5.3. HRMS (ESI-) m/z 467.0948 calculated for
C21H20N4O4PS, found 467.0946 [M-H-]-. 20 21 2 23 24 26
25
2-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-228
27
phenylacetyl)phosphoramidic acid (30): Isolated as a white solid. 1H NMR (500 MHz, DMSO-d6): 29 30
δ 2.36 (s, 3H), 6.09 (brs, 1H), 7.35-7.46 (m, 4H), 7.55-7.58 (m, 3H), 7.73 (s, 1H), 8.24 (s, 1H), 3
32
31
8.38-8.43 (2 x s, 2H), 9.44 (br_s, 1H). 31P NMR (202 MHz, DMSO-d6): δ -5.7. HRMS (ESI-) m/z 35
34
487.0402 calculated for C21H17ClN4O4PS, found 487.0398 [M-H-]-. 36 37 38 39 40
((4-methoxyphenyl)((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic acid 41 42
(31): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 7.79 (s, 1H), 7.52 (s, 1H), 7.41 (s, 45
4
43
1H), 7.25 (s, 1H), 7.08 – 6.97 (m, 2H), 6.78 (s, 2H), 6.29 (s, 2H), 5.56 (d, J = 18.2 Hz, 1H), 47
46
3.91 (s, 3H), 1.82 (s, 3H). 31P NMR (203 MHz, D2O) δ 14.6. HRMS (ESI-) m/z 440.0839 48 50
49
calculated for C21H19N3O4PS; found m/z 440.0853 [M - H]51 52 53 54 5 56 57 58 59
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((3-methoxyphenyl)((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic 5
4
acid
(32): Isolated as a beige solid. 1H NMR (500 MHz, D2O) δ 7.76 (s, 1H), 7.49 – 7.42 (m, 2H), 6 8
7
7.18 (s, 1H), 6.95 (t, J = 7.3 Hz, 2H), 6.83 (s, 2H), 6.31 (s, 2H), 5.48 (d, J = 19.1 Hz, 1H), 3.84 10
9
(s, 3H), 1.80 (s, 3H). 31P NMR (203 MHz, D2O) δ 14.5. HRMS (ESI-) m/z 440.0839 calculated 12
1
for C21H19N3O4PS; found m/z 440.0833 [M - H]13 14 15 16 18
17
((2,3-dihydrobenzofuran-5-yl)((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)19 20
phosphonic acid (33): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 7.71 (s, 1H), 7.55 23
2
21
(s, 1H), 7.46 (s, 1H), 7.29 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 7.4 Hz, 2H), 6.81 (d, J = 8.3 Hz, 1H), 25
24
6.48 (d, J = 7.2 Hz, 2H), 4.96 (d, J = 19.6 Hz, 1H), 4.56 – 4.41 (m, 2H), 3.31 – 3.11 (m, 2H), 26 27
1.92 (s, 3H). 13C NMR (126 MHz, D2O) δ 162.5, 157.4, 155.8 (d, J = 11.0 Hz), 151.9, 140.1, 30
29
28
138.3, 134.0 (d, J = 2.4 Hz), 129.2, 128.9, 127.7, 127.2, 124.7, 118.4, 113.1, 110.0, 108.4, 71.7, 32
31
54.9 (d, J = 133.0 Hz), 29.2, 20.0. 31P NMR (203 MHz, D2O) δ 14.2. HRMS (ESI-) m/z 452.0839 3 35
34
calculated for C22H19N3O4PS; found m/z 452.0846 [M - H]36 37 38 39 41
40
((4-methylthiophen-2-yl)((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic 43
42
acid (34): Isolated as a beige solid (15.0 mg, 80%). 1H NMR (500 MHz, D2O) δ 7.80 (s, 1H), 4 45
7.42 (s, 1H), 6.98 (d, J = 7.9 Hz, 2H), 6.94 (s, 1H), 6.79 (s, 1H), 6.43 (d, J = 7.9 Hz, 2H), 5.21 46 48
47
(d, J = 19.7 Hz, 2H), 2.11 (s, 2H), 1.90 (s, 3H). HRMS (ESI-) m/z 430.0454 calculated for 50
49
C19H17N3O3PS2; found m/z 430.0459 [M - H]51 52 53 54 5 56 57 58 59
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(1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)cyclopentyl)phosphonic acid (35): Isolated 5
4
as a white solid. 1H NMR (500 MHz, D2O) δ 8.02 (s, 1H), 7.54 (s, 1H), 7.35 (d, J = 7.4 Hz, 2H), 6 7
7.03 (d, J = 7.4 Hz, 2H), 2.15 (s, 3H), 2.08 – 1.99 (m, 1H), 1.86 (s, 1H), 1.62 (s, 1H). 31P NMR 10
9
8
(203 MHz, D2O) δ 22.8. HRMS (ESI-) m/z 388.0890 calculated for C18H19N3O3PS; found m/z 12
1
388.0897 [M - H]13 14 15 16 18
17
(2-cyclobutyl-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid (36): 19 20
Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 8.14 (s, 1H), 7.56 (s, 1H), 7.29 (d, J = 23
2
21
7.9 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 4.36 – 4.10 (m, 1H), 2.40 – 2.29 (m, 1H), 2.18 (s, 3H), 25
24
2.09 – 2.02 (m, 1H), 2.01 – 1.95 (m, 1H), 1.86 – 1.77 (m, 2H), 1.75 – 1.63 (m, 4H). 13C NMR 26 27
(126 MHz, D2O) δ 162.8, 156.6 (d, J = 5.9 Hz), 152.6, 140.2, 138.7, 129.8, 129.4, 125.4, 30
29
28
118.2, 113.1, 49.2 (d, J = 140.7 Hz), 38.7, 33.6 (d, J = 12.0 Hz), 28.4 (d, J = 28.9 Hz), 20.2, 32
31
18.2. 31P NMR (203 MHz, D2O) δ 16.9. HRMS (ESI-) m/z 402.1047 calculated for 3 35
34
C19H21N3O3PS; found m/z 402.1048 [M - H]36 37 38 39 40
(2-cyclohexyl-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid (37): 41 43
42
Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 8.17 (s, 1H), 7.52 (s, 1H), 7.15 (d, J = 45
4
7.9 Hz, 2H), 6.82 (d, J = 7.9 Hz, 2H), 4.42 (t, J = 13.5 Hz, 1H), 2.00 (s, 3H), 2.01 – 1.93 (m, 47
46
1H), 1.85 – 1.75 (m, 1H), 1.73 – 1.63 (m, 1H), 1.61 – 1.51 (m, 2H), 1.51 – 1.36 (m, 2H), 1.26 48 50
49
(d, J = 7.3 Hz, 1H), 1.09 – 0.82 (m, 5H). 13C NMR (126 MHz, D2O) δ 162.9, 156.6, 152.7, 52
51
140.2, 138.5, 129.8, 129.3, 125.3, 118.2, 113.0, 48.0(d, J = 141.5 Hz), 39.4, 34.4 (d, J = 11.8 53 54
Hz), 34.2, 32.0, 26.2, 25.8, 20.2. 31P NMR (203 MHz, D2O) δ 17.6. HRMS (ESI-) m/z 57
56
5
430.1360 calculated for C21H25N3O3PS; found m/z 430.1353 [M - H]58 59
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1 2 3 5
4
(2-(3-fluorophenyl)-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid 7
6
(38): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 7.84 (s, 1H), 7.58 (s, 1H), 7.22 – 9
8
7.13 (m, 4H), 7.09 (t, J = 11.0 Hz, 1H), 6.79 (d, J = 7.9 Hz, 2H), 6.74 – 6.69 (m, 1H), 4.63 – 10 12
1
4.46 (m, 1H), 3.45 (d, J = 14.1 Hz, 1H), 2.93 (td, J = 13.5, 6.4 Hz, 1H), 2.13 (s, 3H). 13C NMR 14
13
(126 MHz, D2O) δ 162.7, 162.3 (d, J = 251.3 Hz), 156.6 (d, J = 5.7 Hz), 152.1, 142.7 (dd, J = 15 16
13.7, 7.6 Hz), 140.2, 138.7, 129.7, 129.6 (d, J = 8.5 Hz), 129.3, 125.5, 125.3, 118.1, 116.1 (d, J 17 19
18
= 20.9 Hz), 112.8(d, J = 21.1 Hz), 52.2 (d, J = 139.0 Hz), 48.9, 37.8, 20.2. 31P NMR (203 21
20
MHz, D2O) δ 15.7. HRMS (ESI-) m/z 442.0796 calculated for C21H18FN3O3PS; found m/z 2 23
442.0812 [M - H]24 25 26 27 28 29
2-(pyridin-2-yl)-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid (39): 30 32
31
Isolated as a beige solid. 1H NMR (500 MHz, D2O) δ 7.95 (d, J = 4.6 Hz, 1H), 7.63 (d, J = 34
3
13.9 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.37 (t, J = 7.0 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.13 35 36
(d, J = 8.0 Hz, 2H), 6.94 – 6.83 (m, 1H), 4.47 (t, J = 12.7 Hz, 1H), 3.39 (d, J = 13.5 Hz, 1H), 39
38
37
2.87 (td, J = 12.8, 6.2 Hz, 1H), 2.22 (s, 3H). 31P NMR (202 MHz, D2O) δ 15.0. MS [ESI+] m/z 41
40
427.1[M + H]+ 42 43 4 45 47
46
(2-(pyridin-3-yl)-1-((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)phosphonic acid (40): 48 49
Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 8.28 (s, 1H), 7.95 (d, J = 4.1 Hz, 1H), 52
51
50
7.63 (s, 1H), 7.63 – 7.60 (m, 1H), 7.49 (s, 1H), 7.20 (d, J = 8.1 Hz, 2H), 6.98 (dd, J = 7.7, 5.0 53 54
Hz, 1H), 6.82 (d, J = 8.1 Hz, 2H), 4.38 (ddd, J = 15.5, 12.2, 3.1 Hz, 1H), 3.29 (dd, J = 11.3, 5 56
2.7 Hz, 1H), 2.83 – 2.69 (m, 1H), 2.07 (s, 3H). 13C NMR (126 MHz, D2O) δ 162.9, 156.6, 58
57 59
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152.1, 149.3, 146.1, 140.3, 139.0, 138.5, 136.0 (d, J = 13.8 Hz), 129.8, 129.5, 125.5, 123.5, 5
4
117.9, 113.0, 51.9 (d, J = 137.1 Hz), 35.5, 20.2. 31P NMR (202 MHz, D2O) δ 15.1. HRMS 6 7
(ESI-) m/z 425.0843 calculated for C20H18N3O3PS; found m/z 425.0841[M - H]9
8 10 1 12 14
13
(Pyridin-3-yl((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic acid (41): 16
15
Isolated as cream solid. 1H NMR (500 MHz, D2O) δ 8.72 (s, 1H), 8.48 (d, J = 4.5 Hz, 1H), 8.06 17 19
18
(dd, J = 8.0, 1.3 Hz, 1H), 7.79 (s, 1H), 7.60 (s, 1H), 7.55 (dd, J = 7.8, 5.0 Hz, 1H), 7.01 (d, J = 7.8 21
20
Hz, 2H), 6.56 (d, J = 7.8 Hz, 2H), 5.11 (d, J = 19.8 Hz, 1H), 1.95 (s, 3H). 31P NMR (203 MHz, 23
2
D2O) δ 12.75 (s). 13C NMR (126 MHz, D2O) δ 162.7, 155.7 (d, J = 7.7 Hz), 151.8, 147.8 (d, J = 24 26
25
4.9 Hz), 146.6, 140.3, 138.3, 137.6, 136.6 (d, J = 3.2 Hz), 129.1, 128.9, 124.7, 124.0, 118.4, 28
27
113.0, 53.3(d, J = 131.0 Hz), 20.0. HRMS (ESI+) m/z 457.0471 calculated for C19H16N4Na2O3PS; 29 30
found m/z 457.0480 [M + 2 Na]+ 31 32 3 34 35 37
36
(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(3-fluorophenyl)ethyl)39
38
phosphonic acid (42): Isolated as a white solid (21.7 mg, 64%). 1H NMR (500 MHz, D2O) δ 40 41
7.89 (s, 1H), 7.58 (s, 1H), 7.30 (s, 1H), 7.15 (t, J = 10.1 Hz, 2H), 7.10 – 7.03 (m, 2H), 6.75 – 4
43
42
6.67 (m, 2H), 4.62 (t, J = 12.6 Hz, 1H), 3.46 (d, J = 14.2 Hz, 1H), 3.03 – 2.87 (m, 1H), 2.02 (s, 46
45
3H). 13C NMR (126 MHz, D2O) δ 163.0, 162.3 (d, J = 242.7 Hz), 156.8, 152.5, 142.6 (dd, J = 47 49
48
13.4, 7.5 Hz), 138.4, 136.1, 134.1, 131.7, 131.0, 129.6 (d, J = 8.4 Hz), 125.5, 125.4 (d, J = 5.8 51
50
Hz), 123.6, 117.9, 116.1 (d, J = 20.9 Hz), 113.9, 112.7 (d, J = 20.8 Hz), 52.1 (d, J = 137.5 Hz), 52 54
53
37.9, 18.8.
31
P NMR (203 MHz, D2O) δ 15.6. HRMS (ESI-) m/z 476.0406 calculated for
56
5
C21H17IFN3O3PS; found m/z 476.0406 [M - H]57 58 59
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4
(1-((6-(3-fluoro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(37
6
fluorophenyl)ethyl)phosphonic acid (43): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 9
8
7.85 (s, 1H), 7.63 (s, 1H), 7.18 – 7.08 (m, 4H), 7.00 (dd, J = 7.8, 1.7 Hz, 1H), 6.81 (t, J = 8.0 10 12
1
Hz, 1H), 6.78 – 6.72 (m, 1H), 4.65 – 4.50 (m, 1H), 3.48 – 3.40 (m, 1H), 3.04 – 2.87 (m, 1H), 14
13
2.09 (s, 3H). 15
31
P NMR (203 MHz, D2O) δ 15.5. HRMS (ESI-) m/z 460.0702 calculated for
16
C21H17F2N3O3PS; found m/z 460.0695 [M - H]17 18 19 20 21 2 24
23
(1-((6-(5-fluoro-2-methoxyphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(326
25
fluorophenyl)ethyl)phosphonic acid (44): Isolated as a white solid. 1H NMR (500 MHz, DMSO28
27
d6): δ 3.02 - 3.08 (m, 1H), 3.25 - 3.29 (m, 1H), 3.94 (s, 3H), 5.00 - 5.08 (m, 1H), 6.89 (td, 1H, J1 = 29 31
30
8.4 Hz, J2 = 1.9 Hz), 7.10 - 7.13 (m, 2H), 7.18 - 7.24 (m, 3H), 7.52 (dd, 1H, J1 = 9.8 Hz, J2 = 2.0 3
32
Hz), 7.98 (d, 1H, J = 9.4 Hz), 8.20 (s, 1H), 8.35 (s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 35.3, 34 35
49.3 (d), 57.0, 113.4 (d), 113.9 (d), 114.8 (d), 116.1 (dd), 116.4, 118.5, 123.6 (d), 125.4, 130.3 (d), 36 38
37
132.6, 152.2, 153.9, 156.0, 156.8, 157.9, 161.3, 163.3, 166.2. 31P NMR (202 MHz, DMSO-d6): δ 40
39
19.48. HRMS (ESI-) m/z 476.0651 calculated for C21H17F2N3O4PS; found m/z 476.0635 [M - H]41 42 43 4 45 47
46
(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(pyridin-249
48
yl)ethyl)phosphonic acid (45): Isolated as a white solid. 1H NMR (500 MHz, D2O) δ 8.07 (d, J 50 51
= 4.3 Hz, 1H), 7.73 (s, 1H), 7.57 (s, 1H), 7.41 (td, J = 7.7, 1.7 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J 52 53
= 7.9 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 6.95 – 6.90 (m, 2H), 4.65 – 4.53 (m, 1H), 3.46 (dt, J = 56
5
54
13.6, 2.8 Hz, 1H), 3.02 – 2.88 (m, 1H), 2.06 (s, 3H). 13C NMR (126 MHz, D2O) δ 159.1, 159.0, 57 58 59
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156.9, 152.5, 147.6, 138.4, 137.3, 136.4, 134.2, 131.9, 131.2, 125.7, 124.9, 123.9, 121.9, 117.8, 5
4
114.2, 50.8, 40.1, 18.9. 31P NMR (203 MHz, D2O) δ 15.2. HRMS (ESI-) m/z 459.0453 calculated 6 7
for C20H17ClN4O3PS; found m/z 459.0466 [M - H]9
8 10 1 13
12
(1-((6-(3-chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(pyridin-314 15
yl)ethyl)phosphonic acid (46): Isolated as a yellow solid. 1H NMR (500 MHz, D2O) δ 8.38 (s, 16 18
17
1H), 8.05 (s, 1H), 7.76 (s, 1H), 7.71 (d, J = 6.8 Hz, 1H), 7.54 (s, 1H), 7.34 (s, 1H), 7.09 (d, J = 20
19
29.7 Hz, 2H), 6.85 (s, 1H), 4.50 (t, J = 13.4 Hz, 1H), 3.38 (d, J = 13.7 Hz, 1H), 2.86 (s, 1H), 21 23
2
2.05 (s, 3H). 25
24
31
P NMR (203 MHz, D2O) δ 15.2. HRMS (ESI-) m/z 459.0453 calculated for
C20H17ClN4O3PS; found m/z 459.0433 [M - H]26 27 28 29 31
30
(1-((6-(5-fluoro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)-2-(33
32
fluorophenyl)ethyl)phosphonic acid (47): Isolated as a white solid. 1H NMR (500 MHz, DMSO34 35
d6): δ 2.30 (s, 3H), 3.01-3.08 (m, 1H), 3.24-3.28 (m, 1H), 3.93 (s, 3H), 4.99-5.07 (m, 1H), 6.89 (td, 38
37
36
1H, J1 = 8.4 Hz, J2 = 2.0 Hz), 7.10-7.15 (m, 3H), 7.18-7.23 (m, 1H), 7.46(d, 1H, J1 = 10.6 Hz), 7.91 40
39
(d, 1H, J = 9.5 Hz), 8.20 (s, 1H), 8.28 (s, 1H). 31P NMR (202 MHz, DMSO-d6): δ 19.53. 13C NMR 41 43
42
(125 MHz, DMSO-d6): δ 14.9, 35.3, 49.2 (d), 57.00, 113.3 (d), 113.4 (d), 116.1 (dd), 117.6, 121.0 45
4
(d), 125.4 (d), 125.8 (d), 130.3 (d), 132.8, 142.1, 151.8, 153.8, 154.5, 156.4, 156.7 (d), 161.3, 163.2, 46 47
166.0. HRMS (ESI-) m/z 490.0807 calculated for C22H19F2N3O4PS; found m/z 490.0795 [M - H]48 49 50 51 52 53 54 5 56 57 58 59
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(((6-(5-Fluoro-2-methoxy-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)phosphonic 5
4
acid (48): The product was isolated as a light beige solid. 1H NMR (500 MHz, D2O) δ 8.19 (s, 1H), 6 8
7
7.68 (s, 1H), 7.19 (d, J = 10.9 Hz, 1H), 6.77 (d, J = 6.6 Hz, 1H), 3.97 (s, 3H), 3.60 (d, J = 13.3 Hz, 10
9
2H), 2.24 (s, 3H). 31P NMR (203 MHz, D2O) δ 13.75. 13C NMR (125 MHz, D2O) δ 163.0, 156.3 1 12
(d, J = 8.8 Hz), 156.0, 154.1, 152.2, 151.0, 134.4, 125.7 (d, J = 19.6 Hz), 119.5 (d, J = 8.1 Hz), 13 15
14
116.7, 115.5, 114.8, 112.7 (d, J = 26.1 Hz), 56.2, 40.6 (d, JC-P = 135.7 Hz), 14.0. HRMS (ESI+) 17
16
m/z 428.0217 calculated for C15H14FN3Na2O4PS; found m/z 428.0220 [M + 2 Na]+. 18 19 20 2
21
Synthesis of analogs of 56a or 56b: 24
23
Analogues of 56 were made according to the synthetic procedure previously described with 26
25
slight modifications.26 To a microwave reactor, 4-chloro-thieno[2,3-d]pyrimidine 54 or 55 (1 eq) 27 28
was dissolved in DMSO (~0.2 M). Potassium fluoride dihydrate (2 eq) was added and the 29 31
30
resulting mixture was stirred at 120oC under microwave irradiation for 15 min. It was then cooled 3
32
down to RT, concentrated in vacuo and purified using silica-gel column chromatography (0% 34 35
25% EtOAc in hexanes). 36 37 38 40
39
6-bromo-4-fluorothieno[2,3-d]pyrimidine (56a; R6=Br): Isolated as a yellow solid (85%). 1H 42
41
NMR (500 MHz, CDCl3) δ 8.74 (s, 1H), 7.46 (s, 1H). 43
13
C NMR (126 MHz, CDCl3) δ 172.6 (d,
45
4
J = 6.9 Hz), 162.8, 160.7, 153.2 (d, J = 13.6 Hz), 119.6 (d, J = 5.4 Hz), 118.9 (d, J = 29.6 Hz), 47
46
118.0 (d, J = 1.1 Hz). 19F NMR (471 MHz, CDCl3) δ -61.8. MS [ESI+] m/z: 233.0 [M + H]+ 48 49 50 51 52 53 54 5 56 57 58 59
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4-fluoro-6-(p-tolyl)thieno[2,3-d]pyrimidine (56a; R6=f2): Isolated as a yellow solid (160 mg, 5
4
85%). 1H NMR (500 MHz, CDCl3) δ 8.72 (d, J = 0.8 Hz, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.51 (s, 6 7
1H), 7.29 (d, J = 7.9 Hz, 2H), 2.42 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 171.3 (d, J = 7.4 Hz), 10
9
8
162.9 (d, J = 258.2 Hz), 152.5 (d, J = 13.5 Hz), 146.2, 140.1, 130.0, 129.8, 126.7, 119.5 (d, J = 12
1
29.1 Hz), 110.9 (d, J = 5.6 Hz), 21.4. 19F NMR (471 MHz, CDCl3) δ -63.13. 13 14 15 17
16
6-(3-chloro-4-methylphenyl)-4-fluorothieno[2,3-d]pyrimidine (56a; R6=f10): Isolated as a 19
18
yellow powder (71%). 1H NMR (500 MHz, CDCl3) δ 8.76 (d, J = 0.7 Hz, 1H), 7.71 (d, J = 1.9 20 2
21
Hz, 1H), 7.54 (s, 1H), 7.50 (dd, J = 7.9, 1.9 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 2.45 (s, 3H). 19F 24
23
NMR (471 MHz, CDCl3) δ -62.49 (s). 25 26 27 29
28
4-fluoro-5-methyl-6-(p-tolyl)thieno[2,3-d]pyrimidine (56b; R6=f2): Prepared from 4-chloro-531
30
methyl-6-(p-tolyl)thieno[2,3-d]pyrimidine 55b; the synthesis for 55b with R6=f2 is provided in 32 3
the Supporting Information. Isolated as a white solid (44%). 1H NMR (500 MHz, CDCl3) δ 8.71 36
35
34
(s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 2.58 (d, J = 1.5 Hz, 3H), 2.44 (s, 3H). 38
37
19
39
F NMR (470 MHz, CDCl3) δ -64.15. 13C NMR (125 MHz, CDCl3) δ 170.9 (d, JC-F = 7.4 Hz),
41
40
164.0 (d, JC-F = 258.4 Hz), 152.5 (d, JC-F = 14.0 Hz), 139.2, 129.8, 129.1, 123.6, 119.9 (d, JC-F = 43
42
25.2 Hz), 21.5, 14.4 (d, JC-F = 3.8 Hz). MS (ESI+) m/z: 259.2 [M+H]+. 4 45 46 48
47
Synthesis of α-aminophosphonate ester intermediates (60): 50
49
Method I: A round bottom flask was charged with the aldehyde (1.0 eq), benzylamine (1.0 eq), 51 52
and diethylphosphite (1.0 eq) and the reaction was heated at 80oC for 45 min. It was then cooled 53 5
54
down to RT, concentrated in vacuo and purified using silica-gel column chromatography (0% to 56 57 58 59
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100% EtOAc/hexanes and 0% to 20% MeOH/EtOAc). Alternatively, zirconium (IV) was added 5
4
as the catalyst following the protocol described by Bhagat and Chakraborti.29 Hydrogenolysis of 6 8
7
the diethyl benzyl aminophosphonate intermediate (using MeOH as solvent) to afford the desired 10
9
α-aminophosphonate 60 was done either (a) in the presence of 4.4% formic acid and Pearlman’s 1 12
catalyst (0.50 eq.) under argon for 2 h or (b) using 12 N HCl (1.0 eq) and H2, catalytic amounts 13 15
14
of Pd/C; details provided below in Method B. 17
16
Method II: Based on literature procedures with minor modifications.30 An argon flushed round 18 19
bottom flask was charged with freshly distilled N,N-diisopropylamine (1.1 eq; dissolved in THF) 20 2
21
and cooled to -78oC using an acetone-dry ice bath. n-BuLi (2.5N, 1.2 eq) was slowly added 24
23
dropwise to the solution. The reaction mixture was stirred for 15 min and 58 (refer to the S.I. for 25 27
26
the synthesis of 58) dissolved in THF was added to the reaction mixture dropwise and stirred for 29
28
5 min. An alkyl, or benzylic-type bromide (1.1 eq) was added dropwise to the reaction and 31
30
stirring was continued for 1 h at -78oC. The solution was slowly warmed to room temperature 32 34
3
and quenched by adding saturated NaHCO3 solution. THF was removed via evaporation under 36
35
reduced pressure. The aqueous layer was then extracted with EtOAc, dried over anhydrous 37 38
Na2SO4 and purified by silica-gel column chromatography using a gradient of 0% - 100% 39 41
40
hexane/EtOAc. The dibenzylaminophosphonate intermediate was then dissolved in MeOH and 43
42
Pd on carbon (0.05 – 0.20 eq.) was added to the solution followed by 12 N HCl (1.0 eq). A 4 45
hydrogen balloon was then attached and the reaction was stirred at RT until completion (typically 46 48
47
finished after 3h; monitored by TLC). The crude mixture was neutralized by addition of 10 N 50
49
NaOH and filtered through a bed of celite. The filtrate was concentrated under vacuum to give 51 52
the α-aminophosphonate 60 as an oil. If solid contaminants were observed, the residue was 53 5
54
dissolved in CHCl3 and filtered using a 42.5 μm filter. 56 57 58 59
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Diethyl (amino(4-methoxyphenyl)methyl)phosphonate (60; R1=f3): Method I - Step 1: Benzyl5
4
protected amine intermediate was isolated as a black oil (75%) 6 7 1
10
9
8
H NMR (500 MHz, CDCl3) δ 7.35 – 7.29 (m, 2H), 7.27 – 7.16 (m, 5H), 6.87 (d, J = 8.3 Hz,
2H), 4.08 – 3.99 (m, 2H), 3.97 – 3.87 (m, 2H), 3.81 – 3.76 (m, 1H), 3.75 (s, 3H), 3.49 (d, J = 12
1
13.3 Hz, 1H), 1.23 (t, J = 7.1 Hz, 3H), 1.10 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 13 15
14
159.3 (d, J = 2.9 Hz), 139.3, 129.7 (d, J = 6.2 Hz), 128.3, 128.3, 127.4 (d, J = 4.3 Hz), 127.1, 17
16
113.9 (d, J = 2.3 Hz), 62.8 (d, J = 7.1 Hz), 62.7 (d, J = 6.9 Hz), 58.7 (d, J = 155 Hz), 55.2, 50.9 18 19
(d, J = 18 Hz), 16.5 (d, J = 5.9 Hz), 16.3 (d, J = 5.8 Hz). 20 2
21
Step 2: Building block 60 (R1=f3) was isolated as a black oil (97%). 1H NMR (500 MHz, CDCl3) 24
23
δ 7.45 – 7.34 (m, 2H), 6.88 (d, J = 8.3 Hz, 2H), 4.25 (d, J = 16.4 Hz, 1H), 4.09 – 4.02 (m, 2H), 25 26
4.02 – 3.94 (m, 1H), 3.92 – 3.83 (m, 1H), 3.80 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H), 1.19 (t, J = 7.1 27 29
28
Hz, 3H). 30 31 3
32
Diethyl (amino(2,3-dihydrobenzofuran-5-yl)methyl)phosphonate (60; R1=f26): 35
34
Method I - Step 1: Benzyl-protected amine intermediate was isolated as brown oil (67%): 1H 36 38
37
NMR (500 MHz, CDCl3) δ 7.27 – 7.17 (m, 6H), 7.08 (d, J = 8.2 Hz, 1H), 6.72 (d, J = 8.2 Hz, 40
39
1H), 4.52 (t, J = 8.7 Hz, 2H), 4.11 – 4.00 (m, 2H), 3.97 – 3.87 (m, 2H), 3.84 – 3.72 (m, 2H), 41 42
3.50 (d, J = 13.4 Hz, 1H), 3.17 (t, J = 8.7 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H), 1.11 (t, J = 7.1 Hz, 43 45
4
3H). 13C NMR (126 MHz, CDCl3) δ 160.0 (d, J = 2.9 Hz), 139.4, 128.6 (d, J = 6.9 Hz), 128.3, 47
46
128.29, 127.4 (d, J = 2.4 Hz), 127.3 (d, J = 4.1 Hz), 127.0, 125.0 (d, J = 5.8 Hz), 109.0 (d, J = 48 50
49
2.2 Hz), 71.3, 62.8 (d, J = 7.0 Hz), 62.7 (d, J = 6.9 Hz), 58.9 (d, J = 155 Hz), 50.9 (d, J = 17 52
51
Hz), 29.7, 16.5 (d, J = 5.9 Hz), 16.3 (d, J = 5.8 Hz). 31P NMR (202 MHz, CDCl3) δ 23.9. 54
53
Step 2: Building block 60 (R1=f26) was isolated as a pale yellow oil (95% yield) 1H NMR (500 5 56
MHz, CDCl3) δ 7.33 (s, 1H), 7.18 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 4.57 (t, J = 8.7 58
57 59
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Hz, 2H), 4.23 (d, J = 16.2 Hz, 1H), 4.11 – 4.04 (m, 2H), 4.03 – 3.97 (m, 1H), 3.94 – 3.85 (m, 5
4
1H), 3.20 (t, J = 8.7 Hz, 2H), 1.29 (q, J = 6.9 Hz, 3H), 1.22 – 1.17 (m, 3H). 6 7 8 10
9
Diethyl (amino(pyridin-3-yl)methyl)phosphonate (60; R1=f31): 12
1
Method I - Step 1: Benzyl-protected amine intermediate was isolated as a yellow oil (42%): 1H 13 14
NMR (500 MHz, DMSO-d6) δ 8.54 (t, J = 1.9 Hz, 1H), 8.49 (dt, J = 4.7, 1.7 Hz, 1H), 7.85 (ddd, 17
16
15
J = 7.9, 3.9, 1.9 Hz, 1H), 7.40 (dd, J = 7.8, 4.8 Hz, 1H), 7.32 - 7.29 (m, 2H), 7.27 – 7.21 (m, 3H), 19
18
4.08 - 3.98 (m, 3H), 3.90 – 3.85 (m, 1H), 3.84 - 3.76 (m, 1H), 3.71 (dd, J = 14.1, 5.4 Hz, 1H), 20 2
21
3.45 (dd, J = 13.8, 6.2 Hz, 1H), 3.04 (dt, J = 12.6, 6.2 Hz, -NH), 1.20 (t, J = 7.1 Hz, 3H), 1.05 (t, J 24
23
= 7.0 Hz, 3H). 31P NMR (203 MHz, DMSO-d6) δ 22.70 (s). 25 26
Step 2: Building block 60 (R1=f31) was isolated as a yellow oil (50%).1H NMR (500 MHz, DMSO27 29
28
d6) δ 8.59 (s, 1H), 8.47 - 8.45 (m, 1H), 7.84 (dd, J = 7.9, 2.1 Hz, 1H), 7.37 (dd, J = 7.9, 4.8 Hz, 31
30
1H), 4.33 (d, J = 18.4 Hz, 1H), 4.06 – 4.00 (m, 2H), 3.96 – 3.85 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H), 32 3
1.11 (t, J = 7.0 Hz, 3H). 31P NMR (203 MHz, DMSO-d6) δ 24.52 (s). 35
34 36 37 38
Diethyl (1-amino-2-phenylethyl)phosphonate (60; R1=f22): 39 41
40
Method II - Step 1: The dibenzyl-protected amine intermediate was isolated as a colorless oil 43
42
(92%): 1H NMR (500 MHz, CDCl3) δ 7.33 (s, 1H), 7.33 – 7.30 (m, 3H), 7.29 – 7.24 (m, 4H), 45
4
7.22 – 7.18 (m, 1H), 7.13 – 7.08 (m, 2H), 4.23 – 4.11 (m, 4H), 4.01 (dd, J = 13.8, 4.1 Hz, 2H), 46 48
47
3.94 (d, J = 13.8 Hz, 2H), 3.43 (ddd, J = 16.3, 9.0, 5.6 Hz, 1H), 3.18 – 3.02 (m, 2H), 1.40 (td, J 50
49
= 7.1, 5.6 Hz, 6H). 31P NMR (203 MHz, CDCl3) δ 28.05. 51 52
Step 2: Building block 60 (R1=f22) was isolated as an orange oil (quant. yield). 1H NMR (500 53 5
54
MHz, CDCl3) δ 7.34 – 7.29 (m, 3H), 7.28 – 7.24 (m, 2H), 4.22 – 4.08 (m, 4H), 3.35 (ddd, J = 56 57 58 59
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12.1, 10.5, 4.1 Hz, 1H), 3.26 – 3.16 (m, 1H), 2.78 (dt, J = 13.8, 10.6 Hz, 1H), 1.33 (dt, J = 10.2, 5
4
7.1 Hz, 6H). 31P NMR (203 MHz, CDCl3) δ 26.71 6 7 8 10
9
Diethyl (1-amino-2-cyclobutylethyl)phosphonate (60; R1=f25): 1 12
Method II - Step 1: The dibenzyl-protected amine intermediate was isolated as a colorless oil 13 14
(64%): 1H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 7.3 Hz, 4H), 7.31 (t, J = 7.5 Hz, 4H), 7.23 (t, 17
16
15
J = 7.2 Hz, 2H), 4.18 – 4.03 (m, 4H), 3.90 – 3.83 (m, 4H), 2.90 – 2.81 (m, 1H), 2.65 – 2.52 (m, 19
18
1H), 2.08 – 1.95 (m, 1H), 1.92 – 1.83 (m, 1H), 1.80 – 1.60 (m, 3H), 1.59 – 1.49 (m, 1H), 1.45 – 20 2
21
1.38 (m, 1H), 1.37 – 1.30 (m, 6H), 1.24 – 1.11 (m, 1H). 13C NMR (126 MHz, CDCl3) δ 139.9, 24
23
129.2, 128.1, 126.9, 61.4 (d, J = 7.2 Hz), 61.0 (d, J = 7.6 Hz), 54.9 (d, J = 2.3 Hz), 53.1 (d, J = 25 27
26
131.9 Hz), 35.1 (d, J = 5.6 Hz), 32.7 (d, J = 12.0 Hz), 28.0, 27.5, 16.69 (d, J = 5.6 Hz), 16.64 (d, 29
28
J = 5.8 Hz). 31P NMR (203 MHz, CDCl3) δ 29.5. 31
30
Step 2: Building block 60, R1=f25, was isolated as a transparent oil (93%). 1H NMR (500 MHz, 32 3
CDCl3) δ 4.25 – 4.03 (m, 4H), 3.11 – 2.98 (m, 1H), 1.77 (d, J = 12.9 Hz, 1H), 1.73 – 1.63 (m, 36
35
34
3H), 1.63 – 1.53 (m, 2H), 1.45 – 1.37 (m, 1H), 1.34 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H), 38
37
1.27 – 1.20 (m, 2H), 1.05 – 0.94 (m, 1H), 0.90 – 0.75 (m, 1H). 39 40 41 43
42
Diethyl (1-amino-2-cyclohexylethyl)phosphonate (60; R1=f24): 4 45
Method II - Step 1: The dibenzyl-protected amine intermediate was isolated as a transparent oil 46 48
47
(57%): 1H NMR (500 MHz, CDCl3) δ 7.37 – 7.28 (m, 8H), 7.23 (t, J = 7.1 Hz, 2H), 4.18 – 4.07 50
49
(m, 4H), 3.96 – 3.76 (m, 4H), 3.04 (ddd, J = 15.8, 10.6, 3.2 Hz, 1H), 1.73 – 1.51 (m, 6H), 1.49 51 52
– 1.41 (m, 1H), 1.35 (t, J = 7.1 Hz, 3H), 1.33 (t, J = 7.1 Hz, 3H), 1.29 – 1.18 (m, 1H), 1.11 – 53 5
54
1.00 (m, 1H)z, 1.00 – 0.93 (m, 1H), 0.93 – 0.82 (m, 2H), 0.51 – 0.35 (m, 1H). 13C NMR (126 56 57 58 59
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MHz, CDCl3) δ 140.0, 129.3, 128.1, 126.9, 61.3 (d, J = 7.2 Hz), 61.0 (d, J = 7.6 Hz), 54.8, 52.1 5
4
(d, J = 130.8 Hz), 35.4 (d, J = 5.9 Hz), 34.3, 33.4 (d, J = 11.5 Hz), 31.8, 26.7, 26.6, 26.0, 16.7 6 7
(d, J = 5.5 Hz), 16.7 (d, J = 5.7 Hz). 31P NMR (203 MHz, CDCl3) δ 29.9. 10
9
8
Step 2: Building block 60, R1=f24 was isolated as a colorless oil (92%). 1H NMR (500 MHz, 12
1
CDCl3) δ 4.22 – 4.03 (m, 4H), 2.99 – 2.83 (m, 1H), 2.68 – 2.49 (m, 1H), 2.16 – 1.98 (m, 3H), 13 14
1.91 – 1.77 (m, 3H), 1.73 – 1.53 (m, 4H), 1.34 (t, J = 7.0 Hz, 3H), 1.34 (t, J = 7.0, 3H), 1.26 – 17
16
15
1.15 (m, 2H). 18 19 20 2
21
Diethyl (1-amino-2-(3-fluorophenyl)ethyl)phosphonate (60, R1=f23): 24
23
Method II - Step 1: The dibenzyl-protected amine intermediate was isolated as a colorless oil 25 26
(71%): 1H NMR (500 MHz, CDCl3) δ 7.24 – 7.17 (m, 7H), 7.13 (m, 4H), 6.94 (td, J = 8.5, 2.3 27 29
28
Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.65 (d, J = 9.9 Hz, 1H), 4.21 – 4.06 (m, 4H), 3.96 – 3.82 (m, 31
30
4H), 3.37 – 3.26 (m, 1H), 3.06 – 2.98 (m, 2H), 1.34 (t, J = 7.1 Hz, 3H), 1.34 (t, J = 7.1, 3H). 13C 32 3
NMR (126 MHz, CDCl3) δ 163.7, 161.7, 141.6, 139.1, 129.4 (d, J = 8.2 Hz), 128.9, 128.1, 127.0, 36
35
34
125.3, 116.2 (d, J = 21.0 Hz), 113.1 (d, J = 20.9 Hz), 61.6 (d, J = 7.2 Hz), 61.3 (d, J = 7.7 Hz), 38
37
56.9 (d, J = 135.1 Hz), 54.6, 33.7, 16.7 (d, J = 5.7 Hz), 16.6 (d, J = 5.7 Hz). 31P NMR (203 MHz, 39 41
40
CDCl3) δ 27.7 43
42
Step 2: Building block 60, R1=f23, was isolated as a slightly yellow oil (104.7 mg, 93%). 1H 45
4
NMR (500 MHz, CDCl3) δ 7.31 – 7.27 (m, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.98 – 6.91 (m, 2H), 46 48
47
4.23 – 4.13 (m, 4H), 3.29 – 3.14 (m, 2H), 2.77 – 2.62 (m, 1H), 1.35 (t, J = 7.0 Hz, 3H), 1.35 (t, 50
49
J = 7.1, 3H), 1.25 (dd, J = 14.2, 6.5 Hz, 2H). 51 52 53 54 5 56 57 58 59
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Diethyl (1-amino-2-(pyridin-2-yl)ethyl)phosphonate (60; R1=f29): 5
4
Method II - Step 1: The dibenzyl-protected amine intermediate was isolated as a yellow oil 7
6
(23%): 1H NMR (500 MHz, CDCl3) δ 8.47 – 8.40 (m, 1H), 7.59 – 7.52 (m, 1H), 7.21 – 7.14 (m, 9
8
7H), 7.12 – 7.08 (m, 4H), 7.01 (d, J = 7.8 Hz, 1H), 4.18 – 4.07 (m, 4H), 3.93 (dd, J = 13.6, 4.1 10 12
1
Hz, 2H), 3.84 (d, J = 13.7 Hz, 2H), 3.73 – 3.64 (m, 1H), 3.30 – 3.11 (m, 2H), 1.34 (t, J = 7.1 Hz, 14
13
3H), 1.33 (t, J = 7.1 Hz, 3H). 31P NMR (203 MHz, CDCl3) δ 27.7. MS (ESI+) m/z: 438.9 [M + 15 16
H]+. 17 19
18
Step 2: Building block 60, R1=f29, was isolated as a slightly yellow oil (22.1 mg). 1H NMR 21
20
(400 MHz, CDCl3) δ 8.52 (d, J = 4.7 Hz, 1H), 7.59 (td, J = 7.7, 1.8 Hz, 1H), 7.18 (d, J = 7.7 Hz, 2 23
1H), 7.13 (dd, J = 7.1, 5.2 Hz, 1H), 4.21 – 4.09 (m, 4H), 3.64 (td, J = 11.4, 3.3 Hz, 1H), 3.30 24 26
25
(ddd, J = 14.3, 7.7, 3.3 Hz, 1H), 2.87 (ddd, J = 14.4, 10.8, 8.8 Hz, 1H), 1.54 (br. s, 2H), 1.32 (t, 28
27
J = 7.1 Hz, 3H), 1.31 (t, J = 7.1, 3H). 31P NMR (162 MHz, CDCl3) δ 28.2 29 30 31 32
Diethyl (1-amino-2-(pyridin-3-yl)ethyl)phosphonate (60; R1=f30): 34
3
Method II - Step 1: The dibenzyl-protected amine intermediate was isolated as a yellow oil (33% 35 36
isolated yield): 1H NMR (500 MHz, CDCl3) δ 8.49 (dd, J = 4.7, 1.2 Hz, 1H), 8.32 (d, J = 1.5 Hz, 39
38
37
1H), 7.24 – 7.17 (m, 7H), 7.14 – 7.10 (m, 5H), 4.21 – 4.08 (m, 4H), 3.91 (d, J = 1.7 Hz, 4H), 41
40
3.28 (ddd, J = 16.4, 8.3, 6.3 Hz, 1H), 3.05 – 2.94 (m, 2H), 1.35 (t, J = 7.1 Hz, 3H), 1.34 (t, J = 42 4
43
7.1 Hz, 3H). 46
45
13
C NMR (126 MHz, CDCl3) δ 150.6, 147.6, 138.9, 136.7, 128.8, 128.2, 127.1,
123.1, 61.7 (d, J = 7.2 Hz), 61.3 (d, J = 7.6 Hz), 56.9 (d, J = 134.8 Hz), 54.7 (d, J = 2.3 Hz), 48
47
31.3 (d, J = 7.8 Hz), 16.7 (d, J = 5.6 Hz), 16.6 (d, J = 5.7 Hz). 31P NMR (203 MHz, CDCl3) δ 49 51
50
27.4. MS (ESI+) m/z: 438.9 [M + H]+. 53
52
Step 2: Building block 60, R1=f30, was isolated as a slightly yellow oil (quantitative yield). 1H 5
54
NMR (500 MHz, CDCl3) δ 8.45 (d, J = 1.9 Hz, 1H), 8.44 (dd, J = 4.8, 1.5 Hz, 1H), 7.54 (dt, J = 56 57 58 59
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7.8, 2.0 Hz, 1H), 7.19 (ddd, J = 7.8, 4.8, 0.7 Hz, 1H), 4.15 – 4.06 (m, 4H), 3.20 – 3.07 (m, 2H), 5
4
2.71 – 2.60 (m, 1H), 1.28 (t, J = 7.0 Hz, 3H), 1.27 (t, J = 7.0 Hz, 3H). 6
13
C NMR (126 MHz,
7
CDCl3) δ 150.6, 148.1, 136.7, 133.5 (d, J = 14.8 Hz), 123.3, 62.4 (d, J = 7.1 Hz), 62.3 (d, J = 10
9
8
7.1 Hz), 50.0 (d, J = 152.3 Hz), 35.1 (d, J = 1.2 Hz), 16.5 (d, J = 5.6 Hz). 31P NMR (202 MHz, 12
1
CDCl3) δ 27.3 13 14 15 16 17 19
18
Expression and purification of hFPPS 21
20
A pET-based plasmid (vector p11, SGC Oxford) encoding hFPPS with an N-terminal His-tag was 2 24
23
transformed into E. coli BL21 (DE3) cells. The cells were grown in LB at 37 °C until the OD600 26
25
of 0.6 was reached. Expression of the recombinant enzyme was induced overnight in the presence 27 28
of 1 mM IPTG at 18 °C. The cells were lysed in a buffer containing 50 mM HEPES (pH 7.5), 500 29 30
mM NaCl, 5 mM β-mercaptoethanol, 5 mM imidazole, and 5% (v/v) glycerol. The lysate was 3
32
31
cleared by centrifugation and applied to a metal ion affinity column (Ni-NTA Superflow 34 35
Cartridge, Qiagen), from which hFPPS was eluted with an increasing imidazole gradient. The 36 38
37
enzyme containing fractions were pooled and passed through a size-exclusion column (HiLoad 40
39
26/60 Superdex 200, GE Healthcare Life Sciences). Imidazole was removed from the sample 42
41
buffer during this step, which then consisted of 50 mM HEPES (pH 7.5), 500 mM NaCl, 5 mM β43 45
4
mercaptoethanol, and 5% (v/v) glycerol. The purified enzyme was concentrated to 20 mg/mL by 47
46
ultrafiltration and stored at 4 °C for later use. 48 49 50 51
Crystallization of hFPPS with bound allosteric inhibitors: 52 54
53
Inhibitor compounds were added to the purified hFPPS sample to give the final concentrations of 56
5
1-2 mM inhibitor and 0.25 mM protein. Crystal growth was achieved at 22˚C by vapor diffusion in 57 58 59
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sitting drops composed of 1 µL inhibitor/protein solution and 1 µL crystallization buffer. The 5
4
composition of the crystallization buffer was as follows: 0.6 M KH2PO4, 0.6 M NaH2PO4, 25% 6 8
7
glycerol, and 0.075 M HEPES (pH 7.5) for analogs 29 and 33; 0.425 M (NH4)2SO4, 0.85 M LiSO4, 10
9
15% glycerol, and 0.085 M sodium citrate (pH 5.6) for analog 38; 25.5% PEG 8K, 0.17 M sodium 1 12
acetate, 15% glycerol, and 0.085 M sodium cacodylate (pH 6.5) for analog 42; and 1.6 M 13 15
14
(NH4)2HPO4, 20% glycerol, and 0.08 M Tris (pH 8.5) for analog 49. We note that analogs 33, 38, 17
16
42, and 49 are chiral, and the racemic mixtures of these compounds were used for the crystallization 18 19
trials. 20 21 2 23 24 26
25
X-ray diffraction and structure refinement 28
27
Diffraction data were collected from single crystals under cryogenic conditions with synchrotron 29 31
30
radiation (Canadian Light Source, Saskatoon, SK) and/or with the home source (MicroMax-007 3
32
HF generator, Rigaku). The collected data were indexed and scaled with the xia2 program 35
34
package.43 The initial structure models were built by difference Fourier methods with the PDB entry 36 38
37
4XQR as the starting template. The models were improved through iterative rounds of manual and 40
39
automated refinement with Coot44 and REFMAC5.45 The final models were deposited to the PDB. 41 42
To measure the anomalous scattering by phosphorous, sulfur, and chloride atoms of the bound 43 45
4
compounds, the home source data sets were processed without merging Friedel mates. Anomalous 47
46
signal maps were calculated with the programs SHELXC46 and ANODE.47 The anomalous signal 48 50
49
maps as well as the discovery maps for the bound inhibitors are presented in Figure S1 (Supporting 52
51
Information). The ligand omit maps calculated with phases from the final models are shown in 54
53
Figure S2. Stereochemistry was clear from the refined electron density for inhibitors 33, 38 and 42. 5 57
56
The chirality of 29 was as best estimated based on the refined geometry of the compound as well 58 59
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as the enzyme inhibition data. Data collection and structure refinement statistics and the PDB IDs 5
4
for the final structure models are summarized in Table S1. 6 7 8 10
9
Associated Content 12
1
Supporting Information 13 14
The Supporting Information is available free of charge on the ACS Publications website at DOI: 15 17
16
NMR spectra of key final inhibitors from Table 1, examples of homogeneity data for key final 19
18
inhibitors and additional synthesis protocols for inhibitors (as indicated above) are provided 20 21
(PDF) 2 24
23
Molecular Formula Strings CSV) 26
25
Accession Codes 27 29
28
The PDB ID codes for the co-crystal structures with 29, 33, 38, 42, and 49 are 5KSX, 5JUZ, 31
30
5JV0, 5JV1, and 5JV2, respectively. Authors will release the atomic coordinates and 32 3
experimental data upon article publication. 34 35 36 38
37
Author Information 39 40
Corresponding Author 41 43
42
*Phone 514-398-3638. Fax 514-398-3797. Email:
[email protected] 4 45 46 48
47
Acknowledgments 50
49
Financial support for this work was provided by the Canadian Institute of Health Research (CIHR) 52
51
research grants to A.M. Berghuis and Y.S. Tsantrizos. 53 54 5 56 57 58 59
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4
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