Pharmacophore Mapping of Thienopyrimidine-Based


Feb 16, 2017 - A number of non-bisphosphonate allosteric inhibitors of hFFPs (e.g., compounds 3–5), with good potency in vitro, have been reported, ...
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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

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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

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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



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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2

(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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2

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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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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(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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REFERENCES 5

4

1. Nguyen, U.T.T.; Guo, Z.; Delon, C.; Wu, Y.; Deraeve, C.; Fränzel, B.; Bon, R.S.; 6 8

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Table of Contents Graphic 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 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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