Bisphosphonate esters interact with HMG-CoA reductase membrane domain to induce its degradation

Article abstract of DOI:10.1016/j.bmc.2020.115576

HMG-CoA reductase (HMGCR) is a rate-limiting enzyme in the cholesterol biosynthetic pathway, and its catalytic domain is the well-known target of cholesterol-lowering drugs, statins. HMGCR is subject to layers of negative feedback loops; excess cholesterol inhibits transcription of the gene, and lanosterols and oxysterols accelerate degradation of HMGCR. A class of synthetic small molecules, bisphosphonate esters exemplified by SR12813, has been known to induce accelerated degradation of HMGCR and reduce the serum cholesterol level. Although genetic and biochemical studies revealed that the accelerated degradation requires the membrane domain of HMGCR and Insig, an oxysterol sensor on the endoplasmic reticulum membrane, the direct target of the bisphosphonate esters remains unclear. In this study, we developed a potent photoaffinity probe of the bisphosphonate esters through preliminary structure–activity relationship study and demonstrated binding of the bisphosphonate esters to the HMGCR membrane domain. These results provide an important clue to understand the elusive mechanism of the SR12813-mediated HMGCR degradation and serve as a basis to develop more potent HMGCR degraders that target the non-catalytic, membrane domain of the enzyme.

Full text of DOI:10.1016/j.bmc.2020.115576

Journal Pre-proofs
Bisphosphonate esters interact with HMG-CoA reductase membrane domain
to induce its degradation
Yosuke Toyota, Hiromasa Yoshioka, Ikuya Sagimori, Yuichi Hashimoto,
Kenji Ohgane
PII:
DOI:
Reference:
S0968-0896(20)30406-5
https://doi.org/10.1016/j.bmc.2020.115576
BMC 115576
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Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 April 2020
24 May 2020
26 May 2020
Please cite this article as: Toyota, Y., Yoshioka, H., Sagimori, I., Hashimoto, Y., Ohgane, K., Bisphosphonate
esters interact with HMG-CoA reductase membrane domain to induce its degradation, Bioorganic & Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115576
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1
Bisphosphonate esters interact with HMG-CoA reductase
membrane domain to induce its degradation
Yosuke Toyota, Hiromasa Yoshioka, Ikuya Sagimori, Yuichi Hashimoto, Kenji Ohgane*, **
Institute for Quantitative Biosciences, The University of Tokyo; 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-0032, Japan
* Correspondence e-mail: ohgane@rs.tus.ac.jp
** Current address: Department of Applied Biological Science, Faculty of Science and
Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8519, Japan
Abstract
HMG-CoA reductase (HMGCR) is a rate-limiting enzyme in the cholesterol biosynthetic
pathway, and its catalytic domain is the well-known target of cholesterol-lowering drugs, statins.
HMGCR is subject to layers of negative feedback loops; excess cholesterol inhibits transcription
of the gene, and lanosterols and oxysterols accelerate degradation of HMGCR. A class of
synthetic small molecules, bisphosphonate esters exemplified by SR12813, has been known to
induce accelerated degradation of HMGCR and reduce the serum cholesterol level. Although
genetic and biochemical studies revealed that the accelerated degradation requires the
membrane domain of HMGCR and Insig, an oxysterol sensor on the endoplasmic reticulum
membrane, the direct target of the bisphosphonate esters remains unclear. In this study, we
developed a potent photoaffinity probe of the bisphosphonate esters through preliminary
structure-activity relationship study and demonstrated binding of the bisphosphonate esters to
the HMGCR membrane domain. These results provide an important clue to understand the
elusive mechanism of the SR12813-mediated HMGCR degradation and serve as a basis to
develop more potent HMGCR degraders that target the non-catalytic, membrane domain of the
enzyme.
Keywords
HMG-CoA reductase; Degradation; SR12813; SR45023A (apomine); Bisphosphonate esters;
Photoaffinity labeling
1. Introduction
HMG-CoA reductase (HMGCR) is a rate-limiting enzyme in the cholesterol biosynthetic
pathway, and is responsible for the conversion of 3-hydroxymethyl-glutaryl CoA to mevalonate,
1
which serves as an essential intermediate in synthesizing cholesterol.
Inhibitors of this

2
enzyme, a class of small molecules called statins, have been widely used to lower plasma
cholesterol levels, which is a key risk factor for cardiovascular diseases. Statins mimic the
partial structure of HMG-CoA to fit into the catalytic pocket of the HMGCR, and shut down the
catalytic activity of the enzyme, leading to the uptake of plasma low-density lipoprotein, which
contains a large amount of cholesterol and its esters, into the cells.
Cholesterol is an essential lipid for cells, and therefore, its abundance within cells is
tightly regulated via transcriptional and post-translational mechanisms. With less cholesterol,
transcription of relevant genes, including HMGCR and low-density lipoprotein receptor, is
upregulated via activation of a master transcriptional regulator, sterol-regulatory element binding
2
proteins (SREBPs).
In addition to such transcriptional feedback mechanisms, cells are
3
equipped with another post-translational mechanism that controls the stability of HMGCR.
Late-stage intermediates in the cholesterol biosynthesis, lanosterols, or oxygenated sterols
4,5
induce rapid degradation of HMGCR protein,
regulation, fine-tuning cholesterol homeostasis.
constituting another layer of feedback
Aside from the sterols, geranylgeranyl
6,7
pyrophosphate, a nonsterol metabolite downstream of mevalonate, is also involved in controlling
the endoplasmic reticulum (ER)-associated degradation of polyubiquitinated HMGCR, adding
another layer of complexity to the network of feedback mechanisms. 8,9
Sterol-induced degradation of HMGCR requires only the transmembrane, non-catalytic
domain of HMGCR, and an ER-resident oxysterol sensor Insig protein. 10-13 Oxysterol-binding to
Insig protein induces interaction of HMGCR and Insig, and Insig brings E3 ubiquitin ligases into
proximity of HMGCR, leading to accelerated ubiquitination and proteasomal degradation of
14-16
HMGCR.
Binding of oxysterols to Insig also triggers the transcriptional negative feedback
pathway, via formation of ternary complex of Insig, SREBP, and SREBP-cleavage activating
protein, which prevents proteolytic activation of SREBP and eventually inhibits transcription of
HMGCR and other SREBP target genes. In contrast to oxysterols, lanosterols only trigger
HMGCR degradation, and are postulated to act through a similar but distinct mechanism from
5
that of oxysterols. Considering the presence of sterol-sensing domain within the HMGCR
16,17
membrane domain,
which shares sequence similarity with the sterol-sensing domains of
18-22
23,24
sterol transporters Niemann-Pick type C1
and Ptch1,
a plausible hypothesis is that
lanosterols directly bind to the membrane domain of HMGCR to induce the formation of
HMGCR-Insig-E3 complex and subsequent degradation. 25,26
Similarly to lanosterols, a class of compounds with bisphosphonate esters, including
SR12813 and SR45023A (also known as apomine) (Fig. 1), has been shown to trigger
accelerated degradation of HMGCR without affecting SREBP pathway activity. 27 They were first
28-30
identified by their cholesterol-lowering effect,
and subsequent analyses revealed that the
bisphosphonates-induced degradation also requires the presence of the membrane domain of
31-33
HMGCR and Insig, suggesting the same mechanism as that of lanosterols.
Although the
lack of SREBP inhibitory activity points to the possibility of direct binding of the bisphosphonate
esters to the membrane domain of HMGCR, the direct interaction between the bisphosphonate
esters and HMGCR is yet to be proven. To evaluate their interaction, we aimed at developing a
photoaffinity probe of the bisphosphonate ester. As structure-activity relationship (SAR) of

3
SR12813 derivatives on HMGCR degradation activity has not been reported, we first performed
34
a preliminary SAR study using a luciferase-based HMGCR degradation assay. Through the
SAR, we identified a highly potent derivative, SRP3042, and successfully developed a potent
photoaffinity probe, srpDHY (Fig. 1). Using these compounds and the probe, we provide the
first experimental evidence supporting the direct interaction between the bisphosphonate ester
and the membrane domain of HMGCR.
Figure 1. Bisphosphonate esters that induce degradation of HMG-CoA reductase (HMGCR).
Here, we report SRP3042 as the most potent HMGCR degrader known to date and the
corresponding photoaffinity-labeling probe, srpDHY.
2. Materials and Methods
2.1 Materials
An expression vector (pCMV-Insig1-myc-DDK) that expresses human Insig1 tagged with myc
and DDK (FLAG) under the control of the CMV promoter (Cat# RC200312, NCBI Reference
Sequence NM_005542) was purchased from OriGene Technologies, Inc., Meryland, USA.
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from FUJIFILM Wako Pure
Chemical Industries, Japan.
2.2 Cell lines and cell culture
HEK293 cells were obtained from American ATCC and maintained in DMEM supplemented with
10% fetal bovine serum (FBS) and penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan).
HEK293 cells stably expressing HMGCR-dCat-ELuc, the membrane domain of HMGCR

4
(residue number 1-499) fused with a FLAG tag and emerald luciferase, were maintained in the
same medium in a humidified 5% CO2 incubator as previously described. 34
2.3 Synthesis of bisphosphonate esters
General methods. All chemical reagents and solvents were purchased from Sigma-Aldrich,
Kanto Chemical Industry, and Wako Pure Chemical Industries, and used without further
purification. Moisture-sensitive reactions were performed under an atmosphere of argon, unless
otherwise noted. Reactions were monitored by thin-layer chromatography (TLC; Merck silica gel
60 F254 plates). Bands were visualized using UV light, iodine vapors, acidic phosphomolybdic
acid stain, and/or basic permanganate stain. Flash column chromatography was performed with
silica gel (Silica gel 60 N, 40–50 μm particle size) purchased from Kanto Chemical. NMR
1
spectra were recorded on a JEOL JNM-ECA500 spectrometer at 500 MHz for H NMR, at 125
MHz for 13C NMR, and at 202 MHz for 31P NMR. Proton, carbon, and phosphorus chemical
shifts are expressed in δ values (ppm) relative to internal tetramethylsilane (0.00 ppm) or
1
residual CHCl3 (7.26 ppm) for H NMR, internal tetramethylsilane (0.00 ppm) or CDCl3 (77.16
ppm) for 13C NMR, and external triphenylphosphine (-5.65 ppm) for 31P NMR. Data are reported
as follows: chemical shift, multiplicity (s, singlet: d, doublet; t, triplet: q, quartet; m, multiplet: br,
broad), coupling constants (Hz), and integration. High-resolution mass spectra were recorded
using a Bruker micrOTOF II mass spectrometer. Room temperature or ambient temperature in
the following synthetic procedures represents a range of temperature from 15 to 25 °C.
Tetraethyl 2-(4-hydroxyphenyl)ethenylidene-1,1-bisphosphonate (2). The Knoevenagel
condensation of aldehydes and methylenediphosphonates was performed following the method
35
reported by Lehnert et al. To a solution of 4-hydroxybenzaldehyde 1 (248 mg, 2.03 mmol) in
dry tetrahydrofuran (THF) (6 mL) was added TiCl4 (0.69 mL, 6.1 mmol) slowly at 0 °C under Ar.
Tetraethyl methylenediphosphonate (0.710 mL, 2.84 mmol) was added to the resulting mixture,
followed by the dropwise addition of N-methylmorpholine (1.34 mL, 12.2 mmol) at 0 °C. The
reaction was allowed to warm up to ambient temperature and stirred for 3 h. The reaction was
cooled to 0 °C and quenched by adding ice tips, followed by saturated aqueous NH4Cl solution.
The mixture was extracted with EtOAc, and the organic layer was washed with water and brine,
dried over MgSO4, and concentrated under reduced pressure. The residue was purified by flash
column chromatography (EtOAc/CH2Cl2/MeOH = 10:10:0 to 10:10:1) to afford the title
1
compound (732 mg, 1.87 mmol, 92%) as a pale yellow oil. H-NMR (CDCl3, 500 MHz, δ): 9.64
(s, 1H), 8.24 (dd, J = 49.0, 29.5 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 4.21–
4.15 (m, 4H), 4.12–4.06 (m, 4H), 1.37 (t, J = 7.2 Hz, 6H), 1.24 (t, J = 7.2 Hz, 6H). 13C-NMR
(CDCl3, 125 MHz, δ): 162.80, 161.13, 133.83 (2C), 125.14 (dd, J = 21.6, 8.4 Hz), 115.64 (2C),
113.05 (dd, J = 173.9, 169.1 Hz), 62.73 (d, J = 6.0 Hz, 2C), 62.57 (d, J = 6.0 Hz, 2C), 16.26 (d,
J = 6.0 Hz, 2C), 16.04 (d, J = 6.0 Hz, 2C). 31P-NMR (CDCl3, 202 MHz, δ): 18.62 (d, J = 51.2 Hz,
1P), 13.82 (d, J = 51.2 Hz, 1P). HRMS–ESI (m/z): [M - H]- calcd for C16H26O7P2, 391.1081;
found, 391.1093.

5
3-tert-Butyl-4-hydroxybenzaldehyde (4). To oxidize the methyl group on 2-tert-butyl-p-cresol,
the DDQ/MeOH conditions were applied. 36 Note that the DDQ oxidation of this particular cresol
has been reported to give poor yield of the aldehyde (< 5%), owing to oxidative coupling of the
37
cresol.
A cobalt-catalyzed aerobic oxidation (1 mol% Co(OAc)2, 1 atm O2, 6 eq NaOH in
ethylene glycol, 80 °C for 10 h) was also tested; however, the yield was quite low (4.2%).38
A solution of 2-tert-butyl-p-cresol 3 (386 mg, 2.35 mmol) in MeOH (3 mL) was treated
with a solution of DDQ (1068 mg, 4.70 mmol) in MeOH (5 mL), and the resulting mixture was
stirred at ambient temperature for 3 h. After evaporation of the solvent, the residue was
suspended in CHCl3/MeOH (10:1) and filtered through a silica gel pad to remove insoluble
materials. Purification by flash column chromatography (hexane/EtOAc = 5:1 to 4:1) gave
aldehyde 4 (76.4 mg, 0.429 mmol, 18%) as a pale brown oil. Rf = 0.25 (hexane/EtOAc = 2:1).
1H-NMR (CDCl3, 500 MHz, δ): 9.85 (s, 1H), 7.84 (d, J = 2.3 Hz, 1H), 7.64 (dd, J = 8.0, 2.3 Hz,
1H), 6.83 (d, J = 8.6 Hz, 1H), 6.28 (br s, 1H), 1.44 (s, 9H). 13C-NMR (CDCl3, 125 MHz, δ):
191.64, 160.41, 137.05, 130.09, 129.64, 129.61, 117.00, 34.70, 29.28 (3C).
Tetraethyl 2-(3-tert-butyl-4-hydroxyphenyl)ethenylidene-1,1-bisphosphonate (5). To a
solution of 4 (85.2 mg, 0.478 mmol) in dry THF (4 mL) was added TiCl4 (0.160 mL, 1.46 mmol)
slowly at 0 °C under Ar. To the resulting mixture was added tetraethyl methylenebisphophonate
(0.119 mL, 0.478 mmol), followed by dropwise treatment with N-methylmorpholine (0.315 mL,
2.87 mmol) at 0 °C. The reaction was allowed to warm up to ambient temperature and stirred for
18 h. The reaction was cooled to 0 °C, and quenched by adding ice tips followed by saturated
aqueous NH4Cl solution. The mixture was extracted with EtOAc, and the organic layer was
washed with water and brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (EtOAc/CH2Cl2/MeOH =
100:100:2 to 100:100:5 to 100:100:10) to afford the title compound (46.8 mg, 0.104 mmol, 22%)
1
as red oil. Rf = 0.25 (EtOAc/CH2Cl2/MeOH = 10:10:1). H-NMR (CDCl3, 500 MHz, δ): 9.46 (s,
1H), 8.25 (dd, J = 49.0, 29.5 Hz, 1H), 7.79 (dd, J = 8.6, 2.3 Hz, 1H), 7.57 (d, J = 2.3 Hz, 1H),
6.89 (d, J = 8.6 Hz, 1H), 4.22–4.15 (m, 4H), 4.13–4.08 (m, 4H), 1.37 (t, J = 7.2 Hz, 6H), 1.37 (s,
9H), 1.24 (t, J = 6.9 Hz, 6H). 13C-NMR (CDCl3, 125 MHz, δ): 163.79, 160.25, 135.80, 132.56,
130.91, 124.59 (dd, J = 21.6, 8.4 Hz), 116.84, 111.34 (dd, J = 174.5, 169.7 Hz), 62.63 (d, J =
6.0 Hz, 2C), 62.48 (d, J = 4.8 Hz, 2C), 34.67, 29.25 (3C), 16.29 (d, J = 7.2 Hz, 2C), 16.05 (d, J =
31
7.2 Hz, 2C). P-NMR (CDCl3, 202 MHz, δ): 19.36 (d, J = 52.0 Hz, 1P), 14.34 (d, J = 52.0 Hz,
1P). HRMS–ESI (m/z): [M - H]- calcd for C20H34O7P2, 447.1707; found, 447.1722.
Tetraethyl 2-(3,5-di-tert-butylphenyl)ethenylidene-1,1-bisphosphonate (7). To a solution of
3,5-di-tert-butylbenzaldehyde 6 (218 mg, 1.00 mmol) in dry THF (2 mL) was added TiCl4 (0.329
mL, 3.00 mmol), tetraethyl methylenediphosphonate (0.348 mL, 1.40 mmol), N-
methylmorpholine (0.625 mL, 5.68 mmol) at 0°C under argon atmosphere. After stirring for 4 h,
saturated aqueous NH4Cl was added. THF was removed under reduced pressure. The residue
was extracted with EtOAc and washed brine, combined organic layers were dried over Na2SO4,

6
and concentrated under reduced pressure. The crude product was purified by flash column
chromatography on silica gel (CHCl3/MeOH = 19:1) to afford the title compound 7 (142 mg,
1
0.291 mmol, 29%) as a colorless oil. H-NMR (500 MHz, CDCl3) δ 8.33 (dd, J = 48.1, 29.2 Hz,
1H), 7.64 (d, J = 1.7 Hz, 2H), 7.47 (s, 1H), 4.27–4.15 (m, 4H), 3.95-4.08 (m, 4H), 1.38 (t, J = 7.2
Hz, 6H), 1.33 (s, 18H), 1.11 (t, J = 7.2 Hz, 6H). 13C-NMR (CDCl3, 125 MHz, δ): 162.93, 150.64
(2C), 133.76 (dd, J = 22.2, 7.8 Hz), 125.31 (2C), 124.97, 119.21 (t, J = 168.5 Hz), 62.70 (d, J =
6.0 Hz, 2C), 62.30 (d, J = 7.2 Hz, 2C), 35.06 (2C), 31.45 (6C), 16.45 (d, J = 7.2 Hz, 2C), 16.04
(d, J = 7.2 Hz, 2C). 31P-NMR (CDCl3, 202 MHz, δ): 18.71 (d, J = 54.5 Hz, 1P), 13.03 (d, J = 54.5
Hz, 1P).
Tetraethyl 2-(4-hydroxy-3,5-di-tert-butylphenyl)ethan-1,1-bisphosphonate (8). To a solution
of SR12813 (33.6 mg, 0.0666 mmol) in EtOH (1 mL) was added LiBH4 (20.1 mg, 0.923 mmol)
slowly at 0 °C, and the reaction mixture was stirred for 1 h at ambient temperature. Additional
portion of LiBH4 (24.2 mg, 1.11 mmol) was added at the temperature, and the bright yellow
suspension was stirred for 16 h. The reaction was cooled to 0 °C, and quenched by adding
saturated aqueous NH4Cl solution. The colorless mixture was extracted with EtOAc, and the
organic layer was washed with water and brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by flash silica gel column chromatography
(EtOAc/CH2Cl2/MeOH = 50:50:1 to 10:10:1) to afford the title compound (21.0 mg, 0.0415 mmol,
62%) as colorless oil. Rf = 0.25 (EtOAc/CH2Cl2/MeOH = 10:10:1), slightly more polar than the
1
starting material. H-NMR (CDCl3, 500 MHz, δ): 7.07 (s, 2H), 5.08 (s, 1H), 4.14–4.04 (m, 8H),
3.17 (td, J = 16.6, 6.3 Hz, 2H), 2.64 (tt, J = 24.1, 6.3 Hz, 1H), 1.42 (s, 18H), 1.27 (t, J = 7.2 Hz,
6H), 1.25 (t, J = 7.2 Hz, 6H). 13C-NMR (CDCl3, 125 MHz, δ): 152.29, 135.57 (2C), 130.08 (t, J =
7.2 Hz), 125.44 (2C), 62.46 (d, J = 6.0 Hz, 2C), 62.31 (d, J = 6.0 Hz, 2C), 39.47 (t, J = 131.4
31
Hz), 34.24 (2C), 31.05 (t, J = 4.8 Hz), 30.27 (6C), 16.29 (d, J = 7.1 Hz, 4C). P-NMR (CDCl3,
202 MHz, δ): 23.05. HRMS–ESI (m/z): [M - H]- calcd for C24H44O7P2, 505.2490; found,
505.2483.
Tetraisopropyl
2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenylidene-1,1-bisphosphonate
(10). To a solution of 3,5-di-tert-butyl-4-hydroxybenzaldehyde 9 (469 mg, 2.0 mmol) in dry THF
(4 mL) was added TiCl4 (0.658 mL, 6.0 mmol), tetraisopropyl methylenediphosphonate (0.910
mL, 2.8 mmol), and N-methylmorpholine (1.25 mL, 11.4 mmol) at 0°C under argon atmosphere.
After stirring for 4 h at room temperature, saturated aqueous NH4Cl was added, and THF was
removed under reduced pressure. The residue was extracted with EtOAc, and the organic layer
was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The
crude product was purified by flash column chromatography on silica gel (EtOAc /Hexane = 4:1)
to afford the title compound 10 (831.5 mg, 1.483 mmol, 74%) as a pale yellow solid. The spectra
obtained were consistent with those in the previous report. 39 1H-NMR (500 MHz, CDCl3) δ 8.20
(dd, J = 48.1, 30.4 Hz, 1H), 7.77 (s, 2H), 5.60 (s, 1H), 4.83–4.74 (m, 2H), 4.72–4.63 (m, 2H),
1.44 (s, 18H), 1.39 (d, J = 5.7 Hz, 6H), 1.35 (d, J = 6.3 Hz, 6H), 1.22 (d, J = 5.7 Hz, 6H), 1.16 (d,
J = 5.7 Hz, 6H). 13C-NMR (CDCl3, 125 MHz, δ): 161.29, 156.84, 135.58 (2C), 130.26 (2C),

7
125.92 (dd, J = 21.6, 8.4 Hz), 117.30 (t, J = 169.1 Hz), 71.25 (d, J = 6.0 Hz, 2C), 71.13 (d, J =
6.0 Hz, 2C), 34.69 (s, 2C), 30.43 (s, 6C), 24.17–24.27 (m, 6C), 23.76 (d, J = 6.0 Hz, 2C).
Tetraisopropyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethan-1,1-bisphosphonate (12). To a
solution of tetraisopropyl methylenediphosphonate (347 mg, 1.01 mmol) in dry THF (5 mL) was
added nBuLi (0.61 mL, 0.958 mmol) at -78 °C under argon atmosphere. After 10 min, to the
solution was added dropwise a solution of 3, 5-di-tert-butylbenzyl bromide 11 (90.8 mg, 0.321
mmol) and tetrabutylammonium iodide (TBAI) (22.7 mg, 0.0615 mmol), and the reaction was
stirred at room temperature for 16 h. The reaction mixture was diluted with water and extracted
with EtOAc, and the organic layer was washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure. The crude product was purified by flash column
chromatography (EtOAc/CH2Cl2/MeOH = 100:100:0 to 100:100:1 to 100:100:5) to afford the title
compound (126.5 mg, 0.231 mmol, 72%) as a colorless oil. 1H-NMR (CDCl3, 500 MHz, δ): 7.25
(s, 1H), 7.10 (s, 2H), 4.79–4.70 (m, 4H), 3.21 (td, J = 16.5, 6.0 Hz, 2H), 2.59 (tt, J = 24.0, 6.0
Hz, 1H), 1.31–1.29 (m, 30H), 1.23 (d, J = 6.3, 6H), 1.20 (d, J = 6.3, 6H). 13C-NMR (CDCl3, 125
MHz, δ): 150.36, 138.97 (t, J = 7.2 Hz, 2C), 123.17 (2C), 120.28, 71.11 (t, J = 3.6 Hz, 2C),
70.83 (t, J = 3.0 Hz, 2C), 40.89 (t, J = 133.8 Hz), 34.75 (2C), 32.00 (t, J = 4.8 Hz), 31.49 (6C),
24.17 (4C), 23.88 (m, 2C), 23.70 (m, 2C). 31P-NMR (CDCl3, 202 MHz, δ): 21.05 (s, 2P).
Tetraisopropyl 2-(-2,6-di-tert-butylpyridine)ethan-1,1-bisphosphonate (SRP3042). Step 1.
40
The intermediate 15 was prepared as reported previously, with slight modifications.
To a
solution of 2,6-di-tert-buty-4-methylpyridine 13 (190 mg, 0.925 mmol) in dichloroethane (DCE)
(3 mL) was added N-bromosuccinimide (NBS) (171 mg, 0.961 mmol, 1.04 eq) and 2,2’-
azobis(isobutyronitrile) (AIBN) (30.3 mg, 0.187 mmol, 0.2 eq) at room temperature, and the
reaction was heated to reflux for 1 h with a fluorescent light turned on. An additional portion of
AIBN was added, and the reaction was further refluxed for 1h. Then the reaction mixture was
cooled and diluted with hexane (9 mL). Precipitates was filtered off, and the filtrate was
concentrated under reduced pressure to afford the crude 4-bromomethyl-2,6-di-tert-
butylpyridine 14 (232 mg) as a colorless oil. This crude compound 14 was used in the next
1
reaction without further purification. H-NMR (CDCl3, 500 MHz, δ): 7.08 (s, 2H), 4.38 (s, 2H),
1.34 (s, 18H).
Step 2. To a solution of tetraisopropyl methylenediphosphonate (0.526 mL, 1.62 mmol)
in dry THF (4 mL) was added nBuLi (1.55 M in hexane, 0.94 mL, 1.46 mmol) at -78 °C under
argon atmosphere. After 10 min, a solution of the crude 4-bromomethyl-2,6-di-tert-butylpyridine
14 (230 mg) and TBAI (62.1 mg, 0.168 mmol) in dry THF (4 mL) was added dropwise. The
reaction was stirred at room temperature for 16 h, and partitioned between water and EtOAc.
The organic layer was separated, washed with water and brine, dried over MgSO4, and
concentrated under reduced pressure. Purification by flash column chromatography
(EtOAc/CH2Cl2/MeOH = 100:100:1) gave the title compound (222 mg, 0.405 mmol, 44% over 2
steps) as a pale yellow oil. 1H-NMR (CDCl3, 500 MHz, δ): 6.99 (s, 2H), 4.80–4.71 (m, 4H), 3.15
(td, J = 16.3, 6.3 Hz, 2H), 2.56 (tt, J = 24.1, 6.3 Hz, 1H), 1.33 (s, 18H), 1.31 (d, J = 6.3 Hz, 12H),

8
13
1.23 (d, J = 6.3 Hz, 6H), 1.22 (d, J = 6.3 Hz, 6H). C-NMR (CDCl3, 125 MHz, δ): 167.30 (2C),
148.46 (t, J = 7.2 Hz), 116.01 (2C), 71.37 (t, J = 3.5 Hz, 2C), 71.06 (t, J = 3.5 Hz, 2C), 40.02 (t,
J = 135.0 Hz), 37.50 (2C), 31.67 (t, J = 4.8 Hz), 30.17 (6C), 24.15 (4C), 23.92–23.86 (m, 2C),
23.76–23.71 (m, 2C). 31P-NMR (CDCl3, 202 MHz, δ): 20.47 (s, 2P). Copies of the NMR spectra
can be found in the Supplementary Materials.
3-Iodo-5-tert-butyltoluene (16). The title compound was prepared by following the reported
41
procedure. A 100 mL flask was charged with 3-iodotoluene 15 (10.87 g, 49.9 mmol) and 2-
chloro-2-methylpropane (6.90 g, 74.5 mmol), and cooled to 0°C under argon atmosphere. To
the cooled flask was added AlCl3 (199 mg, 1.49 mmol), and the reaction was stirred at the
temperature for 20 min. To the dark red solution was added ice tips, water, and saturated
aqueous NaHCO3, and the mixture was extracted with CH2Cl2. The dark purple organic layer
was washed with saturated aqueous Na2S2O3 and brine, dried over MgSO4, and concentrated
under reduced pressure. The liquid was purified by vacuum distillation (7 mmHg, fractions
collected between vapor temperature of 110-120°C) to afford the title compound (7.74 g, 28.2
1
mmol, 57%) as a faintly orange liquid which partially crystallizes upon cooling to 4 °C. H-NMR
(CDCl3, 500 MHz, δ): 7.50 (s, 1H), 7.36 (s, 1H), 7.13 (s, 1H), 2.29 (s, 3H), 1.28 (s, 9H). 13C-
NMR (CDCl3, 125 MHz, δ): 153.42, 139.72, 135.15, 131.67, 125.64, 94.56, 34.60, 31.19, 21.20
(3C).
Tetraisopropyl 2-(3-iodo-5-tert-butyl-4-hydroxyphenyl)ethan-1,1-bisphosphonate (18).
Step 1. To a solution of 3-Iodo-5-tertbutyl-methybenzene 16 (1.40 g, 5.10 mmol) in
dichloroethane (10 mL) was added NBS (954 mg, 5.36 mmol, 1.05eq) and AIBN (167 mg, 1.02
mmol, 0.2eq) at room temperature, and the reaction was heated to refulx for 1 h with a
fluorescent light turned on. An additional portion of AIBN was added, and the reaction was
further refulxed for 1.5 h. Then the reaction mixture was cooled. Precipitates was filtered off,
and the filtrate was concentrated under reduced pressure. The crude product was purified by
flash column chromatography on silica gel (EtOAc/Hexane = 1:20) to afford crude 3-iodo-5-tert-
butylbenzyl bromide 17 (975 mg) as a colorless oil. The crude mixture also contains the starting
material in approximately 40 mol% and corresponding dibromide in 10 mol% relative to the
desired monobromide 17. 1H-NMR (CDCl3, 500 MHz, δ): 7.62 (s, 1H), 7.56 (s, 1H), 7.33 (s, 1H),
4.39 (s, 2H), 1.27–1.33 (m, 18H).
Step 2. To a solution of tetraisopropyl methylenediphosphonate (1.79 mL, 5.52 mmol)
in dry THF (11 mL) was added nBuLi (1.55 M in hexane, 3.21 mL, 4.97 mmol) at -78 °C under
argon atmosphere. After 10 min, a solution of 17 (975 mg) and TBAI (192 mg, 0.52 mmol) in dry
THF (11 mL) was added dropwise. The reaction was stirred at room temperature for 24 h. Then
water was added, and the solution was extracted with EtOAc and washed with brine, combined
organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The
crude product was purified by flash column chromatography on silica gel (EtOAc/CH2Cl2 = 1:1 to
EtOAc/CH2Cl2/MeOH = 10:10:1) to afford the title compound 18 (396 mg, 0.643 mmol, 13% over
1
2 steps) as a pale yellow oil. H-NMR (CDCl3, 500 MHz, δ): 7.52 (t, J = 1.7, 1H), 7.43 (s, 1H),

9
7.21 (s, 1H), 4.78–4.69 (m, 4H), 3.12 (td, J = 16.1, 5.8 Hz, 2H), 2.45 (tt, J = 24.1, 6.3 Hz, 1H),
1.31–1.25 (m, 33H). 13C-NMR (CDCl3, 125 MHz, δ): 153.44, 142.10 (t, J = 7.2 Hz), 135.42,
132.63, 125.67, 94.29, 71.44 (t, J = 3.6 Hz, 2C), 71.18 (t, J = 3.6 Hz, 2C), 40.91 (t, J = 133.5
Hz), 34.78, 31.46 (t, J = 4.8 Hz), 31.30 (3C), 24.27 (4C), 23.99 (d, J = 2.4 Hz, 2C), 23.87 (d, J =
2.4 Hz, 2C). 31P-NMR (CDCl3, 202 MHz, δ): 21.34 (s, 2P). HRMS–ESI (m/z): [M +Na]+ calcd for
C24H43O6P2I, 639,1427; found, 639.1445
Tetraisopropyl 2-(3-ethnyl-5-tert-butylphenyl)ethan-1,1-bisphosphonate (19). Step 1. To a
solution of 19 (123.5 mg, 0.200 mmol) in THF (2.0 mL) was added TMS-acetylene (69.2 μL,
0.500 mmol), triethylamine (69.3 μL, 0.500 mmol), PdCl2(PPh3) (11.2 mg, 0.016 mmol), and CuI
(1.5 mg, 0.0080 mmol) at room temperature. After stirring for 24 h, water was added and the
mixture was extracted with CH2Cl2. The organic layer was dried over Na2SO4, and concentrated
under reduced pressure to give the crude TMS-protected intermediate as a brown oil (108.8
1
mg). H-NMR (CDCl3, 500 MHz, δ): 7.31 (s, 1H), 7.24 (s, 2H), 4.79–4.72 (m, 4H), 3.16 (td, J =
16.6, 6.3 Hz, 2H), 2.49 (tt, J = 24.1, 6.3 Hz, 1H), 1.31–1.24 (m, 33H), 0.23 (s, 9H).
Step 2. To a solution of the intermediate (108.8 mg) in MeOH (2.0 mL) was added
K2CO3 (3.0 mg, 0.0217 mmol) at room temperature. After stirring for 24 h, water was added to
the solution, and the mixture was extracted with CH2Cl2. The organic layer was washed with
brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was
purified by flash column chromatography on silica gel (EtOAc/CH2Cl2 = 1:1 to
EtOAc/CH2Cl2/MeOH = 10:10:1) to afford the title compound 19 (22.2 mg, 0.0431 mmol, 22%
1
over 2 steps) as a pale pink oil. H-NMR (CDCl3, 500 MHz, δ): 7.35 (s, 1H), 7.27 (s, 1H), 7.24
(s, 1H), 4.70–4.79 (m, 4H), 3.18 (td, J = 16.6, 6.3 Hz, 2H), 3.01 (s, 1H), 2.50 (tt, J = 24.1, 6.3
Hz, 1H), 1.37–1.27 (21H), 1.27-1.23 (m, 4H), 1.19 (s, 3H). 13C-NMR (CDCl3, 125 MHz, δ):
151.24, 140.02, 130.17, 127.29, 127.06, 121.41, 84.38, 76.23, 71.42 (d, J = 3.6 Hz), 71.10 (d, J
= 7.2 Hz), 40.86 (t, J = 135.0 Hz), 34.70, 31.69 (t, J = 4.8 Hz), 31.31 (3C), 24.25 (4C), 23.99
(2C), 23.84 (2C). 31P-NMR (CDCl3, 202 MHz, δ): 21.47 (s, 2P).
Tetraisopropyl 2-(3-azido-5-tert-butylphenyl)ethan-1,1-bisphosphonate (20)
To a solution of 18 (116.4 mg, 0.189 mmol) in EtOH (2.7 mL) and water (1.8 mL) was added
NaN3 (24.5 mg, 0.378 mmol), trans-N,N’-dimethylcyclohexane-1,2-diamine (0.045 mL, 0.028
mmol), CuI (3.6 mg, 0.019 mmol), and L-sodium ascorbate (1.9 mg, 0.0095 mmol), and then the
solution was degassed by sonication under argon atmosphere. The solution was refluxed for 40
h, diluted with water, and extracted with CHCl3 and EtOAc. The combined organic layers were
dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified
by flash column chromatography on silica gel (EtOAc/CH2Cl2/MeOH = 10:10:1) to afford the title
1
compound 20 (75.3 mg, 0.142 mmol, 75%) as a pale yellow oil. H-NMR (CDCl3, 500 MHz, δ):
7.06 (s, 1H), 6.84 (s, 1H), 6.80 (s, 1H) 4.81–4.70 (m, 4H), 3.18 (td, J = 16.6, 6.3 Hz, 2H), 2.49
13
(tt, J = 36.7, 5.8 Hz, 1H), 1.31–1.25 (m, 33H). C-NMR (CDCl3, 125 MHz, δ): 153.19, 141.78,
139.41, 123.08, 116.93, 114.40, 71.22 (t, J = 3.6 Hz, 2C), 71.18 (t, J = 3.6 Hz, 2C), 40.86 (t, J =
133.5 Hz), 34.92, 31.46 (t, J = 4.8 Hz), 31.32 (3C), 24.27 (4C), 23.99 (d, J = 2.4 Hz, 2C), 23.87

10
(d, J = 2.4 Hz, 2C). 31P-NMR (CDCl3, 202 MHz, δ): 21.49 (s, 2P). ESI-MS (m/z): [M +Na]+
554.24.
3-Azido-5-ethynylbenzyl alcohol (22). A solution of methyl 3-azido-5-ethynylbenzoate 21,
19
which was prepared as previously described, in dry THF (6 mL) was treated dropwise with
diisobutylaluminium hydride (DIBAL) (1.0 M in hexane, 2.4 mL, 2.4 mmol) at 0 °C under argon
atmosphere. After 1 h at the temperature, the reaction was quenched by sequentially adding
MeOH, water, EtOAc, and 1M aqueous HCl. The organic layer was separated, washed with
water and brine, dried over MgSO4, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (hexane/EtOAc = 2:1) to afford the title compound 22
1
(17.3 mg, 0.100 mmol, 21%) as a pale yellow oil. H-NMR (CDCl3, 500 MHz, δ): 7.26 (s, 1H),
7.07 (s, 1H), 7.05 (s, 1H), 4.68 (s, 2H), 3.11 (s, 1H), 1.77 (br s, 1H). 13C-NMR (CDCl3, 125 MHz,
δ): 143.07, 140.61, 126.74, 123.83, 121.50, 117.76, 82.42, 78.19, 64.22.
3-Azido-5-ethynylbenzyl bromide (23). A solution of 23 (17.0 mg, 0.0982 mmol) in CHCl3 was
treated dropwise with a solution of PBr3 (0.1 mM in CHCl3, 0.423 mL, 0.0423 mmol) at 0 °C
under argon atmosphere, and the reaction was stirred at the temperature for 30 min. To the
reaction was added an additional portion of PBr3 (0.192 mmol), and the mixture was further
stirred at room temperature for 30 min. The reaction was quenched with saturated aqueous
NaHCO3, and extracted with CHCl3. The organic layer was washed with brine, dried over
MgSO4, and purified by flash column chromatography (hexane/EtOAc = 5:1) to afford the title
compound 23 (20.6 mg, 0.0873 mmol, 89%) as a yellow oil. 1H-NMR (CDCl3, 500 MHz, δ): 7.28
(t, J = 1.4 Hz, 1H), 7.08 (dd, J = 2.0, 1.4 Hz, 1H), 7.03 (t, J = 1.7 Hz, 1H), 4.40 (s, 2H), 3.13 (1H,
s). 13C-NMR (CDCl3, 125 MHz, δ): 140.85, 139.86, 129.06, 124.25, 122.28, 120.09, 81.95,
78.68, 31.57.
Tetraisopropyl 2-(3-azido-5-ethynylphenyl)ethan-1,1-bisphosphonate (24). To a solution of
tetraisopropyl methylenediphosphonate (0.0890 mL, 0.274 mmol) in dry THF (2.0 mL) was
added nBuLi (1.54 M in hexane, 0.16 mL, 0.25 mmol) at -78 °C under argon atmosphere. After
10 min, a solution of 23 (19.6 mg, 0.0830 mmol) and TBAI (6.5 mg, 0.018 mmol) in dry THF (2.0
mL) was added dropwise. The reaction was stirred at room temperature for 16 h. Then water
was added, and the solution was extracted with EtOAc, and the organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified
by flash column chromatography (EtOAc/CH2Cl2/MeOH = 100:100:0 to 100:100:5) to afford the
1
title compound 24 (5.4 mg, 0.011 mmol, 13%) as a pale brown oil. H-NMR (CDCl3, 500 MHz,
δ): 7.19 (s, 1H), 6.98 (s, 1H), 6.93 (s, 1H), 4.65–4.74 (m, 4H), 4.09–4.13 (m, 1H), 3.15 (td, J =
16.3, 6.0 Hz, 2H), 3.06 (s, 1H), 2.43 (tt, J = 24.1, 6.0 Hz, 1H), 1.30-1.31 (m, 12H), 1.27 (d, J =
6.3 Hz, 6H), 1.25 (d, J = 6.3 Hz, 6H). 31P-NMR (CDCl3, 202 MHz, δ): 21.04 (s, 2P).
Hydrogen triisopropyl 2-(2,6-di-tert-butylpyridine)ethan-1,1-bisphosphonate (25) To a
solution of SRP3042 (323.9mg, 0.591 mmol) in MeCN (1.0 mL) was added LiBr (51.4 mg, 0.591

11
mmol) at room temperature, and the solution was refluxed for 31 h. To the reaction was added 1
M aqueous HCl was added, and the mixture was extracted with CH2Cl2. The organic layer were
washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude
product was purified by flash column chromatography on silica gel (EtOAc/CH2Cl2/MeOH =
5:5:1 to EtOAc/MeOH = 4:1) to afford the title compound 25 (120.4 mg, 0.238 mmol, 46%) as a
1
white solid. H-NMR (CDCl3, 500 MHz, δ): 6.93 (s, 2H), 4.91–4.87 (m, 1H), 4.55 (s, 1H), 4.41–
4.35 (m, 1H), 3.34–3.23 (m, 1H), 2.95–2.86(m, 1H), 1.44–1.14 (m, 30H), 0.89 (d, J = 5.8 Hz,
3H), 0.84 (d, J = 5.7 Hz, 3H). 13C-NMR (CDCl3, 125 MHz, δ): 167.00 (2C), 149.59, 116.36 (2C),
71.97 (d, J = 6.0 Hz), 70.45 (d, J = 6.0 Hz), 68.17 (d, J = 4.8 Hz), 40.35 (t, J = 123.6 Hz), 37.48
(2C), 32.42, 30.25 (6C), 23.43–24.61 (m, 6C). 31P-NMR (CDCl3, 202 MHz, δ): 27.63 (s, 1P),
12.71 (s, 1P).
Propargyl triisopropyl 2-(2,6-di-tert-butylpyridine)ethan-1,1-bisphosphonate (26). To a
solution of compound 25 (64.1 mg, 0.127 mmol) in 1.0 mL CH2Cl2 was added oxalyl chloride
(43.8 μL, 0.51 mmol) and DMF (4.0 μL, 0.051 mmol) at 0 °C, and the reaction was stirred for 30
min. Then the reaction was allowed to warm up to room temperature, and further stirred for 1 h.
The solution was concentrated under reduced pressure to obtain a crude intermediate, which
was used in the next reaction without further purification. To the solution of the intermediate in
1.5 mL CH2Cl2 was added 2-propyn-1-ol (15.0 μL, 0.254 mmol) and triethylamine (70.0 μL,
0.508 mmol) at 0 °C, and the reaction was stirred for 24 h at room temperature. To the reaction
was added 1 M aqueous HCl, and the mixture was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel (EtOAc/Hexane = 6:1) and
preparative thin-layer chromatography (PTLC) (EtOAc/MeOH = 10:1, silica gel 60 PLC glass
plate, 0.5 mm thickness from Merck Millipore) to afford the title compound (6.1 mg, 0.011 mmol,
8.8%) as a colorless oil. Note that NMR spectra indicated the presence of a pair of
diastereomers with 1.3 : 1 ratio. The major isomer was marked as “a” and the minor one as “b”
1
in the following spectra. H-NMR (CDCl3, 500 MHz, δ): 6.98 (s, 2H, b), 6.97 (s, 2H, a), 4.54–
4.85 (m, 5H+5H, a+b), 3.19–3.11 (m, 2H+2H, a+b), 2.72–2.58 (m, 1H+1H, a+b), 2.49 (t, J = 2.3
Hz, 1H, b), 2.47 (t, J = 2.3 Hz, 1H, a), 1.17–1.36 (m, 36H+36H, a+b). 13C-NMR (CDCl3, 125
MHz, δ): 167.51 (s, 2C, b), 167.49 (s, 2C, a), 148.15 (d, J = 7.2 Hz, 1C, b), 148.05 (d, J = 7.2
Hz, 1C, a), 116.03 (s, 2C, b), 116.01 (s, 2C, a), 78.83 (d, J = 7.2 Hz, 1C, a), 78.56 (d, J = 7.2
Hz, 1C, b), 75.37 (s, 1C, b), 75.16 (s, 1C, a), 72.07 (d, J = 7.2 Hz, 1C, b), 71.90 (d, J = 7.2 Hz,
1C, a), 71.78 (d, J = 7.2 Hz, 1C, b), 71.57 (d, J = 7.2 Hz, 1C, a), 71.36 (d, J = 6.0 Hz, 1C, b),
71.27 (d, J = 7.2 Hz, 1C, a), 54.77 (d, J = 6.0 Hz, 1C, a), 54.08 (d, J = 6.0 Hz, 1C, b), 39.84 (t, J
= 134.4 Hz, 1C, b), 39.74 (t, J = 134.4 Hz, 1C, a), 37.59 (s, 2C+2C, a+b), 31.54–31.43 (m,
1C+1C, a+b), 30.25 (s, 6C+6C, a+b), 23.77–24.35 (m, 6C+6C, a+b). 31P-NMR (CDCl3, 202
MHz, δ): 24.00 (d, J = 6.2 Hz, 1P, a), 23.60 (s, 1P, b), 20.80 (d, J = 3.1 Hz, 1P, b), 20.65 (d, J =
4.7 Hz, 1P, a). HRMS–ESI (m/z): [M + Na]+ calcd for C27H47O6NP2,566.2771; found,566.2770.

12
3-Butyn-1-yl triisopropyl 2-(2,6-di-tert-butylpyridine)ethan-1,1-bisphosphonate (27). To a
solution of 25 (55.4 mg, 0.110 mmol) in 1.0 mL CH2Cl2 was added oxalyl chloride (37.7 μL, 0.44
mmol) and DMF (3.4 μL, 0.044 mmol) at 0 °C, and the reaction was stirred at the temperature
for 30 min then at rt for 1 h. The solution was concentrated under reduced pressure, and the
crude intermediat dissolved in 1.5 mL CH2Cl2 was treated with 3-butyn-1-ol (16.6 μL, 0.22
mmol) and triethylamine(61.0 μL, 0.44 mmol) at 0 °C. The reaction was stirred for 24 h at room
temperature, and 1 M aqueous HCl was added to the solution. The mixture was extracted with
EtOAc, and the organic layer was washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash column chromatography
(EtOAc/Hexane = 6:1) and PTLC (EtOAc/MeOH = 10:1) to afford the title compound 27 (5.7 mg,
0.010 mmol, 9.3%) as a colorless oil. Note that NMR spectra indicated the presence of a pair of
diastereomers in 1:0.75 ratio. The major isomer was marked as “a” and the minor one as “b” in
1
the following spectra. H-NMR (CDCl3, 500 MHz, δ): 6.98 (s, 2H, b), 6.97 (s, 2H, a), 4.65–4.84
(m, 3H+3H, a+b), 4.07–4.23 (m, 2H+2H, a+b), 3.12–3.23 (m, 2H+2H, a+b), 2.69–2.56 (m,
2H+2H, a+b), 2.45–2.50 (m, 2H+2H), 1.96 (t, J = 2.6 Hz, 1H, b), 1.95 (t, J = 2.6 Hz, 1H, a),
13
1.20-1.35 (m, 36H+36H, a+b). C-NMR (CDCl3, 125 MHz, δ): 167.51 (s, 2C+2C, a+b), 148.27
(t, J = 7.8 Hz, 1C+1C, a+b), 116.01 (s, 2C+2C, a+b), 80.10 (s, 1C, a), 79.98 (s, 1C, b), 71.27-
71.80 (m, 3C+3C, a+b), 70.20 (s, 1C, b), 70.09 (s, 1C, a), 64.70 (d, J = 6.0 Hz, 1C, b), 64.09 (d,
J = 7.2 Hz, 1C, a), 39.72 (d, J = 135.0 Hz, 1C, b), 39.58 (d, J = 135.0 Hz, 1C, a), 37.61 (s,
2C+2C, a+b), 31.65–31.60 (m, 1C+1C, a+b), 30.26 (s, 6C+6C, a+b), 23.77–24.30 (m, 6C+6C,
31
a+b), 20.93 (d, J = 7.2 Hz, 2C+2C, a+b). P-NMR (CDCl3, 202 MHz, δ): 22.90 (d, J = 6.0 Hz,
1P, a), 22.57 (d, J = 3.1 Hz, 1P, b), 21.05 (d, J = 3.1 Hz, 1P, b), 20.92 (d, J = 6.0 Hz, 1P, a).
HRMS–ESI (m/z): [M + Na]+ calcd for C28H49O6NP2,580.2927; found, 580.2917.
2-(3-But-3-ynyl-3H-diazirin-3-yl)-ethyl p-toluensulfonate (29). The title compound 29 was
42
prepared as previously described.
2-(3-But-3-ynyl-3H-diazirin-3-yl)ethanol 28 (22 μL, 0.172
mmol) was dissolved in 150 μL CH2Cl2 at 0 °C. After the addition of pyridine (41.6 μL, 0.516
mmol), Et3N (71.5 μL, 0.516 mmol), and DMAP (1 mg) to the solution, a solution of p-
toluenesulfonyl chloride (98.3 mg, 0.516 mmol) in 150 μL CH2Cl2 was added dropwise over 10
min, and the mixture was stirred at room temperature for 24 h. After addition of 1 M aqueous
HCl, the mixture was extracted twice with diethyl ether. The organic layer was concentrated,
and the residue was purified by flash column chromatography (hexane/EtOAc = 3:1) to afford
the title compound (23.7 mg, 0.0811 mmol, 47%) as a yellow oil. 1H-NMR (CDCl3, 500 MHz, δ):
7.81 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 3.90 (t, J = 6.3 Hz, 2H), 2.45 (s, 3H), 1.94–
1.97 (m, 3H), 1.77 (t, J = 6.3 Hz, 2H), 1.60 (t, J = 7.2 Hz, 2H).
2-(3-But-3-ynyl-3H-diazirin-3-yl)ethyl triisopropyl 2-(2,6-di-tert-butylpyridine)ethan-1,1-
bisphosphonate (srpDHY). Compound 25 (61.7 mg, 0.122 mmol) and tosylate 29 (23.7 mg,
0.081 mmol) were dissolved in a mixture of CH2Cl2 (0.60 mL) and DMF (0.60 mL), and Cs2CO3
(79.2 mg, 0.243 mmol) was added to the solution. The reaction mixture was stirred at 60 °C for
72 h and quenched with 1 M aqueous HCl. The mixture was extracted with EtOAc, and the

13
organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced
pressure. The crude product was purified by flash column chromatography (EtOAc/hexane =
6:1) to afford the title compound srpDHY (7.6 mg, 0.012 mmol, 15%) as a colorless oil. Note
that NMR spectra indicated the presence of a pair of diastereomers in 1:0.75 ratio. Although the
separation of the major/minor isomers was unclear and their assignment can be ambiguous, the
major isomer was tentatively marked as “a” and the minor one as “b” in the following spectral
1
data, where possible. H-NMR (CDCl3, 500 MHz, δ): 6.99 (s, 2H, b), 6.98 (s, 2H, a), 4.67–4.81
(m, 3H+3H, a+b), 3.92–4.02 (m, 2H+2H, a+b), 3.11–3.20 (m, 2H+2H, a+b), 2.57–2.67 (m,
1H+1H, a+b), 2.00 (td, J = 7.6, 2.7 Hz, 2H, b), 1.99 (td, J = 7.6, 2.7 Hz, 2H, a), 1.95 (t, J = 2.7
Hz, 1H, a), 1.94 (t, J = 2.7 Hz, 1H, b), 1.64-1.77 (m, 4H+4H, a+b), 1.20–1.34 (m, 36H+36H,
a+b). 13C-NMR (CDCl3, 125 MHz, δ): 167.52 (s, 2C+2C, a+b), 148.18–148.36 (m, 1C+1C, a+b),
116.00 (s, 2C+2C, a+b), 82.63 (s, 1C+1C, a+b), 71.24–71.92 (m, 3C+3C, a+b), 69.37 (s, 1C, a),
69.32 (s, 1C, b), 61.70 (d, J = 6.0 Hz, 1C, b), 61.13 (d, J = 6.0 Hz, 1C, a), 39.70 (t, J = 134.4 Hz,
1C, a), 39.54 (t, J = 134.4 Hz, 1C, b), 37.61 (s, 2C+2C, a+b), 34.20 (s, 1C, a), 34.14 (s, 1C, b),
32.24 (s, 1C, a), 32.21 (s, 1C, b), 31.58-31.67 (m, 1C+1C, a+b), 30.27 (s, 6C+6C, a+b), 26.24
(s, 1C, a), 26.22 (s, 1C, b), 23.72–24.28 (m, 6C+6C, a+b), 13.33 (s, 1C+1C, a+b). 31P-NMR
(CDCl3, 202 MHz, δ): 23.03 (d, J = 4.7 Hz, 1P, a), 22.75 (d, J = 3.5 Hz, 1P, b), 20.97 (d, J = 3.5
Hz, 1P, b), 20.87 (d, J = 4.7 Hz, 1P, a). HRMS–ESI (m/z): [M + Na]+ calcd for C31H53O6N3P2,
648.3302; found, 648.3295. Copies of the NMR spectra can be found in the Supplementary
Materials.
2.4 Luciferase-based HMGCR degradation assay
HMGCR degradation activity of SR12813 derivatives was tested using the HEK293 cell line
34
stably expressing HMGCR-dCat-ELuc, as previously described.
Briefly, the HMGCR-dCat-
ELuc expressing cells cultured on a white 96-well clear-bottom plate (Greiner, product number
655098) were treated with test compounds for 4 h, and the remaining amount of HMGCR-dCat-
ELuc proteins were quantified using luciferase assay. For each experiment, luminescence data
was normalized with vehicle (0.1% DMSO)-treated samples and expressed as % of control. To
confirm that the compound treatment does not compromise cell viability, CytoRed viability
assay, which detects cellular esterase activity, was performed from the same samples within the
43
wells just before luciferase activity measurement.
tested did not show cytotoxicity unless otherwise noted.
The compounds at the concentrations
2.5 Photoaffinity labeling of HMGCR-dCat-ELuc and Insig
The protocol for the photoaffinity labeling experiments were adopted from our previous report
and modified slightly. 19,44 HEK293 cells stably expressing HMGCR-dCat-ELuc were cultured to
about 70% confluency, and transiently transfected with pCMV-Insig1-myc-DDK using
Lipofectamine 3000 following the manufacturer’s instructions. After 6 h, the culture medium was
replaced with fresh medium with additional 6 µM compactin, which was added to increase the

14
expression level of HMGCR-dCat-ELuc. After 16 h of compactin treatment, cells were scraped
19,44
into ice-cold PBS and membrane preparation was performed as described previously.
The
membrane fraction was treated with 20 µM srpDHY for 30 min on ice in the absence or
presence of a competitor SRP3042, and irradiated with UV light (approximately 1 cm from the
sample, 365 nm, LED365-SPT/L from OptoCode, Tokyo, Japan) for 3 min on ice. The
membrane fractions were then solubilized and denatured with 1% SDS and 1% Triton X-100,
and biotin was conjugated onto the alkyne on the srpDHY via copper-catalyzed azide-alkyne
45
cycloaddition (CuAAC) reaction. The reaction mixtures were diluted 10-fold and subjected to
anti-FLAG immunoprecipitation with anti-FLAG M2 magnetic beads (4 °C, overnight). The beads
were washed, and eluted with a mixture of TNET, membrane protein solubilization buffer, (62.5
mM Tris pH 6.8, 8 M urea, 100 mM DTT, 15% SDS, 10% glycerol), and Laemmli sample buffer
33,34,46,47
(2:2:1) at 37 °C for 30 min.
The eluted samples were then resolved on SDS-PAGE
(SuperSep Ace 10-20% gel, FUJIFILM-Wako Pure Chemicals), and proteins were transferred
onto a PVDF membrane (Immobilon-P, Millipore). The membrane was probed with ImmunoPure
streptavidin-HRP (Pierce, 1/12000 dilution in TBST) or anti-FLAG M2 antibody (Sigma-Aldrich,
1/2000 dilution in CanGetSignal signal enhancer solution) as previously described. 44
3. Results
3.1 Pharmacophore analysis of SR12813 derivatives as HMGCR degraders
Although the rational design of photoaffinity probes from SR12813 requires detailed SAR
data regarding HMGCR degradation activity, no direct comparison of SR12813 derivatives on
HMGCR degradation activity has been performed until now, and only limited information on
29
cholesterol synthesis inhibitory activity is available. Therefore, we started to evaluate SAR of
SR12813 derivatives on HMGCR degradation activity, aiming at rationally designing
photoaffinity probes.
First, to gain insight into where we could modify and append functional groups required for
photoaffinity labeling probes, we examined the requirement of the bulky tert-butyl groups and
the phenolic hydroxyl group on SR12813 (Scheme 1 and Table 1). Removal of either one or
both of the tert-butyl groups resulted in the complete loss of HMGCR degradation activity
(compare SR12813, 2, and 5), clearly indicating the importance of these tert-butyl groups. In
contrast, the phenolic hydroxyl group was dispensable, as exemplified by the comparable
activity of the compound 7 and SR12813. Although the dispensability of the hydroxyl group
suggested a possibility that we can introduce some functional groups on it, our attempts to
alkylate or acylate the hydroxyl group failed to give sufficient conversion of SR12813.

15
Scheme 1. Synthesis of SR12813 derivatives listed in Table 1
Table 1. Requirement of the tert-butyl groups and dispensability of the phenolic hydroxyl group
for HMG-CoA reductase (HMGCR) degradation activity
EC50 (µM)
(mean ± SD)a
Compounds
R1
R2
R3
SR12813
1.10 ± 0.35
2
5
7
NAb
NAb
1.16 ± 0.15
aThe EC50 values represent mean ± SD from more than three independent dose-response experiments.
bNA, no activity at 10 µM.
Next, we examined the importance of the double bond in SR12813 and alkyl groups (Et or
iPr) on the bisphosphonate esters. Of the four combinations tested (Scheme 2 and Table 2),
SR45023A, which lacks the double bond and has the iPr group on the esters, was found to

16
possess slightly more potent HMGCR degradation activity than SR12813. At higher
concentrations, the bisphosphonates with the double bond tended to be slightly more toxic than
their counterparts with the single bond. Therefore, we selected SR45023A (single bond and iPr
esters) as a new starting point for the next round of SAR examination.
Scheme 2. Synthesis of SR12813 derivatives listed in Table 2
Table 2. HMG-CoA reductase (HMGCR) degradation activity of bisphosphonate esters with
double or single bond and with Et or iPr esters
EC50 (µM)
(mean ± SD)a
Compounds
R
SR12813
1.10 ± 0.35
8
2.77 ± 0.25
1.34 ± 0.14
10
SR45023A
0.876 ± 0.086
aThe EC50 values represent mean ± SD from more than three independent dose-response experiments.

17
Considering the dispensability of the phenolic hydroxyl group on SR12813, we
removed the hydroxyl group from SR45023A to find 3-fold improvement in activity (compound
12 in Scheme 3 and Table 3). Furthermore, when the aromatic C-H was replaced with pyridine
nitrogen (Table 3, SRP3042), an additional 2-fold improvement in HMGCR degradation activity
was observed. Although the reason for the improvement is not clear at this point, differences in
the hydrogen bond donor/acceptor property, steric effect, or electronic effect on the aromatic
ring might contribute to this improvement. As far as the authors are aware, this nitrogen-
containing bisphosphonate ester SRP3042 is the most potent HMGCR degrader reported to
date.
Scheme 3. Synthesis of SRP3042, an SR45023A derivative with a pyridine nitrogen
Table 3. Removal of the phenolic hydroxyl group and introduction of nitrogen into SR45023A
improves HMG-CoA reductase (HMGCR) degradation activity
EC50 (µM)
(mean ± SD)a
Compounds
A
SR45023A
0.876 ± 0.086
12
0.317 ± 0.106
0.153 ± 0.032
SRP3042
aThe EC50 values represent mean ± SD from more than three independent dose-response experiments.

18
3.2 Photoaffinity bisphosphonate esters with HMGCR degradation activity
As is clear from the initial analysis shown in Table 1, the presence of the tert-butyl
group was essential for the HMGCR degradation activity. Here, we examined if replacement of
one or both of the tert-butyl groups with an ethynyl group or an azide, which can serve as a
clickable handle or photo-reactive group, can be tolerated. As shown in Scheme 4 and Table 4,
replacement of one tert-butyl group on compound 12 resulted in 4- to 11-fold lower activity (12,
18-20), and replacement of both groups gave an inactive compound 24.
Scheme 4. Synthesis of the bisphosphonate ester 6 derivatives with tert-butyl group(s) replaced
with probe units, an alkyne and/or an azide
Table 4. Attempts to introduce functional groups required for photoaffinity probes at the tert-
butyl groups
EC50 (µM)
(mean ± SD)a
Compounds
R1
R2
12
0.317 ± 0.106
18
1.46 ± 0.30

19
19
20
24
3.54 ± 1.45
1.66 ± 0.38
NAb
aThe EC50 values represent mean ± SD from more than three independent dose-response experiments.
bNA, no activity at 10 µM.
We next focused our attention on an alkyl group on the bisphosphonate esters, and
introduced alkyne-containing alkyl groups or a minimalist photo-crosslinker that contains both
48
diazirine, a photo-reactive group, and a terminal alkyne (Scheme 5 and Table 5).
Modifications at one of the phosphonate ester were relatively well tolerated (4- to 6-fold loss of
activity for compounds 25, 26, and 27), and we obtained a photoaffinity probe srpDHY with
minimal loss of HMGCR degradation activity (3-fold loss). The loss owing to modification of the
alkyl group was compensated by the improved activity of SRP3042, overall giving a more potent
bisphosphonate ester srpDHY than the original SR12813 or SR45023A (Fig. 2A).
Scheme 5. Synthesis of SRP3042 derivatives with probe units introduced on one of the
phosphonate esters
Table 5. Effect of modifications on the phosphonate ester moiety and identification of a
photoaffinity probe srpDHY
EC50 (µM)
(mean ± SD)a
Compounds
R
25
NAb

20
SRP3042
26
0.153 ± 0.032
0.633 ± 0.150
0.795 ± 0.257
0.417 ± 0.052
27
srpDHY
aThe EC50 values represent mean ± SD from more than three independent dose-response experiments.
bNA, no activity at 10 µM.
3.3 Direct interaction of the bisphosphonate esters with the membrane domain of
HMGCR
With the potent photoaffinity probe srpDHY in hand (Fig. 2A), we set out to test the
interaction between the bisphosphonate and HMGCR or Insig. The membrane fraction was
isolated from the cells expressing both HMGCR-dCat-ELuc tagged with the FLAG tag and
Insig1 tagged with myc and FLAG tags, and the membrane fraction was labeled with the
srpDHY probe in the absence or presence of a competitor, SRP3042. After immunoprecipitation
of both the FLAG-tagged HMGCR-dCat-ELuc and Insig1, the extent of srpDHY-labeling was
examined via biotin introduced on the alkyne through CuAAC reaction (Fig. 2B). UV irradiation
resulted in selective labeling of the HMGCR membrane domain over Insig1, and the extent of
the labeling was reduced by competition with SRP3042, supporting specific interaction between
the bisphosphonate ester and the HMGCR membrane domain.
Figure 2. Photoaffinity labeling of HMGCR and Insig with the srpDHY probe. (A) Dose-
dependent degradation of HMGCR by SRP3042 and srpDHY probes, in comparison with that
by SR45023A. Data represent mean ± SD of more than three independent dose-response
experiments. (B) Photoaffinity labeling of HMGCR-dCat-ELuc and Insig1 by srpDHY probe and
its competition by SRP3042. Cells stably expressing HMGCR-dCat-FLAG-ELuc were transiently
transfected with pCMV-Insig1-myc-FLAG, and the membrane fraction was isolated after pre-

21
treatment with 6 µM compactin for 16 h to facilitate detection of HMGCR. The membrane
fraction was labeled with srpDHY with or without competition from SRP3042, and biotin was
introduced onto the alkyne via click chemistry. After immunoprecipitation of both HMGCR-dCat-
FLAG-ELuc and Insig1-myc-FLAG, the presence of biotin and FLAG tag was probed.
4. Discussion
In this study, we identified a potent photoaffinity probe srpDHY based on a more potent
SR12813/SR45023A derivative SRP3042 and evaluated its binding to the HMGCR membrane
domain. Our data showed that the bisphosphonate esters preferentially interacted with the
HMGCR membrane domain over co-expressed Insig1. Although Insig1 was also slightly labeled
with srpDHY in a UV- and competition-dependent manner, a most likely interpretation would be
that complex formation of HMGCR and Insig1 brought the proteins in close proximity, within
which the photo-activated probe labeled the other protein. Alternatively, we cannot exclude a
possibility that the bisphosphonate esters bind to the interface of the HMGCR and Insig1,
resulting in labeling of both proteins, with the limited spatial resolution of the photoaffinity
labeling technique. Further studies would be needed to clarify the remaining questions: where
the bisphosphonate esters binds to within the membrane domain of HMGCR, how the
compound is recognized, and how the binding of the bisphosphonate esters triggers the
sequence of events that leads to degradation of HMGCR.
Statins have remained to be effective drugs in reducing serum cholesterol level since
1,49
their discovery in the early 1970s.
However, prolonged use of statins sometimes results in
massive increase in HMGCR protein level through SREBP pathway activation and cancellation
15,25
of sterol-induced degradation.
Such compensatory increase gives rise to statin resistance,
necessitates increased dose of statins, and concomitantly leads to increased risk of their side
effects. 47 The increased level of HMGCR also worsens the clinical outcome if statin treatment is
50
interrupted or discontinued, a phenomenon called rebound or withdrawal syndrome.
Therefore, the combined use of HMGCR degraders along with statins is expected to be
synergistically effective in reducing serum cholesterol, decreasing atherosclerosis, and reducing
the risk associated with the statin-induced upregulation of HMGCR proteins. 47
Additionally, a form of immune-mediated necrotizing myopathy, which has been linked
to statin use, is characterized by the presence of autoantibodies against HMGCR and is
51
reported to be progressive even after discontinuation of statin treatment. HMGCR degraders
might be useful in reducing HMGCR proteins and thereby suppressing immune reactions
against HMGCR, although this possibility needs to be experimentally validated.
Overall, through preliminary SAR study starting from SR12813, we identified a more
potent HMGCR degrader SRP3042 and developed a potent photoaffinity probe srpDHY.
Labeling experiments with the probe indicated direct binding of the bisphosphonate esters to the
membrane domain of HMGCR, augmenting our mechanistic understanding of the
bisphosphonate ester-mediated degradation of HMGCR. Additionally, our finding of SRP3042,
the most potent HMGCR degrader reported to date, offers a promising avenue to find better

22
small molecules that may lead to improved treatment of hypercholesterolemia, atherosclerosis,
and immune-mediated necrotizing myopathy.
Acknowledgements
We thank the members of our laboratory for helpful discussions, and Professor Koji Kuramochi
at the Tokyo University of Science for his support. The work described in this article was
supported in part by the Grant-in-Aid for Young Scientists B and Grant-in-Aid for Young
Scientists for K.O., Grants-in-Aid for JSPS Research Fellow for H.Y. and Y.T., and Grants-in-Aid
for Scientific Research B for Y.H. (JSPS KAKENHI Grant number JP17K15487, 20K15949,
JP18J14851, JP18J15022, and JP17H03996). We would like to thank Editage
(www.editage.com) for English language editing.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which
may be considered as potential competing interests:

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