ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126633

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.

Full text of DOI:10.1016/j.bmcl.2019.126633

Bioorganic&MedicinalChemistryLetters29(2019)126633
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors
Nazmul H. Bhuiyana, Michelle L. Varneyb, Deep S. Bhattacharyac, William M. Paynec,
⁎
Aaron M. Mohsc,d,e, Sarah A. Holsteinb,e, David F. Wiemera,f,
a Department of Chemistry, University of Iowa, Iowa City, IA 52242-1294, United States
b Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198, United States
c Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, United States
d Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, United States
e Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, United States
f Department of Pharmacology, University of Iowa, Iowa City, IA 52242-1109, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple mye-
loma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS
results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our
previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective in-
hibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new
analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular ac-
tivity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS in-
hibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid
resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodis-
tribution of HA-conjugated GGDPS inhibitors.
GGDP synthase
Inhibition
Isoprenoid biosynthesis
Triazole
Bisphosphonate
Geranylgeranyl diphosphate (1, GGDP, Fig. 1) represents an im-
portant branch point in isoprenoid biosynthetic pathways. This inter-
mediate is used in plants to afford a tremendous variety of cyclic di-
terpenoids,1 and in mammals it is used primarily for post-translational
modification of proteins.2 Among the proteins modified by reaction
with GGDP are those in the Ras superfamily of small GTPases such as
the Rho proteins which play roles in cancer cell migration and metas-
tasis,3 and the Rab proteins which are essential for intracellular traf-
ficking processes.4
standard doses of statins do not alter protein prenylation8,9 and the
nitrogenous bisphosphonates in clinical use do not have sufficient sys-
temic exposure.10
For some time there has been interest in identification of com-
pounds that directly inhibit the enzyme geranylgeranyl diphosphate
synthase (GGDPS)11,12 to disrupt geranylgeranylation more specifi-
cally.11,13 We and others have focused on the development of GGDPS
inhibitors as anti-myeloma agents because disruption of Rab ger-
anylgeranylation results in impairment of monoclonal protein traf-
ficking within myeloma cells, leading to ER stress and cell death.12,14,15
In this context, we have reported the preparation and biological activity
of a number of compounds with significant activity against this enzyme.
The first was digeranyl bisphosphonate (2, DGBP),16 which showed an
IC50 of ∼260 nM for GGDPS and good selectivity over the related en-
zyme farnesyl diphosphate synthase,17 but cellular activity only at high
concentrations (∼10 μM). Crystallographic studies indicated that
DGBP’s V-shaped structure occupied the enzyme’s active site, with the
phosphonates complexed to magnesium ions and the two isoprenoid
side chains occupying the FPP and GGPP channels.18 More recently we
have focused on monoalkyl compounds assembled through click
chemistry and incorporating a triazole ring system. These efforts first
Isoprenoid biosynthesis in humans already is targeted by at least
two families of blockbuster drugs, the statins and the nitrogenous bi-
sphosphonates. Statins target the enzyme 3-hydroxy-3-methylglutaryl
coenzyme A reductase (HMGCR) which is the rate-limiting step in the
mevalonate pathway to higher isoprenoids, and these drugs are widely
used as cholesterol-lowering agents and to prevent cardiovascular dis-
ease.5 The nitrogenous bisphosphonates target the later enzyme far-
nesyl diphosphate synthase (FDPS), and are widely used to treat os-
teoporosis and other diseases of the bone.6 In vitro, both classes of drugs
can exert anti-cancer activities as a consequence of disruption of protein
prenylation, particularly geranylgeranylation.7 These drugs are not
ideal in the setting of systemic anti-cancer therapy however, because
⁎ Corresponding author.
E-mail address: david-wiemer@uiowa.edu (D.F. Wiemer).
https://doi.org/10.1016/j.bmcl.2019.126633
Received 26 July 2019; Accepted 20 August 2019
Availableonline20August2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.

N.H. Bhuiyan, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126633
O
P
O
P
studies have demonstrated that HA can target both to CD44-over-
expressed solid tumors34–36 and to cells of hematological malig-
nancies37 including myeloma cells.38 As an initial foray into this area,
we describe the synthesis and biological activity of two ω-hydroxy
triazole bisphosphonates. Furthermore, we demonstrate that the ω-OH
modification allows linkage to HA via ester formation and report the
cellular activity of the first HA-GGDPS inhibitor conjugate.
NaO
O
O
NaO NaO
1 (GGDP)
2 (DGBP)
As an initial test of this strategy, the first target chosen was the
triazole bisphosphonate 15 (Scheme 1) which was viewed as reasonably
accessible. The synthesis of this compound started with selenium di-
oxide catalyzed allylic oxidation of commercially available geranyl
acetate (7).39 While oxidation provided a mixture of the desired alcohol
and the corresponding aldehyde, treatment with NaBH4 to reduce that
aldehyde increased the yield of the desired alcohol 8 to an acceptable
level. After protection of the free alcohol 8 as the TBS ether 9, base
catalyzed hydrolysis of the acetate afforded compound 10.40 Conver-
sion to the bromide 1141 was accomplished in near-quantitative yield
by reaction with PBr3. Reaction of the bromide 11 with sodium azide
proceeded cleanly, but gave the allylic azide as a mixture of E and Z
isomers due to a well known [3,3] sigmatropic rearrangement.42 The
azide then was allowed to react with the acetylene 1343 to afford the
TBS protected triazole, which was immediately carried to acidic hy-
drolysis to afford the desired alcohol 14. Standard McKenna hydro-
lysis44 of the tetraethyl ester 14 by treatment with TMSBr followed by
NaOH provided the tetra-sodium salt 15. Based on the 1H NMR spec-
trum of this product, the E/Z ratio was found to be ∼2:1 in favor of the
E isomer.
(NaO)2P P(ONa)2
O O
O
(NaO)2P
N
(NaO)2P
O
N
N
3
4
O
(NaO)2P
N
H
(NaO)2P
N
N
O
O
(NaO)2P
N
CH3
(NaO)2P
N
N
N
O
O
5
6
The second synthetic target in this series was the ω-hydroxy ana-
logue of compound 6, which required a longer synthesis but is one of
the compounds with better cellular activity. This compound could be
envisioned as arising from homonerol through a sequence parallel to
that used to obtain compound 15. However, our previous route to
homonerol employed an 8-step sequence45 and while it gave iso-
merically pure material, to avoid an even longer sequence an alter-
native route to homonerol was developed. For this route, nerol (16) first
was oxidized to neral (17) under either of two reaction conditions
(Scheme 2). A TEMPO oxidation46 gave the desired aldehyde in just
four hours while an MnO2 oxidation required several days but gave the
same aldehyde in nearly quantitative yield. Wittig olefination of neral
produced the isomerically pure triene 18 in high yield. Regioselective
hydroboration-oxidation of the terminal double bond in the triene 18
gave homonerol (19) in modest yield,47 but the brevity of this reaction
sequence made that acceptable.
(NaO)2P
N
CH3
(NaO)2P
O
N
Fig. 1. Geranylgeranyl diphosphate and known inhibitors of geranylgeranyl
diphosphate synthase.
yielded the inhibitor 3, which shows enzyme activity at 2.2 μM and
cellular activity ∼1 μM.19 Surprisingly, as a mixture of olefin isomers
the homologue 4 showed an IC50 of 45 nM against GGDPS, high spe-
cificity for GGDPS over FDPS, and cellular activity at concentrations as
low as 30 nM in multiple myeloma cells.20,21 After preparation of the
individual isomers 5 and 6, and methylation of the alpha position,
bioassays revealed these compounds had IC50 values of 125 and 86 nM
respectively, and cellular activity at 20 and 25 nM levels.21
Once homonerol (19) was in hand, multiple attempts to accomplish
a SeO2 oxidation went unrewarded. Therefore, the reaction sequence
employed in Scheme 1 was reorganized so that the selenium dioxide
oxidation could be pursued at a later stage. Treatment of homonerol
(19) with methanesulfonyl chloride and subsequent reaction of the
mesylate with sodium bromide gave homoneryl bromide (20) in good
yield.21 At this stage of the sequence, the selenium dioxide mediated
allylic oxidation48 was modestly successful, and furnished the desired
alcohol 21 in ∼25% yield. While this yield might still be improved,
once the alcohol 21 was in hand replacement of bromide with azide was
performed and the product immediately was carried to the next step.
For this click reaction, the alkyne 23 was synthesized by a known
procedure from tetraethyl vinyl bisphosphonate (22) through a two-
step process.21 The click reaction then was conducted under standard
conditions to afford the ester 24. McKenna hydrolysis of the phospho-
nate esters provided the desired salt 25.
The potency of these GGDPS inhibitors in both enzyme and cell
assays is relevant from a therapeutic perspective. Preclinical studies of
compounds 4–6 have shown systemic distribution, prolonged half-lives
and metabolic stability,22,23 all of which are important drug-like fea-
tures. The dose-limiting toxicity for these compounds has been hepatic
in nature22,23 and it would be desirable to enhance their anti-myeloma
activity by optimizing drug delivery to the target organs of interest and
thereby minimize off-target effects. A prodrug approach might help in
this regard,24–27 but it may be preferable to utilize GGDPS inhibitors
that can be conjugated to other agents. Hyaluronic acid (HA), a non-
sulfated glycosaminoglycan, can be readily optimized for delivery of
varied cargoes including small molecule chemotherapeutic agents,28–30
and has thus far found clinical use in ophthalmology, rheumatology and
wound healing applications.31,32 Furthermore, work done with fluor-
escent dyes has revealed that conjugation of the dye to HA can lead to
improved tumor uptake relative to surrounding tissue and alter bio-
distribution profiles.33 HA is the native ligand for CD44 and multiple
Once the two new triazoles 15 and 25 were available, they were
tested for their ability to disrupt protein geranylgeranylation in cell
assays and to inhibit GGDPS in enzyme assays. The activity of the new
ω-hydroxy compounds was compared directly to their respective parent
2

N.H. Bhuiyan, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126633
Scheme 1. Synthesis of compound 15, the ω-hydroxy analogue of compound 3.
compounds 3 and 6. As shown in Fig. 2, the disruption of protein
geranylgeranylation was evaluated using two methods: 1) immunoblot
analysis for unmodified Rap1a (a representative substrate for ger-
anylgeranyl transferase (GGTase) I; and, 2) ELISA for intracellular
lambda light chain levels as a marker for disruption of Rab ger-
anylgeranylation.14 Lovastatin was included as a positive control.14
Both compounds 15 and 25 induced concentration-dependent effects in
these assays, consistent with GGDPS inhibitory activity. In both cases,
the addition of the ω-OH group did modestly diminish cellular potency,
with compound 15 approximately 10-fold less potent than 3 and
compound 25 approximately 10-fold less potent than 6. Enzyme assays
utilizing recombinant GGDPS and FDPS confirmed the specificity of
these new compounds as GGDPS inhibitors (Table 1).
In conclusion, after introduction of the ω-hydroxyl group to geranyl
acetate (7), and its immediate protection as a TBS ether, the synthetic
sequence to compound 15 closely parallels our earlier synthesis of
compound 3. In contrast, the preparation of compound 25 employs a
different synthetic sequence for preparation of homonerol (19), a se-
quence that is just three steps long rather than the eight used pre-
viously. As a result, this intermediate was available in sufficient
quantity that the disappointing yield for the selenium dioxide oxidation
to afford compound 21 could be tolerated. The remaining steps in the
sequence then run parallel to our earlier synthesis of compound 6.
While our prior studies have included substantial structure-function
analysis of the triazole bisphosphonate class of GGDPS inhibitors, we
have not previously evaluated the impact of modification of the term-
inal component of the isoprenoid chain. In the studies presented here
we demonstrate that the addition of an ω-hydroxyl group to two of our
previously reported inhibitors results in retention of GGDPS inhibitory
activity. While the potency of these new analogues is diminished re-
lative to the parent compounds, the hydroxy group affords the ability to
conjugate these inhibitors to other agents such as HA with the goal of
modifying drug biodistribution patterns and enhancing the therapeutic
index. Our preliminary studies with the conjugate 27 demonstrate en-
hanced cellular activity of the HA-GGDPS inhibitor conjugate compared
to the free drug. Future studies now can examine the biodistribution
and toxicity profile of the HA-GGDPS inhibitor conjugates in vivo, as
well as explore alternative modifications at the ω-position to create
additional “linkable” inhibitors.
Because it was more readily available, we then joined the alcohol 15
to HA (26, 10 K) via NHS/EDC conjugation chemistry49–52 to prepare
the drug conjugate 27 (Scheme 3). Conjugation was confirmed by the
presence of the aromatic signal from the triazole moiety and acetyl
hydrogens from HA in the 1H NMR spectrum as well as by the presence
of a phosphonate resonance in the 31P NMR spectrum.
Next, we compared the cellular activity of the parent ω-OH com-
pound 15 to the HA conjugate 27. As shown in Fig. 3, the HA-conjugate
27 induced greater accumulation of unmodified Rap1a than the free
drug 15 in two different human myeloma cell lines. This enhanced
cellular potency is likely a consequence of improved cellular uptake due
to CD44 cell surface expression. Presumably cellular esterases53,54 then
hydrolyze the ω-OH-GGDPS inhibitor from the polymer, releasing free
drug in the cell.
3

N.H. Bhuiyan, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126633
TEMPO, PhI(OAc)2
or MnO2
97%
X
Ph3PCH3Br
HO
16
17 X = O
18 X = CH2
t-BuOK
88%
1) 9-BBN
2) H2O2, NaOH
45%
SeO2, tBuOOH
salicylic acid
OH
25%
X
Br
1) MsCl, Et3N
2) NaBr, DMF
21
19 X = OH
20 X = Br
85%
O
O
1) H2, Pd/C (95%)
2) NaH, BrCH2CCH (43%)
1) NaN3, DMF (92%)
2) 23, CuSO4
(EtO)2P
(EtO)2P
Na-ascorbate
tBuOH/H2O (75%)
CH3
(EtO)2P
O
(EtO)2P
O
23
22
O
OH
(RO)2P
N
CH3
(RO)2P
O
N
N
TMSBr, collidine
then NaOH
50%
24 R = CH3CH2
25 R = Na
Scheme 2. Synthesis of compound 25, the ω-hydroxy analogue of compound 6.
Fig. 2. Comparison of the effects of the
novel ω-hydroxyl triazole bisphosphonates
to the parent analogues on protein ger-
anylgeranylation. RPMI-8226 cells were in-
cubated for 48 h in the presence or absence
of lovastatin (Lov, 10 μM) or varying con-
centrations of the test compounds. A)
Immunoblot analysis of uRap1a (antibody
detects only unmodified protein) and β-tu-
bulin (as a loading control). B) Intracellular
lambda light chain concentrations were de-
termined via ELISA. Data are expressed as a
percentage of control (mean
SD, n = 3).
The * denotes p < 0.05 per unpaired two-
tailed t-test.
4

N.H. Bhuiyan, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126633
Table 1
Summary of the bioassay results of the novel ω-OH-triazole bisphosphonates.
Compound
GGDPS IC50 (μM)
FDPS IC50 (μM)
Fold-selectivity for GGDPS compared to FDPS
Cellular LEC1 (μM)
15
25
11.6
0.67
2.6
91.2
81.4
25.0
23.5
8
10
0.30
121
0.25
1
Cellular LEC (lowest effective concentration) is defined as the lowest concentration for which an unmodified Rap1a band is visible in the immunoblot and a
statistically significant increase in intracellular lambda light chain is observed in the ELISA.
Graduate Fellowship Program.
O
OH
OH
N
N
N
P(ONa)2
H
O
O
O
O
HO
HO
Appendix A. Supplementary data
+
P(ONa)2
O
O
HO
OH
NH
15
O
Supplementary data associated with this article, including experi-
mental conditions and NMR spectra can be found in the online version
at https://doi.org/10.1016/j.bmcl.2019.126633.
n
CH3
26 (HA)
NHS, EDC
rt, 24 h
O
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H
N
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