Boron-Pleuromutilins as Anti- Wolbachia Agents with Potential for Treatment of Onchocerciasis and Lymphatic Filariasis

Article abstract of DOI:10.1021/acs.jmedchem.8b01854

A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti-Wolbachia antibiotics and, as such, may be useful in the treatment of filarial

Full text of DOI:10.1021/acs.jmedchem.8b01854

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Article
Boron-Pleuromutilins as Anti-Wolbachia Agents with Potential
for Treatment of Onchocerciasis and Lymphatic Filariasis.
Robert Toms Jacobs, Christopher S. Lunde, Yvonne R. Freund, Vincent Hernandez, Xianfeng Li,
Yi Xia, David S Carter, Pamela Berry, Jason Halladay, Fernando Rock, Rianna Stefanakis, Eric E.
Easom, Jacob J. Plattner, Louise Ford, Kelly L Johnston, Darren A.N. Cook, Rachel Clare, Andrew
Cassidy, Laura Myhill, Hayley Tyrer, Joanne Gamble, Ana F Guimaraes, Andrew Steven, Franziska
Lenz, Alexandra Ehrens, Stefan J Frohberger, Marianne Koschel, Achim Hoerauf, Marc P Hübner,
Case McNamara, Malina A Bakowski, Joseph D Turner, Mark J Taylor, and Stephen A. Ward
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01854 • Publication Date (Web): 07 Feb 2019
Downloaded from http://pubs.acs.org on February 8, 2019
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Koschel, Marianne; Universtiy Hospital Bonn, Institute for Medical
Microbiology, Immunology and Parasitology
Hoerauf, Achim; Universtiy Hospital Bonn, Institute for Medical
Microbiology, Immunology and Parasitology
Hübner, Marc; Universtiy Hospital Bonn, Institute for Medical
Microbiology, Immunology and Parasitology
McNamara, Case; California Institute for Biomedical Research,
Bakowski, Malina; California Institute for Biomedical Research
Turner, Joseph; Liverpool School of Tropical Medicine, Centre for Drugs
and Diagnostics, Department of Parasitology
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Taylor, Mark; Liverpool School of Tropical Medicine, Centre for Drugs and
Diagnostics, Department of Parasitology
Ward, Stephen; Liverpool School of Tropical Medicine,
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Boron-Pleuromutilins as Anti-Wolbachia Agents with Potential for
Treatment of Onchocerciasis and Lymphatic Filariasis.
1*
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Robert T. Jacobs, Christopher S. Lunde, Yvonne R. Freund, Vincent Hernandez,
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Rock, Rianna Stefanakis, Eric Easom, Jacob J. Plattner, Louise Ford, Kelly L.
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Johnston, Darren A.N. Cook, Rachel Clare, Andrew Cassidy, Laura Myhill, Hayley
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Tyrer, Joanne Gamble, Ana F. Guimaraes, Andrew Steven, Franziska Lenz,
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Alexandra Ehrens, Stefan J. Frohberger, Marianne Koschel, Achim Hoerauf, Marc P.
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Hübner, Case W. McNamara, Malina A. Bakowski, Joseph D. Turner, Mark J.
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Taylor, Stephen A. Ward.
1Anacor Pharmaceuticals, 1020 East Meadow Circle, Palo Alto, CA 94303, USA.
2Centre for Drugs and Diagnostics, Department of Parasitology, Liverpool School of
Tropical Medicine, Pembroke Place Liverpool L3 5QA UK
3Institute for Medical Microbiology, Immunology and Parasitology, University Hospital
Bonn, Sigmund Freud Str. 25, 53127 Bonn, Germany
4Calibr, 11119 North Torrey Pines Road, Suite 100, La Jolla, CA 92037, USA.
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Abstract
A series of pleuromutilins modified by introduction of a boron-containing heterocycle on
C(14) of the polycyclic core are described. These analogs were found to be potent anti-
Wolbachia antibiotics, and as such, may be useful in the treatment of filarial infections
caused by Onchocherca volvulus, resulting in Onchocerciasis or river blindness, or
Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in
lymphatic filariasis. These two important Neglected Tropical Diseases (NTDs)
disproportionately impact patients in the developing world. The lead preclinical
candidate compound containing a 7-fluoro-6-oxybenzoxaborole (15, AN11251) was
shown to have good in vitro anti-Wolbachia activity and physicochemical and
pharmacokinetic properties providing high exposure in plasma. The lead was effective in
reducing the Wolbachia load in filarial worms following oral administration to mice.
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Introduction
Parasitic nematodes of the family Filarioidea including Onchocerca volvulus, Wuchereria
bancrofti and Brugia malayi are responsible for significant disease burden in developing
1
countries around the world. Specifically, O. volvulus is the parasite responsible for River
Blindness, and W. bancrofti, B. malayi and B. timori cause lymphatic filariasis
2
(elephantiasis). Lymphatic filariasis is one of the the leading causes of global disability,
and accounts for at least 2.8 million disability-adjusted life years (DALYs).
Treatment/control and elimination programs for these infections have been in place for
many years, but fall short of full effectiveness due to the demanding treatment paradigm
required to break disease transmission.3-5 More specifically, the long life span of adult
worms (up to 15 years) requires annual to bi-annual mass drug administration (MDA) of
therapeutics such as ivermectin which only kills the juvenile microfilariae released by
5
adult worms and sterilizes adult worms but is not macrofilaricidal. Very recently, the
results of a triple-drug treatment (ivermectin, albendazole and diethylcarbamazine)
clinical trial demonstrated that this combination may require less frequent administration
(perhaps once every three years), but only a marginal improvement of macrofilarical
6
effects over a dual-drug therapy were noted. Consequently, new approaches to kill the
adult worms (macrofilaricides) are required in order for elimination time-frames of both
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diseases to be radically reduced. A short course of treatment (7 days or fewer) would
likely be required for ease of implementation in the field.
A unique feature of these parasitic worms is the presence of an obligate symbiotic
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bacteria of the Wolbachia genus. It has been known for some time that classical
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antibacterial agents such as doxycycline can kill the bacteria present in the worms, which
results in a reduced life span of the adult worm itself.8-10 Unfortunately, doxycycline
presents challenges as a drug for mass administration, including the requirement for long
1
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treatment periods (4–6 weeks) and contraindications in pregnancy and in children. An
anti-Wolbachia approach to the treatment of filarial infections has a number of patient
benefits especially as the co-endemic eyeworm Loa loa does not harbor the
endosymbiont and is therefore unaffected by treatment. It has been observed that
concurrent killing of Loa loa microfilariae by directly acting drugs in patients with
>
30,000 microfilariae per mL can have serious side effects including neurologic effects,
12
coma and death. In addition, recent work has suggested that depleting Wolbachia in
worms can also diminish the number of microfilariae that are able to develop in the insect
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vector, thus providing transmission blocking activity. The underlying parasitology
suggests that the discovery and development of new anti-Wolbachia drugs remains an
attractive option for improving our ability to reduce the global burden of River Blindness
and Elephantiasis and accelerate disease elimination goals.
As part of our ongoing effort to capitalize on the unique properties of benzoxaboroles in
modifying pharmacologic, physicochemical and pharmacokinetic properties of existing
drug scaffolds, we prepared analogs of the antibiotic class known as pleuromutilins.14, 15
This class of ribosomal protein synthesis inhibitors predominantly targets Gram-positive
bacteria and had been extensively explored since the 1950s, but had not previously been
shown to have activity against Wolbachia. The pleuromutilins have been shown to
inhibit protein synthesis through binding to the peptidyl-transfer center (PTC) of the
ribosome, with a crystal structure of tiamulin (4) with the 50S subunit of Deinoccus
radiodurans providing the most direct evidence of this mechanism of action.16 Recently,
Nabriva Therapeutics has been developing lefamulin (2), which is currently in clinical
trials for community-acquired bacterial pneumonia, demonstrating the potential of this
class of antibiotic.17 Furthermore, the starting material for our explorations,
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pleuromutilin (1), is readily available and inexpensive with synthetic modification,
particularly of the hydroxyacetate at C(14), being synthetically straightforward. In
addition to boronated analogs of the pleuromutilin core, we also obtained or prepared
several clinically relevant non-boron analogs as summarized in Figure 1.18-21
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Figure 1. Pleuromutilin and clinically relevant derivatives.
Results
The boronpleuromutilins modified at the C(14) position of the pleuromutilin core were
easily prepared via an S 2 displacement reaction of the pleuromutilin tosylate or iodide
N
(Scheme 1). These intermediates were synthesized from the commercially available
pleuromutilin. For compounds wherein the vinyl group at C(12) was modified,
ozonolysis of the pleuromutilin tosylate with a reductive workup provided the C(12)
aldehyde (Scheme 2). Displacement of the C(14) tosylate with 7-fluoro-6-
hydroxybenzoxaborole afforded the boronpleuromutilin, which in turn was condensed
with hydroxyl amines to provide the oximes, or with amines and sodium
cyanoborohydride to provide the amines. Hydrogenation of the C(12) vinyl group of the
boronpleuormutilin afforded the corresponding C(12) ethyl analog.
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Scheme 1. General route to C(14)-modified boronpleuromutilins.
Scheme 2. Route to C(12)-modified boronpleuromutilins.
The boronpleuromutilins with a C(14)-carbamate linker were prepared in two steps from
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-epi-pleuromutilin (72) in a manner analogous to that described in the literature
(
Scheme 3).23 For example, following conversion of 72 to the carbamoyl chloride 73,
treatment with 6-aminobenzoxaborole 78 followed by a zinc chloride mediated 1,5-
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hydride shift gave the C(14)-carbamate 74. Compounds 75 – 77 were also prepared in a
similar manner.
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Scheme 3. Route to C(14) boronpleuromutilin carbamates.
The boron containing heterocycles were prepared by a variety of synthetic sequences that
we have described in previous publications.24 As an example, 7-fluoro-6-
hydroxybenzoxaborole was prepared by the route shown in Scheme 4. Protection of 3,4-
difluorophenol (79) as the methoxymethyl ether was followed by ortho-metallation and
trapping of the intermediate aryllithium with ethyl formate to provide the benzaldehyde
derivative 81. Nucleophilic displacement of the para fluorine with benzyl alcohol under
basic conditions provided 82. Deprotection of the methoxymethyl ether followed by
conversion to triflate 84 permitted introduction of the boron via palladium mediated
borylation. Reduction of the aldehyde 85 with sodium borohydride followed by
treatment with aqueous hydrochloric acid resulted in formation of the benzoxaborole 86.
Finally, hydrogenolysis of the benzyl ether gave the desired 7-fluoro-6-
hydroxybenzoxaborole intermediate 87.
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Scheme 4. Synthesis of 7-fluoro-6-hydroxybenzoxaborole.
We screened the pleuromutilin drug candidates in two primary in vitro assays that
differed in the strain of Wolbachia used, the insect cell line used to host the bacteria, and
in the method of detection.25, 26 We used data from both assays to help ensure robust
selection of compounds for progression to subsequent in vitro ADME and in vivo PK
experiments. While the absolute potency of individual compounds varied between the
two assays in some cases, the SAR trends observed in these assays were generally
similar. Initial screening revealed that several of the clinically relevant pleuromutilins,
e.g., retapamulin (3) and valnemulin (5), showed good activity in in vitro assays;
significantly less activity was observed for lefamulin (2), tiamulin (4), or for the
unadorned pleuromutilin core (1, Table 1). More interestingly, attachment of a
benzoxaborole ring with either an oxygen (8), nitrogen (25) or sulfur (33) linker at the 6-
position to the C(14) sidechain provided compounds (hereafter referred to as
boronpleuromutilins) of similar in vitro potency to valnemulin and retapamulin.
Introduction of a methylene spacer between the pleuromutilin core and the benzoxaborole
ring as in 35 significantly reduced potency. Attachment of the benzoxaborole ring via the
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- position also provided compounds (37 – 39) with significantly reduced in vitro
activity.
We next turned our attention to substitution of the benzoxaborole core, where we found
that introduction of a fluoro substituent at the 4- (13), 5- (14) or 7- (15) positions was
generally tolerated with the C(7) analog 15 exhibiting very potent activity. Other small
substituents at C(7) such as methyl (12), chloro (16) or methoxy (17) were also tolerated,
but did not improve activity beyond the fluoro analog. As the pleuromutilins are
intrinsically very lipophilic, we also examined introduction of an aminomethyl
substituent onto the benzoxaborole core (19 – 23, 26 – 29), but these analogs were
generally less active than the corresponding neutral compounds. Finally, we found that
addition of two methyl groups at C(3) of the benzoxaborole (24, 30), a strategy that had
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improved pharmacokinetic properties in other series, significantly reduced potency.
Table 1. Pleuromutilin Derivatives with Benzoxaboroles Linked via C(14)
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Wolbachia
Wolbachia
infected
C6/36 cells LDW1 cells
wAlb) EC50
infected
Link
atom
ID
X
R
(
(wMel)
(nM)
EC (nM)
5
0
1
NAa
NA
NA
OH
NA
NA
NA
NA
NA
>1000
205
6868
220
91
(pleuromutilin)
2
(lefamulin)
3
NTb
(
retapamulin)
4
(tiamulin)
NA
NA
6
6
6
6
6
6
6
6
6
6
6
6
6
NA
NA
O
O
O
O
O
O
O
O
O
O
O
O
O
NA
NA
H
317
NT
6.3
215
100
11
606
6.1
1.3
24
5 (valnemulin)
8
9
3-Me (R,S)
4-Me
5-Me
7-Me
4-F
1
1
0
1
28
2.8
2.9
19
NT
1.5
14
3.7
1.2
19
12
13
52
158
8.4
15
1
1
1
1
4
5
6
7
5-F
7-F
7-Cl
32
7-OMe
5,7-F2
3-CH NH
NT
NT
104
64
18
1
2
9
0
2
2
2
4-CH NH
18
2
7
-Cl, 3-
2
2
1
2
6
6
6
O
O
O
197
>1000
50
49
317
9
CH NH
7-F, 3-
CH NH
7
2
2
2
2
2
-Cl, 4-
23
CH NH
2
2
2
26
2
2
2
3
4
5
6
6
6
6
6
6
6
O
3,3-Me2
H
>10000
5.0
229
409
302
148
4.1
17
NH
NH
NH
NH
NH
NH
3-CH NH
2
2
2
2
2
7
8
9
0
4-CH NH
44
2
5-CH NH
1182
173
198
2
7-CH NH
257
>1000
2
3,3-Me2
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
3
1
2
3
6
6
6
6
6
6
5
5
5
NH
NH
S
5-F
7-F
H
7-F
H
23
106
123
NT
13
17
34
S
NT
15
3
3
3
3
5
6
7
8
-CH NH-
>1000
>10000
>10000
298
243
1418
NT
39
2
-CH NH-
3,3-Me2
H
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
5-F
7-F
39
149
NT
a
b
NA = not applicable, NT = not tested.
Table 2. Pleuromutilins with Other Boron Heterocycles Linked via C(14)
Wolbachia Wolbachia
infected
C6/36
cells
wAlb)
EC (nM) EC (nM)
infected
LDW1
cells
ID
P
Q-R
R8
(
(wMel)
5
0
50
4
4
4
4
0
1
2
3
CH SO N
N=CH
N=CH
N=CH
N=CH
N=CH
H
H
H
H
H
9
0.8
3
2
CH N
14.8
4
0.7
3.5
1.5
14.2
240
172
107
164
329
26.1
>1000
1.5
0.2
0.2
2.7
0.3
5.1
6.3
11
NT
1.3
27
NT
NT
3
CH C(=O)N
3
Boc-N
HN
44
4
4
4
4
5
6
7
8
CH OC(=O)N N=CH
H
H
3
O
N=CH
N=CH
N=CH
N=CH
CH SO N
8-F
8-F
8-F
8-F
8-F
8-CH3
H
3
2
CH N
3
a
49
50
CH C(=O)N
3
CH OC(=O)N N=CH
3
5
5
5
1
2
3
O
N=CH
N=CH
CH CH
CH C(=O)N
3
O
2
2
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9
aNT = not tested.
Initial results with other boron containing heterocycles linked via C(14) of the
pleuromutilin core were encouraging, as a number of these analogs were very potent in
the in vitro Wolbachia assay (Table 2). In particular, the N-methanesulfonyl (40), N-
acetyl (42) and N-methylcarbamoyl (45) derivatives of the diazaborine scaffold exhibited
low nanomolar to picomolar potency. Unlike the benzoxaboroles, however, inclusion of
a fluorine atom adjacent to the B-OH did not improve potency; in fact, these derivatives
(47 – 51) were significantly less potent. The benzoxaborine 53, wherein the five
membered ring was expanded by one carbon to a six membered ring, was completely
inactive.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We also explored modification of the C(12) vinyl substituent (Table 3). Work in the
classical pleuromutilin literature had shown that this double bond could be reduced to
provide an ethyl group (54), converted to the epoxide (55) or oxidized to an aldehyde
(
58) that could be functionalized in a variety of ways.28 In particular, we sought to
introduce small amines at C(12) to modify physicochemical and pharmacokinetic
properties. Given the interesting properties of 15 (vide supra), this work was performed
with the 7-fluoro-6-oxobenzoxaborole linked via to C(14) of the pleuromutilin core.
Table 3. Boronpleuromutilins Modified at C(12)
Wolbachia
infected
C6/36 cells LDW1 cells
(wAlb) EC50 (wMel) EC50
Wolbachia
infected
ID
Rb
(nM)
38
148
278
>1000
>1000
(nM)
2.7
13
33
242
71
5
5
5
5
4
5
8
9
CH CH -
3
2
epoxide
OHC-
HOCH2-
60
HON=CH-
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
1
2
3
CH ON=CH-
197
NT
>1000
>1000
474
357
372
115
328
39
101
6892
1718
1399
478
243
44
3
a
iso-C H ON=CH-
3
7
H NCH -
2
2
64
CH NHCH -
3 2
C H NHCH -
2 5 2
6
6
6
6
69
7
7
5
6
7
8
n-C H NHCH -
3
7
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
n-C H NHCH
2
4
9
cyclo-C H NHCH
2
3
5
(CH ) NCH -
439
133
29
3
2
2
0
1
CH ONHCH -
NT
>1000
3
2
CH C(=O)NHCH -
3
2
aNT = not tested.
Disappointingly, only the ethyl (54) derivative exhibited potency close to the C(12) vinyl
analog 15; oximes (60 – 62) and amines (63 – 70) were significantly less potent. Some
potency was regained by acylation of the amine (71), but this was not perceived as being
of sufficient advantage to pursue further.
As a final area of exploration, we prepared several boronpleuromutilins where the C(14)-
hydroxyacetate linker was replaced by a shorter carbamate (Table 4). This strategy had
previously been explored in the pleuromutilins as a means to improve oral
2
3
bioavailability. The carbamates prepared (74 – 77) exhibited moderate to good in vitro
potency.
Table 4. Boronpleuromutilin Carbamates
Wolbachia
infected
C6/36 cells LDW1 cells
wAlb) EC50 (wMel) EC50
Wolbachia
infected
ID
X
Ra
(
(nM)
(nM)
7.5
14
7
7
4
5
bond
bond
CH2
H
5-F
H
101
>1000
12
76
3.5
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7
7
CH2
7-F
113
4.9
Concurrent with our evaluation of the in vitro potency of the boronpleuromutilins, we
also selected a number of active analogs for characterization in both in vitro ADME
(
Absorption, Distribution, Metabolism, Excretion) and in vivo pharmacokinetics (PK)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
experiments. It has been suggested that one of the main limitations of the pleuromutilins
is high metabolic instability leading to poor pharmacokinetics.29, 30 In fact, the majority of
the clinically relevant pleuromutilins are of limited oral bioavailability, which has
hampered their utility for human applications. A notable exception is the recently
described lefamulin (2), which is currently in Phase 3 clinical trials as an oral treatment
for a variety of bacterial infections.31, 32 In order to calibrate our expectations for the
boronpleuromutilins, we screened several of the clinically relevant analogs in our in vitro
ADME assays as well (Table 5). As anticipated, tiamulin and retapamulin were found to
be rapidly metabolized by mouse liver S9 fraction, whereas lefamulin was slowly
metabolized. These classical pleuromutilins exhibited a range of binding to mouse
plasma proteins, with free fraction (funbound) from 0.05 – 0.26. Somewhat surprisingly, we
found that lefamulin was not particularly permeable in our MDR1-MDCK assay, though
the high stringency of this assay may underestimate the permeability of this compound
through the gut. We had chosen the MDR1-MDCK assay to assess permeability over the
related Caco2 assay due to throughput and the opportunity to more directly assess the
impact of P-glycoprotein mediated efflux in this cell line. In addition, it has also been
reported that the MDR1-MDCK is an acceptable assay for evaluation of the permeability
of compounds through the intestinal mucosa of humans.33
The neutral boronpleuromutilins evaluated (8 – 12, 14, 25, 33) were generally
metabolized quickly by mouse S9 (Cl > 50 µL/min/mg), were highly protein bound
int
(
funbound < 0.03) and were predicted to have good permeability based on the MDR1-
-
6
MDCK assay (Papp > 5  10 cm/sec). In contrast, boronpleuromutilins bearing an
aminomethyl subsitituent (19 – 21, 23, 26) were less rapidly metabolized (Cl < 10
int
µL/min/mg) much less protein bound (funbound > 0.1), but were predicted to be of low
-6
permeability (Papp < 1  10 cm/sec). Later compounds incorporating boron-containing
heterocycles other than the benzoxaborole (40 – 42, 44- 46, 52, 53) or modification of
C(12) (55, 59) did not significantly change the overall in vitro ADME profile. One
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound that stood out as having an attractive balance of properties was the 7-
fluorobenzoxaborole derivative 15, which exhibited modest clearance (Cl = 33
int
µL/min/mg) and protein binding (funbound = 0.03), and good permeability (Papp = 14.1 
-6
1
0 cm/sec).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. In vitro ADME properties of Boronpleuromutilins.
mouse S9 Clint Mouse protein binding
ID
MDR1-MDCK Pappa
(
L/min/mg)
(funbound @ 2 M)
0.218
2
3
4
5
8
9
4
0.2
6.7
22.1
1.4
41
0.154
b
131
NT
145
168
139
77
136
117
33
0.26
0.05
b
0.003
0.002
0.005
< 0.001
0.012
0.003
0.034
0.008
0.116
0.110
0.119
0.048
0.008
0.306
0.005
0.008
0.058
0.007
0.005
0.008
0.037
< 0.001
0.006
< 0.001
NT
15.1
c
NT
1
1
1
1
0
1
2
4
NT
NT
NT
NT
14.1
NT
0.3
0.1
0.5
0.1
36.5
0.1
22.6
9.0
6.6
NT
NT
2.3
NT
8.8
NT
NT
8.7
3.7
15
1
1
2
2
6
9
0
1
44
NT
7
NT
NT
102
2
NT
142
226
NT
NT
80
NT
NT
217
NT
NT
53
23
2
2
3
3
5
6
2
3
40
41
4
4
4
4
2
4
5
6
52
5
5
5
3
5
9
0.033
a
Permeability measured in a Madin Darby Canine Kidney cell monolayer transfected with
b
the multi-drug resistance 1 (mdr1) gene encoding p-glycoprotein MDR1. Protein binding
measured at 1 M. NT = not tested.
c
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8
9
Based on these in vitro profiles and due to resource constraints, we selected
representative compounds from the various classes (e.g. neutral, aminomethyl), different
linkers (e.g. O, N) and boron heterocycles (e.g. benzoxaboroles, diazaborines) for
progression to in vivo PK experiments as summarized in Table 6. When the in vivo
pharmacokinetics in mice of these representative compounds were measured, we
observed that the PK parameters between them were variable following both IV and oral
administration, consistent with the in vitro properties described above. Several
observations were made from these studies. First, and as expected, clearance following
intravenous dosing was generally quite high, with only compound 15 exhibiting low to
modest clearance. Secondly, incorporation of an aminomethyl group on the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzoxaborole core (19) significantly reduced oral bioavailability, as did replacement of
the benzoxaborole with a diazaborine (45, 52). Finally, oral exposure and bioavailability
was generally good for the 7-fluorobenzoxaborole derivatives tested (15, 32 and 77).
Table 6. In vivo pharmacokinetic properties of lead boronpleuromutilins in BALB/c
mice.
IV at 5 mg/kg,
Vehicle 55/25/20 PEG/PG/H O
PO at 10 mg/kg, Vehicle
55/25/20 PEG/PG/H O
Cmpd
2
2
Cmax
CL
Vss
AUC0-last
Cmax
AUC0-last
%F
(
g/mL) (mL/hr/kg)
(mL/kg)
(hr*g/mL) (g/mL) (hr*g/mL)
8
4.13
3.21
2.48
5.91
7.40
3.22
3.91
2296
505
2716
4186
10435
2231
854
2.18
8.85
2.26
4.60
3.03
1.52
3.71
0.673
1.60
1.54
12.1
35
61
1
1
5
9
2120
1080
1642
3273
1338
0.003
1.66
0.02
<1
32
11.0
~100
7
4
5
5
2
0.262
0.227
1.09
0.425
0.166
8.51
4658
2561
6
77
~100
Given the relatively low clearance and high exposure exhibited by 15, coupled with its
good biological potency, additional PK experiments were performed with this compound.
These confirmed the attractive PK profile following oral administration to mice of 15, as
systemic exposures at doses from 10 – 50 mg/kg were good, with Cmax values increasing
dose-dependently while AUC values remained relatively constant, suggesting
dissolution-limited absorption of compound from the gut (Table 7).
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. In vivo pharmacokinetic properties of 7-fluorobenzoxaborole analog 15 in
BALB/c mice.
1
5 IV
5 mg/kg (Mean, n=3)
Vehicle
55/25/20 PEG/PG/H O
2
Cmax (µg/mL) @ 5 min
CL (mL/h/kg)
3.21 ± 1.3
505
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
V (mL/kg)
4186
8.85
9.91
ss
AUClast (h*µg/mL)
AUC0-inf (h*µg/mL)
Terminal t (h)
9.10
1
/2
1
5 PO
1
0 mg/kg
10 mg/kg
Suspension
25 mg/kg
Suspension
50 mg/kg
Suspension
Solution
(Mean, n=3)
(Mean, n=3) (Mean, n=3) (Mean, n=3)
% CMC, 1% CMC, 1% CMC,
0.1% Tween 0.1% Tween 0.1% Tween
1
55/25/20
Vehicle
PEG/PG/H O
2
8
0 in H O
80 in H O
80 in H O
2
2
2
Cmax (µg/mL)
Tmax (h)
AUClast (h*µg/mL)
AUC0-inf
1.60
0.083
12.1
2.81
0.50
20.7
4.25
0.25
23.7
6.38
0.50
23.0
a
NC
14.0
22.8
24.2
(h*µg/mL)
Terminal t (h)
Bioavailability (%)
8.41
61.1
7.51
NC
4.60
NC
NC
NC
1
/2
aNC = not calculated.
Based on this combination of in vitro potency and in vivo PK data, we next examined the
ability of 15 to deplete the Wolbachia symbiont from filarial worms in vivo. In all in vivo
efficacy assays, depletion of Wolbachia was determined through comparison to the
Wolbachia load found in worms present in animals that were treated with only the dosing
vehicle as described in the Experimental section. For our initial in vivo screen, groups of
three CB.17 SCID mice were inoculated with 50 L3-stage B. malayi larvae via the
peritoneal cavity and treated from point of infection with 15 for 7 or 14 days with oral
doses of 25 mg/kg, BID. At point of necropsy, on day 14, fourth stage (L4) B.malayi
larvae were isolated from the peritoneal cavity. Larvae were pooled per group and intra-
worm Wolbachia titres were quantified from groups of 10 larvae using qPCR. Included
in the same experiment were a vehicle control group and two groups of doxycycline-
treated animals as positive controls. We were encouraged to find that 15 reduced the
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Wolbachia load in the B. malayi larvae by 75.4% and 98.8% in the 7- and 14-day
treatment groups, respectively. In this experiment, doxycycline reduced Wolbachia load
by 76.3% and 96.7% following 7- and 14-day treatment at 50 mg/kg, QD, respectively
(Table 8).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 8. Summary of Efficacy of 15 and doxycycline in reduction of Wolbachia in a
larval B. malayi SCID mouse model.
Dosing
Duration
doxycycline,50
mg/kg QD
7 days
76.3%
75.4%
14 days
96.7%
98.8%
1
5, 25 mg/kg,
BID
Values represent percent (%) median reduction of Wolbachia versus the vehicle control,
as measured by qPCR of Wolbachia surface protein single copy gene (wsp) per B. malayi
L4 larva (n=10 per group) obtained from necropsies undertaken two weeks after first
dose.
Encouraged by the profile of 15, and in preparation for more extensive in vivo
characterization of this molecule, we conducted a number of preliminary in vitro and in
vivo studies to assess the safety of this molecule. In a non-GLP Ames assay (Bioreliance,
Rockville, MD), 15 was determined to be non-mutagenic. Furthermore, 15 was found to
be negative in its potential to induce micronuclei in human peripheral blood lymphocytes
(Bioreliance, Rockville, MD). In a panel of over 50 mammalian receptors, enzymes and
ion channels (Eurofins Cerep, France), 15 was found to be without any significant effect
at 10 M, and it was also shown to have no significant (<10%) effect on the hERG
potassium channel expressed in HEK cells at a concentration of 30 µM (ChanTest,
Cleveland, OH). Finally in a preliminary safety study (Anacor, Palo Alto, CA), no
adverse effects were observed in BALB/c mice treated with 15 at doses up to 200
mg/kg/day for 7 days.
We next progressed 15 to chronic in vivo infection models of adult filarial parasitism (the
target life cycle stage for an anti-Wolbachia indication) ; one using B. malayi and one
using Litomosoides sigmodontis. We initially chose to use the B. malayi SCID mouse
3
4
2
model as this worm species is a causative agent of LF in humans. In this B. malayi
assay, 100 L3-stage larvae, were inoculated via the peritoneal route and adult infections
were allowed to develop. At six-weeks post-infection, the mice were treated with 15 (25
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mg/kg, BID) for 7, 14 or 28 days. At the end of the experiment (12 weeks post
infection), worms were recovered from the mice and Wolbachia load was determined by
qPCR. In this experiment, vehicle and minocycline (25 mg/kg, BID  28 days) groups
were included as negative and positive controls, respectively. Minocycline was chosen as
the positive control for this study based on concurrent results in a Litomosoides
sigmondontis model (vide infra) that suggested this tetracycline antibiotic exhibited
superior efficacy to doxycycline.35 Somewhat disappointingly, efficacy of 15 was not
nearly as good in the adult model, with a 45.5% reduction of Wolbachia load observed
for the 28 day treatment group. By comparison, minocycline provided 98.2% reduction
in this experiment (Table 9). We hypothesize that the failure of 15 to show efficacy at
the dose chosen in the B. malayi adult worm model is likely due to the increased washout
period between treatment start and necropsy in comparison to the B. malayi L3 larval
model, leading to a rebound of the Wolbachia with a suboptimal treatment regimen of 15.
0
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Table 9. Summary of Efficacy of 15 and minocycline against Wolbachia in the adult
B. malayi SCID mouse model.
Dosing Duration
28 days
14 days
7 days
13.4
1
5
2
5 mg/kg, BID
45.5
16.6
minocycline
5 mg/kg, BID
2
98.2
Values represent percent (%) median reduction of Wolbachia versus the vehicle control,
as measured by qPCR of Wolbachia surface protein single copy gene (wsp) per adult
female B. malayi (n=10 per group) obtained from necropsies undertaken six weeks after
first dose.
In a second efficacy model,35, 36 we used mice infected with the rodent filarial nematode
Litomosoides sigmodontis, another helminth species known to host Wolbachia, and
treated these animals with 15 at a dose of 50 mg/kg, BID for 14 or 28 days. The L.
sigmodontis model was chosen as an additional, confirmatory filarial model as it uses
immunocompetent wild-type mice as host and initial studies demonstrated the efficacy of
tetracycline against Wolbachia-containing filariae first in this model.37 In addition, the
infection could be maintained in mice using a natural infection protocol.35, 38-40 Upon
completion of dosing, animals were maintained for a total of 64–77 days (to maintain a
consistent total time following the final dose), at which time worms were recovered from
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the peritoneum and the thoracic cavity. Wolbachia load was determined by qPCR.
Doxycycline was included as a positive control in this experiment. We were encouraged
to find that 15 was able to reduce Wolbachia burden in L. sigmodontis by >99% at this
dose and duration (Table 10). The stronger reduction of the Wolbachia load in the L.
sigmodontis mouse model in comparison to the adult B. malayi mouse model is probably
mostly due to the increased dose used for treatment with 15, although L. sigmodontis
were previously reported to have an increased susceptibility for anti-Wolbachia drugs
compared to B. malayi filariae.41
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9
0
Table 10. Efficacy of 15 and doxycycline against Wolbachia in the adult L.
sigmodontis mouse model.
Dosing Duration
2
8 days
14 days
99.7
1
5
5
0 mg/kg, BID
99.2
Doxycycline
40 mg/kg, BID
99.9
99.9
Values represent percent (%) reduction of Wolbachia versus the untreated control, as
measured by qPCR FtsZ/actin per mouse and were obtained from necropsies performed
one month after first dose.
Discussion and Conclusions
Previous work from our laboratories has demonstrated that boron-containing heterocycles
can be useful in the treatment of various diseases, including infectious diseases of the
developing world such as malaria,42, 43 human African trypanosomiasis27, 44 and
tuberculosis.45, 46 We have extended our work to look at modification of known classes of
antibacterial agents as an approach to treat River Blindness via the bacterial symbiont
(Wolbachia) present in the worms that cause this disease. Clear clinical “proof of
concept” for an anti-Wolbachia approach has been demonstrated with the tetracycline
9
antibiotic doxycycline. The utility of doxycycline is limited, however, by the
requirement that patients must be treated for 4–6 weeks in order to achieve significant
reduction of Wolbachia in the O. volvulus parasite.8-10 More recently, it has been
demonstrated that combination of doxycycline (200 mg/day for 3 weeks) with
albendazole (800 mg/day for 3 days) may provide a shorter treatment course.47 While
moving closer to the desired short course (7 day or fewer) treatment paradigm, this
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combination therapy is still not ideal and does not address tetracycline contraindicated
groups (children ≤8 and pregnant women).
Our work started by evaluating representatives of various classes of antibiotics including
several clinically relevant pleuromutilins. This initial screening revealed that some
activity was present in this class of compounds, and we prepared several analogs
containing our benzoxaborole core. The synthesis of these boronpleuromutilins was
facilitated by the ready availability of the tosylate derivative of the hydroxyacetate ester
at C(14) of the pleuromutilin core. Simple nucleophilic displacement reactions of this to
afford C(14)-functionalized boronpleuromutilins with nitrogen, oxygen or sulfur linker
atoms proceeded in good yield. Early structure-activity relationships (SAR) revealed that
direct attachment of the benzoxaborole ring to the pleuromutilin core provided
compounds with good in vitro potency. In contrast, inclusion of a linker group such as
present in the clinically relevant pleuromutilins between the core and benzoxaborole ring
were of poor activity. Based on our previous work in the benzoxaboroles, we focused
our attention on compounds linked through the 6-position, as these were both
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synthetically accessible and generally had been observed to have good ADME properties.
Inclusion of a variety of substituents such as halogen, small alkyl or aminoalkyl at
positions 3, 4, 5 and 7 of the 6-O linked benzoxaboroles gave compounds of similar in
vitro activity and allowed us to explore the effect of these substituents on ADME and PK.
We were particularly pleased to find that the 7-fluoro analog (15) exhibited a good
balance of potency, physicochemical and ADME properties. In particular, we were
encouraged by the modest metabolic stability in mouse microsomes (Cl = 33
int
µL/min/mg) and good permeability through an MDR1-MDCK monolayer (Papp = 14.1 
-6
1
0 cm/sec). We were somewhat concerned about the high protein binding of 15 (f
unbound
=
0.03), though this was predicted based on the calculated high lipophilicity (logD 4.23)
of this compound. While other compounds, particularly those with an aminomethyl
substituent on the benzoxaborole core (e.g. 20) exhibited better metabolic stability (Cl =
int
6
µL/min/mg), they were compromised by very low permeability (Papp < 0.1  10-6
cm/sec).
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When 15 was dosed to mice by the intravenous route (5 mg/kg), we were encouraged by
the relatively low clearance (Cl = 505 mL/hr/kg), good exposure (AUC
= 8.85
int
0-24h
hr/µg/mL). Following oral administration (10 mg/kg), 15 exhibited good exposure
AUC0-24h = 14.0 hr/µg/mL) and bioavailability (F = 61%). Additional PK studies at
(
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higher oral doses demonstrated that exposure increased with dose, though not dose
proportionally, to very high levels (AUC0-24h = 58.7 hr/µg/mL at 400 mg/kg). In addition
to the parent compound, we also tracked the major (and active) metabolite of 15 (15a,
IC = 193 nM) where hydroxylation at C(2) of the pleuromutilin core had occurred, as
5
0
this represented about 10–15% of the parent dose at most time points. Metabolism of
pleuromutilins at this position of the core is well precedented.48, 49
Based on these observations, 15 was selected for evaluation in several in vivo models that
had been established for evaluation of anti-Wolbachia compounds in mice. In the first
study, we found that 15, when dosed orally at 25 mg/kg, BID, to SCID mice infected with
larval stage B. malayi, efficacy (defined as >99% reduction in Wolbachia load in worms)
was achieved following 14 days of dosing; 7 days dosing did not achieve full efficacy. In
a second B. malayi model employing worms that had been allowed to mature to the adult
stage, we were disappointed to find that 15 did not demonstrate measureable efficacy
when dosed at 25 mg/kg, BID for 28 days. More encouraging data was obtained in the L.
sigmodontis BALB/c mouse model, where 15 was found to show excellent efficacy
(>99% Wolbachia depletion) when dosed at 50 mg/kg, BID for 14 or 28 days. Additional
in vivo studies to address this hypothesis are ongoing and will be reported in due course.
In conclusion, exploration of a series of pleuromutilin derivatives incorporating a novel
boron-containing heterocycle linked to C(14) of pleuromutilin core as anti-Wolbachia
agents has resulted in the identification of 15 as a potential pre-clinical candidate. SAR
developed in this program has demonstrated that optimal activity was obtained by direct
linkage of the benzoxaborole to the pleuromutilin core via C(6) of the benzoxaborole, and
that small substituents on the benzoxaborole ring system had an impact on potency and
pharmacokinetic properties. Good-to-excellent exposure following oral administration of
1
5 to mice was achieved, and this translated into high levels of Wolbachia depletion in
several in vivo models. We were encouraged by the efficacy of 15 in these models,
prompting more extensive evaluation of this compound, both alone and in combination
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with other known anti-Wolbachia drugs. The outcome of these studies will be reported in
due course.
Experimental Section
General
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All chemicals were purchased from commercial suppliers and used as received. 1H
NMR spectra were recorded on a Bruker Avance 400 spectrometer. Chemical shifts are
expressed in  ppm referenced to an internal tetramethylsilane ( = 0 ppm) standard.
Abbreviations used in describing peak signals are br = broad, s = singlet, d = doublet, dd
=
doublet of doublets, t = triplet, q = quartet, m = multiplet. All final compounds were
purified to have purity higher than 95% by reverse phase high-performance liquid
chromatography (HPLC), supercritical fluid chromatography (SFC), normal phase flash
chromatography or crystallization. Preparative HPLC was accomplished using a Luna
C18 250  30 mm, 10 µm column. The purity was assessed by reverse phase HPLC with
a gradient of 5 – 95% acetonitrile in water (with or without acid modifier) and monitored
by diode array ultraviolet detector at 220 and 254 nm. Low resolution mass spectra were
recorded on a liquid chromatography-mass spectrometer in electrospray positive (ESI +)
or negative (ESI -) modes.
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-(tosyloxy)acetate. (6).
p-Toluenesulfonyl chloride (19.1 g, 0.1 mol) in 1,2-dichloroethane (100 mL) was slowly
added to a mixture of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-
oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-hydroxyacetate (35.4 g,
0.1 mol), triethylamine (12.0 g, 0.1 mol) and pyridine (1 mL) in 1, 2-dichloroethane (100
mL). The mixture was stirred at 10–15 °C for 20h, washed with water (3  100 mL),
then concentrated to dryness. Purification was achieved by recrystallization from
dichloromethane/petroleum ether (1:100) to afford (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-
hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-
propanocyclopenta[8]annulen-5-yl 2-(tosyloxy)acetate 6 as a white solid (45.0 g, yield
1
9
0.0%). H NMR (DMSO-d , 400 MHz) δ 7.80 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz,
6
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H), 6.05 (dd, J = 17.8, 11.2 Hz, 1H), 5.53 (d, J = 8.4 Hz, 1H), 5.09-4.96 (m, 2 H), 4.81-
4.59 (m, 2H) , 3.40 (d, J = 5.6 Hz, 1H) , 2.41 (s, 2H), 2.39 (br. s., 1H), 2.24-1.95 (m, 5H),
.75-1.41 (m, 4H), 1.30 (s, 4H), 1.27-1.18 (m, 4H), 1.03 (s, 3H), 0.99-0.92 (m, 2H), 0.81
d, J = 7.2 Hz, 3H), 0.50 (d, J = 7.2 Hz, 3H).
1
(
1
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0
Preparation of (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-
oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-iodoacetate (7).
To a solution of 6 (51.5 g, 96.7 mmol, 1.0 eq.) in acetonitrile (600.0 mL) was added
sodium iodide (87.0 g, 580.1 mmol, 6.0 eq.). The mixture was stirred at 90 °C for 16
hours at which time HPLC indicated the reaction was completed. The reaction mixture
was concentrated under reduced pressure to remove acetonitrile. The residue was diluted
with H O (500 mL) and extracted with dichloromethane (3  500 mL). The combined
2
organic layers were concentrated under reduced pressure to give a residue. The residue
was washed with petroleum ether (200 mL). (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-
4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-
1
iodoacetate 7 (40.0 g, 81.9 mmol, 84.7% yield) was obtained as a yellow solid. H NMR
(DMSO-d , 400 MHz) δ 6.11 (dd, J = 11.2, 17.9 Hz, 1H), 5.52 (d, J = 7.9 Hz, 1H), 5.13-
6
5.01 (m, 2H), 4.55 (d, J = 5.7 Hz, 1H), 3.82-3.75 (m, 1H), 3.73-3.66 (m, 1H), 3.43 (t, J =
5
1
.3 Hz, 1H), 2.45-2.39 (m, 1H), 2.25-2.00 (m, 4H), 1.72-1.57 (m, 2H), 1.53-1.21 (m, 9H),
.11-0.97 (m, 4H), 0.83 (d, J = 6.6 Hz, 3H), 0.64 (d, J = 7.1 Hz, 3H).
Method A. Use of Pleuromutilin tosylate 6. Preparation of
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
(
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (8)
A solution of 6 (1.8 g, 3.3 mmol), benzo[c][1,2] oxaborole-1,6(3H)-diol (0.5 g, 3.3
°
mmol) and K CO (0.7 g, 5.0 mmol) in 20 mL of DMF was heated to 50 C overnight, at
2
3
which time LC/MS indicated the reaction was completed. Water was added and the
mixture was adjust pH<4 with 2N HCl. The solid was filtered and the crude product was
purified by prep HPLC to give (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy -4,7,9,12-
tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl2-((1-
1
hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy) acetate 8 (1.0 g, yield 57.0 %). H
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NMR (DMSO-d , 400 MHz) δ 7.30 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 2.0 Hz, 1H), 7.04
6
(dd, J = 8.4, 2.4 Hz, 1H) 6.10 (dd, J = 17.6, 11.2 Hz, 1H), 5.59 (d, J = 8.4 Hz, 1H), 5.11-
4
2
.97 (m, 2H), 4.91 (s, 2H), 4.76-4.63 (m, 2H), 3.41 (d, J = 5.6 Hz, 1H), 2.40 (br. s., 1H),
.26-1.99 (m, 4H), 1.72-1.43 (m, 4H) , 1.38 (d, J = 13.2 Hz, 1H) , 1.34 (s, 3H), 1.29-1.15
0
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(m, 4H), 1.03 (s, 3H), 1.01 (br. s., 1H), 0.81 (d, J = 6.8 Hz, 3H), 0.63 (d, J = 6.8 Hz, 3H).
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-3-methyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (9).
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 9.00 (s, 1H), 7.28 (d, J
6
= 9.2 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.09 (dd, J = 17.6, 11.4
Hz, 1H), 5.59 (d, J = 8.0 Hz, 1H), 5.15-4.98 (m, 3H), 4.72-4.62 (m, 2H), 4.51 (d, J = 6.0
Hz, 1H), 3.40 (m, 1H), 2.40 (s, 1H), 2.26-1.98 (m, 4H), 1.67-1.26 (m, 14H), 1.07-1.01
(m, 3H), 0.81 (d, J = 6.8 Hz, 3H), 0.63 (d, J = 6.8 Hz, 3H). MS (ESI): mass calcd. for
-
C H BO 524.3, m/z found 523.2 [M-1] . HPLC: 93.3% (220 nm), 100% (254 nm).
30 41
7
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-4-methyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (10).
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 7.02 (d, J = 1.76 Hz,
6
1
5
2
H), 6.83 (d, J = 1.8 Hz, 1H), 6.10 (dd, J = 17.6, 11.2 Hz, 1H), 5.60 (d, J = 8.4 Hz, 1H),
.12-4.96 (m, 2H), 4.87 (s, 2H), 4.75-4.60 (m, 2H), 2.41 (br. s., 1H), 2.17 (s, 3H), 2.00-
.13 (m, 5H), 1.72-1.55 (m, 2H), 1.54-1.39 (m, 2H), 1.35 (s, 3H), 1.32-1.20 (m, 3H), 1.04
(
m, 4H), 0.82 (d, J = 6.4 Hz, 3H), 0.64 (d, J = 7.2 Hz, 3H). MS: 523 (M-1)-
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-5-methyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (11)
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 8.93 (br. s., 1 H), 7.19
6
(s, 1 H), 7.09 (s, 1 H), 6.08 (dd, J = 17.6, 11.2 Hz, 1 H), 5.59 (d, J = 8.0 Hz, 1H), 5.12-
4.95 (m, 2H), 4.87 (s, 2H), 4.71 (s, 2H), 3.40 (d, J = 4.8 Hz, 1H), 2.40 (s, 1H), 2.25 (s,
3
H), 2.20-1.96 (m, 3H), 1.74-1.55 (m, 2H), 1.50-1.45 (m, 1H), 1.37 (s, 3H), 1.45-1.17
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(
m, 6H), 1.10-0.94 (m, 4H), 0.82 (d, J = 6.4 Hz, 3H), 0.62 (d, J = 6.4 Hz, 3H). MS: 523
(M-1)-
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-7-methyl-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (12).
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
3
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6
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 7.09 (d, J = 8.0 Hz,
6
1
5
1
H), 6.95 (d, J = 8.0 Hz, 1H), 6.11 (dd, J = 17.6, 11.2 Hz, 1H), 5.61 (d, J = 8.0 Hz, 1H),
.09-4.98 (m, 2H), 4.87 (s, 2H), 4.72 (d, J = 2.0 Hz, 2H), 3.41 (d, J =5.6 Hz, 1H), 2.36 (s,
H), 2.33 (s, 3H), 2.20-2.10 (m, 1H), 2.10-1.95 (m, 4H), 1.72-1.55 (m, 2H), 1.54-1.36
(m, 2H), 1.34 (s, 3H), 1.31-1.15 (m, 3H), 1.03 (s, 4H), 0.82 (d, J = 6.8 Hz, 3H), 0.63 (d, J
6.8 Hz, 3H). MS: 523 (M-1)-.
=
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((4-fluoro-1-hydroxy-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (13).
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 9.29-9.24 (br. s.,1H),
6
7
.08 (s, 1H), 6.93 (d, J = 11.2 Hz, 1H), 6.15-6.05 (m, 1H), 5.60-5.56 (d, J = 8.8 Hz, 1H),
5.05-4.99 (m, 4H), 4.77-4.75 (d, J = 8.0 Hz, 2H), 2.41 (s, 1H), 2.41-2.04 (m, 4H), 1.63-
1
6
.41 (m, 4H), 1.33-1.28 (m, 7H), 1.04-0.85 (m, 4H), 0.81 (d, J = 6.4 Hz, 3H), 0.63 (d, J =
-
.4 Hz, 3H). MS (ESI): mass calcd. for C H BFO 528.27, m/z found 527.2 [M-1] .
2
9
38
7
HPLC: 96.0% (220 nm), 69.6% (254 nm).
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
(
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((5-fluoro-1-hydroxy-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (14)
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 9.10 (s, 1H), 7.35-7.26
6
(m, 2H), 6.08 (dd, J = 18.0, 11.2 Hz, 1H), 5.58 (d, J = 7.6 Hz, 1H), 5.09-4.96 (m, 2H),
4
2
.90 (s, 2H), 4.85-4.74 (m, 2H), 4.52 (d, J = 6.0 Hz, 1H), 3.43-3.38 (m, 1H), 2.41 (s, 1H),
.25-1.96 (m, 6H), 1.73-1.20 (m, 9H), 1.03 (s, 3H), 0.81 (d, J = 6.8 Hz, 3H), 0.61 (d, J =
6.8 Hz, 3H). MS: 527 (M-1)-.
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Method B. Use of Pleuromutilin tosylate 7. Preparation of
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((7-fluoro-1-hydroxy-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (15)
0
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9
0
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9
0
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9
0
To a solution of 7-fluorobenzo[c][1,2]oxaborole-1,6(3H)-diol 80 (5.0 g, 29.77 mmol, 1.0
eq) and 7 (18.9 g, 38.71 mmol, 1.3 eq) in DMSO (60.00 mL) was added Na CO (9.5 g,
2
3
8
9.32 mmol, 3.0 eq). The mixture was stirred at 35 °C for 14 hours under a nitrogen
atmosphere, after which time HPLC indicated that starting material was consumed
completely. The reaction mixture was quenched by addition H O (200 mL) at 0°C, and
2
then was adjusted to pH = 7 and then filtered to give crude product. Combined four
batches together, and the crude product was purified by prep HPLC, then removed the
acetonitrile, resulting aqueous phase was extracted by dichloromethane (3  1500 mL).
The combined organic layers were concentrated under reduced pressure to give the
product as a light yellow solid. This product was dissolved in dichloromethane, then
MTBE and petroleum ether was added until the product precipitated. The suspension was
filtered and the filtrate was concentrated under reduced pressure to give additional
product as a white solid. (3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-
tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((7-
fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate 15 (15.0 g, 28.2
1
mmol, 24% yield, 99.0% purity) was obtained as a white solid. H NMR (DMSO-d6, 400
MHz) δ 9.26 (br. s., 1H), 7.25-7.17 (m, 1H), 7.09 (d, J = 7.9 Hz, 1H), 6.11 (dd, J = 11.2,
1
7.9 Hz, 1H), 5.60 (d, J = 7.9 Hz, 1H), 5.10-4.98 (m, 2H), 4.92 (s, 2H), 4.86-4.74 (m,
2H), 4.52 (br. s., 1H), 3.41 (br. s., 1H), 2.41 (br. s., 1H), 2.25-2.01 (m, 4H), 1.70-1.57 (m,
2
3
H), 1.48-1.20 (m,8H), 1.15-0.91 (m, 4H), 0.82 (d, J = 6.6 Hz, 3H), 0.62 (d, J = 7.1 Hz,
H). MS (ESI): mass calcd. for C29H38BFO7 528.27, m/z found 527.3[M-H]-. HPLC:
99.0% (220 nm), 100.0% (254 nm).
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
(
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((7-chloro-1-hydroxy-1,3-
dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (16).
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Prepared from 6 by Method A. H NMR (400 MHz, DMSO-d ) δ 9.06 (s, 1H), 7.18 (d, J
6
= 8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.04 (dd, J = 17.6, 11.2 Hz, 1H), 5.53 (d, J = 7.2
Hz, 1H), 5.02 (m, 2H), 4.84 (s, 2H), 4.79 (s, 2H), 4.49 (d, J = 6.4 Hz, 1H), 3.35 (m, 1H),
2
.35 (s, 1H), 2.11-1.96 (m, 4H), 1.61-1.17 (m, 10 H), 0.98-0.93 (m, 4 H), 0.74 (d, J = 6.8
1
1
1
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2
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3
3
3
4
4
4
4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
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8
9
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7
8
9
0
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3
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7
8
9
0
Hz, 3H), 0.58 (d, J = 6.8 Hz, 3H). MS (ESI): mass calcd. For C H BO Cl 544.87, m/z
2
9
38
7
-
found 543.2 [M-H] . HPLC: 100% (220 nm), 100% (254 nm).
(
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-7-methoxy-
1
,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (17).
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ 9.20 - 8.94 (m, 1H),
6
7
.03 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.12 (dd, J = 11.5, 17.6 Hz, 1H), 5.61
d, J = 8.4 Hz, 1H), 5.10-5.00 (m, 2H), 4.89 (s, 2H), 4.74-4.60 (m, 2H), 3.93 (s, 3H), 3.42
(d, J = 6.2 Hz, 1H), 2.41 (br. s., 1H), 2.24-1.99 (m, 4H), 1.71-1.20 (m, 11H), 1.12-0.94
(
(
m, 4H), 0.82 (d, J = 7.1 Hz, 3H), 0.63 (d, J = 6.6 Hz, 3H). MS (ESI): mass calcd. for
-
C H BO 540.3, m/z found 539.3 [M-1] . HPLC: 100% (220 nm), 100% (254 nm).
3
0
41
8
(
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((5,7-difluoro-1-hydroxy-
1
,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (18).
1
Prepared from 6 by Method A. H NMR (DMSO-d , 400 MHz) δ7.16 (d, J = 10.1 Hz,
6
1
4
1
1
H), 6.05 (dd, J = 11.5, 17.6 Hz, 1H), 5.57 (d, J = 7.9 Hz, 1H), 4.97 (d, J = 6.6 Hz, 1H),
.93 (s, 1H), 4.89 (s, 2H), 4.71 (d, J = 19.0 Hz, 2H), 3.36 (d, J = 6.2 Hz, 1H), 2.34 (br. s.,
H), 2.20-2.09 (m, 1H), 2.08-1.94 (m, 3H), 1.67-1.51 (m, 3H), 1.41 (d, J = 17.2 Hz, 1H),
.36-1.14 (m, 6H), 1.03-0.90 (m, 4H), 0.78 (d, J = 7.1 Hz, 3H), 0.53 (d, J = 6.6 Hz, 3H).
-
MS (ESI): mass calcd. For C H BF NO 546.3, m/z found 545.2[M-H] . HPLC: 97.3%
2
9
37
2
7
(220 nm), 94.0% (254 nm).
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((3-(aminomethyl)-1-
hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate hydrochloride (19).
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Prepared from 7 by Method B. H NMR (DMSO-d , 400 MHz) δ 9.47 (br. s., 1H), 8.13
6
(br. s., 3H), 7.44 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.09 (td, J = 2.4, 8.5 Hz,
1
1
2
H), 6.10 (dd, J = 11.2, 17.9 Hz, 1H), 5.60 (d, J = 8.4 Hz, 1H), 5.28 (dd, J = 2.6, 9.3 Hz,
H), 5.12-4.96 (m, 2H), 4.80-4.64 (m, 2H), 4.62-4.48 (m, 1H), 3.42 (d, J = 5.7 Hz, 2H),
.78-2.63 (m, 1H), 2.42 (br. s., 1H), 2.26-2.00 (m, 4H), 1.73-1.18 (m, 7H), 1.05 (s, 2H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.89-0.78 (m, 5H), 0.63 (d, J = 7.2 Hz, 3H). MS (ESI): mass calcd. for C H BNO
7
3
0
42
+
5
39.31, m/z found 540.1[M+H] . HPLC: 99.2% (220 nm), 100.0% (254 nm).
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((4-(aminomethyl)-1-
hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate hydrochloride (20).
1
Prepared from 7 by Method B. H NMR (DMSO-d , 400 MHz)  9.19 (s, 1H), 8.30 (br.
6
s., 3H), 7.26-7.17 (m, 2H), 6.11 (dd, J = 11.2, 17.9 Hz, 1H), 5.62 (d, J = 8.8 Hz, 1H),
5
.15-4.95 (m, 4H), 4.79-4.65 (m, 2H), 4.55 (br. s.,1H), 3.93 (d, J = 5.3 Hz, 2H), 3.43 (br.
s., 2H), 2.44 (d, J = 7.9 Hz, 1H), 2.26-2.01 (m, 4H), 1.73-1.19 (m, 10H), 1.12-0.94 (m,
4
H), 0.82 (d, J = 7.1 Hz, 3H), 0.65 (d, J = 7.1 Hz, 3H). MS (ESI): mass calcd. for
+
C H BClNO 575.3, m/z found 540.4 [M+H] . HPLC: 96.1% (220 nm), 91.0% (weak
3
0
43
7
absorption at 254 nm).
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
(
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((3-(aminomethyl)-7-
chloro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate hydrochloride
(21).
1
Prepared from 7 by Method B. H NMR (400 MHz, DMSO-d ) δ 9.31 (s, 1H), 8.13 (s,
6
3
H), 7.40 (d, J = 8.4 Hz, 1H), 7.18 (t, J = 8.4 Hz, 1H), 6.15-6.08 (m, 1H), 5.60 (d, J=7.2
Hz, 1H), 5.29 (d, J = 8.0 Hz, 1H), 5.14-4.57 (m, 5H), 3.47-3.38 (m, 3H), 2.87 (b, 1H),
2.42 (s, 1H), 2.23-2.02 (m, 4H), 1.67-1.05 (m, 14H), 0.82 (d, J = 6.8 Hz, 3H), 0.63 (d, J =
6
5
.8 Hz, 3H). HPLC purity: 100% (220 nm); MS (ESI): mass calcd. for C H BClNO
30 41 7
73.27, m/z found 574.2 [M+H]+.
(
3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((3-(aminomethyl)-7-
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fluoro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate hydrochloride
(22).
1
Prepared from 7 by Method B. H NMR (DMSO-d , 400 MHz) δ 9.51 (brs., 1H), 8.14
6
(brs, 3H), 7.27-7.22 (m, 2H), 6.11 (dd, J = 10.8, 18.0 Hz, 1H), 5.60 (d, J = 7.6 Hz, 1H),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.30 (d, J = 8.8 Hz, 1H), 5.06 (d, J = 18.0 Hz, 1H), 5.03 (d, J = 10.8 Hz, 1H), 4.86-4.82
(m, 2H), 2.84-2.82 (m, 2H), 2.41 (s, 1H), 2.18-2.05 (m, 4H), 1.64-1.20 (m, 9H), 1.09-
0
.94 (m, 4H), 0.83-0.79 (d, J = 6.8 Hz, 3H), 0.63 (d, J = 6.8 Hz, 3H). MS (ESI): mass
+
calcd. for C H BClFNO 593.3, m/z found 558.0(M+H) . HPLC: 99.0% (220 nm),
3
0
42
7
9
6.3% (254 nm).
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((4-(aminomethyl)-7-
chloro-1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (23).
1
Prepared from 7 by Method B. H NMR (400 MHz, DMSO-d ) δ 9.04 (s, 1H), 7.17 (s,
6
1
H), 6.16-6.13 (m, 1H), 5.61 (d, J=8.0 Hz, 1H), 5.11-5.01 (m, 2H), 4.97 (s, 2H), 4.822 (d,
J= 4.0 Hz, 2H), 4.54 (d, J = 6.0 Hz, 1H), 4.46-4.44 (m, 2H), 3.62 (s, 2H), 2.43 (s, 1H),
.23-2.04 (m, 4H), 1.68-0.97 (m, 14H), 0.81 (d, J = 7.2 Hz, 3H), 0.66 (d, J = 6.8 Hz, 3H).
HPLC purity: 100% (214 nm); MS (ESI): mass calcd. For C H BClNO 573.27, m/z
2
3
0
41
7
found 573.8 [M+H]+.
(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-
vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-((1-hydroxy-3,3-dimethyl-
1
,3-dihydrobenzo[c][1,2]oxaborol-6-yl)oxy)acetate (24).
1
Prepared from 7 by Method B. H NMR (DMSO-d , 400 MHz) δ 8.92 (s, 1 H), 7.29 (d, J
6
=
8.4 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.4, 2.4 Hz, 1H), 6.09 (dd, J = 17.6,
1
6
1.2 Hz, 1H), 5.59 (d, J = 8.4 Hz, 1H), 5.08-4.95 (m, 2H), 4.75-4.61 (m, 2H), 4.51 (d, J =
.0 Hz, 1H), 3.45-3.38 (m, 1H), 2.40 (s, 1H), 2.25-1.98 (m, 5H), 1.73-1.55 (m, 3H), 1.52-
1.15 (m, 12H), 1.08-0.98 (m, 4H), 0.81 (d, J = 7.0 Hz, 3H), 0.62 (d, J = 6.8 Hz, 3H). MS
-
(
ESI): mass calcd. for C H BO7 538.3, m/z found 537.2 [M-1] . HPLC: 99.3% (220
3
1
43
3
nm), 100% (254 nm).
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