Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2)

Article abstract of DOI:10.1016/j.bmcl.2009.02.009

We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In add

Full text of DOI:10.1016/j.bmcl.2009.02.009

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2053–2058
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring the pharmacokinetic properties of phosphorus-containing selective
HDAC 1 and 2 inhibitors (SHI-1:2)
c
a
a
b
Richard W. Heidebrecht Jr. a, , Melissa Chenard , Joshua Close , William K. Dahlberg , Judith Fleming ,
Jonathan B. Grimm a, Julie E. Hamill a, Andreas Harsch a, Brian B. Haines c, Bethany Hughes a, Astrid M. Kral b,
Richard E. Middleton a, Chandrasekhar Mushti a, Nicole Ozerova b, Alexander A. Szewczak a,
Hongmei Wang b, Kevin Wilson a, David J. Witter a, J. Paul Secrist b, Thomas A. Miller a
*
a Department of Drug Design and Optimization, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
b Department of Cancer Biology and Therapeutics, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
c Department of Oncology Pharmacology, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the preparation and structure–activity relationships of phosphorus-containing histone deace-
tylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior
mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor
growth inhibition in xenograft studies.
Received 3 December 2008
Revised 2 February 2009
Accepted 3 February 2009
Available online 8 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pharmacokinetics
Selective histone deacetylase inhibitor
Zolinza
Xenograft
Phosphorus-containing therapeutic
The control of gene expression is maintained by direct chemical
modifications to DNA and control of chromatin structure. The ‘his-
tone code’ of chromatin is represented by differential methylation
of lysine and arginine residues combined with acetylation state of
lysine residues on the four types of histones (H1–H4). These epige-
netic signals, combined with DNA modifications such as CpG island
methylation, represent a complex and tunable mechanism for gene
expression. Analogous to mutations of the primary structure of
DNA, aberrant epigenetic changes have been linked to a growing
number of human diseases, including cancer.1
complex underlying biology of HDACs, combined with the first
clinically successful inhibitor ZolinzaÒ, makes understanding the
systematic modification of the action of these enzymes an impor-
tant field in medicinal chemistry.
ZolinzaÒ became the first HDAC inhibitor to gain approval in the
United States, and is indicated for the treatment of cutaneous man-
ifestations of cutaneous T-cell lymphoma (Fig. 1).3 In the ortho-
amino benzamide class of HDAC inhibitor, MS-275 has entered
clinical trials for several oncological indications.4 Another example
Histone deacetylases (HDACs), in combination with histone
acetylases (HATs), are important regulators of epigenetic equilib-
rium.2 The eleven known zinc-dependent HDACs are divided into
three classes based on sequence homology. Class I enzymes
(HDACs 1–3, 8) are predominantly found in the nucleus and consist
of 350–500 amino acid residues, while class II examples (HDACs 4–
7, 9–10) are tissue specific, shuttle between the cytoplasm and the
nucleus, and are about 1000 amino acids in length. The single
member of the class III HDACs (HDAC 11) contains two active sites,
and displays characteristics of both class I and II enzymes. The
O
H
Zolinza®
MS-275
N
OH
N
H
O
O
N
N
H
N
H
O
NH2
O
* Corresponding author.
E-mail address: richard_heidebrecht@merck.com (R.W. Heidebrecht Jr.).
Figure 1. HDAC inhibitors ZolinzaÒ and MS-275.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.009

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R. W. Heidebrecht et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2053–2058
from this class is 1, a potent ortho-amino benzamide malonate
developed in our laboratory with a structurally distinct surface rec-
ognition domain.5 Our most recent reports are exemplified by
biphenyl SHI-1:2 2 (Fig. 2),6 which projects an aryl ring into the
internal cavity of HDACs 1 and 2 (putatively not accommodated
by other HDAC’s), resulting in excellent selectivity over the HDAC3
isoform.7
O
O
a
OMe
OMe
R2
P
O
R1
Br
3
4
As exemplified by the inhibitors above (Figs. 1 and 2), we looked
to further expand our understanding of moieties that optimize
interactions at the surface recognition domain.8 It has been noted
that phosphonates and malonates can be bioisosteric.9 With this
in mind, we pursued the synthesis of derivatives containing both
phosphorus-based functional groups in the surface recognition do-
main and a biphenyl warhead to facilitate HDAC 1:2 selectivity.
Our initial synthetic efforts were directed toward simple aryl
and benzylically oriented phosphorus-containing inhibitors. The
aryl examples were prepared via nickel-catalyzed cross coupling
(Scheme 1). A combination of methyl 4-bromobenzoate (3), a
phosphonite (PR(OR)2), and catalytic nickel(II) bromide was heated
at elevated temperatures without solvent to afford phosphinate
4.10 Saponification, amide formation, and deprotection led to our
target compounds 6. Phosphonate 6a (R1, R2 = OEt) was prepared
via a truncated route from the corresponding commercially avail-
able substituted benzoate.11
R1 = Alkyl, Alkoxy
R2 = Alkyl
b, c
S
S
O
N
R2
R1
H2N
H
NHY
NHBoc
P
O
7
5, Y = Boc
6, Y = H
d
Scheme 1. The synthesis of aryl phosphorus derivatives 6a, c, d, e, and f. Reagents
and conditions: (a) NiBr2, phosphonite (RP(OR)2), 160 °C, neat; (b) LiOH, THF/
MeOH/water; (c) 7, EDC, DMF; (d) TFA, DCM.
While many of the phosphonites (PR(OR)2) and phosphinites
(PR2(OR)) required to prepare the requisite derivatives were com-
mercially available, the remainder were prepared via displacement
of the corresponding chloride (Scheme 2). Reactants required for
cross-couplings (e.g., Scheme 1) were prepared in pentane, filtered,
and concentrated.12 Those designated for benzylic derivatives (e.g.,
Scheme 3) were synthesized in toluene, filtered to remove amine
salts, and directly used in subsequent reactions.
ROH
RP(OR)2
R2POR
RPCl2
R2PCl
Et3N
ROH
Et3N
The benzylic examples (e.g., Scheme 3) were prepared via a
Michaelis-Arbuzov approach.13 Bromomethylbenzoyl bromide 8
was treated with monoprotected diamine 7 to afford amide 9
(Scheme 3). This key intermediate (9) was reacted with a range
of phosphites, phosphonites, and phosphinites to afford, after
deprotection, target compounds 11.
The phosphonic and phosphinic acid derivatives were prepared
via hydrolysis of the corresponding methoxy and ethoxy phospho-
nates and phosphinates with NaOH in dioxane at elevated temper-
atures.14 This reaction was performed on the deprotected
compounds, affording 6b and 11b/f wherein R2 = OH.
Scheme 2. The preparation of phosphonites (PR(OR)2) and phosphinites P(R)2(OR).
O
S
Br
Br
8
O
7
N
H
Br
a
NHBoc
9
b
O
S
NH
O
N
H
O
NH2
R1, R2 = alkyl, alkoxy,
aryl
O
P
O
R2
R1
N
H
NH
NHY
1
10, Y = Boc
11, Y = H
c
Scheme 3. The synthesis of benzylic phosphorus derivatives 11a, c, e, g–k, m–p.
Reagents and conditions: (a) 8, iPr2NEt, DCM; (b) phosphite (P(OR)3), phosphonite
(RP(OR)2), or phosphinite ((R)2POR), toluene,
D; (c) TFA, DCM.
internal
cavity
2
O
To facilitate late-stage generation of structural diversity, a se-
quence was developed beginning with protected benzylically-ori-
ented intermediate 10 (Scheme 4). Methoxy-substituted
phosphonates and phosphinates (10) were hydrolyzed, and their
corresponding acids were subjected to BOP15 coupling; deprotec-
tion provided target compounds 11.
N
N
H
H
N
O
NH2
O
surface
recognition
linker
metal
binding
Other structurally unique examples were prepared during the
course of this work. For example, amino phosphonate 13 proved
Figure 2. HDAC inhibitors 1 and 2 superimposed with the pharmocophore.

R. W. Heidebrecht et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2053–2058
2055
Gratifyingly, HDAC inhibitors with a phosphorus-based surface
recognition element generally led to compounds with in vitro
activity in the low nanomolar range (Table 1). Both aryl and benzy-
lically-substituted examples were potent inhibitors of HDAC1 en-
zyme and proliferation in HDAC-dependent HCT116 cells17 across
three phosphorus oxidation states: phosphonates, phosphinates,
and phosphine oxides.
Para-substituted benzamides were generally more potent
inhibitors of cell proliferation than their analogous meta examples
with corresponding differences in measured GI50 values; compare
S
O
10
a, b, c
R1 = OMe,
(preferentially)
O
P
N
H
R2
NH2
R1
R2 = alkyl, alkoxy, aryl
11
R1 = alkoxy
Scheme 4. The synthesis of phosphonate and phosphinate ester libraries, including
examples 11a and 11c (GI50 = 0.250, 2.50
and 11g (GI50 = 0.360, 8.60 M, respectively), 11h and 11i
(GI50 = 0.130, 6.00 M, respectively), 11j and 11k (GI50 = 0.200,
>20.0 M, respectively). It was also apparent that the acids of both
lM, respectively), 11e
compound 11l. Reagents and conditions: (a) NaOH, dioxane,
D
; (b) Alcohol R1OH,
l
BOP, iPr2NEt, DMF; (c) TFA, DCM.
l
l
phosphonates (6b, HDAC1 IC50 = 42nM and 11b, HDAC1 IC50 = 38
nM) and phosphinates (11f, HDAC1 IC50 = 38 nM) were potent in
the cell free enzyme assay but were not effective in the inhibition
S
O
EtO P
EtO
O
of immortalized cell growth (GI50 > 20
lack of cell permeability.
lM), presumably due to
NH2
N
H
It should be noted that several examples were prepared that
varied the biphenyl aryl ring. Similar properties were observed be-
tween phenyl, 2-thiophene, and 3-thiophene substituents.18 The 2-
thiophene ‘warhead’ was utilized due to a general trend affording
modest advantages in GI50 in HCT-116 cells.
Br
NHBoc
12
9, para
a, b
With a range of potent compounds in hand, we turned to eval-
uate our leads in pharmacokinetic (PK) and pharmacokinetic/phar-
macodynamic (PK/PD) assays. Compound 11a demonstrated
promising PK in the rat, possessing relatively low plasma clearance
(Clp = 15.8 mL/min/kg) and acceptable oral bioavailability and
S
O
exposure (F = 45%, P.O. AUC(0–1) = 4.40 lM h). These results
O
EtO P
EtO
N
H
H
N
prompted us to assess the acetylation state of H2B histones after
dosing of HCT116 tumor-bearing nude CD-1 mice at 150 mg/kg
qd for three days. Compound 2 was used as a positive control.
Unfortunately, initial lead 11a did not show a significant increase
in acetylation relative to vehicle (Fig. 3).19 This can be explained
by the low exposure of 11a achieved after ip administration
NH2
13
Scheme 5. The synthesis of aminophosphonate 13. Reagents and conditions: (a)
iPr2NEt, DMF, 50 °C; (b) TFA, DCM.
(1.54 lM in plasma, 3 h post-dose), suggesting that the compound
had high clearance in mice. The pharmacokinetics of 11a in mouse
and dog were assessed, revealing clearance greater than hepatic
blood flow in both species.
A significant improvement in mouse PK was required in order to
determine the in vivo potency and efficacy of our phosphorus-
to be particularly promising; its synthesis is depicted in Scheme 5.
Key benzyl bromide 9 (4-substituted) was treated with diethyl
(aminomethyl) phosphonate (12)16 and deprotected to afford tar-
get 13 (Scheme 5).
Table 1
Exploration of phosphorus-based functional groups
S
6, n = 0
O
11, n = 1
O
R2
R1
P
N
H
NH2
n
Compound
n
Substitution
R1
R2
HDAC1 IC50 (nM)
GI50 HCT116 (lM)
Phosphonates
Phosphonites
6a
6b
11a
11b
11c
0
0
1
1
1
4
4
4
4
3
OEt
OEt
OEt
OEt
OEt
OEt
22
42
13
38
23
0.410
>20
0.250
>20
OH
OEt
OH
OEt
2.5
6c
6d
0
0
1
1
1
1
1
4
4
4
4
3
4
3
OEt
Me
Ph
Ph
Ph
Ph
Et
14
29
20
28
140
7
0.46
0.38
0.36
>20
8.6
0.130
6.0
OMe
OMe
OH
OMe
OMe
OMe
11e
11f
11g
11h
11i
Et
90
Phosphine oxides
11j
11k
1
1
4
3
Et
Et
Et
Et
9
270
0.20
>20

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R. W. Heidebrecht et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2053–2058
New chemical entities were dosed orally at 150 mg/kg, and mea-
surements were taken from whole blood collected by nicking the
tail vein at six time points from 0.25 h to 7 h.
An analysis of the mouse pharmacokinetic data from phospho-
rus-containing inhibitors indicated a strong trend where smaller
substituents led to compounds with superior exposure (Table 2).
In the analogs where there is an aryl-phosphorus bond (6e–6f),
Rat (Sprague-Dawley)
AUC: 4.40 µM h
Cl: 15.8 mL/min/Kg
Vdss: 7.27 L/kg
t1/2: 5.5h
S
O
O
EtO P
EtO
N
H
Mouse (Swiss-Webster)
AUC: 0.17 µM h
Cl: >Hept
Vdss: 20.1 L/kg
t1/2: 55 h
NH2
11a
going from a phenyl/ethoxy substitution (6e, AUC = 0.91
lM h) to
a methyl/ethoxy phosphinate (6c, AUC = 44 M h) resulted in a
l
marked improvement in maximum exposure. These aryl examples
did not appear particularly sensitive to the identity of the alkoxy
groups, exemplified by a small difference between methoxy (6f,
HDAC1 IC50: 13 nM
HCT116 GI50: 250 nM
PK/PD(mouse): negative
Dog (Beagle)
AUC: 0.25 µM h
Cl: >Hept
Vdss: 2.85 L/kg
t1/2: 0.43 h
AUC = 60
lM h) and ethoxy (6c, AUC = 44 lM h) phosphinates
when the alkyl group remained constant (Me).
The benzylically-substituted phosphinate examples demon-
strated a similar effect (Table 2, 11l–11o). The largest effect was
observed on the alkoxy group of these phosphinates. As the alkyl
group is held as a methyl, a constant and remarkable improvement
in exposure is observed when going from larger phosphinate esters
Figure 3. Summary of data for compound 11a.
based inhibitors. A streamlined assay to facilitate the weekly
assessment of oral exposure of compounds in mice was designed.
such as 3-pyridylmethyl analog 11l (AUC = 16 lM h) to ethoxy
Table 2
The critical relationship between the groups on phosphorus and mouse PK parameters
S
O
N
H
NH2
R
Compound
R
HDAC1 IC50 (nM)
GI50 HCT116(
l
M)
Cmax
(l
M)
AUC (lM h)
EtO
P
O
Ph
6e
21
29
14
52
3.5
6.5
0.170
0.56
0.91
MeO
Ph
6d
0.380
0.460
0.420
0.130
0.240
11
43
39
12
P
O
EtO
Me
6c
P
44
60
16
17
O
MeO
Me
6f
P
O
O
P
Me
11l
11m
8.8
O
N
O
17
31
88
P
EtO
Et
O
P
Me
11n
11o
11p
11j
16
54
11
8.5
0.330
0.160
0.270
0.200
50
EtO
O
P
115
MeO
Me
O
P
iPr
1.9
1.3
iPr
O
14
17
P
Et
Et

R. W. Heidebrecht et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2053–2058
2057
compound 11n (AUC = 50
tained with methoxy example 11o (AUC = 115 lM h).
l
M h). The most dramatic result was ob-
S
Minor improvements in AUC were observed when smaller
phosphorus-linked alkyl groups were installed. This is exemplified
O
by
a
comparison of ethyl-substituted phosphinate 11m
N
H
(AUC = 17
(AUC = 50
lM h) with the closely related methyl derivative 11n
NH2
R
l
M h). The same trend was observed for phosphine oxi-
des: 4-diisopropyl-phosphinomethyl (AUC = 1.3
much reduced exposure compared to 4-diethylphosphinomethyl
11j (AUC = 17 M h).
Promising compounds were tested in the PK/PD model for his-
tone acetylation (HCT116 tumor-bearing nude CD-1 mice,
150 mg/kg, po, qd, 3 days), and three examples are depicted below
(Fig. 4). Phosphinate 11n did not demonstrate changes to the ratio
of acetylated to deacetylated H2B histones relative to control;
however, a statistically significant response was demonstrated by
both phosphinate 11o and aminophosphonate 13.
Leads 11o and 13 were further tested in a mouse xenograft
model (HCT116 colon) at various daily oral doses for 21 days
(Fig. 5), and dose-dependent inhibition of tumor growth was ob-
served. Unfortunately, mice treated with 11o also experienced sig-
nificant and dose proportionate weight loss (22% loss in the
100 mg/kg cohort after 21 days), affording a very small therapeutic
window. On the other hand, compound 13 was very well tolerated
at all doses, illustrating the potential utility of phosphorus based
HDAC inhibitors for the treatment of cancer.
lM h) 11p gave
Compound
11n
R
Data Summary
l
HDAC1 IC50: 16 nM
O
P
Me
HCT116 GI50: 0.330 µM
PD(mouse): negative
EtO
Time Post Dose (h): 24
HDAC1 IC50: 54 nM
HCT116 GI50: 0.160 µM
PD(mouse): positive
O
P
Me
11o
13
MeO
Time Post Dose (h): 24
HDAC1 IC50: 7.7 nM
HCT116 GI50: 0.300 µM
PD(mouse): positive
O
P
EtO
H
N
EtO
Time Post Dose (h): 8
Figure 4. Summary of data for compounds 11n, 11o, and 13.
410
360
310
260
210
160
110
60
The phosphorus-containing biphenyl inhibitors disclosed in this
work possess the excellent selectivity for HDACs 1 and 2 that was
previously observed.7b Full data for compound 13 is provided as an
example (Fig. 6).20 A minimum of three logs of selectivity separates
the in vitro HDAC1 (IC50 = 7.7 nM) and HDAC2 (IC50 = 14 nM) po-
tency of 13 from other class I isoforms, i.e., the closely related
HDAC3 (IC50 = 12,000 nM) as well as HDAC8 (IC50 = 37,000 nM).
Excellent selectivity was observed over class II and III enzymes
(HDACs 4–7, 11, IC50 = >50,000 nM).
In summary, we observed a disconnect between rat and mouse
pharmacokinetics of phosphorus-containing SHI-1:2 analogs. A
survey of mouse PK parameters showed a strong correlation be-
tween smaller alkyl phosphorus-based groups and superior oral
exposure. Sample compound (13) showed promising and signifi-
cant tumor growth inhibition in a xenograft model without signif-
icant weight loss.21
Vehicle
11o, 50 mg/kg
11o, 100 mg/kg
2-way ANOVA
*** : P < 0.0001
***
1
4
7
10
13
16
19
22
Days after beginning treatment
350
300
250
200
150
100
50
Vehicle, Qd
13, 150 mg/kg
13, 300 mg/kg
2-way ANOVA analysis
*** : P < 0.001
Acknowledgements
We would like to express our appreciation to Sujal Deshmukh
and Dapeng Chen for assistance with pharmacokinetic studies.
***
References and notes
1
4
7
10
13
16
19
22
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S
14 nM
12000 nM
>50000 nM
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>50000 nM
>50000 nM
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O
N
H
O
EtO P
EtO
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:
13
Figure 6. Full HDAC selectivity data for 13.

2058
R. W. Heidebrecht et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2053–2058
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0.460 lM) substituents.
19. Statistical significance was demonstrated via t-test analysis looking for a P-
value of <0.05 relative to vehicle.
20. For assay details, see reference within Jones, P.; Altamura, S.; Chakravarty, P. K.;
Cecchetti, O.; De Francesco, R.; Gallinari, P.; Ingenito, R.; Meinke, P. T.;
Petrocchi, A.; Rowley, M.; Scarpelli, R.; Serafini, S.; Steinkühler, C. Bioorg. Med.
Chem. Lett. 2006, 16, 5948.
21. The compounds described in this paper are the topic of a patent application.
The reader is directed to this publication for experimental information:
Close, J.; Grimm, J.; Heidebrecht, R. W.; Kattar, S.; Miller, T. A.; Otte, K. M.;
Peterson, S.; Siliphaivanh, P.; Tempest, P.; Wilson, K. J.; Witter, D. J.; WO
2008010985.