Lead diversification. Application to existing drug molecules: Mifepristone 1 and antalarmin 8

Article abstract of DOI:10.1016/j.bmcl.2011.10.066

A series of C-H functionalisation plate-based chemical screens and other C-H activation protocols were developed for the chemical diversification of drug molecules. In this Letter, metalloporphyrin and other catalytic oxidation systems are described in ad

Full text of DOI:10.1016/j.bmcl.2011.10.066

Bioorganic & Medicinal Chemistry Letters 22 (2012) 723–728
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead diversification. Application to existing drug molecules: Mifepristone 1
and antalarmin 8
M. Abid Masood , Elizabeth Farrant, Inaki Morao, Marc Bazin 1, Manuel Perez, Mark E. Bunnage,
⇑
Sally-Ann Fancy, Torren Peakman
Worldwide Medicinal Chemistry, Pfizer Worldwide R&D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of C–H functionalisation plate-based chemical screens and other C–H activation protocols were
developed for the chemical diversification of drug molecules. In this Letter, metalloporphyrin and other
catalytic oxidation systems are described in addition to chlorination. Mifepristone and antalarmin are
used as substrates. The products obtained and the biological data demonstrate the potential utility of this
approach.
Received 17 June 2011
Revised 10 October 2011
Accepted 11 October 2011
Available online 25 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Biomimetic
Lead diversification
Metalloporphyrin
Mifepristone
Antalarmin
Structure–activity relationships (SAR) around a given chemical
series is obviously limited to compounds that can actually be
synthesised and tested in biological systems. Novel synthetic
methodology which enables small subtle changes to complex lead
molecules in a single step, for example, through the introduction of
functional groups such as F, Cl, Me, OH, OMe, and CN would there-
fore be an extremely valuable tool. An arsenal of such chemical
transformations could have advantages, such as beneficial tuning
of physiochemical properties, blocking of metabolically vulnerable
sites, or removal of toxicophores. As part of the drive within the
pharmaceutical industry to find more efficient methods for opti-
mising leads and reduce cycle times, the concept of lead diversifi-
cation was explored in a drug discovery paradigm.
Lead diversification (LD) can be defined as the functionalisation
of test compounds of around 450–500 MW using chemical or bio-
chemical means to generate novel close-in analogues and ex-
panded SAR. It is a different approach to chemical synthesis
compared to the more traditional building block methodology
where functionality is built in from the beginning. Typically trans-
formations to introduce small functional groups such as OMe, OH,
CN, F, Cl, Me, and deuterium via C–H activation were investigated.
It was decided to apply this LD approach to late stage lead com-
pounds for several reasons including the previously reported use
of biomimetic oxidation1 of drug molecules using metalloporphy-
rins to generate metabolites.
C–H activation is a rapidly expanding area of research and
some of the novel chemistry described in the literature (e.g.,
Sanford’s palladium-catalysed C–H activation2 or iridium cata-
lysed borylation3) had been reported using simple test systems
such as N-phenylpyrazole. It was necessary to explore the
breadth and limitations of these synthetic transformations in
the context of real drug molecules and on novel substrates.
Our strategy was to find synthetic methodologies ideally for a
plate based approach, but also for use as discrete reactions because
inert atmosphere reactions are often not conducive to plate based
methods. A survey of the literature was carried out to find methods
for oxidation, fluorination, palladium and copper-catalysed C–H
activation and other chemical methods for introduction of CN, Cl,
Me, OMe either via metal-catalysed C–H activation or by electro-
philic substitution.
The first step in the LD platform was to evaluate the structure
of the test compound to determine which LD chemistries could be
used based on synthetic experience and knowledge. For example,
if there were alkyl groups, the BMO screen was used. For aro-
matic groups, the fluorination screen incorporating the chlorina-
tion conditions was used. If there was a Lewis basic sp2 centre
that could be used to coordinate for C–H activation, then copper
or palladium-catalysed chemistry was attempted (Fig. 1).
Followingstructural analysis, therequisite chemistry was carried
out. In the case of the BMO screen and other plate based screens, the
data obtained indicated the conditions and catalyst for a scale-up
⇑
Corresponding author.
E-mail addresses: mabidmasood@gmail.com, abid.masood@pfizer.com (M. Abid
Masood).
1
Current address: Hepatochem, 185 Alewife Brook Parkway, # 410, Cambridge, MA
02138, USA.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.066

724
M. Abid Masood et al. / Bioorg. Med. Chem. Lett. 22 (2012) 723–728
Purification and
Analysis of Compound
Structure
LD protocol
Structure Elucidation
Lewis basic centre
with potential to coordinate
to Pd,Cu, Ru
O
O
O
N
N
H
N
N
N
N
O
N
O
N
N
N
N
Aromatic
F, Cl screen
N
N- demethylation
BMO screen
OH
Biological testing
Figure 1. Process flow diagram for LD.
reaction. The reaction was then scaled up to ca. 100–150 mg scale of
substrate. In the case of the metal-catalysed reactions, the crude
reaction mixture was purified and then structure elucidation was
carried out. The pure components were then submitted for biologi-
cal testing. Chemical transformations that were not conducive to
plate based chemistry including ultrasound mediated reaction
benzylic acetylation and iridium catalysed borylation were also
investigated.
While there are a plethora of oxidation catalysts that have been
developed over the years, little work has been done to compare the
reactivity of these oxidation catalysts over a diverse range of sub-
strates to see if a selective catalyst/transformation can be found.
Metalloporphyrin-catalysed oxidations4 have been extensively
investigated over the past 40 years. They have been used for a
variety of transformations such as alkane hydroxylation,5 drug
metabolite synthesis6 and other reactions such as O-dealkylation,
N-dealkylation, benzylic hydroxylation, sulphur and N-oxidation
as well as epoxidation amongst other transformations.7 Metallo-
porphyrins have been synthesized using every first row transition
metal (Fig. 2) and the sheer number and complexity of these metal-
loporphyrins, as well as their different reactivities8 meant that a
combinatorial 96 well plate based chemical screen was necessary
in order to cover a maximum number of conditions and reagents.
There are over one hundred commercially available metallopor-
phyrins for oxidation reactions, however metal centres such as
iron, manganese, ruthenium and chromium are the most widely
reported.
A review of the literature suggested that 9–10 commercial
metalloporphyrins with the above transition metals would be a
good starting point since these have been reported to be the most
active catalysts displaying broad reactivity over a variety of sub-
strates including drugs.9 All the metalloporphyrins were purchased
from commercial suppliers,10 as were the oxidising agents.11 A to-
tal of nine metalloporphyrins and thirteen oxidants were identified
from literature searches. Initially, dimethyl aminoantipyrene 1
(Fig. 3) was used as a control substrate because metabolite studies
had been reported12 and it had previously been used as a substrate
for metalloporphyrin based oxidations.13
After several iterations using this substrate, an optimised plate
consisting 36 wells and three metalloporphyrins: Fe(2-NO2PP)
(MP1), Mn(TDClPP) (MP2) and Fe(PFPP) (MP3) (Fig. 4) giving thirty
six sets of conditions (2 solvents, 6 oxidants and 3 MP’s) (Fig. 4).
The screen was divided into two halves: one side using aprotic sol-
vent and the other side using protic solvent and this gave results as
informative as the whole 9 Â 13 array. Often the products were de-
tected in higher yields than the original screen (based on LCMS
data).
The importance of identification and testing of drug metabolites
at every stage of the drug discovery process, meant that oxidation
methods to synthesise metabolites was a key area of work.
Our LD protocols were trialled on two internal Pfizer programs
initially; the progesterone antagonist14 and corticotrophin releas-
ing factor-1 (CRF1)15 antagonist programs.
Mifepristone16 2, a known progesterone antagonist, is a very
lipophilic steroid with a clogP of 4.65 (LogD7.4 = 4.94) (Fig. 5).
Mifepristone 2 (1 mg) was reacted in the BMO screen17 and
analysis of the LCMS data showed several wells with multiple
Ar
Ar
N
M2+
N
N
O
N
N
N
Ar
Ar
N
Ar = 1,2,3,4,5-pentafluoropheny, M=Fe : Fe(PFPP)
Ar = 2-Nitropheny, M=Fe : Fe(2NO2PP)
Ar = 2,6-Dichlorophenyl, M=Mn : Mn(DClPP)
Dimethylaminoantipyrene
1
Figure 2. Structure of metalloporphyrins.
Figure 3. Dimethyl aminoantipyrene.

M. Abid Masood et al. / Bioorg. Med. Chem. Lett. 22 (2012) 723–728
725
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
MP1
MP2
MP3
H2O2
H2O2
H2O2
H O2
PhIO
PhIO
PhIO
Oxone
Oxone
Oxone
Cum-OOH mCPBA Cum-OOH PhIO
Cum-OOH mCPBA Cum-OOH PhIO
Cum-OOH mCPBA Cum-OOH PhIO
Oxone Cum-OOH mCPBA Cum-OOH
Oxone Cum-OOH mCPBA Cum-OOH
Oxone Cum-OOH mCPBA Cum-OOH
2
H O2
2
H O2
2
protic solvent
Formic acid, imidazole
Aprotic solvent
Cum-OOH is Cumene hydrogen peroxide
Fe[(TNO )PP]
MP1
2
MP2 Mn[TDClPP]
MP3
Fe[TFPP]
Figure 4. Plate map of biomimetic oxidation screen.
Acid 3 was found to be less potent than the parent mifepristone
2. By cleaving the A-ring, the contribution to binding that the
remainder of the molecule makes can be dissected.
N
O
OH
The second well scaled up was cumene hydrogen peroxide and
Fe(2-NO2)PP in 1,2-dichloroethane, the major product isolated in
19% yield was identified as the N-formyl adduct 4. This was equi-
potent with mifepristone 2 and found to be metabolically more
stable, representing a new piece of SAR. While the yield is low
and the reaction gives access to milligram quantities, this is more
than sufficient material to put into many high throughput screens
and generate initial data.
H
H
Mifepristone
2
PR IC 50 : 10nM
Figure 5.
Other oxidation catalysts were also explored such as DuBois’
oxaziridine catalyst18 5 were found to be selective and showed
comparable reactivity to the metalloporphyrin systems in some
examples including mifepristone 2 (Scheme 3).
The BMO screen demonstrated that polar groups can be intro-
duced to lipophilic molecules, quickly without the need to synthe-
size the molecule from suitable starting fragments. In several cases,
products; the wells which contained 1 major product were scaled
up. The first well scaled up was Fe(PFPP), H2O2 in 1,2-dichloroeth-
ane, the major component was isolated, tested and structure eluci-
dation carried out. It was found to be the ketocarboxylic acid 3
formed by oxidative cleavage of the steroid A ring (Scheme 1).
O
H
N
OH
N
O
OH
i
H
H
H
HO
H
O
O
Mifepristone
Pr IC50 : 10nM
Clint : 92.5
2
(32%)
3
PR IC50 : 430nM
ul/min/mg
ii or iii
cLogP : 4.6
cLog D7.4 : 4.9
O
O
O
S
N
O
H
CF3
OH
N
5
H
H
O
4
Pr IC50 : 10nM
Clint : 81
ul/min/mg
Scheme 1. BMO product. Reagents and conditions: (i) H2O2, Fe(PFPP) (10 mol %), dichloroethane, rt 4 h; (ii) Fe(2-NO2)PP (10 mol %), cumene hydroperoxide, DCE, rt, 6 h; (iii)
5 (5 mol %), UreaÁHOOH, PhSeSePh (3 mol %), Microwave (100 °C), 4 h DCE, rt, 6 h.

726
M. Abid Masood et al. / Bioorg. Med. Chem. Lett. 22 (2012) 723–728
N
O
N
OH
OH
H
i
H
H
H
Mifepristone
H
(41%)
O
2
Pr IC50: 10nM
Clint : 92.5 ul/min/mg
cLogP : 4.6
6
PR IC 50 : 307nM
cLog D7.4 : 4.9
ii
Cl
N
OH
Cl
Cl
O
H
H
7
Pr IC50: 771nM
Clint : 76ul/min/mg
cLogP : 6.4
(41%
cLog D7.4 : 6.8
Scheme 2. Benzylic oxidation and elimination. Reagents and conditions: (i) DDQ, acetic acid, ultrasound, rt, 1 h; (ii) cyanuric chloride, CH3CN, rt, 48 h.
this enabled the research team to explore new chemical space.
Moving beyond neutral, lipophilic steroids, more polar compounds
with basic centres were put through the BMO screen where the ma-
jor product was often the N-oxide. In many cases these N-oxides
were the drug metabolites. The BMO screen has proved to be a ver-
satile and invaluable tool for LD.
A second robust oxidative transformation was benzylic oxida-
tion by reaction with dichlorodicyanoquinone (DDQ) using ultra-
sound.19 This was attempted on mifepristone 2 and gave clean
transformation to the triene 6 in 41% isolated yield. Benzylic acet-
ylation followed by elimination had occurred to give the double
bond (Scheme 2).
Initially a computational analysis was carried out to determine
if the selectivity of benzylic hydroxylation’s could be predicted. An
analysis of the regioisomeric radicals resulting from a hydrogen
abstraction at all benzylic and nitrogen a-positions (x,y) was per-
formed.24 The observed regioselectivity was in exact agreement
with the calculations as the experimental oxidation site was where
the most stable radical had formed25 (Fig. 6).
After running the BMO screen, analysis of the LCMS data
showed three wells that had produced several new polar compo-
nents. However, the well that had the cleanest profile was Fe(PFPP)
using m-CPBA as the oxidant and methanol/acetonitrile (1:1) as the
solvent (Scheme 3).
Purification and structure elucidation were carried out. Biolog-
ical testing showed a loss in potency and this is due to the change
in conformation of mifepristone 2. The dimethylaminobenzene
group moves into the plane of conjugation following introduction
of the double bond.
Later investigations focused on chlorination and fluorination.
These atoms introduce small changes to drugs and have been
widely used to moderate pharmacokinetic properties in drug mol-
ecules. Iodination and bromination was not explored as the
changes in molecular weight were too large. A plate-based combi-
natorial chemical screen for fluorination was developed, which
also incorporating chlorination conditions and this will be reported
in later communications. Chlorination of mifepristone 2 using
cyanuric chloride20 in a mixture of tetrahydrofuran and acetoni-
trile (1:5) gave a mixture of products; the major product being
identified as the trichlorinated steroid 7 in 41% yield.
Scale-up of these best conditions on 200 mg of substrate gave a
crude mixture of products with approximately 20% unreacted start-
ing material. After work-up, purification and structure elucidation, it
was found that the three components were all products of oxidation
of the azaindole core on the 2-methyl position: the hydroxymethyl
compound 9, the aldehyde 10, and the methoxy-substituted ana-
logue 11. The methoxy adduct 11 was formed by displacement of
an activated benzylic intermediate by the methanol solvent. Testing
of the compounds in a CRF-1 assay26 showed that the methoxy 11
and hydroxy 9 derivatives were more potent. Evaluation of the phys-
iochemical properties of 9, 10 and 11 showed them to be more polar
The trichlorinated product 7 was seventy fold potent as a pro-
gesterone antagonist, but was more lipophilic and had greater sta-
bility in human liver microsomes.
Several other catalysts used in the screens that showed promis-
ing transformations21 such as alkane hydroxylation included the
DuBois catalyst 5 and RuCl3.22
Attention then turned to antalarmin23 8, which is a drug that
acts as a CRF-1 antagonist. It is a very lipophilic compound with
a logD7.4 of 8.6 and efforts were centered on making a more polar
compound to improve aqueous solubility and pharmacokinetic
properties.
Figure 6. Relative energies (in kcal/mol) of the corresponding neutral radicals
formed by C–H abstraction at the different benzylic positions.

M. Abid Masood et al. / Bioorg. Med. Chem. Lett. 22 (2012) 723–728
727
N
N
N
N
N
N
O
OH
N
N
i
N
N
N
N
(21%)
(12%)
10
9
Antalarmin
8
CRF-1 Ki : 52nM
clogD7.4 : 6
CRF-1 Ki : 6nM
clogD7.4 : 6.2
CRF-1 Ki : 32nM
clogD7.4 : 8.6
N
N
O
N
N
(9%)
11
CRF-1 Ki : 11nM
clogD7.4 : 6
Scheme 3. BMO scale-up of antalarmin to oxidation products. Reagents and Conditions: (i) mCPBA, Fe(PFPP) (10 mol %), MeOH/MeCN (1:1), 6 h, rt.
than the starting compound 8. The carbonyl derivative 10 was
slightly less potent, but was again a more polar compound. An anal-
ysis of the chemical literature showed that these oxidised com-
pounds occupy new chemical space. CRF-1 has been targeted by
several pharmaceutical companies over the past 10 years and it
has been found to find new chemical space around some of the tem-
plates has been synthetically challenging. This example is a proof of
concept for LD and it has been shown that by taking a lipophilic com-
pound and adding small polar groups using a reaction catalyst
screen, several compounds that were structurally close to the start-
ing drug were made. These compounds were more potent, more po-
lar (2 log units) and opened up new chemical space for this target.
Ki values were determined from the IC50 using the Cheng and
Prussoff relationship and the EC50 for the agonist dose–response
curve which was determined on the same day.
With macromolecules and peptides such as cyclosporin A29 and
FK50630 the chemical yields were low (around 10–20%) and there
were several products that proved too difficult to separate.
Structure elucidation was found to be the biggest bottleneck. As
the molecular weight and hence complexity of the starting mate-
rial and products increased, the more tedious and time consuming
the purification and structure elucidation. However, this was bal-
anced by the argument that mifepristone is synthesised in eleven
chemical steps from (+) estrone.31 Similarly antalarmin analogues
generated by BMO versus direct synthesis from start show that
LD is a viable approach. However, it is not a standalone tool and
is dependant on the quality of the substrate. Other screens and
transformations have been developed in collaboration with aca-
demic groups and will be published elsewhere.
Conclusion
Over the course of several screens and substrates it was found
that in some wells, the same product was formed regardless of
the metalloporphyrin used. This indicated that the oxidising agent
was responsible for the reaction and the metalloporphyrin was not
necessary in these cases.
Chemical transformations to functionalise drug molecules in or-
der to obtain SAR can circumvent a long synthesis to make the de-
sired compound. C–H activation is a rapidly expanding area of
research and so LD-type approaches are attractive as they can
cut down the effort of a multistep synthesis of a target molecule.
The examples shown in this report demonstrate proof of principle
for LD and encouraged us to expand the types of transformations
and screens for this platform technology.
In this report it has been shown that functionalization of late
stage molecules is possible in a drug discovery environment and
can potentially give results/SAR that are unexpected as well as
leading project teams into new avenues and directions. The use
of BMO to generate new close analogues has been discussed.
Two case studies show the utility of LD. From a practical stand-
point the main bottlenecks were: scaling-up of reactions, purifica-
tion and structural elucidation. Metalloporphyrin-catalysed
reactions have been reported over the past 30 years with some
examples showing remarkable catalytic turnover and reactivity.27
It should be noted that the quantities made using LD were
more than sufficient to test in multiple screens against many tar-
gets. However, in our hands scaleability was an issue; working on
10–90 mg gave adequate yields but scaling above this quantity
seldom met with success, the reactions had low yields or no
products were detected. There are many reasons for this includ-
ing ‘bleaching’ of the metalloporphyrin by the oxidant rendering
the catalyst inactive.28
Further work will address factors that will lead to more general
applicability. Ease of purification was dependant on molecular
weight and properties of the molecule. As a general rule of thumb:
as the molecular weight increased, the products and starting mate-
rial eluted closer on HPLC and so purification was difficult and
sometimes impossible.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.066.
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a top final assay concentration of 20 lM. The percent (%)
response of the test compound at different test doses were then fitted to a 4-
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determined from the IC50 using the Cheng and Prussoff relationship and the
EC50 for the agonist dose–response curve which was determined on the same
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