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Fragment‐based drug design facilitatedby dynamic combinatorial chemistryandNMRspectroscopy
Peter Kroon, s1687735
18/03/2015
AbstractDrug development is a challenging process, which often fails. To ameliorate this, we attempted to
develop an efficient methodology for fragment‐based drug design based on dynamic combinatorial
chemistry and NMR spectroscopy. To this end, two libraries were designed based on crystallographic
data from the group of Klebe for the model protein endothiapepsin using computer aided modelling.
The designed compounds all feature an acylhdyrazone linker and at least one fluorine atom. The first
library was discarded due to problems with the synthesis. The second library was analysed using
quantitative 19F‐NMR and 1H‐STD‐NMR spectroscopy, using trifluoroacetic acid as an internal
reference for the fluorine NMR spectra. No binding compounds could be identified, even though one
of the compounds was a known binder. It is hypothesised this is due to the trifluoroacetic acid since
the 1H‐STD‐NMR spectrometry also produced no results. However, it could be concluded that 19F‐NMR spectrometry provides better peak resolution with little loss of sensitivity compared to 1H‐NMR spectrometry when analysing mixtures; and that STD‐NMR requires far less protein than
regular NMR spectroscopy for analysing templated DCLs.
2
TableofContentsAbstract ................................................................................................................................................... 1
Table of Contents .................................................................................................................................... 2
Introduction ............................................................................................................................................. 3
Structure‐based drug design ............................................................................................................... 3
Fragment‐based drug design ............................................................................................................... 4
Dynamic combinatorial chemistry....................................................................................................... 5
NMR techniques .................................................................................................................................. 7
Quantitative NMR............................................................................................................................ 7
Saturation‐transfer difference NMR ............................................................................................... 7
Endothiapepsin .................................................................................................................................... 9
Library design ........................................................................................................................................ 10
Modelling ........................................................................................................................................... 14
Discussion .............................................................................................................................................. 16
Quantitative 19F‐NMR ........................................................................................................................ 16
1H‐STD‐NMR ...................................................................................................................................... 16
19F‐STD‐NMR ...................................................................................................................................... 16
Conclusions ............................................................................................................................................ 17
Acknowledgements ............................................................................................................................... 17
Bibliography ........................................................................................................................................... 17
Supporting Information ..........................................................................................................................S1
Library A — Compounds 1 to 8 ..............................................................................................................S1
Modelling methods ............................................................................................................................S1
Modelling results ................................................................................................................................S2
Synthesis .......................................................................................................................................... S11
Library B — Compounds 10 to 18........................................................................................................ S27
Modelling results ............................................................................................................................. S27
NMR experiments ............................................................................................................................ S32
Preparation of NMR samples ...................................................................................................... S32
Quantitative 19F‐NMR .................................................................................................................. S32
1H‐STD‐NMR ................................................................................................................................ S32
Quantitative 19F‐NMR results ...................................................................................................... S33
1H‐STD‐NMR spectrum ................................................................................................................ S36
IntrodDrug de
number
and ana
dynamic
required
StructuDrug dev
enzymes
these en
selected
dimensio
consider
and opti
Figure 1: T
Medicina
the enzy
started.
heavily
synthesi
enzyme,
crystal s
inhibitor
found.[1,
intricate
consumi
ductionevelopment
of compoun
alyse each o
c combinato
d in synthesis
ure‐basedvelopment is
s involved in
nzymes kills o
an inhibitor
onal structu
red at this st
misation) an
The process fro
al chemistry
yme is obta
By using co
on the intu
sed, and its
, it is attemp
structure is t
r that do no2] This mea
e) compound
ing.
nowadays is
nds that nee
of these com
rial chemist
s, and nuclea
ddrugdes a long and
n the disease
only the pat
r can be desi
ure of the
age.[1] If a po
nd finally sub
m disease to ne
y is involved
ained (usual
mputer‐aide
uition and s
s efficacy is
pted to obta
then used in
t bind efficie
ns that bef
ds have to
s plagued b
ed to be con
mpounds.[1,2
try based on
ar magnetic r
esign costly proce
e need to be
hogen (Targ
gned based
enzyme. C
otent inhibit
bmitted for c
ew drug. Figure
mainly in lea
ly via crysta
ed modelling
skill of the
tested in v
ain a co‐cryst
n silico to ide
ently. The p
ore a suffic
be synthesi
y long deve
nsidered, and2] We hope
n already kn
resonance (N
ess, progress
e identified a
et identifica
on known in
Commonly, a
tor is found,
clinical trials
e taken from Jia
ad optimisat
allography),
g techniques
medicinal c
vitro. If it is
tal structure
entify empty
process is rep
ciently poten
ised, purifie
elopment tim
d the amoun
to ameliora
nown inhibit
NMR) spectr
sing through
and it has to
tion and vali
nhibitors, en
around 100
this lead is f
(Figure 1).[1,2
ang et al.[2]
tion.[1] Once
an iterative
s, a potentia
chemist invo
confirmed
e of the enzy
y pockets in
peated until
nt inhibitor
d and analy
mes, stemm
nt of time re
ate this situ
tors to grea
oscopy to fa
h several disc
o be confirm
idation). Onc
zyme functio
0,000 separa
urther optim2]
a three‐dim
e cycle, dep
l inhibitor is
olved.[3] This
that the com
yme with thi
the enzyme
a sufficient
is found a
ysed. This is
ing mainly f
equired to sy
uation by e
atly reduce
cilitate analy
crete steps.
ed that knoc
ce a target e
on, or ideally
ate compou
mised (Lead d
mensional str
picted in Fig
s designed; t
s compound
mpound inh
is inhibitor.
e, or moietie
tly potent in
multitude o
s usually ve
3
from the
ynthesise
mploying
the time
ysis.
First, the
cking out
enzyme is
y a three‐
unds are
discovery
ucture of
ure 2, is
this relies
d is then
hibits the
This new
es of the
hibitor is
of (often
ery time‐
4
Figure 2: The process of structure‐based drug design.
Fragment‐baseddrugdesignFragment‐based drug design is a methodology that can be used to facilitate structure‐based drug
design, and in particular the lead‐identification and optimisation steps. Classically, many large
compounds are screened against a target, in the hope one or more will bind to the protein
(high‐throughput screening, HTS). However, because the compounds screened are usually rather
large, and enzymes bind compounds very selectively, many compounds often need to be screened to
find a successful candidate.[1] Two alternative approaches can be used when applying fragment‐
based drug design: fragment linking, and fragment growing.
In fragment linking and growing, smaller compounds are screened instead. These will usually bind
the target enzyme only weakly, but chemical space can be sampled far more efficiently. If multiple,
small compounds bind the protein in different pockets, these can be thought of as fragments of the
ultimate inhibitor. In this case, if the binding modes of these fragments can be elucidated, it may be
possible to link the fragments together in order to obtain a highly potent inhibitor in an efficient
manner. However, great care must be exercised: if there is too much strain in the linker between the
fragments, or if it slightly too long or too short, this will prevent the inhibitor from binding like the
fragments, leading to a loss of potency. Because of this strict geometrical requirement on the linker,
fragment linking is usually considered hard, but there are examples in which this methodology has
been successfully applied, resulting in very potent inhibitors.[4–6]
If instead there is only binding information available for one fragment, this fragment may be "grown"
in order to make more beneficial interactions with the protein. This process is generally more
elaborate than fragment linking, but it is easier. There are still pitfalls however. It is often very easy
to identify empty hydrophobic pockets in a three‐dimensional model of an enzyme. Growing the
inhibitor in this direction would probably result in an insoluble compound however. This
Compound synthesis and purification
Evaluation
Crystallography
Modelling and design
methodo
Figure 3.
In practi
not unco
modellin
Figure 3: Flarge comsmall orgamanner. Bpocket in o
DynamDynamic
compou
template
accordin
of chang
affinity;*
noted th
template
change i
* Some cato the cha
ology has a
.
ice, the disti
ommon to g
ng is used to
Fragment‐basedpounds are scranic molecules Bottom panel: order to form a
miccombic combinato
nds is form
e, and will in
ng to Le Chât
ge in conce* this degree
hat the amp
e, and the to
n concentra
are must be eanges in build
lso been us
inction betw
grow one fra
facilitate the
d drug design. Teened against tbind the targeIf only one sma potent inhibit
inatorialcorial chemist
med.[12–14] Ne
n this way be
telier's princi
ntration of
e of change
lification fac
otal concentr
tion, a lot of
exercised sinceding block con
ed successf
ween fragme
agment in o
e process.
Top panel: the the target enzyet protein thesall organic molor. Figure taken
chemistrytry is an app
ext, if a tem
e depleted fr
iple, making
individual li
in concentr
ctor is a func
ration of bui
f template m
e it is possiblencentrations.
ully, resultin
nt linking an
rder to mim
classical approyme, but only ase may be linklecule binds thn from Erlanson
yproach in w
mplate is ad
rom their eq
more of the
brary memb
ation is calle
ction of: the
lding blocks.
may be requir
e that compou
ng in comm
nd fragment
mic a second
oach to drug de few — if any —ked together toe protein, this n.[1]
which a libra
dded, some
quilibrium. Th
e compounds
bers can be
ed an ampli
e affinity of
. This means
red.
unds which do
ercially avai
growing is
.[11] In all th
esign is high‐thr— will bind. Mido form a potenfragment may
ary of dynam
library mem
he library wi
s that bind (F
used as a
fication fact
the binder,
, that in orde
o not bind wil
ilable drugs.
not so clear
hese cases, c
roughput screeddle panel: if twnt inhibitor in y be grown into
mically inter
mbers can
ill then re‐eq
Figure 4). Th
measure for
tor.[12–14] It s
the concent
er to see a s
ll also be amp
5
.[7–10] See
‐cut. It is
computer
ning; many wo or more an elegant o an empty
rchanging
bind this
quilibrate
he degree
r binding
hould be
tration of
ignificant
plified due
DCC has
building
efficient
significa
member
be desig
(few) dif
target pr
Because
there are
Figure 4: Aprotein be
Since th
connecti
forming
Diels‐Ald
conditio
aqueous
stopped
To these
can rev
equilibra
nucleoph
of condit
s the advant
blocks can b
screening o
ntly accelera
r has to be sy
gned around
fferent ways
rotein.
of this, dyn
e numerous
A schematic reest, it is selected
e only requi
ions can be
reactions c
der reaction
ns which ca
s environme
at will by ch
e ends we h
versibly com
ation occurs
hilic catalyst
tions (Schem
tage that rel
be synthesise
of large libra
ates both the
ynthesised,
d one or mo
the system
amic combin
examples in
presentation od from the libra
irement for
used betwe
an be used,
, etc. The o
an be tolera
nt that is sli
hanging the r
ave opted fo
mbine to fo
at an acidi
t such as ani
me 1).
latively few
ed in paralle
ries with rel
e synthesis a
purified and
re fragment
will show wh
natorial chem
which this t
of dynamic comary. Figure take
DCC is that t
een the bui
, such as im
only prerequ
ted by the
ghtly acidic
reaction cond
or acylhydra
orm an acy
c pH. Addit
line,[20] allow
building blo
el, or may ev
atively little
and analysis
analysed se
ts. For exam
hich linker ha
mistry is inc
technique ha
mbinatorial cheen from Mamid
the library c
lding blocks
mine format
uisite is that
template.[12
or basic. La
ditions, since
zones as lin
ylhydrazone
tionally, acyl
wing this rea
ocks need to
ven be comm
effort,[12,14]
of potential
eparately; pa
mple, if two f
as the best o
reasingly be
as been used
mistry. Since thyala and Finn.[1
constituents
s. In principl
ion, transac
the reactio,14,19] In case
stly, it is des
e this facilita
king group.
e. Acylhydra
hydrazone e
action to also
o be synthes
mercially avai
which mean
inhibitors, si
rticularly if a
fragments ca
orientation a
ing used in d
d successfully
he compound A13]
can intercha
e, all revers
ylation, disu
on runs at a
e of a prote
sirable that t
ates analysis.
Here an alde
azones are
exchange ca
o be used un
sised, and th
ilable. This a
ns that this t
ince not eve
a dynamic lib
an link toge
and propertie
drug design,
y.[15–18]
A2‐B3 binds th
ange, many
sible, covale
ulfide forma
n acceptabl
ein this is u
the equilibri
.[12,14]
ehyde and h
rather sta
an be cataly
nder a broad
6
hat these
allows for
echnique
ry library
brary can
ether in a
es for the
[12,14] and
e template
different
nt bond‐
tion, the
e rate in
sually an
ia can be
hydrazide
ble, and
sed by a
der range
Scheme 1:
Classical
(LC‐MS).
be stopp
and a lo
some of
NMRte
QuantitNMR spe1H‐NMR
sensitive
structura
abundan
Secondly
result in
An alter
addition
sensitive
from –20
solvents
referenc
In order
so, two
of releva
spectra.
required
SaturatAn altern
measure
(STD‐NM
which is
been sat
the nucl
saturate
* Note th
: The formation
ly, DCLs are
. These tech
ped, separat
ot of protein
these drawb
echnique
tativeNMRectroscopy i
spectroscop
e nucleus, an
al informatio
nt, many solv
y, 1H‐NMR si
overlapping
rnative to 1H
, its gyroma
e as 1H‐NMR
00 to 20 ppm
do not cont
ce might be d
to analyse a
spectra are
ant peaks a
Since the ch
d.
tion‐transfenative to me
e the bindin
MR) was dev
s not of inte
turated, this
ear Oberhau
ed, and no lo
at all NMR sa
n of an acylhydr
e analysed u
niques have
tion may be
is required
backs by usin
es
s a common
py. There ar
nd 1H atoms
on. Unfortun
vents also co
ignals span o
g signals.
H‐NMR may
gnetic ratio
R. Furthermo
m. This mean
tain 19F atom
desirable.
a DCL with th
recorded, on
are normalis
hange in equ
erdifferenceasuring the
g of ligands
veloped. The
rest, and th
s saturation c
user effect (
onger give a
mples must co
razone from an
using metho
e some draw
hard to ach
since the eq
ng NMR spec
n analytical m
re good reas
are abunda
nately, 1H‐NM
ontain them,
only a narrow
be 19F‐NMR
is 83% as la
ore, 19F‐NM
ns that 19F sig
ms, no isotop
hese techniq
ne with tem
ed against a
uilibrium is m
ceNMRchange in e
s to the tem
e template, i
is saturation
can transfer
(NOE).[21] As
signal in NM
ontain at leas
n aldehyde and
ods such as
wbacks since
ieve, all libra
quilibrium is
ctroscopy.
method in s
sons why 1H
ant in most o
MR has some
, demanding
w spectral ra
R spectrosco
arge as that
R signals sp
gnals are oft
pically enric
ques, a chan
mplate and on
an internal
measured dir
quilibrium c
mplate. To th
in this case
n spreads th
to a bound
a result, inh
MR. This mea
st some deute
a hydrazide un
liquid chro
they are de
ary member
s measured
ynthetic org
H is so popu
organic com
e drawbacks
g the use of (
ange: roughl
opy. 19F is 1
of 1H. This m
pan a much
en well sepa
hed solvent
ge in equilib
ne without,
reference a
rectly, large
aused by ad
his end, sat
a protein, i
hrough the p
inhibitor by
hibitors that
ans that whe
erium for locki
der influence o
matography
estructive, th
s need to ha
directly. We
ganic chemis
ular in NMR
pounds, wh
. Firstly, beca
(expensive) d
y from –2 to
00% natura
means that 1
broader spe
arated. Also,
is needed;*
brium is mea
after which
nd compare
amounts of
dition of a te
uration‐tran
s saturated
protein. Onc
means of sp
bind to the
en a regular
ng and shimm
of catalytic anili
y mass spec
he equilibriu
ave a differe
e hope to cir
stry, and in p
since it is t
ich results in
ause 1H atom
deuterated s
o 12 ppm, w
lly abundan19F‐NMR is a
ectral range
since many
although an
asured direct
the signal in
ed between
template ar
emplate is to
sfer differen
in a spectra
ce the active
pin diffusion
e protein wi1H‐NMR spe
ming.
7
ne.
ctrometry
m has to
ent mass,
rcumvent
particular
the most
n a lot of
ms are so
solvents.*
hich may
t, and in
almost as
: roughly
common
n internal
tly. To do
ntensities
the two
re usually
o directly
nce NMR
al region,
e site has
n through
ll also be
ectrum is
compare
decrease
Because
many lig
methods
analysed
problem
Figure 5: aprotein tomultiple lig
Unfortun
spectros
circumve
saturate
the 1H c
explicitly
using an
capable
Of cours
least one
ed to one wh
e in intensity
of the dyna
gand molecu
s. For this to
d.[19] Also, ra
m.
a schematic depo ligands that gand molecule
nately, 1H‐S
scopy: a sm
ent these hu
ed as before,
channel, it
y transferrin
n INEPT‐like
of measurin
se, when ana
e 19F atom.
here the prot
y; compound
amic binding
les, meaning
o work howe
apid ligand e
piction of STD‐bind. Due to ds.
TD‐NMR sp
all spectral
urdles, the te
, and the sa
is decouple
ng the satura
sequence.[22
g 19F whilst
alysing a DCL
tein was satu
ds that do no
g and unbind
g that this te
ever, extensi
exchange is
NMR spectroscdynamic bindin
ectroscopy
range and
echnique has
turation is t
d, and 19F i
ation from h2] This techn
saturating 1H
L using any 19
urated, the s
ot bind the te
ding of com
echnique req
ive measure
required,[21
copy. Saturationng and unbind
suffers from
the require
s been modif
transferred t
is observed
hydrogens n
nique howev
H.
9F‐NMR tech
signals corre
emplate will
pounds, one
quires far les
ement times ] but for we
n is depicted ining, one prote
m the same
ment for is
fied to 19F‐ST
to the ligand
. Additional
nuclei in the
ver, does req
hnique all lib
sponding to
not be affec
e protein mo
ss protein th
are required
eak binders
blue. Saturatioein molecule ca
e problems
otopically e
TD‐NMR, in w
d. However,
sensitivity
e inhibitor to
quire an NM
rary membe
bound inhib
cted (Figure 5
olecule may
an quantitat
d when mixt
this is usua
on is transferrean transfer sat
as normal
nriched solv
which the te
instead of o
can be obt
o the fluorin
MR instrumen
ers need to c
8
bitors will
5).[21]
saturate
tive NMR
tures are
ally not a
ed from the turation to
1H‐NMR
vents. To
mplate is
observing
ained by
ne nuclei
nt that is
ontain at
EndothIn order
attempt
fungus E
This clas
several s
bonds,[23
catalytic
These as
Some of
binding.[
Figure 6: cartoon. Tblue). The
As a mo
research
complex
which is
chemistr
this enzy
hiapepsinr to demons
to design an
Endothia par
ss of enzyme
serious disea3–25] and the
c dyad: two
spartic acids
f the know[19]
X‐ray crystal The catalytic dya water molecul
odel enzyme
h group of K
x with a seri
beneficial fo
ry is compat
yme.[19]
nstrate the a
n inhibitor fo
asitica, and
es is widespr
ases such as
e inhibitors
aspartic aci
s are tightly
n inhibitors
structure of ead (aspartic acile closely bound
e, endothiap
Klebe et al.[2
ies of fragm
or acylhydraz
tible with en
advantages D
or the enzym
it is a model
ead, and has
s malaria and
known toda
id residues
bound to a w
displace th
ndothiapepsind residues 32 ad to the catalyt
pepsin is re28] has previ
ents; its me
zone exchan
ndothiapepsi
DCC can off
me endothia
enzyme for
s varied biol
d AIDS.[23–25]
ay block the
(Asp‐32 and
water molec
his catalytic
(PDB code: 1Eand 215) is deptic dyad is show
eadily availab
iously publis
echanism is
ge;[23,26] it ha
n;[19] and las
fer in fragm
pepsin. This
aspartic pro
ogical functi
Its function
e active site
d Asp‐215) o
cule through
water mole
ER8[27]). The pricted as a stick
wn as a red sphe
ble and crys
shed co‐crys
fully elucida
as been show
stly, our grou
ment‐based d
protein is o
teases.[23]
ions and play
n in nature is
.[23,26] This a
of which one
h hydrogen b
ecule, but o
rotein backbonmodel (Colour ere.
stallises eas
stal structure
ated;[23,25] its
wn in the pas
up is current
drug design,
originally fou
ys a causativ
s to hydroly
active site fe
e is protona
bonds (Figur
others requi
ne is depicted r code: C: green
sily. Addition
es of this en
s pH optimu
st that acylhy
tly also wor
9
, we will
nd in the
ve role in
se amide
eatures a
ated.[23,25]
e 6).[23,25]
ire it for
as a green n, O: red, N:
nally, the
nzyme in
m is 4.5,
ydrazone
king with
Based on
analysis
designed
developm
LibrarA library
by the g
their ori
structure
order to
Fragmen
substitut
Since it
would h
acylhydr
construc
(Scheme
Predicte
found in
Figure 7: TColour codsurface is water mol
S4
S2
n the require
and contai
d a library o
ment can be
rydesigny of compou
group of G. K
iginal paper
es in Schem
o accommod
nt F284 was
ted with an e
is hard to de
have to be o
razone moie
cted by syste
e 3).
d binding m
the support
The binding mde: O: red, N: bdepicted in grelecule bound to
S1
4
ements that
n at least
of compoun
e greatly acce
nnds was des
Klebe.[28] The
r. The bindin
e 2. Since th
ate an acylh
s reduced to
ethylamine m
esign the ide
oriented, a t
ety, and fo
ematically inc
modes for al
ting informat
odes of the twblue, F: pale cyaey. It can be seeo the active site
all potentia
one acylhyd
ds based on
elerated usin
signed based
e fragments
ng modes o
he fragments
hydrazone lin
o a fluoroph
moiety to pre
eal linker an
total of eigh
our featurin
cluding meth
l these com
tion.
wo fragments can, Caspartic dyad:en that the frage upon binding
S2'
al inhibitors s
drazone mo
n computer
ng DCC and S
d on fragmen
used will b
of these frag
s overlap wh
nker, whilst
henyl moiety
event interfe
d predict ex
ht inhibitors
ng a "backw
hylene space
mpounds, alo
co‐crystallised wgreen, CF109: te
gments bind in d(translucent re
'
S3
S1
should conta
iety to ena
models in o
STD‐NMR spe
nts in comple
e referred t
gments are
hen binding,
preserving k
y, and the h
erence with
xactly in wha
were desig
ward" acylh
ers at either
ong with cal
with endothiapeal, CF284: hot pdifferent pocked sphere).
ain at least o
ble the dyn
order to de
ectroscopy.
ex with endo
o as F109 an
depicted in
they had to
key interactio
hydrazide in
the acylhydr
at way the a
gned: four fe
hydrazone m
end of the a
culated bind
pepsin (PDB copink. Endothiapets, and that fra
one fluorine
namic excha
monstrate t
othiapepsin
nd F284, in
n Figure 7 a
o be made s
ons with the
fragment F
razone excha
acylhydrazon
eaturing a "
moiety. The
acylhydrazon
ding energie
odes: 3PBZ andpepsin's solventagment F109 di
10
atom for
ange, we
that drug
reported
line with
and their
smaller in
e protein.
F109 was
ange.
e moiety
forward"
ese were
ne moiety
es can be
3PMU).[28] t‐accessible splaces the
Scheme 2acylhydrazmoiety.
Scheme 3"forward" of the acyl
Due to p
second l
The R'
endothia
and F284
the pos
substitue
These ni
crystal s
accelera
done fo
enabling
2: After obtainzone linker; red
3: The designed(1–4) or "backlhydrazone mo
problems wi
ibrary was d
fragment w
apepsin in u
4, a new libr
sition of the
ents were al
ine building
structures of
te the drug
r many com
g non‐destru
ing binding mducing F284 to
d inhibitors. Thkward" (5–8) aciety, and is dep
th the synth
esigned, this
was replace
npublished
rary was des
e fluorine w
so designed
blocks were
f endothiape
g‐developme
mpounds in
ctive analysi
modes for fragmo a fluoropheny
hey were formcylhydrazone mpicted in red.
hesis of the
s time consis
ed for imid
work by M.
signed. Since
was varied
(Figure 8 an
e obtained c
epsin in orde
nt process s
one experim
s of the DCL
ments F109 anyl moiety, and
med from fragmmoiety. A methy
R' fragment
sting of comm
dazole alde
Mondal, N.
e there was a
(10–15), a
nd Scheme 4,
commercially
er to demon
since both t
ment. The a
L.
nd F284, they exchanging th
ments F109 anylene spacer w
in Scheme 3
mercially ava
hyde (9), w
Radeva, A.
ample space
nd some co
, 16–18).
y and used t
nstrate that
the synthesi
nalysis was
were modifiehe hydrazide of
d F284 (Schemwas systematica
3 (see Suppo
ailable comp
which was
Hirsch and G
e around F28
ompounds w
to construct
the use of D
s and biolog
done using
ed to make rof F109 for an
me 2) by addinally included at
orting Inform
pounds.
co‐crystallis
G. Klebe. Ba
84 inside the
with trifluo
t a DCL base
DCC can sig
gical analysi
g NMR spect
11
oom for an ethylamine
ng either a either end
mation) a
sed with
ased on 9
e protein,
romethyl
ed on co‐
nificantly
s can be
troscopy,
Figure 8: Ccyan, Caspagrey. It casite via hy
* Work of
S
Co‐crystal struc
artic dyad: green, n be seen that drogen bonds (
f M. Mondal,
S
S4
S2
ctures of F284 Cimidazol aldehyde:imidazole alde(dashed yellow
N. Radeva, A.
S1'
(PDB code: 3PM: orange, CF284:ehyde requires lines).
Hirsch and G
MU)[28] and imi: hot pink. Enda water molecu
. Klebe. To be
S2'
dazole aldehydothiapepsin's sule (red sphere
e published.
S1
de.* Colour codsolvent‐accessibe) in order to bi
de: O: red, N: bble surface is dind to the prot
12
lue, F: pale depicted in ein's active
Scheme 4:F284 was hydrazides
: The building bvaried, and ps 19–27.
blocks used. Theotentially subs
e compounds astituted for a t
are based on F2trifluoromethyl
284 and 9, but tl group in ord
he position of ter to yield acy
the fluorine subylhydrazones 1
13
bstituent in 10–18 from
ModelThe des
inhibitor
Modellin
acylhydr
stationa
acylhydr
Accordin
mediate
der Waa
15 are
Supporti
Each acy
suite.[30]
isomeris
visually i
Figure 9: Tyellow linewater mol
S
S2
llingigned librari
rs and would
ng was perf
razone insid
ry. Water m
razone.
ng to Moloc,
d by a wate
als interactio
shown in F
ing Informat
ylhydrazone
Aspartic aci
sm was cons
inspected.
The predicted bes. Favourablelecule bound cl
4
ies were firs
d bind to the
formed usin
e the prote
olecules we
, all acylhydr
r molecule;
ons were gen
igure 9 and
tion.
was docked
id residue 21
idered. The 3
binding mode o Van der Waaosely to the cat
S1'
st studied in
enzyme like
g the softw
ein, and the
re reset to t
razones sho
and occasion
nerally very f
d 10, respec
d inside the
19 was proto
30 poses wh
of 13. Colour cols interactions talytic dyad is s
n silico, to s
e the fragme
ware program
en optimisin
their position
uld bind to
nally form a
favourable. A
ctively. Bind
enzyme us
onated and
hich were mo
ode: O: red, F: involving fluorshown as a red
S2'
see if the de
nts upon wh
m Moloc[29]
ng its posit
ns according
the catalytic
dditional hy
As an examp
ding modes
ing the Flex
key water m
ost promising
pale cyan, Cprotrine atoms aresphere.
esigned com
ich they wer
by first gen
ion whilst k
to the cryst
c dyad throu
drogen bond
ple, the bind
for 10–18
X docking m
molecules we
g according t
tein: green, C13: depicted as d
S1
mpounds we
re based.
nerating the
keeping the
tal structure
ugh a hydrog
ds. Furtherm
ding modes o
are provide
module in th
ere unrestra
to the softw
purple, H‐bonddashed, salmon
1
14
re viable
e desired
enzyme
for each
gen bond
more, Van
of 13 and
ed in the
he LeadIT
ined; E/Z
ware were
ds: dashed, n lines. The
Figure 10: yellow linewater mol
Unfortun
was bou
FlexX re
scored b
more im
To furth
poses in
yet, it is
bound to
S
S2
The predicted es. Favourablelecule bound cl
nately, FlexX
und to the c
sults were d
by Hyde[30] d
mportantly, th
er analyse t
which the li
s unknown w
o the catalyt
S4
binding mode Van der Waaosely to the cat
X produced n
catalytic dya
discarded. So
directly; how
he ligand wa
this anomaly
igand was bo
why the soft
tic dyad.
S1'
of 15. Colour cls interactions talytic dyad is s
no poses for
d with a hy
ome ligands,
wever, the re
s not bound
y, 9 was doc
ound to the
tware would
code: O: red, F: involving fluorshown as a red
any of the a
drogen bon
, which wer
esulting calcu
to the cataly
cked using F
catalytic dya
d not produ
S2
pale cyan, Cprorine atoms aresphere.
cylhydrazon
d via a wate
e most prom
ulated energ
ytic dyad.
FlexX. In this
ad as indicat
ce poses in
'
tein: green, C15: depicted as d
es in which t
er molecule.
mising accor
gy of binding
s case, the s
ted by the cr
which the a
S
purple, H‐bonddashed, salmon
the imidazol
. For this re
rding to Mol
g was very p
software did
rystal structu
acylhydrazon
S1
15
ds: dashed, n lines. The
le moiety
eason the
loc, were
poor, and
produce
ure. As of
nes were
16
Discussion
Quantitative19F‐NMRTwo spectra were recorded, one of a sample containing a high protein concentration, and one
without protein (experimental details can be found in the Supporting Information). Peaks were well
separated, although less so for trifluoromethylated compounds. The integrals of the peaks from the
library members were normalised against the integral of peak from trifluoroacetic acid and
compared. Since sum of the concentration of acylhydrazone and the concentration the
corresponding hydrazide should be constant, their respective peak areas were summed to 100%. The
result is plotted in Figure 11. Unfortunately, upon addition of protein, no significant change in
concentrations is observed. This means that none of the compounds bind to the protein according to
these data. The raw data and spectra are provided in the Supporting Information.
Figure 11:The difference in peak area for separate library members between a sample containing protein (plus), and one without (minus).
1H‐STD‐NMRNo signal could be observed from the 1H‐STD‐NMR spectrum, not even one originating from 9 (the
spectrum is provided in the Supporting Information). Since 9 is a known inhibitor this could indicate a
problem with library, and it is hypothesised that the trifluoroacetic acid interferes with the binding of
library members to the protein by either denaturing the protein, blocking the active site, or stopping
the acylhydrazone exchange. Therefore, these experiments should be repeated with either a lower
concentration of trifluoroacetic acid, a different reference compound, or with a library of known
binders in order to validate the methodology.
19F‐STD‐NMRDespite our best efforts, no 19F‐STD‐NMR spectrum could be recorded on the NMR spectrometers
available. No machine was available with a probe that could be tuned to 19F and 1H simultaneously.
Tuning a probe to 19F and saturating 1H at −1.2 ppm by directly specifying the frequency produced
too many artefacts to yield a useful spectrum.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Hydrazide
Acylhydrazone
17
ConclusionsIn conclusion, DCC can play a significant role in both fragment linking and fragment growing, and
should be part of every medicinal chemist’s toolkit. However, analysis of the resulting mixtures is
often challenging, but here a variety of NMR techniques can help, providing a way to analyse an
equilibrating library in a non‐destructive manner. STD‐NMR is particularly interesting in this sense,
since it requires very little protein.
As demonstrated, 19F‐NMR could extend these techniques further, granting less background signals
and excellent peak separation. The only additional requirement is that all relevant compounds must
contain at least one 19F atom.
Modelling and the NMR studies are in agreement, and no inhibitor was found in this study. It would
be worthwhile to repeat this methodology with a library of known inhibitors, as well as repeating it
with a different reference compound to make sure trifluoroacetic acid does not influence the
measurements. Once it is conclusively proven that 19F‐STD‐NMR and quantitative 19F‐NMR are
suitable for analysing DCLs, it should be explored how little protein is required as a function of the
free energy of binding of the library members.
AcknowledgementsI would like to particularly thank Pieter van der Meulen for his extensive help with all the NMR
measurements, as well as his limitless patience and perseverance in trying to get 19F‐STD‐NMR to
work on a machine that is not designed to do so. Furthermore, I would like to thank Milon Mondal
for his daily supervision and help with the syntheses; and I would like to thank Anna Hirsch for
supervising this research project in her group.
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S1
SupportingInformationAll figures in this work featuring endothiapepin are generated using PyMOL.[31]
LibraryA—Compounds1to8
ModellingmethodsThe target compounds were first designed inside the protein using Moloc,[29] and their position was
optimised whilst keeping the protein stationary. Each acylhydrazone was docked inside the enzyme
using the FlexX docking module in the LeadIT suite.[30] Aspartic acid residue 219 was protonated and
key water molecules were unrestrained; E/Z isomerism was considered. 30 poses were kept per
ligand.
The resulting poses were visually inspected to ensure they bound to the protein like the original
fragments, and were otherwise discarded. The most promising pose was used for Hyde scoring[30] in
order to obtain a predicted free energy of binding (ΔGHyde).
Colour code for the figures: O: red, N: blue, F: pale cyan, Cprotein: green, Cligand: orange. The solvent‐
accessible surface of the protein is shown in grey. Hydrogen bonds are shown as dashed, yellow
lines.
S2
ModellingresultsTable S1 depicts the binding energies as predicted by Hyde.
Compound ΔGHyde (kJ/mol)
1 −30
2 −39
3 −21
4 −30
5 −32
6 −39
7 −19
8 −17 Table S1: The binding energies for compounds 1–8, predicted by Hyde.
1:(E)‐N
ΔGHyde =
N'‐(2‐(2‐Ami
−30 kJ/mol
S4
S2
noethyl)‐5‐
S1'
(diethylami
ino)benzyliddene)‐2‐fluo
S
orobenzohyd
S2'
S
drazide
3
S1
S3
2:(E)‐N'
ΔGHyde =
S
'‐(2‐(2‐Amin
−39 kJ/mol
S4
S2
noethyl)‐5‐(
S1'
(diethylamin
no)benzyliddene)‐2‐(2‐fl
S2
luorophenyl
2'
S3
l)acetohydr
3
S1
S4
razide
3:(E)‐N
ΔGHyde =
S2
N'‐(2‐(2‐(2‐A
−21 kJ/mol
S4
Aminoethyl)
S1'
‐5‐(diethylaamino)phen
nyl)ethyliden
S2'
ne)‐2‐fluoro
S3
S
obenzohydra
S1
S5
azide
4:(E)‐Ndrazide
ΔGHyde =
S
N'‐(2‐(2‐(2‐A
−30 kJ/mol
S4
S2
Aminoethyl)
S1'
‐5‐(diethyla
amino)phennyl)ethyliden
S2
ne)‐2‐(2‐fluo
2'
S3
orophenyl)a
3
S1
S6
acetohy
5:(E)‐2
ΔGHyde =
‐(2‐Aminoe
−32 kJ/mol
S4
S2
thyl)‐5‐(die
S1'
ethylamino)‐‐N'‐(2‐fluorobenzyliden
S
ne)benzohyd
S2'
S
drazide
S3
S1
S7
6:(E)‐2
ΔGHyde =
‐(2‐Aminoe
−39 kJ/mol
S4
S2
thyl)‐5‐(die
S1'
ethylamino)‐
‐N'‐(2‐(2‐fluuorophenyl)
)ethylidene)
S2'
S
)benzohydra
S3
S1
S8
azide
7:(E)‐2
ΔGHyde =
S
‐(2‐(2‐Amin
−19 kJ/mol
S4
S2
noethyl)‐5‐(
S1'
diethylamin
no)phenyl)‐NN'‐(2‐fluoro
S2
obenzylidene
'
S3
e)acetohydr
S1
S9
razide
8:(E)‐2drazide
ΔGHyde =
S
‐(2‐(2‐Amin
−17 kJ/mol
S4
S2
noethyl)‐5‐(
S1'
diethylamin
no)phenyl)‐NN'‐(2‐(2‐flu
S2
orophenyl)e
'
S3
ethylidene)a
S1
S10
acetohy
SyntheThe plan
obtained
and S2).
Library d
Scheme S1with a boxnight; rt: r
esisnned and att
d starting ma
Since not a
design).
1: The used synx; target comporoom temperat
tempted syn
aterials are s
all desired co
nthetic routes founds are in bure; quant.: qu
nthetic route
surrounded w
ompounds c
or the fluorophlue; failed reacantitative.
es are depic
with a box, a
could be obt
henyl compounctions are show
cted in the f
and target co
tained, a dif
ds. Commerciawn with a red, c
ollowing sch
ompounds ar
ferent librar
lly obtained cocrossed arrow.
hemes. Com
re in blue (Sc
ry was desig
ompounds are s Abbreviations
S11
mercially
cheme S1
gned (see
surrounded : o.n.: over
Scheme S2obtained ccrossed ar
2: The used syncompounds arerrow. Abbreviat
nthetic routes fe surrounded wtions: o.n.: over
for the 2‐(2‐amwith a box; tarr night; rt: room
minoethyl)‐5‐(dirget compoundm temperature.
ethylamino)beds are in blue; .
nzohydrazide cfailed reaction
compounds. Cons are shown w
S12
mmercially with a red,
Methyl2N‐Iodosu
in dry m
stirred i
heptane
mixture
saturate
solvent i
7.94 (td,
δ= −109.
2‐fluorobenuccinimide (1
methanol (25
n the dark
e). Upon com
was extrac
ed aqueous
in vacuo yiel
, 1H), 7.56 –
.50 – −109.7
nzoate(29)[3
1.4 g, 6.22 m
5 mL), and 2
for 2.5 h, a
mpletion, wat
cted with 50
NaCl solutio
ded 29 as a
7.46 (m, 1H
4 (m, 1F).
32]mmol, 2.4 eq
28 (0.41 mL,
and the rea
ter (16 mL) a
0% ether in
on, dried ov
yellow liquid
), 7.20 (td, 1
) and dried K
0.32 g, 2.5
action was f
and sodium
n pentane (4
ver magnesiu
d (0.44 g, 2.8
H), 7.14 (ddd
K2CO3 (1 g, 7
6 mmol, 1 e
followed by
thiosulfate (
4 ×). The o
um sulfate a
85 mmol, 75
d, 1H), 3.93
.24 mmol, 2.
eq) was adde
TLC (SiO2;
(1.6 g) were
rganic phas
and filtered.
5%). 1H‐NMR
(s, 3H). 19F‐N
.8 eq) were
ed. The mix
20% ethylac
added. The
se was wash
. Evaporatio
R(400 MHz, C
NMR(376 MH
S13
dissolved
xture was
cetate in
resulting
hed with
on of the
CDCl3): δ=
Hz, CDCl3)
2‐Fluoro29 (0.4 g
Upon ad
the reac
(aq) wer
and the
filtration
2.13 mm
6.84 (td,
obenzohydrg, 2.60 mmo
ddition of hyd
ction with TL
re added and
e resulting m
n, and the s
mol, 82.5%) a
, 1H), 6.79 –
razide(30)[1
l, 1 eq) was d
drazine the s
LC (SiO2; 30%
d the metha
mixture was
olvent was
as a bright or
6.69 (m, 1H)
19]dissolved in
solution disc
% ethylacetat
nol was rem
s sonicated.
removed fro
range solid. 1
), 2.12 (p, 2H
methanol (1
colored. The
te in heptan
moved in vac
Compound
om the resu1H‐NMR(400
H). 19F‐NMR(3
1.7 mL) and N
solution was
e). Upon co
cuo. 1.25 M e
ds that did
lting solutio
0MHz, CDCl3)
376 MHz, CD
N2H4∙H2O (1.0
s refluxed fo
mpletion a f
ethanolic HC
not dissolve
n in vacuo y
): δ= 7.39 (td
DCl3): δ= −106
05 g, 21 mm
or 6 h whilst f
few drops of
Cl (15 mL) w
e were rem
yielding 30
d, 1H), 7.12 (
6.54 (p, 1F).
S14
mol, 8 eq).
following
f 2 M HCl
as added
moved by
(329 mg,
(tdd, 1H),
Methyl231 (1 g,
with a se
left to s
heptane
amount
3H), 3.5
121.56,
2‐(2‐fluorop6.49 mmol,
eptum and a
stir overnigh
e). Upon com
of pure 32.
8 (s, 2H). 13
121.40, 115.
phenyl)aceta1 eq) was d
acetyl chlorid
t after whic
mpletion th1H‐NMR(40
3C‐NMR(101
.64, 115.43, 5
ate(32)*dissolved in d
de (2.5 mL,
ch the conve
e solvent w
0 MHz, CDC
MHz, CDCl
52.31, 34.44
dry methano
35.16 mmol
ersion was c
was evapora
Cl3): δ= 7.21
l3): δ= 171.2
4. 19F‐NMR(3
ol (30 mL) in
, 5.4 eq) wa
checked with
ated in vacu
– 7.12 (m, 2
28, 162.41,
76 MHz, CDC
n a round‐bo
s added slow
h TLC (SiO2;
uo which yie
2H), 7.05 – 6
159.96, 131
Cl3): δ= −117
ottom flask e
wly. The mix
20% ethyla
elded a qua
6.90 (m, 2H)
1.57, 129.22
7.21 – −117.4
S15
equipped
xture was
cetate in
antitative
), 3.61 (s,
, 124.26,
46 (m).
S16
2‐(2‐Flu32 (0.7 g
8 eq). Th
ethylace
methano
mixture
was rem
white so
(s, 2H), 2
uorophenyl)g, 4.16 mmo
he resulting m
etate in hep
ol was remo
was sonicate
moved from t
olid. 1H‐NMR
2.08 (s, 1H). 1
acetohydral, 1 eq) was
mixture was
ptane). Upon
oved in vac
ed. Compou
the resulting
R(400 MHz, D19F‐NMR(376
zide(33)*dissolved in
refluxed ov
n completio
uo. 1.25 M
nds that did
g solution in
DMSO‐d6): δ=
6 MHz, DMSO
ethanol (1 m
ernight. The
n a few dr
ethanolic H
not dissolve
n vacuo yield
= 11.11 (s), 7
O‐d6): δ= −1
mL) and N2H
conversion
ops of 2 M
HCl (23.3 m
e were remo
ding 30 (454
7.42 – 7.29 (
17.06 (q, 1F)
4∙H2O (1.61
was checked
HCl (aq) w
L) was adde
oved by filtra
mg, 2.70 m
m, 2H), 7.22
).
mL, 1.66 g, 3
d with TLC (S
were added
ed and the
ation, and th
mmol, 65%) a
2 – 7.14 (m,
S17
33 mmol,
SiO2; 30%
and the
resulting
e solvent
as an off‐
2H), 3.65
2‐(2‐Flu31 (0.2 g
trimethy
after wh
The resu
instead,
34 could
correspo
31 (771
triperflu
nitrogen
followed
mixture
magnesi
column c
5‐BromoTo 35 (5
solution
and extr
dried ov
15.3 mm
1H), 3.10
* Reaction
uorophenyl)g, 297 mmol,
ylamine (181
hich it was co
ulting mixtur
only aromat
d not be syn
onding alcoh
mg, 5 mmo
orophenylbo
n line. After
d by 1 M HCl
was extrac
um sulfate,
chromatogra
o‐2,3‐dihyd.27 g, 23 mm
was stirred
racted with D
ver sodium s
mol, 68%) as
0 (t, 2H), 2.7
n performed b
acetaldehyd, 1 eq) was d
1 µL, 131 mg,
ooled to −78
re was stirred
tic hydrogen
nthesised by
ol was obser
l, 1 eq); ben
orane (2.56
1.3 h the c
l (aq, 20 mL)
cted three t
filtered and
aphy (SiO2; 2
ro‐1H‐indenmol, 1 eq) wa
for 1 h at 0°
DCM. The or
sulfate, filter
white needl
7 – 2.69 (m,
by Milon Mon
de(34)*dissolved in D
, 297 mmol,
8°C followed
d for 30 min
atoms are o
y reducing 3
rved.
zene (3 mL);
mg, 5 µmol,
rude reactio
whilst stirri
times with e
the solvent
2% ethylaceta
n‐1‐one(36as added chlo
C after whic
rganic layer w
red and the
les. 1H‐NMR
2H).
ndal.
DCM (2 mL).
1 eq) were a
d by the add
. No formati
observed.
32 with DIBA
; triethylsila
, 0.001 eq) w
on mixture w
ng vigorousl
ether. The
t was remov
ate in penta
6)[33]orosulfonic a
h the reactio
was washed
solvent was
(400 MHz, C
TMSCl (165
added at 0°C
ition of 1.2
ion of aldehy
AL‐H in tolu
ne (1.84 mL,
were added
was dried in
ly for 3 h at
organic laye
ved in vacuo
ne). No prod
acid (40 mL,
on was quen
with concen
s removed in
CDCl3): δ= 7.8
µL, 141 mg,
C. The mixtur
M DIBAL‐H i
yde 34 was o
uene at −78°
, 1.34 g, 11.5
to a dried fl
vacuo. THF
room tempe
ers were co
. The produc
duct formatio
70.1 g, 602 m
ched with ic
ntrated sodiu
n vacuo, whi
88 (d, 1H), 7
297 mmol, 1
re was stirre
in toluene (1
observed by
°C. Reductio
5 mmol, 2.3
lask equippe
F (20 mL) wa
erature. The
ombined, dr
ct was purif
on was obse
mmol, 26 eq
ce‐cold wate
um bicarbon
ich yielded 3
7.69 (dd, 1H)
S18
1 eq) and
d for 1 h;
1.08 mL). 1H‐NMR,
on to the
eq); and
ed with a
as added
resulting
ried over
ied using
rved.
) and the
r (70 mL)
nate (aq),
36 (3.2 g,
, 7.37 (d,
6‐BromoTo a sol
sodium
warm to
1 M sod
organic l
sodium
ethylace1H‐NMR
2H).
o‐3,4‐dihydution of 36
azide (1.39 g
o room temp
dium hydrox
layers were
sulfate, filte
etate or DCM
(400 MHz, C
roisoquinol(3 g, 14.2 m
g, 21.32 mm
perature and
xide (aq, 50
washed sequ
ered, and c
M. Four batch
CDCl3): δ= 8.4
in‐1(2H)‐onmmol, 1 eq) i
mol, 1.5 eq) w
d stirred ove
mL). The a
uentially wit
oncentrated
hes yielded a
46 (s, 1H), 7
ne(37)[34]in DCM (84
was added s
ernight. The
aqueous laye
th water and
d in vacuo.
a total of 2.1
7.11 (dd, 1H)
mL) and me
slowly. The r
mixture wa
er was extra
d saturated a
The resultin
16 g of 37 (9.
), 7.05 – 6.9
ethansulfonic
resulting mix
s partitioned
acted with D
queous NaC
ng solid was
55 mmol, 68
4 (m, 2H), 2
c acid (42 m
xture was al
d between D
DCM. The c
Cl solution, d
s recrystallis
8%) as white
2.93 (t, 2H),
S19
mL) at 0°C
lowed to
DCM and
combined
ried over
sed from
e crystals.
2.64 (dd,
6‐(DiethAn oven
(900 m
2‐dicyclo
was plac
8.748 m
a syringe
(SiO2; 50
with eth
2.1 mmo1H‐NMR
2H), 2.37
hylamino)‐3‐dried Schle
mg, 3.98 m
ohexylphosp
ced under a
L, 8.75 mmo
e. The solutio
0% ethylacet
hylacetate,
ol, 53%) as
(400 MHz, D
7 (t, 2H), 1.0
3,4‐dihydroink flask equi
mmol, 1 eq
hino‐2′,6′‐di
n Argon atm
ol, 2.2 eq) an
on was heat
tate in penta
washed wit
s an orang
DMSO‐d6): δ=
5 (t, 6H).
soquinolin‐ipped with a
q), palladiu
isopropoxyb
mosphere, a
d diethylam
ed to 50°C a
ane). After c
th water, a
ge solid. T
= 9.74 (s, 1H
1(2H)‐one(a magnetic st
um(II) aceta
biphenyl (RuP
nd 1 M lith
ine (0.453 m
and stirred fo
cooling the m
nd concent
he product
H), 6.90 (d, 1
(38)[35]tirring bar an
ate (45 m
Phos, 185.4
ium bis(trim
mL, 17.77 g, 2
or 24 h whils
mixture to r
rated in va
t was used
H), 6.28 – 6
nd a septum
g, 0.2 mm
µg, 0.42 mm
methylsilyl)am
243 mmol, 6
st following t
room tempe
cuo. This y
d without
.12 (m, 2H),
was charged
mol, 0.05
mol, 0.1 eq).
mide in THF
1 eq) were a
the reaction
erature it wa
yielded 38 (
further pur
3.25 (q, 4H)
S20
d with 37
eq) and
The flask
(LHMDS,
added via
with TLC
as diluted
(458 mg,
rification.
), 2.70 (t,
N,N‐Diet38 (358
(35 mL)
Water (3
(10.8 mL
yielded 3
1H), 3.29
* Reaction
thyl‐1,2,3,4‐mg, 1.62 m
over the cou
3.6 mL) was
L). The prec
39 (270 mg,
9 – 3.19 (m,
n performed b
‐tetrahydromol, 1 eq) w
urse of 30 m
s added dro
ipitate was
1.32 mmol,
4H), 2.67 (t,
by Milon Mon
isoquinolin‐was added to
min. The mixt
pwise, follow
removed an
80%). 1H‐NM
2H), 1.97 – 1
ndal.
‐6‐amine(3o a solution
ture was gen
wed by 4 M
nd washed w
MR(400 MHz
1.85 (m, 2H)
9)* of LiAlH4 (6
ntly refluxed
M sodium hy
with THF. Th
z, CDCl3): δ=
, 1.12 (t, 6H)
650 mg, 4.86
overnight, c
droxide (aq,
he filtrate w
6.80 (d, 1H),
).
6 mmol, 3 eq
cooled in an
, 3.6 mL), an
was evapora
, 6.07 (d, 1H
S21
q) in THF
ice bath.
nd water
ted. This
), 5.84 (s,
N,N‐Diet39 (10
0.054 m
tempera
mixture
(aq, 131
extracte
over ma
accordin
A mixtur
stirred a
yielded o
To a fla
dissolved
solution
1.05 eq)
then hea
1.1 eq) w
tempera
bicarbon
layer wa
sulfate, f
44 (50 m
ethylbro
mixture
This yiel
1H), 3.74
thyl‐3,4‐dihmg, 0.049 m
mol, 1.1 eq
ature. To thi
was stirred v
1 µL). The ac
d with DCM
agnesium su
ng to 1H‐NMR
re of 39 (50
at room tem
over‐oxidised
me‐dried fla
d in dry DCL
was cooled
was added
ated to 0°C
was added d
ature and st
nate (aq, 0.2
as extracted
filtered, and
mg, 0.342 m
omide (149 m
was diluted
ded 10% of
4 (t, 2H), 3.1
hydroisoquinmmol, 1 eq
q) was add
is mixture 3
vigorously fo
cidic extract
M. The organ
lfate and filt
R.
mg, 0.245 m
mperature fo
d product. N
ask equipped
L (0.8 mL). 2
d to −78°C a
dropwise via
and stirred
ropwise and
tirred for 5
mL) and dilu
d with DCM
the solvent
mmol, 1 eq)
mg, 1.369 m
d with conce
40. 1H‐NMR
7 (q, 4H), 2.6
nolin‐6‐amin) was disso
ded and the
30% NaOH (a
or 1 h. The or
s were mad
ic layers we
tered. The s
mmol, 1 eq) a
or 40 h. The
No improvem
d with a sep
2‐Fluoropyrid
and stirred f
a a syringe a
for an addit
the mixture
h. The rea
uted with DC
(2 ×). The
was remove
), tertbutyla
mol, 4 eq) w
entrated sod
(400 MHz, C
63 (t, 2H), 1.2
ne(40)*lved in DCM
e reaction
aq, 33 µL, 5
rganic layer
de alkaline to
ere washed w
solvent was
and MnO2 (5
e mixture wa
ment was obs
ptum 38 (44
dine (19 µL,
for 10 min.
and the reac
tional 10 m
e was stirred
action was q
CM (0.8 mL).
organic laye
ed in vacuo. N
mmonium c
were added
dium bicarbo
CDCl3): δ= 9.0
26 (t, 6H).
M (100 µL).
mixture wa
5 eq) was a
was washed
o pH 10 by
with saturat
removed in
53 mg, 0.61 m
as filtered, a
served with s
4 mg, 0.2 m
21 mg, 0.22
Triflic anhyd
tion was stir
in. Triethylsi
for 10 min.
quenched by
The layers w
ers were co
No product w
chloride (35
to a flask an
onate (aq) a
01 (s, 1H), 8
N‐Bromosu
s stirred fo
dded and th
with water
addition of
ed aqueous
vacuo. No
mmol, 2.5 eq
and the filtra
shorter react
mmol, 1 eq)
2 mmol, 1.1
dride (35 µL
rred for 10 m
ilane (35 µL,
The solution
y addition o
were separat
ombined and
was observed
0 mg, 1.26
nd stirred at
and extracte
.29 (d, 1H),
uccinimide (
or 30 min
he resulting
(131 µL) and
1 M NaOH
NaCl soluti
product was
q) in DCM (5
ate evapora
tion times.
was added
eq) was ad
L, 59 mg, 0.2
min. The mix
L, 26 mg, 0.2
n was heated
of saturated
ted, and the
d dried ove
d by DART‐M
mmol, 3.7
t 75°C overn
ed with ethy
7.64 (d, 1H)
S22
19.6 mg,
at room
biphasic
d 1 M HCl
(aq) and
on, dried
s formed
5 mL) was
ated. This
and was
ded. The
21 mmol,
xture was
22 mmol,
d to room
d sodium
aqueous
r sodium
MS.
eq), and
ight. The
ylacetate.
, 7.49 (d,
3,4‐Dihy41 (5 g,
1.1 eq) w
NaOH (a
organic
alkaline
were wa
solvent
(SiO2; 6.
(m, 2H),
* Reaction
ydroisoquin37.5 mmol,
was added sl
aq, 25 mL) wa
layer was w
to pH 10 by
ashed with s
was remove
25% methan
7.17 (d, 1H)
n performed b
noline(42)* 1 eq) was d
lowly and th
as added an
washed with
addition of 1
aturated aq
ed in vacuo,
nol in DCM).
, 3.83 – 3.74
by Milon Mon
dissolved in
e resulting m
d the resulti
water and
1 M NaOH (a
ueous NaCl
and the pr1H‐NMR(40
4 (m, 2H), 2.7
ndal.
DCM (75 m
mixture was
ng biphasic
1 M HCl (a
aq) and extra
solution, dri
oduct was p
00 MHz, CDC
76 (q, 2H).
L). N‐Bromo
stirred for 3
mixture was
aq, 100 mL).
acted with D
ed over mag
purified usin
Cl3): δ= 8.35 (
osuccinimide
0 min at roo
stirred vigor
The acidic
CM. The com
gnesium sulf
ng flash colu
(s, 1H), 7.37
e (7.5 g, 41.2
om temperat
rously overn
extracts we
mbined organ
fate and filte
umn chroma
(td, 1H), 7.3
S23
25 mmol,
ture. 30%
night. The
ere made
nic layers
ered. The
tography
34 – 7.25
6‐Nitro‐Oven dr
mixture
to room
which th
DCM. Th
sulfate a
chromat
product.
3.91 – 3.
‐3,4‐dihydroied KNO3 (2.
42 (2.6 g, 19
temperatur
he mixture w
he organic la
and filtered.
tography (SiO
. 1H‐NMR(40
.82 (m, 2H),
oisoquinolin.02 g, 29.6 m
9.82 mmol, 1
re over 2 h.
was made a
ayers were w
. The solven
O2; 50% eth
00 MHz, CDC
2.87 (t, 2H).
ne(43)*mmol, 1.5 eq
1 eq) was ad
It was then
lkaline to pH
washed with
nt was remo
ylacetate in
Cl3): δ= 8.43
q) was disso
dded at 0°C a
immersed i
H 10 by add
saturated aq
oved in vacu
hexane). Th
(t, 1H), 8.2
lved in 1 M
and the resu
n a preheat
dition of 3 M
queous NaC
uo. The pro
his yielded 6
7 – 8.19 (m
sulfuric acid
lting mixture
ed oil bath a
M NaOH (aq
l solution, dr
duct was pu
67% product
, 1H), 8.14 (
d (aq, 20 mL
e was slowly
at 60°C for 4
) and extrac
ried over ma
urified using
t, and 33%
(d, 1H), 7.35
S24
L). To this
y warmed
4 h, after
cted with
agnesium
g column
hydrated
5 (d, 1H),
3,4‐Dihy43 (0.1 g
treated w
2 mL) an
ice‐cold
residue.
chromat
oil. 1H‐N
2H), 3.67
117.68,
* Reaction
ydroisoquing, 0.56 mmo
with a soluti
nd heated to
water (17 m
The residue
tography (SiO
MR(400 MH
7 (s, 2H), 2.6
113.95, 48.2
n perfomed b
noline‐6‐amiol, 1 eq) was
ion of tin(II)
o 60°C for 1
mL) and made
e was extract
O2; 0.5% am
Hz, CDCl3): δ=
63 (t, 2H). 13
20, 24.37.
by Milon Mond
ine(44)*s dissolved in
chloride deh
h. After coo
e alkaline to
ted into DCM
monia and 4
= 8.23 (t, 1H)
C‐NMR(101
dal.
n ethanol (2
hydrate (0.5
oling to room
pH 9 with p
M and dried i
4.5% methan
), 6.95 (d, 1H
MHz, CDCl3)
5 mL) and h
g, 2.28 mmo
m temperatu
potassium hy
in vacuo. The
nol in DCM).
H), 6.69 (dd,
): δ= 160.59,
eated to 60°
ol, 4 eq) in c
ure the mixtu
ydroxide pell
e product wa
This yielded
1H), 6.62 (d,
, 145.52, 129
0°C. This solu
concentrated
ure was pou
ets, liberatin
as purified b
d 44 as a da
, 1H), 3.77 –
9.27, 128.29
S25
ution was
d HCl (aq,
ured onto
ng an oily
y column
rk yellow
– 3.69 (m,
9, 126.39,
S26
N'‐(4‐Amino‐2‐(2‐aminoethyl)benzylidene)‐2‐fluorobenzohydrazide(45)*44 (50 mg, 0.324 mmol, 1 eq) and 30 (71 mg, 0.389 mmol, 1.2 eq) were dissolved in sodium acetate
buffer (0.6 mL, 0.1 M, pH 4.6). The mixture was heated in a microwave, but no product formation
was observed.
Librar
ModelColour c
solvent‐a
yellow li
10:(E)‐
S2
ryB—Co
llingresulcode for th
accessible su
nes. The wat
N'‐((1H‐Imi
S4
2
ompound
ltshe figures: O
urface of th
ter molecule
idazol‐2‐yl)m
S1'
ds10to1
O: red, N:
e protein is
e bound to th
methylene)‐
18
blue, F: pa
shown in g
he catalytic d
‐2‐fluoroben
S
ale cyan, Cp
grey. Hydrog
dyad is show
nzohydrazid
S2'
rotein: green,
en bonds ar
wn as a red sp
de
, Cligand: pur
re shown as
phere.
S1
S27
rple. The
s dashed,
11:(E)‐
12:(E)‐
S
S2
S4
S2
N'‐((1H‐Imi
N'‐((1H‐Imi
S4
4
idazol‐2‐yl)m
idazol‐2‐yl)m
S1'
S1'
methylene)‐
methylene)‐
‐3‐fluoroben
‐4‐fluoroben
S2'
S2'
nzohydrazid
nzohydrazid
'
de
de
S1
S1
1
S28
13:(E)‐
14:(E)‐
S4
S2
S2
N'‐((1H‐Imi
N'‐((1H‐Imi
4
S4
idazol‐2‐yl)m
idazol‐2‐yl)m
S1'
S1'
methylene)‐
methylene)‐
‐2,4‐difluoro
‐2,5‐difluoro
S2'
S2
obenzohydr
obenzohydr
2'
razide
razide
S1
S
1
S1
S29
15:(E)‐
16:(E)‐
S2
S
S2
N'‐((1H‐Imi
N'‐((1H‐Imi
S4
S4
idazol‐2‐yl)m
idazol‐2‐yl)m
S1'
S1'
methylene)‐
methylene)‐
‐2,6‐difluoro
‐3‐(trifluoro
S2
S2
obenzohydr
omethyl)ben
2'
2'
razide
nzohydrazid
S
S
de
S1
S1
S30
17:(E)‐
18:(E)‐
S
S2
S
N'‐((1H‐Imi
N'‐((1H‐Imi
4
S4
S2
idazol‐2‐yl)m
idazol‐2‐yl)m
S1'
S1'
methylene)‐
methylene)‐
‐4‐(trifluoro
‐3,5‐bis(trif
S2'
omethyl)ben
fluoromethy
S2'
nzohydrazid
yl)benzohyd
S1
de
drazide
1
S1
S31
S32
NMRexperiments
PreparationofNMRsamples
HighproteinconcentrationAn NMR sample was prepared such that the solution contained each hydrazide (200 µM); imidazole
aldehyde (400 µM); protein (100 µM); and DMSO (5% (v/v)) in deuterated sodium acetate buffer
(0.1 M, pH 4.6). Trifluoroacetic acid (0.17% (v/v)) was added as a reference. The sample was
equilibrated overnight by mild shaking at room temperature.
LowproteinconcentrationAn NMR sample was prepared such that the solution contained each hydrazide (400 µM); imidazole
aldehyde (800 µM); protein (4 µM); and DMSO (5% (v/v)) in deuterated sodium acetate buffer
(0.1 M, pH 4.6). Trifluoroacetic acid (0.17% (v/v)) was added as a reference. The sample was
equilibrated overnight by mild shaking at room temperature.
BackgroundAn NMR sample was prepared such that the solution contained each hydrazide (400 µM); imidazole
aldehyde (800 µM); and DMSO (5% (v/v)) in deuterated sodium acetate buffer (0.1 M, pH 4.6).
Trifluoroacetic acid (0.17% (v/v)) was added as a reference. The sample was equilibrated overnight
by mild shaking at room temperature.
ReferencesamplesAn NMR sample was prepared such that the solution contained one hydrazide (400 µM); imidazole
aldehyde (1600 µM); and DMSO (5% (v/v)) in deuterated sodium acetate buffer (0.1 M, pH 4.6).
Trifluoroacetic acid (0.17% (v/v)) was added as a reference. The sample was equilibrated overnight
by mild shaking at room temperature.
Quantitative19F‐NMRThe spectra for quantitative 19F‐NMR were recorded on a 500 MHz Varian Inova NMR spectrometer
equipped with an ID probe at 415 MHz; acquiring 2048 scans with a pulse width of 60° and
referenced against trifluoroacetic acid at −76.5 ppm. The integral of the trifluoroace c acid peak was
set to 1000, and peaks of the library members were assigned using spectra from the reference
samples.
1H‐STD‐NMRThe 1H‐STD‐NMR spectrum was recorded on a 600 MHz Varian Inova NMR spectrometer equipped
with a HCN triple resonance probe; suppressing solvent peaks from water and DMSO using a WET
pulse sequence.
S33
Quantitative19F‐NMRresults Hydrazide Acylhydrazone
10‐minus 4.02 0.7
10‐plus 3.37 0.35
11‐minus 6.16 1.34
11‐plus 6.92 1.55
12‐minus 5.85 1.77
12‐plus 5.88 1.65
13‐F1‐minus 5.75 1.02
13‐F1‐plus 5.54 0.89
13‐F2‐minus 6.55 0.98
13‐F2‐plus 6.24 0.72
14‐F1‐minus 5.75 0.62
14‐F1‐plus 4.37 0.38
14‐F2‐minus 6.04 0.68
14‐F2‐plus 4.58 0.42
15‐minus 10.35 0.78
15‐plus 9.28 0.75
16‐minus 21.21 3.62
16‐plus 10.3 2.38
17‐minus 15.56 0.39
17‐plus 17.83 0.31
18‐minus 26.32 2.84
18‐plus 29.08 0.39 Table S2: Peak areas of all library members compared to trifluoroacetic acid at 1000. X‐minus depict compound X in the sample without protein, X‐plus depicts compound X in the sample with protein.
S34
S35
1H‐STD‐NMRspecttrum
S36