Structural Re-engineering of the α-Helix Mimetic JY-1-106 into Small Molecules: Disruption of the Mcl-1-Bak-BH3 Protein-Protein Interaction with 2,6-Di-Substituted Nicotinates

Article abstract of DOI:10.1002/cmdc.201500461

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: K_i=2.90 μm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D ¹H-¹⁵N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts. 2,6-Di-substituted nicotinates inhibit the myeloid cell leukemia-1 (Mcl-1) oncoprotein with potencies in the single-digit micromolar range, as determined by a fluorescence polarization competition assay. Direct binding to Mcl-1 was confirmed by 2D ¹H-¹⁵N HSQC NMR spectroscopy. Inspired by a fragment of a previously reported α-helix mimetic, the small-molecules reported herein are more druglike and, owing to greater synthetic accessibility, future optimization is expected to be elementary.

Full text of DOI:10.1002/cmdc.201500461

DOI: 10.1002/cmdc.201500461
Communications
Structural Re-engineering of the a-Helix Mimetic JY-1-106
into Small Molecules: Disruption of the Mcl-1–Bak-BH3
Protein–Protein Interaction with 2,6-Di-Substituted
Nicotinates
Brandon Drennen,[a] Jacob A. Scheenstra,[a] Jeremy L. Yap,[a] Lijia Chen,[a]
Maryanna E. Lanning,[a] Braden M. Roth,[b] Paul T. Wilder,[b, c] and Steven Fletcher*[a, c]
The disruption of aberrant protein–protein interactions (PPIs)
with synthetic agents remains a challenging goal in contempo-
rary medicinal chemistry but some progress has been made.
One such dysregulated PPI is that between the anti-apoptotic
Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and
the a-helical Bcl-2 homology-3 (BH3) domains of its pro-apop-
totic counterparts, such as Bak. Herein, we describe the discov-
ery of small-molecule inhibitors of the Mcl-1 oncoprotein
based on a novel chemotype. Particularly, re-engineering of
our a-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates
afforded inhibitors of comparable potencies but with signifi-
cantly decreased molecular weights. The most potent inhibitor
teins.[3] The anti-apoptotic proteins, which include Bcl-2, Bcl-xL,
Bcl-w, Bcl-A1 and Mcl-1, bind to the a-helical Bcl-2 homology-3
(BH3) domains of pro-apoptotic proteins, which include Bax,
Bak and Bim, effectively neutralizing them and inhibiting apop-
tosis.[4,5] In many cancers, the anti-apoptotic Bcl-2 proteins are
upregulated, and in this way the cancer cells can evade apop-
tosis.[2] Accordingly, synthetic small-molecule strategies to in-
hibit the Bcl-2 proteins have been adopted as a new avenue
for cancer therapy.[6]
The Mcl-1 gene is frequently overexpressed in human can-
cers,[7] including lung,[8] breast,[9] pancreatic,[10] cervical cancers,[11]
as well as leukemia.[12] Importantly, genetic mouse models have
shown that Mcl-1 is involved in tumor initiation and develop-
ment.[13] Thus, for these reasons, Mcl-1 has emerged as an at-
tractive target for anticancer therapy. Indeed, in the last few
years, several groups, including ours, have developed inhibitors
of Mcl-1,[14,15] which span a variety of scaffolds such as in-
doles,[16–18] 5-phenylsalicylates, 1-hydroxynaphthalenes,[19] 1-hy-
droxy-2-naphthoates[20] and tetrahydroquinolines,[21] all of which
contain a carboxylic acid to bind R263. In addition, the neutral
pyrogallol MIM1[22] and an 8-hydroxyquinoline derivative[23] have
also been described as Mcl-1 inhibitors, and it is possible these
neutral agents still engage R263 through hydrogen bond(s).
However, no Mcl-1 inhibitor has advanced to the clinic. Finally,
it is especially noteworthy that Leverson and colleagues re-
cently reported that their highly potent and selective Mcl-1 in-
hibitor A-1210477 demonstrates on-target cellular effects, thus
establishing for the first time that Mcl-1 is a viable cell target
for the development of novel anticancer therapies.[24]
2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic
acid
(1r: Ki =2.90 mm) likely binds in the p2 pocket of Mcl-1 and en-
gages R263 in a salt bridge through its carboxylic acid, as sup-
1
ported by 2D H–15N HSQC NMR data. Significantly, inhibitors
were easily accessed in just four steps, which will facilitate
future optimization efforts.
Apoptosis, or programmed cell death, ensures normal tissue
homeostasis, and its dysregulation can lead to several human
pathologies, including cancer.[1,2] Whilst the extrinsic apoptosis
pathway is initiated through the activation of cell-surface re-
ceptors, the intrinsic apoptosis pathway occurs at the mito-
chondrial outer membrane and is governed by the binding in-
teractions between pro- and anti-apoptotic Bcl-2 family pro-
[a] B. Drennen, J. A. Scheenstra, Dr. J. L. Yap, L. Chen, M. E. Lanning,
Dr. S. Fletcher
In addition to conventional small-molecule strategies, Mcl-
1 inhibitors have been fashioned through the structural mimi-
cry of BH3 a-helical domains.[25–31] However, such “a-helix mim-
etics” targeting Bcl-2 proteins have not advanced to the
clinic,[32–34] and this may be due to their high molecular
weights (MWs), which contravene Lipinski’s rule as they are
typically in excess of 500, and/or a lack of specificity. a-Helix
mimetics typically reproduce functionality on only one face of
the a-helix, specifically the i, i+3/4 and i+7 positions. These
are often rather hydrophobic molecules, and their inhibitory
activities might stem from nonspecific hydrophobic effects, at
least in part. We believe that the recognition properties of an
a-helix mimetic can be enhanced by mimicking additional
faces of the a-helix.[28,31,35] On the other hand, a successful a-
helix mimetic might be used as the inspiration from which
Department of Pharmaceutical Sciences
University of Maryland School of Pharmacy
Baltimore, MD 21201 (USA)
E-mail: sfletcher@rx.umaryland.edu
[b] Dr. B. M. Roth, Dr. P. T. Wilder
Department of Biochemistry and Molecular Biology
Center for Biomolecular Therapeutics
University of Maryland School of Medicine
Baltimore, MD 21201 (USA)
[c] Dr. P. T. Wilder, Dr. S. Fletcher
University of Maryland Greenebaum Cancer Center
Baltimore, MD 21201 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500461.
This article is part of a Special Issue on Protein–Protein Interactions. To
view the complete issue, visit: http://onlinelibrary.wiley.com/doi/10.1002/
cmdc.v11.8/issuetoc.
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a more traditional small molecule could be realized. Along
these lines, we herein describe the structural re-engineering of
our a-helix mimetic JY-1-106[26,27] into a series of 2,6-di-substi-
tuted nicotinates that are equipotent at inhibiting Mcl-1 but
bear significantly decreased MWs.
given in Figure 3. The small molecule–Mcl-1 crystal structure
4HW2 was selected for docking studies because our synthetic
ligand is now more akin to a traditional small molecule rather
than an a-helix or a conventional a-helix mimetic.[32] According
to the modeling results, the phenyl ring snuggly fits into the
p2 pocket (partially obscured) and the isobutyl group is point-
ed towards the p3 pocket, docking nicely into a new pocket
bounded by A227 and M231 not found in crystal structures of
Mcl-1 with BH3 ligands.[16] Furthermore, a weak salt bridge
(3.9 Š) is detected between the carboxylic acid and R263, al-
though given the plasticity[16] of the hydrophobic groove of
Mcl-1, as revealed by multiple crystal structures (e.g. PDB ID:
4HW2,[16] 4HW3,[16] 4HW4,[16] 3WIX[36]), we envisage stronger in-
teractions between the acid and R263 may be possible. With
this modeling data in hand, we then prepared a library of ana-
logues of compound 1a in which we modified the alkoxy
(R1O) and aryloxy (R2O) groups.
BH3 a-helices recognize Mcl-1 through binding four hydro-
phobic sub-pockets p1-p4 via conserved hydrophobic residues
at the i, i+3/4 and i+7 and i+11 positions along one face of
the a-helix. Additionally, a conserved aspartate on the oppos-
ing face of the a-helix binds R263. As an example, the co-crys-
tal structure of Mcl-1–Bim-BH3 is given in Figure 1A, and the
key residues of the Bim-BH3 a-helix are shown more clearly in
Figure 1B. We recently reported the discovery of the BH3 a-
helix mimetic JY-1-106 (Figure 1C), which is a dual Bcl-xL/Mcl-
1
inhibitor (Bcl-xL: Ki =179Æ24 nm; Mcl-1: Ki =1.79Æ
0.15 mm).[26,27] The isopropyl groups of JY-1-106 were designed
to target the p2, p3 and p4 pockets of Mcl-1, whilst the role of
the carboxylic acid group was to enhance compound solubility
rather than target any residue in particular.
Accordingly, 2,6-dichloronicotinic acid (2) was regioselective-
ly alkoxylated ortho to the acid with various R1OH alcohols to
afford 3 using chemistry recently developed in our laborato-
ry.[37] Incidentally, the ability to selectively control the displace-
ment of the two chlorines in 2,6-dichloronicotinic acid directly
facilitated the development of a new a-helix mimetic.[38] Subse-
quent esterification of 3 was accomplished with thionyl chlo-
ride and methanol to yield methyl esters 4. A second SNAr re-
action was effected with phenols R2OH to deliver compounds
5, or thiophenols R2SH to yield compounds 6, which were final-
ly saponified to furnish target molecules 1a–1r and 7a–7d, re-
spectively, in just four steps (Scheme 1).
We hypothesized that the central picolinamide subunit of
JY-1-106 could serve as a scaffold to generate synthetically ac-
cessible, small-molecule inhibitors of Mcl-1. We postulated that
retaining but modifying the substitution at both positions
flanking the pyridine nitrogen of the picolinamide fragment
might still permit binding to both the p2 and p3 pockets,
whilst relocation of the carboxylic acid meta to the nitrogen
atom might also allow the formation of a salt bridge with
R263. One such simple molecule, 1a, is shown in Figure 2.
Computational modeling of 1a with Mcl-1 (after ligand extrac-
tion, Mcl-1 coordinates from PDB ID: 4HW2[16]) was conducted
with GOLD (version 5.2.2), and a low-energy docked solution is
In order to quantify the abilities of compounds to inhibit
Mcl-1, a fluorescence polarization competition (FPC) assay was
Figure 1. A) Structure of Mcl-1–Bim–BH3 (PDB ID: 4HW4[16]). Mcl-1 colored by atom type: carbon=grey; blue=nitrogen; red=oxygen; yellow=sulfur. Bim–
BH3 a-helix colored in green. Key residues and binding pockets on Mcl-1 are shown in black. B) Key residues of the Bim–BH3 a-helix. C) The a-helix mimetic
JY-1-106.
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Figure 2. Reduction of the a-helix mimetic JY-1-106 into small molecule 1a. Labels in half-bubbles refer to Mcl-1 residues and subpockets.
Scheme 1. Reagents and conditions: a) R1OH, NaH, THF, 608C, 16 h; b) SOCl2,
MeOH, 608C, 3 h; c) R2OH or R2SH, K2CO3, DMF, 1008C, 72 h; d) LiOH·H2O,
THF/MeOH/H2O (3:1:1), RT, 3 h.
the phenyl R2 moiety with hydrophobic groups always en-
hanced the inhibitory activity over parent compound 1a, para-
substitution led to the greatest increases in inhibition. For ex-
ample, compare the data for 2-chlorophenyl (1c) with 4-chlor-
ophenyl (1e) derivatives, and 3,5-dimethylphenyl (1k) with 4-
methylphenyl (1h) derivatives. In every case, replacement of
the R2 ether oxygen (X group) with a sulfur atom (7a–7d) re-
sulted in an improvement in inhibitory activity of between 2.5-
fold and 12-fold. Compound 7c was the most potent of the
thioether series with a Ki value of 3.69 mm. Finally, we examined
the effect of varying the R1 group. As the size of the hydropho-
bic R1 group was increased, the resulting inhibitor generally
became more potent. In fact, 1r was the most potent com-
pound of the entire series (Ki =2.90 mm).
Figure 3. Low-energy GOLD docking solution of 1a (green carbon atoms;
heteroatoms colored by atom type) bound to Mcl-1 (coordinates from PDB
ID: 4HW2[16]). Mcl-1 (surface representation) is colored by atom type: car-
bon=grey; blue=nitrogen; red=oxygen; yellow=sulfur.
employed in which the compounds competed with a fluores-
cein isothiocyanate (FITC)-labeled Bak-BH3 peptide for binding
to Mcl-1172–327. IC50 data resulting from the analysis were con-
verted to Ki values using the Nikolovska–Coleska equation[39]
and are given in Table 1. For further details, see the Experimen-
tal Section. The simplest compound (1a) exhibited the weakest
binding to Mcl-1 with a Ki value of 261 mm. However, the intro-
duction of halogens into the phenyl R2 group resulted in im-
proved inhibition, and the effect was cumulative, which can be
seen by comparing the data for 1c and 1e with that for 1 f.
Indeed, 1 f (Ki =6.69 mm) was almost 40-fold more potent than
parent compound 1a. Naphthyl R2 derivatives also proved
better inhibitors of Mcl-1 over 1a with the 1-naphthyl deriva-
tive (1b) superior to its regioisomer (1g). Whilst substitution of
Evidence for the direct binding of 1r to Mcl-1 was provided
1
by heteronuclear NMR studies. 2D H–15N heteronuclear single
quantum coherence (HSQC) spectra of [15N]-labelled Mcl-
1 were collected in the absence and presence of 1r. An overlay
of the two spectra (Figure 4) revealed significant chemical shift
changes, particularly for R263 and those residues located
around the p2 pocket; those changes ꢀ0.3 ppm have been
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Table 1. Mcl-1 structure–activity relationships of 2,6-di-substituted nicotinates. IC50 data[a] from a fluorescence polarization competition assay with Mcl-1172–
371
and fluorescein isothiocyanate (FITC)-labeled Bak were converted to Ki values using the Nikolovska–Coleska equation.[39]
Compd
R1
R2
X
Ki [mm]
Compd
R1
R2
X
Ki [mm]
1a
O
261Æ33
1l
O
11.1Æ2.3
1b
O
40.0Æ4.2
7a
S
48.1Æ7.1
1c
O
O
72.8Æ4.6
55.6Æ4.9
7b
7c
S
S
7.11Æ0.78
3.69Æ0.17
1d
1e
1 f
1g
1h
1i
O
O
O
O
O
O
O
39.8Æ2.1
6.69Æ0.71
59.6Æ3.4
87.8Æ18.4
47.0Æ10.6
12.7Æ1.3
123Æ16
7d
1m
1n
1o
1p
1q
1r
S
50.3Æ3.7
25.4Æ1.4
26.9Æ8.6
20.0Æ1.1
4.58Æ1.20
4.62Æ0.77
2.90Æ1.24
O
O
O
O
O
O
1j
1k
[a] Data represent the meanÆSD of three biological replicates, each performed in triplicate.
mapped onto the Mcl-1 crystal structure PDB ID: 4HW2[16] and
are shown in red in Figure 5.
Overall, larger and more hydrophobic R1 and R2 groups af-
forded greater inhibition of Mcl-1. This finding may be due to
more efficient interactions with the hydrophobic p2 pocket,
which is supported by the HSQC NMR data. The inhibitory ac-
tivity was enhanced further still by replacing the R2 phenyl
ether oxygen with a sulfur atom, which can be rationalized by
the greater hydrophobicity of sulfur over oxygen coupled with
the idea that the R2 group is directed into the hydrophobic p2
pocket. In addition, the carboxylic acid of the inhibitors was
critical to activity since the methyl ester derivative of 1r exhib-
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Figure 4. 1H–15N heteronuclear single-quantum correlation (HSQC) spectra overlay of apo-Mcl-1 (black) and 1r-bound Mcl-1 (red).
Figure 5. 1H–15N chemical shift perturbations ꢀ0.3 ppm of Mcl-1 in the presence of 1r (red) mapped onto the Mcl-1 crystal structure (PDB ID: 4HW2[16]).
ited no effect on the Mcl-1–Bak-BH3 PPI (data not shown),
which is consistent with the design rationale wherein the car-
boxylic acid was proposed to form a salt bridge with R263.
Many of the inhibitors described in the present work exhibit-
ed similar Ki values to the a-helix mimetic JY-1-106 (Ki =
1.79 mm), yet they have much lower MWs. Indeed, the two
most potent compounds (7c and 1r) are about two-thirds the
mass of JY-1-106, bringing their MWs within the threshold pro-
posed by Lipinski (MW<500). Furthermore, their lipophilicities
are also significantly decreased. For example, the cLogP and
cLogD values (pH 7.4) for JY-1-106 are 7.37 and 4.32, respec-
tively, whilst the corresponding values for 1r are 6.11 and 2.98,
respectively. Finally, we evaluated the selectivity profile of 1r
for Bcl-2 family members. As shown in Figure 6, the IC50 values
for 1r against Mcl-1 and Bcl-xL were 11.35Æ4.85 mm and
31.53Æ6.19 mm, respectively, which correspond to Ki values of
2.90Æ1.24 mm and 2.04Æ0.40 mm, respectively. It is notewor-
thy that the 10-fold Bcl-xL-selectivity of JY-1-106 has been
almost completely eroded upon its transformation into small-
molecule nicotinates; future work will focus on the acquisition
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tion mixture was partitioned between CH2Cl2 and 1m aq HCl (the
aqueous layer was pH~2). The organic layer was collected, and the
aqueous layer was extracted with CH2Cl2 twice further. The organic
layers were combined, dried with Na2SO4, filtered and concentrated
to yield the crude ortho-substituted nicotinic acid, which was used
without further purification.
Esterification (general procedure B): The ortho-substituted nico-
tinic acid (1 equiv) was dissolved in MeOH (0.10m), and the solu-
tion was cooled to 08C. Thionyl chloride (3 equiv) was slowly
added, and then the mixture was refluxed for 3 h. The reaction
mixture was concentrated to dryness. The crude ester was ad-
sorbed onto silica gel, then purified by flash column chromatogra-
phy (eluent: hexane/EtOAc, 4:1).
Second SNAr Reaction (general procedure C): The methyl ester
(1 equiv) was dissolved in DMF (0.10m). The appropriate phenol
(R2OH) or thiophenol (R2SH) (4 equiv) was added to the solution
followed by K2CO3 (3 equiv). The reaction was heated at 1008C for
72 h. The reaction was partitioned between EtOAc and H2O. The
organic layer was isolated and washed repeatedly (5”) with H2O.
The organic layer was then collected, dried with Na2SO4, filtered
and concentrated under vacuum. The crude material was adsorbed
onto silica gel, then purified by flash column chromatography
(eluent: hexane/EtOAc, 4:1).
Figure 6. Inhibition of Mcl-1 (&) and Bcl-xL (!) by compound 1r, as deter-
mined by a fluorescent polarization competition (FPC) assay with fluorescein
isothiocyanate (FITC)-labeled Bak. Data points are the mean and error bars
the standard deviation of three biological replicates performed in triplicate.
of crystal structures of 1r with Mcl-1 and Bcl-xL to assist in fur-
ther enhancement in affinity and selectivity for Mcl-1.
In summary, using our previously reported a-helix mimetic
JY-1-106 as a starting point, we have developed a new family
of readily accessible Mcl-1 inhibitors based on a 2,6-di-substi-
tuted nicotinic acid core. HSQC NMR data supported the hy-
pothesis that the R1 group and/or the R2 group probe(s) into
the p2 pocket, whilst the carboxylic acid likely engages R263
through a salt bridge, although the exact binding mode re-
mains unknown at this time. It is noteworthy that our most
potent compounds are about as potent as the a-helix mimetic
JY-1-106 yet exhibit significantly decreased MW and cLogD
values. Additional structure–activity studies are currently un-
derway, which, as well as enhancing compound affinity, aim to
gain a better understanding of the specific binding mode and
how this can be exploited towards achieving selectivity for
specific Bcl-2 family members of anti-apoptotic proteins.
Saponification (general procedure D): The o,p-di-substituted nico-
tinate (1 equiv) was dissolved in a 3:1:1 solution of THF/MeOH/H2O
(0.10m). LiOH·H2O (4 equiv) was added, and the reaction mixture
was stirred at RT for 3 h. If necessary, the reaction volume was de-
creased to approximately 10 mL, then partitioned between Et2O
and H2O. The ethereal layer was discarded, and then the aqueous
layer was acidified to pH 2 with 0.1m HCl, then extracted twice
with CH2Cl2. The organic layer was collected, the aqueous layer
was extracted once more with CH2Cl2, then the organic extractions
were combined, dried with Na2SO4, filtered and concentrated to
produce the o,p-di-substituted nicotinic acid. All final molecules
were deemed to exhibit >95% purities by HPLC and were not pu-
rified further.
Fluorescence polarization experiments: Fluorescence polarization
experiments were conducted using a PHERAstar FS multimode mi-
croplate reader (BMG LabTech) equipped with two photomultiplier
tubes (PMTs) for simultaneous measurements of the perpendicular
and parallel fluorescence emission. For the competition assay, in-
hibitors were titrated into a solution of Mcl-1172–327 (or Bcl-xL2–212),
and the fluorescently labeled Bak-BH3 peptide FITC-Ahx-
GQVGRQLAIIGDDINR-CONH2 (hereafter “FITC-Bak”), where FITC is
fluorescein isocyanate and Ahx is 6-aminohexanoyl linker. Regres-
sion analysis was carried out using Origin (OriginLab, Northamp-
ton, USA) to fit the data to the Hill equation to determine the ini-
tial binding affinity (Kd) and the IC50 in the FPC assay. Kd values for
the FITC–Bak peptide to Mcl-1 and Bcl-xL were 33.8Æ0.50 nm and
6.67Æ0.05 nm, respectively. For the fluorescence polarization com-
petition titrations, an equation derived by Nikolovska–Coleska et al.
was used to calculate the Ki values from the IC50 data.[39] All experi-
ments were run in three biological replicates, each performed in
triplicate.
Experimental Section
Supporting Information: Complete protocols for both chemical
syntheses and biological methods together with characterization
data are presented in the Supporting Information available via
Wiley Online Library.
Molecular docking: Compound 1 a was first MM2 energy mini-
mized in ChemBio3D Ultra 14.0. The 4HW2[16] PDB file was upload-
ed into GOLD (version 5.2.2), all the appropriate hydrogen atoms
were added, water molecules were removed, and then the ligand
was extracted. The binding site was defined as 10 Š about the side
chain sulfur of Met231; no further constraints were used.
First SNAr Reaction (general procedure A): Alcohol R1OH (5 equiv)
was dissolved in anhydrous THF (0.10m), and the solution was
cooled to 08C then NaH (5 equiv; 60% dispersion in mineral oil)
was slowly added. The reaction mixture was stirred for 30 min at
08C before 2,6-dichloronicotinic acid (1 equiv) was added portion-
wise, and then the mixture was heated overnight at 608C in an oil
bath. The next day, the reaction was cooled to 08C, quenched with
brine (1 mL), then concentrated to approximately 10 mL. The reac-
1
NMR Spectroscopy: 2D H–15N HSQC spectra were collected with
200 indirect points and 32 scans at 258C with a Bruker AVANCE
800 MHz NMR spectrometer (800.27 MHz for protons) equipped
with pulsed-field gradients, four frequency channels, and triple res-
onance, z-axis gradient cryogenic probes. A one-second relaxation
delay was used, and quadrature detection in the indirect dimen-
sions was obtained with states–time proportional phase incremen-
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were set to give zero- and first-order phase corrections of 908 and
À1808, respectively.[40,41] Data were processed using the processing
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1
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1, 20 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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Published online on February 4, 2016
ChemMedChem 2016, 11, 827 – 833
www.chemmedchem.org
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