Structure-guided design of novel l -Cysteine derivatives as potent KSP inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00221

Kinesin spindle protein (KSP), known as Hs Eg5, a member of the kinesin-5 family, plays an important role in the formation and maintenance of the bipolar spindle. We previously reported S-trityl-l-cysteine derivatives as selective KSP inhibitors. Here, we report further optimizations using docking modeling in the L5 allosteric binding site, which led to the discovery of several high affinity derivatives with two fused phenyl rings in the trityl group giving low nanomolar range KSP ATPase inhibition. The representative derivatives potently inhibited cell growth of HCT116 cells in correlation with KSP inhibitory activities and significantly suppressed tumor growth in the xenograft model in vivo.

Full text of DOI:10.1021/acsmedchemlett.5b00221

Letter
pubs.acs.org/acsmedchemlett
Structure-Guided Design of Novel L‑Cysteine Derivatives as Potent
KSP Inhibitors
Naohisa Ogo,† Yoshinobu Ishikawa,‡ Jun-ichi Sawada,† Kenji Matsuno,†,∥ Akihiro Hashimoto,§
and Akira Asai*,†
†Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
‡Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan
§Tsukuba Research Center, Taiho Pharmaceutical Co., Ltd., 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan
S
* Supporting Information
ABSTRACT: Kinesin spindle protein (KSP), known as Hs
Eg5, a member of the kinesin-5 family, plays an important role
in the formation and maintenance of the bipolar spindle. We
previously reported S-trityl-L-cysteine derivatives as selective
KSP inhibitors. Here, we report further optimizations using
docking modeling in the L5 allosteric binding site, which led to
the discovery of several high affinity derivatives with two fused
phenyl rings in the trityl group giving low nanomolar range
KSP ATPase inhibition. The representative derivatives
potently inhibited cell growth of HCT116 cells in correlation
with KSP inhibitory activities and significantly suppressed tumor growth in the xenograft model in vivo.
KEYWORDS: Kinesin spindle protein, L-cysteine derivative, molecular modeling, differential scanning fluorimetry,
HCT-116 xenograft model
ntimitotic agents such as microtubule stabilizers (e.g.,
Ataxanes) and destabilizers (e.g., vinca alkaloids) have been
clinically validated in the treatment of multiple cancers.1
Eribulin, which is derived from the natural product
haricondolyn B, has recently been approved as a microtubule
inhibitor with a new mode of action for the treatment of
metastatic breast cancer.2 Microtubules play important roles
throughout the cell cycle of cancer cells; however, the
Figure 1. Structure of KSP inhibitors: ispinesib (1), filanesib (2),
STLC (3), and the previously reported derivatives (3a and 3b).
disruption of microtubule dynamics produces undesirable side
effects, such as neurotoxicity and peripheral neuropathy, which
are related to the central role tubulin plays in cell signaling and
vesicular transport.3 Acquired drug resistance is another
limited to date, better results have recently been obtained in the
obstacle that can arise following long-term use of these agents.4
treatment of hematological malignancies with thiadiazole-based
To overcome these limitations, novel anticancer agents that do
not directly act on microtubules are being developed.5 Kinesin
spindle protein (KSP), known as Hs Eg5, a member of the
kinesin-5 family, plays an important role in the formation and
maintenance of the bipolar spindle.6 Inhibition of KSP leads to
cell cycle arrest during mitosis and results in cells with
monopolar spindles (so-called monoasters), followed by
apoptosis.7 As KSP is absent in postmitotic neurons and is
likely to act only in dividing cells, its inhibitors might provide
better specificity than microtubule inhibitors in the treatment of
human malignancies.8 A large number of KSP inhibitors with
various chemical scaffolds have been reported and some of
them, including ispinesib (1) and filanesib (known as ARRY-
520, 2), have been entered into clinical trials (Figure 1).9−13
Although the clinical efficacy of these inhibitors has been
KSP inhibitors, such as filanesib. Currently, filanesib is
undergoing clinical evaluation in combination with proteasome
inhibitors such as bortezomib or carfilzomib.14
Previously, we reported structure−activity relationship
studies (SARs) of S-trityl-L-cysteine (STLC, 3) derivatives as
KSP inhibitors (Figure 1).15 Several STLC derivatives, such as
compounds 3a and 3b with a single lipophilic para substituent
in one phenyl ring of the trityl group, were 4- to 10-fold more
potent than original STLC, in terms of both KSP ATPase
inhibition and cell cytotoxicity. Recently, we showed that
compound 3a induces mitotic arrest by activating the mitotic
Received: June 2, 2015
Accepted: July 22, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00221
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ACS Medicinal Chemistry Letters
Letter
checkpoint, which eventually led to caspase-independent cell
death in K562 chronic myeloid cells,16 as well as demonstrated
the capture of KSP in cells by using 3b immobilized on affinity
beads.17 Kozielski and co-workers, who contributed to the
discovery of STLC as a KSP inhibitor,18,19 have solved the
crystal structure of KSP in complex with STLC, revealing that it
binds in an allosteric loop L5 binding pocket, as is the case for
most other allosteric inhibitors.20−23 Recently, they reported
new STLC-related compounds, where the sulfur atom in STLC
was replaced with CH2, and the triphenylbutanamine
derivatives showed good oral bioavailability and pharmacoki-
netics.24,25 According to their data, the trityl group of STLC
was situated in a predominantly hydrophobic core region and
was involved in hydrophobic interactions. In particular, the
aromaticity and configuration of each phenyl ring in the trityl
group are important for interactions such as π−π, CH−π, and
edge-to-face interactions with the alkyl side chains of Glu215,
Glu116, and Arg119 of KSP, respectively. Based on these
findings, we hypothesized that the locking of two phenyl rings
with appropriate alkyl chains in the trityl group might afford a
configuration that would enable hydrophobic interactions and
potent binding to KSP with reduced loss of entropy. Here, we
describe a novel series of STLC derivatives with conforma-
tionally constrained trityl groups that may contribute to the
development of potent KSP inhibitors. We also performed
docking simulation of these novel derivatives using AMBER12
and differential scanning fluorimetry (DSF) analysis26 to
investigate the novel ligand−KSP direct interactions. Further-
more, we demonstrated that the representative derivatives 5a−
5d and 6a significantly suppressed tumor growth in the
HCT116 xenograft model.
inhibition of KSP ATPase was improved by a conformationally
constrained compound, we determined a putative binding
mode of compound 4 within the well-known allosteric binding
pocket of KSP. The possible binding mode of 4 was
investigated by a molecular docking experiment via
AMBER12 using the previously reported crystal structure of
KSP in complex with a para-chloro substituted STLC derivative
(PDB code: 2xae).22 Compound 4 was found to dock in an
almost completely similar way except for the ethylene linker
(Figure 2B). Interestingly, our modeling indicated that the
ethylene linker of 4 participates in van der Waals interactions
with Tyr211 and Leu214 and CH−π interaction with Tyr211,
participation that was not seen in the previously reported STLC
derivatives. In addition, this binding mode suggested the
feasibility of improving the inhibitory activity by introducing a
substituent into the nonlinked phenyl ring. We therefore
decided to attempt further SARs based on optimization of the
cross-linked ring size and the substituent effects of the
nonlinked phenyl ring in the trityl group (Figure 2A).
As shown in Scheme 1, target compounds 4, 5a−j, 6a−e, 7a,
7b, and 8a−c were synthesized by the formation of tertiary
a
Scheme 1. Synthesis of Novel STLC Derivatives
To confirm our hypothesis, we initially prepared a cross-
linked derivative with an ethylene linker between the phenyl
rings of the trityl group in STLC and evaluated the inhibitory
activity against KSP ATPase. Encouragingly, we found that such
a subtle structural variation resulted in compound 4, which
exhibited a 9-fold increase in KSP ATPase inhibition relative to
STLC (Figure 2A). To enhance our understanding of why the
a
Reagents and conditions: (a) ArBr, n-BuLi, THF, −78 °C, 3 h, then
rt, overnight, 70−95%; (b) L-cysteine, TFA, rt, 3 h, 15−80%; (c)
PhCH2CH2Br, K2CO3, acetone, rt to reflux, 3 h, 81%; (d) PPA, 1,4-
dioxane, 105 °C, 12 h, then 130 °C, 2 h, 67%.
alcohols before thioetherification with cysteine in the presence
of trifluoroacetic acid, as described previously.15 Tertiary
alcohols were prepared by the reaction of commercially
available appropriate ketones with substituted phenyl lithium.
The ethylene linker type derivatives, such as compounds 4 and
6c, were published as glycine transport inhibitors for treating
neurological and neuropsychiatric disorders.27 However, there
have been no reports to date of the antitumor activity with the
KSP inhibition for those compounds. For the intermediate
ketone of compound 8c, thiosalicylic acid 11 was thio-alkylated
by phenethyl bromide and potassium carbonate as a base to
give 2-(phenethylthio)benzoic acid 12, which then underwent
intramolecular Friedel−Crafts acylation using polyphosphoric
acid to afford the target ketone. As for compounds 7b, 8b, and
8c, which incorporate chiral trityl groups, spectral analysis of
the diastereomeric mixtures implied that a 1:1 mixture of the
(2R, 11R) and (2R, 11S) pairs for 7b and 8b, and the (2R, 12R)
and (2R, 12S) pairs for 8c had been formed. Attempts to
separate these using HPLC proved unsuccessful, so we
prepared and assayed them as diastereomeric mixtures.
Comparisons between compounds should therefore be viewed
from a SAR perspective as relative. The synthetic processes and
Figure 2. Design and docking simulation of novel STLC derivatives.
(A) Design of compound 4 and its chemical optimization. (B)
Docking mode of compound 4 (left, gray; right, cyan). The protein
structure from PDB 2xae was used for docking. The oxygen, nitrogen,
and chlorine atoms are colored red, blue, and green, respectively.
Hydrogen bonds are shown as dotted lines. The docked pose of
compound 4 overlaid with a cocrystal structure of para-chloro STLC
derivative (right).
B
DOI: 10.1021/acsmedchemlett.5b00221
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ACS Medicinal Chemistry Letters
Letter
physical data for the intermediate and final compounds are
reported in the Supporting Information.
The newly synthesized compounds were evaluated for KSP
ATPase inhibition and cytotoxicity toward HCT116 cells,
together with the previously reported compounds (3, 3a, and
3b) as controls and the clinical candidate benchmarks (1 and
2) (Table 1). We first investigated the substituent effects on the
activity against HCT116 cells. Although cytotoxicity of
compounds 5a−5d were less potent than that of 1 and 2,
KSP ATPase inhibitory activity of those were comparable. We
then investigated the size and substitution position of the
substituents in 4. Incorporation of a 4-phenyl substituent (5e)
resulted in loss of potency, while derivatives with similar bulky
substituents (4-PhO, 5f; 4-tBu, 5g) were equipotent with 4.
Regarding the positions of the substituents, derivatives with 3-
CF3 (5h) and 3-Cl (5i) were better tolerated compared with 4
but were more than 10 times weaker than the corresponding 4-
substituent derivatives. In addition, 2-Me (5j) was more than
10 times weaker than 4. These results suggest that a small
lipophilic substituent at the para position is essential for a high
level of KSP ATPase inhibition and cytotoxic activities, as
reported in our previous SARs of STLC derivatives.15
Table 1. Inhibitory Activity on KSP ATPase and HCT-116
Cell Proliferation
Next, we investigated modification of the cross-linked alkyl
chain and the ring size. Among derivatives 6a−6d, which have
the same substituents as compounds 5a−5d, respectively,
replacement of the C−C single bond with a CC double bond
also afforded potent inhibitory activity against KSP ATPase. It
should be noted that compounds 6a−6e showed more potent
cytotoxic activity than the corresponding derivatives 5a−5e,
except for 6e, with inhibitory activity in the single-digit
nanomolar range. Although the reasons for this enhanced
cytotoxicity were not obvious, the cytotoxicity of these
derivatives presumably reflected different levels of cell
permeability because the ClogP value of a compound with a
double bond tends to be higher than that of a single bond.28
Replacement of the C−C bond with C−O (7b) and C−S (8b)
bonds slightly reduced the KSP ATPase inhibitory activity but
retained the cell cytotoxicity compared with the corresponding
substituent of 5a. Interestingly, changing the linked ring size in
compound 5a led to a dramatic loss of potency. Replacement of
the two carbons with O (7a), S (8a), and CH2−CH2−S (8c)
resulted in substantial loss of potency. These SARs indicate that
appropriate restriction of the rotatable phenyl ring in the trityl
group has favorable effects on the inhibition of KSP ATPase
and cytotoxic activities. Thus, an optimal size for the linked
ring, which has a cross-linked alkyl chain length of two atoms
and a single lipophilic para substituent in the nonlinked phenyl
ring of the trityl group, is an essential requirement for the
potent inhibition of KSP by the new STLC derivatives.
b
KSP ATPase
a
cytotoxicity
a
compd
X
R
IC50 (nM)
IC50 (nM)
3 (STLC)
H
1900 230
262 17
910 58
216 14
213 4.4
3a
3b
4-CF3
4-
166 8.8
OMe
4
-CH2CH2-
-CH2CH2-
-CH2CH2-
209 34
21 1.7
14 1.2
325 15
11 0.6
28 1.1
5a
5b
4-CF3
4-
OMe
5c
5d
5e
5f
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CH2CH2-
-CHCH-
-CHCH-
4-Me
4-Cl
9.3 0.2
11 1.3
25 1.1
41 0.6
4-Ph
1060 120
306 13
301 13
238 38
160 45
7800 550
36 0.7
1300 54
291 6.3
234 13
123 7.0
637 33
4590 270
5.0 0.3
2.6 0.4
4-PhO
4-tBu
3-CF3
3-Cl
5g
5h
5i
5j
2-Me
4-CF3
6a
6b
4-
8.6 1.1
OMe
6c
6d
6e
7a
7b
8a
8b
8c
-CHCH-
-CHCH-
-CHCH-
-O-
4-Me
4-Cl
14 1.6
12 1.7
1400 300
>20000
3.4 0.1
3.9 0.1
395 4.7
>10000
4-Ph
4-CF3
4-CF3
4-CF3
4-CF3
4-CF3
-CH2O-
-S-
75 4.7
>20000
76 2.4
>10000
We next performed DSF analysis26 to investigate the direct
interactions between the novel ring-linked STLC derivatives
with KSP. The ability to stabilize KSP was assessed by
measuring the thermal stability of the KSP native protein and
the protein complex with the ligand. Complexes of each
compound were heated stepwise in the presence of the
fluorescent dye whose fluorescence increases when it interacts
with the hydrophobic residues of the protein. These residues
become exposed to the dye when the protein is denatured. A
higher positive shift of Tm in comparison to complexes with an
inactive compound, such as dibenzyl-L-cysteine (ΔTm 0.0 °C,
KSP ATPase IC50 > 63.0 μM), means greater stabilization of
the complexes, which is a consequence of a direct inhibitor
binding. The positive control experiment using 3 as a ligand
confirmed a significant improvement in the stability of the
complex (ΔTm 5.0 °C) in the presence of nucleotide as
reported previously.29 Furthermore, as expected, the degree of
rise in the Tm shift value correlated with the potency of KSP
inhibitory activity in the compound series (4, ΔTm 8.0 °C; 3b,
ΔTm 9.0 °C; 5b, ΔTm 12.0 °C; 6b, ΔTm 12.5 °C; Figure 3A).
These results suggest that STLC derivatives in this series
-CH2S-
61 8.7
>20000
15 0.9
>10000
-CH2CH2S-
1
47 0.6
1.1 0.1
(ispinesib)
2 (filanesib)
18 0.4
0.7 0.1
a
IC50 values are shown as means SEM (for potent compounds only)
from at least three independent dose−response curves. HCT116
b
cells.
nonlinked phenyl ring in compound 4. Most encouragingly,
compared with 4, the introduction of a 4-trifluoromethyl
moiety afforded compound 5a, which showed a 10-fold increase
in KSP ATPase inhibition and a 30-fold increase in cytotoxic
activity, while a 4-methoxy moiety afforded compound 5b
giving a 15-fold increase in KSP ATPase inhibition and a 12-
fold increase in cytotoxic activity. Furthermore, compounds 5c
(4-Me) and 5d (4-Cl), with similar size substituents as
compounds 5a and 5b, also displayed excellent KSP ATPase
inhibition and potent cytotoxicity. Compared with 3,
compounds 5a−5d exhibited double-digit increases in KSP
ATPase inhibition and 22- to 83-fold increases in inhibitory
C
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Figure 3. Affinity of the STLC derivatives to KSP motor domain and effect on cell proliferation. (A) Compounds interacted with and thermal-
stabilized KSP motor domain in the presence of ATP. The fluorescence profiles of Sypro Orange obtained using 4 μM of the KSP motor domain in
the presence of various compounds at 100 μM along with 1 mM of ATP by differential scanning fluorimetry. The gray line indicates that no inhibitor
was added. The color of each line indicates the individual compound used. The melting temperatures of the KSP motor domain in the absence and
presence of compounds were presented. The compound-mediated thermal shifts were also shown as Δtemperatures. (B,C) Effect of 5a (left) and 6a
(right) on cell proliferation. Mitotic index dose−response curve (B) and mitotic phenotype (C) of HeLa cells treated with compound 5a (40 nM)
and compound 6a (40 nM). Chromosomes are colored blue and the microtubules red.
directly bind to KSP and that the inhibitory activity depends on
the potency of the KSP-ligand binding affinity.
Table 2. Antitumor Activity in the HCT116 Xenograft
Model upon Treatment with Newly Synthesized Derivatives
We previously demonstrated that 3a and 3b did not inhibit
ATPase activity of other kinesins such as centromere-associated
protein E (CENP-E), kid, KIF4, and mitotic kinesin-like
protein 1 (MKLP-1).15 The ATPase inhibitory activities of
newly synthesized derivatives such as 5a and 5b were evaluated
for those motor proteins. Neither those kinesins nor addition-
ally evaluated kinesins such as KIF15 and KIF3C were inhibited
by 5a or 5b (Supporting Information Figure S1). Furthermore,
an excellent positive correlation between KSP ATPase
inhibition and cell cytotoxicity was observed (Table 1 and
Supporting Information Figure S2). We then investigated the
effects on cell cycle progression. The MI50 values (50% mitotic
index induction concentration) of compounds 5a and 6a were
correlated with that of the level of cytotoxicity (5a, MI50 14
nM; 6a, MI50 12 nM) (Figure 3B). Microscopic analysis of the
mitotic phenotype of the cells treated with 5a or 6a showed the
formation of monopolar spindles, the typical phenotype of KSP
inhibition, in all cells during mitotic arrest (Figure 3C). Taken
together, we demonstrate that the newly designed derivatives
with two fused phenyl rings in the trityl group inhibit KSP in
cells and the inhibition of KSP subsequently induced M-phase
arrest with typical monoastral phenotype.
The in vivo antitumor effects of the representative
compounds were examined in the HCT116 xenograft model
(Table 2 and Supporting Information Figure S3). To improve
the solubility of these candidates, a sodium salt of each
compound was prepared with no differences observed in in
vitro activities (Supporting Information Table S1). Treatment
mice received compounds as intravenous injections on days 1,
3, 5, 8, 10, and 12. Compounds 4, 5a, and 6a demonstrated
dose-dependent antitumor activity in this model. At a dose of
150 mg/kg, compound 4 inhibited tumor growth (T/C: 23%).
At a 75 mg/kg dose, compound 4 showed moderate inhibition
of tumor growth (T/C: 43%). At the same dose, compound 5a,
which exhibited approximately a 30-fold increase in cytotoxicity
against HCT116 cells compared with 4, was more potent (T/
C: 9%). Whereas compound 4 showed weak inhibition of
tumor growth (T/C: 62%) at a dose of 25 mg/kg, compounds
a
compd
dose (mg/kg)
T/C (%)
mortality
c
4
150
75
25
75
25
25
25
25
25
10
23
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
b
4
43
b
4
62
c
5a
5a
5b
5c
5d
6a
6a
9
c
21
c
26
c
19
c
11
c
12
b
31
a
T/C (%) = (mean tumor weight of treated group)/(mean tumor
weight of control group) × 100, n = 6. *p < 0.0001. **p < 0.00001
b
c
vs control.
5a−5d and 6a showed significant suppression of HCT-116 cell
growth (P < 0.00001) on day 15. With regard to body weight
change by the compound treatment, slight to moderate body
weight loss was observed against the vehicle control
(Supporting Information Figure S3). To assess the exact
efficacy and toxicity of those compounds, it is necessary to
optimize the amount, schedule, and root of administration.
However, none of the mice died during the treatment under the
condition used in this study. These pharmacological studies
with HCT-116 xenograft models demonstrated the potent
antitumor efficacy of the STLC derivatives with locked phenyl
rings. The most potent group of the derivatives such as
compounds 5a−5d and 6a in the in vitro studies also exhibited
potent antitumor activity in vivo, suggesting that the antitumor
activity of those derivatives depend on the KSP inhibition in
the tumor. Although the details for the effect of those
derivatives on tumor microenvironment still remained to be
elucidated, the newly identified STLC derivatives could be
potent lead compounds for developing next-generation KSP
inhibitors and probing biological function of KSP in tumor
microenvironment.
In summary, a series of novel STLC derivatives were
designed by a structure-guided approach, and those were
D
DOI: 10.1021/acsmedchemlett.5b00221
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ACS Medicinal Chemistry Letters
Letter
synthesized and biologically evaluated as KSP inhibitors. The
SARs data indicate that the two linked phenyl rings and the
nonlinked phenyl ring with a small lipophilic para substituent in
the trityl group enabled better binding by occupying a
hydrophobic pocket in the STLC binding site. The modeling
results indicated that van der Waals interactions between the
new STLC derivatives and KSP might contribute to the
improvements seen in inhibitory activities. New derivatives,
such as 5a−5d and 6a−6d, displayed potent KSP ATPase
inhibition and cell cytotoxicity in the nanomolar range. In
addition, excellent correlation was observed between the
inhibitory activities. DSF analysis showed direct binding of
the STLC derivatives and KSP and revealed that inhibitory
activity was dependent on the potency of the binding affinity to
the protein. Representative compounds 5a and 6a arrested cells
in mitosis, leading to formation of the monopolar spindle
phenotype. Furthermore, compounds 5a−5d and 6a signifi-
cantly suppressed HCT116 xenograft tumor growth in vivo.
Thus, the STLC derivatives with two linked phenyl rings could
be novel lead compounds in the design of clinical candidates for
next-generation KSP inhibitors as antitumor chemotherapies.
Although limited clinical responses have been reported for
almost all KSP inhibitors examined as monotherapies, the use
of KSP inhibitors, such as 2, in combination with other
anticancer drugs to improve the clinical effects is still an
attractive prospect. Further detailed studies of this novel STLC
series, including X-ray cocrystallization, in vivo evaluation, and
the exploration of predictive biomarkers, are in progress.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00221.
Figures, table, compound characterization, and methods
for syntheses and biological studies (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-54-264-5231. Fax: +81-54-264-5231. E-mail:
aasai@u-shizuoka-ken.ac.jp.
■
Present Address
∥Department of Chemistry and Life Science School of
Advanced Engineering, Kogakuin University, Tokyo, 192−
0015, Japan.
Funding
This work was supported by the Drug Discovery Program of
the Pharma Valley Center and JSPS KAKENHI Grant Number
26460150.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Ms. Chika Tokuyama for excellent technical
assistance in biological evaluations.
■
ABBREVIATIONS
■
KSP, kinesin spindle protein; STLC, S-trityl-L-cysteine; TFA,
trifluoroacetic acid; PPA, polyphosphoric acid; DSF, differential
scanning fluorimetry; CENP-E, centromere-associated protein
E; MKLP-1, mitotic kinesin-like protein 1
E
DOI: 10.1021/acsmedchemlett.5b00221
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ACS Medicinal Chemistry Letters
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