Synthesis, Structural Characterization, and Antiangiogenic Activity of Polyfluorinated Benzamides

Article abstract of DOI:10.1002/cmdc.201800263

The introduction of fluorine into bioactive molecules is a matter of importance in medicinal chemistry. In this study, representatives of various chemical entities of fluoroaromatic compounds were synthesized. Depending on the reaction conditions, either tetrafluorophthalimides or ammonium tetrafluorophthalamates are accessible from tetrafluorophthalic anhydride and primary amines. Tetrafluorophthalamic acids undergo thermal decarboxylation to yield tetrafluorobenzamides. These could be successfully converted upon treatment with primary amines, in the course of an aromatic nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective amino substituents at the 4-position. The five structure types were characterized by means of spectroscopic and crystallographic methods. The synthesized compounds were evaluated as inhibitors of angiogenesis by measuring microvessel outgrowth in a rat aortic ring assay. The biological activity was maintained throughout these different polyfluorinated chemotypes.

Full text of DOI:10.1002/cmdc.201800263

Accepted Article
Title: Synthesis, Structural Characterization and Antiangiogenic Activity
of Polyfluorinated Benzamides
Authors: Christian Steinebach, Agnieszka Ambrozak, Stefan
Dosa, Shaunna L. Beedie, Jonathan D. Strope, Gregor
Schnakenburg, William D. Figg, and Michael Gütschow
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201800263
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10.1002/cmdc.201800263
ChemMedChem
FULL PAPER
Synthesis, Structural Characterization and Antiangiogenic
Activity of Polyfluorinated Benzamides
Christian Steinebach,[a] Agnieszka Ambrożak,[a] Stefan Dosa,[a] Shaunna L. Beedie,[b] Jonathan D.
Strope,[b] Gregor Schnakenburg,[c] William D. Figg,[b] and Michael Gütschow*[a]
The introduction of fluorine into bioactive molecules is a matter of
importance in medicinal chemistry. In this study, representatives of
various chemical entities of fluoroaromatic compounds have been
synthesized. Depending on the reaction conditions, either
tetrafluorophthalimides or ammonium tetrafluorophthalamates are
accessible from tetrafluorophthalic anhydride and primary amines.
Tetrafluorophthalamic acids undergo thermal decarboxylation to
yield tetrafluorobenzamides. These could be successfully converted
upon treatment with primary amines, in the course of an aromatic
nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective
amino substituents at 4-position. The five structure types have been
characterized by means of spectroscopic and crystallographic
methods. The synthesized compounds were evaluated as inhibitors
of angiogenesis by measuring the microvessel outgrowth in the rat
aortic ring assay. The biological activity was maintained throughout
these different polyfluorinated chemotypes.
The importance of tetrafluorination of simple phthalimides to
achieve antiangiogenic activity has been shown.[3] Similarly,
compound I (Figure 1), a polyfluorinated phthalimide derivative,
leads to a strongly enhanced inhibition of the microvessel
outgrowth in an angiogenesis model,[4] and of the TNF-
production by monocytes.[5] It possesses antimyeloma capacity,
activates the stress kinase p38 pathway, affects blood vessels
depending on their state of maturity,[6] and was active in a mouse
xenograft model at low doses in combination with flavopiridol.[7]
Whereas phthalimidobarbituric acids of type II failed to act as
antiangiogenic agents, the fluorinated analogs III showed a
strong decrease in microvessel outgrowth in rat aortic ring
explants.[8,9] The intact fused five-membered ring was not
essential since the activity was retained in case of the
tetrafluorobenzamide derivatives IV.[8,9]
F
O
O
O
R1
F
F
R2
O
N
N
N
F
O
N
H
O
F
O
F
F
Introduction
I
II
Angiogenesis, the formation of new blood vessels from existing
ones, is driven by a finely tuned balance between factors which
constitute a functional network. The process of angiogenesis is
important for several physiological and pathological conditions,
including cancer, where functional vessels are required to supply
nutrients and oxygen, and to sustain tumor growth and
metastatic dissemination. Angiogenesis can be inhibited by
blocking pro-angiogenic or raising antiangiogenic factors or by
killing tumor-activated endothelial cells. Antiangiogenic drugs
have been shown to slow tumor progression and metastasis.[1]
F
F
O
O
O
R1
O
R2
R1
F
F
F
F
R2
O
N
H
N
N
N
O
N
O
O
N
H
H
O
F
IV
III
Figure 1. Structures of (fluorinated) phthalimide and benzamide derivatives as
antiangiogenic agents.
Covalently bound fluorine has attracted much attention in drug
development, although it is an unusual element in natural
products. The substitution of hydrogen by fluorine in bioactive
molecules produces only minor changes in the 3D structure due
to the similar van der Waals radii of fluorine and hydrogen.
Strong dipole moments of the carbon-fluorine bond result from
the large electronegativity of fluorine and may contribute to inter-
and intramolecular interactions.[10]
Accordingly, therapies based on such drugs represent
a
promising approach for the treatment of cancer diseases and
there remains a need for novel agents against angiogenesis.[2]
[a]
C. Steinebach, Dr. A. Ambrożak, Dr. S. Dosa, Prof. Dr. Michael
Gütschow
Pharmaceutical Institute, Pharmaceutical Chemistry I
University of Bonn
The property of covalently bound fluorine in organic molecules
as a hydrogen-bond acceptor has been a controversial issue.
Meanwhile, a number of spectroscopic, crystallographic and
computational studies provided several lines of evidence for
weak C-F…H-X hydrogen bonds. Hydrogen bonds to fluorine
have the potential to effectively alter the binding events of
fluorinated compounds to biological target structures.[11] In
addition to an enhanced target affinity, increased metabolic
stability can be attained; in particular, labile sites of metabolic
oxidation can be blocked by fluorination.[10]
An der Immenburg 4, D-53121 Bonn, Germany
E-mail: guetschow@uni-bonn.de
Dr. S. L. Beedie, J. D. Strope, Dr. W. D. Figg
Molecular Pharmacology Section
National Cancer Institute, NIH
Bethesda, MD 20892, USA
Dr. G. Schnakenburg
Institute of Inorganic Chemistry
University of Bonn
Gerhard-Domagk-Str. 1, D-53121 Bonn, Germany
[b]
[c]
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10.1002/cmdc.201800263
ChemMedChem
FULL PAPER
F
F
F
The high electronegativity of fluorine and the resulting
polarization of the C–F bond lead to a remarkable alteration of
the physicochemical properties, such as acidity or basicity of
adjacent functional groups. Moreover, the addition of fluorine or
trifluoromethyl groups on aryl or heteroaryl groups can increase
the lipophilicity and enhance the membrane permeability in
comparison with the non-fluorinated parent compounds.[10]
In the course of the determination of favorable (fluorophilic) and
unfavorable (fluorophobic) environments within target proteins,
inhibitors of serine and metallo proteases,[12] cysteine
proteases,[13] and protein kinases[14] have been established.
In this study, it was intended to incorporate several simplified
amino components into tetrafluorophthalimides III and
tetrafluorobenzamides IV and to extend the series of test
compounds by two types of related polysubstituted
fluorobenzene derivatives decorated with the same amino
components. The resulting compounds were investigated on the
basis of spectroscopic and crystallographic data. Finally, the
entire library of fluoro compounds was subjected to the
examination of antiangiogenic properties.
O
O
4
F
F
F
(i)
O
N R
1
F
O
O
F
1
2a-f
(ii)
R
R
O
F
F
HN
1
F
F
HN
1
F
F
F
O
(iii)
R
H3N
CO2H
4a-f
(iv)
F
CO2
2
2
3a-f
R
a
b
c
d
e
f
R = n-Pr
R = i-Pr
R = i-Bu
R = cyclohexyl
R = Bn
F
HN
2
F
F
O
1
F
R = tert-Bu
5a-f
Results and Discussion
F
HN
1
Six representatives of the chemotype of tetrafluorophthalimides
(2a-f) were synthesized by condensation of tetrafluorophthalic
anhydride (1) with different primary amines. The ring-
opening/recyclization reactions were performed in AcOH at
118 °C (Scheme 1). Under different conditions, when treating 1
with primary amines in refluxing toluene, the corresponding
ammonium tetrafluorophthalamates (3a-f) were obtained. In the
absence of AcOH, the recyclization step is prevented because
the products accumulate as salts without carbonyl reactivity and
precipitate. Tetrafluorophthalamic acids 4a-f were easily
2
F
5b
O
(v)
R
N
H
F
F
6a-d
R-NH2
HN
2
F
5d
O
1
R
(v)
N
H
accessible by protonation of
3 in an aqueous medium.
F
Chemotypes 3 and 4 constitute intermediate structures of a
previously reported tandem ring opening/decarboxylation
sequence.[9] In the present study, we have isolated and
characterized these compounds and provided those of type 4 to
a biological evaluation.
7a-e
Scheme 1. Synthesis of polyfluorinated compounds 2-7. Conditions (i) RNH2,
AcOH, reflux, 3 h; (ii) RNH2, dry toluene, reflux, 3 h; (iii) H2O, pH 1-2, 4 °C; (iv)
1. dry DMSO, 153 °C, 4 h, 2. H2O, rt, 3 d; (v) , 4 h.
Thermal decarboxylation of tetrafluorophthalamic acids 4 was
achieved in DMSO at 153 °C to successfully produce 5a-f.
These tetrafluorobenzamides represent structurally simplified
analogs of the aforementioned antiangiogenic barbituric acids IV
(Figure 1).
We selected two tetrafluorobenzamides (5b and 5d) for the
reaction with primary amines, the isopropyl derivative 5b
because of its promising antiangiogenic activity (Table 1) and 5d
because the bulky cyclohexyl moiety was expected to be
To introduce a further point of diversity, we considered to subject
tetrafluoro derivatives to a nucleophilic substitution with amines.
The reaction of certain phthalimides of type 2 with primary
amines led to mixtures of 4- and 5- substituted regioisomers,
which influenced cellular vesicular systems, in particular the
generation of lipids droplets.[15] Mixtures of even four isomeric
products might be formed from tetrafluorophthalamic acids 4 and
amines. However, the transformation of tetrafluorobenzamides 5
advantageous.
The
nucleophilic
substitution
occurred
unsurprisingly at the para position. With the so substituted
products 6a-d and 7a-e, we received nine highly functionalized
benzene derivatives. This transformation could also be
accomplished under microwave assisted conditions with similar
yields, as exemplified for 7c and 7d.
The 13C NMR spectra of tetrafluorophthalimides 2 exhibited
three distinct shifts of the carbon resonances of the fused
benzene ring, C-3a/7a, C-4/7, and C5/6. Our assignments are in
agreement with previous work[9] and
investigation including N-phenyl-tetrafluorophthalimide.[17] The
is expected to proceed regioselectively.
A corresponding
conversion has been reported, even though in rare cases.[16]
a
detailed NMR
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10.1002/cmdc.201800263
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magnitude of the C-F coupling of carbons C-3a/7a varied from
7 to 10 Hz, although larger constants could be assumed for
two-bond fluorine couplings, e.g. as described for C-3a/7a in
N-phenyl-tetrafluorophthalimide (2J(C,F) = 13.5 Hz).[17] However,
smaller values (2J(C,F) ~ 7 Hz) have been reported for four
derivatives of N-phenyl-tetrafluorophthalimide.[18] The 2J(C,F)
coupling constants of 4-fluoroisoindoline (8, Figure 2) of ~ 19 Hz
for both C-3a and C-5 indicate that carbons which are engaged
in ring-fusion do not necessarily produce small 2J(C,F) constants.
Thus, the fixed orientation of the carbonyl groups relative to the
C-F bonds at positions 4 and 7 attributes to the small coupling
constants in case of 2a-f.
relative to the aromatic plane by 70°. A sandwich - stacking is
created with alternating orientation of the substituents and a 3.7
Å centroid-distance of the benzene rings.
The C-1 and C-6 signals of ammonium tetrafluorophthalamates
3 derived from 2J(C,F) coupling constants of either around 22 Hz
or 15 Hz could initially not be distinguished. In order to assign
the chemical shifts and the corresponding coupling constants to
carbons C-1 and C-6, we have recorded a 13C NMR spectrum of
2-fluorobenzamide (9, Figure 2).[19] An X-ray crystal structure of
9 revealed the orientation of the NH2 moiety to the fluorine
substituent, while in other ortho-halobenzamides the oxygen
pointed in the direction of the halogen.[20] As in 9, a preferred
intramolecular F∙∙∙H-N interaction was also postulated for our
Figure 3. Left. Molecular plot of 2,3,4,5-tetrafluoro-6-(propylcarbamoyl)
benzoic acid (4a) showing the displacement ellipsoids at the 50% probability
level for the non-H atoms. Right. Packing diagram of 4a. The N–H∙∙∙F and N–
H∙∙∙O contacts are shown as broken lines.[21]
2
compounds 3. We have now considered the J(C-1,F) coupling
constant in 9 of 14.2 Hz as a reference for the 2J(C-6,F) coupling
constants in compounds 3. Accordingly, we could assign the 13
C
The regioselectivity of the nucleophilic substitution was
confirmed by the chemical shifts in the 13C NMR spectra.
Compounds 6 and 7 exhibited 2J(C-1,F) values of ~ 13 Hz,
similar to the coupling constants of the carboxamide-substituted
aromatic carbons in 3 and 4. The unsubstituted aromatic
2
NMR signals with J(C,F) values of around 15 Hz to C-6 ( 120
ppm) and those with 22 Hz to C-1 ( 128 ppm). In
3-fluorobenzamide (10, Figure 2), the 2J(C-2,F) coupling
constant of 22.6 Hz was determined. Thus, the magnitude of the
heteronuclear spin-spin coupling depended not only on the
distance between the carbon and fluorine nuclei, but also on the
orientation of their orbitals and intramolecular interactions.
2
carbons of 5, 6 and 7 showed J(C-6,F) constants in the usual
range of 20-23 Hz. An X-ray refraction of 7e (Figure 4) revealed
a sandwich - stacking (4.5 Å centroid-distance) and that the
residues are pointing in the same direction. The amidic and
every other anilinic NH moiety make intramolecular N–H∙∙∙F
F
F
NH2
O
NH2
O
bonds. Due to further intermolecular N–H∙∙∙O contact,
a
4
2
3
F
bifurcated H-bond motif is formed. The geometrical analysis
demonstrated a chair conformation of the cyclohexane ring.
To determine the antiangiogenic activity of the synthesized
compounds, the rat aortic ring assay was performed and the
compounds were administered at a concentration of 50 µM
(Table 1). 5-Amino-1-{[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]-
1
1
NH
1
8
9
10
Figure 2. Reference compounds.
methyl}-1H-1,2,3-triazole-4-carboxamide (CAI),
cytostatic inhibitor of non-voltage-operated calcium channels
and calcium channel-mediated signaling pathways, as well as O-
chloroacetyl-carbamoyl fumagillol (TNP-470),
a
synthetic,
Tetrafluorophthalamic acids 4 exhibited 2J(C,F) values for the
heteronuclear coupling of C-6 but also of C-1 were between 13
and 19 Hz, indicating a different pattern of intramolecular
interactions in comparison to the corresponding salts 3. The
assignment of the 13C NMR signals was based on the chemical
shifts and in relation to compounds 3 and 5 (see below); hence
C-6 resonates at 123 ppm, C-1 at 118 ppm. A single crystal X-
ray analysis of 4a as a representative compound was carried out
(Figure 3). As in case of 2-fluorobenzamide (9), the NH is
oriented towards the 5-fluorine substituent to form an
intramolecular H∙∙∙F hydrogen bond. It produces, together with
an intermolecular contact to a carbonyl oxygen, a bifurcated
hydrogen bond network and accounts for a dihedral angle of 37°
between the tetrafluorinated benzene ring and the plane through
the carboxamide moiety. The carboxylate plane is twisted
a methionine
aminopeptidase-2 and neovascularization inhibitor, were used
as positive controls. Moreover, data of two particularly potent
representatives of tetrafluorophthalimido and benzamido
barbituric acids (types III and IV) were included in Table 1.
In comparison to the DMSO-treated ring explants, most of the
test compounds of chemotypes 2, 4 and 5 showed moderate
inhibitory activities, with the N-isopropyl-tetrafluorophthalimide
2b as the most potent derivative. Thus, the isopropyl residue
was advantageous for antiangiogenic activity. However, the
strong activity of tetrafluorophthalimides with a barbituric acid
moiety as the N-substituent, as reported previously,[9] was not
reached with compound 2b. The corresponding derivatives of
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chemotypes 4 and 5 bearing the isopropyl moiety, i.e. 4b and 5b, We selected two tetrafluorobenzamides, 5b and 5d, and
showed lower activity, compared to 2b. The antiangiogenic
potential of tetrafluorobenzamides has already been
elucidated,[9] and was confirmed for chemotype 5 in this study.
Tetrafluorophthalamic acids have first been shown as
antiangiogenic agents herein. The exchange of hydrogen by a
carboxyl group did not have a strong impact on angiogenesis
inhibition, as can be seen by a comparison of representatives
5a-f with the analogues 4a-f. Chemotypes 4 and 5 are
considered to be more appropriate for further structural
optimization because these ring-opened derivatives are more
conformationally flexible than phthalimides of type 2. Moreover,
phthalimides 2 are inferior in stability because of the easy
opening of the five-membered ring.
introduced a further point of diversity, i.e. an amino substituent
at position 4. The antiangiogenesis data in response to
trifluorobenzamides 6 and 7 at an inhibitor concentration of 50
µM
are
presented
in
Table
2.
Among
the
N-isopropylcarboxamides 6, the cyclohexylamino derivative 6d
exhibited an improved activity (80% inhibition), when compared
to its precursor 5b (58% inhibition). In case of 5d (35%
inhibition), the exchange of fluorine by all four different amino
groups led to slightly improved inhibitors 7 (> 50% inhibition).
Thus, trifluorobenzamides have been identified as a new
chemotype of antiangiogenetic agents.
The antiangiogenic activity of the two best compounds in the rat
aortic ring model was complemented by a preliminary in vitro
human umbilical vein endothelial cell (HUVEC) cytotoxicity
assay, in which 2b and 6d, at a concentration of 10 µM, inhibited
the survival of HUVECs 74% and 78%, respectively.
Conclusions
In this study, different molecular entities of fluoroaromatic
compounds were synthesized and subjected to detailed
spectroscopic and crystallographic analyses. Such compounds
are potentially bioactive.
We have determined the antiangiogenic activity of
tetrafluorophthalimides with ordinary N-alkyl residues. Although
these
compounds
2
were
less
potent
than
tetrafluorophthalimides with
a
barbituric acid moiety as
N-substituent, they provide a starting point for tailored structural
modifications and a comparative analysis of the resulting
chemotypes. With respect to antiangiogenic activity, ring
opening as realized in 4-7 was tolerated. In particular, we found
that one fluorine substituent can be replaced by an amino group
leading to 6/7 without an essential loss of bioactivity. Thus, the
repertoire of structural frameworks to develop angiogenesis
inhibitors was expanded. Further biological studies are ongoing
to understand the clinical utility of selected compounds from this
cohort.
Future structural modifications are intended as follows. The
carboxamide moiety of 6/7 can be revised and re-equipped with
a barbituric acid unit. The scopes and limitations of the aromatic
amino substitution need to be further explored. It can also be
envisaged to utilize this substitution for the introduction of linkers
which bear a second functionality. Thus, our study might pave
the way for the design of bifunctional molecules with fluorescent
or biotin labels as tool compounds for studying angiogenesis.
Figure 4. Top. Molecular plot of 4-(benzylamino)-N-cyclohexyl-2,3,5-
trifluorobenzamide (7e) showing the displacement ellipsoids at the 50%
probability level for the non-H atoms. Bottom. Packing diagram of 7e. The
N–H∙∙∙F and N–H∙∙∙O contacts are shown as broken lines.[21]
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Experimental Section
Table 1. Inhibitory effects of tetrafluoro derivatives 2, 4 and 5 in the rat aortic
ring assay of angiogenesis.
Melting points were determined on a Gallenkamp capillary melting point
apparatus or on a Rapido Boetius apparatus, and are uncorrected. The
purity of the obtained substances and the composition of the reaction
mixtures were controlled by thin-layer chromatography (TLC) using
Merck aluminum silica gel plates with 60 F254 indicator. Preparative
column chromatography was performed using Merck silica gel 60 (63-
200 mesh). Petroleum ether used was a mixture of alkanes boiling
between 40-60 °C, according to the supplier’s declaration. NMR spectra
were recorded on a Bruker Avance DRX 500 spectrometer at 500 MHz
(1H NMR) and 125 MHz (13C NMR) in DMSO-d6. Chemical shifts (δ) are
reported in ppm. Spin multiplicities are indicated by the following
symbols: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet),
sext (sextet), sept (septet), oct (octet), non (nonet), m (multiplet). All
multiplets related with J(C, F) couplings in 13C NMR spectra are centered.
Mass spectra were recorded on an MS-50 A.E.I. spectrometer under
electron impact ionization (EI) at 70 eV. Elemental analyses were
performed with a Vario EL apparatus.
F
F
F
O
F
O
O
F
F
F
F
R
F
F
R
N
H
N
N R
H
CO2H
O
F
F
5
2
4
Inhibition [%][a]
Substitution
pattern
R
2
4
5
a
b
c
d
e
f
n-Pr
i-Pr
59
81
67
59
10
55
n.i.
50
30
50
46
n.i.[b]
58
i-Bu
31
cyclohexyl
Bn
35
31
tert-Bu
n.d.[c]
Inhibition [%][a]
III[f]
44
Chemistry
Positive
IV[f]
controls[d]
Synthesis of tetrafluorophthalimides 2a-f: General Procedure. A
mixture of the primary amine (1.5 mmol) and tetrafluorophthalic
anhydride (1, 0.40 g, 1.8 mmol) in glacial AcOH (11 mL) was stirred
under reflux for 3 h. The resulting solution was then evaporated to
dryness under reduced pressure. The oily residue was recrystallized from
EtOH.
CAI[e]
73
TNP-470
R1 = Et, R2 = i-Pr
47
>95 90
[a] Compounds were administered as a single dose at a concentration of 50
µM and incubated for 5 days. Outgrowth was compared to microvessel
outgrowth from rings treated with the control (0.5% DMSO). Control outgrowth
is classed at 100% outgrowth. [b] n.i.: no inhibitory activity. [c] n.d.: not
determined. [d] Incubation was conducted for 4 days. [e] A concentration of 30
µM was used. [f] Data from ref. [9].
4,5,6,7-Tetrafluoro-2-propylisoindoline-1,3-dione (2a) was prepared
from 1 and propylamine (89 mg, 0.12 mL); yield 49% (0.19 g); mp 106-
108 °C; 1H NMR (DMSO-d6) δ 0.86 (t, J = 7.4 Hz, 3H, CH3), 1.59 (sext, J
= 7.3 Hz, 2H, CH2CH3) 3.50 (t, J = 7.1 Hz, 2H, NCH2); 13C NMR (DMSO-
d6) δ 11.22 (CH3), 21.12 (CH2CH3), 39.91 (NCH2), 114.30 (m, 3J(C,F) =
9.4 Hz, C-3a, C-7a), 142.50 (m, 1J(C,F) = 264.2 Hz, C-4, C-7), 144.09 (m,
1J(C,F) = 260.9 Hz, C-5, C-6), 162.86 (CO). Anal. calcd for C11H7F4NO2:
C 50.59, H 2.70, N 5.36, found: C 50.65, H 2.61, N 5.40.
Table 2. Inhibitory effects of trifluoro derivatives 6 and 7 in the rat aortic ring
4,5,6,7-Tetrafluoro-2-isopropylisoindoline-1,3-dione
(2b)[22]
was
assay of angiogenesis.
prepared from 1 and isopropylamine (89 mg, 0.13 mL); yield 51% (0.20
g); mp 148-151 °C; 1H NMR (DMSO-d6) δ 1.38 (d, J = 7.0 Hz, 6H, CH3),
4.34 (sept, J = 6.9 Hz, 1H, CH); 13C NMR (DMSO-d6) δ 19.61 (CH3),
43.38 (CH), 114.09 (m, 3J(C,F) = 9.9 Hz, C-3a, C-7a), 142.46 (m, 1J(C,F)
= 264.1 Hz, C-4, C-7), 144.02 (m, J(C,F) = 258.7 Hz, C-5, C-6), 162.62
(CO). Anal. calcd for C11H7F4NO2: C 50.59, H 2.70, N 5.36, found: C
50.59, H 2.90, N 5.36.
F
F
O
F
O
F
F
N
N
H
H
1
R
R
N
H
N
H
6
F
7
Inhibition [%][a]
Substitution
R
4,5,6,7-Tetrafluoro-2-isobutylisoindoline-1,3-dione (2c) was prepared
from 1 and isobutylamine (110 mg, 0.15 mL); yield 99% (0.41 g); mp 136-
138 °C; 1H NMR (DMSO-d6) δ 0.87 (d, J = 6.7 Hz, 6H, CH3), 1.91-2.02
(m, 1H, CH), 3.35 (d, J = 7.3 Hz, 2H, CH2); 13C NMR (DMSO-d6) δ 20.02
(CH3), 27.40 (CH), 45.57 (CH2), 114.11 (m, 3J(C,F) = 7.4 Hz, C-3a, C-7a),
142.55 (m, 1J(C,F) = 263.8 Hz, C-4, C-7), 144.16 (m, 1J(C,F) = 262.5 Hz,
C-5, C-6), 163.06 (CO). Anal. calcd for C12H9F4NO2: C 52.37, H 3.30, N
5.09, found: C 52.63, H 3.21, N 5.09.
pattern
6
7
a
b
c
d
n-Pr
i-Pr
51
32
52
80
54
67
51
54
i-Bu
cyclohexyl
Bn
e
n.a.[b]
n.i.[c]
[a] Compounds were administered as a single dose at a concentration of 50
µM and incubated for 5 days. Outgrowth was compared to microvessel
outgrowth from rings treated with the control (0.5% DMSO). Control outgrowth
is classed at 100% outgrowth. [b] n.a.: not applicable. [c] n.i.: no inhibitory
activity.
4,5,6,7-Tetrafluoro-2-cyclohexylisoindoline-1,3-dione (2d)[22] was
prepared from 1 and cyclohexylamine (150 mg, 0.17 mL); yield 80%
(0.36 g); mp 155-158 °C; 1H NMR (DMSO-d6) δ 1.10-2.04 (m, 10H, CH2),
3.93 (tt, J = 12.5, 3.9 Hz, 1H, CH); 13C NMR (DMSO-d6) δ 24.94 (C-4’),
25.48 (C-3’, C-5’), 29.22 (C-2’, C-6’), 51.12 (C-1’), 113.98 (m, 3J(C,F) =
7.7 Hz, C-3a, C-7a), 142.49 (m, 1J(C,F) = 264.4 Hz, C-4, C-7), 144.06 (m,
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10.1002/cmdc.201800263
ChemMedChem
FULL PAPER
1J(C,F) = 262.5 Hz, C-5, C-6), 162.64 (CO). Anal. calcd for C14H11F4NO2:
C 55.82, H 3.68, N 4.65, found: C 55.58, H 3.76, N 4.72.
(m, 1J(C,F) = 240.6 Hz), 143.69 (m, 1J(C,F) = 247.4 Hz, C-2, C-3, C-4, C-
5), 161.53 (CO), 163.00 (CO). Anal. calcd for C16H22F4N2O3 × H2O: C
50.00, H 6.29, N 7.29, found: C 49.82, H 5.98, N 7.30.
4,5,6,7-Tetrafluoro-2-benzylisoindoline-1,3-dione (2e) was prepared
from 1 and benzylamine (161 mg, 0.16 mL); yield 93% (0.43 g); mp 177-
179 °C; lit. mp 179-181 °C;[3] 1H NMR (DMSO-d6) δ 4.74 (s, 2H, CH2),
7.26-7.35 (m, 5H, Ar-H); 13C NMR (DMSO-d6) δ 41.65 (CH2), 114.19 (m,
3J(C,F) = 8.3 Hz, C-3a, C-7a), 127.73, 128.75 (C-2’, C-6’, C-3’, C-5’),
127.81 (C-4’), 135.92 (C-1’), 142.70 (m, 1J(C,F) = 267.0 Hz, C-4, C-7),
144.27 (m, 1J(C,F) = 263.6 Hz, C-5, C-6), 162.69 (CO). Anal. calcd for
C15H7F4NO2: C 58.26, H 2.28, N 4.53, found: C 58.19, H 2.32, N 4.56.
Cyclohexylammonium 2,3,4,5-tetrafluoro-6-(cyclohexylcarbamoyl)-
benzoate (3d) was prepared from 1 and cyclohexylamine (1.49 g, 1.72
mL); yield 91% (2.86 g); mp 212-214 °C; 1H NMR (DMSO-d6) δ 1.07-1.30
(m, 10H, CH2), 1.51-189 (m, 10H, CH2), 2.89-2.93 (m, 1H, CH), 3.59-3.66
(m, 1H, CH), 8.01 (3H, NH3), 8.25 (d, J = 7.9 Hz, 1H, NH); 13C NMR
(DMSO-d6) δ 23.96 (CH2), 24.44 (CH2), 24.72 (CH2), 25.37 (CH2), 30.55
(CH2), 32.05 (CH2), 48.15 (CH), 49.36 (CH), 119.30 (m, 2J(C,F) = 13.9
Hz, C-6), 128.31 (m, J(C,F) = 22.8 Hz, C-1), 137.66 (m J(C,F) = 241.3
Hz), 139.62 (m, 1J(C,F) = 249.1 Hz), 142.41 (m, 1J(C,F) = 240.4 Hz),
143.78 (m, 1J(C,F) = 245.5 Hz, C-2, C-3, C-4, C-5), 160.65 (CO), 162.88
(CO). Anal. calcd for C20H26F4N2O3: C 57.41, H 6.26, N 6.69, found: C
57.58, H 6.41, N 6.71.
2
1
4,5,6,7-Tetrafluoro-2-tert-butylisoindoline-1,3-dione (2f) was prepared
from 1 and tert-butylamine (110 mg, 0.16 mL); yield 11% (45 mg); mp
1
174-176 °C; H NMR (DMSO-d6) δ 1.59 (s, 9H, CH3); 13C NMR (DMSO-
d6) δ 28.41 (CH3), 59.47 (C(CH3)3), 114.01 (m, 3J(C,F) = 7.2 Hz, C-3a, C-
7a), 142.36 (m, 1J(C,F) = 264.9 Hz, C-4, C-7), 144.06 (m, 1J(C,F) = 262.5
Hz, C-5, C-6), 163.49 (CO). Anal. calcd for C12H9F4NO2: C 52.37, H 3.30,
N 5.09, found: C 52.36, H 3.12, N 5.04.
Benzylammonium 2,3,4,5-tetrafluoro-6-(benzylcarbamoyl)benzoate
(3e) was prepared from 1 and benzylamine (1.61 g, 1.64 mL); yield 61%
(1.98 g); mp 181-183 °C; 1H NMR (DMSO-d6) δ 3.98 (s, 2H, CH2), 4.40
(d, J = 5.9 Hz, 2H, CH2), 7.20-7.45 (m, 10H, 2 × Ar-H), 8.34 (br s, 3H,
NH3), 8.94 (t, J = 5.9 Hz, 1H, NH); 13C NMR (DMSO-d6) δ 42.49 (CH2),
42.71 (CH2), 119.49 (m, 2J(C,F) = 15.4 Hz, C-6), 126.77 (C-Ar), 127.26
(C-Ar), 128.26 (C-Ar), 128.31 (C-Ar), 128.63 (C-Ar), 128.87 (C-Ar),
134.91 (C-Ar), 139.00 (C-Ar), 137.76 (m 1J(C,F) = 251.4 Hz), 139.75 (m,
1J(C,F) = 245.4 Hz), 142.70 (m, 1J(C,F) = 241.1 Hz), 143.68 (m, 1J(C,F) =
245.5 Hz, C-2, C-3, C-4, C-5), 161.91 (CO), 163.00 (CO). One carbon
signal (C-1) was not observed. Anal. calcd for C22H18F4N2O3: C 58.41, H
4.46, N 6.19, found: C 58.47, H 4.60, N 6.19.
Synthesis of ammonium tetrafluorophthalamates 3a-f: General
Procedure.
A mixture of the primary amine (15 mmol) and
tetrafluorophthalic anhydride (1, 1.65 g, 7.5 mmol) in dry toluene (500
mL) was stirred under reflux for 3 h. The mixture was allowed to cool
down to room temperature, the resulting solid was filtered off, washed
with n-hexanes (2 × 20 mL) and dried under reduced pressure.
Propylammonium 2,3,4,5-tetrafluoro-6-(propylcarbamoyl)benzoate
(3a) was prepared from 1 and propylamine (0.89 g, 1.24 mL); yield 65%
1
(1.65 g); mp 160-164 °C; H NMR (DMSO-d6) δ 0.87 (t, J = 7.6 Hz, 3H,
tert-Butylammonium
benzoate (3f) was prepared from 1 and tert-butylamine (1.10 g, 1.58
mL); yield 81% (2.22 g); mp 178-180 °C; H NMR (DMSO-d6) δ 1.24 (s,
9H, CH3), 1.29 (s, 9H, CH3), 7.96 (s, 1H, NH), 8.10 (br s, 3H, NH3); 13C
2,3,4,5-tetrafluoro-6-(tert-butylcarbamoyl)-
CH3), 0.87 (t, 7.3 Hz, 3H, CH3), 1.46 (sext, J = 7.3 Hz, 2H, CH2CH3), 1.53
(sext, J = 7.6 Hz, 2H, CH2CH3), 2.69 (t, J = 7.6 Hz, 2H, NCH2), 3.08-3.12
(m, 2H, NCH2), 8.05 (br s, 3H, NH3), 8.36 (t, J = 5.7 Hz, 1H, NH); 13C
NMR (DMSO-d6) δ 11.01 (CH3), 11.48 (CH3), 20.62 (CH2CH3), 22.19
(CH2CH3), 40.53 (NCH2), 41.02 (NCH2), 119.69 (m, 2J(C,F) = 15.4 Hz, C-
6), 127.96 (m, 2J(C,F) = 21.6 Hz, C-1), 137.75 (m 1J(C,F) = 239.6 Hz),
139.67 (m, J(C,F) = 241.6 Hz), 142.57 (m, J(C,F) = 241.1 Hz), 143.60
(m, 1J(C,F) = 245.6 Hz, C-2, C-3, C-4, C-5), 161.51 (CO), 163.11 (CO).
Anal. calcd for C14H18F4N2O3 × 0.25 H2O: C 49.05, H 5.44, N 8.17, found:
C 48.77, H 5.89, N 8.20.
1
NMR (DMSO-d6) δ 27.34 (CH3), 28.42 (CH3), 50.80 (C(CH3)3), 51.00
2
2
(C(CH3)3), 119.91 (m, J(C,F) = 14.6 Hz, C-6), 127.97 (m, J(C,F) = 22.1
Hz, C-1), 137.67 (m 1J(C,F) = 236.5 Hz), 139.57 (m, 1J(C,F) = 239.3 Hz),
142.39 (m, 1J(C,F) = 241.4 Hz), 143.82 (m, 1J(C,F) = 247.0 Hz, C-2, C-3,
C-4, C-5), 160.94 (CO), 163.18 (CO). Anal. calcd for C16H22F4N2O3: C
52.46, H 6.05, N 7.65, found C 52.03, H 6.80, N 7.17.
1
1
Synthesis of tetrafluorophthalamic acids 4a-f: General Procedure. A
solution of the ammonium tetrafluorophthalamate (5 mmol) in H2O was
acidified with HCl (2 mol/L) to pH 1-2 and kept overnight at 4 °C. The
colorless precipitate was filtered off, washed with H2O and dried under
reduced pressure.
Isopropylammonium
2,3,4,5-tetrafluoro-6-(isopropylcarbamoyl)-
benzoate (3b) was prepared from 1 and isopropylamine (0.89 g, 1.29
mL); yield 88% (2.24 g); mp 178-180 °C; 1H NMR (DMSO-d6) δ 0.87 (d, J
= 6.7 Hz, 6H, CH3), 1.16 (d, J = 6.6 Hz, 6H, CH3), 3.25 (sept, J = 6.6 Hz,
1H, CH), 3.87-3.97 (m, 1H, CH); 8.03 (s, 3H, NH3), 8.24 (d, J = 7.6 Hz,
1H, NH); 13C NMR (DMSO-d6) δ 20.56 (CH3), 22.16 (CH3), 41.21 (CH),
42.96 (CH), 119.77 (m, 2J(C,F) = 15.9 Hz, C-6), 127.43 (m, 2J(C,F) =
21.3 Hz, C-1), 137.87 (m, 1J(C,F) = 229.2 Hz), 139.73 (m, 1J(C,F) =
233.6 Hz), 142.68 (m, 1J(C,F) = 242.9 Hz), 143.66 (m, 1J(C,F) = 244.5 Hz,
C-2, C-3, C-4, C-5), 160.59 (CO), 163.06 (CO). Anal. calcd for
C14H18F4N2O3 × 0.1 H2O: C 49.44, H 5.39, N 8.24, found: C 49.20, H
5.78, N 8.27.
2,3,4,5-Tetrafluoro-6-(propylcarbamoyl)benzoic
acid
(4a)
was
prepared from 3a (1.69 g) in H2O (30 mL); yield 73% (1.02 g); mp 132-
134 °C; 1H NMR (DMSO-d6) δ 0.89 (t, J = 7.3 Hz, 3H, CH3), 1.48 (sext, J
= 7.3 Hz, 2H, CH2CH3), 3.14-3.18 (m, 2H, NCH2), 8.66 (t, J = 5.5 Hz, 1H,
NH), 14.02 (br s, 1H, CO2H); 13C NMR (DMSO-d6) δ 11.42 (CH3), 22.08
(CH2CH3), 41.19 (NCH2), 117.86 (m, 2J(C,F) = 13.6 Hz, C-1), 122.27 (m,
2J(C,F) = 15.4 Hz, C-6), 140.24 (m 1J(C,F) = 252.0 Hz), 140.78 (m,
1J(C,F) = 254.4 Hz), 143.63 (m, 1J(C,F) = 245.0 Hz), 144.88 (m, 1J(C,F) =
250.7 Hz, C-2, C-3, C-4, C-5), 160.10 (CO), 162.63 (CO). MS (EI)
(m/z, %) 279 (M+, 13), 235 (M+ - CO2, 25), 221 (M+ - C3H8N, 61), 177 (M+
- C3H8N – CO2, 100), 149 (M+ - C3H8N – CO2 – CO, 25). Anal. calcd for
C11H9F4NO3: C 47.32, H 3.25, N 5.02, found: C 47.18, H 3.38, N 5.13.
Isobutylammonium
2,3,4,5-tetrafluoro-6-(isobutylcarbamoyl)-
benzoate (3c) was prepared from 1 and isobutylamine (1.10 g, 1.50 mL);
yield 33% (0.92 g); mp 168-170 °C; 1H NMR (DMSO-d6) δ 0.87 (d, J =
6.6 Hz, 6H, CH3), 0.89 (d, J = 6.7 Hz, 6H, CH3), 1.71-1.87 (m, 2H, 2 ×
CH), 2.58 (d, J = 7.0 Hz, 2H, CH2), 2.98 (t, J = 6.0 Hz, 2H, CH2), 8.00 (br
s, 3H, NH3), 8.37 (t, J = 6.0 Hz, 1H, NH); 13C NMR (DMSO-d6) δ 19.87
(CH3), 20.27 (CH3), 26.59 (CH), 28.06 (CH), 45.87 (CH2), 46.78 (CH2),
2,3,4,5-Tetrafluoro-6-(isopropylcarbamoyl)benzoic acid (4b) was
prepared from 3b (1.69 g) in H2O (36 mL); yield 81% (1.13 g); mp 144-
146 °C; 1H NMR (DMSO-d6) δ 1.11 (d, J = 6.6 Hz, 6H, CH3), 3.92-4.02
2
2
119.53 (m, J(C,F) = 15.1 Hz, C-6), 128.12 (m, J(C,F) = 22.1 Hz, C-1),
137.69 (m 1J(C,F) = 242.5 Hz), 139.64 (m, 1J(C,F) = 244.1 Hz), 142.48
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10.1002/cmdc.201800263
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FULL PAPER
(m, 1H, CH), 8.55 (d, J = 7.9 Hz, 1H, NH), 14.00 (br s, 1H, CO2H); 13C
NMR (DMSO-d6) δ 22.04 (CH3), 41.53 (CH), 117.68 (m, 2J(C,F) = 14.6
Hz, C-1), 122.50 (m, 2J(C,F) = 15.9 Hz, C-6), 140.18 (m, 1J(C,F) = 251.4
Hz), 140.80 (m, 1J(C,F) = 255.2 Hz), 143.59 (m, 1J(C,F) = 244.6 Hz),
144.93 (m, 1J(C,F) = 251.4 Hz, C-2, C-3, C-4, C-5), 159.19 (CO), 162.60
(CO). MS (EI) (m/z, %) 279 (M+, 6), 235 (M+ - CO2, 19), 221 (M+ - C3H8N,
38), 177 (M+ - C3H8N – CO2, 100), 149 (M+ - C3H8N – CO2 – CO, 25).
Anal. calcd for C11H9F4NO3 × 0.1 H2O: C 47.02, H 3.30, N 4.98, found: C
46.72, H 3.70, N 5.19.
H2O was added (10 mL). The oil formed in the reaction mixture was
removed and the aqueous solution was kept for 3 d at room temperature.
The precipitate was filtered off, dried under reduced pressure and further
purified as indicated.
2,3,4,5-Tetrafluoro-N-propylbenzamide (5a) was prepared from 4a
(0.42 g) and purified by column chromatography using petroleum
ether/EtOAc (4:1); yield 26% (92 mg); mp 46-50 °C; 1H NMR (DMSO-d6)
δ 0.89 (t, J = 7.4 Hz, 3H, CH3), 1.48-1.55 (m, 2H, CH2CH3), 3.20 (q, J =
6.5 Hz, 2H, NCH2), 7.53-7.59 (m, 1H, 6-H), 8.49 (br s, 1H, NH); 13C NMR
(DMSO-d6) δ 11.43 (CH3), 22.19 (CH2CH3), 41.25 (NCH2), 111.70 (m,
2J(C,F) = 20.3 Hz, C-6), 121.20 (m, C-1), 140.00 (m 1J(C,F) = 247.6 Hz),
2,3,4,5-Tetrafluoro-6-(isobutylcarbamoyl)benzoic acid (4c) was
prepared from 3c (1.83 g) in H2O (100 mL); yield 58% (0.85 g); mp 148-
150 °C; 1H NMR (DMSO-d6) δ 0.88 (d, J = 6.7 Hz, 6H, CH3), 1.74-1.82
(m, 1H, CH), 3.02-3.04 (m, 2H, CH2), 8.67 (t, J = 5.8 Hz, 1H, NH), 14.04
(br s, 1H, CO2H); 13C NMR (DMSO-d6) δ 20.18 (CH3), 28.00 (CH), 46.94
(CH2), 117.90 (m, 2J(C,F) = 14.6 Hz, C-1), 122.27 (m, 2J(C,F) = 19.1 Hz,
C-6), 140.21 (m 1J(C,F) = 249.7 Hz), 140.74 (m, 1J(C,F) = 253.4 Hz),
143.66 (m, 1J(C,F) = 244.2 Hz), 144.82 (m, 1J(C,F) = 250.6 Hz, C-2, C-3,
C-4, C-5), 160.15 (CO), 162.65 (CO). MS (EI) (m/z, %) 293 (M+, 3), 249
(M+ - CO2, 8), 221 (M+ - C4H10N, 36), 177 (M+ - C4H10N – CO2, 100), 149
(M+ - C4H10N – CO2 – CO, 18). Anal. calcd for C12H11F4NO3 × 0.1 H2O: C
48.86, H 3.83, N 4.75, found: C 48.62, H 4.11, N 4.96.
1
1
140.77 (m, J(C,F) = 252.2 Hz), 144.69 (m, J(C,F) = 246.7 Hz), 146.18
(m, 1J(C,F) = 245.5 Hz, C-2, C-3, C-4, C-5), 160.89 (CO). MS (EI)
(m/z, %) 235 (M+, 19), 220 (M+ - CH3, 7), 206 (M+ - C2H5, 11), 177 (M+ -
C3H8N, 100), 149 (M+ - C3H8N – CO, 14). Anal. calcd for C10H9F4NO: C
51.07, H 3.86, N 5.96, found: C 51.32, H 4.44, N 5.69.
2,3,4,5-Tetrafluoro-N-isopropylbenzamide (5b) was prepared from 4b
(0.42 g) and recrystallized from n-hexanes; yield 43% (0.15 g); mp 111-
113 °C; 1H NMR (DMSO-d6) δ 1.14 (d, J = 6.7 Hz, 6H, CH3), 3.97-4.06
(m, 1H, CH), 7.52-7.57 (m, 1H, 6-H), 8.36 (d, J = 6.3 Hz, 1H, NH); 13C
NMR (DMSO-d6) δ 22.18 (CH3), 41.62 (CH), 111.68 (m, 2J(C,F) = 20.1
Hz, C-6), 121.51 (m, C-1), 139.98 (m, 1J(C,F) = 250.9 Hz), 140.68 (m,
1J(C,F) = 252.0 Hz), 144.63 (m, 1J(C,F) = 247.1 Hz), 146.16 (m, 1J(C,F) =
245.0 Hz, C-2, C-3, C-4, C-5), 160.12 (CO). MS (EI) (m/z, %) 235 (M+,
19), 220 (M+ - CH3, 21), 177 (M+ - C3H8N, 100). Anal. calcd for
C10H9F4NO: C 51.07, H 3.86, N 5.96, found: C 50.81, H 4.23, N 5.97.
2,3,4,5-Tetrafluoro-6-(cyclohexylcarbamoyl)benzoic acid (4d)[23] was
prepared from 3d (2.09 g) in H2O (700 mL); yield 85% (1.36 g); mp 174-
178 °C; 1H NMR (DMSO-d6) δ 1.11-1.81 (m, 10H, CH2), 3.64-3.71 (m, 1H,
CH), 8.54 (d, J = 7.9 Hz, 1H, NH), 14.00 (br s, 1H, CO2H); 13C NMR
(DMSO-d6) δ 24.43 (CH2), 25.27 (CH2), 32.00 (CH2), 48.51 (CH), 117.61
2
2
(m, J(C,F) = 13.6 Hz, C-1), 122.54 (m, J(C,F) = 18.6 Hz, C-6), 140.15
(m 1J(C,F) = 252.7 Hz), 140.79 (m, 1J(C,F) = 255.3 Hz), 143.58 (m,
2,3,4,5-Tetrafluoro-N-isobutylbenzamide (5c) was prepared from 4c
(0.44 g); yield 80% (0.30 g); mp 88-90 °C; 1H NMR (DMSO-d6) δ 0.89 (d,
J = 6.6 Hz, 6H, CH3), 1.81 (non, J = 6.7 Hz, 1H, CH), 3.07 (t, J = 6.5 Hz,
2H, CH2), 7.53-7.58 (m, 1H, 6-H), 8.50 (br s 1H, NH); 13C NMR (DMSO-
d6) δ 20.16 (CH3), 28.06 (CH), 46.89 (CH2), 111.67 (m, 2J(C,F) = 20.3 Hz,
C-6), 121.36 (m, C-1), 139.99 (m 1J(C,F) = 248.3 Hz), 140.74 (m, 1J(C,F)
1J(C,F) = 245.2 Hz), 144.92 (m, J(C,F) = 251.3 Hz, C-2, C-3, C-4, C-5),
1
159.19 (CO), 162.54 (CO). MS (EI) (m/z, %) 319 (M+, 5), 275 (M+ - CO2,
18), 221 (M+ - C6H12N, 23), 177 (M+ - C6H12N – CO2, 100), 149 (M+
-
C6H12N – CO2 – CO, 21). Anal. calcd for C14H13F4NO3: C 52.67, H 4.10,
N 4.39, found: C 52.56, H 4.15, N 4.43.
1
1
= 252.2 Hz), 144.64 (m, J(C,F) = 247.8 Hz), 146.21 (m, J(C,F) = 244.5
Hz, C-2, C-3, C-4, C-5), 161.02 (CO). MS (EI) (m/z, %) 249 (M+, 7), 234
(M+ - CH3, 9), 177 (M+ - C4H10N, 100). Anal. calcd for C11H11F4NO: C
53.02, H 4.45, N 5.62, found: C 52.27, H 4.75, N 5.68.
2,3,4,5-Tetrafluoro-6-(benzylcarbamoyl)benzoic
acid (4e) was
prepared from 3e (2.17 g) in H2O (450 mL); yield 68% (1.11 g); mp 153-
156 °C; 1H NMR (DMSO-d6) δ 4.45 (d, J = 5.9 Hz, 2H, CH2), 7.24-7.35
(m, 5H, Ar-H), 9.21 (t, J = 5.9 Hz, 1H, NH), 14.14 (br s, 1H, CO2H); 13C
NMR (DMSO-d6) δ 42.94 (CH2), 117.94 (m, 2J(C,F) = 14.4 Hz, C-1),
121.89 (m, 2J(C,F) = 17.4 Hz, C-6), 127.08 (C-Ar), 127.39 (C-Ar), 128.43
(C-Ar), 138.58 (C-Ar), 140.40 (m 1J(C,F) = 255.3 Hz), 140.83 (m, 1J(C,F)
2,3,4,5-Tetrafluoro-N-cyclohexylbenzamide (5d) was prepared from
4d (0.48 g) and recrystallized from n-hexanes; yield 56% (0.23 g); mp
124-126 °C; 1H NMR (DMSO-d6) δ 1.09-1.82 (m, 10H, CH2), 3.67-3.74
(m, 1H, CH), 7.51-7.56 (m, 1H, 6-H), 8.35 (d, J = 7.6 Hz, 1H, NH); 13C
NMR (DMSO-d6) δ 24.61 (CH2), 25.25 (CH2), 32.16 (CH2), 48.70 (CH),
111.68 (m, 2J(C,F) = 20.3 Hz, C-6), 121.59 (m, C-1), 139.98 (m 1J(C,F) =
251.6 Hz), 140.66 (m, 1J(C,F) = 250.6 Hz), 144.62 (m, 1J(C,F) = 248.6
Hz), 146.15 (m, 1J(C,F) = 244.0 Hz, C-2, C-3, C-4, C-5), 160.13 (CO).
MS (EI) (m/z, %) 275 (M+, 19), 232 (M+ - C3H7, 18), 194 (M+ - C6H9, 64),
177 (M+ - C6H12N, 100). Anal. calcd for C13H13F4NO: C 56.73, H 4.76, N
5.09, found: C 57.10, H 4.96, N 5.21.
1
1
= 253.5 Hz), 143.82 (m, J(C,F) = 245.7 Hz), 144.94 (m, J(C,F) = 249.6
Hz, C-2, C-3, C-4, C-5), 160.40 (CO), 162.71 (CO). MS (EI) (m/z, %) 328
(M+H+, 9), 283 (M+ - CO2, 100), 177 (M+ - C7H8N – CO2, 95), 149 (M+
-
C7H8N – CO2 – CO, 27). Anal. calcd for C15H9F4NO3 × 0.7 H2O: C 53.01,
H 3.08, N 4.12, found: C 53.02, H 2.94, N 4.27.
2,3,4,5-Tetrafluoro-6-(tert-butylcarbamoyl)benzoic acid (4f) was
prepared from 3f (1.83 g) in H2O (115 mL); yield 52% (0.76 g); mp 148-
151 °C; 1H NMR (DMSO-d6) δ 1.31 (s, 9H, CH3), 8.28 (s, 1H, NH), 14.02
(br s, 1H, CO2H); 13C NMR (DMSO-d6) δ 28.35 (CH3), 51.43 (C(CH3)3),
2,3,4,5-Tetrafluoro-N-benzylbenzamide (5e) was prepared from 4e
(0.49 g) and recrystallized from n-hexanes; yield 47% (0.20 g); mp 96-
99 °C; 1H NMR (DMSO-d6) δ 4.46 (d, J = 6.0 Hz, 2H, CH2), 7.23-7.27 (m,
1H, 4’-H), 7.32-7.36 (m, 4H, 2’-H, 3’-H, 5’-H, 6’-H), 7.60-7.66 (m, 1H, 6-
H), 9.06 (t, J = 5.1 Hz, 1H, NH); 13C NMR (DMSO-d6) δ 43.00 (CH2),
111.84 (m, 2J(C,F) = 20.6 Hz, C-6), 120.76 (m, C-1), 127.08 (C-4’),
127.37, 128.48 (C-2’, C-6’, C-3’, C-5’), 138.78 (C-1’), 140.08 (m 1J(C,F) =
249.4 Hz), 140.96 (m, 1J(C,F) = 252.5 Hz), 144.88 (m, 1J(C,F) = 247.7
Hz), 146.23 (m, 1J(C,F) = 244.4 Hz, C-2, C-3, C-4, C-5), 161.08 (CO).
MS (EI) (m/z, %) 283 (M+, 100), 177 (M+ - C7H8N, 80). Anal. calcd for
C14H9F4NO: C 59.37, H 3.20, N 4.95, found: C 58.99, H 3.80, N 5.25.
2
2
117.56 (m, J(C,F) = 12.9 Hz, C-1), 123.17 (m, J(C,F) = 17.1 Hz, C-6),
139.98 (m 1J(C,F) = 251.8 Hz), 140.71 (m, 1J(C,F) = 255.2 Hz), 143.61
(m, 1J(C,F) = 245.7 Hz), 144.83 (m, 1J(C,F) = 251.1 Hz, C-2, C-3, C-4, C-
5), 159.44 (CO), 162.69 (CO). MS (EI) (m/z, %) 293 (M+, 10), 249 (M+
-
CO2, 8), 221 (M+ - C4H10H, 44), 177 (M+ - C4H10N – CO2, 100), 149 (M+ -
C4H10N – CO2 – CO, 21). Anal. calcd for C12H11F4NO3: C 49.15, H 3.78,
N 4.78, found: C 48.83, H 4.06, N 4.98.
Synthesis of tetrafluorobenzamides 5a-f: General Procedure. A
mixture of the tetrafluorophthalamic acid (1.5 mmol) and dry DMSO (1
mL) was heated to 153 °C for 4 h. After cooling to room temperature,
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10.1002/cmdc.201800263
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2,3,4,5-Tetrafluoro-N-tert-butylbenzamide (5f) was prepared from 4f
(0.44 g); yield 56% (0.21 g); mp 62-65-151 °C; 1H NMR (DMSO-d6) δ
1.34 (s, 9H, CH3), 7.49-7.54 (m, 1H, 6-H), 8.06 (br s, 1H, NH); 13C NMR
(DMSO-d6) δ 28.45 (CH3), 51.51 (C(CH3)3), 111.62 (m, 2J(C,F) = 20.1 Hz,
C-6), 122.44 (m, C-1), 139.84 (m 1J(C,F) = 246.3 Hz), 140.46 (m, 1J(C,F)
= 251.7 Hz), 144.48 (m, J(C,F) = 246.5 Hz), 146.12 (m, J(C,F) = 244.0
Hz, C-2, C-3, C-4, C-5), 160.10 (CO). MS (EI) (m/z, %) 234 (M+ -CH3, 42),
177 (M+ - C4H10N, 100). Anal. calcd for C11H11F4NO: C 53.02, H 4.45, N
5.62, found: C 52.61, H 4.73, N 5.51.
2,3,5-Trifluoro-4-(isobutylamino)-N-isopropylbenzamide (6c) was
prepared from 5b and isobutylamine (8 mL) following General Procedure
A at oil bath temperature of 70 °C. The aqueous mixture was extracted
with ethyl acetate (3 × 15 mL), the combined organic layers were dried
(Na2SO4) and the filtrate was evaporated to dryness. The residue was
purified by column chromatography using a gradient of petroleum
ether/CH2Cl2 (1:1) to CH2Cl2; yield 26% (75 mg); mp 42-43 °C; 1H NMR
(DMSO-d6) δ 0.86 (d, J = 6.6 Hz, 6H, CH2CHCH3), 1.12 (d, J = 6.6 Hz,
6H, NHCHCH3), 1.73-1.81 (m, 1H, CH2CH), 3.07-3.10 (m, 2H, CH2),
3.97-4.03 (m, 1H, NHCH), 6.02 (t, J = 6.5 Hz, 1H, 4-NH), 7.11-7.16 (m,
1H, 6-H), 7.77 (d, J = 6.7 Hz, 1H, CONH). 13C NMR (DMSO-d6) δ 19.90
(CH2CHCH3), 22.30 (NHCHCH3), 28.95 (CH2CH), 41.26 (NHCH), 51.90
(CH2), 110.08 (m, 2,3J(C,F) = 12.5, 7.1 Hz, C-1), 110.56 (m, 2J(C,F) =
23.3 Hz, C-6), 129.74 (m, C-4), 139.33 (m, 1,2,3J(C,F) = 238.5, 17.4, 8.4
Hz, C-3), 145.98 (m, 1,2J(C,F) = 243.9, 12.6 Hz, C-2), 146.54 (m,
1,3J(C,F) = 235.1, 7.3 Hz, C-5), 161.14 (CO). MS (EI) (m/z, %) 274 (M+,
30), 245 (M+ - C3H7, 100). Anal. calcd for C14H19F3N2O: C 58.32, H 6.64,
N 9.72, found: C 57.81, H 6.69, N 9.37.
1
1
Synthesis of 4-amino-2,3,5-trifluorobenzamides 6-7. General
Procedure A: A mixture of 1 mmol of the tetrafluorobenzamide 5b (0.24
g) or 5d (0.28 g) and the corresponding primary amine was heated for 4
h at an indicated oil bath temperature. Upon cooling and stirring, 2N HCl
was added until a weak acidic pH was reached. A saturated aqueous
solution of NaHCO3 was then added to adjust a pH value of 7. The
isolation and purification of the product was carried out as indicated.
Synthesis of 4-amino-2,3,5-trifluorobenzamides 7c and 7d. General
4-(Cyclohexylamino)-2,3,5-trifluoro-N-isopropylbenzamide (6d) was
prepared from 5b and cyclohexylamine (8 mL) following General
Procedure A at oil bath temperature of 130 °C. The aqueous mixture was
extracted with ethyl acetate (3 × 20 mL), the combined organic layers
were dried (Na2SO4) and the filtrate was evaporated to dryness. The
residue was recrystallized from n-hexanes; yield 17% (53 mg); mp 83-
85 °C; 1H NMR (DMSO-d6) δ 1.12 (d, J = 6.7 Hz, 6H, CH3), 1.30-2.00 (m,
10H, CH2), 3.41-3.52 (m, 1H, CHCH2), 3.96-4.05 (m, 1H, CHCH3), 5.35
(d, J = 9.2 Hz, 1H, 4-NH), 7.12-7.17 (m, 1H, 6-H), 7.80 (d, J = 6.3 Hz, 1H,
Procedure B:
A mixture of 0.5 mmol of 2,3,4,5-tetrafluoro-N-
cyclohexylbenzamide 5d (0.14 g) and the corresponding primary amine
(1 mmol) in DMSO (3 mL) was reacted in a sealed microwave vessel for
10 min at 110 °C in a CEM Discovery reactor (max. 100 W). After
addition of half-saturated brine (50 mL), it was extracted with ethyl
acetate (2 × 25 mL), washed with 10% KHSO4 (50 mL) and brine (50 mL),
dried (Na2SO4) and the filtrate was evaporated to dryness. The crude
product was purified by column chromatography using a gradient of
petroleum ether/ethyl acetate (19:1 to 10:1).
CONH). 13C NMR (DMSO-d6)
δ
22.30 (CH3), 24.86, 25.31
(CHCH2CH2CH2), 33.68 (CHCH2), 41.29 (CHCH3), 53.55 (CHCH2),
2,3,5-Trifluoro-N-isopropyl-4-(propylamino)benzamide
(6a)
was
2
110.59 (m, J(C,F) = 23.8 Hz, C-6), 110.90 (m, 2,3J(C,F) = 12.6, 7.2 Hz,
prepared from 5b and propylamine (7 mL) following General Procedure A
at oil bath temperature of 55 °C. The aqueous mixture was kept overnight
at 4 °C and the supernatant was decanted. The residue was dried in a
desiccator and recrystallized from n-hexanes; yield 51% (0.14 g); mp 41-
42 °C; 1H NMR (DMSO-d6) δ 0.86 (t, J = 7.3 Hz, 3H, CH2CH3), 1.12 (d, J
= 6.7 Hz, 6H, CHCH3), 1.49-1.54 (m, 2H, CH2CH3), 3.23-3.27 (m, 2H,
NHCH2), 3.98-4.02 (m, 1H, CH), 5.95-5.97 (m, 1H, NHCH2), 7.11-7.16 (m,
1H, 6-H), 7.77 (d, J = 6.3 Hz, 1H, CONH); 13C NMR (DMSO-d6) δ 11.11
(CH2CH3), 22.31 (CHCH3), 23.54 (CH2CH3), 41.26 (CH), 46.14 (NHCH2),
C-1), 128.80 (m, C-4), 139.61 (m, 1,2,3J(C,F) = 239.0, 17.1, 8.4 Hz, C-3),
145.90 (m, 1,2,4J(C,F) = 244.1, 12.8, 2.2 Hz, C-2), 146.81 (m, 1,3J(C,F) =
235.8, 6.3 Hz, C-5), 161.11 (CO). MS (EI) (m/z, %) 314 (M+, 76), 271 (M+
- C3H7, 100), 174 (M+ - C3H8N – C6H11, 42). Anal. calcd for C16H21F3N2O:
C 61.13, H 6.73, N 8.91, found: C 60.72, H 6.91, N 8.83.
N-Cyclohexyl-2,3,5-trifluoro-4-(propylamino)benzamide (7a) was
prepared from 5d and propylamine (9 mL) following General Procedure A
at oil bath temperature of 55 °C. The aqueous mixture was extracted with
ethyl acetate (3 × 15 mL), the combined organic layers were dried
(Na2SO4) and the filtrate was evaporated to dryness. The residue was
recrystallized from n-hexanes; yield 38% (0.12 g); mp 86-88 °C; 1H NMR
(DMSO-d6) δ 0.86 (t, J = 7.4 Hz, 3H, CH3), 1.11-1.79 (m, 10H,
CHCH2CH2CH2), 1.51 (sext, J = 7.3 Hz, 2H, CH2CH3), 3.24 (q, J = 6.9 Hz,
2H, NHCH2), 3.67-3.69 (m, 1H, CHCH2), 5.95 (br s, 1H, 4-NH), 7.10-7.14
(m, 1H, 6-H), 7.76 (d, J = 6.6 Hz, 1H, CONH). 13C NMR (DMSO-d6) δ
11.10 (CH3), 23.52 (CH2CH3), 24.74, 25.30 (CHCH2CH2CH2), 32.29
(CHCH2), 46.14 (NHCH2), 48.41 (CH), 110.15 (m, 2,3J(C,F) = 12.4, 6.9 Hz,
C-1), 110.54 (m, 2J(C,F) = 23.3 Hz, C-6), 129.65 (m, C-4), 139.32 (m,
1,2,3J(C,F) = 238.4, 17.2, 8.6 Hz, C-3), 145.93 (m, 1,2J(C,F) = 242.9, 11.6
Hz, C-2), 146.53 (m, 1,3J(C,F) = 235.1, 6.6 Hz, C-5), 161.09 (CO). MS
(EI) (m/z, %) 314 (M+, 80), 285 (M+ - C2H5, 34), 232 (M+ - C6H10, 100).
Anal. calcd for C16H21F3N2O: C 61.13, H 6.73, N 8.91, found: C 61.00, H
6.96, N 8.80.
2
110.08 (m, 2,3J(C,F) = 12.8, 6.9 Hz, C-1), 110.55 (m, J(C,F) = 23.3 Hz,
C-6), 129.68 (m, C-4), 139.33 (m, 1,2,3J(C,F) = 238.4, 17.2, 8.6 Hz, C-3),
145.97 (m, 1,2J(C,F) = 242.9, 11.6 Hz, C-2), 146.53 (m, 1,3J(C,F) = 235.1,
6.6 Hz, C-5), 161.14 (CO). MS (EI) (m/z, %) 274 (M+, 52), 245 (M+ - C2H5,
100), 216 (M+ -C3H8N, 64). Anal. calcd for C13H17F3N2O: C 56.93, H 6.25,
N 10.21, found: C 56.93, H 6.38, N 10.38.
2,3,5-Trifluoro-N-isopropyl-4-(isopropylamino)benzamide (6b) was
prepared from 5b and isopropylamine (5 mL) following General
Procedure A at oil bath temperature of 55 °C. The aqueous mixture was
extracted with ethyl acetate (3 × 15 mL), the combined organic layers
were dried (Na2SO4) and the filtrate was evaporated to dryness. The
residue was recrystallized from n-hexanes; yield 33% (91 mg); mp 56-
1
59 °C; H NMR (DMSO-d6) δ 1.12 (d, J = 6.3 Hz, 6H, CH3), 1.16 (d, J =
7.3 Hz, 6H, CH3), 3.84-3.90 (m, 1H, CH), 3.97-4.04 (m, 1H, CH), 5.36 (d,
J = 9.2 Hz, 1H, 4-NH), 7.13-7.17 (m, 1H, 6-H), 7.82 (d, J = 7.0 Hz, 1H,
CONH). 13C NMR (DMSO-d6) δ 22.29 (CH3), 23.39 (CH3), 41.26 (CH),
46.19 (CH), 110.58 (m, 2J(C,F) = 23.6 Hz, C-6), 110.99 (m, 2,3J(C,F) =
12.5, 7.2 Hz, C-1), 128.98 (m, C-4), 139.60 (m, 1,2,3J(C,F) = 239.2, 17.1,
8.3 Hz, C-3), 145.88 (m, 1,2J(C,F) = 241.9, 14.0 Hz, C-2), 146.79 (m,
1,3J(C,F) = 235.6, 6.1 Hz, C-5), 161.09 (CO). MS (EI) (m/z, %) 274 (M+,
45), 259 (M+ - CH3, 100), 216 (M+ -C3H8N, 20). Anal. calcd for
C13H17F3N2O: C 56.93, H 6.25, N 10.21, found: C 56.94, H 6.48, N 9.80.
N-Cyclohexyl-2,3,5-trifluoro-4-(isopropylamino)benzamide (7b) was
prepared from 5d and isopropylamine (6 mL) following General
Procedure A at oil bath temperature of 35 °C. The aqueous mixture was
extracted with ethyl acetate (3 × 15 mL), the combined organic layers
were dried (Na2SO4) and the filtrate was evaporated to dryness. The
residue was purified by column chromatography using a gradient of
petroleum ether/CH2Cl2 (1:1) to CH2Cl2; yield 30% (94 mg); mp 83-85 °C;
1H NMR (DMSO-d6) δ 1.16 (d, J = 6.7 Hz, 6H, CH3), 1.24-1.82 (m, 10H,
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10.1002/cmdc.201800263
ChemMedChem
FULL PAPER
CH2), 3.66-3.68 (m, 1H, CHCH2), 3.87 (oct, J = 6.8 Hz, CHCH3), 5.35 (d,
1H, J = 9.1 Hz, 4-NH), 7.12-7.16 (m, 1H, 6-H), 7.80 (d, J = 7.0 Hz, 1H,
139.60 (m, 1,2,3J(C,F) = 239.1, 16.9, 8.7 Hz, C-3), 140.43 (Ar-C), 145.70
(m, 1,2,4J(C,F) = 244.1, 12.8, 1.8 Hz, C-2), 146.81 (m, 1,3J(C,F) = 236.4,
6.8 Hz, C-5), 161.01 (CO). MS (EI) (m/z, %) 362 (M+, 46), 280 (M+
-
CONH). 13C NMR (DMSO-d6)
δ
23.39 (CH3), 24.75, 25.32
(CHCH2CH2CH2), 32.30 (CHCH2), 46.21 (CHCH3), 48.44 (CHCH2),
C6H10, 48), 91 (C7H7+). Anal. calcd for C20H21F3N2O: C 66.29, H 5.84, N
7.73, found: C 66.55, H 5.91, N 7.44.
2
110.39 (m, 2,3J(C,F) = 12.5, 7.2 Hz, C-1), 110.60 (m, J(C,F) = 23.6 Hz,
C-6), 128.95 (m, C-4), 139.64 (m, 1,2,3J(C,F) = 239.2, 17.1, 8.3 Hz, C-3),
145.37 (m, 1,2J(C,F) = 241.9, 14.0 Hz, C-2), 146.81 (m, 1,3J(C,F) = 235.6,
6.1 Hz, C-5), 160.14 (CO). MS (EI) (m/z, %) 314 (M+, 100), 299 (M+ - CH3,
75), 232 (M+ - C6H10). Anal. calcd for C16H21F3N2O: C 61.13, H 6.73, N
8.91, found: C 61.19, H 6.94, N 8.61.
4-Fluoroisoindoline (8). 13C NMR (DMSO-d6) δ 47.32 (C-3), 50.38 (C-1),
114.99 (m, 2J(C,F) =18.9 Hz, C-5), 119.45 (m, 4J(C,F) = 3.0 Hz, C-7),
121.85 (m, 2J(C,F) =18.9 Hz, C-3a), 131.23 (m, 3J(C,F) = 6.9 Hz, C-6),
139.04 (m, 3J(C,F) = 4.7 Hz, C-7a), 157.29 (m, 1J(C,F) = 247.6 Hz, C-4).
2-Fluorobenzamide (9). 13C NMR (DMSO-d6) δ 116.23 (m, 2J(C,F) =
N-Cyclohexyl-2,3,5-trifluoro-4-(isobutylamino)benzamide (7c) was
prepared from 5d and isobutylamine (9 mL) following General Procedure
A at oil bath temperature of 80 °C. The aqueous mixture was extracted
with ethyl acetate (3 × 15 mL), the combined organic layers were dried
(Na2SO4) and the filtrate was evaporated to dryness. The residue was
recrystallized from n-hexanes; yield 37% (0.12 g); mp 102-105 °C; 1H
NMR (DMSO-d6) δ 0.86 (d, J = 7.0 Hz, 6H, CH3), 1.11-1.81 (m, 11H,
CHCH2CH2CH2, CHCH3), 3.08 (t, J = 6.8 Hz, 2H, NHCH2), 3.65-3.72 (m,
1H, NHCH), 6.02 (t, J = 6.5 Hz, 1H, 4-NH), 7.10-7.14 (m, 1H, 6-H), 7.76
(d, J = 6.6 Hz, 1H, CONH). 13C NMR (DMSO-d6) δ 19.89 (CH3), 24.75,
25.32 (CHCH2CH2CH2), 28.94 (CHCH3), 32.29 (CHCH2), 48.41 (NHCH),
51.90 (NHCH2), 110.16 (m, 2,3J(C,F) = 12.5, 7.1 Hz, C-1), 110.56 (m,
2J(C,F) = 23.3 Hz, C-6), 129.74 (m, C-4), 139.34 (m, 1,2,3J(C,F) = 238.5,
17.4, 8.4 Hz, C-3), 145.95 (m, 1,2J(C,F) = 243.9, 12.6 Hz, C-2), 146.54 (m,
1,3J(C,F) = 235.1, 7.3 Hz, C-5), 161.12 (CO). MS (EI) (m/z, %) 328 (M+,
56), 285 (M+ - C3H7, 100), 246 (M+ - C6H10, 55). Anal. calcd for
C17H23F3N2O: C 62.18, H 7.06, N 8.53, found: C 62.01, H 7.32, N 8.24. In
a separate experiment, compound 5d was reacted with isobutylamine
under microwave conditions (General Procedure B). After column
chromatography, the product 7c was obtained in 45% yield.
1
4
22.7 Hz, C-3), 123.99 (m, J(C,F) = 14.2 Hz, C-1), 124.54 (m, J(C,F) =
3.4 Hz, C-5), 130.38 (m, 3J(C,F) = 2.9 Hz, C-6), 132.61 (m, 3J(C,F) = 8.6
Hz, C-4), 159.46 (m, 1J(C,F) = 249.5 Hz, C-2), 165.38 (CO).
3-Fluorobenzamide (10). 13C NMR (DMSO-d6) δ 114.39 (m, 2J(C,F) =
2
4
22.6 Hz, C-2), 118.24 (m, J(C,F) = 21.2 Hz, C-4), 123.78 (m, J(C,F) =
2.9 Hz, C-6), 130.51 (m, 3J(C,F) = 7.9 Hz, C-5), 136.89 (m, 3J(C,F) = 6.7
1
4
Hz, C-1), 162.13 (m, J(C,F) = 244.3 Hz, C-3), 166.68 (m, J(C,F) = 2.5
Hz, CO).
Biological investigations
Rat aortic ring assay. To determine the extent of the antiangiogenic
effects of the test compounds, the rat aortic ring assay was carried out as
described elsewhere.[8] Briefly, 24-well tissue culture plates were covered
with 250 µL of Matrigel (Becton-Dickinson) and allowed to gel for 30 to 45
min at 37 °C and 5% CO2. Sections of thoracic aorta were removed from
8- to 10-week-old male Sprague Dawley rats. Following excision of
fibroadipose tissue, the aortic sections were cut into 1-mm-long cross-
sections, placed on Matrigel-coated wells, and layered with additional
Matrigel (250 µL). These were then allowed to set, after which the cross-
sectional rings were covered with endothelial cell growth media (EGM-II)
and incubated under 5% CO2 at 37 °C overnight. EGM-II consists of
endothelial cell basal medium (EBM-II) and endothelial cell growth
factors. After 24 h, the medium was removed and replaced with EBM-II
(1 mL) with the vehicle (0.5% DMSO), positive control TNP-470 (50 µM),
or test compound (50 µM). This was replicated three times. The area of
angiogenic sprouting was quantified using Adobe Photoshop.
N-Cyclohexyl-2,3,5-trifluoro-4-(cyclohexylamino)benzamide (7d) was
prepared from 5d and cyclohexylamine (6 mL) following General
Procedure A at oil bath temperature of 130 °C. The aqueous mixture was
extracted with ethyl acetate (3 × 15 mL), the combined organic layers
were dried (Na2SO4) and the filtrate was evaporated to dryness. The
residue was recrystallized from n-hexanes; yield 27% (96 mg); mp 98-
102 °C; 1H NMR (DMSO-d6) δ 1.09-1.87 (m, 20H, CH2), 3.40-3.51 (m, 1H,
CH), 3.62-3.70 (m, 1H, CH), 5.34 (d, J = 9.2 Hz, 1H, 4-NH), 7.11-7.15 (m,
1H, 6-H), 7.79 (d, J = 6.3 Hz, 1H, CONH). 13C NMR (DMSO-d6) δ 24.74,
24.85, 25.30, 25.30, (CHCH2CH2CH2), 32.29, 33.67 (CHCH2), 48.43
(CONHCH), 53.55 (4-NHCH), 110.58 (m, 2J(C,F) = 23.8 Hz, C-6), 111.00
(m, 2,3J(C,F) = 12.6, 7.2 Hz, C-1), 128.77 (m, C-4), 139.59 (m, 1,2,3J(C,F)
= 239.0, 17.1, 8.4 Hz, C-3), 145.85 (m, 1,2,4J(C,F) = 241.1, 12.8, 2.2 Hz,
C-2), 146.81 (m, 1,3J(C,F) = 235.8, 6.3 Hz, C-5), 161.07 (CO). MS (EI)
Cytotoxicity assay. This assay was determined as described
elsewhere.[8] Briefly, HUVECs were treated with compounds at 10 µM.
After 24 hours of incubation with compounds, 10 µL of CCK-8 solution
(Dojindo, MD) was added to each well. The plates were left to incubate
for 2 hours, and then read at a wavelength of 450 nm using a microplate
reader.
(m/z, %) 354 (M+, 80), 272 (M+
- C6H10, 100). Anal. calcd for
C19H25F3N2O: C 64.39, H 7.11, N 7.90, found: C 64.56, H 7.32, N 7.90. In
a separate experiment, compound 5d was reacted with cyclohexylamine
under microwave conditions (General Procedure B). After column
chromatography, the product 7d was obtained in 43% yield.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed by the authors.
4-(Benzylamino)-N-cyclohexyl-2,3,5-trifluorobenzamide (7e) was
prepared from 5d and benzylamine (9 mL) following General Procedure
A at oil bath temperature of 130 °C. The aqueous mixture was extracted
with ethyl acetate (3 × 15 mL), the combined organic layers were dried
(Na2SO4) and the filtrate was evaporated to dryness. The residue was
recrystallized from n-hexanes; yield 45% (0.16 g); mp 133-136 °C; 1H
NMR (DMSO-d6) δ 1.09-1.80 (m, 10H, CH2), 3.62-3.70 (m, 1H, CH), 4.47
(d, J = 6.9 Hz, 2H, NHCH2), 6.66-6.69 (m, 1H, 4-NH), 7.18-7.21 (m, 1H,
6-H), 7.27-7.31 (m, 5H, Ar-H), 7.79 (d, J = 6.6 Hz, 1H, CONH). 13C NMR
(DMSO-d6) δ 24.71, 25.31 (CHCH2CH2CH2), 32.32 (CHCH2), 47.63
(NHCH2), 48.41 (NHCH), 110.48 (m, 2J(C,F) = 22.8 Hz, C-6), 111.25 (m,
2J(C,F) = 12.7 Hz, C-1), 126.84, 127.46, 128.34 (Ar-CH), 128.46 (m, C-4),
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Keywords: angiogenesis • fluorine • tetrafluorobenzamides •
tetrafluorophthalimides • trifluorobenzamides
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This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800263
ChemMedChem
FULL PAPER
FULL PAPER
Christian Steinebach,[a] Agnieszka
Ambrożak,[a] Stefan Dosa,[a] Shaunna L.
Beedie,[b] Jonathan D. Strope,[b] Gregor
Schnakenburg,[c] William D. Figg,[b] Michael
Gütschow*[a]
F
F
F
F
CONHR1
CO2H
F
F
CONHR1
F
F
F
F
F
F
O
CONHR1
F
F
F
Page No. – Page No.
N
R1
R2NH
Synthesis, Structural Characterization
and Antiangiogenic Activity of
Polyfluorinated Benzamides
O
Two or one ring – four or three fluorines. A path of stepwise structural modifications led from tetrafluorophthalimides via
tetrafluorophthalamic acids and tetrafluorobenzamides to 4-amino-2,3,5-trifluorobenzamides. The representatives of these
chemotypes were investigated by means of spectroscopic and crystallographic methods and evaluated as inhibitors of
angiogenesis.
This article is protected by copyright. All rights reserved.