Discovery and Characterization of R/S-N-3-Cyanophenyl-N′-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, a New Histone Deacetylase Class III Inhibitor Exerting Antiproliferative Activity against Cancer Cell Lines

Article abstract of DOI:10.1021/acs.jmedchem.7b00533

A new series of N-aryl-N′-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N′-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of >10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.

Full text of DOI:10.1021/acs.jmedchem.7b00533

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Article
Discovery and Characterization of R/S-N-3-Cyanophenyl-N'-
6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-
(
benzopyran-4-yl)urea, a New Histone Deacetylase Class III Inhibitor
Exerting Antiproliferative Activity Against Cancer Cell Lines.
Michael Schnekenburger, Eric Goffin, Jin-Young Lee, Jun Young Jang, Aloran Mazumder, Seungwon Ji,
Bernard Rogister, Nafila Bouider, Florence Lefranc, Walter Miklos, Véronique Mathieu, Pascal De Tullio, Kyu-
Won Kim, Mario Dicato, Walter Berger, Byung Woo Han, Robert Kiss, Bernard Pirotte, and Marc Diederich
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 05 May 2017
Downloaded from http://pubs.acs.org on May 6, 2017
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Discovery and Characterization of R/SꢀNꢀ3ꢀCyanophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ
3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea, a New Histone Deacetylase Class III
Inhibitor Exerting Antiproliferative Activity Against Cancer Cell Lines
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a,‡
b,‡
c
c
c
Michael Schnekenburger, Eric Goffin, JinꢀYoung Lee, Jun Young Jang, Aloran Mazumder,
c
d
b
e
f
Seungwon Ji, Bernard Rogister, Nafila Bouider, Florence Lefranc, Walter Miklos, Véronique
g
b
h
a
f
c
Mathieu, Pascal de Tullio, KyuꢀWon Kim, Mario Dicato, Walter Berger, Byung Woo Han,
h
b,#,*
c,#,*
Robert Kiss, Bernard Pirotte,
Marc Diederich,
*
Corresponding authors.
These authors equally contributed to the work.
‡
#These authors equally supervised the work.
Author’s Adresses:
a
Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9, rue
Edward Steichen, L-2540 Luxembourg, Luxembourg.
b
Laboratory of Medicinal Chemistry, Center for Interdisciplinary Research on Medicines
(
CIRM), University of Liège, 4000 Liège, Belgium.
c
Department of Pharmacy, Research Institute of Pharmaceutical Sciences, College of Pharmacy,
Seoul National University, 1 Gwanak-ro, Gwanak-gu, Korea
d
GIGA Neurosciences, Nervous System Diseases and Treatment, University of Liège, 4000
Liège, Belgium.
e
Service de Neurochirurgie, Hôpital Erasme, Université Libre de Bruxelles, 1070 Brussels,
Belgium.
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Department of Medicine I, Comprehensive Cancer Center and Institute of Cancer Research,
Medical University Vienna, Vienna, Austria.
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SNU-Harvard Neurovascular Protection Center, College of Pharmacy and Research Institute of
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Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Korea
h
Laboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie,
Université Libre de Bruxelles, 1050 Brussels, Belgium.
Keywords
2
,2ꢀDimethylchromans; glioma; anticancer agent; histone deacetylase; sirtuin inhibitor;
apoptosis; quantitative videomicroscopy; multidrug resistance
Abstract
A new series of NꢀarylꢀN'ꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)ureas bearing an
alkoxycarbonylamino group at the 6ꢀposition were synthesized and examined as putative
anticancer agents targeting sirtuins in glioma cells. Based on computational docking combined to
in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/SꢀNꢀ3ꢀcyanophenylꢀN'ꢀ(6ꢀtertꢀ
butoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea] which displays a
potent antiproliferative activity on various glioma cell types, assessed by quantitative
videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower
with a selectivity index > 10. Furthermore, human U373 and Hs683 glioblastoma cell lines
served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III
sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and nonꢀhistone proteins.
The translational potential of 18 was validated by an NCIꢀ60 cell line screen and validation of
growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18
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was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary,
compound 18 is the first representative of a new class of SIRT inhibitors opening new
perspectives in the medicinal chemistry of HDAC inhibitors.
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Introduction
Glioblastoma are associated with dismal prognoses due to their intrinsic resistance to proꢀ
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apoptotic stimuli and temozolomide, a generic compound, remains today the most efficient
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chemotherapeutic agent to fight glioblastoma once maximal surgery and radiotherapy have
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already been performed. Considering the absence of chemotherapeutically active drugs against
glioblastoma, the reduced life expectancy of the patients and the specific molecular
characteristics of this type of cancer, it becomes increasingly clear that new compound groups
and molecular targets need to be investigated. Recently, histone deacetylase (HDAC) and sirtuin
(SIRT) inhibitors (SIRTi) were shown to trigger metabolic effects in glioblastoma both
3
independently or combined with temozolomide allowing speculating about the potential impact
of SIRTi on this cancer type.
+
NAD ꢀdependent deacetylase class III of HDACs comprises a family of 7 members namely
SIRT1ꢀ7 known to deacetylate histones and other target proteins. Intracellular localization
characterizes the different isoforms (SIRT1ꢀ2, nucleus/cytoplasm; SIRT3ꢀ5, mitochondria;
SIRT6, nucleus, and SIRT7, nucleolus) as well as substrate specificity: besides histones, many
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, 5
transcriptional regulators and mitochondrial proteins are deacetylated by SIRTs.
SIRTs are emerging targets for the treatment of various chronic diseases including cancer.
Especially, both SIRT1 and 2 have been reported to play a key role in the development of
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, 7
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, 9
cancer.
Accordingly, first inhibitors comprised compounds resembling the physiological
+
substrates such as NAD analogs that were described as competitive SIRTi that compete with
+
+
NAD for the NAD ꢀbinding domain, thus inhibiting enzymatic activity of multiple SIRTs. Such
compounds showed anticancer activity in Bꢀcell chronic lymphoblastic leukemia or prostate
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cancer. Suramin (1) (Fig. 1), an adenosine receptor antagonist, acts as an SIRTi by binding the
1
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C pocket, B pocket and partially the enzyme's substrateꢀbinding site. 1 was shown to possess
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anticancer potential in combination with cytotoxic agents in xenografted prostate, lung and breast
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cancer cells. Similarly, sirtinol (2) (Fig. 1), a well described SIRT1 and 2 selective inhibitor,
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, 10
displays antiꢀcancer properties against various cancer models.
Although, most SIRTi lack
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selectivity against a specific isoform, EXꢀ527 (3) (Fig. 1) was reported to inhibit SIRT1 but is
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less active against SIRT2 and SIRT3. A combination treatment using 3 and other HDAC
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inhibitors was found to induce apoptosis in acute myeloid leukemia cells. The selective
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inhibition of SIRT2 by AGK2 (4) (Fig. 1) has been reported to induce antiꢀcancer effects by
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5
suppressing tumor cell growth and migration.
Recently, some of our structural analogs of the ATPꢀsensitive potassium (KATP) channel opener
cromakalim (5), 6ꢀhaloꢀsubstituted 3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyrans (or 2,2ꢀ
dimethylchromans), bearing an arylurea or arylthiourea moiety at the 4ꢀposition (6: X = F, Cl, Br)
were described as potent inhibitors of glioma. Here we synthetized a series of NꢀarylꢀN'ꢀ3,4ꢀ
dihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)ureas
and
thioureas
bearing
an
alkoxycarbonylamino group (‘carbamate’ group) at the 6ꢀposition (6: X = NHCOOR) and
investigated their anticancer effects on glial tumor cells (Fig. 2). Our results show that compound
18
[R/SꢀNꢀ3ꢀcyanophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea] inhibits SIRT1/2 activity associated with a potent antiproliferative activity
on glioblastoma cells while having a lower impact on normal glial cells, thus demonstrating an
excellent differential toxicity. For the first time, we document here the in vitro characterization of
18 as a novel SIRT inhibitor, its in cellulo epigenetic effect combined with its anticancer
potential in 2D and 3D cell culture. Our results are validated in vivo by zebrafish glioblastoma
xenografts.
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Results and Discussion
Chemistry
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The synthesis of the key intermediate 9 has been previously described. The target compounds
0ꢀ28 were prepared in good yields by reaction of 9 with the appropriate monosubstituted phenyl
1
isocyanate in methylene chloride at room temperature (Scheme 1).
Starting from the Nꢀ3ꢀcyanophenylꢀsubstituted urea 18, the corresponding Nꢀ3ꢀaminomethylꢀ
substituted analog 22 was obtained after reduction of the cyano group using hydrogen gas in the
presence of RaneyꢀNickel° (Scheme 2). On the other hand, acidꢀcatalyzed deprotection of the
tertꢀbutyl carbamate group of 18 provided the corresponding 6ꢀaminoꢀsubstituted chroman 33,
which by formylation gave access to the 6ꢀformamidoꢀsubstituted analog 34 (Scheme 2).
Reduction of the nitro group of compound 19 into the aminoꢀsubstituted compound 23 was
performed with iron powder in the presence of ammonium chloride in ethanol/water 3:1 (Scheme
3).
17
The target compound 32 was obtained by reaction of the previously described compound 31
with 3ꢀcyanophenyl isocyanate (Scheme 4).
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All the compounds were crystallized from appropriate solvents and characterized by H and C
NMR. Their purity was assessed by elemental analysis to obtain the final materials with the
chemical purity required before pharmacological evaluations.
Growth inhibitory activity against glioma cells
The in vitro effects of the 2,2ꢀdimethylchromans 10ꢀ28 and 32ꢀ37 on the growth of three human
highꢀgrade glioma cell lines, i.e. U373 and T98G from astroglial origin and Hs683 from
oligodendroglial origin, were examined (Tables 1 and 2; see Materials and Methods for details).
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Moreover, the selective toxicity between normal astrocytes and glioma cells regarding in vitro
growth inhibition was also investigated for several compounds listed in Table 1.
The 2,2ꢀdimethylchroman compounds tested on glioma cells were characterized by the presence
of a variety of substituents at the ortho, meta or para position of the phenyl ring in the arylurea or
arylthiourea moiety located at the 4ꢀposition of the benzopyran ring. The monosubstitution was
investigated in these three positions with moderate to strong electron withdrawing (halogen, CN,
NO , CF ) or electron donating (OCH , NH ) groups (Table 1). With a second set of compounds,
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the nature of the substituent in the 6ꢀposition was also examined (Table 2). Particular attention
was paid to the progressive increase of the steric hindrance of the ‘carbamate' moiety in this
position.
From the data reported in Table 1, it was observed that, for the compounds bearing the same
substituent on the phenyl ring, the ortho position was generally found to be less favorable for
growth inhibitory activity on glioma cells than the meta and the para position (compare 10 versus
16 and 24; compare 11 versus 18 and 25; compare 13 versus 20 and 27). However, no general
trend can be formally deduced. The nitro group, for example, was better tolerated in the ortho
position than in the para position, while the meta position remains the best choice for improved
activity against all tumor cell lines (see 12, 19 and 26).
It also appears that for growth inhibitory activity on glioma cells, an electron withdrawing group
was preferred to an electron donating group (i.e. positive mesomeric effect of OCH and NH ), in
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particular in the meta and the para positions (compare 21 and 23 versus 15ꢀ20; compare 28 versus
4ꢀ27).
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Taken as a whole, the two best compounds from Table 1 were the 3ꢀcyano and the 3ꢀnitroꢀ
substituted arylurea compounds 18 and 19, with an equal potency compared to cisplatin in these
three tumor cell lines models and 4ꢀcyanoꢀsubstituted arylthiourea compound 30, previously
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described as an inhibitor of insulin release. Interestingly, the reduction of the cyano group of
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complete loss of biological activity.
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Results reported in Table 2 clearly indicated that the presence of a bulky tertꢀ
butoxycarbonylamino group (see 18) was preferred to less bulky ‘carbamate’ moieties (see 35,
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formamido group (34) was responsible for a complete loss of growth inhibitory activity against
the three tumor cell lines.
Finally, looking at the selectivity index defined as the ratio between the GI values on normal
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promising compound.
Biology
Therapeutic potential of SIRT1 and 2 targeting in glioblastoma
Since the simultaneous inhibition of SIRT1 and 2 has been shown to display promising antiꢀ
8
, 18
cancer properties
, we aimed to assess the therapeutic potential of the pharmacological
targeting of SIRT1 and 2 in glioblastoma. First, we investigated basal SIRT1 and 2 protein
expression levels in U373 and Hs683 (Fig. 3A). Results showed that Hs683 cells expressed a
high constitutive level of SIRT1 compared to U373 cell, whereas SIRT2 was expressed at higher
levels in U373 cells. Accordingly, we then knocked down SIRT1 in HS683 and SIRT1 and
SIRT2 in U373 using siRNAs (Fig. 3B), and followed their effects on cell growth (Fig. 3C) by
videomicroscopy over 72 hours. Results suggested that a depletion of SIRT1 or 2 strongly
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reduced the proliferation of glioblastoma cells thus further validating SIRT1 and 2 as interesting
pharmacological targets.
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Predicting SIRT1/2 binding affinity of 2,2-dimethylchroman derivatives
To combine the differential cytotoxicity of our 2,2ꢀdimethylchromans to their potential to inhibit
SIRT1/2 activities, we compared the binding affinity of selected 2,2ꢀdimethylchroman
derivatives to known SIRTi including 2 as a dual SIRT1/2 inhibitor, 3 (SIRT1 inhibitor) and 4
(SIRT2 inhibitor). Firstly, derivatives were analyzed against the human SIRT1 complex [protein
1
9
data bank (PDB) ID: 4I5I] and SIRT2 complex (PDB ID: 4RMG) using AutoDock Vina as
described in "Materials and Methods". Briefly, the SIRT1 and 2 complexes were divided into
ligand and protein and then the ligands were docked into the protein complexes. In silico docking
results are represented in Table 3 and Fig. SIꢀ1.
Since compound 18 with the Nꢀalkyl-N’ꢀarylurea group in its cis, trans lowꢀenergy conformation
demonstrated best docking affinity against SIRT1/2 (Fig. 4), a qualitative molecular docking was
then performed with compound 18 in comparison to the SIRTi 4, and 2 against 7 available
experimental coꢀcrystallized human SIRT2 complexes. The resulting lowest binding affinities
2
0ꢀ23
were compared (Table 4A).
Overall, compound 18 exhibited a similar affinity to 2 but less
than 4. In Fig. 4A, the lowest affinity pose of compound 18 was selected as the likely binding
modes in the binding pocket of SIRT2 (PDB ID: 4RMG) where compound 18 formed a hydrogen
bond with the backbone carbonyl oxygen of Val233. In addition, similar docking experiments
were performed using compound 18 in comparison with the SIRTi 3 and 2 against 6 available
2
4ꢀ27
experimental coꢀcrystallized human SIRT1 complexes (Table 4B).
Compound 18 exhibited
better binding affinities than 3 and 2. The lowest binding affinity pose of compound 18 was
selected as the likely binding mode in the binding pocket of SIRT1 protein (PDB ID: 4I5I) where
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compound 18 formed two hydrogen bonds with the backbone nitrogen of Ala262 and the Nδ
atom of His363 (Fig. 4B).
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Compound 18 acts as a SIRT1/2 inhibitor
Considering the proficiency of various HDAC inhibitors to impair glioblastoma cell
3
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proliferation,
and the strong effect of the depletion of SIRT1 and 2 on glioblastoma cell
growth, we then validated the potential of various 2,2ꢀdimethylchromans selected based on our
computational docking results to inhibit SIRT activities in vitro (Table 5). In agreement with our
docking data, compound 18 markedly inhibited both SIRT1 (IC : 6.2 ± 1.7 µM) and SIRT2
5
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IC : 4.2 ± 1.6 µM) activities within a concentration range comparable to 1 used as a positive
50
control, even though 1 showed higher selectivity for SIRT1 (2.8 ± 0.3 µM) versus SIRT2 (13 ± 1
µM), whereas other derivatives performed less well or not at all.
Collectively, these data pointed out compound 18 as a good drug candidate for further
investigation. First, we evaluated the selectivity of 18 by testing its activity against total in vitro
HDAC activities (Fig. 5A) as well as against the in vitro activity of various nonꢀsirtuin HDAC
isoenzymes (Fig. 5B). Results showed only residual inhibition of total HDAC activity, whereas
increasing concentrations (0.1ꢀ100 µM) of 18 did not significantly impact HDAC 1, 2, 3, 8, 6, 10
and 11 activities. Therefore, compound 18 appeared essentially as a potent inhibitor of SIRT1/2,
but not of SIRT3. Presently, we cannot exclude that 18 also targets other SIRT isoforms insofar
expressed by glioblastoma.
To validate in vitro results, 18 was then investigated on human U373 and Hs683 glioblastoma to
assess in cellulo effects of SIRT inhibition on acetylation levels of histone and nonꢀhistone
proteins. Cells were treated for 24h at concentrations of 1ꢀ10 µM of 18 compared to the panꢀ
HDAC inhibitor suberoylanilide hydroxamic acid (SAHA, 38; 2 µM) used as a positive control
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(Figs. 1 and 6). We observed a concentrationꢀdependent increase of αꢀtubulin, a wellꢀknown
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protein substrate for SIRT2 , and histone H4 acetylation starting at 5 µM in U373 cells and 1ꢀ2.5
µM in Hs683 cells, respectively. The differential doseꢀresponse of protein acetylation observed in
those two cell lines after 18 exposure depending on the analyzed sirtuin substrate could be
explained by the constitutive SIRT1 and 2 protein expression levels observed in glioblastoma cell
lines (Fig. 3A). Furthermore, 18 increased acetylation levels in both lines for acetylated H4 and
H3K56, reported to serve as substrates for SIRT1 (Fig. SIꢀ2).
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Furthermore, we compared the growth inhibitory activity of 18 to reference SIRTi against U373
and Hs683 cell lines (Table 6). MTT results highlighted 18 as a promising antiꢀcancer drug as it
had the most pronounced growth inhibitory potency against glioblastoma cells; about 6 times
compared to the SIRT1/2 inhibitor 2, and about 20 and 6ꢀ14 times for the SIRT1 and SIRT2
inhibitors 3 and 4, respectively.
Finally, we investigated the effect of the depletion of SIRT1 or SIRT2 on the anticancer effects of
compound 18 in glioblastoma cells. Results demonstrated that the downregulation of either one
of these two HDAC enhanced the growth inhibitory properties of compound 18 on U373 and
Hs683 cells (Fig. 7).
Compound 18 does not exert its growth inhibitory activity through pro-apoptotic effects in
human glioblastoma cells
We then assessed the biological effect of compound 18 by using computerꢀassisted phase contrast
microscopy (quantitative videomicroscopy) to grossly analyze the way by which compound 18
inhibits the growth of human U373 and Hs683 glioma cell lines (Fig. 8). Fig. 8A shows that 10
µM (i.e. ~the GI50 value revealed by the MTT colorimetric assay, Table 1) compound 18
decreased by ~70% the growth of the Hs683 cell line after 72h of culture. While the growth
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inhibitory effects of 18 were pronounced on Hs683 cell line growth, the 18ꢀrelated growth
inhibitory activity rather corresponded to cytostatic than to cytotoxic effects on these Hs683
tumor cells as morphologically illustrated in Fig. 8. The definitions of the global growth and the
global growth ratio are provided in the legend to Fig. 8. The same features were observed on the
U373 glioblastoma cells (Fig. 8B). Remarkably, while U373 glioblastoma cells display certain
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levels of resistance to proꢀapoptotic stimuli, these U373 cells appear as sensitive as Hs683
oligodendroglioma cells to the growth inhibitory effects of compound 18 (Table 1, Fig. 8) with
30, 31
the fact that Hs683 cells are sensitive to proꢀapoptotic stimuli.
The quantitative videomicroscopyꢀrelated analyses suggested that compound 18 is cytostatic
rather than cytotoxic in both Hs683 and U373 glioma cell lines. We thus made use of flow
cytometry analyses to investigate whether compound 18 induced or not significant proꢀapoptotic
effects in these two glioma cell lines. The data obtained with 10 µM compound 18 indicated
modest proꢀapoptotic effects in Hs683 and weak if any effects in U373 glioma cells (Fig. SIꢀ3). It
is thus unlikely that the growth inhibitory effects observed at 10 µM for compound 18 (Table 1;
30, 31
Fig. 8) could relate to cytotoxic proꢀapoptotic effects, even if, as expected,
Hs683
oligodendroglioma cells are more prone to enter apoptosis than U373 glioblastoma cells. Indeed,
while both the MTT colorimetric assay and the quantitative videomicroscopy approach revealed
that 10 µM compound 18 inhibited by ≥ 50% the growth of both Hs683 and U373 glioma cell
lines (Table 1, Fig. 8), flow cytometry analyses revealed that 10 µM compound 14 induced ~25%
and <10% apoptosis in Hs683 and U373 glioma cells, respectively (Fig. SIꢀ3). These results were
confirmed and generalized by the absence of a significant accumulation of cells in the sub G1
population (Fig. SIꢀ4) when cells were treated with various SIRTi at the IC calculated from
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MTT assays (Table 6). Only 4 induced a modest increase of sub G1 population in both cell lines
as observed with the panꢀnonꢀsirtuin HDAC inhibitor 38 in U373 cells.
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Effect of compound 18 on cell cycle and senescence
Considering the strong effect of 18 on glioblastoma cell growth, we then investigated the effect
of various SIRTi on cell cycle distribution. As shown in Fig. 9A, compound 18 induced an
accumulation in G1 in both cell lines comparable to the one observed with the SIRT1 inhibitor 3
as well as with the SIRT1/2 inhibitor 2 and the panꢀnonꢀsirtuin inhibitor 38 in Hs683 cells. In
contrast, the SIRT2 inhibitor 4 triggered G2/M accumulation in both cell lines as observed with 2
and 38 in U373 cells.
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Irreversible cell cycle arrest may result in senescence induction. Therefore, we investigated
whether sustained exposure to subꢀtoxic concentrations of compound 18 may promote
senescenceꢀassociated βꢀgalactosidase (SAꢀβꢀGal) activity. As shown in Fig. 9B, the number of
senescent cells, identified as blueꢀstained cells, was markedly increased in 18ꢀtreated U373 cells
(≅ 20%), whereas no blueꢀstained cells were detected in untreated cells.
Compound 18 inhibits glioblastoma cell growth in vivo
Considering the drastic antiproliferative properties of 18 in vitro, we next investigated whether
this compound could also hamper the spheroidꢀforming capacity of glioblastoma cells. Our
results show that 5 and 10 µM of compound 18 significantly reduced the surface area and volume
of multicellular tumor spheroids from glioma Hs683 (Fig. 10A) and U373 (Fig. SIꢀ5) cell lines.
The propensity of compound 18 to reduce the 3D tumorꢀlike growth of glioblastoma cells was
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also confirmed by decreased ATP levels in compound 18ꢀtreated tumor spheroid glioblastoma
cultures (Figs. 10A and SIꢀ5).
To further extend our evaluation of the growth inhibitory capacities of 18 towards in vivo
settings, we examined the ability of 18 to abrogate tumor development in a zebrafish
xenotransplantation model. Fluorescently labeled tumor cells were preꢀtreated for 30 (Fig. SIꢀ5)
or 36h (Fig. 10B) with compound 18 and then injected into the yolk sacs of zebrafish embryos.
Results revealed that the tumorꢀassociated fluorescence intensity signal was drastically lowered
in the compound 18ꢀtreated zebrafish group compared to the untreated control group for both
glioblastoma Hs683 and U373 cell lines. The capacity of 18 to impede tumor formation in vivo
validated our in vitro results and sustained the potential anticancer activity of 18.
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Compound 18 is active against a broad range of tumor cell lines including drug-resistant
cancer cells harboring different resistance mechanisms
Next, to extend the anticancer potential of 18, Fig. 11 (revealed with the permission of the NCI)
GI50
shows that the mean GI value of compound 18 in the NCIꢀ60 cell line panel is Log
10
= ꢀ
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5.52, i.e. ~ 3 µM, a concentration very close to the one we obtained on the three Hs683, U373
and T98G glioma cell line, i.e. 8 ± 5 µM (Table 1). Fig. 11 further shows that compound 18
displays similar growth inhibitory activity against a broad majority of the NCI cell lines.
Successful cancer therapy by chemotherapeutic agents but also targeted small molecule inhibitors
is frequently limited by drug resistance mechanisms leading to treatment failure and recurrence.
Resistance might be mediated by mutations in sensor genes of DNA damage, like the tumor
3
3
suppressor p53 , or overexpression of pleiotropic efflux pumps for multiple drugs causing a
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broadꢀscale multidrug resistance (MDR) phenotype. Hence we tested whether mutation of p53
or overexpression of the major MDR proteins ABCB1 (Pꢀglycoprotein) and ABCC1 (multidrug
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resistance protein 1, MRP1) significantly reduce the anticancer activity of 18 (Fig. 12). Neither
loss of p53 nor overexpression of these ABC transporters resulted in a significant loss of activity
against the investigated drug resistance models (Table SIꢀ1). In contrast, ABCB1 overexpression
in SW1573 2R160 and KBꢀCꢀ1 lines even resulted in a slight hypersensitivity to 18 (Figs. 12B
and D, respectively and Table SIꢀ1). Additionally, an ovarian cancer cell model with acquired
cisplatin resistance (A2780cis) and a hydroxyureaꢀresistant HeLa derivative overexpressing
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ribonucleotide reductase model (KBꢀHU) were compared to their respective parental cell lines
(Figs. 12C and D, respectively). Also in these two cases, no significant impact of the resistance
phenotype on the cytotoxic activity of 18 was detectable. Collectively these data demonstrate that
18 is not susceptible to several important chemotherapy resistance mechanisms.
Finally, the effect of 18 was tested after 24 and 48h of treatment on the viability of peripheral
blood mononuclear cells (PBMCs) from healthy donors (Fig. 13). Compound 18 does not exhibit
any major acute toxicity on this normal cell model.
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Conclusions
We prepared new series of NꢀarylꢀN'ꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)ureas and
thioureas bearing an alkoxycarbonylamino group (‘carbamate' group) at the 6ꢀposition of the
chroman nucleus. These compounds, structurally related to previously described 2,2ꢀ
dimethylchroman derivatives acting on glioma cells, were examined as putative anticancer
agents. Growth inhibitory activity was measured in three glioma cell lines (U373, T98G and
Hs683), as well as on normal astrocytes. Those data revealed that the compound bearing a cyano
group at the meta position of the phenyl ring of the 4ꢀarylurea moiety, as well as a bulky tertꢀ
butoxycarbonylamino group at the 6ꢀposition (compound 18), exhibited a potent antiproliferative
activity against the tumor glial cells, while having lower impact on normal glial cells (selectivity
index > 10). From the computational docking analysis, compound 18 also showed a potent
binding affinity when compared with other derivatives as observed with our antiproliferative
activity assay. This compound was therefore selected for further investigations to better
understand its mechanism of action. Compound 18 was assayed in the NCIꢀ60 cell line panel
screening demonstrating interesting inhibitory potential in various cell types. 18 was also
submitted to a COMPARE analysis (results not presented) to further decipher the mode of action
of compound 18 as successfully done previously with respect to other compounds but without
providing further clues. The screening confirmed the antiproliferative activity of 18 on a variety
of cancer cell lines (mean GI value = 3 µM), while the COMPARE tool did not allow obtaining
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a clear hypothesis about its potential mechanism of action. Quantitative videomicroscopy
analysis revealed that 18 was a cytostatic rather than a cytotoxic agent.
Furthermore, neither mutation of p53 nor overexpression of MDR efflux pumps including
ABCB1 and ABCC1 significantly reduced the anticancer activity of 18. The same holds true for
acquired cisplatin and hydroxyurea resistance. These data suggest that several major drug
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resistance mechanisms limiting the success of current systemic cancer therapy are not affecting
the activity of 18. Hence this substance might be especially attractive for targeting treatmentꢀ
resistant recurrences.
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Then, 18 was tested against a panel of HDAC activities and a potent inhibitory activity was
observed in vitro on HDAC SIRT1/2 and IC values in the low micromolar range (Table 3).
50
Presently, we cannot exclude that inhibition of other SIRT isoforms lead to growth arrest if
expressed by the targeted cancer type. This observation was validated in cellulo when 18 was
investigated on human U373 and Hs683 glioblastoma cell lines to assess effects of deacetylase
inhibition on acetylation levels of histone and nonꢀhistone proteins. Interestingly, compound 18
acted as an in vitro SIRTi with activities commonly reported in the literature. Importantly we
demonstrated associated biological activities (i.e. increased protein acetylation levels and
important antiproliferative properties) at concentrations in the low micromolar range, which is
usually not the case of SIRTi published over the past years (Table SIꢀ4).
Finally, the antiꢀcancer potential of 18 was further highlighted through the growth inhibition of
multicellular glioblastoma tumor spheroids as well as the reduction of tumor formation in vivo
using zebrafish xenografts.
In conclusion, compound 18 is new anticancer agent active on glioma cells and expressing a
cytostatic rather than a cytotoxic activity. Moreover, this compound is a potent inhibitor of
SIRT1 and 2 and constitutes a model of chemical structure opening new perspectives in the
medicinal chemistry of HDAC inhibitors against class III.
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Experimental Section
Chemistry
Melting points were determined on a Stuart SMP3 capillary apparatus and are uncorrected. The
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H NMR spectra were recorded on a Bruker Avance (500 MHz) instrument using d ꢀDMSO as
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the solvent with TMS as an internal standard; chemical shifts are reported in d values (ppm)
relative to that of internal TMS. The abbreviation s = singlet, d = doublet, t = triplet, q =
quadruplet, m = multiplet, b = broad are used throughout. Elemental analyses (C, H, N, S) were
realized on a Thermo Scientific FlashEA 1112ꢀelemental analyzer and were within ±0.4% of the
theoretical values. This analytical method certified a purity ≥95% for each tested compound. All
reactions were routinely checked by TLC on silica gel Merck 60 F .
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R/SꢀNꢀ2ꢀChlorophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (10)
3ꢀChlorophenyl isocyanate (0.13 g, 0.82 mmol) was added to a solution of 9 (0.2 g, 0.68 mmol)
in methylene chloride (5 mL). After 1 hour, the solvent was removed under vacuum; the title
1
product was obtained from the crude product by DCVC purification: m.p.: 192ꢀ194 °C; H NMR
(
DMSOꢀd ) d 1.25 (s, 3H, CH ), 1.36 (s, 3H, CH ), 1.43 (s, 9H, NHCOOC(CH ) ), 1.66 (dd,
6 3 3 3 3
J=13 Hz/11 Hz, 1H, 3ꢀH), 2.13 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.91 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6,65
(d, J=8.8 Hz, 1H, 8ꢀH), 6.97 (td, J=7.9 Hz/1.5 Hz, 1H, 4'ꢀH), 7.19 (d, J=7.1 Hz, 1H, 7ꢀH), 7.27
(td, J=7.9 Hz/1.5 Hz, 1H, 5'ꢀH), 7.38 (d, J=8.4 Hz, 1H, CHNHCONHAr), 7.41ꢀ7.42 (m, 2H, 5ꢀ
H/3'ꢀH), 8.07 (s, 1H, CHNHCONHAr), 8.26 (dd, J=8.3 Hz/1.2 Hz, 1H, 6'ꢀH), 9.10 (s, 1H,
13
NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.6 (CH ), 28.1 (C(CH ) ), 28.9 (CH ), 40.0 (Cꢀ3),
3
3
6
3
3 3
3
42.4 (Cꢀ4), 74.7 (Cꢀ2), 78.6 (C(CH ) ), 116.7 (Cꢀ8), 117.7 (Cꢀ5), 119.7 (Cꢀ7), 120.7 (Cꢀ6'), 121.1
3 3
(Cꢀ2'), 122.5 (Cꢀ4'), 122.6 (Cꢀ4a), 127.5 (Cꢀ5'), 129.1 (Cꢀ3'), 132.1 (Cꢀ6), 136.6 (Cꢀ1'), 148.6 (Cꢀ
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a), 153.0 (NHCOOC(CH ) ), 154.7 (CHNHCONHAr). Anal. (C H ClN O ) theoretical: C,
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23 28
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1.95; H, 6.33; N, 9.42. Found: C, 61.71; H, 6.33; N, 9.44.
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R/SꢀNꢀ2ꢀCyanophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (11)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 2ꢀ
1
cyanophenyl isocyanate (0.12 g, 0.82 mmol): m.p.: 206ꢀ208 °C; H NMR (DMSOꢀd ) d 1.25 (s,
6
3H, CH ), 1.36 (s, 3H, CH ), 1.43 (s, 9H, NHCOOC(CH ) ), 1.69 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH),
3
3
3 3
2
.14 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.91 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6,65 (d, J=8.8 Hz, 1H, 8ꢀH),
.12 (t, J=8 Hz, 1H, 4'ꢀH), 7.20 (d, J=7.3 Hz, 1H, 7ꢀH), 7.42 (d, J=8.8 Hz, 1H, CHNHCONHAr),
7
7.43 (s, 1H, 5ꢀH), 7.61 (m, 1H, 5'ꢀH), 7.71 (dd, J=7.8 Hz/1.4 Hz, 3'ꢀH), 8.22 (d, J=8.5 Hz, 1H, 6'ꢀ
13
H), 8.54 (s, 1H, CHNHCONHAr), 9.11 (s, 1H, NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.6
3
3
6
(CH ), 28.1 (C(CH ) ), 28.8 (CH ), 40.0 (Cꢀ3), 42.6 (Cꢀ4), 74.7 (Cꢀ2), 78.6 (C(CH ) ), 100.5 (Cꢀ
3 3 3 3 3 3
2'), 116.8 (Cꢀ8), 117.0 (CN), 117.6 (Cꢀ5), 119.7 (Cꢀ7), 120.4 (Cꢀ6'), 122.2 (Cꢀ4'), 122.4 (Cꢀ4a),
1
1
6
32.1 (Cꢀ6), 133.0 (Cꢀ3'), 133.9 (Cꢀ5'), 142.7 (Cꢀ1'), 148.5 (Cꢀ8a), 153.0 (NHCOOC(CH ) ),
3 3
54.4 (CHNHCONHAr). Anal. (C H N O ) theoretical: C, 66.04; H, 6.47; N, 12.84. Found: C,
24 28
4
4
6.12; H, 6.11; N, 13.26.
R/SꢀNꢀ2ꢀNitrophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (12)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 2ꢀ
1
nitrophenyl isocyanate (0.13 g, 0.82 mmol): m.p.: 218ꢀ219 °C; H NMR (DMSOꢀd ) d 1.25 (s,
6
3H, CH ), 1.37 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.69 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH),
3 3 3 3
2
.11 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.92 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6,65 (d, J=8.8 Hz, 1H, 8ꢀH),
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Journal of Medicinal Chemistry
Page 20 of 80
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7
.16 (m, 1H, 4'ꢀH), 7.20 (d, J=6.4 Hz, 1H, 7ꢀH), 7.40 (s, 1H, 5ꢀH), 7.68 (m, 1H, 5'ꢀH), 7.98 (d,
J=8.1 Hz, 1H, CHNHCONHAr), 8.09 (dd, J=8.4 Hz/1.5 Hz, 1H, 6'ꢀH), 8.45 (d, J=8.5 Hz, 1H, 3'ꢀ
1
3
H), 9.09 (s, 1H, NHCOOC(CH ) ) , 9.45 (s, 1H, CHNHCONHAr). C NMR (DMSOꢀd ) d 24.4
3
3
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
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8
9
0
(CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.0 (Cꢀ3), 42.7 (Cꢀ4), 74.7 (Cꢀ2), 78.6 (C(CH ) ), 116.7 (Cꢀ
3 3 3 3 3 3
8
), 117.7 (Cꢀ5), 119.6 (Cꢀ7), 121.5 (Cꢀ4'), 122.1 (Cꢀ3'), 122.4 (Cꢀ4a), 125.4 (Cꢀ6'), 132.0 (Cꢀ6),
1
35.0 (Cꢀ5'), 135.8 (Cꢀ1'), 136.7 (Cꢀ2'), 148.5 (Cꢀ8a), 152.9 (NHCOOC(CH ) ), 154.2
3
3
(CHNHCONHAr). Anal. (C H N O ) theoretical: C, 60.52; H, 6.18; N, 12.27. Found: C, 60.18;
23 28 4 6
H, 6.17; N, 11.86.
R/SꢀNꢀ2ꢀTrifluoromethylphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ
2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea (13)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 2ꢀ
1
trifluoromethylphenyl isocyanate (0.15 g, 0.82 mmol): m.p.: 239ꢀ239.5 °C; H NMR (DMSOꢀd )
6
d 1.25 (s, 3H, CH ), 1.36 (s, 3H, CH ), 1.44 (s, 9H, NHCOOC(CH ) ), 1.67 (dd, J=13 Hz/11 Hz,
3
3
3 3
1H, 3ꢀH), 2.12 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.90 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6.64 (d, J=8.8 Hz, 8ꢀ
H), 7.15 (d, J=7.6 Hz, 1H, 7ꢀH), 7.21 (t, J=7.6 Hz, 1H, 4'ꢀH), 7.38 (d, J=8.3 Hz, 1H,
CHNHCONHAr), 7.48 (s, 1H, 5ꢀH), 7.60 (t, J=7.8 Hz , 1H, 5'ꢀH), 7.63 (d, J=7.9 Hz, 1H, 3'ꢀH),
13
7.83 (s, 1H, CHNHCONHAr), 8.09 (d, J=8.3 Hz, 1H, 6'ꢀH), 9.12 (s, 1H, NHCOOC(CH ) ). C
3
3
NMR (DMSOꢀd ) d 24.6 (CH ), 28.1 (C(CH ) ), 28.9 (CH ), 40.0 (Cꢀ3), 42.6 (Cꢀ4), 74.7 (Cꢀ2),
6
3
3 3
3
78.6 (C(CH ) ), 116.7 (Cꢀ8), 117.8 (Cꢀ5), 119.6 (Cꢀ7), 122.7 (Cꢀ4a), 122.8 (Cꢀ4'), 123.0ꢀ125.1 (d,
3 3
J=273 Hz, CF ), 124.8 (Cꢀ6'), 125.7 (Cꢀ3'), 127.3 (Cꢀ2'), 132.0 (Cꢀ6), 132.7 (Cꢀ5'), 137.2 (Cꢀ1'),
3
148.5 (Cꢀ8a), 152.9 (NHCOOC(CH ) ), 154.9 (CHNHCONHAr). Anal. (C H F N O )
3
3
24 28
3
3
4
theoretical: C, 60.12; H, 5.89; N, 8.76. Found: C, 60.12; H, 5.95; N, 8.73.
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Page 21 of 80
Journal of Medicinal Chemistry
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R/SꢀNꢀ2ꢀMethoxyphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (14)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 2ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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28
29
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
methoxyphenyl isocyanate (0.12 g, 0.82 mmol): m.p.: 204ꢀ205 °C; H NMR (DMSOꢀd ) d 1.25
6
(
s, 3H, CH ), 1.35 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.62 (dd, J=13 Hz/11 Hz, 1H, 3ꢀ
3 3 3 3
H), 2.10 (dd, J=13 Hz/6 Hz, 3ꢀH), 3.83 (s, 3H, OCH ), 4.90 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6,63 (d,
3
J=8.8 Hz, 1H, 8ꢀH), 6.88 (pd, J=7.4 Hz/1.6 Hz, 2H, 4'ꢀH/5'ꢀH), 6.98 (dd, J=7.7 Hz/1.6 Hz, 1H, 3'ꢀ
H), 7.19 (bs, 1H, CHNHCONHAr), 7.20 (d, J=8.5 Hz, 1H, 7ꢀH), 7.40 (s, 1H, 5ꢀH), 8.01 (s, 1H,
13
CHNHCONHAr), 8.18 (dd, J=7.5 Hz/1.9 Hz, 1H, 6'ꢀH), 9.10 (s, 1H, NHCOOC(CH ) ).
C
3
3
NMR (DMSOꢀd ) d 24.6 (CH ), 28.1 (C(CH ) ), 28.9 (CH ), 40.0 (Cꢀ3), 42.2 (Cꢀ4), 55.6
6
3
3 3
3
(OCH ), 74.7 (Cꢀ2), 78.5 (C(CH ) ), 110.5 (Cꢀ3'), 116.6 (Cꢀ8), 117.8 (Cꢀ5/Cꢀ6'), 119.6 (Cꢀ7),
3
3 3
1
20.5 (Cꢀ5'), 121.0 (Cꢀ4'), 122.9 (Cꢀ4a), 129.4 (Cꢀ1'), 132.0 (Cꢀ6), 147.2 (Cꢀ2'), 148.6 (Cꢀ8a),
53.0 (NHCOOC(CH ) ), 155.0 (CHNHCONHAr). Anal. (C H N O ) theoretical: C, 65.29; H,
1
3 3
24 31
3
5
7.08; N, 9.52. Found: C, 65.20; H, 7.09; N, 9.51.
R/SꢀNꢀ3ꢀFluorophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (15)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 3ꢀ
1
fluorophenyl isocyanate (0.11 g, 0.82 mmol): m.p.: 115ꢀ125 °C; H NMR (DMSOꢀd ) d 1.24 (s,
6
3
H, CH ), 1.35 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.70 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH),
3 3 3 3
2
.08 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.92 (dd, J=16 Hz/10 Hz, 1H, 4ꢀH), 6.55 (d, J=8.6 Hz, 1H,
CHNHCONHAr), 6,64 (d, J=8.7 Hz, 1H, 8ꢀH), 6.73 (t, J=7.6 Hz, 1H, 4'ꢀH), 7.06 (d, J=7.8 Hz,
1
H, 6'ꢀH), 7.17 (d, J=7.1 Hz, 1H, 7ꢀH), 7.26 (dd, J=15.2 Hz/7.7 Hz, 1H, 5'ꢀH), 7.41 (s, 1H, 5ꢀH),
1
3
7
.53 (d, J=12 Hz, 1H, 2'ꢀH), 8.73 (s, 1H, CHNHCONHAr), 9.11 (s, 1H, NHCOOC(CH ) ). C
3 3
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Journal of Medicinal Chemistry
Page 22 of 80
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NMR (DMSOꢀd ) d 24.4 (CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.0 (Cꢀ3), 42.3 (Cꢀ4), 74.8 (Cꢀ2),
6
3
3 3
3
7
8.5 (C(CH ) ), 104.4 (d, J=27 Hz, Cꢀ2'), 107.4 (d, J=21 Hz, Cꢀ4'), 113.5 (d, J=2 Hz, Cꢀ6'), 116.7
3 3
(Cꢀ8), 117.6 (Cꢀ5), 119.6 (Cꢀ7), 122.9 (Cꢀ4a), 130.1 (d, J=10 Hz, Cꢀ5'), 132.0 (Cꢀ6), 142.2 (d,
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J=12 Hz, Cꢀ1'), 148.5 (Cꢀ8a), 152.9 (NHCOOC(CH ) ), 154.9 (CHNHCONHAr), 161.5ꢀ163.4 (d,
3
3
J=240 Hz, Cꢀ3'). Anal. (C H FN O ) theoretical: C, 64.32; H, 6.57; N, 9.78. Found: C, 64.68;
23 28
3
4
H, 6.26; N, 10.09.
R/SꢀNꢀ3ꢀBromophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (17)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 3ꢀ
1
bromophenyl isocyanate (0.16 g, 0.82 mmol): m.p.: 209.5ꢀ210.5 °C; H NMR (DMSOꢀd ) d 1.24
6
(
s, 3H, CH ), 1.35 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.71 (dd, J=13 Hz/11 Hz, 1H, 3ꢀ
3 3 3 3
H), 2.08 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.92 (dd, J=16 Hz/10 Hz, 1H, 4ꢀH), 6.57 (d, J=8.6 Hz, 1H,
CHNHCONHAr), 6,63 (d, J=8.7 Hz, 1H, 8ꢀH), 7.09 (d, J=8.5 Hz, 1H, 4'ꢀH), 7.16 (d, J=7.1 Hz,
1H, 7ꢀH), 7.20 (t, J=8 Hz, 1H, 5'ꢀH), 7.26 (d, J=8.7 Hz, 1H, 6'ꢀH), 7.40 (s, 1H, 5ꢀH), 7.90 (t,
13
J=1.9 Hz, 1H, 2'ꢀH), 8.71 (s, 1H, CHNHCONHAr), 9.09 (s, 1H, NHCOOC(CH ) ). C NMR
3 3
(DMSOꢀd ) d 24.4 (CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.0 (Cꢀ3), 42.4 (Cꢀ4), 74.8 (Cꢀ2), 78.5
6 3 3 3 3
(
C(CH ) ), 116.6 (Cꢀ8/Cꢀ6'), 117.5 (Cꢀ5), 119.6 (Cꢀ7), 120.1 (Cꢀ2'), 121.7 (Cꢀ3'), 122.9 (Cꢀ4a),
3
3
123.7 (Cꢀ4'), 130.6 (Cꢀ5'), 132.0 (Cꢀ6), 142.0 (Cꢀ1'), 148.5 (Cꢀ8a), 152.9 (NHCOOC(CH ) ),
3
3
1
54.9 (CHNHCONHAr). Anal. (C H BrN O ) theoretical: C, 56.33; H, 5.76; N, 8.57. Found:
23 28 3 4
C, 56.40; H, 5.83; N, 8.44.
R/SꢀNꢀ3ꢀNitrophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (19)
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Journal of Medicinal Chemistry
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The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 3ꢀ
1
nitrophenyl isocyanate (0.13 g, 0.82 mmol): m.p.: 120ꢀ155 °C; H NMR (DMSOꢀd ) d 1.25 (s,
6
3H, CH ), 1.36 (s, 3H, CH ), 1.41 (s, 9H, NHCOOC(CH ) ), 1.76 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH),
3
3
3 3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
6
1
9
.09 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.95 (dd, J=16 Hz/10 Hz, 1H, 4ꢀH), 6.64 (d, J=8.8 Hz, 1H, 8ꢀH),
.71 (d, J=8.7 Hz, 1H, CHNHCONHAr), 7.17 (bs, 1H, 7ꢀH), 7.41 (s, 1H, 5ꢀH), 7.53 (t, J=8.2 Hz,
H, 5'ꢀH), 7.69 (d, J=7.8 Hz, 1H, 4'ꢀH), 7.78 (d, J=8.1 Hz, 1H, 6'ꢀH), 8.61 (t, J=2 Hz, 1H, 2'ꢀH),
13
.09 (s, 2H, CHNHCONHAr / NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.4 (CH ), 28.1
3
3
6
3
(C(CH ) ), 29.1 (CH ), 40.0 (Cꢀ3), 42.5 (Cꢀ4), 74.8 (Cꢀ2), 78.5 (C(CH ) ), 111.7 (Cꢀ2'), 115.7 (Cꢀ
3
3
3
3 3
6'), 116.7 (Cꢀ8), 117.5 (Cꢀ5), 119.6 (Cꢀ7), 122.8 (Cꢀ4a), 123.9 (Cꢀ4'), 129.9 (Cꢀ5'), 132.0 (Cꢀ6),
141.7 (Cꢀ1'), 148.1 (Cꢀ3'), 148.5 (Cꢀ8a), 152.9 (NHCOOC(CH ) ), 154.9 (CHNHCONHAr).
3
3
Anal. (C H N O ) theoretical: C, 60.52; H, 6.18; N, 12.27. Found: C, 61.01; H, 6.13; N, 12.18.
2
3
28
4
6
R/SꢀNꢀ3ꢀTrifluoromethylphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ
2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea (20)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 3ꢀ
1
trifluoromethylphenyl isocyanate (0.15 g, 0.82 mmol): m.p.: 140ꢀ155 °C; H NMR (DMSOꢀd ) d
6
1.25 (s, 3H, CH ), 1.36 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.74 (dd, J=13 Hz/11 Hz,
3 3 3 3
1H, 3ꢀH), 2.09 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.94 (dd, J=16 Hz/10 Hz, 1H, 4ꢀH), 6.62 (d, J=8.6 Hz,
1H, CHNHCONHAr), 6.64 (d, J=8.8 Hz, 1H, 8ꢀH), 7.17 (bd, J=6.7 Hz, 1H, 7ꢀH), 7.26 (d, J=7.5
Hz, 1H, 4'ꢀH), 7.41 (s, 1H, 5ꢀH), 7.47 (t, J=7.9 Hz, 1H, 5'ꢀH), 7.53 (d, J=8.2 Hz, 1H, 6'ꢀH), 8.07
1
3
(s, 1H, 2'ꢀH), 8.89 (s, 1H, CHNHCONHAr), 9.08 (s, 1H, NHCOOC(CH ) ). C NMR (DMSOꢀ
3 3
d ) d 24.4 (CH ), 28.1 (C(CH ) ), 29.1 (CH ), 40.0 (Cꢀ3), 42.4 (Cꢀ4), 74.8 (Cꢀ2), 78.5 (C(CH ) ),
6
3
3 3
3
3 3
113.8 (Cꢀ2'), 116.6 (Cꢀ8), 117.4 (Cꢀ5/Cꢀ4'), 119.6 (Cꢀ7), 121.3 (Cꢀ6'), 122.9 (Cꢀ4a), 123.2ꢀ125.4
(d, J=272 Hz, CF ), 129.3ꢀ129.6 (d, J=31 Hz, Cꢀ3'), 129.7 (Cꢀ5'), 132.0 (Cꢀ6), 141.2 (Cꢀ1'), 148.5
3
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Journal of Medicinal Chemistry
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(
Cꢀ8a), 153.0 (NHCOOC(CH ) ), 155.0 (CHNHCONHAr). Anal. (C H F N O ) theoretical: C,
3
3
24 28
3
3
4
6
0.12; H, 5.89; N, 8.76. Found: C, 60.19; H, 6.03; N, 8.59.
0
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R/SꢀNꢀ3ꢀMethoxyphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (21)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 3ꢀ
1
methoxyphenyl isocyanate (0.12 g, 0.82 mmol): m.p.: 189ꢀ191 °C; H NMR (DMSOꢀd ) d 1.24
6
(s, 3H, CH ), 1.35 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.68 (dd, J=13 Hz/11 Hz, 1H, 3ꢀ
3
3
3 3
H), 2.08 (dd, J=13 Hz/6 Hz, 3ꢀH), 3.72 (s, 1H, OCH ), 4.91 (dd, J=16 Hz/10 Hz, 1H, 4ꢀH), 6.46
3
(
d, J=8.7 Hz, 1H, CHNHCONHAr), 6.50 (dd, J=8.2 Hz/1.9 Hz, 1H, 4'ꢀH), 6.63 (d, J=8.8 Hz, 1H,
8ꢀH), 6.89 (dd, J=8.1 Hz/1 Hz, 1H, 6'ꢀH), 7.13 (t, J=8 Hz, 1H, 5'ꢀH), 7.17 (d, J=7.8 Hz, 1H, 7ꢀH),
.22 (t, J=2 Hz, 1H, 2'ꢀH), 7.40 (s, 1H, 5ꢀH), 8.52 (s, 1H, CHNHCONHAr), 9.08 (s, 1H,
7
13
NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.5 (CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.0 (Cꢀ3),
3 3
6
3
3 3
3
42.3 (Cꢀ4), 54.8 (OCH ), 74.8 (Cꢀ2), 78.5 (C(CH ) ), 103.4 (Cꢀ2'), 106.7 (Cꢀ4'), 110.1 (Cꢀ6'),
3
3 3
1
16.6 (Cꢀ8), 117.6 (Cꢀ5), 119.6 (Cꢀ7), 123.0 (Cꢀ4a), 129.4 (Cꢀ5'), 132.0 (Cꢀ6), 141.6 (Cꢀ1'),
48.6 (Cꢀ8a), 153.0 (NHCOOC(CH ) ), 155.0 (CHNHCONHAr), 159.7 (Cꢀ3'). Anal.
1
3 3
(C H N O ) theoretical: C, 65.29; H, 7.08; N, 9.52. Found: C, 65.25; H, 7.13; N, 9.43.
24 31 3 5
R/SꢀNꢀ3ꢀAminomethylphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ
Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea (22)
2
RaneyꢀNickel was added to a solution of 18 (0.3 g, 0.69 mmol) and ammonia (0.3 mL) in ethanol
(10 mL). The solution was stirred in a sealed hydrogenator under a hydrogen pressure of 7 bars.
When the reaction was ended (3 h), the catalyst was filtered off and the filtrate was evaporated
under pressure. The title compound was obtained after purification on a DCVC system: m.p.:
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20ꢀ170 °C; H NMR (DMSOꢀd ) d 1.24 (s, 3H, CH ), 1.35 (s, 3H, CH ), 1.42 (s, 9H,
6 3 3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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4
4
4
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4
4
4
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5
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5
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5
5
5
6
NHCOOC(CH ) ), 1.68 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH), 2.09 (dd, J=13 Hz/6 Hz, 3ꢀH), 3.3 (bs,
3
3
2H, CH NH ), 3.67 (s, 2H, CH NH ), 4.91 (dd, J=16 Hz/10 Hz, 1H, 4ꢀH), 6.47 (d, J=8.7 Hz, 1H,
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
CHNHCONHAr), 6.63 (d, J=8.8 Hz, 1H, 8ꢀH), 6.89 (d, J=7.5 Hz, 1H, 4'ꢀH), 7.17 (m, 2H, 7ꢀH/5'ꢀ
H), 7.31 (d, J=8.2 Hz, 1H, 6'ꢀH), 7.37 (s, 1H, 2'ꢀH), 7.41 (s, 1H, 5ꢀH), 8.46 (s, 1H,
13
CHNHCONHAr), 9.08 (s, 1H, NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.5 (CH ), 28.1
3
3
6
3
(
C(CH ) ), 29.0 (CH ), 40.0 (Cꢀ3), 42.3 (Cꢀ4), 45.7 (CH NH ), 74.8 (Cꢀ2), 78.5 (C(CH ) ), 115.8
3 3 3 2 2 3 3
(Cꢀ6'), 116.5 (Cꢀ2'), 116.7 (Cꢀ8), 117.5 (Cꢀ5), 119.6 (Cꢀ7), 120.0 (Cꢀ4'), 123.0 (Cꢀ4a), 128.4 (Cꢀ
'), 132.0 (Cꢀ6), 140.2 (Cꢀ1'), 144.5 (Cꢀ3'), 148.6 (Cꢀ8a), 153.0 (NHCOOC(CH ) ), 155.1
5
3
3
(
CHNHCONHAr). Anal. (C H N O ) theoretical: C, 65.43; H, 7.32; N, 12.72. Found: C, 64.96;
24 32 4 4
H, 7.31; N, 12.70.
R/SꢀNꢀ3ꢀAminophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (23)
Ammonium chloride (0.15 g, 2.83 mmol) and iron (0.6 g, 10,7 mmol) were added to a solution of
19 (0.3 g, 0.66 mmol) in ethanol/water 3:1 (10 mL). The solution was heated at 80°C for 15 min.
After filtration (without cooling), the filtrate was treated with charcoal, filtrated and evaporated
under vacuum. The title compound was obtained after purification on a DCVC system: m.p.:
1
130ꢀ140 °C; H NMR (DMSOꢀd ) d 1.24 (s, 3H, CH ), 1.35 (s, 3H, CH ), 1.43 (s, 9H,
6
3
3
NHCOOC(CH ) ), 1.65 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH), 2.08 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.89 (dd,
3
3
J=16 Hz/10 Hz, 1H, 4ꢀH), 4.95 (s, 1H, NH ), 6.14 (dd, J=7.9 Hz/1.3 Hz, 1H, 4'ꢀH), 6.35 (d, J=8.7
2
Hz, 1H, CHNHCONHAr), 6.57 (dd, J=8.1 Hz/1 Hz, 1H, 6'ꢀH), 6.63 (d, J=8.8 Hz, 1H, 8ꢀH), 6.72
(t, J=1.9 Hz, 1H, 2'ꢀH), 6.85 (t, J=8 Hz, 1H, 5'ꢀH), 7.17 (d, J=6.6 Hz, 1H, 7ꢀH), 7.39 (s, 1H, 5ꢀH),
13
8
.17 (s, 1H, CHNHCONHAr), 9.10 (s, 1H, NHCOOC(CH3)3). C NMR (DMSOꢀd ) d 24.6
6
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(
CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.0 (Cꢀ3), 42.2 (Cꢀ4), 74.8 (Cꢀ2), 78.5 (C(CH ) ), 103.4 (Cꢀ
3
3 3
3
3 3
2
'), 105.8 (Cꢀ6'), 107.6 (Cꢀ4'), 116.6 (Cꢀ8), 117.7 (Cꢀ5), 119.6 (Cꢀ7), 123.1 (Cꢀ4a), 128.9 (Cꢀ5'),
132.0 (Cꢀ6), 140.9 (Cꢀ1'), 148.6 (Cꢀ8a), 149.0 (Cꢀ3'), 153.0 (NHCOOC(CH ) ), 154.9
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(CHNHCONHAr). Anal. (C H N O ) theoretical: C, 64.77; H, 7.09; N, 13.14. Found: C, 64.32;
23 30 4 4
H, 7.06; N, 12.95.
R/SꢀNꢀ4ꢀNitrophenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (26)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 4ꢀ
1
nitrophenyl isocyanate (0.13 g, 0.82 mmol): m.p.: 233ꢀ233.5 °C; H NMR (DMSOꢀd ) d 1.25 (s,
6
3H, CH ), 1.36 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.75 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH),
3
3
3 3
2
.10 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.94 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6,64 (d, J=8.8 Hz, 1H, 8ꢀH),
.82 (d, J=8.6 Hz, 1H, CHNHCONHAr), 7.18 (d, J=6.5 Hz, 1H, 7ꢀH), 7.40 (s, 1H, 5ꢀH), 7.69 (d,
6
J=7.7 Hz, 2H, 2'ꢀH/6'ꢀH), 8.17 (d, J=9.3 Hz , 2H, 3'ꢀH/5'ꢀH), 9.09 (s, 1H, NHCOOC(CH ) ), 9.32
3
3
13
(
(
(
s, 1H, CHNHCONHAr). C NMR (DMSOꢀd ) d 24.4 (CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.4
6 3 3 3 3
Cꢀ3), 42.5 (Cꢀ4), 74.8 (Cꢀ2), 78.6 (C(CH ) ), 116.7 (Cꢀ8), 117.0 (Cꢀ2'/Cꢀ6'), 117.5 (Cꢀ5), 119.6
3 3
Cꢀ7), 122.5 (Cꢀ4a), 125.1 (Cꢀ3'/Cꢀ5'), 132.1 (Cꢀ6), 140.5 (Cꢀ1'), 147.0 (Cꢀ4'), 148.5 (Cꢀ8a),
1
52.9 (NHCOOC(CH ) ), 154.4 (CHNHCONHAr). Anal. (C H N O ) theoretical: C, 60.52; H,
3 3 23 28 4 6
6.18; N, 12.27. Found: C, 59.95; H, 6.16; N, 12.09.
R/SꢀNꢀ4ꢀTrifluoromethylphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ
2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea (27)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 4ꢀ
1
trifluoromethylphenyl isocyanate (0.15 g, 0.82 mmol): m.p.: 186ꢀ188 °C; H NMR (DMSOꢀd ) d
6
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.25 (s, 3H, CH ), 1.36 (s, 3H, CH ), 1.42 (s, 9H, NHCOOC(CH ) ), 1.72 (dd, J=13 Hz/11 Hz,
3
3
3 3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H, 3ꢀH), 2.09 (dd, J=13 Hz/6 Hz, 3ꢀH), 4.94 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6.63ꢀ6.66 (m, 2H,
CHNHCONHAr / 8ꢀH), 7.18 (d, J=7 Hz, 1H, 7ꢀH), 7.40 (s, 1H, 5ꢀH), 7.59 (d, J=8.8 Hz , 2H, 3'ꢀ
H/5'ꢀH), 7.65 (d, J=8.8 Hz, 1H, 2'ꢀH/6'ꢀH), 8.95 (s, 1H, CHNHCONHAr), 9.09 (s, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.4 (CH ), 28.1 (C(CH ) ), 29.0 (CH ), 40.4 (Cꢀ3),
3 3
6
3
3 3
3
4
2.4 (Cꢀ4), 74.8 (Cꢀ2), 78.5 (C(CH ) ), 116.7 (Cꢀ8), 117.4 (Cꢀ5/Cꢀ2'/Cꢀ6'), 119.6 (Cꢀ7), 121.0ꢀ
3 3
1
21.2 (d, J=32 Hz, Cꢀ4'), 122.8 (Cꢀ4a), 123.5ꢀ125.7 (d, J=271 Hz, CF ), 126.0 (Cꢀ3'/Cꢀ5'), 132.0
3
(Cꢀ6), 144.1 (Cꢀ1'), 148.6 (Cꢀ8a), 153.0 (NHCOOC(CH ) ), 154.8 (CHNHCONHAr). Anal.
3
3
(C H F N O ) theoretical: C, 60.12; H, 5.89; N, 8.76. Found: C, 60.08; H, 5.97; N, 8.72.
24 28 3 3 4
R/SꢀNꢀ4ꢀMethoxyphenylꢀN'ꢀ(6ꢀtertꢀbutoxycarbonylaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀ
benzopyranꢀ4ꢀyl)urea (28)
The title compound was obtained as described for 10, starting from 9 (0.2 g, 0.68 mmol) and 4ꢀ
1
methoxyphenyl isocyanate (0.12 g, 0.82 mmol): m.p.: 204ꢀ205 °C; H NMR (DMSOꢀd ) d 1.24
6
(s, 3H, CH ), 1.35 (s, 3H, CH ), 1.43 (s, 9H, NHCOOC(CH ) ), 1.68 (dd, J=13 Hz/11 Hz, 1H, 3ꢀ
3 3 3 3
H), 2.06 (dd, J=13 Hz/6 Hz, 3ꢀH), 3.70 (s, 3H, OCH ), 4.91 (dd, J=16 Hz/9 Hz, 1H, 4ꢀH), 6.47 (d,
3
J=8.7 Hz, 1H, CHNHCONHAr), 6,62 (d, J=8.8 Hz, 1H, 8ꢀH), 6.83 (d, J=9 Hz , 2H, 3'ꢀH/5'ꢀH),
7
.16 (d, J=7.2 Hz, 7ꢀH), 7.35 (d, J=9 Hz, 2H, 2'ꢀH/6'ꢀH), 7.40 (s, 1H, 5ꢀH), 8.41 (s, 1H,
13
CHNHCONHAr), 9.08 (s, 1H, NHCOOC(CH ) ). C NMR (DMSOꢀd ) d 24.4 (CH ), 28.1
3
3
6
3
(C(CH ) ), 29.1 (CH ), 40.0 (Cꢀ3), 42.3 (Cꢀ4), 55.1 (OCH ), 74.8 (Cꢀ2), 78.5 (C(CH ) ), 113.8
3 3 3 3 3 3
(Cꢀ3'/Cꢀ5'), 116.6 (Cꢀ8), 117.7 (Cꢀ5), 119.5 (Cꢀ2'/Cꢀ6'), 119.6 (Cꢀ7), 123.3 (Cꢀ4a), 132.0 (Cꢀ6),
133.6 (Cꢀ1'), 148.6 (Cꢀ8a), 153.0 (NHCOOC(CH ) ), 154.0 (CHNHCONHAr), 155.4 (Cꢀ4').
3
3
Anal. (C H N O ) theoretical: C, 65.29; H, 7.08; N, 9.52. Found: C, 64.88; H, 7.20; N, 9.13.
2
4
31
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R/SꢀNꢀ3ꢀCyanophenylꢀN'ꢀ(3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea (32)
The title compound was obtained as described for 10, starting from 31 (0.2 g, 1.13 mmol) and 3ꢀ
1
cyanophenyl isocyanate (0.18 g, 1.25 mmol): m.p.: 217ꢀ219 °C; H NMR (DMSOꢀd ) d 1.28 (s,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
H, CH ), 1.39 (s, 3H, CH ), 1.77 (t, J=12 Hz, 1H, 3ꢀH), 2.12 (dd, J=13 Hz/6.2 Hz, 3ꢀH), 4.98
3 3
(
dd, J=16.3 Hz/9.2 Hz, 1H, 4ꢀH), 6.74 (d, J=8.7 Hz, 1H, 8ꢀH), 6.76 (d, J=10 Hz, 1H,
CHNHCONHAr), 6.88 (t, J=7.4 Hz, 1H, 6ꢀH), 7.14 (t, J=7.6 Hz, 1H, 7ꢀH), 7.26 (d, J=7.6 Hz, 1H,
ꢀH), 7.37 (d, J=7.5 Hz, 1H, 4'ꢀH), 7.46 (t, J=8 Hz, 1H, 5'ꢀH), 7.64 (d, J=8.3 Hz, 1H, 6'ꢀH), 8.00
5
1
3
(s, 1H, 2'ꢀH), 8.92 (s, 1H, CHNHCONHAr). C NMR (DMSOꢀd ) d 25.0 (CH ), 29.6 (CH ),
6
3
3
4
0.0 (Cꢀ3), 42.8 (Cꢀ4), 75.6 (Cꢀ2), 117.2 (Cꢀ8), 119.4 (CN), 120.4 (Cꢀ6), 120.8 (Cꢀ2'), 122.9 (Cꢀ
'), 123.7 (Cꢀ4a), 125.1 (Cꢀ4'), 128.0 (Cꢀ5), 129.0 (Cꢀ7), 130.6 (Cꢀ5'), 141.8 (Cꢀ1'), 153.8 (Cꢀ8a),
6
155.5 (CHNHCONHAr). Anal. (C H N O ) theoretical: C, 71.01; H, 5.96; N, 13.08. Found: C,
19
19
3
2
70.90; H, 5.97; N, 12.83.
R/SꢀNꢀ3ꢀCyanophenylꢀN'ꢀ(6ꢀaminoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀyl)urea
33)
(
A suspension of 18 (2.85 g, 6.5 mmol) in a 5N hydrochloric acid ethanolic solution (100 mL) was
heated for 5 min. The mixture was then poured into water (200 mL) The title compound was
precipitated by addition of a 20% aqueous solution of sodium hydroxide until pH=10, filtrated,
1
washed with water and dried: m.p.: 176ꢀ178 °C; H NMR (DMSOꢀd ) d 1.21 (s, 3H, CH ), 1.32
6
3
(
s, 3H, CH ), 1.67 (dd, J=13 Hz/11 Hz, 1H, 3ꢀH), 2.03 (dd, J=13 Hz/6.3 Hz, 3ꢀH), 4.57 (s, 2H,
3
NH ), 4.87 (dd, J=15.9 Hz/9.7 Hz, 1H, 4ꢀH), 6.40 (dd, J=8.5 Hz/2.5 Hz, 1H, 7ꢀH), 6.45 (d, J=8.5
2
Hz, 1H, 8ꢀH), 6.51 (d, J=2.2 Hz, 1H, 5ꢀH), 6.63 (d, J=8.7 Hz, 1H, CHNHCONHAr), 7.36 (d,
J=7.7 Hz, 1H, 4'ꢀH), 7.45 (t, J=8 Hz, 1H, 5'ꢀH), 7.63 (d, J=8.3 Hz, 1H, 6'ꢀH), 8.00 (s, 1H, 2'ꢀH),
1
3
8
.86 (s, 1H, CHNHCONHAr). C NMR (DMSOꢀd ) d 24.8 (CH ), 29.6 (CH ), 40.8 (Cꢀ3), 42.9
6
3
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(Cꢀ4), 74.4 (Cꢀ2), 112.0 (Cꢀ3'), 112.7 (Cꢀ5), 115.7 (Cꢀ7), 117.4 (Cꢀ8), 119.5 (CN), 120.7 (Cꢀ2'),
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
22.8 (Cꢀ6'), 123.4 (Cꢀ4a), 125.1 (Cꢀ4'), 130.6 (Cꢀ5'), 141.8 (Cꢀ6), 142.2 (Cꢀ1'), 144.8 (Cꢀ8a),
155.4 (CHNHCONHAr). Anal. (C H N O ) theoretical: C, 67.84; H, 5.99; N, 16.66. Found: C,
19
20
4
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7.47; H, 6.03; N, 16.53.
R/SꢀNꢀ3ꢀCyanophenylꢀN'ꢀ(6ꢀformamidoꢀ3,4ꢀdihydroꢀ2,2ꢀdimethylꢀ2Hꢀ1ꢀbenzopyranꢀ4ꢀ
yl)urea (34)
A mixture of acetic anhydride (2 mL, 21.2 mmol) and formic acid (1 mL, 26.5 mmol) was heated
at 55°C for 2h. The mixture was then cooled to 0°C and anhydrous THF (2mL) was added. A
solution of 33 (0.3 g, 0.89 mmol) in anhydrous THF was added dropwise. When the reaction was
complete, water was added and the resulting precipitate was filtrated, washed with water and
dried. The title product was obtained from the crude product by DCVC purification: m.p.: 200ꢀ
1
2
02 °C; H NMR (DMSOꢀd ) d 1.26 (s, 3H, CH ), 1.37 (s, 3H, CH ), 1.76 (t, J=12 Hz, 1H, 3ꢀH),
6
3
3
2.09 (dd, J=13 Hz/6.2 Hz, 3ꢀH), 4.96 (dd, J=15.3 Hz/10.3 Hz, 1H, 4ꢀH), 6.70 (d, J=8.7 Hz, 1H, 8ꢀ
H), 6.80 (d, J=8.7 Hz, 1H, CHNHCONHAr), 7.37 (d, J=7.7 Hz, 1H, 4'ꢀH), 7.42ꢀ7.48 (m, 3H, 5ꢀ
H/7ꢀH/5'ꢀH), 7.66 (m, 1H, 6'ꢀH), 8.01 (s, 1H, 2'ꢀH), 8.15 (d, J=2 Hz, 1H, NHCHO), 8.96 (s, 1H,
1
3
CHNHCONHAr), 10.00 (s, 1H, NHCHO). C NMR (DMSOꢀd ) d 24.4 (CH ), 29.1 (CH ), 40.0
6
3
3
(Cꢀ3), 42.3 (Cꢀ4), 75.1 (Cꢀ2), 111.5 (Cꢀ3'), 116.8 (Cꢀ8), 118.3 (Cꢀ5), 119.0 (CN), 120.2 (Cꢀ7),
120.3 (Cꢀ2'), 122.4 (Cꢀ6'), 123.2 (Cꢀ4a), 124.0 (), 124.7 (Cꢀ4'), 130.1 (Cꢀ5'), 131.0 (Cꢀ6), 141.2
(Cꢀ1'), 149.5 (Cꢀ8a), 154.9 (CHNHCONHAr), 158.9 (NHCHO). Anal. (C H N OS) theoretical:
19 20 4
C, 65.92; H, 5.53; N, 15.38. Found: C, 65.43; H, 5.55; N, 15.37.
Docking studies
ACS Paragon Plus Environment
29