Cytotoxic triterpenoid–safirinium conjugates target the endoplasmic reticulum

Article abstract of DOI:10.1016/j.ejmech.2020.112920

Safirinium P and Q fluorescence labels were synthesized and conjugated with spacered triterpenoic acids to access hybrid structures. While the parent safirinium compounds were not cytotoxic at all, many triterpenoid safirinium P and Q conjugates showed moderate cytotoxicity. An exception, however, was safirinium P derived compound 30 holding low EC₅₀ = 5.4 μM (for A375 cells) to EC₅₀ = 7.5 μM (for FaDu cells) as well as EC₅₀ = 6.6 μM for non-malignant fibroblasts NIH 3T3. Fluorescence imaging showed that the safirinium core structures cannot enter the cells (not even after a prolonged incubation time of 24 h), while the conjugates (as exemplified for 30) are accumulating in the endoplasmic reticulum but not in the mitochondria. The development of safirinium–hybrids targeting the endoplasmic reticulum can be regarded as a promising strategy in the development of cytotoxic agents.

Full text of DOI:10.1016/j.ejmech.2020.112920

European Journal of Medicinal Chemistry 209 (2021) 112920
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Cytotoxic triterpenoidesafirinium conjugates target the endoplasmic
reticulum
1
1
*
Oliver Kraft , Marie Kozubek , Sophie Hoenke, Immo Serbian, Daniel Major, Ren eꢀ Csuk
MartineLuthereUniversity HalleeWittenberg, Organic Chemistry, KurteMotheseStr. 2, De06120, Halle, Saale, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 August 2020
Received in revised form
Safirinium P and Q fluorescence labels were synthesized and conjugated with spacered triterpenoic acids
to access hybrid structures. While the parent safirinium compounds were not cytotoxic at all, many
triterpenoid safirinium P and Q conjugates showed moderate cytotoxicity. An exception, however, was
4
October 2020
safirinium P derived compound 30 holding low EC50 ¼ 5.4
m
M (for A375 cells) to EC50 ¼ 7.5
mM (for FaDu
Accepted 4 October 2020
Available online 8 October 2020
cells) as well as EC50 ¼ 6.6 M for non-malignant fibroblasts NIH 3T3. Fluorescence imaging showed that
m
the safirinium core structures cannot enter the cells (not even after a prolonged incubation time of 24 h),
while the conjugates (as exemplified for 30) are accumulating in the endoplasmic reticulum but not in
the mitochondria. The development of safiriniumehybrids targeting the endoplasmic reticulum can be
regarded as a promising strategy in the development of cytotoxic agents.
Keywords:
Betulinic acid
Safirinium
Cytotoxicity
© 2020 Elsevier Masson SAS. All rights reserved.
Endoplasmic reticulum
1. Introduction
on the spacer used, rhodamine Betriterpene derivatives are obvi-
ously only cytotoxic if an “open” (fluorescent) form is present. If
Cancer is still the second most leading cause of death. Especially
some types of cancer come with more aggressive phenotypes than
others like prostate and breast cancer. Furthermore hypoxic tumors
lead to an acidification of the tumor microenvironment, which
further increases resistance, invasion and proliferation of the tumor
rhodamine B, on the other hand, is present in a closed/non-
efluorescent lactone or lactam form, the corresponding conjugates
are practically nonecytotoxic [11]. Conjugates holding the rhoda-
mine B residue directly attached to the basic skeleton of the tri-
terpene (e.g. as an ester) are cytotoxic but usually not very selective
[12,13].
[
1]. Recently, mitochondrial targeted antiecancer drugs („mito-
cans“) have gotten more and more attention as these compounds
trigger cell death signals from the mitochondria, where tumor cells
e even tumor cells not responding to death signals due to DNA
damage e trigger apoptosis [2e9]. This has already been shown for
analogs of vitamin E [2e9] as well as for triterpenoids [10e13] and
steroids [14]. For the latter compounds this was mainly achieved by
attaching lipophilic cations (such as rhodamine B [10e13], a
BODIPYemoiety [15], a triphenylphosphonium residue [16e18], an
ammonium salt [19,20]or derivatives of malachite green [15]) to the
triterpenoid or steroidal skeleton [5,21].
We are particularly interested in the development of cytotoxic
triterpeneeconjugates carrying a permanent positively charged
moiety; these compounds should also enable to act as fluorescence
molecular probe [24,25]. This latter property is also associated with
the ability to observe the uptake and intracellular distribution of
the molecules in a more directly way. Thus, valuable information on
molecular mechanisms can be gained quite easily.
Some time ago, Saczewski et al. succeeded in synthesizing
soecalled „safiriniumehybrids“ by tandem Mannicheelectrophilic
amination reactions starting from proefluorogenic isoxazolinones
and secondary amines, such as quinolones or short peptides. The
former hybrids showed higher lipophilicity and the latter were able
to stain spores of Bacillus subtilis [24e31].
However, the presence of a cation is not sufficient to achieve
good cytotoxicity in tumor cells. For example, distal Neoxides and
quaternary ammonium salts of triterpenoids [19,20] are signifi-
cantly less cytotoxic than, e.g. benzylamides [19e23]. Depending
The latter observation, that safirinium hybrids are able to be
well transported into cells, led us to investigate the cytotoxic po-
tential of safiriniumetriterpene hybrids. Hybridization is often
associated with the ability to counterbalance the known problem
*
Corresponding author.
(efficacy, affinity, solubility, unwanted side effects, …) with the
E-mail address: rene.csuk@chemie.uni-halle.de (R. Csuk).
These authors contributed equally.
1
other hybrid part. Lack of solubility in biological systems is a
https://doi.org/10.1016/j.ejmech.2020.112920
223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
0

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
notorious problem often associated with triterpenes. This problem,
however, might be solved by binding to a cationic residue, such as a
safirinium moiety. Hence, we became interested to deduce whether
triterpeneesafirinium hybrids would also be as efficient as rhoda-
mine B conjugates in triggering cell death.
Betulinic acid (BA, Fig. 1), for example, is a triterpene, that holds
promising in vitro and in vivo antitumor activity in melanoma,
neuroeectodermal tumors and glioma cell lines [32e35]. It targets
the mitochondrial permeability transition pore [36]. It also down-
regulates Bcle2, which promotes apoptosis as well as VEGFR and
NFekB, which are both closely related to angiogenesis and the
proliferation of tumors [37].
obtained after precipitation from acetone in yields ranging between
43% and 91%, except for compound 7 (57%).
The synthesis of the safirinium Q derivatives (Scheme 2) fol-
lowed the previously reported strategy.
As far as the synthesis of acetylated triterpenoic acid holding an
ethylenediamine or a piperazinyl spacer is concerned, the corre-
sponding triterpenoic acid (BA, OA, UA, PA; Fig. 1) was acetylated
yielding compounds 17e20 (Scheme 3); these were activated with
oxalyl chloride followed by the addition of the amine. Thus, com-
pounds 21e28 were obtained in good yields. These spacers were
chosen in analogy to our previous studies on spacered triterpenoid
hybrids [10e14].
Based on this strategy, we set out to synthesize different
triterpenoidesafirinium conjugates, and to examine their cytotox-
icity and selectivity for different human tumor cell lines.
The safirinium P fluorophores were activated with oxalyl chlo-
ride followed by their reaction with piperazinylespacered
3eOeacetylebetulinic acid (21) to yield derivatives 29e31
(Scheme 4).
Albeit all of these reactions proceeded well (as observed by TLC),
2
. Results and discussion
the isolated yields of pure products were low. These compounds
tend to “stick” to the silica gel used for chromatographic purifica-
tion. Even elution with MeOH was incomplete. While upon addi-
tion of AcOH to the eluent reasonably high recovery rates were
obtained, this procedure inevitably led to the formation of impu-
rities in the product. Hence, yield was sacrificed for purity, and
most of the compounds were eluted from the column with MeOH.
The synthesis of the safirinium Q derivatives proceeded in an
analogous manner (Scheme 5) and yielded compounds 32e39,
respectively.
2
.1. Synthesis
The synthesis of the safirinium P derivatives (Scheme 1) was
straightforward and followed the synthetic scheme previously
outlined by Saczewski et al. [30,31] Compound 1 was obtained in an
almost quantitative yield from the reaction of ethyl cyanoacetate
with hydroxylamine following the procedure reported by Lee [38].
Compound 2 is a precursor for the safirinium P fluorophores and
was obtained in 73% yield from the reaction of 1 with acetylacetone
in the presence of piperidine followed by a precipitation of the
product with hydrochloric acid. A major advantage of these re-
actions is that no timeeconsuming purifications are necessary at
all.
As exemplified for the safiriniumebetulinic acid hybrids, their
UV/Vis spectra are characterized by maxima at
l
m
ax ¼ 253e254 nm
(
with log ε ¼ 3.92e4.07) and
l
max ¼ 352e354 nm (with
log ε ¼ 3.62e3.82). These values are in perfect agreement with
previously published data for analogs.
The synthesis of the safirinium P derivatives (Scheme 1) pro-
ceeded also nicely as reported, and compounds 3e13 were
Fig. 1. Structure of triterpenoic acids: BA (betulinic acid), OA (oleanolic acid), UA (ursolic acid), and PA (platanic acid).
2

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
ꢀ
ꢀ
Scheme 1. Synthesis of safirinium P derivatives 3e11: a) 1. acetylacetone, piperidine, H
2
O, 100 C, 15 min; 2. 4M HCl, 73%; b) formaldehyde, corresponding amine, MeOH, 4 h, 45 C,
2
h.
ꢀ ꢀ ꢀ
2
Scheme 2. Synthesis of the safirinium Q derivative 16: a) Ac O, acetic acid, reflux, 1 h, 78%; b) 1. DMF, phosphoryl chloride, inert atmosphere, 0 C / 25 C, 4 h 2. 17, 25 C / reflux.
ꢀ
ꢀ
ꢀ
2
9
4 h, 70%; c) nebutanol, NaH PO , NaClO , H O, r.t, 2 h, 87%; d) 1. HOSA, dry DMF, 25 C, 12 h, 2. TEA, H O, 25 C, 12 h, 81%; e) diethylamine, formaldehyde (aqu., 37%), MeOH, 25 C,
2%.
2
4
2
2
2
2.2. Biological evaluation
cells). An unselective cytotoxicity, however, was also observed for
this compound inasmuch as for nonemalignant fibroblasts (NIH
The compounds were subjected to sulforhodamine B assays to
3T3) an EC50 ¼ 6.6
mM was noted. Most of the other safirinium
determine their cytotoxicity employing several human tumor cell
lines and nonemalignant fibroblasts (NIH 3T3). The results from
these assays are summarized in Table 1.
The results from these assays showed safirinium P derivatives
e11 as not cytotoxic up to a concentration of 30 mM, thus paral-
leling both previous findings as well as the also insignificant
cytotoxicity of rhodamine B (Rho). Extra fluorescence imaging ex-
periments revealed that these compounds are not taken up by the
cells; this explains their nonecytotoxicity. Upon derivatization with
triterpenoids, however, a slight increase in cytotoxicity was
observed. Especially, betulinic acid derived 30 showed good cyto-
hybrids were only of moderate cytotoxicity and selectivity was e by
and large e missing at all. Quite on the contrary, compounds 35 and
38 were even more cytotoxic for the fibroblasts than for the tumor
cells incorporated in this study. For comparison, human
nonemalignant HEK293 were incubated with representative
compounds 30e32 and 36 showing these compounds also to be
cytotoxic for these cells.
3
These results were somewhat surprising inasmuch as we ex-
pected the safirinium conjugates to act likewise the rhodamine B
mitocans of previous studies. Therefore, a closer investigation of 30
was called for. Fluorescence imaging experiments showed 30 not to
enter the mitochondria (within the limits of detection) but to
accumulate in the endoplasmic reticulum (ER). Control
toxicity ranging between EC50 ¼ 4.6
mM (for A2780 ovarian carci-
noma cells) to EC50 ¼ 7.5 M (for HT29 colorectal adenocarcinoma
m
3

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
ꢀ
ꢀ
Scheme 3. Synthesis of compounds 17e28: a) Ac
piperazine, DCM, 0 C / r.t. 2 h, yields: 21 (80%), 22 (86%), 23 (84%), 24 (84%); c) (COCl)
2
O, pyridine, DMAP (cat.), 12 h, yields: 17 (90%), 18 (88%), 19 (84%), 20 (87%) b) 1. (COCl)
2
, DCM, DMF, 0 C / 25 C, 4 h 2.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2
, DCM, DMF, 0 C / 25 C, 4 h 2. ethylenediamine, DCM, 0 C / 25 C, 2 h, yields: 25 (83%),
26 (81%), 27 (84%), 28 (86%).
ꢀ ꢀ ꢀ ꢀ
2 3
Scheme 4. Synthesis of betulinic acid derived safirinium hybrids 29e31: a) 1. (COCl) , DCM, DMF 0 C / 25 C, 2 h, 2. 21, NEt , 0 C / 25 C, yields. 29 (57%), 30 (39%), 31 (40%).
experiments were performed employing an established
EReTracker (for the accumulation of 30 in the ER), and a Mito-
Tracker (to exclude an accumulation in the mitochondria). Repre-
sentative images are depicted in Fig. 2.
EC50 ¼ 5.4
m
M (for A375 cells) to EC50 ¼ 7.5
mM (for FaDu cells).
However, safirinium Q hybrids 36 and 39 were even more cytotoxic
for nonemalignant NIH 3T3 fibroblasts than for the human tumor
cell lines included in this study; the same cytotoxicity was also
observed for representative compounds 30e32 and 36 and
HEK293 cells. Fluorescence imaging showed that the safirinium
core structures 3e11 and 16 cannot enter the cells (not even after a
prolonged incubation time of 24 h), while the conjugates (as
exemplified for 30) are accumulating in the endoplasmic reticulum
but not in the mitochondria. This finding might explain their
diminished cytotoxic activity for human tumor cell lines as
compared to rhodamine B analogs that enter the mitochondria.
While many cytostatics trigger an induced cell death via the
activation of mitochondrial apoptosis, an increasing number of
studies already indicated a pivotal role of drugs targeting the
3
. Conclusion
The syntheses of the safirinium fluorescence labels was
straightforward following known procedures to allow an access to
the safirinium P derivatives 3e11 and safirinium Q derivative 16 in
good to excellent yields. The conjugation reaction with spacered
triterpenoic acid derivatives 21e28 proceeded well applying stan-
dard coupling procedures; main obstacle of this reaction was the
purification of the hybrid compounds due to their high affinity to
silica gel during chromatographic purification. The safirinium
compounds were not cytotoxic at all while many triterpenoid
safirinium P and Q conjugates showed only moderate cytotoxicity.
An exception was safirinium P derived compound 30 holding low
endoplasmic
reticulum.
Further
development
of
the
safiriniumetriterpene hybrids can be seen as an encouraging
strategy in the development of cytotoxic agents.
4

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
ꢀ ꢀ ꢀ ꢀ
2 3
Scheme 5. a) 1. (COCl) , DCM, DMF, 0 C / 25 C, 2 h, 2. NEt , 0 C / 25 C, yields: 32 (57%), 33 (47%), 34 (52%), 35 (44%), 36 (44%), 37 (40%), 38 (45%), 39 (44%).
Table 1
Cytotoxicity of compounds 3e11 and rhodamine B (Rho) (EC50 values in
m
M from SRB assays after 72 h of treatment, the values are averaged from three independent ex-
periments performed each in triplicate, confidence interval CI ¼ 95%; mean ± standard mean error). Human cancer cell lines: A375 (epithelial melanoma), HT29 (colorectal
adenocarcinoma), MCFe7 (breast adenocarcinoma), A2780 (ovarian carcinoma), FaDu (squamous cell carcinoma), nonemalignant: NIH 3T3 (mouse fibroblasts), HEK293
(
human embryonic kidney cells); n.d. not determined; doxorubicin (DX) and staurosporine (ST) were used as positive controls.
A375
HT29
MCFe7
A2780
FaDu
NIH3T3
HEK293
Rho
>30
>30
>30
>30
>30
>30
>30
3
2
3
3
3
3
3
3
3
3
3
3
e11
>30
>30
>30
>30
>30
>30
>30
>30
n.d.
n.d.
3.0 ± 0.3
13.3 ± 0.7
16.5 ± 0.8
n.d.
n.d.
n.d.
13.6 ± 1.0
n.d.
n.d.
9
0
1
2
3
4
5
6
7
8
9
26.2 ± 1.3
5.4 ± 0.1
16.8 ± 1.0
20.8 ± 1.7
21.4 ± 0.7
21.4 ± 1.4
15.0 ± 1.2
18.6 ± 2.1
19.0 ± 2.0
13.8 ± 0.8
16.6 ± 2.1
n.d.
26.3 ± 2.1
4.9 ± 0.3
15.7 ± 1.2
18.1 ± 1.0
19.8 ± 1.2
10.2 ± 1.1
13.2 ± 0.7
17.6 ± 1.5
21.8 ± 1.7
11.7 ± 0.7
15.7 ± 1.4
1.1 ± 0.3
0.1 ± 0.01
21.6 ± 2.4
4.6 ± 0.3
11.0 ± 1.2
14.1 ± 2.0
21.4 ± 1.9
9.0 ± 1.2
10.9 ± 1.8
18.3 ± 2.8
24.2 ± 2.6
11.2 ± 1.4
20.8 ± 1.9
0.01 ± 0.01
0.1 ± 0.01
22.4 ± 2.2
6.6 ± 0.3
>30
>30
18.4 ± 2.0
>30
3.0 ± 0.6
17.4 ± 1.7
28.5 ± 1.1
3.6 ± 1.0
21.3 ± 1.3
0.4 ± 0.07
0.008 ± 0.001
7.5 ± 0.3
24.9 ± 1.7
27.0 ± 2.0
26.2 ± 0.7
>30
22.2 ± 0.9
26.8 ± 0.6
25.0 ± 1.0
22.1 ± 0.8
>30
6.0 ± 0.3
10.7 ± 1.0
28.2 ± 2.3
18.1 ± 1.8
18.3 ± 1.1
13.3 ± 0.9
12.3 ± 1.3
27.1 ± 2.0
10.2 ± 0.1
16.1 ± 1.3
n.d.
n.d.
n.d.
n.d.
DX
ST
0.9 ± 0.01
0.2 ± 0.02
n.d.
0.1 ± 0.05
Fig. 2. Fluorescence imaging of living A375 epithelial carcinoma cells incubated with: compound 30 (left, 12 h, 5.4 mM), EReTracker (center) and merged pictures (right).
4
. Experimental
solvents were dried according to usual procedures. The triterpenoic
acid were obtained from “Betulinines” (St ꢁr íbrn ꢀa Skalice, Czech
1
13
4.1. General
Republic) and used as received. The H and C NMR of all new
compounds have been depicted in the Supplementary Materials file
as well as a description of the SRB assay and the staining
experiments.
Equipment and general methods have been used as previously
described [10e15]. All reactions were performed under an argon;
5

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
4
4
.2. Syntheses
n
¼ 3376w, 3004w, 2947m, 2927m, 2867w, 1667m, 1635s, 1587vs,
1
560s, 1531s, 1485w, 1468m, 1439w, 1433w, 1398m, 1373m, 1356m,
.2.1. 3eAminoeNehydroxye3e(hydroxyimino)propanamide (1)
Following the procedure as described by Lee [38], a mixture of
1302w, 1225w, 1211w, 1187m, 1143m, 1055w, 1042w, 1032w, 802m,
729m, 616m, 605 s cm ; H NMR (500 MHz, CD
ꢂ1
1
3
OD):
d
¼ 5.99 (d,
ethyl cyanoacetate (10.0 g, 88.4 mmol) and hydroxylamine (50% in
water, 12.9 g, 194 mmol, 2.2 eq) in EtOH (10 ml) was stirred at room
temperature for 30 min. External cooling was necessary to keep the
J ¼ 1.3 Hz, 1H, 6eH), 5.71 (s, 2H, 3eH), 3.53 (dddd, J ¼ 31.0, 12.6,
10.7, 5.9 Hz, 4H, 13eH þ 13eH’), 2.30 (d, J ¼ 1.0 Hz, 3H, 11eH), 2.20
(s, 3H, 10eH), 1.86e1.75 (m, 4H, 14eH þ 14‘eH), 1.41e1.30 (m, 12H,
15e17eH þ 15‘e17‘eH), 0.91 (t, J ¼ 6.9 Hz, 6H,18eH þ 18‘eH) ppm;
ꢀ
temperature constant. Upon cooling to 10 C, crystals formed and
13
were collected by filtration, washed with ice cold ethanol and dried
in high vacuum to afford 1 as a colorless solid being used without
further purification.
C NMR (125 MHz, CD
3
OD):
d
¼ 170.1 (C-12), 155.7 (C-5), 146.3 (C-
7), 139.1 (C-9), 118.1 (C-8), 110.1 (C-6), 72.9 (C-3), 66.3 (C-13 þ C-
13‘), 31.1 (C-16 þ C-16‘), 25.6 (C-15 þ C-15‘), 22.2 (C-17 þ C-17‘),
2
2.1 (C-14 þ C-14‘), 18.4 (C-10), 16.8 (C-11), 12.9 (C-18 þ C-18‘) ppm;
þ
þ
4
.2.2. 4,6eDimethylisoxazolo[3,4eb]pyridine3(1H)eone (2)
A solution of 1 (20.0 g, 0.15 mol), acetylacetone (15.5 mL,
.15 mol) and piperidine (14.8 mL, 0.15 mol) in water (250 mL) was
MS (ESI, MeOH): m/z (%) ¼ 362.3 ([MþH] , 100%), 384.2 ([MþNa] ,
8%); analysis calcd for C21 (361.53): C 69.77, H 9.76, N 11.62;
found: C 69.50, H 9.96, N 11.57.
35 3 2
H N O
0
heated under reflux for 30 min [15]. After cooling to room tem-
perature, the reaction mixture was acidified with 2 HCl, and the
M
4.2.7. 5,7eDimethyle2,2edioctyle2,3edihydroe1heimidazol
resultant yellow precipitate was filtered off and washed with water
[1,2ea]pyridine4eiume8ecarboxylate (6)
ꢀ
ꢀ
(
2
2 ꢁ 50 mL); yield 17.97 g (73%); m.p. 203 C (lit [31].: m.p.
Following GPA; yield: 83%; m.p. 131 C; IR (ATR):
n
¼ 3386w,
ꢀ
04e207 C);
3002w, 2952m, 2926m, 2854m, 1664m, 1635s, 1587vs, 1560s,
530s, 1484m, 1468m, 1432w, 1398m, 1375m, 1354m, 1306w,
1
ꢂ1
1
4.2.3. General procedure (GPA) for the preparation of safirinium P
1218w, 1184m, 1043w, 1032w, 802m, 725m, 614m, 605 m cm ; H
NMR (500 MHz, CD OD):
derivatives 3e11
3
d
¼ 5.99 (d, J ¼ 1.1 Hz, 1H, 6eH), 5.70 (s,
A solution of 2 (2.0 g, 12.2 mmol), the corresponding amine
2H, 3eH), 3.61e3.43 (m, 4H, 13eH þ 13‘eH), 2.30 (d, J ¼ 1.0 Hz, 3H,
11eH), 2.20 (s, 3H, 10eH), 1.84e1.76 (m, 4H, 14eH þ 14‘eH),
1.40e1.25 (m, 21H, 15eH þ 15‘eH þ 16eH þ 16‘eH þ 17eH þ
17‘eH þ 18eH þ 18‘eH þ 19eH þ 19‘eH), 0.93e0.87 (m, 6H,
(
13.4 mmol) and 37% aqueous formaldehyde (1.0 mL, 13.4 mmol) in
ꢀ
MeOH (40 mL) was heated at 40 C for 3 h. The resulting reaction
mixture was concentrated under reduced pressure, and the solid
residue was precipitated from acetone.
13
20eH þ 20‘eH) ppm; C NMR (125 MHz, CD
3
OD)
d
¼ 170.1 (C-12),
1
55.7 (C-5), 146.3 (ꢂ7), 139.1 (C-9), 118.1 (C-8), 110.1 (C-6), 72.9 (C-
4.2.4. 2,2,5,7eTetramethyle2,3edihydroe1heimidazol[1,2ea]
3), 66.3 (C-13 þ C-13‘), 31.5 (C-16 þ C-16‘), 28.9 (C-18 þ C-18‘ þ C-
15 þ C-15‘), 26.0 (C-17 þ C-17‘), 22.2 (C-19 þ C-19‘ þ C-14 þ C-14‘),
18.4 (C-10), 16.8 (C-11), 13.0 (C-20 þ C-20‘) ppm; MS (ESI, MeOH):
pyridine4eiume8ecarboxylate (3)
ꢀ
Following GPA; yield: 89%; m.p. 227e230
C (lit [29].:
ꢀ
þ
þ
2
1
28e230 C); IR (ATR):
n
¼ 3031w, 2964w, 2882w, 1635s, 1594vs,
m/z (%) ¼ 418.4 ([MþH] , 100%), 440.2 ([MþNa] , 6%); analysis
549vs,1519vs,1499m, 1459m, 1401w, 1382s, 1371m, 1344s, 1288w,
calcd for C25
43 3 2
H N O (417.64): C 71.90, H 10.38, N 10.06; found: C
1
7
241w, 1228w, 1213w, 1181m, 1119w, 1068w, 1047w, 1034w, 798s,
71.64, H 10.60, N 9.84.
ꢂ1
1
06m, 611s, 604 s cm ; H NMR (500 MHz, CD
3
OD):
d
¼ 6.02 (d,
0
J ¼ 1.2 Hz, 1H, 6eH), 5.76 (s, 2H, 3eH), 3.43 (s, 6H, 13eH þ 13 eH),
4.2.8. 2,2eDidodecyle5,7edimethyle2,3edihydroe1heimidazol
[1,2ea]pyridine4eiume8ecarboxylate (7)
13
2
.30 (d, J ¼ 1.0 Hz, 3H, 11eH), 2.21 (s, 3H, 10eH) ppm; C NMR
(
(
1
125 MHz, CD OD):
3
d
¼ 170.0 (C-12), 155.5 (C-5), 146.7 (C-7), 139.5
Following GPA but precipitation from diethyl ether; yield: 57%;
ꢀ
C-9), 118.0 (C-8), 110.4 (C-6), 77.3 (C-3), 55.6 (C-13 þ C-13‘), 18.5 (C-
m.p. 142e145 C; IR (ATR):
n
¼ 3362w, 2956m, 2917vs, 2874w,
þ
0), 16.9 (C-11) ppm; MS (ESI, MeOH): m/z (%) ¼ 222.1 ([MþH] ,
2849s, 1635s, 1583vs, 1485w, 1466m, 1446m, 1435w, 1403m, 1375s,
1347m, 1235w, 1192m, 809m, 720m, 631m, 601 s cm
þ
ꢂ1 1
1
00%), 244.1 ([MþNa] , 18%); analysis calcd for C11
15
H N
3
O
2
; H NMR
(
221.26): C 59.21, H 6.83, N 18.99; found: C 58.97, H 7.01, N 18.77.
(400 MHz, CD OD):
3
d
¼ 5.98 (d, J ¼ 1.2 Hz, 1H, 6eH), 5.70 (s, 2H,
3
eH), 3.63e3.42 (m, 4H, 13eH þ 13‘eH), 2.29 (d, J ¼ 1.0 Hz, 3H,
4.2.5. 2,2eDiethyle5,7edimethyle2,3e1heimidazol[1,2ea]
11eH), 2.20 (s, 3H, 10eH), 1.85e1.74 (m, 4H, 14eH þ 14‘eH),
pyridine4eiume8ecarboxylate (4)
1.40e1.25 (m, 36H, 15e23eH þ 15‘e23‘eH), 0.88 (t, J ¼ 7.0z Hz, 6H,
ꢀ
13
Following GPA; yield: 91%; m.p. 189e191
C
(lit [30].:
24eH þ 24‘eH) ppm; C NMR (101 MHz, CD
3
OD):
d
¼ 170.1 (C-12),
ꢀ
2
00e203 C); IR (ATR):
n
¼ 3367w, 3201w, 2973w, 1632s, 1586vs,
155.6 (C-5), 146.3 (C-7), 139.1 (C-9), 118.1 (C-8), 110.1 (C-6), 73.0 (C-
3), 66.2 (C-13 þ C-13‘), 31.7 (C-22 þ C-22‘), 29.4e28.9 (C-
16eCe21 þ C-16‘e C-21‘), 26.0 (C-15 þ C-15‘), 22.3 (C-23 þ C-23‘),
22.2 (C-14 þ C-14‘), 18.4 (C-10), 16.9 (C-11), 13.0 (C-24 þ C-24‘)
1
1
555vs, 1528s, 1456m, 1438m, 1431m, 1407m, 1398m, 1383s, 1373s,
353s, 1305m, 1247w, 1225m, 1193m, 1185m, 1166m, 1144w, 1121w,
1113w, 1073w, 1043w, 1031m, 802s, 784m, 752m, 687s, 669m,
ꢂ1
1
þ
6
18vs, 606vs, 577s, 561s, 530m, 510 s cm
OD):
¼ 5.99 (d, J ¼ 1.1 Hz,1H, 6eH), 5.70 (s, 2H, 3eH), 3.62 (qq,
J ¼ 12.8, 7.1 Hz, 4H, 13eH þ 13‘eH), 2.31 (d, J ¼ 1.0 Hz, 3H, 11eH),
;
H NMR (400 MHz,
ppm; MS (ESI, MeOH): m/z (%) ¼ 530.4 ([MþH] , 100%), 552.3
þ
CD
3
d
([MþNa] , 2%); analysis calcd for C33
59 3 2
H N O (529.85): C 74.81, H
11.22, N 7.93; found: C 74.73, H 11.02, N 7.68.
13
2
.20 (s, 3H, 10eH), 1.38 (t, J ¼ 7.1 Hz, 6H, 14eH þ 14‘eH) ppm;
NMR (101 MHz, CD OD):
¼ 170.2 (C-12), 155.9 (C-5), 146.2 (C-7),
39.1 (C-9), 118.2 (C-8), 110.1 (C-6), 71.9 (C-3), 61.2 (C-13 þ C-13‘),
8.4 (C-10), 16.8 (C-11), 7.0 (C-14 þ C-14‘) ppm; MS (ESI, MeOH): m/
C
3
d
4.2.9. 5’,7’eDimethyle3’Hespiro[pyrrolidinee1,2’e[1,2,4]triazolo
[4,3ea]pyridin]e1eiume8 ecarboxylate (8)
0
1
1
ꢀ
Following GPA; yield: 76%; m.p. 141e144 C; IR (ATR):
þ
þ
z (%) ¼ 250.1 ([MþH] , 100%), 272.0 ([MþNa] , 4%); analysis calcd
n
¼ 3386w, 3024w, 2963w, 1634m, 1591vs, 1558s, 1520m, 1478w,
for C13 (249.31): C 62.63, H 7.68, N 16.85; found: C 62.47, H
19 3 2
H N O
1457w, 1434m, 1376s, 1345m, 1228w, 1188w, 1051w, 798m, 779m,
605m, 595 m cm
ꢂ1
1
7.90, N 16.52.
;
H NMR (500 MHz, CD
3
OD):
d
¼ 5.99 (d,
J ¼ 1.2 Hz, 1H, 6eH), 5.83 (s, 2H, 3eH), 3.88e3.75 (m, 4H,
4.2.6. 2,2eDihexyle5,7edimethyle2,3edihydroe1heimidazol
13eH þ 13‘eH), 2.40e2.32 (m, 2H, 14eH), 2.30 (d, J ¼ 1.0 Hz, 3H,
13
[1,2ea]pyridine4eiume8ecarboxylate (5)
11eH), 2.24e2.20 (m, 2H, 14‘eH), 2.20 (s, 3H, 10eH) ppm; C NMR
ꢀ
Following GPA; yield: 82%; m.p. 134e135 C; IR (ATR):
(125 MHz, CD
3
OD):
d
¼ 170.2 (C-12), 155.5 (C-5), 146.3 (C-7), 139.4
6

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
(
1
C-9), 118.0 (C-8), 110.1 (C-6), 74.6 (C-3), 68.2 (C-13 þ C-13’), 21.4 (C-
4 þ C-14‘), 18.4 (C-10), 16.9 (C-11) ppm; MS (ESI, MeOH): m/z
4.2.14. 2eChloroe3equinolinecarboxaldehyde (13)
To a mixture of N, Nedimethylformaldehyde (9.6 mL, 0.125 mol)
and phosphoryl chloride (32 mL, 0.35 mol) at 0 C, 1 (6.8 g,
þ
þ
ꢀ
(
C
7
%) ¼ 248.2 ([MþH] , 100%), 270.1 ([MþNa] , 4%); analysis calcd for
(247.30): C 63.14, H 6.93, N 16.99; found: C 62.85, H
.14, N 16.73.
13
H
17
N
3
O
2
0.05 mol) was added, and the resulting mixture was heated under
reflux for 24 h. Usual aqueous workup gave a paleeyellow pre-
cipitate, that was filtered off, washed with water (2 ꢁ 50 mL) and
dried to afford 13 (6.7 g, 70%) as a pale yellow solid; m.p.
4
.2.10. 5’,7’eDimethyle3’Hespiro[piperidinee1,2’e[1,2,4]triazolo
0
ꢀ
ꢀ
[4,3ea]pyridin]e1eiume8 ecarboxylate (9)
152e154 C (lit [40].: 150e151 C); R
formic acid/neheptane, 80:26:5:10); IR (ATR):
2941vw, 2873w, 1687vs, 1579m, 1046s, 761s, 487s cme1; UVeVis
F
¼ 0.69 (toluene/ethyl acetate/
ꢀ
Following GPA; yield: 82%; m.p. 167e171 C; IR (ATR):
¼ 3383m, 3003w, 2957w, 2858w, 1634s, 1580s, 1564vs, 1558vs,
525s, 1482w, 1450m, 1436m, 1381m, 1372s, 1350m, 1305w, 1277w,
n
¼ 3044vw,
n
1
1
1
5
(MeOH):
(400 MHz, CDCl
J ¼ 8.5 Hz, 1H, 5eH), 7.98 (d, J ¼ 8.2 Hz, 1H, 8eH), 7.93e7.84 (m, 1H,
l
max (log ε) ¼ 234 (5.50), 319 (4.53) nm; H NMR
266w, 1232w, 1193m, 1170w, 1029w, 805m, 697m, 667m, 622m,
3
):
d
¼ 10.56 (s, 1H, 9‘eH), 8.75 (s, 1H, 4eH), 8.07 (d,
ꢂ1
1
95s, 478 m cm
;
H NMR (500 MHz, CD
3
OD):
d
¼ 5.98 (d,
13
J ¼ 1.2 Hz, 1H, 6eH), 5.71 (s, 2H, 3eH), 3.63 (dddd, J ¼ 35.4, 12.2, 7.8,
.7 Hz, 4H,13eH þ 13‘eH), 2.30 (d, J ¼ 1.0 Hz, 3H,11eH), 2.20 (s, 3H,
0eH), 2.19e2.14 (m, 2H, H-14), 1.91e1.83 (m, 2H, H-14‘), 1.73e1.67
7eH), 7.69e7.61 (m, 1H, 6eH) ppm; C NMR (100 MHz, CDCl
¼ 150.3 (C-2), 149.8 (Ce8a), 140.4 (C-4), 133.7 (C-7), 129.9 (C-8),
128.8 (C-5), 128.3 (C-6), 126.7 (Ce4a), 126.6 (C-3) ppm; MS (ESI,
3
):
3
1
(
1
3
d
1
3
þ
þ
m, 2H, H-15) ppm; C NMR (125 MHz, CD
3
OD):
d
¼ 170.2 (C-12),
MeOH): m/z (%) 192.1 ([MþH] , 28%), 194.1 ([MþH] , 10%), 224.1
þ
þ
55.3 (C-5), 146.3 (C-7), 139.5 (C-9), 118.3 (C-8), 110.1 (C-6), 75.0 (C-
([M þ H þ MeOH] , 100%), 226.1 ([M þ H þ MeOH] , 34%).
), 65.2 (C-13), 20.8 (C-14), 20.4 (C-15),18.4 (C-10),16.8 (C-11) ppm;
þ
þ
MS (ESI, MeOH): m/z (%) ¼ 262.2 ([MþH] , 100%), 284.1 ([MþNa] ,
%); analysis calcd for C14 (261.33): C 64.35, H 7.33, N 16.08;
found: C 64.17, H 7.51, N 15.88.
4.2.15. Chloroquinolinee3ecarboxylic acid (14)
To a solution of 2 (0.9 g, 4.68 mmol) in nebutanol (90 mL), a
solution of sodium dihydrogen orthophosphate (5.56 g,
4
H
19
N
3
O
2
3
5.61 mmol) and sodium chlorite (4.29 g, 47.47 mmol) in water
ꢀ
4.2.11. 5’,7’eDimethyle3’Hespiro[morpholinee4,2’e[1,2,4]triazolo
(40 mL) was added, and the mixture was stirred at 25 C for 2 h. The
mixture was concentrated under reduced pressure, diluted with
water (70 mL), and ethyl acetate (50 mL) and sodium carbonate
(5.0 g) were added. The aqueous layer was separated, acidified with
[
4,3ea]pyridin]e4eiume8’ecarboxylate (10)
ꢀ
Following GPA; yield: 71%; m.p. 166e168 C; IR (ATR):
¼ 3350w, 3267w, 2982w, 1632m, 1587vs, 1556s, 1514s, 1476m,
452m, 1437m, 1427m, 1418m, 1404w, 1375s, 1348s, 1320w, 1311w,
n
1
2M HCl and 3 (0.85 g, 87%) was obtained as an offewhite solid; m.p.
ꢀ
ꢀ
1
1
274w, 1250w, 1233w, 1222w, 1195s, 1138w, 1120m, 1109m, 1089w,
073m, 845m, 803m, 775m, 689m, 659m, 602s, 559m, 530m,
192e194 C (lit [41].: 200 C decomp.); IR (ATR):
n
¼ 2940w, 1729m,
1566m, 1449m, 1190s, 1023m, 789s, 747vs, 572w cme1; UVeVis
ꢂ1
1
1
513 m cm ; H NMR (500 MHz, CD
3
OD):
d
¼ 6.04 (d, J ¼ 1.2 Hz, 1H,
(DMSO):
l
max (log ε) ¼ 258 (4.83), 287 (4.82) nm; H NMR
6
3
2
2
eH), 5.79 (s, 2H, 3eH), 4.30 (ddd, J ¼ 13.1, 8.9, 2.6 Hz, 2H, 14eH),
(400 MHz, DMSOed
6
):
d
¼ 13.76 (s, 1H, OH), 8.93 (s, 1H, 4eH), 8.17
.93 (dt, J ¼ 13.2, 3.7 Hz, 2H, 14‘eH), 3.78 (ddd, J ¼ 12.3, 8.9, 3.3 Hz,
H, 13eH), 3.69e3.61 (m, 2H, 13‘eH), 2.31 (d, J ¼ 1.0 Hz, 3H, 11eH),
(d, J ¼ 8.2 Hz, 1H, 8eH), 8.00 (d, J ¼ 8.4 Hz, 1H, 5eH), 7.92 (ddd,
J ¼ 8.4, 6.8, 1.5 Hz, 1H, 7eH), 7.72 (ddd, J ¼ 8.1, 6.8, 1.3 Hz, 1H, 6eH)
13
13
.21 (s, 3H, 10eH) ppm; C NMR (125 MHz, CD
3
OD):
d
¼ 170.1 (C-
ppm; C NMR (100 MHz, DMSOed
6
):
d
¼ 165.7 (C-9), 147.3 (Ce8a),
12),155.6 (C-5), 146.8 (C-7), 139.5 (C-9), 118.3 (C-8),110.5 (C-6), 64.0
146.4 (C-2), 141.2 (C-4), 132.7 (C-5), 128.9 (C-7), 128.0 (C-6), 127.6
(
(
C-14 þ C-14‘), 61.6 (C-13 þ C-13‘), 18.4 (C-10), 16.8 (C-11) ppm; MS
(C-8), 125.9 (Ce4a), 125.4 (C-3) ppm; MS (ESI, MeOH): m/z (%) 208.1
([MþH] , 100%), 210.1 [MþH] , 34%).
þ
þ
þ
þ
ESI, MeOH): m/z (%) ¼ 264.2 ([MþH] , 100%), 286.1 ([MþNa] , 8%);
(263.30): C 59.30, H 6.51, N 15.96;
found: C 59.12, H 6.70, N 15.73.
17 3 3
analysis calcd for C13H N O
4.2.16. Isoxazolo[3,4eb]quinoline3(1H)eone (15)
A
solution of 3 (1000 mg, 4.8 mmol) and hydrox-
4
.2.12. 4,5’,7’eTrimethyle3’Hespiro[piperazinee1,2’e[1,2,4]
yamineeOesulfonic acid (2172 mg, 19.2 mmol) in dry DMF (5 mL)
was stirred at 25 C for 12 h. The solid was filtered off, washed with
ꢀ
triazolo[4,3ea]pyridin]e1eiume8’ecarboxylate (11)
ꢀ
Following GPA; yield: 43%; m.p. 187e191 C; IR (ATR):
¼ 3358m, 3175w, 2996w, 2954w, 2942w, 2855w, 2811w, 2785w,
634s, 1586vs, 1561vs, 1531s, 1456m, 1449m, 1433m, 1407m, 1379s,
water (3 ꢁ 3 mL), and triethylamine (2.1 mL, 15.0 mmol) and water
ꢀ
n
(10 mL) were added. After stirring at 25 C for 12 h, the precipitate
1
was filtered off and washed with water (3 ꢁ 3 mL) to yield 4
ꢀ
1355s, 1335m, 1314w, 1291m, 1260w, 1233w, 1222m, 1191s, 1171w,
(726 mg, 81%) as a red solid; m.p. 247e250 C (lit [30].:
ꢀ
1152m, 1129m, 1115w, 1098w, 1078w, 970m, 805m, 790s, 779m,
261e264 C); IR (ATR):
n
¼ 2899w, 1725m, 1566m, 1449m, 1250m,
ꢂ1
1
ꢂ1;
UVeVis (DMSO):
6
CD
3
2
95m, 660m, 605m, 597s, 562 m cm
OD):
;
H NMR (500 MHz,
1190s, 1022s, 941w, 788vs, 747vs 572 m cm
1
3
d
¼ 6.01 (d, J ¼ 1.1 Hz, 1H, 6eH), 5.77 (s, 2H, 3eH),
l
max (log ε) ¼ 259 (4.65), 313 (4.93), 482 (4.87) nm; H NMR
.79e3.72 (m, 2H, 13eH), 3.72e3.65 (m, 2H, 13’eH), 3.10e3.02 (m,
(400 MHz, DMSOed ):
6
d
¼ 12.55 (s, 1H, NH), 8.71 (s, 1H, 4eH), 7.89
0
H, 14eH), 2.81e2.71 (m, 2H, 14 eH), 2.39 (s, 3H, 15eH), 2.31 (d,
(dd, J ¼ 8.0, 1.3 Hz, 1H, 5eH), 7.70 (ddd, J ¼ 8.6, 7.2, 1.5 Hz, 1H, 8eH),
13
13
J ¼ 1.0 Hz, 3H, 11eH), 2.20 (s, 3H, 10eH) ppm; C NMR (125 MHz,
CD OD):
¼ 170.1 (C-12), 155.5 (C-5), 146.5 (C-7), 139.5 (C-9), 118.3
C-8), 110.3 (C-6), 64.0 (C-13 þ C-13‘), 49.0 (C-14 þ C-14‘), 43.9 (C-
5), 18.4 (C-10), 16.9 (C-11) ppm; MS (ESI, MeOH): m/z (%) ¼ 277.2
7.39 (d, J ¼ 8.4 Hz, 1H, 7eH), 7.28e7.20 (m, 1H, 6eH) ppm; C NMR
3
d
(100 MHz, DMSOed
6
):
d
¼ 168.6 (C-9),155.2 (C-2),143.3 (C-4),141.0
(
1
(Ce8a), 135.6 (C-8), 132.2 (C-5), 122.7 (C-6), 119.7 (Ce4a), 116.8 (C-
þ
7), 112.5 (C-3) ppm; MS (ESI, MeOH): m/z (%) 187.1 ([MþH] , 100%);
þ
þ
(
C
7
[MþH] , 100%), 299.1 ([MþNa] , 4%); analysis calcd for
(276.34): C 60.85, H 7.30, N 20.28; found: C 60.61, H
.43, N 20.11.
6 2 2
analysis calcd for C10H N O (186.17): C 64.52, H 3.25, N 15.05;
14
20
H N
4
O
2
found: C 64.31, H 3.48, N 14.81.
4.2.17. 2,2eDiethyle1,2edihydroe[1,2,4]triazolo[4,3ea]
4.2.13. Acetanilide (12)
quinoline2eium 4ecarboxylate (16)
Acetanilide (12) was prepared by usual acetylation of aniline
A solution of 4 (514 mg, 2.76 mmol), diethylamine (0.31 mL,
3.00 mmol) and formaldehyde (37%, 0.22 mL, 2.76 mmol) in MeOH
(10 mL) was stirred at 25 C for 8 h (during this timr, the color of the
ꢀ
with acetic anhydride in 78% yield; m.p. 116e118 C (lit [39].:
ꢀ
þ
ꢀ
1
(
16e117 C); MS (ESI, MeOH): m/z (%) 136.1 ([MþH] , 100%), 158.0
þ
[MþNa] , 48%).
mixture turned from red to slightly yellowegreen). The solvent was
7

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
removed under reduced pressure, and the solid was washed with
acetone (3 ꢁ 2 mL) to yield 16 (780 mg, 92%; m.p. 202e204 C (lit
4.2.27. (3
b
) 3eOeacetyleoleanolic acid 2eaminoethylamide (26)
ꢀ
Reaction of 18 with oxalyl chloride followed by the addition of
ethylenediamine as previously reported gave 26 (81%) as an
offewhite solid; m.p. 190e193 C (lit [15].: 190.5 C).
ꢀ
[
1
l
30].: 202e208 C); IR (ATR):
n
¼ 2961w, 1751m, 1663s, 1614m,
ꢂ1
ꢀ
ꢀ
487m, 1321s, 1052s, 778vs, 603s, 460vs cm ; UVeVis (DMSO):
1
max (log ε) ¼ 257 (5.50), 364 (4.94) nm; H NMR (400 MHz,
DMSOed ):
(
5
6
d
¼ 7.90 (s,1H, 4eH), 7.74e7.68 (m,1H, 5eH), 7.60e7.51
4.2.28. (3 ) 3eOeacetyleursolic acid 2eaminoethylamide (27)
Reaction of 19 with oxalyl chloride followed by the addition of
ethylenediamine as previously reported gave 27 (84%) as an
b
m, 1H, 7eH), 7.26e7.18 (m, 1H, 6eH), 7.09 (d, J ¼ 8.2 Hz, 1H, 8eH),
.77 (s, 2H, 10eH), 3.78e3.58 (m, 4H, 13eH þ 13’eH), 1.27 (t,
0
13
ꢀ
ꢀ
J ¼ 7.1 Hz, 5H,14eH þ 14 eH) ppm; C NMR (100 MHz, DMSOed
¼ 164.0 (C-9), 155.5 (C-2), 137.4 (C-4), 134.1 (Ce8a), 131.1 (C-7),
29.4 (C-5), 127.1 (Ce4a), 123.2 (C-6), 121.8 (C-3), 114.0 (C-8), 71.1
6
):
offewhite solid; m.p. 196e198 C (lit [15].: 198 C).
d
1
(
4.2.29. (3 ) 3eOeacetyleplatanic acid 2eaminoethylamide (28)
Reaction of 20 (1.68 g, 3.4 mmol) with oxalyl chloride followed
by the addition of ethylenediamine as previously reported gave 28
(86%) as an offewhite solid; m.p. 230e233
230e234 C).
b
0
0
C-10), 60.7 (C-13 þ C-13 ), 8.3 (C-14 þ C-14 ) ppm; MS (ESI,
þ
MeOH): m/z (%) 272.13 ([MþH] , 100%); analysis calcd for
ꢀ
C
15
H
17
N
3
O
2
(271.32): C 66.40, H 6.32, N 15.49; found: C 66.19, H
C (lit [44].:
ꢀ
6
.50, N 15.25.
4
.2.18. (3
b
) 3eOeacetylebetulinic acid (17)
4.2.30. General procedure (GPB) for the synthesis of the conjugates
The safirinium dye (1.9 mmol) in dry DCM (20 mL) was activated
with oxalyl chloride (0.4 mL, 4.7 mmol) followed by the addition of
the corresponding acetylated triterpenoid amide (1.9 mmol) and
Acetylation of betulinic acid (BA) 20.0 g, 43.7 mmol) with acetic
anhydride in the presence of DMAP (cat.) as previously reported
ꢀ
[
34] gave 17 (90%) as a colorless solid; m.p. 285e290 C (lit [42].:
ꢀ
ꢀ
2
87e289 C).
triethylamine (0.56 mL, 4.0 mmol). After stirring at 25 C for 12 h
followed by usual aqueous workup and column chromatography
(
silica gel, CHCl
3
/MeOH / MeOH) the conjugates were obtained.
4
.2.19. (3
Acetylation of oleanolic acid (OA), with acetic anhydride in the
presence of DMAP (cat.) as previously reported [15] gave 18 (88%)
b) 3eOeacetyleoleanolic acid (18)
4
.2.31. (3 ) 3eAcetyloxye28e{4e
b
ꢀ
ꢀ
[(2,2ediethyle5,7edimethyle2,3edihydro[1,2,4]triazolo[4,3ea]
pyridine2eiume8eyl)carbonyl]e1epiperazinyl}e
as a colorless solid; m.p. 265e267 C (lit [43].: 264e265 C).
2
8eoxolupe20(29)eene chloride (29)
4
.2.20. (3
Acetylation of ursolic acid (UA) with acetic anhydride in the
presence of DMAP (cat.) as previously reported gave 19 (84%) as a
b) 3eOeacetyleursolic acid (19)
Following GPB 29 (0.42 g, 57%) was obtained as a pale yellow
ꢀ
solid; m.p. 212 C; R
n
F
¼ 0.3 (silica gel, CHCl
3
/MeOH, 9:1); IR (ATR):
ꢀ
ꢀ
¼ 2943m, 2867m, 1729m, 1632s, 1566m, 1535m, 1447m, 1392m,
colorless solid; m.p. 280e283 C (lit [15].: 281e283 C).
1
371m, 1315w, 1282m, 1245s, 1188m, 1107m, 1065m, 1027m, 999s;
1
H NMR (500 MHz, CD
3
OD):
H, 42eH), 4.75 (s, 1H, 29eH
H, 3eH), 3.84e3.73 (m, 5H, 49eHa,b þ 49‘Ha,b þ 12eH
d
¼ 6.22 (s,1H, 45eH), 5.93e5.89 (m,
), 4.64 (s, 1H, 29eH ), 4.50e4.47 (m,
) 3.01e2.89
4
.2.21. 4.2.19 (3
Acetylation of platanic acid (PA) with acetic anhydride in the
presence of DMAP (cat.) as previously reported gave 20 (87%) as a
b) 3eOeacetyleplatanic acid (20)
2
1
a
b
b
(
1
3
1
1
6
1
(
(
m, 2H, 19eH þ 13eH), 2.46 (s, 3H, 48eH), 2.26e2.23 (m, 1H,
6eH ), 2.22 (s, 3H, 47eH), 2.12e2.09 (m, 1H, 22eH ), 2.07 (s, 3H,
2eH), 1.92e1.84 (m, 2H, 21eH), 1.81e1.77 (m, 2H, 1eH ),
þ 12eH
.75 (s, 3H, 30eH) 1.73e1.66 (m, 4H, 18eH þ 16eH þ 2eH),
.61e1.53 (m, 2H, 6eH 22eH ), 1.51e1.36 (m, 12H,
eH þ 7eH þ H-15eH þ 9eH þ 50eH þ 50‘eH),
þ 11eH
.34e1.25 (m, 2H, 11eH ), 1.07 (s, 3H, 27eH), 1.07e1.03
þ 15eH
m, 2H, 12eH ), 1.01 (s, 3H, 26eH), 0.95 (s, 3H, 25eH), 0.91
þ 1eH
s, 3H, 23eH), 0.90 (s, 3H, 24eH 0.89e0.87 (m, 1H, 5eH) ppm;
OD):
¼ 176.0 (C-28), 172.8 (C-31), 165.6 (C-
7), 156.4 (C-44), 152.7 (C-20), 152.4 (C-46), 144.3 (C-39), 112.3 (C-
ꢀ
ꢀ
colorless solid; m.p. 280e283 C (lit [11].: 280e283 C).
a
a
a
a
4.2.22. (3
b) 3eOeacetylebetulinic acid piperazinyl amide (21)
Reaction of 17 (18.0 g, 36.1 mmol) in dry DCM (220 mL) with
a
a
þ
b
oxalyl chloride (15.2 mL, 177.2 mmol) followed by the addition of
dry piperazine (15.6 g, 181.1 mmol) as previously reported gave 21
b
a
a
b
b
ꢀ
(
15.6 g, 80%) as an offewhite solid; m.p. 179e181 C (lit [11].:
b
b
ꢀ
177e181 C).
13
C
NMR (125 MHz, CD
3
3
d
4
.2.23. (3
Reaction of 18 with oxalyl chloride followed by the addition of
dry piperazine as previously reported gave 22 (86%) as an
b) 3eOeacetyleoleanolic acid piperazinyl amide (22)
3
4
8),111.7 (C-45),110.0 (C-29), 82.5 (C-3), 74.0 (C-42), 62.6 (C-49 þ C-
9‘), 56.9 (C-5), 56.1 (C-17), 53.9 (C-18), 52.1 (C-9), 47.3 (C-19), 43.0
ꢀ
ꢀ
offewhite solid; m.p. 173e175 C (lit [15].: 173e175 C).
(
3
2
(
C-14), 42.0 (C-8), 39.6 (C-1), 38.9 (C-4), 38.4 (C-13), 38.3 (C-10),
7.0 (C-22), 35.5 (C-7), 33.4 (C-16), 32.4 (C-21), 31.0 (C-15), 28.5 (C-
4), 26.9 (C-12), 24.7 (C-2), 22.3 (C-11), 21.2 (C-32), 19.8 (C-30), 19.4
C-47), 19.3 (C-6), 18.7 (C-48), 17.0 (C-23), 16.8 (C-25), 16.7 (C-26),
4
.2.24. (3
Reaction of 19 with oxalyl chloride followed by the addition of
dry piperazine as previously reported gave 23 (84%) as an
b) 3eOeacetyleursolic acid piperazinyl amide (23)
15.1 (C-27), 8.7 (C-50), 8.3 (C-50‘) ppm; MS (ESI, MeOH): m/z
ꢀ
ꢀ
offewhite solid; m.p. 186e188 C (lit [15].: 187e188 C).
þ
(
(
%) ¼ 798.6 ([M ꢂ Cl] , 100%); analysis calcd for C49
5 4
H76ClN O
834.63): C 70.52, H 9.18, N 8.39; found: C 70.37, H 9.31, N 8.21.
4
.2.25. (3
Reaction of 20 with oxalyl chloride followed by the addition of
dry piperazine as previously reported gave 24 (84%) as an
b) 3eOeacetyleplatanic acid piperazinyl amide (24)
4.2.32. (3b) 3eAcetyloxye28e{4e
[(2,2edihexyle5,7edimethyle2,3edihydro[1,2,4]triazolo[4,3ea]
pyridine2eiume8eyl)carbonyl]e1epiperazinyl}e
ꢀ
ꢀ
offewhite solid; m.p. 118e121 C (lit [11].: 115e125 C).
28eoxolupe20(29)eene chloride (30)
4
.2.26. (3
b
) 3eOeacetylebetulinic acid 2eaminoethylamide (25)
Following GPB 30 (0.32 g, 39%) was obtained as an yellowish
ꢀ
Reaction of 17 (1.68 g, 3.4 mmol) in dry DCM (20 mL) with oxalyl
solid; m.p. 189 C; R
F
¼ 0.6 (silica gel, CHCl
3
/MeOH, 9:1); IR (ATR):
chloride (1.5 mL, 17.5 mmol) followed by the addition of ethyl-
enediamine (1.2 mL, 18.0 mmol) as previously reported gave 25
n
¼ 2934m, 2868m, 1731m, 1632s, 1565m, 1535m, 1459m, 1371m,
1315m, 1282m, 1245s, 1184s, 1138m, 1108m, 1064m, 1027m, 999s;
1H NMR (500 MHz, CD OD):
¼ 6.21 (m, 1H, 45eH), 5.94e5.84 (m,
ꢀ
ꢀ
(
1.4 g, 83%) as an offewhite solid; m.p.140e142 C (lit [15].: 142 C).
3
d
8

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
ꢂ1
2
1
2
H, 42eHa,b), 4.76 (s, 1H, 29eH
a
), 4.64 (s, 1H, 29eH
b
), 4.49e4.47 (m,
1731s, 1624s, 1575m, 1453m, 1243s, 980s, 751m, 583 m cm
UVeVis (MeOH):
;
H, 3eH), 3.72e3.66 (m, 4H, 49eHa,b þ 49‘eHa,b), 3.03e2.89 (m,
l
max (log ε) ¼ 246 (5.54), 287 (4.93), 371 (4.84)
1
H, 19eH þ 13eH), 2.44 (s, 3H, 48eH), 2.27e2.19 (m, 4H, 16eH
a
þ
nm; H NMR (500 MHz, CDCl ):
3
d
¼ 7.76 (s, 1H, 37eH), 7.66 (t,
47eH), 2.12e2.05 (m, 4H, 22H
a
þ 32eH), 1.88e1.69 (m, 9H,
J ¼ 7.7 Hz, 1H, 41eH), 7.58 (d, J ¼ 7.3 Hz, 1H, 39eH), 7.51 (d,
J ¼ 8.6 Hz, 1H, 42eH), 7.30 (t, J ¼ 7.6 Hz, 1H, 40eH), 6.50 (s, 2H,
21eH
a
þ
50eH
a
þ
50’eH
a
þ
12eH 1eH
a
þ
a
þ
1
8eH þ 16eH
b
þ 2eH), 1.62e1.25 (m, 26H, 6eHa,b þ H-
þ 15eHa,b þ 9eH þ 7eH þ 51eH þ 51‘eH þ 52eH
2‘eH þ 53eH þ 53‘eH þ 12eHb þ 21eHb þ 50eH þ ꢂ50’eH
) 1.09e1.05 (m, 4H, 27eH þ 1eH ), 1.02 (s, 3H, 26eH),
.00e0.96 (m, 6H, 54eH þ 54‘eH), 0.95 (s, 3H, 25eH), 0.91 (d,
a b
44eH), 4.70 (s, 1H, 29eH ), 4.57 (s, 1H, 29eH ), 4.48e4.40 (m, 1H,
2
5
2
b
þ 11eH
a
3eH), 4.39e4.26 (m, 2H, 46eH), 3.91e3.57 (m, 6H, 46’eH þ 34eH),
b
b
þ
3.43 (s, 4H, 33eH), 2.98e2.89 (m, 1H, 19eH), 2.85e2.75 (m, 1H,
11eH
b
b
13eH), 2.08e2.00 (m, 1H, 16eH
a
), 2.01 (s, 3H, 32eH), 1.95e1.76 (m,
1
2H, 22eH ), 1.66 (s, 3H, 30eH), 1.74e1.52 (m, 6H, 12eH
a
þ 21eH
a
a
þ
13
J ¼ 3.5 Hz, 6H, 23eH þ 24eH) ppm; C NMR (125 MHz, CD
3
OD):
1eH
a
þ 2eH þ 16eH
b
þ 18eH), 1.43 (t, J ¼ 6.9 Hz, 6H,
þ 11eH
þ 15eH
0.94 (s, 3H, 27eH), 0.91 (s, 3H, 26eH), 1.00e0.88 (m, 2H,
1eH ), 0.82 (s, 3H, 25eH), 0.81 (s, 3H, 24eH), 0.81 (s, 3H,
þ 12eH
23eH), 0.79e0.74 (m, 1H, 5eH) ppm; C NMR (125 MHz, CDCl
0
d
¼ 176.0 (C-28), 172.81 (C-31),165.6 (C-37), 156.2 (C-44), 152.7 (C-
47eH þ 47 eH), 1.51e1.28 (m, 8H, 6eH þ 22eH
b
a
þ
2
2
0), 152.4 (C-46), 144.2 (C-39), 112.3 (C-38), 111.7 (C-45), 110.1 (C-
21eH
b
þ 7eH þ 15eH), 1.28e1.12 (m, 3H, 9eH þ 11eH
b
b
),
9), 82.4 (C-3), 75.0 (C-42), 68.0 (C-49þ C-49‘), 56.9 (C-5), 56.2 (C-
17), 56.1 (C-14), 53.9 (C-18), 52.1 (C-9), 47.3 (C-19), 42.0 (C-8), 39.6
b
b
13
(
C-1), 38.9 (C-4), 38.4 (C-13), 38.3 (C-10), 37.0 (C-22), 35.5 (C-7),
3
):
3
5
3.4 (C-16), 32.4 (C-21), 28.5 (C-24), 27.1 (C-51 þ C-51‘ þ C-52 þ C-
d
¼ 174.2 (C-28), 171.1 (C-31), 163.1 (C-35), 154.0 (C-45), 151.0 (C-
2‘ þ C-53 þ C-53‘), 26.9 (C-12), 24.7 (C-2), 23.5 (C-50), 22.3 (C-11),
20), 140.7 (C-37), 134.2 (C-36), 134.2 (C-41), 129.9 (C-39), 124.6 (C-
40), 120.3 (C-43) 118.4 (C-38), 115.1 (C-42), 109.6 (C-29), 81.0 (C-3),
21.2 (C-32), 19.8 (C-30), 19.4 (C-47), 19.3 (C-6), 18.6 (C-48), 17.0 (C-
0
2
3), 16.8 (C-25), 16.7 (C-26), 15.2 (C-27), 14.4 (C-54), 14.3 (C-54‘)
72.8 (C-44), 62.6 (C-46 þ C-46 ), 55.6 (C-5), 54.8 (C-17), 52.7 (C-18),
þ
ppm; MS (ESI, MeOH): m/z (%) ¼ 910.4 ([M ꢂ Cl] , 100%); analysis
50.8 (C-9), 47.5 (C-33), 45.7 (C-19), 42.5 (C-34), 42.0 (C1e4), 40.8 (C-
8), 38.5 (C-1), 37.9 (C-4), 37.3 (C-10), 37.0 (C-13), 36.1 (C-22), 34.5
(C-7), 32.7 (C-16), 31.4 (C-21), 30.0 (C-15), 28.0 (C-24), 25.7 (C-12),
calcd for C57
H92ClN
5 4
O (946.84): C 72.31, H 9.79, N 7.40; found: C
72.15, H 9.97, N 7.31.
2
3.8 (C-2), 21.4 (C-1), 21.2 (C-32), 19.7 (C-30), 18.3 (C-6), 16.6 (C-23),
0
4
.2.33. (3
b
) 3eAcetyloxye28e{4e
16.4 (C-25), 16.3 (C-26), 14.8 (C-27), 8.8 (C-47 þ C-47 ) ppm; MS
þ
[(2,2edidodecyle5,7edimethyle2,3edihydro[1,2,4]triazolo [4,3ea]
(ESI, MeOH): m/z (%) 820.4 ([M ꢂ Cl] , 100%); analysis calcd for
pyridine2eiume8eyl)carbonyl]e1epiperazinyl}e
C
51
H72ClN
5 4
O (854.62): C 71.68H 8.49, N 8.19; found: C 71.42, H
28eoxolupe20(29)eene chloride (31)
8.67, N 8.06.
Following GPB 31 (390 mg, 40%) was obtained as a yellow solid;
ꢀ
m.p. 158 C; R
n
F
¼ 0.2 (silica gel, CHCl
3
/MeOH, 95:5); IR (ATR):
¼ 2923s, 2853m, 1732m, 1627s, 1567m, 1530m, 1465m, 1405m,
1
9
371m, 1283m, 1245s, 1186s, 1133w, 1107w, 1065w, 1029m,
ꢂ1
1
99 s cm ; H NMR (500 MHz, CDCl
), 4.58 (s, 1H, 29eH
.99e3.86 (m, 2H, 49eHa,b), 3.79e3.74 (m, 1H, 42eH
þ 49’eH ), 3.47e3.39 (m, 1H, 42eH ), 2.94 (m, 1H,
9eH), 2.81 (m, 1H, 13eH), 2.51 (s, 3H, 48eH), 2.12 (s, 3H, 47eH),
3
):
), 4.47e4.44 (m, 1H, 3eH),
), 3.66e3.61
d
¼ 5.89 (s, 1H, 45eH), 4.71
(
s, 1H, 29eH
a
b
4.2.35. (3b) 3eAcetyloxye28e{4e[(2,2ediethyle1,2edihydro
[1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]e
1epiperazinyl}e28eoxoeoleane12eene chloride (33)
3
a
(
m, 2H, 49eH
b
b
b
1
2
1
1
1
Following GPB 33 (389 mg, 47%) was obtained as a yellow solid;
ꢀ
.02 (s, 4H, 16eH
.73e1.66 (m, 7H, 59eH
a
þ 32eH), 1.92e1.83 (m, 2H, 22eH
a
þ21eH
a
a
a
),
),
),
m.p. 220e224 C; IR (ATR):
n
¼ 3369br, 2942s, 2096m, 1731s, 1625s,
0
ꢂ1
a
þ 59 eH
a
þ 12eH
a
þ 30eH þ 1eH
1466m, 1391m, 1243vs, 1004s, 750m, 458 m cm ; UVeVis
1
.60e1.55 (m, 4H, 2eH þ 16eH
b
þ 18eH), 1.50e1.46 (m, 1H, 6eH
þ 11eH þ 21eH þ 6eH
51eH 51‘eH), 1.27e1.21 (m, 33H,
þ 9eH þ 58eH þ 58‘eH þ 15eH þ 52eHe57eH
þ 27eH), 0.92e0.90 (d,
J ¼ 4.2 Hz, 3H, 26eH), 0.88e0.86 (m, 6H, 60eH þ 60‘eH), 0.84e0.81
(MeOH):
l
max (log ε) ¼ 246 (5.57), 287 (4.98), 373 (4.87) nm;
H
.42e1.29 (m, 15H, 22eH
b
b
b
þ 7eH þ
NMR (500 MHz, CDCl
3
):
d
¼ 7.76 (s,1H, 37eH), 7.65 (t, J ¼ 7.7 Hz,1H,
5
5
0eH
9eH
þ
50eH’
þ
þ
41eH), 7.58 (d, J ¼ 7.2 Hz, 1H, 39eH), 7.50 (d, J ¼ 8.1 Hz, 1H, 42eH),
7.30 (t, J ¼ 7.6 Hz, 1H, 40eH), 6.49 (s, 2H, 44eH), 5.25 (dd, J ¼ 3.3,
3.3 Hz, 1H, 12eH), 4.50e4.43 (m, 1H, 3eH), 4.39e4.27 (m, 2H,
b
þ 59’eH
b
þ 52‘eHe57‘eH), 0.95e0.94 (m, 4H, 12eH
b
0
46eH), 3.92e3.57 (m, 6H, 46 eH þ 33eH þ 34eH), 3.13e2.99 (m,
13
(
d
m, 9H, 25eH þ 24eH þ 23eH) ppm; C NMR (125 MHz, CDCl
3
):
a
1H, 18eH), 2.19e2.09 (m, 1H, 16eH ), 2.02 (s, 3H, 32eH), 1.96e1.79
¼ 174.0 (C-28), 171.1 (C-31), 163.4 (C-37), 155.1 (C-44), 151.0 (C-
(m, 2H, 11eH
2eH þ 15eH
J ¼ 6.7 Hz, 6H, 47eH), 1.46e1.29 (m, 3H, 22eH
1.27e1.15 (m, 3H, 22eH þ 19eH ), 1.12 (s, 3H, 27eH),
þ 21eH
1.10e0.95 (m, 2H, 15eH ), 0.91 (s, 3H, 29eH), 0.90 (s, 3H,
þ 1eH
25eH), 0.89 (s, 3H, 30eH), 0.84 (s, 3H, 24eH), 0.83 (s, 3H, 23eH),
a
þ 2eHa), 1.75e1.46 (m, 11H, 7eH þ 19eH
þ 11eH þ 16eH þ 1eH þ 9eH þ 6eH ), 1.42 (t,
þ 21eH þ 6eH ),
a
þ
2
2
1
0), 150.7 (C-46), 142.7 (C-39), 111.6 (C-38), 110.7 (C-45), 109.6 (C-
a
b
b
a
a
9), 81.1 (C-3), 67.5 (C-49 þ C-49‘), 67.2 (C-42), 55.7 (C-5), 54.8 (C-
a
a
b
7), 54.8 (C-14), 52.8 (C-18), 50.9 (C-9), 45.8 (C-19), 40.8 (C-8), 38.6
b
b
b
(
(
5
5
2
C-1), 37.9 (C-4), 37.3 (C-10), 37.1 (C-13), 36.1 (C-22), 34.5 (C-7), 32.7
b
b
C-16), 32.1 (C-58 þ C-58‘), 31.4 (C-21), 30.0 (C-15), 30.0e29.3 (C-
13
2eCe57 þ C-52‘eCe57‘), 28.1 (C-24), 26.4 (C-50 þ C-50‘ þ C-
0.83e0.78 (m, 1H, 5eH), 0.69 (s, 3H, 26eH) ppm; C NMR
(125 MHz, CDCl ):
1 þ C-51‘), 25.7 (C-12), 23.8 (C-2), 22.8 (C-59 þ C-59‘), 21.4 (C-32),
3
d
¼ 175.8 (C-28), 171.1 (C-31), 163.1 (C-35), 154.0
1.3 (C-11), 19.7 (C-30), 19.62 (C-48), 19.60 (C-47), 18.3 (C-6), 16.6
(C-45), 144.5 (C-13), 140.7 (C-37), 134.2 (C-36), 134.1 (C-41), 129.9
(C-39), 124.6 (C-40), 121.9 (C-12), 120.3 (C-43), 118.4 (C-38), 115.0
(C-42), 81.0 (C-3), 72.9 (C-44), 62.6 (C-46), 62.5 (C-46 ), 55.5 (C-5),
(
C-23),16.4 (C-25),16.2 (C-26),14.7 (C-27),14.2 (C-60 þ C-60‘) ppm;
þ
0
MS (ESI, MeOH): m/z (%) ¼ 1079.0 ([M ꢂ Cl] , 90%); analysis calcd
for C69
H116ClN
5
O
4
(1115.17): C 74.32, H 10.49, N 6.28; found: C
47.7 (C-9), 47.7 (C-17), 46.4 (C-19), 45.4 (C-34), 43.7 (C-18), 42.5 (C-
33), 42.0 (C-14), 39.3 (C-8), 38.2 (C-1), 37.8 (C-4), 37.1 (C-10), 34.1(C-
7
4.03, H 10.71, N 6.05.
21), 33.1 (C-30), 33.0 (C-22), 30.5 (C-20), 30.2 (C-7), 28.1 (C-23),
4
.2.34. (3
b
) 3eAcetyloxye28e{4e[(2,2ediethyle1,2edihydro
28.0 (C-15), 26.0 (C-27), 24.1 (C-29), 23.6 (C-11), 23.5 (C-2), 22.9 (C-
16), 21.4 (C-32), 18.3 (C-6), 17.1 (C-26), 16.8 (C-24), 15.5 (C-25), 8.8
[1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]e
0
þ
1
epiperazinyl}e28eoxalupae12,20(29)ediene chloride (32)
Following GPB 32 (450 mg, 57%) was obtained as a yellow solid;
(C-47 þ C-47 ) ppm; MS (ESI, MeOH): m/z (%) 820.2 ([M ꢂ Cl] ,
100%); analysis calcd for C51 74ClN (856.63): C 71.51, H 8.71, N
8.18; found: C 71.32, H 8.96, N 8.01.
H
5 4
O
ꢀ
m.p. 215e218 C; IR (ATR):
n
¼ 3373br, 2941m, 2869m, 2095m,
9

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
4
.2.36. (3
b
) 3eAcetyloxye28e{4e[(2,2ediethyle1,2edihydro
4.2.38. (3
b
) 3eAcetyloxye28e[2e{[(2,2ediethyle1,2edihydro
[
1
1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]e
epiperazinyl}e28eoxoeurse12eene chloride (34)
[1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]amino}
ethyl)amino]e28eoxolupae12,20(29)ediene chloride (36)
Following GPB 34 (408 mg, 52%) was obtained as a yellow solid;
Following GPB 36 (316 mg, 44%) was obtained as a yellow solid;
ꢀ
ꢀ
m.p. 230e234 C; IR (ATR):
n
¼ 3373br, 2941m, 2096w, 1731m,
m.p. 200e204 C; IR (ATR):
n
¼ 3321br, 2942m, 2095w, 1731m,
ꢂ1
ꢂ1
1
624s, 1576m, 1368m, 1243vs, 1045m, 950m, 751m, 582w cm
;
1661m, 1474m, 1243m, 1172m, 1035s, 980m, 750w, 582 m cm
;
UVeVis (MeOH):
nm; H NMR (500 MHz, CDCl3):
J ¼ 7.8 Hz, 1H, 41eH), 7.58 (d, J ¼ 7.2 Hz, 1H, 39eH), 7.51 (d,
J ¼ 8.2 Hz, 1H, 42eH), 7.30 (t, J ¼ 7.6 Hz, 1H, 40eH), 6.50 (s, 2H,
l
max (log ε) ¼ 246 (5.37), 287 (4.79), 371 (4.67)
UVeVis (MeOH):
388 (4.36) nm; H NMR (400 MHz, CDCl ):
l
max (log ε) ¼ 206 (5.26), 248 (5.13), 296 (4.66),
1
1
d
¼ 7.76 (s, 1H, 37eH), 7.66 (t,
3
d
¼ 8.75 (s, 1H, 37eH),
8.41e8.25 (m, 1H, NH), 7.81e7.71 (m, 2H, 39eH þ 41eH), 7.63e7.50
(m, 1H, 42eH), 7.39 (t, J ¼ 7.6 Hz, 1H, 40eH), 6.70e6.25 (m, 3H,
4
4
3
1
4eH), 5.22e5.18 (m, 1H, 12eH), 4.51e4.44 (m, 1H, 3eH),
NH þ 44eH), 4.65 (s, 1H, 29eH
a
), 4.56 (s, 1H, 29eH
b
), 4.54e4.37 (m,
0
0
.38e4.26 (m, 2H, 46eH), 3.90e3.53 (m, 4H, 46 eH þ 34eH),
3H, 46eH þ 3eH), 4.03e3.84 (m, 2H, 46 eH), 3.74e3.35 (m, 4H,
.49e3.36 (m, 2H, 33eH), 2.48e2.33 (m, 1H, 18eH), 2.23e2.09 (m,
33eH þ 34eH), 3.15e2.98 (m, 1H, 19eH), 2.41e2.27 (m, 1H, 13eH),
H, 16eH
a
), 2.02 (s, 3H, 32eH), 1.96e1.83 (m, 2H, 11eH), 1.82e1.55
), 1.55e1.37
þ 19eH), 1.42 (t, J ¼ 6.7 Hz, 6H,
þ 7eH ), 1.14e0.96 (m, 3H,
a
2.02 (s, 3H, 32eH), 2.06e1.94 (m, 1H, 16eH ), 1.92e1.69 (m, 2H,
(
(
4
1
m, 9H, 22eH þ 16eH
b
þ 21eH þ 1eH
a
þ 2eH þ 15eH
a
21eH
22eH
a
þ 1eH
a
), 1.64 (s, 3H, 30eH), 1.68e1.45 (m, 6H, 12eH
þ 2eH þ 16eH
a
þ
m, 4H, 9eH þ 6eH
a
þ 7eH
a
a
b
þ 18eH), 1.41 (t, J ¼ 7.3 Hz, 3H,
þ 11eH þ 15eH
9eH), 1.16e1.03 (m, 4H,
), 0.89 (s, 3H, 27eH), 1.02e0.81 (m,
b
), 0.78 (s, 3H, 24eH), 0.74 (s, 3H, 23eH), 0.74 (s,
0
0
7eH þ 47 eH), 1.38e1.15 (m, 2H, 6eH
b
b
47eH þ 47 eH), 1.44e1.18 (m, 7H, 22eH
b
a
a
þ
5eH
0eH), 0.91 (s, 3H, 25eH), 0.88 (d, J ¼ 6.4 Hz, 6H, 29eH), 0.85 (s, 3H,
4eH), 0.83 (s, 3H, 23eH), 0.83e0.79 (m, 1H, 5eH), 0.71 (s, 3H,
6eH) ppm; 13C NMR (125 MHz, CDCl
):
¼ 176.0 (C-28), 171.1 (C-
1), 163.1 (C-35), 154.0 (C-45), 138.5 (C-13), 140.7 (C-37), 134.2 (C-
1), 134.1 (C-36), 129.9 (C-39), 125.5 (C-12), 124.6 (C-40), 120.3 (C-
b
þ 1eH
b
þ 20eH), 1.06 (s, 3H, 27eH), 0.93 (d, J ¼ 6.1 Hz, 3H,
21eH
7eH
2H, 12eH
3H, 26eH), 0.72e0.58 (m, 1H, 5eH), 0.64 (s, 3H, 25eH) ppm;
NMR (100 MHz, CDCl ):
b
þ
6eH
þ 11eH þ 6eH
þ 1eH
a
þ
7eH
þ 15eH
a
þ
3
2
2
3
4
b
b
b
b
b
13
3
d
C
3
d
¼ 177.2 (C-28), 171.0 (C-31), 161.5 (C-35),
154.5 (C-45), 150.6 (C-20), 146.1 (C-37), 135.3 (C-41), 134.4 (C-36),
131.1 (C-39), 125.1 (C-40), 120.4 (C-43), 114.9 (C-42), 114.4 (C-38),
4
4
3
3
3), 118.4 (C-38), 115.1 (C-42), 81.0 (C-3), 72.9 (C-44), 62.6 (C-
0
0
6 þ C-46 ), 55.4 (C-5), 55.3 (C-18), 47.7 (C-17), 47.6 (C-9), 47.3 (C-
109.6 (C-29), 80.8 (C-3), 72.3 (C-44), 63.2 (C-46 þ C-46 ), 55.7 (C-
3), 42.4 (C-34), 42.2 (C-14), 39.6 (C-19), 39.3 (C-8), 38.8 (C-20),
8.3 (C-1), 37.8 (C-4), 37.0 (C-10), 34.6 (C-22), 33.1 (C-7), 30.5 (C-
17), 55.3 (C-5), 50.3 (C-9), 50.0 (C-18), 46.9 (C-19), 40.6 (C-14), 40.4
(C-34), 39.7 (C-33), 38.4 (C-1), 38.3 (C-22), 37.7 (C-13), 37.7 (C-10),
37.0 (C-8), 34.2 (C-7), 33.5 (C-16), 30.8 (C-21), 29.5 (C-15), 27.9 (C-
24), 25.4 (C-12), 23.6 (C-2), 21.3 (C-32), 20.9 (C-11), 19.3 (C-30), 17.9
(C-6), 16.5 (C-23), 16.2 (C-25), 16.1 (C-26), 14.5 (C-27), 8.8 (C-47), 8.6
21), 28.3 (C-15), 28.2 (C-24), 23.8 (C-27), 23.6 (C-11), 23.5 (C-16),
2
2
3.4 (C-2), 21.4 (C-32), 21.3 (C-30), 18.3 (C-6), 17.5 (C-29), 17.1 (C-
6), 16.8 (C-23), 15.6 (C-25), 8.8 (C-47 þ C-47 ) ppm; MS (ESI,
0
þ
0
þ
MeOH): m/z (%) 820.2 ([M ꢂ Cl] , 100%); analysis calcd for
(C-47 ) ppm; MS (ESI, MeOH): m/z (%) 794.2 ([M ꢂ Cl] , 100%);
analysis calcd for C49 70ClN (828.58): C 71.03, H 8.52, N 8.45;
found: C 70.76, H 8.69, N 8.26.
C
51
H74ClN
5
O
4
(856.63): C 71.51, H 8.71, N 8.18; found: C 71.26, H
H
5 4
O
8
.90, N 8.03.
4
.2.37. (3
b
) 3eAcetyloxye28e{4e[(2,2ediethyle1,2edihydro
4.2.39. (3b) 3eAcetyloxye28e[2e{[(2,2ediethyle1,2edihydro
[
1
1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]e
epiperazinyl}e20,28edioxoe30enorlupae12eene chloride (35)
[1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]amino}
ethyl)amino]e28eoxoeoleane12eene chloride (37)
Following GPB 35 (348 mg, 44%) was obtained as a yellow solid;
Following GPB 37 (314 mg, 40%) was obtained as a yellow solid;
ꢀ
ꢀ
m.p. 236e240 C; IR (ATR):
n
¼ 3373br, 2941m, 2871m, 2096m,
m.p. 210e214 C; IR (ATR):
n
¼ 3334br, 2941m, 2097w, 1731m,
1
5
3
7
(
4
6
2
(
2
2
731s, 1624s, 1452m, 1242vs, 1170m, 1028m, 980s, 751m,
1661m, 1574m,1366m, 1243m, 1027m, 750m, 583w cme1; UVeVis
(MeOH):
max (log ε) ¼ 213 (5.64), 248 (5.50), 296 (5.00), 387 (4.73)
nm; H NMR (400 MHz, CDCl ):
ꢂ1
82 m cm ; UVeVis (MeOH):
l
max (log ε) ¼ 246 (5.67), 288 (5.09),
l
1
1
74 (4.93) nm; H NMR (500 MHz, CDCl
3
):
d
¼ 7.76 (s, 1H, 36eH),
3
d
¼ 8.72 (s, 1H, 37eH), 8.42e8.37
.69e7.62 (m, 1H, 40eH), 7.58 (d, J ¼ 7.8 Hz, 1H, 38eH), 7.54e7.49
(m, 1H, NH), 7.77e7.69 (m, 2H, 39eH þ 41eH), 7.56 (d, J ¼ 8.3 Hz,
1H, 42eH), 7.36 (t, J ¼ 7.6 Hz, 1H, 40eH), 6.59e6.47 (m, 2H, 44eH),
6.44e6.37 (m, 1H, NH), 5.39 (s, 1H, 12eH), 4.53e4.37 (m, 3H,
m, 1H, 41eH), 7.30 (t, J ¼ 7.5 Hz, 1H, 39eH), 6.50 (s, 2H, 43eH),
.47e4.40 (m, 1H, 3eH), 4.40e4.21 (m, 2H, 45eH), 4.07e3.33 (m,
0
0
H, 45 eH þ 33eH þ 32eH), 3.19 (dd, J ¼ 11.2, 7.3 Hz, 1H, 19eH),
46eH þ 3eH), 4.00e3.80 (m, 1H, 46 eH), 3.73e3.42 (m, 2H, 33eH),
.62 (dd, J ¼ 12.1, 8.5 Hz, 1H, 13eH), 2.14 (s, 3H, 29eH), 2.12e2.00
3.32e3.16 (m, 2H, 34eH), 2.60e2.48 (m, 1H, 18eH), 2.02 (s, 3H,
m, 2H, 18eH þ 16eH
a
), 2.01 (s, 3H, 31eH), 1.97e1.76 (m, 2H,
31eH), 1.97e1.83 (m, 3H, 16eH
1.62e1.50 (m, 6H, 16eH
a
þ 11eH), 1.77e1.65 (m, 1H,19eH
a
),
þ 9eH),
), 1.42 (t, J ¼ 6.9 Hz, 6H,
þ 21eH þ 22eH ),
2eH
a
þ
21eHa), 1.72e1.47 (m, 7H, 1eH
þ 21eH þ 6eH
a
þ
16eH
b
þ
b
þ 22eH
a
þ 2eH þ 1eH
a
0
eH þ 22eH
b
b
a
), 1.43 (t, 6H, 46eH þ 46 eH),
1.50e1.37 (m, 3H, 15eH
a
þ 6eH
a
þ 7eH
a
0
1
(
(
.45e1.13 (m, 8H, 11eH þ 7eH þ 6eH
m, 3H, 12eH þ 1eH ), 0.96 (s, 3H, 27eH), 0.89 (s, 3H, 26eH), 0.82
s, 3H, 25eH), 0.82 (s, 3H, 24eH), 0.81 (s, 3H, 23eH), 0.80e0.73 (m,
H, 5eH) ppm; 13C NMR (125 MHz, CDCl
):
b
þ 15eH þ 9eH), 1.07e0.87
47eH þ 47 eH), 1.36e1.22 (m, 3H, 6eH
b
a
b
b
1.21e1.15 (m, 2H, 7eH þ 19eH
21eH þ 15eH ), 0.87 (s, 3H, 30eH), 0.85 (s, 3H, 25eH),
þ 1eH
0.84 (s, 3H, 29eH), 0.82 (s, 3H, 23eH), 0.81 (s, 3H, 24eH), 0.80e0.76
b
), 1.12 (s, 3H), 1.11e0.94 (m, 3H,
a
a
b
1
3
d
¼ 212.7 (C-20), 174.1
13
(
(
(
(
4
3
1
(
8
1
C-28), 171.0 (C-30), 163.1 (C-34), 154.0 (C-44), 140.8 (C-36), 134.3
(m, 1H, 5eH), 0.71 (s, 3H, 26eH) ppm; C NMR (100 MHz, CDCl
3
):
C-40), 134.2 (C-35), 129.9 (C-38), 124.6 (C-39), 120.3 (C-42), 118.3
d
¼ 179.2 (C-28), 171.1 (C-31), 161.5 (C-35), 154.6 (C-45), 146.0 (C-
0
C-37),115.1 (C-41), 80.9 (C-3), 72.9 (C-43), 62.5 (C-45 þ C-45 ), 55.6
37), 144.6 (C-13), 135.3 (C-39), 134.6 (C-36), 131.1 (C-41), 125.0 (C-
40), 123.0 (C-12), 120.5 (C-43), 115.1 (C-42), 114.4 (C-38), 80.9 (C-3),
72.2 (C-44), 63.3 (C-46), 63.1 (C-46‘), 55.2 (C-5), 47.5 (C-9), 46.7 (C-
19), 46.4 (C-17), 42.1 (C-14), 42.0 (C-18), 40.5 (C-33), 39.8 (C-34),
39.4 (C-8), 38.2 (C-1), 37.8 (C-4), 36.9 (C-10), 34.2 (C-21), 33.0 (C-
30), 32.9 (C-22), 32.4 (C-7), 30.8 (C-20), 28.1 (C-23), 27.4 (C-15),
25.8 (C-27), 23.9 (C-16), 23.7 (C-29), 23.6 (C-11), 23.6 (C-2), 21.4 (C-
32), 18.2 (C-6), 17.0 (C-26), 16.8 (C-24), 15.5 (C-25), 8.9 (C-47 þ C-
C-5), 54.7 (C-17), 52.6 (C-18), 50.7 (C-9), 50.0 (C-19), 47.5 (C-32),
2.5 (C-33), 41.9 (C-14), 40.7 (C-8), 38.5 (C-1), 37.9 (C-4), 37.3 (C-10),
6.1 (C-13), 35.8 (C-22), 34.3 (C-7), 32.3 (C-16), 30.4 (C-29), 30.0 (C-
5), 28.8 (C-21), 28.0 (C-24), 27.5 (C-12), 23.8 (C-2), 21.4 (C-31), 21.3
C-11), 18.3 (C-6), 16.6 (C-23), 16.3 (C-25), 16.2 (C-26), 14.8 (C-27),
0
þ
.8 (C-46 þ C-46 ) ppm; MS (ESI, MeOH): m/z (%) 822.2 ([M ꢂ Cl] ,
70ClN (856.59): C 70.11, H 8.24, N
.18; found: C 69.87, H 8.47, N 8.02.
00%); analysis calcd for C50
H
5 5
O
þ
8
47‘) ppm; MS (ESI, MeOH): m/z (%) 794.1 ([M ꢂ Cl] , 100%); analysis
10

O. Kraft, M. Kozubek, S. Hoenke et al.
European Journal of Medicinal Chemistry 209 (2021) 112920
calcd for C49
H
72ClN
5
O
4
(829.53): C 70.86, H 8.74, N 8.43; found: C
11), 18.0 (C-6), 16.6 (C-23 þ C-26), 16.2 (C-25), 14.7 (C-27), 8.9 (C-
0
þ
7
0.69, H 8.91, N 8.25.
46), 8.9 (C-46 ) ppm; MS (ESI, MeOH): m/z (%) 796.2 ([M ꢂ Cl] ,
5 5
100%); analysis calcd for C48H66ClN O (830.55): C 69.42, H 8.25, N
4
.2.40. (3
b
) 3eAcetyloxye28e[2e{[(2,2ediethyle1,2edihydro
8.43; found: C 69.27, H 8.41, N 8.32.
[1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]amino}
ethyl)amino]e28eoxoeurse12eene chloride (38)
Declaration of competing interest
Following GPB 38 (341 mg, 45%) was obtained as a yellow solid;
ꢀ
m.p. 198e202 C; IR (ATR):
1
n
¼ 3321br, 2940m, 2095m, 1731m,
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
ꢂ1
456m, 1367m, 1243vs, 1147w, 1027m, 982m, 750w, 582w cm
;
UVeVis (MeOH):
nm; H NMR (400 MHz, CDCl
l
max (log ε) ¼ 248 (5.54), 297 (5.04), 387 (4.79)
1
3
):
d
¼ 8.71 (s, 1H, 37eH), 8.43e8.35
(
m, 1H, NH), 7.72 (t, J ¼ 7.0 Hz, 2H, 39eH þ 41eH), 7.53 (d,
J ¼ 8.5 Hz, 1H, 42eH), 7.35 (t, J ¼ 7.6 Hz, 1H, 40eH), 6.54 (s, 2H,
4eH), 6.40e6.31 (m, 1H, NH), 5.33e5.29 (m, 1H, 12eH), 4.52e4.40
m, 2H, 46eH þ 3eH), 3.98e3.84 (m, 2H), 3.67e3.45 (m, 2H, 33eH),
Acknowledgments
4
(
We like to thank Dr. D. Str o€ hl, Y. Schiller and S. Ludwig for the
NMR spectra and the late Dr. R. Kluge for measuring the MS spectra;
some spectra were recorded by Th. Schmidt. Many thanks are also
due to V. Simon and M. Schneider for taking the IR spectra. The
microanalyses have been determined by S. Ludwig and M.
Schneider. The cell lines have been provided by the Department of
Oncology (MartineLuthereUniversity Halle Wittenberg).
3
2
1
7
4
1
2
0
.32e3.20 (m, 2H, 34eH), 2.01 (s, 3H, 32eH), 2.07e1.81 (m, 3H,
eH þ 18eH), 1.77e1.50 (m, 6H, 22eH
eH 1.49e1.16
þ9eH),
eH þ 6eH þ 19eH þ 21eH þ 22eH
7eH 47’eH, 1.05 (s, 3H, 27eH), 1.06e0.95 (m, 2H,
eH ), 0.90 (s, 3H, 30eH), 0.86 (s, 3H, 25eH), 0.84 (s, 3H,
þ 15eH
9eH), 0.82 (s, 3H, 24eH), 0.80 (s, 3H, 23eH), 0.71 (s, 3H, 26eH),
a
þ 11eH þ 15eH
a
þ
a
(m,
8H,
b
), 1.44 (t, J ¼ 6.8 Hz, 6H,
þ
b
b
Appendix A. Supplementary data
13
.74e0.64 (m, 1H, 5eH) ppm; C NMR (100 MHz, CDCl
3
):
d
¼ 179.0
(
(
(
C-28), 170.9 (C-31), 161.2 (C-35), 154.4 (C-45), 145.8 (C-37), 139.4
C-13), 135.2 (C-41), 134.4 (C-36), 131.0 (C-39), 125.7 (C-12), 124.9
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112920.
C-40), 120.4 (C-43), 114.9 (C-42), 114.4 (C-38), 80.7 (C-3), 72.1 (C-
0
4
4), 63.1 (C-46), 63.0 (C-46 ), 55.1 (C-5), 53.5 (C-18), 47.7 (C-17), 47.4
References
(
C-9), 42.3 (C-14), 40.4 (C-33), 39.6 (C-19), 39.5 (C-8), 39.3 (C-34),
3
7
9.1 (C-20), 38.2 (C-1), 37.6 (C-4), 37.4 (C-22), 36.8 (C-10), 32.6 (C-
), 30.8 (C-21), 28.0 (C-24), 27.8 (C-15), 23.5 (C-11), 23.5 (C-16), 23.3
[
1] E.H. Gort, A.J. Groot, E. van der Wall, P.J. van Diest, M.A. Vooijs, Hypoxic
regulation of metastasis via hypoxiaeinducible factors, Curr. Mol. Med. 8
(2008) 60e67.
(
C-2), 23.2 (C-27), 21.3 (C-32), 21.2 (C-30), 18.1 (C-6), 17.3 (C-29),
[
2] L. Dong, J. Rohlena, S. Ralph, J. Neuzil, Mitochondrial targeting of vitamin E
succinate enhances its antiecancer activity via mitochondrial complex II and
presents potential benefit against tumour angiogenesis, Eur. J. Canc. 48 (2012)
S221eS222.
3] L.F. Dong, V.J.A. Jameson, D. Tilly, J. Cerny, E. Mahdavian, A. MarineHernandez,
L. HernandezeEsquivel, S. RodriguezeEnriquez, J. Stursa, P.K. Witting,
B. Stantic, J. Rohlena, J. Truksa, K. Kluckova, J.C. Dyason, M. Ledvina,
B.A. Salvatore, R. MorenoeSanchez, M.J. Coster, S.J. Ralph, R.A.J. Smith,
0
16.9 (C-26), 16.7 (C-23), 15.5 (C-25), 8.8 (C-47), 8.8 (C-47 ) ppm; MS
þ
(
C
ESI, MeOH): m/z (%) 794.2 ([M ꢂ Cl] , 100%); analysis calcd for
49
H72ClN
5 4
O (829.53): C 70.86, H 8.74, N 8.43; found: C 70.58, H
[
8
.93, N 8.21.
4.2.41. (3
b) 3eAcetyloxye28e[2e{[(2,2ediethyle1,2edihydro
J. Neuzil, Mitochondrial targeting of vitamin
E succinate enhances its
[1,2,4]triazolo[4,3ea]quinoline2eiume4eyl)carbonyl]amino}
proeapoptotic and antiecancer activity via mitochondrial complex II, J. Biol.
Chem. 286 (2011) 3717e3728.
ethyl)amino]e20,28edioxoe30enorlupane12eene chloride (39)
[
[
4] J. Neuzil, L.F. Dong, L. Ramanathapuram, T. Hahn, M. Chladova, X.F. Wang,
R. Zobalova, L. Prochazka, M. Gold, R. Freeman, J. Turanek, E.T. Akporiaye,
J.C. Dyason, S.J. Ralph, Vitamin E analogues as a novel group of mitocans:
antiecancer agents that act by targeting mitochondria, Mol. Aspect. Med. 28
(2007) 607e645.
Following GPB 39 (335 mg, 44%) was obtained as a yellow solid;
ꢀ
m.p. 196e200 C; IR (ATR):
547m, 1243vs, 1167m, 1028m, 980m, 750w, 583 cm ; UVeVis
n
¼ 3321br, 2941m, 2095m, 1731s,
ꢂ1
1
(
1
MeOH):
NMR (500 MHz, CDCl
l
max (log ε) ¼ 248 (5.76), 297 (5.27), 385 (5.02) nm;
H
5] J. Neuzil, L.F. Dong, J. Rohlena, J. Truksa, S.J. Ralph, Classification of mitocans,
antiecancer drugs acting on mitochondria, Mitochondrion 13 (2013)
3
):
d
¼ 8.72 (s, 1H, 36eH), 8.30 (t, J ¼ 5.3 Hz,
1
4
H, NH), 7.77e7.70 (m, 2H, 38eH þ 40eH), 7.54 (d, J ¼ 8.1 Hz, 1H,
199e208.
1eH), 7.37 (t, J ¼ 7.6 Hz,1H, 39eH), 6.70e6.64 (m, 1H, NH), 6.60 (d,
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J ¼ 10.0 Hz, 1H, 43eH
a
), 6.47 (d, J ¼ 10.1 Hz, 1H, 43eH
b
), 4.49e4.35
0
(
3
2
3
m, 3H, 3eH þ 45eH), 3.98e3.88 (m, 2H, 45 eH), 3.71e3.48 (m, 2H,
2eH), 3.45e3.29 (m, 3H, 33eH þ 18eH), 2.19e2.08 (m, 1H, 13eH),
.11 (s, 3H, 29eH), 2.06e2.00 (m, 2H, 16eH
þ 19eH), 2.00 (s, 3H,
1eH), 1.98e1.90 (m, 1H, 21eH ), 1.80e1.73 (m, 1H, 22eH ),
.61e1.42 (m, 5H, 1eH þ 2eH þ 22eH ), 1.46 (q,
þ 16eH
þ 11eH
a
a
a
1
a
b
b
0
J ¼ 6.9 Hz, 6H, 46eH þ 46 eH), 1.42e1.19 (m, 6H, 21eH
b
a
þ
1
5eH
a
þ
6eH
a
þ
7eH
a
þ
9eH), 1.19e0.96 (m, 6H,
þ 12eH), 0.92 (s, 3H, 27eH),
1
0
0
1eH
b
þ 6eH þ 7eH
b
b
þ 15eH
b
.95e0.85 (m, 1H, 1eH
b
), 0.76 (s, 3H, 24eH), 0.73 (s, 3H, 26eH),
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129e139.
1
3
C NMR (125 MHz, CDCl
0), 161.8 (C-34), 154.7 (C-44), 146.2 (C-36), 135.2 (C-40), 134.5 (C-
5), 131.2 (C-38), 125.2 (C-39), 120.5 (C-42), 115.0 (C-41), 114.4 (C-
3
):
d
¼ 212.7 (C-20), 176.9 (C-28), 171.0 (C-
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