triterpenoid pyrazines and benzopyrazines with cytotoxic activity

Article abstract of DOI:10.1021/np060436d

Twelve lupane, 18α-oleanane, and des-E-lupane derivatives (1a-5b) were either extracted from natural sources or synthesized from betulinic acid (1a) and betulin (2). Compounds 1b, 1c, 3b, 3c, 4b, 4c, 5a, and 5b were then used as starting materials for further synthesis of a series of pyrazines and benzopyrazines (6a-18); 20 of them are new (6a-6c, 7a-7d, and 10a-18). Activity of pyrazine 6a against the T-lymphoblastic leukemia cell line CEM encouraged us to synthesize several new esters (6b-6d) to study structure-activity relationships with respect to substitution of the carboxyl group at position 28. The synthesized compounds were tested for cytotoxicity against a variety of cancer cell lines of different histogenetic origin, and the results were compared with cytotoxicity of the known starting compounds. Significant cytotoxic activity against A 549, K 562, and multidrug-resistant K 562-tax cell lines was found in pyrazines 6a, 6d, and 6e.

Full text of DOI:10.1021/np060436d

526
J. Nat. Prod. 2007, 70, 526-532
Triterpenoid Pyrazines and Benzopyrazines with Cytotoxic Activity
Milan Urban,†,‡ Jan Sarek,*,† Miroslav Kvasnica,†,‡ Iva Tislerova,† and Marian Hajduch§
Department of Organic and Nuclear Chemistry, Faculty of Science, Charles UniVersity in Prague, HlaVoVa 8, 128 43 Prague 2,
Czech Republic, Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic, FlemingoVo n. 2, 166 10,
Prague 6, Czech Republic, and Laboratory of Experimental Medicine, Department of Pediatrics, Faculty of Medicine, Palacky UniVersity and
Faculty Hospital in Olomouc, PuskinoVa 6, 775 20 Olomouc, Czech Republic
ReceiVed September 6, 2006
Twelve lupane, 18R-oleanane, and des-E-lupane derivatives (1a-5b) were either extracted from natural sources or
synthesized from betulinic acid (1a) and betulin (2). Compounds 1b, 1c, 3b, 3c, 4b, 4c, 5a, and 5b were then used as
starting materials for further synthesis of a series of pyrazines and benzopyrazines (6a-18); 20 of them are new (6a-
6e, 7a-7d, and 10a-18). Activity of pyrazine 6a against the T-lymphoblastic leukemia cell line CEM encouraged us
to synthesize several new esters (6b-6d) to study structure-activity relationships with respect to substitution of the
carboxyl group at position 28. The synthesized compounds were tested for cytotoxicity against a variety of cancer cell
lines of different histogenetic origin, and the results were compared with cytotoxicity of the known starting compounds.
Significant cytotoxic activity against A 549, K 562, and multidrug-resistant K 562-tax cell lines was found in pyrazines
6a, 6d, and 6e.
Heterocyclic compounds occur widely in living organisms, and
many possess a broad range of biological activities. Researchers
have also demonstrated the interesting biological activities of many
natural terpenoids.1 However, terpenoids possessing a nitrogen-
containing heterocycle condensed to an isoprenoid skeleton are not
common. One example of a less common isoprenoid is cephalosta-
tin, isolated1 from the sea worm Cephalodiscus gilchristie. This
bis-steroidal pyrazine is highly cytotoxic,2 which sparked interest
in the synthesis of similar structures.3-5 To date no parallel studies
have been reported in triterpene (especially lupane and oleanane)
chemistry, a fact that motivated us to synthesize a group of novel
pyrazines from betulinic acid (1a) and betulin (2). Acid 1a and
betulin (2) are pentacyclic triterpenes that occur in the bark of
Platanus hispanica and Betula pendula, respectively, and many
other plant sources.6 Betulinic acid (1a) has anti-HIV7,8 activity
and cytotoxic9,10 activity against human melanoma (MEL-2),11 lung
adenocarcinoma12 (A 549), neuroblastoma,13 medulloblastoma,14
glioblastoma,15 ovarian (OVCAR-3),14 colon (HCT-15),14 and
glioma (XF498)14 cell lines. It has also high in ViVo activities against
MEL-215 and other neuroectodermal tumors15,16 including Ewings
sarcoma.16 Although betulin (2) itself does not seem to be cytotoxic,
some derivatives were found to be active.17,18
Several heterocycles,19,20 containing pyrazines,21-23 have previ-
ously been synthesized from allobetulone 4b, although no report
concerning their cytotoxicity has been published. In this paper, we
describe both the synthesis and structure-activity relationships of
20 new (6a-6e, 7a-7d, 10a, 10b, 11a, 11b, and 12-18) and 14
known (1a-5b, 6f, 8, and 9) compounds. These include triterpenoid
derivatives modified with pyrazine and benzopyrazine rings attached
to the A-ring or E-ring of the triterpenes. Compounds 1b, 3b, and
5b have previously shown interesting activities18 and were selected
as leading compounds for further modification. Allobetulon 4b and
diketones 4c and 5a were chosen as examples of inactive triterpenes,
and their derivatives were useful for comparison of cytotoxicity
with pyrazines prepared from active compounds (1b, 3b, and 5b).
3â,28-diol (2) was extracted from the outer layers of birch bark,
Betula pendula (both collected in the Czech Republic). Betulonic
acid (1b), dihydrobetulinic acid (3a), and dihydrobetulonic acid
(3b) were synthesized24 from betulinic acid (1a). Methyl betulonate
(1c) and methyl dihydrobetulonate (3c) were prepared from free
acids 1b and 3b by reaction with diazomethane. Allobetulone 4b
and diketones 5a and 5b were synthesized18 from betulin (2).
Diketone 4c was synthesized from allobetulin (4a)25 (Scheme 1).
The purpose of this work was to synthesize a series of pyrazine
and benzopyrazine derivatives of triterpenes as summarized in
Schemes 2 and 3. The reaction of ketone 4b with ethylenediamine
and sulfur in refluxing morpholine has been previously described.22
We used an analogous procedure to prepare pyrazines 6a, 6b, 6e,
6f, 8, and 17 from ketones 1b, 1c, 3b, 3c, 4b, and 4c, the only
modification being in the amounts of reagents used. Reaction of
ketones 1b, 1c, 3b, 3c, and 4b with 1,2-phenylenediamine under
the same conditions gave benzopyrazines 7a-7d and 9. We present
a shorter alternative procedure for the synthesis of compound 9
than that previously described.21
An analogous procedure for the preparation of pyrazines at the
E-ring (Scheme 3), from diketones 5a and 5b, was not highly
successful. Reaction of 5a with ethylenediamine followed by
acetylation gave pyrazine 12 (47%), along with multiple byproducts.
In an analogous reaction, pyrazine 14 was obtained from 5b as the
major product (57%) instead of the desired compound 15. Mixtures
containing no benzopyrazine derivatives were obtained from
reactions of 5a and 5b with 1,2-phenylenediamine. Since all of
those side reactions could be caused by the basicity of morpholine,
we optimized the reaction solvent. Compounds 13, 15, and 16 were
obtained in yields of 50-70% in refluxing xylene. This approach
led to fewer byproducts than the morpholine procedure and allowed
easier purification. This methodology was then applied for the
preparation of pyrazines at the A-ring. To our surprise, we found
that using morpholine as a solvent is essential for the reaction of
ketones in the A-ring with ethylenediamine, since no pyrazines were
obtained in xylene. It appears that the basic solvent is important
for an oxidative attack at the 2 position. The reaction of diketone
4c with 1,2-phenylenediamine gave rise to compound 18 (63%).
Results and Discussion
3â-Hydroxylup-20(29)-en-28-oic acid (1a) was extracted from
the bark of the plane tree, Platanus hispanica. Lup-20(29)-ene-
Compound 6a was treated with diazomethane, pivaloyloxymethyl
chloride (PomCl), or acetoxymethyl bromide (AcmBr) to give esters
6b-6d. We found previously,26 in another group of triterpene acids,
that Acm esters can be used as a prodrug and that cytotoxicity did
not decrease significantly when compared with methyl and Pom
* To whom correspondence should be addressed. Tel: +420 221 951
332. Fax: +420 221 951 332. E-mail: jan.sarek@gmail.com.
† Charles University in Prague.
‡ Academy of Sciences.
§ Palacky University in Olomouc.
10.1021/np060436d CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/20/2007

Triterpenoid Pyrazines and Benzopyrazines with Cytotoxic ActiVity
Journal of Natural Products, 2007, Vol. 70, No. 4 527
Table 1. Cytotoxicity of Compounds 1a-18 to the CEM Cell
Line
Scheme 1. Preparation of Starting Material for Synthesis of
Heterocycles
IC50 (µmol/La)
IC50 (µmol/La)
compound
CEM
compound
CEM
1a
1b
1c
2
30
20
250
250
7
6f
151
42
7a
7b
7c
7d
8
250
124
250
250
250
101
74
3a
3b
3c
4a
4b
4c
5a
5b
6a
6b
6c
6d
6e
2
173
250
250
250
250
16
9
10a
10b
11a
11b
12
13
14
15
16
17
18
178
35
215
106
244
250
192
250
250
14
250
232
12
8
a The lowest concentration that kills 50% of tumor cells.
(Table 2). Of these tests the activities of compounds 6a, 6d, and
6e against A 549, K 562, and the paclitaxel-resistant and Pgp-
overexpressing21a line K 562-tax (IC50 0.25-8 µmol/L) proved to
be the most interesting. These compounds were preferentially
cytotoxic against human carcinoma cell lines A 549 and HT-29
and less cytotoxic against prostate cancer PC-3 compared to
otherwise highly sensitive CEM leukemia or neuroectodermal
tumors (melanoma SK-MEL-2, glioblastoma U87-Mg). The high
activity of 6a and 6e against the human myeloid leukemia line K
562 suggests that further studies should be performed using these
compounds. These additional assays should pay particular attention
to those bearing bcr-abl translocation. Compounds 6a, 6d, and 6e
were approximately 10 times less active against Pgp-positive
multidrug-resistant cell line K 562-tax. This result suggests that
further compounds having no affinity to multidrug resistance
transporters should be synthesized. Nonetheless, the activity of 6e
was not affected by other multidrug resistance associated genes,
represented by MRP protein in the case of the CEM-DNR-bulk
cell line (Tables 1 and 2). No other derivative showed significant
activity.
In conclusion, pyrazines and benzopyrazines 6a-18 were
synthesized and their cytotoxic activity was compared with that of
starting compounds 1a-5b. The previously known22 procedure for
the preparation of pyrazines and benzopyrazines at the A-ring was
optimized for the synthesis of corresponding heterocycles from 21,-
22-diketones at the E-ring. Esters 6b-6d and 28-hydroxymethyl
derivatives 10a-11b were prepared from pyrazines 6b and 6f and
benzopyrazines 7b and 7d. Among all of the new derivatives,
cytotoxicity of compounds 6a, 6d, and 6e was found in a broad
range of cancer cell lines having different histogenetic origins and
drug sensitivity profiles. We confirmed24,26 that methyl and Pom
esters are less active than the corresponding free acids. The oleanane
heterocycles 8 and 9 and 20(29)-lupene heterocycles 10a-11b were
inactive as well as compounds having pyrazine or benzopyrazine
groups condensed to the five-membered ring (12-18).
esters. Esters 6b, 6f, 7b, and 7d were reduced with LAH under
standard conditions to give pyrazines 10a-11b, which contain a
28-hydroxymethyl group (Scheme 2).
Cytotoxicity of the new pyrazines 6a-18 was then studied.
Pyrazine 6a showed interesting in Vitro activity against the
T-lymphoblastic leukemia CEM cell line (IC50 14 µmol/L), which
motivated us to synthesize a larger group of pyrazines and
benzopyrazines for structure-activity relationship analysis. Among
pyrazines 6a-18 (Table 1) we found that 6e was the most active
(IC50 8 µmol/L) against the CEM cell line. Acm ester 6d was the
most active (IC50 12 µmol/L) compound among esters 6b-6d, with
activity higher than that of free acid 6a. Methyl ester 6b and Pom
ester 6c were inactive. The cytotoxic activity of benzopyrazines
(7a-7d, 9, 11a, 11b, 13, 16, and 18) was in the high micromolar
ranges. All other changes of the terpene skeleton led to significant
decrease of cytotoxicity. Pyrazines and benzopyrazines 8, 9, 17,
and 18 synthesized from inactive 4b and diketone 4c as well as
compounds with a 28-hydroxymethyl group (10a-11b) were also
inactive. Compounds with the heterocycle condensed to the E-ring
(12-16) were less active than the starting diketones 5a and 5b.
The most active of these compounds against T-lymphoblastic
leukemia CEM cells (6a, 6e, and 6d) along with several members
of each structurally similar group of compounds were then tested
on additional cancer cell lines of different histogenetic origin
Experimental Section
General Experimental Procedures. Melting points were determined
using a Kofler block and are uncorrected. Optical rotations were
measured using CHCl3 solutions (unless otherwise stated) on an Autopol
III (Rudolph Research, Flanders, NJ) polarimeter. NMR spectra were
recorded on a Varian Unity INOVA 400 instrument (1H NMR spectra
at 399.95 MHz) using CDCl3 solutions (unless otherwise stated), with
SiMe4 as an internal standard. EIMS were recorded on an INCOS 50
(Finigan MAT) spectrometer at 70 eV and an ion source temperature
of 150 °C. The samples were introduced from a direct exposure probe

528 Journal of Natural Products, 2007, Vol. 70, No. 4
Urban et al.
Scheme 2. Preparation of Pyrazines and Benzopyrazines at Six-Membered Rings
at a heating rate of 10 mA/s. Column chromatography was performed
using silica gel 60 (Merck 7734). The HPLC system consisted of a
high-pressure pump (Gilson model 361), a Rheodyne injection valve,
and a preparative column (25 × 250 mm) filled with silica gel (Biospher
7 µm), The differential refractometer detector (Laboratorni Pristroje,
Praha, CZ) was connected with a PC (software Chromulan) and an
automatic fraction collector (Gilson model 246). A mixture of EtOAc
and hexane was used as the mobile phase, its composition specified in
each experiment. TLC was carried out on Kieselgel 60 F254 plates
(Merck) using toluene-Et2O (5:1), detected by spraying with 10%
aqueous H2SO4 and heating to 150-200 °C.
Workup refers to pouring the reaction mixture into H2O, extraction
of the product with organic solvent, washing the organic layer
successively with H2O, dilute aqueous HCl, H2O, saturated aqueous
NaHCO3, and again H2O, followed by drying over MgSO4, filtration,
and evaporation of the filtrate under reduced pressure. Analytical
samples were dried over P2O5 under diminished pressure. AcmBr,
PomCl, montmorillonite K10, DBU, morpholine, ethylenediamine, 1,2-
phenylenediamine, THF, LAH, and selenium dioxide were purchased
from Sigma-Aldrich Company.
Dihydrobetulinic Acid (3a) and Dihydrobetulonic Acid (3b).
Compounds were prepared according to literature procedures.28,29
Allobetulin (4a). A solution of 2 (20 g, 45.2 mmol) in CHCl3 (1 L)
was vigorously stirred with montmorillonite K10 (50 g) under reflux
for 2 days.30,31 The solution was then filtered, CHCl3 was evaporated,
and the crude 4a was crystallized from butanone to give colorless
crystals (13.2 g, 66%): mp 264-266 °C; [R]25 +40 (c 0.35).30,31
D
Allobetulon 4b. Ac2O (2 mL), AcONa (2 g), and Na2Cr2O7‚2H2O
(3 g, 10.1 mmol) were added to a solution of 4a (10 g, 22.6 mmol) in
a mixture of toluene (70 mL) and AcOH (30 mL). The mixture was
worked up after 1 h. The crude product was chromatographed over
silica gel (150 g) eluted with toluene, and crystallization from toluene-
MeOH gave allobetulon 4b (3.7 g, 37% yield) as a white powder: mp
228-230 °C; [R]25 +83 (c 0.35).32
D
Diketone 4c. This compound was synthesized from 4a according
to literature procedures25 and was identical with an authentic sample.
Diketones 5a and 5b. These compounds were obtained from 2 using
four-step and seven-step reactions according to literature procedures18
and were identical with authentic samples.
General Procedure for Preparation of Pyrazines at the A-Ring.
Sulfur (1.5 g, 47 mmol) and ethylenediamine (1.5 mL, 25 mmol) were
added to a solution of each ketone (1b, 1c, 3b, 3c, 4b, and 4c; 5 mmol)
in morpholine (20 mL). Each mixture was heated under reflux for 2-4
h and then worked up. Crude products were purified by chromatography
on silica gel in a gradient from toluene to toluene-Et2O (10:1) and
then crystallized.
Betulinic Acid (1a) and Betulonic Acid (1b). Those compounds
were prepared according to literature procedures24 and were identical
with authentic samples.
Betulin (2). Bark of Betula pendula (500 g) was extracted with EtOH
in a Soxhlet extractor, and the solvent was evaporated to give crude 2
as a light brown powder (50 g, 10% yield). Several crystallizations
Pyrazine 6a. Pyrazine 6a was prepared from 1b (2.3 g, 5.1 mmol)
by the general procedure. Crystallization from MeOH yielded 6a (1 g,
from EtOH gave a white crystalline powder, mp 256-258 °C; [R]25
D
+16 (c 0.37).27
41%): mp 240-243 °C; [R]25 +27 (c 0.30); IR (CHCl3) νmax 3521,
D

Triterpenoid Pyrazines and Benzopyrazines with Cytotoxic ActiVity
Journal of Natural Products, 2007, Vol. 70, No. 4 529
Scheme 3. Preparation of Pyrazines and Benzopyrazines at Five-Membered Rings
1736, 1695, 1642 cm-1; 1H NMR δ 0.81, 1.02, 1.04, 1.27, 1.30 (15H,
all s, 5 × CH3), 1.72 (3H, s, H-30), 2.23-2.35 (2H), 2.46 (1H, d, J(H-
1a, H-1b) ) 16.6 Hz, H-1a), 3.04 (1H, td, J(H-19â, H-18R) ) 10.5
Hz, J(H-19â, H-21R) ) 10.5 Hz, J(H-19â, H-21â) ) 5.2 Hz, H-19â),
3.05 (1H, d, J(H-1b, H-1a) ) 16.6 Hz, H-1b), 4.65 (1H, m, H-29 pro-
E), 4.77 (1H, bd, J ) 2.3 Hz, H-29 pro-Z), 8.29 (1H, d, J ) 2.4 Hz),
8.42 (1H, dd, J1 ) 2.4 Hz, J2 ) 0.9, 2 × H-pyrazine); EIMS m/z 490
[M]+ (39), 475 (100), 445 (19), 431 (16), 257 (30), 243 (28), 189 (33);
anal C 78.25%, H 9.58%, N 5.58%; calcd for C32H46N2O2, C 78.31%,
H 9.45%, N 5.71%.
H-1a) ) 16.6 Hz, H-1b), 4.64 (1H, m, H-29 pro-E), 4.76 (1H, bd, J )
2.3 Hz, H-29 pro-Z), 5.77 (1H, d, J ) 5.5 Hz), 5.81 (1H, d, J ) 5.5
Hz, CH2-Pom), 8.27 (1H, d, J ) 2.4 Hz), 8.41 (1H, dd, J1 ) 2.4 Hz,
J2 ) 0.9 Hz, 2 × H-pyrazine); EIMS m/z 604 [M]+ (88), 589 (76),
574 (14), 489 (33), 475 (22), 445 (100), 429 (23), 401 (43); anal C
75.39%, H 9.26%, N 4.51%; calcd for C38H56N2O4, C 75.46%, H 9.33%,
N 4.63%.
Acm Ester 6d. AcmBr (0.1 mL, 0.78 mmol) and DBU (0.1 mL,
0.60 mmol) were added to a solution of 6a (150 mg, 0.31 mmol) in
CH2Cl2 (3 mL) and MeCN (1 mL). The reaction was stirred at rt for
14 h and then worked up. The crude product was purified by HPLC in
25% EtOAc in hexane and crystallized from MeOH-pentane to give
Methyl Ester 6b. Methyl ester 6b was prepared by the general
procedure from crude 1c, which was previously obtained from acid 1b
(4.5 g, 9.9 mmol). This yielded 6b (4.2 g, 85%) as colorless crystals:
mp 215-216 °C (MeOH); [R]25D +24 (c 0.38); IR (CHCl3) νmax 1642,
needles of 6d (100 mg, 58%): mp 63-66 °C (MeOH-pentane); [R]25
D
+21 (c 0.24); IR (CHCl3) νmax 1748b, 1643 cm-1; H NMR δ 0.81,
1
1
1720 cm-1; H NMR δ 0.81, 1.00, 1.02, 1.28, 1.30 (15H, all s, 5 ×
1.01, 1.02, 1.28, 1.30, (15H, all s, 5 × CH3), 1.71 (3H, s, H-30) 2.11
(3H, s, CH3-Acm), 2.22-2.33 (2H), 2.45 (1H, d, J(H-1a, H-1b) ) 16.5
Hz, H-1a), 3.02 (1H, td, J(H-19â, H-18R) ) 11.1 Hz, J(H-19â, H-21R)
) 11.1 Hz, J(H-19â, H-21â) ) 4.1 Hz, H-19â), 3.04 (1H, d, J(H-1b,
H-1a) ) 16.6 Hz, H-1b), 4.64 (1H, m, H-29 pro-E), 4.77 (1H, bd, J )
2.3 Hz, H-29 pro-Z), 5.73 (1H, d, J ) 5.5 Hz), 5.82 (1H, d, J ) 5.5
Hz, CH2-Acm), 8.27 (1H, d, J ) 2.4 Hz), 8.41 (1H, dd, J1 ) 2.5 Hz,
J2 ) 0.8 Hz, 2 × H-pyrazine); EIMS m/z 562 [M]+ (74), 547 (56),
502 (100), 489 (85), 475 (31), 445 (21), 429 (17), 401 (20); anal C
74.65%, H 9.07%, N 4.88%; calcd for C35H50N2O4, C 74.70%, H 8.95%,
N 4.98%.
CH3), 1.71 (3H, s, H-30), 2.23-2.33 (2H), 2.45 (1H, d, J(H-1a, H-1b)
) 16.6 Hz, H-1a), 3.02 (1H, td, J(H-19â, H-18R) ) 11.0 Hz, J(H-
19â, H-21R) ) 11.0 Hz, J(H-19â, H-21â) ) 4.3 Hz, H-19â), 3.04
(1H, d, J(H-1b, H-1a) ) 16.5 Hz, H-1b), 3.68 (3H, s, O-CH3), 4.64
(1H, m, H-29 pro-E), 4.77 (1H, bd, J ) 2.3 Hz, H-29 pro-Z), 8.27
(1H, d, J ) 2.4 Hz), 8.41 (1H, dd, J1 ) 2.4 Hz, J2 ) 0.9 Hz, 2 ×
H-pyrazine); EIMS m/z 504 [M]+ (67), 489 (100), 445 (23), 422 (10),
256 (47), 211 (9), 189 (21); anal C 78.52%, H 9.29%, N 5.61%; calcd
for C33H48N2O2, C 78.53%, H 9.59%, N 5.55%.
Pom Ester 6c. PomCl (0.1 mL, 0.59 mmol) and DBU (0.1 mL,
0.60 mmol) were added to a solution of 6a (150 mg, 0.31 mmol) in
CH2Cl2 (3 mL) and MeCN (1 mL). The reaction was stirred at rt for
14 h and then worked up. The crude product was purified by HPLC in
15% EtOAc in hexane. Crystallization from MeOH gave colorless
needles of 6c (130 mg, 70%): mp 88-91 °C (MeOH); [R]25D +22 (c
0.32); IR (CHCl3) νmax 1748b, 1643 cm-1; 1H NMR δ 0.81, 1.01, 1.02,
(9H, all s, 3 × CH3), 1.23 (9H, s, 3 × CH3-Pom), 1.28, 1.30 (6H, all
s, 2 × CH3), 1.71 (3H, m, H-30), 2.45 (1H, d, J(H-1a, H-1b) ) 16.6
Hz, H-1a), 2.99 (1H, td, J(H-19â, H-18R) ) 11.1 Hz, J(H-19â, H-21R)
) 11.1 Hz, J(H-19â, H-21â) ) 4.7 Hz, H-19â), 3.04 (1H, d, J(H-1b,
Pyrazine 6e. This compound was prepared from acid 3b (0.5 g, 1.1
mmol) by the general procedure. Crystallization from CHCl3-MeOH
yielded 6e (0.3 g, 55%) as white crystals: mp 267-269 °C; [R]25D -8
1
(c 0.25); IR (CHCl3) νmax 3518, 1734, 1693 cm-1; H NMR δ 0.78
(3H, d, J ) 6.8 Hz), 0.82, 0.88 (3H, d, J ) 7.2 Hz), 1.02 (6H, m), 1.27
(3H, s), 1.31 (3H, s, 7 × CH3), 2.29 (3H, m), 2.49 (1H, d, J(H-1a,
H-1b) ) 16.8 Hz, H-1a), 3.10 (1H, d, J(H-1b, H-1a) ) 16.6 Hz), 8.30
(1H, d, J ) 2.4 Hz), 8.45 (1H, bd, J ) 2.0 Hz, 2 × H-pyrazine);
FABMS m/z 493 [M + H]+, 477, 428, 303; anal C 78.00%, H 9.82%,
N 5.69%; calcd for C32H48N2O2, C 77.70%, H 9.97%, N 5.75%

530 Journal of Natural Products, 2007, Vol. 70, No. 4
Urban et al.
Table 2. Cytotoxic Activity of Selected Compounds on Additional Cell Lines
IC50 (µmol/La)
compound
A 549
HT 29
K 562
K 562-tax
PC-3
SK-MEL2
U87-Mg
159
B2-4
C-D-B
5
1b
5a
5b
6a
6c
6d
6e
6f
7a
7b
7c
13
15
103
13
0.25
51
17
250
7
16
250
77
2
164
96
250
123
229
6
250
6
0.77
100
2
0.4
201
86
245
113
70
17
250
9
8
149
35
4
91
118
250
117
234
15
208
14
11
250
35
17
227
64
250
103
233
26
170
14
98
250
69
74
1
8
19
231
0.3
94
13
235
220
48
250
122
101
192
231
250
148
47
99
250
198
a The lowest concentration that kills 50% of tumor cells.
Methyl Ester 6f. Ester 6f was prepared by the general procedure
from 3c, which was previously obtained by the reaction of acid 3b
(0.5 g, 1.1 mmol) with diazomethane. Crystallization from MeOH
yielded 6h (0.4 g, 74%) as colorless crystals: mp 121-123 °C (MeOH);
s), 1.44 (3H, s), 1.45 (3H, s), 1.44 (3H, s, 7 × CH3), 2.28 (3H), 2.66
(1H, d, J(H-1a, H-1b) ) 16.8 Hz, H-1a), 3.51 (1H, bd, J(H-1b, H-1a)
) 16.8 Hz, H-1b), 3.66 (3H, s, OCH3), 7.70 (2H, m), 8.06 (1H, m),
8.13 (1H, m, 4 × H-benzopyrazine); FABMS m/z 557 [M + H]+, 477,
471; anal C 79.64%, H 9.64%, N 4.97%; calcd for C37H52N2O2, C
79.81%, H 9.41%, N 5.03%.
[R]25 -7 (c 0.29) (lit.23 mp 220 °C). Although the mp is different
D
1
from the literature value, the H NMR and MS are identical.
General Procedure for Preparation of Benzopyrazines at the
A-Ring. Sulfur (6.0 g, 141 mmol) and 1,2-phenylenediamine (2.0 g,
18.5 mmol) were added to a solution of each ketone 1b, 1c, 3b, 3c,
and 4b (4.5 mmol) in morpholine (20 mL). The mixture was stirred
under reflux for 2-4 h and then worked up. Crude products were
chromatographed on silica gel (150 g) in a gradient from toluene to
toluene-Et2O (10:1) and then crystallized.
Pyrazine 8. This compound was prepared from 4b (5.0 g, 11.3
mmol) by the general procedure. Crystallization from CHCl3-MeOH
yielded 8 (4.1 g, 76%) as pale yellow crystals: mp 270-273 °C ; [R]25
D
+53 (c 0.25); IR (CHCl3) νmax 1452, 1403 cm-1 22
.
Benzopyrazine 9. This compound was prepared from allobetulon
4b (5 g, 11.3 mmol) by the general procedure. The crystallization from
CHCl3-MeOH yielded pyrazine 9 (4.4 g, 74%) as yellow crystals: mp
Benzopyrazine 7a. This compound was prepared from 1b (2.0 g,
4.5 mmol) by the general procedure. Crystallization from CHCl3-
MeOH yielded 7a (1.3 g, 55%) as yellow crystals: mp 305-307 °C;
272 - 275 °C; [R]25D +62 (c 0.35); IR (CHCl3) νmax 1487, 1456 cm-1 21
.
General Procedure for Reduction Methyl Esters. LAH (1.5 g,
39.5 mmol) was added to a solution of each ester (6b, 6f, 7b, 7d; 1.0
mmol) in THF (20 mL), and the mixture was stirred for 30 min under
reflux. Excess LAH was decomposed by addition of EtOAc (5 mL)
and EtOH (5 mL), and the mixture was worked up. Crude hydroxyl
derivatives were chromatographed and crystallized.
[R]25 +49 (c 0.42); IR (CHCl3) νmax 3513, 1736, 1694, 1641 cm-1
;
D
1H NMR δ 0.85, 1.05, 1.06, 1.39, 1.42 (15H, all s, 5 × CH3), 1.73
(3H, s, H-30), 1.97-2.09 (2H), 2.23-2.37 (2H), 2.60 (1H, d, J(H-1a,
H-1b) ) 17.6 Hz, H-1a), 3.04 (1H, td, J(H-19â, H-18R) ) 11.7 Hz,
J(H-19â, H-21R) ) 11.7 Hz, J(H-19â, H-21â) ) 4.7 Hz, H-19â), 3.34
(1H, d, J(H-1b, H-1a) ) 16.2 Hz, H-1b), 4.66 (1H, m, H-29 pro-E),
4.78 (1H, bd, J ) 2.0 Hz, H-29 pro-Z), 7.65 (2H, m), 7.99 (2H, m, 4
× H-benzopyrazine); EIMS m/z 540 [M]+ (41), 525 (100), 496 (19),
481 (27), 427 (16), 413 (6), 368 (8), 307 (24), 293 (20), 237 (24), 223
(33),209 (31), 183 (16); anal C 80.05%, H 8.91%, N 5.18%; calcd for
C36H48N2O2, C 79.96%, H 8.95%, N 5.18%.
Pyrazine 10a. Pyrazine 10a was prepared from ester 6b (0.5 g, 1.0
mmol) by the general procedure. HPLC in a 20% solution of EtOAc
in hexane followed by crystallization from MeOH yielded 10a (210
mg, 44%) as colorless crystals: mp 247-249 °C (MeOH); [R]25D +29
1
(c 0.28); IR (CHCl3) νmax 3625, 1640 cm-1; H NMR δ 0.82, 1.04,
1.11, 1.30, 1.32 (15H, all s, 5 × CH3), 1.71 (3H, s, H-30), 1.80-2.06
(3H), 2.36-2.46 (1H, H-19â), 2.48 (1H, d, J(H-1a, H-1b) ) 16.6 Hz,
H-1a), 3.11 (1H, d, J(H-1b, H-1a) ) 16.6 Hz, H-1b), 3.36 (1H, d, J(H-
28a, H-28b) ) 10.8 Hz, H-28a), 3.82 (1H, d, J(H-28b, H-28a) ) 10.8
Hz, H-28b), 4.62 (1H, m, H-29 pro-E), 4.78 (1H, bd, J ) 2.0 Hz, H-29
pro-Z), 8.29 (1H, bd, J ) 2.4 Hz), 8.48 (1H, bs, 2 × H-pyrazine);
FABMS m/z 477 [M + H]+, 461, 445; anal C 80.47%, H 9.95%, N
5.67%; calcd for C32H48N2O, C 80.62%, H 10.15%, N 5.88%.
Pyrazine 10b. This compound was prepared from ester 6f (0.5 g,
1.0 mmol) by the general procedure. Chromatography on silica gel (50
g) in toluene-Et2O (10:1) and lyophilization from t-BuOH yielded 10b
Methyl Ester 7b. Methyl ester 7b was prepared by the general
procedure from methyl ester 1c, which was previously obtained by the
reaction of acid 1b (2.0 g, 4.3 mmol) with diazomethane. Crystallization
from MeOH yielded 7b (1.3 g, 55%) as needles: mp 161-163 °C;
1
[R]25 + 37 (c 0.34); IR (CHCl3) νmax 1720, 1644 cm-1; H NMR δ
D
0.84, 1.02, 1.04, 1.42, 1.43 (15H, all s, 5 × CH3), 1.72 (3H, s, H-30),
1.84-1.98 (2H), 2.20-2.36 (2H), 2.60 (1H, d, J(H-1a, H-1b) ) 16.5
Hz, H-1a), 3.02 (1H, td, J(H-19â, H-18R) ) 11.0 Hz, J(H-19â, H-21R)
) 11.0 Hz, J(H-19â, H-21â) ) 4.4 Hz, H-19â), 3.34 (1H, bd, J(H-1b,
H-1a) ) 16.0 Hz, H-1b), 3.68 (3H, s, OCH3), 4.65 (1H, m, H-29 pro-
E), 4.78 (1H, bd, J ) 2.0 Hz, H-29 pro-Z), 7.66 (2H, m), 8.01 (2H, m,
4 × H-benzopyrazine); FABMS m/z 555 [M + H]+, 539, 513; anal C
79.87%, H 9.21%, N 5.32%; calcd for C37H50N2O2, C 80.10%, H 9.08%,
N 5.05%.
(340 mg, 72%): mp 231-233 °C, [R]25 +7 (c 0.50); IR (KBr) νmax
D
3626, 1672 cm-1; H NMR δ 0.79 (d, J(H-29, H-20) ) 6.8 Hz), 0.83
1
(s), 0.87 (d, J(H-30, H-20) ) 6.8 Hz), 1.02 (s), 1.11 (s), 1.29 (s), 1.31
(s) (21H, 7 × CH3), 2.47 (1H, d, J (H-1a, H-1b) ) 16.6 Hz, H-1a),
3.04 (1H, d, J(H-1b, H-1a) ) 16.5 Hz, H-1b), 3.33 (1H, d, J(H-28a,
H-28b) ) 11.0 Hz, H-28a), 3.80 (1H, d, J(H-28b, H-28a) ) 10.8 Hz,
H-28b), 8.28 (1H, d, J ) 2.8 Hz, H-pyrazine), 8.42 (1H, m, H-pyrazine);
EIMS m/z 478 [M]+ (24), 463 (23), 447 (100), 433 (41), 421 (19), 417
(10), 413 (82), 405 (74), 393 (51); anal C 80.07%, H 10.32%, N 5.74%;
calcd for C32H50N2O, C 80.28%, H 10.53%, N 5.85%.
Benzopyrazine 7c. This compound was prepared from 3b (1.0 g,
2.2 mmol) by the general procedure. Crystallization from MeOH yielded
7c (0.7 g, 59%) as colorless crystals: mp 204-207 °C; [R]25D +11 (c
1
0.38); IR (CHCl3) νmax 3515, 1732, 1694 cm-1; H NMR δ 0.79 (3H,
d, J ) 6.4 Hz), 0.86 (3H, s), 0.88 (3H, d, J ) 6.4 Hz), 1.03 (3H, s),
1.04 (3H, s), 1.41 (3H, s), 1.44 (3H, s, 7 × CH3), 2.28 (3H), 2.65 (1H,
d, J(H-1a, H-1b) ) 16.4 Hz, H-1a), 3.46 (1H, bd, J(H-1b, H-1a) )
14.4 Hz, H-1b), 7.70 (2H, m), 8.06 (2H, m, 4 × H-benzopyrazine);
FABMS m/z 543 [M + H]+, 527, 497, 457; anal C 79.53%, H 9.34%,
N 5.21%; calcd for C36H50N2O2, C 79.66%, H 9.28%, N 5.16%.
Methyl Ester 7d. This compound was prepared by the general
procedure from crude 3c, which was obtained by reaction of acid 3b
(1.1 g, 2.3 mmol) with diazomethane. This yielded 7d (0.5 g, 38%) as
Benzopyrazine 11a. This compound was prepared from ester 7b
(0.5 g, 0.9 mmol) by the general procedure. Chromatography on silica
gel (50 g) in toluene-Et2O (10:1) and lyophyllization from t-BuOH
yielded 11a (320 mg, 67%): mp 138-142 °C; [R]25D +42 (c 0.25); IR
(CHCl3) νmax 3624, 1637 cm-1; 1H NMR δ 0.85, 1.05, 1.13, 1.43, 1.44
(15H, all s, 5 × CH3), 1.72 (3H, s, H-30), 2.48 (1H, td, J(H-19â, H-18R)
) 11.0 Hz, J(H-19â, H-21R) ) 11.0 Hz, J(H-19â, H-21â) ) 4.4 Hz,
H-19â), 2.61 (1H, d, J(H-1a, H-1b) ) 16.6 Hz, H-1a), 3.39 (1H, vbs,
H-1b), 3.88 (1H, d, J(H-28a, H-28b) ) 11.2 Hz, H-28a), 3.82 (1H, d,
J(H-28b, H-28a) ) 11.2 Hz, H-28b), 4.64 (1H, m, H-29 pro-E), 4.73
(1H, bd, J ) 2.0 Hz, H-29 pro-Z), 7.68 (2H, m), 8.04 (2H, m, 4 ×
colorless crystals: mp 153-155 °C (lyophilized from t-BuOH); [R]25
D
+11 (c 0.27); IR (CHCl3) νmax 1718 cm-1; H NMR δ (50 °C) 0.79
1
(3H, d, J ) 6.8 Hz), 0.86 (3H, s), 0.89 (3H, d, J ) 7.2 Hz), 1.02 (6H,

Triterpenoid Pyrazines and Benzopyrazines with Cytotoxic ActiVity
Journal of Natural Products, 2007, Vol. 70, No. 4 531
H-benzopyrazine); FABMS m/z 527 [M + H]+, 511; anal C 82.46%,
H 9.21%, N 5.59%; calcd for C36H50N2O, C 82.08%, H 9.57%, N
5.32%.
mmol) in xylene (10 mL). The mixture was refluxed for 4 h and then
worked up. Crystallization from CHCl3-MeOH yielded 16 (0.6 g, 53%)
as yellow crystals: mp 215-217 °C; [R]25D -115 (c 0.29); IR (CHCl3)
1
Benzopyrazine 11b. This compound was prepared from ester 7d
(0.5 g, 0.9 mmol) by the general procedure. Chromatography on silica
gel (50 g) in toluene-Et2O (10:1) and crystallization yielded 11b (250
mg, 53%): mp 170-174 °C; [R]25D +4 (c 0.23); IR (CHCl3) νmax 3626
cm-1; 1H NMR δ 0.80 (d, J(H-29, H-20) ) 6.4 Hz), 0.86 (s), 0.88 (d,
J(H30, H-20) ) 6.4 Hz), 1.04 (s), 1.13 (s), 1.43 (s), 1.44 (s) (21H, 7
× CH3), 2.61 (1H, d, J (H-1a, H-1b) ) 16.3 Hz, H-1a), 3.35 (1H, d,
J(H-1b, H-1a) ) 16.3 Hz, H-1b), 3.32 (1H, d, J(H-28a, H-28b) ) 10.8
Hz, H-28a), 3.81 (1H, d, J(H-28b, H-28a) ) 10.8 Hz, H-28b), 7.66
(2H, m), 7.94-8.06 (2H, m, 4 × H-benzopyrazine); EIMS m/z 528
[M]+ (42), 513 (37), 497 (100), 483 (48). C 82.03%, H 9.58%, N 5.61%;
calcd for C36H52N2O, C 81.77%, H 9.91%, N 5.30%.
νmax 1728, 1586, 1567 cm-1; H NMR δ 0.86, 0.92, 0.94, 1.11 (15H,
all s, 5 × CH3), 1.53 (3H, d, J(H-20, H-29) ) 6.8 Hz, H-29), 1.58
(3H, d, J(H-20, H-30) ) 6.8 Hz, H-30), 2.06 (3H, s, Ac), 2.16-2.24
(1H), 2.79 (1H, dd, J1 ) 12.8 Hz, J2 ) 3.2 Hz, H-13), 2.96 (1H, dm,
J ) 12.1 Hz), 3.62 (1H, septet, J ) 6.7 Hz, H-20), 3.63 (3H, s, O-CH3),
4.50 (1H, m, H-3), 7.58-7.69 (2H), 8.05 (2H, dm, J ) 7.9 Hz, 4 ×
H-benzopyrazine); EIMS m/z 612 [M]+ (100), 597 (8), 553 (12), 349
(19), 289 (17),259 (8); anal C 76.52%, H 8.43%, N 4.47%; calcd for
C39H52N2O4, C 76.43%, H 8.55%, N 4.57%.
Pyrazine 17. This compound was prepared from diketone 4c (1.0
g, 2.2 mmol) by the general procedure for a preparation of pyrazines
at the A-ring. This yielded pyrazine 17 (0.61 g, 58% yield): mp 202-
Pyrazine 12. This compound was prepared from diketone 5a (1.5
g, 2.7 mmol) by the general procedure. Before the purification, the
crude product was reacetylated with Ac2O in pyridine. This yielded
203 °C (MeOH); [R]25 -12 (c 0.37); IR (CHCl3) νmax 1619, 1535,
D
1032, 908 cm-1; 1H NMR δ 0.72, 0.76, 0.94, 1.23, 1.29 (15H, all s, 5
× CH3), 1.38 (3H, d, J(H-23, H-4) ) 6.8 Hz, H-23), 1.41 (3H, d, J(H-
24, H-4) ) 6.8 Hz, H-24), 2.40 (1H, td, J1 ) 13.3 Hz, J2 ) 4.1 Hz,
H-6a), 2.51 (1H, m, H-11R), 2.81 (1H, dt, J1 ) 13.3 Hz, J2 ) 3.7 Hz,
H-6b), 3.19 (1H, septet, all J ) 6.9 Hz, H-4), 3.47 (1H, d, J(H-28
pro-R, H-28 pro-S) ) 7.6 Hz, H-28 pro-R), 3.55 (1H, s, H-19R), 3.80
(1H, bd, J(H-28 pro-S, H-28 pro-R) ) 7.6 Hz, H-28 pro-S), 8.07 (1H,
d, J1 ) 2.9 Hz) , 8.27 (1H, d, J1 ) 2.9 Hz, 2 × H-pyrazine); EIMS m/z
474 [M]+ (85), 459 (11), 432 (10), 401 (5), 28 (8), 255 (11), 211 (15),
188 (100); anal C 81.03%, H 9.72%, N 5.93%; calcd for C36H52N2O4,
C 80.96%, H 9.77%, N 5.90%.
pyrazine 12 (0.8 g, 47%): mp 242-246 °C (MeOH); [R]25 -94 (c
D
0.53); IR (CHCl3) νmax 1743, 1728, 1596, 1538 cm-1; 1H NMR δ 0.85,
0.86, 0.96, 1.23 (15H, all s, 5 × CH3), 1.41 (3H, d, J(H-20, H-29) )
6.8 Hz, H-29), 1.44 (3H, d, J(H-20, H-30) ) 6.8 Hz, H-30), 1.78 (3H,
s, Ac), 2.06 (3H, s, Ac), 2.20 (1H), 2.98 (1H, dd, J1 ) 12.7 Hz, J2 )
3.4 Hz, H-13), 3.56 (1H, septet, all J ) 7.0 Hz, H-20), 4.37 (1H, d, J
) 10.8 Hz, H-28a), 4.50 (1H, m, H-3), 4.79 (1H, d, J ) 10.8 Hz,
H-28b), 8.12 (1H, d, J ) 2.9 Hz), 8.32 (1H, d, J ) 2.9 Hz, 2 ×
H-pyrazine); EIMS m/z 576 [M]+ (100), 561 (11), 516 (10), 503 (8),
473 (6), 443 (8), 312 (19), 299 (7), 253 (12), 183 (11); anal C 74.85%,
H 9.07%, N 4.91%; calcd for C36H52N2O4, C 74.96%, H 9.09%, N
4.86%.
Benzopyrazine 18. 1,2-Phenylenediamine (0.3 g, 2.88 mmol) and
sulfur (0.8 g, 25 mmol) were added to a solution of 4c (0.3 g, 0.66
mmol) in xylene (6 mL). The mixture was refluxed for 4 h and then
worked up. Crystallization from CHCl3-MeOH yielded 18 (0.22 g,
Benzopyrazine 13. 1,2-Phenylenediamine (2.0 g, 18.5 mmol) and
sulfur (6.0 g, 141 mmol) were added to a solution of diketone 5a (2.0
g, 3.6 mmol) in xylene (20 mL). The mixture was refluxed for 4 h and
then worked up. The crude product was chromatographed over silica
gel (50 g) eluted with toluene-Et2O (15:1). Crystallization from EtOAc
yielded benzopyrazine 13 (1.6 g, 71%) as colorless needles: mp 280-
63%) as white crystals: mp 197-199 °C; [R]25 -63 (c 0.425); IR
D
(CHCl3) νmax 1619, 1568, 1032 cm-1; 1H NMR δ 0.72, 0.75, 0.94, 1.33,
1.34 (15H, all s, 5 × CH3), 1.45 (3H, d, J(H-23, H-4) ) 6.8 Hz, H-23),
1.49 (3H, d, J(H-24, H-4) ) 6.8 Hz, H-24), 2.49 (1H, td, J1 ) 13.3
Hz, J2 ) 4.1 Hz, H-6a), 2.78 (1H, dq, J1 ) 12.4 Hz, J2 ) 5.8 Hz,
H-11R), 2.89 (1H, dt, J1 ) 13.3 Hz, J2 ) 3.4 Hz, H-6b), 3.30 (1H,
septet, all J ) 6.9 Hz, H-4), 3.47 (1H, d, J(H-28 pro-R, H-28 pro-S)
) 7.6 Hz, H-28 pro-R), 3.57 (1H, s, H-19R), 3.81 (1H, d, J(H-28 pro-
S, H-28 pro-R) ) 7.6 Hz, H-28 pro-S), 7.56-7.65 (2H), 8.00 (2H, d,
J ) 8.6 Hz, 4 × H-benzopyrazine); EIMS m/z 524 [M]+ (80), 509
(66), 481 (73), 373 (7), 319 (8), 261 (24), 236 (96), 221 (100), 196
(91); anal C 76.52%, H 8.43%, N 4.47%; calcd for C39H52N2O4, C
82.44%, H 9.18%, N 5.36%; calcd for C39H52N2O4, C 82.39%, H 9.22%,
N 5.34%.
282 °C; [R]25D -59 (c 0.39); IR (CHCl3) νmax 1727, 1585, 1569 cm-1
;
1H NMR δ 0.86, 0.89, 0.96, 1.24 (15H, all s, 5 × CH3), 1.52 (3H, d,
J(H-20, H-29) ) 6.4 Hz, H-29), 1.54 (3H, d, J(H-20, H-30) ) 6.4 Hz,
H-30), 1.74 (3H, s, Ac), 2.06 (3H, s, Ac), 2.28 (1H), 3.07 (1H, dd, J1
) 12.6 Hz, J2 ) 3.2 Hz, H-13), 3.62 (1H, septet, all J ) 7.0 Hz, H-20),
4.42 (1H, d, J ) 10.9 Hz, H-28a), 4.51 (1H, m, H-3), 5.00 (1H, d, J
) 10.7 Hz, H-28b), 7.64 (2H, m), 8.03 (2H, m, 4 × H-benzopyrazine);
EIMS m/z 626 [M]+ (100), 611 (16), 583, (4), 567 (21), 553 (9), 523
(5), 493, (4), 363 (19), 349 (5); anal C 76.55%, H 8.53%, N 4.52%;
calcd for C40H54N2O4, C 76.64%, H 8.68%, N 4.47%.
Cell Lines. CEM, A 549, HT 29, K 562, PC-3, SK-Mel2, and U87-
Mg cell lines were purchased from the American Tissue Culture
Collection (ATTC). Paclitaxel/daunorubicin-resistant sublines of K 562/
CEM cells were prepared and characterized in our laboratories. The
human T-lymphoblastic leukemia cell line, CEM, was used for routine
screening of compounds. To prove a common mechanism of action,
selected compounds, which showed activity in the screening assay, were
further tested in a panel of cell lines. These lines were of various
histogenetic origin and drug resistance profiles. The cells were
maintained in Nunc/Corning 80 cm2 plastic tissue culture flasks and
cultured in cell culture medium (DMEM/RPMI 1640 with 5 g/L
glucose, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL strepto-
mycin, 10% fetal calf serum, and NaHCO3).
Pyrazine 14. This compound was prepared from diketone 5b (1.5
g, 2.8 mmol) by the general procedure. Before purification, the crude
product was reacetylated with Ac2O in pyridine. Crystallization from
MeOH yielded 14 (0.8 g, 57%): mp 224-226 °C; [R]25D +73 (c 0.42);
1
IR (CHCl3) νmax 1724, 1620 cm-1; H NMR δ 0.34 (3H, d, J(H-20,
H-29) ) 7.2 Hz, H-29), 0.87, 0.93, 0.94, 1.08 (15H, all s, 5 × CH3),
1.42 (3H, d, J(H-20, H-30) ) 6.8 Hz, H-30), 2.06 (3H, s, Ac), 2.30-
2.50 (2H), 2.56-2.73 (2H), 3.35 (1H, m, H-20), 4.49 (1H, m, H-3),
8.14 (1H, d, J ) 3.0 Hz), 8.22 (1H, d, J ) 3.1 Hz, 2 × H-pyrazine);
EIMS m/z 504 [M]+ (80), 489 (3), 461 (12), 444 (9), 429 (7), 419 (3),
401, (10), 309 (4), 174 (100); anal C 78.39%, H 9.51%, N 5.49%;
calcd for C33H48N2O2, C 78.53%, H 9.59%, N 5.55%.
Pyrazine 15. Ethylenediamine (0.7 mL, 11.7 mmol) and sulfur (2.0
g, 47 mmol) were added to a solution of diketone 5b (1.0 g, 1.9 mmol)
in xylene (10 mL). The mixture was refluxed for 4 h and then worked
up. The crude product was chromatographed over silica gel (50 g) eluted
with toluene. Crystallization from EtOAc yielded 15 (0.7 g, 67%) as
colorless crystals: mp 244-246 °C; [R]25D +136 (c 0.30); IR (CHCl3)
Cytotoxicity Assay. Cell suspensions were prepared and diluted
according to the particular cell type and the expected target cell density
(2500-30 000 cells/well based on cell growth characteristics). Cells
were added by pipet (80 µL) into 96-well microtiter plates. Inoculates
were allowed a preincubation period of 24 h at 37 °C and 5% CO2 for
stabilization. Four-fold dilutions, in 20 µL aliquots, of the intended
test concentration were added at time zero to the microtiter plate wells.
All compounds were dissolved in 10% DMSO and tested in quadru-
plicate. Incubation of the cells with the test compounds lasted for 72
h at 37 °C, in a 5% CO2 atmosphere at 100% humidity. At the end of
the incubation period, the cells were assayed using MTT. Aliquots (10
µL) of the MTT stock solution were pipetted into each well and
incubated for a further 1-4 h. After this incubation period the formazan
produced was dissolved by addition of 100 µL/well of 10% aqueous
SDS (pH ) 5.5), followed by a further incubation at 37 °C overnight.
The optical density (OD) was measured at 540 nm with a Labsystem
iEMS Reader MF. Tumor cell survival (TCS) was calculated using
1
νmax 1728, 1596, 1531, 1452 cm-1; H NMR δ 0.86, 0.88, 0.93, 1.09
(15H, all s, 5 × CH3), 1.42 (3H, d, J(H-20, H-30) ) 7.2 Hz, H-30),
1.48 (3H, d, J(H-20, H-29) ) 6.8 Hz, H-29), 2.06 (3H, s, Ac), 2.12-
2.23 (1H), 2.77 (1H, dd, J1 ) 12.8 Hz, J2 ) 3.1 Hz), 2.90 (1H, bd, J
) 13.0 Hz), 3.56 (1H, septet, J ) 7.0 Hz, H-20), 3.65 (3H, s, O-CH3),
4.50 (1H, m, H-3), 8.12 (1H, d, J ) 2.6 Hz), 8.37 (1H, d, J ) 2.8 Hz,
2 × H-pyrazine); EIMS m/z 562 [M]+ (100), 547 (7), 503 (8), 487 (3),
297 (11), 239 (18), 218 (11); anal C 74.79%, H 8.86%, N 4.95%; calcd
for C35H50N2O4, C 74.70%, H 8.95%, N 4.98%.
Benzopyrazine 16. 1,2-Phenylenediamine (1.0 g, 9.3 mmol) and
sulfur (3.0 g, 71 mmol) were added to a solution of 5b (1.0 g, 1.9

532 Journal of Natural Products, 2007, Vol. 70, No. 4
Urban et al.
(12) Fulda, S.; Jeremias, I.; Pietsch, T.; Debatin, K. M. Int. J. Cancer
1999, 82, 435-441.
the following equation: TCS ) (ODdrug-exposed well/mean ODcontrol wells
)
× 100%. The TCS50 value, the drug concentration lethal to 50% of the
(13) Kim, Y. K.; Yoon, S. K.; Ryu, S. Y. Planta Med. 2000, 66, 485-
tumor cells, was calculated from appropriate dose-response curves.
486.
(14) Schmidt, M. L.; Kuzmanoff, K. L.; Ling-Indeck, L.; Pezzuto, J. M.
Eur. J. Cancer 1997, 33, 2007-2010.
Acknowledgment. This study was supported in part by the Ministry
of Education of the Czech Republic MSM 6198959216 and MSM
0021620857, which paid for instrumental equipment, and the Czech
Science Foundation 203/03/D152, from which the chemicals and all
other material support were paid. Biological testing was supported by
the MPO FT-TA/027. All deuterosolvents for NMR spectra measure-
ments were paid from Czech Science Foundation 203/05/P025 and from
the Ministry of Education of the Czech Republic, project LC06070.
We are grateful to Prof. J. Klinot for the interpretations of NMR spectra
and to Dr. S. Hilgard for measurement of IR spectra. Special thanks to
Mgr. B. Sperlichova for measurement of optical rotations.
(15) Fulda, S.; Jeremias, I.; Pietsch, T.; Debatin, K. M. Klin. Pediatr.
1999, 211, 319-322.
(16) Ryu, S. Y.; Choi, S. U.; Lee, S. H.; Lee, Ch. O.; No, Z.; Ahn, J. W.
Arch. Pharm. Res. 1994, 17, 375-377.
(17) Hajduch, M.; Sarek, J. PCT Intl. Patent Appl. Publ. WO 01/90046,
CAN 136:6179, 2001.
(18) Sarek, J.; Klinot, J.; Brazinova, S.; Dzubak, P.; Klinotova, E.;
Noskova, V.; Krecek, V.; Korinkova, J.; Thomson, J. O.; Janostakova,
J.; Wang, S.; Parsons, S.; Fischer, P. M.; Zhelev, N. Z.; Hajduch,
M. J. Med. Chem. 2003, 46, 5402-5415.
(19) You, Y.-J.; Kim, Y; Nam, N.-H.; Ahn, B.-Z. Bioorg. Med. Chem.
Lett. 2003, 13, 3137-3140.
(20) Reddy, R. P.; Ravindranath, A.; Sudhakar, B.; Ramaiah, T. S.; Rao,
M. V. J. Ind. Chem. Soc. 1988, 65, 688-690.
References and Notes
(1) Dzubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.; Kvasnica, M.;
Biedermann, D.; Markova, L.; Urban, M.; Sarek, J. Nat. Prod. Rep.
2006, 23, 394-411.
(21) Huneck, S. Chem. Ber. 1965, 98, 1837-1857.
(22) Noskova, V.; Dzubak, P.; Kuzmina, G.; Ludkova, A.; Stehlik, D.;
Trojanec, R.; Janostakova, A.; Korinkova, G.; Mihal, V.; Hajduch,
M. Neoplasma 2002, 49, 418-425.
(2) Pettit, G. R.; Inoue, M.; Kamano, Y.; Herald, D. L.; Arm, C.;
Dufresne, C.; Christie, N. D.; Schmidt, J. M.; Doubek, D. L.; Krupa,
T. S. J. Am. Chem. Soc. 1988, 110, 2006-2007.
(23) Sejbal, J.; Klinot, J.; Protiva, J.; Vystrcil, A. Collect. Czech. Chem.
Commun. 1986, 51, 118-127.
(3) Heathcock, C. H.; Smith, S. C. J. Org. Chem. 1994, 59, 6828-6839.
(4) Kramer, A.; Ullmann, U.; Winterfeldt, E. J. Chem. Soc., Perkin Trans.
1 1993, 2865-2867.
(24) Pradhan, B. P.; Ghosh, P. Ind. J. Chem., Sect. B 1993, 32, 1068-
1069.
(25) Urban, M.; Sarek, J.; Klinot, J.; Korinkova, G.; Hajduch, M. J. Nat.
Prod. 2004, 67, 1100-1105.
(5) Morzycki, J. W.; Gryszkiewicz, A.; Lotowski, Z.; Szczepek, W. J.
Collect. Czech. Chem. Commun. 1998, 63, 1589-1596.
(6) Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Cosentino, L. M.;
Ballas, L. M.; Jiang, J. B.; Janzen, W. P.; Chen, I. S.; Lee, K. H. J.
Nat. Prod. 1994, 57, 243-247.
(26) Kvasnica, M.; Tislerova, I.; Sarek, J.; Sejbal, J.; Cisarova, I. Collect.
Czech. Chem. Commun. 2005, 70, 1447-1464.
(27) Urban, M.; Sarek, J; Tislerova, I; Dzubak, P.; Hajduch, M. Bioorg.
Med. Chem. 2005, 13, 5527-5535.
(7) Evers, M.; Poujade, C.; Soler, F.; Ribeill, Y.; James, C.; Lelicevre,
Y.; Gueguen, J. C.; Reisdorf, D.; Morize, I.; Pauwels, R.; De Clercq,
E.; Henin, Y.; Bousseau, A.; Mayaux, J. F.; Le Pecq, J. B.; Dereu,
N. J. Med. Chem. 1996, 39, 1056-1068.
(8) Setzer, W. N.; Setzer, M. C.; Bates, R. B.; Jackes, B. R. Planta Med.
2000, 66, 176-177.
(9) Costantini, P.; Jacotot, E.; Decaudin, D.; Kroemer, G. J. Natl. Cancer
Inst. 2000, 92, 1042-1053.
(10) Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.; Farnsworth, N.
R.; Cordell, G. A.; Beecher, C. W. W.; Fong, H. H. S.; Kinghorn,
A. D.; Brown, D. M.; Wani, M. C.; Wall, M. E.; Hieken, T. J.; Gupta,
T. K. D.; Pezzuto, J. M. Nat. Med. 1995, 1, 1046-1051.
(11) Jeong, H. J.; Chai, H. B.; Park, S. Y.; Kim, D. S. H. L. Bioorg.
Med. Chem. Lett. 1999, 9, 1201-1204.
(28) Hui, W.-H.; Li, M.-M. J. Chem. Soc., Perkin Trans. 1 1977, 8, 897-
904.
(29) Otsuka, H.; Fujioka, S.; Komiya, T.; Goto, M.; Hirmatsu, Y.;
Fujimora, H. Chem. Pharm. Bull. 1981, 29, 3099-3104.
(30) Hashimoto, F.; Kashiwada, Y.; Cosentino, L. M.; Chen, C. H.; Garrett,
P. E.; Lee, K. H. Bioorg. Med. Chem. 1997, 5, 2133-2143.
(31) Li, T. S.; Wang, J. X.; Zheng, X. J. Chem. Soc., Perkin Trans. 1
1998, 3957-3965.
(32) Biedermann, D.; Sarek, J.; Klinot, J.; Hajduch, M.; Dzubak, P.
Synthesis 2005, 7, 1157-1163.
(33) Huneck, S. Tetrahedron Lett. 1963, 4, 1977-1980.
NP060436D