Spirobipyridopyrans, spirobinaphthopyrans, indolinospiropyridopyrans, indolinospironaphthopyrans and indolinospironaphtho-1,4-oxazines: Synthesis, study of X-ray crystal structure, antitumoral and antiviral evaluation

Article abstract of DOI:10.1016/j.bmc.2003.12.010

The novel racemic indolinospirobenzopyrans (5-7), indolinospironaphthopyrans (11-14) and indolinospironaphtho-1,4-oxazine (17) were synthesized by an aldol type of condensation of 1′,3′,3′- trimethyl-2 ′-methyleneindoline and its 5-substituted derivatives with an appropriately substituted hydroxybenzaldehyde, hydroxynaphthaldehyde or nitrosonaphthol. An unequivocal proof of the stereostructures of 9 and 17 was obtained by the single-crystal X-ray diffraction method. A substituted indoline ring and the benzopyran ring in 9 and the naphtho-1,4-oxazine moiety in 17 are interconnected via the common chiral atom and positioned almost perpendicularly to each other. The five-membered 2,3-dihydropyrrolo moiety of the indoline ring adopts an envelope conformation in both structures. Of all the compounds of this series, spirobipyridopyran (1) inhibited specifically the growth of human melanoma (HBL) (IC₅₀: 0.9 μM) cells but not the growth of normal fibroblasts (WI38). Indolinospirobenzopyrans (8-10) showed significant cytostatic activities against all tumor cell lines. However, these compounds also exhibited a cytotoxic effect on normal human fibroblasts. The indolinospirobenzopyrans 4, 6-8, 10 and the indolinospironaphtho-1,4-oxazine 16 showed, albeit modest, selectivity as antiviral agents against varicella-zoster virus (VZV) and/or cytomegalovirus (CMV) (EC₅₀ within the concentration range of 1.0-12.6 μM).

Full text of DOI:10.1016/j.bmc.2003.12.010

Bioorganic & Medicinal Chemistry 12 (2004) 1037–1045
Spirobipyridopyrans, spirobinaphthopyrans,
indolinospiropyridopyrans, indolinospironaphthopyrans and
indolinospironaphtho-1,4-oxazines: synthesis, study of X-ray
crystal structure, antitumoral and antiviral evaluation
Silvana Raic-Malic,a Linda Tomaskovic,a,y Draginja Mrvos-Sermek,b
Biserka Prugovecki,b Mario Cetina,c Mira Grdisa,d Kresimir Pavelic,d
Albrecht Mannschreck,e Jan Balzarini,f Erik De Clercqf and Mladen Mintasa,*
a
´
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20,
PO Box 177, HR-10000 Zagreb, Croatia
bLaboratory of General and Inorganic Chemistry, Faculty of Science, University of Zagreb, Zvonimirova 8,
HR-10000 Zagreb, Croatia
cFaculty of Textile Technology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia
d
´
ˇ
Division of Molecular Medicine, Ruder Bosˇkovic Institute, Bijenicka 54, PO Box 1016, HR-10001 Zagreb, Croatia
¨
eInstitute of Organic Chemistry, University of Regensburg, Universitatsstr. 31, D-93040, Regensburg, Germany
fRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Belgium
Received 7 November 2003; accepted 9 December 2003
Abstract—The novel racemic indolinospirobenzopyrans (5–7), indolinospironaphthopyrans (11–14) and indolinospironaphtho-1,4-
0
oxazine (17) were synthesized by an aldol type of condensation of 10,30,30-trimethyl-2 -methyleneindoline and its 5-substituted
derivatives with an appropriately substituted hydroxybenzaldehyde, hydroxynaphthaldehyde or nitrosonaphthol. An unequivocal
proof of the stereostructures of 9 and 17 was obtained by the single-crystal X-ray diffraction method. A substituted indoline ring
and the benzopyran ring in 9 and the naphtho-1,4-oxazine moiety in 17 are interconnected via the common chiral atom and posi-
tioned almost perpendicularly to each other. The five-membered 2,3-dihydropyrrolo moiety of the indoline ring adopts an envelope
conformation in both structures. Of all the compounds of this series, spirobipyridopyran (1) inhibited specifically the growth of
human melanoma (HBL) (IC50: 0.9 mM) cells but not the growth of normal fibroblasts (WI38). Indolinospirobenzopyrans (8–10)
showed significant cytostatic activities against all tumor cell lines. However, these compounds also exhibited a cytotoxic effect on
normal human fibroblasts. The indolinospirobenzopyrans 4, 6–8, 10 and the indolinospironaphtho-1,4-oxazine 16 showed, albeit
modest, selectivity as antiviral agents against varicella-zoster virus (VZV) and/or cytomegalovirus (CMV) (EC50 within the con-
centration range of 1.0–12.6 mM).
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
information. In contrast, there is almost no information
on biological and pharmacological properties of this
class of compounds. The present study deals with var-
ious, structurally closely related types of the spiro-com-
pounds: spirobipyridopyran (1), spirobinaphthopyrans
(2 and 3) (Fig. 1), indolinospirobenzopyrans (4–10),
indolinospironaphthopyrans (11–14), indolinospiropyr-
idopyran (15) and indolinospironaphtho-1,4-oxazines
(16–18) (Fig. 2) and their cytostatic and antiviral activ-
ities. The principal aim of this study was to evaluate the
spiro-compounds (1–18) for their cytostatic and anti-
viral activities.
Chiral spiropyrans have been extensively investigated1À3
largely because of their thermo- and photochromic
properties and application in display and storage of
Keywords: Spiropyrans; 1H and 13C NMR spectra; Single crystal
X-ray analysis; Cytostatic activities; Antiviral activities.
* Corresponding author. Tel.: +385-1-4597-214; fax: +385-1-4597-
250; e-mail: mladen.mintas@fkit.hr
Present address: Pliva Research Institute, Prilaz baruna Filipovica
25, HR-1000 Zagreb.
y
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.12.010

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S. Raic-Malic et al. / Bioorg. Med. Chem. 12(2004) 1037–1045
1038
Figure 1. Spirobipyridopyrans 11 and spirobinaphthopyrans 22 and 32.
Figure 2. Indolinospirobenzopyrans 4–10, indolinospironaphthopyrans 11–14, indolinospiropyridopyran 15 and indolinospironaphtho-1,4-oxazines
16–18.
group and subsequent ring closure by intramolecular
addition of the phenolic oxygen to the immonium group
which gave under elimination of water the spiro-com-
pounds (5–7, 11–14 and 17).6
2. Results and discussion
2.1. Chemistry
The novel indolinospirobenzopyrans 5–7, indolinospir-
onaphthopyrans 11–14 and indolinospironaphtho-1,4-
oxazine 17 (Fig. 2) were synthesized by an aldol type of
condensation of equimolar amounts of 10,30,30-trimethyl-
20-methyleneindoline and its 50-substituted derivatives
with 2-hydroxybenzaldehyde substituted with a nitro or
methoxy group at position 6 or 7, 1-hydroxy-2-naph-
thaldehyde, 2-hydroxy-1-naphthaldehyde or 1-nitroso-
2-naphthol (Scheme 1). This reaction involves nucleo-
philic attack of the enamine to the carbonyl or nitroso
The structures of the novel compounds 5–7, 11–14 and
17 were elucidated by analysis of their one- and two-
1
dimensional H and 13C NMR spectra, mass spectra,
and elemental analyses (see Experimental). The 1H
NMR spectroscopic data of 5–7, 11–14 and 17 are dis-
played in Table 1. The chemical shifts and H–H coupling
constants are consistent with the proposed structures.
The chemical shift assignments are in agreement with
the corresponding ones of the related spiro-systems.1À3

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S. Raic-Malic et al. / Bioorg. Med. Chem. 12(2004) 1037–1045
1039
Scheme 1. Synthesis of indolinospirobenzopyrans 5–7, indolinospironaphtopyrans 11–14 and indolinospironaphtho-1,4-oxazine 17 (c.f. Figs 1 and 2).
2.2. X-Ray crystal structure analysis
the atoms N10/C7A0/C3A0/C30, and the plane defined by
the atoms N10/C20/C30 amounting to 32.7(2)ꢀ in 9 and
24.2(2)ꢀ in 17. This means that the deviation from the
planarity of the five-membered ring is more evident in 9
than in 17. The atoms N10/C7A0/C3A0/C30 of the five-
membered ring and the fused benzene ring via the com-
mon carbon atoms C3A0 and C7A0 are coplanar. The
dihedral angle between these mean planes is equal in
both structures [2.5(2)ꢀ]. The consequence of the five-
membered ring folding is an increase in pyramidality of
the indoline nitrogen atom N10, the sum of the angles
about this atom amounting to 347.4 and 349.7ꢀ in 9 and
17, respectively.
In order to determine the exact structures of the indoli-
nospirobenzopyran 9 and indolinospironaphthoxazine
17, their X-ray crystal structure analyses were per-
formed. The molecular structures of the racemic com-
pounds 9 and 17 with the atom numbering Schemes are
shown in Figures 3 and 4.
The analysis of the X-ray data shows that the skeleton
of the compound 9 consists of a substituted indoline
ring connected to a methoxy-substituted benzopyran
ring via the spiro-C20 atom. Similarly, in the compound
17, a substituted indoline ring is linked via the spiro-C20
atom to a naphtho-1,4-oxazine moiety. The dihedral
angles between the planes defined by the atoms N10/C20/
C30 and O1/C20/C3 amount to 87.4(2)ꢀ in 9 and 86.1(3)ꢀ
in 17. This means that the indoline ring and the benzo-
pyran moiety in 9, and the naphtho-1,4-oxazine moiety
3. Biological results
3.1. Cytostatic activities
in 17 are almost perpendicular to each other. The Cspiro
-
The compounds 1–18 were evaluated for their activities
against malignant tumor cell lines: murine leukemia
(L1210) and human T-lymphocytes (Molt4/C8 and
CEM), melanoma (HBL), cervical carcinoma (HeLa),
breast carcinoma (MCF7), colon carcinoma (HT29 and
SW620), laryngeal carcinoma (Hep2), pancreatic carci-
noma (MiaPaCa2) cell lines as well as normal human
fibroblasts (WI38) (Table 2). Of all compounds in the
series, spyrobipyridopyran 1 had the most pronounced
inhibitory activity against human melanoma (HBL)
cells (IC50: 0.9 mM). This compound also exhibited the
highest cytostatic selectivity: this compound inhibited
specifically the growth of human melanoma (HBL) cells
but not that of normal fibroblasts (WI38).
N bond lengths are equal in both spiro-compounds
[1.447(3) A]; the Cspiro–O bond lengths are also equal
within standard uncertainties amounting to 1.469(3) A
in 9 and 1.464(3) A in 17. These both lengths are in
agreement with those found in the structurally related
10,30,30 - trimethyl - 50 - nitro(indoline - 2,20 - (2H) - spiro-
benzopyran),7 10,30,30-trimethyl-7-methoxy(indoline-2,20-
(2H-1)spirobenzopyran),8 1,3,3-trimethyl(indoline-2,30-
(3H)-naphtho(2,1-b)(1,4)spirooxazine),9 and (1,3,3-tri-
methyl-6-nitroindoline-2,30-3H-naphtho(2,1-b)(1,4)spiro-
oxazine).10 The spiro carbon atom C20 in the five-
membered moiety of the indoline ring is out of the plane
defined by the atoms N10, C7A0, C3A0 and C30. It means
that the 2,3-dihydropyrrole ring in both structures adopts
an envelope conformation. This is shown by the values of
the dihedral angles between the mean plane defined by
Among other classes, indolinospirobenzopyrans (8–10)
showed significant cytostatic activities in all tumor cell

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S. Raic-Malic et al. / Bioorg. Med. Chem. 12(2004) 1037–1045
1040
Table 1. 1H NMR chemical shifts (d/ppm)a and coupling constants (J/Hz)b of compounds 5–7, 11–14 and 17
c
Compd
5
6
711
12
13
14
17
C-(CH3)2
d
d
1.20(s, 3H)
1.35(s,3H)
1.22(s, 3H)
1.34(s,3H)
1.18(s, 3H)
1.25(s,3H)
1.23(s, 3H)
1.35(s,3H)
1.25(s, 3H)
1.35(s,3H)
1.26(s, 3H)
1.40(s,3H)
1.20(s, 3H)
1.30(s,3H)
1.33(s, 3H)
1.36(s,3H)
N-CH3/
H-40
2.83(s,3H)
2.87(s,3H)
7.96(1H)
2.70(s,3H)
2.73(s,3H)
2.72(s,3H)
7.10(s, 1H)
2.85(s,3H)
2.70(s,3H)
2.74(s,3H)
7.03(1H)
d
J
6.1–8.4(1H)
6.3–7.3(1H)
7.12(1H)
6.4(d)
7.99(1H)
2.1(d)
6.2–8.2(1H)
H-50
H-60
d
J
—
—
—
6.88(1H)
6.8(t)
—
—
—
—
d
6.1–8.4(1H)
8.20(1H)
2.3; 8.7(dd)
6.3–7.3(1H)
7.18(1H)
m
7.18(1H)
8.7(d)
8.22(1H)
6.2–8.2(1H)
7.16(1H)
J
2.3;8.7(dd)
8.3(d)
H-70
H-3
H-4
H-5
H-6
H-7
d
J
6.1–8.4(1H)
6.80(1H)
6.3–7.3(1H)
6.54(1H)
7.6(d)
6.47(1H)
8.2(d)
6.51(1H)
8.7(d)
6.2–8.2(1H)
6.47(1H)
d
J
5.64(1H)
10(d)
5.85(1H)
10.4(d)
5.85(1H)
10(d)
5.71(1H)
10.2(d)
5.71(1H)
10.2(d)
5.69(1H)
10.1(d)
5.75(1H)
10.6(d)
—
—
d
J
6.1–8.4(1H)
6.1–8.4(1H)
6.1–8.4(1H)
6.1–8.4(1H)
7.0(1H)
8.03(1H)
—
6.3–7.3(1H)
6.3–7.3(1H)
—
6.97(1H)
10.1(d)
6.70(1H)
10.0(d)
7.04(1H)
10.2(d)
6.2–8.2(1H)
6.2–8.2(1H)
6.2–8.2(1H)
6.2–8.2(1H)
d
J
7.36(1H)
8.1(d)
7.35(1H)
8.5(d)
7.36(m,1H)
7.0(1H)
8.9(d)
d
J
7.39(1H)
8.1(d)
7.42(1H)
8.3(t)
7.42(m,1H)
7.68(1H)
8.9(d)
d
J
8.06(1H)
8.9(d)
6.3–7.3(1H)
7.70(1H)
8.1(d)
7.74(1H)
8.0(d)
7.73(1H)
8.2(d)
7.75(1H)
8.1(d)
H-8
H-9
d
6.1–8.4(1H)
—
6.54(1H)
—
6.3–7.3(1H)
—
7.30(m,1H)
7.9(m,1H)
7.33(m,1H)
7.32(m,1H)
6.2–8.2(1H)
6.2–8.2(1H)
7.40(1H)
d
J
7.21(1H)
8.5(d)
7.21(1H)
8.3(d)
7.58(1H)
8.5(d)
H-10
d
J
—
—
—
7.88(1H)
8.3(d)
7.91(1H)
8.2(d)
7.86(1H)
8.4(d)
6.2–8.2(1H)
8.55(1H)
a CDCl3, chemical shifts referred to TMS. Multiplicity of coupling and number of protons are given in parentheses.
b Digital resolutionÆ0,29 Hz.
c d=7.72 ppm for H-2.
Figure 3. The molecular structure and atom labelling of the indoli-
nospirobenzopyran 9. Displacement ellipsoid are drawn at the 30%
probability level.
Figure 4. The molecular structure and atom labelling of the indoli-
nospironaphtho-1,4-oxazine 17. Displacement ellipsoid are drawn at
the 30% probability level.

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1041
Table 2. Inhibitory effects of spirobipyridopyran 1, spirobinaphthopyrans 2 and 3 (c.f. Fig. 1), indolinospirobenzopyrans 4–10, indolinospiro-
naphthopyrans 11–14, indolinospiropyridopyran 15 and indolinospironaphtho-1,4-oxazines 16–18 (c.f. Fig. 2) on the growth of malignant tumor
cells lines in comparison with their effects on the growth of normal human fibroblasts (WI38)
Tumor cell growth IC50a (mM)
Comp
L1210
Molt4/C8
CEM
HBL
0.9
>1000
40
>1000
>1000
>1000
420
SW620
HeLa
MCF7
HT29
Hep2
MiaPaCa2
WI38
1
2
3
4
5
6
7
8
144.5
>500
>500
31.1
348.3
285.6
26.6
150.3
>500
>500
40.4
317.2
>500
11.5
14.5
>500
>500
24.9
48
>1000
54
>1000
>1000
>1000
380
600
>1000
>1000
105
>1000
>1000
>1000
10
2.0
15.8
>1000
>1000
>1000
890
>100
>1000
>1000
166
>1000
>1000
23.9
35
>1000
>1000
178
>1000
>1000
>1000
25
3.8
41.6
>1000
>1000
400
700
>1000
>1000
178
>1000
>1000
>1000
300
>1000
>1000
199
>1000
>1000
50.1
>1000
>1000
>1000
126
>1000
>1000
72.4
10
5.6
29.5
>1000
>1000
>1000
417
283
>500
10.6
11.1
2.2
19
244.8
>500
>500
80
49.6
57.9
170.5
>500
32.6
4.9
49.6
17.6
2.3
35
14.3
3.5
16.2
13.2
4.5
15.8
34.7
3.1
44.7
15.8
2.5
41.6
5.5
3.2
40.7
9
10
11
12
13
14
15
16
17
18
>500
>500
>500
281.5
120.9
103.7
332.8
>500
156.1
>500
>500
93.8
114.7
54.9
>1000
>1000
>1000
>1000
>1000
35
295
>1000
>1000
>1000
>1000
>1000
427
10
10.9
>1000
>1000
>1000
>1000
>1000
600
>1000
90
100
>1000
>1000
>1000
30
420
>1000
>1000
>1000
>100
>1000
10
>1000
>1000
>1000
>1000
10
>1000
>1000
>1000
10
600
14.8
>1000
>1000
>1000
>1000
>1000
167.8
>500
>1000
>1000
a 50% Inhibitory concentration, or compound concentration required to inhibit tumor cell proliferation by 50%.
lines. However, these compounds also exhibited a cyto-
toxic effect on normal human fibroblasts. The com-
pound 9 of this class containing a methoxy group at the
position 6 of the benzopyran ring exhibited pronounced
inhibitory effects on the growth of all tumor cell lines
(IC50 within the concentration range of 2.0–4.9 mM)
(Fig. 5). Indolinospirobenzopyran 8 with the methoxy
group at position 7 was approximately 2–10 fold less
active against all examined cell lines than 9, while indo-
linospirobenzopyrans 4 and 7 with a nitro group at the
position 6 of the benzopyran ring exhibited more selec-
tive cytostatic activity. These compounds showed better
inhibition of the growth of murine L1210 (IC50: 31.1
mM for 4 and 26.6 mM for 7) and human Molt4/C8
(IC50: 40.4 mM for 4 and 11.5 mM for 7) as well as CEM
(IC50: 24.9 mM for 4 and 10.6 mM for 7) cell lines than
other cells. Furthermore, compound 14 as representa-
tive of the indolinospironaphthopyran class showed
only slight activity against human T-lymphocytes, while
indolinospiropyridopyran 15 containing a nitro group
at position 50, was rather active against all examined
tumor cell lines, particularly against HeLa, MCF7,
HT29 and Hep2 cells (IC50: 10 mM). Among the indoli-
nospironaphtho-1,4-oxazines, compound 16 showed a
moderate cytostatic activity, particularly against human
breast carcinoma (MCF7) cells (IC50: 10.9 mM).
3.2. Antiviral activities
Compounds 4–10, 14 and 16–18 were evaluated against
varicella-zoster virus (VZV) and cytomegalovirus
(CMV) in human embryonic lung (HEL) cells and their
activities were compared with those of acyclovir (ACV),
5-(2-bromovinyl)-20-deoxyuridine (BVDU), ganciclovir
(GCV) and (S)-1-[3-hydroxy-2-(phosphonylmethoxy)-
propyl]cytosine (HPMPC, cidofovir, CDV, Table 3).
Compounds 6 and 7 showed best and selective activity
against VZV, whereas compounds 4, 8, 10 and 16 had
some lower activity against VZV. Thus, 6 showed sig-
nificant activity against thymidine kinase-positive
(TK+) strains (IC50: 1.5 mM against the YS strain and
1.0 mM against the OKA strain) and thymidine kinase-
deficient (TKÀ) (IC50: 1.1 mM) 07/1 strain of VZV.
Indolinospirobenzopyran 10 with the methoxy group at
position 7 of the benzopyran ring and chloro at position
50 of the indolino moiety exhibited selective activity
against CMV (AD-169 strain: IC50: 12.6 mM and Davis
strain: IC50: 7.0 mM). Comparison of antiviral activity
of 10 to that of GCV ascertained that activity against
CMV of compound 10 was close to that of GCV.
However, compound 10 exhibited also a more pro-
nounced cytotoxic effect (Table 3).
Compounds 4–10, 14 and 16–18 were also evaluated for
their activity against human immunodeficiency virus
type 1 and 2 (HIV-1, HIV-2), vaccinia virus, vesicular
stomatitis virus, Coxsackie virus B4, respiratory syncy-
tial virus, parainfluenza-3 virus, reovirus-1, Sindbis
virus and Punta Toro virus. No specific antiviral effects
Figure 5. Cytostatic effect of compound 9 on different human cell
lines. The cells were treated with compound 9 at different concen-
tration and percentage of growth was calculated. Each point repre-
sents a mean value of three parallel samples in three individual
experiments.

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1042
Table 3. Activity of 4–10, 14 and 16–18 against varicella-zoster virus (VZV) and cytomegalovirus (CMV) in human embryonic lung (HEL) cells
Antiviral activity EC50 (mM)a
Cytotoxicity (mM)
c
Comp
TK+ VZV
YS strain OKA strain
TKÀ VZV
CMV
AD-169 strain
Cell morphology (MCC)b
Cell growth (CC50)
07/1 strain
Davis strain
4
5
6
7
8
9
10
14
16
17
18
5.7
ND
1.5
2.7
9.2
ND
4.4
ND
4.7
ND
ND
1.1
0.0033
ND
ND
3.6
>9.9
1.0
1.9
6.0
>2.1
3.7
>44.2
4.5
>8.8
>8.2
0.8
0.0048
ND
ND
3.1
>9.9
1.1
1.7
5.5
>2.1
2.5
>8.8
5.1
>8.8
>8.2
53.3
>150
ND
>12.4
>49.8
>8.7
>1.8
>10.4
>10.4
12.6
>44.2
>9.8
>44
7.5
>49.8
>8.7
>1.8
>10.4
>10.4
7.0
>44.2
>9.8
>44
62.2
248.8
43.5
8.9
52.2
52.2
54.5
220.8
48.8
220
204.8
>222
ND
>196
>179
16.2
141.5
18.5
8.9
18.9
<6.5
32.1
119.2
20.1
107.5
96.3
888
>204.8
ND
ND
>40.9
ND
ND
ACV
BVDU
GCV
CDV
489
674.2
236.3
7.9
2.7
9.1
2.1
ND
ND, not determined.
a Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU).
b Minimum cytotoxic concentration that causes a microscopically detectable alteration of cell morphology.
c Cytotoxic concentration required to reduce cell growth by 50%.
were noted for any of the compounds against any of
those viruses (data not shown).
(Goppingen). Precoated Merck silica gel 60F-254 plates
were used for thin layer chromatography (TLC) and the
spots were detected under UV light (254 nm). Column
chromatography (CLC) was performed using silica gel
(0.063–0.2 mm), a Kemika; glass column being slurry-
packed under gravity. The electron impact mass spectra
were recorded with a Varian MAT 311A spectrometer
with ionising energy 70 eV. Elemental analyses were
performed in the Micro-analytical Laboratory of the
4. Conclusions
The principal aim of the presented work was to evaluate
various but structurally closely related classes of the
chiral spiro-compounds for their cytostatic and antiviral
activities. For this reason the new indolinospiro-
benzopyrans 5–7, indolinospironaphtopyrans 11–14 and
indolinospironaphtho-1,4-oxazine 17 were synthesized.
The exact stereostructures of the representatives of two
classes, namely indolinospirobenzopyran 9 and indoli-
nospironaphtho-1,4-oxazine 17, were determined by
X-ray structural analysis.
1
University of Regensburg. H and 13C NMR spectra
were recorded on Varian Gemini 300, Bruker WM-250
and Bruker ARX-400 spectrometers. The samples were
dissolved in CDCl3 and measured in 5 mm NMR tubes.
The 1H and 13C NMR chemical shift values (d) are
expressed in ppm referred to TMS and coupling con-
stants (J) in Hz. Two-dimensional COSY (Correlated
Spectroscopy), NOESY (Nuclear Overhauser Effect
Spectroscopy) and DEPT (Distortionless Enhancement
by Polarization Transfer) 1H and 13C NMR spectra
were also used for structure elucidation of the novel
compounds. IR spectra were measured on a Beckman
Acculab 1 instrument. UV spectra were recorded on a
Hitachi U 2000 spectrometer.
Among the evaluated compounds, spyrobipyridopyran
1 showed the most pronounced inhibitory activity against
human melanoma (HBL) cells (IC50: 0.9 mM). This
compound also exhibited the highest cytostatic selectiv-
ity. Compound 9, containing a methoxy group at the
position 6 of the benzopyran ring, exhibited significant
cytostatic activities against all examined cell lines (IC50
within the concentration range of 2.0–4.9 mM). How-
ever, this compound also exhibited a cytotoxic effect on
normal human fibroblasts. Compounds 4, 6–8, 10 and
16 showed a selective activity against VZV, whereas
compound 10 also showed selectivity against CMV
(EC50: within the concentration range of 1.0-12.6 mM).
5.2. Compounds preparations
The compounds 1,1 2–3,2 4,4 15,1 165 and 186 had been
prepared as described previously.
5.2.1.
(Æ)-50-Nitro-10,30,30-trimethylspiro(2H-1-benzo-
pyran-2,20-indoline) (5). The mixture of 5-nitro-1,3,3-tri-
methyl-2-methyleneindoline (1.1 g, 5 mmol) and 2-
hydroxybenzaldehyde (0.6 g, 5 mmol) in absolute etha-
nol (20 mL) was heated under reflux for 2 h and the hot
reaction mixture was then filtered. The solidified pro-
duct was obtained by cooling the reaction mixture to
room temperature. Recrystallization from light petro-
leum (80–110 ꢀC) gave yellow-orange crystals. Yield
5. Experimental
5.1. General methods
Melting points (uncorrected) were determined with
Kofler micro hot-stage (Reichert, Wien) and Buchi 530

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S. Raic-Malic et al. / Bioorg. Med. Chem. 12(2004) 1037–1045
1043
ꢀ
30%, mp=178–182 C; UV (MeOH): lmax (log e)=214
5.2.5.
(Æ)-50-Chloro-10,30,30-trimethyspiro[indoline-2,20-
nm (4.55), 255 (4.08), 308 (3.75), 372 (4.20); IR (KBr):
n=1610 cmÀ1 (C¼C), 1500 (C¼Caromat., N-O), 1310
(N-O), 1255 (C-O), 970 (N-Csp.-O); MS m/z 322.4
(M+ ). Anal. calcd for C19H18N2O3: C, 70.79; H, 5.63;
3H-naphtho(1,2-b)pyran] (12). Compound 12 was pre-
pared by reacting equimolar quantities of 1-hydroxy-2-
naphthaldehyde and 5-chloro-1,3,3-trimethyl-2-methyle-
neindoline using the procedure analogous to that for
preparing 11. Recrystallization of the crude product
from ethanol gave crystals of 12. Yield 17%, mp=200–
.
N, 8.69. Found: C, 70.57; H, 5.62; N, 8.71.
5.2.2. (Æ)-50-Nitro-6-nitro-10,30,30-trimethylspiro(2H-1-
benzopyran-2,20-indoline) (6). 5-Nitro-1,3,3-trimethyl-2-
methyleneindoline (2.1 g, 9.62 mmol) and 2-hydroxy-5-
nitrobenzaldehyde (1.67 g, 10 mmol) were dissolved in
ethanol (99%, 35 mL) and heated under reflux for 2 h.
After cooling the reaction mixture to room temperature
yellow-orange crystals were obtained, filtered off,
washed and dried in vaccuo. The crude solidified pro-
duct was recrystallized from ethanol. Yield 67%,
mp=201–204 ꢀC; IR (KBr): n=3060 cmÀ1 (¼C–H),
2980, 2965 (–C–H), 1590, 1495, 1485 (C¼C, N–O), 1300
203 C; UV(MeOH): lmax (log e)=208nm (4,63); 218
ꢀ
(4,57); 260 (4,63); 268 (4,61); 310 (3,93); 354 (3,61); 388
(3,11); 556 (3,90); 592 (3,91). IR(KBr): n=2966 cmÀ1
(C–H), 1605 (C¼C), 1274 (C–O), 934 (N–Cspꢁ–O); 13C
NMR d 28.80 (N–CH3), 105.39 (C-2/20), 51.62 (C-30),
20.09 (30-CH3), 25.61 (30-CH3), 139.13 (C-30a), 122.33
(C-40), 123.90 (C-50), 127.39 (C-60), 107.90 (C-70), 147.03
(C-70a), 117.12 (C-3), 130.26 (C-4), 134.88 (C-4a),
119.70 (C-5), 126.75 (C-6), 112.70 (C-6a), 127.76 (C-7),
125.65 (C-8), 124.72 (C-9), 121.90 (C-10), 123.68 (C-
10a), 149.50 (C-10b); MS(70 eV) m/z=362 M+ꢁ. Anal.
calcd for C23H20 ClNO: C, 76.34; H, 5.57; N, 3.87.
Found: C, 76.11; H, 5.58; N, 3.86.
(N–O); MS m/z 367.4 (M+ ). Anal. calcd for
.
C19H17N3O5: C, 62.12; H, 4.66; N, 11.44. Found: C,
62.18; H, 4.64; N, 11.41.
5.2.6.
3H-naphtho(1,2-b)pyran] (13). Reaction of equimolar
(Æ)-50-Nitro-10,30,30-trimethylspiro[indoline-2,20-
5.2.3. (Æ)-50-Chloro-6-nitro-10,30,30-trimethylspiro(2H-1-
benzopyran-2,20-indoline) (7). Compound 7 was pre-
pared by heating the mixture of 5-chloro-1,3,3-tri-
methyl-2-methyleneindoline (1.05 g, 5 mmol) and 2-
hydroxy-5-nitrobenzaldehyde (835 mg, 5 mmol) in eth-
anol (25 mL) under reflux for 4 h. The solidified crude
product was filtered off and recrystallized from ethanol
to give crystals of 7. Yield 28%, mp=152–154 ꢀC;
UV(MeOH): lmax (log e)=227 nm (4.10), 249 (4.31),
312 (3.87), 334 (3.85); MS m/z 356.8 (M+ꢁ). Anal. calcd
for C19H17ClN2O3: C, 63.96; H, 4.80; N, 7.85. Found:
C, 63.81; H, 4.82; N, 7.83.
quantities of 1-hydroxy-2-naphthaldehyde and 5-nitro-
a procedure
1,3,3-trimethyl-2-methyleneindoline by
analogous to that for 11 gave crystals of 13. Yield 23%,
mp=230–232 ꢀC; UV(MeOH): lmax (log e)=206 nm
(4,51); 218 (4,59); 260 (4,56); 268 (4,62); 358 (4,25).
IR(KBr): n=2924 cmÀ1 (C–H), 1608 (C¼C), 1271/1239
(C–O), 924 (N–Cspꢁ–O); 13C NMR d 28.58 (N–CH3),
105 (C-2/20), 50.82 (C-30), 19.86 (30-CH3), 25.38 (30-
CH3), 137.56 (C-30a), 121.40 (C-40), 153.16 (C-50),
126.76 (C-60), 105.26 (C-70), 140.49 (C-70a), 115.82 (C-3),
130.61 (C-4), 134.75 (C-4a), 118.35 (C-5), 126.23 (C-6),
112.22 (C-6a), 127.64 (C-7), 125.70 (C-8), 124.40 (C-9),
120.11 (C-10), 123.31 (C-10a), 148.74 (C-10b); MS(70
eV) m/z=372 M+ꢁ. Anal. calcd for C23H20N2O3: C,
74.18; H, 5.41; N, 7.52. Found: C, 74.09; H, 5.40; N,
7.50.
Compounds 8–10 were prepared according to S. Kraml,
Diploma Thesis, University of Regensburg, Germany,
1994.
5.2.4. (Æ)-10,30,30-Trimethylspiro[indoline-2,20-3H-naph-
tho(1,2-b)pyran] (11). Compound 11 was prepared by
reacting of 1-hydroxy-2-naphthaldehyde (1 g, 5.8 mmol)
with 1,3,3-trimethyl-2-methyleneindoline (1 mL, 5.8
mmol) in absolute ethanol (15 mL). After refluxing for 2
h, the reaction mixture was cooled to room temperature
and a precipitate was formed. After filtration of this pre-
cipitate, the crude product was purified by column chro-
matography using light petroleum (40–60ꢀ):chloroform=
1:2 as eluent. Recrystallization of the crude crystals
from ethanol gave red crystals. Yield 32%, mp=208–
210ꢀ; UV(MeOH): lmax (log e)=206nm (4,51); 218
(4,40); 260 (4,33); 268 (4,35); 308 (3,82); 386 (3,59); 554
(4,28); 588 (4,32). IR(KBr): n=2926 cmÀ1 (C–H), 1586
(C¼C), 1204 (C–O), 932 (N–Cspꢁ–O); 13C NMR d 28.82
(N–CH3), 105.04 (C-2/20), 51.34 (C-30), 20.19 (30-CH3),
25.68 (30-CH3), 136.80 (C-30a), 121.72 (C-40), 118.96 (C-
50), 127.36 (C-60), 106.72 (C-70), 147.99 (C-7a), 117.42
(C-3), 129.57 (C-4), 134.49 (C-4a), 118.99 (C-5), 126.28
(C-6), 112.50 (C-6a), 127.36 (C-7), 125.17 (C-8), 124.43
(C-9), 121.42 (C-10), 123.43 (C-10a), 149.34 (C-10b);
MS (70 eV) m/z=327 M+ꢁ. Anal. calcd for C23H21NO:
C, 84.37; H, 6.46; N 4.28. Found: C, 84.14; H, 6.47; N
4.27.
5.2.7.
(Æ)-50-Chloro-10,30,30-trimethylspiro[indoline-2,30-
3H-naphtho(1,2-b)pyran] (14). Compound 14 was pre-
pared by reaction of 2-hydroxy-1-naphthaldehyde (0.85
g, 5 mmol) with 5-chloro-1,3,3-trimethyl-2-methyle-
neindoline (1.05g, 5 mmol) in absolute ethanol (25 mL).
After refluxing for 4 h, the reaction mixture was cooled.
A pink solid was precipitated, filtered and washed with
ethanol. The product was purified by recrystalliza-
tion from ethanol (96%). Yield 20%, mp=220–224
ꢀC; UV(MeOH): lmax (log e)=241 nm (4.70), 300
(4.03), 313 (4.02), 339 (3.70), 360 (2.50)sh; IR (KBr):
n=1635 cmÀ1 (C¼C), 1248 (C–O), 990 (N–Cspꢁ–O);
MS m/z 361.9 (M+ꢁ). Anal. calcd for C23H20ClNO: C,
76.34; H, 5.57; N, 3.87. Found: C, 76.39; H, 5.58; N,
3.86.
5.2.8. (Æ)-50-Chloro-10,30,30-trimethylspiro[indoline-2,30-
3H-naphtho(1,2-b)(1,4)oxazine] (17). 1-Nitroso-2-naph-
thol (3 g, 17.3 mmol) reacted with freshly destilled 5-
chloro-1,3,3-trimethyl-2-methyleneindoline (3.59 g, 17.3
mmol) under reflux for 18 h. Purification of crude solid
product by column chromatography (toluene and light
petroleum:ethyl acetate=15:1) gave an oily product.

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1044
Formation of the green crystals was effected by adding
a small amount of acetone. That product was additio-
naly washed with ice cold petrꢀolether and dried in
vacuo. Yield 15%, mp=176–179 C; UV(MeOH): lmax
(log e)=231 nm (4.71), 313 (3.87), 347 (3.58)sh; IR
(KBr): n=3040 cmÀ1 (¼C–H), 2840, 2870, 2905, 2970
(–C–H), 1470, 1573, 1590, 1600 (C¼C, C¼N). MS
m/z 362.9 (M+ꢁ). Anal. calcd for C22H19ClN2O: C,
72.82; H, 5.28; N, 7.72. Found: C, 72.63; H, 5.29; N,
7.73.
for potential antitumor activity. The effects of different
concentrations of each compound on proliferation of
tumor and normal cell lines was examined. The cells
(melanoma, HBL; cervical carcinoma, HeLa; breast
carcinoma, MCF7; colon carcinoma, HT29 and SW620;
laryngeal carcinoma, Hep2; pancreatic carcinoma,
MiaPaCa2 and human normal fibroblasts, WI38) were
seeded in 96-well plates at a concentration of 3ꢃ104 /
mL in D-MEM, supplemented with 10% FBS and glu-
tamine (2 mM) and grown in humidified atmosphere
with 5% CO2. At 24 h later, the test compounds were
added at a final concentration of 10À6, 10À5 and 10À4
M. The number of cells was determined imediatelly
before (day 0) and 72 h after addition of compounds,
using the MTT test.18 A method is based on a reduction
of MTT (yellow) with a mitochondrial dehydrogenase
from alive cells to yield a formazan product (red). The
number of living cells is linearly proportional to the
amount of reduced MTT. The medium was discarded
and MTT (20 mg/40 mL) was added to each well.
Formed precipitates were disolved in 160 mL DMSO
and the absorbance was measured on an ELISA reader
at 570 nm. The compounds were dissolved in DMSO as
a 10À1 M solution and diluted with a medium to the
appropriate concentration. The final concentration of
DMSO was less than 0.1%, and at that concentration it
did not influence cell growth. Control cells were grown
in D-MEM without any addition. The antitumor activ-
ity results were expressed as IC50, that is the concen-
tration required to afford 50% inhibition of cell growth.
Each result was a mean value from three parallel sam-
ples in three individual experiments.
5.3. X-Ray determination
The single crystals of 9 and 17 suitable for X-ray struc-
ture analysis were obtained by growth at room temper-
ature of a very dilute ethanol solution. For compound
9, the intensities were collected at room temperature on
a Philips PW1100 diffractometer updated by Stoe and
Cie11,12 using Mo-Ka radiation (l=0.71073 A) with the
!-scan mode. For compound 17, the intensities were
collected at 100 K on a Nonius KappaCCD dif-
fractometer13,14 using Cu-Ka radiation (l=1.54184 A)
with the o-scan mode. The intensities were corrected for
Lorentz and polarization effects for both compounds.
The crystal structures were solved by direct methods
using SHELXS9715 program. All non-hydrogen atoms
were refined anisotropically by full-matrix least-squares
calculations based on F2 using SHELXL9716 program.
The hydrogen atoms were treated using appropriate
riding models and their coordinates were included in
structure factor calculations. The final difference map
contained no significant features (ꢀrmax./ꢀrmin.
=
0.209/À0.146 eAÀ3 for 9 and 0.257/À0.235 eAÀ3 for
17). The molecular drawings were prepared by PLA-
TON17 program. Crystallographic data (excluding
structure factors) for the structures in this paper have
been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication numbers
CCDC-213813 and CCDC-213814. Copies of the data
can be obtained, free of charge, on application to
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
+44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Antitumor activity against L1210 (murine leukemia),
FM3A (murine mammary carcinoma), Molt4/C8 and
CEM (human T-lymphocytes) cell lines were measured
essentially as originally described for the mouse leuke-
mia (L1210) cell line.19
5.3.2. Antiviral activity assays. Antiviral activity against
VZV, CMV, HIV-1, HIV-2, vaccinia virus, vesicular
stomatitis virus, Coxsackie virus B4, respiratory syncy-
tial virus, parainfluenza-3 virus, reovirus-1, Sindbis
virus and Punta Toro virus was determined essentially
as described previously.20,21 The antiviral activity results
were expressed as EC50, that is the effective concen-
tration required to afford 50% protection against viral
cytopathogenicity or viral plaque formation.
Crystal data for 9: C20H21NO2, Mr=307.38, ortho-
rhombic space group Pbca; a=38.699(5), b=11.972(2),
c=7.215(1) A, V=3342.7(8) A3; Z=8; F(000)=1312;
dx=1.222 g cmÀ3; m(MoKa)=0.078 mmÀ1; S=0.986;
R/wR=0.0594/0.1340 for 210 parameters and 1578
reflections with Iꢂ2ꢀ(I), R/wR=0.1769/0.1791 for all
3990 independent reflections measured in the range
5.42ꢀÀ2yÀ55.96ꢀ.
Acknowledgements
Crystal data for 17: C22H19ClN2O, Mr=362.84, mono-
clinic space group P21/a; a=8.3312(1), b=16.2963(1),
c=12.9924(1) A, b=95.223(6)ꢀ; V=1756.63(1) A3;
Z=4; F(000)=760; dx=1.372 g cmÀ3; m(CuKa)=2.022
mmÀ1; S=1.047; R/wR=0.0474/0.1240 for 238 para-
meters and 1827 reflections with Iꢂ2ꢀ(I), R/
wR=0.0507/0.1266 for all 1993 independent reflections
measured in the range 8.72ꢀÀ2ꢁÀ108.48ꢀ.
Support for this study was provided by the Ministry of
Science of the Republic of Croatia (Projects 0125003
and 00981499). We are grateful to Dr. R. Kiessweter,
Dipl.-Chem. S. Kraml, Dr. A. Leiminer and Dr. B.
Stephan for providing spiropyran samples. We thank
Lizette van Berckelaer for excellent technical assistance
in performing the antitumor cell activity assays, as well
as Ann Absillis, Anita Van Lierde, Frieda De Meyer,
Anita Camps and Lies Vandenheurck for excellent
technical assistance in performing the antiviral activity
assays.
5.3.1. Biological tests antitumor activity assays. Biologi-
cal evaluation of the compounds 1–18 was performed

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1045
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