Discovery of antiglioma activity of biaryl 1,2,3,4-tetrahydroisoquinoline derivatives and conformationally flexible analogues

Article abstract of DOI:10.1021/jm060020x

Cultured rat astrocytes and C6 rat glioma were used as a differential screen for a variety of 1,2,3,4-tetrahydroisoquinoline (THI) derivatives. Compound 1[1-(biphenyl-4-ylmethyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol hydrochloride] selectively blocked t

Full text of DOI:10.1021/jm060020x

J. Med. Chem. 2006, 49, 5845-5848
5845
Discovery of Antiglioma Activity of Biaryl 1,2,3,4-Tetrahydroisoquinoline Derivatives and
Conformationally Flexible Analogues
Michael L. Mohler,† Gyong-Suk Kang,† Seoung-Soo Hong,† Renukadevi Patil,† Oleg V. Kirichenko,† Wei Li,† Igor M. Rakov,†
Eldon E. Geisert,‡ and Duane D. Miller*,†
Department of Pharmaceutical Sciences, College of Pharmacy, and Department of Ophthalmology, College of Medicine, UniVersity of
Tennessee Health Science Center, Memphis, Tennessee 38163
ReceiVed January 9, 2006
Cultured rat astrocytes and C6 rat glioma were used as a differential screen for a variety of 1,2,3,4-
tetrahydroisoquinoline (THI) derivatives. Compound 1 [1-(biphenyl-4-ylmethyl)-1,2,3,4-tetrahydroisoquino-
line-6,7-diol hydrochloride] selectively blocked the growth of C6 glioma leaving normal astrocytes relatively
unaffected. The potential for clinical utility of 1 was further substantiated in human gliomas and other
tumor cell lines. Preliminary SAR of this activity was characterized by synthesis and testing of several THI
and conformationally flexible variants.
Introduction
1. Reaction of 2-(3,4-dibenzyloxyphenyl)ethylamine hydrochlo-
ride 15 with 4-biphenylacetic acid 16 in the presence of diethyl
cyanophosphonate and triethylamine yielded the amide 17. The
amide was cyclized to 18 by reaction with POCl3 followed by
reduction with NaBH4. The lead structure 1 was synthesized
by acidic deprotonation of 18. The N-methyl analogue, 7, was
prepared from 18 by treatment with formaldehyde followed by
sodium cyanoborohydride and zinc chloride to form 26. Acidic
deprotection of 26 to remove the O-benzyl groups protecting
the catechol yielded 7. Two types of analogues of 1 were
synthesized and tested, substituted THI derivatives such as 2
and open chain derivatives such as 3 (Chart 1). The synthesis
of 1-biphenyl-4-ylmethyl-6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinoline hydrochloride 4 is shown in Scheme 2. Initially, the
3,4-dimethoxyphenethylamine 19 was reacted with 4-biphen-
ylacetic acid 16 to form the intermediate amide 21. The amide
21 was cyclized to form the 6,7-dimethoxy analogue 4 by
reaction with POCl3 and NaBH4 followed by 2 M HCl solution
in diethyl ether. Intermediate 21 was also used to synthesize
conformationally flexible analogues 3 and 6. Reaction of 21
Gliomas are among the deadliest of tumors because surgical
excision of these tumors is extremely difficult due to the invasive
nature of tumors derived from glia.1 Very few glioblastoma
patients live more than one year with essentially no long-term
cures.2 The mainstay for chemotherapy is DNA alkylating agents
such as BCNU, procarbazine, temozolomide, and cisplatin.3 We
report here the discovery of a series of 1,2,3,4-tetrahydroiso-
quinolines (THIs) and flexible analogues that have selective
cytotoxic activity against rat C6 glioma cells relative to cultured
rat astrocytes.4 The C6 glioma cytotoxic activity of our lead,
compound 1 (Chart 1), and a series of analogues is reported
and compared to the peripheral benzodiazepine receptor (PBR)
binding agents (9, 10, and 11) that possess similar cytotoxic
activity against C6 glioma cells5 (Chart 1) with IC50 values in
C6 glioma of 73.0 µM, 95.0 µΜ, and 37.5 µΜ, respectively.
Compound 1 is also compared to BCNU, 5-fluorouracil (5FU),
and melphalan, chemotherapeutic agents used clinically. A broad
array of THI compounds bearing a 1-monoaryl group, such as
in 12, 13, and 14,6,7 lacked the cytotoxic activity of 1, providing
the initial SAR observation that directed us toward 1-biaryl
analogues of 1. Correspondingly, we have synthesized novel
1-biaryl compounds 2, 3, 4, 6, and 7 and tested 2-8 as analogues
of 1 to develop a preliminary set of structure-activity relation-
ships (SAR). Cytotoxicity for 1 was also demonstrated in human
gliomas and diverse human tumor cell lines supporting its status
as a preclinical lead for drug development.
.
with BF3 Et2O yielded the linear secondary amine 22. Reaction
of 22 with formaldehyde followed by sodium cyanoborohydride
and ZnCl2 yielded the tertiary amine 3. Reaction of 22 with
boron tribromide in methylene chloride yielded the secondary
amine, catechol 6. The synthesis of 2-(2-biphenyl-4-ylethyl)-
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride 2
is shown in Scheme 3. Reaction of 6,7-dimethoxytetrahydroiso-
quinoline hydrochloride 28 with 4-biphenylacetic acid 16 in the
presence of diethyl cyanophosphonate in DMF followed by
reaction with triethylamine yielded the amide intermediate 27
(Scheme 3). The amide 27 was reduced using BF3‚Et2O in THF
followed by 1 M B2H6 then cold 10% HCl to produce the
N-biphenyl tertiary amine, 2 (Scheme 3). All the analogues of
1 synthesized and tested are summarized in Chart 1.
Antiglioma Activity of (1) Compared to Known Anti-
glioma Agents. An in vitro assay was used to screen compounds
for their effects on the growth of cultured normal astrocytes
and C6 glioma. We discovered that C6 glioma growth was more
sensitive than normal astrocytes to certain 1,2,3,4-tetrahydroiso-
quinoline (THI) compounds, indicating selective cytotoxic
activity in C6 glioma (rat) vs rat astrocyte. The most active
and selective THI screened, compound 1, had IC50 values of
2.6 µΜ in C6 glioma vs 29.0 µΜ in primary astrocytes. To
Chemistry
The synthesis of compound 1 1-biphenyl-4-ylmethyl-1,2,3,4-
tetrahydroisoquinoline-6,7-diol hydrochloride is shown in Scheme
* To whom correspondence should be addressed. College of Pharmacy,
Department Pharmaceutical Sciences, 847 Monroe Ave., Rm. 227C,
Memphis, TN 38163. Phone (901) 448-6026. Fax (901) 448-3446. E-mail
dmiller@utmem.edu.
† Department of Pharmaceutical Sciences.
‡ Department of Ophthalmology.
a Abbreviations: 5FU, 5-fluorouracil; BCNU, 1,3-bis(2-chloroethyl)-1-
nitrosourea; BME, Basal Media Eagle; DMEM, Dulbecco’s Modification
of Eagle’s Medium; DNA, deoxyribonucleic acid; FCS, fetal calf serum;
PBR, peripheral benzodiazepine receptor; PK11195 (1-(2-chlorophenyl(N-
methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide; RO5-4864, 7-chloro-
5-(4-chlorophenyl)-2,3-dihydro-1H-benzo[e][1,4]diazepine; SAR, structure-
activity relationship; THI, 1,2,3,4-tetrahydroisoquinoline.
10.1021/jm060020x CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/30/2006

5846 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Brief Articles
Scheme 1a
a Reagents: (a) Et3N, (EtO)2P(O)CN; (b) POCl3; (c) NaBH4; (d) concentrated HCl; (e) CH2O, NaBH3CN, ZnCl2.
Chart 1. C6 Glioma Cytotoxic Potency of Antiglioma Agentsa
for melphalan, BCNU, and 5FU were 12.6 µΜ, 9.3 µΜ, and
6.1 µΜ, respectively, for C6 glioma; thus 1 demonstrated greater
than 2-fold higher potency and similar to improved selectivity
(Table 1), suggesting that 1 may have advantages over currently
used antiglioma agents. Next we wanted to compare 1 to
reported compounds that have similar structure and activity to
see if compounds such as 1 were known. The closest structural
analogues that reported C6 glioma cytotoxicity were the PBR
ligands. Two commercially available examples, 9 and 10, were
purchased and characterized in our assay but did not exhibit
any cytotoxicity in the dose range used, revealing that 1 and
analogues were much more potent than the PBR ligands. Thus
1 was more potent and at least as selective as all the known
cytotoxic agents that were characterized as reference materials.
These observations suggested to us that THI compounds such
as 1 should be explored for their potential as antiglioma
chemotherapeutic agents and prompted us to try to establish a
preliminary SAR.
Activity in Human Gliomas and Other Human Tumor
Cell Lines. Compound 1 was tested for cytotoxic activity in
T98G human glioblastoma cell line as a proof of principle that
cytotoxicity in C6 glioma was indicative of cytotoxicity in
human glioma cells. No normal fetal human astrocyte cell lines
were readily available, so this experiment did not have a negative
control. Nonetheless, 1 did exhibit activity in T98G cells with
an IC50 ) 10.2 µM ( 1.8 µM. Despite the decline in activity
relative to C6 glioma (approximately 4-fold less active), this
experiment suggests that rat C6 glioma activity is indicative of
cytotoxicity in human gliomas. Given that gliomas are known
to be widely variable,8 we decided to determine the IC50 values
for 1 in an additional human glioma cell line, U87, with an
IC50 value of 25.9 µM (Table 1). Compound 1 was further
characterized with regard to diverse cancer cell type cytotoxicity.
The IC50 values for 1 in A549 lung cancer, MCF7 breast cancer,
LNCaP prostate cancer, and HeLa cervical cancer cell lines were
10.2 µM, 3.8 µM, 10.7 µM, and 3.0 µM, respectively (Table
2). These six cell lines demonstrated that compound 1 has
cytotoxicity in a broad array of cancer cell lines, suggesting
that 1 and analogues may have therapeutic potential in a variety
of cancers.
Synthesis and Testing of THI and Non-THI Analogues.
To expand on 1 as a lead structure, other readily obtainable
THI analogues were tested for activity. Compounds 12, 13, and
14 are examples of compounds that were screened that did not
have activity (Chart 1). The prominent difference between these
molecules and 1 was the biaryl group in 1 which seemed to be
the key structural component that conferred activity to 1. Thus
the biaryl component was taken to be critical to the antiglioma
activity, and this initial SAR observation led us to compile and
characterize a series of biaryl compounds including biaryl
a The C6 glioma cytotoxic activities of several classes of agents are
summarized in Chart 1. The THI and derivative compounds 1-8 and 12-
14 were synthesized and tested by us using the protocol in the Supporting
Information and reported in Table 1 with exception of 12-14 which had
IC50 > 50 µM. Briefly, cells were grown and maintained in 10% FCS BME
(fetal calf serum in Basal Media Eagle). The test compound was incubated
with cells in 200 µL of 2% FCS BME for 96 h at 37 °C in a humid, 5%
CO2 incubator. Cells were fixed and quantitated using 0.1% cresylecth violet
stain whose A560 was determined with a Titertek plate reader. Zisterer et
al. incubated 9, 10, and 11 with cells in 100 µL of 10% FCS DMEM for
48 h, and then cells were quantitated spectrophotometrically (A570) using
MTT stain.5 Compounds 9, 10, and 11 did not demonstrate any activity in
our hands in a dose range comparable to 1. Compounds 5, 8, 12, 13, and
14 were synthesized and reported by us elsewhere.6,7,9
assess the significance of this observation we decided to
determine the IC50 values in our assay for commonly used
chemotherapeutic agents and compare them to 1. The IC50 values

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5847
Scheme 2a
a Reagents: (a) toluene, reflux; (b) POCl3; (c) NaBH4; (d) BF3‚Et2O, 1 M B2H6; (e) CH2O, NaBH3CN, ZnCl2; (f) BBr3.
Scheme 3a
a Reagents: (a) (EtO)2P(O)CN, Et3N; (b) BF3‚Et2O, 1 M B2H6; (c) HCl.
Table 1. C6 Glioma Selective Cytotoxic Activities of 1 and Derivatives
suggests that C6 cytotoxic activity has some degree of tolerance
IC50 (µM)a-c
to modification of the biaryl group at the C-1 position. The
observation of activity in the benzophenone suggested a specific
molecular recognition event that mediates the cytotoxic activity,
suggesting that optimization of this activity should be possible.
compound
C6 glioma
astrocytes
1
2.6 ( 0.6
29.0 ( 0.6
n/a
10.2 ( 5.8 (human T98G)d
25.9 ( 3.3 (human U87)d
4.1 ( 0.2
Other biaryl structural analogues of 1 explored included
O-methylated 1 yielding 4 (Scheme 2), which is less than 2-fold
lower activity than 1. The effect on selectivity is hard to discern
since the astrocyte error is large for this compound. The
O-methyl benzophenone analogue 8 also demonstrated dimin-
ished activity and surprisingly abolished selectivity compared
to 5. The N-methylation of 1 to form 7 resulted in a 3-fold
decrease in activity and significantly reduced selectivity. Varying
the point of attachment of the aromatic moiety also decreased
the activity of 2 which is 2-fold less active than 4. Significant
activity was retained for all these THI variants discussed thus
far, suggesting we could expand our SAR probe to include
analogues that were very different than the lead compound 1.
4
83.8 ( 73.2
21.0 ( 7.2
40.0 ( 0.8
15.0 ( 1.3
15.5 ( 1.7
5e
9.4 ( 0.9
8e
40.0 ( 2.6
7
2
6
3
8.0 ( 0.9
9.3 ( 0.6
8.6 ( 0.5
>100
12.5 ( 0.8
39.0 ( 30.7
34.0 ( 1.6
melphalan
BCNU
5FU
12.6 ( 2.6
9.3 ( 3.9
155.6 ( 104.8
6.1 ( 3.9
>100
a IC50 values were determined in dose-response experiments with
incubation periods of 4 days. b The reported IC50 values and standard
deviation for all compounds except 1 and BCNU were calculated using
WinNonlin as discussed in the Experimental Section. c Note that 1 and
BCNU were further characterized using the computer program Scientist to
derive the reported IC50 values and standard deviations. d Values for IC50
determined in T98G and U87 human glioblastoma cell lines which were
purchased from ATCC. e The synthesis of compounds 5 and 8 was
previously reported.9
Conformationally flexible derivatives were synthesized as
linear secondary or tertiary amines linking the disubstituted
phenyl and biaryl moieties (6 and 3). The rationale of synthesiz-
ing these compounds was to determine whether these linear
analogues could adopt a conformation similar to the THI ring
system and therefore bind to the same target site. These open
chain analogues demonstrate significant albeit diminished
antiglioma activity compared to 1 but in line with the other
analogues. The linear analogue 6 of 1 is more than 3-fold less
active than 1. However, it is noteworthy that the selectivity of
6 is preserved or potentially even enhanced. The tertiary amine
3 demonstrated that the cumulative effect of O- and N-methyl
groups in the open chain analogues such as 6 was a relatively
small decrease in cytotoxicity (only approximately 50%).
O-Methylation alone generally decreases cytotoxicity by ap-
proximately two- to 4-fold. This suggests that the N-methyl
group may contribute favorably to cytotoxic activity (i.e.
Table 2. Activity of 1 in Diverse Human Tumor Cell Linesa,b
A549 (lung)
MCF7 (breast)
LNCaP (prostate)
HeLa (cervical)
10.2 ( 0.9
3.8 ( 2.5
10.7 ( 3.4
3.0 ( 0.05
a A549, MCF7, LNCaP, and HeLa tumor cell lines were purchased from
ATCC cell lines were purchased from ATCC. b The reported IC50 values
and standard errors were derived using the computer program Scientist.
variants (5 and 8), methoxylated variants (2, 3, 4, and 8), a
biaryl attachment point variant (2), flexible non-THI variants
(3 and 6), and tertiary amine variants (3 and 7).
Replacement of the biphenyl group at the 1-position of 1 with
the benzophenone substituent as in 5 resulted in an almost 4-fold
decrease in activity and decreased selectivity (Table 1). This

5848 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Brief Articles
138.8, 135.4, 130.2, 128.9, 127.4, 126.8, 126.5, 122.7, 122.4, 115.2,
113.5, 55.0, 39.1, 24.2; MS (ES) m/z 332 [M + H]+. Anal. (C22H21-
NO2‚HCl‚0.25 H2O) C, H, N.
partially counteracts the loss of cytotoxicity associated with the
O-methyl group). However the cumulative O- and N-methyla-
tion has a detrimental effect on selectivity. The synthesis and
testing of these variants of 1 allowed the observation of the
structural determinants necessary for the cytotoxic activity.
Acknowledgment. The College of Pharmacy, The Center
for Neurobiology of Brain Disorders, The Van Vleet Foundation,
and American Chemical Society Division of Medicinal Chem-
istry (of M.L.M.) are gratefully acknowledged for their support.
The following scientists are gratefully acknowledged for their
contributions: Victor I. Nikulin, Saeed Binaei, Viet Q. Nguyen,
Svetlana N. Kirichenko, and Shelley M. Childers.
Conclusions
Normal cultured rat astrocytes and C6 rat glioma were used
as a differential screen for a variety of 1,2,3,4-tetrahydroiso-
quinoline (THI) derivatives. Compound 1 selectively blocked
the growth of C6 glioma, leaving normal astrocytes relatively
unaffected. Compound 1 also demonstrated improved potency
and improved to equivocal selectivity in our assay, when
compared to other C6 cytotoxic agents such as 9 and 10, and
established chemotherapeutic agents such as BCNU, 5FU, and
melphalan. To establish the potential for clinical utility of this
activity, we demonstrated that 1 also had cytotoxic activity in
human gliomas and other diverse human cancers. Biaryl
analogues were synthesized and tested to develop an initial SAR
including the following observations concerning glioma selective
cytotoxicity: (1) a substituted THI is superior to comparable
conformationally mobile analogues, (2) a biaryl moiety confers
significant cytotoxicity, and (3) a tertiary amine group in linear
compounds is more toxic but perhaps less selective. All the
derivatives of 1 tested in this study were more potent than 9
and 10, suggesting that these molecules represent a new class
of antiglioma molecules worthy of further exploration, and these
preclinical studies have supported 1 as the most potent of these
antiglioma agents.
Supporting Information Available: Characterization of 1-4,
6, 7, 17, 18, 21, 22, 26, and 27 and description of biological assays10
are available free of charge via the Internet at http://pubs.acs.org.
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Experimental Section
1-Biphenyl-4-ylmethyl-1,2,3,4-tetrahydroisoquinoline-6,7-
diol hydrochloride (1). 6,7-Bis-benzyloxy-1-biphenyl-4-ylmethyl-
1,2,3,4-tetrahydroisoquinoline hydrochloride, compound 18 (2.5 g,
4.56 mmol), was stirred in 10 mL of concentrated aqueous HCl
solution and 10 mL of methanol. The reaction mixture was refluxed
for 10 h, concentrated under reduced pressure, and then treated
with 10 mL of ether to give a solid. The solid was collected on a
glass filter funnel, washed with ether (2 × 10 mL), and recrystal-
lized from ether/methanol to give 1.3 g (78%) of 1 as off-white
crystals: mp ) 208-210 °C; 1H NMR (d6-DMSO) δ 9.17 (s, 1H,
ArOH), 9.09 (s, 1H, NH), 8.90 (s, 1H, ArOH), 7.68 (d, J ) 7.9
Hz, 4H, ArH), 7.47 (t, J ) 7.3 Hz, 4H, ArH), 7.36 (t, J ) 7.3 Hz,
1H, ArH), 6.58 (d, J ) 4.0 Hz, 2H, ArH), 4.63 (s, 1H, CH), 3.32
(t, J ) 9.9 Hz, 2H, CH2), 3.17 (q, J ) 7.3 Hz, 2H, CH2), 2.98-
2.73 (m, 2H, CH2); 13C NMR (d6-DMSO) δ 145.1, 144.0, 139.8,
JM060020X