Discovery of 4-(benzylaminomethylene)isoquinoline-1,3-(2H,4H)-diones and 4-[(pyridylmethyl)aminomethylene]isoquinoline-1,3-(2H,4H)-diones as potent and selective inhibitors of the cyclin-dependent kinase 4

Article abstract of DOI:10.1021/jm801026e

The series of 4-(benzylaminomethylene)isoquinoline-1,3-(2H,4H)-dione and 4-[(pyridylmethyl)aminometh-ylene]isoquinoline-1,3-(2H,4H)-dione derivatives reported here represents a novel class of potential antitumor agents, which potently and selectively inhibit CDK4 over CDK2 and CDK1. In the benzylamino headpiece, a 3-OH substituent is required on the phenyl ring for CDK4 inhibitory activity, which is further enhanced when an iodo, aryl, heteroaryl, t-butyl, or cyclopentyl substituent is introduced at the C-6 position of the isoquinoline-1,3-dione core. To circumvent the metabolic liability associated with the phenolic OH group on the 4-substituted 3-OH phenyl headpiece, we take two approaches: first, introduce a nitrogen o- or p- to the 3-OH group in the phenyl ring; second, replace the phenyl headpiece with N-substituted 2-pyridones. We present here the synthesis, SAR data, metabolic stability data, and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.

Full text of DOI:10.1021/jm801026e

J. Med. Chem. 2009, 52, 2289–2310
2289
Discovery of 4-(Benzylaminomethylene)isoquinoline-1,3-(2H,4H)-diones and
4-[(Pyridylmethyl)aminomethylene]isoquinoline-1,3-(2H,4H)-diones as Potent and Selective
Inhibitors of the Cyclin-Dependent Kinase 4
Hwei-Ru Tsou,*,† Xiaoxiang Liu,† Gary Birnberg,† Joshua Kaplan,† Mercy Otteng,† Tritin Tran,† Kristina Kutterer,†
Zhilian Tang,† Ron Suayan,† Arie Zask,† Malini Ravi,† Angela Bretz,† Mary Grillo,‡ John P. McGinnis,‡
Sridhar K. Rabindran,‡,§ Semiramis Ayral-Kaloustian,† and Tarek S. Mansour†
Chemical and Screening Sciences, and Oncology Research, Wyeth Research, 401 N. Middletown Road, Pearl RiVer, New York 10965
ReceiVed August 14, 2008
The series of 4-(benzylaminomethylene)isoquinoline-1,3-(2H,4H)-dione and 4-[(pyridylmethyl)aminometh-
ylene]isoquinoline-1,3-(2H,4H)-dione derivatives reported here represents a novel class of potential antitumor
agents, which potently and selectively inhibit CDK4 over CDK2 and CDK1. In the benzylamino headpiece,
a 3-OH substituent is required on the phenyl ring for CDK4 inhibitory activity, which is further enhanced
when an iodo, aryl, heteroaryl, t-butyl, or cyclopentyl substituent is introduced at the C-6 position of the
isoquinoline-1,3-dione core. To circumvent the metabolic liability associated with the phenolic OH group
on the 4-substituted 3-OH phenyl headpiece, we take two approaches: first, introduce a nitrogen o- or p- to
the 3-OH group in the phenyl ring; second, replace the phenyl headpiece with N-substituted 2-pyridones.
We present here the synthesis, SAR data, metabolic stability data, and a CDK4 mimic model that explains
the binding, potency, and selectivity of our CDK4 selective inhibitors.
Introduction
inhibition of endogenous cyclin D1 or CDK4 expression by
RNA interference in MCF-7 breast cancer cells resulted in the
inhibition of cell growth, hypophosphorylation of Rb, and
accumulation of cells in the G1 phase.8 This result supports
the view that pharmacological inhibition of cyclin D1/CDK4
complexes is a useful strategy to inhibit the growth of tumors.
Consistent with this, microinjection of cyclin D1 antibodies or
antisense oligonucleotides in cells caused G1/S arrest and
decreased tumorigenesis in nude mice.9–12 The importance of
deregulated cyclin D/CDK4 complexes in tumorigenesis is
underscored by the observation that both cyclin D1 and CDK4
knockout mice are resistant to ErbB-2 induced tumorigenesis.13,14
Importantly, this effect is mediated by the catalytic activity of
CDK4 since mice expressing a mutant cyclin D1 that binds to,
but cannot activate CDK4/6, are resistant to ErbB-2-mediated
tumorigenesis.15
Progression of cells through the cell division cycle is regulated
by a family of serine/threonine kinases (CDKsa) and their
activating partners, the cyclins.1 Different combinations of
cyclin/CDK complexes regulate each of the cell cycle transitions.
In the G1-S phase transition, the D-type cyclins (D1, D2, or
D3) in complex with CDK4 (or CDK6 in hematopoietic cells),
along with cyclin E/CDK2 complexes, cooperatively phospho-
rylate the retinoblastoma susceptibility gene family of proteins
(Rb).1–3 Phosphorylation of Rb converts it from a repressor to
an activator of the E2F transcription factors, leading to the
transcription of genes required for DNA synthesis.4 The
expression/activity of G1 cyclin/CDK complexes are positively
regulated by the mitogenic signaling pathways and negatively
regulated by the cyclin-dependent kinase inhibitors (CKIs),
which include p16INK4a, p15INK4b, p21CIP1, and p27KIP1.2 This
coordinated regulation facilitates sequential activation of the G1
cyclin/CDK complexes required for orderly transition into
S-phase.
In the past few years, several small-molecule CDK inhibitors
have been identified, but none have been approved for clinical
use.16 Flavopiridol and CYC-202 (Seliciclib), which inhibit
multiple CDKs, are in clinical trials but have only shown modest
activity.16a Until recently, flavopiridol was reported to demon-
strate a marked clinical efficacy in treating patients with chronic
lymphocytic leukemia by using a pharmacologically derived
schedule for drug administration.16b Among the second genera-
tion of CDK inhibitors, a pyridopyrimidine (PD-0332991),17 a
Aberrant control of cell proliferation is a hallmark of cancer,5
and the p16INK4a-cyclin D1/CDK4-Rb axis has been implicated
in this dysregulation.6,7 CDK4 mutants that are resistant to p16
have been indentified in melanoma, while loss of p16 activity
due to mutations, deletions, or gene silencing has been observed
in glioblastoma, pancreatic cancers, and NSCLC.6 Cyclin D1
is deregulated by chromosomal translocation in B-cell lym-
phoma and parathyroid adenomas, and is overexpressed in
breast, esophagus, head and neck, and colon cancers.6,7 CDK4
is overexpressed in osteosarcomas, and Rb is mutated in
retinoblastomas and SCLC.6,7 We recently reported that the
3-aminothioacridone,18
a
triaminopyrimidine derivative
(CINK4),19 a diarylurea,20 and 3-(R-heteroarylaminobenzylidene)-
2-indolinones21 are reported to be selective for CDK4/6, as
compared with other CDKs. Recently, we reported a novel series
of 4-(phenylaminomethylene)isoquinoline-1,3-diones as potent
and selective inhibitors of CDK4.22 In that series, substitution
with an N-methylpiperazine or a piperidinylmethyl group at the
para position of the aniline headpiece is essential for CDK4
potency. Here, we report a new series, 4-[(3-hydroxybenzy-
lamino)-methylene]-4H-isoquinoline-1,3-diones, as highly potent
and selective CDK4 inhibitors. Compared to our earlier series
where the headpiece was a substituted aniline, this new series
* To whom correspondence should be addressed. Phone: (845) 602-4712.
Fax: (845) 602-5561. E-mail: tsouh@wyeth.com.
†
Chemical and Screening Sciences.
Oncology Research.
Current address: GlaxoSmithKline, 1250 S. Collegeville Road, UP1450,
‡
§
Collegeville, PA 19426.
a Abbreviations: CDK, cyclin-dependent kinase.
10.1021/jm801026e CCC: $40.75
2009 American Chemical Society
Published on Web 03/24/2009

2290 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
Scheme 1
Scheme 2a
carried a substituted 3-hydroxybenzylamino headpiece and
exhibited higher potency for CDK4 and higher activity in
inhibiting cell proliferation. We describe the details of our
optimization efforts and the structure-activity relationships
relevant to the substituents at the C-4, C-5, and C-6 positions
of the isoquinoline-1,3-dione core. Furthermore, we show a
closely related new series, where the benzylamino headpiece
was replaced with the pyridylmethylamino headpieces, to
enhance metabolic stability while maintaining potency as CDK4
selective inhibitors. We also present one of our inhibitors docked
in the ATP binding domain of the CDK4 mimic model, to show
the critical hydrogen bond interactions that may explain the
CDK4 selectivity as well as potency.
a (i) 2e, DMF, Et3N, room temperature; (ii) PhB(OH)2, Pd(PPh3)4,
Na2CO3, DMF, microwave, 150 °C.
Scheme 3a
Chemistry. As shown in Scheme 1, 4-(methoxymethyl-
ene)isoquinoline-1,3-dione (1)22 was reacted with 3,4-dihy-
droxyaniline (2a) or 3,4-dihydroxyphenethylamine (2f) to give
the corresponding aniline derivative (3a) and phenethylamine
derivative (3f), respectively, similar to the protocol used in our
earlier paper.22 The corresponding benzylamine derivatives
(3b-e) were obtained from the reaction of 1 and benzylamines
(2b-e). The early lead compound 3e has a (3-hydroxy-4-
methoxybenzyl)aminomethylene substituent at the C-4 of the
isoquinoline-1,3-dione core, with no substituents on the rest of
the core. We began to explore analogues carrying substituents
at either the C-5 or the C-6 position. The 5-bromo and 6-halogen
analogues, namely, 5c and 5a,b,d,e, were prepared from the
corresponding 5-bromo intermediate (4c) and 6-halogen inter-
mediates (4a,b,d,e),22 respectively, as depicted in Scheme 2.
Similarly, the 6-methoxy derivative (5f), 6-(N-pyrrolyl) deriva-
tive (5g), 6-(3-thienyl) derivative (5h), and 6-(3-furyl) derivative
(5i) were prepared from the corresponding 6-methoxy, 6-(N-
pyrrolyl), 6-(3-thienyl), and 6-(3-furyl) intermediates, namely,
4f-i,22 respectively. The 6-bromo derivative (5d) was further
reacted with phenylboronic acid, under Suzuki coupling condi-
tions, to yield the 6-phenyl derivative (5j).
The linker group at C-4 of 5d was varied by introducing a
methyl group at the methylene or the nitrogen of the benzyl-
amine, namely, 11 and 13, as shown in Scheme 3. The hydroxyl
group of 3-hydroxy-4-methoxybenzaldehyde (6) was protected
as the benzyl ether, and then methyllithium was added to the
aldehyde group of 7 to form the secondary alcohol 8. The
alcohol 8 was treated with (PhO)2P(O)N3 and DBU in toluene
to yield the corresponding azide (9), which was reduced and
debenzylated to yield the amine hydrochloride salt 10. Coupling
of 4d with 10 afforded 11. Derivative 13 was prepared by
reacting 4d with N-methyl-3-hydroxy-4-methoxybenzylamine
(12), which was prepared by reductive amination of 6 with
methylamine.
a (i) PhCH2Br, K2CO3, DMF, room temperature; (ii) THF, MeLi in ethyl
ether, -78 °C; (iii) (PhO)2P(O)N3, toluene, DBU, 0 °C; (iv) Pd/C, H2, EtOH,
HCl; (v) 4d, DMF, Et3N, room temperature; (vi) MeNH2, EtOH, room
temperature; then NaBH4, MeOH; (vii) 4d, THF, room temperature.
Early on, we observed that moving the OH group from C-3
(3d) to C-4 (3c) on the benzylamine, resulted in a dramatic loss
of potency. Additional analogues also supported the importance
of the OH group, at the C-3 position of benzylamine, for activity.
Therefore, in the majority of our analoguing efforts, we kept
this unique structural feature unchanged. We turned our attention
to study the effect of the methoxy group at C-4 of the
benzylamine in 5d, by replacing it with a variety of other
functional groups, as depicted in Scheme 4. Treatment of 3,4-
dihydroxybenzaldehyde (14) and potasium carbonate in DMF,
with 1-bromopropane, or 2-bromoethyl methyl ether, provided
the corresponding 3-hydroxy-4-alkoxybenzaldehydes 15b,c, after
purification by column chromatography. Subsequent reaction
of 15a-c with methoxyamine hydrochloride in pyridine to form
O-methyl-oximes, followed by catalytic hydrogenation, yielded
the corresponding benzylamine hydrochlorides 16a-c. Treat-
ment of 4d with 16a-c gave the desired 4-alkoxy derivatives
17a-c, respectively. Further reaction of 17a with iodoethane
and potassium carbonate in DMF yielded the ethoxy derivative
17d after purification by high performance liquid chromatog-
raphy. The 3-hydroxy-4,5-dimethoxy derivative 18 was prepared
in a similar way from 4d and 3-hydroxy-4,5-dimethoxybenzy-
lamine. An additional analogue of 5d, where the 4-methoxy

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2291
Scheme 5a
Scheme 4a
a (i) RBr, K2CO3, or Na2CO3, DMF, room temperature or 65 °C; (ii)
MeONH2 · HCl, pyridine, room temperature; then Pd/C, H2, EtOH, HCl;
(iii) 4d, DMF, room temperature; (iv) EtI, K2CO3, DMF, 65 °C.
group was replaced with an amine, was prepared starting from
3-hydroxy-4-nitrobenzaldehyde 19. Hydrogenation of the oxime
of 19 followed by reaction with 4d provided the desired 4-amino
analogue 21.
Other methoxy replacements at C-4 of the benzylamine
headpiece in 5d included methyl and phenyl groups. Their
preparations (25 and 34) are shown in Scheme 5. 3-Hydroxy-
4-methylbenzoic acid 22, was converted to the corresponding
benzoyl chloride 23, followed by the addition of ammonium
hydroxide, to provide 3-hydroxy-4-methylbenzamide 24. Reduc-
tion of 24 gave the desired 3-hydroxy-4-methylbenzylamine 25.
3-Hydroxy-4-phenylbenzylamine 34 was prepared from 3-hy-
droxy-4-aminobenzoic acid 26. After 26 was esterified, it was
converted to the diazonium salt, and the latter was treated with
potassium iodide to yield 3-hydroxy-4-iodobenzoate 28. The
3-hydroxy group of 28 was protected as the methyl ether, then
the resulting 29 was treated with phenylboronic acid under
Suzuki coupling conditions to yield 3-methoxy-4-phenylben-
zoate 30. The benzoate group of 30 was reduced to the benzyl
alcohol 31,23 which was then activated as the benzyl chloride
32. Demethylation of 32 with boron tribromide, followed by
treatment with sodium azide, yielded 3-hydroxy-4-phenylben-
zylazide 33, which was converted to the desired 3-hydroxy-4-
phenylbenzylamine 34 under Staudinger reaction conditions.
In addition, heteroaryl groups were introduced as methoxy
replacements at the C-4 of the benzylamine headpiece in 5d.
Compounds 41 and 45a-d were prepared via a common
intermediate, 3-hydroxy-4-iodobenzonitrile 38, which was readily
synthesized from 3-hydroxybenzoic acid 35 using a procedure
modified from Sagi’s route.24a Treatment of 38 with 2-furyl-
tributyltin under Stille coupling conditions, followed by borane
reduction, gave 3-hydroxy-4-(2-furyl)benzylamine 41. The
corresponding 3-furyl derivative 45a was prepared in a different
way. The hydroxyl group of 38 was protected with MOMCl to
provide 42. The latter intermediate was then coupled with
3-furylboronic acid, followed by reduction of the nitrile group
with lithium aluminum hydride, to yield 3-methoxymethoxy-
4-(3-furyl)benzylamine 44a. Refluxing of 44a in methanolic
hydrochloric acid removed the MOM group to give the desired
3-hydroxy-4-(3-furyl)benzylamine 45a. The corresponding three
4-pyridyl regioisomers 45b-d were prepared in a similar
fashion, except that the introductions of the pyridyl groups were
carried out via Stille coupling procedures instead of Suzuki
coupling conditions.
a (i) (COCl)2, 60 °C; (ii) NH4OH, 0 °C to room temperature; (iii) BH3,
THF, 0 °C; (iv) HCl, MeOH, reflux; (v) H2O, HCl, NaNO2, 0 °C; then KI,
H2O, room temperature; (vi) NaH, DMF, then Mel, 0 °C; (vii) PhB(OH)2,
Pd(PPh3)4, Cs2CO3, DMF, 100 °C; (viii) LiAlH4, Et2O; (ix) SOCl2, CH2Cl2,
0 °C; (x) BBr3, CH2Cl2, -78 °C; then NaN3, DMF, room temperature; (xi)
PPh3, THF, H2O, room temperature; (xii) NH4OH, then I2, KI, H2O; (xiii)
Et3N, THF, isobutylchloroformate, 0 °C; then NH3 in THF, room temper-
ature; (xiv) SOCl2, toluene, reflux; (xv) BH3, THF, room temperature; (xvi)
PdCl2(PPh3)2, 2-furylSnBu3, CuI, DMF, 100 °C; (xvii) NaH, MOMCl, DMF,
0 °C; (xviii) (for 43a) 3-furanboronic acid, Pd(PPh3)4, Cs2CO3, DMF, 100
°C; (for 43b-d) XSnBu3, PdCl2(PPh3)2, CuI, DMF, 100 °C.
We then explored ways in our designs of new analogues to
minimize the potential metabolic liability of the 3-OH group in
the benzylamine headpiece while maintaining the potency and
selectivity in the series. One way was to replace the phenyl
ring of the 3-hydroxy-4-methoxybenzylamine headpiece with
pyrimidine ring 51, as shown in Scheme 6. Substituted aceto-
nitrile 4624b was treated with hydrochloric acid in dioxane to
form acetimidate hydrochloride 47. Subsequent treatment of 47
with ammonia gave the tricyclic iminium salt 48, which was
then coupled with the sodium salt of 3-hydroxy-2-methoxy-
acrylic acid methyl ester 4925 to produce the pyrimidinol
derivative 50. Hydrazinolysis of 50 deprotected the phthalimide
to yield the desired 2-aminomethyl-5-methoxy-pyrimidin-4-ol,
51.
Other attempts to enhance metabolic stability of the phenolic
OH in the headpiece involved the preparation of the corre-
sponding 2-pyridinol 55. Nucleophilic displacement of the
chloro group of 2-chloro-4-cyanopyridine, 52, with sodium
methoxide,26 followed by hydrogenation of the nitrile group of
53 with Raney Ni, in the presence of ammonia27 gave 4-(2-
methoxypyridyl)methylamine, 54. Acid hydrolysis of the meth-
oxy group of 54 provided the desired 2-pyridinol derivative 55.
To evaluate the contribution of potency from the 2-pyridinol

2292 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
Scheme 6a
Scheme 7a
a (i) RBr, KI, K2CO3, DMF; (ii) SOCl2, CHCl3; (iii) NaN3, DMF, room
temperature; (iv) Ph3P, THF, then H2O, room temperature; (v) conc. NH4OH
or NH3 in MeOH, 90 °C, sealed tube; (vi) RB(OH)2, Pd(PPh3)4, Cs2CO3,
KBr, dioxane, 60 °C; (vii) n-Bu4F, THF, room temperature; (viii) Ph3P,
DMF, CBr4; then NaN3; (ix) (CH2O)n, NaHCO3; (x) n-Prl, K2CO3,
2-butanone; (xi) (i-Pr)3SiCl, imidazole, CH2Cl2, room temperature; NaOBz,
BzOH, microwave, 120 °C; (xii) Ph3P, THF, H2O; then EtOH, Pd/C, H2.
a (i) HCl, dioxane, MeOH, room temperature; (ii) NH3 in MeOH, 0 °C;
(iii) MeOH, reflux; (iv) EtOH, NH2NH2; (v) NaOMe, MeOH, dioxane; (vi)
RaNi, H2, NH3, MeOH; (vii) aq. HCl, reflux; (viii) O(CO2-t-Bu)2, dioxane,
NaOH, room temperature; (ix) Me3SnPh, Cu(OAc)2, n-Bu4NF in THF,
CH2Cl2, room temperature; (x) 4 N HCl in dioxane; (xi) (3-furyl)Br, Cul,
K2CO3, DMF, 180 °C, microwave, or 150 °C; (xii) (Me2N)2CH(O-t-Bu),
DMF, 100-150 °C; (xiii) NaIO4, aq. THF, room temperature; (xiv)
MeONH2 HCl, pyridine, room temperature; (xv) Zn, HOAc,100 °C.
4-pyrone intermediates 67a-b. Treatment of 67a-b with
ammonia29 in a sealed tube generated the corresponding
2-hydroxymethyl-5-alkoxy-4-pyridinols 71a-b, which were
then converted into 74a-b, under the conditions described for
the preparation of 70a-b. Additional analogues of 74a-b where
the alkoxy group was replaced by aryl and heteroaryl groups
were also prepared. 4-Pyrone-5-triflate 7531 was coupled with
phenylboronic acid or furan-3-boronic acid under Suzuki
coupling conditions, followed by the removal of the silyl
protecting group to yield 5-phenyl-4-pyrone derivative 77a and
5-(3-furyl)-4-pyrone derivative 77b, respectively. The pyrones
77a-b were then converted into pyridinols 78a-b, followed
by transformation of the hydroxymethyl group into the ami-
nomethyl moiety, under the same conditions used to prepare
74a, to yield the desired 5-phenyl- (81a) and 5-(3-furyl)-4-
pyridinol (81b) derivatives. The 6-pyridinol isomer of 74b,
namely, 88, was obtained from 2-chloro-3-pyridinol 82. Treat-
ment of 82 with paraformaldehye in base32 gave 6-hydroxym-
ethyl-2-chloro-3-pyridinol 83, followed by propylation of the
phenolic group to give 84. After the primary alcohol of 84 was
protected as the triisopropylsilyl ether (TIPSO), it was reacted
with sodium benzyloxide in benzyl alcohol under microwave
conditions to give 85. Removal of the TIPS group from the
OH provided 86, which was converted into the corresponding
azide 87. Treatment of 87 under Staudinger conditions, followed
by catalytic hydrogenation, provided the desired 6-pyridinol
derivative 88.
headpiece 55, where N-1 is unsubstituted, we prepared the N-1
substituted 2-pyridones 58 and 65, which no longer carry an
OH group. Boc protection of the primary amine of 55 set the
stage for installation of a phenyl substituent on N-1 of the
pyridinol 56, via treatment with trimethylphenyl tin in the pr-
esence of cupric acetate and n-tetrabutylammonium fluoride to
generate the Boc protected 1-phenyl-2-pyridone derivative 57.
Acid treatment of 57 produced the desired 4-aminomethyl-1-
phenyl-2-pyridone 58. An alternate synthetic approach was used
to synthesize the corresponding 1-(3-furyl)-2-pyridone deriva-
tive, 65. 2-Hydroxy-4-methylpyridine 59 was N-arylated with
3-bromofuran via the copper-catalyzed Ullmann condensation28
to yield 1-(3-furyl)-4-methyl-pyridone 60. The 4-methyl group
of 60 was extended to N,N-dimethylenamine 61, which upon
periodate oxidation produced the aldehyde 62. The aldehyde
62 was then converted into its oxime 63, which upon reduction
with zinc in acetic acid yielded the desired, but impure, amine
65. Because of the difficulty in purifying the free amine, we
converted 65 into its Boc protected derivative 64, which allowed
easy purification. Final deprotection of the Boc group of 64
yielded pure 4-aminomethyl-1-(3-furyl)-2-pyridone, 65.
Several analogues of 5e were prepared with various alkyl,
aryl, heteroaryl, and alkoxy substituents on the C-4 of the
3-hydroxybenzylamino headpiece. Compounds 89a-g were
prepared by the reaction of 4e with the desired 3-hydroxyben-
zylamine hydrochloride in the presence of triethylamine and
DMF at room temperature, as shown in Scheme 8. The
3-hydroxybenzylamines used included 4-methyl (25), 4-phenyl
Additional headpieces, such as substituted 4-pyrones 70a-b,
4-pyridinols 74a-b, 81a-b, and 6-pyridinol 88, were prepared
as shown in Scheme 7. Kojic acid 66 was alkylated,29 and then
the OH group was transformed into the NH2 functionality,30
under the same conditions used to prepare 34, to yield the
desired 2-aminomethyl-5-alkoxy-4-pyrones 70a-b. The corre-
sponding pyridinols 74a-b were prepared starting from the

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2293
Scheme 9a
Scheme 8a
a (i) lithium diisopropylamide, (MeO)2CO, -78 °C; (ii) urea, 150 °C;
(iii) HC(OMe)3, HOAc, 90 °C; (iv) cyclopentylMgBr, Pd(PPh3)4, ethyl ether,
microwave, 75 °C; then THF, H2O, HCl, room temperature; (v) DMF, room
temperature.
cyclized as described before22 to yield the 6-t-butyl core 97.
The corresponding 6-cyclopentyl core 99 was formed by
reaction of bis-TBS protected 6-bromoisoquonline-1,3-diol22
with cyclopenyl magnesium bromide and tetrakis(triphenylphos-
phine)palladium under microwave conditions. Both 97 and 99
were then converted to the corresponding 4-methoxymethylene
derivatives 4k and 4l, respectively, by treatment with trimethy-
lorthoformate in acetic acid.22 Further reaction of 6-t-butyl
intermediate 4k with 5-substituted 4-pyridinols 74b, 81a, and
81b produced the final compounds 100c, 100e, and 100g,
respectively. Similary, the 6-cyclopentyl intermediate 4l was
reacted with 5-substituted 4-pyridinols 74b and 81b to generate
the final compounds 100d and 100h, respectively. 4-Methoxym-
ethylene-6-iodo intermediate 4e was also used to couple with
5-substituted 4-pyridinols 74a, 74b, and 81b to yield 100a,
100b, and 100f, respectively.
a (i) (For 89a, 89d-h, 94b-c) DMF, Et3N, room temperature; (for
89b-c, 90, 92, 93) DMF, room temperature; (for 91) THF, Et3N, room
temperature; (for 94a) THF, DMF, Et3N, room temperature.
(34), 2-furyl (41), 3-furyl (45a), the three possible pyridyl
isomers (45b-d), and O-n-Pr (16b).
The analogue of 5e, wherein the benzylamine was replaced
with the pyrimidinylmethylamine, namely, 90, was prepared by
treatment of 4e with the amine 51. Analogue 92, bearing a
2-aminomethyl-3-propoxy-6-pyridinol headpiece, was obtained
by reacting 4e with the amine 88. Two analogues 91 and 93,
where no hydroxyl group was present in the headpiece, were
prepared by reacting 4d with 2-methoxypyridyl derivative 54
and 5-methoxy-4-pyrone derivative 70a, respectively. The
analogue of 91, where the methoxy was replaced with a
hydroxyl, namely 94a, was obtained by treatment of 2-pyridone
55 with the 6-Br core 4d. Other 6-iodo compounds carrying
N-substituted 2-pyridones 94b-c were formed by reacting 4e
with N-substituted 2-pyridones 58 and 65, respectively.
Two cores 97 and 99, carrying 6-t-butyl and 6-cyclopentyl
substituents, respectively, were prepared, as depicted in Scheme
9. 2-Methyl-4-t-butylbenzoic acid 9533 was treated with lithium
diisopropylamide at -78 °C, followed by the addition of
dimethylcarbonate to yield the diacid 96, which was then
Results and Discussion
In Vitro Activity, and Metabolic Stability. Our synthetic
efforts on this series began with the discovery of 3f, which
carried a 4-(3,4-dihydroxyphenylethylamino)methylene head-
piece and showed moderate CDK4 activity with an IC50 value
of 16.2 µM, as shown in Table 1. Further analogues were
prepared by varying the chain length between the 3,4-dihy-
droxyphenyl ring and the aminomethylene moiety. When the
3,4-dihydroxyphenyl ring was directly linked to the aminom-
ethylene moiety, as shown in 3a, the CDK4 potency was not
much improved (IC50 ) 7.8 µM). However, when the two
groups were linked via a methylene bridge, the resulting
compound 3b showed a 10-fold enhancement in CDK4 potency.
We then explored the importance of the two hydroxyl groups
by preparing the two monohydroxyl isomers 3c and 3d.
Compared to the 3,4-dihydroxy compound, 3b, the 3-hydroxy
derivative, 3d, was equipotent and selective in inhibiting CDK4;

2294 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Table 1. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
Tsou et al.
Table 2. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
kinase assays IC50 (µM)a
kinase assays IC50 (µM)a
compd
n
R1
R2
CDK4
CDK1
CDK2
compd
R
CDK4
CDK1
CDK2
3a
3b
3c
3d
3e
3f
0
1
1
1
1
2
OH
OH
H
OH
OH
OH
OH
OH
OH
H
OMe
OH
7.8
1.2
<50
0.78
0.19
16.2
3e
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
0.19
0.17
0.23
0.94
0.03
0.01
0.031
0.004
0.002
0.002
0.01
48.7
<50
<50
8.1
<50
6
22.3
9.7
23.3
2.5
6.9
6.6
36.6
37.2
<50
48.7
12.5
17.3
16.9
6.9
6-F
6-Cl
5-Br
6-Br
6-I
<50
3.1
<50
0.87
2.9
3.1
18.3
1.1
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate, and repeat values agreed, on average, with a mean 2-fold
difference.
6-OMe
6-(N-pyrrolyl)
6-(3-thienyl)
6-(3-furyl)
6-phenyl
5j
34.7
25.9
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate, and repeat values agreed, on average, with a mean 2-fold
difference.
however, the 4-hydroxy derivative, 3c, was at least 64-fold less
potent. These results clearly show that the 3-hydroxyl moiety
is critical for CDK4 potency and selectivity. Markwalder et al.34a
and Rossi et al.34b reported a similar requirement of m-OH
moiety on the 6-arylmethyl group of pyrazolopyrimidinone, a
different core from ours, for enhancing CDK4 potency; however,
the improvement was not sufficient enough to render their
compound selective for CDK4. Our modeling studies revealed
a key hydrogen bond interaction between the 3-OH group and
the side chain of His 82 of the CDK4 enzyme, which will be
elaborated below. Analogues of 3d were then prepared by
introducing a variety of substituents at the C-4 position next to
the 3-OH group. One of them, the 3-hydroxy-4-methoxy
analogue 3e, showed a 4-fold increase in potency over 3d, with
an IC50 value of 0.19 uM in inhibiting CDK4, with 256- and
36-fold selectivity over CDK1 and CDK2, respectively.
this NH and the backbone carbonyl of Val 83 was shown. A
detailed discussion on the modeling will be presented later.
Introduction of an additional methoxy group, as in 18, resulted
in a 4-fold increase in potency compared to 5d. We then
investigated the effect of varing the 4-methoxy substituent of
5d. Compared to 5d, the 4-ethoxy derivative 17d was equi-
potent, but the 4-n-propoxy derivative 17b was about an order
of magnitude less potent. However, the 4-methoxyethoxy
derivative, 17c was as active as 5d. Interestingly, the 4-amino
derivative 21 was 3-fold less potent than 5d, but was much less
selective versus CDK2, while selectivity over CDK1 was
maintained.
We then investigated substituent effects on the isoquinoline-
1,3-dione core. Relative to 3e, the corresponding 6-fluoro (5a)
and 6-chloro (5b) derivatives showed comparable CDK4
inhibitory activity; however, the 6-bromo (5d) and 6-iodo (5e)
derivatives exhibited enhanced potency in inhibiting CDK4 with
IC50 values of 30 nM and 10 nM, respectively, as shown in
Table 2. In addition, 5d is over 1600-fold selective for CDK4
over CDK1 and CDK2. The position of the substituent on the
core seems to be important for activity. Moving the bromo
substituent from C-6 (5d) to C-5 (5c) resulted in a 30-fold loss
of CDK4 inhibitory activity. Therefore, our synthetic efforts
focused on C-6 substituted analogues. We found that the
6-phenyl derivative (5j) was as potent as the 6-iodo derivative
(5e) in inhibiting CDK4 (IC50 ) 10 nM); however, the former
was more selective than the latter over CDK1 (3400-fold vs
600-fold) and CDK2 (2600-fold vs 80-fold). Compared to 5j,
the corresponding 6-heteroaryl derivatives such as N-pyrrolyl
(5g), 3-thienyl (5h), and 3-furyl (5i) were more potent CDK4
inhibitors with IC50 values of 4, 2,and 2 nM, respectively. In
particular, 5h showed excellent selectivity for CDK4 over CDK1
and CDK2 by 11,000- and 9,000-fold, respectively.
Table 4 shows the effect of varying the 4-methoxy substituent
of 5e. Compared to 5e, the 4-methyl derivative 89a was 3-fold
less potent, whereas the 4-phenyl, 4-(2-furyl), and 4-(3-furyl)
derivatives (89b, 89c, and 89d, respectively) were about 10-
fold less potent. However, they all showed comparable selectiv-
ity for CDK4 over CDK1 and CDK2. One compound, 89d, was
further evaluated for its ability to inhibit a panel of protein
kinases, as shown in Table 5. It is clear that 89d was highly
potent in inhibiting CDK4 among the serine-threonine kinases
and tyrosine kinases tested. The CDK4 inhibitory activity for
the three C-4 pyridyl isomers varied depending upon the position
of the pyridyl nitrogen. Although the 4-(2-pyridyl) derivative
89e was a poor CDK4 inhibitor, the 4-(3-pyridyl) and 4-(4-
pyridyl) derivatives 89f and 89g were as potent as 5e. Among
these three C-4 pyridyl isomers, the pyridyl nitrogen atom from
89e could potentially form a hydrogen bond interaction with
the phenolic OH group, thereby interfering with this critical OH
group interaction with His 82 of the CDK4 enzyme for activity.
In terms of selectivity, both 89f and 89g showed higher
selectivity for CDK4 over CDK1 and CDK2, relative to 5e.
Analogues 89f demonstrated better cellular activity than 89g.
Through our optimization efforts, we have synthesized
selective CDK4 inhibitors with potencies as low as 2 nM.
However, all of these inhibitors bear a phenolic OH group, a
critical group for activity, but also a metabolic liability for the
effectiveness of these inhibitors in vivo. It is well-known that
phenolic OH groups34c are prone to glucuronidation in the
endoplasmic reticulum and sulfation in the liver via phase II
In the study of the 3-hydroxy-4-methoxybenzylamino group,
shown in Table 3, the R-methylbenzylamine derivative 11 was
an order of magnitude less potent than 5d, but the N-
methylbenzylamine derivative 13 showed a dramatic loss of
activity. The importance of the NH group of the benzylamine
for CDK4 inhibitory activity was substantiated by our molecular
modeling studies, wherein a hydrogen bond interaction between

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2295
Table 3. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
kinase assays IC50 (µM)a
compd
X
Y
CDK4
CDK1
CDK2
5d
11
13
17a
17b
17c
17d
18
H
H
H
Me
H
OH
O-n-Pr
0.03
0.37
<50
0.04
0.35
0.04
0.03
0.007
0.08
<50
<50
<50
<50
42.5
<50
<50
<50
<50
<50
<50
44.4
<50
34.5
<50
45.5
<50
6.7
Me
OH
OH
OH
OH
O-(CH2)2OMe
OEt
21
OH
NH2
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%, as determined from the dose-response curve. Determinations were done in
duplicate, and repeat values agreed, on average, with a mean 2-fold difference.
Table 4. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities and Cell Proliferation
kinase assays IC50 (µM)a
cell-based assays IC50 (µM)b
compd
X
Y
Z
R
CDK4
CDK1
CDK2
HCT116
MCF-7
5e
I
I
I
I
I
I
I
I
I
I
I
I
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
OMe
Me
Ph
0.01
0.03
0.13
0.10
0.06
19.30
0.01
0.008
0.02
0.44
0.02
0.01
0.03
0.02
<0.005
0.03
0.03
0.02
<0.005
6.00
>50
47.8
>50
24.4
>50
>50
>50
>50
>50
10.1
14.7
25.3
6.20
3.10
16.3
13.5
9.50
3.10
0.87
13.6
15.1
8.40
4.70
>50
3.60
>50
2.6
>50
0.70
0.50
1.60
0.96
1.20
2.60
2.30
1.90
1.30
2.00
6.70
0.42
0.38
0.48
0.25
0.44
2.30
0.65
>50
0.67
1.30
0.73
0.43
0.15
0.15
0.55
0.48
0.81
3.70
8.20
0.88
0.70
1.13
0.54
1.10
1.50
1.8
89a
89b
89c
89d
89e
2-furyl
3-furyl
2-pyridyl
3-pyridyl
4-pyridyl
O-n-Pr
OMe
O-n-Pr
OMe
O-n-Pr
O-n-Pr
O-n-Pr
Ph
89f
89g
89h
90
>50
92
N
1.40
1.00
0.88
0.70
0.30
0.70
2.70
1.40
0.83
100a
100b
100c
100d
100e
100f
100g
100h
CH
CH
CH
CH
CH
CH
CH
CH
I
t-Bu
cyclopentyl
t-Bu
N
N
N
N
I
3-furyl
3-furyl
3-furyl
t-Bu
cyclpentyl
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%, as determined from the dose-response curve. Determinations were done in
duplicate, and repeat values agreed, on average, with a mean 2-fold difference. b Dose-response curves were determined at five concentrations. The IC50
(µM) values are the concentrations needed to inhibit cell growth by 50%, as determined from these curves.
Table 5. Inhibitory Activity of 89d against a Panel of Protein Kinases
donating substituents (higher pKa) facilitate the conversion of
phenols to the corresponding glucuronides. As expected, 3-hy-
droxybenzylamine derivatives bearing 4-OMe, 4-(3-furyl), and
4-(O-n-Pr) substituents, namely, 5e, 89d. and 89h, respectively,
suffered reduced stability in phase II metabolism using rat
microsomes, as depicted in Table 6.
To address the phase II metabolism issue associated with the
phenolic OH, we introduced one or two nitrogen atoms in the
phenyl ring of the benzylamine headpiece in 5e, as shown in
Table 4. We expect that electronic effects of the nitrogen in the
dihydroxypyrimidine (90), 6-pyridinol (92), and 4-pyridinols
(100a-h) would decrease the nucleophilicity of the phenols,
thereby decreasing the nucleophilic attack on C-1 of the uridine
kinase
IC50 (µM)
kinase
IC50 (µM)
kinase
IC50 (µM)
CDK4
AKT
IGFR
KDR
MK2
PDK1
Tpl2
0.06
10.2
>10
10
19
17
CDK1
Braf
IKK-2
LCK
mTOR
PKCθ
STAT3
24.4
>10
>10
>30
18
CDK2
EGFR
IR
LYN
p70S6
Src
4.7
>10
>10
8.5
>30
>30
25.9
1.7
>4
metabolism, leading to rapid excretion from the body. The rate
of glucuronidation is dependent on the lipophilicity and the
nucleophilicity of the phenols.35,36 As the lipophilicity of phenols
increases, the rate of glucuronidation increases. Electron-

2296 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Table 6. Rat Microsomal Stability Studies
Tsou et al.
Table 7. Inhibition (IC50) of CDK4, CDK1, and CDK2 Activities
t1/2 (min)
kinase assays IC50 (µM)a
compd
X
Y
R
aphase I
bphaseI/II
5e
CH
CH
CH
CH
N
CH
CH
CH
N
CH
CH
CH
OMe
18
6
18
15
>30
>30
>30
22
compd
X
Y
CDK4
CDK1
CDK2
89d
89h
92
100a
100b
100f
3-furyl
O-n-Pr
O-n-Pr
OMe
O-n-Pr
3-furyl
>30
>30
>30
>30
>30
>30
89b
89d
91
I
I
Br
Br
Br
I
Ph
3-furyl
0.13
0.06
31.7
1.3
0.56
0.27
0.06
47.8
24.4
>50
15.1
4.7
6.1
16.9
2.7
41.5
2.6
93
OMe
H
Ph
>50
N
N
94a
94b
94c
>5050
29.6
11.6
a Substrate half-life (min) when incubated at 37 °C for 15 min with SD
rat liver microsomes (0.5 mg/mL protein) and NADPH cofactor. b Substrate
half-life (min) when incubated at 37 °C for 15 min with SD rat liver
microsomes (0.5 mg/mL protein), NADPH, and UDPGA cofactors.
I
3-furyl
a Concentration (µM) needed to inhibit the Rb phosphorylation by 50%,
as determined from the dose-response curve. Determinations were done
in duplicate, and repeat values agreed, on average, with a mean 2-fold
difference.
diphosphate glucuronic acid to generate glucuronidate metabo-
lites. Furthermore, the 4-pyridinols and 6-pyridinols are expected
to be in equilibrium with the corresponding 4-pyridones and
6-pyridones,37a,b which are devoid of the OH group, therefore
reducing the extent of phase II metabolism. The keto-enol
tautomeric equilibrium strongly depends on the substituent, on
its ring position, and on the solvent dielectric constant.38
However, in all cases, the solvent dielectric effect tends to
increase the preponderance of the keto structure. In the
physiological environment where water has a high dielectric
constant, the equilibrium is expected to shift more toward the
keto tautomeric form, leading to metabolically more stable
molecules. We were pleased to see that the 5-(3-furyl)-4-
pyridinol derivative 100f showed improved phase II metabolic
stability and that the 5-(O-n-Pr)-6-pyridinol derivative 92,
5-OMe-4-pyridinol derivative 100a, and 5-(O-n-Pr)-4-pyridinol
derivative 100b showed good stability with t1/2 values greater
than 30 min (Table 6).
These metabolically more stable pyridinol derivatives are also
potent CDK4 inhibitors. As shown in Table 4, the 4-pyridinol
derivative 100a was equipotent with 5e, whereas the corre-
sponding 4-pyrimidinol derivative 90 was 44-fold less active
in inhibiting the CDK4 enzyme. Varying the C-5 substituent at
the 4-pyridinol headpiece of 100a from methoxy to n-propoxy
in 100b and 3-furyl in 100f resulted in a 3-4-fold decrease in
potency, but a 2-fold increase in cellular activities. Comparing
the two pyridine isomers, the 6-pyridinol derivative 92 was as
active and selective as the corresponding 4-pyridinol derivative
100b, with comparable cellular activities. Additional substitution
effects at the C-6 of the isoquinoline-1,3-dione core were
evaluated. The 6-t-butyl derivatives (100c and 100g) were
equipotent with the corresponding 6-iodo derivatives (100b and
100f), whereas the corresponding 6-cyclopentyl derivatives
(100d and 100h) showed at least 6-fold increase in CDK4
inhibitory activity, with IC50 values below 5 nM. Furthermore,
100d and 100h showed good selectivity for CDK4 over CDK1
and CDK2, by more than 620-fold and 240-fold, respectively.
Although both 100d and 100h showed equivalent potency
against CDK4 and selectivity vs CDK1 and CDK2, 100d was
more active than 100h in inhibiting HCT116 and MCF7 cells,
with IC50 values of 0.15 µM and 0.3 µM, respectively. Among
a pair of 6-iodo and 6-t-butyl derivatives (100b and 100c,
respectively) where the headpiece is 5-propoxy-4-pyridinol, both
showed comparable CDK4 potency (IC50 values of 20-30 nM)
and selectivity, similar to the pairs of the corresponding 5-(3-
furyl)-4-pyridinols (100f and 100g). The three 6-t-butyl deriva-
tives 100c, 100e, and 100g, were equipotent against CDK4;
however, 100e was the most active one in inhibiting cells.
Additional derivatives were prepared where the 3-OH group
in the headpiece of the isoquinoline-1,3-dione was replaced with
a methoxy or a carbonyl group. On the basis of the importance
of the 3-OH group on the headpiece for activity, we expected
91 would be a poor CDK4 inhibitor (IC50 value of 31.7 uM) as
shown in Table 7 since the 3-OH was replaced with a 3-OMe
group. However, the 4-pyrone derivative 93 also lacks an OH
group; to our surprise, its ability to inhibit CDK4 was 30-fold
better than that of 91. We were also intrigued to observe that
N-substituted 2-pyridones 94b-c, bearing no OH substituent,
showed reasonably good potency in inhibiting CDK4. In fact,
comparable potency was observed for the pair of N-phenyl-2-
pyridone derivative 94b and 4-phenyl-3-hydroxyphenyl deriva-
tives 89b, as well as for the pair of N-(3-furyl)-2-pyridone
derivative 94c and 4-(3-furyl)-3-hydroxyphenyl derivatives 89d.
This unexpected result seems to contradict what we observed
earlier, i.e., that a 3-OH group on the headpiece is essential for
activity. However, this puzzle was unraveled by our molecular
modeling, as described below in the subsequent discussions.
Molecular Modeling. A binding model for compound 5e at
the ATP binding site is presented in Figure 1. The protein
coordinates used in this study are those reported in the X-ray
crystal structure of the catalytic domain of CDK2, with three
residues of the binding site mutated to mimic CDK4.20 The
CDK4 mimic structure of Ikuta et al. includes three mutations,
at residues F82H, L83V, and K89T. The amino acid numbering
of the CDK4 mimic structure will therefore be that of CDK2,
which is the parent structure. The inhibitor was docked using
the GLIDE docking algorithm in the XP (extra precision)
mode.39 The resulting model successfully identifies key hydro-
gen bond interactions between the ligands and residues of the
protein’s ATP binding pocket. The binding model accounts for
the nanomolar activity as well as the selectivity of the isoquino-
line-1,3-dione inhibitor, in that significant and strong interactions
with residues unique to CDK4 have been identified. The specific
orientation of the ligand in the binding model exploits the range
of residues unique to CDK4, namely, 82 and 83. In the most
favored bound conformation, the NH of the isoquinoline-1,3-

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2297
Figure 1. Proposed binding model for 5e in the ATP binding site of the CDK4 mimic model.
dione core is found to be 2.6 Å from the backbone carbonyl
oxygen of Glu 81. The binding model places a carbonyl oxygen
of the isoquinoline-1,3-dione core at 2.1 Å from the backbone
NH of Val 83, a position in the binding cavity where the L83V
mutation differentiates CDK4 from CDK2. Additionally, the
amino NH of the benzylamino headpiece is found within 2.5 Å
from the backbone carbonyl oxygen of Val 83. As the sequence
of amino acids confers specific spatial features to the protein
cavity, we postulate that the close proximity of these interactions
lends support to the selectivity of the isoquinoline-1,3-dione
core for the CDK4 binding pocket. A second carbonyl oxygen
of the isoquinoline-1,3-dione core is found within 4.2-4.8 Å
of the center of the phenyl ring of Phe 80, the gatekeeper residue.
Short molecular dynamics runs resulted in the placement of this
carbonyl oxygen within 3.5-4.0 Å of various aromatic hydro-
gens of the Phe 80 side chain. It is postulated here that
interactions between at least two of the aromatic hydrogens of
the phenyl ring and the carbonyl oxygen of the isoquinoline-
1,3-dione core play a significant role in supporting inhibitor
binding. These interactions of the core with Phe 80, Glu 81,
and Val 83 of the CDK4 protein in the binding model are quite
similar to the ones we observed in our previous series, the
4-(phenylaminomethylene)isoquinoline-1,3-diones.22 However,
in the headpiece, the binding interactions of the current series
are different from those observed for our previous series. In
the binding model of our previous series, the basic nitrogen
atom of the headpiece was expected to interact with the flexible
side chains of Asp 84 and His 82, two CDK4-unique amino
acid residues, in the solvent exposed regions. In contrast, the
current series bears a 3-hydroxyl substituent in the benzylamino
headpiece. In the current binding model, this 3-hydroxyl group
forms an H-bond with the imidazole nitrogen of the His 82 side
chain, with an interaction distance of within 2.5 Å. This key
interaction is not attainable by a 4-hydroxyl compound 3c, a
poor inhibitor for CDK4, compared to the corresponding
3-hydroxyl compound 3d. Furthermore, placement of an electron
donating substituent such as methoxy (e.g., compound 3e in
Table 1 and compound 5e in Table 2) is found to enhance this
interaction.
In summary, our binding model identifies three key interac-
tions with the backbone groups of hinge region residues as well
as favorable, albeit supportive, electrostatic interactions with
at least two of the aromatic hydrogens of the side chain of Phe
80, the gatekeeper residue. It is noted here that two of these
three hydrogen bonds involve the backbone groups of Val 83,
a residue unique to CDK4. Additionally, the hydrogen bond
interaction between the 3-hydroxyl group on 5e and the side
chain of a CDK4-unique residue, His 82, contributes to and
strengthens the cooperative hydrogen bond network between
5e and CDK4 enzyme. Like 5e, compound 5d is a potent and
selective CDK4 inhibitor. Addition of a methyl group at the R
position of the benzylamino headpiece in 5d would perturb the
overall binding to CDK4, and the resulting compound 11
showed a 10-fold loss of CDK4 potency, though it is still
selective for CDK4. However, when the NH of the benzylamino
headpiece in 5d is methylated, the resulting compound 13 can
no longer interact with the backbone carbonyl oxygen of Val
83. This led to such a disruption of the cooperative hydrogen
bond network with CDK4 that 13 showed a dramatic loss of
potency and selectivity for CDK4. Thus, having the 3-hydroxyl
group is crucial but not sufficient for CDK4 potency and selectivity,
both of which also depend on the optimal and unique orientation
of these inhibitors in the CDK4 binding pocket, resulting in the
formation of a strong and cooperative hydrogen bond network. In
comparison, modeling studies of pyrazolopyrimidinone34a,b identify
a hydrogen bond interaction between the meta-hydroxyl moiety
of the 6-arylmethyl group and the side chain of His 82 of CDK4,
in addition to interactions of the core with Val 83, a total of
two hydrogen bond interactions with the backbone moieties of
the hinge region of CDK4. Although improved potency for
CDK4 was realized in these studies, no selectivity for CDK4
was achieved. The lack of CDK4 selectivity of the pyrazol-

2298 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
opyrimidinone series may lie in the fact that its core is different
from ours; therefore, the two series are expected to be oriented
differently in the ATP-binding site of the CDK4 enzyme. In
comparison to compound 5e, the pyridone derivatives 94b,c lack
the critical 3-hydroxyl group. However, the pyridone moiety
of 94b,c (see Table 7) places the carbonyl oxygen within 3.5 Å
of the flexible side chain of His 82 in the solvent exposed region.
It is expected that the flexibility and the solvent exposed nature
of this interaction could bring the carbonyl oxygen of pyridones
closer to the NH of His 82 side chain and possibly a water-
mediated interaction. There are precedents that showed H-bond
interaction of NH of imidazole or His side chain with carbonyl
oxygen.40a,b This favorable interaction may explain why the
pyridones 94b,c are as potent as the corresponding phenols
89b,d in inhibiting CDK4. However, the His 82 side chain may
be partially protonated. The extent of the protonation depends
on the pKa of the histidine side chain, which varies according
to the environment surrounding the His residue.40c It is possible
that the protonated nitrogen of imidazole could form an H-bond
interaction with the carbonyl oxygen of the pyridones. But the
nature of the interaction can be fully understood when X-ray
crystal structure of CDK4 is available to enable cocrystal
structures with our pyridone derivatives.
Figure 2. Effect of 89d, 94c, and 100c on pRb phosphorylation in
MCF-7 cells. MCF-7 cells were incubated in the presence of 89d, 94c,
or 100c at the indicated concentrations at 37 °C for 24 h. Protein extracts
were analyzed by SDS-PAGE, followed by immunoblotting using
either phospho-Rb antibody to probe ppRb (Ser 807/811, upper panel)
or Rb antibody to probe the total pRb (lower panel). Equivalent protein
loading of lanes was confirmed by Ponceau S staining. Blots were
scanned and quantified using the FluorS multi-image analyzer (BioRad).
The results were normalized to the amount of total pRb, and the IC50
values for ppRb were determined from inhibition plots (Kaleidagraph).
The cellular IC50 values were determined from inhibition curves (LSW
Toolbox) using data points from the SRB cell proliferation assay.
Other researchers such as McInnes,41 Honma,42 and Aubry43
carried out computational studies using a crystal structure of
inhibitor bound CDK2 and a homology model of CDK4, which
was built using both CDK2 and CDK6 as templates. As such,
the amino acid numbering of these homology models differs
from those of the CDK4 mimic models used by our group,
Ikuta20 and Pratt,44 by a value of 13 for the residues of the
ATP binding site. Docking studies with CDK4 selective
compounds were then used to elucidate binding features of the
CDK4 selective compounds in the ATP binding pockets of both
the CDK2 crystal structure and the CDK4 homology model. A
key finding of their study is the presence of a positively charged
moiety, proximal to the Asp and Glu residues that correspond
to residues numbered 86 and 131, respectively, in the CDK2
structure used in our study (Figure 1). Although the 4-(benzy-
laminomethylene)isoquinoline-1,3-(2H,4H)-dione analogues of
our study are not found to interact with Asp 86 (not shown)
and are a little more than 4.0 Å distance from the Glu 131 side
chain, our binding models place the phenol moiety of our
inhibitors within 2.5 Å of the His 82 side chain, a residue unique
to CDK4. The CDK4 mimic structure of Ikuta et al.20 used in
this study contains a Gln residue at position 131. The corre-
sponding residue in CDK4 is Glu. Again, it should be noted
that, in a previous study,22 an in silico mutation of Gln 131 to
Glu 131 with subsequent short molecular dynamics runs was
carried out. Our binding models place the 6-iodo substituent of
compound 5e in close proximity to the Gln 131 residue.
Specifically, Glu 131 is in a region of the binding pocket, where
protein flexibility and the presence of solvent molecules are
expected to be significant. Park et al.45 have shown that protein
flexibility is crucial to inhibitor binding and that CDK4
undergoes significant conformational changes resulting from
solvent and inhibitor interactions. We postulate that detailed
molecular dynamics studies with the residue at position 131
mutated to Glu will identify the key role that Glu 131 plays in
stabilizing the binding of protein to 4-(benzylaminomethyl-
ene)isoquinoline-1,3-(2H,4H)-dione and further enhance our
understanding of the SAR of substituents at position 6 of the
isoquinoline-1,3-dione core (Table 2).
source of CDK2/CDK4 selectivity by several investigators.20,44
Our binding studies, however, do not indicate any involvement
of the threonine residue in ligand binding. Pratt et al.44 have
pointed to the K89T mutation as being the primary source of
CDK4 selectivity for the bisanilino pyrimidine analogues. We
point to intrinsic differences in the shapes of the 4-(benzylami-
nomethylene)isoquinoline-1,3-(2H,4H)-dione analogues, which
result in optimal orientations in the CDK4 binding cavity, that
do not involve the threonine residue in question. Therefore, the
4-(benzylaminomethylene)isoquinoline-1,3-(2H,4H)-dione in-
hibitors of our study represent a class of molecules that form
interactions with several CDK4 specific amino acids that are
described in earlier literature as well as the side chain of His
82, a nonconserved amino acid residue proximal to the hinge
region of the CDK4 catalytic domain. We believe that the
protein-ligand interactions detailed in our binding models
characterize the selectivity and potency of the isoquinoline-1,3-
dione analogues of this study.
Inhibition of pRb Phosphorylation. The biological activity
of 89d, 94c, and 100c was evaluated in MCF-7 cells, which
overexpress cyclin D1 due to gene amplification. We examined
the phosphorylation state of pRb, the physiological substrate
for CDK4/cyclin D complexes, after overnight incubation with
89d, 94c, or 100c. As shown in Figure 2, treatment of MCF-7
cells with each of these compounds resulted in a reduction of
phosphorylation of pRb, as indicated by the decrease of the band
corresponding to ppRb in polyacrylamide-SDS gels (upper
panel). The inhibition was dose-dependent and could be detected
at the lowest concentration tested (0.15 µM). The resulting IC50
values are in agreement with those obtained in the cellular
proliferation assays. Clearly, the biological activities of these
compounds are consistent with the expected effects of CDK4
inhibition.
Conclusions
We report here the discovery of 4-(benzylaminomethyl-
ene)isoquinoline-1,3-(2H,4H)-diones and 4-[(pyridylmethyl)ami-
nomethylene]isoquinoline-1,3-(2H,4H)-diones as potent and
selective inhibitors of the cyclin-dependent kinase 4 (CDK4).
Compared to our earlier series22 where the headpiece is a
The K89T mutation, which places a threonine at the entrance
to the ATP binding cleft of CDK4, is singled out as a significant

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2299
using the GLIDE39 docking algorithm in the XP (extra precision)
mode. Details of the algorithm are found in GLIDE documentation.
Briefly, GLIDE’s proprietary conformational expansion and exhaus-
tive search of the binding site produces a multitude of ligand poses
that undergo an initial refinement, energy minimization on a
precomputed grid, and a final scoring and ranking. GLIDE uses
proprietary scoring functions that are variations of the ChemScore47
empirical scoring function and the OPLS-AA48 force field to
compute van der Waals and electrostatic grids for the receptor. The
final ligand binding poses are ranked according to a computed E
model score that encompasses the grid score, the proprietary Glide
Score, and the internal energy strain.
substituted aniline, this new series carries substituted 3-hy-
droxybenzylamino or (hydroxypyridyl)methylamino headpieces
and exhibits enhanced potency for CDK4 and higher activity
in inhibiting cell proliferation. Furthermore, compounds bearing
the (hydroxypyridyl)methylamino headpieces show good phase
II metabolic stability. The inhibitory activity is enhanced when
an iodo, aryl, heteroaryl, t-butyl, or cyclopentyl substituent is
introduced at the C-6 position of the isoquinoline-1,3-dione core.
We also present a proposed binding model for 5e at the ATP
binding site of a CDK4 mimic structure that contains three
mutations at residues F82H, L83V, and K89T. This binding
model shows a hydrogen bond interaction between the 3-hy-
droxyl group on 5e and a CDK4-unique His 82 as well as
interactions between the core and Phe 80, Glu 81, and Val 83,
a residue unique to CDK4. The crucial interaction of the
3-hydroxyl group with CDK4 contributes to and strengthens
the cooperative hydrogen bond network with CDK4, thus
providing the potency and selectivity of this series of compounds
for CDK4 over CDK2 and CDK1. In cells, selected compounds,
89d, 94c, and 100c, from this series have demonstrated
biological activity: they inhibit phosphorylation of pRb, the
physiological substrate for CDK4/cyclin D1 at the IC50 values
consistent with their respective IC50 values for inhibition of cell
proliferation.
Preparation of the Substituted Cores. 6-tert-Butylisoquinoline-
1,3(2H,4H)-dione (97). To a solution of N,N-diisopropylamine (1.06
g, 10.4 mmol) in THF (10 mL) at 0 °C was added n-butyllithium
(4.2 mL, 2.5 M, 10.5 mmol). After it was stirred for 10 min, it was
cooled to -78 °C, and then a solution of 4-tert-butyl-2-methyl-
benzoic acid (95) (500 mg, 2.6 mmol) and dimethylcarbonate (471
mg, 5 mmol) in THF (5 mL) was added. It was warmed up to
room temperature and stirred overnight. The mixture was cooled,
and water was added. The organic phase was extracted with water.
The combined aqueous phase was washed with hexane to remove
N,N-diisopropylamine. The water solution was acidified with conc.
HCl to pH 2 and extracted with ethyl acetate. The ethyl acetate
layer was dried and evaporated to give 552 mg (90%) of 4-tert-
butyl-2-(carboxymethyl)benzoic acid (96) as a white solid: MS
(ESI) m/z 237.1 (M + 1)+1
.
Compound 96 (90 mg, 0.38 mmol) and urea (46 mg, 0.76 mmol)
were mixed and heated at 145 °C for 1 h. The solid was collected
by filtration, washed with ethyl acetate, water, and methanol, and
air-dried to give 45 mg (55%) of 97 as a solid: MS (ESI) m/z 216.1
(M - 1)-1; HRMS (ESI) m/z calcd for C13H15NO2 218.11756;
Experimental Section
1
Chemistry. H NMR spectra were determined with a Bruker
DRX400 spectrometer at 400 MHZ or a NT-300 WB spectrometer
at 300 MHz. Chemical shifts (δ) are expressed in parts per million
relative to the internal standard tetramethylsilane. Electrospray mass
spectra were recorded in positive mode on a Micromass Platform
spectrometer. Electron impact (EI) and high-resolution mass spectra
(HRMS) were obtained on a Finnigan MAT-90 spectrometer. Some
high-resolution electrospray mass spectra with higher precision were
obtained on a Brucker 9.4T FTMS spectrometer. Chromatographic
purifications were by flash chromatography using Baker 40 µm
silica gel. Melting points were determined in open capillary tubes
on a Meltemp melting point apparatus and are uncorrected.
Semipreparative reverse-phase high-pressure liquid chromtography
(RP-HPLC) was performed using a Gilson Preparatory HPLC. The
sample was dissolved in DMSO, applied on a Phenomenex C18
Luna column (21.2 mm × 60 mm, 5 µm), and eluted at 22.5 mL/
min with a 19 min of gradient, 5% B; 2.5 min, 5% B; 18 min,
95% B; 19 min, 95% B, where solvent A ) water (0.02% TFA
buffer), and solvent B ) acetonitrile (0.02% TFA buffer). Analytical
high-pressure liquid chromatograpy (HPLC) and LC-MS analyses
were conducted using the following two instruments and conditions.
LCMS1: Analytical HPLC was conducted on an HP 1100 liquid
chromatography system over a 2.1 mm × 30 mm xterra C18 Luna
column (5 µm) at 50 °C using multiple wavelength UV detection
(typically 215, 254 nm) and MS detection (API-ES scanning mode
positive/negative 100-1000; fragmentor, positive 140 mV; nega-
tive, 170 mV). A gradient elution of increasing concentrations of
acetonitrile in water containing 0.02% formic acid (10-90% over
3.5 min and was held at 90% for an additional 1.5 min) and a flow
rate of 1 mL/min were employed. LCMS2: Analytical HPLC was
conducted on an Agilent LC-1100-MSD liquid chromatography
system over a 50 × 2.1 mm Aquasil C18 column (Thermo Electron
Corporation, 5 µm) using multiple wavelenth UV detection (typi-
cally 215, 254 nm) and MS detection (single quadrupole mass filter
scanning from 100-1000 Da). A gradient elution of increasing
concentrations of acetonitrile in water containing 0.1% formic acid
(0-100% over 2.5 min and was held at 100% for additional 1.5
min) and a flow rate of 1 mL/min were employed.
found, 218.11765 (M + H)+1
.
(4E)-6-tert-Butyl-4-(methoxymethylene)isoquinoline-1,3(2H,4H)-
dione (4k). A solution of 97 (45 mg, 0.20 mmol) in DMF (1 mL),
acetic anhydride (2 mL), and trimethyl orthoformate (0.2 mL, 1.83
mmol) was heated at 120 °C for 1 h. After the solvents were
evaporated, the precipitate was washed with methanol, and dried
to give 44 mg (85%) of 4k as a yellow solid: MS (ESI) m/z 260.2;
HRMS (ESI) m/z calcd for C15H17NO3 260.12812; found, 260.12825
(M + H)+1
.
6-Cyclopentyl-4H-isoquinoline-1,3-dione (99). To 6-Bromo-1,3-
bis-(tert-butyl-dimethyl-silanyloxy)-isoquinoline22 (1.76 g, 3.75
mmol) was added tetrakis(triphenylphosphine)palladium (110 mg,
0.095 mmol) and 2 M cyclopentyl magnesium bromide in ether
(3.0 mL, 6.0 mmol). This mixture was at 75 °C under microwave
conditions for 10 min. The mixture was cooled to room temperature,
transferred to a flask with THF and water, then 2 M hydrochloric
acid (20 mL) was added and stirred for 4 h at room temperature.
The organic solvents were removed in vacuo, the mixture extracted
with ethyl acetate, the organic layer dried over sodium sulfate,
filtered, evaporated, and chromatographed with hexane-ethyl acetate
on silica gel to give 268 mg (31%) of 99 as a white solid: MS
1
(ESI) m/z 230.2 (M + 1)+1; H NMR (400 MHz, DMSO-d6) δ
1.48 - 1.60 (m, 2H), 1.60 - 1.72 (m, 2H), 1.72 - 1.84 (m, 2H),
1.98 - 2.08 (m, 2H), 2.99 - 3.09 (m, 1H), 3.99 (s, 2H), 7.26 (bs,
1H), 7.34 (dd, J ) 8.3, 1.2 Hz, 1H), 7.92 (d, J ) 8.3 Hz, 1H),
11.20 (s, 1H).
6-Cyclopentyl-4-methoxymethylene-4H-isoquinoline-1,3-dione (4l).
A mixture of 99 (222 mg, 0.97 mmol), 10 mL of acetic acid, and
trimethyl orthoformate (212 mg, 2.0 mmol) was stirred and heated
to 90 °C. After 2 h at that temperature, the reaction mixture was
cooled, and the solvents were removed in vacuo, and the residue
taken up in 4% methanol in dichloromethane, passed through a
short pad of Florisil, and eluted with 4% methanol in dichlo-
romethane. The eluate was evaporated, and the product was treated
with 4:1 hexane-ethyl acetate and collected by filtration to give
165 mg (62%) of 4l as a yellow solid: MS (ESI) m/z 272.2 (M +
1)+1; 1H NMR (400 MHz, DMSO-d6) δ 1.48 - 1.62 (m, 2H), 1.63
- 1.73 (m, 2H), 1.73 - 1.85 (m, 2H), 1.99 - 2.11 (m, 2H), 3.00
Molecular Modeling. The inhibitor structures were minimized
using the MMFF94 force field of SYBYL.46 A public domain
structure of CDK420 was used as the 3-D representation for
molecular docking studies. The inhibitor structures were docked

2300 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
- 3.12 (m, 1H), 4.23 (s, 3H), 7.32 (dd, J ) 8.2, 1.6 Hz, 1H), 7.99
(d, J ) 8.3 Hz, 1H), 8.01 (s, 1H), 8.14 (d, J ) 1.4 Hz, 1H), 11.18
(s, 1H).
amino-ethyl)-2-methoxyphenol hydrochloride (10) as a tan foam:
MS (ESI) m/z 150.9 (M-NH3 + 1)+1
.
2-Methoxy-5-methylaminomethyl-phenol (12). Methylamine (40%
aqueous solution, 2.2 mL, 25 mmol) was added to a solution of
3-hydroxy-4-methoxybenzaldehyde (6) (3.0 g, 20 mmol) in ethanol
(35 mL). The resulting slurry was stirred for 30 min at room
temperature. Then the solvents were evaporated under reduced
pressure. The resulting solid was dissolved in methanol (300 mL),
and sodium borohydride (0.90 g, 24 mmol) was added. The reaction
mixture was stirred for 2 h at room temperature and then
concentrated under reduced pressure. The residue was partitioned
between saturated aqueous potassium carbonate solution and ethyl
acetate. The aqueous phase was extracted thrice with ethyl acetate,
and the combined extracts were concentrated under reduced pressure
to give 2.5 g (75%) of 12 as a granular white solid: MS (ESI) m/z
Preparation of the Headgroups. 3-Hydroxy-benzylamine Hy-
drogen Chloride (2d). An amount of 2 g (16.79 mmol) of
3-cyanophenol was dissolved in THF (40 mL). After cooling to 0
°C, borane tetrahydrofuran complex (32.0 mL, 2.0 M) was added
dropwise to the solution. It was allowed to stir at 0 °C for 15 min,
then at room temperature for 25 min. After refluxing for 3 h, it
was cooled to room temperature and then evaporated to dryness.
Methanol (14 mL) was added and then evaporated to dryness.
Subsequently conc. hydrogen chloride (155 mL) was added and
then evaporated to dryness. The residue was recrystallized from
ethyl acetate to give 2.66 g (100%) of 2d as a white solid. MS
(ESI) m/z 123.15 (M + 1)+1
.
168.2 (M + 1)+1
.
3-Hydroxy-4-methoxy-benzylamine Hydrogen Chloride (2e). An
amount of 1.52 g (10.0 mmol) of 3-hydroxy-4-methoxy-benzyla-
ldehyde was added to ethanol (20 mL) and pyridine (10 mL) at
room temperature, followed by the addition of hydroxylamine
hydrochloride (764.39 mg, 11.0 mmol). The mixture was stirred at
ambient temperature for 24 h, and 200 mL of water was added.
After the solvents were evaporated, the residue was dissolved in
400 mL of ethyl ether, washed successively with 100 mL of aqueous
sodium bicarbonate, 100 mL of sodium bisulfite, and 100 mL of
brine. It was dried over magnesium sulfate, filtered, and evaporated
to give 1.39 g (83%) of 3-hydroxy-4-methoxy-benzylaldehyde
oxime as a white solid.
3-Hydroxy-4-propoxybenzaldehyde (15b). To a solution of
1-bromopropane (1.7 mL, 18.7 mmol) in anhydrous DMF (25 mL)
was added potassium carbonate (5.7 g, 41.0 mmol). The mixture
was stirred at room temperature, and 1,2-dihydroxy-4-benzaldehyde
(14) (1.0 g, 7.2 mmol) was added. After the mixture was stirred at
65 °C for 30 min, the resulting mixture was concentrated, and the
residue was then partitioned between water (25 mL) and ethyl
acetate (25 mL). The organic layer was then dried and purified by
flash chromatography to give 0.2 g (15%) of 15b as a brown solid:
1
MS (ESI) m/z 181.2 (M + 1)+1; H NMR (400 MHz, DMSO-d6)
δ 0.99 (t, J ) 7.3 Hz, 3H), 1.77 (m, 2H), 4.03 (t, J ) 6.6 Hz, 2H),
7.11 (d, J ) 8.3 Hz, 1H), 7.26 (d, J ) 2.0 Hz, 1H), 7.38 (dd, J )
8.3, 2.0 Hz, 1H), 9.49 (s, 1H), 9.76 (s, 1H).
A solution of 3-hydroxy-4-methoxy-benzylaldehyde oxime (1.0
g, 5.98 mmol) was dissolved in ethanol (50 mL). Then conc.
hydrochloric acid (2 mL) was added, followed by 10% Pd/C (200
mg) and hydrogenated at 35 psi for 3 h. The mixture was filtered
through celite and evaporated to dryness. The residue was recrystal-
lized from ethyl acetate to give 1.13 g (91%) of 2e as a white solid
3-Hydroxy-4-(2-methoxyethoxy)benzaldehyde (15c). An amount
of 14 (5.0 g, 36.2 mmol) in 20 mL of DMF was added to
2-bromoethyl methyl ether (3.4 mL, 36.2 mmol) and sodium
carbonate (5.0 g, 72.4 mmol). The reaction mixture was stirred at
room temperature for 3 days. After removal of solids by filtration,
the solution was subsequently evaporated under high-pressure
vacuum to give a dark brown liquid. The residue was treated with
water and acidified with 12 N HCl solution to pH ∼2, then extracted
with 4× 100 mL of EtOAc. The combined organic layer was
washed with brine, dried over Mg2SO4, stirred in Darco, filtered,
and evaporated to give a yellow liquid. Purification was performed
by column chromatography over silica gel using 40% EtOAc/
Hexane as eluent to give 2.13 g (30%) of 15c as a colorless solid:
(HCl/hydrate salt): mp160-161 °C; MS (ESI) m/z 154.2(M + 1)+1
.
5-(1-Amino-ethyl)-2-methoxyphenol hydrochloride (10). To a
solution of 3-hydroxy-4-methoxybenzaldehyde (6) (5.0 g, 33 mmol)
in DMF (165 mL) was added benzyl bromide (4.3 mL, 36 mmol),
followed by potassium carbonate (∼325 mesh, 14 g, 100 mmol).
The reaction mixture was stirred for 4 h at room temperature and
then partitioned between diethyl ether and water. The aqueous phase
was extracted with ether (3 × 50 mL). The combined ethereal
extracts were washed twice with water and once with saturated
aqueous sodium chloride solution, dried over anhydrous magnesium
sulfate, filtered, and concentrated under reduced pressure to give a
golden oil, which was crystallized upon standing to afford 3-ben-
zyloxy-4-methoxybenzaldehyde (7) as a white solid. The material
was used in the following step without further purification.
1
mp 74-75 °C; MS (ESI) m/z 195.1 (M + H)-1; H NMR (400
MHz, DMSO-d6) δ ppm 3.32 (s, 3 H), 3.62 - 3.79 (m, 2 H), 4.03
- 4.34 (m, 2 H), 7.13 (d, J ) 8.3 Hz, 1 H), 7.27 (d, J ) 2.0 Hz,
1 H), 7.38 (dd, J ) 8.3, 2.0 Hz, 1 H), 9.58 (s, 1 H), 9.77 (s, 1 H).
5-(Aminomethyl)-2-propoxyphenol Hydrochloride (16b). An
amount of 15b (150 mg, 0.83 mmol) was added to pyridine (4.0
mL) at room temperature, followed by the addition of methoxy-
lamine hydrogen chloride (101.2 mg, 1.2 mmol). The mixture was
stirred at ambient temperature for 24 h, and 4 mL of water was
added. After the solvents were evaporated, the residue was dissolved
in 8 mL of anhydrous ether and washed successively with 2 mL of
aqueous sodium bicarbonate, 2 mL of sodium bisulfite, and 2 mL
of brine. It was dried over magnesium sulfate, filtered, and
evaporated to give 150 mg (86%) crude 3-hydroxy-4-propoxyben-
zaldehyde O-methyloxime as a yellow solid: MS (ESI) m/z 210.5
A solution of the crude 7 in THF (150 mL) was cooled to -78
°C. Methyllithium (1.6 M solution in diethyl ether, 31 mL, 49
mmol) was added dropwise via a syringe. Following completion
of the addition, the cooling bath was removed, and the reaction
mixture was allowed to warm to room temperature. After 90 min
at that temperature, the mixture was cooled to 0 °C and treated
with saturated aqueous sodium hydrogen carbonate. The quenched
reaction mixture, after warming to room temperature, was acidified
with 1 M hydrochloric acid solution to pH 1 and extracted thrice
with diethyl ether. The combined extracts were washed with
saturated aqueous sodium chloride solution, dried over anhydrous
magnesium sulfate, filtered, and concentrated under reduced pres-
sure to give 1-(3-benzyloxy-4-methoxy-phenyl)-ethanol (8).
To a mixture of the crude 8 and diphenylphosphoryl azide (2.6
mL, 12 mmol) in toluene (18 mL) at 0 °C was added 1,8-
diazabicyclo[5.4.0]undec-7-ene (1.6 mL, 11 mmol). The mixture
was stirred at 0 °C for 2 h and then at room temperature overnight.
The reaction mixture was washed with water and concentrated to
give 4-(1-azido-ethyl)-2-benzyloxy-1-methoxybenzene (9), which
was used in the next step without purification.
(M + 1)+1
.
An amount of 3-hydroxy-4-propoxybenzaldehyde O-methy-
loxime (150 mg, 0.83 mmol) was dissolved in ethanol (4 mL). Then
12 N HCl (0.600 mL) was added, followed by 10% Pd/C (15.0
mg). After hydrogenation at 35 psi for 3 h, the solution was filtered
through Celite and evaporated to dryness. The residue was
recrystallized from ethyl acetate to give 112 mg (62%) of 16b as
1
a white solid: MS (ESI) m/z 182.3 (M + 1)+1; H H NMR (400
MHz, DMSO-d6) δ 0.97 (t, J ) 7.4 Hz, 3H), 1.53 - 1.94 (m, 2H),
3.84 (s, 2H), 3.91 (t, J ) 6.5 Hz, 2H), 6.75 - 6.86 (m, 1H), 6.88
- 6.98 (m, 2H), 8.08 (s, 3H), 9.03 (s, 1H).
Crude 9 was hydrogenated at 50 psi, using 10% Pd/C in ethanolic
hydrogen chloride solution to give a brown syrup, which, when
was dried under vacuum, gave 0.53 g (8% over 4 steps) of 5-(1-
5-(Aminomethyl)-2-(2-methoxyethoxy)phenol (16c). Using the
procedures described for the preparation of 16b, 15c (1.24 g, 6.32
mmol) was reacted to give a coloress oil, which upon recrystalli-

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2301
zation from MeOH/EtOAc yielded 0.45 g (55%) of 16c as a
evaporated, and the residue was chromatographed to provide 60
colorless solid: mp: 89-90 °C; MS (ESI) m/z 198.1 (M + H)+1
.
mg (33%) of 41: MS (ESI) m/z 190 (M + 1)+1
.
2-Amino-5-(aminomethyl)phenol Hydrochloride (20). Using the
procedure described for the preparation of 2e, 4-nitro-3-hydroxy-
benzadehyde O-methyloxime (2.2 g, 16.1 mmol) was hydrogenated
to give 2.5 g (89%) of 20 as a purple solid: MS (ESI) m/z 138.9
4-Iodo-3-methoxymethoxy-benzonitrile (42). 3-Hydroxy-4-io-
dobenzonitrile (38) (500 mg, 2.04 mmol) and MOMCl (350 mg,
4.37 mmol) were dissolved in anhydrous DMF (5 mL) and cooled
to 0 °C. NaH (100 mg, 60% suspension in mineral oil, 2.5 mmol)
was then added. The resulting mixture was allowed to stir at room
temperature for 1 h. Ether was then added and washed with H2O
(3 × 20 mL) and brine. After drying over Na2SO4, ether was
removed, and the residue was purified by column chromatography
to afford 570 mg (96%) of 42: 1H NMR (300 MHz, CDCl3) δ 7.90
(d, J ) 8.0 Hz, 1H), 7.32 (d, J ) 1.7 Hz, 1H), 7.03 (dd, J ) 8.0
and 1.7 Hz, 1H), 5.27 (s, 2H), 3.51 (s, 3H).
5-Aminomethyl-2-furan-3-yl-phenol (45a). 4-Iodo-3-meth-
oxymethoxy-benzonitrile (42) (150 mg, 0.52 mmol), 3-furanboronic
acid (96 mg, 0.86 mmol), Pd(PPh3)4 (60 mg, 0.052 mmol), and
Cs2CO3 (500 mg, 1.53 mmol) were mixed in DMF (10 mL),
degassed, and then heated at 100 °C for 4 h. After the mixture was
cooled to room temperature, an aqueous work up was performed,
and the residue was purified to afford 100 mg (84%) of 4-furan-
3-yl-3-methoxymethoxy-benzonitrile (43a).
Nitrile 43a (120 mg, 0.52 mmol) was dissolved in ether (10 mL).
The resulting solution was slowly added to a suspension of LiAlH4
(100 mg, 2.6 mmol) in ether. After addition, the mixture was stirred
for another 10 min before quenching with water and 5 N NaOH. It
was extracted with ethyl acetate, and the organic layer was washed
with brine, dried over Na2SO4, filtered, and dried to provide 90
mg (74%) of the crude 4-furan-3-yl-3-methoxymethoxy-benzyl-
amine (44a).
(M + 1)+1
.
5-Aminomethyl-2-methyl-phenol hydrogen chloride (25). A
solution of 3-hydroxy-4-methylbenzoic acid (22) (2 g, 13.14 mmol)
and oxalyl chloride (10 mL) was refluxed at 60 °C until all of the
solid was in solution. After cooling, the mixture was evaporated to
dryness to give 3-hydroxy-4-methylbenzoyl chloride (23). It was
cooled to 0 °C, and ammonium hydroxide (20 mL) was added
within 5 min. The mixture was allowed to stir at 0 °C for 30 min,
then at room temperature for an additional 30 min. Water (30 mL)
was added to the mixture. After the white precipitate was filtered,
the aqueous solution was adjusted to pH 3 and then extracted with
ethyl acetate. The organic solution was dried over MgSO4, filtered,
and dried to give 1.4 mg (70%) of 3-hydroxy-4-methylbenzamide
(24) as a white solid.
A solution of 24 (1.4 g, 9.65 mmol) was dissolved in THF (20
mL) and cooled to 0 °C. A solution of borane tetrahydrofuran
complex (25 mL, 2.0 M) was added dropwise over 15 min and
maintained at 0 °C for an additional 10 min. The mixture was stirred
at room temperature for 20 min, refluxed for 3 h, and then cooled
to room temperature, and HCl (15 mL) was added and reflu-
xed overnight. After cooling, the solvent was evaporated, and
methanol (5 mL) was added. After evaporation to dryness, the
residue was crystallized with ethyl acetate to provide 450 mg (28%)
of the desired product 25 as a white solid: MS (ESI) m/z 174.1 (M
The MOM protected amine (44a) (90 mg, 0.39 mmol) was then
dissolved in 2 N aq HCl/MeOH (1:1). The resulting solution was
heated at reflux for 15 min. After cooling to room temperature, the
mixture was basified with aqueous ammonium hydroxide and
extracted with CH2Cl2/MeOH (9:1). The organic layer was dried,
filtered, and evaporated to yield a crude product 45a, which was
+ 1)+1
.
4-Aminomethyl-biphenyl-2-ol (34). (2-Methoxy-biphenyl-4-yl)-
methanol (31) (170 mg, 0.79 mmol)23 was dissolved in CH2Cl2 (2
mL) and cooled to 0 °C. Thionyl chloride (1 mL) was then added
dropwise. The mixture was then allowed to stir till no starting
material was left, ca. 1 h. The volatile was then removed, and the
crude 4-chloromethyl-2-methoxy-biphenyl (32) was employed
directly in the next step.
used directly in the next step. MS (ESI) m/z 190 (M + 1)+1
.
5-Aminomethyl-2-pyridin-2-yl-phenol (45b). 4-Iodo-3-meth-
oxymethoxy-benzonitrile (42) (190 mg, 0.66 mmol), 2-pyridinyl
tributyltin (370 mg, 1 mmol), PdCl2(PPh3)2 (60 mg, 0.084 mmol),
and CuI (40 mg, 0.21 mmol) were mixed in DMF (10 mL). This
mixture was then degassed and heated at 100 °C for 1 h. TLC
suggested full conversion. After aqueous workup, the residue was
purified by column chromatography to yield 162 mg (93%) of
3-methoxymethoxy-4-pyridin-2-yl-benzonitrile (43b): MS (ESI) m/z
The crude product 32 was dissolved in CH2Cl2 (3 mL) and cooled
to -78 °C. Boron tribromide (3 mL, 1 M solution in CH2Cl2, 3
mmol) was added. The cooling bath was then removed, and the
mixture was allowed to stir at room temperature for 1 h. The mixture
was quenched with ice water, and the methylene chloride layer was
dried and concentrated. The residue was purified by column
chormatography to obtain 4-chloromethyl-2-hydroxy-biphenyl. This
product (170 mg) was then dissolved in DMF (5 mL) to which
NaN3 (100 mg, 1.53 mmol) was added at room temperature for
1 h. Ethyl ether and water were then added, and the ether layer
was washed with water and dried over Na2SO4. Removal of ether
provided 100 mg (53% over three steps starting from 31) of the
desired 4-azidomethyl-2-hydroxy-biphenyl (33).
1
241 (M + 1)+1; H NMR (300 MHz, CDCl3) δ 8.74 (m, 1H),
7.26-7.91 (m, 6H), 5.22 (s, 2H), 3.48 (s, 3H).
By the procedure described above for 44a, 160 mg (0.67 mmol)
of 43b was reacted in two steps to give 63 mg (47%) of 45b: MS
(ESI) m/z 201 (M + 1)+1; 1H NMR (300 MHz, CDCl3) δ 8.50 (m,
1H), 7.76-7.92 (m, 3H), 7.24 (m, 1H), 6.86-6.97 (m, 2H), 3.87
(s, 2H).
Triphenylphosphine (100 mg, 0.38 mmol) was added to a solution
of 33 (100 mg, 0.42 mmol) in THF (5 mL) and water (0.5 mL).
The mixture was stirred at room temperature overnight. After the
solvents were evaporated, the residue was purified by column
chromatography to provide 84 mg (100%) of 34: MS (ESI) m/z
5-Aminomethyl-2-pyridin-3-yl-phenol (45c). By the procedure
described above for 45b, 200 mg (0.69 mmol) of 42 was reacted
in three steps to give 53 mg (33%) of 45c: MS (ESI) m/z 201 (M
+ 1)+1
.
5-Aminomethyl-2-pyridin-4-yl-phenol (45d). By the procedure
described above for 45b, 200 mg (0.69 mmol) of 42 was reacted
in three steps to give 40 mg (25%) of 45d: MS (ESI) m/z 201 (M
199.10 (M + 1)+1
.
5-Aminomethyl-2-furan-2-yl-phenol (41). 3-Hydroxy-4-iodo-
benzonitrile (38) (120 mg, 0.49 mmol), PdCl2(PPh3)2 (35 mg, 0.049
mmol), and 2-furyltributyltin (200 g, 0.56 mmol) in DMF (5 mL)
were degassed and heated at 100 °C for 30 min. After the mixture
was cooled to room temperature, an aqueous work up was
performed, and 80 mg (88%) of 4-furan-2-yl-3-hydroxy-benzonitrile
(40) was isolated after column chromatography.
To a solution of 40 (180 mg, 0.97 mmol) in THF (5 mL) was
added BH3 ·THF (5 mL, 1 M solution in THF, 5 mmol) under
nitrogen. The mixture was then allowed to stir at room temperature
for 24 h. The reaction was then quenched with 6 N HCl. THF was
then removed, and the aqueous layer was then neutralized with
ammonium hydroxide to pH 9. The mixture was then extracted
with CHCl3/MeOH (9:1). The organic layer was dried and
+ 1)+1
.
2-Aminomethyl-5-methoxy-pyrimidin-4-ol (51). 1,3-Dioxo-1,3-
dihydro-isoindol-2-yl)-acetonitrile (46)24 (1 g, 5.34 mmol) was
dissolved in 4 N HCl in dioxane (10 mL). The mixture was stirred
at room temperature, and MeOH (1 mL) was added. The solution
turned cloudy shortly. The precipitate was collected and washed
with ether to provide 1 g (86%) of 2-(1,3-dioxo-1,3-dihydro-
isoindol-2-yl)-acetimidic acid methyl ester hydrochloride (47): MS
(ESI) m/z 219 (M + 1)+1
.
A suspension of 47 (1 g, 4.58 mmol) in MeOH (15 mL) was
added to a saturated NH3/MeOH solution. The mixture was stirred
at 0 °C and then room temperature. After all the volatile was
removed, the product 9b-hydroxy-2-imino-1,2,3,9b-tetrahydro-

2302 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
imidazo[2,1-a]isoindol-5-one (48) was directly employed in the next
of stirring at room temperature, the reaction mixture was filtered.
The filtrate was washed thrice with saturated aqueous sodium
hydrogen carbonate solution, dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure to give 2.1 g (53%
over 2 steps) of 62 as a yellow powder: MS (ESI) m/z 190.3 (M +
step: MS (ESI) m/z 204 (M + 1)+1
.
Compound 48 (500 mg, 2.09 mmol) and the sodium salt of
3-hydroxy-2-methoxy-acrylic acid methyl ester (49)25 (500 mg, 3.24
mmol) were mixed in MeOH and stirred at room temperature for
2 h, and then heated at reflux overnight. The crude mixture was
filtered. After the filtrate was concentrated, the resulting residue
was purified by column chromatography to afford 100 mg (16%)
of 2-(4-hydroxy-5-methoxy-pyrimidin-2-ylmethyl)-isoindole-1,3-
1)+1
.
4-Aminomethyl-1-furan-3-yl-1H-pyridin-2-one (65). A solution
of 62 (1.4 g, 7.4 mmol) in pyridine (40 mL) was treated with
methoxylamine hydrochloride (0.68 g, 8.1 mmol). After stirring
overnight at room temperature, the reaction mixture was concen-
trated under reduced pressure. The residue was partitioned between
ethyl acetate and water. The organic phase was washed twice with
water and once with saturated aqueous sodium chloride solution,
dried, and concentrated under reduced pressure to provide 1-furan-
3-yl-2-oxo-1,2-dihydro-pyridine-4-carbaldehyde O-methyl-oxime
(63), which was used in the next step without further purification:
dione (50): MS (ESI) m/z 286 (M + 1)+1
.
Hydrazine (0.5 mL) was added to a suspension of 50 (50 mg,
0.18 mmol) in EtOH (2 mL), and the mixture was stirred till no
starting material was left. The precipitate was filtered and the filtrate
was concentrated to provide 51, which was used directly without
further purification: MS (ESI) m/z 156 (M + 1)+1
.
4-Aminomethyl-1H-pyridin-2-one hydrochloride (55). 2-Meth-
oxy-4-cyanopyridine (53) was prepared from 2-chloro-4-cyanopy-
ridine according to the procedure by Brown et al.26 Reduction of
53 to 4-(2-methoxypyridyl)methylamine (54) was achieved through
the method of Walpole et al.27
MS (ESI) m/z 219.3 (M + 1)+1
.
To a mixture of the crude 63 and glacial acetic acid (77 mL)
was added zinc powder (3.1 g). The reaction mixture was heated
for 45 min at 100 °C in an oil bath and then allowed to cool to
room temperature. The mixture was filtered through a pad of
diatomaceous earth. After the filtrate was concentrated, the residue
was purified by reverse-phase HPLC to give 2.3 g of crude
4-aminomethyl-1-furan-3-yl-1H-pyridin-2-trifluoroacetic acid salt
A solution of 54 (0.19 g, 1.1 mmol) in water (50 mL) and 3 N
aqueous hydrochloric acid (25 mL) was heated at reflux for 5 h.
The solvents were evaporated to give 0.15 g (83%) of 55 as an
off-white solid: MS (ESI) m/z 125.1 (M + 1)+1
.
contaminated with zinc salt: MS (ESI) m/z 191.3 (M + 1)+1
.
2-Oxo-1-phenyl-1,2-dihydropyridin-4-ylmethylamine hydro-
chloride (58). To a solution of 55 (3.68 g, 23 mmol) in 50% aqueous
dioxane (100 mL) was added sodium hydroxide (2.8 g, 69 mmol),
followed by di-tert-butyldicarbonate (5.0 g, 23 mmol). After stirring
overnight at room temperature, the mixture was neutralized with
5% aqueous potassium hydrogen sulfate solution. The mixture was
extracted four times with ethyl acetate. The combined extracts were
dried over anhydrous magnesium sulfate, filtered, and concentrated
under reduced pressure. A sample of crude material was purified
by reverse-phase HPLC to give tert-butyl (2-oxo-1,2-dihydro-
pyridin-4-ylmethyl)-carbamate (56) as a straw colored foam: MS
To a solution of 2.3 g of the crude 4-aminomethyl-1-furan-3-
yl-1H-pyridin-2-one trifluoroacetic acid salt in 50% aqueous dioxane
(20 mL) was added sodium hydroxide (800 mg), followed by an
additional volume of aqueous dioxane (20 mL), and then the
addition of di-tert-butyldicarbonate (600 µL). After the reaction
was completed, the reaction mixture was filtered. The filtrate was
neutralized with 5% aqueous potassium hydrogen sulfate solution
and extracted thrice with ethyl acetate. The combined extracts were
washed with saturated aqueous sodium chloride solution, dried, and
concentrated under reduced pressure to give 0.18 g (8% over 3
steps) of tert-butyl (1-furan-3-yl-2-oxo-1,2-dihydro-pyridin-4-yl-
(ESI) m/z 225.3 (M + 1)+1
.
To a solution of 56 (0.13 g, 0.58 mmol) in dichloromethane (5
mL) was added trimethylphenylstannane (210 µL, 1.2 mmol),
followed successively by copper (II) acetate (0.12 g, 0.64 mmol)
and tetra-n-butylammonium fluoride (1.0 M solution in tetrahy-
drofuran, 1.2 mL). The reaction mixture was stirred for 2 days at
room temperature and then was quenched by the addition of
methanolic ammonia (2 M, 4 mL). The reaction mixture was
concentrated and then purified by flash silica gel chromatography
(methanol/chloroform) to give 88 mg (52%) of 57: MS (ESI) m/z
methyl)-carbamate (64): MS (ESI) m/z 291.3 (M + 1)+1
.
tert-Butyl (1-furan-3-yl-2-oxo-1,2-dihydro-pyridin-4-ylmethyl)-
carbamate (64) (0.20 g, 0.69 mmol) was treated with 4 N hydrogen
chloride in dioxane (5 mL) at room temperature. After the
deprotection was completed, the solution was concentrated to yield
43 mg (27%) of 4-aminomethyl-1-furan-3-yl-1H-pyridin-2-one (65):
MS (ESI) m/z 191.2 (M + 1)+1
.
2-Hydroxymethyl-5-propoxy-pyran-4-one (67b). A mixture of
kojic acid (66) (28.4 g, 0.20 mol), 120 mL of DMF, potassium
carbonate powder (27.6 g, 0.20 mol), potassium iodide (1.66 g,
0.01 mol), and 1-bromopropane (24.6 g, 0.20 mol) was stirred for
15 min at ambient temperature and then stirred at 90 °C for 3 h.
The reaction mixture was cooled, evaporated to dryness in vacuo,
and then partitioned between water and chloroform. The aqueous
layer was extracted with chloroform (3 × 100 mL) and ethyl acetate
(6 × 100 mL). The combined organic solution was dried with
sodium sulfate and passed through a pad of magnesol and silica
gel eluting with ethyl acetate. The eluate was evaporated in vacuo
and crystallized with hexane/ethyl acetate (2:1) to give 22.6 g (61%)
301.3 (M + 1)+1
.
Compound 57 (83 mg, 0.28 mmol) was treated with 4 N
hydrogen chloride (2 mL) in dioxane to remove the Boc protecting
group. The reaction solution was evaporated to dryness to give 66
mg (100%) of 4-aminomethyl-1-phenyl-1H-pyridin-2-one hydro-
chloride (58).
1-Furan-3-yl-4-methyl-1H-pyridin-2-one (60). A mixture of
2-hydroxy-4-methylpyridine (59) (0.66 g, 6.0 mmol), 3-bromofuran
(1.7 g, 12 mmol), copper (I) iodide (0.11 g, 0.60 mmol), and
potassium carbonate (0.84 g, 6.0 mmol) in DMF (12 mL) was
heated at 180 °C for 2 h in a 300 W microwave reactor. The reaction
mixture was then diluted with 10% aqueous ammonium hydroxide
solution and extracted thrice with ethyl acetate. The combined
extracts were washed with saturated aqueous sodium chloride
solution, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure to give 0.65 g (62%) of 60: MS (ESI) m/z
1
of 67b as an off-white solid: MS (ESI) m/z 185.3 (M + 1)+1; H
NMR (400 MHz, CDCl3) δ 1.02 (t, J ) 7.5, 3H), 1.78 - 1.89 (m,
2H), 3.57 (t, J ) 6.5, 1H), 3.81 (t, J ) 6.7, 2H), 4.48 (dd, J ) 6.7,
0.7, 2H), 6.52 (m, 1H), 7.57 (s, 1H).
2-Hydroxymethyl-5-propoxy-pyridin-4-ol (71b). A mixture of
67b (30.0 g, 0.163 mol) and ammonium hydroxide (150 mL) was
stirred and heated in a sealed vessel at 90 °C for 2 h. The reaction
mixture was cooled and evaporated to dryness in vacuo. The residue
was taken up in 15% methanol in chloroform and passed through
a pad of magnesol and silica gel eluting with the same solvent.
The eluate was evaporated, and the residue was treated with acetone,
filtered, washed with acetone, and air-dried to give 8.03 g (80%)
of 71b as a gray solid: mp 159-160 °C; MS (ESI) m/z 184.3 (M
176.1 (M + 1)+1
.
1-Furan-3-yl-2-oxo-1,2-dihydro-pyridine-4-carbaldehyde (62). A
mixture of 60 (3.7 g, 21 mmol), tert-butoxybis(dimethylami-
no)methane (11 g, 63 mmol), and DMF (4 mL) was heated at 150
°C for 2.5 h and then concentrated to dryness under reduced pressure
to yield crude 4-(2-dimethylamino-vinyl)-1-furan-3-yl-1H-pyridin-
2-one (61), which was used without further purification: MS (ESI)
m/z 231.3 (M + 1)+1
.
1
+ 1)+1; H NMR (400 MHz, DMSO-d6) δ 1.00 (t, J ) 7.5, 3H),
To a solution of 61 (21 mmol) in 50% aqueous tetrahydrofuran
1.73 - 1.83 (m, 2H), 4.08 (t, J ) 6.6, 2H), 4.68 (s, 2H), 7.21 (s,
(700 mL) was added sodium periodate (13 g, 63 mmol). After 6 h
1H), 8.19 (s, 1H).

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2303
2-Azidomethyl-5-propoxy-pyridin-4-ol (73b). To a mixture of
71b (5.56 g, 30.3 mmol) and chloroform (30 mL) cooled in an ice
bath was added 30 mL of thionyl chloride. It was stirred for 15
min at ice bath temperature and then refluxed for 1 h. The reaction
mixture was cooled, evaporated to dryness in vacuo, and then treated
with isopropanol. The solid was filtered, washed with fresh
isopropanol, then ether, and air-dried to give 3.4 g (48%) of
2-chloromethyl-5-propoxy-pyridin-4-ol (72b) as an off-white solid:
1)+1; 1H NMR (300 MHz, DMSO-d6) δ 4.36(d, J ) 5.9, 2H), 5.74
(t, J ) 5.9, 1H), 6.39 (s, 1H), 6.93 - 6.97 (m, 1H), 7.72 - 7.56
(m, 1H), 8.40 - 8.43 (m, 1H), 8.70 (s, 1H).
2-Hydroxymethyl-5-(3-furyl)-pyridin-4-ol (78b). A mixture of
2-hydroxymethyl-5-furan-3-yl-pyran-4-one (77b) (1.92 g, 10.0
mmol) and 7 M ammonia in methanol (50.0 mL) was stirred and
heated in a sealed vessel at 90 °C overnight. The reaction mixture
was cooled, evaporated to dryness in vacuo, taken up in 15%
methanol in chloroform, and passed through a pad of magnesol
and silica gel eluting with the same solvent. The eluate was
evaporated, treated with acetone, filtered, washed with acetone, and
air-dried to give 1.21 g (63%) of 78b as a gray solid: MS (ESI)
1
mp 165-167 °C; MS (ESI) m/z 202.3 (M + 1)+1; H NMR (400
MHz, DMSO-d6) δ 0.99 (t, J ) 7.5, 3H), 1.71 - 1.82 (m, 2H),
4.10 (t, J ) 6.6, 2H), 4.95 (s, 2H), 7.83 (s, 1H), 8.34 (s, 1H).
To a suspension of sodium azide (2.82 g, 43.3 mmol) in 30 mL
of DMF was added 72b (8.74 g, 43.3 mmol). It was stirred overnight
at ambient temperature and then quenched into ice water. The solid
formed was filtered, washed with cold water, and dried to give
5.74 g (63%) of 73b as an off white solid: MS (ESI) m/z 209.3 (M
1
m/z 192.1 (M + 1)+1; H NMR (300 MHz, DMSO-d6, w/TFA) δ
4.75 (s, 2H), 7.20 - 7.26 (m, 1H), 7.34 (s, 1H), 7.82 - 7.87 (m,
1H), 8.39 (s, 1H), 8.81 (s, 1H).
2-Aminomethyl-5-furan-3-yl-pyridin-4-ol (81b). To a mixture of
78b (1.148 g, 6.00 mmol) and 30 mL of N,N-dimethylformamide
stirred and cooled with an ice bath to 0 °C was added triph-
enylphosphine (2.361 g, 9 mmol) followed by carbon tetrabromide
(2.988 g, 9 mmol). This was stirred for 15 min, maintaining the
temperature between 0-5 °C. Sodium azide was then added (1.172
g, 18.03 mmol) and the reaction mixture stirred for 24 h at ambient
temperature. The reaction mixture was diluted with water and
extracted with ethyl acetate. The combined extracts were dried over
sodium sulfate, filtered, evaporated, and the residue chromato-
graphed on silica gel eluting with ethyl acetate to give 0.417 g
(32%) of 2-azidomethyl-5-furan-3-yl)-pyridin-4-ol (80b) as a white
1
+ 1)+1; H NMR (400 MHz, DMSO-d6) δ 1.00 (t, J ) 7.5, 3H),
1.73 - 1.83 (m, 2H), 4.09 (t, J ) 6.6, 2H), 4.76 (s, 2H), 7.26 (s,
1H), 8.35 (s, 1H).
2-Aminomethyl-5-propoxy-pyridin-4-ol (74b). Azide 73b (9.70
g, 46.6 mol) was suspended in 120 mL of THF and treated with
triphenylphosphine (12.22 g, 46.6 mmol). After stirring for 10 min
at ambient temperature, water was added (12.6 mL, 700 mmol),
and the reaction mixture was stirred at ambient temperature
overnight. The solid was gradually dissolved, followed by the
formation of a precipitate. The resulting solid was filtered, washed
with THF/water (10:1), and dried to give 5.92 g (69%) of 74b: mp
159-160 °C; MS (ESI) m/z 183.3 (M + 1)+1; 1H NMR (400 MHz,
DMSO-d6) δ 1.00 (t, J ) 7.5, 3H), 1.73 - 1.84 (m, 2H), 4.10 (t,
J ) 6.6, 2H), 4.24 (bs, 2H), 7.32 (s, 1H), 8.44 (s, 1H), 8.51 (bs,
3H).
1
solid: mp 198-200 °C (dec); MS (ESI) m/z 217.3 (M + 1)+1; H
NMR (300 MHz, DMSO-d6) δ 4.87 (s, 2H), 7.23 - 7.25 (m, 1H),
7.40 (s, 1H), 7.81 - 7.83 (m, 1H), 8.42 (bs, 1H), 8.95 (s, 1H).
To a mixture of 80b (216 mg, 1 mmol), 6 mL of tetrahydrofuran
and triphenylphosphine (262 mg, 1 mmol) were added followed
by water (270 µL, 15.0 mmol). It was stirred at ambient temperature
overnight and then at 60 °C overnight. The reaction mixture was
cooled, evaporated to dryness in vacuo, and then treated with warm
toluene. This mixture was cooled and the solid was filtered, washed
with toluene, and dried to give 0.170 g (89%) of 81b as an off-
2-Azidomethyl-5-methoxy-pyridin-4-ol (73a). Using the prece-
dure described for 73b, 2-chloromethyl-5-methoxy-pyridin-4-ol29
(72a) (3.47 g, 20.0 mmol) and sodium azide (1.30 g, 20.0 mmol)
were reacted to give 1.53 g (42%) of 73a as a light brown solid:
mp 111-114 °C (dec); MS (ESI) m/z 179.3 (M - 1)-1
.
2-Aminomethyl-5-methoxy-pyridin-4-ol (74a). Using the proce-
dure described for 74b, 73a (1.45 g, 8.05 mol) and triphenylphos-
phine (2.11 g, 8.05 mmol) were reacted to give 0.897 g (88%) of
white solid: mp 183-7 °C (dec); MS (ESI) m/z 191.3 (M + 1)+1
;
1H NMR (300 MHz, DMSO-d6, w/TFA) δ 4.32 (s, 2H), 7.25 (s,
1H), 7.41 (s, 1H), 7.86 (s, 1H), 8.45 (s, 1H), 8.58 (bs, 3H), 9.00 (s,
1H).
1
74a: mp 196-201 °C (dec); MS (ESI): m/z 155.3 (M + 1)+1; H
NMR (300 MHz, DMSO-d6, w/TFA) δ 4.00 (s, 3H), 4.27 (s, 2H),
7.32 (s, 1H), 8.43 (s, 1H), 8.59 (bs, 3H).
2-Aminomethyl-5-phenyl-pyridin-4-ol (81a). A mixture of 2-hy-
droxymethyl-5-phenyl-pyridin-4-ol (78a) (0.40 g, 2 mmol) and 5
mL of thionyl chloride was heated to a gentle reflux. After 4 h, the
reaction mixture was cooled and evaporated to dryness in vacuo,
and the residue was treated with water and neutralized with sodium
bicarbonate. The resulting solid was collected by filtration, washed
with water, and dried to give 256 mg (58%) of the 2-chloromethyl-
5-phenyl-pyridin-4-ol (79a).
2-Hydroxymethyl-5-furan-3-yl-pyran-4-one (77b). A mixture of
trifluoro-methanesulfonic acid 6-(tert-butyl-dimethyl-silanyloxym-
ethyl)-4-oxo-4H-pyran-3-yl ester31 (75) (6.94 g, 17.8 mmol), furan-
3-boronic acid (4.0 g, 35.7 mmol), tetrakistriphenylphosphine
palladium (1.024 g, 0.87 mmol), cesium carbonate (16.32 g, 50.1
mmol), and potassium bromide (10.63 g, 89.3 mmol) in (250 mL)
dioxane (250 mL) was heated to 60 °C and stirred overnight. The
reaction mixture was cooled to room temperature and diluted with
saturated aqueous ammonium chloride and extracted with ethyl
acetate. The combined ethyl acetate layers were washed with water,
dried over sodium sulfate, filtered, diluted with an equal volume
of hexane, and passed through a short column of magnesol and
silica gel eluting with 1:1 hexane/ethyl acetate. The product was
eluted with 2:1 ethyl acetate/hexane, the solvents were evaporated,
triturated with 1:1 hexane/ethyl acetate, filtered, washed with the
same solvent, and air-dried to give 2.58 g (47%) of 76b as an off
white solid: MS (ESI) m/z 307.3 (M + 1).
Compound 76b was dissolved in tetrahydrofuran, and tetrabu-
tylammonium fluoride solution in tetrahydrofuran (40.0 mL, 40.0
mmol) was added and the mixture stirred at room temperature
overnight. The reaction mixture was diluted with water and
extracted with ethyl acetate. The ethyl acetate layers were combined
and washed with water, dried over sodium sulfate, filtered, diluted
with an equal volume of hexane, and passed through a short column
of magnesol and silica gel eluting with 1:1 hexane/ethyl acetate.
The product was eluted with 2:1 ethyl acetate/hexane, and the
solvents were evaporated, triturated with 1:1 hexane/ethyl acetate,
filtered, washed with the same solvent, and air-dried to give 2.06 g,
(37%) of 77b as an off white solid, MS (ESI) m/z 193.3 (M +
Compound 79a (252 mg, 1.15 mmol) was then stirred with 3
mL of dimethyl formamide, and sodium azide was then added (75
mg, 1.15 mmol) and the reaction mixture stirred for 24 h at ambient
temperature. The solvent was removed in vacuo and the residue
treated with water, filtered, washed with water, and dried to give
231 mg (88%) of 2-azidomethyl-5-phenyl-pyridin-4-ol (80a).
Compound 80a (228 mg, 1.01 mmol) was then suspended in 3
mL of tetrahydrofuran and treated with triphenylphosphine (264
mg, 1.01 mmol). After stirring for 10 min at ambient temperature,
water was added (272 µL, 15 equivalents) and the reaction mixture
warmed with an oil bath at 60 °C, and stirred at that temperature
overnight. The reaction mixture was evaporated to dryness in vacuo
and treated with a 2:1 mixture of ethyl acetate and hexane. The
resulting solid was filtered, washed with fresh 2:1 ethyl acetate and
hexane, and dried to give 116 mg (57%) of 81a as a gray solid:
MS (ESI) m/z 201.1 (M + 1)+1
.
(6-Chloro-5-propoxy-pyridin-2-yl)-methanol (84). A mixture of
2-chloro-6-hydroxymethyl-pyridin-3-ol32 (83) (27.92 g, 0.175 mol),
120 mL of 2-butanone, potassium carbonate powder (48.37 g, 0.35
mol), and 1-iodopropane (37.19 g, 0.219 mol) was stirred for 15
min at ambient temperature and then stirred at 90 °C for 3 h. The
reaction mixture was cooled, evaporated to dryness in vacuo, and

2304 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
then portioned between water and dichloromethane. The organic
layer was dried, filtered, and crystallized with 2/1 hexane/ethyl
acetate to give 5.875 g (16%) of 84 as a pale yellow solid: mp
1H), 7.72 (d, 8.5 Hz, 1H), 8.56 (s, 1H), 8.86 (d, 12.84 Hz, 1H),
11.31 (s, 1H), 12.59 (d, 12.84 Hz, 1H); Anal. (C18H15IN2O4) C,
H, N.
4-[(3,4-Dihydroxy-phenylamino)-methylene]-4H-isoquinoline-
1,3-dione (3a). The title compound was obtained by the procedure
described above for 5e except that no triethylamine was used, and
the reaction was carried out at 115 °C for 1.5 h. 4-Methoxymeth-
ylene-4H-isoquinoline-1,3-dione (1) (40.6 mg, 0.2 mmol) and 3,4-
dihydroxyaniline (2a) (25 mg, 0.2 mmol) were reacted to give 15.8
59-60 °C; MS (ESI) m/z 160.3, 162.3 (M + 1)+1
.
2-Benzyloxy-3-propoxy-6-triisopropylsilanyloxymethyl-pyri-
dine (85). A mixture of 84 (0.93 g, 4.61 mmol), 20 mL of
dichloromethane, tri(isopropyl)silyl chloride (1.0 g, 5.19 mmol),
and imidazole (0.47 g, 6.9 mmol) was stirred overnight at ambient
temperature. The reaction mixture was washed with water, dried
over sodium sulfate, filtered, and evaporated in vacuo to give an
oil which was chromatographed on silica gel with 10:1 hexane/
ethyl acetate to give 2-chloro-3-propoxy-6-triisopropylsilanyloxym-
ethyl-pyridine as a clear liquid (1.06 g) (64%); MS (ESI): m/z 358.2,
360.2 (M + 1). A portion of this (716 mg, 2.0 mmol) and 5 mL of
1 M sodium benzyloxide in benzyl alcohol were heated at 120 °C
under microwave conditions for 5 min. The reaction mixture was
cooled, transferred to a separatory funnel with ethyl acetate and
washed with water, and the organic layer was dried, filtered,
evaporated, and chromatographed on silica gel with hexane/ethyl
acetate to give 524 mg (61%) of 85 as a clear liquid; MS (ESI)
mg (27%) of 3a; LC/MS1 Rt 1.716 min, m/z 297.1 (M + H)+1
;
1
LC/MS2 Rt 1.74 min, m/z 295 (M - H)-1; H NMR (300 MHz,
DMSO-d6) δ ppm 6.55 (s, 1H) 6.60 (d, J ) 8.7 Hz, 1 H) 7.24 (t,
J ) 8.3 Hz, J ) 7.3 Hz, 1 H) 7.39 (d, J ) 8.7 Hz, 1 H) 7.60 (t, J
) 7.8 Hz, 1 H) 8.02 (d, J ) 7.8 Hz, 1 H) 8.11 (d, J ) 8.7 Hz, 1
H) 8.79 (d, J ) 12.8 Hz, 1 H) 9.56 (br s, 2 H) 11.24 (s, 1 H) 12.41
(d, J ) 12.8 Hz, 1 H).
4-[(3,4-Dihydroxy-benzylamino)-methylene]-4H-isoquinoline-1,3-
dione (3b). The title compound was obtained by the procedure
described above for 5e except that the reaction was carried out at
115 °C for 1.5 h. Compound 1 (20.3 mg, 0.1 mmol), 3,4-
dihydroxybenzylamine hydrobromide (2b) (22 mg, 0.1 mmol), and
triethylamine (21 µL) were reacted to give 28.4 mg (91%) of 3b.
LC/MS1 Rt 1.940 min, m/z 311.0 (M + H); LC/MS2 Rt 1.67 min,
m/z 311 (M + H); HRMS (ESI) m/z calcd for N17H14N2O4 311.1031;
1
m/z 430.3 (M + 1)+1; H NMR (400 MHz, CDCl3) δ 1.02 (t, J )
7.4 Hz, 3H), 1.07 - 1.12 (m, 18H), 1.14 - 1.23 (m, 3H), 1.77 (m,
2H), 3.95 (t, J ) 6.7 Hz, 2H), 4.75 (s, 2H), 5.44 (s, 2H), 7.03 (d,
J ) 7.9 Hz, 1H), 7.09 (d, J ) 7.9 Hz, 1H), 7.26 - 7.38 (m, 3H),
7.45 - 7.50 (m, 2H).
1
found, 311.1026 (M + H)+1; H NMR (300 MHz, DMSO-d6) δ
4.52 - 4.54 (m, 2 H), 6.63 (d, J ) 8.0 Hz, 1H), 6.71 (s, 1H), 6.47
(d, J ) 8.0 Hz, 1H), 7.17 (t, J ) 7.4 Hz, J ) 7.9 Hz, 1H), 7.55 (t,
J ) 7.4 Hz, J ) 8.3 Hz, 1H), 7.83 (d, J ) 8.3 Hz, 1H), 7.97 (d, J
) 7.9 Hz, 1H), 8.61 (d, J ) 13.2 Hz, 1H), 8.93 (s, 1H), 8.99 (s,
H), 10.55 - 10.64 (m, 1H), 10.97 (s, 1H).
(6-Benzyloxy-5-propoxy-pyridin-2-yl)-methanol (86). To a solu-
tion of 85 (860 mg, 2.0 mmol) was added 1 M tetrabutylammonium
fluoride solution in tetrahydrofuran (4.0 mL, 4.0 mmol) and the
mixture stirred at room temperature for 4 h. The reaction mixture
was diluted with water and extracted with ether. The ether layers
were combined and washed with water, dried over sodium sulfate,
filtered, and chromatographed on silica gel eluting with 1:1 hexane/
ether to give 355 mg (65%) of 86 as a white solid: mp 50-1 °C;
(4Z)-4-{[(4-Hydroxybenzyl)amino]methylene}isoquinoline-
1,3(2H,4H)-dione (3c). Using the procedure described for the
preparation of 5e except without the addition of triethylamine, 1
(300 mg, 1.48 mmol) and 4-hydroxy-benzylamine (2c) (235 mg,
1.48 mmol) were reacted to give 200 mg (46%) of 3c as a red-
1
MS (ESI) m/z 274.1 (M + 1)+1; H NMR (400 MHz, CDCl3) δ
1.04 (t, J ) 7.5 Hz, 3H), 1.79 - 1.89 (m, 2H), 2.90 (bs, 1H), 3.97
(t, J ) 6.7 Hz, 2H), 4.57 (s, 2H), 5.48 (s, 2H), 6.74 (d, J ) 7.8 Hz,
1H), 7.07 (d, J ) 7.8 Hz, 1H), 7.26 - 7.31 (m, 1H), 7.33 - 7.38
(m, 2H), 7.45 - 7.49 (m, 2H).
6-Azidomethyl-2-benzyloxy-3-propoxy-pyridine (87). Using the
procedure described for the preparation of 80b, 86 (1.09 g, 4.0
mmol) was reacted with triphenylphosphine and carbon tertabro-
mide (1.99 g, 6.0 mmol) to give 1.05 g (88%) of 87 as a clear
liquid: MS (ESI) m/z 299.1 (M + 1)+1; 1H NMR (400 MHz, CDCl3)
δ 1.04 (t, J ) 7.5 Hz, 3H), 1.79 - 1.90 (m, 2H), 3.96 (t, J ) 6.7
Hz, 2H), 4.22 (s, 2H), 5.49 (s, 2H), 6.78(d, J ) 7.8 Hz, 1H), 7.02
(d, J ) 7.8 Hz, 1H), 7.26 - 7.31 (m, 1H), 7.32 - 7.38 (m, 2H),
7.47 - 7.52 (m, 2H).
brown solid: mp 272-273 °C; MS (ESI) m/z 294.31 (M - 1)-1
;
1H NMR (400 MHz, DMSO-d6) δ 4.58 (d, J ) 6.0 Hz, 2H), 6.76
(d, J ) 8.3 Hz, 2H), 6.99 - 7.26 (m, 3H), 7.55 (t, J ) 7.6 Hz,
1H), 7.83 (d, J ) 8.3 Hz, 1H), 7.97 (d, J ) 7.8 Hz, 1H), 8.62 (d,
J ) 13.3 Hz, 1H), 9.44 (s, 1H), 10.00 - 10.77 (m, 1H), 10.96 (s,
1H); Anal. (C17H14N2O3 ·0.2H2O) C, H, N.
(4Z)-4-{[(3-Hydroxybenzyl)amino]methylene}isoquinoline-
1,3(2H,4H)-dione (3d). Using the procedure described for the
preparation of 5e except without the addition of triethylamine, 1
(300 mg, 1.48 mmol) and 3-hydroxy-benzylamine (2d) (235 mg,
1.48 mmol) were reacted to give 200 mg (46%) of 3c as a reddish-
brown solid: mp 261-262 °C; MS (ESI) m/z 294.31 (M - 1); 1H
NMR (400 MHz, DMSO-d6) δ 4.63 (d, J ) 6.0 Hz, 2H), 6.69 (d,
J ) 8.0 Hz, 1H), 6.77 (m, 2H), 7.16 (t, J ) 8.0 Hz, 2H), 7.56 (t,
J ) 7.3 Hz, 1H), 7.83 (d, J ) 7.3 Hz, 1H), 7.98 (d, J ) 7.3 Hz, 1
H), 8.63 (d, J ) 13.3 Hz, 1H), 9.48 (s, 1H), 10.64 (m, 1H), 11.0
(s, 1 H); Anal. (C17H14N2O3 ·0.2H2O) C, H, N.
6-Aminomethyl-3-propoxy-pyridin-2-ol (88). To a mixture of 87
(544 mg, 2.00 mmol), 12 mL of tetrahydrofuran and triphenylphos-
phine (524 mg, 2.00 mmol) were added, followed by water (540
µL, 30.0 mmol). It was stirred at ambient temperature overnight.
The reaction mixture was evaporated to dryness in vacuo and then
washed with 2:1 hexane/ethyl acetate and filtered. This resulting
solid was taken up in ethanol and hydrogenated over 10% palladium
on carbon at 1 atm. The reaction mixture was filtered, washed with
ethanol, and evaporated to give 93 mg (25%) of 88 as a brownish
(4Z)-4-{[(3-Hydroxy-4-methoxybenzyl)amino]methylene}isoquinoline-
1,3(2H,4H)-dione (3e). Using the procedure described for the
preparation of 5e, 1 (0.2 g, 0.98 mmol) and 4-methoxyl-3-hydroxyl-
benzylamine hydrochloride (2e) (0.2 g, 1.08 mmol) were reacted
to give 0.25 g (78%) of 3e as a red-brown solid: mp 192-193 °C;
1H NMR (DMSO-d6) δ 3.75 (s, 3H), 4.56 (d, J ) 4.5 Hz, 2H),
6.78 (m, 2H), 6.91 (d, J ) 6 Hz, 1H), 7.17 (dd, J ) 6, 6 Hz, 1H),
7.55 (dd, J ) 6, 6 Hz, 1H), 7.83 (d, J ) 6 Hz, 1H), 7.98 (d, J )
6 Hz, 1H), 8.62 (d, J ) 9 Hz, 1H), 9.04 (s, 1H), 10.60 (m, 1H),
green solid: MS (ESI) m/z 183.3 (M + 1)+1
.
Preparation of the Final Compounds. (4Z)-6-Iodo-4-{[(3-hy-
droxy-4-methoxybenzyl)amino]methylene}isoquinoline-1,3(2H,4H)-
dione (5e). A mixture of (4Z)-6-iodo-4-{[(4-hydroxy-3-methoxy-
benzyl)amino]methylene}isoquinoline-1,3(2H,4H)-dione (4e) (0.3
g, 0.91 mmol), 4-methoxyl-3-hydroxyl-benzylamine hydrochloride
(2e) (0.19 g, 1 mmol), and triethylamine (0.14 mmol, 1.37 mmol)
in DMF (3 mL) was stirred at room temperature overnight. The
mixture was diluted with diethyl ether, and the solid was
collected by filtration. The crude product was washed with water
and methanol, followed by recrystallization from DMF in ether
to give 0.28 g (68%) of 5e as a tan solid: mp 209-210 °C; MS
10.97 (s, 1H); MS (ESI) m/z 325.1 (M
(C18H16N2O4 ·0.25H2O) C, H, N.
+ ; Anal.
H)+1
4-{[2-(3,4-Dihydroxy-phenyl)-ethylamino]-methylene}-4H-iso-
quinoline-1,3-dione (3f). Using the procedure described for the
preparation of 5e, 1 (20.3 mg, 0.1 mmol) and 3-hydroxytyramine
hydrochloride (2f) (19 mg, 0.1 mmol) were reacted to give 18.1
mg (56%) of 3f as a red-brown solid; LC/MS1 Rt 1.748 min, m/z
1
1
(ESI) m/z 449.0 (M - H)-1; H NMR (400 MHz, DMSO-d6) δ
325.0 (M + H)+1; LC/MS2 Rt 1.73 min, m/z 325 (M + H)+1; H
2.23 (s, 3H) 2.42 - 2.48 (m, 4H), 3.09 - 3.20 (m, 4H), 7.00 (d,
J ) 8.8 Hz, 2H), 7.47 (d, J ) 8.8 Hz, 2H), 7.56 (d, J ) 8.5 Hz,
NMR (300 MHz, DMSO-D6) δ ppm 2.74 (t, J ) 7.1 Hz, 2H),
3.65 (q, J ) 7.1 Hz, J ) 6.7 Hz, 2 H), 6.52 (dd, J ) 9.6 Hz, J )

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2305
1.8 Hz, 1 H), 6.64 (d, J ) 1.8 Hz, 1 H), 6.66 (d, J ) 9.6 Hz, 1 H),
7.14 (t, J ) 7.5 Hz, J ) 7.8 Hz, 1 H), 7.51 (t, J ) 8.4 Hz, 1 H),
7.72 (d, J ) 8.4 Hz, 1 H), 7.95 (d, J ) 7.8 Hz, 1 H), 8.41 (d, J )
13.5 Hz, 1 H), 8.77 (br s, 2 H), 10.37 - 10.45 (m, 1 H), 10.91 (s,
1 H).
(4Z)-6-Fluoro-4-{[(3-hydroxy-4-methoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (5a). Using the procedure described
for the preparation of 5e, (4E)-6-fluoro-4-(methoxymethylene)iso-
quinoline-1,3(2H,4H)-dione (4a) (300 mg, 1.06 mmol) and 3-hy-
droxy-4-methoxy-benzylamine hydrogen chloride (2e) (201.2 mg,
1.06 mmol) were reacted to give 340 mg (94%) of 5a as a light-
(66%) of 5g as a light brown solid: mp 252-253 °C; MS (ESI)
m/z 389.41 (M + 1)+1; 1H NMR (400 MHz, DMSO-d6) δ 3.75 (s,
3H), 4.60 (m, 2H), 6.34 (t, J ) 4.0, 2.0 Hz, 2H), 6.78 (dd, J ) 8.4,
2.0 Hz, 1H), 6.82 (d, J ) 2.0 Hz, 1H), 6.92 (d, J ) 8.4 Hz, 1H),
7.41 (dd, J ) 8.8, 2.0 Hz, 1H), 7.59 (t, J ) 4.0, 2.0 Hz, 2H), 7.92
(d, J ) 2.0 Hz, 1H), 8.02 (d, J ) 8.8 Hz, 1H), 8.80 (m, 1H), 9.07
(s, 1H), 10.37
- 10.78 (m, 1H), 10.96 (s, 1H); Anal.
(C22H19N3O4 ·0.6H2O) C, H, N.
(4Z)-4-{[(3-Hydroxy-4-methoxybenzyl)amino]methylene}-6-thien-
3-ylisoquinoline-1,3(2H,4H)-dione (5h). Using the procedure de-
scribed for the preparation of 5e, (4E)-4-(methoxymethylene)-6-
(3-thienyl)-isoquinoline-1,3(2H,4H-dione (4h) (0.15 g, 0.53 mmol)
and 2e (0.11 g, 0.62 mmol) were reacted to give 0.18 g (86%) of
5h as a tan solid: mp 219-220 °C, MS (ESI) m/z 407.1 (M +
H)+1; Anal. (C22H18N2O4S ·0.8H2O) C, H, N.
brown solid: mp 200-201 °C; MS (ESI) m/z 342.33 (M + 1)+1
;
1H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.56 (m, 2H), 6.77
(dd, J ) 8.0, 2.0 Hz, 1H), 6.81 (d, J ) 2.0 Hz, 1H), 6.91 (d, J )
8.0 Hz, 1H), 6.95-7.0 (m, 1H), 7.68 (dd, J ) 12, 2.0 Hz, 1H),
8.01 (m, J ) 8.0, 1H), 8.65 (d, J ) 12.0 Hz, 1H), 9.05 (s, 1H),
10.62 - 10.65 (m, 1H), 11.01 (s, 1H); Anal. (C18H15FN2O4 ·0.3
H2O) C, H, N.
(4Z)-6-(3-Furyl)-4-{[(3-hydroxy-4-methoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (5i). Using the procedure described
for the preparation of 5e, (4E)-6-(3-furyl)-4-(methoxymethylene)-
isoquinoline-1,3(2H,4H-dione (4h) (160 mg, 0.59 mmol) and 2e
(94.6 mg, 0.59 mmol) were reacted to give 150 mg (65%) of 5i as
(4Z)-6-Chloro-4-{[(3-hydroxy-4-methoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (5b). Using the procedure described
for the preparation of 5e, (4E)-6-chloro-4-(methoxymethylene)-
isoquinoline-1,3(2H,4H)-dione (4b) (0.15 g, 0.63 mmol) and 2e
(0.114 g, 0.756 mmol) were reacted to give 0.182 g (80.2%) of 5b
a brown solid: mp 254-255 °C; MS (ESI) m/z 390.40 (M - 1)-1
;
1H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.60 (m, 2H), 6.78
(dd, J ) 8.0, 2.0 Hz, 1H), 6.82 (d, J ) 2.0 Hz, 1H), 6.92 (d, J )
8.0 Hz, 1H), 7.17 (d, J ) 1.2 Hz, 1H), 7.44 (dd, J ) 8.4, 1.2 Hz,
1H), 7.82 (m, 1H), 7.76 (d, J ) 8.4 Hz, 1H), 8.0 (m, 1H), 8.36 (m,
1H), 8.75 (m, 1H), 9.07 (s, 1H), 10.56 - 10.74 (m, 1H), 10.94 (s,
1H); Anal. (C22H18N2O5 ·0.7H2O) C, H, N.
as a tan solid: mp 265-266 °C; MS (ESI) m/z 357.5 (M - H)-1
;
1H NMR (400 MHz, DMSO-d6) δ ppm 3.75 (s, 3 H), 4.57 (d, J )
6.0 Hz, 2 H), 6.78 (d, J ) 8.1 Hz, 1 H), 6.82 (s, 1 H), 6.91 (d, J
) 8.1 Hz, 1 H), 7.17 (d, J ) 8.3 Hz, 1 H), 7.81 - 8.08 (m, 2 H),
8.71 (d, J ) 13.4 Hz, 1 H), 9.05 (s, 1 H), 10.45 - 10.85 (m, 1 H),
11.07 (s, 1 H); Anal. (C18H15ClN2O4 ·0.8 H2O) C, N. H: calcd, 4.48;
found, 4.06.
4-[(3-Hydroxy-4-methoxy-benzylamino)-methylene]-6-phenyl-
4H-isoquinoline-1,3-dione (5j). To a suspension of 5d (40 mg, 0.1
mmol) in DMF (1 mL) was added phenylboronic acid (14.6 mg,
0.12 mmol), followed by 60 µL of 2 M aqueous cesium carbonate
and tetrakistriphenylphosphine palladium (6 mg, 0.005 mmol). The
reaction mixture was subjected to microwave heating at 150 °C
under microwave conditions for 5 min. The reaction mixture was
then diluted to 2 mL with DMF and purified by C18 reverse phase
HPLC. The pure fractions were combined and concentrated to yield
6.2 mg (15.5%) of 5j; LC/MS1 Rt 2.620 min, m/z 401.0 (M +
H)+1; LC/MS2 Rt 2.36 min, m/z 401 (M + H)+1; HRMS (ESI) m/z
(4Z)-5-Bromo-4-{[(3-hydroxy-4-methoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (5c). Using the procedure described
for the preparation of 5e, (4E)-5-bromo-4-(methoxymethylene)-
isoquinoline-1,3(2H,4H)-dione (4c) (132 mg, 0.468 mmol) and 2e
(88.5 mg, 0.468 mmol) were reacted to give 163 mg (86%) of 5c
as a pale yellow amorphous solid: mp 204-206 °C; HRMS (ESI)
m/e calcd for C25H28N4O3 431.20886; found, 431.20820 (M - H)-1
;
1H NMR (DMSO-d6) δ 3.75 (s, 3H), 4.49 (d, 2H, J ) 5.4 Hz),
6.78 (m, 2H), 6.91 (d, 1H, J ) 8.1 Hz), 7.09 (m, 1H), 7.87 (d, 1H,
J ) 6.9 Hz), 8.05 (d, 1H, J ) 6.9 Hz), 8.93 (d, 1H, J ) 13.6 Hz),
9.08 (s, 1H), 10.56 (m, 1H), 11.11 (s, 1H); Anal.
(C18H15BrN2O4 ·1.25 H2O) C, H, N.
1
calcd for C24H20N2O4 401.1498; found, 401.1496 (M + H)+1; H
NMR (300 MHz, DMSO-d6) δ ppm 3.81 (s, 3H), 4.59 - 4.62 (m,
2H), 6.78 (d, J ) 8.13 Hz, 1H), 6.82 (s, 1H), 6.91 (d, J ) 8.1 Hz,
1H), 7.44 - 7.55 (m, 4H), 7.82 (d, J ) 7.2 Hz, 2H), 8.05 (d, J )
8.2 Hz, 1H), 8.09 (s, 1H), 8.83 (d, J ) 13.2 Hz, 1H), 9.06 (s, 1H),
10.61 - 10.71 (m, 1H), 10.99 (s, 1H).
(4Z)-6-Bromo-4-{[(3-hydroxy-4-methoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (5d). Using the procedure described
for the preparation of 5e, (4E)-6-bromo-4-(methoxymethylene)-
isoquinoline-1,3(2H,4H)-dione (4d) (300 mg, 1.06 mmol) and 2e
(201.2 mg, 1.06 mmol) were reacted to give 340 mg (77%) of 5d
as a light-brown solid: mp 243-244 °C; MS (ESI) m/z 413.23 (M
(4Z)-6-Bromo-4-({[1-(3-hydroxy-4-methoxyphenyl)ethyl]amino}methy-
lene)isoquinoline-1,3(2H,4H)-dione (11). Using the procedure
described for the preparation of 5e, 4d (0.10 g, 0.35 mmol) and
5-(1-amino-ethyl)-2-methoxyphenol hydrochloride (10) (0.142, 0.70
mmol) were reacted to give 0.11 (73%) of 11 as a light tan powder:
1
+ 1)+1; H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.56 (m,
1
2H), 6.77 (dd, J ) 8.4, 2.0 Hz, 1H), 6.81 (d, J ) 2.0 Hz, 1H), 6.91
(d, J ) 8.4 Hz, 1H), 7.30 (dd, J ) 8.4, 1.2 Hz, 1H), 7.87 (d, J )
8.4 Hz, 1H), 8.10 (d, J ) 1.2 Hz, 1H), 8.71 (m, 1H), 9.05 (s, 1H),
10.56 - 10.75 (m, 1H), 11.07 (s, 1H); Anal. (C18H15BrN2O4 ·0.5
H2O) C, N. H: calcd, 3.99; found, 3.56.
MS (ESI) m/z 417.1 (M + 1)+1; H NMR (400 MHz, DMSO-d6)
δ 1.59 (d, J ) 6.8 Hz, 3H), 3.75 (s, 3H), 4.76 - 4.85 (m, 1H),
6.80 (dd, J ) 8.4, 2.0 Hz, 1H), 6.83 (d, J ) 2.0 Hz, 1H), 6.93 (d,
J ) 8.4 Hz, 1H), 7.30 (dd, J ) 8.4, 1.6 Hz, 1H), 7.86 (d, J ) 8.4
Hz, 1H), 8.09 (d, J ) 1.6 Hz, 1H), 8.69 (d, J ) 13.2 Hz, 1H), 9.08
(s, 1H), 10.79 (dd, J ) 13.2, 8.4 Hz, 1H), 11.13 (s, 1H); Anal.
(C19H17BrN2O4) C, H, N.
4-[(3-Hydroxy-4-methoxy-benzylamino)-methylene]-6-methoxy-
4H-isoquinoline-1,3-dione (5f). Using the procedure described for
the preparation of 5e, (4E)-6-methoxy-4-(methoxymethylene)-
isoquinoline-1,3(2H,4H)-dione (4d) (117 mg, 0.50 mmol) and 2e
(145 mg, 0.50 mmol) were reacted to give 161 mg (91%) of 5f as
a light beige solid: mp 240-242 °C (dec); MS (ESI) m/z 355.2 (M
(4Z)-6-Bromo-4-{[(3-hydroxy-4-methoxybenzyl)(methyl)amino]methy-
lene}isoquinoline-1,3(2H,4H)-dione (13). Using the procedure
described for the preparation of 3c, except for replacing DMF with
tetrahydrofuran, 4d (0.15 g, 0.53 mmol) and 2-methoxy-5-methy-
laminomethyl-phenol (12) (80 mg, 0.48 mmol) were reacted to give
0.19 g (95%) of 13 as a yellow solid: MS (ESI) m/z 417.9, 419.9
1
- 1)-1; H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 3.88 (s,
3H), 4.58 (d, J ) 6.0 Hz, 2H), 6.72 - 6.79 (m, 2H), 6.80 (d, J )
2.0 Hz, 1H), 6.91 (d, J ) 8.2 Hz, 1H), 7.27 (d, J ) 2.01 Hz, 1H),
7.90 (d, J ) 9.1 Hz, 1H), 8.63 (d, J ) 13.2 Hz, 1H), 9.07 (bs, 1H),
10.63 (dt, J ) 13.3, 5.8 Hz, 1H), 10.78 (s, 1H); Anal.
(C19H18N2O5 ·0.25H2O) C, H, N.
1
(M + 1)+1; H NMR (400 MHz, DMSO-d6) δ 3.10 (s, 3H), 3.81
(s, 3H), 4.81 (s, 2H), 6.81 (d, J ) 2.0 Hz, 1H), 6.84 (d, J ) 2.0
Hz, 1H), 6.93 (d, J ) 8.8 Hz, 1H), 7.28 (dd, J ) 8.4, 1.6 Hz, 1H),
7.83 (d, J ) 1.6 Hz, 1H), 7.91 (d, J ) 8.4 Hz, 1H), 8.50 (s, 1H),
9.10 (s, 1H), 10.73 (s, 1H); Anal. (C19H17BrN2O4) C, H, N.
(4Z)-6-Bromo-4-{[(3,4-dihydroxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (17a). Using the procedure de-
scribed for the preparation of 5e, 4d (1.21 g, 4.3 mmol) and 3,4-
dihydroxybenzylamine (16a) (0.596 g, 4.3 mmol) were reacted, and
(4Z)-4-{[(3-hydroxy-4-methoxybenzyl)amino]methylene}-6-(1H-
pyrrol-1-yl)isoquinoline-1,3(2H,4H)-dione (5g). Using the procedure
described for the preparation of 5e, (4E)-4-(methoxymethylene)-
6-(1H-pyrrol-1-yl)isoquinoline-1,3(2H,4H)-dione (4g) (100 mg, 0.38
mmol) and 2e (60 mg, 0.35 mmol) were reacted to give 90 mg

2306 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
the crude product was purified by high performance liquid chro-
matography to give 910 mg (54%) of 17a as a white solid: MS
(ESI) m/z 389.7 (M + 1);1H NMR (400 MHz, DMSO-D6) δ ppm
4.53 (d, J ) 6.3 Hz, 2H), 6.63 (dd, J ) 8.0, 1.2 Hz, 1H), 6.72 (d,
J ) 7.5 Hz, 1H), 6.76 (d, J ) 1.7 Hz, 1H), 7.30 (dd, J ) 8.3, 1.6
Hz, 1H), 7.87 (d, J ) 8.3 Hz, 1H), 8.11 (d, J ) 1.5 Hz, 1H), 8.71
(d, J ) 13.3 Hz, 1H), 8.91 (s, 1H), 8.97 (s, 1H), 10.64 (m, 1H),
11.07 (s, 1H); Anal. (C17H13BrN2O4 ·0.5 H2O) C, H, N.
(4Z)-6-Bromo-4-{[(3-hydroxy-4propoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (17b). Using the procedure de-
scribed for the preparation of 5e, 4d (171 mg, 0.607 mmol) and
5-(aminomethyl)-2-propoxyphenol (16b) (100 mg, 0.55 mmol) were
reacted, and the crude product was purified by high performance
liquid chromatography to give 67 mg (28%) of 17b as a white solid:
MS (ESI) m/z 430.8 (M + 1)+1; HRMS (ESI) m/z calcd for
C20H19BrN2O4 431.06010; found, 431.05984 (M + 1)+1; 1H NMR
(300 MHz, DMSO-d6) δ 0.97 (t, J ) 7.4 Hz, 3H), 1.56 - 1.91 (m,
2H), 3.89 (t, J ) 6.6 Hz, 2H), 4.56 (d, J ) 6.0 Hz, 2H), 6.64 -
6.77 (m, 1H), 6.82 (s, 1H), 6.89 (d, J ) 8.3 Hz, 1H), 7.30 (d, J )
8.7 Hz, 1H), 7.87 (d, J ) 8.7 Hz, 1H), 8.11 (s, 1H), 8.72 (d, J )
13.2 Hz, 1H), 8.97 (s, 1H), 10.41 - 10.89 (m, 1H), 11.10 (s, 1H);
Anal. (C20H19BrN2O4) C, H, N.
1H), 10.48 - 10.75 (m, 1H), 11.07 (s, 1H); Anal. (C17H14BrN3-
O3 ·1.4 H2O) C, H, N.
(4Z)-4-{[(4-Methyl-3-hydroxybenzyl)amino]methylene}-6-iodo-
isoquinoline-1,3(2H,4H)-dione (89a). Using the procedure described
for the preparation of 5e, 4e (200 mg, 0.61 mmol) and 5-aminom-
ethyl-2-methyl-phenol hydrogen chloride (25) (105.64 mg, 0.61
mmol) were reacted to give 120 mg (45%) of 89a as an orange
solid: mp 312-313 °C; MS (APCI); HRMS (ESI) m/z calcd for
1
C18H15IN2O3 435.02002; found, 435.02006 (M + 1)+1; H NMR
(400 MHz, DMSO-d6) δ 2.08 (s, 3H), 4.61 (m, 2H), 6.70 (d, J )
7.6 Hz, 1H), 6.75 (m, 1H), 7.1 (d, J ) 7.6 Hz, 1H), 7.5 (dd, J )
8.0, 1.0 Hz, 1H), 7.70 (d, J ) 8.0 Hz, 1H), 8.28 (d, J ) 1.0 Hz,
1H), 8.73 (m, 1H), 9.40 (s, 1H), 10.64 - 10.68 (m, 1H), 11.08 (s,
1H); Anal. (C18H15IN2O3) C, H, N.
4-{[(2-Hydroxy-biphenyl-4-ylmethyl)-amino]-methylene}-6-iodo-
4H-isoquinoline-1,3-dione (89b). Using the procedure described for
the preparation of 5e, except without the addition of triethylamine,
4e (110 mg, 0.34 mmol) and 4-aminomethyl-biphenyl-2-ol (34) (80
mg, 0.4 mmol) were reacted to give 151 mg (89%) of 89b: MS
1
(ESI) m/z 495.1 (M - 1)-1; H NMR (400 MHz, DMSO-d6) δ
4.68 (d, J ) 6.6 Hz, 2H), 6.61 - 6.96 (m, 2H), 7.23 - 7.32 (m,
2H), 7.34 - 7.42 (m, 2H), 7.48 - 7.53 (m, 2H), 7.53 (d, J ) 1.5
Hz, 1H), 7.69 (d, J ) 8.3 Hz, 1H), 8.30 (d, J ) 1.5 Hz, 1H), 8.75
(d, J ) 13.1 Hz, 1H), 9.67 (s, 1H), 10.65 - 10.81 (m, 1H), 11.10
(s, 1H); Anal. (C23H20IN3O4 ·0.7H2O) C, H, N.
(4Z)-6-Bromo-4-({[3-hydroxy-4-(2-methoxyethoxy)benzyl]amino}methyl-
ene)isoquinoline-1,3(2H,4H)-dione (17c). Using the procedure
described for the preparation of 5e, 4d (400 mg, 1.42 mmol) and
5-(aminomethyl)-2-(methoxyethoxy)phenol (16c) (330 mg, 1.67
mmol) were reacted to give 480 mg (76%) of 17c as a tan solid:
mp 179-180 °C; MS (ESI) m/z 447.0-449.0 (M + H)+1;1H NMR
(400 MHz, DMSO-d6) δ ppm 3.30 (s, 3 H), 3.47 - 3.69 (m, 2 H),
3.92 - 4.19 (m, 2 H), 4.57 (d, J ) 6.3 Hz, 2 H), 6.75 (dd, J ) 8.3,
2.0 Hz, 1 H), 6.83 (d, J ) 2.0 Hz, 1 H), 6.92 (d, J ) 8.3 Hz, 1 H),
7.30 (dd, J ) 8.4, 1.7 Hz, 1 H), 7.87 (d, J ) 8.4 Hz, 1 H), 8.11 (d,
J ) 1.7 Hz, 1 H), 8.72 (d, J ) 13.35 Hz, 1 H), 9.06 (s, 1 H), 10.45
- 10.81 (m, 1 H), 11.09 (s, 1 H); Anal. (C20H19BrN2O5) C, H, N.
4-[(4-Furan-2-yl-3-hydroxy-benzylamino)-methylene]-6-iodo-4H-
isoquinoline-1,3-dione (89c). Using the procedure described for the
preparation of 89b, 4e (80 mg, 0.24 mmol) and 5-aminomethyl-
2-furan-3-yl-phenol (41) (50 mg, 0.26 mmol) were reacted to give
1
62 mg (53%) of 89c: MS (ESI) m/z 485.1 (M - 1)-1; H NMR
(400 MHz, DMSO-d6) δ 4.67 (d, J ) 6.3 Hz, 2H), 6.56 (dd, J )
3.3, 1.8 Hz, 1H), 6.88 (dd, J ) 8.1, 1.5 Hz, 1H), 6.90 - 6.94 (m,
2H), 7.50 (dd, J ) 8.3, 1.3 Hz, 1H), 7.57 - 7.79 (m, 3H), 8.16 -
8.35 (m, 1H), 8.73 (d, J ) 13.4 Hz, 1H), 10.23 (s, 1H), 10.47 -
10.82 (m, 1H), 11.08 (s, 1H); Anal. (C21H15IN2O4 ·0.5H2O) C, H.
N: calcd, 5.66; found 5.23.
(4Z)-6-bromo-4-{[(4-ethoxy-3-hydroxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (17d). To a solution of 1-iodoet-
hane (43.3 µL, 0.28 mmol) in anhydrous N,N-dimethylformamide
(2 mL) was added potassium carbonate (207 mg, 1.5 mmol). The
mixture was stirred at room temperature, and 17a (100 mg, 0.26
mmol) was added. After the mixture was stirred at 65 °C for 30
min, the resulting mixture was concentrated, and the residue was
then partitioned between water (50 mL) and ethyl acetate (50 mL).
The organic layer was then dried and purified by high performance
liquid chromatography to give 26 mg (24%) of 17d as a white solid.
4-[(4-Furan-3-yl-3-hydroxy-benzylamino)-methylene]-6-iodo-4H-
isoquinoline-1,3-dione (89d). Using the procedure described for the
preparation of 5e, 4e (85 mg, 0.26 mmol) and 5-aminomethyl-2-
furan-3-yl-phenol (45a) (73 mg, 0.39 mmol) were reacted to give
1
70 mg (56%) of 89d: MS (ESI) m/z 487.1 (M + 1)+1; H NMR
(400 MHz, DMSO-d6) δ 4.66 (d, J )6.6 Hz, 2H), 6.85 (dd, J )
7.9, 1.6 Hz, 1H), 6.90 (d, J ) 1.6 Hz, 1H), 6.99 (d, J ) 1.3 Hz,
1H), 7.49 (dd, J ) 8.1, 1.5 Hz, 1H), 7.54 (d, J ) 8.1 Hz, 1H), 7.67
- 7.72 (m, 2H), 8.12 (s, 1H), 8.24 - 8.37 (m, 1H), 8.74 (d, J )
13.4 Hz, 1H), 10.06 (s, 1H), 10.56 - 10.78 (m, 1H), 11.08 (s, 1H);
Anal. (C21H15IN2O4) C, H, N.
1
MS (ESI) m/z 416.7 (M + 1)+1; H NMR (400 MHz, DMSO-d6)
δ 1.31 (t, J ) 7.0 Hz, 3H), 3.99 (q, J ) 7.0 Hz, 2H), 4.56 (d, J )
6.3 Hz, 2H), 6.75 (dd, J ) 8.3, 2.0 Hz, 1H), 6.82 (d, J ) 2.0 Hz,
1H), 6.90 (d, J ) 8.3 Hz, 1H), 7.30 (dd, J ) 8.5, 1.6 Hz, 1H), 7.87
(d, J ) 8.5 Hz, 1H), 8.11 (d, J ) 1.7 Hz, 1H), 8.71 (d, J ) 13.3
Hz, 1H), 9.00 (s, 1H), 10.45 - 10.94 (m, 1H), 11.09 (s, 1H); Anal.
(C19H17BrN2O4 ·0.3DMF·0.5TFA) C, H, N.
4-[(3-Hydroxy-4-pyridin-2-yl-benzylamino)-methylene]-6-iodo-
4H-isoquinoline-1,3-dione (89e). Using the procedure described for
the preparation of 5e, 4e (98 mg, 0.30 mmol) and 5-aminomethyl-
2-pyridin-2-yl-phenol (45b) (60 mg, 0.30 mmol) were reacted to
(4Z)-6-Bromo-4-{[(3-hydroxy-4,5-dimethoxybenzyl)amino]methylene}-
isoquinoline-1,3(2H,4H)-dione (18). Using the procedure described
for the preparation of 5e, 4d (400 mg, 1.42 mmol) and 3-hydroxy-
4,5-dimethoxybenzylamine (272 mg, 1.15 mmol) were reacted to
give 400 mg (65%) of 18 as a brown solid: mp 262-263 °C; MS
1
give 85 mg (57%) of 89e: MS (ESI) m/z 498.1 (M + 1)+1; H
NMR (400 MHz, DMSO-d6) δ 4.69 (d, J ) 6.3 Hz, 2H), 6.75 -
7.02 (m, 2H), 7.43 (dd, J ) 7.1, 5.5 Hz, 1H), 7.50 (d, J ) 8.3 Hz,
1H), 7.69 (d, J ) 8.3 Hz, 1H), 7.95 - 8.08 (m, 2H), 8.20 (d, J )
7.8 Hz, 1H), 8.28 (s, 1H), 8.61 (d, J ) 5.0 Hz, 1H), 8.71 (d, J )
13.4 Hz, 1H), 10.37 - 11.03 (m, 1H), 11.07 (s, 1H), 14.27 (s, 1H);
Anal. (C22H16IN3O3 ·0.3TFA) C, H, N.
1
(ESI) m/z 433.26 (M - 1)-1; H NMR (400 MHz, DMSO-d6) δ
3.64 (s, 3H), 3.76 (s, 3H), 4.56 (m, 2H), 6.49 (d, J ) 1.2 Hz, 1H),
6.55 (m, 1H), 7.3 (dd, J ) 8.4, 1.2 Hz, 1H), 7.87 (d, J ) 8.4 Hz,
1H), 8.10 (m, 1H) 8.70 (m, 1H), 9.24 (s, 1H), 10.50 - 10.80 (m,
1H), 11.09 (s, 1H); Anal. (C19H17BrN2O5) C, H, N.
4-[(3-Hydroxy-4-pyridin-3-yl-benzylamino)-methylene]-6-iodo-
4H-isoquinoline-1,3-dione (89f). Using the procedure described for
the preparation of 5e, 4e (50 mg, 0.15 mmol) and 5-aminomethyl-
3-pyridin-2-yl-phenol (45c) (53 mg, 0.27 mmol) were reacted to
give 57 mg (76%) of 89f: MS (ESI) m/z 498.0 (M + 1)+1; 1H NMR
(400 MHz, DMSO-d6) δ 4.68 (d, J ) 6.3 Hz, 2H), 6.58 - 7.06
(m, 2H), 7.33 (d, J ) 7.6 Hz, 1H), 7.42 (dd, J ) 7.6, 4.0 Hz, 1H),
7.50 (dd, J ) 8.3, 1.3 Hz, 1H), 7.69 (d, J ) 8.3 Hz, 1H), 7.86 -
8.00 (m, 1H), 8.27 (d, J ) 1.3 Hz, 1H), 8.47 (d, J ) 4.0 Hz, 1H),
8.58 - 8.79 (m, 2H), 9.96 (s, 1H), 10.53 - 10.79 (m, 1H), 11.07
(s, 1H); Anal. (C22H16IN3O3 ·0.4H2O) C, H, N.
(4Z)-4-{[(4-Amino-3-hydroxybenzyl)amino]methylene}-6-bro-
moisoquinoline-1,3(2H,4H)-dione (21). Using the procedure de-
scribed for the preparation of 5e, 4d (500 mg, 1.77 mmol) and
2-amino-5-(aminomethyl)phenol hydrochloride (20) (448 mg, 2.57
mmol) were reacted to give 700 mg (100%) of 21 as a yellowish
1
brown solid: mp 245-246 °C; MS (ESI) m/z 390 (M + 1)+1; H
NMR (400 MHz, DMSO-d6) δ 4.45 (m, 2H), 4.56 (m, 2H), 6.57
(m, 2H), 6.66 (m, 1H), 7.29 (dd, J ) 8.4, 2.0 Hz, 1H), 7.86 (d, J
) 8.4 Hz, 1H), 8.11 (d, J ) 2.0 Hz, 1H), 8.71 (m, 1H), 9.09 (s,

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2307
4-[(3-Hydroxy-4-pyridin-4-yl-benzylamino)-methylene]-6-iodo-
4H-isoquinoline-1,3-dione (89g). Using the procedure described for
the preparation of 5e, 4e (50 mg, 0.15 mmol) and 5-aminomethyl-
4-pyridin-2-yl-phenol (45d) (40 mg, 0.20 mmol) were reacted to
MS (ESI) m/z 372.0, 347.0 (M - 1)-1; 1H NMR (400 MHz, DMSO-
d6) δ 4.51 (d, J ) 6.4 Hz, 2H), 6.17 (dd, J ) 6.8, 2.0 Hz, 1H),
6.20 (s, 1H), 7.32 (dd, J ) 8.4, 2.0 Hz, 1H), 7.36 (d, J ) 6.8 Hz,
1H), 7.87 (d, J ) 8.4 Hz, 1H), 8.07 (d, J ) 2.0 Hz, 1H), 8.63 (d,
J ) 13.2 Hz, 1H), 10.59 (dt, J ) 6.4, 13.2 Hz, 1H), 11.13 (s, 1H),
11.53 (s, 1H); Anal. (C16H12BrN3O3 ·0.22TFA) C, H, N.
(4Z)-6-Iodo-4-({[(2-oxo-1-phenyl-1,2dihydropyridin-4-
yl)methyl]amino}methylene)isoquinoline-1,3(2H,4H)-dione (94b).
Using the procedure described for the preparation of 5e, 4e (92
mg, 0.28 mmol) and 4-aminomethyl-1-phenyl-1H-pyridin-2-one
hydrochloride (58) (66 mg, 0.28) were reacted to give 8.8 mg (6.3%)
of 94b as a pale yellow solid: MS (ESI) m/z 498.2 (M + 1)+1; 1H
NMR (400 MHz, DMSO-d6) δ 4.59 (d, J ) 6.4 Hz, 2H), 6.35 (dd,
J ) 5.2, 1.6 Hz, 1H), 6.37 (s, 1H), 7.37 - 7.54 (m, 6 H), 7.67 (dd,
J ) 6.8, 0.4 Hz, 1H), 7.69 (d, J ) 8.4 Hz, 1H), 8.26 (d, J ) 1.2
Hz, 1H), 8.65 (d, J ) 13.2 Hz, 1H), 10.62 (dt, J ) 13.2, 6.4 Hz,
1H), 11.12 (s, 1H); Anal. (C22H16IN3O3 ·0.51TFA) C, H, N.
(4Z)-4-[({[1-(3-Furyl)-2-oxo-1,2-dihydropyridin-4-yl]methyl}-
amino)methylene]-6-iodoisoquinoline-1,3(2H,4H)-dione (94c). Us-
ing the procedure described for the preparation of 5e, 4e (0.20 g,
0.62 mmol) and 4-aminomethyl-1-furan-3-yl-1H-pyridin-2-one
hydrochloride (65a) (140 mg, 0.62) were reacted to give 0.12 g
1
give 30 mg (40%) of 89g: MS (ESI) m/z 498.1 (M + 1)+1; H
NMR (300 MHz, DMSO-d6) δ 4.69 (d, J ) 6.0 Hz, 2H), 6.88 -
7.06 (m, 2H), 7.39 (d, J ) 7.9 Hz, 1H), 7.50 (d, J ) 8.3 Hz, 1H),
7.58 (d, J ) 5.7 Hz, 2H), 7.69 (d, J ) 8.3 Hz, 1H), 8.28 (s, 1H),
8.55 (d, J ) 5.7 Hz, 2H), 8.73 (d, J ) 13.2 Hz, 1H), 10.07 (s, 1H),
10.71 (d,
(C22H16IN3O3 ·1.5H2O) C, H, N.
J ) 13.2 Hz, 1H), 11.10 (s, 1H); Anal.
(4Z)-4-{[(3-Hydroxy-4-propoxybenzyl)amino]methylene}-6-iodo-
isoquinoline-1,3(2H,4H)-dione (89h). Using the procedure described
for the preparation of 5e, 4e (2.5 g, 7.6 mmol) and 5-(aminomethyl)-
2-propoxyphenol hydrochloride (16b) (1.82 g, 8.36 mmol) were
reacted to give 3.0 g (83%) of 89h as a tan solid: MS (ESI) m/z
1
479.1 (M + 1)+1; H NMR (300 MHz, DMSO-d6) δ 0.97 (t, J )
6.9 Hz, 3H), 1.71 (m, 2H), 3.90 (t, J ) 6.9 Hz, 2H), 4.57 (d, J )
8.6 Hz, 2H), 6.74 (d, J ) 8.8 Hz, 1H), 6.81 (m, 1H), 6.90 (d, J )
7.5 Hz, 1H), 7.48 (d, J ) 8.8 Hz, 1H), 7.68 (d, J ) 8.8 Hz, 1H),
8.27 (s, 1H), 8.73 (m, 1H), 8.94 (s, 1H), 10.66 (m, 1H), 11.05 (s,
1H); HRMS (ESI) m/e calcd for C20H19IN2O4 479.04623; found,
479.04449 (M + H)+1; Anal. (C20H19IN2O4 ·0.2H2O) C, H, N.
4-{[(4-Hydroxy-5-methoxy-pyrimidin-2-ylmethyl)-amino]-meth-
ylene}-6-iodo-4H-isoquinoline-1,3-dione (90). Using the procedure
described for the preparation of 89b, 4e (50 mg, 0.15 mmol) and
2-aminomethyl-5-methoxy-pyrimidin-4-ol (51) (10 mg, 0.064 mmol)
were reacted to give 10 mg (34%) of 90: MS (ESI) m/z 453 (M +
1)+1; 1H NMR (400 MHz, DMSO-d6) δ 3.72 (s, 3H), 4.57 (d, J )
6.0 Hz, 2H), 7.44 - 7.56 (m, 2H), 7.69 (d, J ) 8.3 Hz, 1H), 7.89
(s, 1H), 8.20 (s, 1H), 10.55 (d, J ) 13.1 Hz, 1H), 11.09 (s, 1H),
12.74 (s, 1H).
(40%) of 94c as a brown powder: MS (ESI) m/z 488.1 (M + 1)+1
;
1H NMR (400 MHz, DMSO-d6) δ 4.58 (d, J ) 6.0 Hz, 2H), 6.39
- 6.42 (m, 2H), 7.0 (d, J ) 2.0 Hz, 1H), 7.50 (dd, J ) 8.4, 1.6
Hz, 1H), 7.69 (d, J ) 8.4 Hz, 1H), 7.78 (m, 1H), 7.94 (d, J ) 8
Hz, 1H), 8.25 (d, J ) 0.8 Hz, 1H), 8.38 (d, J ) 0.8 Hz, 1H), 8.64
(d, J ) 13.2 Hz, 1H), 10.60 (dt, J ) 13.2, 6.0 Hz, 1H), 11.12 (s,
1H); Anal. (C20H14IN3O4) C, H, N.
6-Iodo-4-{[(4-hydroxy-5-methoxy-pyridin-2-ylmethyl)-amino]-
methylene}-4H-isoquinoline-1,3-dione (100a). Using the procedure
described for the preparation of 89b, 4e (247 mg, 0.75 mmol) and
2-aminomethyl-5-methoxy-pyridin-4-ol (74a) (116 mg, 0.75 mmol)
were reacted to give 293 mg (87%) of 100a as a pink solid: MS
(4Z)-6-Bromo-4-({[(2-methoxypyridin-4-yl)methyl]amino}methylene)-
isoquinoline-1,3(2H,4H)-dione (91). Using the procedure described
for the preparation of 5e, 4d (0.15 g, 0.53 mmol) and 4-(2-
methoxypyridyl)methylamine hydrochloride (54) (93 mg, 0.53
mmol) were reacted to give 0.13 (62%) of 91 as a golden solid:
MS (ESI) m/z 388.0, 390.0 (M + 1)+1; 1H NMR (400 MHz, DMSO-
d6) δ 3.84 (s, 3H), 4.68 (d, J ) 6.4 Hz, 2H), 6.76 (s, 1H), 6.98 (dd,
J ) 5.2, 1.2 Hz, 1H), 7.32 (dd, J ) 8.4, 1.6 Hz, 1H), 7.87 (d, J )
8.4 Hz, 1H), 8.08 (d, J ) 1.6 Hz, 1H), 8.15 (d, J ) 5.2 Hz, 1H),
8.68 (d, J ) 13.2 Hz, 1H), 10.66 (dd, J ) 13.2, 6.4 Hz, 1H), 11.12
(s, 1H); Anal. (C17H14BrN3O3 ·0.2TFA) C, H, N.
1
(ESI) m/z 451.9 (M + 1)+1; H NMR (400 MHz, DMSO-d6) δ
3.97 (s, 3H), 4.89 (d, J ) 6.0 Hz, 2H), 7.25 (s, 1H), 7.55 (dd, J )
8.3, 1.2 Hz, 1H), 7.73 (d, J ) 8.3 Hz, 1H), 8.22 (s, 1H), 8.41 (s,
1H), 8.64 (d, J ) 13.4 Hz, 1H), 10.56 (dt, J ) 12.8, 6.0, 1H),
11.16 (s, 1H); Anal. (C17H14IN3O4) C, H, N.
4-{[(4-Hydroxy-5-propoxy-pyridin-2-ylmethyl)-amino]-methyl-
ene}-6-iodo-4H-isoquinoline-1,3-dione (100b). Using the procedure
described for the preparation of 89b, 4e (247 mg, 0.75 mmol) and
2-aminomethyl-5-propoxy-pyridin-4-ol (74b) (137 mg, 0.75 mmol)
were reacted to give 313 mg (87%) of 100b as a pink solid: MS
4-{[(6-Hydroxy-5-propoxy-pyridin-2-ylmethyl)-amino]-methyl-
ene}-6-iodo-4H-isoquinoline-1,3-dione (92). Using the procedure
described for the preparation of 89b, 4e (165 mg, 0.50 mmol) and
6-aminomethyl-3-propoxy-pyridin-2-ol (88) (91 mg, 0.50 mmol)
were reacted to give 152 mg (63%) of 92 as a beige solid: mp
1
(ESI) m/z 479.9 (M + 1)+1; H NMR (400 MHz, DMSO-d6) δ
1.01 (t, J ) 7.3 Hz, 3H), 1.74 - 1.84 (m, 2H), 4.10 (t, J ) 6.4 Hz,
2H), 4.86 (d, J ) 6.8 Hz, 2H), 7.22 (s, 1H), 7.54 (dd, J ) 8.4, 1.2
Hz, 1H), 7.72 (d, J ) 8.4 Hz, 1H), 8.20 (d, J ) 1.2 Hz, 1H), 8.61
(d, J ) 12.9 Hz, 1H), 10.54 (dt, J ) 12.9, 6.8 Hz, 1H), 11.16 (s,
1H); Anal. (C19H18IN3O4 ·0.2TFA) C, H, N.
1
275-81 °C (dec); MS (ESI) m/z 478.1 (M + 1)+1; H NMR (400
MHz, DMSO-d6) δ 0.95 (t, J ) 7.4 Hz, 3H), 1.64 - 1.76 (m, 2H),
3.81 (t, J ) 6.6 Hz, 2H), 4.41 (d, J ) 6.1 Hz, 2H), 6.11 (d, J ) 6.8
Hz, 1H), 6.79 (d, J ) 7.3 Hz, 1H), 7.50 (d, J ) 8.1 Hz, 1H), 7.68
(d, J ) 8.31 Hz, 1H), 8.19 (s, 1H), 8.57 (d, J ) 13.3 Hz, 1H),
10.52 (dt, J ) 13.0, 6.1 Hz,, 1H), 11.09 (s, 1H), 11.84 (s, 1H);
Anal. (C19H18IN3O4 ·0.3H2O) C, H, N.
6-tert-Butyl-4-{[(4-hydroxy-5-propoxy-pyridin-2-ylmethyl)-amino]-
methylene}-4H-isoquinoline-1,3-dione (100c). Using the procedure
described for the preparation of 89b, (4E)-6-tert-butyl-4-(meth-
oxymethylene)isoquinoline-1,3(2H,4H)-dione (4k) (50.0 mg, 0.193
mmol) and 2-aminomethyl-5-propoxy-pyridin-4-ol (74b) (35 mg,
0.193 mmol) were reacted to give 52 mg (66%) of 100c as a light
6-Bromo-4-{[(5-methoxy-4-oxo-4H-pyran-2-ylmethyl)-amino]-
methylene}-4H-isoquinoline-1,3-dione (93). Using the procedure
described for the preparation of 89b, 4d (212 mg, 0.75 mmol) and
2-aminomethyl-5-methoxy-pyran-4-one30 (70a) (116 mg, 0.75
mmol) were reacted to give 152 mg (50%) of 93 as a light pink
solid: mp 259-61 °C (dec); MS (ESI) m/z 403 (M - 1)-1; 1H NMR
(400 MHz, DMSO-d6) δ 3.65 (s, 3H), 4.59 (d, J ) 6.3 Hz, 2H),
6.29 (s, 1H), 7.34 (dd, J ) 8.5, 1.7 Hz, 1H), 7.88 (d, J ) 8.6 Hz,
1H), 8.05 (d, J ) 1.7 Hz, 1H), 8.17 (s, 1H), 8.61 (d, J ) 13.2 Hz,
1H), 10.47 (dt, J ) 13.2, 6.3 Hz, 1H), 11.15 (s, 1H); Anal. (C17H13
BrN2O5 ·0.1TFA) C, N. H: calcd, 3.18; found, 2.67.
(4Z)-6-Bromo-4-({[(2-oxo-1,2-dihydropyridin-4-yl)methyl]amino}methy-
lene)isoquinoline-1,3(2H,4H)-dione (94a). Using the procedure
described for the preparation of 5e, 4d (0.26 g, 0.94 mmol) and
4-aminomethyl-1H-pyridin-2-one hydrochloride (55) (0.15 g, 0.94
mmol) were reacted to give 0.19 g (54%) of 94a as a gray powder:
pink solid: mp 162-178 °C (dec); MS (ESI) m/z 410.4 (M + 1)+1
;
1H NMR (400 MHz, DMSO-d6) δ 1.02 (t, J ) 7.4 Hz, 3H), 1.37
(s, 9H), 1.75 - 1.87 (m, 2H), 4.12 (t, J ) 6.6 Hz, 2H), 4.95 (d, J
) 6.1 Hz, 2H), 7.26 (s, 1H), 7.28 (dd, J ) 8.5, 1.51 Hz, 1H), 7.76
(bs, 1H), 7.94 (d, J ) 8.5 Hz, 1H), 8.40 (s, 1H), 8.59 (d, J ) 12.9
Hz, 1H), 10.60 (dt, J ) 12.9, 5.9 Hz, 1H), 11.02 (s, 1H); Anal.
(C23H27N3O4 ·1.0H2O) C, H, N.
6-Cyclopentyl-4-{[(4-hydroxy-5-propoxy-pyridin-2-ylmethyl)-
amino]-methylene}-4H-isoquinoline-1,3-dione (100d). Using the
procedure described for the preparation of 89b, 6-cyclopentyl-4-
methoxymethylene-4H-isoquinoline-1,3-dione (4l) (110 mg, 0.405
mmol) and 2-aminomethyl-5-propoxy-pyridin-4-ol (74b) (74 mg,
0.405 mmol) were reacted to give 162 mg (94%) of 100d as a
pink solid: mp 263-6 °C (dec); MS (ESI): m/z 422.2 (M + 1)+1
;
1H NMR (400 MHz, DMSO-d6) δ 0.99 (t, J ) 7.4 Hz, 3H), 1.56

2308 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
- 1.72 (m, 4H), 1.72 - 1.87 (m, 4H), 1.98 - 2.10 (m, 2H), 2.96
- 3.08 (m, 1H), 4.09 (t, J ) 6.5 Hz, 2H), 4.87 (d, J ) 6.3 Hz,
2H), 7.12 (d, J ) 8.0 Hz, 1H), 7.20 (bd, J ) 1.5 Hz, 1H), 7.63 (s,
1H), 7.92 (d, J ) 8.0 Hz, 1H), 8.39 (s, 1H), 8.56 (d, J ) 12.8 Hz,
1H), 10.49 (dt, J ) 12.8, 6.3 Hz, 1H), 10.99 (s, 1H); Anal.
(C24H27N3O4 ·1.0H2O) C, H, N.
0.1% Tween-20 and 5% nonfat dry milk). Phoshporylation of the
substrate was detected using phospho-Rb specific antibodies (ser-
795) (Cell Signaling Technologies) and antirabbit IgG/horseradish
peroxidase conjugates (Amersham Life Science) using TMB as
substrate. Colorimetric reactions were stopped with 2 N sulfuric
acid, and the absorbance was measured at 450 nm. IC50 values were
determined from inhibition plots.
Cell Culture and Proliferation Assay. HCT-116, LoVo, BT-474,
and MCF-7 cells were cultured in RPMI medium (Invitrogen)
supplemented with 10% fetal bovine serum (Invitrogen) and 50
µg/mL gentamicin (Invitrogen) at 37 °C in a humidified incubator
under 5-7% CO2. Cells were seeded in RPMI medium supple-
mented with 5% fetal bovine serum in 96 well plates at a density
of 5000 to 10000 cells per well depending on the cell line. The
following day, 11 point serial dilutions of the test compound (3-
fold increments) were added to each well. Cells were incubated
with the compound for 3 days (6 days for BT-474) at 37 °C and
cell growth was determined using sulforhodamine B (SRB), a
protein binding dye. Briefly, the surviving cells were fixed with
TCA, and rinsed extensively in water. Cells were stained with 0.4%
sulforhodamine B (Sigma-Aldrich) and washed in 1% acetic acid.
Protein-associated dye was solubilized in 10 mM Tris, and the
absorbance was measured in a Victor fluorescence reader (Wallac/
Perkin-Elmer Life Sciences, Boston, MA).
Preparation of Cell Extracts and Protein Immunoblotting. Cell
lysates were prepared in lysis buffer (25 mM Tris-Cl, pH 7.5, 150
mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium
deoxycholate) supplemented with 0.2 mM PMSF, 1 mM DTT, 0.2
mM sodium fluoride, and 1 mM sodium vanadate (all chemical
reagents were from Sigma-Aldrich, St Louis, MO). Protein con-
centrations were determined by the BioRad protein assay (BioRad,
Hercules, CA). Proteins (10-20 µg) were separated by electro-
phoresis on 8% polyacrylamide-SDS gels (SDS-PAGE) and
transferred to nitrocellulose. Blots were blocked in phosphate
buffered saline (PBS) or tris-buffered saline (TBS) containing 5%
skim milk (or 5% BSA) and 0.1% Tween-20 and incubated with
antibodies in the same buffer. Blots were developed using enhanced
chemiluminescence (ECL, Amersham/GE Healthcare, Piscataway,
NJ). The antibodies used were RB (Santa Cruz Biotechnologies,
Santa Cruz, CA or EMD Biosciences, Darmstadt, Germany) and
phospho-RB (Ser 807/811) (Cell Signaling Technologies, Beverly,
MA).
Metabolic Stability Assays. Substrates were initially dissolved
in HPLC grade DMSO to prepare a 0.5 mM solution, which was
subsequently added to 4 equivalent volumes of acetonitrile. The
resulting 0.1 mM substrate solution in DMSO/acetonitrile was added
to 0.1 M potassium phosphate buffer at pH 7.4 containing 0.5 mg/
mL male SD rat liver microsome protein (BD Gentest). Samples
were preincubated at 37 °C for 10 min and diluted to 1 µM by the
addition of 1 mM NADPH regeneration solution (BD Gentest) for
phase I metabolic stability screening or 1 mM NADPH regeneration
solution containing 2 mM UDPGA (BD Gentest) for phase I and
II metabolic stability screening. The microsomal solution was
incubated at 37 °C for 15 min followed by the addition of 2
equivalent volumes of cold acetonitrile to quench the enzyme
reaction. Nonincubated samples were prepared by adding cold
acetonitrile before adding the regeneration solutions without incuba-
tion. Samples were analyzed by HPLC-MS/MS with a Waters
Quattro Micro mass spectrometer coupled to an Agilent 1100
HPLC. The Agilent 1100 HPLC was equipped with an Aquasil
C18 2.1 × 50 mm, 5 µm column. Samples were injected with a
LEAP Technologies HTS PAL autosampler at 15 µL. The mobile
phase flow rate was 1.0 mL/min with a 3:1 post column split. The
HPLC mobile phase gradient was initiated at 5% eluent B, held at
5% eluent B for 0.1 min, followed by a linear increase to 95%
eluent B over 0.5 min, held at 95% eluent B for 0.4 min, and
reconditioned at 5% eluent B for 0.5 min. Eluent A was 0.1% formic
acid in water and eluent B was 0.1% formic acid in acetonitrile.
HPLC column temperature was maintained at 40 °C. Compounds
were detected by HPLC-MS/MS using multiple reaction monitoring
(MRM) transitions. Automated determination of MRM transitions
6-tert-Butyl-4-{[(4-hydroxy-5-phenyl-pyridin-2-ylmethyl)-amino]-
methylene}-4H-isoquinoline-1,3-dione (100e). Using the procedure
described for the preparation of 89b, 4k (40.0 mg, 0.154 mmol)
and 2-aminomethyl-5-phenyl-pyridin-4-ol (81a) (31 mg, 0.154
mmol) were reacted to give 44 mg (66%) of 100e as a pink solid:
1
mp 195-218 °C (dec); MS (ESI) m/z 428.4 (M + 1)+1; H NMR
(400 MHz, DMSO-d6) δ 1.36 (s, 9H), 5.01 (d, J ) 6.3 Hz, 2H),
7.26 - 7.30 (m, 2H), 7.45 - 7.56 (m, 3H), 7.65 (d, J ) 6.8 Hz,
2H), 7.75 (s, 1H), 7.95 (d, J ) 8.3 Hz, 1H), 8.63 (d, J ) 12.8 Hz,
1H), 8.76 (s, 1H), 10.60 (dt, J ) 12.7, 6.3 Hz, 1H), 11.04 (s, 1H);
Anal. (C26H25N3O3 ·1.0H2O) C, H, N.
(4Z)-{[(5-Furan-3-yl-4-hydroxy-pyridin-2-ylmethyl)-amino]-me-
thylene}-6-iodo-4H-isoquinoline-1,3-dione (100f). Using the pro-
cedure described for the preparation of 89b, 4e (110 mg (0.33
mmol) and 2-aminomethyl-5-furan-3-yl-pyridin-4-ol (81b) (64 mg,
0.33 mmol) were reacted to give 109 mg (67%) of 100f as a light
beige solid: mp 213-228 °C (dec): MS (ESI) m/z 488.0 (M + 1)+1
;
1H NMR (400 MHz, DMSO-d6) δ 4.87 (d, J ) 6.2 Hz, 2H), 7.11
(s, 1H), 7.18 (d, J ) 1.3 Hz, 1H), 7.54 (dd, J ) 8.3, 1.3 Hz, 1H),
7.71 (d, J ) 8.3 Hz, 1H), 7.84 (t, J ) 1.8 Hz, 1H), 7.95 (s, 1H),
8.21 (d, J ) 1.1 Hz, 1H), 8.39 (s, 1H), 8.63 (d, J ) 13.1 Hz, 1H),
8.82 (s, 1H), 10.56 (dt, J ) 13.1, 6.2 Hz, 1H), 11.18 (s, 1H); Anal.
(C20H14IN3O4 ·1.5H2O) C, H, N.
6-tert-Butyl-4-{[(5-furan-3-yl-4-hydroxy-pyridin-2-ylmethyl)-
amino]-methylene}-4H-isoquinoline-1,3-dione (100g). Using the
procedure described for the preparation of 89b, 4k (40.0 mg, 0.154
mmol) and 2-aminomethyl-5-furan-3-yl-pyridin-4-ol (81b) (29 mg,
0.154 mmol) were reacted to give 41 mg (64%) of 100g as a pink
1
solid: mp 215-230 °C (dec); MS (ESI) m/z 418.3 (M + 1)+1; H
NMR (400 MHz, DMSO-d6) δ 1.36 (s, 9H), 5.01 (d, J ) 6.1 Hz,
2H), 7.23 (s, 1H), 7.26 - 7.34 (m, 2H), 7.76 (s, 1H), 7.85 (s, 1H),
7.96 (d, J ) 8.3 Hz, 1H), 8.39 (s, 1H), 8.63 (d, J ) 12.8 Hz, 1H),
8.97 (s, 1H), 10.60 (dt, J ) 12.5, 6.4 Hz, 1H), 11.04 (s, 1H); Anal.
(C24H23N3O4 ·0.3TFA) C, H, N.
6-Cyclopentyl-4-{[(5-furan-3-yl-4-hydroxy-pyridin-2-ylmethyl)-
amino]-methylene}-4H-isoquinoline-1,3-dione (100h). Using the
procedure described for the preparation of 89b, 4l (110 mg, 0.405
mmol) and 2-aminomethyl-5-furan-3-yl-pyridin-4-ol (81b) (77 mg,
0.405 mmol) were reacted to give 152 mg (87%) of 100h as a
pink solid: mp 279-283 °C (dec); MS (ESI) m/z 430.1 (M + 1)+1
;
1H NMR (400 MHz, DMSO-d6) δ 1.55 - 1.74 (m, 4H), 1.74 -
1.88 (m, 2H), 1.98 - 2.10 (m, 2H), 2.96 - 3.09 (m, 1H), 4.95 (d,
J ) 6.3 Hz, 2H), 7.13 (d, J ) 8.6 Hz, 1H), 7.21 - 7.24 (m, 1H),
7.24 - 7.29 (bm, 1H), 7.65 (s, 1H), 7.86 (d, J ) 1.5 Hz, 1H), 7.93
(d, J ) 8.0 Hz, 1H), 8.39 (s, 1H), 8.59 (d, J ) 13.2 Hz, 1H), 8.95
(s, 1H), 10.53 (dt, J ) 13.2, 6.3 Hz, 1H), 11.02 (s, 1H); Anal.
(C25H23N3O4 ·0.5H2O) C, H, N.
Biological Methods. Enzyme Assay. Cyclin D1/CDK4 and cyclin
E/CDK2 were expressed in insect cells (Sf9) infected with
recombinant baculovirus and partially purified using ammonium
sulfate fractionation. Cyclin B1/CDC2 was purchased from New
England BioLabs (Beverly, MA). Test compounds were diluted in
20% DMSO/20 mM HEPES, pH 7.5, and serial dilutions were
prepared (5 concentrations; 0.005-50 µM). High-binding ELISA
microtiter plates (Costar) were coated with the kinase substrate
(glutathione-S-transferase (GST) fusion of C-terminal fragment of
the retinoblastoma susceptibility gene product (Rb)). Nonspecific
binding sites were blocked with Superblock in Tris-buffered saline
(TBS; Pierce). Kinase reactions contained the test inhibitor, 200
µM ATP, 0.5 mg/mL bovine serum albumin (BSA; Sigma), and
0.1 µL of enzyme. Reaction volumes were adjusted to 30 µL with
kinase assay buffer (50 mM HEPES, pH 7.5, 10 mM MgCl 2, 5%
glycerol, 10 mM 2-mercaptoethanol), and plates were incubated at
30 °C for 1 h. Reactions were terminated by aspiration, and
nonspecific sites were blocked with blocking buffer (TBS containing

Potent and SelectiVe Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2309
activity in human tumor xenografts. Mol. Cancer Ther. 2004, 3,
1427–1438.
(18) Kubo, A.; Nakagawa, K.; Varma, R. K.; Conrad, N. K.; Cheng, J. Q.;
lee, W.-C.; Testa, J. R.; Johnson, B. E.; Kaye, F. E.; Kelley, M. J.
The p16 status of tumor cell lines identifies small molecule inhibitors
specific for cyclin-dependent kinase 4. Clin. Cancer Res. 1999, 5,
4279–4286.
were generated using Waters QuanOptimize software. The HPLC-
MS/MS substrate peak area at 15 min was compared to the
nonincubated substrate providing a relative percent remaining from
which an experimental half-life could be calculated assuming
pseudo-first-order reaction rate kinetics.
Acknowledgment. We thank Drs. John Ellingboe and Philip
Frost for their support and encouragement. We are grateful to
Dr. Ping-Zhong Huang for scaling up key intermediates. We
also would like to thank members of the Wyeth Chemical
Technologies group for analytical, spectral, and metabolic
stability determinations.
(19) Soni, R.; O’Reilly, T.; Furet, P.; Muller, L.; Stephan, C.; Zumstein-
Mecker, S.; Fretz, H.; Fabbro, D.; Chaudhuri, B. Selective in vivo
and in vitro effects of a small molecule inhibitor of cyclin-dependent
kinase 4. J. Natl. Cancer Inst. 2001, 93, 436–446.
(20) Ikuta, M.; Kamata, K.; Fukasawa, K.; Honma, T.; Machida, T.; Hirai,
H.; Suzuki-Takahashi, I.; Hayama, T.; Nishimura, S. Crystallographic
approcach to identification of cyclin-dependent kinase 4 (CDK4)-
specific inhibitors by using CDK4 mimic CDK2 protein. J. Biol. Chem.
2001, 276, 27548–27554.
(21) Grell, W.; Walter, R.; Heckel, A.; Himmelsbach, F.; Wittneben, H.;
van Meel, J.; Redemann, N.; Haigh, R. Preparation of 3-(R-Heteroary-
laminobenzylidene)-2-indolinones as Cyclin Dependent Kinase Inhibi-
tors. DE 19815020 A1 19991007, 1999.
Supporting Information Available: Elementary analysis data
for compounds 3c-3e, 5a-5i, 11, 13, 17a-17d, 18, 21, 89a-89h,
91-93, 94a-94c, and 100a-100 h. This material is available free
of charge via the Internet at http://pubs.acs.org.
(22) Tsou, H. R.; Otteng, M.; Tran, T.; Floyd, M. B.; Reich, M.; Birnberg,
G.; Kutterer, K.; Ayral-Kaloustian, S.; Ravi, M.; Nilakantan, R.; Grillo,
M.; McGinnis, J. P.; Rabindran, S. K. 4-(Phenylaminomethylene)iso-
quinoline-1,3(2H,4H)-diones as potent and selective inhibitors of the
cyclin-dependent kinase 4 (CDK4). J. Med. Chem. 2008, 51, 3507–
3525.
(23) De Lombaert, S.; Fink, C. A.; Firoozina, F.; Hoyer, D. W. Certain
Thiol inhibitors of Endothelin-Converting Enzyme. PCT Int. Appl.
WO 9955726 A1 19991104, 1999.
(24) (a) Sagi, K.; Nakagawa, T.; Yamanashi, M.; Makino, S.; Takahashi,
M.; Takayanagi, M.; Takenaka, K.; Suzuki, N.; Ono, S.; Kataoka, N.;
Ishikawa, K.; Shima, S.; Fukuda, Y.; Kayahara, T.; Takehana, S.;
Shima, Y.; Tashiro, K.; Yamamoto, H.; Yoshimoto, R.; Iwata, S.; Tsuji,
T.; Sakurai, K.; Shoji, M. Rational design, synthesis, and structure-
activity relationships of novel factor Xa inhibitors: (2-Substituted-4-
amidinophenyl)pyruvic and -propionic acid. J. Med. Chem. 2003, 46,
1845–1857. (b) Imanishi, N.; Iwaoka, K.; Koshio, H.; Nagashima, S.-
y.; Kazuta, K.-i.; Ohta, M.; Sakamoto, S.; Ito, H.; Akuzawa, S.; Kiso,
T.; Tsukamoto, S.-i.; Mase, T. New thiazole derivatives as potent and
selective 5-hydroxytriptamine 3 (5-HT3) receptor agonists for the
treatment of constipation. Bioorg. Med. Chem. 2003, 11, 1493–1502.
(25) Singh, B.; Lesher, G. Y. Synthesis of aza analogs of Amrinone.
Hereocycles 1990, 31, 2163–2172.
(26) Brown, T. H.; Blakemore, R. C.; Durant, G. J.; Ganellin, C. R.;
Parsons, M. E.; Rasmussen, A. C.,zprint>; DA ]?>; Rawlings, D. A.
Isocytosine H2-receptor histamine antagonists. IV. The synthesis and
biological activity of donetidine (SK&F 93574) and related com-
pounds. Eur. J. Med. Chem. 1993, 28, 601–608.
(27) Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.; Campbell, E. A.;
Dray, A.; James, I. F.; Perkins, M. N.; Reid, D. J.; Winter, J. Analogs
of capsaicin with agonist activity as novel analgesic agents; structure-
activity studies. 1. The aromatic “A-region”. J. Med. Chem. 1993, 36
(16), 2362–2372.
(28) Sugahara, M.; Ukita, T. A facile copper-catalyzed Ullmann condensa-
tion: N-Arylation of heterocyclic compounds containing an -NHCO-
moiety. Chem. Pharm. Bull. 1997, 45 (4), 719–721.
(29) Campbell, K. N.; Ackerman, J. F.; Campbell, B. K. Studies on g-Pyrones.
I. Derivatives of Kojic Acid. J. Org. Chem. 1950, 15, 221–6.
(30) Cizmaricova, Ruzena; Miskikova, E.; Gregan, F. Derivatives of kojic
acid. Pharmazie. 1991, 46 (6), 460.
References
(1) Massague, J. G1 cell cycle and cancer. Nature 2004, 432, 298–306.
(2) Sherr, C. J.; Roberts, J. M. CDK inhibitors: Positive and negative
regulators of G1 progression. Genes DeV. 1999, 13, 1501–1512.
(3) Harbour, J. W.; Luo, R. X.; Dei Santi, A.; Postigo, A. A.; Dean, D. C.
Cdk phosphorylation triggers sequential intramolecular interactions
that progressively block Rb functions as cells move through G1. Cell
1999, 98, 859–869.
(4) Dyson, N. The regulation of E2F by pRB -family proteins. Gene DeV.
1998, 12, 2245–2262.
(5) Hanahan, D.; Weiberg, R. A. The hallmarks of cancer. Cell 2000,
100, 57–70.
(6) Hall, M.; Peters, G. Genetic alterations of cyclins, cyclin-dependent
kinases, and cdk inhibitors in human cancer. AdV. Cancer Res. 1996,
68, 67–108.
(7) Michalides, R. J. A. M. Cell cycle regulators: mechanisms and their
role in aetiology, prognosis, and treatment of cancer. J. Clin. Pathol.
1999, 52, 555–568.
(8) Grillo, M.; Bott, M. J.; Khandke, N.; McGinnis, J. P.; Miranda, M.;
Meyyappan, M.; Rosfjord, E. C.; Rabindran, S. K. Validation of cyclin
D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells:
Effect of regulated overexpression of cyclin D1 and siRNA-mediated
inhibition of endogenous cyclin D1 and CDK4 expression. Breast
Cancer Res. Treat. 2006, 95, 185–194.
(9) Tam, S. W.; Theodoras, A. M.; Shay, J. W.; Draetta, G. F.; Pagano,
M. Differential expression and regulation of cyclin D1 protein in
normal and tumor human cells: association with Cdk4 is required for
cyclin D1 function in G1 progression. Oncogene 1994, 9, 2663–2674.
(10) Arber, N.; Doki, Y.; Han, E. K.-H.; Sgambato, A.; Zhou, P.; Kim,
N.-H.; Delohery, T.; Klein, M. G.; Holt, P. R.; Weinstein, I. B.
Antisense to cyclin D1 inhibits the growth and tumorigenicity of
human colon cancer cells. Cancer Res. 1997, 57, 1569–1574.
(11) Bartkova, J.; Lukas, J.; Muller, H.; Lutzhoft, D.; Strauss, M.; Bartek,
K. Cyclin D1 protein expression and function in human breast cancer.
Int. J. Cancer 1994, 57, 353–361.
(12) Uto, H.; Ido, A.; Moriuchi, A.; Onaga, Y.; Nagata, K.; Onaga, M.;
Tahara, Y.; Hori, T.; Hirono, S.; Hayashi, K.; Tsubouchi, H.
Transduction of antisense cyclin D1 using two-step gene transfer
inhibits the growth of rat hepatoma cells. Cancer Res. 2001, 61, 4779–
4783.
(13) Yu, Q.; Geng, Y.; Sicinski, P. Specific protection against breast cancers
by cyclin D1 ablation. Nature 2001, 411, 1017–1021.
(14) Yu, Q.; Sicinska, E.; Geng, Y.; Ahnstrom, M.; Zagozdzon, A.; Kong,
Y.; Gardner, H.; Kiyokawa, H.; Harris, L. N.; Stal, O.; Sicinski, P.
Requirement for CDK4 kinase function in breast cancer. Cancer Cell
2006, 9, 23–32.
(15) Landis, M. W.; Pawlyk, B. S.; Li, T.; Sicinski, P.; Hinds, P. W. Cyclin
D1-dependent kinase activity in murine development and mammary
tumorigenesis. Cancer Cell 2006, 9, 13–22.
(16) (a) Malumbres, M.; Pevarello, P.; Barbacid, M.; Bischoff, J. R. CDK
inhibitors in cancer therapy: What is next. Trends Pharmacol. Sci.
2007, 29, 16–21. (b) Byrdt, J. C.; Thomas S. Lin, T. S.; Dalton, J. T.;
Wu, D.; Phelps, M. A.; Fischer, B.; Moran, M.; Blum, K. A.; Rovin,
B.; Brooker-McEldowney, M.; Broering, S.; Schaaf, L. J.; Johnson,
A. J.; Lucas, D. M.; Heerema, N. A.; Lozanski, G.; Young, D. C.;
Suarez, J.-R.; Colevas, A. D.; Grever, M. R. Flavopiridol administered
using a pharmacologically derived schedule is associated with marked
clinical efficacy in refractory, genetically high-risk chronic lymphocytic
leukemia. Blood 2007, 109, 399–404.
(31) Kamino, T.; Kuramochi, K.; Kobayashi, S. A concise approach to
5-substituted-4-pyrones from kojic acid. Tet. Lett. 2003, 44, 7349–
7351.
(32) Wishka, D. G.; Graber, D. R.; Seest, E. P.; Dolak, L. A.; Han, F.;
William Watt, W.; Joel Morris, J. Stereoselective synthesis of furo[2,3-
c]pyridine pyrimidine thioethers, a new class of potent HIV-1 non-
nucleoside reverse transcriptase inhibitors. J. Org. Chem. 1998, 63
(22), 7851–7859.
(33) Nightingale, D.; Janes, J. R. The Alkylation of o- and p-Xylene.
J. Amer. Chem, Soc. 1944, 66, 154–155.
(34) (a) Markwalder, J. A.; Arnone, M. R.; Benfield, P. A.; Boisclair, M.;
Burton, C. R.; Chang, C.-H.; Cox, S. S.; Czerniak, P. M.; Dean, C. L.;
Doleniak, D.; Grafstrom, R.; Harrison, B. A.; Kaltenbach, III, R. F.;
Nugiel, D. A.; Rossi, K. A.; Sherk, S. R.; Sisk, L. M.; Stouten, P.;
Trainor, G. L.; Worland, P.; Seitz, S. P. Synthesis and biological
evaluation of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one
inhibitors of cyclin-dependent kinases. J. Med. Chem. 2004, 47, 5894–
5911. (b) Rossi, K. A.; Markwalder, J. A.; Seitz, S. P.; Chang, C.-H.;
Cox, S.; Boisclair, M. D.; Brizuela, L.; Brenner, S. L.; Stouten, P. F. W.
Understanding and modulating cyclin-dependent kinase inhibitor
specificity: molecular modeling and biochemical evaluation of pyra-
zolopyrimidinones as CDK2/cyclin A and CDK4/cyclin D1 inhibitors.
J. Comput.-Aided Mol. Des. 2005, 19, 111–122. (c) Shangari, N.; Chan,
(17) Fry, D. W.; Harvey, P. J.; Keller, P. R. Specific inhibition of cyclin-
dependent kinase 4/6 by PD0332991 and associated antitumor

2310 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Tsou et al.
T. S.; O’Brien, P. J. Sulfation and glucuronidation of phenols:
Implications in coenyzme Q metabolism. Methods Enzymol. 2005, 400,
342–259.
design of selective ATP competitive inhibitors. Chem. Biol. 2004, 11,
525–534.
(42) Honma, T.; Hayashi, K.; Aoyama, T.; Hashimoto, N.; Machida, T.;
Fukasawa, K.; Iwama, T.; Ikeura, C.; Iduta, M.; Suzuki-Takahashi,
I.; Iwasawa, Y.; Hayama, T.; Nishimura, S.; Morishima, H. Structure-
based generation of a new class of potent cdk4 inhibitors: New de
novo design strategy and library design. J. Med. Chem. 2001, 44, 4615–
4627.
(43) Aubry, C.; Wilson, A. J.; Jenkins, P. R.; Mahale, S.; Chaudhuri, B.;
Marechal, J.; Sutcliffe, M. J. Design, synthesis and biological activity
of new CDK4-specific inhibitors, based on fascaplysin. Org. Biomol.
Chem. 2006, 4, 787–801.
(44) Pratt, D. J.; Bentley, J.; Jewsbury, F.; Boyle, T.; Endicott, J. A.; Noble,
M. E. M. Dissecting the determinants of cyclin-dependent kinase 2
and cyclin-dependent kinase 4 inhibitor selectivity. J. Med. Chem.
2006, 49, 5470–5477.
(45) Park, H.; Yeom, M. S.; Lee, S. Loop flexibility and solvent dynamics
as determinants for the selective inhibition of cyclin-dependent kinase
4: Comparative molecular dynamics simulation studies of CDK2 and
CDK4. ChemBioChem. 2004, 5, 1662–1672.
(46) SYBYL, version 7.2; Tripos, Inc.: St. Louis, MO, 2006.
(47) Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee,
R. P. Empirical scoring functions: I. The development of a fast
empirical scoring function to estimate the binding affinity of ligands
in receptor complexes. J. Comput.-Aided Mol. Des. 1997, 11, 425–
445.
(48) Jorgensen, W. L.; Maxwell, D. S.; Triado-Rives, J. Development and
testing of the OPLS all-atom force field on conformational energetics
and properties of organic liquids. J. Am. Chem. Soc. 1996, 118 (45),
11225–11236.
(35) Kim, K. H. Quantitative structure-activity relationships of the me-
tabolism of drugs by uridine diphosphate glucuronosyltransferase. J.
Phamacol. Sci. 1991, 80 (10), 966–970.
(36) Yin, H.; Bennett, G.; Jones, J. P. Mechanistic studies of uridine
diphosphate glucuronosyltransferase. Chem.-Biol. Interact. 1994, 90,
47–58.
(37) (a) Katritzky, A. R.; Lagowski, J. M. Prototropic tautomerism of
heteroaromatic compounds: I. General discussion and methods of
study. AdV. Heterocyclic Chem. 1963, 1, 311–38. (b) Wang, J.; Boyd,
R. J. Tautomeric equilibria of hydroxypyridines in different solvents:
An ab initio study. J. Phys. Chem. 1996, 100, 16141–16146.
(38) De Kowaleski, D. G.; Contreras, R. H.; diez, E.; Esteban, A. NMR
J(C, C) scalar coupling analysis of the effects of substituents on the
keto-enol tautomeric equilibrium in 2-OH-n-X-pyridines. An experi-
mental and DFT study. Mol. Phys. 2004, 23-24, 2607–2615.
(39) GLIDE; Schrodinger Software: Portland, OR (www.schrodinger.com).
(40) (a) Thompson, S. K.; Murthy, K. H. M.; Zhao, B.; Winborne, E.;
Green, D. W.; Fisher, S. M.; DesJarlais, R. L.; Thomszek, T. A., Jr.;
Meek, T. D.; Gleason, J. G.; Abdel-Meguid, S. S. Rational design,
syntheis, and crystallographic analysis of a hydroxyethylene-based
HIV-1 protease inhibitor containing a heterocyclic P1′-P2′ amide bond
isostere. J. Med. Chem. 1994, 37, 3100–3107. (b) Noguchi, T.; Inoue,
Y.; Tang, X.-S. Hydrogen bonding interaction between the primary
quinone acceptor QA and a histidine side chain in photosystem II as
revealed by fourier transform infrared spectroscopy. Biochemistry
1999, 38, 399–403. (c) Edgcomb, S. P.; Murphy, K. P. Variability in
the pKa of histidine side-chains correlates with burial within proteins.
Proteins: Struct., Funct., Bioinf. 2002, 49, 1–6.
(41) McInnes, C.; Wang, S.; Anderson, S.; O’Boyle, J.; Jackson, W.;
Kontopidis, G.; Meades, C.; Mezna, M.; Thomas, M.; Wood, G.; Lane,
D. P.; Fischer, P. M. Structural determinants of CDK4 inhibition and
JM801026E