Total synthesis and protein kinase activity of C(7) methyl derivatives of K252a

Article abstract of DOI:10.1055/s-1999-3652

Recent efforts in these laboratories have resulted in the development of a synthetic approach to C(7) alkyl analogs in the K252a class of indolocarbazole natural products. Synthesis of 7- (R)-methyl K252a (3a) and 7-(S)-methyl K252a (3b) will be described along with their activity against protein tyrosine kinases.

Full text of DOI:10.1055/s-1999-3652

PAPER
1529
Total Synthesis and Protein Kinase Activity of C(7) Methyl Derivatives of
K252a
John L. Wood,a* Dejah T. Petsch,a Brian M. Stoltz,a Elizabeth M. Hawkins,a Daniel Elbaum,b David R. Stoverb
aSterling Chemistry Laboratory, Department of Chemistry, Yale University, New Haven, CT 06520–8107, USA
Fax +1(203)4326144; E-mail: john.wood@yale.edu
bKinetix Pharmaceuticals, Inc., 200 Boston Avenue, Suite 3500, Medford, MA 02155, USA
Fax +1(781)3915771; E-mail: elbaum@kinetixpharm.com
Received 24 March 1999; revised 12 May 1999
of the indolocarbazole class of natural products, have at-
tracted great interest throughout the chemical community.
The furanosylated congener K252a was isolated indepen-
dently by Sezaki from Actinomadura sp. SF-2370,3 and by
Kase from Norcardiopsis sp. K-252.4 Kase subsequently
found K252a to inhibit PKC with an IC50 value of 32 nm.
Abstract: Recent efforts in these laboratories have resulted in the
development of a synthetic approach to C(7) alkyl analogs in the
K252a class of indolocarbazole natural products. Synthesis of 7-
(R)-methyl K252a (3a) and 7-(S)-methyl K252a (3b) will be de-
scribed along with their activity against protein tyrosine kinases.
Key words: indolocarbazole, K252a, protein kinase, K252a ana-
logs, synthesis
Considerable effort has been focused on the production of
numerous analogs of 1, however, the lack of efficient syn-
thetic approaches to this class of molecules has limited the
effort to structures that are readily derived from the natu-
ral material. As summarized in Figure 2, the known ana-
logs of K252a have been prepared via single and double
Friedel–Crafts alkylation/acylation, oxidation of the lac-
tam methylene, and acylation of the lactam nitrogen. In
addition, the literature indicates that derivatization of the
carbohydrate moiety has been limited to manipulation of
the resident functionality.
Protein kinases are vital to the signal transduction path-
ways which carry external signals to the nucleus of the
cell. The disruption of cellular signal transduction via pro-
tein kinase (PK) malfunction has been related to the onset
of several disease states, including rheumatoid arthritis,
systemic lupus erythematosis (SLE), diabetes melitus,
Alzheimer’s disease, and cancer.1 While PK inhibition
seems a logical target for chemotherapeutic intervention,
efforts to prepare PK-specific inhibitors based on ATP
have met with limited success due to sequence homology
among the many PK isozymes and throughout the large
kinase superfamily. However, recent structural data sug-
gests that the homology is limited to the triphosphate
binding region, and not the adenosine pocket.2 Thus, high-
ly specific inhibitors which could take advantage of subtle
differences in the nucleoside binding pocket would be in-
valuable to studies of the cellular roles of individual pro-
tein kinases, and in many cases could have significant
medicinal value.
Acylation / Alkylation
Oxidation
H
Friedel-Crafts
Friedel-Crafts
N
O
N
N
O
H3C
MeO2C
Reduction
Acylation
OH
Due to their potent inhibition of protein kinase C (PKC),
K252a (1), and staurosporine (2) (Figure 1), two members
Figure 2 Summary of known K252a analogs
Recently, we devised an efficient synthesis of K252a that
enables functionalization of the lactam region and is effi-
cient enough to provide realistic access to a variety of an-
alogs that are not available via derivatization of the
natural material.5 Herein we report extension of this syn-
thesis to the production of C(7) methyl derivatives of
K252a (e.g. 3a and 3b, Figure 1).
As outlined in retrosynthetic fashion in Scheme 1, we en-
visioned 3a as arising via cycloglycosidative coupling of
aglycon 4a with our previously prepared carbohydrate
moiety 5. Aglycon 4a was expected to derive from the
coupling of 2,2'-biindole6 (7) with diazolactam 8a. As in
our K252a synthesis, 5 derives from methyl acetoacetate
Figure 1 Indolocarbazole natural products and analogs
Synthesis 1999, No. SI, 1529–1533 ISSN 0039-7881 © Thieme Stuttgart · New York

1530
J. L. Wood et al.
PAPER
(6), and the diazolactam originates from a protected a-
amino acid derivative.
Scheme 2
Scheme 1
In a modification of our previously reported protocol, we
found that equimolar amounts 7 and 8a undergo coupling
in the presence of 1 mol% of Rh2(OAc)4 in pinacolone at
reflux (Scheme 3). Under these conditions, aglycon 4a
was produced in 35% yield (70% based on recovered bi-
indole). Recycling of the recovered biindole has allowed
the ready preparation of multigram quantities of 4a.
In the synthetic sense, D-alanine methyl ester hydrochlo-
ride (9a, Scheme 2) was monoprotected as the dimethox-
ybenzyl (DMB) derivative via reductive amination with
3,4-dimethoxybenzaldehyde (93% yield).7 The protected
amine 10a was then coupled to ethyl hydrogen malonate
under standard coupling conditions (DCC) to yield the cy-
clization precursor 11a in 97% yield. Dieckmann
cyclisation8 of 11a (NaOEt, EtOH, D) furnished the de-
sired lactam which, without further purification, was sub-
jected to decarboethoxylation (CH3CN, H2O, D) to
provide ketone 12 (80% yield, two steps). Diazo transfer
from mesyl azide led to the desired diazolactam 8a in 91%
yield (64% overall from D-alanine methyl ester hydro-
chloride).
Cycloglycosidative coupling of 4a and 5 (CSA, 1,2-
dichloroethane, reflux, 72 h) affords an anticipated 2:1
mixture of regioisomers 13a and 14a (80% yield), which
upon chromatographic separation and deprotection with
TFA provides the desired analog 3a in 85% yield.9 The di-
astereomeric analog 7-(S)-methyl K252a (3b) was simi-
larly prepared using L-alanine methyl ester hydrochloride
(9b) as the point of departure.
Scheme 3
Synthesis 1999, No. SI, 1529–1533 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Total Synthesis and Protein Kinase Activity of C(7) Methyl Derivatives of K252a
1531
Table 1 Activity of 1, 3a, and 3b Against Protein Tyrosine Kinases
using the Bac-to-Bac expression system (Life Technologies, Pais-
ley, UK). The proteins are then purified to near homogeneity by glu-
tathione-affinity chromatography. The kinase is incubated with
[33P]-ATP in a 96-well plate coated with substrate (i.e. poly[Glu,
Tyr]4:1). The kinase activity is then measured by a 96-well scintil-
lation counter (i.e. Microbeta, Wallac or Top-Count, Packard). In-
hibition by compounds is measured by comparing the relative
activity of kinase in the presence and absence of various concentra-
tions of 1, 3a, or 3b. The IC50 represents the concentration where
50% of the phosphorylation activity is inhibited.
m
1
3a
3b
Lck
Fyn
ZAP-70
Syk
Itk
1.25
4.59
0.32
0.04
0.35
5.22
10.71
>10
3.55
2.02
2.70
6.22
4.29
0.19
5.07
Amine 10a
To a solution of D-alanine methyl ester hydrochloride (18.1 g, 130
mmol, 1 equiv.), in MeOH (45 mL) was added Et3N (18.1 mL, 130
mmol, 1 equiv.) followed by addition of 3,4 dimethoxybenzalde-
hyde (15.1 g, 90.8 mmol, 0.7 eq) as a solution in EtOH (230 mL).
To explore the influence of C(7) functionalization we as- The mixture was concentrated under reduced pressure to yield the
crude imine as a white solid. The imine was suspended in EtOH
(310 mL) and NaBH4 (4.9 g, 130 mmol, 1 equiv.) was added in three
to four portions over a 15 minute period. The heterogeneous mix-
ture was stirred for 24 h at r.t. At the end of this period, the EtOH
sayed our compounds against a number of protein tyrosine
kinases (Table). Although 3a was found not to be a very
potent inhibitor, 3b showed IC50 values ranging from high
nM to low mM with the best value being 190 nM against
the kinase Syk. Moreover, compound 3b shows a selectiv-
ity profile different from that of the natural product.
was removed under reduced pressure and the residue was dissolved
in 1 M HCl (200 mL). The acid solution was washed with EtOAc
(100 mL) and then basified to a pH of 10 using 10 M aq NaOH (10
mL). The basic solution was extracted with CH2Cl2 (3 ¥ 100 mL)
and the combined organic layers were dried (MgSO4), filtered, and
concentrated to yield the protected amine 10a (21.3 g, 93%) as a
clear, colorless oil, [a]20D+35.52 (c = 1.00, MeOH).
1H NMR (500 MHz, CDCl3): d = 1.30 (d, J = 7.0 Hz, 3H), 1.81 (br
s, 1H), 3.37 (q, J = 7.0 Hz, 1H), 3.60 (d, J = 12.6 Hz, 1H), 3.72 (m,
4H), 3.84 (s, 3H), 3.87 (s, 3H), 6.78–6.88 (comp m, 3H).
In conclusion, the efficient synthesis of K252a developed
in our laboratory has been extended to allow stereoselec-
tive access to C(7) alkyl derivatives of which 7-(R)-meth-
yl K252a (3a) and 7-(S)-methyl K252a (3b) are
representative. Importantly these studies have shown that
functionalization at the C(7) position does not completely
abrogate kinase activity and can in fact induce changes in
selectivity. Based on these promising preliminary results,
efforts in our laboratories are currently focused on the
preparation of a number of additional C(7) alkyl deriva-
tives of K252a.
13C NMR (125 MHz, CDCl3): d = 18.5, 51.0, 51.1, 55.1, 55.1, 55.2,
110.5, 110.9, 119.7, 131.8, 147.5, 148.4, 175.5.
IR (film): n = 3329 (br w), 2944 (m), 1735 (s), 1513 (s), 1457 (m),
1262 (s), 1199 (s), 1031 (s), 980 (w), 855 cm-1.
HRMS (EI): calcd for C13H19NO4 253.1314; found 253.1315.
Amide 11a
Unless otherwise stated, reactions were performed in flame-dried
glassware under N2, using freshly distilled solvents. Et2O and THF
were distilled from sodium/benzophenone ketyl. CH2Cl2, benzene,
and Et3N were distilled from CaH2. 1,2-dichloroethane and
BF3◊OEt2 were purchased from the Aldrich Chemical Co. in Sure/
Seal containers and used without further purification. All other
commercially obtained reagents were used as received. Unless oth-
erwise stated, all reactions were magnetically stirred and monitored
by thin-layer chromatography (TLC) using E. Merck silica gel 60
F254 pre-coated plates (0.25 mm). Column or flash chromatogra-
phy (silica) was performed with the indicated solvents using silica
gel (particle size 0.032– 0.063 mm) purchased from Fisher Scientif-
ic. In general, the chromatography guidelines reported by Still were
followed. Infrared spectra were recorded on a Midac M-1200 FTIR.
1H and 13C NMR spectra were recorded on a Bruker AM-500 spec-
trometer. Chemical shifts are reported relative to residual solvents
CHCl3 (1H, d 7.27 ppm, 13C, d 77.0 ppm), acetone (1H, d 2.04 ppm,
13C, d 29.9 ppm) or DMSO (1H, d 2.49 ppm, 13C, d 39.5 ppm).
HRMS were performed at The University of Illinois Mass Spec-
trometry Center. High performance liquid chromatography (HPLC)
was performed on a Waters model 510 system using a Rainin Mi-
crosorb 80-199-C5 column, or a Rainin Dynamax SD-200 system
with a Rainin Microsorb 80-120-C5 column. Optical rotations were
measured on a Perkin-Elmer 241 polarimeter.
A flame-dried 500 mL three-necked round bottom flask equipped
with an addition funnel was charged with the amine 10a (25 g, 99
mmol, 1 equiv.), ethyl hydrogen malonate (13.1 g, 99 mmol, 1
equiv.), and CH2Cl2 (400 mL). The flask was cooled to 0 °C and a
solution of 1,3-dicyclohexyl carbodiimide (20.4 g, 99 mmol, 1
equiv.) in CH2Cl2 (160 mL) was added dropwise through the addi-
tion funnel over a 15 min period. The ice bath was removed and the
mixture was stirred for an additional 3 h at r.t. The mixture was fil-
tered to remove the urea byproduct and the filtrate was washed with
H2O (500 mL). The organic layer was separated, and the aqueous
layer was extracted with CH2Cl2 (3 ¥ 100 mL). The combined or-
ganic layers were dried (MgSO4), filtered and concentrated to yield
amide 11a as a clear, light yellow oil (34 g, 94%), [a]20D+39.7 (c =
0.65, MeOH).
1H NMR (500 MHz, CDCl3): d = 1.25 (t, J = 7.1 Hz, 3H), 1.40 (d,
J = 7.2 Hz, 3H), 3.37–3.57 (comp m, 2H), 3.68 (s, 3H), 3.87 (s, 3H),
3.88 (s, 3H), 4.17 (q, J = 7.1 Hz, 2H), 4.38–4.74 (comp m, 3H),
6.76–6.87 (comp m, 3H).
13C NMR (125 MHz, CDCl3): d = 14.0, 14.5, 41.4, 50.6, 52.2, 54.4,
55.8, 61.4, 109.5, 110.9, 111.2, 118.6, 119.6, 128.6, 149.3, 167.0,
167.3, 171.6.
IR (film): n = 2948 (m), 1741 (s), 1654 (s), 1516 (s), 1261 (s), 1149
(s), 1029 (s), 807 (w) cm-1.
Kinase Inhibition Assays
Kinase domains are expressed as Glutathione S-transferase (GST)
fusion proteins in Spodoptera frugiperda (SF9) or High-Five cells,
HRMS (EI): calcd for C18H25NO7 367.1631; found 367.1632.
Synthesis 1999, No. SI, 1529–1533 ISSN 0039-7881 © Thieme Stuttgart · New York

1532
J. L. Wood et al.
PAPER
Lactam 12a
J = 8.3 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 9.53 (d, J = 8.0 Hz, 1H),
A three-necked round bottom flask was charged with EtOH (18
mL). Na metal (293 mg, 12.8 mmol, 0.94 equiv.) was then carefully
added in one portion. When the sodium had completely reacted, es-
ter 11a (5 g, 13.5 mmol, 1 equiv.) was added dropwise to the mix-
ture as a solution in EtOH (17 mL). The mixture was brought to
reflux for 5 min and then allowed to cool to r.t. The EtOH was re-
moved under reduced pressure and the residue was dissolved in H2O
(100 mL). The aqueous layer was washed with EtOAc (50 mL), and
acidified to a pH of 2, with 2M HCl (10 mL). The acidic solution
was extracted with CH2Cl2 (3 ¥ 50 mL). The combined organic lay-
ers were dried (MgSO4), filtered, and concentrated to yield a yellow
oil (3.8 g, 80%). A suspension of this oil in CH3CN (1130 mL) and
H2O (1 mL) was warmed to reflux open to the air for 3 h. The mix-
ture was cooled to r.t. and the CH3CN/H2O mixture was removed at
reduced pressure to yield lactam 12a as a dark yellow oil (2.84 g,
100%) which solidified upon standing, [a]20D+29.1 (c = 0.25,
MeOH).
10.70 (s, 1H), 10.92 (s, 1H).
13C NMR (125 MHz, CDCl3): d = 18.9, 44.0, 56.1, 56.2, 111.9,
112.6, 112.9, 112.9, 115.2, 117.6, 119.5, 120.2, 121.0, 121.1, 122.8,
123.4, 124.5, 126.0, 126.3, 126.8, 126.9, 127.0, 129.3, 132.2, 137.9,
140.9, 140.9, 149.8, 150.6, 170.0.
IR (film): n = 3323 (br w), 2931 (w), 1649 (s), 1514 (s), 1320 (s),
1236 (s), 1231 (s), 1140 (m), 1024 (m) cm-1.
HRMS (EI): calcd for C30H25N3O3 475.1896; found 475.1896.
Indolocarbazoles 13a and 14a
To a refluxing solution of aglycon 4a (700 mg, 1.47 mmol, 1 equiv.)
and camphorsulfonic acid (34 mg, 0.147 mmol, 0.1 equiv.) in 1,2-
dichloroethane (50 mL) was added, via addition funnel over 24h, a
solution of furanose 5 (638 mg, 2.9 mmol, 2 equiv.) in 1,2 dichloro-
ethane (32 mL). Reflux was then continued for an additional 48h.
The crude mixture was washed with a 10% aq NaHCO3 (20 mL).
The aqueous layer was washed with CH2Cl2 (3 ¥ 20 mL) and the
combined organic layers were dried (Na2SO4) and concentrated un-
der reduced pressure. The residue was taken up in a minimum of
CH2Cl2 and purified by flash chromatography (50% EtOAc/hex-
anes eluant) to provide a 2:1 mixture of regioisomers 13a and 14a
(80% yield). Separation of the regioisomers could be achieved us-
ing HPLC (190:10:1 CH2Cl2:EtOAc:MeOH eluent).
1H NMR (500 MHz, CDCl3): d = 1.31 (d, J = 6.9 Hz, 3H), 3.09 (s,
2H), 3.78 (q, J = 6.9 Hz, 1H), 3.85 (s, 3H), 3.86 (s, 3H), 3.97 (d,
J = 14.7 Hz, 1H), 5.17 (d, J = 14.7 Hz, 1H), 6.80 (m, 3H).
13C NMR (125 MHz, CDCl3): d = 15.1, 40.5, 43.6, 55.9, 56.0, 61.7,
111.1, 111.4, 120.7, 127.8, 148.9, 149.4, 168.3, 206.7.
IR (film): n = 2940 (br m), 1766 (m), 1689 (s), 1603 (w), 1512 (m),
1424 (m), 1252 (s), 1147 (m), 916 (w) cm-1.
13a, [a]20D+59 (c = 0.8, MeOH).
HRMS (EI): calcd for C14H17NO4 263.1158; found 263.1160.
1H NMR (500 MHz, acetone-d6): d = 1.78 (d, J = 6.5 Hz, 3H), 2.23
(dd, J = 5.0, 14.1 Hz, 1H), 2.24 (s, 3H), 3.50 (dd, J = 7.4, 14.1 Hz,
1H), 3.76 (s, 3H), 3.77 (s, 3H), 4.01 (s, 3H), 4.45 (d, J = 15.2 Hz,
1H), 5.19 (q, J = 6.5 Hz, 1H), 5.37 (d, J = 15.2 Hz, 1H), 6.92 (m,
1H), 6.99 (m, 1H), 7.08 (m, 1H), 7.16 (dd, J = 4.9, 7.4 Hz, 1H),
7.28–7.33 (comp m, 2H), 7.43 (ddd, J = 1.1, 7.2, 8.3 Hz, 1H), 7.51
(ddd, J = 1.1, 7.2, 8.2 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 8.00 (d,
J = 8.5 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 9.50 (d, J = 7.8 Hz, 1H).
13C NMR (125 MHz, CDCl3): d = 18.9, 23.5, 43.3, 43.9, 53.5, 56.1,
56.1, 56.1, 85.9, 86.4, 100.5, 109.2, 113.0, 115.5, 115.8, 117.6,
120.1, 120.5, 121.1, 121.2, 122.9, 124.2, 124.8, 125.5, 125.7, 126.5,
127.3, 127.4, 129.8, 132.0, 137.7, 138.5, 141.5, 149.8, 150.7, 169.2,
174.0.
Diazo Lactam 8a
A round-bottom flask was charged with lactam 12a (1.5 g, 5.7
mmol, 1 equiv.), MsN3 (763 mg, 6.3 mmol, 1.1 equiv.), and CH3CN
(40 mL). The solution was cooled to 0 °C and Et3N (0.95 mL, 6.8
mmol, 1.2 eq) was added dropwise to the mixture. After gradually
warming to r.t., the mixture was stirred for an additional 3 h and the
volatiles were removed under reduced pressure. The residue was
dissolved in CH2Cl2 (20 mL), and washed with a 1M aq NaOH (20
mL). The organic layer was separated, concentrated, dissolved in
EtOAc (50 mL), and filtered through a plug of silica gel to yield di-
azolactam 8a as a bright yellow foam (1.5 g, 91%), [a]20D+94 (c =
0.8, MeOH).
1H NMR (500 MHz, CDCl3): d = 1.37 (d, J = 6.9 Hz, 3H), 3.76 (q,
J = 6.9, 1H), 3.88 (s, 6H), 3.85 (s, 3H), 4.04 (d, J = 14.9 Hz, 1H),
5.09 (d, J = 14.9 Hz, 1H), 6.79–6.83 (comp m, 3H).
IR (film): n = 3479 (br w), 3361 (br w), 2949 (w), 1728 (s), 1660 (s),
1583 (m), 1516 (m), 1454 (m), 1257 (m), 1140 (m), 1034 (s), 745
(s) cm-1.
13C NMR (125 MHz, CDCl3): d = 15.3, 44.2, 55.9, 55.9, 59.6,
111.1, 111.2, 120.6, 128.0, 148.9, 149.4, 161.3, 189.5.
HRMS (EI): calcd for C37H33N3O7 631.2319; found 631.2314.
14a, [a]20D+62.7 (c = 0.26, MeOH).
1H NMR (500 MHz, acetone-d6): d = 1.78 (d, J = 6.5 Hz, 3H), 2.22
(s, 3H), 2.27 (ddd, J = 1.9, 5.0, 14.0 Hz, 1H), 3.50 (dd, J = 7.5, 14.0
Hz, 1H), 3.75 (s, 3H), 3.77 (s, 3H), 4.01 (s, 3H), 4.45 (d, J = 15.2
Hz, 1H), 5.15 (q, J = 6.5 Hz, 1H), 5.23 (s, 1H), 5.38 (d, J = 15.2 Hz,
1H), 6.91 (m, 1H), 6.99 (m, 1H), 7.08 (m, 1H), 7.15 (dd, J = 4.9, 7.4
Hz, 1H), 7.31 (q, J = 7.7 Hz, 1H), 7.44 (ddd, J = 1.4, 7.1, 8.5 Hz,
1H), 7.51 (ddd, J = 1.0, 7.2, 8.2 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H),
7.94 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 7.9 Hz, 1H), 9.75 (d, J = 8.1
Hz, 1H).
13C NMR (125 MHz, CDCl3): d = 18.7, 23.3, 43.1, 43.7, 53.3, 55.4,
56.0, 56.0, 86.0, 86.4, 100.1, 110.0, 112.7, 114.8, 115.4, 118.1,
119.6, 120.4, 120.9, 121.2, 123.1, 123.2, 125.9, 126.0, 126.1, 127.1,
127.2, 127.5, 127.7, 131.9, 138.0, 138.4, 141.1, 149.6, 150.5, 169.4,
173.7.
IR (film): n = 2928 (br m), 2857 (w), 2125 (s), 1684 (s), 1514 (m),
1401 (m), 1356 (s), 1259 (m), 1025 (w) cm-1.
HRMS (EI): calcd for C14H15N3O4 289.1065; found 263.1063.
Aglycon 4a
To a flame-dried three-necked round bottom flask equipped with a
condenser were added the diazolactam 8a (800 mg, 2.8 mmol, 1
equiv.), 2,2'-biindole (650 mg, 2.8 mmol, 1 equiv.), Rh2(OAc)4 (12
mg, 0.028 mmol, 0.01 eq) and pinacolone (28 mL). The whole was
degassed by bubbling a stream of N2 gas through the solution for 2
h. The mixture was then refluxed for 8 h. The mixture was concen-
trated to a dark brown foamy solid, which was adsorbed onto silica
gel and subjected to flash chromatography (50% EtOAc/hexanes
eluent). Compound 4a was isolated as a pale yellow solid (430 mg,
35%), [a]20D+31.5 (c = 0.45, MeOH).
1H NMR (500 MHz, CDCl3): d = 1.75 (d, J = 6.5 Hz, 3H), 3.73 (s,
3H), 3.74 (s, 3H), 4.46 (d, J = 15.2 Hz, 1H), 5.15 (m, 1H), 5.36 (d,
J = 15.2 Hz, 1H), 6.88 (m, 1H), 6.99 (m, 1H), 7.07 (m, 1H), 7.25 (t,
J = 7.5 Hz, 2H), 7.41 (m, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.64 (d,
IR (film): n = 3479 (w), 3365 (br w), 3057 (w), 2933 (w), 2835 (w),
1738 (s), 1676 (s), 1588 (m), 1511 (m), 1454 (m), 1243 (m), 1140
(m), 1021 (m), 743 (m) cm-1.
HRMS (EI): calcd for C37H33N3O7 631.2319; found 631.2314.
Synthesis 1999, No. SI, 1529–1533 ISSN 0039-7881 © Thieme Stuttgart · New York

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Total Synthesis and Protein Kinase Activity of C(7) Methyl Derivatives of K252a
1533
7-(R)-Methyl K252a 3a
120.6, 121.1, 121.3, 123.4, 125.9, 126.1, 126.1, 126.2, 127.1, 127.3,
127.5, 127.7, 131.9, 138.1, 138.4, 141.1, 149.6, 150.5, 169.5, 173.7.
A solution of protected amide 13a (60 mg, 0.095 mmol, 1 equiv.)
and anisole (1.03 g, 9.5 mmol, 100 eq.) in CH2Cl2 (2 mL) was treat-
ed dropwise with 2,2,2-trifluoroacetic acid (2 mL). After stirring at
r.t. for 12 h, the reaction was quenched with a 20% aq NaHCO3 (2
mL). The aqueous layers was washed with CH2Cl2 (3 ¥ 5 mL) and
the combined organic layers were dried (Na2SO4), filtered and ad-
sorbed onto SiO2. Flash chromatography (50% EtOAc/hexanes elu-
ent) provided 3a as a pale yellow solid (45 mg, 85%), [a]20D+41 (c
= 0.1, MeOH).
1H NMR (500 MHz, acetone-d6): d = 1.76 (d, J = 6.5 Hz, 3H), 2.21
(s, 3H), 2.34 (dd, J = 5.0, 14.1 Hz, 1H), 3.52 (dd, J = 7.5, 14.1 Hz,
1H), 4.02 (s, 3H), 5.39 (s, 1H), 5.45 (q, J = 6.4 Hz, 1H), 7.15 (dd,
J = 5.0, 7.4 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.5 Hz,
1H), 7.44–7.51 (comp m, 2H), 7.65 (s, 1H), 7.79 (d, J = 8.3 Hz, 1H),
8.02 (d, J = 8.5 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 9.41 (d, J = 7.8
Hz, 1H).
IR (film): n = 3479 (w), 3365 (br w), 3057 (w), 2933 (w), 2835 (w),
1738 (s), 1676 (s), 1588 (m), 1511 (m), 1454 (m), 1243 (m), 1140
(m), 1021 (m), 743 (m) cm-1.
HRMS (EI): calcd for C37H33N3O7 631.2319; found 631.2314.
3b, [a]20D –4 (c = 0.1, MeOH).
1H NMR (500 MHz, acetone-d6): d = 1.79 (d, J = 6.5 Hz, 3H), 2.24
(m, 4H), 3.50 (dd, J = 7.4, 14.1 Hz, 1H), 4.01 (s, 3H), 5.28 (s, 1H),
5.43 (q, J = 6.5 Hz, 1H), 7.15 (dd, J = 4.9, 7.4 Hz, 1H), 7.28 (t,
J = 7.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.44–7.50 (comp m, 2H),
7.63 (s, 1H), 7.79 (d, J = 8.3 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 8.19
(d, J = 7.7 Hz, 1H), 9.41 (d, J = 7.9 Hz, 1H).
13C NMR (125 MHz, CDCl3): d = 21.3, 23.4, 43.4, 53.2, 53.3, 53.4,
86.2, 86.5, 100.3, 109.2, 116.0, 117.6, 121.4, 122.7, 124.4, 125.3,
125.5, 125.7, 126.4, 127.4, 127.5, 130.1, 138.4, 139.7, 141.4, 171.6,
174.0.
13C NMR (125 MHz, CDCl3): d = 21.4, 23.5, 43.3, 53.2, 53.3, 53.5,
85.9, 86.4, 100.5, 109.1, 115.8, 117.7, 120.4, 121.2, 122.9, 124.2,
125.1, 125.4, 125.6, 126.4, 127.4, 127.5, 129.9, 138.4, 139.7, 141.5,
171.6, 174.0.
IR (film): n = 3407 (br w), 3053 (w), 2950 (w), 1733 (m), 1679 (s),
1450 (s), 1392 (s), 1308 (m), 1257 (m), 1199 (m), 1140 (m), 1083
(m), 748 (s) cm-1.
IR (film): n = 3407 (br w), 2963 (w), 2928 (m), 2861 (w), 1738 (m),
1678 (s), 1588 (m), 1449 (s), 1392 (m), 1201 (m), 1084 (w), 877
(w), 743 (m) cm-1.
HRMS (EI): calcd for C28H23N3O5 481.1638; found 481.1637.
HRMS (EI): calcd for C28H23N3O5 481.1638; found 481.1633.
Acknowledgement
Compounds from the opposite epimeric series (i.e. 13b, 14b and 3b)
can also be produced using the same experimental procedures de-
scribed above and provide the following data:
13b, [a]20D –14.4 (c = 0.23, MeOH).
We are pleased to acknowledge the support of this investigation by
the NIH (Grant 1RO1GM54131) and ACS (Grant DHP-82223).
Bristol-Myers Squibb, Eli Lilly, Glaxo-Wellcome, Yamanouchi
and Zeneca provided additional support through their Junior Facul-
ty Awards programs. J.L.W is a fellow of the Alfred P. Sloan Foun-
dation.
1H NMR (500 MHz, acetone-d6): d = 1.78 (d, J = 6.5 Hz, 3H), 2.23
(dd, J = 5.0, 14.1 Hz, 1H), 2.23 (s, 3H), 3.50 (dd, J = 7.4, 14.1 Hz,
1H), 3.76 (s, 3H), 3.77 (s, 3H), 4.01 (s, 3H), 4.45 (d, J = 15.2 Hz,
1H), 5.19 (q, J = 6.5 Hz, 1H), 5.37 (d, J = 15.2 Hz, 1H), 6.92 (m,
1H), 6.99 (m, 1H), 7.08 (m, 1H), 7.16 (dd, J = 4.9, 7.4 Hz, 1H),
7.28–7.33 (comp m, 2H), 7.43 (ddd, J = 1.1, 7.2, 8.3 Hz, 1H), 7.51
(ddd, J = 1.1, 7.2, 8.2 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 8.00 (d,
J = 8.5 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H), 9.50 (d, J = 7.8 Hz, 1H).
13C NMR (125 MHz, CDCl3): d = 18.6, 23.2, 43.1, 43.2, 43.6, 53.2,
55.9, 85.7, 86.0, 86.2, 100.0, 109.1, 112.6, 115.2, 115.8, 117.3,
119.7, 120.3, 120.8, 121.2, 122.5, 124.0, 124.9, 125.3, 125.6, 126.3,
127.1, 127.2, 129.8, 131.8, 137.4, 138.2, 141.2, 149.5, 150.4, 169.1,
173.7.
References
(1) Harris, W.; Hill, C. H.; Lewis, E. J.; Nixon, J. S.; Wilkinson,
S. E. Drugs of the Future 1993, 18, 727.
(2) Prade, L.; Engh, R. A.; Girod, A.; Kinzel, V.; Huber, R.;
Bossemeyer, D. Structure 1997, 5, 1627.
(3) Sezaki, M.; Sasaki, T.; Nakazawa, T.; Takeda, U.; Iwata, M.;
Watanabe, T.; Koyama, M.; Kai, F.; Shomura, T.; Kojima, M.
J. Antibiot. 1985, 38, 1437.
(4) Kase, H.; Iwahashi, K.; Matsuda, Y. J. Antibiot. 1986, 39,
1059.
IR (film): n = 3473 (br w), 3003 (w), 2950 (w), 1733 (s), 1670 (s),
1586 (m), 1514 (m), 1455 (s), 1259 (s), 1201 (m), 1138 (m), 1026
(m), 747 (s) cm-1.
(5) Wood, J. L.; Stoltz, B. M.; Dietrich, H. J. J. Am. Chem. Soc.
1995, 117, 10413.
Wood, J. L.; Stoltz, B. M.; Dietrich, H. J.; Pflum, D. A.;
Petsch, D. T. J. Am. Chem. Soc. 1997, 119, 9641.
(6) Bergman, J.; Koch, E.; Pelcman, B. Tetrahedron 1995, 51,
5631.
HRMS (EI): calcd for C37H33N3O7 631.2319; found 631.2314.
14b, [a]20D –15.9 (c = 0.18, MeOH).
1H NMR (500 MHz, acetone-d6): d = 1.75 (d, J = 6.5 Hz, 3H), 2.20
(s, 3H), 2.40 (dd, J = 4.9, 14.1 Hz, 1H), 3.55 (dd, J = 7.5, 14.1 Hz,
1H), 3.76 (s, 3H), 3.78 (s, 3H), 4.01 (s, 3H), 4.46 (d, J = 15.2 Hz,
1H), 5.19 (q, J = 6.5 Hz, 1H), 5.30 (s, 1H), 5.36 (d, J = 15.2 Hz,
1H), 6.93 (m, 1H), 7.01 (m, 1H), 7.09 (m, 1H), 7.15 (dd, J = 4.9, 7.4
Hz, 1H), 7.31 (m, 2H), 7.44 (ddd, J = 1.4, 7.0, 8.5 Hz, 1H), 7.52
(ddd, J = 1.0, 7.3, 8.2 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.94 (d,
J = 8.5 Hz, 1H), 8.13 (d, J = 7.9 Hz, 1H), 9.75 (d, J = 7.7 Hz, 1H).
(7) Boeckmann, R. K., Jr.; Starrett, J. E., Jr.; Nickell, D. G.; Sum,
P.-E. J. Am. Chem. Soc. 1986, 108, 5549.
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Article Identifier:
1437-210X,E;1999,0,SI,1529,1533,ftx,en;C01799SS.pdf
13C NMR (125 MHz, CDCl3): d = 18.8, 23.5, 43.5, 43.6, 43.9, 53.4,
55.7, 56.2, 85.8, 86.4, 100.1, 110.0, 113.0, 115.0, 118.1, 119.6,
Synthesis 1999, No. SI, 1529–1533 ISSN 0039-7881 © Thieme Stuttgart · New York