Discovery of pyrimidine benzimidazoles as Src-family selective Lck inhibitors. Part II

Article abstract of DOI:10.1016/j.bmcl.2009.09.123

A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src-family kinases (SFKs). These type II inhibitors were optimized using a cellular Lck-dependent proliferation assay and are

Full text of DOI:10.1016/j.bmcl.2009.09.123

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6691–6695
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of pyrimidine benzimidazoles as Src-family selective Lck inhibitors.
Part II
*
Guobao Zhang , Pingda Ren, Nathanael S. Gray, Taebo Sim, Xia Wang, Yi Liu, Jianwei Che, Weitong Dong,
Shin-Shay Tian, Mark L. Sandberg, Tracy A. Spalding, Russell Romeo, Maya Iskandar, Zhiliang Wang,
,
*
H. Martin Seidel, Donald S. Karanewsky, Yun He
Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine
kinase (Lck), a member of the Src-family kinases (SFKs). These type II inhibitors were optimized using
a cellular Lck-dependent proliferation assay and are capable of inhibiting Lck at single-digit nanomolar
concentrations. This scaffold is likely to serve a valuable template for developing potent inhibitors of a
number of SFKs.
Received 27 August 2009
Revised 25 September 2009
Accepted 30 September 2009
Available online 12 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Lck inhibitor
Kinase inhibitor
Transplantation rejection
Autoimmune disease
Pyrimidine benzimidazoles
Lymphocyte-specific kinase (Lck) is a member of the Src-family
of protein tyrosine kinases. Lck plays a critical role in the initial
steps of T-cell receptor (TCR) signaling.1 Activation of TCR signaling
by Lck triggers a cascade of downstream signaling pathways, lead-
ing to the production of cytokines such as interleukin-2 (IL-2) and
Both type I and type II Lck inhibitors have been reported in the lit-
erature.6,8 In our previous communication, we reported the design
and synthesis of pyrimidine benzimidazoles as type I Lck inhibi-
tors.9 To take advantage of the known ability of Lck to be inhibited
by ATP-competitive inhibitors that bind to the ‘DFG-out’ conforma-
tion of the activation loop (type II), we extended our effort to ex-
ploit this binding conformation. Herein we report the design,
synthesis, SAR, and kinase selectivity profilings of a series of 4-ami-
no-6-benzimidazole-pyrimidines as type II Lck inhibitors.
To develop type II Lck inhibitors, we followed the strategies de-
scribed by Okram et al.10 Briefly, a R2 group was introduced to tar-
get the Lck hydrophobic binding site (Fig. 1). Compounds 7a–7n
were synthesized according to Scheme 1. First, nucleophilic substi-
tution of 4,6-dichloropyrimidine (1) with 2-chlorobenzimidazole
(2) was carried out in N,N-dimethylforamide in the presence of so-
interferon-c 2,3
Lck is an attractive drug target because of its re-
.
stricted expression in T-cells and natural killer (NK) cells.4 Selec-
tive inhibitors of Lck would be expected to have an improved
safety profile over current immunosuppressive agents, which
invariably have non-lymphocyte related toxicities. Therefore,
development of selective Lck inhibitors offers a promising ap-
proach for treating T-cell mediated autoimmune diseases and
chronic transplantation rejection.5 Selective Lck inhibitors have
been shown to prolong the survival of major histocompatibility
mismatched allografts in preclinic animal models of solid organ
6
transplantation
.
Protein kinase inhibitors are generally classified into three types
based on their binding modes.7 Type I kinase inhibitors bind only
to the ATP binding region. Type II protein kinase inhibitors bind
to both ATP and an adjacent hydrophobic pocket, whereas type
III protein kinase inhibitors bind only to an allosteric binding site.
* Corresponding authors. Tel.: +1 858 812 1614; fax: +1 858 812 1684 (G.Z.); tel.:
+86 10 80768611; fax: +86 10 80703150 (Y.H.).
E-mail addresses: gzhang@gnf.org (G. Zhang), yun.he@bioduro.com (Y. He).
Present address: Bioduro (Beijing) Co. Ltd, Building E, 29 Life Science Park Road,
Changping District, Beijing 102206, China.
Figure 1. General structure of type II Lck inhibitors based on pyrimidine
benzimidazoles.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.123

6692
G. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6691–6695
Cl
Cl
Cl
N
N
N
N
N
N
HN
N
HN
N
N
NO2
a
b
c
+
Cl
N
N
H2N
N
N
Cl
Cl
H2N
N
N
1
2
3
4
5
d
O
N
N
HN
N
R2
N
N
HN
NH2
H
e
H2N
N
N
H2N
N
N
7
6
2
O
7j: R2=
7k: R2=
2
7a
7b
7g: R2=
: R =Ph-3-Cl
7m: R2=
2
N
N
N
N
N
N
N
: R =Ph-3-CF3
CF3
S
7c: R2=Ph-3,5-Cl2
F3C
7h: R2=
N
N
2
7d
7e
: R =Ph-3-Me
2
Cl
N
7n:
R =
2
t
: R =4-Py-3- Bu
2
7i
7l:
: R =
R =
N
7f: R2=4-Py-3-Cl
N
HN
F3C
N
N
Scheme 1. Synthesis of compounds 7a–7n. Reagents and conditions: (a) NaH (1.5 equiv), DMF, 0–25 °C, 12 h, 56%; (b) NH3 (excess), isopropanol, 50 °C, 12 h, 92%; (c) 2-
methyl-5-nitroaniline (1.5 equiv), CH3SO3H (2.0 equiv), N,N-dimethylimidazolinone, 90 °C, 2 h, 83%; (d) H2, Pd/C, ethanol, 25 °C, 4 h, 89%; (e) RCO2H (1.2 equiv), HATU
(1.2 equiv), DIEA (3.0 equiv), DMF, 25 °C, 2 h, 60–95%.
dium hydride to give compound 3 in 56% yield. Selective amination
of the chloropyrimidine ring was readily achieved by reacting com-
pound 3 with excess ammonia in isopropanol at 50 °C to give com-
cellular assays. Large hyrophobic substituents, for example, t-Bu-
tyl (7e, 7h, and 7i), can be tolerated in the ‘DFG-out’ binding
pocket. The Lck inhibition potency is improved as the size of
the substituent becomes bigger (7a, 7d, 7b, 7e, and 7f). Heterocy-
cles are also tolerated in the allosteric binding site (7e, 7f, 7h, 7i,
and 7l). The discrepancy between biochemical and cellular data of
compound 7c is likely due to its poor cell permeability because it
has high Clog P (6.56). The apparent cellular activity of compound
7h is due to its general cytotoxicity (IC50 = 6 nM in Ba/F3 parental
cell line) presumably due to engagement of additional kinases.
Hydrophilic substitutions on the right phenyl ring are also well
tolerated (7g, 7k–7n).
Most of these compounds also display good selectivity over Src
in both biochemical and cellular assays. However, their selectivity
over Hck is very low. Inhibition of Hck might have undesirable
effects on myeloid cell function because it is expressed broadly
in hematopoietic cells.11 Among them, compound 7b shows very
pound
4 in 92% yield. The 2-chlorobenzimidazolegroup in
compound 4 was then reacted with 2-methyl-5-nitroaniline in
the presence of methanesulfonic acid to give compound 5 83%
yield. The nitro group of compound 5 was subsequently reduced
under hydrogenation conditions to give compound 6 in 89% yield.
Compound 6 was then reacted with various commercial and in-
house made benzoic acids in the presence of HATU and diisopro-
pylethylamine to give the target compounds 7a–7n.
Compounds 7a–7n were tested for their Lck inhibitory activi-
ties using both biochemical (LcK Lance) and cellular (BaF3/Tel-
Lck) assays as previously described.9 Activity against several other
protein tyrosine kinases was also measured and the data are sum-
marized in Table 1. In general, these pyrimidine benzimidazole
compounds show strong inhibition of Lck in biochemical and
Table 1
Biochemical and cellular kinase inhibition activity (IC50 nM) of compounds 7a–7n
Compounds
Lck Lance
Hck Lance
Src Lance
BaF3/Tel-LCK
BaF3/Tel-LYN
BaF3/Tel-SRC
BaF3/Tel-KDR
BaF3/Tel-InsR
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
132
11
58
70
10
715
24
43
120
316
4
475
43
121
245
46
>2500
27
84
446
844
9
11
20
1650
450
530
1539
ND*
ND*
233
916
7359
7188
32
84
7
1074
32
7
890
7
4
50
224
5
18
3
133
14
3608
82
7
595
5
5
96
332
10
88
6
650
78
9693
490
56
>10,000
39
4
56
959
47
249
47
57
576
36
1988
661
>10,000
1493
ND*
ND*
746
4
75
1961
63
818
409
588
6365
280
ND*
ND*
66
4
48
451
30
63
8
11
11
177
130
71
43
5
16
4
9
*
ND = not determined.

G. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6691–6695
6693
Cl
N
N
HN
HN
NH
d
HN
NH
a
b
H2N
NH
c
O
+
Cl
N
N
N
O2N
NH2
O
O
O
CF3
8
9
CF3
CF3
11
10
CF3
12
e
13a: R1=OCH3
1
R =NH(CH2)2NH2
R1=NH-c-Hex-trans-4-NH2
R1=NHCH(CH2CH2)2NMe
R1=NHCH2CH2-2-(2-Py)
13b:
13j:
13k:
13l:
13m:
13n:
13o:
13p:
13q:
13c: R1=NH(CH2)3NH2
N
N
13d: R1=NH(CH2)4NH2
HN
NH
1
1
R1
13e:
R =morpholine
R =NH(CH2)2NEt2
N
N
O
R1=NH(CH2)2N(CH2CH2)2NCH3
R1=NH(CH2)3N(CH2CH2)2NCH3
R1=NH(CH2)2OH
13f: R1=NH(CH2)4NMe2
13g: R1=NH(CH2)4NEt2
1
CF3
13h:
R =NH(CH2)3-3-morpholine
13
R1=NH(CH2)2OCH3
13i: R1=NH(CH2)2-2-pyrrolidine
Scheme 2. Synthesis of compounds 12–13q. Reagents and conditions: (a) DIEA (1.5 equiv), DCM, 25 °C, 3 h; (b) H2, Pd/C, ethanol, 25 °C, 4 h, 85% in two steps; (c) 2-chloro-
benzimidazole (1.0 equiv), CH3SO3H (2.0 equiv), N,N-dimethylimidazolinone, 90 °C, 2 h, 88%; (d) 4,6-dichloropyrimidine (2.5 equiv), NaH (1.5 equiv), DMF, 0 °C to 80 °C, 1 h,
80%; e) RNH2 (3.0 equiv), isopropanol, 50 °C, 1 h, 80–95%.
potent Lck inhibition in both biochemical and cellular assays. This
compound also displays 40-fold selectivity against Src and 4-fold
selectivity against Hck in the biochemical (Lance) assay. Therefore,
compound 7b was chosen for further optimization.
tency drop is consistent with our previous results and can be ex-
plained by the conformational analysis described in our previous
communication.9 Molecular modeling studies (Fig. 2) suggest that
the basic nitrogen in compounds 13f, 13g, and 13o can participate
in an electrostatic interaction with the carboxylic acid of Glu320
(Lck numbering) on Lck. Sequence alignment shows that Glu320
is not conserved across other Scr-family protein tyrosine kinases.
Therefore, compounds 13f, 13g, and 13o display better Src-family
kinase selectivity compared to compound 7b. In fact, most of the
compounds with R1 bearing a basic nitrogen atom display better
selectivity over Hck (Lance data) compared to compound 7b. It is
also interesting to note that the electrostatic interaction weakens
as the alkyl spacer is shortened from four carbons to two carbons
(13b–13d, Lck lance data). These data suggest that this non-con-
served Glu320 can provide a valuable handle for improving the
selectivity over Hck.
To explore the SAR on R1 (Fig. 1), with a focus on improving aque-
ous solubility and selectivity against Hck, we fixed R2 as 3-(trifluoro-
methyl)phenyl and synthesized a series of analogues 13a–13q as
outlined in Scheme 2. Briefly, aniline 10 was prepared by coupling
4-methyl-3-nitro-aniline (8) with 3-(trifluoromethyl)benzoyl chlo-
ride (9) in the presence of base followed by Pd/C catalyzed hydroge-
nation. Reaction of 2-chlorobenzimidazole with 10 under acid
catalysis gave aminoimidazole 11 in excellent yield. Compound 11
was then coupled to 4,6-dichloro-pyrimidine (1) in the presence of
sodium hydride to provide key intermediate 12 for derivatization.
Compound 12 was then reacted with various amines or methanol
to give compounds 13a–13q.
Table 2 summarizes the SAR at the R1 position. Not surprisingly,
compounds 12, 13a, and 13m are inactive or have very weak Lck
inhibition because they lack the hydrogen bond-donating NH
group at the pyrimidine 6 position, which is important for binding
to the Lck hinge region. In the Lck Lance assay, compounds 13f,
13g, and 13o are similar in potency to compound 7b. However,
all the other compounds display weaker Lck inhibition. The po-
Unfortunately, all of the basic amine compounds 13a–13q
showed relatively weak cellular activity in comparison to com-
pound 7b. We believe this is due to decreased cellular permeabil-
ity, since the compounds with the largest discrepancy between
enzymatic and cellular activity have polar substituents; for exam-
ple, –OH in compound 13p and –NH2 in compounds 13c, 13d, and
13j (Table 2).
Table 2
Kinase inhibition activity (IC50 nM) of compounds 12–13q
Compounds
Lck Lance
Hck Lance
Src Lance
BaF3/Tel-LCK
BaF3/Tel-LYN
BaF3/Tel-SRC
BaF3/Tel-KDR
BaF3/Tel-InsR
7b
11
43
450
7
14
78
36
661
12
1162
2030
230
23
13
111
5
12
26
38
71
168
204
>2500
29
6
27
3826
>2500
>2500
1960
345
378
65
16,507
>2500
ND
ND
ND
>2500
139
97
2702
>10,000
470
387
366
418
108
41
3296
>10,000
1471
1117
457
642
399
217
67
818
547
1422
259
>10,000
204
149
400
126
1220
>10,000
1288
1322
1661
398
1211
438
600
903
739
1378
524
>10,000
918
9069
>10,000
1748
1472
599
1726
1365
885
318
941
609
1712
549
3332
>10,000
1573
2768
3760
1141
3109
3012
4156
1087
1443
1613
>10,000
>10,000
1435
3162
4401
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
13o
13p
13q
430
49
165
40
805
450
>2500
917
>2500
69
31
192
405
>2500
1722
>2500
2370
>2500
305
128
1920
1688
238
392
881
171
>10,000
56
25
229
132
>10,000
605
582
590
296
601
1698
606
135
>10,000

6694
G. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6691–6695
despite having good in vitro metabolic stability, with mouse
extraction ratio of <0.3 (13g) and 0.383 (13j). However, compound
13q
displayed
much
improved
plasma
exposure
*
(AUC = 2687 h nM) in a mouse PK study following the same dos-
ing. These data further support the hypothesis that compounds
bearing a basic nitrogen atom have low cell permeability. Thus, de-
spite the success in achieving enzymatic Hck selectivity through
inclusion of a basic amine as part of the R1 group, we were forced
to seek an alternative approach to improving Hck selectivity.
By using the versatile intermediate 4, we were able to quickly
synthesize compounds 15a–15k (Scheme 3) to explore how a ‘re-
versed’ amide bond affects Lck inhibition potency and kinase selec-
tivity. These compounds are hereafter referred to as reverse amides
and the previous compounds as normal amides. The Lck inhibition
data of these reverse amides are shown in Table 3. Generally, re-
verse amides have comparable or slightly weaker Lck inhibition
than the normal amides. However, the kinase selectivity profiles
are considerably improved (15a vs 7d, 15g vs 7a, 15f vs 7b). Com-
pound 15a is especially interesting because of its superior kinase
selectivity. The SAR on the right phenyl ring substitutions is quite
similar to that of normal amides, and hydrophobic substitutions
are preferred for inhibitory activity (15b–c vs 15a, 15d vs 15h,
15e vs 15g). The compound without substitution on the right phe-
nyl ring (15i) has very weak Lck inhibition while analogs 15j and
15k which bear hydrophilic substituents, lose Lck inhibitory activ-
ity significantly.
Figure 2. Binding mode of compound 13g to Lck based on modeling studies. The
sequences of several other protein tyrosine kinases of interest are aligned with Lck
and partial sequences around the protein kinase hinge region are shown. The non-
conserved Glu320 in Lck and its corresponding residues in other protein tyrosine
kinases are highlighted.
With R3 fixed as methyl or trifluoromethyl, compounds 22a–
22k (Scheme 4) were synthesized in a similar fashion to com-
pounds 13a–13q except that the amide bond direction was re-
versed. With R3 fixed as methyl, compounds 22a–22d showed
In mouse pharmacokinetics (PK) studies, compounds 13g and
13j showed very low plasma exposure, with AUC value of
368 h nM and 0 h nM, respectively, following 20 mg/kg PO dosing,
R2=Ph-3-Me
R2=Ph-3-iPr
R2=Ph-3-tBu
R2=Ph-3-OCHF2
R2=Ph-3-Br
R2=Ph-3-CF3
R2=Ph-3-Cl
R2=Ph-3-OCH3
R2=Ph
H
N
15a:
15b:
15c:
15d:
15e:
15f:
15g:
15h:
15i:
O
Cl
N
N
HN
N
N
R2
HN
N
N
O
b
a
OH
N
H2N
N
H2N
N
N
N
H2N
N
15
4
14
R2=
15l:
15m:
N
R2=
15j:
2
N
O
R =
15k: R2=
N
N
NH2
F3
C
Scheme 3. Synthesis of reverse amides 15a–15m. Reagents and conditions: (a) 3-amino-4-methyl-benzoic acid (1.2 equiv), CH3SO3H (2.0 equiv), N,N-dimethylimidazolinone,
80 °C, 12 h, 82%; (b) RNH2 (2.0 equiv), DIEA (3 equiv), DMF, 25–60 °C, 2 h, 40–90%.
Table 3
Kinase inhibition activity (IC50 nM) of compounds 15a–15m
Compounds
Lck Lance
Hck Lance
Src Lance
BaF3/Tel-LCK
BaF3/Tel-LYN
BaF3/Tel-SRC
BaF3/Tel-KDR
BaF3/Tel-InsR
15a
15b
15c
15d
15e
15f
15g
15h
15i
52
30
22
17
43
59
63
90
1080
616
>2500
177
238
>2500
151
139
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
83
7
16
47
52
20
76
157
2280
3241
>10,000
702
14
1079
15
16
100
272
15
>10,000
437
5512
7836
>10,000
>10,000
28
7173
21
202
>10,000
5375
>10,000
>10,000
4389
6168
7613
1015
17
52
92
464
55
260
516
8817
10,577
>10,000
135
20
7121
29
1224
>10,000
5629
>10,000
>10,000
5435
5935
5708
>2500
>2500
>2500
>2500
>2500
>2500
>2500
>2500
>2500
904
15j
15k
15l
>10,000
>10,000
107
>10,000
>10,000
7631
15m

G. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6691–6695
6695
O
O
HN
HN
O2N
O
c
a
Cl
b
H2N
HN
HN
O2N
+
H2N
R3
N
HN
O
16
17
18
19
R3
R3
R3
20
X=CH, R3=CH3, R1=NH(CH2)4NMe2
X=CH, R3=CH3, R1=NH(CH2)4NEt2
22a:
22b:
22c:
22d:
22e:
22f:
22g:
22h:
22i:
d
X=CH, R3=CH3, R1=NH(CH2)2N(CH2CH2)2NMe
X=CH, R3=CH3, R1=NH(CH2)3N(CH2CH2)2NMe
X=CH, R3=CF3, R1=NH(CH2)4NMe2
O
O
X=CH, R3=CF3, R1=NH(CH2)4NEt2
HN
N
N
e
HN
N
N
X=CH, R3=CF3, R1=NH(CH2)2N(CH2CH2)2NMe
X=CH, R3=CF3, R1=NH(CH2)3N(CH2CH2)2NMe
X=CH, R3=CF3, R1=NHCH2CH2CN
HN
HN
R1
X
N
N
N
N
Cl
X
R3
X=CCH3, R3=CF3, R1=NH2
22j:
22k:
R3
X=N, R3=CF3, R1=NH(CH2)3N(CH2CH2)2NMe
22
21
Scheme 4. Synthesis of compounds 22a–22k. Reagents and conditions: (a) DIEA (1.5 equiv), DCM, 25 °C, 3 h; (b) H2, Pd/C, ethanol, 25 °C, 4 h, 85–90% in two steps; (c) 2-
chloro-benzimidazole (1.0 equiv), CH3SO3H (2.0 equiv), N,N-dimethylimidazolinone, 90 °C, 2 h, 85–90%; (d) 4,6-dichloropyrimidine or 2,4-dichloro-1,3,5-triazine (2.5 equiv),
NaH (1.5 equiv), DMF, 0–80 °C, 1 h, 60–71%; (e) RNH2 (3.0 equiv), isopropanol, 50 °C, 1 h, 80–95%.
Table 4
Kinase inhibition activity (IC50 nM) of compounds 22a–22k
Compounds
Lck Lance
Hck Lance
Src Lance
BaF3/Tel-LCK
BaF3/Tel-LYN
BaF3/Tel-SRC
BaF3/Tel-KDR
BaF3/Tel-InsR
22a
22b
22c
22d
22e
22f
22g
22h
22i
22
20
38
19
15
18
11
18
>2500
1810
>2500
1875
56
475
130
260
ND
ND
ND
ND
485
274
458
494
ND
531
406
762
337
38
52
45
23
1384
1315
1658
1228
134
140
113
84
1268
1065
1537
1409
556
461
527
364
1497
1364
2094
1113
249
243
185
117
245
2628
2263
3486
3194
1221
1808
1506
1856
1092
>10,000
1365
1367
2400
12
>2500
>2500
54
275
4456
20
468
8012
43
1223
>10,000
117
22j
22k
>2500
ND
8181
133
improved Lck enzymatic inhibition activity compared to com-
pound 15a (Table 4). However, their cellular potencies are de-
creased significantly. This SAR is consistent with the normal
amides series and has been rationalized in a previous publication.9
Compound 22i showed very weak Lck inhibition, while compounds
22e–22h showed improved Lck inhibitory activity over compound
15f in both the enzymatic assay and the cellular assay, suggesting
that the basic amine in the left side is very important for Lck inhi-
bition. Also, compound 22f showed very good Lck inhibition po-
tency as well as protein kinase selectivity.
Finally, we conducted a brief investigation of pyrimidine ring
variants. Triazine 22k has slightly improved Lck inhibition activity
compared with the corresponding pyrimidine 22h. However, the
kinase selectivity of compound 22k is decreased. The apparent cel-
lular activity of compound 22k is partially due to its general cyto-
based drug design. Some of these Lck inhibitors (e.g., 15a and
22f) display good selectivity over non-Src-family kinases as well
as Src-family kinases.
References and notes
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toxicity (IC50 = 1.33 lM in Ba/F3 parental cell line). Introduction of
a methyl group at the C5 position of the pyrimidine (22j) essen-
tially eradicates inhibition activity at Lck and all other kinases as-
sayed. This phenomenon is consistent with our previous results
and has been explained by conformational analysis.9
In summary, a series of very potent type II Lck inhibitors was
discovered through rational design. Highly selective Lck inhibitors
were prepared through SAR-guided optimization and structure-
11. Quintrell, N.; Lebo, R.; Varmus, H.; Bishop, J. M.; Pettenati, M. J.; LeBeau, M. M.;
Diaz, M. O.; Rowley, J. D. Mol. Cell Biol. 1987, 7, 2267.