US 201701 14323A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0114323 A1 Theunissen et al. (43) Pub. Date: Apr. 27, 2017
(54) USES OF KINASE INHIBITORS FOR Related U.S. Application Data INDUCING AND MANTAINING (60) Provisional application No. 62/014,674, filed on Jun. PLURPOTENCY 19, 2014, provisional application No. 62/045,337, filed on Sep. 3, 2014. (71) Applicants: Whitehead Institute for Biomedical Research, Cambridge, MA (US); Publication Classification Dana-Farber Cancer Institute, Inc., (51) Int. Cl. Boston, MA (US) CI2N 5/0735 (2006.01) (52) U.S. Cl. (72) Inventors: Thorold W. Theunissen, Belmont, MA CPC ...... CI2N 5/0606 (2013.01); C12N 2501/11 (US); Nathanael S. Gray, Boston, MA (2013.01); C12N 2501/165 (2013.01); C12N (US); Rudolf Jaenisch, Brookline, MA 2501/115 (2013.01); C12N 2501/727 (US) (2013.01); C12N 2501/999 (2013.01) (73) Assignees: Whitehead Institute for Biomedical (57) ABSTRACT Research, Cambridge, MA (US); The present disclosure provides compounds of any one of Dana-Farber Cancer Institute, Inc., Formulae (A) to (L). The present disclosure also provides Boston, MA (US) compositions, uses, and methods that include or involve a compound described herein, a serine/threonine-protein (21) Appl. No.: 15/318,533 kinase B-Raf (BRAF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial growth (22) PCT Filed: Jun. 19, 2015 factor 1 (VEGFR1) inhibitor, a fibroblast growth factor PCT No.: PCT/US 15/36.692 receptor 1 (FGFR1) inhibitor, or a combination thereof. The (86) compounds, compositions, uses, and methods are useful in S 371 (c)(1), changing the pluripotency state of a vertebrate cell to a more (2) Date: Dec. 13, 2016 naive state. Patent Application Publication Apr. 27, 2017. Sheet 1 of 76 US 2017/0114323 A1
s i
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Figure 1B ------Figure 1C Patent Application Publication Apr. 27, 2017. Sheet 3 of 76 US 2017/0114323 A1
450 . OCT4-APE-GFP 200 4OO o OCT4-GFP 18O 350 160 s 3OO 5 140 C 120 st 250 O 200. 100 80 150. t 60 1OO 40 50 20 O O O 50 1OO GFP 150 200 250 OCT4-GFP OCT4-APE-GFP Figure 1D Patent Application Publication Apr. 27, 2017. Sheet 4 of 76 US 2017/0114323 A1
hESM 2i/L 2i/L/DOX
105 R1=21% 105 in 10 104 Sz. 103 103 102 102 0 R1 R2 O R2 0 102 103 104 105 O 102 103 104 105
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105 R1=50% 105 R2=206 g10 * * 104 104 R2=11% goZ 103 103 102 102 * 102 0 R1 R2 0 R1 0. R2 O 102 103 104 105 O 102 103 104 105 0 102 103 104 105 Figure 1E Patent Application Publication Apr. 27, 2017. Sheet 5 of 76 US 2017/0114323 A1
2i/L/DOX 2i/L (P1-DOX)
3.
3 23
2
o 101 102 103 104 100 101 102 103 104 Figure 1F
Patent Application Publication Apr. 27, 2017. Sheet 7 of 76 US 2017/0114323 A1
Figure 1H Patent Application Publication Apr. 27, 2017. Sheet 8 of 76 US 2017/0114323 A1
2i/L/DOX 2L-pNSC
Figure 1
FUW TetO-KLF2 AFUW FUW-rtTA TetO-NANOG
2/L/DOX
Figure 1.J Patent Application Publication Apr. 27, 2017. Sheet 9 of 76 US 2017/0114323 A1
Maintenance Screen WIBR3 APE-GFP 2i/L/DOX c. 16
Trypsinize + Seed 5000 cells High throughput FACS analysis (d7)
Withdraw DOX Apply Small molecule library (1 uM)
V dy 70 -- a 60 S LL- I s s
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Figure2B Patent Application Publication Apr. 27, 2017. Sheet 11 of 76 US 2017/0114323 A1
AMN-107
A.
BAY-439006
sax E. GDC-0879
Figure2C Patent Application Publication Apr. 27, 2017. Sheet 12 of 76 US 2017/0114323 A1
2iffDOX --AMN-107 --AZ-628 --BAY-439006 --GDCO879 --PD173074 --SB590885 3. 3: 3 3. & 3 : :
OCT4-APE-GFP Figure 2D
Patent Application Publication Apr. 27, 2017. Sheet 14 of 76 US 2017/0114323 A1
SB590885 Figure2F
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&S Yy Y S
Figure 2G Patent Application Publication US 2017/0114323 A1
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Patent Application Publication Apr. 27, 2017. Sheet 16 of 76 US 2017/0114323 A1
NOLIVE/DEADDISCRIMINATION
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WITH LIVE/DEADDISCRIMINATION 2+Dox 2+SB59 = 26.5
rzczaez
2i/L/DOX Figure 3A Patent Application Publication Apr. 27, 2017. Sheet 17 of 76 US 2017/0114323 A1
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Patent Application Publication US 2017/0114323 A1
2/L/DOX 2i/L/SB59+Kinase inhibitor Plate Figure 3C Patent Application Publication Apr. 27, 2017. Sheet 19 of 76 US 2017/0114323 A1
Figure 3D Patent Application Publication Apr. 27, 2017. Sheet 20 of 76 US 2017/0114323 A1
P2-DOX N1 OuMCH 23 uMCH 0.3 uM CH s--~~~~~~~~~~~~<~::~~~~*~ ZOMCH
Joe6e?uedded 10 uMPD03 maeaer:???r.aeramrmrnm,
10 uMSB59 1 uMSB59 0.5uMSB59 0.1 uMSB59
Figure 3E Patent Application Publication Apr. 27, 2017. Sheet 21 of 76 US 2017/0114323 A1
104 < 10 3 10 2 102 56% 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 GFP-A GFP-A GFP-A Figure 3F Patent Application Publication Apr. 27, 2017. Sheet 22 of 76 US 2017/0114323 A1
WBR3 OCT4-APE-GFP WBR3--KN +KN cl.21 (P3-DOX) cl.1
y Š & Š S & Figure 3G
Figure3H Patent Application Publication Apr. 27, 2017. Sheet 23 of 76 US 2017/0114323 A1
Transgene-free conversion
OCT4-APE-GFP primed hESCs Apply 5i/L Trypsinize + Replate 100K cells
Figure 4A
WBR3 OCT4-APE-GFP
; Figure 4B Patent Application Publication Apr. 27, 2017. Sheet 24 of 76 US 2017/0114323 A1
S
S Ss
59%. 101 3% 02103 104 105 101102103 104 105 101102103 104 105 -> OCT 4-APE-GFP Figure 4C Patent Application Publication Apr. 27, 2017. Sheet 25 of 76 US 2017/0114323 A1
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Figure 4E Patent Application Publication Apr. 27, 2017. Sheet 26 of 76 US 2017/0114323 A1
5i/L/FA -PDO325901 -M12
S SS. S S.S S. s SkS Sk& S S S S. S. S. 84% m 82% 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 -FGF -Activin A -hF
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101 102 103 104 105 101 102 103 104 105 101 102 103 104 105
-F-A -M+-CH -F-A-WH
101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 OCT4-NPE-GFP Figure 4F Patent Application Publication Apr. 27, 2017. Sheet 27 of 76 US 2017/0114323 A1
OCT4 NANOG KLF4
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SOX2 REX1 KLF2
Figure 4G Patent Application Publication Apr. 27, 2017. Sheet 28 of 76 US 2017/0114323 A1
JNK p38 PKC ROCK ROCK ROCK BMP BRAF
RT SFSRNRGRSERGEN ŠGFRSSActives ŠRNRSSRNRSSRNRSSRRS Albumax+N2 mTesr1 2096 KSR 20%6. KSR N2B27 f or 20% KSR (bFGF--TGFB) Figure 5A Patent Application Publication Apr. 27, 2017. Sheet 29 of 76 US 2017/0114323 A1
Phase FP
101 1 02 103 104 105
101 102 103 104 105 ?
101 102 103 104 105 Figure 5B OCT4-APE-GFP Patent Application Publication Apr. 27, 2017. Sheet 30 of 76 US 2017/0114323 A1
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N2B27/L/A 20% KSR/L/A 5i-JNKi (6i) 5i-JNKi (6i)
100101 102 103 104 105 100101 102 103 104 105 -> OCT4-APE-GFP Figure 5D Patent Application Publication Apr. 27, 2017. Sheet 31 of 76 US 2017/0114323 A1
SOX2 1 5
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Patent Application Publication Apr. 27, 2017. Sheet 33 of 76 US 2017/0114323 A1
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Patent Application Publication Apr. 27, 2017. Sheet 35 of 76 US 2017/0114323 A1
This study TFCP2L 1 KLF4 DNMT3L HORMAD 1
-log2 fold change
Gafni et al., 2013
-log2 fold change Figure6C Patent Application Publication Apr. 27, 2017. Sheet 36 of 76 US 2017/0114323 A1
Naive vs. Primed (Human) EThis study
Gafnietal of 2013
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Naive vs. Primed (Mouse) =
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Figure 6E Patent Application Publication Apr. 27, 2017. Sheet 37 of 76 US 2017/0114323 A1
NANOG C 12 O 10 (T. 8 4.r 5 4.6 7.% 2. 7.7 is2 S. 2 2 9 0 WBR2 WBR3 WBR3- OCT4-APE tdTomato GFP hESM 6OO STELLA EGafni, 2013 2.5i/L/FA 4 O O
, WBR2 WBR3 WIBR3- OCT4-APE tdTomato GFP Figure 6F Patent Application Publication Apr. 27, 2017. Sheet 38 of 76 US 2017/0114323 A1
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cell no.
5i-A 48 Nanog primed 43 Gafni, 2013 40 5i-A 63 Kf4 primed 81 Gafni, 2013 60 5i-A 63 ReX1 primed 81 Gafni, 2013 6O 5i-A 111 Octa. primed 124 Gafni, 2013 100
O 50 1 OO 150 200 25O 3OO median mRNA/cell Figure 6H Patent Application Publication Apr. 27, 2017. Sheet 40 of 76 US 2017/0114323 A1
HOXA9 10kb.
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Naive NANOGmma 2 kb
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Figure 7E Patent Application Publication Apr. 27, 2017. Sheet 43 of 76 US 2017/0114323 A1
Primed Naive
H3K4me3 H3K27me3 H3K4me.3 H3K27me3
-5kb Figure 7F Patent Application Publication Apr. 27, 2017. Sheet 44 of 76 US 2017/0114323 A1
". H3K4me3 Primed -H3K4me3 Naive
-3 kb TSS End +3 kb
H3K27me3 Primed ^w-H3K27me3 Naive
–3 kb TSS End +3 kb Figure 7G Patent Application Publication Apr. 27, 2017. Sheet 45 of 76 US 2017/0114323 A1
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PEKO:46kb->
Figure 8A Patent Application Publication Apr. 27, 2017. Sheet 46 of 76 US 2017/0114323 A1
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Figure 8C Patent Application Publication US 2017/0114323 A1
Patent Application Publication
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2ifLFDMSO 2ifL/DOX 2i/L+kinase inhibitor plate 3 (1 uM) Figure 9A
Patent Application Publication Apr. 27, 2017. Sheet 51 of 76 US 2017/0114323 A1
2i/L/DOX 25 OK 105 < 2OOK 514 10 <150K , ; 1OOK :310 2 210 5OK 5 101 100 1 0 1 102 103 104 105 101 102 103 104 105 - > --> DAP GFP 2i/L/SB59 (P2-DOX)
105 s
10 9 10 3 102 Q 101
100 101 102 103 104 105 10 1 10 2 10 3 10 4. 10 5 -D -> DAP GFP Figure 9C Patent Application Publication Apr. 27, 2017. Sheet 52 of 76 US 2017/0114323 A1
XMD 8 92 PDO332991 KINOO1 244 SU11274 KINOO1 220 KU55933
85) AÐ 69 |19 2ifL/DOX 2/L/SB59+LINCS plate II (1 uM) Figure 10A-1
Patent Application Publication Apr. 27, 2017. Sheet 53 of 76 US 2017/0114323 A1
PHA-665752* BBF-1120 SU11248
2ifL/DOX 2i/L/SB59+LINCS plate III (1 uM) Figure 10A-2
Apr. 27, 2017. Sheet 54 of 76 US 2017/0114323 A1
2i/L/SB59 2i/L/SB59+LINCS plate IV (1 uM) Figure 10A-3 Patent Application Publication Apr. 27, 2017. Sheet 55 of 76 US 2017/0114323 A1
Compound Target(s)
Figure 10B Patent Application Publication Apr. 27, 2017. Sheet 56 of 76 US 2017/0114323 A1
410 5 d. 104 A. S ... 103 102 s 101 101 102 103 104 105 Comp-GFP-A
s: 10 5
104 V 9 10 102 J 10 1 58% SSSSSS 101 102 103 104 105 Š Comp-GFP-A H) OCT4-NPE-GFP Figure 10C Patent Application Publication Apr. 27, 2017. Sheet 57 of 76 US 2017/0114323 A1
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Patent Application Publication Apr. 27, 2017. Sheet 60 of 76 US 2017/0114323 A1
Control (P3) +JNKi (P3) +p38i (P3) +JNK+p38i (P3) +Insulin (P3)
: Ag9 100101102 103 104 105M 100101102 1030405 100101102 103 10 105 10 100101102 103 104 105r 10 100101102 103 104 105 Figure 12A OCT4-APE-GFP Patent Application Publication Apr. 27, 2017. Sheet 61 of 76 US 2017/0114323 A1
3. KLF2 3 KLF4
2.5 2.5 S 2 2 sh a a 1.5 15 di VD 2 C. - 1 1
O.5 O.5
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20%. KSR/L/A-5i
Control ( P3) +JNKi (P3) --Insulin (P3)
8 |-$
101 102 103 104 105 Figure 12C OCT4-APE-GFP
Patent Application Publication Apr. 27, 2017. Sheet 63 of 76 US 2017/0114323 A1
2. hESM Gafnietal. (2013) 5i/L/FA s 3O KLF4 3 6OO REXT y 2 T. U S.S.ar 20 ga 400 g s 10 S:J 53200 is O & O - O WIBR2 WBR3 WIBR3- OCT4-APE- WBR2 WIBR3 WIBR3- OCT4-APE todTomato GFP toTomato GFP Figure 13A Patent Application Publication Apr. 27, 2017. Sheet 64 of 76 US 2017/0114323 A1
KHDC1L 30000 5000
Patent Application Publication Apr. 27, 2017. Sheet 65 of 76 US 2017/0114323 A1
TFCP2L1
hESM
hESM
Figure 13B Continued Patent Application Publication Apr. 27, 2017. Sheet 66 of 76 US 2017/0114323 A1
DPPA2 2000 33 1500 90 C.2 &V. 1000 W 3 60 59. at 30 Z CU 500 ? O 1 - 'g''g''g''g''g''s's hESM 6i/L/A 35iia 9 sãi a C S. S. S. C S ob Figure 13B Continued
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WBR3 OCT4-APE-GFP (5i/L)
WBR3 OCT4-APE-GFP (5/L/FA)
WBR3 AAVS1-tdTomato (5i/L/FA) S Figure 14A Patent Application Publication Apr. 27, 2017. Sheet 68 of 76 US 2017/0114323 A1
Figure 14B Patent Application Publication Apr. 27, 2017. Sheet 69 of 76 US 2017/0114323 A1
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VGL.?un61 Patent Application Publication Apr. 27, 2017. Sheet 71 of 76 US 2017/0114323 A1
3 passages after DOX withdrawal (1:10 density splitting):
105 105 105 | 10 104 104 : 103 103 |10 102 102 102 is 101 101 11ol S. & O96 & FO 3.:* 310 a. 100101 102 103 104 105 10 100101 102 103 104 105 10 100101 102 103 104 105 100101 102 103 104 105
OCT4-APE-GFP Figure 15B Patent Application Publication Apr. 27, 2017. Sheet 72 of 76 US 2017/0114323 A1
ExO. KLF2 EXO, NANOG VIM 1.2 1.2
O.9
O.6
O.3 WSHU, ?ZZZZZZZZZZZZZZZZZZAXOGI/T/?Z
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Figure 15C Patent Application Publication Apr. 27, 2017. Sheet 73 of 76 US 2017/0114323 A1
Passage 1 Passage 2 Passage 3 --- 2OE-06 2OE-06 1,6E+06 r r 5 16E--06 it 16E--06 T g 12E--06 w 1.E+06 1.2E--O6 38.OE+05 al 8.OE-05 80E-05 - C S 40E+05 4.OE-05 4.OE-05 C l, O.OE+00 OOE-00 O.OE-00 W. SCX s.& s.S s.S is 9> si& s is ita is > |Vd |Ognd||XEÐd NET\/|ZdDEW snoop :JouOC] ZdDEW Patent Application Publication Apr. 27, 2017. Sheet 75 of 76 US 2017/0114323 A1 Naive conversion of GFP'? Tomato primed line: 102 103 104 105 PE-Texas Red-A 103 104 PE-Texas Red-A Figure 17B PE-Texas Red-A Patent Application Publication Apr. 27, 2017. Sheet 76 of 76 US 2017/0114323 A1 Naive conversion of GFP/Tomato primed line: 150 3 1950 O 125 930. E 109 5569970 3S 7539 C 338 23 2.2.2 104;is 102 103 104 105 Mean: 8592 1 O2 103 104 105 PE-Texas Red-A 103 103 104 105 PE-Texas Red-A US 2017/0114323 A1 Apr. 27, 2017 USES OF KINASE INHIBITORS FOR -continued INDUCING AND MANTAINING (B) PLURIPOTENCY RELATED APPLICATIONS (RPI)oing 1^5 (R) XB31S sy 0001. This application claims priority under 35 U.S.C. S119(e) to U.S. provisional applications, U.S. Ser. No. \ / Cl LB2 l XB2 -(R'). 62/014,674, filed Jun. 19, 2014, and U.S. Ser. No. 62/045, 2 337, filed Sep. 3, 2014, each of which is incorporated herein by reference. GOVERNMENT SUPPORT 0002. This invention was made with U.S. Government support under grant number R01-CA084198 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention. BACKGROUND OF THE INVENTION 0003 Human pluripotent stem cells, including embry onic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great promise for regenerative medicine and disease modeling (Hanna et al., 2010b). Full realization of their potential is currently constrained by laborious culture requirements and inconsistencies in developmental potential between lines (Melichar et al., 2011: Osafune et al., 2008). Researchers have had a relatively easy time genetically manipulating and preventing differentiation in mouse ES and iPS cells. However, human ES cells can be more technically demanding to culture and exhibit properties such as slow growth and poor tolerance to passaging as single (F) cells. Thus, there is a need for more effective techniques to isolate and culture human pluripotent stem cells. SUMMARY OF THE INVENTION 0004 One aspect of the disclosure provides methods for changing the pluripotency state of a vertebrate cell to a more nave state, the methods comprising: culturing a pluripotent vertebrate cell in the presence of a serine/threonine-protein kinase B-Raf (BRAF) inhibitor, an epidermal growth factor (G) receptor (EGFR) inhibitor, a vascular endothelial growth factor 1 (VEGFR1) inhibitor, or a fibroblast growth factor receptor 1 (FGFR1) inhibitor; and maintaining the cell in culture under conditions suitable and a time sufficient to convert the pluripotency state of the vertebrate cell to a more nave state than the pluripotency state of the vertebrate cell of culturing step. 0005. Another aspect of the disclosure provides methods for changing the pluripotency state of a vertebrate cell to a (H) more nave state, the method comprising: culturing a pluri (RF) potent vertebrate cell in the presence of a compound of any 21 1s%. N one of Formulae (A) to (L): (RHI)-- l -- (R), N N 4n. 2 (A) RH2 R2 i5 (I) LA1 N N O I R N Yi N R"it 21 21S.n(/ A y1V(R) n4 / \ N R", N N (R)-C/ H R15 US 2017/0114323 A1 Apr. 27, 2017 -continued 0009. Yet another aspect of the disclosure relates to naive (J) pluripotent vertebrate cells, wherein the cells have a global gene expression profile which clusters with naive mouse ESCs as opposed to stem cell lines derived from mouse epiblast (EpiSCs) and/or less naive human ESCs. 0010. Another aspect of the disclosure provides kits for changing the pluripotency state of a vertebrate cell to a more naive state, the kits comprising a pluripotent vertebrate cell; and cell culture medium comprising a serine/threonine protein kinase B-Raf (BRAF) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a vascular endothelial growth factor 1 (VEGFR1) inhibitor, or a fibroblast growth factor receptor 1 (FGFR1) inhibitor. (K) 0011. In yet another aspect, the present disclosure pro O NN1 RK2 vides compounds, compositions, and kits described herein RK11 for use in a method of the present disclosure. 0012. The details of particular embodiments of the inven O 4N tion are set forth herein. Other features, objects, and advan - (R), tages of the invention will be apparent from the Detailed 2. S RK -N Description, the Figures, the Examples, and the Claims. DEFINITIONS N -- (R), 0013 Definitions of specific functional groups and 21 chemical terms are described in more detail below. The chemical elements are identified in accordance with the (L) Periodic Table of the Elements, CAS version, Handbook of (R'), Chemistry and Physics, 75" Ed., inside cover, and specific functional groups are generally defined as described therein. N A Additionally, general principles of organic chemistry, as \ 5 st well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5" Edition, John Wiley & Sons, Inc., New York, 2001: Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers. Some Modern Methods of Organic Synthesis, 3" Edition, Cambridge University Press, Cam and pharmaceutically acceptable salts, Solvates, hydrates, bridge, 1987. polymorphs, co-crystals, tautomers, stereoisomers, isotopi 0014 Compounds described herein can comprise one or cally labeled derivatives, and prodrugs thereof, and main more asymmetric centers, and thus can exist in various taining the cell in culture under conditions Suitable and a Stereoisomeric forms, e.g., enantiomers and/or diastereom time sufficient to convert the pluripotency state of the ers. For example, the compounds described herein can be in vertebrate cell to a more naive state than the pluripotency the form of an individual enantiomer, diastereomer or geo state of the vertebrate cell of culturing step. In some embodi metric isomer, or can be in the form of a mixture of ments, the time sufficient to convert the pluripotency state of Stereoisomers, including racemic mixtures and mixtures the vertebrate cell to a more naive state is at least about 5 enriched in one or more stereoisomer. Isomers can be days (e.g., about 10 days). isolated from mixtures by methods known to those skilled in 0006 Exemplary compounds useful in a system, compo the art, including chiral high pressure liquid chromatography sition, kit, or method described herein include, but are not (HPLC) and the formation and crystallization of chiral salts: limited to, AMG706, AMN-107, AZ-628, BAY 73-4506, or preferred isomers can be prepared by asymmetric Syn BAY-439006, GDC-0879, KIN001-260, SB590885, theses. See, for example, Jacques et al., Enantiomers, Race CHIR99021, KIN001-244, KU55933, SP600125, mates and Resolutions (Wiley Interscience, New York, PD0332991 PD173074, WZ-7043, BIBF-1120, PHA 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. 665752, SU11248, SU11274, KIN001-220, WH-4-023, Stereochemistry of Carbon Compounds (McGrawHill, NY. WH-4-025, XMD 8-92, XMD11-50, XMD 8-85, IM12, 1962); and Wilen, S. H. Tables of Resolving Agents and PD0325901, and Y-27632. Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre 0007 Another aspect of the disclosure provides compo Dame Press, Notre Dame, Ind. 1972). The disclosure addi sitions comprising naive pluripotent vertebrate cells pro tionally encompasses compounds as individual isomers Sub duced by a method described herein. stantially free of other isomers, and alternatively, as mix 0008 Another aspect of the disclosure provides naive tures of various isomers. pluripotent vertebrate cells, wherein the cells have a global 0015. In a formula. --- is absent or a single bond, and gene expression profile which clusters with naive mouse , or is a single or double bond. ESCs as opposed to stem cell lines derived from mouse 0016. The term "heteroatom” refers to an atom that is not epiblast (EpiSCs) and/or less naive human ESCs. hydrogen or carbon. In certain embodiments, the heteroatom US 2017/0114323 A1 Apr. 27, 2017 is nitrogen. In certain embodiments, the heteroatom is parent chain. In certain embodiments, a heteroalkyl group oxygen. In certain embodiments, the heteroatom is Sulfur. refers to a saturated group having from 1 to 10 carbon atoms 0017. When a range of values is listed, it is intended to and 1 or more heteroatoms within the parent chain (“het encompass each value and Sub range within the range. For eroCo alkyl). In some embodiments, a heteroalkyl group example "Ce alkyl is intended to encompass, C, C, C, is a saturated group having 1 to 9 carbon atoms and 1 or C4, Cs, C6, C1-6, C1-s: C-4 C-3; C-2, C2-6, C2-s: C2-4, C2-3. more heteroatoms within the parent chain ("heteroCo. C3-6. Cs-s: C-4 C4-6. CAs, and CS-6 alkyl. alkyl). In some embodiments, a heteroalkyl group is a 0018. The term “aliphatic” refers to alkyl, alkenyl, alky saturated group having 1 to 8 carbon atoms and 1 or more nyl, and carbocyclic groups. Likewise, the term "heteroali heteroatoms within the parent chain ("heteroCs alkyl). In phatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl, Some embodiments, a heteroalkyl group is a Saturated group and heterocyclic groups. having 1 to 7 carbon atoms and 1 or more heteroatoms 0019. The term “alkyl refers to a radical of a straight within the parent chain ("heteroC, alkyl). In some chain or branched saturated hydrocarbon group having from embodiments, a heteroalkyl group is a saturated group 1 to 10 carbon atoms (“Clio alkyl). In some embodiments, having 1 to 6 carbon atoms and 1 or more heteroatoms an alkyl group has 1 to 9 carbon atoms (“Co alkyl). In within the parent chain ("heteroC alkyl). In some Some embodiments, an alkyl group has 1 to 8 carbon atoms embodiments, a heteroalkyl group is a saturated group (“Cs alkyl). In some embodiments, an alkyl group has 1 having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the to 7 carbon atoms (“C, alkyl). In some embodiments, an parent chain ("heteroCs alkyl). In some embodiments, a alkyl group has 1 to 6 carbon atoms ("C. alkyl). In some heteroalkyl group is a Saturated group having 1 to 4 carbon embodiments, an alkyl group has 1 to 5 carbon atoms (“Cs atoms and for 2 heteroatoms within the parent chain (“het alkyl). In some embodiments, an alkyl group has 1 to 4 eroC alkyl). In some embodiments, a heteroalkyl group carbon atoms ("C. alkyl). In some embodiments, an alkyl is a saturated group having 1 to 3 carbon atoms and 1 group has 1 to 3 carbon atoms ("C. alkyl). In some heteroatom within the parent chain ("heteroC alkyl). In embodiments, an alkyl group has 1 to 2 carbon atoms (“C. Some embodiments, a heteroalkyl group is a Saturated group alkyl). In some embodiments, an alkyl group has 1 carbon having 1 to 2 carbon atoms and 1 heteroatom within the atom (“C alkyl). In some embodiments, an alkyl group has parent chain ("heteroC alkyl). In some embodiments, a 2 to 6 carbon atoms ("Ce alkyl). Examples of C alkyl heteroalkyl group is a saturated group having 1 carbon atom groups include methyl (C), ethyl (C), n-propyl (C), iso and 1 heteroatom ("heteroC alkyl). In some embodiments, propyl (C), n-butyl (C), tert-butyl (C), sec-butyl (C), a heteroalkyl group is a saturated group having 2 to 6 carbon iso-butyl (C), n-pentyl (Cs), 3-pentanyl (Cs), amyl (Cs), atoms and 1 or 2 heteroatoms within the parent chain neopentyl (Cs), 3-methyl-2-butanyl (Cs), tertiary amyl (Cs), ("heteroC alkyl). Unless otherwise specified, each and n-hexyl (C). Additional examples of alkyl groups instance of a heteroalkyl group is independently unsubsti include n-heptyl (C7), n-octyl (Cs) and the like. Unless tuted (an "unsubstituted heteroalkyl) or substituted (a “sub otherwise specified, each instance of an alkyl group is stituted heteroalkyl) with one or more substituents. In independently unsubstituted (an “unsubstituted alkyl) or certain embodiments, the heteroalkyl group is an unsubsti substituted (a “substituted alkyl) with one or more sub tuted heteroCo alkyl. In certain embodiments, the het stituents. In certain embodiments, the alkyl group is an eroalkyl group is a substituted heteroCo alkyl. unsubstituted Co alkyl (e.g., —CH). In certain embodi 0022. The term “alkenyl refers to a radical of a straight ments, the alkyl group is a Substituted Co alkyl. chain or branched hydrocarbon group having from 2 to 10 0020. The term “haloalkyl is a substituted alkyl group, carbon atoms and one or more carbon-carbon double bonds wherein one or more of the hydrogen atoms are indepen (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an dently replaced by a halogen, e.g., fluoro, bromo, chloro, or alkenyl group has 2 to 9 carbon atoms (“Coalkenyl). In iodo. “Perhaloalkyl is a subset of haloalkyl, and refers to an Some embodiments, an alkenyl group has 2 to 8 carbon alkyl group wherein all of the hydrogen atoms are indepen atoms (“Cs alkenyl). In some embodiments, an alkenyl dently replaced by a halogen, e.g., fluoro, bromo, chloro, or group has 2 to 7 carbon atoms ("C-7 alkenyl). In some iodo. In some embodiments, the haloalkyl moiety has 1 to 8 embodiments, an alkenyl group has 2 to 6 carbon atoms carbon atoms ("Cs haloalkyl). In some embodiments, the ("Ce alkenyl). In some embodiments, an alkenyl group haloalkyl moiety has 1 to 6 carbon atoms (“Chaloalkyl). has 2 to 5 carbon atoms (“Cs alkenyl). In some embodi In some embodiments, the haloalkyl moiety has 1 to 4 ments, an alkenyl group has 2 to 4 carbon atoms ("Ca carbon atoms (“Chaloalkyl). In some embodiments, the alkenyl). In some embodiments, an alkenyl group has 2 to haloalkyl moiety has 1 to 3 carbon atoms (“Chaloalkyl). 3 carbon atoms (“C- alkenyl). In some embodiments, an In some embodiments, the haloalkyl moiety has 1 to 2 alkenyl group has 2 carbon atoms ("Calkenyl). The one or carbon atoms (“Chaloalkyl). In some embodiments, all more carbon-carbon double bonds can be internal (such as in of the haloalkyl hydrogen atoms are replaced with fluoro to 2-butenyl) or terminal (such as in 1-butenyl). Examples of provide a perfluoroalkyl group. In some embodiments, all of C. alkenyl groups include ethenyl (C), 1-propenyl (C), the haloalkyl hydrogen atoms are replaced with chloro to 2-propenyl (C), 1-butenyl (C), 2-butenyl (C), butadienyl provide a “perchloroalkyl group. Examples of haloalkyl (C), and the like. Examples of Calkenyl groups include groups include —CF, —CFCF —CFCFCF, —CC1, the aforementioned Calkenyl groups as well as pentenyl —CFC1, —CFC1, and the like. (Cs), pentadienyl (Cs), hexenyl (C), and the like. Additional 0021. The term "heteroalkyl refers to an alkyl group, examples of alkenyl include heptenyl (C7), octenyl (Cs), which further includes at least one heteroatom (e.g., 1, 2, 3, octatrienyl (Cs), and the like. Unless otherwise specified, or 4 heteroatoms) selected from oxygen, nitrogen, or Sulfur each instance of an alkenyl group is independently unsub within (i.e., inserted between adjacent carbon atoms of) stituted (an "unsubstituted alkenyl) or substituted (a "sub and/or placed at one or more terminal position(s) of the stituted alkenyl) with one or more substituents. In certain US 2017/0114323 A1 Apr. 27, 2017 embodiments, the alkenyl group is an unsubstituted Co embodiments, an alkynyl group has 2 carbon atoms (“C alkenyl. In certain embodiments, the alkenyl group is a alkynyl). The one or more carbon carbon triple bonds can Substituted Co alkenyl. In an alkenyl group, a C=C be internal (such as in 2-butynyl) or terminal (Such as in double bond for which the stereochemistry is unspecified 1-butynyl). Examples of C alkynyl groups include, with (e.g., —CH=CHCH or out limitation, ethynyl (C), 1-propynyl (C), 2-propynyl (C), 1-butynyl (C), 2-butynyl (C), and the like. Examples of C- alkenyl groups include the aforementioned Ca alkynyl groups as well as pentynyl (Cs), hexynyl (C), and the like. Additional examples of alkynyl include heptynyl WN’) (C7), octynyl (Cs), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsub may be an (E)- or (Z)-double bond. stituted (an “unsubstituted alkynyl) or substituted (a "sub 0023 The term “heteroalkenyl refers to an alkenyl stituted alkynyl) with one or more substituents. In certain group, which further includes at least one heteroatom (e.g., embodiments, the alkynyl group is an unsubstituted Co 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or alkynyl. In certain embodiments, the alkynyl group is a Sulfur within (i.e., inserted between adjacent carbon atoms Substituted Co alkynyl. of) and/or placed at one or more terminal position(s) of the (0025. The term “heteroalkynyl refers to an alkynyl parent chain. In certain embodiments, a heteroalkenyl group group, which further includes at least one heteroatom (e.g., refers to a group having from 2 to 10 carbon atoms, at least 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or one double bond, and 1 or more heteroatoms within the Sulfur within (i.e., inserted between adjacent carbon atoms parent chain ("heteroCo alkenyl). In some embodiments, of) and/or placed at one or more terminal position(s) of the a heteroalkenyl group has 2 to 9 carbon atoms at least one parent chain. In certain embodiments, a heteroalkynyl group double bond, and 1 or more heteroatoms within the parent refers to a group having from 2 to 10 carbon atoms, at least chain ("heteroCo alkenyl). In some embodiments, a het one triple bond, and 1 or more heteroatoms within the parent eroalkenyl group has 2 to 8 carbon atoms, at least one double chain ("heteroCo alkynyl). In some embodiments, a bond, and 1 or more heteroatoms within the parent chain heteroalkynyl group has 2 to 9 carbon atoms, at least one ("heteroCs alkenyl). In some embodiments, a heteroalk triple bond, and 1 or more heteroatoms within the parent enyl group has 2 to 7 carbon atoms, at least one double bond, chain ("heteroCo alkynyl). In some embodiments, a het and 1 or more heteroatoms within the parent chain ("het eroalkynyl group has 2 to 8 carbon atoms, at least one triple eroC, alkenyl). In some embodiments, a heteroalkenyl bond, and 1 or more heteroatoms within the parent chain group has 2 to 6 carbon atoms, at least one double bond, and ("heteroCs alkynyl). In some embodiments, a heteroalky 1 or more heteroatoms within the parent chain ("heteroC nyl group has 2 to 7 carbon atoms, at least one triple bond, alkenyl). In some embodiments, a heteroalkenyl group has and 1 or more heteroatoms within the parent chain (“het 2 to 5 carbon atoms, at least one double bond, and 1 or 2 eroC, alkynyl). In some embodiments, a heteroalkynyl heteroatoms within the parent chain ("heteroCs alkenyl). group has 2 to 6 carbon atoms, at least one triple bond, and In some embodiments, a heteroalkenyl group has 2 to 4 1 or more heteroatoms within the parent chain ("heteroC carbon atoms, at least one double bond, and for 2 heteroa alkynyl). In some embodiments, a heteroalkynyl group has toms within the parent chain ("heteroCalkenyl). In some 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 embodiments, a heteroalkenyl group has 2 to 3 carbon heteroatoms within the parent chain ("heteroCs alkynyl). atoms, at least one double bond, and 1 heteroatom within the In some embodiments, a heteroalkynyl group has 2 to 4 parent chain ("heteroC alkenyl). In some embodiments, carbon atoms, at least one triple bond, and for 2 heteroatoms a heteroalkenyl group has 2 to 6 carbon atoms, at least one within the parent chain ("heteroC alkynyl). In some double bond, and 1 or 2 heteroatoms within the parent chain embodiments, a heteroalkynyl group has 2 to 3 carbon ("heteroC alkenyl). Unless otherwise specified, each atoms, at least one triple bond, and 1 heteroatom within the instance of a heteroalkenyl group is independently unsub parent chain ("heteroC alkynyl). In some embodiments, stituted (an "unsubstituted heteroalkenyl) or substituted (a a heteroalkynyl group has 2 to 6 carbon atoms, at least one “substituted heteroalkenyl) with one or more substituents. triple bond, and 1 or 2 heteroatoms within the parent chain In certain embodiments, the heteroalkenyl group is an ("heteroC alkynyl). Unless otherwise specified, each unsubstituted heteroCo alkenyl. In certain embodiments, instance of a heteroalkynyl group is independently unsub the heteroalkenyl group is a Substituted heteroCo alkenyl. stituted (an "unsubstituted heteroalkynyl) or substituted (a 0024. The term “alkynyl refers to a radical of a straight “substituted heteroalkynyl) with one or more substituents. chain or branched hydrocarbon group having from 2 to 10 In certain embodiments, the heteroalkynyl group is an carbon atoms and one or more carbon-carbon triple bonds unsubstituted heteroCo alkynyl. In certain embodiments, (e.g., 1, 2, 3, or 4 triple bonds) ("Clo alkynyl). In some the heteroalkynyl group is a Substituted heteroCo alkynyl. embodiments, an alkynyl group has 2 to 9 carbon atoms (0026. The term “carbocyclyl or “carbocyclic” refers to a (“Co alkynyl). In some embodiments, an alkynyl group radical of a nonaromatic cyclic hydrocarbon group having has 2 to 8 carbon atoms (“Cs alkynyl). In some embodi from 3 to 14 ring carbon atoms ("C. carbocyclyl) and ments, an alkynyl group has 2 to 7 carbon atoms ("C-7 Zero heteroatoms in the nonaromatic ring system. In some alkynyl). In some embodiments, an alkynyl group has 2 to embodiments, a carbocyclyl group has 3 to 10 ring carbon 6 carbon atoms ("C. alkynyl). In some embodiments, an atoms (“Clio carbocyclyl). In some embodiments, a car alkynyl group has 2 to 5 carbon atoms ("Cs alkynyl). In bocyclyl group has 3 to 8 ring carbon atoms ("Cs carbo Some embodiments, an alkynyl group has 2 to 4 carbon cyclyl). In some embodiments, a carbocyclyl group has 3 to atoms ("C. alkynyl). In some embodiments, an alkynyl 7 ring carbon atoms ("C-7 carbocyclyl). In some embodi group has 2 to 3 carbon atoms (“C- alkynyl). In some ments, a carbocyclyl group has 3 to 6 ring carbon atoms US 2017/0114323 A1 Apr. 27, 2017 ("Ce carbocyclyl). In some embodiments, a carbocyclyl (0027. The term “heterocyclyl or "heterocyclic” refers to group has 4 to 6 ring carbon atoms ("C. carbocyclyl). In a radical of a 3 to 14-membered nonaromatic ring system Some embodiments, a carbocyclyl group has 5 to 6 ring having ring carbon atoms and 1 to 4 ring heteroatoms, carbon atoms ("Cs carbocyclyl). In some embodiments, a wherein each heteroatom is independently selected from carbocyclyl group has 5 to 10 ring carbon atoms (“Cso nitrogen, oxygen, and Sulfur (3-14 membered heterocy carbocyclyl). Exemplary C. carbocyclyl groups include, clyl). In heterocyclyl groups that contain one or more without limitation, cyclopropyl (C), cyclopropenyl (C), nitrogen atoms, the point of attachment can be a carbon or cyclobutyl (C), cyclobutenyl (C), cyclopentyl (Cs), cyclo nitrogen atom, as Valency permits. A heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl) or poly pentenyl (Cs), cyclohexyl (C), cyclohexenyl (C), cyclo cyclic (e.g., a fused, bridged or spiro ring system such as a hexadienyl (C), and the like. Exemplary Cs carbocyclyl bicyclic system (“bicyclic heterocyclyl) or tricyclic system groups include, without limitation, the aforementioned C. (“tricyclic heterocyclyl)), and can be saturated or can carbocyclyl groups as well as cycloheptyl (C7), cyclohep contain one or more carbon carbon double or triple bonds. tenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7). Heterocyclyl polycyclic ring systems can include one or cyclooctyl (Cs), cyclooctenyl (Cs), bicyclo2.2.1]heptanyl more heteroatoms in one or both rings. “Heterocyclyl also (C7), bicyclo[2.2.2]octanyl (Cs), and the like. Exemplary includes ring systems wherein the heterocyclyl ring, as Clo carbocyclyl groups include, without limitation, the defined above, is fused with one or more carbocyclyl groups aforementioned Cls carbocyclyl groups as well as wherein the point of attachment is either on the carbocyclyl cyclononyl (C), cyclononenyl (C), cyclodecyl (C), or heterocyclyl ring, or ring systems wherein the heterocy cyclodecenyl (Co), octahydro-1H-indenyl (Co.), decahy clyl ring, as defined above, is fused with one or more aryl or dronaphthalenyl (C), spiro4.5 decanyl (C), and the like. heteroaryl groups, wherein the point of attachment is on the As the foregoing examples illustrate, in certain embodi heterocyclyl ring, and in Such instances, the number of ring ments, the carbocyclyl group is either monocyclic ("mono members continue to designate the number of ring members cyclic carbocyclyl) or polycyclic (e.g., containing a fused, in the heterocyclyl ring system. Unless otherwise specified, bridged or spiro ring system such as a bicyclic system each instance of heterocyclyl is independently unsubstituted (“bicyclic carbocyclyl) or tricyclic system (“tricyclic car (an "unsubstituted heterocyclyl) or substituted (a “substi bocyclyl)) and can be saturated or can contain one or more tuted heterocyclyl) with one or more substituents. In certain carbon-carbon double or triple bonds. “Carbocyclyl also embodiments, the heterocyclyl group is an unsubstituted includes ring systems wherein the carbocyclyl ring, as 3-14-membered heterocyclyl. In certain embodiments, the defined above, is fused with one or more aryl or heteroaryl heterocyclyl group is a substituted 3-14-membered hetero groups wherein the point of attachment is on the carbocyclyl cyclyl. ring, and in Such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring 0028. In some embodiments, a heterocyclyl group is a system. Unless otherwise specified, each instance of a 5-10-membered nonaromatic ring system having ring car carbocyclyl group is independently unsubstituted (an bon atoms and 1-4 ring heteroatoms, wherein each heteroa “unsubstituted carbocyclyl) or substituted (a "substituted tom is independently selected from nitrogen, oxygen, and carbocyclyl) with one or more substituents. In certain sulfur (“5-10-membered heterocyclyl). In some embodi embodiments, the carbocyclyl group is an unsubstituted ments, a heterocyclyl group is a 5-8-membered nonaromatic C. carbocyclyl. In certain embodiments, the carbocyclyl ring system having ring carbon atoms and 1-4 ring heteroa group is a substituted C. carbocyclyl. In some embodi toms, wherein each heteroatom is independently selected ments, carbocyclyl is a monocyclic, Saturated carbocyclyl from nitrogen, oxygen, and Sulfur ("5-8-membered hetero group having from 3 to 14 ring carbon atoms (“C. cyclyl). In some embodiments, a heterocyclyl group is a cycloalkyl). In some embodiments, a cycloalkyl group has 5-6-membered nonaromatic ring system having ring carbon 3 to 10 ring carbon atoms (“Clio cycloalkyl). In some atoms and 1-4 ring heteroatoms, wherein each heteroatom is embodiments, a cycloalkyl group has 3 to 8 ring carbon independently selected from nitrogen, oxygen, and Sulfur atoms ("Cs cycloalkyl). In some embodiments, a (“5-6-membered heterocyclyl). In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms ("C. 5-6-membered heterocyclyl has 1-3 ring heteroatoms cycloalkyl). In some embodiments, a cycloalkyl group has selected from nitrogen, oxygen, and Sulfur. In some embodi 4 to 6 ring carbon atoms ("Ce cycloalkyl). In some ments, the 5-6-membered heterocyclyl has 1-2 ring heteroa embodiments, a cycloalkyl group has 5 to 6 ring carbon toms selected from nitrogen, oxygen, and Sulfur. In some atoms ("Cs cycloalkyl). In some embodiments, a embodiments, the 5-6 membered heterocyclyl has 1 ring cycloalkyl group has 5 to 10 ring carbon atoms (“Cso heteroatom selected from nitrogen, oxygen, and Sulfur. cycloalkyl). Examples of Cse cycloalkyl groups include 0029. Exemplary 3-membered heterocyclyl groups con cyclopentyl (Cs) and cyclohexyl (Cs). Examples of C. taining 1 heteroatom include, without limitation, azirdinyl, cycloalkyl groups include the aforementioned Cs. oxiranyl, and thiranyl. Exemplary 4-membered heterocy cycloalkyl groups as well as cyclopropyl (C) and cyclobu clyl groups containing 1 heteroatom include, without limi tyl (C). Examples of Css cycloalkyl groups include the tation, aZetidinyl, oxetanyl, and thietanyl. Exemplary aforementioned C. cycloalkyl groups as well as cyclohep 5-membered heterocyclyl groups containing 1 heteroatom tyl (C7) and cyclooctyl (Cs). Unless otherwise specified, include, without limitation, tetrahydrofuranyl, dihydrofura each instance of a cycloalkyl group is independently unsub nyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, stituted (an "unsubstituted cycloalky1') or substituted (a dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5 mem “substituted cycloalkyl) with one or more substituents. In bered heterocyclyl groups containing 2 heteroatoms include, certain embodiments, the cycloalkyl group is an unsubsti without limitation, dioxolanyl, Oxathiolanyl and dithiolanyl. tuted C. cycloalkyl. In certain embodiments, the Exemplary 5-membered heterocyclyl groups containing 3 cycloalkyl group is a Substituted C. cycloalkyl. heteroatoms include, without limitation, triazolinyl, oxadi US 2017/0114323 A1 Apr. 27, 2017 azolinyl, and thiadiazolinyl. Exemplary 6-membered hetero polycyclic ring systems can include one or more heteroa cyclyl groups containing 1 heteroatom include, without toms in one or both rings. “Heteroaryl includes ring sys limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, tems wherein the heteroaryl ring, as defined above, is fused and thianyl. Exemplary 6-membered heterocyclyl groups with one or more carbocyclyl or heterocyclyl groups containing 2 heteroatoms include, without limitation, piper wherein the point of attachment is on the heteroaryl ring, and azinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary in Such instances, the number of ring members continue to 6-membered heterocyclyl groups containing 2 heteroatoms designate the number of ring members in the heteroaryl ring include, without limitation, triaZinanyl. Exemplary 7-mem system. “Heteroaryl also includes ring systems wherein the bered heterocyclyl groups containing 1 heteroatom include, heteroaryl ring, as defined above, is fused with one or more without limitation, azepanyl, oxepanyl and thiepanyl. Exem aryl groups wherein the point of attachment is either on the plary 8-membered heterocyclyl groups containing 1 heteroa aryl or heteroaryl ring, and in Such instances, the number of tom include, without limitation, azocanyl, oxecanyl and ring members designates the number of ring members in the thiocanyl. Exemplary bicyclic heterocyclyl groups include, fused polycyclic (aryl/heteroaryl) ring system. Polycyclic without limitation, indolinyl, isoindolinyl, dihydrobenzo heteroaryl groups wherein one ring does not contain a furanyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetra heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the hydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, like) the point of attachment can be on either ring, i.e., either tetrahydroisoquinolinyl, decahydroquinolinyl, decahy the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that droisoquinolinyl, octahydrochromenyl, octahydroiso does not contain a heteroatom (e.g., 5-indolyl). chromenyl, decahydronaphthyridinyl, decahydro-1.8-naph 0033. In some embodiments, a heteroaryl group is a thyridinyl, octahydropyrrolo3.2-bipyrrole, indolinyl, 5-10-membered aromatic ring system having ring carbon phthalimidyl, naphthalimidyl, chromanyl, chromenyl, atoms and 1-4 ring heteroatoms provided in the aromatic 1H-benzoe 1.4 diazepinyl, 1,4,5,7-tetrahydropyrano3,4- ring system, wherein each heteroatom is independently bipyrrolyl, 5,6-dihydro-4H-furo3,2-bipyrrolyl, 6,7-di selected from nitrogen, oxygen, and sulfur ("5-10 membered hydro-5H-furo3.2-bipyranyl, 5,7-dihydro-4H-thieno2.3-c. heteroaryl'). In some embodiments, a heteroaryl group is a pyranyl, 2,3-dihydro-1H-pyrrolo2,3-bipyridinyl, 2.3- 5-8-membered aromatic ring system having ring carbon dihydrofuro2,3-bipyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo atoms and 1-4 ring heteroatoms provided in the aromatic 2,3-bipyridinyl, 4,5,6,7-tetrahydrofuro3.2-cpyridinyl, ring system, wherein each heteroatom is independently 4,5,6,7-tetrahydrothieno 3.2-bipyridinyl, 1,2,3,4-tetra selected from nitrogen, oxygen, and Sulfur ("5-8-membered hydro-1,6-naphthyridinyl, and the like. heteroaryl). In some embodiments, a heteroaryl group is a 0030 The term “aryl” refers to a radical of a monocyclic 5-6-membered aromatic ring system having ring carbon or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring atoms and 1-4 ring heteroatoms provided in the aromatic system (e.g., having 6, 10, or 14 JC electrons shared in a ring system, wherein each heteroatom is independently cyclic array) having 6-14 ring carbon atoms and Zero selected from nitrogen, oxygen, and Sulfur ("5-6-membered heteroatoms provided in the aromatic ring system (“Ca heteroaryl'). In some embodiments, the 5-6-membered het aryl'). In some embodiments, an aryl group has 6 ring eroaryl has 1-3 ring heteroatoms selected from nitrogen, carbon atoms ("Caryl', e.g., phenyl). In some embodi oxygen, and Sulfur. In some embodiments, the 5-6-mem ments, an aryl group has 10 ring carbon atoms (“Co aryl': bered heteroaryl has 1-2 ring heteroatoms selected from e.g., naphthyl Such as 1-naphthyl and 2-naphthyl). In some nitrogen, oxygen, and Sulfur. In some embodiments, the embodiments, an aryl group has 14 ring carbon atoms ("Ca 5-6-membered heteroaryl has 1 ring heteroatom selected aryl'; e.g., anthracyl). "Aryl also includes ring systems from nitrogen, oxygen, and Sulfur. Unless otherwise speci wherein the aryl ring, as defined above, is fused with one or fied, each instance of a heteroaryl group is independently more carbocyclyl or heterocyclyl groups wherein the radical unsubstituted (an “unsubstituted heteroaryl') or substituted or point of attachment is on the aryl ring, and in Such (a “substituted heteroaryl') with one or more substituents. In instances, the number of carbon atoms continue to designate certain embodiments, the heteroaryl group is an unsubsti the number of carbon atoms in the aryl ring system. Unless tuted 5-14 membered heteroaryl. In certain embodiments, otherwise specified, each instance of an aryl group is inde the heteroaryl group is a substituted 5-14 membered het pendently unsubstituted (an “unsubstituted aryl') or substi eroaryl. tuted (a “substituted aryl') with one or more substituents. In 0034 Exemplary 5-membered heteroaryl groups contain certain embodiments, the aryl group is an unsubstituted ing 1 heteroatom include, without limitation, pyrrolyl, fura C. aryl. In certain embodiments, the aryl group is a nyl, and thiophenyl. Exemplary 5-membered heteroaryl Substituted Caryl. groups containing 2 heteroatoms include, without limitation, 0031 “Aralkyl is a subset of “alkyl and refers to an imidazolyl pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and alkyl group Substituted by an aryl group, wherein the point isothiazolyl. Exemplary 5-membered heteroaryl groups con of attachment is on the alkyl moiety. taining 3 heteroatoms include, without limitation, triazolyl, 0032. The term “heteroaryl” refers to a radical of a oxadiazolyl, and thiadiazolyl. Exemplary 5-membered het 5-14-membered monocyclic or polycyclic (e.g., bicyclic, eroaryl groups containing 4 heteroatoms include, without tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or limitation, tetrazolyl. Exemplary 6-membered heteroaryl 14 JC electrons shared in a cyclic array) having ring carbon groups containing 1 heteroatom include, without limitation, atoms and 1-4 ring heteroatoms provided in the aromatic pyridinyl. Exemplary 6-membered heteroaryl groups con ring system, wherein each heteroatom is independently taining 2 heteroatoms include, without limitation, pyridazi selected from nitrogen, oxygen, and Sulfur ("5-14-mem nyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered bered heteroaryl'). In heteroaryl groups that contain one or heteroaryl groups containing 3 or 4 heteroatoms include, more nitrogen atoms, the point of attachment can be a without limitation, triazinyl and tetrazinyl, respectively. carbon or nitrogen atom, as Valency permits. Heteroaryl Exemplary 7-membered heteroaryl groups containing 1 het US 2017/0114323 A1 Apr. 27, 2017 eroatom include, without limitation, azepinyl, oxepinyl, and Such as nitrogen may have hydrogen Substituents and/or any thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, suitable substituent as described herein which satisfy the without limitation, indolyl, isoindolyl, indazolyl, benzotri valencies of the heteroatoms and results in the formation of azolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl. a stable moiety. benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoX 0041 Exemplary carbon atom substituents include, but azolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, are not limited to, halogen, —CN. —NO. —N, -SOH, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6- - SOH, -OH, -OR", ON(R), N(R), bicyclic heteroaryl groups include, without limitation, naph N(R'),"X, N(OR)R, SH, SR', SSR, thyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, C(=O)R, COH, CHO, C(OR), COR", quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tri OC(=O)R", OCOR', C(=O)N(R), OC cyclic heteroaryl groups include, without limitation, phenan (—O)N(R), NRC(O)R*, NRCR, thridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothi NRC(=O)N(R), C(-NR)R’, C(-NR) azinyl, phenoxazinyl and phenazinyl. OR, OC( NR)R*, OC( NR)OR*, 0035) “Heteroaralkyl is a subset of “alkyl and refers to C(—NR)N(R), OC(—NR)N(R), NRC an alkyl group Substituted by a heteroaryl group, wherein the (—NR)N(R), C(=O)NR'SOR, NRSOR", point of attachment is on the alkyl moiety. - SON(R), -SOR', SOOR", OSOR", 0036. The term “unsaturated bond refers to a double or S(=O)R', OS(=O)R', Si(R'), OSi(R), C triple bond. (—S)N(R), C(=O)SR', C(—S)SR', SC(-S) 0037. The term “unsaturated” or “partially unsaturated” SR, SC(=O)SR, OC(=O)SR', SC(=O)CR, refers to a moiety that includes at least one double or triple SC(=O)R', P(=O)(R') - P(=O)(OR), OP bond. (=O)(R'), OP(=O)(OR), -P(=O)(N(R).), 0038. The term “saturated refers to a moiety that does OP(=O)(N(R).), NRP(=O)(R), NRP(=) not contain a double or triple bond, i.e., the moiety only (OR), NRP(=O)(N(R).), -P(R), -P(OR), contains single bonds. P(R), X, P(OR),"X, P(R) - P(OR), 0039. Affixing the suffix '-ene' to a group indicates the OP(R), OP(R)'X, OP(OR), OP(OR)," group is a divalent moiety, e.g., alkylene is the divalent X, OP(R) - OP(OR), B(R), B(OR), moiety of alkyl, alkenylene is the divalent moiety of alkenyl, —BR“(OR), Co alkyl, Co perhaloalkyl, Co alk alkynylene is the divalent moiety of alkynyl, heteroalkylene enyl, Co alkynyl, heteroCo alkyl, heteroCo alkenyl, is the divalent moiety of heteroalkyl, heteroalkenylene is the heteroCo alkynyl, Co carbocyclyl, 3-14 membered het divalent moiety of heteroalkenyl, heteroalkynylene is the erocyclyl, C. aryl, and 5-14 membered heteroaryl, divalent moiety of heteroalkynyl, carbocyclylene is the wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalk divalent moiety of carbocyclyl, heterocyclylene is the diva enyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and lent moiety of heterocyclyl, arylene is the divalent moiety of heteroaryl is independently substituted with 0, 1, 2, 3, 4, or aryl, and heteroarylene is the divalent moiety of heteroaryl. 5 R“groups; wherein X is a counterion; 0040. A group is optionally substituted unless expressly 0042 or two geminal hydrogens on a carbon atom are provided otherwise. In certain embodiments, alkyl, alkenyl, replaced with the group —O, =S, —NN(R) =NNRC alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocy (—O)R', -NNRC(=O)CR', NNRS(—O).R.", clyl, heterocyclyl, aryl, and heteroaryl groups are optionally —NR, or —NOR; substituted. “Optionally substituted” refers to a group which 0043 each instance of R“ is, independently, selected may be substituted or unsubstituted (e.g., “substituted” or from Co alkyl, Co perhaloalkyl, Co alkenyl, Co “unsubstituted alkyl, “substituted” or “unsubstituted alk alkynyl, heteroC 10 alkyl, heteroCo alkenyl, heteroC enyl, “substituted” or “unsubstituted alkynyl, “substituted alkynyl, Co carbocyclyl, 3-14 membered heterocyclyl, or “unsubstituted heteroalkyl, “substituted” or “unsubsti Caryl, and 5-14 membered heteroaryl, or two R“groups tuted heteroalkenyl, “substituted” or “unsubstituted het are joined to form a 3-14 membered heterocyclyl or 5-14 eroalkynyl, “substituted or “unsubstituted carbocyclyl, membered heteroaryl ring, wherein each alkyl, alkenyl, “substituted' or “unsubstituted heterocyclyl, “substituted alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocy or “unsubstituted aryl or “substituted” or “unsubstituted clyl, heterocyclyl, aryl, and heteroaryl is independently heteroaryl group). In general, the term 'substituted” means substituted with 0, 1, 2, 3, 4, or 5 R' groups; that at least one hydrogen present on a group is replaced with 0044) each instance of R” is, independently, selected a permissible Substituent, e.g., a Substituent which upon from hydrogen, OH, -OR", N(R), —CN, Substitution results in a stable compound, e.g., a compound C(=O)R", C(=O)N(R), COR, -SOR", which does not spontaneously undergo transformation Such C(-NR)OR', C(-NR)N(R), SON(R), as by rearrangement, cyclization, elimination, or other reac - SOR-, -SOOR-, -SOR", C(=S)N(R), tion. Unless otherwise indicated, a “substituted group has a C(=O)SR, C(=S)SR, P(=O)(R'), P(=O) substituent at one or more substitutable positions of the (OR), —P(=O)(N(R)2)2, Co alkyl, Co perha group, and when more than one position in any given loalkyl, Coalkenyl, Co alkynyl, heteroCoalkyl, het structure is substituted, the substituent is either the same or eroCoalkenyl, heteroCoalkynyl, Co carbocyclyl. 3-14 different at each position. The term “substituted' is contem membered heterocyclyl, C. aryl, and 5-14 membered plated to include substitution with all permissible substitu heteroaryl, or two R' groups are joined to form a 3-14 ents of organic compounds, and includes any of the Sub membered heterocyclyl or 5-14 membered heteroaryl ring, stituents described herein that results in the formation of a wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalk stable compound. The present disclosure contemplates any enyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and and all Such combinations in order to arrive at a stable heteroaryl is independently substituted with 0, 1, 2, 3, 4, or compound. For purposes of this disclosure, heteroatoms 5 R“groups; wherein X is a counterion; US 2017/0114323 A1 Apr. 27, 2017 proviso that the nitrogen atom directly attached to the parent limited to, formamide, acetamide, chloroacetamide, trichlo molecule is not substituted with hydrogen. roacetamide, trifluoroacetamide, phenylacetamide, 3-phe 0057 The term “trisubstituted amino” refers to an amino nylpropanamide, picolinamide, 3-pyridylcarboxamide, group wherein the nitrogen atom directly attached to the N-benzoylphenylalanyl derivative, benzamide, p-phenyl parent molecule is Substituted with three groups, and benzamide, o-nitophenylacetamide, o-nitrophenoxyacet includes groups selected from N(R'), and N(R')." amide, acetoacetamide, (N'-dithiobenzyloxyacylamino)ac X, wherein R” and X are as defined herein. etamide, 3-(p-hydroxyphenyl)propanamide, 3-(o- 0058. The term “carbonyl refers a group wherein the nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy) carbon directly attached to the parent molecule is sp? propanamide, 2-methyl-2-(o-phenylaZophenoxy) hybridized, and is Substituted with an oxygen, nitrogen or propanamide, 4-chlorobutanamide, 3-methyl-3- Sulfur atom, e.g., a group selected from ketones (—C(=O) nitrobutanamide, o-nitrocinnamide, N-acetylmethionine R“), carboxylic acids (—COH), aldehydes ( CHO), derivative, o-nitrobenzamide and o-(benzoyloxymethyl) esters ( COR", C(=O)SR", C(=S)SR"), amides benzamide. ( C(=O)N(R), C(=O)NR'SOR, C(—S)N 0064 Nitrogen protecting groups such as carbamate (R),), and imines ( C(=NR)R', C(=NR)OR), groups (e.g., —C(=O)CR') include, but are not limited to, —C(=NR)N(R).), wherein R* and R are as defined methyl carbamate, ethyl carbamate, 9-fluorenylmethyl car herein. bamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2, 0059. The term “silyl refers to the group - Si(R'), 7-dibromo) fluoroenylmethyl carbamate, 2,7-di-t-butyl-9- wherein R' is as defined herein. (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)methyl 0060. The term “oxo” refers to the group —O, and the carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate term “thiooxo” refers to the group —S. (Phenoc), 2.2.2-trichloroethyl carbamate (Troc), 2-trimeth 0061 Nitrogen atoms can be substituted or unsubstituted ylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate as Valency permits, and include primary, secondary, tertiary, (h7), 1-(1-adamantyl)-1-methylethyl carbamate (Adipoc), and quaternary nitrogen atoms. Exemplary nitrogen atom 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-di Substituents include, but are not limited to, hydrogen, —OH, bromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2.2.2- OR, N(R) - CN, C(=O)R", C(=O)N(R) trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-bipheny COR', SOR, C(-NR)R’, C(—NR) lyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1- OR', C(=NR)N(R), SON(R) - SOR, methylethyl carbamate (t-Bumeoc), 2-(2- and 4'-pyridyl) –SOOR-, -SOR', C(=S)N(R), C(=O)SR, ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido) C(=S)SR-, - P(=O)(OR), P(=O)(R'), ethyl carbamate, t-butyl carbamate (BOC or Boc), —P(=O)(N(R)), Co alkyl, Co perhaloalkyl, Co 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl alkenyl, C2-io alkynyl, heteroCo-alkyl, heteroCoalk carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cin enyl, heteroCoalkynyl, Co carbocyclyl, 3-14 membered namyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), heterocyclyl, Caryl, and 5-14 membered heteroaryl, or 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, two R groups attached to an Natom are joined to form a alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxy 3-14 membered heterocyclyl or 5-14 membered heteroaryl benzyl carbamate (MoZ), p-nitobenzyl carbamate, p-bro ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, het mobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlo eroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, robenzyl carbamate, 4-methylsulfinylbenzyl carbamate and heteroaryl is independently substituted with 0, 1, 2, 3, 4, (MSZ), 9-anthrylmethyl carbamate, diphenylmethyl carbam or 5 R“groups, and wherein R", R. R* and R* are as ate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl defined above. carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, 2-(1,3- 0062. In certain embodiments, the substituent present on dithianyl)methyl carbamate (Dmoc), 4-methylthiophenyl the nitrogen atom is an nitrogen protecting group (also carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate referred to herein as an "amino protecting group'). Nitrogen (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenyl protecting groups include, but are not limited to. —OH, phosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2- –OR, N(R), C(=O)R", C(=O)N(R), cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbam COR', SOR", C(-NR)R', C(-NR) ate, p-(dihydroxyboryl)benzyl carbamate, OR', C(=NR)N(R), -SON(R), -SOR, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6- - SOOR, SOR", C(=S)N(R), C(=O)SR, chromonylmethyl carbamate (Tcroc), m-nitrophenyl car —C(=S)SR, Co alkyl (e.g., aralkyl, heteroaralkyl). bamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl car Coalkenyl, C2-io alkynyl, heteroCo alkyl, heteroCo bamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o- alkenyl, heteroCo alkynyl, Co carbocyclyl, 3-14 mem nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl bered heterocyclyl, C. aryl, and 5-14 membered het thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl car eroaryl groups, wherein each alkyl, alkenyl, alkynyl, het bamate, cyclohexyl carbamate, cyclopentyl carbamate, eroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, cyclopropylmethyl carbamate, p-decyloxybenzyl carbam heterocyclyl, aralkyl, aryl, and heteroaryl is independently ate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethyl substituted with 0, 1, 2, 3, 4, or 5 R“groups, and wherein carboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dim R", R, R- and R* are as defined herein. Nitrogen ethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl protecting groups are well known in the art and include those carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl described in detail in Protecting Groups in Organic Synthe carbamate, 2-iodoethyl carbamate, isoborynl carbamate, sis, T. W. Greene and P. G. M. Wuts, 3' edition, John Wiley isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxy & Sons, 1999, incorporated herein by reference. phenylazo)benzyl carbamate, 1-methylcyclobutyl carbam 0063 For example, nitrogen protecting groups such as ate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropy amide groups (e.g., —C(=O)R“) include, but are not lmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl US 2017/0114323 A1 Apr. 27, 2017 carbamate, 1-methyl-1-(p-phenylaZophenyl)ethyl carbam N(R), C(=O)SR', C(=O)R, COR", ate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4- C(=O)N(R), C(—NR)R*, C(—NR)OR*, pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo) C(-NR)N(R), S(—O)R', SOR', Si(R) benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, , - P(R) - P(R),"X, -P(ORc), -P(OR),"X, 4-(trimethylammonium)benzyl carbamate, and 2.4.6-trim - P(=O)(R') - P(=O)(OR), and P(=O)(N(R).) ethylbenzyl carbamate. , wherein X, R", R', and R* are as defined herein. 0065 Nitrogen protecting groups such as sulfonamide Oxygen protecting groups are well known in the art and groups (e.g., —S(=O)R') include, but are not limited to, include those described in detail in Protecting Groups in p-toluenesulfonamide (TS), benzenesulfonamide, 2.3.6.- Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 trimethyl-4-methoxybenzenesulfonamide (Mitr), 2.4.6- edition, John Wiley & Sons, 1999, incorporated herein by trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4- reference. methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- 0068 Exemplary oxygen protecting groups include, but methoxybenzenesulfonamide (Mte), are not limited to, methyl, methoxylmethyl (MOM), meth 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylben ylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsi Zenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzene lyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), sulfonamide (iMds), 2.2.5.7.8-pentamethylchroman-6-sul p-methoxybenzyloxymethyl (PMBM), (4-methoxyphe fonamide (Pmc), methanesulfonamide (Ms), noxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxym B-trimethylsilylethanesulfonamide (SES), 9-anthracenesul ethyl, 4-pentenyloxymethyl (POM), siloxymethyl, fonamide, 4-(4,8-dimethoxynaphthylmethyl)benzenesulfo 2-methoxyethoxymethyl (MEM), 2.2.2-trichloroethoxym namide (DNMBS), benzylsulfonamide, trifluoromethylsul ethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl) fonamide, and phenacylsulfonamide. ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bro 0066 Other nitrogen protecting groups include, but are motetrahydropyranyl, tetrahydrothiopyranyl, not limited to, phenothiazinyl-(10)-acyl derivative, N'-p- 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl toluenesulfonylaminoacyl derivative, N'-phenylaminothioa (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetra cyl derivative, N-benzoylphenylalanyl derivative, N-acetyl hydrothiopyranyl S.S.-dioxide, 1-(2-chloro-4-methyl)phe methionine derivative, 4.5-diphenyl-3-oxazolin-2-one, nyl-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tet N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenyl rahydrofuranyl, tetrahydrothiofuranyl, 2.3.3a,4,5,6,7,7a maleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyld octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, isilylazacyclopentane adduct (STABASE), 5-substituted 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1- 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-ben 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted Zyloxy-2-fluoroethyl, 2.2.2-trichloroethyl, 2-trimethylsilyl 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, ethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, N-2-(trimethylsilyl)ethoxymethylamine (SEM), N-3-ac p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), etoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-py p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, roolin-3-yl)amine, quaternary ammonium salts, N-ben p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cya Zylamine, N-di(4-methoxyphenyl)methylamine, N-5- nobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2- dibenzosuberylamine, N-triphenylmethylamine (Tr), N-(4- picolyl N-oxido, diphenylmethyl, p.p'-dinitrobenzhydryl, methoxyphenyl)diphenylmethylamine (MMTr), N-9- 5-dibenzosuberyl, triphenylmethyl, C.-naphthyldiphenylm phenylfluorenylamine (PhF), N-2,7-dichloro-9- ethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphe fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), nyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4-bro N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyl mophenacyloxyphenyl)diphenylmethyl, 4,4',4'-tris(4,5- eneamine, N-benzylideneamine, N-p-methoxybenzylide dichlorophthalimidophenyl)methyl, 4,4',4'-tris neamine, N-diphenylmethyleneamine, N-(2-pyridyl)mesi (levulinoyloxyphenyl)methyl, 4,4',4'-tris tylmethyleneamine, N—(N',N'-dimethylaminomethylene) (benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4,4'- amine, N,N'-isopropylidenediamine, N-p- dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1- nitrobenzylideneamine, N-Salicylideneamine, N-5- pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl) phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, phenylmethyleneamine. N-cyclohexylideneamine, N-(5.5- benzisothiazolyl S.S.-dioxido, trimethylsilyl (TMS), trieth dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane ylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsi derivative, N-diphenylborinic acid derivative, N-phenyl lyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthex (pentaacylchromium- or tungsten)acylamine, N-copper ylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, amine N-oxide, diphenylphosphinamide (Dpp), dimethylth diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl iophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), (TBMPS), formate, benzoylformate, acetate, chloroacetate, dialkyl phosphoramidates, dibenzyl phosphoramidate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxy diphenyl phosphoramidate, benzenesulfenamide, o-ni acetate, triphenylmethoxyacetate, phenoxyacetate, p-chloro trobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfena phenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (le mide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxy Vulinate), 4.4-(ethylenedithio)pentanoate benzenesulfenamide, triphenylmethylsulfenamide, and (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 3-nitropyridinesulfenamide (Npys). 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2.4.6- 0067. In certain embodiments, the substituent present on trimethylbenzoate (mesitoate), methyl carbonate, 9-fluore an oxygen atom is an oxygen protecting group (also referred nylmethyl carbonate (Fmoc), ethyl carbonate, 2.2.2-trichlo to herein as an “hydroxyl protecting group'). Oxygen pro roethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate tecting groups include, but are not limited to. —R“. (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(tri US 2017/0114323 A1 Apr. 27, 2017 phenylphosphonio)ethyl carbonate (Peoc), isobutyl carbon heteroatoms. However, a non-chain Substituent of an ali ate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC phatic chain may include any atoms, including hydrogen or Boc), p-nitrophenyl carbonate, benzyl carbonate, atoms, carbon atoms, and heteroatoms. For example, ali p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbon phatic chain —CH(C.HCH)— includes one chain atom C, one hydrogen atom on C", and non-chain substituent ate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, -(CH2CH). The term "Caliphatic chain.” wherein X is S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, a positive integer, refers to an aliphatic chain that includes methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, x number of chain atom(s) between the two radicals of the 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, aliphatic chain. If there is more than one possible value of 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, X, the smallest possible value of X is used for the definition 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxym of the aliphatic chain. For example, —CH(CHs)— is a C ethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2.6- aliphatic chain, and dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4- bis(1, 1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- methyl-2-butenoate, o-(methoxyacyl)benzoate, C.-naph thoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiami date, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, is a Caliphatic chain. When a range of values is used, the Sulfate, methanesulfonate (mesylate), benzylsulfonate, and meaning of the range is as described herein. For example, a tosylate (Ts). Coaliphatic chain refers to an aliphatic chain where the 0069. In certain embodiments, the substituent present on number of chain atoms of the shortest chain of carbon atoms a Sulfur atom is a Sulfur protecting group (also referred to as immediately between the two radicals of the aliphatic chain a “thiol protecting group'). Sulfur protecting groups include, is 3, 4, 5, 6, 7, 8, 9, or 10. An aliphatic chain may be but are not limited to R', N(R), C(=O)SR", saturated (e.g., -(CH2) ). An aliphatic chain may also be C(=O)R, COR*, de O)N(R6) C(-NR) unsaturated and include one or more C–C and/or C=C R, C( NR)OR, de NRN(R), —S(=O) bonds anywhere in the aliphatic chain. For instance, R“, SOR', Si(R) P(R) P(R),"X, —CH=CH (CH) , CH C=C CH , and P(OR), -P(OR), X P(=O)(R') - P(=O) —C=C CH=CH- are all examples of a unsubstituted (ORC), and -P(=O)(N(R).), wherein R. R. and and unsaturated aliphatic chain. In certain embodiments, the R* are as defined herein. Sulfur protecting groups are well aliphatic chain is unsubstituted (e.g., -C=C- or —(CH) known in the art and include those described in detail in ). In certain embodiments, the aliphatic chain is Substi Protecting Groups in Organic Synthesis, T. W. Greene and P. tuted (e.g., -CH(CHs)—and —CF ). Any two Substitu G. M. Wuts, 3' edition, John Wiley & Sons, 1999, incor ents on the aliphatic chain may be joined to form an porated herein by reference. optionally substituted carbocyclyl, optionally substituted 0070 A “counterion' or “anionic counterion' is a nega heterocyclyl, optionally Substituted aryl, or optionally Sub tively charged group associated with a positively charged stituted heteroaryl ring. For instance group in order to maintain electronic neutrality. An anionic counterion may be monovalent (i.e., including one formal negative charge). An anionic counterion may also be mul tivalent (i.e., including more than one formal negative charge). Such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F. Cl, Br, I), NO, CIO, OH, HPO, HCO, HSO, sulfonate ions (e.g., meth /c /\H anSulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor Sulfonate, naphthalene-2- N Sulfonate, naphthalene-1-sulfonic acid-5-Sulfonate, ethan-1- Sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tar trate, glycolate, gluconate, and the like), BF, PF., PF. /\ ^\ AsF, SbF, B3.5-(CF).C.H.I., B(CFs), BPh. Al(OC(CF)), and carborane anions (e.g., CBH or (HCBMesBr)). Exemplary counterions which may be multivalent include CO, HPO, PO, B.O., SO, S.O., carboxylate anions (e.g., tartrate, citrate, fumarate, /As and /cyS maleate, malate, malonate, gluconate. Succinate, glutarate, adipate, pimelate, Suberate, aZelate, sebacate, Salicylate, are all examples of an aliphatic chain. In contrast, in certain phthalates, aspartate, glutamate, and the like), and carbo embodiments, aeS. 0071. An “aliphatic chain” refers to a substituted or H unsubstituted divalent alkyl, alkenyl, or alkynyl group. An N N aliphatic chain includes (1) one or more chains of carbon atoms immediately between the two radicals of the aliphatic and / N chain; (2) optionally one or more hydrogen atoms on the N N chain(s) of carbon atoms; and (3) optionally one or more H substituents (“non-chain substituents,” which are not hydro gen) on the chain(s) of carbon atoms. A chain of carbon are not within the scope of the aliphatic chains described atoms consists of consecutively connected carbon atoms herein. A "heteroaliphatic chain” is an aliphatic chain where ("chain atoms) and does not include hydrogen atoms or at least one chain atom of each chain of the aliphatic chain US 2017/0114323 A1 Apr. 27, 2017 is independently replaced with a heteroatom. In certain Representative alkali or alkaline earth metal salts include embodiments, an aliphatic chain described herein is a Ca Sodium, lithium, potassium, calcium, magnesium, and the aliphatic chain, a Caliphatic chain, a C-7 aliphatic chain, like. Further pharmaceutically acceptable salts include, a Caliphatic chain, a Caliphatic chain, or a Caliphatic when appropriate, nontoxic ammonium, quaternary ammo chain. nium, and amine cations formed using counterions such as 0072. In certain embodiments, one, two, or three chain halide, hydroxide, carboxylate, Sulfate, phosphate, nitrate, atoms of an aliphatic chain described herein are indepen lower alkyl Sulfonate, and aryl Sulfonate. dently replaced with —O , —S , NR' , N=, or 0074 The term “solvate” refers to forms of the com =N , wherein each instance of R is independently hydro pound that are associated with a solvent, usually by a gen, Substituted or unsubstituted C. alkyl, or a nitrogen Solvolysis reaction. This physical association may include protecting group. In certain embodiments, one, two, or three hydrogen bonding. Conventional Solvents include water, chain atoms of an aliphatic chain described herein are methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, independently replaced with - O - S -, or - NR'-. In and the like. The compounds described herein may be certain embodiments, the molecular weight of an aliphatic or prepared, e.g., in crystalline form, and may be solvated. heteroaliphatic chain is not more than about 300 g/mol, not Suitable solvates include pharmaceutically acceptable sol more than about 200 g/mol, not more than about 150 g/mol. vates and further include both stoichiometric solvates and not more than about 100 g/mol, not more than about 70 non-stoichiometric Solvates. In certain instances, the Solvate g/mol, not more than about 50 g/mol, or not more than 30 will be capable of isolation, for example, when one or more g/mol. In certain embodiments, an aliphatic or heteroali Solvent molecules are incorporated in the crystal lattice of a phatic chain consists of not more than about 70 atoms, not crystalline solid. “Solvate” encompasses both solution more than about 50 atoms, not more than about 30 atoms, phase and isolatable Solvates. Representative Solvates not more than about 20 atoms, not more than about 15 include hydrates, ethanolates, and methanolates. atoms, or not more than 10 atoms. In certain embodiments, (0075. The term “hydrate” refers to a compound that is an aliphatic or heteroaliphatic chain does not include unsatu associated with water. Typically, the number of the water rated bonds in the shortest chain. In certain embodiments, an molecules contained in a hydrate of a compound is in a aliphatic or heteroaliphatic chain consists of one or two definite ratio to the number of the compound molecules in unsaturated bonds in the shortest chain. In certain embodi the hydrate. Therefore, a hydrate of a compound may be ments, an aliphatic or heteroaliphatic chain includes at least represented, for example, by the general formula R.X HO, one instance of —O as a non-chain Substituent on a chain wherein R is the compound, and X is a number greater than atom (e.g., carbon or Sulfur atom). 0. A given compound may form more than one type of 0073. The term “pharmaceutically acceptable salt” refers hydrate, including, e.g., monohydrates (X is 1), lower to those salts which are, within the scope of sound medical hydrates (X is a number greater than 0 and Smaller than 1, judgment, Suitable for use in contact with the tissues of e.g., hemihydrates (R.0.5 HO)), and polyhydrates (X is a humans and lower animals without undue toxicity, irritation, number greater than 1, e.g., dihydrates (R.2 H2O) and allergic response and the like, and are commensurate with a hexahydrates (R.6 HO)). reasonable benefit/risk ratio. Pharmaceutically acceptable 0076. The term “tautomers' or “tautomeric' refers to two salts are well known in the art. For example, Berge et al., or more interconvertible compounds resulting from at least describe pharmaceutically acceptable salts in detail in J. one formal migration of a hydrogen atom and at least one Pharmaceutical Sciences, 1977. 66, 1-19, incorporated change in Valency (e.g., a single bond to a double bond, a herein by reference. Pharmaceutically acceptable salts of the triple bond to a single bond, or vice versa). The exact ratio compounds of this disclosure include those derived from of the tautomers depends on several factors, including Suitable inorganic and organic acids and bases. Examples of temperature, Solvent, and pH. Tautomerizations (i.e., the pharmaceutically acceptable, nontoxic acid addition salts are reaction providing a tautomeric pair) may catalyzed by acid salts of an amino group formed with inorganic acids such as or base. Exemplary tautomerizations include keto-to-enol, hydrochloric acid, hydrobromic acid, phosphoric acid, Sul amide-to-imide, lactam-to-lactim, enamine-to-imine, and furic acid, and perchloric acid or with organic acids such as enamine-to-(a different enamine) tautomerizations. acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, 0077. It is also to be understood that compounds that Succinic acid, or malonic acid or by using other methods have the same molecular formula but differ in the nature or known in the art such as ion exchange. Other pharmaceu sequence of bonding of their atoms or the arrangement of tically acceptable salts include adipate, alginate, ascorbate, their atoms in space are termed "isomers'. Isomers that aspartate, benzenesulfonate, benzoate, bisulfate, borate, differ in the arrangement of their atoms in space are termed butyrate, camphorate, camphorsulfonate, citrate, cyclopen “stereoisomers'. tanepropionate, digluconate, dodecylsulfate, ethanesul 0078 Stereoisomers that are not mirror images of one fonate, formate, fumarate, glucoheptonate, glycerophos another are termed "diastereomers' and those that are non phate, gluconate, hemisulfate, heptanoate, hexanoate, Superimposable mirror images of each other are termed hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lac “enantiomers’. When a compound has an asymmetric cen tate, laurate, lauryl Sulfate, malate, maleate, malonate, meth ter, for example, it is bonded to four different groups, a pair anesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, of enantiomers is possible. An enantiomer can be charac oleate, oxalate, palmitate, pamoate, pectinate, persulfate, terized by the absolute configuration of its asymmetric 3-phenylpropionate, phosphate, picrate, pivalate, propi center and is described by the R- and S-sequencing rules of onate, Stearate. Succinate, Sulfate, tartrate, thiocyanate, Cahn and Prelog, or by the manner in which the molecule p-toluenesulfonate, undecanoate, Valerate salts, and the like. rotates the plane of polarized light and designated as dex Salts derived from appropriate bases include alkali metal, trorotatory or levorotatory (i.e., as (+) or (-)-isomers respec alkaline earth metal, ammonium and N(C. alkyl) salts. tively). A chiral compound can exist as either individual US 2017/0114323 A1 Apr. 27, 2017 enantiomer or as a mixture thereof. A mixture containing injecting, inhaling, or otherwise introducing a compound or equal proportions of the enantiomers is called a “racemic cell described herein or generated as described herein, or a mixture'. composition thereof, in or on a Subject. 0079. The term “polymorphs' refers to a crystalline form I0085. The terms “treatment,” “treat,” and “treating refer of a compound (or a salt, hydrate, or Solvate thereof) in a to reversing, alleviating, delaying the onset of, or inhibiting particular crystal packing arrangement. All polymorphs have the progress of a disease. In some embodiments, treatment the same elemental composition. Different crystalline forms may be administered after one or more signs or symptoms of usually have different X-ray diffraction patterns, infrared the disease have developed or have been observed. In other spectra, melting points, density, hardness, crystal shape, embodiments, treatment may be administered in the absence optical and electrical properties, stability, and solubility. of signs or symptoms of the disease. For example, treatment Recrystallization solvent, rate of crystallization, Storage may be administered to a susceptible subject prior to the temperature, and other factors may cause one crystal form to onset of symptoms (e.g., in light of a history of symptoms dominate. Various polymorphs of a compound can be pre and/or in light of exposure to a pathogen and/or in light of pared by crystallization under different conditions. detecting that the Subject has a genotype associated with the 0080. The term “prodrugs’ refer to compounds which disease). Treatment may also be continued after symptoms have cleavable groups and become by Solvolysis or under have resolved, for example, to delay or prevent recurrence. physiological conditions the compounds described herein, 0086. The terms “condition,” “disease, and “disorder which are pharmaceutically active in vivo. Such examples are used interchangeably. include, but are not limited to, choline ester derivatives and I0087. A “kinase' is a type of enzyme that transfers the like, N-alkylmorpholine esters and the like. Other phosphate groups from high energy donor molecules. Such derivatives of the compounds described herein have activity as ATP, to specific substrates, referred to as phosphorylation. in both their acid and acid derivative forms, but in the acid Kinases are part of the larger family of phosphotransferases. sensitive form often offer advantages of solubility, tissue One of the largest groups of kinases are protein kinases, compatibility, or delayed release in the mammalian organ which act on and modify the activity of specific proteins. ism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Kinases are used extensively to transmit signals and control Elsevier, Amsterdam 1985). Prodrugs include acid deriva complex processes in cells. Various other kinases act on tives well known to practitioners of the art, such as, for Small molecules Such as lipids, carbohydrates, amino acids, example, esters prepared by reaction of the parent acid with and nucleotides, either for signaling or to prime them for a suitable alcohol, or amides prepared by reaction of the metabolic pathways. Kinases are often named after their parent acid compound with a Substituted or unsubstituted substrates. More than 500 different protein kinases have amine, or acid anhydrides, or mixed anhydrides. Simple been identified in humans. These exemplary human protein aliphatic or aromatic esters, amides, and anhydrides derived kinases include, but are not limited to, AAK1, ABL, ACK, from acidic groups pendant on the compounds described ACTR2, ACTR2B, AKT1, AKT2, AKT3, ALK, ALK1, herein are particular prodrugs. In some cases it is desirable ALK2, ALK4, ALK7, AMPKa1, AMPKa2, ANKRP, to prepare double ester type prodrugs such as (acyloxy)alkyl ANPa, ANPb, ARAF, ARAFps, ARG, Aur A, Aur Aps1, esters or (alkoxycarbonyl)oxy)alkylesters. C-C alkyl, AurAps2. AurB, AurBps 1, Aurc, AXL, BAR^, BARs, C-Cs alkenyl, C-C alkynyl, aryl, C7-C. Substituted aryl, BIKE, BLK, BMPR1A, BMPR1Aps1, BMPR1Aps2, and C7-C arylalkyl esters of the compounds described BMPR1B, BMPR2, BMX, BRAF, BRAFps, BRK, BRSK1, herein may be preferred. BRSK2, BTK, BUB1, BUBR1, CaMK1a, CaMK1b, 0081. The “molecular weight of a monovalent moiety CaMK1d, CaMK1g, CaMK2a, CaMK2b, CaMK2d, —R is calculated by subtracting 1 from the molecular CaMK2g, CaMK4, CaMKK1, CaMKK2, caMLCK, CASK, weight of the compound R. H. The “molecular weight of CCK4, CCRK, CDC2, CDC7, CDK10, CDK11, CDK2, a divalent moiety -L- is calculated by Subtracting 2 from the CDK3, CDK4, CDK4ps, CDK5, CDK5ps, CDK6, CDK7, molecular weight of the compound H-L-H. CDK7ps, CDK8, CDK8ps, CDK9, CDKL1, CDKL2, 0082. The terms “composition' and “formulation' are CDKL3, CDKL4, CDKL5, CGDps, CHED, CHK1, CHK2, used interchangeably. CHK2ps 1, CHK2ps2. CK1a, CK1a2, CKlaps 1, CK1aps2. 0083. A “subject’ to which administration is contem CKlaps3, CK1d, CK1e, CK1 g1, CK1g2. CK1g2ps, CK1 g3, plated includes, but is not limited to, humans (i.e., a male or CK2a1, CK2a1-rs, CK2a2, CLIK1, CLIKIL, CLK1, CLK2, female of any age group, e.g., a pediatric Subject (e.g., CLK2ps, CLK3, CLK3ps, CLK4, COT, CRIK, CR^7, CSK, infant, child, adolescent) or adult Subject (e.g., young adult, CTK, CYGD, CYGF, DAPK1, DAPK2, DAPK3, middle-aged adult, or senior adult)) and/or other non-human DCAMKL1, DCAMKL2, DCAMKL3, DDR1, DDR2, animals, for example, mammals (e.g., primates (e.g., cyno DLK, DMPK1, DMPK2, DRAK1, DRAK2, DYRKIA, molgus monkeys, rhesus monkeys); commercially relevant DYRKIB, DYR^2, DYR^, DYR*, EGFR, EphA1, mammals such as cattle, pigs, horses, sheep, goats, cats, EphA10, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, and/or dogs); and birds (e.g., commercially relevant birds EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, Erk1, Such as chickens, ducks, geese, and/or turkeys). In certain Erk2, Erk3, Erk3ps 1, Erk3ps2, Erk3ps3, Erk3ps4, Erk4, embodiments, the animal is a mammal. The animal may be Erk5, Erk7, FAK, FER, FERps, FES, FGFR1, FGFR2, a male or female at any stage of development. The animal FGFR3, FGFR4, FGR, FLT1, FLT1ps, FLT3, FLT4, FMS, may be a transgenic animal or genetically engineered ani FRK, Fused, FYN, GAK, GCK, GCN2, GCN22, GPR'', mal. In certain embodiments, the Subject is a non-human GPR^, GPR*, GPR^ps, GPR^7, GSK3A, GSK3B, Has animal. In certain embodiments, the animal is a fish or pin, HCK, HER2/ErbB2, HER3/ErbB3, HER4/ErbB4, reptile. HH498, HIPK1, HIPK2, HIPK3, HIPK4, HPK1, HRI, 0084. The term “administer,” “administering,” or HRIps, HSER, HUNK, ICK, IGF1R, IKKa, IKKb, IKKe. “administration” refers to implanting, absorbing, ingesting, ILK, INSR, IRAK1, IRAK2, IRAK3, IRAK4, IRE1, IRE2, US 2017/0114323 A1 Apr. 27, 2017 IRR, ITK, JAK1, JAK2, JAK3, JNK1, JNK2, JNK3, KDR, I0089. By “pluripotency” and pluripotent stem cells it is KHS1, KHS2, KIS, KIT, KSGCps, KSR1, KSR2, LATS1, meant that such cells have the ability under appropriate LATS2, LCK, LIMK1, LIMK2, LIMK2ps, LKB1, LMR1, conditions to differentiate into cells that are derivatives of all LMR2, LMR3, LOK, LRRK1, LRRK2, LTK, LYN, LZK, three embryonic germ layers (endoderm, mesoderm and MAK, MAP2K1, MAP2K1ps, MAP2K2, MAP2K2ps, ectoderm). A pluripotent cell line or cell culture is often MAP2K3, MAP2K4, MAP2K5, MAP2K6, MAP2K7, characterized in that the cells can differentiate into a wide MAP3K1, MAP3K2, MAP3K3, MAP3K4, MAP3K5, variety of cell types in vitro and in vivo. Cells that are able MAP3K6, MAP3K7, MAP3K8, MAPKAPK2, MAP to form teratomas containing cells having characteristics of KAPK3, MAPKAPK5, MAPKAPKps1, MARK1, MARK2, endoderm, mesoderm, and ectoderm when injected into MARK3, MARK4, MARKps01, MARKps02, MARKps03, SCID mice are considered pluripotent. In addition, cells that MARKps04, MARKps05, MARKps07, MARKps08, possess the ability to participate in the formation of chimeras MARKps09, MARKps10, MARKps 11, MARKps12, (upon injection into a blastocyst of the same species that is MARKps13, MARKps 15, MARKps 16, MARKps17. transferred to a suitable foster mother of the same species) MARKps 18, MARKps 19, MARKps20, MARKps21, that survive to term are considered pluripotent. Pluripotent MARKps22, MARKps23, MARKps24, MARKps25, cell types as used in the present invention may be provided MARKps26, MARKps27, MARKps28, MARKps29, in the form of human embryonic stem cells, or human MARKps30, MAST1, MAST2, MAST3, MAST4, MASTL, induced pluripotent cell (iPS cell), or may be derived from MELK, MER, MET, MISR2, MLK1, MLK2, MLK3, a human embryonic stem cell line. MLK4, MLKL, MNK1, MNK1 ps, MNK2, MOK, MOS, 0090. The term “stem cell refers to a vertebrate cell that MPSK1, MPSK1ps, MRCKa, MRCKb, MRCKps, MSK1, has the ability both to self-renew, and to generate differen MSK12, MSK2, MSK22, MSSK1, MST1, MST2, MST3, tiated progeny. The ability to generate differentiated progeny MST3ps, MST4, MUSK, MYO3A, MYO3B, MYT1, may be described as pluripotent (see Morrison et al. (1997) NDR1, NDR2, NEK1, NEK10, NEK11, NEK2, NEK2ps1, Cell 88:287-298). “Embryonic stem cells' (ES cells) are NEK2ps2, NEK2ps3, NEK3, NEK4, NEK4ps, NEK5, pluripotent stem cells derived from the inner cell mass of a NEK6, NEK7, NEK8, NEK9, NIK, NIM1, NLK, NRBP1, blastocyst, an early-stage preimplantation embryo. Pluripo NRBP2, NuaK1, NuaK2, Obscn, Obscn2, OSR1, p38a, tency distinguishes embryonic stem cells from adult stem p38b, p38d, p38g., p70S6K, p70S6Kb, p70S6Kps1, cells found in adults; while embryonic stem cells can p70S6Kps2, PAK1, PAK2, PAK2ps, PAK3, PAK4, PAK5, generate all cell types in the body, adult stem cells are PAK6, PASK, PBK, PCTAIRE1 PCTAIRE2, PCTAIRE3, multipotent and can produce only a limited number of cell PDGFRa, PDGFRb, PDK1, PEK., PFTAIRE1, PFTAIRE2, types. PHKg1, PHKglps 1, PHKglps2, PHKglps3, PHKg2, 0091) “Induced pluripotent stem cells’, abbreviated as PIK3R4, PIM1, PIM2, PIM3, PINK1, PITSLRE, PKACa, iPS cells, are a type of pluripotent stem cell artificially PKACb, PKACg., PKCa, PKCb, PKCd, PKCe, PKCg, derived from a non-pluripotent cell, typically an adult PKCh, PKCi, PKCips, PKCt, PKCz, PKD1, PKD2, PKD3, Somatic cell, by inducing expression of certain genes (e.g., PKG 1, PKG2, PKN1, PKN2, PKN3, PKR, PLK1, injection of an expression construct). Induced pluripotent PLK1ps1, PLK1ps2, PLK2, PLK3, PLK4, PRKX, PRKXps, stem cells are identical in many respects to natural pluripo PRKY, PRP4, PRP4ps, PRPK, PSKH1, PSKH1ps, PSKH2, tent stem cells, such as embryonic stem (ES) cells (e.g., in PYK2, QIK, QSK, RAF1, RAF1 ps, RET, RHOK, RIPK1, their physical properties). They may be the same in their RIPK2, RIPK3, RNAse.I., ROCK1, ROCK2, RON, ROR1, expressions of certain stem cell genes and proteins, chro ROR2, ROS, RSK1, RSK12, RSK2, RSK22, RSK3, matin methylation patterns, doubling time, embryoid body RSK32, RSK4, RSK42, RSKL1, RSKL2, RYK, RYKps, formation, teratoma formation, viable chimera formation, SAKps, SBK, SCYL1, SCYL2, SCYL2ps, SCYL3, SGK, and potency and differentiability. The term “induced pluri SgK050ps, SgK069, SgK071, SgKO85, SgK110, SgK196, potent stem cell encompasses pluripotent cells, that, like SGK2, SgK223, SgK269, SgK288, SGK3, SgK307, embryonic stem (ES) cells, can be cultured over a long SgK384ps, SgK396, SgK424, SgK493, SgK494, SgK495, period of time while maintaining the ability to differentiate SgK496, SIK (e.g., SIK1, SIK2), skMLCK, SLK, Slob, into all types of cells in an organism. However, unlike ES smMLCK, SNRK, SPEG, SPEG2, SRC, SRM, SRPK1, cells (which are typically derived from the inner cell mass of SRPK2, SRPK2ps, SSTK, STK33, STK33ps, STLK3, blastocysts), iPS cells are derived from differentiated STLK5, STLK6, STLK6ps1, STLK6-rs, SuRTK106, SYK, Somatic cells, that is, cells that have a narrower, more TAK1, TAO1, TAO2, TAO3, TBCK, TBK1, TEC, TESK1, defined potential. TESK2, TGFbR1, TGFbR2, TIE1, TIE2, TLK1, TLK1ps, 0092. By “culturing the cell means growing the cells in TLK2, TLK2ps1, TLK2ps2, TNK1, Trad, Trb1, Trb2, Trb3, an artificial, in vitro environment. By “maintaining” means Trio, TRKA, TRKB, TRKC, TSSK1, TSSK2, TSSK3, continuing to grow the cells in culture under Suitable con TSSK4, TSSKps1, TSSKps2, TTBK1, TTBK2, TTK, TTN, ditions until the pluripotency state of the cell is converted to TXK, TYK2, TYK22, TYRO3, TYRO3ps, ULK1, ULK2, a more naive State. ULK3, ULK4, VACAMKL, VRK1, VRK2, VRK3, (0093. A “cell culture medium' (also referred to herein as VRK3ps, Weel, Wee1B, Weel Bps, Weelps 1, Weelps2, a “culture medium' or “medium') is a medium for culturing Wnk1, Wnk2, Wink3, Wink4, YANK1, YANK2, YANK3, cells containing nutrients that maintain cell viability and YES, YESps, YSK1, ZAK, ZAP70, ZC1/HGK, ZC2/TNIK, Support proliferation. The cell culture medium may contain ZC3/MINK, and ZC4/NRK. any of the following nutrients in appropriate amounts and I0088. The term “inhibition”, “inhibiting, “inhibit,” or combinations: salt(s), buffer(s), amino acids, glucose or “inhibitor” refer to the ability of a compound to reduce, other Sugar(s), antibiotics, serum or serum replacement, and slow, halt, or prevent activity of a particular biological other components such as, but not limited to, peptide growth process (e.g., kinase activity) in a cell relative to vehicle. factors, cofactors, and trace elements. Cell culture media US 2017/0114323 A1 Apr. 27, 2017 ordinarily used for particular cell types are known to those BRIEF DESCRIPTION OF THE DRAWINGS skilled in the art. For example, cell culture media of use for 0099 FIG. 1 shows a reporter system for naive human culturing and maintaining pluripotent cells are known in the pluripotency based on endogenous OCT4 distal enhancer art. activity. In FIG. 1A, the proximal enhancer (PE) targeting 0094. In some embodiments, the cell culture medium is strategy in human ESCs containing a 2A-GFP sequence in chemically defined medium. In some embodiments, cell frame with the 3' UTR of OCT4 is shown. FIG. 1B is a culture medium is serum-free medium, e.g. mTeSR1TM Southern blot analysis confirming disruption of the PE in medium (StemCell Technologies, Vancouver, BC). In some OCT4-GFP ESCs. Ndel-digested genomic DNA was embodiments, the culture medium comprises one or more hybridized with 5' and 3' external probes. Expected fragment supplements, such as, but not limited to N2 and B27. In size: WT (wild type)=5.6 kb, T (targeted)=6.4 kb. FIG. 1C Some embodiments, the cell culture medium comprises a shows the images of OCT4-GFP human ESCs before (left) serum replacement composition. In some embodiments, the and after TALEN-mediated deletion of the PE (right). FIG. cell culture medium comprises low amount, such as less than 1D shows the single molecule RNAFISH analysis for OCT4 1% or less than 0.5%, of knock-out serum replacement and GFP transcripts in OCT4-GFP human ESCs before and medium. In some embodiments, the cell culture medium after TALEN-mediated disruption of the PE. FIG. 1E shows does not comprise a serum replacement composition. In the flow cytometric analysis of the proportion of OCT4 Some embodiments, the cell culture medium comprises an APE-GFP+ cells obtained after DOX induction of lentiviral activator of STAT3 pathways, for example but not limited to KLF2, NANOG or KLF2+NANOG. Following primary leukemia inhibitory factor (LIF). In some embodiments, the infection WIBR3 human ESCs containing the OCT4-APE cell culture comprises serum free recombinant human LIF. GFP reporter allele were trypsinized and treated with hESM, 2i/L, or 2i/L/DOX for one week. FIG. 1F are phase and 0.095. In some embodiments, the cell culture medium fluorescence images and flow cytometric analysis of the comprises a basal medium to which one or more Supple proportion of OCT4-APE-GFP+ cells of a clonal line of 38 ments are added, such as: DMEM/F12, Neurobasal, N2 hESCs derived in 2i/L/DOX (left). Phase and fluorescence Supplement, 10 mL B27 Supplement, human LIF, glutamine, images and flow cytometric analysis after replating in the nonessential amino acids, 3-mercaptoethanol, penicillin absence of DOX for one week (right). FIG. 1G shows the streptomycin, and/or BSA (Sigma). In some embodiments, quantitative gene expression analysis for FUW-KLF2. the supplemented basal cell culture medium further com FUW-NANOG, endogenous OCT4, and endogenous KLF4 prises fibroblast growth factor 2 (FGF2) and 1%, 0.8%, in WIBR3 hESCS cultured in hESM and clonal OCT4-APE 0.6%, 0.4%, 0.2%, or 0.1% KSR. GFP+ derivatives generated in 2i/L/DOX. FIG. 1H are phase 0096. In some embodiments, the cell culture medium is and fluorescence images of primitive neural stem cells free or essentially free of components of non-human origin. (pNSCs) derived by treating WIBR3 hESCs containing the In some embodiments, the cell culture medium is free or OCT4-APE-GFP allele with 2i/L for three passages. FIG. 1I essentially free of components isolated from humans or shows immunofluorescence staining for NANOG and NES non-human animals. In some embodiments, the cell culture TIN in a clonal line of OCT4-APE-GFP-positive cells medium uses recombinant human proteins (e.g., recombi derived in 2i/L/DOX, and a clonal line of OCT4-APE-GFP nant human albumin). negative pNSCs derived in 2i/L. FIG. 1J is a model repre senting the distinct phenotypic responses of hESCs to treat 0097 “Cell line” refers to a population of largely or ment with 2i/L and 2i/L/DOX. OCT4-APE-GFP+ cells substantially identical cells, wherein the cells have often generated in 2i/L/DOX do not maintain reporter activity been derived from a single ancestor cell or from a defined upon transgene withdrawal. OCT4-APE-GFP+ cells can and/or Substantially identical population of ancestor cells. revert back to the conventional primed hESC state by For example, a cell line may consist of descendants of a re-exposure to serum and FGF. single cell. A cell line may have been or may be capable of 0.100 FIG. 2 shows the identification of small molecules being maintained in culture for an extended period (e.g., that maintain OCT4-APE-GFP activity after transgene with months, years, for an unlimited period of time). It will be drawal. FIG. 2A shows the strategy for Screening a kinase appreciated that cells may acquire mutations and possibly inhibitor library to identify compounds that maintain OCT4 epigenetic changes over time Such that some individual cells APE-GFP reporter activity upon withdrawal of DOX-depen of a cell line may differ with respect to each other. In some dent KLF2 and NANOG expression. FIG. 2B is the raw data embodiments, at least 80%, 85%, 86%, 87%, 88%, 89%, obtained from high-throughput flow cytometric analysis of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% the proportion of OCT4-APE-GFP+ cells in 96 wells supple of the cells of a cell line or cell culture are at least 95%, 96%, mented with a kinase inhibitor library (n=2). FIG. 2C shows 97%, 98%, or 99% genetically identical. In some embodi the hit compounds from a maintenance screen using a clonal ments, at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, line of WIBR3 OCT4-APE-GFP+ ESCS established in 21/L/ 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells DOX. FIG. 2D are the phase images (top) and flow cyto of a cell line or cell culture express the same set of cell metric analysis of the proportion of OCT4-APE-GFP+ cells surface markers. The set of markers could be markers (bottom) in a clonal line of OCT4-APE-GFP+ cells derived indicative of ground state (naive) pluripotency or cell-type in 2i/L/DOX and maintained for 10 passages without DOX specific markers. in the presence of each candidate compound. FIG. 2E is the 0098. A “clone” refers to a cell derived from a single cell quantitative gene expression analysis for FUW-KLF2. without change. It will be understood that if cells of a clone FUW-NANOG, endogenous OCT4, and EGFP in a clonal are subjected to different culture conditions or if some of the line of OCT4-APE-GFP+ cells maintained in 2i/L/DOX or cells are Subjected to genetic modification, the resulting cells for five passages without DOX in the presence of each may be considered distinct clones. candidate compound. FIG. 2F is the chemical structure of US 2017/0114323 A1 Apr. 27, 2017 the BRAF inhibitor, SB590885. FIG. 2G shows phase 0104 FIG. 4E shows the phase image of a primary images of a clonal line of WIBR3 human ESCs established human ESC line derived in 5i/L/FA conditions from an in 2i/L/DOX and maintained for 8 passages without DOX in explanted human blastocyst. Cell line is called Whitehead 2i/L/SB590885 (1 uM). FIG. 2H is the quantitative gene Institute Naive Human ESC (WIN)-1. FIG. 4F is the flow expression analysis for FUW-KLF2, FUW-NANOG, and cytometric analysis of the proportion of OCT4-APE-GFP+ endogenous OCT4 in two clonal lines of WIBR3 human cells three passages after withdrawal of individual inhibitors ESCs maintained for 8 passages without DOX in 2i/L/ and growth factors. FIG. 4G is the quantitative gene expres SB590885 (1 uM). sion analysis for OCT4, NANOG, KLF4, KLF2, SOX2, and 0101 FIG. 3 shows the optimization of medium for REX1 three passages after withdrawal of individual inhibi maintaining viable OCT4-APE-GFP+ cells. tors and growth factors. 0105 FIG. 5 shows the evaluation of alternative culture 0102 FIG. 3A shows the flow cytometric analyses of the conditions for naive human pluripotency. FIG. 5A is a table proportion of OCT4-APE-GFP+ cells in a 96 well one week comparing the components of four recent protocols for after culture in 2i/L/DOX, 2i/L alone or 2i/L/SB590885 (1 capturing naive-like human ESCs with 5i/L/A medium. FIG. uM). Top panel shows quantification of OCT4-APE-GFP+ 5B show the phase and fluorescence images and flow cells without including live/dead discrimination. Bottom cytometric analyses showing the response of OCT4-APE panel shows quantification of OCT4-APE-GFP+ cells after GFP-negative primed cells to recently reported protocols for gating out DAPI+ cells. FIG. 3B shows the strategy for naive human pluripotency (see FIG. 5A) and 5i/L/A. FIG. screening a kinase inhibitor library to identify compounds 5C shows the quantification of the proportion of GFP that improve the fraction of viable (DAPI-) OCT4-APE positive cells in WIBR3 OCT4-GFP and OCT4-APE-GFP GFP+ cells maintained without DOX for 2 passages in human ESCs upon removal of DOX-inducible KLF2 and 2i/L/SB590995 (1 uM). FIG. 3C is the raw data obtained NANOG expression in primed medium (PM) and four from high-throughput flow cytometric analysis of the pro alternative conditions for naive human pluripotency. FIG. portion of DAPI-/OCT4-APE-GFP+ cells in 96 wells 5D shows the flow cytometric analysis of the proportion of supplemented with one plate of a kinase inhibitor library OCT4-APE-GFP+ cells in 5i/L/A and the JNK inhibitor (n=2). Hit compound WH-4-023 is indicated with chemical SP600 125 (6i/L/A) in serum-free N2B27 basal medium vs. structure. FIGS. 3D-3E show high-throughput flow cyto 20% KSR basal medium. FIG. 5E is the quantitative gene metric quantification of the proportion of DAPI-/OCT4 expression analysis for OCT4, SOX2, KLF2 and NANOG in APE-GFP+ cells in 96 wells cultured for one passage (FIG. human ESCs cultured in 6i/L/A and supplemented with 3D) or two passages (FIG.3E) in 64 different concentrations 1-10% FBS or KSR. FIG.5F are the phase and fluorescence of PD0325901, CHIR99021, and SB590885. All the legends images of induction of OCT4-APE-GFP activity from the in FIG. 3E also apply in FIG. 3D. Asterisk denotes rank of primed state in 6i/L/A, and 6i/L/A supplemented with 0.5- the standard concentration of the three inhibitors used in the 196 KSR. preceding experiments (1 M PD0325901, 0.3 uM 0106 FIG. 6 shows the transcriptional profiling of naive CHIR99021 and 0.5 M SB590885). FIG. 3F are the phase human ESCs in 5i/L/A. FIG. 6A shows the cross-species and fluorescence images (top) and flow cytometric analysis hierarchical clustering of naive and primed pluripotent cells of the proportion of OCT4-APE-GFP+ cells (bottom) in a from mice and humans, as performed previously by Gafni et clonal line of OCT4-APE-GFP+ cells derived in 2i/L/DOX al. (2013). Affymetrix expression data were normalized and maintained for 2 passages without DOX in 2i/L/ using RNA spike-in. Two groups of human ESC samples are SB590885°P/Y-27632 or 2/L/SB590885P/Y-27632/WH included: WIBR2, WIBR3 and WIN1 human ESCs derived 4-023. Opt=optimized concentrations of PD0325901, in the optimized naive medium (5i/L/A or 6i/L/A, as indi CHIR99021, and SB590885 (see FIG. 4E). FIG. 3G are the cated), and parental WIBR2 and WIBR3 human ESCs in phase and fluorescence images of a clonal line of WIBR3 primed human ESC medium. Correlation matrix of gene OCT4-APE-GFP+ cells (left) and a clonal line of wild-type expression was clustered using Pearson correlation coeffi WIBR3 human ESCs generated in 2i/L/DOX (right) and cients (PCCs). The average linkage hierarchical clustering maintained for 3 passages in PD0325901/IM12/SB590885/ of the Pearson correlation is shown in the heatmap. Y-27632/WH-4-023 (5i) and hLIF. FIG. 3H shows the mEpiSCs, mouse EpiSCs: mESC, mouse ESC: miPSC, teratoma generated from wild-type WIBR3 human ESCs mouse iPSC. maintained in PDO325901 FIM12/SEB590885/Y-27632/WH 0107 FIG. 6B shows the gene ontology (GO) analysis 4-023 (5i) and hLIF after transgene withdrawal. Represen showing up- and down-regulated gene categories with most tative tissues of the three germ layers are indicated. significant p values between the naive human conditions and 0103 FIG. 4 shows the direct conversion of conventional primed human ESCs. FIG. 6C is a volcano plot showing fold human ESCs to naive pluripotency in 5i/L. FIG. 4A shows change (X axis) between the naive human ESC samples and the strategy for assessing direct conversion of primed human primed human ESCs on all genes (left); volcano plot show ESCs into OCT4-APE-GFP+ cells under optimized chemi ing previously published fold change (X axis) between the cal conditions. FIG. 4B shows phase and fluorescence naive human ESC samples of Gafni et al. (2013) and primed images of emerging naive colony and expanded cells from human ESCs on all genes (right). The open circles are those WIBR3 OCT4-APE-GFP human ESCS treated with 5i/L for that are log 2 fold change > 1 and <-1 & meet a p<0.05. FIG. 10 days. FIG. 4C are the phase and fluorescence images and 6D shows the fold changes in expression of naive pluripo flow cytometric analyses of the proportion of GFP+ cells tency-associated transcripts in the naive human ESC during conversion experiments in 5i/L Supplemented with samples vs. primed hESCs (blue or black), and the naive FGF and/or Activin A (FA). FIG. 4D are the phase images human samples published by Gafni et al. (2013) vs. primed of wild-type naive WIBR2 human ES cells converted in 5i/L human ESCs (red or grey). FIG. 6E shows, for comparison supplemented with FGF and/or Activin A (FA). with (FIG. 6D), fold changes in expression of naive pluri US 2017/0114323 A1 Apr. 27, 2017 potency-associated transcripts in naive mouse ESCs VS. in 2i/L/DOX or for five passages without DOX in the primed mouse EpiSCs were curated from a previously presence of each candidate compound. FIG. 9C: flow cyto published study (Najm et al., 2011). FIG. 6F shows the metric analyses of the proportion of viable (DAPI-negative) quantitative gene expression analysis for NANOG and and OCT4-APE-GFP+ cells in 2i/L/DOX or two passages STELLA in human ESCs cultured in parallel in primed after DOX withdrawal in 2i/L/SB590885 (1 uM). medium, the medium of Gafni et al. (2013) and 5i/L/FA. 0111 FIG. 10 shows the optimization of medium for FIG. 6G shows single molecule (sm) RNA FISH analysis maintaining viable OCT4-APE-GFP+ cells (associated with using OCT4 and NANOG probes in WIBR2 human ESCs FIG. 3). FIG. 10A: raw data obtained from high-throughput cultured in primed medium, the medium of Gafni et al. flow cytometric analysis of the proportion of DAPI-/OCT4 (2013) or 5i/L/A. FIG. 6H shows single molecule (sm) RNA APE-GFP+ cells in 96 wells supplemented with three plates FISH analysis using OCT4, NANOG, KLF4 and REX1 of a kinase inhibitor library in the presence of the primary hit probes in human ESCs cultured in primed medium, the compound SB590885 (n=2). FIG. 10B: hit compounds from medium of Gafni et al. (2013) or 5i/L/A. a viability screen using a clonal line of WIBR3 OCT4-APE 0108 FIG. 7 shows the chromatin landscape of naive GFP+ ESCs established in 2i/L/DOX. FIG. 10C: phase human pluripotency. FIG. 7A-7E show the ChIP-Seq tracks images and flow cytometric analyses of OCT4-APE-GFP+ for H3K4me3 and H3K27me3 at four classes of genes: (FIG. cells maintained in 2i/L/SB590885 (0.5 M)+ROCK inhibi 7A) developmental genes that are bivalent in the primed tor Y-27632 (10 uM) for two passages. FIG. 10D: phase and state and exhibit loss of H3K27me3 in the naive state; (FIG. GFP images of OCT4-APE-GFP+ cells maintained for four 7B) naive pluripotency genes that are bivalent in the primed passages in 2i/L/DOX, 2i/L/SB590885 (0.5 uM)+ROCK state and exhibit loss of H3K27me3 in the naive state; (FIG. inhibitor Y-27632 (10 uM) or the same medium in which 7C) naive pluripotency genes that acquire H3K4me3 in the CHIR99021 was replaced with an alternative GSK3 inhibi naive state; (FIG. 7D) master transcription factors that have tor, IM-12 (1.0 uM). a signal for H3K4me3, but not H3K27me3, in both naive 0112 FIG. 11 shows the direct conversion of conven and primed states: (FIG. 7E) ChIP-Seq tracks for H3K4me3 tional human ESCs to naive pluripotency in 5i/L associated and H3K27me3 at the DUSP6 and SOX11 loci in WIBR2 with FIG. 4). FIG. 11A: karyotype analysis of naive human human ESCs cultured under primed (red or grey) or naive ESCs WIBR2 (P8 in 5i/L/A). Cytogenetic analysis was 6i/L/A (blue or black) conditions. FIG. 7F shows the ChIP performed on 20 metaphase cells. FIG. 11B: karyotype Seq analysis for H3K4me3 and H3K27me3 at Polycomb analysis of naive human ESCs WIN1 (P7 in 5i/L/FA). target genes in WIBR2 human ESCs cultured in primed Cytogenetic analysis was performed on 20 metaphase cells. medium (left) or naive 6i/L/A medium (right). FIG. 7G FIG. 11C: phase images of OCT4-APE-GFP+ cells three shows average H3K4me3 and H3K27me3 signal at Poly passages after withdrawal of individual inhibitors and comb target genes in WIBR2 human ESCs cultured in growth factors. primed medium (red or grey) or naive 6i/L/A medium (blue 0113 FIG. 12 shows the evaluation of alternative culture or black). conditions for naive human pluripotency associated with 0109 FIG. 8 shows a reporter system for naive human FIG. 5. FIG. 12A; phase and fluorescence images (Top) and pluripotency based on endogenous OCT4 distal enhancer flow cytometric analysis of the proportion of OCT4-APE activity (associated with FIG. 1). FIG. 8A: southern blot GFP+ cells (Bottom) in OCT4-APE-GFP+ cells derived in analysis confirming deletion of the PE in OCT4-GFP ESCs 5i/L/FA and maintained for three passages in the presence of and the removal of floxed PGK-puro cassette. Ndel-digested additional media additives, as indicated. FIG. 12B: quanti genomic DNA was hybridized with 5' and 3' external probes. tative gene expression analysis for KLF2 and KLF4 in Expected fragment size: WT (wild type) 5.6 kb, T (targeted) OCT4-APE-GFP-positive naive human ESCs cultured in =6.4 kb, PEKO (targeted allele after PGK-puro removal) 5i/L/A and supplemented with various components of the =4.6 kb. FIG. 8B: phase and GFP images of OCT4-APE medium of Gafni et al. (2013). FIG. 12C: phase and fluo GFP+ cells obtained after DOX induction of lentiviral rescence images (Top) and flow cytometric analysis of the KLF2+NANOG. Following primary infection, WIBR3 proportion of OCT4-APE-GFP+ cells (Bottom) in OCT4 human ESCs containing the OCT4-APE-GFP reporter allele APE-GFP+ cells derived in 5i/L/FA and maintained for three were trypsinized and treated with hESM, 2i/L or 2i/L/DOX passages in 20% KSR basal medium supplemented with the for one week. FIG. 8C: immunofluorescence staining for media additives described in (FIG. 12A). OCT4 in a clonal line of OCT4-APE-GFP-positive cells 0114 FIG. 13 shows the transcriptional profiling of naive derived in 2i/L/DOX, and a clonal line of OCT4-APE-GFP human ESCs in 5i/L/A and 6i/L/A associated with FIG. 6. negative pNSCs derived in 2i/L. FIG. 8D: quantitative gene FIG. 13A: quantitative gene expression analysis for KLF4 expression analysis for EGFP, SOX2, PRMD14 and PAX6 and REX1 in human ESCs cultured in parallel in primed in clonal OCT4-APE-GFP+ human ESC lines generated in medium, 5i/L/FA and the medium of Gafni et al. (2013). 2i/L/DOX, secondary primed cells generated by withdrawal FIG. 13B: expression profile of transcripts upregulated in of DOX and expansion in conventional hESM, and clonal 6i/L/A during human embryonic development. For each lines of OCT4-APE-GFP-negative pNSCs derived in 2i/L. gene, the normalized expression values in human ESCs 0110 FIG. 9 shows the identification of small molecules cultured in 6i/L/A vs. primed human ESCs are indicated that maintain OCT4-APE-GFP activity after transgene with (Left). An unpaired two-tailed t test was performed to drawal (associated with FIG. 2). FIG.9A: raw data obtained establish the degree of significance. Expression of the cor from high-throughput flow cytometric analysis of the pro responding transcript is shown at nine stages of human portion of OCT4-APE-GFP+ cells in 96 wells supplemented pre-implantation development, as detected by single cell with a kinase inhibitor library (n=2). FIG. 9B: quantitative RNA-Seq profiling (Yan et al., 2013) (Right). This compari gene expression analysis for STELLA, KLF4, PRDM14 and son indicates that naive-associated transcripts upregulated in SOX2 in a clonal line of OCT4-APE-GFP+ cells maintained 6i/L/A are enriched at the morula/epiblast stage of human US 2017/0114323 A1 Apr. 27, 2017 development when compared to human ESCs at passage 0 or tive GSK3 inhibitor CHIR99021 or addition of the Wnt passage 10. FIG. 13C: variability in NANOG expression inhibitor IWR1 did not further stimulate the proliferation of compared between single human ESCs cultured in primed naive human cells. medium, 5i/L/A or the medium of Gafni et al. (2013). 0118 FIG. 17 shows the induction of naive human pluri potency in 5i/L/A or 4i/L/A is associated with X chromo 0115 FIG. 14 shows the developmental potential of naive some reactivation. FIG. 17A: a reporter system for X human ESCs in 5i/L/A. FIG. 14A: teratomas generated from chromosome status of human ES cells was engineered by WIBR3 OCT4-APE-GFP-positive human ESCs derived and TALEN-mediated targeting of both alleles of the X-linked maintained in 5i/LEFA and WIBR3 AAVS1-tdTomato MECP2 gene with fluorescent reporters. FIG. 17B: starting human ESCs in 5i/L/FA. Representative tissues of the three from a single color (GFP-positive) primed line, conversion germ layers are indicated. FIG. 14B: immunofluorescence to the naive state in 5i/L/A or 4i/L/A (-IM12) results in staining for AFP and HNF4a following 20d of hepatic activation of the tdTomato-labelled allele while GFP activity differentiation in naive WIBR2 human ESCs derived and is maintained. FIG. 17C: starting from a single color (tdTo maintained in 5i/L/A. FIG. 14C: table summarizing injec mato-positive) primed line, conversion to the naive state in tions of human ESCs maintained in 5i/L/FA (top) or the 5i/L/A or 4i/L/A (-IM12) results in activation of the GFP medium of Gafni et al. (2013) (bottom) in mouse embryos. labelled allele while tdTomato activity is maintained. These C1-AAVS1-GFP human ESCs in the medium of Gafni et al. results indicate that induction of naive human pluripotency (2013) were cultured on MEFs, gelatin/vitronectin or matri is accompanied by a Switch towards biallelic expression of gel prior to injection. (*) E10.5 embryos injected with X-linked genes, which is confirmed by RNA FISH data (not human ESCs cultured in 5i/L/FA and the medium of Gafni shown). et al. (2013) were mixed during collection, but none were identified as positive. (**) 30 injected embryos were lost DETAILED DESCRIPTION OF THE during transfer. INVENTION 0119 The present disclosure, in one aspect, is based on 0116 FIG. 15 shows that the proliferation of naive the identification of compounds (e.g., compounds of any one human embryonic stem cells is enhanced by reduction or of Formulae (A) to (L), and pharmaceutically acceptable removal of GSK3 inhibition. FIG. 15A: transgene-depen salts, Solvates, hydrates, polymorphs, co-crystals, tautomers, dent naive human embryonic stem (ES) cell line used to Stereoisomers, isotopically labeled derivatives, and prodrugs titrate the concentrations of inhibitors in 5i/L/A (see The thereof) that support self-renewal of naive human ESCs. In unissen et al., Cell Stem Cell, 2014). This subclone of particular, iterative chemical screening identified kinase WIBR3 is dependent on Doxycycline (DOX) to maintain inhibitors that induce and maintain OCT4 distal enhancer expression of two lentiviral transgenes, KLF2 and NANOG. activity, a molecular signature of ground state pluripotency, Left, phase image shows colony morphology in 2i/L/DOX. when applied directly to conventional human ESCs. These Middle, flow cytometric quantification shows the proportion inhibitors generate a homogeneous population of human of cells positive for the OCT4-APE-GFP reporter in 2i/L/ pluripotent stem cells in which transcription factors associ DOX. Right, this cell line has a normal (46.XX) karyotype. ated with the ground state of pluripotency are highly upregu FIG. 15B: titration assay to attain optimal concentrations of lated. Comparison with previously reported naive human Small molecule inhibitors for maintenance of naive human ESCs indicates that the procedures defined herein capture a ES cells. Top, phase images showing colony morphology novel pluripotent state in humans that closely resembles after sequential passaging by single cell dissociation at low mouse ESCs. Accordingly, aspects of the disclosure provide density (1:10) in four different conditions: 2i/L, 5i/L/A, methods for converting the pluripotency state of a vertebrate t5i/L/A (0.2 uM GSK3 inhibitor IM12) and 4i/L/A (removal (e.g., human) cell to a more naive state. Also provided are of GSK3 inhibitor IM12). Bottom, flow cytometric quanti naive pluripotent vertebrate cells (e.g., human) and cell lines fication shows the proportion of cells positive for the OCT4 produced by the methods described herein. Some aspects of APE-GFP reporter in each condition. FIG. 15C: quantitative the invention also involve kits for converting the pluripo RT-PCR (qRT-PCR) analysis confirming the downregulation tency state of a vertebrate (e.g., human) cell to a more naive of exogenous KLF2 and NANOG transgenes and primed State. marker VIMENTIN and upregulation of naive markers I0120 Embryonic stem cells (ESCs) and induced pluri KLF4, REX1 and STELLA in t5i/L/A and 4i/L/A. Error bars potent stem cells (iPSCs) have attracted much attention indicate one standard deviation. because of their potential to mature into virtually any cell 0117 FIG. 16 shows the quantification of the prolifera type in the body. However, mouse ESCs and iPSCs have tion of naive human cells in presence of different Wnt signal different growth factor requirements and provide a more modulators. Quantification of cell number in naive culture reliable vehicle for directed differentiation as compared to conditions Supplemented with distinct Wnt signal modula human ESCs and iPSCs. It was thought for many years that tors after withdrawal of DOX from transgene-dependent these differences reflected variation between species. In naive human ES cells (see FIG. 15A). 1x105 cells were 2007, however, two groups reported that novel stem cell seeded per individual well of a 6 well plate and cells were lines derived from the post-implantation epiblast of mouse dissociated and re-seeded at 1:5 density for three successive embryos, called EpiSCs, have properties similar to human passages. Cell numbers at Successive passages were ESCs (Brons et al., 2007; Tesar et al., 2007). These include recorded in six conditions (n=2): 2i/L/DOX (control), 2i/L. a flat morphology, dependence on bFGF and activin signal 5i/L/A, 4i/L/A (removal of GSK3 inhibitor IM12), 4i/L/A+ ing, and use of the OCT4 proximal enhancer element. The CHIR99021 (1 uM), and 4i/L/A+IWR1 (2.5 uM). At pas inner cell mass (ICM)-like state of mouse ESCs was sages 2 and 3 the proliferation was significantly elevated in described as "naive,” whereas EpiSCs and human ESCs 4i/L/A compared to 5i/L/A. Replacing IM 12 with alterna were designated as “primed” (Nichols and Smith, 2009); the US 2017/0114323 A1 Apr. 27, 2017 implication is that the primed State is prone to differentiate, values.<10 nM. In other embodiments, the compounds whereas the naive condition corresponds to the more imma exhibit IC50 values.<7.5 nM. In other embodiments, the ture “ground state' of pluripotency. compounds exhibit IC50 values.<5 nM. 0121 The present disclosure relates to the discovery that 0.124. In certain embodiments, the BRAF inhibitor is a conventional human ESCs can be converted into a more compound of Formula (B) (e.g., AZ-628 or BAY-439006) or immature/less restricted State (i.e., a more "naive' pluripo Formula (C) (e.g., GDC-0879 or SB590885). In certain tent state) that extensively shares defining features with embodiments, the BRAF inhibitor is Sorafenib; PLX4720; pluripotent mouse ESCs. Described herein are optimized PLX-3603; GSK2118436: N-(3-(5-(4-chlorophenyl)-1H conditions that enable the interconversion between conven pyrrolo 2:3-bipyridine-3-carbonyl)-2:4-difluorophenyl) tional and naive human ESCs in the absence of reprogram propane-1-sulfonamide: Vemurafenib (also known as Zelo ming factors, as well as the direct isolation of genetically brafR and PLX-4032); GSK 2118436; RAF265 (Novartis); unmodified naive ESCs from human blastocysts. The meth XL281; ARQ736; a compound described in international ods described herein capture a distinct and novel state of PCT application publication, WO 2007/002325, WO 2007/ human pluripotency that shares defining features with mouse 002433, WO 2009/111278, WO 2009/111279, WO 2009/ ESCS. 111277, WO 2009/111280, or WO 2011/025927; or a com 0122. Accordingly, some aspects of the disclosure pro pound described in U.S. Pat. No. 7,491,829 or 7,482.367. In vide a method for changing the pluripotency state of a certain embodiments, the VEGFR1 inhibitor is a compound vertebrate cell to a more naive state. The method comprises of Formula (A) (e.g., AMG706), Formula (B) (e.g., BAY (a) culturing a pluripotent vertebrate cell in the presence of 73-4506), or Formula (G) (e.g., BIBF-1120 or SU11248). In a serine/threonine-protein kinase B-Raf (BRAF) inhibitor, certain embodiments, the VEGFR1 inhibitor is SU5416 or a an epidermal growth factor receptor (EGFR) inhibitor, a compound described in U.S. patent application publication, vascular endothelial growth factor 1 (VEGFR1) inhibitor, or US 2006/0030000. In certain embodiments, the FGFR1 a fibroblast growth factor receptor 1 (FGFR1) inhibitor; and inhibitor is a compound of Formula (F) (e.g., PD173074). In (b) maintaining the cell in culture under conditions Suitable certain embodiments, the FGFR1 inhibitor is cediranib; and a time Sufficient to changing the pluripotency state of the brivanib; TSU-68; BIBF1120; dovitinib: Ki23057; vertebrate cell to a more naive state than the pluripotency MK-2461; E7080: SU5402; BGJ398; E-3810; AZD4547: state of the original vertebrate cell of step (a). PLX052; or a compound described in U.S. Pat. No. 8,709, 0123. The kinase inhibitor(s) used herein may be a small 718. In certain embodiments, the MEK inhibitor is a com molecule, antibodies/antibody fragments (at least for those pound of Formula (K) (e.g., PD0325901). In certain embodi kinases that have an extracellular domain), short interfering ments, the MEK inhibitor is a compound described in RNA, and/or aptamers. A “small molecule.” (M) as used international PCT application publication, WO 2010/ herein, refers to an alkyl, alkenyl, alkynyl, aryl, heteroaryl, 138377, WO 2009/153554, WO 2009/093009, WO 2009/ carbocyclic, or heterocyclic moiety, as defined herein, com 0.13462, WO 2009/093013, WO 2008/020206, WO 2008/ prising carbon and hydrogen, and optionally comprising one 078086, WO 2008/120004, WO 2008/125820, WO 2009/ or more heteroatoms as a part of the molecule (in the case 093008, WO 2009/074827, WO 2009/093009, WO 2010/ of heteroaryland heterocyclic groups) and/or attached to the 108652, WO 2010/105110, WO 2010/105082, WO 2009/ molecule selected from oxygen, nitrogen, Sulfur, phospho 129246, WO 2009/018238, WO 2009/018233, WO 2008/ rus, boron, silicon, and selenium. In certain embodiments, 089459, WO 2008/124085, WO 2008/076415, WO 2008/ the specificity of the inhibitors is given by the IC50 value. 021389, WO 2010/051935, WO 2010/051933, WO 2009/ The IC50 value is defined as the concentration of inhibitor 129938, WO 2009/021887, WO 2008/101840, WO 2008/ required to inhibit 50% of the kinase activity. In certain 055236, WO 2010/003025, WO 2010/003022, WO 2007/ embodiments, the compounds of Formula (I) or (II) may 096259, WO 2008/067481, WO 2008/024724, WO 2008/ exhibit IC50 values.<100 uM. In certain other embodiments, 024725, or WO 2010/0145197; or a compound described in the compounds exhibit IC50 values.<50 uM. In certain other U.S. patent application publication, US 2008/0255133, US embodiments, the compounds exhibit IC50 values.<40 uM. 2008/0058340, US 2009/0275606, or US 2009/0246.198. In In certain other embodiments, the compounds exhibit IC50 certain embodiments, the GSK3 inhibitor is a compound of values.<30 uM. In certain other embodiments, the com Formula (C) (e.g., CHIR99021) or Formula (J) (e.g., IM12). pounds exhibit IC50 values.<20 uM. In certain other embodi In certain embodiments, the GSK3 inhibitor is CHIR98014: ments, the compounds exhibit IC50 values<10 uM. In CHIR98023; B1O-acetoxime; BIO: LiCl; SB 216763; SB certain other embodiments, the compounds exhibit IC50 415286; AR-A014418; 1-azakenpaullone; bis-7-indolylma values.<7.5 uM. In certain embodiments, the compounds leimide; kenpaullone; CT99021; CT 20026; SB216763; SB exhibit IC50 values.<5 uM. In certain other embodiments, 415286: TDZD-8; TIBPO (2-thio(3-iodobenzyl)-5-(1- the compounds exhibit IC50 values.<2.5 uM. In certain pyridyl)-1,3,4-oxadiazole); a compound described in U.S. embodiments, the compounds exhibit IC50 values.<1 uM. In patent application publication, US 2013/00593.85, US 2001/ certain embodiments, the compounds exhibit IC50 val 0034051, US 2002/0156087, US 2004/0092535, US 2004/ ues<0.75 uM. In certain embodiments, the compounds 0209878, US 2004/0138273, US 2004/0077707, US 2005/ exhibit IC50 values<0.5 uM. In certain embodiments, the 0.054663, US or 2006/0089369; a compound described in compounds exhibit IC50 values<0.25 uM. In certain U.S. Pat. No. 6,057,117; U.S. Pat. No. 6,608,063: U.S. Pat. embodiments, the compounds exhibit IC50 values.<0.1 uM. No. 6,417,185; U.S. Pat. No. 6,489,344; or U.S. Pat. No. In certain other embodiments, the compounds exhibit IC50 6,153,618; or a compound described in international PCT values<75 nM. In certain other embodiments, the com application publication, WO/2003/049739; WO/2002/ pounds exhibit IC50 values.<50 nM. In certain other embodi 085909; WO/2003/011287, WO/2005/039485, or WO/2006/ ments, the compounds exhibit IC50 values<25 nM. In 091737. In certain embodiments, the ROCK inhibitor is a certain other embodiments, the compounds exhibit IC50 compound of Formula (L) (e.g., Y-27632). In certain US 2017/0114323 A1 Apr. 27, 2017 20 embodiments, the ROCK inhibitor is fasudil (HA-1077); maintaining the cell in culture under conditions suitable and thiazovivin; AMA0076; AR-12286: AMA0076; AR-12286: a time sufficient to change the pluripotency state of the AR-13324; ATS907; DE-104; INS-115644; INS-117548: vertebrate cell to a more naive state. K-115; PG324; Y-39983; RKI-983: SNJ-1656; a compound 0127. The cell is cultured and maintained in a culture described in international PCT application publication, WO medium under Suitable conditions until the pluripotency 2014/068035, WO 2013/030216, WO 2013/030367, WO state of the cell is converted to a more naive state. Condi 2013/030366, WO 2013/030365, WO 2011/107608, WO tions suitable for conversion of the pluripotent state of the 2012/146724, WO 2006/137368, or WO 2005/035506; or a cell to a more naive state include the presence of one or more compound described in U.S. patent application publication, kinase inhibitors in the culture medium. Thus, in some US 2013/196437. In certain embodiments, the Src inhibitor embodiments, the culture medium contains a serine/threo is a compound of Formula (H) (e.g., WH-4-023). In certain nine-protein kinase B-Raf (BRAF) inhibitor, an epidermal embodiments; the Src inhibitor is SKI606 (bosutinib); dasa growth factor receptor (EGFR) inhibitor, a vascular endothe tinib (SPYRCEL); saracatenib (AZD-0530); PP1; PP2: lial growth factor 1 (VEGFR1) inhibitor, or a fibroblast PD173955; AGL1872; PD162531; radicicol R2146; geldan growth factor receptor 1 (FGFR1) inhibitor. In some amycin; or a compound described in U.S. patent application embodiments, the culture medium further contains a mito publication, US 2006/258686, US 2009/0227608, US 2010/ gen-activated protein kinase kinase (MEK) inhibitor. In 0249152, or US 2013/0040972. ESCs are pluripotent cells. Some embodiments, the culture medium further contains a ESCs have been derived from vertebrate animals such as ROCK inhibitor and/or an Src inhibitor. In some embodi mice, primates (including humans), and some other species. ments, the culture medium further contains a glycogen ESCs are often derived from cells obtained from the inner synthase kinase 3 (GSK3) inhibitor, a rho-associated protein cell mass (ICM) of a vertebrate blastocyst but can also be kinase (ROCK) inhibitor, and/or a proto-oncogene tyrosine derived from single blastomeres (e.g., removed from a protein kinase (Src) inhibitor. In some embodiments, the morula). Pluripotent cells can also be obtained using par culture medium contains a BRAF inhibitor, a MEK inhibi thenogenesis, e.g., from germ cells, e.g., oocytes. Other tor, a GSK3 inhibitor, a ROCK inhibitor, and an Src inhibi pluripotent cells include embryonic carcinoma (EC) and tor. In some embodiments, the culture medium further embryonic germ (EG) cells. See, e.g., Yu J. Thomson JA, contains fibroblast growth factor and/or Activin A. In some Genes Dev. Pluripotent stem cell lines. 22(15):1987-97, embodiments, the culture medium contains one or more of 2008. the compounds described herein. In some embodiments, the 0.125 Standard techniques for preparing deriving human culture medium contains a BRAF inhibitor, a MEK inhibi ES cells typically involve the use of a MEF or human cell tor, a ROCK inhibitor, and an Src inhibitor. In some embodi feeder layer and serum or, if cultured in serum-free medium, ments, the culture medium does not contain a GSK3 inhibi compounds such as bFGF. For example, the ICM of a human tOr blastocyst is removed by immunosurgery, dissociated in 0128. The concentration of the kinase inhibitors used in Cat-Mg"-free medium, and plated over mouse embryonic the culture medium will depend on the amount of culture fibroblasts or human feeder cells (Thomson et al., Science medium being generated. In some embodiments, 0.1 uM, 0.2 282, 1145 (1998). The mouse cells are irradiated to suppress uM, 0.3 uM, 0.4 uM, 0.5uM, 1 uM, 1.5uM, 2.0 uM, 2.5uM, their proliferation. See, e.g., B. E. Reubinoff et al., Nature 3.0 uM, 3.5 uM, 4.0 uM, 4.5 uM, 5.0 uM, 5.5 uM, 6.0 uM, Biotechnol. 18, 399, 2000; Mitalipova M & Palmarini G. 6.5uM, 7.0 uM, 8.0 uM, 8.5 uM, 9.0 uM, 9.5 uM, 10.5uM, Isolation and characterization of human embryonic stem 11.0 uM, 12.0 uM 13.0 uM, 14.0 uM, or 15.0 uM of one or cells. Methods Mol. Biol. 331:55-76, 2006; Ilic D, et al., more kinase inhibitors are included in about 500 mL of Derivation of hESC from intact blastocysts. Curr Protoc culture medium. In some embodiments, 0.1-1.0 uM of Stem Cell Biol. Chapter 1:Unit 1 A.2, 2007; Ludwig T. A BRAF inhibitor is used in about 500 ml of culture medium. Thomson J., Defined, feeder-independent medium for In some embodiments, 0.5-1.5uM of MEK inhibitor is used human embryonic stem cell culture. Curr Protoc Stem Cell in about 500 ml of culture medium. In some embodiments, Biol. Chapter 1:Unit 1C.2, 2007. It will be understood that 0.5-1.5uM of Src inhibitor is used in about 500 ml of culture culture conditions can be feeder layer free. It will also be medium. In some embodiments, 5.0-15uM of ROCK inhibi understood that the culture conditions can include the use of tor is used in about 500 ml of culture medium. In some matrices such as laminin, MatrigelTM, and the like. In some embodiments, 1.0-5.0LM of GSK3 inhibitor is used in about embodiments, methods described in Chen A E, et al., Opti 500 ml of culture medium. mal timing of inner cell mass isolation increases the effi 0129. In some embodiments, the concentration of GSK3 ciency of human embryonic stem cell derivation and allows inhibitor is 1 nM to 10 nM. In some embodiments, the generation of sibling cell lines. Cell Stem Cell. 4(2): 103-6, concentration of GSK3 inhibitor is 10 nM to 0.1 uM. In 2009, are used. In some embodiments, mitomycin C-inac some embodiments, the concentration of GSK3 inhibitor is tivated mouse embryonic fibroblast feeder cells are used for 0.1 uM to 0.2 uM. In some embodiments, the concentration culturing human embryonic stem cells. It will be understood of GSK3 inhibitor is 0.2 uM to 0.5 LM. In some embodi that in Some embodiments other methods of inactivating ments, the concentration of GSK3 inhibitor is 0.5uM to 1 feeder cells may be used such as other compounds or gamma LM. In some embodiments, the culture medium contains a irradiation. BRAF inhibitor, a MEK inhibitor, a ROCK inhibitor, and an 0126. A pluripotent vertebrate cell can be converted to a Src inhibitor, and further contains a GSK3 inhibitor in more naive state by culturing the cell in the presence of a concentration range listed above in this paragraph. serine/threonine-protein kinase B-Raf (BRAF) inhibitor, an 0.130. In some embodiments, if a different kinase inhibi epidermal growth factor receptor (EGFR) inhibitor, a vas tor that targets the same kinase is used. Such kinase inhibitor cular endothelial growth factor 1 (VEGFR1) inhibitor, or a may be used at a concentration that provides an approxi fibroblast growth factor receptor 1 (FGFR1) inhibitor; and mately equivalent effect. US 2017/0114323 A1 Apr. 27, 2017 0131. In some embodiments, conditions suitable for con to, a BRAF inhibitor. In some embodiments, the culture version of the pluripotent state of the cell to a more naive medium contains a BRAF inhibitor and a MEK inhibitor. In state include, in addition to the one or more kinase inhibitors some embodiments, the culture medium contains a BRAF described herein, the addition of one or more of the follow inhibitor, a MEK inhibitor, and a ROCK inhibitor. In some ing components to the culture medium: N2 Supplement, B27 embodiments, the culture medium contains a BRAF inhibi Supplement, human LIF, glutamine, nonessential amino tor, a MEK inhibitor, a ROCK inhibitor, and an Src inhibitor. acids, 3-mercaptoethanol, penicillin-Streptomycin, and/or In Some embodiments, the culture medium contains an albumin (e.g., BSA or human albumin). In some embodi inhibitor of a receptor tyrosine kinase and a MEK inhibitor. ments, the culture media comprise DMEM/F 12 or Neu In some embodiments, the culture medium contains a BRAF robasal. In some embodiments, the culture media comprise inhibitor, a MEK inhibitor, and an Src inhibitor. In some DMEM/F12 and Neurobasal in a suitable ratio (e.g., 10:90 embodiments, the culture medium does not contain a GSK3 to 90:10 (e.g., 25:75, 50:50, and 75:25). DMEM/F12: Neu inhibitor. robasal). In some embodiments, the culture media compris 0133. In some embodiments, the concentration of GSK3 ing DMEM/F12 and/or Neurobasal further comprise N2 inhibitor is 1 nM to 10 nM. In some embodiments, the Supplement, B27 Supplement, human LIF, glutamine, non concentration of GSK3 inhibitor is 10 nM to 0.1 M. In some essential amino acids, B-mercaptoethanol, penicillin-strep embodiments, the concentration of GSK3 inhibitor is 0.1 M tomycin, and/or albumin (e.g., BSA or human albumin). In to 0.2 M. In some embodiments, the concentration of GSK3 Some embodiments, the culture medium further comprises inhibitor is 0.2 M to 0.5 M. In some embodiments, the fibroblast growth factor 2 (FGF2) and 1%, 0.8%, 0.6%, concentration of GSK3 inhibitor is 0.5 M to 1 M. In some 0.4%, 0.2%, or 0.1% KSR. In some embodiment, the cells embodiments, the culture medium contains a BRAF inhibi are maintained on mitomycin C-inactivated mouse embry tor, a MEK inhibitor, a ROCK inhibitor, and an Src inhibitor, onic fibroblast feeder cells. In some embodiments, condi and further contains a GSK3 inhibitor in concentration range tions suitable for conversion of the pluripotent state of the listed above in this paragraph. In some embodiments, the cell to a more naive state include growing the cells under culture medium contains a BRAF inhibitor and a MEK physiological oxygen conditions, i.e., about 5% O2. In some inhibitor, and further contains a GSK3 inhibitor in concen embodiments, the cells are grown under between about 1% tration range listed above in this paragraph. In some embodi O and about 5% O2. In some embodiments, the cells are ments, the culture medium contains a BRAF inhibitor, a grown under between about 2% O, and about 5% O. In MEK inhibitor, and a ROCK inhibitor and further contains some embodiments, the cells are grown under between a GSK3 inhibitor in concentration range listed above in this about 5% O, and about 10% O. In some embodiments, the paragraph. In some embodiments, the culture medium con cells are grown under between about 10% O and about 20% tains a receptor tyrosine kinase and a MEK inhibitor, and O. In some embodiments, the cells are grown under 1% O2, further contains a GSK3 inhibitor in concentration range 2% O.3% O, 4% O, 5% O, 6% O, 7% O, 8% O.9% listed above in this paragraph. In some embodiments, the O., 10% O. 11% O. 12% O., 13% O., 14% O, 15% O, culture medium contains BRAF inhibitor, a MEK inhibitor, 16% O, 17% O, 18% O, 19% O, 20% O, 21% O, 22% and an Src inhibitor, and further contains a GSK3 inhibitor O, 23% O, 24% O, or 25% O. in concentration range listed above in this paragraph. 0132) Some aspects of the invention relate to a cell I0134. As used herein, a “basal medium is typically an culture medium comprising: a basal medium; and a serine/ unsupplemented medium (e.g., Eagle’s minimal essential threonine-protein kinase B-Raf (BRAF) inhibitor, an epi medium (EMEM); Dulbecco's modified Eagle's medium dermal growth factor receptor (EGFR) inhibitor, a vascular (DMEM)). As will be appreciated by those of skill in the art, endothelial growth factor 1 (VEGFR1) inhibitor, or a fibro a basal medium can comprises a variety of components such blast growth factor receptor 1 (FGFR1) inhibitor. In some as one or more amino acids (e.g., non-essential amino acids, embodiments, the cell culture medium further comprises essential amino acids), salts (e.g., calcium chloride, potas mitogen-activated protein kinase kinase (MEK) inhibitor. In sium chloride, magnesium sulfate, sodium chloride, and some embodiments, the cell culture medium further com monosodium phosphate), Sugars (e.g., glucose), and Vita prises a glycogen synthase kinase 3 (GSK3) inhibitor, a mins (e.g., folic acid, nicotinamide, riboflavin, B12), iron rho-associated protein kinase (ROCK) inhibitor, and/or a and pH indicators (e.g., phenol red). The basal medium can proto-oncogene tyrosine-protein kinase (Src) inhibitor. In further comprise proteins (e.g., albumin), hormones (e.g., Some embodiments, the culture medium further contains a insulin), glycoproteins (e.g., transferrin), minerals (e.g., ROCK inhibitor and/or an Src inhibitor. In some embodi Selenium), serum (e.g., fetal bovine serum), antibiotics, ments, the cell culture medium comprises a BRAF inhibitor, antimycotics and glycosaminoglycans. a MEK inhibitor, and a GSK3 inhibitor. In some embodi ments, the cell culture medium comprises a BRAF inhibitor, 0.135. In some embodiments, the basal medium is serum a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. free medium. In some embodiments, the basal medium In some embodiments, the cell culture medium comprises a comprises one or more Supplements, such as, but not limited BRAF inhibitor, a MEK inhibitor, a GSK3 inhibitor, a to, supplements such as B27 and/or N2. In some embodi ROCK inhibitor, and an Src inhibitor. In some embodiments, ments, the basal medium is Supplemented with one or more the culture medium contains an inhibitor of a receptor of the following: DMEM/F12, Neurobasal, N2 supplement, tyrosine kinase, a MEK inhibitor, and a GSK3 inhibitor. In 10 mL B27 Supplement, human LIF, glutamine, nonessential some embodiments, the culture medium contains a BRAF amino acids, B-mercaptoethanol, penicillin-Streptomycin, inhibitor, a MEK inhibitor, a GSK3 inhibitor, and an Src and/or BSA (Sigma). In some embodiments, the Supple inhibitor. In some embodiments, any of the culture media mented basal cell culture medium further comprises fibro contains a VEGRF1 inhibitor, an EGFR inhibitor, a FGFR1 blast growth factor 2 (FGF2) and 1%, 0.8%, 0.6%, 0.4%, inhibitor, or a combination thereof, instead of, or in addition 0.2%, or 0.1% KSR. US 2017/0114323 A1 Apr. 27, 2017 22 0136. The pluripotent vertebrate cell is maintained in epidermal growth factor receptor (EGFR) inhibitor, a vas culture for a time Sufficient to change the pluripotency state cular endothelial growth factor 1 (VEGFR1) inhibitor, or a of the cell to a more naive state. In some embodiments, time fibroblast growth factor receptor 1 (FGFR1) inhibitor. The Sufficient to change the pluripotency state of the cell to a vertebrate cells can be naive pluripotent cells, human pluri more naive state is at least 1 day, at least 2 days, at least 3 potent cells, or naive human pluripotent cells. In some days, at least 4 days, at least 5 days, at least 6 days, at least embodiments, the cell culture medium further comprises 7 days, at least 8 days, at least 9 days, at least 10 days, at mitogen-activated protein kinase kinase (MEK) inhibitor. In least 11 days, at least 12 days, at least 13 days, at least 14 some embodiments, the cell culture medium further com days, at least 15 days, at least 16 days, at least 17 days, at prises a glycogen synthase kinase 3 (GSK3) inhibitor, a least 18 days, at least 19 days, or at least 20 days. In some rho-associated protein kinase (ROCK) inhibitor, and/or a embodiments, time Sufficient to change the pluripotency proto-oncogene tyrosine-protein kinase (Src) inhibitor. In state of the cell to a more naive state is between 1-5 days, Some embodiments, the culture medium further contains a 1-10 days, 1-15 days, 1-20 days, 5-10 days, 5-15 days, 5-20 ROCK inhibitor and/or an Src inhibitor. In some embodi days, 10-15 days, 10-20 days, or 15-20 days. In some ments, the cell culture medium comprises a BRAF inhibitor, embodiments, time Sufficient to change the pluripotency a MEK inhibitor, and a GSK3 inhibitor. In some embodi state of the cell to a more naive state is at least about 5 days ments, the cell culture medium comprises a BRAF inhibitor, (e.g., about 10 days). In some embodiments, the culture a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor. medium is replenished as required during this time. In some In some embodiments, the cell culture medium comprises a embodiments, the cell is maintained in culture until the cell BRAF inhibitor, a MEK inhibitor, a GSK3 inhibitor, a has at least one property which is similar to the correspond ROCK inhibitor, and an Src inhibitor. In some embodiments, ing property of mouse embryonic stem cells. In some the culture medium contains a BRAF inhibitor and a MEK embodiments, the at least one property which is similar to inhibitor. In some embodiments, the culture medium con the corresponding property of mouse embryonic stem cells tains a BRAF inhibitor, a MEK inhibitor, and a ROCK is the utilization of the distal OCT4 enhancer element. An inhibitor. In some embodiments, the culture medium con important molecular signature of naive pluripotency in the tains a BRAF inhibitor, a MEK inhibitor, a ROCK inhibitor, mouse system is the use of the distal enhancer (DE) of and an Src inhibitor. In some embodiments, the culture OCT4. Thus, in some embodiments, the cell is maintained in medium does not contain a GSK3 inhibitor. culture until the cell uses the distal Oct4 enhancer element 0140. In some embodiments, the concentration of GSK3 for OCT4 expression. In some embodiments, the cell is inhibitor is 1 nM to 10 nM. In some embodiments, the maintained in culture until the cell uses the endogenous concentration of GSK3 inhibitor is 10 nM to 0.1 uM. In distal Oct4 enhancer element for OCT4 expression. In some some embodiments, the concentration of GSK3 inhibitor is embodiments, the cell is maintained in culture until the cell 0.1 uM to 0.2 uM. In some embodiments, the concentration has enhanced utilization of the distal Oct4 enhancer element of GSK3 inhibitor is 0.2 uM to 0.5 LM. In some embodi for OCT4 expression as compared to the cell prior to the ments, the concentration of GSK3 inhibitor is 0.5uM to 1 culturing/maintenance period. In some embodiments, the LM. In some embodiments, the culture medium contains a cell is maintained in culture until the cell has enhanced BRAF inhibitor, a MEK inhibitor, a ROCK inhibitor, and an utilization of the endogenous distal Oct4 enhancer element Src inhibitor, and further contains a GSK3 inhibitor in for OCT4 expression as compared to the cell prior to the concentration range listed above in this paragraph. In some culturing/maintenance period. The utilization of the distal embodiments, the culture medium contains a BRAF inhibi OCT4 enhancer element can be tested using the OCT4-APE tor and a MEK inhibitor, and further contains a GSK3 GFP reporter system described in the Examples below. inhibitor in concentration range listed above in this para 0.137 In some embodiments, the at least one property graph. In some embodiments, the culture medium contains which is similar to the corresponding property of mouse a BRAF inhibitor, a MEK inhibitor, and a ROCK inhibitor embryonic stem cells is colony morphology. Naive pluripo and further contains a GSK3 inhibitor in concentration range tent cells that correspond to the more immature ground listed above in this paragraph. state' of pluripotency, exhibit a dome-like colony morphol 0.141. The cell culture medium may be a basal medium. ogy. Thus, in certain embodiments, the cell is maintained in As used herein, a "basal medium' is typically an unsupple culture until it exhibits a dome-like colony morphology. mented medium (e.g., Eagle's minimal essential medium 0.138. In some embodiments, the at least one property (EMEM); Dulbecco's modified Eagle's medium (DMEM)). which is similar to the corresponding property of mouse As will be appreciated by those of skill in the art, a basal embryonic stem cells is gene expression profile. The cell is medium can comprises a variety of components such as one maintained in culture until it has a global gene expression or more amino acids (e.g., non-essential amino acids, essen profile which clusters with naive mouse ESCs as opposed to tial amino acids), salts (e.g., calcium chloride, potassium mouse EpiSCs and/or less naive human ESCs. In some chloride, magnesium Sulfate, Sodium chloride, and mono embodiments, the gene expression profile includes markers Sodium phosphate), Sugars (e.g., glucose), and Vitamins of ground State pluripotency, such as, but not limited to, (e.g., folic acid, nicotinamide, riboflavin, B12), iron and pH NANOG, OCT4, DPPA5, DPPA3 (also known as STELLA), indicators (e.g., phenol red). The basal medium can further KLF4, KLF5, TFCP2L1, and/or REX1. In some embodi comprise proteins (e.g., albumin), hormones (e.g., insulin), ments, the cell is maintained in culture until it exhibits a glycoproteins (e.g., transferrin), minerals (e.g., selenium), gene expression profile similar to that shown in FIG. 6. serum (e.g., fetal bovine serum), antibiotics, antimycotics 0.139. Some aspects of the invention relate to a method and glycosaminoglycans. for culturing vertebrate cells, the method comprising grow 0142. In some embodiments, the basal medium is serum ing vertebrate cells in a cell culture medium comprising a free medium. In some embodiments, the basal medium is serine/threonine-protein kinase B-Raf (BRAF) inhibitor, an supplemented with one or more of the following: DMEM/ US 2017/0114323 A1 Apr. 27, 2017 F12, Neurobasal, N2 supplement, 10 mL B27 supplement, protein kinase (Src) inhibitor. In some embodiments, the kit human LIF, glutamine, nonessential amino acids, B-mercap further comprises a ROCK inhibitor and/or an Src inhibitor. toethanol, penicillin-Streptomycin, and/or BSA (Sigma). In In some embodiments, the kit comprises a BRAF inhibitor, Some embodiments, the Supplemented basal medium further a MEK inhibitor, and a GSK3 inhibitor. In some embodi comprises fibroblast growth factor 2 (FGF2) and 1%, 0.8%, ments, the kit comprises a BRAF inhibitor, a MEK inhibitor, 0.6%, 0.4%, 0.2%, or 0.1% KSR. a GSK3 inhibitor, and a ROCK inhibitor. In some embodi 0143 Some aspects of the disclosure relate to naive ments, the kit comprises a BRAF inhibitor, a MEK inhibitor, pluripotent vertebrate cells produced by the methods a GSK3 inhibitor, a ROCK inhibitor, and an Src inhibitor. In described herein. Some aspects of the disclosure relate to some embodiments, the kit comprises a BRAF inhibitor and compositions comprising the naive pluripotent vertebrate a MEK inhibitor. In some embodiments, the kit comprises a cells produced by the methods described herein. The dis BRAF inhibitor, a MEK inhibitor, and a ROCK inhibitor. In closure provides pluripotent cells, cell lines, and cell clones some embodiments, the kit comprises a BRAF inhibitor, a derived or cultured using the methods and/or compositions MEK inhibitor, a ROCK inhibitor, and an Src inhibitor. In described herein. The invention further provides cell cul some embodiments, the kit does not comprise a GSK tures, wherein at least some of the cells in the cell culture are inhibitor. In some embodiments, the kit further comprises derived or cultured using the methods and/or compositions cell culture medium. The vertebrate cells can be naive described herein. Some aspects of the invention relate to a pluripotent cells, human pluripotent cells, or naive human naive pluripotent vertebrate cell line, e.g., a naive pluripo pluripotent cells. The compositions or contents of the kits tent human cell line, produced by the methods described can be provided in one or more containers (e.g., compounds herein. In some aspects, a naive pluripotent vertebrate cell is that are compatible can be provided together in the same provided, wherein the cell uses the distal Oct4 enhancer container). The components of the kit may be sterile. element for OCT4 expression. In some embodiments, the 0147 Some aspects of the invention relate to a kit for cell primarily uses the endogenous distal OCT4 enhancer preparing a cell culture medium. The kit comprises a serine/ element, e.g., the ratio of the cells use of endogenous distal threonine-protein kinase B-Raf (BRAF) inhibitor, an epi OCT4 enhancer to the cells use of endogenous proximal dermal growth factor receptor (EGFR) inhibitor, a vascular OCT4 enhancer is at least about 1:1 (e.g., about 2:1, about endothelial growth factor 1 (VEGFR1) inhibitor, or a fibro 3:1, about 5:1, or about 10:1). In some aspects, a naive blast growth factor receptor 1 (FGFR1) inhibitor; and pluripotent vertebrate cell, e.g., a naive pluripotent human instructions for preparing a cell culture medium. In some cell, is provided, wherein the cell has a global gene expres embodiments, the kit further comprises mitogen-activated sion profile which clusters with naive mouse ESCs as protein kinase kinase (MEK) inhibitor. In some embodi opposed to stem cell lines derived from mouse epiblast ments, the kit further comprises a glycogen synthase kinase (EpiSCs) and/or less naive human ESCs. In some embodi 3 (GSK3) inhibitor, a rho-associated protein kinase (ROCK) ments, these cells are produced by methods described inhibitor, and/or a proto-oncogene tyrosine-protein kinase herein. In some aspects, provided herein are conventional (Src) inhibitor. In some embodiments, the kit further com pluripotent vertebrate cells (e.g., conventional pluripotent prises a ROCK inhibitor and/or an Src inhibitor. In some human cells) with a deletion of the proximal OCT4 enhancer embodiments, the kit comprises a BRAF inhibitor, a MEK element (or other disabling mutation). In some aspects, inhibitor, and a GSK3 inhibitor. In some embodiments, the provided herein are methods of using the conventional kit comprises a BRAF inhibitor, a MEK inhibitor, a GSK3 pluripotent vertebrate cells (e.g., conventional pluripotent inhibitor, and a ROCK inhibitor. In some embodiments, the human cells) with a deletion of the proximal OCT4 enhancer kit comprises a BRAF inhibitor, a MEK inhibitor, a GSK3 element (or other disabling mutation), examples of Such inhibitor, a ROCK inhibitor, and an Src inhibitor. In some methods including methods of identifying compounds that embodiments, the kit comprises a BRAF inhibitor and a induce a naive pluripotent state. MEK inhibitor. 0144. In some embodiments, at least 80% or at least 90% 0.148. In some embodiments, the kit comprises a BRAF of the pluripotent stem cells of a colony, cell line, or cell inhibitor, a MEK inhibitor, and a ROCK inhibitor. In some culture express one or more marker(s), e.g., a set of markers, embodiments, the kit comprises a BRAF inhibitor, a MEK indicative of pluripotency, e.g., a ground state of pluripo inhibitor, a ROCK inhibitor, and an Src inhibitor. In some tency. In some embodiments at least 91%, at least 92%, at embodiments, the kit does not comprise a GSK3 inhibitor. least 93%, at least 94%, at least 95%, at least 96%, at least 0149. In some embodiments, the kit further comprises a 97%, at least 98%, at least 99%, or more of the cells of a basal medium. As used herein, a “basal medium' is typically colony, cell line, or cell culture express the marker(s). an unsupplemented medium (e.g., Eagle’s minimal essential 0145 Some aspects of the invention provide a kit for medium (EMEM); Dulbecco's modified Eagle's medium culturing vertebrate cells. The kit comprises a serine/threo (DMEM)). As will be appreciated by those of skill in the art, nine-protein kinase B-Raf (BRAF) inhibitor, an epidermal a basal medium can comprises a variety of components such growth factor receptor (EGFR) inhibitor, a vascular endothe as one or more amino acids (e.g., non-essential amino acids, lial growth factor 1 (VEGFR1) inhibitor, or a fibroblast essential amino acids), salts (e.g., calcium chloride, potas growth factor receptor 1 (FGFR1) inhibitor. In some sium chloride, magnesium sulfate, sodium chloride, and embodiments, the kit also comprises instructions for cultur monosodium phosphate), Sugars (e.g., glucose), and Vita ing vertebrate cells. mins (e.g., folic acid, nicotinamide, riboflavin, B12), iron 0146 In some embodiments, the kit further comprises and pH indicators (e.g., phenol red). The basal medium can mitogen-activated protein kinase kinase (MEK) inhibitor. In further comprise proteins (e.g., albumin), hormones (e.g., Some embodiments, the kit further comprises a glycogen insulin), glycoproteins (e.g., transferrin), minerals (e.g., synthase kinase 3 (GSK3) inhibitor, a rho-associated protein Selenium), serum (e.g., fetal bovine serum), antibiotics, kinase (ROCK) inhibitor, and/or a proto-oncogene tyrosine antimycotics and glycosaminoglycans.