Improved synthesis of EM-1745, preparation of its C17-ketone analogue and comparison of their inhibitory potency on 17β-hydroxysteroid dehydrogenase type 1

Figures & data

Figure 1.  17β-HSD1 catalyzes the reduction of estrone (E₁) into estradiol (E₂) with the cofactor NADH or NADPH.

Figure 2.  Chemical structure of the bisubstrate inhibitors of 17β-HSD1: EM-1745 (1) and its C17-ketone analogue 2. Illustrated only for 1 and 2, the stereogenic centers are the same for all other steroid derivatives reported in this paper.

Table I.  ¹³C NMR data for compounds 2, 7-11.

C #	2^*	7^†	8^†	9^†	10^†	11^†
C1′	126.0	126.9	126.9	126.9	126.8	126.8
C2′	112.8	117.9	117.8	117.9	113.4	113.4
C3′	155.0	154.0	153.9	153.9	155.7	155.8
C4′	115.0	120.6	120.5	120.5	115.7	115.8
C5′	137.1	138.4	138.4	138.4	138.3	138.2
C6′	29.1	‡	‡	‡	‡	‡
C7′	28.0	28.0	27.9	28.0	28.2	27.32
C8′	c	40.5	40.5	39.2 (40.5)	41.2	38.7
C9′	47.8	44.6 (44.8)	44.2	44.6 (44.76)	44.8	49.4
C10′	130.0	133.9 (134.0)	134.0	133.9 (134.0)	132.0	131.4
C11′	26.3	26.9 (27.0)	26.9 (27.0)	27.1	27.1	26.5
C12′	37.6	38.7 (39.1)	38.7 (39.0)	39.5	38.6	34.1
C13′	48.0	44.6 (44.8)	44.6 (44.7)	44.2 (44.79)	44.7	48.7
C14′	48.5	49.4 (49.6)	49.3 (49.5)	49.4 (49.6)	49.4	49.6
C15′	28.4	32.4 (32.6)	32.8 (32.9)	33.0 (33.1)	33.2	28.5
C16′	43.6	39.2 (39.4)	39.4 (39.5)	39.06 (39.10)	39.3	44.8
C17′	221.7	86.1 (86.4)	86.0 (86.6)	86.1 (86.7)	82.49	221.5
C18′	13.7	13.6	13.4	13.5	12.9	14.2
(CH3)2Si	–	− 4.2	− 4.1	− 4.4	–	–
(CH3)3C-Si	–	18.6	18.5	18.5	–	–
(CH3)3C-Si	–	25.9	25.8	25.9	–	–
CHO of THP	–	98.1 (99.6)	98.0 (99.4)	98.1 (99.4)	–	–
CH2O of THP	–	62.0 (62.9)	61.9 (62.8)	61.9 (62.8)	–	–
CH2CHO of THP	–	31.5 (31.6)	31.3 (31.6)	31.4 (31.6)	–	–
CH2CH2O of THP	–	26.2 (26.3)	26.2 (26.3)	26.2 (26.3)	–	–
CH2CH2CHO of THP	–	20.0 (20.5)	19.9 (20.4)	20.4	–	–
CH = CH	–	130.9 (131.0) 131.3	–	–	–	–
CHO or COO	172.8	202.6	174.4	173.2	173.2	173.2
CH2CHO or CH2COO	33.3	44.2	34.0	38.8	34.1	32.9
C1′ of ribose	87.7	–	–	91.0	91.0	90.9
C2′ of ribose	72.9	–	–	84.7	84.7	84.7
C3′ of ribose	70.3	–	–	82.6	82.46	82.5
C4′ of ribose	81.4	–	–	85.4	85.3	85.3
C5′ of ribose	63.7	–	–	64.3	64.3	64.3
C(CH3)2	–	–	–	25.41 & 27.3	25.4 & 27.3	25.3 & 27.26
C(CH3)2	–	–	–	114.5	114.4	114.4
C adenine	156.1	–	–	157.1	157.0	157.0
C adenine	152.7	–	–	153.6	153.6	153.6
C adenine	149.3	–	–	150.0	150.0	150.0
C adenine	139.7	–	–	140.5	140.6	140.6
C adenine	119.1	–	–	120.7	120.9	120.6
C alkyl chain	31.6	36.3 (36.5)	33.1	34.2	32.52	32.7
C alkyl chain	28.8	33.0	25.5	30.4	32.48	6C^‡
C alkyl chain	28.7	30.5	5C^‡	25.37	30.22	–
C alkyl chain	28.6	22.5	–	4C^‡	29.1	–
C alkyl chain	27.4	1C^‡	–	–	25.3	–
C alkyl chain	25.5	–	–	–	2C^‡	–
C alkyl chain	24.4	–	–	–	–	–

^* DMSO-d₆ as solvent

^† acetone-d₆ as solvent

^‡ under solvent peak.

Scheme 1.  A new procedure for preparing EM-1745 (1) and synthesizing the C17-ketone analogue of EM-1745 (2). Reagents, conditions and yields: (a) NaI, acetone, reflux, 16 h; (b) i. CuI, vinylMgBr, THF, − 40°C, 15 min; ii. HMPA, P(OEt)₃, THF, − 40°C to rt, 5 h (75% for two steps); (c) TPAP, NMO, molecular sieves, DCM, rt, 90 min (51%); (d) 5, 2nd generation Grubbs' catalyst, DCM, reflux, 16 h (50%); (e) 10% Pd/C, H₂, EtOAc, rt, 16 h; (f) NaClO₂, NaH₂PO₄, THF, 2-methyl-2-butene, t-BuOH, rt, 30 min (75% for two steps); (g) 2′,3′-isopropylidene adenosine, PyBOP, HOBt, DIPEA, DMF, rt, 16 h (51%); (h) HCl_g, DCM, rt, 3 h (27%); (i) PPTS, DCM, MeOH, reflux, 5 h; (j) TBAF, THF, 0°C, 10 min (72% for two steps); (k) Jones' reagent, acetone, 0°C, 8 min (52%); (l) TFA/H₂O, 9:1, THF, rt, 30 min (56%).

Figure 3.  Inhibition of the transformation of [¹⁴C]-E₁ into [¹⁴C]-E₂ by EM-1745 (1) and its C17-ketone analogue 2 in homogenated HEK-293 cells everexpressing 17β-HSD1. See experimental section for more details. Data with the same symbol (* or **) are significantly different (P < 0.01).

Table II.  Inhibition of 17β-HSD1 by different kinds of inhibitors.

			Inhibition of 17β-HSD1
Entry	Compounds	C17 (X)	% at 0.01 mM	% at 0.1 mM	% at 1 mM	IC50 (nM)
1^*	1 (EM-1745)	β-OH	73	95	98	4
	2	O	44	84	96	12
2^* [51]	12	β-OH	–	15	46	–
	13	O	–	12	23	–
3^† [53]	14	β-OH	–	77	94	44
	15	O	–	51	88	171
4^‡ [54]	16	β-OH	–	–	–	460
	17	O	–	–	–	310
5^‡ [54]	Unlabeled-E2	β-OH	–	–	–	>1000
	Unlabeled-E1	O	–	–	–	300

^* Inhibition of the transformation of [¹⁴C]-E₁ (100 nM) into [¹⁴C]-E₂ in homogenated HEK-293 cells everexpressing 17β-HSD1. See experimental section for more details

^† Inhibition of the transformation of [¹⁴C]-E₁ (60 nM) into [¹⁴C]-E₂ by 17β-HSD1 in breast cancer T-47D cells

^‡ Inhibition of the transformation of [³H]-E₁ (6 nM) into [³H]-E₂ in partially purified human placenta cytosolic 17β-HSD1.

Figure 4.  A selection of our previously reported inhibitors of 17β-HSD1.

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