Defensive Appeal After Substitutions

 
One out with runners on 3rd base (R3) and 2nd base (R2). The batter (B1) doubles, so R3 scores, but R2 is thrown out at the plate for the 2nd out. However, B1 missed 1st base on his way to 2nd base. The defensive coach calls "time" to visit his pitcher and decides to put a new pitcher in. After the new pitcher throws his warm up pitches, the offensive coach decides to put in a pinch runner for B1 and then decides to put in a pinch hitter for B2. The plate umpire finally puts the ball in play, but now the defensive coach calls "time" and verbally appeals that the original B1 missed 1st base (15 minutes ago!).
 
Can the defense appeal after so much time and so many things have transpired? If the appeal is valid and the runner at 2nd base is called out, are there any other actions?
 
Call
None of the actions by the offense or defensive prohibit an appeal on the missed base. The appeal is proper and upheld, so the batter-runner is out -- for the third out and the inning is over. However, there is another action. Since the third out is a force out, R3's run (which scored 15 minutes before the end of the inning) does NOT count (NF 9-1-1d) -- take the run off the board (but be ready to provide a detailed explanation to the offensive coach).